Article

Covalent conjugation of polyethyleneimine on biodegradable microparticles for delivery of plasmid DNA vaccines

Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX 78712, USA.
Biomaterials (Impact Factor: 8.31). 12/2005; 26(32):6375-85. DOI: 10.1016/j.biomaterials.2005.03.043
Source: PubMed

ABSTRACT Microparticle-based delivery of nucleic acids has gained particular attention in recent years in view of improving the potency of DNA vaccination. Such improvement has been reported by encapsulation of pDNA within biodegradable microparticles or through surface adsorption on cationic microparticles. However, the intrinsic intracellular barriers for gene delivery to antigen presenting cells (APCs) have not been adequately addressed in the rational design of delivery systems for DNA vaccines. Here we report synthesis and characterization of biodegradable microparticles that (a) can passively target phagocytic APCs, (b) have intrinsic buffering ability that might allow for enhanced phagosomal escape, (c) are not cytotoxic and (d) have improved APC transfection efficiency. Branched polyethyleneimine (b-PEI) was covalently conjugated using carbodiimide chemistry to the surface of poly(lactide-coglycolide) (PLGA) microparticles to create cationic microparticles capable of simultaneously delivering both DNA vaccines as well as other immunomodulatory agents (cytokines or nucleic acids) within a single injectable delivery vehicle. Our results indicate that covalent conjugation of b-PEI allows efficient surface loading of nucleic acids, introduces intrinsic buffering properties to PLGA particles and enhances transfection of phagocytic cells without affecting the cytocompatibility of PLGA carriers.

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    • "Kasturi et al. found that pDNA encapsulated within PLGA leads to significant confinement of the complexes within endolysosomes, yet the application of chitosan to the surface of the NPs was projected to improve pDNA-NP release from the endolysosomes and offer a more protected path through the cytoplasm to the nucleus [18]. There are several reports on various aspects of chitosan- PLGA NPs for pDNA delivery, focusing on preparation methods, the final characteristics of synthesized NPs, and their behavior in vivo (i.e., cytotoxicity and transfection efficiency) [15–17, 19–21]. "
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    ABSTRACT: Poly(D,L-lactide-co-glycolide-) (PLGA-)chitosan nanoparticles are becoming an increasingly common choice for the delivery of nucleic acids to cells for various genetic manipulation techniques. These particles are biocompatible, with tunable size and surface properties, possessing an overall positive charge that promotes complex formation with negatively charged nucleic acids. This study examines properties of the PLGA-chitosan nanoparticle/plasmid DNA complex after formation. Specifically, the study aims to determine the optimal ratio of plasmid DNA:nanoparticles for nucleic acid delivery purposes and to elucidate the location of the pDNA within these complexes. Such characterization will be necessary for the adoption of these formulations in a clinical setting. The ability of PLGA-chitosan nanoparticles to form complexes with pDNA was evaluated by using the fluorescent intercalating due OliGreen to label free plasmid DNA. By monitoring the fluorescence at different plasmid: nanoparticle ratios, the ideal plasmid:nanoparticle ration for complete complexation of plasmid was determined to be 1:50. Surface-Enhanced Raman Spectroscopy and gel digest studies suggested that even at these optimal complexation ratios, a portion of the plasmid DNA was located on the outer complex surface. This knowledge will facilitate future investigations into the functionality of the system in vitro and in vivo.
    Journal of Nanomaterials 01/2011; 2011. DOI:10.1155/2011/952060 · 1.61 Impact Factor
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    • "Moreover, also the microsphere biocompatibility may be affected by the presence of the surfactant, thus leading to further criticism for future clinical development [27]. To overcome these problems, PLA or PLGA particles with charged groups covalently bound to the particle surface were developed by chemical modification of preformed particles or by using functional polymers or copolymers during particle synthesis [28] [29]. In addition, to avoid the still unresolved concerns about the presence of surfactants such as SDS or stabilizers such as PVA in microparticle formulations to be used in humans, new anionic surfactant-free nanoparticles containing only PLA polymers with a carboxylic end group were also developed and shown to be efficient vaccine delivery systems [30]. "
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    ABSTRACT: Anionic surfactant-free polymeric core-shell nanospheres and microspheres were previously described with an inner core constituted by poly(methylmethacrylate) (PMMA) and a highly hydrophilic outer shell composed of a hydrosoluble co-polymer (Eudragit L100-55). The outer shell is tightly linked to the core and bears carboxylic groups capable of adsorbing high amounts (antigen loading ability of up to 20%, w/w) of native basic proteins, mainly by electrostatic interactions, while preserving their activity. In the present study we have evaluated in mice the safety and immunogenicity of new vaccine formulations composed of these nano- and microspheres and the HIV-1 Tat protein. Vaccines were administered by different routes, including intramuscular, subcutaneous or intranasal and the results were compared to immunization with Tat alone or with Tat delivered with the alum adjuvant. The data demonstrate that the nano- and microspheres/Tat formulations are safe and induce robust and long-lasting cellular and humoral responses in mice after systemic and/or mucosal immunization. These delivery systems may have great potential for novel Tat protein-based vaccines against HIV-1 and hold promise for other protein-based vaccines.
    Vaccine 07/2009; 27(27):3605-15. DOI:10.1016/j.vaccine.2009.03.047 · 3.49 Impact Factor
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    • "Similarly, Elamanchili et al. reported that PLGA nanoparticles encapsulated with TLR 4 ligand, monophosphoryl lipid A, resulted in increased expression of stimulatory and co-stimulatory molecules on DCs and resulted in a robust Th1 type response [21] [22]. Additionally, antisense oligonucleotides specific for either co-stimulatory molecules or IL-10, loaded into PLGA MPs have been shown to be able to either induce immune suppression or direct specific Th-1 helper-type response respectively [22] [23]. Finally, in addition to directly loading antigenic proteins or peptides into particles, it has been demonstrated that delivery of DNA encoding for antigenic protein via PLGA MPs is a viable immunotherapeutic option [24] [25]. "
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    ABSTRACT: Immunogenomic approaches combined with advances in adjuvant immunology are guiding progress toward rational design of vaccines. Furthermore, drug delivery platforms (e.g., synthetic particles) are demonstrating promise for increasing vaccine efficacy. Currently there are scores of known antigenic epitopes and adjuvants, and numerous synthetic delivery systems accessible for formulation of vaccines for various applications. However, the lack of an efficient means to test immune cell responses to the abundant combinations available represents a significant blockade on the development of new vaccines. In order to overcome this barrier, we report fabrication of a new class of microarray consisting of antigen/adjuvant-loadable poly(D,L lactide-co-glycolide) microparticles (PLGA MPs), identified as a promising carrier for immunotherapeutics, which are co-localized with dendritic cells (DCs), key regulators of the immune system and prime targets for vaccines. The intention is to utilize this high-throughput platform to optimize particle-based vaccines designed to target DCs in vivo for immune system-related disorders, such as autoimmune diseases, cancer and infection. Fabrication of DC/MP arrays leverages the use of standard contact printing miniarraying equipment in conjunction with surface modification to achieve co-localization of particles/cells on isolated islands while providing background non-adhesive surfaces to prevent off-island cell migration. We optimized MP overspotting pin diameter, accounting for alignment error, to allow construction of large, high-fidelity arrays. Reproducible, quantitative delivery of as few as 16+/-2 MPs per spot was demonstrated and two-component MP dosing arrays were constructed, achieving MP delivery which was independent of formulation, with minimal cross-contamination. Furthermore, quantification of spotted, surface-adsorbed MP degradation was demonstrated, potentially useful for optimizing MP release properties. Finally, we demonstrate DC co-localization with PLGA MPs on isolated islands and that DCs do not migrate between islands for up to 24 h. Using this platform, we intend to analyze modulation of DC function by providing multi-parameter combinatorial cues in the form of proteins, peptides and other immuno-modulatory molecules encapsulated in or tethered on MPs. Critically, the miniaturization attained enables high-throughput investigation of rare cell populations by reducing the requirement for cells and reagents by many-fold, facilitating advances in personalized vaccines which target DCs in vivo.
    Biomaterials 06/2009; 30(25):4168-77. DOI:10.1016/j.biomaterials.2009.04.032 · 8.31 Impact Factor
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