PPSA and Pspa polymer-virus complex and pharmaceutical compositions comprising the same

Abstract

The present invention relates to a complex of PPSA or PSPA polymer and a virus, and a pharmaceutical composition including the same. According to the present invention, when a polymer-virus complex formed using PPSA or PSPA polymer is used, transduction efficiency thereof to cells may be enhanced, an excellent therapeutic effect may thus be obtained when used as a pharmaceutical composition, and, therefore, the pharmaceutical agent may be useful as a therapeutic agent.

Claims

1. A bioreducible polymer represented by Formula 2, ##STR00005## where each of x and y independently is an integer from 1 to 5.

2. A polymer-virus complex in which the polymer of Formula 2 of claim 1 is bound to a surface of a virus.

3. The polymer-virus complex of claim 2, wherein the virus is any one selected from the group consisting of adenovirus (Ads), adeno-associated virus (AAVs), retrovirus, lentivirus, herpes simplex virus and vaccinia virus.

4. The polymer-virus complex of claim 2, wherein the virus is an Ad.

5. A pharmaceutical composition, comprising: (a) the polymer-virus complex of claim 2; and (b) a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, wherein the composition further comprises a therapeutic gene.

7. The pharmaceutical composition of claim 5, wherein the virus is any one selected from the group consisting of adenovirus (Ads), adeno-associated virus (AAVs), retrovirus, lentivirus, herpes simplex virus and vaccinia virus.

8. The pharmaceutical composition of claim 5, wherein the virus is an Ad.

9. The pharmaceutical composition of claim 6, wherein the therapeutic gene is a cancer-treating gene selected from the group consisting of a drug-sensitizing gene, a tumor suppressor gene, an antigenic gene, a cytokine gene, a cytotoxic gene, a cytostatic gene, a pro-apoptotic gene, and an anti-angiogenic gene.

Description

DESCRIPTION OF DRAWINGS

(1) FIGS. 1A, 1B and 1C are .sup.1H NMR spectra of samples analyzed by D20: (A) mPEG-PEI; (B) mPEG-PEI-g-Arg; and (C) PPSA.

(2) FIG. 2 is an MALDI-TOF spectrum of PPSA.

(3) FIGS. 3A and 3B show the cytotoxicity of PPSA in A549 and MCF7 cells, assessed by an MIT assay.

(4) FIGS. 4A, 4B, 4C, 4D, 4E and 4F show the cytotoxicity in A549, MCF7 and CT-26 cells according to treating concentrations and time of PEI 25 k, PPSA and PSPA polymers, assessed by an MT assay.

(5) FIGS. 5A, 5B and 5C show characteristics of an Ad/PPSA nanocomplex: (A) The result of gel retardation assay of Ad/PPSA, (B) Average size distribution of naked Ad or Ad/PPSA at various molar ratios, (C) Zeta-potential value of naked Ad or Ad/PPSA at various molar ratios.

(6) FIGS. 6A, 6B and 6C are graphs showing characteristics of the Ad/PPSA complex: (A) Average particle size according to time, (B) Zeta potential according to time, (C) Average size distribution of naked Ad or Ad/PPSA before and after treatment with DTT (5 mM).

(7) FIGS. 7A, 7B and 7C are graphs showing characteristics of the Ad/PSPA nanocomplex: (A) Result of gel retardation assay for the Ad/PAPS complex, (B) Average size distribution and zeta potential of the Ad/PAPS complex, (C) Average size distribution of naked Ad, Ad/PAP and Ad/PAPS before and after treatment with DTT.

(8) FIGS. 8A and 8B are analysis results of transduction efficiency of naked Ad, Ad/25K PEI, Ad/ABP or Ad/PPSA in A549 and MCF7 cells: (A) Fluorescence microscopy images of transduced cells, (B) Transduction efficiency on respective A549 and MCF7 cells, measured by flow cytometry.

(9) FIGS. 9A and 9B are results of a competition assay of naked Ad, Ad/ABP and Ad/PPSA: (A) GFP fluorescence microscopy images and (B) GFP expression levels measured by flow cytometry.

(10) FIGS. 10A, 10B, 10C and 10D show cellular uptake efficiency of naked Ad, Ad/PPS and Ad/PPSA, which are labeled with FITC, observed by confocal microscopy (A, C) and analyzed by FACS (B, D).

(11) FIGS. 11A, 11B and 11C show GFP expression levels of viruses and the virus/polymer complex in respective A549 (FIG. 11A), MCF7 (FIG. 11B) and CT-26 (FIG. 11C) cells.

(12) FIGS. 12A, 12B and 12C are graphs showing GFP expression levels of Ad/PSPA complex, Ad/PPSA complex, and Ad/PEI complex groups in respective A549 (FIG. 12A), MCF7 (FIG. 12B) and CT-26 (FIG. 12C) cells.

(13) FIGS. 13A and 13B are graphs showing a tumor killing effect of DWP418, DWP418/ABP or DWP418/PPSA in A549 (A) and MCF7 (B).

(14) FIGS. 14A, 14B and 14C are graphs showing a tumor killing effect of a virus/polymer complex in A549 (A), MCF7 (B) and CT-26 (C).

(15) FIGS. 15A and 15B show gene expression and increased effects of a virus/polymer complex in cells.

(16) FIGS. 16A and 16B are (A) a graph showing anticancer efficacy of DWP418, DWP418/ABP or DWP418/PPSA in nude mice onto which MCF7 tumors are xenografted, and (B) microscopy images of tumor sections from each group strained with H&E, E1A, PCNA or TUNEL.

(17) FIGS. 17A, 17B and 17C show innate and adaptive immune responses against Ads: (A) Assessment of innate immune response against naked DWP418, DWP418/ABP or DWP418/PPSA by analyzing IL-6 levels in serum by ELISA, and (B, C) Adaptive immune responses against naked Ad and Ad/PPSA by observing a GFP expression level after naked Ad (dE1/GFP) or an Ad/PPSA complex is reacted with serum with or without Ad-specific neutralizing antibody.

(18) FIGS. 18A and 18B show the hepatotoxicity of DWP418, DWP418/ABP and DWP418/PPSA, assessed by measuring ALT (A) and AST (B) levels in serum.

EXAMPLES

(19) Hereinafter, the present invention will be described in further detail with respect to examples. These examples are only provided to more fully describe the present invention, and it is obvious to those of ordinary skill in the art that the scope of the present invention is not limited to these examples, according to the gist of the present invention.

Examples

(20) Test Materials and Method

(21) 1. Test Materials

(22) Methoxyl PEG succinimidyl carbonate NHS was purchased from Nanocs (USA). Arginine, N,N-diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA), triisopropyl silane (TIPS), polyethylenimine (1.8 kDa, 50 wt %), branched polyethylenimine (25 kDa), N-hydroxysuccinimide, 2-imidothiolane hydrochloride (Traut's reagent), DL-dithiothreitol, dimethylsulfoxide (DMSO), 2-imidothiolane, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and dimethylformaldehyde (DMF) were purchased from Sigma (St Louis, USA).

(23) 2-(1-H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) was purchased from Novabiochem (San Diego, Calif.). Fmoc-L-Arg(Pbf)-OH was purchased from Anaspec, Inc. (San Jose, Calif.). Ellman's reagent was purchased from Thermo scientific (Rockford, Ill.). Deuterium oxide was purchased from Cambridge Isotope Laboratories, Inc. (Andover, Mass.).

(24) 2. Synthesis of mPEG-PEI-g-Arg-S-S-Arg-g-PEI-mPEG (PPSA)

(25) (1) Synthesis of Methoxy Poly(Ethylene Glycol)-Polyethylenimine (mPEG-PEI)

(26) PEG-PEI was synthesized as described in the reference (33). Polyethyleneimide was dissolved in 3.0 ml of PBS (pH 7.4). Subsequently, one molar equivalent of methoxy PEF succinimidyl carbonate NHS (mPEG-NHS, 2.0 kDa) was added. The reaction product was stirred at room temperature overnight. The product was dialyzed against double distilled water at room temperature for 2 hours using a Slide-A-Lyzer dialysis cassette (2.0 kDa MWCO, Pierce, Rockford, Ill., USA) and lyophilized, thereby obtaining a pale white substance (75% yield). The chemical structure of the substance was confirmed by .sup.1H NMR observing a D.sub.2O-solubilized sample at 300 MHz (Mercury Plus 300 MHz Spectrometer, Varian, Inc. Vernon Hills, Ill., USA). Characteristic PEG (3.6 ppm, (CH.sub.2CH.sub.2O)) and PEI (2.0 to 3.0 ppm) peaks were observed.

(27) (2) Synthesis of Arginine-Grafted mPEG-PEI (mPEG-PEI-g-Arg)

(28) As described in the reference (28), arginine was grafted onto mPEG-PEI. The grafting was done by combining 9 equivalents of Fmoc-Arg(Pbf)-OH and HBTU with 12 equivalents of DIPEA in DMF (1.0 ml) at room temperature for 48 hours. The resulting product was precipitated in diethyl ether twice to remove unreacted reagents. To remove the Fmoc moiety from the Fmoc-Arg(Pbf)OH, the precipitant was mixed with an equal volume of 30% piperidine solution in DMF (Sigma, St Louis, Mo., USA) at room temperature for 1 hour. The precipitation process was repeated twice. A reagent solution (TFA:TIPS:H20, 95/2.5/2.5 v/v) was added to the precipitate to remove the Pbf group from the arginine residue. The reaction was performed at room temperature for 30 minutes. The polymer was precipitated with ether. The final product, mPEG-PEI-g-Arg, was dialyzed (2.0 kDa MWCO) against double distilled water overnight and lyophilized, thereby obtaining a white product (60% yield). The chemical structure was confirmed by .sup.1H NMR as described above. Characteristic peaks of PEG (3.6 ppm, (CH.sub.2CH.sub.2O)), PEI (2.0 to 3.0 ppm) and arginine (1.66 ppm (HCCH.sub.2CH.sub.2CH.sub.2NH); 1.86 ppm (HCCH.sub.2CH.sub.2CH.sub.2NH); 3.24 ppm (HCCH.sub.2CH.sub.2CH.sub.2NH); 3.86 ppm (HCCH.sub.2CH.sub.2CH.sub.2NH)) were observed.

(29) (3) Synthesis of mPEG-PEI-g-Arg-S-S-Arg-g-PEI-mPEG (PPSA)

(30) mPEG-PEI-g-Arg was dissolved in PBS (2.0 ml, pH 7.4, 4 mg/ml EDTA). 8 equivalents of 2-imidothiolane hydrochloride (Traut's reagent) per surface amine in mPEG-PEI-g-Arg were added and continuously stirred at room temperature for 3 hours. The product was dialyzed against double distilled water (2.0 kDa MWCO) to remove unreacted reagents and was lyophilized.

(31) The lyophilized mPEG-PEI-Arg-SH was dissolved in 1PBS, and 500 l DMSO was added to oxidize the SH group. The reaction product was stirred at room temperature for 48 hours. The product was then dialyzed against double distilled water (2.0 kDa MWCO) again for 24 hours. The final product, mPEG-PEI-g-Arg-S-S-Arg-g-PEI-mPEG (PPSA), was lyophilized, thereby obtaining a white product (80% yields). As described in the reference, the disulfide cross-linking was confirmed by Ellman test (34).

(32) 3. Synthesis of PEI-Arg-mPEG-S-S-mPEG-Arg-PEI (PSPA)

(33) (1) Synthesis of Poly(Ethylenimine)-Arginine (PEI-Arg)

(34) Arginine was conjugated to polyethylenimide according to the procedure reported in the literature Enhanced in-vitro transfection and biocompatibility of L-arginine modified oligo(-alkylaminosiloxanes)-graft-polyethylenimine. The carboxyl group of the amino acid, arginine (350 mg, 2.0 mmol), was activated with a coupling agent, EDC/NHS (EDC, 384 mg, 2.0 mmol and NHS=230 mg 2.0 mmol) in phosphate saline buffer (pH 7.4, 3.0 ml) at 4 C. for 4 hours. Subsequently, polyethylenimine (PEI; 360 mg, 0.2 mmol) was added to the activated arginine, and the reaction was maintained at room temperature for 18 hours. The product was dialyzed (MWCO 1.0 kDa) against double distilled water for a day to remove unreacted compounds and was lyophilized. The chemical structure was confirmed by .sup.1H NMR (300 MHz, D2O). A characteristic PEI peak was observed at 2.0 to 3.0 ppm, and characteristic arginine peaks were observed at 1.66 (HCCH.sub.2CH.sub.2CH.sub.2NH); 1.86 (HCCH.sub.2CH.sub.2CH.sub.2NH); 3.24 (HCCH.sub.2CH.sub.2CH.sub.2NH); and 3.86 (HCCH.sub.2CH.sub.2CH.sub.2NH).

(35) (2) Synthesis of PEI-Arg-mPEG

(36) Arginine-grafted poly(ethyleneimide) was dissolved in 3.0 ml PBS (pH 7.4). Subsequently, one equivalent of methoxy PEG succineimidylcarbonate NHS-2.0 kDa was added. The reaction mixture was stirred at room temperature overnight. The product was dialyzed against double distilled water at room temperature for 24 hours using a Slide-A-Lyzer dialysis cassette (2.0 kDa MWCO, Pierce, Rockford, Ill., USA) and lyophilized, thereby obtaining PEI-Arg-mPEG.

(37) The chemical structure was confirmed by .sup.1H NMR (300 MHz, D.sub.2O). The NMR spectrum showed characteristic PEG peak (3.6 ppm, (CH.sub.2CH.sub.2O)), PEI peak (2.0 to 3.0 ppm) and arginine peaks 1.66-(HCCH.sub.2CH.sub.2CH.sub.2NH); 1.86 (HCCH.sub.2CH.sub.2CH.sub.2NH); 3.24 (HCCH.sub.2CH.sub.2CH.sub.2NH); 3.86 (HCCH.sub.2CH.sub.2CH.sub.2NH).

(38) (3) Synthesis of PEI-Arg-mPEG-S-S-mPEG-Arg-PEI (PSPA)

(39) PEI-Arg-mPEG was dissolved in IX PBS (2.0 mL, pH=7.4, 4 mg/mL EDTA). 8 equivalents or higher of 2-imidothiolane hydrochloride (Traut's reagent) was added per surface imine of PEI-Arg-mPEG and continuously stirred at room temperature for 3 hours. The product was dialyzed against double distilled water using an Slide-A-dialysis cassette (2.0 kDa MWCO) to remove unreacted reagents, and the product, PEI-Arg-mPEG-SH, was lyophilized. In addition, the lyophilized mPEG-PEI-Arg-SH polymer was redissolved in 1PBS, and 500 l DMSO was added to oxidize the SH group. The reaction mixture was stirred at room temperature for 48 hours, and then the product was dialyzed against double distilled water for 24 hours using a Slide-A-dialysis cassette (2.0 kDa MWCO) again. The product was lyophilized to obtain PEI-Arg-mPEG-S-S-mPEG-Arg-PEI (PSPA).

(40) 4. Cell Lines and Cell Culture

(41) The following cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, Va.): HEK293, a human embryonic kidney cell line expressing Ad E1 replication protein; A549, a non-small cell lung cancer cell line; MCF7, a breast cancer cell line; and CT-26, a colorectal cancer cell line. All cell lines were cultured in DMEM (Gibco BRL, Grand Island, N.Y.) containing 10% FBS (Gibco BRL) and penicillin/streptomycin (Gibco BRL) at 37 C. in a humidified 5% CO.sub.2 atmosphere.

(42) 5. Ad Preparation

(43) Replication-incompetent Ad (dEl/GFP) expressing green fluorescent protein (GFP) under the control of a CMV promoter in an E1 region and oncolytic Ad (DWP418 or RdB/IL-12/decorin; oAd) were used basically using the methods described in the previous research that had been conducted by the inventor (35-38). All Ads were propagated in HEK293 cells and then purified by CsCl (Sigma, St Louis, Mich.) density-gradient centrifugation. A viral particle (VP) number was calculated from OD.sub.260 measurement, for which an absorbance of 1 was equivalent to 10.sup.12 VP/ml.

(44) Infectious titers (PFU/mL) were determined using a limiting dilution assay on HEK293 cells. The viral particle/PFU ratios for dEl/GFP and DWP418 were 29:1 and 81:1, respectively. The MOI was calculated from the infectious titers.

(45) 6. Cytotoxicity Analysis

(46) Cytotoxicity of the polymers of the present invention and various cationic polymers was analyzed. Specifically, quantitative cell viability was analyzed on 25 kDa branched polyethylenimine (25 kDa PEI), the previous Ad-binding polymer (ABP), PSPA (PAPS) polymer and PPSA polymer by a method of measuring conversion of MTT to formazan over time (39, 40).

(47) A549 and MCF7 cells were cultured to 50% confluence in 96-well plates, and then each was treated with each of the 25 k PEI polymer, the ABP polymer and the PPSA polymer at concentrations of 0.5 g/ml, 1 g/ml, 5 g/ml and 10 g/ml. Three days after the polymer treatment (72-hour treatment), 100 l of MTT (2 mg/ml) was added to each well, and reacted at 37 C. for 4 hours. The supernatant was discarded, and the precipitate was dissolved in 100 l DMSO. Plates were analyzed on a microplate reader (Bio-Rad, Hercules, Calif.) at 540 nm.

(48) Also, the cytotoxicity of the polymers was measured by the same method as above, except that A549, MCF7 and CT-26 were treated with 0.1 g/ml, 0.5 g/ml, 1 g/ml, 5 g/ml and 10 g/ml of each of the 25 k PEI polymer, the PPSA polymer and the PSPA polymer for 24 hours and 72 hours.

(49) 7. Preparation of Ad/PPSA Complex

(50) To construct the Ad/PPSA complex, Ad particles (210.sup.10 VP/PBS, pH 7.4) were mixed with various concentrations of the PPSA polymer. As a result, PPSA ratios per Ad particle came to 210.sup.4, 110.sup.5, 410.sup.5 and 110.sup.6. The solution was incubated at room temperature for 30 minutes before use.

(51) 8. Preparation of Ad/PSPA Complex

(52) To construct an Ad/PSPA complex, Ad particles (210.sup.10 VP/PBS, pH 7.4) were mixed with various concentrations of the PSPA polymer. As a result, PSPA ratios per Ad particle came to 210.sup.3, 510.sup.3, 110.sup.4, 510.sup.4, 110.sup.5, 510.sup.5, 110.sup.6. The solution was incubated at room temperature for 30 minutes before use.

(53) 9. Measurement of Particle Size and Surface Change

(54) Average particle sizes and surface changes of naked Ad and Ad/PPSA were determined by dynamic laser scattering (DLS) at 488 nm and zeta particle analysis (90 fixed angle scattering) at 633 nm, respectively, at room temperature using Zetasizer 3000HS (Malvern Instrument Inc., Worcestershire, UK) with a HeNe laser.

(55) Also, average particle sizes, surface changes such as zeta potential and changes in average particle size according to DTT treatment were assessed on the Ad/PAPS complex by the same method as described above.

(56) In the specification, the sizes and variations are average values of five independent runs.

(57) 10. Gel Retardation

(58) Gel retardation was performed to examine the encapsulation profiles of the Ad/PSPA and the Ad/PPSA complex. After the construction of the Ad/PPSA complex, a virus lysis buffer (0.1% SDS, 1 mM Tris-HCl (pH7.4), 0.1 mM EDTA) was added to the Ad/PPSA complex and reacted at 56 C. for 30 minutes. The Ad/PPSA complex sample was loaded on a 1% (w/v) agarose gel in 1TAE buffer (10 mM Tris-HCl, 1% (v/v) acetic acid, 1 mM EDTA (w/EtBr)). Electrophoresis was performed at 100 V for 30 minutes in the same buffer. The locations of DNA bands were visualized using a ChemiDoc gel documentation system (Syngene, Cambridge, UK). Gel retardation was also performed on the analyzed Ad/PAPS complex by the same method described above.

(59) 11. Analysis of Transduction Efficiency

(60) Each type of cancer cells (A549, MCF7 and CT-26) were seeded into a 24-well plate and cultured to 60% confluence one day before transduction assay. The cells were treated with naked Ad (dEl/GFP) or Ad (dEl/GFP)/polymer complex (Ad/25 KDa PEI, Ad/ABP, Ad/PSPA or Ad/PPSA).

(61) The transduction efficiency was analyzed by assessing GFP expression levels of the PPSA:Ad complex, 25 k PEI complex and ABP complex having the polymervirus molar ratios of 210.sup.4, 110.sup.5, 410.sup.5 and 110.sup.6 in the A549 and MCF7 cell lines.

(62) Also, the transduction efficiency was analyzed in each of A549, MCF7 and CT-26 cell lines using PAPS:Ad complex, PPSA:Ad complex and 25 kDa PEI:Ad complex molar ratios of 110.sup.3, 510.sup.3, 110.sup.4, 510.sup.4, 110.sup.5, 510.sup.5, 110.sup.6. Due to the varying Ad susceptibility of each cell line, different MOI were applied to A549, CT-26 and MCF7.

(63) Transduced cells were further cultured for 48 hours. The cells were imaged using fluorescence microscopy (Olympus IX81; Olympus Optical, Tokyo, Japan), and the GFP expression levels were quantified using FACS analysis BD FACScan analyzer (Becton Dickinson, San Jose, Calif.) and CellQuest software (Becton-Dickinson). Data from 10,000 events were collected, and the meanstandard deviations of three independent experiments were presented.

(64) 12. Competition Assay

(65) A549 cells (510.sup.4 cells/well) were seeded into a 24-well plate. Following 24-hour culture, the cells were pre-treated with PBS or purified Ad fiber knob protein (2 or 10 mg/ml) for 30 minutes. The cells were washed with PBS three times and then treated with 30 MOI of naked Ad or Ad/PPSA complex (110.sup.6 PPSA:Ad molar ratio) in 5% FBS-supplemented DMEM. The cells were incubated for 2 days, imaged using the fluorescence microscopy (Olympus IX81; Olympus Optical), and analyzed by the BD FACScan analyzer (Beckton-Dickinson) and the CellQuest software (Beckton-Dickinson).

(66) 13. Evaluation of Cancer Cell Killing Effect of Oncolytic Ad

(67) To evaluate the cancer cell killing effect of oncolytic Ad, each type of A549 and MCF7 cell lines were seeded into a 96-well plate, and after 24 hours, naked DWP418 and DWP418/ABP, DWP418/PPSA complexes were treated. After 48 hours, medium was removed, 100 l of MTT (2 mg/ml) was added to each well, and the cells were cultured at 37 C. for 4 hours. Supernatant was discarded, and pellets were dissolved in 100 l of DMSO. Plates were analyzed on a microplate reader (Bio-Rad, Hercules, Calif.) at 540 nm.

(68) Also, the A549, MCF7 and CT-26 cell lines were seeded in 96-well plates, respectively, and after 24 hours, treated with naked RdB/IL-12/decorin; oAd, oAd/PPSA and oAd/PAPS complexes. After 48 hours, medium was removed, 100 d of MT (2 mg/ml) was added to each well, and the cells were cultured at 37 C. for 4 hours. Supernatant was discarded, and pellets were dissolved in 100 l of DMSO. Plates were analyzed on a microplate reader (Bio-Rad, Hercules, Calif.) at 540 nm.

(69) 14. Western Blotting

(70) To validate production of DCN proteins in cells when CT-26 cell lines were infected by oAd/PAPS complex in which the surface of the virus expressing decorin and IL-12 is coated with PAPS, the CT-26 cells were treated with each of 100, 200 and 500 MOI of naked oAd and oAd/PAPS (110.sup.5 polymer:virus molar ratio), and after 48 hours, both of the cell culture and the cells were harvested to perform sodium-dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins in the gel were electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane and reacted with antibodies specifically recognizing decorin as primary antibodies. After being reacted with horse radish peroxidase (HRP)-binding goat anti-mouse IgG as a secondary antibody, the binding of the proteins on the membrane with the antibodies was detected and protein expression patterns were determined using LAS4000 by enhanced chemiluminescence (ECL; Pierce, Rockford, Ill., USA).

(71) 15. ELISA for Detecting Change in IL-12 Expression

(72) Enzyme-linked immunosorbent assay (ELISA) was performed to detect secretion of cytokine to a cell culture when CT-26 cell lines were infected by oAd/PAPS complex in which the surface of a virus expressing decorin and IL-2 is coated with PAPS. One day after the CT-26 cell lines were seeded into a 12-well plate at a density of 510.sup.5 cell/well, the cells were treated with each of 100 and 200 MOI of naked oAd and oAd/PAPS, and after 48 hours, a medium was retrieved from the cells to quantify the IL-12 expression level through ELISA.

(73) 16. In Vivo Anticancer Effect and Histological Analysis

(74) MCF7 cells (510.sup.6) were subcutaneously injected into 6 week-old female nude mice (Orientbio Inc., Gyeonggi-do, Korea). When the tumor volume reached approximately 100 mm.sup.3, the mice were injected with PBS, naked Ad, ABP, PPSA, Ad/ABP, or Ad/PPSA (510.sup.10 VP per injection, 110.sup.6 PPSA:Ad molar ratio) into tumors of the mice every other day for 5 days (total three injections). Tumor growth was assessed every two days by caliper measurement and volume calculation as follows: volume (mm.sup.3)=0.523height (mm)area (mm.sup.2). For histological analyses, three days after the final treatment, tumors were harvested, fixed in 10% formalin, and embedded in paraffin. Tumor sections (5 m thickness) were stained with hematoxylin and eosin (H&E) and examined by light microscopy at 100 magnification.

(75) For immunohistochemical analyses, paraffin-embedded tumor tissues were first deparaffinized by incubation in xylene for 10 minutes and then sequentially incubated with 100%, 900% and 70% ethanol for 5 minutes each.

(76) The tissues were blocked with 3% bovine serum albumin (BSA) at room temperature for 2 hours and stained with Ad E1A-specific antibody (SC-430; Santa Cruz Biotechnology, Santa Cruz, Calif.) or proliferating cell nuclear antigen (PCNA)-specific antibody (Neomarkers, Freemont, Calif.). Sections were counterstained with Mayer's hematoxylin. Apoptosis detection by UNEL analysis was performed using an Apoptag detection kit (Serologicals Corp., Norcross, Ga.) according to the manufacturer's instructions.

(77) 17. Assay for Innate Immune Response

(78) To determine the effects of naked DWP418, DWP418/ABP, or DWP418/PPSA complex on the innate immune response, Balb/C mice were systemically injected with the naked DWP418, DWP418/ABP, or DWP418/PPSA complex (210.sup.10 VP per mouse, 110.sup.6 PPSA:Ad molar ratio). Serum samples were collected 6 hours after injection. IL-6 serum levels were quantified using an IL-6 ELISA kit (R&D Systems, Minneapolis, Minn.) according to the manufacturer's instructions.

(79) 18. Assay for Adaptive Immune Response

(80) For assessing adaptive immune response against Ad, naked Ad (dEl/GFP) was intravenously injected into Balb/c mice at a single dose of 110.sup.10 VP, and 14 days later, Ad was administered again to generate a neutralizing antibody against Ad. 14 days after the second injection, mouse serum immunized with naked Ad (or without naked Ad) was harvested, incubated at 56 C. for 45 minutes to inactivate blood complement, and then stored at 20 C. Naked dEl/GFP (30 MOI) or dEl/GFP coated with PPSA polymer (110.sup.6 molecules/VP; 30 MOI) was exposed to PBS or serum (with or without Ad-specific neutralizing antibody) at 37 C. for 30 minutes and added to human cancer cells (A549). Two days after incubation, GFP expression levels were analyzed by fluorescence (Olympus BX51) and FACScan flow cytometry (Beckton-Dickinson).

(81) 19. In Vivo Toxicity Assessment

(82) To evaluate in vivo potential toxicity, naked DWP418, DWP418/ABP, or DWP418/PPSA (210.sup.10 VP/mouse, 110.sup.6 PPSA:Ad molar ratio) was intravenously injected into Balb/C mice. Three days after injection, serum levels were measured by aspartate aminotransferase (AST) and alanine transaminase (ALT).

(83) 20. Statistical Analysis

(84) Data were expressed as meanstandard deviation (SD). Statistical analyses were performed by a two-tailed Student t test (SPSS 13.0 software; SPSS, Chicago, Ill.), and the P value of less than 0.05 was considered statistically significant.

(85) Test Results

(86) 1. Synthesis and Characterization of Bioreducible Polymer

(87) High molecular weight branched polyethylenimide (25K PEI) is used as the benchmark for non-viral gene transfer due to high in vitro and in vivo transduction efficacy (42). However, the polymer has significant cytotoxicity and is not biodegradable, and thus clinical application is limited. To solve such a problem, the inventors designed and synthesized a novel cationic polymer which has low cytotoxicity and is biodegradable using PEI with a low molecular weight (1.8 kDa) in the previous research. PEG-complexed PEI was reduced in cytotoxicity, compared to PEI alone (43). PEI cross-linked by bioreducible linkages showed reduced cytotoxicity (44).

(88) Meanwhile, it is known that cell-penetrating peptides containing arginine residues effectively transfer nucleic acids through intracellular translocation (26, 45). Based on such findings, improved biopolymers, PPSA and PSPA, were synthesized.

(89) The main synthetic route of mPEG-PEI-g-Arg-S-S-Arg-PEI-mPEG (PPSA) is summarized in Reaction Scheme 1.

(90) ##STR00003##

(91) First, to synthesize mPEG-PEI, PEI 1.8 kDa was reacted with succinimidyl ester methoxy polyethylene glycol (mPEG-NHS) (33). Subsequently, to synthesize mPEG-PEI-g-Arg, arginine was grafted onto the polymer using Fmoc-Arg(Pbf)-OH in the presence of HTBU/DIPEA (28). Afterward, to prepare mPEG-PEI-g-Arg-SH, mPEG-PEI-g-Arg was treated with imidothiolane to link thiol groups to the terminal ends. Finally, a novel bioreducible polymer (mPEG-PEI-g-Arg-S-S-Arg-PEI-mPEG; PPSA) was synthesized by cross-linking the terminal thiol groups using dimethylsulfoxide (DMSO).

(92) The synthesis of PPSA was confirmed by .sup.1H NMR (FIG. 1). The occurrence of spectra peaks at 3.64 and 3.36 ppm indicated the presence of methylene protons corresponding to CH.sub.2CH.sub.2O and OCH.sub.3 PEG end groups. Three peaks observed at 2.2 to 3.0 ppm correspond to the CH.sub.2 NH-methane protons of PEI (FIG. 1A). Such results are consistent with the previous reports (33). Following addition of arginine groups, characteristic arginine peaks appeared at 1.44, 1.70, 3.2 and 3.86 ppm, and were assigned to the methylene and methyne protons of (HCCH.sub.2CH.sub.2CH.sub.2NH), (HCCH.sub.2CH.sub.2CH.sub.2NH), (HCCH.sub.2CH.sub.2CH.sub.2NH) and (HCCH.sub.2CH.sub.2CH.sub.2NH), respectively (FIG. 1B). The amount of the grafted arginine was calculated by integrating the area under the PEI methylene peaks (CH.sub.2CH.sub.2N) at 2.3 to 3.0 ppm and the arginine methylene peak (HCCH.sub.2CH.sub.2CH.sub.2NH) at 1.7 ppm. By the calculations, it was shown that approximately seven arginines were grafted per mPEG-PEI. Also, new characteristic peaks were observed at 1.8 to 2.2 ppm. These peaks correspond to cross-linker, iminothiolane methylene protons (NHCH(NH.sub.2)CH.sub.2CH.sub.2CH.sub.2SS) (FIG. 1C), which showed that (mPEG-PEI-g-Arg-S-S-Arg-g-PEI-mPEG (PPSA) was synthesized. Also, the molecular weight was analyzed by MALDI-TOF-Mass. As a result, it was confirmed that the final polymer molecular weight is approximately 10.6 kDa.

(93) The main synthetic route of PEI-Arg-mPEG-S-S-mPEG-Arg-PEI (PSPA) is summarized in Reaction Scheme 2.

(94) ##STR00004##

(95) PSPA was synthesized by the following procedures: arginine was added to polyethylenimine (PEI) in PBS in the presence of EDC/NHS as a coupling agent at room temperature for 18 hours, thereby synthesizing PEI-Arg. In the .sup.1H NMR spectra for the PEI-Arg, resonance peaks at 1.44, 1.70, 3.2 and 3.86 ppm were assigned to the methyne protons of (HCCH.sub.2CH.sub.2CH.sub.2NH); (HCCH.sub.2CH.sub.2CH.sub.2NH); (HCCH.sub.2CH.sub.2CH.sub.2NH); and (HCCH.sub.2CH.sub.2CH.sub.2NH) of arginines grafted with PEI. Also, the amount of the grafted arginines was calculated by integrating the area under the 2.3 to 3.0 ppm PEI methylene peaks (CH.sub.2CH.sub.2N) and the 1.7 ppm arginine methylene peak (HCCH.sub.2CH.sub.2CH.sub.2NH). By the calculation, it was shown that approximately 5 to 6 arginines per PEI were grafted. To improve biocompatibility and hydrophilic blocks, PEI-Arg was treated with succinimidyl ester methoxy poly(ethylene glycol) (MPEG-NHS) in PBS to create PEI-Arg-mPEG. By .sup.1H NMR for determining the chemical structure, new peaks appeared at 3.4 and 3.6 ppm and assigned to ethylene glycol (CH.sub.2CH.sub.2O) and methyl (OCH.sub.3) protons in addition to the PEI-Arg peaks, which indicated linkage of PEG. Next, PEI-Arg-mPE was treated with iminothiolane to synthesize PEI-Arg-mPEG-SH, and terminal thiol groups were oxidized in the presence of a mixture of PBS and DMSO at room temperature for 48 hours, resulting in synthesis of a bioreducible polymer, PSPA. The chemical structure of the polymer PSPA was confirmed by .sup.1H NMR (300 MHz, D.sub.2O). New characteristic peaks appeared at 1.8 to 2.2 ppm and corresponded to iminothiolane methylene protons (NHCH(NH.sub.2)CH.sub.2CH.sub.2CH.sub.2SS) cross-linked with PEI-Arg-mPEG, which showed synthesis of the PEI-Arg-mPEGS-S-mPEG-Arg-PEI (PSPA).

(96) 2. Cytotoxicity Assays for PPSA and PSPA Polymers

(97) To evaluate the potential cytotoxicity, MTT assays were performed on A549 and MCF7 cells treated with PPSA, a control (Mock), PPSA, 25K PEI or ABP. The cells were treated with each of the polymers at various concentrations of 0.5, 1, 5, and 10 g/ml, incubated for 72 hours to analyze cell viability, and presented as relative values with respect to the control.

(98) As shown in FIGS. 3A and 3B, the 25K PEI decreased cell viability in all concentration ranges tested. The ABP or PPSA did not show cytotoxic effects up to 10 g/ml. When 10 g/ml of the 25K PEI, ABP or PPSA was treated, the cell viability of the A549 cells was approximately 46%, 92% or 97%, respectively. At the same dosage of the 25K PEI, ABP or PPSA, the MCF7 cells showed cell viability of approximately 36%, 94% or 97%, respectively. These results are consistent with the previous reports demonstrating that ABP does not have obvious toxicity to mammal cells (32). Meanwhile, more importantly, PPSA does not show cytotoxicity, either, which seems to be because of the low molecular weight of PEI (1.8 kDa) and PEG conjugation (46).

(99) (2) Cytotoxicity Assays for Polymer According to Time

(100) (1) Cytotoxicity Assay for PPSA

(101) To evaluate the potential cytotoxicity, MTT assays were performed on A549 and MCF7 cells treated with PPSA, a control (Mock), PPSA, 25K PEI or ABP. The cells were treated with each of the polymers at various concentrations of 0.5, 1, 5, and 10 g/ml, incubated for 72 hours to analyze cell viability, and presented as relative values with respect to the control.

(102) As shown in FIGS. 4A, 4B, 4C, 4D, 4E and 4F, when the A549 cell line was treated with 10 g/ml of the PAPS polymer for 24 hours, the cell viability was 83%. In contrast, when the A549 cell line was treated with 10 g/ml of PEI widely used for nucleic acid transfer for 24 hours, the cell viability was 15% (FIG. 4A). Also, when the same amount of the PAPS, PPSA, or PEI polymer is added for 72 hours, the cell viability was 75%, 78% or 21%. These results showed that the PAPS polymer has similar cell viability, compared to the cytotoxicity of the PPSA and has a remarkably lower toxicity than PEI. It is estimated that the biodegradable PAPS is able to be reduced into a lower molecular weight, and thus has a lower cytotoxicity than the non-biodegradable PEI. Accordingly, the PAPS has remarkably increased biocompatibility than 25 kDa PEI. Similar results according to the same concentrations and treating time were also obtained from the other cell lines such as MCF7 and CT-26.

(103) 3. Characterization of Nanocomplex

(104) (1) Characterization of Ad/PPSA Nanocomplex

(105) To evaluate the capability of PPSA to form a complex with Ad, comparative agarose gel retardation electrophoresis assays were performed on the polymer at various molar ratios of 0 (naked Ad), 210.sup.4, 110.sup.5, 410.sup.5, and 110.sup.6 per Ad particles.

(106) As shown in FIGS. 5A, 5B and 5C, Ad migration was gradually increased with increased PPSA:Ad molar ratios. The Ad migration was completely retarded at the molar ratio of 110.sup.6, which indicated that an Ad surface was saturated with the PPSA polymer (FIG. 5A).

(107) It is important for a gene transfer vector to have a proper size (<200 nm) for efficient cellular uptake through a non-specific clathrin-dependent process (47, 48). Also, the complex is required to be overall positively charged for being more effectively attached to a negatively-charged cell membrane. To evaluate the biophysical characteristic of Ad/PPSA nanoparticles, the hydrated size and surface charge were measured by DLS and zeta potential analyzer. The average naked Ad particle size in a solution was 110.8 nm in diameter and increased up to 200 nm (110.sup.6 molar ratio), proportional to an increased molar ratio of PPSA:Ad (FIG. 5B).

(108) In agreement with the DLS data, surface charge was also increased from 19.71.2 mV (naked Ad) to 19.60.9 mV (110.sup.6 molar ratio), proportional to the increased PPSA:Ad molar ratio (FIG. 5C). These results show that, through electrostatic interaction, the Ad surface was successfully coated with PPSA that shielded negative charge and thus had a net positive charge at a molar ratio of 110.sup.5 or higher.

(109) The colloidal stability of Ad/PPSA nanoparticles in PBS buffer was measured at room temperature for up to 72 hours by a method of measuring the average size and surface charge of the nanoparticle of the Ad/PPSA complexes with molar ratios of the polymer per Ad particle of 410.sup.5 and 110.sup.6. Also, the reducibility of the PPSA and non-reducible mPEG-PEI-g-Arg (PPA) was examined by treatment with dithiothreitol (DTT) as a reducing agent. The particle sizes of the naked Ad, Ad/PPSA and Ad/PPA complexes, each of which was either treated or not treated with DTT, were measured by a DLS analyzer.

(110) As shown in FIGS. 6A and 6B, the average size and surface charge of the Ad/PPSA nanoparticle were not significantly changed for 72 hours, which implies that PPSA cationic polymer-coated Ad has excellent colloidal stability.

(111) Also, as shown in FIG. 6C, the size of the naked Ad or Ad/PPA complex was not changed by DTT treatment. However, the average particle size of the PPSA-coated Ad complex was significantly reduced after the DTT treatment and approximated the size of the naked Ad. This result clearly confirmed that PPSA is biodegradable in a reducible microenvironment.

(112) Taken together, the test results show that the Ad/PPSA complex was successfully constructed to form a particle with a diameter of less than 200 nm (FIG. 6A), created a positively charged surface (FIG. 6B), and thus was able to be effectively transduced into cells.

(113) (2) Characterization of Ad/PSPA Nanocomplex

(114) Comparative agarose gel retardation electrophoresis assays were performed to analyze the interaction between the Ad/PAPS complex and Ad according to various concentration ratios. The test was performed on the complex having various molar ratios of the polymer per Ad particle of 110.sup.3, 510.sup.3, 110.sup.4, 110.sup.5, 510.sup.5 and 110.sup.6.

(115) As shown in FIG. 7A, Ad migration through the gel was retarded with an increased polymer ratio, which indicates that the Ad surface charge was converted to be positive. When the polymer and Ad ratios exceed the neutralization point, the surface charge of the complex was converted to be positive, thereby interrupting migration. When the molar ratio was 110.sup.5, Ad was not migrated, which indicates that the Ad surface was saturated by PAPS at the above concentration. Also, from such a result, the Ad band was not observed, indicating that the PAPS polymer effectively forms a complex with Ad.

(116) To evaluate the biophysical characteristic of the Ad/PAPS nanoparticle, the hydrated size and surface charge were measured by DLS and zeta potential analyzer. The average naked Ad particle size in a solution was 124.8 nm in diameter, was maintained below approximately 200 nm for PSPA:Ad molar ratio up to 110.sup.5, and, at a higher molar ratio, increased up to 935.6 nm (110.sup.6 molar ratio), proportional to the molar ratio (FIG. 7B).

(117) In agreement with the DLS data, the surface charge was also increased from 21.80.75 mV (naked Ad) to 19.74.9 mV (110.sup.6 molar ratio), proportional to the increased PPSA:Ad molar ratios (FIG. 7B). These results show that the Ad surface was successfully coated with PAPS through electrostatic interaction and thus finally had a positively charged surface.

(118) Also, the reducibility of the PAPS and non-reducible PEI-Arg-mPEG (PAP) was examined by treatment with dithiothreitol (DTT) as a reducing agent. The particle sizes of the naked Ad, Ad/PAPS and Ad/PAP complexes, each of which was either treated or not treated with DTT, were measured by a DLS analyzer. As a result, the size of the naked Ad or Ad/PAP complex was not changed by DTT treatment (FIG. 7C). However, the average particle size of the PAPS-coated Ad complex was significantly reduced after the DTT treatment and approximated the size of the naked Ad. Such a result proves that the PAPS is biodegradable under a reducible microenvironment.

(119) Taken together, the test results show that the Ad/PAPS complex was successfully constructed to form a particle having a diameter of less than 200 nm for the molar ratio of polymer:Ad of 110.sup.5, created a positively charged surface, and thus was effectively transduced into cells.

(120) 4. Enhanced Transduction Efficiency of Ad/PPSA Complex

(121) Ad-mediated gene transfer is dependent on the CAR expression level on a target cell membrane. However, malignant tumors often down-regulate CAR expression, resulting in poor Ad tumor infectivity (49, 50). Therefore, it is necessary to develop a CAR pathway-independent delivery method in order to ensure the delivery of an effective gene therapeutic agent.

(122) To evaluate the ability of Ad/PPSA to bypass CAR-mediated transfer, Ad/PPSA was transduced into CAR(+) A549 cells and CAR() MCF7 cells, and 25K PEI and Ad/ABP complex were used as controls. The inventors have confirmed in a previous research that Ad/ABP complex enters into cells through a CAR-independent cell transfer pathway, has tolerance to Ad infection, and promotes the gene transfer even in cells with low CAR expression (32).

(123) As shown in FIGS. 8A and 8B, the transduction efficiency of Ad/PPSA was considerably increased in all of the A549 and MCF7 cells, compared to the naked Ad. This shows that Ad/PPSA may be effectively CAR expression-independently transduced into cancer cells. Importantly, the effect of the PPSA complex was shown in CAR() MCF7 cells, and the transduction efficiency was increased 107 times (410.sup.5 PPSA:Ad molar ratio) and 110 times (110.sup.6 PPSA:Ad molar ratio), compared to the naked Ad (P<0.001). More importantly, at the 410.sup.5 polymer:Ad molar ratio, GFP expression in the A549 and MCF7 cells treated with Ad/ABP was increased two-fold higher than that in the Ad/PPSA-treated cells (P<0.001). This shows the superiority of Ad/PPSA in terms of transduction efficiency. Meanwhile, the GFP expression in the Ad/25K PEI-treated cells was lower than those treated with naked Ad, which may be caused by significant cytotoxicity of 25K PEI.

(124) Also, to further confirm CAR-independent cell introduction of Ad/PPSA, competition assays were performed using Ad5 knob protein binding to CAR.

(125) As shown in FIGS. 9A and 9B, when the A549 cells were pretreated with a knob protein, naked Ad-treated cells had significantly decreased GFP expression in a dose-dependent manner, such as decreasing by 56.1% (2 mg/ml knob protein treatment) and 81.1% (10 mg/ml knob protein treatment). Meanwhile, GFP expression of Ad/ABP was decreased by 27.2% (2 mg/ml knob protein treatment) and 53.8% (10 mg/ml knob protein treatment), and GFP expression of Ad/PPSA was decreased by 12.2% (2 mg/ml knob protein treatment) and 23.3% (10 mg/ml knob protein treatment). These results show that the introduction of Ad/ABP and Ad/PPSA into cells was mainly mediated by CAR-dependent cellular uptake, and had a therapeutic value for treating malignant cancer cells in a clinical aspect.

(126) Cellular uptake efficiency of the Ad/PPSA complex was compared to the naked Ad or mPEG-PEI-S-S-PEI-mPEG (PPS)-coated Ad using FITC fluorescence labeling.

(127) As shown in FIGS. 10A, 10B, 10C and 10D, the Ad/PPS or Ad/PPSA complex was considerably improved in cellular uptake efficiency, compared to the naked Ad (P<0.001). Importantly, the cellular uptake efficiency was significantly increased when the cells were treated with Ad/PPSA, compared to when treated with Ad/PPS (P<0.05). This demonstrates that arginine grafting is able to increase the cellular uptake efficiency.

(128) 5. Enhanced Transduction Efficiency of Ad/PSPA

(129) To examine the transduction efficiency of the Ad/PSPA nanocomplex in vitro, GFP expression levels of the Ad/PAPS complex in the MCF7 cell lines, CT-26 cell lines and A549 cell lines having low CAR expression levels were analyzed.

(130) The transduction efficiency of the Ad/PSPA complex was compared to that of the PEI 25 kDa and that of the Ad/PPSA complex which is the other aspect of the present invention. At pH 7.4, 500 MOI (VIRUS OD titer 210.sup.10 VP) of naked Ad, Ad/PSPA nanocomplex, Ad/PPSA nanocomplex and Ad/PEI 25 kDa nanocomplex were transduced into each of the A549, MCF7 and CT-26 cells in 110.sup.3, 510.sup.3, 110.sup.4, 510.sup.4, 110.sup.5, 510.sup.5, and 110.sup.6 polymer:Ad molar ratios for 48 hours. To visualize the transduction efficiency of each treated vector, green fluorescence images of the cells were analyzed by fluorescence microscopy.

(131) As shown in FIGS. 11B, 11C, 12B and 12C, in the MCF7 and CT-26 cells, GFP expression was not observed when treated with the naked Ad but considerably high GFP expression was observed when treated with the polymer-coated Ad nanocomplex. When treated with the Ad/PPSA complex, the cells exhibited no GFP expression at low concentrations but exhibited dose-dependently increase of GFP expression. When treated with the Ad/PEI 25 kDa complex, the cells exhibited high GFP expression at low concentrations but exhibited decreased GFP expression with increasing capacity. This may be caused by the cytotoxicity of the Ad/PEI 25 kDa complex at high concentration. When treated with the Ad/PAPS complex, the cells exhibited high GFP expression in the 110.sup.5 polymer:Ad molar ratio and decreased GFP expression with increasing polymer:Ad molar ratio. These results show that the Ad/PAPS complex is able to considerably increase the transduction efficiency even for a polymer Ad molar ratio that is lower than the Ad/PPSA in CAR() cells.

(132) Also, as shown in FIGS. 11A and 12A, the transduction efficiency of Ad/PAPS was also considerably increased in the A549 cells, compared to the naked Ad. This shows that the Ad/PAPS is also CAR expression-independently and effectively transduced into cancer cells.

(133) 6. Cancer Cell Killing Effect of Virus/Polymer Complex

(134) (1) Anticancer Effect of Virus/PPSA Complex Containing Therapeutic Gene

(135) To further evaluate the potential therapeutic value of PPSA, oncolytic Ad (DWP418) was formed in a complex with PPSA. DWP418 replication is controlled by a modified TERT promoter and contains relaxin as a therapeutic gene. In the previous research, the inventors have confirmed that DWP418 only replicates in cells with high telomerase activity, which is a common feature of the cancer cells, and relaxin expression increases viral spread throughout tumor tissue by reducing extracellular matrix components (36).

(136) As shown in FIGS. 13A and 13B, naked DWP418 induced cell lysis in CAR(+) A549 cells, but not in CAR() MCF7 cells, which means that naked DWP418 is dependent on CAR expression in cell introduction. In contrast, when DWP418 was coated with ABP or PPSA at a 110.sup.6 polymer:Ad molar ratio, cell killing effect was considerably increased by 34% and 80%, respectively, in MCF7 cells (P<0.001). Likewise, increase in cell killing effect of DWP418/ABP (18% increase) and DWP418/PPSA (40% increase), compared to naked DWP418, in the CAR(+) A549 cells were observed (P<0.001). These results are consistent with enhanced gene transfer efficiency of the nanocomplex, compared to the naked Ad (FIGS. 8A and 8B), and show that the therapeutic effect of the oncolytic Ad may be considerably improved by coating the viral surface with PPSA.

(137) (2) Anticancer Efficacy of Virus/Polymer Complex

(138) A surface of RdB/IL-12/decorin, which is oncolytic Ad (oAd), was coated with either a PPSA polymer or a PAPS polymer to construct a complex. The complex was constructed at a polymer:virus molar ratio of 110.sup.5.

(139) As shown in FIGS. 14A, 14B and 14C, when CAR(+) A549 cells were treated with 1, 2, and 5 MOI of test groups, respectively, 41%, 61%, and 69% enhanced cell killing effects were exhibited with respective MOI (P<0.001). Also, it was confirmed that, when CAR () MCF7 and CT-26 cells were treated with the test groups with volumes of 50, 100 and 200 MOI and 100, 500 and 1000 MOI, respectively, and MCF7 cells were treated with 200 MOI each of the test groups, the cell killing effects of oAd/PAPS and oAd/PPSA were increased by 55% and 29%, respectively, compared to naked oAd. It was confirmed that, when the CT-26 cells were treated with 1000 MOI each of the test groups, the cell killing effects of oAd/PAPS and oAd/PPSA were increased by 63% and 45%, respectively, compared to naked oAd. These results are consistent with the enhanced gene transfer efficiency of the Ad/polymer complex, compared to naked Ad, and showed that the therapeutic effect of the oncolytic Ad was considerably increased by coating the Ad surface with PPSA or PAPS.

(140) 7. Confirmation of Increased Gene Expression Efficiency of Ad/PAPS Complex

(141) When CT-26 cell lines were infected by oAd/PAPS complex in which the surface of a virus expressing decorin and IL-12 is coated with PAPS, DCN protein was generated in the cells, and IL-12 cytokine was generated to be secreted to a cell culture. Therefore, in order to confirm increased gene expression efficiency when the complex of the present invention was used, 48 hours after CT-26 cells were treated with 100, 200 or 500 MOI each of naked oAd and oAd/PAPS, both the cell culture and the cells were harvested to perform sodium-dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE). All of infected tumor cells and medium were harvested and subjected to western blotting using a decorin-detectable antibody.

(142) Subsequently, to confirm IL-12 expression, enzyme-linked immunosorbent assay (ELISA) was performed. 48 hours after the CT-26 cell lines were treated with 100 or 200 MOI of naked oAd and oAd/PAPS, the medium was retrieved from the cells, and IL-12 expression levels were quantified by ELISA.

(143) As shown in FIGS. 15A and 15B, an amount of decorin that is enough to be detected was observed from a 500 MOI of oAd/PAPS-treated cell lysate. However, from the cell lysate treated with naked oAd, decorin expression could not be detected. This is because it is impossible to introduce naked oAd into CAR () CT-26 cell lines. Therefore, it was confirmed that oAd/PAPS can also be introduced into CAR () CT-26 cell lines, and decorin is generated in the cells.

(144) Also, it was confirmed that IL-12 expression was not observed when the cells were treated with naked oAd used in the test, but an increased IL-12 expression level was observed as MOI increased, when the cells were treated with oAd/PAPS. This means that the generation of a therapeutic substance can be induced through the expression of a therapeutic gene by CAR-independent introduction of the oAd/PAPS complex into the cells.

(145) 8. Potential Anticancer Efficacy of Ad/PPSA

(146) To validate the therapeutic anticancer efficacy of DWP418/PPSA, MCF7 tumors xenografted onto nude mice were injected every other day for 5 days (total three injections) with PBS, ABP, PPSA, DWP418, DWP418/ABP or DWP418/PPSA.

(147) As shown in FIGS. 16A and 16B, the injection of DWP418/ABP or DWP418/PPSA into tumors significantly reduced tumor growth, compared to naked DWP418. This result shows that the oncolytic anticancer activity of cationic polymer-coated DWP418 was enhanced (P<0.01). Volumes of the MCF7 xenograft tumors treated with PBS, ABP, PPSA, DWP418, DWP418/ABP, or DWP418/PPSA were 152030, 132547, 129791, 108442, 80242, and 48379 mm.sup.3, respectively, at 18 days after treatment (FIG. 16A). The tumor volumes of the mice treated with DWP418, DWP418/ABP or DWP418/PPSA were reduced by 28.7%, 47.2% and 68.2%, respectively, when compared to the PBS-treated control. 19 days after treatment, DWP418/ABP or DWP418/PPSA treatment resulted in 1.3-fold or 2.24-fold decrease in tumor volumes, compared to naked DWP418 (P<0.01). This result demonstrates excellent anticancer efficacy and an improved therapeutic effect of DWP418/PPSA, compared to DWP418/ABP (P<0.01).

(148) For histological and immunohistochemical analysis, MCF7 tumors treated with PBS, ABP, PPSA, DWP418, DWP418/ABP or DWP418/PPSA were harvested three days after the final injection. Tissue sections were then subjected to staining with Ad E1A-specific antibody, PCNA, and TUNEL as well as standard H & E staining (FIG. 16B). DWP418/PPSA-treated tumor tissue showed extensive necrosis and a larger Ad spread compared to DWP418 or DWP418/ABP-treated tumors. Dark staining of Ad E1A in tumor tissue indicated active replication of oncolytic Ad in infected cancer cells according to PPSA release. Also, proliferating cell nuclear antigen (PCNA) expression in DWP418/PPSA-treated tumor tissue was remarkably reduced compared to naked DWP418 or DWP418/ABP-treated tumor tissue. This result demonstrated that DWP418/PPSA is more effective in inhibiting tumor cell proliferation. Likewise, in the DWP418/PPSA-treated group, TUNEL-positive apoptotic cells are abundant in the region such as E1A-positive cells. Taken together, this result demonstrates that the oncolytic Ad/PPSA complex had enhanced infection ability and increased anticancer efficacy, compared to the naked oncolytic Ad and oncolytic Ad/ABP complex.

(149) 9. Innate and Adaptive Immune Response Against Ad

(150) Intravenous Ad injection may activate an innate immune response, which limits the therapeutic efficiency of Ad. To evaluate whether DWP418/PPSA is able to evade the innate immune response, 6 hours after treatment, proinflammatory cytokine IL-6 secretion from mice was measured.

(151) Naked DWP418 induced an increase in IL-6 serum level by 4.87-fold over the base level in Balb/C mice (P<0.01)(FIG. 17A). In remarkable contrast, DWP418/ABP and DWP418/PPSA treatment showed IL-6 serum levels that are almost the same as PBS-treated mice. This result indicates that Ad surface coating with both ABP and PPSA may reduce the innate immune response against Ad.

(152) Also, the potential efficacy of DWP418/PPSA to evade the adaptive immune response against Ad was evaluated. Ad-specific neutralizing antibody-containing serum obtained from a mouse treated with naked Ad (dEl/GFP) reduced the transduction efficiency of naked dEl/GFP by 94.8% (FIGS. 17B and 17C). In contrast, the transduction efficiency of the Ad/PPSA complex was not reduced. This result demonstrates that the PPSA complex can evade preexisting neutralizing antibodies and further shows that the Ad/PPSA nanocomplex can be used in systemic multidose treatment.

(153) 10. In Vivo Hepatotoxicity of Intravenously-Injected Ad/PPSA

(154) To evaluate Ad treatment-related hepatotoxicity, serum ALT and AST levels were measured after intravenous injection of naked DWP418, DWP418/ABP, or DWP418/PPSA.

(155) As shown in FIGS. 18A and 18B, the naked DWP418-treated mice showed a significantly higher transaminase serum level than the PBS-treated control three days after injection (P<0.05). In contrast, no significant increases in ALT and AST levels were observed from the DWP418/PPSA-treated mice. The serum ALT and AST levels were a little decreased in the DWP418/ABP-treated mice, but considerably increased in the PBS-treated mice. These results show that Ad PEGylation induced a decrease in Ad-related hepatotoxicity. The lower hepatotoxicity level in the DWP418/PPSA-treated mice compared to that in the DWP418/ABP-treated mice may be caused by the PEGylated PEI on PPSA.

(156) Above, specific parts of the present invention have been described in detail. It is apparent to those of ordinary skill in the art that such specific descriptions are merely specific embodiments, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention is to be defined by the accompanying claims and equivalents thereof.

REFERENCES

(157) (1) El-Aneed, A. J. Controlled Release 2004, 94, 114. (2) Meng, F.; Hennink, W. E.; Zhong, Z. Biomaterials 2009, 30, 2180-2198. (3) Samal, S. K.; Dash, M.; Van Vlierberghe, S.; Kaplan, D. L.; Chiellini, E.; van Blitterswijk, C.; Moroni, L.; Dubruel, P. Chem. Soc. Rev. 2012, 41, 7147-7194. (4) Park, T. G.; Jeong, J. H.; Kim, S. W. Adv. Drug Delivery Rev. 2006, 58, 467-486. (5) Coughlan, L.; Alba, R.; Parker, A. L.; Bradshaw, A. C.; McNeish, I. A.; Nicklin, S. A.; Baker, A. H. Viruses 2010, 2, 2290-2355. (6) Singh, R.; Kostarelos, K. Trend. Biotechnol. 2009, 27, 220-229. (7) Kasala, D.; Choi, J. W.; Kim, S. W.; Yun, C. O. Expert Opin. Drug Delivery 2014, 11, 379-392. (8) Roelvink, P. W.; Lizonova, A.; Lee, J. G.; Li, Y.; Bergelson, J. M.; Finberg, R. W.; Brough, D. E.; Kovesdi, I.; Wickham, T. J. J. Virol. 1998, 72, 7909-7915. (9) Park, J.; Kim, W. J. J. Drug Target. 2012, 20, 6486466. (10) Neu, M.; Fischer, D.; Kissel, T. J. Gene Med. 2005, 7, 992-1009. (11) Shen, J.; Zhao, D. J.; Li, W.; Hu, Q. L.; Wang, Q. W.; Xu, F. J.; Tang, G. P. Biomaterials 2013, 34, 45204531. (12) Coue, G.; Engbersen, J. F. J. Controlled Release 2010, 148, e911. (13) Esfand, R.; Tomalia, D. A. Drug Discovery Today 2001, 6, 427-436. (14) Arote, R. B.; Jiang, H. L.; Kim, Y. K.; Cho, M. H.; Choi, Y. J.; Cho, C. S. Expert Opin. Drug Delivery 2011, 8, 1237-1246. (15) Green, J. J.; Langer, R.; Anderson, D. G. Acc. Chem. Res. 2008, 41, 749-759. (16) Lin, C.; Blaauboer, C. J.; Timoneda, M. M.; Lok, M. C.; van Steenbergen, M.; Hennink, W. E.; Zhong, Z.; Feijen, J.; Engbersen, J. F. J. Controlled Release 2008, 126, 166-174. (17) Green, J. J.; Zugates, G. T.; Langer, R.; Anderson, D. G. Method. Mol. Biol. 2009, 480, 53-63. (18) Fasbender, A.; Zabner, J.; Chillon, M.; Moninger, T. O.; Puga, A. P.; Davidson, B. L.; Welsh, M. J. J. Biol. Chem. 1997, 272, 6479-6489. (19) Mok, H.; Bae, K. H.; Ahn, C. H.; Park, T. G. Langmuir 2009, 25, 1645-1650. (20) Mok, H.; Park, J. W.; Park, T. G. Bioconjugate Chem. 2008, 19, 797-801. (21) Bolhassani, A. Biochim. Biophys. Acta 2011, 1816, 232-246. (22) Torchilin, V. P. Adv. Drug Delivery Rev. 2008, 60, 548-558. (23) Levchenko, T. S.; Rammohan, R.; Volodina, N.; Torchilin, V. P. Method. Enzymol. 2003, 372, 33949. (24) Nam, H. Y.; Kim, J.; Kim, S.; Yockman, J. W.; Kim, S. W.; Bull, D. A. Biomaterials 2011, 32, 5213-5222. (25) Koren, E.; Torchilin, V. P. Trend. Mol. Med. 2012, 18, 385-393. (26) Zorko, M.; Langel, U. Adv. Drug Delivery Rev. 2005, 57, 529-545. (27) Morris, V. B.; Sharma, C. P. J. Colloid Interface Sci. 2010, 348, 360-368. (28) Kim, T. I.; Ou, M.; Lee, M.; Kim, S. W. Biomaterials 2009, 30, 658-664. (29) Nam, H. Y.; Hahn, H. J.; Nam, K.; Choi, W. H.; Jeong, Y.; Kim, D. E.; Park, J. S. Int. J. Pharm. 2008, 363, 199-205. (30) Kim, W. J.; Christensen, L. V.; Jo, S.; Yockman, J. W.; Jeong, J. H.; Kim, Y. H.; Kim, S. W. Mol. Ther. 2006, 14, 343-350. (31) Won, Y. W.; Yoon, S. M.; Lee, K. M.; Kim, Y. H. Mol. Ther. 2011, 19, 372380. (32) Kim, P. H.; Kim, T. I.; Yockman, J. W.; Kim, S. W.; Yun, C. O. Biomaterials 2010, 31, 1865-1874. (33) Zhan, C.; Qian, J.; Feng, L.; Zhong, G.; Zhu, J.; Lu, W. J. Drug Target. 2011, 19, 573581. (34) Eyer, P.; Worek, F.; Kiderlen, D.; Sinko, G.; Stuglin, A.; Simeon-Rudolf, V.; Reiner, E. Anal. Biochem. 2003, 312, 224-227. (35) Kim, E.; Kim, J. H.; Shin, H. Y.; Lee, H.; Yang, J. M.; Kim, J.; Sohn, J. H.; Kim, H.; Yun, C. O. Hum. Gene Ther. 2003, 14, 1415-1428. (36) Kim, J. H.; Lee, Y. S.; Kim, H.; Huang, J. H.; Yoon, A. R.; Yun, C. O. J. Natl. Cancer Inst. 2006, 98, 1482-1493. (37) Choi, J. W.; Kang, E.; Kwon, O. J.; Yun, T. J.; Park, H. K.; Kim, P. H.; Kim, S. W.; Kim, J. H.; Yun, C. O. Gene Ther. 2013, 20, 880-892. (38) Kim, P. H.; Kim, J.; Kim, T. I.; Nam, H. Y.; Yockman, J. W.; Kim, M.; Kim, S. W.; Yun, C. O. Biomaterials 2011, 32, 9328-9342. (39) Kim, P. H.; Sohn, J. H.; Choi, J. W.; Jung, Y.; Kim, S. W.; Haam, S.; Yun, C. O. Biomaterials 2011, 32, 2314-2326. (40) Kim, J.; Nam, H. Y.; Kim, T. I.; Kim, P. H.; Ryu, J.; Yun, C. O.; Kim, S. W. Biomaterials 2011, 32, 5158-5166. (41) Yun, C. O.; Cho, E. A.; Song, J. J.; Kang, D. B.; Kim, E.; Sohn, J. H.; Kim, J. H. Hum. Gene Ther. 2003, 14, 1643-1652. (42) Schaffert, D.; Wagner, E. G Gene Ther. 2008, 15, 11311138. (43) Kim, W. J.; Yockman, J. W.; Lee, M.; Jeong, J. H.; Kim, Y. H.; Kim, S. W. J. Controlled Release 2005, 106, 224-234. (44) Han, J.; Zhao, D.; Zhong, Z.; Zhang, Z.; Gong, T.; Sun, X. Nanotechnology 2010, 21, 105-106. (45) Futaki, S.; Suzuki, T.; Ohashi, W.; Yagami, T.; Tanaka, S.; Ueda, K.; Sugiura, Y. J. Biol. Chem. 2001, 276, 5836-5840. (46) Huang, F. W.; Wang, H. Y.; Li, C.; Wang, H. F.; Sun, Y. X.; Feng, J.; Zhang, X. Z.; Zhuo, R. X. Acta Biomater. 2010, 6, 4285-4295. (47) Wiethoff, C. M.; Wodrich, H.; Gerace, L.; Nemerow, G. R. J. Virol. 2005, 79, 992-2000. (48) Meier, O.; Greber, U. F. J. Gene Med. 2003, 5, 451462. (49) Matsumoto, K.; Shariat, S. F.; Ayala, G. E.; Rauen, K. A.; Lerner, S. P. Urology 2005, 66, 441-446 (50) Sachs, M. D.; Rauen, K. A.; Ramamurthy, M.; Dodson, J. L.; De Marzo, A. M.; Putzi, M. J.; Schoenberg, M. P.; Rodriguez, R. Urology 2002, 60, 531-536. (51) Lee, C. H.; Kasala, D.; Na, Y.; Lee, M. S.; Kim, S. W.; Jeong, J. H.; Yun, C. O. Biomaterials 2014, 35, 5505-5516.