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Growth factor choice is critical for successful functionalization of nanoparticles.

Pinkernelle J, Raffa V, Calatayud MP, Goya GF, Riggio C, Keilhoff G - Front Neurosci (2015)

Bottom Line: Nanoparticles (NPs) show new characteristics compared to the corresponding bulk material.It also shows that successful functionalization of magnetic NPs with growth factors is dependent on the used binding chemistry and that it is hardly predictable.For use as therapeutics, functionalization strategies have to be reproducible and future studies are needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology and Hypertension, Diabetes and Endocrinology, Otto-von-Guericke University of Magdeburg Magdeburg, Germany ; Institute for Biochemistry and Cell Biology, Otto-von-Guericke University of Magdeburg Magdeburg, Germany.

ABSTRACT
Nanoparticles (NPs) show new characteristics compared to the corresponding bulk material. These nanoscale properties make them interesting for various applications in biomedicine and life sciences. One field of application is the use of magnetic NPs to support regeneration in the nervous system. Drug delivery requires a functionalization of NPs with bio-functional molecules. In our study, we functionalized self-made PEI-coated iron oxide NPs with nerve growth factor (NGF) and glial cell-line derived neurotrophic factor (GDNF). Next, we tested the bio-functionality of NGF in a rat pheochromocytoma cell line (PC12) and the bio-functionality of GDNF in an organotypic spinal cord culture. Covalent binding of NGF to PEI-NPs impaired bio-functionality of NGF, but non-covalent approach differentiated PC12 cells reliably. Non-covalent binding of GDNF showed a satisfying bio-functionality of GDNF:PEI-NPs, but turned out to be unstable in conjugation to the PEI-NPs. Taken together, our study showed the importance of assessing bio-functionality and binding stability of functionalized growth factors using proper biological models. It also shows that successful functionalization of magnetic NPs with growth factors is dependent on the used binding chemistry and that it is hardly predictable. For use as therapeutics, functionalization strategies have to be reproducible and future studies are needed.

No MeSH data available.


Related in: MedlinePlus

SEM image of PC12 cells incubated 72 h with NGF:PEI-NPs (A–D). PC12 cells grew neurites confirming differentiation due to NGF (A–C). Box in (C) points to NPs bound to the cell surface, (D) illustrates higher magnification of NPs and area of EDX analysis shown in (E).
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Figure 2: SEM image of PC12 cells incubated 72 h with NGF:PEI-NPs (A–D). PC12 cells grew neurites confirming differentiation due to NGF (A–C). Box in (C) points to NPs bound to the cell surface, (D) illustrates higher magnification of NPs and area of EDX analysis shown in (E).

Mentions: Experimental results revealed that the fluorescent NGF binds the surface of the PEI-NPs at a ratio of 14 ± 3 μg of fluorescent NGF per mg of PEI-NPs. No statistical difference was found between non-covalent NGF:PEI-NPs and covalent NGF-PEI-NPs (data not shown). PC12 cells were incubated with reduced media modified with NGF:PEI-NPs or NGF-PEI-NPs (NGF concentration 140 ng/ml, NP concentration 10 μg/ml). In Figure 1, PC12 cultures incubated with reduced media (Figure 1A), reduced media and NGF (Figure 1B), reduced media and NGF-PEI-NPs (Figure 1C), and reduced media and NGF:PEI-NPs (Figure 1D) are shown. For NGF-PEI-MNPs, a strong reduction of the neurite number and length was observed compared to the control (NGF 140 ng/ml). Otherwise, for NGF:PEI-NPs cell differentiation proceeded similarly to the control cultures treated with NGF. Confocal imaging demonstrated that the NGF:PEI-NPs are strongly engulfed by cells, being localized in both the cell body and neurite protrusions (Figure 1E). Based on these results, we can conclude that the covalent approach impairs the bio-functionality of the protein. We postulate that the strong interaction between the particles surface and NGF induce the protein wrapping/adsorption onto the particle surface. This could strongly affect the protein mobility, ultimately altering its 3D structure and the interaction with the TrkA receptor. Based on these results, we considered the non-covalent approach to be the most promising. Electron microscopy confirmed that in presence of NGF:PEI-NPs, PC12 cells were properly differentiated. They exhibited long neurites, which were well connected within the network (Figures 2A,B). Figure 2C shows a representative cell with a cluster of NPs bound to the cell surface, which is highlighted in Figure 2D. EDX analysis revealed iron content and therefore confirmed the NGF:PEI-NP–cell interaction (Figure 2E). FIB-SEM analysis of milled PC12 cells, a technique that allows sectioning a cell and acquiring information on the constitutive elements inside (Riggio et al., 2014), illustrated the successful internalization of NGF:PEI-NPs (Figures 3A,B,D). EDX analysis, shown in Figures 3C,E, confirmed that internalized clusters seen in Figures 3A,B,D are from iron and therefore iron oxide PEI-NPs.


Growth factor choice is critical for successful functionalization of nanoparticles.

Pinkernelle J, Raffa V, Calatayud MP, Goya GF, Riggio C, Keilhoff G - Front Neurosci (2015)

SEM image of PC12 cells incubated 72 h with NGF:PEI-NPs (A–D). PC12 cells grew neurites confirming differentiation due to NGF (A–C). Box in (C) points to NPs bound to the cell surface, (D) illustrates higher magnification of NPs and area of EDX analysis shown in (E).
© Copyright Policy
Related In: Results  -  Collection

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Figure 2: SEM image of PC12 cells incubated 72 h with NGF:PEI-NPs (A–D). PC12 cells grew neurites confirming differentiation due to NGF (A–C). Box in (C) points to NPs bound to the cell surface, (D) illustrates higher magnification of NPs and area of EDX analysis shown in (E).
Mentions: Experimental results revealed that the fluorescent NGF binds the surface of the PEI-NPs at a ratio of 14 ± 3 μg of fluorescent NGF per mg of PEI-NPs. No statistical difference was found between non-covalent NGF:PEI-NPs and covalent NGF-PEI-NPs (data not shown). PC12 cells were incubated with reduced media modified with NGF:PEI-NPs or NGF-PEI-NPs (NGF concentration 140 ng/ml, NP concentration 10 μg/ml). In Figure 1, PC12 cultures incubated with reduced media (Figure 1A), reduced media and NGF (Figure 1B), reduced media and NGF-PEI-NPs (Figure 1C), and reduced media and NGF:PEI-NPs (Figure 1D) are shown. For NGF-PEI-MNPs, a strong reduction of the neurite number and length was observed compared to the control (NGF 140 ng/ml). Otherwise, for NGF:PEI-NPs cell differentiation proceeded similarly to the control cultures treated with NGF. Confocal imaging demonstrated that the NGF:PEI-NPs are strongly engulfed by cells, being localized in both the cell body and neurite protrusions (Figure 1E). Based on these results, we can conclude that the covalent approach impairs the bio-functionality of the protein. We postulate that the strong interaction between the particles surface and NGF induce the protein wrapping/adsorption onto the particle surface. This could strongly affect the protein mobility, ultimately altering its 3D structure and the interaction with the TrkA receptor. Based on these results, we considered the non-covalent approach to be the most promising. Electron microscopy confirmed that in presence of NGF:PEI-NPs, PC12 cells were properly differentiated. They exhibited long neurites, which were well connected within the network (Figures 2A,B). Figure 2C shows a representative cell with a cluster of NPs bound to the cell surface, which is highlighted in Figure 2D. EDX analysis revealed iron content and therefore confirmed the NGF:PEI-NP–cell interaction (Figure 2E). FIB-SEM analysis of milled PC12 cells, a technique that allows sectioning a cell and acquiring information on the constitutive elements inside (Riggio et al., 2014), illustrated the successful internalization of NGF:PEI-NPs (Figures 3A,B,D). EDX analysis, shown in Figures 3C,E, confirmed that internalized clusters seen in Figures 3A,B,D are from iron and therefore iron oxide PEI-NPs.

Bottom Line: Nanoparticles (NPs) show new characteristics compared to the corresponding bulk material.It also shows that successful functionalization of magnetic NPs with growth factors is dependent on the used binding chemistry and that it is hardly predictable.For use as therapeutics, functionalization strategies have to be reproducible and future studies are needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology and Hypertension, Diabetes and Endocrinology, Otto-von-Guericke University of Magdeburg Magdeburg, Germany ; Institute for Biochemistry and Cell Biology, Otto-von-Guericke University of Magdeburg Magdeburg, Germany.

ABSTRACT
Nanoparticles (NPs) show new characteristics compared to the corresponding bulk material. These nanoscale properties make them interesting for various applications in biomedicine and life sciences. One field of application is the use of magnetic NPs to support regeneration in the nervous system. Drug delivery requires a functionalization of NPs with bio-functional molecules. In our study, we functionalized self-made PEI-coated iron oxide NPs with nerve growth factor (NGF) and glial cell-line derived neurotrophic factor (GDNF). Next, we tested the bio-functionality of NGF in a rat pheochromocytoma cell line (PC12) and the bio-functionality of GDNF in an organotypic spinal cord culture. Covalent binding of NGF to PEI-NPs impaired bio-functionality of NGF, but non-covalent approach differentiated PC12 cells reliably. Non-covalent binding of GDNF showed a satisfying bio-functionality of GDNF:PEI-NPs, but turned out to be unstable in conjugation to the PEI-NPs. Taken together, our study showed the importance of assessing bio-functionality and binding stability of functionalized growth factors using proper biological models. It also shows that successful functionalization of magnetic NPs with growth factors is dependent on the used binding chemistry and that it is hardly predictable. For use as therapeutics, functionalization strategies have to be reproducible and future studies are needed.

No MeSH data available.


Related in: MedlinePlus