Preparation and characterization of a nanovaccine of pVAX1-NH36 for leishmaniasis

Abstract

Leishmaniasis is an endemic disease mainly in very poor countries. This disease may cause only a cutaneous lesion but also a fatal visceral disease. Vaccines of plasmid DNA (pDNA) are an alternative of conventional treatments of the disease since are considered safer, cheaper and easier to produce than conventional vaccines. However, it is being shown that naked plasmids are very susceptible to degradation and do not enter the cells efficiently. Liposomes may be used for pDNA administration due to their biocompatibility, biodegradability and low immunogenicity. However, they also present problems in reaching the correct cells, such as opsonization, aggregation and phagocytosis. Pegylation of liposomes has proven to increase circulation time and reduce cytotoxicity. This work aimed at the development of a process to prepare lipidic nanoparticles (NPs) loaded with plasmid pVAX1-NH36 for application as leishmaniasis nanovaccine. NPs were prepared by thin film technique using l-α-phosphatidylcholine, octadecylamine and cholesterol in a molar ratio of 7:2:1. An experimental setup was used to analyze the effect of PEG% and molar charge ratio (R) on the physicochemical properties and pDNA carry capacity of the NPs. In the preparation of the nanovaccines 0, 2.5 or 5 mol% of PEG2000-PE was used. The molar charge ratio of pVAX1 to phospholipids was varied between 1 and 11 for each type of nanovaccine. The initial experiments showed that R = 1 and R = 5 nanovaccines present instability and were discarded for further studies. Dynamic light scattering (DLS) analysis showed pDNA-NPs size with an average diameter of 120–160 nm depending on R and PEG2000-PE% and a polydispersity index (PDI) between 0.06-0.16. The Z potentials determined by electrophoretic light scattering were in the range of 15–40 mV. The PEGylated particles showed a PDI and Z potential values more adequate for nanovaccine preparation. The PEGylated and non-PEGylated nanovaccines were visualized by transmission electron microscopy (TEM) and presented a rather spherical shape which is also a desired property. To determine the stability of the nanovaccines their pDNA content was estimated in samples obtained from the reaction solutions at days 0, 1, 2, 7 and 14, measuring the pDNA concentration in the solution by high-performance liquid chromatography on a hydrophobic interaction column (HPLC-HIC). All the samples presented a stability greater than 84% in the period of study. A mixed order lumped parameter model was successfully fitted (with r2 > 0.92 in all the preparations) to the kinetics of plasmid liberation as initial approach to model the phenomena.