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Cytotoxicity of Antimicrobial Peptides and Their Assemblies: The Case of Gramicidin D

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Ana Maria Carmona-Ribeiro, Yunys Pérez-Betancourt, Ricardo Márcio e Silva

Submitted: 20 October 2025 Reviewed: 01 December 2025 Published: 27 February 2026

DOI: 10.5772/intechopen.1014188

Cytotoxicity - Applications in Toxicity Screening IntechOpen
Cytotoxicity - Applications in Toxicity Screening Edited by Sonia Soloneski

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Cytotoxicity - Applications in Toxicity Screening [Working Title]

Dr. Sonia Soloneski

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Abstract

Bioactive molecules, such as antimicrobial peptides (AMPs), are ubiquitous in nature, eventually displaying the ability to synergize with drugs and other biomolecules. They are amenable to synthetic modifications for imparting targeting, self-assembly properties as a protection against proteolysis in vivo, or for facile combinations with drugs or other molecules through physical or covalent binding. Their broad biomedical applications mostly derive from the effective destabilization of microbial or mammalian cells. Among the oldest AMPs reported are the linear gramicidins and melittin, which are presently being investigated as promising anticancer peptides. This review provides an overview of novel formulations for AMPs and their broad perspectives in therapeutics.

Keywords

  • antimicrobial peptides
  • gramicidin D
  • self-assembly of AMPs
  • nanoparticles
  • combinations of AMPs and other bioactive molecules
  • synergism
  • anticancer activity for AMPs

1. Introduction

1.1 The importance of assemblies with antimicrobial peptides in therapeutics

Many mammalian antimicrobial or host defense peptides stimulate the cellular immunity of the host, aiding in the clearance of invading pathogens [1]. In pigs, Staphylococcus epidermidis colonization in wounds increases when antimicrobial peptide (AMP) activation is inhibited [2]. In mice, Salmonella virulence correlates with a natural resistance to AMP action [3]. In humans, Shigella infections correlate with downregulation of enteric cathelicidin and β-defensin-1 expression [4]. In transgenic mice, overexpression of a human AMP gene improves lung clearance of Pseudomonas aeruginosa [5]. In mammals, AMPs are usually called defensins due to their role as part of the innate immune system, ensuring protection against infection. Defensins and cathelicidin peptides act by forming pores in the cell membrane [6].

In biomedical research against cancer, AMPs have often been delivered using passive and/or active targeting [7]. Passive targeting can be achieved by targeting the endothelial cells, tumor cells, or the acidic environment of tumors. Passive targeting agents are mainly nanoparticles (NPs) (1–1,000 nm in size), which accumulate around the tumors with leaky vasculature due to enhanced permeation and retention (EPR) effects in tumors [7]. Active targeting is based on ligand-receptor binding, which improves selective ligand accumulation at targeted sites and thus discriminates between diseased and healthy tissues. The peptide RGD (Arg-Gly-Asp) is an example of a tumor-targeting peptide, which targets integrin receptors on several types of cancer cells [8, 9].

As an instance of EPR passive targeting, the cathepsin B-cleavable peptide FRRG was conjugated to doxorubicin (DOX), yielding the FRRG-DOX prodrug that assembled in aqueous solutions as NPs (213 nm mean diameter). These FRRG-DOX NPs displayed enhanced therapeutic efficiency in mice bearing human colon adenocarcinoma (HT-29). Since the HT-29 tumor overexpresses cathepsin B, the DOX moiety of the FRRG-DOX prodrug was only released in the tumor; thereby, the DOX toxicity against the host’s normal cells was prevented [10].

Folic acid (FA) moiety covalently bound to bioactive molecules or drugs actively targets the folate receptors, which are transmembrane proteins frequently overexpressed in cancer cells [11]. Another peptide construction for both passive and active targeting was based on the covalent binding of an FA moiety to the peptide EEYSV-NH2. The molecules FA-EEYSV-NH2, consisting of a target recognition site FA, a dipeptide linker, and a peptide, could self-assemble into NPs at pH 7.0 and nanofibers at pH 5.0. This endowed the peptide with dual targeting (the passive EPR and the active targeting) and improved therapeutic index due to specific internalization and toxicity toward cancer cells [12]. Figure 1 illustrates the pH-dependent assemblies of FA-EEYSV-NH2 both in the normal cell microenvironment as NPs at pH 7.0 and as nanofibers at pH 5.0. The modification of the self-assemblies from NPs to nanofibers, when pH decreased from 7.0 to 5.0, enhanced the retention and therapeutic index of the peptide specifically in the tumor.

Figure 1.

pH-responsive self-assemblies from the folic acid (FA)-modified peptide derivative FA-EEYSV-NH2. Reprinted from the study by Wang et al. [12] with permission. Copyright 2021 American Chemical Society.

Cancer and infectious diseases cause high mortality rates worldwide, and the discovery of new antitumor and antimicrobial drugs is still challenging for medicine, especially concerning the consequences of side effects [13, 14]. The use of targeting peptides appears as a promising strategy in this context. From this perspective, peptides act by reducing the amount of drug administered, increasing bioavailability, enabling specific targeting, and decreasing drug cytotoxicity against normal cells [15, 16].

AMPs often display a broad range of bioactivity useful in biomedical applications against pathogens and cancer. Both AMPs and anticancer peptides (ACPs) have been formulated as important therapeutic agents, which can either create pores in the cell membrane or hamper the functionality of a vital intracellular target [6]. ACPs, which induce tumor cell death, can modulate the immune response, induce apoptosis, and/or disrupt the cell membrane [15].

Peptides as immune modulators enhance antitumor immunity by activating CD4+ helper T cells and CD8+ cytotoxic T cells. Death receptor-activating peptides engage TNF-R1, Fas, and TRAIL-R2, leading to caspase-8 activation and triggering the extrinsic apoptosis pathway. Mitochondria-disrupting peptides induce mitochondrial membrane destabilization, resulting in caspase-3 activation and intrinsic apoptosis. In addition, membrane-disrupting peptides compromise the integrity of the cancer cell membrane, leading to the loss of the semipermeable character of the cell membrane. The mechanisms of action for ACPs leading to cancer cell death are illustrated in Figure 2 and have been comprehensively reviewed.

Figure 2.

Mechanisms by which anticancer peptides induce tumor cell death. Immune-modulator peptides enhance T cell responses, promoting CD4+ helper T cells and CD8+ cytotoxic T cells. Death receptor-activating peptides bind to TNF-R1, Fas, and TRAIL-R2, triggering caspase-8 activation and initiating the apoptosis pathway. Mitochondria-disrupting peptides destabilize mitochondrial membranes, leading to caspase-3 activation and apoptosis. Additionally, peptides can directly affect the cancer cell membrane as transmembrane channels or pore-forming compounds.

Against infections, the mechanisms by which AMPs exhibit nonlytic membrane activity have been explored [17, 18]. However, it is believed that AMPs penetrate the bacterial membrane and interact directly with internal cellular components such as DNA, RNA, and various enzymes essential for microbial survival and replication. This interaction disrupts metabolic homeostasis, ultimately resulting in bacterial death. AMPs’ sites of action, besides the cell membrane, can be components of intracellular processes essential for microbial survival, such as transcription, translation, cell wall synthesis, cell division, chaperone proteins, enzymes, and channel proteins. Non-membrane-targeting AMPs can also infiltrate bacterial biofilms effectively [19].

Self-assembly of AMPs into nanometer-scale structures can improve their antimicrobial activity, prolong their half-life, and increase their bioavailability. Six different AMPs with nine amino acid residues and central symmetry, containing triple tryptophan (WWW) or double tryptophan (WW) motifs, were tested, pointing out peptide K6 as the most potent against P. aeruginosa and S. aureus thanks to its ability to self-assemble into micelles. The K6 peptide also had potent action against biofilms formed by P. aeruginosa and S. aureus, revealing an increase in its lytic capacity against bacterial cell membranes. The reason why these peptides have this strong antimicrobial capacity is the fact that the WWW or WW motifs were placed in the middle or at the ends of the peptide to provide hydrophobicity and enhance self-assembly into micelles, facilitating their interaction with bacterial membranes and disruption of their integrity [19].

The structure–function relationship for antimicrobial peptides has been extensively reviewed for decades [20–26]. They have performed well regarding their desirable actions, such as membrane-perturbing [22], lytic [23–25, 27], oncolytic [25], antibacterial [20, 28], and/or biofilm-disruptive effects [26, 29, 30]. However, in vivo, common drawbacks have included general cytotoxicity, poor stability due to the proteolytic action of enzymes, and poor bioavailability due to rapid clearance. To circumvent these problems, novel formulations for AMPs have been proposed [31, 32]. In fact, there is still much to be learned and explored regarding the effect of the formulation on peptide action. Another promising area for research pertains to the similar lytic behavior of antimicrobial polymers and peptides [21]. In these systems, the lytic effect can also be controlled by the degree of freedom of the antimicrobial in the formulation [33].

Against cancer, two natural AMPs are among the oldest AMPs studied regarding their therapeutic effects and should be pointed out: melittin (MLT) [34] and gramicidin D (Gr) [35].

MLT is a 26-amino-acid amphiphilic cationic peptide found in the venom of the European honeybee. MLT has been described as a promising agent in anticancer treatment since the 1950s [36]. However, MLT’s indiscriminate lytic cytotoxicity in vivo has been a major drawback impeding its use in the clinic; MLT insertion into the lipid bilayer of membranes leads to the disruption of cells [13, 37, 38], which was reported to be effective even against solid tumors, resulting in oncolytic effects [39]. MLT was also reported to act via cellular apoptosis due to the lysis of mitochondrial membranes [40].

In tumors, due to the disruption of cancer cell membranes, MLT induction of tumor necrosis or apoptosis releases intracellular contents such as whole-tumor antigens and damage-associated molecular patterns (DAMPs) [41]. Recently, in nanometric formulations of MLT with lipid NPs targeted to lymph nodes (LNs), an important and systemic antitumor immune response was achieved; there was whole-tumor antigen release in situ and activation of antigen-presenting cells (APCs) in LNs, accompanied by improved antigen-specific CD8+ T cell responses. Experiments in mouse models with tumors revealed primary and distant tumor growth inhibition of 95% and 92%, respectively [42]. This α-MLT-NPs formulation was an effective LN-targeted whole-cell nano-vaccine against the tumor, representing a valuable immunotherapeutic tool.

The problem of targeting ACPs and reducing their indiscriminate action against all host cells was also addressed using anticancer polymeric prodrug candidates of AMPs [43]. Poly(ethylene glycol) (PEG) moiety in polymer conjugates with a derivative of the P18 peptide, namely, the D–P18 (D–P18 peptide was synthesized from D-amino acids and modified with a leucine residue at position 8), induced the mitochondria-associated pathway of apoptosis in pancreatic carcinoma and cervical cancer cell lines [44]; pegylated prodrugs of the D–P18 also displayed this same mechanism of action [43]. Consistently, an α-helical peptide derived from magainin 2 was pegylated and had its antimicrobial activity reduced and cytotoxicity against CHO-K1 cells abolished, as expected for a prodrug [45]. Oncolytic peptides can also be synthesized from modifications or stapling of other existing peptides, such as the Ano-3/3s peptide produced from wasp venom, which is resistant to proteases, is membranolytic, and induces prolonged immune responses against melanoma cells [46]. In these examples, the prodrugs release the drugs, such as the AMP and doxorubicin, by hydrolysis of the PEG moiety inside the cancer cell.

In another interesting development, pegylated lipids self-assembled as lipodisks [47] for dual delivery of anticancer drugs and the oncolytic peptide MLT; synergistic action for some combinations was obtained [48]. The lipodisks could also carry ligands capable of high-affinity binding to cell-surface receptors upregulated in cancer cells [49]. High doses of added doxorubicin (96.1%) could be carried by the DOX-nanodisks, which showed a pH-dependent release of the drug [50].

In fact, the urgent need to develop carriers for hydrophobic peptides or drugs useful in anti-infective therapy (e.g., amphotericin B) [51] and cancer (e.g., paclitaxel) has been recognized [52]. Cationic lipid disks have been revealing their potential as carriers also for hydrophobic peptides (e.g., Gr) [53, 54]. Pegylated cationic lipids assembled as nanodisks incorporated up to 2.5 mol% paclitaxel and showed significantly higher blood half-life, tumor penetration, and proapoptotic activity, suggesting that PEG coverage of the nano-formulations improved their pharmacokinetics and therapeutic efficacy [52].

ACP-loaded PEG-PLGA NPs, 20 nm in diameter, facilitated the release of the membrane-lytic peptide L-EEK and its perforating action on tumor cells, as demonstrated using fluorescently labeled peptides and measuring intracellular peptide delivery in four different breast cancer cell lines. Mice that received EEK-NP treatment showed significantly inhibited tumor growth, with two of the four treated mice exhibiting complete tumor eradication [55]. In this same work, cancer cell-selective membrane perforation was achieved, with a 200-fold selectivity over non-cancerogenic cells and superior cytotoxicity compared to doxorubicin against breast cancer tumor-spheres.

Combination therapies of two anticancer agents with different mechanisms of ac-tion, despite their complex development, are less prone to drug resistance than treatments with single drugs [56]. For example, combining cancer-growth inhibiting (CGI) siRNA for oncogene silencing with an oppositely charged selective anticancer peptide (SAP), the CGI siRNA complex not only enhanced the stability and delivery efficiency of CGI siRNA but also exhibited a synergistic anticancer effect, as shown from viability studies of specific cancer cell lines and in vivo studies with animals bearing tumors [57]. The activity of AMPs with selective antitumor mechanisms has been reviewed [58].

In an interesting biomimetic anticancer approach, a construction based on self-assembled NPs with amphiphilic penetrating Zein protein from corn and a rabies virus glycoprotein carrying temozolomide (TMZ) was able to treat glioma, the most frequent brain cancer. The self-assembled amphiphilic NPs penetrated the blood–brain barrier (BBB), targeted the tumor with the virus glycoprotein, and released TMZ. These NPs were considered ideal for treating brain diseases due to their biocompatible character, BBB penetration, targeting to the brain, and nanometric size [59].

Several lytic peptides with activity against cancer cells have been reported [23, 60–62]. For example, a short cationic peptide composed of D- and L-leucines, lysines, and arginines displayed selective toxicity toward cancer cells and prevented the formation of lung metastases in mice with no detectable side effects; the mechanism of action involved membrane depolarization of cancer cells at a few micromolar concentrations. The simple peptide structure, high solubility in water, and resistance to degradation in vivo made it a good candidate for treating cancer [61]. In general, lytic anticancer peptides empowered by self-assembly [63], cyclization [64], or diastereoisomerism from the manipulation of the amino acid sequence [65], novel nanoformulations for peptide loading [20, 32, 54, 55, 66] and conjugation of peptide drugs to natural or synthetic polymers have been improving their prospects for use in anticancer therapy.

Peptides in novel supramolecular assemblies may become essential in vaccine de-sign, antimicrobial chemotherapy, cancer immunotherapy, food preservation, organ transplants, design of novel materials for dentistry, formulations against diabetes, and other important applications; often, their therapeutic index can be improved by protecting their activity and increasing their bioavailability [20]. The potential use of AMPs in biomedical applications goes beyond their direct antimicrobial activities for the treatment of antibiotic-resistant infections [67]. Combinations of peptides with lipids, liposomes, NPs, polymers, micelles, and so on, within the limits of nanotechnology, may also provide novel applications going beyond antimicrobial therapy for infectious diseases [68]. The current methods and formulations to enhance the half-life of peptide drugs and improve peptide drug delivery have been reviewed [69].

The LTX-315 is an oncolytic peptide capable of altering the tumor microenvironment (TME) and inducing immune-mediated antitumor action [70]. The TME often hampers the penetration and activity of T-cells that are capable of combating the tumor [71]. Combinatorial strategies have been proposed, such as oncolytic virotherapy, wherein genetically modified or naturally occurring viruses can infect and replicate selectively in cancer cells, inducing not only lysis but also immunogenic cell death (ICD) [72, 73]. Upon oncolytic cell disruption, tumor-associated antigens are released and taken up by dendritic cells, which activate specific antitumor T-cells. Furthermore, the lysis of cancer cells releases factors known as DAMPs that activate ICD. Recently, oncolytic viruses encoding tumor antigens and tumor antigen-decorated adenoviral platforms have been used as cancer vaccines, eliciting local and systemic antitumor responses in a poorly immunogenic melanoma mice model [74]. Additionally, new branched oncolytic peptides, BOP7 and BOP9 have been shown to elicit the release of DAMPs, mediators of ICD, in pancreatic cancer cells [75]. These peptides selectively killed tumor cells but not cells of non-tumor origin. This result was interpreted as being due to their repeated cationic sequences, which enable multivalent binding to heparan sulfate glycosaminoglycans that display multiple anionic charges on cancer cells. BOPs triggered the release of DAMPs by dying cells, particularly HMGB1, IFN-β, and ATP. In vivo, nude mice demonstrated a 20% inhibition of tumor grafting and growth in pancreatic cancer.

Peptide-based strategies for cancer treatment have been reviewed [14–16, 76–78].

Major therapeutic limitations of ACPs and AMPs, such as production cost, biodegradability in vivo, rapid clearance, and their similar cytotoxicity to normal and transformed cells, have been circumvented by appropriate peptide design and novel formulations, thanks to advances in materials science, molecular biology, and nanotechnology, as discussed in the next sections [79].

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2. Overcoming limitations of ACPs and AMPs: The power of assemblies, combinations, and formulations in cancer and infection therapeutics

Supramolecular assemblies with ACPs and AMPs peptides have been designed based on four different basic strategies: (1) peptide self-assembly; (2) peptide incorporation in carriers; (3) design of peptide libraries and screening for selection of maximal desired activity; (4) peptide combination with other bioactive molecules aiming at synergistic action, achieving maximal activity. AMPs can both self-assemble into a variety of architectures such as nanospheres, nanofibers, nets, sheets, tubes, or hydrogels [80] and/or combine with other bioactive molecules to avoid biodegradation, allow targeting to the desired site of action, and reduce production costs and dosages through tailored design of novel formulations [81]. For example, cyclic peptides functionalized through conjugation to polymers generate peptide–-polymer nanotubes able to insert into membranes, act as ion channels, deliver anticancer drugs or genetic materials, or provide applications as antivirals [82]. Sophisticated techniques such as super-resolution microscopy with modern image analysis, small-angle neutron scattering, and solid-state nuclear magnetic resonance have allowed the characterization of nanostructures from short peptides, which have strategic applications in biomedicine and nanotechnology. They can function as antimicrobials, anticancer agents, vehicles for controlled drug release, and responsive cell culture materials [83].

In water solution, peptides adopt a conformation of minimum free energy under equilibrium conditions. Intermolecular weak interactions between peptides in water solution often drive their self-assembly in solution. These interactions involve hydrogen-bonding, π–π, cation–π, electrostatic, hydrophobic, and van der Waals interaction forces. The thermodynamics and kinetics of the self-assembly process for short peptides were comprehensively reviewed [84]. These physical interactions, acting cooperatively, can be reasonably long-ranged and able to yield energies equivalent to a weak covalent bond [85].

The self-assembly of peptides can provide interesting novel materials, such as “hydrogels.” Although classic hydrogels have been made from high molecular weight natural polymers, such as proteins (e.g., gelatin), peptides as building blocks for gels are similarly suitable for obtaining these soft, biodegradable, and biocompatible materials. Peptide-based hydrogels have been used as scaffolds for wound healing, drug and biomolecule release, cell culture, and tissue engineering [86]. The many applications of peptide fibers assembling as gels have been the subject of excellent reviews [86–88]. Bioactive hydrogels based on peptides have also been used for the delivery of antibiotics, AMPs, cationic polymers, photosensitizers, and nanomaterials for photodynamic or photothermal therapy. Multiple wound-repairing effects can be obtained from hydrogels, such as hemostasis, adhesion, wound contraction and closure, antimicrobial effects, anti-oxidative effects, and others [89].

Lysine-based dendritic hydrogels, capable of managing severe trauma or intraoperative bleeding, have been synthesized [90]. Oxidized carboxymethylcellulose (OCMC) and third-generation lysine “peptide dendrimers” (OCMC/G3KP) demonstrated inherent hemostatic ability, antibacterial properties, and high adhesiveness. In vivo, this hydrogel promoted superior wound healing compared to conventional sutures. The oxidation of hydroxyl groups on carboxymethylcellulose (CMC) produced aldehydes on the OCMC polymer chain, which could covalently bind to amino moieties on the wounded tissue, resulting in the observed strong adhesiveness, whereas dendritic poly(lysine) accounted for the high bactericidal efficiency of the hydrogel [91].

Another interesting development has been the hydrogel-based delivery of growth factors, which are important therapeutic agents for tissue engineering and regenerative medicine. Growth factors require protection for in vivo delivery due to side effects and rapid degradation. These limitations have been circumvented by novel hydrogel formula-tions for growth factor delivery [92]. For example, the IKVAV sequence is a biologically recognized peptide epitope from laminin, a protein from the extracellular matrix in the central nervous system. This isoleucine–lysine–valine–alanine–valine sequence was modified by adding aspartic acid to the N-terminus of the peptide, adjusting the apparent pKa of the peptide molecule to ensure that the pH-driven self-assembly would take place at physiological pH. This self-assembled peptide (DIKVAV) formed “nanofibers” (∼10 nm in diameter), and the resulting “hydrogel” was used to stabilize and deliver growth factors such as the glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) [93].

Peptide self-assembly directly depends on the intervening medium. For example, in toluene, short phenylalanine dipeptides made of only two phenylalanine residues self-assemble as “gels,” where the nanofibrils are interconnected by hydrogen bonding. On the contrary, in ethanol, the π–π stacking of phenyl moieties between chains favors the formation of “microcrystals” [94]. In the gel, the molecules adopt antiparallel β-sheet structures, whereas in the crystals, parallel β-sheets occur [94].

Natural AMPs, such as Gr and MLT, also interact in different microenvironments to yield a variety of assemblies [31, 95]. Both Gr and MLT interact with the plasma membranes of eukaryotic cells. Their interaction with the membranes of erythrocytes changes permeability, allowing the passage of ions. A similar mechanism of action was reported for staphylococcal delta-toxin, in which toxin molecules embed themselves in the membrane, where they assemble as “transmembrane channels” [95, 96].

Membrane damage in erythrocytes by MLT involved the accumulation of the peptide molecules predominantly in the outer half of the lipid bilayer. This accumulation induced deformation of the erythrocyte into crenature, destabilizing the membrane structure and releasing membrane fragments; this mechanism of action was similar to that of surfactants [97]. At the very low lipid-to-peptide molar ratio of 50, MLT was found to form pores of 2.5–3.0 nm in diameter in palmitoyl oleoyl phosphatidylcholine (POPC) vesicles [98]. More recently, the therapeutic potential of MLT for treating cancer has been explored, thanks to optimized assemblies this peptide can yield with biocompatible carriers. MLT in stable “supramolecular complexes” of ≈60 nm with the photosensitizer chlorin e6 (Ce6), further coated with hyaluronic acid, displayed reduced hemolysis and selective cytotoxicity against cancer cells. There was a “synergism” of action for MLT’s lytic effect and photosensitization of chlorin e6, leading to increased penetration of the complexes in the tumor [99].

Based on artificial molecular design, protein folds unseen in nature have been created [100]. Interestingly, two-dimensional giant nanosheets were obtained from the self-assembly of d/l-alternating cyclic peptides [101]. These giant nanosheets represent one of the largest two-dimensional (2D) supramolecular materials ever made. Figure 3 illustrates the self-assembly of cyclic peptide nanotubes to yield giant nanosheets; the model for the sequential one-dimensional (1D)-to-2D self-assembly of cyclic peptide CPx involved the secondary parallel β-sheet conformation between CPx backbones within nanotubes, stabilized via eight hydrogen bonds, and the tertiary 1D self-assembly of CPx as amphiphilic peptide nanotubes with hydrophobic (Leu–Trp–Leu in yellow-gray) and hydrophilic (Glu–His–Gln in red-blue-green) surfaces. The 2D self-assembly of CPx into nanosheets comprising nanotube bilayers with hydrophobic cores, composed of leucine zippers (yellow) and tryptophan stacks (gray), and polar residues on their surface can be seen in Figure 3 [101].

Figure 3.

Nanosheets assembled from nanotubes of the cyclic peptide CPx. (a) The chemical structure and self-assembly of the cyclic peptide CPx in water. (b) Formation of amphiphilic nanotubes based on the multiple hydrogen-bonding interactions between the peptide rings. (c) Formation of 2D nanosheet assembly by the amphiphilic nanotubes. Reprinted from the study by Insua and Montenegro [101] with permission. Copyright 2020 American Chemical Society.

Other cyclic AMPs have been described as biosurfactants (BSs). Microorganisms often synthesize a wide range of BSs [102]. For example, the cyclic lipopeptide (CLP) surfactin is a BS that downregulates the expression of several cell surface molecules such as CD40, CD54, CD80, and major histocompatibility complex class II (MHC-II), finding applications for treating autoimmune diseases [102]. BS interactions with cell membranes lead to antimicrobial, antiviral, and anticancer properties for these molecules [103]. Among the BS, CLPs are synthesized and secreted by Bacillus and Pseudomonas. CLPs identified in the genus Bacillus include surfactins, iturins, lichenysins, and fengycins [104, 105]. For example, the self-assembly of a CLP mixture secreted by a Bacillus megaterium strain yielded negatively charged and large aggregates (300–800 nm of mean hydrodynamic radius) [104, 106]. Interestingly, increasing pH and the aggregates’ negative charge promoted disaggregation, leakage of intracellular phosphorylated compounds of Bacillus cereus, and increased CLP lytic power. In addition, removing the cyclic character of the CLP by hydrolysis of the lactone or lactam rings abolished the antibiotic activity [104]. Given the natural functions of lipopeptides from Bacillus and Pseudomonas, they have been recognized as more than surfactants and antibiotics [107]. They are a kind of primeval defense mechanism of a given microbe against competitors in the same niche.

The cyclic peptide cyclo-Histidine-Histidine (Cyclo-HH) yielded noncovalent assemblies with a variety of anticancer drugs; these assemblies were evaluated both theoretically (via molecular dynamics simulations) and experimentally [108]. Most drugs tested required zinc and nitrate to form co-assemblies with the peptide, displaying bio-compatibility and successful intracellular release, except for cisplatin. Drug encapsulation and release were higher for epirubicin, doxorubicin, and methotrexate compared to mitomycin-C and 5-fluorouracil.

Macrocyclic peptides have been studied as alternatives to monoclonal antibodies (mAbs) capable of reversing the immunosuppressive effect of cancers on the oncolytic immune response by T cells [109]. Instead of using entire mAbs, peptides were employed to block the PD-1/PD–L1 pathway and its immune-suppressive effect, which is associated with cancers. PD-1 stands for programmed death 1 protein, and PD–L1 stands for programmed death ligand 1, representing a very important alternative in immunotherapy against cancer. For example, a series of macrocyclic peptides have been developed that exhibit binding strengths to PD–L1 over a range of sub-micromolar to micromolar concentrations; the macrocyclic PD–L1-targeting peptide, pAC65, displayed a potency equivalent to the current FDA-approved mAbs for cancer immunotherapy [109]. Compared to mAbs, peptides are smaller in size, can have their synthesis easily scaled up in the biotechnology industry with reproducible results in different batches, display lower immunogenicity, and have a longer shelf life; the use of peptides in cancer therapy was recently reviewed [110], as was the design of supramolecular assemblies from cyclic peptides [82].

Interestingly, the self-assembly of the cationic and anticancer killerFLIP peptide [62] yielded improved selectivity against cancer cells [111]. Reducing the exposure of peptide hydrophobic moieties from peptide self-assembly in water solution diminished its affinity toward normal cell membranes but improved the electrostatic attraction of the aggregates toward cancer cells [111]. In fact, cationic AMPs are more strongly attracted to cancer cells than to normal cells due to the higher frequency of negative charges of heparan sulfate on cancer cells [112]. The killerFLIP peptide sequence is GRKKRRQRRRFFWSLCTA, displaying practically two moieties: the one on the left with cationic residues and the one on the right with hydrophobic residues. This led to the aggregation of peptide monomers in water, hiding the apolar amino acids and exposing the charged ones to the water phase. The self-assembly of the killerFLIP peptide reduced its selectivity toward normal eukaryotic cell membranes but improved its binding to cancer cells. The effect was explained by considering that cancer cells bear a much higher negative charge than normal cells, which are practically neutral; the hydrophobic attraction between the peptide and the normal cells is reduced due to the inner localization of the hydrophobic moieties in the aggregate [111].

In the next sections, we discuss the importance of self-assembly and synergistic combinations of AMPs with other bioactive molecules. As a case study, novel formulations for Gr and their assemblies are reviewed.

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3. Cytotoxicity of Gr in novel formulations based on self-assembly and/or combination with other bioactive molecules

Gr was first isolated by Dubos from soil samples containing Bacillus brevis [113]. It is a channel-forming polypeptide with 15 L- and D-amino acid residues, including a β-helix secondary structure with an internal pore. Gr molecules combine to form homodimers with β-helix pore structures spanning the cell membrane.

As models for membrane proteins, linear gramicidins have been extensively characterized regarding their organization, dynamics, and function. Gr pores in membranes, resulting from self-assembled dimeric channels, have been described in lipid bilayers, model membranes, and natural membranes [114–119]. Upon insertion into membranes, Gr molecules acquire a helix conformation, and Gr dimeric channels become pores for single-file passive diffusion of Na+ and K+ cations, with Na+ influx and K+ efflux from cells. Gr dimeric channels are cation-selective, form pores in model membranes, and conduct around 107 ions per second [120]. Natural Gr consists mostly of gramicidin A (85%), which has four tryptophan residues at positions 9, 11, 13, and 15, imparting intrinsic fluorescence to the molecule [121]. The hydrophobic amino acid residues in the Gr molecule account for its low solubility in water and drive Gr self-assembly in water dispersions as NPs with about 150 nm of mean hydrodynamic diameter [31, 32, 122–124].

Gr displayed a strict dependency on the formulation for bioactivity [20, 31, 32, 53, 54, 66, 125]. In combination with complementary antimicrobials, such as the cationic polymer PDDA, synergism of action was observed. This complementary action involved PDDA’s ability to withdraw biopolymers from the microbial cell wall, opening the way for Gr NPs to interact with the inner cell membrane. PDDA disrupts the cell wall, and Gr NPs span the cell membrane as dimeric channels [31, 32, 122, 123].

Figure 4 illustrates the activity of Gr NPs. Figure 4a shows the Gr NPs water dispersion in a glass assay tube (insert) and the scanning electron micrograph of these NPs. Figure 4b shows nanodisks displaying an outermost layer of PDDA and a high microbicidal activity against multidrug-resistant P. aeruginosa [126]. Coatings on glass surfaces made by casting and drying combinations of Gr NPs and the nanodisks in dispersion revealed the synergistic action of the combinations (see Figure 4e).

Figure 4.

Gramicidin D nanoparticles in aqueous dispersions and coatings. (a) Macroscopic and microscopic features of Gramicidin D dispersions in water, seen from photos as a turbid dispersion or visualized from scanning electron micrographs (SEM) as nanoparticles, as reprinted from the study by Pérez-Betancourt et al. [32]. (b) Nanodisks prepared from DODAB bilayer fragments (DODAB BF), surrounded by a layer of carboxymethylcellulose (CMC) and an uppermost layer of poly(diallyl dimethylammonium bromide) (PDDA), reprinted from the study by de Melo Carrasco et al. [126]. (c) SEM images for mixtures of PDDA (1.0 mg/mL) and P. aeruginosa multidrug-resistant (MDR) bacteria (8.9 × 105 cells/mL), reprinted from the study by de Melo Carrasco et al. [126]. (d) Coatings cast on glass cover slips from aqueous dispersions of Gr nanoparticles and DODAB BF/CMC/PDDA nanodisks. Reproduced from the study by Zaia et al. [122]. (e) Microbicidal activity of the coatings against bacteria and fungus. Reproduced from the study by Zaia et al. [122].

In our laboratory, novel formulations for Gr have been described based on its self-assembly as NPs in water dispersions [32, 124], formation of dimeric channels in closed, open, or supported cationic bilayers [53, 54, 66, 127, 128] or synergism of antimicrobial action with the cationic polymer poly(diallyldimethylammonium chloride) (PDDA) [33, 126, 129–136]. Gr NPs and the antimicrobial polymer PDDA displayed convenient synergistic action against bacteria and fungi both in water dispersions [32, 122] and in coatings [123]. Successful formulations for antimicrobial peptides involve not only their self-assembly property but also their combination with other bioactive molecules such as cationic lipids, surfactants, and polymers [31].

Similarly to other peptides, the conformation and self-assembly of Gr are highly dependent on the Gr microenvironment [53]. The Gr medium determined not only its conformation but also its self-assembly. Gr intrinsic fluorescence, circular dichroism (CD) spectroscopy, dynamic light scattering, and zeta potential analysis were essential for characterizing Gr conformations and self-assembled structures in different media [53, 54]. Additionally, bilayer phase transition analysis using extrinsic fluorescence and colloidal stability evaluation over time and varying NaCl concentrations could provide further insights into Gr behavior in water dispersions [53].

After describing the preparation and properties of dipalmitoyl phosphatidylcholine (DPPC)/dioctadecyl dimethylammonium bromide (DODAB) 1:1 large vesicles (LVs) [137], studying the Gr/LV interactions revealed that the Gr dimeric channel in LVs yielded CD and intrinsic fluorescence spectra similar to those in trifluoroethanol (TFE); these Gr channels were functional, allowing KCl or glucose permeation through the bilayer [53]. For Gr in the DPPC/DODAB bilayer fragments (BFs), the intertwined dimeric Gr conformation was evidenced by CD and intrinsic fluorescence spectra similar to those in ethanol. Both LVs and BFs protected Gr tryptophan fluorescence against quenching by acrylamide. However, the Stern–Volmer quenching constant was slightly higher for Gr in BFs, showing that Gr intrinsic fluorescence was more exposed to water in BFs than in LVs [53]. Figure 5 illustrates the dimeric channel and the intertwined assemblies of Gr in the closed and open DPPC/DODAB 1:1 bilayer, respectively.

Figure 5.

Assemblies of gramicidin D molecules in different microenvironments, such as lipid large vesicles (LVs) of cationic lipids or cationic lipid bilayer fragments (BFs).

Gramicidin’s effects on water/solute permeation through the DPPC/DODAB LV bilayer were evaluated using turbidity kinetics as a function of time. For the DPPC/DODAB 1:1 LV dispersions prepared in pure water and subjected to a final external concentration of 50 mM KCl or 100 mM glucose, the turbidity at 400 nm increased over time (see Figure 7) [53]. It was previously reported for similar cationic LVs that an increase in turbidity over time was due to vesicle shrinkage, whereas a decrease was associated with vesicle swelling [138–141]. Therefore, while the DPPC/DODAB LV dispersion responded to the hypertonic external solutions by shrinking, the LV/Gr dispersion exhibited vesicle swelling. The Gr channel inserted in the bilayer allowed the entrance of solutes, completely altering the bilayer permeability profile. The typically low permeability of cations and solutes through the DPPC/DODAB LV bilayer increased substantially upon incorporating 10% Gr. In Figure 6, KCl or glucose entered from the outer to the inner vesicle compartment, carrying water and dissipating the solute concentration gradient to the point of changing shrinkage into swelling.

Figure 6.

Turbidity kinetics for DPPC/DODAB large vesicles (LVs) prepared in pure water due to the establishment of KCl (●, ○) or glucose osmotic gradients through the LV bilayer (▲, ∆) in the absence (●, ▲) or in the presence of 10% gramicidin at 1.0 mM lipid and 25°C (○, ∆). Reprinted from the study by Carvalho et al. [53] with. Copyright 2020 Elsevier.

Synthetic cationic vesicles or BFs made of DODAB only were used in novel formulations for Gr [54]. The reason behind this approach was the significant antimicrobial properties of the DODAB cationic lipid [129, 134, 135, 141–144], its differential cytotoxicity [145], its thorough characterization regarding physico-chemical properties [138, 139, 141] and its versatility in combination with other bioactive molecules such as amphotericin B [130, 146–148], rifampicin [149], oligonucleotides [150], nucleic acids [151, 152], polysaccharides [131, 147], proteins [153, 154], biocompatible synthetic polymers [136, 144, 155–158], and antigens in vaccines [128, 150, 159].

In order to formulate Gr in the large bilayer vesicles (DODAB LV), a 1:10 gramicidin:DODAB molar ratio was used. Figure 7a shows the leakage of phosphorylated intracellular compounds induced by these formulations as a function of DODAB concentration. Figure 7b shows the changes in the morphology of bacterial cells in the presence of Gr/DODAB formulations. There are major morphological changes and the appearance of cellular debris, indicative of lysis.

Figure 7.

(a) Leakage of phosphorylated compounds (%) from bacteria for different dispersions at 3.7 × 108 to 4.8 × 109 CFU/mL (Escherichia coli) or 5.2 × 109 to 5.1 × 10¹⁰ CFU/mL (Staphylococcus aureus). The dispersions were tested over a range of DODAB or DODAB/Gr concentrations after one hour of interaction with both types of bacteria. The DODAB/Gr molar ratio was kept at 10/1. As a control, over a range of Gr concentrations (10⁻² to 10⁻⁴ mM), no leakage was detected from bacteria after the same interaction time. (b) Micrographs of Escherichia coli (A–C) or staphylococcus aureus (D–F) cells untreated (A, D) or treated with DODAB BF/Gr (B, E) or DODAB LV/Gr dispersions (C, F), obtained by scanning electron microscopy. Cells appear enlarged by 10,000 × (E. Coli) or 20,000 × (S. Aureus). Against E. Coli cells, in (B), DODAB = 0.005 mM (DODAB BF/Gr) and in (C), DODAB = 0.01 mM (DODAB LV/Gr). Against S. aureus, in (E), DODAB = 0.01 mM (DODAB BF/Gr), and in (F), DODAB = 0.05 mM (DODAB LV/Gr). Abbreviations: DODAB: dioctadecyl dimethylammonium bromide; LV: closed bilayers; BF: bilayer disks; Gr: gramicidin. Reproduced from the study by Regioto et al. [54].

Other Gr formulations have been proposed, involving graphene oxide [160], silver NPs stabilized with dodecane thiol [161], and fluoride for applications against biofilms on teeth [162]. In renal carcinoma, energy depletion in cancer cells induced by Gr led to cancer cell death [163]. In another instance, the systemic toxicity of Gr could be circumvented by intratumoral administration, which was reported to be both safe and effective in murine xenograft studies in the absence of significant side effects [164]. The selective fusion of cancer cell membranes with liposomes containing both Gr and the fusogenic peptide pHLIP® was an interesting strategy applied for Gr delivery to cancer cells [165]. This approach considered that one of the main differences between normal and cancer cells in most solid tumors is the low extracellular pH in cancer cells; the pH-selective transfer of Gr nanopores to cancer cells dysregulated the ionic balance of monovalent cations in the cell, inducing cell death at mildly acidic extracellular pH. Gramicidin channels inserted into the cancer cells open flux of protons into the cytoplasm and disrupt the balance of other monovalent cations across membranes, which induces cell apoptosis.

In a very interesting approach, the gramicidin A unimolecular channel was targeted to cancer cells by covalently binding a galactose molecule to the Gr A N-terminus; thereby, the conjugate containing the galactose moiety recognized the asialo glycoprotein on cancer cells, forming a unimolecular transmembrane channel and inducing cancer cell death by apoptosis [166]. In comparison to cationic AMPs that can be retained by the negative charges of heparan sulfate on cancer cells [112], Gr has the advantage of being a neutral peptide that inserts into cell membranes due to the hydrophobic effect and is not retained by heparan sulfate.

Gramicidin A and doxorubicin were combined and tested against spheroids from colorectal cancer cells (HT-29) [167]. The spheroid evolution, cell viability, and ATP levels were monitored at 24 and 48 hours after the applied treatments, which showed a significant drop in cell viability and cellular ATP levels. Therefore, the use of Gr A/doxorubicin combinations acted synergistically against the spheroids [167].

Acting over a nanomolar range of concentrations against the human breast cancer cell line MCF-7, gramicidin A not only dissipated ion gradients across cancer cells but also became inserted in the inner mitochondrial membrane, reducing the H+ gradient and hampering ATP synthesis. There was a cytostatic action at 1 nanomolar Gr; the mitochondrial dysfunction induced mitophagy, and the lack of ATP caused cell cycle arrest [168]. Gr also inhibited the proliferation of human gastric cancer cells, arrested their cell cycle, and induced apoptosis [169]. Iturin A is a natural BS secreted by Bacillus, which is also able to disrupt the cell membrane due to its cyclic character [104, 106]. In combination with Gr A, there was cell membrane disruption, inhibited proliferation, apoptosis, and regulated inflammation in breast cancer cells [170].

In comparison with the general population of cancer cells, cancer stem cells (CSCs) display a lack of differentiation, self-renewal capability, pluripotency, resistance to chemo and radiotherapy, and higher tumorigenicity. In pancreatic ductal adenocarcinoma, CSCs contribute to an aggressive prognosis for this cancer and treatment resistance [171]. The effect of Gr A at 0.05 μM on pancreatic CSCs was tumor sphere disintegration and a reduction in cell counting; there was CD47 downregulation and modulation of macrophage/tumor cell interaction [172]. In another instance, Gr A inhibited the proliferation of both acute promyelocytic and chronic myeloid leukemia cell lines in the absence of hemolytic effects; combinations of GrA with other anticancer drugs were also evaluated, revealing downregulation of oncogenes such as c-Myc, Eya3, and Axin2 [173].

Table 1 provides some important instances of the broad range of biomedical applications for linear gramicidins and their assemblies. Novel formulations derived from self-assembly or combinations with other bioactive molecules highlight the path to be followed in order to achieve useful applications for these molecules in vivo.

AMP Assembly Biomedical application Refs.
Gramicidin D Self-assembled nanoparticles in water dispersion (150 nm in size) Microbicidal effect against S. aureus and C. albicans [32]
Gramicidin D and cationic polymer PDDA Coatings from nanoparticles: self-assembled NPs of Gr D and self-assembled NPs of bilayer fragments/carboxymethylcellulose (CMC)/(PDDA) Broad-spectrum, high-performance, synergistic activity against bacteria and fungus [122]
Fusogenic peptide pHLIP® /Gr A pHLIP®/ GrA in liposomes Acidic pH transfer of Gr A to cancer cells [89]
galactose–Gr A Galactose conjugated to Gr A N-terminus Targeting galactose–Gr A to asialoglycoprotein in cancer cells [91]
Gr A /doxorubicin Combination Synergism against colorectal cancer cells (HT-29) [92]
Gr A/iturin A Combination Apoptosis of breast cancer cells [95]
Gr A Inhibition of pancreatic cancer stem cell proliferation [97]
Gr A Alone or in combination with anticancer drugs Inhibition of the proliferation of acute promyelocytic and chronic myeloid leukemia cell lines in the absence of hemolytic effects; downregulation of leukemia oncogenes [98]
Gramicidin D and cationic polymer PDDA Coatings from gramicidin D nanoparticles and PDDA polymer Broad-spectrum, high-performance, synergistic activity against bacteria and fungus [123]
Gramicidin D Nanoparticles Cytotoxicity against microbes and mammalian cells [124]

Table 1.

Linear gramicidins, their assemblies, and cytotoxicity against microbes or cancer cells.

Table 2 shows the cytotoxicity of Gr NPs against mammalian and microbial cells (0–5 µM Gr) [124]. Five different mammalian cell lines in culture responded similarly to the interaction with Gr NPs, as depicted by the similar values of IC50. Similar values for IC50 against mammalian cells and minimal microbicidal concentration (MMC) were also obtained. The Gr mechanism of action is the same in microbial and mammalian cells: Gr changes the semi-permeable nature of cell membranes and prevents their function as barriers against ions. The Gr channels dissipate ion concentration gradients across the cell membranes of microbial or mammalian cells. Gr NPs at 2 µM Gr kill C. albicans over seven logarithmic cycles [32]. Microbicidal coatings were prepared from Gr water dispersions by casting and drying these dispersions on hydrophobic or hydrophilic surfaces, providing materials of biomedical importance with antimicrobial properties [122, 123]. The insertion of Gr molecules in cell membranes induced the formation of dimeric channels for single-file diffusion of cations. This affected the ionic balance of the cell, leading to cell disruption and death. This might have been due to the fact that Gr D is a neutral peptide that depends only on the hydrophobic effect to become inserted in the host cell membrane. Thereby, the higher proportion of negatively charged phosphatidylserine reported in cancer cells as compared to normal cells, considered an asset to improving the effects of cationic AMPs [58], would not affect Gr insertion in the cell membrane, which is mostly driven by the hydrophobic effect.

Cells Cell line [Gr]/µM IC50 [Gr]/µM MMC Reference
Macrophages/104 cells J774 3.3 (0.3) [124]
Fibroblasts/104 cells L929 ˃5.0 (0.3) [124]
A31 2.5 (0.3) [124]
SVT2 1.7 (0.3) [124]
HeLa /104 cells HeLa 3.5 (0.3) [124]
C. albicans/107 CFU 2.0 (7) [32]
E. coli/108 CFU 5.0 (0.3) [54]

Table 2.

Cytotoxicity of Gr NPs against microbes and mammalian cells.

The reduction in cell viability is expressed as the number of logarithmic cycles (in between parentheses) beside each minimal microbicidal concentration (MMC) or concentration for killing 50% of the 104 mammalian cells (IC50). One should notice that killing 50% of mammalian cells means reducing cell viability by 0.3 logarithmic cycles.


In vivo, cells actively maintain a high intracellular concentration of K+ and a low intracellular concentration of Na+. In model membranes, such as lipid bilayer vesicles, Gr dimeric channels promote the diffusion of solutes, such as cations and small neutral molecules, which does not occur in the absence of Gr [53, 54]. Gr channels were also reported in the inner mitochondrial membrane. They promoted the dissipation of the proton gradient and hampered ATP biosynthesis, leading to energy depletion, metabolic dysfunction, and cell death, which is useful against renal cell carcinoma [163].

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4. Conclusions

A multidisciplinary approach is needed for fighting drug resistance in infectious diseases and/or cancer. Natural or synthetic AMPs have been intensively and extensively studied for many reasons. They have easily changeable structure-function relationships, often display the property of self-assembly, can be easily endowed with targeting, have unlimited possibilities for combination or conjugation with specific drugs, and disrupt cells via mechanisms difficult to counteract from the microbe or cancer cell’s point of view. The joint venture of nanotechnology, self-assembly, and combinations with other bioactive compounds has been poorly but promisingly explored, and advancements can be foreseen for novel AMP nanometric formulations in biomedical applications. Synergistic action of components in each formulation has been reducing the doses needed for microbial or cancer cell death in many instances. Synergy represents one of the most promising avenues to follow for achieving efficient treatments against infectious diseases and cancer. The clinical application of oncolytic peptides has not been achieved yet due to structural instability, proteolytic degradation, and undesired toxicity when AMPs are administered systemically. The most prolific trend nowadays is mimicking AMPs with other molecules such as polymers. Oncolytic peptides can be copied by oncolytic polymers that are chemically stable, not susceptible to degradation in vivo, and easier to scale up in the biotechnology industry. ICD triggered by cancer cell lysis is one of the most promising approaches for fighting cancer.

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Acknowledgments

Financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) to A.M.C.-R. is gratefully acknowledged (grants 304091/2023-5 and 302758/2019-4). R.M.S. was the recipient of an MSc fellowship from CAPES (grant 88887.829313/2023-00).

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Written By

Ana Maria Carmona-Ribeiro, Yunys Pérez-Betancourt, Ricardo Márcio e Silva

Submitted: 20 October 2025 Reviewed: 01 December 2025 Published: 27 February 2026

© 2026 The Author(s). Licensee IntechOpen. This content is distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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