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# Low frequency vibrating magnetic field-triggered magnetic microspheres with a nanoflagellum-like floor for most cancers remedy | Journal of Nanobiotechnology

### Characterization of Fe3O4/BSA and Fe3O4/BSA/rSiO2

Cubic superparamagnetic Fe3O4 nanoparticles with excessive saturation magnetization (76.25 emu/g) (Fig. 1a and Extra file 1: Figs. S1, S2) had been synthesized. BSA microspheres containing Fe3O4 cores (i.e., Fe3O4/BSA) had been then shaped by spray drying adopted by denaturation of BSA. Within the obtained magnetic microspheres (Fig. 1b, left), numerous Fe3O4 nanoparticles might be clearly noticed within the inner area (Fig. 1b, proper).

SiO2 nanorods may develop on the surfaces of the Fe3O4/BSA microspheres. The obtained Fe3O4/BSA/rSiO2 microspheres had a flagellum-like floor (Fig. 1c). The size of the silica nanorods could possibly be tailor-made by adjusting the water quantity within the response system (see strategies). For instance, when the amount of water added to the response precursor was modified from 2 to 1.3, 0.7, and 0.35 mL, silica nanorods with lengths of 305.3 ± 118.0 nm, 873.7 ± 178.2 nm, 1691.3 ± 302.2 nm, and curled silicon (greater than 1700 nm) grew on the microsphere surfaces (Extra file 1: Fig. S3). Within the following sections, these microspheres are known as Fe3O4/BSA/SrSiO2, Fe3O4/BSA/MrSiO2, Fe3O4/BSA/LrSiO2, and Fe3O4/BSA/CrSiO2, the place the letters S, M, L and C point out quick, medium, lengthy, and curled, respectively. The sizes of Fe3O4/BSA and Fe3O4/BSA/rSiO2 primarily ranged roughly from 0.5 to 2.5 μm and 1 to 4 μm, respectively.

Excessive-resolution transmission electron microscopy (HRTEM) photos clearly confirmed that Fe3O4/BSA/LrSiO2 contained quite a few “darkish” nanocores (Fig. 2a). These cores had been iron oxide nanoparticles, as a result of HRTEM elemental mapping revealed that Fe3O4/BSA/LrSiO2 contained C, O, Fe, Si, N, and S. The Fe atoms had been positioned contained in the microspheres and the microspheres had been capped with a silica shell with a pointy floor (Fig. 2b and c). Vitality-dispersive spectroscopy (EDS) of the entire pattern confirmed that the Fe3O4/BSA/LrSiO2 microspheres had been composed of the above six parts (Fig. 2d) and the mass ratio of Fe atoms was 15.54% (Fig. 2e). The mass of Fe atoms within the last microspheres might be simply adjusted by altering the focus of Fe3O4 nanoparticles within the precursor resolution earlier than spray drying.

The magnetic hysteresis loops of Fe3O4/BSA and Fe3O4/BSA/rSiO2 with 4 kinds of silica rods confirmed that each one the samples maintained superparamagnetic character (the coercive drive was near zero) (Fig. 3a). The saturation magnetization of Fe3O4/BSA was 39.5 emu/g. It decreased after Fe3O4/BSA was coated with a silica shell, and it additionally decreased with growing silica rod size, which is as a result of SiO2 is a non-magnetic materials (Fig. 3a). Fe3O4/BSA/rSiO2 could possibly be properly dispersed in aqueous resolution, which can be owing to that each SiO2 and BSA are hydrophilic supplies. For instance, when Fe3O4/BSA/LrSiO2 had been dispersed in PBS or RPMI-1640 tradition medium, no precipitates could possibly be noticed on the backside of tubes after being saved at room temperature for no less than 5 min (Extra file 1: Fig. S4). After storing in absolute ethanol at 4 °C for 1 12 months, each the magnetism and morphology of Fe3O4/BSA/rSiO2 had been nearly unchanged. For instance, for Fe3O4/BSA/LrSiO2, the saturation magnetization 1 12 months post-synthesis was near 26 emu/g, which was much like that of the as-synthesized pattern (Fig. 3b). The silica rods on the Fe3O4/BSA microsphere floor and the Fe3O4/BSA microspheres didn’t clearly change in form after 1 12 months storage (Fig. 3c), which confirmed their excessive stability throughout storage.

Owing to the magnetism, Fe3O4/BSA/rSiO2 uncovered to a low frequency VMF (2 Hz) vigorously vibrated with vibration of the magnetic subject (Fig. 3d and a video within the Extra file 2), which could possibly be clearly noticed straight. Moreover, the morphology of the Fe3O4/BSA/rSiO2 microspheres didn’t clearly change after the microspheres had been constantly uncovered to a VMF for six h (Fig. 3e). These outcomes indicated that Fe3O4/BSA/rSiO2 might be handled with a VMF for a very long time, and such vibrating microspheres with sharp surfaces below a VMF could act as small weapons to struggle most cancers cells.

### Cytotoxicity of Fe3O4/BSA/rSiO2

#### Impact of Fe 3 O 4 /BSA/rSiO 2 on the cell cycle distribution

After Tu212 cells had been incubated with 0.5 mg/mL of Fe3O4/BSA or Fe3O4/BSA/LrSiO2 for 48 h, the populations of the cells within the G0/G1, S, and G2/M phases had been all much like these of the untreated cells (Fig. 4a and b). The cell cycle distribution was not considerably modified by the existence of Fe3O4/BSA or Fe3O4/BSA/LrSiO2, indicating that each supplies don’t have any affect to the cell life actions.

#### Impact of Fe 3 O 4 /BSA/rSiO 2 on cell viability

When Tu212 cells had been incubated with Fe3O4/BSA or Fe3O4/BSA/LrSiO2 (0.2 or 2 mg/mL) for both 2 or 24 h, the cell viabilities had been near these of the cells incubated with phosphate-buffered saline (PBS) alone (Fig. 4c). This example was related for the opposite Fe3O4/BSA/rSiO2 compounds, together with Fe3O4/BSA/SrSiO2, Fe3O4/BSA/MrSiO2, and Fe3O4/BSA/CrSiO2. That’s, the entire cell viabilities remained at about 100% after incubation with Fe3O4/BSA/rSiO2 containing totally different size silica nanorods (Fig. 4d). It ought to be famous that 2 mg/mL is a excessive focus for evaluating the cytotoxicity of nanoparticles. This indicated that Fe3O4/BSA earlier than and after coating with silica rods with totally different lengths all confirmed no cytotoxicity even at excessive focus. The great biocompatibility of Fe3O4/BSA/rSiO2 could also be as a result of Fe3O4, BSA, and SiO2 are all biocompatible supplies.

### VMF-triggered Fe3O4/BSA/rSiO2 for killing most cancers cells

To find out whether or not Fe3O4/BSA/rSiO2 can kill most cancers cells below the publicity to a VMF, Fe3O4/BSA/rSiO2 microspheres had been added to Tu212 cells after which uncovered to a VMF for 1 h. The outcomes confirmed that Fe3O4/BSA/rSiO2 with 4 lengths of silica rods all effectively induced a lower within the cell viability (Fig. 5a and b). Moreover, the cell killing effectivity of VMF-triggered Fe3O4/BSA/rSiO2 was larger than that of Fe3O4/BSA with out silica rods below publicity to the identical VMF. As well as, extra cells had been killed when the cells had been incubated with Fe3O4/BSA/rSiO2 with longer silica rods below publicity to the identical VMF. These outcomes had been confirmed by each qualitative evaluation with Hoechst 33,342/propidium iodide (PI) double staining reagent (Fig. 5a) and quantitative evaluation with CellTiter-Glo® reagent (Fig. 5b). It is because Fe3O4/BSA/rSiO2 had sharper surfaces than Fe3O4/BSA, and the microspheres with longer silica rods had extra alternative to slice extra cells and/or pierce their cell membranes than these with shorter silica rods when these sharp microspheres had been uncovered to a VMF. For instance, the viabilities of the Tu212 cells handled with Fe3O4/BSA, Fe3O4/BSA/SrSiO2, Fe3O4/BSA/MrSiO2, and Fe3O4/BSA/LrSiO2 (1 mg/mL for all samples) below the identical VMF (2 Hz, 1 h) had been 85.39% ± 4.12%, 76.66% ± 7.37%, 51.37% ± 8.25%, 70.94% ± 9.22%, respectively. The lower of cell viability could also be induced straight by the cell membrane harm, as PI reagent can penetrate into cells solely throughout the broken membranes after which “gentle” the cells with purple fluorescence.

When the silica nanorods on the microspheres had been lengthy however curled, the cell killing effectivity of the pattern (i.e., Fe3O4/BSA/CrSiO2) below VMF publicity was decrease than that induced by the microspheres with lengthy however straight silica nanorods below publicity to the identical VMF (Fig. 5a and b). This can be as a result of the sharp ends of the curled silica nanorods had fewer alternatives to pierce the cells than these of the straight silica nanorods.

When cells are broken by a mechanical drive, the mobile contents, similar to lactic dehydrogenase (LDH), could also be launched. The LDH leakage charges induced by the VMF-triggered Fe3O4/BSA, Fe3O4/BSA/SrSiO2, Fe3O4/BSA/MrSiO2, and Fe3O4/BSA/LrSiO2 elevated so as (Fig. 5c). Nevertheless, the LDH leakage charge was vital decrease for the Fe3O4/BSA/CrSiO2 group than that for the Fe3O4/BSA/LrSiO2 group. These outcomes additional demonstrated that the magnetic microspheres with sharp surfaces exhibited sturdy cell harm capability by way of the magneto-mechanical drive.

Based mostly on the above outcomes, we are able to conclude that the order of the talents of Fe3O4/BSA and Fe3O4/BSA/rSiO2 below VMF publicity for damaging most cancers cells from sturdy to weak is Fe3O4/BSA/LrSiO2 > Fe3O4/BSA/CrSiO2 ≈ Fe3O4/BSA/MrSiO2 > Fe3O4/BSA/SrSiO2 > Fe3O4/BSA (Fig. 5d). Fe3O4/BSA/LrSiO2 was chosen for subsequent cell and animal experiments.

The cell killing effectivity improved with growing magnetic subject frequency from 0.5 to three Hz, however it didn’t additional enhance by constantly growing the frequency when the cells had been incubated with Fe3O4/BSA/LrSiO2 (1 mg/mL) adopted by 1 h of VMF publicity (Fig. 6a). This can be as a result of a few of the magnetic microspheres with excessive frequency oscillation quickly left the cells as quickly as they arrived on the cell surfaces and even didn’t attain the cells. That’s, a few of the microspheres triggered by a excessive frequency (e.g., 4 Hz) VMF didn’t have the chance to strongly impression the cells. When the VMF publicity time was elevated from 0.5 to 1.5 h (Fig. 6b) or the focus of Fe3O4/BSA/LrSiO2 was elevated from 0.2 to 1 mg/mL (Fig. 6c), the viabilities of the Tu212 cells considerably decreased. The useless cells in above teams had been additionally confirmed by the fluorescent imaging (i.e., purple fluorescent cells had been useless or membrane broken) (Extra file 1: Figs. S5–S7).

The diploma of injury of the most cancers cells could possibly be clearly noticed by SEM. Tu212 cells incubated with Fe3O4/BSA/LrSiO2 with out VMF publicity maintained regular morphology (Fig. 6d). Nevertheless, massive holes appeared within the cells after the cells had been uncovered to a VMF, and the quantity and measurement of the holes elevated with growing VMF publicity time (Fig. 6d). For instance, when the Fe3O4/BSA/LrSiO2-incubated cells had been uncovered to a VMF (2 Hz) for 0.5 h, many holes (most not more than ~ 2 μm in diameter) appeared within the cells. For 1.5 h VMF publicity, numerous massive holes (greater than 5 μm in diameter) appeared within the cells, and a few cells had been practically damaged into items. These quite a few massive holes had been generated by the Fe3O4/BSA/LrSiO2-mediated magneto-mechanical drive, which was the direct motion inflicting cell dying.

### VMF-triggered Fe3O4/BSA/rSiO2 for inhibiting mouse tumor progress

To analyze whether or not the VMF-triggered magnetic microspheres can inhibit tumor progress, Tu212 cells (5 × 106) combined with Fe3O4/BSA/LrSiO2 (5 mg/mL) had been subcutaneously injected into nude mice. One group was uncovered to a VMF for 1 h every day for 21 days (Fe3O4/BSA/LrSiO2 + VMF) after which constantly housed for 21 days. The opposite group was not handled with the VMF (Fe3O4/BSA/LrSiO2, no VMF). Tumor progress within the VMF group was considerably inhibited in contrast with the no VMF group (Fig. 7a, b and Extra file 1: Figs. S8–S12). A statistical distinction within the common tumor volumes between these two teams appeared after 14 days of steady VMF publicity. This distinction within the tumor volumes constantly elevated. After 21 days of VMF publicity, the typical tumor quantity within the VMF group was solely a few third of that within the no VMF group. When the VMF was stopped from 22 to 42 days, tumor progress accelerated and the typical tumor quantity reached 613.991 ± 283.09 mm3 on the forty second day. Nevertheless, the expansion charge in today was nonetheless vital slower than that within the no VMF group (tumor quantity on the forty second day of 1650.29 ± 328.47 mm3). It ought to be famous that the most cancers cells in a single mouse within the VMF group had been utterly killed after injection of the pattern, whereas the tumors in the entire mice in no VMF group quickly grew after injection of the identical pattern.

To additional examine the flexibility of the Fe3O4/BSA/LrSiO2-mediated magneto-mechanical drive to inhibit tumor progress, Fe3O4/BSA/LrSiO2 with larger focus (15 mg/mL) along with Tu212 cells (5 × 106) was subcutaneously injected into mice. These mice had been additionally divided into two teams: VMF for 1 h every day for 21 days with no VMF for the next 21 days and no VMF for 42 days. An fascinating discovering was that the tumors in the entire mice within the VMF group may nearly not be detected in the course of the first 21 days after injection, no tumors could possibly be detected in two mice in the course of the subsequent 21 days of no VMF publicity, and the opposite two mice grew solely small tumors in contrast with the management group (Fig. 7a, b and Extra file 1: Figs. S8–S12). Nevertheless, for the no VMF group, the tumors on the authentic injection websites quickly grew in the course of the 42 days after injection. As well as, Fe3O4/BSA/LrSiO2 (5 or 15 mg/mL) with out VMF publicity didn’t inhibit tumor progress in contrast with the PBS group (management group).

At 42 days post-injection, the mice had been sacrificed and the tumors and fundamental organs had been resected for evaluation. The weights (Fig. 7c) and sizes (Fig. 7d) of the resected tumors additional confirmed that VMF-triggered Fe3O4/BSA/LrSiO2 exhibited sturdy capability for inhibiting tumor progress. For instance, the typical tumor weight of the Fe3O4/BSA/LrSiO2 (15 mg/mL) + VMF group was solely 8.13% of that of the PBS group and 22.76% of that of the Fe3O4/BSA/LrSiO2 (5 mg/mL) + VMF group. There was no statistical distinction within the weights of the Fe3O4/BSA/LrSiO2 (no VMF) and PBS teams. The histology photos confirmed that the tumor cells a small distance from Fe3O4/BSA/LrSiO2 within the tumor tissue had been much like these within the PBS group, which would be the purpose why a number of tumors recovered after VMF therapy was stopped (Fig. 7e). For the teams during which no tumors could possibly be detected, no tumor cells within the tissues on the authentic injection websites had been noticed (Fig. 7e). Typically, tumor progress may cause enlargement of the spleen owing to the immune response. As a result of the expansion of the tumors within the two Fe3O4/BSA/LrSiO2 + VMF teams (5 and 15 mg/mL Fe3O4/BSA/LrSiO2) was considerably inhibited, the spleen coefficients (ratios of the spleen weight to the physique weight) in these two teams had been a lot smaller than these within the PBS, two Fe3O4/BSA/LrSiO2 and no VMF teams (Fig. 7f). The coefficients of the opposite fundamental organs, the guts, liver, lungs, and kidneys (Fig. 7f), in addition to the mouse physique weights (Fig. 7g), had been no statistically totally different amongst the entire teams, indicating that Fe3O4/BSA/LrSiO and VMF publicity did no hurt to the mice.

### Magneto-mechanical drive generated by Fe3O4/BSA/LrSiO2 uncovered to a VMF

No temperature improve within the Fe3O4/BSA/LrSiO2 aqueous suspension or mouse tumor containing Fe3O4/BSA/LrSiO2 was detected when the suspension or tumor was uncovered to a VMF (Fig. 8a and b). The magneto-mechanical drive could play an vital function in in vivo tumor inhibition. The method of a Fe3O4/BSA/LrSiO2 microsphere in tumor tissue producing a drive below a VMF is proven in Fig. 8c. When Fe3O4/BSA/LrSiO2 is positioned in the course of the magnetic subject (state I), it generates a minimal drive. When the amplitude of the VMF reaches the utmost worth (state II), the drive additionally reaches the utmost worth. When the VMF returns to its authentic location (state III) and likewise reaches the inverse most amplitude (state IV), the drive reaches a minima and likewise reaches the inverse most worth. After the VMF place returns to state V, the following cycle begins.

For Fe3O4/BSA/LrSiO2 microspheres in mouse tumor tissue, a lot of the vibrating microspheres below VMF publicity are exhausting to go away the most cancers cell floor as a result of small intercellular area. On this case, the utilized magnetic drive (F) on one microsphere is

$$F={mu }_{0}chi V{H}_{t}nabla H$$

(1)

the place μ0 = 4π × 10−7 (T·m·A−1, observe: T, m and A are unites of Tesla, metre and Ampere, respectively) which is the permeability of vacuum, V is the amount of Fe3O4/BSA/LrSiO2 (3.35 × 10−18 m3), and Ht is the magnetic subject energy on the microsphere at a sure time. (nabla H) is the grade of the magnetic subject energy:

$$nabla H = , (H_{max} – H_{min} )/D$$

(2)

the place D = 0.015 m (Fig. 8c), which is the space between a magnet and one Fe3O4/BSA/LrSiO2.

On this research, the utmost and minimal magnetic subject energy values (Hmax and Hmin) had been 400 mT and 0, which had been on the surfaces of the 2 magnets and the center place between the 2 magnets within the VMF gear, respectively. Due to this fact, ΔHmax = 400 mT.

X is the typical magnetic susceptibility of the Fe3O4/BSA/LrSiO2 microsphere below a particular magnetic subject:

$$X , = B/H = [{4}pi rho B^{prime}/({1 } times { 1}0^{{4}} )]/H_{max}$$

(3)

the place ρ is the density of a Fe3O4/BSA/LrSiO2 microsphere (ρ = 2.72 g/cm3).

(Observe: the unit of B and H ought to be the identical since X is nondimensional. The 4πρB/(1 × 104) a part of (3) is to transformed the unit of B from emu/g to tesla).

Based mostly on the magnetic hysteresis loop of Fe3O4/BSA/LrSiO2 (Fig. 3), we calculated that B = 25.4 emu/g when H = 400 mT.

Based mostly on the above evaluation and Eq. (3), X = 1.1726.

The space between a magnet and one Fe3O4/BSA/LrSiO2 microsphere at a sure time level (Dt) is

$$D_{{textual content{t}}} = D – D{textual content{sin}}omega t$$

(4)

the place ω = 2πf (f is the magnetic subject frequency), which is the rotating velocity of the disk triggering the 2 magnets shifting in straight reciprocating mode (see Scheme 1).

Ht might be thought-about to be the linear change between Hmax and Hmin. Due to this fact, when the VMF place modifications from I to III (Fig. 8c),

$$H_{{textual content{t}}} = H_{{{textual content{max}}}} left( {D – D_{{textual content{t}}} } proper)/D = H_{{{textual content{max}}}} {textual content{sin}}omega t$$

(5)

When the VMF place modifications from III to V (see Fig. 8c), the route of the magnetic subject modifications to the other way to the case from I to III. Due to this fact, for the entire working technique of VMF,

$$H_{{textual content{t}}} = H_{{{textual content{max}}}} mid {textual content{sin}}omega t mid$$

(6)

Based mostly on Eqs. (1) to (6) and the values of μ0, X, V, ΔHmax, and D described above, the magneto-mechanical drive is

$$F=frac{{mu }_{0}chi V{H}_{max}^{2}}{D}|{textual content{sin}}omegatext{t}|= 6.17 textual content{pN.}$$

That is the utmost drive generated by one Fe3O4/BSA/LrSiO2 microsphere below a VMF.

In tumor tissue, the Fe3O4/BSA/LrSiO2 microspheres could all the time connect to the most cancers cell floor or solely barely separate from the cell floor in a really quick time throughout VMF publicity, as a result of these microspheres are firmly trapped by surrounding cells. The magnetic subject displays sturdy tissue penetration capability, and the tumor measurement on this research is not more than 150 mm3 throughout VMF publicity. Due to this fact, the drive generated by Fe3O4/BSA/LrSiO2 might be utterly delivered to the encompassing most cancers cells.

One most cancers cell on the injection website within the tumor tissue could connect multiple Fe3O4/BSA/LrSiO2 microsphere. The overall drive (Fcomplete) transferred from these magnetic microspheres to 1 cell floor is

$$F_{textual content{complete}} = 6.17 occasions n{textual content{pN}}$$

(7)

the place n is the variety of Fe3O4/BSA/LrSiO2 microspheres on the cell floor. Due to this fact, the Fe3O4/BSA/LrSiO2-mediated drive appearing on one cell could also be tens or multiple hundred of piconewtons. The most cancers cells shall be induced to dying by such a big drive, because it has been demonstrated that tens of piconewtons [1] and even smaller drive [18] generated by one particle may harm cells.

### Intracellular ROS generated by Fe3O4/BSA/LrSiO2 uncovered to a VMF

It was an fascinating consequence that Fe3O4/BSA/rSiO2 uncovered to a VMF generated intracellular ROS. When the focus of Fe3O4/BSA/LrSiO2 within the cells was 0.2, 0.5, and 1 mg/mL and the VMF (400 mT, 2 Hz) publicity time was 1 h, the ROS ranges had been all larger than that within the management group (cells solely), and the ROS stage elevated with growing Fe3O4/BSA/LrSiO2 focus (Fig. 9). For instance, when 1 mg/mL of Fe3O4/BSA/LrSiO2 was added to Tu212 cells and triggered with the VMF for 1 h, the ROS stage was ~ 2.08 occasions larger than that of 0.2 mg/mL Fe3O4/BSA/LrSiO2 below the identical VMF and ~ 3.08 occasions larger than that of the management. For the instances the place the cells had been incubated with totally different Fe3O4/BSA/LrSiO2 concentrations with out VMF publicity and the cells alone had been uncovered to the VMF for 1 h, practically no ROS had been detected in contrast with the management.

The magneto-mechanical drive induced ROS era, which can be as a result of the cells had been mechanically broken, resulted in an imbalance between manufacturing and elimination of oxygen free radicals within the cells. The irregular cell metabolism could lead to ROS similar to hydrogen peroxide accumulating in cells. The broken mobile microenvironment infiltrated the cells, and Fe3O4 in Fe3O4/BSA/rSiO2 catalyzed hydrogen peroxide to additional generate ROS (hydroxyl radicals). ROS exhibit excessive toxicity to cells and tissues by way of oxidative harm of the proteins and lipids within the cells, similar to chopping off the peptide chains in proteins. Due to this fact, the VMF-triggered Fe3O4/BSA/LrSiO2 killing of most cancers cells and inhibition of tumor progress described above could also be due to the mixture of the magneto-mechanical drive and force-induced ROS (e.g., FDT).

### Toxicity of Fe3O4/BSA/LrSiO2 to mice

The eight parameter ranges of the liver and kidney capabilities of mice injected with Fe3O4/BSA/LrSiO2 at each 1 day and 15 days post-injection had been all within the regular ranges, they usually weren’t statistically vital totally different from these of mice injected with PBS (management) (Fig. 10a). At 1 day post-injection, the typical white blood cell (WBC) depend was considerably larger than that of the management (Fig. 10b). This can be as a result of the native harm brought on by injection of Fe3O4/BSA/LrSiO2 induced an immune response, and extra WBCs had been generated for wound restore. At 15 days post-injection, the WBC depend returned to regular. The opposite eight blood routine parameters at each 1 day and 15 days post-injection had been all not statistically totally different from these of the management group, they usually had been all positioned within the regular ranges (Fig. 10b).

The sizes and weights of the mouse spleens within the Fe3O4/BSA/LrSiO2 group 15 days post-injection had been related with these within the PBS group (Fig. 10c), which indicated that there was no vital inflammatory response at the moment. This consequence additional demonstrated that the WBC improve talked about above was a brief phenomenon within the early stage after injection. There have been no statistically vital variations in the principle organ (together with spleen) coefficients between the Fe3O4/BSA/LrSiO2 and PBS teams (Fig. 10c). These outcomes indicated that the mice injected with Fe3O4/BSA/LrSiO2 had been in good well being.

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