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Back to Journal »International Journal of Nanomedicine» Volume 16
Au catalyzes the release of NO in the body and inhibits tumor growth in the combined treatment of chemo-photothermal
Authors: Zhang Ying, Zhou Tao, Li Jie, Xu Nan, Cai Mei, Zhang Hua, Zhao Q, Wang Shi
Published on March 29, 2021, the 2021 volume: 16 pages 2501-2513
DOI https://doi.org/10.2147/IJN.S270466
Single anonymous peer review
Editor who approved for publication: Dr. Linlin Sun
Zhang Ying, 1 Zhou Tianfu, 1 Li Jian, 1 Xu Nuo, 1 Cai Mingze, 1 Zhang Hong, 2 Zhao Qinfu, 3 Wang Siling 3 1 Key Laboratory of Target Drug Design and Research, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, China People's Republic; 2Van'T Hoff Institute of Molecular Science, University of Amsterdam, Amsterdam, 1098 XH, Netherlands; 3Department of Pharmacy, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 110016 Corresponding author: Zhao Qinfu; Wang Siling Tel/Fax +86 24 43520555 Email [email protected]; [email protected] Introduction: In order to obtain a NO donor that can control the release of NO in the body and has efficient tumor suppression and targeting effects, a structure composed of FA-Fe3O4@mSiO2-Au/DOX was constructed Nano platform. Methods: In vitro, the NO release catalyzed by the nanoplatform at 43°C and pH=5.0 reached a maximum of 4.91 μM within 60 minutes, which increased by 1.14 times at 37°C. In the body, it was found that 11.7 μg Au in the tumor tissue continued to catalyze S-nitrosoglutathione, and 54 μM NO was detected in the urine. Results and discussion: It was found that high concentrations of NO can increase the rate of apoptosis and reduce tumor proliferation. In the combination of chemo-photothermal therapy, the tumor suppression rate increased to 94.3%, and the contribution of Au to catalyze the release of NO was 8.17%. Keywords: hyperthermia, chemotherapy, controlled release of NO, magnetic mesoporous silica nanocomposites
The multifunctional treatment system with targeted drug delivery, imaging and combined treatment modes can greatly improve the treatment effect and minimize damage to normal tissues, and has been extensively studied. 1-3 The integrated gold nanocomposite, 4 carbon nanotubes, 5 and magnetic mesoporous microspheres 6 of the thermochemotherapy system is an effective in vitro and in vivo treatment system. Although some achievements have been made, a comprehensive treatment system usually loaded with exogenous drugs (doxorubicin, paclitaxel, and macromolecular proteins) often brings adverse side effects, cannot be effectively delivered to the tumor site, and tumor cells are resistant to 7- 9 Facing difficult problems. Nitric oxide (NO), as a messenger molecule in the body, plays an important role in a variety of physiological activities such as cardiovascular, respiration, nerves, and immunity. 10 NO exists in a low concentration to perform physiological functions; however, high concentration of nitric oxide forms highly active substances (N2O3, ONOO−, etc.), causing nitrification and oxidative stress, and excessive consumption of intracellular glutathione (GSH), which hinders Mitochondria function normally, damage DNA and induce apoptosis. 11-13 Considering the distinct effects of NO caused by concentration, it is a good strategy to design a controlled release NO donor integrated into a multifunctional treatment system.
Several NO donors have been reported as drugs for studying anti-tumor efficacy. 14-17 Unfortunately, these donors lack a cancer cell targeting design that releases NO in a controlled manner, resulting in unsatisfactory tumor suppression and limiting their practical application in therapy. Recently, some specific catalysts (such as Cu2+, Fe3+, Au, etc.) have been shown to produce NO when in contact with fresh serum. For example, S-nitrososerum albumin and S-nitrosoglutathione (GSNO), which are widely present in the blood, can destroy the S-NO bond and produce NO under the action of a catalyst. 18,19 Among these catalysts, Au nanoparticles have near-infrared light absorption properties, which can convert the absorbed light energy into heat energy, and at the same time activate the heat-related pathways in tumor cells and induce the expression of apoptotic proteins. 20-23 Our research interest. In addition, it is reported that magnetic gold nanocomposites can accurately accumulate in tumors after a long period of blood circulation, and significantly improve anti-tumor drugs (doxorubicin, paclitaxel and macromolecular proteins) in the combination therapy of thermochemotherapy Efficacy. 24-26 Designing a multifunctional treatment system composed of Au is an ideal solution for integrating a controlled NO release donor into a thermochemical treatment system.
In order to prove that Au can release NO through thermal control as a NO donor catalyst for chemotherapy in vivo and achieve satisfactory tumor suppression, a nanoplatform FA-MMSN-Au/ DOX. First, aminated magnetic mesoporous silica nanoparticles (MMSN-NH2) are prepared by thermal decomposition and sol-gel methods, and then assembled with gold nanoparticles (MMSN-Au) by electrostatic adsorption, and finally DOX is loaded and combined with FA molecules Coupling (as shown in Figure 1A). Secondly, in vitro, the relationship between the NO release catalyzed by MMSN-Au particles, temperature and GSNO concentration was measured. Third, under the irradiation of magnetic target and near-infrared light in the body, FA-MMSN-Au/DOX carrier accumulates at the tumor site and generates heat to increase tissue temperature; the amount of NO released in urine is checked. A comparative combination treatment plan was designed to study the effect of Au-catalyzed NO on tumor suppression. Under the same hyperthermia conditions, the effects of FA-MMSN-Au/DOX and FA-MMSN/DOX carriers on tumor growth at different stages were compared, and the contribution of Au as a NO donor catalyst was clarified. This provides a useful supplement to Au's hyperthermia mechanism, and the results have never been reported before. Our work also provides a new strategy for the integration of nitric oxide chemical biology and multimodal combination therapy (such as gold-hot chemotherapy). Figure 1 (A) Multifunctional platform scheme; (B) TEM image of MNP, (C) MMSN, (D) HRTEM image of Au nanoparticles, (E) MMSN-Au nanocomposite; MMSN-Au and different doses EDS (G) 20 μL, (H) 40 μL, (I) 80 μL; (F) MNP, MMSN and MMSN-Au size distribution. The diameter of MNP comes from the reference, 32 (J) zeta potential change.
Figure 1 (A) Multifunctional platform scheme; (B) TEM image of MNP, (C) MMSN, (D) HRTEM image of Au nanoparticles, (E) MMSN-Au nanocomposite material; MMSN-Au and different doses EDS (G) 20 μL, (H) 40 μL, (I) 80 μL; (F) MNP, MMSN and MMSN-Au size distribution. The diameter of MNP comes from the reference, 32 (J) zeta potential change.
FeCl3·6H2O, sodium oleate, and oleic acid were purchased from Tianjin Yongda Chemical Reagent Co., Ltd.; oleylamine (80-90%) was purchased from Shanghai McLean Biochemical Technology Co., Ltd. Cetyltrimethylammonium bromide (CTAB) and ammonium nitrate were purchased from Beijing Chemical Reagent Co., Ltd.; n-hexane, absolute ethanol and sodium hydroxide (95%) were purchased from Tianjin Damao Chemical Reagent Factory; octadecene , N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (EDS) and tetraethoxysilane (TEOS) were purchased from Beijing Balingwei Reagent Co., Ltd.; polyoxyethylene-polypropylene-polyoxy Ethylene copolymer (F127) was purchased from BASF, Germany; chloroauric acid (HAuCl4·3H2O, 99.99%) was purchased from Alpha Aisha (China) Chemical Co., Ltd.; folic acid (FA, ≥97%), doxorubicin hydrochloride (DOX) , ≥98%) and S-nitrosoglutathione (GSNO) were purchased from Aladdin Biochemical Technology Co., Ltd.; Grice reagent Shanghai Biyuntian Biotechnology Co., Ltd. 1-hydroxybenzotriazole (HOBT) and o-benzene Hexatriazole-hexafluorophosphate tetramethylurea (HBTU) was purchased from Gil Biochemical (Shanghai) Co., Ltd. All chemicals are analytically pure and no further purification is required.
Fe3O4 magnetite nanoparticles (MNP) were synthesized using the thermal decomposition method established by Park et al. 27 Fe3O4@mSiO2-NH2 Magnetic Mesoporous Silica Nanocomposite (MMSN-NH2) was prepared according to the method reported by this research group. 28 Au nanoparticles were prepared according to literature. 29 Add the above MMSN-NH2 absolute ethanol solution (1 mL, 20 mg/mL) to the Au solution (10 mL, 2.4 mg/mL) and keep stirring for 1 hour. After centrifugation (4500 rpm/min, 10 min), the magnetic gold nanocomposite material (MMSN-Au) was collected and washed twice with ethanol. Dissolve FA (10 mg) in dimethyl sulfoxide (DMSO, 5 mL). Then, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 200 mg) and n-hydroxysuccinimide (NHS, 160 mg) were added and excited for 3 hours. MMSN-Au (100 mg) nanoparticles dispersed in DMSO (20 mL) were added to the above solution and reacted at room temperature for 24 hours. The final carrier of FA-MMSN-Au was obtained by centrifugation.
Prepare the test solution by dispersing an accurate amount of FA-MMSN-Au composite in a PBS (pH=7.4) solution. The prepared solutions of different concentrations (1 mL) were introduced into the cuvette and exposed to the NIR laser (808 nm, 0.27 W/cm2). For the other group, the FA-MMSN-Au solution (1 mL) of serial concentration was placed in a high-frequency induction heating instrument with different alternating currents. Use a thermal imaging camera to record the temperature of the solution.
First, add the composite of DOX (4 mg) and MMSN-Au (20 mg) to the PBS (2.0 mL, pH=7.4) solution, then mix and vortex to disperse for 24 hours. Second, wash the composite material with pH 7.4 PBS solution to remove DOX adsorbed on the surface of the carrier until it is colorless. The composite material was then collected and recorded as FA-MMSN-Au/DOX; the supernatant was also collected and measured at a wavelength of 480 nm. According to the literature method, the 30 load capacity is calculated as follows:
The drug release behavior study is as follows: Disperse 4 mg MMSN-Au/DOX in two different dispersion media (simulating tumor tissue and normal tissue environment respectively) in 10 mL pH 5.0 and 7.4 PBS, and place it in a 37°C air bath The shaker is shaken at 120 rpm. At specific times (0.5, 1, 2, 3, 4, and 6 hours), measure the absorbance of the release medium at 480 nm. The drug release behavior of MMSN-Au/DOX is measured in the same procedure.
The GSNO PBS solution is used to simulate plasma. The gold nanoparticles in the MMSN-Au composite material can catalyze the release of NO from GSNO, which is converted into nitrite or nitrate in the PBS solution. Griess kit is used to detect the performance of MMSN-Au carrier to catalyze the release of NO.31 from S-nitrosoglutathione (GSNO). According to the instructions of Griess kit, determine the standard curve of NO. Next, mix 2 mL PBS solution containing 300 μL GSNO (1.2 mM) and 1 mL MMSN-Au nanocomposite aqueous solution (12 mg/mL) in a sealed dialysis bag and immerse it in 35 mL at 37°C PBS solution. After dialysis for different time, take out 1 mL of PBS solution outside the dialysis bag for NO test, keeping the volume of PBS solution unchanged. The experiments under the conditions of 2.4 mM and 4.8 mM GSNO, 25°C and 43°C were also measured by the above procedure.
MCF-7 cells were provided by Shanghai Cell Research Institute (Shanghai, China). MCF-7 cells are maintained in RPMI 1640 and supplemented with FBS (10%), penicillin (1%) and streptomycin (1%) in 5% CO2 at 37°C. The medium was changed regularly every 2 days, and the cells were separated by trypsinization before confluence. For microscopic analysis, MCF-7 cells were seeded in a 24-well plate in a round glass cover slip at a rate of 5 × 104 cells/well. Then, add DOX solution and FA-MMSN-Au/DOX (equivalent to 5 µg/mL DOX) into the corresponding wells. After 2 hours, the cells were washed with cold PBS, fixed with formaldehyde, and stained with DAPI. Observe the fixed cells with a confocal laser scanning microscope (CLSM). The cellular uptake was quantitatively evaluated on the flow cytometry technique (FCM). MCF-7 cells were seeded in 6-well plates and incubated with DOX solution and FA-MMSN-Au/DOX (equivalent to 5 µg/mL DOX). Subsequently, the cells were washed, trypsinized and resuspended in 400 μL PBS. The mean fluorescence intensity (MFI) was measured using FCM (FACSCalibur, Becton Dickinson, USA). In the competition experiment, MCF-7 cells were incubated with 2 mg/mL FA for 2 hours before adding nanoparticles.
Use the following information to conduct experiments involving animals. The ethics approval number is SYPU-IACUC-C2017, and the name of the authorized institution is the Animal Ethics Committee of Shenyang Pharmaceutical University. The animals used in the research follow the NC3Rs ARRIVE guidelines.
Mouse sarcoma S180 cells were provided by Shanghai Cell Research Institute (Shanghai, China) and were cultured in RPMI 1640 medium containing 10% FBS and 5% CO2 at 37°C. Kunming mice were inoculated subcutaneously with S180 cells (1×107 cells) into the right flanks. S180 tumor-bearing mice were randomly divided into several groups, with six mice in each group. Blank control group: No treatment was given. DOX control group: 0.2 mg/mL DOX 0.2 mL was injected into the tail vein. FA-MMSN-Au/DOX magnetic targeting chemotherapy experimental group (T): The mice were intraperitoneally injected with 0.2 mL of 3.5% chloral hydrate solution and fixed on the hyperthermia plate. After activating the 0.2 mL 12 mg/mL FA-MMSN-Au/DOX carrier through the tail vein, a 0.3T permanent magnet is used for the 30-minute target. FA-MMSN-Au/DOX magnetic targeted magnetic therapy experimental group (T+MT): adopt the same treatment method as FA-MMSN-Au/DOX(T) experimental group. Then put the mouse into the magnetic induction coil; the tumor site is located in the center of the magnetic induction coil, and keep the temperature at 43°C for 30 minutes at 25A. FA-MMSN/DOX Magnetic Targeting Chemical Magnetocaloric Experimental Group (T+MT): The treatment is the same as FA-MMSN-Au/DOX (T+MT). FA-MMSN-Au/DOX Magnetic Targeting Chemical Photothermal Experimental Group (T+PT): After anesthesia, tail vein injection, and magnetic targeting, the mice are placed under a 808nm near-infrared laser transmitter with a power density of 0.27 W /cm2, the irradiation of the tumor site is heated to 43°C for 30 minutes. The thermal imager records the temperature.
HE staining microscopy of mouse heart, lung, liver, spleen and kidney specimens 24 h after FA-MMSN-Au/DOX (12 mg/mL, 0.2 mL) injection was used as the control group to observe histopathology.
Use Griess kit for measurement. Blank control group: No treatment; 0.5 mL urine was taken every day for measurement. FA-MMSN-Au/DOX Magnetic Targeting Chemical Photothermal Experimental Group (T+PT): Measure 0.5 mL of urine within 4-24 hours after each treatment.
The comprehensive treatment system is synthesized according to the scheme in Figure 1A, and its characterization is described in the supplementary material. It can be seen that MNP magnetic nanoparticles are uniform solid spheres with a particle size of about 14 nm and good dispersion (Figure 1B). The MMSN carrier has a spherical shape and a uniform size of 50 nm. The thickness of the mesoporous silicon shell is about 15-20 nm. Magnetic MNP nanoparticles are embedded in the center of the silica shell. In order to obtain better magnetic and thermal properties, we increased the mass ratio of MNP and TEOS when synthesizing magnetic mesoporous silica nanocomposites, which resulted in the multi-core of MNP embedded in the center of the silica shell (Figure 1C). The MMSN-Au nanocomposite showed a certain degree of aggregation, and a small amount of gold nanoparticles were adsorbed on the surface of MMSN (Figure 1E). Gold nanoparticles with a particle size of approximately 8 nm (Figure 1D) can be adsorbed on the surface of MMSN in a controlled manner. The silane coupling agent N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (EDS) was used as an adjustment reagent to modify the Zeta potential of MMSN (Figure S1). As shown in Figure 1G and H, when the amount of EDS is 20 μL and 40 μL, the number of gold nanoparticles adsorbed by MMSN is similar; when the amount of EDS is 80 μL, the dispersibility of the MMSN-Au carrier decreases, and the Au absorbed by MMSN The amount increased significantly (Figure 1I). The size distribution of MNP, MMSN and MMSN-Au was evaluated by DSL (Figure 1F). The change of particle size during the preparation process is consistent with the composite structure observed by TEM. Zeta potential also proved the synthesis process (Figure 1J). The MSN with hydroxyl is -16.2 mV, and the amino-modified MMSN is 30.0 mV, which does not change much with the amount of EDS; after coupling with citric acid-stabilized Au, the potential of MMSN decreases to -7.6 mV. MMSN-Au load FA and amino group correspondingly causes the potential to increase to -2.877 mV.
Figure 2A shows the XRD patterns of MNP, MMSN and MMSN-Au. The MNP diffraction pattern is consistent with the standard Fe3O4 PDF card (19-0629). The characteristic diffraction peaks of Fe3O4 nanoparticles appeared at 2θ=35.40, 42.92, 56.97 and 62.22°, which proved that Fe3O4 nanoparticles were formed. For the MMSN diffraction pattern, the characteristic peak of Fe3O4 is still maintained, which indicates that the crystal structure of Fe3O4 nanoparticles has not changed during the SiO2 coating process. In the MMSN-Au diffraction pattern, the diffraction peaks of Au nanoparticles appear at 2θ=38.17, 44.24, 64.90, 77.70, and 81.82°, indicating that Au (17-0629) was successfully prepared and the crystal structure of Fe3O4 nanoparticles has the ability to absorb gold. There is no change in the process. The binding energy and atomic percentage of MMSN-Au were analyzed by XPS (Figure 2B and Table 1). The two peaks of 709.2 and 722.3 eV in the carrier coincide with the two standard peaks of Fe2p1/2 and Fe2p3/2 in different states. The binding energy peak at 397.7 eV indicates the binding energy spectrum of N. The peaks at 81.4 and 85.1 eV are characteristic peaks of Au. After the grafting process, the atomic ratio of Si:C decreased slightly, indicating that Au was stabilized by the combination of citric acid and MMSN. In short, a magnetic gold nanocomposite material was successfully constructed. The UV absorption spectrum is shown in Figure 2C. The absorption peak at 480 nm is characteristic of Au nanoparticles. After coupling with MMSN, the absorbance intensity of Au decreased but remained unchanged, indicating that MMSN was assembled with Au. Table 1 Elemental analysis of MMSN and MMSN-Au (atomic percentage) Figure 2 (A) XRD patterns of MNP, MMSN and MMSN-Au; (B) XPS spectrum of MMSN-Au; (C) Au, MNP, MMSN and MMSN -Au's UV-Vis spectrum; (D) N2 adsorption-desorption isotherm; (E) MMSN pore size distribution curve; (F) MNP, MMSN and MMSN-Au nanocomposite hysteresis loop, T=298 K.
Table 1 Elemental analysis of MMSN and MMSN-Au (atomic percentage)
Figure 2 (A) XRD patterns of MNP, MMSN and MMSN-Au; (B) XPS spectrum of MMSN-Au; (C) UV-visible spectra of Au, MNP, MMSN and MMSN-Au; (D) N2 adsorption-desorption Isotherm; (E) the pore size distribution curve of MMSN; (F) the hysteresis loop of MNP, MMSN and MMSN-Au nanocomposites, T=298 K.
The adsorption-desorption isotherm and pore size distribution of MMSN are characterized by N 2 adsorption-desorption analysis, as shown in Figures 2D and E. The N 2 adsorption-desorption isotherm shows a type IV isotherm with a hysteresis loop. The shape and characteristics of the isotherm confirm the typical mesoporous structure inside the carrier. The pore size distribution curve of MMSN shows a sharp peak distribution, indicating that the pore structure of the MMSN carrier is uniform and regular. SBET, pore size distribution (Dp) and total pore volume (Vt) are shown in Table 2. The single-point BET specific surface area of the carrier is 734.956 m2/g, the cumulative pore volume of BJH adsorption is 1.41 cm3/g, and the calculated pore size of the BJH desorption data is 3.2 nm. Figure 2F shows the magnetic properties of MNP, MMSN and MMSN-Au nanocomposites. All MNP, MMSN and MMSN-Au nanocomposites exhibit paramagnetic properties at T=298 K. After coating with SiO2 and Au nanoparticles, the saturation magnetic intensity of MNP nanoparticles decreased from 49.1 to 30.8, and further decreased to 22.3 emu/g. Table 2 Particle size distribution and mesoporous structure parameters of MMSN
Table 2 Particle size distribution and mesoporous structure parameters of MMSN
Under the conditions of simulating tumor acidic environment (pH 5.0 PBS) and plasma (pH 7.4 PBS) in vitro, it can be seen from Figure 3A-E that the NO release reaction of GSNO catalyzed by Au is positively correlated with GSNO concentration and temperature. Under the acidic condition of tumor tissue, as the concentration of GSNO increased from 300 μL to 1200 μL, the amount of NO released in about 60 minutes increased from 4.36 μM to 5.85 μM (Figure 3D); as the temperature increased from 37°C to 43° C. The release rate and amount of NO gradually increased, reaching an equilibrium state of 4.91 μM in about 60 minutes (Figure 3E). For 30 minutes of hyperthermia at 43°C, the total amount of NO produced by the catalytic release of Au was 1.14 times the amount released at normal body temperature. Compared with normal tissue conditions, the release of NO in tumors increased by 28.6%, as shown in Figure 3C. Therefore, the performance of the carrier to catalyze the release of NO from GSNO has a dual response to weak acid conditions and hyperthermia temperature. It can be inferred that in the tumor tissues treated with photothermal treatment, the release of NO within 60 minutes is significantly greater than that of normal tissues without thermal treatment. Figure 3 MMSN-Au (1.2 mg/mL, 1 mL) (A) NO release behavior at different GSNO concentrations in PBS solution at 37°C and pH=7.4; (B) 300 μL GSNO and pH at different temperatures =7.4 PBS solution; (C) DOX release behavior at 37°C, pH=7.4 and 5.0 PBS solution; (D) 37°C and pH=5.0 PBS solution; (E) 300 μL GSNO and pH= 5.0 PBS solution; (F) DOX release behavior.
Figure 3 MMSN-Au (1.2 mg/mL, 1 mL) (A) NO release behavior at different GSNO concentrations in PBS solution at 37°C and pH=7.4; (B) 300 μL GSNO and pH at different temperatures =7.4 PBS solution; (C) DOX release behavior at 37°C, pH=7.4 and 5.0 PBS solution; (D) 37°C and pH=5.0 PBS solution; (E) 300 μL GSNO and pH= 5.0 PBS solution; (F) DOX release behavior.
Calculate the loaded and released DOX from the standard curve of DOX: A=0.0204C-0.0276. The drug encapsulation rate of MMSN-Au for 24 h was 21.0%. The -COOH group of DOX and the Si-OH group on the surface of the mesoporous pore wall may interact in the form of hydrogen bonds to promote the loading of DOX in the mesoporous channel. The in vitro DOX release behavior of MMSN-Au/DOX is shown in Figure 3F. The cumulative release of DOX of MMSN-Au/DOX in pH 5.0 and 7.4 PBS solutions was 27.8% and 19.9%. The DOX release of FA-MMSN-Au/DOX in pH 7.4 PBS solution is 6.5%, which is significantly lower than MMSN-Au/DOX, because the modification of FA molecules has steric hindrance, which slows down the release of DOX.
It can be seen from Figure 4A that the medium with higher MMSN-Au content produces a more significant temperature increase. For example, a 12 mg/mL sample is heated to 29.1°C in 20 minutes at a current of 25 A (Figure 4A). When the current was increased from 20 A to 30 A, the temperature of the sample solution (12 mg/mL) changed from 17.7°C to 43.1°C (Figure 4A). According to the mouse's tolerance current value and MMSN-Au hemolysis rate, a current of 25A and a concentration of 12mg/mL were selected for subsequent magnetic heat treatment. It can be seen from Figure 5B that the medium containing a higher amount of MMSN-Au produces a more significant temperature rise. The 12 mg/mL sample was heated to 33.6°C in 20 minutes at a density of 0.27 W/cm2. When the optical density increased from 0.15 to 0.36 W/cm2, the temperature of the sample solution (12 mg/mL) changed from 28.0°C to 42.7°C (Figure 4B). In general, the MMSN-Au nanocomposite exhibits obvious photothermal performance and magnetocaloric behavior. Figure 4 (A) Magnetocaloric heating curve of MMSN-Au nanocomposite, (B) Photothermal heating curve of MMSN-Au nanocomposite under 808 nm laser radiation, (C) MNP, MMSN and MMSN-Au are at the same The heating curve at a concentration of 12 mg/mL, I=25 A, d=0.27 W/cm2, V=1 mL. Figure 5 (A) CLSM image and (B) FCM analysis of MCF-7 cells incubated with free DOX and FA-MMSN-Au/DOX and pre-incubated with FA for 2 hours before adding FA-MMSN-Au/DOX. Note: **P<0.05.
Figure 4 (A) Magnetocaloric heating curve of MMSN-Au nanocomposite, (B) Photothermal heating curve of MMSN-Au nanocomposite under 808 nm laser radiation, (C) MNP, MMSN and MMSN-Au are at the same The heating curve at a concentration of 12 mg/mL, I=25 A, d=0.27 W/cm2, V=1 mL.
Figure 5 (A) CLSM image and (B) FCM analysis of MCF-7 cells incubated with free DOX and FA-MMSN-Au/DOX and pre-incubated with FA for 2 hours before adding FA-MMSN-Au/DOX.
Note: **P<0.05.
In order to understand the difference between the magnetic and photothermal behavior and its influencing factors, the changes in the magnetic/photothermal properties of MNP and MMSN were also measured, and further compared with the behavior of the MMSN-Au carrier. As shown in Figure 4C, MNP itself has excellent light and heat absorption characteristics and can be quickly heated to 34°C in 8 minutes. After coating the SiO2 shell that does not absorb light, the heating rate of the carrier MMSN decreases (Figure 4C). After Au and MMSN are coupled, the photothermal effect is enhanced to a certain extent, which may be related to the surface electron oscillation after Au absorbs near-infrared light. Because Fe3O4 microspheres have stronger absorption than Au in the near-infrared region, and MNP has better photothermal conversion under near-infrared light, the heating rate of 32,33 MNP under PT is higher than that of MMSN-Au. However, the situation with magnetocaloric properties is different, because gold nanoparticles cannot generate heat in an alternating electric field. The coupling of Au leads to a decrease in the magneto-caloric performance of the carrier. Therefore, for the composite carrier MMSN-Au, the photothermal performance is better than the magnetocaloric performance under the experimental conditions. Calculated according to Figure S2, the light-to-heat conversion efficiency of MMSN-Au is 24.04%.
MCF-7 cells overexpressing FA receptors were used to evaluate the cellular uptake of FA-MMSN-Au/DOX nanoparticles. 34,35 As shown in Figure 5A, due to the high affinity between free Dox and DNA, it is mainly located in the nucleus. The red fluorescence of DOX appears in the cytoplasm of the FA-MMSN-Au/DOX group, and the fluorescence intensity is significant after 2h pre-incubation with FA Decrease, indicating that the target-specific endocytosis of FA-MMSN-Au/DOX is mediated through FA receptors. In addition, FCM is used to quantitatively measure the cellular uptake of FA-MMSN-Au/DOX (Figure 5B). Compared with the untreated FA-MMSN-Au/DOX group, when MCF-7 cells were pretreated with 2 mg/mL FA for 2 hours, MFI was significantly reduced, with a p value of 0.003. These results indicate that FA modified on the surface of MMSN-Au can increase the cellular uptake of MCF-7 cells overexpressing FA receptors.
Before hyperthermia, the histocompatibility of FA-MMSN-Au/DOX carrier was studied by HE staining. As shown in Figure 6, the liver structure of the FA-MMSN-Au/DOX group (Figure 6B) is slightly different from that of the saline control group (Figure 6B1), and FA-MMSN-Au/DOX is in the liver cells. The liver cells are arranged in polygonal stripes around the central vein, and there is no degeneration and necrosis of the liver cells. By comparing Figure 6A and A1, spleen (Figure 6C and C1), lung (Figure 6D and D1), and kidney (Figure 6E and E1), FA-MMSN-Au/DOX showed no obvious cardiac response. Therefore, the carrier is histocompatibility. The constructed FA-MMSN-Au/DOX vector is safe for blood; the adsorption capacity of FA-MMSN-Au/DOX to BSA is 12.25%, which can avoid protein interference to a certain extent; FA-MMSN-Au/DOX The systemic acute toxicity is negative (Figure S3, Table S1 and Table S2). Figure 6 The results of HE staining of cells in the experimental group (AE) and control group (A1-E1) after injection of 0.2 mL 12 mg/mL FA-MMSN-Au/DOX into the tail vein for 24 h. (A heart; (B) liver; (C) spleen; (D) lungs; (E) kidneys of the experimental group and the control group.
Figure 6 The results of HE staining of cells in the experimental group (AE) and control group (A1-E1) after injection of 0.2 mL 12 mg/mL FA-MMSN-Au/DOX into the tail vein for 24 h. (A heart; (B) liver; (C) spleen; (D) lungs; (E) kidneys of the experimental group and the control group.
Various therapies were used on tumor-bearing mice, as shown in Figure 7A. The tumor volume of experimental mice was recorded to evaluate the effect of NO in situ chemotherapy and the effect of combined magneto-optical/photothermal treatment. The inhibition rate was calculated based on the relative change of tumor volume. The change of tumor volume over time in each experimental group is shown in Figure 7B. Compared with the tumor growth in the saline group, the DOX group, FA-MMSN-Au/DOX(T) group, FA-MMSN/DOX(T+MT) group, FA-MMSN-Au/DOX (T+MT) and FA-MMSN-Au/DOX (T+PT) group. As shown in Figure 7C, the tumor inhibition rates of 43.4% in the DOX chemotherapy group, FA-MMSN-Au/DOX(T) group, FA-MMSN-Au/DOX(T+MT) group, and FA-MMSN/DOX group are similar Compared with (T+MT) and FA-MMSN-Au/DOX (T+PT) groups, they showed higher tumor suppression rates, which were 69.0%, 79.7%, 71.6%, and 94.3%, respectively. The tumor inhibition rate of FA-MMSN-Au/DOX (T+MT) is 10.7% higher than that of FA-MMSN-Au/DOX (T), which can be attributed to the contribution of magnetic hyperthermia to tumor suppression. The tumor inhibition rate of FA-MMSN-Au/DOX (T+PT) is 25.3% higher than that of FA-MMSN-Au/DOX (T) and 14.6% higher than that of the magnetocaloric group. This may be due to the characteristics of magnetocaloric and photothermal difference. From the comparison of FA-MMSN-Au/DOX (T+MT) and FA-MMSN/DOX (T+MT) groups, it can be seen that the presence of Au in the combined process of DOX chemotherapy and hyperthermia can promote tumors. Figure 7 (A ) Multiple treatment options, (B) tumor growth curve and (C) tumor suppression curve of different treatments. Note: *P<0.05, **P<0.05, ***P<0.05.
Figure 7 (A) multiple treatment options, (B) tumor growth curve of different treatments and (C) tumor suppression curve.
Note: *P<0.05, **P<0.05, ***P<0.05.
In order to verify that Au can be used as a NO donor catalyst for chemotherapy in vivo by releasing NO in a thermally controlled manner, the distribution of FA-MMSN-Au/DOX carrier and its catalytic ability in the body were studied. As shown in Figure 8A, 30 minutes after the carrier is magnetically targeted to the tumor site, and then hyperthermia is performed, the temperature of the target site rises rapidly. It reaches above 43°C within 300 seconds, and the non-target part changes little, indicating that the distribution of FA-MMSN-Au/DOX vector is mainly at the target tumor site (Figure 8A). During multiple treatments, the Au content in tumor tissue is shown in Figure 8B. The third target and the Au content in tumor tissues increased to 11.7 μg after photothermal treatment. The release of NO catalyzed by the carrier in the body increases with the increase of the number of treatments, which is related to the accumulation of Au in the tumor tissue. After three hyperthermia treatments, 54 μM NO in urine was detected within 96 hours (Figure 8C). In the absence of hyperthermia to trigger the release of NO, the normal metabolic nitrogen content of mice in the blank group was about two-thirds of that of the hyperthermia group. It can be seen that the FA-MMSN-Au carrier exerts NO chemotherapeutic effect by releasing NO in a thermally controlled manner, and exerts a hyperthermic effect in the body by accumulating at the targeted tumor site. Figure 8 (A) Photothermal therapy image; (B) ICP detection of gold ion concentration in tumor tissue; (C) target at different times and the amount of NO in urine within 24 hours after photothermal therapy (T+PT) .
Figure 8 (A) Photothermal therapy image; (B) ICP detection of gold ion concentration in tumor tissue; (C) target at different times and the amount of NO in urine within 24 hours after photothermal therapy (T+PT) .
During 6-14 days of treatment, only the FA-MMSN-Au/DOX (T+PT) and (T+MT) combined chemotherapy and Au group showed a reduction in tumor volume, which is different from other groups. This reduction is related to tumor proliferation. Without inhibiting the ability of tumor proliferation, even short-term combination therapy can cause tumor cell apoptosis within 48 hours after treatment, but as the tumor volume increases, tumor cells will expand and even increase drug resistance in the later stage. Therefore, the presence of Au has an inhibitory effect on tumor proliferation in the combination of hyperthermia and DOX chemotherapy. As mentioned above, Au exerts the effects of NO chemotherapy and hyperthermia in the body through accumulation and catalysis at targeted tumor sites; to be precise, NO enhances the inhibition of the combined proliferation process of DOX chemotherapy and hyperthermia. Without the effect of NO, the tumor volume in the FA-MMSN/DOX (T+MT) group showed a significant increase within 6-14 days. From the report that hyperthermia combined with NO chemotherapy can induce cell apoptosis and inhibit tumor proliferation, we can infer that Au catalyzes the release of NO to help reduce drug resistance and tumor proliferation, resulting in an 8.17% increase in tumor suppression rate.
On day 6, the FA-MMSN-Au/DOX(T+MT) tumor growth curve showed a peak tumor growth inhibition rate lower than that of the FA-MMSN-Au/DOX(T) group, which can be attributed to hyperthermia. Since the heating process of the T+MT group is stable, protein heat tolerance may occur, and the heat-induced tumor damage is less. Therefore, the effect of thermal controlled release of NO on tumor growth can be analyzed by comparing with FA-MMSN-Au/DOX (T) group and (T+MT) group. Since gold-catalyzed NO release in vitro is temperature-dependent, the NO release at 43°C hyperthermia temperature is 1/3 higher than that at 37°C. Therefore, it can be inferred that the amount of NO catalytically released by Au in the T+MT group is greater than that in the T group. The apoptotic rate was higher in the group with higher NO concentration, which is consistent with previous studies. 36 From the comparison of the tumor growth curves of the two groups, it can be seen that the thermally controlled release of NO leads to a higher rate of apoptosis, leading to tumor occurrence. The inhibition rate increased by 10.7%.
On the 14th day, the FA-MMSN-Au/DOX(T+PT) tumor growth curve showed a peak tumor growth inhibition rate lower than that of the FA-MMSN-Au/DOX(T+MT) group, which could be attributed to magneto/photothermal Differences in characteristics. For the MMSN-Au carrier, under the experimental conditions, the heat production rate of the photothermal performance is higher than that of the magnetocaloric performance (Figure 5C). In the body, it takes only 5 minutes for photothermal therapy to reach 43°C for tumor tissue, and 20 minutes for magnetothermal therapy to heat the tumor. In photothermal therapy, the temperature of tumor tissue rises rapidly, which may cause greater damage to tumor cells. However, the warming and slowness of tumor tissues under magnetic heating conditions causes tumor cells to have heat resistance. It is reported that the formation of heat tolerance accompanying BCL-2 gene overexpression reduces heat-induced apoptosis. 38 We can infer that FA-MMSN-Au/DOX (T+PT) therapy with higher heating rate characteristics can increase tumor cell apoptosis compared with FA-MMSN-Au/DOX (T+MT) group with heat resistance. It should also be noted that the tumor growth in the FA-MMSN/DOX (T+MT) group is stable during the 6-14 day period. However, the tumor volume in the FA-MMSN/DOX (T+PT) group continued to decrease. This difference may be related to prolonged tumor survival. As mentioned above, heat resistance may stimulate tumor growth, mainly by prolonging the survival time of cells. 39 We can infer that rapid heat treatment is more helpful to increase tumor suppression rate by shortening the survival time. Combined with rapid heating therapy, the tumor inhibition rate of chemotherapy can be increased by 25.3%.
We constructed the FA-MMSN-Au/DOX platform as a NO donor, combined with targeted chemotherapy and hyperthermia, which can thermally control the release of NO in the body, with a tumor suppression rate of 94.3%. This multifunctional platform catalyzes the release of NO from GSNO in vitro, and causes high NO release concentration in the body during hyperthermia, thereby increasing the rate of cell apoptosis and reducing tumor proliferation. The extremely rapid hyperthermia properties caused by the two components of Fe3O4 and Au also lead to shortened tumor survival time. The contribution of this article is to construct a comprehensive treatment system as a NO donor, which can thermally control the NO released in the body and verify the effect of Au on tumor suppression. Since many key biological functions of nitric oxide are related to its concentration, the real-time detection of different concentrations of nitric oxide in the body and the damage to different tumor cells will be further studied in the future.
This work was funded by National Natural Science Foundation of China 81401501, Liaoning Provincial Department of Education 2019LJC03, LJQ2015110, China Scholarship Council 20180820463, and Liaoning University Student Innovation Funding Project 10330010320. And 201919163050, Career Development Support Plan for Young and Middle-aged Teachers of Shenyang Pharmaceutical University, No. ZQN2015013.
The authors report no conflicts of interest for this work.
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