**1. Introduction**

Nanoencapsulation is making its way into the administration of drugs and subsequent site-specific release at the target cell or tissue through active functionalization of NPs with proteins, antibodies, carbohydrates, and peptides, among other ligands [1]. Ligandfunctionalized NPs enhance uptake extent and can considerably increase the stability and systemic bioavailability of the drug since it protects its physicochemical characteristics, avoids possible degradation before reaching the therapeutic target, can achieve adequate doses at the intracellular level and, for example, facilitate the elimination of pathogens. The release of adequate drugs at the intracellular level is a strategy to avoid generating drug resistance to pathogens such as fungus and prevent non-specific accumulation in other tissues while reducing their toxicity and adverse side effects [2,3]. These adverse effects are due to the drug's action outside macrophages, the most important effector cells in host resistance, as in the case of histoplasmosis (HPM), used herein as a model of intracellular infection.

HPM is an endemic and systemic mycosis of primary pulmonary origin, affecting especially immunosuppressed and non-immunocompetent individuals [4]. HPM is caused by inhaling aerosols that contain the infecting particles (microconidia and small mycelial

**Citation:** Mejía, S.P.; López, D.; Cano, L.E.; Naranjo, T.W.; Orozco, J. Antifungal Encapsulated into Ligand-Functionalized Nanoparticles with High Specificity for Macrophages. *Pharmaceutics* **2022**, *14*, 1932. https://doi.org/10.3390/ pharmaceutics14091932

Academic Editor: Maria Nowakowska

Received: 29 July 2022 Accepted: 2 September 2022 Published: 13 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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fragments) of the dimorphic fungus *Histoplasma capsulatum*. This mycosis has been reported in more than sixty countries on all continents, but it is endemic in the Americas [5]. The clinical presentation of HPM includes the acute, chronic pulmonary and progressive disseminated forms, the latter occurring especially in patients infected with HIV, considered of high prevalence with a 0.9% overall incidence of coinfection, reaching 27% in endemic areas and mortality rates up to 30% [6,7].

Although HPM can be treated with different antifungals, Itraconazole (ITZ) is considered the first choice to treat mild and moderate forms of HPM. Yet, at least twelve months of therapy is required for the acute and chronic forms and even longer times for the progressive disseminated form [8], causing adverse effects in patients. Hepatoxicity has been the most frequent failure, relevant for all antifungal agents, from mild anomalies in liver function to fatal fulminant liver failure [9,10]. Given orally, it causes nausea, vomiting, diarrhea, skin rashes, headache, etc. [11]. ITZ has other limitations such as high lipophilicity, low absorption capacity, low systemic bioavailability, high susceptibility of the active compound to gastrointestinal hydrolysis, and drug interactions, suggesting extreme caution when used as part of multiple drug therapy [12–15]. In addition, HPM treatments generally offer limited efficacy because many drugs degrade before reaching the target tissues or cells, hindering therapeutic levels [16–18]. As an alternative, Sporanox is the only commercial formulation of ITZ for oral administration, encapsulated with hydroxypropylβ-cyclodextrin as an adjuvant that improves its solubility and biodistribution. However, it is contraindicated in patients with renal failure due to inefficient adjuvant removal [11].

It is then imperative to develop more efficient therapeutic strategies that shorten the treatment time and reduce the adverse side effects of drugs used to fight HPM. Different polymeric NPs have been reported in this context, searching for effective treatment with less toxicity and more patient-friendly regimes. These nanosystems have stability and high-load capacity and can transport one or more active principles (with similar or different physicochemical properties) or a combination of therapeutic and contrast image agents to be administered in the same formulation by various routes. Additionally, polymers can be chemically modified to achieve the optimal conditions required as active ingredient transport systems [19–21]. Among the most used, poly-(lactic-co-glycolic acid) (PLGA) is a highly biocompatible polymer approved by the Food and Drug Administration (FDA) that has a rate of adjustable biodegradation and modular mechanical properties [12].

Functionalization of the nanocarrier's surface has been widely explored for diagnosis, drug delivery, and other biomedical applications. Different ligands can be attached to the NPs surface, depending on the expected effect and whether intended for one or more functions [22–24]. For example, ligands containing bulky hydrophobic molecules can be attached to nanomaterial surfaces to prevent core agglomeration or be coated with watersoluble polymers, such as polyethylene glycol (PEG), to increase solubility required in physiological conditions, stealth properties, and biocompatibility. Ligands may also be attached to the surface of NPs to define their properties or act as "tags" for their recognition by target cells, either infected host cells or the microorganism directly [12].

Clinically important antifungals such as clotrimazole and econazole have been nanoencapsulated to improve their oral bioavailability [25]. ITZ encapsulated in PEGfunctionalized poly-lactic acid (PLA) NPs has much better biocompatibility than commercial ITZ-cyclodextrin formulations, increasing drug solubility and stability in a wide range of concentrations and pHs [26]. Additionally, in vitro evaluations of the PLA-PEG-ITZ complex showed sustained drug release and growth inhibition of *H. capsulatum* [16]. Highly hydrophobic amphotericin B (AmtB), used to treat leishmaniasis and fungal infections, is hampered by its high toxicity. Encapsulation of AmptB into chitosan and PLGA-based NPs has reduced the drug's toxicity and increased the antiparasitic effect. Furthermore, AmtB-loaded NPs functionalized with mannan-type carbohydrates have shown to be an effective treatment against leishmaniasis. In addition to its antiparasitic effect, recognizing the carbohydrate by specific macrophage receptors causes an activation of the cell's defense mechanisms, increasing the treatment effectiveness [27,28]. PLGA NPs

loaded with Fluconazole and coated with the cationic polymer polyethyleneimine (PEI) (FLZ-NP-PEI) also improved antifungal activity against four strains of clinically relevant *Candida* spp. [29,30]. Those are examples of antimicrobials encapsulated into functional (or not) polymeric nanocarriers reported in the literature but not in connection to PLGA-based NP-encapsulated ITZ against HPM.

We report a therapeutic solution to fight HPM intracellular infection. The therapeutic uses ITZ encapsulated in PLGA NPs, which functionalized with anti-F4/80 antibodies demonstrated for the first time increased antifungal effect on murine macrophages infected with *H. capsulatum* compared with bare NPs. Overall, results pave the way to design highly efficient nanocarriers for drug delivery against intracellular infections. Current studies are focused on directing the ITZ-loaded NPs to the lungs as the target organ by pegylation of NPs to improve their stealth properties, testing other macrophage-specific ligands and intranasal administration routes that can help increase the amount of NPs in the target organ. Additionally, the immunomodulation effect of functionalized nanoparticles was studied in vivo.

### **2. Materials and Methods**

### *2.1. Reagents*

Evonik (Essen, Germany) generously donated poly-lactic-co-glycolic acid (PLGA); LA: GA 75:25 (RG 752H) with 0.14–0.22 dl/g inherent viscosity (4 to 15 kDa). Sigma-Aldrich provided Nile Red (CAS 7385-67-3), Itraconazole (ITZ, CAS 84625-61-6), poloxamer 188 (CAS 9003-11-6, Kolliphor®), D-α-tocopherol polyethylene glycol 1000 succinate (vitamin E-TPGS, CAS 9002-96-4), voriconazole (CAS 137234-62-9), and phosphate-buffered saline solution (PBS D8573). MTT TOX-1 Kit, HMM Broth (F12 HAM nutrient medium, N6760), Janus Green (CAS 2869-83-2), sodium periodate (CAS 7790-28-5), Hoechst (CAS 23491-45-4), and TRIzol1 (Ref. 15596026) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) (Gibco, Ref. 16000044), Horse serum (HS) (Gibco, Ref. 16050122), Dulbecco's Modified Eagle Medium (DMEM, Ref. 10569010), and the antibiotic penicillinstreptomycin (Ref. 15140122), DNase I (Ref. EN0521), 3,3 -dihexyloxacarbocyanine iodide (DIOC6, CAS 53213-82-4), and RT-qPCR kit (Ref. A46109) were from Gibco (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Acetonitrile (CAS 75-05-8), ethyl acetate (CAS 141-78-6), ethanol (CAS 64-17-5), and dimethylsulfoxide (DMSO, CAS 67-68-5) were purchased from Merck. Citric acid (CAS 77-92-9) and sucrose (CAS 57-50-1) were purchased from VWR Chemicals. Potassium chloride (CAS 7447-40-7), sodium chloride (CAS 7647-14- 5), and monobasic potassium phosphate (CAS 7778-77-0) were provided by JT Baker (JT Baker, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Sodium citrate dihydrate (CAS 6132-04-3) and dibasic sodium phosphate (CAS 7558-79-4) were purchased from Panreac. Anti-F4/80 (ab100790) and Alexa flour 488 (ab150077) were obtained from Abcam (Abcam, Cambridge, UK). Brain Heart Infusion Agar (BHI) was obtained from Difco Laboratories (Ref. 211065, Thermo Fisher Scientific, Inc., Waltham, MA, USA). 0.1 M citrate buffer pH 4.5–5.5, 50 mM MES buffer pH 6.0–6.5, 50 mM HEPES buffer pH 7.0, and 1 M PBS buffer pH 6.5 were prepared by dissolving the reagents as received in Milli-Q water 18 MΩ·cm filtered through a 0.2 μm membrane.
