Chicken embryo model for in vivo acute toxicological and antitumor efficacy evaluation of lipid nanocarrier containing doxorubicin.

Nanoencapsulation of chemotherapeutics, including doxorubicin, can endow the formulations with unique properties, such as a decrease in adverse effects and toxicity. The chicken embryo model is an alternative and well-accepted strategy for evaluating the toxicity and efficacy of drugs and nanoformulations. Therefore, this study proposes the development of a new lipid nanocarrier for doxorubicin delivery (NanoLip-Dox) and posterior evaluation of toxicological profile and antitumoral efficacy against a breast tumor in chicken embryos. NanoLip-Dox showed a unimodal particle size (< 150 nm), negative zeta potential (-19.5 mV), absence of drug crystals, drug content of 0.099 mg·mL-1, and high entrapment efficiency (95%). NanoLip-Dox did not cause toxicity in the chicken embryos; in contrast, doxorubicin hydrochloride induced moderate irritation in the chorioallantoic membrane (at 862.1 µmol·L-1), a survival rate of 50% (at 1.7 µmol·L-1), and an increase in aspartate aminotransferase (at 862.1, 344.8, and 172.4 µmol·L-1). In addition, NanoLip-Dox (at 1.7 µmol·L-1) showed potent antitumor efficacy with a high tumor remission percentage (40.9 ± 9.7%) compared to the control group (8.6 ± 14.8%). These findings together with the absence of toxicity concerning morphological characteristics, weights of embryos and organs, hematologic parameters, and enzymatic activity (alanine aminotransferase, aspartate aminotransferase, and creatinine) suggest the safety and efficacy of NanoLip-Dox.

In vitro and in vivo activity of the essential oil and nanoemulsion of Cymbopogon flexuosus against Trichomonas gallinae.

OBJECTIVE: This study was done to evaluate the in vitro and in vivo effects of the essential oil (OE-CL) and nanoemulsion (N-CL) of Cymbopogon flexuosus against Trichomonas gallinae. MATERIALS AND METHODS: In vitro assays were done with 106 parasites and OE-CL and N-CL in the concentrations: 110, 220, 330, 440, 550, 660, 770 and 880 µg/ml and four controls: CN (culture medium and trophozoites), MTZ (trophozoites plus 800 µg/ml of metronidazole), TW (trophozoites plus vehicles used for solubilization of derivatives (0.01% Tween) and NB (blank nanoemulsion 880 µg/ml). The in vivo assay was done in 35 quails (Coturnix coturnix) infected experimentally 4x104 mg/kg, were divided in seven groups (n=5): A (control-healthy), B (control infected), C (control TW 0.01%), D (NB 0.88 mg/kg), E (drug MTZ 25 mg/kg, F (OE-CL at 0.55 mg/kg) and G (N-CL at 0.44 mg/kg), during 7 consecutive days. RESULTS: The in vitro test showed that the OE-CL (550 µg/ml) and N-CL (440 µg/ml) concentrations reduced the trophozoites viability in 100%. In the in vivo test, the treatment with OE-CL was efficient on the 4th treatment day and the N-CL after the 3rd day, and the MTZ in the therapeutic concentration was efficient on the 7th day. CONCLUSION: It can be observed in this study that the lemon grass has natural potential antitrichomonal activity against T. gallinae in vitro and in vivo.

Evaluation of cytotoxicity, genotoxicity and ecotoxicity of nanoemulsions containing Mancozeb and Eugenol.

Mancozeb is a fungicide widely used in agriculture, mostly against the pathogen Glomerella cingulata responsible for the rot of ripe grape, but presents high toxicity. Strategies are sought to reduce the toxicity of this fungicide and alternative treatments are welcome. An alternative could be the use of clove oil, which has Eugenol as its major compound, and has antifungal potential against G. cingulata, however, Eugenol is susceptible to degradation processes which may compromise its efficacy. The nanoencapsulation of Mancozeb and Eugenol is a possible strategy to overcome the limitations of toxicity, solubility and instability of these compounds. Therefore, the objective of this study is to develop nanoemulsions containing Mancozeb (0.1 mg/mL) and Eugenol (33 mg/mL), isolated or associated, and evaluate the safety of these formulations through cytotoxicity, genotoxicity and ecotoxicity tests. Nanoemulsions were developed by the spontaneous emulsification method, cytotoxicity and genotoxicity were evaluated in healthy human cells through MTT, Dichlorofluorescein diacetate and Picogreen tests, and ecotoxicity assessment was carried out using the chronic toxicity test in springtails. After preparation, the physicochemical characterization of the nanoemulsions were performed which presented mean particle size between 200 and 300 nm, polydispersity index less than 0.3, negative zeta potential and acid pH. The nanoencapsulation was able to avoid the reduction of the cell viability caused by Mancozeb, while Eugenol was shown to be safe for cell use in both free and nanostructured forms, however the association of the two active compounds showed toxicity in the higher doses of Mancozeb. In the ecotoxicity tests, both free Mancozeb and Eugenol forms presented high toxic potential for soil, whereas the nanoencapsulation of these compounds did not cause a reduction in number of springtails. Therefore, from the tests performed, it was possible to observe that nanoencapsulation of Mancozeb and Eugenol is a safe alternative for the application of these compounds mainly in agriculture.

Nanoemulsions containing Cymbopogon flexuosus essential oil: Development, characterization, stability study and evaluation of antimicrobial and antibiofilm activities.

The increase of microbial resistance generates the search for new substances with antimicrobial potential. The essential oil of Cymbopogon flexuosus (Lemongrass) stands out in the literature for its antimicrobial, insecticide and antioxidant properties, but it has high volatilization and low stability, and the nanoencapsulation of this oil could be an alternative to overcome these limitations. Thus, the objective of this study was to develop, for the first time, nanoemulsions containing the essential oil of C. flexuosus, through a method that does not use organic solvent and with temperature control to avoid the volatilization of the oil, characterize and evaluate of stability and the antimicrobial and antibiofilm activities of these nanoemulsions. Nanoemulsions presented adequate physicochemical characteristics (average size less than 200 nm, polydispersity index less than 0.3, negative zeta potential and acid pH) which were maintained during 90 days of storage, and the nanoencapsulation of the C. flexuosus oil enhanced its therapeutic efficacy against the microorganisms evaluated in this study compared to the free oil. These results are very promising because among the microorganisms that the nanoemulsion containing C. flexuosus was able to inhibit the formation of biofilm are the bacteria Pseudomonas aeruginosa and Staphylococcus aureus, which were recently listed by the World Health Organization as priority pathogens for development of new antibiotics.

Antibiofilm activity of nanoemulsions of Cymbopogon flexuosus against rapidly growing mycobacteria.

Rapidly growing mycobacteria (RGM) are opportunistic microorganisms that can cause both local and disseminated infections. When in biofilm, these pathogens become highly resistant to antimicrobials used in clinical practice. Composed abundantly of polymeric substances, biofilms delay the diffusion of antimicrobials, preventing the drug from penetrating the deeper layers and having an effective action. Therefore, the search for new and alternative therapeutic options has become of fundamental importance. Natural products fall into these options, especially essential oils. However, these oils present problems, such as low miscibility in water (which decreases its bioavailability) and degradation by light and temperature. Thus, the objective of this work was to explore the action of free essential oil and nanoemulsions of Cymbopogon flexuosus on strains of RGM, in planktonic and sessile forms. In this work, standard strains of Mycobacterium fortuitum (ATCC 6841), Mycobacterium massiliense (ATCC 48898) and Mycobacterium abscessus (ATCC 19977) were used. The susceptibility of the microorganisms in planktonic form was obtained by conventional microdilution techniques and by cell viability curve. The analysis of the antibiofilm activity was performed by a semi-quantitative macrotechnique. The nanoemulsion exhibited significant antimicrobial activity, with minimum inhibitory concentration values lower than those presented by the free essential oil, against strains in the planktonic state. However, both were efficient in destroying the already formed biofilm, whereas only the free oil inhibited the formation of mycobacterial biofilm. This study demonstrated the therapeutic potential of C. flexuosus essential oil, especially in its nanostructured form, which can be demonstrated against infections caused by rapidly growing mycobacteria.