A cationic gene delivery vector, guanidinylated disulfide-containing poly(amido amine)(CARCBA), was synthesized by Michael addition reaction between N,N′-cystaminebisacrylamide(CBA) and guanidine hydrochloride(CAR). ...A cationic gene delivery vector, guanidinylated disulfide-containing poly(amido amine)(CARCBA), was synthesized by Michael addition reaction between N,N′-cystaminebisacrylamide(CBA) and guanidine hydrochloride(CAR). Gel permeation chromatography(GPC) was used to evaluate the molecular weight of synthesized CAR-CBA. Polyethyleneimine(PEI) with molecular weight of 25 kDa was adopted as a reference, and polyethylene glycols(PEG) with different molecular weights were used to establish a standard curve for determining the molecular weight of CAR-CBA. The effects of two critical factors, namely columns and eluents,on the molecular weight measurement of CAR-CBA were investigated to optimize the GPC quantitative method. The results showed that Ultrahydrogel columns(120, 250) and HAc–NaAc(0.5 M, pH 4.5) buffer solution were the optimal column and GPC eluent, respectively.The molecular weight of the synthesized CAR-CBA was analyzed by the optimized GPC method and determined to be 24.66 kDa.展开更多
Gene-based therapeutics has emerged as a promising approach for human cancer therapy. Among a variety of non-viral vectors, polymer vectors are particularly attractive due to their safety and multivalent groups on the...Gene-based therapeutics has emerged as a promising approach for human cancer therapy. Among a variety of non-viral vectors, polymer vectors are particularly attractive due to their safety and multivalent groups on their surface. This study focuses on guanidinylated O-carboxymethyl chitosan(GOCMCS) along with poly-β-amino ester(PBAE) for si RNA delivery. Binding efficiency of PBAE/si RNA/GOCMCS nanoparticles were characterized by gel electrophoresis. The si RNA-loaded nanoparticles were found to be stable in the presence of RNase A, serum and BALF respectively. Fine particle fraction(FPF) which was determined by a two-stage impinger(TSI) was 57.8% ± 2.6%. The particle size and zeta potential of the nanoparticles were 153.8 ± 12.54 nm and + 12.2 ± 4.94 m V. In vitro cell transfection studies were carried out with A549 cells. The cellular uptake was significantly increased. When the cells were incubated with si Survivin-loaded nanoparticles, it could induce 26.83% ± 0.59% apoptosis of A549 cells and the gene silencing level of survivin expression in A549 cells were 30.93% ± 2.27%. The results suggested that PBAE/GOCMCS nanoparticle was a very promising gene delivery carrier.展开更多
Guanidinylated bioresponsive poly(amido amine)s polymers, CAR-CBA and CHL-CBA, weresynthesized by Michael-type addition reaction between guanidine hydrochloride(CAR) orchlorhexidine(CHL) and N,N-cystaminebisacrylamide...Guanidinylated bioresponsive poly(amido amine)s polymers, CAR-CBA and CHL-CBA, weresynthesized by Michael-type addition reaction between guanidine hydrochloride(CAR) orchlorhexidine(CHL) and N,N-cystaminebisacrylamide(CBA). Previous studies have shownthat both polymers had high transfection efficiencies as gene delivery carriers. In this study,we investigated the nucleolus localization abilities and cellular internalization pathways ofthese two polymers in gene delivery. Each polymer condensed plasmid DNA(p DNA) andformed nanoparticle complexes, and then their transfection studies were performed inMCF-7 cells. Both complexes were found enriched in nucleolus after cellular transfection,and their transfection efficiencies were significantly improved when transfection was per-formed on MCF-7 cells arrested at M phase. The transfection efficiency of CAR-CBA-pDNAwas inhibited by chlorpromazine, and cell endosomes were disrupted after being exposedto CAR-CBA-pDNA. In regards to CHL-CBA-pDNA, its transfection efficiency was not affected by three types of endocytosis inhibitors used in the study, and CHL-CBA-pDNA showed no effect on endosomes. Cellular lactate dehydrogenase release and membrane morphology were changed after cells were transfected by the two complexes. The results indicated that both CAR-CBA and CHL-CBA polymers demonstrated good nucleolus localization abilities. It was beneficial for transfection when cells were arrested at M phase. CAR-CBA-pDNA cellular internalization was involved with clathrin-mediated endocytosis pathway, and escaping from endosomal entrapment, while the cellular uptake of CHL-CBA-pDNA occurs via clathrin-and caveolae-independent mechanism.展开更多
基金the National Natural Science Foundation of China for financial support(No.81373335)
文摘A cationic gene delivery vector, guanidinylated disulfide-containing poly(amido amine)(CARCBA), was synthesized by Michael addition reaction between N,N′-cystaminebisacrylamide(CBA) and guanidine hydrochloride(CAR). Gel permeation chromatography(GPC) was used to evaluate the molecular weight of synthesized CAR-CBA. Polyethyleneimine(PEI) with molecular weight of 25 kDa was adopted as a reference, and polyethylene glycols(PEG) with different molecular weights were used to establish a standard curve for determining the molecular weight of CAR-CBA. The effects of two critical factors, namely columns and eluents,on the molecular weight measurement of CAR-CBA were investigated to optimize the GPC quantitative method. The results showed that Ultrahydrogel columns(120, 250) and HAc–NaAc(0.5 M, pH 4.5) buffer solution were the optimal column and GPC eluent, respectively.The molecular weight of the synthesized CAR-CBA was analyzed by the optimized GPC method and determined to be 24.66 kDa.
基金This work was supported by the 3rd Jiangsu Overseas Research&Training Program for University Prominent Young&Middleaged Teachers and Presidentsthe College Students Innovation Project for the R&D of Novel Drugs[No.J1310032]And we would like to thank cell and molecular biology experiment platform of China Pharmaceutical University for the assistance with relevant test items.
文摘Gene-based therapeutics has emerged as a promising approach for human cancer therapy. Among a variety of non-viral vectors, polymer vectors are particularly attractive due to their safety and multivalent groups on their surface. This study focuses on guanidinylated O-carboxymethyl chitosan(GOCMCS) along with poly-β-amino ester(PBAE) for si RNA delivery. Binding efficiency of PBAE/si RNA/GOCMCS nanoparticles were characterized by gel electrophoresis. The si RNA-loaded nanoparticles were found to be stable in the presence of RNase A, serum and BALF respectively. Fine particle fraction(FPF) which was determined by a two-stage impinger(TSI) was 57.8% ± 2.6%. The particle size and zeta potential of the nanoparticles were 153.8 ± 12.54 nm and + 12.2 ± 4.94 m V. In vitro cell transfection studies were carried out with A549 cells. The cellular uptake was significantly increased. When the cells were incubated with si Survivin-loaded nanoparticles, it could induce 26.83% ± 0.59% apoptosis of A549 cells and the gene silencing level of survivin expression in A549 cells were 30.93% ± 2.27%. The results suggested that PBAE/GOCMCS nanoparticle was a very promising gene delivery carrier.
基金supported by National Natural Science Foundation of China (Grant no. 81373335)
文摘Guanidinylated bioresponsive poly(amido amine)s polymers, CAR-CBA and CHL-CBA, weresynthesized by Michael-type addition reaction between guanidine hydrochloride(CAR) orchlorhexidine(CHL) and N,N-cystaminebisacrylamide(CBA). Previous studies have shownthat both polymers had high transfection efficiencies as gene delivery carriers. In this study,we investigated the nucleolus localization abilities and cellular internalization pathways ofthese two polymers in gene delivery. Each polymer condensed plasmid DNA(p DNA) andformed nanoparticle complexes, and then their transfection studies were performed inMCF-7 cells. Both complexes were found enriched in nucleolus after cellular transfection,and their transfection efficiencies were significantly improved when transfection was per-formed on MCF-7 cells arrested at M phase. The transfection efficiency of CAR-CBA-pDNAwas inhibited by chlorpromazine, and cell endosomes were disrupted after being exposedto CAR-CBA-pDNA. In regards to CHL-CBA-pDNA, its transfection efficiency was not affected by three types of endocytosis inhibitors used in the study, and CHL-CBA-pDNA showed no effect on endosomes. Cellular lactate dehydrogenase release and membrane morphology were changed after cells were transfected by the two complexes. The results indicated that both CAR-CBA and CHL-CBA polymers demonstrated good nucleolus localization abilities. It was beneficial for transfection when cells were arrested at M phase. CAR-CBA-pDNA cellular internalization was involved with clathrin-mediated endocytosis pathway, and escaping from endosomal entrapment, while the cellular uptake of CHL-CBA-pDNA occurs via clathrin-and caveolae-independent mechanism.
