(1) Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Caixa Postal 26077, 05513-970, São Paulo, Brazil
(2) Departamento de Análises Clínicas e Toxicológicas, Faculdade de CiênciasFarmacêuticas, Universidade de São Paulo, São Paulo, Brazil
* Corresponding author Email: firstname.lastname@example.org
Fungicidal assemblies can be built from lipids, polymers and/or drugs to yield optimal activity against fungus in virtual absence of haemolysis.
The major factors determining the activity of fungicidal assemblies are the mobility of the antimicrobial moiety, the charge on the fungus cell and the size and shape of the antifungal assembly.
The development of novel fungicidal assemblies is important to circumvent the generally high toxicity of antifungal drugs and the problem of fungus resistance derived from the widespread use of such drugs in clinics. Drugs of choice for treating fungal infections belong to different classes such as the azoles which inhibit the synthesis of ergosterol, the echinocandins which inhibit the cell wall synthesis, the polienic antibiotics (e.g. amphotericin B and nystatin) which combine with ergosterol in the fungus membrane to form pores thereby altering the membrane permeability, the nucleoside analogues which inhibit the synthesis of nucleic acids, some antibiotics (e.g. griseofulvin) which inhibit cell division by hampering the synthesis of the microtubules and others. Alternative and less toxic antifungal formulations may include natural products, synthetic agents and polymeric materials such as some saponins, alkaloids, peptides, essential oils, polymers with quaternary nitrogen atoms, synthetic amphiphiles, lipids or polymers loaded or not with antifungal compounds[1,2,3].
(A) Transmission electron micrograph of a Saccharomycces cerevisiae cell where some subcellular structures such as the nucleus (a), a vacuole (b), a mitochondria (c), the cytoplasmic membrane (d) and the cell wall (e) can be seen. Adapted from ref. 4. The enlarged cell wall of the yeast cell shows an inner skeletal layer composed of cross-linked and stress-bearing polysaccharides and chitin running parallel to the cell surface and acting as a scaffold for a dense outer layer of glycoproteins (A). The primary targets of several antifungal agents in the yeast cell are in (B). Reprinted from ref. 1.
The authors have referenced some of their own studies in this review. The protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.
The therapeutic index of toxic drugs can be considerably increased by appropriate formulation. Among the classical fungicides, amphotericin B (AB) is a good example of this statement with several improved formulations available such as liposomal AB, AB lipid complex, AB colloidal dispersion, AB in microspheres and nanoparticles. However, the formulation cost is an important issue mainly for neglected infectious diseases in developing countries (e.g. leishmaniasis). In this regard, a low-cost AB lipid formulation using an inexpensive and synthetic cationic lipid (dioctadecyldimethylammonium bromide, DOD) at low drug to lipid molar ratios was developed based on the high affinity of AB for the borders of cationic bilayer fragments (BFs). Despite the relatively high dose of the pro-inflammatory and toxic cationic lipid, the DOD/AB formulation displayed a high therapeutic index
Schematic representations of some fungicidal assemblies based on bilayer fragments (BF) of the cationic lipid DOD prepared from sonication with tip in aqueous solution. DOD BF are unloaded (a), loaded with amphotericin B yielding DOD/AB (b) or assembled as a bilayer coating on amphotericin B aggregates yielding AB/DOD(c). (a) is adapted from ref. 5. Copyright (2001), with permission from Elsevier; (b) is adapted from ref. 10; (c) is adapted from ref. 9, by permission of Oxford University Press. DOD/AB, AB/DOD or unloaded DOD BF further covered by consecutive layers of carboxymethyl cellulose (CMC) and PDDA yielded the DOD/AB/CMC/PDDA (d), AB/DOD/CMC/PDDA (e) and DOD/CMC/PDDA fungicidal assemblies (f), respectively. (d) and (e) are adapted from ref. 10; (f) is adapted with permission from ref. 11. Copyright (2010) American Chemical Society.
Remarkable fungicidal activity (A) in virtual absence of haemolysis (B) for the unloaded DOD/CMC/PDDA discoidal nanoparticles. Adapted with permission from ref. 12.
Among the azoles, miconazole (MCZ) was used as a model drug to obtain DOD/MCZ assemblies at low drug to lipid molar ratios based on MCZ solubilisation in DOD bilayers. At high drug to lipid molar ratios, MCZ cationic aggregates in water solution were covered by anionic sodium dihexadecyl phosphate (DHP) BF. The minimal fungicidal concentrations (MFC) for MCZ in ethanol and in formulations with DOD BF (DOD/MCZ) or DHP BFs (DHP/MCZ) were determined against
The antifungal activity of agents bearing the quaternary ammonium moiety largely depends on molecular structure. While substantial fungicidal activity was described for the micelle-forming quaternary ammonium surfactants, the bilayer forming, double-chained DOD lipid with long C18 hydrocarbon chains did not show the ability to move from the bilayer assembly to the fungus cell membrane; there is a poor fungicidal activity of DOD BF or large vesicles (LV) against
The mode of action of gemini surfactants bearing the quaternary ammonium moiety seems to involve lysis of the cell membrane and organelles with no apparent damage to the fungus cell wall[4,25]. The gemini quaternary salt (gemini-QUAT) containing two pyridinium residues per molecule, 3,3ʹ-(2,7-dioxaoctane)bis (1-decylpyridiniumbromide) (3DOBP-4,10), exerts fungicidal activity against
Chemical structure of the gemini quaternary salt, 3,3ʹ-(2,7-dioxaoctane) bis (1-decylpyridinium bromide) (3DOBP-4,10) (a) and its induction of ATP (b) and potassium ion leakage (c) from Saccharomycces cerevisiae cells after just 2 minutes of interaction with the cells. The sequence of cellular events taking place on increasing the gemini surfactant concentration are shown in (d). Adapted from ref. 4.
The evaluation of the antifungal activity of gemini quaternary ammonium salts over a range of hydrocarbon chain lengths showed that the compound with double 10 carbons chains was the most active one but exhibited significant toxicity against mammalian red blood cells around the minimal inhibitory concentrations (MIC) effective against the fungus. Since
Chemical structure of gemini quaternary ammonium chloride and bromide, with various alkyl chain and spacer lengths, derivatives of N,N,Nʹ,Nʹ-tetramethylethylenediamine (TMEG) or N,N,Nʹ,Nʹ-tetramethyl-1,3- propanediamine (TMPG) (a), their inhibitory effect on filamentous growth of Candida albicans after 6 h incubation at 37°C, at 0.25 of their minimal inhibitory concentrations (MIC) (b) and their MIC as a function of n for TMEG (CH2)n Cl against four different fungus (c).Reprinted from ref. 26, Copyright (2013), with permission from Elsevier.
Recently, C12 single and gemini cationic surfactants derived from arginine with spacers from 6-12 C were combined with a phospholipid (L-dilauroylphosphatidylcholine, DLPC) or cholesterol (CHOL)-forming cationic vesicles or aggregates with variable sizes for which the haemolytic activity and antimicrobial and antifungal activity were affected by the same parameters. Their haemolytic activity is lower than one of the bis(QUATS) gemini surfactants as their antimicrobial activity. For triblock polymers with the quaternary ammonium as the outermost block, simple changes to the core served to direct the self-assembly into distinct morphologies: spheres and rods. Despite the vast majority of material consisting of poly(lactide) (interior block) and cationic polycarbonates (exterior block) testing the spherical and rod-like morphologies for antimicrobial properties showed that both possessed broad-spectrum activity (Gram-negative and Gram-positive bacteria as well as fungi) with minimal haemolysis, although only the rod-like assemblies were effective against
For the quaternary ammonium hybrid assemblies with water soluble polymers, optimal fungicidal and non-haemolytic activities have been achieved using the self-assembly of BFs, CMC and the PDDA antimicrobial polymer[10,11,12]. Table 1 shows the antifungal and haemolytic activity for several fungicides and their assemblies. PDDA as a fungicidal agent has an MFC equal to 0.4 μg/mL and is effective in complete absence of haemolysis, contrasting with the poor activity of DOD against the fungus. Furthermore, PDDA is easily assembled with proteins, for example, albumin to yield non-haemolytic nanoparticles. Important assemblies described for AB were non-hemolytic cross-linked albumin microspheres (5 μ of mean diameter) or hybrid nanoparticles (0.08 μ of mean diameter), self-assembled from lipid and consecutive layers of water soluble hydrophilic polymers, yielding effective AB formulations[10,11].
Comparison between fungicidal and haemolytic activity for some cationic assemblies against C. albicans
Chemical structure and schematic representation of triblock copolymers that were polymerised from an assembly directing a rigid hydrophobic terephthalamide-bisurea core, flanked by hydrophobic poly (lactide) blocks (PLA) along with peripheric hydrophilic polycarbonate blocks having cationic propyl trimethylammonium bromide (PCPAB) as the antimicrobial block. Two different bisurea cores were used: one with and the other without a flexible methylene spacer between terephthalamide and aryl urea groups, in order to direct the self-assembly into the rod (n = 1) or sphere morphology (n = 0). The two shapes were evidenced by molecular modelling and atomic force microscopy. Adapted with permission from ref. 28. Copyright (2012) American Chemical Society.
Fungicidal activity and low toxicity of antifungal assemblies requires the controllable self-assembly of toxic and biocompatible materials. This critical review emphasises how readily available materials including the antimicrobial quaternary ammonium moiety and water soluble hydrophilic polymers can be assembled to yield non-haemolytic and efficient fungicidal nanostructures. Based on the absence of haemolysis only, some promising combinations have been described that should be further tested for antifungal activity. Based on the fungicidal activity only, some promising combinations have been described that should be further tested for haemolytic activity. Among the most promising fungicidal agents is PDDA which is basically a non-haemolytic, water soluble hydrophilic polymer with excellent antimicrobial activity easily assembled with proteins, for example, albumin to yieldnon-haemolytic nanoparticles, although the haemolysis test was not yet performed. Amphotericin B can be incorporated in cross-linked albumin microspheres (5 μ of mean diameter) or in hybrid nanoparticles (0.08 μ of mean diameter), self-assembled from lipid and consecutive layers of water soluble hydrophilic polymers, yielding effective AB formulations. PDDA itself or in nanostructured, hybrid lipid-polymer particles efficiently kills
AB, amphotericin B; BF, bilayer fragment; CMC, carboxymethyl cellulose; CTAB, cetyltrimethylammonium bromide; DHP, dihexadecyl phosphate; DOD, dioctadecyldimethylammonium bromide; LV, large vesicle; MFC, minimal fungicidal concentrations; MCZ, miconazole; MIC, minimal inhibitory concentration; PDDA, poly (diallyldimethy lammonium chloride).
This work was financially supported by research grants from ConselhoNacional de DesenvolvimentoCientıfíco e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo for AMC-R (grant number 2011/00046-5). LDMC is the recipient of a PhD fellowship from FAPESP (grant number 2012/24534-1).
All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.
Comparison between fungicidal and haemolytic activity for some cationic assemblies against C. albicans
|(μg/mL)||(mM)||(μg/mL)||C for 50%H (μM)||%H at (C)|
||9 (0.08 mM)|
||13 (0.04 mM)|
||0(PDDA 103 μg/mL)|
|DOD/ CMC/PDDA||0.32 DOD 0.5 CMC 0.5 PDDA
||0 (12 μg/mL DOD, 20 μg/mL CMC, 20 μg/mL PDDA)|
||< 4 (5 ' 103μg/mL)|
|Triblock 5b-D||75c||< 4 (5 ' 10
||< 4 (5 ' 103μg/mL)|
MIC: minimal inhibitory concentrations; MFC, minimal fungicidal concentrations; CHOL, cholesterol; DLPC, dilauroylphosphatidylcholine; TMEG, tetramethy- lethylenediamine; PDDA, poly(diallyldimethylammonium chloride); DOD, dioctadecyldimethylammonium bromide; CMC, carboxymethyl cellulose; CTAB, cetyltrimethylammonium bromide.