Preparation method and application of injectable hydrogel for colon cancer treatment

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of an injectable hydrogel for the treatment of colon cancer, comprising firstly loading a TPT package into ZIF-8 to keep the lactone ring stable to exert toxicity, and then co-loading the ZIF-8 loaded with the chemotherapy drug TPT and the sonosensitizer riboflavin sodium phosphate VB2 into the injectable ALG hydrogel, utilizing Ca in the tumor microenvironment to 2+ It forms an injectable hydrogel locally in situ; the specific steps are as follows: step 1, preparation of TPT / ZIF‑8; step 2, preparation of GRTZ. The present invention simply dopes two functional materials by using a carrier. First, TPT is encapsulated in the zeolite imidazole framework TPT / ZIF‑8, and then TPT / ZIF‑8 and riboflavin sodium phosphate (VB2) are blended in a sodium alginate solution to form an injectable gel TPT / ZIF‑8@GR (abbreviated as GRTZ). The accumulation and long-term release of drugs at the tumor site are finally achieved; and GRTZ can produce enhanced ROS under ultrasound, realizing chemotherapy-sound dynamic synergistic treatment of tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of an injectable hydrogel for treating colon cancer. Background Art

[0002] Colon cancer, also known as colorectal cancer, is a type of cancer that occurs in the colon or rectum. It has a complex pathogenesis, is prone to metastasis, has a poor prognosis, and is difficult to treat. It is the third most common cause of cancer-related death worldwide. Despite advances in laparoscopic surgery combined with neoadjuvant and adjuvant therapy, the treatment of colon cancer remains challenging in oncology, and recurrence (metastasis) of local or distant organ cancer tissue is the main problem in the treatment of colon cancer. Treatment of colon cancer usually includes a combination of surgery, chemotherapy, and radiotherapy. However, these therapies have side effects and limited therapeutic effects. For example, depending on the location of the cancer, a partial colectomy may be performed to remove part of the colon, or a proctocolectomy may be performed to remove the rectum. Radiotherapy can cause damage to healthy tissues around the tumor, such as fatigue, skin irritation, and organ damage. In addition, chemotherapy for colon cancer may cause significant side effects, including liver toxicity, gastrointestinal toxicity, and hematologic disorders.

[0003] Topotecan (TPT) is a water-soluble compound and a synthetic derivative of camptothecin. TPT has significant antitumor activity against colorectal cancer and small cell lung cancer. It inhibits the proliferation of tumor cells by destroying topoisomerase 1 and inhibiting the replication of rapidly dividing cells. However, one limitation of the application of camptothecin drugs is that their lactone ring is hydrolyzed to an inactive carboxylic acid group (inactive form) at physiological pH (pH = 7.4), which reduces the efficacy of the drug. These compounds exhibit reversible pH-dependent hydrolysis under alkaline and neutral pH conditions. Therefore, it is urgent to design and develop a drug delivery system to maintain the active form of TPT and effective sustained release.

[0004] In recent years, various nanoparticle-mediated drug delivery systems have been designed and developed to maintain the chemical stability of anti-tumor drugs and improve their release. These drug delivery systems include metal organic frameworks, lipid nanoparticles, mesoporous silica nanoparticles, solid lipid nanoparticles, liposomes, etc. It is reported that the metal organic framework ZIF-8 can protect the loaded drug and achieve specific release of the drug at the target. Nanocarrier systems based on ZIF-8 have been shown to solve some of the problems of traditional anticancer therapies, including lack of selectivity, induced tissue toxicity, reduced tumor cell uptake and instability. Although ZIF-8 is very effective for drug delivery, sometimes the loaded drug is released suddenly, causing serious toxic side effects and treatment failure. Based on this, designing a drug delivery system that responds to and locates in the tumor microenvironment is a strategy to maximize the anticancer treatment effect while minimizing the side effects of existing anticancer drugs.

[0005] Hydrogels have attracted great interest in the field of anticancer therapy because they can encapsulate drugs, peptides, and other therapeutic drugs and deliver them to targeted sites in a controlled and sustained manner. Sodium alginate (ALG) is composed of α-L-guluronic acid and β-D-mannuronic acid. It has the advantages of a wide range of raw material sources, low price, good biocompatibility, and biodegradability. It is often used as a carrier for drug delivery and release. ALG has rich functional groups, and its negatively charged carboxyl part can interact with multivalent cations or cationic polymers to construct hydrogels through ionic crosslinking. Ca 2+ ALG is a commonly used cross-linking agent. 2+ Complexation occurs, achieving an in situ sol-gel transition. One of the key advantages of using hydrogels for cancer therapy is the specific delivery of drugs to the tumor site, which can reduce the side effects associated with systemic administration.

[0006] In order to achieve highly effective and low-toxic anti-tumor treatment effects, a single treatment method often cannot meet the needs of clinical treatment effectiveness. Drawing on the advantages of different anti-cancer strategies, such as sonodynamic therapy (SDT), ferroptosis, immunotherapy, etc., and combining multiple medicinal mechanisms and anti-cancer pathways, it is expected to make up for the shortcomings of a single treatment method and achieve better anti-tumor treatment effects. However, there is still a lack of reliable solutions today. Summary of the invention

[0007] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for preparing an injectable hydrogel for the treatment of colon cancer and its application. The injectable hydrogel prepared by the invention can achieve the accumulation and long-term release of drugs at the tumor site, and ultimately realize the chemo-sonodynamic synergistic treatment of the tumor.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for preparing an injectable hydrogel for the treatment of colon cancer comprises firstly loading TPT into ZIF-8 to keep the lactone ring stable to exert toxicity, then co-loading the ZIF-8 loaded with the chemotherapy drug TPT and the sonosensitizer riboflavin sodium phosphate VB2 into the injectable ALG hydrogel, and utilizing Ca in the tumor microenvironment to 2+ The specific steps of forming an injectable hydrogel locally in situ are as follows:

[0010] Step 1. Preparation and characterization of TPT / ZIF-8: TPT was dissolved in 2 mL PBS buffer solution (pH = 5) to prepare a solution of 80 mg / mL, the TPT solution was mixed with Zn(NO3)2·6H2O, and after stirring evenly, the dimethylimidazole solution was rapidly added dropwise to the Zn(NO3)2·6H2O mixed solution, stirred for 5 min, centrifuged at 13000 rpm / min for 10 min, and then washed three times with PBS buffer solution (pH = 5) (13000 rpm / min, 10 min), the supernatant was collected for subsequent determination of the loading rate, and the precipitate was placed in a vacuum drying oven and dried overnight to obtain TPT / ZIF-8 spherical particles.

[0011] The size and morphology of TPT / ZIF-8 were observed under a transmission electron microscope, and the results showed that TPT / ZIF-8 was spherical particles with a particle size of about 180nm. The X-ray diffraction pattern (XRD) showed that TPT / ZIF-8 maintained a crystalline structure, with characteristic diffraction peaks at 7.39°, 10.45°, 12.84° and 16.51°, corresponding to the (110), (200), (211) and (222) planes, respectively.

[0012] In order to calculate the loading amount of TPT, HPLC analysis was performed here. Since TPT is easily hydrolyzed in neutral solution, PBS with pH = 5 was used to prepare standard solution samples of 20, 40, 60, 80, 100 and 120 μg / mL TPT. The concentration of TPT was used as the horizontal axis and the peak area obtained by detection was used as the vertical axis. The standard curve and regression equation of TPT were drawn as the basis for calculating the drug loading amount. The content of TPT in TPT / ZIF-8 was calculated to be 50.29wt%.

[0013] Step 2, preparation and characterization of GRTZ: 2% ALG, 5 mg / mL VB2 and 1.94 mg / mL TPT / ZIF-8 were physically mixed to obtain TPT / ZIF-8@GR, referred to as GRTZ. ALG and VB2 were fully dissolved in 5 mL of triple distilled water and named GR. ALG and TPT were fully mixed in 5 mL of triple distilled water and named TPT@GR, referred to as GRT.

[0014] The present invention also provides an application of the injectable hydrogel prepared by the preparation method in preparing a drug for treating colon cancer.

[0015] Preparation and drug loading analysis of GRTZ

[0016] Reference Figure 1 The integrity of the lactone ring of TPT is the key to its cytotoxicity. It is stable under acidic conditions (pH < 4), but is prone to hydrolysis under physiological conditions (pH = 7.4), reversibly converting to its carboxylate form, thereby losing its inhibitory activity against topoisomerase I. Therefore, TPT was loaded into ZIF-8 and further loaded into a hydrogel containing VB2 to ensure the integrity of its lactone ring before reaching the tumor site.

[0017] Transmission electron microscopy showed that TPT / ZIF-8 was spherical particles with a particle size of about 180 nm. X-ray diffraction (XRD) showed that TPT / ZIF-8 maintained a crystalline structure with characteristic diffraction peaks at 7.39°, 10.45°, 12.84°, and 16.51°, corresponding to the (110), (200), (211), and (222) planes, respectively.

[0018] The drug loading was analyzed by HPLC. Since TPT is easily hydrolyzed in a neutral solution, a PBS buffer solution with pH = 5 was used to prepare standard solution samples of 20, 40, 60, 80, 100 and 120 μg / mL TPT. The concentration of TPT was used as the horizontal axis and the peak area obtained by detection was used as the vertical axis. The standard curve and regression equation of TPT were drawn as the basis for calculating the drug loading. The content of TPT in TPT / ZIF-8 was calculated to be 50.29wt%.

[0019] In vivo and in vitro gelation performance test of GRTZ:

[0020] Reference Figure 2 ALG is a water-soluble natural polysaccharide whose molecule is composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G) units connected by the Na + With Ca in solution 2+ An ion exchange reaction occurs to form an interpenetrating network gel with an "egg-box" structure. Based on the gelling properties of ALG, it is used as a carrier to construct an injectable GRTZ hydrogel. ALG can achieve an in situ "sol-gel" transition in vivo. The resulting cross-linked eggshell structure fixes VB2 and TPT / ZIF-8 in the gel, making it less susceptible to degradation and metabolism, slowly releasing drugs, prolonging the drug action time, and improving drug efficacy. ALG is colorless and transparent, while VB2 can act as a visual indicator of gelling. The prepared GRTZ solution is an orange-yellow flowing liquid with weak fluidity and high viscosity. When injected with Ca2+ When the solution of ALG and Ca 2+ After ionic crosslinking, the GRTZ solution transformed into an orange-yellow translucent hydrogel within seconds.

[0021] GRTZ has injectability and in vivo gelation ability. ALG is in a flowing state before gelation, and can still pass smoothly through a 0.45-gauge needle after mixing with the drug solution. 2+ The sol-gel transition can be achieved quickly, indicating that GRTZ has the ability to deliver injectable gels in situ. At the same time, GRTZ solution was injected subcutaneously in BALB / c mice to verify its in situ gelling performance. It can quickly form hydrogels after injection into the body, thereby improving the in situ retention of drugs.

[0022] Scanning electron microscopy revealed that the GRTZ hydrogel had a multilayer porous structure. 2+ Complexation with ALG causes G units to stack and form a cross-linked network structure, indicating that it can respond to Ca 2+ It achieves sol-gel transition and is therefore suitable as an efficient drug loading platform for in situ drug delivery.

[0023] GRTZ release behavior test:

[0024] Reference Figure 3 The GRTZ hydrogel was placed in a solution filled with Ca 2+ The glass bottles were filled with (1.8mM) buffer and shaken at 37°C to simulate the release of drugs under physiological conditions. The experimental results showed that GRTZ can achieve long-term sustained release, with a 96h release rate of nearly 80%.

[0025] Rheological behavior test of GRTZ:

[0026] Reference Figure 4 The internal interactions of hydrogels are closely related to their rheological properties, which affect whether the hydrogels can effectively release drugs. In order to study the rheological behavior of GRTZ hydrogels, a rotational rheometer was used to test the rheological properties of the material, such as viscosity, G', and G". Over the entire shear rate range, the viscosity of the GRTZ gel continued to decrease, showing the characteristics of a non-Newtonian fluid, indicating that the hydrogel has shear-thinning behavior. It can be observed that within a certain shear rate range, the G' curve of the GRTZ gel is always greater than the G" curve, indicating that the gel has formed a relatively stable network structure.

[0027] GRTZ's ROS generation performance test:

[0028] Reference Figure 5 , 1,3-diphenylisobenzofuran (DPBF) was used to verify the sonosensitivity of VB2 and GRTZ.1 O2) reaction at 410nm absorbance decreases, which can be used to determine whether there is 1 O2 generation. UT1021-ultrasound therapy instrument was used, and the ultrasound intensity was controlled at 160mW / cm 2 , and the two produced 1 O2 capacity detection.

[0029] Compared with the DPBF group alone, the presence of ultrasound and VB2 accelerated the decrease in DPBF absorbance, indicating that VB2 has the ability to produce 1 O2 can produce a certain sonosensitivity effect. On this basis, the consumption of DPBF in the presence of GRTZ was tested under ultrasonic conditions, and the results showed that the rate of decrease in its absorbance was comparable to that of VB2. Each set of data was normalized, and the results showed that the decrease in the content of the GRTZ group under ultrasonic irradiation was consistent with the above corresponding results. In summary, loading VB2 into the gel will not affect its sonosensitivity effect, and it still has the ability to produce under the action of US. 1 O2 capabilities.

[0030] Reference Figure 6 The strong oxidizing property of hydroxyl radical (·OH) can be used to reduce methylene blue (MB) to hydroxylated methylene blue, and the ·OH content is detected. During the reaction, the MB solution gradually changes from blue to colorless, and the ultraviolet absorbance decreases accordingly. Therefore, taking the change in MB absorbance as the starting point, the ability of VB2 and GRTZ to produce ·OH under ultrasonic irradiation was tested. When the ultrasonic intensity was 160mW / cm 2 Under the condition of VB2 and GRTZ, the absorbance of MB solution decreased faster than that of pure MB. In addition, when the US time reached 10min, the MB content in VB2 and GRTZ solution reached the lowest, indicating that they have good ability to generate ·OH.

[0031] Cellular uptake test of GRTZ:

[0032] Reference Figure 7 CT26 cells were treated with VB2, TPT / ZIF-8 and GRTZ respectively. After 4 hours, the cell nuclei were stained with DAPI to study the cell uptake of different treatment groups. The cell uptake behavior was observed through the DAPI channel of the laser confocal microscope. After treatment with VB2 and TPT / ZIF-8, weak green fluorescence appeared around the cell nucleus, proving that VB2 and TPT / ZIF-8 can be effectively taken up by cells. After treatment with GRTZ, the green fluorescence around the cell nucleus was significantly stronger than that of the first two groups, indicating that CT26 cells had a significant uptake of GRTZ.

[0033] Cytotoxicity test:

[0034] Reference Figure 8 , after verifying that the gel system can be effectively absorbed, different concentrations of VB2 or GRTZ were co-incubated with CT26 cells for 24 hours, and the cytotoxicity was detected by MTT method. After incubation with VB2 alone, the cell activity did not change significantly; however, due to the presence of the chemotherapeutic drug TPT, the cell activity of GRTZ-treated cells decreased slightly with the increase of the drug concentration. Subsequently, the cell activity of cells co-incubated with VB2 or GRTZ and irradiated with US was tested. The cell activity of the VB2+US (ultrasound) group showed a downward trend with the concentration gradient. Compared with the GRTZ incubation group alone, the cell activity was further inhibited after GRTZ co-incubation and ultrasound treatment. The above results show that VB2 can play an excellent SDT effect, and GRTZ can synergistically inhibit the growth of tumor cells under ultrasound irradiation through the sonosensitizer VB2 and the chemotherapeutic drug TPT, and has good cytotoxicity.

[0035] Intracellular ROS generation test:

[0036] Reference Fig. 9 , sonodynamic therapy can act on sonosensitizers in target tissues through ultrasound radiation, thereby generating cytotoxic ROS. In addition, ROS and lipid ROS also play a key role in ferroptosis, which increases oxidative damage through excessive ROS accumulation. Based on the relevant ROS characterization of the material, GRTZ has the ability to generate ROS under ultrasound irradiation. Here, the DCFH-DA probe is used to detect the production of ROS at the cellular level. After incubation with GR and US irradiation, the cells showed green fluorescence, indicating that VB2 can produce certain toxic ROS. When only GRT or GRTZ were co-incubated with cells, weak green fluorescence was shown, which may be that TPT destroyed the redox balance and induced intracellular ROS accumulation. However, on this basis, after combined US treatment, the green fluorescence was further enhanced, indicating that SDT and the chemotherapeutic drug TPT can synergistically induce intracellular ROS accumulation. The above experimental results are consistent with the in vitro ROS characterization, further indicating that GRTZ can produce ROS under the action of US, and the accumulation of TPT in the tumor can destroy the redox homeostasis to a certain extent, thereby promoting ROS accumulation, and the two synergistically inhibit tumor cell growth.

[0037] Intracellular LPO production test:

[0038] Reference Fig.10Lipid peroxidation is the process of lipid oxidation and degradation, which can cause damage to the cell membrane. The C11-BODIPY581 / 591 probe can be used to detect the increase of LPO in ferroptosis by ratiometric fluorescence detection. The probe is localized on the membrane of living cells. The oxidation of the polyunsaturated butadiene part of this fatty acid analog can shift the fluorescence emission peak from red to green, so that the level of lipid peroxidation can be detected using confocal microscopy. CT26 cells incubated with GR and irradiated with ultrasound are not much different from the control group. The BODIPY581 / 591-C11 probe shows a certain red fluorescence, indicating that there are fewer intracellular lipid peroxides at this time. When only GRT or GRTZ are co-incubated with cells, weak green fluorescence of LPO is observed, indicating that there is a certain accumulation of lipid peroxides. However, after adding ultrasound treatment on this basis, the green fluorescence signal from LPO is significantly enhanced. The above experimental results show that under ultrasound irradiation, GRTZ can further increase the accumulation of intracellular lipid peroxides and induce cell ferroptosis.

[0039] In vivo tumor inhibition effect test:

[0040] Reference Fig.11 The anti-tumor effect in vivo was preliminarily evaluated in CT26 tumor-bearing mice. 3 At the same time, the mice were randomly divided into 6 groups: PBS group, GR+U group, TPT@GR group, TPT@GR+U group, TPT / ZIF-8@GR group, and TPT / ZIF-8@GR+U group. The drug was administered by intratumoral injection with an injection volume of 100 μL, and the drug was administered twice every other day. Ultrasound irradiation was performed the next day after each administration. The body weight and tumor volume changes of BALB / c mice were monitored every day to evaluate the therapeutic effect of GRTZ.

[0041] Compared with PBS, the effect of TPT@GR treatment group in inhibiting tumor growth was not very ideal, while TPT / ZIF-8@GR group showed a slight tumor growth inhibition effect. This was attributed to the fact that TPT could not stably exist under physiological conditions (pH = 7.4), and its lactone ring (active form) was easily hydrolyzed to carboxylic acid group (inactive form), thereby reducing the therapeutic effect. Loading it into ZIF-8 can achieve pH-responsive release in the tumor tissue microenvironment (pH 4.0-6.5), ensuring its toxic effect. Compared with the PBS group, the tumor growth of the GR+U group also showed a certain degree of inhibition, indicating that VB2 exerted an SDT effect. At the same time, after GRTZ treatment and ultrasound treatment, the tumor inhibition rate reached 84.62%, which was the most significant tumor inhibition effect among the 6 groups. After the treatment, the subcutaneous tumors of mice in each group were dissected and weighed. It was observed that the average tumor weight of the TPT / ZIF-8@GR+U group was the lowest, showing the most significant tumor inhibition effect, and the other groups except the PBS group showed a tumor inhibition effect consistent with the tumor volume. By monitoring the body weight of mice in different treatment groups during the treatment period, it was found that the range of body weight changes tended to be stable and no adverse reactions were observed during the entire treatment process, indicating that the gel system has biosafety.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The present invention simply dopes two functional materials by using a carrier. First, TPT is encapsulated in the zeolite imidazole framework TPT / ZIF-8, and then TPT / ZIF-8 and riboflavin sodium phosphate (VB2) are blended in a sodium alginate solution to form an injectable gel TPT / ZIF-8@GR (abbreviated as GRTZ).

[0044] 2. The present invention has found that GRTZ can be activated in the tumor microenvironment at high concentrations of Ca 2+ The conditions for in situ gelation can be achieved under the conditions of ultrasound, ultimately achieving the accumulation and long-term release of drugs at the tumor site; moreover, GRTZ can produce enhanced ROS under the action of ultrasound, realizing the synergistic chemotherapy-sonodynamic treatment of tumors.

[0045] 3. The preparation conditions of the present invention are mild, the stability is strong, the process operation is simple, and it has shown good therapeutic effects in cell and animal experiments, providing new means and technologies for clinical anti-colon cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The morphology and structure diagram of TPT / ZIF-8 of the present invention; (a) transmission electron microscope and elemental analysis diagram of TPT / ZIF-8; (b) XRD spectrum of TPT / ZIF-8; (c) standard curve of TPT solution.

[0047] Figure 2 The in vivo and in vitro gelation performance diagrams of the GRTZ of the present invention; (a) GRTZ solution and its gel morphology; (b) needle permeability results of the GRTZ solution; (c) in vivo gelation performance of the GRTZ solution; (d) scanning electron microscopy image of GRTZ.

[0048] Figure 3 The in vitro drug release performance diagram of GRTZ of the present invention; (a) drug release schematic diagram and (b) VB2 release of GRTZ gel under physiological conditions.

[0049] Figure 4 The rheological behavior diagram of the GRTZ of the present invention; (a) viscosity test of GRTZ; (b) rheological test of GRTZ.

[0050] Figure 5 The GRTZ of the present invention is produced under ultrasonic conditions 1 Capacity diagram of O2; (a) oxidation reaction based on DPBF molecules; absorbance changes of DPBF at different times under ultrasonic conditions in the presence of (b) PBS, (c) VB2, and (d) GRTZ, and (e) changes in the content of DPBF in different groups.

[0051] Figure 6 Graph showing the ability of GRTZ of the present invention to generate ·OH under ultrasonic conditions; (a) oxidation reaction based on MB molecules; changes in the absorbance of MB under ultrasonic conditions in the presence of (b) PBS (c) VB2 (d) GRTZ at different times and (e) changes in the content of MB in different groups.

[0052] Figure 7 This is a graph showing the cellular uptake of GTRZ of the present invention; scale bar = 25 μm.

[0053] Figure 8 The figure is a graph showing the effect of GTRZ of the present invention on the activity of CT26 cells under ultrasound conditions; (a) relative activity of cells after VB2 / GRTZ treatment; (b) relative activity of cells after GRTZ treatment under ultrasound irradiation.

[0054] Fig. 9 A graph showing the ability of GTRZ of the present invention to generate ROS in CT26 cells under ultrasound conditions; scale bar = 25 μm.

[0055] Fig.10 This is a graph showing the ability of GTRZ of the present invention to produce LPO in CT26 cells under ultrasound conditions; scale bar = 50 μm.

[0056] Fig.11The figure shows the tumor treatment of tumor-bearing mice by GTRZ of the present invention; (a) tumor treatment timeline; (bg) tumor growth curves of individual mice in each group; (h) changes in tumor volume growth in different groups; (i) tumor weights in different groups after treatment; (j) changes in body weight of mice in different groups during treatment. DETAILED DESCRIPTION

[0057] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the protection scope of the present invention. The embodiments described in the present invention are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.

[0058] Embodiment 1:

[0059] 1. Dissolve TPT in 2 mL PBS buffer (pH = 5) to prepare a solution of 80 mg / mL;

[0060] 2. Mix TPT solution with Zn(NO3)2·6H2O and stir evenly;

[0061] 3. Add the dimethylimidazole solution dropwise to the Zn(NO3)2·6H2O mixed solution and stir for 5 minutes;

[0062] 4. Centrifuge at 13000 rpm / min for 10 min, then wash three times with PBS buffer solution (pH=5) (13000 rpm / min, 10 min);

[0063] 5. Collect the supernatant for subsequent determination of the loading rate, and place the precipitate in a vacuum drying oven to dry overnight to obtain TPT / ZIF-8 spherical particles;

[0064] 6. Prepare the prepared TPT / ZIF-8 into a 0.1 mg / mL solution, drop an appropriate amount of the solution onto a copper grid, and observe its morphology using a transmission electron microscope;

[0065] 7. Detect the crystal structure of TPT / ZIF-8 by XRD, with a scanning speed of 10° / min and a scanning range of 5°-40°;

[0066] 8. Prepare standard solutions with TPT concentrations ranging from 20 to 120 μg / mL, using 60% methanol as the mobile phase, an injection volume of 10 μL, a flow rate of 1 mL / min, and test the standard curve of chromatographic peak area and TPT concentration at a detection wavelength of 268 nm. Perform quantitative analysis of the supernatant of TPT / ZIF-8 under the same conditions, and use the standard curve to calculate the concentration of encapsulated TPT.

[0067] Embodiment 2:

[0068] 1. Physically mix ALG (2%), VB2 (5 mg / mL) and TPT / ZIF-8 (1.94 mg / mL) to obtain TPT / ZIF-8@GR, referred to as GRTZ;

[0069] 2. Dissolve ALG and VB2 in 5 mL of triple distilled water to obtain GR;

[0070] 3. ALG and TPT were thoroughly mixed in 5 mL of triple-distilled water to obtain TPT@GR, referred to as GRT.

[0071] Embodiment 3:

[0072] 1. Take a certain amount of GRTZ solution and inject it into CaCl2 solution ([Ca 2+ ]=1.8mM) to observe the gelation properties under physiological conditions. 2+ Ionic cross-linking occurred, and the GRTZ solution transformed into an orange-yellow translucent hydrogel within seconds, indicating that GRTZ has injectability and in vivo gelation ability;

[0073] 2. In the same way, GRTZ solution was injected subcutaneously into BALB / c mice to verify its in situ gelling performance. It can quickly form hydrogels after injection into the body, thereby improving the in situ retention of drugs;

[0074] 3. Seal the prepared GRTZ hydrogel sealing film and place it in a -80℃ refrigerator for 24 hours. Place it in a vacuum freeze dryer, freeze-dry it, and store the obtained sample in a dark and dry place at room temperature;

[0075] 4. Take a certain amount of sample slices and stick them on the conductive tape on the sample stage. After the sample is fixed, it is sprayed with gold. The morphological observation is carried out by scanning electron microscopy. It is found that GRTZ is a hydrogel structure, which indicates that it can respond to Ca 2+ It achieves sol-gel transition and is therefore suitable as an efficient drug loading platform for in situ drug delivery.

[0076] Embodiment 4:

[0077] 1. Place GRTZ (final concentration: VB2 5 mg / mL, TPT 0.97 mg / mL, ALG 2%) in a solution filled with Ca 2+ (1.8 mM) buffer in a glass bottle;

[0078] 2. Place it on a shaker at 37°C at a shaking speed of 100 rpm, and observe and collect the drug supernatant at 24h, 48h, 72h, 96h, 120h, 144h, and 168h after shaking, and add an equal amount of supplementary solution;

[0079] 3. The absorption spectrum curve was detected by UV-visible spectrophotometer. The experimental results showed that GRTZ can achieve long-term sustained release, with a 96h release rate of nearly 80%.

[0080] Embodiment 5:

[0081] 1. Rheological properties were tested on an ARES-G2 rotational rheometer with a plate diameter of 40 mm and a spacing of 1 mm;

[0082] 2. At a shear rate of 0.001 to 1000 s -1 At room temperature, the elastic modulus (G'), viscous modulus (G") and viscosity were measured. Over the entire shear rate range, the viscosity of the GRTZ gel continued to decrease, showing the characteristics of a non-Newtonian fluid, indicating that the hydrogel has shear thinning behavior. Within a certain shear rate range, the GRTZ gel shows that the G' curve is always greater than the G" curve, indicating that the gel has formed a relatively stable network structure.

[0083] Embodiment 6:

[0084] 1. Prepare free VB2 and GRTZ solution in advance (final concentration: VB2 is 5 mg / mL), and mix the freshly prepared DPBF solution (3.7 mM, 100 μL) with the two;

[0085] 2. Use ultrasonic therapy device, and control its ultrasonic frequency and intensity at 1.0MHz and 160mW / cm 2 , adjust different ultrasonic times to process the samples;

[0086] 3. Use UV-visible spectrophotometer to obtain the absorbance of DPBF under different conditions at 412nm to investigate 1 GRTZ has the ability to generate O2 under ultrasonic irradiation conditions. 1 O2's ability can produce a certain sound-sensitive effect.

[0087] Embodiment 7:

[0088] 1. Prepare fresh MB aqueous solution (2.7 mM, 10 μL) in advance, and mix it with the pre-prepared free VB2 and GRTZ solution (5 μg / mL);

[0089] 2. The mixed solution was subjected to ultrasonic treatment, and the ultrasonic frequency and intensity were controlled at 1.0 MHz and 160 mW / cm 2 ;

[0090] 3. The absorbance of MB at 665 nm at the corresponding time point was obtained by ultraviolet measurement to test the ·OH generation performance of the GRTZ gel system. 2 Under the condition of VB2 and GRTZ, the absorbance of MB solution decreased faster than that of pure MB. In addition, when the ultrasonic time reached 10 min, the MB content in VB2 and GRTZ solution reached the lowest, indicating that GRTZ has a good ability to produce ·OH.

[0091] Embodiment 8:

[0092] 1. Place the CT26 cell slides at the bottom of a 6-well plate for UV irradiation. Then, after the logarithmic growth of CT26 cells is digested, 2×10 5 The cell density of cells / well was inoculated on the surface of the cell slide in a 6-well plate;

[0093] 2. After incubation for 24 h, VB2, TPT / ZIF-8, and GRTZ (VB2: final concentration 100 μg / mL, TPT 4 μg / mL) solutions were co-incubated with CT26 cells respectively;

[0094] After 3 or 4 h, the 6-well plate was taken out and the culture medium and materials were gently aspirated, and an appropriate amount of 4% paraformaldehyde was added for fixation. DAPI staining solution was added to each well for incubation, and slides were prepared after the incubation.

[0095] 4. Observe the cells with a laser scanning confocal microscope. After VB2 and TPT / ZIF-8 treatment, the cell nucleus showed weak green fluorescence, proving that both VB2 and TPT / ZIF-8 can be effectively taken up by cells. After GRTZ treatment, the green fluorescence around the cell nucleus was significantly stronger than that of the first two groups, indicating that CT26 cells have a significant uptake of GRTZ.

[0096] Embodiment 9:

[0097] 1. Digest and resuspend CT26 cells in the logarithmic growth phase to prepare a single cell suspension at 1×10 4 The cells were seeded at a density of 10 cells / well in a 96-well plate and cultured for 24 h;

[0098] 2. After the cells are completely attached, discard the old culture medium and add 100 μL of VB2 and GRTZ of different concentrations. The final concentration of VB2 is 0.1, 1, 5, 25, 50, and 100 μg / mL. The culture wells without VB2 or GRTZ are the control group (n=3);

[0099] 3. After 4 h of incubation, the cells were exposed to ultrasound (160 mW / cm 2 , 5min), and continue to culture for 24h;

[0100] 4. Add MTT solution (5 mg / mL), 20 μL per well, and continue to culture for 4 hours. Gently aspirate the culture medium (avoid aspirating the formazan crystals at the bottom), add 150 μL dimethyl sulfoxide to each well, and shake on a shaker for 5 minutes to dissolve the formazan;

[0101] 5. Use an ELISA reader to measure the absorbance of each well at a wavelength of 490nm, and calculate the cell survival rate according to the formula. GRTZ can synergistically inhibit tumor cell growth under ultrasound irradiation through the sonosensitizer VB2 and the chemotherapy drug TPT, and has good cytotoxicity.

[0102] Embodiment 10:

[0103] 1. Select CT26 cells in the logarithmic growth phase, digest and resuspend them to make a single cell suspension, and use 1×10 5 The cells were seeded at a density of 10 cells / well in a confocal dish and cultured in an incubator for 24 h;

[0104] 2. After the cells are completely attached, remove the old culture medium, rinse with PBS three times, add the prepared GR, GRT, GRTZ (final concentration: VB2 100μg / mL, TPT 4μg / mL) and PBS, and incubate for 6h;

[0105] 3. Rinse with PBS three times, and co-stain the cells with 10 μM DCFH-DA probe for 15 min. The corresponding groups were ultrasonically treated with an intensity of 160 mW / cm 2 Irradiation treatment for 5 min;

[0106] 4. Observe cells with a laser scanning confocal microscope. GRTZ can produce ROS under ultrasound, and the accumulation of TPT in the tumor can destroy the redox homeostasis to a certain extent, thereby promoting the accumulation of ROS. The two synergistically inhibit the growth of tumor cells.

[0107] Embodiment 11:

[0108] 1. Select CT26 cells in the logarithmic growth phase, digest and resuspend them to make a single cell suspension, and use 1×10 5 The cells were seeded at a density of 10 cells / well in a confocal dish and cultured in an incubator for 24 h;

[0109] 2. After the cells are completely attached, rinse with PBS three times, and then incubate the cells with the prepared GR, GRT, GRTZ (final concentration: VB2 100μg / mL, TPT 4μg / mL) and PBS for 6h;

[0110] 3. Prepare 2 μM C11-BODIPY581 / 591 probe, discard the old culture medium after 6 hours, add the probe for co-staining for 30 minutes, and ultrasonically treat the corresponding groups with an intensity of 160 mW / cm 2 Irradiation treatment for 5 min;

[0111] 4. After treatment, add the prepared DAPI staining solution and incubate for 5 minutes at room temperature. Gently aspirate the staining solution and rinse with PBS 3 times;

[0112] 5. Use laser scanning confocal microscopy to observe the intracellular LPO production. Among them, DAPI, Rhod and FITC channels correspond to the cell nucleus (blue fluorescence), reduced BODIPY-C11 (red fluorescence) and oxidized BODIPY-C11 (green fluorescence), respectively. Under ultrasound irradiation, GRTZ can further increase the accumulation of intracellular lipid peroxides and induce cell ferroptosis.

[0113] Embodiment 12:

[0114] 1. Select 6-8 week old female BALB / c mice and remove the hair on the right back. Take logarithmically growing CT26 cells and digest them. Add them to the culture medium, centrifuge and resuspend them to make a single cell suspension. Count the cells and dilute them with PBS to 1×10 6 cells / mL, placed on ice until injection;

[0115] 2. PBS containing 100 μL CT26 cells was injected subcutaneously from the right back of the mouse. When the tumor volume reached about 50 mm 3 At , the mice were randomly divided into 6 groups;

[0116] 3. PBS, GR+U, TPT@GR, TPT@GR+U, GRTZ, GRTZ+U (final concentration: VB2 5 mg / mL, TPT 0.97 mg / mL, ALG 2%) were injected into 6 groups of mice by intratumoral injection. The injection volume was 100 μL. The mice were given twice every other day. Ultrasound irradiation (160 mW / cm 2 , 5min);

[0117] 4. During the treatment, the body weight of BALB / c mice in each group and the length and width of the subcutaneous tumor were monitored and recorded every day, and the tumor volume was calculated;

[0118] 5. Tumor volume exceeds 2000mm 3 BALB / c mice need to be euthanized. After the treatment, the subcutaneous tumors of the mice were dissected and weighed for subsequent in vivo anti-tumor studies. It was observed that the average tumor weight of the GRTZ group was the lowest, showing the most significant tumor inhibition effect. The change in the weight of the mice tended to be stable, and no adverse reactions were observed during the entire treatment process, indicating that the GRTZ gel system has biosafety.

[0119] In summary, the present invention uses a carrier to simply dope two functional materials. First, TPT is encapsulated in the zeolite imidazole framework TPT / ZIF-8, and then TPT / ZIF-8 and riboflavin sodium phosphate (VB2) are blended in a sodium alginate solution to form an injectable gel TPT / ZIF-8@GR (abbreviated as GRTZ). Ultimately, the drug is accumulated and released for a long time at the tumor site; and GRTZ can generate enhanced ROS under ultrasound, realizing chemotherapy-sound dynamic synergistic treatment of tumors.

[0120] The description and practice disclosed in the present invention are easy to think and understand for ordinary technicians in the technical field, and several improvements and modifications can be made without departing from the principles of the present invention. Therefore, modifications or improvements made without departing from the spirit of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing an injectable hydrogel for treating colon cancer, characterized in that: The method includes first loading the topotecan TPT package into ZIF-8 to keep the lactone ring stable and exert toxicity, and then co-loading the ZIF-8 loaded with the chemotherapy drug TPT and the sonosensitizer riboflavin sodium phosphate VB2 into the injectable sodium alginate ALG hydrogel. The specific steps are as follows: Step 1, preparation of TPT / ZIF-8: TPT was dissolved in 2 mL PBS buffer solution to prepare a solution of 80 mg / mL, the TPT solution was mixed with Zn(NO3)2•6H2O, and after stirring evenly, the dimethylimidazole solution was quickly added dropwise to the Zn(NO3)2•6H2O mixed solution, stirred for 5 min, centrifuged at 13000 rpm / min for 10 min, and then washed three times with PBS buffer solution, the supernatant was collected for subsequent determination of the loading rate, and the precipitate was placed in a vacuum drying oven and dried overnight to obtain TPT / ZIF-8 spherical particles; Step 2, preparation of GRTZ: 2% ALG, 5 mg / mL VB2 and 1.94 mg / mL TPT / ZIF-8 were physically mixed to obtain TPT / ZIF-8@GR, referred to as GRTZ.

2. The method for preparing an injectable hydrogel for treating colon cancer according to claim 1, characterized in that: In step 1, the pH of the PBS buffer solution is 5.

3. Use of an injectable hydrogel prepared by the preparation method according to any one of claims 1 to 2 in the preparation of a drug for treating colon cancer.

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