Elsevier

Biomaterials

Volume 31, Issue 14, May 2010, Pages 4214-4222
Biomaterials

Gene delivery using dimethyldidodecylammonium bromide-coated PLGA nanoparticles

https://doi.org/10.1016/j.biomaterials.2010.01.143Get rights and content

Abstract

In this present work we describe a poly(lactic-co-glycolic acid) (PLGA) nanoparticle formulation for intracellular delivery of plasmid DNA. This formulation was developed to encapsulate DNA within PLGA nanoparticles that combined salting out and emulsion–evaporation processes. This process reduced the requirement for sonication which can induce degradation of the DNA. A monodispersed nanoparticle population with a mean diameter of approximately 240 nm was produced, entrapping a model plasmid DNA in both supercoiled and open circular structures. To induce endosomal escape of the nanoparticles, a superficial cationic charge was introduced using positively charged surfactants cetyl trimethylammonium bromide (CTAB) and dimethyldidodecylammonium bromide (DMAB), which resulted in elevated zeta potentials. As expected, both cationic coatings reduced cell viability, but at equivalent positive zeta potentials, the DMAB coated nanoparticles induced significantly less cytotoxicity than those coated with CTAB. Fluorescence and transmission electron microscopy demonstrated that the DMAB coated cationic nanoparticles were able to evade the endosomal lumen and localise in the cytosol of treated cells. Consequently, DMAB coated PLGA nanoparticles loaded with a GFP reporter plasmid exhibited significant improvements in transfection efficiencies with comparison to non-modified particles, highlighting their functional usefulness. These nanoparticles may be useful in delivery of gene therapies to targeted cells.

Introduction

The application of nucleic acids as therapeutic agents for gene therapy has been extensively studied in a broad range of diseases [1], [2], [3], [4]. However, a recurrent limitation in these therapies is the efficient delivery of the therapeutic DNA to the disease site. To address this issue, various strategies have been examined including vectors engineered from adeno- or adeno-associated viruses [5], but clinical trials have demonstrated substantial obstacles to their use, such as immunogenicity and inflammatory potential [6].

An alternative strategy is the application of non-viral gene delivery vectors, including liposomes [7], dendrimers [8], polycationic polymers [9], [10] and polymeric nanoparticles (NP) [11] to reduce or avoid immunogenicity and associated risks of toxicity [12]. A frequently employed biodegradable polymer in NP formulation is poly(lactic-co-glycolic acid) (PLGA). PLGA NP have shown particular promise in the delivery of a range of drug molecules to disease sites to improve efficacies [13], [14], [15], [16]. Moreover, the non-toxic biodegradability of PLGA has resulted in FDA approval for the application of this polymer in various medicinal products [17].

One approach in the application of PLGA NP for nucleic acid delivery uses adsorption of the anionic DNA molecules onto cationic NP [18], [19]. Despite this efficient formulation procedure, the peripheral exposure of the labile DNA limits stability and shortens its half-life, particularly in the acidic environment found within late endosomes where the particles will accumulate upon internalisation [20], [21]. Therefore, formulations that can encapsulate and protect the DNA from degradation are attractive for gene therapy approaches.

In addition to the protection of the DNA cargo, the physical characteristics of the nanoparticles can be manipulated to escape the degradative endosomal lumen, resulting in cytosolic localisation. Various strategies have been used to promote this sub-cellular relocalisation including application of fusogenic peptides [22], proton sponge polymers [23], light excitation [24] and cationic coating [18], [19], [25], [26]. It is thought that the presence of a cationic surface charge promotes interaction and binding of the NP to the endosomal membrane, inducing membrane destabilisation and cytosolic relocalisation of the NP [27].On this basis, the objective of this current study was to develop a PLGA NP formulation that would combine the ability to produce NP encapsulating DNA with the capacity to evade endosomal degradation.

Section snippets

Materials

PLGA (Resomer RG 502 H) with an acid value of 9 mg KOH per g PLGA, molecular weight 12 kDa, was a generous gift from Boehringer Ingelheim, Germany. Poly(vinyl alcohol) (PVA), 87–89% hydrolysed with molecular weight 13–23 kDa, and dimethyldidodecylammonium bromide (DMAB) were obtained from Sigma Aldrich, Germany. Magnesium chloride hexahydrate (MgCl2·6H2O), sodium bicarbonate NaHCO3, Tris-EDTA buffer and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT reagent) were obtained

Nanoparticle formulation

Upon internalisation by endocytosis, NP preferentially localise to late endosomes [20]. This localisation can be destructive to cargo DNA, particularly if adsorbed to the surface of cationic NP. Premature dissociation of surface-adsorbed DNA from NP in endosomal compartments following cellular internalisation has previously been observed [21], where degradation occurs rapidly in the acidified environment. Thus, the ability to entrap the nucleic acid inside the PLGA NP is attractive in order to

Conclusion

In summary, the application of a salting out and emulsion–evaporation process has successfully resulted in the formulation of 240 nm DNA-loaded NP. The introduction of a cationic surfactant (DMAB) has facilitated the endosomal escape of endocytosed NP, thus preventing premature degradation of the transfected DNA. This effect was exemplified by the observation of improved transfection efficiencies over anionic NP carrying a reporter plasmid. These DNA delivery modalities require approximately

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