Auayporn Apirakaramwong

Chitosan lactate as a nonviral gene delivery vector in COS-1 cells

The purpose of this research was to evaluate chitosan lactate (CL) of different molecular weights (MWs) as a DNA complexing agent for its efficiency in transfecting COS-1 cells (green monkey fibroblasts) and its effect on cell viability compared with polyethylenimine (PEI), a commercially available cationic polymer. CL and chitosan base dissolved in dilute acetic acid (chitosan acetate, [CA]) of different MWs (20, 45, 200, 460 kDa) and N/P ratios (2∶1, 4∶1, 8∶1, 12∶1, 24∶1) formed complexes with pSV β-galactosidase plasmid DNA. The complexes were characterized by agarose gel electrophoresis and investigated for their ability to transfect COS-1 cells compared with PEI. Additionally, the effect of CL on the viability of COS-1 cells was investigated using 3-(4,5-dimethyliazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The binding of CL/DNA and CA/DNA was dependent on chitosan MWs. The N/P ratio of CL to completely form the complex with the DNA was higher than that of CA. Both CL and CA were comparable in transfection efficiencies at an N/P ratio of 12∶1, but less efficient than PEI (P<.05). The cell viability in the presence of CL and CA at all MWs was over 90%, whereas that of PEI-treated cells was ≈50%. These results suggest the advantage of CL for in vitro gene transfection, with the ease of preparation of polymer/DNA complexes and low cytotoxicity.

Effect of Salt Forms and Molecular Weight of Chitosans on In Vitro Permeability Enhancement in Intestinal Epithelial Cells (Caco-2)

The purpose of this study was to investigate the effect of molecular weight (MW) and salt forms of chitosans (aspartate; CS A, glutamate; CS G, lactate; CS L and hydrochloride, CS HCl) on the transepithelial electrical resistance (TEER) and permeability of Caco-2 cells monolayer, using fluorescein isothiocyanate dextran 4000 (FD-4) as the model compound for paracellular tight junction transport. Chitosan salts were prepared by spray-drying method. FTIR and solid-state 13C NMR spectra showed the functional groups of salts in their molecular structures. Salt form, MW of chitosan, and amount of chitosan influenced the permeation-enhancing effects. These studies showed that chitosan salts appeared to increase cell permeability in a dose-dependent manner and caused relatively reversible effects only at the lower doses of 0.001–0.01% w/v. As the MW of chitosan increased from 20 to 460 kDa, the reduction in TEER significantly decreased in the following order: 20 < 45 < 200 < 460 kDa, observed in CS L and CS HCl. In CS A and CS G, the decrease in TEER was not significantly different in all MW because both chitosan salts showed rapid reduction in TEER within 20 min after the start of the experiment. Among chitosan salts, CS A was the most potent absorption enhancer in acidic (pH 6.2) environment. Cytotoxicity of chitosan salts was concentration dependent and varied slightly among the salt forms of chitosan used. CS HCl (MW 45 kDa) was the most toxic having an IC50 of 0.22 ± 0.06 mg/mL. The ranking of chitosan salts cytotoxicity was CS HCl > CS L> CS G > CS A.

Methylated N-(4-N,N-dimethylaminobenzyl) chitosan coated liposomes for oral protein drug delivery.

In the present study, methylated N-(4-N,N-dimethylaminobenzyl) chitosan (TM(56)Bz(42)CS) was synthesised and investigated for oral protein drug delivery by combining it with liposomes entrapped with fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA), a model protein. Liposomes (LPs) composed of 10:2 molar ratios of egg yolk phosphatidylcholine (EPC) and sodium oleate (NaO) were prepared by a thin film hydration method and coated with TM(56)Bz(42)CS. BSA-loaded, TM(56)Bz(42)CS-coated liposomes (TM(56)Bz(42)CS-coated FITC-BSA-LP) were evaluated for their protein transport efficiencies and cytotoxicities in Caco-2 cells. Moreover, the in vitro stabilities of the TM(56)Bz(42)CS-coated LP-BSA were determined by examining the degradation of the protein in simulated intestinal fluid containing 1% w/v pancreatin porcine pancreas. The mean particle size and zeta-potential of the TM(56)Bz(42)CS-coated LP-BSA were 128 ± 15 nm and 5.38 ± 1.66 mV, respectively. Additionally, the initial FITC-BSA to lipid ratio (2.5% w/w) showed the highest entrapment efficiency percentage (50.13%) and FITC-BSA content (8.08 mg/g of lipid) overall. The results of the FITC-BSA transport showed that the TM(56)Bz(42)CS-coated FITC-BSA-LP enhanced protein permeability across the Caco-2 cell monolayers with low cytotoxicity. In addition, these liposomes protected against protein degradation in pancreatin. Our studies demonstrated that TM(56)Bz(42)CS-coated liposomes have the potential to be used in oral protein drug delivery methods.

Structure Relationship of Cationic Lipids on Gene Transfection Mediated by Cationic Liposomes

The aim of this study was to investigate the transfection efficiency of cationic liposomes formulated with phosphatidylcholine (PC) and novel synthesized diethanolamine-based cationic lipids at a molar ratio of 5:1 in comparison with Lipofectamine™ 2000. Factors affecting transfection efficiency and cell viability, including the chemical structure of the cationic lipids, such as different amine head group (diamine and polyamine; and non-spermine and spermine) and acyl chain lengths (C14, C16, and C18) and the weight ratio of liposomes to DNA were evaluated on a human cervical carcinoma cell line (HeLa cells) using the pDNA encoding green fluorescent protein (pEGFP-C2). Characterizations of these lipoplexes in terms of size and charge measurement and agarose gel electrophoresis were performed. The results from this study revealed that almost no transfection was observed in the liposome formulations composed of cationic lipids with a non-spermine head group. In addition, the transfection efficiency of these cationic liposomes was in the following order: spermine-C14 > spermine-C16 > spermine-C18. The highest transfection efficiency was observed in the formulation of spermine-C14 liposomes at a weight ratio of 25; furthermore, this formulation was safe for use in vitro. In conclusion, cationic liposomes containing spermine head groups demonstrated promising potential as gene carriers.

The development of poly-L-arginine-coated liposomes for gene delivery

In this study, liposomes coated with cationic polymers, poly-L-arginine (PLA), were assessed as a promising gene transfer system in human cervical carcinoma (HeLa) cells and human hepatoma cell line (Huh7) cells. The liposomes were prepared using egg yolk phosphatidylcholine and sodium oleate in the molar ratio of 10:2 with an ultrasonic generator and then coated with PLA. The PLA-coated liposomes (PCLs) formed complexes with plasmid DNA encoding green fluorescent protein. The complexes were characterized by agarose gel electrophoresis and investigated for their transfection efficiency in HeLa and Huh7 cells. The data were compared with PLA/DNA complexes and the positive control Lipofectamine 2000™. The results showed that complete PCL/DNA complexes were formed at weight ratios of more than 0.05. Efficient gene transfer by PCLs was dependent on the cell type. The transfection efficiency of PCLs was about two times higher than that of PLA/DNA complexes in both HeLa cells and Huh7 cells. Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and showed that 80%–100% of both of the cells were viable after treating PCL/DNA complexes. The present results demonstrate that PCLs are a promising, nonviral gene carrier with low toxicity.

Effect of Acyl Chain Length of Spermine Derivatives on Transfection Efficiency

In this study, cationic liposomes prepared from egg phosphatidylcholine (PC) and novel spermine-based cationic lipids at a molar ratio of 5:1 were formulated. The chemical structures of these cationic lipids consisted of spermine head group and four hydrocarbon tails with differences in acyl chain (C14, C16 and C18). The effects of acyl chain and weight ratio of liposomes to DNA on transfection efficiency and cytotoxicity were investigated on a human cervical carcinoma cell line (HeLa cells) using the pDNA encoding green fluorescent protein (pEGFP-C2). The results from agarose gel electrophoresis illustrated that all cationic liposomes were able to condense with pDNA. The transfection efficiency of these cationic liposomes was in the following order: C18 (3,497±120 cells/cm2) > C14 (809±52 cells/cm2) > C16 (91±5 cells/cm2). The highest transfection efficiency was observed in the formulation of cationic liposomes with C18 tail at weight ratio of 15. In cytotoxicity studies, all formulations showed low cytotoxicity. In conclusion, these cationic liposomes containing novel cationic lipids (C18), showed promising potential as a gene carrier by efficient DNA condensation and mediated higher level of gene transfection.

Application of Methylated N-(4-N,N-Dimethylaminocinnamyl) Chitosan for Oral Protein Drug Delivery

In the present study, methylated N-(4-N,N-dimethylaminocinnamyl) chitosan (TM65CM50CS) was synthesized and investigated for oral protein drug delivery by combining with liposomes entrapped bovine serum albumin (FITC-BSA), a model protein. FITC-BSA liposomes composed of egg yolk phosphatidylcholine and sodium oleate in molar ratio of 10:2 were prepared by thin film hydration method. The TM65CM50CS coated liposomal FITC-BSA was evaluated for transport of protein and its cytotoxicity in Caco-2 cells. Moreover, the in vitro stability of BSA in TM65CM50CS coated liposomes was also examined by the degradation of protein from pancreatin. The mean particle size and zeta-potential of liposomes were 101+0.02 nm and -27.44+2.02 mV, respectively. Initial FITC-BSA (2.5% w/w) to lipid showed the highest percentage entrapment efficiency (50.13%) and FITC-BSA content (8.08 mg/g of lipid). The results of FITC-BSA transport showed that TM65CM50CS coated FITC-BSA liposomes enhanced protein permeability across Caco-2 cell monolayers with low cytotoxicity. In addition, these liposomes could protect the degradation of protein from pancreatin. Our studies demonstrated that TM65CM50CS coated liposomes have the potential to be used as an oral protein drug delivery.

Mechanisms of Cellular Uptake with Chitosan/DNA Complex in Hepatoma Cell Line

Chitosan (CS) has a high potential for gene delivery into mammalian cells. However, its uptake mechanism is not well clarified. We investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine), caveolae-mediated endocytosis (genistein), macropinocytosis (LY 29004 and wortmannin), microtubuli polymerization (nocodazole) and of membrane cholesterol recycle (methyl-β-cyclodextrin) on the transfection efficiency with CS/pEGFP complexes and on the internalization of CS/rhodamine-labeled pEGFP complexes by hepatoma cell line (Huh 7 cells). The transfection was blocked by nocodazole, genistein, and methyl-β-cyclodextrin, respectively. CS/DNA complexes internalization was clearly inhibited by genistein. We conclude that the complexes uptake predominantly by caveolin-mediated pathways. In addition, fluorescence colocalization studies with acidotropic probes, LysoSensor dye, illustrated that CS/DNA complexes are targeted to lysosomes for the degradation after internalization.