Preparation and evaluation of curcumin nanoemulsion to inhibit TC-1 cell growth

Preparation and evaluation of curcumin nanoemulsion to inhibit TC-1 cell growth | 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 Preparation and evaluation of curcumin nanoemulsion to inhibit TC-1 cell growth Mehrnaz Karimi, Mahnaz Qomi, Mahsa Hadipour Jahromy, Masoud Parsania, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3859423/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Curcumin (Cur), a substance originating from Curcuma longa, has been comprehensively examined for its anticancer properties. Nonetheless, its clinical application has been restricted by its inadequate solubility, bioavailability, and stability. TC-1 cells have been impressive in understanding HPV biology and developing therapeutic approaches for HPV infection and related cancers, like cervical cancer, offering a close mimicry of HPV-induced carcinogenesis. This study's primary goal is to formulate and optimize curcumin nanoemulsions (Cur-NE) to address these challenges and, secondarily, evaluate their impact on TC-1 cell growth. Characterization of the nanoemulsions was conducted using dynamic light scattering (DLS), transmission electron microscopy (TEM) and High-performance liquid chromatography (HPLC) revealing an average particle size of 52.5 nm, a zeta potential of -13.1 mV, and a drug content of 94.6%. Through the dialysis diffusion technique, drug release profiles demonstrated a sustained, slower release of Cur from Cur-NE compared to free curcumin. According to an MTT assay, Cur-Ne with an IC50 35 µg/ml exhibited an increased inhibitory effect of Cur on TC-1 cancer cells, while showing no inhibitory effects on MC3T3 normal cells at concentrations up to 100 µg/ml. In summary, this study underscores the potential of nanoemulsions as efficient carriers for Cur, with demonstrated safety in both cancer and normal cells. Moreover, Cur-NE displayed substantial inhibitory activity against TC-1 cancer cells, suggesting its promise in treating HPV-associated cancers, particularly cervical cancer. Further research is warranted to evaluate the long-term safety of this nanoemulsion for clinical trials and its efficacy against other cancer cell lines. Curcumin Cytotoxicity Nanoemulsion TC-1 cell HPV Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction According to studies, cervical cancer is a prevalent cancer among women globally. This disease leads to the death of more than 300,000 individuals annually, particularly in developing nations ( 1 , 2 ). Numerous studies conducted on human and animal samples have determined that the principal factor responsible for cervical cancer is the presence of the human papillomavirus (HPV) ( 3 ). Infection with HPV, particularly high-risk strains such as HPV 16, 18, and 14, has been identified as a significant early occurrence in developing cervical cancer. HPV infects nearly all patients with invasive cervical cancer ( 4 ). E6 and E7 oncoproteins are known as the central viral carcinogenic genes of HPV that are responsible for cell transformation ( 5 , 6 ). TC-1 cells are a murine cancer cell line generated by transfecting C57BL/6 mouse lung cells with the HPV-16 E6/E7 oncogene ( 7 ). The importance of TC-1 cell lines lies in their ability to recapitulate many aspects of HPV and related pathologies, making them a valuable model system for studying HPV-related cancers ( 8 ). Curcumin, a polyphenolic compound obtained from the Curcuma longa plant, traditionally has been utilized in Chinese and Southeast Asian countries for its medicinal properties ( 9 ). Curcumin has been the subject of extensive research for over five decades due to its diverse pharmacological characteristics, such as anti-inflammatory, antioxidant, antibacterial, anticancer, antiviral, and antitumor effects. ( 10 ). Nevertheless, the therapeutic effectiveness of curcumin has been hindered by challenges such as low solubility, limited bioavailability, and rapid metabolism ( 11 ). To enhance the bioavailability of curcumin, researchers have investigated various methods to develop appropriate formulations ( 12 ). In the past years, researchers have shown interest in utilizing nanotechnology to enhance the effectiveness of curcumin. Currently, nano-delivery systems are commonly employed to enhance drug dissolution and facilitate the transport of hydrophobic drugs to target tissues ( 13 ). Encapsulating curcumin in nanoscale carrier systems such as nanoemulsion significantly enhances the efficiency of curcumin, including its bioavailability and solubility ( 14 ). The primary classifications of oil-in-water (O/W) and water-in-oil (W/O) nanoemulsions consist of two immiscible liquids with a surfactant for stabilization. Their average droplet diameter is usually less than 500 nm ( 15 – 17 ). The ingredients of the nanoemulsion system include oil, surfactant/co-surfactant, water-soluble co-solvent, and water ( 18 , 19 ). The present study aimed to develop an optimized Cur-NE formulation and conduct toxicity and inhibitory assessments of the developed formulation in the TC-1 cell line. To date, there has been no investigation into the impact of curcumin encapsulated in a nanoemulsion formulation on the TC-1 cell line. Materials and Methods 2.1. Materials Curcumin 95%, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, DMSO (dimethyl sulfoxide) were purchased from Sigma company. Castor oil, tocopherol acetate, tween 80, and PEG400 were provided by Merk Company. DMEM and fetal bovine serum were obtained from Gibco company. 2.2. Preparation To prepare Cur-NE, the method described by Heni Rachmawati and colleagues was used with some modifications ( 20 ). An oil phase consisting of different weight ratios of oil (w/w 20%-10%), surfactant (w/w 10%-90%), and co-surfactant (w/w 30%-0%) was prepared by mixing castor oil, tocopheryl acetate, a surfactant component containing Tween 80, and a co-surfactant part including polyethylene glycol 400. This way, curcumin (10-60mg) was added to the oil phase. The prepared oil phase was mixed at 50 ºC on a stirrer at 500 rpm for 15 minutes. Then, water was added to the obtained mixture in a ratio of 5 to 1. After that, the obtained mixture was sonicated by a probe sonicator for 15 minutes. 2.3. Characterization The morphological characteristics of the Cur-NE specimen were examined using a Zeiss transmission electron microscope (model EM10C, manufactured in Germany), which operated at an acceleration voltage of 100 kilovolts (kV). The attenuate aqueous solution of the specimen was sonicated using an ultrasonic homogenizer (Misonix- S3000, United States) for a duration of 15 minutes. Subsequently, a 20-microliter aliquot of the sample was dispensed onto a formvar-coated carbon film positioned on a copper grid with a 300-mesh structure (EMS-USA) and allowed to desiccate completely at ambient room temperature. The size and zeta potential of prepared nanoemulsions were assessed via dynamic light scattering (DLS) utilizing a Malvern Nano Zetasizer (Malvern Instruments, UK). Before analysis, samples were diluted with deionized distilled water. In order to examine the stability of nanoemulsions, the samples were evaluated during 60 days at room temperature 25 ֯C by microscopic monitoring and DLS in terms of the formation of precipitation and droplet size. 2.4. Cur release To study the release behavior of Cur from Cur-NE, the dialysis method was employed. For this test, 1 ml of Cur-NE or free Cur solution was introduced into a dialysis bag (MW 12 kD) and floated separately into beakers containing 100 ml of pH 7.4 phosphate-buffered saline with stirring at 37°C. At each selected time span (0.5, 1, 2, 4, 6, 8, 24, 48, and 72 h), 1 ml of test solution was removed and substituted with 1 ml of new phosphate buffer. Then, a high-performance liquid chromatography (HPLC, Younglin Acme 9000, South Korea) instrument quantified the concentration of Cur in the samples. The employed HPLC parameters were as follows: a mobile phase composition consisting of 52 (0.1% phosphoric acid):48 (acetonitrile), with a flow rate set at 1 mL/min. The detector was configured to a wavelength of 424 nm, and each analysis run involved an injection volume of 20 ml. 2.5. Cell lines The TC-1 (obtained from the primary lung epithelial cells sourced from C57BL/6 mice.) and MC3T3 (The osteoblastic cell from a C57BL/6 mouse calvaria) Cell lines were acquired from the Pasteur Institute in Iran. The cells were cultured in DMEM (Gibco) culture medium containing 10% fetal bovine serum (Gibco) and maintained at 5% Co2 and 37°C 2.6. MTT assay MTT assay evaluated the impacts of Cur-NE, Cur, and nanoemulsion on TC-1 (cancer cells) and MC3T3 cells (normal cells) viability. Initially, the cell number was quantified using a hemocytometer with trypan blue staining, and subsequently, 15×10 3 cells were seeded in each well of the 96-well microplate. The cells were cultured for a duration of 24 hours, followed by subsequent treatment with Cur-NE (10 µg/ml − 100µg/ml), curcumin (10µg/ml -100µg/ml), and nanoemulsions (10 µg/ml -900 µg/ml) individually in three wells were incubated in a controlled condition with 5% CO2 at 37°C for 48 hours. Three wells of 96-well-microplate were considered the control containing only DMEM medium. After 48 h, the medium of each well was supplanted with 80 µl of DMEM and A volume of 20 µl of MTT solution at a concentration of 0.5 mg ml − 1 , and the incubation period lasted for 4 hours at 37°C under conditions of 5% CO2. By using 100µl of dimethyl sulfoxide (DMSO), formazan was dissolved, and plate Reader (BioTek Company, USA) was utilized to measure the optical density (OD) of tests at 540nm. Three wells were considered blank, containing only 100 µl DMSO. The calculation of cytotoxicity percentage was performed using the following formula: Cytotoxicity% = 1 - (Mean ODtest - Mean OD blank / Mean OD control - Mean OD blank)×100 The inhibitory concentration (IC50) is the amount at which 50% of cellular viability is destroyed. Results 3.1. Screening of Cur-NE formulation and characterizations Different mass ratios of oil, curcumin, surfactant, and co-surfactant were used to optimize stable formulations. Based on particle size and zeta potential, the most optimized formulation consisted of 55 mg curcumin dissolved in a mixture of oil phases, including castor oil and tocopheryl acetate (10% w/w), tween 80 (70% w/w) and PEG 400 (20% w/w) as the oil, surfactant, and co-surfactant respectively. According to the TEM result (Fig. 1A, B), spherical particles having a mean size of 42 nm showed that the nanoparticles have a uniform distribution and morphology. In addition, the absence of curcumin precipitation in the TEM image indicates the stability of the formed nanoemulsions. Based on DLS results (Fig. 1C), Cur-NEs exhibited a mean size of 52.5 nm, with a polydisperse index (PDI) of 0.03 and a zeta potential of − 13.1 mV. The larger size of DLS results compared to TEM is due to the hydrodynamic radius. Based on the zeta potential, the sample has good stability. Figure 1 . A) TEM image, B) particle size distribution of Cur-NE, and (C) DLS of Cur-NE. Visual assessments confirmed an acceptable stability of Cur-NEs, as evidenced by the absence of precipitation and phase separation over at least 60 days. As illustrated in Fig. 2, no remarkable alteration in the size of Cur-NEs was determined by DLS. Figure 2 . Size variations of Cur-NE in 60 days carried out by DLS 3.2. Analysis of drug content The Cur content in the optimized Cur-NE system was determined by HPLC chromatography ( 21 ). Based on the result, the percentage drug (Cur) content was 96.3 for optimized Cur-NE. 3.3. Cur Release The Cur release characteristics of Cur-NE were evaluated by a dialysis method, and the release patterns of free Curcumin and Cur-NE were shown in Fig. 3. Free Cur exhibited an extremely rapid release pattern, whereas Cur-NE demonstrated a considerably slower and sustained release. Free Cur was 100% released at 8 hours, while Cur-NE was 94.8% at 48 hours. Figure 3 . Cur release profile. Cur release data from the optimized Cur-NE formulation were fitted to different release models, including Hill, Weibull, Korsmeyer-Peppas, and third-order (Table 1 ). The best model for the in vitro release of Cur from Cur-NE should have the highest R 2 . Accordingly, the highest value of the correlation coefficient is related to Hill (R 2 = 0.988), followed by Weibull (R 2 = 0.966), Korsmeyer-Peppas (R 2 = 0.90), and third-order. Table 1 The release models fitted to the Cur release results. Relese Model Parameter Formulation Cur-NE Third-order R 2 0.948 Hill R 2 0.988 Weibull R 2 0.966 Korsmeyer-Peppas R 2 0.90 3.4. Cellular cytotoxicity The cytotoxic impacts of Cur-NE, Cur, and nanoemulsion treatments at varying concentrations on the viability of the TC-1 cell line are illustrated in Fig. 4. According to this figure, in the TC-1 cancer cell line, enhancing concentrations of Cur-NE ranging from 10 to 100 µg/ml in the culture medium substantially decreased cell viability after 48 hours. The IC50 measurement of Cur-NE on the TC-1 cell line after a 48-hour exposure was almost 35µg/ml (Fig. 4A). Notably, the inhibitory effect of Cur-NE is significantly less compared to Cur. As shown in Fig. 4B, the IC50 value for TC-1 cells subjected to a 48-hour Curcumin treatment increased to approximately 55µg/ml. Figure 4. Cytotoxicity assay results of TC-1 cells treated with different concentration of (A) curcumin nanoemulsion, (B) Curcumin and (C) Nanoemulsion for 48 h. Average viability percent of three independent experiments are shown in the graph. Error bars indicate standard deviations. The nanoemulsion does not exhibit any inhibitory impact on the growth of TC-1 cells. In this research, TC-1 cells were treated with nanoemulsion concentrations reaching up to 900µg/ml. However, there were no cytotoxic effects of more than 50% in all concentrations (Fig. 4C). Interestingly, Cur-NE does not exert a significant influence on the proliferation of normal MC3T3 cells at concentrations that are cytotoxic to TC-1 cells. (Fig. 5A). At none of the concentrations of Cur-NE (10–100µg/ml), cell survival reached 50%. Moreover, pure Cur and nanoemulsion did not cause a substantial inhibitory effect on the growth of normal cells (Fig. 5B, C). Figure 5 . Cytotoxicity assay results of MC3T3 cells treated with different concentration of A) curcumin nanoemulsion, B) Curcumin and C) Nanoemulsion for 48 h. Average viability percent of three independent experiments are shown in the graph. Error bars indicate standard deviations. Discussion Here, the standards for designing stable Cur-NEs were as follows: the particle size of the emulsion had to be as small as possible (˂100 nm) and a high electrical charge (zeta potential > ± 30 mV). Considering these criteria, various mass ratios of core (oil and curcumin) and coating materials (surfactant and co-surfactant) were employed to optimize the stable formulations. To ensure that as much Cur as possible was incorporated into the oil phase, a 9:1 ratio of castor oil and tocopheryl acetate was selected. Tocopheryl acetate, in fact as a highly effective solvent, exhibited strong compatibility with the oil and significantly improved the solubility of Cur ( 22 , 23 ). Tween 80 and PEG400 were chosen to emulsify fats. As Previous studies showed here, the combination of tween 80 and PEG400 could produce a transparent emulsion ( 24 , 25 ). This combination was able to generate a stable nanoemulsion. The zeta potential indicates the surface charge and the stability of Cur-NE, the higher repulsive force, the lower aggregate of the emulsion droplets ( 26 , 27 ). Thus, the optimized Cur-NE has the highest value of zeta potential (-13.8 mV), which is related to the surfactants, tween80 and PEG400, and Castrol oil and tocopheryl acetate. Producing small zeta potential through the use of non-ionic surfactants results in the reduction of particle sizes. The solubility of Cur increased up to 5.5mg/ml after incorporation into NE, which is higher than previous reports ( 28 ). Not changing the appearance and size of the NE not only confirms the stability of the formulation in a certain period of time but also its possible stability for a long time ( 29 ). Here, visual assessment and DLS analysis performed over 60 days emphasized stability of the optimized Cur-NE. The in vitro drug release profile exhibited a two-phase pattern, marked by an initial burst release followed by a gradual and continuous release. Various factors, such as lipophilicity, drug-surfactant interactions, interfacial barrier rigidity, and diffusion from these barriers (e.g., dialysis bags), can influence the release of drugs from emulsions during in vitro studies ( 22 , 30 ). The initial rapid release could be attributed to the permeation of Cur on the external surface of Cur-NE. Subsequently, the release of drugs from these systems decreases over time, resulting in a sustained release profile. The responsible for this release manner is the lipid phase incorporating the drug during the formation of NE ( 31 , 32 ). Our findings align with the previous studies by Yang et al.( 33 ) and Sari et al ( 34 ). These in vitro release results suggest that the developed nanoemulsion effectively retains Cur and remains stable ( 35 ). According to our cytotoxicity assay in TC-1 cancer cells, both free Cur and Cur-NE revealed cytotoxic functionality within TC-1 cells in a manner that depends on the dosage. Accordingly, the IC50 values for free Cur and Cur-NE were 55 µg/ml and 35 µg/ml, respectively. It is worth noticing the inhibitory concentration of Cur-NE is less than Cur, indicating the enhanced bioavailability and cellular uptake of curcumin when incorporated in NE ( 36 ). The result aligns with findings from earlier research that showed the IC50 of nanocurcumin is less than the IC50 of Cur, which indicates that nanocurcumin has better antioxidant activity ( 37 ). Cheragh and colleagues reported the cytotoxicity of dendrosomal nano-curcumin in Burkitt's lymphoma Daudi cell line with the IC50 30 µg/ml ( 38 ). In another study, Yan-bin GUAN S-yZ and colleagues showed curcumin nanoemulsions could inhibit the proliferation of prostate cells both in vitro and in vivo in a dose and time dependent manner ( 39 ). Treatment of Both TC-1 cancer cells and normal MC3T3 cells with nanoemulsion had no significant effect on cellular viability, indicating this nanocarrier is safe. Moreover, the result of the MTT assessment declared that Cur-NE did not have a significant impact on the viability of normal MC3T3 cells in a manner depending on the dosage. The results of this study align with previous research. The survey carried out by Javidi and colleagues showed that although nanocurcumin could inhibit cancerous cell growth in a depending on both time and dosage manner, it had no notable effects on IPCs normal cells. ( 40 ). Additionally, in vitro investigations conducted by Simón that CUR-NE exhibits safety when tested on non-cancerous human cells (HEK-293T), and displays selective cytotoxicity towards gastric (AGS), colon (HT29-ATCC, HT29-US), breast (MDA-MB-231), and melanoma (B16F10) cells ( 41 ). Conclusion In this study, a stable Cur-NE formulation was successfully prepared and optimized. The composition of the optimal Cur-NE includes castor oil, tocopherol acetate, Tween 80, and PEG 400 (9:1:70:20) emulsified in water to form nanoemulsion. The findings of the cytotoxicity test offer compelling proof that Cur-NE is a highly efficient nano-delivery system that enhances the bioavailability of curcumin. Additionally, our study demonstrates the lack of toxicity of the nanoemulsion formulation in vitro, highlighting its potential as a carrier for hydrophobic drugs. Further research is required to examine the long-term safety of the nanoemulsion for its clinical testing and its cytotoxicity effects in other cancer cells. Declarations Acknowledgments We would like to thank the staff of the medical Genomics Research Center and the Nano-Pharmaceutics Laboratory of the Faculty of Medicine, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran, for their helpful assistance with this study and for providing necessary research facilities to carry out this study, respectively. The authors declare no competing interests. References De Sanjosé S DM, Castellsagué X, Clifford G, Bruni L, Muñoz N, et al. Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: a meta-analysis. The Lancet infectious diseases. 2007;7(7):453-9. Rayner M, Welp A, Stoler MH, Cantrell LA, editors. Cervical Cancer Screening Recommendations: Now and for the Future. Healthcare; 2023: MDPI. Chibwesha CJ SJ. Cervical Cancer as a Global Concern: Contributions of the Dual Epidemics of HPV and HIV. Jama. 2019;322(16):1558-60. Li P, Tan Y, Zhu LX, Zhou LN, Zeng P, Liu Q, et al. 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Investigating curcumin potential for diabetes cell therapy, in vitro and in vivo study. Life Sciences. 2019;239(15). Simón Guerrero MI-R, Pamela Contreras-Orellana, Victor Diaz-Garcia, Pablo Lara, Andrea Vivanco-Palma, Areli Cárdenas, Victor Miranda, Paz Robert,e Lisette Leyton, Marcelo J. Kogan, Andrew F. G. Quest and Felipe Oyarzun-Ampuero. Curcumin-loaded nanoemulsion: a new safe and effective formulation to prevent tumor reincidence and metastasis. Nanoscale. 2018;10:22612–22. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3859423","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267705266,"identity":"387759bf-b39f-4530-8866-29cbef5304de","order_by":0,"name":"Mehrnaz Karimi","email":"","orcid":"","institution":"Tehran Medical Sciences, Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Mehrnaz","middleName":"","lastName":"Karimi","suffix":""},{"id":267705267,"identity":"50379c21-18c2-4368-836d-62cb71856b70","order_by":1,"name":"Mahnaz Qomi","email":"","orcid":"","institution":"Tehran Medical Sciences, Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Mahnaz","middleName":"","lastName":"Qomi","suffix":""},{"id":267705268,"identity":"28a4efcb-ea2c-4e38-b0ef-8f5c41f2bc68","order_by":2,"name":"Mahsa Hadipour Jahromy","email":"","orcid":"","institution":"Tehran Medical Sciences, Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Mahsa","middleName":"Hadipour","lastName":"Jahromy","suffix":""},{"id":267705269,"identity":"ca9bd798-6271-4682-8faa-32ff92cc1d56","order_by":3,"name":"Masoud Parsania","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYBADHgb2xgYEN4EoLTwHGxBKidHCwCCRQKRS3dnNzz7+qLkjwz/zceNn3h/bEhvYDz9geLgHtxazO8eMZ/Mce8YjcTuxWZon4XZiA0+aAUPCMzxabiQYMzOwHeZhACqGaGHIATrwAD4t6Z8Zf/w7zCN/82Dzb7AW/jeEtOQYM/C2HeYxuMHYBrFFgpAtd84UM/P2HeYxPJPYZjkn7bZxm8QzgwN4tdxu38z449the7njxx/feGNzW7afP/nhwx94tDBIoAuwATE+DVi0jIJRMApGwShABwAkflYmS1wMnAAAAABJRU5ErkJggg==","orcid":"","institution":"Tehran Medical Sciences, Islamic Azad University","correspondingAuthor":true,"prefix":"","firstName":"Masoud","middleName":"","lastName":"Parsania","suffix":""},{"id":267705270,"identity":"f22146c0-8ba8-4381-8319-a6551bea5f80","order_by":4,"name":"Negar Motakef Kazemi","email":"","orcid":"","institution":"Tehran Medical Sciences, Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Negar","middleName":"Motakef","lastName":"Kazemi","suffix":""}],"badges":[],"createdAt":"2024-01-13 07:59:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3859423/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3859423/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49822496,"identity":"ff628b44-b259-4fd3-a526-2f468fa576d8","added_by":"auto","created_at":"2024-01-18 15:21:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":289491,"visible":true,"origin":"","legend":"\u003cp\u003eA) TEM image, B) particle size distribution of Cur-NE, and (C) DLS of Cur-NE.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3859423/v1/29a4e29ff7cdb6f7dab97a3e.png"},{"id":49823007,"identity":"0c8860d0-0bdc-4376-b765-d12592548bdc","added_by":"auto","created_at":"2024-01-18 15:29:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":23731,"visible":true,"origin":"","legend":"\u003cp\u003eSize variations of Cur-NE in 60 days carried out by DLS\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3859423/v1/37ef6389ccdf2b24cf6844a5.png"},{"id":49822500,"identity":"53f5e8a6-45d5-4301-98e9-3880a4938212","added_by":"auto","created_at":"2024-01-18 15:21:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":52523,"visible":true,"origin":"","legend":"\u003cp\u003eCur release profile.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3859423/v1/c8a3629a1dfd2adcd2b1ec3a.png"},{"id":49822501,"identity":"aea1bcd4-b1ba-44a7-8ed7-bfb10e065a3c","added_by":"auto","created_at":"2024-01-18 15:21:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":284247,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity assay results of TC-1 cells treated with different concentration of (A) curcumin nanoemulsion, (B) Curcumin and (C) Nanoemulsion for 48 h. Average viability percent of three independent experiments are shown in the graph. Error bars indicate standard deviations.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3859423/v1/ef5f99325e6c2f100d88595b.png"},{"id":49822498,"identity":"d236da91-6039-43ba-a487-9fa59adafd63","added_by":"auto","created_at":"2024-01-18 15:21:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":282262,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity assay results of MC3T3 cells treated with different concentration of A) curcumin nanoemulsion, B) Curcumin and C) Nanoemulsion for 48 h. Average viability percent of three independent experiments are shown in the graph. Error bars indicate standard deviations.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3859423/v1/7c2812999057549d23d19203.png"},{"id":49882654,"identity":"9fb90ad8-5eab-4dfe-b662-956e46b358eb","added_by":"auto","created_at":"2024-01-19 16:40:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":798293,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3859423/v1/90baab5f-eeb8-42ec-9c90-5eac5ea6e18c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preparation and evaluation of curcumin nanoemulsion to inhibit TC-1 cell growth","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAccording to studies, cervical cancer is a prevalent cancer among women globally. This disease leads to the death of more than 300,000 individuals annually, particularly in developing nations (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Numerous studies conducted on human and animal samples have determined that the principal factor responsible for cervical cancer is the presence of the human papillomavirus (HPV) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Infection with HPV, particularly high-risk strains such as HPV 16, 18, and 14, has been identified as a significant early occurrence in developing cervical cancer. HPV infects nearly all patients with invasive cervical cancer (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). E6 and E7 oncoproteins are known as the central viral carcinogenic genes of HPV that are responsible for cell transformation (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). TC-1 cells are a murine cancer cell line generated by transfecting C57BL/6 mouse lung cells with the HPV-16 E6/E7 oncogene (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The importance of TC-1 cell lines lies in their ability to recapitulate many aspects of HPV and related pathologies, making them a valuable model system for studying HPV-related cancers (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurcumin, a polyphenolic compound obtained from the Curcuma longa plant, traditionally has been utilized in Chinese and Southeast Asian countries for its medicinal properties (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Curcumin has been the subject of extensive research for over five decades due to its diverse pharmacological characteristics, such as anti-inflammatory, antioxidant, antibacterial, anticancer, antiviral, and antitumor effects. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Nevertheless, the therapeutic effectiveness of curcumin has been hindered by challenges such as low solubility, limited bioavailability, and rapid metabolism (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). To enhance the bioavailability of curcumin, researchers have investigated various methods to develop appropriate formulations (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In the past years, researchers have shown interest in utilizing nanotechnology to enhance the effectiveness of curcumin. Currently, nano-delivery systems are commonly employed to enhance drug dissolution and facilitate the transport of hydrophobic drugs to target tissues (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Encapsulating curcumin in nanoscale carrier systems such as nanoemulsion significantly enhances the efficiency of curcumin, including its bioavailability and solubility (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The primary classifications of oil-in-water (O/W) and water-in-oil (W/O) nanoemulsions consist of two immiscible liquids with a surfactant for stabilization. Their average droplet diameter is usually less than 500 nm (\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The ingredients of the nanoemulsion system include oil, surfactant/co-surfactant, water-soluble co-solvent, and water (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The present study aimed to develop an optimized Cur-NE formulation and conduct toxicity and inhibitory assessments of the developed formulation in the TC-1 cell line. To date, there has been no investigation into the impact of curcumin encapsulated in a nanoemulsion formulation on the TC-1 cell line.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eCurcumin 95%, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, DMSO (dimethyl sulfoxide) were purchased from Sigma company. Castor oil, tocopherol acetate, tween 80, and PEG400 were provided by Merk Company. DMEM and fetal bovine serum were obtained from Gibco company.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation\u003c/h2\u003e \u003cp\u003eTo prepare Cur-NE, the method described by Heni Rachmawati and colleagues was used with some modifications (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). An oil phase consisting of different weight ratios of oil (w/w 20%-10%), surfactant (w/w 10%-90%), and co-surfactant (w/w 30%-0%) was prepared by mixing castor oil, tocopheryl acetate, a surfactant component containing Tween 80, and a co-surfactant part including polyethylene glycol 400. This way, curcumin (10-60mg) was added to the oil phase. The prepared oil phase was mixed at 50 \u0026ordm;C on a stirrer at 500 rpm for 15 minutes. Then, water was added to the obtained mixture in a ratio of 5 to 1. After that, the obtained mixture was sonicated by a probe sonicator for 15 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Characterization\u003c/h2\u003e \u003cp\u003eThe morphological characteristics of the Cur-NE specimen were examined using a Zeiss transmission electron microscope (model EM10C, manufactured in Germany), which operated at an acceleration voltage of 100 kilovolts (kV). The attenuate aqueous solution of the specimen was sonicated using an ultrasonic homogenizer (Misonix- S3000, United States) for a duration of 15 minutes. Subsequently, a 20-microliter aliquot of the sample was dispensed onto a formvar-coated carbon film positioned on a copper grid with a 300-mesh structure (EMS-USA) and allowed to desiccate completely at ambient room temperature.\u003c/p\u003e \u003cp\u003eThe size and zeta potential of prepared nanoemulsions were assessed via dynamic light scattering (DLS) utilizing a Malvern Nano Zetasizer (Malvern Instruments, UK). Before analysis, samples were diluted with deionized distilled water.\u003c/p\u003e \u003cp\u003eIn order to examine the stability of nanoemulsions, the samples were evaluated during 60 days at room temperature 25 ֯C by microscopic monitoring and DLS in terms of the formation of precipitation and droplet size.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.4. Cur release\u003c/h2\u003e \u003cp\u003eTo study the release behavior of Cur from Cur-NE, the dialysis method was employed. For this test, 1 ml of Cur-NE or free Cur solution was introduced into a dialysis bag (MW 12 kD) and floated separately into beakers containing 100 ml of pH 7.4 phosphate-buffered saline with stirring at 37\u0026deg;C. At each selected time span (0.5, 1, 2, 4, 6, 8, 24, 48, and 72 h), 1 ml of test solution was removed and substituted with 1 ml of new phosphate buffer. Then, a high-performance liquid chromatography (HPLC, Younglin Acme 9000, South Korea) instrument quantified the concentration of Cur in the samples. The employed HPLC parameters were as follows: a mobile phase composition consisting of 52 (0.1% phosphoric acid):48 (acetonitrile), with a flow rate set at 1 mL/min. The detector was configured to a wavelength of 424 nm, and each analysis run involved an injection volume of 20 ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.5. Cell lines\u003c/h2\u003e \u003cp\u003eThe TC-1 (obtained from the primary lung epithelial cells sourced from C57BL/6 mice.) and MC3T3 (The osteoblastic cell from a C57BL/6 mouse calvaria) Cell lines were acquired from the Pasteur Institute in Iran. The cells were cultured in DMEM (Gibco) culture medium containing 10% fetal bovine serum (Gibco) and maintained at 5% Co2 and 37\u0026deg;C\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. MTT assay\u003c/h2\u003e \u003cp\u003eMTT assay evaluated the impacts of Cur-NE, Cur, and nanoemulsion on TC-1 (cancer cells) and MC3T3 cells (normal cells) viability. Initially, the cell number was quantified using a hemocytometer with trypan blue staining, and subsequently, 15\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells were seeded in each well of the 96-well microplate. The cells were cultured for a duration of 24 hours, followed by subsequent treatment with Cur-NE (10 \u0026micro;g/ml \u0026minus;\u0026thinsp;100\u0026micro;g/ml), curcumin (10\u0026micro;g/ml -100\u0026micro;g/ml), and nanoemulsions (10 \u0026micro;g/ml -900 \u0026micro;g/ml) individually in three wells were incubated in a controlled condition with 5% CO2 at 37\u0026deg;C for 48 hours. Three wells of 96-well-microplate were considered the control containing only DMEM medium. After 48 h, the medium of each well was supplanted with 80 \u0026micro;l of DMEM and A volume of 20 \u0026micro;l of MTT solution at a concentration of 0.5 mg ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the incubation period lasted for 4 hours at 37\u0026deg;C under conditions of 5% CO2. By using 100\u0026micro;l of dimethyl sulfoxide (DMSO), formazan was dissolved, and plate Reader (BioTek Company, USA) was utilized to measure the optical density (OD) of tests at 540nm. Three wells were considered blank, containing only 100 \u0026micro;l DMSO. The calculation of cytotoxicity percentage was performed using the following formula:\u003c/p\u003e \u003cp\u003eCytotoxicity% = 1 - (Mean ODtest - Mean OD blank\u003cem\u003e/\u003c/em\u003eMean OD control - Mean OD blank)\u0026times;100\u003c/p\u003e \u003cp\u003eThe inhibitory concentration (IC50) is the amount at which 50% of cellular viability is destroyed.\u003c/p\u003e \u003c/div\u003e"},{"header":" Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Screening of Cur-NE formulation and characterizations\u003c/h2\u003e \u003cp\u003eDifferent mass ratios of oil, curcumin, surfactant, and co-surfactant were used to optimize stable formulations. Based on particle size and zeta potential, the most optimized formulation consisted of 55 mg curcumin dissolved in a mixture of oil phases, including castor oil and tocopheryl acetate (10% w/w), tween 80 (70% w/w) and PEG 400 (20% w/w) as the oil, surfactant, and co-surfactant respectively.\u003c/p\u003e \u003cp\u003eAccording to the TEM result (Fig.\u0026nbsp;1A, B), spherical particles having a mean size of 42 nm showed that the nanoparticles have a uniform distribution and morphology. In addition, the absence of curcumin precipitation in the TEM image indicates the stability of the formed nanoemulsions.\u003c/p\u003e \u003cp\u003eBased on DLS results (Fig.\u0026nbsp;1C), Cur-NEs exhibited a mean size of 52.5 nm, with a polydisperse index (PDI) of 0.03 and a zeta potential of \u0026minus;\u0026thinsp;13.1 mV. The larger size of DLS results compared to TEM is due to the hydrodynamic radius. Based on the zeta potential, the sample has good stability.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;1\u003c/b\u003e. A) TEM image, B) particle size distribution of Cur-NE, and (C) DLS of Cur-NE.\u003c/p\u003e \u003cp\u003eVisual assessments confirmed an acceptable stability of Cur-NEs, as evidenced by the absence of precipitation and phase separation over at least 60 days. As illustrated in Fig.\u0026nbsp;2, no remarkable alteration in the size of Cur-NEs was determined by DLS.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;2\u003c/b\u003e. Size variations of Cur-NE in 60 days carried out by DLS\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Analysis of drug content\u003c/h2\u003e \u003cp\u003eThe Cur content in the optimized Cur-NE system was determined by HPLC chromatography (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Based on the result, the percentage drug (Cur) content was 96.3 for optimized Cur-NE.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Cur Release\u003c/h2\u003e \u003cp\u003eThe Cur release characteristics of Cur-NE were evaluated by a dialysis method, and the release patterns of free Curcumin and Cur-NE were shown in Fig.\u0026nbsp;3. Free Cur exhibited an extremely rapid release pattern, whereas Cur-NE demonstrated a considerably slower and sustained release. Free Cur was 100% released at 8 hours, while Cur-NE was 94.8% at 48 hours.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;3\u003c/b\u003e. Cur release profile.\u003c/p\u003e \u003cp\u003eCur release data from the optimized Cur-NE formulation were fitted to different release models, including Hill, Weibull, Korsmeyer-Peppas, and third-order (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The best model for the in vitro release of Cur from Cur-NE should have the highest R\u003csup\u003e2\u003c/sup\u003e. Accordingly, the highest value of the correlation coefficient is related to Hill (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.988), followed by Weibull (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.966), Korsmeyer-Peppas (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.90), and third-order.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe release models fitted to the Cur release results.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRelese Model\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFormulation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCur-NE\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThird-order\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.948\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHill\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.988\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeibull\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.966\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKorsmeyer-Peppas\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Cellular cytotoxicity\u003c/h2\u003e \u003cp\u003eThe cytotoxic impacts of Cur-NE, Cur, and nanoemulsion treatments at varying concentrations on the viability of the TC-1 cell line are illustrated in Fig.\u0026nbsp;4. According to this figure, in the TC-1 cancer cell line, enhancing concentrations of Cur-NE ranging from 10 to 100 \u0026micro;g/ml in the culture medium substantially decreased cell viability after 48 hours. The IC50 measurement of Cur-NE on the TC-1 cell line after a 48-hour exposure was almost 35\u0026micro;g/ml (Fig.\u0026nbsp;4A). Notably, the inhibitory effect of Cur-NE is significantly less compared to Cur. As shown in Fig.\u0026nbsp;4B, the IC50 value for TC-1 cells subjected to a 48-hour Curcumin treatment increased to approximately 55\u0026micro;g/ml.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;4.\u003c/b\u003e Cytotoxicity assay results of TC-1 cells treated with different concentration of (A) curcumin nanoemulsion, (B) Curcumin and (C) Nanoemulsion for 48 h. Average viability percent of three independent experiments are shown in the graph. Error bars indicate standard deviations.\u003c/p\u003e \u003cp\u003eThe nanoemulsion does not exhibit any inhibitory impact on the growth of TC-1 cells. In this research, TC-1 cells were treated with nanoemulsion concentrations reaching up to 900\u0026micro;g/ml. However, there were no cytotoxic effects of more than 50% in all concentrations (Fig.\u0026nbsp;4C). Interestingly, Cur-NE does not exert a significant influence on the proliferation of normal MC3T3 cells at concentrations that are cytotoxic to TC-1 cells. (Fig.\u0026nbsp;5A). At none of the concentrations of Cur-NE (10\u0026ndash;100\u0026micro;g/ml), cell survival reached 50%. Moreover, pure Cur and nanoemulsion did not cause a substantial inhibitory effect on the growth of normal cells (Fig.\u0026nbsp;5B, C).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;5\u003c/b\u003e. Cytotoxicity assay results of MC3T3 cells treated with different concentration of A) curcumin nanoemulsion, B) Curcumin and C) Nanoemulsion for 48 h. Average viability percent of three independent experiments are shown in the graph. Error bars indicate standard deviations.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, the standards for designing stable Cur-NEs were as follows: the particle size of the emulsion had to be as small as possible (˂100 nm) and a high electrical charge (zeta potential\u0026thinsp;\u0026gt;\u0026thinsp;\u0026plusmn;\u0026thinsp;30 mV). Considering these criteria, various mass ratios of core (oil and curcumin) and coating materials (surfactant and co-surfactant) were employed to optimize the stable formulations. To ensure that as much Cur as possible was incorporated into the oil phase, a 9:1 ratio of castor oil and tocopheryl acetate was selected. Tocopheryl acetate, in fact as a highly effective solvent, exhibited strong compatibility with the oil and significantly improved the solubility of Cur (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Tween 80 and PEG400 were chosen to emulsify fats. As Previous studies showed here, the combination of tween 80 and PEG400 could produce a transparent emulsion (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). This combination was able to generate a stable nanoemulsion.\u003c/p\u003e \u003cp\u003eThe zeta potential indicates the surface charge and the stability of Cur-NE, the higher repulsive force, the lower aggregate of the emulsion droplets (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Thus, the optimized Cur-NE has the highest value of zeta potential (-13.8 mV), which is related to the surfactants, tween80 and PEG400, and Castrol oil and tocopheryl acetate. Producing small zeta potential through the use of non-ionic surfactants results in the reduction of particle sizes. The solubility of Cur increased up to 5.5mg/ml after incorporation into NE, which is higher than previous reports (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Not changing the appearance and size of the NE not only confirms the stability of the formulation in a certain period of time but also its possible stability for a long time (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Here, visual assessment and DLS analysis performed over 60 days emphasized stability of the optimized Cur-NE.\u003c/p\u003e \u003cp\u003eThe in vitro drug release profile exhibited a two-phase pattern, marked by an initial burst release followed by a gradual and continuous release. Various factors, such as lipophilicity, drug-surfactant interactions, interfacial barrier rigidity, and diffusion from these barriers (e.g., dialysis bags), can influence the release of drugs from emulsions during in vitro studies (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The initial rapid release could be attributed to the permeation of Cur on the external surface of Cur-NE. Subsequently, the release of drugs from these systems decreases over time, resulting in a sustained release profile. The responsible for this release manner is the lipid phase incorporating the drug during the formation of NE (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Our findings align with the previous studies by Yang et al.(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) and Sari et al (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). These in vitro release results suggest that the developed nanoemulsion effectively retains Cur and remains stable (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to our cytotoxicity assay in TC-1 cancer cells, both free Cur and Cur-NE revealed cytotoxic functionality within TC-1 cells in a manner that depends on the dosage. Accordingly, the IC50 values for free Cur and Cur-NE were 55 \u0026micro;g/ml and 35 \u0026micro;g/ml, respectively. It is worth noticing the inhibitory concentration of Cur-NE is less than Cur, indicating the enhanced bioavailability and cellular uptake of curcumin when incorporated in NE (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The result aligns with findings from earlier research that showed the IC50 of nanocurcumin is less than the IC50 of Cur, which indicates that nanocurcumin has better antioxidant activity (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Cheragh and colleagues reported the cytotoxicity of dendrosomal nano-curcumin in Burkitt's lymphoma Daudi cell line with the IC50 30 \u0026micro;g/ml (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In another study, Yan-bin GUAN S-yZ and colleagues showed curcumin nanoemulsions could inhibit the proliferation of prostate cells both in vitro and in vivo in a dose and time dependent manner (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Treatment of Both TC-1 cancer cells and normal MC3T3 cells with nanoemulsion had no significant effect on cellular viability, indicating this nanocarrier is safe.\u003c/p\u003e \u003cp\u003eMoreover, the result of the MTT assessment declared that Cur-NE did not have a significant impact on the viability of normal MC3T3 cells in a manner depending on the dosage. The results of this study align with previous research. The survey carried out by Javidi and colleagues showed that although nanocurcumin could inhibit cancerous cell growth in a depending on both time and dosage manner, it had no notable effects on IPCs normal cells. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Additionally, in vitro investigations conducted by Sim\u0026oacute;n that CUR-NE exhibits safety when tested on non-cancerous human cells (HEK-293T), and displays selective cytotoxicity towards gastric (AGS), colon (HT29-ATCC, HT29-US), breast (MDA-MB-231), and melanoma (B16F10) cells (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, a stable Cur-NE formulation was successfully prepared and optimized. The composition of the optimal Cur-NE includes castor oil, tocopherol acetate, Tween 80, and PEG 400 (9:1:70:20) emulsified in water to form nanoemulsion. The findings of the cytotoxicity test offer compelling proof that Cur-NE is a highly efficient nano-delivery system that enhances the bioavailability of curcumin. Additionally, our study demonstrates the lack of toxicity of the nanoemulsion formulation in vitro, highlighting its potential as a carrier for hydrophobic drugs. Further research is required to examine the long-term safety of the nanoemulsion for its clinical testing and its cytotoxicity effects in other cancer cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the staff of the medical Genomics Research Center and the Nano-Pharmaceutics Laboratory of the Faculty of Medicine, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran, for their helpful assistance with this study and for providing necessary research facilities to carry out this study, respectively.\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDe Sanjos\u0026eacute; S DM, Castellsagu\u0026eacute; X, Clifford G, Bruni L, Mu\u0026ntilde;oz N, et al. 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Pharm Dev Technol. 2014;19(6):677-80.\u003c/li\u003e\n\u003cli\u003eSongyot Anuchapreeda YF, Siriporn Okonogi and Hideki Ichikawa. Preparation of Lipid Nanoemulsions Incorporating Curcumin for Cancer Therapy. Journal of Nanotechnology. 2011;2012.\u003c/li\u003e\n\u003cli\u003eLing Wei XL, Fumin Guo, Xin Liu, Zhongni Wang. Structural properties, in vitro release and radical scavenging activity of lecithin based curcumin-encapsulated inverse hexagonal (HII ) liquid crystals. Colloids and Surfaces A: Physicochem Eng Aspects. 2017.\u003c/li\u003e\n\u003cli\u003eMahboobeh Cheragh MS, Mohammad Hassan Pouriayevali, Masoud Parsania. Dendrosomal nano-curcumin reduces VEGF gene expression and with increasing cell apoptosis has an inhibitory effect on the Burkitt lymphoma cell line. J Human Gen Genom. 2021;5(1).\u003c/li\u003e\n\u003cli\u003eYan-bin GUAN S-yZ, Yu-qiong ZHANG, Jia-le WANG, Yu-dong TIAN, Yong-yan JIA, Yan-jun SUN. Therapeutic Effects of Curcumin Nanoemulsions on Prostate Cancer. J Huazhong Univ Sci Technol. 2017;37(7):371-8.\u003c/li\u003e\n\u003cli\u003eMohammad Amin Javidi AK, Seyedeh Sahar Mortazavi Farsani, Sadegh Babashah, Majid Sadeghizadeh. Investigating curcumin potential for diabetes cell therapy, in vitro and in vivo study. Life Sciences. 2019;239(15).\u003c/li\u003e\n\u003cli\u003eSim\u0026oacute;n Guerrero MI-R, Pamela Contreras-Orellana, Victor Diaz-Garcia, Pablo Lara, Andrea Vivanco-Palma, Areli C\u0026aacute;rdenas, Victor Miranda, Paz Robert,e Lisette Leyton, Marcelo J. Kogan, Andrew F. G. Quest and Felipe Oyarzun-Ampuero. Curcumin-loaded nanoemulsion: a new safe and effective formulation to prevent tumor reincidence and metastasis. Nanoscale. 2018;10:22612\u0026ndash;22.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Curcumin, Cytotoxicity, Nanoemulsion, TC-1 cell, HPV","lastPublishedDoi":"10.21203/rs.3.rs-3859423/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3859423/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCurcumin (Cur), a substance originating from Curcuma longa, has been comprehensively examined for its anticancer properties. Nonetheless, its clinical application has been restricted by its inadequate solubility, bioavailability, and stability. TC-1 cells have been impressive in understanding HPV biology and developing therapeutic approaches for HPV infection and related cancers, like cervical cancer, offering a close mimicry of HPV-induced carcinogenesis. This study's primary goal is to formulate and optimize curcumin nanoemulsions (Cur-NE) to address these challenges and, secondarily, evaluate their impact on TC-1 cell growth. Characterization of the nanoemulsions was conducted using dynamic light scattering (DLS), transmission electron microscopy (TEM) and High-performance liquid chromatography (HPLC) revealing an average particle size of 52.5 nm, a zeta potential of -13.1 mV, and a drug content of 94.6%. Through the dialysis diffusion technique, drug release profiles demonstrated a sustained, slower release of Cur from Cur-NE compared to free curcumin. According to an MTT assay, Cur-Ne with an IC50 35 \u0026micro;g/ml exhibited an increased inhibitory effect of Cur on TC-1 cancer cells, while showing no inhibitory effects on MC3T3 normal cells at concentrations up to 100 \u0026micro;g/ml. In summary, this study underscores the potential of nanoemulsions as efficient carriers for Cur, with demonstrated safety in both cancer and normal cells. Moreover, Cur-NE displayed substantial inhibitory activity against TC-1 cancer cells, suggesting its promise in treating HPV-associated cancers, particularly cervical cancer. Further research is warranted to evaluate the long-term safety of this nanoemulsion for clinical trials and its efficacy against other cancer cell lines.\u003c/p\u003e","manuscriptTitle":"Preparation and evaluation of curcumin nanoemulsion to inhibit TC-1 cell growth","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-18 15:21:12","doi":"10.21203/rs.3.rs-3859423/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ad3c8fd7-3c68-4f45-8487-a2fbb87815e2","owner":[],"postedDate":"January 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-13T13:16:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-18 15:21:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3859423","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3859423","identity":"rs-3859423","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}