LL-37 antimicrobial peptide and heterologous prime-boost vaccination regimen significantly induce HIV-1 Nef-Vpr antigen- and virion-specific immune responses in mice.

OBJECTIVES: HIV infection still remains a leading cause of morbidity and mortality worldwide. The inability of highly-active antiretroviral therapy in HIV-1 eradication led to development of therapeutic vaccines. Exploiting effective immunogenic constructs and potent delivery systems are important to generate effective therapeutic vaccines, and overcome their poor membrane permeability. Among HIV-1 proteins, the Nef and Vpr proteins can be considered as antigen candidates in vaccine design. METHODS: In this study, the immunogenicity of Nef-Vpr antigen candidate in different regimens along with antimicrobial peptide LL-37 (as a DNA carrier) and Montanide 720 (as an adjuvant) was studied in mice. Moreover, the secretion of cytokines was assessed in virion-exposed mice lymphocytes in vitro. RESULTS: Our data indicated that groups immunized with the homologous protein + Montanide regimen (group 1), and also the heterologous DNA + LL-37 prime/protein + Montanide boost regimen (group 2) could significantly generate strong immune responses as compared to groups immunized with the DNA constructs (groups 3 & 4). Moreover, immunization of mice with the homologous DNA + LL-37 regimen in low dose of DNA (5 µg) could induce higher immune responses than the homologous naked DNA regimen in high dose of DNA (50 µg) indicating the role of LL-37 as a cell penetrating peptide. Additionally, the heterologous DNA + LL-37 prime/protein + Montanide boost regimen (group 2) induced significantly IFN-gamma secretion from virion-exposed lymphocytes in vitro. CONCLUSION: Generally, the use of LL-37 for DNA delivery, Montanide 720 as an adjuvant, and heterologous DNA prime/protein boost strategy could significantly increase IgG2a, IFN-gamma, and Granzyme B, and maintain cytokine secretion after exposure to virions. Indeed, the heterologous DNA + LL-37 prime/protein + Montanide boost regimen can be considered as a potent strategy for development of therapeutic HIV vaccines.

Sphingosine-1-Phosphate-Triggered Expression of Cathelicidin LL-37 Promotes the Growth of Human Bladder Cancer Cells.

It has been proven that tumour growth and progression are regulated by a variety of mediators released during the inflammatory process preceding the tumour appearance, but the role of inflammation in the development of bladder cancer is ambiguous. This study was designed around the hypothesis that sphingosine-1-phosphate (S1P), as a regulator of several cellular processes important in both inflammation and cancer development, may exert some of the pro-tumorigenic effects indirectly due to its ability to regulate the expression of human cathelicidin (hCAP-18). LL-37 peptide released from hCAP-18 is involved in the development of various types of cancer in humans, especially those associated with infections. Using immunohistological staining, we showed high expression of hCAP-18/LL-37 and sphingosine kinase 1 (the enzyme that forms S1P from sphingosine) in human bladder cancer cells. In a cell culture model, S1P was able to stimulate the expression and release of hCAP-18/LL-37 from human bladder cells, and the addition of LL-37 peptide dose-dependently increased their proliferation. Additionally, the effect of S1P on LL-37 release was inhibited in the presence of FTY720P, a synthetic immunosuppressant that blocks S1P receptors. Together, this study presents the possibility of paracrine relation in which LL-37 production following cell stimulation by S1P promotes the development and growth of bladder cancer.

Nanoantibiotics containing membrane-active human cathelicidin LL-37 or synthetic ceragenins attached to the surface of magnetic nanoparticles as novel and innovative therapeutic tools: current status and potential future applications.

Nanotechnology-based therapeutic approaches have attracted attention of scientists, in particular due to the special features of nanomaterials, such as adequate biocompatibility, ability to improve therapeutic efficiency of incorporated drugs and to limit their adverse effects. Among a variety of reported nanomaterials for biomedical applications, metal and metal oxide-based nanoparticles offer unique physicochemical properties allowing their use in combination with conventional antimicrobials and as magnetic field-controlled drug delivery nanocarriers. An ever-growing number of studies demonstrate that by combining magnetic nanoparticles with membrane-active, natural human cathelicidin-derived LL-37 peptide, and its synthetic mimics such as ceragenins, innovative nanoagents might be developed. Between others, they demonstrate high clinical potential as antimicrobial, anti-cancer, immunomodulatory and regenerative agents. Due to continuous research, knowledge on pleiotropic character of natural antibacterial peptides and their mimics is growing, and it is justifying to stay that the therapeutic potential of nanosystems containing membrane active compounds has not been exhausted yet.

Use of ceragenins as a potential treatment for urinary tract infections.

BACKGROUND: Urinary tract infections (UTIs) are one of the most common bacterial infections. High recurrence rates and the increasing antibiotic resistance among uropathogens constitute a large social and economic problem in current public health. We assumed that combination of treatment that includes the administration ceragenins (CSAs), will reinforce the effect of antimicrobial LL-37 peptide continuously produced by urinary tract epithelial cells. Such treatment might be an innovative approach to enhance innate antibacterial activity against multidrug-resistant E. coli. METHODS: Antibacterial activity measured using killing assays. Biofilm formation was assessed using crystal violet staining. Viability of bacteria and bladder epithelial cells subjected to incubation with tested agents was determined using MTT assays. We investigated the effects of chosen molecules, both alone and in combinations against four clinical strains of E. coli, obtained from patients diagnosed with recurrent UTI. RESULTS: We observed that the LL-37 peptide, whose concentration increases at sites of urinary infection, exerts increased bactericidal effect against E. coli when combined with ceragenins CSA-13 and CSA-131. CONCLUSION: We suggest that the employment of combination of natural peptide LL-37 with synthetic analogs might be a potential solution to treat urinary tract infections caused by drug-resistant bacteria.

Cathelicidin LL-37 induces angiogenesis via PGE2-EP3 signaling in endothelial cells, in vivo inhibition by aspirin.

OBJECTIVE: LL-37, the unique cathelicidin expressed in humans, in addition to acting as an endogenous antibiotic, is an important cell-signaling molecule upregulated in ovarian, breast, and lung tumors. However, the role of LL-37 in tumor microenvironment and its specific actions on the endothelial compartment remain elusive. Prostanoids are key regulators of inflammation, and cyclooxygenases (COXs) display proangiogenic activity in vitro and in vivo, mediated principally through prostaglandin E2 (PGE2). Here, we provide evidence for a novel proangiogenic role of LL-37, exerted via activation of endothelial cells and subsequent PGE2 biosynthesis. APPROACH AND RESULTS: LL-37 triggers PGE2 synthesis in endothelial cells in a dose-dependent manner with maximal induction after 4 hours. Endothelial PGE2 biosynthesis was dependent on COX-1, rather than COX-2, as judged by pharmacological inhibition and gene silencing. In vitro matrigel assays supported these findings because LL-37-induced cord formation was abolished by COX-1, but not COX-2, small interfering RNA, and the angiogenic phenotype could be rescued by addition of exogenous PGE2. We find that LL-37 acts on endothelial cells as a potent calcium agonist, inducing phosphorylation and activation of cytosolic phospholipase A2 (cPLA2), promoting a cPLA2→COX-1→PGE2 biosynthetic pathway and subsequent signaling via PGE2 receptor EP3. Moreover, cathelicidin-related antimicrobial peptide, which is the murine ortholog of LL-37, induced prostaglandin-dependent angiogenesis in vivo, which could be blocked by aspirin. CONCLUSIONS: Our results identify a novel proangiogenic role of LL-37, suggesting that the axis LL-37/COX-1/PGE2 followed by EP3 signaling is amenable to therapeutic intervention in pathological angiogenesis, for instance by aspirin.

Chlorin-e6 conjugated to the antimicrobial peptide LL-37 loaded nanoemulsion enhances photodynamic therapy against multi-species biofilms related to periodontitis.

In our previous studies, Chlorin-e6 (Ce6) demonstrated a significant reduction of microorganisms' viability against multi-species biofilm related to periodontitis while irradiated with blue light. However, the conjugation of Ce6 and antimicrobial peptides, and the incorporation of this photosensitizer in a nanocarrier, is still poorly explored. We hypothesized that chlorin-e6 conjugated to the antimicrobial peptide LL-37 loaded nanoemulsion could inhibit a multi-species biofilm related to periodontitis during photodynamic therapy (PDT), the pre-treatment with hydrogen peroxide was also tested. The nanoemulsion (NE) incorporated with Ce6 was characterized regarding the physiochemical parameters. Images were obtained by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Later, the Ce6 and LL-37 incorporated in NE was submitted to UV-Vis analysis and Reactive Oxygen Species (ROS) assay. Finally, the combined formulation (Ce6+LL-37 in nanoemulsion) was tested against multi-species biofilm related to periodontitis. The formed nanoformulation was kinetically stable, optically transparent with a relatively small droplet diameter (134.2 unloaded and 146.9 loaded), and weak light scattering. The NE system did not impact the standard UV-VIS spectra of Ce6, and the ROS production was improved while Ce6 was incorporated in the NE. The combination of Ce6 and LL-37 in NE was effective to reduce the viability of all bacteria tested. The treatment with hydrogen peroxide previous to PDT significantly impacted bacterial viability. The current aPDT regimen was the best already tested against periodontal biofilm by our research team. Our results suggest that this combined protocol must be exploited for clinical applications in localized infections such as periodontal disease. - Nanoemulsion demonstrated to be an excellent nanocarrier for photodynamic application. - Chlorin-e6 incorporated in nanoemulsion showed great physicochemical and biophotonic parameters. - The combination of chlorin-e6 and LL-37 peptide in nanoemulsion is effective to eliminate periodontal pathogenic bacteria. - The treatment with hydrogen peroxide previous to PDT significantly impacted bacterial viability.

Plant-Derived Polyphenol and LL-37 Peptide-Modified Nanofibrous Scaffolds for Promotion of Antibacterial Activity, Anti-Inflammation, and Type-H Vascularized Bone Regeneration.

The regeneration of oral tissues is a challenging clinical problem because of the complex microbial and biological stress environments. Electrospun fibrous scaffolds have attracted significant interest as effective barrier membranes for guided bone regeneration (GBR); however, no mature strategy yet exists for the surface modification of fibers to provide versatility to satisfy clinical requirements. This study demonstrated a practical biosafety strategy: the combined use of plant polyphenols and LL-37 peptides to modify the fiber surface to endow the fibrous scaffold with antimicrobial activity, immunoregulation, and vascularized bone regeneration. We confirmed that the LL-37 peptides interacted with tannic acid (TA) through noncovalent bonds through experiments and molecular docking simulation analysis. In vitro experiments showed that the TA coating imparted strong antibacterial properties to the fibrous scaffold, but it also caused cytotoxicity. The grafting of LL-37 peptide promoted the spreading, migration, and osteogenic differentiation of mesenchymal stem cells and was also conducive to the M2 polarization of RAW264.7 cells. In vivo experiments further verified that the LL-37 peptide-grafted fibrous scaffold significantly enhanced angiogenesis, anti-inflammatory effects, and type-H vascularized bone regeneration. Overall, the fibrous scaffold modified by the LL-37 peptide through TA grafting has significant potential for GBR applications.

Nanoparticles of VAV1 siRNA combined with LL37 peptide for the treatment of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is among the leading causes of cancer-related death, and it is highly resistant to therapy owing to its unique extracellular matrix. VAV1 protein, overexpressed in several cancer diseases including pancreatic cancer (PC), increases tumor proliferation and enhances metastases formation, which are associated with decreased survival. We hypothesized that an additive anti-tumor effect could be obtained by co-encapsulating in PLGA nanoparticles (NPs), the negatively charged siRNA against VAV1 (siVAV1) with the positively charged anti-tumor LL37 peptide, as a counter-ion. Several types of NPs were formulated and were characterized for their physicochemical properties, cellular internalization, and bioactivity in vitro. NPs' biodistribution, toxicity, and bioactivity were examined in a mice PDAC model. An optimal siVAV1 formulation (siVAV1-LL37 NPs) was characterized with desirable physicochemical properties in terms of nano-size, low polydispersity index (PDI), neutral surface charge, high siVAV1 encapsulation efficiency, spherical shape, and long-term shelf-life stability. Cell assays demonstrated rapid engulfment by PC cells, a specific and significant dose-dependent proliferation inhibition, as well as knockdown of VAV1 mRNA levels and migration inhibition in VAV1+ cells. Treatment with siVAV1-LL37 NPs in the mice PDAC model revealed marked accumulation of NPs in the liver and in the tumor, resulting in an increased survival rate following suppression of tumor growth and metastases, mediated via the knockdown of both VAV1 mRNA and protein levels. This proof-of-concept study validates our hypothesis of an additive effect in the treatment of PC facilitated by co-encapsulating siVAV1 in NPs with LL37 serving a dual role as a counter ion as well as an anti-tumor agent.

The human LL-37 peptide exerts antimicrobial activity against Legionella micdadei interacting with membrane phospholipids.

Legionella micdadei is responsible for community- or nosocomial-acquired pneumonia as well as the influenza-like illness Pontiac fever. The aim of this study was to investigate the ability of L. micdadei to utilize extracellular choline for phosphatidylcholine (PC) synthesis and its consequences for the phospholipid composition of its membrane system and the interaction with the human LL-37 peptide. Comparative analysis of the PC content using isotopic labeling revealed that in presence of exogenous choline 98% of the total PC was synthesized via the Pcs pathway while the remaining 2% were generated via the PE-methylation (PmtA) pathway. PC species were to a greater extent defined by the Pcs pathway in the outer membrane than in the inner membrane. While no major changes in the bacterial lipid content were observed using 31P NMR, indication for utilization of longer acyl chains and slight increase of PG in response to choline addition was observed by a top-down lipidomics screen. The LL-37 peptide inhibited L. micdadei growth in a dose-dependent manner. Bacteria cultured with exogenous choline were more sensitive to the LL-37 peptide when compared to the standard culture condition. Our biophysical investigations show that the peptide perturbs bacterial-derived phospholipid monolayers and this interaction is dependent on the molar portion of PC. This interaction is responsible for the observed changes in the anti-L. micdadei activity of the LL-37 peptide.

Antibacterial and osteogenesis performances of LL37-loaded titania nanopores in vitro and in vivo.

Background: Many studies have shown that the size of nanotube (NT) can significantly affect the behavior of osteoblasts on titanium-based materials. But the weak bonding strength between NT and substrate greatly limits their application. Purpose: The objective of this study was to compare the stability of NT and nanopore (NP) coatings, and further prepare antibacterial titanium-based materials by loading LL37 peptide in NP structures. Methods: The adhesion strength of NT and NP layers was investigated using a scratch tester. The proliferation and differentiation of MC3T3-E1 cells on different substrates were evaluated in vitro by CCK8, alkaline phosphatase activity, mineralization and polymerase chain reaction assays. The antibacterial rates of NP and NP/LL37 were also measured by spread plate method. Moreover, the osteogenesis around NP and NP/LL373 in vivo was further evaluated using uninfected and infected models. Results: Scratch test proved that the NP layers had stronger bonding strength with the substrates due to their continuous pore structures and thicker pipe walls than the independent NT structures. In vitro, cell results showed that MC3T3-E1 cells on NP substrates had better early adhesion, spreading and osteogenic differentiation than those of NT group. In addition, based on the drug reservoir characteristics of porous materials, the NP substrates were also used to load antibacterial LL37 peptide. After loading LL37, the antibacterial and osteogenic induction abilities of NP were further improved, thus significantly promoting osteogenesis in both uninfected and infected models. Conclusion: We determined that the NP layers had stronger bonding strength than NT structures, and the corresponding NP materials might be more suitable than NT for preparing drug-device combined titanium implants for bone injury treatment.

Peptide LL-37 coating on micro-structured titanium implants to facilitate bone formation in vivo via mesenchymal stem cell recruitment.

Titanium (Ti) and Ti-alloys were widely used in clinic orthopedics, however, the insufficient bone formation surrounding Ti-based implants still limited their biological performances. Surface modification of Ti substrates is essential to improve their interactions with bone-forming cells and bone tissue. In this study, we modified Ti substrates by coating peptide LL-37 onto micro-structured Ti substrates and aimed to (i) induce mesenchymal stem cells (MSCs) migration both in vitro and in vivo, (ii) facilitate osteogenic differentiation of MSCs and new bone formation. The surface micro-structured Ti substrates with hydroxyapatite deposition were fabricated by a two-step method including micro-arc oxidation (MAO) and hydrothermal treatment. LL-37 was loaded on micro-structured Ti substrates with the assistance of polydopamine coating. We confirmed that surface-modified Ti substrates benefited viability, adhesion, migration and osteogenic differentiation of MSCs in vitro. In a femur-defect rat model, the surface-modified Ti implants effectively induced CD29+/CD90+ positive cells migration in one week after implantation. According to the results of H&E, Masson's trichrome staining and immunohistochemical staining of OCN, OPN and collagen I, the targeted Ti implants exhibited significant new bone formation after implantation for 4 weeks. These results indicate that the surface modification of Ti samples facilitated bone formation through MSCs recruitment. STATEMENT OF SIGNIFICANCE: The inherent surface bioinertness of titanium (Ti) and Ti-alloys still limits their biological performances in clinical applications. Recently, the strategy of mesenchymal stem cells (MSCs) recruitment has been proposed to improve the osteointegration of bone implants. Herein, we reports the surface modification of Ti implants from the point of MSCs recruitment. Peptide LL-37 was coated on micro-structured Ti substrates to (i) recruit MSCs, (ii) regulate bio-physiological performance of MSCs, and (iii) facilitate bone formation in vivo. Our results improve the understanding of the interaction between Ti implants and MSCs, and provide a promising strategy of MSCs recruitment in the design of bone repair related biomaterials.

Core-shell magnetic nanoparticles display synergistic antibacterial effects against Pseudomonas aeruginosa and Staphylococcus aureus when combined with cathelicidin LL-37 or selected ceragenins.

Core-shell magnetic nanoparticles (MNPs) are promising candidates in the development of new treatment methods against infections, including those caused by antibiotic-resistant pathogens. In this study, the bactericidal activity of human antibacterial peptide cathelicidin LL-37, synthetic ceragenins CSA-13 and CSA-131, and classical antibiotics vancomycin and colistin, against methicillin-resistant Staphylococcus aureus Xen 30 and Pseudomonas aeruginosa Xen 5, was assessed alone and in combination with core-shell MNPs. Fractional inhibitory concentration index and fractional bactericidal concentration index were determined by microdilution methods. The potential of combined therapy using nanomaterials and selected antibiotics was confirmed using chemiluminescence measurements. Additionally, the ability of tested agents to prevent bacterial biofilm formation was evaluated using crystal violet staining. In most conditions, synergistic or additive effects were observed when combinations of core-shell MNPs with ceragenins or classical antibiotics were used. Our study revealed that a mixture of membrane-active agents such as LL-37 peptide or ceragenin CSA-13 with MNPs potentialized their antibacterial properties and might be considered as a method of delaying and overcoming bacterial drug resistance.