Enhanced Antitumor Response Mediated by the Codelivery of

Mar 27, 2013 - Paclitaxel and Adenoviral Vector Expressing IL-12 ... KEYWORDS: co-delivery, adenovirus, interleukin-12, paclitaxel, anionic liposomes,...
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Enhanced Antitumor Response Mediated by the Codelivery of Paclitaxel and Adenoviral Vector Expressing IL-12 Linjie Cao,† Qin Zeng,† Chaoqun Xu,‡ Sanjun Shi,† Zhirong Zhang,† and Xun Sun*,† †

Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, P. R. China ‡ Sichuan Academy of Chinese Medicine Sciences, Chengdu, Sichuan, 610041, P. R. China

ABSTRACT: It has been well-established that chemo-immunotherapy using cytotoxic drugs and appropriate cytokines offers a promising approach for the treatment of neoplastic diseases. In view of this, to improve melanoma treatment effect, our study developed a new codelivery system (AL/Ad5/PTX) that paclitaxel (PTX) and adenovirus encoding for murine interleukin-12 (Ad5-mIL-12) were incorporated into anionic liposomes (AL). First, AL/Ad5/PTX complexes were prepared by incorporating Ad5 into anionic PTX liposomes using calcium-induced phase change. Second, the size distribution and zeta potential of AL/ Ad5/PTX were investigated. Third, the results of in vitro transduction assays showed that PTX introduced into AL/Ad-luc or AL/Ad5-mIL-12 highly enhanced gene transduction efficiency in B16 cells than naked Ad5 or AL/Ad complexes while it had no comparability in A549 cells. Finally, a melanoma-bearing mouse model was established to assess the antitumor effect. Tumor growth inhibition and prolonged survival time, accompanied by increased mIL-12 or interferon-γ (IFN-γ) expression levels in serum or tumor sites, were observed in mice treated with AL/Ad5-mIL-12/PTX, as compared with those treated with either AL/ Ad5-mIL-12 or AL/PTX. In conclusion, these results suggested that codelivery of Ad5-mIL-12 and PTX incorporated into AL could be a relatively efficient strategy for the treatment of melanoma. KEYWORDS: co-delivery, adenovirus, interleukin-12, paclitaxel, anionic liposomes, melanoma

1. INTRODUCTION

has been reported that IL-12 has potent in vivo antitumor and antimetastatic activity against murine tumors.5 In fact, recombinant IL-12 has shown remarkable properties as an anticancer agent in various animal models.6 Subsequent phase I and II studies on various cancer types using intravenous or subcutaneous administration of human rIL-127,8 showed some beneficial responses, but there were also adverse effects that interrupted the clinical trials.9 Based on those problems stated above, alternative approaches, including gene therapy, for the delivery of IL-12 have been pursued. For example, intratumoral administration of a recombinant adenovirus encoding for the IL-12 gene for the treatment of metastatic colon cancer;10 cancer gene therapy by IL-12 gene delivery using liposomal bubbles and tumoral ultrasound exposure;11 transfer of IL-12

Despite advances in the development of new chemotherapeutic drugs and improvements in radiation therapy, conventional cancer therapy often falls short of the goal of controlling tumor progression. Cancer immunotherapy has been considered as a very attractive option among the many approaches to cancer treatment.1 Recent cancer vaccination studies in animal models and humans have demonstrated that IL-12 remains a very promising immunotherapeutic agent.2 IL-12 is a heterodimeric protein composed of two disulfide-linked subunits with molecular weights of 40 kDa (p40) and 35 kDa (p35).3 It is primarily produced by antigen-presenting cells (APCs) such as dendritic cell macrophages.3 IL-12 has an essential role in the interaction between innate and adaptive immune responses, acting as a mediator in the communication between APCs and lymphocytes. It also has a variety of immune-modulatory antitumor effects including inducing interferon-γ (IFN-γ) and promoting maturation of cytotoxic T lymphocytes (CTLs).4 It © 2013 American Chemical Society

Received: Revised: Accepted: Published: 1804

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MDCK and CHO compared to that of naked Ad5.32 In this study we sought to further explore the transduction efficiency of AL/Ad5/PTX in vitro and the combined effect of immunotherapy and chemotherapy on melanomas in vivo. B16 cells (CAR deficient)33 and A549 cells were chosen for in vitro studies. For in vivo, Ad5 encoding mIL-12 and PTX were coencapsulated into anionic liposomes (AL/Ad5-mIL-12/PTX) as a codelivery system and then were injected into melanomabearing mice for determining therapeutic index as compared with the complex composed of either AL/Ad5-mIL-12 or AL/ PTX (Figure 1).

cDNA via gene gun-mediated transfection of skin tissue overlaying subcutaneous melanomas;12 and injection of genetically engineered fibroblasts for the secretion of IL-12 in an orthotopic melanoma model.13 These studies suggest that IL-12 gene therapy could result in tumor regression and suppression of metastasis. Adenovirus vectors (Ads) are nonenveloped double-stranded DNA viruses and have the potential to become the dominant gene delivery vector and anticancer vaccination.14 Several results have suggested that adenoviral vector vectors expressing mouse IL-12 are effective in the treatment of cancer.15,16 However, Sangro et al. demonstrated that intratumorally administered Ad5 expressing IL-12 had low efficacy in a phase I trial for advanced digestive tumors. This low antitumor effect might be due to poor transduction efficiency.7 For entering into target cells, Ad5 needs to initially bind to appropriate receptors on cell members, such as the coxsackievirus and adenovirus receptor (CAR).17 A number of studies have been reported to correlate with CAR expression and the efficiency of adenoviral transgene expression, suggesting that adenoviral binding and entry into target cells is a critical step in achieving successful adenoviral gene expression.18 Unfortunately, the loss of CAR expression has been frequently observed in established cancer cell lines and clinical cancer specimens,19 thus hampering efforts to perform efficient adenoviral gene therapy in cancer patients. With this in mind, a number of efforts have been made to improve the transduction ability of Ad5 into those CAR-negative cells. A number of previous studies using cell lines expressing reduced or no levels of CAR have shown that cationic liposome-Ad5 complexes were able to induce significantly higher levels of transgene expression when compared to uncomplexed adenoviral vectors. These observations highlight some of the benefits of using this gene delivery system.20−22 However, cytotoxicity and inactivation in the presence of serum23 have restricted the potential applications of some cationic liposomal vectors, while anionic lipids in general are less cytotoxic, and some of them are composed of naturally occurring membrane lipids.24 As a result, there have been reports using anionic liposomes as alternatives to cationic liposomes in gene therapy.25,26 It has been established that chemo-immunotherapy using cytotoxic drugs and appropriate cytokines offers a new approach to increasing the therapeutic index in the treatment of neoplastic diseases.27 Moreover, some chemotherapeutics were found to exert their therapeutic effects not only by direct inhibition of the proliferation of, or cytotoxic effect on, tumor cells, but also by modifying the host responses against the tumors.27 For example, paclitaxel (PTX) was reported to activate some functions of macrophages.28 As an anticancer agents, PTX has been successfully utilized in clinical settings and has demonstrated significant antitumor efficacy against breast and ovarian cancers and other solid tumors29 since it was identified in 1971. Moreover, PTX has been reported to significantly augment IL-12 efficacy in tumor models.30 Incorporation of PTX in liposomes not only eliminates the hypersensitivity reactions associated with the Cremophor EL vehicle but also decreases the toxicities that arise from the drug′s pharmacological action.31 Therefore, PTX was our choice as the chemotherapeutic agent. In a previous study, we had prepared one version of Ad5 and anionic liposome complexes (AL/Ad5), which showed a much higher transduction efficiency in the CAR-deficient cell lines

2. MATERIALS 2.1. Biological Materials. Paclitaxel (purity >99.5% by HPLC) was obtained from Hao-xuan Biotechnology Co., Ltd. (Xi’an, China). Cholesteryl hemisuccinate (CHEMS), a negatively charged cholesterol derivative, N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Corp (St. Louis, MO). Egg phosphatidylcholine (PC) was purchased from Doosan Korea. Cholesterol and ethylenediaminetetraacetic acid (EDTA-disodium salt) were purchased from Amersco. Purified antimouse IL-12, antimouse IL-12/IL-23 p40 Biotin, recombinant mouse IL-12 p70, and Avidin-HRP were purchased from eBioscience (San Diego, CA). Purified antimouse IFN-γ, Biotin antimouse IFN-γ, and recombinant mouse IFN-γ (ELISA Std.) were purchased from Biolegend (San Diego, CA). 2.2. Cell Culture and Animals. A549 lung carcinoma and B16 melanoma cell lines were maintained in RPMI-1640 medium (GIBCO-BRL) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin, incubated at 37 °C in a humidified atmosphere with 5% CO2. C57BL/6 mice were purchased from the Laboratory Animal Center of Sichuan University. Six-to-eight-week-old male mice were housed under specific pathogen-free conditions and treated according to institutional animal care and use guidelines. 2.3. Adenoviral Vectors. Replication-deficient adenoviral vectors encoding LacZ reporter gene (Ad5-LacZ) or Photinuspyralis luciferase (Ad5-luc) were constructed and purified as previously described.32 Ad5 encoding mIL-12 (Ad5-mIL-12) was kindly provided by State Key Laboratory of Biotherapy, Sichuan University, in which pORF-mIL-12 was placed under the transcriptional control of the cytomegalovirus immediate-early gene promoter (CMV); this vector mediates transient expression of very high levels of biologically active mIL-12 both in vitro and in vivo.34 3. METHODS 3.1. Preparation of Ad5 and PTX Anionic Liposomes. The blank anionic paclitaxel liposome composition was PTX, CHEMS, PC, and Chol (1.5:4:5:1 molar ratio); liposomes were prepared by the thin film hydration and sonication dispersion technique. Complexes of naked Ad5 and anionic PTX liposomes (AL/ Ad5/PTX) were formed on the basis of a calcium-induced phase change method. First, 100 mM CaCl2 was added to the anionic PTX liposomes to a final concentration of 10 mM and then incubated at 37 °C for 1 h. Next, the resulting precipitate 1805

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the gene transduction efficiency between AL/Ad5/PTX and AL/Ad5. Additionally, in order to make the detection more accurate and convenient, the Ad5 used here encoded luciferase (luc). For the transduction, B16 or A549 cells were seeded in 24-well plates at a density of 1 × 105 cells per well and cultivated in 0.5 mL of 1640 growth medium for gene transduction. After 24 h, naked Ad5-luc, AL/Ad5-luc, or AL/ Ad5-luc/PTX in 0.3 mL of medium, with serum, were gently added to each well, in which Ad5-luc was at a MOI of 25. After 4 h incubation, the cells were washed twice with cold PBS and replaced with fresh culture medium. Cells were assayed for gene expression after two days. Here, we further evaluated the influence of AL/PTX on the transduction efficiency of naked Ad5 (AL/PTX + Ad5-luc) or AL/Ad5-luc (AL/PTX + AL/ Ad5-luc) by adding AL/PTX 1 h prior to the addition of the other treatments, and the process was similar to that described above. For the detection of luciferase gene expression, the cells were washed twice with PBS, and 0.1 mL of cell lysis buffer (Promega) was added to each well, followed by incubation at 4 °C for 20 min. The cell lysates were centrifuged at 4 °C for 5 min at 12 000 r.p.m to pellet any debris; 20 μL of each supernatant was added to a Thermo Immulno/MicroFluor Microlite 96-well plate, and the luciferase activity was detected using a Luciferase Assay System Kit (Promega).The relative light units (RLU) were measured using a Varioskan Flash spectral scanning multimode reader (Thermo Scientific), and the protein contents were detected by the BCA protein assay kit (Pierce). The data were expressed as RLU per mg of protein. 3.4.2. In Vitro mIL-12 Gene Transduction. We further evaluated the transduction efficiency of various samples in B16 cells with the Ad5 encoding mIL-12. The process of transduction was similar to that described in section 3.4.1. After 48 h of transduction, the media were sampled from each of the wells, and mIL-12 levels were assayed by an enzymelinked immunosorbent assay (ELISA). 3.5. In Vivo Experiments. 3.5.1. Therapeutic Experiments. For in vivo therapeutic experiments, B16 cells were trypsinized, harvested by centrifugation at 1500 for 5 min, and washed twice with PBS. A single-cell suspension was prepared in PBS, and the cell density was adjusted to 5 × 106/mL. For tumor inoculation, male C57BL/6N mice weighing 18−22 g were injected with 100 μL of the cell suspension containing 5 × 105 viable B16 cells subcutaneously on the right flank. After ∼10 days, when the tumor reached approximately 2−4 mm in diameter, tumor-bearing mice were randomly divided into six groups (n = 10), and each group were intratumorally administrated with AL/Ad5-mIL-12/PTX, AL/Ad5-mIL-12 + AL/PTX, AL/Ad5-mIL-12, AL/PTX, AL/Ad5-luc, and PBS. The tumors were measured every 3−4 days using digital Vernier calipers along the longest width (W) and the corresponding perpendicular length (L), and the tumor volume (L × (0.5W)2) was calculated.35 The death date of each mouse was recorded for survival studies, and it was counted from the date of tumor inoculation. In this experiment, the pfu of Ad-mIL-12 was 4 × 108, and the dose of PTX was 150 μg per mouse, respectively. 3.5.2. Measurement of mIL-12 and IFN-γ Expression in Tumor Tissue and Serum. To investigate whether PTX could enhance the in vivo gene expression of Ad5/mIL-12, the levels of mIL-12 and IFN-γ in tumor sites or serum were measured. Briefly, when tumors reached the size of 50−80 mm3, animals

was pelleted by centrifuging at 2500g for 10 min, and then, the pellet was resuspended at a lipid concentration of 10 μmol/mL in a concentrated virus solution or TES buffer (100 mM NaCl, 2 mM histidine, 2 mM TES, and 1.5 mM CaCl2 at pH 7.4) by vortex mixing for 10 min at room temperature. EDTA (100 mM) was added directly to this solution at a final concentration of 15 mM, and 10−20 μL of 100 mM NaOH was added to adjust the pH to 7.4. Finally, the solution was vortex-mixed for 10 min at 37 °C followed by incubation for 30 min. All buffers and solutions were sterilized by filtration or autoclaving prior to addition to vesicle preparations.32 3.2. Characterization of Naked Ad5 and Liposomes. 3.2.1. Photon Correlation Spectroscopy and Determination of Zeta Potential. To evaluate the physical characteristics of the complex of Ad5 and anionic PTX liposomes (AL/Ad5/ PTX), the average diameter, polydispersity index, and zeta potential of naked Ad5, AL/PTX, AL/Ad5, or AL/Ad5/PTX were measured by photon correlation spectroscopy (PCS, Malvern Zetasizer Nano ZS90, Malvern Instruments Ltd., U.K.) with a 50 mV laser. Before the measurements, 0.2 mL of each sample was diluted with 1 mL of TES buffer (pH 7.4) before being added into the sample cell. The measurements were performed at 25 °C, and the fixed angle was 90° with a 2 min equilibrium time and automatic measurement cycles. Measurements were carried out in triplicate, and each value is reported as the mean ± standard deviation (SD) of three experiments, with each experiment comprising three measurements of the same sample with at least 10 runs each. 3.2.2. Transmission Electron Microscopy (TEM). The morphology of naked Ad5 and complexes of AL/Ad5/PTX was observed with a transmission electron microscopy (TecnaiG2F-20, FEI, Holland) at an accelerating voltage of 20 kV, using a negative stain technique. The sample solutions were freshly prepared and after being diluted with pure water, they were added to a copper grid and incubated for 3 min. Then the solutions were removed with filter paper and stained with 1% (w/v) aqueous uranyl acetate for 1 min. Afterward, the dye was removed with filter paper, samples were air-dried to completely remove the water, and the samples were imaged. 3.3. In Vitro Cytotoxicity. To determine the appropriate concentrations of PTX for transduction in B16 and A549 cell lines, toxicity studies were performed using MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide). In the assay, MOI of Ad5 encoding mIL-12 (pfu value per cell) was maintained at 25. Briefly, 1 × 104 cells per well were seeded in 96-well plates in a volume of 200 μL. After 24 h incubation, medium was removed, and serial dilutions of AL/PTX or AL/ Ad5/PTX were added at equivalent PTX concentrations per well. After a 24 h-exposure period, the medium was replaced with 200 μL of fresh RPMI-1640 medium containing 5 mg/mL MTT. Four h later, the supernatants were carefully removed from the wells. Next, each well was washed with phosphatebuffered saline (PBS) for twice, and then each well was added in 100 μL of dimethyl sulfoxide (DMSO). The resulting purple solution was analyzed spectrophotometrically at 570 nm using Varioskan Flash spectral scanning multimode reader (Thermo Scientific). The cell viability (%) was calculated according to the formula (Atreated − Abackground) × 100/(Acontrol − Abackground), while the control cells were not exposed to the samples and the background had no cells. The MTT assay was performed in triplicate. 3.4. In Vitro Transduction. 3.4.1. In Vitro Luciferase Gene Transduction. In vitro transduction was performed to compare 1806

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zeta potential increased from −17.1 ± 0.55 mV to −27.5 ± 1.39 mV. As for the AL/Ad5 complex compared with blank AL, the result was similar to the findings with AL/Ad5/PTX, in that the size was unimodal, with a mean diameter that increased from 130.1 ± 2.03 nm to 202.7 ± 5.07 nm, while the zeta potential of AL/Ad5 varied increased from −15.2 ± 0.74 mV to −19.6 ± 3.11 mV. The negative-stain transmission electron microscopy images of naked Ad5 and AL/Ad5/PTX are shown in Figure 1. Figure 1A shows that the average particle size of Ad5 was ∼80 nm, while Figure 1B shows that the naked Ad5 particles were surrounded by lipid membranes, in which the black core was Ad5. Additionally, the size of naked Ad5 or complexes was some lower than that measured by Zetasizer Nano ZS. The results were consistent with our previous study.32 4.2. In Vitro Cytotoxicity Assays. The cytotoxicity of PTX-loaded AL/Ad5/PTX or AL/PTX complexes was carried out to determine the appropriate concentrations of PTX for transduction in CAR-lack B16 and CAR-rich A549 cell lines. As we can see from Figure 2, AL/Ad5/PTX showed PTX-dosedependent cytoxicity for both cell lines, as did AL/PTX. For B16 cells, when the concentration of PTX was at 5, 10, 50, 200, or 400 nM, cell viability with AL/Ad5/PTX was 98.09%, 94.86%, 88.78%, 84.41%, and 74.01%, respectively, and that of AL/PTX was 103.18%, 103.24%, 87.92%, 82.86%, and 76.95%, respectively. There was no significant difference between AL/Ad5/PTX and AL/PTX (P > 0.05). For A549 cells, the results were similar to those in B16 cells. When the concentration of PTX was at 1, 5, 10, 100, or 200 nM, the cell viability of AL/Ad5/PTX or AL/PTX was 103.65%, 104.96%, 98.23%, 84.10%, 68.13% or 104.53%, 103.18%, 99.02%, 88.2%, 70.09%, respectively, and there was also no significant difference between AL/Ad5/PTX and AL/ PTX (P > 0.05). Simultaneously, the low cytotoxicity of naked Ad5 or PTXfree AL/Ad5 (MOI 25) against both cell lines was observed in the study even when the concentration of total lipid reached to 400 μg/mL (data not shown). Therefore, in the subsequent in vitro transduction experiments, the dose of PTX was determined as 100 nM, based on the condition in which the cell viability was >80%. 4.3. In Vitro Transduction. 4.3.1. In Vitro Luciferase (luc) Gene Expression. It has been previously shown that that formulating Ad5 with anionic liposomes could significantly enhance the LacZ gene expression in CAR/integrin-deficient CHO and MDCK cells.32 Therefore, to determine whether AL/Ad5/PTX could still retain the same high transduction

were randomly divided into six groups (n = 6): AL/Ad5-mIL12/PTX, AL/Ad5-mIL-12 + AL/PTX, AL/Ad5-mIL-12, naked Ad5-mIL-12, AL/Ad5-luc, and PBS. For the detection of mIL-12 and IFN-γ in serum and tumor tissues, 7 or 14 days after injection of medication, blood samples were collected by orbital puncture at room temperature. Serum was extracted from blood samples by centrifugation (5000 r.p.m., 20 min) at 4 °C and stored at −80 °C. Then, the mice were sacrificed, and tumor tissues were removed and grinded and liquefied in the cell lysis buffer. The homogenates were centrifuged at 12 000 r.p.m. for 2 min at 4 °C, and the supernatants were collected and stored at −80 °C. Levels of mIL-12 and IFN-γ in supernatants were determined by ELISA. For tumor tissue, cytokine results were normalized relative to the total protein concentration in each tumor and were calculated as pictograms per milligram of total protein; for serum, the results are reported as pictograms per milliliter of serum. 3.6. Statistical Analysis. All statistical analyses were performed by Student’s t test and are presented as mean ± SD. P values less than 0.05 was considered to be statistically significant.

4. RESULTS 4.1. Particle Size, Polydispersity, and Surface Morphology. The size and zeta-potential measurements of naked Ad5, blank AL, AL/PTX, AL/Ad5, or AL/Ad5/PTX were carried out after dilution with purified water and were determined with a Zetasizer Nano ZS. As shown in Table 1, Table 1. Zeta Potential and Size Distribution of Naked Ad5 and Complexesa zeta potential (mV) naked Ad5 blank AL AL/Ad5 AL/PTX AL/Ad5/PTX

−7.62 −15.2 −19.6 −17.1 −27.5

± ± ± ± ±

0.81 0.74 3.11 0.55 1.39

size (nm) 101.5 130.1 202.7 131.8 242.2

± ± ± ± ±

1.97 2.03 1.70 0.80 5.07

PDI 0.065 0.224 0.143 0.230 0.236

± ± ± ± ±

0.011 0.009 0.025 0.009 0.009

a

AL: anionic liposomes, PTX: paclitaxel. Each value represents as the mean ± SD (n = 3).

after incorporation of Ad5 (101.5 ± 1.97 nm) into AL/PTX to form the AL/Ad5/PTX complex, the size increased from 131.8 ± 0.80 nm to 242.2 ± 5.07 nm as compared with the initial AL/ PTX complex. Meanwhile, the presence of Ad5 resulted in a more negatively charged AL/Ad5/PTX complex, in which the

Figure 1. Transmission electron micrograph (TEM) images of naked Ad5 (A) and AL/Ad5/PTX (B). Bar = 100 nm. 1807

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Figure 2. Cytotoxicity of complexes of AL/PTX or AL/Ad5/PTX with various concentrations of PTX. (A) B16 cells and (B) A549 cells in the presence of 10% FCS with increasing amounts of two kinds of complexes (PTX concentrations ranging from 5 to 400 nM for B16 cells and 1 to 200 nM for A549 cells) was measured using the MTT assay (MOI 25). Error bars represent SDs. Each point represents one representative experiment performed in triplicates. AL: anionic liposome, PTX: paclitaxel.

Figure 3. In vitro transfection efficiency assays of (A) B16 cells and (B) A549 cells. The cells were treated with various formulations at an MOI of 25, and the concentration of PTX for B16 was 200 nM and for A549 was 100 nM. After 48 h transfection, the results were detected by Luciferase Assay System Kit (Promega). Each value represents as the mean ± SD (n = 3). ***P < 0.001; **P < 0.01. AL/PTX + AL/Ad5luc: AL/PTX was added 1 h before the complex of AL/Ad5-luc, AL/ PTX + Ad5-luc: AL/PTX was added 1 h before the addition of naked Ad5-luc.

efficiency in CAR-lacking cells, we chose B16 for further study;33 A549 cells, efficiently expressing CAR, were used as a control. Figure 3 shows the results of gene expression by qualitative detection in B16 cells and A549 cells. The incorporation of PTX into AL/Ad5-luc promoted a more significant increase in the luciferase gene expression, especially in B16 cells, in which the transduction efficiency increased approximately 5 times, from 8554112.01 RLU/mg protein (AL/Ad5-luc) to 39861328.17 RLU/mg protein (AL/Ad5-luc/PTX). In A549 cells, however, there was no significant difference between the two groups. On the other hand, in B16 cells both AL/Ad5-luc and AL/Ad5-luc/PTX showed enhanced transduction efficiencies when compared with the naked Ad5-luc. Interestingly, in B16 cells the approach of adding AL/PTX 1 h prior to the addition of other treatments could also enhance the transduction efficiency of AL/Ad5-luc or naked Ad5-luc. The enhancement was approximately 5 times or 3 times, respectively. However, in both cell lines, the groups between AL/Ad5-luc/PTX and AL/PTX + AL/Ad5-luc displayed no significant differences. Those results suggest that AL/PTX could enhance the transduction efficiency of AL/Ad5-luc, even naked Ad5-luc, especially in B16 cells, regardless of whether it was added 1 h before or simultaneously.

4.3.2. In Vitro mIL-12 Gene Expression. B16 cells were transfected with various formulations (AL/Ad5-mIL-12/PTX, AL/PTX + AL/Ad5-mIL-12, AL/Ad5-mIL-12, naked Ad-mIL12), and the ability of the formulations to express mIL-12 was determined by ELISA using supernatants harvested from the wells at 48 h. As can be seen in Figure 4, there were no significant differences between AL/Ad5-mIL-12/PTX and AL/ PTX + AL/Ad5-mIL-12. Compared with AL/Ad5-mIL-12 or naked Ad5-mIL-12, the relative level of IL-12 increased approximately 1.5 times or 2 times, respectively. All of the results obtained here were in consistent with those in Section 4.3.1. 4.4. In Vivo Experiments. 4.4.1. Tumor Inhibition Experiments. The in vivo antitumor efficacy of AL/Ad5-mIL12/PTX, AL/Ad5-mIL-12 + AL/PTX, AL/PTX, AL/Ad5-mIL12, and AL/Ad5-luc were evaluated in B16 melanoma tumorbearing mice following the procedures in Section 3.5.1, and the 1808

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PTX) yielded significantly higher levels of mIL-12 (P < 0.001) than those groups treated with AL/Ad5-mIL-12, naked Ad5mIL-12, AL/Ad5-luc, or PBS, which is consistent with the antitumor effect shown in Figure 5. Since IL-12 is a strong inducer of IFN-γ,36,37 we also measured the production of IFN-γ in serum and tumor tissue. Significantly higher IFN-γ levels (P < 0.05) were observed in serum and tumors treated with AL/Ad5-mIL-12/PTX or AL/ Ad5-mIL-12 + AL/PTX than those treated with other formulations (Figure 6C and D). This result was similar to the observations of mIL-12 levels, and as expected there were no significant differences between the two groups of mice treated with AL/Ad5-mIL-12/PTX and AL/Ad5-mIL-12 + AL/ PTX, indicating that, while not precisely coinciding, the increasing levels of IFN-γ follow a trend similar to that of mIL-12. Finally, in relative comparison, the group of tumor-bearing mice treated with AL/Ad5-mIL-12/PTX produced both IL-12 and IFN-γ significantly higher levels than the other treatment groups.

5. DISCUSSION Tumor chemo-immunotherapy is based on a combination of chemotherapeutics and cytokines and has been demonstrated to be a promising approach to the treatment of many neoplastic diseases.27 Moreover, it is reported that the simultaneous in vivo transfer of cytokine gene and chemotherapeutic agent synergistically induces specific and nonspecific antitumor immune responses, resulting in a robust therapeutic outcome against subcutaneous and metastatic tumors in mice.38 IL-12, a novel heterodimeric cytokine that enhances antitumor cytotoxic responses and a single factor that possesses the potential to correct many tumor-bearing host immune dysfunctions,39 exerts potent antitumor effects in experimentally induced and spontaneous tumors.40 Intratumoral transduction of the IL-12 gene may result in continuous expression of a low dose of the cytokine, enhancing both adaptive and innate antitumor immune responses,41 and this aspect was supported by some reports that demonstrated the therapeutic effectiveness of intratumoral IL-12 gene transfer against B16 melanoma,42 hepatocellular carcinoma,43 and colon and renal cell carcinoma.44 IL-12 not only promotes NK cell cytotoxicity, but also induces the production of multiple other cytokines, while the induction of IFN-γ predominates.45 Importantly, IFN-γ operates in a positive feedback mechanism, as IFN-γ in turn stimulates IL-12 synthesis by phagocytic cells.46 IFN-γ affects mammary carcinoma metastasis47 by acting on host cells that secondarily produce factors to diminish tumor growth. Macrophages, which are the main cell population involved in IFN-γ-mediated innate immunity, exhibit tumoricidal activity by releasing incompletely reduced oxygen intermediates, such as nitric oxide (NO).48 IFN-γ activates the macrophage production of NO, resulting in the direct killing of tumor cells. Furthermore, PTX can increase the production of IL-12 by mimicking bacterial lipopolysaccharide (LPS) via a mechanism that involves a NO-mediated autocrine induction pathway.49,50 Chemotherapeutic PTX regimens also impart a significant but reversible inhibition of lymphocyte populations, and IL-12 may be a useful ancillary immunotherapeutic to overcome PTX-induced modulation of lymphocyte activities.51 Based on the interaction between PTX and IL-12, we hypothesized that the codelivery of IL-12 and PTX would have a synergistic effect on melanomas. To test this hypothesis, we

Figure 4. In vitro transduction efficiency assays of various formulations (AL/Ad5-mIL-12/PTX, AL/PTX + AL/Ad5-mIL-12, AL/Ad5-mIL12, naked Ad-mIL-12) in B16 cells (MOI 25). Supernatants were collected after 48 h and secreted mIL-12 concentration (pg/mL) determined by ELISA. Each value represents the mean ± SD (n = 5). ***P < 0.001; **P < 0.01. AL/PTX + AL/Ad5-mIL-12: AL/PTX was added 1 h before the complex of AL/Ad5-mIL-12.

group of animals treated with PBS served as a control. As shown in Figure 5A, the tumor growth rate in the mice treated with either AL/PTX or AL/Ad5-mIL-12 alone was significantly lower than that treated with PBS or AL/Ad5-luc (P < 0.001), while the combination of both of AL/PTX and AL/Ad5-mIL12 (AL/Ad5-mIL-12/PTX or AL/Ad5-mIL-12 + AL/PTX) showed the highest therapeutic effect. On the other hand, mice treated with AL/Ad5-mIL-12/PTX or AL/Ad5-mIL-12 + AL/ PTX exhibited negligible growth within 21 days, but after that the differences in tumor volumes was significant (P < 0.01). The observed differences between these two groups in survival time displayed in Figure 5B confirmed the efficacy of AL/Ad5mIL-12/PTX treatment. These results indicated that PTX and Ad5-mIL-12 codelivered in liposomes can synergistically inhibit tumor growth and enhance the therapeutic effect, suggesting a significant synergistic/combined effect of codelivery of PTX and mIL-12. 4.4.2. Measurement of mIL-12 and IFN-γ Expression in Tumor Tissue and Serum. To further assess whether AL/Ad5mIL-12/PTX could induce immune responses against B16 melanomas, serum, and tumor tissue were both obtained at days 7 and 14 post-transduction. mIL-12 and IFN-γ levels were separately measured using ELISA. As shown in Figure 6A and B, the serum or tumor tissue levels of mIL-12 in AL/Ad5-mIL12/PTX-treated melanoma-bearing mice were highest (682 pg/ mL in serum and 761 pg/mg in tumor tissues). Even on day 14, this group of mice showed relatively high mIL-12 levels (472 pg/mL in serum and 536 pg/mg in tumor tissue). Furthermore, there was a significant difference (P < 0.05) when compared with the group of mice treated with AL/Ad5-mIL-12 + AL/ PTX. On the other hand, the combination of AL/PTX and AL/ Ad5-mIL-12 (AL/Ad5-mIL-12/PTX or AL/Ad5-mIL-12 + AL/ 1809

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Figure 5. Therapeutic effect of B16 melanoma tumor-bearing mice after treatment with various formulations: PBS, AL/Ad5-luc, AL/Ad5-mIL-12, AL/PTX, AL/Ad5-mIL-12 + AL/PTX, and AL/Ad5-mIL-12/PTX. Growth in tumor volume (A) and survival (B) were monitored on a regular basis (n = 10). AL/Ad5-mIL-12/PTX showed the most robust anticancer effect compared with the other groups. Data are given as the mean ± S.D. *P < 0.05. AL/Ad5-mIL-12 + AL/PTX: AL/PTX and the complex of AL/Ad5-mIL-12 were injected respectively.

mIL-12/PTX or AL/Ad5-mIL-12 + AL/PTX exhibited negligible growth within 21 days. Beyond that, however, there were significant differences between them (P < 0.01), and similarly the group of mice treated with AL/Ad5-mIL-12/PTX had a longer survival time than the group of mice treated with AL/Ad5-mIL-12 + AL/PTX. At the same time, the levels of mIL-12 detected in serum and tumor tissues also confirmed the above results; when compared with the mice treated with AL/ Ad5-mIL-12 + AL/PTX, the AL/Ad5-mIL-12/PTX group expressed a higher level of mIL-12, whether in serum (Figure 6A) or tumor tissues (Figure 6B). One hypothesis to account for this observation is that it would be more advantageous to transport PTX with Ad5-mIL-12 in the same carrier, so that both of them could be delivered into the same part of the tumor for combined actions and synergistic effects. As reported,41 mIL-12 is a pro-inflammatory cytokine that could induce the innate resistance and adaptive immunity and stimulates the production of IFN-γ. Figure 6C and D illustrated that the secretion level of IFN-γ was in a mIL-12-concentration-

chose Ad5 as the vector to deliver mIL-12 gene and encapsulated both Ad5-mIL-12 and PTX within the anionic liposomes in order to enable codelivery. The present study has demonstrated that the AL/Ad5 complex not only compensates for the limitation of viral and nonviral vectors, but also takes advantage of the unique features of these two classical gene carriers. Therefore, a combination of AL and Ad5 might hold great potential for efficient in vivo gene delivery. From Figure 4, we can see that AL/Ad5-mIL-12/PTX could synergistically inhibit tumor growth and enhance the therapeutic effect, suggesting a significant synergistic/combined effect of codelivery of PTX and the mIL-12 gene. This conclusion is consistent with our initial hypothesis, which is also supported by the results shown in Figure 5. In the in vivo therapeutic experiments, we further evaluated the anticancer effect under the condition in which AL/PTX and AL/Ad5-mIL-12 were not prepared in the same system, and were separately injected into tumors virtually without an intervening time interval (AL/Ad5mIL-12 + AL/PTX). As a result, mice treated with AL/Ad51810

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Figure 6. Cytokine (mIL-12 and IFN-γ) production in tumor or serum of tumor-bearing mice on day 7 and day 14 after treatment with AL/Ad5mIL-12/PTX, AL/Ad5-mIL-12 + AL/PTX, AL/Ad5-mIL-12, naked Ad5-mIL-12, AL/Ad5-luc, or PBS. Tumor-bearing mice were respectively intratumorally administrated with the above samples, and on day 7 and day 14, tumor or serum tissue was isolated from the mice (n = 6). The levels of mIL-12 in serum (A) or tumor (B) and IFN-γ in serum (C) or tumor (D) was respectively determined by ELISA. Data are representative of five independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

mice.52 Thus, it could be said that the antitumor efficacy of the IL-12/PTX combination depends on the tumor model used.27 All of the above observations demonstrate strong potentiation of the antitumor effects of IL-12/PTX in some specific tumor models, while the potential augmentation mechanisms, as well as the dependence on the types of tumor models, need to be further studied. While there was an unanticipated result observed in the in vitro studies, in that the group of B16 cells treated with AL/ Ad5-luc/PTX exhibited higher gene expression levels than those with AL/Ad5-luc, we further evaluated the influence of AL/PTX on the transduction efficiency of naked Ad5-luc (AL/ PTX + Ad5-luc) or AL/Ad5-luc (AL/PTX + AL/Ad5-luc) by adding AL/PTX 1 h prior. As a result, we found that PTX could

dependent way. The result heavily supported that PTX and Ad5-mIL-12 codelivered in anionic liposomes has an optimum anticancer effect than other groups. The synergistic/combined effect of codelivery of paclitaxel and the IL-12 gene in cancers has previously been observed. Wang et al. demonstrated that, in 4T1 mouse breast cancer models, the tumor growth rate in the mice treated with PTXloaded nanoparticle/IL-12-encoded plasmid complexes was significantly lower than that in mice treated with either PTXloaded codelivery effect of IL-12 and PTX may depend on the kind of cell lines. It has been reported that the combination of murine recombinant IL-12 + PTX has a significant effect on mice inoculated with murine melanoma B16 cells, while this regimen proved ineffective in the treatment of leukemia-bearing 1811

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ACKNOWLEDGMENTS We are thankful for the financial support of the National Natural and Science Foundation of China (No. 81173011) and Program for New Century Excellent Talents in University (No. NTEC-10-0601).

significantly enhance the AL/Ad5-luc transduction efficiency in B16 cells, a CAR-negative cell line, regardless of whether PTX was incorporated into the AL/Ad5-luc complex and given as a codelivery system, or was pretreated 1 h before the incubation of AL/Ad5-luc (data not shown). In A549 cells however, which efficient express CAR, there was no significant difference. Even for naked Ad5-luc, the transduction efficiency could also be enhanced by adding the appropriate dose of AL/PTX 1 h prior to the addition of Ad5-luc in B16 cells. The results further confirmed that synergistic effect of IL-12 and PTX was tumor cell line-dependent, which suggested the codelivery system of PTX and Ad5 in anionic liposomes may result in higher tissuespecificity, lower toxicity, and increased therapeutical effect for melanoma. It was expected that the presence of paclitaxel would enhance gene expression of DNA-cationic liposomes possibly because of its antimitotic function.53,54 PTX is a microtubule-stabilizing compound with potent antitumor activity. It influences the cytoskeleton equilibrium by increasing the assembly of altered microtubules, thereby inducing cellular modifications that result in reduced proliferation, migration, and signal transduction.55 Local delivery of PTX resulted in enlargement in vessel size.55 Both of these effects contribute to a preservation of vessel shape and are likely to be caused by a structural alteration of the cytoskeleton.56 Wong et al. demonstrated that paclitaxel pretreatment can induce apoptosis, cause expansion of the interstitial space, and thereby improve nanoparticle delivery and transport in tumor interstitium, and thus improved the uptake/ penetration, transduction, and functionality of siRNA loaded in cationic liposomes in two-dimensional monolayers and threedimensional tumor spheroids and histocultures.57 Thus, based on the above observations, it can be concluded that in certain circumstances PTX could enhance the delivery of liposomes, Ad5, siRNA, or other kinds of formulations into some tumor cells, and therefore perhaps it can be developed into a new kind of approach for enhancing anticancer therapy or gene delivery in the future. Taken together, we present a novel antimelanoma delivery system in the form of the AL/Ad5/PTX complex for B16 melanoma chemo-immunotherapy. Incorporation of Ad5 and PTX into anionic liposomes not only facilitated the administration of PTX due to enhanced aqueous solubility, but also achieved highly enhanced mIL-12 gene transduction. More importantly, this codelivery system prolongs the survival time of melanoma-bearing mice in a synerstic way. We expect that this system will be effective in other tumor models, which requires further study. In addition, the mechanism of the synerstic effect of PTX and Ad5-mIL-12 for melanoma treatment is also in our consideration.

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AUTHOR INFORMATION

Corresponding Author

*West China School of Pharmacy, Sichuan University, No. 17, Section 3, Southern Renmin Road, Chengdu 610041, People’s Republic of China. Tel./Fax: +86-28-85502307. E-mail: [email protected]. Author Contributions

L.C. and Q.Z. contributed equally to this work. Notes

The authors declare no competing financial interest. 1812

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