Composite hydrogel drug-loaded stent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of drug-loading stents, and discloses a composite hydrogel drug-loading stent as well as a preparation method and application thereof. The preparation method of the composite hydrogel drug-loading stent comprises the following steps: mixing acrylamide, bacterial cellulose, N, N '-methyl bisacrylamide, potassium persulfate and water, and then adding a fluorouracil solution to obtain a pre-solution; and dipping a GAA hydrogel tube in the pre-solution, and then drying to obtain the composite hydrogel drug-loading stent. According to the fluorouracil-loaded hydrogel drug coating stent, the surface of the stent is covered with a hydrogel-loaded fluorouracil drug, and the hydrogel-loaded fluorouracil drug is controlled in a colorectal cancer focus, so that an emergency operation is converted into a selective operation, and the risk is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of drug-loaded stents, and in particular to a composite hydrogel drug-loaded stent and a preparation method and application thereof. Background Art

[0002] Colorectal cancer (CRC) ranks third in the world in terms of cancer incidence and mortality, and about 7%-28% of cases present with intestinal obstruction as the first symptom. Emergency surgery is the main treatment, but the risk of primary anastomotic leakage, preventive stoma rate, postoperative complications and mortality rate of emergency surgery are all high. Therefore, there is an urgent need to find an effective drug-eluting intestinal obstruction stent to improve the clinical efficacy and prognosis of colorectal cancer patients. Summary of the invention

[0003] The purpose of the present invention is to provide a composite hydrogel drug-loaded stent and a preparation method and application thereof, so as to solve the problem of precise treatment within colorectal cancer lesions and reduce systemic toxic and side effects.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing a composite hydrogel drug-loaded stent, comprising the following steps:

[0006] condensation tube;

[0007] (1) acrylamide, bacterial cellulose, N,N′-methylbisacrylamide, potassium persulfate and water are mixed, and then a fluorouracil solution is added to obtain a pre-solution;

[0008] (2) The GAA hydrogel tube is immersed in the pre-solution and then dried to obtain a composite hydrogel drug-loaded scaffold.

[0009] Preferably, the usage ratio of acrylamide, bacterial cellulose, N,N′-methylbisacrylamide, potassium persulfate and water in step (1) is 4-6 g: 2-3 g: 0.004-0.01 g: 0.01-0.1 g: 10-100 mL;

[0010] and / or, the concentration of the fluorouracil solution is 4 to 10 mg / mL;

[0011] And / or, the usage ratio of the fluorouracil solution to acrylamide is 4-10 mg:4-6 g.

[0012] Preferably, the immersion temperature in step (2) is 20 to 30° C., and the immersion time is 12 to 24 hours.

[0013] The drying temperature in step (2) is 40-60°C.

[0014] The invention also provides a composite hydrogel drug-loaded stent prepared by the method for preparing the composite hydrogel drug-loaded stent.

[0015] The present invention also provides an application of the composite hydrogel drug-loaded stent in drug release.

[0016] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0017] The fluorouracil-loaded hydrogel drug-coated stent of the present invention has a surface coated hydrogel-loaded fluorouracil drug that is controlled to release in colorectal cancer lesions, in order to achieve the goal of converting emergency surgery into elective surgery to reduce risks, and has the following advantages:

[0018] Safety and feasibility: Studies have shown that intestinal stent placement combined with elective surgery is safe and reliable for the treatment of obstructive colorectal cancer. This treatment method can effectively relieve intestinal obstruction and restore intestinal function, and has significant advantages for elective surgery after adequate intestinal preparation.

[0019] Improve surgical results: The use of stents can increase the first-stage anastomosis rate, reduce the stoma rate, and promote the patient's postoperative recovery. After the stent is implanted, it can relieve and improve the patient's obstruction within 48 hours and reduce the patient's pain.

[0020] Reduce complications and improve quality of life: Compared with emergency surgery, stenting has lower mortality, complication rate, and fistula rate. In addition, stenting can be used as a palliative treatment to avoid the trauma of surgical fistula and improve the patient's quality of life.

[0021] Economical: Stent implantation can save medical costs compared to emergency surgery.

[0022] Flexibility in surgical timing: Stent placement provides patients with more time to allow obstructive symptoms to be relieved, intestinal edema to subside, and the patient's general condition to recover, allowing for a more reasonable choice of surgical timing.

[0023] Promote early chemotherapy: Since stent implantation can relieve obstructive symptoms, patients can receive chemotherapy earlier, which indirectly increases the possibility of downstaging the tumor.

[0024] Reduce hospital stay and recovery time: After stent implantation, patients have a shorter postoperative hospital stay and earlier postoperative ventilation, which is beneficial to their postoperative recovery.

[0025] In summary, the use of colorectal cancer stents provides therapeutic advantages at multiple levels, including improving surgical safety, reducing complications, improving quality of life, reducing medical costs, and providing more flexible surgical timing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the description of the embodiments or the prior art are briefly introduced below.

[0027] Figure 1 The mold diagram and the actual diagram of the composite hydrogel drug-loaded stent in Example 1;

[0028] Figure 2 This is a graph showing the fluorouracil loading capacity of the composite hydrogel drug-loaded stent in Example 1;

[0029] Figure 3 is the tumor growth curve in Example 2;

[0030] Figure 4 To cut the drug-loaded gel and blank gel into gel blocks of different areas (S is 1 cm2), the organoids were added to the gel and culture medium and cultured for 2 days, and the results of organoid viability were tested;

[0031] Figure 5 HE staining results of cardiac tumor tissue;

[0032] Figure 6 HE staining results of liver tumor tissue;

[0033] Figure 7 HE staining results of spleen tumor tissue;

[0034] Figure 8 HE staining results of lung tumor tissue;

[0035] Fig. 9 The results of HE staining of renal tumor tissue. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1

[0038] 5g gelatin, 0.5mol calcium chloride and 100mL water can be mixed at 60°C to obtain a mixed solution; the mixed solution is gelled at 4°C for 30min to obtain a gelatin core; the gelatin core is immersed in a 1wt% sodium alginate solution to obtain a GAA hydrogel tube. On this basis, a composite hydrogel drug-loaded scaffold is prepared, including the following steps:

[0039] (1) 4.2 g acrylamide, 2.3 g bacterial cellulose (BC, Hainan Nayang Biopharmaceutical Technology Co., Ltd.), 0.006 g N,N′-methylbisacrylamide, 0.02% potassium persulfate and 10 mL water were mixed, and then 5 mg fluorouracil solution was added to obtain a pre-solution;

[0040] (2) The GAA hydrogel tube was immersed in the pre-solution at 25°C for 12 h and then dried at 60°C to obtain a composite hydrogel drug-loaded scaffold.

[0041] Figure 1 The mold diagram and the actual diagram of the composite hydrogel drug-loaded stent in Example 1 are shown.

[0042] The mold picture and the actual picture of the composite hydrogel drug-loaded stent prepared in Example 1 are as follows: Figure 1 As shown, Figure 1 The left picture is the mold picture. Figure 1 The picture on the right is the actual picture.

[0043] Figure 2 This is a graph showing the fluorouracil loading capacity of the composite hydrogel drug-loaded stent in Example 1.

[0044] like Figure 2 As shown, the fluorouracil loading capacity of the composite hydrogel drug-loaded scaffold in Example 1 was quantitatively analyzed: the supernatant was freeze-dried at 60°C for 48 hours to obtain a freeze-dried powder, which was dissolved in 2 ml of acetonitrile and the content of fluorouracil was determined by high performance liquid chromatography, and the drug loading amount and encapsulation efficiency of the drug-loaded microsphere nanoparticles were calculated.

[0045] Example 2

[0046] In vitro anti-tumor experiment of composite hydrogel drug-loaded stent

[0047] (1) Isolation of mouse colon epithelial cells and organoid culture The mouse colon was obtained and cut longitudinally. After washing three times with penicillin (200U / mL) and streptomycin (200ug / mL) (Invitrogen) in cold PBS, the intestine was cut into 5mm fragments and incubated with 2mm EDTA in pre-cooled PBS on ice for 1h. The intestine was then transferred to new cold PBS. The sample was shaken again for 2min, and intestinal epithelial cells (crypts) were enriched in the supernatant. The supernatant was centrifuged to collect the entire epithelial cells, or filtered through a 70μm cell strainer (Corning). Spin down after centrifugation at 300xg for 5 minutes. The crypts were embedded in 50ul matrix gel (Corning) and seeded in a 24-well plate. The crypts in the matrix gel were then cultured in DMEM / F12 medium (Invitrogen) containing 50 ng mL-1 EGF (Peprotech), 100 ng mL-1 Nogin (Peprotech), 500 ng mL-1 R-spongelin 1, N-acetylcysteine ​​(Sigma-Aldrich), N2, and B27 (Invitrogen).

[0048] (2) In order to explore the tumor inhibition effect of drug-loaded hydrogel, an in vitro tumor inhibition experiment was conducted. 1 ml of drug-loaded hydrogel was placed in a 10 ml test tube, 5 mL of PBS buffer solution (Ph7.4, 0.01 M) was added, and the test tube was placed in a 37°C air bath constant temperature oscillating box. 4 ml of supernatant was taken at the same time every day and the same volume of new PBS buffer solution (pH7.4, 0.01 M) was added; the supernatant was placed in normal cells, colon organoids, and colon organoids for culture, and the tumor volume was measured. The tumor tissue was removed and the cell apoptosis was calculated by flow cytometry.

[0049] For example, 60 eagle-like mice (Balb / c, 6 weeks, 20 g) were divided into two groups. Thirty eagle-like mice in one group were divided into three groups (n=10). 100 uL of 1*10 6 A tumor-bearing mouse model was constructed by injecting 100uL of PBS solution containing 1*106 fluorouracil-resistant DLD1 cells into the back of another group of 30 mice, which were also divided into 3 groups (n=10). A tumor-bearing mouse model was constructed by injecting 100uL of PBS solution containing 1*106 fluorouracil-resistant DLD1 cells into the back of the mice. After culturing the mice for 1 week, mice with tumor volumes of about 100mm were selected for the experiment. There were 6 groups in total, namely:

[0050] The first type of tumor-bearing mice were non-fluorouracil-resistant group and were divided into 3 groups (n=10).

[0051] (1) Subcutaneous injection, 100uL drug-loaded gel;

[0052] (2) Intravenous injection of 100uL TAxol;

[0053] (3) Intravenous injection of 100uL PBS buffer (Ph7.4, 0.01M).

[0054] The second type of tumor-bearing mice was the fluorouracil-resistant group, which was divided into 3 groups (n=10).

[0055] (1) Subcutaneous injection, 100 μl of drug-loaded gel;

[0056] (2) Intravenous injection of 100 μl TAxol;

[0057] (3) Intravenous injection of 100uL PBS buffer (Ph7.4, 0.01M).

[0058] The weight of the mice and the tumor volume were measured every other day. After 30 days, the mice were killed by cervical dislocation, the tumors were removed, and the tumor volume was measured. The sections were made and the necrosis of the tumor tissue was observed under an optical microscope. The other organs of the mice (heart, liver, kidney, spleen, lung) were removed in the same way to observe their tissue conditions. After 30 days of subcutaneous tumor observation, all animals were euthanized to remove the heart, liver, spleen, lung, and kidney tumor tissues, which were fixed and stained with HE. The experimental results are shown in the figure. Figure 3 As shown, the horizontal axis is the fluorouracil density, and the vertical axis is the volume of colorectal cancer cells. The fluorouracil density affects the proliferation of colorectal cancer cells. The effect of the fluorouracil-resistant density on the proliferation of colorectal cancer cells is greater than that of the non-fluorouracil-resistant density on the proliferation of colorectal cancer cells. Moreover, as the fluorouracil-resistant density increases, the effect on the proliferation of colorectal cancer cells becomes greater.

[0059] Figure 4 To cut the drug-loaded gel and blank gel into gel blocks of different areas (S is 1 cm 2 ), after adding the organoids to the gel and culture medium and culturing for 2 days, the organoid viability was detected. Hydrogel was the empty gel group, and Hydrogel+Drug was the drug-loaded gel group.

[0060] HE staining results of heart, liver, spleen, lung and kidney tumor tissues are as follows Figures 5 to 9 As shown. Figures 5 to 9 It can be seen that the drug-loaded hydrogel has an inhibitory effect on the growth of tumor tissues in the heart, liver, spleen, lungs and kidneys.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a composite hydrogel drug-loaded stent, characterized in that: The following steps are involved: (1) acrylamide, bacterial cellulose, N,N′-methylbisacrylamide, potassium persulfate and water are mixed, and then a fluorouracil solution is added to obtain a pre-solution; (2) Immersing the GAA hydrogel tube in the pre-solution and then drying it to obtain a composite hydrogel drug-loaded scaffold.

2. The method for preparing the composite hydrogel drug-loaded stent according to claim 1, characterized in that: The usage ratio of acrylamide, bacterial cellulose, N,N′-methylbisacrylamide, potassium persulfate and water in step (1) is 4-6 g: 2-3 g: 0.004-0.01 g: 0.01-0.1 g: 10-100 mL; and / or, the concentration of the fluorouracil solution is 4 to 10 mg / mL; And / or, the usage ratio of the fluorouracil solution to acrylamide is 4-10 mg:4-6 g.

3. The method for preparing the composite hydrogel drug-loaded stent according to claim 1, characterized in that: The immersion temperature in step (2) is 20 to 30° C., and the immersion time is 12 to 24 hours. The drying temperature in step (2) is 40-60°C.

4. A composite hydrogel drug-loaded stent prepared by the method for preparing a composite hydrogel drug-loaded stent according to any one of claims 1 to 3.

5. Use of the composite hydrogel drug-loaded stent according to claim 4 in drug release.

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