Preparation method and application of targeted pancreatic cancer immune checkpoint diagnosis and treatment integrated nanoprobe

Abstract

The invention relates to a preparation method and application of a targeted pancreatic cancer immune checkpoint diagnosis and treatment integrated nanoprobe, which can realize integration of sonodynamic therapy and MRI imaging of pancreatic cancer cells with high expression of VISTA, realize targeted SDT therapy of pancreatic cancer and visual evaluation of the therapeutic effect, have good biological safety, and can be applied to diagnosis and treatment of pancreatic cancer cells with high expression of VISTA. SDT visual treatment of targeted pancreatic cancer is realized at a cellular level, and SDT combined immunotherapy of pancreatic cancer is realized at an animal level. The method comprises the following steps: (1) preparing Fe3O4 nanoparticles by using a solvothermal method; (2) preparing Fe3O4 (at) SiO2 nano particles; (3) preparing Fe3O4 (at) SiO2 (at) TiO2 nano particles; (4) preparing Fe3O4 (at) TiO2 by adopting an alkaline hydrothermal corrosion auxiliary crystallization method; and (5) modifying the nanoprobe with a VISTA antibody through an electrostatic adsorption method, and finally successfully constructing the garland-shaped nanoprobe.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection and medical image processing, and in particular relates to a method for preparing a nanoprobe for diagnosis and treatment of pancreatic cancer targeting immune checkpoints, and the use of the nanoprobe in preparing drugs for treating pancreatic cancer. Background Art

[0002] Pancreatic cancer is a malignant tumor of the digestive tract that develops insidiously, progresses rapidly, and has extremely poor treatment effects and prognosis. It is more common in people over 40 years old and is more common in men than in women. Currently the fourth most common cause of death from cancer, the 5-year survival rate of pancreatic cancer is less than 10%. Surgical resection is an effective treatment for pancreatic cancer, but due to the difficulty in early detection of pancreatic cancer and the lack of effective screening indicators, the vast majority of patients are already in the middle or late stages when diagnosed and lose the opportunity for surgery. In addition, radiotherapy and chemotherapy are ineffective, making it one of the malignant tumors with the worst prognosis. Therefore, there is an urgent need to find new diagnosis and treatment options for pancreatic cancer.

[0003] In recent years, immunotherapy has become a new breakthrough in tumor treatment and can improve the therapeutic efficacy of some tumors. The main methods include: immune checkpoint therapy, oncolytic virus therapy, tumor vaccine therapy, etc., among which immune checkpoint therapy has its unique advantages. Immune checkpoints are molecules that play a protective role in the human immune system and can prevent inflammatory damage caused by excessive activation of T cells. However, tumor cells can overexpress immune checkpoints, thereby escaping immune surveillance and killing and promoting their own growth. Immune checkpoint therapy (ICT) is to inhibit the activity of this molecule and reactivate the immune response of T cells to tumors, thereby achieving the purpose of anti-tumor. Recent studies have found that the effect of ICT alone in the treatment of pancreatic cancer is not good. The primary reason is that there is a large amount of dense fibrous matrix around pancreatic cancer, which limits the penetration and function of targeted drugs. At the same time, the fibrous matrix is ​​also an important carrier of the immune escape mechanism of pancreatic cancer. Therefore, how to construct a new drug delivery platform, break through the matrix barrier, increase the drug concentration in the tumor area, reverse the immune microenvironment, and more effectively play the role of immune checkpoint inhibitors (ICIs) is the difficulty and hot spot of current pancreatic cancer immunotherapy. Summary of the invention

[0004] In order to overcome the defects of the prior art, the technical problem to be solved by the present invention is to provide a method for preparing a nanoprobe for integrated diagnosis and treatment of pancreatic cancer immune checkpoints, which can realize the integration of sonodynamic therapy and MRI imaging of pancreatic cancer cells with high expression of VISTA (V-domain Ig suppressor of Tcell activation, V-domain immunosuppressor of T cell activation, which is an immune checkpoint specifically highly expressed in pancreatic cancer), realize targeted SDT (Sonodynamic therapy) treatment for pancreatic cancer, and perform visual evaluation of the treatment effect at the same time. The nanoprobe has good biosafety, realizes SDT visualization treatment of targeted pancreatic cancer at the cellular level, and realizes SDT combined with immunotherapy of pancreatic cancer at the animal level.

[0005] The technical solution of the present invention is: a method for preparing a nanoprobe for diagnosis and treatment of pancreatic cancer targeting immune checkpoints, comprising the following steps:

[0006] (1) Preparation of Fe3O4 nanoparticles using a solvothermal method;

[0007] (2) Preparation of Fe3O4@SiO2 nanoparticles: TEOS was hydrolyzed under alkaline conditions as a silicon source to coat the surface of Fe3O4 with silicon dioxide to construct Fe3O4@SiO2 nanoparticles;

[0008] (3) Preparation of Fe3O4@SiO2@TiO2 nanoparticles: Based on the hydrolysis and condensation of TBOT, titanium dioxide TiO2 was coated on the surface of Fe3O4@SiO2 to synthesize Fe3O4@SiO2@TiO2 nanoparticles;

[0009] (4) Fe3O4@TiO2 was prepared by alkaline hydrothermal corrosion assisted crystallization method;

[0010] (5) The VISTA antibody was modified on the nanoprobe by electrostatic adsorption, and finally the wreath-shaped Fe3O4@TiO2@VISTAmAb nanoprobe was successfully constructed.

[0011] The present invention prepares Fe3O4 nanoparticles by a solvothermal method, modifies SiO2 layers and TiO2 layers, adsorbs VISTAmAb after etching the SiO2 layer, constructs a wreath-shaped Fe3O4@TiO2@VISTAmAb nanoprobe, and characterizes the probe by biological transmission electron microscopy, magnetic resonance imaging, ultraviolet spectrophotometer, etc., confirming that the probe can realize the integration of sonodynamic therapy and MRI imaging of pancreatic cancer cells with high expression of VISTA, realize targeted SDT treatment of pancreatic cancer, and perform visual evaluation of the treatment effect. The nanoprobe has good biosafety, realizes SDT visualization treatment of targeted pancreatic cancer at the cellular level, and realizes SDT combined with immunotherapy of pancreatic cancer at the animal level.

[0012] Also provided is a Fe3O4@TiO2@VISTAmAb nanoprobe for use in the preparation of drugs for treating pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Biological transmission electron microscopy images of Fe3O4@TiO2 nanoprobes. Biological transmission electron microscopy images of the products in the synthesis steps (a) TEM images of Fe3O4 (b) Fe3O4@SiO2 (c) Fe3O4@SiO2@TiO2, TEM images of Fe3O4@TiO2 (d, e).

[0014] Figure 2 The figure is a Western Blot diagram of VISTA expression in pancreatic cancer cell lines. The VISTA expression levels of Miapaca-2 pancreatic cancer cells, hTERT-HPNE normal pancreatic ductal epithelial cells, Panc-2 pancreatic cancer cells, and Panc-1 pancreatic cancer cells were detected by Western Blot.

[0015] Figure 3 The present invention is a flow chart of a method for preparing a nanoprobe for integrated diagnosis and treatment of pancreatic cancer targeting immune checkpoints. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0017] In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description of the implementation and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The implementation covers the features of multiple specific embodiments and the method steps and sequences used to construct and operate these specific embodiments. However, other specific embodiments can also be used to achieve the same or equivalent functions and step sequences.

[0018] like Figure 3 As shown, a method for preparing a nanoprobe for diagnosis and treatment of pancreatic cancer targeting immune checkpoints comprises the following steps:

[0019] (1) Preparation of Fe3O4 nanoparticles using a solvothermal method;

[0020] (2) Preparation of Fe3O4@SiO2 nanoparticles: TEOS was hydrolyzed under alkaline conditions as a silicon source to coat the surface of Fe3O4 with silicon dioxide to construct Fe3O4@SiO2 nanoparticles;

[0021] (3) Preparation of Fe3O4@SiO2@TiO2 nanoparticles: Based on the hydrolysis and condensation of TBOT, titanium dioxide TiO2 was coated on the surface of Fe3O4@SiO2 to synthesize Fe3O4@SiO2@TiO2 nanoparticles;

[0022] (4) Fe3O4@TiO2 was prepared by alkaline hydrothermal corrosion assisted crystallization method;

[0023] (5) The VISTA antibody was modified on the nanoprobe by electrostatic adsorption, and finally the wreath-shaped Fe3O4@TiO2@VISTAmAb nanoprobe was successfully constructed.

[0024] The present invention prepares Fe3O4 nanoparticles by a solvothermal method, modifies SiO2 layers and TiO2 layers, adsorbs VISTAmAb after etching the SiO2 layer, constructs a wreath-shaped Fe3O4@TiO2@VISTAmAb nanoprobe, and characterizes the probe by biological transmission electron microscopy, magnetic resonance imaging, ultraviolet spectrophotometer, etc., confirming that the probe can realize the integration of sonodynamic therapy and MRI imaging of pancreatic cancer cells with high expression of VISTA, realize targeted SDT treatment of pancreatic cancer, and perform visual evaluation of the treatment effect. The nanoprobe has good biosafety, realizes SDT visualization treatment of targeted pancreatic cancer at the cellular level, and realizes SDT combined with immunotherapy of pancreatic cancer at the animal level.

[0025] Preferably, in the step (1), 1.62 g of ferric chloride hexahydrate, 1.625 g of sodium citrate and 3.0 g of sodium acetate are added to 50 mL of ethylene glycol and dissolved under magnetic stirring at 50° C. The obtained clear yellow solution is transferred and sealed in a Teflon liner with a capacity of 100 mL, placed in a stainless steel autoclave, heated in an oven at 180° C. for 12 h, then cooled to room temperature, and the black product is collected with a magnet, and washed 3 times with deionized water and ethanol respectively.

[0026] Preferably, in the step (2), 45 mg of Fe3O4 nanoparticles are added to 80 mL of anhydrous ethanol, 16 mL of deionized water and 2.0 mL of 28 wt% concentrated ammonia water and mixed in a three-necked round-bottom flask, ultrasonicated for 15 min, 0.4 mL of TEOS is slowly added dropwise within 5 min, and the mixture is reacted for 10 h at room temperature with continuous mechanical stirring at 150 rpm. The Fe3O4@SiO2 nanoparticles with core-shell structure are separated and collected by a magnet, and then washed three times with deionized water and ethanol, respectively.

[0027] Preferably, in the step (3), 150 mg of the prepared Fe3O4@SiO2 nanoparticles are redispersed in 200 mL of anhydrous ethanol, mixed with 0.9 mL of a 28 wt% concentrated ammonia solution, ultrasonicated for 15 min, 2.0 mL of TBOT is added dropwise within 5 min, and the mixture is reacted at 45°C under continuous mechanical stirring for 24 h. The product is separated and collected with a magnet, and then washed three times with deionized water and ethanol, respectively.

[0028] Preferably, in the step (4), 50 mg of the prepared Fe3O4@SiO2@TiO2 is ultrasonically treated for 5 minutes by an ultrasonic cell crusher, then mixed with 10 mL of a 1.0 M sodium hydroxide aqueous solution and added to a 20 mL polytetrafluoroethylene-lined stainless steel autoclave, the autoclave is heated in an oven at 150° C. for 6 hours, then cooled to room temperature, the obtained product is soaked in 20 mL of a 0.1 M hydrochloric acid aqueous solution for 30 minutes, then washed with deionized water until the pH value is close to 7, and the nanoparticles are dispersed in deionized water.

[0029] Preferably, in step (5), the immune checkpoint inhibitor VISTAmAb is loaded by physical adsorption, Fe3O4@TiO2 and VISTAmAb are mixed in sterile water at a mass ratio of 10:1, and the mixture is mechanically stirred at 250 rpm for 12 hours and then centrifuged to obtain the Fe3O4@TiO2@VISTAmAb nanoprobe.

[0030] Preferably, the prepared Fe3O4@TiO2@VISTAmAb nanoprobe has an average hydrated particle size of 225.3 nm, a Zeta potential of -28.9 mV, and an r2 / r1 value of 32.03.

[0031] Also provided is a Fe3O4@TiO2@VISTAmAb nanoprobe for use in the preparation of drugs for treating pancreatic cancer.

[0032] Preferably, US irradiation and ultrasonic radiation are performed based on the probe to loosen the dense fibrous matrix of pancreatic cancer, promote tumor microangiogenesis, and improve the immunosuppressive microenvironment.

[0033] Preferably, images are acquired by magnetic resonance imaging (MRI) and applied in the preparation of visualization products for early screening of pancreatic cancer.

[0034] The following is a detailed description of embodiments of the present invention.

[0035] Example 1: Preparation and characterization of Fe3O4@TiO2@VISTAmAb probe

[0036] TiO2 has a large energy band gap of about 3.2eV and is a typical inorganic sonosensitizer. Its electrons can jump from the valence band to the conduction band under ultraviolet irradiation. This helps to generate free radicals, including peroxyl radicals and hydroxyl radicals, as well as 1 O2, under the action of ultrasound, the ROS yield is as high as 60%, which is a nanomaterial with excellent SDT function. In order to make TiO2 nanoparticles have the ability to target pancreatic cancer cells, this study selected anti-VISTA antibody (VISTAmAb) to modify the TiO2 surface. VISTA is a protein that is a specific and highly expressed immune checkpoint on cancer cells and stromal cells of pancreatic cancer. It is one of the key targets in the inhibitory immune microenvironment and immune escape of pancreatic cancer. At the same time, Fe3O4 nanoparticles are selected as the core to give the nanoprobe MR imaging function. Therefore, this embodiment designs a nanoprobe with SDT effect and immune activation ability to achieve the effect of MR visualization therapy.

[0037] In this embodiment, an ultrasound-responsive nanoprobe Fe3O4@TiO2@VISTAmAb was constructed to achieve precisely targeted SDT and immunotherapy for pancreatic cancer. Using Fe3O4 nanoparticles as the basic unit, Fe3O4@TiO2 was formed by coating SiO2 and TiO2, etching SiO2, and loading VISTAmAb to prepare a Fe3O4@TiO2@VISTAmAb nanoprobe with a double-layer core-shell structure. Targeting to pancreatic cancer with high expression of VISTA, promoting the phagocytosis of pancreatic cancer cells, and stimulating TiO2 particles to produce ROS under ultrasonic irradiation, achieving the SDT effect while promoting the activation of immune response, and at the same time, Fe3O4 can achieve T2 imaging effect in MR. Finally, MRI-guided SDT and immune synergistic therapy are achieved.

[0038] Preparation of nanoprobes

[0039] (1) Preparation of Fe3O4 nanoparticles

[0040] 1.62g of ferric chloride hexahydrate, 1.625g of sodium citrate and 3.0g of sodium acetate were added to 50mL of ethylene glycol and dissolved under magnetic stirring at 50°C. The obtained clear yellow solution was transferred and sealed in a 100mL Teflon liner and placed in a stainless steel autoclave. Heat in an oven at 180°C for 12h and then cool to room temperature. The black product was collected with a magnet and washed 3 times with deionized water and ethanol respectively.

[0041] (2) Preparation of Fe3O4@SiO2 nanoparticles

[0042] TEOS was hydrolyzed under alkaline conditions as a silicon source to coat the surface of Fe3O4 with silicon dioxide (SiO2) to construct Fe3O4@SiO2 nanoparticles. 45 mg of Fe3O4 nanoparticles were added to a three-necked round-bottom flask containing a mixture of 80 mL of anhydrous ethanol, 16 mL of deionized water, and 2.0 mL of concentrated ammonia (28 wt%), and ultrasonicated for 15 min. 0.4 mL of TEOS was slowly added dropwise within 5 min, and the reaction was carried out at room temperature for 10 h under continuous mechanical stirring (150 rpm). The core-shell Fe3O4@SiO2 nanoparticles were separated and collected by magnet, and then washed 3 times with deionized water and ethanol, respectively.

[0043] (3) Preparation of Fe3O4@SiO2@TiO2 nanoparticles

[0044] Based on the hydrolysis and condensation of TBOT, Fe3O4@SiO2@TiO2 nanoparticles were synthesized by coating titanium dioxide (TiO2) on the surface of Fe3O4@SiO2. The prepared Fe3O4@SiO2 nanoparticles (150 mg) were redispersed in anhydrous ethanol (200 mL), mixed with concentrated ammonia solution (0.9 mL, 28 wt%), and ultrasonicated for 15 min. 2.0 mL of TBOT was added dropwise within 5 min, and the mixture was reacted at 45 ° C for 24 h under continuous mechanical stirring. The product was separated and collected by a magnet, and then washed 3 times with deionized water and ethanol, respectively.

[0045] (4) Preparation of Fe3O4@TiO2 nanoparticles

[0046] Fe3O4@TiO2 was prepared by alkaline hydrothermal corrosion assisted crystallization. 50mg of Fe3O4@SiO2@TiO2 was ultrasonically treated for 5min by ultrasonic cell crusher, mixed with 10mL of sodium hydroxide aqueous solution (1.0M) and added to a 20mL polytetrafluoroethylene-lined stainless steel autoclave, which was heated in an oven at 150℃ for 6h and then cooled to room temperature. The obtained product was soaked in 20mL of hydrochloric acid aqueous solution (0.1M) for 30min, then washed with deionized water until the pH value was close to 7, and the nanoparticles were dispersed in deionized water.

[0047] (5) Synthesis of Fe3O4@TiO2@VISTAmAb nanoprobe

[0048] The immune checkpoint inhibitor VISTAmAb was loaded by physical adsorption. Specifically, Fe3O4@TiO2 and VISTAmAb were mixed in sterile water at a mass ratio of 10:1, and mechanically stirred (250 rpm) for 12 hours and then centrifuged to obtain the Fe3O4@TiO2@VISTAmAb nanoprobe.

[0049] Characterization of the physicochemical properties of nanoprobes

[0050] The particle size and microscopic morphology of the nanoprobes were observed by transmission electron microscope (TEM); the distribution of analytical elements of the nanoprobes was understood by energy dispersive spectrometry (EDS); the hydrated particle size distribution and Zeta potential of the nanoprobes were tested by Nano ZS (Malvern Instruments Ltd, UK). The ultraviolet-visible near-infrared absorption spectra of the nanoprobes were analyzed by spectrophotometer; the infrared spectral characteristics of the nanoprobes were studied by Micro-Fourier transform infrared spectroscopy (micro-FTIR) and the loading of VISTAmAb on the nanoprobes were analyzed; the crystal phase analysis of the nanoprobes was performed by X-ray diffraction (XRD); the MRI imaging system with a magnetic field strength of 0.5T was used to study the MR imaging and relaxation properties of the nanoprobes; and the nanoprobes were ultrasonically excited by an ultrasonic therapeutic device.

[0051] Nanoprobe acoustic dynamic performance detection

[0052] Using singlet oxygen ( 1The photodynamic properties of Fe3O4, Fe3O4@bTiO2, and Fe3O4@TiO2@VISTAmAb materials under 808nm and 660nm laser irradiation were detected by using 1,3-diphenylisobenzofuran (DPBF), a photosensitive agent. Under the excitation of laser, the nanomaterial containing photosensitizer can produce 1 O2, DPBF reacts with it and the fluorescence intensity is weakened. Therefore, this can be used to indirectly react to the material to produce 1 100 μg / mL P25 and Fe3O4@TiO2@VISTAmAb solutions were prepared in ethanol, 100 μL was placed in 1 mL quartz dishes, 20 μL DPBF (1 mg / mL) and 880 μL deionized water were added, and ultrasonic irradiation (1.0 W / cm 2 , 1min, duty cycle 50%) three groups of materials, every 1min to detect the fluorescence intensity with a fluorescence spectrometer, irradiation for a total of 8min; get the emission spectrum in the range of 300 ~ 600nm, take the fluorescence intensity value of each group at 425nm.

[0053] Magnetic resonance properties of nanoprobes

[0054] The synthesized Fe3O4@TiO2@VISTAmAb nanoprobe was scanned and tested using a 0.5T magnetic resonance imaging system to evaluate its MRI imaging and MR relaxation performance. First, the iron ion concentration in Fe3O4@TiO2@VISTAmAb was determined using ICP, and Fe3O4@TiO2@VISTAmAb was prepared into solutions with different iron ion concentrations (0.05mM, 0.10mM, 0.15mM, 0.20mM, 0.25mM) and added to a 1.5mL centrifuge tube. The T1 and T2 relaxation times were obtained by scanning with a 0.5T magnetic resonance scanner, and the corresponding relaxation rate fitting curves were drawn and analyzed. In addition, T2-weighted image scans were performed on Fe3O4@TiO2@VISTAmAb nanoprobes with different concentrations. The parameters were set as follows: T2 spin echo sequence, TR = 2000.00 ms, TE = 40.00 ms, FOV = 100 mm × 100 mm, slice thickness = 2.0 mm, 0.5 T.

[0055] Preparation and characterization of physicochemical properties of nanoprobes

[0056] Fe3O4 is a T2-weighted MRI contrast agent. First, FeCl3·6H2O was used as raw material to prepare magnetic Fe3O4 nanoparticles by solvothermal method, TEOS was used as silicon source to modify silicon dioxide (SiO2) on the surface to form Fe3O4@SiO2, and TBOT was used as titanium source to coat titanium dioxide (TiO2) on the surface of Fe3O4@SiO2 to synthesize Fe3O4@SiO2@TiO2. The middle SiO2 was removed by sodium hydroxide hydrothermal etching, and the outer layer TiO2 continued to grow in an alkaline environment to form a wreath-shaped Fe3O4@TiO2 nanoprobe. VISTAmAb is an immune checkpoint inhibitor that can be loaded onto Fe3O4@TiO2 by electrostatic adsorption. VISTAmAb was adsorbed onto the nanoprobe to construct a wreath-shaped Fe3O4@TiO2@VISTAmAb nanoprobe, and the synthesized nanoparticles had good dispersibility. The TEM results of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@TiO2, and Fe3O4@TiO2 nanoprobes show that Figure 1 (ae), the particle size of the final prepared nanoprobe is about 120 nm.

[0057] The hydrated particle size and Zeta potential of different nanoprobes were tested by dynamic light scattering (DLS) and Zeta potential technology. The average hydrated particle sizes of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@TiO2, Fe3O4@TiO2, and Fe3O4@TiO2@VISTAmAb were 179.8nm, 201.3nm, 213.8nm, 200.9nm, and 225.3nm, respectively. This shows that with the addition of related components during the modification process, the particle size of the nanoprobe gradually increases, but the particle size of the final synthesized probe still meets the needs of precise treatment of biological tumors. The Zeta potentials of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@TiO2, Fe3O4@TiO2, VISTAmAb, and Fe3O4@TiO2@VISTAmAb were approximately -19.88mV, -35.34mV, -35.34mV, -43.47mV, -38.64mV, 0.7647mV, and -28.9mV, respectively. The final synthesized Fe3O4@TiO2@VISTAmAb nanoprobe has a negative potential that is beneficial for circulation in vivo.

[0058] In order to further analyze the structure of Fe3O4@TiO2 nanomaterials, the crystal structure of Fe3O4@TiO2 was analyzed by powder X-ray diffraction (XRD). Fe3O4 corresponds to multiple crystal planes of magnetite Fe3O4, such as (220), (331), (400), (511) and (440). TiO2 corresponds to the (200) crystal plane.

[0059] VISTAmAb, Fe3O4@TiO2, and Fe3O4@TiO2@VISTAmAb were detected by Micro-FTIR. After adsorbing VISTAmAb protein, Fe3O4@TiO2@VISTAmAb showed a higher fluorescence intensity at 1750cm-1 than Fe3O4@TiO2. -1 A new characteristic peak appeared at the position, which was consistent with the peak of VISTAmAb, proving that VISTAmAb was successfully adsorbed on Fe3O4@TiO2 and the Fe3O4@TiO2@VISTAmAb nanoprobe was successfully synthesized.

[0060] Results of the evaluation of the sonodynamic performance of the nanoprobe

[0061] The reactive oxygen generation rate of Fe3O4@TiO2@VISTAmAb was tested by UV-visible spectrophotometer: the generation of reactive oxygen was determined by DPBF. The results showed that with the increase of ultrasound time, the ultraviolet absorption peak of DPBF at 425nm gradually decreased, and it could be reduced to 70% of the original level after 8 minutes of ultrasound. Compared with commercial P25 (TiO2), this material has better sonodynamic effect.

[0062] Results of MRI performance evaluation of nanoprobes

[0063] The magnetic resonance performance of the nanoprobe was evaluated by a 0.5T MRI scanner. At the same Fe3O4 concentration, the longitudinal relaxation rate (r1) value of the Fe3O4@TiO2@VISTAmAb nanoprobe was 1.57 Mm -1 s -1 The transverse relaxation rate (r2) value of Fe3O4@TiO2@VISTAmAb nanoprobe is 50.28Mm -1 s -1 The r2 / r1 value of the Fe3O4@TiO2@VISTAmAb nanoprobe is about 32.03, indicating that the nanoprobe is a good MRI T2 contrast agent. The dark signal of T2 of the Fe3O4@TiO2@VISTAmAb nanoprobe increases with the increase of concentration.

[0064] The experimental results of material performance research show that the average hydrated particle size of the prepared nanoprobe is about 225.3nm, the Zeta potential is -28.9mV, and the negative charge on the surface is more conducive to long-term blood circulation. The longitudinal relaxation rate r2 value of the nanoprobe is about 50.28Mm -1 s -1 , r2 / r1 value is 32.03, and it is an excellent T2 MRI contrast agent. The results of sonodynamic performance show that Fe3O4@TiO2@VISTAmAb has a good sonodynamic effect under ultrasound irradiation, and the production of singlet oxygen increases with the increase of irradiation time and irradiation power. These research results show that Fe3O4@TiO2@VISTAmAb nanoprobe has good SDT potential under ultrasound irradiation and is expected to be used in the treatment of pancreatic cancer.

[0065] Example 2: Study on sonodynamic therapy of pancreatic cancer cells using nanoprobes

[0066] SDT is a very promising non-invasive treatment method. Compared with PTT and PDT, SDT has many unique advantages, including deeper tissue penetration, high precision, low side effects and good patient compliance. Due to the continuous development of nanotechnology, nanoparticle-based sonosensitizers have shown significant advantages in improving SDT efficacy, binding affinity and targeting specificity.

[0067] Ultrasonic cavitation caused by SDT induces free radicals and 1 The generation of O2 has a killing effect on cells. The rupture of cavitation microbubbles will produce strong shock waves, local high temperature and high pressure, and free radicals. These adverse physical conditions will cause damage to the cytoskeleton and cell membrane and ultimately lead to cell death. In addition, in the absence of sonosensitizers, the physical and mechanical effects of US play a more important role in destroying and killing cells.

[0068] In summary, SDT kills cells by producing ROS, thereby achieving the purpose of treating tumors. In this example, the successfully prepared Fe3O4@TiO2@VISTAmAb nanoprobe with good SDT effect was used in the treatment of pancreatic cancer cells stimulated by ultrasound, and its therapeutic effect was evaluated. The experimental method is as follows:

[0069] 1. Cell culture

[0070] Miapaca-2 human pancreatic cancer cell line, hTERT-HPNE human pancreatic ductal epithelial cell line, Panc-2 mouse pancreatic cancer cell line, and Panc-1 human pancreatic cancer cell line were obtained from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Miapaca-2, hTERT-HPNE, Panc-2 cell lines, and Panc-1 cell lines were cultured in complete DMEM medium. The medium contained 10% FBS, 100 U / mL penicillin, and 100 U / mL streptomycin, and was routinely cultured in a 37°C, 5% CO2 constant temperature incubator.

[0071] 2. Cell recovery

[0072] Take out the cryopreservation tube containing cells from the liquid nitrogen tank, place it in a 37℃ constant temperature water bath and shake it to melt it quickly, then transfer it to the clean bench, aspirate all the liquid in the cryopreservation tube into a centrifuge tube, centrifuge it at 1000rpm for 4min, remove the supernatant, add 1-2mL of new culture medium, blow it evenly, transfer it to the culture bottle, add an appropriate amount of culture medium and shake it evenly, while avoiding bubbles. Place it in a 37℃, 5% CO2 cell culture incubator, replace the culture medium once the next day and continue to culture.

[0073] 3. Cell passaging

[0074] Observe the cells. When the cells grow to 80% of the visual field, they need to be subcultured. Remove the old culture medium, wash with PBS 3 times, add 1mL trypsin-EDTA (0.25%), shake evenly, and place in a 37°C, 5% CO2 cell culture incubator. Take out after 3 minutes and observe the cell state under a microscope. When most of the cells are in a floating state, add 1-2mL culture medium and blow to form a uniform cell suspension. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 2mL new culture medium and blow evenly, transfer to a culture bottle, add an appropriate amount of new culture medium, and place it in a 37°C, 5% CO2 cell culture incubator for culture.

[0075] 4. Cell cryopreservation

[0076] Remove the old culture medium, wash with PBS to remove dead cells and metabolites, add 1mL trypsin-EDTA (0.25%), shake evenly, and place in a 37°C, 5% CO2 cell culture incubator. Take out after 3 minutes and observe the cell state under a microscope. When most of the cells are in a floating state, add 1-2mL culture medium and blow to form a uniform cell suspension. Transfer the cell suspension to a centrifuge tube, aspirate the liquid and transfer it to a centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 1mL cell freezing solution and blow evenly, transfer to a cryopreservation tube, seal it, mark the date and model, put it in a cryopreservation box, place it in a -80°C refrigerator, and transfer it to a liquid nitrogen tank for storage after 2 days.

[0077] 5. Screening of pancreatic cancer cell lines with high expression of immune checkpoint VISTA

[0078] Western blot was used to detect the VISTA expression levels of hTERT-HPNE pancreatic ductal epithelial cells, Panc-1 pancreatic cancer cells, Panc-2 pancreatic cancer cells, and Miapaca-2 pancreatic cancer cells. First, the total protein of the above four cell types was extracted and the protein quantification and concentration were determined. Then, according to the molecular weight of the protein, 10% concentration of concentrated gel and separation gel were selected to prepare the gel. The solidified PAGE gel was installed on the electrophoresis tank, and 1× electrophoresis buffer was added to the middle of the two glass plates until it exceeded the gel plane, and the comb was gently pulled out. According to the different protein concentrations, equal mass protein samples were added to each well, and the appropriate protein marker was selected according to the different protein molecular weights and added to the outermost wells, and the protein electrophoresis was performed using the constant pressure method. A PVDF membrane of appropriate size was selected for transfer. After the transfer was completed, the PVDF membrane was carefully clamped out and protein blocking was performed. The PVDF membrane was removed and washed three times. The PVDF membrane was incubated with the prepared primary antibody and placed on a horizontal shaker at 4°C for 6 hours. Finally, the primary antibody was recovered, the PVDF membrane was removed and washed three times. The PVDF membrane was incubated with the prepared secondary antibody and placed on a horizontal shaker at 4°C for 1 hour. After the incubation, the PVDF membrane was removed and washed three times. Then, an appropriate amount of ECL chemiluminescence was prepared according to the size of the membrane and added to the PVDF membrane. The membrane was exposed in a chemiluminescence imager and the results were recorded.

[0079] 6. Cytotoxicity experiment of nanoprobes

[0080] To evaluate the cytotoxicity of Fe3O4@TiO2@VISTAmAb, 100 μL of complete culture medium of Panc-2 mouse pancreatic cancer cells (density of 2×10 5 / mL), 5 parallel samples were set up in each group, and cultured overnight in a 37°C, 5% CO2 cell culture incubator for 12 hours to wait for the cells to adhere. Then, DMEM culture medium with different concentrations (5-150μg / mL) of Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb nanoprobes was added to each well. After incubation for 24 hours, the old culture medium was aspirated, and 100μL of DMEM culture medium containing 10μL CCK-8 solution (5mg / mL) was added to each well, and incubated in a 37°C, 5% CO2 cell culture incubator for 30min. Finally, the absorbance of each well was recorded at a wavelength of 450nm using an ELISA reader, and the cell viability was calculated. It is assumed that the cell survival rate cultured only with complete culture medium is 100%.

[0081] 7. Cellular uptake experiment of nanoprobes

[0082] The phagocytic targeting performance of the nanoprobe on Panc-2 mouse pancreatic cancer cells was detected by laser confocal microscopy. Since the nanoprobe does not have fluorescent properties, the Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb nanoprobes were first fluorescently labeled with rhodamine B: Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb were stirred with excess rhodamine B for 24 hours and then centrifuged and washed several times until the supernatant was colorless to obtain a nanoprobe with fluorescent properties. Panc-2 mouse pancreatic cancer cells were incubated at 1×10 5 The cells were inoculated into confocal culture dishes at a density of 100 μg / mL and cultured in a cell culture incubator at 37°C and 5% CO2 for 12 hours. After waiting for the cells to adhere to the wall and grow, the old culture medium was aspirated, and 50 μg / mL of Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb were added to the experimental group culture dishes and incubated for 4 hours. Fresh complete culture medium was added to the blank group culture dishes. After incubation, the cells were rinsed with PBS several times to remove excess nanoprobes. They were fixed with 4% paraformaldehyde at 4°C for 30 minutes, rinsed with PBS several times, and then the cells were stained with the prepared fluorescent dye Hoechst solution for nuclei. After incubation in a 37°C and 5% CO2 incubator in the dark for 20 minutes, the cells were taken out, the dye was aspirated, and the excess dye was removed by rinsing with PBS several times. Then 1 mL of PBS was added to prevent the cells from drying out. Finally, the samples were observed with a laser confocal microscope. The excitation wavelength of Hoechst dye is 350nm, the emission wavelength is 460nm, and the excitation wavelength of rhodamine B is 552nm, and the emission wavelength is 571nm. ImageJ software was used to analyze the fluorescence intensity of rhodamine B in cells after incubation with Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb nanoprobes for 4 h (n=3).

[0083] Flow cytometry was used to semi-quantitatively analyze the cell uptake of materials. Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb were stirred with excess rhodamine B for 24 h and then centrifuged and washed several times until the supernatant was colorless to obtain a nanoprobe with fluorescent properties. Cells in the logarithmic growth phase were collected and the cell density was adjusted to 5×10 5 / mL, 1mL of cell suspension was inoculated into a six-well plate, and cultured at 37℃ and 5% CO2 for 12h, waiting for the cells to adhere to the wall. The original culture medium was discarded, and a Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb solution with a concentration of 50μg / mL was prepared with fresh complete culture medium, and added to each well. The blank group was added with fresh complete culture medium and incubated for 4h. The original culture medium was discarded, rinsed with PBS 3 times, 1mL of trypsin-EDTA (0.25%) was added, and 1mL of complete culture medium was added to the incubator for 2min to terminate the digestion, transferred to a centrifuge tube, centrifuged at 1000rpm for 5min, and the supernatant was removed. Then PBS was added to blow evenly, centrifuged at 1000rpm for 5min, and the supernatant was removed. Repeat the operation 3 times. Finally, 1.5mL of PBS was added to blow the cells evenly. The fluorescence intensity of rhodamine B in each group of cells was detected by flow cytometry.

[0084] 8. Sonodynamic therapy of nanoprobes for detection of intracellular reactive oxygen species

[0085] The ability of nanoprobes to produce reactive oxygen species after ultrasound treatment of Panc-2 mouse pancreatic cancer cells was evaluated by laser confocal microscopy. The groups were set as follows: a. PBS group, b. Fe3O4@TiO2 group, c. Fe3O4@TiO2@VISTAmAb, d. US group, e. Fe3O4@TiO2+US group, and f. Fe3O4@TiO2@VISTAmAb+US group. Panc-2 mouse pancreatic cancer cells were treated with 2×10 5 The cells were inoculated into confocal culture dishes at a density of 100 μg / mL and cultured in a cell culture incubator at 37°C and 5% CO2 for 12 hours. After waiting for the cells to adhere to the wall and grow, the old culture medium was aspirated, and 50 μg / mL of Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb were added to the experimental group culture dishes and incubated for 2 hours. Fresh complete culture medium was added to the blank group culture dishes. After incubation, the cells were rinsed with PBS several times for 5 minutes each time to remove excess nanoprobes. Prepare ROS probe solution: Add DCFH-DA to DMEM solution to make a 1 μL / mL solution. Except for the blank control group, 1 mL of the prepared probe solution was added to the other experimental groups and incubated in a 37°C, 5% CO2 cell culture incubator away from light for 30 minutes. Then use an ultrasonic device (0.5W / cm 2 , 1MHz, duty cycle 50%,) the cells in the ultrasound treatment group were treated with ultrasound for 1 min. After treatment, they were incubated in a 37℃, 5% CO2 cell culture incubator for 4 h, the old culture medium was aspirated, and then gently rinsed with PBS several times to remove excess ROS probes. Observed under a laser confocal microscope.

[0086] 9. In vitro sonodynamic therapy experiment using nanoprobes

[0087] The cell survival rate of Panc-2 mouse pancreatic cancer cells after ultrasound treatment was evaluated by using an ELISA instrument to detect the nanoprobe. The groups were set as follows: a. PBS group, b. Fe3O4@TiO2 group, c. Fe3O4@TiO2@VISTAmAb, d. US group, e. Fe3O4@TiO2+US group, f. Fe3O4@TiO2@VISTAmAb+US group, with 3 parallel samples in each group. 100 μL of complete culture medium of Panc-2 mouse pancreatic cancer cells (density of 2×10 5 / mL), and cultured overnight in a 37°C, 5% CO2 cell culture incubator for 12 hours to wait for the cells to adhere. The old culture medium was aspirated, and 50μg / mL of Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb were added to the experimental group culture dishes and incubated for 4 hours. The concentration of VISTAmAb in the nanoprobe containing VISTAmAb components was 100μg / mL. Fresh complete culture medium was added to the blank group culture dishes. After incubation, the cells were rinsed with PBS, excess nanoprobes were removed, and fresh complete culture medium was added. Then an ultrasonicator (1.0W / cm 2 , 1MHz, duty cycle 50%) for 1min of ultrasound treatment for the cells. After treatment, incubate in a 37°C, 5% CO2 cell culture incubator for 12 hours, aspirate the old culture medium, add 100μL of DMEM culture medium containing 10μL of CCK-8 solution (5mg / mL) to each well, and incubate in a 37°C, 5% CO2 cell culture incubator for 30min. Finally, the absorbance of each well was recorded at a wavelength of 450nm using an ELISA reader, and the cell viability was calculated. It is assumed that the cell viability of cells cultured only with complete culture medium is 100%.

[0088] The CAM (calcein acetoxymethyl ester) / PI (propidium iodide) live-dead cell staining method was used to directly observe the cell survival of Panc-2 cells after ultrasound irradiation after taking up Fe3O4@TiO2@VISTAmAb. Panc-2 cells were incubated with Fe3O4@TiO2@VISTAmAb with a mass concentration of 50 μg / mL for 12 hours, and then an ultrasonic device (1.0 W / cm 2 ,1MHz,duty cycle 50%,) for 2min of ultrasound treatment on cells. After treatment, incubate in a 37℃,5% CO2 cell culture incubator for 12 hours, aspirate the old culture medium, add a certain proportion of CAM / PI dye solution prepared according to the instructions, and continue to culture in the incubator for 1h. Obtain cell fluorescence staining images under a fluorescence microscope. Live cells are labeled with CAM and excited at 490nm to produce green fluorescence; dead cells are labeled with PI and excited at 545nm to produce red fluorescence.

[0089] 10. Statistical analysis

[0090] Statistical analysis was performed using GraphPad Prism9. All data were tested for normality and homogeneity of variance. If the data met the normal distribution and the variance was homogeneous, the data were expressed as mean ± standard deviation (x ± s). The two-tailed t-test was used to derive the significance of the difference between the two groups, and the one-way ANOVA was used to derive the significance of the pairwise comparisons between multiple groups; the results were plotted using GraphPad Prism 9 and Origin2021 software. The data are expressed as mean ± SD. All experiments were repeated 3 times.

[0091] Experimental results and discussion:

[0092] A. Screening of pancreatic cancer cell lines with high expression of immune checkpoint VISTA

[0093] The expression level of VISTA protein in Miapaca-2 pancreatic cancer cells, hTERT-HPNE normal pancreatic ductal epithelial cells, and Panc-2 pancreatic cancer cells was quantitatively detected by Western blot. Figure 2 As shown in the figure, hTERT-HPNE pancreatic normal ductal epithelial cells express less VISTA, while the VISTA expression levels of Panc-1 pancreatic cancer cells and Panc-2 pancreatic cancer cells are increased to varying degrees compared with hTERT-HPNE pancreatic ductal epithelial cells, which is consistent with the report in the literature that the immune checkpoint VISTA is highly expressed in pancreatic cancer cells and stromal cells, and relatively low in normal pancreas. Among them, among the above four cells, the Fe3O4@TiO2@VISTAmAb nanoprobe has a strong targeting ability for Panc-2 and Panc-1 pancreatic cancer cells. Considering that mice will be used in this study to explore the mechanism of tumor immune microenvironment in the future. Therefore, this study selected Panc-2 pancreatic cancer cells for cell-level experiments.

[0094] B. Cellular uptake studies of nanoprobes

[0095] As mentioned above, there is a dense fibrous matrix in pancreatic cancer. VISTA is highly expressed in pancreatic cancer cells and stromal cells, which requires the nanoprobe to break through the pancreatic cancer matrix and enter the pancreatic cancer cells to work better. In this study, Fe3O4@TiO2@VISTAmAb nanoprobe was constructed by combining Fe3O4@TiO2 with the immune checkpoint inhibitor VISTAmAb, which enabled the nanoprobe to acquire the ability to target VISTA.

[0096] First, the uptake capacity of Panc-2 cells for Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb was observed using laser confocal microscopy. Since Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb themselves do not have fluorescent properties, this study connected rhodamine B on the surface of Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb to make them fluorescent. After Panc-2 cells were incubated with 50μg / mL Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb for 4 hours, different components were stained with fluorescent dyes. Blue fluorescence indicated the cell nucleus labeled with Hoechst, and red fluorescence indicated the nanoprobe labeled with rhodamine B. The results showed that compared with the control group incubated without materials, red fluorescence could be observed in the pancreatic cancer cells of Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb groups, which was the fluorescence emitted by rhodamine B adsorbed on the surface of nanomaterials. As the co-incubation time with nanomaterials increases, the uptake of nanoprobes by Panc-2 cells in the Fe3O4@TiO2 group and the Fe3O4@TiO2@VISTAmAb group continues to increase, and the uptake of Fe3O4@TiO2@VISTAmAb nanoprobe by Panc-2 cells is significantly higher than that of non-targeted Fe3O4@TiO2 nanoprobe. The average fluorescence intensity of the three groups of images was analyzed by ImageJ software (n=3), and the results showed that the fluorescence intensity of the Fe3O4@TiO2@VISTAmAb group was significantly higher than that of the Fe3O4@TiO2 group, and the difference in fluorescence intensity between the two groups was statistically significant (p<0.01). The above results suggest that VISTAmAb can significantly improve the targeting performance of nanoprobes to Panc-2 cells, thereby increasing the uptake of nanoprobes by Panc-2 cells.

[0097] Afterwards, the fluorescence intensity in the cells after Panc-2 cells took up different nanoprobes was detected by flow cytometry. Since the synthesized nanoprobes are not fluorescent, the nanoprobes are used to adsorb the fluorescent dye Rhodamine B to form a fluorescent molecular probe. It can be observed that obvious Rhodamine B fluorescence can be detected in the cells co-incubated with the nanoprobes adsorbed with VISTAmAb, and the fluorescence intensity in the Fe3O4@TiO2@VISTAmAb group is significantly higher than that in the Fe3O4@TiO2 group (p < 0.0001). Therefore, the fluorescence semi-quantitative analysis of the cell uptake of nanoprobes was performed using flow cytometry, which further proved that the Fe3O4@TiO2@VISTAmAb nanoprobe has the ability to target Panc-2 cells. Therefore, the Fe3O4@TiO2@VISTAmAb nanoprobe can enhance the uptake of Panc-2 cells, increase the intracellular accumulation of nanoprobes, and is expected to enhance the effect of sonodynamic therapy.

[0098] C. Cytotoxicity analysis of nanoprobes

[0099] In this study, the CCK-8 experiment was used to detect the effects of different concentrations of Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb nanomaterials on the activity of Panc-2 pancreatic cancer cells. The results showed that when the concentration of Fe3O4@TiO2 nanomaterials was 0-150μg / mL, after incubation with Panc-2 pancreatic cancer cells for 24 hours. The results showed that the cell survival rate of nanomaterials was higher than 80% when the concentration of Fe3O4@TiO2 was in the range of 0-100μg / mL, indicating that the cytotoxicity of the nanoprobes synthesized in this subject was low. When the concentration of Fe3O4@TiO2 was 150μg / mL, the cell survival rate of the nanomaterials was about 73.3%. After the immune checkpoint inhibitor VISTAmAb, the effect of Fe3O4@TiO2@VISTAmAb nanoprobe on the activity of Panc-2 pancreatic cancer cells was detected again. When the concentration of Fe3O4@TiO2@VISTAmAb nanoprobe was between 0 and 100 μg / mL, the cell survival rate was higher than 80% after incubation with Panc-2 pancreatic cancer cells for 24 hours; when the concentration was 150 μg / mL, the survival rate of Panc-2 pancreatic cancer cells was 76%, indicating that VISTAmAb had no biological toxicity, and the Fe3O4@TiO2@VISTAmAb nanomaterial after adsorbing VISTAmAb still had good biocompatibility.

[0100] D. In vitro sonodynamic therapy effect of nanoprobes

[0101] The CCK-8 method was used to detect the sonodynamic therapy effect of Panc-2 pancreatic cancer cells. Panc-2 cells were incubated with DMEM, Fe3O4@TiO2, and Fe3O4@TiO2@VISTAmAb for 2 h. The ultrasound treatment group was given 1.0 W / cm 2 After 1 min of ultrasound irradiation, the effects of Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb nanomaterials on the cell activity of Panc-2 pancreatic cancer cells were evaluated by CCK-8 method. The cell survival rates of the groups without ultrasound treatment were 100±4.6% (DMEM), 91.1±3.3% (Fe3O4@TiO2), and 95.8±2.3% (Fe3O4@TiO2@VISTAmAb). 2 , 1min, duty cycle 50%, 1MHz), the cell survival rates were 77.8±5.5% (US), 66.9±14.5% (Fe3O4@TiO2+US), and 56.7±6.6% (Fe3O4@TiO2@VISTAmAb+US), respectively. Compared with the Control group, the cell survival rate in the Fe3O4@TiO2@VISTAmAb+US group was significantly reduced (56.7%), proving that Fe3O4@TiO2@VISTAmAb can produce cytotoxic ROS under ultrasound irradiation and kill Panc-2 pancreatic cancer cells, p<0.01, with statistical significance. The study found that compared with the control group, simple ultrasound treatment can also reduce cell survival rate (77.8%). This therapeutic effect may come from the cavitation effect produced under ultrasound irradiation, which converts part of the mechanical energy into heat energy, causing some heat-sensitive cells to die due to heat. Due to the small number of parallel groups (n=3), the results showed no statistical difference, p=0.054, and repeated experiments are still needed to verify this. Since there is no antigen presentation and T cell activation to kill tumor cells in in vitro cell experiments, the therapeutic effect of VISTAmAb cannot be evaluated by the CCK-8 method at the in vitro cell level.

[0102] Live / dead cell staining was used to observe the killing of Panc-2 cells by Fe3O4@TiO2@VISTAmAb nanoprobe sonodynamic therapy. Panc-2 cells were incubated with DMEM and Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb at a mass concentration of 50 μg / mL for 4 h. 2Panc-2 cells were irradiated with ultrasound for 2 minutes. Among them, live cells were labeled with Calcein-AM and emitted green fluorescence, and dead cells were labeled with PI and emitted red fluorescence. The live / dead cell staining results were basically consistent with the CCK-8 experimental results. Fe3O4@TiO2@VISTAmAb has a strong ultrasonic killing effect on Panc-2 cells.

[0103] E. Effect of in vitro sonodynamic therapy with nanoprobes on the production of reactive oxygen species

[0104] Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb nanoprobes were co-incubated with Panc-2 cells and ultrasonically treated with 0.5 W / cm 2 , 1min, duty cycle 50%) to detect the generation of reactive oxygen species in cells. No obvious reactive oxygen species were generated in cells without ultrasound irradiation. In the group without incubation material, some cells had green fluorescence after simple ultrasound irradiation, indicating the generation of ROS. However, when the power was 0.5W / cm 2 After 1 minute of ultrasound irradiation, obvious green fluorescence can be observed in most cells in the field of view. This shows that ultrasound irradiation can stimulate the Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb nanoprobes in cells to generate cytotoxic ROS, thereby producing an SDT effect.

[0105] In this example, the Western Blot method was first used to screen the cell line Panc-2 that highly expressed the immune checkpoint VISTA. Secondly, the Fe3O4@TiO2@VISTAmAb nanoprobe was co-incubated with Panc-2 pancreatic cancer cells. The results showed that the probe had no obvious effect on cell activity, indicating that it had good biosafety. After being engulfed by cells, the Fe3O4@TiO2@VISTAmAb nanoprobe was mainly distributed in the cytoplasm, and as the incubation time increased, more nanoprobes were engulfed by cells. Furthermore, laser confocal microscopy and flow cytometry tests showed that compared with Fe3O4@TiO2 nanoparticles, the Fe3O4@TiO2@VISTAmAb nanoprobe adsorbed with VISTAmAb showed the ability to target Panc-2 pancreatic cancer cells and could be taken up more by the cells. Finally, at 0.5W / cm 2 Under ultrasound irradiation, it was found that SDT mediated by Fe3O4@TiO2@VISTAmAb nanoprobe could induce cells to produce more cytotoxic ROS and have a significant killing effect on pancreatic cancer cells.

[0106] Example 3: Nanoprobe sonodynamic therapy study of pancreatic cancer mouse model

[0107] Ultrasound-stimulated Fe3O4@TiO2@VISTAmAb nanoprobes were used to treat subcutaneous pancreatic cancer mouse models in vivo to observe the tumor suppression effect and biological toxicity; evaluate the role and value of VISTAmAb in the tumor microenvironment; observe the immune cell infiltration and immune activation in the tumor site after VISTAmAb treatment; evaluate the role of SDT in stimulating angiogenesis and in converting pancreatic cancer "cold" tumors to "hot" tumors. The experimental methods are as follows:

[0108] 1. Construction of subcutaneous pancreatic cancer tumor model

[0109] Six-week-old female C57BL / 6 (6-8 weeks) mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd.

[0110] For animal experiments, animal care and handling procedures were in accordance with the guidelines of the Regional Ethics Committee for Animal Experimentation of Ningbo University. Mice were housed under barrier conditions and fed with a standard rodent diet and water. Panc-2 murine pancreatic cancer cells were grown at a density of 3 × 10 7 100 μL of ...

[0111] 2. Testing of in vivo magnetic resonance imaging capabilities of nanoprobes

[0112] The MRI imaging performance of the nanoprobe in vivo was evaluated using a 3.0T magnetic resonance imaging scanning system. First, the MR images of subcutaneous tumors in mice were measured, and then PBS, Fe3O4@TiO2, and Fe3O4@TiO2@VISTAmAb PBS solutions were injected into the tumors of mice (10 mg / kg), and MR images were recorded 2 hours later to compare and analyze the MR imaging performance of Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb.

[0113] 3. Sonodynamic combined with immunotherapy in mouse pancreatic cancer model

[0114] C57BL / 6 tumor-bearing mice were randomly divided into 6 groups: a. PBS group, b. Fe3O4@TiO2 group, c. Fe3O4@TiO2@VISTAmAb, d. US group, e. Fe3O4@TiO2+US group, and f. Fe3O4@TiO2@VISTAmAb+US group. PBS and PBS solutions containing Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb (dose of 15 mg / kg) were injected into the subcutaneous tumors of C57BL / 6 mice, respectively. Two hours later, the mice in the ultrasound treatment groups (d, e, and f groups) were treated with ultrasound. The parameters were as follows: 1.5 W / cm 2 , 3min, duty cycle 50%, 1.0MHz. The weight and tumor size of mice were recorded every two days. Mice were killed after 14 days, and the tumors were removed and the weight and size were recorded.

[0115] 4. Biosafety Experiment

[0116] In order to further evaluate the in vitro biological toxicity of the nanoprobe, 6-week-old Babl / c female mice were injected with PBS and PBS solution containing Fe3O4@TiO2 or Fe3O4@TiO2@VISTAmAb (dose of 15 mg / kg, 100 μL) through the tail vein. After 14 days, blood was taken from the eyeball and the mice were killed by cervical dislocation. Blood samples were used for routine blood tests and blood biochemistry tests. Whole blood samples were placed at room temperature for 2 hours or at 4°C overnight, and then at 4°C 3000rpm for 15 minutes. The supernatant was taken and tested with a kit. After setting the corresponding parameters on the fully automatic biochemical analyzer, the samples were loaded and automatically determined by the fully automatic biochemical analyzer. The whole blood samples were loaded with anticoagulant EP tubes and automatically determined using a fully automatic blood routine analyzer. The main organs of the heart, liver, spleen, kidney, and lung were fixed with polyformaldehyde and stained with hematoxylin-eosin, and the tissue specimens were pathologically evaluated.

[0117] 5. Hematoxylin-eosin staining experiment

[0118] Cut formalin-fixed, paraffin-embedded tissue into 5 μm thick slices. Pass through 3 layers of xylene and ethanol. Rinse the slices slowly with running water to wash off the alcohol. Stain with hematoxylin for 3-5 minutes, then wash with tap water. Differentiate with 1% hydrochloric acid alcohol solution and then wash with water. Blue with dilute lithium carbonate aqueous solution for 30 seconds and then wash with water. Add to eosin staining solution (alcohol soluble) and adjust the color in 95% ethanol. Dehydrate in anhydrous ethanol and make transparent in xylene. After sealing, observe the cell nucleus under a microscope and it will be blue-purple, and the cytoplasm, stroma, and various fibers will show different degrees of red.

[0119] 6. Immunohistochemistry of immune checkpoint expression

[0120] Cut 5μm slices from formalin-fixed, paraffin-embedded tissues. Pass through 3 layers of xylene and ethanol. Rinse the slices slowly with running water to wash off the alcohol. Antigen high-pressure repair. Prepare 2L of citrate repair solution, boil the slices in a pressure cooker, and heat repair for 2 minutes. Add 3% hydrogen peroxide to the tissue and incubate at room temperature for 20 minutes. Rinse with PBS 3 times, spin dry the slices, add primary antibody (PD-1CST 84651T 1:100; PD-L1 Servicebio GB11339A 1:200; Vista CST 54979 1:300), and incubate at 37℃ for 60 minutes. Rinse with PBS 3 times. Add secondary antibody, cover the incubation box lid, and incubate at 37℃ for 20 minutes. Rinse with PBS 3 times. Add DAB colorimetric agent, stop color development, and rinse with distilled water. Stain with hematoxylin for 1 minute and then wash with water. Differentiate with 1% hydrochloric acid alcohol and then wash with water. Lithium carbonate bluing for 1 min, washing with water, dehydration, transparency, and sealing. After DAB color development, observe the location of brown (positive) expression under a microscope.

[0121] 7. Fluorescence staining experiment of tumor immune cells

[0122] Cut formalin-fixed, paraffin-embedded tissues into 5 μm thick slices. Dewax the paraffin sections to water, and wash the sections in environmentally friendly dewaxing solution, anhydrous ethanol, and distilled water. For antigen retrieval, immerse the sections in 10 mM sodium citrate, heat in a water bath at 95°C-99°C for 20 minutes, and then cool to room temperature. Place the slides in PBS (PH 7.4) and shake on a decolorizing shaker to wash. Circle drawing: Draw circles around the tissue with a hydrogen peroxide blocking pen, place the sections in a 3% hydrogen peroxide solution, incubate at room temperature in the dark for 25 minutes, block endogenous peroxidase, and place the slides in PBS (PH7.4) on a decolorizing shaker. Serum blocking: Dry PBS, add 10% rabbit serum to block (primary antibody: goat source is blocked with 10% rabbit serum, other sources: blocked with 3% BSA), and block for 30 minutes. Add the first primary antibody (CD4 abcam ab183685 1:1000): remove the blocking solution, add the prepared primary antibody, and incubate it in a humidified box at 4°C overnight. Add the corresponding HRP-labeled secondary antibody: place the slide in PBS (PH 7.4) and wash it on a decolorizing shaker. After the slices are slightly dried, add the corresponding HRP-labeled secondary antibody in the circle and incubate it at room temperature for 50 minutes. Add the corresponding TSA dye: place the slide in PBS (PH 7.4) and wash it on a decolorizing shaker. Add TSA in the circle and incubate it at room temperature for 10 minutes in the dark. Place the slide in TBST and wash it on a decolorizing shaker. Microwave treatment: place the tissue section in a repair box filled with antigen retrieval buffer and heat it in a microwave oven, medium heat for 8 minutes, stop the heat for 8 minutes, and switch to medium-low heat for 7 minutes. Add the second primary antibody (CD8CST 98941 1:400): add the prepared primary antibody and incubate it at 4°C overnight. Add the corresponding secondary antibody: Place the slide in PBS (PH 7.4) and wash on a decolorizing shaker. Add the corresponding fluorescent secondary antibody and incubate at room temperature in the dark for 50 minutes. After incubation, place the slide in PBS (PH 7.4) and wash on a decolorizing shaker. DAPI counterstaining of cell nuclei: After the slices are slightly dried, add DAPI staining solution in the circle and incubate at room temperature in the dark for 10 minutes. Autofluorescence quenching: Place the slide in PBS (PH 7.4) and wash on a decolorizing shaker. After the slices are slightly dried, add autofluorescence quencher B solution in the circle, incubate for 5 minutes, and rinse for 10 minutes. Sealing: Sealing with anti-fluorescence quenching sealing agent. Under fluorescence microscopy, the cell nucleus is blue, the CD4 positive expression position is green, and the CD8 positive expression position is red.

[0123] 8. Tumor collagen fiber staining experiment

[0124] Formalin-fixed, paraffin-embedded tissues were sliced ​​5 μm thick. The sections were dewaxed in xylene and rehydrated in a series of graded ethanol. Stained with Weigert iron hematoxylin staining solution for 5-10 min, differentiated in acidic ethanol differentiation solution and then washed with water, bluing in Masson blueing solution for 3-5 min and then washed with water, stained with Ponceau fuchsin staining solution for 5-10 min, and washed with weak acid working solution (distilled water: weak acid solution = 2:1) for 1 min. Washed with phosphomolybdic acid solution for 1-2 min. Washed with the prepared weak acid working solution for 1 min. Stained directly in aniline blue staining solution for 1-2 min. Washed with weak acid working solution for 1 min. Dehydrated with anhydrous ethanol. Transparented with xylene 3 times. Finally, sealed with neutral gum and observed under an ordinary optical microscope.

[0125] 9. Tumor vascular immunofluorescence staining experiment

[0126] Cut formalin-fixed, paraffin-embedded tissue into 5 μm thick slices. Pass through xylene three times in sequence. Pass through ethanol three times. Place the slices in a beaker and rinse slowly to remove the alcohol until the slices are clean and transparent. Antigen high-pressure repair. Prepare 2L of citrate repair solution, boil the slices in a pressure cooker, and heat repair for 2 minutes. Block with goat serum for 30 minutes. Add primary antibody (CD31 Servicebio GB12063 1:200) and incubate at 37°C for 1 hour. After rinsing with PBS three times, shake dry the slices and add fluorescent secondary antibody (1:300), incubate at 37°C in the dark for 30 minutes. Rinse with PBS three times. Counterstain with DAPI for 5 minutes. Rinse with PBS three times. Seal the slices with anti-fluorescence quenching sealing agent. Image and observe with a fluorescent slice scanner. The nucleus is blue and the antibody positive expression is green.

[0127] 10. Statistical analysis

[0128] Statistical analysis was performed using GraphPad Prism 9. All data were tested for normality and homogeneity of variance. If the data met the normal distribution and homogeneity of variance, the data were expressed as mean ± standard deviation (x ± s). The two-tailed t-test was used to determine the significance of the difference between the two groups, and the one-way ANOVA was used to determine the significance of the pairwise comparison between multiple groups; the results were plotted using GraphPad Prism 9 and origin2021 software. The data are expressed as mean ± SD.

[0129] Experimental results and discussion:

[0130] A. In vivo biological toxicity analysis results of nanoprobes

[0131] To further study the in vivo biosafety of the nanoprobe, PBS and PBS solutions containing Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb were injected into the tail vein of Babl / c mice (dose of 15 mg / kg). There was no significant difference in the body weight of mice among the groups within 14 days. After 14 days, the main organs such as heart, lung, liver, kidney, spleen, etc. were collected for histopathological examination, and blood samples were collected for blood biochemistry and blood routine tests. The results of blood biochemistry and blood routine tests showed that there was no significant difference in the indicators between the control group and the injection material group. The pathological results showed that the organs of mice injected with PBS solutions containing Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb had no obvious tissue cell level damage compared with the control group PBS. Therefore, Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb have ideal biosafety.

[0132] B. Results of in vivo MRI performance evaluation of nanoprobes

[0133] A 3.0T MR scanning system was used to evaluate the in vivo MRI performance of the nanoprobe. Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb were injected into the tumors of different mice (10 mg / Kg), and the MR imaging performance of Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb was compared and analyzed. 2 hours after the injection of the two nanomaterials, it can be observed that the tumor site on the T2 imaging picture becomes darker, showing a strong dark signal, which can accurately show the size of the tumor and its relationship with the surrounding tissues. Therefore, the results prove that the Fe3O4@TiO2 and Fe3O4@TiO2@VISTAmAb nanomaterials have good MRI T2-weighted imaging performance.

[0134] C. Results of the in vivo sonodynamic combined immunotherapy efficacy evaluation of nanoprobes

[0135] The anti-tumor effect of Fe3O4@TiO2@VISTAmAb nanoprobe-mediated sonodynamic synergistic immunotherapy was used in subcutaneous tumor C57BL / 6 mice as treatment subjects. The pancreatic cancer subcutaneous tumor model was established and the treatment process was as follows: 30 mice were randomly divided into 6 groups and received different treatments. The weight and tumor volume of the mice were recorded every two days. The results showed that there was no significant difference in the weight of mice between the groups within 14 days. After the 14-day treatment period, the mice were killed, the tumors were removed and the weight was recorded.

[0136] Tumor inhibition was found in mice treated with Fe3O4@TiO2@VISTAmAb nanoprobes, indicating that the VISTA-targeted nanoprobes provided a therapeutic effect in a pancreatic cancer model. Notably, the tumor growth inhibition in the Fe3O4@TiO2@VISTAmAb+US group was significantly stronger than that in the Fe3O4@TiO2@VISTAmAb group, indicating that ultrasound irradiation significantly improved the anti-tumor effect. Compared with the control group, the tumor growth in the Fe3O4@TiO2@VISTAmAb+US group was significantly inhibited, with the tumor weight being only 14.64% of that in the control group, and the inhibition rate being 85.36%. The tumor weight inhibition rate in the Fe3O4@TiO2+US group was 67.86%, which was significantly different from that in the Fe3O4@TiO2@VISTAmAb+US group, p=0.0021. However, this study found that subcutaneous tumor growth in mice was also inhibited after simple ultrasound irradiation, which may be due to the damage of tumor cells caused by the mechanical waves generated by ultrasound.

[0137] The results showed that compared with sonodynamic therapy alone, combined immunotherapy can improve the tumor growth inhibition rate and effectively reduce tumor volume. The tumor growth of mice in the Fe3O4@TiO2@VISTAmAb+US group was significantly inhibited after a single treatment, and even the tumor volume was reduced, indicating that the nanoprobe sonodynamic combined immunotherapy achieved a good therapeutic effect on mice by generating reactive oxygen species to kill tumor cells and activate adaptive immune responses.

[0138] After 14 days of treatment, the subcutaneous tumor was peeled off, and the combined sonodynamic and immunotherapy had the best inhibitory effect on the tumor. The subcutaneous tumors in each group were fixed and sliced ​​by HE staining to observe the tumor tissue sections of the mice. The chromatin in the nucleus and the nucleic acid in the cytoplasm were purple-blue; the components in the cytoplasm and the extracellular matrix were red. Therefore, the fewer blue-purple areas and the more pink areas without nuclei or fragmented nuclei represent the higher degree of tumor necrosis. In the control group, the cell nucleus morphology was clear and complete, without necrosis and apoptosis; the cell morphology of the Fe3O4@TiO2 group was relatively complete. After treatment, the tumor tissues of the Fe3O4@TiO2@VISTAmAb group, the US group, the Fe3O4@TiO2+US group, and the Fe3O4@TiO2@VISTAmAb+US group all showed varying degrees of necrosis, and the cell apoptosis in the Fe3O4@TiO2@VISTAmAb+US group was more obvious, indicating that the sonodynamic combined immunotherapy of the nanoprobe can cause more tumor cell necrosis, thereby achieving a better anti-tumor effect.

[0139] D. Analysis of vascular density and fibrous matrix in in vivo sonodynamic therapy using nanoprobes

[0140] The tumor tissue was stained with Masson staining and CD31 fluorescence staining to characterize the collagen fibers and blood vessels of the constructed mouse subcutaneous pancreatic cancer model. In the Masson staining results, the blue part is collagen fiber, which shows that the constructed pancreatic cancer model has abundant collagen fibers, and a large amount of matrix is ​​seen in the stroma around the tumor cells. The blood vessels were further characterized by immunofluorescence. There was almost no green fluorescence in the control group, that is, there were no capillaries, which was consistent with the clinical pathological characteristics of pancreatic cancer. Therefore, the establishment of this model is suitable for this study.

[0141] The collagen fiber level of the pancreatic cancer tumor model was characterized after ultrasound. The results showed that under ultrasound, the dense fibrous tissue in pancreatic cancer can be loosened and the tight connections between cancer cells are destroyed. This may be due to the mechanical force generated by ultrasound, which mechanically damaged the tumor tissue and then affected the collagen fibers and cell arrangement in the tissue. At the same time, it can be observed that compared with the control group, the vascular density in the ultrasound group increased by more than two times, indicating that under the action of ultrasound, the angiogenesis of tumor tissue can be increased, and the central vascular density of tumor tissue can be increased, which will help nanomedicines reach the inside of the tumor and increase the effective accumulation of drugs.

[0142] E. Analysis of the immune microenvironment of nanoprobe-based in vivo sonodynamic combined immunotherapy

[0143] In this study, pancreatic cancer mice were immunotherapyed by VISTA antibody loaded in Fe3O4@TiO2@VISTAmAb. VISTAmAb antagonized the VISTA receptor protein on the tumor cell membrane, blocking the immune checkpoint and blocking its binding to the ligand on T lymphocytes, thereby activating the immune response mediated by T lymphocytes, preventing immune escape, and reversing the local inhibitory immune microenvironment of pancreatic cancer. Therefore, the construction of a subcutaneous pancreatic cancer model with high expression of VISTA is a prerequisite for immunotherapy in this study. The model was constructed using the Panc-2 pancreatic cancer cell line, and the tumor tissue was verified by immunohistochemical VISTA staining. The results showed that the subcutaneous tumor model of C57BL / 6J mice constructed by Panc-2 pancreatic cancer cells in the control group had strong VISTA positive expression, so the model can be used in Fe3O4@TiO2@VISTAmAb nanoprobe-mediated sonodynamic synergistic immunotherapy in the future.

[0144] The subcutaneous pancreatic cancer mouse model was treated with Fe3O4@TiO2, Fe3O4@TiO2@VISTAmAb and ultrasound irradiation. Compared with the treatment with Fe3O4@TiO2 nanoprobe alone, the expression of VISTA in the yellow-brown particles was observed to be reduced after Fe3O4@TiO2@VISTAmAb nanoprobe treatment, indicating that the positive expression of VISTA in the tumor tissue site decreased after antibody blocking. The positive expression of VISTA increased slightly after ultrasound irradiation alone or Fe3O4@TiO2+US treatment. However, after Fe3O4@TiO2@VISTAmAb+US treatment, the positive expression of VISTA in the tumor tissue site decreased. Immunotherapy combined with VISTAmAb can downregulate the positive expression of VISTA, thereby reducing immune escape and enhancing T lymphocyte-mediated immune response, thereby achieving the purpose of anti-tumor.

[0145] Secondly, immunohistochemical PD-L1 and PD-1 staining was performed on the tumor tissue constructed by the Panc-2 pancreatic cancer cell line model. The results showed that there was no positive expression of PD-L1 and PD-1 in the C57BL / 6 mouse subcutaneous tumor model constructed by Panc-2 pancreatic cancer cells in the control group. Because of the low PD-L1 and PD-1 expression in the C57BL / 6 mouse pancreatic cancer model, it is difficult to achieve a good immunotherapy effect using anti-PD-L1 and anti-PD-1 antibody treatment. After VISTAmAb blocked VISTA treatment, compensatory expression of PD-L1 and PD-1 increased, which also provides new ideas for subsequent combined anti-PD-L1 / PD-1 and VISTAmAb drug treatment.

[0146] After blocking with VISTAmAb, it will stimulate the activation and maturation of APC cells (such as DCs) in the tumor site, thereby activating T lymphocytes, causing the initial T lymphocytes to further differentiate into cytotoxic T lymphocytes with anti-tumor activity, which then kill tumor cells by secreting cytokines such as IFN-γ and TNF-α. Therefore, this study used double immunofluorescence to detect CD4 + T cells and CD8 + T cells were fluorescently stained to analyze their infiltration in the tumor site.

[0147] The results show that blue is the cell nucleus and green is CD4 + T cells, red is CD8 + Green and red fluorescence were almost not observed in the slices of the T cell control group, indicating that there was almost no infiltration of immune cells in the tumor tissue of the subcutaneous pancreatic cancer model constructed in this study, and it was in an immunosuppressive microenvironment. Compared with other groups, a higher frequency of CD8+ T cells (red fluorescence) and CD4 + T cells (green), indicating high levels of CD8 + Effector T cells and CD4 + T cell infiltration leads to a strong anti-tumor immune response.

[0148] The levels of red fluorescence and green in the slices of mice injected with Fe3O4@TiO2@VISTAmAb after US irradiation were higher than those in the group without US irradiation. This may be because the dense tissue structure of the tumor becomes loose under US irradiation, which is more conducive to drug penetration into the deep part of the tumor. + T cells and CD4 + The infiltration of T cells is higher and the degree of immune activation is also higher, achieving a better tumor suppression effect.

[0149] In this example, an in vivo pancreatic cancer animal model was constructed, and the tumor tissue of the model was subjected to immunohistochemical analysis of VISTA, PD-L1 and PD-1 expression levels, which verified that it had VISTA positivity and low PD-1 / PD-L1 expression, which met the premise of using VISTAmAb for immune checkpoint blockade therapy. It also explains that the previous immunotherapy for pancreatic cancer was not effective, and the reason may be the lack of specific immune checkpoint drugs with high expression in the tumor.

[0150] First, this study evaluated the biosafety of Fe3O4@TiO2@VISTAmAb nanoprobe combined with sonodynamic immunotherapy at the animal level. By comparing the weight changes of mice, blood biochemistry and routine blood tests, and HE staining results of major organs, it was found that the probe has low biotoxicity and good biocompatibility.

[0151] Next, the tumor inhibition effect of the nanoprobe in vivo was evaluated at the animal level. The two nanomaterials were injected into the mouse tumor by in situ administration of subcutaneous tumors in the immune-competent C57BL / 6 mouse model, and then the mice were treated with sonodynamic combined immunotherapy. The results showed that the Fe3O4@TiO2@VISTAmAb nanoprobe had the most obvious anti-tumor effect under sonodynamic combined immunotherapy under US irradiation. Although the Fe3O4@TiO2 nanomaterial can show a good tumor inhibition effect under US irradiation, its immune infiltration is low, so it may lack the activation and killing effect of immune cells. The effect of ultrasound on blood vessels in the tumor microenvironment was studied. The results showed that under the action of ultrasound, the dense collagen fibers of pancreatic cancer were broken, the tight junctions between cancer cells were destroyed, the angiogenesis of tumor tissue increased, and the central blood vessel density increased, which was conducive to the penetration of subsequent nanomedicines.

[0152] The present invention successfully constructed a novel multifunctional Fe3O4@TiO2@VISTAmAb nanoprobe, which has good sonodynamic performance and MRI imaging function. The sonodynamic combined immunotherapy effect at the cell and animal levels was studied, and the following conclusions were finally obtained:

[0153] 1) Successfully constructed Fe3O4@TiO2@VISTAmAb nanoprobe with sonodynamic-immunotherapy and MRI function. Fe3O4 nanoparticles were prepared by solvothermal method, and SiO2 and TiO2 were modified on the surface. Fe3O4@TiO2 nanoprobes were formed by hydrothermal etching, and VISTAmAb was coupled to the nanoprobe to construct a wreath-shaped Fe3O4@TiO2@VISTAmAb nanoprobe. The experimental results of material performance research showed that the average hydrated particle size of the prepared nanoprobe was about 225.3nm, the Zeta potential was -28.9mV, and the negative charge on the surface was more conducive to long-term blood circulation. The longitudinal relaxation rate (r2) value of the nanoprobe was about 50.28Mm -1 s -1 , r2 / r1 value is 32.03, which is an excellent T2 MRI contrast agent. The ROS yield of the material was tested by DPBF. The experimental results showed that the power of the probe solution with a concentration of Fe3O4@TiO2@VISTAmAb of 100μg / mL was 1.0W / cm 2 After 8 min of ultrasonic irradiation with a duty cycle of 50%, the absorption peak of DPBF at 425 nm can be reduced to 70% of the original value, indicating that the nanoprobe has good acoustic dynamic performance.

[0154] 2) The results of sonodynamic therapy and cytotoxicity experiments of Fe3O4@TiO2@VISTAmAb nanoprobe on pancreatic cancer cells showed that after 24 hours of co-incubation of Panc-2 cells with Fe3O4@TiO2@VISTAmAb nanoprobes in the concentration range of 25-150μg / mL, the cell survival rate was still higher than 80%, and no obvious damage to the main organs of mice was observed 14 days after the tail vein injection of 15mg / kg of nanoprobes, indicating that the nanoprobe has good biocompatibility. The results of laser confocal microscopy showed that the nanoprobe was taken up by Panc-2 cells after being co-incubated with Panc-2 cells for 4 hours, and Fe3O4@TiO2@VISTAmAb could be taken up more by Panc-2 cells. The sonodynamic therapy experiment showed that the Fe3O4@TiO2@VISTAmAb probe with a concentration of 50μg / mL was co-incubated with Panc-2 cells for 12 hours at 1.0W / cm 2 After 1 minute of ultrasound irradiation, the cell survival rate was about 56.7%, showing a strong killing therapeutic effect.

[0155] 3) The results of in vivo treatment studies with Fe3O4@TiO2@VISTAmAb nanoprobes showed that after ultrasound irradiation, the tumors in the treatment group were significantly inhibited. The tumor growth inhibition in the Fe3O4@TiO2@VISTAmAb+US group was significantly stronger than that in the Fe3O4@TiO2@VISTAmAb group, indicating that US irradiation significantly improved the anti-tumor effect. The Fe3O4@TiO2@VISTAmAb+US group had a stronger therapeutic effect than the Fe3O4@TiO2 group, and VISTAmAb was able to achieve a certain immunotherapy effect.

[0156] 4) The results of the immune microenvironment evaluation of Fe3O4@TiO2@VISTAmAb nanoprobe combined with sonodynamic immunotherapy showed that after VISTAmAb treatment, the positive expression of VISTA decreased, proving that the immune checkpoint therapy reached the expected level. And after anti-VISTA treatment, the positive expression of PD-L1 / PD-1 was slightly upregulated by immunohistochemical analysis, indicating that when VISTA is inhibited, tumor cells will evade immunity by regulating other immune checkpoints.

[0157] 5) The cascade targeting function of the Fe3O4@TiO2@VISTAmAb nanoprobe was confirmed by in vivo experiments, that is, on the basis of VISTAmAb simultaneously targeting pancreatic cancer stroma and cells, the ultrasound effect of the nanoprobe was used to study the effect on vascular density. The expression level of VISTAmAb, stromal fibroblasts and collagen fiber levels of pancreatic cancer tumor models were characterized. The results showed that under the action of ultrasound, the dense fibrous tissue of pancreatic cancer can be loosened, the close arrangement between cancer cells is destroyed, the angiogenesis of tumor tissue is increased, and the central blood vessel density is increased.

[0158] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a nanoprobe for diagnosis and treatment of pancreatic cancer targeting immune checkpoints, characterized in that: It includes the following steps: (1) Preparation of Fe3O4 nanoparticles using a solvothermal method; (2) Preparation of Fe3O4@SiO2 nanoparticles: TEOS was hydrolyzed under alkaline conditions as a silicon source to coat the surface of Fe3O4 with silicon dioxide to construct Fe3O4@SiO2 nanoparticles; (3) Preparation of Fe3O4@SiO2@TiO2 nanoparticles: Based on the hydrolysis and condensation of TBOT, titanium dioxide TiO2 was coated on the surface of Fe3O4@SiO2 to synthesize Fe3O4@SiO2@TiO2 nanoparticles; (4) Fe3O4@TiO2 was prepared by alkaline hydrothermal corrosion assisted crystallization method; (5) The VISTA antibody was modified on the nanoprobe by electrostatic adsorption, and finally the wreath-shaped Fe3O4@TiO2@VISTAmAb nanoprobe was successfully constructed.

2. The method for preparing a nanoprobe for diagnosis and treatment of pancreatic cancer targeting immune checkpoints according to claim 1, characterized in that: In the step (1), 1.62 g of ferric chloride hexahydrate, 1.625 g of sodium citrate and 3.0 g of sodium acetate were added to 50 mL of ethylene glycol and dissolved under magnetic stirring at 50 °C. The obtained clear yellow solution was transferred and sealed in a Teflon liner with a capacity of 100 mL, placed in a stainless steel autoclave, heated in an oven at 180 °C for 12 h, and then cooled to room temperature. The black product was collected with a magnet and washed three times with deionized water and ethanol, respectively.

3. The method for preparing a nanoprobe for diagnosis and treatment of pancreatic cancer targeting immune checkpoints according to claim 2, characterized in that: In the step (2), 45 mg of Fe3O4 nanoparticles are added to 80 mL of anhydrous ethanol, 16 mL of deionized water and 2.0 mL of 28 wt% concentrated ammonia water and mixed in a three-necked round-bottom flask, ultrasonicated for 15 min, 0.4 mL of TEOS is slowly added dropwise within 5 min, and the mixture is reacted for 10 h at room temperature with continuous mechanical stirring at 150 rpm. The Fe3O4@SiO2 nanoparticles with a core-shell structure are separated and collected by a magnet, and then washed three times with deionized water and ethanol, respectively.

4. The method for preparing a nanoprobe for diagnosis and treatment of pancreatic cancer targeting immune checkpoints according to claim 3, characterized in that: In the step (3), 150 mg of the prepared Fe3O4@SiO2 nanoparticles are redispersed in 200 mL of anhydrous ethanol, mixed with 0.9 mL of a 28 wt% concentrated ammonia solution, ultrasonicated for 15 min, 2.0 mL of TBOT is added dropwise within 5 min, and reacted at 45° C. under continuous mechanical stirring for 24 h. The product is separated and collected by a magnet, and then washed three times with deionized water and ethanol, respectively.

5. The method for preparing a nanoprobe for diagnosis and treatment of pancreatic cancer targeting immune checkpoints according to claim 4, characterized in that: In the step (4), 50 mg of Fe3O4@SiO2@TiO2 was ultrasonically treated with an ultrasonic cell crusher for 5 min, mixed with 10 mL of 1.0 M sodium hydroxide aqueous solution and added to a 20 mL polytetrafluoroethylene-lined stainless steel autoclave. The autoclave was heated in an oven at 150 °C for 6 h and then cooled to room temperature. The resulting product was soaked in 20 mL of 0.1 M hydrochloric acid aqueous solution for 30 min and then washed with deionized water until the pH value was close to 7, thereby dispersing the nanoparticles in deionized water.

6. The method for preparing a nanoprobe for diagnosis and treatment of pancreatic cancer targeting immune checkpoints according to claim 5, characterized in that: In the step (5), the immune checkpoint inhibitor VISTAmAb is loaded by physical adsorption, Fe3O4@TiO2 and VISTAmAb are mixed in sterile water at a mass ratio of 10:1, and the mixture is mechanically stirred at 250 rpm for 12 hours and then centrifuged to obtain the Fe3O4@TiO2@VISTAmAb nanoprobe.

7. The method for preparing a nanoprobe for diagnosis and treatment of pancreatic cancer targeting immune checkpoints according to claim 6, characterized in that: The average hydrated particle size of the prepared Fe3O4@TiO2@VISTAmAb nanoprobe was 225.3 nm, the Zeta potential was -28.9 mV, and the r2 / r1 value was 32.

03.

8. Application of Fe3O4@TiO2@VISTAmAb nanoprobe, characterized by: The invention is used in preparing medicine for treating pancreatic cancer.

9. The use of the Fe3O4@TiO2@VISTAmAb nanoprobe according to claim 8, characterized in that: Based on this probe, US irradiation and ultrasonic radiation are performed to loosen the dense fibrous matrix of pancreatic cancer, promote tumor microangiogenesis, and improve the immunosuppressive microenvironment.

10. The use of the Fe3O4@TiO2@VISTAmAb nanoprobe according to claim 9, characterized in that: It acquires images through magnetic resonance imaging (MRI) and is used in the preparation of visualization products for early screening of pancreatic cancer.

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