- Email: [email protected]
concanavalin A for insulin delivery
Abstracts / Journal of Controlled Release 152 (2011) e133–e191
grafting, a new broad band at 3100–3500 cm–1 centered at ~3300 cm–1 characteristic of O-H stretching was clearly observed. This is strong evidence that there is a high density of carboxylic acid groups on the surface of CPLGA films. Furthermore, in order to obtain quantitative results of carboxyl density of PLGA films, a colorimetric staining assay with toluidine blue O was carried out. The density of carboxylic acid groups on the surface of CPLGA films was 0.892 ±0.11 nmol/cm2. The immobilization of PEI/DNA complexes on the surface of CPLGA films was evaluated by ATR-FTIR (Fig. 3). In ATR-FTIR spectra of activated CPLGA films conjugated with PEI/DNA complexes, two absorption bands around 1646 and 1540 cm–1 were observed, which can be assigned to amide I and amide II, respectively. SEM images showed the conjugation of PEI/DNA complexes on the surface of CPLGA films (Fig. 4). It can be seen that PEI/DNA complexes were irregularly aggregated on the CPLGA film in the control groups by the physical adsorption method, while they are uniformly deposited on the chemically activated CPLGA film preserving their spherical morphology and size. It was previously reported that PEI/DNA polyplexes nonspecifically adsorbed on polymer surfaces were highly aggregated, resulting in reduced gene transfection efficiency. Surface deposited PEI/DNA complexes which are were less aggregated and better distributed would have more chance to be transported into adhered cells through substrate-mediated gene transfection than aggregated and ill distributed complexes. Conclusion In this study, we have developed a novel strategy to covalently immobilize gene vectors onto functionalized PLGA films via -CONHcross-links. The cationic PEI/DNA complexes were successfully immobilized onto the functionalized PLGA films and characterized by ATR-FTIR and SEM analysis. This method has the potential to be an effective tool in surface-mediated gene delivery. These gene-activated PLGA film surfaces are being evaluated for their efficacy in localized gene delivery both in cell culture and in vivo. Our group will further investigate the hypothesis that this technique could deliver gene
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vectors in a site-specific manner and provide relatively high levels of local gene expression with minimal distal spread. Acknowledgments The authors are grateful to the Natural Science Foundation of China (Nos. 50903093, 50830106). References [1] L. Mei, X. Jin, C.X. Song, M.Y. Wang, R.J. Levy, Immobilization of gene vectors on polyurethane surfaces using a monoclonal antibody for localized gene deliveryJ. Gene Med. 8 (2006) 690–698. [2] X. Jin, L. Mei, C.X. Song, L.X. Liu, X.G. Leng, H.F. Sun, D.L. Kong, R.J. Levy, Immobilization of plasmid DNA on an anti-DNA antibody modified coronary stent for intravascular site-specific gene therapy, J. Gene Med. 10 (2008) 421–429.
doi:10.1016/j.jconrel.2011.08.063
Glucose-responsive microhydrogels based on methacrylate modified dextran/concanavalin A for insulin delivery Ruixue Yin1, Zi Tong1, Dongzhi Yang1, Jun Nie1,2 1 State Key Laboratory of Chemical Resource Engineering, Key Lab of Beijing City on Preparation and Processing of Novel Polymer Materials, Beijing University of Chemical Technology, Beijing 100029, China 2 College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072, China E-mail address: [email protected] (J. Nie). Summary Glucose-responsive microhydrogels based on methacrylate derivatives of dextran and concanavalin A have been prepared and used for self-regulated insulin delivery. Insulin release from these microhydrogels was in response to different glucose concentrations in the medium and the glucose sensitivity was reversible. The released insulin was proved to remain active without destroying the tertiary structure. The degree of substitution (DS) of the dextran methacrylate derivative had effect on the glucose sensitivity of the microhydrogels. Keywords: Glucose-responsive, Microhydrogel, Concanavalin A, Insulin delivery
Fig. 3. ATR-FTIR spectra of (A): CPLGA films and (B): CPLGA films with immobilized PEI/DNA complexes.
Fig. 4. SEM micrographs of PEI/DNA complexes immobilized onto the surface of CPLGA films. (A) physical adsorption method; and (B) chemical immobilization method.
Introduction Self-regulated insulin delivery systems are very significant for the treatment of insulin-dependent diabetes mellitus to control the blood glucose level, in which insulin can be released in response to glucose concentrations in the blood [1]. Glucose-responsive hydrogels or microhydrogels are very useful for the development of self-regulated insulin delivery systems. Four types of glucose-sensitive systems have been intensively investigated, which are on the basis of glucose oxidase, concanavalin A (Con A), phenylboronic acid and glucose binding protein [2–5]. The specific saccharide-binding properties of Con A make it capable of causing affinity gelation of polysaccharide or glucose moieties containing polymer. However, this system is vulnerable to component loss, especially Con A loss, which could lead to weak glucose sensitivity and undesirable biocompatibility. Therefore, it is necessary to develop an efficiently crosslinked network and covalently immobilize Con A to the polymer matrix. In this study, methacrylate derivatives of dextran (Dex-G) and concanavalin A (Con A-E), were synthesized, then the microhydrogels using Dex-G, Con A-E and the crosslinker poly (ethylene glycol) dimethacrylate (PEGDMA) were prepared. Insulin release in response to different glucose concentrations and the influence of the DS of Dex-G on the glucose sensitivity were investigated.
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Abstracts / Journal of Controlled Release 152 (2011) e133–e191
Experimental methods Preparation of microhydrogels. In our previous study, Dex-G was synthesized through ring-opening reaction of glycidyl methacrylate (GMA) and dextran, while Con A-E was obtained by a Michael addition reaction under mild condition using ethylene glycol acrylate methacrylate (EGAMA) and Con A. The insulin-loaded microhydrogels were prepared through reversed-phase emulsion crosslinking initiated by (NH4)2S2O8 and Na2SO3 in cyclohexane stabilized by Span 80 for 24 h, before which insulin was dissolved in the mixed solution of Dex-G, Con A-E and PEGDMA. The resulting mixture was left standing for a while and then centrifuged to remove the upper oil. The obtained microhydrogels were washed with distilled water and freeze dried before use. In vitro release of insulin. Insulin release was analyzed by incubating insulin-loaded microhydrogels at 37 °C (±0.5 °C) in PBS (pH 7.4) while shaking (100 r/min) as a function of time and a stepwise change in glucose concentration (0, 4, 10 mg/mL). Insulin release in response to step changes in glucose concentrations (0 and 10 mg/mL) in several alternated cycles was also tested. The released insulin concentration and activity were measured by a fluorescence spectrophotometer at an emission wavelength of 304 nm. Influence of the DS of Dex- on Insulin release. Insulin release profiles in the medium of 0 and 10 mg/mL glucose solutions with different DS of Dex-G (15, 21, 32) were depicted and compared. Results and discussion The Dex-G precursor was prepared through a ring-opening reaction, while Con A-E was obtained from Michael addition reaction at room temperature. FT-IR spectra confirmed the structures of the prepolymers by the appearance of peaks of methacrylate carbonyl and vinyl groups (1725 and 810 cm–1). The DS of Dex-G was measured by 1H NMR. The morphology of the obtained microhydrogels was observed by optical microscopy, scanning electron microscopy and fluorescence microscopy. All of the images demonstrated that the microhydrogels existed as individual particles with a sphere like shape (average size about 5 μm) and a dense structure displaying no gaps on the surface. Insulin release from microhydrogels (DS of Dex-G was 15) in response to a stepwise change in glucose concentration (0, 4, 10 mg/ mL) is revealed in Fig. 1. With the increase of glucose concentration, the cumulative released amount of insulin increased and had a dramatic change at each moment of replacing the glucose concentration. Besides, compared to the release profile in glucose 0 mg/mL alone, the cumulative released amount of insulin in response to the stepwise change in glucose level was much higher, owing to the disconnection between the sugar-binding site of Con A-E and Dex-G, indicating that the microhydrogels could effectively respond to
Fig. 1. Insulin release in response to change of stepwise glucose concentration (upper) and in the medium without glucose (lower).
Fig. 2. Insulin release in response to an abrupt change in glucose concentration in three alternated cycles.
different glucose levels. Fig. 2 shows insulin release in response to step changes in glucose concentrations (0 and 10 mg/mL) in three alternated cycles. The microhydrogels responded quickly to changes in glucose concentration. Except for the first 20 min, resulting from the burst release of the surface, the amount of released insulin increased with increasing glucose concentration from 0 to 10 mg/mL and decreased when glucose was reduced to 0 mg/mL, owing to the reversible affinity of Con A-E and Dex-G, which indicated the reproducibility of the microhydrogels. The released insulin and standard insulin formulations displayed similar peaks with an emission maximum at 304 nm, indicating that the released insulin stayed active with undestroyed tertiary structure. Fig. 3 displays the comparison of insulin release profiles in the medium of glucose 0 and 10 mg/mL with different DS of Dex-G (15, 21, 32). Increasing of the DS of Dex-G allowed more permanent covalent cross-links, making it more difficult for the polymer chains to move along each other, which could prevent the loss of active components, but inversely reduced the specific binding sites of Dex-G to Con A-E, thus decreased the glucose sensitivity of the microhydrogels. When the DS of Dex-G was 32 (Fig. 3C), there was no obvious difference between insulin release profiles in the medium of glucose 0 and 10 mg/mL, because the specific affinity of Con A-E for Dex-G became weak and the predominant force for insulin release was swelling of the microhydrogels by water absorption.
Fig. 3. Insulin release in the medium of glucose 0 and 10 mg/mL with different DS of Dex-G: (A) 15, (B) 21 and (C) 32.
Abstracts / Journal of Controlled Release 152 (2011) e133–e191
Conclusion Glucose-responsive microhydrogels based on methacrylate derivates of Dex-G and Con A-E have been prepared. The release profiles of insulin revealed that the insulin release could respond quickly to different glucose concentrations in the medium and the glucose sensitivity was reversible. The released insulin was proved to remain active. The glucose sensitivity of microhydrogels became less significant with increase of the DS of Dex-G and extremely weak when the DS reached to 32. All the results suggested that the microhydrogels might be a promising system for self-regulated insulin delivery. References [1] G.M. Steil, A.E. Panteleon, K. Rebrin, Closed-loop insulin delivery-the path to physiological glucose controlAdv. Drug Deliv. Rev. 56 (2004) 125–144. [2] W. Qi, X. Yan, L. Duan, Y. Cui, Y. Yang, J. Li, Glucose-sensitive microcapsules from glutaraldehyde cross-linked hemoglobin and glucose oxidase, Biomacromolecules 10 (2009) 1212–1216. [3] A.F. Che, Z.M. Liu, X.J. Huang, Z.G. Wang, Z.K. Xu, Chitosan-modified poly(acrylonitrileco-acrylic acid) nanofibrous membranes for the immobilization of concanavalin a, Biomacromolecules 9 (2008) 3397–3403. [4] X. Jin, X. Zhang, Z. Wu, D. Teng, X. Zhang, Y. Wang, Z. Wang, C. Li, Amphiphilic random glycopolymer based on phenylboronic acid: synthesis, characterization, and potential as glucose-sensitive matrix, Biomacromolecules 10 (2009) 1337–1345. [5] D.E. Jason, R.L. Matthew, K. Santoshkumar, W. Yinan, K.D. Sapna, G.B. Leonidas, D. Sylvia, Glucose responsive hydrogel networks based on protein recognition, Macromol. Biosci. 9 (2009) 864–868.
doi:10.1016/j.jconrel.2011.08.064
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hypothesized that an introduction of oligoarginine to chitosan could improve the ability of complex formation with negatively charged siRNA. A peptide with nine repeating units of arginine (9R) was chemically coupled to the chitosan backbone and various characteristics of 9R-chitosan/siRNA nanoparticles were investigated.
Experimental methods Preparation of 9R-chitosan/siRNA nanoparticles. Chitosan glutamate was dissolved in MES buffer solution and 9R peptide was added to the chitosan solution in the presence of water soluble carbodiimide. The 9R-modified chitosan was purified by extensive dialysis against deionized water for 4 days (molecular weight cutoff = 3,500), activated charcoal treatment, and sterilized through a 0.22 μm filter. Nanoparticles were prepared by adding a chitosan solution to an equal volume of polyguluronate (PG) solution containing siRNA [3], followed by incubation at room temperature for 30 min before use or further analysis. Characterization of nanoparticles. Chemical conjugation between 9R and chitosan was confirmed by FT-IR. The mean diameter and surface charge of nanoparticles were determined at 25 °C by Nano ZS Zetasizer. The binding of siRNA with 9R-chitosan was confirmed by electrophoresis using 3% agarose gel. Samples were loaded into the gel, and electrophoresis was carried out at 100 V for 30 min running with a TBE buffer. Ethidium bromide was used to visualize siRNA bands using a UV transilluminator at 365 nm.
Oligoarginine-modified chitosan for siRNA delivery Soyeon Park, Sang Kyung Lee, Kuen Yong Lee Department of Bioengineering, Hanyang University, Seoul 133-791, Republic of Korea E-mail address: [email protected] (S. Park). Summary Small interfering RNA (siRNA) has been widely investigated as a potential therapeutic for treatment of various diseases. However, naked siRNA is rapidly degraded by nucleases, showing poor cellular uptake and low transfection efficiency. Chitosan-based nanoparticles have been extensively exploited as a gene delivery carrier due to low toxicity and positively charged amino groups of chitosan. In this study, we synthesized 9R-modified chitosan and used it to form stable nanoparticles in the presence of siRNA. Various physicochemical properties of the nanoparticles, including size, surface charge, and complex forming ability, were investigated.
Results and discussion We first confirmed conjugation of 9R to chitosan using FT-IR. The absorption band at 1640 cm–1, assigned to primary amines of chitosan, significantly diminished for 9R-modified chitosan. In addition, a peak for the amide bond at 1550 cm–1 was clearly observed for 9R-modified chitosan, indicating successful linkage between oligoarginine and chitosan. The complex formation of 9R-chitosan with siRNA was next confirmed by gel electrophoresis. 9R-chitosan/siRNA nanoparticles were prepared at a weight ratio of 20 (Fig. 1). Movement of siRNA was substantially retarded compared to control siRNA. The mean diameter and zeta potential of nanoparticles increased when siRNA was complexed with 9R-chitosan, compared to non-modified chitosan (Table 1). These values were varied depending on the weight ratio
1
2
3
4
Keywords: Chitosan, Arginine, siRNA, Gene delivery Introduction RNA interference (RNAi) has raised much attention to date as it allows developing a new class of therapeutics to treat various diseases, including infectious diseases [1]. In the process of RNAi, double stranded small interfering RNA (siRNA) consisting of 21–23 nucleotides degrade target mRNA with the help of RNA-induced silencing complex (RISC) and finally inhibits the synthesis of protein encoded by the mRNA. However, the delivery of siRNA has raised several issues, including rapid enzymatic degradation, low intracellular uptake, and limited blood stability. To overcome these limitations, siRNA has been combined with cationic polymers for enhancing intracellular uptake and increasing stability against nuclease. Chitosan is a naturally existing cationic polysaccharide obtained from crustacean shells. Chitosan is known to be biocompatible and biodegradable. Chitosan has been widely used in many drug delivery applications, especially in gene delivery systems [2]. In this study, we
Fig. 1. Gel retardation assay of chitosan/siRNA nanoparticles (lane 1, DNA markers; lane 2, siRNA only; lane 3, chitosan/siRNA; and lane 4, 9R-chitosan/siRNA).
Table 1 Characteristics of chitosan/siRNA nanoparticles.
Non-modified chitosan R-chitosan
Size (nm)
Zeta potential (mV)
213 309
7.5 ± 1.1 13.9 ± 0.9
grafting, a new broad band at 3100–3500 cm–1 centered at ~3300 cm–1 characteristic of O-H stretching was clearly observed. This is strong evidence that there is a high density of carboxylic acid groups on the surface of CPLGA films. Furthermore, in order to obtain quantitative results of carboxyl density of PLGA films, a colorimetric staining assay with toluidine blue O was carried out. The density of carboxylic acid groups on the surface of CPLGA films was 0.892 ±0.11 nmol/cm2. The immobilization of PEI/DNA complexes on the surface of CPLGA films was evaluated by ATR-FTIR (Fig. 3). In ATR-FTIR spectra of activated CPLGA films conjugated with PEI/DNA complexes, two absorption bands around 1646 and 1540 cm–1 were observed, which can be assigned to amide I and amide II, respectively. SEM images showed the conjugation of PEI/DNA complexes on the surface of CPLGA films (Fig. 4). It can be seen that PEI/DNA complexes were irregularly aggregated on the CPLGA film in the control groups by the physical adsorption method, while they are uniformly deposited on the chemically activated CPLGA film preserving their spherical morphology and size. It was previously reported that PEI/DNA polyplexes nonspecifically adsorbed on polymer surfaces were highly aggregated, resulting in reduced gene transfection efficiency. Surface deposited PEI/DNA complexes which are were less aggregated and better distributed would have more chance to be transported into adhered cells through substrate-mediated gene transfection than aggregated and ill distributed complexes. Conclusion In this study, we have developed a novel strategy to covalently immobilize gene vectors onto functionalized PLGA films via -CONHcross-links. The cationic PEI/DNA complexes were successfully immobilized onto the functionalized PLGA films and characterized by ATR-FTIR and SEM analysis. This method has the potential to be an effective tool in surface-mediated gene delivery. These gene-activated PLGA film surfaces are being evaluated for their efficacy in localized gene delivery both in cell culture and in vivo. Our group will further investigate the hypothesis that this technique could deliver gene
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vectors in a site-specific manner and provide relatively high levels of local gene expression with minimal distal spread. Acknowledgments The authors are grateful to the Natural Science Foundation of China (Nos. 50903093, 50830106). References [1] L. Mei, X. Jin, C.X. Song, M.Y. Wang, R.J. Levy, Immobilization of gene vectors on polyurethane surfaces using a monoclonal antibody for localized gene deliveryJ. Gene Med. 8 (2006) 690–698. [2] X. Jin, L. Mei, C.X. Song, L.X. Liu, X.G. Leng, H.F. Sun, D.L. Kong, R.J. Levy, Immobilization of plasmid DNA on an anti-DNA antibody modified coronary stent for intravascular site-specific gene therapy, J. Gene Med. 10 (2008) 421–429.
doi:10.1016/j.jconrel.2011.08.063
Glucose-responsive microhydrogels based on methacrylate modified dextran/concanavalin A for insulin delivery Ruixue Yin1, Zi Tong1, Dongzhi Yang1, Jun Nie1,2 1 State Key Laboratory of Chemical Resource Engineering, Key Lab of Beijing City on Preparation and Processing of Novel Polymer Materials, Beijing University of Chemical Technology, Beijing 100029, China 2 College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072, China E-mail address: [email protected] (J. Nie). Summary Glucose-responsive microhydrogels based on methacrylate derivatives of dextran and concanavalin A have been prepared and used for self-regulated insulin delivery. Insulin release from these microhydrogels was in response to different glucose concentrations in the medium and the glucose sensitivity was reversible. The released insulin was proved to remain active without destroying the tertiary structure. The degree of substitution (DS) of the dextran methacrylate derivative had effect on the glucose sensitivity of the microhydrogels. Keywords: Glucose-responsive, Microhydrogel, Concanavalin A, Insulin delivery
Fig. 3. ATR-FTIR spectra of (A): CPLGA films and (B): CPLGA films with immobilized PEI/DNA complexes.
Fig. 4. SEM micrographs of PEI/DNA complexes immobilized onto the surface of CPLGA films. (A) physical adsorption method; and (B) chemical immobilization method.
Introduction Self-regulated insulin delivery systems are very significant for the treatment of insulin-dependent diabetes mellitus to control the blood glucose level, in which insulin can be released in response to glucose concentrations in the blood [1]. Glucose-responsive hydrogels or microhydrogels are very useful for the development of self-regulated insulin delivery systems. Four types of glucose-sensitive systems have been intensively investigated, which are on the basis of glucose oxidase, concanavalin A (Con A), phenylboronic acid and glucose binding protein [2–5]. The specific saccharide-binding properties of Con A make it capable of causing affinity gelation of polysaccharide or glucose moieties containing polymer. However, this system is vulnerable to component loss, especially Con A loss, which could lead to weak glucose sensitivity and undesirable biocompatibility. Therefore, it is necessary to develop an efficiently crosslinked network and covalently immobilize Con A to the polymer matrix. In this study, methacrylate derivatives of dextran (Dex-G) and concanavalin A (Con A-E), were synthesized, then the microhydrogels using Dex-G, Con A-E and the crosslinker poly (ethylene glycol) dimethacrylate (PEGDMA) were prepared. Insulin release in response to different glucose concentrations and the influence of the DS of Dex-G on the glucose sensitivity were investigated.
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Abstracts / Journal of Controlled Release 152 (2011) e133–e191
Experimental methods Preparation of microhydrogels. In our previous study, Dex-G was synthesized through ring-opening reaction of glycidyl methacrylate (GMA) and dextran, while Con A-E was obtained by a Michael addition reaction under mild condition using ethylene glycol acrylate methacrylate (EGAMA) and Con A. The insulin-loaded microhydrogels were prepared through reversed-phase emulsion crosslinking initiated by (NH4)2S2O8 and Na2SO3 in cyclohexane stabilized by Span 80 for 24 h, before which insulin was dissolved in the mixed solution of Dex-G, Con A-E and PEGDMA. The resulting mixture was left standing for a while and then centrifuged to remove the upper oil. The obtained microhydrogels were washed with distilled water and freeze dried before use. In vitro release of insulin. Insulin release was analyzed by incubating insulin-loaded microhydrogels at 37 °C (±0.5 °C) in PBS (pH 7.4) while shaking (100 r/min) as a function of time and a stepwise change in glucose concentration (0, 4, 10 mg/mL). Insulin release in response to step changes in glucose concentrations (0 and 10 mg/mL) in several alternated cycles was also tested. The released insulin concentration and activity were measured by a fluorescence spectrophotometer at an emission wavelength of 304 nm. Influence of the DS of Dex- on Insulin release. Insulin release profiles in the medium of 0 and 10 mg/mL glucose solutions with different DS of Dex-G (15, 21, 32) were depicted and compared. Results and discussion The Dex-G precursor was prepared through a ring-opening reaction, while Con A-E was obtained from Michael addition reaction at room temperature. FT-IR spectra confirmed the structures of the prepolymers by the appearance of peaks of methacrylate carbonyl and vinyl groups (1725 and 810 cm–1). The DS of Dex-G was measured by 1H NMR. The morphology of the obtained microhydrogels was observed by optical microscopy, scanning electron microscopy and fluorescence microscopy. All of the images demonstrated that the microhydrogels existed as individual particles with a sphere like shape (average size about 5 μm) and a dense structure displaying no gaps on the surface. Insulin release from microhydrogels (DS of Dex-G was 15) in response to a stepwise change in glucose concentration (0, 4, 10 mg/ mL) is revealed in Fig. 1. With the increase of glucose concentration, the cumulative released amount of insulin increased and had a dramatic change at each moment of replacing the glucose concentration. Besides, compared to the release profile in glucose 0 mg/mL alone, the cumulative released amount of insulin in response to the stepwise change in glucose level was much higher, owing to the disconnection between the sugar-binding site of Con A-E and Dex-G, indicating that the microhydrogels could effectively respond to
Fig. 1. Insulin release in response to change of stepwise glucose concentration (upper) and in the medium without glucose (lower).
Fig. 2. Insulin release in response to an abrupt change in glucose concentration in three alternated cycles.
different glucose levels. Fig. 2 shows insulin release in response to step changes in glucose concentrations (0 and 10 mg/mL) in three alternated cycles. The microhydrogels responded quickly to changes in glucose concentration. Except for the first 20 min, resulting from the burst release of the surface, the amount of released insulin increased with increasing glucose concentration from 0 to 10 mg/mL and decreased when glucose was reduced to 0 mg/mL, owing to the reversible affinity of Con A-E and Dex-G, which indicated the reproducibility of the microhydrogels. The released insulin and standard insulin formulations displayed similar peaks with an emission maximum at 304 nm, indicating that the released insulin stayed active with undestroyed tertiary structure. Fig. 3 displays the comparison of insulin release profiles in the medium of glucose 0 and 10 mg/mL with different DS of Dex-G (15, 21, 32). Increasing of the DS of Dex-G allowed more permanent covalent cross-links, making it more difficult for the polymer chains to move along each other, which could prevent the loss of active components, but inversely reduced the specific binding sites of Dex-G to Con A-E, thus decreased the glucose sensitivity of the microhydrogels. When the DS of Dex-G was 32 (Fig. 3C), there was no obvious difference between insulin release profiles in the medium of glucose 0 and 10 mg/mL, because the specific affinity of Con A-E for Dex-G became weak and the predominant force for insulin release was swelling of the microhydrogels by water absorption.
Fig. 3. Insulin release in the medium of glucose 0 and 10 mg/mL with different DS of Dex-G: (A) 15, (B) 21 and (C) 32.
Abstracts / Journal of Controlled Release 152 (2011) e133–e191
Conclusion Glucose-responsive microhydrogels based on methacrylate derivates of Dex-G and Con A-E have been prepared. The release profiles of insulin revealed that the insulin release could respond quickly to different glucose concentrations in the medium and the glucose sensitivity was reversible. The released insulin was proved to remain active. The glucose sensitivity of microhydrogels became less significant with increase of the DS of Dex-G and extremely weak when the DS reached to 32. All the results suggested that the microhydrogels might be a promising system for self-regulated insulin delivery. References [1] G.M. Steil, A.E. Panteleon, K. Rebrin, Closed-loop insulin delivery-the path to physiological glucose controlAdv. Drug Deliv. Rev. 56 (2004) 125–144. [2] W. Qi, X. Yan, L. Duan, Y. Cui, Y. Yang, J. Li, Glucose-sensitive microcapsules from glutaraldehyde cross-linked hemoglobin and glucose oxidase, Biomacromolecules 10 (2009) 1212–1216. [3] A.F. Che, Z.M. Liu, X.J. Huang, Z.G. Wang, Z.K. Xu, Chitosan-modified poly(acrylonitrileco-acrylic acid) nanofibrous membranes for the immobilization of concanavalin a, Biomacromolecules 9 (2008) 3397–3403. [4] X. Jin, X. Zhang, Z. Wu, D. Teng, X. Zhang, Y. Wang, Z. Wang, C. Li, Amphiphilic random glycopolymer based on phenylboronic acid: synthesis, characterization, and potential as glucose-sensitive matrix, Biomacromolecules 10 (2009) 1337–1345. [5] D.E. Jason, R.L. Matthew, K. Santoshkumar, W. Yinan, K.D. Sapna, G.B. Leonidas, D. Sylvia, Glucose responsive hydrogel networks based on protein recognition, Macromol. Biosci. 9 (2009) 864–868.
doi:10.1016/j.jconrel.2011.08.064
e165
hypothesized that an introduction of oligoarginine to chitosan could improve the ability of complex formation with negatively charged siRNA. A peptide with nine repeating units of arginine (9R) was chemically coupled to the chitosan backbone and various characteristics of 9R-chitosan/siRNA nanoparticles were investigated.
Experimental methods Preparation of 9R-chitosan/siRNA nanoparticles. Chitosan glutamate was dissolved in MES buffer solution and 9R peptide was added to the chitosan solution in the presence of water soluble carbodiimide. The 9R-modified chitosan was purified by extensive dialysis against deionized water for 4 days (molecular weight cutoff = 3,500), activated charcoal treatment, and sterilized through a 0.22 μm filter. Nanoparticles were prepared by adding a chitosan solution to an equal volume of polyguluronate (PG) solution containing siRNA [3], followed by incubation at room temperature for 30 min before use or further analysis. Characterization of nanoparticles. Chemical conjugation between 9R and chitosan was confirmed by FT-IR. The mean diameter and surface charge of nanoparticles were determined at 25 °C by Nano ZS Zetasizer. The binding of siRNA with 9R-chitosan was confirmed by electrophoresis using 3% agarose gel. Samples were loaded into the gel, and electrophoresis was carried out at 100 V for 30 min running with a TBE buffer. Ethidium bromide was used to visualize siRNA bands using a UV transilluminator at 365 nm.
Oligoarginine-modified chitosan for siRNA delivery Soyeon Park, Sang Kyung Lee, Kuen Yong Lee Department of Bioengineering, Hanyang University, Seoul 133-791, Republic of Korea E-mail address: [email protected] (S. Park). Summary Small interfering RNA (siRNA) has been widely investigated as a potential therapeutic for treatment of various diseases. However, naked siRNA is rapidly degraded by nucleases, showing poor cellular uptake and low transfection efficiency. Chitosan-based nanoparticles have been extensively exploited as a gene delivery carrier due to low toxicity and positively charged amino groups of chitosan. In this study, we synthesized 9R-modified chitosan and used it to form stable nanoparticles in the presence of siRNA. Various physicochemical properties of the nanoparticles, including size, surface charge, and complex forming ability, were investigated.
Results and discussion We first confirmed conjugation of 9R to chitosan using FT-IR. The absorption band at 1640 cm–1, assigned to primary amines of chitosan, significantly diminished for 9R-modified chitosan. In addition, a peak for the amide bond at 1550 cm–1 was clearly observed for 9R-modified chitosan, indicating successful linkage between oligoarginine and chitosan. The complex formation of 9R-chitosan with siRNA was next confirmed by gel electrophoresis. 9R-chitosan/siRNA nanoparticles were prepared at a weight ratio of 20 (Fig. 1). Movement of siRNA was substantially retarded compared to control siRNA. The mean diameter and zeta potential of nanoparticles increased when siRNA was complexed with 9R-chitosan, compared to non-modified chitosan (Table 1). These values were varied depending on the weight ratio
1
2
3
4
Keywords: Chitosan, Arginine, siRNA, Gene delivery Introduction RNA interference (RNAi) has raised much attention to date as it allows developing a new class of therapeutics to treat various diseases, including infectious diseases [1]. In the process of RNAi, double stranded small interfering RNA (siRNA) consisting of 21–23 nucleotides degrade target mRNA with the help of RNA-induced silencing complex (RISC) and finally inhibits the synthesis of protein encoded by the mRNA. However, the delivery of siRNA has raised several issues, including rapid enzymatic degradation, low intracellular uptake, and limited blood stability. To overcome these limitations, siRNA has been combined with cationic polymers for enhancing intracellular uptake and increasing stability against nuclease. Chitosan is a naturally existing cationic polysaccharide obtained from crustacean shells. Chitosan is known to be biocompatible and biodegradable. Chitosan has been widely used in many drug delivery applications, especially in gene delivery systems [2]. In this study, we
Fig. 1. Gel retardation assay of chitosan/siRNA nanoparticles (lane 1, DNA markers; lane 2, siRNA only; lane 3, chitosan/siRNA; and lane 4, 9R-chitosan/siRNA).
Table 1 Characteristics of chitosan/siRNA nanoparticles.
Non-modified chitosan R-chitosan
Size (nm)
Zeta potential (mV)
213 309
7.5 ± 1.1 13.9 ± 0.9