Evaluation and comparison of cytotoxicity, genotoxicity, and apoptotic effects of poly-l-lysine/plasmid DNA micro- and nanoparticles.

The principal impediment to gene therapy is the development of efficient, nontoxic gene carriers that can handle and deliver foreign genetic materials into various cell types, including healthy and cancerous cells. Poly-l-lysine (PLL) polymers are one of the most favorable gene carriers among nonviral vectors, and PLL had low transfection and safety issues. The purpose of this study was to measure cellular toxicity, DNA damage, and apoptotic effects of PLL nanoparticles. Neuro2A mammalian cells were cultured and exposed to PLL/DNA complexes at different polymer/DNA ratios (C/P ratio 2 and 6) for 24 h. To evaluate metabolic activity, genotoxicity, and apoptotic influences of PLL nanoparticle, the following experimental methods were employed, in order: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), DNA damage (COMET analysis) assay, and sub-G1 peak apoptosis assay. Our data indicate that toxicity is concentration dependent and a high concentration of polymer declined the metabolic activity. In addition, largest complexes (C/P 6 in HEPES buffered saline buffer) have slighter negative impact on metabolic activity. In agreement with our cytotoxicity data, apoptotic assay result represented that increase in size of PLL/DNA complexes decrease the number of apoptotic cells. Also, there was a remarkable increase in percent tail DNA of Neuro2A cells treated with higher concentration of PLL and its polyplexes. The present study demonstrated that PLL/DNA complexes caused cytotoxic, apoptotic, and genotoxic effects in a dose-dependent and weight ratio-dependent manner, which also affected the size of polyplexes.

Double loading of cyclosporine A in liposomes using cyclodextrin complexes.

When liposomes are used as drug delivery systems, it is important that the therapeutic agent is efficiently and adequately encapsulated. In this study, cyclosporine A (CyA) was chosen as a model drug for two reasons. First, liposomes are a potential delivery system for CyA, as it has been shown that CyA has decreased side effects when encapsulated in liposomes. Secondly, if the aqueous solubility of a lipophilic drug can be increased, then it is possible to increase liposomal encapsulation by additionally loading the drug into the liposomes' aqueous compartments. Therefore, we investigated the use cyclodextrins (CDs) for complexing CyA to increase aqueous solubility as a strategy to increase liposomal loading. The effect of CyA loading on the liposomes' characteristics, stability and rigidity of the bilayer, and also the drug release profile were evaluated. Liposomes encapsulating CyA, liposomes containing CyA-CD complexes, and liposomes loaded with both plain drug and complex (double-loaded liposomes) were prepared. For evaluation of the effect of CD on bilayer rigidity and integrity, the permeability of the liposomal membrane in terms of carboxyfluorescein (CF) leakage was studied. Among liposomal formulations containing only CyA, 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC):cholesterol (CHOL) (1:1) and hydrogenated soybean phosphatidylcholine (HSPC):CHOL (1:1) formulations demonstrated maximum drug entrapments of 65.94 +/- 4.68% and 75.03 +/- 4.87%, respectively. There was no significant difference in encapsulation efficiencies between different liposomal formulations for those containing CyA-CD complexes (P > 0.05). Measurement of encapsulation efficiency showed that the amount of drug entrapped in the lipid bilayers was identical when prepared in the form of CyA inclusion complexes. Drug entrapment in double-loaded liposomes was increased by approximately 2-fold. The release profile of all liposomal formulations was biphasic, with an initial rapid phase during the first 5 h followed by a continuous and slower release thereafter. During the first 5 h, CyA used as the complex was released to a greater extent than free CyA. Leakage of CF from liposomes was affected by the inclusion of CD. The leakage rate was minimum for CyA liposomes and maximum for double-loaded (CyA and CyA-CD) liposomes. In conclusion, it is possible to encapsulate CyA both in the aqueous and lipidbilayers of liposomes if the aqueous solubility of CyA is increased by complexation with CD. Although entrapment of a higher amount of drug was achieved, the stability of the liposomes was compromised and should therefore be considered.