Table 1.
Properties of PLGA NPs constructs used in this study.
Fig 1.
(A) Morphological analysis of PLGA NP/OVA/MPLA by scanning electron microscopy (SEM). (B) Schematic representation of the PLGA/OVA/MPLA NP and (C) SEM of a single PLGA NP/OVA/MPLA. Scale bar = 100 nm and 100 μ. PLGA: poly-lactic-co-glycolic acid, MPLA: Monophosphoryl Lipid A, OVA: ovalbumin
Fig 2.
In vitro release profile of OVA from PLGA NPs.
One milligram of the PLGA/OVA/MPLA NPs were dispersed in 4 ml of PBS containing 0.02% sodium azide, and then were kept at 37°C. At the indicated weeks, the supernatants were collected after centrifugation and the protein contents were analyzed.
Fig 3.
In vitro generation of canine monocyte-derived macrophages.
Monocytes were obtained by positive selection using CD14 monoclonal antibody coated columns. CD14+ monocytes were then seed in 12 or 24 wells plates at 1x106 cell/mL in DMEM enriched with 50 ng/mL of canine GM-CSF. Macrophages were allowed to mature for 7 days upon which were used for the experiments. Flow cytometry after monocyte isolation: (A) Control and (B) CD14+ cells. (C) Morphology of adherent macrophages after 7 days of incubation with DMEM and GM-CSF by light microscopy. Scale bar = 20 μm. Similar data were obtained in three independent experiments using macrophages from different animals.
Fig 4.
Flow cytometric evaluation of cellular uptake of PLGA NP preparations by canine macrophages.
Flow cytometry analysis of canine macrophages treated with fluorescent PLGA/OVA/MPLA NPs. Day 7 canine macrophages cultures (1x106) were incubated with 50 μg/mL of OVA-FITC or PLGA/OVA-FITC/MPLA NP. After 24 hours, nonadherent cells were harvested and analyzed by flow cytometry. One representative experiment is shown. (A) Histogram of macrophages incubated with 50 μg/mL OVA-FIT for 24 hours. (B) Histogram of macrophages incubated with 50 μg/mL PLGA/OVA-FITC/MPLA for 24 hours.
Fig 5.
Transmission electron microscopy (TEM) evaluation of cellular uptake of PLGA NP preparations by canine macrophages.
Day 7 canine macrophages cultures (1x106) were incubated with 50 μg/mL of PLGA/OVA/MPLA NPs. After 24 hours, nonadherent cells were harvested and analyzed by TEM. The macrophages without treatment (A) and (B) contain empty phagocytic vacuoles while the macrophages incubated with PLGA NP/OVA/MPLA (C) and (D) demonstrated absence of vacuoles and the presence of electron-dense material reflecting uptake of the NPs. (A) scale bar = 2 μm, (B) and (C) scale bar = 1 μm, and (D) scale bar = 500 nm. Similar data were obtained in three independent experiments.
Fig 6.
Cellular localization of PLGA/OVA-FITC/MPLA NP uptake by canine macrophages and evaluated by confocal laser scanning microscopy after 2 hours of incubation.
Day 7 canine macrophages cultures (1x106) were incubated with 50 μg/mL of PLGA/OVA-FITC/ MPLA NPs. After 2 hours, macrophages were harvested and analyzed by confocal microscopy. One representative experiment is shown. (A) Nucleus of macrophages stain blue by DAPI. (B) PLGA/OVA-FITC/MPLA NP stain green. (C) Canine macrophages. (D) Merge of A&B demonstrating internalization of particles. Scale bar = 20 μm. DAPI = 4',6-Diamidino-2-Phenylindole, Dihydrochloride; FITC = Fluorescein isothiocyanate. Similar data were obtained in three independent experiments.
Fig 7.
Evaluation of canine macrophage viability incubated with PLGA NP formulations.
Day 7 canine macrophages cultures (1x106) were incubated with 50 μg/mL of different NP formulations for 2 hours or 24 hours at 37°C. Macrophages were then stained with Trypan Blue and evaluated microscopically. (A) Cells that take the stain have impaired membranes and are considered dead cells. (B) Day 7 canine macrophages (1x106), were cytocentrifuged and evaluated under light microscopy for evidence of pyknosis or cellular necrosis. Similar data were obtained in three independent experiments.
Fig 8.
Effects of incubation of PLGA NP preparations on TNF-alpha (A), IL-12p40 (B), and IL-10 (C), gene expression by canine macrophages.
Day 7 canine macrophages (1x106/mL) were pretreated with 50 μg/mL of PLGA NP, PLGA NP/MPLA, PLGA NP/OVA/MPLA or LPS (1 μg/mL) for 24 hours or 36 hours. GAPDH was used to normalize the results. * Statistically significant difference when compared to PBS and PLGA NP-treated group (P<0.05). ** Statistically significant when compared with PLGA NP/OVA/ MPLA treated macrophages (P < 0.05). Similar data were obtained in three independent experiments.
Fig 9.
Flow cytometric evaluation of MHC-I and MHC-II phenotype in canine macrophages exposed to PLGA NP preparations.
Day 7 canine macrophages (1x106) were incubated with 50 μg/mL of PLGA NP or PLGA/OVA/MPLA NP. After 24 hours, nonadherent cells were harvested and analyzed by flow cytometry. FSC (forward scatter) and SSC (side scatter) profiles of PBS and PLGA NP-incubated with canine macrophages are shown. One color flow cytometry dot plots gated indicating the phenotype of macrophages incubated with PLGA NPs. One representative experiment is shown.