Highly Potent Peptide Therapeutics To Prevent Protein Aggregation in Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disorder resulting from a significant amplification of CAG repeats in exon 1 of the Huntingtin (Htt) gene. More than 36 CAG repeats result in the formation of a mutant Htt (mHtt) protein. These amino-terminal mHtt fragments lead to the formation of misfolded proteins, which then form aggregates in the relevant brain regions. Therapies that can delay the progression of the disease are imperative to halting the course of the disease. Peptide-based drug therapies provide such a platform. Inhibitory peptides were screened against monomeric units of both wild type (Htt(Q25)) and mHtt fragments, Htt(Q46) and Htt(Q103). Fibril kinetics was studied by utilizing the Thioflavin T (ThT) assay. Atomic force microscopy was also used to study the influence of the peptides on fibril formation. These experiments demonstrate that the chosen peptides suppress the formation of fibrils in mHtt proteins and can provide a therapeutic lead for further optimization and development.

Monitoring Molecular Assembly of Biofilms Using Quartz Crystal Microbalance with Dissipation (QCM-D).

The structure and the functionality of biofilm proteins, the main components of the extracellular matrix, can be tuned by protein engineering. The use of binding kinetics data has been demonstrated in the characterization of recombinantly produced biofilm proteins to control their behavior on certain surfaces or under certain conditions. Quartz crystal microbalance with dissipation monitoring (QCM-D) allows measuring the change in resonance frequency and the energy loss and distribution upon the interaction of molecules with the surface. The characterization of the molecular assembly of curli biofilm proteins on different surfaces using QCM-D is presented here as a detailed protocol. The experimental procedure detailed in this chapter can be applied and modified for other biofilm proteins or subunits to determine their surface adsorption and kinetic binding characteristics.

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.

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.

Interaction of microbial functional amyloids with solid surfaces.

HYPOTHESIS: Self-assembling protein subunits hold great potential as biomaterials with improved functions. Among the self-assembled protein structures functional amyloids are promising unique properties such as resistance to harsh physical and chemical conditions their mechanical strength, and ease of functionalization. Curli proteins, which are functional amyloids of bacterial biofilms can be programmed as intelligent biomaterials. EXPERIMENTS: In order to obtain controllable curli based biomaterials for biomedical applications, and to understand role of each of the curli forming monomeric proteins (namely CsgA and CsgB from Escherichia coli) we characterized their binding kinetics to gold, hydroxyapatite, and silica surfaces. FINDINGS: We demonstrated that CsgA, CsgB, and their equimolar mixture have different binding strengths for different surfaces. On hydroxyapatite and silica surfaces, CsgB is the crucial element that determines the final adhesiveness of the CsgA-CsgB mixture. On the gold surface, on the other hand, CsgA controls the behavior of the mixture. Those findings uncover the binding behavior of curli proteins CsgA and CsgB on different biomedically valuable surfaces to obtain a more precise control on their adhesion to a targeted surface.

Bioinspired multifunctional adhesive system for next generation bio-additively designed dental restorations.

Resin-based composite has overtaken dental amalgam as the most popular material for the repair of lost or damaged tooth structure. In spite of the popularity, the average composite lifetime is about half that of amalgam restorations. The leading cause of composite-restoration failure is decay at the margin where the adhesive is applied. The adhesive is intended to seal the composite/tooth interface, but the adhesive seal to dentin is fragile and readily degraded by acids, enzymes and other oral fluids. The inherent weakness of this material system is attributable to several factors including the lack of antimicrobial properties, remineralization capabilities and durable mechanical performance - elements that are central to the integrity of the adhesive/dentin (a/d) interfacial seal. Our approach to this problem offers a transition from a hybrid to a biohybrid structure. Discrete peptides are tethered to polymers to provide multi-bio-functional adhesive formulations that simultaneously achieve antimicrobial and remineralization properties. The bio-additive materials design combines several functional properties with the goal of providing an adhesive that will serve as a durable barrier to recurrent decay at the composite/tooth interface. This article provides an overview of our multi-faceted approach which uses peptides tethered to polymers and new polymer chemistries to achieve the next generation adhesive system - an adhesive that provides antimicrobial properties, repair of defective dentin and enhanced mechanical performance.

Antimicrobial Peptide-Polymer Conjugates for Dentistry.

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.

Repeatedly Applied Peptide Film Kills Bacteria on Dental Implants.

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, nonsur-gical 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.

Biomineralization of Calcium Phosphate Crystals Controlled by Protein-Protein Interactions.

Hydroxyapatite (HAP) is the major biomineral of bone. Despite the large number of studies addressing HAP formation, a fundamental understanding of the critical roles of HAP-forming proteins in vitro is needed. Effects of two HAP-interacting proteins, osteocalcin (OCN) and osteopontin (OPN), on HAP formation was investigated via in vitro biomineralization experiments, and their outcomes on the crystal structure of calcium phosphate (CaP) was revealed. Our data suggest that OCN concentration is negatively correlated with crystal formation rate and crystal size, yet the presence of OCN leads to a more ordered HAP crystal formation. On the other hand, OPN protein promotes faster formation of CaP crystals potentially working as a growth site for mineral formation, and it decreases the Ca:P ratio. This effect results in a shift from HAP-type minerals to less ordered crystals. The crystal size, shape, and Ca:P ratio can be tuned to design improved mammalian hard tissue environment-mimicking matrices by taking advantage of the OCN and OPN proteins on crystal formation. We believe our current findings will lead to innovative approaches for bone biomineralization in regenerative medicine.

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.

Self Assembled Recombinant Proteins on Metallic Nanoparticles As Bimodal Imaging Probes.

Combining multiple modalities is at the center of developing 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 owning 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 to assemble selective functional nano-entities or proteins onto nanoparticles surfaces. Herein we explored formation of self-assembled hybrid metallic nano-architectures that are 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 been 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 biological tissues as well as providing high signal-to-noise ratio and sensitivity.

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.

Self-assembly of bacterial amyloid protein nanomaterials on solid surfaces.

HYPOTHESIS: Amyloid-forming biofilm proteins of Escherichia coli, namely CsgA and CsgB, can form self-assembled nanofibers on solid surfaces. These proteins can be programmed to form bio-nanomaterials for functional applications. EXPERIMENTS: In this study, the assembly of the CsgA and CsgB protein on solid surfaces was investigated in real time using a quartz crystal microbalance instrument with dissipation monitoring. The assembly kinetics of the CsgA and CsgB proteins in various settings on solid surfaces were investigated. Protein nanowires were investigated using electron microscopy. FINDINGS: CsgA protein polymers and CsgB-added CsgA polymers form densely packed biofilm on gold surfaces, whereas CsgB polymers and CsgA-added CsgB polymers form biofilms with high water-holding capacity according to the dissipation data. Electron microscopy images of nanofibers grown on gold surfaces showed that CsgA and CsgB polymers include thicker nanofibers compared to the nanofibers formed by CsgA-CsgB protein combinations. The resulting nano/microstructures were found to have strong fluorescence signals in aqueous environments and in chloroform while conserving the protein nanowire network.

Engineered Peptide Repairs Defective Adhesive-Dentin Interface.

Failure of dental composite restorations is primarily due to recurrent decay at the tooth-composite interface. At this interface, the adhesive and its bond with dentin is the barrier between the restored tooth and the oral environment. In vivo degradation of the bond formed at the adhesive/dentin (a/d) interface follows a cascade of events leading to weakening of the composite restoration. Here, a peptide-based approach is developed to mineralize deficient dentin matrices at the a/d interface. Peptides that have an inherent capacity to self-assemble on dentin and to induce calcium-phosphate remineralization are anchored at the interface. Distribution of adhesive, collagen, and mineral is analyzed using micro-Raman spectroscopy and fluorescence microscopy. The analysis demonstrates remineralization of the deficient dentin matrices achieved throughout the interface with homogeneous distribution of mineral. The peptide-based remineralization demonstrated here can be an enabling technology to design integrated biomaterial-tissue interfaces.

Biosilver nanoparticle interface offers improved cell viability.

Silver nanoparticles (AgNP) are promising candidates for fighting drug-resistant infections because of their intrinsic antimicrobial effect. The design of high-yield antimicrobial molecules may inadvertently cause variation in host cells' biological responses. While many factors affect AgNPs' efficacy, their surface is exposed to the biological environment and thus plays a critical role in both the preservation of antimicrobial efficacy against pathogens and the modulation of host cells cytotoxicity. This work investigated an engineered biomimetic interface approach to controlling AgNP surface properties to provide them a competitive advantage in a biological environment. Here, a fusion protein featuring a silver-binding peptide (AgBP) domain was engineered to enable self-assembly and track assembly by a green fluorescent protein (GFP) reporter. Following AgNP functionalisation with GFP-AgBP, their antimicrobial and cytotoxic properties were evaluated. GFP-AgBP binding affinity to AgNPs was evaluated using localized surface plasmon resonance sensing. The GFP-AgBP biomimetic interface on AgNPs' surfaces provided sustained antibacterial efficacy at low concentrations based on bacterial growth inhibition assays. Viability and cytotoxicity measurements in fibroblast cells exposed to GFP-AgBP protein-functionalised AgNPs showed significant improvement compared to controls. Biointerface engineering offers promise towards tailoring AgNP antimicrobial efficacy while addressing safety concerns to maintain optimum cellular interactions.

Coupling Infusion and Gyration for the Nanoscale Assembly of Functional Polymer Nanofibers Integrated with Genetically Engineered Proteins.

Nanofibers featuring functional nanoassemblies show great promise as enabling constituents for a diverse range of applications in areas such as tissue engineering, sensing, optoelectronics, and nanophotonics due to their controlled organization and architecture. An infusion gyration method is reported that enables the production of nanofibers with inherent biological functions by simply adjusting the flow rate of a polymer solution. Sufficient polymer chain entanglement is obtained at Berry number > 1.6 to make bead-free fibers integrated with gold nanoparticles and proteins, in the diameter range of 117-216 nm. Integration of gold nanoparticles into the nanofiber assembly is followed using a gold-binding peptide tag genetically conjugated to red fluorescence protein (DsRed). Fluorescence microscopy analysis corroborated with Fourier transform infrared spectroscopy (FTIR) data confirms the integration of the engineered red fluorescence protein with the nanofibers. The gold nanoparticle decorated nanofibers having red fluorescence protein as an integral part keep their biological functionality including copper-induced fluorescence quenching of the DsRed protein due to its selective Cu(+2) binding. Thus, coupling the infusion gyration method in this way offers a simple nanoscale assembly approach to integrate a diverse repertoire of protein functionalities into nanofibers to generate biohybrid materials for imaging, sensing, and biomaterial applications.

Addressable self-immobilization of lactate dehydrogenase across multiple length scales.

Successful nanobiotechnology implementation largely depends on control over the interfaces between inorganic materials and biological molecules. Controlling the orientations of biomolecules and their spatial arrangements on the surface may transform many technologies including sensors, to energy. Here, we demonstrate the self-organization of L-lactate dehydrogenase (LDH), which exhibits enhanced enzymatic activity and stability on a variety of gold surfaces ranging from nanoparticles to electrodes, by incorporating a gold-binding peptide tag (AuBP2) as the fusion partner for Bacillus stearothermophilus LDH (bsLDH). Binding kinetics and enzymatic assays verified orientation control of the enzyme on the gold surface through the genetically incorporated peptide tag. Finally, redox catalysis efficiency of the immobilized enzyme was detected using cyclic voltammetry analysis in enzyme-based biosensors for lactate detection as well as in biofuel cell energy systems as the anodic counterpart. Our results demonstrate that the LDH enzyme can be self-immobilized onto different gold substrates using the short peptide tag under a biologically friendly environment. Depending on the desired inorganic surface, the proposed peptide-mediated path could be extended to any surface to achieve single-step oriented enzyme immobilization for a wide range of applications.

In vitro labeling of hydroxyapatite minerals by an engineered protein.

Biological and biomimetic synthesis of inorganics have been a major focus in hard tissue engineering as well as in green processing of advanced materials. Among the minerals formed by organisms, calcium phosphate mineralization is studied extensively to understand the formation of mineral-rich tissues. Herein, we report an engineered fusion protein that not only targets calcium phosphate minerals but also allows monitoring of biomineralization. To produce the bi-functional fusion protein, nucleotide sequence encoding combinatorially selected hydroxyapatite-binding peptides (HABP) was genetically linked to the 3' end of the open reading frame of green fluorescence protein (GFPuv) and successfully expressed in Escherichia coli. The fluorescence and binding activities of the bi-functional proteins were characterized by, respectively, using fluorescence microscopy and quartz crystal microbalance spectroscopy. The utility of GFPuv-HABP fusion protein was assessed for both time-wise monitoring of mineralization and the visualization of the mineralized tissues. We used an alkaline phosphatase-based reaction to control phosphate release, thereby mimicking biological processes, to monitor calcium phosphate mineralization. The increase in mineral amount was observed using the fusion protein at different time points. GFPuv-HABP1 was also used for efficient fluorescence labeling of mineralized regions on the extracted human incisors. Our results demonstrate a simple and versatile application of inorganic-binding peptides conjugated with bioluminescence proteins as bi-functional bioimaging molecular probes that target mineralization, and which can be employed to a wide range of biomimetic processing and cell-free tissue engineering.