Encapsulation of plasmid DNA into colloidal carrier of poly(D.L-lactide-co-glycolide)/chitosan/nigella sativa intended for gene delivery to central nervous system

Abstract

Delivery of plasmid DNA (pDNA) to the central nervous system (CNS) is challenging in gene therapy due to the presence of cellular and biochemical barriers, which restrict the passage of most substances into the brain. Nigella Sativa oil (NSO) is a lipophilic material and has been reported to have neurotherapeutics effects such as neuroprotective and neuroregenerative. Owing to its lipophilicity, NSO-incorporated carrier system could enhance penetration of the gene into the brain. Here, we aimed to amplify and isolate pDNA from Escherichia coli (E. coli) DH5?. We had attempted to co-encapsulate the pDNA and NSO into biodegradable poly(D,L-lactide-co-glycolide) (PLGA) and chitosan using diffusion-solvent evaporation technique. Firstly, commercial pDNA was amplified and isolated using conventional isolated/purification of pDNA, namely ‘IIUM Concentrated Alkaline Lysis’ (iCALL); a modified Sambrook and Russell’s protocol in order to obtain high quality and plasmid yield for our study. Secondly, the pDNA and NSO were co-encapsulated into PLGA/chitosan by manipulating several variables i.e. fabrication techniques (single or double emulsion solvent evaporation); volume ratio of solvent to co-solvent, concentration of chitosan and NSO; and mixing rate/type (homogenizer and sonicator processor) in order to investigate their effects on the particles’ characteristics and to obtain optimized formulations. The optimized formula that was prepared by single emulsion (o/w), volume ratio of solvent to co-solvent at 1:3 and sonicated at 15 sec time was selected. To improve the encapsulation efficiency of pDNA using single emulsion, pre-complexation between pDNA and cetyltrimethylammonium bromide (CTAB) prior encapsulation was introduced. Further optimization of co-encapsulation of NSO and pDNA in PLGA NPs were carried out by employing two independent variables namely, molecular weights (MW) of PLGA 50:50 (14 and 34 kDa) and chitosan (50-190 and 190-310 kDa).These NPs were thereafter called “Neurobionanoparticles” (NBPs). Size, surface morphology, zeta potential, pDNA in vitro release profile, cell viability and transfectibility were characterized as a function of those multiple variables. The selected NBPs were subjected to stability studies by investigating the effect of excipients, namely human serum albumin, glycine and potassium chloride with different NBP/excipients weight ratio systems on stability of lyophilized NBP upon three months’ storage. Our data revealed that iCALL method gave the highest value for the pDNA yield compared with other commercial methods. The resultant NBPs showed an encapsulation efficiency of ~99%, particle sizes around 400 nm and positive zeta potential. These optimized preparations showed sustained release rate of pDNA over 5 weeks and was capable of expressing pGL3 gene in Neuro-2A (N2a) cell line. Interestingly, preparation with PLGA 50:50 (14 kDa) and chitosan (190 – 310 kDa) showed higher gene expressions in N2a cells. None of these NPs were toxic for N2a cells after incubation for 48 h in the tested dose ranges (0.01, 0.1 and 0.2 mg/ml). Furthermore, the co-lyophilized of NBP with HSA at ratios 10:1 and 2:1 abled to stabilize lyophilized particles for three months storage and exhibit better enhancement in the transfection activity of gene delivery when compared with NBP alone and combination of NBP with other excipients. In conclusion, the fabricated NBPs may be used as promising non-viral gene delivery to the CNS.