Journal of Controlled Release 142 (2010) 238–244

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Journal of Controlled Release

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GENEDELIVERY

Physical adsorption of PEG grafted and blocked poly-L-lysine copolymers onadenovirus surface for enhanced gene transduction

Ji Won Park, Hyejung Mok, Tae Gwan Park ⁎Department of Biological Sciences and Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea

⁎ Corresponding author. Tel.: +82 42 350 2621; fax:E-mail address: tgpark@kaist.ac.kr (T.G. Park).

0168-3659/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.jconrel.2009.11.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 June 2009Accepted 1 November 2009Available online 12 November 2009

Keywords:AdenovirusPLL-g-PEGPLL-b-PEGSurface modificationTransduction efficiencyCytotoxicity

Polyethylene glycol (PEG) has been chemically immobilized onto the surface of adenoviruses (ADVs) toreduce non-specific immune response and extend blood circulation time while maintaining the hightransduction efficiency of a foreign gene into cells. In this study, ADVs encoding an exogenous greenfluorescent protein (GFP) were physically coated with PEG grafted and blocked poly-L-lysine (PLL-g-PEG andPLL-b-PEG) copolymers via ionic interactions to comparatively evaluate their gene transduction efficiency.The surface immobilization of ADVs with the two types of PLL–PEG copolymers exhibited significantlyincreased GFP transduction activity, compared to that of naked ADVs. ADVs coated with PLL-b-PEG showedhigher extent of GFP expression than those with PLL-g-PEG under serum conditions. For PLL-g-PEGcopolymers, the substitution degree of PEG in the PLL backbone greatly influenced the gene expression level.Additionally, ADVs modified with PLL-b-PEG exhibited greater transduction efficiency for bone marrowderived human mesenchymal stem cells compared to naked or PLL coated ADVs in the serum condition. Thisstudy suggests that enhanced ADV gene transduction efficiency can be attained for various cells by simplycoating PLL-b-PEG on the surface.

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© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Viral gene delivery systems exhibit far enhanced gene transduc-tion efficiency for a broad spectrum of cell types, as compared to non-viral systems [1]. Among them, adenoviruses (ADVs) have beenwidely used as a powerful gene carrier for clinical gene therapy trials.However, ADVs have several inherent problems such as innateimmune responses induced by neutralizing antibodies and non-specific toxicities caused by accumulation of viral particles in the liver[2,3]. Moreover, non-specific cellular internalization of ADVs viacoxsackie adenovirus receptor (CAR)-mediated endocytosis maycause serious side effects. To overcome these problems, chemically[4] and genetically modified ADVs [5,6] were reported. The surfacemodification of ADVs with cell-specific targeting moieties such asproteins, antibodies, and vitamins could enable target-specific cellularuptake and gene transduction [7–9]. Recently, ADVs have also beensurface PEGylated to substantially reduce unwanted immuneresponses and to ultimately achieve systemic gene therapy by in-creasing the blood circulation time both in vitro and in vivo [10–12].The covalent immobilization of PEG chains onto the viral surface notonly maintained physical stability of ADVs under harsh formulationconditions such as encapsulation within biodegradable microspheres,but also prolonged their half-life in the serum-containing media [13].

However, chemically PEGylated ADVs normally exhibited varyingdegrees of gene transduction efficiency and immune response level,depending on the mode of PEGylation that was largely affected byvarious factors such as types of terminal reactive groups in PEGderivatives, PEG molecular weight, and PEG architecture [14].Moreover, the chemical immobilization of PEG on the surface ofADVs deactivates viral surface proteins, resulting in the irreversibleloss of innate viral functions. It was previously shown that the genetransduction efficiency and the reducing extent of immune responsewere oppositely influenced by the degree of PEGylation on the surfaceof ADVs, suggesting that optimally PEGylatd ADVs are required toachieve high gene transduction efficiency with low immune response[15].

Surface characteristics of ADVs could also be readily engineered bycoating cationic polymers and lipids via electrostatic interactions.Previous studies demonstrated that ADVs coated with cationicpolymers could increase viral gene transduction efficiency via aCAR-independent cellular uptake pathway [16–18]. It was reportedthat ADVs coated with PLL enhanced the level of gene expression forboth CAR over-expressing cells, COS-7 and HeLa cells, and CAR-deficient cells, NIH-3T3 and 9L gilosarcoma cells [18]. In addition,ADVs complexed with PLL exhibited high expression of transgene tofibroblast cells in vitro and in vivo [19]. However, the extent of ADVgene expression greatly varied with the types of cationic polymers,their molecular weight, and polymer concentrations. ADVs coatedwith PLL showed enhanced gene expression only under an optimalPLL concentration. When an excessive amount of PLL was used for

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GENEDELIVERY

coating, ADVs were severely aggregated due to the PLL-inducedcrosslinking between ADV particles, suppressing gene transductionefficiency to a great extent [18,19]. Furthermore, cationic polymers athigh concentrations elicit undesirable cytotoxicity. Thus, it is highlydesirable to additionally introduce PEG chains on the cationicallymodified ADVs to maintain sufficient serum stability with concom-itantly attaining maximal transduction efficiency.

In this study, two types of cationic copolymers, PEG grafted PLL(PLL-g-PEG) and PEG blocked PLL (PLL-b-PEG) were synthesized andused for surface coating of ADVs by physical adsorption (Fig. 1A).Three kinds of PLL-g-PEG copolymers with different PEG graftpercentages were synthesized to comparatively evaluate their genetransduction efficiencies, as compared to that of PLL-b-PEG copoly-mer. It was hypothesized that the cationic PLL parts in the graft orblock copolymers were electrostatically anchored on the anionic ADVsurface, while grafted or blocked PEG chains were oriented outside inaqueous medium. The physical and non-covalent PEG immobilizationmethod is postulated to be more straightforward and less destructive

Fig. 1. (A) Schematic representation of ADVs coated with PLL-b-PEG and PLL-g-PEGcopolymers via electrostatic interactions. Chemical structures of (B) PLL-b-PEG and(C) PLL-g-PEG.

to diverse biological functions of capsid proteins on the surface ofADVs than the chemical and covalent PEG immobilization method[9,15,20]. The surface charge and particle size of ADVs coated with thetwo copolymers were characterized. After the copolymers werephysically immobilized on the surface of green fluorescent protein(GFP) encoding ADVs, the level of viral gene expression wasquantitatively analyzed using various CAR positive/negative cells,such as human epidermal carcinoma cells (KB), human breastadenocarcinoma cells (MCF), mouse fibroblast cells (NIH3T3), andprimary human mesenchymal stem cells (hMSCs). The effect of PEGsubstitution degree in the graft copolymer on the viral geneexpression was also investigated. The extent of intracellular uptakeand cytotoxicity of ADVs modified with PLL-g-PEG and PLL-b-PEGcopolymers was examined as compared to those of ADVs modifiedwith PLL alone.

2. Materials and methods

2.1. Materials

E1-deleted ADV encoding GFP gene was obtained from QBIOgene(AD5.CMV5-GFP) (Montreal, Canada). Poly(ε-carbobenzyoxy-L-ly-sine) (ε-CBZ-PLL, MW 1000), poly-L-lysine hydrobromide (MW30,000), rhodamine B isothiocyanate (MW 536.08), CsCl2, sucrose,and Triton-X 100 were purchased from Sigma (St. Louis, MO). mPEG-succinimidyl-succinate (mPEG-SS; MW 2000) was obtained fromSunbio (Anyang, Korea). Human embryonic kidney (HEK 293) andMCF-7 cells were purchased from the Korea Cell Line Bank (Seoul,Korea), and KB (CCL-7) and NIH3T3 cells were purchased from theATCC (Manassas, VA). Human mesenchymal stem cells (hMSCs) wereobtained from Lonza Walkersville (Maryland, USA). Fetal bovineserum (FBS), Dulbecco's Modified Eagle's Medium (DMEM), RPMI1640 medium, α-Modified Eagle's Medium (α-MEM), Dulbecco'sPhosphate Buffered Saline (PBS), penicillin, streptomycin, and trypsinwere the products of Gibco-BRL(Grand Island, NY). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc(MD, USA). Dialysis membrane (MW cut-off: 10,000) was obtainedfrom Spectrum (Houston, Texas, USA).

2.2. Cells

HEK293, MCF-7, and NIH3T3 cells were cultured in DMEMsupplemented with 10% FBS. KB cells were maintained in RPMI1640 medium containing 10% FBS. hMSCs (Human mesenchymalstem cells) were cultured in α-MEM supplemented with 10% FBS. Themedia contained 100 U/ml of penicillin G sodium, and 100 µg/ml ofstreptomycin sulfate. The cells were maintained at 37 °C in ahumidified atmosphere of 5% of CO2.

2.3. Preparation of PLL–PEG block and graft copolymers

To synthesize PLL-b-PEG, an active terminal amine group of ε-CBZ-PLL (MW 1000, 100 mg) was coupled with mPEG-SS (MW 2000,66 mg) in DMSO. After 5 h of reaction, the conjugate was dialyzedagainst deionized water and freeze-dried. For deprotecting ε-CBZgroups, the conjugate (50 mg) was added into 2 ml of acetic acidsolution containing hydrogen bromide (30 wt.%) with gentle stirring.Following the reaction, PLL-b-PEG copolymer was purified by dialysisand lyophilized. Synthesis of the final product was confirmed by 1HNMR in d6-DMSO (Brucker DRX 400 spectrometer operating at400 MHz).

PLL-g-PEG copolymers were prepared and characterized accordingto the previous method [21]. PLL hydrobromide (MW 24,000, 50 mg)dissolved in 1 ml of PBS (pH 7.4) solution was added to 1 ml of PBSsolution containing 20 mg, 68 mg, and 340 mg of mPEG-SS (MW2000, molar ratios of lysine to mPEG=1:0.03, 1:0.1, and 1:0.5) and

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then reacted for 12 h at room temperature. After reaction, unreactedmPEG was removed by dialysis against deionized water (MW cut-off:3500) and subsequently lyophilized. The product was analyzed by 1HNMRwith D2O as a solvent (Brucker DRX 400 spectrometer operatingat 400 MHz) to determine the graft percentage of PEG chain per PLL[21,22].

2.4. Preparation of ADV particles and synthesis of ADV-rhodamine

E1-deleted ADVs expressing an exogenous GFP with a cytomeg-alovirus (CMV) promoter was produced by the previous procedure[8]. ADV particles (1×1010 pts) were infected to HEK293 cells whichwere cultured in DMEM containing 10% FBS, 100 U/ml of penicillin Gsodium, and 100 µg/ml of streptomycin sulfate using a 150 mm tissueculture dish at 37 °C under 5% CO2 atmosphere. When over 60% ofcells was detached from the culture dish after 4–5 days of infection,the cells were harvested for lysis. To collect the supernatant con-taining ADV pts, the cell lysate were centrifuged at 1500×g for10 min. After CsCl2 gradient ultracentrifugation, the purified ADVswere dialyzed in 3% sucrose/PBS solution at 4 °C. The number of ADVpts was determined by a spectrophotometric method, as previouslyreported [23]. To synthesize rhodamine-labeled ADVs, amine groupson the surface of ADV (5×1011 pts) were reacted with rhodamine Bisothiocyanate (9 µg) in 150 µl of PBS solution at 4 °C overnight. Theresulting rhodamine-labeled ADVs were dialyzed against PBS solutioncontaining 3% sucrose (MW cut-off: 3500) for 2 days and stored at−20 °C until use.

2.5. Characterization of ADVs coated with PLL–PEG copolymers

The size and surface charge of ADVs coated with cationiccopolymers of PLL-b-PEG (PLL MW: 1000) and PLL-g-PEG (PLL MW:30,000) were measured using a dynamic light scattering (DLS)instrument (Zetaplus, Brookhaven, NY) equipped with a He–Nelaser at a wavelength of 677.0 nm. The DLS instrument operatedwith a multimodal particle size distribution pattern was calibrated byusing a Nanosphere™ size standard (Duke scientific Corporation, CA,USA) with a mean diameter of 96±3.1 nm and a size distribution of8.3 nm. PLL–PEG copolymers (5 µg/ml) were added into 10 µl of PBSsolution containing 5×109 ADV pts and then incubated for 30 min atroom temperature for the DLSmeasurement. ADVs coatedwith PLL-b-PEG and PLL-g-PEG copolymers were visualized by atomic forcemicroscopy (AFM). An aliquot containing ADVs coated with PLL–PEGcopolymers was deposited onto a clean mica surface and dried in avacuum oven at room temperature. Scanned images from a 5×5 µmarea were obtained by using a PSIA XE-100 AFM system (Santa Clara,CA, USA) in a non-contact mode.

2.6. Intracellular uptake of ADVs coated with PLL–PEG copolymers

To visualize the cellular uptake of ADVs modified with cationiccopolymers, rhodamine-labeled ADVs (5×109 pts) were incubatedwith 1 µg/ml of PLL (MW 1500), PLL-b-PEG, or PLL-g-PEG (degree ofPEG substitution per PLL: 3.5%, 8.4%, and 32.3%) for 30 min at roomtemperature. The resulting complexes were infected to KB cellscultured in a four-chamber tissue culture slide at a density of 2×105

cells/well. After infecting for 3 h, the culture slides were washed usingPBS solution and fixed with 1% formaldehyde in PBS solution for30 min. The fixed cells were observed using confocal laser microscopy(LSM510, Ziess Pascal, Jene, Germany). Rhodamine-labeled ADVsalone and ADVs (5×109 pts) coated with 5 µg/ml of PLL (MW 1500)and PLL-b-PEG were also treated into NIH3T3 cells seeded in a four-chamber tissue culture slide at 2×105 cells/well by the method asdescribed above. To quantify the extent of cellular uptake in KB cells,rhodamine-labeled ADVs (7.5×109 pts) were coated with 2.5 µg/mlof PLL (MW 1500), PLL-b-PEG, or PLL-g-PEG (degree of PEG

substitution per PLL: 3.5%, 8.4%, and 32.3%). After incubating for30 min at room temperature, the complexes were infected into KBcells seeded at the density of 5×105 cells/well in a 6-well plate andincubated for 3 h. The cells were washed by PBS solution and lysedwith 1.0% Triton X-100 in PBS solution. The intensity of rhodaminewas detected by a spectrofluorotometer with excitation and emissionwavelength at 545 and 580 nm, respectively.

2.7. Transduction of ADVs coated with PLL–PEG copolymers

KB cells were maintained in RPMI 1640 medium containing 10%fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin.Cells were seeded in a 12-well plate at a density of 2×105 cells/well inRPMI medium and incubated for 24 h at 37 °C. ADVs (5×109 pts)were coatedwith various amounts of PLL (MW1500 andMW24,000),PLL-b-PEG copolymer, or three kinds of PLL-g-PEG copolymers(degree of PEG substitution per PLL: 3.5%, 8.4%, and 32.3%). The MOI(multiplicity of infection) value to infect 2×105 cells was estimated tobe about 500, assuming that VP (virus particle)/PFU (plaque formingunit) ratio was 50:1 as reported in the manufacturer's manual. Afterincubating for 6 h, cells were replaced with fresh medium andincubated for 2 days. The cells were treated with 1% Triton X-100 inPBS solution for lysis. The extent of GFP expressionwas determined bya spectrofluorotometer with excitation and emission wavelength at488 and 509 nm, respectively.

MCF-7, NIH3T3, and hMSC cells were prepared to evaluate theextent of relative GFP expression of ADVs coated with PLL (MW 1500)or PLL-b-PEG. MCF-7 and NIH3T3 cells were cultured in DMEMmedium containing 10% FBS, 100 U/ml penicillin, and 100 µg/mlstreptomycin at 37 °C in 5% CO2 humidified atmosphere. Toinvestigate transduction efficiency, MCF-7 and NIH3T3 cells wereseeded on a 12-well plate at a density of 2×105 cells/well andincubated at 37 °C for 24 h. Various amounts (0, 0.05, 0.5, 5, and 50 µg/ml of PLL concentration) of PLL (MW 1500) or PLL-b-PEG were addedto the solution containing ADVs, and incubated at room temperaturefor 30 min. ADVs coated with copolymers were treated to MCF-7 cellsin the absence and presence of serum and incubated for 48 h, but weretreated to NIH3T3 cells in the absence of serum for 3 days since theyneeded a sufficient incubating time compared to other cell lines. Afterincubation, the intensity of GFP expression was measured by themethod as described above. hMSCs were plated in α-MEM with 10%FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. hMSCs(5×104) were seeded in a 6-well culture slide with α-MEM con-taining 10% FBS. After 24 h, ADVs coatedwith different amounts of PLL(MW 1500) or PLL-b-PEG were treated. The GFP expression levelof the cells treated with ADVs coated with 1 µg/ml of PLL or PLL-b-PEG per ADV particle was visualized by confocal laser microscopy(LSM510, Ziess Pascal, Jene, Germany).

2.8. Cytotoxicity assay

For cytotoxicity assay, KB cells were prepared onto a 96-well plateat a density of 1×104 cells/well and maintained for 24 h. ADVs(2.5×108 pts per well) were complexed with 0–10 µg/ml of variouscationic polymers (PLL (MW 1500), PLL(MW 24,000), or PLL-b-PEG,and PLL-g-PEG) for 30 min and then treated to the cells. After 24 hincubation, the cytotoxicity of ADV complexes was measured by theCCK-8 cell viability assay according to the manufacturer's instruction.

3. Results and discussion

Chemical structures of PLL-b-PEG and PLL-g-PEG copolymers areshown in Fig. 1B and C. In order to synthesize PLL-b-PEG, N-terminalα-amino group of poly(ε-carbobenzyoxy-L-lysine) (ε-CBZ-PLL, MW1000) was coupled with an active NHS ester group of mPEG-succinimidyl-succinate (mPEG-SS, MW 2000). The protecting ε-CBZ

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groups in the PLL part were removed by acidic hydrolysis. From 1HNMR spectra, almost 100% of deprotection degree was confirmed(data not shown). PLL-g-PEG copolymers were also synthesized byconjugating NHS-activated mPEG (MW 2000) to ε-amine groups inthe PLL, as previously described [21]. In this study, three kinds of PLL-g-PEG copolymers were synthesized with different PEG substitutiondegrees. The grafting percentages of PEG on the PLL backbone were3.5%, 8.4%, and 32.3%, as calculated from 1H NMR spectra analysis(data not shown), based on the relative integration ratio of a single –

CH2− proton peak in PEG (s, 3.7–3.8 ppm) and multiple –CH– protonpeaks in PLL (m, 4.3–4.4 ppm). The PLL-g-PEG copolymers with PEGgrafting ratios of 3.5%, 8.4%, and 32.3% have ca. 7, 16, and 61 PEGchains per PLL (MW 24,000), respectively. Both of the PLL-b-PEG andthe PLL-g-PEG with a grafting ratio of 8.4% have a similar PLLmolecular weight value per PEG chain, enabling a parallel comparisonfor the transduction efficiency of ADVs between the two differenttypes of copolymer based on the equivalent molar ratio of PLL to PEG.

ADVs were coated with PLL-b-PEG and PLL-g-PEG copolymers byionic interactions. Particle sizes and surface charge values of ADVs,ADV/PLL-b-PEG, and ADV/PLL-g-PEG complexes were measured byDLS, as shown in Fig. 2A. The average sizes of ADV and ADV/PLL-b-PEGwere 72.1±5.2, and 240.2±4.6 nm, respectively. The sizes of ADV/PLL-g-PEG with grafting ratios of 3.5%, 8.4%, and 32.3% were 132.1±3.0, 239.8±7.2, and 230.2±9.3 nm, respectively. The ADVs coatedwith PLL–PEG copolymers showed increased particle sizes ascompared to that of naked ADVs, suggesting that the copolymerswere ionically anchored on the viral surface by slightly inducingaggregation of ADVs. The surface zeta potential values of ADVs andADV/PLL-b-PEG were −21.3±4.2, and +22.5±2.2 mV, respectively,whereas those of ADV/PLL-g-PEG with grafting ratios of 3.5%, 8.4%,and 32.3% were +23.1±2.4, +22.2±1.7, and +23.7±2 mV,

Fig. 2. (A) DLS size distribution and (B) AFM image of ADVs coated with PLL–PEGcopolymers.

respectively. AFM image analysis in Fig 2B revealed that ADVs coatedwith PLL–PEG copolymers were individually separated withoutshowing any severe aggregation. Negatively charged ADVs becamepositively charged by the surface coating of PLL–PEG copolymers.When PLL homopolymer (MW 1500) was used to coat on the surfaceof ADVs, they aggregated and precipitated with increasing amount ofPLL, resulting in unreliable and highly fluctuated DLS data. Thus, PLL–PEG copolymers on the surface of ADV played important roles instabilizing individual ADV particles by the stealth effect of PEG chains.

Cellular uptake of ADVs coated with PLL, PLL-b-PEG, and PLL-g-PEG was visualized by conjugating rhodamine dye to the aminegroups of the viral surface. After transduction of rhodamine-labeledADV complexes with PLL, PLL-b-PEG, or three kinds of PLL-g-PEG for3 h in KB cells, the cellular uptake of ADV complexes was observed byconfocal microscopy. As shown in Fig. 3A, the cellular uptake extent ofADVs coated with PLL-g-PEG (8.4%) was significantly higher than thatof ADVs with PLL-b-PEG, PLL-g-PEG (3.5% and 32.3%) or PLL alone. Thequantitative analysis of cellular uptake extent, as determined from themeasurement of fluorescence intensity after cell lysis, also indicatedthat ADVs coated with PLL-g-PEG (8.4%) had the highest level asshown in Fig. 3B. This suggests that an optimal PLL molecular weightper PEG chain in the copolymer is crucially required for maximizingcellular uptake. It appears that various formulation parameters, suchas PEG surface density, PEG molecular weight, PLL–PEG copolymerarchitecture, and PLL molecular weight per PEG chain on the ADVsurface, could contribute to the non-specific endocytic uptake ofphysically PEGylated ADVs in varying degrees.

GFP gene transduction efficiencies were also investigated fortransfecting ADVs coated with PLL, PLL-b-PEG and PLL-g-PEG to KBcells in the absence of serum, which are shown in Fig. 3C and D. It canbe seen that with increasing amounts of PLL, ADVs coated with PLL-b-PEG and PLL-g-PEG copolymers exhibit gradually increased geneexpression levels, followed by saturated or slightly decreased levelsabove a certain PLL concentration range. ADVs coated with PLL alsoshowed marginally enhanced gene expression levels, but far lowergene expression than those coated with PLL–PEG copolymers. Inparticular, ADVs coated with PLL-b-PEG and PLL-g-PEG (8.4%), both ofwhich have almost the same PLL molecular weight value per PEGchain, similarly demonstrated much higher GFP expression levelsthan those with PLL. The gene transduction results were consistentwith the cellular uptake extents as shown in Fig. 3A and B. RelativeGFP expression levels of ADVs coated with PLL-g-PEG copolymers(grafting percentages: 0%, 3.5%, 8.4%, and 32.3%) were 165, 135, 370,and 225%, respectively at the PLL concentration of 1 µg (Fig. 3D). ADVscoated with PLL-g-PEG (8.4%) showed the highest GFP genetransduction efficiency among the three PLL-g-PEG copolymers. Thelower PEG substitution degree in the PLL backbone might beinsufficient for the stabilization of individual ADV particles, whereasthe higher PEG substitution degree could retard the internalization ofexcessively PEGylated ADVs into the cells to some extent. PLL-g-PEGwith an 8.4% grafting ratio may be the optimal copolymer for surfacecoating of ADVs. It was also reported that for chemically PEGylatedADVs, an optimal surface PEGylation density of ADV was required tomaximize gene transduction efficiency [9]. Since both PLL–PEGcopolymers exhibited the greatest gene expression level at a certainPLL concentration, it can be concluded that the physical immobiliza-tion of PEG chains on the ADV surface in an appropriate surfacedensity could produce cationic, but stable ADV particles withoutsacrificing the capability of non-specific intracellular uptake, resultingin enhanced cellular uptake with concomitantly increased genetransduction efficiency.

Gene transduction efficiencies of ADVs coated with PLL-b-PEG andPLL-g-PEG (8.4%) copolymers were also examined in the presence ofserum, because physically PEGylated ADVs were expected to bedePEGylated by negatively charged serum proteins via an ionexchange mechanism. As shown in Fig. 4A, it can be seen that ADVs

Fig. 4. (A) Comparative GFP transduction efficiencies of ADVs coated with PLL-b-PEGand PLL-g-PEG (grafting ratio of 8.4%). ADVs (5×109 pts) were modified with differentamount of PLL in the present of serum. (B) Cytotoxicities of PLL (MW 1500 and 24,000),PLL-b-PEG, and PLL-g-PEG (grafting ratio of 8.4%) copolymers.

Fig. 3. (A) Confocal microscopy image and (B) the quantitative cellular uptake extent ofrhodamine-labeled ADVs coated with PLL–PEG copolymers after treating to KB cells.GFP transduction efficiencies of ADVs (5×109 pts) coated with (C) PLL (MW 1500) andPLL-b-PEG, and (D) PLL (MW 24,000) and three different PEG-g-PLL copolymers (3.5%,8.4%, and 32.3%) as a function of PLL concentration.

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coated with PLL-b-PEG demonstrate about 6-fold higher geneexpression that those with PLL-g-PEG at the PLL concentration of5 µg/ml in the presence of serum, in contrast to those in the absence ofserum (Fig. 3C, D). Although both of PLL-b-PEG and PLL-g-PEG hadsimilar PLL molecular weights per PEG chain, only PLL-b-PEGexhibited greatly enhanced gene expression efficiency at higher PLLconcentration range in the presence of serum. This might beattributed to the fact that PLL-b-PEG could more effectively protectADVs from the spatial access of serum proteins than the PLL-g-PEG byproducing a more densely PEGylated surface shielding layer aroundthe ADV surface. It is conceivable that the block copolymer can bemore tightly and stably anchored onto the ADV surface than the graftcopolymer due to the different architecture.

Cytotoxicities of ADVs coated with PLL (MW 1500 and 24,000),PLL-b-PEG, and PLL-g-PEG were determined. As shown Fig. 4B, lowmolecular weight PLL shows no cytotoxicity similar to PEGylated PLLcopolymers, whereas high molecular weight PLL exhibits graduallyincreased toxicity with increasing PLL concentration. Both PLL-b-PEGand PLL-g-PEG copolymers did not show any cytotoxicities over thePLL concentration range used for surface coating of ADVs. To furtherconfirm superior gene expression efficiency of ADVs coated with PLL-b-PEG for other cancer cells (MCF-7 cells), ADVs (2×105 pts) coatedwith PLL-b-PEG was used to test their gene expression levels atdifferent PLL concentrations in the absence and presence of serum. Asshown in Fig. 5A, with increasing the PLL concentration up to 5 µg/ml,both PLL and PLL-b-PEG showed gradually enhanced gene expressionpercentages, while both showed reduced gene expression percen-tages at the PLL concentration of 50 µg/ml, suggesting again thatexcessively PEGylated ADV were not readily internalized within cellsdue to the presence of sterically repulsive PEG chains on the ADVsurface. There was no significant difference in gene transductionefficiency between PLL and PLL-b-PEG for MCF-7 cells in the serumfree condition (Fig. 5A). However, PLL-b-PEG showed far superior

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gene expression levels compared to PLL in the presence of serum(Fig. 5B), consistent with the results in Fig. 4A.

The cellular entry of ADVs occurs by initial attachment of an ADVsurface fiber protein moiety to the CAR on the cells, and docked ADVsare internalized by interaction between an RGD peptide at the pentonbase of ADV and a cellular integrin molecule [24]. To demonstrateCAR-independent cellular uptake pathway of ADVs coated withcopolymers, intercellular uptake and gene transduction of nakedADV, ADV/PLL, and ADV/PLL-b-PEG were tested with NIH3T3 cells, awell-known CAR-deficient cell line [25]. Rhodamine-labeled-ADVsalone were very slightly taken up by NIH3T3 cells, indicating theabsence of CAR on the cell surface. In contrast, ADVs coated with PLLand PLL-b-PEG at the PLL concentration of 5 µg/ml were readilyinternalized with cells as shown in Fig. 5C. The relative GFP expressionextent was also increased in a dose dependent manner up to theconcentration of 5 µg/ml. However, at 50 µg/ml of PLL-b-PEG, severecytotoxicity was observed for NIH3T3 cells, suggesting that theNIH3T3 cells responded sensitively to excess concentration of PLL-b-PEG as compared to the other cancer cells. ADVs coated with PLL-b-PEG copolymers at the optimal concentration could be sufficientlydelivered within cells without the CAR-mediated binding process.

hMSCs, humanmesenchymal stem cells, have been popularly usedfor regeneration of multi-potent precursor cells [26]. hMSCs havebeen known to be poorly transfected by ADVs due to their insufficientexpression of CAR onto the cellular membrane [27]. ADVs coated withPLL-b-PEG copolymer were used to enhance the gene transductionefficiency for hMSCs in the presence of serum. The GFP expressionlevels of ADV/PLL and ADV/PLL-b-PEG complexes were 3-fold and 4.3-fold higher than naked ADVs, respectively at the PLL concentration of1 µg/ml. Compared to ADVs coated with PLL, ADVs coated with PLL-b-PEG showed greatly enhanced GFP expression at the PLL concentra-tions of 0.1 µg/ml and 1 µg/ml, but showed much reduced geneexpression levels at higher PLL concentrations (Fig. 5D top). The GFPexpression extents of hMSCs transfected with ADVs, ADV/PLL, andADV/PLL-b-PEG complexes at the PLL concentration of 1 µg/ml werevisualized (Fig. 5D bottom). It can be observed that limited ADVtransduction into hMSCs can be significantly improved by surfacecoating of ADVs with PLL-b-PEG copolymer.

Since the main cellular entry route of naked ADVs is a CAR-mediated endocytic pathway for CAR over-expressing cells, it can bededuced that physically PEGylated ADVs were readily taken up bydifferent cells including mesenchymal stems cells mainly via a CAR-independent and non-specific endocytic pathway. In the currentstudy, ADVs coated with PLL-b-PEG copolymer, rather than thosecoated with PLL-g-PEG graft copolymers, exhibited far superior genetransduction efficiencies for various cells at an optimal PEG seedingdensity. This was possibly caused by different PEG anchoring modeson the surface of ADV depending on the two different copolymerarchitectures. Densely PEGylated ADVs, having similar characteristicsto nano-sized non-viral polyplexes and lipoplexes, weremost likely tobe non-specifically endocytosed within cells. Physically PEGylatedADVs formulated in this study demonstrated far superior in vitro genetransduction efficiencies to chemically PEGylated ADVs reported inprevious studies [9], suggesting that many viral vectors could beeasily PEGylated by facile physical adsorption of PEGylated catio-nic polymers on the surface for enhancing gene transduction andreducing innate immune response. More rigorous in vitro and in vivo

Fig. 5. Relative GFP expression extents of ADVs coatedwith PLL and PLL-b-PEG for MCF-7 cells (A) in the absence of serum and (B) in the presence of serum. The difference inthe relative GFP expression of ADVs at 5 µg/ml and 50 µg/ml of PLL concentration wasstatistically significant (**pb0.001 and *pb0.004). (C) Intracellular uptake images ofADVs alone and ADVs coated with PLL and PLL-b-PEG at 5 µg/ml of PLL concentrationand the relative GFP expression extent of ADV, ADV/PLL and ADV/PLL-b-PEG for NIH3T3cells. (D) GFP expression extent of ADVs coated with PLL and PLL-b-PEG for hMSCs.Fluorescent images of GFP expression for naked ADVs and ADVs coated with PLL andPLL-b-PEG.

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studies are in progress to examine whether physically PEGylatedADVs show reduced immune responses and exhibit a passive tumortargeting ability when systemically administered.

4. Conclusion

We demonstrated here that surface coating of ADVs with PLL-b-PEG copolymer could significantly enhance gene transductionefficiency for cancer and mesenchemal stem cells, compared tonaked ADVs and PLL coated ADVs. While both PLL-g-PEG and PLL-b-PEG copolymers could improve the extent of GFP gene expression ofADVs in the absence of serum, only PLL-b-PEG showed superior geneexpression in the presence of serum possibly due to the formation ofdensely seeded PEG shielding layer on the ADV surface. PhysicallyPEGylated ADV can be prepared in a simple manner; as compared tochemically PEGylated ADVs. Optimally formulated ADVs with PLL-b-PEG copolymer could be usefully applied to enhance gene transduc-tion efficiency for a variety of CAR-deficient cells.

Acknowledgements

This study was supported by the grants of the National CancerCenter (0620240-1) and the WCU program, Republic of Korea.

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