Molecular Interaction of Poly(acrylic acid) Gold Nanoparticles with Human Fibrinogen

The binding of fibrinogen to various nanoparticles can result in protein unfolding and exposure of cryptic epitopes that subsequently interact with cell surface receptors. This response is dependent on the size, charge, and concentration of the nanoparticle. Here we examine the binding kinetics of human fibrinogen to negatively charged poly(acrylic acid)-coated gold nanoparticles ranging in size from 7 to 22 nm. These particles have previously been shown to elicit an inflammatory response in human cells. The larger nanoparticles bound fibrinogen with increasing affinity and a slower dissociation rate. Each fibrinogen molecule could accommodate two 7 nm nanoparticles but only one when the diameter increased to 10 nm. Nanoparticles larger than 12 nm bound multiple fibrinogen molecules in a positively cooperative manner. However, in the presence of excess nanoparticle, fibrinogen induced aggregation of the larger particles that could bind more than one protein molecule. This is consistent with interparticle bridging by the fibrinogen. Taken together, these results demonstrate that subtle changes in nanoparticle size can influence protein binding both with the surface of the nanoparticle and within the protein corona

Schematic model of the SjIRs with approximate locations of mapped peptide analogues 1-22.

<p>Peptides 1-10 (in red) are located on SjIR-1; peptides 11, 13-22 (in blue) are located on SjIR-2. Peptide 9 is located on the HIR (its approximate location relative to the sequence in the SjIRs is indicated). Peptide 12 is the reverse analogue of peptide 9. L1, L1 subdomain; L2, L2 subdomain; CR, cysteine rich domain; FnIII-1-3, fibronectin domains 1, 2 and 3; TM, transmembrane domain; TK, tyrosine kinase domain.</p

Design of Fully Synthetic, Self-Adjuvanting Vaccine Incorporating the Tumor-Associated Carbohydrate Tn Antigen and Lipoamino Acid-Based Toll-like Receptor 2 Ligand

Overexpression of certain tumor-associated carbohydrate antigens (TACA) caused by malignant transformation offers promising targets to develop novel antitumor vaccines, provided the ability to break their inherent low immunogenicity and overcome the tolerance of the immune system. We designed, synthesized, and immunologically evaluated a number of fully synthetic new chimeric constructs incorporating a cluster of the most common TACA (known as Tn antigen) covalently attached to T-cell peptide epitopes derived from polio virus and ovalbumin and included a synthetic built-in adjuvant consisting of two 16-carbon lipoamino acids. Vaccine candidates were able to induce significantly strong antibody responses in mice without the need for any additional adjuvant, carrier protein, or special pharmaceutical preparation (e.g., liposomes). Vaccine constructs were assembled either in a linear or in a branched architecture, which demonstrated the intervening effects of the incorporation and arrangement of T-cell epitopes on antibody recognition

The binding affinity between human insulin and analogues 3, 15 and 29 derived from SjIR-1, SjIR-2 and HIR, respectively, measured using the Octet RED system.

<p>Binding between human insulin and analogues 3 (A), 15 (B) and 29 (C) is shown at different concentrations (μM). The real time binding response (nm) was measured in seconds (sec). The parameters of the binding response (nm) and the KD value (M) of the binding between insulin and the analogues at different concentrations are shown in the tables on the right. The coefficient of determination (R^2) for all these interactions was close to 1.0, indicating a good curve fit.</p

The binding affinity between human insulin and analogues 1, 13 and 28 derived from SjIR-1, SjIR-2 and HIR, respectively, determined using the Octet RED system.

<p>Binding is shown between human insulin and analogues 1 (A), 13 (B) and 28 (C) at different peptide concentrations (μM). The real time binding response (nm) was measured in seconds (sec). The parameters of the binding response (nm) and the KD value (M) of the binding between insulin and the analogues at different concentrations are shown. The coefficient of determination (R^2) of these interactions was close to 1.0, indicating a good curve fit. The left panels show that an increased binding response was observed when the concentration of peptide analogues was increased from low to high, as shown on the right in the tables.</p

Details of peptide analogues screened for insulin binding activity including sequence information and their location within the SjIR proteins.

<p>Details of peptide analogues screened for insulin binding activity including sequence information and their location within the SjIR proteins.</p

Details of the peptide analogues screened for human insulin binding activity.

<p>Details of the peptide analogues screened for human insulin binding activity.</p

Site-Specific Incorporation of Three Toll-Like Receptor 2 Targeting Adjuvants into Semisynthetic, Molecularly Defined Nanoparticles: Application to Group A Streptococcal Vaccines

Subunit vaccines offer a means to produce safer, more defined vaccines compared to traditional whole microorganism approaches. Subunit antigens, however, exhibit weak immunity, which is normally overcome through coadministration with adjuvants. Enhanced vaccine properties (e.g., improved potency) can be obtained by linking antigen and adjuvant, as observed for synthetic peptide antigens and Toll-like receptor 2 (TLR2) ligands. As few protective peptide antigens have been reported, compared to protein antigens, we sought to extend the utility of this approach to recombinant proteins, while ensuring that conjugation reactions yielded a single, molecularly defined product. Herein we describe the development and optimization of techniques that enable the efficient, site-specific attachment of three synthetic TLR2 ligands (lipid core peptide (LCP), Pam2Cys, and Pam3Cys) onto engineered protein antigens, permitting the selection of optimal TLR2 agonists during the vaccine development process. Using this approach, broadly protective (J14) and population targeted (seven M protein N-terminal antigens) multiantigenic vaccines against group A streptococcus (GAS; Streptococcus pyogenes) were produced and observed to self-assemble in PBS to yield nanoparticules (69, 101, and 123 nm, respectively). All nanoparticle formulations exhibited self-adjuvanting properties, with rapid, persistent, antigen-specific IgG antibody responses elicited toward each antigen in subcutaneously immunized C57BL/6J mice. These antibodies were demonstrated to strongly bind to the cell surface of five GAS serotypes that are not represented by vaccine M protein N-terminal antigens, are among the top 20 circulating strains in developed countries, and are associated with clinical disease, suggesting that these vaccines may elicit broadly protective immune responses

Summary of <i>in vitro</i> results.

[a]<p>The apparent permeability, <i>P</i>_app (cm/s), across Caco-2 cell monolayers. Analysis were done using LC/MS and each point is expressed as mean ± SEM.</p>[b]<p>The <i>K</i>_iμ values of <b>1</b>, <b>2</b>, <b>3</b> and <b>4</b> for the MOP receptor were obtained using competitive radioligand binding assay. Competitive displacement of the MOP selective radioligand, [³H]DAMGO, was determined from whole SH-SY5Y cells in the presence of DPDPE (1 µM). Binding affinity values (<i>K</i>_iμ) were determined using seven concentrations of each compound in the range 10 pM to 1 µM performed in three independent experiments, each in triplicate. Data expressed as mean ± SEM. Competitive binding curves are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041909#pone-0041909-g002" target="_blank">Fig. 2</a>.</p>[c]<p>The IC₅₀ values (nM) were estimated from dose-response curves shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041909#pone-0041909-g003" target="_blank">Fig. 3</a>. Inhibition of forskolin-stimulated cAMP formation was measured to determine the agonist activity of the compounds. Experiments were performed three independent times, each in triplicate.</p>[d]<p>These values are obtained from the same experiment. ND, not determined. Each value represents mean ± SEM. *<i>p</i><0.05, compared to compound <b>1</b> in the same experiment.</p

Insignificant antinociception produced by compounds 1 and 2.

<p>The highest doses of compounds <b>1</b> and <b>2</b> did not produce pain relief at 16 µmol/kg after single bolus i.v. administration to CCI-rats. Each value represents mean (± SEM) (<i>n</i> = 6–8).</p