lipoplexes elicit in situ oncolytic viral replication and potent antitumor efficacy via systemic delivery

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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l

Viral genome DNA/lipoplexes elicit in situ oncolytic viral replication and potent antitumor efficacy via systemic delivery Oh-Joon Kwon a, 1, Eunah Kang a, 1, Sungwan Kim c, Chae-Ok Yun a, b,⁎ a b c

Nanomedical Science, Yonsei University College of Medicine, Seoul, Republic of Korea Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Republic of Korea Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, USA

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Article history: Received 29 April 2011 Accepted 3 June 2011 Available online 14 June 2011 Keywords: Oncolytic adenovirus Systemic delivery Ad genome lipoplex Orthotopic lung tumor model

a b s t r a c t Modifying the viral genome to express potent and cancer-selective therapeutic genes has enhanced the role of adenoviruses (Ads) in cancer molecular therapeutics. However, the efficacy of Ad systemic delivery in vivo is limited by neutralizing antibodies, short blood circulation time, and high levels of nonspecific liver uptake resulting in hepatotoxicity. We therefore investigated the systemic delivery of tumor necrosis factor-related apoptosis-inducing ligand-expressing oncolytic Ad genome DNA (pmT-d19/stTR) via lipid envelopment as an alternative approach for cancer virotherapy in an orthotopic lung cancer model. Cationic liposomes (DOTAP/DOPE) were complexed with pmT-d19/stTR to generate pmT-d19/stTR + DOTAP/DOPE with the average diameter of which was 143.3 ± 5.7 nm at the optimal DNA:lipid ratio (1:6). Systemic administration of pmT-d19/stTR + DOTAP/DOPE elicited highly effective antitumor responses in vivo, with tumor volumes decreasing 94.5%, 90.5%, and 92.4% compared to phosphate buffered saline-, naked Ad (mT-d19/stTR)-, or pmT-d19/stTR-treated groups, respectively. Additionally, innate immune responses and Ad-specific neutralizing antibodies were significantly decreased in pmT-d19/stTR + DOTAP/DOPE-treated mice compared to those in the mT-d19/stTR-treated group. The biodistribution profile analyzed by quantitative-PCR and immunohistochemical analysis demonstrated that viral replication occurred preferentially in tumor tissues. Moreover, the viral genome tumor-to-liver ratio was significantly elevated in pmT-d19/stTR + DOTAP/DOPE-treated mice, which was 934- and 27-fold greater than the mT-d19/stTR- and pmT-d19/stTRtreated mice, respectively. These results demonstrate that systemic delivery of oncolytic viral genome DNA with liposomes is a powerful alternative to naked Ad, overcoming the limited clinical applicability of conventional Ads and enabling effective treatment of disseminated metastatic tumors. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Adenovirus (Ad) is currently the most commonly used vector in clinical trials investigating cancer gene therapy [1]. Ad vectors have many advantages over other vectors, including the ability to grow to very high titers, large capacity for insertion of foreign genes, easy genetic manipulation, and ability to efficiently transduce both dividing and quiescent cells without risk of viral-induced insertional mutagenesis [2–4]. Replication-incompetent Ads were first developed to express therapeutic genes, but the therapeutic efficacy of these Ads was limited by low transgene expression and outgrowth of untransduced cancer cells. To overcome these limitations, replicationcompetent Ads were investigated [5,6]. Replication-competent Ads replicate and lyse infected cells, resulting in transgene expression that

⁎ Corresponding author at: Department of Bioengineering, College of Engineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea. Tel.: + 82 2 2228 8040; fax: + 82 2 2227 7751. E-mail address: [email protected] (C.-O. Yun). 1 These authors contributed equally to this work. 0168-3659/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2011.06.014

is significantly increased over replication-incompetent Ads [7]. Oncolytic Ads selectively replicate in and lyse cancer cells as a result of selective E1A expression using tumor-specific promoters or attenuation of endogenous adenoviral genes [8–11]. Moreover, released progeny viruses infect the surrounding uninfected cancer cells, thereby enhancing the therapeutic effect of oncolytic Ads. Most current preclinical and clinical trials have used loco-regional injection as the principle means of oncolytic Ad delivery. This mode of delivery has primarily been used due to the several limitations of systemic administration, such as pre-existing and developing Adspecific neutralizing antibodies, rapid blood clearance rate, and nonspecific liver uptake causing liver toxicity [12–14]. However for effective treatment of metastatic or dispersed cancers, a systemicallyinjectable Ad vector must be developed. For the development of oncolytic Ads that can be delivered systemically, genetic modification of Ads and conjugation with non-viral vectors has been extensively studied [15–17]. In recent years, encapsulation methods with various polymers through electrostatic interaction-mediated physical absorption [18,19], chemical conjugation via amine-terminated Ad surface [20,21], electrospinning-mediated Ad encapsulation [22], liposomal

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encapsulation [23–25], and antibody attachment for active targeting [21] have been investigated. Originally, these nonviral delivery systems had been studied to deliver plasmid DNA and siRNA [26– 28]. The advantages of nonviral vectors over viral vectors include easier large-scale production and lower immunogenicity, allowing for repeated treatments to maximize therapeutic efficacy. However, nonviral vectors escape endosomes at a lower frequency, resulting in low gene transfer efficiency compared to viral vectors [29]. To take advantages of both viral and nonviral properties, delivery of oncolytic viral genomes with a nonviral carrier has been assessed as an alternative approach of systemic cancer virotherapy. Combined hybrid delivery systems can effectively synergize oncolytic Ad-mediated high therapeutic efficacy and non-viral vector-mediated systemic delivery. Oncolytic viral genomes first arrive at tumor foci via systemic blood circulation with the aid of a nonviral carrier, and then translocate into the nucleus and subsequently replicate, generating infectious oncolytic Ad progenies within tumor cells. These newly generated oncolytic Ads subsequently lyse the cancer cells and infect neighboring cancer cells, augmenting the efficacy of cancer cell killing. This approach is also advantageous over nonviral vector-encapsulated conventional oncolytic Ads in that the viral genome DNA and nonviral vector complex do not contain any viral capsid proteins, whereas encapsulated Ads by nonviral vectors still express viral proteins that can induce nondesirable cytotoxic and immunogenic effects. Therefore, this attractive combination of features is safer as a result of a reduced propensity for inducing humoral and cellular adaptive immune responses to Ad capsid proteins. In addition, the robust efficacy of this approach lies in its ability to induce active viral replication in local tumor tissues via systemic delivery, thereby eliciting the full potential of systemic antitumor therapeutic effects. In this study, the antitumor efficacy of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-expressing armed oncolytic Ad genome DNA encapsulated in cationic liposomes delivered via systemic administration was investigated in an orthotopic lung cancer model. Efficacy of this approach was compared to that of naked oncolytic Ad genome DNA and conventional oncolytic Ad. Tumor development or regression in the orthotropic cancer model was visualized via bioluminescence imaging. In addition, induction of immune responses, in vivo biodistribution profile, and local viral replication in tumor tissues were also evaluated.

with 50 or 100 mT-d19/stTR oncolytic Ad viral particles (VP)/cell. At 48 h after infection, culture media was collected and each sample separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electrotransferred onto a polyvinylidene difluoride membrane (IPVH00010; Millipore, Bedford, MA). The membrane was incubated with anti-TRAIL (sc-7877; Santa Cruz Biotechnology, Santa Cruz, CA), and the blot was developed with the enhanced chemiluminescence system (LR01-01; Animal Genetics Inc., Hwaseong, Korea), according to the manufacturer's instructions. GFP-expressing replication-defective Ads (dE1/GFP) were also used to evaluate the immune response upon Ad transduction. Propagation of mT-d19/stTR and dE1/GFP Ads was performed in HEK293 cells and Ads were purified using the CsCl gradient method. For titration, VP numbers were measured by optical density at 260 nm, for which an absorbance value of 1 is equivalent to 1 × 10 12 VP/mL. The total Ad plasmid of the oncolytic mT-d19/stTR (pmT-d19/stTR) was amplified in DH5α Escherichia coli and purified using a QIAGEN Endofree Plasmid Kit (QIAGEN, Hilden, Germany). DNA concentration was measured by NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE) at 260 nm. The optimal conditions to form complexes of liposomes and DNA were determined by the following method: stock solutions (1 mM) of DOTAP and DOPE were prepared in chloroform, and the lipid mixtures of DOTAP/DOPE were prepared at a molar ratio of 1:1. The mixture was gently evaporated under argon gas, and the residue of solvent in the lipid film was dried under a vacuum for 3 h. Lipid films were then hydrated with distilled water for 24 h. Suspended lipids were then vortexed and subjected to three freeze–thaw cycles. To form bilayered liposome vesicles, the suspension was extruded 20 times through a polycarbonate membrane (100-nm pore size; Avanti Polar Lipids) fitted into an extruder. The liposome/DNA complexes were formed at DNA:lipid ratios ranging between 1:0.5 and 1:10. DNA was mixed with the liposome solution (100 μL) and incubated for 15 min to form the complexes, and DNA/liposome complexes were then loaded onto a 0.5% agarose gel in the presence of ethidium bromide. DNA was visualized using a UV transilluminator. The particle size and zeta potential of the DNA/lipid complexes were measured with dynamic laser scattering (DLS) at 488 nm and a MALVERN zetasizer Nano ZS particle analyzer (Malvern Instruments Ltd., Worcestershire, UK), respectively.

2. Materials and methods

2.3. MTT assay

2.1. Cell lines and materials

To evaluate the oncolytic Ad-induced cytopathic effects, 3 × 104 A549 or H1975 cells were grown to approximately 30–70% confluence in 24-well plates. Each well was treated with mT-d19/stTR (50 VP/cell), pmT-d19/stTR (4 μg), or pmT-d19/stTR (4 μg) + DOTAP/DOPE (24 μg) complexes in triplicate for 12 h at 37 °C, after which culture media was exchanged with fresh media. At 2, 3, 4, 5, 6, and 7 days post-treatment, 200 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma Chemical Corp, St. Louis, MO) solution (2 mg/mL) in phosphate buffered saline (PBS) was added to each well. Cells were incubated at 37 °C for 4 h, after which the supernatant was discarded, and the precipitate was dissolved in 1 mL dimethylsulfoxide. Plates were then read on a microplate reader at 540 nm. The number of living cells in a PBS-treated sample was similarly analyzed and the results were used as a 100% viability control.

Cell lines (HEK293, A549, and H1975) were purchased from American Type Culture Collection (ATCC, Manassas, VA). HEK293, A549, and firefly luciferase expressing A549 (A549-Fluc) cells were cultured in Dulbecco's modified Eagle's medium (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin–streptomycin (100 U/mL). H1975 cells were cultured in RPMI1650 medium (Hyclone). Avanti Polar Lipids (Alabaster, AL) was the source of 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). 2.2. Adenovirus and DNA/liposome lipoplex characterization and preparation

2.4. Inflammatory cytokine assay The mT-d19/stTR oncolytic Ad expresses E1A under the control of modified human telomerase reverse transcriptase (hTERT) promoter for selective replication in cancer cells, as well as secretable trimeric tumor necrosis factor-related apoptosis-inducing ligand (stTRAIL), which enhances apoptosis. The oncolytic Ad-mediated expression of stTRAIL was detected by western blot (Fig. 1B). A549 cells were cultured on 10 cm culture dishes for 24 h, and subsequently treated

To assess the in vivo inflammatory immune response induced by the vectors, PBS, mT-d19/stTR (1× 1010 VP), pmT-d19/stTR (30 μg), or pmTd19/stTR (30 μg)+ DOTAP/DOPE (180 μg) complexes were injected intravenously into each BALB/c mouse (Orientbio Inc, Seongnam, Korea). At 6 h post-injection, blood was harvested by retro-orbital bleeding, and enzyme-linked immunosorbent assay (ELISA) was used to

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manufacturer's instructions. The Ad genomes were quantitatively measured by quantitative real-time polymerase chain reaction (Q-PCR; TaqMan PCR detection; Applied Biosystems, Foster City, CA). A fluorescence probe (FAM-5′-CCTGAGCCCGAGCCAG-3′-NFQ) was designed so that the sense primer (5′-GGTCCTGTGTCTGAACCTGAG-3′) and the antisense primer (5′-GGCGGGTAGGTCTTGCA-3′) annealed to DNA within the Ad E1A gene region. DNA was amplified in duplicate for 40 cycles (ABI 7500; Applied Biosystems), and data was processed by the SDS 19.1 software package (Applied Biosystems). 2.7. Assessment of in vivo anti-tumor effects and histological and immunohistological analysis

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Fig. 1. The schematic representation showing adenoviral genome lipoplexes administered systematically (a) and the expression of TRAIL (b). The modified hTERT promoter was used to control the expression of E1A protein in a cancer cell-specific manner. An stTRAIL-expressing gene cassette was inserted into Ad E3 region. The expression of stTRAIL was verified by western blot in A549 cancer cells after infection with mT-d19/stTR oncolytic Ad at indicated VPs (50 and 100 VP/cell).

detect serum interleukin-6 (IL-6; DY406; R&D System, Minneapolis, MN). 2.5. Neutralizing antibody assays To assess Ad-specific immune response, serum Ad-neutralizing antibodies (Abs) were measured based on neutralization of GFPexpressing Ad (dE1/GFP). After intravenous injection of a single dose of mT-d19/stTR (1 × 10 10 VP), pmT-d19/stTR (30 μg), or pmTd19/stTR (30 μg) + DOTAP/DOPE (180 μg) complexes into male BALB/c mice (Orientbio Inc), the same amount of each vector was re-administered 14 days later. Whole blood was collected from the retro-orbital vein 14 days after the second injection. Mouse serum complement proteins were inactivated by heating serum samples for 45 min at 56 °C. After diluting the heat-inactivated serum in PBS (1:50), diluted serum was then mixed with dE1/GFP and incubated for 1 h at 37 °C. U343 cells were treated with these serum-treated Ads at 10,000 VP/cell and incubated for 48 h at 37 °C. The degree of GFP expression was then observed by fluorescence microscopy (Olympus BX51; Olympus Optical, Tokyo, Japan) and analyzed with the Metamorphic Imaging System (Molecular Devices, Sunnyvale, CA). In addition, GFP expression was quantitatively measured by flow cytometry (LSR2; Beckton-Dickinson, San Jose, CA) and data were collected from 15,000 cells and analyzed with CellQuest software (Beckton-Dickinson).

To establish the human orthotopic lung tumor model, A549-Fluc cells (3 × 10 6), which stably express firefly luciferase, were intravenously injected into 7 week-old male nude mice (Orientbio Inc). At three-weeks post-injection, optical imaging was performed with IVIS II (Caliper Life Sciences; Hopkinton, MA) and mice were divided into four separate systemic treatment groups (n = 6, each group): PBS, mT-d19/stTR (5 × 10 9 VP), pmT-d19/stTR (30 μg), or pmT-d19/stTR (30 μg) + DOTAP/DOPE (180 μg). Each treatment modality was administered over 3 consecutive days. Tumor growth was assessed by optical imaging on day 0, 7, 14, 21, and 28, and the signal intensity of the region of interest was quantitatively analyzed with IGOR-PRO Living Image software (Caliper Life Sciences). After in vivo imaging of lung and tumor tissues on day 28, these tissues were harvested and weighed. For histological and immunohistological analysis, tumor-bearing lungs were harvested 28 days after first systemic treatment, fixed in 10% formalin, and embedded in paraffin (Wax-it; Vancouver, Canada). Tumor sections (5 μm thick) were stained with rabbit anti-Ad E1A (sc-430; Santa Cruz Biotechnology) or mouse anti-TRAIL monoclonal antibody (556468; Beckton-Dickinson). The in situ ApopTag kit (S7100; Chemicon International) was used to detect apoptotic cells via detection of cleaved deoxyribonucleic acid. 2.8. Assessment of Ad biodistribution by quantitative PCR When A549-Fluc orthotopic tumor volume had grown to approximately 50 mm 3, tumor-bearing mice were injected intravenously with PBS, mT-d19/stTR (1 × 10 10 VP), pmT-d19/stTR (30 μg), or pmTd19/stTR (30 μg) + DOTAP/DOPE (180 μg) complexes three times every other day. At 24 h after last treatment, tumor and liver tissues were harvested. DNA was extracted from harvested tissues using the GENEALL Clinic SV mini kit (General Biosystem, Seoul, Korea). Q-PCR was used to assess the number of viral genomes in each sample. 2.9. Statistical analysis All data are expressed as mean ± SE. The Mann–Whitney test (nonparametric rank-sum test) was applied with Stat View software (Abacus Concepts Inc., Berkeley, CA) in order to make statistical comparisons. Results were compared with the log-rank test (SPSS software v.13.0; SPSS Inc., Chicago, IL). Differences were considered statistically significant when P b 0.05. 3. Results

2.6. Determination of Ad clearance from blood 3.1. TRAIL expression To assess the clearance rate of vector from the blood, 50 μL of blood was harvested through retro-orbital bleeding at 5 min, 10 min, 20 min, 30 min, and 1 h after intravenous administration of mT-d19/stTR (1 × 10 10 VP), pmT-d19/stTR (30 μg), or pmT-d19/stTR (30 μg) + DOTAP/DOPE (180 μg) complexes to BALB/c mice (Orientbio Inc). DNA was extracted from blood samples and resuspended to a final volume of 100 μL using the QIAamp DNA blood mini kit (QIAGEN) according to the

A diagram showing the experimental plan for administering oncolytic Ad genome DNA/lipoplexes is shown in Fig. 1a. For selective Ad replication in cancer cells, the mT-d19/stTR oncolytic Ad expresses E1A under the control of modified hTERT promoter (Fig. 1b) [8]. The mT-d19/stTR oncolytic Ad also expresses stTRAIL, which enables induction of apoptosis. TRAIL expression from mT-d19/stTR oncolytic

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To determine the optimal conditions for the generation of DNA and liposome complexes, the ratio of DNA and lipids was altered by a sequential increase in lipid concentration (DNA:lipid ratio range: 1:0.5 to 1:10). The results of a gel retardation assay revealed that DNA lipoplex retardation began at a DNA:lipid weight ratio of 1:2 and were completely retarded at a weight ratio of 1:8. This retardation is due to the decreased free form of DNA, demonstrating strong interactions between DNA and lipids, generating tight complex formation (Fig. 2a). After incubation with HindIII restriction enzyme for 4 h, DNA lipoplex retardation was detectable at a DNA:lipid ratio of 1:4 and were completely retarded at the same weight at a ratio of 1:8, indicating that the Ad DNA complexed with liposomes was protected from degradation by this restriction enzyme. We infer that, because DNA was absorbed on the liposome surface, HindIII did not have access to bind near the DNA cleavage site. Based on these results, we generated DNA lipoplexes at a weight ratio of 1:6 (DNA:lipid) for the experiments that follow. The viral DNA lipoplexes generated at a weight ratio of 1:6 (DNA: lipid) were then measured for average size and surface charge using DLS and a zeta potentiometer, respectively (Fig. 2b). As viral DNA was added to the DOTAP/DOPE lipids to generate complexes, the hydrodynamic diameter increased from 88.3 ± 0.3 nm to 144.3 ± 5.7 nm. Concurrently, the zeta potential as the viral DNA combined with DOTAP/DOPE decreased from 45.9± 1.9 mV to 39.5 ± 0.5 mV, demonstrating that positive-charged liposomes were well complexed with negativelycharged Ad plasmid DNA.

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Fig. 2. Gel retardation assay and pmT-d19/stTR + DOTAP/DOPE complex characterization. (a) Optimal conditions for formation of Ad genome lipoplex complexes were determined by gel retardation assay. Ad genome DNA was combined with liposomes at varied weight ratios of DNA to lipid without (first panel) and with (second panel) restriction enzyme, Hind III, incubation. The complexes were then loaded on a 0.5% agarose gel and electrophoresis was performed. (b) The average particle size and surface charge of mT-d19/stTR, pmT-d19/stTR, and pmT-d19/stTR + DOTAP/DOPE at a DNA:lipid ratio of 1:6 were measured by DLS and zeta potential analyzer, respectively.

Ad was confirmed by western blot in A549 cells infected with mTd19/stTR (50 or 100 VP/cell). As viral titers of mT-d19/stTR oncolytic Ad increased, the amount of TRAIL expressed also increased, demonstrating dose-dependent TRAIL expression.

To assess the cancer cell-killing effects of oncolytic viral DNA lipoplexes, cell viability was measured via MTT assay in A549 and H1975 lung cancer cell lines at varied incubation time points (Fig. 3). Cells were treated with mT-d19/stTR oncolytic Ad, pmT-d19/stTR Ad genome DNA, or pmT-d19/stTR+ DOTAP/DOPE complexes. The viability of cells incubated with PBS, which served as a negative control, was set as 100% for both cell lines. Cells treated with pmT-d19/stTR naked plasmid DNA demonstrated no cell killing effect until 6–7 days after treatment, while both oncolytic virus (mT-d19/stTR) and viral genome DNA lipoplexes (pmT-d19/stTR+ DOTAP/DOPE) exhibited potent killing effects, with cell killing of 70.4% for mT-d19/stTR and 61.2% for pmTd19/stTR + DOTAP/DOPE in A549 cells on day 7. A cancer cell killing assay with H1975 cells demonstrated similar results. Interestingly, the onset of A549 cancer cell killing mediated by the oncolytic Ad began early after treatment, while pmT-d19/stTR+ DOTAP/DOPE demonstrated effective cell killing at later time points: mT-d19/stTR demonstrated 29.0% cell killing versus only 6.6% cell killing for pmT-d19/stTR+ DOTAP/DOPE at 2 days after treatment. The oncolytic cell killing effects in the same cell line treated with Ad DNA lipoplexes steadily increased to 61.2% on day 7, which is close to that of oncolytic Ad (70.4%), demonstrating that Ad DNA in the lipoplexes effectively generates active oncolytic Ad progenies after time was allowed for viral replication. Importantly, the oncolytic effects of Ad DNA lipoplexes was as high as those elicited from the efficient infection of oncolytic Ad in CAR-positive A549 and H1975 cells, suggesting that delivery of oncolytic viral DNA in the form of lipoplexes can overcome low cellular uptake efficiency of non-viral vectors. These effects are likely due to the prevention of Ad DNA degradation and continuous Ad replication within cancer cells. 3.4. Pharmacokinetic profiles of vectors We then examined the pharmacokinetics of oncolytic Ad, oncolytic Ad genome DNA, or oncolytic Ad genome DNA lipoplexes after systemic administration into BALB/c mice. After administration of mT-d19/stTR (1 × 10 10 VP), pmT-d19/stTR (30 μg), or pmT-d19/stTR (30 μg) + DOTAP/DOPE (180 μg) complexes, the number of viral DNA

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Days Fig. 3. In vitro cytotoxicity of naked oncolytic Ads, oncolytic Ad plasmids, and oncolytic viral DNA lipoplexes in A549 and H1975 lung cancer cells. Cells were treated with PBS (♦), mT-d19/stTR (■), pmT-d19/stTR (▲), or pmT-d19/stTR + DOTAP/DOPE (●), followed by cell viability determination at the day indicated. At each time point, the cell viability of PBS-treated cells was set at 100%, and cell viability of each Ad was normalized to PBS-treated cells.

The Ad vector-mediated innate inflammatory immune response is the primary limiting factor in systemically administered Ad vectors. We therefore assessed the innate immune response after systemic administration of naked Ad, viral plasmid, or viral DNA lipoplexes. Blood samples were taken at 6 h post-systemic injection of each Ad formulation into BALB/c mice and IL-6 was measured purified serum by ELISA. As shown in Fig. 5a, serum IL-6 increased approximately 5-fold in the mT-d19/stTR-treated group (40.0 pg/mL) compared to the PBS-treated group (8.3 pg/mL). In marked contrast, serum IL-6 in pmT-d19/stTR-treated group was the 18.4 pg/mL, which was 2.2-fold less than that of naked Ad-treated group. Moreover, serum IL-6 expression in pmT-d19/stTR + DOTAP/DOPE-treated mice was approximately 2.3-fold lower (17.4 pg/mL) than that of naked Ad-treated group. These results indicate that Ad genome lipoplexes can significantly reduce the innate immune response mounted against Ad in vivo. The adaptive immune response induced by Ad DNA lipoplexes was then examined in BALB/c mice. Naked Ads, Ad plasmids, or viral DNA lipoplexes was intravenously administered twice, 14 days apart. Fourteen days following the second injection, Ad-specific neutralizing antibodies present in the serum were measured based on reduction in GFP expression by dE1/GFP Ad. These results revealed that the infectivity of dE1/GFP pre-incubated with serum from mT-d19/stTRtreated mice was reduced by 97.5% compared to that from PBS-treated mice (vs. PBS group, P b 0.001; Fig. 5b), indicating that naked mTd19/stTR-treated mice produced large amounts of neutralizing antibodies that recognize and inactivate Ad. In contrast, the transduction level of dE1/GFP was not significantly reduced in cells treated with the sera of pmT-d19/stTR- or mT-d19/stTR + DOTAP/DOPE-treated mice, resulting in a GFP level that was 82.6% and 79.6%, respectively, of that resulting from sera of PBS-treated mice (vs. mT-d19/stTR group, P b 0.001; Fig. 5b). These data indicate that complex formation of viral DNA with liposome can prevent the generation of Ad-specific neutralizing Abs in vivo. Consistent with the innate immune response data, these results show that DNA lipoplexes are a safe formulation that prevents induction of both innate and adaptive immune response against Ad. 3.6. Therapeutic efficacy of DNA lipoplexes in the A549 orthotopic lung cancer model

copies in the blood was measured by real-time Q-PCR at various time periods (5 min to 1 h) in order to detect the clearance of vectors from the blood. The number of Ad genome copies measured at each time point was normalized to the number of Ad genome copies measured at 5 min, which was considered 100%. Rapid clearance in blood over time was evident in the mT-d19/stTR oncolytic Ad-treated mice (Fig. 4). At 10 min post-injection, only 24.3% of injected oncolytic Ads (5.81 × 10 7 VP) was retained in blood, which fell to 6% (1.44 × 10 7 VP) at 30 min post-injection. It has been previously demonstrated that the hexon capsid protein on the Ad surface interacts with factor X (FX) in the blood, resulting in rapid localization of Ad to the liver and liver toxicity [30]. These previous results support the short blood circulation time of the oncolytic Ads detected in this study. The level of naked Ad plasmid in the pmT-d19/stTR-treated group was 78.4% of injected plasmid at 10 min and 61.9% at 20 min, thus demonstrating a longer blood circulation time compared to the oncolytic Ad-treated group (Fig. 4). However, naked Ad genome DNA was completely cleared from the blood at 1 h post-injection, requiring more sustained circulation. Lipoplex-treated mice also demonstrated higher levels of blood circulation compared to naked Ad at early time points and at 1 h after injection (22.6% of injected dose; 8.56 × 107 VP). These results demonstrate that the lipoplex formulation of naked Ad

To compare the anti-tumor efficacy of viral DNA lipoplexes relative to naked oncolytic Ads or oncolytic Ad plasmids, tumor growth was measured by tracking luciferase-expressing A549 cells. Bioluminescence images of whole mice treated with PBS, mT-d19/stTR, pmT-d19/stTR, or pmT-d19/stTR+ DOTAP/DOPE are shown in Fig. 6a. As the tumor grew, the luminescence signal increased significantly in mice treated with PBS, mT-d19/stTR, or pmT-d19/stTR for 28 days after systemic administration of vectors. In contrast, the luciferase signal intensity in mice treated with pmT-d19/stTR+ DOTAP/DOPE was similar to that in these mice at day 0, demonstrating the latter as the most effective formulation to inhibit tumor growth in A549 orthotopic lung cancer xenografts. The bioluminescence signal intensity was quantitatively analyzed by measuring the total photon flux (Fig. 6b). The total photon flux of the PBS-treated group increased 20-fold from day 0 (7.54 × 10 5 photon/s) to day 28 (1.47 × 10 7 photon/s). The photon flux of mice treated with pmTd19/stTR and mT-d19/stTR increased 14.0-fold (1.05 × 107 photon/s) and 13.6-fold (8.42× 106 photon/s), respectively, from day 0 to day 28. The group treated with the viral plasmids did not demonstrate statistically significant antitumor effects, probably due to low rate of cancer cell transfection, rapid blood clearance, and DNA degradation in the blood. Naked oncolytic Ads also induced weak antitumor effects,

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Fig. 4. Pharmacokinetics of mT-d19/stTR, pmT-d19/stTR, and pmT-d19/stTR + DOTAP/ DOPE. BALB/c mice were intravenously injected with mT-d19/stTR (1 × 1010 VP), pmT-d19/stTR (30 μg), or pmT-d19/stTR (30 μg) + DOTAP/DOPE (180 μg) and blood samples were taken at 5 min, 10 min, 20 min, 30 min, and 1 h. The number of Ad genomes present in the blood was measured by Q-PCR. The number of viral genomes at 5 min after systemic injection was set as 100% in each group.

*** probably due to the nonspecific uptake by the liver, induction of immune responses, and rapid blood clearance. Notably, the bioluminescence tumor signal in pmT-d19/stTR+ DOTAP/DOPE-treated mice increased 1.2-fold (8.01× 105 photon/s) from day 0 to day 28, demonstrating inhibition of tumor growth compared to PBS-, pmT-d19/stTR-, or mTd19/stTR-treated groups of 94.5%, 92.4%, and 90.5%, respectively (vs. PBS group, P b 0.01; vs. pmT-d19/stTR, P b 0.05; vs. mT-d19/stTR, P b 0.05). Tumor-bearing lung tissues were harvested at 28 days after the first injection of each vector and bioluminescence signals in the lungs were measured ex vivo, followed by quantitative analysis. Merged bright field and luminescence images are shown in Fig. 6c and the total photon flux of whole ex vivo tumors is shown in Fig. 6d. The bioluminescence signal intensity in pmT-d19/stTR + DOTAP/DOPEtreated lung tissue was reduced by 93.9% compared to that of lung tissues from PBS-treated mice (P b 0.01), whereas total photon flux of ex vivo lung tissues from mice treated with mT-d19/stTR or pmTd19/stTR showed a signal intensity similar to that from PBS-treated groups. Additionally, the signal intensity from lungs of pmT-d19/stTR + DOTAP/DOPE-treated mice was reduced by 91.4% compared to lungs of mice treated with mT-d19/stTR naked oncolytic Ad (P b 0.05), demonstrating the superior antitumor effects of oncolytic Ad DNA lipoplexes over naked Ad.

3.7. Ad genome DNA lipoplexes improve tumor-to-liver biodistribution ratio The in vivo biodistribution of naked oncolytic Ad, Ad genome DNA, and oncolytic Ad DNA lipoplexes were examined in mice bearing A549 orthotopic lung tumors. At 24 h after the third administration of each vector, Ad genome content in liver and lung tumor tissues were measured by Q-PCR and normalized to cellular genomic DNA (Fig. 7a). As expected, the highest Ad genome copy numbers were detected in liver of mT-d19/stTR-treated mice (2.6× 107 VP/50 ng DNA), whereas the amount of pmT-d19/stTR (6.6× 104 VP/50 ng DNA) and pmT-d19/stTR + DOTAP/DOPE (1.3× 106 VP/50 ng DNA) in the liver were significantly

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Fig. 5. Assessment of immune response against Ad. (a) Serum levels of the proinflammatory cytokine IL-6. At 6 h after systemic injection of mT-d19/stTR (1× 1010), pmT-d19/stTR (30 μg), or pmT-d19/stTR (30 μg)+ DOTAP/DOPE (180 μg), blood samples were taken, and serum IL-6 was measured by ELISA. *P b 0.05 vs. mT-d19/stTR-treated group. (b) The level of Ad-specific neutralizing antibodies presents in the blood based on inhibition of GFP expression (see Materials and Methods). GFP expression was assessed by fluorescence microscopy and FACS. ***P b 0.001 vs. mT-d19/stTR treatment. The error bars represent± SE. Original magnification: ×100.

lower than in the mT-d19/stTR-treated mice (Pb 0.01). In marked contrast, significantly higher number of viral genomes were detected in tumor tissues of mice injected with pmT-d19/stTR + DOTAP/DOPE (4.2 ×106 VP/50 ng DNA) compared to naked Ads (9.10 × 104 VG/50 ng DNA) or naked Ad genome plasmids (7.9 × 10 3 VG/50 ng DNA). Importantly, the tumor-to-liver ratio, which is an important indicator of therapeutic efficacy as well as clinical safety, in pmT-d19/stTR + DOTAP/DOPE-treated mice was significantly elevated by 934- and 27-fold compared to mice treated with mT-d19/stTR or pmT-d19/stTR, respectively (Fig. 7b). These results indicate that Ad DNA-liposome hybrid vectors exhibit reduced non-specific liver uptake and enhanced accumulation of Ad genome in tumor tissues.

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Fig. 6. In vivo and ex vivo optical imaging to assess anticancer efficacy in orthotopic lung tumor xenograft models. (a) Luciferase-expressing A549 (A549-Fluc) orthotopic lung tumorbearing mice were monitored by bioluminescence imaging. At 2 weeks after A549-Fluc cell implantation, PBS (♦), mT-d19/stTR (▲), pmT-d19/stTR (■), or pmT-d19/stTR + DOTAP/DOPE (×) were intravenously injected and luciferase expression was monitored every week following treatment. (c) Tumor-bearing lungs were harvested at 28 days after first vector treatment and subsequently imaged. In vivo (b) and ex vivo (d) average optical signal intensity, expressed as photons acquired per second in regions of interest. Optical signals from cancer cells are expressed as mean ± SE. *P b 0.05, pmT-d19/stTR + DOTAP/DOPE vs. mT-d19/stTR or vs. pmT-d19/stTR treatment.

3.8. Immunohistochemical analysis and apoptosis We then examined Ad distribution, therapeutic effects, and TRAIL gene expression in tumor-bearing lung tissues. Tumor-bearing lungs were harvested from mice in each treatment group at 28 days after the first administration of each vector type. PBS-, mT-d19/stTR-, and

pmT-d19/stTR-treated mice developed lung tumors with broad distribution in exterior lung tissue, but normal tumor-free tissue located in the inner lung (Fig. 8; hematoxylin-eosin staining). Conversely, only few, small tumor nodules were detected in the pmT-d19/stTR +DOTAP/DOPE-treated group, with large areas of porous tissues indicative of normal lung tissue. Importantly, dark

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Fig. 8. Histological and immunohistological analysis of tumor-bearing lung sections. Representative light micrographs of hematoxylin and eosin (H & E) staining, Ad E1A expression, and TRAIL expression in tumor-bearing lungs tissue. (N, normal lung tissue; T, tumor tissue). Abundant Ad E1A and TRAIL expression was detected in lungs of the pmT-d19/stTR + DOTAP/DOPE-treated group, demonstrating active and persistent Ad replication in tumor tissues. TUNEL staining indicates apoptotic cells. Colocalization of E1A-, TRAIL-, and TUNEL-positive apoptotic cell populations is apparent. Original magnification: × 40, ×400.

complexes of oncolytic Ad genomes and liposomes utilize advantages of both viral and non-viral systems. Further investigation is needed for the therapeutic application of this approach to treat metastatic and disseminated cancers. Fig. 7. Biodistribution of luciferase-expressing A549 orthotopic lung tumor-bearing mice. BALB/c mice were systemically injected with mT-d19/stTR (1× 1010 VP), pmT-d19/stTR (30 μg), or pmT-d19/stTR (30 μg)+ DOTAP/DOPE (180 μg) three times every other day. (a) Ad genome content of liver and tumor tissues at 24 h after final treatment. DNA was extracted from tissue and viral genomes were quantified using Q-PCR. Data are expressed as mean ± SE and n = 3 for each experimental condition. (b) The tumor-to-liver ratio of viral genomes, which was normalized to the mT-d19/stTR-treated group.

brown spots representing E1A and TRAIL proteins were broadly distributed throughout the lung tumor tissues of mice in the pmTd19/stTR + DOTAP/DOPE-treated group, indicating active virus replication with concurrent high expression of the therapeutic gene TRAIL. In pmT-d19/stTR + DOTAP/DOPE-treated mice, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells were also abundant in the same region of tissue that included E1A- and TRAIL-positive cells. Taken together, these data indicate that the systemic administration of oncolytic viral DNA with lipoplexes (pmT-d19/stTR + DOTAP/DOPE) induced potent therapeutic effects due to a reduction in innate and adaptive immune responses, prolonged blood circulation time, improved biodistribution profile, active and continuous replication of oncolytic Ad in tumor tissues, and stable, high TRAIL expression. Systemic delivery of non-viral Ad genome lipoplexes results in the selective replication of Ads in cancer cells in situ, subsequent active oncolysis of tumor cells, and high expression of therapeutic genes. These properties overcome the common limitations of systemic administration of oncolytic viruses. The hybrid

4. Conclusion The genome of oncolytic virus was systemically delivered with liposomes and the therapeutic efficacy of these complexes was examined in an orthotopic lung tumor xenograft model. Oncolytic Ad genome lipoplex-treated mice mounted significantly reduced innate and adaptive immune responses against Ads, as well as subsequent prolonged stability and blood circulation time. The bioluminescence signal of tumor tissues was the lowest in mice treated with pmTd19/stTR + DOTAP/DOPE, demonstrating a higher therapeutic efficacy than those of naked oncolytic Ads or Ad genome plasmids. Moreover, high tumor-to-liver ratios in mice treated with pmT-d19/stTR + DOTAP/DOPE indicate that oncolytic viral genomes were accumulated at high concentrations in tumor tissues after systemic administration by enhanced permeability and retention-mediated preferential tumor targeting and subsequent active in situ viral replication. Furthermore, high TRAIL gene expression and a high proportion of TUNEL-positive apoptotic tumor cells indicate potent antitumor effects. In summary, these results emphasize the advantages of combining oncolytic viral gene delivery with a nonviral carrier, demonstrating the potential use of oncolytic Ad genome lipoplexes as an alternative to naked oncolytic Ad for the systemic treatment of metastatic cancers. Acknowledgments This research was supported by grants from the Ministry of Knowledge Economy (10030051 to Dr. C-O Yun); the Korea Science

and Engineering Foundation (R15-2004-024-02001-0, 2009 K001644, 2010-0029220 to Dr. C-O Yun); the Korea Food and Drug Administration (KFDA-11172-358 to Dr. C-O Yun); a Faculty Research Grant from Yonsei University College of Medicine (6-2010-0052 to Dr. C-O Yun).

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Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10. 1016/j.jconrel.2011.06.014.

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