WO2019068161A1 - Method for obtaining nanostructured lipid carriers, the nanostructured lipid carriers obtained and use thereof - Google Patents

Abstract

The present invention relates to a method for obtaining nanostructured lipid carriers comprising buparvaquone coated with cationic and anionic polymers associated with a polypeptide using high-pressure homogenisation technology to promote electrostatic interaction between the components thereof in an aqueous medium. The present invention also relates to the aforementioned nanostructured lipid carriers obtained, which comprise 0.5 to 50% w/v oil phase, 1 to 10% w/v surfactant, 50 to 500,000 IU/ml of a polypeptide, 0.01 to 2% w/v of a cationic polymer; 0.04 to 5% w/v of an anionic polymer, and purified water QSP, thereby enabling site-specific release in macrophages, enabling the development of innovative medicines to improve the treatment of leishmaniasis.

Description

 PROCESS OF OBTAINING NANOESTRUTURED LIPID CAREERS, NANOESTRUTURED LIPID CAREERS

 OBTAINED AND USED

 Field of the Invention:

 The present invention is within the scope of medical science, more specifically, in the field of preparations for medical or veterinary purposes, since it relates to a process for the preparation of nanostructured lipid carriers comprising buparvaquone coated with cationic polymers and anionic compounds associated with a polypeptide, as well as nanocarriers obtained for the improvement of the treatment of leishmaniasis.

 State of the art:

 [002] Leishmaniasis is among the major neglected tropical diseases. These diseases account for the second highest number of deaths due to parasitic infection in the world and are extremely associated with poverty. They are prevalent in 98 countries, on three of the five continents. About 1.3 million new cases occur annually and the estimated number of deaths from visceral leishmaniasis ranges from 20,000 to 50,000 per year.

The classical treatment of leishmaniasis requires the administration of poorly tolerated and highly toxic drugs, since the intracellular localization of leishmaniasis parasites hinders the access of chemotherapeutics to the site of action, which requires the administration of high and repeated doses of which is responsible for its high toxicity. The pentavalent antimonials, meglumine antimoniate (Glucantime®) and sodium stibogluconate (Pentostam®) are the first-line compounds used to treat leishmaniasis.

 Other compounds that may be used include pentamidine and amphotericin B. However, resistance of the parasite greatly reduces the effectiveness of such conventional medicaments. Miltefosine is the only oral medication for the treatment of leishmaniasis, however, this drug has serious adverse effects such as teratogenic effect and gastrointestinal disorders. In the last 15 years, the inadequate prescription of these drugs, as well as the patient's low adherence to treatment, has caused the development of generalized resistance to these agents. In view of this scenario, the search for new drugs and new forms of administration of these chemotherapeutic agents is urgent.

 In order to meet the needs of leishmaniasis therapy, the present invention proposes the development, optimization and evaluation of novel formulations of nanostructured lipid carriers coated with polymyxin B, chitosan and dextran for encapsulation of buparvaquone in order to improve the treatment of leishmaniasis.

Some prior art documents describe nanostructured formulations comprising buparvaquone, for example, IN02166MU2012A discloses a process for the preparation of solid nanoparticles (SLN) containing buparvaquone by ultrasound method. In contrast, the present invention proposes a method of preparing lipid carriers nanostructured (CLN) comprising buparvaquone by homogenization at high pressure; and the functionalization of these nanoparticles for site-specific release of the drug.

 CLN are considered the second generation of lipid nanoparticles and are constituted by colloidal particles that present matrix composed by binary mixture of solid lipid with liquid lipid. Such a special structure has advantages compared to SLNs, such as increased encapsulation efficiency, increased physical stability and reduced risk of drug release during storage. In addition, the amount of drug can be increased by the use of liquid lipid, which buparvaquone has shown to be more soluble. In addition to using more advanced technology, the present invention describes the coating of the nanoparticles, incorporating a second drug (polymyxin B) and molecules that enable drug internalization and release selectively into phagocytic cells. Additionally, it presents the CLN performance in parasites of Leíshmanía ínfantum and its safety using cytotoxicity tests.

US2003059470A1 describes the preparation of conventional, non-nanostructured emulsions comprising buparvaquone. As described above, the CLN's consist of a mixture of solid lipid and liquid lipid. This blend provides novel properties, such as improved formulation stability and enhanced encapsulation efficiency. US2003059470A1 teaches only conventional emulsions prepared with liquid lipid requiring only large amount of surfactants. Accordingly, the US document differs from the present invention in that it presents a formulation in the form of an emulsion which does not have any nanostructure, which has no site-specific action promoting the unexpected improved effect and the action of intracellular buparvaquone as proposed by the CLNs of the present invention.

 The document BR102014023050A2 refers to the obtaining of lipid nanostructure employing high pressure homogenization and coating of this nanostructure employing polymyxin B. However, there is no mention in this document of the use of cationic polymers (chitosan) and anionic (dextran) polymers associated with nanostructure coated with the polypeptide (polymyxin B). The association of dextran sulfate with the nanostructure, as proposed by the present invention, was of fundamental importance for the internalization of this, in the macrophage.

 The present invention therefore provides the use of buparvaquone by means of nanocarriers in order to reduce the systemic toxicity of drugs present on the market, which would facilitate the acceptance of this drug by the patients to be treated and by the clinical staff.

Thus, the specific site action of the proposed buparvaquone CLNs is attributed to the coating by a combination of chitosan, dextran and dextran sulfate compounds, which allow the recognition by the cellular receptors of said CLN that allows site-specific action which promotes the unexpected improved effect and the action of intracellular buparvaquone.

 It is understood, therefore, that the synergistic effect between the action of polyximine B, dextran sulfate, chitosan and buparvaquone is responsible for achieving the achievement of the unexpected result of the present invention.

 BRIEF DESCRIPTION OF THE INVENTION:

 The present invention relates to a method of obtaining nanostructured lipid carriers comprising buparvaquone coated with cationic and anionic polymers associated with a polypeptide by high pressure homogenization technology promoting the electrostatic interaction between its components in aqueous medium.

 In addition, the present invention relates to such nanostructured lipid carriers obtained, which comprise from 0.5 to 50% w / v oil phase; 1 to 10% w / v surfactant; 50 to 500,000 IU / ml of a polypeptide; from 0.01 to 2% w / v of a cationic polymer; from 0.04 to 5% w / v of an anionic polymer; and purified water q.s.p.

 [015] Therefore, the nanostructured lipid carriers obtained allow the site-specific release in macrophages, thus enabling the development of innovative drugs for the advancement of the treatment of leishmaniasis.

 Brief description of the figures:

 [016] For the full and complete visualization of the object of this invention, the reference figures are shown, as follows.

[017] Figure 1 shows the diagram of steps ("g", "h", "i") of the coating of nanostructured lipid carriers containing buparvaquone (A) by polymyxin B (B), chitosan (C) and dextran sulfate (D).

 Figure 2 shows the flowchart of the process of obtaining the nanostructured lipid carriers of the present invention.

 Figure 3 shows a photograph of the nanostructured lipid carrier formulation for the encapsulation of buparvaquone, wherein (A) refers to the formulation before and (B) refers to the formulation after high pressure homogenization.

 Figure 4 graphically depicts the validation of the mathematical model of the mean particle hydrodynamic diameter (DHM) of nanostructured lipid carriers for the encapsulation of buparvaquone of formulation 1 (VI).

 Figure 5 graphically depicts the validation of the mathematical model of the mean particle hydrodynamic diameter (DHM) of nanostructured lipid carriers for the encapsulation of buparvaquone of formulation 2 (V2).

 Figure 6 shows the contour plots in the assay for evaluation of the mean particle hydrodynamic diameter (DHM) of nanostructured lipid carriers for the encapsulation of buparvaquone.

Figure 7 graphically represents the validation of the zeta potential mathematical model of nanostructured lipid carriers for the encapsulation of buparvaquone coated with polymyxin, chitosan and dextran of formulation 1 (VI). Figure 8 graphically represents the validation of the mathematical model of the zeta potential of nanostructured lipid carriers for the encapsulation of buparvaquone coated with polymyxin, chitosan and dextran of formulation 2 (V2).

 Figure 9 graphically depicts the mean dissolution (DP) (n = 6) of free buparvaquone (BPQ) and nonstructured lipid carriers with pancrelipase (gray triangle) and no pancrelipase (black circle) containing 4 mg of BPQ using 0.05M pH 7.4 phosphate buffer, 0.07% tween 80, wherein (A) represents Formulation VI, and (B) represents Formulation V2.

 Figure 10 graphically depicts the mean (SD) (n = 6) dissolution of free buparvaquone (BPQ) (black circles) containing 4 mg of BPQ using phosphate buffer pH 7, 40.05 M, dodecyl sulfate 1% sodium (w / v), wherein (A) refers to formulation VI (gray inverted triangle); and (B) refers to the formulation V2 - Z mean: 230.7 nm (black diamond).

 Figure 11 graphically depicts the leishmanicidal activity in L.infantum (n = 3) free buparvaquone (BPQ) amastigotes (light gray circle, VI (black square), VI coated with polymyxin B, chitosan and anionic dextran ( asterisk black) correlating the measured viability of cell death of L. infantile amastigotes with the concentration of buparvaquone.

 Detailed description of the invention:

The present invention relates to a process for the preparation of nanostructured lipid carriers comprising buparvaquone coated with polymers cationic and anionic compounds associated with a polypeptide.

 [029] Accordingly, said method comprises the steps of:

 a) Prepare from 0.5 to 50% (w / v) oil phase;

 b) Add 0.01 to 5% (w / v) buparvaquone;

 c) Prepare the aqueous phase;

 d) Heat and mix the oily and aqueous phases under stirring;

 e) Agitate the mixture of the oily and aqueous phases obtained;

 f) Homogenize at high pressure;

 g) Cool the obtained carrier at room temperature;

 h) Dilute the carrier obtained in step "g" with up to 1: 3 parts of purified water;

 i) Add 50 to 500,000 IU / mL of a polypeptide under agitation;

 j) Add 0.01 to 2.0% w / v of a cationic polymer under stirring;

 k) Add 0.04 to 5.0% w / v of anionic polymer under stirring; and

 1) Carry out the potting.

 [030] In step "a", the liquid and solid lipids are mixed in the preparation of the oil phase until homogenisation thereof. Thus, said oily phase comprises 0.5 to 50% (w / v), preferably 5 to 15% (w / v), of a ratio of 0.1 (10: 1) to 10.0 (1 : 10) of liquid and solid lipids (LL / LS), respectively, preferably 0.5 (2: 1) to 3 (1: 3).

[031] Liquid lipids are selected from a group consisting of caprylic and caprylic acid triglycerides, glyceryl monocaprylate, safflower oil, corn oil, olive oil, sesame oil, cottonseed oil, soybean oil, oleic acid, preferably triglycerides of capric and caprylic acids.

The solid lipids are selected from the group consisting of hydrogenated palm oil, hydrogenated coconut mono, di and triglycerides (Witepsol ^® E85), stearoyl macrogol-32-glycerides (Gelucire ^® 50/13), distearate of glyceryl (Precirol ^® ATO 5), hydrogenated soybean oil (Sterotex ^® HM), triglycerides of palmitic and stearic acids (Dynasan ^® P60), glyceryl behenate (Compritol ^® 888), preferably hydrogenated palm oil.

 In step "b", the buparvaquone is added in the oil phase obtained in the previous step, in the concentration of 0.01 to 5%, preferably 0.3% (w / v).

 The aqueous phase, which comprises the mixture of purified water qsp and from 1 to 10% (w / v) surfactant, is then prepared in step c, subjected to magnetic stirring until complete homogenization, preferably 2 to 4% (w / v).

The surfactants are selected from the group consisting of ethylene oxide and propylene oxide poloxamer copolymers 188, 407, 101, 105, 108, 122, 123, 124, 181, 182, 183, 184, 185, 212, 215, 217, 231, 234, 235, 237, 238, 282, 284, 288, 331, 333, 334, 335, 338, 401, 402, 403; sorbitan stearate, sorbitan monooleate, sorbitan trioleate, sorbitan tristearate, polysorbate 80, polysorbate 60, preferably poloxamer 188.

 In step "d", the mixture of the oily and aqueous phases is carried out at a temperature ranging from 45 to 85 ° C, preferably 70 ° C, with stirring ranging from 150 to 800 rpm, preferably 500 rpm, for 2 hours. to 45 minutes, preferably 10 minutes.

 Next in step "e", the preemulsion is formed by employing suitable equipment, such as a high performance disperser, at a rotation ranging from 500 to 14000 rpm, preferably 8000 rpm, at a temperature which varies 40-80 ° C, preferably 70 ° C, for 2 to 45 minutes, preferably 10 minutes.

[038] In step "f", the high pressure homogenization is performed for one to ten cycles IO ⁶ to IO ⁷ Pa, at the temperature ranging from 45 to 85 ° C, preferably 70 ° C.

 [039] Subsequently, in step "g" the cooling is carried out at room temperature (20-25 ° C).

 [040] Next, in step "h", carrier dilution is performed in up to 1: 3 parts of purified water.

 [041] Next, in the step "i" the addition of 50 to 500,000 IU / ml of a polypeptide, preferably 1000 to 9000 IU / ml, is added to the carrier at a temperature ranging from 20 to 25 ° C, homogenization by magnetic stirring ranging from 50 to 250 rpm, preferably 100 rpm, at a pH ranging from 3.5 to 9.0, preferably 5.0 to 7.0, for 50 to 120 minutes, preferably 60 minutes.

 [042] Said polypeptide is preferably polymyxin B and may be substituted for polymyxin E (colistin).

[043] Subsequently, in step "j" of 0.01 to 2% w / v of a cationic polymer, preferably 0.05 to 0.2%, homogenization by magnetic stirring ranging from 50 to 1000 rpm, preferably 100 rpm, at a pH ranging from 3.5 at 9.0, at a temperature ranging from 20 to 25øC, for 50 to 120 minutes, preferably 60 minutes.

 [044] Said cationic polymer is preferably chitosan, which may range from low to medium molecular weight (50 to 200 kDa), with a degree of deacetylation of at least 75%.

 Next, in step "k" from 0.04 to 5% w / v, preferably from 0.4 to 0.55% w / v, of an anionic polymer is added to the homogenization by magnetic stirring which varies 50 to 150 rpm, preferably 100 rpm, at a pH ranging from 3.5 to 9.0, at a temperature ranging from 20 to 25øC, for 50 to 120 minutes, preferably 60 minutes.

 [046] Said anionic polymer is preferably dextran sulfate, which may be substituted by D-mannose-6-phosphate.

 [047] Figure 1 shows the diagram of steps

("g", "h", "i") of the coating of nanostructured lipid carriers containing buparvaquone (A) by polymyxin B (B), chitosan (C) and dextran sulfate (D).

 [048] Finally, in step "1" the packaging of the obtained formulation is carried out for subsequent application in pharmaceutical vehicles.

[049] It is to be noted that, in order to exemplify the invention, all processes were carried out in suitable containers, such as beakers, volumetric flasks, erlenmeyers, falcon tubes and test tubes. However, it should be noted that the present invention is not limited by the foregoing examples, and other equipment, utensils and containers for reproduction on an industrial scale may be used.

 [050] For better visualization, Figure 2 shows the flowchart of the process of the present invention.

 Therefore, at the end of the process described herein, nanostructured lipid carriers comprising buparvaquone coated with cationic and anionic polymers associated with a polypeptide are obtained, enabling the recognition by their cellular receptors that they allow the site-specific action that promotes the effect and the action of intracellular buparvaquone in the treatment of visceral leishmaniasis and prevention of recidivism of cutaneous leishmaniasis.

 Accordingly, in addition, the present invention relates to the obtained nanostructured lipid carriers which comprise:

 - from 0.5 to 50% w / v oil phase;

 - from 1 to 10% w / v surfactant;

 50 to 500,000 IU / ml of a polypeptide;

 - from 0.01 to 2% w / v of a cationic polymer;

 - from 0.04 to 5% w / v of an anionic polymer; and

 - purified water q.s.p.

 [053] Said oily phase comprises from 0.5 to

50% (w / v) of a ratio of 0.1 (1:10) to 10,0 (1:10) liquid and solid lipids (LL / LS), respectively, preferably 0.5 (2: 1) ) to 3 (1: 3), and buparvaquone from 0.1 to 5.0%, preferably 0.3% (w / v). Liquid lipids are selected from the group consisting of triglycerides of caprylic and caprylic acids, glyceryl monocaprylate, safflower oil, corn oil, olive oil, sesame oil, cottonseed oil, soybean oil, oleic acid , preferably triglycerides of capric and caprylic acids.

Solid lipids are selected from the group consisting of hydrogenated palm oil, hydrogenated coconut mono, di and triglycerides (Witepsol ^® E85), stearoyl macrogol-32-glycerides (Gelucire ^® 50/13), distearate of glyceryl (Precirol ^® ATO 5), hydrogenated soybean oil (Sterotex ^® HM), triglycerides of palmitic and stearic acids (Dynasan ^® P60), glyceryl behenate (Compritol ^® 888), preferably hydrogenated palm oil.

 Preferably, said surfactant is in a concentration of 2 to 4% (w / v), and it is selected from the group consisting of ethylene oxide and propylene oxide (poloxamer) copolymers 188, 407, 101, 001, 234, 235, 237, 238, 282, 284, 288, 331, 333, 334, 335, 338, 401, 402, 403; sorbitan stearate, sorbitan monooleate, sorbitan trioleate, sorbitan tristearate, polysorbate 80, polysorbate 60, preferably poloxamer 188.

 [057] Preferably, said polypeptide is in a concentration of 1000 to 9000 IU / ml, which is preferably polymyxin B and may be substituted for polymyxin E (colistin).

[058] Preferably, said polymer cationic acid is in a concentration between 0.05 and 0.2%, which is preferably chitosan, which may range from low to medium molecular weight (50 to 200 kDa), with a degree of deacetylation of at least 75%.

 [059] Preferably, the anionic polymer is in a concentration of 0.4 to 0.55% w / v, which is preferably dextran sulfate, which may be substituted by D-mannose-6-phosphate.

 [060] In addition, said nanostructured lipid carriers have a mean hydrodynamic diameter (DHM) ranging from 100 to 350 nm; zeta potential (mV) less than -10, preferably less than -20; and polydispersity index less than 0.3.

 [061] Thus, said nanostructured lipid carriers have novel features that allow nanoparticles to be internalized in the macrophage cell membrane through the direct action of polymyxin B, combined with the interaction of polysaccharides with SIGN-R1 receptors. Thus, administration of such carriers directed to the lymphatic system may be an innovative approach for the treatment of leishmaniasis.

 In this regard, in addition, the present invention relates to the use of the nanostructured lipid carriers obtained for the manufacture of a medicament for treating leishmaniasis.

 [063] In order to evaluate the potential of the nanostructured lipid carriers of the present invention, the results of the tests performed are presented below.

 Tests :

Example of the invention: [064] Hydrogenated palm oil (Softisan ^® 154) was chosen for the preparation of the nanoparticles, since buparvaquone (BPQ) presented greater solubility in this lipid and because its melting point varies from 53-58 ° C, characteristic that allows the addition of a greater amount of liquid lipid to the solid lipid while maintaining the solid characteristics of the mixture.

[065] In assessing the solubility of BPQ in liquid lipids, caprylic and caprylic acid triglycerides (Miglyol ^® 182) have been shown to be lipid which best solubilizes BPQ, which lipid is widely used for oral and topical formulations, is resistant to oxidation and is synthesized industrially and, therefore, was chosen as liquid lipid to compose formulations of CLNs.

 [066] The preparation of nanostructured lipid carriers for the encapsulation of buparvaquone by homogenization at high pressure proved to be simple, rapid and without intercurrences. It was possible to verify that the drug was encapsulated soon after the preparation; Figure 3A, prior to homogenization, shows the yellowing of the drug. This characteristic color was not observed after the process (Figure 3B). Thus, it is concluded that the drug remained localized within the lipid structure, therefore, encapsulated.

[067] The first step in the preparation of the CLNs consisted of heating and stirring the oily and aqueous phases at 70 ± 5 ° C, at 500 ± 50 rpm for 10 minutes. The second step consisted of pre-emulsion formation using ultra-turrax disperser at 8,000 rpm for 10 minutes. THE third and final stage consists of homogenization at high pressure for five cycles at 600 bar and 70 ± 5 ° C.

 Factorial study to evaluate the significant variables in the preparation of the nanostructured lipid carrier for the encapsulation of buparvaquone:

[068] Factorial study ²³ was applied in the method of preparing nanostructured lipid carriers to select concentrations of excipients. The variables evaluated were the ratio of liquid to solid lipid (LL / LS) in the range of 0.5 (2: 1) to 3.0 (1: 3); the percentage of oil phase between 5 to 15% (w / v) and the concentration of poloxamer between 1 and 4% (w / v). The measured response was mean hydrodynamic diameter (DHM) and the results are described in Table 1.

 Table 1 - Matrix of experiments and values of mean hydrodynamic diameter (DHM) in the statistical evaluation of the preparation of nanostructured lipid carriers for the encapsulation of buparvaquone (BPQ).

 LS: solid lipid. LL: liquid lipid. LS / LL: ratio of solid and liquid lipid

 [069] By the results of the analysis of variance

(Table 2 and 3), the mathematical model proved to be adequate due to the absence of a lack of adjustment (p-value> 0.05) and values of adjusted R, R and prediction R of the adjusted model, are greater than 80% and do not vary abruptly, which indicates that the DHM data are well described by the mathematical model and can be used to predict values of DHM when the factors studied are modified, at the time of optimization / validation of the model.

 Table 2 - Analysis of variance to test the significance of the regression for the data obtained in the assay for the evaluation of the mean particle hydrodynamic diameter of nanostructured lipid carriers for the encapsulation of buparvaquone.

 GL: degrees of freedom; CT: contribution; SQ seq: sum of squares; SQ aj: sum of squares adjusted; Test F: statistics F; MQ (aj): adjusted quadratic mean. P-value: level of significance.

 [070] Equation 1 that describes the influence of the factors in the process is presented below. With this equation it is possible to calculate the DHM values from the variation of the concentrations of poloxamer, oil phase and LS / LL.

 Equation 1

 DHM = 173.8 +9.00 LS / LL-5.58 POL + 15.68 FO -3.183

 POL * FO

on what : LS / LL: ratio of solid and liquid lipids;

 POL: poloxamer concentration (w / v); and

 FO: oily phase concentration (w / v).

 Optimization and validation of the mathematical model of the preparation of nanostructured lipid carriers for the encapsulation of buparvaquone:

 [071] For validation of the mathematical model, it is necessary to challenge it with the preparation of at least two formulations calculated from the variations in the main factors. In the present invention two exemplary embodiments have been proposed: Formulations VI and V2 having the parameters shown in Figures 6 and 7. These formulations present optimized excipient concentrations to obtain DHMOOO nm.

 [072] In LV, LS / LL was optimized to 0.5 (2: 1); the poloxamer concentration was 4% (w / w) and the oil phase concentration was 5% (w / w). With these parameters, the mathematical model provided the theoretical DHM value of 170.7 nm with a 95% confidence interval of 139.9 to 201.5 nm (Table 3). Figure 4 shows the graph of formulation validation 1 (VI) of the mathematical model of the mean particle hydrodynamic diameter (DHM) of nanostructured lipid carriers for the encapsulation of buparvaquone.

[073] For V2, the modified parameter was the 2% (w / w) poloxamer concentration. The model provided the theoretical DHM value of 213.7 nm with a confidence interval of 189.4 to 238.0 nm (Table 3). Figure 5 shows the graph of the validation formulation 2 (V2) of the mathematical model of the mean particle hydrodynamic diameter (DHM) of nanostructured lipid carriers for the encapsulation of buparvaquone.

 [074] The practical DHM values are presented in Table 4, it is possible to verify that both formulations are within the confidence interval and, therefore, the mathematical model of DHM for that process / product is validated.

Table 3 - Validation of the mathematical model of the mean particle hydrodynamic diameter of the production process of nanostructured lipid carriers for the encapsulation of buparvaquone.

 [075] Therefore, to obtain the CLNs, the concentrations of the materials should be among the studied tracks. For the ratio of liquid to solid (LL / LS) lipid the range should be 0.5 (2: 1) to 3 (1: 3). For the oil phase percentage, the range should be between 5 to 15% (w / v). For the poloxamer concentration, it should remain between 1 and 4% (w / v), as previously described.

 [076] The theoretical basis of the coating of nanostructured lipid carriers containing buparvaquone by polymyxin B, chitosan and dextran sulfate is the electrostatic interaction between these components.

 [077] Carriers have negative charges

(zeta potential of approximately -15 mV) that interact electrostatically with the positive charges of polymyxin B (MW: 1301.6 g / mol). Therefore, the first step of the coating is the addition of polymyxin B solution to the carrier in order to deposit the drug on the surface but to avoid the complete neutralization of negative charges from the carrier. Chitosan (PM: 50000-150000 g / mol, 75-90% deacetylation), in turn, presents positive charges, which interact with the liquid negative charges of the first step, by reversing the zeta potential to positive.

 [078] The third step is similar to that described in the literature, it relates to the preparation of particles formed with chitosan nucleus and coated with dextran. Due to the positive net charge of the carriers, dextran sulfate (PM ~ 40,000 g / mol), of negative charge, can be deposited on the surface of the nanostructured lipid carrier.

 The coating process was started by preparing stock solutions of polymyxin B: 100,000 IU / ml, chitosan: 2% (w / v) and 2% dextran sulfate (w / v) and the dilution of a part of the nanostructured carrier to three parts purified water.

The first step consists of the addition of the solution of polymyxin B to the diluted carrier, the homogenization was carried out by magnetic stirring at 100 rpm for 1 hour. In the second step, the chitosan is added and the stirring is continued for another hour. In the third and last step, the dextran sulfate is added and the system is maintained for another hour at 100 rpm. After each step, a small aliquot is removed for verification of the zeta potential as process control. The coating process was carried out in the following conditions: pH 5, 0 - 7.0, temperature 25 ° C.

 Factorial study for the evaluation of significant variables in nanostructured lipid carrier coating for encapsulation of buparvaquone:

 [081] The Plackett-Burman factorial study was applied to the coating method of the nanostructured lipid carriers, the variables evaluated were: concentrations of polymyxin, chitosan and dextran. The measured response was the zeta potential. Table 4 shows the experiment matrix and zeta potential results. It was observed that at concentrations of 0.35% (w / v) chitosan and 9,000 IU / ml of polymyxin, the system showed aggregation, indicating that lower concentrations of chitosan should be used to contain more polymyxin.

 Table 4 - Matrix of experiments and zeta potential values (PZ) of the factorial study of the preparation of nanostructured lipid carriers for the encapsulation of buparvaquone, coated with polymyxin (POL), chitosan

(QUI) and dextran (DEX).

 Assay POL (Ul / mL) QUI (%) w / v DEX (%) w / v PZ (mV) Obs.

 1 1000 0.090 0.55 -18.3

 2 9000 0, 35 0, 05 30.2 Aggregation

 3 1000 0.05-0.05 -13.6

 4,900,0,0,0,0,0,0,63

 5 1000 0, 350, 55 -21.1

 6 5000 0.2 0.3 -15.5

 7 9000 0.35 0, 05 10.7

 8 1000 0.05-0.05 -8.15

 9 9000 0.05 0, 55 -27.0

 10 9000 0, 35 0, 55 -19.0 Aggregation

 11 9000 0.090, 55-26.9

 12 1000 0, 35 0.55 -23.5

 13 5000 0.2 0.3 -16.2

 14 5000 0.2 0.3 -16.9

15 1000 0, 35 0.05 32.5 [082] According to Table 5, the main factors are the concentration of chitosan and dextran, the p-value for both factors is <0.05 and therefore are significant for the zeta potential, as well as, the interaction between them. In the same table, we can verify the non-significance of the lack of adjustment (p> 0.5). Thus, the mathematical model is adequate, since it represents well the data collected. The adequacy of the model can be corroborated by the values of r ² presented in Table 6. The values of r ² , r ² adjusted and ² of forecast are above 80% and there is no great discrepancy between these coefficients.

 Table 5 - Analysis of variance to test the significance of the regression for the data obtained in the assay for the evaluation of the zeta potential of nanostructured lipid carriers for the encapsulation of buparvaquone coated with polymyxin, chitosan and dextran.

 GL: degrees of freedom; SQ seq: sum of squares; SQ aj: sum of squares adjusted; Test F: statistics F; MQ (aj): adjusted quadratic mean. P-value: level of significance.

Table 6 - Test for significance of the coded regression coefficients and adjustment indices of the selected model in the zeta potential test of nanostructured lipid carriers for the encapsulation of buparvaquone coated with polymyxin, chitosan and dextran. Terms Coefficients Statistic T p-value

Constant -11, 962 -13, 42,000

 Polymyxin 0.87 0.82 0.430

Chitosan 7, 332 7.36 0.000

Dextran -13.97 -13.15,000

Chitosan * Dextran -4.24 -4.01 0.002

DP: 3.45133 R ² : 96.13% R ² (aj): R ² (prev):

94, 58% 92, 04% adjustment indices of the model: R ² : coefficient of determination; R ² (aj): adjusted coefficient of determination; R ² (prev): coefficient of prediction determination of the adjusted model; SD = standard deviation; P-value: level of significance C-DP: coefficients of standard deviation.

 [083] Another important contribution presented in the

Table 6 addresses the interaction between chitosan and dextran. This interaction contributes to the reduction of zeta potential (negative coefficient -4,24). This result reveals how the application of a factorial study is a necessary tool to describe and understand a process or product and even to provide a theoretical basis for the continuous improvement of these.

 [084] Equation 2 presents the mathematical model that describes the coating process of the nanostructured lipid carrier for encapsulation of buparvaquone. This equation was used to optimize this process in order to reduce the zeta potential to approximately - 30 mV. These formulations with reduced zeta potential have loads necessary for repulsion between particles, which contributes to the long-term stability of the coated carriers.

 Equation 2:

 PZ (mv) = -7.02 + 0.000826 POL +40.10 QUI-56.86 DEX-113.1

 QUI * DEX

on what :

 PZ: zeta potential in mV (millivolts);

 POL: concentration of polymyxin B in IU / mL;

QUI: chitosan concentration in% (w / v); and DEX: dextran concentration in% (w / v).

 [085] Figure 6 shows the behavior of the zeta potential for each pair of factors. The graphs show that the concentration of chitosan should be less than 0.2% w / v and that of dextran should be above 0.4% w / v for formulations of zeta potential less than -20 mV can be achieved. Therefore, in the preparation of coated CLNs, the concentrations should be the following, as previously described: polymyxin B between 1,000 and 9,000 IU / ml, chitosan between 0.05 and 0.2% (w / v) and dextran between 0.4 to 0.55% (w / v).

Table 7 - Validation of the zeta potential (PZ) model of the production process of nanostructured lipid carriers for the encapsulation of buparvaquone coated with polymyxin, chitosan and dextran.

 Optimization and validation of the mathematical model of the coating of the nanostructured lipid carrier for the encapsulation of buparvaquone:

[086] Figures 7 and 8 present, respectively, formulations VI and V2 for the validation of the mathematical model in the coating process of nanostructured lipid carriers with polymyxin B, chitosan and dextran. Said concentrations refer to the optimized formulations. The reduced concentration of chitosan in V2 reflects calculated zeta potential lower than VI. Dextran concentrations were maintained at the upper limit of the model to obtain carriers with the lowest possible zeta potential. Table 7 shows the results practical and theoretical aspects of validation formulations. In both formulations, the value obtained was within the predicted range (95% confidence interval). Therefore, the mathematical model is validated and can be used to predict zeta potential values according to the modifications in the desired main factors.

 Determination of the encapsulation efficiency of buparvaquone in nanostructured lipid carriers:

 [087] Encapsulation efficiency was calculated by subtracting the amount of drug in the supernatant from the total drug concentration in the sample. Two uncoated samples were tested and the results are summarized in Table 8 and show that the encapsulation efficiency was satisfactory because it has values close to 100%.

Table 8 - Efficacy test of buparvaquone encapsulation in nanostructured lipid carrier by high performance liquid chromatography.

- Evaluation of the cytotoxicity of the nanostructured iipid carrier containing buparvaquone coated polymyxin B _r chitosan and dextran:

[088] The evaluation of cytotoxicity was performed in two cell types, THP-1 and mouse peritoneum macrophages. The first cell type was cultured in RPMI 1640 medium and treated with PMA (phorbol myristate acetate) ΙΟηΜ for 24 hours for monocyte differentiation to human macrophages. In both tests, 2x 10 ⁵ cells were incubated for 24 h at 37 ° C with final formulation VI. The concentrations used were 0.88; 1.75; 3.5; 7.0; and 14.00 μ b buparvaquone.

 [089] Cell survival results are presented in Tables 9 and 10, which shows the low in vitro toxicity of these carriers because they did not present a mean or survival sample below 70%. Thus, these coated carriers can be safely used in future in vitro and in vivo leishmanicidal activity tests.

 Table 9 - Test of cytotoxicity of nucostructured lipid carrier containing buparvaquone (BPQ) and coated with polymyxin, chitosan and dextran on THP-1 cells, activated by PMA (24 h, 37 ° C).

 Table 10 - Test of cytotoxicity of nanostructured lipid carrier containing buparvaquone (BPQ) and coated with polymyxin, chitosan and dextran in mouse peritoneal macrophages (24 h, 37 ° C).

 Concentration BPQ 0.88 μΜ 1.75 μΜ 3.50 μΜ 7.00 μΜ 14.00 μΜ

Aml 75.74 98.02.28, 89 98, 0294, 46

Am2 85.06 96.26 94.98 96.26 84.27

Am3 98, 95 88, 95 91.50 88, 95 103, 67

Am4 95, 30 81.30 90.20 88.43 99.90

Am5 81.70 101.10 99.20 99.21 101.20

Am6 99.00 98.20 89.90 91.20 99.80

Mean 89, 29 93, 97 91, 45 93, 68 97, 22 DPR 1.10 0.79 0.60 0.51 0.72

 - Evaluation of the solubility of buparvaquone-containing nanostructured lipid carriers (BPQ):

 Table 11A shows the solubility of uncoated CLNs. The coated nanoparticles should be employed in injectable formulations. All formulations showed higher solubility in water when compared to free drug. In general, the lower the DHM, the greater the solubility. Formulation VI, which has the lowest DHM (170.4 nm), showed solubility of 611 times greater when compared to the free drug in the simulated body fluid. Even in the presence of high concentration of sodium dodecyl sulfate (1% w / v) pH 7.4, the solubility was 3.2 times higher.

 [091] Phosphate buffer pH 7.4 with 1% (w / v) sodium dodecylsulfate was tested to simulate the fed state and phosphate buffer pH 7.4 with 0.07% w / v Tween 80 to simulate the release of BPQ in the fasted state. Since the drug BPQ showed similar solubility in these media (Table 11).

 Table 11A - Mean solubility (SD) (n = 3) of free buparvaquone and buparvaquone-containing nanostructured lipid carriers.

 Solubility (g / ml) BPQ VI V2

Simulated gastric fluid pH 1.2 0.055 3.564 3.64

 (0.01) (1.72) (2.20)

0.05 M phosphate buffer pH 4.5 0.06 3.30 2.78

 (0.04) (0.98) (0.90)

0.05 M phosphate buffer pH 6.8 0.08 2.82 2.77

 (0.02) (1.41) (3.05)

0.05 M phosphate buffer pH 7.4 0.19 12.62 2.16

 (0.10) (1.52) (0.65)

Simulated intestinal fluid 12.53 24.98 25.56 fed pH 5.0 (1.85) (2.04) (1.12)

Simulated intestinal fluid fasting pH 4.0 3.39 26, 30 2.28

(0.24) (2.66) (0.85) Simulated body fluid 0, 02 12,22 15, 32

 (0.01) (6.53) (22, 36)

Phosphate buffer 0.05 M 11.68 37.74 17.08 Solium dodecylsulfate% (w / v) (0.78) (10.66) (4.5)

0.05 M phosphate buffer pH 7.4 3.39 15.31 9.19 Tween 80 0.07% (0.30) (7.05) (2.02)

Additionally, stability of uncoated formulations VI and V2 to the wet sterilization process, 121 ° C for 30 minutes, were evaluated. The values set forth in Tables 11B and 11C show that DHM and polydispersity in both formulations did not vary considerably, indicating suitability of the process for the sterilization of the formulations.

 Table 11C - V2 wet sterilization test.

 Dissolution profiles of buparvaquone-containing nanostructured lipid carriers (BPQ):

[093] Table 12 shows the dissolution values of free BPQ in pH 7.0, pH 4.0, 0.05M phosphate buffer with 0.07% w / v Tween 80. Even after four hours, only 2.89% of 4 mg of free drug was dissolved. Such a result revealed that the drug did not reach the solubility value (3.39 g / ml) in that medium. During the development of the dissolution method, pancrelipase was tested to mimic the degradation of the nanoparticles from intestinal lipases. [094] Figure 9 shows the dissolution profiles in 0.05M phosphate buffer pH 7.4 with 0.07% tween 80 of formulations VI and V2. Table 13 and Table 14 show mean dissolution values. VI, in the non-pancrelipase assay, released 65.43% of the 4 mg dose after 90 minutes. With the use of the enzyme, the dissolution reached 77.34%. The V2 formulation had similar behavior, but the increase in dissolution was more prominent with pancrelipase. Thereafter, after 60 minutes,

, 25% of the dose of 4 mg.

 Table 12 - Dissolution profile of free buparvaquone

 Table 13 - Mean dissolution (DP) (n = 6) of the formulation

VI with and without pancrelipase containing 4 mg of BPQ in 0.05M phosphate buffer pH 7.4, 0.07% tween 80.

 Table 14 - Mean (SD) (n = 6) dissolution of the V2 formulation with and without pancrelipase, containing 4 mg of BPQ, in 0.05M phosphate buffer pH 7.4, 0.07% tween 80.

Time (min) Dissolution (%) (DP) Dissolution (%) (DP) with pancrelipase 5 39.78 15.11 11 70.37 26.99

8 35.0 04 19.20 72.23 19.30

12 38.31 3. 03 78, 14 14, 12

15 49, 72 18, 33 78.11 8, 62

30 50.21 16.10, 82.30, 2.41

60 39.07 10.00 81.25 6.55

Table 15 shows the dissolution of 4 mg of

Free BPQ in pH 4.0, 4.00M phosphate buffer with 1% (w / v) sodium dodecyl sulfate. Although the concentration of the surfactant increased when compared to the previous method (tween 80 0.07%), the free drug did not dissolve in this medium. This can be explained by the precipitation of BPQ due to interaction with the sodium salt, which may occur in vivo. Therefore bile salts, such as sodium taurocholate, can precipitate the drug in the gastrointestinal system.

 Table 15 - Dissolution of free buparvaquone (BPQ) (4 mg) in phosphate buffer pH 7.4 with 1% (w / v) sodium dodecyl sulfate.

[096] Figure 10 and Table 16 show the dissolution profiles of formulations VI and V2 in pH 7.0, 4.00 M phosphate buffer with 1% (w / v) sodium dodecyl sulfate. For VI, the dissolution reached 83.71% at 5 minutes of the test, although some precipitation was observed at 20 minutes, the dissolution was 75.12%. The extent of precipitation and the variation between the samples (DP) were minimized when compared to the Tween 80 medium profiles. For V2, the dissolution reached 79, 84% of the 4 mg dose after 60 minutes. As found in VI, precipitation and standard deviation were reduced.

 Table 16 - Mean dissolution (SD) (n = 6) of free buparvaquone (4 mg) (black circle) and nanostructured lipid carriers in phosphate buffer pH 7.4 0.05 M and sodium dodecylsulfate 1% (w / v ), (A) Formulation VI; (B) Formulation V2.

 [097] The evaluation of the dissolution profiles shows the importance of the surfactant for the release, dissolution and, therefore, absorption of the BPQ. In the case of administration of CLNs orally, the drug would not be released until the nanoparticles reach the duodenum, where the bile salts and pancrelipase are secreted. Consequently, the drug could be absorbed in the intestines.

 [098] Furthermore, the formulations developed may constitute a drug delivery system directed to the lymphatic system. The drug can reach the lymphatic vessels due to the small size of the nanoparticles or even the drug dissolved in the lipids, which can be hydrolyzed by pancrelipase.

[099] Drug administration directed to the lymphatic system may be an innovative approach for the treatment of leishmaniasis. After the bite, the parasites are disseminated through the vascular and lymphatic systems, and infect monocytes and macrophages of the mononuclear system. The spleen, liver and lymph nodes are the organs most affected by visceral leishmaniasis. Thus, absorption and distribution of BPQ through the lymphatic system may increase drug availability at the site of action.

 [100] As a result of intestinal absorption and distribution of CLNs through the lymphatic system and systemic circulation, BPQ may reach the spleen, liver and lymph nodes after oral administration. Such a feature is not possible by employing the conventional veterinary formulation or the liposome. Consequently, developed CLNs have potential application for the treatment of visceral leishmaniasis and prevention of recurrence of cutaneous leishmaniasis.

 - Evaluation of the leishmanial activity of BPQ liyre and BPQ-CLNs in macrophages infected by amastigotes of L. infantum

 [101] Figure 11 shows the activity of the formulation

VI with and without coating against amastigotes of L. infantum. Table 17 shows the EC ₅₀ values for each condition. All presented improved leishmanicidal activity when compared to free BPQ. VI showed EC ₅₀ of 229.0 nM, which represents a 2.0-fold increase. The coated formulation showed increase in EC ₅₀ (150.5 nM) 3.0 fold compared to free BPQ (456.5 nM). This difference can be explained by the direct action of polymyxin B, combined with the interaction of polysaccharides with SIGN-R1 receptors on the macrophage cell membrane, which would favor internalization of the nanoparticles.

Table 17 - Leishmanicidal activity of amintigotes of L.ínfantum (n = 3) of free buparvaquone (BPQ), formulation VI and VI coated with polymyxin B, chitosan and anionic dextran.

 [102] The increase in the leishmanicidal activity of the in vitro tests of CNLs shows a solid perspective for its in vivo parenteral application. In the literature it is described that the serum proteins present in the interactions of nanoparticles and macrophages form a "corona" (opsonization) around the nanoparticle, making them more "visible" for the recognition and internalization of macrophages (endocytosis).

 [103] Endocytosis is a process of eukaryotic cells, which consists of the internalization of extracellular substances typically by the invagination of the membrane forming vesicles, known as phagosomes. After internalization, the lysosome fuses with the phagosome and releases its hydrolases to degrade the content in amino acids, fatty acids and glucose. This resulting structure is known as phagolysosome.

[104] It is therefore possible to develop specific drug delivery systems for the treatment of leishmaniasis using the endocytosis mechanism. The parasites can survive and multiply within the phagolysosomes. However, the nanoparticles also end in phagolysosomes. Thus, the encapsulated drug can be released by the degradation of the nanoparticle and reach the parasite.

 [105] Since the in vitro method does not present all of the compartments of an organism, the internalization of the nanoparticles can be reinforced by opsonization and, therefore, developed CNLs may have even more pronounced leishmanicidal activity.

 [106] Due to the results of the present invention, developed CLNs have potential application to improve drug efficacy, reduce treatment toxicity, and improve patient compliance. Such formulations may be used as oral and parenteral medicaments to fill the gaps of conventional treatment of leishmaniasis. As a consequence, the cost can be minimized by reducing hospitalization for medication administration and monitoring of side effects.

 Those skilled in the art will appreciate the knowledge presented herein and may reproduce the invention in the embodiments presented and in other embodiments within the scope of the appended claims.

Claims

 A process for obtaining nanostructured lipid carriers characterized in that it comprises the steps of:  a) Prepare from 0.5 to 50% (w / v) oil phase;  b) Add 0.01 to 5% (w / v) buparvaquone;  c) Prepare the aqueous phase;  d) Heat and mix the oily and aqueous phases under stirring;  e) Agitate the mixture of the oily and aqueous phases obtained;  f) Homogenize the high pressure;  g) Cool the obtained carrier at room temperature;  h) Dilute the carrier obtained in step "g" in up to 1: 3 parts of purified water;  i) Add 50 to 500,000 IU / mL of a polypeptide under agitation;  j) Add 0.01 to 2.0% w / v of a cationic polymer under stirring;  k) Add 0.04 to 5.0% w / v of anionic polymer under stirring; and  1) Carry out the potting.  Process according to claim 1, characterized in that in step "a" the oil phase comprises 0.5 to 50% (w / v), preferably 5 to 15% (w / v) , of a ratio of 0.1 (10: 1) to 10.0 (1:10) of liquid and solid lipids (LL / LS), respectively, preferably 0.5 (2: 1) to 3 (1: 3). A process according to claim 2, characterized in that the liquid lipids are selected from the group consisting of triglycerides of caprylic and caprylic acids, glyceryl monocaprylate, cardiac oil, corn oil, olive oil, sesame oil, cottonseed oil, soybean oil, oleic acid, preferably triglycerides of capric and caprylic acids.  Process according to claim 2, characterized in that the solid lipids are selected from the group consisting of hydrogenated palm oil, hydrogenated coconut mono, di and triglycerides, stearoyl macrogol 32-glycerides, glyceryl distearate , hydrogenated soybean oil, triglycerides of palmitic and stearic acids, glyceryl behenate, preferably hydrogenated palm oil.  Process according to claim 1, characterized in that, in step b, the buparvaquone is added in the oil phase obtained in the previous step in the concentration of 0.01 to 5%, preferably 0.3% ( w / v).  A process according to claim 1, characterized in that in step c the aqueous phase is prepared, which comprises the mixture of purified water qsp and from 1 to 10% (w / v) of surfactant, subjected to magnetic stirring until complete homogenization, preferably from 2 to 4% (w / v). A process according to claim 6, characterized in that the surfactants are selected from the group consisting of ethylene oxide and propylene oxide copolymers 188, 407, 101, 105, 108, 122, 123, 124, 181, 182, 183, 184, 185, 212, 215, 217, 231, 234, 235, 237, 238, 282, 284, 288, 331, 333, 334, 335, 338, 401, 402, 403; sorbitan stearate, sorbitan monooleate, sorbitan trioleate, sorbitan tristearate, polysorbate 80, polysorbate 60, preferably poloxamer 188.  Process according to claim 1, characterized in that in step "d" the mixture of the oily and aqueous phases is carried out at a temperature ranging from 45 to 85 ° C, preferably 70 ° C, with stirring which ranges from 150 to 800 rpm, preferably 500 rpm, for 2 to 45 minutes, preferably 10 minutes.  A process according to claim 1, characterized in that, in step "e", the pre-emulsion formation is carried out employing suitable equipment, such as a high performance disperser, at a rotation ranging from 500 to 14000 rpm, preferably 8000 rpm, the temperature ranging from 40 to 80øC, preferably 70øC, for 2 to 45 minutes, preferably 10 minutes. 10. Process according to claim 1, characterized in that in step "f", the high pressure homogenization is performed for one to ten cycles IO ⁶ to IO ⁷ Pa, at a temperature ranging 45-85 Preferably 70 ° C.  A process according to claim 1, characterized in that, in step "g", the cooling is carried out at ambient temperature ranging from 20-25 ° C.  A process according to claim 1, characterized in that, in step "h", the carrier is diluted in up to 1: 3 parts of purified water. A process according to claim 1, characterized in that, in step "i", 50 to 500,000 IU / ml of a polypeptide, preferably 1000 to 9000 IU / ml, is added to the carrier at a temperature ranging from 20 to 25 ° C, homogenization by magnetic stirring ranging from 50 to 250 rpm, preferably 100 rpm, at a pH ranging from 3.5 to 9.0, preferably 5.0 to 7.0, for 50 to 120 minutes, preferably 60 minutes.  A process according to claim 13, characterized in that said polypeptide is preferably polymyxin B, where it is alternatively substituted by polymyxin E (colistin).  A process according to claim 1, characterized in that in step "j", from 0.01 to 2% w / v of a cationic polymer, preferably from 0.05 to 0.2%, is added, homogenization by magnetic stirring ranging from 50 to 1000 rpm, preferably 100 rpm, at a pH ranging from 3.5 to 9.0, at a temperature ranging from 20 to 25Â ° C, for 50 to 120 minutes, preferably 60 minutes.  A process according to claim 15, characterized in that said cationic polymer is preferably chitosan, in which it ranges from 50 to 200 kDa, with a degree of deacetylation of at least 75%. A process according to claim 1, characterized in that, in step "k", from 0.04 to 5% w / v, preferably from 0.4 to 0.55% w / v, of an anionic polymer, homogenization by magnetic stirring ranging from 50 to 150 rpm, preferably 100 rpm, at a pH ranging from 3.5 to 9.0, at a temperature ranging from 20 to 25 ° C, for 50 to 120 minutes, preferably 60 minutes.  A process according to claim 17, characterized in that said anionic polymer is preferably dextran sulfate, in which the latter is substituted by D-mannose-6-phosphate.  Nanostructured lipid carriers obtained as defined in any one of claims 1 to 18, characterized in that it comprises:  - from 0.5 to 50% w / v oil phase;  - from 1 to 10% w / v surfactant;  50 to 500,000 IU / ml of a polypeptide;  - from 0.01 to 2% w / v of a cationic polymer;  - from 0.04 to 5% w / v of an anionic polymer; and  - purified water q.s.p.  Nanostructured lipid carriers according to claim 19, characterized in that said oil phase comprises 0.5 to 50% (w / v) of a ratio of 0.1 (1:10) to 10.0 (1:10) liquid and solid lipids (LL / LS), respectively, preferably 0.5 (2: 1) to 3 (1: 3), and 0.1 to 5.0% buparvaquone, preferably 0 , 3% (w / v). Nanostructured lipid carriers according to claim 20, characterized in that the liquid lipids are selected from the group consisting of caprylic and capric acid triglycerides, glyceryl monocaprylate, cardboard oil, corn oil, olive oil, sesame oil, cotton oil, soybean oil, oleic acid, preferably triglycerides of capric and caprylic acids. Nanostructured lipid carriers according to claim 20, characterized in that the solid lipids are selected from the group consisting of hydrogenated palm oil, hydrogenated coconut mono, di and triglycerides, stearoyl macrogol 32-glycerides, distearate of glyceryl, hydrogenated soybean oil, triglycerides of palmitic and stearic acids, glyceryl behenate, preferably hydrogenated palm oil.  Nanostructured lipid carriers according to claim 19, characterized in that said surfactant preferably is in a concentration of 2 to 4% (w / v), and is selected from the group consisting of ethylene oxide and propylene oxide 188, 407, 101, 123, 124, 181, 182, 183, 184, 185, 212, 215, 217, 231, 234, 235, 237, 238, 282, 284, 288, 331, 333, 334, 335, 338, 401, 402, 403 sorbitan stearate, sorbitan monooleate, sorbitan trioleate, sorbitan tristearate, polysorbate 80, polysorbate 60, preferably poloxamer 188.  Nanostructured lipid carriers according to claim 19, characterized in that preferably said polypeptide is in a concentration of 1000 to 9000 IU / ml, and is preferably polymyxin B, where it is alternatively substituted by polymyxin E ( colistin). Nanostructured lipid carriers according to claim 19, characterized in that said cation polymer preferably is in a concentration between 0.05 and 0.2%; and is preferably chitosan, which ranges from 50 to 200 kDa in weight with a degree of deacetylation of at least 75%.  Nanostructured lipid carriers according to claim 19, characterized in that preferably the anionic polymer is in a concentration of 0.4 to 0.55% w / v; and is preferably dextran sulfate, where it is alternatively substituted by D-mannose-6-phosphate.  Nanostructured lipid carriers according to claim 19, characterized in that they have a mean hydrodynamic diameter (DHM) ranging from 100 to 350 nm; zeta potential (mV) less than -10, preferably less than -20; and polydispersity index less than 0.3.  Use of the nanostructured lipid carriers as defined in any one of claims 19 to 27, characterized in that it is in the preparation of a medicament for treating leishmaniasis.  Use according to claim 28, characterized in that it is directed to the lymphatic system.