(489ae) PLGA Nanocarriers for Lymphatic Nasal Delivery of Proteins

Vaccination is the most effective way of fighting infectious diseases like HIV, malaria, influenza etc. Among the potential needle-free routes, nasal vaccination is particularly attractive. The nose is easily accessible and the nasal cavity is equipped with a high density of dendritic cells (DC) that can mediate strong systemic and local immune responses against pathogens that invade the human body through the respiratory tract. Furthermore, the enzymatic activity in the nose is relatively slow, which is favorable for antigen stability at the administration site. In addition, much lower doses of antigen are required for the intranasal route because there is no significant dilution of the vaccine formulations by nasal fluids and no exposure to low pH or a broad range of secreted degradative enzymes.

The formulation of antigens in particulate delivery systems can lead to protection of the antigen, co-encapsulation of antigen and adjuvant, potential increase of retention time at the nasal mucosa via bioadhesion, facilitation of the transport of the encapsulated antigen by microfold (M) epithelial cells to the nasal-associated lymphoid tissue (NALT) from where it can be delivered to a lymphatic environment and sustained release of antigen and thus increased presentation time to antigen presenting cells (APCs).

Poly(lactide-co-glycolide) (PLGA) and its various derivatives attract a lot of attention for the synthesis of nano-, microparticles encapsulating therapeutic drugs for controlled release applications due to their biocompatibility / biodegradability, extended release rates and ease of administration via injection.

In the present study, PLGA nanoparticles (NPs) containing the model antigen ovalbumin (OVA) and the adjuvant monophosphoryl lipid A (MPLA) were prepared by the method of double emulsion as potential nanocarriers for nasal vaccination. A water-in-oil (w/o) emulsion was initially formed by adding an aqueous OVA solution into a PLGA chloroform solution previously mixed with MPLA solution in methanol:chloroform (1:4 v/v). A double emulsion (w/o/w) was subsequently formed by the addition of the w/o emulsion to an aqueous PVA solution. Following solvent evaporation, the NPs were collected, purified by means of four successive centrifugation / redispersion cycles and subsequently lyophilized. NPs with different OVA and MPLA concentrations were prepared by varying the initial amounts of OVA (e.g., 0.15, 1, 2.5 and 10mg OVA per 90mg PLGA) and MPLA (e.g., 200 μg, 500μg, 1mg, 1.5mg and 2mg MPLA per 90mg PLGA). Finally, the NPs were functionalized for M cells targeting by coupling wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC) via the carbodiimide method. OVA loaded and blank PLGA NPs were also prepared as controls. Endotoxin-free PLGA (Resomer RG752H, Boehringer Ingelheim) and OVA (Profos AG EndoGradeTM Ovalbumin, <1EU/mg) were used in order to be able to quantify MPLA in the NPs. The morphology of the PLGA NPs was examined by scanning electron microscopy (JEOL JSM 6300), their size distribution was determined by dynamic light scattering and their zeta potential (æ) was calculated from aqueous electrophoresis measurements (Malvern Nano ZS90). The %wt of OVA and MPLA loaded in the PLGA NPs were determined by the bicinchoninic acid (BCA) protein assay and the Limulus Amebocyte Lysate (LAL) kit respectively whereas, the %wt of WGA-FITC on the PLGA NPs was assayed spectrophotometrically at 490 nm. The stability of the NPs was examined in vitro in 9%wt sucrose solution at 25^oC and 4^oC whereas the in vitro release of OVA from the NPs was tested in PBS at 37^oC. The amount of OVA released was estimated by the BCA protein assay and the antigen integrity was examined by gel electrophoresis.

The synthesized NPs had average sizes between 300 and 350nm and negative zeta potential values (e.g., -12 to -25mV). It was shown that, depending on the initial OVA and MPLA concentrations, the respective OVA and MPLA loadings varied from 0.1 to 10% and from 0.18 to 1.75% wt. High encapsulation efficiency of OVA and MPLA was achieved in each case. The amount of WGA-FITC anchored on the surface of the PLGA NPs was found to be ~0.14%wt. The PLGA NPs were found to be stable, with respect to particle aggregation during their storage in 9%wt sucrose solution at 4^oC for four weeks whereas limited aggregation was observed during their storage at 25^oC. In addition, the gel electrophoresis experiments showed that the antigen integrity was preserved during the NPs storage for four weeks. The examination of the release profile of OVA from the PLGA NPs in PBS at 37^oC revealed an initial burst release (>20%wt in 2hrs), attributed to the surface-associated OVA, which was followed by a lag phase of minimum release. The latter is known to depend on polymer degradation and to be prolonged for polymers with a low glycolide content and a high molecular mass. The lag phase is typically followed by a continuous release, which is initiated when sufficient polymer degradation has occurred.