Colloids and Surfaces B: Biointerfaces 115 (2014) 349–358

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Micelles of enzymatically synthesized PEG-poly(amine-co-ester) block copolymers as pH-responsive nanocarriers for docetaxel delivery Xiaofang Zhang a,1 , Bo Liu b,1 , Zhe Yang a , Chao Zhang a , Hao Li a , Xingen Luo a , Huiyan Luo a,c , Di Gao a , Qing Jiang a , Jie Liu a,∗ , Zhaozhong Jiang d,∗ a

Department of Biomedical Engineering, School of Engineering, Sun Yat-sen University, Guangzhou, Guangdong 510006, China Department of General Surgery, The Ling Nan Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, China c Department of Medical Oncology, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in Southern China, Guangzhou, Guangdong 510060, China d Molecular Innovations Center, Yale University, 600 West Campus Drive, West Haven, CT 06516, United States b

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Article history: Received 3 October 2013 Received in revised form 10 December 2013 Accepted 16 December 2013 Available online 24 December 2013 Keywords: Lipase Poly(amine-co-ester) Micelle Docetaxel delivery pH-responsive

a b s t r a c t A series of PEGylated poly(amine-co-ester) terpolymers were successfully synthesized in one step via lipase-catalyzed copolymerization of ␻-pentadecalactone (PDL), diethyl sebacate (DES), and Nmethyldiethanolamine (MDEA) comonomers in the presence of poly(ethylene glycol) methyl ether as a chain-terminating agent. The resultant amphiphilic poly(ethylene glycol)-poly(PDL-co-MDEA-cosebacate) (PEG-PPMS) block copolymers consisted of hydrophilic PEG chain segments and hydrophobic random PPMS chain segments, which self-assembled in aqueous medium to form stable, nanosized micelles at physiological pH of 7.4. Upon decreasing the medium pH from 7.4 to 5.0, the copolymer micelles swell significantly due to protonation of the amino groups in the micelle PPMS cores. Correspondingly, docetaxel (DTX)-encapsulated PEG2K-PPMS copolymer micelles showed gradual sustained drug release at pH of 7.4, but remarkably accelerated DTX release at acidic pH of 5.0. The drug-loaded micelle particles were readily internalized by SK-BR-3 cancer cells and, compared to free DTX drug, DTXloaded micelles of the copolymers with optimal compositions exhibited enhanced potency against the cells. Biodegradable PEG-PPMS copolymer micelles represent a new type of promising, pH-responsive nanocarriers for anticancer drug delivery, and the drug release rate from the micelles can be systematically controlled by both pH and the copolymer composition. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Chemotherapy remains as a primary procedure to treat malignant tumors. However, many therapeutic anticancer drugs are limited in clinical applications due to their severe side effects and lack of optimal formulations for their efficient delivery. Nanotechnology has been evaluated in recent years and has shown great potential to improve cancer chemotherapy treatments by enhancing anticancer drug efficacy and by reducing the side effects of the treatments [1–4]. It is known that rapid vascularization takes place at tumor sites, resulting in leaky, defective structure and impaired lymphatic drainage. This characteristic tumor neovascular architecture allows passive targeted delivery of drugloaded nanocarriers to tumor sites via enhanced permeation and

∗ Corresponding authors. Tel.: +86 20 39332145, +1 2037373262. E-mail addresses: [email protected] (J. Liu), [email protected] (Z. Jiang). 1 These authors contributed equally to this work. 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.12.029

retention (EPR) effect [2,4,5]. To achieve desirable EPR effect, the size of the nanocarriers needs to be controlled in the range approximately between 30 and 200 nm in diameter [3,6–9]. Among various nanocarriers that include nanoparticles [10], micelles [11], hydrogels [12], liposomes [13], dendrimers [14], and capsules [15], amphiphilic copolymer micelles are particularly attractive as they are capable of encapsulating drugs, subsequently releasing the drugs in a controlled fashion, and effectively escaping from uptake and clearance by reticuloendothelial system (RES) to prolong their circulation in the blood [3,16,17]. In addition to their defective neovascular structures, typical solid tumors possess a weakly acidic extracellular pH ranging from 5.7 to 7.0 as the result of lactic acid accumulation due to poor oxygen perfusion [18,19]. In contrast, the extracellular pH of the normal tissue and blood is slightly basic (pH of 7.2–7.4). Thus, enhanced drug delivery efficiency is anticipated for anticancer drug-loaded micelles that are pH-responsive and can be triggered by acidic pH to accelerate the drug release [10,20–22]. Furthermore, even more acidic conditions (pH = 4.0–6.0) are encountered in endosomes and lysosomes after uptake of the micelles by tumor cells via

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endocytosis pathways [23], which may further increase the cytotoxicity of the drug-encapsulated micelles. Various pH-responsive micelle nanocarriers have been investigated in the past decade. Such micelles are often formed via self-assembly of amphiphilic block copolymers and consist of a hydrophilic (e.g. PEG) outer shell and a hydrophobic inner core capable of response to medium pH. Typically, upon changing the medium pH from neutral or slightly basic to mildly acidic, the micelle cores undergo accelerated degradation [24,25], become completely soluble in water [10], or swell substantially in aqueous medium [26]. As the result, the drug-encapsulated micelles with a slow drug-release rate at the physiological pH can be triggered by an acidic pH to rapidly unload the drug molecules. The polymer segments constituting the micelle cores in previous reports include poly(ortho esters) [24], poly(ˇ-amino esters) [10,26–31], poly(l-histidine) [11,23], and others [16,32,33]. These studies have made significant progress in developing pH-responsive polymeric micelles for drug delivery and cancer treatment. The disadvantages with many previous pH-responsive micelle systems are the multiple steps required for preparing the copolymers and the difficulty of systematically controlling the hydrophobicity of the copolymer hydrophobic segments during the polymer synthesis [26,34–36]. Furthermore, since in vivo pH cannot be externally adjusted, the drug release rate cannot be controlled using those previous drug-carriers that solely rely on pH to regulate the release rate. Herein, we wish to report lipase-catalyzed, one-step synthesis of poly(ethylene glycol)-poly(␻-pentadecalactone-co-Nmethyldiethyleneamine-co-sebacate) (PEG-PPMS) block copolymers from readily available substrates and the results on evaluation of the new micelle system based on the biodegradable copolymers as pH-responsive nanocarriers for controlled release delivery of anticancer drug docetaxel (DTX). DTX is a commercial anti-mitotic chemotherapy medicine that is normally administered after anthracycline-based chemotherapy drugs (e.g., doxorubicin) fail to stop cancer progression in a patient. In a previous report, poly(␻-pentadecalactone-co-Nmethyldiethyleneamine-co-sebacate) (PPMS) terpolymers were employed as non-viral vectors for gene delivery by forming nanometer-sized polyplexes with DNA [37]. However, PPMS copolymers alone do not self-assemble to form stable nanoparticles in aqueous medium. In this work, stable nanosized micelles were prepared from PEG-PPMS copolymers, in which the degree of hydrophobicity of the PPMS segments can be tuned by adjusting the ␻-pentadecalactone content in the chain segments. The PEG-PPMS micelles differ from previous pH-responsive nanocarriers in that the drug release rate from PEG-PPMS micelles can be controlled by both pH and the copolymer composition (or PPMS hydrophobicity). Thus, for in vivo applications where pH is not controllable, the drug release rate can be regulated by adjusting the PEG-PPMS copolymer composition. Attempts were made previously with limited success to achieve similar results by using micelles from two structurally different copolymers with the drug paclitaxel covalently linked to the polymer chains via pH-sensitive hydrazone bonds [38]. The current studies were designed to explore how the composition of the new PEG-PPMS micelle system influences their pH-response behavior, stability, biocompatibility and cellular uptake properties, and to evaluate their potential to serve as nanocarriers for delivery of anticancer drug DTX.

2. Experimental 2.1. Materials ␻-Pentadecalactone (PDL, 98%), N-methyldiethanolamine (MDEA, 99+%), diethyl sebacate (DES, 98%), diphenyl ether (99%),

poly(ethylene glycol) methyl ether (MeO-PEG-OH, Mn = 2000 Da and 5000 Da, Mw /Mn = 1.2 for both polymers), and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich Chemical Co. and were used as received. Immobilized CALB (Candida antarctica lipase B supported on acrylic resin) catalyst or Novozym 435, chloroform (HPLC grade), acetonitrile (HPLC grade), chloroform-d, and n-hexane (97+%) were also obtained from Sigma–Aldrich Chemical Co. The lipase catalyst was dried at 40 ◦ C under 2.0 mmHg for 20 h prior to use. Docetaxel (DTX) was purchased from Beijing Huafeng United Technology Co. SK-BR-3 cells were acquired from Shanghai cell bank of Chinese Academy of Science (Shanghai, China) and were maintained in RPMI-1640 (Gibco) containing 10% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillin-streptomycin at 37 ◦ C under a 5% CO2 humidified atmosphere. 2.2. Instrumental methods 1 H and 13 C NMR spectra were recorded on a Bruker AVANCE 500 spectrometer. The chemical shifts reported were referenced to internal tetramethylsilane (0.00 ppm) or to the solvent resonance at the appropriate frequency. The number and weight average molecular weights (Mn and Mw , respectively) of copolymers were measured by gel permeation chromatography (GPC) using chloroform as the eluent and narrow polydispersity polystyrenes as the standards according to previously reported procedures [39]. The average size, size polydispersity, and zeta-potential of micelles were measured by dynamic light scattering (DLS) in aqueous medium at 25 ◦ C using a Malvern Zetasizer Nano ZS90. All samples were analyzed in triplicate at 633 nm and at 90◦ fixed scattering angle.

2.3. Synthesis and purification of poly(ethylene glycol)-poly(␻pentadecalactone-co-N-methyldiethyleneamine-co-sebacate) (PEG-PPMS) block copolymers The block copolymers were synthesized via copolymerization of ␻-pentadecalactone (PDL), diethyl sebacate (DES), and N-methyldiethanolamine (MDEA) using MeO-PEG-OH as the chainterminating agent and Novozym 435 as the catalyst. The molar ratios of the comonomers and the PEG substrates are reported in Table 1. The amount of MeO-PEG-OH with Mn of 5000 Da or 2000 Da (PEG5K or PEG2K) was selected to form PEG-PPMS block copolymers with approximately 40 wt% PEG upon complete conversion of the feeds. The PDL content in the PPMS blocks of the copolymers is controlled by adjusting the molar ratio of PDL/DES/MDEA comonomers. Thus, PDL, DES, MDEA, and PEG5K or PEG2K in various ratios (Table 1) were blended with Novozym 435 (10 wt% vs. total substrates) and diphenyl ether solvent (200 wt% vs. total substrates) to form reaction mixtures. All copolymerization reactions were carried out in two stages: first stage oligomerization at 90 ◦ C under 1 atm pressure of nitrogen gas for 20 h, and second stage polymerization at 90 ◦ C under 1.4 mmHg vacuum for 72 h. At the end of the reactions, n-hexane was added to the product mixtures to precipitate the resultant copolymers. The obtained crude products were then washed with fresh n-hexane twice to extract and remove the residual diphenyl ether solvent in the polymers. Subsequently, the copolymers were dissolved in chloroform and were filtered to remove the catalyst particles. Complete evaporation and removal of the chloroform solvent from the filtrates at 30 ◦ C under high vacuum (