Self-Assembled Recombinant Proteins on Metallic Nanoparticles as Bimodal Imaging Probes

Combining multiple modalities is central to developing the new methods for sensing and imaging that are required for comprehensive understanding of events at the molecular level. Various imaging modalities have been developed using metallic nanoparticles owing to their exceptional physical and chemical properties. Due to their localized surface plasmon resonance characteristics, gold and silver nanoparticles exhibit unique optoelectronic properties commonly used in biomedical sciences and engineering. Self-assembled monolayers or physical adsorption have previously been adapted to functionalize the surfaces of nanoparticles with biomolecules for targeted imaging. However, depending on differences among the functional groups used on the nanoparticle surface, wide variation in the displayed biomolecular property to recognize its target may result. In the last decade, the properties of inorganic binding peptides have been proven advantageous for assembling selective functional nano-entities or proteins onto nanoparticle surfaces. Herein we explored the formation of self-assembled hybrid metallic nano-architectures composed of gold and silver nanoparticles with fluorescent proteins for use as bimodal imaging probes. We employed metal-binding peptide-based assembly to self-assemble green fluorescence protein onto metallic substrates of various geometries. Assembly of the green fluorescent proteins, genetically engineered to incorporate gold- or silver-binding peptides onto metallic nanoparticles, resulted in the generation of hybrid-, biomodal-imaging probes in a single step. Green fluorescent activity on gold and silver surfaces can be monitored using both plasmonic and fluorescent signatures. Our results demonstrate a novel bimodal imaging system that can be finely tuned with respect to nanoparticle size and protein concentration. Resulting hybrid probes may mitigate the limitation of depth penetration into biologic tissues and provide a high signal-to-noise ratio and sensitivity

Repeatedly Applied Peptide Film Kills Bacteria on Dental Implants

This is a post-peer-review, pre-copyedit version of an article published in JOM Journal of the Minerals, Metals and Materials Society. The final authenticated version is available online at: http://dx.doi.org/10.1007/s11837-019-03334-w.The rising use of titanium dental implants has increased the prevalence of peri-implant disease that shortens their useful life. A growing view of peri-implant disease suggests that plaque accumulation and microbiome dysbiogenesis trigger a host immune inflammatory response that destroys soft and hard tissues supporting the implant. The incidence of peri-implant disease is difficult to estimate, but with over 3 million implants placed in the USA alone, and the market growing by 500,000 implants/year, such extensive use demands additional interceptive approaches. We report a water-based, nonsurgical approach to address peri-implant disease using a bifunctional peptide film, which can be applied during initial implant placement and later reapplied to existing implants to reduce bacterial growth. Bifunctional peptides are based upon a titanium binding peptide (TiBP) optimally linked by a spacer peptide to an antimicrobial peptide (AMP). We show herein that dental implant surfaces covered with a bifunctional peptide film kill bacteria. Further, using a simple protocol for cleaning implant surfaces fouled by bacteria, the surface can be effectively recoated with TiBP-AMP to regain an antimicrobial state. Fouling, cleansing, and rebinding was confirmed for up to four cycles with minimal loss of binding efficacy. After fouling, rebinding with a water-based peptide film extends control over the oral microbiome composition, providing a novel nonsurgical treatment for dental implants

Reconfigurable Dual Peptide Tethered Polymer System Offers a Synergistic Solution for Next Generation Dental Adhesives

Resin-based composite materials have been widely used in restorative dental materials due to their aesthetic, mechanical, and physical properties. However, they still encounter clinical shortcomings mainly due to recurrent decay that develops at the composite-tooth interface. The low-viscosity adhesive that bonds the composite to the tooth is intended to seal this interface, but the adhesive seal is inherently defective and readily damaged by acids, enzymes, and oral fluids. Bacteria infiltrate the resulting gaps at the composite-tooth interface and bacterial by-products demineralize the tooth and erode the adhesive. These activities lead to wider and deeper gaps that provide an ideal environment for bacteria to proliferate. This complex degradation process mediated by several biological and environmental factors damages the tooth, destroys the adhesive seal, and ultimately, leads to failure of the composite restoration. This paper describes a co-tethered dual peptide-polymer system to address composite-tooth interface vulnerability. The adhesive system incorporates an antimicrobial peptide to inhibit bacterial attack and a hydroxyapatite-binding peptide to promote remineralization of damaged tooth structure. A designer spacer sequence was incorporated into each peptide sequence to not only provide a conjugation site for methacrylate (MA) monomer but also to retain active peptide conformations and enhance the display of the peptides in the material. The resulting MA-antimicrobial peptides and MA-remineralization peptides were copolymerized into dental adhesives formulations. The results on the adhesive system composed of co-tethered peptides demonstrated both strong metabolic inhibition of S. mutans and localized calcium phosphate remineralization. Overall, the result offers a reconfigurable and tunable peptide-polymer hybrid system as next-generation adhesives to address composite-tooth interface vulnerability

Antimicrobial Peptide–Polymer Conjugates for Dentistry

This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Applied Polymer Materials, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see doi.org/10.1021/acsapm.9b00921.Bacterial adhesion and growth at the composite/adhesive/tooth interface remain the primary cause of dental composite restoration failure. Early colonizers, including Streptococcus mutans, play a critical role in the formation of dental caries by creating an environment that reduces the adhesive’s integrity. Subsequently, other bacterial species, biofilm formation, and lactic acid from S. mutans demineralize the adjoining tooth. Because of their broad spectrum of antibacterial activity and low risk for antibiotic resistance, antimicrobial peptides (AMPs) have received significant attention to prevent bacterial biofilms. Harnessing the potential of AMPs is still very limited in dentistry—a few studies have explored peptide-enabled antimicrobial adhesive copolymer systems using mainly nonspecific adsorption. In the current investigation, to avoid limitations from nonspecific adsorption and to prevent potential peptide leakage out of the resin, we conjugated an AMP with a commonly used monomer for dental adhesive formulation. To tailor the flexibility between the peptide and the resin material, we designed two different spacer domains. The spacer-integrated antimicrobial peptides were conjugated to methacrylate (MA), and the resulting MA–AMP monomers were next copolymerized into dental adhesives as AMP–polymer conjugates. The resulting bioactivity of the polymethacrylate-based AMP conjugated matrix activity was investigated. The antimicrobial peptide conjugated to the resin matrix demonstrated significant antimicrobial activity against S. mutans. Secondary structure analyses of conjugated peptides were applied to understand the activity differential. When mechanical properties of the adhesive system were investigated with respect to AMP and cross-linking concentration, resulting AMP–polymer conjugates maintained higher compressive moduli compared to hydrogel analogues including polyHEMA. Overall, our result provides a robust approach to develop a fine-tuned bioenabled peptide adhesive system with improved mechanical properties and antimicrobial activity. The results of this study represent a critical step toward the development of peptide-conjugated dentin adhesives for treatment of secondary caries and the enhanced durability of dental composite restorations

Kinetic Release Studies of Antibiotic Patches for Local Transdermal Delivery

This study investigates the usage of electrohydrodynamic (EHD)-3D printing for the fabrication of bacterial cellulose (BC)/polycaprolactone (PCL) patches loaded with different antibiotics (amoxicillin (AMX), ampicillin (AMP), and kanamycin (KAN)) for transdermal delivery. The composite patches demonstrated facilitated drug loading and encapsulation efficiency of drugs along with extended drug release profiles. Release curves were also subjected to model fitting, and it was found that drug release was optimally adapted to the Higuchi square root model for each drug. They performed a time-dependent and diffusion-controlled release from the patches and followed Fick’s diffusion law by the Korsmeyer–Peppas energy law equation. Moreover, produced patches demonstrated excellent antimicrobial activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) strains, so they could be helpful in the treatment of chronic infectious lesions during wound closures. As different tests have confirmed, various types of antibiotics could be loaded and successfully released regardless of their types from produced BC/PCL patches. This study could breathe life into the production of antibiotic patches for local transdermal applications in wound dressing studies and improve the quality of life of patients

Peptide Mediated Antimicrobial Dental Adhesive System

The most common cause for dental composite failures is secondary caries due to invasive bacterial colonization of the adhesive/dentin (a/d) interface. Innate material weakness often lead to an insufficient seal between the adhesive and dentin. Consequently, bacterial by-products invade the porous a/d interface leading to material degradation and dental caries. Current approaches to achieve antibacterial properties in these materials continue to raise concerns regarding hypersensitivity and antibiotic resistance. Herein, we have developed a multi-faceted, bio-functionalized approach to overcome the vulnerability of such interfaces. An antimicrobial adhesive formulation was designed using a combination of antimicrobial peptide and a ε-polylysine resin system. Effector molecules boasting innate immunity are brought together with a biopolymer offering a two-fold biomimetic design approach. The selection of ε-polylysine was inspired due to its non-toxic nature and common use as food preservative. Biomolecular characterization and functional activity of our engineered dental adhesive formulation were assessed and the combinatorial formulation demonstrated significant antimicrobial activity against Streptococcus mutans. Our antimicrobial peptide-hydrophilic adhesive hybrid system design offers advanced, biofunctional properties at the critical a/d interface

Autonomous Synthesis of Fluorescent Silica Biodots Using Engineered Fusion Proteins

Formation of biological materials is a well-controlled process that is orchestrated by biomolecules such as proteins. Proteins can control the nucleation and mineralization of biomaterials, thereby forming the hard tissues of biological organisms, such as bones, teeth, and shells. In this study, the design and implementation of multifunctional designer proteins are demonstrated for fluorescent silica micro/nanoparticle synthesis. The R5 motif of silaffin polypeptide, which is known for its silicification capability, was fused genetically into three spectrally distinct fluorescent proteins with the intention of forming modified fluorescent proteins. The bifunctional R5 peptide domain served as a tag to provide silica synthesis at ambient conditions. Three functional fusion constructs have been prepared, including GFPmut3-R5, Venus YFP-R5, and mCherry-R5. Recombinant fluorescent proteins were purified using silica-binding peptide tag through silica gel resin. Purified proteins were tested for their binding affinity to silica using quartz crystal microbalance with dissipation monitoring to make sure they can interact strong enough with the silica surfaces. Later, engineered fluorescent proteins were used to synthesize silica nano/microparticles using silica precursor materials. Synthesized silica particles were investigated for their fluorescence properties, including time-resolved fluorescence. Additionally, elemental analysis of the particles was carried out using electron energy loss spectroscopy and energy-filtered transmission electron microscopy. Last, they were tested for their biocompatibility. In this study, we aimed to provide a biomimetic route to synthesize fluorescent silica nanoparticles. Recombinant fluorescent proteins-directed silica nanoparticles synthesis offers a one-step, reliable method to produce fluorescent particles both for biomaterial applications and other nanotechnology applications