Implantable in-situ crosslinking hydrogel and application thereof

Abstract

The invention relates to the technical field of medical apparatus and instruments, and discloses implantable in-situ cross-linked hydrogel and application thereof in preparation of radiotherapy protection isolation hydrogel. The implantable in-situ cross-linked hydrogel comprises a solution A and a solution B, wherein the solution A is a polyethylene glycol derivative solution containing SOD (superoxide dismutase); the solution B is prepared by dissolving polyethyleneimine and epsilon-polylysine in a borate buffer solution with the pH value of 9-10, superoxide dismutase SOD loaded on the solution B can quickly release SOD after being injected into gel between a tumor and surrounding normal tissues, oxygen free radicals generated by radiotherapy dosage in the normal tissues during radiotherapy are captured, and the treatment effect is improved. Compared with the prior art, the implantable in-situ cross-linked hydrogel has the advantages that the effect of protecting normal tissues around a tumor is improved, and radiotherapy injury is reduced, so that when the implantable in-situ cross-linked hydrogel provided by the invention is used for preparing radiotherapy protection and isolation hydrogel, the physical distance between the tumor and the surrounding normal tissues can be increased, the irradiation dose of the normal tissues can be reduced, and SOD can be locally released to the normal tissues; and oxygen free radicals generated by radiotherapy are captured, so that the radiotherapy injury is further reduced, and the radiotherapy protection effect is improved.

Description

Technical Field

[0001] The invention relates to the technical field of medical devices, and in particular to an implantable in-situ cross-linked hydrogel and an application thereof in the preparation of a radiotherapy protective isolation hydrogel. Background Art

[0002] Hydrogel materials are considered to be the most biologically functional materials that are closest to living tissues due to their high water content, good water retention, excellent biocompatibility, and microstructures similar to the extracellular matrix. At the same time, hydrogels have good permeability to small molecule water-soluble solutes, which can keep the loaded substances active for a long time while ensuring the release of drugs in the body. Superoxide dismutase (SOD) can catalyze the dismutation of superoxide anion free radicals into hydrogen peroxide and oxygen. When free radicals accumulate excessively in the body, the lipids in the cell membrane will peroxidize and cause membrane fission, leading to cell damage and even death. SOD is the most important and best free radical scavenger in the body, maintaining the body's metabolic balance. SOD also has a strong anti-inflammatory effect and has a therapeutic and ameliorative effect on idiopathic radiation damage.

[0003] Radiotherapy is a method of treating tumors by using the ionizing radiation of high-energy rays to kill cancer cells. It is listed as one of the three major means of tumor treatment along with surgery and chemotherapy. It can directly damage DNA, and it can also indirectly damage DNA by producing charged groups (such as free radicals) in cells. When tumors grow close to surrounding normal tissues or important organs, radiation usually also damages surrounding normal tissues or organs. The hydrogel material embedded with SOD drugs is transplanted or injected into the designated location of the surrounding tissue, which can not only push normal tissue away from the high-dose target area, but also alleviate radiation damage by neutralizing free radicals caused by radiation. This new type of radiotherapy protective hydrogel material is expected to be used in implantable medical devices, radiotherapy protection and other fields.

[0004] As an implantable radiotherapy protection isolation hydrogel, when used clinically, after being implanted into the corresponding position, the shorter the gelation time, the more conducive it is to its molding and fixation, and it generally needs to be controlled within 1 minute; in order to cooperate with the cycle of radiotherapy, which is generally about 1 month, therefore, as an implantable radiotherapy protection isolation hydrogel, the degradation cycle after implantation in the body is preferably about 1 month; the loaded SOD needs to maintain good enzyme activity; on the basis of meeting these basic properties, the higher the cumulative drug release of the hydrogel, the better the effect of alleviating radiation damage.

[0005] The hydrogel products currently provided in the prior art for radiotherapy isolation protection only play a physical isolation role, including the patent CN109646723B previously applied for by the applicant, which discloses a medical hydrogel, which is formed by in situ cross-linking of aldehyde-terminated star-shaped multi-arm polyethylene glycol and polyamino compounds. The prepared hydrogel gels quickly and has long-term stability in aqueous solution. It still has good swelling performance and stability after multiple irradiations, and can be used as a medical radiotherapy protective gasket for radiation protection.

[0006] The hydrogels with SOD are mainly used for skin surface anti-oxidation. For example, CN115381735A discloses a hydrogel dressing for skin anti-oxidation and a preparation method thereof. The main material of the hydrogel is non-ionic polyacrylamide. The non-ionic polyacrylamide with a molecular weight of 10,000,000 to 30,000,000 is used to prepare the hydrogel base. The hydrogel base contains salicylic acid, superoxide dismutase (SOD), vitamin C, vitamin E, and cationic hyaluronic acid. The pH value of the hydrogel dressing is 5 to 6. It can be applied as a dressing on the disinfected skin to prevent skin oxidation and hydrate the skin to keep the skin breathable.

[0007] The prior art has not provided an implantable hydrogel loaded with SOD and having good gelling properties, and it is unpredictable whether the medical hydrogel previously disclosed by the applicant can maintain the original good properties after being loaded with SOD. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides an implantable in situ cross-linked hydrogel, which can be used as a medical hydrogel to increase the radiotherapy protection effect. Without affecting the good gelation properties of the hydrogel, the drug SOD loaded can significantly reduce radiation damage, so that the hydrogel loaded with SOD can achieve a significant increase in the radiotherapy protection effect.

[0009] The specific technical solutions of the present invention are as follows:

[0010] In a first aspect, the present invention provides an implantable in situ cross-linked hydrogel, the implantable in situ cross-linked hydrogel comprising liquid A and liquid B, the liquid A being a polyethylene glycol derivative solution containing SOD; the liquid B being prepared by dissolving polyethyleneimine and ε-polylysine in a borate buffer solution at pH 9 to 10;

[0011] The A solution is prepared by mixing SOD and a polyethylene glycol derivative solution. In the A solution, the concentration of superoxide dismutase is 1000-100000U / g, preferably 2000-5000U / g; the polyethylene glycol derivative solution as a precursor component is composed of a 6-arm polyethylene glycol terminated with a benzaldehyde group, i.e., 6-arm PEG-CHO, and a phosphate buffer, specifically, the 6-arm PEG-CHO is dissolved in a phosphate buffer at pH 5-6; the molecular weight of the 6-arm PEG-CHO is 15000Da, and the chemical structure of the 6-arm PEG-CHO is as follows:

[0012]

[0013] The ratio of the 6-arm PEG-CHO to the total mass of the precursor component, i.e., the mass concentration, is 10-30%, preferably 20%;

[0014] The proportion of the polyethyleneimine to the total mass of liquid B, that is, the mass concentration, is 5-10%, and preferably 6.078%; the mass ratio of the polyethyleneimine to the ε-polylysine is in the range of 1:1-4.5, and preferably 1:1.6.

[0015] Preferably, the pH of the phosphate buffer is 5.6; the pH of the borate buffer is 9.2.

[0016] After mixing liquid A and liquid B, the polyethylene glycol derivative containing superoxide dismutase in liquid A and the two-component crosslinking agent polyethylene imine and ε-polylysine in liquid B are crosslinked in situ to form a hydrogel loaded with superoxide dismutase. The chemical crosslinking reaction between the polyethylene glycol derivative and the crosslinking agent does not affect the stability of superoxide dismutase, and the bioavailability of superoxide dismutase within 24 hours is greater than 80%.

[0017] The implantable in-situ cross-linked hydrogel provided by the present invention can achieve the physical isolation of medical hydrogels while neutralizing the oxygen free radicals generated by radiation irradiation tissues, thereby improving the purpose of radiotherapy protection effect. Therefore, in the second aspect, the present invention also provides the application of the above-mentioned implantable in-situ cross-linked hydrogel in the preparation of radiotherapy protection isolation hydrogel, and the application step includes: injecting and mixing liquid A and liquid B to obtain a radiotherapy protection isolation hydrogel loaded with SOD through in-situ cross-linking. Further, the liquid A and liquid B are injected and mixed in equal volumes, that is, the volume ratio of the two is 1:1. The drug SOD is used to neutralize the free radicals generated in the cell caused by radiation, effectively increasing the radiotherapy protection effect of the hydrogel. And the radiotherapy protection isolation hydrogel loaded with SOD is in line with the intended use. Although SOD is loaded, it does not affect the gelation time and degradation of the hydrogel, and can maintain the degradation time at about one month on the basis of not affecting the gelation time, and has good enzyme activity after gelation, and can further increase the SOD release amount, which can better match clinical needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The graphs are the release curves of SOD loaded in chemically cross-linked hydrogels of Examples 1 to 4.

[0019] Figure 2 The release curves of SOD loaded in thermosensitive hydrogels of comparative examples 4 to 7;

[0020] Figure 3 The graph is a release curve of SOD loaded in the chemically cross-linked hydrogel prepared with single component B solution in comparative examples 8 to 13. DETAILED DESCRIPTION

[0021] The specific steps of the present invention are described below by way of examples, but are not limited by the examples. The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0022] The present invention is further described in detail below in conjunction with specific examples and with reference to data. It should be understood that these examples are only intended to illustrate the present invention, and are not intended to limit the scope of the present invention in any way.

[0023] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0024] According to the synthesis process of benzaldehyde-terminated four-arm polyethylene glycol reported in the open document Adv Healthc Mater. 2016 Nov; 5 (21): 2813-2822. doi: 10.1002 / adhm.201600720. Epub 2016 Sep 26., section 2.1, 6-arm-PEG-CHO capped with benzaldehyde was prepared by replacing 4-arm PEG in the raw material with 6-arm PEG. The other preparation conditions were exactly the same as those in the above document. The prepared 6-arm-PEG-CHO was obtained.

[0025] 6-arm PEG, 6-arm Poly(ethylene glycol)(DP)[6-arm PEG-OH(DP)], with a molecular weight of 15k, was purchased from Xiamen Sinobond Biotechnology, catalog number 06020800109;

[0026] Polyethyleneimine (MW 1.8K) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., catalog number E808878;

[0027] ε-Poly-lysine was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., catalog number P875277;

[0028] The following tests or calculations involve:

[0029] Average gelation time test and calculation method: Place liquid A, liquid B and vial in a 37±0.5℃ water bath. After standing for 5 minutes, use a pipette to take 200μL of liquid A into the vial, then use another pipette tip to take 200μL of liquid B and add it to the vial containing liquid A. Vibrate the vial with your wrist for 1s, then tilt the vial repeatedly to observe until the liquid stops flowing. Start timing at the same time as adding reagent B, and end timing when the liquid stops flowing. The timing duration is the curing time. Measure three groups of samples and take the average value.

[0030] 37°C in vitro degradation time test and calculation method: Take the prepared gel for gelation time, weigh the gel mass, add PBS buffer at a ratio of 1:10, put it in a 37°C constant temperature water bath oscillator, replace the PBS buffer every 3 days, and observe the gel degradation every day.

[0031] The drug release was tested by UV spectrophotometer. Test group: the hydrogel and PBS buffer were mixed at a volume ratio of 1:10, placed in a 37°C water bath constant temperature oscillator, and the liquid was taken out according to the time point and placed in a UV spectrophotometer for testing. The release amount can be determined by comparing with the standard curve. Standard curve preparation: SOD was dissolved in PBS, and 5 points were tested in a gradient form. The blank sample was the same A / B solution concentration of the gel without SOD in PBS.

[0032] Enzyme activity test: Determine according to the national standard "GB / T5009.171-2003" (Marklund modified method).

[0033] It should be noted that the percentages in the following examples and comparative examples are the percentages of the mass of the corresponding substances to the total mass of the solution;

[0034] Example 1

[0035] Step 1: Preparation of hydrogel precursor solution: dissolve 200 mg of 6-arm-PEG-CHO in a phosphate buffer solution at pH 5.6 as liquid A, the total mass of liquid A is 1 g, that is, the mass percentage of 6-arm-PEG-CHO in the total mass of liquid A is 20%;

[0036] Step 2: Weigh 0.06078 g polyethyleneimine (MW 1.8K) and 0.10152 g ε-polylysine, that is, the mass ratio of the two is 3:5, and dissolve the prepared polyethyleneimine and ε-polylysine in a borax buffer solution at pH 9.2 as solution B, and the total mass of solution B is 1 g.

[0037] Step 3: Dissolve 5000U superoxide dismutase in solution A to obtain solution A loaded with SOD;

[0038] Step 4: Inject and mix the prepared SOD-loaded liquid A and liquid B to obtain the superoxide dismutase-loaded radiotherapy protective isolation hydrogel.

[0039] Example 2

[0040] The only difference between Example 2 and Example 1 is that in step 1, the dissolved amount of 6-Arm-PEG-CHO is 100 mg, that is, in liquid A, the mass percentage of 6-Arm-PEG-CHO in the total mass of liquid A is 10%.

[0041] Example 3

[0042] The only difference between Example 3 and Example 1 is that in step 1, the dissolved amount of 6-Arm-PEG-CHO is 300 mg, that is, in liquid A, the mass percentage of 6-Arm-PEG-CHO in the total mass of liquid A is 30%.

[0043] Example 4

[0044] The only difference between Example 4 and Example 1 is that in step 3, 10000 U of superoxide dismutase is dissolved in liquid A.

[0045] In order to compare the performance of the above hydrogel before and after drug loading, comparative examples 1 to 3 were set up;

[0046] Comparative Example 1

[0047] The difference between Comparative Example 1 and Example 1 is that in Comparative Example 1, the liquid A prepared in step 1 and the liquid B prepared in step 2 are directly mixed according to step 4;

[0048] Comparative Example 2

[0049] The difference between Comparative Example 2 and Example 2 is that in Comparative Example 2, the liquid A prepared in step 1 and the liquid B prepared in step 2 are directly mixed according to step 4;

[0050] Comparative Example 3

[0051] The difference between Comparative Example 3 and Example 3 is that in Comparative Example 3, the liquid A prepared in step 1 and the liquid B prepared in step 2 are directly mixed according to step 4;

[0052] The test results of the average gelation time and 37°C in vitro degradation time of Examples 1 to 4 and Comparative Examples 1 to 3 are shown in Table 1 below;

[0053] Table 1:

[0054]

[0055] The average turnover time of liquid A and liquid B in Examples 1 to 3 is within 10 seconds, which meets the use requirements of rapid gelation, and the in vitro degradation time of the hydrogels after gelation in Examples 1 to 3 at 37°C is about 1 month, which is conducive to reducing foreign body stimulation, and this degradation cycle meets the duration of radiotherapy for most cancers and meets the clinical use requirements of a wider range of radiotherapy. By comparing with Comparative Examples 1 to 3, it is found that SOD encapsulation has only a slight effect on the gelation time and degradation cycle. Therefore, after SOD encapsulation, the hydrogel products provided in Examples 1 to 3 can still meet the requirements of rapid gelation and degradation within a specific cycle;

[0056] like Figure 1 The figures show the cumulative drug release rate test results of Examples 1 to 4. Examples 1 to 4 can all achieve a cumulative drug release of more than 80% in 24 hours, and the release amounts of Examples 1 and 4 can best reach more than 95%.

[0057] In addition, the enzyme activity of Example 1 and Example 4 after chemical cross-linking and gelation for 24 hours was tested. The test results showed that the enzyme activity was 4726U and 9782U, respectively, indicating that the A liquid and B liquid formula provided by the present invention had no significant effect on the enzyme activity of SOD after mixing and gelation.

[0058] In order to compare the performance differences between thermally cross-linked hydrogels and chemically cross-linked hydrogels, superoxide dismutase-loaded thermally initiated cross-linked hydrogels of Comparative Examples 4 to 7 were set up;

[0059] Comparative Example 4

[0060] Dissolve 200 mg of polyethylene glycol diacrylate PEGDA with a molecular weight of 700 and 30 mg of ammonium persulfate in purified water, and after preparation, the mass concentration of polyethylene glycol diacrylate PEGDA accounts for 20% of the total mass of the solution; the mass concentration of ammonium persulfate accounts for 3% of the total mass of the solution;

[0061] After dissolution, 5000U SOD was added, and finally the mixture was placed in a 60°C oven. After about 30 minutes, a SOD-loaded heat-induced cross-linked hydrogel was obtained.

[0062] Comparative Example 5

[0063] The difference between Comparative Example 5 and Comparative Example 4 is that in Comparative Example 5, the molecular weight of polyethylene glycol diacrylate is 6000, the mass concentration of polyethylene glycol diacrylate is 10%, and the mass concentration of ammonium persulfate is 1%.

[0064] Comparative Example 6

[0065] The only difference between Comparative Example 6 and Comparative Example 4 is that the molecular weight of polyethylene glycol diacrylate in Comparative Example 6 is 10,000, and the mass concentration of ammonium persulfate is 1%.

[0066] Comparative Example 7

[0067] The only difference between Comparative Example 7 and Comparative Example 4 is that the molecular weight of polyethylene glycol diacrylate in Comparative Example 7 is 40,000, and the mass concentration of ammonium persulfate is 1%.

[0068] The test results of the average gelation time and in vitro degradation time at 37°C of Comparative Examples 4 to 7 are shown in Table 2 below;

[0069] Table 2:

[0070]

[0071] The thermosensitive hydrogels in Comparative Examples 4 to 7 have too long a gelling time and cannot be used clinically.

[0072] like Figure 2As shown in the figure, the molecular weight of PEGDA affects the release of SOD. When the molecular weight is higher than 700, the release rate decreases significantly. The maximum release rate of SOD-loaded thermosensitive hydrogel can only reach about 60%. It is speculated that light intensity irradiation and high temperature increase the activity of SOD, causing SOD failure or denaturation, resulting in the problem of low SOD release and reducing the therapeutic effect.

[0073] In order to verify the effects of the two-component B liquid formula and the one-component B liquid formula in Examples 1 to 4 on the performance of the hydrogel, Comparative Examples 8 to 13 were set up;

[0074] Comparative Example 8

[0075] The difference between Comparative Example 8 and Example 1 is only that the B solution is different, specifically:

[0076] Step 2: 81.04 mg of polyethyleneimine (MW 1.8K) was dissolved in a borax buffer solution at pH 9.2 as solution B; accordingly, the mass concentration of polyethyleneimine was 8.104%.

[0077] Comparative Example 9

[0078] The difference between Comparative Example 9 and Comparative Example 8 is that the amount of polyethyleneimine dissolved is 60.78 mg, and the mass concentration of polyethyleneimine is 6.078% accordingly;

[0079] Comparative Example 10

[0080] The only difference between Comparative Example 10 and Comparative Example 8 is that the dissolved amount of polyethyleneimine raw material is 30.39 mg, and accordingly, the mass concentration of polyethyleneimine is 3.039%.

[0081] Comparative Example 11

[0082] The difference between Comparative Example 11 and Example 1 is that the B solution is different, specifically:

[0083] Step 2: Dissolve 135.36 mg of ε-polylysine in a borax buffer solution at pH 9.2 as solution B; the mass concentration of ε-polylysine is 13.536%;

[0084] Comparative Example 12

[0085] The only difference between Comparative Example 12 and Comparative Example 11 is that the dissolved amount of the ε-polylysine raw material is 101.52 mg, and accordingly, the mass concentration of the ε-polylysine is 10.152%.

[0086] Comparative Example 13

[0087] The only difference between Comparative Example 13 and Comparative Example 11 is that the dissolved amount of the ε-polylysine raw material is 76.14 mg, and accordingly, the mass concentration of the ε-polylysine is 7.614%.

[0088] Comparative Example 14

[0089] The only difference between Comparative Example 14 and Comparative Example 11 is that the dissolved amount of the ε-polylysine raw material is 160 mg, and accordingly, the mass concentration of the ε-polylysine is 16%.

[0090] The test results of the average gelation time of Comparative Examples 8 to Comparative Examples 14 and the in vitro degradation time at 37°C of Comparative Examples 8 to Comparative Examples 13 are shown in Table 3 below;

[0091] Table 3:

[0092]

[0093] According to the test results in the above table, it is found that the degradation time of single-component polyethyleneimine is fast, and the degradation time of single-component ε-polylysine is slow. Further, the above comparative examples 8 to 13 are used for drug loading experiments, and the results are as follows: Figure 3 It can be seen that when the concentration of chemical cross-linked single-component B liquid polyethyleneimine is 6.078%, the release rate is as high as 90%, and when the concentration of chemical cross-linked single-component B liquid ε-polylysine is 10.152%, the release rate is as high as 52%. Therefore, the test results can verify that it is difficult to achieve a balance in performance in terms of degradation cycle and SOD release for single-component B liquid, whether it is polyethyleneimine or ε-polylysine.

[0094] In summary, the two-component B liquid has a longer in vitro degradation time than the single-component polyethyleneimine; and a shorter in vitro degradation time than the single-component ε-polylysine. The implantable in situ cross-linked hydrogel formulas provided in Examples 1 to 4 meet the intended use, and the loading of SOD does not affect the gelation time and degradation of the hydrogel. In particular, the formulas of Examples 1 and 4 can maintain the degradation time at about one month without affecting the gelation time, have good enzyme activity after gelation, and can further increase the SOD release amount, which can better match clinical needs.

Claims

1. An implantable in situ cross-linked hydrogel, characterized in that: The implantable in situ cross-linked hydrogel comprises liquid A and liquid B, wherein the liquid A is a polyethylene glycol derivative solution containing SOD; and the liquid B is prepared by dissolving polyethyleneimine and ε-polylysine in a borate buffer solution with a pH of 9 to 10; The A solution is prepared by mixing SOD and a polyethylene glycol derivative solution. In the A solution, the concentration of superoxide dismutase is 1000-100000 U / g; the polyethylene glycol derivative solution is composed of 6-arm PEG-CHO and a phosphate buffer solution with a pH of 5-6; the molecular weight of the 6-arm PEG-CHO is 15000 Da, and the chemical structure of the 6-arm PEG-CHO is as follows: The proportion of the 6-arm PEG-CHO to the total mass of the polyethylene glycol derivative solution, i.e., the mass concentration, is 10 to 30%; The proportion of the polyethyleneimine to the total mass of liquid B, that is, the mass concentration, is 5 to 10%; the mass ratio of the polyethyleneimine to the ε-polylysine is in the range of 1:1 to 4.

5.

2. An implantable in situ cross-linked hydrogel according to claim 1, characterized in that: In solution A, the concentration of superoxide dismutase is 2000-5000 U / g.

3. The implantable in situ cross-linked hydrogel according to claim 1, characterized in that: The proportion of the 6-arm PEG-CHO to the total mass of the precursor components, ie, the mass concentration, is 20%.

4. The implantable in situ cross-linked hydrogel according to claim 1, characterized in that: The proportion of the polyethyleneimine to the total mass of liquid B, ie, the mass concentration, is 6.078%.

5. The implantable in situ cross-linked hydrogel according to claim 1, characterized in that: The mass ratio of the polyethyleneimine to the ε-polylysine is in the range of 1:1.

6.

6. The implantable in situ cross-linked hydrogel according to claim 1, characterized in that: The pH of the phosphate buffer is 5.

6.

7. The implantable in situ cross-linked hydrogel according to claim 1, characterized in that: The pH of the borate buffer was 9.

2.

8. Use of the implantable in situ cross-linked hydrogel according to claim 1 in preparing a radiotherapy protective isolation hydrogel, the application step comprising: The A solution and the B solution are mixed by injection, and then the radiotherapy protection isolation hydrogel loaded with SOD can be obtained through in situ cross-linking.

9. The use according to claim 8, characterized in that The A solution and the B solution are mixed into equal volumes for injection.

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