Topically Applied Chitosan-Coated Poly(isobutylcyanoacrylate

Article Cite This: ACS Appl. Bio Mater. 2019, 2, 2573−2586

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Topically Applied Chitosan-Coated Poly(isobutylcyanoacrylate) Nanoparticles Are Active Against Cutaneous Leishmaniasis by Accelerating Lesion Healing and Reducing the Parasitic Load Sophia Malli,†,‡ Sebastien Pomel,‡ Yasmine Ayadi,†,‡ Claudine Deloménie,§ Antonio Da Costa,∥ Philippe M. Loiseau,‡ and Kawthar Bouchemal*,⊥

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†

Institut Galien Paris Sud, UMR CNRS 8612, Univ. Paris-Sud, Université Paris-Saclay, Faculté de Pharmacie, 5, rue J.B. Clément, 92296 Cedex Châtenay-Malabry, France ‡ BioCIS Biomolécules: Conception, Isolement, Synthèse, Chimiothérapie Antiparasitaire, UMR CNRS 8076, Univ. Paris-Sud, Université Paris-Saclay, Faculté de Pharmacie, 5, rue J.B. Clément, 92296 Cedex Châtenay-Malabry, France § Faculté de Pharmacie, Institut Paris Saclay d’Innovation Thérapeutique, UMS Inserm CNRS UPSud, Université Paris-Saclay, 92296 Cedex Châtenay-Malabry, France ∥ Université d’Artois, CNRS, Centrale Lille, ENSCL, Université Lille, UMR 8181, Unité de Catalyse et de Chimie du Solide (UCCS), Faculté Jean-Perrin, Rue Jean Souvras − SP 18, 62307 Lens, France ⊥ Institut Galien Paris Sud, Junior Member of the Institut Universitaire de France, UMR CNRS 8612, Univ. Paris-Sud, Université Paris-Saclay, Faculté de Pharmacie, 5, rue J.B. Clément, 92296 Cedex Châtenay-Malabry, France S Supporting Information *

ABSTRACT: Parenteral administration of amphotericin B-deoxycholate (AmB-DOC) or pentavalent antimonials to cure cutaneous leishmaniasis (CL) results in severe adverse reactions, while topically applied antileishmanial drugs are ineffective despite their good tolerance. This work is aimed to investigate whether poly(isobutylcyanoacrylate) nanoparticles coated with chitosan (Cs-NPs) could provide intrinsic antileishmanial activity after topical application. In vitro evaluations revealed that nanoparticles were active against the promastigote, axenic amastigote, and intramacrophage forms of Leishmania major. In vivo evaluations after repetitive topical applications on the skin of mice infected with L. major showed that Cs-NPs combined or not with AmB-DOC allowed partial healing of the lesion characterized by histological analyses. The parasitic load of skin specimens collected from mice was significantly reduced compared with that from nontreated mice, as analyzed by quantitative polymerase chain reaction (q-PCR). Ultrastructure characterizations by electron microscopy of L. major promastigotes after incubation with Cs-NPs showed morphological alterations, including aberrant shape and swelling of mitochondria and parasitic vacuoles. KEYWORDS: leishmaniasis, chitosan, nanoparticles, Leishmania major, poly(isobutylcyanoacrylate)

1. INTRODUCTION According to the WHO estimates, of the 0.7−1 million annual cases of cutaneous leishmaniasis (CL) worldwide, 90% occur in Afghanistan, Syria, Iran, Saudi Arabia, Brazil, Peru, Pakistan, and Algeria.1−3 During recent years, CL has emerged in nonendemic countries because of forced migration.4−6 Although CL lesions can self-heal over 3−18 months, CL can also lead to substantial mutilation of skin, morbidity, disfigurement, stigmatization, and psychological consequences. More severe manifestations known as disseminated CL, diffuse CL, © 2019 American Chemical Society

mucocutaneous leishmaniasis, and refractory leishmaniasis could occur depending on the parasite species. The decision to treat CL depends on different criteria, such as large individual lesions, multiple lesions, the location of the lesion (the face or joints), and a duration of more than 6 months.7,8 Treatment could reduce scarring as well as accelerate the cure Received: March 27, 2019 Accepted: May 8, 2019 Published: May 8, 2019 2573

DOI: 10.1021/acsabm.9b00263 ACS Appl. Bio Mater. 2019, 2, 2573−2586

Article

ACS Applied Bio Materials

Figure 1. Topography of diluted Cs-NPs obtained in the low-amplitude noncontact mode of atomic force microscope at scan ranges of 5 μm (a) and 1 μm (b and c). Panels (b) and (c) represent magnifications of two zones of (a). Panel (d) represents cross sections of Cs-NPs from AFM line analysis, from which the diameters were calculated using WSxM software (d). The imaging was performed after drying for 10 min at 20 °C. (e) 3D image of the Cs-NP suspension. (f) Comparative data of diameters of Cs-NPs from AFM and quasi-elastic light scattering (QELS). The diameters calculated from software-derived cross sections of the nanoparticles at maximum diameter representative of 33 nanoparticles.

Fungizone), or liposomal AmB.12−16 Miltefosine is the only oral treatment approved by the FDA in 2014 against leishmaniasis. However, miltefosine is teratogenic and induces gastro-intestinal side effects. Other strategies for the treatment of CL are cryotherapy, local thermotherapy, or topical application of paromomycin-containing formulations.3 Comparison of systemic antimonials to thermotherapy in a clinical trial showed that the overall efficacy of systemic antimonials was found to be 74%, whereas that of thermotherapy was 82.5%.17 Although well tolerated, topical application of paromomycincontaining formulations showed low efficacy to eradicate the infection. In a meta-analysis, the efficacy of an ointment-containing

and limit the risk of dissemination. Intralesional (IL) injection of pentavalent antimony (mainly sodium stibogluconate), which is the first-line treatment of CL, results from pain, particularly when the lesions are located in sensitive areas and for pediatric CL. Furthermore, a poor response to sodium stibogluconate has been revealed in different clinical studies (the cure rate ranged from 72 to 97%), highlighting the urgent need for alternative treatments. Systemic treatment for CL is usually reserved for mucocutaneous leishmaniasis, extensive lesions, and immunosuppressed patients.3 Those treatments include oral miltefosine,9−11 oral fluconazole, parenteral pentavalent antimonials, amphotericin B-deoxycholate (AmB-DOC; 2574


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ACS Applied Bio Materials Table 1. In Vitro Evaluation of the Antileishmanial Activity of Cs-NPs and F68-NPsa formulations

L. major promastigotes IC50 (μg/mL) ± SEMb

L. major axenic amastigotes IC50c (μg/mL) ± SEM

L. major intramacrophage IC50 (μg/mL) ± SEM

Cs-NPsd F68-NPse AmB−DOCf native chitosan DOC isobutanol

1.14 ± 0.11 1.24 ± 0.07 0.25 ± 0.05 >100 >100 >100

0.53 ± 0.07 0.46 ± 0.09 0.24 ± 0.16 >100 >100 >100

7.15 ± 2.01 10.61 ± 2.01 0.42 ± 0.17 >100 >100 >100

a

IC50 were evaluated in L. major promastigotes, axenic amastigotes and intra-macrophage forms. AmB-DOC was used as an anti-leishmanial reference drug. bSEM: Standard error of measurement. cIC50: Inhibitory concentration 50. dCs-NPs: Chitosan-coated poly(isobutylcyanoacrylate) nanoparticles. eF68-NPs: poly(isobutylcyanoacrylate) nanoparticles without chitosan, stabilized by F68. fAmB−DOC: Amphotericin B−deoxycholate (water-soluble form, commercial Fungizone).

Figure 2. High-resolution scans of HES-stained skin samples collected from a noninfected (A) and an infected mouse without treatment (B). In (A), the epidermis (1), the dermis (2), and the hypodermis (3) were identified. (C) and (D) are high magnifications of an infected mouse skin containing Leishmania-packed macrophagic cytoplasm. In (D), 5−9 amastigotes were observed inside each infected macrophage. Legend to arrows: Thin epithelium (black arrow); hair follicles (blue arrow); hypodermis composed of adipocytes (white oval or round shaped, gray arrows); and muscles (stained pink, red arrows); L. major infected macrophages (green arrow).

paromomycin methylbenzethonium chloride twice a day for 10−20 days to cure L. major CL was comparable to antimonial IL injections.18 In another study, paromomycin ointment containing 0.5% gentamycin has led to a similar efficacy for the treatment of CL.19 In this phase 3 investigation conducted in Tunisia, the cure rate of CL caused by L. major was 81−82%.19 The cure rates reached 80% for CL due to L. braziliensis and L. panamensis in Panama (NCT01790659). However, these results, compared with a placebo cure rate of 58%, revealed almost no difference between formulations of paromomycin alone or combined with gentamicin. In summary, available drugs to treat CL are mainly toxic, ineffective, and/or mostly expensive. To cure CL, an ideal formulation should be applied topically. It should also have intrinsic antileishmanial activity and accelerate the healing of the lesion. In this context, chitosan-based nanoparticles (NPs) seem to be a good strategy to reach these

objectives because previous work from our group has demonstrated that nanoparticles comprising chitosan-coated poly(isobutylcyanoacrylates) (Cs-NPs) had strong intrinsic antiparasitic activity toward Trichomonas vaginalis, another Protozoan parasite.20,21 Poly(isobutylcyanoacrylate) nanoparticles, without chitosan stabilized by pluronic F68 (F68-NPs), also showed intrinsic antiparasitic activity against Trypanosomes, other kinetoplastids close to Leishmania sp.22 Moreover, chitosan formulations are known to have wound healing23−25 and antibacterial activities.26 This work aimed to evaluate the antileishmanial activity of chitosan-coated poly(isobutylcyanoacrylate) nanoparticles in vitro and in vivo after topical application. Our hypothesis is that the cationic charge and small size of nanoparticles make them susceptible to be internalized by the parasites, leading to antiparasitic activity. Thus, internalization of nanoparticles by L. major was monitored 2575


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Figure 3. HES-stained high-resolution scans of skin sections collected from mice infected by L. major and treated with IL injections of AmB− DOCIL (A), Cs-NPsIL (B), and (AmB−DOC/Cs-NPs)IL (C). Legend to arrows: adipocytes (gray arrows); safranin-stained fibrosis (orange arrows); and muscle fibers (red arrows). works.20,21,27 The combination of AmB−DOC suspension (final concentration of 5 mg/mL in AmB) and nanoparticles (final concentration of 20 mg/mL) was prepared by adapting a previous method developed in our research group,28 where AmB and DOC were added to nanoparticle suspension. Pluronic F127 (20 wt %) hydrogels containing the suspensions were prepared by adding under magnetic stirring at 4 °C pluronic F127 powder into nanoparticle suspension, AmB−DOC, or their combination. 3.2. In Vitro Antileishmanial Activity Evaluation. Leishmania major promastigotes and axenic amastigotes were cultured according to our previous works28,29 and detailed in the Supporting Information. Formulation activity in promastigotes in axenic and intramacrophage amastigotes was performed as previously described28,30−32 with slight modifications and detailed in the Supporting Information. The tested formulations were as follows: Cs-NPs, F68-NPs, and AmB−DOC.

by transmission electron microscopy (TEM) at different contact times. Nanoparticles were gelified with a thermosensitive hydrogel to increase their residence time on the skin.

2. MATERIALS Pluronic F127 (Poloxamer P407) was from BASF ChemTrade GmbH (Ludwigshafen, Germany). Amphotericin B was from Carbosynth (Berkshire, UK). All other reagents were from Sigma-Aldrich (SaintQuentin Fallavier, France).

3. METHODS 3.1. Formulation. Nanoparticles coated with chitosan (Cs-NPs) or stabilized by F68 (F68-NPs) were prepared and characterized as detailed in the Supporting Information, according to previous 2576


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Figure 4. HES-stained high-resolution scans of skin sections collected from mice infected by L. major and treated with (A) F127 and (B) AmB− DOCF127. Legend to arrows: L. major containing macrophages (green arrow); safranin-stained fibrosis corresponds to collagen fibers (orange arrows); muscle fibers (red arrows). 3.5. Histology. Skin sections were prepared and stained with HES (hematoxilin-eosin-safranin) as described in our previous work.28 Then, representative sections of skin collected from each animal were selected and digitized.28 3.6. Quantification of L. major Genomes by q-PCR. 3.6.1. Genomic DNA Preparation. Total DNA was isolated from skin sections previously fixed and embedded in paraffin as detailed in the Supporting Information by adapting the methods reported by previous works.33,34 For negative control, and to check the absence of cross-contamination, blocks of paraffin without skin samples were analyzed.33 Standard genomic DNA was similarly prepared from a culture containing 106 L. major parasites/mL. 3.6.2. q-PCR Analysis. The quality of DNA samples was first checked regarding the risk of false-negative results that could be due to DNA degradation and/or from the presence of PCR-inhibiting contaminants in the DNA extracts. The reproducibility of the amplification signal provided by the ACTA1 mouse gene was checked in all samples. These q-PCR analyses were performed as described in the Supporting Information by adapting previously described realtime PCR assays.28 The copy number of L. major genomes was measured from each skin sample against a standard curve generated from the standard L. major genomic DNA ranging from 10 to 106 genomes per PCR. The PCR efficiencies calculated for each gene from the slopes of the calibration curves generated from a standard mouse or L. major DNA sample were above 95%. 3.6.3. Statistical Analysis. Data of parasite genome counts were analyzed wit hGraphPad Prism using one-way ANOVA. Differences were considered significant when p-values < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001).

DOC, native chitosan, and isobutanol from the degradation of poly(isobutylcyanoacrylate) nanoparticles were also evaluated as controls. Experiments were performed in triplicate. 3.3. Investigation of the L. major Ultrastructure after Incubation with Cs-NPs Using TEM. L. major ultrastructure was determined by TEM after incubation of Cs-NPs with L. major promastigotes for 10 min, 20 min, 30 min, 1 h, or 2 h. After each incubation time, samples were prepared as described in our previous work21 and reported in the Supporting Information. 3.4. In Vivo Experiments. Animals (female BALB/c mice28 (Janvier France 18−20 g)) were infected at the base of the tail with a suspension of L. major promastigotes as previously described by Malli et al.28 After 9 to 11 weeks postinfection, the lesions reached 25 mm2 of surface area.28 The mice were separated in different cages and treated by either IL injections or topical applications (n = 6). IL Injections. Typically, 100 μL of suspensions (AmB−DOCIL, CsNPsIL, or their combination (Cs-NPs/AmB−DOC)IL) was injected at the periphery of the lesion every 2 days for 13 days.28 The animals were kept for an additional 2 weeks before euthanasia.28 Two control groups were used: noninfected mice and untreated infected mice group. Topical Application. Each formulation (200 μL) was applied topically using a glove-coated finger on each lesion daily for 3 consecutive weeks. Four formulations were applied: AmB−DOCF127, Cs-NPsF127, and the combination of (Cs-NPs/AmB−DOC)F127 and F127. Animals were euthanatized immediately after the end of treatment. Two control groups were used: noninfected mice and untreated infected mice. F68-NPs were considered as not stable enough in vivo to be used as a control for in vivo experiments. 2577


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Figure 5. HES-stained high-resolution scans of skin sections collected from mice infected by L. major and treated topically with (A) Cs-NPsF127 and (B) (Cs-NPs/AmB-DOC)F127. Safranin-stained fibrosis corresponding to collagen fibers is indicated by orange arrows.

4. RESULTS 4.1. Physico-Chemical Characterization of Nanoparticles. Chitosan-coated nanoparticles (Cs-NPs) had a positive zeta potential (+53.8 ± 2.8 mV), while nanoparticles stabilized by F68 (F68-NPs) exhibited a negative zeta potential (−4.0 ± 0.6 mV), in agreement with previous data.21 However, nanoparticles without chitosan aggregated immediately after preparation, and sedimentation followed by phase separation occurred after a few hours of storage (data not shown). Thus, the noncontact mode of atomic force microscopy was conducted only on Cs-NPs. The suspension was diluted, deposited on a mica surface, and dried for 10 min at 20 °C as described in the Supporting Information, allowing the observation of untreated surface structures in air, determination of the Cs-NP diameter, and high-resolution topographic imaging in 2D and 3D. The images in Figure 1 revealed that most of the nanoparticles had diameters less than 150 nm (Figure 1a and 1f). However, in some cases, individual nanoparticles with diameters of approximately 250 nm were observed (arrow in Figure 1b). Interestingly, the mean hydrodynamic diameter of Cs-NPs measured in water by DLS was in the range of an arithmetic average of the two populations (187 ± 6 nm) (Figure 1f and Figure S1 in the Supporting Information). Image cross sections of typical small-sized Cs-NPs are given in Figures 1c and 1d and showed that the diameters of this small-sized nanoparticle population ranged from 100 to

150 nm. A 3D image of the Cs-NP suspension is shown in Figure 1e. 4.2. In Vitro Evaluation of Antileishmanial Activity. Evaluation showed that nanoparticles of poly(isobutylcyanoacrylate) coated with chitosan had potent antileishmanial activity (Table 1). The IC50 values evaluated in L. major promastigote and axenic amastigote forms were 1.14 ± 0.11 μg/mL and 0.53 ± 0.07 μg/mL, respectively. The IC50 of AmB−DOC used as an antileishmanial reference drug was 0.25 ± 0.05 μg/mL in promastigotes and 0.24 ± 0.16 μg/mL in axenic amastigotes. To understand whether the antileishmanial activity of CsNPs was due to the chitosan shell or nanoparticle core composed of poly(isobutylcyanoacrylates), the activities of native chitosan and nanoparticles uncoated with chitosan were investigated separately. The results in Table 1 showed that native chitosan was inactive, while nanoparticles without chitosan stabilized by F68 (F68-NPs) exhibited intrinsic antiparasitic activity, which was comparable to that obtained with Cs-NPs. Finally, antileishmanial activity in infected macrophages was higher when nanoparticles were coated with chitosan. However, the activity of AmB−DOC was far stronger in infected macrophages than in Cs-NPs (Table 1). 4.3. In Vivo Antileishmanial Activity. The activity of chitosan-coated nanoparticles to cure infection was then investigated in vivo in a mouse experimental model. Lesions were induced by the subcutaneous administration of L. major promastigotes at the base of the mouse tail.28 After 4 weeks of 2578


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ACS Applied Bio Materials parasite administration, pink or red papules were observed at the site of administration. Next, 9−12 weeks after the injection, the lesions reached a surface of 25 mm2 and became mutilating. The periphery was moist and exudative. 4.3.1. Histopathology. Histopathology of Healthy Noninfected Mouse Skin. High-resolution scan images of healthy noninfected mice skin in Figure 2A and Figure S2 in the Supporting Information revealed normal structures of the epidermis, dermis, and hypodermis: a thin cornified layer (black arrow), regularly distributed hair follicles (blue arrows), and the cells in the dermis, followed by the hypodermis comprising adipocytes (gray arrow) and muscles (red arrows). Histopathology of Infected Mouse Skin without Treatment. For infected mouse skin, an extensive granuloma (stained purple) was observed in Figure 2B and Figure S3 in the Supporting Information. The granuloma was diffuse, involving the dermis, striated muscle fibers (stained pink on Figure S3), and adipocytes of the hypodermis that are infiltrated and partially destroyed. The adipocytes appeared white oval or round shaped (gray arrows in Figure 2B). The architecture was completely altered, and necrotic tissue was observed. Skin necrosis is a form of cell injury that results in cell death in living tissue. The necrotic zone, which appears without cell structures, is indicated by black arrows in Figure S3 (Supporting Information). The hair follicles and the glands were quasi-absent, in agreement with our previous observations for the skin of mice infected with L. major.28 At higher magnifications in Figure 2C and 2D, macrophages filled with amastigote Leishmania parasites stained purple (big green arrows) were observed. Another skin specimen collected from an infected mouse also showed ulcerated and invasive granuloma (Figure S3, lower panel). Histopathology of Mice Treated by IL Injections of the Formulations. In a first set of experiments, the mice were treated by IL injections of Cs-NPsIL, AmB−DOCIL, or their combination at the periphery of the lesions. Skin samples were collected after mice euthanasia, fixed, and embedded in paraffin before HES staining and analyses. IL injections of AmB− DOCIL did not heal the lesions as shown in Figure 3A and Figure S4 in the Supporting Information. The inflammatory granuloma was continuous and invasive, infiltrating the striated muscle (red arrow in Figures 3B and S4A) and hypodermis of the mouse. In Figure 3A, the adipocytes (white round or oval shaped, indicated by gray arrows) were dissociated by the granuloma and muscle fibers, and hair follicles were quasiabsent. This granuloma was not yet necrotic as revealed by the general view of the high-resolution scan of skin lesions (Figure S4A). Specimens collected from mice treated with IL injections of Cs-NPsIL showed numerous necrotic areas (Figure S5, black arrows). A zoom of a non-necrotic skin in Figure 3B and Figure S5B showed the presence of deeply located granuloma infiltrating the muscle fibers (red arrows). The granuloma is stained purple and indicated by a blue dashed line in Figure S5B. However, the hypodermis and muscle fibers were less disorganized than those in mice treated with IL injections of AmB−DOCIL. Similar observations were reported for mice treated with AmB−DOCIL combined with chitosan-coated nanoparticles (Cs-NPs/AmB−DOC)IL (Figures 3C and S6). In the specimen observed in Figure S6B, the muscle fibers (stained pink and indicated by red arrows) were infiltrated by the granuloma. Histopathology of Mice Treated with Topical Applications of the Formulations. Next, we investigated whether

Figure 6. Comparison of number of L. major copies of mice skin infected and treated with IL injections (higher panel) or topically (lower panel) with AmB−DOCIL (A), Cs-NPsIL (B), (Cs-NPs/ AmB−DOC)IL (C), F127 20 wt % hydrogel (D), AmB−DOCF127 (E), Cs-NPsF127 (F), and (Cs-NPs/AmB−DOC)F127 (G). Skin specimens collected from infected and nontreated mice were used as controls (NT1 and NT2). Statistic comparisons with healthy mice skin (C1) are presented (ANOVA, *p < 0.05; ***p < 0.001).

topical application of the formulations succeeded to treat the infection compared with IL injections. Treatments were applied to mouse lesions daily for 3 weeks. Regardless of the formulations used (AmB−DOC, Cs-NPs, or their combinations), they were gelified with pluronic F127 powder.28 Notably, samples collected from mice treated with F127 hydrogel used as a control showed significant necrotic tissue (Supporting Information Figure S7A, black arrows). In some areas, the granuloma was deep and diffuse, dissociating the muscles and adipocytes (Supporting Information Figure S7B). However, skin fragments with less diffuse granuloma and recognizable skin architecture were also observed in Figure 4A and Figure S7C. The adipocytes (round or oval shaped) and some muscle fibers (stained pink) are dissociated by the granuloma (Figure 4A). At a higher magnification, macrophages loaded with parasites were observed (green arrow in Figure 4A, high magnification). Skin sections treated with different formulations (AmB− DOCF127, Cs-NPsF127, and the combination of (Cs-NPs/AmB− DOC)F127) revealed tissue necrosis, cutaneous structural changes, and the presence of moderate to severe granulomatous chronic inflammation depending on the formulation. Major structural changes were observed, mainly for the tissues treated with AmB−DOCF127 (Figure 4B and Figure S8 in the Supporting Information). The deep part of the tissues was the most affected in all specimens with invasion of the muscle fibers by the granuloma that causes their dissociation. For mice treated with Cs-NPsF127 and the combination (Cs-NPs/AmB− DOC)F127, the architecture of the skin was preserved (Figure 5 and Figures S9 and S10 in the Supporting Information). The presence of collagen fibers stained orange in the hypodermis for Cs-NP treatments, as well as the (Cs-NPs/AmB−DOC)F127 2579


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Figure 7. Representative transmission electron microscopy images of L. major promastigote specimen after 10 min incubation with chitosan-coated nanoparticles Cs-NPs showing aberrant parasite shapes in (A) and (C). Polvacuoles (V) were observed in (A). Image in (B) represents a partial view of nanoparticles inside a swelled vacuole. Two large mitochondria (M) with a double membrane were observed in (C). Disorganized large cytoplasmic vacuoles were observed in (D). In (A) and (C), cell debris are indicated by red arrows. (E) is a magnification of (C) showing microtubules (arrows) distributed along the parasite membrane. g: Golgi complex, N: the nucleus. Scale bar in (A) and (C): 1 μm and in (D) 500 nm.

with that in the nontreated mouse group. Interestingly, the lowest parasitic load was found with the hydrogel containing the combination of (Cs-NPs/AmB−DOC)F127 (Figure 6G). Notably, quantification of the mouse DNA in all the samples demonstrated that the difference between the groups was not significant (p-value ANOVA = 0.1441), constituting proof of the reproducibility of the technique (Figure S11 in the Supporting Information section). 4.4. TEM. The effect of chitosan-coated nanoparticles on the ultrastructure of L. major promastigotes was monitored by TEM at different time intervals ranging from 10 min to 2 h. Control untreated samples revealed L. major specimens with a typical elongated shape and organelles with a normal morphology (Figure S12), in agreement with previous TEM observations.35,36 After 10 min incubation of L. major with Cs-NPs, the specimens had disorganized aberrant shapes, and many vacuoles were present in some specimens (Figure 7A and 7D), while swelled mitochondria were observed for other parasites

combination, indicate tissue repair. Notably, safranin-stained fibrosis (orange arrows) was also observed for F127 alone (Figure 4A). 4.3.2. q-PCR. Parasitic load in skin fragments collected from mice after euthanasia was first quantified by q-PCR for CsNPsIL, AmB−DOCIL, and their combination applied by IL injections (Figure 6, higher panel). Parasitic load in skin lesions collected from mice treated with IL injections of AmB− DOCIL was as high as that for untreated mice. Cs-NPIL, alone or combined with AmB−DOC, showed a decrease in the number of L. major copies in the mouse skin. The parasitic load in skin collected from mice after euthanasia was then quantified by q-PCR for Cs-NPsF127 and AmB−DOCF127 and their combination (Figure 6, lower panel). The results highlighted, unexpectedly, a significant decrease in the parasitic load for F127 hydrogel compared with nontreated mice. Topical application of AmB−DOCF127 or Cs-NPsF127 also reduced the parasitic load in collected skin samples compared 2580


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Figure 8. Representative TEM images of L. major promastigotes after 20 min incubation with chitosan-coated nanoparticles Cs-NPs. Two swelled mitochondria (M) were observed in (A). Polyvacuoles (V) and a large vacuole-containing membranous material (V′) were observed in (B). AC: acidocaldisome, N: the nucleus, K: kinetoplast. Scale bars: 1 μm.

(Figure 7C). Nanometer-sized particles were clearly seen inside the vacuoles (Figure 7B). However, these particles could not correspond to Cs-NPs because their size was too small (9− 38 nm) compared with Cs-NPs (higher than 100 nm according to AFM and quasi-elastic light-scattering analyses). Numerous cellular debris were observed, suggesting parasite lysis (red arrows on Figure 7A and 7C). After 20 min incubation with Cs-NPs, the observed parasites exhibited significant shape deformation (Figure 8A and B), and swelled mitochondria were also observed. Some vacuoles were large, reaching a 1.5 μm size containing membranous material (V′ in Figure 8B). Large cytoplasmic vacuoles were also observed by previous works on antileishmanial drugs such as essential oils.35 Acidocalcisomes appearing as rounded organelles containing electron-dense material were seen close to the mitochondria (Figure 8A).37 A large view of the sample showed numerous abnormal morphologies of the parasites in the Supporting Information (Figure S13). After 30 min incubation, again, aberrant parasite shapes were observed (Figure 9A and 9B), and internal structures were disorganized and even absent in some specimens (Figure 9B). Cellular debris was abundant (red arrows on Figure 9). Noteworthy, flagella, flagellar pockets, and kinetoplasts were observed for some parasites without apparent alteration (Figure 8B after 20 min incubation and Figure 9A after 30 min incubation). After 1 h incubation, Figure 10A shows two deformed mitochondria and large vacuoles. Magnification of one vacuole highlights the presence of nanometer-sized particles (20−25 nm) (Figure 10B) in the same size range as those observed after 10 min incubation in Figure 7B. Some specimens had a

cytoplasm where the internal structures are quasi-absent (Figure S14). After 2 h incubation, specimens with altered nuclear and plasma membranes were observed (Figure 11). Other internal structures of the parasite, such as the kinetoplast, flagellar pocket, and Golgi complex, were not detected. Notably, microtubules were regularly distributed along the parasite membrane regardless of the contact time with nanoparticles (arrows in Figure 7E and Figure 11).

5. DISCUSSION When the skin is infected by Leishmania. sp, IL injections have the advantage to administer the drug directly into the lesion, loaded with the parasites. However, besides being painful, histological examinations of skin specimens collected from L. major infected mice and treated by IL injections of AmB− DOC showed that the parasitic load was as high as that in the nontreated control mouse group (Figure 6A). Topical application of AmB−DOC on infected mouse skin daily for 3 weeks also failed to cure the lesion, while the parasitic load was reduced compared with that in infected and nontreated mouse skin (Figure 6B). Although safranin-stained fibrosis was seen from histology (orange arrows on Figure 4B), indicating a healing process, inflammatory granuloma was deep and invasive, dissociating the adipocytes and muscle fibers (Figure 4B). Previous work on an ointment containing Fungizone applied topically twice daily in mice for 12 days showed slight activity.38 In another study, Fungizone was active in vitro but was ineffective in a mouse experimental model after daily topical application for 15 days.39 Failure of the topical application of drugs to cure CL lesions was attributed to low diffusion of the 2581


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Figure 9. Representative TEM images of L. major promastigotes after 30 min incubation with chitosan-coated nanoparticles Cs-NPs. In (A), Fl, FP, and K were observed, while internal structures are disorganized and even absent in (B). FP: flagellar pocket, M: mitochondria, Fl: flagella, K: kinetoplast. Cellular debris are indicated by red arrows. Scale bar: 1 μm.

makes them more active than native chitosan toward Trypanosome parasites such as T. vaginalis.20 Due to their small size (∼187 nm) and cationic charge (+53 mV), Cs-NPs were internalized by the trophozoite after 10 min of incubation.21 Interestingly, a previous study observed no toxicity of Cs-NPs evaluated ex vivo.20 Cs-NPs showed no significant toxicological behavior using fully differentiated human cells.48 Observation of treated Leishmania ultrastructures revealed major swelling of mitochondria after 10 min incubation with Cs-NPs. To our knowledge, this is the first report on the ultrastructure changes induced by chitosan-coated poly(isobutylcyanoacrylate) nanoparticles in L. major. Mitochondria swelling of Leishmania was also reported using other antileishmanial drugs, such as 22,26-azasterol.49 Furthermore, treated parasites exhibited an increased number of acidocalcisomes and vacuoles. A similar effect on internal organelles was noticed after treatment with other drugs such as terbinafine and ketoconazole.50 In Leishmania, nutrients and

drug through the skin. Indeed, topical application of paramomycin cream also failed to cure the lesions,40−43 while a high cure rate (74%) was obtained when quaternary ammonium surfactants used as penetration enhancers were added.44 Unfortunately, severe irritation and intolerance due to surfactants were reported. Due to the low efficacy of topically applied AmB−DOCF127 in mice, we investigated its activity when combined with formulations comprising chitosan. This cationic biopolymer already showed antimicrobial activity, mainly due to the electrostatic interactions with the membranes of microorganisms. The antimicrobial activity of chitosan toward various microorganisms has been demonstrated by several studies in fungi and bacteria.45−47 In vitro experiments of chitosan performed in L. major promastigotes, axenic amastigotes, and infected macrophages show no activity (Table 1). Thus, chitosan was formulated as nanoparticles using the emulsion polymerization of isobutylcyanoacrylates. Previous studies showed that the formulation of chitosan as nanoparticles 2582


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Figure 10. Representative TEM images of L. major promastigotes after 1 h incubation with chitosan-coated nanoparticles Cs-NPs. Image (B) represents a magnification of (A). On A, two mitochondria (M) and large vacuoles (V) were observed. In (A) the parasite morphology was altered, and cellular debris are indicated by a red arrow. Scale bar: 1 μm for (A) and 200 nm for (B).

macromolecules are ingested by the flagellar pocket through a clathrin-dependent endocytosis process.36 This could explain the absence of endocytosis vesicles on the Leishmania membrane in transmission electron micrographs, in contrast to what we reported for T. vaginalis incubated with similar nanoparticles.21 Nanoparticles could also be internalized by infected macrophages through an endocytosis process. Endocytosis of nanoparticles by macrophages has been demonstrated for a while for materials comprising poly(alkylcyanoacrylates).51,52 This internalization could be higher with chitosan-coated nanoparticles resulting from higher antileishmanial activity in infected macrophages compared with F68-NPs (Table 1). Additionally, poly(alkylcyanoacrylate) nanoparticles can diffuse through the skin.53 Thus, the small size and amphiphilic nature of chitosan-coated poly(isobutylcyanoacrylate) nanoparticles are favorable to their diffusion through the skin. For specimens treated with either IL injections or topical applications of chitosan nanoparticles and their combination with AmB−DOC, a fibrosis was observed (stained orange in Figure 3B and C). Fibrosis indicates a healing process, probably due to chitosan, which has healing properties. For topically applied formulations, a thermosensitive and mucoadhesive hydrogel was used. This hydrogel, which is particularly adapted for mucosal and cutaneous applications, is liquid at temperatures below the gelling temperature (Tgel = 22 °C),54 and gels upon temperature increase after topical application, allowing a prolonged residence time. For example, the residence time of F127 hydrogel was detected by video-rate near-infrared fluorescence imaging after 6 h of vaginal administration in mice, while no signal was found for hydroxyethylcellulose hydrogel (1.5 wt %) after 3 h administration.55 Unexpectedly, fibrosis was also observed for an F127 hydrogel (Figure 4A and Figure S7C, orange arrows), and the parasitic load was significantly decreased compared with that in nontreated mice, likely due to a physical effect of the hydrogel. Previous data have shown that occlusive conditions benefited wound healing because water evaporation was limited;

consequently, hydration of the skin was improved. Previous works also demonstrated that topical application of F127 hydrogel in an open-excision wounded rat enhances the healing.56 Although the F127 hydrogel showed its ability to enhance lesion healing, its benefit to cure CL was demonstrated for the first time in this work. One hypothesis is that, besides enhancing lesion healing, the high viscosity of the hydrogel could limit parasite spreading through the skin. In this case, it would be better if the treatment was started at the early stages of infection. In summary, when applied topically, Cs-NPs could diffuse through infected skin, where they were internalized by both free L. major amastigotes and infected macrophages. Furthermore, the presence of chitosan could accelerate the healing of lesions. The superior activity of the association (Cs-NPs/ AmB−DOC) F127 compared with the two components administered separately could be due to a dual effect of CsNPs and AmB−DOC. The two components could act through separate mechanisms. While AmB acts on ergosterol present in the parasitic membrane, Cs-NPs could be internalized and degraded by the parasites. Thus, the degradation products of nanoparticles consisting of isobutanol release from the poly(cyanoacrylic acid) chain induce the antileishmanial activity. Numerous cell debris observed in TEM images suggest parasite lysis. The nonactivity of isobutanol suggests that, to be active, the nanoparticles should be internalized by the parasites. However, our studies did not formally confirm that Cs-NPs were internalized by the parasites, and the exact mechanism of activity is not fully understood. Further investigations using labeled nanoparticles and Leishmania should be performed to follow the internalization process.

6. CONCLUSIONS AND PERSPECTIVES This investigation highlighted the interest in chitosan-coated poly(isobutyanoacrylate) nanoparticles as an antileishmanial agent without the addition of drugs. The separate determination of the antiparasitic activity of each component revealed 2583


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Figure 11. Partial views of the body of L. major promastigotes after 2 h incubation with chitosan-coated nanoparticles Cs-NPs. The plasma membrane is disrupted. Nuclear membrane (N) is also altered. Cellular debris are indicated by a red arrow. Scale bar: 1 μm.

that the efficacy of nanoparticles was due to the poly(isobutyanoacrylate) core rather than to the presence of chitosan on the nanoparticle shell. The second major conclusion of this work was that the F127 hydrogel alone results in a decreased parasitic load. Finally, the combination of AmB−DOC and chitosan-coated nanoparticles enhanced the histological status of the skin and reduced the parasitic load. These results will be followed by the investigation of the mechanism of antileishmanial activity of nanoparticles coated or not with chitosan using confocal laser scanning microscopy and flow cytometry techniques. Furthermore, other formulation parameters should be considered, such as the salinity. In fact, 7% hypertonic saline has been shown to be effective and safe against CL caused by L. major and L. tropica in Iraq57,58 and L. donovani in Sri Lanka,2 with cure rates of 92−96% within one to ten IL injections. Another study conducted in Sri Lanka on 444 patients (643 lesions) demonstrated that 10% hypertonic saline IL injection showed a cure rate of 93% within one to ten injections.59 Therefore, chitosan-coated poly(isobutylcyanoacrylate) nanoparticles in hypertonic saline medium should be evaluated to obtain a synergistic effect. This work opens the way for other applications, such as the treatment of visceral leishmaniasis, because nanoparticles of poly(isobutylcyanoacrylate) coated with chitosan are injectable by the parenteral routes. After intravenous injection, it is likely that these nanoparticles accumulate preferentially in the liver, making them interesting candidates to treat visceral leishmaniasis.

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L. major promastigote after different incubation times with Cs-NPs are also reported (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Sebastien Pomel: 0000-0002-9294-3967 Kawthar Bouchemal: 0000-0003-2274-8725 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has benefited from the facilities and expertise of MIMA2MET and MEB-GABI, INRA, Agroparistech, 78352 Jouy-en-Josas, France. Author K.B. received funding from the Institut Universitaire de France and the ANR-17-CE09-0038-1.

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ABBREVIATIONS AFM, atomic force microscopy; AmB, Amphotericin B; AmBDOCF127, Amphotericin B-deoxycholate gelified by pluronic F127 and topically applied on infected mice; AmB-DOCIL, Amphotericin B-deoxycholate administered to infected mice by intralesional injections; DOC, deoxycholate; Chito20, chitosan 20 kDa; CL, cutaneous leishmaniasis; Cs-NPs, chitosan-coated poly(isobutylcyanoacrylate) nanoparticles; Cs-NPsF127, chitosan-coated poly(isobutylcyanoacrylate) nanoparticles gelified by pluronic F127 and topically applied on infected mice; CsNPsIL, chitosan-coated poly(isobutylcyanoacrylate) nanoparticles administered to infected mice by intralesional injections; QELS, quasi-elastic light scattering; DOC, deoxycholate; F68NPs, poly(isobutylcyanoacrylate) nanoparticles stabilized by pluronic F68; FFPE, formalin-fixed, paraffin-embedded; HES,

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.9b00263. Nanoparticle formulation and characterization, additional images of histological examinations of a healthy noninfected mouse, and infected and treated mice and specimens collected from mice infected and treated by different formulations. Additional TEM images of 2584


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hematoxylin-eosin-safranin; IBCA, isobutylcyanoacrylates; IC50, inhibitory concentration 50; IL, intralesional; MTT, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NPs, nanoparticles; PFA, paraformaldehyde; PIBCA, poly(isobutylcyanoacrylates); q-PCR, quantitative polymerase chain reaction; Tgel, gelling temperature; TEM, transmission electron microscopy; TGF-β1, transforming growth factorbeta1; VEGF, vascular endothelial growth factor

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