Amphotericin B Aggregation Research Articles

“Click” amphotericin B in prodrug nanoformulations for enhanced systemic fungemia treatment

Severe nephrotoxicity and infusion-related side effects pose significant obstacles to the clinical application of Amphotericin B (AmB) in life-threatening systemic fungal infections. In pursuit of a cost-effective and safe formulation, we have introduced multiple phenylboronic acid (PBA) moieties onto a linear dendritic telodendrimer (TD) scaffold, enabling effective AmB conjugation via boronate chemistry through a rapid, high yield, catalysis-free and dialysis-free “Click” drug loading process. Optimized AmB-TD prodrugs self-assemble into monodispersed micelles characterized by small particle sizes and neutral surface charges. AmB prodrugs sustain drug release in circulation, which is accelerated in response to the acidic pH and Reactive Oxygen Species (ROS) in the infection and inflammation. Prodrugs mitigate the AmB aggregation status, reduce cytotoxicity and hemolytic activity compared to Fungizone®, and demonstrate superior antifungal activity to AmBisome®. AmB-PEG5kBA4 has a comparable maximum tolerated dose (MTD) to AmBisome®, while over 20-fold increase than Fungizone®. A single dose of AmB-PEG5kBA4 demonstrates superior efficacy to Fungizone® and AmBisome® in treating systemic fungal infections in both immunocompetent and immunocompromised mice.

Exploring the permeability of Amphotericin B trough serum albumin dispersions and lipid nanocarriers for oral delivery

Amphotericin B (AmB) is a potent polyenic antifungal agent with leishmanicidal activity. Due to its low solubility and permeability in the gastrointestinal tract, AmB is usually administered intravenously. In this context, various approaches have been used to try to improve these properties. Some of the systems developed have shown proven successful, but there is still a lack of knowledge about the pathways AmB takes after oral administration. Therefore, the aim of this work was not only to obtain aqueous dispersions containing AmB at different aggregation states, but also to entrap this molecule in nanocarriers, and evaluate the influence of these conditions on the jejunal permeability of AmB. To observe the aggregation states of AmB, physicochemical characterization of AmB-albumin complexes and AmB-loaded formulations was performed. Different degrees of AmB aggregation states were obtained. Thus, permeability tests were performed in the Ussing chamber and a decrease in AmB concentration in the donor compartment was observed. Electrophysiological measurements showed different responses depending on the AmB formulation. In conclusion, although control of the AmB aggregation state was observed by physicochemical characterization, this approach does not seem to have a sufficient effect on AmB permeability, but on its toxicity. For a complete understanding of AmB-loaded nanocarriers, other pathways, such as lymphatic absorption, should also be investigated.

Engineering Telodendrimer Nanocarriers for Monomeric Amphotericin B Delivery

Systemic fungal infections are an increasingly prevalent health problem. Amphotericin B (AmB), a hydrophobic polyene antibiotic, remains the drug of choice for life-threatening invasive fungal infections. However, it has dose-limiting side effects, including nephrotoxicity. The efficacy and toxicity of AmB are directly related to its aggregation state. Here, we report the preparation of a series of telodendrimer (TD) nanocarriers with the freely engineered core structures for AmB encapsulation to fine-tune AmB aggregation status. The reduced aggregation status correlates well with the optimized antifungal activity, attenuated hemolytic properties, and reduced cytotoxicity to mammalian cells. The optimized TD nanocarrier for monomeric AmB encapsulation significantly increases the therapeutic index, reduces the in vivo toxicity, and enhances antifungal effects in mouse models with Candida albicans infection in comparison to two common clinical formulations, i.e., Fungizone and AmBisome.

Liposomal Amphotericin B for Treatment of Leishmaniasis: From the Identification of Critical Physicochemical Attributes to the Design of Effective Topical and Oral Formulations.

The liposomal amphotericin B (AmB) formulation, AmBisome®, still represents the best therapeutic option for cutaneous and visceral leishmaniasis. However, its clinical efficacy depends on the patient's immunological status, the clinical manifestation and the endemic region. Moreover, the need for parenteral administration, its side effects and high cost significantly limit its use in developing countries. This review reports the progress achieved thus far toward the understanding of the mechanism responsible for the reduced toxicity of liposomal AmB formulations and the factors that influence their efficacy against leishmaniasis. It also presents the recent advances in the development of more effective liposomal AmB formulations, including topical and oral liposome formulations. The critical role of the AmB aggregation state and release rate in the reduction of drug toxicity and in the drug efficacy by non-invasive routes is emphasized. This paper is expected to guide future research and development of innovative liposomal formulations of AmB.

Solubility, aggregation and stability of Amphotericin B drug in pure organic solvents: Thermodynamic analysis and solid form characterization

The solubility of amphotericin B (AmB) has been studied between temperatures of 298–343 K in a range of pure organic solvents including methanol, ethanol, isopropanol, butanol, acetone, tetrahydrofuran, and 1,4-dioxane. The initial solid form used in solubility determinations has been characterized by a series of techniques including powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) and Raman spectroscopy, thermogravimetric analysis and differential scanning calorimetry. Pure AmB melts at 444.5 K with an associated enthalpy of fusion of 177.2 kJ/mol. The equilibrated solids in suspension have been characterized by SEM and PXRD and additionally, the equilibrated saturated solutions have been characterized by in-situ UV–vis to probe the saturation and the aggregation state of AmB. In the explored solvents, in terms of mass ratio, the solubility has been found to be remarkably low, decreasing in the order: ethanol > tetrahydrofuran > butanol ≃ 1,4-dioxane ≃ isopropanol > acetone. The relative order of solubility obtained in the alcohols correlates well with their polarity, revealing important interactions of the solvents’ hydroxyl group with the polyol chain and the micosamine sugar carboxylic moiety of amphotericin B. Except in dioxane and isopropanol, the obtained Van’t Hoff enthalpies of solution reveal an inverse proportionality to the experimental molar fraction solubility values obtained experimentally, indicating a larger energy requirement to solvate AmB molecules. Aggregation of AmB in the equilibrated solutions was not detected in any of the solvents studied yet HPLC analyses revealed that the API degraded in methanol during the equilibration time for the solubility determinations.

Cholesterol improves stability of amphotericin B nanoemulsion: promising use in the treatment of cutaneous leishmaniasis.

Aim: Amphotericin B (AmB) is an antileishmanial drug with high toxicity; however, this drawback might overcome by decreasing the AmB self-aggregation state. This work aimed at evaluating the influence of cholesterol on the aggregation state of AmB loaded in a nanoemulsion (NE-AmB) for the treatment of cutaneous leishmaniasis. NE-AmB (1, 4 and 8mg/kg/day) was administered intravenously to animals infected by Leishmania major every 2days for a total of five injections. Results: Ultraviolet-visible spectroscopy and circular dichroism studies demonstrated that cholesterol reduced AmB aggregation state in NE. NE-AmB was stable after 180days, and its hemolytic toxicity was lower than that observed for the conventional AmB. NE-AmB administered intravenously into animals infected by Leishmania major at 8mg/kg was capable of stabilizing the lesion size and reducing the parasitic load. Conclusion: These findings support the NE potential as a stable nanocarrier for AmB in the treatment of cutaneous leishmaniasis.

Amphotericin B release rate is the link between drug status in the liposomal bilayer and toxicity

Amphotericin B (AmB) is an amphiphilic drug commonly formulated in liposomes and administered intravenously to treat systemic fungal infections. Recent studies on the liposomal drug product have shed light on the AmB aggregation status in the bilayer, which heat treatment (curing) modifies. Although toxicity was found related to aggregation status - loose aggregates significantly more toxic than tight aggregates - the precise mechanism linking aggregation and toxicity was not well understood. This study directly measured drug release rate from various AmB liposomal preparations made with modified curing protocols to evaluate correlations among drug aggregation state, drug release, and in vitro toxicity. UV–Vis spectroscopy of these products detected unique curing-induced changes in the UV spectral features: a ∼25 nm blue-shift of the main absorption peak (λmax) in aqueous buffer and a decrease in the OD346/OD322 ratio upon thermal curing, reflecting tighter aggregation. In vitro release testing (IVRT) data showed, by applying and fitting first-order release kinetic models for one or two pools, that curing impacts two significant changes: a 3–5-fold drop in the overall drug release rate and a ten-fold decrease in the ratio between the loosely aggregated and the tightly aggregated, more thermodynamically stable drug pool. The kinetic data thus corroborated the trend independently deduced from the UV–Vis spectral data. The in vitro toxicity assay indicated a decreased toxicity with curing, as shown by the significantly increased concentration, causing half-maximal potassium release (TC50). The data suggest that the release of AmB requires dissociation of the tight complexes within the bilayer and that the reduced toxicity relates to this slower rate of dissociation. This study demonstrates the relationship between AmB aggregation status within the lipid bilayer and drug release (directly measured rate constants), providing a mechanistic link between aggregation status and in vitro toxicity in the liposomal formulations.

Cyclodextrin-based Pickering nanoemulsions containing amphotericin B: Part II. Formulation, antifungal activity, and chemical stability

In this study, cyclodextrin (CD)-stabilized, amphotericin B (AmB)-loaded Pickering and non-Pickering nanoemulsions (NEs) were developed using a high-pressure homogenization technique. The physicochemical and chemical properties of the formulations, including pH, osmolality, viscosity, particle size, size distribution, zeta potential, drug content, and entrapment efficiency, were acceptable for eye drops. The developed formulations showed a lower degree of AmB aggregation and lower hemolytic activity than commercial Amphotericin-B®. Because of the high AmB entrapment in their inner core, Pickering NEs displayed sustained in vitro drug release. The antifungal activity of AmB-loaded NEs was superior to that of intact AmB, comparable to that of Amphotericin-B® against C. albicans, and poorer than that of Amphotericin-B® against filamentous fungi. Stability studies showed that CD-based Pickering NEs exhibited good physical and chemical stability compared with their respective non-Pickering NEs. Thus, αCD-based Pickering NEs are a promising platform for loading AmB and should undergo in vivo studies for the treatment of fungal keratitis.

Improved In Vitro Anti-Mucorales Activity and Cytotoxicity of Amphotericin B with a Pegylated Surfactant.

The aim of this study was to evaluate the effect of the combination of amphotericin B (AmB) and various non-ionic surfactants on the anti-Mucorales activity of AmB, the toxicity of the combination on eukaryotic cells and the modification of AmB aggregation states. Checkerboards were performed on five genera of Mucorales (12 strains) using several combinations of different surfactants and AmB. These data were analyzed by an Emax model. The effect of surfactants on the cytotoxic activity of AmB was then evaluated for red blood cells and two eukaryotic cell lines by absorbance and propidium iodide internalization. Finally, the effect of polyethylene glycol (15)-hydroxystearate (PEG15HS) on the aggregation states of AmB was evaluated by UV-visible spectrometry. PEG15HS increased the efficacy of AmB on four of the five Mucorales genera, and MICs of AmB were decreased up to 68-fold for L. ramosa. PEG15HS was the only surfactant to not increase the cytotoxic activity of AmB. Finally, the analysis of AmB aggregation states showed that the increased efficacy of AmB and the absence of toxicity are related to an increase in monomeric and polyaggregated forms of AmB at the detriment of the dimeric form. In conclusion, PEG15HS increases the in vitro efficacy of AmB against Mucorales at low concentration, without increasing its toxicity; this combination could therefore be evaluated in the treatment of mucormycosis.

Self-assembling, supramolecular chemistry and pharmacology of amphotericin B: Poly-aggregates, oligomers and monomers

Antifungal drugs such as amphotericin B (AmB) interact with lipids and phospholipids located on fungal cell membranes to disrupt them and create pores, leading to cell apoptosis and therefore efficacy. At the same time, the interaction can also take place with cell components from mammalian cells, leading to toxicity. AmB was selected as a model antifungal drug due to the complexity of its supramolecular chemical structure which can self-assemble in three different aggregation states in aqueous media: monomer, oligomer (also known as dimer) and poly-aggregate. The interplay between AmB self-assembly and its efficacy or toxicity against fungal or mammalian cells is not yet fully understood. To the best of our knowledge, this is the first report that investigates the role of excipients in the supramolecular chemistry of AmB and the impact on its biological activity and toxicity. The monomeric state was obtained by complexation with cyclodextrins resulting in the most toxic state, which was attributed to the greater production of highly reactive oxygen species upon disruption of mammalian cell membranes, a less specific mechanism of action compared to the binding to the ergosterol located in fungal cell membranes. The interaction between AmB and sodium deoxycholate resulted in the oligomeric and poly-aggregated forms which bound more selectively to the ergosterol of fungal cell membranes. NMR combined with XRD studies elucidated the interaction between drug and excipient to achieve the AmB aggregation states, and ultimately, their diffusivity across membranes. A linear correlation between particle size and the efficacy/toxicity ratio was established allowing to modulate the biological effect of the drug and hence, to improve pharmacological regimens. However, particle size is not the only factor modulating the biological response but also the equilibrium of each state which dictates the fraction of free monomeric form available. Tuning the aggregation state of AmB formulations is a promising strategy to trigger a more selective response against fungal cells and to reduce the toxicity in mammalian cells.

Development of amphotericin B nanosuspensions for fungal keratitis therapy: effect of self-assembled γ-cyclodextrin

Fungal keratitis is one of the major causes in loss of vision and blindness. Amphotericin B (AmB) is the first-line treatment of keratitis caused by Candida spp. and related fungi. Due to its high molecular weight and low aqueous solubility, it hampers the ocular bioavailability. It is well-known that γ-cyclodextrin (γCD) can enhance AmB solubility. The aim of this study was to investigate the effect of the additives on γCD solubilization of AmB and develop AmB eye drop nanosuspensions. 0.18% (w/v) AmB nanosuspensions containing either 0.25% (w/v) chitosan or 0.10% (w/v) phospholipids (PL) in aqueous γCD eye drop medium were prepared by using high pressure homogenization technique. Physicochemical properties, in vitro mucoadhesive, in vitro permeation, degree of AmB aggregation, hemolytic activity and in vitro antifungal test were determined. The physicochemical properties of the nanosuspensions containing AmB/γCD nanoaggregates were in the acceptable range. The particle size of all formulations was in nanometer range. The degree of aggregation was less than 2.0 that meant the drug was not in the aggregate form when solubilized in γCD. These formulations demonstrated relatively low hemolytic activity and effectively improved the antifungal activity. AmB/γCD nanosuspension stabilized by 0.10% (w/v) PL is the promising formulation for further studies due to that it was excellent physical appearance, had mucoadhesive property, enhanced in vitro permeation, had low toxicity and improved the antifungal activity.

Amphotericin B Loaded Nanostructured Lipid Carriers for Parenteral Delivery: Characterization, Antifungal and In vitro Toxicity Assessment.

Amphotericin B (AmB) is important for the treatment of systemic fungal infections. Nowadays, only intravenous administration (IV) of AmB has been available due to its low aqueous solubility. Two forms of AmB are available. The first is Fungizone®, a mixture of AmB and sodium deoxcycholate that produces severe nephrotoxicity. The second are lipid-based formulations that reduce nephrotoxicity, but they are costly and require higher dose than Fungizone®. Thus, a cheaper delivery system with reduced AmB toxicity is required. To develop and characterize AmB loaded-nanostructured lipid carriers (AmB-loaded NLCs) for IV administration to reduce AmB toxicity. AmB-loaded NLCs with different solid lipids were prepared by the high-pressure homogenization technique. Their physicochemical properties and the drug release profile were examined. The molecular structure of AmB, antifungal and hemolysis activities of developed AmB-loaded NLCs were also evaluated. AmB-loaded NLCs ~110 to ~140 nm in diameter were successfully produced with a zeta potential of ~-19 mV and entrapment efficiency of ~75%. In vitro release showed fast release characteristics. AmB-loaded NLCs could reduce the AmB molecular aggregation as evident from the absorbance ratio of the first to the fourth peak showing a partial aggregation of AmB. This result suggested that AmB-loaded NLCs could offer less nephrotoxicity compared to Fungizone®. In vitro antifungal activity of AmB-loaded NLCs showed a minimum inhibitory concentration of 0.25 µgmL-1. AmB-loaded NLCs present high potential carriers for effective IV treatment with prolonged circulation time and reduced toxicity.

Preparation and In vitro Evaluation of Efficacy and Toxicity of Polysorbate 80-coated Bovine Serum Albumin Nanoparticles containing Amphotericin B.

In this study, bovine serum albumin (BSA) nanoparticles coated with polysorbate- 80 (PS-80) and containing amphotericin B (AmB) were developed using a coacervation method. The nanoparticles were spherical, had a uniform size distribution (polydispersity < 0.25), a small mean size (185 ± 5 nm), a high zeta potential (-38.0 ± 0.7 mV), and a high AmB encapsulation efficiency (93 ± 1%). The AmB release profile was prolonged and diffusion-controlled, resulting in a low degree of AmB aggregation in solution. The physicochemical characteristics of these AmB containing nanoparticles were evaluated by X-ray diffraction, Fourier transform infrared spectroscopy, differential scanning calorimetry, and derivative thermogravimetry and showed that the nanoencapsulation process lead to AmB amorphization while maintaining its chemical integrity. In a hemolysis assay, AmB-loaded PS-80-coated BSA nanoparticles demonstrated an absence of cytotoxicity toward erythrocytes, whereas pure and commercial AmB were highly hemolytic. In an assay to assess antifungal activity against Cryptococcus neoformans, AmB-charged PS-80-coated BSA nanoparticles were effective, however, due to the prolonged AmB release from the nanoparticles, the MIC was higher than for pure or commercial AmB. PS-80-coated BSA nanoparticles are potential carriers for the delivery of AmB for the treatment of Cryptococcus sp infections.

Method validation and nanoparticle characterization assays for an innovative amphothericin B formulation to reach increased stability and safety in infectious diseases

Drug Delivery Systems (DDS) of known drugs are prominent candidates towards new and more-effective treatments of various infectious diseases as they may increase drug bioavailability, control drug delivery and target the site of action. In this sense, the encapsulation of Amphotericin B (AmB) in Nanostructured Lipid Carriers (NLCs) designed with pH-sensible phospholipids to target infectious tissues was proposed and suitable analytical methods were validated, as well as, proper nanoparticle characterization were conducted. Characterization assays by Dinamic Light Scattering (DLS) and Atomic Force Microscopy demonstrated spherical particles with nanometric size 268.0±11.8nm and Zeta Potential −42.5±1.5mV suggestive of important stability. DSC/TGA and FT-IR assessments suggested mechanical encapsulation of AmB. The AmB aggregation study indicated that the encapsulation provided AmB at the lowest cytotoxic form, polyaggregate. Analytical methods were developed and validated according to regulatory agencies in order to fast and assertively determine AmB in nanoparticle suspension and, in Drug Encapsulation Efficiency (EE%), release and stability studies. The quantification method for AmB in NLC suspension presented linearity in 5.05–60.60μgmL−1 range (y=0.07659x+0.05344) and for AmB in receptor solution presented linearity in 0.15–10.00μgmL−1 range (y=54609x+263.1), both with r≥0.999. EE% was approximately 100% and according to the release results, at pH 7.4, a sustained controlled profile was observed for up 46h. In the meantime, a micellar AmB solution demonstrated an instability pattern after 7h of contact with the medium. Degradation and release studies under acid conditions (infectious condition) firstly depicted a prominent degradation of AmB (raw-material), with 20.3±3.5% at the first hour, reaching 43.3±7.0% after 7h of study. Next, particles faster disruption in acid environment was evidenced by measuring the NLC size variation by DLS and by the loss of the bluish sheen, characteristic of the nanostructured system macroscopically observed. Finally, safety studies depicted that NLC-AmB presented reduced toxicity in fibroblast cells, corroborating with AmB aggregated form study. Therefore, an innovative AmB formulation was fully characterized and it is a new proposal for in vivo investigations.

Unique aggregation of conjugated amphotericin B and its interaction with lipid membranes.

The purpose of this paper was to investigate the aggregation of amphotericin B (AMB) and AMB-arabinogalactan conjugate (AMB-AGC), and the interactions of these drugs with free and membrane-embedded sterols. Aggregation of AMB and AMB-AGC was studied by circular dichroic (CD) and UV absorbance spectroscopic techniques. The effect of liposomes on the spectra was utilized to investigate the interactions of aggregates with membrane-embedded sterols. Interaction with free sterols was studied by measuring sterols' effect on AMB/AMB-AGC susceptibility test. The results demonstrated that AMB-AGC forms unique aggregates in aqueous solution which differ from those formed by free AMB. Ergosterol and cholesterol embedded in liposomes, affected the CD spectra obtained for both AMB and AMB-AGC, indicating interactions of these sterols with both drugs. Interaction with both cholesterol and ergosterol resulted in an increase of AMB-AGC's minimal inhibitory concentration (MIC) in Candida albicans. In conclusion, AMB-AGC forms unique aggregates in aqueous solution; these aggregates interact with membrane-embedded cholesterol and ergosterol and with free sterols. These results indicate that the selectivity of AMB-AGC to fungal cells may not occur due to inability to bind cholesterol but probably as a result of this unique aggregation. Understanding this mechanism may help to develop a safer AMB formulation for therapy.

Biodegradable functional polycarbonate micelles for controlled release of amphotericin B.

There is a pressing need for a novel and cost-effective delivery system to reduce the toxicity induced by the antifungal agent, amphotericin B (AmB). In this study, phenylboronic acid-functionalized polycarbonate/PEG diblock copolymers were used to fabricate micelles for improved AmB-micelle interaction via the manipulation of hydrogen-bonding, boronate ester bond, ionic and hydrophobic interactions. Compared to free AmB and Fungizone®, the resultant micellar systems displayed improved stability while reducing non-specific toxicities without a compromise in antifungal activity. These findings demonstrate the potential of biodegradable functional polycarbonate micellar systems as promising carriers of AmB for the treatment of systemic fungal infections.

A study on characteristic of different sample pretreatment methods to evaluate the entrapment efficiency of liposomes.

To examine how methods affect the evaluation of entrapment efficiency (EE) of liposomes, four different sample pretreatment methods were adopted in the experiment. The four sample pretreatment methods were size-exclusion chromatography (SEC), solid-phase extraction (SPE), centrifugation ultrafiltration (CF-UF) and hollow fiber centrifugal ultrafiltration (HF-CF-UF). Amphotericin B (AmB), which could self-associate to form aggregates in water is adopted as the model drugs in this paper. In the present work, it was found that the characterization results of four methods were quite different. The EE of liposome by SEC was about 93%, only 5-13% using C18 or HLB columns, and approximately 100% by CF-UF. The EE of HF-CF-UF reached up to nearly 99.0%. Further, this paper revealed the reasons making the difference of EE among four methods. Conventional SEC may distort the authentic of EE of liposomes with mainly employing some small liposomes or excessive water as eluent. For SPE, cholesterol on liposome surface could interact with the stationary phase making it hard to elute with water, and increase the risk of liposome leakage. While for CF-UF, concentration polarization was a main limitation hindering unentrapped drug to pass through membrane, making unentrapped drug undetectable in liposome. HF-CF-UF could truly reflect EE of liposomes with the concentration of unentrapped AmB lower than 25.0μg/mL. However, when the concentration was higher than 25.0μg/mL, AmB aggregates could be entrapped by hollow fiber. From the above analysis, this paper came to the conclusion that each method had its own feature in characterization. This study provided a reasonable guideline for choosing methods to character the EE of liposome.

Amphotericin B-silver hybrid nanoparticles: synthesis, properties and antifungal activity

High antifungal activity is reported, in comparison with commercially available products, of a novel hybrid system based on silver nanoparticles synthesized using a popular antifungal macrocyclic polyene amphotericin B (AmB) acting both as a reducing and stabilizing/capping agent. The synthesis reaction proceeds in an alkaline environment which prevents aggregation of AmB itself and promotes nanoparticle formation. The innovative approach produces monodisperse (PDI=0.05), AmB-coated silver nanoparticles (AmB-AgNPs) with the diameter ~7nm. The products were characterized using imaging (electron microscopy) and spectroscopic (UV–vis and infrared absorption, dynamic light scattering and Raman scattering) methods. The nanoparticles were tested against Candida albicans, Aspergillus niger and Fusarium culmorum species. For cytotoxicity studies CCD-841CoTr and THP-1 cell lines were used. Particularly high antifungal activity of AmB-AgNPs is interpreted as the result of synergy between the antifungal activity of amphotericin B and silver antimicrobial properties (Ag+ ions release). From the Clinical EditorAmphotericin B (AmB) is a common agent used for the treatment against severe fungal infections. In this article, the authors described a new approach in using a combination of AmB and silver nanoparticles, in which the silver nanoparticles were synthesized and stabilized by AmB. Experimental data confirmed synergistic antifungal effects between amphotericin B and silver. This novel synthesis process could potentially be important in future drug development and fabrication.

A novel performing PEG-cholane nanoformulation for Amphotericin B delivery

A novel formulation for amphotericin B (AmB) delivery has been developed using micelle-forming 5kDa monomethoxy-polyethylene glycol functionalized with cholanic acid (PEG5kDa-cholane). This polymer was found to increase 103 times the AmB solubility with a 12:1 AmB/PEG5kDa-cholane molar ratio (2:1 w/w ratio). Dynamic light scattering and transmission electron microscopy analyses showed that PEG5kDa-cholane associated with AmB to form 30nm micelles. Isothermal titration calorimetry analyses performed at different pH showed that PEG5kDa-cholane interacts with AmB according to multiple-site association profiles. Affinity constants and enthalpy and entropy changes were found to depend on pH, suggesting that the polymer interaction depends on the AmB ionization and aggregation. The freeze-dried product could be promptly re-dispersed forming a colloidal dispersion with the biopharmaceutical features of the freshly prepared micelles, namely AmB solubility and micelle size. The dispersion was stable over one month incubation at room temperature. FT-infrared spectrometry, differential scanning calorimetry and X-ray diffractometry showed that in the freeze-dried product, AmB intimately interacts with PEG5kDa-cholane. In presence of serum albumin, AmB formulated with PEG5kDa-cholane was found to undergo less extensive and slower disaggregation than in Fungizone®. Antifungal activity studies performed using Candida albicans showed that AmB/PEG5kDa-cholane was 15% more active than AmB in buffer.

Efficacy and toxicity evaluation of new amphotericin B micelle systems for brain fungal infections

The aim of this work is to study the micelle systems of amphotericin B (AmB) and surfactant sodium deoxycholate (NaDC) as possible formulations to treat brain fungal infections. Fungizone® and Ambisome® were used as AmB references. The particle size, aggregation state, toxicity and efficacy of AmB:NaDC micelles were studied with increasing proportions of NaDC. Differences in the size and aggregation state of the reference formulations and micellar NaDC formulations might explain the differences in their distribution and therefore in their toxicity and efficacy. AmB:NaDC 1:0.8 and 1:1.5 nano-sized micelle systems showed a poly-aggregated form of AmB and small mean particle size (450–750nm). The AmB:NaDC 1:0.8 and AmB:NaDC 1:1.5 micelle systems studied showed an 8-fold lower toxicity than Fungizone®. Efficacy was examined in a murine candidiasis model by determining the survival rate and tissue burden reduction in kidneys and brain. The AmB:NaDC 1:1.5 micellar system at 5mg/kg of AmB and the highest amount of NaDC (7.5mg/kg) presented a good survival rate, and induced a major clearance of brain infection. The new AmB:NaDC 1:1.5 nano-sized micelle system is a promising formulation with a good efficacy/toxicity ratio, which can be attributed to its particle size, AmB aggregation state and NaDC content.