Anti-biofilm activity of a sophorolipid-amphotericin B niosomal formulation against Candida albicans

Sophorolipids (SLs) have gained interest in the pharmaceutical industries due to their anti-microbial, anti-adhesive and anti-biofilm properties. In the present study, the production of SL was increased by using low-cost media components. The potential of a SL-based niosomal formulation of amphotericin B (AmB) was determined against biofilm of the opportunistic fungal pathogen Candida albicans. In-house prepared SL-AmB niosomes were characterized by different microscopic techniques. The mean entrapment efficiency of AmB within SL-AmB niosome was 63.20% ± 3.86. The cytotoxicity of SL-AmB on mature C. albicans biofilm was compared with an expensive, marketed drug, viz. phosome (a liposomal formulation of AmB). Fewer hyphae were observed in C. albicans biofilm treated with SL-AmB niosome whereas more budding cells were found in phosome treated biofilm. The present study has established the affordable production of SL and the suitability of this approach for delivery of poorly soluble drugs such as AmB against candidiasis infections.     

Physical biochemistry of a liposomal amphotericin B mixture used for patient treatment

There seems little doubt now that intravenous liposomal amphotericin B can be a useful treatment modality for the management of immunocompromised patients with suspected or proven disseminated fungal infections. Interestingly, the very significant reduction in toxicity reported when amphotericin B is part of a bilayer membrane is closely tied to the physical characteristics of the liposomes involved, although these are poorly understood at the molecular level. We record here an examination by spectroscopy and freeze-etch electron microscopy of unsonicated amphotericin B multilamellar vesicles prepared along the lines that we and others have followed for samples used in clinical trials and preclinical in vivo or in vitro studies. Our study has focussed on liposomes of 7:3 dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bearing 0-25 mol% amphotericin B, since this lipid mixture has been the choice for the first clinical trials. Phase transition behaviour of these liposomes was examined by electron paramagnetic resonance (EPR) spectroscopy of a nitroxide spin label partitioning into the bilayers. The same experiments were then performed on similarly prepared liposomes of the disaturated species, dipalmitoylphosphatidylcholine (DPPC), and the diunsaturated species, dielaidoylphosphatidylcholine (DEPC). Partial phase diagrams were constructed for each of the lipid/drug mixtures. Melting curves and derived phase diagrams showed evidence that amphotericin B is relatively immiscible with the solid phase of bilayer membranes. The phase diagram for DEPC/amphotericin B was very similar to that of DPPC/amphotericin B, and both exhibited less extensive temperature ranges of phase separation than did the 7:3 DMPC/DMPG mixture with amphotericin B. Between 25 and 37 degrees C the measured fluidity of the 7:3 DMPC/DMPG liposomes was similar to that of the (unsaturated fatty acid) DEPC liposomes, and considerably higher than that seen for (saturated fatty acid) DPPC liposomes. Preparations of 7:3 DMPC/DMPG, DPPC, and DEPC containing 0-25 mol% amphotericin B were examined by freeze-etch electron microscopy at 35 and 22 degrees C (to cover the temperature range of the mammalian body core and periphery). The same liposome features were present in all three liposome types studied. The appearance of individual liposomes at x 100,000 magnification reflected their molecular characteristics, which were found to be significantly heterogeneous within each batch. The lipid/drug structures were bilayer in nature, although liposomes showing considerable disruption were common, particularly at the highest drug concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of antibody covalently coupled to liposomes

We have explored the covalent coupling of fatty acids to immunoglobulin G(IgG). N-hydroxysuccinimide ester of palmitic acid (NHSP) was used to couple palmitic acid to either a mouse monoclonal antibody to the major histocompatibility antigen, H-2^k, or goat antibody to the major glycoprotein of the Molony Leukemia Virus, gp-70. The reaction was characterized in terms of the time course, input ratio of NHSP to IgG, stoichiometry of the coupling, distribution of palmitic acid in the IgG subunits, and the antigen binding capacity of the coupled antibody. Incorporation of the fatty acid modified IgG into liposomal membranes using a detergent-dialysis method was studied as a function of extent of fatty acid coupling. Finally, the binding of IgG-coated liposomes with cells expressing proper antigens was characterized. The major conclusions were: (1) the optimal molar ratio of NHSP to IgG in the reaction was between 10 and 20, which yields about 4–5 palmitoyl chains per IgG molecule; (2) at this level of coupling, the antigen binding capacity of the IgG antibody decreased about 3–4-fold; (3) incorporation of the coupled antibody into unilamellar liposomes (about 1000 Å diameter) can be achieved with a deoxycholate-dialysis method with an optimal lipid-to-protein ratio of 10:1 (w/w); (4) there were about 48 IgG molecules incorporated per liposome under these conditions; (5) the apparent dissociation constant of the liposome-bound antibody under the optimal condition was about 6–7-fold higher than that of the native antibody; (6) binding of antibody to the target cells was accompanied by binding of liposomal lipids; both bindings could be blocked by pretreatment of cell with unmodified antibody.     

Latrotoxin-induced fusion of liposomes with bilayer phospholipid membranes

Liposomes containing amphotericin B as ionophoric marker were used to investigate the fusion of bilayer phospholipid membranes with liposomes. It was found that latrotoxin isolated from black widow spider venom induced the fusion of liposomes with planar bilayer when liposomes and latrotoxin were administered at opposite sides of the membrane.     

Phosphatidylcholine liposomes containing cholesterol analogues with side chains of various lengths

The effect of the length of the side chain of sterols on their interaction with phosphatidylcholine was studied by measuring the permeability properties of liposomes constituted with sterol analogues with side chains of various lengths. The sensitivities of liposomes constituted with these sterol analogues toward digitonin and polyene antibiotics were also examined.The effects of sterols on phase transition of phosphatidylcholine were examined by measuring their effects on permeability increase due to perturbation of phase equilibrium and by differential scanning calorimetry. An analogue with a short side chain, isopropyl (C-22), had a very similar effect to cholesterol in suppressing the permeability increase, suggesting that the full length of the side chain is not necessary for this effect.The permeability of egg yolk phosphatidylcholine at 42°C was suppressed as much by the analogue C-22 as by cholesterol. Androstene-3-β-ol, an analogue without a side chain, however, had little suppressive effect. Thus it is concluded that the condensing effect of sterol requires a side chain, but not the full length of side chain.Liposomes constituted with analogues having a side chain with more than 5 carbon atoms showed maximum reactivity with a polyene antibiotic, amphotericin B, whereas those constituted with analogues having a side chain with less than 4 carbon atoms showed weaker reactivity. These findings indicate that a side chain with more than 5 carbon atoms is essential for the maximum interaction of liposomes with amphotericin B. Unlike amphotericin B, filipin reacted almost equally well with liposomes containing C-22 and with those containing cholesterol. Thus the chain length of the side chain of sterol is less important for interaction of liposomes with filipin than for their interaction with amphotericin B.Liposomes containing analogues having a side chain with more than 6 carbon atoms showed maximum reactivity with digitonin. Thus for the maximum interaction of liposomes with digitonin, the side chain of sterol should be longer than 6 carbon atoms.     

Latrotoxin-induced fusion of liposomes with bilayer phospholipid membranes

Liposomes containing amphotericin B as ionophoric marker were used to investigate the fusion of bilayer phospholipid membranes with liposomes. It was found that latrotoxin isolated from black widow spider venom induced the fusion of liposomes with planar bilayer when liposomes and latrotoxin were administered at opposite sides of the membrane.     

Biofilm formation by <Emphasis Type="Italic">Candida albicans</Emphasis> isolated from intrauterine devices

Our survey revealed that infected intrauterine devices (IUDs) recovered from patients suffering from reproductive tract infections (RTIs) were tainted with Candida biofilm composed of a single or multiple species. Scanning electron microscopy (SEM) analysis of C. albicans biofilm topography showed that it consists of a dense network of mono- or multilayer of cells embedded within the matrix of extracellular polymeric substances (EPS). Confocal scanning laser microscopy (CSLM) and atomic force microscopy (AFM) images depicted that C. albicans biofilms have a highly heterogeneous architecture composed of cellular and noncellular elements with EPS distributed in the cell-surface periphery or at cell-cell interface. Biochemical analysis showed that EPS produced by C. albicans biofilm contained significantly reduced total carbohydrate (40%), protein (5%) and enhanced amount of hexosamine (4%) in contrast to its planktonic counterparts. The in vitro activity of antifungal agents amphotericin B, nystatin, fluconazole and chlorhexidine against pre-formed C. albicans biofilm, assessed using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay revealed increased resistance of these infectious biofilm (50% reduction in metabolic activity at a concentration of 8, 16, 64, 128 μg/ml respectively) in comparison to its planktonic form.     

Candida albicans and Candida tropicalis in oral candidosis: quantitative analysis, exoenzyme activity, and antifungal drug sensitivity

Candida albicans and C. tropicalis obtained from whole saliva of patients presenting signs of oral candidosis were assayed for quantification of colony forming units, exoenzyme activity (phospholipase and proteinase) and antifungal drug sensitivity (amphotericin B, fluconazole and itraconazole) by the reference method of the Clinical and Laboratory Standards Institute. The number of colony forming units per milliliter varied according to the Candida species involved and whether a single or mixed infection was present. Proteinase activity was observed in both C. albicans and C. tropicalis, but phospholipase activity was noted only in C. albicans. In vitro resistance to antifungals was verified in both species, but C. tropicalis appears to be more resistant to the tested antifungals than C. albicans.     

The impact of polyene,azole, and DNA analogue antimycotics on the cell surface hydrophobicity of Candida albicans and Candida tropicalis in HIV infection

Oral candidiasis is the most common opportunistic infection in individuals infected with the human immunodeficiency virus. Though Candida albicans is the major aetiological agent, non-albicans species such Candida tropicalis are now emerging as important agents of such infection. The Candida cell surface hydrophobicity (CSH) is considered a critical factor contributing to its colonization potential and virulence. It is also known that brief exposure to sub-cidal concentrations of antifungal agents is a likely scenario in the oral environment where the administered drugs are diluted continuously due to the flushing action of saliva. Hence the objective of the present study was to compare the CSH of 10 isolates each of C. albicans and C. tropicalis from HIV-infected individuals following brief exposure (1hour) of isolates to sub-therapeutic concentrations of nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine. The CSH was assessed by a previously described biphasic aqueous-hydrocarbon assay. The mean percentage reduction of CSH of C. albicans following brief exposure to nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine was27.33 (p < 0.001), 21.34 (p < 0.05), 11.74 (p > 0.05), 18.4 (p > 0.05) and 14.64 (p > 0.05) respectively. The mean percentage reduction of CSH of C. tropicalis following brief exposure to nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine was 33.81 (p < 0.01), 28.88 (p < 0.01), 12.6 (p > 0.05), 21.53(p > 0.05) and 17.68 (p > 0.05) respectively. A significant inter-species variation in CSH was observed for nystatin and amphoterecin B. Overall the results reveal that the CSH of C. albicans is affected to a significantly lesser degree compared with C. tropicalis when exposed to the antifungals. These data further illustrate another mode of action of antifungals on Candida leading to a reduction in the CSH and thereby the yeast adherence to host tissues. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro and in vivo antifungal activities of liposomal amphotericin B,and amphotericin B lipid complex

The in vitro and in vivo antifungal activities of liposomal amphotericin B (L-AMPH) and amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, were compared with those of conventional amphotericin B (Fungizone®, AMPH). The acute intravenous toxicity was markedly lower in BALB/c mice; 50% lethal doses (LD_50s) were 2.75 mg/kg in AMPH, 32.9 mg/kg in L-AMPH and >75 mg/kg in ABLC. In vitro antifungal activities againstCandida albicans, C. parapsilosis, C. tropicalis, C. glabrata, andC. krusei were evaluated by the agar plate dilution method. The activities were unchanged againstC. albicans, but MICs increased more than four fold in 18 of the 20 strains other thanC. albicans in L-AMPH and in 9 of the 20 in ABLC. L-AMPH and ABLC were as efficacious as AMPH in the treatment of mice infected withC. albicans, and at a dose of 0.5 and 1.0 mg/kg of body weight, ABLC was more efficacious on survival. A ten-times larger dose (10 mg/kg) of L-AMPH and ABLC was administered to mice with 100% survival, suggesting improved tolerability as compared to amphotericin B.     

Synergistic effects of low doses of histatin 5 and its analogues on amphotericin B anti-mycotic activity

The increase in the use of antifungal agents for prophylaxis and therapy has led to the development of antifungal drug resistance. Drug combinations may prevent or delay resistance development. The aim of the present study was to investigate whether naturally and designed cationic antifungal peptides act synergistically with commonly used antimycotics. No enhanced activity was found upon addition of dhvar4, a designed analogue of the human salivary peptide histatin 5, or PGLa to fluconazole or 5–flucytosine, respectively. In contrast, strong synergism of amphotericin B with the peptides was found against several Aspergillus, Candida, and Cryptococcus strains, and against an amphotericin B-resistant C. albicans laboratory mutant in the standardised broth microdilution assays according to the NCCLS standard method M27–T. Amphotericin B showed synergism with dhvar5, another designed analogue of histatin 5, and with magainin 2 against all seven tested strains. Combinations of amphotericin B with histatin 5, dhvar4, and PGLa showed synergism against four of the seven strains. The growth inhibitory activity of amphotericin B was enhanced by sub-MIC concentrations of peptide, but its haemolytic activity remained unaffected, suggesting that its cytotoxicity to host cells was not increased and that peptides may be suitable candidates for combination therapy.     

Disseminated cutaneous and peritoneal hyalohyphomycosis caused by Fusarium species: Three cases and review of the literature

Three recent cases of hyalohyphomycosis caused by Fusarium sp. illustrate differing aspects of infections produced by these organisms. One patient was undergoing continuous ambulatory peritoneal dialysis when peritonitis developed caused by Fusarium moniliforme. Removal of the catheter and amphotericin B were used in successful management. In a neutropenic patient on therapy for leukemia, multiple persistent infections occurred including JK diptheroids, and Candida albicans sepsis. Finally, numerous florid skin lesions caused by Fusarium oxysporum developed even while the patient was receiving amphotericin B and he died. In a second neutropenic patient on treatment for leukemia, sinus and cutaneous lesions developed due to Fusarium. These resolved on amphotericin B therapy following the return of circulating neutrophils. The literature on Fusarium infections and aspects of the biology of the organism are reviewed.     

Targeting of monoclonal antibody-coated liposomes to sheep red blood cells

A monoclonal mouse anti-sheep red blood cell specific antibody IgG_2b was esterified with palmitic acid which served as a hydrophobic anchor for successfull incorporation into the liposomal membrane. The formation of coated liposomes by dialyzing the mixed antibody/lipid/detergent micelles against phosphate buffer was simplified b by using the same detergent as for the antibody derivatization. No purification step of any intermediate product was necessary. Targeting of the resulting vesicles to sheep red blood cells occured with high efficiency compared with control liposomes. The uptake was fast and specific as demonstrated with sheep and horse red blood cells by the use of radioactively labelled liposomes and by scanning electron microscopy.     

Biotransformation of pseudolaric acid B by Chaetomium globosum

Pseudolaric acid B (1) is a natural product with potent antifungal activity. We discovered that pseudolaric acid B did not kill but only suppress the growth of the filamentous fungus Chaetomium globosum. It was proposed that pseudolaric acid B was converted to metabolites with decreased antifungal activities. In this study, a scaled-up biotransformation of pseudolaric acid B by C. globosum produced five metabolites, including three new compounds, pseudolaric acid I (2), pseudolaric acid B 18-oyl-alanine (4) and pseudolaric acid B 18-oyl-serine (6), together with two known compounds, pseudolaric acid F (3) and pseudolaric acid B 18-oyl-glycine (5). The structures were characterized by NMR and MS spectroscopy. The major biotransformation reaction was conjugation with amino acids. None of the metabolites showed inhibitory effects on the growth of Candida albicans. The results suggested that biotransformation might be a detoxification process for fungi to resist antifungal drugs.     

Molecular Analysis and Susceptibility Profiling of Candida albicans Isolates from Immunocompromised Patients in South India

The genetic diversity and in vitro antifungal susceptibility profiles of 55 Candida albicans from immunocompromised patients were studied. PCR based analysis of the transposable intron in the 25S rDNA revealed 39 genotype A, 4 genotype B and 12 genotype C isolates. Serotype analysis categorized 52 isolates as serotype A and 3 as serotype B. All strains were susceptible to micafungin, 5-flucytosine and miconazole, whereas resistance against amphotericin B (3.6%), fluconazole (3.6%), itraconazole (7.3%) and voriconazole (5.5%) was observed. No association was seen between antifungal resistance and genotype/serotype status.     

Small liposomes are better than large liposomes for specific drug delivery in vitro

We have compared drug transfer into target cells in vitro from liposomes of different sizes. Liposomes of mean diameter 800 Å, 2000 Å or 4000 Å, containing the folate analogue, methotrexate, and the fluorophore, carboxyfluorescein, were covalently coupled to Staphylococcus aureus protein A. Cells of the murine k haplotype were preincubated with an anti-H-2K^k monoclonal antibody. Excess antibody was removed and then cells were incubated with liposomes. The number of cell-bound liposomes was determined by fluorimetry. The drug effect was assayed by the methotrexate-mediated inhibition of radiolabeled deoxyuridine uptake. The drug effect was more important in the case of the 800 Å vesicles than for the larger liposomes, despite the fact that the quantity of drug bound to cells was several-fold greater for large liposomes than for small ones. Since fusion is excluded by the non-proportionality of drug binding and drug effect, the predominant manner of liposome entry seems to be endocytosis. At least for these in vitro studies, the endocytosis by target cells of small liposomes seems to be more efficient than that of large liposomes.     

Comparative efficacy of amphotericin B,clotrimazole and itraconazole againstAspergillus spp.

The susceptibilities of two isolates ofAspergillus flavus, one from a human case of recalcitrant mycotic keratitis, and an environmental isolate ofA. fumigatus, to itraconazole, clotrimazole and amphotericin B were measured. Observations of macroscopic growth and microscopic evaluations of conidia germination both indicated that the two isolates ofA. flavus were markedly more resistant to amphotericin B than to itraconazole and clotrimazole. Itraconazole was more effective than clotrimazole for all isolates. Ourin vitro susceptibility results suggest the use of itraconazole should be a primary consideration in the treatment ofAspergillus keratitis.     

Incorporation of influenza virus M-protein into liposomes.

M-protein from influenza virus vaccine was purified by sodium dodecyl sulfate-gel chromatography and incorporated into liposomes by solubilization with octylglucoside and subsequent dialysis. Liposomes containing M-protein formed a distinct population with a density of 1.22 g/ml on sucrose-gradient centrifugation, regardless of the net charge on the liposomes. Treatment of the liposomes by freeze-fracture followed by electron microscopic examination showed multilamellar structures in those liposomes without M-protein; liposomes containing M-protein were mulberry-like structures which appeared unilamellar. These studies show incorporation of M-protein into the lipid bilayer.     

Investigations on feasibility of in situ development of amphotericin B liposomes for industrial applications

Amphotericin B (AmB) liposome formulations are very successful in the treatment of fungal infections and leishmaniasis. But higher cost limits its widespread use among people in developing countries. Therefore, we have developed a modified ethanol-injection method for the preparation of AmB liposomes. Two liposomal formulations were developed with dimyristoyl phosphatidylcholine [F-1a] and soya phosphatidylcholine [F-2a], along with egg phosphatidyl glycerol and cholesterol. AmB was dissolved in acidified dimethyl acetamide and mixed with ethanolic lipid solution and rapidly injected in 5% dextrose to prepare liposomes. Liposomes were characterized on the basis of size (~100?nm), zeta (–43.3?±?2.8 mV) and percent entrapment efficiency (>95%). The in vitro release study showed an insignificant difference (P?≥?0.05) for 24-hour release between marketed AmB liposomes (AmBisome) and F-1a and F-2a. Proliposome concentrate, used for the preparation of in situ liposomes, was physically stable for more than 3 months at experimental conditions. Similarly, AmB showed no sign of degradation in reconstituted liposomes stored at 2–8°C for more than 3 months. IC₅₀ value of Ambisome (0.18 µg/mL) was comparatively similar to F-1a (0.17 µg/mL) and F-2a (0.16 µg/mL) against intramacrophagic amastigotes. Under experimental conditions, a novel modified method for AmB liposomes is a great success and generates interest for development as a platform technology for many therapeutic drug products.     

The action of ascorbate in vesicular systems

Many effects of ascorbate center on its interactions with membranes from plant and animal cells. These actions can be studied using vesicles produced from phospholipid components (liposomes), by isolating naturally occurring vesicles, or by purifying particular membranes that form vesicles during the extraction process. Liposomes have provided information concerning the anti- and prooxidant properties of ascorbate and about how the water-soluble vitamin can have effects within the phospholipid bilayer. The involvement of ascorbate in transmembrane electron transport has been characterized in vesicles normally found in certain cells, such as, chromaffin granules, synaptosomes, glyoxisomes, peroxisomes, and clathrincoated vesicles. Redox activity using reducing power associated with ascorbate/ascorbate free radical (AFR) has been characterized in some of these vesicles and it appears to be mediated by ab-type cytochrome. Ascorbate also participates in the reduction of iron within clathrin-coated vesicles. Vesicles appearing during purification of plasma membranes have transmembrane electron transport, oxidoreductase activity with ascorbate/AFR as redox agents, and an ascorbate-reducibleb-type cytochrome. It is also possible that ascorbate-related redox activity exists at the tonoplast of plant cells.