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.

PepBiotics, novel cathelicidin-inspired antimicrobials to fight pulmonary bacterial infections.

BACKGROUND: Antimicrobial peptides are considered potential alternatives to antibiotics. Here we describe the antibacterial properties of a family of novel cathelicidin-related (CR-) peptides, which we named PepBiotics, against bacteria typically present in cystic fibrosis (CF) patients. METHODS: Broth dilution assays were used to determine antibacterial activity of PepBiotics under physiological conditions, as well as development of bacterial resistance against these peptides. Toxicity was tested in mice and cell cultures while molecular interactions of PepBiotics with bacterial membrane components was determined using CD, ITC and LPS/LTA induced macrophage studies. RESULTS: A relatively small number of PepBiotics remained highly antibacterial against CF-related respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus, at high ionic strength and low pH. Interestingly, these PepBiotics also prevented LPS/LTA induced activation of macrophages and was shown to be non-toxic to primary human nasal epithelial cells. Furthermore, both P. aeruginosa and S. aureus were unable to induce resistance against CR-163 and CR-172, two PepBiotics selected for their excellent antimicrobial and immunomodulatory properties. Toxicity studies in mice indicated that intratracheal administration of CR-163 was well tolerated in vivo. Finally, interaction of CR-163 with bacterial-type anionic membranes but not with mammalian-type (zwitterionic lipid) membranes was confirmed using ITC and 31P solid state NMR. CONCLUSIONS: PepBiotics are a promising novel class of highly active antimicrobial peptides, of which CR-163 showed the most potential for treatment of clinically relevant (CF-) pathogens in physiological conditions. GENERAL SIGNIFICANCE: These observations emphasize the therapeutic potential of PepBiotics against CF-related bacterial respiratory infections.

Spray-Dried Inhalable Powder Formulations of Therapeutic Proteins and Peptides.

Respiratory diseases are among the leading causes of morbidity and mortality worldwide. Innovations in biochemical engineering and understanding of the pathophysiology of respiratory diseases resulted in the development of many therapeutic proteins and peptide drugs with high specificity and potency. Currently, protein and peptide drugs are mostly administered by injections due to their large molecular size, poor oral absorption, and labile physicochemical properties. However, parenteral administration has several limitations such as frequent dosing due to the short half-life of protein and peptide in blood, pain on administration, sterility requirement, and poor patient compliance. Among various noninvasive routes of administrations, the pulmonary route has received a great deal of attention and is a better alternative to deliver protein and peptide drugs for treating respiratory diseases and systemic diseases. Among the various aerosol dosage forms, dry powder inhaler (DPI) systems appear to be promising for inhalation delivery of proteins and peptides due to their improved stability in solid state. This review focuses on the development of DPI formulations of protein and peptide drugs using advanced spray drying. An overview of the challenges in maintaining protein stability during the drying process and stabilizing excipients used in spray drying of proteins and peptide drugs is discussed. Finally, a summary of spray-dried DPI formulations of protein and peptide drugs, their characterization, various DPI devices used to deliver protein and peptide drugs, and current clinical status are discussed.

Immunotherapeutic effect of adenovirus encoding antimicrobial peptides in experimental pulmonary tuberculosis.

As components of the innate immune response, antimicrobial peptides (AMPs) efficiently contribute to infection control and maintenance of a latent state in pulmonary tuberculosis (TB). As a therapeutic strategy, the administration of recombinant AMPs could be limited by enzymatic degradation and high production costs. Likewise, strategies based on the induction of AMPs have generated controversial results. In this study, 2 recombinant type-5 adenoviruses (Ad) expressing the human ß-defensin 3 (HßD3) or cathelicidin (LL37) were assessed in a murine pulmonary TB model. Mice infected with either a high dose of a drug-sensitive (H37Rv) or a multidrug-resistant (MDR) strain of Mycobacterium tuberculosis (Mtb) were treated with a single administration of AdHßD3, AdLL37, AdGFP (control vector expressing a green fluorescent protein), or saline solution (SS). Lungs were obtained to determine the bacterial burden, histologic damage, and cytokine expression at different time points. Mice treated with AdHßD3 or AdLL37 showed significantly lower bacterial load and pneumonia, and higher proinflammatory cytokine expression than the control groups AdGFP and SS. A synergistic therapeutic effect could be observed when first- or second-line antibiotics (ABs) were administered with adenoviral therapy in animals infected with H37Rv or MDR strains, respectively. Adenovirus-delivered AMP's administration constitutes a promising adjuvant therapy for current anti-TB drugs by enhancing a protective immune response and potentially reducing current AB regimes' duration.

Direct antimicrobial activity of cationic amphipathic peptide WLBU2 against Staphylococcus aureus biofilms is enhanced in physiologic buffered saline.

Periprosthetic joint infection of total knee arthroplasties represents a major challenge to the field of orthopedic surgery. These infections are commonly associated with antibiotic-tolerant Staphylococcus aureus biofilms. Engineered cationic amphipathic peptide WLBU2 has shown the ability to kill antibiotic-resistant pathogens and drug-tolerant bacterial biofilms. The novelty of using WLBU2 during the direct irrigation and debridement of periprosthetic joint infections led our group to investigate the optimal washout conditions for treatment of S. aureus biofilms. S. aureus mature biofilms were grown on metal implant material and treated with WLBU2 dissolved in differing irrigation solvents. Mature biofilms were treated both in vitro as well as in a periprosthetic joint infection murine model. WLBU2 activity against S. aureus biofilms was increased when dissolved in diphosphate-buffered saline (dPBS) with pH of 7.0 compared with normal saline with pH of 5.5. WLBU2 activity was decreased in acidic dPBS and increased in alkaline dPBS. WLBU2 activity could be decreased in hypertonic dPBS and increased in hypotonic dPBS. WLBU2 dissolved in less acidic dPBS displayed increased efficacy in treating periprosthetic joint infection (PJI) implants ex vivo. WLBU2 demonstrated the ability to eliminate PJI associated S. aureus biofilms on arthroplasty material. The efficacy of engineered cationic amphipathic peptide WLBU2 for intraoperative elimination of S. aureus biofilms can be further optimized when kept in a less acidic and more physiologic pH adjusted saline. Understanding optimal physical washout conditions are vital for the success of WLBU2 in treating S. aureus biofilms in PJI clinical trials going forward.

Ototopical drops containing a novel antibacterial synthetic peptide: Safety and efficacy in adults with chronic suppurative otitis media.

OBJECTIVE: Chronic suppurative otitis media (CSOM) is a chronic infectious disease with worldwide prevalence that causes hearing loss and decreased quality of life. As current (antibiotic) treatments often unsuccessful and antibiotic resistance is emerging, alternative agents and/or strategies are urgently needed. We considered the synthetic antimicrobial and anti-biofilm peptide P60.4Ac to be an interesting candidate because it also displays anti-inflammatory activities including lipopolysaccharide-neutralizing activity. The aim of the present study was to investigate the safety and efficacy of ototopical drops containing P60.4Ac in adults with CSOM without cholesteatoma. METHODS: We conducted a range-finding study in 16 subjects followed by a randomized, double blinded, placebo-controlled, multicentre phase IIa study in 34 subjects. P60.4Ac-containing ototopical drops or placebo drops were applied twice a day for 2 weeks and adverse events (AEs) and medication use were recorded. Laboratory tests, swabs from the middle ear and throat for bacterial cultures, and audiometry were performed at intervals up to 10 weeks after therapy. Response to treatment was assessed by blinded symptom scoring on otoscopy. RESULTS: Application of P60.4Ac-containing ototopical drops (0.25-2.0 mg of peptide/ml) in the ear canal of patients suffering from CSOM was found to be safe and well-tolerated. The optimal dose (0.5 mg of peptide/ml) was selected for the subsequent phase IIa study. Safety evaluation revealed only a few AEs that were unlikely related to study treatment and all, except one, were of mild to moderate intensity. In addition to this excellent safety profile, P60.4Ac ototopical drops resulted in a treatment success in 47% of cases versus 6% in the placebo group. CONCLUSION: The efficacy/safety balance assessed in the present study provides a compelling justification for continued clinical development of P60.4Ac in therapy-resistant CSOM.

Relationship between rosacea and sleep.

Rosacea is a chronic facial skin disease involved in neurovascular dysregulation and neurogenic inflammation. Behavioral factors such as stress, anxiety, depression and sleep were identified to be associated with other inflammatory skin diseases. Few studies have reported sleep status in rosacea. Aiming to investigate the relationship between rosacea and sleep, a case-control survey was conducted, enrolling 608 rosacea patients and 608 sex- and age-matched healthy controls. Sleep quality was assessed through the Pittsburgh Sleep Quality Index (PSQI) questionnaire. Diagnosis and severity grading of rosacea were evaluated under the standard guidelines of the National Rosacea Society. More rosacea patients (52.3%, n = 318) suffered poor sleep quality (PSQI, >5) than the healthy controls (24.0%, n = 146), displaying a much higher PSQI score (rosacea vs control, 6.20 vs 3.95). There was a strong association between sleep quality and rosacea (odds ratio [OR], 3.525; 95% confidence interval [CI], 2.759-4.519). Moreover, the severity of rosacea was also associated with sleep quality (OR, 1.847; 95% CI, 1.332-2.570). Single nucleotide polymorphisms in hydroxytryptamine receptor 2A and adrenoceptor-ß1 genes, which are associated with sleep behaviour, were detected and revealed to be associated with rosacea. Furthermore, the LL-37-induced rosacea-like phenotype and sleep-deprivation mice models were applied, revealing that sleep deprivation aggravated the rosacea-like phenotype in mice, with higher expression of matrix metallopeptidase 9, Toll-like receptor 2, cathelicidin antimicrobial peptide and vascular endothelial growth factor. In conclusion, rosacea patients presented poorer sleep quality, as well as a higher propability of genetic background with sleep disturbance. In addition, poor sleep might aggravate rosacea through regulating inflammatory factors, contributing to a vicious cycle in the progression of disease.

Omiganan Enhances Imiquimod-Induced Inflammatory Responses in Skin of Healthy Volunteers.

Omiganan (OMN; a synthetic cationic peptide) and imiquimod (IMQ; a TLR7 agonist) have synergistic effects on interferon responses in vitro. The objective of this study was to translate this to a human model for proof-of-concept, and to explore the potential of OMN add-on treatment for viral skin diseases. Sixteen healthy volunteers received topical IMQ, OMN, or a combination of both for up to 4 days on tape-stripped skin. Skin inflammation was quantified by laser speckle contrast imaging and 2D photography, and molecular and cellular responses were analyzed in biopsies. IMQ treatment induced an inflammatory response of the skin. Co-treatment with OMN enhanced this inflammatory response to IMQ, with increases in perfusion (+17.1%; 95% confidence interval (CI) 5.6%-30%; P < 0.01) and erythema (+1.5; 95% CI 0.25%-2.83; P = 0.02). Interferon regulatory factor-driven and NFκB-driven responses following TLR7 stimulation were enhanced by OMN (increases in IL-6, IL-10, MXA, and IFNÉ£), and more immune cell infiltration was observed (in particular CD4+, CD8+, and CD14+ cells). These findings are in line with the earlier mechanistic in vitro data, and support evaluation of imiquimod/OMN combination therapy in human papillomavirus-induced skin diseases.

Vitamin lipid nanoparticles enable adoptive macrophage transfer for the treatment of multidrug-resistant bacterial sepsis.

Sepsis, a condition caused by severe infections, affects more than 30 million people worldwide every year and remains the leading cause of death in hospitals1,2. Moreover, antimicrobial resistance has become an additional challenge in the treatment of sepsis3, and thus, alternative therapeutic approaches are urgently needed2,3. Here, we show that adoptive transfer of macrophages containing antimicrobial peptides linked to cathepsin B in the lysosomes (MACs) can be applied for the treatment of multidrug-resistant bacteria-induced sepsis in mice with immunosuppression. The MACs are constructed by transfection of vitamin C lipid nanoparticles that deliver antimicrobial peptide and cathepsin B (AMP-CatB) mRNA. The vitamin C lipid nanoparticles allow the specific accumulation of AMP-CatB in macrophage lysosomes, which is the key location for bactericidal activities. Our results demonstrate that adoptive MAC transfer leads to the elimination of multidrug-resistant bacteria, including Staphylococcus aureus and Escherichia coli, leading to the complete recovery of immunocompromised septic mice. Our work provides an alternative strategy for overcoming multidrug-resistant bacteria-induced sepsis and opens up possibilities for the development of nanoparticle-enabled cell therapy for infectious diseases.

Superoxide Dismutase 3 Inhibits LL-37/KLK-5-Mediated Skin Inflammation through Modulation of EGFR and Associated Inflammatory Cascades.

The expressions of LL-37 and KLK-5 were found to be altered in various dermatoses, including atopic dermatitis, psoriasis, and rosacea. However, the downstream inflammatory effect of LL-37 and KLK-5 is not as well studied. In addition, there is little high-quality evidence for the treatment of LL-37- and KLK-5-mediated inflammation. In this study, we investigated the effect of superoxide dismutase 3 (SOD3) on LL-37- or KLK-5-induced skin inflammation in vitro and in vivo and its underlying anti-inflammatory mechanisms. Our data showed that SOD3 significantly reduced both LL-37- and KLK-5-induced expression of pro-inflammatory mediators and suppressed the activation of EGFR, protease-activated receptor 2, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3, and p38/extracellular signal-regulated kinase signaling pathways in human keratinocytes. Moreover, SOD3 suppressed LL-37-induced expression of inflammatory mediators, reactive oxygen species production, and p38/extracellular signal-regulated kinase activation in mast cells. In addition, subcutaneous injection of KLK-5 in SOD3 knockout mice exhibited erythema with increased epidermal thickness, mast cell and neutrophil infiltration, expression of inflammatory mediators, and activation of EGFR, protease-activated receptor 2, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3, and downstream mitogen-activated protein kinase pathways. However, treatment with SOD3 in SOD3 knockout mice rescued KLK-5-induced inflammatory cascades. Similarly, KLK-5-induced inflammation in wild-type mice was also ameliorated when treated with SOD3. Taken together, our data suggest that SOD3 is a potentially effective therapy for both LL-37-and KLK-5-induced skin inflammation.

The inhibitory effect of tachyplesin I on thrombosis and its mechanisms.

Thrombotic diseases are major cause of cardiovascular diseases. This study was designed to investigate the effect of tachyplesin I on platelet aggregation and thrombosis. Platelet aggregation was analysed with a whole blood aggregometer. The mice were employed to investigate the effect of tachyplesin I on thrombosis in vivo. Tachyplesin I inhibited thrombin-induced platelet aggregation in a dose-dependent manner. Furthermore, tachyplesin I significantly reduced thrombosis in carrageenan-induced tail thrombosis model by intraperitoneal injection (0.1, 0.2 or 0.4 mg/kg) or intragastric administration (15, 30 or 60 mg/kg). Tachyplesin I also prolonged the bleeding time (BT) and clotting time (CT). The results revealed that tachyplesin I inhibited platelet aggregation and thrombosis by interfering the PI3K/AKT pathway. Tachyplesin I did not show significantly toxicity to mice under 300 mg/kg via intravenous injection. The results show that tachyplesin I inhibits thrombosis and has low toxicity. It is suggested that tachyplesin I has the potential to develop a new anti-thrombotic drug.

Sea snake cathelicidin (Hc-cath) exerts a protective effect in mouse models of lung inflammation and infection.

We investigated the anti-inflammatory and antibacterial activities of Hc-cath, a cathelicidin peptide derived from the venom of the sea snake, Hydrophis cyanocyntus, using in vivo models of inflammation and infection. Hc-cath function was evaluated in in vitro, in vivo in the wax moth, Galleria mellonella, and in mouse models of intraperitoneal and respiratory Pseudomonas aeruginosa infection. Hc-Cath downregulated LPS-induced pro-inflammatory responses in macrophages and significantly improved the survival of P. aeruginosa infected G. mellonella over a 5-day period. We also demonstrated, for the first time, that Hc-cath can modulate inflammation in a mouse model of LPS-induced lung inflammation by significantly reducing the release of the pro-inflammatory cytokine and neutrophil chemoattractant, KC, resulting in reduced cellular infiltration into the lungs. Moreover, Hc-cath treatment significantly reduced the bacterial load and inflammation in mouse models of P. aeruginosa intraperitoneal and respiratory infection. The effect of Hc-cath in our studies highlights the potential to develop this peptide as a candidate for therapeutic development.

Poly(lactide- co-glycolide) Nanoparticles for Prolonged Therapeutic Efficacy of Esculentin-1a-Derived Antimicrobial Peptides against Pseudomonas aeruginosa Lung Infection: in Vitro and in Vivo Studies.

Due to their excellent in vitro activity against multidrug resistant bacteria, antimicrobial peptides (AMPs) hold promise for treatment of Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) sufferers. In this work, poly(lactide- co-glycolide) (PLGA) nanoparticles for lung delivery of AMPs deriving from the frog-skin esculentin-1a, namely, Esc(1-21) and Esc(1-21)-1c (Esc peptides), were successfully developed. Improved peptide transport through artificial CF mucus and simulated bacterial extracellular matrix was achieved in vitro. The formulations were effectively delivered through a liquid jet nebulizer already available to patients. Notably, Esc peptide-loaded nanoparticles displayed an improved efficacy in inhibiting P. aeruginosa growth in vitro and in vivo in the long term. A single intratracheal administration of Esc peptide-loaded nanoparticles in a mouse model of P. aeruginosa lung infection resulted in a 3-log reduction of pulmonary bacterial burden up to 36 h. Overall, results unravel the potential of PLGA nanoparticles as a reliable delivery system of AMPs to lungs.

Multiple cargo deliveries of growth factors and antimicrobial peptide using biodegradable nanopolymer as a potential wound healing system.

BACKGROUND: Treatment of wounds with the help of nanoparticles (NPs) is more effective and superior in comparison to traditional wound healing methods as it protects and sustains active drug release at the wound site thus enhancing the safety of the drug and reducing the possibility of side effects. The advantages of this method are the possibility of allowing a reduction in administered dose, limiting toxicity levels to the minimum, and increasing safety of topical delivery of the drug. MATERIALS AND METHODS: We report the synthesis of a novel poly (lactic-co-glycolic acid) (PLGA) NP-based multicargo delivery system for growth factors and antimicrobial peptide. Growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were entrapped in PLGA NPs by solvent diffusion method and an antimicrobial peptide (K4) was conjugated to the NP by carbodiimide chemistry. The developed multiple cargo delivery systems with growth factors (VEGF and bFGF) and an antimicrobial peptide (K4) were investigated and optimized for potential wound healing. RESULTS: The system showed a sustained release of growth factors and was evaluated for cytotoxicity by MTT and live/dead assay, which revealed that the bioactivity of the growth factor-entrapped NPs was higher than that of free growth factors, and it also induced enhanced cell proliferation in vitro. CONCLUSION: The development of a system for the codelivery of growth factors (VEGF and bFGF) and an antimicrobial peptide (K4) was investigated for potential wound healing application. The entrapment of growth factors with very high efficiency is an advantage in this method along with its sustained release from the nanoparticulate system, which will enhance the angiogenesis. Our system also displayed broad-spectrum antimicrobial activity against both gram-positive and gram-negative bacteria.

The Immune Activity of PT-Peptide Derived from Anti-Lipopolysaccharide Factor of the Swimming Crab Portunus trituberculatus Is Enhanced when Encapsulated in Milk-Derived Extracellular Vesicles.

PT-peptide is derived from the anti-lipopolysaccharide factor of the swimming crab Portunus trituberculatus. The peptide, consisting of 34 amino acids, contains a lipopolysaccharide binding domain. In this study, we investigated the effect of PT-peptide encapsulated in raw milk-derived extracellular vesicles (EVs), designated as EVs-PT peptide, on immune regulation. The results showed that raw milk-derived EVs efficaciously delivered the PT-peptide into monocytes and elevated immune activity, including reactive oxygen species level, superoxide anion production, and phagocytosis. PT-peptide and EVs-PT peptide also elevated the secretion of cytokines, such as interferon-γ, interleukin-6, and tumor necrosis factor-α in human monocytic THP-1 cells. These results suggest that the PT-peptide could be developed as an immune stimulator.

Botulinum toxin blocks mast cells and prevents rosacea like inflammation.

BACKGROUND: Rosacea is a chronic inflammatory skin condition whose etiology has been linked to mast cells and the antimicrobial peptide cathelicidin LL-37. Individuals with refractory disease have demonstrated clinical benefit with periodic injections of onabotulinum toxin, but the mechanism of action is unknown. OBJECTIVES: To investigate the molecular mechanism by which botulinum toxin improves rosacea lesions. METHODS: Primary human and murine mast cells were pretreated with onabotulinum toxin A or B or control. Mast cell degranulation was evaluated by ß-hexosaminidase activity. Expression of botulinum toxin receptor Sv2 was measured by qPCR. The presence of SNAP-25 and VAMP2 was established by immunofluorescence. In vivo rosacea model was established by intradermally injecting LL-37 with or without onabotulinum toxin A pretreatment. Mast cell degranulation was assessed in vivo by histologic counts. Rosacea biomarkers were analyzed by qPCR of mouse skin sections. RESULTS: Onabotulinum toxin A and B inhibited compound 48/80-induced degranulation of both human and murine mast cells. Expression of Sv2 was established in mouse mast cells. Onabotulinum toxin A and B increased cleaved SNAP-25 and decreased VAMP2 staining in mast cells respectively. In mice, injection of onabotulinum toxin A significantly reduced LL-37-induced skin erythema, mast cell degranulation, and mRNA expression of rosacea biomarkers. CONCLUSIONS: These findings suggest that onabotulinum toxin reduces rosacea-associated skin inflammation by directly inhibiting mast cell degranulation. Periodic applications of onabotulinum toxin may be an effective therapy for refractory rosacea and deserves further study.

A novel antimicrobial peptide-derived vehicle for oligodeoxynucleotide delivery to inhibit TNF-α expression.

Indolicidin (IL), an antimicrobial peptide, was investigated as a vehicle to promote oligodeoxynucleotides (ODNs) delivery. To increase charge density, IL was dimerized by adding a cysteine to its C or N terminus, which was denoted as ILC or CIL, respectively. In contrast to IL, cytotoxicity of ILC and CIL was significantly reduced because these dimeric peptides were longer than IL, which restricted their insertions to cell membrane. In contrast to ILC, CIL displayed well loading efficiency. These peptides were applied to deliver ODNs against tumor necrosis factor-α (TNF-α) because TNF-α is a pro-inflammatory cytokine which plays an important role in immunological diseases. Although IL/ODN slightly reduced TNF-α expression, the high cytotoxicity restricted its application window. Furthermore, ILC/ODN was incapable of inducing gene silence due to its low encapsulation efficiency and poor endosomal escape. In contrast, CIL exhibited excellent ODN transportation and the internalized CIL/ODN complexes may escape from endosomes. Therefore, TNF-α expression can be specifically reduced by CIL/ODN complexes, and the silence effect was maintained longer than 14 h. This study provides a useful strategy of peptide vehicle design, which may facilitate the delivery of not only ODN but also other oligonucleotides, including siRNA and miRNA, to promote gene silence application.

Characterization of Host Responses during Pseudomonas aeruginosa Acute Infection in the Lungs and Blood and after Treatment with the Synthetic Immunomodulatory Peptide IDR-1002.

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial pneumonia and infects patients with cystic fibrosis. P. aeruginosa lung infections are difficult to treat due to bacterial resistance to antibiotics, and strains with multidrug resistance are becoming more prevalent. Here, we examined the use of a small host defense peptide, innate defense regulator 1002 (IDR-1002), in an acute P. aeruginosa lung infection in vivo IDR-1002 significantly reduced the bacterial burden in bronchoalveolar lavage fluid (BALF), as well as MCP-1 in BALF and serum, KC in serum, and interleukin 6 (IL-6) in BALF. Transcriptome sequencing (RNA-Seq) was conducted on lungs and whole blood, and the effects of P. aeruginosa, IDR-1002, and the combination of P. aeruginosa and IDR-1002 were evaluated. Differential gene expression analysis showed that P. aeruginosa increased multiple inflammatory and innate immune pathways, as well as affected hemostasis, matrix metalloproteinases, collagen biosynthesis, and various metabolism pathways in the lungs and/or blood. Infected mice treated with IDR-1002 had significant changes in gene expression compared to untreated infected mice, with fewer differentially expressed genes associated with the inflammatory and innate immune responses to microbial infection, and treatment also affected morphogenesis, certain metabolic pathways, and lymphocyte activation. Overall, these results showed that IDR-1002 was effective in treating P. aeruginosa acute lung infections and associated inflammation.

Aurein-Derived Antimicrobial Peptides Formulated with Pegylated Phospholipid Micelles to Target Methicillin-Resistant Staphylococcus aureus Skin Infections.

Antimicrobial peptides have been the focus of considerable research; however, issues associated with toxicity and aggregation have the potential to limit clinical applications. Here, a derivative of a truncated version of aurein 2.2 (aurein 2.2Δ3), namely peptide 73, was investigated, along with its d-amino acid counterpart (D-73) and a retro-inverso version (RI-73). A version that incorporated a cysteine residue to the C-terminus (73c) was also generated, as this form is required to covalently attach antimicrobial peptides to polymers (e.g., polyethylene glycol (PEG) or hyperbranched polyglycerol (HPG)). The antimicrobial activity of the 73-derived peptides was enhanced 2- to 8-fold, and all the derivatives eradicated preformed Staphylococcus aureus biofilms. Formulation of the peptides with compatible polyethylene glycol (PEG)-modified phospholipid micelles alleviated toxicity toward human cells and reduced aggregation. When evaluated in vivo, the unformulated d-enantiomers aggregated when injected under the skin of mice, but micelle encapsulated peptides were well absorbed. Pegylated micelle formulated peptides were investigated for their potential as therapeutic agents for treating high-density infections in a murine cutaneous abscess model. Formulated peptide 73 reduced abscess size by 36% and bacterial loads by 2.2-fold compared to the parent peptide aurein 2.2Δ3. Micelle encapsulated peptides 73c and D-73 exhibited superior activity, further reducing abscess sizes by 85% and 63% and lowering bacterial loads by 510- and 9-fold compared to peptide 73.

Effects of antimicrobial peptides on serum biochemical parameters, antioxidant activity and non-specific immune responses in Epinephelus coioides.

Antimicrobial peptides (AMPs) are small proteins showing broad-spectrum antimicrobial activity that have been known to be powerful agents against a variety of pathogens (bacteria, fungi and viruses). In this study, the effects of AMPs from Bacillus subtilis on Epinephelus coioides were examined. E. coioides were fed with diets containing AMPs (0, 100, 200, 400 or 800 mg/kg) for four weeks. Results showed that the levels of total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and blood glucose (GLU) and lipopolysaccharide (LPS) in the serum of E. coioides changed than those of the control group; compared to the control group, the levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and lysozyme (LZM) levels in E. coioides fed with different dosages AMP diets were also different; in addition, the mRNA expression of tumor necrosis factor alpha (TNF-α), interleukin-1-beta (IL-1ß), and heat shock protein 90 (Hsp90) in the tissues of E. coioides were measured, the three genes in the tissues examined were significantly upregulated. The results demonstrated that diets containing AMPs can enhance the antioxidant capacity and innate immune ability of E. coioides, indicating that AMPs might be a potential alternative to antibiotics in E. coioides.