LL-37: Practical Research and Usage Guide

LL-37, the human cathelicidin antimicrobial peptide, is a 37-amino acid cationic peptide with broad applications in antimicrobial research, immunology, wound healing, and cancer biology. This guide provides practical protocols for research use, drawing on established methodologies from the antimicrobial peptide research community. LL-37 is supplied as a synthetic lyophilized peptide with the sequence LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES and a molecular weight of 4,493.33 daltons. Research-grade LL-37 is available from specialized peptide synthesis companies at purities of 95 percent or greater by HPLC, typically in quantities of 1 mg, 5 mg, or 10 mg per vial. The peptide may be supplied as the trifluoroacetate (TFA) salt or acetate salt. For cell-based and in vivo research, the acetate salt is preferred because TFA can cause cytotoxicity at higher peptide concentrations. If only TFA-salt peptide is available, TFA can be removed by repeated lyophilization from dilute hydrochloric acid (0.1 M HCl) or by anion exchange. For reconstitution of lyophilized LL-37, allow the vial to reach room temperature for 15 to 20 minutes. LL-37 is soluble in water, saline, and buffered solutions, but its solubility and biological activity are pH-dependent and ionic-strength-dependent. For stock solution preparation, dissolve LL-37 in sterile endotoxin-free water to a concentration of 1 to 5 mg per mL (approximately 220 to 1,110 micromolar). At these concentrations, LL-37 may self-associate into oligomeric structures, which is normal and does not indicate degradation. For working solutions in antimicrobial assays, dilute the stock in the appropriate assay buffer or medium. Important: LL-37 adsorbs readily to plastic and glass surfaces, particularly at low concentrations. To minimize adsorption losses, use low-protein-binding tubes (polypropylene or siliconized), prepare working solutions at concentrations above 1 micromolar whenever possible, and add carrier protein (0.01 percent bovine serum albumin) to very dilute solutions. For injection purposes, dissolve in sterile bacteriostatic water or sterile physiological saline. The reconstituted solution should be clear. Slight opalescence at high concentrations is normal and reflects oligomeric association. Dosing protocols for LL-37 research vary substantially depending on the application and model system. The following sections describe established protocols for major research categories. For in vitro antimicrobial testing, the standard methodology follows Clinical and Laboratory Standards Institute (CLSI) guidelines for antimicrobial susceptibility testing, adapted for peptide agents. Minimum inhibitory concentration (MIC) determination uses broth microdilution in 96-well polypropylene plates (not polystyrene, which adsorbs cationic peptides). Mueller-Hinton broth is the standard medium, but note that its divalent cation content affects LL-37 activity. Some investigators use cation-adjusted Mueller-Hinton broth (CAMHB) for standardization. LL-37 is tested in two-fold serial dilutions from 128 mcg per mL to 0.25 mcg per mL. Typical MIC values for LL-37 against common pathogens are: Escherichia coli, 2 to 16 mcg per mL; Staphylococcus aureus, 16 to 64 mcg per mL; Pseudomonas aeruginosa, 16 to 64 mcg per mL; Candida albicans, 8 to 32 mcg per mL. For time-kill assays, bacteria at mid-logarithmic phase (approximately 10 to the sixth colony forming units per mL) are exposed to LL-37 at 1x, 2x, and 4x MIC, with samples taken at 0, 15, 30, 60, 120, and 240 minutes for viable count determination. For anti-biofilm research, LL-37 is particularly noteworthy because it inhibits biofilm formation at sub-MIC concentrations. Biofilm prevention assays use LL-37 at 0.5 to 16 mcg per mL added at the time of bacterial inoculation in 96-well plates. Biofilm mass is quantified after 24 hours by crystal violet staining. For disruption of established biofilms, mature 24 to 48 hour biofilms are treated with LL-37 at 32 to 256 mcg per mL (2x to 16x MIC) for 24 hours, with biofilm viability assessed by LIVE/DEAD staining and confocal microscopy or by colony count of dispersed biofilm bacteria. For immunomodulatory research in cell culture, LL-37 is used at concentrations of 1 to 50 micromolar (approximately 4.5 to 225 mcg per mL) to study its effects on immune cells. Chemotaxis assays employ modified Boyden chambers or transwell inserts (8-micron pore size) with LL-37 at 1 to 10 micromolar in the lower chamber and monocytes, neutrophils, or T cells in the upper chamber. Migration is assessed after 2 to 4 hours. For dendritic cell studies, monocyte-derived dendritic cells are generated by standard GM-CSF and IL-4 differentiation and then exposed to LL-37 at 5 to 20 micromolar during lipopolysaccharide stimulation to study its effects on dendritic cell maturation and cytokine production. For the LL-37-self-DNA complex studies relevant to psoriasis research, LL-37 at 10 to 40 micromolar is complexed with human genomic DNA or CpG oligonucleotides at a 1:1 to 10:1 peptide-to-DNA mass ratio, and the complexes are applied to plasmacytoid dendritic cells to measure type I interferon production by ELISA. For wound healing research in vitro, keratinocyte migration assays (scratch wound assay) use LL-37 at 1 to 20 micromolar applied to confluent HaCaT keratinocyte monolayers after creating a defined scratch with a pipette tip. Wound closure is monitored by time-lapse microscopy over 24 to 48 hours. Endothelial tube formation assays use LL-37 at 1 to 10 micromolar applied to human umbilical vein endothelial cells (HUVECs) plated on Matrigel-coated wells, with tube network formation quantified after 6 to 18 hours. For EGFR transactivation studies, keratinocytes or epithelial cells are treated with LL-37 at 5 to 20 micromolar, and EGFR phosphorylation is assessed by western blot at 5, 15, 30, and 60 minute time points. For in vivo research in animal models, LL-37 dosing varies by route of administration and research objective. In murine wound healing models, LL-37 is applied topically at 50 to 200 mcg per wound in a gel or solution vehicle, applied once or twice daily. In systemic infection models, LL-37 is administered intraperitoneally at doses of 2 to 10 mg per kg body weight. In murine sepsis models, LL-37 at 4 mg per kg intraperitoneally has demonstrated protective effects when administered concurrent with bacterial challenge. For intratracheal delivery in pneumonia models, LL-37 at 25 to 100 mcg per mouse is delivered in 50 microliters of saline. It is important to note that LL-37 is a human-specific peptide and the murine homolog is CRAMP (cathelicidin-related antimicrobial peptide, mCRAMP). Some researchers use CRAMP in mouse models for physiological relevance, while others use human LL-37 to evaluate the specific human peptide's therapeutic potential. Administration route selection for LL-37 research depends on the target tissue. Topical application is the most practical for skin, corneal, and mucosal infections and is the route used in clinical development programs. LL-37 formulations for topical use include aqueous solutions, hydrogel preparations (using carbomer or hyaluronic acid bases), and lipid-based formulations. Intranasal delivery has been studied for respiratory infection applications, with nebulized LL-37 solutions at 50 to 250 mcg per mL demonstrating antimicrobial activity in airway models. Subcutaneous and intraperitoneal injection routes are used for systemic delivery in animal models. Intravenous delivery is theoretically possible but limited by rapid serum protease degradation and potential hemolytic activity at high concentrations. Storage protocols for LL-37 are critical for maintaining peptide integrity. Lyophilized LL-37 should be stored at minus 20 degrees Celsius (stable for at least 24 months) or minus 80 degrees Celsius (stable for at least 36 months). Protect from light and moisture. Reconstituted LL-37 stock solutions should be stored at minus 20 degrees Celsius in single-use aliquots to avoid repeated freeze-thaw cycles. At 2 to 8 degrees Celsius, reconstituted solutions maintain stability for approximately 1 to 2 weeks. Avoid repeated freeze-thaw cycles (more than 3) as this can lead to peptide aggregation and loss of activity. For short-term storage during experiments, keep solutions on ice. LL-37 is susceptible to oxidation (particularly at the methionine residue if present in analogs) and proteolytic degradation. Include protease inhibitors in biological samples intended for LL-37 quantification. For long-term storage of stock solutions, adding 10 percent DMSO can improve stability, but DMSO must be diluted below 0.1 percent in working solutions to avoid cytotoxicity. Quality control for research-grade LL-37 should verify the following parameters. Identity confirmation by mass spectrometry with expected molecular weight of 4,493.33 daltons (monoisotopic) or approximately 4,496 daltons (average mass). Purity by HPLC of greater than 95 percent. Endotoxin content below 0.1 EU per mg by LAL assay (critical for immunological research, as LPS contamination confounds all innate immunity studies). Peptide content by amino acid analysis. Net peptide content of lyophilized peptide (accounting for counter-ions and moisture; typically 70 to 85 percent of total weight). Confirmation of correct sequence by tandem mass spectrometry or Edman degradation. Safety considerations for LL-37 research include the following. LL-37 is cytotoxic to mammalian cells at high concentrations (typically above 50 micromolar for most cell types), so working concentrations must be carefully titrated. The therapeutic index (ratio of cytotoxic concentration to antimicrobial concentration) varies by cell type and should be established for each experimental system. For in vivo research, high systemic doses of LL-37 can cause hemolysis and renal toxicity; dosing should follow established preclinical protocols with appropriate monitoring. Researchers with psoriasis or rosacea should be aware that LL-37 is implicated in these conditions and should use appropriate personal protective equipment during handling. For researchers interested in vitamin D-mediated LL-37 induction as an alternative to exogenous peptide administration, the standard protocol involves treating monocytes, macrophages, or epithelial cells with 1,25-dihydroxyvitamin D3 at concentrations of 10 to 100 nanomolar for 24 to 48 hours, followed by quantification of LL-37 mRNA by RT-qPCR and protein by ELISA or western blot. This approach studies the physiological regulation of endogenous LL-37 production and is relevant to vitamin D supplementation strategies for infection prevention. Common controls for LL-37 experiments include scrambled LL-37 (sLL-37, a peptide with the same amino acid composition but randomized sequence) as a sequence-specificity control, heat-denatured LL-37 (boiled for 10 minutes) as a structural control, and polymyxin B or colistin as positive controls for membrane-targeting antimicrobial activity. For immunomodulatory experiments, include lipopolysaccharide as a positive control for innate immune activation and synthetic TLR ligands (CpG for TLR9, imiquimod for TLR7) for nucleic acid-sensing pathway controls. In summary, LL-37 is a versatile research tool with applications spanning antimicrobial testing, immunology, wound healing, and cancer biology. Success in LL-37 research requires attention to peptide quality, proper handling to minimize adsorption and degradation, appropriate assay conditions that account for salt and serum sensitivity, and rigorous controls to distinguish specific LL-37 effects from nonspecific cationic peptide activities.