In Vitro Evaluation of the Biological Responses of Canine Macrophages Challenged with PLGA Nanoparticles Containing Monophosphoryl Lipid A

Abstract

Poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) have been considerably studied as a promising biodegradable delivery system to induce effective immune responses and to improve stability, safety, and cost effectiveness of vaccines. The study aimed at evaluating early inflammatory effects and cellular safety of PLGA NPs, co-encapsulating ovalbumin (PLGA/OVA NPs), as a model antigen and the adjuvant monophosphoryl lipid A (PLGA/MPLA NPs) as an adjuvant, on primary canine macrophages. The PLGA NPs constructs were prepared following the emulsion-solvent evaporation technique and further physic-chemically characterized. Peripheral blood mononuclear cells were isolated from canine whole blood by magnetic sorting and further cultured to generate macrophages. The uptake of PLGA NP constructs by macrophages was demonstrated by flow cytometry, transmission electron microscopy and confocal microscopy. Macrophage viability and morphology were evaluated by trypan blue exclusion and light microscopy. Macrophages were immunophenotyped for the expression of MHC-I and MHC-II and gene expression of Interleukin-10 (IL-10), Interleukin-12 (IL-12p40), and tumor necrosis factor alpha (TNF-α) were measured. The results showed that incubation of PLGA NP constructs with macrophages revealed effective early uptake of the PLGA NPs without altering the viability of macrophages. PLGA/OVA/MPLA NPs strongly induced TNF-α and IL-12p40 expression by macrophages as well as increase relative expression of MHC-I but not MHC-II molecules. Taken together, these results indicated that PLGA NPs with addition of MPLA represent a good model, when used as antigen carrier, for further, in vivo, work aiming to evaluate their potential to induce strong, specific, immune responses in dogs.

A nano particle vector comprised of poly lactic-co-glycolic acid and monophosphoryl lipid A and recombinant Mycobacterium avium subsp paratuberculosis peptides stimulate a pro-immune profile in bovine macrophages

“…; Guldner et al . ). Daily visual evaluation of the MoMΦ in culture for up to 5 days postviability analysis did not demonstrate cytomorphological changes that could be consistent with cell death.…”

“…; Guldner et al . ). Physicochemical analysis of the PLGA/MPLA NP constructs in solution demonstrated that NPs in solution were dispersed with limited aggregation of particles.…”

“…; Kanchan and Panda ; Guldner et al . ). Examination by TEM showed the PLGA/MPLA NPs containing peptides were comprised of an outer ring of MPLA and an inner ring of PLGA with a dense peptide central core.…”

“…; Guldner et al . ). The present results support they can also be used ex vivo , expanding opportunities for further studies of Anaplasmosis and especially for ongoing studies focused on developing a peptide‐based vaccine for Paratuberculosis.…”

“…Externally PLGA NPs were loaded with MPLA at 1% w/v. PLGA NPs were generated using the double emulsion water-in-oil-in-water (w/o/w) technique as previously described by our group (Guldner et al 2016a). Briefly, 9 g l À1 of PLGA 75:25 (PolySciTech â , IN) at a molecular weight of 25-35 kDa were dissolved in 450 ll of chloroform; 50 ll of PBS was then added to create a water-in-oil (w/o) emulsion.…”