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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

Flow cytometry analysis of cellular uptake.Notes: Monocyte-derived macrophages were exposed to various concentrations of fluorescent BSA alone or PLGA-FITC-BSA NPs for 3 hours. Then cells were washed and analyzed by flow cytometry.Abbreviations: PLGA, poly(lactic-co-glycolic) acid; NPs, nanoparticles; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; FU, fluorescence unit.
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f3-ijn-10-5965: Flow cytometry analysis of cellular uptake.Notes: Monocyte-derived macrophages were exposed to various concentrations of fluorescent BSA alone or PLGA-FITC-BSA NPs for 3 hours. Then cells were washed and analyzed by flow cytometry.Abbreviations: PLGA, poly(lactic-co-glycolic) acid; NPs, nanoparticles; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; FU, fluorescence unit.

Mentions: In order to evaluate cellular uptake, THP-1 MDM cells were treated with different concentrations of PLGA-FITC-BSA NPs for 3 hours, and fluorescence intensity of the resulting labeled cells was measured by flow cytometry. Results are shown in Figure 2. Flow cytometry data demonstrated that PLGA-FITC-BSA NPs are rapidly and efficiently taken up by THP-1 MDMs (Figure 2A). A significant dose-dependent increase in fluorescence was observed from 20 up to 200 µg/mL and then increased more slowly up to the highest concentration tested, 1,000 µg/mL (Figure 2B). We next compared the cellular uptake of PLGA-FITC-BSA with free FITC-BSA. THP-1 MDM cells were exposed to different concentrations of free FITC-BSA or PLGA-FITC-BSA for 3 hours, and then the fluorescence was measured by flow cytometry. Data are shown in Figure 3. In contrast to Figure 2 in which the concentration represented the amount of NPs, in this study, the data are expressed in terms of the amount of BSA. As shown in Figure 3, cell-associated fluorescence increased up to fivefold when the protein was incorporated into PLGA NPs compared with free delivery, demonstrating that uptake of BSA is more efficient when loaded into PLGA NPs.


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)

Flow cytometry analysis of cellular uptake.Notes: Monocyte-derived macrophages were exposed to various concentrations of fluorescent BSA alone or PLGA-FITC-BSA NPs for 3 hours. Then cells were washed and analyzed by flow cytometry.Abbreviations: PLGA, poly(lactic-co-glycolic) acid; NPs, nanoparticles; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; FU, fluorescence unit.
© Copyright Policy
Related In: Results  -  Collection

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

f3-ijn-10-5965: Flow cytometry analysis of cellular uptake.Notes: Monocyte-derived macrophages were exposed to various concentrations of fluorescent BSA alone or PLGA-FITC-BSA NPs for 3 hours. Then cells were washed and analyzed by flow cytometry.Abbreviations: PLGA, poly(lactic-co-glycolic) acid; NPs, nanoparticles; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; FU, fluorescence unit.
Mentions: In order to evaluate cellular uptake, THP-1 MDM cells were treated with different concentrations of PLGA-FITC-BSA NPs for 3 hours, and fluorescence intensity of the resulting labeled cells was measured by flow cytometry. Results are shown in Figure 2. Flow cytometry data demonstrated that PLGA-FITC-BSA NPs are rapidly and efficiently taken up by THP-1 MDMs (Figure 2A). A significant dose-dependent increase in fluorescence was observed from 20 up to 200 µg/mL and then increased more slowly up to the highest concentration tested, 1,000 µg/mL (Figure 2B). We next compared the cellular uptake of PLGA-FITC-BSA with free FITC-BSA. THP-1 MDM cells were exposed to different concentrations of free FITC-BSA or PLGA-FITC-BSA for 3 hours, and then the fluorescence was measured by flow cytometry. Data are shown in Figure 3. In contrast to Figure 2 in which the concentration represented the amount of NPs, in this study, the data are expressed in terms of the amount of BSA. As shown in Figure 3, cell-associated fluorescence increased up to fivefold when the protein was incorporated into PLGA NPs compared with free delivery, demonstrating that uptake of BSA is more efficient when loaded into PLGA NPs.

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