Versatility of biodegradable poly(d,l-lactic-co-glycolic acid) microspheres for plasmid DNA delivery

Abstract

In this study, we have optimized different formulations of DNA encapsulated into PLGA microspheres by correlating the protocol of preparation and the molecular weight and composition of the polymer, with the main characteristics of these systems in order to design an efficient non-viral gene delivery vector. For that, we prepared poly(d,l-lactic-co-glycolic acid) (PLGA) microparticles with an optimized water–oil–water double emulsion process, by using several types of polymers (RG502, RG503, RG504, RG502H and RG752), and characterized in terms of size, zeta potential, encapsulation efficiency (EE%), morphology, DNA conformation, release kinetics, plasmid integrity and erosion. The size of the particles ranged between 0.7 and 5.7 μm depending on the protocol of formulation and the molecular mass of the polymer used. The microspheres prepared by using in their formulation polymers of high molecular weight (RG503 and RG504) were bigger in size than in the case of using a lower molecular weight polymer (RG502). The EE (%) of plasmid DNA increased with increasing the molecular mass of the polymer and by using the most hydrophilic polymer RG502H, which contains terminal acidic groups in its structure. The plasmid could be encapsulated without compromising its structural and functional integrity. Also a protective effect of PLGA on endonuclease digestion is observed. Plasmid DNA release from microspheres composed of low molecular weight or hydrophilic polymers, like RG502H, was faster than from particles containing high molecular weight or hydrophobic polymers. These PLGA microspheres could be an alternative to the viral vectors used in gene therapy, given that may be used to deliver genes and other bioactive molecules, either very rapidly or in a controlled manner.

Introduction

Gene therapy is a form of molecular medicine that has the potential to influence significantly human health in this century. It promises to provide new treatments for a large number of inherited and acquired diseases. However, to achieve this goal, gene therapy requires technologies capable of gene transfer into a wide variety of cells, tissues and organs without causing cytotoxicity. Despite that naked DNA has been used successfully when injected locally, it is highly prone to tissue clearance and totally inefficient for systemic delivery. Because of that, it is important to design appropriate DNA carriers, which protect the plasmid from degradation and allow efficient gene delivery.

The molecular form of the DNA has been reported to affect the efficiency with which the pDNA will transfect cells. Linear pDNA is much less efficient in transfection than open circle or supercoiled DNA, and there is little difference between the supercoiled and open circle forms [1], [2]. Therefore, it is desirable to have the plasmid DNA encapsulation conditions optimized for minimal effect on the supercoiled DNA topology. In this respect, it is important to note that the double emulsion technique commonly used for DNA microencapsulation utilizes shear forces typically thought to be harmful to the structural integrity and expression capacity of plasmid DNA. This effect has been studied by different authors [3], [4], [5].

There are two main groups of vectors used in gene delivery: viral and non-viral vectors. Toxicity and immunogenicity concerns associated with viral vectors have led to an active interest in non-viral systems for gene delivery [6], [7], such as liposomes, cationic blok copolymers, polymer complexes and polymeric micro- and nanoparticles, because they are relatively safe and are easy to formulate [8], [9]. Specifically, much attention has been focused on biocompatible, biodegradable polymers, such as PLGA, for the encapsulation of genes [10], [11], [12], [13], [14], [15]. Also, chitosan [16], [17] and gelatin [18] have been used to encapsulate pDNA. On the other hand, the composition and molecular weight of the polymers not only determines the release pattern of encapsulated material, but it is suspected to also considerably affect the hydrophobicity of the resulting microspheres and therefore may influence the behavior [12], [19]. It should be also considered that, although controlled release polymeric systems offer the advantage of sustained pDNA activity [20], in some cases a rapid release of DNA is of interest. For example, it is known that the ability of pDNA to induce an immune response in vivo will decrease as a function of the time of release.

Taking all this into account, the aim of this study was to optimize different formulations of PLGA/DNA microspheres to be used as an efficient non-viral gene delivery system, by correlating the properties of the microparticles with their capabilities to encapsulate and release DNA. For that, microspheres were prepared and characterized in the presence of different PLGA polymers (RG502, RG503, RG504, RG502H and RG752), by using two protocols of formulation.