Abstracts / Journal of Controlled Release 152 (2011) e1–e132
Experimental methods Degradation studies were performed on amino acid based polymers in phosphate buffer at 37 °C and in the presence of various enzymes such as chymotrypsin and esterases. In these studies mass loss and decrease in molecular weight determined by GPC as well as changes in pH were monitored. Cell signaling studies on coatings based on lysine based polyester urethanes where the lysine was chemically modified with arginine– glycine–aspartic acid (RGD) were performed with human foreskin derived fibroblasts with a control where cyclic RGD was introduced into the growth medium. Drug release studies were performed with polyesterurethane acrylate films [1] and polyesteramides [2] using bupivacaine and dexamethasone as model drugs.
Gravimetric Weight Loss (%)
Results and discussion Degradation studies performed on lysine based polyester urethanes revealed that there is a lower pH drop when compared to the lactide/glycolide based polymers of equivalent chemical structure when degradation was performed in phosphate buffer at 37 °C. In the case of polyesteramides containing lysine and leucine, degradation is mainly enzymatic. An example of the degradation profiles in the presence of chymotrypsin is given in Fig. 1. This controlled degradation has been used for controlled release of bupivacaine as shown in Fig. 2.
Biodegradation - PEA.Ac.Bz
-5 0 5
% Bupivacaine (30wt%) Released Over Time
70 60
% Drug Eluted
Introduction The evolution of resorbable degradable polymers from aliphatic polyesters to nitrogen bearing polymers and eventually to amino acid based polymers such as polyurethanes, polyester amides, polyureas and polycarbonates has been accompanied with better control over degradation and release properties. Amino acid containing polymers have emerged as novel biodegradable materials in the drive to find materials that breakdown into safe, metabolizable and/or excretable building blocks. However the effect of using amino acids is that in many cases it involves their incorporation into the polymer via ester, amide, urethane and urea bonds. Most of these bonds do not readily hydrolyze by purely chemical means and may require biocatalytic or cell mediated reactions in order to ensure complete degradation. In this study, we explored the effects of using amino acid building blocks on hydrolytic, enzymatic and macrophage mediated degradation. This has been done with novel polyester amides and polyester urethanes.
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500
1000
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1500 8 weeks
Fig. 2. Drug release of bupivacaine from polyesteramide films. In the absence of a top coat (diamond) and with drug loaded in a primer (square) and base coat (triangle).
derived fibroblasts with a control where cyclic RGD was introduced into the growth medium. Preliminary results indicated that coatings of the polyester urethanes modified with RGD stimulate fibroblast attachment. Conclusion Amino acid based polymers provide a means to reduce a pH drop upon degradation. They can be tailored to degrade hydrolytically or enzymatically. The amino acid building block provides a site for attachment of chemical moieties for biological and cellular response. Acknowledgements Medivas LLC, San Diego, USA, TNO Zeist, The Netherlands. References [1] A. Dias et al., Microparticles comprising a cross-linked polymer WO2007107358 and carbamate, thiocarbamate or carbamide comprising a biomolecular moiety WO2008055666. [2] K.M. DeFife, et al., Poly(ester amide) co-polymers promote blood and tissue compatibility, J. Biomater. Sci. 20 (2009) 1495.
doi:10.1016/j.jconrel.2011.08.090
pH and dual redox responsive nanogel based on poly(l-glutamic acid) as potential intracellular drug carrier Jianxun Ding1,2, Chunsheng Xiao1,2, Lesan Yan1,2, Zhaohui Tang1, Xiuli Zhuang1, Xuesi Chen1, Xiabin Jing1 1 Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China 2 Graduate University of the Chinese Academy of Sciences, Beijing 100039, China E-mail address:
[email protected] (X. Chen).
10 15
Chymotrypsin Elastase I Elastase IIa PBS
20 25 30 0
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35
Time (days)
Abstract summary A triple responsive nanogel was prepared by the crosslinking of poly (ethylene glycol)–b-poly(l-glutamic acid-co-γ-2-chloroethyl-l-glutamate) (mPEG–b-P(LG-co-CELG)) micelle using the dual redox responsive diselenide bond. Preliminary MTT assay and doxorubicin (DOX) release studies revealed that the nanogel was biocompatible and the DOX release could be accelerated by glutathione (GSH), rendering this nanogel's potential application as intracellular drug carrier.
Fig. 1. Degradation of polyesteramides in the presence of chymotrypsin at 37 °C.
Keywords: Diselenide bond, Drug delivery, Nanogel, Poly(l-glutamic acid), Responsive Cell signaling studies on coatings based on the lysine based polyesterurethanes where the lysine was chemically modified with arginine–glycine–aspartic acid were performed with human foreskin
Introduction Within the last decade, various carriers for drug delivery have been developed to improve the efficacy of chemotherapy. Stimuli-
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Abstracts / Journal of Controlled Release 152 (2011) e1–e132
responsive drug delivery systems, especially releasing drugs triggered by intracellular stimuli, such as pH, temperature, redox, and enzyme etc., have been considered as one kind of most attractive carriers [1]. As soon as reaching the targeted tumor, such carriers can be rapidly provoked by the stimuli to release the drug, leading to aggressive activity within tumor cells and maximal therapeutic efficacy with reduced side effects [2–3]. The reducible carriers involving disulfide bond have received considerable attention for drug and gene delivery. However, carriers responsive to both oxidants and reductants under mild physiological conditions are scarcely investigated. In this study, a pH and dual redox responsive nanogel was prepared by diselenide bond crosslinking. Diselenide bond can be cleaved and reduced to selenol in the presence of reductants and oxidized to seleninic acid in oxidative conditions [4]. Preliminary in vitro experiments showed that the nanogel was biocompatible and drug release could be induced by GSH, indicating the nanogel as a highly promising carrier for intracellular drug delivery.
The diameters of the nanogel and its precursor were 162 nm and 75.4 nm, respectively, determined by DLS. An initial in vitro DOX release study showed that minimal drug release (b20%) was observed within 48 h from DOX-loaded mPEG-bP(LG-co-Se2ELG) nanogel in the absence of GSH. However, with only 0.5 mmol L− 1 GSH, a weaker reductive environment comparable to the intracellular compartments, the nanogel released DOX rapidly and quantitatively with 57% DOX release in 48 h (Fig. 1). It should be further noted that the initially burst release was observed in 7 h and then DOX was released from the nanogel in a zero order manner. These release results indicate that DOX release was controlled most likely by the combination of diffusion and degradation of diselenide bond. The in vitro cytotoxicity of copolymer and blank nanogel to HeLa cells was evaluated using the MTT assay. As shown in Fig. 2, the viabilities of HeLa cells were around 90–100% at all test concentrations up to 0.5 g L− 1, showing the low toxicity and good compatibility to cells of the nanogel.
Experimental methods Polymer synthesis. Firstly, mPEG-b-P(BLG-co-CELG) was synthesized by the ring-opening polymerization (ROP) of γ-benzyl-lglutamate-N-carboxyanhydride (BLG-NCA) and γ-2-chloroethyl-lglutamate-N-carboxyanhydride (CELG-NCA) with amino group terminated poly(ethylene glycol) monomethyl ether (mPEG, Mn = 5000) as the macroinitiator. The polymerization was performed in DMF at 25 °C for 3 d, yielding mPEG-b-P(BLG-co-CELG) copolymer with prescribed compositions. Subsequently, mPEG-b-P(BLG-coCELG) was deprotected with HBr/acetic acid (33 wt.%) in dichloroacetic acid at 25 °C for 1 h, which led to mPEG-b-P(LG-co-CELG). Nanogel preparation. mPEG-b-P(LG-co-Se2ELG) nanogel was prepared by the crosslinking of mPEG-b-P(LG-co-CELG) micelle with Na2Se2 at 25 °C for 12 h in aqueous solution. In vitro DOX release and MTT assay. The in vitro DOX release profiles from mPEG-b-P(LG-co-Se2ELG) nanogel were studied using dialysis tubes (MWCO 12000) at 37 °C in PBS (pH 7.4) with and without 0.5 mmol L− 1 GSH. The relative cytotoxicity of mPEG-b-P(LG-coCELG) copolymer and blank nanogel was assessed with the MTT viability assay using HeLa cells.
Fig. 1. In vitro release of DOX from DOX-loaded nanogel in PBS (pH 7.4) at 37 °C with or without GSH treatment.
Results and discussion mPEG-b-P(LG-co-CELG) was synthesized through ROP of CELGNCA and BLG-NCA, and subsequent deprotection with HBr/acetic acid (33 wt.%). 1H NMR, FT-IR and GPC analyses confirmed that the copolymer had controlled compositions and relatively low polydispersity index (PDI = Mw/Mn, 1.50). The degree of polymerization (DP) of LG and DP of CELG were 77 and 36, respectively, calculated from 1H NMR spectrum. mPEG-b-P(LG-co-Se2ELG) nanogel was prepared from the crosslinking of micelle with Na2Se2 (Scheme 1). The formation of the nanogel was confirmed by 1H NMR, ion chromatography, and TEM. Fig. 2. Cell viability of HeLa cells after incubation with mPEG-b-P(LG-co-CELG) copolymer and blank nanogel.
Scheme 1. Schematic illustration of pH-dependent micellization of mPEG-b-P(LG-coCELG) copolymer and the formation of a nanogel.
Conclusion mPEG-b-P(LG-co-Se2ELG) nanogel with GSH-responsive properties possessed low toxicity in vitro. The nanogel was stable and only little DOX release was observed under non-reductive environment, while the DOX released rapidly in the presence of reductive GSH. The in-depth studies of this triple responsive nanogel, including the oxidative, pH-sensitive properties, and the further application as intracellular drug delivery carrier are in progress.
Abstracts / Journal of Controlled Release 152 (2011) e1–e132
Acknowledgements We are grateful for the financial support from National Natural Science Foundation of China (Key Project 50733003, Project 20904053 and 50973108, A3 Foresight Program 50425309) and the Knowledge Innovation Program of the Chinese Academy of Sciences, Grant No. KJCX2-YW-H19. References [1] J.Z. Du, T.M. Sun, W.J. Song, J. Wu, J. Wang, A tumor-acidity-activated chargeconversional nanogel as an intelligent vehicle for promoted tumoral-cell uptake and drug delivery, Angew. Chem. Int. Ed. 49 (2010) 3621–3626. [2] L.Y. Tang, Y.C. Wang, Y. Li, J.Z. Du, J. Wang, Shell-detachable micelles based on disulfide-linked block copolymer as potential carrier for intracellular drug delivery, Bioconjug. Chem. 20 (2009) 1095–1099. [3] Y.L. Li, L. Zhu, Z.Z. Liu, R. Cheng, F.H. Meng, J.H. Cui, S.J. Ji, Z.Y. Zhong, Reversibly stabilized multifunctional dextran nanoparticles efficiently deliver doxorubicin into the nuclei of cancer cells, Angew. Chem. Int. Ed. 48 (2009) 9914–9918. [4] N. Ma, Y. Li, H.P. Xu, Z.Q. Wang, X. Zhang, Dual redox responsive assemblies formed from diselenide block copolymers, J. Am. Chem. Soc. 132 (2010) 442–443.
doi:10.1016/j.jconrel.2011.08.091
Stimuli-responsive polypeptide-based reverse micellar hydrogel Chang-Ming Dong, Yi Chen Department of Polymer Science & Engineering, School of Chemistry & Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China E-mail address:
[email protected] (C.-M. Dong). Abstract summary A new concept—the reverse micellar hydrogel—is introduced, and a versatile strategy is provided for fabricating supramolecular polypeptide-based normal and reverse micellar hydrogels from the same polypeptide-based copolymer via the cooperation of host–guest chemistry and hydrogen-bonding interactions [1]. These hydrogels can respond to dual stimuli—temperature and pH—and their mechanical and controlled drug-release properties can be tuned by the copolymer topology and the polypeptide composition.
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Experimental methods DOX-loaded hydrogel. The general protocol for the DOX-loaded hydrogel formation is exemplified by the following procedure. For the DOX-loaded normal micellar hydrogel, 2 mg of DOX was added to a stirred solution containing of 50 mg of the PLG10-b-PEO copolymer in 0.5 mL of distilled water, and then 60 mg of α-CD was added under vigorous stirring followed by sonication for 5 min. For the DOXloaded reverse micellar hydrogel, 2 mg of DOX was added to a stirred solution containing of 50 mg of the PLG35-b-PEO copolymer in 0.5 mL of distilled water at pH 8, and then 60 mg of α-CD was added under vigorous stirring followed by sonication for 5 min; finally acid (HCl) was added up to pH 5. The mixed solution was then incubated at 25 ° C, allowing the mixture to form the hydrogel. Results and discussion As for PLG-b-PEO with a long PLG chain, a novel class of supramolecular reverse micellar hydrogel formed between PLG35-bPEO and α-CD in aqueous solution at room temperature (Scheme 1). The PLG35-b-PEO copolymer could not form the normal micellar hydrogel with α-CD in neutral solution because of its lower solubility. Fortunately, the suspension (e.g., at 40 mg/mL) turned transparent at pH 8 after the addition of less amount of NaOH. This is because the PLG segment within the PLG35-b-PEO copolymer formed an anionic polyelectrolyte at pH 8, and the copolymer basically existed as a unimer. After α-CD was added into the alkaline PLG35-b-PEO solution, the mixed solution turned turbid and formed micelles, as confirmed by fluorescence spectroscopy and DLS. The self-assembly of α-CD/ PLG35-b-PEO complex was triggered by the supramolecular inclusion complexation between α-CD and PEO, and the complex micelles had a crystalline α-CD/PEO polypseudorotaxane core and an anionic PLG corona in alkaline solution. Compared with the normal micelle (i.e., the micelle with a PEO corona and a PLG core), we denote this as the reverse micelle, a term coined by Armes et al. Then, a smaller amount of HCl was added into the suspension at pH 5; the gelation immediately occurred, and the hydrogel formed within 5 min. We term this as a reverse micellar hydrogel, because the gelation was induced through the formation of the reverse micelles with a polypeptide corona via the supramolecular inclusion complexation
Keywords: Host–guest chemistry, Polypeptide, Reverse micellar hydrogel, Self-assembly Introduction Synthetic polypeptide or protein hybrid copolymers recently received much attention for the fabrication of stimuli-responsive micelles and hydrogels for drug/gene delivery and tissue engineering [2]. Particularly, intensive efforts are being made on the hierarchical self-assembly of synthetic polymer–polypeptide block copolymers with varying hydrophobic blocks and the polypeptide blocks. However, the aqueous self-assembly of dendritic or branched polypeptide hybrid copolymers is rarely studied, which might provide a platform for developing stimulus-responsive drug delivery vesicles. Recently, cyclodextrins-based host–guest chemistry proved to be a powerful tool for self-assembling nanoparticles and hydrogels [3]. Herein we discover a new class of reverse micellar hydrogel, and propose a versatile strategy for fabricating supramolecular polypeptide-based normal and reverse micellar hydrogel from the same polypeptide-based copolymer, via the cooperation of the host–guest chemistry and the hydrogen-bonding interactions (Scheme 1) [1]. The supramolecular hydrogels and their formation mechanism were thoroughly characterized by means of UV–vis and fluorescent spectroscopy, wide-angle X-ray diffraction, dynamic light scattering, transmission electron microscopy, and rheological measurements.
Scheme 1. The illustration of the proposed structures and gelation mechanism of supramolecular hydrogels: a) normal micellar hydrogel, b) reverse micellar hydrogel. Conditions: 1) the micellization of copolymer, 2) the normal micellar hydrogel induced by supramolecular inclusion complexation, 3) the reverse micellization of copolymer, 4) the reverse micellar hydrogelation [1].
