RESUMEN
Nanostructures formed by self-assembled peptides have been increasingly exploited as functional materials for a wide variety of applications, from biotechnology to energy. However, it is sometimes challenging to assemble free short peptides into functional supramolecular structures, since not all peptides have the ability to self-assemble. Here, we report a self-assembly mechanism for short functional peptides that we derived from a class of fiber-forming amyloid proteins called curli. CsgA, the major subunit of curli fibers, is a self-assembling ß-helical subunit composed of five pseudorepeats (R1-R5). We first deleted the internal repeats (R2, R3, R4), known to be less essential for the aggregation of CsgA monomers into fibers, forming a truncated CsgA variant (R1/R5). As a proof-of-concept to introduce functionality in the fibers, we then genetically substituted the internal repeats by a hydroxyapatite (HAP)-binding peptide, resulting in a R1/HAP/R5 construct. Our method thus utilizes the R1/R5-driven self-assembly mechanism to assemble the HAP-binding peptide and form hydrogel-like materials in macroscopic quantities suitable for biomineralization. We confirmed the expression and fibrillar morphology of the truncated and HAP-containing curli-like amyloid fibers. X-ray diffraction and TEM showed the functionality of the HAP-binding peptide for mineralization and formation of nanocrystalline HAP. Overall, we show that fusion to the R1 and R5 repeats of CsgA enables the self-assembly of functional peptides into micron long fibers. Further, the mineral-templating ability that the R1/HAP/R5 fibers possesses opens up broader applications for curli proteins in the tissue engineering and biomaterials fields.
Asunto(s)
Durapatita/metabolismo , Proteínas de Escherichia coli/metabolismo , Péptidos/metabolismo , Durapatita/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Nanoestructuras/química , Péptidos/genética , Plásmidos/genética , Plásmidos/metabolismo , Agregado de Proteínas , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
Bacterial infection is a serious medical problem leading to implant failure. The current antibiotic based therapies rise concerns due to bacterial resistance. The family of antimicrobial peptides (AMP) is one of the promising candidates as local therapy agents due to their broad-spectrum activity. Despite AMPs receive increasing attention to treat infection, their effective delivery to the implantation site has been limited. Here, we developed an engineered dual functional peptide which delivers AMP as a biomolecular therapeutic agent onto calcium phosphate (Ca-P) deposited nanotubular titanium surfaces. Dual functionality of the peptide was achieved by combining a hydroxyapatite binding peptide-1 (HABP1) with an AMP using a flexible linker. HABP functionality of the peptide provided a self-coating property onto the nano-topographies that are designed to improve osteointegration capability, while AMP offered an antimicrobial protection onto the implant surface. We successfully deposited calcium phosphate minerals on nanotubular titanium oxide surface using pulse electrochemical deposition (PECD) and characterized the minerals by XRD, FT-IR, FE-SEM. Antimicrobial activity of the engineered peptide was tested against S. mutans (gram- positive) and E. coli (gram-negative) both in solution and on the Ca-P coated nanotubular titanium surface. In solution activity of AMP and dual functional peptide have the same Minimum Inhibitory Concentration (MIC) (32â¯mg/mL). The peptide also resulted in the reduction of the number of bacteria both for E.coli and S. mutans compare to control groups on the surface. Antimicrobial features of dual functional peptides are strongly correlated with their structures suggesting tunability in design through linkers regions. The dual-function peptide offers single-step solution for implant surface functionalization that could be applicable to any implant surface having different topographies.
Asunto(s)
Antiinfecciosos/farmacología , Fosfatos de Calcio/química , Materiales Biocompatibles Revestidos/química , Nanotubos/química , Péptidos/farmacología , Titanio/química , Secuencia de Aminoácidos , Adhesión Bacteriana/efectos de los fármacos , Durapatita/química , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Nanotubos/ultraestructura , Péptidos/química , Estructura Secundaria de Proteína , Staphylococcus aureus/efectos de los fármacosRESUMEN
BACKGROUND: Although microcalcifications of hydroxyapatite can be found in both benign and malignant osteotropic tumors, they are mostly seen in proliferative lesions, including carcinoma. The aim of this present study is to develop a molecular imaging contrast agent for selective identification of hydroxyapatite calcification in human osteotropic tumor tissues ex vivo and in human osteosarcoma cells in vitro. METHODS: A bioinspired biomarker, hydroxyapatite binding peptide (HABP), was designed to mimic natural protein osteocalcin property in vivo. A fluorescein isothiocyanate dye conjugated HABP (HABP-19) was utilized to characterize hydroxyapatite on human osteotropic tumor tissue sections ex vivo and to selectively image hydroxyapatite calcifications in human osteosarcoma cells in vitro. RESULTS: Using a HABP-19 molecular imaging probe, we have shown that it is possible to selectively image hydroxyapatite calcifications in osteotropic cancers ex vivo and in human SaOS-2 osteosarcoma cells in vitro. CONCLUSION: Hydroxyapatite calcifications were selectively detected in osteotropic tissues ex vivo and in the early stage of the calcification process of SaOS-2 human osteosarcoma in vitro using our HABP-19 molecular imaging probe. This new target-selective molecular imaging probe makes it possible to study the earliest events associated with hydroxyapatite deposition in various osteotropic cancers at the cellular and molecular levels. GENERAL SIGNIFICANCE: It potentially could be used to diagnose and treat osteotropic cancer or to anchor therapeutic agents directing the local distribution of desired therapy at calcified sites.