Pharmaceutical nanotechnology
PEG/RGD-modified magnetic polymeric liposomes for controlled drug release and tumor cell targeting

https://doi.org/10.1016/j.ijpharm.2012.01.013Get rights and content

Abstract

Polymeric liposomes (PEG/RGD-MPLs), composed of amphiphilic polymer octadecyl-quaternized modified poly (γ-glutamic acid) (OQPGA), PEGylated OQPGA, RGD peptide grafted OQPGA and magnetic nanoparticles, was prepared successfully. These PEG/RGD-MPLs could be used as a multifunctional platform for targeted drug delivery. The results showed that PEG/RGD-MPLs were multilamellar spheres with nano-size (50–70 nm) and positive surface charge (28–42 mV). Compared with magnetic conventional liposomes (MCLs), PEG/RGD-MPLs exhibited sufficient size and zeta potential stability, low initial burst release and less magnetic nanoparticles leakage. The cell uptake results suggested that the PEG/RGD-MPLs (with RGD and magnetic particles) exhibited more drug cellular uptake than non RGD and non magnetism carriers in MCF-7 cells. MTT assay revealed that PEG/RGD-MPLs showed lower in vitro cytotoxicity to GES-1 cells at ≤100 μg/mL. These data indicated that the multifunctional PEG/RGD-MPLs may be an alternative formulation for drug delivery system.

Introduction

Currently, a critical challenge in developing drug delivery system was how to achieve an optimal pharmacokinetic profile to allow sufficient targeting and avoid rapid clearance by the reticuloendothelial system (RES) simultaneously. Many studies have been done on developing drug delivery systems in order to achieve long-circulation (Xiong et al., 2005). Huang and Li have developed PEGylated liposome by surface incorporation of polyethylene glycol (PEG) in liposome to minimize the clearance by the RES and prolong circulation time of liposome (Li and Huang, 2009, Klibanov et al., 1990, Blume and Cevc, 1990, Maruyama et al., 1991, Allen et al., 1991, Maeda et al., 2000). Various efforts have been undertaken to enable drug to accumulate in target tumor sites, among which magnetic drug targeting (MDT) and receptor-specific targeting were developed. Using paramagnetic particles as drug carriers, MDT can guide their accumulation in target tissues under strong local magnetic fields, and has been applied in the treatment of cancer patients with some success (Lubbe et al., 2001, Ito et al., 2006, Hirao et al., 2003). As conventional paramagnetic nanoparticles, superparamagnetic ferroferric oxide (Fe3O4) was encapsulated in nanocarriers for magnetic separation and magnetic tissue targeting (Benyettou et al., 2009). Another mechanism used in drug targeting involves receptor-specific targeting which takes advantage of over-expressed surface receptors on tumor cells. Receptor-targeted nanocarriers can be used to load drugs thereby facilitating delivery of high drug payloads to tumors. Meanwhile, nanocarriers can protect non-target normal organs from the toxic drug effects (Gabizon et al., 1982, Park et al., 2005, Zhang et al., 2010a, Zhang et al., 2010b). Recently, RGD (arginine–glycine–aspartic acid)-modified liposomes have been developed as drug delivery system. The RGD sequence was known to serve as a recognition motif for integrin ανβ3 over-expressed on tumor cells surface, which enhanced the opportunities of liposomes binding to tumor cells (Xiong et al., 2005, Ruoslahti, 1996).

It has become the hotspot in nowadays studies that how to construct an effective delivery nanocarriers with sufficient stability. Self-assembled liposomes were used as a delivery system combining receptor-specific targeting and magnetic targeting (Zhu et al., 2010). However, liposomes were prone to adhere to each other and fuse to form larger vesicles in suspension, which restrict its application in therapy (Zhang and Granick, 2006). Toward this end, many efforts have been made to design a sufficiently stable drug delivery nanocarrier. Among various nanocarriers, polymeric liposomes (PLs) showed promising ability because of good physical and thermal stability, excellent solubility in water, and high effectiveness in drug encapsulation (Liang et al., 2008a, Liang et al., 2008b). For the past several years, PLs were self-assembled by amphiphilic polymers base on chitosan derivatives in our lab with some success. Chitosan is a natural cationic polymer which has been under investigation for various biomedical and pharmaceutical applications. However, its poor aqueous solubility is major drawbacks for its use at physiological conditions (Verheul et al., 2008). In this case, our lab developed hydrophilic carboxymethyl chitosan (CMC) (Liang et al., 2008a, Liang et al., 2008b) and lysine modified chitosan (LCS) (Wang et al., 2010) successfully in past years. The synthetic routes were so complicated that the repeatability of experiments decreased and the cost of production enhanced.

In this study, a new amphiphilic polymer octadecyl-quaternized modified poly (γ-glutamic acid) (OQPGA) were synthesized by poly-γ-glutamic acid (PGA) and octadecyl dimethylammonium chloride (QA). PGA was water-soluble, biocompatibility, biodegradable and non-toxicity (Prencipe et al., 2009). We simplified the experimental procedures and improved the repeatability of experiments after PGA has been substituted for chitosan derivatives (CMC and LCS). In order to construct a sufficiently stable tumor targeting delivery system combining tumor accumulating and enhanced intracellular delivery, polymeric liposomes (PEG/RGD-MPLs) were developed by OQPGA, mPEG modified OQPGA (mPEG-OQPGA), RGD modified OQPGA (RGD-OQPGA) and magnetic nanoparticles. Their particle size, zeta potential, morphology and magnetic characterization were studied. To research its drug release effect, epidoxorubicin (EPI) as a model drug was encapsulated into the PEG/RGD-MPLs. The influences of samples/MCF-7 cells incubation time on cellular uptake were investigated. The cell-viability test of GES-1 cells was assessed by MTT assay.

Section snippets

Materials

PGA with a molecular weight (MW) of 5 × 104 was supplied by Guilin peptide Technology Limited (Guangxi, China). RGD peptide of the arginine–glycine–aspartic acid sequence was obtained by GL Biochem (Shanghai) Ltd. N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were obtained from DingguoBiotechnology Co. Ltd. (Shanghai, China). mPEG with a molecular weight (MW) of 2 × 103 was purchased from Aldrich. Pyrene was purchased from Sigma–Aldrich. EPI

Structural characterization of OQPGA and its derivatives

The synthesis of OQPGA and its derivatives were shown in Fig. 1. The results of FT-IR analysis were shown in Fig. 2a. In the spectrum of OQPGA, the appearance of new intensive peak at 721.48 cm−1 (n  4) corresponding to methylene planar rocking vibration which can be attributed to the long carbon segment of the quaternary ammonium salt (Qin et al., 2002). The two peaks at 2918.01 cm−1 (belongs to antisymmetrical stretching) and at 2850.11 cm−1 (belongs to symmetrical stretching) corresponded to

Conclusion

In this paper, new amphiphilic OQPGA, PEG-OQPGA, RGD-OQPGA were synthesized and their carboxyl groups amounts were measured. Self-assembled PEG/RGD-MPLs carriers were produced successfully. Compared to MCLs, PEG/RGD-MPLs exhibited sufficient size and zeta stability, controlled drug release and less magnetic nanoparticles leakage. The cellular uptake results suggested that the PEG/RGD-MPLs (with RGD and magnetic particles) exhibited more uptake than non RGD and non magnetism carriers in MCF-7

Acknowledgments

The authors gratefully acknowledge National Natural Science Foundation of China (50873076), National High Technology Program of China (863 Program) (2007AA021808), National High Technology Program of China (863 Program) (2007AA021802) and Tianjin Science and Technology Program (09ZCGYSF00900).

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Both authors contributed equally to this work.

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