Research paper
Targeted delivery of RGD-modified liposomes encapsulating both combretastatin A-4 and doxorubicin for tumor therapy: In vitro and in vivo studies

https://doi.org/10.1016/j.ejpb.2010.01.002Get rights and content

Abstract

Arg–Gly–Asp (RGD) modified doxorubicin-loaded liposomes could improve anticancer effect, and vascular disrupting agents (VDAs) could induce a rapid and selective shutdown of the blood vessels of tumors. We propose that RGD-modified liposomes for co-encapsulation and sequential release of vascular disrupting agent combretastatin A-4 (CA-4) and cytotoxic agent doxorubicin (Dox) could enhance tumor inhibition responses. In this study, we encapsulated Dox and CA-4 in RGD-modified liposomes. The release rate of Dox was proved to be much slower than that of CA-4 in vitro. Flow cytometry and laser confocal scanning microscopy clearly showed that RGD-modification promoted intracellular uptake of liposomal drugs by B16/B16F10 melanoma tumor cells and human umbilical vein endothelial cells (HUVECs). Cytotoxicity assay showed that the IC50 of RGD-modified liposomes was lower than that of the corresponding unmodified liposomes. Therapeutic benefits were examined on B16F10 melanoma tumors subcutaneously growing in C57BL/6 mice. In vivo study demonstrated that RGD-modified liposomes exhibited the most pronounced tumor regression effect when both CA-4 and Dox were co-encapsulated. These results suggest that the targeted drug delivery system for co-encapsulation of vascular disrupting agents and anticancer agents may be a promising strategy for cancer treatment.

Introduction

The superiority of combination therapy for cancer treatment has been well known for several decades. Antivascular therapy and cytotoxic therapy are complementary: circulating tumor cells, very early metastases and the dividing rim of mature tumors are sensitive to tumor-targeted therapies, whereas angiogenic metastases and more mature tumors are most sensitive to antivascular therapies [1]. However, the combination of these two drugs, typically when given separately [2], faced several problems: (i) In a long-term treatment which is needed to suppress tumor growth, anti-angiogenesis agent impairs blood flow inside the tumor microenvironment, precluding the access of cytotoxic agent and accumulation of next dosages. (ii) Activation of hypoxic response leads to increased metastases and resistance to chemotherapy [3]. An effective solution is to choose an agent which causes reversible vascular disruption and deliver the cytotoxic drug into tumor site before vascular collapse caused by antivascular agent. For instance, Sengupta et al. developed a vehicle called “nanocell” [4], a multidrug-loaded delivery system which releases the drugs sequentially. Focal drug release within tumor results in improved therapeutic index with reduced toxicity. This study may represent a successful attempt of sequential release strategy for combination chemotherapy.

Liposomal delivery has become a well-established method in cancer treatment [5], [6]. While sterically stabilized liposomes (SSL) can passively accumulate into tumor tissue due to the effect of enhanced permeability and retention (EPR), the subsequent intracellular uptake of the entrapped anticancer drugs by the tumor cells remains an inefficient step [7]. Based on the fact that the integrin (RGD-dependent receptor) is overexpressed in the melanoma tumor cells and tumor endothelial cells [1], we recently validated that RGD-modified micelles significantly facilitated the intracellular delivery of the encapsulated agents into human umbilical vein endothelial cells (HUVEC) and melanoma B16 cells via integrin-mediated endocytosis [8].

Aiming at establishing an “integrative” drug delivery system for sequential release of CA-4 and Dox, we developed RGD-modified liposomes loaded with both CA-4 and Dox. One potential advantage of our liposomes compared to “nanocell” was the surface modification with RGD peptide, which may enhance the intracellular drug uptake by tumor endothelial cells and melanoma tumor cells. Another benefit would come from a more complete release of doxorubicin from liposomes than from doxorubicin-PLGA oligomers, which is not bioactive until decomposed [4]. To test its targeting effect, the specific cell internalization of liposomes was investigated on B16F10 melanoma tumor cells and human umbilical vein endothelial cells (HUVECs) in vitro. In addition, the anti-tumor activity was evaluated via i.v. injection in C57BL/6 mice bearing B16F10 melanoma.

Section snippets

Materials

Arginine–glycine–aspartic acid, RGD (Zhongkeyaguang Biotechnology Co., Ltd., Beijing, China), egg phosphatidylcholine (EPC) (Lipoid GmbH, Co., Germany), Cholesterol (Chol) and Sephadex G50 from Pharmacia Biotech (Piscataway, NJ, USA). Doxorubicin hydrochloride (Dox) was obtained from Haizheng Pharmaceutical Co. (Zhejiang Province, China). Combretastatin A-4 was supplied by Fude Chemical Co., Ltd. (Shanghai, China). (Methoxypolyetheleneglycol (Mw = 2000)–distearylphosphatidylethanolamine

Synthesis of RGD–PEG-DSPE

Preparation of DSPE-PEG-RGD was performed as described previously [12]. Chromatographic analysis of the reaction mixture at different time points (Fig. 1) showed that the RGD was consumed almost completely under the conditions of reaction. Around 50% of input DSPE-PEG-NHS in reaction system was conjugated with RGD peptide. The resulting product was then used for preparing liposomes without further purification.

Characterization of drug loaded liposomes

The particle sizes of various liposomes were around 90 nm (PDI < 0.3) (Table 1), and all

Discussion

Increased expression of αVβ3 integrins was seen in melanoma cells [16]. In addition, endothelial cells in tumor blood vessels also overexpress αV integrins [17]. The RGD peptide specifically binds to the αVβ3 integrins on melanoma tumor cells and endothelial cells. Due to their significant role in tumor growth and metastasis, those two types of cells have been shown to be promising targets for cancer treatment.

Up to now, few studies have been reported on co-delivery of multiple agents in

Acknowledgements

This work was supported by the National High Technology Research and Development Program of China (863 program No. 2007AA021811) and National Basic Research Program of China (973 Program No. 2007CB935800) and National Key Program of New Drug innovation (No. 2009zx09310-001).

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