Biodistribution of the cationic host defense peptide LL-37 using SPECT/CT.

Human cathelicidin LL-37, a cationic host defense peptide (CHDP), has several important physiological roles, including antimicrobial activity, immune modulation, and wound healing, and is a being investigated as a therapeutic candidate for several indications. While the effects of endogenously produced LL-37 are well studied, the biodistribution of exogenously administered LL-37 are less known. Here we assess the biodistribution of a gallium-67 labeled variant of LL-37 using nuclear imaging techniques over a 48 h period in healthy mice. When administered as an intravenous bolus just over 20 µg, the LL-37-based radiotracer was rapidly cleared from the blood, largely by the liver, while an appreciable fraction of the dose temporarily distributed to the lungs. When administered subcutaneously at the same dose level, the radiotracer was absorbed systemically following a two-phase kinetic model and was predominately cleared renally. Uptake into sites rich in immune cells, such as the lymph nodes and the spleen, was observed for both routes of administration. Scans of free gallium-67 were also performed as controls. Important preclinical insights into the biodistribution of exogenously administered LL-37 were gained from this study, which can aid in the understanding of this and related cationic host-defense peptides.

Nasal cathelicidin is expressed in early life and is increased during mild, but not severe respiratory syncytial virus infection.

Respiratory syncytial virus is the major cause of acute lower respiratory tract infections in young children, causing extensive mortality and morbidity globally, with limited therapeutic or preventative options. Cathelicidins are innate immune antimicrobial host defence peptides and have antiviral activity against RSV. However, upper respiratory tract cathelicidin expression and the relationship with host and environment factors in early life, are unknown. Infant cohorts were analysed to characterise early life nasal cathelicidin levels, revealing low expression levels in the first week of life, with increased levels at 9 months which are comparable to 2-year-olds and healthy adults. No impact of prematurity on nasal cathelicidin expression was observed, nor were there effects of sex or birth mode, however, nasal cathelicidin expression was lower in the first week-of-life in winter births. Nasal cathelicidin levels were positively associated with specific inflammatory markers and demonstrated to be associated with microbial community composition. Importantly, levels of nasal cathelicidin expression were elevated in infants with mild RSV infection, but, in contrast, were not upregulated in infants hospitalised with severe RSV infection. These data suggest important relationships between nasal cathelicidin, upper airway microbiota, inflammation, and immunity against RSV infection, with interventional potential.

Milk-derived extracellular vesicles functionalized with anti-tumour necrosis factor-α nanobody and anti-microbial peptide alleviate ulcerative colitis in mice.

Ulcerative colitis (UC) manifests clinically with chronic intestinal inflammation and microflora dysbiosis. Although biologics can effectively control inflammation, efficient delivery to the colon and colon epithelial cells remains challenging. Milk-derived extracellular vesicles (EV) show promise as an oral delivery tool, however, the ability to load biologics into EV presents challenges to therapeutic applications. Here, we demonstrate that fusing cell-penetrating peptide (TAT) to green fluorescent protein (GFP) enabled biologics loading into EV and protected against degradation in the gastrointestinal environment in vitro and in vivo after oral delivery. Oral administration of EV loaded with anti-tumour necrosis factor-α (TNF-α) nanobody (VHHm3F) (EVVHH) via TAT significantly reduced tissue TNF-α levels and alleviated pathologies in mice with acute UC, compared to VHH alone. In mice with chronic UC, simultaneously introducing VHH and an antimicrobial peptide LL37 into EV (EVLV), then administering orally improved intestinal barrier, inflammation and microbiota balance, resulted in relief of UC-induced depression and anxiety. Collectively, we demonstrated that oral delivery of EVLV effectively alleviated UC in mice and TAT efficiently loaded biologics into EV to confer protection from degradation in the gastrointestinal tract. This therapeutic strategy is promising for UC and is a simple and generalizable approach towards drug-loaded orally-administrable EV treatment for other diseases.

Investigation of a novel bilayered PCL/PVA electrospun nanofiber incorporated Chitosan-LL37 and Chitosan-VEGF nanoparticles as an advanced antibacterial cell growth-promoting wound dressing.

Chronic wounds have become a growing concern as they can have a profound impact on individuals, potentially resulting in mortality. It is crucial to prevent and manage bacterial infections, particularly drug-resistant ones. Antimicrobial peptides, such as LL-37, can firmly eliminate pathogens. Additionally, the process of angiogenesis, facilitated by growth factors like VEGF, is essential for tissue repair and wound healing. To enhance the stability and bioavailability of therapeutic agents, targeted delivery strategies utilizing Chitosan-based carriers have been employed. Electrospun biopolymers in advanced wound dressings have revolutionized wound care by providing a more effective and efficient solution for promoting tissue regeneration and speeding up the healing process. The present investigation utilized Chitosan nanoparticles to encapsulate the recombinant LL37 peptide and VEGF. An in-depth investigation was carried out to analyze the biophysical and morphological traits of the LL37-CSNPs and VEGF-CSNPs. The first support layer consisted of PCL electrospun nanofiber, followed by the electrospinning of PVA/CsLL37, PVA/CsVEGF, and PVA/CsLL37/CsVEGF onto the PCL layer. An in vitro examination assessed the fabricated nanofibers' morphological, mechanical, and biological characteristics. The antimicrobial effects were tested on methicillin-resistant Staphylococcus aureus (MRSA). The in vivo experiments assessed the antibacterial and wound-healing capabilities of the nanofibers. The findings validated the continuous release of LL37 and VEGF. The composite material PCL/PVA/CsLL37/CsVEGF demonstrated potent bactericidal and antioxidant characteristics. The cytotoxic assay demonstrated the biocompatibility of the fabricated nano mats and their potential to accelerate fibroblast cell proliferation. The efficacy of PVA/CsLL37/CsVEGF in promoting wound healing was confirmed through an in vivo wound healing assay. Furthermore, the histological analysis provided evidence of faster epidermal formation and improved antibacterial activity in wounds covered with PVA/CsLL37/CsVEGF. Adding LL37 and VEGF to the composite material improves the immune response and promotes blood vessel formation, accelerating wound healing and decreasing inflammation.

LL37/self-DNA complexes mediate monocyte reprogramming.

LL37 alone and in complex with self-DNA triggers inflammatory responses in myeloid cells and plays a crucial role in the development of systemic autoimmune diseases, like psoriasis and systemic lupus erythematosus. We demonstrated that LL37/self-DNA complexes induce long-term metabolic and epigenetic changes in monocytes, enhancing their responsiveness to subsequent stimuli. Monocytes trained with LL37/self-DNA complexes and those derived from psoriatic patients exhibited heightened glycolytic and oxidative phosphorylation rates, elevated release of proinflammatory cytokines, and affected naïve CD4+ T cells. Additionally, KDM6A/B, a demethylase of lysine 27 on histone 3, was upregulated in psoriatic monocytes and monocytes treated with LL37/self-DNA complexes. Inhibition of KDM6A/B reversed the trained immune phenotype by reducing proinflammatory cytokine production, metabolic activity, and the induction of IL-17-producing T cells by LL37/self-DNA-treated monocytes. Our findings highlight the role of LL37/self-DNA-induced innate immune memory in psoriasis pathogenesis, uncovering its impact on monocyte and T cell dynamics.

Autoreactivity to self-antigens LL37 and ADAMTSL5 influences the clinical response to risankizumab in psoriatic patients.

The autoantigens LL37 and ADAMTSL5 contribute to induce pathogenetic T-cells responses in a subset of psoriatic patients. Whether the presence of LL37-and/or ADAMTS5-reactive T-cells influences the clinical response to treatment is still unknown. The aim of the study is to evaluate the clinical responses to the anti-IL-23 risankizumab in LL37 and/or ADAMTSL5-reactive patients in comparison with non-reactive ones and to assess whether genetics (HLA-Cw06.02) or BMI influences the response to treatment. Patients were screened at baseline for the presence of circulating LL37 or/and ADAMTSL5-reactive T-cells and were treated as per protocol with risankizumab. Effectiveness data (PASI scores) were collected at weeks 4, 16, 28, 40 and 52. Data were also analyzed based on HLA-Cw06.02 status and BMI. The overall response to treatment of patients with autoreactivity to LL37 or ADAMTSL5 did not differ compared to the non-reactive cohort as measured as PASI75/90/100 at different time points; however, subjects that had autoreactive T-cells to both LL37 and ADAMTS5 demonstrated suboptimal response to treatment starting at week16. HLA-Cw06:02+ patients demonstrated faster response to risankizumab at week 4 compared to HLA-Cw06:02-. Additionally, the response to treatment was influenced by the BMI with slower responses seen in overweight and obese patients at week 4 and week16. In conclusion, while the presence of either LL37-and ADAMTS5-reactive circulating T-cells do not influence the clinical response to risankizumab, the presence of the double reactivity to both LL37 and ADAMTS5 decreases the clinical responses. Moreover, we evidenced that HLA-Cw06+ respond faster to IL-23 inhibition and that BMI, associated to autoreactivity, can influence the speed in response.

LL37 Microspheres Loaded on Activated Carbon-chitosan Hydrogel: Anti-bacterial and Anti-toxin Wound Dressing for Chronic Wound Infections.

Antimicrobial peptide LL37 is a promising antibacterial candidate due to its potent antimicrobial activity with no known bacterial resistance. However, intrinsically LL37 is susceptible to degradation in wound fluids limits its effectiveness. Bacterial toxins which are released after cell lysis are found to hinder wound healing. To address these challenges, encapsulating LL37 in microspheres (MS) and loading the MS onto activated carbon (AC)-chitosan (CS) hydrogel. This advanced wound dressing not only protects LL37 from degradation but also targets bacterial toxins, aiding in the healing of chronic wound infections. First, LL37 MS and LL37-AC-CS hydrogel were prepared and characterised in terms of physicochemical properties, drug release, and peptide-polymer compatibility. Antibacterial and antibiofilm activity, bacterial toxin elimination, cell migration, and cell cytotoxicity activities were investigated. LL37-AC-CS hydrogel was effective against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. LL37-AC-CS hydrogel bound more endotoxin than AC with CS hydrogel alone. The hydrogel also induced cell migration after 72 h and showed no cytotoxicity towards NHDF after 72 h of treatment. In conclusion, the LL37-AC-CS hydrogel was shown to be a stable, non-toxic advanced wound dressing method with enhanced antimicrobial and antitoxin activity, and it can potentially be applied to chronic wound infections to accelerate wound healing.

Susceptibility of Legionella gormanii Membrane-Derived Phospholipids to the Peptide Action of Antimicrobial LL-37-Langmuir Monolayer Studies.

LL-37 is the only member of the cathelicidin-type host defense peptide family in humans. It exhibits broad-spectrum bactericidal activity, which represents a distinctive advantage for future therapeutic targets. The presence of choline in the growth medium for bacteria changes the composition and physicochemical properties of their membranes, which affects LL-37's activity as an antimicrobial agent. In this study, the effect of the LL-37 peptide on the phospholipid monolayers at the liquid-air interface imitating the membranes of Legionella gormanii bacteria was determined. The Langmuir monolayer technique was employed to prepare model membranes composed of individual classes of phospholipids-phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL)-isolated from L. gormanii bacteria supplemented or non-supplemented with exogenous choline. Compression isotherms were obtained for the monolayers with or without the addition of the peptide to the subphase. Then, penetration tests were carried out for the phospholipid monolayers compressed to a surface pressure of 30 mN/m, followed by the insertion of the peptide into the subphase. Changes in the mean molecular area were observed over time. Our findings demonstrate the diversified effect of LL-37 on the phospholipid monolayers, depending on the bacteria growth conditions. The substantial changes in membrane properties due to its interactions with LL-37 enable us to propose a feasible mechanism of peptide action at a molecular level. This can be associated with the stable incorporation of the peptide inside the monolayer or with the disruption of the membrane leading to the removal (desorption) of molecules into the subphase. Understanding the role of antimicrobial peptides is crucial for the design and development of new strategies and routes for combating resistance to conventional antibiotics.

[Progress of researches on the antiparasitic activity of antimicrobial peptide LL-37].

Parasitic diseases caused by protozoan and helminth infections are still widespread across the world, notably in tropical and subtropical areas, which threaten the children and adult health. Long-term use of anti-parasitic drugs may result in reduced drug susceptibility and even drug resistance. Antimicrobial peptides have been demonstrated to inhibit parasite growth and development, which has potential antiparasitic values. LL-37, the only human antimicrobial peptide in the cathelicidin family, has been widely investigated. This paper reviews the progress of researches on the antiparasitic activity of LL-37, and discusses the prospects of LL-37 in the research of parasites.

A Comprehensive Review of Recent Research into the Effects of Antimicrobial Peptides on Biofilms-January 2020 to September 2023.

Microbial biofilm formation creates a persistent and resistant environment in which microorganisms can survive, contributing to antibiotic resistance and chronic inflammatory diseases. Increasingly, biofilms are caused by multi-drug resistant microorganisms, which, coupled with a diminishing supply of effective antibiotics, is driving the search for new antibiotic therapies. In this respect, antimicrobial peptides (AMPs) are short, hydrophobic, and amphipathic peptides that show activity against multidrug-resistant bacteria and biofilm formation. They also possess broad-spectrum activity and diverse mechanisms of action. In this comprehensive review, 150 publications (from January 2020 to September 2023) were collected and categorized using the search terms 'polypeptide antibiotic agent', 'antimicrobial peptide', and 'biofilm'. During this period, a wide range of natural and synthetic AMPs were studied, of which LL-37, polymyxin B, GH12, and Nisin were the most frequently cited. Furthermore, although many microbes were studied, Staphylococcus aureus and Pseudomonas aeruginosa were the most popular. Publications also considered AMP combinations and the potential role of AMP delivery systems in increasing the efficacy of AMPs, including nanoparticle delivery. Relatively few publications focused on AMP resistance. This comprehensive review informs and guides researchers about the latest developments in AMP research, presenting promising evidence of the role of AMPs as effective antimicrobial agents.

Vitamin D triggers hCAP18/LL-37 production: Implications for LL-37-induced human osteoblast cytotoxicity.

The human cathelicidin LL-37 shows activity against microorganisms, but it is also cytotoxic to host cells. The CAMP gene codes for the LL-37 precursor hCAP18 which is processed extracellularly to active LL-37. It has previously been shown that vitamin D stimulates CAMP gene activity, but less information is available demonstrating that vitamin D also can increase hCAP18/LL-37 protein production. Here, we show with RT-qPCR that a physiological concentration of vitamin D (50 nM) enhances CAMP mRNA levels by about 170 times in human THP-1 monocyte cells. Stimulation with 50 nM vitamin D increases hCAP18/LL-37 protein contents 3-4 times in THP-1 cell lysates demonstrated by both dot blot analysis and ELISA applying two different hCAP18/LL-37 antibodies. Treatment with the proteasome inhibitor MG132 enhances hCAP18/LL-37 levels, suggesting that turnover of hCAP18/LL-37 protein is regulated by the proteasome. The hCAP18/LL-37 concentration in vitamin D-stimulated THP-1 cells corresponds to 1.04 µM LL-37. Interestingly, synthetic LL-37, at this concentration, reduces viability of human osteoblast-like MG63 cells, whereas the THP-1 cells are less sensitive as demonstrated by the MTT assay. In summary, we show that vitamin D enhances hCAP18/LL-37 production, and that this effect can be of physiological/pathophysiological relevance for LL-37-induced human osteoblast toxicity.

Dectin-1/SYK Activation Induces Antimicrobial Peptide and Negative Regulator of NF-κB Signaling in Human Oral Epithelial Cells.

BACKGROUND/AIM: Oral epithelial cells serve as the primary defense against microbial exposure in the oral cavity, including the fungus Candida albicans. Dectin-1 is crucial for recognition of ß-glucan in fungi. However, expression and function of Dectin-1 in oral epithelial cells remain unclear. MATERIALS AND METHODS: We assessed Dectin-1 expression in Ca9-22 (gingiva), HSC-2 (mouth), HSC-3 (tongue), and HSC-4 (tongue) human oral epithelial cells using flow cytometry and real-time polymerase chain reaction. Cell treated with ß-glucan-rich zymosan were evaluated using real-time polymerase chain reaction. Phosphorylation of spleen-associated tyrosine kinase (SYK) was analyzed by western blotting. RESULTS: Dectin-1 was expressed in all four cell types, with high expression in Ca9-22 and HSC-2. In Ca9-22 cells, exposure to ß-glucan-rich zymosan did not alter the mRNA expression of chemokines nor of interleukin (IL)6, IL8, IL1ß, IL17A, and IL17F. Zymosan induced the expression of antimicrobial peptides ß-defensin-1 and LL-37, but not S100 calcium-binding protein A8 (S100A8) and S100A9. Furthermore, the expression of cylindromatosis (CYLD), a negative regulator of nuclear factor kappa B (NF-κB) signaling, was induced. In HSC-2 cells, zymosan induced the expression of IL17A. The expression of tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a negative regulator of NF-κB signaling, was also induced. Expression of other cytokines and antimicrobial peptides remained unchanged. Zymosan induced phosphorylation of SYK in Ca9-22 cells, as well as NF-κB. CONCLUSION: Oral epithelial cells express Dectin-1 and recognize ß-glucan, which activates SYK and induces the expression of antimicrobial peptides and negative regulators of NF-κB, potentially maintaining oral homeostasis.

Antileishmanial activity of cathelicidin and its modulation by Leishmania donovani in a CREM-dependent manner for establishing infection.

Concerns regarding toxicity and resistance of current drugs have been reported in visceral leishmaniasis. Anti-microbial peptides are considered as new promising candidates and amongst them, human cathelicidin hCAP18/LL-37 showed significant parasite killing on drug-sensitive and resistant Leishmania promastigotes, coupled with its apoptosis-inducing role. Administration of hCAP18/LL-37 in infected macrophages also decreased parasite survival and increased the host favorable cytokine IL-12. However, 1,25-dihydroxyvitamin D3 (VitD3)-induced endogenous hCAP18/LL-37 production was hampered in infected THP-1 cells. Infection also suppressed the VitD3-receptor (VDR), transcription factor of hCAP18/LL-37. cAMP response element modulator (CREM), the repressor of VDR, was induced in infection resulting in suppression of both VDR and cathelicidin expression. PGE2/cAMP/PKA axis was found to regulate CREM induction during infection and silencing CREM in infected cells and BALB/c mice led to decreased parasite survival. Present study thus documents the anti-leishmanial potential of cathelicidin and further identifies CREM as a repressor of cathelicidin in Leishmania infection.

LL-37: Structures, Antimicrobial Activity, and Influence on Amyloid-Related Diseases.

Antimicrobial peptides (AMPs), as well as host defense peptides (HDPs), constitute the first line of defense as part of the innate immune system. Humans are known to express antimicrobial precursor proteins, which are further processed to generate AMPs, including several types of α/ß defensins, histatins, and cathelicidin-derived AMPs like LL37. The broad-spectrum activity of AMPs is crucial to defend against infections caused by pathogenic bacteria, viruses, fungi, and parasites. The emergence of multi-drug resistant pathogenic bacteria is of global concern for public health. The prospects of targeting antibiotic-resistant strains of bacteria with AMPs are of high significance for developing new generations of antimicrobial agents. The 37-residue long LL37, the only cathelicidin family of AMP in humans, has been the major focus for the past few decades of research. The host defense activity of LL37 is likely underscored by its expression throughout the body, spanning from the epithelial cells of various organs-testis, skin, respiratory tract, and gastrointestinal tract-to immune cells. Remarkably, apart from canonical direct killing of pathogenic organisms, LL37 exerts several other host defense activities, including inflammatory response modulation, chemo-attraction, and wound healing and closure at the infected sites. In addition, LL37 and its derived peptides are bestowed with anti-cancer and anti-amyloidogenic properties. In this review article, we aim to develop integrative, mechanistic insight into LL37 and its derived peptides, based on the known biophysical, structural, and functional studies in recent years. We believe that this review will pave the way for future research on the structures, biochemical and biophysical properties, and design of novel LL37-based molecules.

Self-assembly of PEG-PPS polymers and LL-37 peptide nanomicelles improves the oxidative microenvironment and promotes angiogenesis to facilitate chronic wound healing.

Refractory diabetic wounds are associated with high incidence, mortality, and recurrence rates and are a devastating and rapidly growing clinical problem. However, treating these wounds is difficult owing to uncontrolled inflammatory microenvironments and defective angiogenesis in the affected areas, with no established effective treatment to the best of our knowledge. Herein, we optimized a dual functional therapeutic agent based on the assembly of LL-37 peptides and diblock copolymer poly(ethylene glycol)-poly(propylene sulfide) (PEG-PPS). The incorporation of PEG-PPS enabled responsive or controlled LL-37 peptide release in the presence of reactive oxygen species (ROS). LL-37@PEG-PPS nanomicelles not only scavenged excessive ROS to improve the microenvironment for angiogenesis but also released LL-37 peptides and protected them from degradation, thereby robustly increasing angiogenesis. Diabetic wounds treated with LL-37@PEG-PPS exhibited accelerated and high-quality wound healing in vivo. This study shows that LL-37@PEG-PPS can restore beneficial angiogenesis in the wound microenvironment by continuously providing angiogenesis-promoting signals. Thus, it may be a promising drug for improving chronic refractory wound healing.

Construction of exosome-loaded LL-37 and its protection against zika virus infection.

Zika virus (ZIKV) is an enveloped, single-stranded and positive-stranded RNA virus of the genus Flavivirus in the family Flaviviridae. ZIKV can cross the placental barrier and infect the fetus, causing microcephaly, congenital ZIKV syndrome, and even fetal death. ZIKV infection can also lead to testicular damage and male sterility. But no effective drugs and vaccines are available up to now. Previous studies have shown that the cathelicidin antimicrobial peptide LL-37 can protect against ZIKV infection. However, LL-37 is a secreted peptide, which can be easily degraded in vivo. We herein constructed exosome-loaded LL-37 (named LL-37-TM-exo and TM-LL-37-exo) using the transmembrane protein TM to load LL-37 onto the membrane of exosome. We found that exosome-loaded LL-37 could significantly inhibit ZIKV infection in vitro and in vivo, and LL-37-TM-exo had stronger antiviral activity than that of TM-LL-37-exo, which could significantly reduce ZIKV-induced testicular injury and sperm injury, and had broad-spectrum antiviral effect. Compared to free LL-37, exosome-loaded LL-37 showed a better serum stability, higher efficiency to cross the placental barrier, and stronger antiviral activity. The mechanism of exosome-loaded LL-37 against ZIKV infection was consistent with that of free LL-37, which could directly inactivate viral particles, reduce the susceptibility of host cells, and act on viral replication stage. Our study provides a novel strategy for the development of LL-37 against viral infection.

Human Host Defense Peptide LL-37 Suppresses TNFα-Mediated Matrix Metalloproteinases MMP9 and MMP13 in Human Bronchial Epithelial Cells.

INTRODUCTION: TNFα-inducible matrix metalloproteinases play a critical role in the process of airway remodeling in respiratory inflammatory disease including asthma. The cationic host defense peptide LL-37 is elevated in the lungs during airway inflammation. However, the impact of LL-37 on TNFα-driven processes is not well understood. Here, we examined the effect of LL-37 on TNFα-mediated responses in human bronchial epithelial cells (HBECs). METHODS: We used a slow off-rate modified aptamer-based proteomics approach to define the HBEC proteome altered in response to TNFα. Abundance of selected protein candidates and signaling intermediates was examined using immunoassays, ELISA and Western blots, and mRNA abundance was examined by qRT-PCR. RESULTS: Proteomics analysis revealed that 124 proteins were significantly altered, 12 proteins were enhanced by ≥2-fold compared to unstimulated cells, in response to TNFα. MMP9 was the topmost increased protein in response to TNFα, enhanced by ∼10-fold, and MMP13 was increased by ∼3-fold, compared to unstimulated cells. Furthermore, we demonstrated that LL-37 significantly suppressed TNFα-mediated MMP9 and MMP13 in HBEC. Mechanistic data revealed that TNFα-mediated MMP9 and MMP13 production is controlled by SRC kinase and that LL-37 enhances related upstream negative regulators, namely, phospho-AKT (T308) and TNFα-mediated TNFAIP3 or A20. CONCLUSIONS: The findings of this study suggest that LL-37 may play a role in intervening in the process of airway remodeling in chronic inflammatory respiratory disease such as asthma.

Boosting Membrane Interactions and Antimicrobial Effects of Photocatalytic Titanium Dioxide Nanoparticles by Peptide Coating.

Photocatalytic nanoparticles offer antimicrobial effects under illumination due to the formation of reactive oxygen species (ROS), capable of degrading bacterial membranes. ROS may, however, also degrade human cell membranes and trigger toxicity. Since antimicrobial peptides (AMPs) may display excellent selectivity between human cells and bacteria, these may offer opportunities to effectively "target" nanoparticles to bacterial membranes for increased selectivity. Investigating this, photocatalytic TiO2 nanoparticles (NPs) are coated with the AMP LL-37, and ROS generation is found by C11 -BODIPY to be essentially unaffected after AMP coating. Furthermore, peptide-coated TiO2 NPs retain their positive ζ-potential also after 1-2 h of UV illumination, showing peptide degradation to be sufficiently limited to allow peptide-mediated targeting. In line with this, quartz crystal microbalance measurements show peptide coating to promote membrane binding of TiO2 NPs, particularly so for bacteria-like anionic and cholesterol-void membranes. As a result, membrane degradation during illumination is strongly promoted for such membranes, but not so for mammalian-like membranes. The mechanisms of these effects are elucidated by neutron reflectometry. Analogously, LL-37 coating promoted membrane rupture by TiO2 NPs for Gram-negative and Gram-positive bacteria, but not for human monocytes. These findings demonstrate that AMP coating may selectively boost the antimicrobial effects of photocatalytic NPs.

Discovery of novel antibacterial agent for the infected wound treatment: all-hydrocarbon stapling optimization of LL-37.

Rationale: Antimicrobial peptide LL-37 has been recognized as a favorable alternative to antibiotics due to its broad antibacterial spectrum, low resistance development and diverse biological activities. However, its high manufactory cost, poor proteolytic stability, and unpredictable cytotoxicity seriously hindered its medical translation. Methods: To push the frontiers of its clinical application, all-hydrocarbon stapling strategy was exploited here for the structural modification of KR-12, the core and minimal fragment of LL-37. Results: Based on a library of KR-12 derivatives that designed and synthesized to be stapled at positions of either i, i+4 or i, i+7, structure to activity relationship was investigated. Among them, KR-12(Q5, D9) with the glutamine and aspartic acid residues stapled displayed increased helical content and positive charge. The reinforced α-helical conformation not only protected it from proteolytic hydrolysis but also improved its antibacterial efficacy via effective membrane perturbation and anti-inflammatory efficacy via compact LPS binding. Besides, the increased positive charge endowed it with an enhanced therapeutic index. On infected wound mouse model, it was demonstrated to eliminate bacteria and promote wound closure and regeneration effectively. Conclusion: Overall, the all-hydrocarbon stapling was proven to lay the foundation for the future development of antibacterial agents. KR-12(Q5, D9) could serve as a lead compound for the clinical treatment of bacterial infections.

Expression of the Antimicrobial Peptide SE-33-A2P, a Modified Analog of Cathelicidin, and an Analysis of Its Properties.

In this study, we developed a method for the expression of the antimicrobial peptide SE-33-A2P in E. coli bacterial cells. The SE-33-A2P peptide consists of A2P and SE-33 peptides and is a retro analog of cathelicidin possessing antimicrobial activity against both Gram-positive and Gram-negative bacteria. Furthermore, the A2P peptide is a self-cleaving peptide. For an efficient expression of the SE-33-A2P peptide, a gene encoding several repetitive sequences of the SE-33 peptide separated by A2P sequences was created. The gene was cloned into a plasmid, with which E. coli cells were transformed. An induction of the product expression was carried out by IPTG after the cell culture gained high density. The inducible expression product, due to the properties of the A2P peptide, was cleaved in the cell into SE-33-A2P peptides. As the next step, the SE-33-A2P peptide was purified using filtration and chromatography. Its activity against both Gram-positive and Gram-negative bacteria, including antibiotic-resistant bacteria, was proved. The developed approach for obtaining a prokaryotic system for the expression of a highly active antimicrobial peptide expands the opportunities for producing antimicrobial peptides via industrial methods.