Estudos pr??-cl??nicos com nanopart??culas de antim??nio pentavalente no tratamento da Leishmaniose cut??nea

• Author(s): Soares, Fabiane Veloso
• Date: 2017
• Origin: Oasisbr
• Subject(s): Antimonial pentavalente; Nanopart??culas; Leishmania amazonensis; CIENCIAS BIOLOGICAS
• Document Type: Doctoral thesis
• Language: Portuguese

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CAPES - Coordena????o de Aperfei??oamento de Pessoal de N??vel Superior

Cutaneous leishmaniasis is a neglected disease, endemic in tropical and subtropical areas, and Brazil is one of the countries most affected by the disease. Treatment with pentavalent antimonials has been used for over 70 years as the first choice drug in the country and has been shown to be ineffective against some Leishmania species. In addition to toxic, it presents in injectable form, offers a wide spectrum of adverse effects and still represents a high acquisition cost for the country. Consequently, there is an immediate need for research into new antileishmania compounds in order to combat resistance to the drugs used and to demonstrate efficacy and safety in order to facilitate the treatment of the disease. Nanoparticles (NPs) promise an alternative approach to new drugs in the treatment of infectious diseases because their small size and large surface area provide an opportunity for them to interact with vital components of infectious agents, and are more effective than traditional therapies. In this study, pentavalent antimony NPs (Sb+5 NPs) were produced and characterized in order to evaluate their effect in vitro and in vivo in the treatment of cutaneous leishmaniasis. The concentration of Sb+5 was determined by graphite furnace atomic absorption spectrometry; The NPs were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), submitted to in vitro bioassays against promastigotes and amastigotes of Leishmania amazonensis, by the direct counting method in the Neubauer chamber and To cytotoxic activity in J774 lineage macrophages. In the in vivo assay, the hamsters (Mesocricetus auratus) experimentally inoculated with L. amazonensis were grouped to receive intralesional (snout) treatment, with subsequent euthanasia for removal of tissue fragments for analysis by impression by apposition, culture of lesion biopsy, histopathology And MET. The results obtained in this study showed NPs with mean size of 42 nanometers and zeta potential around -49.8 mV, spherical shape, smooth surface and dispersed in suspensions. Bioassays indicated that these NPs showed cytotoxicity at the highest concentrations tested. The most promising suspension was ETSbL2, which had the highest antileishmania activity, with EC50 of 1.05 mg/mL in 72h. The persistence of infection in experimentally infected macrophages when treated with Sb5 + NPs was not noted. After 26 days of experimental treatment, the animals had statistically significant differences between the control group and the groups treated with NPs, regarding the area and clinical aspect of the lesion, and regarding the parasitic control. The histological findings of the lesions of the groups treated with these Sb+5 NPs showed intense inflammatory infiltrate, compromising both the dermosuperficial layer and the dermoprofound of the tissue. Histological changes were also observed in the liver, spleen and kidney. However, ETSbL1[b] (18.6 mg/Sb5+/kg/day) showed greater efficiency in clinical cure regarding reduction in lesion size and parasitic load among the NPs tested. These results suggest the continuity of this study with new tests in animals less susceptible to infection (mice) aiming at a possible clinical and parasitological cure, as well as the evaluation of toxicological and biochemical parameters after treatment.

A leishmaniose tegumentar ?? uma doen??a negligenciada, sendo end??mica em ??reas tropicais e subtropicais do mundo. O Brasil ?? um dos pa??ses mais acometidos pela doen??a. O tratamento com antimoniais pentavalente ?? utilizado h?? mais de 70 anos como medicamento de primeira escolha no pa??s e tem se mostrado ineficaz contra algumas esp??cies de Leishmania. Al??m de t??xico, este se apresenta na forma injet??vel, oferece um amplo espectro de efeitos adversos e representa ainda alto custo de aquisi????o para o pa??s. Consequentemente, existe uma necessidade imediata de pesquisas para novos compostos antileishmania, a fim de combater a resist??ncia aos medicamentos utilizados, e que demonstre efic??cia e seguran??a, visando de facilitar o tratamento da doen??a. Nanopart??culas (NPs) prometem uma abordagem alternativa para novas drogas no tratamento de doen??as infecciosas, pois sua pequena dimens??o e grande ??rea de superf??cie proporcionam uma oportunidade para que elas interajam com componentes vitais de agentes infecciosos, apresentando-se mais eficazes que as terapias tradicionais. Neste estudo NPs de antim??nio pentavalente (NPs Sb+5) em fase aquosa foram produzidas e caracterizadas, com o objetivo de avaliar seu efeito in vitro e in vivo no tratamento da leishmaniose cut??nea. A concentra????o do Sb+5 foi determinada por espectrometria de absor????o at??mica em forno de grafite; as NPs foram caracterizadas por espalhamento din??mico de luz (Dinamic Light Scattering ??? DLS) e microscopia eletr??nica de transmiss??o (MET) e submetidas a bioensaios in vitro contra formas promastigotas e amastigotas de Leishmania amazonensis, pelo m??todo da contagem direta em c??mara de Neubauer e quanto ?? atividade citot??xica em macr??fagos de linhagem J774. No ensaio in vivo, os hamsters (Mesocricetus auratus) experimentalmente inoculados com L. amazonensis foram agrupados para receber tratamento intralesional (focinho), com posterior eutanasia para retirada de fragmentos de tecidos para an??lise por impress??o por aposi????o, cultivo de bi??psia de les??es, histopatologia e MET. Os resultados obtidos neste estudo mostraram NPs com tamanho m??dio de 42 nanom??tros e potencial zeta em torno de -49,8 mV, formato esf??rico, superf??cie lisa e dispersas nas suspens??es. Os bioensaios indicaram que essas NPs apresentaram citotoxicidade nas concentra????es mais altas testadas. A suspens??o mais promissora foi a ETSbL2 que apresentou maior atividade antileishmania, com CE50 de 1.05 mg/mL em 72h. N??o foi notada a perman??ncia de infec????o nos macr??fagos experimentalmente infectados quando tratados com NPs Sb5+. Ap??s 26 dias de tratamento experimental, os animais apresentaram diferen??as estat??sticas significantes entre o grupo controle e os grupos tratados com NPs, quanto ?? ??rea e aspecto cl??nico da les??o, e quanto ao controle parasit??rio. Os achados histol??gicos das les??es dos grupos tratados com essas NPs Sb+5mostraram infiltrado inflamat??rio intenso, comprometendo tanto a camada dermosuperficial como a dermoprofunda do tecido. Altera????es histol??gicas tamb??m foram observadas no f??gado, ba??o e rim. Contudo, ETSbL1[b] (18,6mg/Sb5+/kg/dia) apresentou maior efici??ncia quanto a cura cl??nica referente a redu????o no tamanho da les??o e na carga parasit??ria entre as NPs testadas. Estes resultados sugerem a continuidade deste estudo com novos testes em animais menos suscet??veis a infec????o (camundongos) objetivando uma poss??vel cura cl??nica e parasitol??gica, bem como a avalia????o dos par??metros toxicol??gicos e biquimicos ap??s o tratamento.