Conjugation Of Antimicrobial Peptides Research Articles

Effective Immobilization of Novel Antimicrobial Peptides via Conjugation onto Activated Silicon Catheter Surfaces

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.

Size-Controlled Synthesis of Gold Nanoparticles Tethering Antimicrobial Peptides with Potent Broad-Spectrum Antimicrobial and Antibiofilm Activities.

New antimicrobials are urgently needed to combat the rising global health concern of antibiotic resistance. Antimicrobial peptides (AMPs) are one of the leading candidates as new antimicrobials since they target bacterial membranes and are therefore less prone to bacterial resistance. However, poor enzymatic stability, high production costs, and toxicity are drawbacks that limit their clinical use. Conjugation of AMPs to gold nanoparticles (NPs) may help to improve enzymatic stability and, thus, their overall antimicrobial efficiency. We did a one-pot synthesis of size-controlled (10 nm) gold NPs selectively conjugated to lipopeptides and determined their antibacterial activity. The conjugates exhibited potent (0.13-1.25 μM) antimicrobial activity against clinical isolates, including Gram-positive methicillin-resistant Staphylococcus aureus (S. aureus) ATCC33593, Gram-negative Escherichia coli (E. coli) CTX-M-14, multidrug-resistant Pseudomonas aeruginosa LESB58 and Acinetobacter baumannii ATCC19606, and showed promising activity (90% inhibition of initial biofilms and 80% reduction of preformed biofilms) against S. aureus and E. coli DH5α biofilms at low micromolar concentrations. The conjugates were stable in rat serum and not toxic to representative mammalian cell lines in vitro (≤64 μM) and in vivo (≤100 μM).

Effects of Conjugation of Ferrocene and Gallic Acid On desCys11/Lys12/Lys13-(p-BthTX-I)2K Peptide: Structure, Permeabilization and Antibacterial Activity.

Antimicrobial resistance is an emerging global health challenge that has led researchers to study alternatives to conventional antibiotics. A promising alternative is antimicrobial peptides (AMPs), produced as the first line of defense by almost all living organisms. To improve its biological activity, the conjugation of AMPs is a promising approach. In this study, we evaluated the N-terminal conjugation of p-Bt (a peptide derived from Bothrops Jararacuçu`s venom) with ferrocene (Fc) and gallic acid (GA). Acetylated and linear versions of p-Bt were also synthesized to evaluate the importance of N-terminal charge and dimeric structure. The compounds were obtained using solid-phase peptide synthesis. Circular dichroism, vesicle permeabilization, antimicrobial activity, and cytotoxicity studies were conducted. No increase in antibacterial activity against Escherichia coli was observed by adding either Fc or GA to p-Bt. However, Fc-p-Bt and GA-p-Bt exhibited improved activity against Staphylococcus aureus. No cytotoxicity upon fibroblast was observed for GA-p-Bt. On the other hand, conjugation with Fc increased cytotoxicity. This toxicity may be related to the membrane permeabilization capacity of this bioconjugate, which showed the highest carboxyfluorescein leakage in vesicle permeabilization experiments. Considering these observations, our findings highlight the importance of adding bioactive organic compounds in the N-terminal position as a tool to modulate the activity of AMPs.

Exploring the mechanism of photosensitizer conjugation on membrane perturbation of antimicrobial peptide: A multiscale molecular simulation study

Antimicrobial peptides (AMPs) exert their biological functions by perturbation with cellular membrane. Conjugation of AMPs with photosensitizer (PS) is a promising strategy for enhancing the efficacy and reducing systemic toxicity of AMPs. However, it is still elusive how the conjugated PS impacts the perturbation of AMPs on cell membrane from molecular level. Here, we addressed this issue by a multiscale computational strategy on pyropheophorbide-a (PPA) conjugated K6L9 (PPA-K6L9), a PS-AMP conjugate developed by us previously. Our atomistic molecular dynamics (MD) simulations revealed that the porphyrin moiety of PPA enhanced the stability of the conjugate in a lipid bilayer membrane model. Moreover, such moiety also maintained the amphipathic structure of K6L9, which is crucial for membrane pore formation. Coarse-grained MD simulations further showed that the conjugates aggregated in membrane environment and formed more stable toroidal pores with respect to K6L9 alone, suggesting the conjugation of PPA may enhance the membrane-disruption activity of K6L9. Consistent with this, our cellular experiments confirmed that PPA-K6L9 was more toxic to 4 T1 tumor cells than K6L9. This study provides insights into the mechanism by which PS-AMP conjugates disrupt cellular membranes and could aid in the design of more potent AMP conjugates.

Rapid Assembly of Infection-Resistant Coatings: Screening and Identification of Antimicrobial Peptides Works in Cooperation with an Antifouling Background.

Bacterial adhesion and the succeeding biofilm formation onto surfaces are responsible for implant- and device-associated infections. Bifunctional coatings integrating both nonfouling components and antimicrobial peptides (AMPs) are a promising approach to develop potent antibiofilm coatings. However, the current approaches and chemistry for such coatings are time-consuming and dependent on substrates and involve a multistep process. Also, the information is limited on the influence of the coating structure or its components on the antibiofilm activity of such AMP-based coatings. Here, we report a new strategy to rapidly assemble a stable, potent, and substrate-independent AMP-based antibiofilm coating in a nonfouling background. The coating structure allowed for the screening of AMPs in a relevant nonfouling background to identify optimal peptide combinations that work in cooperation to generate potent antibiofilm activity. The structure of the coating was changed by altering the organization of the hydrophilic polymer chains within the coatings. The coatings were thoroughly characterized using various surface analytical techniques and correlated with the efficiency to prevent biofilm formation against diverse bacteria. The coating method that allowed the conjugation of AMPs without altering the steric protection ability of hydrophilic polymer structure results in a bifunctional surface coating with excellent antibiofilm activity. In contrast, the conjugation of AMPs directly to the hydrophilic polymer chains resulted in a surface with poor antibiofilm activity and increased adhesion of bacteria. Using this coating approach, we further established a new screening method and identified a set of potent surface-tethered AMPs with high activity. The success of this new peptide screening and coating method is demonstrated using a clinically relevant mouse infection model to prevent catheter-associated urinary tract infection (CAUTI).

Surface modification strategies for hemodialysis catheters to prevent catheter-related infections: A review.

Insertion of a central venous catheter is one of the most common invasive procedures applied in hemodialysis therapy for end-stage renal disease. The most important complication of a central venous catheter is catheter-related infections that increase hospitalization and duration of intensive care unit stay, cost of treatment, mortality, and morbidity rates. Pathogenic microorganisms, such as, bacteria and fungi, enter the body from the catheter insertion site and the surface of the catheter can become colonized. The exopolysaccharide-based biofilms from bacterial colonies on the surface are the main challenge in the treatment of infections. Catheter lock solutions and systemic antibiotic treatment, which are commonly used in the treatment of hemodialysis catheter-related infections, are insufficient to prevent and terminate the infections and eventually the catheter needs to be replaced. The inadequacy of these approaches in termination and prevention of infection revealed the necessity of coating of hemodialysis catheters with bactericidal and/or antiadhesive agents. Silver compounds and nanoparticles, anticoagulants (e.g., heparin), antibiotics (e.g., gentamicin and chlorhexidine) are some of the agents used for this purpose. The effectiveness of few commercial hemodialysis catheters that were coated with antibacterial agents has been tested in clinical trials against catheter-related infections of pathogenic bacteria, such as Staphylococcus aureus and Staphylococcus epidermidis with promising results. Novel biomedical materials and engineering techniques, such as, surface micro/nano patterning and the conjugation of antimicrobial peptides, enzymes, metallic cations, and hydrophilic polymers (e.g., poly [ethylene glycol]) on the surface, has been suggested recently.

Inorganic Gold and Polymeric Poly(Lactide-co-glycolide) Nanoparticles as Novel Strategies to Ameliorate the Biological Properties of Antimicrobial Peptides.

Cationic antimicrobial peptides (AMPs) are an interesting class of gene-encoded molecules endowed with a broad-spectrum of anti-infective activity and immunomodulatory properties. They represent promising candidates for the development of new antibiotics, mainly due to their membraneperturbing mechanism of action that very rarely induces microbial resistance. However, bringing AMPs into the clinical field is hampered by some intrinsic limitations, encompassing low peptide bioavailability at the target site and high peptide susceptibility to proteolytic degradation. In this regard, nanotechnologies represent an innovative strategy to circumvent these issues. According to the literature, a large variety of nanoparticulate systems have been employed for drug-delivery, bioimaging, biosensors or nanoantibiotics. The possibility of conjugating different types of molecules, including AMPs, to these systems, allows the production of nanoformulations able to enhance the biological profile of the compound while reducing its cytotoxicity and prolonging its residence time. In this minireview, inorganic gold nanoparticles (NPs) and biodegradable polymeric NPs made of poly(lactide-coglycolide) are described with particular emphasis on examples of the conjugation of AMPs to them, to highlight the great potential of such nanoformulations as alternative antimicrobials.

Thiol-Reactive Polymers for Titanium Interfaces: Fabrication of Antimicrobial Coatings

Infection associated with surgical implants is a major cause of their failure. Often in such cases, the implant has to be removed and replaced, which causes undesirable patient discomfort and complications. Bacterial adhesion and growth on implant surface is the primary reason for such infections. Among the approaches to prevent implant associated infections, the conjugation of antimicrobial peptide (AMP) onto the surface of the implant is a very promising approach. In this study, we describe a facile method for the surface modification of titanium (Ti), a widely used material in dental and orthopedic implants, to prevent bacterial adhesion and growth. Thin polymeric films were synthesized on the Ti surface by using a copolymer containing the maleimide group as a thiol-reactive handle to enable the conjugation of AMPs. Robust attachment of the polymeric coating on Ti surfaces was ensured through installation of catechol moieties on the polymer as surface anchoring groups and the variation of the amount of...

Recent Advances in the Development of Antimicrobial Peptides (AMPs): Attempts for Sustainable Medicine?

Since the first isolation of antimicrobial peptides (AMPs) they have attracted extensive interest in medicinal chemistry. However, only a few AMP-based drugs are currently available on the market. Despite their effectiveness, biodegradability, and versatile mode of action that is less likely to induce resistance compared to conventional antibiotics, AMPs suffer from major issues that need to be addressed to broaden their use. Notably, AMPs can lack selectivity leading to side effects and cytotoxicity, and also exhibit in vivo instability. Several strategies are being actively considered to overcome the limitations that restrain the success of AMPs. In the current work, recent strategies reported for improving AMPs in the context of drug design and delivery were surveyed, and also their possible impact on patients and the environment was assessed. As a major advantage AMPs possess an easily tunable skeleton offering opportunities to improve their properties. Strategic structural modifications and the beneficial properties of cyclic or branched AMPs in term of stability have been reported. The conjugation of AMPs with nanoparticles has also been explored to increase their in vivo stability. Other techniques such as the coupling of AMPs with specific antibodies aim to increase the selectivity of the potential drug towards the target. These strategies were evaluated for their effect on the environment highlighting green technologies. Although further research is needed taking into account both environmental and human health consequences of novel AMPs, several of these compounds are promising drug candidates for use in sustainable medicine.

Synthesis, Self-Assembly, and Biomedical Applications of Antimicrobial Peptide-Polymer Conjugates.

Antimicrobial peptides (AMPs) have been attracting much attention due to their excellent antimicrobial efficiency and low rate in driving antimicrobial resistance (AMR), which has been increasing globally to alarming levels. Conjugation of AMPs into functional polymers not only preserves excellent antimicrobial activities but reduces the toxicity and offers more functionalities, which brings new insight toward developing multifunctional biomedical materials such as hydrogels, polymer vesicles, polymer micelles, and so forth. These nanomaterials have been exhibiting excellent antimicrobial activity against a broad spectrum of bacteria including multidrug-resistant (MDR) ones, high selectivity, and low cytotoxicity, suggesting promising potentials in wound dressing, implant coating, antibiofilm, tissue engineering, and so forth. This Perspective seeks to highlight the state-of-the-art strategy for the synthesis, self-assembly, and biomedical applications of AMP-polymer conjugates and explore the promising directions for future research ranging from synthetic strategies, multistage and stimuli-responsive antibacterial activities, antifungi applications, and potentials in elimination of inflammation during medical treatment. It also will provide perspectives on how to stem the remaining challenges and unresolved problems in combating bacteria, including MDR ones.

Functionalisation of Ti6Al4V and hydroxyapatite surfaces with combined peptides based on KKLPDA and EEEEEEEE peptides

Surface modifications are usually performed on titanium alloys to improve osteo-integration and surface bioactivity. Modifications such as alkaline and acid etching, or coating with bioactive materials such as hydroxyapatite, have previously been demonstrated. The aim of this work is to develop a peptide with combined titanium oxide and hydroxyapatite binders in order to achieve a biomimetic hydroxyapatite coating on titanium surfaces. The technology would also be applicable for the functionalisation of titanium and hydroxyapatite surfaces for selective protein adsorption, conjugation of antimicrobial peptides, and adsorption of specialised drugs for drug delivery. In this work, functionalisation of Ti6Al4V and hydroxyapatite surfaces was achieved using combined titanium-hydroxyapatite (Ti-Hap) peptides based on titanium peptide binder (KKLPDA) and hydroxyapatite peptide binder (EEEEEEEE). Homogeneous peptide coatings on Ti6Al4V surfaces were obtained after surface chemical treatments with a 30wt% aqueous solution of H2O2 for 24 and 48h. The treated titanium surfaces presented an average roughness of Sa=197nm (24h) and Sa=128nm (48h); an untreated mirror polished sample exhibited an Sa of 13nm. The advancing water contact angle of the titanium oxide layer after 1h of exposure to 30wt% aqueous solution of H2O2 was around 65°, decreasing gradually with time until it reached 35° after a 48h exposure, suggesting that the surface hydrophilicity increased over etching time. The presence of a lysine (L) amino acid in the sequence of the titanium binder resulted in fluorescence intensity roughly 16% higher compared with the arginine (R) amino acid analogue and therefore the lysine containing titanium peptide binder was used in this work. The Ti-Hap peptide KKLPDAEEEEEEEE (Ti-Hap1) was not adsorbed by the treated Ti6Al4V surfaces and therefore was modified. The modifications involved the inclusion of a glycine spacer between the binding terminals (Ti-Hap2) and the addition of a second titanium binder (KKLPDA) (Ti-Hap3 and Ti-Hap4). The combined Ti-Hap peptide which exhibited the strongest intensity after the titanium surface dip coating was KKLPDAKKLPDAEEEEEEEE (Ti-HAp4). On the other hand, hydroxyapatite surfaces, exhibiting an average roughness of Sa=1.42μm, showed a higher fluorescence for peptides with a higher negative net charge.

Antimicrobial peptide-gold nanoscale therapeutic formulation with high skin regenerative potential

Chronic skin wounds affect ≈3% of persons aged >60years (Davies et al., 2007) [1]. These wounds are typically difficult to heal by conventional therapies and in many cases they get infected making even harder the regeneration process. The antimicrobial peptide (AMP) LL37 combines antimicrobial with pro-regenerative properties and thus represents a promising topical therapy to address both problems. Here, we investigated the wound healing potential of soluble and immobilized LL37 (LL37-conjugated gold nanoparticles, LL37-Au NPs), both in vitro (migration of keratinocytes) and in vivo (skin wound healing). Our results show that LL37-Au NPs, but not LL37 peptide, have the capacity to prolong the phosphorylation of EGFR and ERK1/2 and enhance the migratory properties of keratinocytes in a large in vitro wound model. We further report that both LL37 and LL37-Au NPs promote keratinocyte migration by the transactivation of EGFR, a process that seems to be initiated at the P2X7 receptor, as confirmed by chemical and genetic inhibition studies. Finally, we show in vivo that LL37-Au NPs have higher wound healing activity than LL37 peptide in a splinted mouse full thickness excisional model. Animal wounds treated by LL37-Au NPs have higher expression of collagen, IL6 and VEGF than the ones treated with LL37 peptide or NPs without LL37. Altogether, the conjugation of AMPs to NPs offers a promising platform to enhance their pro-regenerative properties.

Gold-nanoparticles coated with the antimicrobial peptide esculentin-1a(1-21)NH2 as a reliable strategy for antipseudomonal drugs.

Despite conjugation of AMPs to AuNPs represents a worthwhile solution to face some limitations for their development as new therapeutics, only a very limited number of studies is available on peptide-coated AuNPs. Importantly, this is the first report showing that a covalent binding of a linear AMP via a poly(ethylene glycol) linker to AuNPs highly enhances antipseudomonal activity, preserving the same mode of action of the free peptide, without being harmful. Furthermore, AuNPs@Esc(1-21) are expected to accelerate recovery of an injured skin layer. All together, these findings suggest our peptide-coated AuNPs as attractive novel nanoscale formulation to treat bacterial infections and to heal the injured tissue.

Molecular Validation of Putative Antimicrobial Peptides for Improved Human Immunodeficiency Virus Diagnostics via HIV Protein p24

Objective: Various HIV diagnostic systems are available however; the p24 antigen detection assay is preferred due to its ability to decrease the window period for HIV detection. The p24 antigen assay has been associated with low sensitivity. Antimicrobial Peptides (AMPs) display huge potential in development of more effective diagnostics tools. The aim of this study was to design derivative AMPs from AMPs identified previously, using an in silico approach, as templates, with higher predicted binding affinity for the p24 protein and implement the best suited AMPs in a Point-Of- Care (POC) device for detection of HIV-1 and HIV-2. Method: This study firstly used an in silico approach to identify derivative AMPs which bind the N-terminal domain of the p24 antigen with increased predicted binding affinity. The in silico approach used the parental AMPs as templates for generation of the derivative AMPs through site-directed mutagenesis, 3-D structure prediction and docking studies. Secondly, the binding between the synthetically synthesized AMPs and the HIV antigen p24 was validated by an in house Lateral Flow Device (LFD) “on/off” binding experiment. The most promising AMPs were conjugated to gold nanoparticles (AuNPs) and implemented in a LFD to detect HIV-1 and HIV-2 using patient sera as well as a panel of international HIV standards (panel C10). Result: With conjugation of AMPs to gold nanoparticles, parent AMP 1 and derivative AMP 1.1 used in combination most sensitively detected the recombinant protein p24 and was therefore selected for development of a LFD prototype for HIV diagnostics. Global HIV-1 (C10) and HIV-2 (C10) standards were successfully detected by the LFD prototype as well as HIV infected serum samples. Conclusion: A sensitive HIV-1 and HIV-2 LFD prototype was developed which would within 15 minutes provide patients with accurate and sensitive diagnosis of HIV.

Synthesis and Evaluation of Biological Activity of Antimicrobial--Pro-Proliferative Peptide Conjugates.

Skin represents the largest organ of the human body and plays a crucial role in its protection from the negative impact of the outside environment, maintains its homeostasis, enables sensory interaction and thermoregulation. The traumatized skin tissue undergoes several phenotype switches due to progressive reoxygenation and release of cytokine and growth factors, that activate mechanisms of reparative processes. However, in case of wounds colonized with pathogenic microflora natural regenerative mechanisms become substantially impaired, that could lead to chronic inflammatory states with non-healing skin lesions. Herein, we present the initial results of our studies aimed at the design of bifunctional peptide-based compounds. The chemical approach, that was utilized in this work, was based on the conjugation of antimicrobial peptides with the peptides, that have potential pro-proliferative and/or cytoprotective activity towards human keratinocytes and fibroblasts, in order to obtain antimicrobials with reduced cytotoxicity or compounds that maintain both activities, i.e. inhibit bacterial or fungi growth and activate cell proliferation/migration in in vitro tests. As a result, we obtained a group of peptide conjugates that effectively inhibited the growth of selected bacterial and fungi strains and were able to stimulate proliferation and migration of keratinocytes and fibroblasts under their effective microbicidal concentrations.

Magnetic Nanoparticles: New Players in Antimicrobial Peptide Therapeutics.

Antimicrobial peptides are distributed in all forms of life presenting activity against bacteria, fungi, viruses, parasites and cancer. In spite of the tremendous potential of these molecules, very few of them have been successfully developed into therapeutics. The major problems associated with this new class of antimicrobials are molecule stability, toxicity in host cells and production cost. A novel strategy to overcome these obstacles is conjugation to nanomaterials. Magnetic nanoparticles have been widely studied in biomedicine due to their physicochemical properties. The conjugation of antimicrobial peptides to magnetic nanoparticles could combine the best properties of both, generating an improved antimicrobial nanoparticle. Here we provide an overview and discuss the potential application of magnetic nanoparticles conjugated to antimicrobial peptides in overcoming diseases.

Enhancement of cytotoxicity of antimicrobial peptide magainin II in tumor cells by bombesin-targeted delivery

To investigate whether the conjugation of magainin II (MG2), an antimicrobial peptides (AMPs), to the tumor-homing peptide bombesin could enhance its cytotoxicity in tumor cells. A magainin II-bombesin conjugate (MG2B) was constructed by attaching magainin II (MG2) to bombesin at its N-terminus. The peptides were synthesized using Fmoc-chemistry. The in vitro cytotoxicity of the peptide in cancer cells was quantitatively determined using the CCK-8 cell counting kit. Moreover, the in vivo antitumor effect of the peptide was determined in tumor xenograft models. The IC(50) of MG2B for cancer cells (10-15 μmol/L) was at least 10 times lower than the IC(50) of unconjugated MG2 (125 μmol/L). Moreover, the binding affinity of MG2B for cancer cells was higher than that of unconjugated MG2. In contrast, conjugation to a bombesin analog lacking the receptor-binding domain failed to increase the cytotoxicity of MG2, suggesting that bombesin conjugation enhances the cytotoxicity of MG2 in cancer cells through improved binding. Indeed, MG2B selectively induced cell death in cancer cells in vitro with the IC(50) ranging from 10 to 15 μmol/L, which was about 6-10 times lower than the IC(50) for normal cells. MG2B (20 mg/kg per day, intratumorally injected for 5 d) also exhibited antitumor effects in mice bearing MCF-7 tumor grafts. The mean weights of tumor grafts in MG2B- and PBS-treated mice were 0.21±0.05 g and 0.59±0.12 g, respectively. The results suggest that conjugation of AMPs to bombesin might be an alternative approach for targeted cancer therapy.