Fluconazole and propolis co-encapsulated in chitosan nanoparticles for the treatment of vulvovaginal candidiasis in a murine model.

Vulvovaginal candidiasis (VVC) is a fungal infection caused mainly by Candida albicans. The treatment of VVC with azoles has been impaired due to the increased cases of resistance presented by this pathogen. The aim of the present study was to investigate the antifungal activity of mucoadhesive chitosan nanoparticles encapsulating both green propolis and fluconazole for topical use in the treatment of VVC. The nanoparticles were prepared by the ionic gelation method, resulting in a size of 316.5 nm containing 22 mg/kg of green propolis and 2.4 mg/kg of fluconazole. The nanoparticles were non-toxic in vitro using red blood cells or in vivo in a Galleria mellonella toxicity model. The treatment of female BALB/c mice infected by C. albicans ATCC 10231 with topical nanoparticles co-encapsulating fluconazole and green propolis was effective even using a fluconazole amount 20 times lower than the amount of miconazole nitrate 2% cream. Considering that the mucoadhesive property of chitosan, which is known to allow a prolonged retention time of the compounds at the mucous epithelia, the antifungal potential of the phenols and flavonoids present in green propolis may have favored the effectiveness of this treatment. These results indicate that this formulation of topical use for fluconazole associated with green propolis can be used as a promising approach to therapy for the treatment of VVC, thus contributing to reducing the development of resistance to azoles.

Vulvovaginal candidiasis is a fungal infection for which we search for alternatives for its treatment. Thus, a nanoparticle formulation based on fluconazole and green propolis was developed. These nanoparticles were tested, and we obtained adequate results in laboratory tests.

Topical application of melatonin accelerates the maturation of skin wounds and increases collagen deposition in a rat model of diabetes.

AIMS: This study aimed to evaluate the cicatricial potential of melatonin when applied to wounds of diabetic rats. MATHERIALS AND METHODS: The formulation containing melatonin was developed and applied topically to cutaneous wounds of diabetic rats. 48 Wistar rats were used, divided into two groups of 24 diabetic animals each: (i) control group (CG), the animals received topical application of the no-melatonin formulation; (ii) treatment group (TG), the animals received topical application of the melatonin-containing formulation. All animals in each group were treated at four time points: 3, 7, 14, and 21 days. Each subgroup consisted of six animals. RESULTS: The treatment with melatonin improved wound healing by promoting wound closure earlier than the control group evaluated. Also improved a better resolution of the inflammatory phase observed mainly at 7 days, higher tissue maturation and expressive collagen deposition. CONCLUSION: The observed data reveal that the use of melatonin topically could be a promising strategy for the healing of wounds in diabetes. The results of this study elucidate the effects of previously described pathways in which it is proposed that melatonin acts promoting wound healing in diabetes.

Farnesol: An approach on biofilms and nanotechnology.

Biofilms are important virulence factor in infections caused by microorganisms because of its complex structure, which provide resistance to conventional antimicrobials. Strategies involving the use of molecules capable of inhibiting their formation and also act synergistically with conventional drugs have been explored. Farnesol is a molecule present in essential oils and produced by Candida albicans as a quorum sensing component. This sesquiterpene presents inhibitory properties in the formation of microbial biofilms and synergism with antimicrobials used in clinical practice, and can be exploited even for eradication of biofilms formed by drug-resistant microorganisms. Despite this, farnesol has physical and chemical characteristics that can limit its use, such as high hydrophobicity and volatility. Therefore, nanotechnology may represent an option to improve the efficiency of this molecule in high complex environments such as biofilms. Nanostructured systems present important results in the improvement of treatment with different commercial drugs and molecules with therapeutic or preventive potential. The formation of nanoparticles offers advantages such as protection of the incorporated drugs against degradation, improved biodistribution and residence time in specific treatment sites. The combination of farnesol with nanotechnology may be promising for the development of more effective antibiofilm therapies, as it can improve its solubility, reduce volatility, and increase bioavailability. This review summarizes existing data about farnesol, its action on biofilms, and discusses its encapsulation in nanostructured systems. LAY SUMMARY: Farnesol is a natural compound that inhibits the formation of biofilms from different microbial species. The encapsulation of this molecule in nanoparticles is a promising alternative for the development of more effective therapies against biofilms.

HPLC Method Validated for Quantification of Fluconazole Co-Encapsulated with Propolis Within Chitosan Nanoparticles.

Considering the cases of fungal resistance to classic antifungals, it is necessary to develop more efficient and innovative therapies capable of reversing this situation. Fluconazole is an antifungal frequently used in the treatment of mycosis and some fungi developed resistance to its mechanism of action. In this work, fluconazole and green propolis were co-encapsulated in chitosan nanoparticles to be explored in order to promote a synergistic effect to enhance its therapeutic efficacy. However, because of the complexity of the chemical composition of green propolis, it was necessary to develop a simple and precise methodology to quantify fluconazole in the formulation. High Efficiency Liquid Chromatography methodology was developed and validated following the Brazilian regulatory guidelines (ANVISA, RDC 166/2017) for the separation of co-eluted peaks of fluconazole and green propolis in the nanoparticle supernatant. Applying the method developed, it was possible to quantify fluconazole in the same sample containing propolis. Thus, the results allow to affirm that it is a specific test, effective, precise and robust, which helped to determine the efficiency of association of the compounds within the nanoparticle. The method can be applied to quantify compounds that have similar chromatographic retention times. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00954-2.

Is it possible to track intracellular chitosan nanoparticles using magnetic nanoparticles as contrast agent?

Drug delivery systems prepared with nanostructures are able to overcome biological barriers. However, one of the main challenges in the use of these nanosystems is their internalization by macrophages. This study aims to prepare and characterize chitosan nanoparticles incorporating maghemite nanoparticles and investigate their intracellular tracking in RAW 264.7 macrophages in vitro. Then, maghemite nanoparticles were encapsulated within chitosan nanoparticles by ionotropic gelification method. The images from transmission electron microscopy were used to investigate the intracellular penetration of conjugated nanoparticles by macrophages using different times. Our data suggests that magnetic nanoparticles are suitable to act as a contrast agent to investigate the cellular internalization of chitosan nanoparticles.

Antifungal Resistance, Metabolic Routes as Drug Targets, and New Antifungal Agents: An Overview about Endemic Dimorphic Fungi.

Diseases caused by fungi can occur in healthy people, but immunocompromised patients are the major risk group for invasive fungal infections. Cases of fungal resistance and the difficulty of treatment make fungal infections a public health problem. This review explores mechanisms used by fungi to promote fungal resistance, such as the mutation or overexpression of drug targets, efflux and degradation systems, and pleiotropic drug responses. Alternative novel drug targets have been investigated; these include metabolic routes used by fungi during infection, such as trehalose and amino acid metabolism and mitochondrial proteins. An overview of new antifungal agents, including nanostructured antifungals, as well as of repositioning approaches is discussed. Studies focusing on the development of vaccines against antifungal diseases have increased in recent years, as these strategies can be applied in combination with antifungal therapy to prevent posttreatment sequelae. Studies focused on the development of a pan-fungal vaccine and antifungal drugs can improve the treatment of immunocompromised patients and reduce treatment costs.

Distinct patterns of yeast cell morphology and host responses induced by representative strains of Paracoccidioides brasiliensis (Pb18) and Paracoccidioides lutzii (Pb01).

Paracoccidioidomycosis (PCM) is a systemic mycosis, widespread in Latin America. PCM is a granulomatous disease characterized by a polymorphism of lesions depending on the pathogen's virulence, the immune status of the host and its genetic susceptibility. The thermodimorphic fungus Paracoccidioides brasiliensis was considered the only etiologic agent of PCM, yet recent works have shown significant genetic diversity among different strains of P. brasiliensis. Therefore, it has been proposed for a new species within the Paracoccidioides genus, named Paracoccidioides lutzii. To better understand the fungus-host interactions elicited by strains Pb01 and Pb18 as key representatives of P. lutzii and P. brasiliensis, respectively, we carried out studies to investigate differences in morphology, induced immune response, virulence and pathology between these two Paracoccidioides species. Our results demonstrate distinct patterns of host-parasite interaction and pathology caused by Pb18 and Pb01. These results open up new fronts for NEW: clinical studies, which may result in significant consequences for the diagnosis and treatment of PCM. Considering that our results cannot be extended to all strains of both species, more studies about the virulence among Paracoccioides must be explored in the future.

Production of cyclodextrin glycosyltransferase by immobilized Bacillus sp. on chitosan matrix.

The whole-cell immobilization on chitosan matrix was evaluated. Bacillus sp., as producer of CGTase, was grown in solid-state and batch cultivation using three types of starches (cassava, potato and cornstarch). Biomass growth and substrate consumption were assessed by flow cytometry and modified phenol-sulfuric acid assays, respectively. Qualitative analysis of CGTase production was determined by colorless area formation on solid culture containing phenolphthalein. Scanning electron microscopy (SEM) analysis demonstrated that bacterial cells were immobilized on chitosan matrix efficiently. Free cells reached very high numbers during batch culture while immobilized cells maintained initial inoculum concentration. The maximum enzyme activity achieved by free cells was 58.15 U ml(-1) (36 h), 47.50 U ml(-1) (36 h) and 68.36 U ml(-1) (36 h) on cassava, potato and cornstarch, respectively. CGTase activities for immobilized cells were 82.15 U ml(-1) (18 h) on cassava, 79.17 U ml(-1) (12 h) on potato and 55.37 U ml(-1) (in 6 h and max 77.75 U ml(-1) in 36 h) on cornstarch. Application of immobilization technique increased CGTase activity significantly. The immobilized cells produced CGTase with higher activity in a shorter fermentation time comparing to free cells.

Principles, techniques, and applications of biocatalyst immobilization for industrial application.

Immobilization is one of the most effective and powerful tools used in industry, which has been studied and improved since the last century. Various immobilization techniques and support materials have been used on both laboratory and industrial scale. Each immobilization technique is applicable for a specific production mostly depending on the cost and sensibility of process. Compared to free biocatalyst systems, immobilization techniques often offer better stability, increased activity and selectivity, higher resistance, improved separation and purification, reuse of enzymes, and consequently more efficient process. Recently, many reviews have been published about immobilization systems; however, most of them have focused on a specific application or not emphasized in details. This review focuses on most commonly used techniques in industry with many recent applications including using bioreactor systems for industrial production. It is also aimed to emphasize the advantages and disadvantages of the immobilization techniques and how these systems improve process productivity compared to non-immobilized systems.

Chitosan nanoparticles encapsulating farnesol evaluated in vivo against Candida albicans.

Farnesol is a natural essential oil with antimicrobial properties. Complexation of farnesol in chitosan nanoparticles can be useful to improve its bioavailability and potentiate its antifungal capabilities such as inhibition of hyphal and biofilm formation. The aim of this study was to develop and characterize chitosan nanoparticles with farnesol (NF) and evaluate their toxicity and antifungal action on C. albicans in vivo. The NF were prepared by the ionic gelation method and showed physicochemical characteristics such as diameter less than 200 nm, monodisperse distribution, positive zeta potential, spherical morphology, and stability after 120 days of storage. In the evaluation of toxicity in Galleria mellonella, NF did not reduce the survival rate, indicating that there was no toxicity in vivo at the doses tested. In the assays with G. mellonella infected by C. albicans, the larvae treated with NF had a high survival rate after 48 h, with a significant reduction of the fungal load and inhibition of the formation of biofilms and hyphae. In the murine model of vulvovaginal candidiasis (VVC), histopathological analysis showed a reduction in inflammatory parameters, fungal burden, and hyphal inhibition in mice treated with NF. The produced nanoparticles can be a promising alternative to inhibit C. albicans infection.

Paracoccidioides lutzii Infects Galleria mellonella Employing Formamidase as a Virulence Factor.

The formamidase (FMD) enzyme plays an important role in fungal thriving by releasing a secondary nitrogen source as a product of its activity. In Paracoccidioides species, previous studies have demonstrated the upregulation of this enzyme in a wide range of starvation and infective-like conditions. However, Paracoccidioides lutzii formamidase has not yet been defined as a virulence factor. Here, by employing in vivo infections using an fmd-silenced strain in Galleria mellonella larvae model, we demonstrate the influence of formamidase in P. lutzii's immune stimulation and pathogenicity. The formamidase silencing resulted in improper arrangement of the nodules, poor melanogenesis and decreased fungal burden. Thus, we suggest that formamidase may be a piece composing the process of molecular recognition by Galleria immune cells. Furthermore, formamidase silencing doubled the observed survival rate of the larvae, demonstrating its importance in fungal virulence in vivo. Therefore, our findings indicate that formamidase contributes to Galleria's immune incitement and establishes the role of this enzyme as a P. lutzii virulence factor.

A Revision of Polymeric Nanoparticles as a Strategy to Improve the Biological Activity of Melatonin.

Drug delivery systems based on nanotechnology exhibit a number of advantages over traditional pharmacological formulations. Polymeric nanoparticles are commonly used as delivery systems and consist of synthetic or natural polymers that protect drugs from degradation in physiological environments. In this context, indolamine melatonin has been associated with several biological functions, including antioxidant, antitumor, immunoregulatory, neuroprotective, and cardioprotective effects. However, its availability, half-life, and absorption depend upon the route of administration, and this can limit its therapeutic potential. An alternative is the use of polymeric nanoparticle formulations associated with melatonin to increase its bioavailability and therapeutic dose at sites of interest. Thus, the objective of this review is to provide a general and concise approach to the therapeutic association between melatonin and polymeric nanoparticles applied to different biological disorders and to also highlight its advantages and potential applications compared to those of the typical drug formulations that are available.

Biodistribution and Adjuvant Effect of an Intranasal Vaccine Based on Chitosan Nanoparticles against Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a fungal infection caused by the thermodimorphic Paracoccidioides sp. PCM mainly affects the lungs, but, if it is not contained by the immune response, the disease can spread systemically. An immune response derived predominantly from Th1 and Th17 T cell subsets facilitates the elimination of Paracoccidioides cells. In the present work, we evaluated the biodistribution of a prototype vaccine based on the immunodominant and protective P. brasiliensis P10 peptide within chitosan nanoparticles in BALB/c mice infected with P. brasiliensis strain 18 (Pb18). The generated fluorescent (FITC or Cy5.5) or non-fluorescent chitosan nanoparticles ranged in diameter from 230 to 350 nm, and both displayed a Z potential of +20 mV. Most chitosan nanoparticles were found in the upper airway, with smaller amounts localized in the trachea and lungs. The nanoparticles complexed or associated with the P10 peptide were able to reduce the fungal load, and the use of the chitosan nanoparticles reduced the necessary number of doses to achieve fungal reduction. Both vaccines were able to induce a Th1 and Th17 immune response. These data demonstrates that the chitosan P10 nanoparticles are an excellent candidate vaccine for the treatment of PCM.

Increased production of chitinase by a Paenibacillus illinoisensis isolated from Brazilian coastal soil when immobilized in alginate beads.

The accumulation of chitin waste from the seafood industry is a serious environmental problem. However, this residue can be degraded by chitinases and its subproducts, such as chitosan, economically exploited. In this study, a chitinase producer bacteria, identified as Paenibacillus illinoisensis, was isolated from the Brazilian coastal city of Terra de Areia - Rio Grande Do Sul (RS) and was immobilized within alginate beads to evaluate its chitinase production. The alginate beads containing cells presented an average size of 4 mm, 99% of immobilization efficiency and increased the enzymatic activity in 40.71% compared to the free cells. The biomass during enzymatic production increased 62.01% and the total cells leaked from the alginate beads corresponded to 6.46% after 96 h. Immobilized cells were reused in a sequential batch system and remained stable for production for up to four 96-h cycles, decreasing only 21.04% of the initial activity at the end of the fourth cycle. Therefore, the methodology used for cell immobilization resulted in adequate beads to maintain cell viability during the enzymatic production, increasing enzymatic activity, showing low cell leakage from the support and appropriate recyclable capacity.

Dopamine-loaded nanoparticle systems circumvent the blood-brain barrier restoring motor function in mouse model for Parkinson's Disease.

Parkinson's disease (PD) is a progressive and chronic neurodegenerative disease of the central nervous system. Early treatment for PD is efficient; however, long-term systemic medication commonly leads to deleterious side-effects. Strategies that enable more selective drug delivery to the brain using smaller dosages, while crossing the complex brain-blood barrier (BBB), are highly desirable to ensure treatment efficacy and decrease/avoid unwanted outcomes. Our goal was to design and test the neurotherapeutic potential of a forefront nanoparticle-based technology composed of albumin/PLGA nanosystems loaded with dopamine (ALNP-DA) in 6-OHDA PD mice model. ALNP-DA effectively crossed the BBB, replenishing dopamine at the nigrostriatal pathway, resulting in significant motor symptom improvement when compared to Lesioned and L-DOPA groups. Notably, ALNP-DA (20 mg/animal dose) additionally up-regulated and restored motor coordination, balance, and sensorimotor performance to non-lesioned (Sham) animal level. Overall, ALNPs represent an innovative, non-invasive nano-therapeutical strategy for PD, considering its efficacy to circumvent the BBB and ultimately deliver the drug of interest to the brain.

ß2 Integrin-Mediated Susceptibility to Paracoccidioides brasiliensis Experimental Infection in Mice.

The earliest interaction between macrophages and Paracoccidioides brasiliensis is particularly important in paracoccidioidomycosis (PCM) progression, and surface proteins play a central role in this process. The present study investigated the contribution of ß2 integrin in P. brasiliensis-macrophage interaction and PCM progression. We infected ß2-low expression (CD18low) and wild type (WT) mice with P. brasiliensis 18. Disease progression was evaluated for fungal burden, lung granulomatous lesions, nitrate levels, and serum antibody production. Besides, the in vitro capacity of macrophages to internalize and kill fungal yeasts was investigated. Our results revealed that CD18low mice infected with Pb18 survived during the time analyzed; their lungs showed fewer granulomas, a lower fungal load, lower levels of nitrate, and production of high levels of IgG1 in comparison to WT animals. Our results revealed that in vitro macrophages from CD18low mice slowly internalized yeast cells, showing a lower fungal burden compared to WT cells. The migration capacity of macrophages was compromised and showed a higher intensity in the lysosome signal when compared with WT mice. Our data suggest that ß2 integrins play an important role in fungal survival inside macrophages, and once phagocytosed, the macrophage may serve as a protective environment for P. brasiliensis.

Wound healing treatment using insulin within polymeric nanoparticles in the diabetes animal model.

The aim of this work was to prepare chitosan nanoparticles containing insulin and to evaluate its therapeutic activity during wound healing in diabetic rats. The hypothesis that guided this study was that the combination of insulin within chitosan nanoparticles could stimulate the signaling pathway for wound healing. The chitosan nanoparticles were prepared by the ionotropic gelation method presenting average size of 183.3 ± 8.32 nm, polydispersity index (PDI) 0.397 ± 0.07 and zeta potential of 33.7 ± 2.45 mV for empty chitosan nanoparticles (EC) and 245.9 ± 25.46 nm and PDI 0.463 ± 0.01, and zeta potential of 39.3 ± 4.88 mV for chitosan nanoparticles containing insulin (IC). The insulin association efficiency was 97.19% ± 2.18. These nanoparticles and free insulin (FI) were incorporated within a hydrogel (Sepigel®) for topical application in the wound of 72 diabetic rats distributed in four groups: Sepigel® (S, control), free insulin (FI), empty chitosan nanoparticles (EC), and chitosan nanoparticles containing insulin (IC). The animals in each group were reorganized into three subgroups (n = 6) to assess their clinical signs after days 3, 7, and 14 from the beginning of treatments. Intense fibroplasias were observed in the free or insulin-chitosan nanoparticles groups. In the latter, a large number of blood vessels were observed at day 7th. Our data indicated that both empty and insulin-containing chitosan nanoparticles were able to stimulate inflammatory cell proliferation, and angiogenesis, followed by wound maturation.

Investigation of thiosemicarbazide free or within chitosan nanoparticles in a murine model of vulvovaginal candidiasis.

Vulvovaginal candidiasis is a serious health problem affecting numerous women around the world. Its treatment is based on antifungals which may not provide an effective cure because of the resistance presented by its etiological pathogens Candida spp. Candida albicans is the most prevalent species related to vulvovaginal candidiasis. Here, we evaluated the in vivo antifungal potential of thiosemicarbazide and thiosemicarbazide encapsulated within chitosan nanoparticles in a murine model of vulvovaginal candidiasis. The results demonstrated the antifungal capacity of free or nanoencapsulated thiosemicarbazide within chitosan to reduce the fungal load in the vaginal tissue of infected mice. In addition, histological analyses indicated the absence or a mild to moderate infection in thiosemicarbazide-treated groups. Statistical tests confirmed the existence of significant differences between the treated and the control groups. Therefore, our results suggest a potential application of thiosemicarbazide and encapsulated thiosemicarbazide as an alternative vulvovaginal candidiasis therapy.

Miconazole loaded chitosan-based nanoparticles for local treatment of vulvovaginal candidiasis fungal infections.

OBJECTIVES: In this study, polymeric nanoparticles based on chitosan incorporating the antifungal miconazole nitrate were fabricated and testedin vivo using murine vulvovaginal candidiasis. METHODS: Nanoparticles prepared by the ionotropic gelation method presented 200 to 300 nm diameter and polydispersity indexes ranging from 0.2 to 0.4. The nanoparticles were prepared to incorporate 63.9 mg/mL of miconazole nitrate to be testedin vivo. Murine vulvovaginal candidiasis was standardized using estradiol valerate before the animals were challenged by Candida albicans. RESULTS: The treatment using chitosan nanoparticles within miconazole nitrate presented the same therapeutic efficacy as miconazole nitrate in a commercial cream formulation, however using the antifungal content about seven-fold lower. This increase in the miconazole nitrate's therapeutic efficacy is may be due to the down-regulation of interleukin 10 (IL-10) expression. CONCLUSIONS: Our data represent a proof of concept that can be exploited to achieve an alternative and promising therapy for the treatment of vulvovaginal candidiasis.

Therapeutic treatment with scFv-PLGA nanoparticles decreases pulmonary fungal load in a murine model of paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic mycosis with lymphatic dissemination that is caused by Paracoccidioides species. Treatment of PCM consists of chemotherapeutics such as itraconazole, trimethoprim, sulfamethoxazole or amphotericin B. However, several studies are aiming to develop therapeutic alternatives for the treatment of fungal infection using new molecules as adjuvants. The single-chain variable fragments (scFv) from an antibody that mimics the main fungal component incorporated within poly(lactide-co-glycolic) acid (PLGA) nanoparticles helped treat the fungal disease. After expressing the scFv in Picchia pastoris (P. pastoris), the recombinant molecules were coupled with PLGA, and the BALB/c mice were immunized before or after infection with yeast Paracoccidioides brasiliensis (P. brasiliensis). Our results showed decreased disease progression and decreased fungal burden. Taken together, our results showed an increased of IFN-γ and IL-12 cytokine production and an increased number of macrophages and dendritic cells in the pulmonary tissue of BALB/c mice treated with a high concentration of our molecule. Our data further confirm that the scFv plays an important role in the treatment of experimental PCM.