Synergistic action of curcumin and cisplatin on spinel ferrite/hierarchical MCM-41 nanocomposite against MCF-7, HeLa and HCT 116 cancer cell line

Platinum-based drugs are widely used in cancer therapy, but are known for toxic side effects and resistance. Combinational drug delivery represents an effective chemotherapeutic strategy, but often leads to an increased toxicity. Aim of this study is to test the co-delivery of cisplatin with natural antioxidants on hierarchial porous large surface area hexagonal nanocarriers for synergistic action. A series of structured mesoporous materials were impregnated with magnetic spinel ferrite (30% CuFe2O4) and then coated with curcumin (25% wt/wt). Mesosilicalite and MCM-41 with high curcumin release abilities were functionalized with cisplatin (5% wt/wt) for synergistic effect of combinational drugs. The cytotoxic efficiency of our nanocomposites was tested on cell viability of MCF7 (human breast cancer), human cervical cancer (HeLa), colorectal cancer (HCT116), and HFF (human foreskin fibroblasts) cell lines using the MTT cell viability assay. At a concentration of 0.1 mg/ml, CuFe2O4/mesosilicalite/curcumin/cisplatin resulted in 89.53% reduction in viability in MCF7, 94.03% in HeLa, 64% in HCT116 and 87% in HFF; whereas, CuFe2O4/MCM-41/curcumin/cisplatin resulted in 76% reduction in viability in MCF7, 64.46% in HeLa, 64% in HCT116 and 24% in HFF. The EC50 for CuFe2O4/mesosilicalite/curcumin/cisplatin was 81.23 µg/ml in MCF7, 47.55 µg/ml in HeLa, 48.96 µg/ml in HCT116 and 76.83 µg/ml in HFF. The EC50 for CuFe2O4/MCM-41/curcumin/cisplatin was 72.51 µg/ml in MCF7, 58.6 µg/ml in HeLa, 62.58 µg/ml in HCT116 and 154.2 µg/ml in HFF. Furthermore, cells treated with both nanocomposites had a high number of cleaved Caspase 3-positive cells suggesting that the reduction in cell viability was triggered by activating the apoptotic signaling pathway. Our results show that CuFe2O4/MCM-41/curcumin/cisplatin is a better candidate for combinational drug therapy due to its lowest EC50 value and the wider difference in EC50 (a fold change) between cancerous and non-cancerous cell line.


Introduction
In recent years, magnetic nanoparticles in nanomedicine research have been steadily rising for developing targeted drug delivery system for various biomedical applications (Amiri et al. 2019). Spinel ferrite-based nanocomposites have been widely studied in biomedical applications such as hyperthermia, contrasting agents in tumor imaging, drug delivery and magnetic separation of biological specimens (Kefeni and Mamba 2020). The use of magnetic nanocomposites in drug delivery has been reported to increase the targeted drug delivery, reduce the dose, and enhance patient compliance (Ghosh et al. 2019). Among the different types of spinel ferrites, CuFe 2 O 4 is an interesting magnetic nanoparticle due to its superparamagnetic nature, strong T2 MRI contrast, ability to be guided by an external magnetic field, tunable particle size, low synthesis cost, and its eco-friendly characteristics (Khanna et al. 2019). CuFe 2 O 4 was composed of a cubic inverse spinel structure in the Fd3m space group (Dippong et al. 2021). Several nanocarriers with functionalization property based on hydrogels, alginate, clay, liposomes and polymeric species based therapeutic tools have been reported for treating chronic diseases (Li et al. 2019;Mantha et al. 2019;Abduljauwad and Ahmed 2019;de Lima et al. 2018). Mesoporous hexagonal structured silica MCM-41 with superior textural features with high surface area (~ 500-1000 m 2 /g) has been used as catalyst in fine chemical synthesis and gas adsorption (Beck et al. 1992;Lanzafame et al. 2020;Chen et al. 2020). The large hexagonal shaped pore size of Si-MCM-41 is built with amorphous silica framework. ZSM-5/MCM-41 hybrid composite material that are derived from zeolite contains crystalline framework with presence of micropore and mesopores Odedairo et al. 2012). The framework of such hierarchical hexagonal structure of composite is stable due to nanozeolitic building units .
Recently, using multiple drug-loaded nanoparticles had attracted interest as a cancer treatment option to overcome drug resistance. Xylan, a polysaccharide biopolymer modified with a redox-sensitive conjugate disulfide linker, has been explored as a drug delivery system. The delivery of curcumin and 5-fluorouracil using xylan nanoparticles resulted in a significant inhibition of cell viability in the colon carcinoma cells HT-29 and HCT-15 (Sauraj et al. 2020). Biocompatible polymers involving polyethylene glycol and poly(lactic-co-glycolic acid) exhibited a superior release of antineoplastic drug (7-ethyl-10-hydroxycamptothecin) and curcumin. The nanocomposite formulation with hydrophobic coating (dioleoylphosphatidic acid) has shown significant reduction in cell viability of cervical and ovarian cell lines (HeLa, A2780) (Li and Gao 2020). Layerby-layer deposition of natural polysaccharide chitosan and negatively charged dextran sulfate has been reported using double-emulsion cross-linking technique. Chitosanpaclitaxel was prepared using internal oil in water emulsion. After addition of external water to oil emulsion, dextran sodium and chitosan bound 5-fluorouracil were added and then lyophilized. The dual drug-loaded nanoparticles with particle diameter of about 292 nm were reported to induce significant reduction in cell viability of HepG2 cells (Wang et al. 2020). A bilayer structured vesicle Niosome functionalized with folic acid was found to be effective as a dual drug carrier of curcumin and letrozole. Nonionic surfactant Span 80 and cholesterol (lipid) to drug ratio optimization resulted in high drug encapsulation. The synergism between both drugs was observed to inhibit the viability of breast cancer cells  leading to downregulation of Bcl2, cyclin D and cyclin E genes and upregulation of p53, Bax, caspase-3 and caspase-9 genes (Akbarzadeh et al. 2020). We have synthesized several zeolites and expanded their applications from traditional petrochemical applications to biomedical applications including drug delivery and diagnostic applications Jermy and Ravinayagam 2019). Curcumin is less-soluble antioxidant and therefore has lower bioavailability that prevents the advantages of curcumin for therapeutics (Karthikeyan et al. 2020). The presence of curcumin along with Pt compound over mesoporous drug delivery system has multiple advantages. The encapsulation of curcumin inside the nanopores is expected to improve the solubility, bioavailability and target specificity. The presence of cisplatin can influence a synergistic effect with curcumin and spinel is expected to be beneficial for imaging. Till now, the combinational effect of curcumin and cisplatin was not explored on spinel-based hierarchical micro/mesoporous materials like mesosilicalite and mesoporous MCM-41.
In the present study, the effect of copper spinel impregnation into mesosilicalite and MCM-41 was explored for dual release of curcumin and cisplatin. The study shows that spinel, curcumin and cisplatin loading varies with pore structures of mesosilicalite and MCM-41. Micro-and meso-pores containing mesosilicalite is more potent as a cytotoxic agent in both cell lines. However, mesoporous MCM-41 is a better candidate due to the wider difference in cytotoxic capability in cancerous and non-cancerous cell lines.

Preparation of 30%CuFe 2 O 4 /mesosilicalite and 30%CuFe2O4/MCM-41
Mesosilicalite was prepared by disintegrating silicalite crystals in alkaline medium following the top-down approach. A detailed synthesis procedure for mesosilicalite, MCM-41, SBA-16, and mesobeta are provided in our previous publication (Ravinayagam and Jermy 2017). The copper ferrite-impregnated mesosilicalite and MCM-41 were prepared by dry mixing. Briefly, 0.61 g of copper nitrate trihydrate, 1.01 g of iron nitrate nonahydrate and 1.4 g of predried structured silica were physically mixed for 30 min using mortar pistol. The obtained mixture was calcined at 850 ℃ for 6 h.

Curcumin loading over 30%CuFe 2 O 4 /structured silicas
In the first step, curcumin was loaded on a series of copper spinel-loaded structured silicas. 40 mg of curcumin was dissolved in 10 ml of methanol for 10 min. Then 160 mg of respective spinel-loaded structured silicas (mesosilicalite, MCM-41, SBA-16, Q10 silica, hydrophobic silica, mesobeta, mesoZSM-5) was added and the mixture was sonicated for 2 min. Then the solvent was evaporated using rotary evaporator.
(10 ml) and stirred to form a clear solution. Then, CuFe 2 O 4 /mesosilicalite/curcumin or CuFe 2 O 4 /MCM-41/curcumin (600 mg) was added and stirred overnight under ice cold dark environment. The solution was then filtered, washed and dried. The functionalized cisplatin was estimated using UV-visible spectroscopy at 208 nm.

Curcumin release study
In the first step, curcumin release was studied over different copper spinel-loaded structured silicas. The release study was carried out by dissolving 30 mg of curcumin-loaded sample in 50 ml of PBS (pH 5.6). The curcumin release was monitored at 37 °C. At regular time interval, 10 ml of solution was withdrawn and replaced with equal volume of fresh solution. The curcumin release content was identified at specific wavelength of 428 nm. CuFe 2 O 4 /mesosilicalite/curcumin/cisplatin and CuFe 2 O 4 /MCM-41/curcumin/ cisplatin were chosen for cisplatin release. The cisplatin release was measured at 208 nm using UV-visible spectroscopy.

Cell culture
We used human mammary adenocarcinoma (MCF7)), human cervical cancer (HeLa), colorectal cancer (HCT116), and the non-cancerous human foreskin fibroblasts (HFF) cell lines to assess the cytotoxic effect of our compounds. Cells were cultured in a DMEM culture medium containing 10% HI-FBS, 1% penicillin streptomycin, and 1% MEM NEAA. Cells were maintained in a humified setting at 37 °C with 5% CO 2 . For cell viability assay, cells were seeded in a 96-well plate with a density of 20,000 cells/well. For immunofluorescent staining, 50,000 cells/well were plated on eight-well chamber slides. On the following day, cells were switched to the starve culture medium that contains 0.5% HI-FBS. On the next day, cells were treated with our nanocomposites as described in the following section. /mesosilicalite concentration was 0.5 mg/ml, there is 0.125 mg/ml of adsorbed curcumin and 0.0225 mg/ ml of functionalized cisplatin. Therefore, the treatment concentrations of curcumin were: 0.00625, 0.0125, 0.025 and 0.125 mg/ml and the treatment concentrations of cisplatin were: 0.001125, 0.00225, 0.0045 and 0.0225 mg/ml.

Cell viability (MTT) and EC50
To assess the cytotoxicity of our compounds, we used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a cell viability assay. It measures the cell viability by assessing the ability of mitochondria to convert yellow MTT solution into purple formazan insoluble crystals. Following Mosmann (1983) protocol, 5 mg/ml of MTT powder was dissolved in PBS, and 0.5 mg/ml of MTT working solution was prepared. Cells were washed with PBS and followed by the addition of 100 µl MTT working solution. All the experimental conditions were run in triplicates (technical repeats) with five biological repeats (n=5). The 96-well plate was incubated for 3 h at 37 °C. An MTT negative control (background control) was included in the experimental setting by adding MTT working solution to wells that contain no cells. After the incubation time, 0.04 N HCl isopropyl alcohol was added to solubilize the formazan crystals. The difference in color intensity was measured by  BioTek ELISA reader at 570 nm. The technical triplicate readings of each condition were averaged, and the absorbance from MTT negative control was deducted from these readings. An initial reading was measured before MTT addition to exclude background interference. The initial reading was subtracted from the final reading. Then, the treatment groups were analyzed by comparing them to the control (untreated cells). Cell viability was calculated using the following equation: Data from the cell viability assay were used to calculate the half-maximal effective concentration (EC 50 ) using Prism 9 software (GraphPad, La Jolla, CA). Concentration values % Cell viability = ×100.
were transformed to logarithmic data. Then, a sigmoidal curve was selected to fit the data and generate the calculated log EC 50 , EC 50 , and R 2 values for each condition.

Immunofluorescent staining
As mentioned above, cells were plated in an 8-well chamber slide at a concentration of 50,000 cells/well. Cells were then treated for 48 h with the following conditions: CuFe 2 O 4 /mesosilicalite/curcumin, CuFe 2 O 4 /mesosilicalite/curcumin/cisplatin, and CuFe 2 O 4 /MCM-41/curcumin/cisplatin at a concentration of 0.5 mg/ml. Cells were fixed and stained with the apoptotic marker cleaved Caspase 3 (c-Caspase 3) antibody (1:200, Cell Signaling Technology) and incubated at 4 °C overnight. After PBS washing, Alexa Fluor 594-conjugated secondary antibody (Invitrogen, Thermo Fisher Scientific) was added to the cells at a final concentration of 1:1000 for 1 h at room temperature. Cells were then washed and stained with the nuclear stain Hoechst 33,342 (Thermo Fisher Scientific) at a concentration of 2 µg/ml and incubated at room temperature for 20 min. After staining, immunofluorescent images were taken using a confocal fluorescent microscope-Zeiss LSM 700. Bright-field images were captured using an inverted microscope-Nikon Eclipse TS100. Although both light and fluorescent pictures were taken from the same sample, they were not taken from the same field of view.

Statistics
The cell viability assay was performed in five independent experiments (n=5). Statistical analysis was performed using Prism 9 software (GraphPad, La Jolla, CA). The analysis was performed using one-way ANOVA with Dunnett's post hoc test. Error bars ± S.E.M. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 versus control. In case there was no indication of significance, it means that results were non-significant. The data analysis of drug delivery was done using Prism 8 software and SPSS software version 20.0.

Results and discussion
The X-ray diffraction patterns of meso-and micro-phases of two supports were identified at low and high angle of MCM-41 and mesosilicalite (Fig. 1). The XRD pattern of CuFe 2 O 4 impregnated over different structured nanocarriers is shown in Additional file 1: Fig. S1. Conventional MCM-41 showed a typical hexagonal peaks at low angle corresponding to the plane (100), (110) and (200). Mesosilicalite showed the presence of MCM-41 and high silica zeolite peaks at 2 theta value of 7.9° and 8.7° corresponding to plane (101) and (200), respectively. In the higher angle (20-60°), a broad peak of MCM-41 indicates the presence of amorphous framework bound to hexagonal phase. Mesosilicalite showed the typical characteristics peaks of MFI structure with corresponding to (321), (113), (501), (422) and (313) plane. In case of spinel impregnated MCM-41 and mesosilicalite, the presence of cubic phase of copper spinel appears with intense peak at 35.6° corresponding to (103) plane. A trace of α-Fe 2 O 3 appears along with a less intense peak corresponding to CuO at 38.7°.
The characteristics of textural surface, pore volume and average pore size of (a) MCM-41, (b) CuFe2O4/MCM-41, (c) mesosilicalite and (d) CuFe2O4/mesosilicalite are shown in Fig. 2. The surface area and pore size distribution of CuFe 2 O 4 impregnated over different structured materials are shown in Additional file 1: Figs. S2 and S3. Before, spinel ferrite impregnation, MCM-41 showed a typical type IV isotherm indicating the presence of uniform pore size distributions (3.7 nm) with high surface area of 914 m 2 /g and pore volume of 0.85 cc/g (Additional file 1: Table S1). In case of pure hexagonal mesopores of MCM-41, surprisingly, impregnation of spinel, only slightly reduced the surface area to 885 m 2 /g, while pore volume reduced to 0.52 cc/g. In case of mesosilicalite, which contains micro-and mesopores, the initial surface area of 804 m 2 /g reduced significantly after impregnation to 74 m 2 /g. An increase in dual pore sizes from 3.0 nm to 5.9 nm indicates the pore-filling effect and formation of external pores in the hierarchical micro-and meso-pores after spinel ferrite loading. Pore volume reduction from 0.6 cc/g to 0.1 cc/g reflects the pore-filling effect. Overall, the result shows unique deposition of spinel occurs over two supports. The textural characteristics vary depending on the structure of different shaped materials (Additional file 1: Table S1). The rearrangement of curcumin and cisplatin over CuFe 2 O 4 /mesosilicalite was analyzed using TEM, FTIR and diffuse reflectance spectra analysis ( Fig. 3A-F). The morphological feature of mesosilicalite/Pt without curcumin coating and after coating sample CuFe 2 O 4 /mesosilicalite/curcumin/cisplatin was analyzed using transmission electron microscope at different scale bar of 100 nm, 50 nm and 10 nm (Fig. 3A-D). In case of mesosilicalite/Pt sample, the presence of micro-and meso-phase is clearly seen. The presence of discontinuity as white dots indicates the nanozeolitic microphase, while continued pore channels shows the hexagonal mesopore ordering (Fig. 3A). After curcumin coating, the images shows the presence of curcumin at the external surface of MCM-41 ( Fig. 3B and C). Further magnification to 10 nm, a uniformly layered hexagonal pore channels can be clearly seen to coexist with curcumin (Fig. 3D). The FT-IR spectra of cisplatin, curcumin, CuFe 2 O 4 /mesosilicalite, CuFe 2 O 4 /mesosilicalite/curcumin and CuFe 2 O 4 /mesosilicalite/curcumin/cisplatin are shown in Fig. 3E(a-e). Drug cisplatin showed a characteristic functional group related to NH group of platinum complex between 400 and 1800 cm −1 . A symmetric and asymmetric bending of NH 2 group was clearly observed between 1300 and 1600 cm −1 , while plane bending of cisplatin can be seen at about 800 cm −1 (Fig. 3Ea). In case of curcumin, the presence of carbonyl (C=O), carbon-carbon double bond (C=C), and methylene (CH 2 ) bending peaks are observed between 1625 and 1450 cm −1 . Both symmetric and asymmetric vibrations corresponding to ether bond (C-O-C) are observed between 1300 and 1000 cm-1 (Bhandari et al. 2016) (Fig. 3Eb). A peak corresponding to enolic OH group of curcumin appears clearly at about 960 cm −1 . In case of CuFe 2 O 4 /mesosilicalite, a zeolitic peak indicating hybrid formation between mesoporous and microporous zeolite appears at about 550 cm −1 (Fig. 3Ec). After curcumin coating (curcumin loading step 1), the sample CuFe 2 O 4 /mesosilicalite/curcumin showed no distinct peaks of curcumin. However, a significant reduction in hydroxyl group of enol indicates an effective interaction inside the mesosilicalite pores and curcumin (Fig. 3Ed). Unexpectedly, the CuFe 2 O 4 /mesosilicalite/curcumin/cisplatin (cisplatin loading step 2) sample, the loading of cisplatin showed the peaks of curcumin. It indicates that during cisplatin functionalization step, curcumin present inside the pores of mesosilicalite diffuses out and present at the external surface of mesosilicalite (Fig. 3Ee). In addition, a peak at about 1023 cm −1 indicates the C-O-C stretching of C 6 H 5 -O-CH 3 group (Mohan et al. 2012). Also, the presence of cisplatin peaks reveals an effective functionalization of drug at external surface of mesosilicalite.  Fig. 3Fa and b). The support SiMCM-41 showed the absorption bands at about 210 and 260 nm, indicating the framework coordinated siliceous species (Fig. 3Fc). In case of CuFe 2 O 4 /MCM-41 and CuFe 2 O 4 /mesosilicalite, the spectra showed a weak absorption below 230 nm and a strong and broad absorption between 240 and 800 nm ( Fig. 3Fd and e). The peaks of CuFe 2 O 4 /MCM-41 and CuFe 2 O 4 / mesosilicalite correlate with cubic spinel exhibits tetrahedral (215 nm) and octahedral (440-700 nm) crystalline coordination sites (Najmoddin et al. 2014). The presence of such peak absorption indicates the dispersion and integrated spinel ferrites over both supports. Importantly, in case of mesosilicalite support, an enhanced intense broad peak shows the presence of higher crystalline mixed phase of oxides due to octahedral coordinated spinel species compared to the MCM-41 support. This can be attributed mainly due to presence of micropores, which tends to accommodate spinel species at the external surface of mesosilicalite. After loading of curcumin and cisplatin, the absorption maximum increases significantly over both CuFe 2 O 4 /mesosilicalite and CuFe 2 O 4 / MCM-41 nanocomposites. However, in case with CuFe 2 O 4 /mesosilicalite/curcumin, a two distinct band absorptions at about 400 nm and 500 nm clearly indicates the distributed curcumin and Pt species (Fig. 3Ff ). However, with CuFe 2 O 4 /MCM-41/curcumin, a broadness of absorption peak indicates cohabitation of curcumin and Pt species over MCM-41 (Fig. 3Fg). Such increase in homogenous expansion behavior clearly indicates the composite formation over mesoporous MCM-41 support than with mesosilicalite. As detected in XRD and DRS-UV analysis (Figs. 1 and 3F), though the active spinel metal components were not observed in TEM analysis, the migration of curcumin can be clearly observed as a surface coating at the external surface of mesosilicalite.
The magnetic characteristics and saturation value of CuFe 2 O 4 /MCM-41 and CuFe2O4/ mesosilicalite nanocomposites were analyzed by vibrating sample magnetometer at room temperature (Additional file 1: Fig. S4a and b). A distribution of cations at different coordination sites of A and B characterize the magnetic property. CuFe 2 O 4 /MCM-41 and CuFe 2 O 4 /Mesosilicalite showed ferromagnetic property with saturation magnetization value of about 0.9 emu/g. The saturation magnetization value was reported to be related to the magnetic phase concentration. Fe loaded on high surface area MCM-41 was reported to exhibit magnetization of 3.86 emu/g (Kiatphuengporn et al. 2016). In our previous study, spinel impregnation over spherical silica with lower surface area of 178 m 2 /g was found to exhibit similar ferromagnetism with magnetic value of about 7.6 emu/g . Further, it has been reported that superparamagnetic effect was due to anti parallel spins of Fe 3+ species in tetrahedral coordination site. In present study, the influence of support has shown to influence the magnetic property. The loading of spinel over mesosilicalite support tends to generate different types of nanoclusters at the pore walls (as evidenced by increased pore diameter from 3.0 to 5.9 nm). The generation of small sized nanoclusters reported to generate super paramagnetic Fe 3+ ions, while larger nanoclusters generates ferromagnetic behavior (Cuello et al. 2017). In line with the diffuse reflectance spectra, presence of tetrahedral and octahedral species over high surface area parent mesosilicalite and MCM-41 is proposed to lead the broad hysteresis structure characteristics of ferromagnetism. The observed reduction in saturation magnetization value is mainly attributed due to presence of siloxane layers on copper spinel ferrites.
Nanostructured porous materials as drug delivery system facilitates the targeted drug delivery, reduce the in vivo dosage fluctuation, reduce drug disintegration and side effects of drugs (Ghaferi et al. 2020). In present study, a different structured nanocarriers for drug delivery was evaluated by studying the release trend of curcumin under acidic pH (pH=5.6) condition for 72 h (Fig. 4). CuFe 2 O 4 impregnated samples were based on mesosilicalite (hexagonal micro/mesopore), SBA-16 (cubic), MCM-41 (hexagonal mesopore), Q-10 (large pore), hydrophobic silica, mesobeta (BEA large pore) and mesoZSM-5 (MFI). The percentage cumulative release profile of curcumin over hexagonal shaped silica (MCM-41 and mesosilicalite) was found to be superior and high of about 40-50% followed by cubic shaped SBA-16. CuFe 2 O 4 /SBA-16, which contains a 3D pore architecture showed a release of about 25% for 72 h. This indicates the ink shaped pores of SBA-16 (about 3.3 nm) are slightly restricted with spinel ferrite impregnation and showed a sustained release behavior with respect to curcumin. MesoZSM-5 with MFI structure consisting of sinusoidal pores of about 0.56 nm (micropore) and spherical silica with hydrophobic character also showed a release ability at about 20%. Q-10 silica with pore size of about 18 nm and mesobeta with medium pore size of 3 nm showed comparatively a less curcumin release of 10%. The release trend shows the aluminosilicate zeolite based structured silica with medium and large pore shapes profoundly affects and induces slow release of curcumin. For quick release, hexagonal shaped channel pores of MCM-41 and mesosilicalite (micro/meso) can be utilized than cubic shaped pores of SBA-16 and aluminosilicates. Based on the present requirement, cisplatin was loaded on curcumin/CuFe 2 O 4 /mesosilicalite and curcumin/CuFe 2 O 4 /MCM-41.
In case of parent cisplatin, a quick release was reported of about 76% within 1 h. However, loading cisplatin on biodegradable polybutylcyanoacrylate polymer shown to significantly reduce such burst release and enhance both in vitro and in vivo studies (Ghaferi et al. 2020). In a similar stance, the free curcumin release was completed (100%) within 24 h. However, using solid lipid nanoparticles as nanocarriers tends to extend the drug release up to 120 h (Gupta et al. 2020). In our present study, the mesosilicalite and MCM-41 were found to promote release of cisplatin for longer duration. For instance, mesosilicalite showed about 88% of cisplatin release for 72 h, while mesopores of MCM-41 showed a release of about 63% for 72 h. This suggests that cisplatin tends to functionalize on the external micropores of mesosilicalite, while MCM-41 is able to accommodate the cisplatin inside the mesopores. The release trend of cisplatin and curcumin with the presence of both nanocarriers clearly shows the functionalization ability and advantageous of mesopores than cisplatin and curcumin alone. For different structured mesoporous nanocarrier, data analysis was done using Prism 8 software and SPSS software version 20.0 (Additional file 1: Tables S2-S5).
The Additional file 1: Table S2 shows the mean and standard deviation of curcumin drug release at pH 5. While reviewing the results, CuFe 2 O 4 /MCM-41 showed a high mean score of 40.67, whereas CuFe2O4/Q-10 Silica/Curcumin demonstrated a low mean score of 6.38. The maximum score of curcumin drug release at pH 5.6 was observed in CuFe 2 O 4 /MCM-41/Curcumin and CuFe 2 O 4 /mesoZSM-5/curcumin, however the minimum curcumin release was observed in CuFe 2 O 4 /hydrophobic silica/ curcumin. From Additional file 1: Table S3, the results showed that there is significant difference between the groups with respect to curcumin release at pH 5.6 (p < 0.05). As significant difference was found, Scheffe's post hoc test was calculated to find out the significant difference between two groups at the same time. It is observed that the mean difference in the curcumin release between the groups such as  Table S5). On the other hand, CuFe 2 O 4 /Q-10 silica/curcumin described a significant moderate and positive correlation with CuFe 2 O 4 /mesosilicalite/curcumin, CuFe 2 O 4 /SBA-16/curcumin, and CuFe 2 O 4 /MCM-41/curcumin (p < 0.05). A significant moderate and positive correlation was also observed between CuFe 2 O 4 / mesoZSM-5/curcumin and CuFe 2 O 4 /hydrophobic silica/curcumin (p < 0.05) (Additional file 1: Table S5). To test the efficiency of our nanocomposites as potential chemotherapeutic agents, we investigated their effects on the cell viability of MCF7 (human breast cancer), HeLa (human cervical cancer), HCT116 (human colorectal. Cancer) and HFF (human foreskin fibroblast) cell lines. The cell viability assay, MTT, was performed after 48 h of treatment with the following conditions: CuFe 2 O 4 /mesosilicalite, curcumin, cisplatin, curcumin/cisplatin, CuFe 2 O 4 /mesosilicalite/curcumin, CuFe 2 O 4 /mesosilicalite/curcumin/cisplatin, and CuFe 2 O 4 /MCM-41/curcumin/cisplatin nanocomposites (Fig. 5). Treatment concentrations of CuFe 2 O 4 /mesosilicalite, CuFe 2 O 4 /mesosilicalite/curcumin, CuFe 2 O 4 /mesosilicalite/curcumin/cisplatin, and CuFe 2 O 4 /MCM-41/curcumin/cisplatin nanocomposites were: 0.025, 0.05, 0.1, and 0.5 mg/ml. While treatment concentrations for curcumin were: 0.00625, 0.0125, 0.025 and 0.125 mg/ml and that for the cisplatin group: 0.001125, 0.00225, 0.0045, and 0.0225 mg/ml. As detailed in the Materials and methods section, treatment concentrations of curcumin and cisplatin were adjusted to reflect the actual concentration adsorbed onto the mesosilicalite nanocomposites. Our results were analyzed by comparing data from the treated cells with the control group (untreated cells) (Additional file 1: Table S6). Cells treated with CuFe 2 O 4 /mesosilicalite nanocomposites had no effect on either MCF7, HeLa, HCT116, or HFF cells suggesting that the CuFe 2 O 4 /mesosilicalite nanocarrier did not interfere with cell viability. Pure curcumin reduced cell viability only at the highest concentration in MCF7; however, it minimally reduced the cell viability in HFF cell lines. As anticipated, pure cisplatin resulted in a reduction in cell viability in both MCF7 and HFF cell lines. Cells treated with free curcumin and cisplatin resulted in a combined effect and a greater cytotoxicity than the either one of them. Furthermore, cells that were treated with our nanocomposites CuFe 2 O 4 /mesosilicalite/curcumin, CuFe 2 O 4 /mesosilicalite/curcumin/ cisplatin, and CuFe 2 O 4 /MCM-41/curcumin/cisplatin all resulted in a significant reduction in cell viability in a dose-dependent manner with CuFe 2 O 4 /MCM-41/curcumin/cisplatin having the highest effect (Fig. 5).
Upon close investigation at the third dose of treatment in MCF7 cells, the pure curcumin reduced cell viability to 80.71%, while the nanocomposites that were coated with curcumin either with or without cisplatin significantly reduced the cell viability to 20.98% (group D in Fig. 5A), and 8.96% (group E in Fig. 5A). In HeLa cells, the pure curcumin reduced cell viability to 63.07%, while the nanocomposites that were coated with curcumin without cisplatin significantly reduced the cell viability to 56.13%. In HCT116, cell viability was 65.64% and 59.5% in free curcumin and CuFe 2 O 4 /mesosilicalite/curcumin, respectively. In contrast, using the same dose on the non-cancerous cell line HFF, the pure curcumin reduced cell viability to 84.12%, whereas the nanocomposites that were coated with curcumin did not result in a significant reduction in cell viability (72.88%; group D in Fig. 5B); whereas HFF treated with nanocomposites that were coated with curcumin and functionalized with cisplatin resulted in a significant reduction in cell viability of 12.93% (group E in Fig. 5B). Interestingly, the CuFe 2 O 4 / MCM-41/curcumin/cisplatin nanocomposite (group F in Fig. 5) resulted in a significant reduction of cell viability of 24.44% in MCF7, 5.9% in HeLa, 36% in HCT116, and an insignificant reduction in viability of 75.53% in HFF. Using a drug combination of cisplatin and curcumin nanocomposites will increase the cumulative cytotoxic effects of both compounds, solve the problem of cisplatin drug resistance in tumors, and increase the bioavailability of curcumin. The CuFe 2 O 4 /mesosilicalite/curcumin/cisplatin nanocomposite had a stronger effect even at lower concentrations on all cell lines. However, using the MCM-41/mesosilicalite in our cisplatin/curcumin nanocomposite had a significant effect on MCF7, HeLa, and HCT116, while having a minimal effect on HFF. Our results suggest that using MCM-41 with our cisplatin/curcumin drug combination has the potential of affecting cancerous cells while sparing normal ones.
To calculate the EC50 of treatment conditions, we used the data from Fig. 5 to calculate the EC50 (Fig. 6). Treatment with cisplatin resulted in an EC50 of 4.425, 6.48, 6.73, and 3.763 µg/ml in MCF7, HeLa, HCT116, and HFF, respectively. Coating the CuFe 2 O 4 / mesosilicalite with curcumin resulted in an EC50 of 80.2 µg/ml in MCF7; 102.1 µg/ml in HeLa; 117.6 µg/ml in HCT116; and 141.0 µg/ml in HFF. Moreover, functionalizing the CuFe 2 O 4 /Mesosilicalite with curcumin and cisplatin resulted in an EC50 of 81.23 µg/ ml in MCF7; 47.55 µg/ml in HeLa; 48.96 µg/ml in HCT116; and 76.83 µg/ml in HFF. However, functionalizing curcumin and cisplatin onto CuFe 2 O 4 /MCM-41 nanocomposite resulted in an EC50 of 72.51 µg/ml in MCF7; 58.6 µg/ml in HeLa; 62.58 µg/ml in HCT116; and 154.2 µg/ml in HFF. These results show that while using the mesosilicalite resulted in a similar EC50, using the MCM-41 support resulted in a 1 to 1.5  (Fig. 7). For the latter, cells were stained with the apoptotic marker cleaved-Caspase 3 (c-Caspase 3), and Hoechst, which is a nuclear marker. Our images clearly showed cells stained with c-Caspase 3 (magenta), which is the activated form of the protein, after treatment with our nanocomposites for 48 h. These results suggest that our nanocomposites significantly reduce cell viability by activating apoptosis.
Here, we explored merging two compounds that have potential benefits, but suffer from several side effects: curcumin and cisplatin. Curcumin has been extensively investigated as a chemotherapeutic agent in breast cancer, hepatocellular carcinoma, pancreatic cancer, gastric cancer, osteoclastoma, and bladder cancer (Syng-Ai et al. 2004;Zhu and Bu 2017;Li et al. 2017;Cao et al. 2015;Zhang et al. 2018). It has been found to activate JNK pathway, induce the generation of reactive oxygen species (ROS), and subsequently apoptosis (Syng-Ai et al. 2004;Zhu and Bu 2017). However, curcumin has some problems that hinder its full potential such as low solubility, low bioavailability, and rapid elimination from the body (Anand et al. 2007). Research is being conducted to increase the bioavailability of curcumin. On the other hand, cisplatin is a well-established chemotherapeutic drug. Unfortunately, it is fraught with several issues such as drug resistance and systemic toxicity (Dasari and Tchounwou 2014). The mechanism of action of cisplatin is by activating the JNK pathway and inducing oxidative stress, DNA damage, and apoptosis. However, cisplatin will also result in increased levels of Glutathione S transferase (GST), resulting in a reduction in ROS, and resistance to cisplatin. Therefore, a combination treatment of cisplatin and curcumin, which increases ROS, might augment the cytotoxic effect and prevent cisplatin-related drug resistance (Townsend and Tew 2003 In addition, loading of such dual combinational drugs on a biodegradable polymer based on polycaprolactone, linked with Pt using amine based polymer and PEG shell exhibited a synergistic effect (combination index = 0.4-0.8) with dual delivery in one nanocarrier against multidrug resistant cancer. The use of polymeric nanocarrier improves the transport of curcumin and cisplatin in to the cells by endocytosis (Scarano et al. 2015). Our results show that a simple coating of curcumin and functionalization of cisplatin into hexagonal pore structural framework of silica had a significant effect on the cytotoxic effectiveness of nanocomposites on various cancer cells.
We previously tested cisplatin-functionalized cubic spinel CuFe 2 O 4 loaded on monodispersed spherical hydrophilic silica (HYPS) nanoparticles on MCF7 breast cancer cells ). In our current work, we investigated two nanoformulations using the hexagonally shaped silica: (a) the mesosilicalite with zeolite (strong) framework, and (b) the MCM-41 with amorphous (weak) framework. Both of which were coated with curcumin and functionalized with cisplatin. While the EC50 of our previous spherical shaped silica nanoformulation was equal to 180.89 µg/ml ), our current hexagonal shaped silica nanocomposites were 81.23 µg/ml (mesosilicalite) and 72.51 µg/ ml (MCM-41).
The present study clearly indicates that changing the structural framework from spherical to hexagonally shaped silica increased the chemotherapeutic efficiency and reduced the EC50 values. The improved effectiveness is most likely attributed to the increased loading capacity in the hexagonally shaped nanocomposites as indicated in the drug release studies (Fig. 4).
CuFe 2 O 4 /mesosilicalite/curcumin/cisplatin, and CuFe 2 O 4 /MCM-41/curcumin/cisplatin nanocomposites utilize the benefits of curcumin and cisplatin. This combination resulted in novel nanocomposites that significantly reduced cell viability of the MCF7 breast cancer cell line by activating the apoptotic pathway as indicated by c-caspase 3. Our results show that both CuFe 2 O 4 /mesosilicalite/curcumin/cisplatin and CuFe 2 O 4 / MCM-41/curcumin/cisplatin nanocomposites are potential novel chemotherapeutic options. However, CuFe 2 O 4 /MCM-41/curcumin/cisplatin nanocomposites offer an added advantage of a wider gap in cytotoxicity between cancerous and non-cancerous cell lines (Scheme 1).

Conclusions
Synergistic action of curcumin and cisplatin on structured magnetically active hexagonal nanocarriers was studied for targeted combinational therapeutics. Mesoporous MCM-41 and mesosilicalite with micro/mesopores were developed and impregnated with copper spinel ferrite. The textural characteristics have been analyzed with various physico-chemical techniques. A significant difference in curcumin release was observed among the different CuFe 2 O 4 /structured silica-based nanoformulations. Also, those CuFe 2 O 4 /MCM-41 structured silica-based nanoformulations showed a significant positive relationship with each other. CuFe 2 O 4 /mesosilicalite followed by CuFe 2 O 4 /MCM-41 with high curcumin release ability was further functionalized with cisplatin. The effects of our nanocomposite on cell viability of MCF7 and HFF cell lines were tested. Treatment with either CuFe 2 O 4 /mesosilicalite/curcumin/cisplatin or CuFe 2 O 4 /MCM-41/ curcumin/cisplatin nanocomposites resulted in a significant reduction in cell viability that was greater than treatment with either curcumin or cisplatin. Furthermore, cells treated with our two nanocomposites had a higher number of c-Caspase 3-positive cells than the untreated control. These results suggest that the reduction in cell viability is triggered by activating the apoptotic signaling pathway. The EC50 for CuFe 2 O 4 /mesosilicalite/curcumin/cisplatin was 81.23 µg/ml in MCF7; 47.55 µg/ml in HeLa; 48.96 µg/ ml in HCT116 and 76.83 µg/ml in HFF. The EC50 for CuFe 2 O 4 /MCM-41/curcumin/ cisplatin was 72.51 µg/ml in MCF7; 58.6 µg/ml in HeLa; 62.58 µg/ml in HCT116; and 154.2 µg/ml in HFF. Our results show that both nanocomposites can be further explored