Melittin, the main component of bee venom, hyaluronidase and PLA
2 are the three major causes of allergic reactions to this venom [
38]. An amphiphilic peptide comprising 26 amino acid residues, and in which the amino-terminal region is predominantly hydrophobic and, the carboxyl-terminal region is hydrophilic. Melittin is the principal active component of apitoxin and is responsible for breaking up and killing cells. When several melittin peptides accumulate in the cell membrane, phospholipid packing is severely disrupted, thus leading to cell lysis [
39]. Melittin triggers not only the lysis of a wide range of plasmatic membranes but also of intracellular ones such as those found in mitochondria. PLA
2 and melittin act synergistically, breaking up membranes of susceptible cells and enhancing their cytotoxic effect [
40]. This cell damage, in turn, may lead to the release of other harmful compounds, such as lysosomal enzymes from leukocytes, serotonin from thrombocytes, and histamine from mast cells, which can all lead to pain.
Although melittin is the most studied and known bee venom peptide, its development for clinical applications remains mainly in preclinical phases. At the moment of writing, no products for human use are available on the market. Some patents and promising studies have focused on bacterial and viral infections, immunologic adjuvants, rheumatoid arthritis, arteriosclerosis, cancer, and endosomolytic properties for drug delivery.
2.1.1. Antimicrobial Properties of Melittin for Therapeutic Use
Antimicrobial peptides (AMPs) have been widely studied as an alternative to conventional antibiotics, especially for the treatment of drug-resistant infections [
41]. Hundreds of AMPs have been isolated, and several thousand have been
de novo designed and synthesized. Despite displaying extensive sequence heterogeneity, most of these peptides share two functionally important features, namely a net positive charge and the ability to adopt an amphipathic structure. Melittin is considered to show strong antimicrobial properties and it also has hemolytic activity and marked allergenic properties. Early studies using individual peptide analogs of melittin showed that the initial step of the mechanism underlying the hemolytic and antimicrobial activity of this venom peptide involves interactions with the lipid groups of the membrane [
42]. The structural requirements for the action of melittin, its orientation, aggregation state, current view of pore formation, and also its various cellular actions are discussed in detail in an excellent review by Dempsey [
43]. Bruce Merrifield performed pioneering work on improving the features of antimicrobial peptides, shortening their sequences and increasing their activity. In particular, a hybrid undecapeptide derived from the well-known cecropin A and melittin was found to be sufficient for antifungal and antibacterial activities, while displaying low cytotoxicity [
44]. This hybrid version was later improved with retro and retroenantio analogs [
45]. Indeed, a patent of several active
d-peptides with antibiotic and antimalarial activity was even filed [
46]. Despite the therapeutic efficacy of antimicrobial peptides, their use is limited due to poor
in vivo bioavailability caused by instability, cytotoxicity, hydrophobicity, in addition, the cost production is an issue [
47]. In parallel to antimicrobial peptides for therapeutic use in humans, these peptides can be applied to fight economically important plant pathogens, which are currently one of the major factors limiting crop production worldwide [
48]. A library of linear undecapeptides derived from cecropin-melittin hybrids have been tested against phytopathogenic bacteria and patented for future use in phytosanitary compositions [
49]. In this regard, a promising peptide called BP76 has been identified for this purpose [
50].
2.1.7. Cancer Applications of Melittin
Many studies report that melittin inhibits tumor cell growth and induces apoptosis, thereby indicating a potential application of this venom peptide as an alternative or complementary medicine for the treatment of human cancers. A valuable review describing the mechanisms underlying the anticancer effects of melittin has been published [
65]. Cells in several types of cancer, such as renal, lung, liver, prostate, bladder, breast, and leukemia, can be targeted by melittin. It is well-known that melittin is a natural detergent with the capacity to form tetramer aggregates on membranes, which lead to disorders in the structure of phospholipid bilayers, changes in membrane potential, aggregation of membrane proteins, as well as the induction of hormone secretion [
66]. Furthermore, this membrane disruption directly or indirectly leads to alterations in enzymatic systems, such as G-protein [
67], protein kinase C [
68], adenylate cyclase [
69], and phospholipase A [
70]. Melittin can even inhibit calmodulin, a calcium-binding protein that plays a crucial role in cell proliferation [
71]. Tumoral cells expose anionic phospholipids, mainly phosphatidylserine, on the external leaflet of the plasma membrane [
72], and this feature can allow the preferential binding of cationic peptides, like melittin, relative to normal cells. Melittin studies with numerous types of cancer cells and
in vivo animal models have demonstrated its antiproliferative activity [
73,
74]. Furthermore, recent studies have demonstrated that melittin has anti-angiogenesis properties [
75,
76,
77].
However, when a therapeutic dose of melittin is injected in vivo, some side effects, such as liver injury and hemolysis, were observed. To minimize these emerging lesions in off-target tissues, the following three strategies have been designed: (1) conjugaton of melittin to an antibody or a targeting component; (2) development of shielded pro-cytolytic melittin systems; and (3) synthesis of melittin-transporting carriers.
With regard to the first approach, a melittin-based recombinant immunotoxin obtained by fusion of genes that encoded an antibody fragment derived from the murine monoclonal antibody K121 with an oligonucleotide encoding melittin was tested successfully
in vitro [
78]. Another study was based on a recombinant immunotoxin of melittin fused to an anti-asialoglycoprotein receptor (ASGPR) single-chain variable fragment antibody (Ca) which conferred targeting and ASGPR-specific cytotoxicity to hepatocellular carcinoma cells [
79]. Finally, a recent study characterized a CTLA-4-targeted scFv-melittin fusion protein as a potential immunosuppressive agent for organ transplant. In this regard, the selective cytotoxicity of the peptide construction was confirmed in preliminary biological activity assays [
80].
Related to the pro-cytolytic melittin, by taking advantage of tumor matrix metalloproteinase 2 (MMP2) overexpressed on cancer cell membranes, an MMP2 cleavable melittin/avidin conjugate was built. Melittin coupled to avidin becomes inactive, but when released from the conjugate it induces immediate cell lysis [
81]. A similar idea was published years later, this time using avadin, the latency-associated peptide (LAP) domain of the transforming growth factor beta (TGF-β). In this approach, LAP dimerization conferred latency to the MMP2-cleavable melittin-LAP fusion protein [
82].
Regarding pro-cytotoxic melittin systems, a design was based on the mixture of melittin with the anionic detergent sodium dodecyl sulfate formulated into poly(D,L-lactide-co-glycolide acid) nanoparticles by an emulsion solvent diffusion method. The inhibitory
in vitro effects of these 130 nm-diameter melittin-loaded nanoparticles on breast cancer MCF-7 cells were promising [
83]. Another interesting carrier was a pegylated immunoliposome coupled to a humanized antihepatocarcinoma single-chain antibody variable region fragment and loaded with a bee venom peptide fraction [
84]. A similar pegylated immunoliposome but using only melittin as cargo and the complete antibody trastuzumab as targeting component was designed to combat HER2-overexpressing human breast cancer cell lines [
85]. The three aforementioned nanoparticles are not suitable for systemic administration because melittin can be released in blood vessels during transport, particularly in liposomes, which can be disrupted by the lytic peptide [
86]. To overcome this drawback, Samuel A. Wickline’s group developed a perfluorocarbon nanoemulsion vehicle incorporating melittin into its outer lipid monolayer [
87]. This nanocarrier of approximately 270 nm in diameter presented favorable pharmacokinetics, accumulating melittin in murine tumors
in vivo and causing a dramatic reduction in tumor growth without any apparent signs of toxicity [
88,
89]. Finally, the most recent ultra-small diameter melittin-nanoparticle (<40 nm) successfully tested
in vivo with few side effects is the patented α-melittin-NP [
90,
91]. This nanoparticle comprise 1,2 dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) decorated with the hybrid peptide formed by peptide D-4F and melittin via a GSG linker, the peptide D-4F being a peptide that mimics a high-density lipoprotein (HDL) [
92].
2.1.8. Endosomolytic Properties of Melittin
The strategy of packing and carrying small interference RNA (siRNA) using a wide variety of systems for gene therapy has been increasingly followed in recent years. The efficiency mediated by these drug delivery systems is strongly dependent on their endosomal escape capability, otherwise the siRNA would be degraded in endolysosomes [
93]. One mechanism designed for endosomal release is the use of fusogenic peptides, which are generally short amphipathic sequences between 20 and 30 amino acids in length and capable of disrupting biological membranes at endosomal pH [
94,
95]. One of the first highly innovative studies using melittin consisted of reversibly masking the membrane-active peptide using maleic anhydride derivative [
96]. At neutral pH, the lysine residues of melittin were covalently acylated with anhydride, thereby inhibiting the membrane disruption activity of the peptide. Under acidic conditions such as those present within endosomes, the amide bond of the maleamate was cleaved, thus unmasking melittin. Similar studies performed by Ernest Wagner
et al. showed that melittin analogs with high lytic activity at acid pH enhance the transfection of oligonucleotides in cell cultures and in
in vivo mouse models [
82,
97,
98,
99]. Very recently, a derivative of melittin (p5RHH) was reported to successfully trigger siRNA release into the cellular cytoplasm [
100,
101]. The company Arrowhead Therapeutics is currently developing ARC-520 as a novel siRNA-based therapeutic to knock down the expression of viral RNAs of chronic hepatitis B virus. They describe the use of a coinjection of a hepatocyte-targeted,
N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugate siRNA (chol-siRNA) targeting coagulation factor VII [
102,
103]. Preclinical studies with animals as well as Phase I assays have revealed that melittin promotes delivery without generating anti-melittin antibodies. In March 2014, Phase II trials of ARC-520 were started for patients with chronic hepatitis B virus [
104].