Figure 1(a–c) shows the XRD diffraction patterns of SPIONs loaded on different structured silicas S-16, HYPS and MSU-Foam. 10 wt% of SPIONs loadings was carried out using enforced adsorption technique. The presence of characteristics broad peak due to amorphous siliceous framework was observed between 15–30°. The presence of Fe3O4 was expected to be observed at 2 theta value of 35.45°. In case of 10 wt%SPIONs/S-16 and 10 wt%SPIONs/HYPS, the presence of weak diffraction peaks was observed, which corresponding to cubic structure of Fe3O4 (magnetite, PDF # 88-0866) (Figure 1a,b). The presence of such weak peaks with increased broadness is due to small nanosized Fe3O4 indicating lack of crystallization inside cubic cage and spherical nanopores (nanocarriers). The presence of broad peak in the spectra is in good correlation with the high surface area of S-16 that reflects finer particles dispersed on the 3D cage type pores (Table 1). However, the peaks intensity at 35.4°, 43.1°, 53.4°, and 62.6° increased over 10 wt%SPIONs/MSU-F corresponding to (311), (400), (422), and (440) (Fig. 1c). This shows that surface modification of Fe3O4 particles tends to change depending on the nature of structured silica. In case of Cp, crystalline phase of Pt complex was clearly observed with characteristics diffraction patterns (Fig. S1a). In case of Fe/S-16-APAA-Cp, a broad diffraction peak of amorphous phase of silica was observed. The absence of Cp peaks in the diffraction patterns shows the transformation of crystalline to amorphous phase of Cp due to confinement in the cubic pores of SBA-16, beneficial to enhance the solubility of Cp (Fig. S1b).
X-ray diffraction pattern of (a) 10 wt%SPIONs/S-16 (b) 10 wt%SPIONs/HYPS and (c) 10 wt%SPIONs/MSU-F.
The surface area and pore size distributions of parent and SPIONs loaded samples were analyzed using nitrogen adsorption technique. Fig. S2(a–f) shows the isotherms for (a) S-16 (b) 10 wt%SPIONs/S-16, (c) HYPS, (d) 10 wt%SPIONs/HYPS, (e) MSU-F, and (f) 10 wt%SPIONs/MSU-F. Textural results are presented in Table 1. The isotherm pattern of S-16 shows a typical spinodal hysteresis pattern for ink bottle type of pores. In case of SPIONs loading over S-16, a significant decrease observed in the textural characteristics. Specifically, a decrease in the specific surface area from 988 m2/g to 471 m2/g occurs, while cumulative surface area reduces from 590 m2/g to 297 m2/g (Table 1). The observed reduction was about 50% after iron oxide impregnation. The cumulative pore volume showed a similar occupation (46%) compared to parent S-16. Monodispersed spherical silica HYPS show a type IV isotherm corresponding to the presence of mesopores. The silica hysteresis loop tends to be present at higher relative pressure of p/p0 >8. Among the three supports, spherical silica texture was lower with less surface area of 170 m2/g, pore volume of 0.35 cm3/g with intermediate average pore size distributions of 8.3 nm. After iron oxide loading over such lower texture HYPS, about 33% reduction in BET surface area was observed, while a significant increase in the pore size observed from 8.3 nm to 15.5 nm. MSU-F shows that iron oxide was inducted at the pores of foam leading to reduction in surface area (26% reduction) with an increase in open window type of pores (40 nm). The trend clearly shows the accumulation of iron oxide nanoparticles at the external pores of HYPS and MSU-F.
The surface morphology of parent and 10wt.% SPIONs loaded silica S-16, HYPS, and MSU-F were analyzed through SEM (Fig. S3(a–f)). The parent S-16 was found to be composed of micron sized spherical spheres of about 4 µm. The iron oxide loading over S-16 showed no significant changes in the morphological characteristics compared to parent substance, which might be due to presence of high surface area (Fig. S3a,b). On the contrary, the 10 wt%SPIONs/HYPS showed the presence of monodispersed spherical silicas distributed uniformly in the range of about 80 nm. In case of 10 wt%SPIONs/HYPS, the presence of hetero nano-sized clusters were observed to be spread over spherical silica (Fig. S3c,d). In case of MSU-Foam, presence of agglomerated silica in irregular forms were observed. 10 wt%SPIONs/MSU-F showed some porous morphological characteristics changes with agglomerated nanospheres structures at lower scale bar of 3 μm (Fig. S3e,f). The TEM analysis at two different magnification scale bar 100 and 200 nm shows that SPIONs deposition are unique and depends on the nature of support, where the dispersion and agglomeration vary based on the nanocarriers pore architecture (Fig. 2(a–f). For instance, with three-dimensional cage type of SBA-16 pores, the presence of agglomerated forms of SPIONs as nanoclusters were observed along the pore channels (Fig. 2a,b). The presence of cage type of porous layer of SBA-16 appeared to be homogeneous with a constant thickness, and particles were found connected to the layers. In the case of 10 wt%SPIONs/HYPS, presence of uniform spherical silica was clearly observed, where a clear external agglomeration of SPIONs as nanosize clusters was observed to be distributed over spheres (Fig. 2c,d). In case of 10 wt%SPIONs/MSU-F, the presence of nanosized Fe3O4 was observed to be spread across the silica foam (Fig. 2e,f). The average particle size distribution of SPIONs over three supports were calculated (Fig. S4a–d). The particles were divided into different groups based on their size; 0–20, 21–30, 31–40, 41–50, 51–60, 61–70 and 71–80 nm. At constant loading (10 wt%), the nanoparticle size distribution of SPIONs was in the following order: HYPS > Si-SBA-16 > MSU-Foam. The average SPIONs particle size of the 10 wt%SPIONs/HYPS measured from TEM images was found to be high of about 51.0 ± 1.07 nm, followed by S-16 support in the range of 17 ± 1.83 nm. In support MSU-F, small sized agglomerated SPIONs in the range of 13 ± 0.92 nm was observed.
Surface morphology examined using TEM of as-prepared products; (a,b) 10 wt%SPIONs/S-16, (c,d) 10 wt%SPIONs/HYPS and (e,f) 10 wt%SPIONs/MSU-F.
The magnetic properties of designed nanoparticle composites were measured using vibrating sample magnetometer (VSM). Figure 3(a–c) shows the VSM of (a) 10 wt%SPIONs/S-16 (b) 10 wt%SPIONs/HYPS and (c) 10 wt%SPIONs/MSU-F. 10 wt% SPIONs over three different structured silica showed different magnitude of superparamagnetization. The SPIONs magnetization generated over S-16, HYPS and MSU-F was in the following order: HYPS (4.08 emug−1) > S-16 (2.39 emug−1) > MSU-F (0.23 emug−1). It has been shown that presence of small sized nanoclusters at the walls of hexagonal shaped MCM-41 tends to form super paramagnetic interactions among Fe3+ species, while large nanoclusters contribute towards ferromagnetic property18. In the present study, an enforced impregnation of iron oxides into the mesocellular foam consisting large window pore sizes tends to generate such small nanosized Fe3+ clusters. As evidenced from TEM study (Fig. S4), the average nanoparticle of SPIONs over MSU-F was of 13 nm. In line with TEM analysis, the VSM spectra of MSU-F showed super paramagnetic behavior with narrow hysteresis. 10 wt% SPIONs impregnation over S-16 consisting ink bottle shaped pores generates medium nanoclusters with average particle size of 17 nm (Fig. 4a). SPIONs loading over monodisperse spherical silica HYPS generated the highest magnetization, while formation of large nanoclusters with average particle of 51 nm, tends to broaden the hysteresis loop indicating a shift towards ferromagnetic behaviour compared to MSU-F (Fig. 3b,c).
Vibrating sample magnetometer analysis of (a) 10 wt%SPIONs/S-16, (b) 10 wt%SPIONs/HYPS, and (c) 10 wt%SPIONs/MSU-F.
Drug release profile of different nanoformulations S-16, HYPS, MSU-F loaded with 10 wt% SPIONs, APTES, polyacrylic acid, copolymer F127, cisplatin in pH 5 and selected 10 wt%SPIONs/S-16-APAA-Cp in pH 7 at 37 °C for 72 h.
FT-IR spectroscopy was used to investigate the complexation nature of Cp over 10 wt%SPIONs/S-16 after functionalization with silane and polyacrylic acid (Fig. S5a–e). Spectra analysis of Cp showed an intense amine stretching bands at 3218 cm−1 and 3281 cm−1, while a broad peak at 1640 cm−1 was attributed to the characteristic peak of Cp (Fig. S5a). Figure S5(b,c) showed the FT-IR profile of nanocarrier S-16 and S-16 after iron oxide impregnation followed by silane functionalization (10 wt%SPIONs/S-16-A). In case of 10 wt%SPIONs/S-16-A-Cp (d) and 10 wt%SPIONs/S-16-APAA-Cp (e), the presence of weak amine band showed the conjugation between silane NH2 and Cl bond of Cp. Importantly, bending vibration at 1640 cm−1 indicates the functionalization of Cp and reaction between silica and PtII complex (Fig. S5d,e).
The Cp adsorption efficacy was studied for three nanoformulations 10 wt%SPIONs/S-16-A-Cp, 10 wt% SPIONs/HYPS-A-Cp and 10 wt%SPIONs/MSU-F-Cp. Among three functionalized nanocarriers, 10 wt%SPIONs/S-16-A-Cp showed the highest Cp adsorption of 91%. 10 wt% SPIONs/HYPS-A-Cp showed similar adsorption capacity to that of S-16 nanocarrier. The observed pattern indicated the increased solubility of drug at accessible functional groups located at cubic cage type pores of S-16 and spherical pores of HYPS. 10 wt%SPIONs/MSU-F-Cp showed slightly a lesser Cp adsorption of 86%.
The drug release of profile of various SPIONs/nanocarriers/Cp nanoformulations were studied using simulated tumor acid pH condition (pH 5) at 37 °C for 72 h (Fig. 4). Among the different nanoformulations, 10 wt%SPIONs/S-16-APAA-Cp showed highest Cp release of 65% for 72 h. In case of 10 wt% SPIONs/HYPS-A-Cp, a reasonable Cp release (58%) was observed followed by S-16 support. The result indicates that HYPS can be the other potential nanocarrier and can be exploited for drug delivery application. Mesocellular foam (MSU-F-A-Cp) consisting large widow size pores functionalized with APTES showed Cp release of about 63% at 72 h. However, 10 wt% SPIONs/MSU-F-Cp showed the lowest percentage cumulative release of 41% at 72 h. The trend signifies the importance of high textural properties of nanocarrier S-16, which helps to accommodate SPIONs, functionalization with APTES, polyacrylic acid and to release Cp (Table 1). The sustainable release of Cp shows the effectiveness of neutral type of silane APTES that will be charged positive at low pH (acidic condition) and proposed to coordinate with chloride ligand of Cp and help to attain plateau with time. The release trend clearly shows the immediate release formulation rather than burst release that can be aptly used for acute drug delivery targeting diseases. However, Cp over difunctional copolymer functionalized SBA-16 (S-16-F127-Cp) clearly showed an initial burst release of 75% at about 30 min, which then reduces to 60% at 72 h. Such pattern shows the inability of non-ionic polymers to coordinate with Cp at the surface of S-16. The order of Cp release among different nanoformulations are in the following order: 10 wt% SPIONs/S-16-APAA-Cp > 10 wt% SPIONs/HYPS-A-Cp > 10 wt%SPIONs/S-16-A-Cp > S-16-F127-Cp > MSU-Foam-A-Cp > 10 wt%SPIONs/MSU-F-Cp.
In order to study the oral route Cp delivery, pH variation study was performed simulating the strongly acidic pH of intestine and weak basic condition of colon. The capping property of 10wtFe/S-16-APAA-Cp was verified with the Cp release profile at pH 7 (Fig. 4). Our results indicate that 10wtFe/S-16-APAA-Cp showed an exemplary drug release capability (98%) at pH 7. The Cp release decreases from 55% at pH 5 to 30% at acidic pH of 2 over 72 h. The observed trend revealed that at low pH, the release ability of Cp is controlled by the electrostatic interactions between Cp/S-16 and acidic condition. Based on the earlier reported study at lower acidic pH = 5.6, the COOH group of polyacrylic acid is shown to collapses and forms intramolecular hydrogen bond in between COOH groups. Such behavior of polymer is reported to safeguard DOX by blocking inside the pore channels through encapsulation technique19. On the other hand, at pH 7.0, ionization of acid group of polyacrylic acid occurs leading to the formation of -COO- anions. Anion repulsion process was reported to swell the core of polymer, eventually leading to expansion of core and release of Cp. Polyacrylic acid complexation with Cp was reported to be critical for sustained release of Cp (80–90%) at two different pH conditions (7.4 and 5)20. In the present study, Cp release was significantly high of 98% at pH 7. The trend clearly indicates the presence of nano Cp at the cage type of pores of SBA-16, easily accessible to dissolved chloride solution. XRD analysis showed an increased Cp dissolution with S-16 support (Fig. S1). FT-IR confirms the complexation phenomenon of Cp with functionalized S-16 through ligand exchange process with available chloride ion (Fig. S5) thereby facilitating high Cp release from pH 2 to 7. Therefore, the stable release profile was attributed to the complexation phenomenon with copolymer through ligand exchange process with available chloride ion in physiological solution.
In vitro anti-cancer study
The LC50 values varies between the drug Cp and nano formulated drugs (Group-IV, Group-V, Group-VI and their controls (Group-I, Group-II and Group-III) (Fig. 5). The LC50 of Group-I, Group-II and Group-III were very high (Table 2) indicating their zero effect in the cell killing when they included in the nanoparticles construction. HeLa cells were the most sensitive cell line towards Group-IV, Group-V, and Group-VI. The sensitivity of HeLa cells toward Group-VI is approximately 7.5 folds higher than its sensitivity toward Group-V, Whereas its more sensitive toward Cp by 3 folds than Group-VI. In HCT116, the sensitivity towards Group-VI was approximately 13.2 folds higher than its sensitivity toward Group-V, Whereas it was more sensitive toward Cp by 6.3 folds than Group-VI. In normal cell line HEK293 the cells were more resistant toward Group-V, the sensitivity to Group-VI was 69.5 folds higher than Group-V, but less by 9.5 folds than Cp. HEK293 showed a resistant toward Group-V compare to HCT116 and HeLa cancer lines. The effect of Group-VI on HCT116 and HEK293 were same but its more effective by 2.9 folds on HeLa cells.
Cytotoxic effect of (1) S-16 (G-I), (2) SPIONs (G-II), (3) 10 wt%SPIONs/S-16 (G-III), (4) cisplatin (G-IV), (5) 10 wt%SPIONs/S-16-A-Cp (G-VI) and (6) 10 wt%SPIONs/S-16-APAA-Cp (G-V) on cancer and normal cell lines. (A) HCT116, (B) HeLa, (C) HEK293. These graphs were plotted to obtain LC50 values for each of the drugs tested on different cell lines (refer to Table 1). (D) Plotting the LC50 of effective drugs against the three cell lines (*p < 0.05, **p < 0.01 and ***p < 0.001, n = 3). Different concentrations of each nanoformulated drug and support were applied on each cell line then the cytotoxicity was measured by MTT assay, the resulted day was plotted in excel and calculated the LC50 using the equation of the logarithmic trendline. The p value and standard deviation were measured by one way ANOVA in GraphPad prism software.
The apoptotic induction property of cisplatin (Cp), 10 wt%SPIONs/S-16-A-Cp, and 10 wt%SPIONs/S-16-APAA-Cp nanoformulations were studied on treating with HeLa and HCT116 cells using DAPI staining Fig. 6 shows the confocal microscopic images of treated HCT116. HCT116 cells were treated with Group-IV, Group-V and Group-VI for 24 h and stained with DAPI. This study elucidated the anti-cancer effect of nanoformulation based drug on nuclear condensation of cancer cells. Study showed reduction in cell number compare to untreated cancer cells. HCT 116 treated with Group-V, which consists SPIONs, APTES, polyacrylic acid showed no structural changes in the nucleus of HCT116 cells compared to the untreated cancer cells but showed significant reduction in cell number. Cp is a well-known chemotherapeutic drug used for treatment of many cancer types such as breast cancer and ovarian cancer showed DNA fragmentation and high reduction in cell number. However, Group-VI, which consists SPIONs, APTES and Cp interestingly decreased the quantitation of HCT116 cells and showed disintegrated nuclear morphology. These results indicate that Group-VI based nanoformulation is effective against colorectal cancer.
DAPI staining of treated HCT116 cells, the density stained areas are cell nuclei (blue color). (A) Control cancer cells. (B) Cisplatin treatment (G-IV), (C) 10 wt%SPIONs/S-16-APAA-Cp treatment (G-V)(0.188 mg/ml), (D) 10 wt%SPIONs/S-16-A-Cp treatment (G-VI) (0.188 mg/mL). All the scale bars are 100 µm. The arrow indicates the nucleus structural changes.
Figure 7 shows the confocal microscopic images of treated HeLa cells. The cells were treated with Group-IV, Group-V and Group-VI for 24 h and stained with DAPI to evaluate the effect of Group-VI on HeLa cells. Group-V showed a slight reduction in cell quantification compared to control cells. As expected Cp (Group-IV) killed 90% of HeLa cells since it used to treat cervical cancer by inhibiting the replication of DNA. However, HeLa cells proliferation was dramatically affected by Group-VI treatment with clear DNA fragmentation. This indicate that HeLa cells were more sensitive to Group-VI than HCT116 cells.
DAPI staining of treated HELA cells, the density stained areas are cell nuclei (blue color). (A) Control. (B) Cisplatin treatment (G-IV) (0.0342 mg/ml) (C) 10 wt%SPIONs/S-16-APAA-Cp treatment (G-V) (0.188 mg/ml), (D) 10 wt%SPIONs/S-16-A-Cp treatment (G-VI) (0.188 mg/ml). All the scale bars are 100 µm.
Figure 8 shows the cell morphology of treated HeLa cells using inverted microscope. The morphological pattern of treated HCT116 with Group-IV showed that almost all cells were completely dead after 24 h. Treatment of HCT 116 with Group-V showed the entry of the SPIONs particles inside the cells, which were indicated by the characteristics iron red to brown color. However, the presence of cells without kill indicates the poor release of Cp, which might be due to presence of pH sensitive polyacrylic acid. Whereas, in the Group-VI treatment, more than 80% of the cells are found to be dead. The presence of SPIONs were not easy to detect inside the cells indicating that the cells were taken in SPIONs, S-16, APTES, Cp nanoformulation (Group-VI) and were dead as the number of cells decreased dramatically. This indicate the effective release of Cp in Group-VI rather than in the presence of polyacrylic acid at pH 7.4.
Cell morphology of treated Hela cells. (A) Control, (B) Cisplatin treatment (G-IV) (0.0342 mg/ml) (C) 10 wt%SPIONs/S-16-APAA-Cp treatment (G-V) (0.188 mg/ml), (D) 10 wt%SPIONs/S-16-A-Cp treatment (G-VI) (0.188 mg/ml). Whit arrow show cell debris and red arrow shows the SPIONs inside the cells. All the scale bars are 100 µm.
To visualize the effect of different drug combination on cells morphology and number. cells were pictured before and after treatment using inverted microscope (Fig. 9). The control cells showed normal healthy cells, whereas, cells treated with Cp were almost completely dead after 24 h. In Group-V treatment, the cell debris was noticed as well as the entry of SPIONs into the cells (red arrow). But with very low release of Cp from SPIONs, S-16, APTES, polyacrylic acid nanoformulations, only 10% reduction of cell number occurs. Whereas, the Group-VI treatment was more effective and resulted in more than 60% of dead cells indicating effective release of Cp from SPIONs, S-16, APTES nanoparticle formulations. The images are visible evidence that clearly show the cell permeation by brown colored SPIONs impregnated S-16 nanocarrier and cell death shows the release of cisplatin. Overall, the high Cp adsorption efficiency, sustained pH stimuli drug release was advantage of 10 wt%SPIONs/S-16-Cp nanoformulation. The anticancer efficiency of 10 wt%SPIONs/S-16-Cp in cancerous cell lines (HeLa and HCT116) can be the most suitable for multifunctional stimuli responsive nanotherapeutics (Fig. 10).
Cell morphology of treated HCT116 cells. (A) Control, (B) Cisplatin treatment (G-IV) (0.0342 mg/ml) (C) 10 wt%SPIONs/S-16-APAA-Cp treatment (G-V) (0.188 mg/ml), (D) 10 wt%SPIONs/S-16-A-Cp treatment (G-VI) (0.188 mg/ml). Whit arrow show cell debris and red arrow shows the SPIONs inside the cells. All the scale bars are 100 µm.