Synergistic activity of gold nanoparticles with amphotericin B on persister cells of Candida tropicalis biofilms.

AIM: The antifungal activity was studied on sessile and persister cells (PCs) of Candida tropicalis biofilms of gold nanoparticles (AuNPs) stabilized with cetyltrimethylammonium bromide (CTAB-AuNPs) and those conjugated with cysteine, in combination with Amphotericin B (AmB). MATERIALS/METHODS: The PC model was used and synergistic activity was tested by the checkerboard assay. Biofilms were studied by crystal violet and scanning electron microscopy. RESULTS/CONCLUSIONS: After the combination of both AuNPs and AmB the biofilm biomass was reduced, with significant differences in architecture being observed with a reduced biofilm matrix. In addition, the CTAB-AuNPs-AmB combination significantly reduced PCs. Understanding how these AuNPs aid in the fight against biofilms and the development of new approaches to eradicate PCs has relevance for chronic infection treatment.

Synthesis and biological evaluation of nectriatide derivatives, potentiators of amphotericin B activity.

Nectriatide 1a, a naturally occurring cyclic tetrapeptide, has been reported to a potentiator of amphotericin B (AmB) activity. In order to elucidate its structure-activity relationships, we synthesized nectriatide derivatives with different amino acids in solution-phase synthesis and evaluated AmB-potentiating activity against Candida albicans. Among them, C-and N-terminal protected linear peptides were found to show the most potent AmB-potentiating activity.

Dectin-3-targeted antifungal liposomes efficiently bind and kill diverse fungal pathogens.

DectiSomes are anti-infective drug-loaded liposomes targeted to pathogenic cells by pathogen receptors including the Dectins. We have previously used C-type lectin (CTL) pathogen receptors Dectin-1, Dectin-2, and DC-SIGN to target DectiSomes to the extracellular oligoglycans surrounding diverse pathogenic fungi and kill them. Dectin-3 (also known as MCL, CLEC4D) is a CTL pathogen receptor whose known cognate ligands are partly distinct from other CTLs. We expressed and purified a truncated Dectin-3 polypeptide (DEC3) comprised of its carbohydrate recognition domain and stalk region. We prepared amphotericin B (AmB)-loaded pegylated liposomes (AmB-LLs) and coated them with this isoform of Dectin-3 (DEC3-AmB-LLs), and we prepared control liposomes coated with bovine serum albumin (BSA-AmB-LLs). DEC3-AmB-LLs bound to the exopolysaccharide matrices of Candida albicans, Rhizopus delemar (formerly known as R. oryzae), and Cryptococcus neoformans from one to several orders of magnitude more strongly than untargeted AmB-LLs or BSA-AmB-LLs. The data from our quantitative fluorescent binding assays were standardized using a CellProfiler program, AreaPipe, that was developed for this purpose. Consistent with enhanced binding, DEC3-AmB-LLs inhibited and/or killed C. albicans and R. delemar more efficiently than control liposomes and significantly reduced the effective dose of AmB. In conclusion, Dectin-3 targeting has the potential to advance our goal of building pan-antifungal DectiSomes.

A Theoretical Analysis of Interaction Energies and Intermolecular Interactions between Amphotericin B and Potential Bioconjugates Used in the Modification of Nanocarriers for Drug Delivery.

Amphotericin B (AmB) is an antibiotic with a wide spectrum of action and low multidrug resistance, although it exhibits self-aggregation, low specificity, and solubility in aqueous media. An alternative for its oral administration is its encapsulation in polymers modified with bioconjugates. The aim of the present computational research is to determine the affinity between AmB and six bioconjugates to define which one could be more suitable. The CAM-B3LYP-D3/6-31+G(d,p) method was used for all computational calculations. The dimerization enthalpy of the most stable and abundant systems at pH = 7 allows obtaining this affinity order: AmB_1,2-distearoyl-sn-glycerol-3-phosphorylethanolamine (DSPE) > AmB_γ-cyclodextrin > AmB_DSPEc > AmB_retinol > AmB_cholesterol > AmB_dodecanol, where DSPEc is a DSPE analog. Quantum theory of atoms in molecules, the non-covalent interactions index, and natural bond orbital analysis revealed the highest abundance of noncovalent interactions for AmB-DSPE (51), about twice the number of interactions of the other dimers. Depending on the interactions' strength and abundance of the AmB-DSPE dimer, these are classified as strong: O-H---O (2), N-H---O (3) and weak: C-H---O (25), H---H (18), C-H---C (3). Although the C-H---O hydrogen bond is weak, the number of interactions involved in all dimers cannot be underestimated. Thus, non-covalent interactions drive the stabilization of copolymers, and from our analysis, the most promising candidates for encapsulating are DSPE and γ-cyclodextrin.

Alginate-amphotericin B nanocomplexes covered by nanocrystals from bacterial cellulose: physico-chemical characterization and in vitro toxicity.

Nanocomplexes systems made up natural poylymers have pharmacotechnical advantages such as increase of water solubility and a decrease of drugs toxicity. Amphotericin B (AmB) is a drug apply as anti-leishmanial and anti-fungal, however it has low water solubility and high toxicity, limiting its therapeutic application. With this in mind, the present study aimed to produce nanocomplexes composed by alginate (Alg), a natural polymer, with AmB covered by nanocrystals from bacterial cellulose (CNC). For this reason, the nanocomplexes were produced utilizing sodium alginate, amphotericin B in a borate buffer (pH 11.0). The CNC was obtained by enzymatic hydrolysis of the bacterial cellulose. To CNC cover the nanocomplexes 1 ml of the nanocomplexes was added into 1 ml of 0.01% CNC suspension. The results showed an ionic adsorption of the CNC into the Alg-AmB nanocomplexes surface. This phenomena was confirmed by an increase in the particle size and PDI decrease. Besides, nanocomplexes samples covered by CNC showed uniformity. The amorphous inclusion of AmB complex into the polysaccharide chain network in both formulations. AmB in the nanocomplexes was in supper-aggregated form and showed good biocompatibility, being significantly less cytotoxic in vitro against kidney cells and significantly less hemolytic compared to the free-drug. The in vitro toxicity results indicated the Alg-AmB nanocomplexes can be considered a non-toxic alternative to improve the AmB therapeutic effect. All process to obtain nanocomplexes and it coat was conduce without organic solvents, can be considered a green process, and allowed to obtain water soluble particles. Furthermore, CNC covering the nanocomplexes brought additional protection to the system can contribut advancement in the pharmaceutical.

Lyophilized Drug-Loaded Solid Lipid Nanoparticles Formulated with Beeswax and Theobroma Oil.

Solid lipid nanoparticles (SLNs) have the potential to enhance the systemic availability of an active pharmaceutical ingredient (API) or reduce its toxicity through uptake of the SLNs from the gastrointestinal tract or controlled release of the API, respectively. In both aspects, the responses of the lipid matrix to external challenges is crucial. Here, we evaluate the effects of lyophilization on key responses of 1:1 beeswax-theobroma oil matrix SLNs using three model drugs: amphotericin B (AMB), paracetamol (PAR), and sulfasalazine (SSZ). Fresh SLNs were stable with sizes ranging between 206.5-236.9 nm. Lyophilization and storage for 24 months (4-8 °C) caused a 1.6- and 1.5-fold increase in size, respectively, in all three SLNs. Zeta potential was >60 mV in fresh, stored, and lyophilized SLNs, indicating good colloidal stability. Drug release was not significantly affected by lyophilization up to 8 h. Drug release percentages at end time were 11.8 ± 0.4, 65.9 ± 0.04, and 31.4 ± 1.95% from fresh AMB-SLNs, PAR-SLNs, and SSZ-SLNs, respectively, and 11.4 ± 0.4, 76.04 ± 0.21, and 31.6 ± 0.33% from lyophilized SLNs, respectively. Thus, rate of release is dependent on API solubility (AMB < SSZ < PAR). Drug release from each matrix followed the Higuchi model and was not affected by lyophilization. The above SLNs show potential for use in delivering hydrophilic and lipophilic drugs.

Influence of gamma radiation on Amphotericin B incorporated in PVP hydrogel as an alternative treatment for cutaneous leishmaniosis.

Amphotericin B (Amph-B) is an antifungal drug used intravenously for the treatment of leishmaniasis. Side-effects from Amph-B treatment can arise such as cardiac arrhythmia and renal dysfunctions, which will lead to discontinuation of treatment. Unfortunately, patients in endemic countries do not have access to alternative therapies. The objective of this study was to analyze the effects of Cobalt-60 gamma irradiation on crosslinking polymeric hydrogels (Hydg) and the incorporation of Amph-B into the gel as a controlled-release drug delivery alternative. Polyvinylpyrrolidone (PVP)/Amph-B solutions were irradiated with 15 kGy at 0 °C and 25 °C. The drug's stability was ascertained by UV-visible spectrometry, liquid chromatography/mass spectrometry and proton nuclear magnetic resonance. Irradiated Hydg/Amph-B achieved similar stability to the standard Amph-B solution and was enough to promote hydrogel crosslinking. In vitro trials were carried out to ensure Amph-B was still biologically active after irradiation. The results from flow cytometry and MTT assay show that Amph-B had an IC50 = 16.7 nM. A combination of Hydg at 1.324 gmL-1 and Amph-B at 25.1 nM for 24 h lead to the greatest inhibition of L. amazonensis promastigotes, and could be used as an alternative treatment method for cutaneous leishmaniosis.

Chitosan Contribution to Therapeutic and Vaccinal Approaches for the Control of Leishmaniasis.

The control of leishmaniases, a complex parasitic disease caused by the protozoan parasite Leishmania, requires continuous innovation at the therapeutic and vaccination levels. Chitosan is a biocompatible polymer administrable via different routes and possessing numerous qualities to be used in the antileishmanial strategies. This review presents recent progress in chitosan research for antileishmanial applications. First data on the mechanism of action of chitosan revealed an optimal in vitro intrinsic activity at acidic pH, high-molecular-weight chitosan being the most efficient form, with an uptake by pinocytosis and an accumulation in the parasitophorous vacuole of Leishmania-infected macrophages. In addition, the immunomodulatory effect of chitosan is an added value both for the treatment of leishmaniasis and the development of innovative vaccines. The advances in chitosan chemistry allows pharmacomodulation on amine groups opening various opportunities for new polymers of different size, and physico-chemical properties adapted to the chosen routes of administration. Different formulations have been studied in experimental leishmaniasis models to cure visceral and cutaneous leishmaniasis, and chitosan can act as a booster through drug combinations with classical drugs, such as amphotericin B. The various architectural possibilities given by chitosan chemistry and pharmaceutical technology pave the way for promising further developments.

Lipid nanoparticles for amphotericin delivery in the treatment of American tegumentary leishmaniasis.

Leishmaniasis occurs in the five continents and represents a serious public health challenge, but is still a neglected disease, and the current pharmacological weaponry is far from satisfactory. Triglyceride-rich nanoparticles mimicking chylomicrons (TGNP) behave metabolically like native chylomicrons when injected into the bloodstream. Previously we have shown that TGNP as vehicle to amphothericin B (AB) for treatment of fungi infection showed reduced renal toxicity and lower animal death rates compared to conventional AB. The aim of the current study was to test the tolerability and effectiveness of the TGNP-AB preparation in a murine model of Leishmania amazonensis infection. The in vitro assays determined the cytotoxicity of TGNP-AB, AB, and TGNP in macrophages and promastigote forms and the leishmanicidal activity in infected macrophages. The in vivo toxicity tests were performed in healthy mice with increasing doses of TGPN-AB and AB. Then, animals were treated with 2.5 mg/kg/day of AB, 17.5 mg/kg/day of TGNP-AB, or TGNP three times a week for 4 weeks. TGNP-AB formulation was less cytotoxic for macrophages than AB. TGNP-AB was more effective than AB against the promastigotes forms of the parasite and more effective in reducing the number of infected macrophages and the number of amastigotes forms per cell. TGNP-AB-treated animals showed lower hepatotoxicity. In addition, TGNP-AB group showed a marked reduction in lesion size on the paws and parasitic load. The TGNP-AB preparation attained excellent leishmanicidal activity with remarkable lower drug toxicity at very high doses that, due to the toxicity-buffering properties of the nanocarrier, become fully tolerable.

Drug Delivery of Amphotericin B through Core-Shell Composite Based on PLGA/Ag/Fe3O4: In Vitro Test.

This research aimed at developing and designing a slow and targeted delivery of Amphotericin B (AmB) antibiotic by placing three types of shells containing different ratios of biodegradable and biocompatible polymers poly (D, L-lactide)-co-(glycolide) (PLGA), polyethylene glycol (PEG), and polyvinyl pyrrolidone (PVP) on core-shell structures including silver nanoparticles that were activated with magnetic nanoparticles (MNPs). Emulsion solvent evaporation technique was employed to synthesize three types of shells: (i) (PVP-PEG) (100:20, w/w), (ii) (PLGA-PEG) (100:20, w/w), and (iii) (PLGA-PEG) (50:10, w/w) introduced as D1, D2, and D3 respectively. The in vitro release of AmB was examined in aqueous medium phosphate buffer saline (PBS) in pH~ 7.2. Several spectroscopy methods characterized the structure and properties of the nanoparticles. In vitro antifungal activity of pure AmB and D1, D2, and D3 was studied against Candida albicans (C. albicans). The results explained that frequency of drug released from D2 at the first 10 h was (18%) that was compared with D1 (30%) and D3 (24%) at the same time. D2 had more efficient and longer targeted controlled release. The findings showed that D2 can be used as an effective carrier for in vitro targeted controlled release and D2 and D3 had powerful activity against C. albicans.

Amphotericin B containing microneedle ocular patch for effective treatment of fungal keratitis.

Topical application of poorly water-soluble antibiotics cannot achieve the desired therapeutic concentration within cornea. The purpose of this study was to fabricate, characterize and evaluate in-vivo effectiveness of amphotericin B (AmB) containing microneedle ocular patch (MOP) against fungal keratitis. MOP containing free or liposomal AmB was fabricated using micromolding technique to mimic contact lens. MOPs were prepared using dissolvable polymeric matrix including polyvinyl alcohol and polyvinyl pyrrolidone. AmB loaded MOP were studied for their physical and mechanical properties, drug loading and dissolution rate, corneal insertion and drug permeability. MOP loaded with 100 µg AmB had a compression strength of 35.1 ±â€¯6.7 N and required an insertional force of 1.07 ±â€¯0.17 N in excised human cornea. Ex-vivo corneal permeation studies revealed significant enhancement in AmB corneal retention with the application of MOP compared with free AmB or liposomal AmB application. Furthermore, AmB loaded MOP application significantly (P < 0.05) reduced the Candida albicans load within cornea as evaluated in both ex-vivo model and in-vivo rabbit infection model. Histological examination showed that AmB MOP treatment improved the epithelial and stromal differentiation of corneal membrane. AmB containing MOPs can be developed as minimally invasive corneal delivery device for effective treatment of fungal keratitis.

Synthesis, characterization, and mechanistic studies of a gold nanoparticle-amphotericin B covalent conjugate with enhanced antileishmanial efficacy and reduced cytotoxicity.

BACKGROUND: Amphotericin B (AmB) as a liposomal formulation of AmBisome is the first line of treatment for the disease, visceral leishmaniasis, caused by the parasite Leishmania donovani. However, nephrotoxicity is very common due to poor water solubility and aggregation of AmB. This study aimed to develop a water-soluble covalent conjugate of gold nanoparticle (GNP) with AmB for improved antileishmanial efficacy and reduced cytotoxicity. METHODS: Citrate-reduced GNPs (~39 nm) were functionalized with lipoic acid (LA), and the product GNP-LA (GL ~46 nm) was covalently conjugated with AmB using carboxyl-to-amine coupling chemistry to produce GNP-LA-AmB (GL-AmB ~48 nm). The nanoparticles were characterized by dynamic light scattering, transmission electron microscopy (TEM), and spectroscopic (ultraviolet-visible and infrared) methods. Experiments on AmB uptake of macrophages, ergosterol depletion of drug-treated parasites, cytokine ELISA, fluorescence anisotropy, flow cytometry, and gene expression studies established efficacy of GL-AmB over standard AmB. RESULTS: Infrared spectroscopy confirmed the presence of a covalent amide bond in the conjugate. TEM images showed uniform size with smooth surfaces of GL-AmB nanoparticles. Efficiency of AmB conjugation was ~78%. Incubation in serum for 72 h showed <7% AmB release, indicating high stability of conjugate GL-AmB. GL-AmB with AmB equivalents showed ~5-fold enhanced antileishmanial activity compared with AmB against parasite-infected macrophages ex vivo. Macrophages treated with GL-AmB showed increased immunostimulatory Th1 (IL-12 and interferon-γ) response compared with standard AmB. In parallel, AmB uptake was ~5.5 and ~3.7-fold higher for GL-AmB-treated (P<0.001) macrophages within 1 and 2 h of treatment, respectively. The ergosterol content in GL-AmB-treated parasites was ~2-fold reduced compared with AmB-treated parasites. Moreover, GL-AmB was significantly less cytotoxic and hemolytic than AmB (P<0.01). CONCLUSION: GNP-based delivery of AmB can be a better, cheaper, and safer alternative than available AmB formulations.

Red-Emissive Guanylated Polyene-Functionalized Carbon Dots Arm Oral Epithelia against Invasive Fungal Infections.

Oral candidiasis as a highly prevalent and recurrent infection in medically compromised individuals is mainly caused by the opportunistic fungal pathogen Candida albicans. This epithelial infection, if not controlled effectively, can progress to life-threatening systemic conditions and complications. The efficacy of current frontline antifungals is limited due to their poor bioavailability and systemic toxicity. As such, an efficient intervention is essential for controlling disease progression and recurrence. Herein, a theranostic nanoplatform (CD-Gu+-AmB) was developed to track the penetration of antifungals and perturb the invasion of C. albicans at oral epithelial tissues, via decorating the homemade red-emissive carbon dots (CD) with positively charged guanidine groups (Gu+) followed by conjugation with antifungal polyene (amphotericin B, AmB) in a reacting site-controllable manner. The generated CD-Gu+-AmB favorably gathered within the Candida cells and exhibited potent antifungal effects in both planktonic and biofilm forms. It selectively accumulated in the nuclei of human oral keratinocytes and exhibited undetectable toxicity to the host cells. Moreover, we reported for the first time the penetration and exfoliation profiles of CD in a three-dimensional organotypic model of human oral epithelial tissues, demonstrating that the extra- and intracellular accumulation of CD-Gu+-AmB effectively resisted the invasion of C. albicans by forming a "shielding" layer throughout the entire tissue. This study establishes a multifunctional CD-based theranostic nanoplatform functioning as a traceable and topically applied antifungal to arm oral epithelia, thereby shedding light on early intervention of mucosal candidiasis for oral and general health.

Dectin-2-Targeted Antifungal Liposomes Exhibit Enhanced Efficacy.

Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus cause life-threatening candidiasis, cryptococcosis, and aspergillosis, resulting in several hundred thousand deaths annually. The patients at the greatest risk of developing these life-threatening invasive fungal infections have weakened immune systems. The vulnerable population is increasing due to rising numbers of immunocompromised individuals as a result of HIV infection or immunosuppressed individuals receiving anticancer therapies and/or stem cell or organ transplants. While patients are treated with antifungals such as amphotericin B, all antifungals have serious limitations due to lack of sufficient fungicidal effect and/or host toxicity. Even with treatment, 1-year survival rates are low. We explored methods of increasing drug effectiveness by designing fungicide-loaded liposomes specifically targeted to fungal cells. Most pathogenic fungi are encased in cell walls and exopolysaccharide matrices rich in mannans. Dectin-2 is a mammalian innate immune membrane receptor that binds as a dimer to mannans and signals fungal infection. We coated amphotericin-loaded liposomes with monomers of Dectin-2's mannan-binding domain, sDectin-2. sDectin monomers were free to float in the lipid membrane and form dimers that bind mannan substrates. sDectin-2-coated liposomes bound orders of magnitude more efficiently to the extracellular matrices of several developmental stages of C. albicans, C. neoformans, and A. fumigatus than untargeted control liposomes. Dectin-2-coated amphotericin B-loaded liposomes reduced the growth and viability of all three species more than an order of magnitude more efficiently than untargeted control liposomes and dramatically decreased the effective dose. Future efforts focus on examining pan-antifungal targeted liposomal drugs in animal models of fungal diseases.IMPORTANCE Invasive fungal diseases caused by Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals in current use have serious limitations due to host toxicity and/or insufficient fungal cell killing that results in recurrent infections. Few new antifungal drugs have been introduced in the last 2 decades. Hence, there is a critical need for improved antifungal therapeutics. By targeting antifungal-loaded liposomes to α-mannans in the extracellular matrices secreted by these fungi, we dramatically reduced the effective dose of drug. Dectin-2-coated liposomes loaded with amphotericin B bound 50- to 150-fold more strongly to C. albicans, C. neoformans, and A. fumigatus than untargeted liposomes and killed these fungi more than an order of magnitude more efficiently. Targeting drug-loaded liposomes specifically to fungal cells has the potential to greatly enhance the efficacy of most antifungal drugs.

Improved Oral Bioavailability and Gastrointestinal Stability of Amphotericin B through Fatty Acid Conjugation Approach.

Amphotericin B (AmB) is one of the most effective drugs used in the treatment of leishmaniasis and systemic fungal infections. Considering the global burden of leishmaniasis, ∼90% of disease cases occur in developing countries, suggestive of the need for an affordable AmB therapy. However, owing to the physicochemical properties of AmB, all the clinically available formulations must be administered by intravenous route, thereby creating a significant hurdle in patients' access to AmB due to pharmacoeconomic considerations. We have previously demonstrated that lipid conjugation (e.g., fatty acids) to AmB significantly decreases the toxicity of resulting prodrug by a favorable alteration in the aggregation pattern. The hypothesis of the present work was to investigate the potential of the previously established AmB-lipid conjugate [AmB-oleyl conjugate (AmB-OA)] in improving the physicochemical properties such as gastric instability and lower intestinal permeability that otherwise limits the oral delivery of AmB. The synthesized AmB-OA conjugate was remarkably stable at gastric pH in contrast to AmB and exhibited significantly higher permeation across the Caco-2 monolayer (indicative of intestinal permeability). Mechanistic studies revealed that AmB-OA retained an equivalent antifungal activity. Also, AmB-OA was found to interact preferentially with intracellular membranes of Saccharomyces cerevisiae, while AmB interacted with the plasma membrane. The results of Caco-2 monolayer permeation experiments were further confirmed by in vivo pharmacokinetics, which showed that AmB-OA exhibited a 3.13-fold increase in the Cmax and a 4.88-fold increase in AUCTot as compared to AmB. In conclusion, the lipid conjugation approach may provide an effective solution for current challenges in designing drug delivery systems intended for oral AmB therapy.

Efficacy of Oral Encochleated Amphotericin B in a Mouse Model of Cryptococcal Meningoencephalitis.

Cryptococcus neoformans is an encapsulated yeast responsible for approximately a quarter of a million deaths worldwide annually despite therapy, and upwards of 11% of HIV/AIDS-related deaths, rivaling the impact of tuberculosis and malaria. However, the most effective antifungal agent, amphotericin B, requires intravenous delivery and has significant renal and hematopoietic toxicity, making it difficult to utilize, especially in resource-limited settings. The present studies describe a new nanoparticle crystal encapsulated formulation of amphotericin B known as encochleated amphotericin B (CAmB) that seeks to provide an oral formulation that is low in toxicity and cost. Using a 3-day delayed model of murine cryptococcal meningoencephalitis and a large inoculum of a highly virulent strain of serotype A C. neoformans, CAmB, in combination with flucytosine, was found to have efficacy equivalent to parental amphotericin B deoxycholate with flucytosine and superior to oral fluconazole without untoward toxicity. Transport of fluorescent CAmB particles to brain as well as significant brain levels of amphotericin drug was demonstrated in treated mice, and immunological profiles were similar to those of mice treated with conventional amphotericin B. Additional toxicity studies using a standardized rat model showed negligible toxicity after a 28-day treatment schedule. These studies thus offer the potential for an efficacious oral formulation of a known fungicidal drug against intrathecal cryptococcal disease.IMPORTANCECryptococcus neoformans is a significant global fungal pathogen that kills an estimated quarter of a million HIV-infected individuals yearly and has poor outcomes despite therapy. The most effective therapy, amphotericin B, is highly effective in killing the fungus but is available only in highly toxic, intravenous formulations that are unavailable in most of the developing world, where cryptococcal disease in most prevalent. For example, in Ethiopia, reliance on the orally available antifungal fluconazole results in high mortality, even when initiated as preemptive therapy at the time of HIV diagnosis. Thus, alternative agents could result in significant saving of lives. Toward this end, the present work describes the development of a new formulation of amphotericin B (CAmB) that encapsulates the drug as a crystal lipid nanoparticle that facilitates oral absorption and prevents toxicity. Successful oral absorption of the drug was demonstrated in a mouse model that, in combination with the antifungal flucytosine, provided efficacy equal to a parental preparation of amphotericin B plus flucytosine. These studies demonstrate the potential for CAmB in combination with flucytosine to provide an effective oral formulation of a well-known, potent fungicidal drug combination.

Effect of Chemically Transformed Macrocyclic Polyene Antibiotics on Tumor Cells.

Comparative analysis of the effects of chemically transformed polyene antibiotics pimaricin, nystatin, lucensomycin, amphotericin B, and levorin on biological objects in vivo and in vitro revealed the greatest biological activity of original amphotericin B and levorin with its derivatives. The study also examined the effects of alkyl derivatives of amphotericin B and levorin modified in certain parts of the lactone ring on the lipid and biological membranes. It is established that methylated levorin possesses larger biological activity than the original antibiotic. Examination of the effects of alkyl derivatives of levorin and amphotericin B on cell cultures C6 (rat glioma) and HeLa (human cervical carcinoma) in vitro revealed the antitumor action of methylated levorin and original amphotericin B.

Macrophage ghost entrapped amphotericin B: a novel delivery strategy towards experimental visceral leishmaniasis.

Leishmania donovani, an intracellular parasite, poses many challenges against attempted chemotherapy. After the resistance towards the first-line antileishmanial drug, amphotericin B has become the treatment of choice against visceral leishmaniasis, a fatal tropical disease. However, unfavorable toxicity profile, severe side effects, and prolonged parenteral administration limit its use. Lack of available specific delivery system also makes this drug unsafe for long-term use. In the current study, a "ghost cell" strategy based on macrophage membrane-derived nanovesicle has been introduced as a specific carriage for amphotericin B. Membrane proteins of macrophage ghost play a crucial role in the dissemination of infection in host by communicating between infected neutrophil-macrophage system and non-infected macrophages. These membrane proteins are the basis of specificity of the drug delivery to the infected tissues in this current macrophage ghost cell carrier. This cheap and biocompatible delivery vehicle has significantly improved the toxicity profile and lowered LD50 value of the drug compared to traditional way of its direct administration and widely accepted antileishmanial therapy, AmBisome.

Preliminary investigation of novel tetra-tailed macrocycle amphiphile based nano-vesicles for amphotericin B improved oral pharmacokinetics.

Supramolecular macrocycles-based drug delivery systems are receiving wider recognition due to their self-assembly into nanostructures with unique characteristics. This study reports synthesis of resorcinarene-based novel and biocompatible amphiphilic supramolecular macrocycle that self-assembles into nano-vesicular system for Amphotericin B (Am-B) delivery, a model hydrophobic drug. The macrocycle was synthesized through a two-step reaction and was characterized with 1 H NMR and mass spectrometric techniques. Its biocompatibility was assessed in cancer cell lines, blood and animals. Its critical micelle concentration (CMC) was determined using UV spectrophotometer. Am-B loaded in novel macrocycle-based vesicles were examined according to their shape, size, surface charge, drug entrapment efficiency and excepients compatibility using atomic force microscope (AFM), Zetasizer, HPLC and FT-IR spectroscopy. Drug-loaded vesicles were also investigated for their in-vitro release, stability and in-vivo oral bioavailability in rabbits. The macrocycle was found to be nontoxic against cancer cells, haemo-compatible and safe in mice and revealed lower CMC. It formed mono-dispersed spherical shape vesicles of 174.4 ± 3.78 nm in mean size. Vesicles entrapped 92.05 ± 4.39% drug and were stable upon storage with gastric-simulated fluid and increased the drug oral bioavailability in rabbits. Results confirmed novel macrocycle as biocompatible vesicular nanocarrier for enhancing the oral bioavailability of lipophilic drugs.

Amphotericin B-albumin conjugates: Synthesis, toxicity and anti-fungal activity.

Amphotericin B (AmB), a hydrophobic drug with negligible aqueous solubility was conjugated to bovine serum albumin (BSA) via amide bond coupling to give 6 to 8 wt% drug payload. The resulting conjugate was characterized using SDS-PAGE and UV-visible, FTIR and CD spectroscopy. The conjugate was water-soluble to the extent of 150 mg/ml, was non-toxic to HEK 293 T cells at a concentration of 500 µg/ml (equivalent to ~30 µg AmB) and showed hemolysis of <5% at 200 µg/ml (equivalent to ~12 µg AmB) against human erythrocytes in vitro. In vitro release studies at 37 °C demonstrated steady release of AmB up to 20% from the conjugate with little burst effect in phosphate buffered saline whereas thrice the amount was released in human plasma in 72 h. AmBisome® used as a reference showed a very similar release profile in plasma. The conjugate exhibited potential anti-fungal activity against yeast strains such as C. albicans, C. neoformans and C. parapsilosis with the minimum inhibitory concentration (MIC) equivalent to AmB ranging from 0.7 to 1.1 µg/ml while AmBisome® and AmB alone showed the MIC between 0.78 and 1.5 and 0.53-0.78 µg/ml respectively. Although AmB has been conjugated to various natural and synthetic polymers to improve its solubility and reduce its toxicity, the results obtained in this study using the model protein BSA as a carrier point to the possibility of taking this pro-drug approach to human clinical use using human serum albumin (HSA) as the carrier, since HSA has emerged as a versatile drug carrier for treating diabetes and cancer and improving the pharmacokinetic profile of many drugs with US FDA approving HSA as a drug carrier for the anti-cancer drug paclitaxel (Abraxane®) for human use.