LL-37

Research Overview

LL-37 serves as a valuable research tool for investigating innate immune defense mechanisms and antimicrobial peptide biology. This synthetic peptide represents the biologically active C-terminal fragment of human cathelicidin antimicrobial peptide (hCAP-18), released through proteolytic cleavage by proteinase-3. Research applications span antimicrobial activity studies, immune modulation investigations, wound healing research, and host-pathogen interaction studies.

The peptide’s designation LL-37 reflects its composition: beginning with two leucine residues (LL) and consisting of 37 amino acids. LL-37 represents the sole member of the cathelicidin family in humans, distinguished from multiple cathelicidins found in other mammalian species. The peptide plays crucial roles in innate immunity as a first-line defense against microbial pathogens while simultaneously modulating adaptive immune responses.

LL-37 research has contributed significantly to understanding antimicrobial peptide mechanisms, innate immunity pathways, and the bridge between innate and adaptive immune systems. The peptide’s multifunctional properties make it valuable for investigating host defense mechanisms beyond direct antimicrobial activity, including inflammation modulation, angiogenesis, and tissue repair.

Molecular Characteristics

Complete Specifications:

• CAS Registry Number: 154947-66-7
• Molecular Weight: 4,493.34 Da
• Molecular Formula: C₂₀₅H₃₄₀N₆₀O₅₃
• Amino Acid Sequence: LLGDFFRKSKEK-IGKEFKRIVQRIKDFLRNLVPRTES
• Structure: Amphipathic α-helix
• Peptide Classification: Cationic antimicrobial peptide, cathelicidin
• Appearance: White to off-white lyophilized powder
• Solubility: Water, PBS, physiological buffers

The peptide’s 37-amino acid sequence adopts an amphipathic α-helical conformation in membrane-mimetic environments. This structural organization creates distinct hydrophobic and cationic faces enabling membrane interaction and disruption. The sequence contains multiple positively charged residues (lysine, arginine) providing net positive charge (+6 at physiological pH), crucial for electrostatic interaction with negatively charged bacterial membranes.

Antimicrobial and Immunomodulatory Mechanisms

LL-37 demonstrates multiple mechanisms of action:

Direct Antimicrobial Activity:

• Membrane disruption through electrostatic interaction and pore formation
• Broad-spectrum activity against gram-positive and gram-negative bacteria
• Antifungal activity against Candida species and other fungi
• Antiviral effects through envelope disruption and immune modulation
• Biofilm disruption and prevention

Immunomodulatory Functions:

• Chemotactic activity for neutrophils, monocytes, T cells, and mast cells
• Cytokine and chemokine production modulation
• Dendritic cell maturation and antigen presentation regulation
• Inflammatory response balancing (pro- and anti-inflammatory effects)
• Autophagy induction in macrophages

Tissue Repair and Regeneration:

• Angiogenesis promotion through VEGF receptor transactivation
• Keratinocyte and fibroblast migration stimulation
• Wound closure acceleration
• Extracellular matrix remodeling
• Re-epithelialization promotion

Research Applications

Antimicrobial Activity Studies

LL-37 serves as a primary research tool for investigating antimicrobial peptide mechanisms:

• Bacterial Pathogen Research: Investigation of antibacterial activity against diverse gram-positive (S. aureus, MRSA, Streptococcus) and gram-negative (E. coli, P. aeruginosa, K. pneumoniae) bacteria
• Mechanism of Action Studies: Research on membrane disruption, pore formation, intracellular targeting, and bacterial killing kinetics
• Antibiotic Resistance Research: Investigation of activity against antibiotic-resistant strains and mechanisms preventing resistance development
• Biofilm Studies: Examination of biofilm disruption, prevention, and penetration capabilities
• Minimum Inhibitory Concentration: Determination of MIC/MBC values against various pathogens

Research protocols employ bacterial culture assays, membrane permeabilization studies, scanning electron microscopy, and time-kill kinetics experiments.

Innate Immunity and Host Defense Research

LL-37 enables investigation of innate immune mechanisms:

• Pattern Recognition: Research on LL-37 release triggers, including PAMPs and DAMPs recognition
• Neutrophil Function: Studies on neutrophil chemotaxis, activation, and antimicrobial function
• Monocyte/Macrophage Research: Investigation of monocyte recruitment, macrophage polarization, and phagocytic function
• Mast Cell Studies: Research on mast cell degranulation, histamine release, and inflammatory mediator production
• Complement System Interactions: Examination of complement pathway modulation and antimicrobial synergy

Laboratory models include leukocyte migration assays, phagocytosis assays, cytokine measurements, and in vivo infection models.

Wound Healing and Tissue Repair Studies

LL-37 research extends to tissue repair mechanisms:

• Keratinocyte Migration: Investigation of re-epithelialization mechanisms and epithelial cell migration
• Fibroblast Function: Research on fibroblast migration, proliferation, and collagen synthesis
• Angiogenesis Studies: Examination of blood vessel formation through VEGFR2 transactivation and EGFR signaling
• Wound Healing Models: Studies in acute wounds, chronic wounds, and diabetic wound healing models
• Extracellular Matrix Remodeling: Investigation of matrix metalloproteinase modulation and ECM reorganization

Research protocols utilize scratch assays, wound healing animal models, tube formation assays, and histological analysis.

Inflammatory Response Modulation Research

LL-37 demonstrates complex immunomodulatory effects:

• Cytokine Regulation: Investigation of pro-inflammatory (IL-8, TNF-α) and anti-inflammatory (IL-10) cytokine modulation
• NF-κB Pathway Studies: Research on transcription factor activation and inflammatory gene expression
• Inflammasome Regulation: Examination of NLRP3 inflammasome modulation and IL-1β production
• Resolution of Inflammation: Studies on inflammation resolution mechanisms and anti-inflammatory effects
• Balance Effects: Research on context-dependent pro- or anti-inflammatory activities

Laboratory models include inflammatory cell cultures, cytokine assays, inflammasome activation studies, and inflammatory disease models.

Viral and Fungal Pathogen Research

Beyond antibacterial activity, LL-37 demonstrates broader antimicrobial properties:

• Antiviral Mechanisms: Investigation of activity against enveloped viruses (influenza, HIV, HSV) and non-enveloped viruses
• Viral Entry Inhibition: Research on envelope disruption and receptor binding interference
• Antifungal Activity: Studies on activity against Candida species, dermatophytes, and other pathogenic fungi
• Fungal Cell Wall Interactions: Examination of chitin binding and cell wall disruption mechanisms
• Combination Studies: Investigation of synergistic effects with conventional antimicrobial agents

Research protocols employ viral infection models, plaque reduction assays, fungal growth inhibition studies, and mechanism-of-action investigations.

Laboratory Handling and Storage Protocols

Lyophilized Powder Storage:

• Store at -20°C to -80°C in original sealed vial
• Desiccated storage environment required
• Protect from repeated temperature fluctuations
• Stability data available for 12+ months at -20°C

Reconstitution Guidelines:

• Reconstitute with sterile water, PBS (pH 7.4), or appropriate buffer
• Add solvent slowly down vial side
• Gentle mixing recommended (avoid vigorous vortexing)
• Allow complete dissolution (may require 5-10 minutes)
• Final concentration typically 0.1-1 mg/mL for stock solutions

Solution Storage:

• Short-term storage: 4°C for up to 7 days
• Long-term storage: -20°C to -80°C in aliquots
• Avoid repeated freeze-thaw cycles (maximum 2-3 cycles)
• Aliquot into single-use portions
• Sterile filtration recommended for cell culture applications (0.22μm)

Salt Sensitivity Considerations:
LL-37’s antimicrobial activity is sensitive to salt concentration. High ionic strength reduces electrostatic interactions with bacterial membranes, decreasing antimicrobial potency. Researchers should consider physiological salt concentrations when designing experiments and interpreting results.

Quality Assurance and Analytical Testing

Each LL-37 batch undergoes comprehensive analytical characterization:

Purity Analysis:

• High-Performance Liquid Chromatography (HPLC): ≥95% purity
• Analytical method: Reversed-phase HPLC with UV detection at 220nm
• Multiple peak integration to ensure accurate purity determination

Structural Verification:

• Electrospray Ionization Mass Spectrometry (ESI-MS): Confirms molecular weight 4,493.34 Da
• Amino acid analysis: Verifies sequence composition
• Peptide content determination: Quantifies actual peptide content by weight

Contaminant Testing:

• Bacterial endotoxin: <5 EU/mg (LAL method)
• Heavy metals: Below detection limits per USP standards
• Residual solvents: TFA and acetonitrile within acceptable limits
• Water content: Karl Fischer titration (<8%)

Functional Testing:

• Antimicrobial activity verification against control strains
• Confirmatory activity assays to ensure biological function

Documentation:

• Certificate of Analysis (COA) provided with each batch
• Third-party analytical verification available upon request
• Stability data documented for recommended storage conditions
• Batch-specific QC results traceable by lot number

Research Considerations

Experimental Design Factors:

Researchers should consider several factors when designing LL-37 experiments:

1. Salt Sensitivity: LL-37’s antimicrobial activity decreases with increasing ionic strength. Consider physiological salt concentrations and low-salt conditions for maximal activity assessment.

2. Concentration Selection: Antimicrobial assays typically use 0.5-50 μM LL-37. Cell culture studies for immunomodulation often use 1-10 μg/mL. Optimize concentrations for specific applications.

3. pH Considerations: LL-37 maintains activity across physiological pH range (6.5-7.5) but activity may vary with pH changes affecting charge state.

4. Serum Effects: Serum proteins may bind LL-37 and reduce activity. Consider serum-free or low-serum conditions for in vitro antimicrobial assays.

5. Structural Considerations: LL-37 requires amphipathic α-helical structure for membrane interaction. Organic solvents or denaturing conditions may affect activity.

Mechanism Investigation:

LL-37’s mechanisms involve multiple pathways:

• Direct membrane disruption (carpet model, toroidal pore formation)
• Intracellular targeting (DNA/RNA binding, enzyme inhibition)
• Chemotaxis through formyl peptide receptor-like 1 (FPRL1) and other receptors
• EGFR and VEGFR2 transactivation for cell migration and angiogenesis
• Inflammatory signaling modulation (NF-κB, MAPK pathways)
• Autophagy induction through receptor-mediated pathways

Compliance and Safety Information

Regulatory Status:
LL-37 is provided as a research chemical for in-vitro laboratory studies and preclinical research only. This product has not been approved by the FDA for human therapeutic use, diagnostic purposes, or medical applications.

Intended Use:

• In-vitro antimicrobial studies
• In-vivo preclinical research in approved animal models
• Laboratory investigation of innate immunity mechanisms
• Academic and institutional research applications

NOT Intended For:

• Human consumption or administration
• Therapeutic treatment of infections
• Diagnostic purposes
• Dietary supplementation
• Veterinary therapeutic applications without appropriate oversight
• Any medical applications

Safety Protocols:
Researchers should follow standard laboratory safety practices when handling LL-37:

• Use appropriate personal protective equipment
• Handle in well-ventilated areas
• Follow institutional biosafety guidelines for antimicrobial agent handling
• Dispose of waste according to local regulations
• Consult material safety data sheet (MSDS) for additional safety information

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