Qiu et al Nanoscale Research Letters 2012 7666httpwwwnanoscalereslettcomcontent71666

NANO EXPRESS Open Access

Co-delivery of docetaxel and endostatin by abiodegradable nanoparticle for the synergistictreatment of cervical cancerBo Qiu13dagger Minghui Ji2dagger Xiaosong Song34 Yongqiang Zhu34 Zhongyuan Wang34 Xudong Zhang34 Shu Wu34Hongbo Chen34 Lin Mei345 and Yi Zheng345

Abstract

Cervical cancer remains a major problem in womens health worldwide In this research a novel biodegradable d-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) (TPGS-b-(PCL-ran-PGA))nanoparticle (NP) was developed as a co-delivery system of docetaxel and endostatin for the synergistic treatmentof cervical cancer Docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs were prepared and further modified bypolyethyleneimine for coating plasmid pShuttle2-endostatin All NPs were characterized in size surface chargemorphology and in vitro release of docetaxel and pDNA The uptake of coumarin 6-loaded TPGS-b-(PCL-ran-PGA)PEI-pDsRED by HeLa cells was observed via fluorescent microscopy and confocal laser scanning microscopyEndostatin expression in HeLa cells transfected by TPGS-b-(PCL-ran-PGA)PEI-pShuttle2-endostatin NPs was detectedusing Western blot analysis and the cell viability of different NP-treated HeLa cells was determined by MTT assayThe HeLa cells from the tumor model nude mice were treated with various NPs including docetaxel-loaded-TPGS-b-(PCL-ran-PGA)PEI-endostatin NPs and their survival time tumor volume and body weight were monitoredduring regimen process The tumor tissue histopathology was analyzed using hematoxylin and eosin staining andmicrovessel density in tumor tissue was evaluated immunohistochemically The results showed that the TPGS-b-(PCL-ran-PGA)PEI NPs can efficiently and simultaneously deliver both coumarin-6 and plasmids into HeLa cells andthe expression of endostatin was verified via Western blot analysis Compared with control groups the TPGS-b-(PCL-ran-PGA)PEI-pShuttle2-endostatin NPs significantly decreased the cell viability of HeLa cells (p lt 001)inhibited the growth of tumors and even eradicated the tumors The underlying mechanism is attributed tosynergistic anti-tumor effects by the combined use of docetaxel endostatin and TPGS released from NPs TheTPGS-b-(PCL-ran-PGA) NPs could function as multifunctional carrier for chemotherapeutic drugs and geneticmaterial delivery and offer considerable potential as an ideal candidate for in vivo cancer therapy

Keywords TPGS-b-(PCL-ran-PGA) nanoparticles cervical cancer endostatin docetaxel

BackgroundCervical cancer caused by high-risk human papilloma-virus (HPV) persistent infection is the second mostcommon cancer in women worldwide [12] Fortunatelytwo HPV vaccines (Gardasil (Merck Sharp amp DohmeCorp NJ USA) and Cervarix (GlaxoSmithKline TW

Correspondence meilinsztsinghuaeducn zhengysztsinghuaeducndaggerEqual contributors3The Shenzhen Key Lab of Gene and Antibody Therapy Graduate School atShenzhen Tsinghua University Shenzhen 518055 Peoplersquos Republic of China4School of Life Sciences Tsinghua University Beijing 100084 PeoplersquosRepublic of ChinaFull list of author information is available at the end of the article

copy 2012 Qiu et al licensee Springer This is an OAttribution License (httpcreativecommonsorin any medium provided the original work is p

UK)) have been approved for use in many countries thatwould effectively reduce the incidence of cervical cancergenesis However these two vaccines have not shownany therapeutic effect against current cervical cancerHPV infections or associated lesions [3] Thus there areurgent needs to develop new specific drugs or noveltherapeutic methods for cervical cancer treatmentIt has been well established that the progression of

solid tumors including cervical cancer is criticallydependent on neoangiogenesis for nutrition and oxy-gen supply Therefore blockade of neoangiogenesis hasbeen regarded as a promising strategy for tumor

pen Access article distributed under the terms of the Creative Commonsglicensesby20) which permits unrestricted use distribution and reproductionroperly cited

Qiu et al Nanoscale Research Letters 2012 7666 Page 2 of 11httpwwwnanoscalereslettcomcontent71666

therapy [4-8] Endostatin a 20 kDa C-terminal proteo-lytic fragment of collagen XVIII has received thegreatest attention for its broad-spectrum and low-toxicanti-angiogenesis ability [6910] These advantages ac-celerate the investigation process of endostatin into theclinical trial [1112] However as a protein endostatinhas many challenges in its clinical application such asshort half-life and instability Recently gene therapythe use of DNA as a pharmaceutical agent has beenextensively studied in a broad range of diseases includ-ing tumors which can achieve a relative long-termstable expression of therapeutic proteins [13-16] Amajor limitation of gene therapy is the efficient deliv-ery of therapeutic DNA to the target cells and tissuesThe main vectors for DNA delivery are viral vectorsand non-viral vectors having great advantages in safetyconvenient large-scale production physiological stabil-ity and no immunogenicityCurrently a variety of particulate drug delivery sys-

tem based on synthetic and natural materials has beeninvestigated as DNA carriers including liposomes[17] dendrimers [18] polycationic polymers [1920]and polymeric nanoparticles (NPs) [21] Among themPEI-based NPs are considered as one of the most ef-fective carrier since it can complex with pDNA orsiRNA and exhibit a unique lsquoproton sponge effectrsquo forendosomal release of the nano-complexes into cytosol[22] On the other hand many chemotherapeuticdrugs such as paclitaxel doxorubicin and mitomycinare also limited because of low water solubility acutetoxicity to normal tissues due to lack of targetingand multi-drug resistance Several researches showedthat the combination of endostatin and chemothera-peutic drugs such as docetaxel can result in better in-hibitory effects for tumor growth than single therapy[23] However the clinical applications of these com-bined drugs are needed to develop new efficient drugdelivery systemA novel biodegradable copolymer d-α-tocopheryl

polyethylene glycol 1000 succinate-b-poly(ε-caprolac-tone-ran-glycolide) (TPGS-b-(PCL-ran-PGA)) has beensuccessfully synthesized in our laboratory [24] Ourprevious research has confirmed that docetaxel-loadedTPGS-b-(PCL-ran-PGA) NPs can be efficiently uptakenby MCF-7 cells and effectively inhibit the growth oftumor over a longer period of time than commercialTaxotereW (Sanofi-Aventis US LLC NJ USA) at thesame dose To overcome the limitations of single ther-apy we modified the TPGS-b-(PCL-ran-PGA) NPswith a polyplexed PEI and evaluated its ability to sim-ultaneously deliver DNA and a chemotherapeutic drugin cells Most importantly we examined its therapeuticeffect on xenograft models bearing cervical cancercells

MethodsPolymers and reagentsTPGS-b-(PCL-ran-PGA) copolymer (Mw approximately24000) was synthesized in our laboratory (TsinghuaUniversity China) Poly (vinyl alcohol) (PVA (80hydrolyzed) dichloromethane branched polyethyleni-mine (MW approximately 25000 BPEI25k) and 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) kit were purchased from Sigma-Aldrich Corpor-ation (MO USA) 46-diamidino-2-phenylindole (DAPI)was purchased from VECTOR (Burlingame USA) Dul-beccos Modified Eagles Medium (DMEM) penicillin-streptomycin fetal bovine serum (FBS) and Dulbeccosphosphate buffered saline (DPBS) were purchased fromInvitrogen-Gibco (Carlsbad CA) Plasmid vectors pShut-tle2 pIRES-EGFP and pDsRED-E1 were acquired fromInvitrogen Corporation (Clontech USA)

Preparation of expression vectorsHuman endostatin gene was amplified by PCR using thefollowing primers hEndostatin-F 50-GCTCTAGA(XbaІ)gccaccatgggaattcatgcacagccaccgcgacttcc-30 and hEndos-tatin-R 50-GGGGTACC (KpnІ)ttacttggaggcagtcatg-30PCR products were digested with Xba I and KpnІ andinserted into pShuttle2 vector (Clontech) The recom-binant plasmid pShuttle2-endostatin was verified byDNA sequencing The expression of endostatin in HeLacells transfected with PEI or TPGS-b-(PCL-ran-PGA)-based NPs was analyzed by Western blot analysis

Preparation of docetaxel-loaded TPGS-b-(PCL-ran-PGA)NPs and determination of drug contentsDocetaxel-loaded and blank TPGS-b-(PCL-ran-PGA)NPs were prepared using a modified water-in-oil-in-water solvent evaporation as described previously [25]Briefly TPGS-b-(PCL-ran-PGA) copolymer (50 mg) anda certain amount of docetaxel (0 to 25mg) were dis-solved in 2 ml methylene chloride followed by 30-ssonication to emulsify the mixture (UW 70HD 70 tipMS 72D Bandelin Electronic GmbH amp Co BerlinGermany) After the addition of 3 ml of 7 (wv) aque-ous solution of PVA the emulsion was sonicated againfor 20 s The resulting double emulsion was then pouredinto 50 ml 1 (wv) aqueous PVA solution containing2 isopropanol and then maintained under mechanicalstirring for 1 h at 600 rpm The residual methylenechloride was then evaporated by vacuum Next the 2 mlaliquots of the nanosphere suspension were washedtwice with 20 mM 4-(2-hydroxyethyl)-1-piperazineetha-nesulfonic acid (HEPES)NaOH (pH 70) and the NPswere harvested by centrifugation at 7000 rpm for 10min In addition the fluorescent coumarin-6 loadedTPGS-b-(PCL-ran-PGA) NPs were prepared in the same

Qiu et al Nanoscale Research Letters 2012 7666 Page 3 of 11httpwwwnanoscalereslettcomcontent71666

way except that 01 (wv) coumarin-6 was entrappedinstead of docetaxelThe docetaxel-loading content defined as the mass

percentage of docetaxel entrapped in nanoparticles wasquantified using UVndashvis analysis (UV-2000 UVndashvis spec-trophotometer Unico Inc WI USA) First docetaxel-loaded nanoparticle solutions were lyophilized to yield thesolid nanoparticle samples Then the dried nanoparticlesamples were weighed and redissolved in a mixture ofchloroform and DMSO (11 vv) The absorbance of doce-taxel at 4825 nm was measured and the drug content inthe solution was calculated based on a previously estab-lished calibration curve

Preparation of PEI-modified TPGS-b-(PCL-ran-PGA) NPsDNA complexesThe NPs were prepared as described previously [25]Briefly the particles were formulated with a ratio of thepolymer nitrogen to the DNAphosphate (NP) equal toTPGS-b-(PCL-ran-PGA) NP solution (02 ml) and weremixed with 2 mg of PEI in sterile HEPES-buffered salineThe PEI-modified TPGS-b-(PCL-ran-PGA) NP solutionwas then added to the plasmid DNA solution at differentNP ratios and vortexed gently The PEI-modified TPGS-b-(PCL-ran-PGA) NPDNA complexes were incubated insterile PBS for 20 min at room temperature

Characterization of nanoparticlesThe mean particle size and size distribution were mea-sured using dynamic light scattering (DLS) (ZetasizerNano ZS90 Malvern Instruments Ltd Malvern UK) Inbrief the NPs were suspended in deionized water at aconcentration of 01 mgml The mean hydrodynamicdiameter was determined via cumulative analysis TheDLS determinations were predicated based on the elec-trophoretic mobility of the NPs in an aqueous mediumThe electrophoretic mobility was evaluated using foldedcapillary cells in an automatic mode Zeta potential ofthe NPs was detected using laser Doppler anemometry(LDA Zetasizer Nano ZS90 Malvern Instruments Ltd)Samples were prepared in PBS and diluted 13 with deio-nized water to ensure that the measurements were per-formed under conditions of low ionic strength where thesurface charge of the particles can be measured accuratelyThe final concentration of the polymer was 001 mgmlAll data represent five measurements from one sample

Gel retardation assayThe binding of pDNA with free TPGS-b-(PCL-ran-PGA)PEI NPs was determined by 08 agarose gel elec-trophoresis A series of different weight ratios (ww) ofpDNA to NPs was loaded on the agarose gel (10 μl ofthe sample containing different amounts of pDNA) The

pDNA bands were then visualized under a UV transillu-minator at a wavelength of 365 nm

Evaluation of loading efficiency of pDNA to the NPsThe loading efficiency of pDNA to the NPs was evalu-ated by measuring unbound pDNA in the NPs solutionThe supernatant in the loaded docetaxel or free TPGS-b-(PCL-ran-PGA)PEI NPs solution was recovered byremoving the NPs by centrifugation Free pDNA concen-tration in the supernatant was determined by measuringabsorbance at 260 nm using a UV-2550 spectrophotom-eter (Shimadzu Corp Kyoto Japan) Loading efficiencyof pDNA in NPs was determined by subtracting theamount of pDNA recovered in the washing solutionsfrom initial amount of pDNA added Encapsulation effi-ciency was defined as the percentage of pDNA encapsu-lated in NPs with respect to the initially added amountof pDNA

In vitro drug releaseNPs with 15 mg docetaxel-loaded TPGS-b-(PCL-ran-PGA) were dispersed in 5 ml release medium (phosphatebuffer solution of pH 74 containing 01 wv Tween 80)to form a suspension Tween 80 was used to increasethe solubility of docetaxel in the buffer solution andavoid the adherence of docetaxel to the tube wall Thesuspension was transferred into a dialysis membrane bag(SpectraPor 6 MWCO = 1000 Spectrum LaboratoriesInc TX USA) Then the closed bag was put into a cen-trifuge tube and immersed in 15-ml release mediumThe tube was put in an orbital water bath at 37degC undera 120 rpm horizontal shaking Ten milliliters of solutionwas periodically removed for analysis and was replacedwith fresh release medium The collected samples weremixed in a mixture of chloroform and DMSO (11 vv)The analysis procedure was the same as describedaboveTo investigate the in vitro release of pDNA 5 mg of

TPGS-b-(PCL-ran-PGA)PEI-25K (n = 6) and coumarin-6-loaded TPGS-b-(PCL-ran-PGA)PEI (n = 6) wereincubated in 1 ml of DPBS buffer (pH 74) in a micro-centrifuge tube in shaking incubator at 37degC After in-cubating for 24 h half of the samples (n = 3) weretransferred into a 25 mM sodium acetate buffer (pH 50)to simulate acidication of the endolysosome of the cellSamples were taken periodically in microcentrifuge tubesand were centrifuged at 14000 rpm for 10 min to obtainpellet NPs The supernatants were removed and replacedwith fresh buffer and NPs were resuspended by vigorouspipetting The supernatants were stored at minus70degC untilanalysis by UV measurement Unmodified TPGS-b-(PCL-ran-PGA) NPs were also analyzed as a backgroundcontrol

Qiu et al Nanoscale Research Letters 2012 7666 Page 4 of 11httpwwwnanoscalereslettcomcontent71666

Cell cultureHeLa and 293T cells (ATCC VA USA) were cultured inDMEM pH 74 supplemented with 25 mM NaHCO310 μgml streptomycin sulfate 100 μgml penicillin Gand 10(vv) FBS Cells were maintained at 37degC in a 5CO2 95 air incubator

Cell viabilityThe cytotoxicity of the NPs was determined using MTTassay The culture medium was removed and replacedwith 20 μlwell MTT (5 mgml) solution in each wellfollowed by 4-h incubation at 37degC in a fully humidifiedatmosphere with 5 CO2 MTT was taken up by activecells and transformed into insoluble purple formazangranules in the mitochondria Subsequently the mediumwas discarded the precipitated formazan was dissolvedin DMSO (150 μlwell) and optical density of the solu-tion was evaluated using a microplate spectrophotom-eter at a wavelength of 570 nm The analytic assays wereperformed every day and at least 4 wells were randomlytaken into examination each time The determination ofcell viability depends on these physical and biochemicalproperties of cells

Cellular uptake of PEI-modified TPGS-b-(PCL-ran-PGA) NPsThe cells were plated in a 6-well (3 times 105 per well) platecultured at 37degC in 5 CO2 rinsed twice and pre-incubated for 1 h with 2 ml of serum-free medium at37degC Coumarin-6-loaded TPGS-b-(PCL-ran-PGA)PEINPs with or without pDsRED or pIRES-EGFP wereadded in a particle concentration of 001 to 02 mgmland incubated for 1 to 4 h at 37degC The cells were thenwashed three times with 1 ml of PBS (pH 74) to removefree PEIplasmids-modified TPGS-b-(PCL-ran-PGA)NPs resuspended in PBS and then analyzed by fluores-cent microscopyFor confocal laser microscopy analysis cells were pre-

incubated for 1 h at 37degC in serum-free medium andthen for 30 min to 4 h in the presence of PEIplasmidspIRES-EGFP orand pDsRED gene-modified TPGS-b-(PCL-ran-PGA) NPs with a final particle concentrationof 005 mgml The samples were mounted in fluores-cent mounting medium (Dako Glostrup Denmark) andfluorescence was monitored Images were acquired usinga confocal laser microscope (Eclipse TE 2000 NikonCorporation Tokyo Japan)

Western blotThe cells were seeded into 6-well plates overnightallowed to attach the bottom Cells were cultured at 37degCin 5 CO2 rinsed twice and pre-incubated for 1 h with2 ml of serum-free medium at 37degC Endostatin gene-modified TPGS-b-(PCL-ran-PGA) NPs were added ina particle concentration of 001 to 02 mgml and

incubated for 1 to 4 h at 37degC The cells were thenwashed three times with 1 ml PBS (pH 74) to removeany free PEIplasmid-modified TPGS-b-(PCL-ran-PGA)NPs After being cultured in fresh complete medium for48 h the cells were lysed with lysis buffer (Beyotime In-stitute of Biotechnology Haimen Jiangshu China) con-taining PMSF (Sigma Chemical Co St Louis MI USA)for 30 min at 4degC The lysate was then centrifuged for 20min at 13000 rpm and 4degC The proteins were then sepa-rated through SDS-PAGE and transferred onto the PVDFmembrane (Immobion-P Transfer Membrane MilliporeCorp Billerica MA USA) Membranes were blocked ina tris-buffered saline with 01 Tween 20 solution con-taining 5 non-fat dry milk and incubated overnight withanti-human endostatin antibody as primary antibody at4degC Anti-β-actin antibody was used as loading control

Mice maintenance and subcutaneous tumor modelFemale BALBc-nunu mice were supplied by the Med-ical Experimental Animal Center of Guangdong Prov-ince (Guangdong China) A subcutaneous tumor modelof nude mice was constructed Six-week-old female nudemice (18 plusmn 2 g) were subcutaneously inoculated with 15to 2 times 106 HeLa cells When the tumor size reachedabout 100 mm3 the mice were randomly divided intosix groups with six mice each PBS TPGS-b-(PCL-ran-PGA) (group ANP) TPGS-b-(PCL-ran-PGA)PEI(groupBNP) TPGS-b-(PCL-ran-PGA)PEI-pEndostatin (groupCNP) docetaxel-loaded TPGS-b-(PCL-ran-PGA) (groupDNP) and docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pEndostatin (group FNP) of which each dose ofNPs contained 02 mg PCL-PGA-TPGS 10 μg PEI and50 μg pDNA for treatment The groups were treatedonce every 3 days with intratumoral injections of PBS orNPs Tumor size was measured with a caliper and theweight of each mouse was measured with a scale Tumorvolume was calculated as the equation lsquovolume = lengthtimes width2 2rsquo The mean tumor volume and mouseweight were used to construct the curves of tumorgrowth versus mouse growth to evaluate therapeutic effi-ciency and toxicityAt the end of the treatment the mice in each

group were killed and dissected The heart liverlungs spleen kidneys and other organs werechecked for signs of toxicity Tumor specimens werethen prepared as paraffin-embedded sections forhistopathological analysis The study was carried outin strict accordance with the recommendations inthe Guide for the Care and Use of Laboratory Ani-mals of Institutional Animal Care and Use Commit-tee of Graduate School at Shenzhen TsinghuaUniversity The protocol was approved by the Ani-mal Welfare and Ethics Committee of Graduate

Figure 1 Expression of endostatin in HeLa cells using Westernblot analysis Lane 1 HeLa cells were transfected by pShuttle2-endostatin using PEI lane 2 transfected by pShuttle2-endostatinusing TPGS-b-(PCL-ran-PGA)PEI lane 3 transfected by pShuttle2using TPGS-b-(PCL-ran-PGA)PEI

Qiu et al Nanoscale Research Letters 2012 7666 Page 5 of 11httpwwwnanoscalereslettcomcontent71666

School at Shenzhen Tsinghua University China (no2011-YZ-BG)

Measurement of microvessel density in tumor tissuesMicrovessel density (MVD) in tumor tissue was evalu-ated immunohistochemically using a monoclonal anti-

Figure 2 The size of different NPs (mean plusmn SD) (A) ANPs TPGS-b-(PCL-docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI The results showed that the simodified by PEI The zeta potential of the different NPs (B) The zeta potenby PEI was positive and reduced after complexed with pDNA The results sretardation assay of pDNA complexed with TPGS-b-(PCL-ran-PGA)PEI (C) Emodified TPGS-b-(PCL-ran-PGA) NPs was examined by FESEM (D)

mouse CD31 antibody (rat anti-mouse CD31 monoclo-nal antibody clone MEC 133 BD Biosciences NJUSA) Tumor samples were collected after the therapyregimen was finished Immunohistochemical stainingwas performed as described previously [26] MVD ()was calculated from the ratio of the CD31-positive stain-ing area to the total observation area in the viable re-gion Three to six fields per section were randomlyanalyzed excluding necrotic areas Positive stainingareas were calculated using imaging analysis software(Win Roof Mitani Corporation Fukui Japan)

Statistics and data analysisAll cell experiments were repeated at least three timesunless otherwise indicated Images of Western blotresults from representative experiments were presentedThe figures were created using Adobe Photoshop CSgraphics program All data were analyzed by paired t testusing SPSS 110 software Differences were consideredstatistically significant at P lt 005

ran-PGA) NPs DNPs docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs ENPsze of NPs increased because of loading docetaxel andor beingtial of blank NPs was negative the zeta potential of the NPs modifiedhowed that TPGS-b-(PCL-ran-PGA) blank NPs could be modified Gelach lane contained the same pDNA Surface morphology of the PEI-

Figure 3 The in vitro release profile of pDNA from various NPsat pH 74 and pH 50

Figure 4 The in vitro release curve of docetaxel from differentNPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 6 of 11httpwwwnanoscalereslettcomcontent71666

Results and discussionsThe expression of pShuttle2-endostatinProtein expression of endostatin was analyzed by West-ern blot using cell lysate after transfection of HeLa cellsusing PEI (Figure 1) These results showed thatpShuttle2-endostatin was successfully constructed andexpressed in HeLa cells

Characterization of nanoparticlesRecently a novel biodegradable copolymer TPGS-b-(PCL-ran-PGA) has been successfully synthesized inour laboratory [24] Our previous study showed that theNPs made of this novel copolymer can improve drug per-meability solubility degradation and avoid the disadvan-tages of single component [24] More importantly TPGSthe degradation products of TPGS-b-(PCL-ran-PGA)showed synergistic anticancer activity in the presence ofanticancer agents [24] In this research to co-deliverychemotherapeutic drugs and genetic materials into cellsTPGS-b-(PCL-ran-PGA) NPs were modified with cat-ionic polymer PEI (25 K) DLS assay showed that thehydrodynamic diameter of the polyplexed blank TPGS-b-(PCL-ran-PGA) NPs (ANPs) was approximately 215 nmwhile the diameters of the docetaxel-loaded TPGS-b-(PCL-ran-PGA) and docetaxel-loaded TPGS-b-(PCL-ran-PGA) PEI-coated without pDNA NPs were approxi-mately 242 and approximately 270 nm respectively(Figure 2A) The FESEM images showed that the morph-ologies of TPGS-b-(PCL-ran-PGA)PEI NPs weresphere-like in shape and the size was about 250 nm(Figure 2D) consistent with the DLS data The resultsuggested that PEI modification may increase the size ofthe NPs from TPGS-b-(PCL-ran-PGA) copolymer Inaddition the surface charge of the unmodified TPGS-b-(PCL-ran-PGA) NPs was approximately minus1825 mVdetected by LDA assay while the surface charge of the PEI-modified NPs was significantly increased in zeta potentialsuggesting that the surface charge of the TPGS-b-(PCL-ran-PGA) NPs was altered from negative to positive due to thePEI coating However a reduction in surface charge of thePEI-modified NPs was observed after adding pDNAsuggesting PEI condenses negatively charged DNA intopositively charged particles (Figure 2B) Agarose gel electro-phoresis assay showed that the amount of NP-binding DNAincreased as the amount of TPGS-b-(PCL-ran-PGA)PEINPs increased (Figure 2C) In a recent report the DNAencapsulated in the polymeric (PLGA) NPs is in a con-densed form which could protect itself from denaturationand allow it to be efficiently taken up by MSCs [25] An-other study also showed that PLGAPEI NPs can protectDNA against degradation and reduce the cytotoxicity causedby PEI [25] Together results showed that the TPGS-b-(PCL-ran-PGA) NPs modified by PEI could encapsulatedocetaxel and DNA with a high loading efficiency

In vitro releasePrevious study showed that the kinetic of NP degrad-ation and DNA release appears to have a significantregulatory effect on the gene expression [26] In thisstudy we compared the in vitro release of pDNA fromdocetaxel-loaded or doceraxel-free TPGS-b-(PCL-ran-PGA)PEI-pDNA (DNPs or ENPs) formulations at pH74 and pH 50 To simulate acidification in the endo-lysosome of cells following intracellular uptake NPswere first incubated in a solution at pH 74 for 24 hthen one-half of the NPs continued to be incubated at

Figure 5 The fluorescence microscopic images of 293T cells This is after the incubation with plasmids pIRES-EGFP-loaded and pdSRED-loaded TPGS-b-(PCL-ran-PGA)PEI NPs (A) from transmitted light channel (B) from FITC channel (C) from PI channel

Figure 6 Confocal laser scanning microscopy of HeLa cells This is after incubation with loaded-coumarin-6-TPGS-b-(PCL-ran-PGA)PEI-pDSRED NPs at 37degC The cells were stained by DAPI (blue) and the plasmids pDsRED-loaded-TPGS-b-(PCL-ran-PGA)PEI NPs are red The cellularuptake was visualized by overlaying images obtained (A) Coumarin-6 (B) RED (C) DAPI and (D) co-merge

Qiu et al Nanoscale Research Letters 2012 7666 Page 7 of 11httpwwwnanoscalereslettcomcontent71666

Figure 7 Viability of HeLa cells after 24- and 48-h incubations(n = 5) Single asterisk denotes p lt 005 (compared with PBS) whiledouble asterisk indicates p lt 001 (compared with PBS)

Figure 8 The tumor volume curve after animal models weretreated by different NPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 8 of 11httpwwwnanoscalereslettcomcontent71666

pH 74 and another half was transferred to a solution ofpH 50 pDNA release was monitored by measuring UVabsorption at 260 nm at predetermined time points Inthe pH 74 group 405 and 431 of the total pDNAwere released rapidly from docetaxel-loaded NPsPEIand empty NPsPEI respectively at 48 h followed by aslow release until day 7 (Figure 3) These two formula-tions had no significant difference either at initial burstphase or slow release phase at pH 50 suggesting thatthe loaded docetaxel had no significant influence onpDNA release Zolniks group reported that cleavage ofthe ester linkage of the PLGA polymer backbone at lowpH can accelerate the polymer erosion [27] However inthis study the release kinetics of pDNA at pH 74 wassimilar to that at pH 50 but the release of pDNA fromNPs from day 7 at pH 50 was more rapid than that atpH 74 The reason was the degradation of surface ero-sion [27] Being a hydrophilic molecule located at thesurface of the TPGS-b-(PCL-ran-PGA) matrix PEI mayfacilitate the degradation of the NPs by increasing hydra-tion and thereby accelerating hydrolysis [28]In order to investigate the docetaxel release profiles

the three formulations of NPs docetaxel-loaded TPGS-b-(PCL-ran-PGA) docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI and docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pDNA were incubated in 37degC aqueous solutions atpH 74 and pH 50 respectively As shown in Figure 4 atypical release profile was observed for all six testednano-complex solutions including an initial rapid re-lease followed by a sustained slow release over a pro-longed time up to several weeks Furthermore thedocetaxel release at pH 50 was faster than the othernano-assemblies Similar pH-dependent trend was alsoobserved in other docetaxel-loaded polymeric micellarsystems [29] and such behavior was assumed to improveintracellular drug release once the nano-complexes enterthe tumor cells via endocytosis and trapped within theacidic endosomallysosomal compartments In additionboth pDNA complexion and PEI coating were found toprolong docetaxel release which is consistent with theresults for a paclitaxel and Herceptin (Genentech IncCA USA) co-delivery system using cationic micellar NPs[30] These results implied that the release profile of PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPswas prolonged and its toxicity might be decreased

Cellular uptakeTo evaluate cellular uptake of nanoparticles 293T orHeLa cells were incubated with PEI-modified TPGS-b-(PCL-ran-PGA) NPs and coumarin-6-loaded TPGS-b-(PCL-ran-PGA) NPs for 48 h The fluorescence emittedby coumarin-6 and RED protein was observed after NP in-cubation which suggested that the NPs efficiently enteredthe cells The 293T cells took up the particles in a dose-

dependent manner and EGFP and RED expressionachieved peak levels with 15 mgml of PEI complexed with100 mg of NPs (Figure 5) These findings demonstratedthat PEI-modified NPs may be good candidates for thedelivering of genes into tumor cells and PEI-modifiedTPGS-b-(PCL-ran-PGA) nanospheres may increase thegene binding ability which causes the endosomal escapeeasily and lowers the PEI cytotoxicity [25] We also foundthat more PEI-modified coumarin-6-loaded NPs wereuptaken by HeLa cells than unmodified NPs which is inconsistency with other reports [31] It was probably be-cause the PEI-modified NPs had positive charge on surface

Figure 9 The histopathological analysis of the tumor tissues from different treatment groups (A) PBS (B) ANPs (TPGS-b-(PCL-ran-PGA))(C) CNPs (TPGS-b-(PCL-ran-PGAPEIpEndo) (D) DNPs (E) ENPs and (F) FNPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 9 of 11httpwwwnanoscalereslettcomcontent71666

which provided good affiliation with cells which had nega-tive charge on the surface In addition our data alsoshowed that coumarin-6 and RED protein can co-expressin HeLa cells transfected by coumarin-6-loaded TPGS-b-(PCL-ran-PGA)PEI-pDSRED (Figure 6) suggesting thatPEI-modified TPGS-b-(PCL-ran-PGA) can co-deliver che-motherapeutic drugs and genetic materials into cells

Figure 10 MVD of tumor tissue in different treatment groups Single adenotes p lt 001 (compared with PBS)

Cell viabilityCytotoxicity of all NPs was determined in various con-centrations using MTT assay The cell viability was notaffected by TPGS-b-(PCL-ran-PGA) NPs in all concen-trations relative to the control (treated with PBS) after72 h in medium However increasing dosage of PEI-modified TPGS-b-(PCL-ran-PGA) NPs was associated

sterisk indicates p lt 005 (compared with PBS) while double asterisk

Qiu et al Nanoscale Research Letters 2012 7666 Page 10 of 11httpwwwnanoscalereslettcomcontent71666

with increased cytotoxicity because of the increase ofPEI (Figure 7) As expected for TPGS-b-(PCL-ran-PGA) TPGS-b-(PCL-ran-PGA)PEI NPs and coumarin-6-loaded NPs the cell survival rates were greater than84 whereas cell survival rate of the cells treated withequal dose of PEI was less than 70 (data not shown)suggesting that TPGS-b-(PCL-ran-PGA) could reducethe cytotoxicity of PEI (Figure 7) On the other handdocetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pEndos-tatin NPs appeared to have more cytotoxicity than thatof the other control NPs including docetaxel-loadedNPs this may be because endostatin could induce cellapoptosis [32] Among all NPs docetaxel-load NPsPEI-endostatin had the highest cytotoxicityThe cell viability of HeLa cells transfected with

docetaxel-loaded NPs was decreased after 6 h whichwas dose- and time-dependent These results indicatedthat PEI-modified TPGS-b-(PCL-ran-PGA) NPs cansimultaneously delivery docetaxel and genetic materialsinto cancer cells and kill them

In vivo studiesTo investigate the therapeutic effect of TPGS-b-(PCL-ran-PGA)-based NPs on tumors the anti-tumor efficacyand angiogenesis inhibition of various NPs includingdocetaxel-loaded NPs-PEIpEndostatin were further eval-uated in vivo Subcutaneous tumor models were con-structed in nude mice by being inoculated with 1 to 2 times106 HeLa cells The results showed that CNPs ENPs andFNPs containing docetaxel significantly inhibited tumorgrowth compared with PBS ANPs and DNPs (p lt 001)(Figure 8) The tumor volume of the mice treated withFNPs appeared to keep growing in the initial 5 days aftertreatment and then the tumor sites presentedinflammatory-like response and ulcer After 3 weeks thetumor growth was significantly inhibited and 80 tumorwas eradicated Similar trend was also found in the groupstreated with CNPs and ENPs the inhibitory effect wasbetter than other groups except of FNP treatment groupTreatment of mice with docetaxel-loaded NPs and PEI-modified docetaxel-loaded NPs inhibited 84 and 87in tumor growth inhibition efficiency respectively des-pite no significant difference (p gt 005)Compared with the PBS group the tumor growth inhib-

ition of the groups treated with empty NPs and NPsPEI-pEndostatin was 10 and 38 respectively which againproved that endostatin could inhibit tumor growth Theinhibitory effect of empty NPs may be caused by the TPGSdegradation from the NP matrix (Figure 8) The histo-pathological analysis of the tumor tissues also revealedthat NPs containing docetaxel orand endostatin inducedcell necrosis and apoptosis leading to suppression oftumor progression (Figure 9) MVD in FNP DNP andCNP groups was relatively lower (p lt 001) (Figure 10)

compared with PBS group MVD in DNP group was alsolower in contrast with PBS group (p lt 005) (Figure 10)This is due to the cell necrosis and apoptosis caused bydocetaxel and endostatin These results are consistentwith histopathological analysis of the tumor tissues Nosignificant difference in mouse weight was observedamong each NP treatment group (data not shown) At theend of the experiment dissection results revealed thatthere were no obvious signs of toxicity in the heart liverlungs spleen kidneys and other organs in each groupThese results implied that TPGS-b-(PCL-ran-PGA)-basedNPs could serve as multifunctional carrier of docetaxeland endostatin In addition during the regimen processthe release of TPGS may help the anticancer effect of doc-etaxel and endostatin because TPGS possesses the func-tions to improve drug permeability enhance absorption ofdrugs and reduce P-glycoprotein-mediated multi-drugresistance in cancer cells by inhibition of P-glycoproteinactivity [3334]

ConclusionsIn this research PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs polyplexed with endostatin geneshowed an optimizing potential in delivering genes andchemotherapeutic drugs The co-delivery of endostatinand docetaxel by PEI-modified TPGS-b-(PCL-ran-PGA)NPs significantly inhibited the tumor growth of nudemouse models (even regress 80 established tumor) Inconclusion our study suggested that PEI-modified TPGS-b-(PCL-ran-PGA) NPs could function as an optimizingcarrier for chemotherapeutic drugs and genetic materials

Competing interestsThe authors declare that they have no competing interests

Authorsrsquo contributionsBQ carried out the in vivo studies and drafted the manuscript MJ carried outcell studies XS carried out the preparation of nanoparticles YoZ carried outcharacterization of nanoparticles ZW carried out the in vitro drug releasestudies XZ participated in the in vivo studies SW participated in the cellstudies HC participated in the preparation of the nanoparticles LMparticipated in the design of the study and performed the statistical analysisYiZ conceived the study and participated in its design and coordination Allauthors read and approved the final manuscript

Authorsrsquo informationBQ is an associate chief physician of the Department of Orthopedics RenminHospital of Wuhan University MJ is a PhD student of the Southern MedicalUniversity XS is a Postdoctoral Fellow Tsinghua University YoZ ZW XZ andSW are PhD students of Tsinghua University HC and YiZ are assistantprofessors of the Tsinghua University LM is an associate professor of theTsinghua University

AcknowledgmentsThe authors are grateful for the financial support from Shenzhen Bureau ofScience Technology amp Information (JC200903180532AJCYJ20120614191936420 KQC201105310021A and JC201005270308A) theDoctoral Fund of Ministry of Education of China (20090002120055) theNational Natural Science Foundation of China (grant nos 31270019 and81071494) the Natural Science Foundation of Guangdong Province (No

Qiu et al Nanoscale Research Letters 2012 7666 Page 11 of 11httpwwwnanoscalereslettcomcontent71666

10451805702004178 and S2012010010046) and the Program for NewCentury Excellent Talents in University (NCET-11-0275)

Author details1Department of Orthopedics Renmin Hospital of Wuhan University Wuhan430060 Peoplersquos Republic of China 2Southern Medical UniversityGuangzhou 510515 Peoplersquos Republic of China 3The Shenzhen Key Lab ofGene and Antibody Therapy Graduate School at Shenzhen TsinghuaUniversity Shenzhen 518055 Peoplersquos Republic of China 4School of LifeSciences Tsinghua University Beijing 100084 Peoplersquos Republic of China5L401 Tsinghua Campus Xili University Town Shenzhen GuangdongProvince 518055 China

Received 11 October 2012 Accepted 12 November 2012Published 6 December 2012

References1 IARC Monographs on the evaluation of carcinogenic risks to humans

human papillomaviruses vol 90 httpmonographsiarcfr2 WHO (World Health Organization) Human papillomaviruses httpwww

who intvaccine_research diseasesviral_cancersenindex3html3 Schiller JT Castellsagueacute X Villa LL Hildesheim A An update of prophylactic

human papillomavirus L1 virus-like particle vaccine clinical trial resultsVaccine 2008 26(Suppl 10)K53ndash61

4 Winlaw DS Angiogenesis in the pathobiology and treatment of vascularand malignant diseases Ann Thorac Surg 1997 641204ndash1211

5 Folkman J Is angiogenesis an organizing principle in biology andmedicine J Pediatr Surg 2007 421ndash11

6 Folkman J Antiangiogenesis in cancer therapy-endostatin and itsmechanisms of action Exp Cell Res 2006 312594ndash607

7 Folkman J Angiogenesis an organizing principle for drug discovery NatRev Drug Discov 2007 6273ndash286

8 Michael SO Antiangiogenesis and vascular endothelial growth factorvascular endothelial growth factor receptor targeting as part of acombined-modality approach to the treatment of cancer Int J RadiatOncol Biol Phys 2007 69S64ndashS66

9 OReilly MS Boehm T Shing Y Fukai N Vasios G Lane WS Flynn E BirkheadJR Olsen BR Folkman J Endostatin an endogenous inhibitor ofangiogenesis and tumor growth Cell 1997 88277ndash285

10 Boehm T Folkman J Browder T OReilly MS Antiangiogenic therapy ofexperimental cancer does not induce acquired drug resistance Nature1997 390404ndash407

11 Herbst RS Lee AT Tran HT Abbruzzese JL Clinical studies of angiogenesisinhibitors the University of Texas MD Anderson Center Trial of HumanEndostatin Curr Oncol Rep 2001 3131ndash140

12 Zhuang HQ Yuan ZY Process in the mechanisms of endostatincombined with radiotherapy Cancer Letters 2009 2829ndash13

13 Alexander BL Ali RR Alton EW Bainbridge JW Braun S Cheng SH Flotte TRGaspar HB Grez M Griesenbach U Kaplitt MG Ott MG Seger R Simons MThrasher AJ Thrasher AZ Ylauml-Herttuala S Progress and prospects genetherapy clinical trials (part 1) Gene Ther 2007 201439ndash1447

14 Guinn BA Mulherkar R International progress in cancer gene therapyCancer Gene Ther 2008 12765ndash775

15 Scherer L Rossi JJ Weinberg MS Progress and prospects RNA-basedtherapies for treatment of HIV infection Gene Ther 2007 141057ndash1064

16 Androic I Kraumlmer A Yan R Roumldel F Gaumltje R Kaufmann M Strebhardt KYuan J Targeting cyclin B1 inhibits proliferation and sensitizes breastcancer cells to taxol BMC Cancer 2008 8391ndash401

17 Gao Y Liu XL Research progress on siRNA delivery with nonviral carriersInt J Nanomedicine 2011 61017ndash1025

18 Zhou J Wu J Hafdi N PAMAM dendrimers for efficient siRNA deliveryand potent gene silencing Chem Commun (Camb) 2006 222362ndash2364

19 Mao SR Sun W Kissel T Chitosan-based formulations for delivery of DNAand siRNA Adv Drug Deliv Rev 2010 6212ndash27

20 Son S Kim WJ Biodegradable nanoparticles modified by branchedpolyethylenimine for plasmid DNA delivery Biomaterials 201031133ndash143

21 Blum JS Saltzman WM High loading efficiency and tunable release ofplasmid DNA encapsulated in submicron particles fabricated from PLGAconjugated with poly-L-lysine J Control Release 2008 12966ndash72

22 Boussif O Lezoualch F Zanta MA Mergny MD Scherman D Demeneix BBehr JP A versatile vector for gene and oligonucleotide transfer intocells in culture and in vivo polyethylenimine Proc Natl Acad Sci USA 1995927297ndash7301

23 Zhu LP Xing J Wang QX Kou L Li C Hu B Wu ZW Wang JJ Xu GXTherapeutic efficacy of recombinant human endostatin combined withchemotherapeutics in mice-transplanted tumors Eur J Pharmacol 200961723ndash27

24 Huang L Chen H Zheng Y Song X Liu R Liu K Zeng X Mei LNanoformulation of D-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) diblock copolymer for breast cancertherapy Integr Biol 2011 3993ndash1002

25 Kim JH Park JS Yang HN Woo DG Jeon SY Do HJ Lim HY Kim JM ParkKH The use of biodegradable PLGA nanoparticles to mediate SOX9 genedelivery in human mesenchymal stem cells (hMSCs) and inducechondrogenesis Biomaterials 2011 32268ndash278

26 Wang C Ge Q Ting D Nguyen D Shen HR Chen J Eisen HN Heller JLanger R Putnam D Molecularly engineered poly (ortho ester)microspheres for enhanced delivery of DNA vaccines Nat Mater 20043190ndash196

27 Zolnik BS Burgess DJ Effect of acidic pH on PLGA microspheredegradation and release J Control Release 2007 122338ndash344

28 Yin Y Chen D Qiao M Wei X Hu H Lectin-conjugated PLGAnanoparticles loaded with thymopentin ex vivo bioadhesion and in vivobiodistribution J Control Release 2007 12327ndash38

29 Kataoka K Matsumoto T Yokoyama M Okano T Sakurai Y Fukushima SOkamoto K Kwon GS Doxorubicin-loaded poly(ethylene glycol)-poly(beta-benzyl-L-aspartate) copolymer micelles their pharmaceuticalcharacteristics and biological significance J Control Release 200064143ndash153

30 Lee AL Wang Y Cheng HY Pervaiz S Yang YY The co-delivery ofpaclitaxel and Herceptin using cationic micellar nanoparticlesBiomaterials 2009 30919ndash927

31 Fay F Quinn DJ Gilmore BF McCarron PA Scott CJ Gene delivery usingdimethyldidodecylammonium bromide-coated PLGA nanoparticlesBiomaterials 2011 314214ndash4222

32 Wang L Yao B Li Q Mei K Xu JR Li HX Wang YS Wen YJ Wang XD YangHS Li YH Luo F Wu Y Liu YY Yang L Gene therapy with recombinantadenovirus encoding endostatin encapsulated in cationic liposome incoxsackievirus and adenovirus receptor-deficient colon carcinomamurine models Hum Gene Ther 2011 22(9)1061ndash1069

33 Collnot EM Baldes C Wempe MF Kappl R Huumlttermann J Hyatt JA Edgar KJSchaefer UF Lehr CM Mechanism of inhibition of P-glycoproteinmediated efflux by vitamin E TPGS influence on ATPase activity andmembrane fluidity Mol Pharm 2007 4(3)465ndash474

34 Dintaman JM Silverman JA Inhibition of P-glycoprotein by Dalpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) Pharm Res 1999161550ndash1556

doi1011861556-276X-7-666Cite this article as Qiu et al Co-delivery of docetaxel and endostatin bya biodegradable nanoparticle for the synergistic treatment of cervicalcancer Nanoscale Research Letters 2012 7666

Submit your manuscript to a journal and benefi t from

7 Convenient online submission

7 Rigorous peer review

7 Immediate publication on acceptance

7 Open access articles freely available online

7 High visibility within the fi eld

7 Retaining the copyright to your article

Submit your next manuscript at 7 springeropencom

Qiu et al Nanoscale Research Letters 2012 7666 Page 3 of 11httpwwwnanoscalereslettcomcontent71666

way except that 01 (wv) coumarin-6 was entrappedinstead of docetaxelThe docetaxel-loading content defined as the mass

percentage of docetaxel entrapped in nanoparticles wasquantified using UVndashvis analysis (UV-2000 UVndashvis spec-trophotometer Unico Inc WI USA) First docetaxel-loaded nanoparticle solutions were lyophilized to yield thesolid nanoparticle samples Then the dried nanoparticlesamples were weighed and redissolved in a mixture ofchloroform and DMSO (11 vv) The absorbance of doce-taxel at 4825 nm was measured and the drug content inthe solution was calculated based on a previously estab-lished calibration curve

Preparation of PEI-modified TPGS-b-(PCL-ran-PGA) NPsDNA complexesThe NPs were prepared as described previously [25]Briefly the particles were formulated with a ratio of thepolymer nitrogen to the DNAphosphate (NP) equal toTPGS-b-(PCL-ran-PGA) NP solution (02 ml) and weremixed with 2 mg of PEI in sterile HEPES-buffered salineThe PEI-modified TPGS-b-(PCL-ran-PGA) NP solutionwas then added to the plasmid DNA solution at differentNP ratios and vortexed gently The PEI-modified TPGS-b-(PCL-ran-PGA) NPDNA complexes were incubated insterile PBS for 20 min at room temperature

Characterization of nanoparticlesThe mean particle size and size distribution were mea-sured using dynamic light scattering (DLS) (ZetasizerNano ZS90 Malvern Instruments Ltd Malvern UK) Inbrief the NPs were suspended in deionized water at aconcentration of 01 mgml The mean hydrodynamicdiameter was determined via cumulative analysis TheDLS determinations were predicated based on the elec-trophoretic mobility of the NPs in an aqueous mediumThe electrophoretic mobility was evaluated using foldedcapillary cells in an automatic mode Zeta potential ofthe NPs was detected using laser Doppler anemometry(LDA Zetasizer Nano ZS90 Malvern Instruments Ltd)Samples were prepared in PBS and diluted 13 with deio-nized water to ensure that the measurements were per-formed under conditions of low ionic strength where thesurface charge of the particles can be measured accuratelyThe final concentration of the polymer was 001 mgmlAll data represent five measurements from one sample

Gel retardation assayThe binding of pDNA with free TPGS-b-(PCL-ran-PGA)PEI NPs was determined by 08 agarose gel elec-trophoresis A series of different weight ratios (ww) ofpDNA to NPs was loaded on the agarose gel (10 μl ofthe sample containing different amounts of pDNA) The

pDNA bands were then visualized under a UV transillu-minator at a wavelength of 365 nm

Evaluation of loading efficiency of pDNA to the NPsThe loading efficiency of pDNA to the NPs was evalu-ated by measuring unbound pDNA in the NPs solutionThe supernatant in the loaded docetaxel or free TPGS-b-(PCL-ran-PGA)PEI NPs solution was recovered byremoving the NPs by centrifugation Free pDNA concen-tration in the supernatant was determined by measuringabsorbance at 260 nm using a UV-2550 spectrophotom-eter (Shimadzu Corp Kyoto Japan) Loading efficiencyof pDNA in NPs was determined by subtracting theamount of pDNA recovered in the washing solutionsfrom initial amount of pDNA added Encapsulation effi-ciency was defined as the percentage of pDNA encapsu-lated in NPs with respect to the initially added amountof pDNA

In vitro drug releaseNPs with 15 mg docetaxel-loaded TPGS-b-(PCL-ran-PGA) were dispersed in 5 ml release medium (phosphatebuffer solution of pH 74 containing 01 wv Tween 80)to form a suspension Tween 80 was used to increasethe solubility of docetaxel in the buffer solution andavoid the adherence of docetaxel to the tube wall Thesuspension was transferred into a dialysis membrane bag(SpectraPor 6 MWCO = 1000 Spectrum LaboratoriesInc TX USA) Then the closed bag was put into a cen-trifuge tube and immersed in 15-ml release mediumThe tube was put in an orbital water bath at 37degC undera 120 rpm horizontal shaking Ten milliliters of solutionwas periodically removed for analysis and was replacedwith fresh release medium The collected samples weremixed in a mixture of chloroform and DMSO (11 vv)The analysis procedure was the same as describedaboveTo investigate the in vitro release of pDNA 5 mg of

TPGS-b-(PCL-ran-PGA)PEI-25K (n = 6) and coumarin-6-loaded TPGS-b-(PCL-ran-PGA)PEI (n = 6) wereincubated in 1 ml of DPBS buffer (pH 74) in a micro-centrifuge tube in shaking incubator at 37degC After in-cubating for 24 h half of the samples (n = 3) weretransferred into a 25 mM sodium acetate buffer (pH 50)to simulate acidication of the endolysosome of the cellSamples were taken periodically in microcentrifuge tubesand were centrifuged at 14000 rpm for 10 min to obtainpellet NPs The supernatants were removed and replacedwith fresh buffer and NPs were resuspended by vigorouspipetting The supernatants were stored at minus70degC untilanalysis by UV measurement Unmodified TPGS-b-(PCL-ran-PGA) NPs were also analyzed as a backgroundcontrol

Qiu et al Nanoscale Research Letters 2012 7666 Page 4 of 11httpwwwnanoscalereslettcomcontent71666

Cell cultureHeLa and 293T cells (ATCC VA USA) were cultured inDMEM pH 74 supplemented with 25 mM NaHCO310 μgml streptomycin sulfate 100 μgml penicillin Gand 10(vv) FBS Cells were maintained at 37degC in a 5CO2 95 air incubator

Cell viabilityThe cytotoxicity of the NPs was determined using MTTassay The culture medium was removed and replacedwith 20 μlwell MTT (5 mgml) solution in each wellfollowed by 4-h incubation at 37degC in a fully humidifiedatmosphere with 5 CO2 MTT was taken up by activecells and transformed into insoluble purple formazangranules in the mitochondria Subsequently the mediumwas discarded the precipitated formazan was dissolvedin DMSO (150 μlwell) and optical density of the solu-tion was evaluated using a microplate spectrophotom-eter at a wavelength of 570 nm The analytic assays wereperformed every day and at least 4 wells were randomlytaken into examination each time The determination ofcell viability depends on these physical and biochemicalproperties of cells

Cellular uptake of PEI-modified TPGS-b-(PCL-ran-PGA) NPsThe cells were plated in a 6-well (3 times 105 per well) platecultured at 37degC in 5 CO2 rinsed twice and pre-incubated for 1 h with 2 ml of serum-free medium at37degC Coumarin-6-loaded TPGS-b-(PCL-ran-PGA)PEINPs with or without pDsRED or pIRES-EGFP wereadded in a particle concentration of 001 to 02 mgmland incubated for 1 to 4 h at 37degC The cells were thenwashed three times with 1 ml of PBS (pH 74) to removefree PEIplasmids-modified TPGS-b-(PCL-ran-PGA)NPs resuspended in PBS and then analyzed by fluores-cent microscopyFor confocal laser microscopy analysis cells were pre-

incubated for 1 h at 37degC in serum-free medium andthen for 30 min to 4 h in the presence of PEIplasmidspIRES-EGFP orand pDsRED gene-modified TPGS-b-(PCL-ran-PGA) NPs with a final particle concentrationof 005 mgml The samples were mounted in fluores-cent mounting medium (Dako Glostrup Denmark) andfluorescence was monitored Images were acquired usinga confocal laser microscope (Eclipse TE 2000 NikonCorporation Tokyo Japan)

Western blotThe cells were seeded into 6-well plates overnightallowed to attach the bottom Cells were cultured at 37degCin 5 CO2 rinsed twice and pre-incubated for 1 h with2 ml of serum-free medium at 37degC Endostatin gene-modified TPGS-b-(PCL-ran-PGA) NPs were added ina particle concentration of 001 to 02 mgml and

incubated for 1 to 4 h at 37degC The cells were thenwashed three times with 1 ml PBS (pH 74) to removeany free PEIplasmid-modified TPGS-b-(PCL-ran-PGA)NPs After being cultured in fresh complete medium for48 h the cells were lysed with lysis buffer (Beyotime In-stitute of Biotechnology Haimen Jiangshu China) con-taining PMSF (Sigma Chemical Co St Louis MI USA)for 30 min at 4degC The lysate was then centrifuged for 20min at 13000 rpm and 4degC The proteins were then sepa-rated through SDS-PAGE and transferred onto the PVDFmembrane (Immobion-P Transfer Membrane MilliporeCorp Billerica MA USA) Membranes were blocked ina tris-buffered saline with 01 Tween 20 solution con-taining 5 non-fat dry milk and incubated overnight withanti-human endostatin antibody as primary antibody at4degC Anti-β-actin antibody was used as loading control

Mice maintenance and subcutaneous tumor modelFemale BALBc-nunu mice were supplied by the Med-ical Experimental Animal Center of Guangdong Prov-ince (Guangdong China) A subcutaneous tumor modelof nude mice was constructed Six-week-old female nudemice (18 plusmn 2 g) were subcutaneously inoculated with 15to 2 times 106 HeLa cells When the tumor size reachedabout 100 mm3 the mice were randomly divided intosix groups with six mice each PBS TPGS-b-(PCL-ran-PGA) (group ANP) TPGS-b-(PCL-ran-PGA)PEI(groupBNP) TPGS-b-(PCL-ran-PGA)PEI-pEndostatin (groupCNP) docetaxel-loaded TPGS-b-(PCL-ran-PGA) (groupDNP) and docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pEndostatin (group FNP) of which each dose ofNPs contained 02 mg PCL-PGA-TPGS 10 μg PEI and50 μg pDNA for treatment The groups were treatedonce every 3 days with intratumoral injections of PBS orNPs Tumor size was measured with a caliper and theweight of each mouse was measured with a scale Tumorvolume was calculated as the equation lsquovolume = lengthtimes width2 2rsquo The mean tumor volume and mouseweight were used to construct the curves of tumorgrowth versus mouse growth to evaluate therapeutic effi-ciency and toxicityAt the end of the treatment the mice in each

group were killed and dissected The heart liverlungs spleen kidneys and other organs werechecked for signs of toxicity Tumor specimens werethen prepared as paraffin-embedded sections forhistopathological analysis The study was carried outin strict accordance with the recommendations inthe Guide for the Care and Use of Laboratory Ani-mals of Institutional Animal Care and Use Commit-tee of Graduate School at Shenzhen TsinghuaUniversity The protocol was approved by the Ani-mal Welfare and Ethics Committee of Graduate

Figure 1 Expression of endostatin in HeLa cells using Westernblot analysis Lane 1 HeLa cells were transfected by pShuttle2-endostatin using PEI lane 2 transfected by pShuttle2-endostatinusing TPGS-b-(PCL-ran-PGA)PEI lane 3 transfected by pShuttle2using TPGS-b-(PCL-ran-PGA)PEI

Qiu et al Nanoscale Research Letters 2012 7666 Page 5 of 11httpwwwnanoscalereslettcomcontent71666

School at Shenzhen Tsinghua University China (no2011-YZ-BG)

Measurement of microvessel density in tumor tissuesMicrovessel density (MVD) in tumor tissue was evalu-ated immunohistochemically using a monoclonal anti-

Figure 2 The size of different NPs (mean plusmn SD) (A) ANPs TPGS-b-(PCL-docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI The results showed that the simodified by PEI The zeta potential of the different NPs (B) The zeta potenby PEI was positive and reduced after complexed with pDNA The results sretardation assay of pDNA complexed with TPGS-b-(PCL-ran-PGA)PEI (C) Emodified TPGS-b-(PCL-ran-PGA) NPs was examined by FESEM (D)

mouse CD31 antibody (rat anti-mouse CD31 monoclo-nal antibody clone MEC 133 BD Biosciences NJUSA) Tumor samples were collected after the therapyregimen was finished Immunohistochemical stainingwas performed as described previously [26] MVD ()was calculated from the ratio of the CD31-positive stain-ing area to the total observation area in the viable re-gion Three to six fields per section were randomlyanalyzed excluding necrotic areas Positive stainingareas were calculated using imaging analysis software(Win Roof Mitani Corporation Fukui Japan)

Statistics and data analysisAll cell experiments were repeated at least three timesunless otherwise indicated Images of Western blotresults from representative experiments were presentedThe figures were created using Adobe Photoshop CSgraphics program All data were analyzed by paired t testusing SPSS 110 software Differences were consideredstatistically significant at P lt 005

ran-PGA) NPs DNPs docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs ENPsze of NPs increased because of loading docetaxel andor beingtial of blank NPs was negative the zeta potential of the NPs modifiedhowed that TPGS-b-(PCL-ran-PGA) blank NPs could be modified Gelach lane contained the same pDNA Surface morphology of the PEI-

Figure 3 The in vitro release profile of pDNA from various NPsat pH 74 and pH 50

Figure 4 The in vitro release curve of docetaxel from differentNPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 6 of 11httpwwwnanoscalereslettcomcontent71666

Results and discussionsThe expression of pShuttle2-endostatinProtein expression of endostatin was analyzed by West-ern blot using cell lysate after transfection of HeLa cellsusing PEI (Figure 1) These results showed thatpShuttle2-endostatin was successfully constructed andexpressed in HeLa cells

Characterization of nanoparticlesRecently a novel biodegradable copolymer TPGS-b-(PCL-ran-PGA) has been successfully synthesized inour laboratory [24] Our previous study showed that theNPs made of this novel copolymer can improve drug per-meability solubility degradation and avoid the disadvan-tages of single component [24] More importantly TPGSthe degradation products of TPGS-b-(PCL-ran-PGA)showed synergistic anticancer activity in the presence ofanticancer agents [24] In this research to co-deliverychemotherapeutic drugs and genetic materials into cellsTPGS-b-(PCL-ran-PGA) NPs were modified with cat-ionic polymer PEI (25 K) DLS assay showed that thehydrodynamic diameter of the polyplexed blank TPGS-b-(PCL-ran-PGA) NPs (ANPs) was approximately 215 nmwhile the diameters of the docetaxel-loaded TPGS-b-(PCL-ran-PGA) and docetaxel-loaded TPGS-b-(PCL-ran-PGA) PEI-coated without pDNA NPs were approxi-mately 242 and approximately 270 nm respectively(Figure 2A) The FESEM images showed that the morph-ologies of TPGS-b-(PCL-ran-PGA)PEI NPs weresphere-like in shape and the size was about 250 nm(Figure 2D) consistent with the DLS data The resultsuggested that PEI modification may increase the size ofthe NPs from TPGS-b-(PCL-ran-PGA) copolymer Inaddition the surface charge of the unmodified TPGS-b-(PCL-ran-PGA) NPs was approximately minus1825 mVdetected by LDA assay while the surface charge of the PEI-modified NPs was significantly increased in zeta potentialsuggesting that the surface charge of the TPGS-b-(PCL-ran-PGA) NPs was altered from negative to positive due to thePEI coating However a reduction in surface charge of thePEI-modified NPs was observed after adding pDNAsuggesting PEI condenses negatively charged DNA intopositively charged particles (Figure 2B) Agarose gel electro-phoresis assay showed that the amount of NP-binding DNAincreased as the amount of TPGS-b-(PCL-ran-PGA)PEINPs increased (Figure 2C) In a recent report the DNAencapsulated in the polymeric (PLGA) NPs is in a con-densed form which could protect itself from denaturationand allow it to be efficiently taken up by MSCs [25] An-other study also showed that PLGAPEI NPs can protectDNA against degradation and reduce the cytotoxicity causedby PEI [25] Together results showed that the TPGS-b-(PCL-ran-PGA) NPs modified by PEI could encapsulatedocetaxel and DNA with a high loading efficiency

In vitro releasePrevious study showed that the kinetic of NP degrad-ation and DNA release appears to have a significantregulatory effect on the gene expression [26] In thisstudy we compared the in vitro release of pDNA fromdocetaxel-loaded or doceraxel-free TPGS-b-(PCL-ran-PGA)PEI-pDNA (DNPs or ENPs) formulations at pH74 and pH 50 To simulate acidification in the endo-lysosome of cells following intracellular uptake NPswere first incubated in a solution at pH 74 for 24 hthen one-half of the NPs continued to be incubated at

Figure 5 The fluorescence microscopic images of 293T cells This is after the incubation with plasmids pIRES-EGFP-loaded and pdSRED-loaded TPGS-b-(PCL-ran-PGA)PEI NPs (A) from transmitted light channel (B) from FITC channel (C) from PI channel

Figure 6 Confocal laser scanning microscopy of HeLa cells This is after incubation with loaded-coumarin-6-TPGS-b-(PCL-ran-PGA)PEI-pDSRED NPs at 37degC The cells were stained by DAPI (blue) and the plasmids pDsRED-loaded-TPGS-b-(PCL-ran-PGA)PEI NPs are red The cellularuptake was visualized by overlaying images obtained (A) Coumarin-6 (B) RED (C) DAPI and (D) co-merge

Qiu et al Nanoscale Research Letters 2012 7666 Page 7 of 11httpwwwnanoscalereslettcomcontent71666

Figure 7 Viability of HeLa cells after 24- and 48-h incubations(n = 5) Single asterisk denotes p lt 005 (compared with PBS) whiledouble asterisk indicates p lt 001 (compared with PBS)

Figure 8 The tumor volume curve after animal models weretreated by different NPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 8 of 11httpwwwnanoscalereslettcomcontent71666

pH 74 and another half was transferred to a solution ofpH 50 pDNA release was monitored by measuring UVabsorption at 260 nm at predetermined time points Inthe pH 74 group 405 and 431 of the total pDNAwere released rapidly from docetaxel-loaded NPsPEIand empty NPsPEI respectively at 48 h followed by aslow release until day 7 (Figure 3) These two formula-tions had no significant difference either at initial burstphase or slow release phase at pH 50 suggesting thatthe loaded docetaxel had no significant influence onpDNA release Zolniks group reported that cleavage ofthe ester linkage of the PLGA polymer backbone at lowpH can accelerate the polymer erosion [27] However inthis study the release kinetics of pDNA at pH 74 wassimilar to that at pH 50 but the release of pDNA fromNPs from day 7 at pH 50 was more rapid than that atpH 74 The reason was the degradation of surface ero-sion [27] Being a hydrophilic molecule located at thesurface of the TPGS-b-(PCL-ran-PGA) matrix PEI mayfacilitate the degradation of the NPs by increasing hydra-tion and thereby accelerating hydrolysis [28]In order to investigate the docetaxel release profiles

the three formulations of NPs docetaxel-loaded TPGS-b-(PCL-ran-PGA) docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI and docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pDNA were incubated in 37degC aqueous solutions atpH 74 and pH 50 respectively As shown in Figure 4 atypical release profile was observed for all six testednano-complex solutions including an initial rapid re-lease followed by a sustained slow release over a pro-longed time up to several weeks Furthermore thedocetaxel release at pH 50 was faster than the othernano-assemblies Similar pH-dependent trend was alsoobserved in other docetaxel-loaded polymeric micellarsystems [29] and such behavior was assumed to improveintracellular drug release once the nano-complexes enterthe tumor cells via endocytosis and trapped within theacidic endosomallysosomal compartments In additionboth pDNA complexion and PEI coating were found toprolong docetaxel release which is consistent with theresults for a paclitaxel and Herceptin (Genentech IncCA USA) co-delivery system using cationic micellar NPs[30] These results implied that the release profile of PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPswas prolonged and its toxicity might be decreased

Cellular uptakeTo evaluate cellular uptake of nanoparticles 293T orHeLa cells were incubated with PEI-modified TPGS-b-(PCL-ran-PGA) NPs and coumarin-6-loaded TPGS-b-(PCL-ran-PGA) NPs for 48 h The fluorescence emittedby coumarin-6 and RED protein was observed after NP in-cubation which suggested that the NPs efficiently enteredthe cells The 293T cells took up the particles in a dose-

dependent manner and EGFP and RED expressionachieved peak levels with 15 mgml of PEI complexed with100 mg of NPs (Figure 5) These findings demonstratedthat PEI-modified NPs may be good candidates for thedelivering of genes into tumor cells and PEI-modifiedTPGS-b-(PCL-ran-PGA) nanospheres may increase thegene binding ability which causes the endosomal escapeeasily and lowers the PEI cytotoxicity [25] We also foundthat more PEI-modified coumarin-6-loaded NPs wereuptaken by HeLa cells than unmodified NPs which is inconsistency with other reports [31] It was probably be-cause the PEI-modified NPs had positive charge on surface

Figure 9 The histopathological analysis of the tumor tissues from different treatment groups (A) PBS (B) ANPs (TPGS-b-(PCL-ran-PGA))(C) CNPs (TPGS-b-(PCL-ran-PGAPEIpEndo) (D) DNPs (E) ENPs and (F) FNPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 9 of 11httpwwwnanoscalereslettcomcontent71666

which provided good affiliation with cells which had nega-tive charge on the surface In addition our data alsoshowed that coumarin-6 and RED protein can co-expressin HeLa cells transfected by coumarin-6-loaded TPGS-b-(PCL-ran-PGA)PEI-pDSRED (Figure 6) suggesting thatPEI-modified TPGS-b-(PCL-ran-PGA) can co-deliver che-motherapeutic drugs and genetic materials into cells

Figure 10 MVD of tumor tissue in different treatment groups Single adenotes p lt 001 (compared with PBS)

Cell viabilityCytotoxicity of all NPs was determined in various con-centrations using MTT assay The cell viability was notaffected by TPGS-b-(PCL-ran-PGA) NPs in all concen-trations relative to the control (treated with PBS) after72 h in medium However increasing dosage of PEI-modified TPGS-b-(PCL-ran-PGA) NPs was associated

sterisk indicates p lt 005 (compared with PBS) while double asterisk

Qiu et al Nanoscale Research Letters 2012 7666 Page 10 of 11httpwwwnanoscalereslettcomcontent71666

with increased cytotoxicity because of the increase ofPEI (Figure 7) As expected for TPGS-b-(PCL-ran-PGA) TPGS-b-(PCL-ran-PGA)PEI NPs and coumarin-6-loaded NPs the cell survival rates were greater than84 whereas cell survival rate of the cells treated withequal dose of PEI was less than 70 (data not shown)suggesting that TPGS-b-(PCL-ran-PGA) could reducethe cytotoxicity of PEI (Figure 7) On the other handdocetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pEndos-tatin NPs appeared to have more cytotoxicity than thatof the other control NPs including docetaxel-loadedNPs this may be because endostatin could induce cellapoptosis [32] Among all NPs docetaxel-load NPsPEI-endostatin had the highest cytotoxicityThe cell viability of HeLa cells transfected with

docetaxel-loaded NPs was decreased after 6 h whichwas dose- and time-dependent These results indicatedthat PEI-modified TPGS-b-(PCL-ran-PGA) NPs cansimultaneously delivery docetaxel and genetic materialsinto cancer cells and kill them

In vivo studiesTo investigate the therapeutic effect of TPGS-b-(PCL-ran-PGA)-based NPs on tumors the anti-tumor efficacyand angiogenesis inhibition of various NPs includingdocetaxel-loaded NPs-PEIpEndostatin were further eval-uated in vivo Subcutaneous tumor models were con-structed in nude mice by being inoculated with 1 to 2 times106 HeLa cells The results showed that CNPs ENPs andFNPs containing docetaxel significantly inhibited tumorgrowth compared with PBS ANPs and DNPs (p lt 001)(Figure 8) The tumor volume of the mice treated withFNPs appeared to keep growing in the initial 5 days aftertreatment and then the tumor sites presentedinflammatory-like response and ulcer After 3 weeks thetumor growth was significantly inhibited and 80 tumorwas eradicated Similar trend was also found in the groupstreated with CNPs and ENPs the inhibitory effect wasbetter than other groups except of FNP treatment groupTreatment of mice with docetaxel-loaded NPs and PEI-modified docetaxel-loaded NPs inhibited 84 and 87in tumor growth inhibition efficiency respectively des-pite no significant difference (p gt 005)Compared with the PBS group the tumor growth inhib-

ition of the groups treated with empty NPs and NPsPEI-pEndostatin was 10 and 38 respectively which againproved that endostatin could inhibit tumor growth Theinhibitory effect of empty NPs may be caused by the TPGSdegradation from the NP matrix (Figure 8) The histo-pathological analysis of the tumor tissues also revealedthat NPs containing docetaxel orand endostatin inducedcell necrosis and apoptosis leading to suppression oftumor progression (Figure 9) MVD in FNP DNP andCNP groups was relatively lower (p lt 001) (Figure 10)

compared with PBS group MVD in DNP group was alsolower in contrast with PBS group (p lt 005) (Figure 10)This is due to the cell necrosis and apoptosis caused bydocetaxel and endostatin These results are consistentwith histopathological analysis of the tumor tissues Nosignificant difference in mouse weight was observedamong each NP treatment group (data not shown) At theend of the experiment dissection results revealed thatthere were no obvious signs of toxicity in the heart liverlungs spleen kidneys and other organs in each groupThese results implied that TPGS-b-(PCL-ran-PGA)-basedNPs could serve as multifunctional carrier of docetaxeland endostatin In addition during the regimen processthe release of TPGS may help the anticancer effect of doc-etaxel and endostatin because TPGS possesses the func-tions to improve drug permeability enhance absorption ofdrugs and reduce P-glycoprotein-mediated multi-drugresistance in cancer cells by inhibition of P-glycoproteinactivity [3334]

ConclusionsIn this research PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs polyplexed with endostatin geneshowed an optimizing potential in delivering genes andchemotherapeutic drugs The co-delivery of endostatinand docetaxel by PEI-modified TPGS-b-(PCL-ran-PGA)NPs significantly inhibited the tumor growth of nudemouse models (even regress 80 established tumor) Inconclusion our study suggested that PEI-modified TPGS-b-(PCL-ran-PGA) NPs could function as an optimizingcarrier for chemotherapeutic drugs and genetic materials

Competing interestsThe authors declare that they have no competing interests

Authorsrsquo contributionsBQ carried out the in vivo studies and drafted the manuscript MJ carried outcell studies XS carried out the preparation of nanoparticles YoZ carried outcharacterization of nanoparticles ZW carried out the in vitro drug releasestudies XZ participated in the in vivo studies SW participated in the cellstudies HC participated in the preparation of the nanoparticles LMparticipated in the design of the study and performed the statistical analysisYiZ conceived the study and participated in its design and coordination Allauthors read and approved the final manuscript

Authorsrsquo informationBQ is an associate chief physician of the Department of Orthopedics RenminHospital of Wuhan University MJ is a PhD student of the Southern MedicalUniversity XS is a Postdoctoral Fellow Tsinghua University YoZ ZW XZ andSW are PhD students of Tsinghua University HC and YiZ are assistantprofessors of the Tsinghua University LM is an associate professor of theTsinghua University

AcknowledgmentsThe authors are grateful for the financial support from Shenzhen Bureau ofScience Technology amp Information (JC200903180532AJCYJ20120614191936420 KQC201105310021A and JC201005270308A) theDoctoral Fund of Ministry of Education of China (20090002120055) theNational Natural Science Foundation of China (grant nos 31270019 and81071494) the Natural Science Foundation of Guangdong Province (No

Qiu et al Nanoscale Research Letters 2012 7666 Page 11 of 11httpwwwnanoscalereslettcomcontent71666

10451805702004178 and S2012010010046) and the Program for NewCentury Excellent Talents in University (NCET-11-0275)

Author details1Department of Orthopedics Renmin Hospital of Wuhan University Wuhan430060 Peoplersquos Republic of China 2Southern Medical UniversityGuangzhou 510515 Peoplersquos Republic of China 3The Shenzhen Key Lab ofGene and Antibody Therapy Graduate School at Shenzhen TsinghuaUniversity Shenzhen 518055 Peoplersquos Republic of China 4School of LifeSciences Tsinghua University Beijing 100084 Peoplersquos Republic of China5L401 Tsinghua Campus Xili University Town Shenzhen GuangdongProvince 518055 China

Received 11 October 2012 Accepted 12 November 2012Published 6 December 2012

References1 IARC Monographs on the evaluation of carcinogenic risks to humans

human papillomaviruses vol 90 httpmonographsiarcfr2 WHO (World Health Organization) Human papillomaviruses httpwww

who intvaccine_research diseasesviral_cancersenindex3html3 Schiller JT Castellsagueacute X Villa LL Hildesheim A An update of prophylactic

human papillomavirus L1 virus-like particle vaccine clinical trial resultsVaccine 2008 26(Suppl 10)K53ndash61

4 Winlaw DS Angiogenesis in the pathobiology and treatment of vascularand malignant diseases Ann Thorac Surg 1997 641204ndash1211

5 Folkman J Is angiogenesis an organizing principle in biology andmedicine J Pediatr Surg 2007 421ndash11

6 Folkman J Antiangiogenesis in cancer therapy-endostatin and itsmechanisms of action Exp Cell Res 2006 312594ndash607

7 Folkman J Angiogenesis an organizing principle for drug discovery NatRev Drug Discov 2007 6273ndash286

8 Michael SO Antiangiogenesis and vascular endothelial growth factorvascular endothelial growth factor receptor targeting as part of acombined-modality approach to the treatment of cancer Int J RadiatOncol Biol Phys 2007 69S64ndashS66

9 OReilly MS Boehm T Shing Y Fukai N Vasios G Lane WS Flynn E BirkheadJR Olsen BR Folkman J Endostatin an endogenous inhibitor ofangiogenesis and tumor growth Cell 1997 88277ndash285

10 Boehm T Folkman J Browder T OReilly MS Antiangiogenic therapy ofexperimental cancer does not induce acquired drug resistance Nature1997 390404ndash407

11 Herbst RS Lee AT Tran HT Abbruzzese JL Clinical studies of angiogenesisinhibitors the University of Texas MD Anderson Center Trial of HumanEndostatin Curr Oncol Rep 2001 3131ndash140

12 Zhuang HQ Yuan ZY Process in the mechanisms of endostatincombined with radiotherapy Cancer Letters 2009 2829ndash13

13 Alexander BL Ali RR Alton EW Bainbridge JW Braun S Cheng SH Flotte TRGaspar HB Grez M Griesenbach U Kaplitt MG Ott MG Seger R Simons MThrasher AJ Thrasher AZ Ylauml-Herttuala S Progress and prospects genetherapy clinical trials (part 1) Gene Ther 2007 201439ndash1447

14 Guinn BA Mulherkar R International progress in cancer gene therapyCancer Gene Ther 2008 12765ndash775

15 Scherer L Rossi JJ Weinberg MS Progress and prospects RNA-basedtherapies for treatment of HIV infection Gene Ther 2007 141057ndash1064

16 Androic I Kraumlmer A Yan R Roumldel F Gaumltje R Kaufmann M Strebhardt KYuan J Targeting cyclin B1 inhibits proliferation and sensitizes breastcancer cells to taxol BMC Cancer 2008 8391ndash401

17 Gao Y Liu XL Research progress on siRNA delivery with nonviral carriersInt J Nanomedicine 2011 61017ndash1025

18 Zhou J Wu J Hafdi N PAMAM dendrimers for efficient siRNA deliveryand potent gene silencing Chem Commun (Camb) 2006 222362ndash2364

19 Mao SR Sun W Kissel T Chitosan-based formulations for delivery of DNAand siRNA Adv Drug Deliv Rev 2010 6212ndash27

20 Son S Kim WJ Biodegradable nanoparticles modified by branchedpolyethylenimine for plasmid DNA delivery Biomaterials 201031133ndash143

21 Blum JS Saltzman WM High loading efficiency and tunable release ofplasmid DNA encapsulated in submicron particles fabricated from PLGAconjugated with poly-L-lysine J Control Release 2008 12966ndash72

22 Boussif O Lezoualch F Zanta MA Mergny MD Scherman D Demeneix BBehr JP A versatile vector for gene and oligonucleotide transfer intocells in culture and in vivo polyethylenimine Proc Natl Acad Sci USA 1995927297ndash7301

23 Zhu LP Xing J Wang QX Kou L Li C Hu B Wu ZW Wang JJ Xu GXTherapeutic efficacy of recombinant human endostatin combined withchemotherapeutics in mice-transplanted tumors Eur J Pharmacol 200961723ndash27

24 Huang L Chen H Zheng Y Song X Liu R Liu K Zeng X Mei LNanoformulation of D-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) diblock copolymer for breast cancertherapy Integr Biol 2011 3993ndash1002

25 Kim JH Park JS Yang HN Woo DG Jeon SY Do HJ Lim HY Kim JM ParkKH The use of biodegradable PLGA nanoparticles to mediate SOX9 genedelivery in human mesenchymal stem cells (hMSCs) and inducechondrogenesis Biomaterials 2011 32268ndash278

26 Wang C Ge Q Ting D Nguyen D Shen HR Chen J Eisen HN Heller JLanger R Putnam D Molecularly engineered poly (ortho ester)microspheres for enhanced delivery of DNA vaccines Nat Mater 20043190ndash196

27 Zolnik BS Burgess DJ Effect of acidic pH on PLGA microspheredegradation and release J Control Release 2007 122338ndash344

28 Yin Y Chen D Qiao M Wei X Hu H Lectin-conjugated PLGAnanoparticles loaded with thymopentin ex vivo bioadhesion and in vivobiodistribution J Control Release 2007 12327ndash38

29 Kataoka K Matsumoto T Yokoyama M Okano T Sakurai Y Fukushima SOkamoto K Kwon GS Doxorubicin-loaded poly(ethylene glycol)-poly(beta-benzyl-L-aspartate) copolymer micelles their pharmaceuticalcharacteristics and biological significance J Control Release 200064143ndash153

30 Lee AL Wang Y Cheng HY Pervaiz S Yang YY The co-delivery ofpaclitaxel and Herceptin using cationic micellar nanoparticlesBiomaterials 2009 30919ndash927

31 Fay F Quinn DJ Gilmore BF McCarron PA Scott CJ Gene delivery usingdimethyldidodecylammonium bromide-coated PLGA nanoparticlesBiomaterials 2011 314214ndash4222

32 Wang L Yao B Li Q Mei K Xu JR Li HX Wang YS Wen YJ Wang XD YangHS Li YH Luo F Wu Y Liu YY Yang L Gene therapy with recombinantadenovirus encoding endostatin encapsulated in cationic liposome incoxsackievirus and adenovirus receptor-deficient colon carcinomamurine models Hum Gene Ther 2011 22(9)1061ndash1069

33 Collnot EM Baldes C Wempe MF Kappl R Huumlttermann J Hyatt JA Edgar KJSchaefer UF Lehr CM Mechanism of inhibition of P-glycoproteinmediated efflux by vitamin E TPGS influence on ATPase activity andmembrane fluidity Mol Pharm 2007 4(3)465ndash474

34 Dintaman JM Silverman JA Inhibition of P-glycoprotein by Dalpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) Pharm Res 1999161550ndash1556

doi1011861556-276X-7-666Cite this article as Qiu et al Co-delivery of docetaxel and endostatin bya biodegradable nanoparticle for the synergistic treatment of cervicalcancer Nanoscale Research Letters 2012 7666

Submit your manuscript to a journal and benefi t from

7 Convenient online submission

7 Rigorous peer review

7 Immediate publication on acceptance

7 Open access articles freely available online

7 High visibility within the fi eld

7 Retaining the copyright to your article

Submit your next manuscript at 7 springeropencom

Qiu et al Nanoscale Research Letters 2012 7666 Page 4 of 11httpwwwnanoscalereslettcomcontent71666

Cell cultureHeLa and 293T cells (ATCC VA USA) were cultured inDMEM pH 74 supplemented with 25 mM NaHCO310 μgml streptomycin sulfate 100 μgml penicillin Gand 10(vv) FBS Cells were maintained at 37degC in a 5CO2 95 air incubator

Cell viabilityThe cytotoxicity of the NPs was determined using MTTassay The culture medium was removed and replacedwith 20 μlwell MTT (5 mgml) solution in each wellfollowed by 4-h incubation at 37degC in a fully humidifiedatmosphere with 5 CO2 MTT was taken up by activecells and transformed into insoluble purple formazangranules in the mitochondria Subsequently the mediumwas discarded the precipitated formazan was dissolvedin DMSO (150 μlwell) and optical density of the solu-tion was evaluated using a microplate spectrophotom-eter at a wavelength of 570 nm The analytic assays wereperformed every day and at least 4 wells were randomlytaken into examination each time The determination ofcell viability depends on these physical and biochemicalproperties of cells

Cellular uptake of PEI-modified TPGS-b-(PCL-ran-PGA) NPsThe cells were plated in a 6-well (3 times 105 per well) platecultured at 37degC in 5 CO2 rinsed twice and pre-incubated for 1 h with 2 ml of serum-free medium at37degC Coumarin-6-loaded TPGS-b-(PCL-ran-PGA)PEINPs with or without pDsRED or pIRES-EGFP wereadded in a particle concentration of 001 to 02 mgmland incubated for 1 to 4 h at 37degC The cells were thenwashed three times with 1 ml of PBS (pH 74) to removefree PEIplasmids-modified TPGS-b-(PCL-ran-PGA)NPs resuspended in PBS and then analyzed by fluores-cent microscopyFor confocal laser microscopy analysis cells were pre-

incubated for 1 h at 37degC in serum-free medium andthen for 30 min to 4 h in the presence of PEIplasmidspIRES-EGFP orand pDsRED gene-modified TPGS-b-(PCL-ran-PGA) NPs with a final particle concentrationof 005 mgml The samples were mounted in fluores-cent mounting medium (Dako Glostrup Denmark) andfluorescence was monitored Images were acquired usinga confocal laser microscope (Eclipse TE 2000 NikonCorporation Tokyo Japan)

Western blotThe cells were seeded into 6-well plates overnightallowed to attach the bottom Cells were cultured at 37degCin 5 CO2 rinsed twice and pre-incubated for 1 h with2 ml of serum-free medium at 37degC Endostatin gene-modified TPGS-b-(PCL-ran-PGA) NPs were added ina particle concentration of 001 to 02 mgml and

incubated for 1 to 4 h at 37degC The cells were thenwashed three times with 1 ml PBS (pH 74) to removeany free PEIplasmid-modified TPGS-b-(PCL-ran-PGA)NPs After being cultured in fresh complete medium for48 h the cells were lysed with lysis buffer (Beyotime In-stitute of Biotechnology Haimen Jiangshu China) con-taining PMSF (Sigma Chemical Co St Louis MI USA)for 30 min at 4degC The lysate was then centrifuged for 20min at 13000 rpm and 4degC The proteins were then sepa-rated through SDS-PAGE and transferred onto the PVDFmembrane (Immobion-P Transfer Membrane MilliporeCorp Billerica MA USA) Membranes were blocked ina tris-buffered saline with 01 Tween 20 solution con-taining 5 non-fat dry milk and incubated overnight withanti-human endostatin antibody as primary antibody at4degC Anti-β-actin antibody was used as loading control

Mice maintenance and subcutaneous tumor modelFemale BALBc-nunu mice were supplied by the Med-ical Experimental Animal Center of Guangdong Prov-ince (Guangdong China) A subcutaneous tumor modelof nude mice was constructed Six-week-old female nudemice (18 plusmn 2 g) were subcutaneously inoculated with 15to 2 times 106 HeLa cells When the tumor size reachedabout 100 mm3 the mice were randomly divided intosix groups with six mice each PBS TPGS-b-(PCL-ran-PGA) (group ANP) TPGS-b-(PCL-ran-PGA)PEI(groupBNP) TPGS-b-(PCL-ran-PGA)PEI-pEndostatin (groupCNP) docetaxel-loaded TPGS-b-(PCL-ran-PGA) (groupDNP) and docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pEndostatin (group FNP) of which each dose ofNPs contained 02 mg PCL-PGA-TPGS 10 μg PEI and50 μg pDNA for treatment The groups were treatedonce every 3 days with intratumoral injections of PBS orNPs Tumor size was measured with a caliper and theweight of each mouse was measured with a scale Tumorvolume was calculated as the equation lsquovolume = lengthtimes width2 2rsquo The mean tumor volume and mouseweight were used to construct the curves of tumorgrowth versus mouse growth to evaluate therapeutic effi-ciency and toxicityAt the end of the treatment the mice in each

group were killed and dissected The heart liverlungs spleen kidneys and other organs werechecked for signs of toxicity Tumor specimens werethen prepared as paraffin-embedded sections forhistopathological analysis The study was carried outin strict accordance with the recommendations inthe Guide for the Care and Use of Laboratory Ani-mals of Institutional Animal Care and Use Commit-tee of Graduate School at Shenzhen TsinghuaUniversity The protocol was approved by the Ani-mal Welfare and Ethics Committee of Graduate

Figure 1 Expression of endostatin in HeLa cells using Westernblot analysis Lane 1 HeLa cells were transfected by pShuttle2-endostatin using PEI lane 2 transfected by pShuttle2-endostatinusing TPGS-b-(PCL-ran-PGA)PEI lane 3 transfected by pShuttle2using TPGS-b-(PCL-ran-PGA)PEI

Qiu et al Nanoscale Research Letters 2012 7666 Page 5 of 11httpwwwnanoscalereslettcomcontent71666

School at Shenzhen Tsinghua University China (no2011-YZ-BG)

Measurement of microvessel density in tumor tissuesMicrovessel density (MVD) in tumor tissue was evalu-ated immunohistochemically using a monoclonal anti-

Figure 2 The size of different NPs (mean plusmn SD) (A) ANPs TPGS-b-(PCL-docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI The results showed that the simodified by PEI The zeta potential of the different NPs (B) The zeta potenby PEI was positive and reduced after complexed with pDNA The results sretardation assay of pDNA complexed with TPGS-b-(PCL-ran-PGA)PEI (C) Emodified TPGS-b-(PCL-ran-PGA) NPs was examined by FESEM (D)

mouse CD31 antibody (rat anti-mouse CD31 monoclo-nal antibody clone MEC 133 BD Biosciences NJUSA) Tumor samples were collected after the therapyregimen was finished Immunohistochemical stainingwas performed as described previously [26] MVD ()was calculated from the ratio of the CD31-positive stain-ing area to the total observation area in the viable re-gion Three to six fields per section were randomlyanalyzed excluding necrotic areas Positive stainingareas were calculated using imaging analysis software(Win Roof Mitani Corporation Fukui Japan)

Statistics and data analysisAll cell experiments were repeated at least three timesunless otherwise indicated Images of Western blotresults from representative experiments were presentedThe figures were created using Adobe Photoshop CSgraphics program All data were analyzed by paired t testusing SPSS 110 software Differences were consideredstatistically significant at P lt 005

ran-PGA) NPs DNPs docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs ENPsze of NPs increased because of loading docetaxel andor beingtial of blank NPs was negative the zeta potential of the NPs modifiedhowed that TPGS-b-(PCL-ran-PGA) blank NPs could be modified Gelach lane contained the same pDNA Surface morphology of the PEI-

Figure 3 The in vitro release profile of pDNA from various NPsat pH 74 and pH 50

Figure 4 The in vitro release curve of docetaxel from differentNPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 6 of 11httpwwwnanoscalereslettcomcontent71666

Results and discussionsThe expression of pShuttle2-endostatinProtein expression of endostatin was analyzed by West-ern blot using cell lysate after transfection of HeLa cellsusing PEI (Figure 1) These results showed thatpShuttle2-endostatin was successfully constructed andexpressed in HeLa cells

Characterization of nanoparticlesRecently a novel biodegradable copolymer TPGS-b-(PCL-ran-PGA) has been successfully synthesized inour laboratory [24] Our previous study showed that theNPs made of this novel copolymer can improve drug per-meability solubility degradation and avoid the disadvan-tages of single component [24] More importantly TPGSthe degradation products of TPGS-b-(PCL-ran-PGA)showed synergistic anticancer activity in the presence ofanticancer agents [24] In this research to co-deliverychemotherapeutic drugs and genetic materials into cellsTPGS-b-(PCL-ran-PGA) NPs were modified with cat-ionic polymer PEI (25 K) DLS assay showed that thehydrodynamic diameter of the polyplexed blank TPGS-b-(PCL-ran-PGA) NPs (ANPs) was approximately 215 nmwhile the diameters of the docetaxel-loaded TPGS-b-(PCL-ran-PGA) and docetaxel-loaded TPGS-b-(PCL-ran-PGA) PEI-coated without pDNA NPs were approxi-mately 242 and approximately 270 nm respectively(Figure 2A) The FESEM images showed that the morph-ologies of TPGS-b-(PCL-ran-PGA)PEI NPs weresphere-like in shape and the size was about 250 nm(Figure 2D) consistent with the DLS data The resultsuggested that PEI modification may increase the size ofthe NPs from TPGS-b-(PCL-ran-PGA) copolymer Inaddition the surface charge of the unmodified TPGS-b-(PCL-ran-PGA) NPs was approximately minus1825 mVdetected by LDA assay while the surface charge of the PEI-modified NPs was significantly increased in zeta potentialsuggesting that the surface charge of the TPGS-b-(PCL-ran-PGA) NPs was altered from negative to positive due to thePEI coating However a reduction in surface charge of thePEI-modified NPs was observed after adding pDNAsuggesting PEI condenses negatively charged DNA intopositively charged particles (Figure 2B) Agarose gel electro-phoresis assay showed that the amount of NP-binding DNAincreased as the amount of TPGS-b-(PCL-ran-PGA)PEINPs increased (Figure 2C) In a recent report the DNAencapsulated in the polymeric (PLGA) NPs is in a con-densed form which could protect itself from denaturationand allow it to be efficiently taken up by MSCs [25] An-other study also showed that PLGAPEI NPs can protectDNA against degradation and reduce the cytotoxicity causedby PEI [25] Together results showed that the TPGS-b-(PCL-ran-PGA) NPs modified by PEI could encapsulatedocetaxel and DNA with a high loading efficiency

In vitro releasePrevious study showed that the kinetic of NP degrad-ation and DNA release appears to have a significantregulatory effect on the gene expression [26] In thisstudy we compared the in vitro release of pDNA fromdocetaxel-loaded or doceraxel-free TPGS-b-(PCL-ran-PGA)PEI-pDNA (DNPs or ENPs) formulations at pH74 and pH 50 To simulate acidification in the endo-lysosome of cells following intracellular uptake NPswere first incubated in a solution at pH 74 for 24 hthen one-half of the NPs continued to be incubated at

Figure 5 The fluorescence microscopic images of 293T cells This is after the incubation with plasmids pIRES-EGFP-loaded and pdSRED-loaded TPGS-b-(PCL-ran-PGA)PEI NPs (A) from transmitted light channel (B) from FITC channel (C) from PI channel

Figure 6 Confocal laser scanning microscopy of HeLa cells This is after incubation with loaded-coumarin-6-TPGS-b-(PCL-ran-PGA)PEI-pDSRED NPs at 37degC The cells were stained by DAPI (blue) and the plasmids pDsRED-loaded-TPGS-b-(PCL-ran-PGA)PEI NPs are red The cellularuptake was visualized by overlaying images obtained (A) Coumarin-6 (B) RED (C) DAPI and (D) co-merge

Qiu et al Nanoscale Research Letters 2012 7666 Page 7 of 11httpwwwnanoscalereslettcomcontent71666

Figure 7 Viability of HeLa cells after 24- and 48-h incubations(n = 5) Single asterisk denotes p lt 005 (compared with PBS) whiledouble asterisk indicates p lt 001 (compared with PBS)

Figure 8 The tumor volume curve after animal models weretreated by different NPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 8 of 11httpwwwnanoscalereslettcomcontent71666

pH 74 and another half was transferred to a solution ofpH 50 pDNA release was monitored by measuring UVabsorption at 260 nm at predetermined time points Inthe pH 74 group 405 and 431 of the total pDNAwere released rapidly from docetaxel-loaded NPsPEIand empty NPsPEI respectively at 48 h followed by aslow release until day 7 (Figure 3) These two formula-tions had no significant difference either at initial burstphase or slow release phase at pH 50 suggesting thatthe loaded docetaxel had no significant influence onpDNA release Zolniks group reported that cleavage ofthe ester linkage of the PLGA polymer backbone at lowpH can accelerate the polymer erosion [27] However inthis study the release kinetics of pDNA at pH 74 wassimilar to that at pH 50 but the release of pDNA fromNPs from day 7 at pH 50 was more rapid than that atpH 74 The reason was the degradation of surface ero-sion [27] Being a hydrophilic molecule located at thesurface of the TPGS-b-(PCL-ran-PGA) matrix PEI mayfacilitate the degradation of the NPs by increasing hydra-tion and thereby accelerating hydrolysis [28]In order to investigate the docetaxel release profiles

the three formulations of NPs docetaxel-loaded TPGS-b-(PCL-ran-PGA) docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI and docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pDNA were incubated in 37degC aqueous solutions atpH 74 and pH 50 respectively As shown in Figure 4 atypical release profile was observed for all six testednano-complex solutions including an initial rapid re-lease followed by a sustained slow release over a pro-longed time up to several weeks Furthermore thedocetaxel release at pH 50 was faster than the othernano-assemblies Similar pH-dependent trend was alsoobserved in other docetaxel-loaded polymeric micellarsystems [29] and such behavior was assumed to improveintracellular drug release once the nano-complexes enterthe tumor cells via endocytosis and trapped within theacidic endosomallysosomal compartments In additionboth pDNA complexion and PEI coating were found toprolong docetaxel release which is consistent with theresults for a paclitaxel and Herceptin (Genentech IncCA USA) co-delivery system using cationic micellar NPs[30] These results implied that the release profile of PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPswas prolonged and its toxicity might be decreased

Cellular uptakeTo evaluate cellular uptake of nanoparticles 293T orHeLa cells were incubated with PEI-modified TPGS-b-(PCL-ran-PGA) NPs and coumarin-6-loaded TPGS-b-(PCL-ran-PGA) NPs for 48 h The fluorescence emittedby coumarin-6 and RED protein was observed after NP in-cubation which suggested that the NPs efficiently enteredthe cells The 293T cells took up the particles in a dose-

dependent manner and EGFP and RED expressionachieved peak levels with 15 mgml of PEI complexed with100 mg of NPs (Figure 5) These findings demonstratedthat PEI-modified NPs may be good candidates for thedelivering of genes into tumor cells and PEI-modifiedTPGS-b-(PCL-ran-PGA) nanospheres may increase thegene binding ability which causes the endosomal escapeeasily and lowers the PEI cytotoxicity [25] We also foundthat more PEI-modified coumarin-6-loaded NPs wereuptaken by HeLa cells than unmodified NPs which is inconsistency with other reports [31] It was probably be-cause the PEI-modified NPs had positive charge on surface

Figure 9 The histopathological analysis of the tumor tissues from different treatment groups (A) PBS (B) ANPs (TPGS-b-(PCL-ran-PGA))(C) CNPs (TPGS-b-(PCL-ran-PGAPEIpEndo) (D) DNPs (E) ENPs and (F) FNPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 9 of 11httpwwwnanoscalereslettcomcontent71666

which provided good affiliation with cells which had nega-tive charge on the surface In addition our data alsoshowed that coumarin-6 and RED protein can co-expressin HeLa cells transfected by coumarin-6-loaded TPGS-b-(PCL-ran-PGA)PEI-pDSRED (Figure 6) suggesting thatPEI-modified TPGS-b-(PCL-ran-PGA) can co-deliver che-motherapeutic drugs and genetic materials into cells

Figure 10 MVD of tumor tissue in different treatment groups Single adenotes p lt 001 (compared with PBS)

Cell viabilityCytotoxicity of all NPs was determined in various con-centrations using MTT assay The cell viability was notaffected by TPGS-b-(PCL-ran-PGA) NPs in all concen-trations relative to the control (treated with PBS) after72 h in medium However increasing dosage of PEI-modified TPGS-b-(PCL-ran-PGA) NPs was associated

sterisk indicates p lt 005 (compared with PBS) while double asterisk

Qiu et al Nanoscale Research Letters 2012 7666 Page 10 of 11httpwwwnanoscalereslettcomcontent71666

with increased cytotoxicity because of the increase ofPEI (Figure 7) As expected for TPGS-b-(PCL-ran-PGA) TPGS-b-(PCL-ran-PGA)PEI NPs and coumarin-6-loaded NPs the cell survival rates were greater than84 whereas cell survival rate of the cells treated withequal dose of PEI was less than 70 (data not shown)suggesting that TPGS-b-(PCL-ran-PGA) could reducethe cytotoxicity of PEI (Figure 7) On the other handdocetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pEndos-tatin NPs appeared to have more cytotoxicity than thatof the other control NPs including docetaxel-loadedNPs this may be because endostatin could induce cellapoptosis [32] Among all NPs docetaxel-load NPsPEI-endostatin had the highest cytotoxicityThe cell viability of HeLa cells transfected with

docetaxel-loaded NPs was decreased after 6 h whichwas dose- and time-dependent These results indicatedthat PEI-modified TPGS-b-(PCL-ran-PGA) NPs cansimultaneously delivery docetaxel and genetic materialsinto cancer cells and kill them

In vivo studiesTo investigate the therapeutic effect of TPGS-b-(PCL-ran-PGA)-based NPs on tumors the anti-tumor efficacyand angiogenesis inhibition of various NPs includingdocetaxel-loaded NPs-PEIpEndostatin were further eval-uated in vivo Subcutaneous tumor models were con-structed in nude mice by being inoculated with 1 to 2 times106 HeLa cells The results showed that CNPs ENPs andFNPs containing docetaxel significantly inhibited tumorgrowth compared with PBS ANPs and DNPs (p lt 001)(Figure 8) The tumor volume of the mice treated withFNPs appeared to keep growing in the initial 5 days aftertreatment and then the tumor sites presentedinflammatory-like response and ulcer After 3 weeks thetumor growth was significantly inhibited and 80 tumorwas eradicated Similar trend was also found in the groupstreated with CNPs and ENPs the inhibitory effect wasbetter than other groups except of FNP treatment groupTreatment of mice with docetaxel-loaded NPs and PEI-modified docetaxel-loaded NPs inhibited 84 and 87in tumor growth inhibition efficiency respectively des-pite no significant difference (p gt 005)Compared with the PBS group the tumor growth inhib-

ition of the groups treated with empty NPs and NPsPEI-pEndostatin was 10 and 38 respectively which againproved that endostatin could inhibit tumor growth Theinhibitory effect of empty NPs may be caused by the TPGSdegradation from the NP matrix (Figure 8) The histo-pathological analysis of the tumor tissues also revealedthat NPs containing docetaxel orand endostatin inducedcell necrosis and apoptosis leading to suppression oftumor progression (Figure 9) MVD in FNP DNP andCNP groups was relatively lower (p lt 001) (Figure 10)

compared with PBS group MVD in DNP group was alsolower in contrast with PBS group (p lt 005) (Figure 10)This is due to the cell necrosis and apoptosis caused bydocetaxel and endostatin These results are consistentwith histopathological analysis of the tumor tissues Nosignificant difference in mouse weight was observedamong each NP treatment group (data not shown) At theend of the experiment dissection results revealed thatthere were no obvious signs of toxicity in the heart liverlungs spleen kidneys and other organs in each groupThese results implied that TPGS-b-(PCL-ran-PGA)-basedNPs could serve as multifunctional carrier of docetaxeland endostatin In addition during the regimen processthe release of TPGS may help the anticancer effect of doc-etaxel and endostatin because TPGS possesses the func-tions to improve drug permeability enhance absorption ofdrugs and reduce P-glycoprotein-mediated multi-drugresistance in cancer cells by inhibition of P-glycoproteinactivity [3334]

ConclusionsIn this research PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs polyplexed with endostatin geneshowed an optimizing potential in delivering genes andchemotherapeutic drugs The co-delivery of endostatinand docetaxel by PEI-modified TPGS-b-(PCL-ran-PGA)NPs significantly inhibited the tumor growth of nudemouse models (even regress 80 established tumor) Inconclusion our study suggested that PEI-modified TPGS-b-(PCL-ran-PGA) NPs could function as an optimizingcarrier for chemotherapeutic drugs and genetic materials

Competing interestsThe authors declare that they have no competing interests

Authorsrsquo contributionsBQ carried out the in vivo studies and drafted the manuscript MJ carried outcell studies XS carried out the preparation of nanoparticles YoZ carried outcharacterization of nanoparticles ZW carried out the in vitro drug releasestudies XZ participated in the in vivo studies SW participated in the cellstudies HC participated in the preparation of the nanoparticles LMparticipated in the design of the study and performed the statistical analysisYiZ conceived the study and participated in its design and coordination Allauthors read and approved the final manuscript

Authorsrsquo informationBQ is an associate chief physician of the Department of Orthopedics RenminHospital of Wuhan University MJ is a PhD student of the Southern MedicalUniversity XS is a Postdoctoral Fellow Tsinghua University YoZ ZW XZ andSW are PhD students of Tsinghua University HC and YiZ are assistantprofessors of the Tsinghua University LM is an associate professor of theTsinghua University

AcknowledgmentsThe authors are grateful for the financial support from Shenzhen Bureau ofScience Technology amp Information (JC200903180532AJCYJ20120614191936420 KQC201105310021A and JC201005270308A) theDoctoral Fund of Ministry of Education of China (20090002120055) theNational Natural Science Foundation of China (grant nos 31270019 and81071494) the Natural Science Foundation of Guangdong Province (No

Qiu et al Nanoscale Research Letters 2012 7666 Page 11 of 11httpwwwnanoscalereslettcomcontent71666

10451805702004178 and S2012010010046) and the Program for NewCentury Excellent Talents in University (NCET-11-0275)

Author details1Department of Orthopedics Renmin Hospital of Wuhan University Wuhan430060 Peoplersquos Republic of China 2Southern Medical UniversityGuangzhou 510515 Peoplersquos Republic of China 3The Shenzhen Key Lab ofGene and Antibody Therapy Graduate School at Shenzhen TsinghuaUniversity Shenzhen 518055 Peoplersquos Republic of China 4School of LifeSciences Tsinghua University Beijing 100084 Peoplersquos Republic of China5L401 Tsinghua Campus Xili University Town Shenzhen GuangdongProvince 518055 China

Received 11 October 2012 Accepted 12 November 2012Published 6 December 2012

References1 IARC Monographs on the evaluation of carcinogenic risks to humans

human papillomaviruses vol 90 httpmonographsiarcfr2 WHO (World Health Organization) Human papillomaviruses httpwww

who intvaccine_research diseasesviral_cancersenindex3html3 Schiller JT Castellsagueacute X Villa LL Hildesheim A An update of prophylactic

human papillomavirus L1 virus-like particle vaccine clinical trial resultsVaccine 2008 26(Suppl 10)K53ndash61

4 Winlaw DS Angiogenesis in the pathobiology and treatment of vascularand malignant diseases Ann Thorac Surg 1997 641204ndash1211

5 Folkman J Is angiogenesis an organizing principle in biology andmedicine J Pediatr Surg 2007 421ndash11

6 Folkman J Antiangiogenesis in cancer therapy-endostatin and itsmechanisms of action Exp Cell Res 2006 312594ndash607

7 Folkman J Angiogenesis an organizing principle for drug discovery NatRev Drug Discov 2007 6273ndash286

8 Michael SO Antiangiogenesis and vascular endothelial growth factorvascular endothelial growth factor receptor targeting as part of acombined-modality approach to the treatment of cancer Int J RadiatOncol Biol Phys 2007 69S64ndashS66

9 OReilly MS Boehm T Shing Y Fukai N Vasios G Lane WS Flynn E BirkheadJR Olsen BR Folkman J Endostatin an endogenous inhibitor ofangiogenesis and tumor growth Cell 1997 88277ndash285

10 Boehm T Folkman J Browder T OReilly MS Antiangiogenic therapy ofexperimental cancer does not induce acquired drug resistance Nature1997 390404ndash407

11 Herbst RS Lee AT Tran HT Abbruzzese JL Clinical studies of angiogenesisinhibitors the University of Texas MD Anderson Center Trial of HumanEndostatin Curr Oncol Rep 2001 3131ndash140

12 Zhuang HQ Yuan ZY Process in the mechanisms of endostatincombined with radiotherapy Cancer Letters 2009 2829ndash13

13 Alexander BL Ali RR Alton EW Bainbridge JW Braun S Cheng SH Flotte TRGaspar HB Grez M Griesenbach U Kaplitt MG Ott MG Seger R Simons MThrasher AJ Thrasher AZ Ylauml-Herttuala S Progress and prospects genetherapy clinical trials (part 1) Gene Ther 2007 201439ndash1447

14 Guinn BA Mulherkar R International progress in cancer gene therapyCancer Gene Ther 2008 12765ndash775

15 Scherer L Rossi JJ Weinberg MS Progress and prospects RNA-basedtherapies for treatment of HIV infection Gene Ther 2007 141057ndash1064

16 Androic I Kraumlmer A Yan R Roumldel F Gaumltje R Kaufmann M Strebhardt KYuan J Targeting cyclin B1 inhibits proliferation and sensitizes breastcancer cells to taxol BMC Cancer 2008 8391ndash401

17 Gao Y Liu XL Research progress on siRNA delivery with nonviral carriersInt J Nanomedicine 2011 61017ndash1025

18 Zhou J Wu J Hafdi N PAMAM dendrimers for efficient siRNA deliveryand potent gene silencing Chem Commun (Camb) 2006 222362ndash2364

19 Mao SR Sun W Kissel T Chitosan-based formulations for delivery of DNAand siRNA Adv Drug Deliv Rev 2010 6212ndash27

20 Son S Kim WJ Biodegradable nanoparticles modified by branchedpolyethylenimine for plasmid DNA delivery Biomaterials 201031133ndash143

21 Blum JS Saltzman WM High loading efficiency and tunable release ofplasmid DNA encapsulated in submicron particles fabricated from PLGAconjugated with poly-L-lysine J Control Release 2008 12966ndash72

22 Boussif O Lezoualch F Zanta MA Mergny MD Scherman D Demeneix BBehr JP A versatile vector for gene and oligonucleotide transfer intocells in culture and in vivo polyethylenimine Proc Natl Acad Sci USA 1995927297ndash7301

23 Zhu LP Xing J Wang QX Kou L Li C Hu B Wu ZW Wang JJ Xu GXTherapeutic efficacy of recombinant human endostatin combined withchemotherapeutics in mice-transplanted tumors Eur J Pharmacol 200961723ndash27

24 Huang L Chen H Zheng Y Song X Liu R Liu K Zeng X Mei LNanoformulation of D-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) diblock copolymer for breast cancertherapy Integr Biol 2011 3993ndash1002

25 Kim JH Park JS Yang HN Woo DG Jeon SY Do HJ Lim HY Kim JM ParkKH The use of biodegradable PLGA nanoparticles to mediate SOX9 genedelivery in human mesenchymal stem cells (hMSCs) and inducechondrogenesis Biomaterials 2011 32268ndash278

26 Wang C Ge Q Ting D Nguyen D Shen HR Chen J Eisen HN Heller JLanger R Putnam D Molecularly engineered poly (ortho ester)microspheres for enhanced delivery of DNA vaccines Nat Mater 20043190ndash196

27 Zolnik BS Burgess DJ Effect of acidic pH on PLGA microspheredegradation and release J Control Release 2007 122338ndash344

28 Yin Y Chen D Qiao M Wei X Hu H Lectin-conjugated PLGAnanoparticles loaded with thymopentin ex vivo bioadhesion and in vivobiodistribution J Control Release 2007 12327ndash38

29 Kataoka K Matsumoto T Yokoyama M Okano T Sakurai Y Fukushima SOkamoto K Kwon GS Doxorubicin-loaded poly(ethylene glycol)-poly(beta-benzyl-L-aspartate) copolymer micelles their pharmaceuticalcharacteristics and biological significance J Control Release 200064143ndash153

30 Lee AL Wang Y Cheng HY Pervaiz S Yang YY The co-delivery ofpaclitaxel and Herceptin using cationic micellar nanoparticlesBiomaterials 2009 30919ndash927

31 Fay F Quinn DJ Gilmore BF McCarron PA Scott CJ Gene delivery usingdimethyldidodecylammonium bromide-coated PLGA nanoparticlesBiomaterials 2011 314214ndash4222

32 Wang L Yao B Li Q Mei K Xu JR Li HX Wang YS Wen YJ Wang XD YangHS Li YH Luo F Wu Y Liu YY Yang L Gene therapy with recombinantadenovirus encoding endostatin encapsulated in cationic liposome incoxsackievirus and adenovirus receptor-deficient colon carcinomamurine models Hum Gene Ther 2011 22(9)1061ndash1069

33 Collnot EM Baldes C Wempe MF Kappl R Huumlttermann J Hyatt JA Edgar KJSchaefer UF Lehr CM Mechanism of inhibition of P-glycoproteinmediated efflux by vitamin E TPGS influence on ATPase activity andmembrane fluidity Mol Pharm 2007 4(3)465ndash474

34 Dintaman JM Silverman JA Inhibition of P-glycoprotein by Dalpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) Pharm Res 1999161550ndash1556

doi1011861556-276X-7-666Cite this article as Qiu et al Co-delivery of docetaxel and endostatin bya biodegradable nanoparticle for the synergistic treatment of cervicalcancer Nanoscale Research Letters 2012 7666

Submit your manuscript to a journal and benefi t from

7 Convenient online submission

7 Rigorous peer review

7 Immediate publication on acceptance

7 Open access articles freely available online

7 High visibility within the fi eld

7 Retaining the copyright to your article

Submit your next manuscript at 7 springeropencom

Figure 1 Expression of endostatin in HeLa cells using Westernblot analysis Lane 1 HeLa cells were transfected by pShuttle2-endostatin using PEI lane 2 transfected by pShuttle2-endostatinusing TPGS-b-(PCL-ran-PGA)PEI lane 3 transfected by pShuttle2using TPGS-b-(PCL-ran-PGA)PEI

Qiu et al Nanoscale Research Letters 2012 7666 Page 5 of 11httpwwwnanoscalereslettcomcontent71666

School at Shenzhen Tsinghua University China (no2011-YZ-BG)

Measurement of microvessel density in tumor tissuesMicrovessel density (MVD) in tumor tissue was evalu-ated immunohistochemically using a monoclonal anti-

Figure 2 The size of different NPs (mean plusmn SD) (A) ANPs TPGS-b-(PCL-docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI The results showed that the simodified by PEI The zeta potential of the different NPs (B) The zeta potenby PEI was positive and reduced after complexed with pDNA The results sretardation assay of pDNA complexed with TPGS-b-(PCL-ran-PGA)PEI (C) Emodified TPGS-b-(PCL-ran-PGA) NPs was examined by FESEM (D)

mouse CD31 antibody (rat anti-mouse CD31 monoclo-nal antibody clone MEC 133 BD Biosciences NJUSA) Tumor samples were collected after the therapyregimen was finished Immunohistochemical stainingwas performed as described previously [26] MVD ()was calculated from the ratio of the CD31-positive stain-ing area to the total observation area in the viable re-gion Three to six fields per section were randomlyanalyzed excluding necrotic areas Positive stainingareas were calculated using imaging analysis software(Win Roof Mitani Corporation Fukui Japan)

Statistics and data analysisAll cell experiments were repeated at least three timesunless otherwise indicated Images of Western blotresults from representative experiments were presentedThe figures were created using Adobe Photoshop CSgraphics program All data were analyzed by paired t testusing SPSS 110 software Differences were consideredstatistically significant at P lt 005

ran-PGA) NPs DNPs docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs ENPsze of NPs increased because of loading docetaxel andor beingtial of blank NPs was negative the zeta potential of the NPs modifiedhowed that TPGS-b-(PCL-ran-PGA) blank NPs could be modified Gelach lane contained the same pDNA Surface morphology of the PEI-

Figure 3 The in vitro release profile of pDNA from various NPsat pH 74 and pH 50

Figure 4 The in vitro release curve of docetaxel from differentNPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 6 of 11httpwwwnanoscalereslettcomcontent71666

Results and discussionsThe expression of pShuttle2-endostatinProtein expression of endostatin was analyzed by West-ern blot using cell lysate after transfection of HeLa cellsusing PEI (Figure 1) These results showed thatpShuttle2-endostatin was successfully constructed andexpressed in HeLa cells

Characterization of nanoparticlesRecently a novel biodegradable copolymer TPGS-b-(PCL-ran-PGA) has been successfully synthesized inour laboratory [24] Our previous study showed that theNPs made of this novel copolymer can improve drug per-meability solubility degradation and avoid the disadvan-tages of single component [24] More importantly TPGSthe degradation products of TPGS-b-(PCL-ran-PGA)showed synergistic anticancer activity in the presence ofanticancer agents [24] In this research to co-deliverychemotherapeutic drugs and genetic materials into cellsTPGS-b-(PCL-ran-PGA) NPs were modified with cat-ionic polymer PEI (25 K) DLS assay showed that thehydrodynamic diameter of the polyplexed blank TPGS-b-(PCL-ran-PGA) NPs (ANPs) was approximately 215 nmwhile the diameters of the docetaxel-loaded TPGS-b-(PCL-ran-PGA) and docetaxel-loaded TPGS-b-(PCL-ran-PGA) PEI-coated without pDNA NPs were approxi-mately 242 and approximately 270 nm respectively(Figure 2A) The FESEM images showed that the morph-ologies of TPGS-b-(PCL-ran-PGA)PEI NPs weresphere-like in shape and the size was about 250 nm(Figure 2D) consistent with the DLS data The resultsuggested that PEI modification may increase the size ofthe NPs from TPGS-b-(PCL-ran-PGA) copolymer Inaddition the surface charge of the unmodified TPGS-b-(PCL-ran-PGA) NPs was approximately minus1825 mVdetected by LDA assay while the surface charge of the PEI-modified NPs was significantly increased in zeta potentialsuggesting that the surface charge of the TPGS-b-(PCL-ran-PGA) NPs was altered from negative to positive due to thePEI coating However a reduction in surface charge of thePEI-modified NPs was observed after adding pDNAsuggesting PEI condenses negatively charged DNA intopositively charged particles (Figure 2B) Agarose gel electro-phoresis assay showed that the amount of NP-binding DNAincreased as the amount of TPGS-b-(PCL-ran-PGA)PEINPs increased (Figure 2C) In a recent report the DNAencapsulated in the polymeric (PLGA) NPs is in a con-densed form which could protect itself from denaturationand allow it to be efficiently taken up by MSCs [25] An-other study also showed that PLGAPEI NPs can protectDNA against degradation and reduce the cytotoxicity causedby PEI [25] Together results showed that the TPGS-b-(PCL-ran-PGA) NPs modified by PEI could encapsulatedocetaxel and DNA with a high loading efficiency

In vitro releasePrevious study showed that the kinetic of NP degrad-ation and DNA release appears to have a significantregulatory effect on the gene expression [26] In thisstudy we compared the in vitro release of pDNA fromdocetaxel-loaded or doceraxel-free TPGS-b-(PCL-ran-PGA)PEI-pDNA (DNPs or ENPs) formulations at pH74 and pH 50 To simulate acidification in the endo-lysosome of cells following intracellular uptake NPswere first incubated in a solution at pH 74 for 24 hthen one-half of the NPs continued to be incubated at

Figure 5 The fluorescence microscopic images of 293T cells This is after the incubation with plasmids pIRES-EGFP-loaded and pdSRED-loaded TPGS-b-(PCL-ran-PGA)PEI NPs (A) from transmitted light channel (B) from FITC channel (C) from PI channel

Figure 6 Confocal laser scanning microscopy of HeLa cells This is after incubation with loaded-coumarin-6-TPGS-b-(PCL-ran-PGA)PEI-pDSRED NPs at 37degC The cells were stained by DAPI (blue) and the plasmids pDsRED-loaded-TPGS-b-(PCL-ran-PGA)PEI NPs are red The cellularuptake was visualized by overlaying images obtained (A) Coumarin-6 (B) RED (C) DAPI and (D) co-merge

Qiu et al Nanoscale Research Letters 2012 7666 Page 7 of 11httpwwwnanoscalereslettcomcontent71666

Figure 7 Viability of HeLa cells after 24- and 48-h incubations(n = 5) Single asterisk denotes p lt 005 (compared with PBS) whiledouble asterisk indicates p lt 001 (compared with PBS)

Figure 8 The tumor volume curve after animal models weretreated by different NPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 8 of 11httpwwwnanoscalereslettcomcontent71666

pH 74 and another half was transferred to a solution ofpH 50 pDNA release was monitored by measuring UVabsorption at 260 nm at predetermined time points Inthe pH 74 group 405 and 431 of the total pDNAwere released rapidly from docetaxel-loaded NPsPEIand empty NPsPEI respectively at 48 h followed by aslow release until day 7 (Figure 3) These two formula-tions had no significant difference either at initial burstphase or slow release phase at pH 50 suggesting thatthe loaded docetaxel had no significant influence onpDNA release Zolniks group reported that cleavage ofthe ester linkage of the PLGA polymer backbone at lowpH can accelerate the polymer erosion [27] However inthis study the release kinetics of pDNA at pH 74 wassimilar to that at pH 50 but the release of pDNA fromNPs from day 7 at pH 50 was more rapid than that atpH 74 The reason was the degradation of surface ero-sion [27] Being a hydrophilic molecule located at thesurface of the TPGS-b-(PCL-ran-PGA) matrix PEI mayfacilitate the degradation of the NPs by increasing hydra-tion and thereby accelerating hydrolysis [28]In order to investigate the docetaxel release profiles

the three formulations of NPs docetaxel-loaded TPGS-b-(PCL-ran-PGA) docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI and docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pDNA were incubated in 37degC aqueous solutions atpH 74 and pH 50 respectively As shown in Figure 4 atypical release profile was observed for all six testednano-complex solutions including an initial rapid re-lease followed by a sustained slow release over a pro-longed time up to several weeks Furthermore thedocetaxel release at pH 50 was faster than the othernano-assemblies Similar pH-dependent trend was alsoobserved in other docetaxel-loaded polymeric micellarsystems [29] and such behavior was assumed to improveintracellular drug release once the nano-complexes enterthe tumor cells via endocytosis and trapped within theacidic endosomallysosomal compartments In additionboth pDNA complexion and PEI coating were found toprolong docetaxel release which is consistent with theresults for a paclitaxel and Herceptin (Genentech IncCA USA) co-delivery system using cationic micellar NPs[30] These results implied that the release profile of PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPswas prolonged and its toxicity might be decreased

Cellular uptakeTo evaluate cellular uptake of nanoparticles 293T orHeLa cells were incubated with PEI-modified TPGS-b-(PCL-ran-PGA) NPs and coumarin-6-loaded TPGS-b-(PCL-ran-PGA) NPs for 48 h The fluorescence emittedby coumarin-6 and RED protein was observed after NP in-cubation which suggested that the NPs efficiently enteredthe cells The 293T cells took up the particles in a dose-

dependent manner and EGFP and RED expressionachieved peak levels with 15 mgml of PEI complexed with100 mg of NPs (Figure 5) These findings demonstratedthat PEI-modified NPs may be good candidates for thedelivering of genes into tumor cells and PEI-modifiedTPGS-b-(PCL-ran-PGA) nanospheres may increase thegene binding ability which causes the endosomal escapeeasily and lowers the PEI cytotoxicity [25] We also foundthat more PEI-modified coumarin-6-loaded NPs wereuptaken by HeLa cells than unmodified NPs which is inconsistency with other reports [31] It was probably be-cause the PEI-modified NPs had positive charge on surface

Figure 9 The histopathological analysis of the tumor tissues from different treatment groups (A) PBS (B) ANPs (TPGS-b-(PCL-ran-PGA))(C) CNPs (TPGS-b-(PCL-ran-PGAPEIpEndo) (D) DNPs (E) ENPs and (F) FNPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 9 of 11httpwwwnanoscalereslettcomcontent71666

which provided good affiliation with cells which had nega-tive charge on the surface In addition our data alsoshowed that coumarin-6 and RED protein can co-expressin HeLa cells transfected by coumarin-6-loaded TPGS-b-(PCL-ran-PGA)PEI-pDSRED (Figure 6) suggesting thatPEI-modified TPGS-b-(PCL-ran-PGA) can co-deliver che-motherapeutic drugs and genetic materials into cells

Figure 10 MVD of tumor tissue in different treatment groups Single adenotes p lt 001 (compared with PBS)

Cell viabilityCytotoxicity of all NPs was determined in various con-centrations using MTT assay The cell viability was notaffected by TPGS-b-(PCL-ran-PGA) NPs in all concen-trations relative to the control (treated with PBS) after72 h in medium However increasing dosage of PEI-modified TPGS-b-(PCL-ran-PGA) NPs was associated

sterisk indicates p lt 005 (compared with PBS) while double asterisk

Qiu et al Nanoscale Research Letters 2012 7666 Page 10 of 11httpwwwnanoscalereslettcomcontent71666

with increased cytotoxicity because of the increase ofPEI (Figure 7) As expected for TPGS-b-(PCL-ran-PGA) TPGS-b-(PCL-ran-PGA)PEI NPs and coumarin-6-loaded NPs the cell survival rates were greater than84 whereas cell survival rate of the cells treated withequal dose of PEI was less than 70 (data not shown)suggesting that TPGS-b-(PCL-ran-PGA) could reducethe cytotoxicity of PEI (Figure 7) On the other handdocetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pEndos-tatin NPs appeared to have more cytotoxicity than thatof the other control NPs including docetaxel-loadedNPs this may be because endostatin could induce cellapoptosis [32] Among all NPs docetaxel-load NPsPEI-endostatin had the highest cytotoxicityThe cell viability of HeLa cells transfected with

docetaxel-loaded NPs was decreased after 6 h whichwas dose- and time-dependent These results indicatedthat PEI-modified TPGS-b-(PCL-ran-PGA) NPs cansimultaneously delivery docetaxel and genetic materialsinto cancer cells and kill them

In vivo studiesTo investigate the therapeutic effect of TPGS-b-(PCL-ran-PGA)-based NPs on tumors the anti-tumor efficacyand angiogenesis inhibition of various NPs includingdocetaxel-loaded NPs-PEIpEndostatin were further eval-uated in vivo Subcutaneous tumor models were con-structed in nude mice by being inoculated with 1 to 2 times106 HeLa cells The results showed that CNPs ENPs andFNPs containing docetaxel significantly inhibited tumorgrowth compared with PBS ANPs and DNPs (p lt 001)(Figure 8) The tumor volume of the mice treated withFNPs appeared to keep growing in the initial 5 days aftertreatment and then the tumor sites presentedinflammatory-like response and ulcer After 3 weeks thetumor growth was significantly inhibited and 80 tumorwas eradicated Similar trend was also found in the groupstreated with CNPs and ENPs the inhibitory effect wasbetter than other groups except of FNP treatment groupTreatment of mice with docetaxel-loaded NPs and PEI-modified docetaxel-loaded NPs inhibited 84 and 87in tumor growth inhibition efficiency respectively des-pite no significant difference (p gt 005)Compared with the PBS group the tumor growth inhib-

ition of the groups treated with empty NPs and NPsPEI-pEndostatin was 10 and 38 respectively which againproved that endostatin could inhibit tumor growth Theinhibitory effect of empty NPs may be caused by the TPGSdegradation from the NP matrix (Figure 8) The histo-pathological analysis of the tumor tissues also revealedthat NPs containing docetaxel orand endostatin inducedcell necrosis and apoptosis leading to suppression oftumor progression (Figure 9) MVD in FNP DNP andCNP groups was relatively lower (p lt 001) (Figure 10)

compared with PBS group MVD in DNP group was alsolower in contrast with PBS group (p lt 005) (Figure 10)This is due to the cell necrosis and apoptosis caused bydocetaxel and endostatin These results are consistentwith histopathological analysis of the tumor tissues Nosignificant difference in mouse weight was observedamong each NP treatment group (data not shown) At theend of the experiment dissection results revealed thatthere were no obvious signs of toxicity in the heart liverlungs spleen kidneys and other organs in each groupThese results implied that TPGS-b-(PCL-ran-PGA)-basedNPs could serve as multifunctional carrier of docetaxeland endostatin In addition during the regimen processthe release of TPGS may help the anticancer effect of doc-etaxel and endostatin because TPGS possesses the func-tions to improve drug permeability enhance absorption ofdrugs and reduce P-glycoprotein-mediated multi-drugresistance in cancer cells by inhibition of P-glycoproteinactivity [3334]

ConclusionsIn this research PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs polyplexed with endostatin geneshowed an optimizing potential in delivering genes andchemotherapeutic drugs The co-delivery of endostatinand docetaxel by PEI-modified TPGS-b-(PCL-ran-PGA)NPs significantly inhibited the tumor growth of nudemouse models (even regress 80 established tumor) Inconclusion our study suggested that PEI-modified TPGS-b-(PCL-ran-PGA) NPs could function as an optimizingcarrier for chemotherapeutic drugs and genetic materials

Competing interestsThe authors declare that they have no competing interests

Authorsrsquo contributionsBQ carried out the in vivo studies and drafted the manuscript MJ carried outcell studies XS carried out the preparation of nanoparticles YoZ carried outcharacterization of nanoparticles ZW carried out the in vitro drug releasestudies XZ participated in the in vivo studies SW participated in the cellstudies HC participated in the preparation of the nanoparticles LMparticipated in the design of the study and performed the statistical analysisYiZ conceived the study and participated in its design and coordination Allauthors read and approved the final manuscript

Authorsrsquo informationBQ is an associate chief physician of the Department of Orthopedics RenminHospital of Wuhan University MJ is a PhD student of the Southern MedicalUniversity XS is a Postdoctoral Fellow Tsinghua University YoZ ZW XZ andSW are PhD students of Tsinghua University HC and YiZ are assistantprofessors of the Tsinghua University LM is an associate professor of theTsinghua University

AcknowledgmentsThe authors are grateful for the financial support from Shenzhen Bureau ofScience Technology amp Information (JC200903180532AJCYJ20120614191936420 KQC201105310021A and JC201005270308A) theDoctoral Fund of Ministry of Education of China (20090002120055) theNational Natural Science Foundation of China (grant nos 31270019 and81071494) the Natural Science Foundation of Guangdong Province (No

Qiu et al Nanoscale Research Letters 2012 7666 Page 11 of 11httpwwwnanoscalereslettcomcontent71666

10451805702004178 and S2012010010046) and the Program for NewCentury Excellent Talents in University (NCET-11-0275)

Author details1Department of Orthopedics Renmin Hospital of Wuhan University Wuhan430060 Peoplersquos Republic of China 2Southern Medical UniversityGuangzhou 510515 Peoplersquos Republic of China 3The Shenzhen Key Lab ofGene and Antibody Therapy Graduate School at Shenzhen TsinghuaUniversity Shenzhen 518055 Peoplersquos Republic of China 4School of LifeSciences Tsinghua University Beijing 100084 Peoplersquos Republic of China5L401 Tsinghua Campus Xili University Town Shenzhen GuangdongProvince 518055 China

Received 11 October 2012 Accepted 12 November 2012Published 6 December 2012

References1 IARC Monographs on the evaluation of carcinogenic risks to humans

human papillomaviruses vol 90 httpmonographsiarcfr2 WHO (World Health Organization) Human papillomaviruses httpwww

who intvaccine_research diseasesviral_cancersenindex3html3 Schiller JT Castellsagueacute X Villa LL Hildesheim A An update of prophylactic

human papillomavirus L1 virus-like particle vaccine clinical trial resultsVaccine 2008 26(Suppl 10)K53ndash61

4 Winlaw DS Angiogenesis in the pathobiology and treatment of vascularand malignant diseases Ann Thorac Surg 1997 641204ndash1211

5 Folkman J Is angiogenesis an organizing principle in biology andmedicine J Pediatr Surg 2007 421ndash11

6 Folkman J Antiangiogenesis in cancer therapy-endostatin and itsmechanisms of action Exp Cell Res 2006 312594ndash607

7 Folkman J Angiogenesis an organizing principle for drug discovery NatRev Drug Discov 2007 6273ndash286

8 Michael SO Antiangiogenesis and vascular endothelial growth factorvascular endothelial growth factor receptor targeting as part of acombined-modality approach to the treatment of cancer Int J RadiatOncol Biol Phys 2007 69S64ndashS66

9 OReilly MS Boehm T Shing Y Fukai N Vasios G Lane WS Flynn E BirkheadJR Olsen BR Folkman J Endostatin an endogenous inhibitor ofangiogenesis and tumor growth Cell 1997 88277ndash285

10 Boehm T Folkman J Browder T OReilly MS Antiangiogenic therapy ofexperimental cancer does not induce acquired drug resistance Nature1997 390404ndash407

11 Herbst RS Lee AT Tran HT Abbruzzese JL Clinical studies of angiogenesisinhibitors the University of Texas MD Anderson Center Trial of HumanEndostatin Curr Oncol Rep 2001 3131ndash140

12 Zhuang HQ Yuan ZY Process in the mechanisms of endostatincombined with radiotherapy Cancer Letters 2009 2829ndash13

13 Alexander BL Ali RR Alton EW Bainbridge JW Braun S Cheng SH Flotte TRGaspar HB Grez M Griesenbach U Kaplitt MG Ott MG Seger R Simons MThrasher AJ Thrasher AZ Ylauml-Herttuala S Progress and prospects genetherapy clinical trials (part 1) Gene Ther 2007 201439ndash1447

14 Guinn BA Mulherkar R International progress in cancer gene therapyCancer Gene Ther 2008 12765ndash775

15 Scherer L Rossi JJ Weinberg MS Progress and prospects RNA-basedtherapies for treatment of HIV infection Gene Ther 2007 141057ndash1064

16 Androic I Kraumlmer A Yan R Roumldel F Gaumltje R Kaufmann M Strebhardt KYuan J Targeting cyclin B1 inhibits proliferation and sensitizes breastcancer cells to taxol BMC Cancer 2008 8391ndash401

17 Gao Y Liu XL Research progress on siRNA delivery with nonviral carriersInt J Nanomedicine 2011 61017ndash1025

18 Zhou J Wu J Hafdi N PAMAM dendrimers for efficient siRNA deliveryand potent gene silencing Chem Commun (Camb) 2006 222362ndash2364

19 Mao SR Sun W Kissel T Chitosan-based formulations for delivery of DNAand siRNA Adv Drug Deliv Rev 2010 6212ndash27

20 Son S Kim WJ Biodegradable nanoparticles modified by branchedpolyethylenimine for plasmid DNA delivery Biomaterials 201031133ndash143

21 Blum JS Saltzman WM High loading efficiency and tunable release ofplasmid DNA encapsulated in submicron particles fabricated from PLGAconjugated with poly-L-lysine J Control Release 2008 12966ndash72

22 Boussif O Lezoualch F Zanta MA Mergny MD Scherman D Demeneix BBehr JP A versatile vector for gene and oligonucleotide transfer intocells in culture and in vivo polyethylenimine Proc Natl Acad Sci USA 1995927297ndash7301

23 Zhu LP Xing J Wang QX Kou L Li C Hu B Wu ZW Wang JJ Xu GXTherapeutic efficacy of recombinant human endostatin combined withchemotherapeutics in mice-transplanted tumors Eur J Pharmacol 200961723ndash27

24 Huang L Chen H Zheng Y Song X Liu R Liu K Zeng X Mei LNanoformulation of D-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) diblock copolymer for breast cancertherapy Integr Biol 2011 3993ndash1002

25 Kim JH Park JS Yang HN Woo DG Jeon SY Do HJ Lim HY Kim JM ParkKH The use of biodegradable PLGA nanoparticles to mediate SOX9 genedelivery in human mesenchymal stem cells (hMSCs) and inducechondrogenesis Biomaterials 2011 32268ndash278

26 Wang C Ge Q Ting D Nguyen D Shen HR Chen J Eisen HN Heller JLanger R Putnam D Molecularly engineered poly (ortho ester)microspheres for enhanced delivery of DNA vaccines Nat Mater 20043190ndash196

27 Zolnik BS Burgess DJ Effect of acidic pH on PLGA microspheredegradation and release J Control Release 2007 122338ndash344

28 Yin Y Chen D Qiao M Wei X Hu H Lectin-conjugated PLGAnanoparticles loaded with thymopentin ex vivo bioadhesion and in vivobiodistribution J Control Release 2007 12327ndash38

29 Kataoka K Matsumoto T Yokoyama M Okano T Sakurai Y Fukushima SOkamoto K Kwon GS Doxorubicin-loaded poly(ethylene glycol)-poly(beta-benzyl-L-aspartate) copolymer micelles their pharmaceuticalcharacteristics and biological significance J Control Release 200064143ndash153

30 Lee AL Wang Y Cheng HY Pervaiz S Yang YY The co-delivery ofpaclitaxel and Herceptin using cationic micellar nanoparticlesBiomaterials 2009 30919ndash927

31 Fay F Quinn DJ Gilmore BF McCarron PA Scott CJ Gene delivery usingdimethyldidodecylammonium bromide-coated PLGA nanoparticlesBiomaterials 2011 314214ndash4222

32 Wang L Yao B Li Q Mei K Xu JR Li HX Wang YS Wen YJ Wang XD YangHS Li YH Luo F Wu Y Liu YY Yang L Gene therapy with recombinantadenovirus encoding endostatin encapsulated in cationic liposome incoxsackievirus and adenovirus receptor-deficient colon carcinomamurine models Hum Gene Ther 2011 22(9)1061ndash1069

33 Collnot EM Baldes C Wempe MF Kappl R Huumlttermann J Hyatt JA Edgar KJSchaefer UF Lehr CM Mechanism of inhibition of P-glycoproteinmediated efflux by vitamin E TPGS influence on ATPase activity andmembrane fluidity Mol Pharm 2007 4(3)465ndash474

34 Dintaman JM Silverman JA Inhibition of P-glycoprotein by Dalpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) Pharm Res 1999161550ndash1556

doi1011861556-276X-7-666Cite this article as Qiu et al Co-delivery of docetaxel and endostatin bya biodegradable nanoparticle for the synergistic treatment of cervicalcancer Nanoscale Research Letters 2012 7666

Submit your manuscript to a journal and benefi t from

7 Convenient online submission

7 Rigorous peer review

7 Immediate publication on acceptance

7 Open access articles freely available online

7 High visibility within the fi eld

7 Retaining the copyright to your article

Submit your next manuscript at 7 springeropencom

Figure 3 The in vitro release profile of pDNA from various NPsat pH 74 and pH 50

Figure 4 The in vitro release curve of docetaxel from differentNPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 6 of 11httpwwwnanoscalereslettcomcontent71666

Results and discussionsThe expression of pShuttle2-endostatinProtein expression of endostatin was analyzed by West-ern blot using cell lysate after transfection of HeLa cellsusing PEI (Figure 1) These results showed thatpShuttle2-endostatin was successfully constructed andexpressed in HeLa cells

Characterization of nanoparticlesRecently a novel biodegradable copolymer TPGS-b-(PCL-ran-PGA) has been successfully synthesized inour laboratory [24] Our previous study showed that theNPs made of this novel copolymer can improve drug per-meability solubility degradation and avoid the disadvan-tages of single component [24] More importantly TPGSthe degradation products of TPGS-b-(PCL-ran-PGA)showed synergistic anticancer activity in the presence ofanticancer agents [24] In this research to co-deliverychemotherapeutic drugs and genetic materials into cellsTPGS-b-(PCL-ran-PGA) NPs were modified with cat-ionic polymer PEI (25 K) DLS assay showed that thehydrodynamic diameter of the polyplexed blank TPGS-b-(PCL-ran-PGA) NPs (ANPs) was approximately 215 nmwhile the diameters of the docetaxel-loaded TPGS-b-(PCL-ran-PGA) and docetaxel-loaded TPGS-b-(PCL-ran-PGA) PEI-coated without pDNA NPs were approxi-mately 242 and approximately 270 nm respectively(Figure 2A) The FESEM images showed that the morph-ologies of TPGS-b-(PCL-ran-PGA)PEI NPs weresphere-like in shape and the size was about 250 nm(Figure 2D) consistent with the DLS data The resultsuggested that PEI modification may increase the size ofthe NPs from TPGS-b-(PCL-ran-PGA) copolymer Inaddition the surface charge of the unmodified TPGS-b-(PCL-ran-PGA) NPs was approximately minus1825 mVdetected by LDA assay while the surface charge of the PEI-modified NPs was significantly increased in zeta potentialsuggesting that the surface charge of the TPGS-b-(PCL-ran-PGA) NPs was altered from negative to positive due to thePEI coating However a reduction in surface charge of thePEI-modified NPs was observed after adding pDNAsuggesting PEI condenses negatively charged DNA intopositively charged particles (Figure 2B) Agarose gel electro-phoresis assay showed that the amount of NP-binding DNAincreased as the amount of TPGS-b-(PCL-ran-PGA)PEINPs increased (Figure 2C) In a recent report the DNAencapsulated in the polymeric (PLGA) NPs is in a con-densed form which could protect itself from denaturationand allow it to be efficiently taken up by MSCs [25] An-other study also showed that PLGAPEI NPs can protectDNA against degradation and reduce the cytotoxicity causedby PEI [25] Together results showed that the TPGS-b-(PCL-ran-PGA) NPs modified by PEI could encapsulatedocetaxel and DNA with a high loading efficiency

In vitro releasePrevious study showed that the kinetic of NP degrad-ation and DNA release appears to have a significantregulatory effect on the gene expression [26] In thisstudy we compared the in vitro release of pDNA fromdocetaxel-loaded or doceraxel-free TPGS-b-(PCL-ran-PGA)PEI-pDNA (DNPs or ENPs) formulations at pH74 and pH 50 To simulate acidification in the endo-lysosome of cells following intracellular uptake NPswere first incubated in a solution at pH 74 for 24 hthen one-half of the NPs continued to be incubated at

Figure 5 The fluorescence microscopic images of 293T cells This is after the incubation with plasmids pIRES-EGFP-loaded and pdSRED-loaded TPGS-b-(PCL-ran-PGA)PEI NPs (A) from transmitted light channel (B) from FITC channel (C) from PI channel

Figure 6 Confocal laser scanning microscopy of HeLa cells This is after incubation with loaded-coumarin-6-TPGS-b-(PCL-ran-PGA)PEI-pDSRED NPs at 37degC The cells were stained by DAPI (blue) and the plasmids pDsRED-loaded-TPGS-b-(PCL-ran-PGA)PEI NPs are red The cellularuptake was visualized by overlaying images obtained (A) Coumarin-6 (B) RED (C) DAPI and (D) co-merge

Qiu et al Nanoscale Research Letters 2012 7666 Page 7 of 11httpwwwnanoscalereslettcomcontent71666

Figure 7 Viability of HeLa cells after 24- and 48-h incubations(n = 5) Single asterisk denotes p lt 005 (compared with PBS) whiledouble asterisk indicates p lt 001 (compared with PBS)

Figure 8 The tumor volume curve after animal models weretreated by different NPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 8 of 11httpwwwnanoscalereslettcomcontent71666

pH 74 and another half was transferred to a solution ofpH 50 pDNA release was monitored by measuring UVabsorption at 260 nm at predetermined time points Inthe pH 74 group 405 and 431 of the total pDNAwere released rapidly from docetaxel-loaded NPsPEIand empty NPsPEI respectively at 48 h followed by aslow release until day 7 (Figure 3) These two formula-tions had no significant difference either at initial burstphase or slow release phase at pH 50 suggesting thatthe loaded docetaxel had no significant influence onpDNA release Zolniks group reported that cleavage ofthe ester linkage of the PLGA polymer backbone at lowpH can accelerate the polymer erosion [27] However inthis study the release kinetics of pDNA at pH 74 wassimilar to that at pH 50 but the release of pDNA fromNPs from day 7 at pH 50 was more rapid than that atpH 74 The reason was the degradation of surface ero-sion [27] Being a hydrophilic molecule located at thesurface of the TPGS-b-(PCL-ran-PGA) matrix PEI mayfacilitate the degradation of the NPs by increasing hydra-tion and thereby accelerating hydrolysis [28]In order to investigate the docetaxel release profiles

the three formulations of NPs docetaxel-loaded TPGS-b-(PCL-ran-PGA) docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI and docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pDNA were incubated in 37degC aqueous solutions atpH 74 and pH 50 respectively As shown in Figure 4 atypical release profile was observed for all six testednano-complex solutions including an initial rapid re-lease followed by a sustained slow release over a pro-longed time up to several weeks Furthermore thedocetaxel release at pH 50 was faster than the othernano-assemblies Similar pH-dependent trend was alsoobserved in other docetaxel-loaded polymeric micellarsystems [29] and such behavior was assumed to improveintracellular drug release once the nano-complexes enterthe tumor cells via endocytosis and trapped within theacidic endosomallysosomal compartments In additionboth pDNA complexion and PEI coating were found toprolong docetaxel release which is consistent with theresults for a paclitaxel and Herceptin (Genentech IncCA USA) co-delivery system using cationic micellar NPs[30] These results implied that the release profile of PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPswas prolonged and its toxicity might be decreased

Cellular uptakeTo evaluate cellular uptake of nanoparticles 293T orHeLa cells were incubated with PEI-modified TPGS-b-(PCL-ran-PGA) NPs and coumarin-6-loaded TPGS-b-(PCL-ran-PGA) NPs for 48 h The fluorescence emittedby coumarin-6 and RED protein was observed after NP in-cubation which suggested that the NPs efficiently enteredthe cells The 293T cells took up the particles in a dose-

dependent manner and EGFP and RED expressionachieved peak levels with 15 mgml of PEI complexed with100 mg of NPs (Figure 5) These findings demonstratedthat PEI-modified NPs may be good candidates for thedelivering of genes into tumor cells and PEI-modifiedTPGS-b-(PCL-ran-PGA) nanospheres may increase thegene binding ability which causes the endosomal escapeeasily and lowers the PEI cytotoxicity [25] We also foundthat more PEI-modified coumarin-6-loaded NPs wereuptaken by HeLa cells than unmodified NPs which is inconsistency with other reports [31] It was probably be-cause the PEI-modified NPs had positive charge on surface

Figure 9 The histopathological analysis of the tumor tissues from different treatment groups (A) PBS (B) ANPs (TPGS-b-(PCL-ran-PGA))(C) CNPs (TPGS-b-(PCL-ran-PGAPEIpEndo) (D) DNPs (E) ENPs and (F) FNPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 9 of 11httpwwwnanoscalereslettcomcontent71666

which provided good affiliation with cells which had nega-tive charge on the surface In addition our data alsoshowed that coumarin-6 and RED protein can co-expressin HeLa cells transfected by coumarin-6-loaded TPGS-b-(PCL-ran-PGA)PEI-pDSRED (Figure 6) suggesting thatPEI-modified TPGS-b-(PCL-ran-PGA) can co-deliver che-motherapeutic drugs and genetic materials into cells

Figure 10 MVD of tumor tissue in different treatment groups Single adenotes p lt 001 (compared with PBS)

Cell viabilityCytotoxicity of all NPs was determined in various con-centrations using MTT assay The cell viability was notaffected by TPGS-b-(PCL-ran-PGA) NPs in all concen-trations relative to the control (treated with PBS) after72 h in medium However increasing dosage of PEI-modified TPGS-b-(PCL-ran-PGA) NPs was associated

sterisk indicates p lt 005 (compared with PBS) while double asterisk

Qiu et al Nanoscale Research Letters 2012 7666 Page 10 of 11httpwwwnanoscalereslettcomcontent71666

with increased cytotoxicity because of the increase ofPEI (Figure 7) As expected for TPGS-b-(PCL-ran-PGA) TPGS-b-(PCL-ran-PGA)PEI NPs and coumarin-6-loaded NPs the cell survival rates were greater than84 whereas cell survival rate of the cells treated withequal dose of PEI was less than 70 (data not shown)suggesting that TPGS-b-(PCL-ran-PGA) could reducethe cytotoxicity of PEI (Figure 7) On the other handdocetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pEndos-tatin NPs appeared to have more cytotoxicity than thatof the other control NPs including docetaxel-loadedNPs this may be because endostatin could induce cellapoptosis [32] Among all NPs docetaxel-load NPsPEI-endostatin had the highest cytotoxicityThe cell viability of HeLa cells transfected with

docetaxel-loaded NPs was decreased after 6 h whichwas dose- and time-dependent These results indicatedthat PEI-modified TPGS-b-(PCL-ran-PGA) NPs cansimultaneously delivery docetaxel and genetic materialsinto cancer cells and kill them

In vivo studiesTo investigate the therapeutic effect of TPGS-b-(PCL-ran-PGA)-based NPs on tumors the anti-tumor efficacyand angiogenesis inhibition of various NPs includingdocetaxel-loaded NPs-PEIpEndostatin were further eval-uated in vivo Subcutaneous tumor models were con-structed in nude mice by being inoculated with 1 to 2 times106 HeLa cells The results showed that CNPs ENPs andFNPs containing docetaxel significantly inhibited tumorgrowth compared with PBS ANPs and DNPs (p lt 001)(Figure 8) The tumor volume of the mice treated withFNPs appeared to keep growing in the initial 5 days aftertreatment and then the tumor sites presentedinflammatory-like response and ulcer After 3 weeks thetumor growth was significantly inhibited and 80 tumorwas eradicated Similar trend was also found in the groupstreated with CNPs and ENPs the inhibitory effect wasbetter than other groups except of FNP treatment groupTreatment of mice with docetaxel-loaded NPs and PEI-modified docetaxel-loaded NPs inhibited 84 and 87in tumor growth inhibition efficiency respectively des-pite no significant difference (p gt 005)Compared with the PBS group the tumor growth inhib-

ition of the groups treated with empty NPs and NPsPEI-pEndostatin was 10 and 38 respectively which againproved that endostatin could inhibit tumor growth Theinhibitory effect of empty NPs may be caused by the TPGSdegradation from the NP matrix (Figure 8) The histo-pathological analysis of the tumor tissues also revealedthat NPs containing docetaxel orand endostatin inducedcell necrosis and apoptosis leading to suppression oftumor progression (Figure 9) MVD in FNP DNP andCNP groups was relatively lower (p lt 001) (Figure 10)

compared with PBS group MVD in DNP group was alsolower in contrast with PBS group (p lt 005) (Figure 10)This is due to the cell necrosis and apoptosis caused bydocetaxel and endostatin These results are consistentwith histopathological analysis of the tumor tissues Nosignificant difference in mouse weight was observedamong each NP treatment group (data not shown) At theend of the experiment dissection results revealed thatthere were no obvious signs of toxicity in the heart liverlungs spleen kidneys and other organs in each groupThese results implied that TPGS-b-(PCL-ran-PGA)-basedNPs could serve as multifunctional carrier of docetaxeland endostatin In addition during the regimen processthe release of TPGS may help the anticancer effect of doc-etaxel and endostatin because TPGS possesses the func-tions to improve drug permeability enhance absorption ofdrugs and reduce P-glycoprotein-mediated multi-drugresistance in cancer cells by inhibition of P-glycoproteinactivity [3334]

ConclusionsIn this research PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs polyplexed with endostatin geneshowed an optimizing potential in delivering genes andchemotherapeutic drugs The co-delivery of endostatinand docetaxel by PEI-modified TPGS-b-(PCL-ran-PGA)NPs significantly inhibited the tumor growth of nudemouse models (even regress 80 established tumor) Inconclusion our study suggested that PEI-modified TPGS-b-(PCL-ran-PGA) NPs could function as an optimizingcarrier for chemotherapeutic drugs and genetic materials

Competing interestsThe authors declare that they have no competing interests

Authorsrsquo contributionsBQ carried out the in vivo studies and drafted the manuscript MJ carried outcell studies XS carried out the preparation of nanoparticles YoZ carried outcharacterization of nanoparticles ZW carried out the in vitro drug releasestudies XZ participated in the in vivo studies SW participated in the cellstudies HC participated in the preparation of the nanoparticles LMparticipated in the design of the study and performed the statistical analysisYiZ conceived the study and participated in its design and coordination Allauthors read and approved the final manuscript

Authorsrsquo informationBQ is an associate chief physician of the Department of Orthopedics RenminHospital of Wuhan University MJ is a PhD student of the Southern MedicalUniversity XS is a Postdoctoral Fellow Tsinghua University YoZ ZW XZ andSW are PhD students of Tsinghua University HC and YiZ are assistantprofessors of the Tsinghua University LM is an associate professor of theTsinghua University

AcknowledgmentsThe authors are grateful for the financial support from Shenzhen Bureau ofScience Technology amp Information (JC200903180532AJCYJ20120614191936420 KQC201105310021A and JC201005270308A) theDoctoral Fund of Ministry of Education of China (20090002120055) theNational Natural Science Foundation of China (grant nos 31270019 and81071494) the Natural Science Foundation of Guangdong Province (No

Qiu et al Nanoscale Research Letters 2012 7666 Page 11 of 11httpwwwnanoscalereslettcomcontent71666

10451805702004178 and S2012010010046) and the Program for NewCentury Excellent Talents in University (NCET-11-0275)

Author details1Department of Orthopedics Renmin Hospital of Wuhan University Wuhan430060 Peoplersquos Republic of China 2Southern Medical UniversityGuangzhou 510515 Peoplersquos Republic of China 3The Shenzhen Key Lab ofGene and Antibody Therapy Graduate School at Shenzhen TsinghuaUniversity Shenzhen 518055 Peoplersquos Republic of China 4School of LifeSciences Tsinghua University Beijing 100084 Peoplersquos Republic of China5L401 Tsinghua Campus Xili University Town Shenzhen GuangdongProvince 518055 China

Received 11 October 2012 Accepted 12 November 2012Published 6 December 2012

References1 IARC Monographs on the evaluation of carcinogenic risks to humans

human papillomaviruses vol 90 httpmonographsiarcfr2 WHO (World Health Organization) Human papillomaviruses httpwww

who intvaccine_research diseasesviral_cancersenindex3html3 Schiller JT Castellsagueacute X Villa LL Hildesheim A An update of prophylactic

human papillomavirus L1 virus-like particle vaccine clinical trial resultsVaccine 2008 26(Suppl 10)K53ndash61

4 Winlaw DS Angiogenesis in the pathobiology and treatment of vascularand malignant diseases Ann Thorac Surg 1997 641204ndash1211

5 Folkman J Is angiogenesis an organizing principle in biology andmedicine J Pediatr Surg 2007 421ndash11

6 Folkman J Antiangiogenesis in cancer therapy-endostatin and itsmechanisms of action Exp Cell Res 2006 312594ndash607

7 Folkman J Angiogenesis an organizing principle for drug discovery NatRev Drug Discov 2007 6273ndash286

8 Michael SO Antiangiogenesis and vascular endothelial growth factorvascular endothelial growth factor receptor targeting as part of acombined-modality approach to the treatment of cancer Int J RadiatOncol Biol Phys 2007 69S64ndashS66

9 OReilly MS Boehm T Shing Y Fukai N Vasios G Lane WS Flynn E BirkheadJR Olsen BR Folkman J Endostatin an endogenous inhibitor ofangiogenesis and tumor growth Cell 1997 88277ndash285

10 Boehm T Folkman J Browder T OReilly MS Antiangiogenic therapy ofexperimental cancer does not induce acquired drug resistance Nature1997 390404ndash407

11 Herbst RS Lee AT Tran HT Abbruzzese JL Clinical studies of angiogenesisinhibitors the University of Texas MD Anderson Center Trial of HumanEndostatin Curr Oncol Rep 2001 3131ndash140

12 Zhuang HQ Yuan ZY Process in the mechanisms of endostatincombined with radiotherapy Cancer Letters 2009 2829ndash13

13 Alexander BL Ali RR Alton EW Bainbridge JW Braun S Cheng SH Flotte TRGaspar HB Grez M Griesenbach U Kaplitt MG Ott MG Seger R Simons MThrasher AJ Thrasher AZ Ylauml-Herttuala S Progress and prospects genetherapy clinical trials (part 1) Gene Ther 2007 201439ndash1447

14 Guinn BA Mulherkar R International progress in cancer gene therapyCancer Gene Ther 2008 12765ndash775

15 Scherer L Rossi JJ Weinberg MS Progress and prospects RNA-basedtherapies for treatment of HIV infection Gene Ther 2007 141057ndash1064

16 Androic I Kraumlmer A Yan R Roumldel F Gaumltje R Kaufmann M Strebhardt KYuan J Targeting cyclin B1 inhibits proliferation and sensitizes breastcancer cells to taxol BMC Cancer 2008 8391ndash401

17 Gao Y Liu XL Research progress on siRNA delivery with nonviral carriersInt J Nanomedicine 2011 61017ndash1025

18 Zhou J Wu J Hafdi N PAMAM dendrimers for efficient siRNA deliveryand potent gene silencing Chem Commun (Camb) 2006 222362ndash2364

19 Mao SR Sun W Kissel T Chitosan-based formulations for delivery of DNAand siRNA Adv Drug Deliv Rev 2010 6212ndash27

20 Son S Kim WJ Biodegradable nanoparticles modified by branchedpolyethylenimine for plasmid DNA delivery Biomaterials 201031133ndash143

21 Blum JS Saltzman WM High loading efficiency and tunable release ofplasmid DNA encapsulated in submicron particles fabricated from PLGAconjugated with poly-L-lysine J Control Release 2008 12966ndash72

22 Boussif O Lezoualch F Zanta MA Mergny MD Scherman D Demeneix BBehr JP A versatile vector for gene and oligonucleotide transfer intocells in culture and in vivo polyethylenimine Proc Natl Acad Sci USA 1995927297ndash7301

23 Zhu LP Xing J Wang QX Kou L Li C Hu B Wu ZW Wang JJ Xu GXTherapeutic efficacy of recombinant human endostatin combined withchemotherapeutics in mice-transplanted tumors Eur J Pharmacol 200961723ndash27

24 Huang L Chen H Zheng Y Song X Liu R Liu K Zeng X Mei LNanoformulation of D-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) diblock copolymer for breast cancertherapy Integr Biol 2011 3993ndash1002

25 Kim JH Park JS Yang HN Woo DG Jeon SY Do HJ Lim HY Kim JM ParkKH The use of biodegradable PLGA nanoparticles to mediate SOX9 genedelivery in human mesenchymal stem cells (hMSCs) and inducechondrogenesis Biomaterials 2011 32268ndash278

26 Wang C Ge Q Ting D Nguyen D Shen HR Chen J Eisen HN Heller JLanger R Putnam D Molecularly engineered poly (ortho ester)microspheres for enhanced delivery of DNA vaccines Nat Mater 20043190ndash196

27 Zolnik BS Burgess DJ Effect of acidic pH on PLGA microspheredegradation and release J Control Release 2007 122338ndash344

28 Yin Y Chen D Qiao M Wei X Hu H Lectin-conjugated PLGAnanoparticles loaded with thymopentin ex vivo bioadhesion and in vivobiodistribution J Control Release 2007 12327ndash38

29 Kataoka K Matsumoto T Yokoyama M Okano T Sakurai Y Fukushima SOkamoto K Kwon GS Doxorubicin-loaded poly(ethylene glycol)-poly(beta-benzyl-L-aspartate) copolymer micelles their pharmaceuticalcharacteristics and biological significance J Control Release 200064143ndash153

30 Lee AL Wang Y Cheng HY Pervaiz S Yang YY The co-delivery ofpaclitaxel and Herceptin using cationic micellar nanoparticlesBiomaterials 2009 30919ndash927

31 Fay F Quinn DJ Gilmore BF McCarron PA Scott CJ Gene delivery usingdimethyldidodecylammonium bromide-coated PLGA nanoparticlesBiomaterials 2011 314214ndash4222

32 Wang L Yao B Li Q Mei K Xu JR Li HX Wang YS Wen YJ Wang XD YangHS Li YH Luo F Wu Y Liu YY Yang L Gene therapy with recombinantadenovirus encoding endostatin encapsulated in cationic liposome incoxsackievirus and adenovirus receptor-deficient colon carcinomamurine models Hum Gene Ther 2011 22(9)1061ndash1069

33 Collnot EM Baldes C Wempe MF Kappl R Huumlttermann J Hyatt JA Edgar KJSchaefer UF Lehr CM Mechanism of inhibition of P-glycoproteinmediated efflux by vitamin E TPGS influence on ATPase activity andmembrane fluidity Mol Pharm 2007 4(3)465ndash474

34 Dintaman JM Silverman JA Inhibition of P-glycoprotein by Dalpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) Pharm Res 1999161550ndash1556

doi1011861556-276X-7-666Cite this article as Qiu et al Co-delivery of docetaxel and endostatin bya biodegradable nanoparticle for the synergistic treatment of cervicalcancer Nanoscale Research Letters 2012 7666

Submit your manuscript to a journal and benefi t from

7 Convenient online submission

7 Rigorous peer review

7 Immediate publication on acceptance

7 Open access articles freely available online

7 High visibility within the fi eld

7 Retaining the copyright to your article

Submit your next manuscript at 7 springeropencom

Figure 5 The fluorescence microscopic images of 293T cells This is after the incubation with plasmids pIRES-EGFP-loaded and pdSRED-loaded TPGS-b-(PCL-ran-PGA)PEI NPs (A) from transmitted light channel (B) from FITC channel (C) from PI channel

Figure 6 Confocal laser scanning microscopy of HeLa cells This is after incubation with loaded-coumarin-6-TPGS-b-(PCL-ran-PGA)PEI-pDSRED NPs at 37degC The cells were stained by DAPI (blue) and the plasmids pDsRED-loaded-TPGS-b-(PCL-ran-PGA)PEI NPs are red The cellularuptake was visualized by overlaying images obtained (A) Coumarin-6 (B) RED (C) DAPI and (D) co-merge

Qiu et al Nanoscale Research Letters 2012 7666 Page 7 of 11httpwwwnanoscalereslettcomcontent71666

Figure 7 Viability of HeLa cells after 24- and 48-h incubations(n = 5) Single asterisk denotes p lt 005 (compared with PBS) whiledouble asterisk indicates p lt 001 (compared with PBS)

Figure 8 The tumor volume curve after animal models weretreated by different NPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 8 of 11httpwwwnanoscalereslettcomcontent71666

pH 74 and another half was transferred to a solution ofpH 50 pDNA release was monitored by measuring UVabsorption at 260 nm at predetermined time points Inthe pH 74 group 405 and 431 of the total pDNAwere released rapidly from docetaxel-loaded NPsPEIand empty NPsPEI respectively at 48 h followed by aslow release until day 7 (Figure 3) These two formula-tions had no significant difference either at initial burstphase or slow release phase at pH 50 suggesting thatthe loaded docetaxel had no significant influence onpDNA release Zolniks group reported that cleavage ofthe ester linkage of the PLGA polymer backbone at lowpH can accelerate the polymer erosion [27] However inthis study the release kinetics of pDNA at pH 74 wassimilar to that at pH 50 but the release of pDNA fromNPs from day 7 at pH 50 was more rapid than that atpH 74 The reason was the degradation of surface ero-sion [27] Being a hydrophilic molecule located at thesurface of the TPGS-b-(PCL-ran-PGA) matrix PEI mayfacilitate the degradation of the NPs by increasing hydra-tion and thereby accelerating hydrolysis [28]In order to investigate the docetaxel release profiles

the three formulations of NPs docetaxel-loaded TPGS-b-(PCL-ran-PGA) docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI and docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pDNA were incubated in 37degC aqueous solutions atpH 74 and pH 50 respectively As shown in Figure 4 atypical release profile was observed for all six testednano-complex solutions including an initial rapid re-lease followed by a sustained slow release over a pro-longed time up to several weeks Furthermore thedocetaxel release at pH 50 was faster than the othernano-assemblies Similar pH-dependent trend was alsoobserved in other docetaxel-loaded polymeric micellarsystems [29] and such behavior was assumed to improveintracellular drug release once the nano-complexes enterthe tumor cells via endocytosis and trapped within theacidic endosomallysosomal compartments In additionboth pDNA complexion and PEI coating were found toprolong docetaxel release which is consistent with theresults for a paclitaxel and Herceptin (Genentech IncCA USA) co-delivery system using cationic micellar NPs[30] These results implied that the release profile of PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPswas prolonged and its toxicity might be decreased

Cellular uptakeTo evaluate cellular uptake of nanoparticles 293T orHeLa cells were incubated with PEI-modified TPGS-b-(PCL-ran-PGA) NPs and coumarin-6-loaded TPGS-b-(PCL-ran-PGA) NPs for 48 h The fluorescence emittedby coumarin-6 and RED protein was observed after NP in-cubation which suggested that the NPs efficiently enteredthe cells The 293T cells took up the particles in a dose-

dependent manner and EGFP and RED expressionachieved peak levels with 15 mgml of PEI complexed with100 mg of NPs (Figure 5) These findings demonstratedthat PEI-modified NPs may be good candidates for thedelivering of genes into tumor cells and PEI-modifiedTPGS-b-(PCL-ran-PGA) nanospheres may increase thegene binding ability which causes the endosomal escapeeasily and lowers the PEI cytotoxicity [25] We also foundthat more PEI-modified coumarin-6-loaded NPs wereuptaken by HeLa cells than unmodified NPs which is inconsistency with other reports [31] It was probably be-cause the PEI-modified NPs had positive charge on surface

Figure 9 The histopathological analysis of the tumor tissues from different treatment groups (A) PBS (B) ANPs (TPGS-b-(PCL-ran-PGA))(C) CNPs (TPGS-b-(PCL-ran-PGAPEIpEndo) (D) DNPs (E) ENPs and (F) FNPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 9 of 11httpwwwnanoscalereslettcomcontent71666

which provided good affiliation with cells which had nega-tive charge on the surface In addition our data alsoshowed that coumarin-6 and RED protein can co-expressin HeLa cells transfected by coumarin-6-loaded TPGS-b-(PCL-ran-PGA)PEI-pDSRED (Figure 6) suggesting thatPEI-modified TPGS-b-(PCL-ran-PGA) can co-deliver che-motherapeutic drugs and genetic materials into cells

Figure 10 MVD of tumor tissue in different treatment groups Single adenotes p lt 001 (compared with PBS)

Cell viabilityCytotoxicity of all NPs was determined in various con-centrations using MTT assay The cell viability was notaffected by TPGS-b-(PCL-ran-PGA) NPs in all concen-trations relative to the control (treated with PBS) after72 h in medium However increasing dosage of PEI-modified TPGS-b-(PCL-ran-PGA) NPs was associated

sterisk indicates p lt 005 (compared with PBS) while double asterisk

Qiu et al Nanoscale Research Letters 2012 7666 Page 10 of 11httpwwwnanoscalereslettcomcontent71666

with increased cytotoxicity because of the increase ofPEI (Figure 7) As expected for TPGS-b-(PCL-ran-PGA) TPGS-b-(PCL-ran-PGA)PEI NPs and coumarin-6-loaded NPs the cell survival rates were greater than84 whereas cell survival rate of the cells treated withequal dose of PEI was less than 70 (data not shown)suggesting that TPGS-b-(PCL-ran-PGA) could reducethe cytotoxicity of PEI (Figure 7) On the other handdocetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pEndos-tatin NPs appeared to have more cytotoxicity than thatof the other control NPs including docetaxel-loadedNPs this may be because endostatin could induce cellapoptosis [32] Among all NPs docetaxel-load NPsPEI-endostatin had the highest cytotoxicityThe cell viability of HeLa cells transfected with

docetaxel-loaded NPs was decreased after 6 h whichwas dose- and time-dependent These results indicatedthat PEI-modified TPGS-b-(PCL-ran-PGA) NPs cansimultaneously delivery docetaxel and genetic materialsinto cancer cells and kill them

In vivo studiesTo investigate the therapeutic effect of TPGS-b-(PCL-ran-PGA)-based NPs on tumors the anti-tumor efficacyand angiogenesis inhibition of various NPs includingdocetaxel-loaded NPs-PEIpEndostatin were further eval-uated in vivo Subcutaneous tumor models were con-structed in nude mice by being inoculated with 1 to 2 times106 HeLa cells The results showed that CNPs ENPs andFNPs containing docetaxel significantly inhibited tumorgrowth compared with PBS ANPs and DNPs (p lt 001)(Figure 8) The tumor volume of the mice treated withFNPs appeared to keep growing in the initial 5 days aftertreatment and then the tumor sites presentedinflammatory-like response and ulcer After 3 weeks thetumor growth was significantly inhibited and 80 tumorwas eradicated Similar trend was also found in the groupstreated with CNPs and ENPs the inhibitory effect wasbetter than other groups except of FNP treatment groupTreatment of mice with docetaxel-loaded NPs and PEI-modified docetaxel-loaded NPs inhibited 84 and 87in tumor growth inhibition efficiency respectively des-pite no significant difference (p gt 005)Compared with the PBS group the tumor growth inhib-

ition of the groups treated with empty NPs and NPsPEI-pEndostatin was 10 and 38 respectively which againproved that endostatin could inhibit tumor growth Theinhibitory effect of empty NPs may be caused by the TPGSdegradation from the NP matrix (Figure 8) The histo-pathological analysis of the tumor tissues also revealedthat NPs containing docetaxel orand endostatin inducedcell necrosis and apoptosis leading to suppression oftumor progression (Figure 9) MVD in FNP DNP andCNP groups was relatively lower (p lt 001) (Figure 10)

compared with PBS group MVD in DNP group was alsolower in contrast with PBS group (p lt 005) (Figure 10)This is due to the cell necrosis and apoptosis caused bydocetaxel and endostatin These results are consistentwith histopathological analysis of the tumor tissues Nosignificant difference in mouse weight was observedamong each NP treatment group (data not shown) At theend of the experiment dissection results revealed thatthere were no obvious signs of toxicity in the heart liverlungs spleen kidneys and other organs in each groupThese results implied that TPGS-b-(PCL-ran-PGA)-basedNPs could serve as multifunctional carrier of docetaxeland endostatin In addition during the regimen processthe release of TPGS may help the anticancer effect of doc-etaxel and endostatin because TPGS possesses the func-tions to improve drug permeability enhance absorption ofdrugs and reduce P-glycoprotein-mediated multi-drugresistance in cancer cells by inhibition of P-glycoproteinactivity [3334]

ConclusionsIn this research PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs polyplexed with endostatin geneshowed an optimizing potential in delivering genes andchemotherapeutic drugs The co-delivery of endostatinand docetaxel by PEI-modified TPGS-b-(PCL-ran-PGA)NPs significantly inhibited the tumor growth of nudemouse models (even regress 80 established tumor) Inconclusion our study suggested that PEI-modified TPGS-b-(PCL-ran-PGA) NPs could function as an optimizingcarrier for chemotherapeutic drugs and genetic materials

Competing interestsThe authors declare that they have no competing interests

Authorsrsquo contributionsBQ carried out the in vivo studies and drafted the manuscript MJ carried outcell studies XS carried out the preparation of nanoparticles YoZ carried outcharacterization of nanoparticles ZW carried out the in vitro drug releasestudies XZ participated in the in vivo studies SW participated in the cellstudies HC participated in the preparation of the nanoparticles LMparticipated in the design of the study and performed the statistical analysisYiZ conceived the study and participated in its design and coordination Allauthors read and approved the final manuscript

Authorsrsquo informationBQ is an associate chief physician of the Department of Orthopedics RenminHospital of Wuhan University MJ is a PhD student of the Southern MedicalUniversity XS is a Postdoctoral Fellow Tsinghua University YoZ ZW XZ andSW are PhD students of Tsinghua University HC and YiZ are assistantprofessors of the Tsinghua University LM is an associate professor of theTsinghua University

AcknowledgmentsThe authors are grateful for the financial support from Shenzhen Bureau ofScience Technology amp Information (JC200903180532AJCYJ20120614191936420 KQC201105310021A and JC201005270308A) theDoctoral Fund of Ministry of Education of China (20090002120055) theNational Natural Science Foundation of China (grant nos 31270019 and81071494) the Natural Science Foundation of Guangdong Province (No

Qiu et al Nanoscale Research Letters 2012 7666 Page 11 of 11httpwwwnanoscalereslettcomcontent71666

10451805702004178 and S2012010010046) and the Program for NewCentury Excellent Talents in University (NCET-11-0275)

Author details1Department of Orthopedics Renmin Hospital of Wuhan University Wuhan430060 Peoplersquos Republic of China 2Southern Medical UniversityGuangzhou 510515 Peoplersquos Republic of China 3The Shenzhen Key Lab ofGene and Antibody Therapy Graduate School at Shenzhen TsinghuaUniversity Shenzhen 518055 Peoplersquos Republic of China 4School of LifeSciences Tsinghua University Beijing 100084 Peoplersquos Republic of China5L401 Tsinghua Campus Xili University Town Shenzhen GuangdongProvince 518055 China

Received 11 October 2012 Accepted 12 November 2012Published 6 December 2012

References1 IARC Monographs on the evaluation of carcinogenic risks to humans

human papillomaviruses vol 90 httpmonographsiarcfr2 WHO (World Health Organization) Human papillomaviruses httpwww

who intvaccine_research diseasesviral_cancersenindex3html3 Schiller JT Castellsagueacute X Villa LL Hildesheim A An update of prophylactic

human papillomavirus L1 virus-like particle vaccine clinical trial resultsVaccine 2008 26(Suppl 10)K53ndash61

4 Winlaw DS Angiogenesis in the pathobiology and treatment of vascularand malignant diseases Ann Thorac Surg 1997 641204ndash1211

5 Folkman J Is angiogenesis an organizing principle in biology andmedicine J Pediatr Surg 2007 421ndash11

6 Folkman J Antiangiogenesis in cancer therapy-endostatin and itsmechanisms of action Exp Cell Res 2006 312594ndash607

7 Folkman J Angiogenesis an organizing principle for drug discovery NatRev Drug Discov 2007 6273ndash286

8 Michael SO Antiangiogenesis and vascular endothelial growth factorvascular endothelial growth factor receptor targeting as part of acombined-modality approach to the treatment of cancer Int J RadiatOncol Biol Phys 2007 69S64ndashS66

9 OReilly MS Boehm T Shing Y Fukai N Vasios G Lane WS Flynn E BirkheadJR Olsen BR Folkman J Endostatin an endogenous inhibitor ofangiogenesis and tumor growth Cell 1997 88277ndash285

10 Boehm T Folkman J Browder T OReilly MS Antiangiogenic therapy ofexperimental cancer does not induce acquired drug resistance Nature1997 390404ndash407

11 Herbst RS Lee AT Tran HT Abbruzzese JL Clinical studies of angiogenesisinhibitors the University of Texas MD Anderson Center Trial of HumanEndostatin Curr Oncol Rep 2001 3131ndash140

12 Zhuang HQ Yuan ZY Process in the mechanisms of endostatincombined with radiotherapy Cancer Letters 2009 2829ndash13

13 Alexander BL Ali RR Alton EW Bainbridge JW Braun S Cheng SH Flotte TRGaspar HB Grez M Griesenbach U Kaplitt MG Ott MG Seger R Simons MThrasher AJ Thrasher AZ Ylauml-Herttuala S Progress and prospects genetherapy clinical trials (part 1) Gene Ther 2007 201439ndash1447

14 Guinn BA Mulherkar R International progress in cancer gene therapyCancer Gene Ther 2008 12765ndash775

15 Scherer L Rossi JJ Weinberg MS Progress and prospects RNA-basedtherapies for treatment of HIV infection Gene Ther 2007 141057ndash1064

16 Androic I Kraumlmer A Yan R Roumldel F Gaumltje R Kaufmann M Strebhardt KYuan J Targeting cyclin B1 inhibits proliferation and sensitizes breastcancer cells to taxol BMC Cancer 2008 8391ndash401

17 Gao Y Liu XL Research progress on siRNA delivery with nonviral carriersInt J Nanomedicine 2011 61017ndash1025

18 Zhou J Wu J Hafdi N PAMAM dendrimers for efficient siRNA deliveryand potent gene silencing Chem Commun (Camb) 2006 222362ndash2364

19 Mao SR Sun W Kissel T Chitosan-based formulations for delivery of DNAand siRNA Adv Drug Deliv Rev 2010 6212ndash27

20 Son S Kim WJ Biodegradable nanoparticles modified by branchedpolyethylenimine for plasmid DNA delivery Biomaterials 201031133ndash143

21 Blum JS Saltzman WM High loading efficiency and tunable release ofplasmid DNA encapsulated in submicron particles fabricated from PLGAconjugated with poly-L-lysine J Control Release 2008 12966ndash72

22 Boussif O Lezoualch F Zanta MA Mergny MD Scherman D Demeneix BBehr JP A versatile vector for gene and oligonucleotide transfer intocells in culture and in vivo polyethylenimine Proc Natl Acad Sci USA 1995927297ndash7301

23 Zhu LP Xing J Wang QX Kou L Li C Hu B Wu ZW Wang JJ Xu GXTherapeutic efficacy of recombinant human endostatin combined withchemotherapeutics in mice-transplanted tumors Eur J Pharmacol 200961723ndash27

24 Huang L Chen H Zheng Y Song X Liu R Liu K Zeng X Mei LNanoformulation of D-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) diblock copolymer for breast cancertherapy Integr Biol 2011 3993ndash1002

25 Kim JH Park JS Yang HN Woo DG Jeon SY Do HJ Lim HY Kim JM ParkKH The use of biodegradable PLGA nanoparticles to mediate SOX9 genedelivery in human mesenchymal stem cells (hMSCs) and inducechondrogenesis Biomaterials 2011 32268ndash278

26 Wang C Ge Q Ting D Nguyen D Shen HR Chen J Eisen HN Heller JLanger R Putnam D Molecularly engineered poly (ortho ester)microspheres for enhanced delivery of DNA vaccines Nat Mater 20043190ndash196

27 Zolnik BS Burgess DJ Effect of acidic pH on PLGA microspheredegradation and release J Control Release 2007 122338ndash344

28 Yin Y Chen D Qiao M Wei X Hu H Lectin-conjugated PLGAnanoparticles loaded with thymopentin ex vivo bioadhesion and in vivobiodistribution J Control Release 2007 12327ndash38

29 Kataoka K Matsumoto T Yokoyama M Okano T Sakurai Y Fukushima SOkamoto K Kwon GS Doxorubicin-loaded poly(ethylene glycol)-poly(beta-benzyl-L-aspartate) copolymer micelles their pharmaceuticalcharacteristics and biological significance J Control Release 200064143ndash153

30 Lee AL Wang Y Cheng HY Pervaiz S Yang YY The co-delivery ofpaclitaxel and Herceptin using cationic micellar nanoparticlesBiomaterials 2009 30919ndash927

31 Fay F Quinn DJ Gilmore BF McCarron PA Scott CJ Gene delivery usingdimethyldidodecylammonium bromide-coated PLGA nanoparticlesBiomaterials 2011 314214ndash4222

32 Wang L Yao B Li Q Mei K Xu JR Li HX Wang YS Wen YJ Wang XD YangHS Li YH Luo F Wu Y Liu YY Yang L Gene therapy with recombinantadenovirus encoding endostatin encapsulated in cationic liposome incoxsackievirus and adenovirus receptor-deficient colon carcinomamurine models Hum Gene Ther 2011 22(9)1061ndash1069

33 Collnot EM Baldes C Wempe MF Kappl R Huumlttermann J Hyatt JA Edgar KJSchaefer UF Lehr CM Mechanism of inhibition of P-glycoproteinmediated efflux by vitamin E TPGS influence on ATPase activity andmembrane fluidity Mol Pharm 2007 4(3)465ndash474

34 Dintaman JM Silverman JA Inhibition of P-glycoprotein by Dalpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) Pharm Res 1999161550ndash1556

doi1011861556-276X-7-666Cite this article as Qiu et al Co-delivery of docetaxel and endostatin bya biodegradable nanoparticle for the synergistic treatment of cervicalcancer Nanoscale Research Letters 2012 7666

Submit your manuscript to a journal and benefi t from

7 Convenient online submission

7 Rigorous peer review

7 Immediate publication on acceptance

7 Open access articles freely available online

7 High visibility within the fi eld

7 Retaining the copyright to your article

Submit your next manuscript at 7 springeropencom

Figure 7 Viability of HeLa cells after 24- and 48-h incubations(n = 5) Single asterisk denotes p lt 005 (compared with PBS) whiledouble asterisk indicates p lt 001 (compared with PBS)

Figure 8 The tumor volume curve after animal models weretreated by different NPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 8 of 11httpwwwnanoscalereslettcomcontent71666

pH 74 and another half was transferred to a solution ofpH 50 pDNA release was monitored by measuring UVabsorption at 260 nm at predetermined time points Inthe pH 74 group 405 and 431 of the total pDNAwere released rapidly from docetaxel-loaded NPsPEIand empty NPsPEI respectively at 48 h followed by aslow release until day 7 (Figure 3) These two formula-tions had no significant difference either at initial burstphase or slow release phase at pH 50 suggesting thatthe loaded docetaxel had no significant influence onpDNA release Zolniks group reported that cleavage ofthe ester linkage of the PLGA polymer backbone at lowpH can accelerate the polymer erosion [27] However inthis study the release kinetics of pDNA at pH 74 wassimilar to that at pH 50 but the release of pDNA fromNPs from day 7 at pH 50 was more rapid than that atpH 74 The reason was the degradation of surface ero-sion [27] Being a hydrophilic molecule located at thesurface of the TPGS-b-(PCL-ran-PGA) matrix PEI mayfacilitate the degradation of the NPs by increasing hydra-tion and thereby accelerating hydrolysis [28]In order to investigate the docetaxel release profiles

the three formulations of NPs docetaxel-loaded TPGS-b-(PCL-ran-PGA) docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI and docetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pDNA were incubated in 37degC aqueous solutions atpH 74 and pH 50 respectively As shown in Figure 4 atypical release profile was observed for all six testednano-complex solutions including an initial rapid re-lease followed by a sustained slow release over a pro-longed time up to several weeks Furthermore thedocetaxel release at pH 50 was faster than the othernano-assemblies Similar pH-dependent trend was alsoobserved in other docetaxel-loaded polymeric micellarsystems [29] and such behavior was assumed to improveintracellular drug release once the nano-complexes enterthe tumor cells via endocytosis and trapped within theacidic endosomallysosomal compartments In additionboth pDNA complexion and PEI coating were found toprolong docetaxel release which is consistent with theresults for a paclitaxel and Herceptin (Genentech IncCA USA) co-delivery system using cationic micellar NPs[30] These results implied that the release profile of PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPswas prolonged and its toxicity might be decreased

Cellular uptakeTo evaluate cellular uptake of nanoparticles 293T orHeLa cells were incubated with PEI-modified TPGS-b-(PCL-ran-PGA) NPs and coumarin-6-loaded TPGS-b-(PCL-ran-PGA) NPs for 48 h The fluorescence emittedby coumarin-6 and RED protein was observed after NP in-cubation which suggested that the NPs efficiently enteredthe cells The 293T cells took up the particles in a dose-

dependent manner and EGFP and RED expressionachieved peak levels with 15 mgml of PEI complexed with100 mg of NPs (Figure 5) These findings demonstratedthat PEI-modified NPs may be good candidates for thedelivering of genes into tumor cells and PEI-modifiedTPGS-b-(PCL-ran-PGA) nanospheres may increase thegene binding ability which causes the endosomal escapeeasily and lowers the PEI cytotoxicity [25] We also foundthat more PEI-modified coumarin-6-loaded NPs wereuptaken by HeLa cells than unmodified NPs which is inconsistency with other reports [31] It was probably be-cause the PEI-modified NPs had positive charge on surface

Figure 9 The histopathological analysis of the tumor tissues from different treatment groups (A) PBS (B) ANPs (TPGS-b-(PCL-ran-PGA))(C) CNPs (TPGS-b-(PCL-ran-PGAPEIpEndo) (D) DNPs (E) ENPs and (F) FNPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 9 of 11httpwwwnanoscalereslettcomcontent71666

which provided good affiliation with cells which had nega-tive charge on the surface In addition our data alsoshowed that coumarin-6 and RED protein can co-expressin HeLa cells transfected by coumarin-6-loaded TPGS-b-(PCL-ran-PGA)PEI-pDSRED (Figure 6) suggesting thatPEI-modified TPGS-b-(PCL-ran-PGA) can co-deliver che-motherapeutic drugs and genetic materials into cells

Figure 10 MVD of tumor tissue in different treatment groups Single adenotes p lt 001 (compared with PBS)

Cell viabilityCytotoxicity of all NPs was determined in various con-centrations using MTT assay The cell viability was notaffected by TPGS-b-(PCL-ran-PGA) NPs in all concen-trations relative to the control (treated with PBS) after72 h in medium However increasing dosage of PEI-modified TPGS-b-(PCL-ran-PGA) NPs was associated

sterisk indicates p lt 005 (compared with PBS) while double asterisk

Qiu et al Nanoscale Research Letters 2012 7666 Page 10 of 11httpwwwnanoscalereslettcomcontent71666

with increased cytotoxicity because of the increase ofPEI (Figure 7) As expected for TPGS-b-(PCL-ran-PGA) TPGS-b-(PCL-ran-PGA)PEI NPs and coumarin-6-loaded NPs the cell survival rates were greater than84 whereas cell survival rate of the cells treated withequal dose of PEI was less than 70 (data not shown)suggesting that TPGS-b-(PCL-ran-PGA) could reducethe cytotoxicity of PEI (Figure 7) On the other handdocetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pEndos-tatin NPs appeared to have more cytotoxicity than thatof the other control NPs including docetaxel-loadedNPs this may be because endostatin could induce cellapoptosis [32] Among all NPs docetaxel-load NPsPEI-endostatin had the highest cytotoxicityThe cell viability of HeLa cells transfected with

docetaxel-loaded NPs was decreased after 6 h whichwas dose- and time-dependent These results indicatedthat PEI-modified TPGS-b-(PCL-ran-PGA) NPs cansimultaneously delivery docetaxel and genetic materialsinto cancer cells and kill them

In vivo studiesTo investigate the therapeutic effect of TPGS-b-(PCL-ran-PGA)-based NPs on tumors the anti-tumor efficacyand angiogenesis inhibition of various NPs includingdocetaxel-loaded NPs-PEIpEndostatin were further eval-uated in vivo Subcutaneous tumor models were con-structed in nude mice by being inoculated with 1 to 2 times106 HeLa cells The results showed that CNPs ENPs andFNPs containing docetaxel significantly inhibited tumorgrowth compared with PBS ANPs and DNPs (p lt 001)(Figure 8) The tumor volume of the mice treated withFNPs appeared to keep growing in the initial 5 days aftertreatment and then the tumor sites presentedinflammatory-like response and ulcer After 3 weeks thetumor growth was significantly inhibited and 80 tumorwas eradicated Similar trend was also found in the groupstreated with CNPs and ENPs the inhibitory effect wasbetter than other groups except of FNP treatment groupTreatment of mice with docetaxel-loaded NPs and PEI-modified docetaxel-loaded NPs inhibited 84 and 87in tumor growth inhibition efficiency respectively des-pite no significant difference (p gt 005)Compared with the PBS group the tumor growth inhib-

ition of the groups treated with empty NPs and NPsPEI-pEndostatin was 10 and 38 respectively which againproved that endostatin could inhibit tumor growth Theinhibitory effect of empty NPs may be caused by the TPGSdegradation from the NP matrix (Figure 8) The histo-pathological analysis of the tumor tissues also revealedthat NPs containing docetaxel orand endostatin inducedcell necrosis and apoptosis leading to suppression oftumor progression (Figure 9) MVD in FNP DNP andCNP groups was relatively lower (p lt 001) (Figure 10)

compared with PBS group MVD in DNP group was alsolower in contrast with PBS group (p lt 005) (Figure 10)This is due to the cell necrosis and apoptosis caused bydocetaxel and endostatin These results are consistentwith histopathological analysis of the tumor tissues Nosignificant difference in mouse weight was observedamong each NP treatment group (data not shown) At theend of the experiment dissection results revealed thatthere were no obvious signs of toxicity in the heart liverlungs spleen kidneys and other organs in each groupThese results implied that TPGS-b-(PCL-ran-PGA)-basedNPs could serve as multifunctional carrier of docetaxeland endostatin In addition during the regimen processthe release of TPGS may help the anticancer effect of doc-etaxel and endostatin because TPGS possesses the func-tions to improve drug permeability enhance absorption ofdrugs and reduce P-glycoprotein-mediated multi-drugresistance in cancer cells by inhibition of P-glycoproteinactivity [3334]

ConclusionsIn this research PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs polyplexed with endostatin geneshowed an optimizing potential in delivering genes andchemotherapeutic drugs The co-delivery of endostatinand docetaxel by PEI-modified TPGS-b-(PCL-ran-PGA)NPs significantly inhibited the tumor growth of nudemouse models (even regress 80 established tumor) Inconclusion our study suggested that PEI-modified TPGS-b-(PCL-ran-PGA) NPs could function as an optimizingcarrier for chemotherapeutic drugs and genetic materials

Competing interestsThe authors declare that they have no competing interests

Authorsrsquo contributionsBQ carried out the in vivo studies and drafted the manuscript MJ carried outcell studies XS carried out the preparation of nanoparticles YoZ carried outcharacterization of nanoparticles ZW carried out the in vitro drug releasestudies XZ participated in the in vivo studies SW participated in the cellstudies HC participated in the preparation of the nanoparticles LMparticipated in the design of the study and performed the statistical analysisYiZ conceived the study and participated in its design and coordination Allauthors read and approved the final manuscript

Authorsrsquo informationBQ is an associate chief physician of the Department of Orthopedics RenminHospital of Wuhan University MJ is a PhD student of the Southern MedicalUniversity XS is a Postdoctoral Fellow Tsinghua University YoZ ZW XZ andSW are PhD students of Tsinghua University HC and YiZ are assistantprofessors of the Tsinghua University LM is an associate professor of theTsinghua University

AcknowledgmentsThe authors are grateful for the financial support from Shenzhen Bureau ofScience Technology amp Information (JC200903180532AJCYJ20120614191936420 KQC201105310021A and JC201005270308A) theDoctoral Fund of Ministry of Education of China (20090002120055) theNational Natural Science Foundation of China (grant nos 31270019 and81071494) the Natural Science Foundation of Guangdong Province (No

Qiu et al Nanoscale Research Letters 2012 7666 Page 11 of 11httpwwwnanoscalereslettcomcontent71666

10451805702004178 and S2012010010046) and the Program for NewCentury Excellent Talents in University (NCET-11-0275)

Author details1Department of Orthopedics Renmin Hospital of Wuhan University Wuhan430060 Peoplersquos Republic of China 2Southern Medical UniversityGuangzhou 510515 Peoplersquos Republic of China 3The Shenzhen Key Lab ofGene and Antibody Therapy Graduate School at Shenzhen TsinghuaUniversity Shenzhen 518055 Peoplersquos Republic of China 4School of LifeSciences Tsinghua University Beijing 100084 Peoplersquos Republic of China5L401 Tsinghua Campus Xili University Town Shenzhen GuangdongProvince 518055 China

Received 11 October 2012 Accepted 12 November 2012Published 6 December 2012

References1 IARC Monographs on the evaluation of carcinogenic risks to humans

human papillomaviruses vol 90 httpmonographsiarcfr2 WHO (World Health Organization) Human papillomaviruses httpwww

who intvaccine_research diseasesviral_cancersenindex3html3 Schiller JT Castellsagueacute X Villa LL Hildesheim A An update of prophylactic

human papillomavirus L1 virus-like particle vaccine clinical trial resultsVaccine 2008 26(Suppl 10)K53ndash61

4 Winlaw DS Angiogenesis in the pathobiology and treatment of vascularand malignant diseases Ann Thorac Surg 1997 641204ndash1211

5 Folkman J Is angiogenesis an organizing principle in biology andmedicine J Pediatr Surg 2007 421ndash11

6 Folkman J Antiangiogenesis in cancer therapy-endostatin and itsmechanisms of action Exp Cell Res 2006 312594ndash607

7 Folkman J Angiogenesis an organizing principle for drug discovery NatRev Drug Discov 2007 6273ndash286

8 Michael SO Antiangiogenesis and vascular endothelial growth factorvascular endothelial growth factor receptor targeting as part of acombined-modality approach to the treatment of cancer Int J RadiatOncol Biol Phys 2007 69S64ndashS66

9 OReilly MS Boehm T Shing Y Fukai N Vasios G Lane WS Flynn E BirkheadJR Olsen BR Folkman J Endostatin an endogenous inhibitor ofangiogenesis and tumor growth Cell 1997 88277ndash285

10 Boehm T Folkman J Browder T OReilly MS Antiangiogenic therapy ofexperimental cancer does not induce acquired drug resistance Nature1997 390404ndash407

11 Herbst RS Lee AT Tran HT Abbruzzese JL Clinical studies of angiogenesisinhibitors the University of Texas MD Anderson Center Trial of HumanEndostatin Curr Oncol Rep 2001 3131ndash140

12 Zhuang HQ Yuan ZY Process in the mechanisms of endostatincombined with radiotherapy Cancer Letters 2009 2829ndash13

13 Alexander BL Ali RR Alton EW Bainbridge JW Braun S Cheng SH Flotte TRGaspar HB Grez M Griesenbach U Kaplitt MG Ott MG Seger R Simons MThrasher AJ Thrasher AZ Ylauml-Herttuala S Progress and prospects genetherapy clinical trials (part 1) Gene Ther 2007 201439ndash1447

14 Guinn BA Mulherkar R International progress in cancer gene therapyCancer Gene Ther 2008 12765ndash775

15 Scherer L Rossi JJ Weinberg MS Progress and prospects RNA-basedtherapies for treatment of HIV infection Gene Ther 2007 141057ndash1064

16 Androic I Kraumlmer A Yan R Roumldel F Gaumltje R Kaufmann M Strebhardt KYuan J Targeting cyclin B1 inhibits proliferation and sensitizes breastcancer cells to taxol BMC Cancer 2008 8391ndash401

17 Gao Y Liu XL Research progress on siRNA delivery with nonviral carriersInt J Nanomedicine 2011 61017ndash1025

18 Zhou J Wu J Hafdi N PAMAM dendrimers for efficient siRNA deliveryand potent gene silencing Chem Commun (Camb) 2006 222362ndash2364

19 Mao SR Sun W Kissel T Chitosan-based formulations for delivery of DNAand siRNA Adv Drug Deliv Rev 2010 6212ndash27

20 Son S Kim WJ Biodegradable nanoparticles modified by branchedpolyethylenimine for plasmid DNA delivery Biomaterials 201031133ndash143

21 Blum JS Saltzman WM High loading efficiency and tunable release ofplasmid DNA encapsulated in submicron particles fabricated from PLGAconjugated with poly-L-lysine J Control Release 2008 12966ndash72

22 Boussif O Lezoualch F Zanta MA Mergny MD Scherman D Demeneix BBehr JP A versatile vector for gene and oligonucleotide transfer intocells in culture and in vivo polyethylenimine Proc Natl Acad Sci USA 1995927297ndash7301

23 Zhu LP Xing J Wang QX Kou L Li C Hu B Wu ZW Wang JJ Xu GXTherapeutic efficacy of recombinant human endostatin combined withchemotherapeutics in mice-transplanted tumors Eur J Pharmacol 200961723ndash27

24 Huang L Chen H Zheng Y Song X Liu R Liu K Zeng X Mei LNanoformulation of D-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) diblock copolymer for breast cancertherapy Integr Biol 2011 3993ndash1002

25 Kim JH Park JS Yang HN Woo DG Jeon SY Do HJ Lim HY Kim JM ParkKH The use of biodegradable PLGA nanoparticles to mediate SOX9 genedelivery in human mesenchymal stem cells (hMSCs) and inducechondrogenesis Biomaterials 2011 32268ndash278

26 Wang C Ge Q Ting D Nguyen D Shen HR Chen J Eisen HN Heller JLanger R Putnam D Molecularly engineered poly (ortho ester)microspheres for enhanced delivery of DNA vaccines Nat Mater 20043190ndash196

27 Zolnik BS Burgess DJ Effect of acidic pH on PLGA microspheredegradation and release J Control Release 2007 122338ndash344

28 Yin Y Chen D Qiao M Wei X Hu H Lectin-conjugated PLGAnanoparticles loaded with thymopentin ex vivo bioadhesion and in vivobiodistribution J Control Release 2007 12327ndash38

29 Kataoka K Matsumoto T Yokoyama M Okano T Sakurai Y Fukushima SOkamoto K Kwon GS Doxorubicin-loaded poly(ethylene glycol)-poly(beta-benzyl-L-aspartate) copolymer micelles their pharmaceuticalcharacteristics and biological significance J Control Release 200064143ndash153

30 Lee AL Wang Y Cheng HY Pervaiz S Yang YY The co-delivery ofpaclitaxel and Herceptin using cationic micellar nanoparticlesBiomaterials 2009 30919ndash927

31 Fay F Quinn DJ Gilmore BF McCarron PA Scott CJ Gene delivery usingdimethyldidodecylammonium bromide-coated PLGA nanoparticlesBiomaterials 2011 314214ndash4222

32 Wang L Yao B Li Q Mei K Xu JR Li HX Wang YS Wen YJ Wang XD YangHS Li YH Luo F Wu Y Liu YY Yang L Gene therapy with recombinantadenovirus encoding endostatin encapsulated in cationic liposome incoxsackievirus and adenovirus receptor-deficient colon carcinomamurine models Hum Gene Ther 2011 22(9)1061ndash1069

33 Collnot EM Baldes C Wempe MF Kappl R Huumlttermann J Hyatt JA Edgar KJSchaefer UF Lehr CM Mechanism of inhibition of P-glycoproteinmediated efflux by vitamin E TPGS influence on ATPase activity andmembrane fluidity Mol Pharm 2007 4(3)465ndash474

34 Dintaman JM Silverman JA Inhibition of P-glycoprotein by Dalpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) Pharm Res 1999161550ndash1556

doi1011861556-276X-7-666Cite this article as Qiu et al Co-delivery of docetaxel and endostatin bya biodegradable nanoparticle for the synergistic treatment of cervicalcancer Nanoscale Research Letters 2012 7666

Submit your manuscript to a journal and benefi t from

7 Convenient online submission

7 Rigorous peer review

7 Immediate publication on acceptance

7 Open access articles freely available online

7 High visibility within the fi eld

7 Retaining the copyright to your article

Submit your next manuscript at 7 springeropencom

Figure 9 The histopathological analysis of the tumor tissues from different treatment groups (A) PBS (B) ANPs (TPGS-b-(PCL-ran-PGA))(C) CNPs (TPGS-b-(PCL-ran-PGAPEIpEndo) (D) DNPs (E) ENPs and (F) FNPs

Qiu et al Nanoscale Research Letters 2012 7666 Page 9 of 11httpwwwnanoscalereslettcomcontent71666

which provided good affiliation with cells which had nega-tive charge on the surface In addition our data alsoshowed that coumarin-6 and RED protein can co-expressin HeLa cells transfected by coumarin-6-loaded TPGS-b-(PCL-ran-PGA)PEI-pDSRED (Figure 6) suggesting thatPEI-modified TPGS-b-(PCL-ran-PGA) can co-deliver che-motherapeutic drugs and genetic materials into cells

Figure 10 MVD of tumor tissue in different treatment groups Single adenotes p lt 001 (compared with PBS)

Cell viabilityCytotoxicity of all NPs was determined in various con-centrations using MTT assay The cell viability was notaffected by TPGS-b-(PCL-ran-PGA) NPs in all concen-trations relative to the control (treated with PBS) after72 h in medium However increasing dosage of PEI-modified TPGS-b-(PCL-ran-PGA) NPs was associated

sterisk indicates p lt 005 (compared with PBS) while double asterisk

Qiu et al Nanoscale Research Letters 2012 7666 Page 10 of 11httpwwwnanoscalereslettcomcontent71666

with increased cytotoxicity because of the increase ofPEI (Figure 7) As expected for TPGS-b-(PCL-ran-PGA) TPGS-b-(PCL-ran-PGA)PEI NPs and coumarin-6-loaded NPs the cell survival rates were greater than84 whereas cell survival rate of the cells treated withequal dose of PEI was less than 70 (data not shown)suggesting that TPGS-b-(PCL-ran-PGA) could reducethe cytotoxicity of PEI (Figure 7) On the other handdocetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pEndos-tatin NPs appeared to have more cytotoxicity than thatof the other control NPs including docetaxel-loadedNPs this may be because endostatin could induce cellapoptosis [32] Among all NPs docetaxel-load NPsPEI-endostatin had the highest cytotoxicityThe cell viability of HeLa cells transfected with

docetaxel-loaded NPs was decreased after 6 h whichwas dose- and time-dependent These results indicatedthat PEI-modified TPGS-b-(PCL-ran-PGA) NPs cansimultaneously delivery docetaxel and genetic materialsinto cancer cells and kill them

In vivo studiesTo investigate the therapeutic effect of TPGS-b-(PCL-ran-PGA)-based NPs on tumors the anti-tumor efficacyand angiogenesis inhibition of various NPs includingdocetaxel-loaded NPs-PEIpEndostatin were further eval-uated in vivo Subcutaneous tumor models were con-structed in nude mice by being inoculated with 1 to 2 times106 HeLa cells The results showed that CNPs ENPs andFNPs containing docetaxel significantly inhibited tumorgrowth compared with PBS ANPs and DNPs (p lt 001)(Figure 8) The tumor volume of the mice treated withFNPs appeared to keep growing in the initial 5 days aftertreatment and then the tumor sites presentedinflammatory-like response and ulcer After 3 weeks thetumor growth was significantly inhibited and 80 tumorwas eradicated Similar trend was also found in the groupstreated with CNPs and ENPs the inhibitory effect wasbetter than other groups except of FNP treatment groupTreatment of mice with docetaxel-loaded NPs and PEI-modified docetaxel-loaded NPs inhibited 84 and 87in tumor growth inhibition efficiency respectively des-pite no significant difference (p gt 005)Compared with the PBS group the tumor growth inhib-

ition of the groups treated with empty NPs and NPsPEI-pEndostatin was 10 and 38 respectively which againproved that endostatin could inhibit tumor growth Theinhibitory effect of empty NPs may be caused by the TPGSdegradation from the NP matrix (Figure 8) The histo-pathological analysis of the tumor tissues also revealedthat NPs containing docetaxel orand endostatin inducedcell necrosis and apoptosis leading to suppression oftumor progression (Figure 9) MVD in FNP DNP andCNP groups was relatively lower (p lt 001) (Figure 10)

compared with PBS group MVD in DNP group was alsolower in contrast with PBS group (p lt 005) (Figure 10)This is due to the cell necrosis and apoptosis caused bydocetaxel and endostatin These results are consistentwith histopathological analysis of the tumor tissues Nosignificant difference in mouse weight was observedamong each NP treatment group (data not shown) At theend of the experiment dissection results revealed thatthere were no obvious signs of toxicity in the heart liverlungs spleen kidneys and other organs in each groupThese results implied that TPGS-b-(PCL-ran-PGA)-basedNPs could serve as multifunctional carrier of docetaxeland endostatin In addition during the regimen processthe release of TPGS may help the anticancer effect of doc-etaxel and endostatin because TPGS possesses the func-tions to improve drug permeability enhance absorption ofdrugs and reduce P-glycoprotein-mediated multi-drugresistance in cancer cells by inhibition of P-glycoproteinactivity [3334]

ConclusionsIn this research PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs polyplexed with endostatin geneshowed an optimizing potential in delivering genes andchemotherapeutic drugs The co-delivery of endostatinand docetaxel by PEI-modified TPGS-b-(PCL-ran-PGA)NPs significantly inhibited the tumor growth of nudemouse models (even regress 80 established tumor) Inconclusion our study suggested that PEI-modified TPGS-b-(PCL-ran-PGA) NPs could function as an optimizingcarrier for chemotherapeutic drugs and genetic materials

Competing interestsThe authors declare that they have no competing interests

Authorsrsquo contributionsBQ carried out the in vivo studies and drafted the manuscript MJ carried outcell studies XS carried out the preparation of nanoparticles YoZ carried outcharacterization of nanoparticles ZW carried out the in vitro drug releasestudies XZ participated in the in vivo studies SW participated in the cellstudies HC participated in the preparation of the nanoparticles LMparticipated in the design of the study and performed the statistical analysisYiZ conceived the study and participated in its design and coordination Allauthors read and approved the final manuscript

Authorsrsquo informationBQ is an associate chief physician of the Department of Orthopedics RenminHospital of Wuhan University MJ is a PhD student of the Southern MedicalUniversity XS is a Postdoctoral Fellow Tsinghua University YoZ ZW XZ andSW are PhD students of Tsinghua University HC and YiZ are assistantprofessors of the Tsinghua University LM is an associate professor of theTsinghua University

AcknowledgmentsThe authors are grateful for the financial support from Shenzhen Bureau ofScience Technology amp Information (JC200903180532AJCYJ20120614191936420 KQC201105310021A and JC201005270308A) theDoctoral Fund of Ministry of Education of China (20090002120055) theNational Natural Science Foundation of China (grant nos 31270019 and81071494) the Natural Science Foundation of Guangdong Province (No

Qiu et al Nanoscale Research Letters 2012 7666 Page 11 of 11httpwwwnanoscalereslettcomcontent71666

10451805702004178 and S2012010010046) and the Program for NewCentury Excellent Talents in University (NCET-11-0275)

Author details1Department of Orthopedics Renmin Hospital of Wuhan University Wuhan430060 Peoplersquos Republic of China 2Southern Medical UniversityGuangzhou 510515 Peoplersquos Republic of China 3The Shenzhen Key Lab ofGene and Antibody Therapy Graduate School at Shenzhen TsinghuaUniversity Shenzhen 518055 Peoplersquos Republic of China 4School of LifeSciences Tsinghua University Beijing 100084 Peoplersquos Republic of China5L401 Tsinghua Campus Xili University Town Shenzhen GuangdongProvince 518055 China

Received 11 October 2012 Accepted 12 November 2012Published 6 December 2012

References1 IARC Monographs on the evaluation of carcinogenic risks to humans

human papillomaviruses vol 90 httpmonographsiarcfr2 WHO (World Health Organization) Human papillomaviruses httpwww

who intvaccine_research diseasesviral_cancersenindex3html3 Schiller JT Castellsagueacute X Villa LL Hildesheim A An update of prophylactic

human papillomavirus L1 virus-like particle vaccine clinical trial resultsVaccine 2008 26(Suppl 10)K53ndash61

4 Winlaw DS Angiogenesis in the pathobiology and treatment of vascularand malignant diseases Ann Thorac Surg 1997 641204ndash1211

5 Folkman J Is angiogenesis an organizing principle in biology andmedicine J Pediatr Surg 2007 421ndash11

6 Folkman J Antiangiogenesis in cancer therapy-endostatin and itsmechanisms of action Exp Cell Res 2006 312594ndash607

7 Folkman J Angiogenesis an organizing principle for drug discovery NatRev Drug Discov 2007 6273ndash286

8 Michael SO Antiangiogenesis and vascular endothelial growth factorvascular endothelial growth factor receptor targeting as part of acombined-modality approach to the treatment of cancer Int J RadiatOncol Biol Phys 2007 69S64ndashS66

9 OReilly MS Boehm T Shing Y Fukai N Vasios G Lane WS Flynn E BirkheadJR Olsen BR Folkman J Endostatin an endogenous inhibitor ofangiogenesis and tumor growth Cell 1997 88277ndash285

10 Boehm T Folkman J Browder T OReilly MS Antiangiogenic therapy ofexperimental cancer does not induce acquired drug resistance Nature1997 390404ndash407

11 Herbst RS Lee AT Tran HT Abbruzzese JL Clinical studies of angiogenesisinhibitors the University of Texas MD Anderson Center Trial of HumanEndostatin Curr Oncol Rep 2001 3131ndash140

12 Zhuang HQ Yuan ZY Process in the mechanisms of endostatincombined with radiotherapy Cancer Letters 2009 2829ndash13

13 Alexander BL Ali RR Alton EW Bainbridge JW Braun S Cheng SH Flotte TRGaspar HB Grez M Griesenbach U Kaplitt MG Ott MG Seger R Simons MThrasher AJ Thrasher AZ Ylauml-Herttuala S Progress and prospects genetherapy clinical trials (part 1) Gene Ther 2007 201439ndash1447

14 Guinn BA Mulherkar R International progress in cancer gene therapyCancer Gene Ther 2008 12765ndash775

15 Scherer L Rossi JJ Weinberg MS Progress and prospects RNA-basedtherapies for treatment of HIV infection Gene Ther 2007 141057ndash1064

16 Androic I Kraumlmer A Yan R Roumldel F Gaumltje R Kaufmann M Strebhardt KYuan J Targeting cyclin B1 inhibits proliferation and sensitizes breastcancer cells to taxol BMC Cancer 2008 8391ndash401

17 Gao Y Liu XL Research progress on siRNA delivery with nonviral carriersInt J Nanomedicine 2011 61017ndash1025

18 Zhou J Wu J Hafdi N PAMAM dendrimers for efficient siRNA deliveryand potent gene silencing Chem Commun (Camb) 2006 222362ndash2364

19 Mao SR Sun W Kissel T Chitosan-based formulations for delivery of DNAand siRNA Adv Drug Deliv Rev 2010 6212ndash27

20 Son S Kim WJ Biodegradable nanoparticles modified by branchedpolyethylenimine for plasmid DNA delivery Biomaterials 201031133ndash143

21 Blum JS Saltzman WM High loading efficiency and tunable release ofplasmid DNA encapsulated in submicron particles fabricated from PLGAconjugated with poly-L-lysine J Control Release 2008 12966ndash72

22 Boussif O Lezoualch F Zanta MA Mergny MD Scherman D Demeneix BBehr JP A versatile vector for gene and oligonucleotide transfer intocells in culture and in vivo polyethylenimine Proc Natl Acad Sci USA 1995927297ndash7301

23 Zhu LP Xing J Wang QX Kou L Li C Hu B Wu ZW Wang JJ Xu GXTherapeutic efficacy of recombinant human endostatin combined withchemotherapeutics in mice-transplanted tumors Eur J Pharmacol 200961723ndash27

24 Huang L Chen H Zheng Y Song X Liu R Liu K Zeng X Mei LNanoformulation of D-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) diblock copolymer for breast cancertherapy Integr Biol 2011 3993ndash1002

25 Kim JH Park JS Yang HN Woo DG Jeon SY Do HJ Lim HY Kim JM ParkKH The use of biodegradable PLGA nanoparticles to mediate SOX9 genedelivery in human mesenchymal stem cells (hMSCs) and inducechondrogenesis Biomaterials 2011 32268ndash278

26 Wang C Ge Q Ting D Nguyen D Shen HR Chen J Eisen HN Heller JLanger R Putnam D Molecularly engineered poly (ortho ester)microspheres for enhanced delivery of DNA vaccines Nat Mater 20043190ndash196

27 Zolnik BS Burgess DJ Effect of acidic pH on PLGA microspheredegradation and release J Control Release 2007 122338ndash344

28 Yin Y Chen D Qiao M Wei X Hu H Lectin-conjugated PLGAnanoparticles loaded with thymopentin ex vivo bioadhesion and in vivobiodistribution J Control Release 2007 12327ndash38

29 Kataoka K Matsumoto T Yokoyama M Okano T Sakurai Y Fukushima SOkamoto K Kwon GS Doxorubicin-loaded poly(ethylene glycol)-poly(beta-benzyl-L-aspartate) copolymer micelles their pharmaceuticalcharacteristics and biological significance J Control Release 200064143ndash153

30 Lee AL Wang Y Cheng HY Pervaiz S Yang YY The co-delivery ofpaclitaxel and Herceptin using cationic micellar nanoparticlesBiomaterials 2009 30919ndash927

31 Fay F Quinn DJ Gilmore BF McCarron PA Scott CJ Gene delivery usingdimethyldidodecylammonium bromide-coated PLGA nanoparticlesBiomaterials 2011 314214ndash4222

32 Wang L Yao B Li Q Mei K Xu JR Li HX Wang YS Wen YJ Wang XD YangHS Li YH Luo F Wu Y Liu YY Yang L Gene therapy with recombinantadenovirus encoding endostatin encapsulated in cationic liposome incoxsackievirus and adenovirus receptor-deficient colon carcinomamurine models Hum Gene Ther 2011 22(9)1061ndash1069

33 Collnot EM Baldes C Wempe MF Kappl R Huumlttermann J Hyatt JA Edgar KJSchaefer UF Lehr CM Mechanism of inhibition of P-glycoproteinmediated efflux by vitamin E TPGS influence on ATPase activity andmembrane fluidity Mol Pharm 2007 4(3)465ndash474

34 Dintaman JM Silverman JA Inhibition of P-glycoprotein by Dalpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) Pharm Res 1999161550ndash1556

doi1011861556-276X-7-666Cite this article as Qiu et al Co-delivery of docetaxel and endostatin bya biodegradable nanoparticle for the synergistic treatment of cervicalcancer Nanoscale Research Letters 2012 7666

Submit your manuscript to a journal and benefi t from

7 Convenient online submission

7 Rigorous peer review

7 Immediate publication on acceptance

7 Open access articles freely available online

7 High visibility within the fi eld

7 Retaining the copyright to your article

Submit your next manuscript at 7 springeropencom

Qiu et al Nanoscale Research Letters 2012 7666 Page 10 of 11httpwwwnanoscalereslettcomcontent71666

with increased cytotoxicity because of the increase ofPEI (Figure 7) As expected for TPGS-b-(PCL-ran-PGA) TPGS-b-(PCL-ran-PGA)PEI NPs and coumarin-6-loaded NPs the cell survival rates were greater than84 whereas cell survival rate of the cells treated withequal dose of PEI was less than 70 (data not shown)suggesting that TPGS-b-(PCL-ran-PGA) could reducethe cytotoxicity of PEI (Figure 7) On the other handdocetaxel-loaded TPGS-b-(PCL-ran-PGA)PEI-pEndos-tatin NPs appeared to have more cytotoxicity than thatof the other control NPs including docetaxel-loadedNPs this may be because endostatin could induce cellapoptosis [32] Among all NPs docetaxel-load NPsPEI-endostatin had the highest cytotoxicityThe cell viability of HeLa cells transfected with

docetaxel-loaded NPs was decreased after 6 h whichwas dose- and time-dependent These results indicatedthat PEI-modified TPGS-b-(PCL-ran-PGA) NPs cansimultaneously delivery docetaxel and genetic materialsinto cancer cells and kill them

In vivo studiesTo investigate the therapeutic effect of TPGS-b-(PCL-ran-PGA)-based NPs on tumors the anti-tumor efficacyand angiogenesis inhibition of various NPs includingdocetaxel-loaded NPs-PEIpEndostatin were further eval-uated in vivo Subcutaneous tumor models were con-structed in nude mice by being inoculated with 1 to 2 times106 HeLa cells The results showed that CNPs ENPs andFNPs containing docetaxel significantly inhibited tumorgrowth compared with PBS ANPs and DNPs (p lt 001)(Figure 8) The tumor volume of the mice treated withFNPs appeared to keep growing in the initial 5 days aftertreatment and then the tumor sites presentedinflammatory-like response and ulcer After 3 weeks thetumor growth was significantly inhibited and 80 tumorwas eradicated Similar trend was also found in the groupstreated with CNPs and ENPs the inhibitory effect wasbetter than other groups except of FNP treatment groupTreatment of mice with docetaxel-loaded NPs and PEI-modified docetaxel-loaded NPs inhibited 84 and 87in tumor growth inhibition efficiency respectively des-pite no significant difference (p gt 005)Compared with the PBS group the tumor growth inhib-

ition of the groups treated with empty NPs and NPsPEI-pEndostatin was 10 and 38 respectively which againproved that endostatin could inhibit tumor growth Theinhibitory effect of empty NPs may be caused by the TPGSdegradation from the NP matrix (Figure 8) The histo-pathological analysis of the tumor tissues also revealedthat NPs containing docetaxel orand endostatin inducedcell necrosis and apoptosis leading to suppression oftumor progression (Figure 9) MVD in FNP DNP andCNP groups was relatively lower (p lt 001) (Figure 10)

compared with PBS group MVD in DNP group was alsolower in contrast with PBS group (p lt 005) (Figure 10)This is due to the cell necrosis and apoptosis caused bydocetaxel and endostatin These results are consistentwith histopathological analysis of the tumor tissues Nosignificant difference in mouse weight was observedamong each NP treatment group (data not shown) At theend of the experiment dissection results revealed thatthere were no obvious signs of toxicity in the heart liverlungs spleen kidneys and other organs in each groupThese results implied that TPGS-b-(PCL-ran-PGA)-basedNPs could serve as multifunctional carrier of docetaxeland endostatin In addition during the regimen processthe release of TPGS may help the anticancer effect of doc-etaxel and endostatin because TPGS possesses the func-tions to improve drug permeability enhance absorption ofdrugs and reduce P-glycoprotein-mediated multi-drugresistance in cancer cells by inhibition of P-glycoproteinactivity [3334]

ConclusionsIn this research PEI-modified docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs polyplexed with endostatin geneshowed an optimizing potential in delivering genes andchemotherapeutic drugs The co-delivery of endostatinand docetaxel by PEI-modified TPGS-b-(PCL-ran-PGA)NPs significantly inhibited the tumor growth of nudemouse models (even regress 80 established tumor) Inconclusion our study suggested that PEI-modified TPGS-b-(PCL-ran-PGA) NPs could function as an optimizingcarrier for chemotherapeutic drugs and genetic materials

Competing interestsThe authors declare that they have no competing interests

Authorsrsquo contributionsBQ carried out the in vivo studies and drafted the manuscript MJ carried outcell studies XS carried out the preparation of nanoparticles YoZ carried outcharacterization of nanoparticles ZW carried out the in vitro drug releasestudies XZ participated in the in vivo studies SW participated in the cellstudies HC participated in the preparation of the nanoparticles LMparticipated in the design of the study and performed the statistical analysisYiZ conceived the study and participated in its design and coordination Allauthors read and approved the final manuscript

Authorsrsquo informationBQ is an associate chief physician of the Department of Orthopedics RenminHospital of Wuhan University MJ is a PhD student of the Southern MedicalUniversity XS is a Postdoctoral Fellow Tsinghua University YoZ ZW XZ andSW are PhD students of Tsinghua University HC and YiZ are assistantprofessors of the Tsinghua University LM is an associate professor of theTsinghua University

AcknowledgmentsThe authors are grateful for the financial support from Shenzhen Bureau ofScience Technology amp Information (JC200903180532AJCYJ20120614191936420 KQC201105310021A and JC201005270308A) theDoctoral Fund of Ministry of Education of China (20090002120055) theNational Natural Science Foundation of China (grant nos 31270019 and81071494) the Natural Science Foundation of Guangdong Province (No

Qiu et al Nanoscale Research Letters 2012 7666 Page 11 of 11httpwwwnanoscalereslettcomcontent71666

10451805702004178 and S2012010010046) and the Program for NewCentury Excellent Talents in University (NCET-11-0275)

Author details1Department of Orthopedics Renmin Hospital of Wuhan University Wuhan430060 Peoplersquos Republic of China 2Southern Medical UniversityGuangzhou 510515 Peoplersquos Republic of China 3The Shenzhen Key Lab ofGene and Antibody Therapy Graduate School at Shenzhen TsinghuaUniversity Shenzhen 518055 Peoplersquos Republic of China 4School of LifeSciences Tsinghua University Beijing 100084 Peoplersquos Republic of China5L401 Tsinghua Campus Xili University Town Shenzhen GuangdongProvince 518055 China

Received 11 October 2012 Accepted 12 November 2012Published 6 December 2012

References1 IARC Monographs on the evaluation of carcinogenic risks to humans

human papillomaviruses vol 90 httpmonographsiarcfr2 WHO (World Health Organization) Human papillomaviruses httpwww

who intvaccine_research diseasesviral_cancersenindex3html3 Schiller JT Castellsagueacute X Villa LL Hildesheim A An update of prophylactic

human papillomavirus L1 virus-like particle vaccine clinical trial resultsVaccine 2008 26(Suppl 10)K53ndash61

4 Winlaw DS Angiogenesis in the pathobiology and treatment of vascularand malignant diseases Ann Thorac Surg 1997 641204ndash1211

5 Folkman J Is angiogenesis an organizing principle in biology andmedicine J Pediatr Surg 2007 421ndash11

6 Folkman J Antiangiogenesis in cancer therapy-endostatin and itsmechanisms of action Exp Cell Res 2006 312594ndash607

7 Folkman J Angiogenesis an organizing principle for drug discovery NatRev Drug Discov 2007 6273ndash286

8 Michael SO Antiangiogenesis and vascular endothelial growth factorvascular endothelial growth factor receptor targeting as part of acombined-modality approach to the treatment of cancer Int J RadiatOncol Biol Phys 2007 69S64ndashS66

9 OReilly MS Boehm T Shing Y Fukai N Vasios G Lane WS Flynn E BirkheadJR Olsen BR Folkman J Endostatin an endogenous inhibitor ofangiogenesis and tumor growth Cell 1997 88277ndash285

10 Boehm T Folkman J Browder T OReilly MS Antiangiogenic therapy ofexperimental cancer does not induce acquired drug resistance Nature1997 390404ndash407

11 Herbst RS Lee AT Tran HT Abbruzzese JL Clinical studies of angiogenesisinhibitors the University of Texas MD Anderson Center Trial of HumanEndostatin Curr Oncol Rep 2001 3131ndash140

12 Zhuang HQ Yuan ZY Process in the mechanisms of endostatincombined with radiotherapy Cancer Letters 2009 2829ndash13

13 Alexander BL Ali RR Alton EW Bainbridge JW Braun S Cheng SH Flotte TRGaspar HB Grez M Griesenbach U Kaplitt MG Ott MG Seger R Simons MThrasher AJ Thrasher AZ Ylauml-Herttuala S Progress and prospects genetherapy clinical trials (part 1) Gene Ther 2007 201439ndash1447

14 Guinn BA Mulherkar R International progress in cancer gene therapyCancer Gene Ther 2008 12765ndash775

15 Scherer L Rossi JJ Weinberg MS Progress and prospects RNA-basedtherapies for treatment of HIV infection Gene Ther 2007 141057ndash1064

16 Androic I Kraumlmer A Yan R Roumldel F Gaumltje R Kaufmann M Strebhardt KYuan J Targeting cyclin B1 inhibits proliferation and sensitizes breastcancer cells to taxol BMC Cancer 2008 8391ndash401

17 Gao Y Liu XL Research progress on siRNA delivery with nonviral carriersInt J Nanomedicine 2011 61017ndash1025

18 Zhou J Wu J Hafdi N PAMAM dendrimers for efficient siRNA deliveryand potent gene silencing Chem Commun (Camb) 2006 222362ndash2364

19 Mao SR Sun W Kissel T Chitosan-based formulations for delivery of DNAand siRNA Adv Drug Deliv Rev 2010 6212ndash27

20 Son S Kim WJ Biodegradable nanoparticles modified by branchedpolyethylenimine for plasmid DNA delivery Biomaterials 201031133ndash143

21 Blum JS Saltzman WM High loading efficiency and tunable release ofplasmid DNA encapsulated in submicron particles fabricated from PLGAconjugated with poly-L-lysine J Control Release 2008 12966ndash72

22 Boussif O Lezoualch F Zanta MA Mergny MD Scherman D Demeneix BBehr JP A versatile vector for gene and oligonucleotide transfer intocells in culture and in vivo polyethylenimine Proc Natl Acad Sci USA 1995927297ndash7301

23 Zhu LP Xing J Wang QX Kou L Li C Hu B Wu ZW Wang JJ Xu GXTherapeutic efficacy of recombinant human endostatin combined withchemotherapeutics in mice-transplanted tumors Eur J Pharmacol 200961723ndash27

24 Huang L Chen H Zheng Y Song X Liu R Liu K Zeng X Mei LNanoformulation of D-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) diblock copolymer for breast cancertherapy Integr Biol 2011 3993ndash1002

25 Kim JH Park JS Yang HN Woo DG Jeon SY Do HJ Lim HY Kim JM ParkKH The use of biodegradable PLGA nanoparticles to mediate SOX9 genedelivery in human mesenchymal stem cells (hMSCs) and inducechondrogenesis Biomaterials 2011 32268ndash278

26 Wang C Ge Q Ting D Nguyen D Shen HR Chen J Eisen HN Heller JLanger R Putnam D Molecularly engineered poly (ortho ester)microspheres for enhanced delivery of DNA vaccines Nat Mater 20043190ndash196

27 Zolnik BS Burgess DJ Effect of acidic pH on PLGA microspheredegradation and release J Control Release 2007 122338ndash344

28 Yin Y Chen D Qiao M Wei X Hu H Lectin-conjugated PLGAnanoparticles loaded with thymopentin ex vivo bioadhesion and in vivobiodistribution J Control Release 2007 12327ndash38

29 Kataoka K Matsumoto T Yokoyama M Okano T Sakurai Y Fukushima SOkamoto K Kwon GS Doxorubicin-loaded poly(ethylene glycol)-poly(beta-benzyl-L-aspartate) copolymer micelles their pharmaceuticalcharacteristics and biological significance J Control Release 200064143ndash153

30 Lee AL Wang Y Cheng HY Pervaiz S Yang YY The co-delivery ofpaclitaxel and Herceptin using cationic micellar nanoparticlesBiomaterials 2009 30919ndash927

31 Fay F Quinn DJ Gilmore BF McCarron PA Scott CJ Gene delivery usingdimethyldidodecylammonium bromide-coated PLGA nanoparticlesBiomaterials 2011 314214ndash4222

32 Wang L Yao B Li Q Mei K Xu JR Li HX Wang YS Wen YJ Wang XD YangHS Li YH Luo F Wu Y Liu YY Yang L Gene therapy with recombinantadenovirus encoding endostatin encapsulated in cationic liposome incoxsackievirus and adenovirus receptor-deficient colon carcinomamurine models Hum Gene Ther 2011 22(9)1061ndash1069

33 Collnot EM Baldes C Wempe MF Kappl R Huumlttermann J Hyatt JA Edgar KJSchaefer UF Lehr CM Mechanism of inhibition of P-glycoproteinmediated efflux by vitamin E TPGS influence on ATPase activity andmembrane fluidity Mol Pharm 2007 4(3)465ndash474

34 Dintaman JM Silverman JA Inhibition of P-glycoprotein by Dalpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) Pharm Res 1999161550ndash1556

doi1011861556-276X-7-666Cite this article as Qiu et al Co-delivery of docetaxel and endostatin bya biodegradable nanoparticle for the synergistic treatment of cervicalcancer Nanoscale Research Letters 2012 7666

Submit your manuscript to a journal and benefi t from

7 Convenient online submission

7 Rigorous peer review

7 Immediate publication on acceptance

7 Open access articles freely available online

7 High visibility within the fi eld

7 Retaining the copyright to your article

Submit your next manuscript at 7 springeropencom

Qiu et al Nanoscale Research Letters 2012 7666 Page 11 of 11httpwwwnanoscalereslettcomcontent71666

10451805702004178 and S2012010010046) and the Program for NewCentury Excellent Talents in University (NCET-11-0275)

Author details1Department of Orthopedics Renmin Hospital of Wuhan University Wuhan430060 Peoplersquos Republic of China 2Southern Medical UniversityGuangzhou 510515 Peoplersquos Republic of China 3The Shenzhen Key Lab ofGene and Antibody Therapy Graduate School at Shenzhen TsinghuaUniversity Shenzhen 518055 Peoplersquos Republic of China 4School of LifeSciences Tsinghua University Beijing 100084 Peoplersquos Republic of China5L401 Tsinghua Campus Xili University Town Shenzhen GuangdongProvince 518055 China

Received 11 October 2012 Accepted 12 November 2012Published 6 December 2012

References1 IARC Monographs on the evaluation of carcinogenic risks to humans

human papillomaviruses vol 90 httpmonographsiarcfr2 WHO (World Health Organization) Human papillomaviruses httpwww

who intvaccine_research diseasesviral_cancersenindex3html3 Schiller JT Castellsagueacute X Villa LL Hildesheim A An update of prophylactic

human papillomavirus L1 virus-like particle vaccine clinical trial resultsVaccine 2008 26(Suppl 10)K53ndash61

4 Winlaw DS Angiogenesis in the pathobiology and treatment of vascularand malignant diseases Ann Thorac Surg 1997 641204ndash1211

5 Folkman J Is angiogenesis an organizing principle in biology andmedicine J Pediatr Surg 2007 421ndash11

6 Folkman J Antiangiogenesis in cancer therapy-endostatin and itsmechanisms of action Exp Cell Res 2006 312594ndash607

7 Folkman J Angiogenesis an organizing principle for drug discovery NatRev Drug Discov 2007 6273ndash286

8 Michael SO Antiangiogenesis and vascular endothelial growth factorvascular endothelial growth factor receptor targeting as part of acombined-modality approach to the treatment of cancer Int J RadiatOncol Biol Phys 2007 69S64ndashS66

9 OReilly MS Boehm T Shing Y Fukai N Vasios G Lane WS Flynn E BirkheadJR Olsen BR Folkman J Endostatin an endogenous inhibitor ofangiogenesis and tumor growth Cell 1997 88277ndash285

10 Boehm T Folkman J Browder T OReilly MS Antiangiogenic therapy ofexperimental cancer does not induce acquired drug resistance Nature1997 390404ndash407

11 Herbst RS Lee AT Tran HT Abbruzzese JL Clinical studies of angiogenesisinhibitors the University of Texas MD Anderson Center Trial of HumanEndostatin Curr Oncol Rep 2001 3131ndash140

12 Zhuang HQ Yuan ZY Process in the mechanisms of endostatincombined with radiotherapy Cancer Letters 2009 2829ndash13

13 Alexander BL Ali RR Alton EW Bainbridge JW Braun S Cheng SH Flotte TRGaspar HB Grez M Griesenbach U Kaplitt MG Ott MG Seger R Simons MThrasher AJ Thrasher AZ Ylauml-Herttuala S Progress and prospects genetherapy clinical trials (part 1) Gene Ther 2007 201439ndash1447

14 Guinn BA Mulherkar R International progress in cancer gene therapyCancer Gene Ther 2008 12765ndash775

15 Scherer L Rossi JJ Weinberg MS Progress and prospects RNA-basedtherapies for treatment of HIV infection Gene Ther 2007 141057ndash1064

16 Androic I Kraumlmer A Yan R Roumldel F Gaumltje R Kaufmann M Strebhardt KYuan J Targeting cyclin B1 inhibits proliferation and sensitizes breastcancer cells to taxol BMC Cancer 2008 8391ndash401

17 Gao Y Liu XL Research progress on siRNA delivery with nonviral carriersInt J Nanomedicine 2011 61017ndash1025

18 Zhou J Wu J Hafdi N PAMAM dendrimers for efficient siRNA deliveryand potent gene silencing Chem Commun (Camb) 2006 222362ndash2364

19 Mao SR Sun W Kissel T Chitosan-based formulations for delivery of DNAand siRNA Adv Drug Deliv Rev 2010 6212ndash27

20 Son S Kim WJ Biodegradable nanoparticles modified by branchedpolyethylenimine for plasmid DNA delivery Biomaterials 201031133ndash143

21 Blum JS Saltzman WM High loading efficiency and tunable release ofplasmid DNA encapsulated in submicron particles fabricated from PLGAconjugated with poly-L-lysine J Control Release 2008 12966ndash72

22 Boussif O Lezoualch F Zanta MA Mergny MD Scherman D Demeneix BBehr JP A versatile vector for gene and oligonucleotide transfer intocells in culture and in vivo polyethylenimine Proc Natl Acad Sci USA 1995927297ndash7301

23 Zhu LP Xing J Wang QX Kou L Li C Hu B Wu ZW Wang JJ Xu GXTherapeutic efficacy of recombinant human endostatin combined withchemotherapeutics in mice-transplanted tumors Eur J Pharmacol 200961723ndash27

24 Huang L Chen H Zheng Y Song X Liu R Liu K Zeng X Mei LNanoformulation of D-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) diblock copolymer for breast cancertherapy Integr Biol 2011 3993ndash1002

25 Kim JH Park JS Yang HN Woo DG Jeon SY Do HJ Lim HY Kim JM ParkKH The use of biodegradable PLGA nanoparticles to mediate SOX9 genedelivery in human mesenchymal stem cells (hMSCs) and inducechondrogenesis Biomaterials 2011 32268ndash278

26 Wang C Ge Q Ting D Nguyen D Shen HR Chen J Eisen HN Heller JLanger R Putnam D Molecularly engineered poly (ortho ester)microspheres for enhanced delivery of DNA vaccines Nat Mater 20043190ndash196

27 Zolnik BS Burgess DJ Effect of acidic pH on PLGA microspheredegradation and release J Control Release 2007 122338ndash344

28 Yin Y Chen D Qiao M Wei X Hu H Lectin-conjugated PLGAnanoparticles loaded with thymopentin ex vivo bioadhesion and in vivobiodistribution J Control Release 2007 12327ndash38

29 Kataoka K Matsumoto T Yokoyama M Okano T Sakurai Y Fukushima SOkamoto K Kwon GS Doxorubicin-loaded poly(ethylene glycol)-poly(beta-benzyl-L-aspartate) copolymer micelles their pharmaceuticalcharacteristics and biological significance J Control Release 200064143ndash153

30 Lee AL Wang Y Cheng HY Pervaiz S Yang YY The co-delivery ofpaclitaxel and Herceptin using cationic micellar nanoparticlesBiomaterials 2009 30919ndash927

31 Fay F Quinn DJ Gilmore BF McCarron PA Scott CJ Gene delivery usingdimethyldidodecylammonium bromide-coated PLGA nanoparticlesBiomaterials 2011 314214ndash4222

32 Wang L Yao B Li Q Mei K Xu JR Li HX Wang YS Wen YJ Wang XD YangHS Li YH Luo F Wu Y Liu YY Yang L Gene therapy with recombinantadenovirus encoding endostatin encapsulated in cationic liposome incoxsackievirus and adenovirus receptor-deficient colon carcinomamurine models Hum Gene Ther 2011 22(9)1061ndash1069

33 Collnot EM Baldes C Wempe MF Kappl R Huumlttermann J Hyatt JA Edgar KJSchaefer UF Lehr CM Mechanism of inhibition of P-glycoproteinmediated efflux by vitamin E TPGS influence on ATPase activity andmembrane fluidity Mol Pharm 2007 4(3)465ndash474

34 Dintaman JM Silverman JA Inhibition of P-glycoprotein by Dalpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) Pharm Res 1999161550ndash1556

doi1011861556-276X-7-666Cite this article as Qiu et al Co-delivery of docetaxel and endostatin bya biodegradable nanoparticle for the synergistic treatment of cervicalcancer Nanoscale Research Letters 2012 7666

Submit your manuscript to a journal and benefi t from

7 Convenient online submission

7 Rigorous peer review

7 Immediate publication on acceptance

7 Open access articles freely available online

7 High visibility within the fi eld

7 Retaining the copyright to your article

Submit your next manuscript at 7 springeropencom