Poly(lactic acid)-poly(ethylene glycol) nanoparticles as new carriers for the delivery of plasmid DNA

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Abstract

The purpose of the present work was to produce and characterize poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles (size lower than 300 nm) containing a high loading of plasmid DNA in a free form or co-encapsulated with either poly(vinyl alcohol) (PVA) or poly(vinylpyrrolidone) (PVP). The plasmid alone or with PVA or PVP was encapsulated by two different techniques: an optimized w/o/w emulsion-solvent evaporation technique as well as by a new w/o emulsion-solvent diffusion technique. Particle size, ζ potential, plasmid DNA loading and in vitro release were determined for the three plasmid-loaded formulations. The influence of the initial plasmid loadings (5, 10, 20 μg plasmid DNA/mg PLA-PEG) on those parameters was also investigated. The plasmid loaded into the nanoparticles and released in vitro was quantified by fluorimetry and the different molecular forms were identified by gel electrophoresis. PLA-PEG nanoparticles containing plasmid DNA in a free form or co-encapsulated with PVA or PVP were obtained in the range size of 150–300 nm and with a negative ζ potential, both parameters being affected by the preparation technique. Encapsulation efficiencies were high irrespective of the presence of PVA or PVP (60–90%) and were slightly affected by the preparation technique and by the initial loading. The final plasmid DNA loading in the nanoparticles was up to 10–12 μg plasmid DNA/mg polymer. Plasmid DNA release kinetics varied depending on the plasmid incorporation technique: nanoparticles prepared by the w/o diffusion technique released their content rapidly whereas those obtained by the w/o/w showed an initial burst followed by a slow release for at least 28 days. No significant influence of the plasmid DNA loading and of the co-encapsulation of PVP or PVA on the in vitro release rate was observed. In all cases the conversion of the supercoiled form to the open circular and linear forms was detected. In conclusion, plasmid DNA can be very efficiently encapsulated, either in a free form or in combination with PVP and PVA, into PLA-PEG nanoparticles. Additionally, depending on the processing conditions, these nanoparticles release plasmid DNA either very rapidly or in a controlled manner.

Introduction

At present, it is broadly accepted that plasmid DNA has potential not only as a therapeutic agent but also as a new vaccination approach [1]. However, as research in this field advances, it becomes evident that the use of plasmid DNA-based pharmaceuticals as drugs and vaccines will largely depend on the development of efficient delivery systems [2]. Naked plasmid DNA is highly susceptible to nuclease degradation and, therefore, is rapidly cleared from the bloodstream when injected intravenously [3]. Additionally, because of its large size and negative charge plasmid DNA is not able to enter cells and, hence, not able to cross epithelia. An exception is represented by muscle and epidermal cells, which have been shown to produce detectable levels of gene expression following intramuscular injection or by gene gun delivery [4]. Nevertheless, the access of plasmids to other population of cells is highly restricted. For example, in the case of a DNA vaccine, it would be critical to target the plasmid to the antigen presenting cells (APCs) in order to generate a T-cell response. These cells are in the blood lymphocyte pool and particularly concentrated in the sub-mucosal tissues. Therefore, the design of a carrier appropriately tailored for delivering plasmid DNA to the APCs, following mucosal administration, appears to be a very promising but challenging approach towards improving DNA vaccination. In addition, the possibility to provide a long-term sustained delivery of plasmid DNA would represent a major advance for the use of DNA vaccines.

One strategy to achieve targeted and controlled delivery of DNA vaccines has been based on the use of biodegradable polymer particles. Interesting results were found following oral administration of mucoadhesive particles such as polyanhydride microspheres [5] and chitosan nanoparticles [6]. In both cases, the model plasmids associated to the particles elicited significant levels of transfection. However, probably because of their safety profile, microspheres made of PLGA are those which have received the greatest deal of attention over the last few years [7]. Research in this area has also been encouraged by the protective humoral and mucosal immune responses obtained following oral administration of plasmid DNA encapsulated in PLGA microspheres [8] and also by the evidence of the ability of these plasmid containing particles to elicit T-cell responses [9], [10].

Because of their extremely large size and polar character, the encapsulation of plasmid DNA molecules within hydrophobic PLGA microspheres appears to be a challenge for researchers interested in the formulation of delicate macromolecules. One of the approaches to associate plasmid DNA to PLA/PLGA nano/microparticles has focused on the use of PLA grafted with cationic polysaccharides or cationic surfactants which provide a positive charge to the particles, thereby facilitating the adsorption of the negatively charged plasmid DNA molecules [10], [11]. Nevertheless, in order to achieve a better protection of the plasmid and a more precise control of the release process, attention has been specially addressed to the encapsulation of the plasmid within PLGA microspheres. Using three model plasmids, Luo et al. showed that prolonged plasmid DNA release could be achieved by adjusting the polymer molecular weight and composition of PLA/PLGA microspheres [12]. A limitation of some of these microspheres with respect to their utility for vaccine delivery is their large size (100 μm). In fact, is has been generally accepted that only particles of a size smaller than 10 μm are able to cross mucosal surfaces and enter the APCs, significantly. Successful approaches for encapsulating plasmid DNA within PLGA microspheres have been the spray-drying [13] and the water–oil–water solvent evaporation techniques [12], [14]. However, what these authors discovered while developing these microspheres is that the encapsulated plasmid is degraded not only during the encapsulation process but mainly in the course of the polymer degradation [13], [14]. The inactivation of the plasmid during encapsulation was addressed by Ando et al. [15] who proposed a cryopreparation technique in order to prevent the exposure of the plasmid to shear forces created during the homogenization process. Nevertheless, the stabilization of the plasmid in a long-term release process requires further investigation. A possibility could rely on the encapsulation of plasmids previously complexed with other hydrophilic polymers. For example, Capan et al. observed that the complexation of the plasmid with poly(l-lysine) (PLL) improved its encapsulation and release in the supercoiled form [16], [17]. However, a limitation of these small microspheres was their low encapsulation efficiency and, hence, their low final loading.

In the present work, taking advantage of our experience on the encapsulation and stabilization of proteins in PLGA and PLA-PEG micro/nanoparticles, we attempt to explore the potential of PLA-PEG nanoparticles as carriers for the controlled delivery of plasmid DNA. The selection of this carrier was based on its capacity to encapsulate antigens efficiently, i.e., tetanus toxoid, and to transport them across mucosal surfaces, after either nasal [18] or oral administration [19]. Furthermore, recent studies have revealed their ability to elicit long lasting humoral and mucosal immune responses following nasal immunization [20]. Based on this information, the goal of this study is to evaluate the potential of these nanoparticles as carriers for the controlled release of plasmid DNA, using pCMVLuc as a model plasmid. From a practical standpoint, the objectives are: (i) to encapsulate a high amount of plasmid DNA in a free form or in combination with PVA or PVP within very small PEG-coated PLA nanoparticles (ii) to evaluate the controlled release properties of this new plasmid DNA delivery device.

Section snippets

Materials

Methoxy PEG-(d,l-lactide) (PLA-PEG) with a PLA and PEG molecular weight (Mw) of 46 and 5 kDa, respectively, was purchased from Alkermes (Cincinnati, OH). Luciferase reporter plasmid (pBK-Luc) driven by the CMV promoter was a kind gift from Zycos, (Cambridge, MA). Plasmid was harvested from STBL cells by ion-exchange chromatography (Qiagen, Chatsworth, CA). One-kb DNA ladder was obtained from Life Technologies (Barcelona, Spain). PicoGreen® quantitation kit was acquired from Molecular Probes

Results and discussion

Several articles published recently have shown the potential value of PLGA microspheres as plasmid DNA controlled release systems [7], [12], [13], [16], [17], [24]. Interestingly, our previous attempts to create new mucosal vaccine carriers led us to consider that nanoparticles may be preferable to microparticles, and also that a PEG coating around the nanoparticles has a positive effect on their interaction with mucosal surfaces [18], [19]. Besides the importance of the particle size and the

Conclusions

We have adopted encapsulation approaches that allowed us to produce PLA-PEG nanoparticles containing high loadings of plasmids. The plasmids could be encapsulated equally efficiently either alone or in combination with PVP or PVA. Depending on the processing conditions these nanoparticles released the encapsulated plasmid either very rapidly or in a long-term continuous fashion. Despite the mild encapsulation conditions, the supercoiled plasmid was converted into open circular and linear forms.

Acknowledgements

This work has been supported by grants from the Fullbright Commission (Ref. 99112) and the Xunta de Galicia (Ref. PGIDT00BIO20301PR) (Spain).

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