Anti-tumor metastasis hydrogel loaded with doxorubicin liposome and CXCL12 as well as preparation method and application of anti-tumor metastasis hydrogel

Abstract

The invention discloses an anti-tumor metastasis hydrogel loaded with adriamycin liposome and CXCL12 as well as a preparation method and application of the anti-tumor metastasis hydrogel. The hydrogel comprises CXCL12 with the effect of recruiting tumor cells and adriamycin lipidosome capable of killing the tumor cells. According to the preparation method, a thickening agent and a cross-linking agent solution containing adriamycin liposome and CXCL12 are mixed, and the anti-tumor metastasis hydrogel loaded with the adriamycin liposome and the CXCL12 is prepared. According to the hydrogel prepared by the invention, by loading CXCL12 and adriamycin lipidosome, metastatic tumor cells can be trapped, and enriched tumor cells can be efficiently killed; the bottleneck problem of how to sensitively recognize and capture metastatic tumor cells, efficiently kill tumor cells and prevent omission and escape of the tumor cells in the design of the existing anti-tumor metastasis drugs is solved, a new drug treatment strategy is provided for preventing and treating tumor cell metastasis, and the drug has wide market application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12, and a preparation method and application thereof. Background Art

[0002] Cancer is one of the diseases with the highest morbidity and mortality in the world. Although humans have made significant progress in early cancer screening and clinical treatment, patients with advanced cancer still face a grim prognosis, mainly because tumor cells have a strong ability to invade surrounding tissues and metastasize to distant sites. The cause of death of about 90% of cancer patients is closely related to tumor escape and metastasis. Chemotherapy is the most commonly used treatment method in tumor treatment. It can induce cell death through multiple mechanisms such as regulating cell metabolism and blocking cell mitosis. However, while chemotherapy kills tumor cells, it is accompanied by the destruction of the homeostatic structure of tumor tissue and stimulates the proliferation of immunosuppressive cells, thereby triggering tumor cell invasion and metastasis.

[0003] Currently, there is a lack of targeted drugs for the prevention and treatment of tumor metastasis in clinical practice. The drug strategies in the preclinical research and clinical trial stages mainly include reshaping the pre-metastatic microenvironment and killing metastatic cells.

[0004] Remodeling the pre-metastatic microenvironment uses chemical interference or gene regulation to reduce the content of tumor-promoting cells in the pre-metastatic lesion or activate the microenvironmental immune response, which can interfere with the colonization process of tumor cells and thus hinder the formation of metastatic lesions. However, this strategy requires predicting the location of the pre-metastatic lesion to avoid treatment side effects caused by improper regulation.

[0005] Killing metastatic tumor cells uses specific antibodies or immune cell membrane components to modify the killing drug, which increases the drug's ability to recognize tumor cells and improves the killing efficiency of metastatic tumor cells, thereby reducing the tumor metastasis rate. However, tumor cell phenotypes are variable and heterogeneous, and the effectiveness of drug recognition of metastatic tumor cells and the completeness of tracking and killing one by one will greatly affect the development of strategies.

[0006] Therefore, how to sensitively identify and capture metastatic tumor cells and how to efficiently kill tumor cells and prevent them from escaping are two bottleneck issues in the design of anti-tumor metastasis drugs. Summary of the invention

[0007] The purpose of the present invention is to provide an anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12, and a preparation method and application thereof. The present invention constructs a "tumor killing trap" to trap metastatic tumor cells, allowing the doxorubicin liposomes in the trap to efficiently kill the enriched tumor cells, thereby achieving the therapeutic goal of preventing and treating tumor metastasis.

[0008] In order to solve the above technical problems, the technical solution to be adopted in the present invention is: The invention provides an anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12. The preparation method of the anti-tumor metastasis hydrogel comprises the following steps: dissolving the doxorubicin liposomes and CXCL12 in a cross-linking agent solution, and then adding the cross-linking agent solution containing the doxorubicin liposomes and CXCL12 into a thickener solution.

[0009] The present invention provides a method for preparing the anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12, and the preparation method specifically comprises the following steps: (1) dissolving the lipid material and doxorubicin in an organic solvent, removing the organic solvent by vacuum rotary evaporation to obtain a uniform lipid film, adding PBS solution for hydration, homogenizing the mixture by ultrasonication, and filtering and removing impurities to obtain doxorubicin liposomes encapsulating doxorubicin; (2) Dissolving CXCL12 and the doxorubicin liposomes in a crosslinker solution, and rapidly stirring the crosslinker solution with the dissolved thickener solution to obtain an anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12.

[0010] Furthermore: in the step (1), the lipid material is a mixture of lecithin and cholesterol, and the mass ratio of lecithin to cholesterol is 3-6:1.

[0011] Furthermore: in the step (2), the concentration of CXCL12 in the mixed solution with the cross-linking agent is greater than 6 μg / mL, and the concentration of the active drug doxorubicin in the mixed solution is greater than 900 μg / mL.

[0012] Furthermore: in the step (2), the thickener is polyvinyl alcohol, and the mass ratio of polyvinyl alcohol in the hydrogel is 1-8%.

[0013] Furthermore: in step (2), the cross-linking agent is borax, and the mass ratio of borax in the hydrogel is 0.01-0.1%.

[0014] The present invention also provides the use of the hydrogel or the anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12 obtained by the preparation method in the preparation of anti-tumor metastasis preparations.

[0015] Furthermore: the tumor is lung cancer, breast cancer, liver cancer, ovarian cancer, gastric cancer, or myeloma.

[0016] Furthermore: CXCL12 released from the hydrogel can attract tumor cells, causing the tumor cells to actively migrate toward the gel.

[0017] Furthermore: the hydrogel significantly reduced liver damage and significantly reduced cardiac toxicity caused by free Dox Lipo.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The "bait" CXCL12 released from the anti-tumor metastasis hydrogel (BH-gel) loaded with doxorubicin liposomes and CXCL12 can recruit tumor cells and attract tumor cells to actively migrate to the gel; 2. The release of doxorubicin liposomes from anti-tumor metastasis hydrogels loaded with doxorubicin liposomes and CXCL12 can kill recruited tumor cells in a concentrated and efficient manner, minimize systemic toxicity and limit cancer metastasis; 3. Compared with the same dose of doxorubicin liposomes, local injection of the BH-gel has better anti-tumor activity against lung tumors; 4. The BH-gel not only reduces the risk of tumor cell escape, but also reduces the adverse side effects associated with doxorubicin.

[0019] The anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12 constructed by the present invention comprises CXCL12 having the function of recruiting tumor cells and doxorubicin liposomes capable of killing tumor cells. The present invention firstly prepares doxorubicin liposomes by thin film hydration method (commercially available doxorubicin liposomes can also be used), and then physically mixes polyvinyl alcohol with a borax solution containing doxorubicin liposomes and CXCL12 to prepare the anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12.

[0020] The anti-tumor metastasis hydrogel can solve the bottleneck problem of how to sensitively identify and capture metastatic tumor cells and efficiently kill tumor cells and prevent them from escaping through the enrichment effect of CXCL12 to induce tumor cells to actively approach and the drug killing effect mediated by doxorubicin liposomes. It provides a new drug treatment strategy for preventing and treating tumor cell metastasis and has broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0022] Figure 1 This is the average particle size and average Zeta potential change graph of Dox Lipo within 14 days; Figure 2 This is the field emission transmission electron microscopy image of Dox Lipo; Figure 3 It is the Fourier transform infrared absorption spectra of polyvinyl alcohol, borax and blank gel formed after cross-linking of polyvinyl alcohol and borax; Figure 4 is the field emission SEM image of the blank gel; Figure 5 It is a schematic diagram of BH-gel recruitment to A549 cells, and the fluorescence and quantitative graphs of the lower chamber of the transwell experiment; Figure 6 is the in vitro degradation rate of BH-gel and the cumulative release rate of Dox Lipo from BH-gel; Figure 7 This is a laser confocal image of tumor cells taking up free Dox and Dox Lipo; Figure 8 The MTT assay was used to detect the effects of free Dox and Dox Lipo on A549 cell viability; Fig. 9 is the result of apoptosis detection of A549 cells by free Dox and Dox Lipo; Fig.10 It is the steps to construct the BALB / c nude mouse tumor model and the treatment plan; Fig.11 is the weight of tumor tissue in mice of each group; Fig.12 It is the fluorescence quantification of the distribution of A549-GFP in mice over 28 days; Fig.13 is the number of tumor metastatic nodules in each group of mice; Fig.14 is the weight of the heart, liver, spleen, lung, and kidney of mice in each group; Fig.15 It is the liver function index test of each group of mice; Fig.16 These are HE sections of heart tissues in each group. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with embodiments. The following embodiments are for explanation of the present invention but the present invention is not limited to the following embodiments.

[0024] Example 1: Preparation of anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12 In order to overcome the research bottleneck of anti-tumor metastasis drug design, the present invention uses polyvinyl alcohol gel with biosafety and tissue compatibility as a matrix, and forms a three-dimensional network structure through cross-linking with borax. By uniformly dispersing CXCL12 protein and doxorubicin liposomes therein, the anti-tumor metastasis drug proposed by the present invention is obtained simply and efficiently.

[0025] The specific preparation process includes the following steps: 1. Preparation of fat-soluble doxorubicin (Dox) Weigh 20 mg of water-soluble doxorubicin hydrochloride DoxHCl into a 50 ml round-bottom flask, add an appropriate amount of dichloromethane to dissolve evenly, then add 14.3 μL of triethylamine solution to make the molar ratio of triethylamine to DoxHCl 3:1, and stir in a 25°C water bath to react overnight in the dark. Then transfer the reaction solution to an activated dialysis bag (MWCO=3500), dialyze with deionized water for 3 days, collect the liquid sample in the bag and freeze-dry it in a freeze dryer for 48 hours to obtain fat-soluble doxorubicin Dox.

[0026] 2. Preparation of Dox liposomes Dissolve 150 mg of lecithin, 50 mg of cholesterol, and 5.4 mg of doxorubicin in 15 mL of dichloromethane and stir in a water bath at 60°C for 30 min until completely dissolved. Then transfer the mixture to a 250 mL round-bottom flask and remove dichloromethane using a rotary evaporator at 40-45°C under vacuum conditions. A uniform film can be observed to form at the bottom of the round-bottom flask.

[0027] Then, 6 ml of preheated phosphate buffer (PBS, pH 7.4) was added, and the round-bottom flask was transferred to a 60°C water bath for hydration for 1 h to ensure that the lipid film at the bottom of the flask was completely hydrated. The mixture was then homogenized using a cell ultrasonic disruptor (7 min, 30% power, on 1 s, off 1 s) to form liposomes of uniform size. Finally, the mixture was passed through 0.8 µm, 0.45 µm, and 0.22 µm needle filters to obtain doxorubicin liposomes, which were stored at 4°C for later use.

[0028] 3. Preparation of anti-tumor metastasis hydrogel (BH-gel) loaded with doxorubicin liposomes and CXCL12 BH-gel is prepared by mixing 1% polyvinyl alcohol (PVA, thickener, 1% is the mass ratio of PVA to hydrogel), 0.01% borax (cross-linking agent, 0.01% is the mass ratio of borax to hydrogel), Dox Lipo and CXCL12 at room temperature, specifically comprising the following steps: 1. Dissolve 135 mg PVA in double distilled water to a total volume of 9 mL. Stir magnetically in a 90°C oil bath for 60 min until the PVA solution is completely dissolved. Cool to room temperature for later use. 2. Prepare borax solution by dissolving 1.35 mg borax in double distilled water; 3. Dissolve Dox Lipo and CXCL12 in the borax solution to make the total volume 4.5 mL, the concentration of CXCL12 in the mixed solution is 6 μg / mL, and the concentration of doxorubicin in the mixed solution is 900 μg / mL; 4. Add 4.5 mL of borax solution containing DoxLipo and CXCL12 to the PVA solution, and stir the mixture rapidly for 5 s to promote cross-linking to form BH-gel.

[0029] Example 2: Preparation of anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12 The Dox Lipo used in this example can be commercially available doxorubicin liposomes, or the doxorubicin liposomes prepared in Example 1.

[0030] In this embodiment, BH-gel is prepared by mixing 1.3% polyvinyl alcohol (PVA, thickener), 0.026% borax (cross-linking agent), Dox Lipo and CXCL12 at room temperature, specifically comprising the following steps: 1. Dissolve 175.5 mg PVA in double distilled water to a total volume of 9 mL. Stir magnetically in a 90°C oil bath for 60 min until the PVA solution is completely dissolved. Cool to room temperature for later use. 2. Prepare borax solution by dissolving 3.51 mg borax in double distilled water; 3. Dissolve Dox Lipo and CXCL12 in the borax solution to make the total volume 4.5 mL, and the concentration of CXCL12 in the mixed solution is 6 μg / mL; 4. Add 4.5 mL of borax solution containing DoxLipo and CXCL12 to the PVA solution, and stir the mixture rapidly for 5 s to promote cross-linking to form BH-gel.

[0031] Example 3: Preparation of anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12 The Dox Lipo used in this example can be commercially available doxorubicin liposomes, or the doxorubicin liposomes prepared in Example 1.

[0032] In this embodiment, BH-gel is prepared by mixing 3% polyvinyl alcohol (PVA, thickener), 0.05% borax (cross-linking agent), DoxLipo and CXCL12 at room temperature, specifically comprising the following steps: 1. Dissolve 405 mg PVA in double distilled water to a total volume of 9 mL. Stir magnetically in a 90°C oil bath for 60 min until the PVA solution is completely dissolved. Cool to room temperature for later use. 2. Prepare borax solution by dissolving 6.75 mg of borax in double distilled water; 3. Dissolve Dox Lipo and CXCL12 in the borax solution to make the total volume 4.5 mL, and the concentration of CXCL12 in the mixed solution is 6 μg / mL; 4. Add 4.5 mL of borax solution containing DoxLipo and CXCL12 to the PVA solution, and stir the mixture rapidly for 5 s to promote cross-linking to form BH-gel.

[0033] Example 4: Preparation of anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12 The Dox Lipo used in this example can be commercially available doxorubicin liposomes, or the doxorubicin liposomes prepared in Example 1.

[0034] In this embodiment, BH-gel is prepared by mixing 4% polyvinyl alcohol (PVA, thickener), 0.08% borax (cross-linking agent), DoxLipo and CXCL12 at room temperature, specifically comprising the following steps: 1. Dissolve 540 mg PVA in double distilled water to a total volume of 9 mL. Stir magnetically in a 90°C oil bath for 60 min until the PVA solution is completely dissolved. Cool to room temperature for later use. 2. Dissolve 10.8 mg of borax in double distilled water to prepare borax solution; 3. Dissolve Dox Lipo and CXCL12 in the borax solution to make the total volume 4.5 mL, and the concentration of CXCL12 in the mixed solution is 6 μg / mL; 4. Add 4.5 mL of borax solution containing DoxLipo and CXCL12 to the PVA solution, and stir the mixture rapidly for 5 s to promote cross-linking to form BH-gel.

[0035] Example 5: Preparation of anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12 The Dox Lipo used in this example can be commercially available doxorubicin liposomes, or the doxorubicin liposomes prepared in Example 1.

[0036] In this embodiment, BH-gel is prepared by mixing 8% polyvinyl alcohol (PVA, thickener), 0.1% borax (cross-linking agent), DoxLipo and CXCL12 at room temperature, specifically comprising the following steps: 1. Dissolve 1.08 g PVA in double distilled water to a total volume of 9 mL. Stir magnetically in a 90°C oil bath for 60 min until the PVA solution is completely dissolved. Cool to room temperature for later use. 2. Prepare borax solution by dissolving 13.5 mg borax in double distilled water; 3. Dissolve Dox Lipo and CXCL12 in the borax solution to make the total volume 4.5 mL, and the concentration of CXCL12 in the mixed solution is 6 μg / mL; 4. Add 4.5 mL of borax solution containing DoxLipo and CXCL12 to the PVA solution, and stir the mixture rapidly for 5 s to promote cross-linking to form BH-gel.

[0037] Example 6: Particle Characterization of Doxorubicin Liposomes In this example, the average particle size and Zeta potential of the Dox Lipo prepared in Example 1 were measured using a Malvern particle size analyzer using a dynamic light scattering method. The stability of Dox Lipo was evaluated within 14 days by monitoring the changes in particle size and potential. The morphology of Dox Lipo was observed using a field emission transmission electron microscope using a 1% phosphotungstic acid negative staining technique.

[0038] (1) Changes in particle size and average Zeta potential of Dox Lipo within 14 days Figure 1 The average particle size of Dox Lipo measured by dynamic light scattering was 141.2±2.7 nm, and the average Zeta potential was -1.394±0.017 mV. There was no significant change in the average particle size and average potential within 14 days, which proved that Dox Lipo had good stability.

[0039] (2) TEM image of Dox Lipo Figure 2 As shown, Dox Lipo has a typical cup-shaped structure.

[0040] Example 7: Fourier transform infrared absorption spectrum of BH-gel In order to study the cross-linking mechanism of hydrogel, a certain amount of polyvinyl alcohol, borax and blank gel were prepared and freeze-dried. They were ground and pressed into tablets with potassium bromide, and the infrared absorption of the samples was measured at a wavelength range of 4000 ~ 400 cm⁻¹. Figure 3 As shown in the figure, FT-IR showed that the blank hydrogel had a higher peak at 1394 cm-1 compared with PVA and borax. −1 The characteristic peak shift is due to the stretching vibration of the B─O bond, proving the formation of borate ester bonds.

[0041] Example 8: Observation of the morphology and structure of BH-gel The cross section of the freeze-dried hydrogel prepared in Example 7 was mounted on a metal base and sprayed with gold under vacuum, and its morphology and structure were observed using a field emission scanning electron microscope. The SEM image is shown in FIG. Figure 4As shown, the blank gel has a three-dimensional porous structure of 300-500 nm and is an ideal carrier for loading CXCL12 and Dox Lipo.

[0042] Example 9: Study on tumor cell migration dynamics The BH-gel prepared in the present embodiment was placed in the lower chamber of the CIM-16 plate, and 100 μL of A549 cell suspension (2×10 4 After a period of time, the cells in the lower chamber were stained with Calcein-AM, and live cell images were obtained using a cell imaging system. Figure 5 As shown in the cell migration experiment, the number of A549 cells migrating in the BH-gel group increased significantly, and its fluorescence intensity was about 15 times that of the blank gel group. This indicates that the "bait" CXCL12 released from the BH-gel can attract tumor cells and prompt them to actively migrate to the gel.

[0043] Example 10: In vitro degradation of BH-gel and in vitro release of Dox lipo The BH-gel (W0) was weighed and immersed in 10 mL PBS (pH 7.4), and the sample was placed in a constant temperature shaker (37°C, 100 rpm) for 48 h. The gel was taken out and weighed at different time points (W t ), the weight ratio is calculated as follows:

[0044] BH-gel containing 100 µg of Dox was immersed in 10 mL PBS (pH 7.4) and gently shaken in a constant temperature shaker at 37°C and 100 rpm for 48 h. At different time points, 1 mL of the extract was taken out and 1 mL of fresh PBS was added to keep the total volume at 10 mL. Dox solutions of different concentrations were prepared with DMSO, and the characteristic absorption of Dox was measured at 481 nm using a UV spectrophotometer to establish a standard curve. The absorbance measured at 481 nm of the extract was substituted into the standard curve to calculate the Dox concentration in the extract, and the release rate of Dox Lipo in the gel was calculated based on the total amount of Dox.

[0045] like Figure 6As shown in the figure, the in vitro degradation experiment of BH-gel and the in vitro release experiment of Dox Lipo showed that within 48 hours, nearly 80% of BH-gel was degraded in PBS. BH-gel showed excellent degradability, indicating that it is safe and degradable in vivo. Dox Lipo can be detected to be released rapidly from BH-gel within the first 12 hours, followed by a slow and continuous release until 48 hours, which confirms that Dox Lipo can be effectively and fully released from BH-gel to improve the therapeutic effect.

[0046] Example 11: Cellular uptake assay Laser scanning confocal microscopy was used to evaluate the cellular uptake of Dox Lipo by A549 cells. A549 cells were seeded in glass-bottom culture dishes and incubated at 37°C to 60% - 80% confluence. The medium was replaced with fresh medium containing Dox Lipo (Dox content 32 μM), and the medium was discarded after 4 h of incubation. Hoechst 33342 was added and incubated for 10 minutes. The cells were then washed three times with PBS and imaged under a laser scanning confocal microscope.

[0047] Laser scanning confocal microscopy was used to investigate the efficiency of tumor cells taking up Dox Lipo released from BH-gel. Figure 7 As shown in the figure, the uptake of Dox Lipo by tumor cells A549 at 4 h was significantly higher than that of free Dox. Among them, the cell nucleus was stained with Hoechst33342, and Dox was autofluorescent. This shows that administration in the form of liposomes significantly increased the uptake of Dox by tumor cells.

[0048] Example 12: Cytotoxicity test A549 cells were seeded in a 96-well plate at a density of 5000 cells / well and incubated for 24 h. When the cells reached about 80% confluence, the medium was replaced with fresh medium containing Dox Lipo. After another 24 h of incubation, 20 μL of MTT solution (5 mg / mL) was added to 100 μL of medium per well. Then, the plates were incubated at 37°C for 4 h until purple formazan crystals were clearly visible, then the medium was discarded, 150 μL of DMSO was added to each well to dissolve the formazan, and the absorbance was measured at 570 nm using a microplate reader.

[0049] like Figure 8 As shown in the figure, the MTT results showed that the inhibition rate of free Dox on the growth of A549 cells was about 15%. Dox Lipo significantly inhibited the growth of A549 cell tumors with an inhibition rate of about 45%. The addition of liposomes enhanced the killing effect of doxorubicin on tumor cells by about 3 times.

[0050] Example 13: Cell apoptosis detection A549 cells were collected at 3×10 4 The cells were seeded in a 24-well plate at a density of 1.5 cells / well. Dox and Dox Lipo were added and incubated for 2 h, respectively. The cells were treated according to the Annexin V-FITC apoptosis detection kit protocol, and the treated cells were analyzed by flow cytometry.

[0051] like Fig. 9 As shown in the figure, the apoptosis rate induced by Dox Lipo was about 2.2 times higher than that of free Dox. Among them, the early apoptosis rate and late apoptosis rate increased by 1.8% and 4.01%, respectively, and the necrosis rate increased by 35.4%. Therefore, Dox Lipo was completely released from BH-gel within 48 h and was rapidly internalized by tumor cells, inducing a strong apoptotic response.

[0052] Example 14: In vivo tumor model The BALB / c nude mouse tumor model was established by intraperitoneal injection of A549-GFP cells. 6 A549-GFP cells were resuspended in 25 μL DMEM medium and matrigel and then injected into the peritoneal cavity near the intestine. After injection of tumor cells, the mice were placed in the right lateral position and monitored for 30 minutes until full recovery. Subsequently, tumor-bearing mice were intraperitoneally injected with normal saline, Dox Lipo and the BH-gel prepared by the present invention (2 mg / kg, injected once every 3 days), and body weight was recorded. In vivo fluorescence imaging was performed using the Night OWLIILB 983 system on days 1, 7, 14, 21 and 28 to track tumor migration, with an exposure time of 5s. After 4 weeks, the mice were killed, and the main organs (heart, lung, liver, spleen, kidney) and tumor tissues were collected and weighed. The heart was fixed with 10% neutral formalin and stained with H&E.

[0053] BH-gel can significantly enhance the anti-tumor effect of doxorubicin and effectively inhibit tumor metastasis, providing a new strategy for the treatment of metastatic lung cancer.

[0054] (1) Experimental steps and treatment plans for tumor induction Fig.10 shown.

[0055] (2) After 28 days, the intraperitoneal tumors in different treatment groups were collected, weighed and photographed. Fig.11 As shown, the tumor weight of the BH-gel group was significantly smaller than that of the control group and the Dox Lipo group, demonstrating that BH-gel has good anti-tumor metastasis and killing effects in vivo.

[0056] (3) Quantification of A549-GFP fluorescence intensity in tumor-bearing mice. Fig.12As shown in the figure, the A549-GFP fluorescence signal in the Dox Lipo group was weaker than that in the control group, but Dox Lipo could not prevent the fluorescence signal from increasing over time. In contrast, the A549-GFP fluorescence signal in the BH-gel group was weak and difficult to detect throughout the 28 days, indicating that BH-gel has an excellent tumor eradication effect.

[0057] (4) If Fig.13 As shown in the figure, the number of tumor metastasis nodules showed that compared with the Dox Lipo group (15.8±6.8 / mouse) and the control group (10.5±4.3 / mouse), only a small number of nodules were observed in the BH-gel group (2.2±2.3 / mouse), indicating that BH-gel has a strong anti-metastasis ability.

[0058] (5) If Fig.14 As shown, organ weight analysis showed that the weight of all organs of mice given Dox Lipo was reduced compared with the control group, while the organ weight of the BH-gel group was not significantly different from that of the control group, indicating the significant systemic toxicity of free Dox Lipo and the safety of BH-gel in vivo.

[0059] Example 15: Blood biochemical analysis The mouse orbital blood sampling method was used to collect about 1 mL of blood samples from each mouse. The blood samples were coagulated at room temperature for 15-30 minutes, and then centrifuged at 3000 rpm for 20 minutes to separate the serum. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin (TBIL) in the serum were measured using an automatic biochemical analyzer to evaluate whether liver function was normal.

[0060] like Fig.15 As shown in the figure, blood biochemical tests showed that compared with the control group, the levels of aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in the serum of mice in the Dox Lipo group were significantly increased, indicating that free Dox Lipo may cause a certain degree of liver damage. However, there was no significant difference in the indicators of the BH-gel group compared with the control group. In addition, there was no difference in the levels of alanine aminotransferase (ALT) and total bilirubin (TBIL) between the groups, indicating that BH-gel can significantly reduce liver damage.

[0061] Example 16: Histological Analysis The heart was fixed in 10% neutral formalin, dried and embedded in paraffin, and each organ was cut into 3 mm thick sections, processed by standard HE staining procedures, and then observed using a cell imaging system.

[0062] like Fig.16As shown, H&E sections of heart tissue showed that mice in the Dox Lipo group had obvious heart tissue damage, indicating that free Dox Lipo was cardiotoxic, while the heart tissue damage in the BH-gel group was significantly reduced, indicating that BH-gel significantly reduced the cardiotoxicity caused by free Dox Lipo.

[0063] The hydrogel prepared by the present invention is loaded with the chemokine CXCL12 that can induce metastatic tumor cells to spread everywhere. Tumor cells have stronger invasive metastasis after surgery. Tumor cells are dispersed, with many and hidden lesions, and often have the ability to invade blood vessels for distal metastasis. Traditional targeted drugs are difficult to track completely, and it is difficult to achieve the blood drug concentration required for treatment through the dispersed identification of multiple lesions. The present invention releases the chemokine CXCL12 to induce tumor cells to converge toward the hydrogel site, thereby solving the problem that metastatic tumor cells cannot be captured in the existing system.

[0064] The invention loads adriamycin liposomes which can efficiently kill the enriched tumor cells and mobilize the immune clearance effect. At present, while chemotherapeutic drugs kill tumor cells, they have the potential risk of inducing changes in tumor cell characteristics and then escaping and metastasizing. Adriamycin liposomes can exert stronger killing ability, improve the treatment effect, and significantly inhibit the escape of tumor cells induced by chemotherapeutic drugs.

[0065] In addition, it should be noted that the shapes and names of the parts and components of the specific embodiments described in this specification may be different. Any equivalent or simple changes made based on the structure, features and principles described in the patent concept of the present invention are included in the protection scope of the patent of the present invention. The technicians in the technical field of the present invention can make various modifications or supplements to the specific embodiments described or replace them in a similar manner, as long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. An anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12, characterized in that: The preparation method of the anti-tumor metastasis hydrogel is as follows: dissolving doxorubicin liposomes and CXCL12 in a crosslinking agent solution, and then adding the crosslinking agent solution containing doxorubicin liposomes and CXCL12 into a thickener solution.

2. The method for preparing the anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12 according to claim 1, characterized in that: The preparation method specifically comprises the following steps: (1) dissolving the lipid material and doxorubicin in an organic solvent, removing the organic solvent by vacuum rotary evaporation to obtain a uniform lipid film, hydrating, homogenizing the mixture by ultrasonication, and filtering to remove impurities to obtain doxorubicin liposomes encapsulating doxorubicin; (2) Dissolving CXCL12 and the doxorubicin liposomes in a crosslinker solution, and rapidly stirring the crosslinker solution with the dissolved thickener solution to obtain an anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12.

3. The preparation method according to claim 2, characterized in that: In the step (1), the lipid material is a mixture of lecithin and cholesterol, and the mass ratio of lecithin to cholesterol is 3-6:

1.

4. The preparation method according to claim 2, characterized in that: In the step (2), the concentration of CXCL12 in the mixed solution with the cross-linking agent is greater than 6 μg / mL, and the concentration of the active drug doxorubicin in the mixed solution is greater than 900 μg / mL.

5. The preparation method according to claim 2, characterized in that: In the step (2), the thickener is polyvinyl alcohol, and the mass ratio of polyvinyl alcohol in the hydrogel is 1-8%.

6. The preparation method according to claim 2, characterized in that: In the step (2), the cross-linking agent is borax, and the mass ratio of borax in the hydrogel is 0.01-0.1%.

7. Use of the hydrogel according to claim 1 or the anti-tumor metastasis hydrogel loaded with doxorubicin liposomes and CXCL12 obtained by the preparation method according to any one of claims 2 to 6 in the preparation of an anti-tumor metastasis preparation.

8. The use according to claim 7, characterized in that: The tumor is lung cancer, breast cancer, liver cancer, ovarian cancer, gastric cancer, or myeloma.

9. The use according to claim 7, characterized in that: The CXCL12 released from the hydrogel can attract tumor cells and cause the tumor cells to actively migrate toward the gel.

10. The use according to claim 7, characterized in that: The hydrogel significantly reduces the damage of liver function and significantly reduces the cardiac toxicity caused by free Dox Lipo.

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