A Novel Synthesis of Fe 3 O 4 @SiO 2 @Au@Porous SiO 2 Structure for NIR Irradiation-Induced DOX Release and Cancer Treatment

Abstract

Doxorubicin (DOX) alone or in combination has been widely used for numerous cancers, including breast, lung, bladder, and so on. In this article, a core/shell/shell structured Fe₃O₄@SiO₂@Au@porous SiO₂ particles for the drug delivery and release of DOX was demonstrated, with the aid of near-infrared irradiation. Fe₃O₄ was used to direct the transportation and delivery of the drug-loaded composite to the target tissues and organs under an external magnetic field, the first layer of SiO₂ was used for Au nanoparticle attachment, Au acted as the agent for light–thermal conversion, and the porous SiO₂ was used to load DOX. The morphology of the nanoparticles was studied by transmission electron microscopy, and the porous structure was characterized by N₂ adsorption/desorption curves. The drug delivery system displayed high drug loading capacity, and the release behavior was largely impacted by the environmental pH. Furthermore, the cytotoxicity of Fe₃O₄@SiO₂@Au@porous SiO₂ and DOX loaded Fe₃O₄@SiO₂@Au@porous SiO₂ was studied through in vitro 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assay.

Introduction

Cancer has been one of the most devastating diseases nowadays, which has resulted in millions of death all over the world each year. ^{1 -4} At the current stage, there are still many huge challenges for the treatment of various cancers. Chemotherapy has been considered as an effective method to treat cancers. ^{5 -7} Among the drugs used for chemotherapy treatment, doxorubicin (DOX) alone or in combination has been widely used for numerous cancers, including breast, lung, bladder, and so on. ^{8 -14} Doxorubicin can intercalate into DNA and prevent the macromolecular biosynthesis and the growth of cancer cells. ¹⁵ However, the side effect of chemotherapy includes high toxicity and damages to healthy tissues and organs. Moreover, another difficulty with conventional chemotherapy is to accumulate chemotherapeutic agents within the cancer tissue and exert the treatment specifically for cancer cells. ¹⁵

To solve this problem, the drug delivery system has been proposed and applied to accumulate drugs within the cancer tissues. ^{16 -22} During the past decades, various nanoparticle-based drug delivery systems incorporating Au nanoparticles have been reported. ²³ Within these systems, Au nanoparticles are sensitive to light and display superb light–thermal conversion ability, which makes them a great candidate for light-induced drug release. ²³ Different Au nanostructures that absorb near-infrared (NIR) irradiation have been synthesized, including nanorods, nanocages, nanostars, and so on, and have been prepared. ²⁴ These structures have shown the capabilities of penetrating deep tissues for NIR-induced drug release. For example, Liu et al delivered Au nanoparticle-functioned peptides with loaded drugs to liver cancer cells and investigated the drug release under NIR. ²⁵ Besides, Au nanoparticle-decorated liposomes and polyelectrolytes with various drug lodgings have been synthesized and applied for drug delivery and release. ^{26 -30} However, the size of most of these capsules is too large for in vivo testing, which limits their further applications within biomedical fields. ^{31 -34}

In this report, we synthesized a novel nanosized Fe₃O₄@SiO₂@Au@porous SiO₂ structure as a drug carrier. Fe₃O₄, SiO₂, and Au all possess low toxicity and high biocompatibility, which make them safe to be used within human organ tissues. The magnetization from Fe₃O₄ could be used to direct the drug delivery with the aid of an external magnetic field. Due to the inert surface of Fe₃O₄, another layer of SiO₂ layer was coated so that Au nanoparticles could be attached. The Au nanoparticles could generate heat within the structure under irradiation due to its superb light–thermal conversion ability. A final layer of porous SiO₂ was coated to prevent the detachment of Au nanoparticles and load drugs. A porous structure was able to provide a large surface area for drug loading. The impacts of pH and irradiation power on the drug release of Fe₃O₄@SiO₂@Au@porous SiO₂-DOX are studied. Finally, the toxicity of Fe₃O₄@SiO₂@Au@porous SiO₂ and Fe₃O₄@SiO₂@Au@porous SiO₂-DOX are studied through in vitro 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay.

Experiment

Synthesis of Fe₃O₄@SiO₂@Au

Fe₃O₄ nanoparticles were synthesized through hydrolysis reaction at 220°C. ³⁵ SiO₂ was coated on Fe₃O₄ particles through a modified Stober method. Specifically, Fe₃O₄ (0.1 g) was mixed in ethanol (100 mL) and ammonia solution (28%, 5 mL) with mechanical stirring. Tetraethyl orthosilicate (0.1 mL) was introduced into the solution every 10 minutes until the total volume reaches 1 mL. After cleaning with ethanol and DI water, the particles with SiO₂ layer of ∼20 nm were dispersed in isopropanol (80 mL) and 3-aminopropyl-triethoxysilane (APTS, 200 µL) at 75°C for 1 hour so that the amino groups could be grafted to SiO₂ surface. Separately, the Au nanoparticles were synthesized through a Turkevich synthesis. ³⁶ Fe₃O₄@SiO₂@Au particles were synthesized by mixing amine group–modified Fe₃O₄@SiO₂ nanoparticles with Au nanoparticles in DI water under ultrasonication. The mass ratio of Fe₃O₄@SiO₂ to Au was 20 to 1.

Synthesis of Fe₃O₄@SiO₂@Au@SiO₂

Another layer of SiO₂ was coated on Fe₃O₄@SiO₂@Au surface by a sol-gel method. ³⁷ Briefly, Fe₃O₄@SiO₂@Au (0.1 g) and polyvidone (PVP, (C6H9NO)_n, 1 g, molecular weight [Mw]: ∼60 000) were mixed in 100 mL water and ultrasonicated for 10 minutes so that the polymer could be adsorbed on the surface of the particles. The colloids were then washed with DI water and introduced into a solution of ethanol (100 mL) and ammonia solution (28%, 5 mL). Tetraethyl orthosilicate (0.1 mL) was introduced into the dispersion every 10 minutes until the total volume reaches 1 mL. Finally, Fe₃O₄@SiO₂@Au@SiO₂ nanoparticles with external SiO₂ layer of ∼20 nm were obtained.

Synthesis of Fe₃O₄@SiO₂@Au@p-SiO₂

The external layer of SiO₂ in Fe₃O₄@SiO₂@Au@SiO₂ was etched into a porous structure so that Fe₃O₄@SiO₂@Au@p-SiO₂ could be prepared. Specifically, Fe₃O₄@SiO₂@Au@SiO₂ (0.1 g) and PVP (1 g, Mw: ∼10 000) were mixed in 100 mL DI water and refluxed at 100°C for 5 hours. After cooling down to room temperature, NaOH solution (0.32 g/mL, 5 mL) was introduced to the suspension under magnetic stirring. After stirring for 10 minutes, the solution pH was quickly regulated to 7 with dilute HCl to quench the etching reaction. The particles were washed with DI water, and Fe₃O₄@SiO₂@Au@p-SiO₂ particles were obtained.

Synthesis of Fe₃O₄@SiO₂@Au@p-SiO₂-DOX

Fe₃O₄@SiO₂@Au@p-SiO₂ particles (0.1 g) and DOX water solution (500 μg/mL, 50 mL) were stirred and shaken at room temperature for 24 hours in the dark. The final concentration of DOX could be determined by ultraviolet–visible (UV-Vis) spectroscopy. The loaded DOX could be calculated with the concentration difference.

Materials Characterization

The morphology of the particles was studied by transmission electron microscopy (TEM; Hitachi HT7700), and the crystal structure was determined by X-ray diffraction (XRD; Bruker D8). The surface area of the particles was investigated by N₂ adsorption–desorption isotherm curves. The existence of DOX on particle surface was determined by fluorescence spectroscopy (USB2000+; Ocean Optics).

Near-Infrared-Induced Drug Release With Fe₃O₄@SiO₂@Au@p-SiO₂-DOX

Near-infrared-induced DOX dry delivery and release were studied by the dialysis method. Briefly, Fe₃O₄@SiO₂@Au@p-SiO₂-DOX (40 mg) was filled in a dialysis bag, which was then exposed in a phosphate buffer saline (0.1 mol/L, 100 mL) at 37°C, and the pH was regulated based on the experimental design. The DOX-loaded system was exposed to NIR laser at 808 nm (P = 5, 7.5, and 10 W/cm²) under stirring so that light–thermal conversion could be achieved. During the drug release, a sample solution (5 mL) was taken out every 25 minutes to determine the DOX concentration by UV-Vis spectroscopy. After the measurement, the sample solution was returned to the solution container.

In Vivo MTT Assay

C6 glioma cells were selected for cytotoxicity testing due to its wide availability and widely used for cytotoxicity investigation. ³⁸ C6 glioma cells were seeded in a 96-well culture plate with 10% fetal bovine serum for 12 hours. The chamber was heated to 37°C and filled with 5% CO₂, and the cell density was controlled at 10 000 cells/well. The medium was 100 µL of Dulbecco Modified Eagle medium. Both DOX and Fe₃O₄@SiO₂@Au@p-SiO₂-DOX were dispersed in 100 mL culture medium, respectively, and then introduced into each well. Six wells without DOX or Fe₃O₄@SiO₂@Au@p-SiO₂-DOX were used as the control experiments. After incubation for 72 hours, the cytotoxicity was studied by MTT assay. The cell survival rate was calculated. The cytotoxicity of Fe₃O₄@SiO₂@Au@p-SiO₂ alone was also evaluated in a similar way.

Results and Discussion

The TEM image of Fe₃O₄ particles is presented in Figure 1A. It is observed that the particles are spherical, with a size around 50 nm. After coating a thin layer of SiO₂ and coupling agent of APTS on the surface of Fe₃O₄, Au nanoparticles were adsorbed on the SiO₂ surface due to the chemical interaction between Au and amine functional groups from APTS, and the corresponding TEM image of Fe₃O₄@SiO₂@Au nanoparticles is presented in Figure 1B. With the presence of PVP, another layer of SiO₂ was coated on the outer layer of Fe₃O₄@SiO₂@Au so that Au nanoparticles are fully encapsulated. Mesoporous structure of the outer SiO₂ is generated by protecting the near-surface layer of SiO₂ by PVP and etching the outer layer of SiO₂ in NaOH solution. Refluxing the particles in PVP solution results in the penetration of PVP into the SiO₂ surface by generating hydrogen bonds between carbonyl groups and silanol groups. ³⁹ The polymer chains within SiO₂ could dramatically increase its stability in NaOH solution, while the regions without PVP protections would be easily etched by NaOH. Therefore, a porous structure of the outer SiO₂ layer is formed. Finally, Fe₃O₄@SiO₂@Au@p-SiO₂ is synthesized, as shown in Figure 1C. A porous structured SiO₂ at the outer layer could provide a larger surface area for DOX loading. The general nanoparticle synthesis and DOX loading process are summarized in Figure 1D.

Figure 1.

A, B, and D, Transmission electron microscopy (TEM) images for Fe3O4, Fe3O4@SiO2@Au, and Fe3O4@SiO2@Au@SiO2. D, Schematic for the synthesis procedure of Fe3O4@SiO2@Au@SiO2 doxorubicin (DOX).

The porous nature of the outer SiO₂ layer could provide a larger surface area for DOX loading. To verify the surface area change before and after the formation of the porous structure, the N₂ adsorption/desorption curves for Fe₃O₄@SiO₂@Au@SiO₂ and Fe₃O₄@SiO₂@Au@p-SiO₂ are presented in Figure 2A. In Fe₃O₄@SiO₂@Au@p-SiO₂, the large hysteresis between adsorption and desorption curves indicates its porous structure. The specific surface area of Fe₃O₄@SiO₂@Au@SiO₂ and Fe3O₄@SiO₂@Au@p-SiO₂ is calculated as 87.5 and 143.5 m²/g, respectively. It is observed that the porous structure could increase the specific surface area by 64%. The corresponding pore size distribution is presented in Figure 2B. The peak pore size is increased from 3.8 to 9.8 nm. Previous research shows that the diameter of DOX molecules is ∼1.5 nm. ⁴⁰ Therefore, the pore size is large enough to hold DOX molecules.

Figure 2.

A, N2 adsorption/desorption curves for Fe3O4@SiO2@Au and Fe3O4@SiO2@Au@p-SiO2. B, Pore size distribution for Fe3O4@SiO2@Au and Fe3O4@SiO2@Au@p-SiO2. C, Electron paramagnetic resonance (EPR) spectra of Fe3O4, Fe3O4@SiO2@Au, and Fe3O4@SiO2@Au@p-SiO2. D, Fluorescence spectra of Fe3O4@SiO2@Au@p-SiO2 and Fe3O4, Fe3O4@SiO2@Au@p-SiO2-DOX, and doxorubicin (DOX).

The electron paramagnetic resonance spectroscopy is used to study the magnetic properties of Fe₃O₄, Fe₃O₄@SiO₂@Au, and Fe₃O₄@SiO₂@Au@p-SiO₂, and the corresponding spectra are presented in Figure 2C. It shows that, after introducing different materials into Fe₃O₄, both peak intensity and width change, indicating the change in spin states within the particles, are attributed to the different environment of the magnetic phase, while the magnetization is still available within the composites, which is featured with the existence of peak in the spectra.

Fluorescence spectrum is applied to investigate the DOX loading behavior on Fe₃O₄@SiO₂@Au@p-SiO₂, and the corresponding fluorescence spectra for Fe₃O₄@SiO₂@Au@p-SiO₂ and Fe₃O₄@Au@p-SiO₂-DOX and DOX are presented in Figure 2A. Pure DOX shows a strong characteristic peak at around 600 nm. Meanwhile, Fe₃O₄@SiO₂@Au@p-SiO₂-DOX also shows a similar peak in a similar position, with a decreased intensity. This indicates that DOX is successfully introduced into Fe₃O₄@SiO₂@Au@p-SiO₂. As a reference and comparison, the fluorescence spectrum of Fe₃O₄@SiO₂@Au@p-SiO₂ is also presented in Figure 2D. It can be seen that there is no peak observed due to the absence of DOX.

The magnetic properties of Fe₃O₄, Fe₃O₄@SiO₂@Au, and Fe₃O₄@SiO₂@Au@p-SiO₂ are further characterized by VSM, and their magnetic hysteresis loops are presented in Figure 3A. It can be seen that pure Fe₃O₄ shows the highest magnetization of 47.3 emu/g, and Fe₃O₄@SiO₂@Au and Fe₃O₄@SiO₂@Au@p-SiO₂ have the magnetization of 38.5 and 37.2 emu/g, respectively. The decreased magnetization could be attributed to the introduction of nonmagnetic phases, while the magnetization is still high enough to direct drug transportation.

Figure 3.

A, Magnetic hysteresis loops of Fe3O4, Fe3O4@SiO2@Au, and Fe3O4@SiO2@Au@p-SiO2. B, X-ray diffraction (XRD) patterns for Fe3O4, Fe3O4@SiO2@Au, and Fe3O4@SiO2@Au@p-SiO2.

The crystal structure of Fe₃O₄, Fe₃O₄@SiO₂@Au, and Fe₃O₄@SiO₂@Au@p-SiO₂ is studied by XRD, and the diffraction patterns are presented in Figure 3B. For pure Fe₃O₄, its diffraction peaks match well with that displayed in the JCPDS card (NO. 75-0033), indicating that Fe₃O₄ is well crystallized. Compared to Fe₃O₄, an extra Au peak (111) is observed in Fe₃O₄@SiO₂@Au and Fe₃O₄@SiO₂@Au@p-SiO₂. This could be attributed to the introduction of Au nanoparticles. However, the peaks corresponding to SiO₂ nanoparticles are not detected. This is because SiO₂ synthesized by the Stober method is in the amorphous phase.

To investigate the application of NIR-induced drug release, the temperature under different power of laser and irradiation time is presented in Figure 4A-C. In each experiment, the Fe₃O₄@SiO₂@Au@p-SiO₂-DOX concentration is controlled as 25, 50, and 100 μg/mL. Under a specific laser power, it is observed that a higher concentration of Fe₃O₄@SiO₂@Au@p-SiO₂-DOX results in a higher temperature. This could be attributed to the enhanced concentration of Fe₃O₄@SiO₂@Au@p-SiO₂-DOX which absorbs greater NIR light and therefore generates more heat. This also indicates that Au nanoparticles within Fe₃O₄@SiO₂@Au@p-SiO₂-DOX could quickly convert the photon energy from the NIR irradiation into heat. Besides, when the NIR power is increased, the corresponding temperature is also increased. This is because more irradiation energy is supplied to Fe₃O₄@SiO₂@Au@p-SiO₂-DOX, and therefore, more heat is generated. This result demonstrates that Fe₃O₄@SiO₂@Au@p-SiO₂ platform is suitable for the delivery and controlled release of drugs under NIR irradiation.

Figure 4.

A-C, Temperature variation curves of Fe3O4@SiO2@Au@p-SiO2-DOX (25, 50, and 100 µg/mL) under 5, 7.5, and 10 W/cm² (the starting temperature was room temperature (23°C, pH = 7), the irradiation time was 36 minutes, and the temperature was measured every 3 minutes). D, Accumulative doxorubicin (DOX) released from various pH solutions and irradiation conditions (starting temperature of 37°C, Fe3O4@SiO2@Au@p-SiO2-DOX concentration of 25 µg/mL, and layer power of 10 W/cm²).

The impacts of pH on DOX release is also studied, and the results are presented in Figure 4D. First, the DOX release is studied under different pH without NIR irradiation, and the pH is controlled as 5.4, 6.4, and 7.4. It is observed that the DOX release is fast at the earlier stage and then slows down gradually. After releasing for 300 minutes, the accumulative DOX release reaches 18.3%, 31.2%, and 37.7% under the pH of 7.4, 6.4 and 5.4, respectively. This result indicates that a lower pH is beneficial for the fast delivery of DOX with the platform of Fe₃O₄@SiO₂@Au@p-SiO₂. This discovery could be used to guide the local pH regulation when releasing DOX into tumor tissue in the pathological process.

After that, NIR irradiation (10 W/cm²) is applied to Fe₃O₄@SiO₂@Au@p-SiO₂-DOX release under the pH of 5.4. After 300 minutes of irradiation, the accumulative DOX release reaches 65.3%, which is almost twice of the condition without NIR irradiation. This result concludes that NIR irradiation plays a critical role in releasing DOX from Fe₃O₄@SiO₂@Au@p-SiO₂ platform.

As a platform for drug delivery and release, its toxicity needs to be well evaluated, which is performed through standard MTT cell viability assay. As shown in Figure 5A, the cytotoxicity of Fe₃O₄@SiO₂@Au@p-SiO₂ is impacted by its concentration but is generally limited. The C6 cell viability could be maintained as high as ∼80% when Fe₃O₄@SiO₂@Au@p-SiO₂concentration is 250 µg/mL. It is concluded that Fe₃O₄@SiO₂@Au@p-SiO₂ does not have appreciable toxicity to C6 cells even after the incubation for 72 hours. The cytotoxicity of DOX and Fe₃O₄@SiO₂@Au@p-SiO₂-DOX with different concentrations in C6 cells is tested and presented in Figure 4B. Generally, Fe₃O₄@SiO₂@Au@p-SiO₂-DOX has lower toxicity than pure DOX. This is could be attributed to the loading ratio in Fe₃O₄@SiO₂@Au@p-SiO₂-DOX (the mass ratio of Fe₃O₄@SiO₂@Au@p-SiO₂), and the real DOX concentration in Fe₃O₄@SiO₂@Au@p-SiO₂-DOX experiment is much lower than that in pure DOX. In Fe₃O₄@SiO₂@Au@p-SiO₂-DOX experiment, even when its concentration reaches 100 µg/mL, the C6 cell viability could be maintained as high as ∼50%, indicating its relatively low toxicity to the cells.

Figure 5.

Results from in vitro cytotoxicity experiment. A, C6 cell viability after being incubated with different concentrations of Fe₃O₄@SiO₂@Au@p-SiO₂ for 72 hours. B, C6 cell viability after being incubated with different concentrations of doxorubicin (DOX) and Fe₃O₄@SiO₂@Au@p-SiO₂-DOX for 72 hours.

Conclusions

Fe₃O₄@SiO₂@Au@p-SiO₂ nanoparticles were synthesized, and DOX was attached to the particle surface. The BET method study indicated that the particles have a porous structure, which was beneficial to load DOX. The drug delivery and release were studied under NIR irradiation. The impacts of laser power and environmental pH on drug delivery and release were investigated. The study indicated that a lower pH and higher power of laser were beneficial for DOX drug release. Finally, the cytotoxicity of both Fe₃O₄@SiO₂@Au@p-SiO₂ and Fe₃O₄@SiO₂@Au@p-SiO₂-DOX was studied through in vitro MTT assay with C6 glioma cells. The results indicated that both Fe₃O₄@SiO₂@Au@p-SiO₂ and Fe₃O₄@SiO₂@Au@p-SiO₂-DOX have limited cytotoxicity to the cells.