Research paperVersatility of biodegradable poly(d,l-lactic-co-glycolic acid) microspheres for plasmid DNA delivery
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.
Section snippets
Materials
Poly(d,l-lactic-co-glycolic acid) (PLGA) polymers of different molecular weight and co-polymerization rate (lactic:glycolic), Resomer® RG502 (Mw 12,000 Da, 50:50), Resomer® RG503 (Mw 34,000 Da, 50:50), Resomer® RG504 (Mw 65,000 Da, 50:50), Resomer® RG502H (Mw 12,000 Da, 50:50) and Resomer® RG752 (Mw 12,000 Da, 75:25), were purchased from Boehringer Ingelheim (Germany). The plasmid pCMVLuc (BioServe Biotechnologies, USA) encoding for luciferase and the lipids 1,2-dioleoyl-3-(trimethylammonium)
Physical characterization of microparticles
DNA-loaded microparticles by using polymers of different composition and molecular weights (RG502, RG503, RG504, RG752 and RG502H) were formulated and characterized for physical properties, such as particle size and surface charge. The influence of the method of formulation, by changing the homogenization rate in the preparation of the second emulsion, as described in Section 2, was also analyzed.
As shown in Table 1, particles prepared by protocol 2 (high homogenization rate) resulted to be
Discussion
In this study, we have investigated the feasibility of DNA encapsulation and release in PLGA microparticles, formulated with two protocols in the presence of different PLGA polymers (RG502, RG503, RG504, RG502H and RG752). Following preparation of the PLGA–DNA microspheres, by using a double emulsion technique, the particles were characterized in terms of size, zeta potential, encapsulation efficiency, morphology, DNA conformation, release kinetics, integrity and erosion.
As shown in Table 1,
Acknowledgements
We acknowledge Dr. R. Jordana for the help with the morphological studies. This work was financially supported by the University of Navarra Foundation (FUN), the Government of Navarra (Department of Education), the “Ministerio de Sanidad y Consumo”, Instituto de Salud Carlos III (Grant RTI Cancer C03/10) and the Roviralta Foundation.
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2019, Regenerative TherapyCitation Excerpt :However, they are required to exclude after the complete release of incorporated drug. On the other hand, various biodegradable polymers have been used as carriers for the controlled release of nucleic acids, such as poly(lactic acid) [67], polyanhydride [68], poly(orthoester) [69], PLGA [70–80], poly(d,l-lactide-co-4-hydroxy-l-proline) [81], poly(1,8-octanediol-co-citrate) [82], oligo(poly(ethylene glycol) fumarate) [83], poly(2-aminoethyl propylene phosphate) [84], polypseudorotaxane [85], polysaccharide [86], silk elastin-like polymer [87], and atelocollagen [88–90]. It has been reported that some ceramics, such as calcium phosphate and calcium carbonates, are used for the controlled release of nucleic acid [91,92].
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