A controllable degradable biomimetic hydrogel and its application

Abstract

The present invention discloses a controllable degradable bionic hydrogel and its application. It belongs to the field of medical material technology. The present invention successfully achieves the controllable degradation of the hydrogel by precisely controlling the composition ratio of low-viscosity sodium alginate, high-viscosity sodium alginate and oxidized high-viscosity sodium alginate in the mixed sodium alginate. Mixed with decellularized matrix, gelatin and PBS, a bionic artificial organ is constructed to achieve the controllable degradation of the organ, effectively avoiding the problem of cell loss and functional impairment caused by the loss of structural support of the artificial organ due to excessive material degradation; it also avoids the problem of inflammation and foreign body reaction caused by slow material degradation; and improves the stability of the artificial organ structure and the durability of its function. In addition, the present invention also carries out bionic design, and the bionic hydrogel developed is suitable for the survival of organ cells, reduces liver damage in mice with liver fibrosis, prolongs survival, and has the potential for artificial liver transplantation therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomaterials, and more particularly to a controllably degradable biomimetic hydrogel and applications thereof. Background Art

[0002] Sodium alginate (SA) is a natural polysaccharide extracted from brown algae. Due to its excellent biocompatibility, gel-forming properties, and functionality, it has a wide range of applications in the food industry, pharmaceuticals and biomedicine, cosmetics and personal care, industrial fields, and agricultural environmental protection. In the pharmaceutical and biomedical fields, sodium alginate can be used as a carrier material in drug sustained-release systems. By controlling the degradation rate of the alginate gel, the drug can be released at varying rates. It can also be used to construct artificial organs or tissue engineering scaffolds, providing a growth environment for cells. It can also be used for cell encapsulation to protect cells from immune rejection. However, the degradation rate of sodium alginate is affected by various factors (such as environmental pH and the presence of enzymes). This uncontrollable nature can affect its effectiveness in drug sustained-release systems, artificial organ construction, or tissue engineering.

[0003] Especially in the process of constructing artificial organs, the following technical problems often arise due to the uncontrollable degradation of hydrogels: (1) Uncontrollable material degradation: After transplantation, artificial organs often lose their structural support due to rapid material degradation, resulting in the loss of organ cells and impaired function; (2) Slow material degradation: If the material remains in the body for a long time, it may cause chronic inflammation and foreign body reaction, and even interfere with the normal organ function of the host; (3) Limited duration of function: Since the survival and functional expression of cells in the artificial matrix are limited by the characteristics of the material, most artificial organs find it difficult to achieve long-term biological functions.

[0004] Therefore, how to provide a controllably degradable biomimetic hydrogel and apply it to the construction of artificial organs to improve the stability of the artificial organ structure and the durability of its function is a technical problem that needs to be urgently solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a controllable degradable bionic hydrogel and its application. By precisely controlling the composition ratio of low-viscosity sodium alginate, high-viscosity sodium alginate and oxidized high-viscosity sodium alginate in the mixed sodium alginate, the controllable degradation of the hydrogel is successfully achieved. Its application in the construction of artificial organs can significantly improve the structural stability and functional durability of artificial organs, solve the problem of poor therapeutic effect after artificial organ transplantation, prolong the survival period, and provide technical support for the treatment of various organ diseases.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A controllable degradable biomimetic hydrogel comprising a mixed sodium alginate; the mixed sodium alginate comprises high-viscosity sodium alginate, low-viscosity sodium alginate and oxidized high-viscosity sodium alginate;

[0008] The oxidized high-viscosity sodium alginate is prepared by the following method:

[0009] An oxidant is added to a high-viscosity sodium alginate solution, and the solution is oxidized for 6-12 hours under low temperature, sealed, and light-proof conditions. Then, ethylene glycol is added to neutralize excess oxidant, and the solution is precipitated and dried to prepare oxidized high-viscosity sodium alginate.

[0010] Another object of the present invention is to provide: applications of the above-mentioned controllable degradable biomimetic hydrogel, wherein the applications are in any of the following directions:

[0011] (1) Application in the preparation of biological scaffold materials;

[0012] (2) Application in the preparation of drug sustained-release carriers;

[0013] (3) Application in the preparation of cell encapsulation materials.

[0014] Another object of the present invention is to provide: a controllably degradable biomimetic hydrogel for preparing artificial organs, comprising mixed sodium alginate, a decellularized matrix, PBS, and gelatin;

[0015] The mixed sodium alginate includes high-viscosity sodium alginate, low-viscosity sodium alginate and oxidized high-viscosity sodium alginate;

[0016] The oxidized high-viscosity sodium alginate is prepared by the following method:

[0017] An oxidant is added to a high-viscosity sodium alginate solution, and the solution is oxidized for 6-12 hours under low temperature, sealed, and light-proof conditions. Then, ethylene glycol is added to neutralize excess oxidant, and the solution is precipitated and dried to prepare oxidized high-viscosity sodium alginate.

[0018] Preferably, the concentration of the decellularized matrix is 4-8% w / v; the concentration of the gelatin is 8-16% w / v; the concentration of the mixed sodium alginate is 4-5% w / v; and the volume ratio of the decellularized matrix, PBS, gelatin and mixed sodium alginate is 1:1:2:1.

[0019] Preferably, the viscosity of the high-viscosity sodium alginate is in the range of 15-25 cp; and the viscosity of the low-viscosity sodium alginate is in the range of 4-12 cp.

[0020] Preferably, when the mass percentage of low-viscosity sodium alginate in the mixed sodium alginate is 0-20%, the mass percentage of high-viscosity sodium alginate is 60-100%, and the mass percentage of oxidized high-viscosity sodium alginate is 0-20%, the degradation time of the artificial organ is ≥45 days.

[0021] When the mass percentage of low-viscosity sodium alginate in the mixed sodium alginate is 20-40%, the mass percentage of high-viscosity sodium alginate is 20-60%, and the mass percentage of oxidized high-viscosity sodium alginate is 20-40%, the degradation time of the artificial organ is 15-45 days.

[0022] When the mass percentage of low-viscosity sodium alginate in the mixed sodium alginate is 40-50%, the mass percentage of high-viscosity sodium alginate is 0-20%, and the mass percentage of oxidized high-viscosity sodium alginate is 40-50%, the degradation time of the artificial organ is 5-15 days.

[0023] Another object of the present invention is to provide: use of the above-mentioned controllable degradable biomimetic hydrogel for preparing artificial organs in preparing controllable degradable artificial organs.

[0024] Another object of the present invention is to provide: a controllably degradable artificial organ, wherein the artificial organ is prepared using the controllably degradable biomimetic hydrogel for preparing the artificial organ.

[0025] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention provides a controllable degradation biomimetic hydrogel and its application. By precisely controlling the composition ratio of low-viscosity sodium alginate, high-viscosity sodium alginate and oxidized high-viscosity sodium alginate in the mixed sodium alginate, the controllable degradation of the hydrogel is successfully achieved. The hydrogel is applied to the construction of artificial organs and mixed with acellular matrix, gelatin and PBS to construct biomimetic artificial organs. The controllable degradation of the organ is achieved, which can effectively avoid the problem of cell loss and functional impairment caused by the loss of structural support of the artificial organ due to excessive material degradation; it also avoids the problem of inflammation and foreign body reaction caused by slow material degradation; and significantly improves the stability of the artificial organ structure and the durability of its function. In addition, the present invention also carries out biomimetic design. The biomimetic hydrogel used to construct the artificial organ is more suitable for the survival of organ cells, provides a suitable environment for their growth, and significantly improves the therapeutic effect of the functional sustainability of the artificial organ.

[0027] (2) The present invention applies the above-mentioned biomimetic hydrogel to the construction process of artificial liver, and verifies its effect of reducing liver damage and prolonging survival in a mouse model of liver fibrosis, and has the potential for artificial liver transplantation treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0029] Figure 1 In vitro degradation rates of artificial livers with different components.

[0030] Figure 2 Figure 3: Degradation of artificial livers with different components in vivo.

[0031] Figure 3 For: In vitro functional characterization of the bionic artificial liver (glycogen staining).

[0032] Figure 4 This is the survival curve of mice with liver fibrosis treated with bionic artificial liver.

[0033] Figure 5 For: Body weight changes in mice with liver fibrosis treated with bionic artificial liver.

[0034] Figure 6 Figure 3: Changes in the number of hepatocytes in different groups at different culture times. DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] Example 1

[0037] A controllable degradable biomimetic hydrogel, comprising mixed sodium alginate, is prepared by the following method:

[0038] (1) Preparation of oxidized high-viscosity sodium alginate: Add 10g of high-viscosity sodium alginate (15-25cp, Sigma, Product No. 180947) to 900ml of water and stir magnetically for about 8h. After the sodium alginate is completely dissolved, place it in a refrigerator for 2h to reduce its temperature to 4°C. Dissolve 10.7g of potassium periodate in 100ml of water and quickly mix it with the dissolved sodium alginate solution. Oxidize it under low temperature, sealed, and light-proof conditions for 6-12h, then add 3ml of ethylene glycol to neutralize the excess oxidant (potassium periodate) to terminate the oxidation reaction. Add this reaction solution to an ethanol-salt mixture (500ml ethanol-1g sodium chloride) in a volume ratio of 1:1 to precipitate the oxidized alginic acid. After standing for 10min, pour off the supernatant, take out the precipitate and put it into a freeze dryer for drying. Repeat this purification process twice to obtain dry oxidized high-viscosity sodium alginate solid;

[0039] (2) High-viscosity sodium alginate (15-25 cp, sigma, product number 180947), low-viscosity sodium alginate (4-12 cp, sigma, product number A1112) and the oxidized high-viscosity sodium alginate solid dried in step (1) were mixed in different mass percentages to prepare a mixed sodium alginate; the mixed sodium alginate was then dissolved in PBS at a ratio of 4% w / v, sterilized, and stored at 4°C to prepare a controllably degradable biomimetic hydrogel, which was heated to 37°C to melt before use.

[0040] Example 2

[0041] A controllably degradable biomimetic hydrogel for preparing an artificial liver comprises a liver decellularized matrix, PBS, gelatin, and mixed sodium alginate. The specific preparation method is as follows:

[0042] (1) Preparation of liver decellularized matrix:

[0043] Liver tissue from 6-8 week old C57 mice was surgically isolated and perfused, and then surface blood was removed using PBS. The liver tissue was then incubated in PBS containing 1% sodium dodecyl sulfate (SDS) and 1% Triton X-100 for 72 hours for decellularization. The decellularized liver tissue was lyophilized, and the lyophilized powder was dissolved in PBS containing 3% acetic acid (Merck Millipore, USA) and 1 mg / ml pepsin (Sigma Aldrich, USA) and shaken at 37°C for 72 hours. Finally, the pH of the dissolved liver decellularized matrix was adjusted to neutral using NaOH, corresponding to a liver decellularized matrix concentration of 8% w / v.

[0044] (2) Preparation of gelatin solution: Dissolve gelatin powder in PBS at a ratio of 16% (w / v), sterilize and store at 4°C. Heat the liquid to 37°C until it melts before use.

[0045] (3) Preparation of mixed sodium alginate: Same as steps (1) and (2) of Example 1; high viscosity sodium alginate (15-25 cp, Sigma, Product No. 180947), low viscosity sodium alginate (4-12 cp, Sigma, Product No. A1112) and oxidized high viscosity sodium alginate were mixed in different mass percentages, and the final concentration of the mixed sodium alginate was 4% w / v;

[0046] (4) 1 ml of the liver decellularized matrix prepared in step (1), 1 ml of PBS, 2 ml of the gelatin prepared in step (2), and 1 ml of the mixed sodium alginate prepared in step (3) were mixed to prepare a controllably degradable biomimetic hydrogel for preparing an artificial liver.

[0047] Example 3

[0048] Study on the degradation of bioartificial livers with different components

[0049] (1) Construction of artificial livers with different components: According to the preparation method of Example 2, different proportions of high-viscosity sodium alginate, low-viscosity sodium alginate and oxidized high-viscosity sodium alginate were prepared to prepare controllable biomimetic hydrogels for the preparation of artificial livers, and the effects of the proportions of different types of sodium alginate on the degradation properties of the hydrogels were explored. The prepared hydrogels were mixed with liver cells and refrigerated at 4°C for 15 minutes. Then, a suitable extrusion nozzle diameter was selected, the printing nozzle temperature was adjusted to 10-15°C, and the printing platform temperature was adjusted to 5-10°C. 1-2 mm was used. 3 The controllable degradable biomimetic hydrogel containing cells was printed in a 6 cm sterile culture dish at an extrusion speed of / s;

[0050] (2) Study on the in vitro degradation of artificial livers with different components

[0051] The artificial livers were divided into multiple groups according to their components, and two artificial livers were printed in each group with the same requirements. The printed livers were placed in a 3% CaCl2 solution for rapid cross-linking. After cross-linking, one of the artificial livers in each group was freeze-dried and the original weight (weight before culture) was weighed. The other one was added with liver cell culture medium and cultured at 37°C. After 14 days of culture, the culture medium was aspirated, the remaining hydrogel was freeze-dried, and the remaining weight was weighed. The gel degradation before and after culture was calculated by the dry weight ratio = remaining weight / original weight. The experimental results are shown as follows: Figure 1 As shown ( Figure 1The horizontal axis is the proportion of oxidized high-viscosity sodium alginate, the vertical axis is the proportion of low-viscosity sodium alginate, and the proportion of high-viscosity sodium alginate is 100%).

[0052] Result analysis: Figure 1 It can be seen that the degradation rate of artificial liver is different when the composition is different, and the degradation rate of artificial liver is proportional to the proportion of low-viscosity sodium alginate and oxidized high-viscosity alginate.

[0053] (3) Study on the in vivo degradation of artificial livers with different components

[0054] An easily degradable artificial liver (40% oxidized high-viscosity sodium alginate + 40% low-viscosity sodium alginate + 20% high-viscosity sodium alginate) and a difficult-to-degrade artificial liver (20% oxidized high-viscosity sodium alginate + 20% low-viscosity sodium alginate + 60% high-viscosity sodium alginate) were prepared, and 0.01 mg / ml of cy7 fluorescent dye was added during gel preparation. In order to prevent the gel from degrading too slowly and affecting the function of the native liver, the easily degradable artificial liver was transplanted in situ in the liver. In order to prevent the gel from degrading too quickly and losing the compensatory function of the liver, the difficult-to-degrade artificial liver was transplanted into the abdominal mesentery. After different periods of time, the degradation of the transplanted artificial liver was observed using a small animal in vivo imaging device. The experimental results are shown as follows. Figure 2 shown.

[0055] The results analysis, by Figure 2 It can be seen that the easily degradable artificial liver can be rapidly degraded within about 7 days when transplanted in situ in the liver, and will not affect the growth and function of the native liver; the difficult-to-degrade artificial liver still exists 21 days after transplantation in the abdominal cavity, and can continue to maintain the artificial liver morphology and exert the liver's compensatory function.

[0056] Example 4

[0057] Liver function tests in vivo transplantation

[0058] Based on Example 3, a biodegradable artificial liver (40% oxidized high-viscosity sodium alginate + 40% low-viscosity sodium alginate + 20% high-viscosity sodium alginate) was selected for in situ transplantation and cultured in vitro for 2 days. Glycogen staining was performed to observe its glycogen secretion and storage. The experimental results are as follows: Figure 3 shown.

[0059] Result analysis: Figure 3 It can be seen that the artificial liver showed obvious glycogen accumulation after in vitro culture, which means that the artificial liver has acquired mature liver function during in vitro culture.

[0060] Example 5

[0061] Detection of survival and body weight of transplanted mice

[0062] Six-week-old C57 mice were intraperitoneally injected with carbon tetrachloride to establish a liver fibrosis mouse model. A controlled-degradable in vivo artificial liver (40% oxidized high-viscosity sodium alginate + 40% low-viscosity sodium alginate + 20% high-viscosity sodium alginate) was transplanted into the liver fibrosis mouse model (in situ). A blank control group and a sham operation group were set up for comparison. The survival time and weight changes of the control and experimental groups were recorded. The experimental results are shown in the figure below. Figure 4 and Figure 5 shown.

[0063] Result analysis: Figure 4 and Figure 5 It can be seen that the survival time of mice with liver fibrosis that underwent orthotopic liver transplantation was prolonged, and there was no significant change in body weight, indicating that artificial liver can effectively alleviate the damage caused by liver fibrosis after orthotopic liver transplantation and provide liver compensatory function.

[0064] Example 6

[0065] Comparison of biomimetic hydrogel cell culture

[0066] A controllable biomimetic hydrogel containing acellular matrix (the concentration of the acellular matrix was 8% w / v; the concentration of the gelatin was 16% w / v; the concentration of the mixed sodium alginate was 4% w / v; the volume ratio of the acellular matrix, PBS, gelatin, and mixed sodium alginate was 1:1:2:1) and a gelatin-alginate hydrogel without acellular matrix (the concentration of the gelatin was 16% w / v; the concentration of the mixed sodium alginate was 4% w / v; the volume ratio of the acellular matrix, PBS, gelatin, and mixed sodium alginate was 0:1:2:1) were prepared. The two hydrogels were mixed with the same number of hepatocytes and the changes in the number of hepatocytes were observed after culture for different time periods. The results are shown in Figure 2. Figure 6 shown.

[0067] Result analysis: Figure 6 It can be seen that after adding decellularized matrix, the proliferation rate of liver cells is higher than that without adding decellularized matrix, which shows that the biomimetic hydrogel with added decellularized matrix is more suitable for cell survival and proliferation.

[0068] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0069] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A controllable degradable biomimetic hydrogel for preparing artificial organs, characterized in that: Including mixed sodium alginate, also includes decellularized matrix, PBS and gelatin; The mixed sodium alginate includes high-viscosity sodium alginate, low-viscosity sodium alginate and oxidized high-viscosity sodium alginate; The oxidized high-viscosity sodium alginate is prepared by the following method: adding potassium periodate as an oxidant to a high-viscosity sodium alginate solution, oxidizing for 6-12 hours at a low temperature of 4°C, in a sealed state, and in the dark, then adding ethylene glycol to neutralize excess oxidant, precipitating, and drying to prepare the oxidized high-viscosity sodium alginate; The viscosity of the high-viscosity sodium alginate is in the range of 15-25 cp; the viscosity of the low-viscosity sodium alginate is in the range of 4-12 cp; When the mass percentage of low-viscosity sodium alginate in the mixed sodium alginate is 0-20%, the mass percentage of high-viscosity sodium alginate is 60-100%, and the mass percentage of oxidized high-viscosity sodium alginate is 0-20%, the degradation time of the artificial organ is ≥45 days; When the mass percentage of low-viscosity sodium alginate in the mixed sodium alginate is 20-40%, the mass percentage of high-viscosity sodium alginate is 20-60%, and the mass percentage of oxidized high-viscosity sodium alginate is 20-40%, the degradation time of the artificial organ is 15-45 days; When the mass percentage of low-viscosity sodium alginate in the mixed sodium alginate is 40-50%, the mass percentage of high-viscosity sodium alginate is 0-20%, and the mass percentage of oxidized high-viscosity sodium alginate is 40-50%, the degradation time of the artificial organ is 5-15 days.

2. The controllable degradable biomimetic hydrogel for preparing artificial organs according to claim 1, characterized in that: The concentration of the decellularized matrix is 4-8% w / v; the concentration of the gelatin is 8-16% w / v; the concentration of the mixed sodium alginate is 4-5% w / v; and the volume ratio of the decellularized matrix, PBS, gelatin and mixed sodium alginate is 1:1:2:

1.

3. Use of the biomimetic hydrogel according to any one of claims 1-2 in the preparation of controllable degradable artificial organs.

4. A controllable degradable artificial organ, characterized in that: The artificial organ is prepared by using the bionic hydrogel according to any one of claims 1-2.

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