The influence of formulation variables on in vitro transfection efficiency and physicochemical properties of chitosan-based polyplexes

Purpose: In gene delivery, a fusogenic lipid such as dioleyl phosphatidylethanolamine (DOPE) which is a component of cationic liposomal vector is important factor for effective transfection efficiency. We investigated the effect of penetration enhancers as alternative helper-lipids to DOPE. Methods: Transdermal penetraion enhancers such as N-lauroylsarcosine (LS), (R)-(+)-limonene (LM), vitamin E (VE), and phosphatidyl choline from eggs (EggPC) were used in this experiments as helper-lipids with N-[1-(2, 3-dioleyloxy) propyl]-N, N, N-trimethlylammonium chloride (DOTMA) and cholesterol (CHOL). We examined in vitro transfection efficiency, cytotoxicity, hematotoxicity, and in vivo transfection efficiency of plasmid DNA/cationic liposomes complexes. Results: In transfection experiments in vitro, the cationic lipoplexes containing LS had highest transfection efficiency among the other lipoplexes independently of FBS. Furthermore, the lipoplexes containing LS had lowest cell toxicity among the other lipoplexes in the presence of FBS. As the results of erythrocytes interaction experiment, DOTMA/LS/CHOL, DOTMA/VE/CHOL, and DOTMA/EggPC/CHOL lipoplexes showed extremely lower hematotoxicity. On the basis of these results, the in vivo transfection efficiencies of the lipoplexes were examined. The lipoplexes containing LS had the highest transfection activity among the other lipoplexes. Conclusion: In conclusion, several transdermal penetration enhancers are available for alternative helper-lipids to DOPE in cationic liposomal vectors. Among them, DOTMA/LS/CHOL lipoplexes showed superior characteristics in in vitro transfection efficiency, cell toxicity, hematotoxicity, and in vivo transfection efficiency.

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Formulation and in vitro transfection efficiency of poly (D, L-lactideco-glycolide) microspheres containing plasmid DNA for gene delivery

AAPS PharmSciTech ◽

10.1208/pt010428 ◽

2000 ◽

Vol 1 (4) ◽

pp. 17-25 ◽

Cited By ~ 16

Author(s):

Sisay Gebrekidan ◽

Byung H. Woo ◽

Patrick P. DeLuca

Keyword(s):

Gene Delivery ◽

Plasmid Dna ◽

Transfection Efficiency ◽

In Vitro Transfection

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Development of Lyophilized Gemini Surfactant-Based Gene Delivery Systems: Influence of Lyophilization on the Structure, Activity and Stability of the Lipoplexes

Journal of Pharmacy & Pharmaceutical Sciences ◽

10.18433/j3x60d ◽

2012 ◽

Vol 15 (4) ◽

pp. 548 ◽

Cited By ~ 13

Author(s):

Waleed Mohammed-Saeid ◽

Deborah Michel ◽

Anas El-Aneed ◽

Ronald E Verrall ◽

Nicholas H Low ◽

...

Keyword(s):

Conformational Changes ◽

Gemini Surfactant ◽

Gemini Surfactants ◽

Transfection Efficiency ◽

Gene Transfection ◽

Physiochemical Properties ◽

Cationic Gemini Surfactants ◽

The Stability ◽

In Vitro Transfection

Purpose. Cationic gemini surfactants have been studied as non-viral vectors for gene therapy. Clinical applications of cationic lipid/DNA lipoplexes are restricted by their instability in aqueous formulations. In this work, we investigated the influence of lyophilization on the essential physiochemical properties and in vitro transfection of gemini surfactant-lipoplexes. Additionally, we evaluated the feasibility of lyophilization as a technique for preparing lipoplexes with long term stability. Methods. A gemini surfactant [12-7NH-12] and plasmid DNA encoding for interferon-γ were used to prepare gemini surfactant/pDNA [P/G] lipoplexes. Helper lipid DOPE [L] was incorporated in all formulation producing a [P/G/L] system. Sucrose and trehalose were utilized as stabilizing agents. To evaluate the ability of lyophilization to improve the stability of gemini surfactant-based lipoplexes, four lyophilized formulations were stored at 25˚C for three months. The formulations were analyzed at different time-points for physiochemical properties and in vitro transfection. Results. The results showed that both sucrose and trehalose provided anticipated stabilizing effect. The transfection efficiency of the lipoplexes increased 2-3 fold compared to fresh formulations upon lyophilization. This effect can be attributed to the improvement of DNA compaction and changes in the lipoplex morphology due to the lyophilization/rehydration cycles. The physiochemical properties of the lyophilized formulations were maintained throughout the stability study. All lyophilized formulations showed a significant loss of gene transfection activity after three months of storage. Nevertheless, no significant losses of transfection efficiency were observed for three formulations after two months storage at 25 ˚C. Conclusion. Lyophilization significantly improved the physical stability of gemini surfactant-based lipoplexes compared to liquid formulations. As well, lyophilization improved the transfection efficiency of the lipoplexes. The loss of transfection activity upon storage is most probably due to the conformational changes in the supramolecular structure of the lipoplexes as a function of time and temperature rather than to DNA degradation. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

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