enhanced gene transfection with addition of a cell-penetrating peptide in substrate-mediated gene...

THE JOURNAL OF GENE MEDICINE R E S E A R C H A R T I C L EJ Gene Med 2010; 12: 705–713.Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jgm.1488

Enhanced gene transfection with addition of acell-penetrating peptide in substrate-mediatedgene delivery

Si ChenRen-Xi ZhuoSi-Xue Cheng*

Key Laboratory of BiomedicalPolymers of Ministry of Education,Department of Chemistry, WuhanUniversity, Wuhan, People’s Republicof China

*Correspondence to: Si-Xue Cheng,Key Laboratory of BiomedicalPolymers of Ministry of Education,Department of Chemistry, WuhanUniversity, Wuhan 430072, People’sRepublic of China. E-mail:[email protected];[email protected]

Received: 12 March 2010Revised: 27 June 2010Accepted: 28 June 2010

Abstract

Background To overcome extracellular barriers in gene therapy, controlledrelease technology has been adapted to protect DNA from degradation andmaintain elevated DNA concentration. Although cell-penetrating peptides(CPPs) have been widely utilized to enhance the transfection efficiency ofsolution-based transfections, as far as we are aware, the effect of thesepeptides on substrate-mediated transfection has not yet been reported.

Methods In the present study, the effect of a CPP, KALA, on thesubstrate-mediated gene delivery was studied. A fast degrading cholic acidfunctionalized star poly(DL-lactide) was used to fabricate Ca-P/DNA/KALAco-precipitates-deposited films and Ca-P/DNA/KALA co-precipitates-encapsulated films for mediating substrate-mediated transfection. The in vitrogene transfections in HEK293T and HepG2 cells mediated by the differentfilms were investigated. The effect of KALA content on gene transfection wasstudied.

Results Gene expression of film-mediated transfections could be signifi-cantly enhanced by the addition of KALA at a quite low content with aDNA/KALA ratio of 10 : 1. As a result of the surface erosion mechanismof the functionalized star poly(DL-lactide), our substrate-mediated transfec-tion system could rapidly release Ca-P/DNA/KALA to mediate efficient geneexpression. During the transfection, the degradation of the polymer filmscould be observed and the degradation did not show any unfavorable effectson gene expression.

Conclusions The presence of a CPP could significantly enhance the geneexpression of substrate-mediated transfections. Copyright 2010 John Wiley& Sons, Ltd.

Keywords calcium phosphate; functional polymers; gene delivery; peptide;substrate-mediated delivery

Introduction

Substrate-mediated gene delivery systems have attracted great interest ingene therapy because of their advantageous aspects in overcoming extracel-lular barriers in gene delivery [1]. Using these systems, DNA immobilizedin polymeric scaffolds can be localized to a target tissue to avoid potentialside-effects, prevent the rapid clearance of the DNA from the application site,

Copyright 2010 John Wiley & Sons, Ltd.

706 S. Chen et al.

and maintain the DNA concentration in a desirable rangefor a prolonged time [2,3]. This technique is also knownas ‘solid-phase transfection’.

Cellular internalization of therapeutic agents is a crit-ical issue in gene delivery because the plasma mem-branes are almost impermeable barriers [4–6]. Tocross these barriers, cell-penetrating peptides (CPPs)have been developed as delivery agents for varioustherapeutic agents [4–7]. Among these CPPs, KALA,a cationic endosomolytic and fusogenic peptide, hasbeen extensively investigated as a gene vector to con-dense pDNA [8–10] and small-interfering RNA [11,12],and an additive to be incorporated into vector/DNAcomplexes to achieve enhanced transfection efficiency[13–16]. Cationic KALA and KALA derivatives suchas PEG-KALA could be incorporated into polymer/DNAcomplexes, including polyethylenimine/DNA [13,14],PEG-g-polylysine [15] and poly(DMAEMA-NVP)-b-PEG-galactose [16], to increase the transfection efficiency toa great extent. Although CPPs have been widely utilizedto enhance the transfection efficiency of solution-basedtransfections and are well-established for their impor-tance in gene delivery, as far as we are aware, the effectof these peptides on substrate-mediated transfection hasnot yet been reported.

The present study aimed to investigate the effectof a CPP on substrate-mediated gene delivery. Usingcholic acid functionalized star poly(DL-lactide) with a fastdegradation rate and surface erosion mechanism as thematrix of the substrate-mediated delivery, we fabricatedCa-P/DNA/KALA co-precipitates-deposited films and Ca-P/DNA/KALA co-precipitates-encapsulated films. Becausethe calcium phosphate transfection method is one of themost common nonviral methods used for the introductionof foreign DNA into the cells, in the present study, calciumphosphate (Ca-P) was used to condense the complexes ofplasmid DNA and peptide KALA into Ca-P/DNA/KALA co-precipitates for gene transfection. The transfection activityof different films was evaluated by the expression ofpGL3-Luc in different cells. The results obtained indicatedthat these Ca-P/DNA/KALA co-precipitates-loaded filmscould effectively mediate sustained transfections. Thegene expression of film-mediated transfections could besignificantly enhanced by the addition of KALA at alow content with a DNA/KALA ratio of 10 : 1, whichwas much lower than the values for the solution-basedtransfections mediated by other polycation/DNA/KALAcomplexes. Furthermore, during the cellular transfection,the degradation of the star poly(DL-lactide) film couldbe detected and the degradation did not show anyunfavorable effects on gene expression.

In the present study, the films were studied as amodel to investigate the effect of a CPP on the substrate-mediated gene transfection. Practical applications of thesubstrate-mediated gene transfection devices include genedelivery in tissue engineering and gene therapy (e.g.controlled release of DNA from the polymer coating layerson expandable stents for coronary disease treatment,and sustained release of therapeutic DNA through local

injection of microspheres directly to the tumor site forcancer treatment).

Materials and methods

Materials

Cholic acid (Acros, Geel, Belgium) was purified byrecrystallization from methanol. DL-Lactide (Aldrich, StLouis, MO, USA) was purified by recrystallization fromethyl acetate. Stannous octoate (Sigma, St Louis, MO,USA) was distilled under reduced pressure and thendissolved in dry toluene prior to use. Other reagents wereof analytical grade and used without further purification.

KALA peptide (WEAKLAKALAKALAKHLAKALAKALKA-CEA) with Mw = 3131 g/mol [10] was obtained fromGL Biochem (Shanghai, China). Plasmid DNA encod-ing for luciferase (pGL3-Luc) with Mw = 3.3 × 106 g/molwas purchased from Promega (Madison, WI, USA). Plas-mids were amplified in Escherichia coli and extracted andpurified by NucleoBond Xtra Maxi Plus EF kit (Macherey-Nagel, Duren, Germany). Plasmids were suspended inwater and stored at −20 ◦C.

HEK293T and HepG2 cells were obtained from ChinaCenter for Typical Culture Collection (Wuhan, China).The medium for cell culture was Dulbecco’s ModifiedEagle’s Medium (DMEM) (Gibco, Gaithersburg, MD,USA) supplemented with 10% fetal bovine serum (FBS),2 mg/ml NaHCO3 and 100 U/ml penicillin/streptomycin.Cells were incubated at 37 ◦C in humidified air/5% CO2.

Polymer synthesis and characterization

Cholic acid functionalized star poly(DL-lactide), CA-PDLLA, was synthesized by the ring-opening polymeriza-tion of DL-lactide using cholic acid with hydroxyl groups asan initiator, as described previously [17]. Briefly, cholicacid, DL-lactide with a cholic acid/DL-lactide feed ratio(mol/mol) of 1 : 15, and catalyst solution [Sn(Oct)2 intoluene] were placed in a thoroughly dried silanized glassflask with a magnetic stirring bar. The flask was evac-uated, purged with nitrogen three times, sealed undervacuum, immersed in an oil bath at 200 ◦C for 5 min toallow cholic acid to melt quickly, and then immersed inanother oil bath at 150 ◦C for 12 h. Then the productwas dissolved in tetrahydrofuran (THF), precipitated intoethanol/water (3 : 1, v/v), filtrated, and dried under vac-uum to obtain CA-PDLLA (Mw = 5.4 × 103 g/mol withMw/Mn = 1.37).

The molecular weight of the polymer was determinedby combined size-exclusion chromatography and multi-angle laser light scattering (SEC-MALLS) analysis. A dualdetector system, consisting of a MALLS device (DAWNEOS; Wyatt Technology, Santa Barbara, CA, USA) andan interferometric refractometer (Optilab DSP; WyattTechnology) was used. The polymer concentration was10 mg/ml and THF was used as the eluent at a flow rate

Copyright 2010 John Wiley & Sons, Ltd. J Gene Med 2010; 12: 705–713.DOI: 10.1002/jgm

Enhanced transfection with addition of a CPP 707

of 0.55 ml/min. The MALLS detector was operated at alaser wavelength of 690.0 nm.

Preparation of Ca-P/DNA/KALAco-precipitates

Plasmid DNA solution containing 10 µg of DNA wasmixed with KALA solution containing a particular amountof KALA (0, 1, 10 and 100 µg, respectively) to formDNA/KALA complexes and then diluted with de-ionizedwater to a volume of 440 µl. Ten microliters of 2 M

CaCl2 solution was added and mixed gently, and thenanother 50 µl of 2 M CaCl2 solution was added and mixedgently. After that, the mixture was added to 500 µl of4.8 mM Na3PO4 solution dropwise under gentle stirring.The mixture was incubated at room temperature for20 min to obtain the Ca-P/DNA/KALA co-precipitatescontained solution. The sizes and zeta potentials ofDNA/KALA complexes, Ca-P/DNA and Ca-P/DNA/KALAco-precipitates in aqueous media were measured by aZetasizer Nano ZS (Malvern Instruments, Malvern, UK).The data are given as the mean ± SD based on threeindependent measurements.

Preparation of Ca-P/DNA/KALAco-precipitates-deposited films andCa-P/DNA/KALAco-precipitates-encapsulated films

To prepare Ca-P/DNA/KALA co-precipitates-depositedfilm, CA-PDLLA was dissolved in chloroform. Then,40 µl of CA-PDLLA solution (10 mg/ml) was depositedon a glass slide with a diameter of 14 mm. Aftersolvent evaporation, the CA-PDLLA film attached on theglass slide was dried under vacuum. The sample wassterilized by ultraviolet light irradiation for 1 h. Anda particular amount (Table 1) of the freshly-preparedCa-P/DNA/KALA co-precipitates contained solution wasoverlaid on the surface of the CA-PDLLA film and driedunder vacuum at room temperature.

Table 1. Structures of the films for substrate-mediatedtransfection

Samplecode

Filmstructure

DNAcontent

(µg)

DNA/KALAratio(w/w)

Film 1a Co-precipitates-depositedfilm

1 1 : 0

Film 1b 1 10 : 1Film 1c 1 1 : 1Film 1d 1 1 : 10Film 1e 0.1 10 : 1Film 2a Co-precipitates-encapsulated

film1 1 : 0

Film 2b 1 10 : 1Film 2c 1 1 : 1Film 2d 1 1 : 10Film 2e 0.1 10 : 1

To prepare Ca-P/DNA/KALA co-precipitates-encapsu-lated film, 40 µl of CA-PDLLA solution (10 mg/ml) wasoverlaid on the on the surface of Ca-P/DNA/KALA co-precipitates-deposited film. The sample was dried in alaminar flow hood. After solvent evaporation, the Ca-P/DNA/KALA co-precipitates were physically entrappedin the polymer film.

These film samples were placed in the wells of a 24-wellcell culture plate and kept in a desiccator until required.

In vitro transfections

For film-mediated transfection, the Ca-P/DNA/KALA co-precipitates-deposited or encapsulated film on the glassslide was placed in the well of a 24-well plate. Then, thecells in 1 ml of complete medium (DMEM containing 10%FBS) with a density of 5 × 104 cells/ml were seeded onthe film in the well of a 24-well plate (5 × 104 cells perwell). Gene expression was assayed after the cells wereincubated at 37 ◦C for predetermined times.

For solution-based transfections, the cells in 1 ml ofcomplete medium (DMEM containing 10% FBS) witha density of 5 × 104 cells/ml were seeded directlyin the well of a 24-well plate (5 × 104 cells perwell). Then, the freshly-prepared Ca-P/DNA/KALA co-precipitates contained solution was immediately addeddropwise. The gene expression was assayed after the cellswere incubated at 37 ◦C for predetermined times.

To assay the expression of luciferase, the medium wasremoved and the cells were rinsed gently by phosphate-buffered saline (PBS, 0.1 M, pH 7.4). After thoroughlysis of the cells with reporter lysis buffer (Promega)(200 µl/well), the luciferase activity was determinedby detecting the light emission from 20 µl cell lysateincubated with 100 µl of luciferin substrate (Promega)in a luminometer (Lumat LB9507; Berthold TechnologiesGmbH, Bad Wildbad, Germany). The protein content ofthe cell lysate was determined by BCA protein assay kit(Pierce, Rockford, IL, USA). The optical density (OD)value was determined at 570 nm using a microplatereader (Bio-Rad 550; Bio-Rad, Hercules, CA, USA). Thedata are given as the mean ± SD based on threeindependent measurements.

Cell viability measurement

After the transfection, the medium was removed, andfresh medium (1 ml) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (60 µl, 5 mg/ml) wereadded to each well, followed by incubation at 37 ◦Cfor 4 h. Then the supernatant was carefully removed,and 1 ml of dimethysulfoxide was added to each wellto dissolve the formazan crystals produced by viablecells. The absorbance of the solution was measured usingmicroplate reader (Bio-Rad 550) at 570 nm to determinethe OD value. The data are given as the mean ± SD basedon three independent measurements.


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Cellular uptake study

One microgram of pGL3-Luc was mixed with 2.5 µl ofYOYO-1 water stock solution (10 µM) and incubatedfor 15 min at 37 ◦C. Then the YOYO-1 labeled pGL3-Luc was used to prepare Ca-P/DNA/KALA co-precipitates(DNA/KALA 10 : 1, w/w). 293T cells in 1 ml of completemedium (DMEM containing 10% FBS) were seededdirectly in the well of a 24-well plate (5 × 104 cellsper well). After incubation at 37 ◦C for 24 h, the Ca-P/DNA/KALA co-precipitates contained solution (100 µlcontaining 1 µg of YOYO-1 labeled pGL3-Luc) was added.During the co-incubation, the free YOYO-1 dye moleculescould not stain living cells because YOYO-1 was a cellmembrane-impermeant dye and healthy cell membranesprevented YOYO-1 molecules from entering cells. Afterincubation for 4 h at 37 ◦C, the medium was removedand the cells were washed twice with PBS to removethe co-precipitates and free YOYO-1 staying outside thecells. Then, 1 ml of complete medium was added. Thecells were observed under excitation at 488 nm usingconfocal laser scanning microscopy (Nikon C1-si TE2000;Nikon, Tokyo, Japan). The magnification of objectivelens used was ×20 with a numerical aperture of 0.75,and the magnification of ocular lens used was ×10. Thecontrol experiment was also carried out under the samecondition by the use of Ca-P/DNA co-precipitates. Allconfocal images were slice images to distinguish YOYO-1labeled pGL3-Luc internalized from that adhered to theoutside of the cellular membranes.

Film degradation study

The morphologies of the films before mediating trans-fection and after mediating transfection for 48 h wereobserved by scanning electron microscopy (SEM) (HitachiX650; Hitachi, Tokyo, Japan). After mediating trans-fection, the medium was removed. Trypsin (Invitrogen,Carlsbad, CA, USA) was added to detach the cells. Thefilms were washed by water three times, left to dry natu-rally, and then dried under vacuum at room temperaturefor 24 h before SEM observation.

Measurement of DNA release

The film containing 1 µg of pGL3-Luc (DNA/KALA10 : 1) was immersed in 1 ml of TE buffer (pH 7.5)(Molecular Probes, Carlsbad, CA, USA) at 37 ◦C. Afterpredetermined intervals, the sample was vortexed gentlyand 100 µl of supernatant was removed and replaceby 100 µl of fresh TE buffer. The amount of DNAin the supernatant was determined by the Quant-iT

PicoGreen dsDNA Assay Kit (Molecular Probes) inaccordance with the manufacturer’s instructions using aspectrofluorophotometer (RF-5301 PC; Shimadzu Corp.,Kyoto, Japan). Data are given as the mean ± SD based onthree independent measurements.

Result and Discussion

Polymers used in film-mediatedtransfection

In the present study, a fast degrading star poly(DL-lactide), CA-PDLLA, was used as the matrix to supportor encapsulate the Ca-P/DNA/KALA co-precipitates tomediate gene expression. CA-PDLLA was synthesized bythe ring-opening polymerization of DL-lactide using cholicacid as an initiator. According to our previous studies,CA-PDLLA had an accelerated degradation rate becauseof its branched structure, and the degradation of CA-PDLLA was dominated by surface erosion mechanismsince its molecular weight (Mw = 5.4 × 103 g/mol) wasnot very high [17]. Compared with the conventional linearpolylactide with bulk degradation mechanism, duringwhich the degradation did not result in obvious weightloss in the initial degradation stage and thus the DNA wasdelivered with low bioavailability and the autocatalyticeffect in the later stage caused unfavorable effects on theactivity of DNA, our star poly(DL-lactide) could uniformlyrelease vector/DNA complexes to mediate efficient genetransfection, and the degradation products did not causeadditional cytotoxicity during gene transfection [18–20].

Effect of KALA content onsubstrate-mediated transfection

In the present study, we prepared two series of filmsfor substrate-mediated transfections: Ca-P/DNA/KALAco-precipitates-deposited films (films 1a-e) and Ca-P/DNA/KALA co-precipitates-encapsulated films (films2a-e) with different co-precipitate compositions. For Ca-P/DNA/KALA co-precipitates-deposited films, during genetransfection, the cells seeded on the film surface couldcontact Ca-P/DNA/KALA co-precipitates because the co-precipitates were directly deposited on the film surfaces.For Ca-P/DNA/KALA co-precipitates-encapsulated films,the co-precipitates were encapsulated inside the polymerfilms through physical entrapment. During gene transfec-tion, initially, the cells seeded on the film surface couldnot directly contact the Ca-P/DNA/KALA co-precipitatesbecause the co-precipitates were embedded inside thepolymer matrix. Furthermore, the cells could accessthe Ca-P/DNA/KALA co-precipitates only after polymerdegradation.

The sizes and zeta potentials of DNA/KALA complexes,Ca-P/DNA and Ca-P/DNA/KALA co-precipitates in theaqueous media before film preparation were measured bya particle analyzer and the results are shown in Table 2.For the DNA/KALA complexes with different DNA/KALAratios, the largest size and lowest zeta potential wereobserved for DNA/KALA complexes with a DNA/KALAratio of 10 : 1 because the amount of positively chargedKALA was not sufficient to bind negatively charged DNAand thus the complexes still existed in a relatively loosestate with a negative surface charge. With an increase



Table 2. Sizes and zeta potentials of DNA/KALA complexes, andCa-P/DNA and Ca-P/DNA/KALA co-precipitates

SampleDNA/KALA

wt/wtSize(nm)

Zetapotential (mV)

DNA/KALA complex 10 : 1 5438 ± 2229 −15.6 ± 1.2DNA/KALA complex 1 : 1 395 ± 33 −11.3 ± 2.3DNA/KALA complex 1 : 10 440 ± 59 4.1 ± 0.7Ca-P/DNA co-precipitate 1 : 0 113 ± 29 −4.29 ± 0.6Ca-P/DNA/KALAco-precipitate

10 : 1 260 ± 70 −5.12 ± 0.4

Ca-P/DNA/KALAco-precipitate

1 : 1 91 ± 32 −6.63 ± 0.7

Ca-P/DNA/KALAco-precipitate

1 : 10 51 ± 18 −1.53 ± 0.2

in KALA content, for DNA/KALA complexes with aDNA/KALA ratio of 1 : 1, the complex size decreasedsharply and the zeta potential increased because moreKALA could bind and condense DNA. With a furtherincrease in KALA content, for DNA/KALA complexes witha DNA/KALA ratio of 1 : 10, the complex size slightlyincreased and the zeta potential increased further andbecame positive because of the excess amount of KALA.These particle size and zeta potential data indicate thatKALA could efficiently bind and condense DNA andform DNA/KALA complexes. As shown in Table 2, thesizes of Ca-P/DNA and Ca-P/DNA/KALA co-precipitateswere in the range 50–300 nm under the experimentalconditions. The Ca-P co-precipitation technique suffersfrom a difficulty in controlling the size of the Ca-P/DNA co-precipitates, which is greatly influenced by thepreparation conditions. Thus, in the present study, thesize of Ca-P/DNA/KALA co-precipitates did not exhibita clear trend. The zeta potentials of Ca-P/DNA and Ca-P/DNA/KALA co-precipitates at DNA/KALA ratios of 10 : 1and 1 : 1 exhibited similar negative values because theexistence of Ca-P in the co-precipitates played a dominantrole. When the DNA and KALA form complexes, thesurface charge of the complexes approaches zero at aDNA/KALA weight ratio of 1 : 2.5 [15]. In the presentstudy, the DNA/KALA complexes at DNA/KALA ratios of10 : 1 and 1 : 1 still had negative zeta potentials and thusthe Ca-P/DNA/KALA co-precipitates also had negativezeta potentials. When the DNA/KALA ratio reached 1 : 10,the DNA/KALA complexes had a positive surface charge.As a result, the zeta potential of Ca-P/DNA/KALA co-precipitates with DNA/KALA ratios of 1 : 10 obviouslyincreased compared to the Ca-P/DNA co-precipitates. Yetthe zeta potential value was still negative as a result ofthe presence of Ca-P.

To study the effect of KALA content on substrate-mediated transfection, we compared gene transfectionsmediated by the films loaded with Ca-P/DNA/KALAco-precipitates with different DNA/KALA ratios. Theluciferase expressions in 293T cells and HepG2 cells underdifferent conditions are shown Figure 1. For HEK293Tcells (Figure 1A), the presence of KALA resulted inan obvious enhancement in gene expression when theratios of DNA/KALA were 10 : 1 and 1 : 1. For the

samples with a DNA/KALA ratio of 10 : 1, the transfactionefficiencies of both solution-based and film-mediatedtransfections were enhanced greatly. With an increase inKALA content (DNA/KALA ratio of 1 : 1), gene expressionlevels were further increased. However, when the KALAcontent further increased (DNA/KALA ratio of 1 : 10), animprovement in gene expression could not be observedfor both solution-based and film-mediated transfections.

HEK293T cells are relatively easy to transfect. Thus, inthe present study, we also investigated the film-mediatedtransfections for HepG2 cells (Figure 1B). Compared withHEK293T cells, the luciferase expression levels for HepG2cells were relatively low. The difference in expressionlevels in different cells was a result of the specificationsof the different cell lines. For HepG2 cells, a similartrend in the improvement of transfaction activity with thepresence of KALA could be observed. By adding KALA at aDNA/KALA ratio of 10 : 1, maximum transfection activitiescould be achieved for both solution-based transfection andfilm-mediated transfections. Gene expression levels forthe solution-based transfection, co-precipitates-deposited

Figure 1. Effect of DNA/KALA weight ratio on luciferaseexpression in HEK293T cells (A) and HepG2 cells (B) aftertransfection for 48 h. The transfections were mediated bydifferent films compared to solution-based transfections. Acomparison between the samples without KALA and the sampleswith a DNA/KALA ratio of 10 : 1 demonstrates a statisticallysignificant difference (p < 0.05) for both HEK293T cells andHepG2 cells)


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film and co-precipitates-encapsulated film increasedsignificantly compared to the samples with an absenceof KALA. Similar to HEK293T cells, the improvement ingene expression decreased with a further increase in KALAcontent.

The highest improvement occurred at differentDNA/KALA ratios for 293T cells and HepG2 cells. Thepossible reason is that HepG2 cells are much more dif-ficult to be transfected compared to 293T cells. As aresult, the addition of KALA could result in a signifi-cant improvement. A comparison of the samples withoutKALA and the samples with a DNA/KALA ratio of 10 : 1demonstrated a statistically significant difference (Stu-dent’s t-test, p < 0.05) using both HEK293T cells andHepG2 cells.

According to previous studies, KALA could interact withand destabilize lipid membranes and facilitate cellularentry as a result of its ability to form an amphipathicα-helical structure and thus induce a marked increasein transfection efficiency when used as a componentto form composite gene delivery systems with otherpolycation vectors [13–16]. With a suitable amount ofKALA presented in the transfection system, enhancedgene expression could be achieved. The addition of excessKALA could not increase the transfection activity further.A possible explantion for this is that excess KALA couldnot bind with DNA efficiently so that the improvementin gene expression through facilitating the cellular entryand endosomal escape could not be further enhanced.

On the basis of previous studies, with the additionof KALA to the vector/DNA complex systems, the trans-fection efficiency increased with increasing KALA/DNAratio up to 10 : 1 when using polycations (PEI [14] andPLL [15]) as gene vectors, although with a subsequentdecrease. In the present study, the high transfection effi-ciency could be obtained at much lower KALA content(KALA/DNA at a ratio of 1 : 1 for HEK293T cells andKALA/DNA at a ratio of 1 : 10 for HepG2 cells). Thisresult implied that Ca-P used as the gene vector in thepresent study was different from the polycation vectorsexplored in other studies.

To confirm the cell-penetrating function of KALA, theconfocal images of HEK293T cells after being transfectedwith Ca-P/DNA co-precipitates and Ca-P/DNA/KALA co-precipitates in the solution-based transfections werecompared. As shown in Figure 2, after co-incubationwith Ca-P/DNA co-precipitates for 4 h, only a few greenfluorescence dots could be observed (Figure 2A). Bycontrast, the intensity of fluorescence was much higher forcells treated with Ca-P/DNA/KALA co-precipitates with aDNA/KALA ratio of 10/1, indicating that the existenceof KALA played an important role in cell uptake. For thefilm-mediated transfections, clear confocal images couldnot be obtained because the films were not transparent,which caused difficulties with respect to the observationby confocal microscopy.

The cell viabilities of HEK293T cells (Figure 3A)and HepG2 cells (Figure 3B) after being transfectedunder different conditions for 48 h were assessed

Figure 2. Confocal images of HEK293T cells after being tran-fected with Ca-P/DNA co-precipitates (A) and Ca-P/DNA/KALAco-precipitates with a DNA/KALA ratio of 10 : 1 (w/w) (B). Thesolution-based transfections were carried out for 4 h

Figure 3. Viabilities of HEK293T cells (A) and HepG2 cells(B) after transfection for 48 h under different conditions

using the cells cultured on tissue culture polystyrenewithout transfection as a control. As shown in Figure 3,the transfections mediated by Ca-P/DNA/KALA co-precipitates in both solution-based and film-mediatedtransfections did not show obvious inhibition effects oncell viability, indicating that low cytotoxicity of the Ca-P/DNA/KALA co-precipitates and the polymer used in thepresent study. This result was in accordance with theprevious studies on low-cytotoxicity KALA compared toother polycation gene vectors [21].



Film degradation study

The morphologies of the films before and after degra-dation in cell culture medium were observed by SEM(Figure 4). Before degradation, both co-precipitates-deposited film and co-precipitates-encapsulated filmexhibited smooth surfaces because the Ca-P/DNA/KALAco-precipitates were very small and could not be observedat the magnification used for the co-precipitates-depositedfilm (Figure 4A1), and Ca-P/DNA/KALA co-precipitateswere encapsulated inside the polymer matrix for the co-precipitates-encapsulated film (Figure 4B1). Comparedwith the films with smooth surfaces before degradation,after mediating transfection for 48 h, many pores andprotuberances appeared on the film surfaces, indicatingthat polymer degradation occurred during transfection.

The molecular weight of CA-PDALA after degradationin the cell culture medium was determined by SEC-MALLS. The Mw value after degradation for 48 h was5.3 × 103 g/mol with Mw/Mn = 1.25, which was veryclose to the Mw value (Mw = 5.4 × 103 g/mol, Mw/Mn =1.37) before degradation. Because the degradation of thepolymer films was clearly observed by SEM, the surfaceeroding degradation mechanism of CA-PDLLA could beconfirmed.

In vitro DNA release

The release of DNA from the films was measured.As shown in Figure 5, the DNA released from theco-precipitates-deposited film was faster than thatfrom the co-precipitates-encapsulated film because DNArelease was controlled by desorption between the

Figure 4. Surface morphologies of co-precipitates-depositedfilm (A1, before mediating transfection; A2, after mediatingtransfection for 48 h) and co-precipitates-encapsulated film (B1,before mediating transfection; B2, after mediating transfectionfor 48 h) as observed by SEM

co-precipitates and the polymer matrix for the Ca-P/DNA/KALA co-precipitates-deposited film and DNArelease was controlled by polymer degradation forthe Ca-P/DNA/KALA co-precipitates-encapsulated film.As mentioned earlier, many pores appeared on thesurface of co-precipitates-encapsulated film after themediating transfection for 48 h. This implied that theCa-P/DNA/KALA co-precipitates could be released easily.

Film-mediated gene expression atdifferent times

One of most important purposes of substrate-mediatedtransfection technique is to maintain the DNA concen-tration in a desirable range for a prolonged period,thus achieving improved gene expression. In the presentstudy, we measured gene expression mediated by Ca-P/DNA/KALA co-precipitates-deposited films and Ca-P/DNA/KALA co-precipitates-encapsulated films at dif-ferent times. Because a significant improvement of genetransfection could be achieved at a DNA/KALA ratio of10 : 1, we fixed the DNA/KALA ratio at 10 : 1 for thepresent study. For both HEK293T cells (Figure 6) andHepG2 cells (Figure 7), gene expression levels mediatedby 1 µg of DNA were higher than that mediated by 0.1 µgof DNA at each particular time. When the DNA contentwas 1 µg, gene expression levels at different time pointswere almost the same. However, when DNA content was0.1 µg, the expression mediated by the Ca-P/DNA/KALAco-precipitates-encapsulated film increased with a pro-longed transfection time. This phenomenon implied thatthe DNA content of 1 µg was quite high for the transfectionand, even at 24 h, the amount of released DNA or the DNAthat could be accessed by the cells was already sufficientfor mediating transfection. Accordingly, gene expressionat different time points did not differ to a great extent.However, on decreasing the DNA content to 0.1 µg, theDNA released from the co-precipitates-encapsulated filmor the DNA that could be accessed by the cells was not suf-ficient to mediate effective transfection and thus the gene

Figure 5. Accumulative DNA release from co-precipitates-deposited film (film 1B) and co-precipitates-encapsulated film(film 2B)


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Figure 6. Luciferase expression in HEK293T cells after beingtransfected for different times with 1 µg of DNA (A) and 0.1 µgof DNA (B)

expression level increased with increasing transfectiontime.

It should be noted that, compared with the solution-based transfection, the co-precipitates-deposited film-mediated transfection exhibited higher gene expressionlevels for all time points. According to previousstudies, vector/DNA complexes on the film surfacecould directly enter the cells seeded on the film viaendocytosis to mediate gene transfection, and transfectionefficiency could be improved through an increasing DNAconcentration at the target cell surface [18,22]. In otherwords, it is not necessary for the Ca-P/DNA/KALA co-precipitates to be released to the cell culture mediumfirst and then to be uptaken by the cells. In thispresent study, the Ca-P/DNA/KALA co-precipitates mightalso be endocytosed by the cells seeded on the filmsurfaces to mediate transfection directly. Furthermore,the increased gene expression level of the film-mediatedtransfection was a result of the increased density ofthe Ca-P/DNA/KALA co-precipitates on the film surfacescompared to the solution-based transfection.

Conclusions

In the present study, Ca-P/DNA/KALA co-precipitates-deposited films and Ca-P/DNA/KALA co-precipitates-encapsulated films were fabricated to study the effect

Figure 7. Luciferase expression in HepG2 cells after beingtransfected for different times with 1 µg of DNA (A) and 0.1 µgof DNA (B)

of a cell-penetrating peptide, KALA, on substrate-mediated gene delivery. Gene expression of film-mediatedtransfections could be significantly enhanced by theaddition of KALA at a quite low content with a DNA/KALAratio of 10 : 1. As a result of the surface erosion mechanismof the functionalized star poly(DL-lactide), our substrate-mediated transfection system could rapidly release Ca-P/DNA/KALA to mediate efficient gene expression. Duringthe transfection, the degradation of the polymer filmscould be observed and the degradation did not show anyunfavorable effects on gene expression.

Acknowledgements

Financial support provided by the National Natural ScienceFoundation of China (20774070) and the Ministry of Scienceand Technology of China (National Basic Research Program ofChina 2009CB930300) is gratefully acknowledged.

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