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    International Journal of Pharmaceutics 441 (2013) 30 39

    Contents lists available at SciVerse ScienceDirect

    International Journal of Pharmaceutics

    jo ur n al homep age: www.elsev ier .com/ locate / i jpharm

    emperature- and pH-responsive nanoparticles of biocompatible polyurethanesor doxorubicin delivery

    nning Wanga, Hui Gaoa, , Yanfang Suna, Yu-long Sunb, Ying-Wei Yangb, Guolin Wuc, Yinong Wangc,unge Fanc, Jianbiao Maa,

    School of Chemistry and Chemical Engineering, Tianjin University of Technology, Tianjin 300384, ChinaState Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, ChinaKey Laboratory of Functional Polymer Materials (Ministry of Education), Institute of Polymer Chemistry, Nankai University, Tianjin 300071, China

    r t i c l e i n f o

    rticle history:eceived 18 October 2012eceived in revised form0 November 2012ccepted 12 December 2012vailable online xxx


    a b s t r a c t

    A series of temperature- and pH-responsive polyurethanes based on hexamethylene diisocyanate (HDI)and 4,4-diphenylmethane diisocyanate (MDI) were synthesized by a coupling reaction with bis-1,4-(hydroxyethyl) piperazine (HEP), N-methyldiethanolamine (MDEA) and N-butyldiethanolamine (BDEA),respectively. The chemical structure, molecular weight, thermal property and crystallization proper-ties were characterized by Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance(NMR) spectroscopy, gel permeation chromatography (GPC), differential scanning calorimetry (DSC) andX-ray diffraction (XRD) spectroscopy. The resulting polyurethanes were then used to prepare nanopar-ticles either by direct dispersion method or dialysis method. Their pH and temperature responsibilitieswere evaluated by optical transmittance and size measurement in aqueous media. Interestingly, HDI-based and MDI-based polyurethanes exhibited different pH and temperature responsive properties.Nanoparticles based on HDI-HEP and HDI-MDEA were temperature-responsive, while MDI-based bio-materials were not. All of them showed pH-sensitive behavior. The possible responsive mechanism


    was investigated by H NMR spectroscopy. The cytotoxicity of the polyurethanes was evaluated usingmethylthiazoletetrazolium (MTT) assay in vitro. It was shown that the HDI-based polyurethanes werenon-toxic, and could be applied to doxorubicin (DOX) encapsulation. The experimental results indicatedthat DOX could be efficiently encapsulated into polyurethane nanoparticles and uptaken by Huh-7 cells.The loaded DOX molecules could be released from the drug-loaded polyurethane nanoparticles upon pHand temperature changes, responsively.

    . Introduction

    Intelligent polymers, whose characteristics changes in responseo various external stimuli, such as light, pH, electric poten-ial, magnetic field, temperature, etc., are spotlighted in materialcience, tissue engineering and drug delivery systems (Leet al., 2007). Especially, pH and temperature changes as spe-ial triggers have drawn much attention in recent years,wing to their physiological importance in the human bodynd practical advantages both in vitro and in vivo (Fu et al.,011).

    As a class of important smart material, pH-sensitive polymers

    an respond to pH changes in the surrounding medium to adjustheir structures and conformations, due to the disequilibrium ofonizationdeionization of polymers in aqueous solution caused by

    Corresponding authors. Tel.: +86 2260214259; fax: +86 2260214251.E-mail addresses: [email protected] (H. Gao), [email protected] (J. Ma).

    378-5173/$ see front matter 2012 Elsevier B.V. All rights reserved.ttp://

    2012 Elsevier B.V. All rights reserved.

    ionizable functional groups (a weak acid or a weak base) (Filippovet al., 2008). It is well known that different parts of the body havedifferent optimal pH levels (Bae et al., 2005a, 2005b; Zhou et al.,2012). The slightly acidic microenvironment of tumors could beemployed as targeting sites for pH-sensitive drug delivery (Aryalet al., 2010; Engler et al., 2011; Haining et al., 2004; Poon et al.,2011; Wang et al., 2004). On the other hand, temperature is also oneof the most commonly used stimuli due to its easy operation andmany other practical advantages both in vitro and in vivo (He et al.,2008). Temperature-sensitive nanoparticles show great potentialas cancer drug carriers, because the temperature in a specific patho-logical site is usually higher or can be externally manipulated tohigher values (Chen et al., 2007). Taking advantage of this prop-erty, drug delivery can be intelligently triggered by temperaturechanges. Inspired by these, nanoparticles responsive to the fluctu-

    ation of both pH and temperature in physiological conditions canbe more attractive as advanced drug carriers for cancer therapy dueto possible synergistic advantages (Akiyoshi et al., 1997; Qiu et al.,1997).[email protected]:[email protected]/10.1016/j.ijpharm.2012.12.021

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    A. Wang et al. / International Jour

    Recently, researchers developed pH/temperature multiple sen-itive polymers for drug delivery in the form of beads (Kimt al., 1994) or hydrogels (Dayananda et al., 2008). However,H/temperature-responsive polymeric nanoparticles are rarelytudied, to the best of our knowledge. Nanoparticles will endowhe polymer specific properties that are quite different from bulk

    aterials (Shah et al., 2012). Kang et al. (2003) synthesized aeries of polymers by the copolymerization of methacryloyl poly(N-sopropylacrylamide-co-N,N-dimethylacrylamide) and methacry-oyl sulfamethoxypyridazine telomers. The resulting polymers

    ere then used to fabricate nanoparticles loaded with doxoru-icin (DOX), showing a pH and temperature-sensitive releaserofile. However, polymers composed of acylamide were notiodegradable and their cytotoxicities were not considered in theirtudy.

    Polyurethanes as important biomaterials have been widelysed in stimuli-responsive drug delivery systems (DDS) for con-rolled drug release, tissue engineering scaffolds, artificial muscles,tc., due to their attractive physical properties and good bio-ompatibility (Chen et al., 2000; Gavini et al., 2009; Huynht al., 2010; Lan et al., 1996; Loh et al., 2008; Zhang et al.,008). Recently, pH-sensitive polyurethane micelles based on car-oxylic groups (Ding et al., 2009), amino groups (Huynh et al.,010) and acidic cleavage hydrazone linkage (Bae et al., 2003,005a, 2005b) have been developed for drug delivery applica-ions. The biologically active drug is released by the disassemblyf micelle in response to external pH change (Sun et al., 2011).ery recently, temperature-responsive polyurethanes were devel-ped by our research groups (Fu et al., 2011). Herein, HDI andDI bearing an alkyl and aromatic chain, as well as three diols,

    is-1,4-(hydroxyethyl) piperazine (HEP), N-methyldiethanolamineMDEA) and N-butyldiethanolamine (BDEA), composed of pH-ensitive amino groups bearing different carbon chain lengthnd steric structure were chosen to prepare polyurethanes. Thisariation in structure will influence the hydrophilichydrophobicalance of the polymer, and thus pH and temperature sen-itivity. Nanoparticles were successfully fabricated from theseolymers and their pH/temperature-responsive properties wereystematically investigated. In addition, the cytotoxicity of theseanoparticles was evaluated against Human Umbilical Veinndothelial Cells (HUVEC cells), and the potential use of these poly-eric nanoparticles as drug delivery platform was also explored,

    mploying DOX as a model anti-cancer drug (Fig. 1).

    . Experimental

    .1. Materials

    Hexamethylene diisocyanate (HDI, 99%), 4,4-diphenylmethaneiisocyanate (MDI, 99%), N-methyldiethanolamine (MDEA, 99%), N-utyldiethanolamine (BDEA, 99%) and dibutyltin dilaurate (DBTDL,5%) were purchased from Aladdin Reagent Co., Ltd. (Shanghai,hina). Bis-1,4-(hydroxyethyl) piperazine (HEP, 99%) was obtainedrom Nanjing Chemlin Chemical Industry Co., Ltd. (Nanjing, China).-Dimethylformamide (DMF, 99%) was freshly distilled under

    educed pressure before subjecting to any reactions. Toluene andichloroethane were distilled over sodium or calcium hydridet ambient pressure, dried over activated 4 A molecular sievesefore use. Doxorubicin hydrochloride (DOX) was purchased

    rom Beijing Huafeng United Technology Co., Ltd. All othereagents and organic solvents obtained from Tianjin Chemicaleagent Co. (Tianjin, China) were of reagent grade and used aseceived.

    Pharmaceutics 441 (2013) 30 39 31

    2.2. Synthesis of polyurethanes

    The two series of polyurethanes were synthesized by the con-densation reaction via the coupling reaction between terminalhydroxyl groups of HEP, MDEA and BDEA and isocyanate groupsof HDI and MDI, respectively. As for HDI-based polyurethanes,dibutyltin dilaurate was used as catalyst, while no catalyst wasemployed for MDI-based polyurethanes due to its high reactionactivity. The stoichiometric ratio of OH and NCO groups wasadjusted to NCO/OH = 1.0 (in the case of HDI) or 1.2 (in the caseof MDI). Briefly, HEP (1.76 g, 10 mmol) was added into a dry three-neck r