Localization of Therapeutic Fab-CHP Conjugates to Sites of Denatured Collagen for the Treatment of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in synovial joints and protease-induced cartilage degradation. Current biologic treatments for RA can effectively reduce symptoms, primarily by neutralizing the proinflammatory cytokine TNFα; however, continued, indiscriminate overinhibition of inflammatory factors can significantly weaken the host immune system, leading to opportunistic infections and interrupting treatment. We hypothesize that localizing anti-TNFα therapeutics to denatured collagen (dCol) present at arthritic joints, via conjugation with collagen-hybridizing peptides (CHPs), will reduce off-site antigen binding and maintain local immunosuppression. We isolated the antigen-binding fragment of the clinically approved anti-TNFα therapeutic infliximab (iFab) and prepared iFab-CHP conjugates via lysine-based conjugation with an SMCC linker. After successful conjugation, confirmed by LC-MS, the binding affinity of iFab-CHP was characterized by ELISA-like assays, which showed comparable antigen binding relative to infliximab, comparable dCol binding relative to CHP, and the hybrid ability to bind both dCol and TNFα simultaneously. We further demonstrated localization of Fab-CHP to areas of high dCol in vivo and promising therapeutic efficacy, assessed by histological staining (Safranin-O and H&E), in a pilot mouse study.

Visualizing collagen proteolysis by peptide hybridization: From 3D cell culture to in vivo imaging.

Degradation of the extracellular matrix (ECM) is one of the fundamental factors contributing to a variety of life-threatening or disabling pathological conditions. However, a thorough understanding of the degradation mechanism and development of new ECM-targeting diagnostics are severely hindered by a lack of technologies for direct interrogation of the ECM structures at the molecular level. Previously we demonstrated that the collagen hybridizing peptide [CHP, sequence: (GPO)9, O: hydroxyproline] can specifically recognize the degraded and unfolded collagen chains through triple helix formation. Here we show that fluorescently labeled CHP robustly visualizes the pericellular matrix turnover caused by proteolytic migration of cancer cells within 3D collagen culture, without the use of synthetic fluorogenic matrices or genetically modified cells. To facilitate in vivo imaging, we modified the CHP sequence by replacing each proline with a (2S,4S)-4-fluoroproline (f) residue which interferes with the peptide's inherent propensity to self-assemble into homo-triple helices. We show that the new CHP, (GfO)9, tagged with a near-infrared fluorophore, enables in vivo imaging and semi-quantitative assessment of osteolytic bone lesions in mouse models of multiple myeloma. Compared to conventional techniques (e.g., micro-CT), CHP-based imaging is simple and versatile in vitro and in vivo. Therefore, we envision CHP's applications in broad biomedical contexts ranging from studies of ECM biology and drug efficiency to development of clinical molecular imaging.

Self-Assembled Water-Soluble Nanofibers Displaying Collagen Hybridizing Peptides.

Collagen hybridizing peptides (CHP) have been demonstrated as a powerful vehicle for targeting denatured collagen (dColl) produced by disease or injury. Conjugation of ß-sheet peptide motif to the CHP results in self-assembly of nonaggregating ß-sheet nanofibers with precise structure. Due to the molecular architecture of the nanofibers which puts high density of hydrophilic CHPs on the nanofiber surface at fixed distance, the nanofibers exhibit high water solubility, without any signs of intramolecular triple helix formation or fiber-fiber aggregation. Other molecules that are flanked with the triple helical forming GlyProHyp repeats can readily bind to the nanofibers by triple helical folding, allowing facile display of bioactive molecules at high density. In addition, the multivalency of CHPs allows the nanofibers to bind to dColl in vitro and in vivo with extraordinary affinity, particularly without preactivation that unravels the CHP homotrimers. The length of the nanofibers can be tuned from micrometers down to 100 nm by simple heat treatment, and when injected intravenously into mice, the small nanofibers can specifically target dColl in the skeletal tissues with little target-associated signals in the skin and other organs. The CHP nanofibers can be a useful tool for detecting and capturing dColl, understanding how ECM remodelling impacts disease progression, and development of new delivery systems that target such diseases.

Molecular level detection and localization of mechanical damage in collagen enabled by collagen hybridizing peptides.

Mechanical injury to connective tissue causes changes in collagen structure and material behaviour, but the role and mechanisms of molecular damage have not been established. In the case of mechanical subfailure damage, no apparent macroscale damage can be detected, yet this damage initiates and potentiates in pathological processes. Here, we utilize collagen hybridizing peptide (CHP), which binds unfolded collagen by triple helix formation, to detect molecular level subfailure damage to collagen in mechanically stretched rat tail tendon fascicle. Our results directly reveal that collagen triple helix unfolding occurs during tensile loading of collagenous tissues and thus is an important damage mechanism. Steered molecular dynamics simulations suggest that a likely mechanism for triple helix unfolding is intermolecular shearing of collagen α-chains. Our results elucidate a probable molecular failure mechanism associated with subfailure injuries, and demonstrate the potential of CHP targeting for diagnosis, treatment and monitoring of tissue disease and injury.

Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide.

Decellularized extracellular matrix (ECM) derived from tissues and organs are emerging as important scaffold materials for regenerative medicine. Many believe that preservation of the native ECM structure during decellularization is highly desirable. However, because effective techniques to assess the structural damage in ECM are lacking, the disruptive effects of a decellularization method and the impact of the associated structural damage upon the scaffold's regenerative capacity are often debated. Using a novel collagen hybridizing peptide (CHP) that specifically binds to unfolded collagen chains, we investigated the molecular denaturation of collagen in the ECM decellularized by four commonly used cell-removing detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium deoxycholate (SD), and Triton X-100. Staining of the detergent-treated porcine ligament and urinary bladder matrix with carboxyfluorescein-labeled CHP demonstrated that SDS and Triton X-100 denature the triple helical collagen molecule while CHAPS and SD do not, although second harmonic generation imaging and transmission electron microscopy (TEM) revealed that all four detergents disrupt collagen fibrils. Our findings from the CHP staining were further confirmed by the circular dichroism spectra of intact triple helical collagen molecules in CHAPS and SD solutions, and the TEM images of CHP-conjugated gold nanoparticles binding only to the SDS and Triton X-100 treated collagen fibrils. CHP is a powerful new tool for direct and reliable measurement of denatured collagen molecules in decellularized tissues. It is expected to have wide applications in the development and standardization of the tissue/organ decellularization technology. STATEMENT OF SIGNIFICANCE: Preservation of the native ECM structure in decellularized tissues is highly desirable, since denaturation of ECM molecules (e.g., collagen) during decellularization can strongly influence the cellular response. Unfortunately, conventional techniques (SEM, SHG) are not conducive to identifying denatured collagen molecules in tissues. We demonstrate the first investigation into the molecular denaturation of collagen in decellularized ECM enabled by a novel Collagen Hybridizing Peptide (CHP) that specifically binds to unfolded collagen chains. We show that SDS and Triton X-100 denature collagen molecules while CHAPS and SD cannot. Such detection has been nearly impossible with other existing techniques. The CHP technique will advance our understanding about the effect of the cell-removing process on ECM, and lead to development of the decellularization technology.

Bioinorganic Nanohybrid Catalyst for Multistep Synthesis of Acetaminophen, an Analgesic.

A bioinorganic nanohybrid catalyst was synthesized by combining esterase with a platinum nanoparticle (PtNP). The combination of two catalysts resulted in enhanced catalytic activities, esterase hydrolysis, and hydrogenation in PtNPs, as compared to each catalyst alone. This hybrid catalyst can be successfully used in the multistep synthesis of acetaminophen (paracetamol), an analgesic and antipyretic drug, in a one-pot reaction with high yield and efficacy within a short time, demonstrating that the nanobiohybrid catalyst offers advantages in the synthesis of fine chemicals in industrial applications.

Nanoparticle Assembly and Gelatin Binding Mediated by Triple Helical Collagen Mimetic Peptide.

Peptide-conjugated nanoparticles (NPs) have promising potential for applications in biosensing, diagnosis, and therapeutics because of their appropriate size, unique self-assembly, and specific substrate-binding properties. However, controlled assembly and selective target binding are difficult to achieve with simple peptides on NP surfaces because high surface energy makes NPs prone to self-aggregate and adhere nonspecifically. Here, we report the self-assembly and gelatin binding properties of collagen mimetic peptide (CMP) conjugated gold NPs (CMP-NPs). We show that the orientation of CMPs displayed on the NP surface can control NP assembly either by promoting or hindering triple helical folding between CMPs of neighboring NPs. We also show that CMP-NPs can specifically bind to denatured collagen by forming triple-helical hybrids between denatured collagen strands and CMPs, demonstrating their potential use for detection and selective removal of gelatin from protein mixtures. CMP conjugated NPs offer a simple and effective method for NP assembly and for targeting denatured collagens with high specificity. Therefore, they may lead to new types of functional nanomaterials for detection and study of denatured collagen associated with diseases characterized by high levels of collagen degradation.

Photocurrent enhancement of SiNW-FETs by integrating protein-shelled CdSe quantum dots.

We proposed a new strategy to increase the photoresponsivity of silicon NW field-effect transistors (FETs) by integrating CdSe quantum dots (QDs) using protein shells (PSs). CdSe QDs were synthesized using ClpP, a bacterial protease, as protein shells to control the size and stability of QD and to facilitate the mounting of QDs on SiNWs. The photocurrent of SiNW-FETs in response to light at a wavelength of 480 nm was enhanced by a factor of 6.5 after integrating CdSe QDs because of the coupling of the optical properties of SiNWs and QDs. As a result, the photoresponsivity to 480 nm light reached up to 3.1 × 10(6), the highest value compared to other SiNW-based devices in the visible light range.

Non-covalent photo-patterning of gelatin matrices using caged collagen mimetic peptides.

To address the downside of conventional photo-patterning which can alter the chemical composition of protein scaffolds, we developed a non-covalent photo-patterning strategy for gelatin (denatured collagen) hydrogels that utilizes UV activated triple helical hybridization of caged collagen mimetic peptide (caged CMP). Here we present 2D and 3D photo-patterning of gelatin hydrogels enabled by the caged CMP derivatives, as well as creation of concentration gradients of CMPs. CMP's specificity for binding to gelatin allows patterning of almost any synthetic or natural gelatin-containing matrix, such as gelatin-methacrylate hydrogels and corneal tissues. This is a radically new tool for immobilizing drugs to natural tissues and for functionalizing scaffolds for complex tissue formation.

Combining protein-shelled platinum nanoparticles with graphene to build a bionanohybrid capacitor.

The electronic properties of biomolecules and their hybrids with inorganic materials can be utilized for the fabrication of nanoelectronic devices. Here, we report the charge transport behavior of protein-shelled inorganic nanoparticles combined with graphene and demonstrate their possible application as a bionanohybrid capacitor. The conductivity of PepA, a bacterial aminopeptidase used as a protein shell (PS), and the platinum nanoparticles (PtNPs) encapsulated by PepA was measured using a field effect transistor (FET) and a graphene-based FET (GFET). Furthermore, we confirmed that the electronic properties of PepA-PtNPs were controlled by varying the size of the PtNPs. The use of two poly(methyl methacrylate) (PMMA)-coated graphene layers separated by PepA-PtNPs enabled us to build a bionanohybrid capacitor with tunable properties. The combination of bioinorganic nanohybrids with graphene is regarded as the cornerstone for developing flexible and biocompatible bionanoelectronic devices that can be integrated into bioelectric circuits for biomedical purposes.

Radiofrequency treatment enhances the catalytic function of an immobilized nanobiohybrid catalyst.

Biocatalysis, the use of enzymes in chemical transformation, has undergone intensive development for a wide range of applications. As such, maximizing the functionality of enzymes for biocatalysis is a major priority to enable industrial use. To date, many innovative technologies have been developed to address the future demand of enzymes for these purposes, but maximizing the catalytic activity of enzymes remains a challenge. In this study, we demonstrated that the functionality of a nanobiocatalyst could be enhanced by combining immobilization and radiofrequency (RF) treatment. Aminopeptidase PepA-encapsulating 2 nm platinum nanoparticles (PepA-PtNPs) with the catalytic activities of hydrolysis and hydrogenation were employed as multifunctional nanobiocatalysts. Immobilizing the nanobiocatalysts in a hydrogel using metal chelation significantly enhanced their functionalities, including catalytic power, thermal-stability, pH tolerance, organic solvent tolerance, and reusability. Most importantly, RF treatment of the hydrogel-immobilized PepA-PtNPs increased their catalytic power by 2.5 fold greater than the immobilized PepA. Our findings indicate that the catalytic activities and functionalities of PepA-PtNPs are greatly enhanced by the combination of hydrogel-immobilization and RF treatment. Based on our findings, we propose that RF treatment of nanobiohybrid catalysts immobilized on the bulk hydrogel represents a new strategy for achieving efficient biocatalysis.

Investigation of the heating properties of platinum nanoparticles under a radiofrequency current.

PURPOSE: For the potential application of platinum nanoparticles (PtNPs) in hyperthermia therapy, the heating efficiency of PtNPs in the presence of radiofrequency (RF) current generated by a capacitive electric transfer (CET) system was compared with that of gold nanoparticles (AuNPs). MATERIALS AND METHODS: PtNPs and AuNPs synthesised by citrate capping (5 nm) were exposed to an RF current of 0.35 ± 0.05 MHz in a CET system. The temperature of the solution containing various concentrations of platinum or gold NPs was monitored for 5 min at various power ranges. RESULTS: When both NP solutions were exposed to an RF field at a fixed power, the temperature of the NP solution increased continuously over the 5 min of measurement. In contrast, the NP-free solutions did not show any temperature change. Both PtNPs and AuNPs can be heated in a concentration- and power-dependent manner. However, PtNPs showed a higher efficiency in generating heat compared with AuNPs in both water and the physiological buffer. CONCLUSIONS: The heat generating efficiency of 5-nm PtNPs was about 50% higher than that of AuNPs when they were exposed to electric current through RF. This result suggests that PtNPs are promising nanomaterials for RF-induced hyperthermia therapy.

Size-controlled synthesis and characterization of CoPt nanoparticles using protein shells.

Nanostructured magnetic materials such as iron oxide and bimetallic nanoparticles can be potentially applied to a variety of fields, including electronics and nanomedicine. To develop these applications, it is important to control their particle size which affects their magnetic properties. In particular, it is a major challenge to synthesize small-sized nanoparticles with high reproducibility. In this study, we synthesized cobalt-platinum nanoparticles (CoPt NPs) in an ambient solution phase using PepA, a bacterial aminopeptidase, as a protein shell, and investigated the physicochemical and magnetic properties of NPs with and without encapsulating proteins. The size of CoPt NPs encapsulated by PepA was stringently controlled from 1.1 to 2.8 nm, and their magnetic property was related to the size. The CoPt NPs with the diameter of 1.1 nm showed a superparamagnetic behavior only at low temperatures, while 2.1 and 2.8 nm CoPt NPs were ferromagnetic below the blocking temperature. PepA had no deleterious effects on the coercivity of CoPt NPs, as evidenced by the marginal effect of PepA on the coercivity of CoPt NPs. This study demonstrated that the particle size and magnetic property of CoPt NPs can be controlled by using PepA as a protein shell. Encapsulation by PepA will aid the development of multifunctional magnetic materials, since the biocompatibility and modification capability of PepA can be synergistically combined with the advanced functionalities of CoPt NPs.

Structural basis for the substrate specificity of PepA from Streptococcus pneumoniae, a dodecameric tetrahedral protease.

Regulated cytosolic proteolysis is one of the key cellular processes ensuring proper functioning of a cell. M42 family proteases show a broad spectrum of substrate specificities, but the structural basis for such diversity of the substrate specificities is lagging behind biochemical data. Here we report the crystal structure of PepA from Streptococcus pneumoniae, a glutamyl aminopeptidase belonging to M42 family (SpPepA). We found that Arg-257 in the substrate binding pocket is strategically positioned so that Arg-257 can make electrostatic interactions with the acidic residue of a substrate at its N-terminus. Structural comparison of the substrate binding pocket of the M42 family proteases, along with the structure-based multiple sequence alignment, argues that the appropriate electrostatic interactions contribute to the selective substrate specificity of SpPepA.