Article pubs.acs.org/molecularpharmaceutics

Versatile Reticular Polyethylenimine Derivative-Mediated Targeted Drug and Gene Codelivery for Tumor Therapy Xuefang Ding,†,∥ Wei Wang,†,∥ Yazhe Wang,† Xiuli Bao,† Yu Wang,*,‡ Cheng Wang,† Jian Chen,§ Fangrong Zhang,† and Jianping Zhou*,† †

State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China ‡ Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Department of Pharmacology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China § School of Pharmacy, Fu Dan University, 826 Zhangheng Road, Shanghai 201203, China S Supporting Information *

ABSTRACT: The study is aimed to develop a versatile reticular polyethylenimine (PEI) derivative eprosartan-g-PEI (ESP) conjugatemediated targeted drug and gene codelivery system for tumor therapy. Eprosartan (ES), an angiotensin II type 1 receptor blocker (ARB), which has been proven to exert beneficial effects on tumor progression, vascularization, and metastasis as the conventional antihypertensive drug, was conjugated with PEI-1.8K chains into ESP via a bis-amide bond of pH-sensitivity to overcome high cytotoxicity and nontargeted gene delivery of PEI-25K. P53 gene was encapsulated in the ESP to form the codelivery system of ESP/p53 complexes, and this system was comprehensively characterized. In vitro ESP/p53 complexes had a significant effect on inhibiting angiogenesis by reducing the expression and secretion of VEGF. In vivo the effective antitumor activity of ESP/ p53 complexes was observed on nude mice bearing PANC-1 xenografts, and the microvessel density (MVD) examination demonstrated that ESP/p53 complex-produced antitumor efficacy was closely correlated with the efficient angiogenesis repression. These findings disclosed that the multifunctional ESP/p53 complexes might be a promising dual anticancer drug and gene codelivery system. KEYWORDS: polyethylenimine, eprosartan, p53, codelivery, angiotensin II type-1 receptor, tumor therapy

1. INTRODUCTION The most challenging issues in successful application of gene therapy to human neoplasms are the choice of a relevant therapeutic gene and an effective vector for delivering the transgene into tumor cells. Therapeutic gene and vector features determine transfection efficiency, duration of transgene expression, and the eventual appearance of side effects.1 Recent attempts in restoring malfunctioning p53 gene through nonviral vector-mediated p53 gene delivery have been reported to sensitize cancer cells toward anticancer drugs, induce apoptosis, and suppress angiogenesis.2,3 More interestingly, tumor angiogenesis was repressed by the introduction of wild-type p53 (wt p53) via downregulation of vascular endothelial growth factor (VEGF) expression.4 Nonviral gene delivery systems that utilize cationic polymers to form pDNA complexes have been thoroughly investigated in the past years as excellent alternatives to viral vectors, which offers certain advantages in terms of safety, stability, and cost-effectiveness leading to efficient gene delivery.5,6 Among all the nonviral vectors, branched polyethylenimine (PEI) based vectors have been considered as the gold standard in © 2014 American Chemical Society

cationic polymer-based gene delivery because of its high transfection efficiency both in vitro and in vivo.7,8 Not only does PEI prevent DNA degradation during transfection but also its strong buffering capacity allows the PEI/DNA complexes to follow an endosome-escape mechanism.9 However, a major concern of using a PEI polymer as a gene carrier is its dosedependent cytotoxicity due to nonbiodegradability inside cells, which can cause the formation of aggregates with negatively charged intracellular proteins. The molecular weight of PEI is a critical factor that influences its toxicity and transfection efficiency. The high-molecular-weight (HMW) PEI exhibits higher transfection efficiency, but its toxicity is also higher than low-molecular-weight (LMW) PEI, so its application is limited.5 LMW PEI has been proven to be nontoxic, but it cannot Special Issue: Recent Molecular Pharmaceutical Development in China Received: Revised: Accepted: Published: 3307

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Scheme 1. Schematic Illustration of the Self-Assembly, Accumulation at Tumor Tissue, and Intracellular Trafficking Pathway of a Multifunctional Nanoscaled Codelivery System for Drug and pDNA; the Intracellular Trafficking Pathway Includes Steps of AT1 Receptor-Meditated Cellular Internalization, Endosome Escape, and Release of Drug and pDNA

carboxyls of ES and the amino groups of PEI, in order to link the LMW PEI into HMW reticular PEI (ESP) to increase transfection efficiency. In the structure of ES, there is an imidazolyl that can perform proton sponge effect as well as the amino groups of PEI. So enhanced transfection efficiency could be reached resulting from the enhanced proton sponge effect. Second, ES is a targeting ligand strongly binding to the AT1 receptors, which highly express in some cancers, so it could further enhance the targetability and transfection efficiency of ESP. Finally, ES itself is a drug used for treatment and chemoprevention of cancer, and it could play a therapeutic role in the ESP-based codelivery system. Therefore, ES here was multifunctional and played an important role in the design of codelivery system. Codelivery system, as an emerging therapeutic strategy, not only could implement the simultaneous transport of drug and gene in the same target cell but also is capable of enhancing gene expression or obtaining the synergistic effect of drug and therapeutic gene.17 Hence, the aim of this study was to develop eprosartan-g-PEI (ESP) conjugate as a platform for the codelivery of ES and pDNA to AT1 receptor-overexpressed tumor cells (Scheme 1). In this work, the ESP conjugate, a versatile reticular PEI derivative, was synthesized and characterized. Subsequently, the codelivery system of ESP/pDNA complexes was well characterized in terms of its buffering capacity, pDNA condensation capability and loading efficiency, DNase I protection properties, particle size, zeta potential, morphology, in vitro pDNA release efficiency, pH-responsive ES release behavior, cytotoxicity, and transfection efficiency of ESP.

condense pDNA completely and has very poor (even no) transfection activity.10 Therefore, modification of LMW PEI polymers has been also studied extensively to improve gene transfer efficiency while keeping low cytotoxicity.11,12 It is well-known that angiotensin II (Ang II), a biologically active peptide of the renin-angiotensin system (RAS), plays a fundamental role not only as a vasoconstrictor in controlling blood pressure and electrolyte/fluid homeostasis but also as a mitogenic factor through the angiotensin II type-1 (AT1) receptor in smooth muscle cells and cardiac myocytes. However, recently many encouraging results have demonstrated that Ang II could modulate tumor progression and angiogenesis by regulation of angiogenesis-associated genes, such as VEGF and angiopoietin 1 and 2, via AT1 receptor.13 Interestingly, numerous recent studies have shown that AT1 receptor overexpression frequently occurs in various aspects of neoplastic cells, such as human pancreatic, breast and ovarian carcinoma cells.14 Herein, AT1 receptor might be a noteworthy molecular target in anticancer therapy. There is increasing evidence that AT1 receptor blockers (ARBs) exert beneficial effects on tumor progression, vascularization, and metastasis in basic science studies.15,16 Eprosartan (ES), an ARB, has the inhibitory effect on renin secretion and vascular proliferation. More interestingly, ES elicits multifactorial changes in cell proliferation, angiogenesis, and fibro-genesis in cancer tissues.13 In this study, ES was used as a multifunctional material. First of all, it is a linkage to cross-link the LMW PEI. Since ES has two carboxyls on its two ends, the conjugates could be synthesized by amide reactions between the 3308

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pH values were recorded after each addition using a pH meter. Similarly, the buffering assay was repeated with each polymer (PEI-1.8K and PEI-25K). 2.5. Preparation of ESP/pDNA Complexes. Complex formation of ESP and plasmid DNA (pDNA) was examined by the electrophoresis experiment. Briefly, the polymer solution (0.3 mg/mL) was mixed with the pDNA solution (0.3 mg/mL) at various N/P ratios by gentle vortex for 30 s while keeping the amount of pDNA constant, and the final volume was 30 μL. The complexes were incubated for 30 min at room temperature, loaded onto 0.7% agarose gels containing 5 μL/100 mL Goldview, and electrophoresed at 90 mV for 45 min. Finally, the gels were analyzed under a UV illuminator, and the pDNA was visualized. 2.6. Determination of pDNA Loading Efficiency. The pDNA loading efficiency of ESP was calculated from the determination of free pDNA concentration in the supernatant recovered after complex centrifugation (13,000g, 15 min) by measuring the absorbance at 260 nm. Different N/P ratios were prepared while keeping the amount of pDNA constant. The supernatant with pDNA only was used as control. The loading efficiency (LE%) was calculated as the equation:

Further to this, the therapeutic efficacy of ESP/p53 complexes in aspects of VEGF reduction and angiogenesis inhibition was investigated in vitro. Finally, the biodistribution and targetability of Cy-7-labled ESP/pDNA complexes were explored in PANC-1 tumor xenograft models by in vivo imaging, and the antitumor efficacy of ESP/p53 complexes against PANC-1 tumors was evaluated to further demonstrate their potential applicability toward clinical use.

2. EXPERIMENTAL SECTION 2.1. Materials. Branched polyethylenimines (PEI, 1.8K and 25K) and eprosartan (ES) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-Ethyl-3(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Aladdin Reagent Database Inc. (Shanghai, China). The plasmids pCMV-EGFP (pEGFP, pDNA, ∼4.7 kb) encoding enhanced green fluorescent protein (GFP) and pCMV-Neo-Bam-p53wt (p53, ∼8.4 kb) encoding wild-type p53 tumor suppressor protein from Addgene were propagated in DH-5α Escherichia coli and purified by Endo-Free Plasmid Maxi Kit (Qiagen, Hilden, Germany). DNase I was offered by Invitrogen Co. (CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) was purchased from Fluka (MO, USA). RevertAid Reverse Transcriptase kit and SYBR Green RT-PCR Kit were purchased from Thermo Fisher Scientific Inc. (Shanghai, China). Matrigel was purchased from Becton Dickinson (Bedford, MA, USA), and the near-infrared dye Cy7 SE was obtained from Beijing Fanbo Science and Technology Co., Ltd. (Beijing, China). CD31-antibody was supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other reagents were of analytical grade and used without further purification. 2.2. Cell Culture. Human pancreatic adenocarcinoma cell line PANC-1 and human umbilical vein endothelial cells HUVECs were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM culture medium (Gibco, USA) supplemented with 10% FBS (HyClone, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 95% air/5% CO2 incubator at 37 °C. All experiments were performed on cells in the logarithmic phase of growth. 2.3. Synthesis and Characterization of Eprosartan-gPEI (ESP) Conjugates. The eprosartan-g-PEI (ESP) conjugates were prepared through the amidation reaction between ES and PEI. First, ES (52 mg, 0.1 mmol) was dissolved in anhydrous DMF. Then EDC (76 mg, 0.4 mmol) and NHS (46 mg, 0.4 mmol) were added to the clear solution of ES and stirred under nitrogen for 2 h to activate the carboxyl groups of ES at room temperature. After that, the reaction mixture was added dropwise into the PEI (PEI-1.8K) dissolved in DMF. The reaction continuously proceeded for 24 h at room temperature with gentle agitation. The resulting solution was dialyzed against distilled water using a dialysis membrane (MWCO 1000) for 2 days to remove the unreacted EDC and NHS. Finally, the solution were lyophilized to obtain ESP as a light yellow floc powder and stored at 4 °C until further use. The degree of ES grafted to PEI-1.8K was determined by 1H NMR analysis (AvaceAV-500, Bruker, Germany). 2.4. Determination of Buffering Capacity. The buffering capacity of the ESP conjugates were determined by acid−base titration as described by Sun et al.18 Briefly, the ESP (0.1 mg/ mL) solution was adjusted to pH 10 with 0.1 M NaOH, then the solution was titrated with 0.1 M HCl in 25 μL increments. The

LE% = (1 − A 260sample /A260control ) × 100

where A260sample represents the absorbance of ESP/pDNA supernatant and A260control is the absorbance of supernatant with pDNA only. 2.7. Nuclease Resistance. The extent and effectiveness of pDNA condensation by ESP was evaluated by a gel retardation assay. Briefly, ESP/pDNA complexes at various N/P ratios were separately incubated with the DNase I buffer at 37 °C for 30 min. For DNase I inactivation, all samples were treated with 10 μL of EDTA (250 mM) for 10 min, followed by heat denaturation at 80 °C for 10 min. Then pDNA molecules were dissociated from complexes using heparin (100 mg/mL) for 10 min, and the results were analyzed by the agarose gel electrophoresis experiment, in which free pDNA was used as control. 2.8. Measurement of Particle Size and Zeta Potential. The ESP/pDNA complexes were prepared at various N/P ratios and vortexed. After 10 min incubation at room temperature, the size and zeta potential of the complexes in PBS solution (pH 7.4) were measured by a Malvern Zetasizer 3000HS (Malvern, U.K.). 2.9. Atomic Force Microscopy (AFM). The morphology and size distribution of ESP/pDNA complexes were determined by atomic force microscopy (AFM, Nano ScopeIIIa, Veeco, USA). The ESP/pDNA complexes were prepared as the method above. Two to three microliters of this suspension was deposited on a cleaned glass slide and allowed to dry for overnight at room temperature. Subsequently, the image was obtained from the glass surface containing the ESP/pDNA complexes. The height differences on the surface are indicated by the color code, and lighter regions indicated higher heights. 2.10. Release of pDNA from Complexes. The reversible nature of the ESP/pDNA complexes was investigated by the addition of heparin to release pDNA. PEI-1.8K, ESP, and PEI25K were complexed with pDNA at the N/P ratio of 12 and incubated for 30 min. Subsequently, the complexes were incubated for 20 min with heparin solutions with increasing concentrations, and the released pDNA was analyzed by 0.7% agarose gel electrophoresis as described above. The amount of pDNA released from complexes after heparin treatment was estimated by densitometry using Gel-Pro analyzer (Genegenius, Syngene). 3309

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2.11. Evaluation of pH Sensitivity. The pH-responsive release behavior of ESP was determined by UV/vis spectroscopy by measuring the absorbance at 303 nm. The release studies were carried out in a glass beaker at 37 °C in acetate buffer (pH 5.0 and 5.8) and phosphate buffer (pH 7.4) solutions, respectively. First, 10 mg of ESP was dispersed in 5 mL of water and placed in a dialysis bag with a molecular weight cutoff of 1000 Da. Then the dialysis bag was immersed in 45 mL of the release medium and kept in a horizontal laboratory shaker maintained at 100 rpm and 37 °C. At predetermined time points, a sample (2 mL) of the medium was removed, the same volume of fresh medium was added, and the release amount of ES was analyzed with a spectrophotometer at 303 nm. In the end, two drops of concentrated HCl were added to completely hydrolyze all the samples, and the absorbance at 100% hydrolysis was measured to calculate the extent of ESP hydrolysis. 2.12. Cytotoxicity Studies. The in vitro cytotoxicity of ESP was studied using MTT colorimetric assay. In brief, PANC-1 cells were seeded at a density of 1 × 105 cells/well in a 96-well plate and incubated for 24 h. Various concentrations of polymers (PEI-1.8K, ESP, and PEI-25K) were prepared, respectively. Twenty microliters of each polymer solution was added to each well, and the plate was incubated for an additional 48 h at 37 °C. Then 20 μL of 5 mg/mL MTT solution was added and incubated for 4 h. The supernatant was removed, and 100 μL of DMSO was added to each well to dissolve the formazan crystals. The optical density (OD) of each well at 570 nm was recorded by a microplate reader (EL800, BIO-TEK Instruments Inc.). 2.13. In Vitro Cell Transfection Studies. The transfection efficiency of the complexes was studied and the enhanced green fluorescent protein (EGFP) was used as the reporter gene. First, PANC-1 cells were seeded at 1 × 104 cells/well in a 24-well plate and grown overnight in the DMEM media (10% FBS) at 37 °C (to reach 70−80% confluence at the time of transfection). Subsequently, the primary growth medium was removed and replaced with serum-free media. One hundred microliters of complexes diluted with serum-free medium were added to the wells and allowed to incubate for 6 h. Complexes were then removed and replaced with fresh culture medium. After incubation for 24 h, the expression of EGFP in PANC-1 cells was observed under the confocal laser scanning microscopy (CLSM, Leica TCS SP5), and the transfection efficiency of the complexes was quantified for EGFP-positive cells using a flow cytometer (BD FACS Calibur, USA). Furthermore, the interaction of ES and AT1 receptor was investigated. The ES of various concentrations was added in the transfection medium for 30 min to saturate the ES receptors of cell surface before adding the complexes. 2.14. Quantitative Real-Time Reverse TranscriptionPCR (qRT-PCR). Several studies have suggested that Ang II could play a fundamental role in upregulating the levels of VEGF and promoting tumor invasion, migration, and angiogenesis via AT1 receptor.19−21 Similarly, Ang II (100 nM) exerted the strongest stimulation effect on VEGF mRNA after 24 h addition into media in our preliminary experiment. Hence, to evaluate the effect of ESP/p53 (8 μg) complexes on VEGF mRNA expression, PANC-1 cells were seeded into 6-well plates for overnight, stimulated by Ang II for 24 h to imitate the actual tumor microenvironment in vivo and transfected with ESP/p53 complexes for 48 h as described above. Cells were also treated with ESP/pDNA complexes in the presence of Ang II to elucidate the synergistic efficacy of ES and p53. Untreated cells group was used as control. Hereafter, total RNA was immediately

extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol and subjected to reverse transcription followed by PCR amplification and fluorescent quantitation for specific expressed genes. First-strand cDNA synthesis was performed using RevertAid Reverse Transcriptase kit and an Oligo (dT) 18 primer (Sangon Biotech, Shanghai, China). SYBR Green RTPCR Kit was utilized to perform qRT-PCR on an Applied Biosystems 7300 Sequence Detection system (Applied Biosystems, Foster City, CA). A housekeeping gene, β-actin was used as a reference control to normalize the other gene relative transcripts. Specific primers used for PCR amplification were designed as follows: VEGF forward, 5′-CGAAGTGGTGAAGTTCATGGATG-3′, and reverse, 5′- TTCTGTATCAGTCTTTCCTGGTGAG-3′; β-actin forward, 5′TCATGTTTGAGACCTTCAA-3′, and reverse, 5′-GTCTTTGCGGATGTCCACG-3′. 2.15. ELISA for VEGF. Briefly, cells seeded in 24-well plates were treated in the same way as for qRT-PCR. The VEGF protein in complete supernatant media collected by centrifugation was quantified using a human-specific VEGF ELISA kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions. All experiments were performed in triplicate and quantitated using the supplied VEGF standard. 2.16. In Vitro Angiogenesis Assay. Angiogenesis assay in vitro was conducted with a gel of basement membrane to evaluate the ability of endothelial cells to form tube-like structures after treatment. HUVEC cells were transfected with PEI-25K/pDNA, PEI-25K/p53, ESP/pDNA and ESP/p53 complexes as described above, respectively. Forty-eight hours later, the cells were collected with trypsin, resuspended in growth media, and plated onto the precoated 96-well plates at a density of 3 × 104 cells/ well for 24 h to induce angiogenesis of HUVECs. Before HUVECs suspended in PBS were seeded into plates, liquefied matrigel was placed in precooled 96-well plates (50 μL/well), and then allowed to polymerize at 37 °C for 30 min. Capillarylike structures were photographed using an inverted bright field microscope (Nikon ECLIPSE TE-2000U, Tokyo, Japan) and the images were processed by Image-Pro Plus 6.0 software from Media Cy-bernetics (Rockville, MD, USA). The untreated cells were set as a control and each experiment was repeated at least thrice. The inhibition rate was calculated using the following formula: inhibition rate (%) = (TLcontrol − TLsample)/TLcontrol × 100%

where TL is the tube length. 2.17. In Vivo Antitumor Studies of ESP/p53 Complexes. 2.17.1. Animal and Tumor Xenograft Models. BALB/c nude mice (male, 5−6 weeks, 18−20 g) purchased from Shanghai Laboratory Animal Center (SLAC, China) were pathogen free and allowed to access food and water ad libitum. All experiments on animals were subjected to the National Institute of Health Guide for the Care and Use of Laboratory Animals. To establish PANC-1 tumor-bearing nude mice model, mice were inoculated s.c. in the left flank with the suspension of PANC-1 cells (1 × 106 cells in 100 mL of PBS). Tumor-bearing mice were used as palpable tumors (100 mm3 in volume) generated within 3 or 4 weeks. Tumor size was measured in two dimensions using a caliper, and tumor volume (mm3) was calculated as V = a2 × b/2 mm3 (a, minor axis; b, major axis). 3310

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Figure 1. Synthetic scheme of eprosartan-g-PEI (ESP) conjugates.

2.17.2. In Vivo Imaging Analysis. For in vivo imaging analysis, the NIR dye Cy7 SE was linked onto polymers PEI-25K and ESP, respectively. The biodistribution and tumor targeting efficacy of Cy7-labeled PEI-25K/pDNA and Cy7-labeled ESP/pDNA complexes in PANC-1 tumor-bearing mice following intravenous administration at a dose of 5 mg Cy7-labeled ESP/kg were investigated via an in vivo imaging system (FX PRO, Kodak, USA). The fluorescent images were taken at the predetermined time points (1, 3, 6, and 12 h), and images were analyzed using the Kodak Molecular Imaging Software 5.X. 2.17.3. Antitumor Efficacy. In vivo antitumor efficacy of the different formulations was evaluated on PANC-1 tumor xenograft models. All PANC-1 tumor-bearing nude mice were weighed and randomly divided into five groups (n = 6): (1) saline

as control; (2) PEI-25K/pDNA complexes at 4 mg of PEI-25K/ kg; (3) PEI-25K/p53 complexes at 4 mg of PEI-25K/kg and 1 mg of p53/kg; (4) ESP/pDNA complexes at 2 mg of ES/kg; (5) ESP/p53 complexes at 2 mg of ES/kg and 1 mg of p53/kg. The initial day of i.v. administration was defined as Day 0. Administration was then repeated seven times at a 2-day interval until Day 14, and tumor sizes were measured every other day. The survival rates were monitored throughout the study. Furthermore, at Day 14 after administration of various formulations, mice in each group were sacrificed to extract tumor tissues. Resected tumors were homogenized and lysed in TNE lysis buffer (1 M Tris-HCl, pH 7.6, 0.5 M EDTA, 10% Nonidet P-40). The lysates were centrifuged at 14,000g for 10 min, and the 3311

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protein concentration in the supernatant was measured by the BCA protein assay. Equivalent volumes of lysates containing 50 μg of protein of each sample were loaded and size-fractionated by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes and blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk for 1 h at room temperature. Subsequently, the membranes were then incubated with the primary antibody (anti-p53 and antibcl-2 at a 1:1000 dilution and antiactin at 1:5000 dilution) overnight at 4 °C. After rinsing the membranes to remove unbound primary antibody, the membranes were incubated with a secondary antibody (1:3000 diluted horseradish peroxidase-conjugated antibody) for 1 h and rinsed. The target proteins were detected by enhanced chemiluminescence Western blotting detection kit (Pierce Biotech, USA). In vivo apoptotic analysis of PANC-1 xenografted nude mice after different treatments was investigated using Annexin-VFITC assay based on binding of Annexin-V with phosphatidyl serine of early apoptotic cells. The single cell suspension from resected tumors was prepared and stained with Annexin-V-FITC for 15 min. The fluorescence of the cells was immediately measured by flow cytometer (FACS Caliber, BD Biosciences, USA). Tumors resected from mice were fixed with 10% Formalin and embedded in paraffin blocks to prepare anti-CD31 antibody stained tumor sections, and then microvessel density (MVD) was determined by light microscopy after immunostaining of sections with anti-CD31 antibodies using the procedure of Wilhelm et al.22 Tissue images were captured at 400× magnification with optical microscope (Olympus, Japan). 2.18. Statistical Analysis. Data were expressed as means ± SD from triplicate experiments performed in a parallel manner unless otherwise indicated. The results were analyzed using Student’s t-test or ANOVA. All comparisons were made relative to corresponding controls, and the significance of difference was indicated as #P < 0.05, *P < 0.05, **P < 0.01, or ***P < 0.001.

Figure 2. 1H NMR of PEI and ESP in D2O.

As seen in Figure 3A, the ESP possessed obviously higher buffering capability than PEI-1.8K. Excitingly, the buffering capability of ESP was similar to that of PEI-25K and even higher. It has been approved that PEI is one of the most effective gene vectors resulting from the strong proton sponge effect due to its high buffering capacity. The endosome acidification promoted PEI to binding protons, inducing osmotic pressure increase and leading to swelling and subsequent disruption of the endosomes and lysosomes, so the PEI-based complexes could escape and release into the cytoplasm or in the nucleus for further transcription.27−29 The proton sponge effect of ESP was increased as a consequence of the imidazole group from ES that could also absorb more protons. Hence, ESP was able to achieve a comparable and even higher proton sponge effect compared with HMW PEI-25K. 3.3. Complex Formation between ESP and pDNA. One prerequisite for good gene carrier was efficient DNA condensation.30 The condensation capability of ESP with pDNA was evaluated by an agarose gel electrophoresis from the retardation of pDNA mobility. As shown in Figure 3B, complexes were formed at various N/P ratios, and the migrating of pDNA was completely retarded when the N/P ratios of PEI1.8K/pDNA, ESP/pDNA, and PEI-25K/pDNA complexes were 4, 4, and 2, respectively. 3.4. Loading Efficiency of pDNA in ESP. The amount of pDNA complexed with ESP was determined by spectrophotometrically measuring the optical density of the supernatants. According to the calculating result, the loading efficiency (LE%) was about 80% at the N/P ratio of 12. Moreover, as the N/P ratio continued increasing, there was almost no change in the loading efficiency (Figure 3C). 3.5. DNase I Digestion Assay. A successful gene delivery system must have the ability to protect DNA from degradation by nucleases presenting in serum, the extracellular matrix, and the mucosal surfaces.30,31 As seen in Figure 3D, the effect of ESP on the protection of pDNA from DNase I degradation was investigated. The ESP/pDNA complexes showed good DNA protection at N/P ratio of 8 and 10, which was certified by the appearance of pDNA band with similar fluorescent intensity as naked pDNA. 3.6. Characterization of ESP/pDNA Complexes. Figure 4A showed the particle size and zeta potential of ESP/pDNA

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of ESP Conjugates. The reticular ESP conjugates were prepared by an amino reaction between the carboxyl groups of eprosartan (ES) and amine groups of PEI, and ESP synthetic scheme is shown in Figure 1. As shown in Figure 2, the chemical structures of PEI and ESP were confirmed using 1H NMR. The proton peaks of PEI (−NHCH2CH2−) from the products appeared at 2.7−3.4 ppm, whereas the PEI only appeared at about 2.6 ppm.23,24 Furthermore, the products had the alkyl peaks of ES at 0.730, 1.184, and 1.469 ppm and heterocyclic ring (phenyl ring, imidazole ring, and thiophene ring) peaks of ES at 6.778−8.050 ppm. The results indicated that ES was successfully grafted to the PEI chain, and the degree of grafted ES in ESP was 31.8%. Moreover, the UV/vis spectrum showed that in comparison with PEI, the product ESP had an absorption peak of 303 nm from ES, which also certified that ES was grafted to the PEI chain (data not shown). 3.2. Buffering Capacity of ESP Conjugates. It is wellknown that good buffering capability would make the complexes escape from endosomes easier and faster, contributing to the release of pDNA into the cytoplasm. Hence, for PEI-mediated gene delivery system, high transfection efficiency was usually extremely associated with the pH buffering capacity of PEI solution.25 In this study, acid−base titration experiments were carried out to evaluate the buffering capacity of PEI and ESP.26 3312

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Figure 3. (A) Acid−base titration profiles of PEI-1.8K, ESP, and PEI-25K. Titration curve of 0.15 M NaCl is presented as a blank control. (B) Agarose gel electrophoresis retardation assay of polymer/pDNA complexes at various N/P ratios of PEI-1.8K (a), ESP (b), and PEI 25K (c) to pDNA. (C) Loading efficiency of pDNA in ESP. (D) Stability of ESP/pDNA complexes against DNase I degradation was evaluated by electrophoretic analysis. Lane 1, pDNA alone; lanes 2−6, : ESP/pDNA complexes at N/P ratios of 1, 4, 6, 8, and 10. Data were shown as mean ± SD (n = 3).

Figure 4. Characterization of ESP/pDNA complexes. (A) Particle size and (B) zeta potential of ESP/pDNA complexes in PBS buffer (pH 7.4) at various N/P ratios. (C) AFM images of ESP/pDNA complexes. Data were shown as mean ± SD (n = 3). 3313

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Figure 5. (A) DNA release assay of ESP/pDNA complexes. (B) pH-responsive release behavior of ESP at pH 5.0, 5.8, and 7.4, simulating the acidic lysosome, the acidic endosome, and the normal physiological environment, respectively. (C) Viability of PANC-1 cells incubated with ESP for 48 h at varying concentrations. Data were expressed as mean ± SD (n = 3). **P < 0.01 vs PEI-25K group.

molecules into the HMW reticular PEI derivative, which could provide a more solid structure of pDNA-loaded complexes by condensing pDNA more efficiently than PEI-1.8K. 3.7. Release of pDNA from Complexes. Release of pDNA from the complexes is a necessary step for the delivered gene to be expressed in the target cells. Physical integrity of pDNA after release is a prerequisite for successful transfection.30,31 The release is believed to occur as a result of interactions between the cationic ESP and other negatively charged macromolecules or cellular components such as mRNA, sulfated sugars, and nuclear chromatin in the cells instead of ionic pDNA.35 Figure 5A showed the results of an experiment simulating the release of pDNA after incubating ESP/pDNA complexes with the model molecule of heparin. Interestingly, it is demonstrated that with two units of heparin the amount of pDNA released from ESP was much larger than that of PEI-1.8K and PEI-25K. 3.8. Analysis of pH-Responsive Degradation of ESP. The pH-responsive release behavior of ES from the ESP conjugate was investigated at pH 5.0, 5.8, and 7.4, simulating the acidic lysosome, the acidic endosome, and the normal physiological environment, respectively. The extent of ESP hydrolysis was conveniently determined using UV/vis spectroscopy by monitoring the absorbance at 303 nm, which was the characteristic absorbance of ES. As shown in Figure 5B, the results showed that the hydrolysis rate of ESP was pH dependent.

complexes measured in PBS at various N/P ratios. As presented, a steady decrease of particle sizes of ESP/pDNA complexes could be seen along with the N/P ratio increased. When the N/P ratio is 12, the size of ESP/pDNA complexes was approximately 150 nm, and no obvious decrease of size was obtained with further increasing N/P ratios. As shown in Figure 4B, the zeta potential of the complexes was investigated. At low N/P ratios, the complexes could not form completely (Figure 3B) and the zeta potential was negative. However, when the N/P ratio exceeded 4, the zeta potential of ESP/pDNA complexes turned to be positive. Furthermore, the zeta potential remained stable (18−20 mV) at N/P ratios beyond 12. Taking the results from the analysis above into consideration, we chose the optimal N/P ratio of 12 to prepare ESP/pDNA complexes in the following study. AFM investigation of ESP/pDNA complexes showed well-shaped, spherical, and condensed complexes with an average size of 150 nm (Figure 4C). The three-dimensional image revealed homogeneous population with a clear absence of aggregates. The AFM images indicated the complete complexation of DNA molecules by ESP. We believed that the specific cross-linked structure of ESP might account for the findings. Earlier reports have pointed out that the poor performance of LMW PEI in gene delivery could be ascribed to unstable constructs with pDNA.32−34 Owing to the structure of ES, two carboxyl groups of ES conjugated PEI-1.8K 3314

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Figure 6. EGFP expression in PANC-1 cells transfected with polymer/DNA complexes. (A) Confocal images of (a) cells transfected with ESP/pDNA complexes at different N/P ratios and (b) cells transfected with different polymers/pDNA complexes at their optimal charge ratios. (B) FACS analysis of EGFP expression in PANC-1 cells transfected with polymer/pDNA complexes. (a) Transfection efficiency of the complexes determined at different N/ P ratio. (b) Transfection efficiency of ESP/pDNA complexes determined after the pretreatment of ES for 30 min at the N/P ratio of 12. Results were expressed as means ± SD (n = 3). **P < 0.01 vs PEI-25K/pDNA (N/P ratio = 20) group and ***P < 0.001 vs PEI-1.8K/pDNA (N/P ratio = 12) group.

At pH 7.4, the release of ES was very slow, with the low release level (only 11.7%) of ES found within 48 h. This result suggested that ESP maintained structural integrity by the amide bond under normal physiological conditions. At pH 5.0 and 5.8, rapid hydrolysis of ESP took place, and approximately 98% and 72% of

amide bonds were hydrolyzed with corresponding half-lives of 3.4 and 11.2 h, respectively. It indicated that the release of ES from ESP was pH dependent and that the linkage between ES and PEI backbone was acid cleavable. Thus, under acidic conditions ES could be released from ESP by hydrolysis of the 3315

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Figure 7. VEGF at mRNA and protein levels in PANC-1 cells treated by Ang II (100 nM), ESP/pDNA, and ESP/p53 complexes in vitro. (A) Relative transcriptional levels of VEGF in cells treated with different formulations for 48 h were detected by qRT-PCR. β-actin mRNA was utilized to normalize gene expression. Results were represented as mean ± SD (n = 3). **P < 0.01 and ***P < 0.001 vs Ang II group. (B) The secretion of VEGF protein in culture media tested by human-specific ELISA kit after 48 h of addition of different formulations. Results were expressed as mean ± SD (n = 3). **P < 0.01 and ***P < 0.001 vs Ang II group. (C,D) Inhibitory effects of PEI-25K/pDNA, PEI-25K/p53, ESP/pDNA, and ESP/p53 complexes against tubule formation on matrigel at an equivalent ES concentration of 5 μM. Results were expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 vs control group.

amide bond. This pH-dependent characteristic could withhold ES release from ESP/pDNA complexes in the plasma under normal physiological conditions (pH 7.4), so the side effects to normal tissues were greatly reduced. However, once ESP/pDNA complexes were taken up via an endocytosis process by tumor cells, ES could be released from the complexes in the endosomes/lysosomes (pH 5.0−5.8) to exert its anticancer effect. 3.9. Cytotoxicity Studies of ESP. The cytotoxicity of the polymers was investigated on PANC-1 cells on the basis of MTT assay. Cells were treated with PEI-1.8K, ESP, and PEI-25K for 48 h at various concentrations. Figure 5C showed that the polymers exhibited a dose-dependent cytotoxicity with increasing concentrations. The PEI-25K exhibited the highest cytotoxicity, reducing cell viability to less than 30%. In comparison with PEI25K, PEI-1.8K and ESP exerted a significantly low cytotoxicity (P < 0.01), showing more than 70% cell viability even at the highest concentration. Therefore, the ESP exhibited a cytotoxicity profile that was drastically different from that of PEI-25K and comparably low and rather similar to that of PEI-1.8K. It has

been reported that HMW PEI was more significantly toxic than LMW PEI.36 Our results also confirmed this observation for PEI1.8K and PEI-25K. Moreover, the concentration of ESP at the optimal N/P ratio displayed almost noncytotoxicity (Over 90% cell viability), which might be related to our results, that the ESP could be degraded into small fragments (PEI-1.8K) via the hydrolysis of the amide bond. Thus, the cytotoxicity of ESP was kept as low as PEI-1.8K. 3.10. In Vitro Cell Transfection Studies. Transfection efficiency studies were carried out in PANC-1 cells with each polymer/pDNA complex prepared at a series of N/P ratios. First, the effectiveness of ESP for gene delivery in comparison to PEI was evaluated by comparing the fluorescence intensities of EGFP expression in PANC-1 cells. As shown in Figure 6A(a), the transfection efficiency of ESP was dependent on the N/P ratio. With increasing N/P ratio, ESP showed the improved transfection efficiency, and the nearly highest transfection efficiency was obtained at the N/P ratio of 12. However, there was no obvious change in the transfection efficiency with further increasing N/P ratio. Then the transfection efficiency for 3316

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Figure 8. In vivo time-dependent tumor-targeting images after intravenous injection of Cy7-labled ESP/pDNA in PANC-1 tumor-bearing mice.

a lack of cellular uptake of ESP/pDNA complexes. The ability to block intracellular uptake of ESP/pDNA complexes with excessfree ES confirmed the receptor-mediated uptake nature of the complexes. Thus, it was suggested that ES could represent an efficacious targeting ligand of AT1 receptors on the surface of tumor cells. 3.11. Effects of ESP/p53 Complexes on VEGF Gene and Protein in Vitro. VEGF, a potent and specific mitogen for endothelial cells, is considered to be the most important protein in angiogenesis. It has been proved that inhibition of VEGF expression was a good target for human cancer therapy. To validate the effects of ESP/p53 complexes on VEGF expression in vitro, PANC-1 cells overexpressing AT1 receptor15 were used to perform qRT-PCR and ELISA. We first determined whether exposure of PANC-1 cells to Ang II (100 nM) caused alterations in mRNA levels of VEGF. The results confirmed that upregulation of Ang II-induced VEGF mRNA in PANC-1 cells was remarkably suppressed by ESP/pDNA complexes (P < 0.01) or ESP/p53 complexes (P < 0.001) after 48 h of incubation, respectively. It was interesting to note that ESP/p53 complexes showed stronger repression effects on VEGF expression at mRNA level compared with ESP/pDNA complexes (Figure 7A). To further verify the inhibitory effect of ESP/p53 complexes on VEGF expression, the level of VEGF protein was determined by human ELISA kit after exposure to different formulations for 48 h. As shown in Figure 7B, a high-level expression of VEGF protein was detected when treated with Ang II (100 nM) compared to untreated cells. Moreover, ESP/pDNA complexes (P < 0.01) or ESP/p53 complexes (P < 0.001) extremely reduced increased-VEGF secretion from cells stimulated by Ang II. Taken together, these findings validated that ES and p53 gene could inhibit the expression of Ang II-induced angiogenesis related gene VEGF. Moreover, significant inhibition of Ang IIinduced VEGF expression by ESP/p53 complexes in AT1

different vectors was determined at their optimal charge ratios (PEI-1.8K/pDNA at the N/P ratio of 20, PEI-25K/pDNA at the N/P ratio of 10,37−39 and ESP/pDNA at the N/P ratio of 12). As seen in Figure 6A(b), the gene expression of ESP could be significantly enhanced as compared with PEI-1.8K, and even a little higher than that of PEI-25K, which was a common commercial transfection agent. The results showed that PANC-1 cells could uptake ESP/pDNA complexes more efficiently than PE1.8K/pDNA and PEI-25K/pDNA complexes. The direct explanation for high transfection efficiency of ESP is efficient cellular uptake capability of ESP/pDNA complexes resulting from AT1 receptor-mediated endocytosis because ES can strongly and specially combine with AT1 receptors on the surface of some cancer cells such as breast cancer, ovarian cancer, laryngeal carcinoma, and pancreatic cancer cells.29 The transfection efficiencies of PEI-1.8K, ESP, and PEI-25K at N/P ratios ranging from 4 to 20 were evaluated in PANC-1 cells by flow cytometry (Figure 6B(a)). Data showed that the EGFP expression of ESP/pDNA complexes was significantly higher than that of PEI-1.8K/pDNA complexes at the N/P ratio of 12 (P < 0.001), and the transfection efficiencies were both improved with increasing charge ratio. At the N/P ratio of 20, the transfection efficiency of PEI-25K/pDNA complexes was significantly lower than that of ESP/pDNA complexes as a result of increased cytotoxicity of PEI-25K (P < 0.01). To explore the in vitro targeted capability of ESP, PANC-1 cells were pretreated with ES. The ESP presented gradually reduced EGFP expression with the increasing concentrations of ES. The down-regulation of EGFP expression was about 17.2% (0.5 μg/mL of ES), 43.4% (1.0 μg/mL of ES), 60.8% (2.0 μg/mL of ES), and 82.6% (4.0 μg/mL of ES), respectively (Figure 6B(b)). The results demonstrated that when the PANC-1 cells were treated with an increasing dose of free ES to block AT1 receptors, EGFP expression was obviously decreased, suggesting 3317

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Figure 9. Tumor growth curve (A), survival rates (B), and representative sections for immunohistochemical staining using CD31 antibody (magnification, 400×) (C) of PANC-1 tumor-bearing mice treated with saline, PEI-25K/pDNA, PEI-25K/p53, ESP/pDNA, and ESP/p53 complexes. Results were represented as mean ± SD (n = 6). **P < 0.01 and ***P < 0.001 vs saline group; #P < 0.05 vs ESP/pDNA group.

pDNA complexes showed almost no inhibitory effect on tubular structure formation, while PEI-25K/p53 and ESP/pDNA complexes exerted a remarkably higher inhibition than control group, respectively (P < 0.05 or P < 0.01). By comparison, the treatment of ESP/p53 complexes resulted in the unchanged morphology of HUVECs and almost no tube formation. In particular, ESP/p53 complexes exerted an inhibition rate of 88.36% for tube formation in HUVECs (P < 0.001), which may contribute to its increased antineovasculature activity in vivo. These observations suggested that ESP/p53 complexes as a codelivery system were more effective in inhibiting morphological differentiation of the HUVECs and angiogenesis, which derived from both ES-dependent angiogenesis repression and the release of inhibitors of angiogenesis induced by p53. 3.13. In Vivo Imaging Analysis. ESP/pDNA complexes have exhibited preferable AT1 receptor-targetting and considerable downregulation on VEGF expression in vitro. Subsequently, the tumor targeting efficiency of ESP/pDNA

receptor-positive PANC-1 cells corroborated a combined function of ES and p53 gene because the introduction of wt p53 gene into tumor cells harboring or expressing p53 mutations would restrain the expression of VEGF.40,41 In all, these results established a direct regulatory relationship between ESP/p53 complexes and Ang II-induced VEGF expression in vitro. 3.12. In Vitro Angiogenesis Assay. The capillary-like structure formation of endothelial cells is a multistep process involving cell adhesion, migration, differentiation, and growth and is an important step in angiogenesis.42 To assess the ability of HUVECs to respond to different formulations regarding angiogenic inhibition and to form microvessel-like structures, HUVECs cocultured with PEI-25K/pDNA, PEI-25K/p53, ESP/ pDNA, or ESP/p53 complexes for 48 h were plated onto matrigel for 24 h. As shown in Figure 7C,D, in the investigated time, the cells aligned themselves end-to-end, elongated, and formed interconnecting networks of capillary tubes because the matrix components influenced the behavior of cells. PEI-25K/ 3318

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than that in the ESP/pDNA group (P < 0.05). According to our results, the treatment of ESP/p53 codelivery system could significantly inhibit the tumor size when compared with single p53 gene or ES therapy. The survival rates of the tumor-bearing mice were also recorded. As seen in Figure 9B, the survival rates of PEI-25K/ pDNA and PEI-25K/p53 groups were lower than those of ESP/ pDNA and ESP/p53 groups, respectively, and mice injected with ESP/p53 complexes survived obviously longer compared to those with saline. This results revealed a clear advantage of ESP over PEI-25K and a distinguished enhancement of antitumor effect resulting from ESP/p53 codelivery system. The in vitro studies mentioned above have demonstrated that constructed ESP/p53 complexes exerted combined VEGFsuppressive and angiogenesis-inhibitory effects. Moreover, angiogenesis, the formation of new blood vessels from preexisting ones, is essential for the progression of most solid tumors.43 Hence, the expression of endothelial PANC-1 tumor markers (CD31) was measured to evaluate the degree of angiogenesis defined as microvessel density (MVD). As shown in Figure 9C, the density of CD31-positive tumor microvessels in tumor xenograft mice treated with different formulations followed the order ESP/p53 complexes < ESP/pDNA complexes < PEI-25K/p53 complexes < PEI-25K/pDNA complexes < saline, which presented persuasive evidence for the significant inhibition of neovascularization in the PANC-1 xenograft models treated with ESP/p53 complexes. The reasons for such powerful tumor angiogenesis inhibition were listed as follows: (1) preferential accumulation at the tumor site and superior targeting ability in the tumor cells; (2) higher transfection efficiency of internalized ESP/p53 complexes owing to stronger buffering capacity of ESP conjugates; (3) synergetic efficacy of potential anticancer agents (ES) and powerful wt p53 gene in antiangiogenic therapy. This powerful tumor angiogenesis repression caused by ESP/p53 complexes ultimately led to an anticipated enhanced antitumor growth efficacy in vivo. In summary, the smartly designed p53-complexed ESP nanosystem could be used as an effective tumor-targeting and antiangiogenesis codelivery system, holding a great potential to achieve more efficient anticancer activity.

complexes in PANC-1 tumor-bearing nude mice was explored using an in vivo fluorescence imaging system, which could monitor nanoparticle tumor penetration with high spatial and temporal resolution. The tumor-bearing mice were injected with Cy7-labled PEI-25K/pDNA and Cy7-labled ESP/pDNA complexes, respectively. As shown in Figure 8, at 1 h postinjection, the difference in the Cy7 signal of the liver and tumor was small in tumor-bearing mice treated with Cy7-labled PEI-25K/pDNA or Cy7-labled ESP/pDNA complexes, suggesting that these nanoparticles mainly were distributed to these two organs. Notably, compared to Cy7-labled PEI-25K/pDNA group, preferential accumulation of Cy7-labled ESP/pDNA complexes in tumor was obviously increased in the nude mice, and a signal enhancement localized in the tumor regions at progressing time (3, 6, and 12 h) after administration was visualized. The finding provided decisive evidence that ESP/pDNA complexes were suitable for tumor-specific drug and gene codelivery. This powerful tumor targetability of complexes might be ascribed to a combination of preferable EPR effect and strong affinity of ES with AT1 receptors overexpressed in tumor for active tumor targeting. Above all, in vivo biodistribution studies indicated that ESP/pDNA complexes could be expected to be a highly efficient drug delivery system to achieve targeted codelivery of anticancer drug and gene. 3.14. In Vivo Delivery Efficacy and Apoptotic Analysis. The expression level of p53 protein and bcl-2 protein in tumor tissues from PANC-1 xenografted nude mice was detected by Western blotting analysis. As shown in Figure S1A, Supporting Information, the p53 protein was expressed in every group since PANC-1 cells are p53 mutant cell type and the p53 antibody in this study can recognize both wild type and mutants. Compared with control group, obvious up-regulation of p53 protein was investigated in mice receiving PEI-25K/p53 and ESP/p53, suggesting the successful expression of p53 gene in vivo. In addition, the strongest protein band of p53 was observed in mice receiving ESP/p53 rather than other groups, indicating that ESP could better potentiate the delivery of p53 gene. On the contrary, the level of bcl-2 protein expression in mice receiving ESP/p53 was much lower than other groups. It has been reported that the excellent antitumor effect of p53 gene is resulted from the critical role in initiating apoptosis of tumor cells, and one of the pathways of p53 gene trigged apoptosis is the suppression of bcl2 gene expression, which acted effectively against cell apoptosis. The Annexin-V-FITC assay was performed for quantitatively determining the cells apoptosis in tumor tissues of mice receiving different treatments. The results were consistent with Western blotting analysis. As seen in Figure S1B, Supporting Information, the mice receiving p53 (PEI-25K/p53 or ESP/p53) treatment showed obvious increase in Annexin-V positive cells when compared with control group. Furthermore, the mice receiving ESP/p53 treatment achieved the highest cell apoptosis up to 23.1% in the tumor tissue, confirming the successful p53 gene therapy in vivo. 3.15. In Vivo Antitumor Efficacy and Angiogenesis Suppression. To further reveal the potential of ESP-based codelivery system in cancer therapy, the antitumor efficacy of ESP/p53 complexes was assessed in PANC-1 xenografted nude mice. As shown in Figure 9A, tumors grew rapidly in the control and PEI-25K/pDNA groups, while a notably strong inhibition of tumor growth was demonstrated in ESP/pDNA group (P < 0.01). Especially, ESP/p53 complexes exhibited significant tumor growth inhibition efficacy (P < 0.001), and the final tumor volume in the ESP/p53 group was below 500 mm3, far less

4. CONCLUSIONS In this study, the eprosartan (ES) cross-linked PEI (ESP) conjugate was complexed with p53 to form a targeted drug and gene codelivery system for tumor therapy. The results demonstrated that ESP had relatively lower cytotoxicity and higher transfection efficiency and tumor targetability as compared with PEI-25K resulting from the special structure and multifunction of ES. We therefore suggested that ESP would be a highly desirable vector. More importantly, wild-type p53 gene-complexed ESP nanoparticle system codelivering both ES and p53 gene was capable of significantly reducing the expression and secretion of VEGF in AT1 receptor-overexpressed PANC-1 cells and inhibiting angiogenesis in vitro. In vivo investigation on nude mice bearing PANC-1 xenografts confirmed that ESP/p53 codelivery system possessed very high tumor-targeting capacity, strong antitumor efficacy, and synergistic antiangiogenesis effect. Taken together, the combined ES and p53 gene therapy using the versatile reticular ESP conjugate may be an effective strategy for cancer treatment, which deserves further investigations toward the action mechanism in vivo and potential clinical application. 3319

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ASSOCIATED CONTENT

S Supporting Information *

Details of the expression levels of p53 protein and bcl-2 protein in tumor tissues from PANC-1 xenografted nude mice by Western blotting analysis and in vivo apoptotic analysis of mice treated with saline, PEI-25K/pDNA, PEI-25K/p53, ESP/pDNA, and ESP/p53 complexes by FACS analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(Yu.W.) E-mail: [email protected]. *(J.Z.) E-mail: [email protected]. Author Contributions ∥

X.D. and W.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by the National Natural Science Foundation of China (Nos. 81102398 and 81273469), the Natural Science Foundation of Jiangsu Province (No. BK2011624), the Ministry of Education Doctoral Program of Higher Specialized Research Fund project (Nos. 20110096120003 and 20113234120008), the Fundamental Research Funds for the Central Universities (No. JKVD2013011), the Graduate Cultivation Innovative Project of Jiangsu Province (No. CXLX13_30), School of Pharmacy, Fudan University and The Open Project Program of Key Lab of Smart Drug Delivery (Fudan University), Ministry of Education, China (No. SDD2012-03), the Open Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (Nos. SKLNMKF201305 and SKLNMKF201215), and the National Found for Fostering Talents of Basic Science (No. J1030830).

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dx.doi.org/10.1021/mp5001263 | Mol. Pharmaceutics 2014, 11, 3307−3321