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Tuning of nanoparticle biological functionality through controlled surface chemistry and characterisation at the bioconjugated nanoparticle surface.

Hristov DR, Rocks L, Kelly PM, Thomas SS, Pitek AS, Verderio P, Mahon E, Dawson KA - Sci Rep (2015)

Bottom Line: We have used a silica - PEG based bionanoconjugate synthetic scheme to study the subtle connection between cell receptor specific recognition and architecture of surface functionalization chemistry.Extensive physicochemical characterization of the grafted architecture is capable of capturing significant levels of detail of both the linker and grafted organization, allowing for improved reproducibility and ultimately insight into biological functionality.Our data suggest that scaffold details, propagating PEG layer architecture effects, determine not only the rate of uptake of conjugated nanoparticles into cells but also, more significantly, the specificity of pathways via which uptake occurs.

View Article: PubMed Central - PubMed

Affiliation: Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland.

ABSTRACT
We have used a silica - PEG based bionanoconjugate synthetic scheme to study the subtle connection between cell receptor specific recognition and architecture of surface functionalization chemistry. Extensive physicochemical characterization of the grafted architecture is capable of capturing significant levels of detail of both the linker and grafted organization, allowing for improved reproducibility and ultimately insight into biological functionality. Our data suggest that scaffold details, propagating PEG layer architecture effects, determine not only the rate of uptake of conjugated nanoparticles into cells but also, more significantly, the specificity of pathways via which uptake occurs.

No MeSH data available.


Bioconjugate Characterization (a).Protein concentration determined by BCA assay and Circular Dichroism (b). Overlapped curves for concentration normalized Circular Dichroism (H, M, L, VL and Tf), averaged over five separate batches for each of H, M, L and VL (mean ± std. dev). (c). Temperature denaturation curves plotting tryptophan emission maximum against temperature. (d). Representative full DCS dispersion size distribution shifts with increasing concentration of soluble TfR for nanoparticles (H,M,L and VL) with a shift in the particle size obtained by incubation (1 h at 37 °C) with TfR at varied concentration (see methods) (e). corresponding averaged binding curves (error bars indicate points averaged from a minimum of three experiments).
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f3: Bioconjugate Characterization (a).Protein concentration determined by BCA assay and Circular Dichroism (b). Overlapped curves for concentration normalized Circular Dichroism (H, M, L, VL and Tf), averaged over five separate batches for each of H, M, L and VL (mean ± std. dev). (c). Temperature denaturation curves plotting tryptophan emission maximum against temperature. (d). Representative full DCS dispersion size distribution shifts with increasing concentration of soluble TfR for nanoparticles (H,M,L and VL) with a shift in the particle size obtained by incubation (1 h at 37 °C) with TfR at varied concentration (see methods) (e). corresponding averaged binding curves (error bars indicate points averaged from a minimum of three experiments).

Mentions: We next coupled the biological moiety (Tf) to the terminal maleimide groups in conditions of ten times protein monolayer excess. We determined the efficiency of centrifugal washing procedures (Fig. S15) and the concentration of surface bound Tf by colorimetric protein assay (microBCA) and Circular Dichroism (CD) (Fig. 3a, S15 and S17). Interestingly, we noted across all methods (further supported by the fluorescence intensities in our tryptophan denaturation studies) that Tf coupled at a saturating concentration almost independent of PEG surface density (90–110 μg/mg NP by microBCA assay). This observation can be explained by the availability of a large excess of reactive maleimide functions at the surface, with the limiting factor being steric crowding of the protein itself.


Tuning of nanoparticle biological functionality through controlled surface chemistry and characterisation at the bioconjugated nanoparticle surface.

Hristov DR, Rocks L, Kelly PM, Thomas SS, Pitek AS, Verderio P, Mahon E, Dawson KA - Sci Rep (2015)

Bioconjugate Characterization (a).Protein concentration determined by BCA assay and Circular Dichroism (b). Overlapped curves for concentration normalized Circular Dichroism (H, M, L, VL and Tf), averaged over five separate batches for each of H, M, L and VL (mean ± std. dev). (c). Temperature denaturation curves plotting tryptophan emission maximum against temperature. (d). Representative full DCS dispersion size distribution shifts with increasing concentration of soluble TfR for nanoparticles (H,M,L and VL) with a shift in the particle size obtained by incubation (1 h at 37 °C) with TfR at varied concentration (see methods) (e). corresponding averaged binding curves (error bars indicate points averaged from a minimum of three experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664868&req=5

f3: Bioconjugate Characterization (a).Protein concentration determined by BCA assay and Circular Dichroism (b). Overlapped curves for concentration normalized Circular Dichroism (H, M, L, VL and Tf), averaged over five separate batches for each of H, M, L and VL (mean ± std. dev). (c). Temperature denaturation curves plotting tryptophan emission maximum against temperature. (d). Representative full DCS dispersion size distribution shifts with increasing concentration of soluble TfR for nanoparticles (H,M,L and VL) with a shift in the particle size obtained by incubation (1 h at 37 °C) with TfR at varied concentration (see methods) (e). corresponding averaged binding curves (error bars indicate points averaged from a minimum of three experiments).
Mentions: We next coupled the biological moiety (Tf) to the terminal maleimide groups in conditions of ten times protein monolayer excess. We determined the efficiency of centrifugal washing procedures (Fig. S15) and the concentration of surface bound Tf by colorimetric protein assay (microBCA) and Circular Dichroism (CD) (Fig. 3a, S15 and S17). Interestingly, we noted across all methods (further supported by the fluorescence intensities in our tryptophan denaturation studies) that Tf coupled at a saturating concentration almost independent of PEG surface density (90–110 μg/mg NP by microBCA assay). This observation can be explained by the availability of a large excess of reactive maleimide functions at the surface, with the limiting factor being steric crowding of the protein itself.

Bottom Line: We have used a silica - PEG based bionanoconjugate synthetic scheme to study the subtle connection between cell receptor specific recognition and architecture of surface functionalization chemistry.Extensive physicochemical characterization of the grafted architecture is capable of capturing significant levels of detail of both the linker and grafted organization, allowing for improved reproducibility and ultimately insight into biological functionality.Our data suggest that scaffold details, propagating PEG layer architecture effects, determine not only the rate of uptake of conjugated nanoparticles into cells but also, more significantly, the specificity of pathways via which uptake occurs.

View Article: PubMed Central - PubMed

Affiliation: Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland.

ABSTRACT
We have used a silica - PEG based bionanoconjugate synthetic scheme to study the subtle connection between cell receptor specific recognition and architecture of surface functionalization chemistry. Extensive physicochemical characterization of the grafted architecture is capable of capturing significant levels of detail of both the linker and grafted organization, allowing for improved reproducibility and ultimately insight into biological functionality. Our data suggest that scaffold details, propagating PEG layer architecture effects, determine not only the rate of uptake of conjugated nanoparticles into cells but also, more significantly, the specificity of pathways via which uptake occurs.

No MeSH data available.