Macromolecular Research, Vol. 20, No. 3, pp 292-301 (2012) DOI 10.1007/s13233-012-0051-0
www.springer.com/13233 pISSN 1598-5032 eISSN 2092-7673
pH-Responsive Drug Delivery Systems Based on Clickable Poly(L-glutamic acid)-Grafted Comb Copolymers Jianxun Ding1,2, Chaoliang He1, Chunsheng Xiao1,2, Jie Chen1, Xiuli Zhuang1, and Xuesi Chen*,1 1
Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China 2 Graduate University of Chinese Academy of Sciences, Beijing 100039, P. R. China Received October 30, 2011; Revised November 15, 2011; Accepted November 15, 2011 Abstract: Five pH-responsive alkyne-poly(2-aminoethyl methacrylate)-graft-poly(L-glutamic acid) (alkynePAMA-g-PLGA) comb copolymers were synthesized through the ring-opening polymerization (ROP) of γ-benzylL-glutamate N-carboxyanhydride (BLG NCA) and the subsequent deprotection of benzyl group from BLG unit. The chemical structures of copolymers were confirmed by proton nuclear magnetic resonance spectra (1H NMR) and Fourier transform infrared spectroscopy (FTIR). The pyrene-probe-based fluorescence technique and transmission electron microscopy (TEM) measurements revealed that the comb copolymers could spontaneously self-assemble into micellar or vesicular nanoparticles in phosphate buffered saline (PBS) at pH 7.4. Doxorubicin (DOX), an anthracycline anticancer drug, was loaded into nanoparticles as a model anticancer drug. The in vitro release results showed that the release behaviors could be altered by adjusting the composition of the comb copolymer and pH of the release medium. In vitro methyl thiazolyl tetrazolium (MTT) assays demonstrated that the copolymers were biocompatible, and DOX-loaded nanoparticles showed effective inhibition of cellular proliferation. Hemolysis tests indicated that the copolymers were also hemocompatible, and that the presence of the copolymers could reduce the hemolysis ratio (HR) of the DOX significantly. In addition, the comb copolymers could be modified through versatile Cu(I)-catalyzed “click chemistry” between the terminal alkyne group and azide-modified functional agents. These properties indicate that the pH-responsive clickable comb copolymers are promising candidates for multifunctional nanocarriers in cancer diagnosis and therapy. Keywords: comb copolymer, drug delivery, nanoparticle, pH-responsive, polypeptide.
Introduction
triggered by the specific microenvironments of tumor site or intracellular space, leading to aggressive anticancer activity and maximal chemotherapeutic efficacy with fewer side effects.23,24 For the nanoparticles to be ideal carriers, they should meet the following two requisites: 1) intelligent drug release behaviors, and 2) good biocompatibility and appropriate biodegradability.20,25 Synthetic polypeptides are one of the most important biocompatible and biodegradable polymers, which have precise secondary conformations and have been widely studied for various biomedical applications, such as drug and gene delivery, biosensors and diagnostics.26-29 Poly(L-glutamic acid) (PLGA) and its derivatives are one of the most widely investigated synthetic polypeptides as biomedical materials due to their modifiable carboxyl side group and pH-responsive property (pKa ~4.5).19,30-33 In this work, alkyne-poly(2-aminoethyl methacrylate hydrochloride) (alkyne-PAMA) macroinitiators were first prepared by atom transfer radical polymerization (ATRP) of 2-aminoethyl methacrylate hydrochloride (AMA) monomers. PLGA grafted
Over recent decades, various systems, such as polymeric micelles,1-9 vesicles,10-13 liposomes,14-17 and nanogels18,19 have been developed as nanocarriers that can effectively achieve the controlled delivery of anticancer drugs, genetic agents and so on. Nanocarriers have expressed many benefits for cancer chemotherapy due to the minimization of serious side effects as well as effective resistance to rapid renal clearance and nonspecific uptake by the reticuloendothelial system (RES), resulting in improved therapeutic efficacy.20 Of these systems, stimuli-responsive carriers that sharply respond to changes in microenvironments of some pathological sites, e.g. tumor tissular or to intracellular stimuli, such as, pH, temperature, redox, enzyme etc., have received considerable attention as fascinated potential drug delivery vehicles.4,21,22 As soon as reaching targeted tumor sites or entering tumor cells, the smart nanocarriers can fast release drug *Corresponding Author. E-mail:
[email protected] The Polymer Society of Korea
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pH-Responsive Drug Delivery Systems Based on Clickable Poly(L-glutamic acid)-Grafted Comb Copolymers
comb copolymers were synthesized through ring-opening polymerization (ROP) of BLG NCA monomer using alkynePAMA as macroinitiator and subsequent deprotection of benzyl group from BLG unit (“grafting from” approach). The molecular weight of short nonbiodegradable PAMA backbone was controlled (Mn CP4 > CP5 > CP1. The release rate of DOX from nanoparticles was affected by the electrostatic interaction between DOX and the carboxyl groups. The slowest DOX release from CP5 was relevant to the highest percent of LGA unit in comb copolymers, which enhanced the electrostatic attraction interaction between nanoparticle and DOX. In addition, the in vitro DOX release behaviors of DOX-loaded nanoparticles were investigated at pH 6.8 and 8.0. As shown in Figure 3, DOX release from the CP4 micelle was pH-dependent, and the release rate was in order of pH 6.8 > pH 7.4 > pH 8.0. The accelerated DOX release at acidic pH was probably due to the protonation of carboxylic groups of nanoparticle, which reduced the interaction between nanoparticle and DOX. Such a phenomenon has also been reported by Lecommandoux et al., when DOX was released from polymeric micelles with PLGA core.25 Lastly, it needs to be pointed out that all DOX-loaded nanoparticles displayed relatively severe burst release, which might indicate that the electrostatic interaction between DOX and nanoparticle is susceptible to environmental pH and the small electrolyte molecules in buffer.53 Fortunately, the burst drug release from nanoparticle may be overcome by conjugation of drug molecules to nanoparticle through an acid-labile bond.54,55 The cellular uptake and intracellular release behavior of DOX-loaded nanoparticle towards HeLa cells were monitored by confocal laser scanning microscopy (CLSM). HeLa cells were incubated with DOX-loaded CP4 micelle and free DOX for 5 h (10 mg L-1 DOX). As expected, the nearly same strong intracellular DOX fluorescence in HeLa cells
Figure 4. Representative CLSM images of intracellular DOX release from the pH-responsive DOX-loaded CP4 NPs using HeLa cells after 5 h incubation (A) with free DOX as control (B). For each panel, images from left to right show cell nuclei stained by DAPI (left, blue), DOX fluorescence in cells (middle, red), and overlays of two images. The bar represents 20 µm. 298
Figure 5. In vitro cytotoxicities of alkyne-PAMA-g-PLGA comb copolymers to HeLa cells with SDS as positive control (A) and DOX-loaded nanoparticles with free DOX as control (B). Data were presented as the mean±standard deviation (n=6).
was observed after 5 h incubation with DOX-loaded nanopartice and free DOX (Figure 4). Almost all researchers including our group have reported that free DOX have stronger fluorescence compared with the DOX in the nanoparticles at the same concentration due to the self-quenching effect of DOX.13,24,56 Therefore, the enhanced fluorescence intensity in the HeLa cells incubated with DOX-loaded nanopartice should be the result of high endocytosis efficiency and enhanced intracellular DOX release of DOXloaded nanoparticle. In vitro Cytotoxicities of Alkyne-PAMA-g-PLGA and DOX-Loaded Nanoparticles. The in vitro cytotoxicities of comb copolymers and DOX-loaded nanoparticles toward HeLa cells were evaluated using MTT assays. As shown in Figure 5(A), the viabilities of HeLa cells treated with copolymers for 24 h were over 85% at all test concentrations up to 0.1 g L-1, revealing the low toxicities and good compatibilities of copolymers toward cells and rendering their potential as drug delivery vehicles. To determine the inhibition of HeLa cells proliferation in vitro, the cell viabilities of HeLa cells were evaluated after 24 h incubation with DOX-loaded nanoparticles. Free DOX was used as control. As shown in Figure 5(B), DOX-loaded nanoparticles showed the effective inhibitory effect on the proliferation of HeLa cells. The somewhat lower toxicities of DOX-loaded nanoparticles compared to free DOX was probably due to the gradual Macromol. Res., Vol. 20, No. 3, 2012
pH-Responsive Drug Delivery Systems Based on Clickable Poly(L-glutamic acid)-Grafted Comb Copolymers
release of DOX within the cells. The benefit not only for minimizing drug loss in blood circulation but also for selective accumulation in tumor tissue by enhanced permeability and retention (EPR) effect may enhance its overall therapeutic efficacy in vivo relative to free DOX. The blood compatibilities of comb copolymers and DOXloaded nanoparticles were assessed by a hemolysis assay (Figure 6). The HR represents the degree of RBC membranes destroyed by the substance in contact with blood. A smaller HR value represents better blood compatibility of biomaterial. RBCs and comb copolymers were coincubated at different polymer concentrations for 1 h, then the test result can be visually sorted out (Figure 6(A)) and HR values were determined with spectrophotometer (Figure 6(B)). As shown in Figure 6(B), the representative CP3 and 5 did not show conspicuous hemolytic activities on RBC even at a very high concentration of ~2 g L-1 indicating that copolymers with PLGA side chains were hemocompatible for potential biomedical application. In addition, the hemolytic activities of DOX-loaded nanoparticles were established with free DOX as control. As shown in Figure 6(C), the presence of comb copolymers could lower the HR of DOX significantly indicating the promising use as drug carriers. Functionalization of Alkyne-PAMA-g-PLGA Comb Copolymers. As biological and drug delivery research progressing, comprehensive strategies such as use of multi-functional delivery systems have emerged. Through a simultaneous or sequential synergistic effect, multi-functional nanocarriers are capable of overcoming distinct physiological barriers and delivering therapeutic payloads and/or image contrast enhancement agents to target tumor sites in the body.15,36,37 In this work, an attempt was performed to modify the comb copolymers through versatile Cu(I)-catalyzed “click chemistry” between the terminal alkyne group and azide modified functional agents (RhB-N3 as a model dye). As shown in Figure 7, the emission spectra of RhB and the derivatives confirmed the successful synthesis of RhB modified comb copolymers (i.e. RhB-CP4 and RhB-CP5), although the
Figure 6. Photographs of RBCs hemolysis in the presence of CP3, 5 and DOX-loaded nanoparticles with free DOX as control (A), percentage of RBCs hemolysis incubated with CP3, 5 (B), and DOX-loaded nanoparticles with free DOX as control (C). Physiological saline (-) and Triton X-100 (10 g L-1) (+) were used as negative and positive controls, respectively. Data represent as the mean±standard deviation from three independent experiments. Macromol. Res., Vol. 20, No. 3, 2012
Figure 7. Fluorescence emission spectra of RhB, RhB-N3, RhBCP4, and RhB-CP5 in PBS at pH 7.4 (0.5 mg L-1 RhB). 299
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fluorescence intensity of RhB-CP4 and RhB-CP5 was only ~3-fold of free RhB with the same RhB concentration (0.5 mg L-1). The result revealed that the modification of alkynePAMA-g-PLGA comb copolymers through “click chemistry” was feasible, and the copolymers can be further modified with more available functional substances, such as targeting groups (e.g. folic acid) and near-infrared fluorescent imaging probes (e.g. Cy5.5), etc.. It indicated that the clickable comb copolymers are promising materials to fabricate multifunctional formations for the diagnosis, and treatment of a wide spectrum of cancers.
Conclusions Five pH-responsive alkyne-PAMA-g-PLGA comb copolymers were successfully synthesized through ROP of BLG NCA with alkyne-PAMA as macroinitiators and subsequent deprotection of benzyl groups. The comb copolymers could self-assemble into micellar or vesicular nanoparticles in PBS at pH 7.4 depending on the composition of comb copolymer spontaneously. DOX was loaded into nanoparticles as a model anticancer drug. The decrease in either length of PLGA side chain or pH led to an accelerated DOX release. All DOXloaded nanoparticles exhibited significant burst release, and thus some further technical improvement is required. The comb copolymers are biocompatible, while the DOX-loaded nanoparticles could inhibit cellular proliferation effectively. The HR of DOX could be obviously reduced by the presence of hemocompatible copolymers. Furthermore, the terminal alkyne group could be further modified through versatile Cu(I)-catalyzed “click chemistry” with azide modified functional agents, e.g. RhB-N3. Therefore, the pH-responsive clickable comb copolymers are promising materials to design potential multi-functional nanocarriers for efficient cancer diagnosis and treatment. Acknowledgment. This research was financially supported by National Natural Science Foundation of China (Key Project 50733003, Project 20904053, 51003103, and 50973108), Ministry of Science and Technology of China (International Cooperation and Communication Program 2010DFB50890), Scientific Development Program of Jilin Province (Project 20090135 and 201101082), Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No. KJCX2YW-H19).
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