Toxoplasma gondii is a protozoan parasite that infects more than a third of the world's population. The drugs used today to treat toxoplasmosis cause severe side effects in many people and have poor success in treating chronic infections. In the current study, extracted oil from tea leaf was loaded into solid lipid nanoparticles (SLNs) and its anti-Toxoplasma properties were analyzed. Double emulsification technique was employed to provide SLNs and its physical criteria was calculated by transmission electron microscope (TEM) and dynamic light scattering (DLS). Cell toxicity and anti-intracellular Toxoplasma activity were investigated by a MTT assay. The anti-Toxoplasma activity of TTO-SLNs was evaluated by trypan-blue staining. The TTO-SLNs were round with a mean particle size of 85.23 nm and clear and stable margins. An association was seen between the cell toxicity of TTO-SLNs with the concentration of the component (P-value = 0.009). The cytotoxic concentration (CC50) against Toxoplasma was > 10 mg/mL, while it was concentration-dependent (P-value < 0.0001). the viability of T. gondii- infected Vero cells was higher in lower concentrations of TTO-SLNs (P-value = 0.0174), while at least 80% of T. gondii- infected Vero cells remained alive in the concentration ˃1 mg/mL. Overall, our findings demonstrated high anti-T. gondii properties of TTO-SLNs, suggesting the promising role of SLNs to carry TTO. In addition, our findings showed prolonged release of the TTO from SLNs capsulation of the can lead to, suggesting the capability of TTO-SLNs to be employed for chronic phase (cyst stages), which should be further investigated in animal models.
Research Article
Evaluation of anti-Toxoplasma effects of lipid nanoparticles carrying Tea tree oil on Toxoplasma gondii tachyzoites in Vero Cells
https://doi.org/10.21203/rs.3.rs-3652981/v1
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Toxoplasma gondii causes toxoplasmosis, which is a common parasitic disease in almost all warm-blooded animals (Levine 2018, de Barros, Torrecilhas et al. 2022). The main symptoms of the disease, especially in patients with compromised immune systems, range from mild flu-like symptoms to severe complications (Joynson 2001). Congenital transmission, ingestion of undercooked food, accidental ingestion of sporulated oocysts from contaminated soil or unwashed fruits and vegetables, and blood transfusion are the main routes of transmission of T. gondii (Pinto-Ferreira, Caldart et al. 2019, E 2021).
For those with weakened immune systems, and children under the age of five, toxoplasmosis treatment is necessary (Konstantinovic, Guegan et al. 2019). Pyrimethamine is the most effective drug for treating toxoplasmosis (Dunay, Gajurel et al. 2018). The current suggested method for treating toxoplasmosis comprises using pyrimethamine and sulfadiazine, commonly referred to as pyr-sulf (Konstantinovic, Guegan et al. 2019). The administering of pyrimethamine and sulfadiazine has been associated with occurrences of severe adverse reactions, such as the death of liver tissue, a low platelet count, and the potential for congenital defects. (Wei, Wei et al. 2015, Montazeri, Sharif et al. 2017). Due to the unfavorable side effects of the currently marketed toxoplasmosis medications, researchers have focused their efforts on creating novel therapeutic agents to efficiently treat conditions like encephalitis, chorioretinitis, and congenital toxoplasmosis (Torre, Casari et al. 1998, McFadden, Tomavo et al. 2000, Aspinall, Joynson et al. 2002, Meneceur, Bouldouyre et al. 2008, Béraud, Pierre-François et al. 2009, Doliwa, Escotte-Binet et al. 2013, Doliwa, Xia et al. 2013, Ben-Harari, Goodwin et al. 2017, Shammaa, Powell et al. 2021).
For countless years throughout history, medicinal plants have served as the primary source of medicine for treating human illnesses. Organic compounds derived from these plants have been shown to be able to be used in the pharmaceutical industry as replacements for synthetic compounds. (Petrovska 2012, Loetscher, Kreuzer et al. 2013, Sofowora, Ogunbodede et al. 2013). According to researchers, natural plant extracts have emerged in recent years as interesting candidates to use as anti-Toxoplasma medications, (Kavitha, Noordin et al. 2012, Choi, Jiang et al. 2013, Mohammad Rahimi, Khosravi et al. 2020, Khamesipour, Pourmohammad et al. 2022, Nemati, Mohammad Rahimi et al. 2022). Significant interest has been shown in clinical studies that concentrate on new treatment approaches employing these substances. Tea tree oil (TTO), an essential oil predominantly derived from the Australian native plant Melaleuca alternifolia, is one such substance and is well known for its antibacterial qualities (Łysakowska, Sienkiewicz et al. 2015). TTO is produced from the leaves of Melaleuca alternifolia through a procedure called steam distillation. This plant species is listed on ("The Plant List" [www.theplantlist.org]) website. Melaleuca alternifolia is used in traditional fever to treat skin infections, insect bites, bruises, vertigo, convulsions, toothache, rheumatism, colds, and flu (Usia, Iwata et al. 2006), and TTO is thought to be effective in treating diseases caused by bacteria, fungi, and viruses due to its broad-spectrum antibacterial properties (Budhiraja, Cullum et al. 1999, Packer, Brouwer et al. 2012, Sharifi-Rad, Salehi et al. 2017). It has also been observed that TTO has anti-cancer properties (Budhiraja, Cullum et al. 1999). TTO has the power to fight against oral microbes and can be used to prevent a variety of oral problems, including dental caries, periodontal disease, and improve the condition of the oral mucosa (Patri and Sahu 2017).
TTO has the ability to activate monocytes by activating some mechanisms, which it causes antibacterial and anti-inflammatory properties (Hart, Brand et al. 2000, Halcón and Milkus 2004, Sharifi-Rad, Salehi et al. 2017, Lam, Long et al. 2020, Qi, Gong et al. 2021). Recent studies have provided evidence that, in addition to TTO also has antibacterial, antifungal (Inouye, Tsuruoka et al. 2000, Inouye, Uchida et al. 2001, Hammer, Carson et al. 2002), antiviral (Schnitzler, Schön et al. 2001, Lu, Han et al. 2013) and anti-protozoa activity (Pena 1962, Mikus, Harkenthal et al. 2000).
Further research, especially large-scale clinical trials, is needed to evaluate the effectiveness of tea tree oil as an additional wound treatment (Halcón and Milkus 2004). The wide rang activities of TTO on bacterial, fungal, viral, and protozoal infections have been explained (Carson, Hammer et al. 2006). Also, a broad spectrum of microorganism has now been exanimated for their susceptibilities to TTO (Chan and Loudon 1998, Banes-Marshall, Cawley et al. 2001, Carson, Hammer et al. 2006). TTO is mostly bactericidal in nature, while it has the potential to be bacteriostatic at low levels. While most bacteria can be effectively targeted by concentrations of 1.0% or less, some species, including Enterococcus faecalis, Pseudomonas aeruginosa, and commensal skin staphylococci and micrococci, have been observed to exhibit minimum inhibitory concentrations (MICs) that exceed 2% (Hammer, Carson et al. 1996, Banes-Marshall, Cawley et al. 2001).
The delivery of therapeutic agent has already improved significantly thanks to nanotechnology. Nanoparticles not only enhance the effectiveness, the accuracy, and the uptake, but also decrease probable toxicity of drugs (Haleem, Javaid et al. 2023). Nanotechnology employs in medicine to increase feeling of hope for achieving major improvements in disease diagnosis and therapy (Hobson 2016). Nanoparticles consist of one or more therapeutic medications which are capable of binding or controlled release in drug delivery systems. These nanoparticles are incorporated inside polymer matrices by adsorption (Shafi, Raina et al. 2018). in recent years, development of nano-drugs for treatment and diagnosis has advanced significantly. Enhancing the bioavailability of targeted delivery, extending the half-life of injectable drugs, and enabling oral administration of pharmaceuticals are the primary objectives of nano-drug systems (Invernizzi and Foladori 2005, Hofmann-Amtenbrink, Hofmann et al. 2014). The development of the novel drug delivery vehicles as a result of recent advances in nanotechnology has found application in the manufacture of pharmaceutical and cosmetic-sanitary goods (Cai and Chen 2007, Zhang, Gu et al. 2008). Common drug delivery vehicles include liposomes, microemulsions, multiple emulsions, and solid particles. Solid lipid nanoparticles (SLNs), with particle sizes ranging from 50 to 1000 nm, were developed as colloidal carriers in the 1990s (Müller, Radtke et al. 2002). In an aqueous surfactant solution, SLNs that are made from physiological lipids disperse efficiently (Musielak, Feliczak-Guzik et al. 2022). SLNs have a number of benefits, including their small size, optimal surface area to volume ratio, interactions at the particle level, sizable drug loading capacity, appropriate release characteristics, applicability in targeted drug delivery, outstanding physical stability, and significant biocompatibility (Uner and Yener 2007, Ezzati Nazhad Dolatabadi, Valizadeh et al. 2015). In addition to reducing the toxicity of an active component of a drug agent, SLNs may also slow down the degradation of therapeutic agents and increase a drug's clearance rate, which helps a drug be effectively eliminated from the body via the reticuloendothelial system (Ghasemiyeh and Mohammadi-Samani 2018).
The mechanism by which tea tree facilitates the elimination of intracellular parasites such as T. gondii remains uncertain. In this study, the effect of TTO-SLNs on the Vero cell line and the RH strain of T. gondii was evaluated.
Preparation of TTO-loaded SLNs
The TTO was loaded into SLNs by an emulsification probe sonication (Vase et al. 2023, Nemati, Mohammad Rahimi et al. 2022). Finally, the organic solvent was evaporated by placing the solvent in a rota evaporator at 30°C (Nemati, Mohammad Rahimi et al. 2022).
Physicochemical evaluations of nanoparticles
Particle size, Polydispersity index (PDI) measurement, and Zeta potential
Dynamic light scattering (DLS) technique using a Zetasizer 1033439 (Malvern Instrument, UK) was analyzed for determination of the particle size, size distribution, and zeta potential of nanoparticles, which were maintained in 25°C distilled water. To compute the average particle size, the Ver.6.01 software was used. Afterwards, the polydispersity index (PDI) and zeta-potential measurements were performed (Vase et al. 2023)
Transmission Electron Microscopy (TEM) and Fourier Transform Infra-Red (FTIR) analysis
After preparing samples, the shape, size, and morphology of TTO-SLN were analyzed using a Zeiss EM10C TEM (Zeiss-EM10C-100 KV, Germany) operating at an accelerator voltage of 80 kV. The inconsistency between TTO and incorporated materials were analyzed by FTIR spectrometry (PerkinElmer ES Version 10.5.3, USA). The average accumulation was registered in 3 spots (16 scans per spot) at a resolution of 4 cm− 1 at wavelengths ranging from 400 cm− 1 to 4000 cm− 1.
Encapsulation Efficiency (EE)
The encapsulation efficiency of TTO-SLNs were analyzed as described elsewhere (Nemattalab, Rohani et al. 2022; Pinheiro, Granja et al. 2020). The encapsulation efficiency (EE%) formula was as follow:
EE (%) = (Total amount of TTO-Free amount of TTO)/ Total amount of TTO ×100
In-vitro release and release kinetic study
The release profile of TTO from SLN was performed with the dialysis method (Vase et al. 2023, Wakade and Shende 2021), using a 14 kDa dialysis bag (Sigma, Steinheim, Germany) based on the mathematical models of Zero-order, First-order, Higuchi, and Korsmeyer-Peppas (Mortazavi et al.2020).
Biology lab experiments
The biological procedures were briefly described in Fig. 1 (Fig. 1).
The Vero cell line
The Vero cells (kidney fibroblast from the African green monkey; ATCC: CCL-81) were cultured at 37°C in Dulbecco’s Modified Eagle Medium (DMEM; Biosera, Arya Tous, Iran) containing 100 U/ml of penicillin and 100 µg/ml streptomycin, and supplemented with 10% FBS (Gibco, Thermo Fisher Scientifc, MA, USA). Vero cells were grown in 25 cm3 flask until confluence.
Investigation of cell toxicity of the TTO- SLNs on Vero cell lines
The cell toxicity assays were performed as frequently described elsewhere (Khosravi, Mohammad Rahimi et al. 2020, Nemati, Mohammad Rahimi et al. 2022). Briefly, 4×104 Vero cells were treated with six different TTO-SLNs concentrations, log− 10 from 1 mg/mL to 100µg/mL and their viability was evaluated using MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The absorbance intensity was determined at a wavelength of 570. According to accepted procedures, the cell viability percentage was calculated (Lee, Mahmud et al. 2012) (Fig. 1).
Investigation of anti Toxoplasma activity
In order to evaluate the efficacy of TTO-SLNs against Toxoplasma, the log− 10 concentrations from 1 mg/mL to 100µg/mL of TTO-SLNs were added to individual tubes containing 2×104 live tachyzoites and after incubation at 37°C for 2 h the number of alive T. gondii tachyzoites were counted using a hemocytometer slide, light microscopy, and methylene blue vital staining (Nemati, Mohammad Rahimi et al. 2022).
Investigation of anti-intracellular Toxoplasma activity
A total of 1×104 Vero cells were infected by 1×104 T. gondii RH strain tachyzoites (MOI = 1) and after 24 h, the cells were treated by six concentrations of TTO-SLNs for further 24 h. The viability of Vero cells was evaluated by MTT assay (Lee, Mahmud et al. 2012, Sharif, Unyah et al. 2019 Nemati, Mohammad Rahimi et al. 2022). All tests were carried out in duplicate.
Statistical analysis
GraphPad Prism software version 8.3.0.538 was used for statistical analysis. P-values ≤ 0.05 were considered statistically significant. One-sample t-test was used to analyze the statistical correlations between the obtained results.
TTO nanoparticles
The morphological characteristics of the TTO-SLNs showed that the nanoparticles were well dispersed with a spherical shape. The average size of the TTO-SLNs and Z-potential were 85.23 nm and − 9.6 mV, respectively with a PDI of 0.67 (Fig. 2).
Encapsulation entrapment and release kinetic
The EE percentage of TTO was 75.3 ± 2.8%, indicating the suitability of the preparation method. The release of TTO-SLNs reach to 82.93% in 72 indicating suitable controlled release of the content for long time (Fig. 3).
Cell viability assay
The TTO-SLNs exhibited a CC50 value ˃10 mg/ml; 10 mg/ml (25.270 ± 4.653%), 100 mg/ml (12.270 ± 5.812%) concentrations resulting in the viability of at less 20% of Vero cells at concentrations above this threshold. The range of cell viability for TTO-SLNs, from 1 µg/mL to 100 mg was observed to be 106.525 ± 6.003% (95% CI: 100.642–112.408) to 12.270 ± 5.812% (95% CI: 6.574–17.966%). One-sample t-test, revealed a significant cytotoxicity reduction of TTO-SLNs which depends on concentration changes (P-value = 0.009), as indicated in the Fig. 4a and Table 1.
| Concentrations mg/mL | Anti-Toxoplasma activity | P-value | Cell viability | P-value | Ratio | ||
| Mean ± SD (%) | 95% CI | 0.0001 | Mean ± SD (%) | 95% CI (%) | 0.009 | ||
| 100 | 93.050 ± 9.829 | 83.418–102.682 | 12.270 ± 5.812 | 6.574–17.966 | 7.62 | ||
| 10 | 93.100 ± 0.000 | 0.00 | 25.270 ± 4.653 | 20.710–29.830 | 3.68 | ||
| 1 | 95.900 ± 1.980 | 93.960–97.840 | 95.080 ± 3.974 | 91.186–98.974 | 1.00* | ||
| 0.1 | 91.700 ± 1.980 | 89.760–93.640 | 104.075 ± 0.106 | 103.978–104.172 | 0.88 | ||
| 0.01 | 90.300 ± 0.000 | 0.00 | 104.155 ± 2.524 | 101.681–106.629 | 0.86 | ||
| 0.001 | 81.900 ± 0.000 | 0.00 | 106.525 ± 6.003 | 100.642 – 112.408 | 0.76 | ||
Anti Toxoplasma activity of the TTO-SLNs assay
TTO-SLNs showed anti-Toxoplasma activity. Results showed an IC50 value ˃1 µg/mL suggesting the inhibitory effect of all concentrations on at least 80% of T. gondii. The inhibitory effect of TTO-SLNs was between the high concentration of 100 mg/mL (93.050 ± 9.829%) and the low concentration of 1 µg/mL (81.900 ± 0.000%). Statistical analysis showed a significant concentration-dependent anti-Toxoplasma effect of TTO-SLN (P value < 0.0001) as depicted in Fig. 5 and Table 1.
Intracellular anti Toxoplasma activity assay
The efficacy of TTO-SLNs in killing intracellular Toxoplasma was evaluated and found to survive over 80% of Toxoplasma-infected Vero cells at a concentration ˃1 mg/ml. However, when Vero cells were infected with T. gondii and treated with TTO-SLNs, cell viability percent were (6.365 ± 2.298),( 15.210 ± 5.134) and (45.565 ± 6.767) at concentrations of 100, 10 and 1 mg/mL, respectively (P-value = 0.0174) (Fig. 4b, Table 2).
| Concentrations mg/mL | Mean ± SD (%) | 95% CI (%) | P-value |
|---|---|---|---|
| 100 | 6.365 ± 2.298 | 4.113–8.617 | 0.0174 |
| 10 | 15.210 ± 5.134 | 10.179–20.241 | |
| 1 | 45.565 ± 6.767 | 38.933–52.197 | |
| 0.1 | 88.620 ± 10.663 | 78.170–99.070 | |
| 0.01 | 90.190 ± 7.849 | 82.498–97.882 | |
| 0.001 | 93.365 ± 12.636 | 80.982–105.748 |
T. gondii belongs to the Apicomplexa, which lives as its obligate intracellular host and it can infect all human cells (Dubey, Lindsay et al. 1998, D'Ambrosio, Keeler et al. 2023). T. gondii is an important public health issue worldwide, which frequently reported from the human population, with an estimated infection rate of about 30% worldwide. This parasite is associated with a wide range of clinical symptoms in humans and affects both healthy and immunocompromised individuals (de Lima Bessa, de Almeida Vitor et al. 2021). Toxoplasmosis can cause serious eye disease, and if maternal-to-fetal infection occurs during pregnancy, it can cause fetal malformations, miscarriage, and stillbirth. Also, in people with defective immune systems more severe complications occur, and can cause encephalitis, respiratory failure, damage to other organs, and death in severely active infections (Ahmadpour, Daryani et al. 2014, Dubey, Murata et al. 2021). Therefore, the treatment of human toxoplasmosis is an urgent need for sensitive groups.
The current standard therapy for toxoplasmosis consists of a combination of pyrimethamine and sulfadiazine (pyr-sulf), which targets the active stage of infection, but significant failures of these drugs are still reported. Other regimens such as pyrimethamine plus atovaquone, clindamycin, clarithromycin, or azithromycin, or monotherapy with atovaquone or trimethoprim-sulfamethoxazole (TMP-SMX) are also used to treat toxoplasmosis, but none are more effective than pyr-sulf and no regimen is effective against the latent stage of infection (Dunay, Gajurel et al. 2018). Due to the unavailability of effective and safe drugs with low side effects, as well as the observation of drug-resistant strains, there is a need to develop new drugs with high efficacy (Montazeri, Mehrzadi et al. 2018).
In recent decades, many alternative medicines, including herbal medicines, have become increasingly popular. Since ancient times, medicinal plants have been used to treat or improve bacterial and parasitic infections in humans and animals (Martin and Ernst 2004, Sharif, Sarvi et al. 2016, Patri and Sahu 2017). Essential oils are distilled from plants and in most cases are considered alternative medicines and have been used for thousands of years to treat various ailments. Essential oils (EOs) are pure liquids that may be extracted from various plant components and have anti-inflammatory, antiviral, and antibacterial properties. Numerous research has shown that using essential oils in endodontic therapy has potential since no cases of resistance have been identified (Łysakowska, Sienkiewicz et al. 2015). In the past, Tea tree oil (TTO) has been used in the treatment of many diseases and recent studies have shown promising evidence of it for the treatment of various diseases. TTO is used as an active ingredient in many topical medications for the treatment of microbial infections, skin infections, and also in the management of inflammatory disorders (Banes-Marshall, Cawley et al. 2001, Mockdeci, Junqueira et al. 2023). In addition to the broad-spectrum antibacterial activity of TTO, its anti-tumor activity is also important, which prevents the migration, growth and invasion of drug-sensitive and drug-resistant melanoma cells (Greay, Ireland et al. 2010). This oil has anti-cancer agents and various antioxidants. Terpene hydrocarbons, such as monoterpenes, sesquiterpenes, and associated alcohols, make up the chemical makeup of TTO. Among other useful compounds of TTO, we can mention terpinen-4-ol, which has strong anti-inflammatory, anti-microbial and anti-tumor properties. The anti-inflammatory activity of TTO reduces TNF, IL-8, IL-1, superoxidase and prostaglandin E (Mondello, De Bernardis et al. 2006), which, in addition to affecting the parasite, can reduce inflammation in different stages of T. gondii infection. Because it has already been proven that T. gondii increases inflammation through the production of IL-1b and IL-18 cytokines (Nemati, Pazoki et al. 2021, Pazoki, Rahimi et al. 2021, Pazoki, Mirjalali et al. 2023), and this feature of TTO in people with healthy immunity and people with immune system disorders may have a good effect on reducing acute and chronic toxoplasmosis inflammations. If experts realize the valuable uses of this herbal substance, TTO can play an important role in the treatment of many diseases, including infectious diseases. Romeo et al 2022, explored the potential use of TTO as a disinfectant agent against coronaviruses Furthermore, three of its major components (terpinen-4-ol, γ-terpinene and 1,8-cineole) was assessed (Romeo, Iacovelli et al. 2022).
The effects of herbal medicines on T. gondii have vastly investigated (Sharif, Sarvi et al. 2016, Montazeri, Mehrzadi et al. 2018, Khosravi, Mohammad Rahimi et al. 2020, Nemati, Mohammad Rahimi et al. 2022). Our previous studies showed positive effects of SLNs encapsulation on drugs (Khosravi, Mohammad Rahimi et al. 2020, Nemati, Mohammad Rahimi et al. 2022). Solid lipid nanoparticles are functional nanoparticles that control the release and delivery of drugs and increase the efficiency of natural products [63]. In these studies, Neem oil-SLNs encapsulation and paromycin-SLNs encapsulation increased drug delivery, increased anti-Toxoplasma activity and decreased cytotoxicity of these drugs. Similar to our study, Mockdeci et al observed that TTO-SLNs has satisfactory and stable physicochemical properties, which improve the activity against oropharyngeal candidiasis, especially against Candida albicans. In addition to C. albicans, this developed formulation showed fungicidal effect for C. guilliermondii, C. glabrata and C. krusei (Mockdeci, Junqueira et al. 2023).
In this study, it was observed that with increasing concentration of TTO-SLNs such as 10–100 mg/mL, the death of cells increases, which is consistent with the results of the study of Söderberg et al (Söderberg, Johansson et al. 1996). They also stated that the cytotoxicity of TTO varies with concentration and exposure time. In other studies, they also indicated that TTO encapsulated in nanoparticles may have a greater capacity due to their lipophilicity with the plasma membrane of cells, and thus potentially penetrate cells more easily and become more cytotoxic (Söderberg, Johansson et al. 1996, Elmi, Ventrella et al. 2019, Mockdeci, Junqueira et al. 2023). In line with these studies, our findings showed a good anti-T. gondii activity at a concentration of 100 µg/ml TTO-SLNs due to lipid nanoparticles carrying tea tree oil, and low cytotoxicity for Vero cells was also observed at this concentration. Also, these findings showed that TTO-SLNs limited the intracellular development of T. gondii in Vero cells at a concentration of 100 µg/mL. Our previous study showed the effect of NeO-SLNs on T. gondii tachyzoites, which is completely consistent with the present study (Nemati, Mohammad Rahimi et al. 2022), however, we observed more toxicity of TTO-SLNs on T.gondii tachyzoites than NeO-SLNs. In this study, as in the previous study, we concluded that the high entrapment and satisfactory cumulative release of nanoparticles in SLNs can be used as an alternative treatment not only for T. gondii tachyzoites in Vero cells, but also can be effective for treating the acute phase of toxoplasmosis. Therefore, this developed formulation is a promising tool for the treatment of acute toxoplasmosis.
According our results, we proposed the use of TTO-SLNs as a novel natural agent against T.gondii, Indeed, TTO-SLNs at the low concentration of TTO, exhibits strong anti-toxoplasma effects (81.900%). We also recommend researchers to investigate the effect of TTO-SLNs on chronic phase cysts in animal models and non-lethal (cyst-forming) strains in future studies. If TTO-SLN is effective on cystic forms of toxoplasma in future studies, it can be introduced as an alternative drug to spiramycin. Because no cases of its teratogenicity have been observed during pregnancy (Pazyar, Yaghoobi et al. 2013, Vostinaru, Heghes et al. 2020), and many cases of spiramycin resistance have been reported (Montazeri, Mehrzadi et al. 2018).
Here, we demonstrated that TTO-SLNs were able to kill T. gondii tachyzoites. Our findings also showed that the encapsulation of SLNs into TTO can make it easier to diffuse into the cell and have a good effect on the parasite. Therefore, the developed formulation of TTO-SLNs can be introduced as a promising tool for the treatment of acute toxoplasmosis caused by T. gondii. In addition, we suggest that researchers investigate the effect of TTO-SLNs on chronic phase cysts in animal models in future studies.
Acknowledgments
The authors would like to thank all the members of the Foodborne and Waterborne Diseases Research Centre for their cooperation.
Authors’ contributions
SN designed the study. ZH provided natural products and synthesized nanoparticles. FAK, HMR and HM contributed in performing the experiments and analyzing the generated data. SN HP contributed in writing the manuscript. SN FH supported and supervised the study. The author(s) read and approved the final manuscript.
Funding
The Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran funded this project.
Availability of data and materials
The article's supplementary files include all the data generated for this study.
Ethics approval and consent to participate
All procedures performed in this study were in accordance with the ethical standards released by the Ethical Review Committee of the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran. As well, the study was approved by the ethics committee/institutional review board of the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Written consent was not applicable to be taken.
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