Polymeric micelles with a pH-responsive structure as intracellular drug carriers

doi:10.1016/j.jconrel.2005.07.017

Journal of Controlled Release

Volume 108, Issue 1, 2 November 2005, Pages 140-149

https://doi.org/10.1016/j.jconrel.2005.07.017 Get rights and content

Abstract

Polymeric micelles based on poly(l-lactide)-b-poly(2-ethyl-2-oxazoline)-b-poly(l-lactide) (PLLA-PEOz-PLLA) ABA triblock copolymers were designed as intracellular drug carriers. The PLLA-PEOz-PLLA micelles adopt a “flower-like” arrangement with A-blocks at the core and a B-block on the shell under neutral condition. The deformation of the core-shell structure is then promoted by the aggregation of PEOzs due to the formation of inter- and intra-hydrogen bonding between protonated nitrogen and carbonyl groups. The experiments on in vitro release have confirmed that the release of doxorubicin (DOX) from micelles was successfully inhibited at pH 7.4. In contrast, an accelerated release of DOX from micelles was observed at acidic conditions. The results of growth inhibition assay indicated that the cell-killing rate of DOX-loaded micelles gradually approached that of free DOX as increasing the concentration and the incubation time. The overlay of fluorescent images on CLSM observation clearly demonstrated the colocalization of DOX with acidic compartments, suggesting that the drug release was successfully triggered in the acidic organelles by means of micelle deformation.

Introduction

Polymeric micelles seem to be one of the best carriers for delivering hydrophobic drugs. The functional polymer micelles possess several advantages such as high drug efficiency, targeted delivery and minimized cytotoxicity. The synthesis of block copolymers using nano-structured templates has emerged as a useful and versatile approach for preparing drug carriers. In aqueous solution, amphiphilic copolymers self-assemble in a core-shell structure that provides a loading space to accommodate mainly hydrophobic drugs. Additionally, the nano-scaled polymer micelles exhibit prolonged systemic circulation times and reduced uptake by the mononuclear phagocyte system (MPS). [1], [2], [3] Anticancer drugs that are incorporated into micelles were shown to improve their stability and efficiency [4], [5].

Recently, water-soluble block copolymers that can exist in different states in aqueous solution have been extensively investigated [6], [7]. The “schizophrenic” block copolymer based on poly[4-vinylbenzoic acid (VBA) and 2-N-(morpholino)ethyl methacrylate (MEMA) was dissolved molecularly above pH 6. Below pH 6, VBA-core micelles were formed, whereas MEMA-core micelles were formed in alkaline media [8]. Triblock copolymers such as poly(acrylic acid)-b-polystyrene-b-poly(4-vinyl pyridine) (PAA-b-PS-b-P4VP) have also been shown to change the aggregate morphologies by adjusting the solution pH. Vesicles with PAA on the outside were inverted to P4VP on the outside due to the difference in repulsive interaction of PAA and P4VP under different pH [9]. Poly[2-(dimethylamino)ethyl methacrylate]-block-poly[2-(N-morpholino)ethyl methacrylate] (DEA-MEMA) formed MEMA-core micelles at pH 6.5 in the presence of 1 M Na₂SO₄, whereas the DEA block formed a micelle core at pH 1 [10]. These unique properties are responsible for the special advantage of using micelles of amphiphilic block copolymers for various applications, including drug delivery systems.

This study proposes a new delivery system, which is potentially useful for targeted drug delivery, and rapidly changes the micelle structure in response to changes in intracellular pH. Conventional pH-sensitive polymers work limitedly under acidic and alkaline conditions. PEOz was used as a pH-sensitive functional polymer because of its low toxicity and favorable pK_a value near neutral pH [11], [12]. PLLA is an extensively investigated biodegradable polymer in the field of drug delivery [13], [14], [15]. Following intravenous administration, polymeric micelles are taken up to cells via endocytosis. The pH value of endocytic vesicles is gradually decreased from 7.4 to 5 because protons are pumped into endocytic vesicles [16]. The content of flower-like micelles based on poly(l-lactide)-b-poly(2-ethyl-2-oxazoline)-b-poly(l-lactide) (PLLA-PEOz-PLLA) can be protected in the hydrophobic inner core when they circulate in blood. The deformation of the micelle structure is then promoted due to the decrease of the pH in acidic organelles following endocytosis. In this study, the pH sensitivity and incorporation of an anti-cancer drug, doxorubicin, into polymeric micelles were investigated. The in vitro drug release, cytotoxicity, growth inhibition and internalization of PLLA-PEOz-PLLA micelles were also studied.

Section snippets

Materials

l-Lactide (Aldrich) and 1,4-dibromo-2-butene (Aldrich) were recrystallized from tetrahydrofuran and n-hexane, respectively. 2-Ethyl-2-oxazoline (Aldrich) was purified by vacuum distillation over CaH₂. Acetonitrile and chlorobenzene was dried over CaH₂ and distilled under nitrogen. Stannous octoate (Sigma), polyethylenimine (M_n 10,000, Aldrich), triethylamine (Tedia), dichloromethane (Tedia) and diethyl ether (Tedia) were used as received. Doxorubicin-HCl was provided by TTY Biopharm, Taiwan.

Preparation of PLLA-PEOz-PLLA micelles

The ABA triblock copolymer consists of biodegradable PLLA A-blocks and water-soluble PEOz B-block (Scheme 1). Characterizations of the products are summarized in Table 1. The composition and molecular weight of PLLA-PEOz-PLLA were determined by comparing the integral peak area associated with the methyl groups of PEOz and that of PLLA [19]. Polymeric micelles were prepared by sonicating a DCM solution of the corresponding copolymers in water. After completely removing the DCM, micelle formation

Conclusion

This study considers the use of pH-sensitive micelles for intracellular drug delivery in cancer therapy. Biomolecular devices triggered by a pH change in intracellular components are used in the design of drug carriers [29]. Biodegradable triblock copolymers with a large drug capacity and effective sensitivity to environmental stimuli are regarded as good candidates for drug delivery [30], [31]. The combined mechanisms of pH-triggered release and biodegradability emerge as having great

Acknowledgment

The authors thank Prof. Jia-Ling Yang at the Institute of Biotechnology of National Tsing Hua University for the assistance with CLSM observation and TTY Biopharm Taiwan for kindly providing Doxorubicin-HCl. This study was supported by National Science Council of ROC (NSC 92-2320-B-007-007) and Industrial Technology Research Institute of Taiwan (A341XS6510).

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Polymeric micelles with a pH-responsive structure as intracellular drug carriers

Abstract

Introduction

Section snippets

Materials

Preparation of PLLA-PEOz-PLLA micelles

Conclusion

Acknowledgment

Adv. Drug Deliv. Rev.

Eur. J. Pharm. Biopharm.

Biochim. Biophys. Acta

J. Pharm. Sci.

Adv. Drug Deliv. Rev.

Colloids Surf., A Physicochem. Eng. Asp.

J. Control. Release

J. Control. Release

J. Control. Release

J. Control. Release

J. Control. Release

J. Control. Release

Biochem. Biophys. Res. Commun.

J. Control. Release

Characterization and anticancer activity of the micelle-forming polymeric anticancer drug adriamycin-conjugated poly(ethylene glycol)-poly(aspartic acid) block copolymer

Cancer Res.

Toxicity and antitumor-activity against solid tumors of micelle-forming polymeric anticancer drug and its extremely long circulation in blood

Cancer Res.