Strategies for Antimicrobial Peptides Immobilization on Surfaces to Prevent Biofilm Growth on Biomedical Devices.
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ABSTRACT:
SUBMITTER: Nicolas M
PROVIDER: S-EPMC8772980 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Immobilizing a protein, that is fully compatible with the patient, on the surface of a biomedical device should make it possible to avoid adverse responses such as inflammation, rejection, or excessive fibrosis. A surface that strongly binds and does not denature the compatible protein is required. Hydrophilic surfaces do not induce denaturation of immobilized protein but exhibit a low binding affinity for protein. Here, we describe an energetic ion-assisted plasma process that can make any surface hydrophilic and at the same time enable it to covalently immobilize functional biological molecules. We show that the modification creates free radicals that migrate to the surface from a reservoir beneath. When they reach the surface, the radicals form covalent bonds with biomolecules. The kinetics and number densities of protein molecules in solution and free radicals in the reservoir control the time required to form a full protein monolayer that is covalently bound. The shelf life of the covalent binding capability is governed by the initial density of free radicals and the depth of the reservoir. We show that the high reactivity of the radicals renders the binding universal across all biological macromolecules. Because the free radical reservoir can be created on any solid material, this approach can be used in medical applications ranging from cardiovascular stents to heart-lung machines.
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Project description:: Bacterial biofilm on medical devices is difficult to eradicate. Many have capitalized the anti-infective capability of silver ions (Ag+) by incorporating nano-silver (nAg) in a biodegradable coating, which is then laid on polymeric medical devices. However, such coating can be subjected to premature dissolution, particularly in harsh diseased tissue microenvironment, leading to rapid nAg clearance. It stands to reason that impregnating nAg directly onto the device, at the surface, is a more ideal solution. We tested this concept for a corneal prosthesis by immobilizing nAg and nano-hydroxyapatite (nHAp) on poly(methyl methacrylate), and tested its biocompatibility with human stromal cells and antimicrobial performance against biofilm-forming pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. Three different dual-functionalized substrates-high Ag (referred to as 75:25 HAp:Ag); intermediate Ag (95:5 HAp:Ag); and low Ag (99:1 HAp:Ag) were studied. The 75:25 HAp:Ag was effective in inhibiting biofilm formation, but was cytotoxic. The 95:5 HAp:Ag showed the best selectivity among the three substrates; it prevented biofilm formation of both pathogens and had excellent biocompatibility. The coating was also effective in eliminating non-adherent bacteria in the culture media. However, a 28-day incubation in artificial tear fluid revealed a ~40% reduction in Ag+ release, compared to freshly-coated substrates. The reduction affected the inhibition of S. aureus growth, but not the P. aeruginosa. Our findings suggest that Ag+ released from surface-immobilized nAg diminishes over time and becomes less effective in suppressing biofilm formation of Gram-positive bacteria, such as S. aureus. This advocates the coating, more as a protection against perioperative and early postoperative infections, and less as a long-term preventive solution.
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Project description:Antibiotic-resistant microorganisms have become a serious threat to public health, resulting in hospital infections, the majority of which are caused by commonly used urinary tract catheters. Strategies for preventing bacterial adhesion to the catheters' surfaces have been potentially shown as effective methods, such as coating thesurface with antimicrobial biomolecules. Here, novel antimicrobial peptides (AMPs) were designed as potential biomolecules to prevent antibiotic-resistant bacteria from binding to catheter surfaces. Thiolated AMPs were synthesized using solid-phase peptide synthesis (SPPS), and prep-HPLC was used to obtain AMPs with purity greater than 90%. On the other side, the silicone catheter surface was activated by UV/ozone treatment, followed by functionalization with allyl moieties for conjugation to the free thiol group of cystein in AMPs using thiol-ene click chemistry. Peptide-immobilized surfaces were found to become more resistant to bacterial adhesion while remaining biocompatible with mammalian cells. The presence and site of conjugation of peptide molecules were investigated by immobilizing them to catheter surfaces from both ends (C-Pep and Pep-C). It was clearly demonstrated that AMPs conjugated to the surface via theirN terminus have a higher antimicrobial activity. This strategy stands out for its effective conjugation of AMPs to silicone-based implant surfaces for the elimination of bacterial infections.
| S-EPMC11360073 | biostudies-literature
Project description:Short antimicrobial peptides are essential to keep us healthy and their lasting potency can inspire the design of new types of antibiotics. This study reports the design of a family of eight-residue tryptophan-rich peptides (TetraF2W) obtained by converting the four phenylalanines in temporin-SHf to tryptophans. The temporin-SHf template was identified from the antimicrobial peptide database (http://aps.unmc.edu/AP). Remarkably, the double arginine variant (TetraF2W-RR) was more effective in killing methicillin-resistant Staphylococcus aureus (MRSA) USA300, but less cytotoxic to human skin HaCat and kidney HEK293 cells, than the lysine-containing dibasic combinations (KR, RK and KK). Killing kinetics and fluorescence spectroscopy suggest membrane targeting of TetraF2W-RR, making it more difficult for bacteria to develop resistance. Because established biofilms on medical devices are difficult to remove, we chose to covalently immobilize TetraF2W-RR onto the polyethylene terephthalate (PET) surface to prevent biofilm formation. The successful surface coating of the peptide is supported by FT-IR and XPS spectroscopies, chemical quantification, and antibacterial assays. This peptide-coated surface indeed prevented S. aureus biofilm formation with no cytotoxicity to human cells. In conclusion, TetraF2W-RR is a short Trp-rich peptide with demonstrated antimicrobial and anti-biofilm potency against MRSA in both the free and immobilized forms. Because these short peptides can be synthesized cost effectively, they may be developed into new antimicrobial agents or used as surface coating compounds.Statement of significanceIt is stunning that the total deaths due to methicillin-resistant Staphylococcus aureus (MRSA) infection are comparable to AIDS/HIV-1, making it urgent to explore new possibilities. This study deals with this problem by two strategies. First, we have designed a family of novel antimicrobial peptides with merely eight amino acids, making it cost effective for chemical synthesis. These peptides are potent against MRSA USA300. Our study uncovers that the high potency of the tryptophan-rich short peptide is coupled with arginines, whereas these Trp- and Arg-rich peptides are less toxic to select human cells than the lysine-containing analogs. Such a combination generates a more selective peptide. As a second strategy, we also demonstrate successful covalent immobilization of this short peptide to the polyethylene terephthalate (PET) surface by first using a chitosan linker, which is easy to obtain. Because biofilms on medical devices are difficult to remove by traditional antibiotics, we also show that the peptide coated surface can prevent biofilm formation. Although rarely demonstrated, we provide evidence that both the free and immobilized peptides target bacterial membranes, rendering it difficult for bacteria to develop resistance. Collectively, the significance of our study is the design of novel antimicrobial peptides provides a useful template for developing novel antimicrobials against MRSA. In addition, orientation-specific immobilization of the same short peptide can prevent biofilm formation on the PET surface, which is widely used in making prosthetic heart valves cuffs and other bio devices.
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Project description:Catheter-associated infections in bladder cancer patients, following radical cystectomy or ureterocutaneostomy, are very frequent, and the development of antibiotic resistance poses great challenges for treating biofilm-based infections. Here, we characterized bacterial communities from catheters of patients who had undergone radical cystectomy for muscle-invasive bladder cancer. We evaluated the efficacy of conventional antibiotics, alone or combined with the human ApoB-derived antimicrobial peptide r(P)ApoBLAla, to treat ureteral catheter-colonizing bacterial communities on clinically isolated bacteria. Microbial communities adhering to indwelling catheters were collected during the patients' regular catheter change schedules (28 days) and extracted within 48 h. Living bacteria were characterized using selective media and biochemical assays. Biofilm growth and novel antimicrobial strategies were analyzed using confocal laser scanning microscopy. Statistical analyses confirmed the relevance of the biofilm reduction induced by conventional antibiotics (fosfomycin, ceftriaxone, ciprofloxacin, gentamicin, and tetracycline) and a well-characterized human antimicrobial peptide r(P)ApoBLAla (1:20 ratio, respectively). Catheters showed polymicrobial communities, with Enterobactericiae and Proteus isolates predominating. In all samples, we recorded a meaningful reduction in biofilms, in both biomass and thickness, upon treatment with the antimicrobial peptide r(P)ApoBLAla in combination with low concentrations of conventional antibiotics. The results suggest that combinations of conventional antibiotics and human antimicrobial peptides might synergistically counteract biofilm growth on ureteral catheters, suggesting novel avenues for preventing catheter-associated infections in patients who have undergone radical cystectomy and ureterocutaneostomy.
| S-EPMC9225455 | biostudies-literature
Project description:Staphylococcus aureus infections represent the major cause of titanium based-orthopaedic implant failure. Current treatments for S. aureus infections involve the systemic delivery of antibiotics and additional surgeries, increasing health-care costs and affecting patient's quality of life. As a step toward the development of new strategies that can prevent these infections, we build upon previous work demonstrating that the colonization of catheters by the fungal pathogen Candida albicans can be prevented by coating them with thin polymer multilayers composed of chitosan (CH) and hyaluronic acid (HA) designed to release a ?-amino acid-based peptidomimetic of antimicrobial peptides (AMPs). We demonstrate here that this ?-peptide is also potent against S. aureus (MBPC?=?4??g/mL) and characterize its selectivity toward S. aureus biofilms. We demonstrate further that ?-peptide-containing CH/HA thin-films can be fabricated on the surfaces of rough planar titanium substrates in ways that allow mammalian cell attachment and permit the long-term release of ?-peptide. ?-Peptide loading on CH/HA thin-films was then adjusted to achieve release of ?-peptide quantities that selectively prevent S. aureus biofilms on titanium substrates in vitro for up to 24?days and remained antimicrobial after being challenged sequentially five times with S. aureus inocula, while causing no significant MC3T3-E1 preosteoblast cytotoxicity compared to uncoated and film-coated controls lacking ?-peptide. We conclude that these ?-peptide-containing films offer a novel and promising localized delivery approach for preventing orthopaedic implant infections. The facile fabrication and loading of ?-peptide-containing films reported here provides opportunities for coating other medical devices prone to biofilm-associated infections. STATEMENT OF SIGNIFICANCE: Titanium (Ti) and its alloys are used widely in orthopaedic devices due to their mechanical strength and long-term biocompatibility. However, these devices are susceptible to bacterial colonization and the subsequent formation of biofilms. Here we report a chitosan and hyaluronic acid polyelectrolyte multilayer-based approach for the localized delivery of helical, cationic, globally amphiphilic ?-peptide mimetics of antimicrobial peptides to inhibit S. aureus colonization and biofilm formation. Our results reveal that controlled release of this ?-peptide can selectively kill S. aureus cells without exhibiting toxicity toward MC3T3-E1 preosteoblast cells. Further development of this polymer-based coating could result in new strategies for preventing orthopaedic implant-related infections, improving outcomes of these titanium implants.
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Project description:Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms.
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Project description:Polydimethylsiloxane (PDMS), a silicone elastomer, is increasingly being used in health and biomedical fields due to its excellent optical and mechanical properties. Its biocompatibility and resistance to biodegradation led to various applications (e.g., lung on a chip replicating blood flow, medical interventions, and diagnostics). The many advantages of PDMS are, however, partially offset by its inherent hydrophobicity, which makes it unsuitable for applications needing wetting, thus requiring the hydrophilization of its surface by exposure to UV or O2 plasma. Yet, the elastomeric state of PDMS translates in a slow, hours to days, process of reducing its surface hydrophilicity-a process denominated as hydrophobic recovery. Using Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM), the present study details the dynamics of hydrophobic recovery of PDMS, on flat bare surfaces and on surfaces embedded with hydrophilic beads. It was found that a thin, stiff, hydrophilic, silica film formed on top of the PDMS material, following its hydrophilization by UV radiation. The hydrophobic recovery of bare PDMS material is the result of an overlap of various nano-mechanical, and diffusional processes, each with its own dynamics rate, which were analyzed in parallel. The hydrophobic recovery presents a hysteresis, with surface hydrophobicity recovering only partially due to a thin, but resilient top silica layer. The monitoring of hydrophobic recovery of PDMS embedded with hydrophilic beads revealed that this is delayed, and then totally stalled in the few-micrometer vicinity of the embedded hydrophilic beads. This region where the hydrophobic recovery stalls can be used as a good approximation of the depth of the resilient, moderately hydrophilic top layer on the PDMS material. The complex processes of hydrophilization and subsequent hydrophobic recovery impact the design, fabrication, and operation of PDMS materials and devices used for diagnostics and medical procedures. Consequently, especially considering the emergence of new surgical procedures using elastomers, the impact of hydrophobic recovery on the surface of PDMS warrants more comprehensive studies.
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Project description:The interface between implanted devices and their host tissue is complex and is often optimized for maximal integration and cell adhesion. However, this also gives a surface suitable for bacterial colonization. We have developed a novel method of modifying the surface at the material-tissue interface with an antimicrobial peptide (AMP) coating to allow cell attachment while inhibiting bacterial colonization. The technology reported here is a dual AMP coating. The dual coating consists of AMPs covalently bonded to the hydroxyapatite surface, followed by deposition of electrostatically bound AMPs. The dual approach gives an efficacious coating which is stable for over 12 months and can prevent colonization of the surface by both Gram-positive and Gram-negative bacteria.
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