AVS2013 Session AS+BI+EM+NL+NS+SS-ThM: Nanoparticle Surface Chemistry

Difficulties assessing human exposure, safety, and possible toxicity from nanotechnologies have prompted questions about how to characterize nanomaterials in various experimental test beds for predictive use. While little consensus is published about human risk/benefit analysis, this is confounded by lack of accepted, sensitive and reliable characterization methods of practical value for nanomaterials in physiological milieu. Relatively few studies are conducted on these materials in biologically relevant media to understand their surface properties and physical states (i.e., sedimentation, aggregation) prior to in vitro or in vivo exposures. Few studies have standard reference materials and analytical protocols established for comparisons to other studies. The current understanding of the fate of nanomaterials of most any size and shape, both in cell culture mediawith serum or inside the mammalian body, is poor at best. Additionally, the collective published scientific record documenting fate of nanomaterials in vivo is consistent with long known tissue-based particle filtration for micro-colloids, with far less success deliberately targeting particles to specific tissue or disease sites (i.e., <5% of a nanoparticle dose reaches a disease site).

To date, most data suggest that size reductions to the nanometer dimension have not significantly changed how nanomaterials interact with physiological systems in vivo, despite in vitro distinctions observed with proteins and in cell cultures. Connecting nanomaterials properties with how they interact with proteins and cells in vitro to affect their biodistribution in vivo allows a more rational approach to designing nanomaterials with specific biomedical and toxicity properties, and to avoid the ubiquitous non-specific tissue scavenging. This is related to materials interactions with whole blood components, including platelets, cells, and plasma proteins, producing fluid transport to tissue sites, particle binding, opsonization and aggregation. However, analytical methods for nanomaterialsare not sufficiently sensitive to study these effects in vivo to alter nanomaterials biodistribution patterns. Additionally, understanding how surface coatings, ligands and contaminants change physiological behavior requires careful analysis.

The nanotechnology field must develop improved, sensitive analytical tools and methods to drive a consensus for how nanomaterials (1) should be fully and reliably characterized for biological and biomedical purposes, and (2) how different nanomaterials properties produce either beneficial (i.e., therapeutic) or toxic responses inside complex physiological systems.

The importance of surface characterization of nano-objects in dimensions below 50 nm is well recognized. Indeed the most pronounced changes in chemical reactivity are expected to occur on the smallest size nano-objects. Yet, as their size shrinks, measurement techniques are lagging. A further concern, when seeking insight into size-composition-reactivity relationships, is population heterogeneity. We present here a technique for assaying individual nano-objects. The method involves bombarding dispersed nano-objects one-by-one with nanoprojectiles specifically Au₄₀₀⁴⁺, at hypervelocity. Their impact causes abundant emission of ionized ejecta which are identified individually for each impact by time-of-flight mass spectrometry. We will describe the characterization of surfaces and environments of nano-objects as revealed by selected grazing projectile impacts.

The ability to quantitatively describe protein coronas of nanoparticles in biological fluids is highly sought for to understand protein corona formation, nanoparticle fate and their interaction with biological systems. A quantitative description of the nanoparticle biomolecular interface is challenging and chemical information along with structural and biofunctional characterization requires the use of complementary techniques.

The parallel use of different techniques provides in fact a range of complementary information of the materials under study, each technique being based on a specific physical principle. Here, we combine the use of liquid-based size measurement techniques such as Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA) and Differential Centrifugal Sedimentation (DCS), with vacuum techniques such as X-ray Photoelectron Spectroscopy (XPS) to provide quantitative information of core/shell nanoparticle systems.

We used a set of spherical gold nanoparticles having diameters from 20 nm to 80 nm and coated with different amount of Immunoglobulin G (IgG) antibodies as a model system. The shift of the nanoparticle Localized Surface Plasmon Resonance (LSPR) frequency measured by UV-Vis spectroscopy is known to relate to the amount of molecules adsorbed at nanoparticles’ interface. We measured the LSPR shifts along with the nanoparticle sizes in liquid by using DLS, NTA and DCS. When the nanoparticles had a complete protein shell, shell thickness measurements were consistent for all the techniques. DCS sedimentation times showed excellent correlation with LSPR frequency shifts, indicating that analytical centrifugation can provide precise measurement of the thickness of complete protein shells on nanoparticles. However, in the low coverage regime, NTA and DLS techniques provided the best correlation with LSPR frequency shifts. By combining the information from the different techniques we estimated the amount of IgG molecules per nanoparticle as a function of the IgG concentration in solution. Data analysis in the high concentration regime suggested that nanoparticle curvature strongly influences the ability of a surface to allow the further adsorption of IgG.

Core/shell nanoparticle systems were also characterized by XPS. A methodology for the preparation of nanoparticle samples for analysis in vacuum was developed. XPS data was collected from nanoparticles of different core size and analysis provided quantitative chemical information of the nanoparticle. The combined use of XPS with liquid-based techniques for particle characterization provided quantitative chemical and structural information of core/shell nanoparticle systems.

Nanoparticles exhibit unique surface properties and require well-controlled surface properties to achieve optimum performance in complex biological or physiological fluids. Thus, there is a need to develop rigorous and detailed surface analysis methods for their characterization. The surface chemistries of oligo(ethylene glycol) (OEG) self-assembled monolayers (SAMs) on Au nanoparticle (AuNP) surfaces were characterized with x-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), Fourier transform IR spectroscopy and high-sensitivity, low-energy ion scattering (HS-LEIS). The size, shape, and size distribution of the AuNPs was determined by transmission electron microscopy (TEM).

Both methoxy (CH₃O-) and hydroxyl (HO-) terminated OEG SAMs with chains containing 11 methylene and 4 ethylene glycol units were examined. ToF-SIMS clearly differentiates the two OEG SAMs based on the C₃H₇O⁺ peak attributed to the CH₃ terminated SAM, while XPS didn’t detect a significant difference between the two SAMs on the same surface. However, XPS did show a significant difference between the same SAM on different sized AuNPs. Both OEG SAMs were more densely packed on the 40 nm diameter AuNPs compared to the 14 nm diameter AuNPs. FTIR experiments indicates the methylene backbone groups are well-ordered on all gold surfaces, but the OEG groups are more ordered on the 40 nm diameter AuNPs. Together the XPS and FTIR results suggest the OEG SAMs form a thicker and/or higher density SAMs on the 40 nm AuNPs compared to the 14nm AuNPs. HS-LEIS experiments showed the OEG SAMs on the 40 nm AuNPs were significantly thicker (2.6 nm) than the OEG SAMs on the 14 nm AuNPs (2.0 nm) and the flat Au surface (1.9 nm). The 2.6 nm thickness measured on the 40 nm AuNPs is consistent with thickness expected for a well-order OEG SAM (2.7 nm). TEM showed the 40 nm AuNPs had a larger size distribution and were less spherical compared to the 14 nm AuNPs, suggesting the shape of the AuNPs can have a significant effect on the structure and thickness of the OEG SAMs.

Protein G was immobilized onto the HO-terminated OEG SAMs via carbonyl diimidazole chemistry. ToF-SIMS analysis showed the relative intensities of characteristic amino acid fragments from Protein G varied with both the protein solution concentration and the type of surface.

The surfaces of chemically synthesized Au nanoparticles have been modified with D- or L-cysteine to render them chiral and enantioselective for adsorption of chiral molecules. Their enantioselective interaction with chiral compounds has been probed by optical rotation measurements when exposed to racemic propylene oxide. The ability of optical rotation to detect enantiospecific adsorption arises from the fact that the specific rotation of polarized light by R- and S-propylene oxide is enhanced by interaction Au nanoparticles. This effect is related to previous observations of enhanced circular dichroism by Au nanoparticles modified by chiral adsorbates. More importantly, chiral Au nanoparticles modified with either D- or L-cysteine selectively adsorb one enantiomer of propylene oxide from a solution of racemic propylene oxide, thus leaving an enantiomeric excess in the solution phase. Au nanoparticles modified with L-cysteine (D-cysteine) selectively adsorb the R-propylene oxide (S-propylene oxide). A robust model based on optical rotation data has been developed that allows extraction of the enantiospecific equilibrium constants for R- and S-PO adsorption on the chiral Au nanoparticles.

[1] N. Shukla, M.A. Bartel, A.J. Gellman “Enantioselective separation on chiral Au nanoparticles” Journal of the American Chemical Society, 132(25), (2010), 8575–8580

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