Lipid microbubble loaded with anti-fibrosis drug Nintedanib as well as preparation method and application of lipid microbubble

Abstract

The invention discloses a lipid microbubble loaded with an anti-fibrosis drug Nintedanib as well as a preparation method and application of the lipid microbubble, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: mixing DPPC, DSPE-PEG, DSPG, cholesterol and an anti-fibrosis drug Nintedanib according to a ratio, adding the mixture into a flask, dissolving with chloroform, and carrying out rotary evaporation at 55-60 DEG C to obtain a dry film mixture; adding a hydration solution into the film mixture for hydration, and repeatedly extruding the obtained lipid suspension by a phospholipid extruder to form a homogeneous lipid suspension; and filling the homogenized lipid suspension into a sealed bottle, pumping out upper-layer air, filling SF6 gas, and carrying out mechanical oscillation to form the lipid microbubble Nin-MBs loaded with the anti-fibrosis drug Nintedanib. The anti-fibrosis drug Nintedanib and the ultrasonic microbubble are prepared into the lipid microbubble, the compact ECM structure of the HCC is changed by targeting the CAF, the barrier hindering effect is overcome, the activity of the CAF is reduced, the tumor promoting effect of the CAF is weakened at the same time, the ECM is adjusted, and a new strategy is provided for treatment of the HCC.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a lipid microbubble loaded with an anti-fibrotic drug Nintedanib, a preparation method thereof, and an application thereof. Background Art

[0002] In the past two decades, the incidence rate of liver cancer has been showing a continuous upward trend. The pathological type is mainly hepatocellular carcinoma (HCC), which is the fourth leading cause of cancer-related deaths globally, and its five-year survival rate is only 18%. The continuous progression of the liver cancer process and the recurrence and metastasis of the lesions are the main reasons for the poor prognosis at present. Currently, the treatments for HCC include radical treatments such as surgical resection, radiofrequency ablation, and liver transplantation, as well as palliative treatments - interventional therapy, radiotherapy, chemotherapy, molecular targeted therapy, and immunotherapy. With the rapid development of nanotechnology, preparing cytotoxic drugs into nanoparticles (NPs) has become an effective method to increase the specific accumulation of drugs in tumors. Generally, in the nanotherapy of liver cancer, researchers pay more attention to the research and development of drug delivery carriers targeting cancer cells, aiming to improve the survival rate of patients by inhibiting tumor cells. However, this nanotherapy centered only on cancer cells is often not sufficient to completely eradicate malignant tumors.

[0003] In addition to heterogeneous tumor cells in liver cancer tissues, there are also many stromal components. The tumor stroma consists of a large amount of extracellular matrix (ECM), blood vessels, lymphatic vessels, nerves, other non-cellular components, and stromal cells. Activated cancer-associated fibroblasts (CAFs) in the liver cancer stroma will produce a large amount of ECM components, forming a fibrous physical barrier, which hinders the delivery of cytotoxic chemotherapeutic agents, molecular targeted biological agents, and nano-drugs to tumor cells, and weakens the targeting, permeability, and anti-tumor efficacy of these agents. Therefore, there is an urgent need to seek barrier penetration strategies to enhance drug perfusion in tumors to improve the treatment effect of liver cancer. A large number of studies have confirmed that the degree of deterioration of the tumor microenvironment (TME) has a significant impact on the efficacy of anti-tumor nanotherapy. The liver cancer TME is an important internal factor affecting the occurrence, development, invasion, and metastasis of HCC. As the main component of the TME, ECM is an important regulatory factor affecting the development of HCC. Therefore, seeking a method for appropriate TME remodeling, while considering the reversal of tumor-promoting effects and the improvement of drug deep penetration, is the key to effectively improving the current situation of HCC treatment and can also provide a promising strategy for HCC treatment. Summary of the Invention

[0004] In view of the above problems, the present invention aims to provide a lipid microbubble loaded with the anti-fibrotic drug Nintedanib, its preparation method and application. The anti-fibrotic drug Nintedanib and ultrasonic microbubbles are prepared into lipid microbubbles, which can change the dense ECM structure of HCC by targeting CAF, overcome the "barrier" hindrance effect, reduce the activity of CAF while weakening the tumor-promoting effect of CAF to regulate ECM, and provide a new strategy for the treatment of HCC.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] On the one hand, the present invention provides a preparation method of a lipid microbubble loaded with the anti-fibrotic drug Nintedanib, including the following steps:

[0007] S1: Mix DPPC, DSPE-PEG, DSPG, cholesterol and the anti-fibrotic drug Nintedanib, add them to a flask, dissolve with chloroform, and perform rotary evaporation at 55°C - 60°C to obtain a dry film mixture;

[0008] S2: Add a hydration solution to the film mixture for hydration, and the obtained lipid suspension is repeatedly extruded through a phospholipid extruder to form a homogeneous lipid suspension;

[0009] S3: Fill the homogeneous lipid suspension into a sealed bottle, evacuate the upper air, fill with SF6 gas, and perform mechanical oscillation to form a lipid microbubble Nin-MBs loaded with the anti-fibrotic drug Nintedanib.

[0010] Specifically, in step S1, the mass ratio of DPPC, DSPE-PEG, DSPG, cholesterol and Nintedanib is 10:4:4:3:5.

[0011] Specifically, in step S1, the volume ratio of the total mass of DPPC, DSPE-PEG, DSPG, cholesterol and the anti-fibrotic drug Nintedanib to chloroform is 2.6:1 mg / ml.

[0012] Specifically, in step S2, the hydration solution includes 10% glycerol and 90% 1×PBS solution.

[0013] Specifically, in step S2, the volume ratio of the hydration solution to the chloroform solution in step S1 is 1:2.

[0014] Specifically, the hydration condition in step S2 is: hydrate at 37°C for 30 minutes.

[0015] On the other hand, the present invention also provides a lipid microbubble loaded with the anti-fibrotic drug Nintedanib prepared by the preparation method as described above.

[0016] Specifically, the lipid microbubbles can burst under low-intensity focused ultrasound (LIFU) irradiation to release the drug Nintedanib.

[0017] On the other hand, the present invention also provides the use of the lipid microbubbles as described above in the preparation of inhibitors of tumor-associated fibroblast activity.

[0018] On the other hand, the present invention also provides the use of the lipid microbubbles as described above in the preparation of drugs for diagnosing and / or treating liver cancer.

[0019] The beneficial effects of the present invention are as follows:

[0020] 1. The present invention successfully prepared a kind of lipid microbubble Nin-MBs loaded with the anti-fibrotic drug Nintedanib, which is in the shape of a regular sphere, has a smooth surface, good dispersibility and good imaging effect. Under real-time ultrasound monitoring, low-intensity focused ultrasound (LIFU) irradiation can be used to target and precisely burst to release the drug, realizing the integration of diagnosis and treatment.

[0021] 2. The Nin-MBs prepared in the present invention combined with ultrasound microbubble targeted burst (UTMD) can target tumor-associated fibroblasts (CAF cells), inhibit the activation and proliferation of CAF cells, reduce the production of Collagen Iα, Elastin, and Fibronectin, and break the tumor obstacle barrier of drug delivery to enhance tumor penetration.

[0022] 3. The Nin-MBs prepared in the present invention combined with UTMD, while inhibiting the activation and proliferation of CAF cells and enhancing tumor delivery, also successfully reverses the CAF tumor-promoting effect, reduces tumor cell stemness, inhibits tumor cell migration and invasion, inhibits the occurrence of EMT, delays the tumor growth rate, and provides a new strategy for the treatment of HCC. Description of the Drawings

[0023] Figure 1 It is the characterization result of Nin-MBs prepared in the present invention; wherein, A is the optical microscope image of Nin-MBs (400×); B is the transmission electron microscope of Nin-MBs (3.0k×); C is the Zeta potential of blank lipid microbubbles MBs and Nin-MBs; D is the particle size distribution of blank lipid microbubbles MBs; E is the particle size distribution of Nin-MBs; F is the detection result of the particle size stability of Nin-MBs.

[0024] Figure 2Detection results of the encapsulation efficiency and drug loading of Nin-MBs in the present invention; among them, A is the ultraviolet absorption curves of Nintedanib, MBs and Nin-MBs; B is the standard ultraviolet absorption curve of Nintedanib.

[0025] Figure 3 Drug release of Nin-MBs and Nin-MBs+UTMD in the present invention.

[0026] Figure 4 Ultrasonic imaging of in vitro gels with different concentrations of Nin-MBs (microbubbles / ml) in the present invention.

[0027] Figure 5 In vivo ultrasonic imaging of Nin-MBs in the present invention; among them, A is the injection of cell suspension under ultrasonic guidance; B is the PW detection result; C is the CEUS imaging detection result of primary liver cancer.

[0028] Figure 6 Morphology and identification results of CAF cells in the present invention; among them, A is the light microscope photograph of CAF cells (100×); B is the immunofluorescence detection of CAF cells (100×).

[0029] Figure 7 Toxicity study results of Nintedanib on CAF cells and HCCLM3 cells in the present invention; among them, A is the toxicity evaluation result of Nintedanib on CAF cells; B is the toxicity evaluation result of Nintedanib on HCCLM3 cells; (*, P<0.05; **, P<0.01; ***, P<0.001).

[0030] Figure 8 Western blot was used to evaluate the expression of the action targets PDGFR-β and FGFR-2 of Nintedanib drug in hepatocellular carcinoma HCCLM3 and CAF.

[0031] Figure 9 In vitro CAF cell uptake results of free Rb, Rb-MBs and Rb-MBs+UTMD in the present invention.

[0032] Figure 10 Nin-MBs+MTMD inhibits the proliferation of CAF cells in the present invention; among them, A is the analysis of the proliferation of CAF cells in different groups by CCK8 method; B is the colony formation assay to verify the proliferation of CAF cells treated in different groups; (*, P<0.05).

[0033] Figure 11 Flow cytometry analysis results of the cell cycle of CAF cells treated differently in the present invention.

[0034] Figure 12 This is for detecting the expression of circulating-related proteins in CAF cells after different treatments in the present invention by Western blot.

[0035] Figure 13 This is the result of the effect of Nin-MBs combined with UTMD on the activation of CAF cells in the present invention.

[0036] Figure 14 This is the Western blot detection result (200×) of Nin-MBs combined with UTMD reducing the production of ECM-related proteins in the present invention.

[0037] Figure 15 This is that Nin-MBs combined with UTMD enhanced the permeability of multicellular tumor spheroids in the present invention.

[0038] Figure 16 This is that Nin-MBs combined with UTMD inhibited the migration and invasion of hepatocellular carcinoma in the present invention; among them, A is the result of detecting the migration ability of HCCLM3 cells by different treatment methods through scratch test; B is the result of detecting the migration and invasion abilities of different treatment groups on HCCLM3 cells by migration and invasion experiments; (*, P<0.05; **, P<0.01; ***, P<0.001).

[0039] Figure 17 This is the light microscope (100×) detection result of the effect of CAF-CM on the morphology of HCCLM3 in the present invention; among them, A is the morphology of HCCLM3 liver cancer cells, and B is the stromal cells with HCCLM3 liver cancer cells elongated to spindle shape.

[0040] Figure 18 This is the Western blot detection result of the inhibitory effect of Nin-MBs combined with UTMD on HCC stem cells and EMT in the present invention.

[0041] Figure 19 This is the growth condition of tumor spheroids after different intervention methods intervened in tumor spheroids in the present invention.

[0042] Figure 20 This is the Western blot detection result of the combination of Nin-MBs and UTMD inhibiting the secretion of matrix regulatory factors by CAF in the present invention.

[0043] Figure 21 This is the USI monitoring result of HCC during the treatment of different groups of nude mice in the present invention, including B-mode, CDFI, CPA and USE.

[0044] Figure 22These are the MRI evaluation results of HCC in different groups of nude mice during treatment in the present invention, including T1WI, T2WI, and T2-mDIXON.

[0045] Figure 23 This shows the effect of Nin-MBs combined with UTMD on HCC tumor growth in the present invention; among them, A is the size comparison between different groups; B is the tumor volume growth curve; C is the curve of the change in the weight of orthotopic tumors in nude mice; (*, P<0.05; **, P<0.01; ***, P<0.001).

[0046] Figure 24 These are the results of IHC staining (400×) of primary liver cancer sections in the present invention.

[0047] Figure 25 These are the results of H&E staining (200×) of the heart, liver, lung, spleen, and muscle of mice in different groups in the present invention. Detailed implementation manners

[0048] In order to enable those of ordinary skill in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0049] The experimental antibodies used in this study include: Collagen Iα, Fibroenctin, CD31, α-SMA, Vimentin, PDGFR-β, FGFR-2, PCNA, CD133, SOX2, E-cadherin, N-cadherin were purchased from Proteintech Group. MMP-2, MMP-9, FAP, P53 were purchased from Cell Signaling Technology. HGF, IL-6, Cyclin D1 were purchased from Santa Cruz Biotechnology. Goat anti-mouse / rabbit IRDye 800CW secondary antibody was purchased from Licor.

[0050] The hepatocellular carcinoma tissue specimens used in this study were sourced from: collecting tissue specimens of 10 HCC patients who underwent surgical treatment in the Department of Hepatobiliary Surgery of the First Affiliated Hospital of Harbin Medical University from 2020 to 2021. The patients had not received other treatments before surgery. This study obtained the review approval of the Ethics Committee of the First Affiliated Hospital of Harbin Medical University, and all patients signed informed consent forms. The tumor specimens used were all pathologically confirmed as hepatocellular carcinoma, and all specimens were stored in DF-12 culture medium on ice after being removed from the body and transported to the laboratory.

[0051] The HCC cell line HCCLM3 used in this study was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Hepatocellular carcinoma CAF cells were obtained from specimen tissues.

[0052] The in vivo animal experiments of this study were approved by the Animal Ethics Committee of the First Affiliated Hospital of Harbin Medical University. Thirty-six 6-week-old male BALB / c nude mice were housed in the animal laboratory of the First Affiliated Hospital of Harbin Medical University, and the feeding feed and drinking water were strictly disinfected.

[0053] The specific preparation methods of the cell culture reagents used in this study are as follows:

[0054] (1) PBS buffer: Add 20 g of PBS preparation to 2000 ml of deionized water, stir magnetically at room temperature until fully dissolved, filter twice through a filter in a sterile laminar flow hood, and store at 4°C.

[0055] (2) Trypsin solution: Dissolve 0.5 g of trypsin powder plus 0.04 g of EDTA in 200 ml of PBS buffer, stir magnetically overnight, filter through a filter in a sterile laminar flow hood, and store at 4°C for later use.

[0056] (3) 0.15% type IV collagenase solution: Add 15 mg of type IV collagenase powder to 10 ml of DF-12 solution, fully dissolve it, filter through a filter in a sterile laminar flow hood, store at 4°C, and it is valid within one week.

[0057] (4) Prepare cell culture medium: In a sterile laminar flow hood sterilized by ultraviolet light, filter 55 ml of fetal bovine serum and 5.5 ml of penicillin-streptomycin mixed solution into 500 ml of high-glucose DMEM culture medium in turn; filter 88 ml of fetal bovine serum and 5.8 ml of penicillin-streptomycin mixed solution into 500 ml of DF-12 culture medium in turn, mix well and store at 4°C in the refrigerator.

[0058] (5) Protein lysate: The preparation ratio of the lysate is as follows: RIPA lysate: phosphatase inhibitor: protease inhibitor = 100:10:1, add them to an EP tube in turn, shake well and set aside.

[0059] (6) Membrane-breaking agent 0.5% TritonX-100: Add 250 μl of TritonX-100 to 50 ml of prepared PBS solution, disperse in a 37°C water bath for 2 hours, mix well and set aside.

[0060] The specific preparation methods of the Western blot-related reagents used in this study are as follows:

[0061] (1) Protein electrophoresis buffer: Weigh and add 7.2 g of glycine, 1.5 g of Tris-base powder, and 0.5 g of SDS into 500 ml of deionized water in sequence, stir magnetically until fully dissolved, and store at room temperature for later use.

[0062] (2) Protein electrotransfer buffer: Weigh and add 1.5 g of Tris base powder and 7.2 g of glycine into 400 ml of deionized water, and add 100 ml of methanol additionally. Stir magnetically until fully dissolved, and store at room temperature for later use.

[0063] (3) PBST solution: Add 20 g of powder into 2000 ml of deionized water, then add 2 ml of Tween 20, and stir magnetically at room temperature until fully dissolved for later use.

[0064] (4) Protein blocking solution: Measure 2.5 g of skim milk powder and add it into 50 ml of PBST, stir evenly to prepare a 5% skim milk powder blocking solution. Note that it should be prepared and used immediately.

[0065] Example 1:

[0066] Example 1 provides a preparation method of lipid microbubbles loaded with the anti-fibrotic drug Nintedanib, and the prepared Nin-MBs are characterized and detected. The specific contents are as follows:

[0067] 1.1 Experimental method

[0068] 1.1.1 Preparation of Nin-MBs

[0069] First, weigh 10 mg of DPPC, 4 mg of DSPE-PEG, 4 mg of DSPG, 3 mg of cholesterol, and 5 mg of Nintedanib in sequence, mix and add them into a 50 ml flask, dissolve with 10 ml of chloroform, and perform rotary evaporation at 55 °C - 60 °C to obtain a dry film mixture. Add 5 ml of solution (10% glycerol, 90% 1×PBS solution) to the dry film mixture and hydrate at 37 °C for 30 minutes. The lipid suspension is repeatedly extruded through a 400 nm phospholipid extruder to form a homogeneous lipid suspension. Subsequently, it is filled into a sealed bottle, the upper air is evacuated, SF6 gas is filled, and it is placed on a dental amalgam mixer and mechanically oscillated for 60 seconds to form drug-loaded microbubbles (Nin-MBs).

[0070] As a comparison, this example also prepared simple blank lipid microbubbles (MBs) and fluorescently labeled lipid microbubbles (Rb-MBs).

[0071] During the preparation of Nin-MBs, simple blank lipid microbubbles (MBs) are generated without adding the anti-fibrotic drug Nintedanib, and the prepared microbubbles are stored at 4 °C.

[0072] When preparing fluorescently labeled lipid microbubbles (Rb-MBs), 5 mg of rhodamine dye (Rb) should be used to replace Nintedanib, and care should be taken to avoid light during the operation process.

[0073] 1.1.2 Microbubble Characterization Detection

[0074] (1) Morphology and structure: Observe the distribution of Nin-MBs in PBS solution using an optical microscope. After mixing the prepared Nin-MBs evenly, use a pipette to aspirate a small amount of the microbubble suspension and drop it onto a glass slide. Cover it with another glass slide and then observe it under the optical microscope. Observe the morphology and structure of Nin-MBs using a transmission electron microscope. Drop the suspended Nin-MBs sample onto a copper grid, allow it to dry naturally at room temperature to form a film, and then observe the morphology of Nin-MBs on the machine and take pictures for recording.

[0075] (2) Detect particle size and potential: After evenly suspending the prepared Nin-MBs sample, use a Malvern nano particle size instrument and a Zeta potential analyzer to detect the particle size and Zeta potential of Nin-MBs and MBs respectively, and record the results. Store the prepared Nin-MBs sample in the refrigerator at 4 °C and continuously detect the particle size change of the microbubbles for 4 days.

[0076] (3) Encapsulation efficiency and drug loading of Nin-MBs: Prepare Nintedanib standard solutions with different concentration gradients, use ultraviolet spectrophotometry to measure the absorbance standard curve of Nintedanib, calculate the mass of Nintedanib in Nin-MBs based on the Nintedanib standard curve and the absorbance of Nin-MBs, and then calculate the encapsulation efficiency and drug loading of Nin-MBs respectively.

[0077] Encapsulation efficiency (%) = (mass of Nintedanib encapsulated in lipid microbubbles / mass of Nintedanib initially added) × 100%.

[0078] Drug loading (%) = (mass of Nintedanib encapsulated in lipid microbubbles / (total mass of phospholipids + mass of Nintedanib initially added)) × 100%.

[0079] 1.1.3 Drug Release Experiment of Nin-MBs

[0080] The release of nintedanib in drug-loaded microbubbles Nin-MBs was detected by dialysis. Grouping was carried out according to the following situations: (a) Nin-MBs, (b) Nin-MBs + ultrasound-targeted microbubble destruction (UTMD). Take 1 ml of the pre-suspended Nin-MBs solution and place it in different dialysis membranes respectively. After the device in group (b) is completed, low-intensity focused ultrasound (LIFU) irradiation (1 W / cm 2 , 60 seconds) was carried out. Immediately after the irradiation, the dialysis bag was placed into a beaker pre-filled with PBS solution. The beaker was placed on a thermostatic shaker and shaken. At the 2nd, 4th, 6th, 8th, 12th, 24th, and 48th hours after shaking, 1 ml of the sample was taken from the beaker respectively. After sampling, an equal volume of PBS liquid at a constant temperature was replenished simultaneously. The obtained samples were measured with an ultraviolet spectrophotometer, and the cumulative release curve of the drug was plotted.

[0081] 1.1.4 Detection of in vitro ultrasound imaging function of Nin-MBs

[0082] (1) Prepare an agarose model with a concentration of 1%. Weigh 5 g of agarose powder and add it to 500 ml of deionized aqueous solution. Heat it on an induction cooker for 5 - 10 minutes, and keep stirring during the addition. Stop heating after it is fully dissolved, pour it into a 1 ml pipette tip box, insert the pipette tips while it is still hot, and pull out the pipette tips after it completely solidifies at room temperature for later use.

[0083] (2) Suspend the prepared Nin-MBs with PBS solution, count with a cell counting chamber and dilute according to the concentration ratio. Take 1 ml of the pre-diluted and pre-suspended Nin-MBs solutions with different concentrations and add them to the pre-prepared 1% agarose gel model in sequence. Degassed saline solution was used as a blank control. The imaging effect was detected using a Canon Aplio500 ultrasound diagnostic imager (PLT-1005BT linear array probe, probe frequency: 7.5 MHz, mechanical index: 0.08), and the images in the stored grayscale mode and contrast-enhanced ultrasound mode were recorded. Then, LIFU (1.0 W / cm 2 , 60 seconds) was used to irradiate from the side of the model, and the images after LIFU irradiation were recorded and stored.

[0084] 1.1.5 Detection of in vivo ultrasound imaging function of Nin-MBs

[0085] When the tumors of nude mice reached a certain volume, the nude mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium. After the anesthesia was completed, the nude mice were fixed on a pre-sterilized operating table. The abdomen of the nude mice was evenly smeared with coupling agent. Under the real-time monitoring of US, 100 μl of the pre-prepared Nin-MBs suspension was injected into the tail vein of the nude mice, and the imaging situation was continuously observed with a Canon Aplio500 ultrasound diagnostic imager in the grayscale and contrast-enhanced modes.

[0086] 1.1.6 Statistical methods

[0087] The experimental results were expressed as mean ± standard deviation, and all experiments were repeated 3 times or more. Among them, the t-test was used to compare between two groups of samples, and one-way ANOVA was used for comparison among multiple groups of samples. The SPSS 23.0 software was used to perform statistical analysis on the obtained data. P < 0.05 indicated statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

[0088] 1.2 Experimental results

[0089] 1.2.1 Preparation and basic characterization of Nin-MBs

[0090] The lipid ultrasound microbubbles Nin-MBs loaded with the anti-fibrotic drug Nintedanib were successfully prepared by thin-film hydration combined with mechanical oscillation. The blank lipid microbubbles MBs appeared as a milky white suspension (as shown in D of the appendix Figure 1 ); Nin-MBs appeared as a light yellow suspension (as shown in E of the appendix Figure 1 ), and it was observed under a light microscope that the distribution of Nin-MBs microbubbles was relatively uniform without obvious adhesion and aggregation, as shown in A of the appendix Figure 1 . Under TEM, Nin-MBs showed a regular spherical structure with a smooth surface and good dispersion without aggregation, as shown in B of the appendix Figure 1 . The sizes of the prepared MBs and Nin-MBs measured by a Malvern particle size analyzer were approximately 397 nm ± 23 nm and 425 nm ± 18 nm respectively, as shown in D and E of the appendix Figure 1 , and the Zeta potentials were approximately -4.73 ± 0.36 mV and -3.32 ± 0.26 mV respectively, as shown in C of the appendix Figure 1 . Continuous monitoring of the particle size of Nin-MBs found that there was no significant change in the particle size within the first 3 days, and the size began to increase on the 4th day, and the concentration also decreased, as shown in F of the appendix Figure 1 .

[0091] The detection results of the encapsulation efficiency and drug loading rate of Nin-MBs are shown in the appendix Figure 2 . The ultraviolet spectrophotometer showed that Nin-MBs had characteristic absorption peaks of both Nintedanib and MBs, as shown in A of the appendix Figure 2 . The drug standard curve of Nintedanib was Y = 1.0219X + 0.0377 (R 2 = 0.99259), as shown in B of the appendix Figure 2 . By calculation, the encapsulation efficiency of Nintedanib in Nin-MBs was 67.1% and the drug loading rate was 10.9%.

[0092] 1.2.2 Drug release of Nin-MBs

[0093] The drug release of Nin-MBs and Nin-MBs+UTMD is shown in the appendix Figure 3 As shown, from the appendix Figure 3 It can be seen that without LIFU irradiation, Nin-MBs generally showed a slow release effect. The amount of Nintedanib released at 6 hours was only about 25.74%, and the amount released at 48 hours was about 94.35%. After Nin-MBs were irradiated by LIFU and acted on by UTMD, the drug release amount increased significantly, achieving rapid drug release in a short time. The release amount at 6 hours could reach 72.41%, and the release amount at 48 hours was about 97.5%.

[0094] 1.2.3 In vitro and in vivo ultrasound imaging of Nin-MBs

[0095] 1.2.3.1 In vitro gel ultrasound imaging of Nin-MBs

[0096] The ultrasound imaging of Nin-MBs (microbubbles / ml) in vitro gel at different concentrations is shown in the appendix Figure 4 As shown. From the appendix Figure 4 It can be seen that the blank control group, that is, normal saline, showed anechoic in both grayscale and contrast states. The echo of Nin-MBs was relatively uniform. As the concentration of microbubbles increased, the echo signal intensity in both grayscale and contrast modes gradually increased. After LIFU ultrasound irradiation, the intensity of the echo signal in the contrast mode decreased compared with that before irradiation.

[0097] 1.2.3.2 In vivo ultrasound imaging of Nin-MBs

[0098] The in vivo ultrasound imaging of Nin-MBs is shown in the appendix Figure 5 As shown. From the appendix Figure 5 It can be seen that a nude mouse orthotopic HCC model was successfully constructed by the method of injecting cell suspension percutaneously under US guidance (as shown in A of the appendix Figure 5 ); the orthotopic tumor model constructed in this study had rich blood supply compared with the traditional subcutaneous tumor, and feeding arteries could be seen around the tumor (as shown in B of the appendix Figure 5 ); after injecting 100 μl of Nin-MBs suspension into the tail vein under the contrast-enhanced ultrasound (CEUS) mode, good imaging effects could be seen in the tumor (as shown in C of the appendix Figure 5 ).

[0099] Example 2:

[0100] Cell experiments were carried out on the drug-loaded microbubbles Nin-MBs prepared in Example 1 to study their effects on the proliferation of CAF cells. Specifically, this part of the research included the following contents:

[0101] 2.1 Experimental methods

[0102] 2.1.1 Extraction and purification of CAF cells

[0103] The freshly obtained surgically resected tumor tissue samples were washed three times with PBS solution containing 1% penicillin-streptomycin double antibody, and the necrosis and fascia in the tissue blocks were removed. The obtained tissue samples were minced with ophthalmic scissors to tissue blocks with a volume of about 1 mm 3 . They were digested with pre-prepared 0.15% type IV collagenase (37 °C, 30 minutes). The digested suspension was passed through a 400-mesh cell sieve and centrifuged in a 4 °C centrifuge (300 g, 10 minutes). After centrifugation, the supernatant was discarded. After washing twice with PBS, the obtained primary CAF cells were resuspended in DF-12 complete medium containing 15% FBS and 1% penicillin-streptomycin double antibody, and cultured in a 37 °C, 5% CO2 cell culture incubator. When the cell fusion reached 70%-80%, trypsin was used to digest the cells by the "differential adhesion" method for cell purification, and cells of passages 3-8 were selected for subsequent experiments.

[0104] 2.1.2 Cell culture

[0105] (1) Cell resuscitation: The cells cryopreserved in the liquid nitrogen tank were taken out and immediately placed in a 37 °C water bath and gently shaken to melt. After the liquid in the cryopreservation tube was completely melted and the surface was sprayed with alcohol for disinfection, it was transferred into a sterile ultra-clean bench that had been ultraviolet disinfected in advance. After using a dropper to suck the liquid in the cryopreservation tube and adding it to a centrifuge tube containing 8 ml of complete culture medium prepared in advance, it was blown and mixed evenly and then centrifuged (1000 r / min, 3 minutes). After centrifugation, the supernatant was poured out, 5 ml of complete culture medium was sucked to resuspend the cells at the bottom of the centrifuge tube, blown and mixed evenly, placed in a culture flask, and transferred to a 37 °C, 5% CO2 cell culture incubator for continued culture. The medium was changed 24 hours later.

[0106] (2) Cell passage: When the cell confluence reached 70%-80%, passage operation was carried out. After washing with PBS solution, it was digested with trypsin. When the cells became round and detached from the bottom, digestion was stopped by neutralizing with complete culture medium, and then it was mixed evenly and centrifuged (1000 r / min, 3 minutes). After centrifugation, the supernatant was discarded. After resuspending the cells at the bottom with complete culture medium and mixing evenly, they were successively subpackaged into different culture flasks as needed, the liquid in the flasks was supplemented to 5 ml, mixed evenly, and transferred to the cell culture incubator for continued culture.

[0107] (3) Cell cryopreservation: Rinse with PBS solution, digest with trypsin, neutralize with complete culture medium to stop digestion, then mix well and centrifuge (1000 r / min, 3 minutes). After centrifugation, discard the supernatant, aspirate 1 ml of cell cryopreservation solution to resuspend the cells at the bottom, mix well and transfer them into a pre-prepared sterile cryopreservation tube. Label the name of the cells, time and operator's name on the tube body. Seal the tube with sealing film and put it into a cell cryopreservation box, then place it in an -80°C refrigerator. After 24 hours, transfer it to a liquid nitrogen tank for storage.

[0108] 2.1.3 Immunofluorescence identification of CAF cells

[0109] Place a sterile glass slide dedicated for cell culture inserts at the bottom of a 12-well plate. Inoculate the pre-digested and resuspended CAF cell suspension into the 12-well plate (1×10 4 cells / well). Incubate overnight in a cell culture incubator until the cells adhere. Discard the culture medium, gently rinse 3 times with PBS solution, and then fix with 4% paraformaldehyde at room temperature for 20 minutes. After rinsing the fixed cells 3 times with PBS solution, perform membrane permeabilization treatment at room temperature (0.5% TritonX-100, 10 minutes). After membrane permeabilization, rinse 3 times with PBS solution and block with 3% BSA on a horizontal shaker at room temperature for 1 hour. After blocking, add the pre-diluted primary antibody and incubate overnight at 4°C in the refrigerator. The next day, after recovering the primary antibody, wash it on a shaker with PBS solution (3 times, 5 minutes each time). Incubate the corresponding fluorescent secondary antibody in the dark at room temperature for 1 hour. After discarding the secondary antibody, wash it with PBS solution (3 times, 5 minutes each time), and pay attention to avoiding light during the operation. After washing, add anti-fluorescence quenching DAPI for nuclear staining, and after staining, perform cover slip mounting and take pictures with a fluorescence microscope.

[0110] 2.1.4 Cytotoxicity experiment of Nintedanib at different concentrations

[0111] Prepare a standard stock solution of Nintedanib drug with DMSO at a concentration of 5 mg / ml and store it at room temperature. To prevent crystal precipitation, incubate it in a 37°C incubator for 30 minutes before adding the drug. Filter it aseptically with a 0.22 μm filter before adding the drug.

[0112] Respectively, inoculate the CAF and HCCLM3 cell suspensions digested with trypsin into the 12-well plate at 3×10 3 cells / well and 4×10 3Cells were seeded at a density of [X] cells / well in a 96-well plate. Different concentrations of Nintedanib (0 μmol / L, 0.5 μmol / L, 1 μmol / L, 2 μmol / L, 2.5 μmol / L, 3 μmol / L, 3.5 μmol / L, 4 μmol / L, 5 μmol / L) were added to the wells in sequence. Six parallel wells were set up for each drug concentration, and the average value was calculated. The OD values at 24 and 48 hours after the intervention were measured by the CCK8 method. The effects of different concentrations of Nintedanib on the proliferation of CAF and HCCLM3 cells were calculated.

[0113] 2.1.5 Cellular uptake experiment of Rb-MBs combined with UTMD

[0114] CAF cells were seeded in a 6-well plate at a density of 2×10 4 cells / well. After the cells adhered to the wall, the intervention was carried out. The experiment was divided into four groups: Con group, free Rb group, Rb-MBs group, and Rb-MBs+UTMD group. The Rb concentration in all groups was 5 μg / mL. The Rb-MBs+UTMD group was irradiated with LIFU ultrasound (1.0 W / cm 2 , for 60 seconds). After 24 hours of the experiment intervention, the cells were rinsed 3 times with pre-cooled PBS solution to terminate cell uptake. After the rinsing, the cells were digested with trypsin, centrifuged, and collected. The fluorescence intensity in the cells was detected by flow cytometry. The above operation process needed to be carried out in the dark.

[0115] 2.1.6 Experimental grouping and the effect of Nin-MBs combined with UTMD on the proliferation of CAF cells

[0116] The CAF cell suspension digested with trypsin was seeded at a density of 3×10 3 cells / well in a 96-well plate. After overnight incubation in the incubator until the cells adhered to the wall, the intervention was carried out according to different groups. The cells were divided into the following five groups: (a) only medium (Con group); (b) MBs+UTMD group; (c) Nin-MBs group; (d) Nin-MBs+UTMD group; (e) Nintedanib group. The Nintedanib concentration in each experimental group was 1 μmol / L. Among them, groups (b) and (d) were irradiated with ultrasound LIFU (1.0 W / cm 2 , for 60 seconds) immediately after drug administration. Then, the OD values of the cells at 24, 48, and 72 hours after the intervention were measured by the CCK8 method, and the proliferation curve of CAF cells was drawn according to the measured results.

[0117] (1) Colony formation assay

[0118] CAF cells were seeded in a six-well plate at a density of 5×10 2(Cells / well). After the cells adhered to the wall, they were intervened according to the above groups. After 48 hours, the culture medium was changed, and then changed again every 72 hours. After 7-15 days, when the cells formed larger colonies, they were fixed with methanol, stained with crystal violet, washed with distilled water, air-dried, and photographed to observe the colony formation.

[0119] (2) Cell cycle detection

[0120] Seed CAF cells into a 6-cm culture dish (3×10 5 (Cells / dish). After the cells adhered to the wall, they were intervened separately according to the above groups. 48 hours after the intervention was completed, the cells were washed and digested successively with PBS solution and trypsin digestion solution. After the digested cell suspension was centrifuged, the supernatant was discarded. Then, it was washed again with PBS solution, centrifuged, and the supernatant was discarded. Add 1 ml of DNA Staining solution and 10 μl of Permeabilizatiob solution into the centrifuge tube, gently pipette and mix well, and incubate in the dark at room temperature for 30 minutes. After the incubation, collect through a filter mesh into a flow tube and detect on a flow cytometer to obtain the DNA content and percentage of cells in different cycles. All operations were carried out in a laminar flow hood.

[0121] 2.1.7 Effect of Nin-MBs targeting CAF cells on the changes in the components of the tumor extracellular matrix (ECM)

[0122] Seed CAF cells evenly in a 10-cm dish. When the cell confluence reached 80%, they were intervened separately according to the groups described in 2.1.6. 48 hours after the intervention, proteins were extracted respectively, and the protein expression levels of Collagen Iα, Elastin, and Fibronectin were detected by Western blot technology.

[0123] 2.1.8 Effect of Nin-MBs targeting CAF cells on co-cultured cell spheres

[0124] (1) Preparation of multi-cellular co-cultured cell spheres

[0125] To evaluate the inhibitory effect of Nin-MBs combined with UTMD on CAF cell barrier, multicellular tumor spheroids were constructed by mixing CAF cells and HCCLM3 cells. The 1% agarose solution prepared with pure DMEM solution was added to the 96-well plates while it was still hot (50 μl / well). After adding, gently shake the 96-well plates horizontally and vertically to remove air bubbles. Disinfect with ultraviolet light in the laminar flow hood for 30 minutes and wait for it to cool and solidify naturally for later use. The well-grown CAF cells and HCCLM3 cells were digested into single-cell suspensions respectively. After mixing them according to the ratio of 1:1, add them to the prepared 96-well plates (200 μl, 4×10³ cells / well), place them in the incubator for continuous culture, observe the growth of tumor spheroids under the microscope, and change the medium every 3 days. When changing the medium, operate gently and carefully to avoid dispersing the tumor spheroids.

[0126] (2) Evaluation of the penetration of co-cultured cell spheroids by Nin-MBs combined with UTMD

[0127] When the diameter of the co-cultured tumor spheroids of CAF cells and HCCLM3 cells constructed in advance reached about 150 μm, different intervention treatments were performed on the tumor spheroids according to the group settings. After the intervention, the tumor spheroids were continuously cultured for 72 hours, then the culture medium was discarded. After washing with PBS solution, add Rb solution (10 μg / mL) and incubate in the dark for 12 hours. Fix with 4% paraformaldehyde and wash three times with PBS solution. Perform Z-stack scanning on the tumor spheroids with a laser confocal microscope to observe the effect of different intervention methods on the penetration of the fluorescent dye Rb in the tumor spheroids.

[0128] (3) Effect of Nin-MBs combined with UTMD on the growth rate of co-cultured cell spheroids

[0129] After performing intervention treatments on the tumor spheroids according to the group settings, transfer them to the cell incubator for continuous culture. After 7 days, observe the effect of different intervention methods on the growth rate of the tumor spheroids. Observe the size of the tumor spheroids under the microscope and take pictures respectively.

[0130] 2.1.9 Effect of Nin-MBs combined with UTMD on the tumor-promoting effect of CAF cells

[0131] (1) Preparation of conditioned medium

[0132] Collect the culture media of CAF cells after being intervened for 72 hours in different groups (Con, Nin-MBs, Nin-MBs+UTMD, and Nintedanib groups), centrifuge (1500 r / min, 10 minutes), filter the supernatant collected after centrifugation with a filter in the laminar flow hood, add an appropriate amount of serum to the filtered liquid, mix well, and store at -80 °C for later use.

[0133] (2) Cell scratch assay

[0134] HCCLM3 cells were seeded in a 6-well plate (6×10 5 cells / well). Before seeding, the cell suspension was thoroughly pipetted to ensure uniform distribution of the cells. After seeding, the plate was transferred to an incubator and incubated overnight. When the cells reached confluence, a 1-ml pipette tip was used to make a scratch perpendicular to the center of the well with uniform force. After making the scratch, the cells were gently rinsed 2-3 times with PBS solution. Then, the conditioned media of different groups obtained in advance were added successively. Photos were taken at 0, 24, and 48 hours after the procedure to observe the healing of the scratch.

[0135] (3) Transwell assay

[0136] Migration assay: HCCLM3 cells were seeded in a 6-well plate. When the cells reached 60% confluence overnight, the prepared conditioned media were added respectively. After 24 hours of intervention, the media were discarded, and the cells were washed with PBS solution, digested with trypsin, centrifuged, and collected. The cells were resuspended in pure DMEM culture medium and counted to adjust the cell concentration (1.0×10 5 cells / ml). 700 μl of DMEM complete culture medium containing 10% FBS was added to the lower chamber, and 200 μl of the single-cell suspension of different groups was added successively to the upper chamber. After the procedure, the plate was transferred to an incubator for continued culture. After 48 hours of culture, the media were removed, and the cells were gently washed with PBS solution. Then, 1 ml of methanol solution was added to the lower chamber for fixation for 20 minutes. After removing the methanol from the chamber, the cells were stained with 0.5% crystal violet solution for 20 minutes. The crystal violet was washed off with distilled water, and the cells on the inner side of the upper chamber were gently wiped off with a dry cotton swab. After air-drying, the cells were photographed under a microscope.

[0137] Invasion assay: Pipette tips, chambers, and serum-free DMEM medium were pre-cooled in advance. The frozen Matrgel was melted in a 4°C refrigerator and diluted with serum-free medium at a ratio of 1:7. It was gently pipetted to mix well, and the generation of bubbles was avoided as much as possible. 50 μl of the diluted and suspended liquid was slowly added to the upper chamber of the chamber, and the chamber was gently tapped to remove the interference of bubbles. Then, it was carefully transferred to an incubator and allowed to solidify for standby. The remaining steps were the same as those of the above migration assay.

[0138] 2.1.10 Extraction of total cellular proteins

[0139] (1) Extraction of total cellular proteins

[0140] Cells with good growth status were seeded in a 10-cm culture dish. When the cell confluence reached 80%, the cells were treated according to the experimental groups.

[0141] After 48 hours of the intervention, the culture medium was removed, and the cells digested with trypsin were centrifuged. After washing with PBS solution, the pre-prepared lysis buffer was added to the cell pellet at the bottom of the centrifuge tube. After pipetting and blowing to mix evenly, it was transferred to a 1.5 ml EP tube and placed on ice for lysis. During lysis, it was shaken on a vortex mixer once every 5 minutes for a total of 6 times. After shaking, it was centrifuged in a 4 °C centrifuge (14000 g, 15 minutes). After centrifugation, the volume of the supernatant was measured, and the liquid was transferred to a new labeled 1.5 ml EP tube and stored in an ice box.

[0142] (2) Protein sample concentration determination by BCA method

[0143] Measure and plot the protein concentration standard curve using the protein standard in the kit according to the instructions of the manual.

[0144] Prepare the BCA working solution in advance according to the ratio (Solution A: Solution B = 50:1) for standby. Add 2 μl of the protein sample to be tested, 18 μl of PBS solution, and 200 μl of the pre-prepared BCA working solution to each well of the 96-well plate in sequence. Operate gently to avoid foaming, and incubate at 37 °C for 30 minutes. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value of the protein sample to be tested at a wavelength of 490 nm, and calculate the concentration of the protein sample to be tested according to the obtained standard protein concentration curve in sequence. To the protein sample whose concentration has been measured and the volume has been calculated, add 5×SDS protein loading buffer according to a volume ratio of 4:1, mix evenly, then boil in boiling water for 10 minutes. After boiling, take it out and place it on ice, and transfer it to a -20 °C refrigerator for storage and standby.

[0145] 2.1.11 Western blot experiment

[0146] (1) SDS-PAGE gel electrophoresis

[0147] First, check for leakage of the gel preparation device, and prepare the gel according to the formula described in the PAGE gel kit instructions. Carefully remove the prepared gel and fix it in the electrophoresis tank, noting that the glass plate should be inserted to the bottom, and then add the pre-prepared electrophoresis buffer. Take out the pre-extracted protein sample, shake it evenly on a vortex mixer, carefully pull out the comb, and add equal-mass protein samples of different groups and marker into the wells in sequence. Adjust the voltage to 80 V to start electrophoresis, and observe the electrophoresis process carefully. When the protein sample is between the upper stacking gel and the lower separating gel, adjust the voltage to 120 V and continue. When it is observed that the protein sample reaches the bottom of the separating gel, turn off the machine.

[0148] (2) Electrotransfer, blocking, and antibody incubation

[0149] Place the pre-cut NC membrane in a water tank filled with electrotransfer buffer. Place the black side of the electrotransfer clip downward in the tank, and cover its surface with a sponge gasket and filter paper in sequence. Carefully take out the electrophoresis gel glass plate, cut the gel to an appropriate size, place it above the filter paper on the black side, and then cover the NC membrane and filter paper on the surface in sequence. After aligning them in sequence, gently roll a small roller to remove air bubbles. Finally, cover the surface with a sponge gasket, clamp it, and place it in the electrotransfer tank according to the electrode direction, and add an appropriate amount of electrotransfer buffer. Perform electrotransfer at a current intensity of 300 mA under ice bath conditions for 2 hours. If you want to obtain protein bands with large molecular weights, appropriately increase the electrotransfer time.

[0150] After the electrotransfer is completed, place the transferred NC membrane as a whole into the pre-prepared 5% skim milk blocking solution and place it on a horizontal shaker for blocking, ensuring that the blocking solution can completely cover the NC membrane. Block at room temperature for 2 hours.

[0151] (3) Antibody incubation

[0152] The stock solutions of antibodies for different detection indicators are diluted in sequence with antibody diluent according to the instructions and diluted in advance for standby. The blocked NC membrane is cut in sequence according to the molecular weight indicated by the target protein. The cut protein bands are placed in the antibody solutions diluted in advance for the corresponding indicators, marked, incubated on a horizontal shaker at room temperature for 1 hour, and then placed in a 4°C refrigerator and incubated overnight. During the incubation process, pay attention to placing it horizontally to ensure that the bands can be completely immersed.

[0153] Take out the bands from the refrigerator the next day and rewarm them at room temperature for 30 minutes. Wash the bands incubated with the primary antibody three times with PBST solution (10 minutes each time), and place them in the corresponding secondary antibody of goat anti-rabbit IRDye 800CW or goat anti-mouse IRDye 800CW diluted in advance according to the source of the primary antibody. Incubate on a horizontal shaker at room temperature for 1 hour, and strictly pay attention to avoiding light during the operation process of secondary antibody incubation.

[0154] (4) Development

[0155] Wash the protein bands incubated with the secondary antibody three times with PBST solution (10 minutes each time) and then perform development. Pay attention to avoiding light during the band washing process. When developing, wipe the developing panel clean with a cotton ball moistened with distilled water. Place the protein bands face down on the developing panel. After placing the bands, use a roller to roll to remove air bubbles. Set the parameters according to the range where the bands are placed, and start the machine for exposure and development.

[0156] 2.1.12 Nin-MBs combined with UTMD in vivo treatment effect

[0157] (1) Establishment of a nude mouse orthotopic HCC model under ultrasound guidance

[0158] BALB / c male nude mice aged 4 - 6 weeks with a body weight of 18 - 20 g were used. HCCLM3 and CAF cells were cultured in 10-cm culture dishes respectively. When the cell confluence reached 90%, the cells were digested with trypsin and collected, and a mixed cell suspension (4×10 6 cells / ml) was prepared according to a 1:1 cell number ratio; after anesthesia by intraperitoneal injection of 150 μl of 1% sodium pentobarbital, 100 μl of the HCCLM3 and CAF mixed cell suspension was aspirated with a microsyringe. Under gray-scale ultrasound guidance, the microsyringe was used to puncture percutaneously to the designated position in the liver. During the puncture process, important organs and vascular structures should be avoided. After reaching the designated position, the cell suspension was slowly injected. After the injection was completed, the needle was withdrawn under negative pressure to avoid needle tract metastasis. USI was used to monitor the tumor size and calculate the tumor volume. Tumor volume (mm 3 ) = 1 / 2 × length (mm) × width (mm 2 ). When the tumor volume grew to 80 - 100 mm 3 , they were randomly divided into 5 groups (n = 6), namely the Con group, the MBs + UTMD group, the Nin-MBs group, the Nin-MBs + UTMD group, and the Nintedanib group.

[0159] (2) Monitoring of the in vivo treatment of Nin-MBs combined with UTMD

[0160] Thirty BALB / c nude mice were selected and randomly divided into 5 groups. In the Nin-MBs, Nin-MBs + UTMD, and Nintedanib intervention groups, drug injection was performed via the tail vein every 2 days (Nintedanib at 3 mg / kg). The Con group and the MBs + UTMD group were given an injection of an equal amount of normal saline and blank lipid MBs. The LIFU irradiation (2.0 W / cm 2, (120 seconds) was performed immediately after microbubble injection, and the treatment lasted for 2 weeks. USI was used to monitor the tumor size and estimate the tumor volume on the 3rd, 7th, 12th, and 14th days after treatment, and the body weight of the mice was recorded at the same time. The body weight change curve of nude mice and the tumor growth curve of nude mice were plotted. USI was performed under anesthesia before treatment (day 0) and weekly (days 7 and 14) after treatment; gray-scale imaging, color Doppler flow imaging (CDFI), color power angiography (CPA), and ultrasonic elasticity (USE) were used to detect the changes in tumor size, blood flow, and hardness. At the same time, MRI was performed, and the nuclear magnetic resonance used conventional T1WI, T2WI, and T2 mDIXON detection sequences. After 2 weeks of treatment, the nude mice were sacrificed by spinal cord transection, the liver was dissected, and the size and number of tumor nodules on the liver were compared among groups to evaluate the effect of Nin-MBs combined with UTMD on tumor growth and intrahepatic metastasis.

[0161] (3) Histological study of tumors after Nin-MBs combined with UTMD treatment

[0162] The tumor tissues of five groups of nude mice after 2 weeks of intervention treatment were dissected and made into paraffin frozen sections for immunohistochemical staining and hematoxylin-eosin (H&E) staining. The immunohistochemical detection indexes included: α-SMA, CollagenIα, HA, Fibronectin, CD31. Among them, α-SMA reflects the activity of CAF, Collagen Ia, HA, and Fibronectin represent the changes in ECM components, and CD31 is used to analyze tumor blood vessels. At the same time, the hearts, livers, spleens, kidneys, and lungs of nude mice in different groups were taken for H&E staining to observe whether there were abnormal areas for safety assessment.

[0163] 2.1.13 Statistical methods

[0164] The same statistical methods as those in 1.1.6 were used.

[0165] 2.2 Experimental results

[0166] 2.2.1 Extraction and identification of CAF cells

[0167] In this study, CAF cells were successfully extracted from surgically resected HCC tissues. Under light microscopy, the adherent CAF cells showed a typical irregular spindle-shaped structure, as shown in Figure 6 Figure A. Immunofluorescence staining identification of the isolated CAF cells was performed according to the surface marker protein of CAF, and the results are shown in Figure 6 Figure B. From Figure 6As can be seen from Figure B, the activation of CAF surface markers α-SMA, FAP, and Vimentin all showed positive expression.

[0168] 2.2.2 Cytotoxicity of Nintedanib

[0169] The effects of different concentrations of Nintedanib on CAF cells were detected by the CCK8 method, and the results are shown in Appendix Figure 7 As shown in Appendix Figure 7 As can be seen from Appendix A, Nintedanib can inhibit the cell viability of CAF cells, and it shows concentration-dependent and time-dependent effects. Appendix Figure 7 Appendix B shows that the same drug concentration and action time did not show obvious inhibitory effects on the proliferation of HCCLM3 cells.

[0170] The expression levels of the action targets PDGFR-β and FGFR-2 of Nintedanib drug in hepatocellular carcinoma HCCLM3 and CAF were evaluated by Western blot, and the results are shown in Appendix Figure 8 As shown. The Western blot results showed that the expression levels of PDGFR-β and FGFR-2 in CAF cells were much higher than those in HCCLM3.

[0171] 2.2.3 Inhibition of the proliferation and activation of CAF cells by Nin-MBs combined with UTMD

[0172] 2.2.3.1 Nin-MBs combined with UTMD enhances cell uptake

[0173] Rb-MBs were prepared by replacing Nintedanib with Rb. After 48 hours of intervention, the uptake of CAF cells among different groups was analyzed and compared by flow cytometry fluorescence quantitative software. The results are shown in Appendix Figure 9 As shown, from Appendix Figure 9 it can be seen that the fluorescence intensity of the Rb-MBs+UTMD group was significantly higher than that of the Rb group and the Rb-MBs group. The signal of free Rb was slightly stronger than that of the Rb-MBs group, but the difference was not statistically significant.

[0174] 2.2.3.2 Nin-MBs combined with UTMD inhibits the proliferation of CAF cells

[0175] The CAF cells were divided into 5 groups: (a) the Con group with only medium; (b) the MBs+UTMD group; (c) the Nin-MBs group; (d) the Nin-MBs+UTMD group; (e) the Nintedanib group. The drug concentration of Nintedanib in each group was the same, which was 1 μmol / L. After the intervention of different groups, the OD values of the cells were detected by the CCK8 method at 24, 48, and 72 hours respectively, and the growth curve of the CAF cells was plotted. The results are as shown in Figure 10 Appendix Figure 10 A. As can be seen from Appendix A, compared with the Con and MBs+US groups, the Nintedaib, Nin-MBs, and Nin-MBs+UTMD groups could significantly inhibit the growth rate of CAF cells. Among them, the inhibitory effect of the Nin-MBs+UTMD group was more significant than that of the free drug Nintedanib and the drug-loaded microbubbles Nin-MBs. There was no significant difference between the Nintedanib and Nin-MBs groups.

[0176] Meanwhile, the results of the colony formation assay are as shown in Figure 10 Appendix Figure 10 B. As can be seen from Appendix B, the Nin-MBs, Nintedanib, and Nin-MBs+UTMD groups could all inhibit the number of CAF cell colonies formed. Among them, the Nin-MBs+UTMD group had the best inhibitory effect and the least number of colonies formed, followed by Nintedanib and Nin-MBs.

[0177] 2.2.3.3 Effect of Nin-MBs combined with UTMD on the cell cycle of CAF cells

[0178] The results of flow cytometry analysis of the cell cycle of CAF cells treated differently are as shown in Figure 11 Appendix Figure 11 . As can be seen from Appendix, Nintedanib, Nin-MBs, and Nin-MBs+UTMD could arrest the cell cycle of CAF cells in the G1 phase, significantly increase the number of G1-phase cells, and at the same time reduce the number of S-phase cells. There was no significant change in cell cycle arrest in the MBs+UTMD group compared with the Con group. The cell cycle arrest effect of the Nin-MBs+UTMD group was significantly better than that of the Nintedanib and Nin-MBs groups, and the difference was statistically significant. There was no significant difference in the effects between the Nintedanib and Nin-MB groups.

[0179] The expression of cycle-related proteins in CAF cells after different treatments was detected by Western blot. The results are as shown in Figure 12 Appendix Figure 12It can be seen that after different intervention methods of Nintedanib, Nin-MBs, and Nin-MBs+UTMD, the expression levels of the cell cycle G1-phase regulatory protein Cyclin D1 and the proliferation protein index PCNA decreased, while the expression level of the G1-phase marker protein P53 increased. Among them, the Nin-MBs+UTMD group had the most significant effect, which was consistent with the results of the flow cytometry cycle experiment.

[0180] 2.2.3.4 Effect of Nin-MBs combined with UTMD on the activation of CAF cells

[0181] To evaluate the effect of Nintedanib-MBs combined with UTMD on the activation of CAF cells, this study used Western blot to examine the expression of the activation CAF surface marker proteins α-SMA, FAP, and Vimentin. The results are shown in the appendix Figure 13 as follows. As can be seen from the appendix Figure 13 It can be seen that Nin-MBs, Nintedanib, and Nin-MBs+UTMD can reduce the expression levels of the CAF cell activation indicators α-SMA, FAP, and Vimentin. Among them, the Nin-MBs+UTMD group has a better effect. At the same time, the Western blot results show that the protein expression levels of the molecular target points PDGFR-β and FGFR-2 of Nintedanib also decrease, and the Nin-MBs+UTMD group has a better effect.

[0182] 2.2.4 In vitro study of Nin-MBs combined with UTMD in remodeling ECM

[0183] 2.2.4.1 Nin-MBs combined with UTMD reduces the production of ECM components

[0184] Activated CAFs are the main producers of ECM components. It has been found that while Nintedanib inhibits the activation and proliferation of CAF cells, it can also reduce the generation of ECM components, as shown in the appendix Figure 13 as follows.

[0185] Furthermore, Western blot shows that the Nintedanib, Nin-MBs+UTMD, and Nin-MBs groups can significantly reduce the contents of the ECM components collagen Iα, Elastin, and Fibronectin. The Nin-MBs+UTMD group has the most significant effect. The immunofluorescence results show that compared with the control group, the fluorescence intensity of the Nin-MBs+UTMD intervention group decreases. At the same time, it can be observed that with the decrease of the fluorescence intensity, the cell morphology also changes, gradually changing from an irregular fusiform shape to a long fusiform shape. This shows that the activation of CAF cells is inhibited, as shown in the appendix Figure 14 as follows.

[0186] 2.2.4.2 Effect of Nin-MBs combined with UTMD on the penetration of multicellular tumor spheroids

[0187] In this study, multicellular tumor spheroids were successfully constructed, and the effects of different intervention methods on the penetration ability of multicellular tumor spheroids were evaluated by Z-stack scanning of confocal microscopy. The results are shown in the appendix Figure 15 as follows. As can be seen from the appendix Figure 15 , the penetration ability of the tumor spheroids in the Nin-MBs combined with UTMD intervention group was the strongest. The fluorescence intensity at a thickness of 100 μm was significantly higher than that of other intervention groups and was relatively evenly distributed. When the thickness was 150 μm, there was a trend of decreasing fluorescence. The effects of the Nintedanib group and the Nin-MBs group were second. Compared with the Con group, the MBs+UTMD group increased the fluorescence penetration in the tumor spheroids to a certain extent at thicknesses of 50 μm and 100 μm. The above experiments proved that Nin-MBs combined with UTMD could significantly enhance the penetration of tumor spheroids

[0188] 2.2.5 Effect of Nin-MBs combined with UTMD on the tumor-promoting effect of CAF cells

[0189] To verify whether Nin-MBs combined with UTMD can inhibit the tumor-promoting effect of CAF while inhibiting the proliferation and activation of CAF cells, conditioned media after different group interventions of CAF cells were obtained in this study, and HCCLM3 cells were continuously intervened with the conditioned media for verification

[0190] 2.2.5.1 Effect of Nin-MBs combined with UTMD on the migration and invasion ability of hepatocellular carcinoma

[0191] The appendix Figure 16 shows the effect of Nin-MBs combined with UTMD on the migration and invasion ability of hepatocellular carcinoma. The experimental results showed that activated CAF cells could significantly promote the migration and invasion ability of HCCLM3 cells. Nintedanib, Nin-MBs+UTMD, and Nin-MBs could significantly reduce this promoting effect of CAF on migration and invasion while inhibiting the activation and proliferation of CAF cells. The results of the scratch experiment (as shown in A of the appendix Figure 16 ) showed that the reversal effect of the Nin-MBs+UTMD group was the most obvious compared with the Nintedanib and Nin-MBs groups. The results of Transwell (as shown in B of the appendix Figure 16 ) were consistent with the scratch results, and the number of cells passing through the chamber in the Nin-MBs+UTMD group was the least

[0192] 2.2.5.2 Effect of Nin-MBs combined with UTMD on HCC EMT and tumor stemness

[0193] CAF cells can promote the transformation of hepatocellular carcinoma cells into mesenchymal cells. It was observed under light microscopy that the conditioned medium of CAF (CAF-CM) could cause the HCCLM3 hepatocellular carcinoma cells, which were originally polarized epithelial cells, to gradually elongate (as shown in A of the appendix Figure 17 ), and transform into spindle-shaped mesenchymal cells (as shown in B of the appendix Figure 17 ), thus promoting the occurrence of EMT in hepatocellular carcinoma.

[0194] To verify whether Nin-MBs combined with UTMD can affect the promotion of tumor cell EMT while inhibiting CAF activation, in this study, the obtained conditioned medium was used to intervene in HCCLM3 cells for 48 hours, and the main markers of EMT were verified by Western blot assay. The results are shown in the appendix Figure 18 . As can be seen from the appendix Figure 18 , compared with the CAF-CM and (MBs+UTMD)-CM groups, Nin-CM, (Nin-MBs+UTMD)-CM, and (Nin-MB)-CM could reduce the expression of mesenchymal markers N-cadherin and Vimentin to a certain extent, and increase the expression of epithelial marker E-cadherin. Among them, the (Nin-MBs+UTMD)-CM group had the most significant effect. Compared with the CAF-CM and (MBs+UTMD)-CM groups, the expression levels of tumor stemness-related indicators CD133 and SOX2 in these three groups were also decreased, and the expression of MMP-2 and MMP-9 was also reduced. Among them, the intervention effect of the Nin-MBs group under LIFU irradiation was the most significant.

[0195] 2.2.5.3 Effect of Nin-MBs combined with UTMD on the growth rate of tumor spheres

[0196] After intervening in tumor spheres with different intervention methods, the growth of tumor spheres was continuously observed in this study. The results are shown in the appendix Figure 19 . As can be seen from the appendix Figure 19 , it was found that after 7 days of intervention in different groups, the growth rate of tumor spheres in the Con and MBs+UTMD groups was faster than that in the other three groups. After drug intervention with Nintedanib, Nin-MBs, and Nin-MBs+UTMD, the growth rate of tumor spheres could be significantly delayed, and the (Nin-MBs+UTMD) group had the strongest effect.

[0197] 2.2.5.4 Effect of Nin-MBs combined with UTMD on the main factors secreted by CAF cells

[0198] The appendix Figure 20 shows the Western blot detection results of the effect of Nin-MBs combined with UTMD on the main factors secreted by CAF cells. As can be seen from the appendixFigure 20 It can be seen that after the drug interventions of Nintedanib, Nin-MBs, and Nin-MBs+UTMD, while reducing the activation and proliferation of CAF cells, they can inhibit the secretion of HGF, TGF-β, IL-6, HIF-Iα, and VEGFA factors by CAF cells. Among them, the inhibitory effect of the Nin-MBs combined with UTMD group is the most significant.

[0199] 2.2.6 In vivo effect study of Nin-MBs combined with UTMD in remodeling tumor ECM

[0200] 2.2.6.1 Detection of the tumor treatment effect of Nin-MBs combined with UTMD

[0201] Before the intervention (day 0) and on the 7th and 14th days after the intervention in different experimental groups, the size, blood flow, and hardness of the tumors in nude mice were monitored using USI (including two-dimensional, CDFI, CPA, USE). MRI detection (including T1WI, T2WI, and T2 mDIXON sequences) was performed at the same time.

[0202] The USI detection results are as shown in the appendix Figure 21 as follows. It can be seen from the appendix Figure 21 that the HCC orthotopic tumors constructed in this study showed slightly hypoechoic on the USI gray scale, with medium hardness (blue-green intermingled). As time increased, the hardness of the tumors gradually increased (the proportion of blue became larger and larger), and the blood flow also gradually increased. After the interventions of the Nin-MBs, Nintedanib, and Nin-MBs+UTMD groups, the hardness and blood supply of the tumors decreased, and the growth rate of the tumors slowed down compared with the Con and MBs+UTMD groups. Among them, the effect of the Nin-MBs+UTMD group was the most significant. Compared with the Con group, the MBs+UTMD group had a certain degree of reduction in tumor hardness, but there were no significant differences in the tumor growth rate and blood supply.

[0203] The MRI detection results are as shown in the appendix Figure 22 as follows. It can be seen from the appendix Figure 22 that the HCC tumors showed isointense or slightly hypointense on T1 and slightly hyperintense on T2. As time extended, the tumor signal of the Con group gradually decreased compared with the experimental intervention groups. The growth rate of the tumors in the control group was significantly higher than that of the Nin-MBs, Nintedanib, and Nin-MBs+UTMD experimental intervention groups, which was consistent with the USI observation results.

[0204] When the nude mice in different intervention groups were given 14 days of intervention treatment, the size of the tumors was regularly measured using ultrasound and the tumor weights were recorded at the same time. After the treatment ended, the nude mice were sacrificed, and the growth conditions of various tumors were visually compared after dissection (as shown in the appendix Figure 23As shown in Figure A, the body weight changes of nude mice and the tumor growth curves were plotted (as shown in the appendix). Figure 23 As shown in Figures B and C. Through detection, it was found that there were no significant differences in the body weight changes of nude mice in each experimental group. Compared with Con and MBs+UTMD, Nin-MBs, Nintedanib, and Nin-MBs+UTMD could significantly delay the tumor growth rate. Among them, the effect of Nin-MBs+UTMD was the most significant. In the Con and MBs+UTMD groups, liver metastasis occurred while the tumors were growing rapidly. No metastatic lesions were observed in the three groups of Nin-MBs, Nintedanib, and Nin-MBs+UTMD.

[0205] 2.2.6.2 Nin-MBs combined with UTMD targets CAF to remodel ECM

[0206] After the nude mice were sacrificed, the tumors were dissected from the body and paraffin tissue sections of the tumors were obtained for immunohistochemical staining. The staining indicators were α-SMA, collagen Iα, Elastin, Fibronectin, and CD31. The staining results are as shown in the appendix Figure 24 As shown. From the appendix Figure 24 It can be seen that Nin-MBs, Nintedanib, and Nin-MBs+UTMD can reduce the contents of the main components of ECM in the tumor, CollagenIα, Elastin, and Fibronectin. There was no significant difference between MBs+UTMD and the Con group. The same results were also shown in the expression levels of the CAF activation index α-SMA and the vascular index CD31.

[0207] 2.2.6.3 In vivo biosafety assessment of Nin-MBs combined with UTMD

[0208] The H&E staining results of the main organs (heart, liver, lung, spleen, and kidney) of nude mice in different experimental intervention groups are as shown in the appendix Figure 25 As shown. From the appendix Figure 25 It can be seen that no obvious tissue damage or necrosis areas were found in each organ, indicating that the experimental intervention group did not cause the degradation and damage of the fibrous matrix of other organs in the body while anti-tumor fibrosis, and had good in vivo safety.

[0209] In summary, in this study, the drug-loaded microbubbles Nin-MBs were successfully prepared, which had good imaging effects. UTMD improved the uptake ability of CAF cells and enhanced the therapeutic effect of Nintedanib, realizing the integration of diagnosis and treatment. Nin-MBs combined with UTMD inhibited the activation and proliferation of CAF cells, remodeled the tumor ECM, improved the diffusion of drugs in the tumor, and at the same time reversed the tumor-promoting effect of CAF, providing a new strategy for the treatment of HCC.

[0210] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A method for preparing lipid microbubbles loaded with the anti-fibrosis drug Nintedanib, characterized in that: The following steps are included: S1: DPPC, DSPE-PEG, DSPG, cholesterol and the anti-fibrotic drug Nintedanib were mixed and added to a flask, dissolved in chloroform, and rotary evaporated at 55°C-60°C to obtain a dry film mixture; S2: adding a hydration solution to the film mixture for hydration, and repeatedly extruding the obtained lipid suspension through a phospholipid extruder to form a homogeneous lipid suspension; S3: The homogenized lipid suspension is placed in a sealed bottle, the upper air is evacuated, SF6 gas is filled in, and mechanical oscillation is performed to form lipid microbubbles Nin-MBs loaded with the anti-fibrotic drug Nintedanib.

2. The method for preparing lipid microbubbles loaded with the anti-fibrosis drug Nintedanib according to claim 1, characterized in that: The mass ratio of DPPC, DSPE-PEG, DSPG, cholesterol and Nintedanib in step S1 is 10:4:4:3:

5.

3. The method for preparing lipid microbubbles loaded with the anti-fibrosis drug Nintedanib according to claim 2, characterized in that: In step S1, the volume ratio of the total mass of DPPC, DSPE-PEG, DSPG, cholesterol and the anti-fibrotic drug Nintedanib to chloroform is 2.6:1 mg / ml.

4. The method for preparing lipid microbubbles loaded with the anti-fibrosis drug Nintedanib according to claim 3, characterized in that: The hydration solution described in step S2 includes 10% glycerol and 90% 1×PBS solution.

5. The method for preparing lipid microbubbles loaded with the anti-fibrosis drug Nintedanib according to claim 4, characterized in that: The volume ratio of the hydrated solution in step S2 to the chloroform solution in step S1 is 1:

2.

6. The method for preparing lipid microbubbles loaded with the anti-fibrosis drug Nintedanib according to claim 5, characterized in that: The hydration condition in step S2 is: hydration at 37° C. for 30 minutes.

7. Lipid microbubbles loaded with the anti-fibrosis drug Nintedanib prepared by the preparation method according to any one of claims 1 to 6.

8. The lipid microbubble loaded with the anti-fibrosis drug Nintedanib according to claim 7, characterized in that: The lipid microbubbles can burst and release the drug Nintedanib under low-intensity focused ultrasound (LIFU) irradiation.

9. Use of the lipid microvesicles according to claim 8 in the preparation of a tumor-associated fibroblast activity inhibitor.

10. Use of the lipid microvesicles according to claim 8 in preparing drugs for diagnosing and / or treating liver cancer.

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