Microencapsulation of inorganic nanocrystals into PLGA microsphere vaccines enables their intracellular localization in dendritic cells by electron and fluorescence microscopy

Abstract

Biodegradable poly-(D,L-lactide-co-glycolide) microspheres (PLGA-MS) are approved as a drug delivery system in humans and represent a promising antigen delivery device for immunotherapy against cancer. Immune responses following PLGA-MS vaccination require cross-presentation of encapsulated antigen by professional antigen presenting cells (APCs). While the potential of PLGA-MS as vaccine formulations is well established, the intracellular pathway of cross-presentation following phagocytosis of PLGA-MS is still under debate. A part of the controversy stems from the difficulty in unambiguously identifying PLGA-MS within cells. Here we show a novel strategy for the efficient encapsulation of inorganic nanocrystals (NCs) into PLGA-MS as a tool to study their intracellular localization. We microencapsulated NCs as an electron dense marker to study the intracellular localization of PLGA-MS by transmission electron microscopy (TEM) and as fluorescent labels for confocal laser scanning microscopy. Using this method, we found PLGA-MS to be rapidly taken up by dendritic cells and macrophages. Co-localization with the lysosomal marker LAMP1 showed a lysosomal storage of PLGA-MS for over two days after uptake, long after the initiation of cross-presentation had occurred. Our data argue against an escape of PLGA-MS from the endosome as has previously been suggested as a mechanism to facilitate cross-presentation of PLGA-MS encapsulated antigen.

Polylactide-co-glycolide-rifampicin-nanoparticles efficiently clearMycobacterium bovisBCG infection in macrophages and remain membrane-bound in phago-lysosomes

“…Collectively, our data support the conclusions of (Schliehe et al, 2011) who found that larger PLGA MPs remained in LAMP 1-positive lysosomes in dendritic cells and macrophages for at least two days after phagocytosis. They provided indirect evidence that ovalbumin encapsulated in the MPs was able to enter the cytoplasm of these cells and could activate the endoplasmic reticulum based MHC-1 pathway of antigen presentation to CD8+ T cells ('cross-presentation').…”

“…These studies relied on light microscopy immunofluorescence analyses and another EM study reached the same conclusion (Mathiowitz et al, 1997). In contrast, (Schliehe et al, 2011) concluded that in dendritic cells, PLGA MPs remained in phago-lysosomes for many days. Given these contradictory results we wanted to establish more definitively here whether or not PLGA NPs of different sizes remain long-term in membrane-enclosed phagocytic/endocytic compartments, or whether they are able to disrupt the enclosing membrane and escape into the cytoplasm.…”

Targeting human dendritic cells via DEC-205 using PLGA nanoparticles leads to enhanced cross-presentation of a melanoma-associated antigen

“…The HLA-A*0201-restricted peptide ELAGIGILTV (derived from the melanoma-associated differentiation antigen Melan-A/MART-1; A to L substitution at position 2 compared with its natural counterpart for higher immunogenicity and referred to as MART-1 [27][28][29][30][31][32][33][34][35] ), as well as the peptide labeled with a COOH-terminus FITC fluorochrome were produced by the Keck Facility (Yale University).…”

“…The human CD8 + T-cell receptor transgenic cell line DMF5, reactive with both the MART-1 (26)(27)(28)(29)(30)(31)(32)(33)(34)(35) and MART-1 (27L- 35) epitopes, was kindly provided by John R Wunderlich, Surgery Branch, National Cancer Institute. This line, originally derived from a high avidity tumor-infiltrating lymphocyte clone that mediated tumor regression clinically, has been extensively described previously.…”

“…Among them, the MART-1 (melanoma antigen recognized by T-cells) gene is expressed by normal melanocytes, as well as by a majority of fresh tumor isolates and melanoma cell lines. The MART-1 [27][28][29][30][31][32][33][34][35] peptide (corresponding to residues 27-35 of the non-mutated MART-1 protein) is a well characterized immune-dominant epitope for human leukocyte antigen (HLA)-A2-restricted melanoma-specific CTLs and can be used for the development of immunotherapeutic strategies. 10 Thus far, however, therapeutic benefits associated with DC/ peptide vaccination alone in patients with melanoma have been limited.…”