Promising use of nanotechnology in Pythium insidiosum: a systematic review

ABSTRACT: The aquatic oomycete Pythium insidiosum is an emerging pathogen highly relevant in human and veterinary medicine and an etiologic agent of pythiosis, a disease of worldwide distribution mainly affecting horses, dogs, and humans, presenting cutaneous, subcutaneous, ocular, gastrointestinal, and systemic forms. The available therapeutic methods to treat this disease and its forms are not entirely effective, thus highlighting the need to investigate the forms of treatments with better efficacy, such as compounds from different pharmacological classes, compounds of natural origin, and new technological alternatives, including nanotechnology. Therefore, this study evaluated scientific publications regarding the use of nanotechnology in P. insidiosum treatment. For this, a systematic literature review, was carried out on articles published from 2010 to 2022 on the LILACS, MEDLINE, Google Scholar, PubMed, and SciELO databases using the descriptors ‘Pythium insidiosum,’ ‘pythiosis,’ ‘nanotechnology,’ ‘nanoparticles,’ ‘nanoemulsion,’ and ‘treatment.’ We reported 162 articles for the researched theme; although, only four studies were included because they met the criteria established herein. A meta-analysis was used for the statistical analysis of the data obtained in vitro studies, and we reported the use of nanotechnology can be a promising alternative in developing antimicrobial compounds with anti-P. insidiosum activity. Nevertheless, additional research is needed to verify the potential use of this technology in clinical therapy against P. insidiosum infections.

Due to the morpho-physiological differences between the oomycete P. insidiosum and members of the kingdom Fungi; the main components of the fungal cell wall and plasma membrane are chitin and ergosterol, respectively, in addition to oomycetes having cellulose, β-glucans, and hydroxyproline as constituents of the cell wall and an incomplete pathway of ergosterol biosynthesis, being ergosterol the main target of action of antifungal drugs (GAASTRA et al., 2010;LERKSUTHIRAT et al., 2017).Thus, pythiosis is a complex disease to treat because the commercially available drugs have limitations, making it essential to improve the therapeutic alternatives to control this disease that affects human and animals.
Nanotechnology is the area of science that studies materials at the nanometer scale and is a multidisciplinary innovation that has gained worldwide notoriety in recent decades.In the health field, nanotechnology has shown significant advances, primarily in therapeutic applicability, as nanostructured systems have been used to improve drug delivery, increase bioavailability and therapeutic index, control the release of the active ingredient, and reduce toxic effects (ERDOĞAR et al., 2018;IANISKI et al., 2022).In fact, new drug delivery systems (e.g., nanostructures) have been researched to develop alternative methods against oomycete infections (ZABRIESKI et al., 2015;VALENTE et al., 2016b), and further research is pivotal for advancing pythiosis treatment.
This carried out a systematic literature review seeking articles from 2010 to 2022 that describe the use of compounds developed in nanotechnology and with anti-P.insidiosum activity.

MATERIALS AND METHODS
This study was composed of a systematic literature review that was carried out according to the PRISMA flow chart, as suggested by PAGE et al. (2021).A bibliographic search was performed in the following databases: Latin American and Caribbean Literature on Health Sciences (LILACS), Online Literature and Recovery System (MEDLINE), Google Scholar , PubMed and Scientific Electronic Library Online (SciELO).In addition, we used combinations of Health Science Descriptors (DeCS) and Medical Subject Headings (MeSH) to assist in the search: 'Pythium insidiosum', 'pythiosis', 'nanotechnology', 'nanoparticles', 'nanoemulsion' and 'treatment'.The inclusion criteria included articles available in the aforementioned databases in Portuguese, English and/ or Spanish.The period from 2010 to 2022 was used as a time frame.Articles that did not provide information on the use of nanotechnology in the treatment of pythiosis, that contained duplicate information or that were out of the selected period were excluded.
Data analysis was performed by first logit transformation of proportion data.Data from Pythium insidiosum isolates were obtained by metaanalysis under a random-effects model.The graphical representation of the meta-analysis was done using forest plots.The meta-analysis was performed in the "Metaprop" package of the R 3.4.2software ® .

RESULTS AND DISCUSSION
After searching the databases, 162 articles were obtained.After reading the titles and abstracts, 68 articles did not meet the inclusion criteria and were excluded, and 94 articles remained.These articles after reading the titles and abstracts, four of them were selected and read in full (Figure 1).The studies involving nanotechnology applied to the oomycete P. insidiosum are summarized in table 1.
Nanotechnology is the science that refers to manipulating matter at the nanometer scale and producing structures between 1 and 100 nm, thus enabling innovation in the areas of biotechnology, industry, electronics, agriculture, disease diagnosis, and pharmacological therapies (PELAZ et al., 2017;IANISKI et al., 2022).Drug delivery systems using nanoparticles have numerous advantages over conventional drugs because they enable the delivery of unstable and insoluble drugs, maintain active ingredient concentrations at the expected site of action, and reduce systemic toxicity.Hence, nanoparticle formulations are used at lower therapeutic doses, making it possible to minimize adverse effects and reduce treatment costs (PARVEEN et al., 2012).
Successfully treating infections by P. insidiosum poses numerous variables, such as size and time of lesion, patient age, and the nutritional status of the affected individual (GAATRA et al., 2010).Additionally, there are limitations when using chemical treatments, which are mainly due to the difference in cellular constituents of the oomycete compared to members of the kingdom Fungi (SANTURIO et al., 2006;LERKSUTHIRAT et al., 2017).
In this regard, VALENTE et al.The meta-analysis showed that the total number of P. insidiosum isolates collected in vitro studies was 412, 130 of which were treated with M. alternifolia free oil, 130 with M. alternifolia nanoemulsion and 152 with Bio-AgNP.Forest plot analysis (Figure 2) revealed that Bio-AgNP-treated isolates had the best results, with a 22% growth Ciência Rural, v.53, n.7, 2023.
inhibition of P. insidiosum isolates, followed by the treatment with M. alternifolia nanoemulsion with a 16% reduction in P. insidiosum isolates.Nonetheless, M. alternifolia free oil treatment only inhibited 8% of the isolates.These results highlighted that the use of nanostructured compounds has better anti-P.insidiosum activity, suggesting that developing antimicrobial compounds using nanotechnology To analyze the activity of biogenic silver nanoparticles (bio-AgNP) in vivo pythiosis treatment.
Gel formulation administered topically and aqueous formulation administered intralesionally, both containing 1 μg/mL of Bio-AgNP, significantly reduced the size of pityriasis skin lesions in the study animals.One animal treated with the topical formulation showed a clinical cure.
In vivo experiment proved the anti-P.may be a promising therapy to control P. insidiosum infections.It is emphasized that the advantages of using nanotechnology include improving drug delivery, increasing bioavailability and therapeutic index, controlling the release of active ingredients, and reducing the toxic effects (ERDOĞAR et al., 2018;IANISKI et al., 2022).Given the number of experiments and similar responses, it was not possible to calculate the variance for the critical analysis of in vivo treatments, suggesting the need for further in vivo studies to compose a meta-analysis.
Notably, the studies evaluated herein were developed in Brazil, a country with the highest occurrence of equine pythiosis cases (SANTURIO et al., 1998;LEAL et al., 2001;MARCOLONGO-PEREIRA et al., 2012;SANTOS et al., 2014;WEIBLEN et al., 2016;REIS-GOMES et al., 2018;SOUTO et al., 2021), thereby demonstrating the importance of Brazilian science in developing cutting-edge research with this mammalian pathogen oomycete.Nonetheless, the development and application of nanotechnology tools in veterinary medicine in Brazil are on the rise, primarily in the therapeutic area, developing products to treat relevant diseases (IANISKI et al., 2022).

CONCLUSION
Based on the studies evaluated, nanotechnology is a promising alternative in developing nanostructured antimicrobial compounds to be explored in pythiosis treatment.Only four studies with nanotechnology and P. insidiosum were reported, all of which were performed by Brazilian researchers, thereby demonstrating the relevance of Brazilian science in the world context and the development of research on this important disease.Moreover, additional in vitro and in vivo studies must be developed to shed more light on the action of this new and prosperous technology against the relevant pathogenic microorganism P. insidiosum.
(2016b) described in vitro anti-P.insidiosum activity of Melaleuca alternifolia essential oil in the free oil and nanoemulsion forms.In another study, VALENTE et al. (2019) demonstrated in vitro susceptibility of biogenic silver nanoparticles (Bio-AgNP) against P. insidiosum isolates.The authors, using scanning electron microscopy (SEM) and transmittance microscopy (MET), verified that Bio-AgNP affected the ultrastructures and cells of the oomycete.Additionally, VALENTE et al. (2020) and SILVEIRA et al. (2022) assessed in vivo anti-P.insidiosum activity of Bio-AgNP and nanoemulsion of M. alternifolia, respectively, using an experimental model in rabbits.

Figure 1 -
Figure 1 -Flowchart of the articles published from 2010 to 2022 and selected in the systematic literature review on nanotechnology and Pythium insidiosum.

Table 1 -
Studies including nanotechnology applied to the oomycete Pythium insidiosum.