Is Micro and Nanotechnology Helping Us Fight Histoplasmosis?

Abstract

Histoplasmosis is an airborne systemic infection, with varied clinical manifestations, from asymptomatic infection to disseminated disease with a deadly outcome. Due to a growing number of immunosuppressed people, this mycosis has become more prevalent and thus, a cause for concern within the medical community. In fact, this fungal infection can be quite serious for children, elderly, people who have had an organ transplant, HIV-positive or people taking immunosuppressants. There has been a limited number of research articles suggesting polymeric, metallic, or lipid micro and nanotechnology-based approaches as a potential way to carry antifungal drugs to treat histoplasmosis. These new drug delivery systems present a variety of means of administration, thereby allowing a more targeted treatment to the lungs, skin, or eyes, according to the infection site. In this review, the aim was to explore these new therapies that have been emerging which hold great potential in comparison to regular antifungal treatments, not only due to their safety but also due to their drug release profile.

1. Introduction

Histoplasmosis is the most common respiratory fungal disease and displays the highest endemicity in North and South America [1, 2]. Nonetheless, tourism and migration have stimulated the worldwide growth of histoplasmosis in nonendemic areas, for instance, China, South Africa, India, and Southeast Asia [3, 4]. This disease triggers symptoms and signs, such as fever, weight loss, headache, abdominal pain, chills, fatigue, chest discomfort, diarrhea, and dry cough [5, 6, 7].

The infection is caused by inhaling aerosols that contain the infecting particles of the dimorphic fungus, Histoplasma capsulatum and it affects most frequently the lung [8]. Nevertheless, it can also affect the skin [9, 10, 11], and the central nervous system (CNS) causing meningitis [12] or even evolve into a progressive disseminated infection that may trigger an inflammatory response and bring rheumatological and heart complications (pericarditis), with high morbidity rates [13, 14].

Upon spores’ inhalation, the mycelial form goes through a dimorphic transition to yeast to infiltrate the host macrophages in almost any organ, granting its intracellular replication and survival [15, 16, 17]. Researchers have proved that the H. capsulatumyeasts facility on colonizing and adhering to different organ cryosections (lung, spleen, liver, gut, and trachea) is due to a well-known survival strategy of microorganisms: biofilm formation [18, 19, 20].

The at-risk population includes immunocompromised patients or those under immunosuppressive or biological regimens, as well as workers with occupational exposure to spore-laden soil [21].

The disease severity spectrum ranges from asymptomatic or mild lung disease to severe pneumonitis with respiratory compromise, depending on inoculum amount, exposure intensity, and host’s immunity [9, 14, 15].

Acute or chronic systemic disease may occur and is associated with immunodepression, particularly acquired immunodeficiency syndrome (AIDS) [3]. In fact, disseminated histoplasmosis among AIDS patients is a rapidly progressing, life-threatening illness that requires prompt treatment with antifungal medication [6]. In Latin America, histoplasmosis is often listed as the number one death cause in patients with advanced AIDS [15].

Infectious diseases caused by intracellular microorganisms, such as histoplasmosis, are described as capable of altering host defense mechanisms and hence allowing these microorganisms to survive inside mononuclear phagocytes, such as macrophages and dendritic cells [22]. These kinds of diseases are regarded as medical challenges due to drug–drug interactions during coinfections and resistance emergencies, which evidently narrows available therapies [23].

Currently, the gold standard treatment for moderate-to-severe disseminated histoplasmosis is liposomal amphotericin B (L-AmB) [1, 6]. The liposomal formulation is preferred to the conventional deoxycholate one, due to decreased nephrotoxicity, lower mortality rates in HIV patients, and overall improved clinical success [3, 24].

On the other hand, for mild and moderate forms of infection, the most appropriate choice is itraconazole. Alternatives to itraconazole include posaconazole, fluconazole, ketoconazole, and voriconazole [1, 13].

Considering the hepatoxicity, limited efficacy due to deficient absorption, low bioavailability, drug degradation, long treatment duration, and frequent drug interactions of the traditional antifungal drugs, it is imperative to develop more efficient strategies for this disease, which would be able to overcome these hurdles [8].

The functionalization of nanocarriers for drug delivery has been ceaselessly disclosing its potential as an alternative and versatile technological platform for the management and treatment of intracellular infections caused by fungi from the H. capsulatum species [23, 25]. Indeed, the encapsulation of antifungal agents into nanoparticles to selectively target pathogens has shed light on improving treatment’s efficacy and efficiency [8]. Some of these novel drug delivery approaches, such as AmBisome® and Visudyne®, are already commercialized and now serve as benchmark treatments and proof-of-concept of the usefulness of nanotechnology in antifungal drug delivery [26, 27, 28].

However, between clear written clinical guidelines and actual clinical practice, there is sometimes a huge gap. In Latin America, for instance, the frequent lack of physician awareness about histoplasmosis and the shortage of accessible diagnostic methods translates into thousands of annual deaths amid advanced-HIV patients, which could have been prevented. It is likewise important to stress that the feasibility of implementing some of these novel treatment options, along with the therapeutic drug monitoring that they require, greatly depends on resource availability, which tends to be scarce in impoverished settings [5, 6].

2. Lipid formulations of Amphotericin B

The burden of invasive fungal infections has grown in the last years, leading to higher morbidity and mortality, especially among immunosuppressed individuals. Amphotericin B deoxycholate (AmBD) (Figure 1) is still regarded as one of the most important antifungals of the last 60 years and has been the foundation to treat these infections, showing efficient fungicidal activity against candidiasis, cryptococcosis, aspergillosis, histoplasmosis, blastomycosis, coccidioidomycosis, zygomycosis, sporotrichosis, fusariosis, and phaeohyphomycosis [29, 30].

Figure 1.

Chemical structure of amphotericin B (C₇₁H₁₁₂NNaO₂₁). Drawn using ChemDraw Professional 22.0 from PerkinElmer Informatics, Inc.

Nonetheless, given the adverse effects in 50–90% of cases, efforts were made to reformulate the first amphotericin B formulation (Fungizone®) with equivalent efficacy yet reduced toxicity.

The commercially available formulations for this effect are Albelcet® (lipid complex), Amphotec® / Amphocil® (colloidal dispersion), and AmBisome® (liposomal formulation) [27, 29]. On account of their superior safety profiles and higher drug therapeutic index, these lipid-based preparations can nowadays be regarded as worthy substitutes for AmBD. Currently, they are first-line therapy for numerous invasive fungal infections in routine medical practice and clinical investigation, for instance, for disseminated histoplasmosis and AIDS, Candida meningitis, or endophthalmitis [27, 31, 32, 33].

Even in pregnancy, the lipid formulations of AmB are the cornerstone treatment of any invasive fungal infection and deemed as safe. On the contrary, azoles present complications in view of their teratogenicity, embryotoxicity, and of transplacental infection transmission to the fetus, consequently being contraindicated in this group. Their use during pregnancy should be restricted to superficial infections [14, 34].

Currently, only AmBisome® has been evaluated in the treatment of disseminated histoplasmosis [2, 31]. Besides, some case reports have also stressed its clinical efficacy in the rare primary cutaneous form of the disease in immunocompetent patients [9, 10, 11].

AmBisome® received its FDA approval in 1997 and is formed of small spherical unilamellar liposomes with a size inferior to 100 nm, where AmB is encapsulated. L-Amb is represented in Figure 2. Hydrogenated soy phosphatidylcholine, cholesterol, and phosphatidylglycerol are composition elements of these liposomes [30].

Figure 2.

Schematic representation of liposomal amphotericin B. Parts of the figure were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License. (https://creativecommons.org/licenses/by/3.0/).

In a 2002 multicenter randomized, double-blind, prospective clinical trial, with 81 participants, intravenous infusion doses of 3.0 mg/kg of body weight liposomal amphotericin B (L-AmB) and 0.7 mg/kg of AmBD were compared. The purpose was to evaluate both their safety and efficacy for induction therapy of moderate to severe disseminated Histoplasmosis in patients with AIDS [24]. A higher treatment response of 88% was achieved for L-AmB counter to 64% for AmBD, as well as lower mortality rates (2% vs. 13%). Furthermore, nephrotoxicity (assessed through an increase in serum creatinine level) was reported for 9% of patients treated with L-AmB, in opposition to 37% for AmBD, along with fewer infusion-related side effects (25% vs. 63%). Taking all these trial findings into account, L-AmB has clearly revealed to be an upgraded choice to the first standardized treatment AmBD, therefore modifications on therapy recommendations were undertaken after this study. Despite being more costly, L-AmB’s attractiveness lies in its less toxic profile, superior efficacy, and improved survival rates for moderate-to-severe invasive histoplasmosis [24, 31].

Nevertheless, clearance rates of fungemia and H. capsulatum antigen from serum and urine were alike with the two treatments. For this reason, 2 weeks after the beginning, induction therapy was replaced by itraconazole, for another 10 weeks of consolidation therapy [24, 35].

With the goal of clearly proving the benefits of L-AmB as the initial treatment of moderate-to-severe histoplasmosis, another study was carried out. It comprised two separate closed clinical trials and aimed to compare the clearance of fungal burden (correlated with survival) in patients with disseminated histoplasmosis treated with L-AmB (n = 51) versus itraconazole (n = 59). The clinical response rates were similar: 86% for L-AmB and 85% for itraconazole group. However, after 2 weeks of treatment, fungemia, antigenemia, and antigenuria cleared more rapidly with L-AmB than with itraconazole. This quicker fungemia clearance justifies the use of L-AmB, in opposition to itraconazole, as the initial treatment of moderate-to-severe histoplasmosis [35].

To address the issue of continuously high morbidity and mortality rates brought about by fungal infections, a preclinical study comparing different prophylactic agents was conducted (AmBisome® and Fungizone®). A single high dose of AmBisome® was able to deliver sufficient concentrations of AmB in tissue in immunocompetent and immunosuppressed murine while keeping a safety standard. It effectively inhibited the growth of Candida albicans in the kidneys and prevented the growth of H. capsulatum in the spleen of mice [36].

3. Drug delivery systems loaded with itraconazole

Itraconazole (Figure 3) is an antifungal drug from the azole group, widely used in the treatment of aspergillosis, cryptococcosis, candidiasis, blastomycosis, and mild histoplasmosis. Its action mechanism encompasses the disruption of ergosterol synthesis to avoid the formation of the fungal cell membrane [37, 38].

Figure 3.

Chemical structure of itraconazole (C₃₅H₃₈CI₂N₈O₄). Drawn using ChemDraw Professional 22.0 from PerkinElmer Informatics, Inc.

3.1 Polymeric nanoparticles of itraconazole

Polymeric nanoparticles are capable of not only safely carrying drugs for specific target organs but also of effectively permeating cellular membranes [39].

A striking example of a novel nano-based delivery system is the encapsulation of itraconazole in nanosphere polymeric nanoparticles (NP) based on poly-(lactic-co-glycolic acid) (PLGA) and functionalized with F4/80 antibodies and mannose. The optimized NP was made up of PLGA 75:25 and a mix of surfactants (Kolliphor P188 and vitamin E-TPGS) at pH 5. It showed optimal drug-loading capacity (6.6%), high encapsulation efficiency (80%), and fitted well with the Fickian diffusion model [23]. A schematic representation of this system is outlined in Figure 4.

Figure 4.

Schematic representation of the functionalized PLGA NPs loaded with itraconazole and their interaction with F4/80 antibodies receptor in macrophages. Created with BioRender.com.

This study has demonstrated increased J774 macrophage uptake in vitro and more efficacy in eliminating the H. capsulatum on murine macrophages compared with bare NP. Moreover, these NP did not affect the viability in macrophages at different concentrations, which proves they are not cytotoxic. A successful antibody-NP surface binding was achieved while keeping its stability and avoiding aggregation. The 200 nm size is adequate to prevent rapid elimination by the endothelial reticulum system. H. capsulatum induces IFN-γ expression in the macrophages and the functionalized NPs developed were able to reduce the expression of this cytokine, emphasizing the role of this antibody as a binding molecule with immunomodulatory properties. In addition, the treatment with these nano-formulations also significantly reduced IL-6 and IL-10 expression compared to free itraconazole. All in all, this research supports the idea that the encapsulation of itraconazole into NP allows a controlled and targeted drug release into macrophages, along with enhanced efficacy and efficiency in battling the fungal intracellular infection [8, 23, 25, 40].

In line with this research was the report of a new polymeric drug delivery system for targeted brain delivery. The authors achieved a stable linkage between RVG29 peptide (a brain-targeting ligand) and itraconazole-loaded albumin nanoparticles by means of the biotin-binding crosslinker streptavidin. This conjugation simplified the intracellular delivery of NPs and enhanced drug distribution in mice brain [41]. Albeit the non-specificity to H. capsulatum of either in vitro or in vivo conducted studies, there is undeniable potential to be exploited herein. In fact, CNS histoplasmosis, despite being rare, is of difficult diagnosis and quickly escalates to disseminated infection with a lower chance of recovery [12].

3.2 Lipid nanoparticles of itraconazole

Nanostructured lipid carriers (NLCs) are attractive drug carriers thanks to their unstructured matrix, which grants enhanced drug loading capacity and long-term drug stability, thereby constituting an upgrade of solid lipid nanoparticles (SLNs) [38, 42].

Itraconazole-NLCs were successfully formulated for brain-targeted delivery. The formulation included Precirol® ATO 5 and Transcutol® HP as lipid phase and Tween 80 and Solutol® HS15 as surfactants. A sustained release was achieved along with an almost double increase in the drug’s concentration in the brain, compared to pure itraconazole alone, which in turn showed much lower permeability across the blood–brain barrier [38]. Even though the conducted in vitro cellular studies have not targeted Histoplasma strains, these itraconazole-NLCs can be further exploited as nanocarriers for brain delivery in SNC histoplasmosis.

4. Drug delivery systems loaded with verteporfin

Verteporfin (Figure 5) is a lipophilic benzoporphyrin derivative synthesized from protoporphyrin IX dimethyl ester. It shares several beneficial properties of a photosensitizer: chemical stability, efficient generation of singlet oxygen species, strong absorption of red light at 692 nm, and diminished skin photosensitivity, due to faster body extravasation [43].

Figure 5.

Chemical structure of verteporfin (C₄₁H₄₂N₄O₈). Drawn using ChemDraw Professional 22.0 from PerkinElmer Informatics, Inc.

4.1 Liposomal formulation of verteporfin

In 2000, both Food and Drug Administration (FDA) and European Medicines Agency (EMA) have approved a liposomal formulation of verteporfin [44]. In fact, encapsulating drugs in liposomes shield drugs from degradation by metabolic enzymes on conjunctiva, cornea, and in tear fluids. Furthermore, liposomes can enhance intraocular penetration and retard the drug’s clearance, leading to a higher concentration in the vitreous humor and an increment in the drug’s half-life [33].

Since verteporfin is a highly hydrophobic drug, encapsulation in liposomes can control its drug release and enhance its in vivo distribution upon intravenous injection [45].

The liposomal formulation was developed by Bausch & Lomb® and is currently marketed by the commercial name Visudyne®. Ophthalmologists consider this formulation a remarkable advance in vision sciences in view of its capability of reducing the magnitude of vision loss for at least 1 year, thus boosting the life quality of these patients [28].

Visudyne® is a light-activated nanomedicine and the first and only clinically approved photosensitizer, being applied in photodynamic therapy (PDT) to eliminate abnormal blood vessels in the eye’s retina and choroid (Figure 6) [43, 45, 46]. Briefly, it accumulates in these vessels and, when exposed to red light in the presence of oxygen, produces highly reactive free oxygen radicals, which impose local damage to the endothelium and vessel blockage [45, 47, 48]. It has become a milestone in the ophthalmology field for the treatment of patients with subfoveal choroidal neovascularization derived either from age-related macular degeneration, secondary to pathological myopia or from ocular histoplasmosis syndrome [44, 45, 48].

Figure 6.

Comparison between a healthy eye and an eye with choroidal neovascularization, a condition observed in ocular histoplasmosis with the formation of abnormal blood vessels in retina and choroid. Created with BioRender.com.

The lipid bilayer of Visudyne® liposomal formulation is made of a synthetic saturated phospholipid DMPC (dimyristoylphosphatidylcholine) and egg yolk phosphatidylglycerol EGPC (comprised of unsaturated multiple species) in the 5:3 ratio [47, 49]. The encapsulation of verteporfin in liposomes was a resourceful way to deliver the drug intravenously, thereby evading the natural predisposition of hydrophobic molecules to self-aggregate in aqueous media [43].

4.2 A theranostic liposome of verteporfin

More recently, in 2020, a group of Brazilian researchers designed a smart theranostic verteporfin-loaded lipid-polymer liposome for PTD. This study proposes the loading of the aforementioned verteporfin liposomes in a theranostic system. Shortly, it consists of lipid-polymer liposomes obtained from DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine) coated with triblock copolymer Pluronic® F127 covalently functionalized with 5 [6]-carboxyfluorescein fluorescent probe. An illustration of this delivery system is depicted in Figure 7. This innovative formulation yielded 100 nm vesicles, 0.15 polydispersity index, outstanding stability, and encapsulation efficiency higher than 90%. Despite these encouraging results, the designed system’s efficiency was only proven in a glioblastoma cancer cell line, leaving its efficiency in ophthalmic diseases open for further future investigation [49].

Figure 7.

Schematic representation of a theranostic system to load verteporfin liposomes. Created with BioRender.com.

5. Magnetic nanoparticles

The insurgence of antimicrobial resistance is the greatest promoter of the fabrication of cutting-edge drug delivery systems. Iron oxide nanoparticles have always merited an outstanding place in drug delivery, thanks to their recognized biomedical importance, precise targeting, and biocompatibility [50].

Bearing this in mind, groups of investigators have recently published promising studies endorsing the development of superparamagnetic, fluorescent, noncytotoxic nano systems with antifungal activity against some fungal strains, namely H. capsulatum. In a summarized manner, iron oxide nanoparticles (IONPs) were synthesized using macromolecular stabilizing starch, noteworthy for their biocompatibility and agglomeration prevention. Upon encapsulation with starch, a shift in magnetic behavior takes place, converting the once weakly ferromagnetic IONPs into superparamagnetic (SPIONs) [50, 51]. The SPIONs were successfully incorporated in a fluorescently modified carrier system, enabling not only an easy identification of the system in a living body but also the application of this system in photodynamic therapy. An alarming discovery of this research is that H. capsulatum was found to be highly susceptible to the designed nano system in PTD studies, whereas it was resistant to the antifungal griseofulvin [51].

Specific drug targeting to the choroid has lately aroused special interest, on behalf of the rising blindness figures in the aged population and the choroid’s unique architecture as one of the most vascularized tissues in the human body.

Magnetic iron oxide nanoparticles stabilized with carboxylic acid, have been covalently functionalized with a recombinant VEGF (vascular endothelial growth factor) permitting the preferential release of the drug into the choroid layer [52]. Albeit the study’s core focus was angiogenesis and so they evaluated a monoclonal antibody (bevacizumab), the information conveyed is transposable to histoplasmosis infection. As a matter of fact, and as previously stated, ocular histoplasmosis instigates neovascularization of choroidal vessels, as can be noticed in Figure 6.

All in all, magnetic iron oxide nanoparticles can be a powerful strategy for cell-specific eye targeting. Their potential against Candida sp. has already been proven and amphotericin B has also been encapsulated in this nano system with enhanced fungicidal activity and reduced side effects [53]. In addition, these systems are biodegradable, intrinsically safe, exhibit increased half-life and their release localization can be controlled [52].

6. Vaccines for histoplasmosis

The creation of vaccination strategies for clinically relevant fungi has been a long-sought ambition for investigators, even with the difficulties assigned to these organisms’ complex eukaryotic cells and their similarity to human proteins. Preventing or diminishing the severity of histoplasmosis through targeted vaccine development is then deemed to be an important scientific breakthrough [54, 55].

Glucan particles (GPs) are hollow, porous microspheres with an average diameter of 2–4 μm, derived from baker’s yeast (Saccharomyces cerevisiae) purified cell walls and composed of 1,3-D-glucan and trace sums of chitin [56]. They are considered innovative and promising vaccine delivery systems, due to the possibility of encapsulating, transporting, delivering, and releasing protein antigens in their inner void cavity. In addition, the GPs delivery system retains the intrinsic immunostimulatory properties of 1,3-D-glucan on the surface. This polysaccharide functions as a ligand for receptor-mediated cell uptake by phagocytic cells bearing β-glucan receptors, for example, macrophages and dendritic cells in the immune system. Hence, GPs act as antigen-presenting phagocytic cells-selective-targeted delivery systems with adjuvant properties [55, 56].

A recent preclinical study was able to produce an extract from H. capsulatum yeast cells with the ability to convene protective immunity when encapsulated in GPs [21]. Succinctly, the GP vaccine consisted of Histoplasma alkaline extract, mouse serum albumin, and yeast RNA complexed with the glucan cells. Overall, the developed alkaline extract packaged in GP conferred vaccine-induced immunity, along with a reduced fungal burden by roughly 80% and improved survival in mice [56].

These data overlooks GP as useful vaccine delivery vehicles and may serve as a platform for the identification of proteins to include in GPs that both enhance protective immunity and modify immune responses to the agent [21]. On the other hand, this opens doors to the development of new GP nanoparticle-loaded formulations, which take advantage not only from the drug encapsulation assets of NPs but also from the macrophage-targeting properties of GPs [56].

Another ground-breaking study combining immunoproteomic and immunopeptidomic methods was able to map H. capsulatum peptide epitopes for the first time using murine dendritic cells and macrophages. After selecting and synthesizing the four most promising peptides, the incorporation into GPs took place. Efficient induction of CD4⁺ and CD8⁺ T lymphocytes was observed, as well as a production stimulation of IFN-γ, IL-17, and IL-2. The selected epitopes are derived from enolase (a heat shock protein 60) and the ATP-dependent molecular chaperone HSC82, which share a great degree of similarity with proteins expressed by other clinically relevant pathogenic fungi. Ergo, the authors preconize these promiscuous epitopes as the steppingstone for the creation of a multi-epitope peptide vaccine against histoplasmosis and other fungi [54].

7. Conclusion

Nanotechnology is still an emerging field in fungal vaccinology and pharmacology, yet there are many studies underlining its improved safety and tolerability. In fact, these innovative approaches have already replaced the original gold standard treatments for several forms of histoplasmosis disease, as outlined in this chapter.

Notwithstanding that, there are still a few therapeutic options to prevent and fight this neglected and potentially fatal disease. Therefore, the development of new drug delivery systems for the treatment and management of histoplasmosis is of utmost importance, particularly for AIDS patients.

Current investigation in antifungal drug delivery needs to put special emphasis on overcoming the challenges that deter the translation of nanoparticle-based systems into clinical practice.

Acknowledgments

This work was supported by the Foundation for Science and Technology, I.P. (FCT), Portugal, through grant reference 2020.05884.BD, and by the Applied Molecular Biosciences Unit – UCIBIO, which receives financial support from Portuguese national funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through grant UIDB/04378/2020.

Conflict of interest

The authors declare no conflict of interest.