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The translation of nanomaterial-based therapeutics to clinical applications remains an elusive target. Many challenges remain, including potential toxicity, immunogenicity, and decreased efficacy. For these reasons, we have focused on elucidating fundamental interactions between nanomaterials and the biological environment. Specifically, we have begun to study the influence of nanomaterial topography and crystal structure (Figure 1) on adsorbed protein behavior, including protein orientation, conformation and resulting biomedical function.

We have studied the effect of gold nanoparticle morphology on the structure and function of adsorbed lysozyme (Lyz) and α-chymotrypsin (ChT). Gold nanospheres (AuNS; Figure 1a) were synthesized with diameters 10.6 ± 1 nm, and gold nanorods (AuNR; Figure 1b) were synthesized with dimensions of (10.3 ± 2) × (36.4 ± 9) nm. Under saturating conditions, proteins adsorb with a higher surface density on AuNR when compared to AuNS. In the case of Lyz, adsorption on AuNS and AuNR resulted in a 10% and 15% loss of secondary structure, respectively, leading to conjugate aggregation and greatly reduced enzymatic activity. ChT retained most of its secondary structure and activity on AuNS and AuNR at low surface coverages; however, as protein loading approached monolayer conditions on AuNR, a 40% loss in secondary structure and 86% loss of activity was observed. Subsequent adsorption of ChT in multilayers on the AuNR surface allowed the conjugates to recover activity and remain stable. It is clear that AuNP morphology does affect adsorbed protein structure.

Further studies were directed at more unique nanoscale topographies; namely, gold nano-octahera (AuNO, Figure 1c) and gold nanocubes (AuNC, Figure 1d). Both nanoparticles have flat surfaces, although with {111} and {100} surface crystallography, respectively. As illustrated in Figure 2, proteins achieve higher packing density on the {100} facet, accompanied by greater loss in native structure and enzymatic activity. It is clear that AuNP morphology and crystallography do affect adsorbed protein structure; a better understanding of these differences will be essential to engineer fully functional nan-bio conjugates. We have also investigated protein orientation on nanoscale surfaces. We developed a methodology of chemical modification combined with mass spectroscopy (Figure 3) to elucidate the orientation of cytochrome c, lysozyme and ribonuclease adsorbed onto silica nanoparticles. We discovered that protein orientation is also affected by nanoparticle surface curvature and protein structure. Along these lines, we are engaged in developing tools and techniques that can further enhance our understanding of nano-bio interaction and would also help in developing nanomaterials and eventually nano-bio conjugates with customized properties.

 

Figure 1. Transmission electron microscopy images of gold (A) nanospheres and (B) nanorods, and scanning electron micrographs of (C) nanooctahedra, (D) nanocubes, (E) nanocages, and (F) nanoplates.

 

Figure 2. Schematic representation of lysozyme (Lyz, blue) and α-chymotrypsin (ChT, green) adsorption onto gold nano-octahedra (AuNO) and gold nanocube (AuNC). In Region I, protein adsorbed onto the nanoparticle; for octahedra, protein initially adsorbs onto the relatively flat Au{111} facet, which is then followed by adsorption onto the edges. ChT-AuNO has relatively low surface coverage and experiences no change in protein structure upon adsorption. Lyz-AuNO develops monolayer coverage (Region IIa) and then undergoes aggregation (Region IIb), as shown in the final conjugate form; both steps are mediated by protein-surface and protein-protein interactions. Lyz-AuNO interactions are similar, except there is only one site of adsorption on AuNC, so both proteins bind indiscriminately. Lyz-AuNC undergoes a similar process as AuNO; however, CHT-AuNC develops a higher surface coverage, potentially on the flat Au{100} facets, such that both protein-surface and protein-protein interactions affect protein structure (Region IIa). It is possible that ChT adsorbed on the edges of AuNC retains most of its structure and relative activity. The final conjugate structures indicate that Lyz interaction with AuNC and AuNO produces unstable conjugates, while ChT-AuNO remains stable, and ChT-AuNC remains stable with some association between small groups of particles.

 

Figure 3. Illustration of using chemical modification combined with proteolysis-mass spectrometry to determine protein orientation on nanoparticles. The proteins interacted with silica nanoparticles (SNPs) through preferential adsorption sites, which are dependent on SNP diameter; 4-nm SNPs induce greater structural stabilization than 15-nm particles, presumably due to greater surface curvature of the former. These results suggest that nanoparticle size and protein structure influence protein orientation on SNPs.

 

Reference

  1. J.E. Gagner, M.M. Lopez, J.S. Dordick, and R.W. Siegel (2011) “Effect of gold nanoparticle morphology on adsorbed protein structure and function”, Biomaterials 32, 7241-7252.
  2. J.E. Gagner, X. Qian, M.M. Lopez, J.S. Dordick, and R.W. Siegel (2012) “Effect of gold nanoparticle structure on the conformation and function of adsorbed proteins”, Biomaterials 33, 8503-8516.
  3. S. Shrivastava, J.H. Nuffer, R.W. Siegel and J.S. Dordick (2012) “Position-Specific Chemical Modification and Quantitative Proteomics Disclose Protein Orientation Adsorbed on Silica Nanoparticles”, Nano Letters 12, 1583–1587.
  4. J.E. Gagner, S. Shrivastava, X. Qian, J.S. Dordick, and R.W. Siegel (2012) “Engineering Nanomaterials for Biomedical Applications Requires Understanding the Nano-Bio Interface: A Perspective”, Journal of Physical Chemistry Letters 3, 3149–3158.

 


Preparation of functional MWNT-Protein-Silver Nanoparticle

 

 

Carbon nanotube-nanoparticle conjugates are widely studied for their interesting electrical, mechanical, thermal and optical properties. To preserve the intrinsic properties of nanotubes, it is important to develop methods for synthesizing such hybrids under mild conditions. Herein, we prepare silver nanoparticle-nanotube conjugates by utilizing the ability of proteins to control and direct the formation of silver nanoparticles on nanotube surface. Proteins such as bovine serum albumin and poly-L-lysine mediated the formation of silver nanoparticles whereas glycosylated proteins such as a1-acid glycoprotein (AGP) and enzyme soybean peroxidase (SBP) mediated the formation of silver nanoparticles only following their enzymatic deglycosylation. Furthermore, deglycosylated SBP (d-SBP) retained enzymatic activity after nanoparticle formation. Thus, we have developed a route for forming hybrid multifunctional nanostructures under mild conditions. Proteins also present a rich variety of functional groups that may be used as orthogonal reactive handles for further nanotube functionalization.

 

Figure 1. TEM characterization of MWNT-silver nanoparticle conjugates

 

We have recently discovered what appears to be a unique property of nanomaterials – their ability to enhance protein activity and stability in strongly denaturing environments. Specifically, we have observed that single-walled carbon nanotubes (SWNTs) can significantly enhance enzyme function and stability in strongly denaturing environments. For instance, the half-life of soybean peroxidase (SBP) adsorbed onto SWNTs at 95 oC was ~90 min, 10-fold greater than that of the enzyme in solution and ~2-times that of SBP adsorbed on graphite flakes or other flat supports. Further enhancement in enzyme stability was achieved by using spherical nanosupports such as C60 fullerenes, gold, and silica nanoparticles (curved in all dimensions) than on cylindrical supports such as SWNTs (curved in only one dimension) and flat supports. We hypothesize this stability enhancement to be due to reduced lateral interactions between adjacent adsorbed proteins which contribute to protein deactivation in harsh environments and that these unfavorable interactions are suppressed on highly curved nanomaterials relative to flat surfaces.

 

Reference

  1. S. S. Bale, P. Asuri, S. S. Karajanagi, J. S. Dordick, and R. S. Kane (2007) "Protein-Directed Formation of Silver Nanoparticles on Carbon Nanotubes", Adv. Mater., 19, 3167-3170.

 


 Nanotube-Mediated Protein Stabilization

 

Figure 1. Left. Schematic showing the hypothesized interactions between enzyme molecules on surfaces. Decreased lateral interactions on a curved surface result into reduced deactivation and hence relatively higher stabilization on nanoscale materials. Right. Enhanced stability of enzymes on nanomaterials conjugates – (●) SBP on SWNTs, (■) SBP on micron-sized graphite flakes, and (o) SBP in solution.

 

We have recently discovered what appears to be a unique property of nanomaterials – their ability to enhance protein activity and stability in strongly denaturing environments. Specifically, we have observed that single-walled carbon nanotubes (SWNTs) can significantly enhance enzyme function and stability in strongly denaturing environments. For instance, the half-life of soybean peroxidase (SBP) adsorbed onto SWNTs at 95 oC was ~90 min, 10-fold greater than that of the enzyme in solution and ~2-times that of SBP adsorbed on graphite flakes or other flat supports. Further enhancement in enzyme stability was achieved by using spherical nanosupports such as C60 fullerenes, gold, and silica nanoparticles (curved in all dimensions) than on cylindrical supports such as SWNTs (curved in only one dimension) and flat supports. We hypothesize this stability enhancement to be due to reduced lateral interactions between adjacent adsorbed proteins which contribute to protein deactivation in harsh environments and that these unfavorable interactions are suppressed on highly curved nanomaterials relative to flat surfaces.

 

Reference

  1. P. Asuri, S. S. Karajanagi, H. Yang, T-J Yim, R. S. Kane, and J. S. Dordick (2006) "Increasing Protein Stability through Control of the Nanoscale Environment", Langmuir, 22, 5833-5836.
  2. P. Asuri, S. S. Karajanagi, A. A. Vertegel, J. S. Dordick, and R. S. Kane (2007) "Enhanced Stability of Enzymes Adsorbed onto Nanoparticles", J. Nanosci. Nanotech., 7, 1675-1678.
  3. P. Asuri, S. S. Krajanagi, J. S. Dordick, and R. S. Kane (2006) "Directed Assembly of Carbon Nanotubes at Liquid-Liquid Interfaces: Nanoscale Conveyors for Interfacial Biocatalysis", Journal of American Chemical Society, 128, 1046-1047.
  4. P. Asuri, S. S. Karajanagi, E. Sellitto, D-Y Kim, R. S. Kane and J. S. Dordick (2006) "Water-Soluble Carbon Nanotube-Enzyme Conjugates as Functional Biocatalytic Formulations", Biotechnology and Bioengineering, 95, 804-811.