Chitosan N-betainates/DNA self-assembly nanoparticles for gene delivery: in vitro uptake and transfection efficiency (original) (raw)
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Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency
Journal of Controlled Release, 2001
Chitosan-DNA nanoparticles were prepared using a complex coacervation process. The important parameters for the nanoparticle synthesis were investigated, including the concentrations of DNA, chitosan and sodium sulfate, temperature of the solutions, pH of the buffer, and molecular weights of chitosan and DNA. At an amino group to phosphate group ratio (N / P ratio) between 3 and 8 and a chitosan concentration of 100 mg / ml, the size of particles was optimized to |100-250 nm with a narrow distribution, with a composition of 35.6 and 64.4% by weight for DNA and chitosan, respectively. The surface charge of these particles was slightly positive with a zeta potential of 112 to 118 mV at pH lower than 6.0, and became nearly neutral at pH 7.2. The chitosan-DNA nanoparticles could partially protect the encapsulated plasmid DNA from nuclease degradation as shown by electrophoretic mobility analysis. The transfection efficiency of chitosan-DNA nanoparticles was cell-type dependent. Typically, it was three to four orders of magnitude, in relative light units, higher than background level in HEK293 cells, and two to ten times lower than that achieved by LipofectAMINEE-DNA complexes. The presence of 10% fetal bovine serum did not interfere with their transfection ability. Chloroquine could be co-encapsulated in the nanoparticles at 5.2%, but with negligible enhancement effect despite the fact that chitosan only showed limited buffering capacity compared with PEI. The present study also developed three different schemes to conjugate transferrin or KNOB protein to the nanoparticle surface. The transferrin conjugation only yielded a maximum of four-fold increase in their transfection efficiency in HEK293 cells and HeLa cells, whereas KNOB conjugated nanoparticles could improve gene expression level in HeLa cells by 130-fold. Conjugation of PEG on the nanoparticles allowed lyophilization without aggregation, and without loss of bioactivity for at least 1 month in storage. The clearance of the PEGylated nanoparticles in mice following intravenous administration was slower than unmodified nanoparticles at 15 min, and with higher depositions in kidney and liver. However, no difference was observed at the 1-h time point.
Journal of Applied …, 2011
The molecular weight (MW) of chitosan (CS) was determined by viscometric method (using Mark-Houwink equation) as well as by gel permeation chromatography, and the degree of deacetylation (DDA) of CS was measured by potentiometric titration method and Gran-type linearization method. The values of DDA were obtained � 83% (by potentiometric titration method) and � 86% (by Gran-type linearization method). The self-assembled nanoparticles of CS/plasmid DNA (pDNA) complex were prepared by varying the concentration of CS. The formation of CS/pDNA complex was confirmed by 0.8% agarose gel electrophoresis and the particle size of the self-assembled nanoparticle was determined by dynamic light scattering, atomic force microscopy and scanning electron microscopy. The stability of CS/pDNA complexes was determined by turbidity test with the help of UV-Vis spectroscopy. The effect of ionic strength on the complexes was also observed by means of fluorescence spectroscopy. The cytotoxicity of CS on the HeLa cell line was observed by absorbance of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay and showed that CS has lower cytotoxicity in HeLa cells compared with that of poly (L-lysine) in 293T cells.
International Journal of Nanomedicine, 2019
The objective of this work was to formulate a delivery system of pDNA encoded p53 gene-loaded chitosan-sodium deoxycholate (CS-DS) nanoparticles, and to evaluate their influence on in vitro cytotoxicity and transfection efficiency of p53 gene. Methods: The prepared pDNA-loaded CS-DS nanoparticles were evaluated for morphology, particle size, zeta potential, entrapment efficiency %, in vitro release, in vitro cytotoxicity, and transfection efficiency. Results: The mean particle size ranged from from 96.5 ± 11.31 to 405 ± 46.39 nm. All nanoparticles had good positive zeta potential values. Entrapment efficiency % ranged from 38.25 ± 3.25 to 94.89 ± 5.67. The agarose gel electrophoresis confirmed the strong binding between plasmid and CS. The in vitro pDNA release from nanoparticles exhibited an initial burst effect followed by a sustained drug release over a period of 6 days. In vitro cytotoxicity against human Caco-2 cells showed low cell cytotoxicity of plain CS-DS nanoparticles, while pDNA-loaded CS-DS nanoparticles showed a cytotoxic effect with increasing nanoparticles' concentration. Gene transfection, analyzed by PCR and ELISA, showed a direct correlation between gene expression and concentration of pDNA. The highest expression of the gene was achieved with pDNA concentration of 9 µg/mL with 5.7 times increase compared to naked pDNA of the same concentration. Conclusion: The obtained results were very encouraging and offer an alternative approach to enhancing the transfection efficiency of genetic material-loaded chitosan-based delivery systems.
Carbohydrate Polymers, 2018
The aim of this study is to prepare the long linear aliphatic amine pendant group-functionalized chitosan based nanoparticulate gene carrier system with improved properties for the efficient transfection. The amine functionalized chitosan (MChi) was synthesized by using N-(2-hydroxyethyl)ethylenediamine (HE-EDA) and characterized for the first time. The nanoparticles of MChi (nMChi) were prepared by ionic gelation method, and their particle size, polydispersity (PDI), zeta potential (mV), gene binding capacity and cytotoxicity were determined. Green Fluorescent Protein circular plasmid DNA (pEGFN1) loaded nanoparticles (gnMChi) were used in the transfection studies. The results showed that nMChi with a particle size of 102.9 nm and zeta potential of 41.9 ± 5.63 mV was non-toxic, had high transfection efficiency in Human Embryonic Kidney 293 and Primary Ovine Fibroblast cell lines and would be used as an efficient gene carrier system.
Cytotoxic and cytostatic side effects of chitosan nanoparticles as a non-viral gene carrier
International journal of pharmaceutics, 2016
Although chitosan nanoparticles (CNs) became a promising tool for several biological and medical applications owing to their inherent biocompatibility and biodegradability features, studies regarding their effects on cytotoxic and cytostatic properties still remain insufficient. Therefore, in the present study, we decided to perform comprehensive analysis of the interactions between CNs-pKindling-Red-Mito (pDNA) and different cell line models derived from blood system and human solid tissues cancers. The resulting CNs-pDNA was investigated in terms of their cellular uptake, transfection efficiency, and physico-chemical, cytotoxic and cytostatic properties. The nanoparticles showed high encapsulation efficiency and physical stability for various formulations even after two days time period. Moreover, high gene expression levels were observed after 96h of transfection. CNs-pDNA treatment, despite the absence of oxidative stress induction, caused cell cycle arrest in G0/G1 phase and as...
Improved stability and efficacy of chitosan/pDNA complexes for gene delivery
Biotechnology Letters, 2014
Among polymeric polycations, chitosan has emerged as a powerful carrier for gene delivery. Only a few studies have focused on the stability of the chitosan/DNA complex under storage, although this is imperative for nanomedicinal applications. Here, we synthesized polyelectrolyte complexes at a charge ratio of 10 using 50 kDa chitosan and plasmid (p)DNA that encodes a GFP reporter. These preparations were stable up to 3 months at 4°C and showed reproducible transfection efficiencies in vitro in HEK293 cells. In addition, we developed a methodology that increases the in vitro transfection efficiency of chitosan/pDNA complexes by 150 % with respect to standard procedures. Notably, intracellular pDNA release and transfected cells peaked 5 days following transection of mitotically active cells. These new developments in formulation technology enhance the potential for polymeric nanoparticle-mediated gene therapy.
Pharmaceutics
Chitosan is a naturally occurring polymer derived from the deacetylation of chitin, which is an abundant carbohydrate found mainly in the shells of various marine and terrestrial (micro)organisms. Chitosan has been extensively used to construct nanoparticles (NPs), which are biocompatible, biodegradable, non-toxic, easy to prepare, and can function as effective drug delivery systems. Moreover, chitosan NPs have been employed in gene and vaccine delivery, as well as advanced cancer therapy, and they can also serve as new therapeutic tools against viral infections. In this review, we summarize the most recent developments in the field of chitosan-based NPs intended as nucleic acid delivery vehicles and gene therapy vectors. Special attention is given to the technological aspects of chitosan complexes for nucleic acid delivery.
N-hexanoyl, N-octanoyl and N-decanoyl chitosans: Binding affinity, cell uptake, and transfection
Carbohydrate Polymers, 2012
Low transfection efficiency of chitosan limits its use as a non-viral vector for practical purposes. This study was designed to investigate the effect of fatty acyl chain length on physicochemical properties, pDNA binding affinity, cell uptake, and in vitro transfection efficiency of N-acyl chitosan (NAC). NAC polymers were synthesized by carbodiimide mediated coupling reaction of chitosan with n-hexanoic, noctanoic, and n-decanoic acid, respectively. These NAC polymers effectively condensed pDNA resulting in the size range of 220-342 nm with net positive charge. NAC polymers also showed good pDNA binding capacity, high protection of pDNA from nuclease degradation and excellent biocompatibility. Transfection efficiency of chitosan, in HEK 293 cells, was enhanced 15-25-fold after coupling with fatty acid and increased with a decrease in fatty acyl chain length of NAC. Thus, the present study demonstrates that the NAC polymers hold great potential as novel non-viral gene delivery vector.