TiO 2-x @GL NPs and their applications

Abstract

The present invention relates to the field of biomedicine technology and specifically discloses TiO 2‑x @GL NPs and their applications, the present invention uses aluminum reduction method to synthesize TiO 2‑x NPs, and then TiO 2‑x The surface of NPs was modified to obtain TiO 2‑x @GL NPs. The TiO prepared by the present invention 2‑x @GL NPs can inhibit tumor cell migration and proliferation, and can also synergize with laser and ultrasound to inhibit tumor proliferation, providing a material basis for the preparation of drugs for treating cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to TiO 2-x @GL NPs and their applications. Background Art

[0002] Titanium dioxide nanoparticles (TiO2NPs) have good biocompatibility, high photoactivity, excellent stability and low toxicity. TiO2NPs have become a promising anticancer material and have been intensively studied. It is well known that TiO2NPs can induce the production of a large number of reactive oxygen species (ROS) in various types of cancer cells after ultraviolet irradiation, ultimately causing cell death, and therefore are widely used in photodynamic therapy (PDT) of malignant tumors. TiO2NPs and synthetic materials based on TiO2NPs have been widely studied as photosensitizers in PDT. Most of the research focuses on improving its ability to generate ROS and expanding the spectral response by doping with various metal or non-metal dopants. TiO2NPs also improve the delivery efficiency by binding to various molecules or antibodies.

[0003] Tumor metastasis is the main harmful feature of malignant tumors and the main cause of death in postoperative patients. Therefore, preventing tumor cell migration is a potential anti-cancer method. Although TiO2NPs have potential anti-cancer ability, they can also cause adverse reactions, and the concentration at which they reach tumor tissue to exert their effects is still not ideal.

[0004] Previous studies have shown that tumor-bearing organisms lack glutamine (GL), and exogenously supplemented glutamine is preferentially delivered to tumor tissues. However, adding glutamine in vitro promotes tumor cell proliferation and tumor growth. Therefore, how to deliver glutamine to tumor tissues while inhibiting tumor cell proliferation and growth is a difficult problem. Currently, no product has been found that can deliver glutamine to tumor tissues while inhibiting tumor proliferation. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides TiO 2-x @GL NPs and their applications, the TiO prepared by the present invention 2-x @GL NPs can inhibit tumor cell migration and proliferation, and can also synergize with laser and ultrasound to inhibit tumor proliferation, providing a material basis for the preparation of drugs for treating cancer.

[0006] The first object of the present invention is to provide a TiO 2-x @GL NPs were prepared by the following steps:

[0007] S1, TiO 2-xPreparation of NPs: TiO was synthesized by aluminum reduction method using titanium dioxide and aluminum as raw materials. 2-x NPs;

[0008] S2, TiO 2-x @GL NPs preparation: TiO 2-x The surface of NPs was modified to obtain TiO 2-x @GLNPs.

[0009] Furthermore, in S1, the specific process of the aluminum reduction method is: aluminum is heated to melt at 700-800°C, mixed with the TiO2 sample, and then heated to reduce at 300-500°C for 6-10h to obtain 500°C-Al-TiO 2-x , then 500℃-Al-TiO 2-x After annealing for 10-12h, TiO 2-x NPs.

[0010] Furthermore, in S2, glutamine has an effect on TiO 2-x The surface modification of NPs is as follows: 40-50 mg of glutamine is added to 8-10 mL of TiO with a mass concentration of 1-1.5 mg / mL. 2-x NPs solution, then ultrasonically treated at 120W for 3-5 hours in an ice bath, centrifuged three times at 10000r / min for 10min, and washed with deionized water to obtain TiO 2-x @GL NPs.

[0011] Furthermore, in S2, glutamine has an effect on TiO 2-x The surface modification of NPs was as follows: 50 mg of glutamine was added to 10 mL of TiO 2-x NPs solution, and then treated with 120W ultrasound in an ice bath for 4 hours, centrifuged three times at 10000r / min for 10min, and washed with deionized water to obtain TiO 2-x @GL NPs.

[0012] Furthermore, the TiO 2-x The hydrated particle size of @GL NPs is 164.2 nm.

[0013] The second object of the present invention is to provide the TiO 2-x @GL Application of NPs in the preparation of drugs for treating tumors.

[0014] Furthermore, the TiO 2-x @GL NPs can be used to prepare inhibitors of tumor cell migration.

[0015] Furthermore, the TiO2-x @GL NPs can be used to prepare inhibitors of tumor cell proliferation.

[0016] Furthermore, the TiO 2-x @GL NPs can be used to prepare synergistic agents for laser and ultrasound tumor inhibition.

[0017] Furthermore, the TiO 2-x The photothermal properties of @GL NPs at a concentration of 300 ppm are able to kill cancer cells.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention uses glutamine to treat TiO 2-x The surface of NPs was modified to obtain TiO 2-x @GL NPs, found TiO 2-x The structure of NPs was well preserved after being encapsulated on the surface of GL. GL had no effect on the NIR absorption capacity and photothermal performance. GL made TiO 2-x @GL content is higher than TiO 2-x The accumulation amount increased nearly 3 times.

[0020] 2. TiO prepared by the present invention 2-x @GL NPs can not only deliver GL to tumor tissues, but also inhibit tumor cell migration and proliferation. At the same time, they can also cooperate with laser and ultrasound to inhibit tumor proliferation, providing a material basis for the preparation of drugs for treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] It should be noted that Figure 1-Figure 6 Medium TiO 2-x @GL and TiO 2-x @GL NPs represent the same substance, TiO 2-x @GL is TiO 2-x @GL Abbreviation for NPs.

[0023] Figure 1 The TiO prepared by the present invention 2-x @GL NPs and TiO 2-x TEM images and SAED pattern images of NPs;

[0024] Among them, Figure a is TiO 2-x Low-magnification TEM image of NPs;

[0025] Figure b is TiO 2-x @Low magnification TEM image of GL NPs;

[0026] Figure c is TiO 2-x High-resolution TEM images of NPs;

[0027] Figure d shows TiO 2-x High-resolution TEM images of @GL NPs;

[0028] Figure e is TiO 2-x SAED pattern images of NPs;

[0029] Figure f is TiO 2-x @GL SAED pattern image of NPs;

[0030] Figure g is TiO 2-x DLS curves of NPs;

[0031] Figure h is TiO 2-x @GL DLS curve of NPs;

[0032] Figure 2 The TiO prepared by the present invention 2-x @GL NPs and TiO 2-x SEM, EDS, Zeta potential, XRD, and UV and NIR absorption spectra of NPs;

[0033] Among them, Figure a is TiO 2-x SEM images of NPs.

[0034] Figure b is TiO 2-x @GL SEM image of NPs;

[0035] Figure c shows the TiO measured based on the purple rectangular area. 2-x EDS of NPs;

[0036] Figure d shows TiO 2-x @GL EDS of NPs;

[0037] Figure e is TiO 2-x NPs and TiO 2-x @GL Zeta potential of NPs;

[0038] Figure f is TiO 2-x NPs and TiO 2-x @GL XRD of NPs;

[0039] Figure g is TiO 2-x NPs and TiO 2-x @GL Absorption spectra of ultraviolet and near-infrared light of NPs;

[0040] Figure h is TiO 2-x NPs and TiO 2-x @GL Photothermal heating curves of NPs.

[0041] Figure 3 TiO 2-x @GL NPs in vitro sonodynamic and photothermal effects;

[0042] Figure a shows the TiO 2-x @GL NPs produced reactive oxygen species in vitro;

[0043] Figure b shows the TiO under continuous ultrasonic irradiation (1.5W / cm2) 2-x @GL NPs’ cumulative reactive oxygen species concentration curve;

[0044] Figure c is a comparison of the effects of different particle concentrations on the production of reactive oxygen species;

[0045] Figure d shows different concentrations of TiO 2-x @GL NPs toxicity to normal epithelial cells;

[0046] Figure e shows different concentrations of TiO 2-x @GL NPs (0, 6.25, 12.5, 25, 50, 100 and 300 ppm) photothermal heating curves under near-infrared (1064 nm) irradiation with a power intensity of 1.5 W / cm2;

[0047] Figure f is 50ppm TiO 2-x @GL NPs at different power densities (0, 0.5, 1.0 and 1.5 W / cm 2 ) under photothermal heating curve.

[0048] Figure 4 Based on the synergistic tumor treatment effect of in vitro SDT / PTT,

[0049] Among them, Figure a is TiO 2-x @GL NPs (50ppm) aqueous dispersion after ultrasound with a power intensity of 1.5W / cm 2 The photothermal properties of the aqueous dispersion under near-infrared light irradiation show that when the light source is turned off, the temperature of the aqueous dispersion tends to be stable.

[0050] Figure b is TiO 2-x @GL NPs aqueous dispersion at a power intensity of 1.5 W / cm 2Three cyclic heating curves of aqueous dispersion in water under the conditions of;

[0051] Figure c shows the relative cell viability of 4T1 cells after different treatments, including control (untreated), TiO 2-x @GL NPs treatment, ultrasonic treatment only, TiO 2-x @GL NPs combined with light, TiO 2-x @GL NPs combined with ultrasound irradiation, and TiO 2-x @GL NPs were irradiated with light and ultrasound.

[0052] Figure d shows the CLSM images of 4T1 cells after different treatments, stained with PI (red fluorescence) and calcein-AM (green fluorescence);

[0053] Figure e is TiO 2-x @GL NPs, variance contribution of ultrasound and light;

[0054] Figure f is the control, TiO 2-x and TiO 2-x @GL Plate counting experiment of NPs.

[0055] Figure 5 This is the research result of SDT / PTT synergistically inhibiting tumors in vivo;

[0056] Figure a shows TiO 2-x @GL Schematic diagram of NPs used for tumor eradication under the synergy of SDT and PTT;

[0057] Figure b shows intravenous injection of TiO 2-x or TiO 2-x @GL NPs Titanium accumulation in tumor tissue after 4h;

[0058] Figure c is the control group, TiO 2-x @GL NPs group, TiO 2-x @GL NPs combined with ultrasound group, TiO 2-x @GL NPs combined with laser group, TiO 2-x @GL NPs combined with laser + ultrasound group of 4T1 tumor-bearing mice with time-dependent body weight curve;

[0059] Figure d shows the tumor weights of different groups on day 15;

[0060] Figure e shows the time-dependent tumor volume curve within 15 days;

[0061] Figure f shows the survival curve of 4T1 tumor-bearing mice after different treatments;

[0062] Figure g shows HE staining, Tunel staining and immunohistochemical Ki67 antigen staining of the tumor area in each group after treatment.

[0063] Figure 6 TiO 2-x @GL Safety evaluation of NPs;

[0064] Figure a shows the TiO 2-x @GL H&E staining images of major organ tissues in the NPs+laser+ultrasound group.

[0065] Figure b shows the blood routine from day 0 to day 28: white blood cells, platelets, and hemoglobin;

[0066] Figure c shows the biochemical indicators from day 0 to day 28: urea nitrogen, C-reactive protein, and globulin. DETAILED DESCRIPTION

[0067] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0068] Example 1

[0069] This embodiment provides TiO 2-x @GL NPs and their preparation methods and applications.

[0070] 1. TiO 2-x @GL NPs preparation method

[0071] TiO 2-x NPs were synthesized by the aluminum (Al) reduction method reported in previous literature. The specific process is as follows:

[0072] The titanium dioxide sample and aluminum were placed in a double-zone tube furnace, and then evacuated to a base pressure of less than 0.5 Pa. Subsequently, the aluminum was heated and melted at 800°C, mixed with the TiO2 sample and heated at 300°C for 6 hours to generate 500°C-Al-TiO 2-x 500℃-Al-TiO 2-x The sample was annealed for 12 h to obtain TiO 2-x NPs.

[0073] In order to increase the TiO 2-xTo investigate the tumor targeting ability of NPs, we used glutamine (Glu, abbreviated as GL) to bind TiO 2-x The surface of NPs was modified. 50 mg GL was added to TiO 2-x NPs solution (1 mg / mL, 10 mL) was then subjected to ultrasonic treatment (120 W) for 4 h in an ice bath. TiO 2-x @GL NPs (also abbreviated as TiO 2-x @GL).

[0074] 2. TiO 2-x @GL NPs characterization

[0075] 1. Transmission electron microscope (TEM) observation TEM image

[0076] X-ray diffraction (XRD) patterns were obtained on a Rigaku D / MAX-2200PX XRD system with the parameters set to Cu, Kα, 40 mA and 40 kV.

[0077] 2. Titanium, oxygen and nitrogen elements were determined using segmented energy dispersive spectroscopy (EDS) technology, and corresponding chromatographic analysis was performed

[0078] 3. Particle size distribution and zeta potential were measured on a Zetasizer system (Nano ZS90, Malvern Instruments Ltd.).

[0079] 4. The absorption spectra of ultraviolet and near-infrared light were recorded using a Shimadzu UV-3600 spectrometer.

[0080] 5. A 1064 nm multimode pump laser (Shanghai Link Fiber Optics Co., Ltd.) was used as the irradiation source for photothermal therapy, and an intelligent transmission ultrasound system (Chattanooga Group, USA) was used as the sound source for ultrasound therapy.

[0081] 6. The content of nanoparticles was quantitatively analyzed using an Agilent 725 inductively coupled plasma optical emission spectrometer (Agilent Technologies).

[0082] 7. Confocal laser scanning microscopy images were recorded by FV1000 (Olympus Corporation, Japan).

[0083] 8. Observe the cell uptake of nanomaterials and cell apoptosis by flow cytometry (Becton, Dickinson, USA).

[0084] 3. Application of TiO2-x@GL NPs in the preparation of tumor therapeutic drugs

[0085] 1. Cell culture

[0086] 4T1 cells were purchased from ATCC: Global Bioresource Center (#CRL-2539). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in 5% CO2 at 37°C, with the cell culture medium replaced daily and treated when the cell concentration reached 60%.

[0087] 2. In vitro sonodynamics and photothermal treatment

[0088] Commercial CCK-8 detection kit is used to evaluate the efficiency of ultrasound and photothermal therapy in killing cancer cells. 2×10 4 / ml4T1 were inoculated into 96-well plates and cultured in RPMI-1640 medium containing 10% fetal bovine serum for 12 hours, and then TiO 2-x @GL (Ti concentration 50ppm) co-incubation. The cell viability of each group was measured by comparison with the control group. The laser power intensity was set to 1.5W / cm 2 , the ultrasonic intensity parameters were set to 40kHz, and the power duration was 180s.

[0089] 3. Reactive oxygen species (ROS) detection

[0090] The cultured 4T1 cells were incubated at 37°C with TiO 2-x @GL was incubated for 4 hours or sonicated for 5 minutes. After the incubation was completed, the medium was removed, the cells were washed three times with PBS, and then DCFHDA was added and incubated for another hour. The production of ROS was detected by flow cytometry, and the cells were collected and the intracellular fluorescence intensity of DCF was detected.

[0091] 4. TiO 2-x Evaluation of the photothermal effect of @GL NPs in vitro

[0092] The TiO 2-x The photothermal effect of @GL was evaluated by recording the temperature changes during laser irradiation in the NIR-II biological channel (1064 nm). 2-x @GL was dispersed in deionized water and the Ti concentration was adjusted to 0, 6.25, 12.5, 25, 50, 100, and 300 ppm, respectively, and exposed to 1064 nm laser radiation with a laser intensity of 1.5 W / cm 2 In addition, TiO 2-x The temperature rise of @GL was observed at different power intensities (0, 0.5, 1.0 and 1.5 W / cm) with 1064 nm laser at a Ti concentration of 400 ppm. 2) for testing.

[0093] 5. Assessment of cell migration ability

[0094] Resuspend 4T1 cells in serum-free medium, and then add 100 mL of cell suspension at 2 × 10 4 / ml density was inoculated in the chamber of the transwell plate, and then the chamber was placed in the well containing serum-containing medium. After continuing to culture for 12 hours, the medium was discarded, the cells were fixed with 4% paraformaldehyde, and then stained with 0.1% crystal violet for 15 minutes at room temperature. After wiping the cells on the upper side of the chamber, the migrated cells were observed and counted under a light microscope. The cell count was taken from four different fields of view, top, bottom, left, and right.

[0095] 6. Differentiation between living cells and dead cells

[0096] Live cells and dead cells were distinguished by a commercial Ca-AM / PI detection kit (#04511, Sigma-Aldich). Live cells were stained green, and dead cells were stained red by PI. After staining, cells were observed using a confocal laser scanning microscope (CLSM).

[0097] 7. Establishment of tumor-bearing model and in vivo SDT / PTT synergistic tumor treatment

[0098] To establish a tumor-bearing model, 4T1 cells (1×10 6 The cells were suspended in 100 μL PBS and injected into the right back of the mouse. Finally, 40 female 4T1 tumor-bearing mice were successfully established in the Experimental Animal Center. 3 Afterwards, the mice were divided into 5 groups (n=8): (a) control group (treated with saline), (b) TiO 2-x @GL group, (c) TiO 2-x @GL+laser group (injection of TiO 2-x @GL, then irradiated with a 1064 nm light source), (d) TiO2-x@GL+ultrasound (with TiO 2-x @GL treatment + ultrasonic irradiation), and (e) TiO 2-x @GL+laser+ultrasound group (injection of TiO 2-x @GL, followed by laser and ultrasound irradiation). TiO 2-x The injection dose of @GL was 15 mg / mL, the time of 4T1 cell tumor formation was recorded as -7 days, and treatment was received on day 0. 2-x @GL for 4 hours, 1064nm laser (1.5W / cm 2 , 10 min) and ultrasound (1 MHz, 50% duty cycle, 1.0 W / cm 2) for subsequent treatment. When combined treatment was performed, laser + ultrasound treatment was given on day 0, and ultrasound treatment was given on days 3 and 5. The weight, length, and width of the tumor were measured every 2 days using a digital scale and caliper, respectively. The tumor volume was calculated as follows: tumor volume (mm 3 )=ab 2 / 2, a=maximum length (mm), b=minimum width (mm).

[0099] 8. TiO 2-x Distribution of @GL in tumor tissue

[0100] To verify the TiO 2-x @GL can penetrate the barrier and accumulate in tumor tissues. We measured the TiO 2-x and TiO 2-x The distribution of @GL. 4T1 female tumor-bearing mice were randomly divided into three groups and intravenously injected with saline, TiO 2-x and TiO 2-x @GL, the dose was 50ppm. Mice were killed 4h after injection, and tumor tissues were collected by dissection. Tumor tissues were weighed, homogenized, and dissolved in aqua regia. The content of Ti in tumor tissues was then determined by inductively coupled plasma optical emission spectrometry (ICP-OES), and its distribution was calculated based on the original weight per gram of tissue.

[0101] 9. Pathological changes, cell apoptosis and cell proliferation after different treatments

[0102] Hematoxylin-eosin (HE) staining, TUNEL assay, and Ki67 antibody were used to detect pathological changes, apoptosis, and cell proliferation of cells, respectively. HE staining was performed according to previous reports, and TUNEL staining was performed using a commercial detection kit according to the manufacturer's instructions. Immunohistochemical staining was performed using Ki67 antibody combined with goat anti-rabbit secondary antibody to show cell proliferation.

[0103] 10. Statistical analysis

[0104] The data are expressed as mean ± standard deviation (SD), and the differences between the two groups were tested using a two-sided t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

[0105] IV. Experimental Results

[0106] 1. TiO 2-x NPs, TiO 2-x @GL Characterization of NPs

[0107] Transmission electron microscopy was used to observe TiO 2-x NPs and TiO 2-x@GL NPs morphology, the results are as follows Figure 1 As shown in Figure a, TiO 2- x NPs are uniform spherical structures with an average size of 50 nanometers. 2-x @GL NPs morphology and TiO 2-x There was no significant change in NPs ( Figure 1 Figure b. From TiO 2-x NPs and TiO 2-x High-resolution TEM imaging results of @GL NPs ( Figure 1 Figure c and d) and the corresponding SAED images ( Figure 1 Figure e and g) show that the coating of GL did not change the TiO 2-x The crystal structure of NPs. DLS results show that GL coating makes TiO 2-x The hydrated particle size of NPs increased from 122.4 nm to 164.2 nm ( Figure 1 Figure g and h).

[0108] SEM images ( Figure 2 Figure a and Figure b) and EDS results ( Figure 2 Figure c, Figure 2 Figure d and Figure S1) show that with TiO 2- x Compared with the NPs group, TiO 2-x The nitrogen (N) content of the @GL NPs group increased significantly, indicating that GL was successfully loaded into the TiO 2-x NPs surface. Although the zeta potential changed before and after GL encapsulation (from -36.1 to -9.1 mV) ( Figure 2 Figure e), but in XRD measurement, TiO 2-x NPs and TiO 2-x The crystal peaks of @GL NPs and TiO 2-x The standard crystal structure of NPs is well matched (JCPDS No.21-1272), indicating that TiO 2-x The structure of NPs was well preserved after surface encapsulation on GL ( Figure 2 In addition, from TiO 2-x NPs and TiO 2-x @UV-Vis-NIR optical absorption curve of GLNPs( Figure 2 g) and the photothermal heating curve ( Figure 2 From Figure h), we can see that the NIR absorption capacity and photothermal performance are not affected. These results show that TiO 2-x NPs and TiO 2-x@GL NPs can treat tumors via PTT.

[0109] 2. In vitro sonodynamics and photothermal effects of TiO2-x@GL NPs

[0110] The generation of ROS may be affected by many factors, such as the power and duration of ultrasound and the concentration of sonosensitizer. First, the generation of ROS increases with the increase of ultrasound intensity. Therefore, the ultrasound duration was set to 180 s and the TiO 2-x @GLNPs concentration is 50ppm( Figure 3 a). To determine the effect of 40 kHz ultrasound on ROS generation, we measured 50 ppm TiO 2-x @GL NPs concentration ROS generation ( Figure 3 b). Using 1.0W / cm 2 Ultrasonic irradiation of TiO 2-x @GL NPs continuously release ROS. Figure 3 c, at 1.0W / cm 2 Under the condition of ultrasonic treatment for 180 s, ROS generation increased slightly with the increase of nanoparticle concentration. 2-x @GL NPs inhibited cell growth (<75%) at high concentrations (>100 ppm). Based on the previous and above results, an optimized procedure (1.0 W / cm 2 Ultrasonic intensity, 180 s, and 50 ppm TiO 2-x @GL NPs) to achieve high levels of ROS generation. 2-x Photothermal performance of @GL NPs at different concentrations (0, 6.25, 12.5, 25, 50, 100, and 300 ppm) and 1.5 W / cm 2 The study found that at a concentration of 300 ppm, the temperature could reach 59.2°C, which is enough to kill cancer cells through heat. The photothermal effect related to laser power (0, 0.5, 1.0 and 1.5 W / cm 2 ) has also been confirmed ( Figure 3 f).

[0111] 3. In vitro synergistic cancer therapy based on SDT / PTT

[0112] In this study, 4T1 cells were treated with SDT and PTT (sequential treatment). 2-x The NPs were incubated together and treated with ultrasound and NIR-II laser irradiation for 4 h. The ultrasound parameters were set to 1 MHz, 50% duty cycle, 1.0 W / cm 2, 240s. The power of the NIR-II laser was set to 1.5W / cm 2 The study found that TiO 2-x @GL NPs concentration of 300ppm, the temperature rises rapidly, up to 66℃, which is enough to kill cancer cells through hyperthermia ( Figure 4 a), when the laser is turned off, the temperature drops rapidly. It is worth noting that under NIR-laser irradiation, the extremely high temperature produces obvious ablation and smoking phenomena. During the three laser on / off cycles, the photothermal performance of TiO2-x@GL NPs has no obvious degradation, showing that the TiO 2-x High photothermal stability for photothermal therapy ( Figure 4 b) CCK-8 experiment is to study TiO 2-x In vitro killing effect of @GL NPs on 4T1 cells under 1064 nm laser, ultrasound, and laser + ultrasound treatment. In the SDT and PTT treatment groups, TiO 2-x After incubation of @GL NPs with cancer cells for 4 h, the cell survival rate dropped to 19.2% ( Figure 4 c). In addition, after various treatments, the cell killing effect was directly observed by CLSM, and the live and dead cells were stained with calcein-am (green) and PI (red), respectively. 2-x A large number of dead cells were observed in the group treated with GL NPs combined with ultrasound and laser, indicating that SDT and PTT synergistically caused extensive cell apoptosis and death ( Figure 4 d) From the perspective of the average contribution rate of regional differences, TiO 2-x @GL NPs contribute a lot, averaging 44.95%. US and NIR contribute 23.33% and 31.72% of the variance, respectively. Figure 4 e) Consistent with the results of previous studies, TiO2 can inhibit the migration of tumor cells. Cell migration experiments show that TiO 2-x @GL NPs and TiO 2-x The number of viable cells in each group was significantly lower than that in the control group. This means that TiO 2-x @GL NPs, like TiO2, can also inhibit the migration of tumor cells ( Figure 4 f).

[0113] 4. In vivo verification of SDT / PTT synergistic treatment of cancer

[0114] Cooperative treatment regimens such as Figure 5 As shown in a, the tumor-bearing model was successfully established at day -7, when TiO was injected 2-x @GL NPs (abbreviated as TiO 2-x@GL) 4h after laser irradiation and ultrasound treatment was used and recorded as day 0. Ultrasound irradiation was then continued on days 3 and 5. The Ti element concentration in the tumor tissue was 2-x or TiO 2-x @GL group increased significantly, and GL made TiO 2-x @GL content is higher than TiO 2-x The accumulation increased nearly 3 times ( Figure 5 b). During the treatment period, there was no significant change in the body weight of mice in each group ( Figure 5 c) TiO 2-x The tumor weight in the GL+laser+ultrasound group was significantly lower (P<0.01) than that in the TiO 2-x @GL group, and on the 15th day, it was also significantly lower (P<0.05) than TiO 2-x @GL+Laser group or TiO 2-x @GL+ultrasound group( Figure 5 d), at the same time, TiO 2-x @ There was no increase in tumor volume in the GL+laser+ultrasound group ( Figure 5 e), synergistic treatment helps prolong death-free survival ( Figure 5 f) HE staining shows TiO 2-x @GL+laser+ultrasound group had obvious pathological changes, mainly manifested as tumor cell swelling, Tunel test showed that TiO 2-x @GL+laser+ultrasound group had increased tumor cell apoptosis. Ki67 antibody localization showed downregulation of tumor cell proliferation. All evidence showed that TiO 2-x @GL+laser+ultrasound can inhibit tumors in vivo.

[0115] 5. TiO 2-x Safety evaluation of @GL NPs

[0116] TiO 2-x @GL The safety evaluation of NPs is shown by pathological changes in important organs and key blood and biochemical parameters. HE staining showed no significant changes in the heart, liver, kidney, and lung. White blood cells (WBC), platelets (PLT), and hemoglobin (HGB) on the first, seventh, and 28th days did not change significantly between the different groups. At the same time, biochemical indicators such as urea nitrogen (BUN), C-reactive protein (Cr), and globulin (GLB) did not change compared with the control group.

[0117] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0118] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. TiO 2-x @GL NPs are used in the preparation of drugs for treating tumors, characterized in that: The TiO 2-x @GL NPs were prepared by the following steps: S1, TiO 2-x Preparation of NPs: TiO2 and aluminum were used as raw materials and TiO 2-x NPs; The specific process of aluminum reduction method is: aluminum is heated to melt at 700-800℃, mixed with TiO2 sample and then heated to reduce at 300-500℃ for 6-10h to obtain 500℃-Al-TiO 2-x , then 500℃-Al-TiO 2-x After annealing for 10-12h, TiO 2-x NPs; S2, TiO 2-x @GL NPs preparation: TiO 2-x The surface of NPs was modified to obtain TiO 2-x @GLNPs; Glutamine on TiO 2-x The surface modification of NPs was carried out as follows: 50 mg of glutamine was added to 10 mL of TiO 2-x NPs solution, and then treated with 120W ultrasound in an ice bath for 4 hours, centrifuged three times at 10000 r / min for 10 min, and washed with deionized water to obtain TiO 2-x @GL NPs; the TiO 2-x @GL NPs hydrated particle size is 164.2 nm; The tumor cells of the tumor are 4T1 cells.

2. A TiO 2-x @GL NPs, characterized by The TiO 2-x @GL NPs were prepared by the following steps: S1, TiO 2-x Preparation of NPs: TiO2 and aluminum were used as raw materials and TiO 2-x NPs; The specific process of aluminum reduction method is: aluminum is heated to melt at 700-800℃, mixed with TiO2 sample and then heated to reduce at 300-500℃ for 6-10h to obtain 500℃-Al-TiO 2-x , then 500℃-Al-TiO 2-x After annealing for 10-12h, TiO 2-x NPs; S2, TiO 2-x @GL NPs preparation: TiO 2-x The surface of NPs was modified to obtain TiO 2-x @GLNPs; Glutamine on TiO 2-x The surface modification of NPs was carried out as follows: 50 mg of glutamine was added to 10 mL of TiO 2-x NPs solution, and then treated with 120W ultrasound in an ice bath for 4 hours, centrifuged three times at 10000 r / min for 10 min, and washed with deionized water to obtain TiO 2-x @GL NPs; the TiO 2-x The hydrated particle size of @GL NPs is 164.2 nm.

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