A Biomimic Reconstituted High-Density-Lipoprotein-Based Drug and p53 Gene Co-delivery System for Effective Antiangiogenesis Therapy of Bladder Cancer

Ouyang et al. Nanoscale Research Letters (2015) 10:283 DOI 10.1186/s11671-015-0965-5 NANO EXPRESS Open Access A Biomimic Reconstituted High-DensityLipoprotein-Based Drug and p53 Gene Co-delivery System for Effective Antiangiogenesis Therapy of Bladder Cancer Qiaohong Ouyang*, Zhongxiang Duan, Guangli Jiao and Jixiao Lei Abstract A biomimic reconstituted high-density-lipoprotein-based drug and p53 gene co-delivery system (rHDL/CD-PEI/p53 complexes) was fabricated as a targeted co-delivery nanovector of drug and gene for potential bladder cancer therapy. Here, CD-PEI was utilized to effectively condense the p53 plasmid, to incorporate the plasmid into rHDL, and to act as an antitumor drug to suppress tumor angiogenesis. The rHDL/CD-PEI/p53 complexes exhibited desirable and homogenous particle size, neutral surface charge, and low cytotoxicity in vitro. The results of confocal laser scanning microscopy and flow cytometry confirmed that SR-BI-targeted function induced specific cytoplasmic delivery and high gene transfection efficiency in MBT-2 murine bladder cells. In addition, rHDL/CD-PEI/p53 complexes co-delivering CD and p53 gene achieved synergistic angiogenesis suppression by more effectively downregulating the expression of vascular endothelial growth factor (VEGF) messenger RNA (mRNA) and protein via different pathways in vitro. In vivo investigation on C3H/He mice bearing MBT-2 tumor xenografts revealed that rHDL/CD-PEI/p53 complexes possessed strong antitumor activity. These findings suggested that rHDL/CD-PEI/p53 complexes could be an ideal tumor-targeting system for simultaneous transfer of drug and gene, which might be a new promising strategy for effective bladder cancer therapy. Keywords: Bladder cancer; Reconstituted high-density lipoprotein; Candesartan; p53; Co-delivery; Antiangiogenesis therapy Background Bladder cancer is one of the most common cancers. There were approximately 70,980 cases diagnosed with bladder cancer in the USA in 2009, of which 14,330 patients would likely succumb to the disease [1]. Gene therapy has garnered significant attention as a therapeutic approach for bladder cancer. From a clinical point of view, this disease is an ideal target for gene therapy [2]. Efficient delivery of genetic material to the required cells within a patient without significant toxicity and side effect in gene therapy requires an ideal delivery vector, which has been extensively studied for several decades [3]. Reconstituted high-density lipoprotein (rHDL) is the synthetic form of the endogenous human HDL. Both * Correspondence: Department of Nuclear Medicine, The First Affiliated Hospital, Chinese PLA General Hospital, Beijing, China rHDL and endogenous human HDL possess similar physicochemical properties. In the past decades, rHDL has been successfully developed as a scavenger receptor class B type 1 (SR-BI)-targeting gene carrier [4], which displayed promising application potential in vivo. Antiangiogenic therapy has been well recognized as an effective antitumor strategy [5]. Among various angiogenesis-related growth factors, vascular endothelial growth factor (VEGF) has been demonstrated to be a major contributor to angiogenesis that occurs in many solid tumors including breast, bladder, and prostate cancers [6]. It has been reported that p53 gene therapy could inhibit tumor-associated angiogenesis by downregulating VEGF expression [7]. On the other hand, angiotensin II type 1 receptor (AT1R), a widely overexpressed receptor in various neoplastic cells, was recognized to play an important role in tumor angiogenesis and progression. There © 2015 Ouyang et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Ouyang et al. Nanoscale Research Letters (2015) 10:283 was increasing evidence that the AT1R blocker (ARB) candesartan (CD) exerted beneficial effects on tumor progression by competitively inhibiting the AT1R signaling pathway and downregulating VEGF expression [8]. Co-delivery systems simultaneously transporting anticancer drug and gene into the same cancer cells by multifunctional nanovectors may provide a new paradigm in cancer treatment. In this study, an rHDL-based system was developed for effective p53 gene delivery and combined antiangiogenesis therapy in a bladder cancer model. CD-PEI was first synthesized and then employed to construct a lipophilic core of rHDL (Lipos/CD-PEI). The cationic CD-PEI was served to condense the p53 plasmid to formulate Lipos/CD-PEI/p53 complexes. Finally, functional protein apoA-I was introduced to eventually assemble the delivery system (rHDL/CD-PEI/p53 complexes). Methods Cell Culture and Animal Model The MBT-2 murine bladder cell line was cultured in DMEM (Gibco, USA) supplemented with 10 % FBS (HyClone, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of a 95 % air/5 % CO2 incubator at 37 °C. Male C3H/He mice (7 weeks old) were maintained at standard conditions with free access to food and water. All animal experiments were conducted in strict accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The MBT-2-tumor-bearing mice models were established by subcutaneous inoculation of the MBT-2 cell suspension to the flank of mice. Synthesis of CD-PEI CD-PEI was prepared according to a previous report [9] and characterized by 1H NMR. Preparation of rHDL/CD-PEI/p53 Complexes Preparation of Lipos/CD-PEI A thin-film dispersion method was employed to construct Lipos/CD-PEI as previously reported [10] with some modifications. Briefly, 60 mg of PC, 6 mg of cholesterol, and 12 mg of CE were dissolved in 2 ml of organic solvent (chloroform:methanol = 1:1, v/v), and the solvent of lipid solutions was evaporated with a rotary evaporator at 30 °C until a thin film was formed. The trace solvent residue was finally removed with a stream of nitrogen gas. Five hundred microliters of CD-PEI solution (10 mg/ml), 50 μl of sodium cholate solution (30 mg/ml in phosphate-buffered saline (PBS) buffer), and Tris buffer (0.1 M KCl, 10 mM Tris, 1 mM EDTA, pH 8.0) were added to dissolve the thin film. The mixture was vortexed thoroughly for 5 min, followed by ultrasonication using an ultrahomogenizer (JY92II, Ningbo, China) until a clear suspension was obtained. The dispersion was Page 2 of 8 then filtered through a 0.22-μm filter and dialyzed to remove the free sodium cholate (MWCO 7500 Da, 2 L × 3). Finally, the prepared Lipos/CD-PEI complexes were collected and stored at 4 °C until further use. Preparation of Lipos/CD-PEI/p53 Complexes The p53 plasmid was dissolved in PBS buffer to obtain a fi (...truncated)