Article pubs.acs.org/molecularpharmaceutics
Multifunctional PLGA-Based Nanoparticles Encapsulating Simultaneously Hydrophilic Antigen and Hydrophobic Immunomodulator for Mucosal Immunization Charlotte Primard, Johanna Poecheim, Simon Heuking, Emmanuelle Sublet, Farnaz Esmaeili, and Gerrit Borchard* School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 1211 Geneva, Switzerland S Supporting Information *
ABSTRACT: We describe here the development of nanoparticles made from poly(lactic-co-glycolic acid) (PLGA) able to deliver an encapsulated antigen with a Toll-Like Receptor-7 (TLR-7) agonist as immunostimulatory signal and coated with a muco-adhesive chitosan-derivate layer. The potential to stimulate an immune response of these vaccine formulations in the absence or presence of the TLR-7 agonist at the systemic and mucosal level were evaluated in mice following subcutaneous or nasal administrations. Intranasally immunized mice developed a high systemic immune response equivalent to mice injected subcutaneously. However, mucosal immune responses were only induced at local and distal sites in mucosally immunized animals. The adjuvant effect of imiquimod on the polarization of the immune response was only detected at local sites, which tends to increase safety of this vaccine delivery system. KEYWORDS: PLGA nanoparticles, imiquimod, mucosal vaccine the development of effective delivery systems and adjuvants.4 Several strategies can be considered to enhance cellular immune responses, e.g., (i) extending the antigen exposure time or (ii) targeting of antigen presenting cell (APC) stimulation.5 By using nanotechnology, these two strategies can be combined. We designed a system encapsulating the antigen with an additional immunostimulatory signal (a Toll-like receptor agonist) into poly(lactic-co-glycolic acid) (PLGA) nanoparticles. Finally, for an efficient delivery to APC of the nasal mucosa, these particles were coated with a cationic polymer to favor particle adherence to the nasal epithelium and uptake by microfold (M)-cells, which are specialized in pathogen transport.6−8 TLR are pathogenic pattern recognition receptors (PRR), which recognize specific molecular patterns present in bacteria, virus, or fungi. TLR are expressed in cells that are located and involved in the first line of defense, such as mucosal epithelial cells, macrophages, dendritic cells (DC), dermal endothelial cells, and neutrophils. Imiquimod (IMQ), an imidazoquinoline amine, is an agonist of TLR-7, located in intracellular endosomes and is physiologically involved in viral recognition.9 IMQ is already approved for topical treatment in humans and has been shown to activate the TLR-7 receptor in
1. INTRODUCTION New vaccine strategies focus on eliciting protective immune responses at the portal of pathogen entry, i.e., the mucosae for most infectious diseases. Mucosal immune responses are induced by direct contact between the antigen and the mucosa, while systemic immunizations elicit merely weak mucosal immunity.1 Therefore, mucosal vaccination represents a challenge to the induction of protective immunity, but successes (oral Polio vaccine and nasal-spray flu vaccine) are moderated by failed attempts (HIV, Mycobacterium tuberculosis). As a mucosal route, the nasal delivery of vaccines combines many advantages, such as a rapid onset of action, the absence of antigen degradation due to acidic pH as present, e.g., in the stomach and vaginal mucosa, or due to enzymatic digestion in the intestine. Moreover, mucosal vaccination potentially induces the development of local humoral and cellular mucosal immune responses, as well as systemic immunity. Live attenuated vaccines favor the induction of cellular responses characterized by Th1-polarized lymphocyte or cytotoxic activities, but are fraught with safety concerns such as infectivity reversion or risks of recombination.2 On the contrary, safer current strategies tend to use subunit protein vaccines, which only induce weak Th2-biased responses, characterized by high antibody levels and the absence of a cellular immune response. However, for viral, parasitic, or chronic diseases, effective and/or protective T-cell immune responses are needed.3 Thus, full potential of vaccines requires © 2013 American Chemical Society
Received: Revised: Accepted: Published: 2996
February 19, 2013 June 24, 2013 June 24, 2013 June 24, 2013 dx.doi.org/10.1021/mp400092y | Mol. Pharmaceutics 2013, 10, 2996−3004
Molecular Pharmaceutics
Article
vitro10 and in vivo,11 inducing the secretion of IL-12 by macrophages and dendritic cells, acting on CD4+ T cells for IFN-γ production, and leading to the induction of a Th1 response. However, the systemic delivery of free-form IMQ has been shown to be toxic in human,12 associated with IFN-like side effects.13 As a consequence, a topical application of IMQ and its encapsulation into a vector of vaccination should reduce its toxicity. In this article, we report the development of a vaccine delivery system intended for mucosal delivery, composed of particles made from PLGA containing both the antigen and an immunostimulatory molecule encapsulated and surrounded by a muco-adherent polymer. The influence of IMQ has been tested in vivo on the systemic and mucosal immune responses at systemic and mucosal levels.
Aldrich, Switzerland) was used, a molecule able to react with the free amino groups of CM-TMC to form a fluorescent derivative. The supernatant was centrifuged once more at 15 000g for 10 min to remove the NP, and 100 μL of this solution were added to 300 μL of fluorescamine dissolved in acetonitrile at 0.5 mg/mL. This solution was incubated for 5 h in the dark before being distributed in duplicate in wells of a black 96-wells plate (100 μL/well). A calibration curve was made, following the same protocol and using CM-TMC solutions of known concentrations. The fluorescence intensity was read at excitation/emission fluorescence wavelengths of 380/495 nm, using a multiwell plate reader (Safire, TECAN, Switzerland). 2.4. Physico-Chemical Characterization of the Particles. The hydrodynamic diameter and the size polydispersity of the highly diluted particles in a 1 mM NaCl solution were determined by dynamic light scattering, using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, U.K.). The zeta potential was determined by electrophoresis and laser light scattering using the same equipment. The results are expressed as the mean of three measurements. The nanoparticles were examined for their shape and surface morphology using scanning electron microscopy. The particles were diluted in water at concentrations of 10 and 5 mg/mL, and a drop of those solutions was deposed on a grid. After two days of drying under vacuum, the samples were sputtered with 20 nm of gold with the metallizer Leica EM SCD 500 (gold 42 mA for 57 s) under vacuum prior to scanning electron microscopy (SEM), using a Jeol JSM-7001 electron microscope (15 kV, 10 mm). 2.5. Loading Capacity and Encapsulation Efficiency. For IMQ extraction, 2 mg of freeze-dried IMQ-BSAencapsulated-PLGA particles were dissolved in 200 μL of 1% acetic acid and vortexed until complete dispersion. The same volume of trichloroacetic acid 50% (w/v) was added and the mixture stirred until a clear solution was obtained. After 10 min centrifugation at 18 000g, the solubilized IMQ and polymer were separated from the precipitated BSA and added to the same volume of methanol to precipitate the PLGA. After centrifugation, the solubilized IMQ was quantified by HPLC. A water 600E multisolvent delivery pump coupled to an inline degasser AF (sprage set to 50), a separation module, an autosampler, and a fluorescent detector (all from Waters, Milford, USA) were used to perform IMQ analysis. The stationary phase consisted of a Supelcosil LC-ABZ 15 cm × 4.6 mm silica gel column (SupelCo, Sigma-Aldrich, Germany), while the mobile phase was a mixture of acetate buffer 0.1 M, pH 5, and methanol (50:50) delivered at a constant flow rate of 0.5 mL/min. IMQ fluorescence was detected at excitation/ emission wavelengths of 260/339 nm. A peak was visible after 8 min of retention time, and the calibration curve was linear between 0.1 and 2.5 μg/mL, with a correlation coefficient (R2) ≥ 0.99. Drug loading (DL) and encapsulation efficiency (EE) were calculated using eqs 1 and 2, respectively:
2. MATERIALS AND METHODS 2.1. Materials. Poly(lactic-co-glycolic acid) (PLGA, Resomer RG 503H (50:50), Mw 31−36 kD) was purchased from Boehringer Ingelheim (Biberach, Germany); dichloromethane (DCM), polyvinhyl alcohol (PVA, Mw 31 kD, 88% hydrolyzed), dimethyl sulfoxyde (DMSO, cellular grade), and bovine serum albumin (BSA, endotoxin
