Abstract
IntroductionColorectal cancer (CRC) is the third most commonly diagnosed cancer and the fourth cause of cancer death worldwide. It is respossible for approximately 7 00 000 deaths per year. This work focused on the development of a combinatorial multivalent nanoplatform for CRC immunotherapy and immunomodulation based on the design of polymeric nanoparticles (NP) able to deliver a combination of CRC-associated antigen, adjuvants and gene regulators according to targeted cells, dendritic cell (DC) and CRC cells.Material and methodsPoly(lactic-co-glycolic) (PLGA)-based NP were prepared by the double emulsion (w/o/w) solvent evaporation method. NP were physicochemically characterised in terms of size, zeta potential and surface morphology. CRC antigen loadings were quantified by fluorescence. Immature DC (ATCC® CRL-11904TM) were used to evaluate the in vitro NP cytotoxicity by the propidium iodide assay, as well as NP cellular uptake profile by flow cytometry. In vivo biodistribution assay of plain NP was also performed using the IVIS Lumina® Bioimaging system. NP uptake in vivo by myeloid antigen presenting cells and the expression of maturation and co-stimulatory molecules at the surface of these cells sorted within draining lymph nodes, were also evaluated by flow cytometry.Results and discussionsPLGA-based NP presented a mean size diameter close to 200 nm, with low polydispersity index (PdI) (≤0.200), a surface charge close to neutrality, as well as, a spherical shape and smooth surface. These multivalent delivery systems presented high loadings for antigen and adjuvants. No cytotoxic effect was observed on immature DC up to 48 hour of incubation. NP were extensively internalised by immature DC in vitro after 48 hour incubation, and by migratory DC in vivo 17 hour after animal immunisation. In vivo real-time monitoring of NP accumulation in mice whole bodies and dissected organs showed a fluorescent signal at 17 hour close to the site of immunisation and in the lymph nodes. No significant differences in the expression of the co-stimulatory CD80, CD86 and MHC class I markers on CD11b+CD11c+MHCII+ population at lymph nodes were observed among different polymeric combinations upon mice immunisation with NP carrying CRC antigen and adjuvant.ConclusionAccording to NP physicochemical characteristics, internalisation and biodistribution patterns, this innovative nanoplatform can lead to a safe multivalent nanomedicine able to modulate dendritic cell activity and T cell expansion against tumour cells expressing entrapped antigens.
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