We were able to manufacture the spheres to have specific mean diameters of any size ranging from 1 to 20 μM, with a tight size distribution about the mean using a precision spray drying technique [15]. The geometric

standard deviation (GSD) of diameter was typically 1.3–1.4 throughout the manufacturing process for each of the particle sizes produced in our experiments (Supplementary Fig. 1). We confirmed that PLGA microspheres were taken up by both mouse IOX1 and human DCs. Time-lapse videos of human dendrocyte phagocytosis events after incubation with 8 μM diameter spheres and 11 μM diameter PLGA microspheres respectively were qualitatively evaluated. Dendrocytes were observed to phagocytose up to three of the 8 μM spheres (Fig. 1a, b, and Supplementary Video 1) and a maximum of one of the 11 μM spheres (Fig. 1c, d, and Supplementary Video 2), consistent with their relative volumes.

A time lapse video of C57BL/6 dendrocytes incubated with 10 μM standard size polystyrene spheres was similarly prepared to ensure that the size of the C57BL/6 dendrocytes was similar to that of the human cells (Fig. 1e, f, and Supplementary Video 3). Qualitative analysis of the C57BL/6 video showed Z-VAD-FMK clinical trial a maximum of one 10 μM polystyrene microsphere phagocytosed by a given C57BL/6 dendrocyte suggesting that the C57BL/6 dendrocytes were similar in size to their human counterparts. We performed our studies with 11 μM spheres, the Thymidine kinase largest to be phagocytosed and thus capable of delivering large doses of epitope. The largest amount of peptide that could be loaded homogenously distributed in a sphere was

0.5% by weight. Spheres were loaded with ovalbumin (OVA) peptide (SIINFEKL) and vesicular stomatitis virus (VSV) peptide (RGYVYQGL), known mouse CTL epitopes [12]. C57BL/6 mice were inoculated with a single inter-dermal injection at the base of the tale and sacrificed after 14 days. Fresh splenocytes were harvested and subjected to IFN gamma ELISPOT analysis by strict Streeck, Frahm Walker criteria [16] against the same epitopes used in the inoculation. No inflammation at the injection site of any mouse was noted. We evaluated various adjuvants for use in the spheres themselves and in the solution surrounding the spheres loaded with the OVA epitope. For use in the carrier solution, we considered Monophosphoryl Lipid A (MPLA), a less toxic derivative of lipopolysaccharide that has been approved for use by the US FDA as an adjuvant for a marketed HPV product. MPLA acts as an immune-stimulant by signaling through the Toll-Like Receptor (TLR) pathway, specifically TLR4 [17]. MPLA has been used in commercial vaccine formulations as a viable alternative to LPS, the lipid A portion of Salmonella Minnesota Re595 lipopolysaccharide which is far too toxic for use in a vaccine [18] and [19].