- Email: [email protected]
Synthesis and characterization of amphiphilic chitosan derivatives as a nano-carrier for paclitaxel delivery
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Abstracts / Journal of Controlled Release 152 (2011) e1–e132
survival times displayed the following order: composite micelles group N PTX micelles group N Taxol group N control group, in agreement with the in vitro observations [5]. We observed that the tumors in the control group became ulcerated at the end of 20 days, possibly due to aggressive growth.
Scheme 1. Structure of PTX–polymer conjugate (1) and FA–polymer conjugate (2).
Drug release profiles (Fig. 1) from conjugate micelles showed that only about 20% of the drug was released from micelles in 6 h and over 80% of PTX was released in 6 days in PBS of pH 7.4. Fast drug release occurred in CPBS pH 4.0 in 24 h, while nearly no more drug was released in the following days. In vivo studies showed that composite micelles resulted in significantly slower tumor growth than that observed with other treatments. PTX micelles also resulted in slightly better tumor growth inhibition compared to control and Taxol groups [5]. As shown in Fig. 2, animal
90
[1] A. Singla, A. Garg, D. Aggarwal, Paclitaxel and its formulationInt. J. Pharm. 235 (2002) 179–192. [2] Z. Xie, X. Hu, X. Chen, T. Lu, S. Liu, X. Jing, A biodegradable diblcok copolymer poly (ethylene glycol)-block-poly(l-lactide-co-2-methyl-2-carboxyl-propylene carbonate): docetaxel and RGD conjugation, J. Appl. Polym. Sci. 110 (2008) 2961–2970. [3] Z. Xie, H. Guan, L. Chen, H. Tian, X. Chen, X. Jing, Novel biodegradable poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate) copolymers: synthesis, characterization and micellization, Polymer 46 (2005) 10523–10530. [4] X. Hu, S. Liu, X. Chen, G. Mo, Z. Xie, X. Jing, Biodegradable amphiphilic block copolymers bearing protected hydroxyl groups: synthesis and characterization, Biomacromolecules 9 (2008) 553–560. [5] Y. Wan, Y. Zheng, X. Song, X. Hu, S. Liu, T. Tong, X. Jing, Anti-tumor activity of biodegradable polymer–paclitaxel conjugate micelles on Lewis lung cancer mice models, J. Biomater. Sci. Polym. Ed. (2010)doi:10.1163/092050610X500570.
70
Drug released (%)
Acknowledgements Financial support was provided by the National Natural Science Foundation of China (project nos. 20674084, 50733003), and by the Ministry of Science and Technology of China (“973 Project”, no. 2009CB930102; “863 Project”, no. 2007AA03Z535.) References
pH7.4 pH4.0
80
Conclusion FA- and PTX–conjugate composite micelles of spherical shape and of 40–60 nm diameter are successfully prepared by self-assembling or co-assembling related amphiphilic block copolymers. The hydrophilic PEG segments constitute the corona of the micelles, leading to effective solubilization of the PTX and protection of the micelle system against the immunological system. Therefore, they can be administered smoothly via tail vein injection into Lewis lung tumorbearing mice. At a dose of 20 mg/kg, the FA–PTX composite micelles exhibit significant anti-tumor effects and higher efficacy than other drug groups without FA because of the FA-receptor-mediated tumor targeting.
60 50 40 30 20 10 0
doi:10.1016/j.jconrel.2011.08.165
-20
0
20
40
60
80
100
120
140
160
Time(hr) Fig. 1. In vitro release of paclitaxel, from conjugate micelles in PBS (pH 7.4) or CPBS (pH 4.0) containing 0.5% (w/v) Tween 80 at 37 °C.
Control Taxol PTX micell FA-PTX micell
100
%Survival
80
60
40
20
0 0
5
10
15
20
25
30
35
40
45
Time(Day) Fig. 2. The survival of C57bl/6 mice bearing Lewis lung cancer with different treatments.
Synthesis and characterization of amphiphilic chitosan derivatives as a nano-carrier for paclitaxel delivery Huofei Zhou1,2, Xiudong Liu3, Xin Guo1, Nan Li1,2, Weiting Yu1, Ying Zhang1, Xiaojun Ma1 1 Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China 2 Graduate School of the Chinese Academy of Sciences, Beijing 100039, China 3 College of Environment and Chemical Engineering, Dalian University, Dalian Economic Technological Development Zone, Dalian 116622, China E-mail addresses: [email protected] (X. Liu), [email protected] (X. Ma). Abstract summary A series of amphiphilic chitosan derivatives has been synthesized by grafting deoxycholic acid and hydrophilic molecules containing glycidyl groups for application in drug delivery. These chitosan derivatives formed stable nanoparticles with sizes ranging from 160 to 240 nm and zeta potentials ranging from 0 to 25 mV in PBS. Paclitaxel (PTX) loaded nanoparticles showed a sustained and controlled release behavior. Furthermore, the nanoparticles can be easily delivered into tumor cells. Keywords: Chitosan, Amphiphilic, Nanoparticles, PTX, Controlled release
Abstracts / Journal of Controlled Release 152 (2011) e1–e132
Introduction Chitosan (CS), a copolymer of N-acetylglucosamine and glucosamine derived from chitin, which is extracted from crustaceans' shells and the only naturally cationic polysaccharide in the world, has been utilized as carrier for drugs, DNA, siRNA, protein and peptide and as tissue engineering material [1–4]. In this study, a series of amphiphilic chitosan derivatives has been synthesized by grafting deoxycholic acid and the hydrophilic molecules glycidyltrimethylammounium chloride (GTMAC) or glycidol successively. Paclitaxel, a hydrophobic antitumor drug was encapsulated into the CS derivatives. The drug release and cellular uptake of PTX loaded CS nanoparticles were studied. Results and discussion Water-soluble amphiphilic CS derivatives were synthesized by grafting deoxycholic acid and a hydrophilic molecule (GTMAC or glycidol). HTsGs-CS-DCAs formed stable nanoparticles (critical aggregation concentration: 0.013–0.079 mg/mL) with particle sizes ranging from 160 to 240 nm and zeta potentials ranging from 0 to 25 mV in PBS (Fig. 1). Both the size and zeta potential of HTsGs-CSDCAs nanoparticles were influenced by the DS of deoxycholic acid, GTMAC and glycidol. The drug loading and encapsulation efficiency of PTX, and PTX release behavior from PTX-HTsGs-CS-DCAs nanoparticles were studied using HPLC. The results showed that the drug loading content was up to 19 wt.% and drug encapsulation efficiency was around 80%. The PTX release study was carried out by adding nanoparticles in 10 mL of buffer solution at 37 °C through 12 d. Fig. 2 showed that PTX was released in a sustained manner. The release rate could be adjusted by changing the DS of deoxycholic acid, GTMAC and glycidol and the pH of the release medium (data not shown). Confocal experiments showed that doxorubicin (DOX) loaded HTsGs-CS-DCAs nanoparticles easily delivered DOX into MCF-7 cells (Fig. 3).
e125
Fig. 3. Confocal microscopic images of MCF-7 cells incubated with DOX loaded HT31G36-CS-DCA3.0 nanoparticles for 5 h.
Conclusion We have developed a series of new chitosan-based nanoparticles. Hydrophobic drugs can be easily and effectively encapsulated into these nanoparticles. The hydrophobic model drug PTX encapsulated in nanoparticles is released in a sustained manner, and the release rate can be controlled by adjusting the DS of grafting molecules. The imaging study indicates that these nanoparticles can easily deliver drugs into tumor cells. Acknowledgements The authors greatly acknowledge the financial support from theNational Natural Science Foundation of China (no. 20876018), National Key Technology R&D Program in the 11th Five-year Plan of China (2006BAD27B04), Knowledge Innovation Project of the Chinese Academy of Sciences (KJCX2.YW.M02 and KJCX2-YW-21002), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. References [1] X.D. Liu, K.A. Howard, M.D. Dong, M.O. Andersen, U.L. Rahbek, M.G. Johnsen, O.C. Hansen, F. Besenbacher, J. Kjems, The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencingBiomaterials 28 (2007) 1280–1288. [2] G. Saravanakumar, K.H. Min, D.S. Min, A.Y. Kim, C.-M. Lee, Y.W. Cho, S.C. Lee, K. Kim, S.Y. Jeong, K. Park, J.H. Park, I.C. Kwon, Hydrotropic oligomer-conjugated glycol chitosan as a carrier of paclitaxel: synthesis, characterization, and in vivo biodistribution, J. Control. Release 140 (2009) 210–217. [3] S.R. Mao, W. Sun, T. Kissel, Chitosan-based formulations for delivery of DNA and siRNA, Adv. Drug Deliv. Rev. 62 (2010) 12–27. [4] L.C. Yin, J.Y. Ding, C.B. He, L.M. Cui, C. Tang, C.H. Yin, Drug permeability and mucoadhesion properties of thiolated trimethyl chitosan nanoparticles in oral insulin delivery, Biomaterials 30 (2009) 5691–5700.
doi:10.1016/j.jconrel.2011.08.166
Thermosensitive pluronic F127-b-poly(ε-caprolactone) mixed micelles
Accumulative release of PTX (%)
Fig. 1. TEM image and size distribution of HT31G36-CS-DCA3.0 nanoaggregates in PBS (0.02 M, pH 7.4) at a concentration of 2 mg/mL.
80 PTX-HT31G36-CS-DCA3.0
70 60 50 40 30 20 10 0 0
50
100
150
200
250
300
Time (hour) Fig. 2. In vitro release of PTX from PTX-HT31G36-CS-DCA3.0 nanoparticles in pH 7.4 PBS containing 0.1% Tween 80 (w/v) at 37 °C (mean ± SD, n = 3).
Qi Zhou, Zhao Zhang, Tao Chen, Shaobing Zhou Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China E-mail addresses: [email protected] (Q. Zhou), [email protected] (S. Zhou). Abstract summary Pluronic F127-b-poly(ε-caprolactone) (F127-CLx) copolymers with three different chain lengths of poly(ε-caprolactone) were synthesized through ring-opening polymerization. The resulting copolymers were characterized by 1H NMR, FT-IR, DSC, XRD, POM and GPC. Blank and mixed micelles with hydrophilic protein and hydrophobic low molecular mass polylactide were prepared by the solvent evaporation method, and the physicochemical properties of the micelles were further investigated by atomic force microscopy (AFM) and transmission electron microscopy (TEM) and dynamic light scattering measurements (DLS). Doxorubicin⋅HCl (DOX⋅HCl) was selected as the model anticancer drug. In vitro release and the cytotoxicity of DOX⋅HCl loaded blank and mixed micelles were investigated, which indicates that the polymeric micelles are attractive vehicles for DOX·HCl delivery
Abstracts / Journal of Controlled Release 152 (2011) e1–e132
survival times displayed the following order: composite micelles group N PTX micelles group N Taxol group N control group, in agreement with the in vitro observations [5]. We observed that the tumors in the control group became ulcerated at the end of 20 days, possibly due to aggressive growth.
Scheme 1. Structure of PTX–polymer conjugate (1) and FA–polymer conjugate (2).
Drug release profiles (Fig. 1) from conjugate micelles showed that only about 20% of the drug was released from micelles in 6 h and over 80% of PTX was released in 6 days in PBS of pH 7.4. Fast drug release occurred in CPBS pH 4.0 in 24 h, while nearly no more drug was released in the following days. In vivo studies showed that composite micelles resulted in significantly slower tumor growth than that observed with other treatments. PTX micelles also resulted in slightly better tumor growth inhibition compared to control and Taxol groups [5]. As shown in Fig. 2, animal
90
[1] A. Singla, A. Garg, D. Aggarwal, Paclitaxel and its formulationInt. J. Pharm. 235 (2002) 179–192. [2] Z. Xie, X. Hu, X. Chen, T. Lu, S. Liu, X. Jing, A biodegradable diblcok copolymer poly (ethylene glycol)-block-poly(l-lactide-co-2-methyl-2-carboxyl-propylene carbonate): docetaxel and RGD conjugation, J. Appl. Polym. Sci. 110 (2008) 2961–2970. [3] Z. Xie, H. Guan, L. Chen, H. Tian, X. Chen, X. Jing, Novel biodegradable poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate) copolymers: synthesis, characterization and micellization, Polymer 46 (2005) 10523–10530. [4] X. Hu, S. Liu, X. Chen, G. Mo, Z. Xie, X. Jing, Biodegradable amphiphilic block copolymers bearing protected hydroxyl groups: synthesis and characterization, Biomacromolecules 9 (2008) 553–560. [5] Y. Wan, Y. Zheng, X. Song, X. Hu, S. Liu, T. Tong, X. Jing, Anti-tumor activity of biodegradable polymer–paclitaxel conjugate micelles on Lewis lung cancer mice models, J. Biomater. Sci. Polym. Ed. (2010)doi:10.1163/092050610X500570.
70
Drug released (%)
Acknowledgements Financial support was provided by the National Natural Science Foundation of China (project nos. 20674084, 50733003), and by the Ministry of Science and Technology of China (“973 Project”, no. 2009CB930102; “863 Project”, no. 2007AA03Z535.) References
pH7.4 pH4.0
80
Conclusion FA- and PTX–conjugate composite micelles of spherical shape and of 40–60 nm diameter are successfully prepared by self-assembling or co-assembling related amphiphilic block copolymers. The hydrophilic PEG segments constitute the corona of the micelles, leading to effective solubilization of the PTX and protection of the micelle system against the immunological system. Therefore, they can be administered smoothly via tail vein injection into Lewis lung tumorbearing mice. At a dose of 20 mg/kg, the FA–PTX composite micelles exhibit significant anti-tumor effects and higher efficacy than other drug groups without FA because of the FA-receptor-mediated tumor targeting.
60 50 40 30 20 10 0
doi:10.1016/j.jconrel.2011.08.165
-20
0
20
40
60
80
100
120
140
160
Time(hr) Fig. 1. In vitro release of paclitaxel, from conjugate micelles in PBS (pH 7.4) or CPBS (pH 4.0) containing 0.5% (w/v) Tween 80 at 37 °C.
Control Taxol PTX micell FA-PTX micell
100
%Survival
80
60
40
20
0 0
5
10
15
20
25
30
35
40
45
Time(Day) Fig. 2. The survival of C57bl/6 mice bearing Lewis lung cancer with different treatments.
Synthesis and characterization of amphiphilic chitosan derivatives as a nano-carrier for paclitaxel delivery Huofei Zhou1,2, Xiudong Liu3, Xin Guo1, Nan Li1,2, Weiting Yu1, Ying Zhang1, Xiaojun Ma1 1 Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China 2 Graduate School of the Chinese Academy of Sciences, Beijing 100039, China 3 College of Environment and Chemical Engineering, Dalian University, Dalian Economic Technological Development Zone, Dalian 116622, China E-mail addresses: [email protected] (X. Liu), [email protected] (X. Ma). Abstract summary A series of amphiphilic chitosan derivatives has been synthesized by grafting deoxycholic acid and hydrophilic molecules containing glycidyl groups for application in drug delivery. These chitosan derivatives formed stable nanoparticles with sizes ranging from 160 to 240 nm and zeta potentials ranging from 0 to 25 mV in PBS. Paclitaxel (PTX) loaded nanoparticles showed a sustained and controlled release behavior. Furthermore, the nanoparticles can be easily delivered into tumor cells. Keywords: Chitosan, Amphiphilic, Nanoparticles, PTX, Controlled release
Abstracts / Journal of Controlled Release 152 (2011) e1–e132
Introduction Chitosan (CS), a copolymer of N-acetylglucosamine and glucosamine derived from chitin, which is extracted from crustaceans' shells and the only naturally cationic polysaccharide in the world, has been utilized as carrier for drugs, DNA, siRNA, protein and peptide and as tissue engineering material [1–4]. In this study, a series of amphiphilic chitosan derivatives has been synthesized by grafting deoxycholic acid and the hydrophilic molecules glycidyltrimethylammounium chloride (GTMAC) or glycidol successively. Paclitaxel, a hydrophobic antitumor drug was encapsulated into the CS derivatives. The drug release and cellular uptake of PTX loaded CS nanoparticles were studied. Results and discussion Water-soluble amphiphilic CS derivatives were synthesized by grafting deoxycholic acid and a hydrophilic molecule (GTMAC or glycidol). HTsGs-CS-DCAs formed stable nanoparticles (critical aggregation concentration: 0.013–0.079 mg/mL) with particle sizes ranging from 160 to 240 nm and zeta potentials ranging from 0 to 25 mV in PBS (Fig. 1). Both the size and zeta potential of HTsGs-CSDCAs nanoparticles were influenced by the DS of deoxycholic acid, GTMAC and glycidol. The drug loading and encapsulation efficiency of PTX, and PTX release behavior from PTX-HTsGs-CS-DCAs nanoparticles were studied using HPLC. The results showed that the drug loading content was up to 19 wt.% and drug encapsulation efficiency was around 80%. The PTX release study was carried out by adding nanoparticles in 10 mL of buffer solution at 37 °C through 12 d. Fig. 2 showed that PTX was released in a sustained manner. The release rate could be adjusted by changing the DS of deoxycholic acid, GTMAC and glycidol and the pH of the release medium (data not shown). Confocal experiments showed that doxorubicin (DOX) loaded HTsGs-CS-DCAs nanoparticles easily delivered DOX into MCF-7 cells (Fig. 3).
e125
Fig. 3. Confocal microscopic images of MCF-7 cells incubated with DOX loaded HT31G36-CS-DCA3.0 nanoparticles for 5 h.
Conclusion We have developed a series of new chitosan-based nanoparticles. Hydrophobic drugs can be easily and effectively encapsulated into these nanoparticles. The hydrophobic model drug PTX encapsulated in nanoparticles is released in a sustained manner, and the release rate can be controlled by adjusting the DS of grafting molecules. The imaging study indicates that these nanoparticles can easily deliver drugs into tumor cells. Acknowledgements The authors greatly acknowledge the financial support from theNational Natural Science Foundation of China (no. 20876018), National Key Technology R&D Program in the 11th Five-year Plan of China (2006BAD27B04), Knowledge Innovation Project of the Chinese Academy of Sciences (KJCX2.YW.M02 and KJCX2-YW-21002), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. References [1] X.D. Liu, K.A. Howard, M.D. Dong, M.O. Andersen, U.L. Rahbek, M.G. Johnsen, O.C. Hansen, F. Besenbacher, J. Kjems, The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencingBiomaterials 28 (2007) 1280–1288. [2] G. Saravanakumar, K.H. Min, D.S. Min, A.Y. Kim, C.-M. Lee, Y.W. Cho, S.C. Lee, K. Kim, S.Y. Jeong, K. Park, J.H. Park, I.C. Kwon, Hydrotropic oligomer-conjugated glycol chitosan as a carrier of paclitaxel: synthesis, characterization, and in vivo biodistribution, J. Control. Release 140 (2009) 210–217. [3] S.R. Mao, W. Sun, T. Kissel, Chitosan-based formulations for delivery of DNA and siRNA, Adv. Drug Deliv. Rev. 62 (2010) 12–27. [4] L.C. Yin, J.Y. Ding, C.B. He, L.M. Cui, C. Tang, C.H. Yin, Drug permeability and mucoadhesion properties of thiolated trimethyl chitosan nanoparticles in oral insulin delivery, Biomaterials 30 (2009) 5691–5700.
doi:10.1016/j.jconrel.2011.08.166
Thermosensitive pluronic F127-b-poly(ε-caprolactone) mixed micelles
Accumulative release of PTX (%)
Fig. 1. TEM image and size distribution of HT31G36-CS-DCA3.0 nanoaggregates in PBS (0.02 M, pH 7.4) at a concentration of 2 mg/mL.
80 PTX-HT31G36-CS-DCA3.0
70 60 50 40 30 20 10 0 0
50
100
150
200
250
300
Time (hour) Fig. 2. In vitro release of PTX from PTX-HT31G36-CS-DCA3.0 nanoparticles in pH 7.4 PBS containing 0.1% Tween 80 (w/v) at 37 °C (mean ± SD, n = 3).
Qi Zhou, Zhao Zhang, Tao Chen, Shaobing Zhou Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China E-mail addresses: [email protected] (Q. Zhou), [email protected] (S. Zhou). Abstract summary Pluronic F127-b-poly(ε-caprolactone) (F127-CLx) copolymers with three different chain lengths of poly(ε-caprolactone) were synthesized through ring-opening polymerization. The resulting copolymers were characterized by 1H NMR, FT-IR, DSC, XRD, POM and GPC. Blank and mixed micelles with hydrophilic protein and hydrophobic low molecular mass polylactide were prepared by the solvent evaporation method, and the physicochemical properties of the micelles were further investigated by atomic force microscopy (AFM) and transmission electron microscopy (TEM) and dynamic light scattering measurements (DLS). Doxorubicin⋅HCl (DOX⋅HCl) was selected as the model anticancer drug. In vitro release and the cytotoxicity of DOX⋅HCl loaded blank and mixed micelles were investigated, which indicates that the polymeric micelles are attractive vehicles for DOX·HCl delivery