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«Kinetic Investigations of Thiolate Protected Gold Nanoparticles: Protein Interactions, Electron Transfer, and Precursor Formation By Brian N. Turner ...»

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Another study from our group focused on a tiopronin gold nanoparticle with a well characterized epitope from the hemagglutanin (HA) protein of influeneza. 112 The 10amino acid peptide was synthesized with a terminating cysteine residue to promote placeexchange chemistry. The specific peptide sequence was selected because it is present in a neutralizing site for influenza and there is a commercially available monoclonal antibody specific for this epitope. The same study also compared 2-D SAMs to 3-D nanoparticles, as illustrated in Figure 17. The HA-MPC was more efficient in presenting the peptide to the antibody as it resulted in a higher ratio of antibody to peptide binding when compared to the 2-D surface.

Figure 17: 10-amino acid HA peptide on a 3-D gold tiopronin nanoparticle (at left) and the same 10-amino acid sequence as a 2-D SAM with tiopronin spacers (at right). 112 Reprinted with permission. Copyright 2005 of the American Chemical Society, reprinted with permission.

An important aspect of nanoparticles presenting peptide epitopes is whether the nanoparticle bound peptide conformation reflects that of the peptide in the native antigen.

Specifically, in our research group, Gerdon and coworkers studied the protective antigen (PA) of B. anthracis110, which is one of three precursors of the anthrax toxin. PA was selected for because it precedes the other two proteins (edema factor and lethal factor) in their transport for infection, which makes it an ideal target for neutralizing antibodies.

Specifically, the C-terminus and two internal loops of the PA protein were identified as cell-receptor sites, making them ideal candidates for the study. As in previous studies in our group, tiopronin MPCs were place-exchanged with three relevant peptides from the regions on PA mentioned previously. As two of the three PA epitopes selected were loop regions, corresponding peptides were designed so that they could mimic the epitope’s native conformation by integrating cysteine residues on both the N and C-terminus.

Cysteine residues at both termini allowed for bidentate attachment across the nanoparticle surface to reconstruct the natural loop. Previous work by Murray and coworkers had reported that ligands attached to the surface will migrate across the MPC to find the most stable conformation possible, as they are in a dynamic equilibrium with the solution phase.33, 58, 113 Migration of peptides at a nanoparticle surface suggested that the bidentate peptide was capable of “walking” itself into a conformation similar to the native antigen. Other nanoparticles were synthesized using the same peptide, except with a single cysteine residue on the C-terminus for monodentate attachment, allowing for comparison with the bidentate peptide nanoparticle. Figure 18 illustrates the bidentate versus the monodentate attachment strategy.

Figure 18: Loop presenting MPCs (left) shown compared to the native protein (middle) and the linear presenting MPCs (right). 110 Reprinted with permission of John Wiley & Sons, Inc.

Nanoparticle peptide conjugates were analyzed using a quartz crystal microbalance platform. The results from this study were that the loop-presenting (bidentate peptide) nanoparticle was strongly recognized by one of seven commercially available monoclonal antibodies, while the linear epitope nanoparticle was recognized only weakly. The loop epitope had a higher affinity (Ka) for this particular antibody than the linear epitope, especially at physiological pH and ionic strength, confirming that the looppresenting nanoparticle was more readily recognized by the antibody. Preferential recognition of the loop epitope suggests that the commercial antibody has a more structurally constrained paratope, or that the loop presented less steric interference between the antibody and the critical binding residues.

Given their high density of functional groups, peptides are not limited to use as only a biological recognition agent. For example, Naik and co-workers used multifunctional peptides as both the reducing agent, gold-protecting ligand, and presented epitope.114 Peptide A3 was selected from a phage display library and found to both bind to gold and reduce it. Flg, a peptide commonly used in tagging proteins with a biomolecular recognition domain, was also found to reduce gold. Resulting Flg-A3 and A3-Flg conjugated gold nanoparticles prepared in a one pot synthesis were capable of binding to anti-Flg IgG on glass slides.

As previously mentioned, nanoparticles are useful scaffolds for holding multiple molecules into one supramolecular structure. As an extension of peptide epitope protected gold MPCs, in collaboration with the research group of David Wright, tiopronin MPCs containing, in their monolayer, flag epitope (flag-MPC), HA epitope (HA-MPC), flag and HA epitope (flag/HA-MPC), or no epitope were synthesized.115 The peptide epitopes were attached to the cluster via a cysteine terminated polyethylene glycol hexamer, via place exchange, to provide enhanced accessibility. QCM immunosensors, as previously described, using either anti-flag or anti-HA IgG were used to evaluate the immunological activity of the mimics synthesized. Detection of the HA-MPC and flag/HA-MPC using the anti-HA immunosensor, and the flag-MPC and HA/flag-MPC using the anti-flag immunosensor was subsequently accomplished. Neither one detected the tiopronin MPCs without peptide epitopes. In all these trials, biological recognition serves as a quick means to identify useful peptide nanoparticles and to determine binding constants.





More recently, Mancin and coworkers described a variety of metal cores coated with a helical 11 amino acid peptide.116 H-Ala3-(Aib-Ala)4-OMe, where Aib is αaminoisobutyric acid, was grafted onto a variety of metal cores (with tritylmercaptopropionic acid) and onto silica nanoparticles. The specific primary sequence has been known for holding an α-helical structure in a variety of media. In Mancin’s experiments, the peptide held the helical structure after grafting onto all of the tested nanoparticles, as was confirmed by circular dichroism. For the metal nanoparticles, the peptide was used as the primary protecting ligand during reduction with sodium borohydride. These nanoparticles were recognized effectively by immune cells, according to observations with confocal microscopy. These peptide conjugated nanoparticles, and control nanoparticles with a non-helical pentapeptide were taken up equally as well by macrophages. When tested with monocytes, the undecapeptide outperformed its uncoiled counterpart by a factor of two or three in terms of amount of material taken up in cells. The conducted experiments are an interesting example of how these conjugates can be employed in living systems.

The research group of David Cliffel is currently engaged in adapting nanoparticle mimics in vivo. Particularly, much of the research focuses on toxicity and antigenicity. Simpson and coworkers showed that while gold-tiopronin nanoparticles are toxic in mice, the addition of a small amount of tetraethylene glycol into the nanoparticle monolayer eliminates this toxicity.117 Antigenicity is currently being investigated, and there has yet to be a published study showing the generation of native antigen specific sera production in mice after challenge with a peptide-nanoparticle conjugate.

The current investigation utilizes water soluble tiopronin nanoparticles, bidentate and monodentate place exchange strategies with peptides, QCM immunosensing, and information gathered from epitope mapping (discussed in the previous chapter). The resulting data supports the synthesized nanomaterial as a further example of how straightforward methods can be used to mimic the effects of complex biological agents.

Quartz Crystal Microbalance Immunosensing The QCM instrument has been described previously in Chapter II. A useful review on piezoelectrics in analytical life sciences, written by Janshoff and coworkers,94 describes piezoelectric immunosensors, such as the QCM, and their advantages and disadvantages.

Summarily, the QCM is a scaled down version of the SPR in terms of analytical capability in biosensing, with somewhat lower sensitivity, and more medium-related complications (signals affected by solution conductivity, viscosity, non-rigid interactions, etc.). Ultimately, it is still a sensitive, label-free technique that has been well-established for the detection of antigens. The strategies described have been useful to our research group in a number of other disease detection studies118-120 and in the validation of antigenic nanoparticle mimics.92, 105, 110, 112 Adapting some of these strategies, the construction of a QCM immunosensor platform for HRSV detection, centered on the commercial IgG Palivizumab(PZ), was undertaken. This work was extended to validate a gold nanoparticle antigenic mimic of HRSV F protein.

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Materials HAuCl4- • 3H2O was synthesized according to standard methods from electrochemically purified Canadian gold maple leaf coins (99.99%).121 Reagent and optima grade solvents, N-2-mercaptopropionylglycine (tiopronin), bovine serum albumin (fraction V, 96%), sodium borohydride, thioanisole, and anisole were purchased from Sigma-Aldrich.

Common laboratory salts and concenetrated ammonium hydroxide were reagent grade and purchased from Fisher scientific. Concentrated sulfuric acid was purchased from EMD. Absolute ethanol was purchased from Pharmco-AAPER. Hydrogen peroxide (30% v/v) was purchased from Acros. Peptide synthesis materials (f-moc protected and side-chain protected amino acids, coupling reagents, and resins) were provided generously by the David Wright research group. 18 MΩ Water was obtained from a U.S.

Filter Modulab water system with a 0.2 µm external filter, or from a Barnstead NANOpure Diamond water purification system. Dry nitrogen is provided in house.

Monoclonal antibody 1214, 1129, Palivizumab, and human respiratory syncytial virus were provided generously by the Dr. James Crowe, Jr. research group. Work areas that contained HRSV were cleaned thoroughly with 70% isopropyl alcohol solution. Amicon Ultra molecular weight cut-off centrifuge tubes were obtained from the molecular biology core at Vanderbilt University. Snakeskin pleated dialysis tubing (10,000 MWCO) was purchased from Thermo Scientific. Deuterated solvents (99.9% D) were purchased from Cambridge Isotope Laboratories.

Peptide Synthesis Peptides were synthesized as discussed in Chapter III. Some peptides were purified by size exclusion chromatography instead of semi-prep HPLC. Peptides used in this study are displayed in Table 6.

Table 6. Peptides that were conjugated to nanoparticles in this study.

Series represents the set of experiments in which they were used. Each series evolved based on the results of the last series, as discussed later. The ID will be used to refer to peptides later in shorthand (e.g. peptide 2-L represents the peptide with ID “L” from series 2). Nterminus describes whether or not the N terminus was acetylated (AcNH is acetylated and NH2 is not acetylated), if a cysteine residue (C) was added here for place exchange, or whether serine-glycine-serine-glycine (SGSG) or hexaethylene glycol (PEG6) spacer was incorporated. Primary epitope sequence indicates the amino acid sequence that was adapted from HRSV F antigenic site A. C-terminus indicates whether or not the Cterminus was amidated (CONH2 is amidated and COOH is not), whether a cysteine residue was incorporated, and if a spacer was used. Projection follows from the location of the cysteine residue, indicating whether the peptide is immobilized by monodentate attachment at the N-terminus thus projecting the C-terminus away from the nanoparticle surface (N to C) or vice versa (C to N), or whether it is immobilized by bidentate attachment at both termini (looped).

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Synthesis of Au216Tiopronin129 Nanoparticles Tiopronin MPCs were synthesized as previously described. 122 5.25 g (15.4 mmol) HAuCl4  3H2O was dissolved in 500 mL 6:1 methanol:acetic acid and chilled in an ice bath for 30 minutes, giving a yellow solution. 7.56 g (46.2 mmol) tiopronin was then added to the solution, forming a ruby red intermediate. Upon dissolution of the tiopronin,

5.84 g (154 mmol) sodium borohydride in approximately 10 mL water was immediately and rapidly added to the mixture. A violent, exothermic reaction results, leaving a black solution. The solution is stirred overnight, yielding polydisperse nanometer sized clusters. The resulting solution is rotary evaporated in vacuo to remove the methanol.

The resulting precipitate/viscous acetic acid solution is redissolved in water, pH adjusted to 1 with concentrated hydrochloric acid and placed in dialysis tubing (cellulose ester, MWCO = 10 kDa). The dialysis proceeds for 2 weeks changing the water at least twice daily until the water does visually turn a color. 1H NMR: 4.00 ppm broad singlet (tiopronin methylene on cluster), 3.76 ppm broad singlet (tiopronin CH on cluster), 1.66 ppm very broad singlet (tiopronin methyl on cluster). TEM: average particle diameter of

2.2 nm  0.6 nm. UV/Visible spectroscopy: no surface plasmon resonance band

observable. TGA: 33 weight percent organic (tiopronin). Average formula:

Au216Tiopronin129.

Synthesis of Au118Tiopronin71 Nanoparticles Tiopronin MPCs were synthesized as previously described. 122 500 mg HAuCl4  3H2O was dissolved in 70 mL 6:1 methanol:acetic acid and chilled in an ice bath for 30 minutes, giving a yellow solution. 620 mg tiopronin was then added to the solution, forming a ruby red intermediate. Upon fading of the solution to near colorless, 500 mg sodium borohydride in a minimum of water was immediately and rapidly added to the mixture. A violent and exothermic reaction results, leaving a black suspension. The solution is stirred overnight, yielding polydisperse nanometer sized particles. The resulting solution is centrifuged, and the solvent decanted. The particles were then resuspended in methanol, and centrifuged a second time. 1H NMR: 4.00 ppm broad singlet (tiopronin methylene on cluster), 3.76 ppm broad singlet (tiopronin CH on cluster), 1.66 ppm very broad singlet (tiopronin methyl on cluster). TEM: average particle diameter of 1.8 nm  0.7 nm. UV/Visible spectroscopy: no surface plasmon

resonance band observable. TGA: 33 % by mass organic (tiopronin). Average formula:

Au118Tiopronin71.



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