Dual tumor targeting with pH-sensitive and bioreducible polymer-complexed oncolytic adenovirus

Biomaterials 41 (2015) 53e68

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Dual tumor targeting with pH-sensitive and bioreducible polymer-complexed oncolytic adenovirus Chang Yoon Moon a, 1, Joung-Woo Choi a, 1, Dayananda Kasala a, 1, Soo-Jung Jung a, Sung Wan Kim a, b, Chae-Ok Yun a, * a b

Department of Bioengineering, College of Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, Republic of Korea Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 August 2014 Accepted 8 November 2014 Available online 5 December 2014

Oncolytic adenoviruses (Ads) have shown great promise in cancer gene therapy but their efficacy has been compromised by potent immunological, biochemical, and specific tumor-targeting limitations. To take full advantage of the innate cancer-specific killing potency of oncolytic Ads but also exploit the subtleties of the tumor microenvironment, we have generated a pH-sensitive and bio-reducible polymer (PPCBA)-coated oncolytic Ad. Ad-PPCBA complexes showed higher cellular uptake at pH 6.0 than pH 7.4 in both high and low coxsackie and adenovirus receptor-(CAR)-expressing cells, thereby demonstrating Ad-PPCBA's ability to target the low pH hypoxic tumor microenvironment and overcome CAR dependence for target cell uptake. Endocytic mechanism studies indicated that Ad-PPCBA internalization is mediated by macropinocytosis instead of the CAR-dependent endocytic pathway that internalizes naked Ad. VEGF-specific shRNA-expressing oncolytic Ad complexed with PPCBA (RdB/shVEGF-PPCBA) elicited much more potent suppression of U87 human brain cancer cell VEGF gene expression in vitro, and human breast cancer MCF7 cell/Matrigel plug vascularization in a mouse model, when cancer cells had been previously infected at pH 6.0 versus pH 7.4. Moreover, intratumorally and intravenously injected RdB/shVEGF-PPCBA nanocomplexes elicited significantly higher therapeutic efficacy than naked virus in U87-tumor mouse xenograft models, reducing IL-6, ALT, and AST serum levels. These data demonstrated PPCBA's biocompatibility and capability to shield the Ad surface to prevent innate immune response against Ad after both intratumoral and systemic administration. Taken together, these results demonstrate that smart, tumor-specific, oncolytic Ad-PPCBA complexes can be exploited to treat both primary and metastatic tumors. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Cancer gene therapy Oncolytic adenovirus pH-sensitive Bioreducible polymer Systemic administration Tumor microenvironment

1. Introduction In the last two decades, oncolytic adenoviral (Ad) approaches have evolved as promising therapeutic strategies against cancer because of their numerous biochemical advantages [1e4]. A primary advantage is that Ads are cancer-specific. Innate defense mechanisms against viral replication inside normal cells include endogenous tumor suppression proteins such as p53, pRb, and p14ARF. These proteins are defunct in certain cancer cells, establishing the selectivity of Ad against these tumors [5]. Not only do Ads destroy their host cells directly at the end of their lytic * Corresponding author. Department of Bioengineering, College of Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, Republic of Korea. Tel.: þ82 2 2220 0491; fax: þ82 2 2220 4850. E-mail address: [email protected] (C.-O. Yun). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.biomaterials.2014.11.021 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

cycles, but they also facilitate the spread of their viral progeny throughout the tumor through cell lysis, leading to further infection and subsequent eradication of neighboring cancerous cells [6,7]. In addition to their oncolytic properties, Ads are among the most potent viral vectors in gene therapeutics. Ads have high gene transduction efficiency in both dividing and non-dividing cells, an effective nuclear entry mechanism, and the capacity to be concentrated at high titers [8]. As a result, several clinical trials have reported success using local Ad administration for cancer gene therapy [9e11]. Despite their favorable characteristics, Ad-based treatments face numerous challenges in realizing maximum therapeutic potential with systemic administration. When delivered systemically, Ad is rapidly cleared from the bloodstream through three major immunological mechanisms: 1) innate immune response induced by macrophages and dendritic cells, 2) adaptive immune response by

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neutralizing anti-Ad antibody, and 3) recruitment of viral particles by Kupffer cells in the liver, causing hepatocyte infection and acute liver toxicity. Moreover, Ad interacts with platelets and erythrocytes, potentially causing further side effects and toxicity [12]. In addition to immunological barriers, Ad's transduction efficiency is severely limited by the level of coxsackie and adenovirus receptor (CAR) expression in tumor cells. The CAR is a 46 kDa transmembrane protein that specifically interacts with Ad surface proteins and is the primary mechanism through which Ad enters target cells. Secondary interactions in Ad infection involve virus binding via Arg-Gly-Asp (RGD) motifs on their capsid penton bases with target cell surface receptors, especially avb3 and avb5 integrins. Viral binding lead to clathrin-coated pit formation on the target cell surface, which facilitates virus internalization inside an endosome [13]. Due to Ad dependency on the CAR for infecting cells, the therapeutic efficacy of Ad intratumoral and systemic injections are compromised in cancer types whose cells express low levels of CAR. One strategy that has been devised to overcome those biochemical and immunological barriers inherent to using the Ad vector for anti-cancer gene therapy is to develop “smart” Ad nanocomplexes by modifying the viral surface with non-viral systems [14e17]. These non-viral additions advantageously compensate for native Ad vector limitations by conferring the virus with increased tumor-specificity, reduced immunogenicity, effective reproducibility, and a simple quality control process [3]. Several Ad nanocomplexes have been developed to maximize the targeting efficiency while minimizing harmful side effects [18,19]. Polyethylene glycol (PEG) has been one of the prominent polymers to be conjugated with Ad to reduce liver uptake and extend its half-life in the circulation. Further, PEGylation of Ad has demonstrated not only to reduce innate immune response against Ad but also to prevent host antibodies from neutralizing Ad [20]. Ad encapsulation with a cationic polymer is another effective strategy to overcome CAR dependence and immunological barriers. Because cell plasma membranes and Ad are both negatively charged, the positively charged polymer is ionically attracted to the virus and the net positive Ad surface charge facilitates viral entry into host cells via electrostatic interaction. Poly(ethylenimine) [21] and poly(L-lysine) [22] have been the two cationic polymers frequently used to improve Ad transduction in gene therapy approaches. However, these polymer-coated Ads remain inefficient in terms of tumor selectivity because their positive charge also increases their nonspecific uptake into normal cells. In addition, these cationic polymers are nondegradable in vivo and induce severe cytotoxicity due to the strong adsorption of positively charged polymer with negatively charged cell membranes [23]. Hypoxia is a pathological characteristic of solid tumor local environments, and decreased oxygen availability negatively impacts cancer gene therapy and also chemotherapy. In hypoxic tissues such as solid tumors, anaerobic glycolysis is upregulated to compensate for reduced ATP production by oxidative phosphorylation. Lactic acid accumulates in and acidifies the local extracellular environment [24]. Thus, a “smart” Ad nanocomplex that targets specific aspects of the tumor microenvironment such as the local tissue pH can enhance vector tumor-selectivity and safety. In this study, we designed and synthesized a pH-sensitive polymer with bioreducible disulfide bond (mPEG-piperazine-N, N0 -cystaminebisacrylamide; PPCBA), in which piperazine and N, N0 -cystaminebisacrylamide acts as a pH-sensitive and bioreducible moiety, respectively. We then investigated the in vitro transduction efficacy and cellular uptake mechanism of Ad complexed with PPCBA and the in vivo therapeutic efficacy of oncolytic Ad encapsulated by PPCBA.

2. Materials and methods 2.1. Cell culture and Ad generation All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (GIBCO-BRL) at 37  C in a humidified atmosphere containing 5% CO2. A human embryonic kidney cell line (HEK 293) expressing the Ad E1 region, human brain cancer cell lines (U343, U87), and human breast cancer cell line (MCF7) were purchased from the American Type Culture Collection (Manassas, VA). Green fluorescence protein (GFP)-expressing replication-incompetent Ad (dE1/GFP) and VEGFtargeted replication-competent Ad mutated in E1A and deleted in E1B regions (RdB/shVEGF) were generated in HEK 293 cells and purified by the CsCl gradient centrifugation [25,26]. Viral particle (VP) numbers were calibrated to optical density measured at 260 nm (OD260) and 1 absorbance unit equaled 1012 VP/mL. Purified viruses were stored at 80  C until use. 2.2. Synthesis of pH-sensitive and bioreducible polymer (PPCBA) To generate the pH-sensitive and bioreducible polymer, mPEG-acrylate (Mn ¼ 2.0 kDa, 0.10 mmol) (SigmaeAldrich, St. Louis, MO) and N, N0 -cystaminebisacrylamide (1.0 mmol) (PolySciences Inc., Warrington, PA) were dissolved in methanol (5.0 mL). Subsequently piperazine (1.0 mmol) (SigmaeAldrich) was added, and the reaction mixture was stirred at 50  C for 48 h in the dark under a 100% nitrogen atmosphere, cooled to room temperature, and the polymer (PPCBA) was precipitated in excess diethyl ether for two times and dried under vacuum at room temperature for 2 days. Finally, PPCBA was characterized by 1H NMR in CDCl3 (Mercury Plus 300 MHz; Varian Inc., Palo Alto, CA). Further, gel permeation chromatography analysis (GPC) showed average molecular weight Mw ¼ 2.85 kDa, PDI ¼ 1.87. GPC measurements were performed using a Waters-GPC, Water 515 pump, Water 410 RI, and thermostated (35  C), TSK gel G3000 þ G5000 columns, 0.3 M NaAc aqueous solution (pH 4.4) plus methanol (70/30 v/v) was used as eluent at a flow rate of 1.0 mL/min. The average molecular weights were calculated against low polydispersity PEG standards. In addition, the matrix assisted laser desorption/ ionization-time of flight mass spectrum (MALDI-TOF) was performed. It showed that the molecular weight of PPCBA polymer was 4.04 kDa. 2.3. Characterization of naked Ad and PPCBA-complexed Ad The average size and zeta potential of dE1/GFP-PPCBA complex were measured with a Zetasizer 3000HS (Malvern Instruments Inc., Worcestershire, UK) with a HeeNe laser beam (633 nm, fixed scattering angle of 90 ) at 25  C. To characterize the electrostatic interaction of dE1/GFP with PPCBA polymer, various concentrations of PPCBA polymer (5e50 mg/mL) were gently pipette-mixed with dE1/GFP Ad (2  1010 VP) and incubated at room temperature for 30 min. After the formation of dE1/GFP-PPCBA complex, phosphate-buffered saline (PBS, pH 7.4) was added to final volume of 1.0 mL before analysis. The sizes and zeta potential values were presented as average values of three measurements. 2.4. Gel-retardation assay To assess the PPCBA-encapsulation of Ad, various concentrations (5e50 mg/mL) of PPCBA-coated dE1/GFP were electrophoresed (80V, 30 min) in 0.8% (w/v) agarose gel in 1 TAE buffer (10.0 mM Tris/HCl, 1% (v/v) acetic acid, and 1.0 mM EDTA) containing ethidium bromide. Viral DNA was visualized using a ChemiDoc gel documentation system (Syngene, Cambridge, UK). 2.5. Transmission electron microscopy (TEM) imaging The morphology of naked dE1/GFP and dE1/GFP-PPCBA complexes were characterized by TEM (JEM-2000EXll, JEPL; Nikon, Tokyo, Japan) at 200 kV after incubating for 30 min at different pH conditions (7.4 and 6.0). 2.6. Transduction efficiency assay Transduction efficiency of PPCBA-complexed Ad was assessed by quantifying GFP expression by fluorescence-activated cell sorting (FACS) analysis in CAR (þ) (U343) and CAR () (MCF7) cells. Each cell line was seeded at a density of 1  105 cells/well in 12-well plates for 24h before Ad transduction. Cells were transduced with naked dE1/GFP or dE1/GFP-PPCBA complexes at 20 (U343) or 200 (MCF7) MOI, in both pH 7.4 and pH 6.0, for 30 min. After 48 h of incubation at 37  C, cells were observed by fluorescence microscopy (Olympus IX81; Olympus Optical, Tokyo, Japan). Cells were also assessed with a BD FACScan analyzer (BectoneDickinson, San Jose, CA) using CellQuest software (BectoneDickinson); data from 10,000 events were collected for further analysis and represented the values of relative fluorescence intensities. Data represent the means and standard deviations of three measurements. 2.7. Competition assay with adenoviral fiber protein U343 cells were plated at 1  105 cells/well in 12-well plates. After 24 h, cells were incubated in serum-free DMEM with Ad fiber protein (0.2 and 2 mg/mL) or PBS (vehicle) at 4  C for 1 h. Naked dE1/GFP or dE1/GFP-PPCBA complexes (50 mg/mL

C.Y. Moon et al. / Biomaterials 41 (2015) 53e68 PPCBA) were added to the cells at an MOI of 50 (dE1/GFP) or 20 (dE1/GFP-PPCBA) and incubated at 37  C for 1 h cells were then washed with ice-cold PBS and replaced with DMEM/5% FBS. After incubating for 48 h, cells were observed by fluorescence microscopy, and GFP expression was quantified by flow cytometry and analyzed with CellQuest software. 2.8. Cellular uptake mechanism/endosomal escape of the Ad-PPCBA complexes

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2.13. Measurement of inflammatory cytokines and toxicity studies The inflammatory immune response and toxicity were examined after intravenous injection of PBS, PPCBA (50 mg/mL), dE1/GFP (2  1010 VP), or dE1/GFPPPCBA (50 mg/mL PPCBA; 2  1010 VP) into BALB/c mice (8 weeks, Charles River Korea Inc., Seoul, Korea). At 6 h post-injection, serum was harvested by retroorbital bleeding and interleukin 6 (IL-6) levels were quantified using an IL-6 ELISA kit (R&D Systems, Minneapolis, MN). At 3 days post-injection with PBS, naked dE1/GFP (2  1010 VP), or dE1/GFP-PPCBA complex (50 mg/mL PPCBA; 1  1010 VP), mice were sacrificed and serum was collected for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, which were determined at Neodin Corporation (Seoul, Korea) as a measure of hepatic toxicity.

Cells were pre-treated with chlorpromazine (CPZ; clathrin-mediated endocytosis inhibitor; 0.2 and 1 mM), genistein (caveolin-mediated endocytosis inhibitor; 1.25 and 5 mM), NH4Cl (macropinocytosis inhibitor; 0.5 and 1 mM), or bafilomycin A1 (Bf-A) endosomal escape inhibitor; 5 and 20 mM) at pH 6.0 and 7.4 for 30 min. Naked dE1/GFP (50 MOI for U343 and 500 MOI for MCF7) or dE1/GFP-PPCBA complexes (20 MOI for U343 and 200 MOI for MCF7) (50 mg/mL PPCBA) were added in the presence or absence of the inhibitors for an additional 2 h. Then, cells were washed with PBS and incubated for 48 h in DMEM/5% FBS. Cells were then observed by fluorescence microscopy, and GFP expression was quantified by flow cytometry and analyzed with CellQuest software.

Data are expressed as the mean ± standard deviation (SD). The ManneWhitney test was performed for statistical comparisons (SPSS v.18.0 software; SPSS/IBM, Chicago, IL). Differences were considered statistically significant when P-values were <0.05.

2.9. VEGF quantification

3. Results

Human VEGF-A was quantified using the human VEGF Quantikine Immunoassay kit (R&D Systems, Minneapolis, MN), according to manufacturer recommendations. Serial dilutions of a known concentration of purified recombinant human VEGF-A were used to establish a standard curve. Briefly, U87 cells were plated onto sixwell plates in DMEM/5% FBS. At 50% confluence, cells were infected with PBS, RdB/shVEGF, or RdB/shVEGF-PPCBA (50 mg/mL PPCBA; pH 6.0 or 7.4) at 1 or 10 MOIs. Conditioned media were harvested 48 h after infection, and secreted VEGF was measured by ELISA [26]. 2.10. MTT assay To evaluate the cancer cell-killing effect of RdB/shVEGF or RdB/shVEGF-PPCBA at pH 6.0 or 7.4, U343 and MCF7 cells grown to 50% confluence in 24-well plates were infected with naked RdB/shVEGF or RdB/shVEGF-PPCBA (50 mg/mL PPCBA) at 20 MOI for U343 and 200 MOI for MCF7 cells, and incubated at 37  C. Two days post infection, 250 mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; SigmaeAldrich, St. Louis, MO) at 2 mg/mL in PBS was added to each well and incubated at 37  C for 4 h, then the supernatant was discarded and the precipitate was dissolved in 1.0 mL dimethylsulfoxide (DMSO). Optical density of the dissolved formazan product was then read on a microplate reader at 540 nm. Non-infected cells served as negative controls. 2.11. Ex vivo Matrigel® plug assay MCF7 cells (2  105) were plated in 6-well plates and infected with naked RdB/ shVEGF or RdB/shVEGF-PPCBA (50 mg/mL PPCBA), or PBS (vehicle, negative control) at either pH 6.0 or 7.4. After 6 h, cells were trypsin-harvested and washed three times with 5 mL of Hank's buffered saline. Cells were then mixed with 600 mL of cold Matrigel® (BD-bioscience, Sanjose, and CA) and injected subcutaneously above the flank region of the male athymic nude mice (Charles River Korea Inc., Seoul, Korea). The injected Matrigel® rapidly formed a single solid gel plug. After 14 days, animals were sacrificed and the skin of each mouse was pulled back to expose the intact Matrigel® plug. To quantify blood vessel formation, Matrigel® plugs were fixed with zinc fixation solution, paraffin-embedded in paraffin, and sections were treated with purified monoclonal rat anti-mouse CD31 (platelet/endothelial cell adhesion molecule 1; BD Biosciences Pharmingen, San Diego, CA), and then with goat anti-rat IgG-HRP. All slides were counterstained with Meyer's hematoxylin. The expression levels of CD31 were analyzed semi-quantitatively with MetaMorph® image analysis software (Universal Image Corp., Westchester, PA). 2.12. Assessment of antitumor efficacy To compare the anti-tumor efficacy of naked RdB/shVEGF and RdB/shVEGFPPCBA, U87 tumor xenografts were established subcutaneously by injecting 1  107 cells under the abdominal skin of 6- to 8-week-old female athymic nude mice (Charles River Korea Inc.). Once tumors size reached approximately 100 mm3 in volume, mice were randomized into three groups (PBS, RdB/shVEGF, and RdB/shVEGF-PPCBA) and injected intratumorally with 30 mL PBS or an equivalent volume of PBS containing 5  109 VP of RdB/shVEGF or RdB/shVEGFPPCBA (50 mg/mL PPCBA) three times every other day. To evaluate antitumor efficacy via systemic administration, U87 tumor-bearing mice were injected with 200 mL PBS, RdB/shVEGF (2  1010 VP/200 mL), or RdB/shVEGF-PPCBA (2  1010 VP/200 mL) via tail vein on three occasions spaced every other day. The length (L) and width (W) of the tumor were measured every other day with a caliper to calculate tumor growth. Tumor volume was calculated according to the following formula: tumor volume ¼ 0.523 LW2. All animal studies were conducted “under the auspices and approval” of Hanyang University institutional animal care and use committee.

2.14. Statistical analysis

3.1. Synthesis of PPCBA polymer The synthesis of pH-sensitive and bioreducible polymer (PPCBA) is illustrated in Fig. 1A. The PPCBA polymer was synthesized through Michael-type addition polymerization [27]. The reaction of mPEG-acrylate and N, N0 -cystaminebisacrylamide with piperazine formed mPEG-b-Pip-CBA (PPCBA). The chemical structure of PPCBA was confirmed by 1H NMR (300 MHz, CDCl3) with its typical resonance peaks at 2.3e2.8, and 3.6 ppm which corresponds to eN (CH2eCH2) methylene protons of piperazine, and -S-CH2CH2eNHeCOe protons of CBA, respectively, along with characteristic peaks of mPEG (Fig. 1B). The absence of any proton signals between 5 and 6.5 ppm indicates that no residual of acrylamide or acrylate monomers are present in finally synthesized PPCBA polymer. Further, the average molecular weight was determined by gel permeation chromatography (GPC). The observed average molecular weight was Mw ¼ 2.85 kDa with a PDI of 1.87. Furthermore, the matrix-assisted laser desorption/ionization-time of flight mass (MALDI-TOF) spectrum showed that the molecular weight of PPCBA copolymer was 4.04 kDa (Fig. S1). This increased molecular weight of PPCBA in comparison to mPEG (2 kDa) confirms the formation of PPCBA copolymer. The ratios of eOCH3 of PEG and eSeSeCH2eCH2eNHeCOe of alkyl chain were calculated by integrals of the two peaks, and value was identified as 1:6 (Fig. 1B) [28]. These results suggest that the polymer is close to the expected composition, in a good agreement with MALDI-TOF data. 3.2. Physico-chemical characterization of PPCBA and Ad-PPCBA complexes Potential cytotoxicity of the PPCBA polymer was evaluated by MTT assay. Fig. 2A shows that PPCBA is not cytotoxic to mammalian MCF7 cells, even at high concentrations (i.e., 100 mg/mL). GFPexpressing non-replicating Ad (dE1/GFP) was then complexed with PPCBA polymer by ionic interactions between the carboxylic acid groups of external amino acids in the hexon of Ad capsids and tertiary amino groups of the PPCBA copolymer. The average size and surface charge of the complexes with increasing concentrations of PPCBA were assessed by dynamic light scattering (DLS) and zeta potential analysis (Fig. 2B). The diameter of the naked Ad increased from 104 ± 6.64 nm to 175 ± 5.2 nm when Ad was complexed with 50 mg/mL of PPCBA. The surface charge of the complex also increased from the negative value of 17.53 mV ± 0.40 mV of naked Ad to the positive value of 7.31 ± 0.78 mV after coating Ad with PPCBA at 50 mg/mL. These results indicate that coating of Ad viral particles with cationic PPCBA polymer concentration-dependently increased the particle

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Fig. 1. Schematic representation of PPCBA structure and characterization. (A) Schematic representation of synthesis of pH-sensitive and bioreducible polymer (PPCBA). (B) 1H NMR spectrum of PPCBA polymer in CDCl3 (300 MHz).

diameter and zeta potential value, confirming that Ad adequately complexes with PPCBA through electrostatic interaction. In order to find the optimal PPCBA concentration for Ad complexing, we performed a gel retardation assay (Fig. 2C). Retardation of dE1/GFP-PPCBA complexes began at 25 mg/mL PPCBA and particles were completely immobilized at 50 mg/mL PPCBA. Based on this result, the PPCBA concentration of 50 mg/mL was used to make a complex with Ad for subsequent experiments, unless otherwise stated. The morphology of naked Ad and dE1/GFP-PPCBA complexes was examined by TEM. Fig. 2D shows photomicrographs of naked Ad at pH 7.4 and dE1/GFP-PPCBA complex at PPCBA concentration of 50 mg/mL at both pH 7.4 and 6.0. The naked Ad shows the characteristic hexon structure and icosahedral shape of the viral particle. On the other hand, we observed a tendency for clusters of Ad particles to be enveloped by a “tensile chunk” of PPCBA polymer, leading to generation of Ad-PPCBA complexes. This observation suggests that the cationic PPCBA polymer is encapsulating Ad and linking the negative surface charge of multiple viral particles [19,29,30]. Colloidal stability of dE1/GFP-PPCBA complexes was next evaluated in PBS buffer after incubating at room temperature for 3 h, 9 h, 18 h, and 24 h. As shown in Fig. S2, the average size of dE1/GFPPPCBA (175.1 nm) was not changed for 24 h, suggesting that PPCBA

polymer-coated Ad complex has a good colloidal stability under the physiological saline condition. We also examined the bioreducibility of the dE1/GFP-PPCBA complexes by treating with reducing agent dithiothreitol (DTT) at 37  C, for 2 h. As shown in Fig. S3, the average size of dE1/GFP-PPCBA complexes after treatment with DTT was reduced to 116.1 nm, which is close to that of naked Ad. In contrast, the treatment of naked Ad with DTT did not change the size of the naked Ad. These results clearly demonstrate that CBA block in PPCBA copolymer can be degraded upon exposure to redox microenvironment, subsequently release Ad in cytosol. 3.3. Enhanced and pH-dependent transduction efficiency of AdPPCBA To evaluate the transduction efficiency of dE1/GFP-PPCBA complexes in comparison to naked Ad, we transduced CAR (þ) U343 and CAR () MCF7 cancer cells at pH 7.4 and 6.0 with naked Ad or dE1/GFP-PPCBA complexes at an MOI of 20 (U343) or 200 (MCF7). As shown in Fig. 3A, the transduction efficiency of dE1/ GFP-PPCBA complex escalated by increasing the concentration of PPCBA polymer used to make the Ad complex, at both pH conditions. Importantly, dE1/GFP-PPCBA complex showed higher GFP expression at pH 6.0 than at pH 7.4 in both cell types, demonstrating enhanced transduction at lower pH. However, no pH-

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Fig. 2. Cytotoxicity of PPCBA polymer and physico-chemical properties of Ad-PPCBA complexes. (A) Cell cytotoxicity of the PPCBA polymer in MCF-7 cells was evaluated by MTT assay. (B) The average size (nm) and zeta potential value (mV) of the PPCBA-complexed Ad were measured at various concentrations of PPCBA polymer (5e50 mg/mL) and 2  1010 VP in 1 mL PBS, at room temperature. The data are representatives of three independent experiments performed in triplicate. Data represents the mean ± SD for three replicates. (C) Gel retardation assay of the Ad-PPCBA complex with various concentrations of PPCBA polymer. (D) TEM images of naked Ad and Ad-PPCBA complex. The Ad and Ad-PPCBA were incubated with PBS at pH 7.4 and 6.0 for 30 min.

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Fig. 3. Enhanced and pH-dependent transduction efficiency of Ad-PPCBA. (A) Fluorescence images of U343 and MCF7 cells transduced with naked dE1/GFP or dE1/GFP-PPCBA complex (5, 10, 25, and 50 mg/mL of PPCBA polymer) at pH 7.4 and 6.0. Original magnification: 100. (B) At 48 h post transduction, cells were analyzed for GFP expression by flow cytometry analysis. The data are representatives of three independent experiments performed in triplicate. Bars represent mean ± SD.

dependent differences were observed for naked Ad. This observation was supported by quantitative FACS analysis of GFP, in which PPCBA-complexing enhanced Ad transduction efficiency by 3.6-fold at pH 6.0 and 2.7-fold at pH 7.4 relative to naked Ad in U343 cells treated with dE1/GFP-PPCBA (50 mg/mL) (Fig. 3B). An even more profound pH-dependent increase in transduction efficiency was observed in dE1/GFP-PPCBA-treated MCF7 cells, which

showed a 475.2-fold increase at pH 6.0 and a 4.4-fold increase at pH 7.4 relative to naked Ad, at 50 mg/mL concentration. Taken together, these results imply that dE1/GFP-PPCBA complex can enhance Ad transduction efficiency regardless of CAR expression by cancer cells, and that transduction efficiency is further enhanced under acidic conditions reflective of the tumor environment.

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3.4. Cellular uptake mechanism studies of Ad-PPCBA complex We then sought to elucidate the cellular uptake mechanism of dE1/GFP-PPCBA complexes. First, a CAR competition assay was performed to investigate the extent of CAR dependency of the nanocomplex in its target cell uptake. Since the transduction efficient of naked Ad was significantly lower than dE1/GFP-PPCBA complex, we used 50 MOI for naked Ad and 20 MOI for dE1/GFP-PPCBA complexes to compensate the low transduction efficiency of naked Ad. Pretreatment of U343 cells with CAR-binding Ad fiber protein substantially reduced subsequent GFP transduction efficiency of naked Ad in a concentration-dependent manner (67% and 90% decreases with 0.2 and 2 mg/ml Ad fiber protein pretreatment, respectively) (Fig. 4A). Cell pretreatment with Ad fiber protein also inhibited dE1/GFP-PPCBA transduction efficiency when performed at pH 7.4. In contrast, GFP expression in cells transduced with dE1/GFP-PPCBA at pH 6.0 was not blocked by Ad fiber protein, indicating that cellular uptake of dE1/ GFP-PPCBA was not mediated by CAR/Ad fiber interactions at low pH. Next, we pretreated U343 cells with various cellular uptake inhibitors: 1) CPZ: an inhibitor of clathrin-mediated endocytosis [31] (0.2 and 1 mM); 2) genistein, an inhibitor of caveolae-mediated endocytosis [32] (1.25 and 5 mM); and 3) NH4Cl, an inhibitor of macropinocytosis [33] (0.5 and 1 mM). As shown in Fig. 4B, dE1/GFP-PPCBA complex at both pH 7.4 and pH 6.0 were not affected by CPZ, which correlated with unchanged GFP expression levels in cells pretreated with CPZ before transduction. In contrast, CPZ concentrationdependently decreased GFP expression in U343 cells transduced with naked Ad, confirming Ad's dependence on clathrin-mediated endocytosis for cell entry [34]. Genistein treatment had no inhibitory effect on the cellular uptake of either naked Ad at pH 7.4 or of dE1/ GFP-CBA complexes at either pH 6.0 or 7.4, indicating that none of these vectors use caveolin-mediated endocytosis for cellular uptake. Cell pretreatment with NH4Cl revealed that macropinocytosis is a major endocytic pathway for both naked and polymer-complexed dE1/GFP-PPCBA (Fig. 4C). At 1 mM, NH4Cl significantly decreased GFP expression in cells transduced with naked Ad at pH 7.4 and by dE1/GFP-PPCBA at both pH 6.0 and 7.4. 3.5. Evaluation of endosome escaping capacity of Ad-PPCBA complex The vacuolar Hþ ATPase inhibitor bafilomycin A1 (Bf-A) prevents, acidification of Ad, which is a crucial factor for viral escape from endosomes. To assess the endosome-escaping capability of AdPPCBA complexes, U343 and MCF-7 cell lines were pre-treated with 5 and 20 mM of Bf-A for 30 min and then transduced with naked dE1/GFP (50 MOI for U343 and 500 MOI for MCF7) at pH 7.4 or dE1/GFP-PPCBA complexes (20 MOI for U343 and 200 MOI for MCF7) at either pH 6.0 or 7.4. As shown in Fig. 5A, Bf-A significantly inhibited U343 cell transduction by both naked Ad and dE1/GFPPPCBA complexes, independent of extracellular pH, by z 85% versus respective non-Bf-A-treated controls. In MCF7 cells, 20 mM BfA potently inhibited GFP expression induced by both naked Ad and dE1/GFP-PPCBA treatment at pH 6.0 (Fig. 5B). This suggests that endosome acidification is critical for efficient gene transfer efficiency of both dE1/GFP-PPCBA complexes and naked Ad. We presume that rapid endosomal escape allows Ad-PPCBA complexes to efficiently avoid lysosomal degradation, resulting in enhanced gene transfer efficiency. 3.6. In vitro tumor cell killing and VEGF suppression efficacy of oncolytic Ad complexed with PPCBA (RdB/shVEGF-PPCBA) After elucidating its transduction efficiency and uptake mechanism, we evaluated the effect of oncolytic Ad complexed with

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PPCBA on cancer cell killing. RdB/shVEGF is an oncolytic Ad that expresses VEGF-specific shRNA, and has previously been demonstrated to have efficient anti-angiogenic and anti-tumor activity by suppressing VEGF expression in target cancer cells [26]. Infection of U87 cells with naked RdB/shVEGF naked oncolytic Ad suppressed VEGF expression by these cells in a viral concentration-dependent manner (Fig. 6A). Similarly, RdB/shVEGF-PPCBA complex decreased the VEGF level in a viral dose-dependent manner, demonstrating that RdB/shVEGF complexed in PPCBA is functionally active in expressing VEGF-specific shRNA, which inhibits angiogenic VEGF secretion. Importantly, RdB/shVEGF-PPCBA complex more potently suppressed VEGF gene expression at pH 6.0 compared to pH 7.4 (p < 0.05), confirming that enhanced infection efficiency of RdB/shVEGF-PPCBA under acidic conditions enhances therapeutic siRNA functionality. Because CAR expression is often lost as cancers progress, and this loss has been one of major hurdles to use anti-cancer Ad vectors in the clinical setting, we compared the oncolytic effect of RdB/ shVEGF-PPCBA complexes in both CAR-positive U343 and CARnegative MCF7 cell lines. As shown in Fig. 6B, naked RdB/shVEGF killed U343 cells very efficiently, whereas MCF7 cells were resistant to naked RdB/shVEGF. This demonstrated that naked oncolytic Ad depends on tumor cell CAR expression for oncolytic activity. Similarly, at pH 7.4, RdB/shVEGF-PPCBA complexes effectively killed CARþ U343 cells but not MCF7 cells, implying that at this pH, PPCBA-Ad complex uptake by tumor cells relies on target cell CAR expression. In marked contrast, RdB/shVEGF-PPCBA complex at pH 6.0 elicited potent cell killing efficacy in both U343 and MCF7, implying that RdB/shVEGF-PPCBA complex can bypass the requirement for tumor cell CAR expression to infect these cells. This suggests that, in the hypoxic and acidified tumor microenvironment, even CAR-deficient tumor cells can be efficiently infected by PPCBA-Ad complexed vectors.

3.7. In vivo therapeutic efficacy of RdB/shVEGF-PPCBA complexes To confirm whether RdB/shVEGF-PPCBA complexes' in vitro anti-tumor therapeutic potency translates to the in vivo scenario, we first evaluated the anti-angiogenic effect of RdB/shVEGF-PPCBA by the Matrigel® plug assay. MCF7 cells were infected with either naked RdB/shVEGF or RdB/shVEGF-PPCBA either at pH 6.0 or at pH 7.4, and were mixed with cold Matrigel® and injected into mice subcutaneously above the flank region. After 14 days, Matrigel® plugs were excised and analyzed for vessel quantification. Matrigel® plugs mixed with cells transduced with PBS, naked RdB/ shVEGF, or RdB/shVEGF-PPCBA (pH 7.4) showed similar amounts of neovascularization. However, plugs containing MCF7 cells that had been infected with RdB/shVEGF-PPCBA at pH 6.0 exhibited dramatically reduced vascularization (p < 0.001) (Fig. 7A). Next, we assessed the in vivo tumor-killing ability of RdB/ shVEGF-PPCBA. Mice with U87 xenograft tumors were established and were treated intratumorally with PBS, naked Ad, or RdB/ shVEGF-PPCBA. As shown in Fig. 7B, tumor growth was significantly inhibited in the RdB/shVEGF-PPCBA group compared to the naked RdB/shVEGF group (p < 0.01). The respective mean tumor sizes at Day 23 after treatment with PBS, naked RdB/shVEGF Ad, and RdB/shVEGF-PPCBA were 1984 ± 439 mm3, 720 ± 119 mm3, and 177 ± 73 mm3. At Day 23, the corresponding tumor growth inhibition percentages for RdB/shVEGF and RdB/shVEGF-PPCBA groups were 63.7% and 91.1% of PBS controls. Taken together, these results demonstrated potent in vivo anti-angiogenic and tumor-killing activities of RdB/shVEGF-PPCBA's, thereby highlighting their potential therapeutic efficacy as anti-cancer treatments.

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Fig. 4. Cellular uptake mechanism studies of Ad-PPCBA complex. (A) CAR competition assay. U343 cells were transduced naked dE1/GFP or dE1/GFP-PPCBA complexes (50 mg/mL PPCBA) in the presence and absence of fiber protein (0.2 and 2 mg/mL) specific to CAR. At 48 h post infection, cells were analyzed for GFP expression by flow cytometry analysis. The data are representatives of three independent experiments performed in triplicate. Bars represent mean ± SD. ***P < 0.001 versus naked dE1/GFP-treated group. Original magnification: 100. (B, C) Endocytosis pathway analysis with chlorpromazine (CPZ), genistein, or ammonium chloride. U343 cells were pre-treated for 30 min with CPZ, genistein, or ammonium chloride at indicated concentrations. Naked dE1/GFP or dE1/GFP-PPCBA complex (50 mg/mL PPCBA) was added in the absence or presence of the inhibitors for an additional 2 h. Cells were then observed for GFP expression by fluorescence microscopy and flow cytometry. Original magnification: 100. Bars represent mean ± SD. **P < 0.01 versus naked dE1/GFP-treated group.

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To evaluate the therapeutic efficacy of systemicallyadministered oncolytic Ad-PPCBA complexes, nude mice bearing U87 xenograft tumors were injected intravenously with PBS, naked RdB/shVEGF, or RdB/shVEGF-PPCBA. As shown in Fig. 7C, tumor growth was significantly inhibited in the RdB/shVEGF-PPCBA group versus the naked RdB/shVEGF group (p < 0.001). The respective mean tumor sizes at Day 27 after intravenous injection with PBS, RdB/shVEGF, or RdB/shVEGF-PPCBA were 2144 ± 167 mm3, 1777 ± 484 mm3, and 752 ± 35 mm3. This corresponded to tumor growth inhibitions for RdB/shVEGF and RdB/shVEGF-PPCBA groups by 17.1% and 65.0%, respectively versus PBS controls. These results not only confirm the immune evading effect of encapsulating Ad with PPCBA, but also demonstrate the potent therapeutic antitumor potential of RdB/shVEGF-PPCBA after systemic adminis tration.

3.8. Evasion of immune response and attenuated liver toxicity in vivo One of the challenges inherent to administering therapeutic viral gene vectors in vivo is innate immune system activation after recognizing Ad particles in the blood. Based on the gel retardation assay and the TEM images of dE1/GFP-PPCBA complexes, we hypothesized that PPCBA encapsulation of Ad particles might enable Ad-PPCBA complexes to evade this immune response by creating a “stealth” Ad. To evaluate the evasive character of Ad-PPCBA complexes, we measured serum levels of the inflammatory cytokine IL6 as a proxy for innate immune system activation against Ad. BALB/ c mice were injected intravenously with PBS only or an equivalent volume containing 2  1010 VP of naked dE1/GFP or dE1/GFPPPCBA, and measured serum IL-6 by ELISA at 6 h after injection

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1: PBS 2: RdB/shVEGF 3: RdB/shVEGF-PPCBA pH(7.4) 4: RdB/shVEGF-PPCBA pH(6.0) Fig. 6. VEGF suppression and cancer cell killing efficacy of RdB/shVEGF-PPCBA complexes. (A) U87 cells were infected with PBS, RdB/shVEGF, or RdB/shVEGF-PPCBA complex at 1 or 10 MOIs. VEGF concentration was measured in the culture supernatant at 48 h after infection by ELISA. The data are representatives of three independent experiments performed in triplicate. Bars represent mean ± SD. *P < 0.05 versus naked RdB/shVEGF-treated group. (B) U343 and MCF7 cells were treated with PBS, RdB/shVEGF, or RdB/shVEGF-PPCBA complex. At 48 h post infection, cell viability was then assessed by MTT assay. The PBS-treated group was set at 100%. The data are representatives of three independent experiments performed in triplicate. Bars represent mean ± SD. *P < 0.05, ***P < 0.001 versus naked RdB/shVEGF-treated group.

(Fig. 8). Expectedly, systemic injection of naked Ad increased serum IL-6 levels over two-fold compared to PBS controls. Intravenous injection of PPCBA or dE1/GFP-PPCBA resulted in serum IL-6 levels that were similar to levels in the PBS group (p > 0.05), demonstrating PPCBA's biocompatibility and capability to shield the Ad surface from innate immune system recognition of, and response against, Ad after systemic administration. Another contributing factor in Ad's rapid clearance from the blood is the recruitment and scavenger receptor-mediated phagocytosis of viral particles by hepatic Kupffer cells [35]. Cytokines released by these specialized macrophages after Ad liver transduction (TNF and IL-6) cause early hepatotoxicity, which is

enhanced by hepatic chemokine-mediated neutrophil accumulation [36,37]. The extent of Ad-polymer complex-induced hepatotoxicity was assessed in vivo by serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which were measured in BALB/c mice 3 days after intravenous injection with PBS, naked dE1/GFP, or dE1/GFP-PPCBA (Fig. 8). Control serum liver-enzyme levels were low but were markedly elevated by naked Ad injection, whereby ALT and AST were respectively increased by 49.9- and 7.6-fold versus the PBS group (p < 0.01 in both cases). However, ALT and AST levels in dE1/GFP-PPCBA-treated mice were not significantly different from levels in PBS control animals. In sum, the reduced circulating IL-6, ALT, and AST levels in

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1: PBS 2: RdB/shVEGF 3: RdB/shVEGF-PPCBA pH(7.4) 4: RdB/shVEGF-PPCBA pH(6.0) Fig. 7. Potent antiangiogenic and antitumor efficacy of RdB/shVEGF-PPCBA. (A) Immunohistochemical staining of sections of matrigel plugs with anti-CD31 antibody. Representative photomicrographs are shown. Original magnification: 200. Blood vessels were counted in tumor tissues for each treatment group. Six images were analyzed per group. Bars represent mean ± SD. ***P < 0.001 versus naked RdB/shVEGF or RdB/shVEGF-PPCBA at pH 7.4. (B) Antitumor therapeutic efficacy of naked RdB/shVEGF or RdB/shVEGF-PPCBA complex in U87 glioblastoma xenograft established in nude mice through intratumoral (IT) injection. U87 tumor-bearing mice were injected with PBS, RdB/shVEGF (5  109 VP), or RdB/shVEGF-PPCBA (5  109 VP) complex three times spaced every other day. The arrow indicates administration of treatment. Results are expressed as mean ± SD (each group, n ¼ 6). ***P < 0.001 versus PBS-treated group (RdB/shVEGF) or RdB/shVEGF-treated group (RdB/shVEGF-PPCBA) (C) Antitumor therapeutic efficacy of naked RdB/shVEGF or RdB/shVEGF-PPCBA complex in U87 glioblastoma xenograft established in nude mice through intravenous (IV) injection. U87 tumor-bearing mice were injected with 200 mL PBS, RdB/shVEGF (2  1010 VP), or RdB/shVEGF-PPCBA (2  1010 VP) via tail vein on three occasions spaced every other day. Tumor volume was measured every 2 days following treatment. The arrow indicates administration of treatment. Results are expressed as mean ± SD (each group, n ¼ 6). ***P < 0.001 for versus PBS- or RdB/shVEGF-treated group.

Ad-PPCBA-injected mice versus the naked Ad-injected animals attest to PPCBA's immune-escaping property. 4. Discussion In considering the efficacy of viral or polymeric nanoparticle gene therapy vectors to target metastatic tumors, three major criteria require consideration: selectivity, transduction efficiency, and immunogenicity. Numerous vector modifications have been

developed to maximize the vector's tumor targeting while decreasing immunogenic side effects. Recent approaches include non-specific Ad surface masking polymers such as poly(ethylenimine) (PEI) [21], PEG [22,38], N-(2-hydroxypropyl) methacrylamide (HPMA) [39,40], arginine-grafted bioreducible polymer [18,30,41] and chitosan [42], alteration of endogenous Ad tropism by ablation of CAR or integrin avb5 and avb5 binding properties [43], and by attaching targeting moieties (e.g., tumor-homing peptides and antibodies) [44e46]. With respect to Ad-polymer

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complexes, however, most combinations fail to take full advantage of their potential benefits. For instance, while PEGylation of Ad endows the virus with excellent shielding properties from immune response, PEG also decreases transduction efficiency by obstructing specific Ad fiber-cell receptor interactions [20]. Furthermore, the polymers are reported not efficiently biodegraded in vivo (i. e. 25 kDa PEI), thus inducing adverse effects. To this end, we have introduced disulfide bond in the CBA moiety of PPCBA block copolymer to be degraded after cellular uptake and release the viral vector in cytoplasm. In cytotoxicity assay, PPCBA polymer was demonstrated not cytotoxic even at high concentration (100 mg/mL; Fig. 2A), whereas 25 kDa PEI shows cytotoxicity as low as 10 mg/mL [30], demonstrating the good safety profile of PPCBA polymer. After release from PPCBA polymer by reduction (dithols (eSeSe) to eSH) of CBA, Ad capsid traffics along microtubules towards the nucleus by interactions with the motor protein dynein. Then, the partially disassembled virion particle is transported to the nuclear membrane to release viral DNA into the nucleus [17].

CAR dependence for Ad's cell entry remains an important issue when considering systemic administration of Ad-polymer complexes. A benefit of the CAR ablation strategy was reduced Ad liver tropism and toxicity, suggesting that any vector that fully relies on the CAR transduction pathway risks increased immunogenicity caused by Kupffer cell recruitment, activation, and release of inflammatory chemokines and cytokines. However, bypassing Ad vector reliance on CAR-mediated target cell transduction without equipping the vector with alternative endocytic pathway that is cancer or cancer-environment specific would decrease Ad's excellent transduction capabilities, especially in CAR-positive tumors. Furthermore, a degree of CAR reliance for Ad-polymer transduction can serve as a tumor-selective feature and prevent Ad-polymer infection of normal cells because most non-tumor cells do not express CARs. The ideal Ad-polymer complex would thus infect tumors via alternative cancer-specific transduction pathways that can potentially function in conjunction with CAR, but also should remain nonreactive to normal cells and should be encapsulated against innate immune attack.

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The daunting task of engineering the ideal Ad nanocomplex therefore demands finesse and versatility in vector design, so that it is ideally only active in the tumor microenvironment. Idiosyncratic features of the tumor environment include hypoxia and acidification which can be used to target and activate suitably adapted vectors. For instance, the acidic tumor environment has been exploited in generating various pH-sensitive polymers for efficient drug and gene delivery [24,47]. In addition, various proteins unique to the hypoxic tumor such as hypoxia-inducible factor 1-a have been targeted for enhanced Ad tumor recruitment and killing [10]. Despite the innovative development of various pH-sensitive polymers, they still suffer from lack of specificity and impeded interactions between the ligand and their target cell receptor possibly due to functional group modifications or steric hindrance from conjugating targeting moieties with polymeric carriers. As a compensatory strategy for the shortcomings of existent pHresponsive drug carriers, we engineered Ads ionically crosslinked to a pH-sensitive, bio-reducible polymer, PPCBA. Our data demonstrate enhanced transduction efficacy of Ad-PPCBA complexes in vitro and elucidate Ad-polymer cellular trafficking mechanisms, but also highlight the vector's in vivo therapeutic antitumor killing efficiency when administered either intratumorally or and intravenously.

Physical and chemical changes of Ad when conjugated with PPCBA include: 1) a 1.7-fold increased particle size; 2) cationic charge reversal of the capsid surface; and 3) encapsulation of Ad particles by the polymer. Encapsulation of Ad by a cationic polymer, as demonstrated with ABP, chitosan, and PNLG [18,19,42], is a useful strategy to improve the Ad transduction efficiency and DNA delivery because the positive charge of the polymer not only enables the ionic interaction between the polymer and the virus but also facilitates viral entry into host cells via electrostatic interaction. By using this strategy, Ad-PPCBA complexes benefit from enhanced transduction efficiency in both CAR-positive and -negative cells. Transduction efficiency studies revealed that the Ad-PPCBA vector transduction efficiency was modulated by extracellular pH, with enhanced infectivity at pH 6.0 versus 7.4. Interestingly, the transduction efficiency studies also demonstrated the vector's ability to overcome CAR dependence, whereby transduction of CAR-negative cancer cells in acidic conditions was significantly elevated over infectivity at physiological pH. Cellular uptake mechanism studies revealed that dE1/GFPPPCBA enters cells via a macropinocytosis-mediated endocytosis, which is a different route than naked Ad. In agreement with this finding, macropinocytosis has been previously implicated as a pivotal pathway in inducing CAR-independent endocytosis and consequentially higher efficiency of infection of CAR-negative cell lines by Ad-polymer complexes [48]. Cell treatment with Bf-A, which prevents viral escape from endosomes, significantly decreased gene transfer efficiency of Ad-PPCBA complex (Fig. 5), suggesting that Ad-PPCBA complexes require endosomal acidification for successful gene transduction, similar to what is required by naked Ad. Cancer cell killing efficacy was significantly increased when oncolytic Ad and PPCBA were complexed (RdB/shVEGF-PPCBA) compared to killing by naked oncolytic Ad alone. This was most likely due to enhanced cellular uptake and was not limited by target cell CAR expression (Fig. 6). The RdB/shVEGF-PPCBA complex decreased tumor cell VEGF expression in viral dose- and pHdependent manners; thus RdB/shVEGF complexed with PPCBA is functionally active and reduces VEGF expression with higher efficiency under acidic conditions characteristic of the tumor environment. Consistent with these findings, RdB/shVEGF-PPCBA complexes elicited a much greater antitumor efficacy versus naked RdB/shVEGF-Ad in U87 xenografts, demonstrating the potent therapeutic value of oncolytic Ad-PPCBA complexes. A key feature of oncolytic Ad is its successive secondary replication in tumor tissues. Therefore, the excellent antitumor activity of RdB/shVEGFPPCBA complexes may be attributable to the active replication of oncolytic Ad in tumor tissues following efficient cancer cell infection in the hypoxic tumor microenvironment. Concurrent expression of siRNA specific against VEGF during viral replication suppresses tumor-driven angiogenesis. Importantly, amplifying therapeutic siRNA in a cancer cell-specific manner would decrease potential side effects of siRNA while maximizing its therapeutic value. Dual tumor targeting strategy by oncolytic Ad-mediated cancer cell specific killing efficacy and tumor-microenvironment (hypoxia)-specific Ad infection enhancement endowed by PPCBA complex can further increase the therapeutic efficacy of Admediated cancer gene therapeutics with decrease of unwanted side-effect. Systemic Ad administration, in contrast to intratumoral injection, presents entirely different challenges: short blood retention time, acute liver tropism and toxicity, and innate immune system activation. In fact, the efficacy and safety of any Ad-conjugate vector engineered for systemic administration have been primarily dependent on the vector's immunogenicity. A leading strategy to reduce immune responses to the Ad vector has been viral

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encapsulation [3]. Conjugation of various polymers on the Ad surface such as PEGylation has fashioned “stealth” Ad particles by masking Ad capsid molecules and reducing absorption of host antibody to the virus [49]. Encapsulation of Ad with PPCBA results in pH-dependent particle morphologies. By TEM, the polymer is tightly wound around the Ad at pH 7.4, but appears loosely expanded in acidic conditions (i.e., pH 6.0). Based on the TEM images, we investigated whether cloaking of Ad with PPCBA at neutral conditions produces the similar “stealth” effects as does PEGylation. As an indicator of innate immune activation after Ad systemic administration, serum IL-6 levels were significantly reduced in AdPPCBA-injected mice versus animals that received naked Ad. This decreased IL-6 following Ad-PPCBA complex administration may be due to reduced vector uptake by Kupffer cells and polymershielding of the Ad surface to the immune system. Reduced immunogenicity of Ad-PPCBA complexes was supported by in vivo studies in which intravenously-injected dB/ shVEGF-PPCBA's showed therapeutic efficacy against established tumors in a mouse xenograft model. Conversely, the therapeutic efficacy of naked oncolytic Ad was significantly attenuated when it was administered systemically, even though same oncolytic Ad elicited marked antitumor efficacy when it was administered intratumorally. This observation confirmed that systemically delivered naked Ad is inactivated and cleared by immune system and non-specific liver uptake, results in low anti-tumor efficacy. Importantly, Ad-related liver toxicity as measured by serum ALT and AST levels was nonexistent in animals that received systemic administration of RdB/shVEGF-PPCBA. Taken together, this pHsensitive, PPCBA shielded, oncolytic Ad shows negligible immunogenicity and liver toxicity, and potent antitumor activity, which confers great potential for in vivo clinical applications to selectively target and kill both primary and metastatic tumors. 5. Conclusions We have synthesized a pH-sensitive and bio-reducible polymer, mPEG-Pip-CBA (PPCBA), and engineered a dual tumor targeting polymer-complexed Ad vector system (Ad-PPCBA) that targets tumors via electrostatic interaction between the virus and target cell. By taking advantage of dual tumor targeting ability from oncolytic Ad's innate oncolytic prowess and the flexibility of pH-sensitive polymer, the oncolytic Ad-PPCBA complex demonstrated enhanced therapeutic efficiency in both CAR-positive and -negative cells, which was significantly increased in the low pH environment reflective of the tumor milieu. The potent therapeutic potential and minimal toxicity of Ad-PPCBA should encourage the further development of other smart, tumor-selective oncolytic Ad complexes that exploit targetable features of the tumor microenvironment. Acknowledgments This work was supported by grants from the Ministry of Trade, Industry & Energy (10030051 Dr. C-O. Yun), the National Research Foundation of Korea (2010-0029220, 2013M3A9D3045879, 2013K1A1A2A02050188, Dr. C-O. Yun), The Korea Food and Drug Administration (KFDA-13172-306, Dr. C-O. Yun), Basic Research Programs by National Research Foundation of Korea (2013R1A1A2012483, Dr. D. Kasala) and the National Institutes of Health, USA (CA 107070, Dr. S-W. Kim). Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2014.11.021.

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