Nattisa Niyomtham

Synthesis and in vitro transfection efficiency of spermine-based cationic lipids with different central core structures and lipophilic tails

Twelve spermine-based cationic lipids with four different central core structures (di(oxyethyl)amino, di(oxyethyl)amino carboxy, 3-amino-1,2-dioxypropyl and 2-amino-1,3-dioxypropyl) and three hydrophobic tails (lauric acid, myristic acid and palmitic acid) were synthesized. The liposomes containing lipids and DOPE showed moderate to good in vitro DNA delivery into HeLa cells. GFP expression experiments revealed that liposomes composed of lipids with 3-amino-1,2-dioxypropyl as a central core structure exhibited highest transfection efficiency under serum-free condition. Whereas, lipid with 2-amino-1,3-dioxypropyl core structure showed highest transfection under 10% serum condition. Moreover, the liposomes and lipoplexes composted of these cationic lipids exhibited low cytotoxicity.

Cationic niosomes composed of spermine-based cationic lipids mediate high gene transfection efficiency

Non-ionic surfactant-based vesicles (niosomes) composed of non-ionic surfactants (i.e., Tween and Span) and cholesterol were formulated, and their turbidity and particle size assayed. The most appropriate niosomes formulation was mixed with novel synthesized spermine-based cationic lipids to prepare cationic niosomes that could act as gene carriers. Factors affecting gene transfection and cell viability including differences in the acyl chain length (C14, C16 and C18) of cationic lipids and the weight ratio of niosomes to DNA were evaluated on a human cervical carcinoma cell line (HeLa cells) using pDNA encoding green fluorescent protein (pEGFP-C2). The morphology, size and charge of the niosomes were also characterized, and a gel retardation assay to determine complex formation was performed. The results revealed that the transfection efficiency of the Span 20-niosomes was the highest for the spermine-C14 formulation and decreased as follows: spermine-C14 > spermine-C16 > spermine-C18. In addition to the highest transfection efficiency, there was also no serum effect on transfection efficiency of the spermine-C14 niosomes at a weight ratio of 10. This formulation was safe in vitro and had good physical stability for at least 1 month at 4°C. In conclusion, the cationic niosomes may constitute a good alternative carrier for gene transfection.

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.

High transfection efficiency of cationic lipids with asymmetric acyl-cholesteryl hydrophobic tails.

The ability of a nonviral gene delivery system to overcome extra- and intracellular barriers is a critical issue for the future clinical applications of gene therapy. In recent years much effort has been focused on the development of a variety of DNA carriers, and cationic liposomes have become the most common nonviral gene delivery system. One hundred and eighty novel cationic lipids with asymmetric acyl-cholesteryl hydrophobic tails were synthesized by parallel solid-phase chemistry. The liposomes were prepared and gel retardation assays were used to study the binding efficiency between the prepared liposome and the DNA. Transfection efficiencies of the lipids were evaluated against various mammalian cells, such as human embryonic kidney (HEK293), human cervical adenocarcinoma (HeLa), canine osteosarcoma (D17), colorectal adenocarcinoma (COLO 205), and human prostate adenocarcinoma (PC3) cells. The lipids with an acyl portion at the terminal part of the polyamine backbone exhibited higher transfection efficiency than those with the acyl portion as an internal part of the backbone. These compounds also showed higher transfection efficiency and lower cytotoxicity than the commercially available agents, Effectene, DOTAP, and DC-Chol.

Cholesterol-based cationic liposome increases dsRNA protection of yellow head virus infection in Penaeus vannamei.

Protection of shrimp from yellow head virus (YHV) infection has been demonstrated by injection and oral delivery of dsRNA-YHV protease gene (dsYHV) or shrimp endogenous gene (dsRab7). However, to achieve complete viral suppression and to prolong dsRNA activity, the development of an effective dsRNA delivery system is required. In this study, four cationic liposomes were synthesized and tested for their ability to increase dsRNA efficiency. The results demonstrated that entrapping dsYHV in a cholesterol-based cationic liposome gave the best protection against YHV infection when compared with other cationic lipids. The cholesterol-based cationic liposome-dsYHV (Chol-dsYHV) complex conferred YHV protection in a dose-dependent manner. Injection with Chol-dsYHV at 0.05μg dsYHV/g shrimp could give comparable level of YHV protection to the injection with 1.25μg naked dsYHV/g shrimp. The shrimp injected with Chol- dsYHV at 1.25μg dsRNA/g shrimp showed only 50% mortality at 60days post injection whereas the naked dsYHV at the same concentration gave 90% mortality. Thus, the liposome-entrapped dsYHV could lower an effective dsRNA concentration in viral protection and prolong dsRNA activity. In addition, encapsulating dsRab7 in the cholesterol-based cationic liposome could protect the dsRab7 from enzymatic digestion, and continuous feeding the shrimp with the diet formulated with the liposome-entrapped dsRab7 for 4days in the total of 960μg dsRab7/g shrimp could enhance YHV protection efficiency compared with the naked dsRab7. Our studies reveal that cholesterol-based cationic liposome is a promising dsRNA carrier to enhance dsRNA efficiency in both injection and oral delivery systems.

Cationic niosomes an effective gene carrier composed of novel spermine-derivative cationic lipids: effect of central core structures

Abstract Context: Cationic niosomes formulated from Span 20, cholesterol (Chol) and novel spermine-based cationic lipids of multiple central core structures (di(oxyethyl)amino, di(oxyethyl)amino carboxy, 3-amino-1,2-dioxypropyl and 2-amino-1,3-dioxypropyl) were successfully prepared for improving transfection efficiency in vitro. The niosomes composed of spermine cationic lipid with central core structure of di(oxyethyl)amino revealed the highest gene transfection efficiency. Objectives: To investigate the factors affecting gene transfection and cell viability including differences in the central core structures of cationic lipids, the composition of vesicles, molar ratio of cationic lipids in formulations and the weight ratio of niosomes to DNA. Methods: Cationic niosomes composed of nonionic surfactants (Span20), cholesterol and spermine-based cationic lipids of multiple central core structures were formulated. Gene transfection and cell viability were evaluated on a human cervical carcinoma cell line (HeLa cells) using pDNA encoding green fluorescent protein (pEGFP-C2). The morphology, size and charge were also characterized. Results and discussion: High transfection efficiency was obtained from cationic niosomes composed of Span20:Chol:cationic lipid at the molar ratio of 2.5:2.5:0.5 mM. Cationic lipids with di(oxyethyl)amino as a central core structure exhibited highest transfection efficiency. In addition, there was also no serum effect on transfection efficiency. Conclusions: These novel cationic niosomes may constitute a good alternative carrier for gene transfection.

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.