Human Cathelicidin and LLKKK18 Derivative

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Figure 2.6 – Mechanisms of LL37 antimicrobial actions.

LL37 can also exert antimicrobial activity indirectly through immunomodulation (Figure 2.7), by acting as a chemoattractant of immune cells to the infected or damaged sites and influencing the production of inflammatory mediators ⁶⁹. Cathelicidin acts on, and is expressed by, several cell types, particularly epithelial and immune cells: neutrophils, epithelial, dendritic, monocytes and macrophages, mast cells, natural killer cells, MSCs, and even lymphocytes, acting as a bridge between the innate and adaptive immune systems ^69,70. The peptide can stimulate the differentiation of immune cells and the secretion of proinflammatory cytokines, chemokines, or costimulatory factors, synergizing with other active substances to promote immune responses. Both pro-inflammatory and anti-inflammatory responses may be stimulated, depending on the microenvironment. Although immunomodulation is not subject of study of this thesis work, it is a noteworthy rational basis for cathelicidin use in therapy design.

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Figure 2.7 – Immunomodulatory actions of LL37 ⁶⁹.

Of note, although regarded as less prone to bacterial resistance than antibiotics, prolonged bacterial exposure to LL37 have shown to result in increased MRSA tolerance ⁷¹. However, more understanding is needed, and the use of cathelicidin in novel treatment approaches is worthwhile, given its superiority compared to other agents. Indeed, cathelicidin is proposed as a smart option for bone infections therapy for holding potent antistaphylococcal activity ^72,73, the most causative species in bone infections ^25,74-76, for inhibiting biofilm formation ^72,77 and LPS activity ⁷⁸. The bactericidal effect of LL37 against both extra and intracellular S. aureus has been reported as being even superior to conventional antibiotics in eliminating intracellular bacteria ⁷³. Additionally, owing to a pro-angiogenic activity ^79-81, and stimulation of the migration of undifferentiated rat stem cells, the peptide has demonstrated to promote wound healing ^80,81 and bone regeneration ^82,83 in vivo.

The first-in-human trial using LL37 as a topical treatment for chronic leg ulcers demonstrated safety and markedly healing rates ⁸⁴. LL37 has entered phase II clinical studies for further investigation of its antimicrobial activity and its ability to modulate inflammation and healing of diabetic foot ulcers (NCT04098562). Moreover, a 24-aminoacid cathelicidin-derived AMP, OP-145 ⁸⁵, has successfully completed a safety and tolerability study in patients with chronic otitis media and has initiated phase II clinical trial in chronic middle ear infection (ISRCTN 84220089). Authors claim OP-145 offers benefits to antibiotic-unresponsive patients, opening a window of acceptance for novel peptide products designed with improved mechanism of action. LL37 has also been used as novel therapy for melanoma delivered as intratumorally injections (NCT02225366) ⁸⁶, and significantly improved symptoms of COVID-19 patients by oral administration of capsules bearing Lactococcus lactis genetically modified to produce

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LL37 ⁸⁷. However, LL37 exogenous administration is discouraged by a costly long peptide length and early degradation, requiring higher doses and frequent administration.

LLKKK18 (LL18) is an 18-lenght amino acid peptide designed from LL37 displaying higher cationicity and hydrophobicity. This results in higher attachment to pathogen membrane (i.e. higher antimicrobial activity), being three-fold more effective in the killing of mycobacteria than LL37 ⁸⁸, exhibiting higher chemoattractant activity, decreased toxicity and biding to plasma protein ⁸⁹. At neutral pH, LL37 is amphipathic and cationic with a net charge of +6 and 37.8 % hydrophobic amino acids, whereas LL18 has +8 and 44.4 %, respectively. In solution, they adopt α-helical structure (Figure 2.8). LL18 retains most of the LL37 residues involved in peptide–lipid (i.e. bacterial membrane) and in peptide–peptide interactions ⁶⁴.

Figure 2.8 – (A) Amino acid sequence projections for LL37 and its LLKKK18 (LL18) peptide derivative, N-terminal (left) to C-terminal (right). To increase cationicity, Q22, D26, and N30 in LL37 were replaced by K22, K26, and K30 LL18. (B) α-helical peptide structures N-terminal (bottom) to C-terminal (up):

LL37 has 37.8 % hydrophobic amino acids and a net charge of +6 at pH 7, whereas LL18 has 44.4 % and +8, respectively. At left, hydrophobic residues are shown in red, hydrophilic residues are shown in white, at right positive charges are highlighted in blue and negative charges in red (PyMOL software).

As an in vivo demonstration of the angiogenic and chemoattractant potential, LL18 has been reported to improve burn wound healing in a rat model, by topically applying a dose of 15 µg of LL18 every 3 days during the first 9 days post-burn ⁹⁰. Treatment with LL18 increased VEGF, and microvessel development,

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reduced oxidative stress and inflammation, specifically, displaying low neutrophil and macrophage infiltration and pro-inflammatory cytokines levels, and induced more collagen deposition. However, the peptide showed no improvements in the regeneration of cranial defects in a goat model ⁹¹, which advocates the importance of more in-depth studies. Early degradation, a common limitation, may diminish its actions in vivo, particularly in the case of a single administration. LL18 also increased tissue damage as a function of concentration in an OM rat model ⁹², revealing another critical parameter. Nevertheless, LL18 is mostly unexplored. While manufacturing costs can be reduced by shorter derivatives, toxicity and early degradation may be circumvented by sustained delivery platforms.

2.3.1 Encapsulation Mechanisms

Numerous nanocarriers have been designed for the delivery of cathelicidin by a range of mechanisms, particularly tailored for antimicrobial and tissue healing applications. The outcomes may vary in encapsulation efficiency (EE) %, ease of preparation, peptide stabilization and prolonged release, all influencing the final peptide biological activity. LL37 was entrapped into chitosan NPs through strong hydrogen bonds with 86.9 % EE ⁹³. NPs were prepared by ionic gelation of chitosan cross-linked by multivalent anions (sodium tripolyphosphate), displaying 68 % biofilm formation inhibition compared to the free peptide. In another work, using the same approach, chitosan NPs had an EE of 78.52 %, enhanced stability under thermal, salts, and acidic pH, subsequently improving MRSA infection and wound healing in rats ⁹⁴. In another approach, LL37 was attached onto the surface of a covalently conjugated vancomycin-NPs formulation, with a loading efficiency of 66.5 % ⁹⁵. The peptide was adsorbed by blending with NPs, owing to their high surface area and suitable pore size, resulting in efficient anti-staphylococcal activity, and excellent wound healing in a mouse model. LL37 was incorporated in the inner water phase of poly(lactic-co-glycolic acid) acid (PLGA) NPs by W/O/W emulsion–solvent evaporation technique, with an EE of 70.2 % and drug loading (DL) of 1.02 (μg LL37/mg NP), showing a sustained in vitro release for 14 days ⁹⁶. Intradermal injection of LL37-PLGA-NPs displayed significant higher collagen deposition, re-epithelialized and neovascularized composition, resulting in complete wound closure after 13 days, unlike the free peptide condition. It increased angiogenesis, up-regulated IL-6 and VEGF expression, as well. In another approach, cationic LL37 was electrostatically attached to a negatively-charged lipid-based nanocarrier by simple mixing, with an EE of 60 % at pH 3.0 ⁹⁷. This pH-sensitive construct, could associate LL37 at pH ≤ 4.5, but dissociated it at pH 6.0, which may hinder its application in certain scenarios. Other nanocarriers may retain LL37 at higher pH, and release it at acidic

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pH, as convenient. Electrostatic attraction is a simple preparation method offering a pH-sensitive triggered release.

Chemical conjugation is another usually employed mechanism. Lipid nanocapsules were used to compare three different peptide incorporation strategies: peptide encapsulation, surface adsorption and covalent attachment ⁹⁸. Three AMPs were used as models, including LL37, and compared in terms of EE, peptide antimicrobial activity and protection against proteases. Adsorption onto the surface of carriers, implying predominantly electrostatic, but also Van der Waals or hydrophobic interactions, provided partial protection, resulting in a more potent antimicrobial activity. Encapsulation into reversed micelles by phase inversion process, provided higher stability and a higher EE that culminated in the preservation of peptide activity. Covalent conjugation by transacylation, linking the pegylated hydroxystearate from the carrier shell and the functional amino groups of peptides, exposing the peptide at the surface, resulted in peptide inactivation, despite a high EE. Although other nanosystems may behave differently, this study provides a useful insight on the critical influence of different LL37 loading mechanisms in functional biological outcomes (Table 2.1).

Table 2.1 – Summary of the outcomes influenced by three different LL37 loading mechanisms ⁹⁸.

Formulation EE

General Antimicrobial

activity

Stabilization against proteases

MIC S. aureus

(µg/mL)

MIC MRSA (µg/mL)

Adsorption + (34.6 %) + + 8–16 4

Encapsulation ++ (99.4 %) + ++ 32 8

Conjugation ++ (82.5 %) – ND >128 >128

Free LL37 * * 8–16 8–16

EE, encapsulation efficiency; MIC, minimal inhibitory concentration; MRSA, methicillin-resistant Staphylococcus aureus; ND, not determined; (–) indicates absence; * a symbol (+/–) was not assigned by authors. LL37 MIC varied between 8 to 16, and displayed considerably lower stabilization against protease compared to all loaded LL37 formulations.