Engineering Nanomaterials for Biomedical Applications Requires

Perspective pubs.acs.org/JPCL

Engineering Nanomaterials for Biomedical Applications Requires Understanding the Nano-Bio Interface: A Perspective Jennifer E. Gagner,†,‡,⊥ Siddhartha Shrivastava,†,⊥ Xi Qian,†,‡,⊥ Jonathan S. Dordick,†,‡,§,∥,⊥ and Richard W. Siegel*,†,‡,⊥ †

Rensselaer Nanotechnology Center, Rensselaer Polytechnic Institute, Troy, New York 12180, United States Department of Materials Science and Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, United States § Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, United States ∥ Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180, United States ⊥ Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, United States ‡

ABSTRACT: The promise of nanobiomaterials for diagnostic and therapeutic biomedical applications has been widely reported throughout the scientific community, and great strides have been made in those directions. And yet, the translation of nanomaterial-based therapeutics to clinical applications remains an elusive target. Many challenges have blocked the usage of nanomaterials in biomedicine, including potential toxicity, immunogenicity, and decreased efficacy. In order to overcome some of these issues, detailed studies have been undertaken to understand fundamental interactions between nanomaterials and the biological environment. In particular, recent developments in nanoparticle synthesis, a better understanding and control over nanoparticle surface chemistry, as well as the organization of that chemistry on the nanoparticle surface, has allowed researchers to begin to understand how spatial arrangement of atomic and molecular species at an interface can affect protein adsorption, structure, and subsequent biological outcomes. This perspective strives to identify ways in which the nanomaterial interface can be controlled to affect interactions with biomolecules for beneficial biomedical applications.

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standing of polymer-nanocomposites can also be applied to understanding the interactions of nanobiocomposites, and vice versa. Controlling the interaction between biological molecules and materials is the perspective of this article; understanding and controlling the subsequent action of these nanobioconjugates is an ongoing topic in the scientific community. Recent developments in our understanding of nanoparticle synthesis and chemical modification19 have allowed unprecedented control over nanoparticle properties, allowing identification of how specific parameters can affect protein adsorption20 and subsequent structural changes.21 In particular, controlling protein orientation and changes in conformation can lead to control over resulting nanobiomaterial function.22 Furthermore, recent studies have shown that fine control over nanoparticle surface chemistry23 can affect nanoparticle assembly,24,25 protein attachment,26 and further interactions.27,28 All of these parameters need to be controlled in order to not only prevent nanobiobiomaterial toxicity, but also to ensure beneficial biological outcomes. This article will review some recent advances in the application of nanomaterials to biomedicine, as well as the suite of nanostructures available for different applications. We then explore the current understanding of

aterials scientists operate in a unique area of the scientific environment. Under the correct lens, almost anything may be considered a material. With the wide variety of material characterization techniques available, much can be understood about the structure and resulting properties of the material in question. Biological molecules add an extra level of complexity to this fundamental idea, as the unique structure of many biological entities such as proteins, enzymes, DNA, and carbohydrates confer exquisite functionality that scientists hope to emulate. Our understanding thus far has allowed the manipulation of these “biopolymers” and their conjugation to other materials in order to improve on their desired properties. This may combine the functionality of both materials, i.e., confer biological function on an inorganic material, or the converse. Initial studies in this area noted that the nano length-scale is ideal for effecting change at the biomolecular level.1,2 Currently, many biomedical applications of nanomaterials rely on the functionality of attached biomolecules in order to target,3−6 deliver,7−9 image,10−13 or otherwise mediate interactions with the biological environment. However, attachment to the nanomaterial, whether by physical adsorption or by covalent attachment, results in changes to the structure of the biological molecule. This can result in loss or alteration of biological function,14−16 or the presentation of new epitopes,17,18 creating an entirely new nanobiomaterial that can interact with the biological milieu in unanticipated ways. Although the principal repeat components of biomolecules differ from other “artificial” polymeric materials, our under© 2012 American Chemical Society

Received: August 23, 2012 Accepted: October 11, 2012 Published: October 11, 2012 3149

dx.doi.org/10.1021/jz301253s | J. Phys. Chem. Lett. 2012, 3, 3149−3158

The Journal of Physical Chemistry Letters

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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. (F) Reprinted with permission from ref 53.

et al.39 have developed a sensitive DNA biosensor based on multilayer metal−molecule−metal nanojunctions that exploit surface-enhanced Raman techniques to detect concentrations of DNA as low as 10−19 M. Many of the diagnostic and therapeutic applications of colloidal nanoparticles require the targeting of the nanoparticle to the organ, cell, protein, or other site of interest. This is accomplished by attachment, through either covalent attachment or physical adsorption of a “targeting” biomolecule to the nanoparticle surface. In many cases, these applications are foiled by a variety of issues, in particular, the relatively low nanoparticle concentration at the target site or the accumulation of the particle in other nontargeted organs, such as the liver or spleen. Surface functionalization of the nanoparticles with polyethylene glycol can increase circulation time and nanoparticle stability,12 but may interfere with the targeting moiety attached to the nanoparticle surface. Furthermore, upon introduction to the biological environment, more proteins will adsorb onto the nanoparticle surface, forming a protein “corona” that has been widely investigated.40,41 It is known that in most cases a cell’s interaction with the nanoparticle is actually mediated by the protein layer adsorbed on the nanoparticle surface.42 Upon adsorption, the protein may change conformation, altering its function and in effect creating a new biomaterial that combines the material properties of the nanostructure and the new biological function of the protein. This function can be beneficial, such as increased interaction with cells to promote phenomena of osteoblast adhesion35 or increased enzymatic activity.15 However, in many cases, changes to protein function can result in the formation of aggregates,20,43 fibrillation,16 or immunogenicity,28 which can have unintended and harmful effects. In order to control these interactions, it is essential to understand how specific nanoparticle properties affect changes in protein structure and function, and then to continue to study how these changes in function affect the biological environment. A wide variety of different nanostructures have been employed for biomedical applications. In particular, silica, iron oxide, and gold nanoparticles offer the benefit of increased biocompatibility over other nanoparticles such as quantum dots, as well as ease of synthesis and particularly useful properties. Due to the number of different synthetic methods to precisely control size, morphology, and crystallography, gold nanoparticles (AuNP) in particular present a unique system in which to determine the effect of each of these parameters on adsorbed biomolecules.19 “Finely divided gold particles” were first investigated by Faraday

how nanoparticle properties can affect interactions with biomolecules, how these properties can be engineered, and the challenges to creating nanobiomaterials with desired biological functions. The potential myriad applications of nanostructures in biomedicine have been the subject of much excitement over the past two decades. Although nanomaterials such as colloidal gold have been prescribed for a variety of ailments such as ulcers, syphilis, epilepsy,29 and arthritis30 for thousands of years, more recent work has outlined the unique influence of nanomaterials on the biological environment. Length scales incorporated into nanostructured materials, here defined as approximately 100 nm or less, are uniquely suited to interacting with biological molecules, which are designed to detect features in that size regime. Indeed, the interactions between proteins and colloidal particles are usually limited to long-range van der Waals forces, electrostatic interactions, solvation and solvophobic forces, and depletion forces.31−33 Furthermore, many nanoparticle properties, which can vary dramatically from the properties of the bulk material, may affect protein adsorption and conformation. In particular, specific properties, including size and curvature, internal and external crystallinity,34 and surface chemistry, can be unique on the nanoscale. These properties may be influenced by the environment, which can in turn effect selective binding reactions as well as temperature or pH-dependent amphoteric or amphiphilic behavior. Additional stabilizing ligands on the nanomaterial surface may further complicate the interaction between the nanostructure surface and the biomolecule. In many cases, the incorporation of inorganic nanomaterials into the biological environment is necessary in order to confer specific properties onto the conjugate that are not entirely or easily accessible via organic species. Implant structures such as orthopedic prosthetics have been shown to benefit greatly from nanostructured surfaces that promote osteoblast integration and limit bacterial adhesion and inflammation, while at the same time matching the material properties of the surrounding bone system.35 Colloidal nanoparticles have been used in a variety of diagnostic and therapeutic applications, which make use of the specific material properties of the nanoparticle, in particular strong fluorescence,36 resistance to photobleaching,13 and sensitive detection based on properties such as surface plasmon resonance (SPR).37 The SPR associated with gold nanoparticles is extremely sensitive to particle size, shape and the local dielectric environment,38 and allows probing of the nanoscale volume immediately surrounding the particle. For example, Hu 3150



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in 1857,44 and a detailed method to precisely control the size of gold nanospheres was first delineated by Turkevich45 in 1953 and Frens46 in 1973. Since that time, a variety of synthetic methods for AuNP morphologies, including nanorods (AuNR),47,48 nanocubes (AuNC), nanooctahedra (AuNO),49,50 nanocages (AuNG),51 and nanoplates,52,53 have been described (Figure 1). Nanorods have been synthesized primarily through seedmediated, surfactant-controlled methods that employ cetyltrimethylammonium bromide to control the AuNR aspect ratio. More complex shapes such as nanocubes and nanooctahedra can be easily synthesized via a polyol process, in which underpotential deposition of silver on the AuNP surface controls the rate of gold facet growth to produce the desired shape.50,54 AuNGs have been extensively investigated by Xia et al.,55 and may be produced with fine control over their size and pore thickness by a galvanic replacement method, with silver nanocubes acting as a sacrificial substrate. Although approximated as specific shapes, AuNPs are crystalline in nature, and the variety of different nanoparticle morphologies available are made possible due to control over the crystal growth process. Gold is a face-centered cubic crystal with a lattice parameter of 4.08 Å and principal low-index crystallographic planes {111}, {100} and {110} usually presented on the surface of each nanoparticle. Each crystal facet possesses a different surface energy (γ), specifically γ{111} < γ{100 } < γ{110}. During nanoparticle formation, the rate of growth along any particular crystallographic direction can be modified by the adsorption of various additives, such as silver. Commonly, AuNRs are bounded by {100/110} facets on the sides and {100} facets on the ends, while shapes such as AuNC are enclosed in {100} facets, and AuNO present {111} facets on each face. Higher-index planes on AuNP have recently been synthesized using further underpotential growth methods, allowing new, even concave morphologies to be synthesized.56 Some form of stabilization is required to prevent AuNPs from aggregating to decrease the overall surface area and energy. Generally, the nanoparticles are protected via strong thiolate chemistry, with alkanethiols stabilizing the AuNP surface, usually possessing an additional functional group such as −COOH or −NH2 for further chemistry. A significant amount of work has been undertaken to understand the nature of the thiol-Au bond, particularly in the formation of self-assembled monolayers.57,58 Previous literature fully detailed the ligand structure of selfassembled monolayers (SAMs) on specific gold crystallographic planes. In particular, early diffraction studies of thiolates adsorbed on Au{111} surfaces indicate that the symmetry of the chemisorbed sulfur atom is hexagonal in nature with a spacing of 4.97 Å,59 primarily binding in the hollows of the energetically heterogeneous, close-packed Au{111} surface. The final packing structure of the ligand layer is also dictated by van der Waals interactions between the alkyl chains and the chain length.60 Alkyl chains chemisorbed onto the Au{100} surface tend to form a square array with a spacing of 4.54 Å.61 These studies indicate that the surface protection of Au by thiolates is a complex topic, with the final monolayer arrangement dependent upon the underlying structure of the Au crystal as well as the chemistry and length of the ligand. When determining the surface arrangement of ligands on a gold nanoparticle, additional considerations such as particle defect structures and surface curvature must be taken into account.62 The effect of mixing ligand chemistry and chain length on the nanoparticle surface has been investigated by Jackson et al.,63 beginning with the synthesis of mixed-monolayer protected

nanoparticles in 2004. Extensive characterization of the chemically functionalized nanoparticle surface showed that a SAM of hydrophobic and anionic ligands coated on an ∼6 nm particle would arrange into regular ribbon-like domains of alternating composition. 64 This phenomenon was characterized via scanning tunneling microscopy and later confirmed by molecular dynamics simulation (Figure 2A).65

Figure 2. (A) Scanning tunnelling microscopy investigation of mixedmonolayer protected 6 nm gold particles, with overlaid schematic to illustrate ligand stripes. (B) Computer simulation of ligand stripe formation on AuNRs, confirmed by atomic force microscopy (C) (all scale bars: 3 nm). (D) Schematic illustrating increased conformational entropy afforded by ligand stripe formation. (A) Reprinted by permission from MacMillan Publishers Ltd.: Nature Materials, ref 23, copyright 2009. (B−D) Adapted from ref 67 with permission from the Royal Chemical Society.

Further investigation of SAMs led to the understanding that the striping was due to differences in configurational entropy between the two ligands.65 In all cases, a binary mixture of a hydrophobic ligand, such as 1-octanethiol (OT), and an anionic ligand (6-mercaptohexan-1-ol, MUS) were adsorbed on the nanoparticle surface in different ratios. It was found that the stripes depend on the degree of incompatibility and length difference between the two ligands. The bending of long ligand chains over short ones allows a gain in conformational entropy, making separation into 2−5 nm striped domains energetically favorable.23 Mixtures of ligands with equal lengths or a small difference in lengths showed interface-minimizing macrophase separation. This effect was found to be reproducible on AuNRs,66 with the stripe formation and placement dependent upon the diameter of the rod (Figure 2B,C). As the nanorod diameter increased, the free volume associated with the ligand arising from the surface curvature is minimal, and interfaces are required for the longer ligands to maximize their entropy while minimizing the system overall free energy (Figure 2D). It has been hypothesized that this effect could be extended to many nanoparticle morphologies, where the longer ligand would phase-separate to areas of higher curvature, and smaller ligands would remain on the relatively flat surfaces. Such information can be combined with other studies looking at the effect of nanoparticle size, curvature, and ligand charge to determine the best parameters for a designed nanobioconjugate. However, in order to understand how each of these parameters affects protein adsorption, it is necessary to thoroughly characterize the nanoparticle sample. Several national and international organizations have begun to develop guidelines and definitions that will assist normalization of standards across the field of nanobiotechnology, such as those set out by the National Nanotechnology Initiative (NNI) in the United States.67 Although several nanobased formulations are currently on the market, these products are being evaluated using the same regulatory requirements as other therapeutics. It is evident that 3151



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Figure 3. (A) Schematic assembly and chemical structure of linear dendritic polymers (LDPs) into “patchy” micelles, where PBLA = poly(benzyl-Laspartic acid), PED = polyester dendron, and PEG = (polyethylene glycol). (B) Characterization of folate clusters on LDP mixed micelle system, with clusters of DTPA-Fe3+ on the surface showing the areas with free folic acid. Reprinted with permission from ref 6. Copyright Wiley-VCH, 2010.

like interactions with specific glycoproteins and glycolipids, which rely on a higher density and controlled spatial organization of the receptor ligand. Although the idea of polyvalency has been exploited to enhance target binding and delivery efficacy, only recently has the unique opportunity to effect and control biological interactions through spatial arrangement of specific ligands on a nanoparticle surface come to be explored. In a recent study by Poon et al.,6 the effect of organization of folate clusters on “patchy” micellar structures on cellular uptake and targeting was undertaken. Folate was conjugated to polyethylene glycol and incorporated into a poly benzyl-Laspartate polypeptide chain with a polyester dendron, at different concentrations. Micelles were formed (80−90 nm) that had the same amount, but different spatial arrangements of folate in variable size clusters (Figure 3). This allowed comparisons to determine the effect of folate spatial presentation on cellular internalization. It was found that a folate ligand cluster of ∼3 ligands enhanced uptake of the nanoparticles due to the resulting higher avidity and longer residence times on the cell membrane.6 Furthermore, it was shown that spatial arrangement can be optimized to increase ligand−receptor interactions, increasing overall binding cooperativity. Studies such as this outline how nanoparticle polyvalency can be used to control biological interactions with cells, but it is possible that more fundamental control over aspects of protein orientation and conformation can be controlled through the same concept.

additional characterization is needed to ascertain the safety and efficacy of nanotherapeutics,68 and to address these issues facilities such as the Nanotechnology Characterization Laboratory (NCL) have been developed to assist in the accelerated clinical translation of nanotechnology-derived formulations. Further information regarding clinical translation of nanobased therapeutics and working with regulatory agencies such as the Food and Drug Administration can be found at the NCL Web site.69 In order to understand protein−nanoparticle interactions and thus advance our understanding of both possible applications and possible toxicity of nanobioconjugates, it is essential that nanomaterials are characterized sufficiently prior to experimentation and that sufficient information is provided to allow assessment of the validity and suitability of the characterization methods. For more information on this important subject, the authors would direct readers to the recent review by Krug and Wick.70 While the potential benefits of nanotechnology for various drug delivery systems has been identified, many hurdles remain before nanotechnology-based therapeutics can be successfully translated to the clinic. Many physical properties of nanoparticles such as the inherent curvature and surface chemistry are within the unique size regime that can easily be “read” by the biological environment. Many studies have investigated the effect of nanoparticle size and curvature on a variety of biological interactions, including protein adsorption,20,71 changes to protein conformation,15,72 uptake into cells,73,74 and inherent toxicity.75 Further studies have investigated the effect of nanoparticle ligand charge,76−78 and hydrophobicity.79 These studies have been conducted in both physiological80 and denaturing conditions.81,82 Despite all of this information, designing perfectly engineered nanobioconjugates for specific biomedical applications remains elusive. Recent advances in applying careful characterization to protein− nanoparticle interactions have begun to elucidate how nanomaterial properties can really be used to control proteinnanoparticle interactions and subsequent function. Many biological systems interact through multiple specific, simultaneous association events, a phenomenon known as polyvalency.83 This set of interactions can have dramatic effects on ligand−receptor binding kinetics and the resulting biological responses, and can depend greatly on the spatial conformation of the reacting ligand. Many viruses, such as the influenza virus,84 and bacteria such as Escherichia coli,85 utilize polyvalent interactions to attach to tissues such as epithelial cells. Some degree of specificity in these interactions is conferred by lectin-

Controlling nanoparticle morphology creates surfaces with specific energetics and chemical structures, which can then be utilized to control assembly and ligand presentation or spatial arrangement on the nanoparticle surface. As previously mentioned, in-depth studies of SAMs on gold surfaces and nanoparticles have shown that the ligand itself may present a structure-packing density, organization, or charge distribution with which a protein could then interact. Interest is beginning to turn in the direction of understanding how atomicscale changes on the surface may affect adsorbed protein orientation and structure. An initial study by Hung et al.26 3152



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Figure 4. (A) Adsorption isotherms for α-chymotrypsin (ChT) adsorbed on AuNO and AuNC, with adsorption on AuNO fit with a Bilangmuir isotherm and adsorption on AuNC fit with a Langmuir isotherm. (B) Relative specific activity of ChT adsorbed on AuNOs and AuNCs at different estimated surface coverages. It is worth noting that AuNOs saturated at relatively low surface coverage compared to AuNCs. Figure adapted from ref 82.

conditions and on relatively flat surfaces, appears to be greatly dependent on the protein density (i.e., coverage) on the nanoparticle surface. Controlling nanoparticle morphology creates surfaces with specific energetics and chemical structures, which can then be utilized to control assembly and ligand presentation or spatial arrangement on the nanoparticle surface. Utilizing nanoparticles with controlled variations in curvature, chemical structure such as “stripes” or “patches”, will allow a greater understanding of how these properties will affect adsorbed protein orientation and conformation. By controlling interactions with this level of specificity and spatial arrangement, it is possible that nanoparticles can be targeted to biological receptors with greater affinity. Furthermore, an enhanced understanding of how specific nanobioconjugates can promote toxicity or unintended events in the biological environment can be investigated. Finally, control over biomolecule placement and orientation could allow development of nanobioconjugates that are more capable of responding to biological stimuli such as changes in pH, hypoxia, and others, in order to effect a change in the biological environment.

examines, through both protein assays and computational molecular dynamics simulation, the effect of nanoscale ordering on protein adsorption. Mixed monolayer-protected metallic nanoparticles were synthesized with different ratios of 6mercapto-1-hexanol (MH) and OT, resulting in ligand structures with different charge densities and contact angles dependent upon the ratio of the two ligands. The adsorption of cytochrome C (Cyt C), increased with increasing hydrophilicity (%MH content) but the protein did not undergo significant disruption to its secondary structure upon adsorption to the nanoparticle. The amphipathic character of lysine residues, which were instrumental in the protein binding, enabled the protein to form close contacts with both polar and nonpolar solvents depending on the width of the stripe formation of the nanoparticle surface. Stronger binding affinities were obtained on the “thicker” stripes in the 1:1 MH:OT surface. Protein orientation would be altered to accommodate “matching” of the hydrophobic/hydrophilic patches, which can result in changes to the secondary and tertiary structure of the protein as it attempts to “lattice match” the ligand structure. Recent work within our group86 has identified that protein adsorption onto nanoparticles can be sensitive not only to size and morphology, but also to nanoparticle crystallography, and potentially the resulting ligand density. In our study, carefully controlled populations of AuNCs and AuNOs were synthesized to create nanoparticle populations that differed primarily in crystallography. Evidence from adsorption isotherms of lysoyzme (Lyz) and α-chymotrypsin (ChT) onto the nanoparticle surface indicates that protein may adsorb with a higher binding affinity to Au{111} over Au{100}, with adsorption occurring initially on the relatively flat crystal facets of the two particle types before proceeding to their edges and corners (Figure 4A). Lysozyme adsorbed on both AuNO and AuNC formed a dense protein layer, resulting in significant structural perturbation and loss of activity regardless of surface coverage. ChT adsorbed on AuNO showed no evidence of loss of structure or activity, indicating that adsorption and protein−surface interactions did not significantly impact the protein. However, ChT adsorption on AuNC, which had a much higher proportion of relatively flat surface, resulted in significant loss of structure and activity at 30−40% surface coverage (Figure 4B). This may correspond to protein adsorption on the relatively flat Au{100} surfaces; subsequent protein adsorption on the AuNC edges and corners, although possessing about the same affinity as the sides, allowed a recovery of ChT structure and activity. Therefore, changes to protein structure and function, in biological

Control over biomolecule placement and orientation will allow development of conjugates more capable of responding to biological stimuli. While the toolbox of characterization techniques used to determine nanoparticle morphology, surface chemistry, and even ligand structure has rapidly expanded over the past decade, it is still very difficult to directly characterize the nature of the protein−nanoparticle interaction. Many methods currently employed, such as circular dichroism, activity assays, and even highly sensitive methods such as Fourier transform infrared analysis and isothermal titration calorimetry, do not capture localized, specific information about which amino acid residues are interacting with the nanoparticle surface. Circumstantial experiments such as changes in activity triggered by pH and nanoparticle charge can be used to determine the position of the active site, but in these studies one cannot absolutely identify interacting residues and points of attachment. Current methods such as Raman spectroscopy and solid-state NMR may be able to identify with specificity the points of interaction and, in turn, 3153



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Figure 5. Schematic illustrating the parameters that can be designed to control protein adsorption, subsequent structure, and orientation. The nanomaterial is usually selected specific to a particular application; control over synthesis can produce nanoparticles with specific crystal facets due to their morphology. These morphologies can then possess curvatures that, combined with their crystal structure, can then control the placement of ligands on the nanoparticle surface. The terminal groups on these ligands can then be used to adsorb proteins in specific locations or orientations on the nanomaterial surface.

specific nanobiomaterial’s effect on the binding and conformation of fibrinogen, and then followed subsequent consequences of that interaction. Fibrinogen was found to exhibit high-affinity binding to negatively charged poly(acrylic acid)-conjugated gold nanoparticles (PAA-GNP) with diameters of 5, 10, and 20 nm, remaining tightly bound after extensive washing. Low stoichiometric binding on the 5 nm particle (0.5 particles/protein) suggested that the nanoparticle may interact with specific sites on the protein. The protein is negatively charged at physiological pH (pI = 5.5),89 so its high-affinity interaction with the negatively charged 5 nm PAA-GNP was unexpected. One sequence in particular, amino acids 377−395 in the C-terminus of the γ chain (γ377−395), is located in the D domain and is responsible for the recruitment of macrophages and leucocytes, and is thus implicated in inflammation.90 This is achieved by the interaction of γ377−395 with the Mac-1 receptor; the positively charged Cterminus of the α chain is located adjacent to the D domain. It was found that 5 nm PAA-GNP induces changes in the fibrinogen structure that expose the γC terminus, similar to previous reports for flat surfaces, resulting in increased binding to the Mac-1 receptor. However, similar results were not found for the larger nanoparticles (10 and 20 nm). Although binding to the 20 nm particle similarly perturbed the secondary structure of fibrinogen, the resulting binding to the Mac-1 receptor was dependent on protein loading. At saturation (∼7 fibrinogen molecules/particle), steric hindrance prevented the exposed C-terminus of the γ chain from interacting with the receptor; lower loadings (∼33%) did result in an increase in receptor binding. Decreasing the surface density of negative charges on the 5 nm PAA-GNPs by including increasing proportions of the neutral polymer poly(2,3-hydroxy-propylacrylamide) (PDHA) inhibited fibrinogen binding to the nanoparticles, and the complex did not bind to the Mac-1 receptor. These results showed that surface charge density, in addition to size, is a critical factor for fibrinogen binding and that

determine how changing nanoparticle properties can affect that interaction. Further techniques used by the polymer nanocomposite community to characterize nanoparticle−polymer surface interactions could also be explored. Shrivastava et al.87 recently developed a simple and sensitive approach to estimate the orientation of adsorbed proteins on nanoscale surfaces. Cyt c, RNase A, and Lyz were adsorbed onto silica nanoparticles (SNPs) 4−15 nm in diameter and used to evaluate the effect of nanoparticle size and surface curvature on protein orientation. Although fundamentally similar to isotopic-coded affinity tagging (ICAT), a technique used to quantify protein expression and abundance between different cell states,88 the described method did not rely on an ICAT linker. Instead, lysine residues of proteins were modified with light (AcL) or heavy (AcH) acetic anhydride followed by proteolytic cleavage and analysis by matrix assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) to compare and quantify solution and adsorbed proteins on the SNPs. In the future, this simple and sensitive approach holds considerable potential to be utilized for the analysis of other proteins bound to nanomaterials and their interactions. Despite our current understanding and control over nanoparticle properties, the other side of the equation necessitates understanding how changes in biomolecule structure affect its resulting function. While improperly folded proteins may elicit an immunogenic response, partial unfolding or the presentation of “new” epitopes after binding to a nanoparticle can result in outcomes such as overexpression of inflammatory factors or reactive oxygen species, or lead to uncontrolled aggregation or amyloidosis diseases.18 The complexity of the biological mileu makes it very difficult to compare proteins or determine how general nanostructure surface properties will affect the conformation of a specific protein and the resulting biological effects. Specific and careful studies are needed, such as those carried out by Deng et al.,28 which allowed the full evaluation of a 3154



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engineered nanobiomaterials for biomedical applications can be realized.

small changes in charge density can influence the ability of fibrinogen−nanoparticle complexes to interact with the Mac-1 receptor. Careful characterization of the nanoparticle samples and well-planned controls validated the results, providing a path forward in studies to determine the final biological influences of nanoparticles in vivo. While advances have clearly been made, further experiments are required in order to understand how slight changes in protein conformation can result in the presentation of new cryptic epitopes that will determine the overall function of the new nanobioconjugate material. As all these elements come together, it will be possible to create engineered nanobiomaterials that can fully exploit the properties of the material, the biopolymer, and the biological environment to achieve the desired physiological response. In conclusion, the application of nanomaterials to biomedicine has reached an exciting juncture, with the suite of available characterization tools allowing a thorough grasp of and control over specific nanoparticle properties, as illustrated in Figure 5. The choices made when structuring a nanomaterial begin with material selection, based on the specific biomedical application such as drug delivery, photothermal91 or radiofrequency92 ablation, diagnostic, or imaging, and follow with the precise synthesis method, which controls the crystal structure, morphology, and surface chemistry of the nanomaterial. Further chemical modification allows engineered placement and control over the spatial arrangement of ligands on the nanoparticle surface. It has been shown that each of the above individual parameters can have a significant effect on the structure and function of adsorbed biomolecules, resulting in changes to the nanobiomaterial’s function, potential toxicity, and/or immunogenicity. A more advanced understanding of how these properties are interrelated will allow the precise and controlled engineering of novel nanobiomaterials that will be able to mimic biological structures in areas such as polyvalency and orientation.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (518) 276-8846, Fax: (518) 276-6540, E-mail: rwsiegel@ rpi.edu. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. Biographies Jennifer E. Gagner received her B.S. in Materials Science and Engineering from MIT in 2007 and obtained her Ph.D. at Rensselaer Polytechnic Institute with Prof. Richard W. Siegel and Prof. Jonathan S. Dordick in 2012. Presently, she is working as a postdoctoral researcher with Dr. Elliot Chaikof at the Beth Israel Deaconess Medical Center. Siddhartha Shrivastava received his Ph.D. from Banaras Hindu University, India, and is currently a postdoctoral researcher with Prof. Richard W. Siegel and Prof. Jonathan S. Dordick at Rensselaer Polytechnic Institute, New York. Xi Qian studied Materials Science and Engineering at Shanghai Jiao Tong University and received his Bachelor of Science degree in 2009. Presently, he is a Ph.D. candidate advised by Prof. Richard W. Siegel and Prof. Jonathan S. Dordick, and his research is focused on nanostructurebiomolecule interactions. Jonathan S. Dordick is the Vice President for Research and the Howard P. Isermann Professor of Chemical and Biological Engineering at Rensselaer Polytechnic Institute. Dordick’s research focuses on gaining a quantitative understanding of biological principles and applying them to advance bioengineering, nanobiotechnology, and the biotic−abiotic interface, high-throughput discovery, and manufacturing.

An advanced understanding of how crystal structure, morphology, and surface chemistry are interrelated will allow precise engineering of novel nanobiomaterials that will be able to mimic biological structures.

Richard W. Siegel is the Robert W. Hunt Professor of Materials Science and Engineering at Rensselaer Polytechnic Institute and Director of the Rensselaer Nanotechnology Center. His research involves synthesis, processing, characterization, properties, and applications of nanostructured ceramics, metals, composites, and biomaterials. He has published and spoken widely on these subjects.

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ACKNOWLEDGMENTS This work was supported by the Nanoscale Science and Engineering Initiative of the National Science Foundation under Grant No. DMR-0642573.

Much remains to be understood about the final form and subsequent interactions of a biomolecule-nanomaterial conjugate; often, changes to the structure of the biomolecule result in unintended and not entirely beneficial consequences. New characterization techniques and control over synthesis has created a rich new environment for determining how specific nanomaterial parameters can influence the biological “identity” of the final construct. As the field moves forward, controlled orientation and spatial arrangement of targeting ligands on the nanoparticle surface will allow manipulation of the physiological response. A complementary understanding from the biopolymer/biology community of how biomolecule residue-nanosurface interactions result in changes to the tertiary and quaternary structures of a protein, as well as the biological outcomes associated with those changes, will complete the picture. With cooperation between these disciplines, the full potential of

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REFERENCES

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