Optimization and Internalization Mechanisms of PEGylated

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Optimization and Internalization Mechanisms of PEGylated Adenovirus Vector with Targeting Peptide for Cancer Gene Therapy Xing-Lei Yao,†,‡ Yasuo Yoshioka,‡,§ Gui-Xin Ruan,† Yu-Zhe Chen,† Hiroyuki Mizuguchi,∥,⊥ Yohei Mukai,‡ Naoki Okada,‡ Jian-Qing Gao,*,†,# and Shinsaku Nakagawa*,‡,§ †

Institute of Pharmaceutics and #Soft Matter Research Center, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, People's Republic of China ‡ Departments of Biotechnology and Therapeutics and ∥Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, and §The Center for Advanced Medical Engineering and Informatics, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan ⊥ Laboratory of Stem Cell Regulation, National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan S Supporting Information *

ABSTRACT: We have previously developed a novel adenovirus vector (Adv) that targeted tumor tissues/ vasculatures after systemic administration. The surface of this Adv is conjugated with CGKRK tumor homing peptide by the cross-linking reaction of polyethyleneglycol (PEG). In this study, we showed that the condition of PEG modification was important to minimize the gene expression in normal tissues after systemic treatment. When Adv was modified only with PEG-linked CGKRK, its luciferase expression was enhanced even in the liver tissue, as well as the tumor tissue. However, in the reaction with the mixture of non-cross-linking PEG and PEG-linked CGKRK, we found out that the best modification could suppress its gene expression in the liver, without losing that in the tumor. We also studied the internalization mechanisms of CGKRK-conjugated Adv. Results suggested that there is a specific interaction of the CGKRK peptide with a receptor at the cell surface enabling efficient internalization of CGKRK-conjugated Adv. The presence of cell-surface heparan sulfate is important receptor for the cellular binding and uptake of CGKRK-conjugated Adv. Moreover, macropinocytosis-mediated endocytosis is also important in endocytosis of CGKRK-conjugated Adv, aside from clathrin-mediated and caveolae-mediated endocytosis. These results could help evaluate the potentiality of CGKRK-conjugated Adv as a prototype vector with suitable efficacy and safety for systemic cancer gene therapy.

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INTRODUCTION Gene therapy is expected to play a key role in next-generation cancer therapy. The adenovirus vector (Adv) is one of the most frequently used vectors in cancer gene therapy because of its many useful features.1,2 In several clinical trials, local injection of Adv resulted in therapeutic efficacy against primary tumors.3−6 However, cancer gene therapy with Adv is limited to local administration because systemic administration of Adv produces weak therapeutic effect and severe side effects, such as hepatotoxicity attributed to high accumulation and transgene expression in the liver.7−9 For this reason, clinical application of systemically administered Adv as gene therapeutic vectors has been limited. The conjugation of Adv with ligands has potentially broad application for targeted therapy. Several groups have reported that Adv linked to targeting ligands such as RGD peptide, FGF2 ligand, and E-selectin antibody were retargeted to tumor cells with these alternative receptors.10−17 In our previous study, CGKRK peptide was used as a targeting ligand to conjugate with Adv by the cross-linking reaction of polyethyleneglycol (PEG; CGKRK-conjugated Adv). 18 CGKRK peptide was used because it accumulates both on © 2012 American Chemical Society

the surface of tumor vessels and within tumor tissues after intravenous injection.19,20 Systemic administration of CGKRKconjugated Adv expressing the herpes simplex virus thymidine kinase (HSVtk) gene showed superior antitumor effects against primary tumors and metastases with negligible side effects in terms of both direct cytotoxic effects and inhibition of tumor angiogenesis.18 The results suggested that CGKRK peptide has potential as an active targeting ligand. In the construction of CGKRK-conjugated Adv, PEG was used not just as cross-linking reagent, but also to control the in vivo pharmacokinetics of Adv. Covalent conjugation to PEG (PEGylation) has been used frequently in pharmaceutical preparation such as liposomes, proteins, and virus vectors since the late 1970s.21 PEGylation can prolong the plasma half-life, prevent hepatic uptake, and alter the tissue distribution of therapeutic molecules because of the steric hindrance of the PEG.2,22−26 One important finding was that the characteristics of PEGylated liposomes or proteins were significantly affected Received: April 27, 2012 Revised: June 28, 2012 Published: June 30, 2012 2402

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cross-linking PEG and incubated at room temperature for 1 h. Unreacted maleimide was blocked by incubation with 2-mercaptoethanol at room temperature for 1 h. All of the reaction was under the medium of 5 mM EDTA (ethylenediaminetetraacetic acid) in PBS (phosphate buffered saline; pH = 7.4). Finally, free peptides and free PEGs were removed by dialysis with PBS using 1000 kDa molecular weight cutoff membranes (Spectrum Laboratories, Rancho Dominguez, CA) to purify PEGylated Adv and CGKRK-conjugated Adv. The concentration of CGKRK-conjugated Adv was measured via picogreen assay (Invitrogen, Carlsbad, CA). In Vitro Gene Transduction. B16BL6 cells (2 × 104 cells/well) were seeded into 48-well plates. The following day, each well was treated with 2 × 108 vp (104 vp/cell) of luciferase-encoding Adv. After 24 h, luciferase activity was determined using the luciferase assay system (Promega, Madison, WI) and a Lumat LB 9507 luminometer (EG&G Berthold, Bad Wildbad, Germany) in accordance with the manufacturers’ instructions. Luciferase activity was calculated as relative light units (RLU)/well. In Vivo Gene Transduction. C57BL/6 mice (purchased from SLC Inc., Hamamatsu, Japan) underwent intradermal inoculation in the flank with 3 × 105 B16BL6 cells. After the tumor diameter reached approximately 7−8 mm, each mice was administered intravenously with 1 × 1010 vp or 5 × 1010 vp of luciferase-encoding Adv. The tissues were harvested 48 h after administration, and luciferase activity was measured. Peptide-Dependency of Cellular Uptake. B16BL6 cells (2 × 104 cells/well) were seeded into 48-well plates. The following day, the cells were pretreated with or without 1 mg/mL peptide CGKRK, KCRGK, or CKGRK (synthesized by Sangon Biotech Shanghai, China; purity > 98%) for 1 h. The concentrations of inhibitors were selected as maximum concentration without toxicity. Each well was treated with 2 × 108 vp (104 vp/cell) of luciferase-encoding Adv. After 1 h, the virus solution was replaced with fresh culture medium. After 24 h of cultivation, luciferase activity was determined using the luciferase assay system. HSPG-Dependency of Cellular Uptake. B16BL6 cells (2 × 104 cells/well) were seeded into separate 48-well plates. The following day, the cells were pretreated with or without 10 μg/mL heparan sulfate (Sigma-Aldrich, U.S.A.) or 100 μg/mL heparin sodium salt (Bio Basic Inc., Canada) for 1 h. The concentrations of inhibitors were selected as maximum concentration without toxicity. Each well was treated with 2 × 108 vp (104 vp/cell) of luciferase-encoding Adv. After 1 h, the virus solution was replaced with fresh culture medium. After 24 h of cultivation, luciferase activity was determined using the luciferase assay system. Endocytosis-Dependency of Cellular Uptake. B16BL6 cells (2 × 104 cells/well) were seeded into separate 48-well plates. The following day, the cells were pretreated with or without endocytosis inhibitor 2.5 mM amiloride HCl hydrate (Sigma-Aldrich, U.S.A.), 5 mM methyl-β-cyclodextrin (MBCD; Kaiyang Bio Co., Shanghai, China), or 30 μM chlorpromazine (CPZ; Kaiyang Bio Co., Shanghai, China) for 1 h. The concentrations of inhibitors were selected as maximum concentration without toxicity. Each well was treated with 2 × 108 vp (104 vp/cell) of luciferase-encoding Adv. After 1 h, the virus solution was replaced with fresh culture medium. After 24 h of cultivation, luciferase activity was determined using the luciferase assay system. Statistical Analysis. All results were expressed as mean ± SD. Differences were compared using Bonferroni’s method after analysis of variance.

by the amount of PEG on the surface (i.e., the PEG modification ratio).27,28 Our group also demonstrated that molecular weight and modification ratio of PEG would yield difference viral distribution and transgene expression pattern of Adv.22,26 In our previous study, we showed that systemic administration of Adv PEGylated with 20 kDa PEG at 45% modification ratio resulted in higher tumor-selective transgene expression compared with unmodified Adv.26 Therefore, we considered that the amount of PEG might also play an important role in the construction of ligand-conjugated Adv that used PEG as cross-linking reagent. Although more groups paid attention to conjugate active targeting ligand with Adv by the cross-linking reaction of PEG, few reports highlighted the importance of the balance between cross-linking PEG and noncross-linking PEG in efficient tumor-specific transgene expression through systemic administration. In this study, we constructed CGKRK-conjugated Advs with different modification ratios of PEG-linked CGKRK and noncross-linking PEG. We also investigated whether the amount of PEG and peptide could affect the transgene expression of CGKRK-conjugated Adv in tumor tissue and in other normal tissues after systemic administration. The internalization mechanisms of CGKRK-conjugated Adv were also studied.

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MATERIALS AND METHODS

Mice and Cell Lines. Five-week-old female C57BL/6 mice were used in this study. All of the animal experimental procedures were performed in accordance with the Osaka University and Zhejiang University guidelines for the welfare of animals. B16BL6 (mouse melanoma) cells were obtained from RIKEN Cell Bank (Tsukuba, Japan). The B16BL6 cells were cultured in Minimal Essential Medium (Sigma-Aldrich, St. Louis, MO) containing 7.5% FBS and antibiotics. Vectors. The E1/E3-deleted adenovirus type 5 expressing firefly luciferase, which is under the control of the cytomegalovirus promoter, was constructed with an improved in vitro ligation method, as previously reported.26,29−31 Adv was amplified in 293 cells using established methods22 and was purified by cesium chloride stepgradient ultracentrifugation. The virus particles (vp) and biological titer were determined via spectrophotometrical method32 and AdenoX Rapid Titer protocol (Clontech Laboratories, Mountain View, CA, U.S.A.), respectively. The ratio of the particle-to-biological titer was between 10 and 30. Preparation of CGKRK-Conjugated Adv. A cross-linking PEG (Sunbright MA-200TS; heterobifunctional PEG: maleimide-PEGcarbonate-N-hydroxylsuccinimide (NHS); molecular weight, 20000; NOF Corporation, Tokyo, Japan) and a non-cross-linking PEG (mPEG-SPA; monofunctional PEG: methoxy-PEG-succinimidyl proprionate; molecular weight, 20000; Nektar, San Carlos, CA) were used for PEGylation. Adv was incubated with the cross-linking PEG at 50or 100-fold molar excess to viral lysine residues (7500 lysine residues in one viral particle) at 37 °C for 45 min and then incubated with or without the non-cross-linking PEG at 50-, 100-, or 150-fold molar excess to viral lysine residues at 37 °C for 45 min to construct PEGylated Adv. The excess NHS group of free PEG was blocked by incubation with 6-aminocaproic acid (Sigma-Aldrich) at 37 °C for 30 min. The modification ratio of PEGylated Adv was determined by confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. SDS-PAGE was carried out in a gradient gel containing 4−20% polyacrylamide (PAG Mini 4/20, Daiichi Pure Chemicals, Tokyo, Japan) as previously reported.12,33 The PEGylated hexon band was separated from the unmodified hexon band. The signal intensity of each band was measured using NIH Image software, and the PEG modification ratio was calculated as: signal intensity of PEGylated hexon/(signal intensity of PEGylated + signal intensity of unmodified hexon) × 100. CGKRK-conjugated Adv was prepared by adding CGKRK peptide (synthesized by Sangon Biotech Shanghai, China. Purity is >98%) at 5-fold molar excess in

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RESULTS Construction of CGKRK-Conjugated Adv. A crosslinking PEG with maleimide and NHS was used to conjugate the CGKRK peptide with PEGylated Adv. We constructed PEGylated Adv using 50- or 100-fold molar cross-linking PEG for the modification of adenoviral lysine residues (Adv-PEG50 and Adv-PEG100). The modification ratio of Adv by PEG was approximately 19 or 32%, as confirmed by SDS-PAGE (Table

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1). Excess cysteine of the CGKRK peptide (10-fold molar) was then reacted with maleimide of the cross-linking PEG chain Table 1. Molecular Ratio of Adv to PEG and Modification Ratio of PEG-Adv heterobifunctional activated PEG Adv-PEG50 AdvPEG100 Adv-PEG 50/50 Adv-PEG 50/100 Adv-PEG 50/150 a

monofunctional activated PEG

modification ratio (%)

1:50a 1:100a

1:0a 1:0a

19 32

1:50a

1:50a

34

1:50a

1:100a

41

a

a

45

1:50

1:150

Refers to the molar ratio of viral lysine residue to PEG. Figure 2. In vitro transgene expression of CGKRK-conjugated Adv. B16BL6 cells (2 × 104 cells/well) were transduced with the indicated Advs. After culturing for 24 h, luciferase expression was measured (n = 4). All data are represented as the means ± SD (**P < 0.01).

conjugated with PEGylated Adv to construct Adv-PEG50CGKRK and Adv-PEG100CGKRK (Figure 1A). Transgene Expression of CGKRK-Conjugated Adv. B16BL6 cells were selected for in vitro and in vivo evaluation of transgene expression of CGKRK-conjugated Adv given that gene expression by Adv is very low in B16BL6 cells because of a lack of coxackie-adenovirus receptor (CAR) expression.29 Luciferase expression from Adv-PEG50 and Adv-PEG100 was lower than that of unmodified Adv in B16BL6 cells (Figure 2). In contrast, luciferase expression from Adv-PEG50CGKRK and Adv-PEG100CGKRK was 36- and 2747-fold higher than the expression from Adv-PEG50 and Adv-PEG100, respectively (Figure 2). Next, we evaluated in vivo transgene expression in the tumor and liver after intravenous injection at 1 × 1010 vp Adv into B16BL6 primary tumor model mice (Figure 3). Luciferase expression from Adv-PEG50 in tumor tissue was 2fold higher compared with that from unmodified Adv and approximately 72-fold lower in the liver tissue compared with that from unmodified Adv (Figure 3A). Furthermore, luciferase expression in tumor tissue with administration of Adv-

PEG50CGKRK was 100- and 60-fold higher than with the administration of unmodified Adv and Adv-PEG50, respectively (Figure 3A). In liver tissue, luciferase expression after AdvPEG50CGKRK administration was 9-fold lower and 8-fold higher than with the administration of unmodified Adv and AdvPEG50, respectively (Figure 3B). We also examined in vivo transgene expression of AdvPEG100 and Adv-PEG100CGKRK (Figure 3). Luciferase expression from Adv-PEG100 in tumor tissue was approximately 6-fold higher than that from unmodified Adv and approximately 30-fold lower in liver tissue compared with that from unmodified Adv (Figure 3). Furthermore, luciferase expression from Adv-PEG100CGKRK was 257- and 67-fold higher in tumor tissue than that with unmodified Adv and AdvPEG100 (Figure 3A), respectively. Luciferase expression from Adv-PEG100CGKRK in liver tissue was 154-fold and even 6000-

Figure 1. Schematic diagram of CGKRK-conjugated Adv. 2404

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after the administration of PEGylated Adv.22,26,34 However, the amount of cross-linking PEG could not be increased; instead, non-cross-linking PEG with NHS only (mPEG-SPA) could be used. In this study, we constructed PEGylated Adv using 50fold molar cross-linking PEG and then using 0-, 50-, 100-, and 150-fold molar non-cross-linking PEG for the modification of adenoviral lysine residues (Adv-PEG50, Adv-PEG50/50, AdvPEG50/100, and Adv-PEG50/150). The modification ratio of Adv in Table 1 was confirmed by the description of SDSPAGE.26 Excess cysteine of the CGKRK peptide (10-fold molar) was reacted with maleimide of the cross-link PEG chain conjugated with PEGylated Adv to construct CGKRKconjugated Adv (Adv-PEG50CGKRK, Adv-PEG50/50CGKRK, Adv-PEG50/100CGKRK, and Adv-PEG50/150CGKRK; Figure 1B). The modification ratio of Adv-PEG50/150CGKRK was the same with which used in our previous study.18 The high transgene expression of CGKRK-conjugated Adv was also confirmed in A549 cell line (Supporting Information, Figure). Transgene Expression of CGKRK-Conjugated Adv with Optimum Modification. First, transgene expression of CGKRK-conjugated Adv was evaluated using B16BL6 cells. Luciferase expressions from Adv-PEG50, Adv-PEG50/50, AdvPEG50/100, and Adv-PEG50/150 were decreased, accompanied by the increased modification of non-cross-linking PEG (Figure 4). Moreover, luciferase expressions from Adv-

Figure 3. In vivo transgene expression in a primary tumor model. B16BL6-bearing mice were intravenously administered with 1 × 1010 vp of the indicated Advs. After 48 h, the luciferase expression in tumors (A) and livers (B) was measured (n = 5). All data are represented as the means ± SD (*P < 0.05, **P < 0.01). Figure 4. In vitro transgene expression of CGKRK-conjugated Adv with optimized modification. B16BL6 cells (2 × 104 cells/well) were transduced with the indicated Advs. After culturing for 24 h, luciferase expression was measured (n = 3). All data are represented as the means ± SD (**P < 0.01).

fold higher than that of unmodified Adv and Adv-PEG100, respectively (Figure 3B). However, this finding is not relative to the decrease in luciferase expression from Adv-PEG100. These results indicate that increased cross-linking PEG, which attached with CGKRK peptide, not only increases transduction in the tumor but also increases transgene expression in normal tissue, such as the liver. Therefore, cross-linking PEG could not be more than 50-fold molar in the modification of Adv. However, transgene expression of Adv-PEG50CGKRK in the liver is still very high, almost equal to that in the tumor. Considering safety in clinical application, studies should be conducted to maintain high transgene expression in the tumor and reduce the expression in normal tissues, such as the liver. Optimized Construction of CGKRK-Conjugated Adv. According to observations in our previous study, increased PEG modification could reduce transgene expression in the liver

PEG50CGKRK, Adv-PEG50/50CGKRK, Adv-PEG50/100CGKRK, and Adv-PEG50/150CGKRK were significantly higher than the expressions from Adv-PEG50, Adv-PEG50/50, Adv-PEG50/ 100, and Adv-PEG50/150, respectively (Figure 4). We then evaluated the in vivo transgene expression of CGKRKconjugated Adv in the tumor and other normal tissues after intravenous injection into B16BL6 primary tumor model mice (Figure 5). A 5 × 1010 vp dose was chosen because of the possibility of a low luciferase expression from Adv-PEG. Luciferase expressions from Adv-PEG50, Adv-PEG50/50, Adv-PEG50/100, and Adv-PEG50/150 in the tumor were 2405

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Figure 5. In vivo transgene expression of CGKRK-conjugated Adv with optimized modification. B16BL6-bearing mice were intravenously administered with 5 × 1010 vp of the indicated Advs. After 48 h, the luciferase expression in the tumors (A), liver (B), spleen (C), and lung (D) was measured (n = 4). All data are represented as the means ± SD (*P < 0.05, **P < 0.01; †P < 0.05, ††P < 0.01 versus value for Adv-treated group).

Adv in the liver (Figure 5B). Meanwhile, similar tendency was observed in the spleen and lung as that in the liver. Luciferase expression from Adv-PEG50/150CGKRK was lowest in the spleen and lung, which was, respectively, 3- and 14-fold lower than that from unmodified Adv in the same tissues (Figure 5C and 5D). Therefore, we obtained the best PEG modification ratio from the construction of CGKRK-conjugated Adv. The ratio is 50-fold molar cross-linking PEG and 150-fold molar noncross-linking PEG for the modification of adenoviral lysine residues. The optimum ratio was in accordance with which used in our previous study.18 However, it is the first report in the construction of PEGylated vector system to focus on the relationship among the proportion of different PEGs, the amount of ligand peptide, and the transgene expression in different tissues. Internalization Mechanisms of CGKRK-Conjugated Adv. To explain the high transgene expression of CGKRKconjugated Adv, we focused on unmodified Adv and Adv-

increased slightly, accompanied by the increased modification of non-cross-linking PEG (Figure 5A). In contrast, luciferase expressions from Adv-PEG50CGKRK, Adv-PEG50/50CGKRK, Adv-PEG50/100CGKRK, and Adv-PEG50/150CGKRK in the tumor were evidently increased, accompanied by the increased modification of non-cross-linking PEG (Figure 5A). Luciferase expression from Adv-PEG50/150CGKRK was the highest among all groups. The expression was approximately 400- and 100-fold higher than that from unmodified Adv and Adv-PEG50/150 (Figure 5A). Luciferase expressions from Adv-PEG50, AdvPEG50/50, Adv-PEG50/100, and Adv-PEG50/150 in the liver tissue were evidently decreased, accompanied by the increased modification of non-cross-linking PEG (Figure 5B). Furthermore, luciferase expression from CGKRK-conjugated Adv was almost the same as that from PEGylated Adv with the same ratio (Figure 5B). Luciferase expression from Adv-PEG50/ 150CGKRK was particularly the lowest among all groups, which was approximately 400-fold lower than that from unmodified 2406

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PEG50/150CGKRK. This finding indicated that only CGKRK peptide could conjugate with the cell receptor while blocking the targeting ligand of Adv-PEG50/150CGKRK from conjugating with the cell receptor. If the sequence order of CGKRK is changed, such as KCRGK or CKGRK, the internalization of Adv-PEG50/150CGKRK into cells could not be blocked. Although the targeting receptor of CGKRK peptide was not clearly explained, binding may occur because of its positive charge and its composition to conjugate with cell surface heparan sulfates.19 We then compared the transduction efficiency of unmodified Adv and Adv-PEG50/150CGKRK in the presence of heparan sulfates or its homologue heparin (Figure 6B). Heparan sulfates or heparin significantly inhibited Adv-PEG50/150CGKRK-mediated transgene expression, whereas the unmodified Adv-mediated transgene expression was not affected. This result suggested that the presence of cell-surface heparan sulfate is very important in the cellular binding and uptake of Adv-PEG50/150CGKRK. The endocytosis pathway of unmodified Adv and AdvPEG50/150CGKRK were compared to study further the mechanisms of internalization in the presence of endocytosis inhibitors (Figure 6C). The transduction of unmodified Adv was inhibited by clathrin-mediated endocytosis inhibitor CPZ and lipid raft inhibitor MBCD but was not inhibited by macropinocytosis-mediated endocytosis amiloride. However, the transduction of Adv-PEG50/150CGKRK was inhibited by all three inhibitors. The results suggested that the endocytosis of Adv-PEG50/150CGKRK was different with unmodified Adv.

PEG50/150CGKRK and compared their mechanisms of internalization. We first investigated whether the transduction of AdvPEG50/150CGKRK was dependence on the ligand peptide CGKRK (Figure 6A). As a result, the treatment of cells with excess free peptide CGKRK significantly reduced the transduction efficiency of Adv-PEG50/150CGKRK. In contrast, the treatment of cells with homologous peptides KCRGK or CKGRK did not reduce the transduction efficiency of Adv-

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DISCUSSION PEGylation is a promising approach for overcoming several limitations of Adv for systemic therapy.22−24,26,34,35 We have previously examined the correlation between PEG modification and gene expression patterns. Different modification ratios of PEG would yield difference in the gene expression of PEGylated Adv in the tumor and the liver tissues.26 Based on the passive targeting effect of PEGylation strategy, we attached a tumor-targeting ligand CGKRK peptide to Adv-PEG to achieve a tumor-specific gene transfer. Although systemic administration of CGKRK-conjugated Adv with therapeutic gene showed remarkable therapeutic effects with negligible side effects in our previous study,18 the importance of the balance of PEG-linked CGKRK and non-cross-linking PEG in constructing a successful vector system of CGKRK-conjugated Adv has not been studied previously. In this study, we at first selected a relatively lower modification ratio of approximately 19 or 32% PEGylation because an extremely high modification ratio of PEGylation would decrease the transgene expression of Adv in the tumor.26 In a preliminary experiment, we found that the in vitro transgene expression of Adv reached its peak after 24 h transduction in cell lines, but in vivo transgene expression of Adv reached its peak after 48 h intravenous injection (data not shown). It is possibly caused by more complex pharmacokinetic system in vivo. The optimum in vitro and in vivo conditions were used in study, respectively. The vector (Adv-PEG50CGKRK or Adv-PEG100CGKRK) was constructed with 50- or 100-fold molar PEG-linked CGKRK. Adv-PEG100CGKRK showed encouraging transgene expression in vitro, which was even higher than that in unmodified Adv (Figure 2). However, in vivo transgene expression from Adv-PEG100CGKRK was enhanced not only in the expected tumor tissue, but also in the liver (Figure 3). This expression may yield high therapeutic effects in

Figure 6. Internalization mechanisms of CGKRK-conjugated Adv. (A) Peptide-dependency of cellular uptake of CGKRK-conjugated Adv. B16BL6 cells (2 × 104 cells/well) were pretreated with or without 1 mg/mL peptide CGKRK, KCRGK, or CKGRK for 1 h. The cells were then transduced with the indicated Adv. After culturing for 24 h, luciferase expression was measured (n = 6). (B) HSPG-dependency of cellular uptake of CGKRK-conjugated Adv. B16BL6 cells (2 × 104 cells/well) were pretreated with or without 10 μg/mL Heparan sulfate or 100 μg/mL heparin sodium salt for 1 h. The cells were then transduced with the indicated Adv. After culturing for 24 h, luciferase expression was measured (n = 6). (C) Endocytosis-dependency of cellular uptake of CGKRK-conjugated Adv. B16BL6 cells (2 × 104 cells/well) were pretreated with or without amiloride HCl hydrate, MBCD, or CPZ for 1 h. The cells were then transduced with the indicated Adv. After culturing for 24 h, luciferase expression was measured (n = 6). All data are represented as the means ± SD (*P < 0.05, **P < 0.01). 2407

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studied the corresponding receptor of CKGRK ligand. We demonstrated that Adv-PEG50/150CGKRK induces a high level of transgene expression in CAR-negative B16BL6 cells via heparan sulfates-mediated pathway (Figure 6B). One of the hurdles confronting Adv-mediated gene transfer is that infection with Adv is dependent on the presence of CAR receptors on the target cells.37 A large number of tumor cells targeted for gene therapy, such as melanoma cells, express little or no CAR, making the achievement of sufficient gene expression and therapeutic effect difficult.37,38 Heparan sulfate proteoglycans are involved in several aspects of cancer biology, including tumor progression, angiogenesis, and metastasis.39,40 Adv-PEG50/150CGKRK can target a heparin sulfate receptor and control the in vivo pharmacokinetics by PEGylation. AdvPEG50/150CGKRK also has the potential to be a wide-ranging tumor-targeting vector that overcomes the problem of conventional Adv. Finally, we study endocytosis pathway of Adv-PEG50/150CGKRK (Figure 6C). Given that B16BL6 cells are CAR-negative cells, clathrin-mediated endocytosis inhibitor CPZ, which primarily affects receptor-mediated endocytosis, could only inhibit the transduction of 46% unmodified Adv. In contrast, CPZ could inhibit the transduction of 75% AdvPEG50/150CGKRK. This coincides with the result in Figure 6B, wherein the receptor of Adv-PEG50/150CGKRK was heparan sulfates and not CAR. Cell surface heparan sulfates are also reported to be indispensable in the induction of actin organization and the macropinocytic uptake.41 This finding was consistent with the inhibition of the transduction of AdvPEGCGKRK with macropinocytosis-mediated endocytosis. The inhibition was performed by amiloride but not with that of unmodified Adv. MBCD could selectively extract cholesterol to organize sphingolipid rafts, which is related with caveolaemediated and macropinocytosis-mediated endocytosis.42 The transduction of 79% unmodified Adv was inhibited with MBCD, but not inhibited completely with amiloride. This result indicated that unmodified Adv was allowed entry into the cells through caveolae-mediated endocytosis. In contrast, the transduction of only 53% Adv-PEG50/150CGKRK was inhibited with MBCD. This result indicated that macropinocytosismediated endocytosis is more important in the endocytosis of Adv-PEGCGKRK than caveolae-mediated endocytosis. These results help explain the high transduction of CGKRKconjugated Adv.

the clinical study, but may also induce high hepatotoxicity risks or even death. CGKRK-conjugated Adv may bind to cell surface heparan sulfates with ligand CGKRK peptide as shown in Figure 6A,B. Although heparan sulfate proteoglycans are used substantially for cancer treatment, heparan sulfate proteoglycans are also expressed by endothelial cells that line the hepatic sinusoids and the underlying hepatocytes, which are present in the perisinusoidal space (space of Disse).36 This condition might explain the high transgene expression in the liver after systemic administration of Adv-PEG100CGKRK. In contrast, in vivo transgene expression from AdvPEG50CGKRK was enhanced only in tumor tissue and was decreased in the liver compared with that from unmodified Adv (Figure 3). Although compared with Adv-PEG50, AdvPEG50CGKRK still enhanced transgene expression in the liver; it showed significantly higher enhancement in the tumor. We have demonstrated in our previous work that an appropriate increase in non-cross-linked PEG could reduce the distribution of Adv particles in the liver.26 Non-cross-linked PEG could only conjugate with Adv but not with ligand peptides. Therefore, the next construction of CGKRK-conjugated Adv added 50-, 100-, or 150-fold molar non-cross-linked PEG based on AdvPEG50CGKRK (Adv-PEG50/50CGKRK, Adv-PEG50/100CGKRK, and Adv-PEG50/150CGKRK). Based on the observations in our previous study,26 more PEGylation would impair the transduction efficiency of PEGylated Adv. Therefore, we no longer added non-cross-linking PEG in amounts greater than 150-fold. Increased PEGylation with non-cross-linking PEG reduced transgene expression from CGKRK-conjugated Adv in normal tissues especially in the liver, as shown in Figure 5A,B. The transgene expression from CGKRK-conjugated Adv in the tumor was slightly increased with the increase in PEGylation with monofunctional PEG. Therefore, we achieved the optimum condition for the construction of CGKRK-conjugated Adv. Previously, we found that the amount of viral particles of Adv-PEG50/150CGKRK in tumor tissue was 60- and 3-fold higher than that of unmodified Adv and Adv-PEG50/150, respectively. Moreover, the amount of Adv-PEG50/150CGKRK in liver tissue was 15-fold less than that of unmodified Adv and almost the same as that of Adv-PEG50/150.18 These results indicate that systemic administration of CGKRK-conjugated Adv enabled tumor targeting due to active targeting of CGKRK peptide and passive targeting of PEG chain and decreased distribution in the liver. In the present study, we demonstrated the importance of choosing the appropriate amount of PEG and targeting ligand to construct a successful vector system of CGKRK-conjugated Adv. It is considered that different amounts of PEG and targeting ligand might lead to quite different tissue distribution of viral particles. After optimized construction of CGKRK-conjugated Adv, we furthermore investigated internalization mechanisms to explain the high transduction efficiency of CGKRK-conjugated Adv. First, we demonstrated that the treatment of cells with excess free peptide CGKRK, unlike similar peptides, KCRGK or CKGRK, significantly reduced the transduction efficiency of Adv-PEG50/150CGKRK (Figure 6A). This result suggested there is a specific interaction of the CGKRK peptide with a receptor at the cell surface enabling efficient internalization of AdvPEG50/150CGKRK, whereas the two other peptides has no affinity to the receptor and thus do not show any concurrent inhibition of the internalization of the conjugate and related transgene expression. After confirming the function of CKGRK ligand in the construction of Adv-PEG50/150CGKRK, we then

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CONCLUSIONS In this study, we optimized the conditions for constructing a successful vector system of CGKRK-conjugated Adv, which targeted tumor after systemic administration. We also studied the internalization mechanisms of CGKRK-conjugated Adv to explain the high transgene expression in the tumor. These results help in determining the potential of CGKRK-conjugated Adv as a prototype vector with suitable efficacy and safety for systemic cancer gene therapy.

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ASSOCIATED CONTENT

S Supporting Information *

Additional supporting figure. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. 2408

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by grants from the Ministry of Health, Labor, and Welfare in Japan; the Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation; the Global COE Program “In Silico Medicine” at Osaka University; the National Natural Science Foundation of China (81101719), China Postdoctoral Science Foundation (20100481438); and the Fundamental Research Funds for the Central Universities, China.

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dx.doi.org/10.1021/bm300665u | Biomacromolecules 2012, 13, 2402−2409