Anticancer potential of chalcones loaded on mesoporous silica nanoparticles

Anticancer potential of chalcones loaded on mesoporous silica nanoparticles | 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 Anticancer potential of chalcones loaded on mesoporous silica nanoparticles Guilherme Jose Schwarzt Sampaio, Rodrigo de Almeida Romagna, Reginaldo Bezerra dos Santos, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4676216/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Mar, 2025 Read the published version in Journal of Nanoparticle Research → Version 1 posted 7 You are reading this latest preprint version Abstract The difficulty in treating cancer has led to several studies on the development of systems that perform targeted drug delivery, with the aim of increasing the effectiveness of treatment and reducing adverse effects. In this study, a series of chalcones were tested for cytotoxic action on gastric adenocarcinoma cells (AGS) and breast cancer cells (MCF-7) using the MTT-tetrazolium method, and significant cytotoxicity was demonstrated for 3-hydroxychalcone (CHO). The synthesis of mesoporous silica nanoparticles (MSNs) and their surface modification with 3-aminopropyltriethoxysilane (APTES) were carried out, and 3-hydroxychalcone was then incorporated into these nanomaterials. Mesoporous silica nanoparticles were characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), elemental analysis (CHN), scanning electron microscopy (SEM), transmission electron microscopy (TEM), zeta potential and nitrogen adsorption. In addition, in vitro release tests were carried out to verify the release profile of 3-hydroxychalcone from mesoporous silica samples. The results obtained showed that the mesoporous silica nanoparticles exhibited a gradual and prolonged release profile. In the cytotoxicity test with silica samples incorporated with 3-hydroxychalcone, significant cytotoxic activity was observed against AGS and MCF-7 cells, with the MSN-CHO sample exhibiting a better cytotoxic effect (IC 50 of 12.93 to 22.30 μM) than 3-hydroxychalcone (IC 50 of 47.58 to 47.97 μM). The results showed that the nanoparticles positively influenced the interaction of 3-hydroxychalcone with tumor cells. This is therefore an unprecedented study on the incorporation of 3-hydroxychalcone into mesoporous silica nanoparticles and its promising results in terms of cytotoxic activity against breast and gastric cancer cells. 3-hydroxychalcone anticancer mesoporous silica nanoparticle MSN-chalcone MSN-APTES-chalcone. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights Series of synthetized chalcones showed antitumor activity against AGS and MCF-7 lineage cells. Synthesis and functionalization of mesoporous silica nanoparticles were carried out. 3-hydroxychalcone was incorporated into mesoporous silica nanoparticles. Prolonged release profile and superior cytotoxic activity of 3-hydroxychalcone-loaded silica were demonstrated. 1. Introduction Cancer, a disease characterized by abnormal and uncontrolled growth of cells that can invade neighboring tissues or distant organs, affects millions of people worldwide each year [ 1 ] and is considered one of the main public health problems in the world. Each year, the high number of new cases and the high mortality rates continue to concern experts and organizations involved in research on the subject. According to the International Agency for Research on Cancer (IARC), there will be 19,965,054 new cases of cancer worldwide in 2022 [ 2 ]. Furthermore, cancer treatment involves the use of drugs known as chemotherapeutic agents, which have low specificity, act on both tumor and normal cells and are associated with several problems, such as the development of resistance and serious side effects—which ultimately reduce the quality of life of patients and limit the use of these medications [ 3 , 4 ]. The difficulty in treating cancer has led to several studies aimed at developing more effective drugs with a lower incidence of side effects. Among the various compounds studied, chalcones have shown promising antitumor activity against different types of cancer, including those resistant to currently used chemotherapeutic agents [ 5 , 6 , 7 ]. Chalcones (1,3-diaryl-prop-2-en-1-ones) are an important class of natural products (Fig. 1 ). They are important precursors in the biosynthesis of flavonoids and exhibit multiple biological activities, including anticancer, antibacterial, anti-inflammatory and antioxidant effects [ 8 , 9 ]. Chalcones and their derivatives have increasingly attracted the attention of researchers due to their numerous pharmacological applications, which are related to the ability of chalcones to bind with high affinity to several classes of receptors and enzymes. Furthermore, chalcones can be easily prepared using the Claisen-Schmidt condensation reaction with acid or base catalysis [ 10 ]. Despite numerous published studies reporting the pharmacological potential of chalcones, few studies have reported the application of these compounds in nanotechnology drug delivery systems. Recently, several approaches have been taken to improve the distribution and efficacy of drugs used in cancer treatment, and one of these strategies involves the loading of bioactive compounds into nanocarriers, which can be prepared from different materials, such as polymers or lipids [ 4 , 11 ]. Among the nanocarriers used, mesoporous silica has stood out due to its important physicochemical properties, such as the possibility of controlled drug release, high degree of purity, good chemical and biological compatibility, large surface area and adjustable pore volume, in addition to the possibility of specific targeting of tumor cells [ 12 ]. Mesoporous silicas are silicate-based compounds with highly organized structures. Porous materials can be classified into three groups based on the size of the nanoparticle pores: microporous, mesoporous, and macroporous, with pore sizes smaller than 2 nm, from 2 to 50 nm, and larger than 50 nm, respectively [ 13 ]. Mesoporous silica nanoparticles have a large surface area, well-structured porosity and multiple options for surface functionalization. The functionalization of mesoporous silica nanoparticles can be carried out using various molecules, both organic and inorganic. The silanol groups on the surface of the nanoparticles facilitate the functionalization process. The hydroxyl, carboxyl, polyethylene glycol (PEG) and amine functional groups, among others, have been widely used in the functionalization process. Mesoporous materials functionalized with amino groups show controlled drug release properties due to electrostatic interactions with the carrier molecules. Furthermore, the functionalization process assists in the substance incorporation process and in the specific site targeting process [ 14 , 15 ]. Thus, mesoporous silica nanoparticles with chalcones may be potential drug candidates for the treatment of various diseases, such as cancer, because nanoparticles provide the possibility of targeted treatment, which can increase the specificity and efficacy of chalcones. In this study, a series of chalcones were tested for cytotoxic effect against gastric adenocarcinoma cells (AGS) and breast cancer cells (MCF-7). The most active chalcones were selected and incorporated into mesoporous silica nanoparticles (MSNs). The synthesis and characterization of MSNs and their surface modification with 3-aminopropyltriethoxysilane (APTES) were carried out and, subsequently, the most active chalcone was incorporated into these materials. Lastly, cytotoxicity assays against AGS and MCF-7 cells were performed to evaluate the cytotoxic activity of silica samples incorporated with the most active chalcone. 2. Materials and methods 2.1. Materials Tetraethyl orthosilicate (TEOS), Pluronic® block copolymer - F127, phosphate-buffered saline (PBS), 3-aminopropyltriethoxysilane, solvents and reagents were purchased from Sigma-Aldrich (São Paulo, Brazil). DMEM F-12 (Dulbecco’s modified eagle medium Nutrient Mixture F-12), DMEM high glucose and fetal bovine serum were purchased from Vitrocell (São Paulo, Brazil). NCTC clone 929 (L-929) fibroblast cells (ATCC CCL-1™), breast cancer cells (MCF-7) (ATCC HTB-22™) and human gastric adenocarcinoma (AGS) cells (ATCC CRL – 1739 ™) were obtained from the Rio de Janeiro Cell Bank. 2.2. Experimental procedure for chalcone synthesis To prepare the substituted chalcones, the methodology described by NGO et al. was used. [ 16 ]. In a 50 mL Erlenmeyer flask equipped with magnetic stirring, 5 mmol aromatic aldehyde, 5 mmol aromatic ketone, 10 mL methanol and 1 mL 30% sodium hydroxide solution were added. The resulting mixture was magnetically stirred for approximately 1.5 h at room temperature. Subsequently, the reaction mixture was left in the refrigerator overnight. The resulting product was vacuum filtered with a Buchner funnel and washed with water and ice-cold ethanol. 2.3. Cell culture AGS cells were maintained in DMEM F-12 medium (Dulbecco’s modified eagle medium Nutrient Mixture F-12) supplemented with 10% fetal bovine serum (FBS) (v/v), and L-929 and MCF-7 cells were maintained in DMEM high glucose medium supplemented with 10% and 20% fetal bovine serum, respectively, and incubated in an oven at 37°C with a 5% CO 2 atmosphere for different periods of time until they reached approximately 80–90% confluence. The cells were then removed with trypsin solution and counted in a Neubauer chamber to obtain cell concentration values. 2.4. Cytotoxicity assay The cytotoxicity of the compounds was determined using the MTT assay [ 17 ]. 100 µL of DMEM supplemented with approximately 1.2 x 10 4 cells/mL (AGS) and 1 x 10 5 cells/mL (L-929 and MCF-7) were distributed in 96-well microplates for culture and incubated at 37°C/5% CO 2 for 24 hours. After this period, the medium was removed and the adherent cells were treated with 100 µL of different concentrations of chalcone and silica samples in supplemented DMEM and incubated at 37°C/5% CO 2 for 48 hours (for AGS cells) or 24 hours (for L-929 and MCF-7 cells). After incubation, MTT tetrazolium reagent was added to each well and the plate was incubated for another 2 hours. After this period, 100 mL of DMSO was added to each well to dissolve the formazan crystals, and cell viability was measured in a spectrophotometer at 540 nm. Cisplatin was used as a negative control (cytotoxic standard). The tests were accompanied by positive control growth (no treatment), performed in triplicate and repeated at least three times. 2.5. Selectivity index The selectivity index (SI) indicates the selectivity of a substance between neoplastic and non-neoplastic cells and is determined by the relationship between the IC 50 (half maximal inhibitory concentration) values obtained for these lineages. This relationship is determined using the following formula: IS = IC 50 non-neoplastic cell/IC 50 neoplastic cell in which “non-neoplastic cell” is the fibroblast cell line NCTC clone 929 (L-929) and “neoplastic cell” is the breast cancer cell line (MCF-7) and gastric adenocarcinoma (AGS). The IS indicates the potential of these substances for preclinical and clinical testing. An IS value greater than or equal to 2.0 is considered significant and indicates that the substance is more active in neoplastic cells [ 18 ]. 2.6. Statistical analysis Descriptive statistical analysis was performed to verify the reproducibility and validation of the results, and analysis by ANOVA combined with Dunnet’s test was performed to investigate statistically significant differences in the results obtained after treatment with the sample and control (no treatment). Four-parameter nonlinear regression analysis and normalized response were used to calculate the half maximal inhibitory concentration (IC 50 ). 2.7. Synthesis of mesoporous silica nanoparticles (MSNs) Mesoporous silica nanoparticles were prepared according to a procedure previously described by SHI et al. [ 19 ], with some improvements, using commercial F127 as a template agent under neutral conditions. Copolymer (1.8 g) was dissolved in NH 4 F solution (pH ~ 7) at 12 g/L concentration under constant stirring at 80°C. Subsequently, 40.3 mmol of tetraethyl orthosilicate (TEOS, Sigma-Aldrich) was added dropwise and the resulting solution was kept under constant stirring at 80°C for 1 hour. After this period, the mixture was collected by centrifugation, washed with water, ethanol and acetone and dried at 37°C for 24 hours. After removal of surfactant F127 by solvent extraction method using ethanol and 1% v/v hydrochloric acid, mesoporous silica nanoparticles were obtained. The extraction method was used because it causes less silanol condensation compared to calcination. The presence of silanol groups is an important issue for the functionalization process. 2.8. Surface modification of matrices with 3-aminopropyltriethoxysilane (APTES) The post-synthesis method was used to perform surface modification of the studied nanomaterials. Mesoporous silica was superficially modified via reaction with 3-aminopropyltriethoxysilane - APTES. For functionalization, the same methodology used in previous research [ 20 ] was used, in which X-ray photoelectron spectroscopy (XPS) analysis confirmed that the bond between the silanol groups present on the silica surface and the 3-aminopropyltriethoxysilane molecules was covalent. In this step, 500 mg of previously dried MSN mesoporous silica was placed in a three-necked round-bottom flask with mechanical stirring in 30 mL of toluene. Subsequently, 4 mEq of APTES functionalizing agent was added to this suspension. This mixture was refluxed for 24 h at 80°C. The resulting solid was filtered through a porous plate funnel, washed with toluene and acetone. The filtrate was dried at 60°C for 24 h. 2.9. Physicochemical characterization of mesoporous silica nanoparticles The nanoparticles were characterized by Fourier Transform Infrared Spectroscopy (FTIR), thermogravimetric analysis (TGA), elemental analysis (CHN), scanning electron microscopy (SEM), transmission electron microscopy (TEM), zeta potential and nitrogen adsorption. Fourier Transform Infrared Spectroscopy (FTIR) was performed using a Thermo Scientific Nicolet 6700 spectrophotometer. Spectra were obtained at room temperature on solid powder pellets containing KBr and were acquired at a resolution of 4 cm − 1 and 64 scans/min. Thermogravimetric analysis (TGA) was performed on the TGA 50WS instrument (Shimadzu). The samples were analyzed under the following conditions: heating rate 5°C.min − 1 , temperature range from 25°C to 750°C, an N 2 atmosphere with a flow of 50 mL.min − 1 and an open alumina crucible. The elemental analysis technique (CHN) was used to analyze the carbon content of the silica samples on a Perkin-Elmer model 2400 CHNSO instrument. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of mesoporous silica were obtained using the scanning electron microscope model JSM 6610 LV (JEOL) and the transmission electron microscope model JEM 1400 (JEOL) from the Carlos Alberto Redins Cellular Ultrastructure Laboratory (LUCCAR), respectively. Zeta potential analysis was performed in triplicate, with mesoporous silica samples diluted in 0.9 M NaCl solution using the Litesizer 500 instrument (Anton Paar). Nitrogen adsorption analyses were carried out using the Quantachrome SiQwin™ instrument, which consists of an automated physical adsorption system that provides adsorption and desorption balance data. The mesoporous silica samples were degassed at 120°C for 2 hours prior to testing. 2.10. Incorporation of 3-hydroxychalcone into mesoporous silica nanoparticles and in vitro release assay A 3-hydroxychalcone solution at a concentration of 2 mg/mL was prepared in acetonitrile. Silica was weighed in the amount of 50 mg in an amber glass bottle with a lid, and the chalcone solution was added to this bottle. The solution was kept under magnetic stirring, and after two days of incorporation, it was filtered and dried at room temperature. CHN analyses were performed on the samples to evaluate the percentage of 3-hydroxychalcone adsorbed. For the release test, 10 mg of the incorporated silica sample was added to 10 mL of pH 7.4 PBS buffer solution. The prepared solution was placed in an orbital shaker incubator (INNOVA 4200 brand) at 37°C and agitated for up to 54 hours. To quantify the concentration of 3-hydroxychalcone released from the silica samples, aliquots were taken at specific time points and analyzed on a Shimadzu UV-VIS V-2401 spectrophotometer at a wavelength of 305 nm. 3. Results and discussion 3.1. Assessment of the cytotoxicity of chalcones in vitro During the study, 11 chalcones with different substituents were subjected to cytotoxicity tests against gastric adenocarcinoma (AGS) and breast cancer (MCF-7) cell lines in order to identify the most cytotoxic chalcone for subsequent incorporation into mesoporous silica nanoparticles. The results obtained showed that the chalcones exhibited significant cytotoxicity, with some showing better activity than cisplatin. Table 1 shows the IC 50 values and selectivity of the chalcones against AGS and MCF-7 cells versus fibroblast cells (L-929). It was observed that most of the chalcones were more active against AGS and MCF-7 cells than against fibroblast cells (L-929), which shows that chalcones have greater selectivity for cancer cells than for normal cells, an important characteristic for anticancer agents. Furthermore, some of the chalcones showed a cytotoxic effect close to that of cisplatin against AGS and MCF-7 cells, with greater selectivity against these cell types than cisplatin. In general, the effects of the substituents on the cytotoxic activity of chalcones against AGS and MCF-7 cells were similar. Compound 1 , the unsubstituted chalcone (benzalacetophenone), showed good cytotoxic activity, with IC 50 ranging from 53.11 ± 1.63 to 57.93 ± 19.59 µM, which led us to investigate the influence of different substituents on the cytotoxicity of chalcones. Methoxyl and hydroxyl groups are known to positively influence the cytotoxic activity of chalcones [ 5 , 21 ]. The presence of the methoxy substituent in position 3 of ring B had a positive effect on the cytotoxic activity of chalcone 4 in both cancer cell lines (IC 50 from 44.55 ± 0.09 to 48.12 ± 2.21 µM), while the methoxy group in position 2 of ring B (compound 3 ) was only beneficial for cytotoxicity against AGS cells (IC 50 46.96 ± 3.38 µM), and its effect against MCF-7 cells (IC50 of 81.04 ± 16.52 µM) was negative compared with compound 1 . Other tested chalcones with methoxy groups on the aromatic rings (compounds 2 , 8 and 10 ) showed higher IC 50 values, indicating that the position of the methoxy group on the aromatic ring significantly influences the activity of these compounds. Compound 11 (3-hydroxychalcone) showed significant cytotoxic activity against AGS and MCF-7 cells (IC 50 from 47.58 ± 0.16 to 47.97 ± 2.56 µM), showing that the presence of the hydroxyl group at position 3 of the ring has an important effect on the cytotoxicity of chalcones. When analyzing chalcones with electron-withdrawing groups (chlorine, nitro) in their structure, it was found that chalcone 6 (3-chlorochalcone) showed good cytotoxic activity (IC 50 from 44.54 ± 1.24 to 45.58 ± 4.66 µM), while chalcones 5 (3-chloro-4’-nitrochalcone), 7 (4’-nitrochalcone), 9 (4-nitrochalcone) and 10 (4-methoxy-4’-nitrochalcone) showed moderate cytotoxic activity. The presence of the chlorine group in position 3 of ring B exerts an important influence on the activity of chalcones, while the presence of the nitro group in position 4 of ring A and ring B appears to have a negative effect on the cytotoxic activity, as can be observed in compounds 5 , 7 , 9 and 10 . In general, the chalcones showed significant cytotoxic activity against AGS and MCF-7 cells, with chalcones 4 (3-methoxychalcone), 6 (3-chlorochalcone) and 11 (3-hydroxychalcone) standing out. Considering the cytotoxicity profile in both tumor cell lines and its physicochemical characteristics, 3-hydroxychalcone was selected for incorporation into mesoporous silica nanoparticles. It is worth highlighting that the hydroxyl group present in 3-hydroxychalcone could favor interaction with the silanol groups of silica though hydrogen interactions. The functionalization process was subsequently carried out with the purpose of adding amino groups to the silica surface. The presence of these groups on the silica surface could further favor the incorporation process by increasing the hydrogen interactions between the nanoparticles and 3-hydroxychalcone, with the possibility of incorporating a greater amount of chalcone into the silica. 3.2. Scanning electron microscopy (SEM) Scanning electron microscopy (SEM) is an important technique for studying the morphology (texture, shape and size) of materials. From SEM images, it was possible to estimate the shape of the mesoporous silica nanoparticles. The MSN and MSN-APTES silica (Fig. 2 ) showed spherical particles, which is consistent with the results described in the literature [ 22 ]. However, it was possible to observe aggregates of different sizes, which are common in nanometer-sized particles. 3.3. Transmission electron microscopy (TEM) Transmission electron microscopy (TEM) was used to characterize the pore structure of MSN and MSN-APTES mesoporous silica. The images show that there were pores, which appeared in a spherical shape and in a lighter tone due to the silica nanoparticles (Fig. 3 ). As observed in the SEM analysis, the silica particles were agglomerated, which can be considered a limiting factor in this type of analysis, as it makes it difficult to visualize the silica structures more clearly. 3.4. Fourier transform infrared spectroscopy (FTIR) FTIR analysis was used to determine the chemical composition of the obtained mesoporous silica nanoparticles and the presence of chemical groups of the functionalizing agent on the silica surface. Figure 4 shows the FTIR spectra of MSN and MSN-APTES nanoparticles. The characteristic bands at 1080, 810 and 460 cm − 1 are related to the asymmetric stretching, bending (δ) and out-of-plane Si-O bonds, respectively. The infrared spectrum of the MSN sample showed peaks of hydroxyl stretching band for O-H axial deformation vibration mode of the Si-O-H group and water molecules at 3550 cm − 1 and 1640 cm − 1 . The band at 960 cm − 1 is typical of the silanol groups (Si-OH) present in the silica structure [ 23 ]. After the functionalization process with APTES, this band showed a change in intensity. A decrease in the intensity of the band at 960 cm − 1 was observed, which can be attributed to the reduction in the number of silanol groups present in the silica structure due to the process of binding of the functionalizing agent to these functional groups. For silica functionalized with APTES, a peak at 1560 cm − 1 corresponding to the NH 2 deformation vibration was observed, indicating the presence of functionalizing amino groups on the surface of the mesoporous silica nanoparticles. Furthermore, the bands observed at 1460 − 1410 cm − 1 correspond to CH-CH 2 bending [ 24 ]. These results are consistent with elemental analysis (CHN), in which an increase in the percentage of carbon and nitrogen was observed after the functionalization process. 3.5. Zeta potential analysis Zeta potential analysis was used to evaluate the surface electric charge of particles of the MSN and MSN-APTES samples and of the samples incorporated with 3-hydroxychalcone (MSN-CHO and MSN-APTES-CHO). Table 2 shows the results. Table 2 Zeta potential analysis Sample Zeta potential ± DP (mV) MSN -6.65 ± 0.93 MSN-CHO -3.37 ± 0.92 MSN-APTES -2.93 ± 0.99 MSN-APTES-CHO -2.89 ± 0.95 MSN silica has a negative charge (-6.65 mV) due to the silanol groups present on its surface. After the functionalization process with APTES, a decrease in the negative charge of the functionalized particles was observed due to the contribution of the amino groups of the functionalizing agent. The functionalization of MSN silica with APTES promotes the formation of covalent bonds between the molecules of the silanol groups present on the silica surface and the aminosilane groups of the functionalizing agent. As a result, the negative charge of the nanoparticles decreased from − 6.65 mV in the MSN sample to -2.93 mV in the MSN-APTES sample. These results show that the functionalization process was efficient and are in line with what was described by Andrade et al. [ 20 ]. For the samples incorporated with 3-hydroxychalcone, a decrease in the negative charge of the particles was observed. This result can be attributed to the presence of chemical groups in the 3-hydroxychalcone molecule, which contribute to the reduction of the negative charge of the sample. 3.6. Nitrogen adsorption Nitrogen adsorption analysis was used to obtain information such as specific surface area, pore diameter, distribution and total volume by measuring the area occupied by nitrogen molecules adsorbed on the silica surface [ 25 ]. The specific surface area values of the MSN silica sample were calculated using the BET method, and the total volume and pore diameter values were calculated using the BJH method (Table 3 ). Table 3 Nitrogen adsorption/desorption Sample Surface area m².g − 1 Pore diameter BJH/DFT Volume cc/g MSN 115 3.5 nm 0.391 MSNAPTES 20 3.3 nm 0.060 The values obtained are characteristic of mesoporous materials, confirmed by pore diameter values from 3.3 to 3.5 nm, which are within the expected standard for mesoporous materials (2 to 50 nm) [ 13 ]. The small variation in the pore diameters of the MSN (3.5 nm) and MSN-APTES (3.3 nm) samples evidences the occupation of the pores by the amino groups after the functionalization process. The surface area and pore volume were 115 m 2 /g − 1 and 0.391 cc 3 .g − 1 for the MSN sample and 20 m 2 /g − 1 and 0.060 cc 3 .g − 1 for the MSN APTES sample, respectively, indicating that the functionalization process of MSN silica with APTES was successful, given the decrease in surface area and pore volume. Although smaller, the surface area of MSN-APTES silica is large enough to incorporate molecules. Furthermore, the isotherms of the MSN and MSN-APTES samples showed adsorption and desorption branches that formed a type H1 hysteresis, characteristic of mesoporous materials (Fig. 5 ). A smaller volume of adsorbed nitrogen was found for the MSN-APTES sample than for the MSN silica in almost all relative pressures, indicating that the functionalization process was successful due to pore filling by the functionalizing agent, corroborating the results shown in Table 3 , in which smaller pore diameter and volume values can be observed for the MSN-APTES sample compared with the MSN sample. 3.7. Thermogravimetric analysis (TGA) and elemental analysis (CHN) Figure 6 shows the mass loss curves of the MSN, MSN-CHO, MSN-APTES and MSN-APTES-CHO samples. For the MSN sample, a mass loss of 3.7% was observed in the temperature range of 25°C to 150°C, which is related to the adsorption of water molecules on silica. From 150°C to 750°C, the mass loss was 9.5%, corresponding to the condensation of silanol groups [ 26 ]. An increase in mass loss was also observed for the MSN-CHO sample from 150°C to 750°C (11.3%), which can be attributed to the thermal degradation of the incorporated 3-hydroxychalcone. This 1.8% increase in the percentage of mass loss in the sample incorporated with 3-hydroxychalcone (MSN-CHO) compared with the unloaded sample (MSN) indicates the presence of chalcone in this sample and also shows that the incorporation process was successful. For samples MSN-APTES and MSN-APTES-CHO, the mass loss in the temperature range of 150 to 750°C was 20.2% and 25.8%, respectively. The substantial mass loss for these samples in this temperature range can be attributed to the presence of amino groups of the functionalizing agent attached to the silica surface and the presence of 3-hydroxychalcone for the loaded sample, indicating that the functionalization and incorporation process was successful. The greater mass loss of MSN-APTES-CHO (5.6%) than MSN-CHO (1.8%) means that the incorporation process is more efficient in functionalized silica, due to the presence of amino groups on the silica surface, which favors the incorporation process by increasing the number of hydrogen interactions between silica and 3-hydroxychalcone. The results obtained by thermogravimetric analysis are in line with those obtained using the elemental analysis technique (CHN). These results provided additional evidence that the silica samples were loaded with 3-hydroxychalcone and that the functionalization process was successful (Table 4 ). Table 4 Mass loss and elemental analysis (CHN) of mesoporous silica nanoparticle samples Sample Weight loss (% m/m) 25–150ºC Weight loss (% m/m) 150–750ºC Residue (%m/m) > 800ºC % Carbon % Nitrogen MSN 3.7 9.5 86.8 5.43 ± 0.04 -0.18 ± 0.011 MSN-CHO 3.4 11.3 85.3 5.91 ± 0.04 -0.64 ± 0.0025 MSN-APTES 7.6 20.2 72.2 13.02 ± 0.06 2.70 ± 0.055 MSN-APTES-CHO 7.0 25.8 67.2 14.30 ± 0.06 2.55 ± 0.025 The MSN sample presented a carbon percentage of 5.43%, this percentage can be explained by the presence of the copolymer F127 that was not completely removed during the extraction process. After the incorporation process with 3-hydroxychalcone, the MSN sample (5.43%) presented an increase in the carbon percentage (5.91%), indicating that the incorporation process was successful. However, the carbon variations between the MSN and MSN-CHO samples were minimal, indicating that 0.48% (9.6 µg) of 3-hydroxychalcone was incorporated into the mesoporous silica nanoparticles at the tested concentration (2 mg/mL). Furthermore, the MSN sample showed an increase in the percentage of carbon and nitrogen after the functionalization process (MSN-APTES), indicating that this process was successful and that the binding of the functionalizing agent 3-aminopropyltriethoxysilane occurred on the silica surface. An increase in the percentage of carbon was also observed in the functionalized sample incorporated with 3-hydroxychalcone (MSN-APTES-CHO). For APTES silica, the incorporation rate was found to be 1.28% or 25.6 µg—almost three times higher than the rate found for the MSN-CHO sample (0.48%). Functionalization with APTES favors the incorporation process due to hydrogen interactions between the amino groups of the functionalized silica and the 3-hydroxychalcone molecules, resulting in a higher incorporation rate. 3.8. 3-hydroxychalcone loading and release After the incorporation process, the amount of 3-hydroxychalcone loaded was 0.48% or 9.6 µg for the MSN-CHO sample and 1.28% or 25.6 µg for the MSN-APTES-CHO. These values were obtained using the elemental analysis (CHN). Figure 7 shows the release profile of the MSN-CHO and MSN-CHO-APTES samples. For MSN-CHO, an initial rapid release of 3-hydroxychalcone can be observed, around 30% (2.88 µg) in one hour, followed by a gradual and prolonged release, with 52.60% (5.05 µg) of chalcone released after 54 hours. These results can be explained by the interaction between the silanol groups of silica and the hydroxyl and carbonyl groups of 3-hydroxychalcone via hydrogen bonding, resulting in a slower release of 3-hydroxychalcone from the silica matrix. The MSN-APTES-CHO sample showed a slower release, with 16.84% (4.31 µg) of 3-hydroxychalcone released in 54 hours. This characteristic can be explained by a possible more intense interaction between 3-hydroxychalcone and functionalized silica. The addition of amino groups to the silica surface can promote a greater number of hydrogen interactions between the nanoparticles and 3-hydroxychalcone, leading to slower release rates. These findings show that mesoporous silica nanoparticles can be adapted to provide an initial release of an adequate dose of the bioactive compound in a short period of time, followed by a slow and gradual release of the drug over a long period of time, which can be useful for maintaining constant compound levels and action and reducing side effects. 3.9. Evaluation of the cytotoxicity of silica samples in vitro Silica samples incorporated with 3-hydroxychalcone (MSN-CHO and MSN-APTES-CHO) and isolated silica samples (MSN and MSN-APTES) were subjected to cytotoxicity tests against gastric adenocarcinoma (AGS) and breast cancer (MCF-7) to verify the effectiveness of these systems in enhancing the cytotoxic activity of 3-hydroxychalcone. Table 5 shows the IC 50 values and selectivity of the silica samples against AGS, MCF-7 and fibroblast cells (L-929). Table 5 IC 50 (µM) and selectivity index (SI) of silica samples Sample IC 50 L-929 IC 50 AGS IC 50 MCF-7 IS AGS IS MCF-7 3-hydroxychalcone 275.03 ± 5.96* 47.58 ± 0.16 47.97 ± 2.56 5.78* 5.75* MSN > 6000 > 6000 > 6000 1.00 1.00 MSN-CHO 49.99 ± 1.11 12.93 ± 0.94 22.30 ± 4.15 4.16** 2.06 MSN-APTES 1619 ± 69.30 > 7300 > 7300 0.22 0.22 MSN-APTES-CHO 73.67 ± 8.07 106.67 ± 7.80 80.98 ± 14.76 0.82 0.83 Cisplatin 24.03 ± 7.42 39.59 ± 0.66 52.85 ± 4.80 0.61 0.46 IC 50 and IS of chalcones and cisplatin (*: p < 0.0001; **: p < 0.001 compared with cisplatin). The silica samples loaded with 3-hydroxychalcone showed significant cytotoxic activity (IC 50 from 12.93 ± 0.94 to 106.67 ± 7.80 µM) against AGS and MCF-7 cells, with the MSN-CHO sample showing a better cytotoxic effect (IC 50 from 12.93 ± 0.94 to 22.30 ± 4.15 µM) than free 3-hydroxychalcone (IC 50 from 47.58 ± 0.16 to 47.97 ± 2.56 µM). These results indicate that the loaded mesoporous silica nanoparticles significantly influenced the interaction of chalcone with tumor cells, requiring lower concentrations of 3-hydroxychalcone to exert greater cytotoxic activity compared with free chalcone. While the mechanism of cellular uptake of 3-hydroxychalcone can occur by passive diffusion [ 27 ], for mesoporous silica nanoparticles, absorption occurs by endocytosis. Studies have reported that mesoporous silica nanoparticles have a high affinity for plasma membrane phospholipids, and the adsorption of these molecules on cell surfaces would lead to absorption via endocytosis [ 28 ]. As can be seen, the silica samples showed variations in cytotoxic activities. Even with higher incorporation rates, the MSN-APTES-CHO sample showed higher IC 50 values (80.98 ± 14.76 to 106.67 ± 7.80 µM) than the MSN-CHO sample (12.93 ± 0.94 to 22.30 ± 4.15 µM), which had a low incorporation rate. These differences in cytotoxic activity can be explained by the different release profiles of the silica samples. As described in the release test, the MSN-CHO sample showed a better release profile, with a greater amount of 3-hydroxychalcone released over time than the MSN-APTES-CHO sample, due to more intense interactions between 3-hydroxychalcone and functionalized silica by hydrogen bonds. This type of interaction affects the release profile of the functionalized nanoparticles and consequently their cytotoxic activity, resulting in higher IC 50 values. In general, the silica samples showed greater cytotoxic activity against AGS and MCF-7 cells than against fibroblast cells (L-929), with the exception of the MSN-APTES and MSN-APTES-CHO samples, which showed greater cytotoxic activity against fibroblast cells than against cancer cells. These results can be explained by the characteristics of the cells tested. The APTES silica surface is normally positively charged under the conditions in which the tests were carried out, promoting greater interaction with the plasma membranes of normal cells, which are negatively charged and attract the functionalized nanoparticles via electrostatic interactions. These interactions may cause a destabilization of the plasma membrane, increasing its permeability and allowing for a greater effect of 3-hydroxychalcone on fibroblast cells. The plasma membranes of tumor cells show changes in the composition and organization of lipids, which cause several changes in cellular functions such as permeability and transport of substances across the plasma membrane [ 29 ]. These changes may result in less intense interactions between functionalized silica nanoparticles and tumor cells, which would explain the results found in the cytotoxicity test. 4. Conclusions The results showed that synthetic chalcones exhibited significant cytotoxic activity against AGS and MCF-7 cells, especially 3-hydroxychalcone, which showed the best antitumor activity and was selected for incorporation studies with mesoporous silica nanoparticles. The synthesis, functionalization and characterization of the nanoparticles and the 3-hydroxychalcone incorporation process were successful. Furthermore, the functionalization of nanoparticles was found to influence the incorporation process and the release profile of the samples due to the intense interactions of 3-hydroxychalcone with the functionalized silica. Based on the cytotoxicity test, the combination of 3-hydroxychalcone incorporated in silica matrix showed effective cytotoxic activity against AGS and MCF-7 cells. The MSN-CHO sample showed better cytotoxic activity than free 3-hydroxychalcone, demonstrating that the incorporation into the nanoparticles enhances cytotoxicity against tumor cells. Our findings highlight that MSN-CHO is a potentially effective agent against tumor cells. Declarations Acknowledgements: The authors thank Fundação de Amparo à Pesquisa e Inovação do Espírito Santo (FAPES) for financial support (grant number 2022-78KWB) and technical support and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for scholarship to RRK (process number 314276/2021-1). Federal University of Espírito Santo for providing the structure necessary to accomplish this work and technical support of Laboratory Ultra Cellular Structure Carlos Alberto Redins (LUCCAR/ UFES) and Laboratory of Nanostructured Materials for Bioapplications (LMNB - CDTN/CNEN). AUTHOR CONTRIBUTIONS Conceptualization: RRK, GFA, GJSS. Formal Analysis: GJSS, RAR. Investigation: GJSS, RAR Methodology: GJSS, RAR, RBS. Project administration: RRK, GFA. Resources: RRK, RCRG. Visualization: GJSS, RRK, GFA. Writing-original draft: GJSS, RRK, GFA. Writing - review & editing: GJSS, RRK, GFA. Supervision: RRK and GFA. FUNDING This study was supported by the “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES)” – [grant number 2144/2022]; Fundação de Amparo à Pesquisa e Inovação do Espírito Santo (FAPES) – [grant numbers 2022-78KWB]. Conflicts of interest: The authors declare that they have no competing interests. References Ouyang Y, Li J, Chen X, Fu X, Sun S, Wu Q (2021) Chalcone derivatives: Role in anticancer therapy. Biomolecules 11(6):894. https://doi.org/10.3390/biom11060894 GLOBOCAN – The Global Cancer Observatory (2024) Cancer Today: IARC. https://gco.iarc.fr/today/en/dataviz/pie?mode=cancer&group_populations=1&types=0. Accessed 10 Feb 2024. Mansouri V, Beheshtizadeh N, Gharibshahian M, Sabouri L, Varzandeh M, Rezaei N (2021) Recent advances in regenerative medicine strategies for cancer treatment. 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Acta Pharm Sin B 8(2):165-177. https://doi.org/10.1016/j.apsb.2018.01.007 Ngo D, Kalala M, Hogan V, Manchanayakage R (2014) One-pot synthesis of chalcone epoxides—A green chemistry strategy. Tetrahedron Lett 55(32):4496-4500. https://doi.org/10.1016/j.tetlet.2014.06.057 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1-2):55-63. https://doi.org/10.1016/0022-1759(83)90303-4 Badisa RB, Darling-Reed SF, Joseph P, Cooperwood JS, Latinwo LM, Goodman CB (2009) Selective cytotoxic activities of two novel synthetic drugs on human breast carcinoma MCF-7 cells. Anticancer Res 29(8):2993-2996. Shi M, Chen Z, Farnaghi S, Friis T, Mao X, Xiao Y, Wu C (2016) Copper-doped mesoporous silica nanospheres, a promising immunomodulatory agent for inducing osteogenesis. Acta Biomater 30:334-344. https://doi.org/10.1016/j.actbio.2015.11.033 Andrade GF, Lima GDS, Gastelois PL, Assis GD, Macedo WADA, de Sousa EMB (2020) Surface modification and biological evaluation of kojic acid/silica nanoparticles as platforms for biomedical systems. Int J Appl Ceram Technol 17(1):380-391. https://doi.org/10.1111/ijac.13376 Juvale K, Pape VF, Wiese M (2012) Investigation of chalcones and benzochalcones as inhibitors of breast cancer resistance protein. Bioorg Med Chem 20(1):346-355. https://doi.org/10.1016/j.bmc.2011.10.074 Wang L, Fan J, Tian B, Yang H, Yu C, Tu B, Zhao D (2004) Synthesis and characterization of small pore thick-walled SBA-16 templated by oligomeric surfactant with ultra-long hydrophilic chains. Microporous Mesoporous Mater 67(2-3):135-141. https://doi.org/10.1016/j.micromeso.2003.10.015 Duran A, Serna C, Fornes V, Navarro JF (1986) Structural considerations about SiO 2 glasses prepared by sol-gel. J Non-Cryst Solids 82(1-3):69-77. https://doi.org/10.1016/0022-3093(86)90112-2 Shi B, Wang Y, Guo Y, Wang Y, Wang Y, Guo Y, Zhang Z, Liu X, Lu G (2009) Aminopropyl-functionalized silicas synthesized by W/O microemulsion for immobilization of penicillin G acylase. Catal Today 148(1-2):184-188. https://doi.org/10.1016/j.cattod.2009.02.014 Sing KS (1989) The use of gas adsorption for the characterization of porous solids. Colloids Surf 38(1):113-124. https://doi.org/10.1016/0166-6622(89)80148-9 Freitas LBO, Corgosinho LM, Faria JAQA, dos Santos VM, Resende JM, Leal AS, Gomes DA, de Sousa EMB (2017) Multifunctional mesoporous silica nanoparticles for cancer-targeted, controlled drug delivery and imaging. Microporous Mesoporous Mater 242:271-283. https://doi.org/10.1016/j.micromeso.2017.01.036 Singh N, Kumar N, Rathee G, Sood D, Singh A, Tomar V, Dass SK, Chandra R (2020) Privileged scaffold chalcone: synthesis, characterization and its mechanistic interaction studies with BSA employing spectroscopic and chemoinformatics approaches. ACS Omega 5(5):2267-2279. https://doi.org/10.1021/acsomega.9b03479 Jia L, Shen J, Li Z, Zhang D, Zhang Q, Liu G, Zheng D, Tian X (2013) In vitro and in vivo evaluation of paclitaxel-loaded mesoporous silica nanoparticles with three pore sizes. Int J Pharm 445(1-2):12-19. https://doi.org/10.1016/j.ijpharm.2013.01.058 Preta G (2020) New insights into targeting membrane lipids for cancer therapy. Front Cell Dev Biol 8:571237. https://doi.org/10.3389/fcell.2020.571237 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 28 Mar, 2025 Read the published version in Journal of Nanoparticle Research → Version 1 posted Editorial decision: Revision requested 04 Mar, 2025 Reviews received at journal 17 Aug, 2024 Reviewers agreed at journal 08 Aug, 2024 Reviewers invited by journal 08 Jul, 2024 Editor assigned by journal 05 Jul, 2024 Submission checks completed at journal 04 Jul, 2024 First submitted to journal 02 Jul, 2024 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. We do this by developing innovative software and high quality services for the global research community. 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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-4676216","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":326450506,"identity":"7d1cc227-3942-40bc-95e5-8fadcf719aea","order_by":0,"name":"Guilherme Jose Schwarzt Sampaio","email":"","orcid":"","institution":"Universidade Federal do Espírito Santo","correspondingAuthor":false,"prefix":"","firstName":"Guilherme","middleName":"Jose Schwarzt","lastName":"Sampaio","suffix":""},{"id":326450507,"identity":"27a9cb63-5f63-4455-872a-9c048ab656f6","order_by":1,"name":"Rodrigo de Almeida 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19:44:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4676216/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4676216/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11051-025-06292-4","type":"published","date":"2025-03-28T15:57:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61328333,"identity":"b3606040-3074-4767-b127-825a5fdbcf5c","added_by":"auto","created_at":"2024-07-29 14:23:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5769,"visible":true,"origin":"","legend":"\u003cp\u003eGeneral structure of chalcones.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4676216/v1/76c59c1205443ddd178a2beb.png"},{"id":61328334,"identity":"b757e726-9824-4635-8bbb-a5b0ff9550c7","added_by":"auto","created_at":"2024-07-29 14:23:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":286832,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy of MSN (left) and MSN-APTES (right).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4676216/v1/4bcd8738cfb3beacefe52204.png"},{"id":61328338,"identity":"f1228bf9-bd3d-4ad0-8915-e649fb55832f","added_by":"auto","created_at":"2024-07-29 14:23:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":334379,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron microscopy of MSN (A), MSN-APTES (B) and average particle size distribution graph of MSN (C).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4676216/v1/66accc68609eb06930e9bb6d.png"},{"id":61328336,"identity":"a9ec0408-ba55-4c9b-94da-93afdabfcaee","added_by":"auto","created_at":"2024-07-29 14:23:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":29628,"visible":true,"origin":"","legend":"\u003cp\u003eInfrared spectrum of mesoporous silica nanoparticles (MSNs) and mesoporous silica nanoparticles functionalized with APTES (MSN APTES).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4676216/v1/67ede7f2b58f2965381720c4.png"},{"id":61329065,"identity":"458da345-03de-4f0e-817f-a894c19b73c5","added_by":"auto","created_at":"2024-07-29 14:31:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":21154,"visible":true,"origin":"","legend":"\u003cp\u003eNitrogen adsorption isotherms of MSN and APTES.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4676216/v1/f3abfeb68fd25a098059ccfd.png"},{"id":61328332,"identity":"2b17c432-f4c5-4145-8bfb-aea28ecdb020","added_by":"auto","created_at":"2024-07-29 14:23:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":23811,"visible":true,"origin":"","legend":"\u003cp\u003eThermogravimetric curve for MSN, MSN-CHO, MSN-APTES, MSN-APTES-CHO and CHO.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4676216/v1/0c0a8387e458fa13e2163254.png"},{"id":61328339,"identity":"7f34150c-b98f-42cc-896b-f28194d6b16c","added_by":"auto","created_at":"2024-07-29 14:23:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":18675,"visible":true,"origin":"","legend":"\u003cp\u003eRelease profile of the MSN-CHO and MSNA-APTES-CHO samples.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4676216/v1/60832448c16dbf2b0fb6d32a.png"},{"id":79604965,"identity":"c25a33fa-0816-4879-8960-f74ed845224c","added_by":"auto","created_at":"2025-03-31 16:09:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1965413,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4676216/v1/0d865c19-fe1a-4a4b-87f9-5c2662fad067.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anticancer potential of chalcones loaded on mesoporous silica nanoparticles","fulltext":[{"header":"Highlights","content":"\u003cp\u003eSeries of synthetized chalcones showed antitumor activity against AGS and MCF-7 lineage cells.\u003c/p\u003e\n\u003cp\u003eSynthesis and functionalization of mesoporous silica nanoparticles were carried out.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3-hydroxychalcone was incorporated into mesoporous silica nanoparticles.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProlonged release profile and superior cytotoxic activity of 3-hydroxychalcone-loaded silica were demonstrated.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eCancer, a disease characterized by abnormal and uncontrolled growth of cells that can invade neighboring tissues or distant organs, affects millions of people worldwide each year [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and is considered one of the main public health problems in the world. Each year, the high number of new cases and the high mortality rates continue to concern experts and organizations involved in research on the subject. According to the International Agency for Research on Cancer (IARC), there will be 19,965,054 new cases of cancer worldwide in 2022 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Furthermore, cancer treatment involves the use of drugs known as chemotherapeutic agents, which have low specificity, act on both tumor and normal cells and are associated with several problems, such as the development of resistance and serious side effects\u0026mdash;which ultimately reduce the quality of life of patients and limit the use of these medications [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe difficulty in treating cancer has led to several studies aimed at developing more effective drugs with a lower incidence of side effects. Among the various compounds studied, chalcones have shown promising antitumor activity against different types of cancer, including those resistant to currently used chemotherapeutic agents [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChalcones (1,3-diaryl-prop-2-en-1-ones) are an important class of natural products (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). They are important precursors in the biosynthesis of flavonoids and exhibit multiple biological activities, including anticancer, antibacterial, anti-inflammatory and antioxidant effects [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Chalcones and their derivatives have increasingly attracted the attention of researchers due to their numerous pharmacological applications, which are related to the ability of chalcones to bind with high affinity to several classes of receptors and enzymes. Furthermore, chalcones can be easily prepared using the Claisen-Schmidt condensation reaction with acid or base catalysis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Despite numerous published studies reporting the pharmacological potential of chalcones, few studies have reported the application of these compounds in nanotechnology drug delivery systems.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRecently, several approaches have been taken to improve the distribution and efficacy of drugs used in cancer treatment, and one of these strategies involves the loading of bioactive compounds into nanocarriers, which can be prepared from different materials, such as polymers or lipids [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Among the nanocarriers used, mesoporous silica has stood out due to its important physicochemical properties, such as the possibility of controlled drug release, high degree of purity, good chemical and biological compatibility, large surface area and adjustable pore volume, in addition to the possibility of specific targeting of tumor cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMesoporous silicas are silicate-based compounds with highly organized structures. Porous materials can be classified into three groups based on the size of the nanoparticle pores: microporous, mesoporous, and macroporous, with pore sizes smaller than 2 nm, from 2 to 50 nm, and larger than 50 nm, respectively [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMesoporous silica nanoparticles have a large surface area, well-structured porosity and multiple options for surface functionalization. The functionalization of mesoporous silica nanoparticles can be carried out using various molecules, both organic and inorganic. The silanol groups on the surface of the nanoparticles facilitate the functionalization process. The hydroxyl, carboxyl, polyethylene glycol (PEG) and amine functional groups, among others, have been widely used in the functionalization process. Mesoporous materials functionalized with amino groups show controlled drug release properties due to electrostatic interactions with the carrier molecules. Furthermore, the functionalization process assists in the substance incorporation process and in the specific site targeting process [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThus, mesoporous silica nanoparticles with chalcones may be potential drug candidates for the treatment of various diseases, such as cancer, because nanoparticles provide the possibility of targeted treatment, which can increase the specificity and efficacy of chalcones.\u003c/p\u003e \u003cp\u003eIn this study, a series of chalcones were tested for cytotoxic effect against gastric adenocarcinoma cells (AGS) and breast cancer cells (MCF-7). The most active chalcones were selected and incorporated into mesoporous silica nanoparticles (MSNs). The synthesis and characterization of MSNs and their surface modification with 3-aminopropyltriethoxysilane (APTES) were carried out and, subsequently, the most active chalcone was incorporated into these materials. Lastly, cytotoxicity assays against AGS and MCF-7 cells were performed to evaluate the cytotoxic activity of silica samples incorporated with the most active chalcone.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eTetraethyl orthosilicate (TEOS), Pluronic\u0026reg; block copolymer - F127, phosphate-buffered saline (PBS), 3-aminopropyltriethoxysilane, solvents and reagents were purchased from Sigma-Aldrich (S\u0026atilde;o Paulo, Brazil). DMEM F-12 (Dulbecco\u0026rsquo;s modified eagle medium Nutrient Mixture F-12), DMEM high glucose and fetal bovine serum were purchased from Vitrocell (S\u0026atilde;o Paulo, Brazil). NCTC clone 929 (L-929) fibroblast cells (ATCC CCL-1\u0026trade;), breast cancer cells (MCF-7) (ATCC HTB-22\u0026trade;) and human gastric adenocarcinoma (AGS) cells (ATCC CRL \u0026ndash; 1739 \u0026trade;) were obtained from the Rio de Janeiro Cell Bank.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental procedure for chalcone synthesis\u003c/h2\u003e \u003cp\u003eTo prepare the substituted chalcones, the methodology described by NGO et al. was used. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In a 50 mL Erlenmeyer flask equipped with magnetic stirring, 5 mmol aromatic aldehyde, 5 mmol aromatic ketone, 10 mL methanol and 1 mL 30% sodium hydroxide solution were added. The resulting mixture was magnetically stirred for approximately 1.5 h at room temperature. Subsequently, the reaction mixture was left in the refrigerator overnight. The resulting product was vacuum filtered with a Buchner funnel and washed with water and ice-cold ethanol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Cell culture\u003c/h2\u003e \u003cp\u003eAGS cells were maintained in DMEM F-12 medium (Dulbecco\u0026rsquo;s modified eagle medium Nutrient Mixture F-12) supplemented with 10% fetal bovine serum (FBS) (v/v), and L-929 and MCF-7 cells were maintained in DMEM high glucose medium supplemented with 10% and 20% fetal bovine serum, respectively, and incubated in an oven at 37\u0026deg;C with a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere for different periods of time until they reached approximately 80\u0026ndash;90% confluence. The cells were then removed with trypsin solution and counted in a Neubauer chamber to obtain cell concentration values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cytotoxicity assay\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of the compounds was determined using the MTT assay [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. 100 \u0026micro;L of DMEM supplemented with approximately 1.2 x 10\u003csup\u003e4\u003c/sup\u003e cells/mL (AGS) and 1 x 10\u003csup\u003e5\u003c/sup\u003e cells/mL (L-929 and MCF-7) were distributed in 96-well microplates for culture and incubated at 37\u0026deg;C/5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours. After this period, the medium was removed and the adherent cells were treated with 100 \u0026micro;L of different concentrations of chalcone and silica samples in supplemented DMEM and incubated at 37\u0026deg;C/5% CO\u003csub\u003e2\u003c/sub\u003e for 48 hours (for AGS cells) or 24 hours (for L-929 and MCF-7 cells). After incubation, MTT tetrazolium reagent was added to each well and the plate was incubated for another 2 hours. After this period, 100 mL of DMSO was added to each well to dissolve the formazan crystals, and cell viability was measured in a spectrophotometer at 540 nm. Cisplatin was used as a negative control (cytotoxic standard). The tests were accompanied by positive control growth (no treatment), performed in triplicate and repeated at least three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Selectivity index\u003c/h2\u003e \u003cp\u003eThe selectivity index (SI) indicates the selectivity of a substance between neoplastic and non-neoplastic cells and is determined by the relationship between the IC\u003csub\u003e50\u003c/sub\u003e (half maximal inhibitory concentration) values obtained for these lineages. This relationship is determined using the following formula:\u003c/p\u003e \u003cp\u003eIS\u0026thinsp;=\u0026thinsp;IC\u003csub\u003e50\u003c/sub\u003e non-neoplastic cell/IC\u003csub\u003e50\u003c/sub\u003e neoplastic cell\u003c/p\u003e \u003cp\u003ein which \u0026ldquo;non-neoplastic cell\u0026rdquo; is the fibroblast cell line NCTC clone 929 (L-929) and \u0026ldquo;neoplastic cell\u0026rdquo; is the breast cancer cell line (MCF-7) and gastric adenocarcinoma (AGS).\u003c/p\u003e \u003cp\u003eThe IS indicates the potential of these substances for preclinical and clinical testing. An IS value greater than or equal to 2.0 is considered significant and indicates that the substance is more active in neoplastic cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eDescriptive statistical analysis was performed to verify the reproducibility and validation of the results, and analysis by ANOVA combined with Dunnet\u0026rsquo;s test was performed to investigate statistically significant differences in the results obtained after treatment with the sample and control (no treatment). Four-parameter nonlinear regression analysis and normalized response were used to calculate the half maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Synthesis of mesoporous silica nanoparticles (MSNs)\u003c/h2\u003e \u003cp\u003eMesoporous silica nanoparticles were prepared according to a procedure previously described by SHI et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], with some improvements, using commercial F127 as a template agent under neutral conditions. Copolymer (1.8 g) was dissolved in NH\u003csub\u003e4\u003c/sub\u003eF solution (pH\u0026thinsp;~\u0026thinsp;7) at 12 g/L concentration under constant stirring at 80\u0026deg;C. Subsequently, 40.3 mmol of tetraethyl orthosilicate (TEOS, Sigma-Aldrich) was added dropwise and the resulting solution was kept under constant stirring at 80\u0026deg;C for 1 hour. After this period, the mixture was collected by centrifugation, washed with water, ethanol and acetone and dried at 37\u0026deg;C for 24 hours. After removal of surfactant F127 by solvent extraction method using ethanol and 1% v/v hydrochloric acid, mesoporous silica nanoparticles were obtained. The extraction method was used because it causes less silanol condensation compared to calcination. The presence of silanol groups is an important issue for the functionalization process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Surface modification of matrices with 3-aminopropyltriethoxysilane (APTES)\u003c/h2\u003e \u003cp\u003eThe post-synthesis method was used to perform surface modification of the studied nanomaterials. Mesoporous silica was superficially modified via reaction with 3-aminopropyltriethoxysilane - APTES. For functionalization, the same methodology used in previous research [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] was used, in which X-ray photoelectron spectroscopy (XPS) analysis confirmed that the bond between the silanol groups present on the silica surface and the 3-aminopropyltriethoxysilane molecules was covalent.\u003c/p\u003e \u003cp\u003eIn this step, 500 mg of previously dried MSN mesoporous silica was placed in a three-necked round-bottom flask with mechanical stirring in 30 mL of toluene. Subsequently, 4 mEq of APTES functionalizing agent was added to this suspension. This mixture was refluxed for 24 h at 80\u0026deg;C. The resulting solid was filtered through a porous plate funnel, washed with toluene and acetone. The filtrate was dried at 60\u0026deg;C for 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Physicochemical characterization of mesoporous silica nanoparticles\u003c/h2\u003e \u003cp\u003eThe nanoparticles were characterized by Fourier Transform Infrared Spectroscopy (FTIR), thermogravimetric analysis (TGA), elemental analysis (CHN), scanning electron microscopy (SEM), transmission electron microscopy (TEM), zeta potential and nitrogen adsorption. Fourier Transform Infrared Spectroscopy (FTIR) was performed using a Thermo Scientific Nicolet 6700 spectrophotometer. Spectra were obtained at room temperature on solid powder pellets containing KBr and were acquired at a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 64 scans/min. Thermogravimetric analysis (TGA) was performed on the TGA 50WS instrument (Shimadzu). The samples were analyzed under the following conditions: heating rate 5\u0026deg;C.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, temperature range from 25\u0026deg;C to 750\u0026deg;C, an N\u003csub\u003e2\u003c/sub\u003e atmosphere with a flow of 50 mL.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and an open alumina crucible. The elemental analysis technique (CHN) was used to analyze the carbon content of the silica samples on a Perkin-Elmer model 2400 CHNSO instrument. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of mesoporous silica were obtained using the scanning electron microscope model JSM 6610 LV (JEOL) and the transmission electron microscope model JEM 1400 (JEOL) from the Carlos Alberto Redins Cellular Ultrastructure Laboratory (LUCCAR), respectively. Zeta potential analysis was performed in triplicate, with mesoporous silica samples diluted in 0.9 M NaCl solution using the Litesizer 500 instrument (Anton Paar). Nitrogen adsorption analyses were carried out using the Quantachrome SiQwin\u0026trade; instrument, which consists of an automated physical adsorption system that provides adsorption and desorption balance data. The mesoporous silica samples were degassed at 120\u0026deg;C for 2 hours prior to testing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Incorporation of 3-hydroxychalcone into mesoporous silica nanoparticles and \u003cem\u003ein vitro\u003c/em\u003e release assay\u003c/h2\u003e \u003cp\u003eA 3-hydroxychalcone solution at a concentration of 2 mg/mL was prepared in acetonitrile. Silica was weighed in the amount of 50 mg in an amber glass bottle with a lid, and the chalcone solution was added to this bottle. The solution was kept under magnetic stirring, and after two days of incorporation, it was filtered and dried at room temperature. CHN analyses were performed on the samples to evaluate the percentage of 3-hydroxychalcone adsorbed. For the release test, 10 mg of the incorporated silica sample was added to 10 mL of pH 7.4 PBS buffer solution. The prepared solution was placed in an orbital shaker incubator (INNOVA 4200 brand) at 37\u0026deg;C and agitated for up to 54 hours. To quantify the concentration of 3-hydroxychalcone released from the silica samples, aliquots were taken at specific time points and analyzed on a Shimadzu UV-VIS V-2401 spectrophotometer at a wavelength of 305 nm.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Assessment of the cytotoxicity of chalcones \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eDuring the study, 11 chalcones with different substituents were subjected to cytotoxicity tests against gastric adenocarcinoma (AGS) and breast cancer (MCF-7) cell lines in order to identify the most cytotoxic chalcone for subsequent incorporation into mesoporous silica nanoparticles. The results obtained showed that the chalcones exhibited significant cytotoxicity, with some showing better activity than cisplatin. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the IC\u003csub\u003e50\u003c/sub\u003e values and selectivity of the chalcones against AGS and MCF-7 cells versus fibroblast cells (L-929).\u003c/p\u003e\u003cp\u003e\u003cimg 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\" style=\"width: 850px; height: 769.371px;\" width=\"850\" height=\"769.371\"\u003e\u003cbr\u003e\u003c/p\u003e \u003cp\u003eIt was observed that most of the chalcones were more active against AGS and MCF-7 cells than against fibroblast cells (L-929), which shows that chalcones have greater selectivity for cancer cells than for normal cells, an important characteristic for anticancer agents. Furthermore, some of the chalcones showed a cytotoxic effect close to that of cisplatin against AGS and MCF-7 cells, with greater selectivity against these cell types than cisplatin.\u003c/p\u003e \u003cp\u003eIn general, the effects of the substituents on the cytotoxic activity of chalcones against AGS and MCF-7 cells were similar. Compound \u003cb\u003e1\u003c/b\u003e, the unsubstituted chalcone (benzalacetophenone), showed good cytotoxic activity, with IC\u003csub\u003e50\u003c/sub\u003e ranging from 53.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63 to 57.93\u0026thinsp;\u0026plusmn;\u0026thinsp;19.59 \u0026micro;M, which led us to investigate the influence of different substituents on the cytotoxicity of chalcones.\u003c/p\u003e \u003cp\u003eMethoxyl and hydroxyl groups are known to positively influence the cytotoxic activity of chalcones [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The presence of the methoxy substituent in position 3 of ring B had a positive effect on the cytotoxic activity of chalcone \u003cb\u003e4\u003c/b\u003e in both cancer cell lines (IC\u003csub\u003e50\u003c/sub\u003e from 44.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 to 48.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21 \u0026micro;M), while the methoxy group in position 2 of ring B (compound \u003cb\u003e3\u003c/b\u003e) was only beneficial for cytotoxicity against AGS cells (IC\u003csub\u003e50\u003c/sub\u003e 46.96\u0026thinsp;\u0026plusmn;\u0026thinsp;3.38 \u0026micro;M), and its effect against MCF-7 cells (IC50 of 81.04\u0026thinsp;\u0026plusmn;\u0026thinsp;16.52 \u0026micro;M) was negative compared with compound \u003cb\u003e1\u003c/b\u003e. Other tested chalcones with methoxy groups on the aromatic rings (compounds \u003cb\u003e2\u003c/b\u003e, \u003cb\u003e8\u003c/b\u003e and \u003cb\u003e10\u003c/b\u003e) showed higher IC\u003csub\u003e50\u003c/sub\u003e values, indicating that the position of the methoxy group on the aromatic ring significantly influences the activity of these compounds.\u003c/p\u003e \u003cp\u003eCompound \u003cb\u003e11\u003c/b\u003e (3-hydroxychalcone) showed significant cytotoxic activity against AGS and MCF-7 cells (IC\u003csub\u003e50\u003c/sub\u003e from 47.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 to 47.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.56 \u0026micro;M), showing that the presence of the hydroxyl group at position 3 of the ring has an important effect on the cytotoxicity of chalcones.\u003c/p\u003e \u003cp\u003eWhen analyzing chalcones with electron-withdrawing groups (chlorine, nitro) in their structure, it was found that chalcone \u003cb\u003e6\u003c/b\u003e (3-chlorochalcone) showed good cytotoxic activity (IC\u003csub\u003e50\u003c/sub\u003e from 44.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24 to 45.58\u0026thinsp;\u0026plusmn;\u0026thinsp;4.66 \u0026micro;M), while chalcones \u003cb\u003e5\u003c/b\u003e (3-chloro-4\u0026rsquo;-nitrochalcone), \u003cb\u003e7\u003c/b\u003e (4\u0026rsquo;-nitrochalcone), \u003cb\u003e9\u003c/b\u003e (4-nitrochalcone) and \u003cb\u003e10\u003c/b\u003e (4-methoxy-4\u0026rsquo;-nitrochalcone) showed moderate cytotoxic activity. The presence of the chlorine group in position 3 of ring B exerts an important influence on the activity of chalcones, while the presence of the nitro group in position 4 of ring A and ring B appears to have a negative effect on the cytotoxic activity, as can be observed in compounds \u003cb\u003e5\u003c/b\u003e, \u003cb\u003e7\u003c/b\u003e, \u003cb\u003e9\u003c/b\u003e and \u003cb\u003e10\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn general, the chalcones showed significant cytotoxic activity against AGS and MCF-7 cells, with chalcones \u003cb\u003e4\u003c/b\u003e (3-methoxychalcone), \u003cb\u003e6\u003c/b\u003e (3-chlorochalcone) and \u003cb\u003e11\u003c/b\u003e (3-hydroxychalcone) standing out. Considering the cytotoxicity profile in both tumor cell lines and its physicochemical characteristics, 3-hydroxychalcone was selected for incorporation into mesoporous silica nanoparticles. It is worth highlighting that the hydroxyl group present in 3-hydroxychalcone could favor interaction with the silanol groups of silica though hydrogen interactions. The functionalization process was subsequently carried out with the purpose of adding amino groups to the silica surface. The presence of these groups on the silica surface could further favor the incorporation process by increasing the hydrogen interactions between the nanoparticles and 3-hydroxychalcone, with the possibility of incorporating a greater amount of chalcone into the silica.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Scanning electron microscopy (SEM)\u003c/h2\u003e \u003cp\u003eScanning electron microscopy (SEM) is an important technique for studying the morphology (texture, shape and size) of materials. From SEM images, it was possible to estimate the shape of the mesoporous silica nanoparticles. The MSN and MSN-APTES silica (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) showed spherical particles, which is consistent with the results described in the literature [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, it was possible to observe aggregates of different sizes, which are common in nanometer-sized particles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Transmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eTransmission electron microscopy (TEM) was used to characterize the pore structure of MSN and MSN-APTES mesoporous silica. The images show that there were pores, which appeared in a spherical shape and in a lighter tone due to the silica nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). As observed in the SEM analysis, the silica particles were agglomerated, which can be considered a limiting factor in this type of analysis, as it makes it difficult to visualize the silica structures more clearly.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Fourier transform infrared spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eFTIR analysis was used to determine the chemical composition of the obtained mesoporous silica nanoparticles and the presence of chemical groups of the functionalizing agent on the silica surface. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the FTIR spectra of MSN and MSN-APTES nanoparticles. The characteristic bands at 1080, 810 and 460 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are related to the asymmetric stretching, bending (δ) and out-of-plane Si-O bonds, respectively. The infrared spectrum of the MSN sample showed peaks of hydroxyl stretching band for O-H axial deformation vibration mode of the Si-O-H group and water molecules at 3550 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1640 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The band at 960 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is typical of the silanol groups (Si-OH) present in the silica structure [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. After the functionalization process with APTES, this band showed a change in intensity. A decrease in the intensity of the band at 960 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was observed, which can be attributed to the reduction in the number of silanol groups present in the silica structure due to the process of binding of the functionalizing agent to these functional groups.\u003c/p\u003e \u003cp\u003eFor silica functionalized with APTES, a peak at 1560 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponding to the NH\u003csub\u003e2\u003c/sub\u003e deformation vibration was observed, indicating the presence of functionalizing amino groups on the surface of the mesoporous silica nanoparticles. Furthermore, the bands observed at 1460\u0026thinsp;\u0026minus;\u0026thinsp;1410 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond to CH-CH\u003csub\u003e2\u003c/sub\u003e bending [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These results are consistent with elemental analysis (CHN), in which an increase in the percentage of carbon and nitrogen was observed after the functionalization process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Zeta potential analysis\u003c/h2\u003e \u003cp\u003eZeta potential analysis was used to evaluate the surface electric charge of particles of the MSN and MSN-APTES samples and of the samples incorporated with 3-hydroxychalcone (MSN-CHO and MSN-APTES-CHO). Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the results.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eZeta potential analysis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZeta potential\u0026thinsp;\u0026plusmn;\u0026thinsp;DP (mV)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-6.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSN-CHO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-3.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSN-APTES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-2.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSN-APTES-CHO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-2.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMSN silica has a negative charge (-6.65 mV) due to the silanol groups present on its surface. After the functionalization process with APTES, a decrease in the negative charge of the functionalized particles was observed due to the contribution of the amino groups of the functionalizing agent. The functionalization of MSN silica with APTES promotes the formation of covalent bonds between the molecules of the silanol groups present on the silica surface and the aminosilane groups of the functionalizing agent. As a result, the negative charge of the nanoparticles decreased from \u0026minus;\u0026thinsp;6.65 mV in the MSN sample to -2.93 mV in the MSN-APTES sample. These results show that the functionalization process was efficient and are in line with what was described by Andrade et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. For the samples incorporated with 3-hydroxychalcone, a decrease in the negative charge of the particles was observed. This result can be attributed to the presence of chemical groups in the 3-hydroxychalcone molecule, which contribute to the reduction of the negative charge of the sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Nitrogen adsorption\u003c/h2\u003e \u003cp\u003eNitrogen adsorption analysis was used to obtain information such as specific surface area, pore diameter, distribution and total volume by measuring the area occupied by nitrogen molecules adsorbed on the silica surface [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The specific surface area values of the MSN silica sample were calculated using the BET method, and the total volume and pore diameter values were calculated using the BJH method (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNitrogen adsorption/desorption\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurface area m\u0026sup2;.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePore diameter BJH/DFT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVolume cc/g\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.391\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSNAPTES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.3 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.060\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe values obtained are characteristic of mesoporous materials, confirmed by pore diameter values from 3.3 to 3.5 nm, which are within the expected standard for mesoporous materials (2 to 50 nm) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The small variation in the pore diameters of the MSN (3.5 nm) and MSN-APTES (3.3 nm) samples evidences the occupation of the pores by the amino groups after the functionalization process.\u003c/p\u003e \u003cp\u003eThe surface area and pore volume were 115 m\u003csup\u003e2\u003c/sup\u003e/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.391 cc\u003csup\u003e3\u003c/sup\u003e.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the MSN sample and 20 m\u003csup\u003e2\u003c/sup\u003e/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.060 cc\u003csup\u003e3\u003c/sup\u003e.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the MSN APTES sample, respectively, indicating that the functionalization process of MSN silica with APTES was successful, given the decrease in surface area and pore volume. Although smaller, the surface area of MSN-APTES silica is large enough to incorporate molecules.\u003c/p\u003e \u003cp\u003eFurthermore, the isotherms of the MSN and MSN-APTES samples showed adsorption and desorption branches that formed a type H1 hysteresis, characteristic of mesoporous materials (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). A smaller volume of adsorbed nitrogen was found for the MSN-APTES sample than for the MSN silica in almost all relative pressures, indicating that the functionalization process was successful due to pore filling by the functionalizing agent, corroborating the results shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, in which smaller pore diameter and volume values can be observed for the MSN-APTES sample compared with the MSN sample.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Thermogravimetric analysis (TGA) and elemental analysis (CHN)\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the mass loss curves of the MSN, MSN-CHO, MSN-APTES and MSN-APTES-CHO samples. For the MSN sample, a mass loss of 3.7% was observed in the temperature range of 25\u0026deg;C to 150\u0026deg;C, which is related to the adsorption of water molecules on silica. From 150\u0026deg;C to 750\u0026deg;C, the mass loss was 9.5%, corresponding to the condensation of silanol groups [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. An increase in mass loss was also observed for the MSN-CHO sample from 150\u0026deg;C to 750\u0026deg;C (11.3%), which can be attributed to the thermal degradation of the incorporated 3-hydroxychalcone. This 1.8% increase in the percentage of mass loss in the sample incorporated with 3-hydroxychalcone (MSN-CHO) compared with the unloaded sample (MSN) indicates the presence of chalcone in this sample and also shows that the incorporation process was successful.\u003c/p\u003e \u003cp\u003eFor samples MSN-APTES and MSN-APTES-CHO, the mass loss in the temperature range of 150 to 750\u0026deg;C was 20.2% and 25.8%, respectively. The substantial mass loss for these samples in this temperature range can be attributed to the presence of amino groups of the functionalizing agent attached to the silica surface and the presence of 3-hydroxychalcone for the loaded sample, indicating that the functionalization and incorporation process was successful. The greater mass loss of MSN-APTES-CHO (5.6%) than MSN-CHO (1.8%) means that the incorporation process is more efficient in functionalized silica, due to the presence of amino groups on the silica surface, which favors the incorporation process by increasing the number of hydrogen interactions between silica and 3-hydroxychalcone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results obtained by thermogravimetric analysis are in line with those obtained using the elemental analysis technique (CHN). These results provided additional evidence that the silica samples were loaded with 3-hydroxychalcone and that the functionalization process was successful (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMass loss and elemental analysis (CHN) of mesoporous silica nanoparticle samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWeight loss (% m/m)\u003c/p\u003e \u003cp\u003e25\u0026ndash;150\u0026ordm;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWeight loss (% m/m)\u003c/p\u003e \u003cp\u003e150\u0026ndash;750\u0026ordm;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eResidue (%m/m)\u0026thinsp;\u0026gt;\u0026thinsp;800\u0026ordm;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e% Carbon\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e% Nitrogen\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e86.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e-0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSN-CHO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e-0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSN-APTES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e13.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.055\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSN-APTES-CHO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e67.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e14.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe MSN sample presented a carbon percentage of 5.43%, this percentage can be explained by the presence of the copolymer F127 that was not completely removed during the extraction process. After the incorporation process with 3-hydroxychalcone, the MSN sample (5.43%) presented an increase in the carbon percentage (5.91%), indicating that the incorporation process was successful. However, the carbon variations between the MSN and MSN-CHO samples were minimal, indicating that 0.48% (9.6 \u0026micro;g) of 3-hydroxychalcone was incorporated into the mesoporous silica nanoparticles at the tested concentration (2 mg/mL).\u003c/p\u003e \u003cp\u003eFurthermore, the MSN sample showed an increase in the percentage of carbon and nitrogen after the functionalization process (MSN-APTES), indicating that this process was successful and that the binding of the functionalizing agent 3-aminopropyltriethoxysilane occurred on the silica surface. An increase in the percentage of carbon was also observed in the functionalized sample incorporated with 3-hydroxychalcone (MSN-APTES-CHO). For APTES silica, the incorporation rate was found to be 1.28% or 25.6 \u0026micro;g\u0026mdash;almost three times higher than the rate found for the MSN-CHO sample (0.48%). Functionalization with APTES favors the incorporation process due to hydrogen interactions between the amino groups of the functionalized silica and the 3-hydroxychalcone molecules, resulting in a higher incorporation rate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.8. 3-hydroxychalcone loading and release\u003c/h2\u003e \u003cp\u003eAfter the incorporation process, the amount of 3-hydroxychalcone loaded was 0.48% or 9.6 \u0026micro;g for the MSN-CHO sample and 1.28% or 25.6 \u0026micro;g for the MSN-APTES-CHO. These values were obtained using the elemental analysis (CHN).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the release profile of the MSN-CHO and MSN-CHO-APTES samples. For MSN-CHO, an initial rapid release of 3-hydroxychalcone can be observed, around 30% (2.88 \u0026micro;g) in one hour, followed by a gradual and prolonged release, with 52.60% (5.05 \u0026micro;g) of chalcone released after 54 hours. These results can be explained by the interaction between the silanol groups of silica and the hydroxyl and carbonyl groups of 3-hydroxychalcone via hydrogen bonding, resulting in a slower release of 3-hydroxychalcone from the silica matrix.\u003c/p\u003e \u003cp\u003eThe MSN-APTES-CHO sample showed a slower release, with 16.84% (4.31 \u0026micro;g) of 3-hydroxychalcone released in 54 hours. This characteristic can be explained by a possible more intense interaction between 3-hydroxychalcone and functionalized silica. The addition of amino groups to the silica surface can promote a greater number of hydrogen interactions between the nanoparticles and 3-hydroxychalcone, leading to slower release rates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings show that mesoporous silica nanoparticles can be adapted to provide an initial release of an adequate dose of the bioactive compound in a short period of time, followed by a slow and gradual release of the drug over a long period of time, which can be useful for maintaining constant compound levels and action and reducing side effects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Evaluation of the cytotoxicity of silica samples \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eSilica samples incorporated with 3-hydroxychalcone (MSN-CHO and MSN-APTES-CHO) and isolated silica samples (MSN and MSN-APTES) were subjected to cytotoxicity tests against gastric adenocarcinoma (AGS) and breast cancer (MCF-7) to verify the effectiveness of these systems in enhancing the cytotoxic activity of 3-hydroxychalcone.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the IC\u003csub\u003e50\u003c/sub\u003e values and selectivity of the silica samples against AGS, MCF-7 and fibroblast cells (L-929).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M) and selectivity index (SI) of silica samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e L-929\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e AGS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e MCF-7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIS AGS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIS MCF-7\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3-hydroxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e275.03\u0026thinsp;\u0026plusmn;\u0026thinsp;5.96*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.78*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.75*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;6000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;6000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;6000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSN-CHO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.30\u0026thinsp;\u0026plusmn;\u0026thinsp;4.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.16**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSN-APTES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1619\u0026thinsp;\u0026plusmn;\u0026thinsp;69.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;7300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;7300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSN-APTES-CHO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.67\u0026thinsp;\u0026plusmn;\u0026thinsp;8.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e106.67\u0026thinsp;\u0026plusmn;\u0026thinsp;7.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80.98\u0026thinsp;\u0026plusmn;\u0026thinsp;14.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCisplatin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.03\u0026thinsp;\u0026plusmn;\u0026thinsp;7.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.85\u0026thinsp;\u0026plusmn;\u0026thinsp;4.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e and IS of chalcones and cisplatin (*: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; **: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared with cisplatin).\u003c/p\u003e \u003cp\u003eThe silica samples loaded with 3-hydroxychalcone showed significant cytotoxic activity (IC\u003csub\u003e50\u003c/sub\u003e from 12.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 to 106.67\u0026thinsp;\u0026plusmn;\u0026thinsp;7.80 \u0026micro;M) against AGS and MCF-7 cells, with the MSN-CHO sample showing a better cytotoxic effect (IC\u003csub\u003e50\u003c/sub\u003e from 12.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 to 22.30\u0026thinsp;\u0026plusmn;\u0026thinsp;4.15 \u0026micro;M) than free 3-hydroxychalcone (IC\u003csub\u003e50\u003c/sub\u003e from 47.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 to 47.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.56 \u0026micro;M). These results indicate that the loaded mesoporous silica nanoparticles significantly influenced the interaction of chalcone with tumor cells, requiring lower concentrations of 3-hydroxychalcone to exert greater cytotoxic activity compared with free chalcone. While the mechanism of cellular uptake of 3-hydroxychalcone can occur by passive diffusion [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], for mesoporous silica nanoparticles, absorption occurs by endocytosis. Studies have reported that mesoporous silica nanoparticles have a high affinity for plasma membrane phospholipids, and the adsorption of these molecules on cell surfaces would lead to absorption via endocytosis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs can be seen, the silica samples showed variations in cytotoxic activities. Even with higher incorporation rates, the MSN-APTES-CHO sample showed higher IC\u003csub\u003e50\u003c/sub\u003e values (80.98\u0026thinsp;\u0026plusmn;\u0026thinsp;14.76 to 106.67\u0026thinsp;\u0026plusmn;\u0026thinsp;7.80 \u0026micro;M) than the MSN-CHO sample (12.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 to 22.30\u0026thinsp;\u0026plusmn;\u0026thinsp;4.15 \u0026micro;M), which had a low incorporation rate. These differences in cytotoxic activity can be explained by the different release profiles of the silica samples. As described in the release test, the MSN-CHO sample showed a better release profile, with a greater amount of 3-hydroxychalcone released over time than the MSN-APTES-CHO sample, due to more intense interactions between 3-hydroxychalcone and functionalized silica by hydrogen bonds. This type of interaction affects the release profile of the functionalized nanoparticles and consequently their cytotoxic activity, resulting in higher IC\u003csub\u003e50\u003c/sub\u003e values.\u003c/p\u003e \u003cp\u003eIn general, the silica samples showed greater cytotoxic activity against AGS and MCF-7 cells than against fibroblast cells (L-929), with the exception of the MSN-APTES and MSN-APTES-CHO samples, which showed greater cytotoxic activity against fibroblast cells than against cancer cells. These results can be explained by the characteristics of the cells tested. The APTES silica surface is normally positively charged under the conditions in which the tests were carried out, promoting greater interaction with the plasma membranes of normal cells, which are negatively charged and attract the functionalized nanoparticles via electrostatic interactions. These interactions may cause a destabilization of the plasma membrane, increasing its permeability and allowing for a greater effect of 3-hydroxychalcone on fibroblast cells. The plasma membranes of tumor cells show changes in the composition and organization of lipids, which cause several changes in cellular functions such as permeability and transport of substances across the plasma membrane [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These changes may result in less intense interactions between functionalized silica nanoparticles and tumor cells, which would explain the results found in the cytotoxicity test.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThe results showed that synthetic chalcones exhibited significant cytotoxic activity against AGS and MCF-7 cells, especially 3-hydroxychalcone, which showed the best antitumor activity and was selected for incorporation studies with mesoporous silica nanoparticles.\u003c/p\u003e \u003cp\u003eThe synthesis, functionalization and characterization of the nanoparticles and the 3-hydroxychalcone incorporation process were successful. Furthermore, the functionalization of nanoparticles was found to influence the incorporation process and the release profile of the samples due to the intense interactions of 3-hydroxychalcone with the functionalized silica.\u003c/p\u003e \u003cp\u003eBased on the cytotoxicity test, the combination of 3-hydroxychalcone incorporated in silica matrix showed effective cytotoxic activity against AGS and MCF-7 cells. The MSN-CHO sample showed better cytotoxic activity than free 3-hydroxychalcone, demonstrating that the incorporation into the nanoparticles enhances cytotoxicity against tumor cells. Our findings highlight that MSN-CHO is a potentially effective agent against tumor cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThe authors thank Fundação de Amparo à Pesquisa e Inovação do Espírito Santo (FAPES) for financial support (grant number 2022-78KWB) and technical support and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for scholarship to RRK (process number 314276/2021-1).\u0026nbsp;Federal University of Espírito Santo for providing the structure necessary to accomplish this work and technical support of Laboratory Ultra Cellular Structure Carlos Alberto Redins (LUCCAR/ UFES) and Laboratory of Nanostructured Materials for Bioapplications (LMNB - CDTN/CNEN).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: RRK, GFA, GJSS. Formal Analysis: GJSS, RAR. Investigation: GJSS, RAR Methodology: GJSS, RAR, RBS. Project administration: RRK, GFA. Resources: RRK, RCRG. Visualization: GJSS, RRK, GFA. Writing-original draft: GJSS, RRK, GFA. Writing - review \u0026amp; editing: GJSS, RRK, GFA.\u0026nbsp;Supervision: RRK and GFA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES)” – [grant number 2144/2022];\u0026nbsp;Fundação de Amparo à Pesquisa e Inovação do Espírito Santo\u0026nbsp;(FAPES) – [grant numbers\u0026nbsp;2022-78KWB].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOuyang Y, Li J, Chen X, Fu X, Sun S, Wu Q (2021) Chalcone derivatives: Role in anticancer therapy. 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Front Cell Dev Biol 8:571237. https://doi.org/10.3389/fcell.2020.571237\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":"journal-of-nanoparticle-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nano","sideBox":"Learn more about [Journal of Nanoparticle Research](http://link.springer.com/journal/11051)","snPcode":"11051","submissionUrl":"https://submission.nature.com/new-submission/11051/3","title":"Journal of Nanoparticle Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"3-hydroxychalcone, anticancer, mesoporous silica nanoparticle, MSN-chalcone, MSN-APTES-chalcone.","lastPublishedDoi":"10.21203/rs.3.rs-4676216/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4676216/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe difficulty in treating cancer has led to several studies on the development of systems that perform targeted drug delivery, with the aim of increasing the effectiveness of treatment and reducing adverse effects. In this study, a series of chalcones were tested for cytotoxic action on gastric adenocarcinoma cells (AGS) and breast cancer cells (MCF-7) using the MTT-tetrazolium method, and significant cytotoxicity was demonstrated for 3-hydroxychalcone (CHO). The synthesis of mesoporous silica nanoparticles (MSNs) and their surface modification with 3-aminopropyltriethoxysilane (APTES) were carried out, and 3-hydroxychalcone was then incorporated into these nanomaterials. Mesoporous silica nanoparticles were characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), elemental analysis (CHN), scanning electron microscopy (SEM), transmission electron microscopy (TEM), zeta potential and nitrogen adsorption. In addition, \u003cem\u003ein vitro\u003c/em\u003e release tests were carried out to verify the release profile of 3-hydroxychalcone from mesoporous silica samples. The results obtained showed that the mesoporous silica nanoparticles exhibited a gradual and prolonged release profile. In the cytotoxicity test with silica samples incorporated with 3-hydroxychalcone, significant cytotoxic activity was observed against AGS and MCF-7 cells, with the MSN-CHO sample exhibiting a better cytotoxic effect (IC\u003csub\u003e50\u003c/sub\u003e of 12.93 to 22.30 μM) than 3-hydroxychalcone (IC\u003csub\u003e50\u003c/sub\u003e of 47.58 to 47.97 μM). The results showed that the nanoparticles positively influenced the interaction of 3-hydroxychalcone with tumor cells. This is therefore an unprecedented study on the incorporation of 3-hydroxychalcone into mesoporous silica nanoparticles and its promising results in terms of cytotoxic activity against breast and gastric cancer cells.\u003c/p\u003e","manuscriptTitle":"Anticancer potential of chalcones loaded on mesoporous silica nanoparticles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 14:23:50","doi":"10.21203/rs.3.rs-4676216/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-04T16:05:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-17T17:53:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178620829743796285564037745257016803202","date":"2024-08-08T20:45:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-08T09:09:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-05T20:48:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-04T14:18:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanoparticle Research","date":"2024-07-02T19:42:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanoparticle-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nano","sideBox":"Learn more about [Journal of Nanoparticle Research](http://link.springer.com/journal/11051)","snPcode":"11051","submissionUrl":"https://submission.nature.com/new-submission/11051/3","title":"Journal of Nanoparticle Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b2641533-9b05-447c-b118-0650a8d06360","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-31T16:04:35+00:00","versionOfRecord":{"articleIdentity":"rs-4676216","link":"https://doi.org/10.1007/s11051-025-06292-4","journal":{"identity":"journal-of-nanoparticle-research","isVorOnly":false,"title":"Journal of Nanoparticle Research"},"publishedOn":"2025-03-28 15:57:23","publishedOnDateReadable":"March 28th, 2025"},"versionCreatedAt":"2024-07-29 14:23:50","video":"","vorDoi":"10.1007/s11051-025-06292-4","vorDoiUrl":"https://doi.org/10.1007/s11051-025-06292-4","workflowStages":[]},"version":"v1","identity":"rs-4676216","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4676216","identity":"rs-4676216","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}