Final Report Summary - PROBI (Protein coatings to prevent bacterial infections)
Bacterial infections are the first cause of failure of biomedical implanted devices as prostheses, heart valves, catheters and intraocular lenses. Often the only solution to remove the infection is the surgical removal of the implanted devices.
The most common bacteria associated with infections on indwelling or implanted biodevices are S. epidermidis, S. aureus, Pseudomonas aeruginosa, E.coli Streptococci and Candida species. Amongst them, S. epidermidis has revealed to be an extremely successful pathogen due to its ability to adhere to surfaces. Specifically, bacteria can contaminate surfaces during the implantation of the device, coming from the patient's skin, from the hands of surgeons or from infected surgery tools. Contamination can also occur post operatively during hospitalisation.
Initially, bacteria adhere to the bio-implant surface. Then, they start to proliferate and colonise the surface producing extracellular polymeric substances and forming a biofilm. Biofilm growth offers protection against the host defence and the presence of antibiotics. Many processes are involved in bacterial deposition including Brownian motion, sedimentation and hydrodynamic forces, while actual adhesion of microorganisms to a substrate surface is mediated by a complex interplay of Lifshitz - van der Waals, electrostatic, acid-base, and hydration (including hydrophobic) interaction forces that are modulated by the ions in solution.
In principle, it should be possible to retard, if not prevent, the formation of biofilms on substrates by using materials to which bacteria cannot initially attach. In practice, however, synthetic materials that are capable of preventing bacterial adsorption are proved difficult to design, despite the huge research effort. Properties of the bio-implant material, such as hydrophobicity, hydrophilicity, steric hindrance and roughness are all thought to be important in the initial cell attachment process. These properties can be tailored coating the implants with suitable materials. Biomaterial surfaces can be modified by a variety of different methods, such as the application of a surface chemical gradient, self-assembled films, surface-active bulk additive and surface chemical reaction. Surface modifications can be achieved easily by the use of self-assembled monolayers which are surface coatings that form highly ordered structures on specific substrates. Surfaces have been modified with various functional groups such as methyl-, hydroxyl, amino- and carboxyl-, all of which can be found on natural biological surfaces.
Based on the knowledge accumulated in recent years about the characteristics that make a successful biocompatible interface we have proposed to research on the properties of amphipatic fungal proteins, hydrophobins, as plausible candidates to modulate the biomaterial interface. In particular, we have investigated a class I hydrophobin extracted from the basidiomycete fungus Pleurotus ostreatus, vmh2, whose properties are only partially investigated. Class I Hydrophobins form amphiphilic membranes able to modify the physico-chemical properties - e.g. wettability - of the surfaces on which they are deposited. Several studies have shown that class I hydrophobins do not seem to be toxic or cytotoxic or immunogenic, thus they are susceptible to be used in various medical and technical applications. We have proposed to coat model surfaces with thin layers of native hydrophobin and to investigate their adhesion properties. Investigation of the interaction of bacteria with hydrophobin-coated surfaces was also foreseen. The goal is to obtain information about the factors that may reduce the incidence of infections due to bacterial colonisation on biomaterials. Modulation of the surfaces wetting properties, using a hydrophobin layer, could help in changing adhesiveness of bacteria to biomedical devices, lowering the occurrence of infections. For the proposed research the main investigation technique is one of the most widely adopted techniques in nanoscience, i.e. atomic force microscopy (AFM), exploited to study the basic interaction mechanisms at the biomaterial interface.
Principal scientific achievements:
- Homogeneous and reproducible depositions of hydrophobin vmh2 on polymer surfaces as poly (dimethylsiloxane), used in biomedical implants, and Cytop, a fluoropolymer used for biosensors and in microfluidics. Uniform depositions of hydrophobins on inorganic substrates as mica and graphite to be used as control surfaces.
- High-resolution AFM imaging in buffer solutions of self-assembled vmh2 layers.
- Quantitative measurements on the mechanical and adhesion properties of an hydrophobin layer deposited on different surfaces obtained using force spectroscopy technique in buffer solution at different pH and salt concentrations.
- Assessment of polymers resistance (poly(dimethylsiloxane), poly(methyl methacrylate), Cytop) to exposure to solutions containing an high percentage of ethanol.
Due to their amphiphilic nature and self-assembling properties, hydrophobins are suitable for a wide range of applications: as surfactants, emulsifiers in food processing, in surface coating and immobilisation applications. Unfortunately, the yield of vmh2 hydrophobin is still not sufficient for extensive application tests. In this particular case, issues regarding their use in a solution containing ethanol make them not suitable for deposition on polymers for prolonged exposure times. Nevertheless, this hydrophobin has different properties with respect to the most studied ones and its investigation can give new insights in understanding the dynamics of their self-assembly as well as assessing the adhesion and mechanical properties of a self-assembled layer. The investigation on using hydrophobins for coating of biomedical devices to reduce bacterial adhesion will benefit of the results obtained so far.
The most common bacteria associated with infections on indwelling or implanted biodevices are S. epidermidis, S. aureus, Pseudomonas aeruginosa, E.coli Streptococci and Candida species. Amongst them, S. epidermidis has revealed to be an extremely successful pathogen due to its ability to adhere to surfaces. Specifically, bacteria can contaminate surfaces during the implantation of the device, coming from the patient's skin, from the hands of surgeons or from infected surgery tools. Contamination can also occur post operatively during hospitalisation.
Initially, bacteria adhere to the bio-implant surface. Then, they start to proliferate and colonise the surface producing extracellular polymeric substances and forming a biofilm. Biofilm growth offers protection against the host defence and the presence of antibiotics. Many processes are involved in bacterial deposition including Brownian motion, sedimentation and hydrodynamic forces, while actual adhesion of microorganisms to a substrate surface is mediated by a complex interplay of Lifshitz - van der Waals, electrostatic, acid-base, and hydration (including hydrophobic) interaction forces that are modulated by the ions in solution.
In principle, it should be possible to retard, if not prevent, the formation of biofilms on substrates by using materials to which bacteria cannot initially attach. In practice, however, synthetic materials that are capable of preventing bacterial adsorption are proved difficult to design, despite the huge research effort. Properties of the bio-implant material, such as hydrophobicity, hydrophilicity, steric hindrance and roughness are all thought to be important in the initial cell attachment process. These properties can be tailored coating the implants with suitable materials. Biomaterial surfaces can be modified by a variety of different methods, such as the application of a surface chemical gradient, self-assembled films, surface-active bulk additive and surface chemical reaction. Surface modifications can be achieved easily by the use of self-assembled monolayers which are surface coatings that form highly ordered structures on specific substrates. Surfaces have been modified with various functional groups such as methyl-, hydroxyl, amino- and carboxyl-, all of which can be found on natural biological surfaces.
Based on the knowledge accumulated in recent years about the characteristics that make a successful biocompatible interface we have proposed to research on the properties of amphipatic fungal proteins, hydrophobins, as plausible candidates to modulate the biomaterial interface. In particular, we have investigated a class I hydrophobin extracted from the basidiomycete fungus Pleurotus ostreatus, vmh2, whose properties are only partially investigated. Class I Hydrophobins form amphiphilic membranes able to modify the physico-chemical properties - e.g. wettability - of the surfaces on which they are deposited. Several studies have shown that class I hydrophobins do not seem to be toxic or cytotoxic or immunogenic, thus they are susceptible to be used in various medical and technical applications. We have proposed to coat model surfaces with thin layers of native hydrophobin and to investigate their adhesion properties. Investigation of the interaction of bacteria with hydrophobin-coated surfaces was also foreseen. The goal is to obtain information about the factors that may reduce the incidence of infections due to bacterial colonisation on biomaterials. Modulation of the surfaces wetting properties, using a hydrophobin layer, could help in changing adhesiveness of bacteria to biomedical devices, lowering the occurrence of infections. For the proposed research the main investigation technique is one of the most widely adopted techniques in nanoscience, i.e. atomic force microscopy (AFM), exploited to study the basic interaction mechanisms at the biomaterial interface.
Principal scientific achievements:
- Homogeneous and reproducible depositions of hydrophobin vmh2 on polymer surfaces as poly (dimethylsiloxane), used in biomedical implants, and Cytop, a fluoropolymer used for biosensors and in microfluidics. Uniform depositions of hydrophobins on inorganic substrates as mica and graphite to be used as control surfaces.
- High-resolution AFM imaging in buffer solutions of self-assembled vmh2 layers.
- Quantitative measurements on the mechanical and adhesion properties of an hydrophobin layer deposited on different surfaces obtained using force spectroscopy technique in buffer solution at different pH and salt concentrations.
- Assessment of polymers resistance (poly(dimethylsiloxane), poly(methyl methacrylate), Cytop) to exposure to solutions containing an high percentage of ethanol.
Due to their amphiphilic nature and self-assembling properties, hydrophobins are suitable for a wide range of applications: as surfactants, emulsifiers in food processing, in surface coating and immobilisation applications. Unfortunately, the yield of vmh2 hydrophobin is still not sufficient for extensive application tests. In this particular case, issues regarding their use in a solution containing ethanol make them not suitable for deposition on polymers for prolonged exposure times. Nevertheless, this hydrophobin has different properties with respect to the most studied ones and its investigation can give new insights in understanding the dynamics of their self-assembly as well as assessing the adhesion and mechanical properties of a self-assembled layer. The investigation on using hydrophobins for coating of biomedical devices to reduce bacterial adhesion will benefit of the results obtained so far.