Izabele de Souza Araújo1 Camila de Oliveira Melo2, Aléxia Gonçalves Dias2, Klinger Antonio da Franca Rodrigues2, Francisco Jaime Bezerra Mendonça Junior3, Élquio Eleamen Oliveira3.
INTRODUCTION Leishmaniasis is a group of neglected parasitic diseases, caused by protozoa of the Trypanosomatidae family, of the genus Leishmania, transmitted by the bite of an infected female vector insect, a sand fly. According to the World Health Organization (WHO), it is estimated that 30,000 new cases of visceral leishmaniasis and more than 1 million new cases of cutaneous leishmaniasis occur annually. The medications available for visceral and cutaneous leishmaniasis are pentavalent antimonials, meglumine antimoniate (Glucantime®), paromomycin, miltefosine, pentamidine isethionate and amphotericin B (deoxycholate amphotericin B and liposomal amphotericin B) (Choi et al., 2021). However, these medications have serious adverse effects, notably cardiotoxicity, pancreatitis, and nephrotoxicity, and are also costly and difficult to access for the public affected by the disease (Nafari et al., 2020). Therefore, the therapy is unsatisfactory as there are reports of resistance among the parasites and there is still no vaccine available (Ospina-villa et al., 2019). The compound 2-[(5-bromo-indol-3-yl)-amino]-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carbonitrile (SB-83) is a derivative of the 2-amino-thiophenes class, whose in vitro anti-leishmanial activity was discovered by our group in 2015 (Rodrigues et al., 2015), where it was observed that this compound was the most active of the series, having superior activity to the reference drug meglumine antimoniate (Glucantime®), presenting IC50 values equal to 3.37 and 18.5 µM, against, respectively, the promastigote and amastigote forms of Leishmania amazonensis, cytotoxicity in macrophages (CC50 = 113.4 µM) and selectivity index equal to 33.6. This same work indicated that the in vitro anti-leishmanial activity of SB-83 is associated with cell death associated with apoptosis and immunomodulation, especially of the cytokines TNF-? and IL12 and nitric oxide (NO) (Rodrigues et al., 2015). Nanoparticles stand out because they have the advantage of being able to increase the cellular permeability of the drug, reduce the quantity and frequency of doses, increase the half-life, in addition to increasing the effectiveness and controlling the release of the nanoencapsulated active ingredient (El-say; El-sawy, 2017; Souza et al., 2018; George; Shah; Shrivastav, 2019). Macrophages take up the nanoparticles through phagocytic mechanisms through receptors that internalize the pathogens into the macrophage, where Leishmania resides (Mosaiab et al., 2019). Therefore, for the treatment of leishmaniasis, nanoparticles are captured by phagocytes and accumulated in the liver and spleen (organs affected mainly in visceral leishmaniasis), therefore, the delivery of nanoparticles directed to macrophages causes the drug to accumulate in the affected organs, strategically reaching the parasite (Sarwar et al., 2017).
OBJECTIVES Develop a nanoparticle system for delivering SB-83 and carry out in vitro susceptibility tests for SB-83 against promastigotes and amastigotes of Leishmania amazonenses.
METHODOLOGY Development of PLA Nanospheres containing SB-83 by the Nanoprecipitation method: PLA nanospheres containing SB-83 were obtained by the nanoprecipitation method described by Fessi et al., 1998. Morphological and physicochemical characterization of nanoparticles: For 90 days, the suspension of nanoparticles containing SB-83 were stored at 4oC± 2 oC and the average size, polydispersity index, zeta potential and encapsulation efficiency were analyzed in order to evaluate the physical stability of the system. Analysis of the average diameter, polydispersity index and zeta potential: The hydrodynamic radius and polydispersity index of the nanoparticles were determined by dynamic light scattering (DLS, Dynamic Light Scattering) in Zetasizer ultra red equipment (Nano ZS, Malvern). In vitro tests: Formulation of the PLA nanoparticle containing SB-83 (NP-SB83), lyophilized NP-SB83 with and without trehalose, free SB-83 and the reference medicine, methylphosine were evaluated, in triplicate, against the promastigotes and axenic amastigotes forms of Leishmania amazonensis (IFLA/BR/67/PH8). Cytotoxicity was evaluated against RAW 264.5 macrophages.
RESULTS AND DISCUSSION Determination of mean diameter, zeta potential and polydispersity index: The results found before and after encapsulation were -19.3 ±0.35 mV and -20.4 ±0.65 mV respectively, suggesting a negative zeta potential. Furthermore, according to the zeta potential classification, values of ± 0-10 mV stand out as highly unstable, ±10-20 mV relatively stable, ±20-30 mV moderately stable and >± 30 mV highly stable (Bhattacharjee, 2016; Patel; Agrawal, 2011). When determining the diameter of the particles (nm), it was verified that the addition of SB-83 contributes to the increase in the average size of the nanoparticle and this size found favors uptake by macrophages. And finally, the nanoparticles with the drug obtained a polydispersity index (P.D.I) of less than 0.3, indicating homogeneity in the diameter of the particles in suspension. Encapsulation efficiency analysis: The nanoparticle encapsulation rate with SB-83 was carried out using the direct and indirect method. Thus, using the indirect and direct method, the encapsulation efficiency values obtained were 62.7% (±0.12) and 88.9% (±0.87) respectively, using a wavelength of 392nm. Therefore, the results suggest that these rates obtained for the encapsulation rate were satisfactory by both methods analyzed. In vitro assays: The concentration of drug necessary to cause cell death in 50% of cells (CC50) was tested in RAW 264.5 macrophages, demonstrating that nanoparticles containing SB83 (lyophilized NPSB-83 (CC50 = 194 µM), NPSB83 lyophilized with trehalose (CC50 = 190 µM) were the least toxic when compared to the drug Miltefosine (CC50 = 119 µM) and the free drug (SB-83) (CC50 = 105 µM), as a higher concentration was needed to cause significant cell damage. tests on promastigotes, the free drug SB-83 obtained the lowest IC50, (IC50 = 5.72 µM) when compared to the drug encapsulated in nanoparticles (IC50 = 8.03 µM) and the drug Miltefosine (IC50 = 17.25 µM) , suggesting that the drug has the greatest potential to inhibit the activity of parasites, being promising as a candidate to become a medicine against leishmaniasis, as they demonstrate a high potential to combat the infection. The effective concentration of NP-SB83 to produce a response biological in 50% of the amastigote forms of L. amazonensis (EC50) were higher (EC50 = 19.6 µM) when compared to the free drug (SB-83) (EC50 = 17.4 µM) and the reference medicine (EC50 = 18.5 µM), that is, they were more effective for axenic amastigotes. However, the highest selectivity indices (SI), both in the promastigote and in the amastigote forms, were higher for the SB-83 nanoformulations, when compared to the medication miltefosine and SB-83, indicating that the treatment with the nanoparticles with SB-83 is more specific in targeting parasite killing, while minimizing adverse effects on Leishmania amazonensis strains. Furthermore, in cytotoxicity studies, NP-SB83 obtained a higher CC50, suggesting that SB-83 encapsulated in nanotechnological systems reduces cytotoxicity parameters, and are more promising for future in vivo tests.
CONCLUSION SB-83 nanoparticles were successfully developed using the nanoprecipitation technique. In the morphological and physical-chemical characterization, it was found that the nanoformulation presented a negative zeta potential, which guaranteed stability and demonstrated homogeneity in diameter. In in vitro tests, nanoformulations containing SB-83, when compared to the free drug and the reference drug, despite being less active against the amastigote and promastigote forms of Leishmania amazonensis, demonstrated to be a more promising drug candidate in cytotoxicity studies, however, in vivo studies are needed to confirm these effects.
ACKNOWLEDGMENT We thank CNPq for financing this study and the partnership between the Molecule Synthesis and Vectorization Laboratory (UEPB) and the Infectious Diseases Laboratory (UFDPar) for the development of biological tests.
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Comissão Organizadora
Francisco Mendonça Junior
Pascal Marchand
Teresinha Gonçalves da Silva
Isabelle Orliac-Garnier
Gerd Bruno da Rocha
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Ricardo Olimpio de Moura
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