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Preparation, characterization, and safety evaluation of poly(lactide-co-glycolide) nanoparticles for protein delivery into macrophages.

Guedj AS, Kell AJ, Barnes M, Stals S, Gonçalves D, Girard D, Lavigne C - Int J Nanomedicine (2015)

Bottom Line: In vitro and in vivo toxicological studies were performed to further determine the safety profile of PLGA NPs including inflammatory effects.PLGA NPs were not cytotoxic for THP-1 MDM cells, did not modulate neutrophil apoptosis in vitro, and did not show inflammatory effect in vivo in the murine air pouch model of acute inflammation.We demonstrated that PLGA-based nanopolymer carriers are good candidates to efficiently and safely enhance the transport of active molecules into human MDMs. In addition, we further investigated their inflammatory profile and showed that PLGA NPs have low inflammatory effects in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada.

ABSTRACT
Following infection, HIV establishes reservoirs within tissues that are inaccessible to optimal levels of antiviral drugs or within cells where HIV lies latent, thus escaping the action of anti-HIV drugs. Macrophages are a persistent reservoir for HIV and may contribute to the rebound viremia observed after antiretroviral treatment is stopped. In this study, we further investigate the potential of poly(lactic-co-glycolic) acid (PLGA)-based nanocarriers as a new strategy to enhance penetration of therapeutic molecules into macrophages. We have prepared stable PLGA nanoparticles (NPs) and evaluated their capacity to transport an active molecule into the human monocyte/macrophage cell line THP-1 using bovine serum albumin (BSA) as a proof-of-concept compound. Intracellular localization of fluorescent BSA molecules encapsulated into PLGA NPs was monitored in live cells using confocal microscopy, and cellular uptake was quantified by flow cytometry. In vitro and in vivo toxicological studies were performed to further determine the safety profile of PLGA NPs including inflammatory effects. The size of the PLGA NPs carrying BSA (PLGA-BSA) in culture medium containing 10% serum was ~126 nm in diameter, and they were negatively charged at their surface (zeta potential =-5.6 mV). Our confocal microscopy studies and flow cytometry data showed that these PLGA-BSA NPs are rapidly and efficiently taken up by THP-1 monocyte-derived macrophages (MDMs) at low doses. We found that PLGA-BSA NPs increased cellular uptake and internalization of the protein in vitro. PLGA NPs were not cytotoxic for THP-1 MDM cells, did not modulate neutrophil apoptosis in vitro, and did not show inflammatory effect in vivo in the murine air pouch model of acute inflammation. In contrast to BSA alone, BSA encapsulated into PLGA NPs increased leukocyte infiltration in vivo, suggesting the in vivo enhanced delivery and protection of the protein by the polymer nanocarrier. We demonstrated that PLGA-based nanopolymer carriers are good candidates to efficiently and safely enhance the transport of active molecules into human MDMs. In addition, we further investigated their inflammatory profile and showed that PLGA NPs have low inflammatory effects in vitro and in vivo. Thus, PLGA nanocarriers are promising as a drug delivery strategy in macrophages for prevention and eradication of intracellular pathogens such as HIV and Mycobacterium tuberculosis.

No MeSH data available.


Related in: MedlinePlus

Induction of leukocyte infiltration by PLGA NPs in vivo.Notes: Murine air pouches were raised in CD-1 mice before the injection of water (control) or the indicated concentrations of (A) PLGA NPs, (B) PLGA-BSA NPs, and (C) BSA protein alone. Exudates were harvested after 6 hours, and the total number of leukocytes was calculated. Results are the mean ± SEM (n≥4). *P<0.05 versus the control, **P<0.01 versus the control.Abbreviations: PLGA, poly(lactic-co-glycolic) acid; NPs, nanoparticles; BSA, bovine serum albumin; SEM, standard error of the mean.
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f7-ijn-10-5965: Induction of leukocyte infiltration by PLGA NPs in vivo.Notes: Murine air pouches were raised in CD-1 mice before the injection of water (control) or the indicated concentrations of (A) PLGA NPs, (B) PLGA-BSA NPs, and (C) BSA protein alone. Exudates were harvested after 6 hours, and the total number of leukocytes was calculated. Results are the mean ± SEM (n≥4). *P<0.05 versus the control, **P<0.01 versus the control.Abbreviations: PLGA, poly(lactic-co-glycolic) acid; NPs, nanoparticles; BSA, bovine serum albumin; SEM, standard error of the mean.

Mentions: The inflammatory profile of the PLGA NPs was performed with a neutrophil apoptosis assays. As illustrated in Figure 6, neither PLGA nor PLGA-BSA NPs affected apoptosis of human neutrophils up to the highest concentration tested (1,000 µg/mL). As expected, V. album agglutinin increased and GM-CSF inhibited neutrophil apoptosis when compared with the controls.51,52 To further investigate the pro-inflammatory property of the PLGA NPs, we also conducted in vivo assays using the murine air pouch model. This model has been commonly used to determine pro-inflammatory effects of molecules including the evaluation of acute pro-inflammatory activity of NPs.53–55 As illustrated in Figure 7, administration of 10, 100, 250, 500, or 1,000 µg/mL of unloaded PLGA NPs or 10, 100, 250, 500 µg/mL of PLGA-BSA NPs did not significantly increase the total number of attracted leukocytes into the air pouch 6 hours postinjection when compared with the control mice. However, administration of 1,000 µg/mL of PLGA-BSA NPs significantly increased leukocyte infiltration into the air pouch to the same extent of theTiO2 positive control NPs (Figure 7B). However, when BSA alone was administered into the air pouch at concentrations equivalent to the amount of BSA found in 1,000, 2,000, or 4,000 µg/mL of PLGA-BSA NPs, we did not observe increased leukocyte infiltration into the air pouch (Figure 7C). Cytology examinations revealed that 75% of the leukocytes attracted into the air pouch by 1,000 µg/mL of PLGA-BSA NPs were neutrophils compared with 56% in control mice or 93% when TiO2 NPs were injected (Figure 8B). The remainders of the leukocytes were mononuclear cells. Although the total number of leukocytes attracted into the air pouch did not increase when unloaded PLGA NPs were administered, the proportion of PMNs was slightly higher (72%) compared with the control mice (63%), but this difference was not found statistically significant (Figure 8A). Finally, BSA alone at any of the concentrations evaluated showed the same proportion of cell subpopulations as in the control mice (Figure 8C).


Preparation, characterization, and safety evaluation of poly(lactide-co-glycolide) nanoparticles for protein delivery into macrophages.

Guedj AS, Kell AJ, Barnes M, Stals S, Gonçalves D, Girard D, Lavigne C - Int J Nanomedicine (2015)

Induction of leukocyte infiltration by PLGA NPs in vivo.Notes: Murine air pouches were raised in CD-1 mice before the injection of water (control) or the indicated concentrations of (A) PLGA NPs, (B) PLGA-BSA NPs, and (C) BSA protein alone. Exudates were harvested after 6 hours, and the total number of leukocytes was calculated. Results are the mean ± SEM (n≥4). *P<0.05 versus the control, **P<0.01 versus the control.Abbreviations: PLGA, poly(lactic-co-glycolic) acid; NPs, nanoparticles; BSA, bovine serum albumin; SEM, standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4590413&req=5

f7-ijn-10-5965: Induction of leukocyte infiltration by PLGA NPs in vivo.Notes: Murine air pouches were raised in CD-1 mice before the injection of water (control) or the indicated concentrations of (A) PLGA NPs, (B) PLGA-BSA NPs, and (C) BSA protein alone. Exudates were harvested after 6 hours, and the total number of leukocytes was calculated. Results are the mean ± SEM (n≥4). *P<0.05 versus the control, **P<0.01 versus the control.Abbreviations: PLGA, poly(lactic-co-glycolic) acid; NPs, nanoparticles; BSA, bovine serum albumin; SEM, standard error of the mean.
Mentions: The inflammatory profile of the PLGA NPs was performed with a neutrophil apoptosis assays. As illustrated in Figure 6, neither PLGA nor PLGA-BSA NPs affected apoptosis of human neutrophils up to the highest concentration tested (1,000 µg/mL). As expected, V. album agglutinin increased and GM-CSF inhibited neutrophil apoptosis when compared with the controls.51,52 To further investigate the pro-inflammatory property of the PLGA NPs, we also conducted in vivo assays using the murine air pouch model. This model has been commonly used to determine pro-inflammatory effects of molecules including the evaluation of acute pro-inflammatory activity of NPs.53–55 As illustrated in Figure 7, administration of 10, 100, 250, 500, or 1,000 µg/mL of unloaded PLGA NPs or 10, 100, 250, 500 µg/mL of PLGA-BSA NPs did not significantly increase the total number of attracted leukocytes into the air pouch 6 hours postinjection when compared with the control mice. However, administration of 1,000 µg/mL of PLGA-BSA NPs significantly increased leukocyte infiltration into the air pouch to the same extent of theTiO2 positive control NPs (Figure 7B). However, when BSA alone was administered into the air pouch at concentrations equivalent to the amount of BSA found in 1,000, 2,000, or 4,000 µg/mL of PLGA-BSA NPs, we did not observe increased leukocyte infiltration into the air pouch (Figure 7C). Cytology examinations revealed that 75% of the leukocytes attracted into the air pouch by 1,000 µg/mL of PLGA-BSA NPs were neutrophils compared with 56% in control mice or 93% when TiO2 NPs were injected (Figure 8B). The remainders of the leukocytes were mononuclear cells. Although the total number of leukocytes attracted into the air pouch did not increase when unloaded PLGA NPs were administered, the proportion of PMNs was slightly higher (72%) compared with the control mice (63%), but this difference was not found statistically significant (Figure 8A). Finally, BSA alone at any of the concentrations evaluated showed the same proportion of cell subpopulations as in the control mice (Figure 8C).

Bottom Line: In vitro and in vivo toxicological studies were performed to further determine the safety profile of PLGA NPs including inflammatory effects.PLGA NPs were not cytotoxic for THP-1 MDM cells, did not modulate neutrophil apoptosis in vitro, and did not show inflammatory effect in vivo in the murine air pouch model of acute inflammation.We demonstrated that PLGA-based nanopolymer carriers are good candidates to efficiently and safely enhance the transport of active molecules into human MDMs. In addition, we further investigated their inflammatory profile and showed that PLGA NPs have low inflammatory effects in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada.

ABSTRACT
Following infection, HIV establishes reservoirs within tissues that are inaccessible to optimal levels of antiviral drugs or within cells where HIV lies latent, thus escaping the action of anti-HIV drugs. Macrophages are a persistent reservoir for HIV and may contribute to the rebound viremia observed after antiretroviral treatment is stopped. In this study, we further investigate the potential of poly(lactic-co-glycolic) acid (PLGA)-based nanocarriers as a new strategy to enhance penetration of therapeutic molecules into macrophages. We have prepared stable PLGA nanoparticles (NPs) and evaluated their capacity to transport an active molecule into the human monocyte/macrophage cell line THP-1 using bovine serum albumin (BSA) as a proof-of-concept compound. Intracellular localization of fluorescent BSA molecules encapsulated into PLGA NPs was monitored in live cells using confocal microscopy, and cellular uptake was quantified by flow cytometry. In vitro and in vivo toxicological studies were performed to further determine the safety profile of PLGA NPs including inflammatory effects. The size of the PLGA NPs carrying BSA (PLGA-BSA) in culture medium containing 10% serum was ~126 nm in diameter, and they were negatively charged at their surface (zeta potential =-5.6 mV). Our confocal microscopy studies and flow cytometry data showed that these PLGA-BSA NPs are rapidly and efficiently taken up by THP-1 monocyte-derived macrophages (MDMs) at low doses. We found that PLGA-BSA NPs increased cellular uptake and internalization of the protein in vitro. PLGA NPs were not cytotoxic for THP-1 MDM cells, did not modulate neutrophil apoptosis in vitro, and did not show inflammatory effect in vivo in the murine air pouch model of acute inflammation. In contrast to BSA alone, BSA encapsulated into PLGA NPs increased leukocyte infiltration in vivo, suggesting the in vivo enhanced delivery and protection of the protein by the polymer nanocarrier. We demonstrated that PLGA-based nanopolymer carriers are good candidates to efficiently and safely enhance the transport of active molecules into human MDMs. In addition, we further investigated their inflammatory profile and showed that PLGA NPs have low inflammatory effects in vitro and in vivo. Thus, PLGA nanocarriers are promising as a drug delivery strategy in macrophages for prevention and eradication of intracellular pathogens such as HIV and Mycobacterium tuberculosis.

No MeSH data available.


Related in: MedlinePlus