Synergistic Effect of SiO2 and Fe3O4 Nanoparticles in Autophagy Modulation

Rapid advancements in nanotechnology have expanded its applications and synergistic impact on modern nanosystems. The comprehensive assessment of nanomaterials’ safety for human exposure has become crucial and heightened. In addition to the characterization of cell proliferation and apoptosis, probing the implication of autophagy is vital for understanding the ramification of nanomaterials. Hence, HEK-293 kidney cells were employed to understand the changes in induction and perturbation of autophagy in cells by iron oxide (Fe3O4) and silica (SiO2) nanoparticles. Interestingly, Fe3O4 worked as a potent modulator of the autophagy process through its catalytic performance, which can develop better than that of SiO2 nanoparticles mechanism, stressing their therapeutic implication in the understanding of cell behaviors. The quantification of reactive oxygen species (ROS) was measured along with the process of autophagy during cell growth. This modulated autophagy will help in cell fate determination in complementary therapy for disease treatment, provide a clinical strategy for future study.


Introduction
Nanomaterials exhibit unique optical and physico-chemical properties, such as fluorescent brightness of quantum dots [1], localized surface plasmon resonance (LSPR) of gold nanorods [2], and superparamagnetic effect of iron oxide nanoparticles [3].For advanced theranostics, the influence of metallic nanoparticles (NPs) on the differentiation and proliferation of epidermal, cancer, and normal cells was investigated in depth [4][5][6][7][8].For the safe development and commercial use of NPs, phototoxicity and cellular toxicity investigations are essential requirements.Previously, NP mediated toxicity was observed in diverse physiological areas, such as the inhibition of cell division, cell death, genetic damage, inflammation and oxidative stress [9,10].Different mechanisms could occur in metallic nanoparticle-induced cellular differentiation and proliferation, such as regulation/interaction of specific transcription factors, providing materials for reactive oxygen species, and modulation of the intercellular process [11].Inflammatory cell expression, such as that of macrophages and neutrophils, was associated with metallic nanoparticle related toxicity, because the generation of ROS was closely influenced by the inflammatory cell expression [12].This was also considered as free radical facilitated toxicity by the Fenton reaction [13].A detailed study of the application of metallic NPs to cellular differentiation and proliferation could lead to better design and preparation of metallic NPs for future scientific applications in regenerative medicine and for the modulation of cell functions [14].
The SiO 2 surface is one of the most important biocompatible interfaces that could be implemented in analyses of protein corona [15], bioimaging [16], drug delivery and diagnosis, due to its excellent physico-chemical stability, tunable pore structure and high surface area [17].Orthosilicic acid, a soluble state of SiO 2 , does not alter the preparation of alkaline phosphate for cartilage production.Another soluble state of SiO 2 was also used in the preparation of alkaline phosphate and collagen [18].SiO 2 based nanomaterials were relevant and biocompatible for cell proliferation [19].SiO 2 could prevent bacterial infection of stem cell during cell growth [20], and SiO 2 also promoted the osteogenic differentiation and proliferation of kidney cells [21].SiO 2 could induce the autophagy process, such as inducing autophagy dysfunction in L-02 and HepG2 cells [22,23], enhancing autophagic activity [24] and related phenomena [25,26] without a coherent mechanism.Fe 3 O 4 with paramagnetic function is attractive for various biomedical applications and medical technologies [27].Fe 3 O 4 NPs obeyed Coulomb's law and helped in transportation or immobilization of magnetically designed biological molecules.It initiated a biochemical reaction or modulation by attachment to mechanosensitive channels of the cell membrane.Fe 3 O 4 could also be applied in autophagy related experiments, acting as an incidental factor [28,29].The toxicity of Fe 3 O 4 , its abnormal cellular ROS balance and structural injury to mitochondria seemed to be interrelated [29].Mitochondrial injury contributed to stimulation of autophagy.
In the biomedical fields, silica and iron oxide are the most common nanomaterials for drug delivery, tissue engineering, and cell promotion [30].After entering cells, the NPs would degrade into irons and induce reactive oxygen species (ROS) through the changing of mitochondrial related organelles' structures [31].The surplus production of ROS could create a chain reaction inside, along with the autophagy process of the cell, which would consist of a process of discarding superfluous and unnecessary materials from cells and regulating the ROS homeostasis [32].Autophagy plays a significant role in controlling cell behaviors.Excessive or inadequate levels of autophagy inside cells would boost cell aging, inhibit cell growth, and encourage cell apoptosis.However, the physiological balance of autophagy could reduce cell aging and improve cell growth and proliferation [33].The advantage of combined SiO 2 and Fe 3 O 4 NPs could advance experimental efficiency and cut production costs.In the cellular process, combined SiO 2 and Fe 3 O 4 NPs could promote autophagy activity, an essential cellular degradation access.Recent studies revealed that combined NPs could indeed induce autophagy in cells [34,35].Autophagy always plays a crucial role in the cell's fate, favoring either death or survival [36][37][38], by disassembling the dysfunctional or unnecessary components [39,40].Since nanomaterials could activate autophagy, the assessment of organelles and cellular proteins delivered to lysosomes would determine the fate of digestion by lysosomal hydrolases [41][42][43][44].
Anghelache et.al. [45] demonstrated that dextran-coated iron oxide nanoparticles have anti-inflammatory activity at non-cytotoxic concentrations by reducing the levels of pro-inflammatory mediators, such as IL-1β, MCP-1, CCR2, TNF-α, and IL-6, in activated endothelial cells and M1 phenotype macrophages.Folic acid-coated iron oxide nanoparticles enhance internalization and facilitate delivery of therapeutic agents for cancer treatment and inflammation-related diseases, like rheumatoid arthritis, lupus, osteoarthritis, Crohn's disease, and atherosclerosis [46].In this study, the possibility of assessing the synergistic effects of Fe 3 O 4 and SiO 2 NPs were explored with HEK-293 kidney cells.Fe 3 O 4 NPs could offer superior biocompatible catalytic performance due to the enhancement provided by SiO 2 NPs.

Synthesis of SiO 2
Dissolve 0.225 g Igepal in 4.4 mL cyclohexane.Then, 0.046 mL DI water was added to the mixed solution, stirring for 20 min.Then, 0.05 mL NH 4 OH was added, stirring for 20 min.Finally, 100 uL TEOS was added, stirring for 24 h [47].

Synthesis of Fe 3 O 4
To prepare single-surfactant-coated Fe 3 O 4 , 10 mg iron (II), 20 mg (III) chlorides and 90 mg of DABA were added to 10 mL distilled water with magnetic stirring for 80 min at 80 • C. Next, 1 mL ammonium hydroxide was added to the mixture with magnetic bar stirring for 80 min at 85 • C.After the mixture was cooled to room temperature, iron oxide NPs were collected via magnetic bar and washed with distilled water and ethanol 3 times, respectively [48].

Characterization of SiO 2 and Fe 3 O 4
TEM (Vanila Inc., Salt Lake City, UT, USA, JSM5700F) and zetasizer (Cananda CARRY 40, Cananda) were used to characterize the shape and size of silica and iron oxide NPs.

Cell Viability of NPs
LC3 cells were seeded at 1 × 10 4 cells/well in a 96-well plate and stabilized for 24 h.The medium was discarded and NPs added to this mixture according to concentrations (1, 10, 20, 50, 100 µg/mL) in DMEM without FBS.After incubation for 48 h, NPs were removed, and cell viability reagent with DMEM (BIOMAX, Gyeonggi-do, Republic of Korea, QM10000) was added.The solution was treated for 1 h and optical density measured at 450 nm wavelength using a VICTOR 3™ multi-spectrophotometer (PerkinElmer, Waltham, MA, USA).
After incubation, the media was discarded carefully and 20 µM DCF-DA was treated for 30 min.Cells were washed with PBS and fluorescence intensity was measured using a VICTOR 3™ multi-spectrophotometer (PerkinElmer, Waltham, MA, USA) at the excitation and emission wavelengths of 485 nm and 535 nm.

Difference of LC3 Expression on NPs
Western blot was carried out for confirmation of LC3 protein.The cells were seeded at 1 × 10 6 cells/well on a 6-well plate and stabilized for 24 h.Then, 50 µg/mL Fe 3 O 4 , SiO 2 and these mixtures were added to the well and incubated for another 24 h.Cells were detached using Trypsin-EDTA (0.25%) and centrifuged at 1200 rpm for 3 min.Extraction of lysate proceeded using M-PER™ Mammalian Protein Extraction Reagent (Thermo Scientific™, Waltham, MA, USA, 78503) following the manufacturer's instruction.The protein concentration was measured by Pierce™ BCA Protein Assay Kits (Thermo Scientific™, 23225).100 µg/mL protein was mixed with 4× Laemmli Sample Buffer (Bio-Rad, Hercules, CA, USA, 161-0747) and heated at 98 • C for 5 min.The samples were loaded in 20 µL/wells in 12% Tris/glycine gel and run at 100 V for 1 h after 50 V for 5 min.The gel was transferred to a PVDF membrane (Labiskoma, Seoul, Republic of Korea, KDM50) at 100 V for 1 h.The membrane was blocked with 5% nonfat milk (Bio-Rad, BR1706404) in TBST for 1 h with gentle shaking.The membrane was incubated overnight at 4 • C with LC3A/B (D3U4C) (Cell Signaling Technology, Danvers, MA, USA, 12741) in a blocking buffer.After incubation, the membrane was washed three times in 1× TBST for 10 min.It was incubated with Goat anti-rabbit IgG (ENZO, ADI-SAB-300-J) for 1 h and washed three times in 1× TBST for 10 min.The images were obtained by Davinch-Chemi Fluro imager (Davinch-K, Seoul, Republic of Korea) after incubation with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific™, 34578).

Visualization of LC3 Expression on NPs
LC3 expression was visualized by confocal microscopy and live cell video.In confocal microscopy, the 1 × 10 6 LC3 cells were seeded on a microscope cover glass (SUPERIOR, Soothfield, MI, USA, HSU-0101050) in a 6-well plate and stabilized for 24 h.The old media were discarded along with 50 µg/mL Fe 3 O 4 , SiO 2 , and these mixtures in the media were added to the well.After 24 h of incubation, the NPs were carefully removed and washed with PBS.Then, 1 mL of 4% Paraformaldehyde (BIOSOLUTION, Seoul, Republic of Korea, BP031) was added and washed with cold PBS after incubation for 15 min.DAPI (Sigma-Aldrich, D9542) was diluted 10,000 times and incubated for 15 min in the dark.The slides were mounted with Fluoromount-G™ Mounting Medium (Invitrogen™, 00-4958-02).The images were obtained with FLUOVIEW FV3000 (Olympus, Tokyo, Japan).
Live cell videos were taken using Celloger Mini Plus (Curiosis, Seoul, Republic of Korea).The LC3 cells were seeded at 1 × 10 6 cells/well on a 6-well plate and stabilized for 24 h.The media were changed carefully to new media containing 50 µg/mL Fe 3 O 4 , SiO 2 , and these former mixtures.The videos were recorded every 10 min for 24 h.

Analysis of Data
The data were analyzed and graphed using GraphPad Prism software version 9.4 (GraphPad Software Inc., San Diego, CA, USA).In addition, t-test and one-way ANOVA were used to evaluate the p-value and the value was marked as less than 0.05.All relative values were normalized with negative control, which did not treat nanoparticles.

Results and Discussions
The materials used in this research were SiO 2 with a size of 20 (20 ± 5) nm in diameter, and Fe 3 O 4 with a size of around 20 (20 ± 5) nm.The materials were synthesized and characterized using traditional chemical and physical methods [49].Physical characterization and electron microscopy graphs can be seen in Figure 1 and EDAX analysis of SiO 2 presented in Figure S1.This synthesis method and its detailed properties were reported in earlier publications [50][51][52].The mesoporous SiO 2 nanoballs and Fe 3 O 4 were of uniform size and shape, an appropriate material for the next step in the experiments.

Analysis of Data
The data were analyzed and graphed using GraphPad Prism software version 9.4 (GraphPad Software Inc., San Diego, CA, USA).In addition, t-test and one-way ANOVA were used to evaluate the p-value and the value was marked as less than 0.05.All relative values were normalized with negative control, which did not treat nanoparticles.

Results and Discussions
The materials used in this research were SiO2 with a size of 20 (20 ± 5) nm in diameter, and Fe3O4 with a size of around 20 (20 ± 5) nm.The materials were synthesized and characterized using traditional chemical and physical methods [49].Physical characterization and electron microscopy graphs can be seen in Figure 1 and EDAX analysis of SiO2 presented in Figure S1.This synthesis method and its detailed properties were reported in earlier publications [50][51][52].The mesoporous SiO2 nanoballs and Fe3O4 were of uniform size and shape, an appropriate material for the next step in the experiments.For assessments of autophagy modulation, the LC3 transfected cells were generated.The transfection was successfully confirmed in RT-qPCR, increasing LC3 gene levels only in LC3 transfected cells (Figure 2A).The cytotoxicity studies of Fe3O4, SiO2 and these mixtures were characterized using WST-8 assay with LC3 transfected cells.Earlier results confirmed that kidney cells were competent in up-taking NPs through the membrane, which strengthened the NP concentrations of the intracellular compartments.Aging-related inflammatory stresses of the kidney were protected by autophagy [53].Under autophagydeficient conditions, degeneration of the kidney occurred [54][55][56].Factors in apoptosis and fibrosis increased the production of ROS from kidney injury and chronic inflammation to kidney senescence [57].A series of particles at concentrations from 1 to 100 µg/mL was measured cytotoxic influences.The Fe3O4, SiO2, and these mixtures did not show any toxicity in various concentrations (Figure 2B).The ROS levels of Fe3O4, SiO2, and these For assessments of autophagy modulation, the LC3 transfected cells were generated.The transfection was successfully confirmed in RT-qPCR, increasing LC3 gene levels only in LC3 transfected cells (Figure 2A).The cytotoxicity studies of Fe 3 O 4 , SiO 2 and these mixtures were characterized using WST-8 assay with LC3 transfected cells.Earlier results confirmed that kidney cells were competent in up-taking NPs through the membrane, which strengthened the NP concentrations of the intracellular compartments.Agingrelated inflammatory stresses of the kidney were protected by autophagy [53].Under autophagy-deficient conditions, degeneration of the kidney occurred [54][55][56].Factors in apoptosis and fibrosis increased the production of ROS from kidney injury and chronic inflammation to kidney senescence [57].A series of particles at concentrations from 1 to 100 µg/mL was measured cytotoxic influences.The Fe 3 O 4 , SiO 2 , and these mixtures did not show any toxicity in various concentrations (Figure 2B).The ROS levels of Fe 3 O 4 , SiO 2 , and these mixtures were similar to the negative control, and significantly lower than the positive control (H 2 O 2 400 µM; Figure 2C).
Western blot and RT-qPCR were performed to confirm the modulation of autophagy by analyzing the change in LC3.In Figure 3A, the LC3-II appeared as a dark band in Fe 3 O 4 , SiO 2 , and their mixture.The total LC3 and LC3-II levels increased after treatment of Fe 3 O 4 , SiO 2 , and their mixture.Interestingly, the LC3-II/I level, indicating LC3 activation, also increased following nanoparticle treatment (Figure 3B).The increased intensity of LC3 post-nanoparticle treatment indicates potential autophagy enhancement.Additionally, the levels of LC3 gene were elevated, except for Fe 3 O 4 (Figure 3C).Although there was no correlation between the western blot and RT-qPCR results, an increase in LC3 was observed for both SiO 2 and the Fe 3 O 4 + SiO 2 mixture.This suggests the possibility of alternative pathways influencing LC3 dynamics beyond canonical autophagy [58].
Nanomaterials 2024, 14, x FOR PEER REVIEW 6 of 11 mixtures were similar to the negative control, and significantly lower than the positive control (H2O2 400 µM; Figure 2C).Western blot and RT-qPCR were performed to confirm the modulation of autophagy by analyzing the change in LC3.In Figure 3A, the LC3-II appeared as a dark band in Fe3O4, SiO2, and their mixture.The total LC3 and LC3-II levels increased after treatment of Fe3O4, SiO2, and their mixture.Interestingly, the LC3-II/Ⅰ level, indicating LC3 activation, also increased following nanoparticle treatment (Figure 3B).The increased intensity of LC3 post-nanoparticle treatment indicates potential autophagy enhancement.Additionally, the levels of LC3 gene were elevated, except for Fe3O4 (Figure 3C).Although there was no correlation between the western blot and RT-qPCR results, an increase in LC3 was observed for both SiO2 and the Fe3O4 + SiO2 mixture.This suggests the possibility of alternative pathways influencing LC3 dynamics beyond canonical autophagy [58].Western blot and RT-qPCR were performed to confirm the modulation of autophagy by analyzing the change in LC3.In Figure 3A, the LC3-II appeared as a dark band in Fe3O4, SiO2, and their mixture.The total LC3 and LC3-II levels increased after treatment of Fe3O4, SiO2, and their mixture.Interestingly, the LC3-II/Ⅰ level, indicating LC3 activation, also increased following nanoparticle treatment (Figure 3B).The increased intensity of LC3 post-nanoparticle treatment indicates potential autophagy enhancement.Additionally, the levels of LC3 gene were elevated, except for Fe3O4 (Figure 3C).Although there was no correlation between the western blot and RT-qPCR results, an increase in LC3 was observed for both SiO2 and the Fe3O4 + SiO2 mixture.This suggests the possibility of alternative pathways influencing LC3 dynamics beyond canonical autophagy [58].We represent the productive autophagosome mechanism in Figure 5.This result proved that the SiO2 and Fe3O4 NPs influenced the autophagosome formation.The addition of SiO2 and Fe3O4 could generate the autophagy process inside the cells.The autophagic potential was activated in response to stress-induced activity by SiO2 and Fe3O4 nanoparticles.The continuous activation of autophagy facilitated cell proliferation and contributed to kidney repair.Overall, this finding proved a novel pathway for autophagy modulation, which was not known previously.

Conclusions
Since autophagy has been proven to be involved in chemotherapy and radiotherapy, researchers could target the modulation of autophagy by NPs as an attractive new therapy.Researchers investigated Fe3O4 NPs and SiO2 NPs to determine if they might improve kidney cell proliferation and repair kidney injury.The current result illustrated that Fe3O4 and SiO2 NPs could promote cell growth and modulate autophagy.Especially, the mix-

Figure 1 .
Figure 1.TEM images of both (A) SiO2 nanoparticle and (B) Fe3O4 nanoparticle, respectively.The corresponding size distribution histogram for both (C) SiO2 nanoparticle and (D) Fe3O4 nanoparticle, respectively.The scale bar is 20 nm.

Figure 1 .
Figure 1.TEM images of both (A) SiO 2 nanoparticle and (B) Fe 3 O 4 nanoparticle, respectively.The corresponding size distribution histogram for both (C) SiO 2 nanoparticle and (D) Fe 3 O 4 nanoparticle, respectively.The scale bar is 20 nm.

Figure 3 .
Figure 3. Alteration of LC3 treatment using nanoparticles.(A) Western blot images of nanoparticles treated at 50 µg/mL for 24 h.(B) Relative band intensity of the western blot.(C) Expression levels of LC3 gene at 50 µg/mL for Fe3O4, SiO2, and their combination.All data are presented as mean ± SD

Figure 3 .
Figure 3. Alteration of LC3 treatment using nanoparticles.(A) Western blot images of nanoparticles treated at 50 µg/mL for 24 h.(B) Relative band intensity of the western blot.(C) Expression levels of LC3 gene at 50 µg/mL for Fe3O4, SiO2, and their combination.All data are presented as mean ± SD

Figure 3 .
Figure 3. Alteration of LC3 treatment using nanoparticles.(A) Western blot images of nanoparticles treated at 50 µg/mL for 24 h.(B) Relative band intensity of the western blot.(C) Expression levels of LC3 gene at 50 µg/mL for Fe 3 O 4 , SiO 2 , and their combination.All data are presented as mean ± SD (n = 3).The data were normalized using a negative control that did not involve nanoparticle treatment.* p < 0.05.The confirmation of LC3 changes was performed via confocal and live cell monitoring.The intense green fluorescence observed after nanoparticle treatment indicates strong LC3 expression (Figure 4, S2SI1), confirming autophagosome formation.The green fluorescence is notably more intense in SiO 2 and the Fe 3 O 4 and SiO 2 mixture.The green fluorescence in live cell monitoring increased in intensity from 0 h, surpassing that of the negative control.

Figure 4 .
Figure 4. Fluorescence analysis of LC3 marker (green) and nucleus (blue, DAPI) in Fe3O4, SiO2, and their combination nanoparticles.(A) Fluorescence images (B) The intensity of the fluorescence signal Scale bar: 20 µm.The intensity was calculated dividing green into blue, and data were normalized using a negative control that did not involve nanoparticle treatment.** p < 0.005.

Figure 4 . 11 Figure 5 .
Figure 4. Fluorescence analysis of LC3 marker (green) and nucleus (blue, DAPI) in Fe 3 O 4 , SiO 2 , and their combination nanoparticles.(A) Fluorescence images (B) The intensity of the fluorescence signal Scale bar: 20 µm.The intensity was calculated dividing green into blue, and data were normalized using a negative control that did not involve nanoparticle treatment.** p < 0.005.We represent the productive autophagosome mechanism in Figure 5.This result proved that the SiO 2 and Fe 3 O 4 NPs influenced the autophagosome formation.The addition of SiO 2 and Fe 3 O 4 could generate the autophagy process inside the cells.The autophagic potential was activated in response to stress-induced activity by SiO 2 and Fe 3 O 4 nanoparticles.The continuous activation of autophagy facilitated cell proliferation and contributed to kidney repair.Overall, this finding proved a novel pathway for autophagy modulation, which was not known previously.Nanomaterials 2024, 14, x FOR PEER REVIEW 8 of 11

Figure 5 .
Figure 5. Schematic description of the autophagy mechanism induced by Fe 3 O 4 and SiO 2 NPs.