Injectable Silk Protein Microparticle-based Fillers: A Novel Material for Potential Use in Glottic Insufficiency.

OBJECTIVES AND HYPOTHESIS: A novel, silk protein-based injectable filler was engineered with the intention of vocal fold augmentation as its eventual intended use. This injectable filler leverages the unique properties of silk protein's superior biocompatibility, mechanical tunability, and slow in vivo degradation to one day better serve the needs of otolaryngologists. This paper intends to demonstrate the mechanical properties of the proposed novel injectable and to evaluate its longevity in animal models. MATERIALS AND METHODS: Experimental. The mechanical properties of silk bulking agents were determined to characterize deformation resistance and recovery compared with commercially available calcium hydroxylapatite through rheologic testing. Fresh porcine vocal fold tissue was used for injectable placement to simulate the mechanical outcomes of native tissue after bulking procedures. In vivo subcutaneous rodent implantation examined immune response, particle migration, and volume retention. RESULTS: Porous, elastomeric silk microparticles demonstrate high recovery (>90% original volume) from compressive strain and mimic the native storage modulus of soft tissues (1-3 kPa). Injectable silk causes only a slight increase in porcine vocal fold stiffness immediately after injection (20%), preserving the native mechanics of bulked tissue. In the subcutaneous rat model, silk demonstrated biocompatibility and slow degradation, thus enabling host cell integration and tissue deposition. CONCLUSIONS: The presented novel silk injectable material demonstrates favorable qualities for a vocal fold injection augmentation material. An in vivo long-term canine study is planned.

A novel silk-based vocal fold augmentation material: 6-month evaluation in a canine model.

OBJECTIVES: Ideal long-term vocal fold augmentation materials should be biocompatible, easily administered, allow tissue integration for long-term effect, and remain at the site of injection. A novel silk protein particle suspended in hyaluronic acid (Silk-HA) has been developed specifically for vocal fold augmentation to address this unmet need. This article presents the 6-month, preclinical findings of a canine vocal fold injection trial for Silk-HA. METHODS: Twelve beagle dogs were injected transorally in the lateral/deep aspect of their right thyroarytenoid muscles with 0.3 cc of Silk-HA or calcium hydroxylapatite in carboxymethyl cellulose (CaHA-CMC). The Silk-HA particle injectable was delivered via a custom catheter, whereas CaHA-CMC was delivered through a commercially available malleable needle. The six dogs from each material group were sacrificed 6 months from the injection date for the evaluation of implant longevity, immune response, and material migration. RESULTS: Silk-HA provides immediate medialization of the right vocal fold, lasting for a minimum of 6 months in a canine model. Silk-HA and CaHA-CMC both demonstrate similar inflammatory responses. The Silk-HA was shown to remain without migration at the site of injection in all six canine subjects, whereas CaHA-CMC demonstrated migration in four of the six canines. In two canines implanted with CaHA-CMC, material was discovered to migrate to the retropharyngeal lymph nodes. CONCLUSION: In a canine subject model, the Silk-HA material compares favorably in terms of longevity and immune response to CaHA-CMC. The lack of migration of the Silk-HA material demonstrates a promising potential for vocal fold injection in the clinic. LEVEL OF EVIDENCE: NA Laryngoscope, 129:1856-1862, 2019.

Broadly neutralizing antibodies from human survivors target a conserved site in the Ebola virus glycoprotein HR2-MPER region.

Ebola virus (EBOV) in humans causes a severe illness with high mortality rates. Several strategies have been developed in the past to treat EBOV infection, including the antibody cocktail ZMapp, which has been shown to be effective in nonhuman primate models of infection 1 and has been used under compassionate-treatment protocols in humans 2 . ZMapp is a mixture of three chimerized murine monoclonal antibodies (mAbs)3-6 that target EBOV-specific epitopes on the surface glycoprotein7,8. However, ZMapp mAbs do not neutralize other species from the genus Ebolavirus, such as Bundibugyo virus (BDBV), Reston virus (RESTV) or Sudan virus (SUDV). Here, we describe three naturally occurring human cross-neutralizing mAbs, from BDBV survivors, that target an antigenic site in the canonical heptad repeat 2 (HR2) region near the membrane-proximal external region (MPER) of the glycoprotein. The identification of a conserved neutralizing antigenic site in the glycoprotein suggests that these mAbs could be used to design universal antibody therapeutics against diverse ebolavirus species. Furthermore, we found that immunization with a peptide comprising the HR2-MPER antigenic site elicits neutralizing antibodies in rabbits. Structural features determined by conserved residues in the antigenic site described here could inform an epitope-based vaccine design against infection caused by diverse ebolavirus species.

Metal Affinity-Enabled Capture and Release Antibody Reagents Generate a Multiplex Biomarker Enrichment System that Improves Detection Limits of Rapid Diagnostic Tests.

Multi-antigen rapid diagnostic tests (RDTs) are highly informative, simple, mobile, and inexpensive, making them valuable point-of-care (POC) diagnostic tools. However, these RDTs suffer from several technical limitations-the most significant being the failure to detect low levels of infection. To overcome this, we have developed a magnetic bead-based multiplex biomarker enrichment strategy that combines metal affinity and immunospecific capture to purify and enrich multiple target biomarkers. Modifying antibodies to contain histidine-rich peptides enables reversible loading onto immobilized metal affinity magnetic beads, generating a novel class of antibodies coined "Capture and Release" (CaR) antibody reagents. This approach extends the specificity of immunocapture to metal affinity magnetic beads while also maintaining a common trigger for releasing multiple biomarkers. Multiplex biomarker enrichment is accomplished by adding magnetic beads equipped with CaR antibody reagents to a large sample volume to capture biomarkers of interest. Once captured, these biomarkers are magnetically purified, concentrated, and released into a RDT-compatible volume. This system was tailored to enhance a popular dual-antigen lateral flow malaria RDT that targets Plasmodium falciparum histidine-rich protein-II (HRPII) and Plasmodium lactate dehydrogenase (pLDH). A suite of pLDH CaR antibody reagents were synthesized, characterized, and the optimal CaR antibody reagent was loaded onto magnetic beads to make a multiplex magnetic capture bead that simultaneously enriches pLDH and HRPII from Plasmodium falciparum parasitized blood samples. This system achieves a 17.5-fold improvement in the dual positive HRPII/pan-pLDH detection limits enabling visual detection of both antigens at levels correlating to 5 p/µL. This front-end sample processing system serves as an efficient strategy to improve the sensitivity of RDTs without the need for modifications or remanufacturing.

Detergent-Mediated Formation of ß-Hematin: Heme Crystallization Promoted by Detergents Implicates Nanostructure Formation for Use as a Biological Mimic.

Hemozoin is a unique biomineral that results from the sequestration of toxic free heme liberated as a consequence of hemoglobin degradation in the malaria parasite. Synthetic neutral lipid droplets (SNLDs) and phospholipids were previously shown to support the rapid formation of ß-hematin, abiological hemozoin, under physiologically relevant pH and temperature, though the mechanism by which heme crystallization occurs remains unclear. Detergents are particularly interesting as a template because they are amphiphilic molecules that spontaneously organize into nanostructures and have been previously shown to mediate ß-hematin formation. Here, 11 detergents were investigated to elucidate the physicochemical properties that best recapitulate crystal formation in the parasite. A strong correlation between the detergent's molecular structure and the corresponding kinetics of ß-hematin formation was observed, where higher molecular weight polar chains promoted faster reactions. The larger hydrophilic chains correlated to the detergent's ability to rapidly sequester heme into the lipophilic core, allowing for crystal nucleation to occur. The data presented here suggest that detergent nanostructures promote ß-hematin formation in a similar manner to SNLDs and phospholipids. Through understanding mediator properties that promote optimal crystal formation, we are able to establish an in vitro assay to probe this drug target pathway.

Spontaneous self-assembly and disassembly of colloidal gold nanoparticles induced by tetrakis(hydroxymethyl) phosphonium chloride.

Upon reacting with tetrakis(hydroxymethyl) phosphonium chloride, 15 nm citrate gold nanoparticles rapidly assemble into linear chains, followed by slowly disassembling into monodisperse components. This work highlights the first example of (31)P NMR on gold particles of this size and suggests that the phosphonium is oxidized on-particle, contributing to particle disassembly.

Ni(II)NTA AuNPs as a low-resource malarial diagnostic platform for the rapid colorimetric detection of Plasmodium falciparum Histidine-Rich Protein-2.

Diagnosing infectious diseases remains a challenge in the developing world where there is a lack of dependable electricity, running water, and skilled technicians. Although rapid immunochromatographic tests (RDTs) have been deployed to diagnose diseases such as malaria, the extreme climate conditions encountered in these regions compounded with the discrepancies in test manufacturing have yielded varying results, so that more innovative and robust technologies are sought. Devoid of antibodies and thermally sensitive materials, we present a robust, colorimetric diagnostic platform for the detection of a malarial biomarker, Plasmodium falciparum Histidine-Rich Protein 2 (PfHRP-II). The assay exploits the optical properties of gold nanoparticles, covalently coupling them to a Ni(II)NTA recognition element specific for PfHRP-II. In the presence of the recombinant malarial biomarker (rcHRP-II), the Ni(II)NTA AuNPs begin to crosslink and aggregate in as little as one minute, triggering a red-to-purple color change in solution. To increase assay sensitivity and platform stability suitable for low-resource regions, the Ni(II)NTA AuNPs were assembled with varying spacer ligands in a mixed monolayer presentation. When assembled with a negatively charged Peg4-thiol ligand, the Ni(II)NTA AuNPs demonstrate low nanomolar limits of rcHRP-II detection in physiological concentrations of human serum albumin and maintain excellent stability at 37°C when stored for four weeks. Detection of the malaria biomarker is also measured by capturing and processing images of aggregated gold nanoparticles with a smartphone camera. By utilizing a smartphone camera and image processing application, there is no significant difference in assay sensitivity and rcHRP-II limit of detection in comparison to a spectrophotometer, further making this diagnostic platform applicable for use in low-resource regions.

Coffee rings as low-resource diagnostics: detection of the malaria biomarker Plasmodium falciparum histidine-rich protein-II using a surface-coupled ring of Ni(II)NTA gold-plated polystyrene particles.

We report a novel, low-resource malaria diagnostic platform inspired by the coffee ring phenomenon, selective for Plasmodium falciparum histidine-rich protein-II (PfHRP-II), a biomarker indicative of the P. falciparum parasite strain. In this diagnostic design, a recombinant HRP-II (rcHRP-II) biomarker is sandwiched between 1 µm Ni(II)nitrilotriacetic acid (NTA) gold-plated polystyrene microspheres (AuPS) and Ni(II)NTA-functionalized glass. After rcHRP-II malaria biomarkers had reacted with Ni(II)NTA-functionalized particles, a 1 µL volume of the particle-protein conjugate solution is deposited onto a functionalized glass slide. Drop evaporation produces the radial flow characteristic of coffee ring formation, and particle-protein conjugates are transported toward the drop edge, where, in the presence of rcHRP-II, particles bind to the Ni(II)NTA-functionalized glass surface. After evaporation, a wash with deionized water removes nonspecifically bound materials while maintaining the integrity of the surface-coupled ring produced by the presence of the protein biomarker. The dynamic range of this design was found to span 3 orders of magnitude, and rings are visible with the naked eye at protein concentrations as low as 10 pM, 1 order of magnitude below the 100 pM PfHRP-II threshold recommended by the World Health Organization. Key enabling features of this design are the inert and robust gold nanoshell to reduce nonspecific interactions on the particle surface, inclusion of a water wash step after drop evaporation to reduce nonspecific binding to the glass, a large diameter particle to project a large two-dimensional viewable area after ring formation, and a low particle density to favor radial flow toward the drop edge and reduce vertical settling to the glass surface in the center of the drop. This robust, antibody-free assay offers a simple user interface and clinically relevant limits of biomarker detection, two critical features required for low-resource malaria detection.

Development of a histidine-targeted spectrophotometric sensor using Ni(II)NTA-functionalized Au and Ag nanoparticles.

An antibody-free diagnostic reagent has been developed based on the aggregation-induced colorimetric change of Ni(II)NTA-functionalized colloidal gold and silver nanoparticles. This diagnostic strategy utilizes the high binding affinity of histidine-rich proteins with Ni(II)NTA to capture and cross-link the histidine-rich protein mimics with the silver and gold nanoparticles. In model studies, the aggregation behavior of the Ni(II)NTA nanoparticles was tested against synthetic targets including charged poly(amino acid)s (histidine, lysine, arginine, and aspartic acid) and mimics of Plasmodium falciparum histidine-rich protein 2 (pfHRP-II). Aggregation of the nanoparticle sensor was induced by all of the basic poly(amino acid)s including poly(l-histidine) within the pH range (5.5-9.0) tested, which is likely caused by the coordination between the multivalent polymer target and Ni(II)NTA groups on multiple particles. The peptide mimics induced aggregation of the nanoparticles only near their pK(a)'s with higher limits of detection. In addition, monomeric amino acids do not show any aggregation behavior, suggesting that multiple target binding sites are necessary for aggregation. Long-term stability studies showed that gold but not silver nanoparticles remained stable and exhibited similar aggregation behavior after 1 month of storage at room temperature and 37 °C. These results suggest that Ni(II)NTA gold nanoparticles could be further investigated for use as a sensor to detect histidine-rich proteins in biological samples.