Nereide S. Santos-Magalhães

Nanotechnology applied to the treatment of malaria

Despite the fact that we live in an era of advanced technology and innovation, infectious diseases, like malaria, continue to be one of the greatest health challenges worldwide. The main drawbacks of conventional malaria chemotherapy are the development of multiple drug resistance and the non-specific targeting to intracellular parasites, resulting in high dose requirements and subsequent intolerable toxicity. Nanosized carriers have been receiving special attention with the aim of minimizing the side effects of drug therapy, such as poor bioavailability and the selectivity of drugs. Several nanosized delivery systems have already proved their effectiveness in animal models for the treatment and prophylaxis of malaria. A number of strategies to deliver antimalarials using nanocarriers and the mechanisms that facilitate their targeting to Plasmodium spp.-infected cells are discussed in this review. Taking into account the peculiarities of malaria parasites, the focus is placed particularly on lipid-based (e.g., liposomes, solid lipid nanoparticles and nano and microemulsions) and polymer-based nanocarriers (nanocapsules and nanospheres). This review emphasizes the main requirements for developing new nanotechnology-based carriers as a promising choice in malaria treatment, especially in the case of severe cerebral malaria.

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.

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.

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.

Nanoencapsulation of quercetin and resveratrol into elastic liposomes.

Based on the fact that quercetin (QUE) and resveratrol (RES) induce a synergic inhibition of the adipogenesis and increase apoptosis in adipocytes, and that sodium deoxycholate (SDC) has necrotic effects, the nanoencapsulation of QUE and RES into SDC-elastic liposomes is proposed as a new approach for dissolving the subcutaneous fat. The concentration of constituents and the effect of the drug incorporation into cyclodextrin inclusion complexes on the stability of QUE/RES-loaded liposomes were studied. The best liposomal formulation reduced the use of phosphatidylcholine and cholesterol in 17.7% and 68.4%, respectively. Liposomes presented a mean diameter of 149nm with a polydispersion index of 0.3. The zeta potential of liposomes was slightly negative (-13.3mV) due to the presence of SDC in the phospholipid bilayer. Encapsulation efficiency of QUE and RES into liposomes was almost 97%. To summarize, QUE/RES-loaded elastic liposomes are stable and suitable for subcutaneous injection, thereby providing a new strategy for reducing subcutaneous fat.

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 µm. The usnic acid encapsulation efficiency was ∼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 µg 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.

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.

The encapsulation of β-lapachone in 2-hydroxypropyl-β-cyclodextrin inclusion complex into liposomes: a physicochemical evaluation and molecular modeling approach.

The aim of this study was to encapsulate lapachone (β-lap) or inclusion complex (β-lap:HPβ-CD) in liposomes and to evaluate their physicochemical characteristics. In addition, the investigation of the main aspects of the interaction between β-lap and 2-hydroxypropyl-β-cyclodextrin (HPβ-CD), using both experimental and molecular modeling approaches was discussed. Furthermore, the in vitro drug release kinetics was evaluated. First, a phase solubility study of β-lap in HPβ-CD was performed and the β-lap:HPβ-CD was prepared by the freeze-drying technique. A 302-fold increase of solubility was achieved for β-lap in HPβ-CD solution with a constant of association K(1:1) of 961 M(-1) and a complexation efficiency of β-lap of 0.1538. (1)H NMR, TG, DSC, IR, Raman and SEM indicated a change in the molecular environment of β-lap in the inclusion complex. Molecular modeling confirms these results suggesting that β-lap was included in the cavity of HPβ-CD, with an intermolecular interaction energy of -23.67 kJ mol(-1). β-lap:HPβ-CD and β-lap-loaded liposomes presented encapsulation efficiencies of 93% and 97%, respectively. The kinetic rate constants of 183.95±1.82 μg/h and 216.25±2.34 μg/h were calculated for β-lap and β-lap:HPβ-CD-loaded liposomes, respectively. In conclusion, molecular modeling elucidates the formation of the inclusion complex, stabilized through hydrogen bonds, and the encapsulation of β-lap and β-lap:HPβ-CD into liposomes could provide an alternative means leading eventually to its use in cancer research.

Elucidation of the complexation mechanism between (+)-usnic acid and cyclodextrins studied by isothermal titration calorimetry and phase-solubility diagram experiments

In the present work the complexation mechanism between (+)‐usnic acid (UA) and cyclodextrins (CDs) has been investigated by isothermal titration calorimetry (ITC) and phase‐solubility diagrams using pH as a tool for modifying the molecule ionization. ITC experiments have been employed to evaluate the stoichiometry of interaction (N), affinity constants (K), and thermodynamic parameter variation associated with complexation between (+)‐UA and α‐, β‐, HP‐β‐, SBE‐β‐, and γ‐CD. It was shown that (+)‐UA did not interact with α‐CD and tended to interact more favorably with γ‐CD (K = 1030 M−1, ΔG = −17.18 kJ · mol−1) than β‐CD (K = 153 M−1, ΔG = −12.46 kJ · mol−1) forming 1:1 complexes. It was also demonstrated using ITC and solubilization experiments that chemical modifications of the parent β‐CD resulted in stronger and more spontaneous interactions (K = 281 M−1, ΔG = −13.97 kJ · mol−1 for SBE‐β‐CD and K = 405 M−1, ΔG = −14.87 kJ · mol−1 for HP‐β‐CD). Analysis of the thermodynamic data suggested that van der Waals forces and hydrogen bonds were responsible for the formation of complexes with a predominance of van der Waals forces. Finally, pH induced modifications of (+)‐UA ionization provided important informations relative to the topology of the interaction between (+)‐UA molecule and the γ‐CD cavity, which were confirmed by molecular modeling. Copyright

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.