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Nanomaterial-Protein Interactions

The translation of nanomaterial-based therapeutics to clinical applications remains a challenge. Many hurdles 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 NM-1) on adsorbed protein behavior, including protein orientation, conformation and resulting biomedical function.

Enzyme Structure and Function on Nanoscale Materials

We have studied (in collaboration with Prof. Richard Siegel) the effect of gold nanoparticle morphology on the structure and function of adsorbed lysozyme (Lyz) and α-chymotrypsin (ChT)1. Gold nanospheres (AuNS; Figure NM-1a) were synthesized with diameters 10.6 ± 1 nm, and gold nanorods (AuNR; Figure NM-1b) were synthesized with dimensions of (10.3 ± 2) × (36.4 ± 9) nm1. 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-octahedra (AuNO, Figure NM-1c) and gold nanocubes (AuNC, Figure NM-1d)2,3. Both nanoparticles have flat surfaces, although with {111} and {100} surface crystallography, respectively. As illustrated in Figure NM-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 surfaces4,5. We developed a methodology of chemical modification combined with mass spectroscopy (Figure NM-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.

Nanoscale curvature plays a critical role in nanostructure-biomolecule interactions, yet the understanding of such effects in concave nanostructures is still very limited. Because concave nanostructures usually possess convex surface curvatures as well, it is challenging to selectively study the proteins on concave surfaces alone. In this work, we have developed a novel and facile method to address this issue by desorbing proteins on the external surfaces of hollow gold nanocages (AuNG), allowing the selective characterization of retained proteins immobilized on their internal concave surfaces. The selective desorption of proteins was achieved via varying the solution ionic strength, and was demonstrated by both calculation and experimental comparison with non-hollow nanoparticles. This method has created a new platform for the discrete observation of proteins adsorbed inside AuNG hollow cores, and this work suggests an expanded biomedical application space for hollow nanomaterials6.

Nanobiotechnology Fig1

Figure NM-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.

Nanobiotechnology Fig2

Figure NM-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.


Nanobiotechnology Fig3

Figure NM-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.

Protein-Nanomaterial Conjugates

Our earlier research in collaboration with Prof. Ravi Kane (now at Georgia Tech) was focused on unique properties of carbon nanotube-nanoparticle conjugates. To preserve the intrinsic properties of nanotubes, it is important to develop methods for synthesizing such hybrids under mild conditions. We have prepared silver nanoparticle-nanotube conjugates by utilizing the ability of proteins to control and direct the formation of silver nanoparticles on nanotube surfaces7. 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 (Figure NM-4). Proteins also present a rich variety of functional groups that may be used as orthogonal reactive handles for further nanotube functionalization.

Nanobiotechnology Fig4

Figure NM-4. TEM characterization of MWNT-silver nanoparticle conjugates.

We have also discovered what appears to be a unique property of nanomaterials – their ability to enhance protein activity and stability in strongly denaturing environments8-11. 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 95oC 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 nanoscale supports 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 (Figure NM-5). 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.

Nanobiotechnology Fig5

Figure NM-5. 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.


  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 Lett. 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”, J. Phys. Chem. Lett. 3, 3149–3158.
  5. X. Qian, A. Levenstein, J.E. Gagner, J.S. Dordick, and R.W. Siegel (2014), “Protein immobilization in hollow nanostructures and investigation of adsorbed protein behavior”, Langmuir 30, 1295-1303.
  6. X. Qian, U. Rameshbabu, J.S. Dordick, and R.W. Siegel (2015), “Selective characterization of proteins on nanoscale concave surfaces,” Biomaterials 75, 305-312.
  7. 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.
  8. 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.
  9. 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.
  10. 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", J. Am. Chem. Soc., 128, 1046-1047.
  11. 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", Biotechnol. Bioeng., 95, 804-811.