The intracellular drug delivery and anti tumor activity of doxorubicin loaded poly(γ-benzyl l-glutamate)-b-hyaluronan polymersomes
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 (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.
References (46)
- et al.
Polymersomes: a new multi-functional tool for cancer diagnosis and therapy
Methods
(2008) - et al.
Polymersome delivery of siRNA and antisense oligonucleotides
J Control Release
(2009) - et al.
Toward 'smart' nano-objects by self-assembly of block copolymers in solution
Prog Polym Sci
(2005) - et al.
Hyaluronic acid-paclitaxel: antitumor efficacy against CD44(+) human ovarian carcinoma xenografts
Neoplasia
(2007) - et al.
Micellar carriers based on block copolymers of poly(epsilon-caprolactone) and poly(ethylene glycol) for doxorubicin delivery
J Control Release
(2004) - et al.
Novel micelles from graft polyphosphazenes as potential anti-cancer drug delivery systems: drug encapsulation and in vitro evaluation
Int J Pharm
(2009) - et al.
Repetitive doxorubicin treatment of glioblastoma enhances the PGP expression–a special role for endothelial cells
Exp Toxicol Pathol
(2003) - et al.
A comparison of commonly used polyethoxylated pharmaceutical excipients on their ability to inhibit P-glycoprotein activity in vitro
J Pharm Sci
(2002) - et al.
Pluronic block copolymers for overcoming drug resistance in cancer
Adv Drug Deliv Rev
(2002) - et al.
Folate-conjugated amphiphilic hyperbranched block copolymers based on Boltorn H40, poly(l-lactide) and poly(ethylene glycol) for tumor-targeted drug delivery
Biomaterials
(2009)
The efflux of anthracyclines in multidrug-resistant cell lines
Biochem Pharmacol
Daunorubicin cardiotoxicity: evidence for the importance of the quinone moiety in a free-radical-independent mechanism
Biochem Pharmacol
Synthesis and protective effects of coumarin derivatives against oxidative stress induced by doxorubicin
Bioorg Med Chem Lett
Polymer vesicles
Science
Role of block copolymer nanoconstructs in cancer therapy
Crit Rev Ther Drug Carrier Syst
Shrinkage of a rapidly growing tumor by drug-loaded polymersomes: pH-triggered release through copolymer degradation
Mol Pharm
Biomimetic doxorubicin loaded polymersomes from hyaluronan-block-poly(gamma-benzyl glutamate) copolymers
Biomacromolecules
Polysaccharide-block-polypeptide copolymer vesicles: towards synthetic viral capsids
Angew Chem Int Ed Engl
Vesicles and liposomes: a self-assembly principle beyond lipids
Adv Mater
Diffusion studies of nanometer polymersomes across tissue engineered human oral mucosa
Pharm Res
Liposome-encapsulated doxorubicin targeted to CD44: a strategy to kill CD44-overexpressing tumor cells
Cancer Res
Tumor-targeted hyaluronan nanoliposomes increase the antitumor activity of liposomal doxorubicin in syngeneic and human xenograft mouse tumor models
Neoplasia
Hyaluronic acid-N-hydroxysuccinimide: a useful intermediate for bioconjugation
Bioconjug Chem
Cited by (311)
Helix-specific properties and applications in synthetic polypeptides
2023, Current Opinion in Solid State and Materials ScienceTargeted therapy of breast tumor by PLGA-based nanostructures: The versatile function in doxorubicin delivery
2023, Environmental ResearchStrategies to prevent water soluble drug leakage from nanovesicles in blood circulation: A coarse-grained molecular study
2023, Chemical Engineering ScienceSelf-assembling nanowires from a linear L,D-peptide conjugated to the dextran end group
2022, International Journal of Biological Macromolecules