Collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula and preparation method of collagen-based penicillin G nano-drug composite hydrogel

Abstract

The invention discloses collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula, which is prepared by dispersing a penicillin G coordination polymer nano-drug prepared by self-assembly induced by Co / Cu coordination in a modified recombinant collagen solution, and then mixing the modified recombinant collagen solution with an oxidized pullulan solution. The invention further discloses a preparation method of the composite hydrogel. Pulullan, recombinant collagen CF-1552 and choline-PEI coated Pg / NM-Cu (II) / Co (II) nano-drugs are creatively introduced into a hydrogel dressing system, so that the problem that protein-based high pancreatic wound repair promotion activity and a long-time effective physical barrier are difficult to consider at the same time can be solved in a targeted manner; according to the present invention, the problems of non-wire-holding pancreatic tissue reversible fibrosis adjustment and difficult non-wire-holding pancreatic tissue reversible fibrosis adjustment are solved, and the prepared composite hydrogel has characteristics of injectable shape self-adaption and self-leveling, is more suitable for irregular wounds of pancreatic fistula patients, and can effectively promote the healing of the wounds.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and particularly relates to a collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula and a preparation method thereof. Background Art

[0002] Pancreatic fistula refers to a pathological phenomenon and a common complication caused by rupture of the main pancreatic duct and its branches due to pancreatic disease, surgery and trauma, resulting in pancreatic fluid leakage. Postoperative pancreatic fistula (POPF) is the main cause of pancreatic fistula. The incidence of pancreatic fistula after pancreaticoduodenectomy is 5-20%, and the incidence of pancreatic fistula after distal pancreatectomy due to benign or malignant pancreatic tumors is as high as 60%. Therefore, inhibiting the occurrence of pancreatic fistula and improving the prognosis level are important issues to improve the survival rate of patients undergoing pancreatic surgery.

[0003] Methods used to prevent POPF include main pancreatic duct ligation, stapler closure, pancreaticojejunostomy, use of biocompatible sealants on the pancreatic stump, patch reinforcement, and drugs such as somatostatin analogs. However, the current clinical management efficiency of pancreatic fistula is as low as 20%, and there is no management strategy with outstanding efficacy to date. The difficulties in clinical prevention of pancreatic fistula are: 1. Drug therapy has poor efficacy, and systemic administration is limited in the number of drug molecules that reach the target organ due to capture and clearance by the endothelial reticular system. 2. Surgical treatment (such as ligation and stapler closure) has a high risk of host reaction to implants and is an invasive treatment method with large surgical trauma and high reliance on the operator's skills. In addition, pancreatic fistulas usually present abnormal pathological characteristics such as soft texture, enzyme environment, ionic strength, pH value, and oxidative stress. These unique pathological characteristics greatly increase the difficulty of pancreatic fistula prevention and treatment.

[0004] Finding safer and more effective postoperative intervention methods to prevent the occurrence of pancreatic fistula has gradually become a research focus of clinical treatment. At present, multifunctional collagen hydrogels are receiving widespread attention because they integrate multiple therapeutic functions and can meet the treatment needs of various diseases. However, existing hydrogels still face many challenges in the treatment of pancreatic fistula. It is difficult to balance the pancreatic wound repair activity and long-term physical barrier function in a high enzyme activity environment. At the same time, they lack asymmetric tissue adhesion properties, making it difficult to achieve stable adhesion repair on the pancreatic fistula surface and anti-adhesion on the non-lesion surface. In addition, existing materials generally lack the ability to reversibly regulate fibrosis for "non-sustained" pancreatic tissues, and lack inherent antibacterial properties, and cannot effectively deal with the high risk of postoperative infection in the alkaline environment of pancreatic fistula. Therefore, an innovative hydrogel that can combine anti-enzymatic self-leveling, instantaneous unilateral adhesion, reversible fibrosis regulation and antibacterial properties is designed to break through the limitations of traditional materials and provide a better solution for preventing and treating pancreatic fistula. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula and its preparation method in view of the deficiencies of the above-mentioned prior art. Introducing pullulan and unique collagen CF-1552 and choline-PEI@Pg / NM-Cu(II) / Co(II) nano-drugs into the hydrogel dressing system can specifically solve the problem of the difficulty in balancing the high pancreatic wound repair activity of the protein-based and the long-term effective physical barrier, as well as the difficulty in regulating the reversible fibrosis of "non-sustainable" pancreatic tissue.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula, characterized in that the composite hydrogel is prepared by dispersing penicillin G coordination polymer nano-drug prepared by Co / Cu coordination-induced self-assembly in a modified recombinant collagen solution, and then mixing with an oxidized pullulan solution.

[0007] Furthermore, the present invention provides a method for preparing the composite hydrogel, characterized in that it comprises the following steps:

[0008] Step 1, mixing the ethanol solution of penicillin G and the ethanol solution of nafamostat mesylate, then dropping the ethanol solution of cobalt chloride and copper chloride into the mixed solution, stirring evenly, to obtain a premixed solution;

[0009] Step 2, mixing the surfactant solution and the Tris hydrochloride solution, then adding the mixed solution into the premixed solution in step 1, and stirring to obtain a penicillin G coordination polymer nano drug solution;

[0010] Step 3, dissolving the recombinant collagen CF-1552 in deionized water, and then adding triethylamine, Tween-20 and glycidyl methacrylate to obtain a CF-1552-containing reactant system; adjusting the pH of the CF-1552-containing reactant system to 8-10, and stirring the reaction at room temperature for 20h-30h, collecting the reaction product in a dialysis bag, dialyzing for 4-6 days, and freeze-drying to obtain modified recombinant collagen CF-1552;

[0011] Step 4: adding sodium periodate to the pullulan solution, stirring the reaction at room temperature for 2 h to 4 h, then adding ethylene glycol to terminate the reaction, collecting the product in a dialysis bag, dialyzing it for 2 to 3 days, and freeze-drying it to obtain oxidized pullulan;

[0012] Step 5, sterilizing the penicillin G coordination polymer nano drug solution in step 2, the modified recombinant collagen CF-1552 in step 3, and the oxidized pullulan in step 4 by Co60 irradiation respectively;

[0013] Step 6: dissolving the sterilized modified recombinant collagen CF-1552 in water to prepare a modified recombinant collagen solution with a mass concentration of 10% to 15%; dissolving the sterilized oxidized pullulan in water to prepare an oxidized pullulan solution with a mass concentration of 10% to 15%;

[0014] Step 7: Disperse the sterilized penicillin G coordination polymer nano drug solution in the modified recombinant collagen solution described in step 6, and then mix it with the oxidized pullulan solution described in step 6 to obtain a collagen-based penicillin G nano drug composite hydrogel.

[0015] The above method is characterized in that the concentration of penicillin G in the penicillin G ethanol solution described in step 1 is 3 mg / mL to 5 mg / mL, the concentration of nafamostat mesylate in the ethanol solution of nafamostat mesylate is 3 mg / mL to 5 mg / mL, the concentration of cobalt chloride in the ethanol solution of cobalt chloride and copper chloride is 5 mg / mL to 15 mg / mL, the concentration of copper chloride is 5 mg / mL to 15 mg / mL, the volume ratio of the ethanol solution of penicillin G to the ethanol solution of nafamostat mesylate is 1:1, and the volume of the ethanol solution of cobalt chloride and copper chloride is 4% to 10% of the volume of the ethanol solution of penicillin G.

[0016] The above method is characterized in that the surfactant in step 2 is choline-PEI, the concentration of the surfactant solution is 5g / L to 15g / L, and the concentration of the Tris hydrochloride solution is 2g / L to 5g / L; the volume ratio of the surfactant solution, the Tris hydrochloride solution and the premix is ​​1:1:(0.1 to 0.3).

[0017] The above method is characterized in that, in step 3, the ratio of the mass of recombinant collagen CF-1552 to the volume of deionized water is 1:(80-120), wherein the unit of mass is g and the unit of volume is mL; the ratio of the volume of triethylamine to the mass of recombinant collagen CF-1552 is 1:(2-5), wherein the unit of mass is g and the unit of volume is mL; the volume of Tween-20 is 30%-40% of the volume of triethylamine, and the volume of glycidyl methacrylate is 70%-90% of the volume of triethylamine.

[0018] The above method is characterized in that the molecular weight cutoff of the dialysis bag in step three is 8000-14000.

[0019] The above method is characterized in that the concentration of the pullulan solution in step 4 is 25g / L to 50g / L, the mass of sodium periodate is 8% to 16% of the mass of pullulan, and the volume of ethylene glycol is 3.5 to 6.5 times the mass of sodium periodate, wherein the unit of mass is g and the unit of volume is mL; the molecular weight cutoff of the dialysis bag is 8000 to 14000.

[0020] The above method is characterized in that in step seven, the volume ratio of the modified recombinant collagen solution, the oxidized pullulan solution and the penicillin G coordination polymer nano drug solution is (3-1):(1-2):0.0035.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] 1. The present invention creatively introduces the natural polysaccharide pullulan, the unique collagen CF-1552 and the choline-PEI@Pg / NM-Cu(II) / Co(II) nanomedicine into the hydrogel dressing system, which can specifically solve the problem of the difficulty in balancing the high pancreatic wound repair activity of the protein-based material and the long-term effective physical barrier, as well as the difficulty in regulating the reversible fibrosis of the "non-sustainable" pancreatic tissue.

[0023] 2. The present invention prepares penicillin G into a nano drug, the method is simple and rapid, the drug loading rate and encapsulation rate are both above 95%, the water solubility and stability of penicillin G are increased, the action time of the drug is prolonged, the therapeutic effect is improved, good antibacterial effect is shown, and the fibrosis effect is reversible. At the same time, nafamostat mesylate (NM) and Co / Cu can reduce the activity of pancreatic enzymes, thereby achieving a double effect of reducing the occurrence of pancreatic fistula.

[0024] 3. The material collagen used in the collagen-based penicillin G nano-drug composite hydrogel of the present invention is a pure natural product with multiple pharmacological activities, and acute toxicity experiments show that it is completely non-toxic.

[0025] 4. The penicillin G composite metal ion Co / Cu nanodrug used in the collagen-based penicillin G nanodrug composite hydrogel of the present invention not only functions to regulate enzyme activity and promote reversible fibrosis, but the amino groups on the outer layer of the nanodrug can also cross-link with the uncross-linked aldehyde groups on the oxidized pullulan, thereby increasing the mechanical properties of the hydrogel.

[0026] 5. The collagen-based penicillin G nano-drug composite hydrogel of the present invention has the characteristics of injectable shape adaptation and self-leveling, and is more suitable for irregular wounds of patients with pancreatic fistula. It can enter deep wounds to ensure effective coverage of irregular wounds and more effectively promote wound healing.

[0027] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a TEM image of the choline-PEI@Pg / NM-Cu(II) / Co(II) nanomedicine prepared in Example 1 of the present invention.

[0029] Figure 2 This is the XPS graph of the choline-PEI@Pg / NM-Cu(II) / Co(II) nanomedicine prepared in Example 1 of the present invention.

[0030] Figure 3 This is the Fourier transform infrared absorption spectrum of the modified CF-1552 (CF-GMA) prepared in Example 1 of the present invention.

[0031] Figure 4 This is a Fourier transform infrared absorption spectrum of the oxidized pullulan (OP) prepared in Example 1 of the present invention.

[0032] Figure 5 This is the hydrogen nuclear magnetic resonance spectrum of the modified CF-1552 (CF-GMA) prepared in Example 1 of the present invention.

[0033] Figure 6 This is a gel formation diagram of the collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula prepared in Example 1 of the present invention.

[0034] Figure 7 This is a SEM image of the collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula prepared in Example 1 of the present invention.

[0035] Figure 8 The figure shows the results of cytotoxicity experiment.

[0036] Fig. 9 This is a diagram showing the effect of testing the preventive and therapeutic effects of pancreatic fistula in SD rats.

[0037] Fig.10 This is a SEM image of the collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula prepared in Example 2 of the present invention.

[0038] Fig.11 This is a SEM image of the collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula prepared in Example 3 of the present invention.

[0039] Fig.12 This is a SEM image of the collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula prepared in Example 4 of the present invention. DETAILED DESCRIPTION

[0040] The present invention is described in detail below through examples, which are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. The recombinant collagen CF-1552 in the following examples is provided by Juzi Biogene Technology Co., Ltd. The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer; the equipment, materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0041] Example 1

[0042] This embodiment provides a collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula. The composite hydrogel is prepared by dispersing penicillin G coordination polymer nano-drug prepared by Co / Cu coordination-induced self-assembly in a modified recombinant collagen solution, and then mixing with an oxidized pullulan solution.

[0043] The specific preparation method comprises the following steps:

[0044] Step 1, mixing an ethanol solution of penicillin G and an ethanol solution of nafamostat mesylate, then dripping an ethanol solution of cobalt chloride and cupric chloride into the mixed solution, stirring evenly, to obtain a premixed solution; the concentration of penicillin G in the ethanol solution of penicillin G is 4 mg / mL, the concentration of nafamostat mesylate in the ethanol solution of nafamostat mesylate is 4 mg / mL, the concentration of cobalt chloride in the ethanol solution of cobalt chloride and cupric chloride is 10 mg / mL, the concentration of cupric chloride is 10 mg / mL, the volume ratio of the ethanol solution of penicillin G to the ethanol solution of nafamostat mesylate is 1:1, and the volume of the ethanol solution of cobalt chloride and cupric chloride is 6% of the volume of the ethanol solution of penicillin G;

[0045] Step 2: Mix the surfactant solution and the Tris hydrochloride solution, then add the mixed solution to the premixed solution in step 1, and stir to obtain a penicillin G coordination polymer nanodrug (choline-PEI@Pg / NM-Cu(II) / Co(II) nanodrug) solution; the surfactant is choline-PEI, the concentration of the surfactant solution is 10 g / L, and the concentration of the Tris hydrochloride solution is 3 g / L; the volume ratio of the surfactant solution, the Tris hydrochloride solution and the premixed solution is 1:1:0.2;

[0046] Step 3: Dissolve the recombinant collagen CF-1552 in deionized water, then add triethylamine, Tween-20 and glycidyl methacrylate to obtain a CF-1552 reactant system; adjust the pH of the CF-1552 reactant system to 9 with 0.1M sodium hydroxide, and stir the reaction at 700 rpm for 24 hours at room temperature, collect the reaction product in a dialysis bag, dialyze for 5 days, and freeze-dry to obtain modified recombinant collagen CF-1552 (CF-GMA); The ratio of the mass of recombinant collagen CF-1552 to the volume of deionized water is 1:100, wherein the unit of mass is g and the unit of volume is mL; the ratio of the volume of triethylamine to the mass of recombinant collagen CF-1552 is 1:3, wherein the unit of mass is g and the unit of volume is mL; the volume of Tween-20 is 35% of the volume of triethylamine, and the volume of glycidyl methacrylate is 80% of the volume of triethylamine; the molecular weight cutoff of the dialysis bag is 8000-14000;

[0047] Step 4: adding sodium periodate to the pullulan solution, stirring at 700 rpm for 3 hours at room temperature, then adding ethylene glycol to terminate the reaction, collecting the product in a dialysis bag for 3 days, and freeze-drying to obtain oxidized pullulan; the concentration of the pullulan solution is 40 g / L, the mass of sodium periodate is 12% of the mass of pullulan, and the volume of ethylene glycol is 5 times the mass of sodium periodate, wherein the unit of mass is g and the unit of volume is mL; the molecular weight cutoff of the dialysis bag is 8000-14000;

[0048] Step 5, sterilizing the penicillin G coordination polymer nano drug solution in step 2, the modified recombinant collagen CF-1552 in step 3, and the oxidized pullulan in step 4 by Co60 irradiation respectively;

[0049] Step 6: Dissolve the sterilized modified recombinant collagen CF-1552 in water to prepare a modified recombinant collagen solution with a mass concentration of 12%; dissolve the sterilized oxidized pullulan in water to prepare an oxidized pullulan solution with a mass concentration of 13%;

[0050] Step 7. Disperse the sterilized penicillin G coordination polymer nanodrug solution in the modified recombinant collagen solution described in step 6, and then mix it with the oxidized pullulan solution described in step 6 to obtain a collagen-based penicillin G nanodrug composite hydrogel (choline-PEI@Pg / NM-Cu(II) / Co(II)-CGO hydrogel); the volume ratio of the modified recombinant collagen solution, the oxidized pullulan solution and the penicillin G coordination polymer nanodrug solution is 2:1:0.0035.

[0051] Figure 1This is a TEM image of the choline-PEI@Pg / NM-Cu(II) / Co(II) nanodrug prepared in step 2 of this embodiment. It can be seen from the figure that the particle size of the prepared choline-PEI@Pg / NM-Cu(II) / Co(II) nanodrug micelles is 2.48±0.67nm, and the scale is 20nm; at the same time, the morphology and size of the nanodrug micelles are uniform and the structure is good. It is calculated that the drug loading rate and encapsulation rate of the polymer nanodrug of Co / Cu self-assembled penicillin G composite NM are 95.1% and 97.28%, respectively.

[0052] Figure 2 This is the XPS image of the choline-PEI@Pg / NM-Cu(II) / Co(II) nanomedicine nanoparticles prepared in step 2 of this example. As can be seen from the figure, the elemental composition analysis further confirmed the coordinated self-assembly chemical composition of choline-PEI@Pg / NM-Cu(II) / Co(II).

[0053] Figure 3 This is the Fourier transform infrared absorption spectrum of the modified CF-1552 prepared in step 3 of this embodiment. It can be seen that at the wave number of 1731cm -1 The peak at the center is the characteristic absorption peak of carbon-carbon double bond, indicating that glycidyl methacrylate was successfully grafted onto CF-1552.

[0054] Figure 4 The Fourier transform infrared absorption spectrum analysis diagram of the oxidized pullulan prepared in step 4 of this embodiment is shown in the figure. -1 and 1444cm -1 The absorption peak at the center is the characteristic absorption peak of aldehyde group, which indicates that the oxidized pullulan modification is successful.

[0055] Figure 5 This is the hydrogen nuclear magnetic resonance spectrum of the modified CF-1552 prepared in step 3 of this embodiment. It can be seen that two characteristic peaks appear between 5.5-6.5 ppm, indicating that after reacting with excess glycidyl methacrylate, the double bond is successfully grafted onto the main chain of CF-1552.

[0056] The collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula prepared in this embodiment was subjected to an inverted non-flow gelation experiment. The experimental method comprises: preparing a modified CF-1552 solution containing choline-PEI@Pg / NM-Cu(II) / Co(II) nano-drug and an oxidized pullulan solution containing choline-PEI@Pg / NM-Cu(II) / Co(II) nano-drug according to the proportions in step seven of this embodiment, and then taking 1 mL of the modified CF-1552 solution containing choline-PEI@Pg / NM-Cu(II) / Co(II) nano-drug and the oxidized pullulan solution containing choline-PEI@Pg / NM-Cu(II) / Co(II) nano-drug respectively and adding them into a glass vial. When the two solutions were inverted, it was found that the above two solutions were liquids with fluidity. When the collagen-based penicillin G nano-drug composite hydrogel prepared in this embodiment was inverted, it was found that the solution did not flow. Figure 6 , indicating that the corresponding hydrogel can be prepared by the method of the present invention.

[0057] Figure 7 This is the SEM image of the collagen-based penicillin G nanodrug composite hydrogel prepared in this example. It can be seen from the figure that the pore size of the prepared composite hydrogel is 11.42μm, and the scale is 30μm; at the same time, it shows that the morphology and pore size of the composite hydrogel are uniform and the structure is good.

[0058] Cytotoxicity assay:

[0059] The modified recombinant collagen solution and the oxidized pullulan solution of Example 1 were mixed in a volume ratio of 1:1 to prepare the hydrogel, and the collagen-based penicillin G nano-drug composite hydrogel prepared in this example was sterilized with Co60 and then immersed in sterile test tubes containing RPMI-1640 culture medium. The concentration of the culture medium added was 0.1 g / mL. The test tubes were then placed in a 37°C constant temperature incubator and cultured for 72 h to obtain CGO hydrogel extracts and choline-PEI@Pg / NM-Cu(II) / Co(II)-CGO hydrogel extracts. L929 cells were counted with a blood cell plate at 1×10 4 The cells / mL density was inoculated in each well of a 96-well plate at 100 μL. After the cells adhered to the wall, the culture medium was removed. The control group was cultured with RPMI-1640 culture medium, the CGO hydrogel group was cultured with CGO hydrogel extract, and the choline-PEI@Pg / NM-Cu(II) / Co(II)-CGO hydrogel group was cultured with choline-PEI@Pg / NM-Cu(II) / Co(II)-CGO hydrogel extract. After 24 hours of co-culture, AO / EB working solution was added to each well, incubated at 37°C for 15 to 20 minutes, and photographed with a fluorescence microscope. The results are shown in the figure. Figure 8The three groups had relatively low toxicity to L929 cells, and penicillin G-loaded hydrogel promoted the proliferation of L929 cells.

[0060] Test on the preventive and therapeutic effect of hydrogel on pancreatic fistula in SD rats:

[0061] Establishment of pancreatic fistula SD rat model: 60 SPF-grade 8-week-old male SD rats with a body weight of 200-250 g were selected, given a standard diet, and adaptively raised at 22-23°C for 1 week; according to the random number table method, the rats were divided into a blank model group, a commercially available dressing (Tisseel) treatment group, a CGO hydrogel treatment group, and a choline-PEI@Pg / NM-Cu(II) / Co(II)-CGO hydrogel treatment group, respectively recorded as: Model, Tisseel, CGO, choline-PEI@Pg / NM-Cu(II) / Co(II)-CGO group, to establish a grade C pancreatic fistula rat model. Before surgery, the rats were fasted overnight (12 hours, but water was not allowed), and anesthetized by intraperitoneal injection of 3% sodium pentobarbital at 300 mg / kg. After successful anesthesia, the rats' abdominal hair was removed with a depilatory cream, the rats' abdomen was disinfected with 75% alcohol, and an 8 cm incision was made along the middle of the abdomen to expose the pancreas. The pancreatic duct (called the splenic duct) in the pancreas facing the spleen was transected under sterile conditions. The Model group did not use dressings for treatment, the Tisseel group used commercially available dressings Tisseel on the rat wounds, the CGO group used CGO hydrogel on the rat wounds, and the choline-PEI@Pg / NM-Cu(II) / Co(II)-CGO group used the collagen-based penicillin G nanodrug composite hydrogel prepared in Example 1 on the rat wounds. Subsequently, the rats' body weights were measured every day, and sodium pentobarbital was used for anesthesia on the third day after surgery. Open abdomen and take photos to observe whether pancreatic fistula occurred. The results are shown in the table below. Fig. 9 As can be seen from the figure, 3 days after the operation, a large amount of saponified adhesion, ascites, organ failure and other phenomena appeared in the abdominal cavity of the Model group; partial saponified adhesion and a small amount of ascites appeared in the Tisseel group; a small amount of saponified adhesion appeared in the CGO group, but no ascites appeared; the choline-PEI@Pg / NM-Cu(II) / Co(II)-CGO group did not show abnormal conditions such as saponified adhesion, indicating that the composite hydrogel of the present invention showed significantly better efficacy than other groups in inhibiting bleeding, abscesses and fat saponified adhesion in abdominal tissues and organs.

[0062] Example 2

[0063] This embodiment provides a collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula. The composite hydrogel is prepared by dispersing penicillin G coordination polymer nano-drug prepared by Co / Cu coordination-induced self-assembly in a modified recombinant collagen solution, and then mixing with an oxidized pullulan solution.

[0064] The specific preparation method comprises the following steps:

[0065] Step 1, mixing the ethanol solution of penicillin G and the ethanol solution of nafamostat mesylate, then dripping the ethanol solution of cobalt chloride and cupric chloride into the mixed solution, stirring evenly, to obtain a premixed solution; the concentration of penicillin G in the ethanol solution of penicillin G is 5 mg / mL, the concentration of nafamostat mesylate in the ethanol solution of nafamostat mesylate is 3 mg / mL, the concentration of cobalt chloride in the ethanol solution of cobalt chloride and cupric chloride is 5 mg / mL, the concentration of cupric chloride is 15 mg / mL, the volume ratio of the ethanol solution of penicillin G to the ethanol solution of nafamostat mesylate is 1:1, and the volume of the ethanol solution of cobalt chloride and cupric chloride is 10% of the volume of the ethanol solution of penicillin G;

[0066] Step 2: Mix the surfactant solution and the Tris hydrochloride solution, then add the mixed solution into the premixed solution in step 1, and stir to obtain a penicillin G coordination polymer nano drug solution; the surfactant is choline-PEI, the concentration of the surfactant solution is 15 g / L, and the concentration of the Tris hydrochloride solution is 2 g / L; the volume ratio of the surfactant solution, the Tris hydrochloride solution and the premixed solution is 1:1:0.3;

[0067] Step 3: Dissolve the recombinant collagen CF-1552 in deionized water, then add triethylamine, Tween-20 and glycidyl methacrylate to obtain a CF-1552 reactant system; adjust the pH of the CF-1552 reactant system to 8 with 0.1M sodium hydroxide, and stir the reaction at 600rpm for 30h at room temperature, collect the reaction product in a dialysis bag, dialyze for 4 days, and lyophilize to obtain modified recombinant collagen CF-1552; recombinant collagen The ratio of the mass of protein CF-1552 to the volume of deionized water is 1:80, wherein the unit of mass is g and the unit of volume is mL; the ratio of the volume of triethylamine to the mass of recombinant collagen CF-1552 is 1:2, wherein the unit of mass is g and the unit of volume is mL; the volume of Tween-20 is 30% of the volume of triethylamine, and the volume of glycidyl methacrylate is 70% of the volume of triethylamine; the molecular weight cutoff of the dialysis bag is 8000-14000;

[0068] Step 4: adding sodium periodate to the pullulan solution, stirring at 600 rpm for 4 hours at room temperature, then adding ethylene glycol to terminate the reaction, collecting the product in a dialysis bag for 3 days, and freeze-drying to obtain oxidized pullulan; the concentration of the pullulan solution is 25 g / L, the mass of sodium periodate is 8% of the mass of pullulan, and the volume of ethylene glycol is 3.5 times the mass of sodium periodate, wherein the unit of mass is g and the unit of volume is mL; the molecular weight cutoff of the dialysis bag is 8000-14000;

[0069] Step 5, sterilizing the penicillin G coordination polymer nano drug solution in step 2, the modified recombinant collagen CF-1552 in step 3, and the oxidized pullulan in step 4 by Co60 irradiation respectively;

[0070] Step 6: Dissolve the sterilized modified recombinant collagen CF-1552 in water to prepare a modified recombinant collagen solution with a mass concentration of 10%; dissolve the sterilized oxidized pullulan in water to prepare an oxidized pullulan solution with a mass concentration of 15%;

[0071] Step 7. Disperse the sterilized penicillin G coordination polymer nano-drug solution in the modified recombinant collagen solution described in step 6, and then mix it with the oxidized pullulan solution described in step 6 to obtain a collagen-based penicillin G nano-drug composite hydrogel; the volume ratio of the modified recombinant collagen solution, the oxidized pullulan solution and the penicillin G coordination polymer nano-drug solution is 1:1:0.0035.

[0072] The effect of the collagen-based penicillin G nano-drug composite hydrogel dressing for pancreatic fistula of this embodiment is basically the same as that of Example 1. Fig.10 This is the SEM image of the collagen-based penicillin G nanodrug composite hydrogel prepared in this example. It can be seen that the pore size of the prepared composite hydrogel is 16.60 μm, and the scale is 30 μm; at the same time, it shows that the morphology and pore size of the composite hydrogel are uniform and the structure is good.

[0073] Example 3

[0074] This embodiment provides a collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula. The composite hydrogel is prepared by dispersing penicillin G coordination polymer nano-drug prepared by Co / Cu coordination-induced self-assembly in a modified recombinant collagen solution, and then mixing with an oxidized pullulan solution.

[0075] The specific preparation method comprises the following steps:

[0076] Step 1, mixing an ethanol solution of penicillin G and an ethanol solution of nafamostat mesylate, then dripping an ethanol solution of cobalt chloride and cupric chloride into the mixed solution, stirring evenly, to obtain a premixed solution; the concentration of penicillin G in the ethanol solution of penicillin G is 3 mg / mL, the concentration of nafamostat mesylate in the ethanol solution of nafamostat mesylate is 5 mg / mL, the concentration of cobalt chloride in the ethanol solution of cobalt chloride and cupric chloride is 15 mg / mL, the concentration of cupric chloride is 5 mg / mL, the volume ratio of the ethanol solution of penicillin G to the ethanol solution of nafamostat mesylate is 1:1, and the volume of the ethanol solution of cobalt chloride and cupric chloride is 4% of the volume of the ethanol solution of penicillin G;

[0077] Step 2: Mix the surfactant solution and the Tris hydrochloride solution, then add the mixed solution into the premixed solution in step 1, and stir to obtain a penicillin G coordination polymer nano drug solution; the surfactant is choline-PEI, the concentration of the surfactant solution is 5 g / L, and the concentration of the Tris hydrochloride solution is 5 g / L; the volume ratio of the surfactant solution, the Tris hydrochloride solution and the premixed solution is 1:1:0.1;

[0078] Step 3: Dissolve the recombinant collagen CF-1552 in deionized water, then add triethylamine, Tween-20 and glycidyl methacrylate to obtain a CF-1552-containing reactant system; adjust the pH of the CF-1552-containing reactant system to 10 with 0.1M sodium hydroxide, and stir the reaction at 800rpm for 20h at room temperature, collect the reaction product in a dialysis bag, dialyze for 6 days, and lyophilize to obtain modified recombinant collagen CF-1552; recombinant collagen The ratio of the mass of protein CF-1552 to the volume of deionized water is 1:120, wherein the unit of mass is g and the unit of volume is mL; the ratio of the volume of triethylamine to the mass of recombinant collagen CF-1552 is 1:5, wherein the unit of mass is g and the unit of volume is mL; the volume of Tween-20 is 40% of the volume of triethylamine, and the volume of glycidyl methacrylate is 90% of the volume of triethylamine; the molecular weight cutoff of the dialysis bag is 8000-14000;

[0079] Step 4: adding sodium periodate to the pullulan solution, stirring at 800 rpm for 2 hours at room temperature, then adding ethylene glycol to terminate the reaction, collecting the product in a dialysis bag for 2 days, and freeze-drying to obtain oxidized pullulan; the concentration of the pullulan solution is 50 g / L, the mass of sodium periodate is 16% of the mass of pullulan, and the volume of ethylene glycol is 6.5 times the mass of sodium periodate, wherein the unit of mass is g and the unit of volume is mL; the molecular weight cutoff of the dialysis bag is 8000-14000;

[0080] Step 5, sterilizing the penicillin G coordination polymer nano drug solution in step 2, the modified recombinant collagen CF-1552 in step 3, and the oxidized pullulan in step 4 by Co60 irradiation respectively;

[0081] Step 6: Dissolve the sterilized modified recombinant collagen CF-1552 in water to prepare a modified recombinant collagen solution with a mass concentration of 15%; dissolve the sterilized oxidized pullulan in water to prepare an oxidized pullulan solution with a mass concentration of 10%;

[0082] Step 7, dispersing the sterilized penicillin G coordination polymer nano drug solution in the modified recombinant collagen solution described in step 6, and then mixing it with the oxidized pullulan solution described in step 6 to obtain a collagen-based penicillin G nano drug composite hydrogel; the volume ratio of the modified recombinant collagen solution, the oxidized pullulan solution and the penicillin G coordination polymer nano drug solution is 1:2:0.0035.

[0083] The effect of the collagen-based penicillin G nano-drug composite hydrogel dressing for pancreatic fistula of this embodiment is basically the same as that of Example 1. Fig.11 This is the SEM image of the collagen-based penicillin G nanodrug composite hydrogel prepared in this example. It can be seen that the pore size of the prepared composite hydrogel is 19.46 μm and the scale is 30 μm; at the same time, it shows that the morphology and pore size of the composite hydrogel are uniform and the structure is good.

[0084] Example 4

[0085] This embodiment provides a collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula. The composite hydrogel is prepared by dispersing penicillin G coordination polymer nano-drug prepared by Co / Cu coordination-induced self-assembly in a modified recombinant collagen solution, and then mixing with an oxidized pullulan solution.

[0086] The specific preparation method comprises the following steps:

[0087] Step 1, mixing the ethanol solution of penicillin G and the ethanol solution of nafamostat mesylate, then dripping the ethanol solution of cobalt chloride and cupric chloride into the mixed solution, stirring evenly, to obtain a premixed solution; the concentration of penicillin G in the ethanol solution of penicillin G is 5 mg / mL, the concentration of nafamostat mesylate in the ethanol solution of nafamostat mesylate is 5 mg / mL, the concentration of cobalt chloride in the ethanol solution of cobalt chloride and cupric chloride is 10 mg / mL, the concentration of cupric chloride is 10 mg / mL, the volume ratio of the ethanol solution of penicillin G to the ethanol solution of nafamostat mesylate is 1:1, and the volume of the ethanol solution of cobalt chloride and cupric chloride is 5% of the volume of the ethanol solution of penicillin G;

[0088] Step 2: Mix the surfactant solution and the Tris hydrochloride solution, then add the mixed solution into the premixed solution in step 1, and stir to obtain a penicillin G coordination polymer nano drug solution; the surfactant is choline-PEI, the concentration of the surfactant solution is 10 g / L, and the concentration of the Tris hydrochloride solution is 4 g / L; the volume ratio of the surfactant solution, the Tris hydrochloride solution and the premixed solution is 1:1:0.2;

[0089] Step 3: Dissolve the recombinant collagen CF-1552 in deionized water, then add triethylamine, Tween-20 and glycidyl methacrylate to obtain a CF-1552-containing reactant system; adjust the pH of the CF-1552-containing reactant system to 9 with 0.1 M sodium hydroxide, and stir the reaction at 600 rpm for 24 hours at room temperature, collect the reaction product in a dialysis bag, dialyze for 5 days, and lyophilize to obtain modified recombinant collagen CF-1552; recombinant collagen The ratio of the mass of protein CF-1552 to the volume of deionized water is 1:100, wherein the unit of mass is g and the unit of volume is mL; the ratio of the volume of triethylamine to the mass of recombinant collagen CF-1552 is 1:4, wherein the unit of mass is g and the unit of volume is mL; the volume of Tween-20 is 40% of the volume of triethylamine, and the volume of glycidyl methacrylate is 70% of the volume of triethylamine; the molecular weight cutoff of the dialysis bag is 8000-14000;

[0090] Step 4: adding sodium periodate to the pullulan solution, stirring at 600 rpm for 3 hours at room temperature, then adding ethylene glycol to terminate the reaction, collecting the product in a dialysis bag for 3 days, and freeze-drying to obtain oxidized pullulan; the concentration of the pullulan solution is 30 g / L, the mass of sodium periodate is 10% of the mass of pullulan, and the volume of ethylene glycol is 4 times the mass of sodium periodate, wherein the unit of mass is g and the unit of volume is mL; the molecular weight cutoff of the dialysis bag is 8000-14000;

[0091] Step 5, sterilizing the penicillin G coordination polymer nano drug solution in step 2, the modified recombinant collagen CF-1552 in step 3, and the oxidized pullulan in step 4 by Co60 irradiation respectively;

[0092] Step 6: Dissolve the sterilized modified recombinant collagen CF-1552 in water to prepare a modified recombinant collagen solution with a mass concentration of 12%; dissolve the sterilized oxidized pullulan in water to prepare an oxidized pullulan solution with a mass concentration of 12%;

[0093] Step 7. Disperse the sterilized penicillin G coordination polymer nano-drug solution in the modified recombinant collagen solution described in step 6, and then mix it with the oxidized pullulan solution described in step 6 to obtain a collagen-based penicillin G nano-drug composite hydrogel; the volume ratio of the modified recombinant collagen solution, the oxidized pullulan solution and the penicillin G coordination polymer nano-drug solution is 3:1:0.0035.

[0094] The effect of the collagen-based penicillin G nano-drug composite hydrogel dressing for pancreatic fistula of this embodiment is basically the same as that of Example 1. Fig.12 This is the SEM image of the collagen-based penicillin G nanodrug composite hydrogel prepared in this example. It can be seen that the pore size of the prepared composite hydrogel is 122.18 μm, and the scale is 300 μm; at the same time, it shows that the morphology and pore size of the composite hydrogel are uniform and the structure is good.

[0095] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A collagen-based penicillin G nano-drug composite hydrogel for pancreatic fistula, characterized in that: The composite hydrogel is prepared by dispersing penicillin G coordination polymer nano-drug prepared by Co / Cu coordination-induced self-assembly in a modified recombinant collagen solution and then mixing the solution with oxidized pullulan.

2. A method for preparing the composite hydrogel as claimed in claim 1, characterized in that: The following steps are involved: Step 1, mixing the ethanol solution of penicillin G and the ethanol solution of nafamostat mesylate, then dropping the ethanol solution of cobalt chloride and copper chloride into the mixed solution, stirring evenly, to obtain a premixed solution; Step 2, mixing the surfactant solution and the Tris hydrochloride solution, then adding the mixed solution into the premixed solution in step 1, and stirring to obtain a penicillin G coordination polymer nano drug solution; Step 3, dissolving the recombinant collagen CF-1552 in deionized water, and then adding triethylamine, Tween-20 and glycidyl methacrylate to obtain a CF-1552-containing reactant system; adjusting the pH of the CF-1552-containing reactant system to 8-10, and stirring the reaction at room temperature for 20h-30h, collecting the reaction product in a dialysis bag, dialyzing for 4-6 days, and freeze-drying to obtain modified recombinant collagen CF-1552; Step 4: adding sodium periodate to the pullulan solution, stirring the reaction at room temperature for 2 h to 4 h, then adding ethylene glycol to terminate the reaction, collecting the product in a dialysis bag, dialyzing it for 2 to 3 days, and freeze-drying it to obtain oxidized pullulan; Step 5, sterilizing the penicillin G coordination polymer nano drug solution in step 2, the modified recombinant collagen CF-1552 in step 3, and the oxidized pullulan in step 4 by Co60 irradiation respectively; Step 6: dissolving the sterilized modified recombinant collagen CF-1552 in water to prepare a modified recombinant collagen solution with a mass concentration of 10% to 15%; dissolving the sterilized oxidized pullulan in water to prepare an oxidized pullulan solution with a mass concentration of 10% to 15%; Step 7: Disperse the sterilized penicillin G coordination polymer nano drug solution in the modified recombinant collagen solution described in step 6, and then mix it with the oxidized pullulan solution described in step 6 to obtain a collagen-based penicillin G nano drug composite hydrogel.

3. The method according to claim 2, characterized in that The concentration of penicillin G in the penicillin G ethanol solution described in step 1 is 3 mg / mL to 5 mg / mL, the concentration of nafamostat mesylate in the nafamostat mesylate ethanol solution is 3 mg / mL to 5 mg / mL, the concentration of cobalt chloride in the cobalt chloride and copper chloride ethanol solution is 5 mg / mL to 15 mg / mL, the concentration of copper chloride is 5 mg / mL to 15 mg / mL, the volume ratio of the penicillin G ethanol solution to the nafamostat mesylate ethanol solution is 1:1, and the volume of the cobalt chloride and copper chloride ethanol solution is 4% to 10% of the volume of the penicillin G ethanol solution.

4. The method according to claim 2, characterized in that: The surfactant in step 2 is choline-PEI, the concentration of the surfactant solution is 5g / L to 15g / L, and the concentration of the Tris hydrochloride solution is 2g / L to 5g / L; the volume ratio of the surfactant solution, the Tris hydrochloride solution and the premix is ​​1:1:(0.1 to 0.3).

5. The method according to claim 2, characterized in that: In step 3, the ratio of the mass of recombinant collagen CF-1552 to the volume of deionized water is 1:(80-120), wherein the unit of mass is g and the unit of volume is mL; the ratio of the volume of triethylamine to the mass of recombinant collagen CF-1552 is 1:(2-5), wherein the unit of mass is g and the unit of volume is mL; the volume of Tween-20 is 30%-40% of the volume of triethylamine, and the volume of glycidyl methacrylate is 70%-90% of the volume of triethylamine.

6. The method according to claim 2, characterized in that The molecular weight cut-off of the dialysis bag in step 3 is 8000-14000.

7. The method according to claim 2, characterized in that The concentration of the pullulan solution in step 4 is 25g / L to 50g / L, the mass of sodium periodate is 8% to 16% of the mass of pullulan, and the volume of ethylene glycol is 3.5 to 6.5 times the mass of sodium periodate, wherein the unit of mass is g and the unit of volume is mL; the molecular weight cutoff of the dialysis bag is 8000 to 14000.

8. The method according to claim 2, characterized in that: In step seven, the volume ratio of the modified recombinant collagen solution, the oxidized pullulan solution and the penicillin G coordination polymer nano drug solution is (3-1):(1-2):0.0035.

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