Elsevier

Journal of Controlled Release

Volume 130, Issue 2, 10 September 2008, Pages 161-167
Journal of Controlled Release

Surface coating of PLGA microparticles with protamine enhances their immunological performance through facilitated phagocytosis

https://doi.org/10.1016/j.jconrel.2008.06.003Get rights and content

Abstract

Surface modifications of poly(lactide-co-glycolide) microparticles with different polycationic electrolytes have mainly been studied for conjugation to antigens and/or adjuvants. However, the in vivo immunological effects of using surface charged particles have not been address yet. In this study, microparticles were coated or not with protamine, a cationic and arginine-rich electrolyte that confers microparticles with a positively surface charge. We then evaluated the potential of protamine-coatings to assist the induction of immune responses in mice. Interestingly, enhanced antibodies and T-cell responses were observed in mice treated with the coated particles. In vitro studies suggested that the improved immunological performance was mediated by an increased uptake. Indeed, protamine-coated particles that carried a plasmid were even internalised into non-phagocytic cells and to cause their transfection. These results open the way for further research into a novel technology that combines the use protamine for facilitated cell penetration of that and biodegradable microparticles for prolonged antigen or drug release.

Introduction

Polymeric biodegradable microparticles have been widely studied for a broad variety of pharmaceutical and biomedical applications [1], [2], [3]. Commonly used poly(lactide-co-glycolide) (PLGA) types are available in medical grade and approved for use in humans, which makes them attractive for developing new drug and antigen delivery systems [4], [5], [6]. With a size range similar to that of microorganisms, polymeric particles can be easily taken up by antigen-presenting cells (APCs) [7], a process that enables potent cellular as well as humoral immune responses [8], [9], [10], [11], [12].

Physico-chemical properties such as the molecular weight and monomer composition of the polymer, and the size and surface charge of the microparticles determine the antigen release rate [12] and may also affect the type of immune response elicited by the delivery system [6], [13]. To add further flexibility and control of antigen and adjuvant delivery, the surface properties of PLGA microparticles have been modified [14], [15], [16]. Surface modifications have been achieved with anionic electrolytes such as sodium dioctylsulfosuccinate [17], [18] and cationic electrolytes such as chitosan, poly(ethylene imine), or protamine [14], [15], [16], [19]. Protamine belongs to a group of low molecular weight (MW: 4000–4250 Da), arginine-rich, basic proteins, which condense DNA in the nucleus and are involved in spermatogenesis [20]. Protamine is a FDA-approved compound, which has found applications in, e.g., stabilising DNA [21], insulin complexation and formulation, and in reverting the anticoagulant effect of heparin [22], [23], [24]. Protamine has been combined into PLGA microparticles [14], [26] and used as a coating agent to bind nucleic acid on the surface of controlled-release particles [27], and on one occasion, the use of protamine-containing PLGA microparticles in mice is reported [25].

The aim of this study was to evaluate the properties of protamine-coated PLGA microparticles with regard to particle uptake in cells, transfection of cells, antigen presentation, and the induction of T-cell and antibody responses in mice. Interestingly, the protamine coating enhanced particle uptake even by non-phagocytic cells and promoted the stimulation of much stronger immune responses as compared to uncoated particles.

Section snippets

Materials

Purified phospholipase A2 (PLA2) from bee venom and chicken egg albumin (OVA; grade V) were purchased from Sigma-Aldrich (Buchs, Switzerland), and soy bean lecithin (Epikuron 200) from Degussa (Hamburg, Germany). Phosphorothioate-modified CpG oligodeoxynucleotide 1668 pt (5′-TCC-ATG-ACG-TTC-CCT-GAC-GTT-3′) was synthesised by Microsynth (Balgach, Switzerland). The 35 kDa poly(lactide-co-glycolide) (PLGA 50:50) with uncapped end-groups (Resomer RG503H) was from Boehringer-Ingelheim (Ingelheim,

Microparticle characterisation

Three different groups of microparticles, all with and without protamine coating, were prepared (Table 1). The formulations with encapsulated pGFP, named pGFP-MP or pGFP-MP/protamine, had a very similar size distribution. Fifty percent of the particles were smaller than 3.2 μm or 3.5 μm, respectively. For both OVA- and PLA2-containing formulations, the size of the protamine-coated particles was larger than that of the non-coated particles. This was most evident for the OVA-containing

Discussion

Microparticles of PLGA have been widely studied for their use as drug and antigen delivery systems [3], [31], [6]. These particulate systems are able to deliver drugs or antigens such as DNA, proteins, and peptides over prolonged periods of time, and induce strong immune responses against encapsulated antigens [13], which makes them attractive candidates for vaccine development.

Stability issues with encapsulated biologicals have led to an alternative method for associating antigens and DNA with

Acknowledgements

The authors thank María J. Pena Rodríguez (Department of Dermatology, University Hospital Zurich) for technical assistance, Silvina Alejandra Bravo (Institute of Pharmaceutical Sciences, ETH Zurich) for helpful discussions, and Martin Bachmann (Cytos Biotechnology) and Tobias Suter (Institute of Clinical Immunology, University of Zurich) for providing the transgenic mice.

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