Synthesis and Evaluation of a Hybrid Miltefosine-Silver Nanoparticle Complex: Synergistic Interaction with Benznidazole Against Trypanosoma cruzi

Synthesis and Evaluation of a Hybrid Miltefosine-Silver Nanoparticle Complex: Synergistic Interaction with Benznidazole Against Trypanosoma cruzi | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis and Evaluation of a Hybrid Miltefosine-Silver Nanoparticle Complex: Synergistic Interaction with Benznidazole Against Trypanosoma cruzi Yener ÖZEL, İbrahim ÇAVUŞ, Feyzullah TOKAY, Sema BAĞDAT, Ahmet ÖZBİLGİN This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6294121/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Jun, 2025 Read the published version in Acta Parasitologica → Version 1 posted 11 You are reading this latest preprint version Abstract Objective : Chagas disease is an infectious disease classified under neglected tropical diseases and caused by the protozoan parasite Trypanosoma cruzi . This study aimed to investigate the cytotoxic activity, antitrypanosomal efficacy, and combination effects with benznidazole of hybrid silver nanoparticles (AgNPs) synthesized with miltefosine. Methods : In this study, a hybrid miltefosine (Mil)-silver nanoparticle (OA-MilAgNP) complex was synthesized. The nanoparticles were characterized using FT-IR spectroscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) analyses. The cytotoxicity of the nanoparticles was assessed in L929 fibroblast cells, while their antitrypanosomal activity was evaluated against a Trypanosoma cruzi strain using the liquid microdilution method. The interaction between the nanoparticle complex or miltefosine and benznidazole was analyzed using the checkerboard method. Results : FT-IR analysis demonstrated that the amylose surface was successfully coated with silver and miltefosine, confirming the successful synthesis of the hybrid complex. SEM analysis revealed that the nanoparticles exhibited a spherical morphology with varying sizes, while TEM analysis determined their sizes ranged between 10.14 and 18.42 nm. The OA-MilAgNP complex exhibited high antitrypanosomal activity and a selectivity index twice as high as that of miltefosine. Synergistic interactions were observed in the combinations of the OA-MilAgNP complex or miltefosine with benznidazole. Conclusion : The development of novel bioactive compounds with lower toxicity compared to traditional drugs has become essential for the treatment of Chagas disease. Drug repurposing combined with nanotechnology applications holds significant potential for improving therapeutic outcomes. The hybridization of miltefosine with silver nanoparticles, demonstrating strong antitrypanosomal activity and synergistic effects with benznidazole, may fill critical gaps in the literature. Silver nanoparticles Miltefosine Synergy T. cruzi Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Chagas disease is an infectious disease categorized under neglected tropical diseases, caused by the protozoan parasite Trypanosoma cruzi . The infection can be transmitted through Triatomine bugs (vector-borne), oral routes (foodborne), during pregnancy or childbirth (congenital), blood or blood products, organ transplantation, and laboratory accidents. It is estimated that approximately 6–7 million people worldwide are infected with T. cruzi , and the disease causes around 12,000 deaths annually. Despite its increasing global presence, Chagas disease is primarily observed in the endemic regions of 21 Latin American countries, where transmission largely depends on the presence of vectors. Currently, it is estimated that approximately 75 million people are at risk of infection [1]. The high levels of global human mobility due to migration, natural disasters, war, and tourism may lead to the emergence of neglected tropical diseases like Chagas disease in non-endemic regions as well [2]. Most acute infections are asymptomatic or present with non-specific symptoms such as fever, lymphadenopathy, and hepatosplenomegaly. Regardless of the mode of transmission, severe manifestations include myocarditis and encephalitis, with the severity of the disease likely associated with the parasitic load. Most acute infections go unrecognized and are rarely diagnosed. Approximately 30% of chronically infected patients develop Chagas cardiomyopathy years later. Chagas cardiomyopathy can lead to stroke, heart failure, thromboembolic events, and death [3]. Nanotechnology is a scientific discipline based on the synthesis and utilization of particles at the nanometer scale for various purposes. The physicochemical and biological properties of nanoparticles (NPs) vary depending on their morphological and structural characteristics, particle size, and surface area [4]. Recently, scientists have focused extensively on research related to nanotechnology applications. Nanotechnology is employed in the execution of precise medical procedures and contributes to fields such as diagnostics, disease treatment, regenerative medicine, gene therapy, dentistry, oncology, the cosmetic industry, drug delivery, and therapeutics [5]. Chagas disease, caused by Trypanosoma cruzi , is treated with two drugs: benznidazole and nifurtimox. However, these drugs have several disadvantages, including their effectiveness being limited to the acute or early stages of infection, the occurrence of side effects during treatment, and the parasite developing resistance to their efficacy. Therefore, it is essential to identify new, safe, and effective therapeutic alternatives for the treatment of Chagas disease. Scientists are working on several strategies to address this issue. These include modifying traditional drug dosages, redesigning drugs, combination therapies, and nanotechnology [6,7]. Some studies in the literature report remarkable results regarding inorganic nanoparticles with antimicrobial properties, such as gold, silver, zinc oxide, and titanium dioxide nanoparticles [7,8]. In our previous study, we determined that hybrid curcumin silver nanoparticles exhibited strong efficacy against Leishmania species, which are closely related to T. cruzi [7]. Based on the findings of that study, the present work aims to synthesize a hybrid complex by combining miltefosine the first and only orally effective drug for the treatment of leishmaniasis with silver nanoparticles, evaluate the antitrypanosomal activity of this novel hybrid complex, and investigate its combination with benznidazole. Material And Methods Ethical Approval No human or animal materials were used in this study. The experiments were conducted using parasite strains preserved in liquid nitrogen. Therefore, ethical approval is not required. Trypanosoma cruzi Strain and Cell Line The Trypanosoma cruzi ATCC 50825 reference strain and the L929 mouse fibroblast cell line (American Type Culture Collection, USA) used in this study were obtained from the Parasite Bank of Manisa Celal Bayar University Faculty of Medicine. Drugs Miltefosine (B2693-072166) was obtained from BOCScience (USA), and benznidazole (419656) was obtained from Sigma-Aldrich (USA). Synthesis of Nanoparticle Complexes Synthesis of Oxidized Amylose 15 mL of 30% H₂O₂, 2 mL of 0.05% CuSO₄, and 60 g of amylose were combined and stirred at 40°C for 15 minutes. At the end of this period, 400 mL of boiling water was added to the mixture, which was then stirred at 75°C for an additional 15 minutes. After this step, another 400 mL of boiling water was added, and the mixture was stirred at 100°C for 30 minutes. The solution was cooled to room temperature and centrifuged at 3000 rpm for 20 minutes. To remove copper ions from the resulting supernatant, dialysis was performed. The dialysis bag was dialyzed against distilled water at room temperature for two days, protected from light [9]. Synthesis of Hybrid Oxidized Amylose Miltefosine Silver Nanoparticle (OA-MilAg-NP) Complex To the dialyzed oxidized amylose supernatant, 100 mL of boiling water and 75 mL of 0.02 mol/L AgNO₃ were added and kept at 100°C in a dark environment for 120 minutes. Subsequently, 1 g of miltefosine was added to the mixture, which was cooled to room temperature with constant stirring. The mixture was centrifuged at 12,000 rpm for 15 minutes to remove excess miltefosine. Similarly, the excess AgNO₃ in the mixture was removed by dialysis [10] . The synthesized hybrid nanoparticle complex was lyophilized using the Christ Alpha 1-2 LD Plus lyophilization device at the Balikesir University Science and Technology Application and Research Center. FT-IR, SEM, and TEM Characterization of OA-MilAg-NP Complex The FT-IR spectra of the hybrid nanoparticle complexes were recorded using a Perkin Elmer Spectrum 65 model device with a wavenumber range of 4000 cm⁻¹ to 400 cm⁻¹, located at Balikesir University, the Department of Analytical Chemistry, Faculty of Science and Letters. Scanning electron microscopy (SEM) images were obtained using the JEOL JSM-7100F model device, and transmission electron microscopy (TEM) images were acquired using the JEOL JEM-1400 Plus model device, both located at the Çanakkale Onsekiz Mart University, Science and Technology Application and Research Center. Preparation of OA-MilAg-NP Complex, Miltefosine, and Benznidazole Stock Solutions The hybrid nanoparticle stock solution was prepared at a concentration of 10,000 μg/mL using sterile distilled water, the miltefosine stock solution at 1024 μg/mL using sterile distilled water, and the benznidazole stock solution at 1240 μg/mL using ethanol + water (solvent + diluent). Determination of Cytotoxic Activity of OA-MilAg-NP Complex, Miltefosine, and Benznidazole Fibroblasts preserved in liquid nitrogen were treated with RPMI-1640 and passaged into RPMI-1640 medium containing 10% FBS (fetal bovine serum). The cells were incubated at 37°C with 5% CO₂ for 48 hours. The number and viability of proliferating cells were determined using Trypan Blue staining and a hemocytometer. A 100 µL cell suspension containing 10⁵ cells/mL was distributed into 96-well microplates. Serial dilutions of the OA-MilAg-NP suspension (final concentration range: 5000–39 µg/mL), miltefosine (final concentration range: 512–0.125 µg/mL), and benznidazole (final concentration range: 640–0.60 µg/mL) were prepared in a separate microplate and transferred to the microplates containing the cells. The plates were incubated at 37°C with 5% CO₂ for 48 hours. Cell viability was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [11], and absorbance values were measured at 570 nm using a Thermo Varioskan model spectrophotometer (USA). IC 50 values indicating cytotoxic activity were calculated using GraphPad Prism 8.4.2 software. The cytotoxic activity tests were repeated three times on different days [7]. Determination of Antitrypanosomal Activity of OA-MilAg-NP Complex, Miltefosine, and Benznidazole The Trypanosoma cruzi (ATCC 50828) strain, thawed from liquid nitrogen, was cultured in LIT and NNN medium [12] and brought to the logarithmic phase in RPMI-1640 medium containing 10% FBS. For the determination of antitrypanosomal activity, 100 μL of RPMI-1640 medium was distributed into each well of sterile, flat-bottom, 96-well microplates. Serial dilutions of the hybrid NP complex were prepared in the range of 2500–19.5 μg/mL, miltefosine in the range of 128–1 μg/mL, and benznidazole in the range of 320–0.30 μg/mL. A 100 μL epimastigote suspension at a concentration of 1×10⁵/mL was added to all wells except the negative control, and the plates were incubated at 26±1°C for 24, 48, and 72 hours [13]. The morphological structure and motility of the epimastigotes in all wells were observed under an inverted microscope. The lowest concentration of the active substance at which all parasites were immobile and their morphology was disrupted was determined as the minimum parasiticidal concentration (MPC). Epimastigote viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [11]. Absorbance values were measured at 570 nm using a Thermo Varioskan model spectrophotometer (USA). IC 50 values indicating antitrypanosomal activity were calculated using GraphPad Prism 8.4.2 software. Efficacy tests were repeated three times on different days [7]. Determination of Selectivity Index (SI) Values for OA-MilAg-NP Complex, Miltefosine, and Benznidazole The SI value was calculated using the following formula by dividing the IC 50 value obtained for fibroblast cells (L929) by the IC 50 value obtained for T. cruzi epimastigotes: SI = IC 50 value for fibroblasts / IC 50 value for T. cruzi epimastigotes SI values greater than 1.00 indicate that the tested substance exhibits higher selectivity against epimastigotes than fibroblasts [14]. Determination of Interactions Between OA-MilAg-NP Complex and Miltefosine, with Benznidazole The interaction between the OA-MilAg-NP complex and miltefosine with benznidazole was determined using the checkerboard method. Two 96-well microplates were used for each combination. In the first microplate, 100 μL of RPMI-1640 medium was distributed into all wells. Serial dilutions of the OA-MilAg-NP complex and miltefosine were performed vertically, starting from three dilutions above their IC 50 values and proceeding five dilutions below. In the second microplate, 110 μL of RPMI-1640 medium was distributed into all wells up to the 8th column. A 110 μL solution of benznidazole was added to the wells in the 8th column, and horizontal serial dilutions were performed from right to left. The dilutions in the second microplate were then transferred to the corresponding wells in the first microplate in 100 μL volumes [15]. In all wells of the first microplate, except for the medium and sterility control wells, 100 μL of a parasite suspension at a concentration of 10⁵ epimastigotes/mL was added. Four wells were used for each control: growth control (RPMI-1640 + epimastigotes), medium control (RPMI-1640), and sterility control (RPMI-1640 + hybrid NP complex/miltefosine/benznidazole). The microplates were incubated at 26°C for 24, 48, and 72 hours. The morphological structure and motility of the epimastigotes in all wells were observed under an inverted microscope. The lowest concentration of the active substance at which all parasites were immobile and their morphology disrupted was determined as the minimum parasiticidal concentration (MPC) [7]. The interaction between the OA-MilAg-NP complex, miltefosine (Mil), and benznidazole (Benz) was determined by calculating the fractional inhibitory concentration (FIC) index using the following formula with MPC values: FIC index= FIC OA-MilAg-NP/Mil + FIC Ben FIC indeX= (MPC OA-MilAg-NP/Mil in comnbination /MPC OA-MilAg-NP/Mil alone ) + (MPC Benz in comnbination /MPC Benz alone ) The calculated FIC index values were interpreted based on the following thresholds: FIC index ≤ 0.5: synergy; FIC index = 0.50–0.75: partial synergy; FIC index = 0.75–1: additive; FIC index = 1.00–4.00: indifferent; FIC index ≥ 4.00: antagonism [16]. Statistical Analyses The obtained data were analyzed using IBM SPSS 25.0 statistical software (IBM SPSS Inc., Chicago, IL, USA). Significant differences between groups were analyzed using one-way ANOVA, followed by multiple comparisons evaluated with Tukey's test. Differences were considered statistically significant when p < 0.05. Results FT-IR Characterization of the OA-MilAg-NP Complex Characterization of the synthesized OA-MilAgNP was achieved using FTIR (Fourier transform infrared spectroscopy) analysis. Structural differences of OA-MilAgNP and bare oxidized amylose were monitored using FTIR spectra to prove synthesize of hybrid nanoparticle complex. Accordingly, the peaks at 1365 cm -1 , 1296 cm -1 , 1022 cm -1 , 942 cm -1 and 611 cm- 1 wavenumbers were newly appeared on OA-MilAgNP spectrum. The peak observed at 1365 cm -1 was attributed to methyl group in phosphocholine structure of miltefosine. Additionally, the peaks 1296 cm -1 and 1022 cm -1 corresponds P=O and P-O-C stretching vibrations, respectively. The peak 942 cm -1 was associated with C-N stretching vibration. The vibration at 611 cm -1 may be attributed to P-O bending. Moreover, the peaks 2851 cm -1 and 2921 cm -1 which were sharper and clear on OA-MilAgNP spectrum, corresponds the asymmetric and symmetric C-H stretching vibrations of the long alkyl chain of miltefosine. Those abovementioned results strongly emphasize that synthesize of hybrid OA-MilAgNP complex was successfully performed. SEM and TEM Characterization of the OA-MilAg-NP Complex SEM analyses of the hybrid nanoparticle complex revealed that silver ions and miltefosine molecules were successfully incorporated into the surface of oxidized amylose, which forms the core of the structure, confirming the successful synthesis of the nanoparticle complex. The obtained images showed that the nanoparticles have a rounded morphological appearance, vary in size, and are successfully attached to the surface of oxidized amylose. TEM analysis revealed that the nanoparticle sizes ranged between 10.14 and 18.42 nm (Figure 2) . Antitrypanosomal Activity Results of OA-MilAg-NP Complex, Miltefosine, and Benznidazole A time-dependent decrease in the IC 50 values of the OA-MilAg-NP complex, miltefosine, and benznidazole was observed, corresponding to an increase in antitrypanosomal activity (Table I and Figure 3). While the MPC values, where all parasites were killed, remained constant at 24 and 48 hours, the effective concentration decreased by one dilution at 72 hours. It was determined that the IC 50 and MPC values of benznidazole and miltefosine were close to each other (p>0.05) and that they exhibited stronger antitrypanosomal activity compared to the OA-MilAg-NP complex. The strongest antitrypanosomal activity was observed in the order of miltefosine, benznidazole, and the OA-MilAg-NP complex. While there was no statistically significant difference between the activities of benznidazole and miltefosine (p>0.05), the difference between each of these compounds and the OA-MilAg-NP complex was found to be significant (p<0.05). All tested compounds were determined to have strong antitrypanosomal activity (Table I and Figure 3). Table I. IC 50 and MPC values (µg/mL) of OA-MilAg-NP complex, miltefosine and benznidazole against T. cruzi strain Active Ingredients 24h 48h 72h IC 50 MPC IC 50 MPC IC 50 MPC OA-MilAg-NP 196.6 625 171.5 625 94.82 312.5 Miltefosine 26.17 128 14.98 128 6.42 64 Benznidazole 67.11 155 44.95 155 34.53 77.5 Mil: Miltefosine; NP: Nanoparticle; IC 50 : 50% inhibitory concentration; MPC : Minimum parasiticidal concentration Cytotoxic Activity Results and Selectivity Index (SI) Values of OA-MilAg-NP Complex, Miltefosine, and Benznidazole The cytotoxic activities of the OA-MilAg-NP complex, miltefosine, and benznidazole against fibroblast cells were determined to range between 1101–764 µg/mL, 38.05–27 µg/mL, and 1011–806.9 µg/mL, respectively, at 24, 48, and 72 hours. When SI values were calculated, miltefosine showed positive selectivity (SI>1), whereas the OA-MilAg-NP complex and benznidazole exhibited high selectivity (SI>3) (Table II and Figure 4). Table II. Cytotoxicity and selectivity index values (µg/mL) of OA-MilAg-NP complex, miltefosine and benznidazole Active Ingredients 24h (IC 50 ) 48h (IC 50 ) 72h (IC 50 ) T.cruzi L929 SI T.cruzi L929 SI T.cruzi L929 SI OA-MilAg-NP 196.6 1101 5.6 171.5 945 5.5 94.82 764 8 Miltefosine 26.17 38.05 1.5 14.98 33.51 2.2 6.42 27 4.2 Benznidazole 67.11 1011 15 44.95 822.1 18.2 34.53 806.9 23 Mil : Miltefosine; Nanoparticles IC 50 : % 50 inhibition concentration; L929 : Fibroblast cell line; S I: Selectivity index Interactions Between OA-MilAg-NP Complex, Miltefosine, and Benznidazole Synergistic interactions were observed for all incubation periods in the combinations of the OA-MilAg-NP complex and miltefosine with benznidazole (Table III). Table III. Interaction of OA-MilAg-NP Complex and Miltefosine and benznidazole combinations against T. cruzi Combinations 24h 48h 72h FIC index Interaction FIC index Interaction FIC index Interaction OA-MilAgNP+Benz 0.499 Synergy 0.374 Synergy 0.312 Synergy Mil+Benz 0.375 Synergy 0.375 Synergy 0.375 Synergy Ag : Silver, NP : Nanoparticle, Benz: Benznidazole, Mil: Miltefosine, FIC : Fractional inhibitory concentration Discussion Chagas disease, caused by Trypanosoma cruzi , is considered a neglected tropical disease due to the lack of safe and effective treatments. Current treatment options are limited to nifurtimox and benznidazole, both of which were developed over 40 years ago [17]. While etiological treatment in the acute phase leads to high rates of parasite clearance and seronegative results, treatment success in the chronic phase remains debatable. Adverse events, particularly in adults, are common and often lead to discontinuation of treatment [18]. Pharmaceutical companies have shifted their focus to long-term use drugs rather than investing in costly R&D for new molecule development [19]. This situation underscores the need for strategies aimed at discovering new antimicrobial drugs. Drug repurposing is the process of using an existing drug or drug candidate for a new therapeutic indication or medical condition not originally intended. In this process, the unintended side effects of drug molecules can serve as markers for discovering their effectiveness in entirely different medical conditions. Typically, drugs with proven safety in humans are tested and developed for efficacy in diseases other than the one for which they were originally designed. This process bypasses early drug development stages, thereby reducing risks and costs [20]. However, like traditional drugs, repurposed drugs face challenges related to formulation and delivery, such as poor aqueous solubility, limited stability, and/or incompatibility with new delivery routes. Nanotechnology plays a significant role in promoting the efficient use of highly active but poorly delivered drugs. It is frequently explored as a means to overcome challenges such as poor solubility, toxicity, and multidrug resistance in drug delivery systems [21]. Miltefosine is considered a protein kinase B (PKB) inhibitor, a key regulator of intracellular signaling essential for cell viability [22]. However, the mechanisms underlying its antiprotozoal activity remain incompletely understood. Some evidence suggests that it inhibits phosphatidylcholine and sphingomyelin synthesis, induces apoptosis [23], or inhibits mitochondrial cytochrome c oxidase and alters the organelle membrane [22]. Similarly, it has been proposed that miltefosine inhibits parasite phosphatidylcholine biosynthesis through the transmethylation pathway [24]. Recently, it has been suggested that miltefosine targets a sphingosine-activated plasma membrane Ca²⁺ channel in both Leishmania donovani and T. cruzi , affecting intracellular calcium homeostasis [25]. Several studies in the literature demonstrate the efficacy of miltefosine against T. cruzi in both in vitro and in vivo [17,26,27]. A recent study by Gulin et al. (2022) reported IC 50 values of miltefosine against T. cruzi amastigote and trypomastigote forms as 0.48 µM and 0.55 µM, respectively, and LC 50 (lethal concentration) values as 29.56 µM and 32.87 µM, respectively. They also found that the combination of miltefosine and benznidazole exhibited synergistic and additive interactions against trypomastigotes and amastigotes, respectively. Additionally, they reported that the combination was more effective than miltefosine alone in reducing parasitemia in an in vivo rodent model. In our study, unlike those in the literature, we synthesized a hybrid oxidized amylose miltefosine silver nanoparticle (OA-MilAg-NP) complex by combining miltefosine with silver and compared its efficacy against T. cruzi with that of miltefosine and benznidazole. We also investigated the in vitro interactions of both miltefosine and the OA-MilAg-NP complex with benznidazole. In our previous study, we demonstrated that a natural active component (curcumin) and silver NP complex exhibited strong antileishmanial activity with low cytotoxicity against Leishmania species [7]. Based on this, we aimed to enhance the efficacy of miltefosine while reducing its toxicity against T. cruzi , a parasite closely related to Leishmania species, using nanoparticle formulations and to identify synergistic interactions in combination with benznidazole. The synthesized miltefosine-silver NP complex was prepared using a method involving oxidized amylose as the core and miltefosine and silver ions at the nanoparticle level on the surface, as in our previous study [7,9,10]. FTIR spectra of the OA-MilAg-NP complex revealed peak shifts and splitting, indicating the successful attachment of functional groups to the oxidized amylose surface. SEM and TEM imaging showed that the OA-MilAg-NP complex had a round morphology, variable sizes, and a size range of 10.14–18.42 nm. A recent study by Latifi et al. (2024) synthesized miltefosine as chitosan nanoparticles with an average size of 46.61 nm and reported that the formulation was more effective against Acanthamoeba trophozoites and cysts than miltefosine alone. The same study also indicated that the NP formulation exhibited significantly lower cytotoxicity on Vero cell lines compared to miltefosine [28]. In our study, we compared the toxicity of the OA-MilAg-NP complex to L929 mouse fibroblasts with that of miltefosine and benznidazole and calculated the selectivity index (SI) for each compound. Benznidazole, as a conventional drug, exhibited the highest SI values during 24, 48, and 72-hour incubation periods. Notably, the OA-MilAg-NP complex showed at least twice the selectivity of miltefosine alone based on SI values (Table II). The SI values suggest that our synthesized NP complex is suitable for further in vivo studies (SI>3). A study investigating the efficacy of miltefosine formulated with lipid NPs against cutaneous leishmaniasis reported that lipid NPs had an average size of 160.8 nm, six times lower cytotoxicity against macrophage cell lines, and strong antileishmanial activity in vivo [29]. We believe that the much smaller NP size (10.14–18.42 nm) achieved with our synthesis method compared to other studies [30] may be a significant factor in enhancing antimicrobial activity. There are limited studies in the literature on the combination of benznidazole and miltefosine against T. cruzi. Gulin et al. (2022) noted that the synergistic interaction of benznidazole and miltefosine observed in vitro was also effective in vivo . In our study, a synergistic interaction was observed between miltefosine and benznidazole. Furthermore, unlike the literature, our study demonstrated a synergistic interaction between the OA-MilAg-NP complex and benznidazole. The data we obtained represent the first evidence demonstrating the hybrid synthesis of the OA-MilAg-NP complex based on oxidized amylose and its efficacy against T. cruzi. The data obtained on the efficacy of our oxidized amylose-based OA-MilAg-NP complex against T. cruzi and its interaction with benznidazole represent a novel contribution to the literature and provide valuable insights for the treatment of Chagas disease. Limitations The most significant limitation of this study is that the antitrypanosomal activity and interactions of the hybrid NP complex and miltefosine with benznidazole, which exhibited acceptable SI values, were not evaluated in experimental animal models. Conclusion Investigating and developing new bioactive compounds with lower toxicity compared to conventional drugs for Chagas disease treatment through nanotechnology applications can provide significant contributions to therapeutic efficacy in the literature. The hybridization of miltefosine with silver nanoparticles, which has demonstrated efficacy against leishmaniasis, and the strong antitrypanosomal activities of these novel molecules along with their synergistic interactions with existing antitrypanosomal drugs, can address significant gaps in the literature. These findings pave the way for designing new drug combinations, reducing side effects, and preventing resistance development, offering valuable insights for future studies. Declarations Conflict Of Interest The authors declare no conflict of interest. Author Contribution Y.O, IC and AO wrote the main manuscript, made the analyses and prepare figures and tables. FT and SB synthesized nanoparticles and performed FTIR characterization. All authors reviewed the manuscript. Acknowledgement We would like to thank the Parasite Bank of Manisa Celal Bayar University Faculty of Medicine for providing the parasite strain and cell line used in this study. 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BMC Infect Dis 13:548. https://doi.org/10.1186/1471-2334-13-548 Gulin JEN, Bisio MMC, Rocco D, Altcheh J, Solana ME, García-Bournissen F (2022Miltefosine and Benznidazole Combination Improve Anti- Trypanosoma cruzi In Vitro and In Vivo Efficacy. Front Cell Infect Microbiol 12:855119. https://doi.org/10.3389/fcimb.2022.855119 Pérez-Molina J, Molina I. (2018Chagas Disease. Lancet 39: 82–94. doi: 10.1016/S0140-6736(17)31612-4 Kayserili A, Kıyak M (2019Assessment Of R&D Actıvıtıes In The Pharmaceutıcal Sector. J Fac Pharm Ankara, 43:239-258. https://doi.org/10.33483/jfpau.546047 Kulkarni VS, Alagarsamy V, Solomon VR, Jose PA, Murugesan S (2023Drug Repurposing: An Effective Tool in Modern Drug Discovery. Russ J Bioorg Chem 49:157–166. https://doi.org/10.1134/S1068162023020139 Najlah M (2021Drug repurposing supported by nanotechnology: A promising strategy to fight cancer. Ther Deliv 12:267–269. https://doi.org/10.4155/tde-2021-0009 Dorlo T, Balasegaram M, Beijnen J, de Vries P (2012Miltefosine: A review of its pharmacology and therapeutic efficacy in the treatment of leishmaniasis. J Antimicrob Chem, 67:2576–2597. https://doi.org/10.1093/jac/dks275 Maya J, Cassels B, Iturriaga-Vásquez P, Ferreira J, Faúndez M, Galanti N, et al. (2007Mode of action of natural and synthetic drugs against Trypanosoma cruzi and their interaction with the mammalian host. Comp Biochem Physiol A Mol Integr Physiol, 146:601–620. https://doi.org/10.1016/j.cbpa.2006.03.004 Lira R, Contreras L, Rita R, Urbina J (2001Mechanism of action of anti-proliferative lysophospholipid analogues against the protozoan parasite Trypanosoma cruzi : Potentiation of in vitro activity by the sterol biosynthesis inhibitor ketoconazole. J Antimicrob Chem, 47:537–546. https://doi.org/10.1093/jac/47.5.537 Benaim G, Paniz-Mondolfi A, Sordillo E, Martinez-Sotillo N (2020Disruption of intracellular calcium homeostasis as a therapeutic target against Trypanosoma cruzi . Front Cell Infect Microbiol 10:46. https://doi.org/10.3389/fcimb.2020.00046 Saraiva V, Gibaldi D, Previato J, Mendonça-Previato L, Bozza M, Freire-De-Lima C, et al. (2002Proinflammatory and cytotoxic effects of hexadecylphosphocholine (miltefosineagainst drug-resistant strains of Trypanosoma cruzi . Antimicrob Agents Chemother, 46:3472–3477. https://doi.org/10.1128/AAC.46.11.3472-3477.2002 Martinez-Peinado N, Cortes-Serra N, Sherman J, Rodriguez A, Bustamante J, Gascon J, et al. (2021Identification of Trypanosoma cruzi growth inhibitors with activity in vivo within a collection of licensed drugs. Microorganisms 9:406. https://doi.org/10.3390/microorganisms9020406 Latifi A, Esmaeili F, Mohebali M, Yasami-Khiabani S, Rezaeian M, Soleimani M, et al. (2024Chitosan nanoparticles improve the effectivity of miltefosine against Acanthamoeba. PLoS Negl Trop Dis 18:e0011976. https://doi.org/10.1371/journal.pntd.0011976 Khan AS, Ud Din F, Ali Z, Bibi M, Zahid F, Zeb A, Mujeeb-Ur-Rehman, Khan GM (2021Development, in vitro and in vivo evaluation of miltefosine loaded nanostructured lipid carriers for the treatment of Cutaneous Leishmaniasis. Inter J Pharm 593:120109. https://doi.org/10.1016/j.ijpharm.2020.120109 Paiva DF, Matos APDS, Garófalo DA, do Nascimento T, Monteiro MSSB, Santos-Oliveira R, Ricci-Junior E (2023Use of Nanocarriers Containing Antitrypanosomal Drugs for the Treatment of Chagas Disease. Pharmaceuticals (Basel16:1163. https://doi.org/10.3390/ph16081163 Additional Declarations No competing interests reported. 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10:08:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6294121/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6294121/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11686-025-01074-3","type":"published","date":"2025-06-12T15:57:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81185051,"identity":"886b1f38-8aad-4f14-94d5-35e4d15a4789","added_by":"auto","created_at":"2025-04-23 08:17:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119304,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectrum of OA-MilAg-NP complex\u003c/p\u003e","description":"","filename":"FIGURE1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6294121/v1/214d460afe10a7fa86ace744.jpg"},{"id":81184685,"identity":"31ceded9-12bb-4b8b-892a-3ccd69934f95","added_by":"auto","created_at":"2025-04-23 08:09:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":708928,"visible":true,"origin":"","legend":"\u003cp\u003eSEM (A) and TEM (B) images of OA-MilAg-NP Complex\u003c/p\u003e","description":"","filename":"FIGURE2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6294121/v1/e6de47c04761b4cfed8a1475.jpg"},{"id":81184688,"identity":"a34307df-99ae-4558-a765-f72bdd9b558e","added_by":"auto","created_at":"2025-04-23 08:09:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":343560,"visible":true,"origin":"","legend":"\u003cp\u003eGraphs showing IC\u003csub\u003e50\u003c/sub\u003e values of OA-MilAg-NP complex, miltefosine and benznidazole\u003c/p\u003e","description":"","filename":"FIGURE3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6294121/v1/5765070e25196d1b5d882a88.jpg"},{"id":81185055,"identity":"519f636b-7068-4f5a-892e-4629850de188","added_by":"auto","created_at":"2025-04-23 08:17:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":333075,"visible":true,"origin":"","legend":"\u003cp\u003eGraphs showing IC\u003csub\u003e50\u003c/sub\u003e values of OA-MilAg-NP complex, miltefosine and benznidazole against L929 Fibroblasts\u003c/p\u003e","description":"","filename":"FIGURE4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6294121/v1/2d47aec3d8d66dfd12f88c36.jpg"},{"id":84726560,"identity":"18928df1-b7a7-4b98-969e-e9be63e4e0ea","added_by":"auto","created_at":"2025-06-16 16:07:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2469996,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6294121/v1/e5c8fe1b-5b6b-4212-8bb5-3f173af76a78.pdf"},{"id":81184686,"identity":"abc58f86-6456-4ef3-9d23-bfc5d9302d15","added_by":"auto","created_at":"2025-04-23 08:09:35","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":86401,"visible":true,"origin":"","legend":"","description":"","filename":"GraficalAbstractOAMilAgNP300.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6294121/v1/384e5aee711d0466fac44490.jpg"},{"id":81184682,"identity":"7c57b54f-9f03-45e9-8061-3bf2b35d4609","added_by":"auto","created_at":"2025-04-23 08:09:35","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14283,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstracttext.docx","url":"https://assets-eu.researchsquare.com/files/rs-6294121/v1/0037d866bbfcc7209f8bd02d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis and Evaluation of a Hybrid Miltefosine-Silver Nanoparticle Complex: Synergistic Interaction with Benznidazole Against Trypanosoma cruzi","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChagas disease is an infectious disease categorized under neglected tropical diseases, caused by the protozoan parasite \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e. The infection can be transmitted through Triatomine bugs (vector-borne), oral routes (foodborne), during pregnancy or childbirth (congenital), blood or blood products, organ transplantation, and laboratory accidents. It is estimated that approximately 6\u0026ndash;7 million people worldwide are infected with \u003cem\u003eT. cruzi\u003c/em\u003e, and the disease causes around 12,000 deaths annually. Despite its increasing global presence, Chagas disease is primarily observed in the endemic regions of 21 Latin American countries, where transmission largely depends on the presence of vectors. Currently, it is estimated that approximately 75 million people are at risk of infection [1]. The high levels of global human mobility due to migration, natural disasters, war, and tourism may lead to the emergence of neglected tropical diseases like Chagas disease in non-endemic regions as well [2].\u003c/p\u003e\n\u003cp\u003eMost acute infections are asymptomatic or present with non-specific symptoms such as fever, lymphadenopathy, and hepatosplenomegaly. Regardless of the mode of transmission, severe manifestations include myocarditis and encephalitis, with the severity of the disease likely associated with the parasitic load. Most acute infections go unrecognized and are rarely diagnosed. Approximately 30% of chronically infected patients develop Chagas cardiomyopathy years later. Chagas cardiomyopathy can lead to stroke, heart failure, thromboembolic events, and death [3].\u003c/p\u003e\n\u003cp\u003eNanotechnology is a scientific discipline based on the synthesis and utilization of particles at the nanometer scale for various purposes. The physicochemical and biological properties of nanoparticles (NPs) vary depending on their morphological and structural characteristics, particle size, and surface area [4]. Recently, scientists have focused extensively on research related to nanotechnology applications. Nanotechnology is employed in the execution of precise medical procedures and contributes to fields such as diagnostics, disease treatment, regenerative medicine, gene therapy, dentistry, oncology, the cosmetic industry, drug delivery, and therapeutics [5].\u003c/p\u003e\n\u003cp\u003eChagas disease, caused by \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e, is treated with two drugs: benznidazole and nifurtimox. However, these drugs have several disadvantages, including their effectiveness being limited to the acute or early stages of infection, the occurrence of side effects during treatment, and the parasite developing resistance to their efficacy. Therefore, it is essential to identify new, safe, and effective therapeutic alternatives for the treatment of Chagas disease. Scientists are working on several strategies to address this issue. These include modifying traditional drug dosages, redesigning drugs, combination therapies, and nanotechnology [6,7]. Some studies in the literature report remarkable results regarding inorganic nanoparticles with antimicrobial properties, such as gold, silver, zinc oxide, and titanium dioxide nanoparticles [7,8].\u003c/p\u003e\n\u003cp\u003eIn our previous study, we determined that hybrid curcumin silver nanoparticles exhibited strong efficacy against \u003cem\u003eLeishmania\u003c/em\u003e species, which are closely related to \u003cem\u003eT. cruzi\u003c/em\u003e [7]. Based on the findings of that study, the present work aims to synthesize a hybrid complex by combining miltefosine the first and only orally effective drug for the treatment of leishmaniasis with silver nanoparticles, evaluate the antitrypanosomal activity of this novel hybrid complex, and investigate its combination with benznidazole.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo human or animal materials were used in this study. The experiments were conducted using parasite strains preserved in liquid nitrogen. Therefore, ethical approval is not required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTrypanosoma cruzi\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eStrain and Cell Line\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e ATCC 50825 reference strain and the L929 mouse fibroblast cell line (American Type Culture Collection, USA) used in this study were obtained from the Parasite Bank of Manisa Celal Bayar University Faculty of Medicine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrugs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMiltefosine (B2693-072166) was obtained from BOCScience (USA), and benznidazole (419656) was obtained from Sigma-Aldrich (USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of Nanoparticle Complexes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of Oxidized Amylose\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e15 mL of 30% H₂O₂, 2 mL of 0.05% CuSO₄, and 60 g of amylose were combined and stirred at 40°C for 15 minutes. At the end of this period, 400 mL of boiling water was added to the mixture, which was then stirred at 75°C for an additional 15 minutes. After this step, another 400 mL of boiling water was added, and the mixture was stirred at 100°C for 30 minutes. The solution was cooled to room temperature and centrifuged at 3000 rpm for 20 minutes. To remove copper ions from the resulting supernatant, dialysis was performed. The dialysis bag was dialyzed against distilled water at room temperature for two days, protected from light [9].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of Hybrid Oxidized Amylose Miltefosine Silver Nanoparticle (OA-MilAg-NP) Complex\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo the dialyzed oxidized amylose supernatant, 100 mL of boiling water and 75 mL of 0.02 mol/L AgNO₃ were added and kept at 100°C in a dark environment for 120 minutes. Subsequently, 1 g of miltefosine was added to the mixture, which was cooled to room temperature with constant stirring. The mixture was centrifuged at 12,000 rpm for 15 minutes to remove excess miltefosine. Similarly, the excess AgNO₃ in the mixture was removed by dialysis [10] . The synthesized hybrid nanoparticle complex was lyophilized using the Christ Alpha 1-2 LD Plus lyophilization device at the Balikesir University Science and Technology Application and Research Center.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFT-IR, SEM, and TEM Characterization of OA-MilAg-NP Complex\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe FT-IR spectra of the hybrid nanoparticle complexes were recorded using a Perkin Elmer Spectrum 65 model device with a wavenumber range of 4000 cm⁻¹ to 400 cm⁻¹, located at Balikesir University, the Department of Analytical Chemistry, Faculty of Science and Letters. Scanning electron microscopy (SEM) images were obtained using the JEOL JSM-7100F model device, and transmission electron microscopy (TEM) images were acquired using the JEOL JEM-1400 Plus model device, both located at the Çanakkale Onsekiz Mart University, Science and Technology Application and Research Center.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of OA-MilAg-NP Complex, Miltefosine, and Benznidazole Stock Solutions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe hybrid nanoparticle stock solution was prepared at a concentration of 10,000 μg/mL using sterile distilled water, the miltefosine stock solution at 1024 μg/mL using sterile distilled water, and the benznidazole stock solution at 1240 μg/mL using ethanol + water (solvent + diluent).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of Cytotoxic Activity of OA-MilAg-NP Complex, Miltefosine, and Benznidazole\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFibroblasts preserved in liquid nitrogen were treated with RPMI-1640 and passaged into RPMI-1640 medium containing 10% FBS (fetal bovine serum). The cells were incubated at 37°C with 5% CO₂ for 48 hours. The number and viability of proliferating cells were determined using Trypan Blue staining and a hemocytometer. A 100 µL cell suspension containing 10⁵ cells/mL was distributed into 96-well microplates. Serial dilutions of the OA-MilAg-NP suspension (final concentration range: 5000–39 µg/mL), miltefosine (final concentration range: 512–0.125 µg/mL), and benznidazole (final concentration range: 640–0.60 µg/mL) were prepared in a separate microplate and transferred to the microplates containing the cells. The plates were incubated at 37°C with 5% CO₂ for 48 hours. Cell viability was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [11], and absorbance values were measured at 570 nm using a Thermo Varioskan model spectrophotometer (USA). IC\u003csub\u003e50\u003c/sub\u003e values indicating cytotoxic activity were calculated using GraphPad Prism 8.4.2 software. The cytotoxic activity tests were repeated three times on different days [7].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of Antitrypanosomal Activity of OA-MilAg-NP Complex, Miltefosine, and Benznidazole\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e (ATCC 50828) strain, thawed from liquid nitrogen, was cultured in LIT and NNN medium [12] and brought to the logarithmic phase in RPMI-1640 medium containing 10% FBS. For the determination of antitrypanosomal activity, 100 μL of RPMI-1640 medium was distributed into each well of sterile, flat-bottom, 96-well microplates. Serial dilutions of the hybrid NP complex were prepared in the range of 2500–19.5 μg/mL, miltefosine in the range of 128–1 μg/mL, and benznidazole in the range of 320–0.30 μg/mL. A 100 μL epimastigote suspension at a concentration of 1×10⁵/mL was added to all wells except the negative control, and the plates were incubated at 26±1°C for 24, 48, and 72 hours [13]. The morphological structure and motility of the epimastigotes in all wells were observed under an inverted microscope. The lowest concentration of the active substance at which all parasites were immobile and their morphology was disrupted was determined as the minimum parasiticidal concentration (MPC). Epimastigote viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [11]. Absorbance values were measured at 570 nm using a Thermo Varioskan model spectrophotometer (USA). IC\u003csub\u003e50\u003c/sub\u003e values indicating antitrypanosomal activity were calculated using GraphPad Prism 8.4.2 software. Efficacy tests were repeated three times on different days [7].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of Selectivity Index (SI) Values for OA-MilAg-NP Complex, Miltefosine, and Benznidazole\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SI value was calculated using the following formula by dividing the IC\u003csub\u003e50\u003c/sub\u003e value obtained for fibroblast cells (L929) by the IC\u003csub\u003e50\u003c/sub\u003e value obtained for \u003cem\u003eT. cruzi\u003c/em\u003e epimastigotes:\u003c/p\u003e\n\u003cp\u003eSI = IC\u003csub\u003e50\u003c/sub\u003e value for fibroblasts / IC\u003csub\u003e50\u003c/sub\u003e value for \u003cem\u003eT. cruzi\u003c/em\u003e epimastigotes\u003c/p\u003e\n\u003cp\u003eSI values greater than 1.00 indicate that the tested substance exhibits higher selectivity against epimastigotes than fibroblasts [14].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of Interactions Between OA-MilAg-NP Complex and Miltefosine, with Benznidazole\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe interaction between the OA-MilAg-NP complex and miltefosine with benznidazole was determined using the checkerboard method. Two 96-well microplates were used for each combination. In the first microplate, 100 μL of RPMI-1640 medium was distributed into all wells. Serial dilutions of the OA-MilAg-NP complex and miltefosine were performed vertically, starting from three dilutions above their IC\u003csub\u003e50\u003c/sub\u003e values and proceeding five dilutions below. In the second microplate, 110 μL of RPMI-1640 medium was distributed into all wells up to the 8th column. A 110 μL solution of benznidazole was added to the wells in the 8th column, and horizontal serial dilutions were performed from right to left. The dilutions in the second microplate were then transferred to the corresponding wells in the first microplate in 100 μL volumes\u0026nbsp;[15].\u0026nbsp;In all wells of the first microplate, except for the medium and sterility control wells, 100 μL of a parasite suspension at a concentration of 10⁵ epimastigotes/mL was added. Four wells were used for each control: growth control (RPMI-1640 + epimastigotes), medium control (RPMI-1640), and sterility control (RPMI-1640 + hybrid NP complex/miltefosine/benznidazole). The microplates were incubated at 26°C for 24, 48, and 72 hours. The morphological structure and motility of the epimastigotes in all wells were observed under an inverted microscope. The lowest concentration of the active substance at which all parasites were immobile and their morphology disrupted was determined as the minimum parasiticidal concentration (MPC) [7].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe interaction between the OA-MilAg-NP complex, miltefosine (Mil), and benznidazole (Benz) was determined by calculating the fractional inhibitory concentration (FIC) index using the following formula with MPC values:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFIC index= FIC\u003csub\u003eOA-MilAg-NP/Mil\u0026nbsp;\u003c/sub\u003e+ FIC\u003csub\u003eBen\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eFIC indeX= (MPC\u003csub\u003e\u0026nbsp;OA-MilAg-NP/Mil in comnbination\u0026nbsp;\u003c/sub\u003e/MPC\u003csub\u003e\u0026nbsp;OA-MilAg-NP/Mil alone\u003c/sub\u003e) + (MPC\u003csub\u003eBenz in comnbination\u0026nbsp;\u003c/sub\u003e/MPC\u003csub\u003eBenz alone\u003c/sub\u003e)\u003c/p\u003e\n\u003cp\u003eThe calculated FIC index values were interpreted based on the following thresholds:\u003c/p\u003e\n\u003cp\u003eFIC index ≤ 0.5: synergy; FIC index = 0.50–0.75: partial synergy; FIC index = 0.75–1: additive; FIC index = 1.00–4.00: indifferent; FIC index ≥ 4.00: antagonism [16].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe obtained data were analyzed using IBM SPSS 25.0 statistical software (IBM SPSS Inc., Chicago, IL, USA). Significant differences between groups were analyzed using one-way ANOVA, followed by multiple comparisons evaluated with Tukey's test. Differences were considered statistically significant when p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eFT-IR Characterization of the OA-MilAg-NP Complex\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCharacterization of the synthesized OA-MilAgNP was achieved using FTIR (Fourier transform infrared spectroscopy) analysis. Structural differences of OA-MilAgNP and bare oxidized amylose were monitored using FTIR spectra to prove synthesize of hybrid nanoparticle complex. Accordingly, the peaks at 1365 cm\u003csup\u003e-1\u003c/sup\u003e, 1296 \u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e, 1022 cm\u003csup\u003e-1\u003c/sup\u003e, 942 cm\u003csup\u003e-1\u003c/sup\u003e and 611 cm-\u003csup\u003e1\u003c/sup\u003e wavenumbers were newly appeared on OA-MilAgNP spectrum. The peak observed at 1365 cm\u003csup\u003e-1\u003c/sup\u003e was attributed to methyl group in phosphocholine structure of miltefosine. Additionally, the peaks 1296 cm\u003csup\u003e-1\u003c/sup\u003e and 1022 cm\u003csup\u003e-1\u003c/sup\u003e corresponds P=O and P-O-C stretching vibrations, respectively. The peak 942 cm\u003csup\u003e-1\u003c/sup\u003e was associated with C-N stretching vibration. The vibration at 611 cm\u003csup\u003e-1\u003c/sup\u003e may be attributed to P-O bending. Moreover, the peaks 2851 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eand 2921 cm\u003csup\u003e-1\u003c/sup\u003e which were sharper and clear on OA-MilAgNP spectrum, corresponds the asymmetric and symmetric C-H stretching vibrations of the long alkyl chain of miltefosine. Those abovementioned results strongly emphasize that synthesize of hybrid OA-MilAgNP complex was successfully performed. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSEM and TEM Characterization of the OA-MilAg-NP Complex\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSEM analyses of the hybrid nanoparticle complex revealed that silver ions and miltefosine molecules were successfully incorporated into the surface of oxidized amylose, which forms the core of the structure, confirming the successful synthesis of the nanoparticle complex. The obtained images showed that the nanoparticles have a rounded morphological appearance, vary in size, and are successfully attached to the surface of oxidized amylose. TEM analysis revealed that the nanoparticle sizes\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eranged between 10.14 and 18.42 nm (Figure 2)\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntitrypanosomal Activity Results of OA-MilAg-NP Complex, Miltefosine, and Benznidazole\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA time-dependent decrease in the IC\u003csub\u003e50\u003c/sub\u003e values of the OA-MilAg-NP complex, miltefosine, and benznidazole was observed, corresponding to an increase in antitrypanosomal activity (Table I and Figure 3). While the MPC values, where all parasites were killed, remained constant at 24 and 48 hours, the effective concentration decreased by one dilution at 72 hours. It was determined that the IC\u003csub\u003e50\u003c/sub\u003e and MPC values of benznidazole and miltefosine were close to each other (p\u0026gt;0.05) and that they exhibited stronger antitrypanosomal activity compared to the OA-MilAg-NP complex. The strongest antitrypanosomal activity was observed in the order of miltefosine, benznidazole, and the OA-MilAg-NP complex. While there was no statistically significant difference between the activities of benznidazole and miltefosine (p\u0026gt;0.05), the difference between each of these compounds and the OA-MilAg-NP complex was found to be significant (p\u0026lt;0.05). All tested compounds were determined to have strong antitrypanosomal activity (Table I and Figure 3).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"603\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"bottom\" style=\"width: 603px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable I.\u0026nbsp;\u003c/strong\u003eIC\u003csub\u003e50\u003c/sub\u003e and MPC values (\u0026micro;g/mL) of OA-MilAg-NP complex, miltefosine and benznidazole against \u003cem\u003eT. cruzi\u003c/em\u003e strain\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 189px;\"\u003e\n \u003cp\u003eActive Ingredients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003e24h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003e48h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003e72h\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eMPC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eMPC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eMPC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eOA-MilAg-NP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e196.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e171.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e94.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e312.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eMiltefosine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e26.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e14.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e6.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eBenznidazole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e67.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e44.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e34.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e77.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"bottom\" style=\"width: 603px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMil:\u003c/strong\u003e Miltefosine; \u003cstrong\u003eNP:\u003c/strong\u003e Nanoparticle; \u003cstrong\u003eIC\u003csub\u003e50\u003c/sub\u003e:\u003c/strong\u003e 50% inhibitory concentration; \u003cstrong\u003eMPC\u003c/strong\u003e: Minimum parasiticidal concentration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxic Activity Results and Selectivity Index (SI) Values of OA-MilAg-NP Complex, Miltefosine, and Benznidazole\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cytotoxic activities of the OA-MilAg-NP complex, miltefosine, and benznidazole against fibroblast cells were determined to range between 1101\u0026ndash;764 \u0026micro;g/mL, 38.05\u0026ndash;27 \u0026micro;g/mL, and 1011\u0026ndash;806.9 \u0026micro;g/mL, respectively, at 24, 48, and 72 hours. When SI values were calculated, miltefosine showed positive selectivity (SI\u0026gt;1), whereas the OA-MilAg-NP complex and benznidazole exhibited high selectivity (SI\u0026gt;3) (Table II and Figure 4).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"627\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\" valign=\"top\" style=\"width: 627px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable II.\u0026nbsp;\u003c/strong\u003eCytotoxicity and selectivity index values (\u0026micro;g/mL) of OA-MilAg-NP complex, miltefosine and benznidazole\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 107px;\"\u003e\n \u003cp\u003eActive Ingredients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 168px;\"\u003e\n \u003cp\u003e24h (IC\u003csub\u003e50\u003c/sub\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 183px;\"\u003e\n \u003cp\u003e48h (IC\u003csub\u003e50\u003c/sub\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 169px;\"\u003e\n \u003cp\u003e72h (IC\u003csub\u003e50\u003c/sub\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cem\u003eT.cruzi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003eL929\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003eSI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cem\u003eT.cruzi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003eL929\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003eSI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003eT.cruzi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003eL929\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46px;\"\u003e\n \u003cp\u003eSI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003eOA-MilAg-NP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e196.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e1101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e5.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e171.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e945\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e94.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e764\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003eMiltefosine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e26.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e38.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e14.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e33.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e6.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46px;\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003eBenznidazole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e67.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e1011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e44.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e822.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e18.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e34.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e806.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\" valign=\"top\" style=\"width: 627px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMil\u003c/strong\u003e: Miltefosine; Nanoparticles \u003cstrong\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003c/strong\u003e: % 50 inhibition concentration; \u003cstrong\u003eL929\u003c/strong\u003e: Fibroblast cell line; \u003cstrong\u003eS\u003c/strong\u003eI: Selectivity index\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eInteractions Between OA-MilAg-NP Complex, Miltefosine, and Benznidazole\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSynergistic interactions were observed for all incubation periods in the combinations of the OA-MilAg-NP complex and miltefosine with benznidazole (Table III).\u003c/p\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"640\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"bottom\" style=\"width: 640px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable III.\u0026nbsp;\u003c/strong\u003eInteraction of OA-MilAg-NP Complex and Miltefosine and benznidazole combinations against \u003cem\u003eT. cruzi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 153px;\"\u003e\n \u003cp\u003eCombinations\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 170px;\"\u003e\n \u003cp\u003e24h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 170px;\"\u003e\n \u003cp\u003e48h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 146px;\"\u003e\n \u003cp\u003e72h\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eFIC index\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003eInteraction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eFIC index\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003eInteraction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eFIC index\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003eInteraction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003eOA-MilAgNP+Benz\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003eSynergy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.374\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003eSynergy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.312\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003eSynergy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003eMil+Benz\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003eSynergy\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003eSynergy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003eSynergy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" style=\"width: 640px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAg\u003c/strong\u003e: Silver, \u003cstrong\u003eNP\u003c/strong\u003e: Nanoparticle, \u003cstrong\u003eBenz:\u003c/strong\u003e Benznidazole, \u003cstrong\u003eMil:\u003c/strong\u003e Miltefosine, \u003cstrong\u003eFIC\u003c/strong\u003e: Fractional inhibitory concentration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eChagas disease, caused by \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e, is considered a neglected tropical disease due to the lack of safe and effective treatments. Current treatment options are limited to nifurtimox and benznidazole, both of which were developed over 40 years ago [17]. While etiological treatment in the acute phase leads to high rates of parasite clearance and seronegative results, treatment success in the chronic phase remains debatable. Adverse events, particularly in adults, are common and often lead to discontinuation of treatment [18]. Pharmaceutical companies have shifted their focus to long-term use drugs rather than investing in costly R\u0026amp;D for new molecule development [19]. This situation underscores the need for strategies aimed at discovering new antimicrobial drugs.\u003c/p\u003e\n\u003cp\u003eDrug repurposing is the process of using an existing drug or drug candidate for a new therapeutic indication or medical condition not originally intended. In this process, the unintended side effects of drug molecules can serve as markers for discovering their effectiveness in entirely different medical conditions. Typically, drugs with proven safety in humans are tested and developed for efficacy in diseases other than the one for which they were originally designed. This process bypasses early drug development stages, thereby reducing risks and costs [20]. However, like traditional drugs, repurposed drugs face challenges related to formulation and delivery, such as poor aqueous solubility, limited stability, and/or incompatibility with new delivery routes. Nanotechnology plays a significant role in promoting the efficient use of highly active but poorly delivered drugs. It is frequently explored as a means to overcome challenges such as poor solubility, toxicity, and multidrug resistance in drug delivery systems [21].\u003c/p\u003e\n\u003cp\u003eMiltefosine is considered a protein kinase B (PKB) inhibitor, a key regulator of intracellular signaling essential for cell viability [22]. However, the mechanisms underlying its antiprotozoal activity remain incompletely understood. Some evidence suggests that it inhibits phosphatidylcholine and sphingomyelin synthesis, induces apoptosis [23], or inhibits mitochondrial cytochrome c oxidase and alters the organelle membrane [22]. Similarly, it has been proposed that miltefosine inhibits parasite phosphatidylcholine biosynthesis through the transmethylation pathway [24]. Recently, it has been suggested that miltefosine targets a sphingosine-activated plasma membrane Ca\u0026sup2;⁺ channel in both \u003cem\u003eLeishmania donovani\u003c/em\u003e and \u003cem\u003eT. cruzi\u003c/em\u003e, affecting intracellular calcium homeostasis [25].\u003c/p\u003e\n\u003cp\u003eSeveral studies in the literature demonstrate the efficacy of miltefosine against \u003cem\u003eT. cruzi\u003c/em\u003e in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [17,26,27]. A recent study by Gulin et al. (2022) reported IC\u003csub\u003e50\u003c/sub\u003e values of miltefosine against \u003cem\u003eT. cruzi\u003c/em\u003e amastigote and trypomastigote forms as 0.48 \u0026micro;M and 0.55 \u0026micro;M, respectively, and LC\u003csub\u003e50\u003c/sub\u003e (lethal concentration) values as 29.56 \u0026micro;M and 32.87 \u0026micro;M, respectively. They also found that the combination of miltefosine and benznidazole exhibited synergistic and additive interactions against trypomastigotes and amastigotes, respectively. Additionally, they reported that the combination was more effective than miltefosine alone in reducing parasitemia in an \u003cem\u003ein vivo\u003c/em\u003e rodent model.\u003c/p\u003e\n\u003cp\u003eIn our study, unlike those in the literature, we synthesized a hybrid oxidized amylose miltefosine silver nanoparticle (OA-MilAg-NP) complex by combining miltefosine with silver and compared its efficacy against \u003cem\u003eT. cruzi\u003c/em\u003e with that of miltefosine and benznidazole. We also investigated the \u003cem\u003ein vitro\u003c/em\u003e interactions of both miltefosine and the OA-MilAg-NP complex with benznidazole. In our previous study, we demonstrated that a natural active component (curcumin) and silver NP complex exhibited strong antileishmanial activity with low cytotoxicity against \u003cem\u003eLeishmania\u003c/em\u003e species [7]. Based on this, we aimed to enhance the efficacy of miltefosine while reducing its toxicity against \u003cem\u003eT. cruzi\u003c/em\u003e, a parasite closely related to \u003cem\u003eLeishmania\u003c/em\u003e species, using nanoparticle formulations and to identify synergistic interactions in combination with benznidazole.\u003c/p\u003e\n\u003cp\u003eThe synthesized miltefosine-silver NP complex was prepared using a method involving oxidized amylose as the core and miltefosine and silver ions at the nanoparticle level on the surface, as in our previous study [7,9,10]. FTIR spectra of the OA-MilAg-NP complex revealed peak shifts and splitting, indicating the successful attachment of functional groups to the oxidized amylose surface. SEM and TEM imaging showed that the OA-MilAg-NP complex had a round morphology, variable sizes, and a size range of 10.14\u0026ndash;18.42 nm.\u003c/p\u003e\n\u003cp\u003eA recent study by Latifi et al. (2024) synthesized miltefosine as chitosan nanoparticles with an average size of 46.61 nm and reported that the formulation was more effective against Acanthamoeba trophozoites and cysts than miltefosine alone. The same study also indicated that the NP formulation exhibited significantly lower cytotoxicity on Vero cell lines compared to miltefosine [28].\u003c/p\u003e\n\u003cp\u003eIn our study, we compared the toxicity of the OA-MilAg-NP complex to L929 mouse fibroblasts with that of miltefosine and benznidazole and calculated the selectivity index (SI) for each compound. Benznidazole, as a conventional drug, exhibited the highest SI values during 24, 48, and 72-hour incubation periods. Notably, the OA-MilAg-NP complex showed at least twice the selectivity of miltefosine alone based on SI values (Table II). The SI values suggest that our synthesized NP complex is suitable for further \u003cem\u003ein vivo\u003c/em\u003e studies (SI\u0026gt;3).\u003c/p\u003e\n\u003cp\u003eA study investigating the efficacy of miltefosine formulated with lipid NPs against cutaneous leishmaniasis reported that lipid NPs had an average size of 160.8 nm, six times lower cytotoxicity against macrophage cell lines, and strong antileishmanial activity \u003cem\u003ein vivo\u003c/em\u003e [29]. We believe that the much smaller NP size (10.14\u0026ndash;18.42 nm) achieved with our synthesis method compared to other studies [30] may be a significant factor in enhancing antimicrobial activity.\u003c/p\u003e\n\u003cp\u003eThere are limited studies in the literature on the combination of benznidazole and miltefosine against \u003cem\u003eT. cruzi.\u003c/em\u003e Gulin et al. (2022) noted that the synergistic interaction of benznidazole and miltefosine observed in vitro was also effective \u003cem\u003ein vivo\u003c/em\u003e. In our study, a synergistic interaction was observed between miltefosine and benznidazole. Furthermore, unlike the literature, our study demonstrated a synergistic interaction between the OA-MilAg-NP complex and benznidazole. The data we obtained represent the first evidence demonstrating the hybrid synthesis of the OA-MilAg-NP complex based on oxidized amylose and its efficacy against \u003cem\u003eT. cruzi.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe data obtained on the efficacy of our oxidized amylose-based OA-MilAg-NP complex against \u003cem\u003eT. cruzi\u003c/em\u003e and its interaction with benznidazole represent a novel contribution to the literature and provide valuable insights for the treatment of Chagas disease.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe most significant limitation of this study is that the antitrypanosomal activity and interactions of the hybrid NP complex and miltefosine with benznidazole, which exhibited acceptable SI values, were not evaluated in experimental animal models.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eInvestigating and developing new bioactive compounds with lower toxicity compared to conventional drugs for Chagas disease treatment through nanotechnology applications can provide significant contributions to therapeutic efficacy in the literature. The hybridization of miltefosine with silver nanoparticles, which has demonstrated efficacy against leishmaniasis, and the strong antitrypanosomal activities of these novel molecules along with their synergistic interactions with existing antitrypanosomal drugs, can address significant gaps in the literature. These findings pave the way for designing new drug combinations, reducing side effects, and preventing resistance development, offering valuable insights for future studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eConflict Of Interest\u003c/strong\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.O, IC and AO wrote the main manuscript, made the analyses and prepare figures and tables. FT and SB synthesized nanoparticles and performed FTIR characterization. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank the Parasite Bank of Manisa Celal Bayar University Faculty of Medicine for providing the parasite strain and cell line used in this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWHO. Chagas disease (also known as American trypanosomiasisAvailable online: https://www.who.int/news-room/fact-sheets/detail/chagas-disease-(american-trypanosomiasis(Erişim Tarihi: 29.12.2024).\u003c/li\u003e\n\u003cli\u003eRuh E, Taylan \u0026Ouml;zkan A (2023Outbreaks Due to Parasites: Examples from the World and T\u0026uuml;rkiye. Mikrobiyol bul 57:317\u0026ndash;329. https://doi.org/10.5578/mb.20239926\u003c/li\u003e\n\u003cli\u003eHochberg NS, Montgomery SP (2023Chagas Disease. 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(2021Identification of \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e growth inhibitors with activity in vivo within a collection of licensed drugs. Microorganisms 9:406. https://doi.org/10.3390/microorganisms9020406\u003c/li\u003e\n\u003cli\u003eLatifi A, Esmaeili F, Mohebali M, Yasami-Khiabani S, Rezaeian M, Soleimani M, et al. (2024Chitosan nanoparticles improve the effectivity of miltefosine against Acanthamoeba. PLoS Negl Trop Dis 18:e0011976. https://doi.org/10.1371/journal.pntd.0011976\u003c/li\u003e\n\u003cli\u003eKhan AS, Ud Din F, Ali Z, Bibi M, Zahid F, Zeb A, Mujeeb-Ur-Rehman, Khan GM (2021Development, in vitro and in vivo evaluation of miltefosine loaded nanostructured lipid carriers for the treatment of Cutaneous Leishmaniasis. Inter J Pharm 593:120109. https://doi.org/10.1016/j.ijpharm.2020.120109\u003c/li\u003e\n\u003cli\u003ePaiva DF, Matos APDS, Gar\u0026oacute;falo DA, do Nascimento T, Monteiro MSSB, Santos-Oliveira R, Ricci-Junior E (2023Use of Nanocarriers Containing Antitrypanosomal Drugs for the Treatment of Chagas Disease. Pharmaceuticals (Basel16:1163. https://doi.org/10.3390/ph16081163\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"acta-parasitologica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"actp","sideBox":"Learn more about [Acta Parasitologica](http://link.springer.com/journal/11686)","snPcode":"11686","submissionUrl":"https://submission.springernature.com/new-submission/11686/3","title":"Acta Parasitologica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Silver nanoparticles, Miltefosine, Synergy, T. cruzi","lastPublishedDoi":"10.21203/rs.3.rs-6294121/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6294121/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e: Chagas disease is an infectious disease classified under neglected tropical diseases and caused by the protozoan parasite \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e. This study aimed to investigate the cytotoxic activity, antitrypanosomal efficacy, and combination effects with benznidazole of hybrid silver nanoparticles (AgNPs) synthesized with miltefosine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: In this study, a hybrid miltefosine (Mil)-silver nanoparticle (OA-MilAgNP) complex was synthesized. The nanoparticles were characterized using FT-IR spectroscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) analyses. The cytotoxicity of the nanoparticles was assessed in L929 fibroblast cells, while their antitrypanosomal activity was evaluated against a \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e strain using the liquid microdilution method. The interaction between the nanoparticle complex or miltefosine and benznidazole was analyzed using the checkerboard method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: FT-IR analysis demonstrated that the amylose surface was successfully coated with silver and miltefosine, confirming the successful synthesis of the hybrid complex. SEM analysis revealed that the nanoparticles exhibited a spherical morphology with varying sizes, while TEM analysis determined their sizes ranged between 10.14 and 18.42 nm. The OA-MilAgNP complex exhibited high antitrypanosomal activity and a selectivity index twice as high as that of miltefosine. Synergistic interactions were observed in the combinations of the OA-MilAgNP complex or miltefosine with benznidazole.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: The development of novel bioactive compounds with lower toxicity compared to traditional drugs has become essential for the treatment of Chagas disease. Drug repurposing combined with nanotechnology applications holds significant potential for improving therapeutic outcomes. The hybridization of miltefosine with silver nanoparticles, demonstrating strong antitrypanosomal activity and synergistic effects with benznidazole, may fill critical gaps in the literature.\u003c/p\u003e","manuscriptTitle":"Synthesis and Evaluation of a Hybrid Miltefosine-Silver Nanoparticle Complex: Synergistic Interaction with Benznidazole Against Trypanosoma cruzi","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-23 08:09:30","doi":"10.21203/rs.3.rs-6294121/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-20T08:57:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-19T03:37:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182323568079093824343334598843269902132","date":"2025-05-19T02:59:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-26T22:30:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"115307917093669352214616926322815404218","date":"2025-04-04T11:56:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22719499873786758575779318804901584118","date":"2025-04-02T22:22:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160335080051494713619371111699677039238","date":"2025-04-02T11:57:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-02T11:11:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-24T15:50:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-24T15:46:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Parasitologica","date":"2025-03-24T09:58:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-parasitologica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"actp","sideBox":"Learn more about [Acta Parasitologica](http://link.springer.com/journal/11686)","snPcode":"11686","submissionUrl":"https://submission.springernature.com/new-submission/11686/3","title":"Acta Parasitologica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f357eaa4-104f-43f9-b413-a2df87cfd2c9","owner":[],"postedDate":"April 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-16T16:02:20+00:00","versionOfRecord":{"articleIdentity":"rs-6294121","link":"https://doi.org/10.1007/s11686-025-01074-3","journal":{"identity":"acta-parasitologica","isVorOnly":false,"title":"Acta Parasitologica"},"publishedOn":"2025-06-12 15:57:53","publishedOnDateReadable":"June 12th, 2025"},"versionCreatedAt":"2025-04-23 08:09:30","video":"","vorDoi":"10.1007/s11686-025-01074-3","vorDoiUrl":"https://doi.org/10.1007/s11686-025-01074-3","workflowStages":[]},"version":"v1","identity":"rs-6294121","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6294121","identity":"rs-6294121","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}