NL2034406A

NL2034406A - The tio2-x@gl nps and application thereof

Info

Publication number: NL2034406A
Application number: NL2034406A
Authority: NL
Inventors: Wan Ting; Wang Huaiyan; Zhang Lina; Hu Haiting; Mao Zhilei; Zhu Pengfeng
Original assignee: Changzhou Maternal And Child Health Care Hospital
Priority date: 2023-02-15
Filing date: 2023-03-22
Publication date: 2023-05-15
Also published as: NL2034406B1; CN116098999A

Abstract

The invention relates to the technical field of biological medicine, in particular to the TiOZ-X@GL NPS and application thereof. In the invention, synthesizing TiOZ—x NPS by aluminum reduction. method, and obtaining TiOZ-X@GL NPS by modifying the surface of TiOZ—x NPS with glutamine. The TiOZ-X@GL NPS prepared by the invention can inhibit the metastasis and proliferation of tumor cells, meanwhile, can inhibit the proliferation of the tumor cooperated with laser and ultrasound, which provides the material basis for the preparation of drugs to treat cancer.

Description

P1747 /NLpd

THE TIO2-X@GL NPS AND APPLICATION THEREOF

Technical field

The invention relates to the technical field of biological medicine, in particular to the Ti02-x@GL NPs and application thereof.

Background technology

Titanium dioxide nano-particles(Ti02 NPs) boasts the charac- teristics of bio-compatibility, high illumination activity, excel- lent stability and low toxicity, which has become a promising an- ti-cancer material and has been intensively studied. As is well- known, Ti02 NPs can induce production of large amounts of reac- tive oxygen species{ROS)} in various types of cancer cells after ultraviolet irradiation, resulting in the death of the cells, therefore it is applied widely in photo-dynamic therapy (PDT) for malignant tumors. Ti©2 NPs and the synthetic material based on it as photo-sensitizers in PDT have been studied more. Most of stud- ies concentrate on improving the ROS generation capacity and spreading spectral response by mingling various metal or non-metal dopants. TiO2 NPs improves the transmission efficiency by cooper- ating various molecules or antibodies.

Tumor metastasis is the main harmful characteristic of the malignant tumor, and the main reason of death in patients after operation. Therefore, to prevent the tumor cell metastasis is a potential anti-cancer method. Although Ti02 NPs has potential an- ti-cancer capacity, it also has bad effect, and when it reaches the tumor tissue, the concentration that can play a role is still not ideal.

The previous studies have shown that glutamine (GL) deficiency is found in tumor-bearing organism and exogenous supplementation of glutamine is preferentially transported to tumor tissue. Howev- er, adding glutamine in-vitro promotes tumor cell proliferation and tumor growth, therefore, it is difficult to inhibit tumor cell proliferation and growth when transport glutamine to the tumor tissue. No product has been found that can deliver glutamine to tumor tissue without inhibiting tumor proliferation.

Summary of the invention

To solve the technical problem above, the invention provides the TiO2-x@GL NPs and application thereof. The Ti0O2-x@GL NPs pre- pared by the invention can inhibit the metastasis and prolifera- tion of tumor cells, meanwhile, inhibiting the proliferation of the tumor cooperated with laser and ultrasound, which provides the material basis for the preparation of drugs to treat cancer.

The first aim of the invention is to provide the Ti02-x@GL

NPs, which prepared by the following steps:

Sl. Preparing TiO2-x NPs: taking titanium dioxide and alumi- num as raw materials to synthesize Ti02-x NPs by aluminum reduc- tion method; 32. Preparing TiO2-x@GL NPs: obtaining Ti02-x@GL NPs by modi- fying the surface of Ti02-x Nps with glutamine.

Further, in 81, the specific process of the aluminum reduc- tion method is: aluminum melts when heated at the temperature of 700-800°C, and is heated and reduced at the temperature of 300- 500°C for 6-10h after mixing with the TiC2 sample to obtain 500°C-

Al-Ti02-x, which is annealed for 10-12h in argon at the tempera- ture of 800-900°C to obtain TiO2-x NPs.

Further, in the S82, the surface modification to Ti02-x NPs by glutamine is: putting 40-50mg glutamine into 8-10mL Ti02-x NPs so- lution with the mass concentration of 1-1.5 mg/mL, then is treated for 3-5h with 120W ultrasound in an ice bath, and is centrifuged three times for 10min with the rotation of 10,000 r/min, whereby the Ti02-x@GL NPs is obtained by removing the ionized water and washing.

Further, in the S2, the surface modification to Ti02-x NPs by glutamine is: putting 50mg glutamine into 10mL Ti02-x NPs solution with the mass concentration of 1mg/mL, then is treated for 4h with 120W ultrasound in an ice bath, and is centrifuged three times for 4h with the rotation of 10,000 r/min, whereby the Ti02-x@GL NPs is obtained by removing the ionized water and washing.

Further, the hydration particle size of the Ti0O2-x@GL NPs is 164.2nm.

The second aim of the invention is to provide the applica- tion of the TiO2-x@GL NPs in the preparation of tumor drugs.

Further, the Ti02-xQ@GL NPs can be applied to prepare the in- hibitor for tumor cell metastasis.

Further, the Ti02-x@GL NPs can be applied to prepare the in- hibitor for tumor cell proliferation.

Further, the Ti02-x@GL NPs can be applied to prepare the syn- ergist for inhibiting tumor by laser and ultrasonic.

Preferably, the photo-thermal performance at a concentration of 300ppm of the TiO2-xEGL NPs can kill the cancer cells.

Compared with the prior art, the beneficial effect of the in- vention is: 1. In the invention, by obtaining the Ti02-x@GL NPs through modifying the surface of the Ti02-x NPs with glutamine, the struc- ture of the Ti02-x NPs is well preserved after surface encapsula- tion of GL, GL has no effects on the wave-absorbing ability and photo-thermal performance of the NIR, and makes the contents of

TiO2-x@GL increase nearly 3 times compared with the accumulation of the Ti02-x. 2. The Ti02-x@GL NPs prepared by the invention not only can transport the GL to the tumor tissue, but also can inhibit the me- tastasis and proliferation of tumor cells, meanwhile, can inhibit the proliferation of the tumor cooperated with laser and ultra- sound, which provides the material basis for the preparation of drugs to treat cancer.

Description of attached drawings

To state the example in the invention or the technical scheme of the prior art, a brief description of the attached drawings needed in the example or prior art is given following. It is obvi- ous that the attached drawings described below are only several examples of the invention, for the ordinary technicians in the field, other drawings can be obtained from these drawings without any creative effort.

What should be noted is, in figure 1 to 6, the Ti02-x@GL and

TiO2-x@GL NPs are represented as the same substance, and the

Ti02-x@GL is simplified form of the Ti02-x@GL NPs.

Figure 1 is the TEM image and SAED mode image of the Ti02-

%x@GL NPs and Ti02-xNPs prepared by the invention;

Wherein, figure a is the TEM image with low range of the

TiO2-x NPs;

Figure b is the TEM image with low range of the Ti02-x@GL

NPs;

Figure c is the TEM image with high resolution of the TiO2-x

NPs;

Figure d is the TEM image with high resolution of the Ti02-x @ GL NPs;

Figure e is the SAED mode image of the Ti02-x NPs;

Figure f is the SAED mode image of the TiO2-x@GL NPs;

Figure g is the DLS curve of the Ti02-x NPs;

Figure h is the DLS curve of the Ti02-x@GL NPs;

Figure 2 is the SEM, EDS, Zeta potential, XRD and absorption spectra of ultraviolet and near-infrared illumination of the Ti02- %x@GL NPs and Ti02-xNPs prepared by the invention;

Wherein, figure a is the SEM image of the Ti0O2-x NPs;

Figure b is the SEM image of the Ti0O2-x@GL NPs;

Figure c is the measured EDS of the TiO2-x Nps based on the purple rectangular region;

Figure d is the EDS of the TiO2-x@GL NPs;

Figure e is the Zeta potential of the Ti02-x NPs and TiO2- xOGL NPs;

Figure f is the XRD of the Ti02-x NPs and Ti02-x@GL NPs;

Figure g is the absorption spectra of ultraviolet and near- infrared illumination of the Ti02-xNPs and TiO2-xz@GL NPs;

Figure h is the photo-thermal heating curve of the Ti02-x NPs and TiO2-xz@GL NPs.

Figure 3 is the in-vitro acoustic dynamic and photo-thermal effects of the Ti02-x@GL NPs;

Wherein, figure a is the reactive oxygen species produced in- vitro of Ti02-x@GL NPs at different ultrasonic intensities;

Figure b is the cumulative reactive oxygen species concentra- tion curve of the Ti02-x@GL NPs under continuous ultrasonic irra- diation (1.5 W/cm2);

Figure c is the comparison of the effects of different parti- cle concentrations on reactive oxygen species;

Figure d is the toxicity of Ti02-x@GL NPs at different con- centrations to normal epithelial cells;

Figure e is the photo-thermal heating curve of the Ti02-xQGL

NPs at different concentrations (0, 6.25, 12.5, 25, 50, 100 and 5 300 ppm) under near-infrared ray (1,064nm) irradiation with power intensity of 1.5 W/cm2;

Figure f is the photo-thermal heating curve of 50 ppm Ti02-

X@GL NPs at different power densities (0, 0.5, 1.0 and 1.5 W/cm2).

Figure 4 is the effect of tumor synergistic therapy based on in-vitre SDT/PTT,

Wherein, figure a is the photo-thermal properties of Ti02- x@GL NPs (50ppm) aqueous dispersion after ultrasonic irradiation under near-infrared illumination with a power intensity of 1.5

W/cm2, when the illumination source is extinguished, the tempera- ture of the aqueous dispersion tends to be stable;

Figure b is the three recycle heating curves of the aqueous dispersion of the Ti02-x@GL NPs under the power intensity of 1.5 w/cm2;

Figure c is the relative cell viability of 4T1 cells after different treatments, including contrast (untreated), only TiO2- x@GL NPs treatment, only ultrasound treatment, TiO2-x@GL NPs + il- lumination, combined irradiation of Ti02-x@GL NPs + ultrasound, and combined irradiation of Ti02-x@GL NPs + illumination + ultra- sound.

Figure d is the CLSM image of 4T1 cells after different treatments, staining with PI (red fluorescence) and fluorexon-AM (green fluorescence);

Figure e is the variance contribution rates of the Ti02-x@GL

NPs, ultrasound and illumination;

Figure f is the contrast of the plate counting experiment of the Ti02-x and Ti02-x@GL NPs.

Figure 5 is the study results of the SDT/PTT synergistic tu- mor inhibition in-vivo;

Wherein, figure a is the schematic diagram of the Ti02-xQGL

NPs used to eradicate tumor in collaboration with SDT and PTT;

Figure b is the accumulation of titanium in tumor tissue af- ter 4h of intravenous injection of Ti02-x or Ti02-x@GL NPs;

Figure c is the contrast group, shows the time-dependent body weight curve of 4T1 tumor-bearing mice in Ti02-x@GL NPs group,

TiO2-x@GL NPs + ultrasound group, Ti02-x@GL NPs + laser group,

TiO2-x@GL NPs + laser + ultrasound group;

Figure d is the tumor weights of different groups on day 15;

Figure e is the time-dependent tumor volume within 15 days;

Figure f is the survival curve of the tumor-bearing mice af- ter different treatments;

Figure g is the HE staining, Tunel staining and immunohisto- chemical Ki67 antigen staining.

Figure 6 is the safety evaluation of the Ti02-x@GL NPs;

Wherein, figure a is the H&E staining images of major organ tissues in Ti02-x@GL NPs + laser + ultrasound group at day 1, 7 and 28;

Figure b is the blood routine from day 0 to day 28: white blood cells, platelets and hemoglobin;

Figure c is the biochemical criterion from day 0 to day 28: blood urea nitrogen, c-reactive protein and globulin.

Specific embodiments

The detailed embodiments in the invention are described in the following, but it should be understood that the protection scope of the invention is not limited by the embodiments. Based on the example of the invention, all the other examples obtained by the ordinary technicians in the field without creative labor be- long to the protection scope of the invention. The experimental methods of the examples in the invention, unless otherwise speci- fied, are conventional methods. The materials, reagents, etc. used in the following examples, unless otherwise specified, are com- mercially available.

Example 1

The example provides the Ti02-x@GL NPs and preparation and application thereof.

The preparation method of the TiO02-x@GL NPs;

TiO2-x NPs is synthesized by the aluminum{(Al) reduction meth- od reported by previous literature. The specific process is as follows:

Placing titanium dioxide sample and aluminum into two zone tube furnace respectively, which is evacuated until the base pres- sure is lower than 0.5 Pa. Then the aluminum melts when heated at the temperature of 800°C, and is heated at the temperature of 300°C for eh after mixing with TiC2 sample to obtain 500°C-Al-TiO2-x, which is annealed for 12h in argon at the temperature of 800°C to obtain TiO2-x NPs.

To improve the tumor targeting ability of the TiO2-x NPs, the surface of Ti02-x NPs is modified with glutamine (Glu, simplified as GL). Putting 50mg GL into TiO2-x NPs solution (1 mg/mL, 10 mL) , then is treated for 4h with ultrasound {120W ) in an ice bath, and is centrifuged three times (10,000 r/min, 10min) , whereby the

Ti02-x@GL NPs (can be abbreviated as Ti02-xQ@GL) is obtained by re- moving the ionized water and washing.

Characterization of the Ti02-xQGL NPs 1. Observing the TEM image through a transmission electron microscope (TEM)

Obtaining X-ray diffraction (XRD) pattern in Rigaku D/MAX-2200

PX XRD system, and parameters are set to Cu, Ko, 40 mA, and 40 kV. 2. Determining the titanium, oxygen and nitrogen with the segmented energy dispersion spectrum (EDS), and the corresponding chromatographic analysis is carried out. 3. Measuring the particle size distribution and { potential on a Zetasizer system (Nano ZS90, Malvern Instruments LTD.) . 4. Recording the absorption spectra of ultraviolet and near- infrared illumination with Shimadzu UV-3600 spectrometer. 5. Adopting 1,064nm multi-mode pumped laser (Shanghai Con- necting Fiber Co., LTD.) as the irradiation source of photo- thermal hyperthermia, and adopting the intelligent transmission ultrasonic system (Chattanooga Group, America) as the sound source for ultrasound therapy. 6. Quantitatively analyzing the content of nano-particles with Agilent 725 inductively coupled plasma optical emission spec- trometer (Agilent Technologies, Inc.). 7. Confocal laser scanning microscope images are recorded by

FV1,000 (Olympus Corporation, Japan). 8. Observing the cell uptake of nanc-materials and apoptosis through flow cytometry (America Becton, Dickinson Corporation).

The application of the Ti02-x@GL NPs in the preparation of tumor drugs 1. Culture of the cells 4T1 cells are purchased from ATCC: Global Biological Resource

Center (#CRL-2539). Culturing the cells in the RPMI-1,640 culture medium with 10% fetal bovine serum(FBS) and 5% C02 at 37°C. The cell medium is changed daily and treated when the cell concentra- tion reached 60%. 2. In-vitro acoustic dynamics and photo-thermal treatment

The commercial CCK-8 test kit is used to evaluate the effec- tiveness of ultrasound and photo-thermal treatments in killing cancer cells. Inoculating 2x104 /ml 4T1 in the plate with 936 holes, and culturing for 12h in the RPMI-1, 640 culture medium with 10% fetal bovine serum, then adding Ti02-x@GL (concentration of Ti is 50 ppm) for co-incubation. Determining the cell viability of each group compared with the contrast group. The laser power in- tensity is set to 1.5W /cm2, the ultrasonic strength parameter is set to 40 kHz, and the power duration is set to 180s. 3. The detection of reactive oxygen species (ROS)

Taking the cultured 4T1 cells, and co-incubating with

Ti02-x%Q@GL at 37°C for 4 hours, or treating 5min under ultrasound.

After incubation, removing the culture medium, and washing the cells three times with PBS, and adding DCFHDA to incubate for an- other 1h. Detecting the ROS generation through flow cytometry, and collecting cells to detect the intracellular luorescence intensity of DCF. 4. The evaluation of in-vitro acoustic dynamic and photo- thermal effects of the Ti02-x@GL NPs;

Evaluating the photo-thermal effect of the Ti02-x@GL with in- frared thermal imaging recorder (FLIR TM A325SC camera) through recording the temperature changes during laser irradiation in the

NIR-II biological channel (1,064nm) . Ti2-x@GL is dispersed in de- ionized water, and the Ti concentration is adjusted to 0, 6.25, 12.5, 25, 50, 100, 300 ppm respectively, and is exposed to the la- ser intensity with 1,064 nm and with 1.5 W/cm2 laser intensity. In addition, when the Ti concentration is 400 ppm, the temperature of the Ti02-x@GL increases, which is tested under a 1,064nm laser at different power intensities (0, 0.5, 1.0 and 1.5W/cm2). 5. The evaluation of the cells metastasis ability 4T1 cells are suspended in the culture medium without serum, and then the 100mL cell suspension are inoculated in the upper chamber of the cross-hole plate at a density of 2x104/ml, and then the cabin is put into the culture medium with serum. After cultur- ing for another 12h, removing the culture medium; Fixing the cells with 4% paraformaldehyde, and staining for 15min with 0.1% crystal violet at the room temperature. After erasing the cells in upper side of the cabin, observing and counting the migrated cells under the light microscope. Cell counts are taken from four different visual fields of up, down, left and right. 6. The distinguish between living and dead cells

Living and dead cells are distinguished by the commercial Ca-

AM/PI test kit (#04511, Sigma-Aldich). Living cells are stained into green, and the dead cells are stained into red by PI. After staining, observing the cells through a confocal laser scanning microscopy (CLSM). 7. The establishment of tumor-bearing model and in-vivo

SDT/PTT synergistic tumor therapy

To establish the tumor-bearing model, 4T1 cells (1x106 cells) are suspended in 100 pL PBS and are injected in the right back of the mice; 40 female 4T1 tumor-bearing mice are successfully estab- lished in the laboratory animal center finally. After the tumor had grown to nearly 50mm3, dividing the mice into 5 groups (n=8): (a) contrast group (normal saline treatment), (b) Ti02-x@GL group, (c) Ti02-x@GL+laser group (injecting Ti02-x@GL, and irradiating with 1,064 nm light source), (d) TiO2-x@GL+ultrasound (Ti02-xQ@GL treatment + ultrasonic irradiation), and (e)

TiO2-x@GL+lasertultrasound group (injecting Ti02-x@GL, and irradi- ating with laser and ultrasound). The injection dose of the

Ti02-x@GL is 15mg/mL, and the tumorigenesis time of injection of 4T1 cells is recorded as -7 days with 0 days of treatment. 4 hours after intravenous injection of Ti02-x@GL, 1,064nm laser ((1.5W/cm2 , 10min) and ultrasound (1Mhz, 50% duty ratio, 1.0W/cm2) are used for later therapy. When synergistic therapeutic is carried out,

laser + ultrasound therapy is used on day 0, and the following ul- trasound therapy is given on day 3 and day 5. Measuring the weight, length and width of the tumors with digital scales and calipers every 2 days. The tumor volume is calculated as follows: tumor volume (mm3)=ab2/2, a=max length (mm), b=minimum width (mm). 8. The distribution of the Ti02-x@GL in tumor tissue

To prove the TiO2-x@GL can penetrate barrier and can accumu- late in the tumor tissue, the distribution of the Ti02-x and

TiO2-xQGL of 5 tumor-bearing mice are measured. 4T1 female tumor- bearing mice are randomly divided into three groups, which are in- jected intravenously in normal saline, Ti02-x and Ti02-x@GL at a dose of 50ppm. Killing the mice after injecting for 4h, and dis- secting and collecting the tumor tissue. Tumor tissue is weighed, homogenized, and dissolved in aqua regia. Then an inductively cou- pled plasma optical emission spectrometer (ICP-OES) is used to de- termine the content of Ti in tumor tissue, and the distribution is calculated based on the original weight per gram of tissue. 9. The pathological change, cell apoptosis and cell prolifer- ation after different treatments

Adopting hematoxylin-eosin (HE) staining, tunel experiment and

Ki67 antibody to detect the pathological change, cell apoptosis and cell proliferation respectively. HE staining is performed ac- cording to previous research reports and tunel staining is per- formed using a commercial test kit according to the manufacturer's instructions. Ki67 antibody combined with goat anti-rabbit second antibody is used for immunchistochemical staining to show cell proliferation. 10. Statistic analysis

The data is represented with mean + standard deviation (Sd), the difference between the two groups are detected with two-sided t test (*, p< 0.05;**, p < 0.01;***, p < 0.001).

The experimental results 1. The represent of the Ti02-x NPs and TiO2-x@GL NPs

Observing the form of Ti02-xNPs and Ti02-x@GL NPs using a transmission electron microscope, the results are shown in figure a of figure 1, TiO2-x NPs is the uniform spherical structure with the average size of 50nm. After adding GL, there is no obvious changes of the morphology of the prepared Ti02-xQ@GL NPs compared with the Ti02-x NPs (figure b in figure 1). From the high- resolution TEM imaging results of the Ti02-x NPs and TiO2-x@GL NPs (figures c and d of figure 1) and the corresponding SAED images (figures e and g of figure 1), the crystal structure of Ti0Q2-x

NPs is not changed by the coating of GL. The result of DLS shows that the coating of GL makes the hydration particle size of the

TiO2-x Nps from 122.4nm to 164.2nm (figure g and h of figurel).

SEM images (figure a and b of figure 2) and EDS results ( figure c of figure 2, figure d and figure S1 of figure 2) show that, compared with the TiO2-x NPs group, the nitrogen{N) content in Ti02-x@GL NPs group increases remarkably, which indicates that

GL is successfully loaded onto the surface of the Ti02-x NPs. Alt- hough the Z potential changes before and after GL encapsulation {from -36.1 to -9.1mV) (figure e of figure 2), in the XRD measure- ment, the crystal peaks of TiO2-x NPs and Ti02-xQ@GL NPs match very well with the standard crystal structure of TiO2-x NPs (JCPDS

No.21-1272), which indicates the structure of the Ti02-x NPs is preserved well after the surface encapsulation of Gl (figure f of figure 2). In addition, from the UV-Vis-NIR optical absorption curve (figure g of figure 2) and the photo-thermal heating curve (figure g of figure 2) of the Ti02-xNPs and Ti02-xlGL NPs, the NIR wave-absorbing ability and photo-thermal performance is no effect- ed, which indicates the Ti02-x NPs and Ti02-x@GL NPs can be used to treat tumors by PTT. 2. In-vitro acoustic dynamic and photo-thermal effects of the

Ti02-x@GL NPs

The ROS generation can be effected by many factors such as the ultrasonic power, duration and concentration of acoustic sen- sitizer. First, the ROS generation increases with the increasing of the ultrasonic strength, therefore, the ultrasound duration is set at 180s and the Ti02-z@GL NPs concentration is 50ppm (figure 3a). To determine the effect of 40khz ultrasound on ROS genera- tion, the ROS generation at a concentration of 50 ppm Ti02-x@GL

NPs is measured (figure 3b). Continuous release of INS from the

Ti02-x@GL NPs is achieved by ultrasonic irradiation of 1.0W/cm2.

As shown in figure 3, the ROS generation slightly increases with the increasing of nano-particle concentration in the condition of treating 180s by a 1.0W/cm2 ultrasound. On the contrary, the Ti02- x@GL NPs will inhibit the cells growth (<75%) in high concentra- tion (>100 ppm). According to the previous and above results, an optimized program (the Ti02-x@GL NPs with 50ppm, under 1.0W/cm2 ultrasonic intensity for 180s) to achieve the high level of ROS generation. In addition, further evaluating the photo-thermal properties of the TiO2-xEGL NPs at different concentration (0, 6.25, 12.5, 25, 50, 100 and 300ppm) under 1.5W/cm2. The study has found that the temperature can reaches to 59.2°C when the concen- tration is 300ppm, which is enough to kill the cancer cells through heat. The photo-thermal effects associated with laser pow- er (0, 0.5, 1.0 and 1.5W/cm2) are also demonstrated (figure 3f). 3. In-vitro synergistic cancer therapy based on SDT/PTT

In the study scheme, 4T1 cells are treated with SDT and PTT respectively (sequential processing). Specifically, 4T1 cancer cells are incubated with the TiO2-x@GL NPs, and is processed for 4h in the irradiation of ultrasound and NIR-II laser. The ultra- sonic parameters are set as 1MHz and 50% duty ratio, 1.0W/cm2, 240s. The power of the NIR-II laser is set as 1.5W/cm2. The study has found that when the concentration of the Ti0O2-x@GL NPs is 300ppm, the temperature increases rapidly, reaching as high as 66°C, which is enough to kill the cancer cells through heating therapy (figure 4a), and when the laser is closed, the temperature decreases rapidly. What is worth to be noted is under the irradia- tion of the NIR-laser, the extremely high temperature produces ob- vious ablation and smoke inhalation phenomenon. In the three laser open/close periods, the photo-thermal performance of the Ti02-x@GL

NPs has no obvious reduction, which indicates the high illumina- tion thermal stability of the Ti02-x for photo-thermal hyperther- mia (figure 4b). The CCK-8 experiment is to investigate the in- vitro killing effect of the Ti02-x@GL NPs on 4T1 cells treated with 1,064nm laser, ultrasound, and laser + ultrasound. In the SDT and PTT treatment groups, after the Ti02-x@GL NPs is incubated with cancer cells for 4h, the survival rate decreases to 19.2% {figure 4c). In addition, after various treatments, the killing effect is observed directly through CLSM, the living cells and dead cells are stained with fluorexon-am (green) and PI (red). A large amount of dead cells are observed in the Ti02-x@GL

NPs+ultrasound+laser treatment group, which indicates SDT and PTT synergistic effect caused widespread apoptosis and death of cells (figure 4d). From the average contribution rate of inter-regional differences, the contribution rate of the Ti02-x@GL NPs is great with an average of 44.95%. The variance contribution rates of US and NIR are 23.33% and 31.72% respectively (figure 4e). Ti02 can inhibit the metastasis of tumor cells as the same with previous study results. Cell metastasis experiment shows that the number of living cells in Ti02-x@GL NPs and TiO2-x groups are significantly lower than that in contrast groups. This means the Ti02-x@GL NPs also can inhibit the metastasis of tumor cells as Ti02 (figure 41). 4. Verification of in-vivo SDT/PTT synergistic therapy for cancer

The synergistic therapy scheme is shown in figure 5a, the es- tablishment of tumor-bearing model is recorded as day -7, after injecting TiC2-x@GL NPs (abbreviated as Ti02-x@GL) for 4h, laser irradiation and ultrasound are used and recorded as day 0, and the ultrasonic irradiation is continued in day 3 and day 5, the con- centration of Ti in tumor tissue increases remarkably in the group of injecting TiO2-X or Ti02-z@GL groups, and GL makes the contents of Ti02-x@GL increase nearly 3 times compared with the accumula- tion of the TiO2-x (figure 5b). There is no obvious changes in weight of mice in each group during the therapy (figure 5c). The tumor weight in TiO2-x{@GL+laser+ultrasound group is significant lower than (P<0.01) that of Ti02-x@GL group, and is significant lower than (P<0.05) that of Ti02-x@GL+laser or

Ti02-x@GL+ultrasound group (figure 5d) in day 15. Meanwhile, the tumor weight in TiO2-x@GL+laser+ultrasound group has no increase (figure 5e), so synergistic therapy contributes to prolong surviv- al without death (figure 5f). HE staining shows that obvious path- ologic changes, mainly is swelling of tumor cells, appear in

Ti02-x@GL+lasertultrasound group. Tunel detection shows that tumor cell apoptosis increases in TiO2-x@GL+laser+ultrasound group. The location of Ki67 antibody shows that the tumor cell proliferation is reduced. All evidence shows that Ti02-x@GL+laser+tultrasound can inhibit tumor in-vivo. 5. The safety evaluation of the TiO2-x@GL NPs

The safety evaluation of the Ti02-x@GL NPs is showed through the pathological changes of vital organs, critical blood and bio- chemical parameters. HE staining shows that heart, liver, kidney and lung have no obvious changes, and there are no significant changes in white blood cells (WBC), platelets (PLT) and hemoglo- bin (HGB) on day 1, day 7 and day 28 among different groups. At the same time, biochemical indexes such as blood urea nitrogen (BUN),

C-reactive protein (Cr) and globulin(GLB) also have no changes com- pared with the contrast group.

Although the preferred examples have been described, addi- tional alterations and modifications on these examples can be made once the technicians in the field knows the basic creative con- cepts. Whereby, the attached claims are intended to be construed to include the preferred examples and all alterations and modifi- cations falling within the scope of the invention.

Obviously, various alterations and modifications on the in- vention can be made by the technicians of the field without de- parting from the spirit and scope of the invention. In this way, if these alterations and modifications belong to the scope of the claims and equivalent thereof of the invention, the invention is also intended to include such alterations and modifications.

Claims

CONCLUSIONS

A method for preparing TiO2-x@GL NPs, characterized in that it comprises the following steps:

S1. Preparation of TiO2-x NPs: using titanium dioxide and aluminum as raw materials to synthesize TiO2-x NPs by aluminum reduction method;

S2. Prepare TiO2-x0GL NPs: Obtain TiO2-z@GL NPs by modifying the surface of TiO2-x NPs with glutamine.

A method for preparing TiO2-x@GL NPs in claim 1, characterized in that, in the 51, the specific process of the aluminum reduction method is: aluminum melts when heated at a temperature of 700-800° C, and is heated and reduced at a temperature of 300-500°C for 6-10 hours after mixing with the TiO2 sample to obtain 500°C-Al-TiO2-x, which is heated in argon for 10-12 hours at temperature of 800-900°C is annealed to obtain TiO2-x NPs.

3. Method for preparing TiO2-x@GL NPs, characterized in that, in the S2, the surface modification of TiO2-x NPs by glutamine is: 40-50mg glutamine in 8-10mL TiO2-x NP- solution with the mass concentration of 1-1.5 mg/mL, then treated with 120W ultrasound in an ice bath for 3-5h, and centrifuged three times for 10min with a rotation of

10,000 r/min, with the TiO2-x@GL NPs obtained by removing and washing the ionized water.

A method for preparing TiO2-x@GL NPs in claim 3, characterized in that, in the S2, the surface modification of TiO2-x NPs by glutamine is: 50mg glutamine in 10mL TiO2-x NP solution with the mass concentration of 1mg/mL, then treated with 120W ultrasound in an ice bath for 4 hours, and centrifuged three times for 4 hours with a rotation of

10,000 rpm, where the Tio2-x@GL NPs are obtained by removing and washing the ionized water.

The method for preparing TiO2-x@GL NPs in claim 4, characterized in that the hydration particle size of the TiO2-x@GL NPs is 164.2nm.

Use of the TiO2-xQGL NPs obtained by the method according to any one of claims 1-5 in the preparation of tumor drugs.

Use according to claim 5, characterized in that the Ti0Q2-x@GL NPs can be used to prepare the tumor cell metastasis inhibitor.

Use according to claim 5, characterized in that the TiO 2 -x@GL NPs can be used to prepare the tumor cell proliferation inhibitor.

Use according to claim 5, characterized in that the TiO 2 -x@GL NPs can be used to prepare the laser and ultrasonic tumor inhibition synergist.

Use according to claim 5, characterized in that the photothermal performance at a concentration of 300ppm of the TiO 2 -x@GL NPs can kill the cancer cells.