- Email: [email protected]
Oligoarginine-modified chitosan for siRNA delivery
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
e166
Abstracts / Journal of Controlled Release 152 (2011) e133–e191
between 9R-chitosan and siRNA as well as on the degree of substitution of 9R to chitosan. Conclusion Chitosan-based nanoparticles were prepared using a coacervation method in the presence of 9R-modified chitosan and siRNA, which had a mean diameter of approximately 300 nm and surface charge of 13.9 ± 0.9 mV. This approach to developing delivery systems with biocompatible and low toxic natural polymers may find useful applications in gene delivery for therapeutic purposes. Acknowledgment This research was supported by National Research Foundation of Korea Grant funded by the Korean Government (2010-0012608) and also by Korea Ministry of Knowledge Economy under the KORUS Tech Program (KT-2008-NT-APFS0-0001). References [1] P. Shankar, N. Manjunath, J. Lieberman, The prospect of silencing disease using RNA interferenceJAMA 293 (2005) 1367–1373. [2] H. Katas, H.O. Alpar, Development and characterisation of chitosan nanoparticles for siRNA delivery, J. Control. Release 115 (2006) 216–225. [3] D.W. Lee, K.S. Yoon, H.S. Ban, W.C. Choe, S.K. Lee, K.Y. Lee, Preparation and characterization of chitosan/polyguluronate nanoparticles for siRNA delivery, J. Control. Release 139 (2009) 146–152.
Experimental methods UV–Vis spectrophotometry (UV Perkin-Elmer Lambda Bio40) was employed to monitor the buildup of the film by exploiting the characteristic absorbance of plasmid DNA at 260 nm. The thickness of micelles/DNA films with 10 bilayers on quartz substrates was determined using profilometry (Taylor-Hobson S4C-3D). A quartz substrate coated with micelles/DNA film was placed in a plastic tube. Glutathione solution (2.5 mM) in phosphate buffer (PBS, 0.1 M, pH 7.4) was added to cover the substrate and incubated at 37 °C. The residual plasmid DNA on the film was determined by UV absorbance at 260 nm after predetermined intervals. Transfection of 293 T cells with micelles/pGL-3 multilayered films was performed as reported previously [2]. Results and discussion The amphiphilic polycation, as shown in Fig. 1, was synthesized via quaternization of N-Boc disulfide-containing poly(amidoamine)s reacting with 1-bromohexadecane, followed by removal of the N-Boc protective groups. The cationic micelles/DNA films were fabricated from micelles, formed from amphiphilic disulfide-containing poly (amidoamine)s, and DNA by alternately dipping the substrates into the solutions of micelles and pGL-3. Micelle 1, micelle 2 and micelle 3 (shortened M 1, M 2, and M 3) formed from polymer 1, polymer 2 and polymer 3, respectively.
doi:10.1016/j.jconrel.2011.08.065
Bioreducible cationic micelle/DNA multilayered films for localized gene delivery Na Peng, Ya-Nan Xue, Xi-Ming Xia, Shi-Wen Huang, Ren-Xi Zhuo Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, China E-mail addresses: [email protected], [email protected] (S.2W Huang). Summary Multilayered films composed of bioreducible cationic micelles, formed from amphiphilic disulfide-containing poly(amidoamine)s, and DNA were prepared via layer-by-layer assembly for localized gene delivery. The thickness of cationic micelles/DNA films with 10 bilayers increased with increasing the grafting degree of alkyl groups in poly(amidoamine)s. Meanwhile, films with 10 bilayers prepared from bioreducible polycations with 28% of alkyl side chains, showed the fastest release of DNA in the presence of 2.5 mM glutathione and highest transfection efficiency when 293T cells were cultured on the surface of films.
Fig. 1. Structure of the amphiphilic copolymer.
Fig. 2A shows the representative plot of absorbance versus bilayer numbers (micelles/DNA films and PEI/DNA film). For micelles/DNA films, the amount of DNA deposited on quartz substrates increased with increasing the substitution degree of alkyl groups in the polycation. Fig. 2B provides the thicknesses of films with 10 bilayers, which is consistent with the results in Fig. 2A. The disulfide bonds in the polycation are stable during preparation and storage, however, cleavable via thiol-disulfide exchange. Cleavage of the films under reductive conditions was carried out by incubating the films in PBS buffer containing 2.5 mM glutathione. The
Keywords: Multilayered films, Bioreducible, Cationic micelles, Disulfide, Transfection Introduction Layer-by-layer (LBL) multilayered films composed of biodegradable polycations and plasmid DNA have been widely applied for gene delivery due to its systematical control over the thickness and the chemical composition of the films [1]. Often used approaches for the release of incorporated DNA from LBL films included hydrolysis [2], bio-cleavage [3,4], enzymatic degradation, and pH alternation. In all above cases, water soluble polycations were used for the construction of DNA-containing films. We here report a novel strategy to fabricate cationic micelles/DNA multilayered films from amphiphilic disulfide-containing poly(amidoamine)s for localized gene delivery.
Fig. 2. (A) Plot of absorbance at 260 nm as a function of bilayer numbers. (B) Thicknesses of (bPEI/DNA)10 films and (micelles/DNA)10 films deposited on quartz substrates.
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
e166
Abstracts / Journal of Controlled Release 152 (2011) e133–e191
between 9R-chitosan and siRNA as well as on the degree of substitution of 9R to chitosan. Conclusion Chitosan-based nanoparticles were prepared using a coacervation method in the presence of 9R-modified chitosan and siRNA, which had a mean diameter of approximately 300 nm and surface charge of 13.9 ± 0.9 mV. This approach to developing delivery systems with biocompatible and low toxic natural polymers may find useful applications in gene delivery for therapeutic purposes. Acknowledgment This research was supported by National Research Foundation of Korea Grant funded by the Korean Government (2010-0012608) and also by Korea Ministry of Knowledge Economy under the KORUS Tech Program (KT-2008-NT-APFS0-0001). References [1] P. Shankar, N. Manjunath, J. Lieberman, The prospect of silencing disease using RNA interferenceJAMA 293 (2005) 1367–1373. [2] H. Katas, H.O. Alpar, Development and characterisation of chitosan nanoparticles for siRNA delivery, J. Control. Release 115 (2006) 216–225. [3] D.W. Lee, K.S. Yoon, H.S. Ban, W.C. Choe, S.K. Lee, K.Y. Lee, Preparation and characterization of chitosan/polyguluronate nanoparticles for siRNA delivery, J. Control. Release 139 (2009) 146–152.
Experimental methods UV–Vis spectrophotometry (UV Perkin-Elmer Lambda Bio40) was employed to monitor the buildup of the film by exploiting the characteristic absorbance of plasmid DNA at 260 nm. The thickness of micelles/DNA films with 10 bilayers on quartz substrates was determined using profilometry (Taylor-Hobson S4C-3D). A quartz substrate coated with micelles/DNA film was placed in a plastic tube. Glutathione solution (2.5 mM) in phosphate buffer (PBS, 0.1 M, pH 7.4) was added to cover the substrate and incubated at 37 °C. The residual plasmid DNA on the film was determined by UV absorbance at 260 nm after predetermined intervals. Transfection of 293 T cells with micelles/pGL-3 multilayered films was performed as reported previously [2]. Results and discussion The amphiphilic polycation, as shown in Fig. 1, was synthesized via quaternization of N-Boc disulfide-containing poly(amidoamine)s reacting with 1-bromohexadecane, followed by removal of the N-Boc protective groups. The cationic micelles/DNA films were fabricated from micelles, formed from amphiphilic disulfide-containing poly (amidoamine)s, and DNA by alternately dipping the substrates into the solutions of micelles and pGL-3. Micelle 1, micelle 2 and micelle 3 (shortened M 1, M 2, and M 3) formed from polymer 1, polymer 2 and polymer 3, respectively.
doi:10.1016/j.jconrel.2011.08.065
Bioreducible cationic micelle/DNA multilayered films for localized gene delivery Na Peng, Ya-Nan Xue, Xi-Ming Xia, Shi-Wen Huang, Ren-Xi Zhuo Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, China E-mail addresses: [email protected], [email protected] (S.2W Huang). Summary Multilayered films composed of bioreducible cationic micelles, formed from amphiphilic disulfide-containing poly(amidoamine)s, and DNA were prepared via layer-by-layer assembly for localized gene delivery. The thickness of cationic micelles/DNA films with 10 bilayers increased with increasing the grafting degree of alkyl groups in poly(amidoamine)s. Meanwhile, films with 10 bilayers prepared from bioreducible polycations with 28% of alkyl side chains, showed the fastest release of DNA in the presence of 2.5 mM glutathione and highest transfection efficiency when 293T cells were cultured on the surface of films.
Fig. 1. Structure of the amphiphilic copolymer.
Fig. 2A shows the representative plot of absorbance versus bilayer numbers (micelles/DNA films and PEI/DNA film). For micelles/DNA films, the amount of DNA deposited on quartz substrates increased with increasing the substitution degree of alkyl groups in the polycation. Fig. 2B provides the thicknesses of films with 10 bilayers, which is consistent with the results in Fig. 2A. The disulfide bonds in the polycation are stable during preparation and storage, however, cleavable via thiol-disulfide exchange. Cleavage of the films under reductive conditions was carried out by incubating the films in PBS buffer containing 2.5 mM glutathione. The
Keywords: Multilayered films, Bioreducible, Cationic micelles, Disulfide, Transfection Introduction Layer-by-layer (LBL) multilayered films composed of biodegradable polycations and plasmid DNA have been widely applied for gene delivery due to its systematical control over the thickness and the chemical composition of the films [1]. Often used approaches for the release of incorporated DNA from LBL films included hydrolysis [2], bio-cleavage [3,4], enzymatic degradation, and pH alternation. In all above cases, water soluble polycations were used for the construction of DNA-containing films. We here report a novel strategy to fabricate cationic micelles/DNA multilayered films from amphiphilic disulfide-containing poly(amidoamine)s for localized gene delivery.
Fig. 2. (A) Plot of absorbance at 260 nm as a function of bilayer numbers. (B) Thicknesses of (bPEI/DNA)10 films and (micelles/DNA)10 films deposited on quartz substrates.