Elsevier

Biomaterials

Volume 31, Issue 10, April 2010, Pages 2882-2892
Biomaterials

The intracellular drug delivery and anti tumor activity of doxorubicin loaded poly(γ-benzyl l-glutamate)-b-hyaluronan polymersomes

https://doi.org/10.1016/j.biomaterials.2009.12.043Get rights and content

Abstract

We have investigated the intracellular delivery of doxorubicin (DOX) loaded poly(γ-benzyl l-glutamate)-block-hyaluronan (PBLG-b-HYA) based polymersomes (PolyDOX) in high (MCF-7) and low (U87) CD44 expressing cancer cell models. DOX was successfully loaded into polymersomes using nanoprecipitation method and in vitro drug release pattern were achieved at pH 5.5 and 7.4 up to 10 days. Block copolymer vesicles without loaded DOX were non cytotoxic in both cells at concentration 150–650 μg/mL. Flow cytometry data suggested successful uptake of PolyDOX in cells and high accumulation was found in MCF-7 than U87 cells. Microscopy imagings revealed that in MCF-7 cells PolyDOX was more in cytoplasm and free DOX in nuclei, whereas in U87 cells free DOX was also found in the cytoplasm. Cytotoxicity of the drug was concentration and exposure time dependent. In addition, PolyDOX significantly enhanced reactive oxygen species (ROS) level in both cells. PolyDOX also suppressed growth of breast tumor on female Sprague–Dawley (SD) rats as compared to phosphate buffer saline pH 7.4 (PBS) control group. In addition reduced level of serum enzymes (LDH and CPK) by PolyDOX formulation indicated less cardiotoxicity of DOX after loading in polymersomes. Results suggest that intracellular delivery of PolyDOX was depended on the CD44 expression level in cells due to presence of hyaluronic acid on the surface of polymersomes, and could be used as a self-targeting drug delivery cargo in over-expressed CD44 glycoprotein cells of breast cancer.

Introduction

Increased attention has been paid to develop vesicular systems based on block copolymers, named polymersomes, as competent drug carriers in recent years, with some remarkable, attractive and feasible characteristics [1], [2], [3], [4], [5], [6], [7]. Compared to liposomes, polymersomes have some advantages such as high membrane stability and low membrane permeability that can be controlled and modulated by varying block lengths. The control of these properties is mainly correlated to the membrane thickness that can be varied to a larger extent in polymersomes (5–20 nm) compared to liposome (3–5 nm). These characteristics can overcome most of the stability problems (mainly drug leakage and disintegration of membrane) encountered in lipidic vesicles due to the high fluidity of their bilayers. In addition, it has been evidenced that the release profiles and kinetics of polymersomes can be controlled in vitro and in vivo [1], [8]. Furthermore, desirable sized polymersomes could be obtained depending on the composition and copolymer chain length, which develop towards smart drug delivery systems [9], [10].

We have investigated here the biomedical applications of a new generation of poly(γ-benzyl l-glutamate)-block-hyaluronan (PBLG-b-HYA) polypeptide-block-polysaccharide copolymers. Hyaluronan (HYA) is a water-soluble, non-immunogenic, natural glycosaminoglycan composed of the repeating disaccharide unit [(1,3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1,4)-O-β-d-glucopyranuronosyl]n. Here, HYA was used as the hydrophilic and stabilizing segment of polymersomes, as well as targeting bio-receptor block [5]. Indeed, HYA is a major ligand for CD44 receptor and it can be used to target cancer cells having over-expressed CD44 glycoproteins [11], [12], [13], [14].

Poly(γ-benzyl l-glutamate)-block-hyaluronan block copolymer has recently reported as self-targeted polymersomes having hyaluronan rich surface for targeting CD44 positive C6 glioma cells [5]. Other strategies on hyaluronan mediated tumor targeting include drug conjugation with HYA [15], HYA attached on the preformed liposomes [13], [16] and micelles based on HYA graft copolymer [17]. Several authors proved the efficacy of HYA targeting in over-expressing CD44 cells and less efficiency in cells having less or no expression of CD44 [11], [18], [19], [20].

In this report, we aimed to demonstrate the in vitro and in vivo efficacy of a formulation of poly(γ-benzyl l-glutamate)-block-hyaluronan (PBLG23-b-HYA10) based polymersomes encapsulating DOX anticancer drug as a chemotherapeutic agent (named PolyDOX). We evaluated the targeting and therapeutic potential of the PolyDOX in cancer cells such as human glioblastoma cell line (U87) [21] and human breast cancer cell line (MCF-7) [22] expressing different level of CD44 receptors, by assessing in vitro drug release kinetics, cell cytotoxicity, cellular uptake efficiency, and reactive oxygen species (ROS) level to address the concept of self-targeted polymersomes. In addition, in vivo antitumor activity was also investigated on 7,12-dimethylbenz[α]anthracene (DMBA) induced breast tumor bearing rats.

Section snippets

Materials

Hyaluronic acid has been purchased from Life core company (Mw¯=5140g/mol,Ip=1.4) (USA) and γ-benzyl-l-glutamate N-carboxyanhydride (NCA-BLG) was purchased from Isochem (France). Doxorubicin hydrochloride (DOX) was purchased from RPG Life Sciences Limited, Mumbai (India). Dulbecco's Modified Eagles Medium (DMEM), antibiotics (Penicillin G, Streptomycin and Nystatin), Dichlorofluorescin diacetate (DCFH-DA) dye and MTT dye were purchased from Sigma (INDIA). Cells from in-house stock in INMAS,

Doxorubicin loaded polymersomes

An amphiphilic polypeptide-block-polysaccharide copolymer was synthesized by coupling an hydrophilic HYA block with a low degree of polymerization DP = 10 (polydispersity PDI = 1.4) to a hydrophobic poly(γ-benzyl l-glutamate) (PBLG) segment (DP = 23, polydispersity = 1.1) using a Huisgen 1,3-dipolar cycloaddition, or “click chemistry” approach. Hydrophilic weight fraction of about 40% together with the intrinsic rigidity of PBLG in α-helical conformation in block copolymer was selected to

Conclusion

In summary, DOX was successfully loaded in PBLG23-b-HYA10 copolymer based polymersomes. The DOX released for more than a week when evaluated in vitro. Hyaluronan based polymersomes efficiently delivered DOX intracellularly in CD44 expressing cancer cells, but to a larger extent in cancer cells that are over-expressed CD44 receptor resulting in a higher cytotoxicity with reduced cardiotoxicity. Selective and enhanced accumulation of PolyDOX in cancer cells may be due to combined enhanced

Acknowledgement

This work was supported by a grant from the Science and technology Service, Embassy of France in India, INDIA for K.K.U. and from internal funds by LCPO. Authors would also thanks to AICTE, TIFAC CORE in NDDS and Director, INMAS for generous funding and extension of laboratory facilities for the work. Authors would also thanks to Mr. Amit Verma (INMAS, Delhi) for their valuable discussions. Correspondence and requests for materials should be addressed to AM and SL.

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