Helder Ferreira Teixeira

Development and characterization of parenteral nanoemulsions containing thalidomide.

This study reports the development of nanoemulsions intended for intravenous administration of thalidomide (THD). The formulations were prepared by spontaneous emulsification method and optimized with respect to thalidomide (0.01-0.05%, w/w), and hydrophilic emulsifier (polysorbate 80; 0.5-4.0%, w/w) content. The formulations were evaluated concerning physical appearance and drug crystallization; droplet size; zeta potential and drug assay. Only the formulation containing 0.01% THD and 0.5% polysorbate kept its properties in a satisfactory range over the evaluated period (60 days), i.e. droplet size around 200nm, drug content around 95% and zeta potential around -30mV. The transmission electron microscopy revealed emulsion droplets almost spherical in shape confirming the results obtained by photon correlation spectroscopy. Drug crystallization observed for higher content (THD 0.05%, w/w) nanoemulsions was investigated. The crystals observed at optical microscopy presented a different crystal habit compared to that of the raw material used. It was speculated whether the kind of THD polymorph employed could influence nanoemulsion formulation. Formulations were prepared with either one of THD polymorphs (β- or α-) and crystals were characterized by fourier transformed infrared spectroscopy (FTIR) and X-ray diffraction (XRD). It was observed that regardless of the polymorph employed (β- or α-), drug crystallization occurs in the α-form. THD solubility in oils was not influenced by the polymorphic form. In addition, the in vitro dissolution profile of the selected formulation (THD 0.01%, w/w; polysorbate 0.5%, w/w) was assessed by bulk-equilibrium reverse dialysis sac technique and demonstrated a release profile similar to that of a THD acetonitrile solution, with around 95% THD being dissolved within 4h. Finally, a pharmacokinetic simulation of an intravenous infusion of 250mL of the selected nanoemulsion suggests that the parenteral administration of a dose as low as 25mg might lead to therapeutic plasma concentrations of thalidomide.

Daidzein/cyclodextrin/hydrophilic polymer ternary systems

Objective: To evaluate the effect of different cyclodextrins (β-cyclodextrin [β-CD], methyl-β-cyclodextrin [Mβ-CD], or hydroxypropyl-β-cyclodextrin [HPβ-CD]) and/or hydrophilic polymers (carboxymethylcellulose, hydroxypropylmethylcellulose [HPMC], polyethyleneglycol, or polyvinylpyrrolidone [PVP]) on daidzein solubility in water. Materials and methods: The corresponding associations were characterized in aqueous media using phase-solubility studies. The morphology of daidzein/cyclodextrin freeze-dried complexes was characterized using scanning electron microscopy, and their spatial configuration was proposed by means of nuclear magnetic resonance spectroscopy. Results and discussion: In the presence of 6 mM of cyclodextrins, the solubility of daidzein in water was significantly enhanced: 5.7-fold (β-CD), 7.2-fold (Mβ-CD), and 9.4-fold (HPβ-CD). The analysis of the three solid complexes proved that the formation of inclusion complexes occurred through the insertion of the B and C rings of daidzein molecule into the cyclodextrins cavity. The association of daidzein/cyclodextrin complexes to the hydrophilic polymers HPMC or PVP (1%, w/w) was able to improve the solubility of daidzein even further. Conclusion: The highest solubilizing effect was obtained for daidzein/HPβ-CD/PVP ternary system (12.7-fold).

Optimization of headspace solid-phase microextraction for analysis of β-caryophyllene in a nanoemulsion dosage form prepared with copaiba (Copaifera multijuga Hayne) oil

Recent studies have shown the anti-inflammatory activity of Copaiba oils may be addressed to the high content of β-caryophyllene, the most common sesquiterpene detected, especially in the Copaifera multijuga Hayne species. In the present study, nanoemulsions were proposed as a delivery system for copaiba oil in view to treat locally inflamed skin. This article describes the optimization and validation of a stability-indicating SPME-GC method, for β-caryophyllene analysis in the nanoemulsions produced by high pressure homogenization. SPME methods are performed with PDMS (polydimethylsiloxane) fiber (100 μm). Three SPME parameters were evaluated by a three-level-three-factor Box-Behnken factorial design as potentially affecting the technique efficiency. According to the results obtained, the best conditions to extract β-caryophyllene were: (i) sampling temperature of 45°C, (ii) sampling time of 20 min and (iii) no NaCl addition. Results coming from the forced degradation tests showed a reduction of β-caryophyllene peak area when both caryophyllene methanolic solution and nanoemulsions were exposed to acid hydrolysis, UV-A irradiation, oxidative (H(2)O(2)) and thermolitic (60°C) conditions. Such reduction occurred in lower extent in the nanoemulsions, suggesting a protective effect of the formulation to β-caryophyllene content. Since no degradation products were detected in the same retention time of β-caryophyllene, the specificity of the method was demonstrated. The method was linear in the range of 0.14-0.68 μg mL(-1) of β-caryophyllene (r(2)>0.999), and was also validated for precision (R.S.D.≤5.0%), accuracy (97.85-101.87%) and robustness. Finally, the method was applied to quantification of β-caryophyllene content in the developed formulations.

Nanoemulsões como sistemas de liberação parenteral de fármacos

Lipid nanoemulsions have recently been proposed as parenteral delivery systems for poorly-soluble drugs. These systems consist of nanoscale oil/water dispersions stabilized by an appropriate surfactant system in which the drug is incorporated into the oil core and/or adsorbed at the interface. This article reviews technological aspects of such nanosystems, including their composition, preparation methods, and physicochemical properties. From this review, it was possible to identify five groups of nanoemulsions based on their composition. Biopharmaceutical aspects of formulations containing some commercially available drugs (diazepam, propofol, dexamethasone, etomidate, flurbiprofen and prostaglandin E1) were then discussed.

Cationic nanoemulsion as a delivery system for oligonucleotides targeting malarial topoisomerase II

A promising strategy based on the antisense oligonucleotides against the Plasmodium falciparum topoisomerase II has been considered using cationic nanoemulsion as oligonucleotide delivery system. Phosphodiester and chemically modified phosphorothioate oligonucleotides bearing negative charges were adsorbed on positively charged emulsion composed of medium chain triglycerides, egg lecithin, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and water, at different +/- charge ratios (positive charges from cationic lipid/negative charges from oligonucleotide): +0.5/-, +2/-, +4/- and +6/-. The physicochemical properties of the complexes were determined, as well as their stability in culture medium. Their interaction with erythrocytes through hemolysis, binding experiments and confocal microscopy were also evaluated. Finally, the in vitro evaluation of parasite growth and reinfection capacity was performed. The overall results showed that antisense oligonucleotides against P. falciparum topoisomerase II gene can be efficiently adsorbed onto a cationic nanoemulsion forming complexes. Whereas unloaded nanoemulsion displayed an hemolytic effect due to the presence of the cationic lipid, this was not the case of loaded nanoemulsion at low +/- ratios. Oligonucleotide-loaded nanoemulsions were found to be located inside the infected erythrocytes, inhibiting efficiently parasite growth (until 80%) and causing a delay in P. falciparum life cycle.

Pharmacokinetic study of a carbamazepine nanoemulsion in beagle dogs

This work describes the pharmacokinetics of a novel carbamazepine nanoemulsion. The plasma concentration profiles were determined in beagle dogs after i.v. bolus administration of a 5 mg/kg carbamazepine nanoemulsion and compared to the corresponding carbamazepine/hydroxypropyl-beta-cyclodextrin complex solution. Both formulations showed similar pharmacokinetic profiles and could represent valuable formulations in case of emergencies, when a rapid action in the central nervous system is desirable.

Physicochemical properties of lecithin-based nanoemulsions obtained by spontaneous emulsification or high-pressure homogenization

Nanoemulsions composed of a medium-chain triglyceride oil core stabilized by rapeseed or sunflower lecithins were prepared by spontaneous emulsification and high-pressure homogenization. These nanoemulsions are compared with formulations stabilized by egg lecithin. Nanoemulsions obtained by high-pressure homogenization display larger droplet size (230 to 440 nm) compared with those obtained by spontaneous emulsification (190 to 310 nm). The zeta potentials of the emulsions were negative and below -25 mV. Zeta potential inversion occurred between pH 3.0 and 4.0. The results demonstrate the feasibility of preparing lipid emulsions comprising rapeseed or sunflower lecithins by spontaneous emulsification and high-pressure homogenization.

Nanoemulsions containing a synthetic chalcone as an alternative for treating cutaneous leshmaniasis: optimization using a full factorial design

Nanoemulsions are drug delivery systems that may increase the penetration of lipophilic compounds through the skin, enhancing their topical effect. Chalcones are compounds of low water solubility that have been described as promising molecules for the treatment of cutaneous leishmaniasis (CL). In this context, the aim of this work was to optimize the development of a nanoemulsion containing a synthetic chalcone for CL treatment using a 22 full factorial design. The formulations were prepared by spontaneous emulsification and the experimental design studied the influence of two independent variables (type of surfactant – soybean lecithin or sorbitan monooleate and type of co-surfactants – polysorbate 20 or polysorbate 80) on the physicochemical characteristics of the nanoemulsions, as well as on the skin permeation/retention of the synthetic chalcone in porcine skin. In order to evaluate the stability of the systems, the antileishmanial assay was performed against Leishmania amazonensis 24 hours and 60 days after the preparation of the nanoemulsions. The formulation composed of soybean lecithin and polysorbate 20 presented suitable physicochemical characteristics (droplet size 171.9 nm; polydispersity index 0.14; zeta potential −39.43 mV; pH 5.16; and viscosity 2.00 cP), drug content (91.09%) and the highest retention in dermis (3.03 µg·g−1) – the main response of interest – confirmed by confocal microscopy. This formulation also presented better stability of leishmanicidal activity in vitro against L. amazonensis amastigote forms (half maximal inhibitory concentration value 0.32±0.05 µM), which confirmed the potential of the nanoemulsion soybean lecithin and polysorbate 20 for CL treatment.

Influence of phospholipid composition on cationic emulsions/DNA complexes: physicochemical properties, cytotoxicity, and transfection on Hep G2 cells

Background Cationic nanoemulsions have been recently considered as potential delivery systems for nucleic acids. This study reports the influence of phospholipids on the properties of cationic nanoemulsions/DNA plasmid complexes. Methods Nanoemulsions composed of medium-chain triglycerides, stearylamine, egg lecithin or isolated phospholipids, ie, DSPC, DOPC, DSPE, or DOPE, glycerol, and water were prepared by spontaneous emulsification. Gene transfer to Hep G2 cells was analyzed using real-time polymerase chain reaction. Results The procedure resulted in monodispersed nanoemulsions with a droplet size and zeta potential of approximately 250 nm and +50 mV, respectively. The complexation of cationic nanoemulsions with DNA plasmid, analyzed by agarose gel retardation assay, was complete when the complex was obtained at a charge ratio of ≥1.0. In these conditions, the complexes were protected from enzymatic degradation by DNase I. The cytotoxicity of the complexes in Hep G2 cells, evaluated by MTT assay, showed that an increasing number of complexes led to progressive toxicity. Higher amounts of reporter DNA were detected for the formulation obtained with the DSPC phospholipid. Complexes containing DSPC and DSPE phospholipids, which have high phase transition temperatures, were less toxic in comparison with the formulations obtained with lecithin, DOPC, and DOPE. Conclusion The results show the effect of the DNA/nanoemulsion complexes composition on the toxicity and transfection results.

A versatile, stability-indicating and high-throughput ultra-fast liquid chromatography method for the determination of isoflavone aglycones in soybeans, topical formulations, and permeation assays

There is a growing interest in the pharmaceutical field concerning isoflavones topical delivery systems, especially with regard to their skin care properties and antiherpetic activity. In this context, the present work describes an ultra-fast liquid chromatography method (UFLC) for determining daidzein, glycitein, and genistein in different matrices during the development of topical systems containing isoflavone aglycones (IA) obtained from soybeans. The method showed to be specific, precise, accurate, and linear (0.1 to 5 µg mL(-1)) for IA determination in soybean acid extract, IA-rich fraction obtained after the purification process, IA loaded-nanoemulsions, and topical hydrogel, as well as for permeation/retention assays in porcine skin and porcine esophageal mucosa. The matrix effect was determined for all complex matrices, demonstrating low effect during the analysis. The stability indicating UFLC method was verified by submitting IA to acidic, alkaline, oxidative, and thermal stress conditions, and no interference of degradation products was detected during analysis. Mass spectrometry was performed to show the main compounds produced after acid hydrolysis of soybeans, as well as suggest the main degradation products formed after stress conditions. Besides the IA, hydroxymethylfurfural and ethoxymethylfurfural were produced and identified after acid hydrolysis of the soybean extract and well separated by the UFLC method. The methods robustness was confirmed using the Plackett-Burman experimental design. Therefore, the new method affords fast IA analysis during routine processes, extract purification, products development, and bioanalytical assays.