In vitro and in vivo evaluation of a somatostatin analogue released from PLGA microspheres

The purpose of this study was to design poly(lactide-co-glycolide) (PLGA) microspheres for the continuous delivery of the somatostatin analogue, vapreotide, over 2–4 weeks. The microspheres were produced by spray-drying and the desired characteristics, i.e. high encapsulation efficiency and controlled release over 2–4 weeks, achieved through optimizing the type of polymer, processing solvent, and co-encapsulated additive. The in vitro release was tested in fetal bovine serum preserved with 0.02% of thiomersal. Furthermore, formulations were injected intramuscularly into rats to obtain pharmacokinetic profiles. Encapsulation efficiency was between 34 and 91%, depending on the particular formulation. The initial peptide release (within 6 h) was lowest, i.e. 1 ng/ml) over 21–28 days in rats was the one made with end-group uncapped PLGA 50:50, the solvent acetic acid and the additive polyethyleneglycol. In conclusion, the optimization of formulation parameters allowed us to produce vapreotide-loaded PLGA microspheres of suitable characteristics for therapeutic use

Particulate formulations for the delivery of poly(I:C) as vaccine adjuvant.

Current research and development of antigens for vaccination often center on purified recombinant proteins, viral subunits, synthetic oligopeptides or oligosaccharides, most of them suffering from being poorly immunogenic and subject to degradation. Hence, they call for efficient delivery systems and potent immunostimulants, jointly denoted as adjuvants. Particulate delivery systems like emulsions, liposomes, nanoparticles and microspheres may provide protection from degradation and facilitate the co-formulation of both the antigen and the immunostimulant. Synthetic double-stranded (ds) RNA, such as polyriboinosinic acid-polyribocytidylic acid, poly(I:C), is a mimic of viral dsRNA and, as such, a promising immunostimulant candidate for vaccines directed against intracellular pathogens. Poly(I:C) signaling is primarily dependent on Toll-like receptor 3 (TLR3), and on melanoma differentiation-associated gene-5 (MDA-5), and strongly drives cell-mediated immunity and a potent type I interferon response. However, stability and toxicity issues so far prevented the clinical application of dsRNAs as they undergo rapid enzymatic degradation and bear the potential to trigger undue immune stimulation as well as autoimmune disorders. This review addresses these concerns and suggests strategies to improve the safety and efficacy of immunostimulatory dsRNA formulations. The focus is on technological means required to lower the necessary dosage of poly(I:C), to target surface-modified microspheres passively or actively to antigen-presenting cells (APCs), to control their interaction with non-professional phagocytes and to modulate the resulting cytokine secretion profile

Modulation of allergic responses in mice by using biodegradable poly(lactide-co-glycolide) microspheres.

BACKGROUND: Biodegradable poly(lactide- co -glycolide) (PLGA) microspheres are a promising carrier for vaccine delivery capable of maturing antigen-presenting cells to stimulate T-cell-mediated immune responses. However, the potential of microspheres to downregulate an allergic response in vivo is unknown. OBJECTIVE: The aim of this study was to determine whether microspheres could potentiate DNA vaccination against allergy and to evaluate the immunomodulatory properties of microspheres alone. METHODS: Mice were treated prophylactically with DNA-loaded plain PLGA microspheres before sensitization with phospholipase A2 (PLA2), the major allergen of bee venom. PLA2-specific IgG1, IgG2a, IgE in serum were measured for 8.5 months, and splenocyte proliferative responses and cytokine profiles were determined. Protection against anaphylaxis was evaluated after injection of an otherwise lethal dose of PLA2. RESULTS: Phospholipase A2-specific IgG1 and IgG2a production turned out to be 2 times higher using cationic microspheres compared with anionic microspheres, but was not influenced by the presence of DNA. In contrast, reduction in IgE production and T-cell hyporesponsiveness were observed with all microsphere formulations. Recall challenge with PLA2 triggered combined expression of both IL-4 and IFN-gamma, together with sustained expression of IL-10 that can explain the protective effect against anaphylaxis. CONCLUSION: Our data suggest a dual mechanism that does initially rely on a TH2 to TH1 immune deviation and then on IL-10-mediated suppression. This is the first physiological demonstration that plain PLGA microspheres can induce tolerance in mice for as long as 6 months postsensitization

Surface-assembled poly(I:C) on PEGylated PLGA microspheres as vaccine adjuvant: APC activation and bystander cell stimulation.

Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres are potential vehicles to deliver antigens for vaccination. Because they lack the full capacity to activate professional antigen presenting cells (APCs), combination with an immunostimulatory adjuvant may be considered. A candidate is the synthetic TLR3 ligand polyriboinosinic acid-polyribocytidylic acid, poly(I:C), which drives cell-mediated immunity. However, poly(I:C) has also been linked to the pathogenesis of autoimmunity, as affected by widespread stimulation of non-hematopoietic bystander cells. To address this aspect, we propose to minimize the poly(I:C) dose as well as to control the stimulation of non-immune bystander cells by poly(I:C). To facilitate the maturation of APCs with minimal poly(I:C) doses, we surface-assembled poly(I:C) onto PLGA microspheres. The microspheres' surface was further modified by poly(ethylene glycol) (PEG) coronas with varying PEG-densities. PLGA microspheres loaded with tetanus toxoid (tt) as model antigen were manufactured by microextrusion-based solvent extraction. The negatively charged PLGA(tt) microspheres were coated with polycationic poly(l-lysine) (PLL) polymers, either PLL itself or PEG-grafted PLL (PLL-g-PEG) with varying grafting ratios (g=2.2 and g=10.1). Stable surface assembly of poly(I:C) was achieved by subsequent incubation of polymer-coated PLGA microspheres with aqueous poly(I:C) solutions. We evaluated the immunostimulatory potential of such PLGA(tt) microsphere formulations on monocyte-derived dendritic cells (MoDCs) as well as human foreskin fibroblasts (HFFs) as model for non-hematopoietic bystander cells. Formulations with surface-assembled poly(I:C) readily activated MoDCs with respect to the expression of maturation-related surface markers, proinflammatory cytokine secretion and directed migration. When surface-assembled, poly(I:C) enhanced its immunostimulatory activity by more than one order of magnitude as compared to free poly(I:C). On fibroblasts, surface-assembled poly(I:C) upregulated class I MHC but not class II MHC. Phagocytosis of PLGA(tt) microsphere formulations by MoDCs and HFFs remained mostly unaffected by PEG-grafted PLL coatings. In contrast, high concentrations of free poly(I:C) led to a marked drop of microsphere phagocytosis by HFFs. Overall, surface assembly on PEGylated PLGA microspheres holds promise to improve both efficacy and safety of poly(I:C) as vaccine adjuvant

Tuning the immune response of dendritic cells to surface-assembled poly(I:C) on microspheres through synergistic interactions between phagocytic and TLR3 signaling.

The artificial dsRNA polyriboinosinic acid-polyribocytidylic acid, poly(I:C), is a potent adjuvant candidate for vaccination, as it strongly drives cell-mediated immunity. However, because of its effects on non-immune bystander cells, poly(I:C) administration may bear danger for the development of autoimmune diseases. Thus poly(I:C) should be applied in the lowest dose possible. We investigated microspheres carrying surface-assembled poly(I:C) as a two-in-one adjuvant formulation to stimulate maturation of monocyte-derived dendritic cells (MoDCs). Negatively charged polystyrene microspheres were equipped with a poly(ethylene glycol) corona through electrostatically driven surface assembly of a library of polycationic poly(l-lysine)-graft-poly(ethylene glycol) copolymers, PLL-g-PEG. Stable surface assembly of poly(I:C) was achieved by incubation of polymer-coated microspheres in an aqueous poly(I:C) solution. Surface-assembled poly(I:C) exhibited a strongly enhanced efficacy to stimulate maturation of MoDCs by up to two orders of magnitude, as compared to free poly(I:C). Multiple phagocytosis events were the key factor to enhance the efficacy. The cytokine secretion pattern of MoDCs after exposure to surface-assembled poly(I:C) differed from that of free poly(I:C), while their ability to stimulate T cell proliferation was similar. Overall, phagocytic signaling plays an important role in defining the resulting immune response to such two-in-one adjuvant formulations

Release of tetanus toxoid from adjuvants and PLGA microspheres: how experimental set-up and surface adsorption fool the pattern.

The classical adjuvants alum and Freund's Incomplete Adjuvant (IFA) are frequently used as references for the design of new adjuvants and antigen delivery systems, e.g., microspheres (MS). Poly(dl-lactic-co-glycolic acid) (PLGA) MS have been proposed for delivering antigen booster doses in vivo after a single injection. However, as antigen release kinetics from conventional adjuvants are generally unknown, it appears presumptuous to propose a desired antigen release pattern from PLGA MS. Therefore, we have studied the tetanus toxoid (Ttxd) in vitro release from alum, IFA formulations and MS in four different test systems. The results showed a stronger Ttxd association to alum than to IFA, and the release from both formulations lasted between 3-9 days. The total of ELISA-responsive antigen released was 60-85% of the actual dose. Both the total amount and the prolongation of release depended on the Ttxd dose. Furthermore, the incomplete in vitro release of Ttxd from the adjuvants and also from PLGA 50:50 MS was shown to be partly due to experimental conditions. Typically, Ttxd adsorbed on the glass vials used for the release test and also on the surface of the PLGA 50:50 MS, wherefrom it was released. In conclusion, the test system depending rate and quantity of release observed evidence the limitations of in vitro release data. Finally, for mimicking conventional vaccination schedules, i.e. injections typically at time points 0, 1, 3, and 12-24 months, PLGA MS should release antigen doses at the corresponding time points, and the release pulse should only last for a few days