Defining Endocytic Pathways of Fucoidan-Coated PIBCA Nanoparticles from the Design of their Surface Architecture.

PURPOSE: This work investigated the endocytic pathways taken by poly(isobutylcyanoacrylate) (PIBCA) nanoparticles differing in their surface composition and architecture, assuming that this might determine their efficiency of intracellular drug delivery. METHODS: Nanoparticles (A0, A25, A100, R0, R25 ) were prepared by anionic or redox radical emulsion polymerization using mixtures of dextran and fucoidan (0, 25, 100 % in fucoidan). Cell uptake was evaluated by incubating J774A.1 macrophages with nanoparticles. Endocytic pathways were studied by incubating cells with endocytic pathway inhibitors (chlorpromazine, genistein, cytochalasin D, methyl-ß-cyclodextrin and nocodazole) and nanoparticle uptake was evaluated by flow cytometry and confocal microscopy. RESULTS: The fucoidan-coated PIBCA nanoparticles A25 were internalized 3-fold more efficiently than R25 due to the different architecture of the fucoidan chains presented on the surface. Different fucoidan density and architecture led to different internalization pathway preferred by the cells. Large A100 nanoparticles with surface was covered with fucoidan chains in a loop and train configuration were internalized the most efficiently, 47-fold compared with A0, and 3-fold compared with R0 and R25 through non-endocytic energy-independent pathways and reached the cell cytoplasm. CONCLUSION: Internalization pathways of PIBCA nanoparticles by J774A.1 macrophages could be determined by nanoparticle fucoidan surface composition and architecture. In turn, this influenced the extent of internalization and localization of accumulated nanoparticles within cells. The results are of interest for rationalizing the design of nanoparticles for potential cytoplamic drug delivery by controlling the nature of the nanoparticle surface.

Antimicrobial activity of ß-lapachone encapsulated into liposomes against meticillin-resistant Staphylococcus aureus and Cryptococcus neoformans clinical strains.

The aim of this study was to determine whether encapsulation of ß-lapachone (ß-lap) into liposomes interferes with its in vitro antimicrobial activity against meticillin-resistant Staphylococcus aureus (MRSA) and Cryptococcus neoformans clinical strains. Liposomes (ß-lap:lipo or ß-lap:HPß-CD-lipo) were prepared using the hydration of thin lipid film method followed by sonication. The in vitro antimicrobial activities of ß-lap-loaded liposomes against MRSA and C. neoformans were evaluated using the microdilution method according to the Clinical and Laboratory Standards Institute (CLSI). The liposomes presented a mean particle size ranging from 88.7±1.5nm to 112.4±1.9nm with a polydispersity index ranging from 0.255 to 0.340, zeta potential from -0.26±0.01mV to +0.25±0.05mV and drug encapsulation efficiency from 97.4±0.3% to 98.9±0.4%. ß-Lap and ß-lap:HPß-CD had minimum inhibitory concentrations (MICs) ranging from 2mg/L to 4mg/L, whereas the MICs of ß-lap-lipo or ß-lap:HPß-CD-lipo ranged from 4mg/L to 16mg/L for the MRSA strains tested. ß-Lap and ß-lap:HPß-CD were able to inhibit fungal growth [MIC=2-8mg/L and minimum fungicidal concentration (MFC)=4-8mg/L]. However, ß-lap-lipo and ß-lap:HPß-CD-lipo were more efficient, with MICs and MFCs of <4mg/L. These findings suggest that the liposomal formulations tested do not interfere significantly with ß-lap antibacterial activity against MRSA and improve its antifungal properties against C. neoformans.

Encapsulation of trans-dehydrocrotonin in liposomes: an enhancement of the antitumor activity.

The aim of this study was the encapsulation of trans-dehydrocrotonin (t-DCTN) and its inclusion complexes with hydropropyl-beta-cyclodextrin (HP-beta-CD) in liposomes to improve t-DCTN antitumor activity. The in vitro kinetic profiles of t-DCTN-loaded liposomes (LD) and t-DCTN:HP-beta-CD-loaded liposomes (LC) were evaluated using the dialysis technique. The antitumor activity of LD and LC were investigated against Sarcoma 180 in Swiss mice. Histopathological and hematological analyses were carried out. The amounts of t-DCTN and t-DCTN:HP-beta-CD inclusion complex encapsulated in liposomes were equivalent to 1 mg of t-DCTN. The encapsulation efficiencies of LD and LC were 95.0 +/- 3.8% and 91.1 +/- 5.6%, respectively. In relation to kinetics, the drug release profiles of t-DCTN are in substantial agreement with the Fickian model. The treatment of animals with LD and LC produced tumor inhibitions of 79.4 +/- 9.6% and 63.5 +/- 5.5%, respectively. The liposomal encapsulation of t-DCTN by entrapment in the phospholipid bilayer increased at twice the antitumor activity. Moreover, the liposomal formulations reduced the hepatotoxicity effect of the drug and no significant hematological toxicity was observed in the treated animals. However, the counting of platelets was slightly decreased. Thus, the results show that the development of liposomal formulations containing t-DCTN or t-DCTN:HP-beta-CD is an important advance for enabling this drug to be use in therapy.

Evaluation of the antischistosomal activity of sulfated α-D-glucan from the lichen Ramalina celastri free and encapsulated into liposomes

The antischistosomal activity of the sulfated polysaccharide α-D-glucan (Glu.SO4) extracted from Ramalina celastri was evaluated after encapsulation into liposomes (Glu.SO4-LIPO) in Schistosoma mansoni-infected mice. The effect of treatment with Glu.SO4 and Glu.SO4-LIPO (10 mg/kg) on egg elimination, worm burden and hepatic granuloma formation was assessed using female albino Swiss mice, 35-40 days of age, weighing 25 ± 2 g, infected with 150 cercariae/animal (Biomphalaria glabrata, BH strain). Four groups (N = 10) were studied, two controls (empty liposomes and NaCl) and two treated groups (Glu.SO4-LIPO and Glu.SO4) using a single dose. Parasitological analysis revealed that Glu.SO4-LIPO was as efficient as Glu.SO4 in reducing egg elimination and worm burden. Treatment with free Glu.SO4 and Glu.SO4-LIPO induced a statistically significant reduction in the number of granulomas (62 and 63 percent, respectively). Lectin histochemistry showed that wheat germ agglutinin intensely stained the egg-granuloma system in all treated groups. On the other hand, peanut agglutinin stained cells in the control groups, but not in the treated groups. The present results suggest a correlation between the decreasing number of hepatic egg-granulomas and the glycosylation profile of the egg-granuloma system in animals treated with free Glu.SO4 or Glu.SO4-LIPO.

Cytotoxicity and cellular uptake of newly synthesized fucoidan-coated nanoparticles.

The aim was to synthesize and characterize fucoidan-coated poly(isobutylcyanoacrylate) nanoparticles. The nanoparticles were prepared by anionic emulsion polymerization (AEP) and by redox radical emulsion polymerization (RREP) of isobutylcyanoacrylate using fucoidan as a new coating material. The nanoparticles were characterized, and their cytotoxicity was evaluated in vitro on J774 macrophage and NIH-3T3 fibroblast cell lines. Cellular uptake of labeled nanoparticles was investigated by confocal fluorescence microscopy. Results showed that both methods were suitable to prepare stable formulations of fucoidan-coated PIBCA nanoparticles. Stable dispersions of nanoparticles were obtained by AEP with up to 100% fucoidan as coating material. By the RREP method, stable suspensions of nanoparticles were obtained with only up to 25% fucoidan in a blend of polysaccharide composed of dextran and fucoidan. The zeta potential of fucoidan-coated nanoparticles was decreased depending on the percentage of fucoidan. It reached the value of -44 mV for nanoparticles prepared by AEP with 100% of fucoidan. Nanoparticles made by AEP appeared more than four times more cytotoxic (IC(50) below 2 µg/mL) on macrophages J774 than nanoparticles made by RREP (IC(50) above 9 µg/mL). In contrast, no significant difference in cytotoxicity was highlighted by incubation of the nanoparticles with a fibroblast cell line. On fibroblasts, both types of nanoparticles showed similar cytotoxicity. Confocal fluorescence microscopy observations revealed that all types of nanoparticles were taken up by both cell lines. The distribution of the fluorescence in the cells varied greatly with the type of nanoparticles.

Evaluation of the antischistosomal activity of sulfated α-D-glucan from the lichen Ramalina celastri free and encapsulated into liposomes.

The antischistosomal activity of the sulfated polysaccharide α-D-glucan (Glu.SO(4)) extracted from Ramalina celastri was evaluated after encapsulation into liposomes (Glu.SO(4)-LIPO) in Schistosoma mansoni-infected mice. The effect of treatment with Glu.SO(4) and Glu.SO(4)-LIPO (10 mg/kg) on egg elimination, worm burden and hepatic granuloma formation was assessed using female albino Swiss mice, 35-40 days of age, weighing 25 ± 2 g, infected with 150 cercariae/animal (Biomphalaria glabrata, BH strain). Four groups (N = 10) were studied, two controls (empty liposomes and NaCl) and two treated groups (Glu.SO(4)-LIPO and Glu.SO(4)) using a single dose. Parasitological analysis revealed that Glu.SO(4)-LIPO was as efficient as Glu.SO(4) in reducing egg elimination and worm burden. Treatment with free Glu.SO(4) and Glu.SO(4)-LIPO induced a statistically significant reduction in the number of granulomas (62 and 63%, respectively). Lectin histochemistry showed that wheat germ agglutinin intensely stained the egg-granuloma system in all treated groups. On the other hand, peanut agglutinin stained cells in the control groups, but not in the treated groups. The present results suggest a correlation between the decreasing number of hepatic egg-granulomas and the glycosylation profile of the egg-granuloma system in animals treated with free Glu.SO(4) or Glu.SO(4)-LIPO.

Does usnic acid affect microtubules in human cancer cells? / O ácido úsnico pode afetar microtúbulos em células cancerosas humanas?

Usnic acid, a lichen metabolite, is known to exert antimitotic and antiproliferative activities against normal and malignant human cells. Many chemotherapy agents exert their activities by blocking cell cycle progression, inducing cell death through apoptosis. Microtubules, protein structure involved in the segregation of chromosomes during mitosis, serve as chemotherapeutical targets due to their key role in cellular division as well as apoptosis. The aim of this work was to investigate whether usnic acid affects the formation and/or stabilisation of microtubules by visualising microtubules and determining mitotic indices after treatment. The breast cancer cell line MCF7 and the lung cancer cell line H1299 were treated with usnic acid 29 µM for 24 hours and two positive controls: vincristine (which prevents the formation of microtubules) or taxol (which stabilizes microtubules). Treatment of MCF7 and H1299 cells with usnic acid did not result in any morphological changes in microtubules or increase in the mitotic index. These results suggest that the antineoplastic activity of usnic acid is not related to alterations in the formation and/or stabilisation of microtubules.

O ácido úsnico, um metabólito de liquens, é conhecido por sua atividade antimitótica e antiproliferativa em células humanas normais e malignas. Muitos quimioterápicos exercem suas atividades bloqueando a progressão do ciclo celular e induzindo morte celular por apoptose. Os microtúbulos, estruturas protéicas envolvidas na segregação dos cromossomos durante a mitose, servem como alvo quimioterapêutico devido ao seu importante papel tanto na divisão celular quanto nos mecanismos de morte celular por apoptose. O objetivo deste trabalho foi investigar se o ácido úsnico afeta a formação e/ou estabilização dos microtúbulos, a partir da visualização de microtúbulos e determinação de índices mitóticos após o tratamento. Células de câncer de mama MCF7 e de câncer de pulmão H1299 foram tratadas por 24 horas com 29 µM de ácido úsnico e dois controles positivos: vincristina (que impede a formação de microtúbulos) e taxol (que estabiliza microtúbulos). O tratamento das células MCF7 e H1299 com o ácido úsnico não resultou em aumento do índice mitótico. Os resultados sugerem que a atividade antineoplásica do ácido úsnico não está relacionada a alterações na formação e/ou estabilização de microtúbulos.

Does usnic acid affect microtubules in human cancer cells?

Usnic acid, a lichen metabolite, is known to exert antimitotic and antiproliferative activities against normal and malignant human cells. Many chemotherapy agents exert their activities by blocking cell cycle progression, inducing cell death through apoptosis. Microtubules, protein structure involved in the segregation of chromosomes during mitosis, serve as chemotherapeutical targets due to their key role in cellular division as well as apoptosis. The aim of this work was to investigate whether usnic acid affects the formation and/or stabilisation of microtubules by visualising microtubules and determining mitotic indices after treatment. The breast cancer cell line MCF7 and the lung cancer cell line H1299 were treated with usnic acid 29 microM for 24 hours and two positive controls: vincristine (which prevents the formation of microtubules) or taxol (which stabilizes microtubules). Treatment of MCF7 and H1299 cells with usnic acid did not result in any morphological changes in microtubules or increase in the mitotic index. These results suggest that the antineoplastic activity of usnic acid is not related to alterations in the formation and/or stabilisation of microtubules.

Stationary cuvette: a new approach to obtaining analytical curves by UV-VIS spectrophotometry.

BACKGROUND: Investigations in the field of pharmaceutical analysis and quality control of medicines require analytical procedures that achieve suitable performance. An analytical curve is one of the most important steps in the chemical analysis presenting a direct relationship to features such as linearity. OBJECTIVE: This study has the aim of developing a new methodology, the stationary cuvette, to derive analytical curves by spectroscopy for drug analysis. METHODOLOGY: The method consists basically of the use of a cuvette with a path length of 10 cm, containing a constant volume of solvent in which increasing amounts of a stock solution of the sample are added, droplet by droplet. After each addition, the cuvette is stirred and the absorbance is measured. This procedure was compared with the currently used methodology, which requires a labour-intensive dilution process, and possible sources of variation between them were evaluated. RESULTS: The results demonstrated that the proposed technique presented high sensitivity and similar reproducibility compared with the conventional methodology. In addition, a number of advantages were observed, such as user-friendliness, cost-effectiveness, accuracy, precision and robustness. CONCLUSION: The stationary cuvette approach may be considered to be an appropriate alternative to derive analytical curves for analysing drug content in raw materials and medicines through UV-VIS spectrophotometry.

Dental enamel birefringence for a wide mineral content range and for different immersion media's refractive indexes: an improved mathematical interpretation.

The aim of this study is to extend, for a larger range of mineral volume (V(1)) and different aqueous immersion media, the model for calculating the refractive index of the non-mineral phase of the human dental enamel proposed by Sousa et al. Published experimental birefringence data of carious and developing human enamel in air and aqueous immersion media with different refractive indexes were interpreted. For 48%

Purification of a lectin from Eugenia uniflora L. seeds and its potential antibacterial activity.

AIMS: The aim of this work was to analyse the antimicrobial properties of a purified lectin from Eugenia uniflora L. seeds. METHODS AND RESULTS: The E. uniflora lectin (EuniSL) was isolated from the seed extract and purified by ion-exchange chromatography in DEAE-Sephadex with a purification factor of 11.68. The purified lectin showed a single band on denaturing electrophoresis, with a molecular mass of 67 kDa. EuniSL agglutinated rabbit and human erythrocytes with a higher specificity for rabbit erythrocytes. The haemagglutination was not inhibited by the tested carbohydrates but glycoproteins exerted a strong inhibitory action. The lectin proved to be thermo resistant with the highest stability at pH 6.5 and divalent ions did not affect its activity. EuniSL demonstrated a remarkable nonselective antibacterial activity. EuniSL strongly inhibited the growth of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella sp. with a minimum inhibitory concentration (MIC) of 1.5 microg ml(-1), and moderately inhibited the growth of Bacillus subtilis, Streptococcus sp. and Escherichia coli with a MIC of 16.5 microg ml(-1). CONCLUSIONS: EuniSL was found to be effective against bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The strong antibacterial activity of the studied lectin indicates a high potential for clinical microbiology and therapeutic applications.

A new approach for improving the birefringence analysis of dental enamel mineral content using polarizing microscopy.

The main problem in interpreting birefringence of dental enamel under polarizing microscopy is the lack of physical constants able to allow the Wiener equation to be applied directly to the composition of such tissue. The present study introduces a new approach to circumvent this constraint. Because the nonmineral phase of enamel is heterogeneous, its refractive index can be computed in terms of its components (namely, water, which is partially replaced by the immersion medium, and organic matter), thereby providing a more acceptable refractive index to be used in the Wiener equation. Furthermore, the enamel mineral volume is ordinarily calculated on the basis of the density 3.15 g cm(-3). The density 2.99 g cm(-3) has been, however, reported to be more accurate for enamel hydroxyapatite, so enamel mineral volumes from selected published data were converted using such a density. The birefringence of mature enamel computed by the Wiener equation, taking into account the above refinements, matched, for the first time, published experimental birefringence values. The theoretical water and organic contents were also consistent with published experimental data. Thus, a direct application of the Wiener equation to the enamel composition has now been achieved. It is speculated that quantitative data on the mineral, the water and the organic contents of mature dental enamel can be derived from interpretation of birefringence in two immersion media (obtained before and after extraction of the organic matter) with this new approach.

In vitro and in vivo properties of usnic acid encapsulated into PLGA-microspheres.

Microparticles will probably play a promising role in the future of chemotherapy. These polymeric delivery systems are capable of maximizing the therapeutic activity while reducing side effects of anti-cancer agents. Usnic acid (UA) is a secondary metabolite produced by lichens, which exhibits an anti-tumour activity. In this study, PLGA-microspheres containing usnic acid from Cladonia substellata were prepared by the double emulsion method, with or without PEG as stabilizer. The morphology of the microspheres was examined by optical and scanning electron microscopy. The in vitro kinetic profile of usnic acid loaded-microspheres was carried out by dissolution testing. The usnic acid content was analysed by HPLC. The cytotoxicity of free and encapsulated usnic acid was evaluated against HEp-2 cells using the MTT method. The anti-tumour assay was performed in mice against Sarcoma-180 tumour (UA 15 mg kg(-1) weight body/day) during 7 days. Animals were then sacrificed and tumour and organs were excised for histopathological analysis. Microspheres presented a smooth spherical surface with a mean diameter of 7.02 +/- 2.72 microm. The usnic acid encapsulation efficiency was approximately 100% (UA 10 mg 460 mg(-1) microspheres). A maximum release of 92% was achieved at the fifth day. The IC50 values for free and encapsulated usnic acid were 12 and 14 microg ml(-1), respectively. The encapsulation of usnic acid into microspheres promoted an increase of 21% in the tumour inhibition as compared with the free usnic acid treatment. In summary, usnic acid was efficiently encapsulated into PLGA-microspheres and the microencapsulation improved its anti-tumour activity.

Novel core(polyester)-shell(polysaccharide) nanoparticles: protein loading and surface modification with lectins.

This study describes new lectin-decorated or protein-loaded nanoparticles with a hydrophobic poly(epsilon-caprolactone) (PCL) core and a hydrophilic dextran (Dex) corona. In this view, a family of block Dex-PCLn copolymers was first synthesized, consisting of a Dex backbone to which n preformed PCL blocks were grafted. The ability of these new copolymers to form nanoparticles was evaluated in comparison with a series of PCL homopolymers of various molecular weights (2000, 10,000 and 40,000 g/mole). Two different nanoparticle preparation methods have been developed and tested for their efficacy to incorporate proteins. For this, three proteins were used: a model protein, bovine serum albumin (BSA), a lectin from leaves of Bauhinia monandra (BmoLL) and Lens culinaris (LC) lectin. All these proteins were successfully incorporated in nanoparticles with a mean diameter around 200 nm. Lectins could also be adsorbed onto the surface of Dex-PCLn nanoparticles. Surface-bound BmoLL conserved its hemagglutinating activity, suggesting the possible application of this type of surface-modified nanoparticles for targeted oral administration. Caco-2 cellular viability was higher than 70% when put in contact with Dex-PCLn nanoparticles, even at concentrations as high as 660 microg/ml.

Bioequivalence of two lamivudine tablet formulations.

The present study describes the determination of the bioavailability of a new commercial tablet formulation of lamivudine (CAS 134678-17-4) compared with a reference formulation. The comparative bioequivalence of the test and a reference formulation (each 3 x 150 mg) was assessed in 24 healthy volunteers by means of a randomized two-way crossover design. Prior to the study both the test and reference formulations were examined for conformation to chromatographic purity and drug content. Each volunteer received the test (T) and the reference formulation (R) with a one-week drug-free interval between administrations. The plasma concentrations of T were monitored over a period of 12 h after drug administration using a sensitive HPLC method. Pharmacokinetic parameters for T were determined from plasma concentration-time data. Statistical tests were carried out at 90% confidence intervals using a parametric method (three-way ANOVA) for AUC and Cmax, and non-parametric method for Tmax. The present study showed that both formulations were bioequivalent for the geometric mean of AUC(0-12), AUC0-infinity), Cmax, and Tmax at the 90% confidence interval. The bioavailability of the test (%) was 96.7, 93.3, 99.7, 100.3, respectively. The T:R ratio was, in each case, well within the acceptable range of 100 +/- 20%.

Colloidal carriers for benzathine penicillin G: nanoemulsions and nanocapsules.

The main purpose of this work is to formulate benzathine penicillin G nanoemulsion and nanocapsules, to evaluate their physicochemical and stabilising characteristics, and to determine their antimicrobial activity and penicillin in vitro release kinetics. Nanoemulsions were produced by the spontaneous emulsification approach and nanocapsules of poly (D,L-lactic acid-co-glycolic acid) polymer (PLGA) were prepared by the method of interfacial deposition of a pre-formed polymer. A 207+/-8 nm mean diameter nanoemulsion formulation maintained stability for more than 5 months at 4 degrees C. Stable nanocapsules with 224+/-58 nm mean diameter were obtained, which remained stabilised over 120 days at 4 degrees C. The penicillin encapsulation ratio in the nanocapsules was 85%. The in vitro release profiles indicated that penicillin released from the nanoemulsion was similar to the one observed from nanocapsules. However it can be clearly deduced from the in vitro kinetic analysis that the antibiotic cannot be protected in colloidal delivery systems. Nevertheless, stable formulations obtained in this investigation supply a potential dosage form to encapsulate more easily soluble drugs.

[Oral emulsions: preparation and bioavailability]. / Les émulsions orales: obtention et biodisponibilité.

Although oral L/H emulsions are rare, they constitute interesting pharmaceutical forms. Such systems are easy to administer, are good to protect a fragile lipophilic active substance and are able to modify drug release. After a recall on the principal components frequently used in these forms, such as oils surface active agents and active drugs, the mechanism of bioavailability and influencing factors are detailed. Finally, evaluation methods in order to analyse in vitro release are discussed.