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Improved biocompatibility and efficient labeling of neural stem cells with poly(L-lysine)-coated maghemite nanoparticles

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ABSTRACT

Background:: Cell tracking is a powerful tool to understand cellular migration, dynamics, homing and function of stem cell transplants. Nanoparticles represent possible stem cell tracers, but they differ in cellular uptake and side effects. Their properties can be modified by coating with different biocompatible polymers. To test if a coating polymer, poly(L-lysine), can improve the biocompatibility of nanoparticles applied to neural stem cells, poly(L-lysine)-coated maghemite nanoparticles were prepared and characterized. We evaluated their cellular uptake, the mechanism of internalization, cytotoxicity, viability and proliferation of neural stem cells, and compared them to the commercially available dextran-coated nanomag®-D-spio nanoparticles.

Results:: Light microscopy of Prussian blue staining revealed a concentration-dependent intracellular uptake of iron oxide in neural stem cells. The methyl thiazolyl tetrazolium assay and the calcein acetoxymethyl ester/propidium iodide assay demonstrated that poly(L-lysine)-coated maghemite nanoparticles scored better than nanomag®-D-spio in cell labeling efficiency, viability and proliferation of neural stem cells. Cytochalasine D blocked the cellular uptake of nanoparticles indicating an actin-dependent process, such as macropinocytosis, to be the internalization mechanism for both nanoparticle types. Finally, immunocytochemistry analysis of neural stem cells after treatment with poly(L-lysine)-coated maghemite and nanomag®-D-spio nanoparticles showed that they preserve their identity as neural stem cells and their potential to differentiate into all three major neural cell types (neurons, astrocytes and oligodendrocytes).

Conclusion:: Improved biocompatibility and efficient cell labeling makes poly(L-lysine)-coated maghemite nanoparticles appropriate candidates for future neural stem cell in vivo tracking studies.

No MeSH data available.


Macropinocytosis is the mechanism of cellular uptake of PLL-γ-Fe2O3 and nanomag®-D-spio nanoparticles. The internalization mechanism of PLL-γ-Fe2O3 (A) and nanomag®-D-spio (B) nanoparticles in NSCs measured by flow cytometry of side scatter (SSC) after treatment of NSCs with different inhibitors: phenylarsine oxide (PAO), cytochalasin D (cytoD), nocodazole (noco) and filipin (fil).
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Figure 7: Macropinocytosis is the mechanism of cellular uptake of PLL-γ-Fe2O3 and nanomag®-D-spio nanoparticles. The internalization mechanism of PLL-γ-Fe2O3 (A) and nanomag®-D-spio (B) nanoparticles in NSCs measured by flow cytometry of side scatter (SSC) after treatment of NSCs with different inhibitors: phenylarsine oxide (PAO), cytochalasin D (cytoD), nocodazole (noco) and filipin (fil).

Mentions: To determine the mechanism of the uptake of PLL-γ-Fe2O3 and nanomag®-D-spio nanoparticles, NSCs were treated with different endocytotic inhibitors, incubated with nanoparticles and subsequently, flow cytometry analysis of the labeled cells was performed (Fig. 7,B). The inhibitors were cytochalasine D (blocks actin-dependent process such as macropinocytosis), nocodazole (inhibits microtubule function involved in intracellular vesicle trafficking), phenylarsine oxide (inhibits the clathrin-mediated endocytotic pathway) and filipin (inhibits caveolae pathways). Flow cytometry analysis showed that NSCs treated with cytochalasine D and incubated with PLL-γ-Fe2O3 or nanomag®-D-spio nanoparticles exhibited a left shift in the cell granularity distribution compared with non-treated control (Fig. 7). No change in labeling was observed in the phenylarsine oxide-, nocodazole- or filipin-treated NSCs when the nanoparticles were used. This indicated that actin-dependent process, e.g., macropinocytosis, was the mechanism of nanoparticle uptake for both types of nanoparticles.


Improved biocompatibility and efficient labeling of neural stem cells with poly(L-lysine)-coated maghemite nanoparticles
Macropinocytosis is the mechanism of cellular uptake of PLL-γ-Fe2O3 and nanomag®-D-spio nanoparticles. The internalization mechanism of PLL-γ-Fe2O3 (A) and nanomag®-D-spio (B) nanoparticles in NSCs measured by flow cytometry of side scatter (SSC) after treatment of NSCs with different inhibitors: phenylarsine oxide (PAO), cytochalasin D (cytoD), nocodazole (noco) and filipin (fil).
© Copyright Policy - Beilstein
Related In: Results  -  Collection

License 1 - License 2
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Figure 7: Macropinocytosis is the mechanism of cellular uptake of PLL-γ-Fe2O3 and nanomag®-D-spio nanoparticles. The internalization mechanism of PLL-γ-Fe2O3 (A) and nanomag®-D-spio (B) nanoparticles in NSCs measured by flow cytometry of side scatter (SSC) after treatment of NSCs with different inhibitors: phenylarsine oxide (PAO), cytochalasin D (cytoD), nocodazole (noco) and filipin (fil).
Mentions: To determine the mechanism of the uptake of PLL-γ-Fe2O3 and nanomag®-D-spio nanoparticles, NSCs were treated with different endocytotic inhibitors, incubated with nanoparticles and subsequently, flow cytometry analysis of the labeled cells was performed (Fig. 7,B). The inhibitors were cytochalasine D (blocks actin-dependent process such as macropinocytosis), nocodazole (inhibits microtubule function involved in intracellular vesicle trafficking), phenylarsine oxide (inhibits the clathrin-mediated endocytotic pathway) and filipin (inhibits caveolae pathways). Flow cytometry analysis showed that NSCs treated with cytochalasine D and incubated with PLL-γ-Fe2O3 or nanomag®-D-spio nanoparticles exhibited a left shift in the cell granularity distribution compared with non-treated control (Fig. 7). No change in labeling was observed in the phenylarsine oxide-, nocodazole- or filipin-treated NSCs when the nanoparticles were used. This indicated that actin-dependent process, e.g., macropinocytosis, was the mechanism of nanoparticle uptake for both types of nanoparticles.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background:: Cell tracking is a powerful tool to understand cellular migration, dynamics, homing and function of stem cell transplants. Nanoparticles represent possible stem cell tracers, but they differ in cellular uptake and side effects. Their properties can be modified by coating with different biocompatible polymers. To test if a coating polymer, poly(L-lysine), can improve the biocompatibility of nanoparticles applied to neural stem cells, poly(L-lysine)-coated maghemite nanoparticles were prepared and characterized. We evaluated their cellular uptake, the mechanism of internalization, cytotoxicity, viability and proliferation of neural stem cells, and compared them to the commercially available dextran-coated nanomag®-D-spio nanoparticles.

Results:: Light microscopy of Prussian blue staining revealed a concentration-dependent intracellular uptake of iron oxide in neural stem cells. The methyl thiazolyl tetrazolium assay and the calcein acetoxymethyl ester/propidium iodide assay demonstrated that poly(L-lysine)-coated maghemite nanoparticles scored better than nanomag®-D-spio in cell labeling efficiency, viability and proliferation of neural stem cells. Cytochalasine D blocked the cellular uptake of nanoparticles indicating an actin-dependent process, such as macropinocytosis, to be the internalization mechanism for both nanoparticle types. Finally, immunocytochemistry analysis of neural stem cells after treatment with poly(L-lysine)-coated maghemite and nanomag®-D-spio nanoparticles showed that they preserve their identity as neural stem cells and their potential to differentiate into all three major neural cell types (neurons, astrocytes and oligodendrocytes).

Conclusion:: Improved biocompatibility and efficient cell labeling makes poly(L-lysine)-coated maghemite nanoparticles appropriate candidates for future neural stem cell in vivo tracking studies.

No MeSH data available.