Elsevier

Biomaterials

Volume 29, Issue 18, June 2008, Pages 2783-2793
Biomaterials

Enhancement of poly(orthoester) microspheres for DNA vaccine delivery by blending with poly(ethylenimine)

https://doi.org/10.1016/j.biomaterials.2008.03.011Get rights and content

Abstract

Poly(orthoester) (POE) microspheres have been previously shown to possess certain advantages for the in vivo delivery of DNA vaccines. In particular, timing of DNA release from POE microspheres in response to acidic phagosomal pH was shown to be an important factor in determining immunogenicity, which was hypothesized to be linked to the natural progression of antigen-presenting cell uptake, transfection, maturation, and antigen presentation. Here we report in vitro characterization of the enhanced efficacy of POE microspheres by blending poly(ethylenimine) (PEI), a well-characterized cationic transfection agent, into the POE matrix. Blending of a tiny amount of PEI (approximately 0.04 wt%) with POE caused large alterations in POE microsphere properties. PEI provided greater control over the rate of pH-triggered DNA release by doubling the total release time of plasmid DNA and enhanced gene transfection efficiency of the microspheres up to 50-fold without any significant cytotoxicity. Confocal microscopy results of labeled PEI and DNA plasmids revealed that PEI caused a surface-localizing distribution of DNA and PEI within the POE microsphere as well as focal co-localization of PEI with DNA. We provide evidence that upon degradation, the microspheres of POE–PEI blends released electrostatic complexes of DNA and PEI, which are responsible for the enhanced gene transfection. Furthermore, blending PEI into the POE microsphere induced 50–60% greater phenotypic maturation and activation of bone marrow-derived dendritic cells in vitro, judged by the up-regulation of co-stimulatory markers on the cell surface. Physically blending PEI with POE is a simple approach for modulating the properties of biodegradable microspheres in terms of gene transfection efficiency and DNA release kinetics. Combined with the ability to induce maturation of antigen-presenting cells, POE–PEI blended microspheres may be excellent carriers for DNA vaccines.

Introduction

Whereas traditional vaccination strategies entail direct administration of an antigen, DNA vaccines are composed of a plasmid carrying a gene for the desired antigen. Plasmid-based vaccination can safely activate both the humoral and cell-mediated immune response pathways leading to antibody and cytotoxic T-lymphocyte (CTL) responses as well as strong memory induction [1]. Additionally, DNA vaccines allow for both prophylactic and therapeutic vaccination strategies and have been targeted against infectious agents, various cancers, and other diseases related to immune dysfunction [2]. An amazing variety of materials, physical formulations, and vector design strategies exist for the development of non-viral delivery systems for nucleic acid-based therapeutics including DNA vaccines. Non-viral strategies for plasmid transfection have included electroporation and physical methods such as ballistic “gene gun” [3], microparticles such as PLGA microspheres [4], nanoparticle formulations in liposomes [5] or polymer nanoparticles [6], [7], and recently, polyelectrolyte multilayered materials [8], [9]. However, very few non-viral vectors have progressed to the stage of clinical trials [10]. The poor in vivo performance of non-viral delivery systems has been generally attributed to poor transfection efficiency leading to massive dosing requirements, lack of targeting, toxicity, and non-specific effects [6], [10], [11].

Polymer microspheres, mostly based on poly(lactic-co-glycolic acid) (PLGA), have been developed to protect plasmids from degradation, control DNA release, and target phagocytic antigen-presenting cells (APCs) on the basis of micrometer (1–10 μm) size [12], [13], [14]. However, these formulations often suffer from low gene transfection efficiency [4]. Efforts to increase the transfection efficiency of PLGA have mainly focused on introducing modifications with cationic surfactants such as cetyl-trimethyl-ammonium-bromide (CTAB) [15] and cationic polymers such as PEI (polyethylenimine) [16], [17], [18], and PBAE (poly(beta-aminoesters)) [19], [20]. In these reports, incorporation of the cationic molecules increased transfection efficiency and boosted immune responses. Yet the use of PLGA microspheres for gene delivery is still not optimal due to low DNA loading [12], undesirably slow release rates [21], [22], and DNA-damaging acidic degradation by-products [23], [24].

Poly(orthoester)s (POEs) are a class of pH-sensitive polymeric materials originally developed in 1970s to be biodegradable and non-toxic alternatives to poly(lactic acid) and poly(glycolic acid) for polymer-mediated drug delivery [25]. We previously reported the development of two 4th-generation POE polymer specifically designed for DNA delivery [26], herein referred to as P1 and P2. Both polymers have similar backbone components to achieve similar polymer physical properties (rigidity, hydrophilicity, latent acid content), but in addition, P2 incorporates tertiary amines in the backbone. These polymers functioned as effective DNA vaccine carriers in vivo. Antibodies to the model antigen β-galactosidase were induced in a gene-specific manner. Furthermore, activation of cytotoxic T-lymphocytes (CTLs) was gene-specific, induced memory responses, and resulted in tumor regression when mice were vaccinated against the model SIY antigen and then challenged with an SIY-bearing syngeneic tumor. The potential of these POE microspheres to stimulate the immune system was indeed polymer-specific. The P1 polymer, which did not contain tertiary amine groups, did not achieve immune responses as robust as the amine-containing P2 polymer. We hypothesized that this disparate immune function was correlated to the timing of DNA release. The addition of a tertiary amine in the polymer backbone, which would be protonated and charged at the acidic pH of the late endosome, slowed the release of DNA from the degrading matrix. We proposed a model that immune responses will be enhanced if DNA release and gene expression are more closely matched to the natural progression of APC migration to the draining lymph nodes, APC maturation, and antigen presentation.

Here we hypothesized that the blending of a cationic polymer into the POE microsphere matrix could further enhance DNA vaccine delivery by POE microspheres. PEI is a well-characterized, commercially available, cationic transfection reagent that forms stable polyplexes via electrostatic interaction with DNA and exhibits very high transfection efficiency [27], [28]. In this paper, we hypothesized that blending limited amounts of PEI into the POE matrix would increase DNA loading, increase transfection efficiency, and modulate release kinetics to be better tuned with the natural progression of immune responses. Furthermore, we investigated the ability of POE and POE–PEI blends to induce APC maturation.

Section snippets

Materials

POE was provided as a gift by AP Pharma (Redwood, CA) as previously characterized [26]. Poly(lactic-co-glycolic acid) acid (PLGA) (Resomer RG503) was from Boehringer Ingelheim Chemicals with a 50:50 ratio of lactide:glycolide and molecular weight of ∼35 kD. Branched PEI of 25 kD was purchased from Sigma. Poly(vinyl alcohol) (PVA) of ∼25 kD was purchased from Polysciences, Inc. Heparin sodium was purchased from Celsus Laboratories. Endotoxin-free plasmid DNA was expanded and purified by Elim

Microsphere formulation and physical properties

Physical properties of representative batches of microspheres are summarized in Table 1. PLGA microspheres were formulated for comparison to POE microspheres. Blending PEI into the POE or PLGA matrix achieved higher (more positive) zeta-potentials indicating the presence of PEI at the surface of the microsphere. The surface charge was further increased by the inclusion of DNA in the microsphere. Total yields of DNA were typically between 60% and 70%. All POE microspheres exhibited similar

Discussion

The POE polymer is acid-labile at the orthoester linkage, and the inclusion of latent acid monomers in the POE chain provides a self-catalyzing degradation mechanism [25]. This pH-sensitive character of POEs is advantageous for gene delivery to phagocytic APCs such as macrophages and dendritic cells, where upon acidification in the late phagosome POE particles should rapidly degrade and release their cargo. Our previous work indicated a role for timing of DNA release in the overall

Conclusion

POE microspheres have already been shown to be a promising means for the purpose of delivering DNA vaccines in a mouse model [26]. The methods described herein, for blending PEI with a POE matrix can serve multiple purposes in the goal of increasing the immunogenicity of POE-based microspheres for DNA vaccines. PEI slows the release rate of DNA, which could synchronize gene transfection with the natural progression of immune responses. PEI increases POE microsphere gene transfection efficiency

Acknowledgements

The authors would like to acknowledge support from NIH Grant # EB000244, the NSF Graduate Research Fellowship, and the Whitaker Foundation Graduate Research Fellowship.

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