article: Most treatments of leishmaniasis require hospitalization and present side effects or parasite resistance; innovations in drug formulation/reposition can overcome these barriers and must be pursued to increase therapeutic alternatives. Therefore, we tested polymyxin B (polB) potential to kill Leishmania amazonensis, adsorbed or not in PBCA nanoparticles (PBCAnp), which could augment polB internalization in infected macrophages. PBCAnps were fabricated by anionic polymerization and analyzed by Dynamic Light Scattering (size, ζ potential), Nanoparticle Tracking Analysis (size/concentration), vertical diffusion cell (release rate), drug incorporation (indirect method, protein determination) and in vitro cell viability. Nanoparticles coated with polB (PBCAnp-polB) presented an adequate size of 261. 5 ± 25. 9 nm, low PDI and ζ of 1. 79 ± 0. 17 mV (stable for 45 days, at least). The 50% drug release from PBCAnp-polB was 6–7 times slower than the free polB, which favors a prolonged and desired release profile. Concerning in vitro evaluations, polB alone reduced in vitro amastigote infection of macrophages (10 μg/mL) without complete parasite elimination, even at higher concentrations. This behavior limits its future application to adjuvant leishmanicidal therapy or antimicrobial coating of carriers. The nanocarrier PBCAnp also presented leishmanicidal effect and surpassed polB activity; however, no antimicrobial activity was detected. PolB maintained its activity against E. coli, Pseudomonas and Klebsiella, adding antimicrobial properties to the nanoparticles. Thus, this coated drug delivery system, described for the first time, demonstrated antileishmanial and antimicrobial properties. The bactericidal feature helps with concomitant prevention/treatment of secondary infections that worst ulcers induced by cutaneous L. amazonensis, ultimately ending in disfiguring or disabling lesions.
  Leishmaniasis is a public health problem in tropical countries, classified as a neglected disease by World Health Organization (WHO) [1]. Phlebotomine sandflies transmit over 20 species of protozoa from the genus Leishmania, an intracellular parasite which hosts mammalian macrophages and establishes disease [2]. WHO divides leishmaniasis into three types of infections: visceral, mucocutaneous and cutaneous leishmaniasis (CL) [3]. Nowadays, 70–75% of CL cases are present in 10 tropical countries of the world [4]. CL causes ulcerated lesions in the skin, which can evolve to disfiguration, mutilation and allow secondary infection by bacteria [5]. CL treatment relies on meglumine antimoniate, sodium stibogluconate, pentamidine, amphotericin B deoxycholate and liposomal amphotericin B [6]. However, these drugs present relevant side effects, require hospitalization for intravenous administration and present some reports on drug resistance [7]. Fluconazole, ketoconazole, and miltefosine can also be administrated as an oral treatment. Newer drugs, such as paromomycin are an alternative to topical treatment [6]. With higher disease incidence in low-income regions, industrial investments in drug innovation are less attractive, such as alternative dosage forms [1]. The use of nanoparticles (NPs) as carriers can overcome drug resistance, side effects and unspecific biodistribution of microbicidal agents, which reduce high drug dosage, loss of activity and the need for chemical modifications [8]. Macrophage increased uptake and residence time can also be accomplished with NPs, a desired feature for a carrier to deliver leishmanicidal drugs. Poly butyl-cyanoacrylate nanoparticles (PBCAnp), for instance, are based on a low toxicity synthetic polymer with adequate biodegradation. Moxifloxacin loaded in PBCAnp triplicated drug accumulation inside macrophages, besides the high increase in residence time [9]; gentamicin uptake was quintuplicated with the PBCAnp given to peritoneal macrophages [10]. PBCAnp also holds another important feature: molecules can be adsorbed on its surface by covalent attachment or internalized during polymerization. This flexibility allows surface functionalization or multiple drug loading with different kinetic release profiles [11]. Even with technological approaches (such as NPs) to enhance the leishmanicidal effect, the treatment for CL does not rely solely on parasite elimination. Most secondary infections in CL ulcers present Gram-negative bacteria, as Klebsiella pneumoniae and Pseudomonas aerugionosa [5], which do not impair leishmaniasis healing; however, Pseudomonas secondary infection can be a co-factor in mutilation induced by CL [12], besides inhibition of phagocytosis due to alginate supported biofilms [13]. Treatment for dermal infection caused by Pseudomonas and other Gram-negative bacteria includes the antimicrobial peptides called polymyxins, which are also active against few strains of Gram-positive bacteria [14]. Among polymyxins, polymyxin B (polB) is used against Pseudomonas infections and briefly reported as an inhibitor of Leishmania growth [15]; other cationic antimicrobial peptides were classified as leishmanicidal, but none of them are available in the market [16]. Since peptide activity can decrease due to cell culture medium additives, we fabricated PBCAnp to adsorb the antimicrobial and deliver it inside macrophages. Considering all previous statements, we produced PBCAnp adsorbed with polB to evaluate its leishmanicidal effect and its viability as a functionalized antibacterial carrier.
  N-butyl-cyanoacrylate monomer (10 mg/mL, BBraun, Germany). Dextran sulphate (70,000 Da, Sigma-Aldrich, Denmark). Albumin, bicinchoninic kit (QuantiPro BCA Assay Kit), Dimethyl sulfoxide, ethanol, hydrochloric acid, isopropanol, methanol, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) and sodium hydroxide were from Sigma-Aldrich, MO, USA. Cellulose membrane (Spectra/por 2,12 to 14 kDa, Spectrapore, USA). M199 medium, RPMI 1640 medium and Müller-Hinton broth were from Vitrocell, Brazil. Rapid panoptic dye (Laborclin, Brazil). Amphotericin B was donated by Cristália, Brazil. Polymyxin B sulphate (500,0000 UI) was donated by Química Haller, Brazil. Distilled water was used for chemical assays and ultrapure water (Milli Q, 18. 2 MΩ. cm at 25°C and a TOC value below 5 ppb) for biological experiments and formulations. The PBCAnp synthesis was performed by the emulsion polymerization method [17,18]. 100 μL of n-butyl-cyanoacrylate monomer was added dropwise, under vigorous stirring (800 rpm), to a 10 mL aqueous solution of HCl 0. 1 M (pH 2. 5) containing 100 mg of dextran 70,000 Da. After 4 hours of stirring, the pH was adjusted to 7. 0 ± 0. 3 with NaOH 0. 1 M. The nanosuspension was filtered (1 μm filter membrane) and centrifuged (14,000 x g, 30 minutes, 25°C) to remove polymer that did not retained a particle structure; the pellet was then re-suspended in ultra purified water for the control carrier formulation (PBCAnp) or in 5 mg/mL polB sulphate solution (PBCAnp-polB), resulting in formulation of nanoparticle suspensions.
  The formulations showed reproducible size (Table 1), around 200 nm, regardless of polB addition (t = 0 days, t-test, p = 0. 51). Size distribution presented a polydispersity index (PDI) of less than 0. 2, adequate for pharmaceutical formulations. NPs maintained their size during the stability study, performed up to 45 days, with no significant statistical difference between intragroup means (Anova test, PBCAnp p = 0. 11; PBCAnp-polB p = 0. 34). According to literature, this size range is adequate to enhance macrophage phagocytic activity and activation of the complement system [22]. The D10 value is higher than 100 nm, which means that 10% of total particles are under the reported this particle size value. In our case, this is a desirable D10, since particles smaller than 100 nm may have modified physical and chemical properties, leading to a greater potential for toxicity [23,24]. Morphological analysis showed that the PBCAnp has a spherical shape (Fig 1), validating the use of DLS measurements that are based in the spherical model for calculations. The spherical shape observed by SEM is in agreement with that found by Bootz and colleagues, as well as the decrease in particle size when compared to that measured by DLS [25]. The size decrease happens due to the removal of water (drying) in sample preparation. The medium Zeta potential goes from negative (-18. 11 ± 5. 97 PBCAnp) to positive values (1. 79 ± 0. 17 PBCAnp-polB) (Table 1) indicating adsorption of the cationic drug on the anionic surface of the NPs and the presence of its free form in solution. The potential shift was expected in polB NPs, and the resulted Zeta potential is not considered enough for pure electric colloidal stability [26]. However, steric stabilization with dextran is sufficient to conserve physical stability [27], confirmed with size maintenance for up to 45 days (Table 1). NTA size measurements were similar among formulations (Table 2; t-test, p = 0. 18), with D10 values above 100 nm, as observed with DLS technique. The mean particle size and D values were higher when measured by DLS (around 200 nm) than those by NTA (around 170 nm), as expected. DLS measures size by volume, whereas larger particles largely contribute to the scattering plot. NTA traces individual particles, and a few larger particles has little effect on the precision of the size measure [28]. Despite the significant statistical difference (t-test, p = 0. 66), the values of particles/mL are in the same order of magnitude. NTA does not offer high precision in this concentration measurement [28]. PolB was incorporated in NPs by electrostatic adsorption. In this case, the more drug in the external medium the higher the adsorption [29]. In accordance, previous tests with 1 mg/mL of drug presented a low adsorption to the NPs (216 μg/mL, IE = 21,6%), but 5mg/mL resulted in approximately four times the amount of bounded drug, with a small decrease in incorporation efficiency index (885 μg/mL, IE = 17. 7%). However, as the concentration gets too high, adsorption gets close to saturation and encapsulation rate drops more, such as we observed with 10 mg/mL of polB (1050 μg/mL, IE = 10. 5%). Therefore, our studies continued with 5 mg/mL of polB added to nanoparticles (named PBCAnp-polB). The efficiency of incorporation was stable: PBCAnp-polB presented 17. 7% (885 μg/mL) on t = 1 day, and 16. 8% (840 μg/mL) on t = 45 days (t-test, p = 0. 99). PBCAnp-polB resulted in approximately 2. 4 x 105 molecules of polB per nanoparticle. The experimental drug loading for PBCAnp-polB was 10. 53% as compared to a theoretical value of 8. 76%, due to unbound polymer (removed in the filtration step). We evaluated drug release profile of PBCAnp-polB in PBS buffer pH 7. 4 at 37° C (Fig 2). Noteworthy, free polB had no immediate release profile. The cellulose membrane used to separate the test compartments is widely used with proteins and described as having low binding with them, in addition to having 12–14 kDa membrane pores, which are at least 12 times larger than polB (1,023 kDa). Therefore, delayed time can be attributed to drug diffusion rate through the membrane. 50% of free polB was detected in the acceptor compartment after 1 hour of the experiment, while polB in PBCAnp-polB took 6 hours to reach this value. This release profile confirms a sustained release of polB from NPs, adequate for prolonged antimicrobial effect. Leishmanicidal activity tests were performed in vitro with Leishmania amazonensis promastigotes. The IC50 value found for free polB was 77. 1 μg/mL (59. 2 μM) (Fig 3). As a reference, the standard drug meglumine antimoniate presented an average IC50 value of 49 to 60 μg/mL, which was measured in reference strains of the same species. It is important to note that the current model of in vitro susceptibility testing may undermine leishmanicidal efficacy, since these peptides can be inactivated by fetal bovine serum and high saline concentration in culture media [30–32]. Unfortunately, high concentration of bovine serum is essential for Leishmania amazonensis adequate growth. Previous attempts from our group to diminish serum concentration did not reach satisfactory growth density for the used strain. PolB binding to serum also favours its cell entrance without its membrane activity; consequently, degradation can occur and aminoacids serve as nutritional molecules, which may explain the increased growth and high standard deviation of the less concentrated samples. As the quantity increases, free polB probably predominates and inhibits growth (S5 Fig). In addition, several drugs had lower activity in extracellular Leishmania forms: amphotericin B had half potency in promastigotes when compared to amastigotes, whereas sodium stibogluconate presented 3 times less potency in the same comparison [33]. Fig 3 shows that PBCAnp-polB formulation reached IC50 of 6. 0 μg/mL (4. 6 μM, NP dilution = 1: 830), a high IC50 decrease, compared with the free drug. However, PBCAnp also killed protozoa, (dilution of NP at IC50 = 1: 620) and was largely responsible for the leishmanicidal effect of PBCAnp-polB. In accordance with our results, control PBCAnp were described as active against promastigotes of Leishmania amazonensis [34], but the related paper did not evaluate coated NPs, like ours. Leishmanicidal activity of NPs tailored with a similar polymer (poly isohexylcyanoacrylate) was also described in infected macrophages with amastigotes from Leishmania donovani [35]. Firstly, we performed cell viability tests in non-infected J774 macrophages with 10 and 20 μg/mL of polB, free or loaded in NPs, which are equivalent to 2 and 4-fold the IC50 of PBCAnp-polB (Fig 4, values above the 100% line graph). Samples with 10 μg/mL of polB were well tolerated by the cells, but the 20 μg/mL ones presented a 30% decline (free polB) to 81% decline (PBCAnp-polB) in macrophage viability. Although extensively used in macrophage cultures to neutralize lipopolysaccharides, polB lipopolysaccharide-binding concentration is lower than the ones used in this study [36]. The enhanced citotoxicity of the nanostructured drug at the highest concentration derived from the carrier, since control NPs at the respective dilution also presented similar decline (PBCAnp 1: 250,88%). Published papers showed that PBCAnp was not cytotoxic at 15 μg/mL to rat glioma cell lines [37], equivalent to what we found for J744 macrophages (16,8 μg/mL of PBCAnp, equivalent to 1: 500 dilution in Fig 4). The same macrophage citotoxicity was found for uninfected murine peritoneal macrophages (50% viability at 16 μg/mL of PBCAnp) in a previous work from our group [35]; other researchers also detected a moderate cytotoxic effect (approximately 60% viability) to THP-1 macrophages at concentrations above 50 μg/mL [9]. Macrophages without treatment (negative leishmanicidal activity, “control”, Fig 4) ) presented 58% of highly infected cells (with up to 7 parasites per cell), and only 6% of cells without infection. Amphotericin B, the second choice treatment of leishmaniasis [37], was used as a positive leishmanicidal control and resulted in 85% of uninfected cells, with practically no highly infected macrophages (1%). We found no reports of leishmanicidal profile of amphotericin B regarding macrophage infection levels for comparison. All treatments decreased highly infected macrophages and increased non-infected cell numbers, compared to the infection control (t test, p < 0. 05). Nanoparticle samples (1: 500 dilution) reached the average of cleared cells from the one obtained with amphotericin B; the polB adsorption did not changed counting averages. Higher nanoparticle concentrations were toxic to macrophages, therefore were not tested. PolB treatment with 10 μg/mL had similar behaviour of 20 μg/mL, with no statistical difference, except for low infected counting. Therefore, our results show that increments in polB concentration were not enough to increase its leishmanicidal efficacy. As described before, a cutaneous lesion caused by Leishmania are frequently contaminated by bacteria. This contamination can lead to a more serious secondary infection, or even worse the leishmaniasis, like Pseudomonas aeruginosa. Therefore, the microbicidal activity of the formulations was tested. The minimum inhibitory concentration used of polB and PBCAnp-polB was 2 μg/mL, in triplicate (P. aeruginosa ATCC 27853, E. coli ATCC 25922, and K. pneumoniae clinical isolate), by visual determination. Since is a two-fold dilution method, the accurate MIC value is bigger than 1, up to 2 μg/mL (1< MIC ≤ 2 μg/mL), as previously reported by other groups [38,39]. All visual determinations corroborated with O. D. analysis (S4 Fig). Incorporation of polB in NPs did not change MIC results, which is in accordance with the lack of antimicrobial activity from the carrier (at the same dilutions tested for the loaded version). Noteworthy, the incorporation of polB was close to 20%, which still leaves 80% of free drug; the bound polB was released after 6–7 hours in PBS, but it could present different kinetics when applied to microbial culture media. Therefore, this test confirms that polB remains active and bound polB is released fast enough to maintain the 24-hour result from the polB alone. The IC50 (against promastigotes) of free polymyxin B (polB) was reported for the first time, such as its intracellular antiparasitic efficacy. The antimicrobial greatly decreased intracellular protozoa number in macrophages, which shows its potential as adjuvant therapy, but it was not capable of a relevant macrophage clearance. PBCAnp formulations had a low PDI, with a reproducible and stable size, adequate for macrophage targeting. PBCAnp-polB presented a coating of approximately 2. 4 x 105 molecules of polB per unit, with a sustained release profile. PolB did not change NP toxicity to protozoa or macrophages. The optimal PBCAnp in pharmaceutics aspects present antileishmanial activity by itself, superposing polB activity. However, as discussed, serum presence in culture media could have underestimated polB efficacy. PBCAnp did not modify the antibacterial activity of polB at safe dilutions to macrophages, which makes it possible to use them in antibacterial therapeutics for Gram-negative infections or even contaminated ulcers caused by cutaneous and mucosal Leishmania. As an example, K. pneumoniae has been found in lesions caused by leishmaniasis, as well as P. aeruginosa, which may be related to disease morbidity and require specific treatment [5]. Considering that PBCAnp can also carry drugs inside their cores, this formulation can easily serve as a carrier for leishmanicidal drugs, helping to prevent or treat concomitant bacterial infections.
summary: Cutaneous leishmaniasis form disfiguring lesions that leads to depression and social isolation. Both consequences result often in economic and education loss for communities already short on resources. The actual treatment aims to eliminate the protozoa disease-vector. Yet, it presents undesired effects or parasite resistance, besides the need for intravenous infusions and hospitalization. There is a few topical leishmanicidals but none with prolonged action. As highlighted, no ideal therapy exists; we must keep searching for alternatives, which includes new drugs, vehicles or therapies. For drugs, the most cost-effective way comprises testing approved molecules for other diseases, such as the antimicrobial polymyxin B (polB). Repositioning a drug has the added benefit of known safety profile and drawbacks, shortening and narrowing the research path. In turn, carriers can decrease side-effects and multiple dosing. They shield and/or improve drug targeting, which in this case means delivering inside macrophages infected with Leishmania. An example is poly (n-butyl cyanoacrylate) nanoparticles (PBCAnp), shown as an efficient carrier for targeting an extended drug release. Therefore, this work aimed to evaluate leishmanicidal and adjuvant properties of polB, alone and absorbed onto PBCAnp.
keywords: blood cells
  antimicrobials
  medicine and health sciences
  immune cells
  engineering and technology
  drugs
  immunology
  microbiology
  parasitic diseases
  protozoan life cycles
  bacterial diseases
  developmental biology
  nanoparticles
  antibiotics
  nanotechnology
  pseudomonas infections
  pharmacology
  promastigotes
  polymyxins
  infectious diseases
  white blood cells
  antimicrobial resistance
  animal cells
  life cycles
  cell biology
  microbial control
  biology and life sciences
  cellular types
  protozoology
  macrophages
title: Biodegradable nanocarriers coated with polymyxin B: Evaluation of leishmanicidal and antibacterial potential