Chitosan‐coated antifungal formulations for nebulisation

Objectives The aim of this study was to produce and characterise amphotericin B (AmB) containing chitosan‐coated liposomes, and to determine their delivery from an air‐jet nebuliser. Methods Soya phosphatidylcholine : AmB (100 : 1) multilamellar vesicles were generated by dispersing ethanol‐based proliposomes with 0.9% sodium chloride or different concentrations of chitosan chloride. These liposomes were compared with vesicles produced by the film hydration method and micelles. AmB loading, particle size, zeta potential and antifungal activity were determined for formulations, which were delivered into a two‐stage impinger using a jet nebuliser. Key findings AmB incorporation was highest for liposomes produced from proliposomes and was greatest (approximately 80% loading) in chitosan‐coated formulations. Following nebulisation, approximately 60% of the AmB was deposited in the lower stage of the two‐stage impinger for liposomal formulations, for which the mean liposome size was reduced. Although AmB loading in deoxycholate micellar formulations was high (99%), a smaller dose of AmB was delivered to the lower stage of the two‐stage impinger compared to chitosan‐coated liposomes generated from proliposomes. Chitosan‐coated and uncoated liposomes loaded with AmB had antifungal activities against Candida albicans and C. tropicalis similar to AmB deoxycholate micelles, with a minimum inhibitory concentration of 0.5 µg/ml. Conclusions This study has demonstrated that chitosan‐coated liposomes, prepared by an ethanol‐based proliposome method, are a promising carrier system for the delivery of AmB using an air‐jet nebuliser, having a high drug‐loading that is likely to be effectively delivered to the peripheral airways for the treatment of pulmonary fungal infections.     

Antifungal and immunomodulatory activity of a novel cochleate for amphotericin B delivery against Sporothrix schenckii

Introduction: Sporotrichosis is an emergent subcutaneous mycoses caused by species of the Sporothrix schenckii complex. Amphotericin B (AmB) remains the main antifungal drug for the treatment of systemic infections, but its use is limited by toxicity reasons. AFCo3 is a novel cochleate containing detoxified LPS, which exhibits drug delivery and immunomodulating properties. Here, AFCo3 was used as the vehicle for AmB to evaluate the immunomodulatory and antifungal efficacy against S. schenckii in vitro and in vivo. Methods and results: The minimum inhibitory concentrations of AFCo3-AmB and AmB were 0.25 and 1 μg/mL respectively. The minimum fungicidal concentration was 0.5 μg/mL for AFCo3-AmB and 2 μg/mL for AmB. AFCo3-AmB was less cytotoxic than AmB for peritoneal macrophages, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and reduced the AmB-induced hemolysis in murine erythrocytes. AFCo3-AmB improved the intracellular killing of phagocytized yeast and it enhanced the in vitro production of IL-1β, TNF-α and NO in peritoneal macrophages. Moreover, AFCo3-AmB was more effective than AmB in reducing spleen and liver fungal burden after repeated (five days) intraperitoneal administration of 5 mg/kg of AmB, in a Balb/c model of systemic infection, associated to a significant induction of Th1/Th17 response. Finally, blood chemistry revealed that AFCo3-AmB did not cause changes suggestive of nephrotoxicity, such as increases in total proteins, albumin, creatinine and blood urea nitrogen that were caused by free AmB. Conclusions: AFCo3-AmB exhibited a significant immunomodulator action, reduced toxicity and improved antifungal action against S. schenckii, suggesting a potential use as AmB delivery for systemic sporotrichosis treatment.     

Release of Prednisolone and Inulin from a New Calcium-Alginate Chitosan-Coated Matrix System for Colonic Delivery

Putative colonic release formulations of calcium (Ca)-alginate coated with chitosan containing two different actives, prednisolone and inulin, were prepared in three different sizes, beads (D₅₀ = 2104 μm) and microparticles (D₅₀ = 354 and 136 μm). The formulations were tested in standard phosphate buffer and biorelevant Krebs bicarbonate buffer at pH 7.4, and were further evaluated in the presence of the bacterium E. coli. Product yield and encapsulation were higher with prednisolone than with inulin. In Krebs bicarbonate buffer, a clear relationship between particle size and prednisolone release was observed. In contrast, release of inulin was independent of the particle size. In phosphate buffer, the particles eroded quickly, whereas in Krebs buffer, the particles swelled slowly. The difference in behavior can be attributed to the formation of calcium phosphate in the phosphate buffer medium, which in turn weakens the Ca-alginate matrix core. In the presence of E. coli, the formulations were fermented and the release of prednisolone was accelerated. In conclusion, the buffer media affects formulation behavior and drug release, with the bicarbonate media providing a better simulation of in vivo behavior. Moreover, the susceptibility of the formulations to bacterial action indicates their suitability as carriers for colonic drug delivery.     

In vitro analysis of flufenamic acid activity against Candida albicans biofilms

In a recent high-throughput screen against specific Candida albicans drug targets, several compounds that exhibited non-specific antifungal activity were identified, including the non-steroidal anti-inflammatory drug flufenamic acid (FFA). This study sought to determine the effect of different doses of FFA, alone or in combination with fixed concentrations of the standard antifungal agents amphotericin B (AmB), caspofungin (CAS) or fluconazole (FLU), for the prevention and treatment of C. albicans biofilms. Biofilms were formed in a 96-well microplate followed by evaluation of antifungal activity using the XTT assay. FFA concentrations of ≥512 mg/L demonstrated >80% prevention of biofilm formation. FFA concentrations of 1024 mg/L demonstrated >85% reduction of mature biofilms. When FFA (≥8 mg/L) was used in combination with FLU (32 mg/L), antifungal activity increased to 99% for the prevention of biofilm formation. Similarly, when a FFA concentration of ≥8 mg/L was used in combination with either AmB (0.25 mg/L) or CAS (0.125 mg/L), antifungal activity also increased up to 99% for the prevention of biofilm formation. The inhibitory effect of FFA on C. albicans biofilms has not been reported previously, therefore these findings suggest that FFA in combination with traditional antifungals might be useful for the treatment and prevention of C. albicans biofilms.     

Enhanced Oral Absorption of Paclitaxel in N-Deoxycholic Acid-N,O-Hydroxyethyl Chitosan Micellar System

The overall goal of this study was to develop a micellar system of paclitaxel (PTX) to enhance its oral absorption. An amphiphilic chitosan derivative, N-deoxycholic acid-N, O-hydroxyethyl chitosan (DHC), was synthesized and characterized by FTIR, 1H NMR, elemental analysis, and X-ray diffraction (XRD) techniques. The degree of substitution (DS) of hydroxyethyl group and deoxycholic acid group ranged from 89.5–114.5% and 1.11-8.17%, respectively. The critical micelle concentration (CMC) values of DHC decreased from 0.26 to 0.16 mg/mL as the DS of deoxycholic acid group increased. PTX was successfully loaded in DHC micelles with a high drug loading (31.68 ± 0.14%) and entrapment efficiency (77.57 ± 0.51%). The particle size of PTX-loaded DHC micelles ranged from 203.35 ± 2.19 to 236.70 ± 3.40 nm as the DS of deoxycholic acid group increased. After orally administration of PTX-loaded DHC micelles, the bioavailability was threefold compared with that of an orally dosed Taxol®. The single-pass intestinal perfusion studies (SPIP) showed that the intestinal absorption of micelles was via endocytosis involving a saturable process and a p-glycoprotein (P-gp)-inde-pendent way. All these indicated that the DHC micelles might be a promising tool for oral delivery of poorly water-soluble drugs. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:4543–4553, 2010     

Supersaturated polymeric micelles for oral cyclosporine A delivery

Polymeric micelles provide a promising platform for improving oral absorption of poorly soluble drugs. However, improved understanding of how drug retention within the hydrophobic micelle core can reduce drug absorption is required. We designed supersaturated polymeric micelles (Super-PMs) to increase molecularly dissolved drug concentration and gain an insight into the effect of the degree of supersaturation on oral absorption of cyclosporine A (CsA) in rats. The drug release from Super-PMs increased with an increase in initial supersaturation degrees in micelles. The cellular uptake of coumarin-6 was reduced by the retention of drug in polymer micelles. The transport flux of CsA across Caco-2 monolayer was increased with initial supersaturation degrees of 0.81–3.53 (p < 0.05). However, increase in supersaturation to 5.64 actually resulted in decreased CsA transport. The same trend was observed in a rat in vivo absorption study, in which the highest bioavailability of 134.6 ± 24.7% (relative to a commercial product, Sandimmun Neoral®, p < 0.01) was achieved when the supersaturation degree was 3.53. These results demonstrated that Super-PMs were a promising drug delivery system for compounds with low aqueous solubility. This study also provided an experimental proof for the hypothesis that moderately supersaturated formulations are valuable alternative to high supersaturation formulations, resulting in optimal in vivo performance, and the degree of supersaturation should be carefully controlled to optimize drug absorption.     

Design,synthesis and in vitro evaluation of mucoadhesive p-coumarate-thiolated-chitosan as a hydrophobic drug carriers

A hydrophobic mucoadhesive thiolated chitosan for hydrophobic drug delivery was designed and prepared by conjugating p-coumaric acid (pCA) to increase hydrophobic compatibility with drug via pi–pi interaction and then covalently linking homocysteine thiolactone (HT) to the pCA-chitosan to increase the mucoadhesive properties. The degree of substituted phenolics in the modified chitosan was about 7.21 ± 0.05 mg gallic acid equivalents (GAE)/g. The pCA-HT-chitosan formed from a 24 h HT conjugation reaction time showed the highest yield of grafted thiol groups (∼17.6 μmol/g) and the strongest mucoadhesive property, being about 10-, 2- and 1.6-fold more than that for the unmodified chitosan at pH 1.2, 4.0 and 6.4, respectively. Piperine (PIP) as a model hydrophobic drug was encapsulated in pCA-HT-chitosan microparticles via electrospray ionization with an encapsulation efficiency of over 80%. In vitro release studies showed a sustained release of PIP to >75% over 12 h between pH 1.2 and 6.4.     

PEGylated chitosan-based polymer micelle as an intracellular delivery carrier for anti-tumor targeting therapy

Stearic acid-grafted chitosan oligosaccharide (CSO-SA) micelles presented a potential candidate for intracellular drug delivery carrier due to its special spatial structure. In this article, CSO-SA was further modified by polyethylene glycol (PEG). The physicochemical properties of PEGylated CSO-SA (PEG-CSO-SA) micelles were characterized. After PEGylation, the critical micelle concentration (CMC) of PEG-CSO-SA had no significant change; the micelle size increased; and the zeta potential decreased. The cellular uptake of CSO-SA micelles before and after PEGylation in macrophage RAW264.7, immortalized rat liver cells BRL-3A and human liver tumor cells HepG2 was studied. About 58.4 ± 0.63% of CSO-SA micelles were uptaked by RAW264.7 in 24 h, however, only 17.7 ± 0.94% of PEG-CSO-SA micelles were internalized into RAW264.7 after the CSO-SA was modified with PEG in five molar times. Meanwhile, there were no changes in the uptake after PEGylation of CSO-SA in BRL-3A and HepG2. Using mitomycin C as a model drug, the in vitro anti-tumor activities of the drug loaded in the micelles were investigated. The 50% cellular growth inhibition (IC₅₀) of the drug decreased from 1.97 ± 0.2 to 0.13 ± 0.02 μg/mL after mitomycin C was loaded into CSO-SA micelles, and the IC₅₀ value of the drug had no obvious change when the CSO-SA was modified by PEG.     

The chemotherapeutic potential of doxorubicin-loaded PEG-b-PLGA nanopolymersomes in mouse breast cancer model

Vesicles of mPEG-PLGA block copolymer were developed to deliver a therapeutic quantity of doxorubicin (DOX) for breast cancer treatment. The DOX-loaded nanoparticles (NPs) were prepared by the pH-gradient method and then evaluated in terms of morphology, size, DOX encapsulation efficiency and in vitro drug release mechanism.The PEG-PLGA nanopolymersomes were 134 ± 1.2 nm spherical NPs with a narrow size distribution (PDI = 0.121). DOX was entrapped in mPEG-PLGA nanopolymersomes with an encapsulation efficiency and a loading content of 91.25 ± 4.27% and 7.3 ± 0.34%, respectively. The DOX-loaded nanopolymersomes were found to be stable, demonstrating no significant change in particle size and encapsulation efficiency (EE%) during the 6-month storage period of the lyophilized powder at 4 °C. The nanopolymersomes sustained the release of DOX. In cytotoxicity studies of 4T1 cell line samples, free DOX showed a higher cytotoxicity (IC50 = 1.76 μg/mL) than did DOX-loaded nanopolymersomes (15.82 μg/mL) in vitro. In order to evaluate the antitumor efficacy and biodistribution of DOX-loaded nanopolymersomes, murine breast tumors were established on the BALB/c mice, and in vivo studies were performed. The obtained results demonstrated that the prepared drug delivery system was highly effective against a murine breast cancer tumor model and successfully accumulated in the tumor site through an enhanced permeation and retention mechanism.In vivo studies also proved that DOX-loaded nanopolymersomes are stable in blood circulation and could be considered a promising and effective DOX delivery system for breast cancer treatment.     

Self-Assembled Methoxy Poly(Ethylene Glycol)-Cholesterol Micelles for Hydrophobic Drug Delivery

To promote the application of methoxy poly(ethylene glycol)-cholesterol (mPEG—Chol), mPEG–Chol was used to prepare core-shell micelles encapsulating poorly water-soluble docetaxel (DTX-PM) by modified cosolvent evaporation method. Approaches to enhance DTX entrapment efficiency (EE) and minimize particle size were investigated in detail, including organic and aqueous phase composition, organic/aqueous phase ratio, and polymer concentration. In optimal formulation, micelles had higher EE (97.6%) and drug loading (4.76%) with the diameter of 13.76 ± 0.68 nm and polydispersity index of 0.213 ± 0.006. Transmission electron microscopy (TEM) showed that the micelles were spherical, and differential scanning calorimetry (DSC) analysis proved that DTX was successfully entrapped into mPEG–Chol micelles. The in vitro cytotoxicity experiments displayed that blank micelles had no effect on the growth of SKOV-3, BXPC-3, A549, and HepG-2 cells, demonstrating that mPEG–Chol was one of the biocompatible biomaterials. The half inhibition concentration of DTX-PM on SKOV-3, BXPC-3, A549, and HepG-2 cells were 10.08, 7.6, 28.37, and 125.75 ng/mL, respectively. DTX-PM had the similar antitumor activity to free DTX, indicating that mPEG–Chol was a promising micellar vector for hydrophobic drug delivery. In addition, this work provided a new and facile approach to prepare drug-loaded micelles with controllable performances. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1054–1062, 2013     

Lipid nanoparticles containing oryzalin for the treatment of leishmaniasis

Oryzalin is a dinitroaniline drug that has attracted recent interest for the treatment of leishmaniasis. Its use as an antiparasitic therapeutic agent is limited by the low water solubility associated with an in vivo rapid clearance, leading to the administration of larger and possibly toxic doses in in vivo studies, and the use of solvents that may lead to undesirable side effects. In the present work oryzalin-containing lipid nanoparticles were produced by a emulsion–solvent evaporation technique using a composition suitable for parenteral administration, i.e., tripalmitin (solid lipid) and a complex mixture of three emulsifying agents (soya lecithin, Tween® 20 and sodium deoxycholate). Physicochemical characterization included the determination of mean particle size, polydispersity index, zeta potential, encapsulation efficiency and DSC studies. Final formulations revealed values of <140 nm (PI < 0.2) and zeta potential of ≈?35 mV, as well as encapsulation efficiency >75%. The effects of various processing parameters, such as lipid and surfactant and composition and concentration, as well as the stability during the harsh procedures of autoclaving (121 °C/15 min) and freeze-drying were also evaluated. Formulations revealed to be stable throughout freeze-drying and moist-heath sterilization without significant variations on physicochemical properties and no significant oryzalin losses. The use of a complex surfactant mixture proved crucial for preserving formulation stability. Particularly, lecithin appears as a key component in the stabilization of tripalmitin-based oryzalin-containing lipid nanoparticles. Finally, cell viability studies demonstrated that the incorporation of oryzalin in nanoparticles decreases cytotoxicity, thus suggesting this strategy may improve tolerability and therapeutic index of dinitroanilines.     

Pharmaceutical Nanotechnology Nanoparticle-Based Topical Ophthalmic Formulations for Sustained Celecoxib Release

Celecoxib-loaded NPs were prepared from biodegradable polymers such as poly- e-caprolactone (PCL), poly(L-lactide) (PLA), and poly(D,L-lactide-co-glycolide) (PLGA) by spontaneous emulsification solvent diffusion method. Different concentrations of polymers, emulsifier, and cosurfactants were used for formulation optimization. Nanoparticles (NPs) were characterized regarding their particle size, PDI, zeta potential, shape, morphology, and drug content. Celecoxib-loaded NPs were incorporated into eye drops, in situ gelling system, and gel and characterized regarding their pH, viscosity, uniformity of drug content, in vitro release, and cytotoxicity. The results of optimized celecoxib-loaded PCL-, PLGA-, and PLA-NPs, respectively, are particle size 119 ± 4, 126.67 ± 7.08, and 135.33 ± 4.15 nm; zeta potential ? 22.43 ± 2.91, ? 25.46 ± 2.35, and ? 31.81 ± 2.54 mV; and encapsulation efficiency 93.44 ± 3.6%, 86.00 ± 1.67%, and 79.04 ± 2.6%. TEM analyses revealed that NPs have spherical shapes with dense core and distinct coat. Formulations possessed uniform drug content with pH and viscosity compatible with the eye. Formulations showed sustained release without any burst effect with the Higuchi non-Fickian diffusion mechanism. Cytotoxicity studies revealed that all formulations are nontoxic. Our formulations provide a great deal of flexibility to formulation scientist whereby sizes and zeta potentials of our NPs can be tuned to suit the need using scalable and robust methodologies. These formulations can thus serve as a potential drug delivery system for both anterior and posterior eye diseases. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1036–1053, 2013     

PHARMACEUTICAL NANOTECHNOLOGY: A Novel Composite Hydrogel Based on Chitosan and Inorganic Phosphate for Local Drug Delivery of Camptothecin Nanocolloids

In attempt to overcome the problem of low water solubility and severe toxicity of camptothecin (CPT) after intravenous administration, a novel drug carrier system based on chitosan (CS) and dibasic sodium phosphate (DSP) has been developed in this paper to encapsulate CPT intending for local administration. Nanocolloids of CPT with size about 500 nm were first prepared, followed by encapsulation in the chitosan/dibasic sodium phosphate (CS/DSP) formulation. The formulation was sol state below 37°C and transformed to nonflowing gel state at 37°C. Encapsulation of CPT nanocolloids had greatly effect on the gelling time as well as the micro-structure of hydrogel. In vitro and in vivo degradation studies revealed that the developed CS/DSP hydrogel was biodegradable and biocompatible. In vitro release study revealed that CPT released from CS/DSP hydrogel in an extended period with about 70% of total CPT released from hydrogel after 18 days. Furthermore, nearly 90% of CPT in the chitosan hydrogels could be preserved in the lactone form (active form) even after 7 days's storage at 37°C. Furthermore, in vitro cytotoxicity of CPT nanocolloids on SKOV3 human ovarian cancer cells suggested the well anti-tumor cell efficiency could be gained at a lower concentration.     

Formulation and evaluation of a bioadhesive patch for buccal delivery of tizanidine

Tizanidine hydrochloride (THCl) is an antispasmodic agent which undergoes extensive first pass metabolism making it a possible candidate for buccal delivery. The aim of this study was to prepare a monolayered buccal patch containing THCl using the emulsification solvent evaporation method. Fourteen formulations were prepared using the polymers Eudragit^® RS 100 or Eudragit^® RL 100 and chitosan. Polymer solutions in acetone were combined with a THCl aqueous solution (in some cases containing chitosan) by homogenization at 9000 rpm for 2 min in the presence of triethyl citrate as plasticizer and cast in novel Teflon molds. Physicochemical properties such as film thickness, in vitro drug release and in vitro mucoadhesion were evaluated after which permeation across sheep buccal mucosa was examined in terms of flux and lag time. Formulations prepared using a Eudragit^® polymer alone exhibited satisfactory physicomechanical properties but lacked a gradual in vitro drug release pattern. Incorporation of chitosan into formulations resulted in the formation of a porous structure which did exhibit gradual release of drug. In conclusion, THCl can be delivered by a buccal patch formulated as a blend of Eudragit^® and chitosan, the latter being necessary to achieve gradual drug release.     

Nanosized ethosomes bearing ketoprofen for improved transdermal delivery

The potential of ethosomes for delivering ketoprofen via skin was evaluated. The ethosomes were prepared, optimized and characterized. Vesicular shape, size and entrapment efficiency were determined by transmission electron microscopy, dynamic light scattering and minicolumn centrifugation technique, respectively. Vesicle sizes varied from 120.3±6.1 to 410.2±21.8 nm depending on the concentrations of soya phosphatidyl choline (SPC) and ethanol. Entrapment efficiency increased with concentrations of SPC and ethanol. The formulations exhibited entrapment efficiencies of 42–78%. In vitro release through cellophane membrane showed sustained release of drug from ethosomal formulations in contrast to hydroalcoholic drug solution (HA), which released most of the drug within 2–3 h. In vitro drug permeation across human skin revealed improved drug permeation and higher transdermal flux with ethosomal formulations compared to hydroethanolic drug solution. Kinetics of in vitro skin permeation showed zero order drug release from formulations. Based on in vitro transdermal flux, the estimated steady state in vivo plasma concentration from ethosomes attained therapeutic drug levels whereas hydroalcoholic drug solution exhibited sub therapeutic drug concentration with a patch size of 50 cm². Skin permeation of ethosomal formulations assessed by confocal microscopy revealed enhanced permeation of Rhodamine 123 loaded formulation in comparison to the hydroalcoholic solution.     

Amphotericin B releasing nanoparticle topical treatment of Candida spp. in the setting of a burn wound

Candida spp. infection in the context of burn wounds leads to invasive disease with a 14–70% mortality rate. Unfortunately, current administrations of AmB, an important therapeutic demonstrating minimal resistance, are only available via potentially cytotoxic IV infusions. In order to circumvent these sequelae, we investigated the efficacy of nanoparticle encapsulated AmB (AmB-np) as a topical therapeutic against Candida spp. (drug release equilibrated solubilized AmB [AmB-sol] included as control). Clinical strains demonstrated equal or enhanced killing efficacy with 72.4–91.1% growth reduction by 4 hours. AmB-nps resulted in statistically significant reduction of fungal biofilm metabolic activity ranging from 80% to 95% viability reduction (P< 0.001). Using a murine full-thickness burn model, AmB-np exhibited a quicker efficiency in fungal clearance versus AmB-sol by day three, although wound healing rates were similar. These data support the concept that AmB-np can function as a topical antifungal in the setting of a burn wound.From the Clinical EditorThe control of fungal infections with Candida species remains a challenge in the context of burn wounds. A nanoencapsulated topical amphotericin-B compound was studied in a murine model of full thickness burn injury, showing remarkable efficacy in controlling Candida infection. This may become a viable alternative to the potentially toxic intravenous formulations.     

Cytotoxic effect of Amphotericin B in a myofibroblast cell line

In this study we investigate whether Amphotericin B (AmB), a widely used antifungal agent, could decrease the proliferation of a myofibroblast cell line – GRX, a model of activated hepatic stellate cells (HSC). Three different hepatic cell lines (GRX, Hep G2 and ARL-6) were treated with two concentrations of AmB (1.25 μg/mL or 2.50 μg/mL). Cytotoxicity was assessed by MTT assay. The effects of AmB on GRX migration was evaluated by Wound-healing Assay. Cell cycle arrest was investigated by flow cytometry. Apoptosis and autophagy were analyzed by Caspase 3 and LC3 immunostaining, respectively. Treatment with AmB 1.25 or 2.50 μg/mL showed a decrease in viability of GRX cells. This decrease was not observed for Hep G2 or ARL-6 in any of the two AmB concentrations tested. GRX cells treated with 1.25 μg/mL AmB were unable to close the wound after 96 h. Cell cycle analysis showed an increase in sub-G1 population and a decrease in G2/M population in AmB-treated cells. In addition, AmB-treated GRX cells showed increased expression of LC-3 and Caspase-3 by immunohistochemistry, suggesting an increase in both autophagy and apoptosis. Here we show that AmB is cytotoxic for GRX cells, a model of activated HSC, but not for hepatic lineages HepG2 and ARL6.     

Investigation of the micellar effect of pluronic P85 on P-glycoprotein inhibition: Cell accumulation and equilibrium dialysis studies

The objective of this study was: (1) to characterize the P-gp inhibitory effect of different concentrations of Pluronic P85 on anti-HIV-1 drug cellular accumulation, and (2) to investigate the relationship between cellular accumulation and free fraction of drug. Cellular accumulation studies in MDCKII-WT and MDCKII-MDR1 cell monolayers showed a biphasic dose response characterized by decline in accumulation at Pluronic concentrations greater than the CMC. This phenomenon was independent of the inhibition of P-gp efflux by Pluronic. Cell-free equilibrium dialysis was used to determine the effect of Pluronic P85 on drug free fraction and the affinity of Pluronic micelles for drug was modeled. Nelfinavir and saquinavir associated extensively with micelles and equilibrium free fractions were low at P85 concentrations above the CMC, with association constants being in the order nelfinavir > saquinavir ? abacavir. Abacavir, a P-gp substrate, showed no association with micelles yet showed a biphasic response in cellular accumulation. These data suggest that, above the CMC, inhibition of P-gp is not affected but rather factors such as micellar trapping could contribute to decreased accumulation. Therefore, the in vitro evaluation of the effect of Pluronic formulations on active transport should take into account both the physicochemical properties of drug and the composition of Pluronic. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4170–4190, 2009     

Antitumoral activity of camptothecin-loaded nanoparticles in 9L rat glioma model

Camptothecin (CPT), a plant alkaloid, is a potent anticancer drug in cell culture studies but it is clinically inactive due to rapid hydrolysis under physiological conditions. The drug exists in two forms depending on the pH value, an active lactone form at pH below 5 and an inactive carboxylate form at basic pH and this is a reversible reaction. In this study, nanoparticulate delivery systems were developed with either amphiphilic cyclodextrins, poly(lactide-co-glycolide) or poly-?-caprolactone in order to maintain the active lactone form and prevent the drug from hydrolysis. All nanoparticles were prepared with nanoprecipitation technique. Mean particle sizes were 130–280 nm and surface charges were negative. The encapsulation efficiency was significantly higher for amphiphilic cyclodextrin nanoparticles when compared to polymeric nanoparticles. Nanoparticle formulations based on cyclodextrins showed a controlled release profile extended up to 12 days. 6-O-Capro-β-cyclodextrin (1.44 μg/60 μL CPT) and concentrated 6-O-Capro-β-cyclodextrin (2.88 μg/60 μL CPT) nanoparticles significantly modified the growth or lethality of the 9L gliomas, since the median survival time was 26 days for the untreated group and between 27 and 33 days for amphiphilic cyclodextrin nanoparticle groups. These results indicate that, CPT-loaded amphiphilic cyclodextrin nanoparticles may provide a promising carrier system for the effective delivery of CPT in comparison to polymeric analogues.     

Amphotericin B-loaded polymeric nanoparticles: formulation optimization by factorial design

In this study, PLGA or PLGA-PEG blend nanoparticles were developed loading amphotericin B (AmB), an antifungal agent broadly used in therapy. A 2²?×?3¹ factorial experimental design was conducted to indicate an optimal formulation of nanoparticles containing AmB and demonstrate the influence of the interactions of components on the mean particle size and drug encapsulation efficiency. The independent variables analyzed were polymer amount (two levels) and organic phase (three factors in one level). The parameters methanol as cosolvent and higher polymer amount originated from the higher AmB encapsulation, but with the larger particle size. The selected optimized parameters were set as the lower polymer amount and ethyl acetate as cosolvent in organic phase, for both PLGA and PLGA-PEG nanoparticles. These parameters originated from nanoparticles with the size of 189.5?±?90?nm and 169?±?6.9?nm and AmB encapsulation efficiency of 94.0?±?1.3% and 92.8?±?2.9% for PLGA and PLGA-PEG nanoparticles, respectively. Additionally, these formulations showed a narrow size distribution indicating homogeneity in the particle size. PLGA and PLGA-PEG nanoparticles are potential carrier for AmB delivery and the factorial design presented an important tool in optimizing nanoparticles formulations.