A T cell hybridoma, B3Z86/90.14 (B3Z), was kindly provided by Dr. Nilabh Shastri (University of California, Berkeley, CA) and T cell hybridomas, CD8 OVA1.3, were kindly provided by Dr. Clifford V. Harding (Case Western Reserve University, Cleveland, OH) (15,16). Bone marrow (BM)-derived DCs were generated from mouse bone marrow cells, as described previously (8). Briefly, BM cells obtained from the femurs of BALB/c mouse were cultured in a 6-well plate (5×10⁶/well) in a culture medium supplemented with 200 U/ml rmGM-CSF. At days 3 and 4 from the initiation of culture, the non-adherent cells were discarded by replacing the culture medium with fresh medium containing the cytokines after gentle shaking. The DCs were harvested by gentle pipetting at day 6.

Particles containing OVA were prepared, as described previously with minor modifications (12). Briefly, 400µl of OVA (100 mg/ml) was added to 2 ml of ethyl acetate containing 200 mg of poly(lactic-co-glycolic acid) (PLGA, Sigma-Aldrich). The mixture was then stirred at 20,000 rpm (Homogeniser, IKA, Japan) in the presence or absence of simultaneous sonication (Ultrasonic bath: 500 W, 30 kHz, BRANSON). After 3 min of emulsification, 8 ml of an aqueous solution of polyvinyl alcohol (PVA, 5%) was added to the w/o emulsion to form a w/o/w double emulsion, which was stirred for 5 min. To solidify the nanoparticles, the organic solvent was evaporated by stirring the double emulsion with 200 ml of an aqueous solution of 0.1% PVA at 500 rpm for 2 h. For the complete removal of ethyl acetate, the dispersion of nanoparticles was concentrated to approximately half the volume using a rotary evaporator at 40℃. The resulting nanoparticles were centrifuged at 3,000 rpm for 20 min, and washed twice with phosphate-buffered saline (PBS). The mean size of the particles was measured using a particle size analyzer (ELS-Z, Otsuka, Japan). The OVA concentration was determined using a micro-bicinchoninic acid assay kit (Pierce, Rockford, IL) after lysing the nanoparticles in a lysis buffer containing 0.1% SDS and 0.1 N NaOH. Nanoparticles containing both OVA and fluorescein isothiocyanate (FITC) were prepared by adding FITC (final, 5 mg/ml) to ethyl acetate along with PLGA (final, 5%). The surface morphology of the formulated NPs was visualized by scanning electron microscopy (LEO-1530, Carl Zeiss, Germany).

OVA-specific mouse IgG (mIgG) was attached covalently to the nanoparticles using (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) (EDC, Pierce, Rockford, IL, USA) as previously described (12). Briefly, 4.5µg of EDC was added to a 360µl mixture of 400µg nanoparticles (as OVA concentration) and 400µg mIgG. The reaction mixture was incubated for 2 h by tapping at room temperature. An excess linking reagent and soluble byproducts were separated by centrifugation at 15,000 rpm for 10 min, and the nanoparticles were washed three times with 1 ml PBS, pH 7.4.

The DCs were added to a 96-well microtiter plate (1×10⁵/well) and were then added with the nanoparticles. The plate was incubated for 2 or 18 h at 37℃,washed twice with 300 µl/well of pre-warmed PBS and fixed with 100µl/well of ice-cold 1.0% paraformaldehyde for 5 min at room temperature. The plate was then washed three times with 300 µl/well of PBS. Class I MHC-complexed OVA peptide quantities were assessed using LacZ T cell activation assays or by IL-2 secretion assays after culturing the paraformaldehyde-fixed DCs with B3Z cells (2×10⁵/well) for 4 h or with CD8 OVA1.3 cells (2×10⁵/well) for 18 h, as described previously (8,17).

Four milligrams of OVA-NPs were dispersed in 4 ml of PBS (pH 7.2) containing 0.02% sodium azide. The suspension was then aliquoted to a 500µl/microfuge tube, and the microfuge tubes were kept at 37℃ or 4℃. At the indicated days, the microfuge tubes were centrifuged at 15,000 rpm for 10 min, and the supernatants were transferred to new tubes. The supernatants were filtered through a membrane filter (pore size 0.22µm), and then the protein content was analyzed using a micro-bicinchoninic acid assay kit (Pierce).

C57BL/6 mice were injected i.p. with 100µg of NPs containing both OVA and FITC. The mice were sacrificed 2 h later, and mononuclear cell suspensions were prepared from the spleens and lymph nodes (popliteal, inguinal, mesenteric, and axillary). The single cell suspensions were fixed with paraformaldehyde and analyzed for FITC-positive cells by flow cytometry, or stained with phycoerythrin-labeled anti-mouse CD11b, CD11c, or Gr-1 monoclonal antibodies (BD pharmingen, San Diego, CA), after blocking the FcR-binding anti-CD16/CD32 monoclonal antibody (clone 2.4G2), as described previously (18). Flow cytometry was performed on a FACS Cantor (Becton-Dickinson).

Table I lists the different formulation conditions used to prepare nano- and microparticles along with their mean particle size, polydispersity index, zeta potential and protein loading efficiency. As expected, increasing the homogenization speed resulted in smaller sized NPs. The application of sonication energy (500 W, 30 kHz) during homogenizing of the two phases, the ethyl acetate phase containing PLGA and water phase containing OVA, further decreased the size of the resulting NPs. The mean OVA content was determined using a micro-bicinchoninic acid assay kit after lysing the NPs in a lysis buffer, and was 22.0~24.8%. The surface morphology of the NPs was visualized by scanning electron microscopy, and Fig. 1A shows a representative image of the NPs with an average size of 1.1µm. The stability of the NPs was examined by assessing the amount of soluble OVA released from the NP. Fig. 1B gives a representative experimental result, which was performed using the NPs with a mean size of 1.1µm. The OVA entrapped within the NPs was not released from the NPs for at least one week from the NPs if the NPs were stored at 4℃.

To examine the relationship between the NP size and cross-presentation-inducing activity, the NPs were incubated with BM-DCs for 2 h and the DCs were fixed with paraformaldehyde. The amounts of H-2K^b-OVA peptides complexes on DCs were assessed using OVA-specific CD8 T cell hybridomas, B3Z cells, which express β-galactosidase in response to T cell receptor (TCR) stimulation. As shown in Fig. 2, DCs phagocytosed NPs in the range of 1.11~1.44µm in size were almost equally efficient in cross-presenting OVA peptides via H-2K^b molecules. The cross-priming-inducing activity of NPs decreased dramatically when the mean size of the NPs was reduced to 0.56µm. Therefore, NPs with the average size of 1.11µm were chosen for the experiments described hereafter.

The NPs (average size, 1.11µm) were opsonized with mouse IgG, and the amounts and time kinetics of OVA peptide presentation were examined in BM-DCs. In this experiment, the amounts of H-2K^b-OVA peptide complexes on DCs were assessed using OVA-specific CD8 T cell hybridomas, CD8 OVA1.3 cells, which express IL-2 in response to H-2K^b-OVA peptide (SIINFEKL) complexes. As shown in Fig. 3, DCs incubated with the NPs containing OVA (NP[OVA]) were much more efficient in cross-presenting OVA peptides than those incubated with soluble OVA. In particular, the DCs incubated with mouse IgG-opsonized NPs (mIgG-NP[OVA]) were much more potent in cross-presenting OVA peptides than those incubated with (NP[OVA]). Moreover, the duration of cross-presenting OVA peptides was also extended markedly in DCs phagocytosed mIgG-NP[OVA]. The DCs phagocytosed mIgG-NP[OVA] could present H-2K^b-OVA peptide (SIINFEKL) complexes for 24 h, whereas the DCs incubated with saturation amounts of synthetic OVA peptide, SIINFEKL, could present H-2K^b-SIINFEKL complexes for only 6 h.

Two hours after an intraperitoneal injection of a single dose of 100µg of FITC-loaded NPs, biodistribution analysis was performed using mononuclear cells isolated from the spleens and lymph nodes. As shown in Fig. 4, the percentage of FITC-positive cells in the spleen and lymph node were increased dramatically when the mice were injected with mouse IgG-opsonized FITC-loaded NPs, compared to the mice injected with unopsonized FITC-loaded NPs. Most of the FITC-positive cells were in the spleen (approximately 20% in the spleen vs. approximately 0.8% in the lymph nodes). To examine the cell type that is positive for FITC, the mononuclear cells isolated from the spleens and lymph nodes were stained with PE-conjugated anti-mouse CD11b, CD11c, or Gr-1 monoclonal antibodies. As shown in Fig. 5, macrophages were the major cell type of FITC-positive cells in the spleen, whereas DCs were the major type in the lymph nodes.