Synthesis and Characteristics of Biodegradable and Temperature Responsive Polymeric Micelles Based On Poly (Aspartic Acid) - G-Poly (N-Isopropylacrylamide-co-N, N-Dimethylacrylamide)
Synthesis and Characteristics of Biodegradable and Temperature Responsive Polymeric Micelles Based on Poly(Aspartic Acid)-G-poly(N-Isopropylacrylamide-co-N,N-dimethylacrylamide)
Synthesis and Characteristics of Biodegradable and Temperature Responsive Polymeric Micelles Based On Poly (Aspartic Acid) - G-Poly (N-Isopropylacrylamide-co-N, N-Dimethylacrylamide)
Title: Synthesis and characteristics of biodegradable and
temperature responsive polymeric micelles based on poly(aspartic acid)-g-poly(N-isopropylacrylamide-co-N,N- dimethylacrylamide) Authors: Jih-Chao Yeh, Huei-Hung Yang, Ya-Ting Hsu, Chao-Ming Su, Tsong-Hai Lee, Shyh-Liang Lou PII: S0927-7757(12)00874-6 DOI: doi:10.1016/j.colsurfa.2012.12.014 Reference: COLSUA 18077 To appear in: Colloids and Surfaces A: Physicochem. Eng. Aspects Received date: 13-5-2012 Revised date: 11-12-2012 Accepted date: 12-12-2012 Please cite this article as: Jih-Chao Yeh, Huei-Hung Yang, Ya- Ting Hsu, Chao-Ming Su, Tsong-Hai Lee, Shyh-Liang Lou, Synthesisandcharacteristicsofbiodegradableandtemperatureresponsivepolymericmicellesbasedonpoly(asparticacid)- g-poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide), Colloids and Surfaces A: Physicochemical and Engineering Aspects doi:10.1016/j.colsurfa.2012.12.014 This is a PDF le of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and reviewof the resulting proof before it is published in its nal form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Page 1 of 31 A c c e p t e d
M a n u s c r i p t *Graphical Abstract (for review) Page 2 of 31 A c c e p t e d
M a n u s c r i p t We synthesized PASp-g-PND possessing temperature response and biodegradability The micelles phase transition temperature is about 41.9 o C The micelles degraded within three days is about 25% The micelles contains free amino groups to be able to conjugate with bio-molecules The micelles has potential to be used as a drug carrier for targeting treatments
*Highlights (for review) Page 3 of 31 A c c e p t e d
M a n u s c r i p t Synthesis and characteristics of biodegradable and temperature responsive polymeric micelles based on poly(aspartic acid)-g- poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) Jih-Chao Yeh a,b , Huei-Hung Yang a , Ya-Ting Hsu a , Chao-Ming Su a , Tsong-Hai Lee b,* , Shyh- Liang Lou a,**
a Department of Biomedical Engineering, Chung-Yuan Christian University, Chung Li 32023, Taiwan, ROC b Department of Neurology and Stroke center, Chang Gung Memorial Hospital, Linkou Medical Center and College of Medicine, Chang Gung University, Taoyuan, Taiwan, ROC.
Both Tsong-Hai Lee and Shyh-Liang Lou act as co-corresponding authors to this manuscript.
Acknowledgment and reprint request to Shyh-Liang Lou Tel.: +886 3 2654517fax: +886 3 2654599. E-mail addresses: lou@cycu.edu.tw 200, Chung Pei Road, Chung Li 32023, Taiwan, R.O.C
*Manuscript Page 4 of 31 A c c e p t e d
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Abstract Temperature responsive polymeric micelles can release drug at controlled rate and minimize the side effects, and have been widely used for drug delivery system. In this study, biodegradable and temperature responsive micelles comprised of poly(aspartic acid)-g-poly(N- isopropylacryamide-co-N,N-dimethylacrylamide) (PASp-g-poly(NIPAAm-co-DMAAm)) were successfully synthesized by grafting poly(NIPAAm-co-DMAAm) onto poly(succinimide) and followed by aminolysis with ammonia hydroxide. The micelles had free amine group and exhibited a phase transition temperature above normal body temperature, which was suitable for targeting drug delivery. In addition, PASp-g-poly(NIPAAm-co-DMAAm) was stable in phosphate buffer solution at 37 o C, and 25% of micelles could degrade within 3 days. Based on these results, the present study demonstrated that PASp-g-poly(NIPAAm-co-DMAAm) can be used as a drug carrier for targeting treatment.
Keywords: biodegradable, temperature responsive polymeric micelles, phase transition temperature, drug carrier
1. Introduction Polymeric micelles have become an interesting system for delivery of poorly water-soluble drugs and are increasingly used in different medical applications, e.g., drug carriers for cancer therapy[1], gene[2], protein[3] and imaging agents[4, 5]. These micelles are block or graft copolymers that are made up of hydrophobic core and hydrophilic shell and can self-assemble in aqueous solution by hydrophobic/hydrophilic interaction[6, 7], hydrogen bonding[8], and electrostatic interactions[9]. Hydrophobic inner core can accommodate hydrophobic drugs, and Page 5 of 31 A c c e p t e d
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hydrophilic outer shell makes particles stable in the blood stream to avoid being recognized by reticuloendothelial system (RES) to increase treatment efficiency[10]. The stimuli-responsive micelles can sense the surrounding environment change, such as pH, temperature, enzyme, and ion change, and response as a phase transition to control drug release. In addition, stimuli-responsive micelles enable drugs to accumulate at the desired site either passively by enhanced permeation and retention effect or actively through the conjugation of recognition signal onto the surface of the micelles[11, 12]. Therefore, stimuli-responsive polymeric micelles have been paid much attention in drug delivery system, because these micelles can release drug at controlled rate and minimize the side effects[13]. Poly(N-isopropylacryamide), PNIPAAm, is one of the most commonly investigated temperature responsive amphiphilic polymer, which exhibits lower critical solution temperature (LCST) at about 32 o C in aqueous media. Below the LCST, PNIPAAm is hydrophilic and extended in the solution, which forms hydrogen bonds between the water and the amide side chain. When above the LCST, they become water-insoluble and undergo volume transition to aggregate in the solution. In addition, the LCST of PNIPAAm can be easily modified by introducing hydrophilic or hydrophobic segments. Using temperature responsive micelles for drug delivery at specific sites, the LCST of micelles needs modification to become slightly higher than the body temperature by incorporating hydrophilic co-monomers, such as N,N- (dimethyl amino)ethyl methacrylate[14], methyacrylic acid[15], acrylic acid[16] and polyethylene glycol[17]. The release profile of doxorubicin in temperature responsive micelles made from poly(N-isopropylacryamide-co-N,N-dimethylacrylamide-co-10-undecenoic acid) with various compositions has been investigated. The micelles in phosphate buffer solution exhibited LCST from 33 to 43 o C. Also, the micelles can be stable in the blood stream at 37 o C, Page 6 of 31 A c c e p t e d
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but can be deformed to release the encapsulated drugs above the LCST by breaking the hydrophilic/hydrophobic balance of micelles[18]. Although PNIPAAm based polymeric micelles offer the advantage of temperature responsive behavior and can act as a potential candidate for drug delivery system, they are not biodegradable. Recently, there is methodology to improve the effectiveness of PNIPAAm based copolymers by grafting onto biodegradable polymers. The biodegradable and temperature responsive polymer micelles made from dextran-g-poly(NIPAAm-co-DMAAm) and chitosan-g- poly(NIPAAm-co-DMAAm) have been studied[19-21]. The LCST of micelles can be increased to 38 o C by grafting poly(NIPAAm-co-DMAAm) onto the main chain of dextran and chitosan. Chitosan has reactive amine groups which serve to conjugate with specific antibody for targeting delivery. However, antidextran antibodies in human limit the cell uptake and reduce the therapeutic effectiveness for targeting treatment. Poly(amino acid) and its derivatives have high biocompatibility, biodegradability and diversity in the various side chain groups for conjugating to antibodies, such as poly(aspartic acid)[22], poly(glutamic acid)[23] and polylysine[6], which have been used for drug delivery system. Poly(aspartic acid), PASp, is a typical poly(amino acid) derivative and is a biodegradable, nontoxic, nonantigenic material. It can be synthesized by thermal polycondensation of L-aspartic acid and is followed by aminolysis with amino hydroxide. The synthesis and characteristics of amphiphilic biodegradable poly(aspartic acid-co-lactic acid) has been investigated[24]. The degradation rate of copolymer can be increased with higher aspartic acid content. This study thus consists of three parts: (1) In order to increase the LCST of temperature responsive micelles, DMAAm was used to adjust the LCST of poly(NIPAAm-co-DMAAm). Page 7 of 31 A c c e p t e d
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poly(NIPAAm-co-DMAAm) with three different compositions including PN35D5 (the feed ratio of NIPAAm and DMAAm =35:5), PN35D10 (35:10), and PN35D15 (35:15) which was synthesized by radical copolymerization. (2) The biodegradable and temperature responsive micelles were prepared by grafting the poly(NIPAAm-co-DMAAm) onto the main chain of poly(aspartic acid) via aminolysis of polysuccinimide (PSI) through amino-terminated poly(NIPAAm-co-DMAAm), followed with aminolysis of residual PSI by ammonium hydroxide. (3) The structure analysis, LCST, biodegradable ratio in PBS and cytotoxicity of PASp-g-poly(NIPAAm-co-DMAAm) were also discussed.
2. Materials and methods 2.1 Materials N-Isopropylacrylamide was received from Tokyo chemical industry (TCI, Tokyo, Japan). N,N-dimethylacrylamide and hydrazine (98%) were purchased from Alfa Aesar (MA, USA). Methyl 3-mercaptopropionate (98%), L-aspartic acid (98%), thiazolyl blue tetrazolium bromide (97.5%), dimethyl sulfoxide, and 1,6-diphenyl-1,3,5-hexatriene (98%) were supplied by Sigma (MO, USA). Potassium peroxydisulfate, N,N-dimethyl methanamide (99.9%), and ammonium hydroxide (30%) were obtained from J. T. Baker (NJ, USA). N-methylprollidone (99.9%) was received from Showa Denko (Tokyo, Japan). Dimethyl sulfoxide-d 6 (99.9%) was purchased from Merck (Darmstadt, Germany). Orthophosphoric acid (80%) was supplied by Riedel-de Haen (USA). Potassium bromide was obtained from Scharlan (Barcelona, Spain). Modified eagles medium was received from GIBCO (Life Technology, NY, USA). All chemicals, reagents and solvents were used without further purification.
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2.2 Synthesis of amino-terminated poly(N-isopropylacrylamide-co-N,N-dimethylmethanamide) poly(NIPAAm-co-DMAAm) Briefly, 0.41 g of NIPAAm, 0.096 mL of DMAAm and 0.02764 mL of methyl 3-mercapto propionate were dissolved in 40 mL of deionized water and degassed with nitrogen gas for 30 min. The mixture solution was heated to 50 o C under nitrogen gas. Then, 0.03 g of potassium peroxydisulfate was dissolved in 10 mL of deionized water. The solution was added into the monomer solution and reacted at 50 o C for 2 h with magnetic stirring. Upon completion, the resulted solution was dialyzed against deionized water using a dialysis membrane (MWCO < 1200) for 24 h and was freeze-dried. Subsequently, an appropriate amount of poly(NIPAAm-co- DMAAm)-COOCH 3 was dissolved in 40 mL of methanol solution. Hydrazine monohydrate was dropwised into the solution and refluxed for 5 h with magnetic stirring. Upon completion, the solution was dialyzed against deionized water using MWCO <1200 dialysis membrane and then freeze-dried to obtain amino-terminated poly(NIPAAm-co-DMAAm).
2.3 Synthesis of poly (succinimide) (PSI) 10 g of L-aspartic acid and 1 g of orthophosphoric acid were mixed and heated to 200 o C in an oil bath under nitrogen gas for 24 h. The resulted off-white fine powder was filtered and washed with deionized water for five times to remove orthophosphoric acid. The obtained fine powder was dried at 50 o C for 12 h to get PSI powder.
2.4 Synthesis of PASp-g-poly(NIPAAm-co-DMAAm) Amino-terminated poly(NIPAAm-co-DMAAm) and PSI were separately dissolved in 10 mL of N,N-dimethylmethanamide. Then, amino-terminated poly(NIPAAm-co-DMAAm) solution was added dropwise into the PSI solution and heated to 60 o C for 24 h with magnetic stirring. Page 9 of 31 A c c e p t e d
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After 24 h, the mixture solution was cooled down to room temperature; the ammonium hydroxide solution was added into the reaction solution and reacted at room temperature for 5 h with stirring. The resulting solution was dialyzed against deionized water using a dialysis membrane (MWCO <1200) for 48 h. Finally, the solution was freeze-dried to obtain PASp-g- poly(NIPAAm-co-DMAAm) powder.
2.5 Characterization of polymer Amino-terminated poly(NIPAAm-co-DMAAm), PSI and PASp-g-(NIPAAm-co-DMAAm) were analyzed by Fourier transform infrared spectroscopy (FTIR, IR-4200, Jasco, Japan) in the region of 400-4000cm -1 . All samples were mixed with potassium bromide and pressed into pellets for analysis. The morphological properties of polymer were observed by a transmission electron microscope (TEM, H7500, Hitachi, Japan) at 80 kV. A drop of nanoparticle suspension was placed on a copper grid and dried at room temperature overnight. The composition of P(NIPAAm-co-DMAAm) was analyzed by a 300 MHz spectrometer (NMR, DRX300, Bruker, Germany) and the polymer was dissolved in DMSO-D 6 for analysis. The molecular weights and polydispersity index (PDI) of polymer were characterized using gel permeation chromatography (GPC, 150C, Waters, USA). The flow rate of mobile phase was 1 mL/min and N- methylprollidone was used as an eluent.
2.6 Lower critical solution temperature (LCST) measurement The LCST of polymer was measured for the transmittance of polymer solution at 500 nm using a UV-Vis spectrophotometer (V-530, Jasco, Japan) and fiber optic thermometry (Luxtron, ROC). The solution was first heated to 50 o C and then measured for the absorbance at different Page 10 of 31 A c c e p t e d
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temperature from 25 to 50 o C. The LCST of the polymer was defined as the temperature decreasing a half of absorbance of polymer solution at 50 o C. 2.7 Biodegration evaluation 25 mg of PASp-g-poly(NIPAAm-co-DMAAm) was accurately weighted and dissolved in 10 mL of 0.1 M phosphate buffer saline (pH 7.4). The solution was placed in a dialysis membrane (MWCO <12000-14000) against 500 mL of PBS at 37 o C in an incubator for 3, 7, 14, 21 and 28 days. Then, the solution was dialyzed against deionized water using a dialysis membrane (MWCO <1200) for 24 h. Finally, the solution was freeze-dried and weighted. The biodegradable ratio of the polymer was calculated by equation (1) :
, where W 0 is the weight of the polymer before degration and W a is the weight of the polymer after degration.
2.8 Cytotoxicity testing The biocompatibility of the polymer was evaluated by MTT cell viability assay using L929 fibroblast cells. 410 -6 L929 cells were cultured in a 24-well culture plate with 1 mL of modified eagle medium (MEM, 10% horse serum, 1% penicillin-streptomycin) at 37 o C, under 5% CO 2 . After 24 h, the culture medium was replaced and 5 mg of polymer was added into the fresh medium. The cells were incubated for another 24, 48 and 72 h. Then, medium with polymer was substituted by fresh medium and 100 L of MTT solution was added. After incubation for 3 h at 37 o C, the medium was removed. In order to dissolve the internalized purple formazan crystals, 1 mL of DMSO was added. 100 L of resulting solution was transferred to a 96-well plate, and an ELISA reader (1500-490, Thermo, USA) was used to determine the absorbance at 570 nm. The cell viability was calculated according to equation (2) : (1) Page 11 of 31 A c c e p t e d
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, where OD polymer is the absorbance obtained in the presence of polymer and OD control is the absorbance obtained without polymer.
3. Results and discussion 3.1 Synthesis and characteristics of PASp-g-poly(NIPAAm-co-DMAAm) First, PSI could be prepared by bulk polymerization of aspartic acid using phosphoric acid as a catalyst. Then, amino-terminated poly(NIPAAm-co-DMAAm) could be synthesized by surfactant-free radical polymerization using KPS as an initiator and methyl 3-meracptopropinate as a chain transfer agent. Finally, the imide cyclic structure of PSI could be easily opened by an amiable reaction. Therefore, PASp-g-poly(NIPAAm-co-DMAAm) could be prepared by nucleophilic opening of PSI using amino-terminated poly(NIPAAm-co-DMAAm) and followed by hydrolysis with ammonium hydroxide. The scheme of PASp-g-poly(NIPAAm-co-DMAAm) is shown in Figure 1.
3.1.1 1 H NMR analysis The chemical structures of amino-terminated poly(NIPAAm-co-DMAAm) and PSI were investigated by 1 H NMR spectroscopy. The 1 H NMR of amino-terminated poly(NIPAAm-co- DMAAm) is shown in Figure 2(a). The characteristic peaks at 1.04 and 3.83 ppm were assigned to the isopropyl group of NIPAAm. The peaks at 2.80 to 2.88 ppm were assigned to methyl group on the DMAAm. In addition, the broad peak at 1.23 to 2.08 ppm was assigned to -CH 2 - CH- on the polymer [25, 26]. This indicated successful copolymerization of amino-terminated poly(NIPAAm-co-DMAAm)-HNHN 2 . Based on the integration of the peak area of signal a and signal f, the actual molar ratio of NIPAAm to DMAAm could be calculated. Table 1 shows the (2) Page 12 of 31 A c c e p t e d
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actual molar ratios of NIPAAm: DMAAm were approximately equaled to the feed ratio. The 1 H NMR spectra of PSI is shown in Figure 2(b), the characteristic peaks at 2.7 to 3.21 ppm and 5.26 ppm was assigned to the protons of the methylene and methyne group on the succinimide units of PSI [27]. The feed molar ratio of amino-terminated poly(NIPAAm-co-DMAAm) and PSI was 1:1. The chemical structure of PASp-g-poly(NIPAAm-co-DMAAm) was analyzed by 1 H NMR. As shown in Figure 2 (c), the NMR spectra of PASp-g-poly(NIPAAm-co-DMAAm) is similar to that of poly(NIPAAm-co-DMAAm). Upon comparison with PSI, the disappearance of the peak at 5.26 ppm and appearance of the peak at 4.52 ppm indicated the rings in PSI were totally opened by the amino-terminated poly(NIPAAm-co-DMAAm) and ammonium hydroxide. According to the results of NMR analysis, the poly(NIPAAm-co-DMAAm) was successfully grafted to the polyaspartic acid backbone chain.
3.1.2 FT-IR analysis The FT-IR spectrum of amino-terminated poly(NIPAAm-co-DMAAm) is shown in Figure 3(a). The characteristic absorption bands at 1645 and 1550 cm -1 were assigned to the absorbance of bending frequency of amide N-H bond, and at 3444 and 3311cm -1 were assigned to the absorbance of N-H bond in poly(NIPAAm-co-DMAAm)[19,28,29]. The characteristic absorption bands from the methyl 3-mercaptopropionate at 663 cm -1 was assigned to the absorption bands of C-S bond. These results suggested there was successful polymerization of poly(NIPAAm-co-DMAAm) and the methyl end group could be replaced by hydrazine monohydrate. The FT-IR spectrum of PSI is shown in Figure 3(b). The characteristic absorption bands at 1397, 1718, and 1792 cm -1 were assigned to the absorbance of methylene group, carbonyl group Page 13 of 31 A c c e p t e d
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and cyclic imide of succinimide group of PSI [30,31]. Grafting of amino-terminated poly(NIPAAm-co-DMAAm) onto the backbone of PSI followed by hydrolysis with ammonium hydroxide was also confirmed by FT-IR spectrum of PASp-g-poly(NIPAAm-co-DMAAm) (Figure 3(c)). The spectrum of PASp-g-poly(NIPAAm-co-DMAAm) was similar to that of poly(NIPAAm-co-DMAAm). The characteristic peak at 1792 cm -1 assigned to the succinimide group of PSI was disappeared. In addition, the peak at 1718 cm -1 was significantly smaller than PSI. These results indicated there was full ring-opening of PSI by amino-terminated poly(NIPAAm-co-DMAAm) and hydrolysis with ammonium hydroxide.
3.1.3 Molecular weight High molecular weight and conversion of PSI was prepared by bulk polycondensation of aspartic acid with o-phosphoric acid as a catalyst [27]. The molecular weight of PSI determined by GPC was 30,000 Da. The conversion of aspartic acid was around 90% [32]. The LCST of PNIPAAm was around 32 o C and could be wildly used in medical applications. In addition, DMAAm is more hydrophilic than PNIPAAm and can be used to increase the LCST of the copolymer [17]. In this study, DMAAm is used to adjust the LCST of PNIPAAm with three different compositions. The molecular weight of various composition poly(NIPAAm-co- DMAAm) is summarized in Table 1. The molecular weights of PN35D5, PN35D10, and PN35D15 determined by GPC were 7946, 10582, and 12317 Da, respectively. Using PN35D10 to conjugate with PSI, the molecular weights of PASp-g-poly(NIPAAm-co-DMAAm) was 32,130 Da. The entire polymer has good polydispersity.
3.1.4 Thermal property and miceller morphology Page 14 of 31 A c c e p t e d
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The thermal weight loss curve of poly(NIPAAm-co-DMAAm), pure poly(aspartic acid), and PASp-g-poly(NIPAAm-co-DMAAm) are shown in Figure 4. The onset of thermal degradation of poly(NIPAAm-co-DMAAm) occurred at about 298 C and the pure poly(aspartic acid) had multi-decomposition steps [33]. As shown in figure 4(b), PASp-g-poly(NIPAAm-co- DMAAm) also had multi-decomposition steps. In addition, the mass loss of approximately 43% on heating from 298 to 458 o C implied that PASp-g-poly(NIPAAm-co-DMAAm) consisted of 43% poly(NIPAAm-co-DMAAm) and 48% PASp. Based on the thermal decomposition temperature of PASp-g-poly(NIPAAm-co-DMAAm) obtained from TGA, these micelles can be sterilized by autoclaving. The particle size and morphology of PASp-g-poly(NIPAAm-co-DMAAm) was observed by DLS and TEM microscopy. TEM image of PASp-g-poly(NIPAAm-co-DMAAm) micelles is shown in Figure 5. All PASp-g-poly(NIPAAm-co-DMAAm) micelles showed spherical shape and the average particle size of micelles was approximately 89.1 nm. However, the particle size analyzed by DLS was 195 nm, which was due to the dehydration of the polymer for the measurement of TEM microscopy. Based on the above results of FTIR, NMR, thermal property and morphology, the biodegradable and temperature responsive polymer PASp-g-P(NIPAAm-co-DMAAm), could be successfully synthesized and sterilized by autoclave.
3.2 Lower critical solution temperature (LCST) of polymer The LCST of different composition of P(NIPAAm-co-DMAAm) in phosphate buffered saline (pH 7.4) including PN35D5, PN35D10 and PN35D15 was 37.2, 40.7 and 45.7 o C, respectively (Table 1). The LCST of poly(NIPAAm-co-DMAAm) could be increased with Page 15 of 31 A c c e p t e d
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increasing DMAAm monomer. In addition, the LCST of PN35D10 and PN35D15 was above normal body temperature, which is more suitable to be used for drug releasing. After grafting of PN35D10 onto the PSI, as shown in Figure 6, the LCST of PASp-g-PN35D10 is slightly increased to 41.9 o C. This can be explained as due to the ring-opening of PSI by PN35D10 and ammonium hydroxide resulting in increase of hydrogen bonding between the hydrophilic segment of PASp and water molecular, therefore, increasing the LCST. However, the LCST of PN35D15 without grafting onto the PSI was 45.7 o C, which is much higher than body temperature. Therefore, PASp-g-PN35D10 is more suitable for drug releasing in living bodies. In pursuance of the observations, we focused on PASp-g-PN35D10 for following investigations.
3.3 Biodegradable rate of polymer The biodegradable rate of PASp-g-poly(NIPAAm-co-DMAAm) was studied in Phosphate buffered saline (pH 7.4) at 37 o C. As shown in Figure 7, approximately 25% of micelle weight disappeared in the PBS within 3 days. The copolymer composition of poly(NIPAAm-co- DMAAm)/PSI was 1. After 7 days, about 40% of micelle weight disappeared in the PBS. The structure of degraded micelles determined by FT-IR was poly(NIPAAm-co-DMAAm). These results suggested that the temperature responsive micelles were degraded causing chain scission at the primary amine site in PASp-g-poly(NIPAAm-co-DMAAm). In addition, we used PN35D10 conjugated to low molecular weight PSI (MW=10,000, poly(NIPAAAm-co- DMAAm)/PSI=1) and investigated the biodegradable rate in PBS at 37 o C (data not shown). Approximately 40 % of micelles disappeared in the PBS within 24 h suggesting a reduced molecular weight of PSI could be used to increase the degradable rate. Based on these research results, it is suggested that the kidney cells can filtrate the particles with molecular weight Page 16 of 31 A c c e p t e d
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smaller than 40k Da [34], which means that PASp-g-poly(NIPAAm-co-DMAAm) can be filtrated through blood stream by kidney. 3.4 Cytotoxicity of polymer For the ultimate of PASp-g-poly(NIPAAm-co-DMAAm) micelles as drug carrier for drug delivery, it is important that these micelles and its degradation products retain low toxicity. L929 celles were incubated with poly(NIPAAm-co-DMAAm)(PN35D10), polyaspartic acid (PASp) and PASp-g-poly(NIPAAm-co-DMAAm) in cultured medium containing 5mg of polymer for 24, 48, and 72 h, respectively. Figure 8 shows that the variability was above 95% and there was no significant difference from that in the control experiment. After 72 h, PASp-g-poly(NIPAAm- co-DMAAm) was degraded about 20% and its degradation products could be dissolved in the cultured medium. There is no significant difference between the PASp-g-poly(NIPAAm-co- DMAAm) and control experiment. Thus, it can be concluded that both PASp-g-poly(NIPAAm- co-DMAAm) and its degradation products have very low or no cytotoxicity for using as drug carriers.
4. Conclusions The present study demonstrated that a biodegradable and temperature responsive micelle, polyaspartic acid-g-poly(N-isopropylacryamide-co-N,N-dimethylacrylamide) (PASp-g- poly(NIPAAm-co-DMAAm)), with a LCST at 41.6 o C could be synthesized for the use as drug delivery system. These micelles were stable in the phosphate buffered saline (pH 7.4) at 37 o C, but could be deformed and aggregated above its LCST. In addition, the micelles contained free amine groups, which allowed further conjugation with specific antibodies for targeting treatment and could be degraded after drug delivery. Thus, these micelles may contribute to the selective accumulation and release of drugs in the desired site. Page 17 of 31 A c c e p t e d
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Acknowledgement The study was supported by the National Science Council, Taiwan (Contract No. NSC 98- 2314-B-182-054-MY2, NSC 99-2628-B-182-027-MY3) and Chang Gung Memorial Hospital under the Medical Research Project (Contract No. Animal Molecular Imaging Center CMRPG340203). And we thank Microscopy Core Laboratory of Chang Gung Memorial Hospital at Linkou for the use of transmission electron microscopy. Page 18 of 31 A c c e p t e d
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M a n u s c r i p t Table 1 Page 24 of 31 A c c e p t e d
M a n u s c r i p t Figure 1 Synthetic scheme Page 25 of 31 A c c e p t e d
M a n u s c r i p t Figure 2 NMR Page 26 of 31 A c c e p t e d
M a n u s c r i p t Figure 3 FTIR Page 27 of 31 A c c e p t e d
M a n u s c r i p t Figure 4 TGA Page 28 of 31 A c c e p t e d
M a n u s c r i p t Figure 5 TEM of PASPPND Page 29 of 31 A c c e p t e d
M a n u s c r i p t Figure 6 LCST Page 30 of 31 A c c e p t e d
M a n u s c r i p t Figure 7 percent degrgradation Page 31 of 31 A c c e p t e d
Synthesis and Characterization of Poly (Amino Urea Urethane) - Based Block Copolymer and Its Potential Application As Injectable PH-temperature-sensitive Hydrogel For Protein Carrier
Rapid Synthesis and Characterization of Chitosan G Poly (D, L Lactide) Copolymers With Hydroxyethyl Chitosan As A Macroinitiator Under Microwave Irradiation
Rapid Synthesis and Characterization of Chitosan G Poly (D, L Lactide) Copolymers With Hydroxyethyl Chitosan As A Macroinitiator Under Microwave Irradiation
Preparation and Characterization of Folate-Poly (Ethylene Glycol) - Grafted-Trimethylchitosan For Intracellular Transport of Protein Through Folate Receptor-Mediated Endocytosis