Immune Netw.  2011 Jun;11(3):163-168. 10.4110/in.2011.11.3.163.

Formulation and Characterization of Antigen-loaded PLGA Nanoparticles for Efficient Cross-priming of the Antigen

• Lee YR
• Lee YH
• Im SA
• Kim K
• Lee CK

• KMID: 2150703
• DOI: http://doi.org/10.4110/in.2011.11.3.163

Abstract

BACKGROUND
Nanoparticles (NPs) prepared from biodegradable polymers, such as poly (D,L-lactic acid-co-glycolic acid) (PLGA), have been studied as vehicles for the delivery of antigens to phagocytes. This paper describes the preparation of antigen-loaded PLGA-NPs for efficient cross-priming.
METHODS
NPs containing a similar amount of ovalbumin (OVA) but different sizes were produced using a micromixer-based W/O/W solvent evaporation procedure, and the efficiency of the NPs to induce the cross-presentation of OVA peptides were examined in dendritic cells (DCs). Cellular uptake and biodistribution studies were performed using fluorescein isothiocyanate (FITC)-loaded NPs in mice.
RESULTS
The NPs in the range of 1.1~1.4microm in size were the most and almost equally efficient in inducing the cross-presentation of OVA peptides via H-2Kb molecules. Cellular uptake and biodistribution studies showed that opsonization of the NPs with mouse IgG greatly increased the percentage of FITC-positive cells in the spleen and lymph nodes. The major cell type of FITC-positive cells in the spleen was macrophages, whereas that of lymph nodes was DCs.
CONCLUSION
These results show that IgG-opsonized PLGA-NPs with a mean size of 1.1microm would be the choice of biodegradable carriers for the targeted-delivery of protein antigens for cross-priming in vivo.

Keyword

PLGA; Nanoparticle; Opsonization; Cross-priming

MeSH Terms

Animals
Cross-Priming
Dendritic Cells
Fluorescein
Immunoglobulin G
Isothiocyanates
Lactic Acid
Lymph Nodes
Macrophages
Mice
Nanoparticles
Ovalbumin
Ovum
Peptides
Phagocytes
Polyglycolic Acid
Polymers
Spleen
Fluorescein
Immunoglobulin G
Isothiocyanates
Lactic Acid
Ovalbumin
Peptides
Polyglycolic Acid
Polymers

Figure

• 

  Figure 1 Release profile of OVA from OVA-loaded NPs. (A) Scanning electron microscopy showing the surface morphology of NPs with a mean size of 1.1µm. (B) Four milligrams of the OVA-NPs were dispersed in 4 ml of PBS containing 0.02% sodium azide, and then were kept at 37℃, or 4℃. At the indicated days, the supernatants were collected after centrifugation, and then the protein contents were analyzed using a micro-bicinchoninic acid assay.

• 

  Figure 2 Effects of the size of NPs on the cross-presentation. DCs were incubated with NPs with different mean sizes for 2 h, washed with PBS, fixed with paraformaldehyde, and the amounts of OVA peptides presented on MHC class I molecules were assessed by a LacZ T cell activation assay using OVA specific CD8 T cell hybridomas, B3Z cells.

• 

  Figure 3 Time-kinetics for the cross-presentation of OVA peptides. DCs were incubated with soluble OVA (50µg/ml), OVA-loaded NPs (50µg/ml), IgG-opsonized OVA-loaded NPs (50µg/ml), or 1 nM synthetic OVA peptide (SIINFEKL) for 2 h. The DCs were then washed with PBS, and were cultured in a medium containing 10% fetal bovine serum. At the indicated time points, the DCs were fixed with paraformaldehyde, and the amounts of OVA peptides presented on MHC class I molecules were assessed by a LacZ T cell activation assay using OVA specific CD8 T cell hybridomas, B3Z cells.

• 

  Figure 4 The uptake and biodistribution of NPs injected into the mouse peritoneum. C57BL/6 mice were injected i.p. with 100µg of NPs containing both OVA and FITC. Mice were sacrificed 2 h later, and mononuclear cell suspensions were prepared from the spleens (A) and lymph nodes (popliteal, inguinal, mesenteric, and axillary) (B). The single cell suspensions were fixed with paraformaldehyde and analyzed for FITC-positive cells by flow cytometry.

• 

  Figure 5 Cell type analysis for the FITC-positive cells in the spleen and lymph nodes. 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 (A) and lymph nodes (B), stained with phycoerythrin-labeled anti-mouse CD11b, CD11c, or Gr-1 monoclonal antibodies, and analyzed for FITC-positive cells by flow cytometry.

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