Journal of Controlled Release 151 (2011) 295–301

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Journal of Controlled Release

j ourna l homepage: www.e lsev ie r.com/ locate / jconre l

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Development of efficient acid cleavable multifunctional prodrugs derived fromdendritic polyglycerol with a poly(ethylene glycol) shell☆

Marcelo Calderón a, Pia Welker b, Kai Licha b, Iduna Fichtner c, Ralph Graeser d,Rainer Haag a,⁎, Felix Kratz d,⁎⁎a Institut für Chemie und Biochemie, Freie Universität Berlin, Takustrasse 3, D-14195 Berlin, Germanyb mivenion GmbH, Robert-Koch-Platz 4, D-10115 Berlin, Germanyc Max-Delbrück Center, Robert-Rössle-Straße 10, D-13122 Berlin, Germanyd Tumor Biology Center and Proqinase GmbH, Breisacher Strasse 117, D-79106 Freiburg, Germany

☆ Dedicated to Prof. Kazunori Kataoka´s 60th birthday⁎ Corresponding author. Tel.: +49 30 838 52633; fa

⁎⁎ Corresponding author. Tel.: +49 761 2062930; faxE-mail addresses: haag@chemie.fu-berlin.de (R. Haa

kratz@tumorbio.uni-freiburg.de (F. Kratz).

0168-3659/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.jconrel.2011.01.017

a b s t r a c t

a r t i c l e i n f o

Article history:Received 17 September 2010Accepted 17 January 2011Available online 21 January 2011

Keywords:PolyglycerolDrug deliveryPolymer therapeuticsDoxorubicinProdrug

In an attempt to explore the potential of dendritic systems for the development of effective anticancer drugdelivery systems, we explored a simple modular approach of preparing polyglycerol doxorubicin prodrugs,with flexibility for drug loading using an acid-sensitive hydrazone linker and further post-modification withpoly(ethylene glycol) shell. The resulting drug polymer conjugates showed optimal properties for in vitro andin vivo applications because of their high water solubility, an appropriate size for passive tumor targeting, ahigh stability at physiological conditions, pronounced acid-sensitive properties, cellular internalization, and afavorable toxicity profile. Doxorubicin polyglycerol conjugates with a high drug loading ratio showed clearlyimproved antitumor efficacy over doxorubicin in an ovarian xenograft tumor model (A2780) inducingtransient complete remissions thus demonstrating the potential of developing efficient multifunctionaldendritic drug delivery using our modular approach.

.x: +49 30 838 53357.: +49 761 2062905.g),

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© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Research in drug targeting is currently one of the major sources ofinnovation in many therapeutic areas such as cancer, rheumaticdiseases, and immunosuppression. The vast majority of clinically useddrugs have a low-molecular weight that makes them diffuse rapidlyinto healthy tissues and distribute evenly throughout the body. As aconsequence, relatively small amounts of the drugs reach the targetsite and therapy is associated with serious side effects. These sideeffects are often dose limiting and thus prevent effective treatment.

Inorder to circumvent these limitations and to improve thepotentialof the respective drug, several carrier technologies are currentlyavailable or under development, e.g. drug conjugates using monoclonalantibodies, synthetic polymers or serum proteins as carriers, and drugsencapsulated in liposomes or other micro- or nanoparticles [1]. Theaccumulation ofmacromolecules in solid tumors forms the rationale fordeveloping polymer-based drug delivery systems and is due to a leakycapillary combined with an absent or defective lymphatic drainagesystem (enhanced permeation and retention effect, EPR effect) [2]. Inaddition, the combination of bioactive molecules with polymers may

reduce their toxicity, eliminate undesirable body interactions, andimprove their solubility, bioavailability, immunogenicity, stability, andhalf-life. Ideally, cleavage of the drug polymer conjugate at the tumorsite is triggered by a biochemical or physiological property unique forthe individual tumor. Although such truly tumor-specific features arerarely encountered, the over-expression of certain enzymes, an acidicand hypoxic environment in solid tumors, as well as the endocytoticpathway of macromolecules offer several options for designing drugpolymer conjugates that are preferentially cleaved within the tumor[3–7].

Due to their low degree of molecular weight dispersity, flexibledesign, and biocompatible nature [8–12], dendritic polyglycerols (PGs)have a broad range of potential applications in medicine andpharmacology [13]. The versatility of the polyglycerol scaffold forapplication in the biomedical field has been recently reviewed [14], andseveral examples described, e.g. smart and stimuli-responsive deliveryand the release of bioactive molecules. Recently, we demonstrated thatchemically post-modified hyperbranched polyglycerol presents suffi-ciently low zeta potentials, lower interactions with serum albumin, andan enhanced cellular uptake by human hematopoietic U-937 cells,which makes it a good candidate for delivering therapeutic agentssystemically [15]. The dendritic systems have the added advantagethat a high local drug concentration could translate into a potentialincrease in efficacy as has beendemonstrated in vitro [16]. However, thedevelopment of tailor-made dendritic drug conjugates with idealantitumor properties in vivo has not been sufficiently addressed.

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Thus, we set out to fine-tune the architecture of polyglycerol drugconjugates for optimal tumor targeting and designed a simplemodularapproach to prepare multifunctional polyglycerol doxorubicin con-jugates with flexibility for drug loading and further post-modificationwith a poly(ethylene glycol) (PEG) shell.

Covalent attachment of drugs to an amino- or thiol-bearingdendritic polymer through a cleavable linker has the advantage thatdrug release can be fine-tuned for a site-specific release of drugs witha narrow therapeutic index such as anticancer drugs. A logicallyconsistent option was to mask remaining and potentially toxic aminogroups with the polymer poly(ethylene glycol) which would have theadditional advantage of increasing the molecular weight of the drugpolyglycerol conjugate to N40 kDa which is a pre-requisite for passivetargeting. Pegylation has been a successful strategy for stabilizing andreducing the immunogenicity of therapeutic relevant proteins andextending the half-life and tumor targeting properties of liposomaldoxorubicin formulations (i.e., Doxil) [17] and polymers [13,18]. Anadditional advantage is that a PEG shell increases the water-solubilityof the formed drug conjugates and can reduce their systemic toxicity.

Herein, we report on the orthogonal synthesis of a macromolecularprodrug derived from hyperbranched amino-bearing polyglycerol (PG–NH2, Fig. 1a) that was partially thiolated in a first step followed by aMichael addition to the maleimide group of the 6-maleimidocaproyl(hydrazone) derivative of doxorubicin (DOXO-EMCH) (Fig. 1b), analbumin binding prodrug that is under clinical development [19]. In asecond step the maleimide group of 2 or 5 kDa PEG chains was reactedwith the remaining thiol groups (Fig. 1c). The synthesis of thehyperbranched amino-bearing polyglycerol (PG–NH2, Fig. 1a) consistedof three steps as reported earlier for the synthesis of polyglycerols withan average molecular weight of 10 kDa and 20% of introduced aminogroups [20,21]. The resulting drug polymer conjugates showed optimalproperties for in vitro and in vivo applications because of their highwater solubility, an appropriate size for passive tumor targeting, a highstability at physiological conditions, pronounced acid-sensitive proper-ties, cellular internalization, and a favorable toxicity profile.

2. Materials and methods

All chemicals were of analytical grade and purchased from Fluka(Germany), Aldrich (Germany), and Merck (Germany), respective-

Fig. 1. Schematic pathway for the synthesis of doxorubicin-polyglycerol conjugates. i) phoiii) phosphate/EDTA buffer, pH 7.0, r.t. 60 min. a) Schematic representation of amino-beadoxorubicin (DOXO-EMCH), a thiol-binding doxorubicin prodrug bearing a maleimide moiet2 or 5 kDa.

ly. Polyglycerol (MW=10 kDa, PD=1.6) was prepared accordingto published procedures [22]. Polyglycerol amine with 20% of thetotal hydroxyl groups bearing amino groups was prepared aspreviously described [20]. Briefly, polyglycerol aminewas preparedby a three-step protocol starting from hyperbranched polyglycerol,conversion of OH groups into mesyl (Ms) groups followed bytransformation of Ms groups into azide (N3) functionalities, and finallyreduction of the N3 groups to primary amino (NH2) groups usingthiphenylphosphine as a reducing agent (Scheme S1, SupportingInformation). Maleimido-poly(ethylene glycol) (2 kDa and 5 kDa) waspurchased from Rapp Polymer, Germany. The (6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH • HCl) was preparedas described previously [23]. Water of Millipore quality (resistivity~18 MΩ cm−1, pH=5.6±0.2) was used in all experiments and forpreparation of all samples. If not otherwise specified, sodium phosphatebuffer (10 mM)was used for the pH range of 7.4–5.8, for acidic pH values50 mM sodium acetate buffer was employed. All measurements werecarried out with freshly prepared solutions at 25 °C. pH-values weremeasured with a Piccolo Plus ATC pH/C-meter at 25 °C.

2.1. Preparation of the doxorubicin polyglycerol conjugatesPG-Doxon-PEGmkDa

For the synthesis of the thiolated derivatives, three differentpathways were previously studied using 3-(tritylthio)propionic acid,2-iminothiolane, or acetyl-thiopropionic acid. In each case the optimalconditions for synthesis and purification were studied as a function ofthe reaction time, solvent, stoichiometry, and purification methodusing UV–Visible and 1H NMR spectroscopy [21]. Among thethiolation methods studied, the 2-iminothiolane pathway was themost reproducible for the in situ Michael reaction with maleimide.The doxorubicin polyglycerol conjugates with polyglycerolamine (PG10 kDa and 20% of amine loading) and the (6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH) were preparedusing iminothiolane as the thiolating agent and maleimido-polyeth-ylene glycol (PEG 2 kDa and 5 kDa) as the decorating agent. Thegeneral reaction scheme is presented in Scheme S2, SupportingInformation. The conjugation reaction was performed at roomtemperature with vigorous stirring for 90 min. To four different flasks,containing 21 ml of a solution of polyglycerolamine (10 mg/ml)

sphate/EDTA buffer, pH 7.0, r.t., 20 min, ii) phosphate buffer pH 5.8, r.t., 10 min, andring hyperbranched polyglycerol. b) (6-maleimidocaproyl-(hydrazone) derivative ofy as the thiol-binding group and a hydrazone bond as a pH-sensitive linker. c) PEG-Mal,

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in sterile 50 mM sodium phosphate (pH 7.0) containing 5 mM EDTAwere added 43 ml of a solution of 2-iminothiolane (2 mg/ml in thesame solvent system). After 20 min, a solution of DOXO-EMCH(10 mg/ml in 10 mM sodium phosphate buffer, pH 5.8) was added(for PG-Doxo5-PEG2k: 8.2 ml; for PG-Doxo10-PEG5k: 16.4 ml; for PG-Doxo5-PEG5k: 8.2 ml; for PG-Doxo1-PEG5k: 1.6 ml). After 10 min,solutions with a concentration of 200 mg/ml of the PEG maleimidederivative dissolved in sterile 50 mM sodium phosphate (pH 7.0)containing 5 mM EDTA were added (for PG-Doxo5-PEG2k: 6.3 ml PEG2 kDa; for PG-Doxo10,5,1-PEG5k: 15 ml PEG 5 kDa), and the resultingsolutions were stirred for 1 h. The solutions were concentrated withCENTRIPREP-10-concentrators from Amicon, FRG (20 min at 4 °C and4000 rpm) to a volume of approximately 5 ml. The PG-doxorubicinconjugates were purified by gel filtration using a Sephadex G-25column (Amersham) with 10 mM sodium phosphate buffer (pH 7.0)yielding 20–50 ml of a red solution. A second concentration withCENTRIPREP-10-concentrators was carried out, and finally theconjugates were lyophilized at −60 °C for 16 h to yield a red powder.Yields obtained (between 54% and 74%), weight composition, andidealized structure of each conjugate are presented in Table S1,Supporting Information.

Conjugate formation was confirmed by chromatography onreverse phase TLC (70% CH3CN/20 mM sodium phosphate pH 7),appearance of a faster band on a Sephadex G-25 column, and by SEC-HPLC. As an example, representative chromatogrammes of PG-Doxo10-PEG5k are depicted in the Figs. S1 and S2 in SupportingInformation showing a broad peak for the polyglycerol doxorubicinconjugate at a retention time of ~6.7 min (Fig. S1). Absence of physicalencapsulation was ruled out by performing the same couplingprocedure with polyglycerolamine alone. By analysis of the cleavageprofile at pH 4 an additional peak was observed at intermediateretention times (at ~13.2 min) between the free drug doxorubicin(retention time~27.5 min) and the PG-drug conjugate (see chroma-togramme A in Fig. S2, Supporting Information). This species wasisolated and identified as a PEG unit that had reacted with adoxorubicin molecule thiolated with iminothiolane at the 3 -aminoposition of doxorubicin as interpreted by MALDI-TOF analysis. Inorder to prevent the formation of this by-product, the excess of 2-iminothiolane was removed after the first step (described in theScheme S1) by Sephadex size exclusion (G-25) prior to drugconjugation. This purification step inhibited the thiolation ofdoxorubicin at the 3 -amino position and its further reaction withPEG-maleimide. As an example, chromatogramme B in Fig. S2,Supporting Information, depicts the chromatogramme of PG-Doxo10-PEG5k at pH 4.0 after 24 h that primarily showed theremaining native peak of the PG-doxorubicin conjugate (~6.7 min)and liberated doxorubicin (~27.5 min).

2.2. Surface charge and size measurements

Zeta potential and size measurements were carried out on aZetasizer Nano ZS analyzer with an integrated 4 mW He–Ne laser,λ=633 nm (Malvern Instruments Ltd, U.K.). Polymer solutions werefreshly prepared by dissolving an appropriate amount of dry polymer(1 mg/ml) in 10 mM sodium phosphate buffer. The sample solutionswere then stirred thoroughly to ensure dissolution of the polymers.The zeta potential was measured by applying an electric field acrossthe conjugate solutions using the technique of laser Doppleranemometry. All measurements were carried out at 25 °C using astandard rectangular quartz cell and at pH=7.4.

2.3. pH-dependent stability

pH-dependent stability studies were carried out with HPLC at pH 4(50 mM sodium acetate buffer) and pH 7.4 (50 mM sodiumphosphate buffer). A 730 μM solution of each polymer conjugate

(concentration stated in doxorubicin equivalents) was incubated atroom temperature in the respective buffer system and analyzed bysize-exclusion HPLC over 22 h using a Kontron-HPLC system withGeminyxSystem software; column: BioSil SEC250 [300×7.8 mm],with a pre-column [80×7.8 mm] from Biorad, Germany; flow: 1.2 ml/min, isocratic; injection: 50 μl; mobile phase: 10% acetonitrile/90%10 mM sodium phosphate buffer, 0.15 NaCl, pH 7.0, detection at220 nm and 495 nm. Half-lives were determined by evaluation of thedecrease of the native peak of the respective doxorubicin polyglycerolconjugate over time. An extended analysis of the pH-dependentrelease profile was carried out at four different pH values for theconjugate PG-Doxo5-PEG5k at pH values of 4.0 and 5.0 (50 mM sodiumacetate buffer), and 6.0 and 7.0 (10 mM sodium phosphate buffer) byincubation at 37 °C over 24 h.

2.4. Cellular uptake and cytotoxicity studies

2.4.1. Cell cultureThe human A2780 ovarian carcinoma cell line was routinely

cultured in RPMI medium, with 10% fetal calf serum (FCS), 2%glutamine, and penicillin/streptomycin (all from PAN Biotech) added.Cells were seeded into medium at 1×105cells/ml, cultured at 37 °Cwith 5% CO2, and split 1:5 twice a week.

2.4.2. Fluorescence microscopy studies — cytochemical stainingIn the present study, cells were seeded at 2×105 cells/ml in a 24-

well culture plate on glass coverslips (Sigma) and cultured for 24 h at37 °C. Thereafter, cells were culturedwithmedium containing 10−5Mdoxorubicin (Mr. 580 gmol−1), doxorubicin polyglycerol conjugatesor 10−5M glycerol-ICC (control) for 4 or 6 h at 37 °C. Afterwards, cellswere fixedwith cold acetone, rinsed and coveredwith Alexa Fluor 488Phalloidin (1:300, Molecular Probes, USA) for staining of the actincytosceleton. Image acquisition was performed using a Leica DMRBmicroscope (Leica, Germany). The results are shown in Figs. S3 and S4,Supporting Information.

2.4.3. Cytotoxicity assaysDrug cytotoxicity was assessed in vitro using theMTT assay (cellular

reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) as a test for metabolic activity of the cells. Briefly, 1×104

cells per well were plated in 96-well plates in 100 μl culture mediumcontaining increasing concentration of the test substance in 4 wells perconcentration (concentrations ranged from 10−6M to 10−9M). Afterthree days of culture, 10 μl MTT (5 mg/ml in PBS, obtained from Sigma,Germany) were added to each well and the plates were incubated for4 h. The resulting formazan product was dissolved with acid isopropa-nol and the absorbance at awavelength of 570 nm(Ex 570)was read ona Microplate Spectrophotometer (Anthos htII, Microsystems). IC50values and standard deviations were determined by plotting doseresponse curves using Microsoft Excel.

2.4.4. Fluorescence Activated Cell Sorting (FACS)2×105 cells/ml cells were cultured in 24-well-plates with normal

culture medium or medium containing different concentrations oftest substance for 30 min or 3 h. Thereafter, cells were washed withPBS and detached with 200 μl/well accutase (PAA) and washed twicewith PBS. Cells were fixed with 500 μl 3% paraformaldehyde for10 min at 4 °C, 2 ml PBS were then added and centrifuged with 250 gfor 10 min at 4 °C. Supernatants were removed and cells weresuspended in 200 μl PBS with 0.5% bovine serum albumin (Roth).Fixed cells were kept at 4 °C until analyzed in a FACS Calibur analysisinstrument (Becton-Dickinson, USA).

First, the uptake of free doxorubicin and doxorubicin-conjugatesby A2780 cells after 30 min and 3 h was quantified (Table S2). Thehighest fluorescence values were detected after incubation with free

Table 1Loading ratios, diameter, z-potential, and half-lives of conjugates at pH 4.0 of the PGdoxorubicin conjugates.

Conjugate DOXO-EMCH[a]

MW PEGchain[b]

Diameter[c] z-potential[d] t ½(h) [e]

PG-Doxo5-PEG2k 5 2 11.6 −1.8 3.3PG-Doxo10-PEG5k 10 5 15.3 −1.0 2.1PG-Doxo5-PEG5k 5 5 16.4 −1.6 1.9PG-Doxo1-PEG5k 1 5 15.6 −2.0 2.7

[a] Average number of DOXO-EMCH conjugated to the polyglycerol scaffold, [b] kDa, [c]Mean diameter in PBS solution pH 7.4, [d] mV with I=0.01, ±0.2 and concentration2 mg ml−1, and [e] half-life of conjugate at pH 4.0.

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doxorubicin and PG-Doxo10-PEG5k, a result that corroborates thefindings of the fluorescence microscopy studies.

Furthermore, the uptake mechanism was studied. When incubat-ing A2780 cells at 4 °C compared to 37 °C both the uptake by freediffusion and endocytosis was decreased compared to doxorubicin asshown for PG-Doxo5-PEG2k as a representative example (Fig. S5,Supporting Information). The difference of mean fluorescence (DMF)of cells incubated with test substance (black line) and cells in medium(grey) correlates with the drug loading ratio of the PG doxorubicinconjugate.

The invagination of clathrin-coated pits during endocytosis wasstrongly inhibited by removing cholesterol from the membrane ofcultured cells. The water-soluble methyl-β-cyclodextrin (MβCD) isknown to form soluble inclusion complexes with cholesterol, therebyenhancing its solubility in aqueous solution. The cellular uptakemechanism for doxorubicin is due to free diffusion with marginalshifting of fluorescence intensities after pretreatment with MβCD(from DMF 100 to 110) compared to cellular uptake of PG-Doxo5-PEG2k in cells pretreated with MβCD (from DMF 5.3 to 1.8) indicatingthe role of endocytosis in this process.

2.5. Xenograft experiments

In vivo efficacy studies were carried out with doxorubicin and thePG drug conjugates in the ovarian carcinoma A2780 xenograft model.For the in vivo testing, female NMRI: nu/nu mice (Taconic, Denmark)were used. The mice were held in individually ventilaged cages (IVC)under sterile and standardized environmental conditions (25±2 °Croom temperature, 50±10% relative humidity, 12 hour light–dark-rhythm). They received autoclaved food and bedding (ssniff, Soest,Germany) and acidified (pH 4.0) drinking water ad libitum. A2780tumor fragments were transplanted subcutaneously (s.c.) into the leftflank region of anaesthetized (40 mg/kg i.p. Radenarkon, Asta Medica,Frankfurt, Germany) mice on day zero. Mice were randomlydistributed to the experimental groups (8 mice per group). Whenthe tumors were grown to a palpable size, treatment was initiated.Mice were treated intravenously with either glucose phosphate buffer(10 mM sodium phosphate, 5% D-(+)-glucose, pH 5.8), doxorubicin(from Pfizer, 2×8 mg/kg), or PG doxorubicin conjugates (PG-Doxo5-PEG2k, PG-Doxo10-PEG5k, PG-Doxo5-PEG5k, PG-Doxo1-PEG5k

(3×24 mg/kg doxorubicin equivalents, dissolved in 10 mM sodiumphosphate, 5% D-(+)-glucose, pH 5.8) at weekly intervals on days 6,13, and 20. The injection volume was 0.2 ml/20 g body weight.

Tumor size was measured twice weekly with a caliper-likeinstrument in two dimensions. Individual tumor volumes (V) werecalculated by the formula V=(length×[width]2)/2 and related to thevalues on the first day of treatment (relative tumor volume, RTV).Statistical analysis was performed with the U-test (Mann andWhitney) with pb0.05. The body weight of mice was determinedevery 3 to 4 days. The body weight change curves (BWC in %) areshown in Fig. S6, Supporting Information.

3. Results and discussion

The conjugation between thiolated polyglycerol and the prodrugas well as the PEG-maleimide was performed sequentially in a one-pot process. The reaction of the polymer with iminothiolane wasfollowed by a selective Michael addition between the maleimidegroup of the prodrug or PEG and the sulfhydryl groups from thiolatedpolyglycerol in PBS solution at pH 7 (see scheme in Fig. 1). The thiolgroup adds to the double bond of the maleimide group in a fast andselective reaction at room temperature forming a stable thioetherbond [21]. In a second step, a solution of PEG-maleimide (MW 2 kDaor 5 kDa) was added. Conjugate formation was confirmed bychromatography on reverse phase TLC, appearance of a faster bandon a Sephadex G 25 column, and by size-exclusion HPLC. Absence of

physical encapsulation was ruled out by performing the samecoupling procedure with polyglycerolamine alone. Thus, the drugwas covalently attached to the hyperbranched polyglycerol core andsurrounded by a “shell” of long, solubilizing PEG chains which radiatefrom the core as shown in Fig. 1.

By varying the amount of the doxorubicin prodrug and of the PEG-maleimide, PG-drug conjugateswith different loading propertieswereobtained (Table 1). The drug concentration of the conjugates wasdetermined photometrically at 495 nm (ε495=10645 M−1 cm−1) forthe doxorubicin conjugates after reconstitution of the lyophilizedconjugates in glucose solution of pH 5.8. Analysis by dynamic lightscattering measurements (DLS) and determination of z-potentialsshowed that average diameters of these macromolecular prodrugswere in the range of 12 to 16 nm and the conjugates showed a slightlynegative surface charge (see Table 1).

The resulting drug conjugates showed optimal properties for invitro and in vivo studies because of their suitable size for passivetargeting to solid tumors and their water solubility; once dissolved,they could be sterile-filtered through a 0.2 μm filter, an important pre-requisite for developing a galenic formulation. In addition, the drugrelease profile for the conjugates was studied by size-exclusion HPLC.The in vitro stability studies showed that the release of doxorubicinwas minimal at pH 7.4 after 24 h (less than 5% — data not shown),while at acidic pH half-lives were below 3.5 h at pH 4.0 at roomtemperature (see Table 1). An additional release was observed at pH4.0 of a 2 or 5 kDa PEG-maleimide doxorubicin derivative that hadreacted with a doxorubicin molecule thiolated with iminothiolane atthe 3 -amino position of doxorubicin. This side reaction could beavoided by removing the excess of 2-iminothiolane after the first stepprior to drug.

An extended analysis of the pH-dependent release profile at 37 °Cwas carried out at four different pH values for the conjugate PG-Doxo5-PEG5k.over 24 h demonstrating a dramatic change in drugrelease between pH 5.0 and 6.0, a new important insight into the newdrug delivery system, suggesting that doxorubicin is primarilyreleased after endocytosis in the acidic endosomes and/or lysosomeswhere the pH range is 4.0–6–0 and not extracellularly where non-invasive techniques with pH-electrodes have demonstrated that thepH-value in tumor tissue is approximately 0.5–1.0 units lower than innormal tissue [24]. Cellular uptake of the PEGylated doxorubicinpolyglycerols is demonstrated by fluorescence imaging studies — seeFig. 3 below and Fig. 3S in the supporting information.

In order to investigate the cellular uptake and intracellulardistribution of doxorubicin and the acid-sensitive doxorubicin con-jugates, human ovarian cancer A2780 cells were incubated with thedrug conjugates for 4 and 6 h, and doxorubicin was subsequentlydetected by fluorescence microscopy. Results are depicted in Fig. 3 forthe incubation of A2780 cells with doxorubicin and PG-Doxo10-PEG5k

as a comparative and representative example. Doxorubicin is localizedin the cell nucleus after incubation for 4 h (see Fig. 2a) which isconfirmed by merging fluorescence microscopy pictures with andwithout nucleus staining with DAPI (see Fig. 3b). In contrast, whencells were incubated with PG-Doxo10-PEG5k for 4 h, fluorescence was

Fig. 2. Representative release profile of PG-Doxo5-PEG5k incubated at pH 4.0, 5.0, 6.0,and 7.0 at 37 °C over 24 h. The doxorubicin release (%) was quantified on a HPLC size-exclusion column (BioSil SEC-250) with UV–Vis detection at 495 nm.

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observed primarily in the cytoplasm with decreased signals in thenucleus (Fig. 3c–d). An increased nuclear accumulation of doxorubicinwas confirmedwhen the conjugateswere incubated for 6 h (Fig. 3e–f),which point towards intracellular drug release and subsequentmigration to the nucleus. The differences in the intracellular kinetics

Fig. 3. Representative fluorescence merge images of human ovarian cancer A2780 cell incubaf). Merge pictures show red (doxorubicin), blue (nuclei stained with DAPI), and pink (cpredominantly found in the cytoplasm, after 6 h in the nucleus. Actin cytoskeleton was sta

between free doxorubicin and the PG doxorubicin conjugates are inaccordance with our earlier work on acid-sensitive doxorubicinconjugates with albumin, transferrin, and PEG which showed aprimarily location of doxorubicin in the mitochondria and Golgiapparatus using confocal fluorescence microscopy and double label-ling experiments with fluorescent markers for these organelles[25,26]. FACS analysis at different temperatures (4 and 37 °C) andin the presence of ß-methyl-cyclodextrin demonstrated that themechanism of uptake of the conjugates was through endocytosis(see Supporting Information).

The antiproliferative activity of doxorubicin and the PG doxoru-bicin conjugates was assessed against the human ovarian cancerA2780 cell line (see Table 2). The IC50 value for doxorubicin at around20 nM was 4–12 times lower than for any of the PG drug conjugates.This is a typical observation when a free drug, which enters cells bydiffusion, is compared to a drug polymer conjugate that is taken up byendocytosis [25,26]. The IC50 values of PG-Doxo5-PEG2k, PG-Doxo10-PEG5k, and PG-Doxo5-PEG5k were in the range of 86–165 nM, whereasPG-Doxo1-PEG5k with only one DOXO-EMCH moiety per PG-scaffoldhad an IC50 value of 266 nM, which is significantly (*p=0.01–0.04)higher than any of the other three. The comparison of the IC50 values,as well as the differences in mean fluorescence found in the FACSanalysis (see Supporting Information) indicating that high local drugconcentrations could translate in a potential increase in efficacy in

ted at 37 °C with doxorubicin for 4 h (a, b), and PG-Doxo10-PEG5k for 4 h (c, d) or 6 h (e,olocalization) color. Conjugate PG-Doxo10-PEG5k signals after 4 h of incubation areined using phalloidin in green color (f). 400× magnification.

Table 2Comparison of IC50 values of doxorubicin and PG doxorubicin conjugates againsthuman ovarian cancer A2780 cells.

Compound IC50 (nM) in A2780 cells

Doxorubicin 21±8PG-Doxo5-PEG2k 93±25PG-Doxo10-PEG5k 86±17PG-Doxo5-PEG5k 165±35PG-Doxo1-PEG5k 266±53

Analyses performed with cells cultured in 24-well-plates. 2×105 cells/ml wereincubated in 1 ml culture medium containing an increasing concentration of testsubstance (n=4). After three days of culture, drug cytotoxicity was assessed in vitrousing the MTT assay as a test for metabolic activity of the cells.

Table 3Comparison of doses, mortality, relative tumor volumes, body weight change ofdoxorubicin and PG doxorubicin conjugates against human ovarian cancer xenografts(A2780).

Substance [a] Dose[b] Toxicdeath

BWC[c] RTV[d] RTV[d]

day 20 day 30

Glucose-phosphate buffer – 0 −2 50.3Doxorubicin 8 0 −21 (d26) 7.4 23.9PG-Doxo5-PEG2k 24 0 −15 (d16) 0.3* 0.1+

PG-Doxo10-PEG5k 24 0 −12 (d13) 0.6* 0.9+

PG-Doxo5-PEG5k 24 0 −6 (d13) 0.6* 0.7+

PG-Doxo1-PEG5k 24 0 −5 (d13) 4.4 37.2

[a] 8 animals were treated i.v. in each group on days 6, 13, and 20, [b] mg/kg (i.v.)doxorubicin equivalents, [c] % body weight change; and [d] relative tumor volume;statistically significant to control* and doxorubicin+ group, respectively, pb0.05 (U-test).

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vitro. In summary, all conjugates were active in the low micromolarrange and were therefore evaluated for antitumor efficacy in vivo.

The antitumor efficacy of the novel PG doxorubicin conjugates anddoxorubicin was compared at equitoxic dose in the ovarian carcinomaA2780 xenograft model. We and others have shown that 2×8 mg/kgdoxorubicin is the MTD in nude mice models and higher doses, e.g.2×12 mg/kg leads to unacceptable toxicity and mortality. Orientatingtoxicity studies showed that the PG doxorubicin conjugates could beadministered at 3×24 mg/kg doxorubicin equivalents without pro-ducing mortality or severe body weight loss. Thus the standard andmaximum tolerated dose of doxorubicin (2×8 mg/kg) was comparedto compounds PG-Doxo5-PEG2k, PG-Doxo10-PEG5k, PG-Doxo5-PEG5k,and PG-Doxo1-PEG5k at 3×24 mg/kg (doxorubicin equivalents).

In contrast to the vitro data, in vivo studies carried out in theovarian carcinoma A2780 xenograft model showed a distinct increasein tolerability and antitumor efficacy for three of the PG doxorubicinconjugates; i.e. PG-Doxo5-PEG2k, PG-Doxo10-PEG5k, and PG-Doxo5-PEG5k which displayed drug loading ratios of 5 to 10 per PG-scaffold incomparison to the parent drug (see Fig. 4).

With respect to antitumor efficacy, doxorubicin only showedmoderate antitumor efficacy. In contrast, treatment with compoundsPG-Doxo5-PEG2k, PG-Doxo10-PEG5k, and PG-Doxo5-PEG5k producedexcellent antitumor effects, statistically significant to the control anddoxorubicin-treated group, with tumor remissions for up to 30 days(see Fig. 4).

Also, compared to the doxorubicin treated animals, less bodyweight change (BWC) was observed, indicating better tolerability(see Table 3). Indeed, examination of the body weight curves overtime of the groups treated with the active PG doxorubicin conjugatesPG-Doxo5-PEG2k, PG-Doxo10-PEG5k, and PG-Doxo5-PEG5k shows thatthe body weights actually increase again after the end of therapy onday 30 indicating a low degree of systemic toxicity (see Fig. S6 of the

0,0

5,0

10,0

15,0

20,0

25,0

30,0

35,0

40,0

45,0

50,0

0 10 20Days after tum

(Med

ian

) R

elat

ive

tum

or

volu

me

Fig. 4. Curves depicting tumor growth inhibition of subcutaneously growing A2780 xenogrPEG5k, and PG-Doxo5-PEG5k were statistically significant to control (day 20) and doxorubic

Supporting information). There is a trend that the conjugate with thehighest loading ratio, PG-Doxo10-PEG5, showed the best antitumorefficacy. PG-Doxo1-PEG5k, the conjugate with the lowest loading ratioof doxorubicin showed only comparable activity to doxorubicin.

Interestingly, a comparison with acid-sensitive PEG doxorubicinconjugates that we developed previously and to which only one ortwo doxorubicin molecules were bound showed comparable in vitroactivity [26] but only moderate antitumor efficacy in vivo which wasnot better than doxorubicin [27] substantiating the importance of ahigh loading ratio with doxorubicin when linked to the syntheticpolymer through an acid-sensitive linker.

In recent years, several attempts were performed using dendri-mers or dendritic polymers as drug carriers with a high loadingcapacity for anticancer drugs [28], but not all of them proved to bebeneficial. Problems associated with the use of perfect (monodis-perse) dendrimers are related to the synthetic difficulties of achievingsufficiently high molecular masses for passive tumor targeting.Furthermore, attaching drugs at the periphery of the dendrimer canlead to unpredictable aggregation [29]. Recent research efforts tocombine the advantages of linear poly(ethylene glycol) and dendriticstructures resulted in the development of interesting hybrid materials[30–32] of different architectures such as dendronized linear poly-mers [33], starlike PEG with terminal dendrons [34], or so-called“bow-tie” hybrids [35,36]. Due to their lack of toxicity and anadvantageous biodistribution profile (urinary excretion and a signif-icant accumulation in tumor tissue), some of these polymers wereconsidered as suitable carriers for anticancer drugs [37]. The bow-tiedendrimers synthesized by Gillies et al. consist of two covalentlyattached polyester dendrons, each of them bearing different terminal

30 40 50or transplantation

5% glucose buffer

Doxorubicin

PG-Doxo5-PEG2k

PG-Doxo10-PEG5k

PG-Doxo5-PEG5k

PG-Doxo1-PEG5k

afts under therapy with doxorubicin and the conjugates; PG-Doxo5-PEG2k, PG-Doxo10-in treated group (day 30); pb0.05 (U-test).

301M. Calderón et al. / Journal of Controlled Release 151 (2011) 295–301

NANOMEDICIN

E

groups. The drug conjugate is based on a pegylated dendritic scaffold(45 kDa) that contains eight PEG chains (5 kDa) and up to sixteenmolecules of the drug (8–10% w/w), the latter attached to thedendritic core via acid-sensitive carboxylic hydrazone bonds andexhibited convincing antitumor efficacy in a C-26 tumor bearing micemodel.

In contrast to these polymer therapeutics, a potential advantage ofour dendritic drug delivery system is that the polymer-bound drug isshielded by a coat of PEG chains to avoid uptake by the reticuloen-dothelial system. The design of our multifunctional approach allowsus to fine-tune the drug PG conjugates with respect to drug loadingand molecular weight and to prepare macromolecular prodrugs withsimilar surface charge and diameter, but different drug loading.Furthermore, the multifunctional drug delivery system can easily beadapted to incorporate targeting ligand such as folated-tethered PEGs.

4. Conclusions

The simplicity of the synthetic strategy described as well as thelow cost of available starting materials is an attractive approach fordeveloping and optimizing efficient anticancer drug delivery systems.In addition, due to certain similarities to pegylated liposomaldoxorubicin it will be of interest to assess the differences regardingbiodistribution in respect to uptake in the reticuloendoethelial systemand with respect to dematotoxicity of the new pegylated dendriticcarrier during further preclinical development.

In our opinion, the significantly better tolerability and antitumorefficacy of high loaded dendritic drug conjugates than the clinicalstandard doxorubicin as well as the fact that complete remissionswere observed are an important milestones for continuing thepreclinical development of our drug carrier system which couldpotentially induce partial or in the best case complete remissions in aclinical setting.

Acknowledgement

We thank the Ministry of Science for their continuing support ofthis work for a NanoFuture award for R. Haag and F. Kratz (BMBF03X5501).

Appendix A. Supplementary data

Supplementary data to this article can be found online atdoi:10.1016/j.jconrel.2011.01.017.

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