Thermo- and pH-responsive copolymers based on PLGA-PEG-PLGA and poly(l-histidine): Synthesis and in vitro characterization of copolymer micelles

doi:10.1016/j.actbio.2013.12.033

Acta Biomaterialia

Volume 10, Issue 3, March 2014, Pages 1259-1271

https://doi.org/10.1016/j.actbio.2013.12.033 Get rights and content

Abstract

A series of novel thermo- and pH-responsive block copolymers of PHis-PLGA-PEG-PLGA-PHis composed of poly(ethylene glycol) (PEG), poly(d,l-lactide-co-glycolide) (PLGA) and poly(l-histidine) (PHis) were synthesized and used for the construction of stimuli-responsive copolymer micelles. The starting polymers of PLGA-PEG-PLGA and PHis were synthesized by ring-opening polymerization of dl-lactide and glycolide with PEG as an initiator and l-histidine N-carboxylanhydride with isopropylamine as an initiator, respectively. The final copolymer was obtained by the coupling reaction of PHis with PLGA-PEG-PLGA. The copolymer micelles were constructed to have an inner core consisting of two hydrophobic blocks (PLGA and deprotonated PHis) and an outer hydrophilic PEG shell. The temperature- and pH-induced structure changes of the micelles were characterized by an alteration in particle size, a decrease in pyrene florescence intensity, and a variation of ¹H NMR spectra in D₂O. It was speculated that the hydrophobic–hydrophilic transitions of PEG and PHis in response to temperature and pH variations accounted for the destabilization of micelles. In vitro release profiles, cell cytotoxicity and intracellular location studies further confirmed the temperature- and pH-responsive properties of the copolymer micelles. These results demonstrate the potential of the developed copolymers to be stimuli-responsive carriers for targeted delivery of anti-cancer drugs.

Introduction

Polymeric micelles from amphiphilic block copolymers have attracted growing interest for anticancer drug delivery applications due to their various advantages over other types of nanoparticulates [1], [2]. The principal advantages of such micelles are their high stability, high loading capacity for hydrophobic drugs, controlled drug release and potential for tumor targeting. Despite the efficient tumor targeting achieved by micelles, the relatively slow drug release kinetics from micelles at target sites has been considered a main problem compromising their therapeutic efficacy, especially for drug-resistant tumor cells [3], [4]. It may also pose a risk to the development of multidrug resistance of initially drug-sensitive tumor cells.

Micelles with drug release mechanisms triggered by a variety of physical or chemical stimuli, such as high temperature [5], pH [6] and ultrasound [7], have been developed to overcome the above-mentioned problem. Among these stimuli, high temperature has been recognized as one of the best options because of its easy and safe medical application. The merits of high temperature are due to the substantial strides that have been made in applications of the hyperthermia therapy to solid tumors and the synergistic effect of hyperthermia and chemotherapy [8]. Studies have demonstrated that the use of local hyperthermia in combination with thermosensitive drug delivery resulted in significantly enhanced antitumor efficacy [9], [10]. Besides temperature, pH has also proved another attractive stimulus because of the acidic prototype of tumor tissue [11].

Numerous thermoresponsive micelles have been reported over the past decade mainly based on the thermosensitive polymer poly(N-isopropylacrylamide) (PNIPAAm) [12]. PNIPAAm exhibits a lower critical solution temperature (LCST) of approximately 33 °C in aqueous solution, above which its solubility changes from hydrophilic to hydrophobic [13]. The change of solubility around the LCST destabilizes PNIPAAm-based micelles and triggers a burst-like release of the incorporated drug. However, the primary factors limiting the applications of PNIPAAm-based micelles were lack of biocompatibility and biodegradability as well as the poor micellar stability [4]. This makes biodegradable thermoresponsive copolymer of poly(dl-lactide-co-glycolide)–poly(ethyleneglycol)–poly(dl-lactide-co-glycolide) (PLGA-PEG-PLGA) more attractive than PNIPAAm. However, studies related to PLGA-PEG-PLGA copolymers have been mainly focused on the physical form of hydrogel rather than on micelles.

A number of pH-responsive micelles based on poly(l-histidine) have been developed, such as poly(l-histidine) (polyHis, M_n 5 K)-poly(ethylene glycol) (PEG, M_n 2 K) (PHis-PEG) diblock copolymer micelles [14], mixed micelles of PHis-PEG and poly(l-Lactide)-poly (ethylene glycol) (PLLA-PEG) [15] and the flower-like micelle constructed from poly(l-lactic acid) (PLA, M_n 3 K)-poly(ethylene glycol) (PEG, M_n 2 K)-poly(l-histidine) (polyHis, M_n 5 K) [16]. These micelles were found to undergo structural destabilization at slightly acidic pH due to the protonation of polyHis. However, it is very difficult to design pH-responsive micelles with a precise response to tumor acidic pH because of the subtle and complicated pH difference between the tumor and normal tissues. For example, the diblock copolymer of PHis-PEG had a higher triggering pH than extracellular tumor pH and began to dissociate below pH 7.4 [17].

More advanced polymers with both thermo- and pH-responsive properties were required to circumvent the above-mentioned problems. The dual-response properties of such copolymers would not only enable better on-demand drug release at the target site than solely thermo- or pH-responsive polymer but would also offer a promising synergistic effect of hyperthermia and chemotherapy. In this study, a new class of thermo- and pH-responsive pentablock copolymers of PHis-PLGA-PEG-PLGA-PHis was designed with the knowledge that a dual stimuli-responsive copolymer can be tailored that comprises both a thermoresponsive block and a pH-responsive block. The copolymer was expected to inherit its thermoresponsiveness from the PLGA-PEG-PLGA, and its pH-responsiveness from PHis. Compared to the previously reported PNIPAAm-based dual thermo- and pH-responsive polymers [18], [19], [20], the copolymers were superior in biodegradability and biocompatibility due to their composition.

PLGA-PEG-PLGA copolymer end-capped with N-Boc-histidine was recently synthesized and applied as tumor extracellular pH-responsive micelles for drug delivery [21]. However, the synthesized copolymer has not previously exhibited thermoresponsiveness. The dual thermo- and pH-responsive properties of the PHis-PLGA-PEG-PLGA-PHis copolymers as well as their potential for the delivery of anticancer drugs have not been investigated to date. In addition, the copolymer featured a large proportion of hydrophobic blocks (PLGA and PHis) in its molecular structure, which could enable good drug-loading capacity and micellar stability and pH responsive property. The main objectives of this study were to synthesize the copolymer PHis-PLGA-PEG-PLGA-PHis, and to characterize the thermo- and pH-responsive properties of the copolymer-based micelles by particle size measurements, fluorescent probe technique, ¹H nuclear magnetic resonance (NMR) study, in vitro drug release and cell cytotoxicity. In addition, the mechanisms of the temperature- and pH-responsiveness of the copolymer-based micelles were proposed.

Section snippets

Materials

dl-Lactide and glycolide were obtained from Beijing CONAN Polymer R&D Center (Beijing, PR China). Polyethylene glycol (PEG 2000), stannous 2-ethylhexanoate and 2-mercaptoethanol were purchased from Sigma–Aldrich (St Louis, MO, USA). Diethyl ether, methylene chloride, pyridine, succinic anhydride, thionyl chloride, isopropylamine, dimethylformamide (DMF) and dimethylsulfoxide (DMSO) were purchased from Tianjin Bodi Chemical Co. Ltd. (Tianjin, PRChina). N,N′-Carbonyldiimidazole (CDI) was

Characterization of PHis-PLGA-PEG-PLGA-PHis copolymer

The ¹H NMR spectra of the intermediates and the final pentablock copolymer are shown in Fig. 2A–F. All the chemical shifts were expressed in parts per million (δ) relative to the solvent signal. The ¹H NMR spectrum (CDCl₃) of PLGA-PEG-PLGA showed peaks at δ_a 5.21 ppm ( single bond COCH(CH₃)O), δ_b 4.82 ppm (COCH₂O), δ_c 3.68 ppm (OCH₂CH₂O) and δ_d 1.58 ppm (COCH(CH₃)O) (Fig. 2A). The ¹H NMR spectrum (CDCl₃) of CDI-PLGA-PEG-PLGA-CDI showed peaks at δ_e 8.23 ppm (the proton “e” on the imidazole moiety), δ_f 7.45 ppm

Conclusion

A series of thermo- and pH-responsive amphiphilic pentablock copolymers of PHis-PLGA-PEG-PLGA-PHis were developed for constructing smart micelles in response to tumor pH and local heating. The thermo- and pH-responsive properties of the copolymer were characterized by an alteration in micelle particle size, a decrease in pyrene fluorescence intensity and accelerated DOX release profiles. The ¹H NMR study indicated that the hydrophilic–hydrophobic transitions of PEG and PHis blocks in response

Acknowledgements

The authors are grateful for financial support from the National Natural Science Foundation of China (30801456) and the Natural Science Foundation of Heibei Province (C2011319009).

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Thermo- and pH-responsive copolymers based on PLGA-PEG-PLGA and poly(l-histidine): Synthesis and in vitro characterization of copolymer micelles

Abstract

Introduction

Section snippets

Materials

Characterization of PHis-PLGA-PEG-PLGA-PHis copolymer

Conclusion

Acknowledgements

Adv Drug Deliv Rev

J Control Release

Prog Polym Sci

J Control Release

J Control Release: Off J Control Release Soc

J Control Release

Prog Polym Sci

Prog Polym Sci

J Control Release

J Control Release

J Control Release

J Control Release

J Control Release

J Colloid Interface Sci