Photo-thermal antibacterial material based on acellular adipose tissue as well as preparation method and application of photo-thermal antibacterial material

Abstract

The invention relates to a photo-thermal antibacterial material based on acellular adipose tissue, a preparation method and application, and the preparation method comprises the following steps: adjusting pH to alkalescence, and modifying the surface of the acellular adipose tissue with polydopamine to obtain the photo-thermal antibacterial material. The photo-thermal antibacterial material has the advantages that the photo-thermal antibacterial material is composed of natural components, is wide in source and has excellent biocompatibility and activity; under the action of exciting light, the photo-thermal antibacterial material can accurately regulate and control a local microenvironment in real time and promote tissue repair; under the action of exciting light, the photo-thermal antibacterial material can induce local heat and has certain antibacterial performance.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a photothermal antibacterial material based on decellularized adipose tissue, a preparation method and an application thereof. Background Art

[0002] Tissue defects seriously damage the quality of life and health of patients. In recent years, the number of patients with tissue defects caused by trauma, disease and population aging has increased year by year, which has become a major clinical problem. Therefore, there is a huge demand for tissue repair materials in clinical practice. Tissue defects are rich in a large amount of reactive oxygen and inflammatory mediators, and are often accompanied by infection, which will seriously hinder the repair and reconstruction of tissues. Therefore, tissue repair materials are required to have both microenvironment regulation capabilities and antibacterial properties. At present, there are endless tissue repair materials in clinical practice, however, the biological activity and local microenvironment regulation capabilities of tissue repair materials still need to be further improved.

[0003] At present, no effective solution has been proposed for the problems existing in related technologies, such as the lack of microenvironment regulation ability and poor antibacterial ability of tissue repair materials. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies in the prior art and provide a photothermal antibacterial material based on decellularized adipose tissue, a preparation method and an application thereof, so as to solve the problems existing in the related art that tissue repair materials do not have the ability to regulate the microenvironment and have poor antibacterial ability.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In a first aspect, a method for preparing a photothermal antibacterial material based on decellularized adipose tissue is provided, comprising:

[0007] The pH is adjusted to weak alkalinity, and polydopamine is modified on the surface of the decellularized adipose tissue to obtain a photothermal antibacterial material.

[0008] In some of the embodiments, it also includes:

[0009] dissolving the decellularized adipose tissue in deionized water to obtain a decellularized adipose tissue solution;

[0010] dissolving polydopamine in the decellularized adipose tissue solution to obtain a mixed solution;

[0011] Adjusting the pH of the mixed solution to weak alkalinity;

[0012] The mixed solution is separated to obtain the photothermal antibacterial material.

[0013] In some of the embodiments, it also includes:

[0014] Dissolving the photothermal antibacterial material in deionized water to obtain a photothermal antibacterial material solution;

[0015] The photothermal antibacterial material solution is freeze-dried to obtain a photothermal antibacterial scaffold.

[0016] In some embodiments, the mass ratio of polydopamine to decellularized adipose tissue is 0.05 to 1:1.

[0017] In some embodiments, the pH is adjusted to 7-9.

[0018] In some embodiments, the concentration of the decellularized adipose tissue solution is 1 mg / ml to 10 mg / ml.

[0019] In some embodiments, the polydopamine concentration of the mixed solution is 0.5 mg / ml to 4 mg / ml.

[0020] In some of the embodiments, the separation method of the mixed solution includes centrifugation, suction filtration, and ultrafiltration.

[0021] In some embodiments, the concentration of the photothermal antibacterial material solution is 0.5 mg / ml to 4 mg / ml.

[0022] In some of the embodiments, including:

[0023] (I) Preparation of decellularized adipose tissue

[0024] The animal-derived adipose tissue was frozen and thawed in a freezing buffer containing 10 mM Tris and 5 mM EDTA using three cycles, wherein the cycle temperature ranged from -80°C to 37°C;

[0025] Wash the adipose tissue three times with PBS buffer;

[0026] Digest the adipose tissue using an enzymatic digestion solution, wherein the enzymatic digestion solution consists of 0.25% trypsin / 0.02% EDTA;

[0027] Incubate overnight on a shaker at 37°C at 1000 rpm;

[0028] Adipose tissue was subjected to polar solvent extraction using 99.9% isopropyl alcohol for 12 h to remove the adipose liquid fraction;

[0029] The adipose tissue was rinsed three times with PBS buffer, wherein the pH of the PBS buffer was 7.4;

[0030] The adipose tissue was incubated again in the enzyme digestion solution for 4 h;

[0031] The adipose tissue was washed three times with PBS buffer;

[0032] The adipose tissue was incubated in a solution containing 10 mg / mL DNase enzyme, 10 mg / mL RNase enzyme, and 2000 U / mL lipase at 37°C for 6 h;

[0033] The adipose tissue was rinsed three times with PBS buffer;

[0034] The adipose tissue was treated again in 99.9% isopropanol for 8 h as the final polar solvent extraction to fully remove fat to obtain decellularized adipose tissue;

[0035] The decellularized adipose tissue was washed three times with 75% ethanol and PBS buffer respectively;

[0036] The decellularized adipose tissue was dissolved using acetic acid, washed, adjusted to a pH value of 7.4, and then freeze-dried to obtain the decellularized adipose tissue;

[0037] (II) Preparation of polydopamine-modified decellularized adipose tissue

[0038] The decellularized adipose tissue was placed in anhydrous ethanol and washed three times with deionized water to obtain pure decellularized adipose tissue;

[0039] dissolving purified decellularized adipose tissue in deionized water to obtain a decellularized adipose tissue solution;

[0040] Fully dissolving polydopamine in the decellularized adipose tissue solution to obtain a mixed solution, wherein the concentration of polydopamine in the mixed solution is 0.5 mg / ml to 4 mg / ml;

[0041] The pH of the mixed solution was adjusted to 7-9 using Tris buffer and stirred vigorously at room temperature for 1 h;

[0042] (III) Preparation of photothermal antibacterial materials

[0043] The mixed solution was centrifuged and washed three times with ultrapure water to obtain the photothermal antibacterial material.

[0044] In some of the embodiments, it also includes:

[0045] (IV) Preparation of photothermal antibacterial scaffolds

[0046] Dissolving the photothermal antibacterial material in deionized water to obtain a photothermal antibacterial material solution, wherein the concentration of the photothermal antibacterial material solution is 0.5 mg / ml to 4 mg / ml;

[0047] The photothermal antibacterial material solution is freeze-dried to obtain a photothermal antibacterial scaffold.

[0048] In a second aspect, a photothermal antibacterial material based on decellularized adipose tissue is provided, which is prepared by the preparation method described in the first aspect and includes decellularized adipose tissue and polydopamine.

[0049] In some embodiments, the mass ratio of polydopamine to decellularized adipose tissue is 0.05 to 1:1.

[0050] In a third aspect, there is provided a photothermal antibacterial material prepared by the preparation method described in the first aspect or the use of the photothermal antibacterial material described in the second aspect in the preparation of a bioremediation material.

[0051] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0052] The present invention discloses a photothermal antibacterial material based on decellularized adipose tissue, a preparation method and an application thereof. The photothermal antibacterial material is composed of natural ingredients, has a wide range of sources, and has excellent biocompatibility and activity. Under the action of excitation light, the photothermal antibacterial material can accurately regulate the local microenvironment in real time and promote tissue repair. Under the action of excitation light, the photothermal antibacterial material can cause local heat and has certain antibacterial properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Histochemical staining of the extracted decellularized adipose tissue (DAT) demonstrated complete removal of cellular and lipid components;

[0054] Figure 2 A mixed solution modified with different concentrations of polydopamine;

[0055] Figure 3 The photothermal antibacterial scaffolds were modified with different concentrations of polydopamine;

[0056] Figure 4 The photothermal performance images of photothermal antibacterial scaffolds modified with different concentrations of polydopamine;

[0057] Figure 5 The photothermal performance curves of photothermal antibacterial scaffolds modified with different concentrations of polydopamine;

[0058] Figure 6 The scanning electron microscopy image of the photothermal antibacterial scaffold (pDAT) modified with 1 mg / ml polydopamine shows that it has a porous structure;

[0059] Figure 7 The figure shows the cell compatibility experiment between 1 mg / ml polydopamine-modified photothermal antibacterial scaffold (pDAT) and decellularized adipose tissue under different environments;

[0060] Figure 8The antibacterial performance experimental diagram of decellularized adipose tissue using the coating plate method, 1 mg / ml polydopamine modified photothermal antibacterial scaffold (pDAT), and 1 mg / ml polydopamine modified photothermal antibacterial scaffold (pDAT) combined with near infrared;

[0061] Fig. 9 These are the antibacterial quantitative images of decellularized adipose tissue, 1 mg / ml polydopamine modified photothermal antibacterial scaffold (pDAT), and 1 mg / ml polydopamine modified photothermal antibacterial scaffold (pDAT) combined with near-infrared. DETAILED DESCRIPTION

[0062] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0063] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0064] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0065] Example 1

[0066] This embodiment relates to the photothermal antibacterial material based on decellularized adipose tissue, its preparation method and application.

[0067] An illustrative embodiment of the present invention is a method for preparing a photothermal antibacterial material based on decellularized adipose tissue, comprising:

[0068] Step S102, adjusting the pH to weak alkalinity, and modifying the surface of the decellularized adipose tissue (DAT) with polydopamine (PDA) to obtain a photothermal antibacterial material (pDAT).

[0069] Wherein, in step S102, the mass ratio of polydopamine to decellularized adipose tissue is 0.05-1:1.

[0070] Wherein, in step S102, the pH is adjusted to 7-9.

[0071] In step S102, the source of the decellularized fat tissue is an animal source, such as a pig source.

[0072] Among them, in step S102, the method for obtaining decellularized adipose tissue is a prior art (refer to Han TT, Toutounji S, Amsden BG, Flynn LE. Adipose-derived stromal cells mediate in vivo adipogenesis, angiogenesis and inflammation in decellularized adipose tissue bioscaffolds. Biomaterials. 2015; 72: 125-137. doi: 10.1016 / j.biomaterials.2015.08.053), which will not be repeated here.

[0073] In the present invention, the photothermal antibacterial material has good biocompatibility, photothermal performance and antibacterial performance.

[0074] Specifically, step S102 includes:

[0075] Step S1021, dissolving the decellularized adipose tissue in deionized water to obtain a decellularized adipose tissue solution;

[0076] Step S1022, dissolving polydopamine in the decellularized adipose tissue solution to obtain a mixed solution;

[0077] Step S1023, adjusting the pH of the mixed solution to be weakly alkaline;

[0078] Step S1024, separating the mixed solution to obtain the photothermal antibacterial material.

[0079] Wherein, in step S1021, the concentration of the decellularized fat tissue solution is 1 mg / ml to 10 mg / ml. Preferably, the concentration of the decellularized fat tissue solution is 2 mg / ml to 8 mg / ml. More preferably, the concentration of the decellularized fat tissue solution is 4 mg / ml to 6 mg / ml.

[0080] Wherein, in step S1022, the polydopamine concentration of the mixed solution is 0.5 mg / ml to 4 mg / ml.

[0081] Wherein, in step S1024, the separation method of the mixed solution includes centrifugation, suction filtration, and ultrafiltration.

[0082] Furthermore, step S102 further includes:

[0083] Step S1025, dissolving the photothermal antibacterial material in deionized water to obtain a photothermal antibacterial material solution;

[0084] Step S1026: freeze-dry the photothermal antibacterial material solution to obtain a photothermal antibacterial stent.

[0085] Wherein, in step S1025, the concentration of the photothermal antibacterial material solution is 0.5 mg / ml to 4 mg / ml.

[0086] The photothermal antibacterial material prepared by the present invention can be used to prepare a biorepair material, which can be used to repair infectious tissues.

[0087] The advantages of the present invention are as follows:

[0088] 1) The photothermal antibacterial material is composed of natural ingredients, has a wide range of sources, and has excellent biocompatibility and activity;

[0089] 2) Under the action of excitation light, the photothermal antibacterial material can accurately regulate the local microenvironment in real time and promote tissue repair;

[0090] 3) Under the action of excitation light, the photothermal antibacterial material can cause local heat and has certain antibacterial properties.

[0091] Example 2

[0092] This embodiment is a specific preparation example of the photothermal antibacterial material of the present invention.

[0093] A method for preparing a photothermal antibacterial material, comprising:

[0094] (I) Preparation of decellularized adipose tissue

[0095] The porcine adipose tissue was frozen and thawed in a freezing buffer containing 10 mM Tris and 5 mM EDTA using three cycles, wherein the cycle temperature ranged from -80°C to 37°C;

[0096] Wash the adipose tissue three times with PBS buffer;

[0097] Digest the adipose tissue using an enzymatic digestion solution, wherein the enzymatic digestion solution consists of 0.25% trypsin / 0.02% EDTA;

[0098] Incubate overnight on a shaker at 37°C at 1000 rpm;

[0099] Adipose tissue was subjected to polar solvent extraction using 99.9% isopropyl alcohol for 12 h to remove the adipose liquid fraction;

[0100] The adipose tissue was rinsed three times with PBS buffer, wherein the pH of the PBS buffer was 7.4;

[0101] The adipose tissue was incubated again in the enzyme digestion solution for 4 h;

[0102] The adipose tissue was washed three times with PBS buffer;

[0103] The adipose tissue was incubated in a solution containing 10 mg / mL DNase enzyme, 10 mg / mL RNase enzyme, and 2000 U / mL lipase at 37°C for 6 h;

[0104] The adipose tissue was rinsed three times with PBS buffer;

[0105] The adipose tissue was treated again in 99.9% isopropanol for 8 h as the final polar solvent extraction to fully remove fat to obtain decellularized adipose tissue;

[0106] The decellularized adipose tissue was washed three times with 75% ethanol and PBS buffer respectively;

[0107] dissolving the decellularized adipose tissue using acetic acid, washing and freeze-drying to obtain the decellularized adipose tissue;

[0108] (II) Preparation of polydopamine-modified decellularized adipose tissue

[0109] The decellularized adipose tissue was placed in anhydrous ethanol and washed three times with deionized water to obtain pure decellularized adipose tissue;

[0110] The purified decellularized adipose tissue was dissolved in deionized water to obtain a 5 mg / ml decellularized adipose tissue solution;

[0111] Fully dissolving polydopamine in a decellularized adipose tissue solution (e.g., 10 ml) to obtain a mixed solution, wherein the concentration of polydopamine in the mixed solution is 0.5 mg / ml;

[0112] The pH of the mixed solution was adjusted to 8.5 using Tris buffer and stirred vigorously at room temperature for 1 h;

[0113] (III) Preparation of photothermal antibacterial materials

[0114] The mixed solution was centrifuged and washed three times with ultrapure water to obtain the photothermal antibacterial material;

[0115] (iv) dissolving the photothermal antibacterial material in deionized water to obtain a photothermal antibacterial material solution, wherein the concentration of the photothermal antibacterial material solution is 2 mg / ml;

[0116] The photothermal antibacterial material solution is freeze-dried to obtain a photothermal antibacterial scaffold.

[0117] The relevant parameters of the photothermal antibacterial stent prepared in this embodiment are as follows:

[0118] The excitation light is 808 nm and the intensity is 0.75 W / cm 2 Under the action of, the temperature can be raised by 11-13℃.

[0119] Example 3

[0120] This example is a specific preparation example of the photothermal antibacterial material of the present invention. The difference between this example and Example 2 is that: in the mixed solution, the concentration of polydopamine is 1 mg / ml.

[0121] The relevant parameters of the photothermal antibacterial stent prepared in this embodiment are as follows:

[0122] The excitation light is 808 nm and the intensity is 0.75 W / cm 2 Under the action of, the temperature can rise by 24-26℃.

[0123] Example 4

[0124] This example is a specific preparation example of the photothermal antibacterial material of the present invention. The difference between this example and Example 2 is that: in the mixed solution, the concentration of polydopamine is 2 mg / ml.

[0125] The relevant parameters of the photothermal antibacterial material prepared in this embodiment are as follows:

[0126] The excitation light is 808 nm and the intensity is 0.75 W / cm 2 Under the action of, the temperature can be raised by 30-32℃.

[0127] Example 5

[0128] This example is a specific preparation example of the photothermal antibacterial material of the present invention. The difference between this example and Example 2 is that in the mixed solution, the concentration of polydopamine is 4 mg / ml.

[0129] The relevant parameters of the photothermal antibacterial material prepared in this embodiment are as follows:

[0130] The excitation light is 808 nm and the intensity is 0.75 W / cm 2 Under the action of, the temperature can rise to 34-36℃.

[0131] Example 6

[0132] This embodiment involves the verification of various performance indicators of the photothermal antibacterial materials of Embodiments 2 to 5.

[0133] In order to avoid immune response, adipose tissue needs to be decellularized. Figure 1It can be seen that the extracted decellularized adipose tissue components were characterized after decellularization. H&E and Masson showed that a large amount of collagen matrix components existed after treatment. Oil-red staining proved that the fat components were basically removed, and the cell components on the DAPI surface were basically removed. Therefore, the decellularized adipose tissue components are non-immunogenic and will not cause immune response.

[0134] Verify the effect of different concentrations of polydopamine on photothermal antibacterial materials. Figure 2 As shown in Figure 2, as the concentration of polydopamine increases, the color of the mixed solution gradually darkens and turns black. Figure 3 As shown in the figure, with the increase of the concentration of polydopamine, the color of the photothermal antibacterial scaffold gradually deepens and turns black. Figure 4-5 As shown, with the increase of the concentration of polydopamine, the photothermal performance of the photothermal antibacterial scaffold also improves.

[0135] Depend on Figure 6 It can be seen that the photothermal antibacterial scaffold modified with 1 mg / ml polydopamine has a porous structure, and a polydopamine coating can be observed to be formed on the surface of the decellularized adipose tissue.

[0136] like Figure 7 As shown, bone marrow mesenchymal stem cells grow well on the surface of the photothermal antibacterial scaffold modified with 1 mg / ml polydopamine, and it can be observed that the bone marrow mesenchymal stem cells adhere and extend well on the scaffold surface.

[0137] like Figure 8 As shown, the coating plate method proved that the photothermal antibacterial scaffold modified with 1 mg / ml polydopamine could reduce the colony formation of Escherichia coli under the action of excitation light.

[0138] like Fig. 9 As shown, the coated plate quantitatively proved that the antibacterial rate of the photothermal antibacterial scaffold modified with 1 mg / ml polydopamine against Escherichia coli under the action of excitation light was 90.6%±4.6%.

[0139] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a photothermal antibacterial material based on decellularized adipose tissue. It is characterized in that include: The pH is adjusted to weak alkalinity, and polydopamine is modified on the surface of the decellularized adipose tissue to obtain a photothermal antibacterial material.

2. The preparation method according to claim 1, It is characterized in that Also includes: dissolving the decellularized adipose tissue in deionized water to obtain a decellularized adipose tissue solution; dissolving polydopamine in the decellularized adipose tissue solution to obtain a mixed solution; Adjusting the pH of the mixed solution to weak alkalinity; The mixed solution is separated to obtain the photothermal antibacterial material.

3. The preparation method according to claim 2, It is characterized in that Also includes: Dissolving the photothermal antibacterial material in deionized water to obtain a photothermal antibacterial material solution; The photothermal antibacterial material solution is freeze-dried to obtain a photothermal antibacterial scaffold.

4. The preparation method according to any one of claims 1 to 3, It is characterized in that The mass ratio of polydopamine to decellularized adipose tissue is 0.05 to 1:1; and / or Adjust pH to 7-9.

5. The preparation method according to claim 2, It is characterized in that The concentration of the decellularized adipose tissue solution is 1 mg / ml to 10 mg / ml; and / or The polydopamine concentration of the mixed solution is 0.5 mg / ml to 4 mg / ml; and / or The separation methods of mixed solutions include centrifugation, suction filtration and ultrafiltration.

6. The preparation method according to claim 3, It is characterized in that The concentration of the photothermal antibacterial material solution is 0.5 mg / ml to 4 mg / ml.

7. The preparation method according to claim 1, It is characterized in that include: (I) Preparation of decellularized adipose tissue The animal-derived adipose tissue was frozen and thawed in a freezing buffer containing 10 mM Tris and 5 mM EDTA using three cycles, wherein the cycle temperature ranged from -80°C to 37°C; Wash the adipose tissue three times with PBS buffer; Digest the adipose tissue using an enzymatic digestion solution, wherein the enzymatic digestion solution consists of 0.25% trypsin / 0.02% EDTA; Incubate overnight on a shaker at 37°C at 1000 rpm; Adipose tissue was subjected to polar solvent extraction using 99.9% isopropyl alcohol for 12 h to remove the adipose liquid fraction; The adipose tissue was rinsed three times with PBS buffer, wherein the pH of the PBS buffer was 7.4; The adipose tissue was incubated again in the enzyme digestion solution for 4 h; The adipose tissue was washed three times with PBS buffer; The adipose tissue was incubated in a solution containing 10 mg / mL DNase enzyme, 10 mg / mL RNase enzyme, and 2000 U / mL lipase at 37°C for 6 h; The adipose tissue was rinsed three times with PBS buffer; The adipose tissue was treated again in 99.9% isopropanol for 8 h as the final polar solvent extraction to fully remove fat to obtain decellularized adipose tissue; The decellularized adipose tissue was washed three times with 75% ethanol and PBS buffer respectively; The decellularized adipose tissue was dissolved using acetic acid, washed, adjusted to a pH value of 7.4, and then freeze-dried to obtain the decellularized adipose tissue; (II) Preparation of polydopamine-modified decellularized adipose tissue The decellularized adipose tissue was placed in anhydrous ethanol and washed three times with deionized water to obtain pure decellularized adipose tissue; dissolving purified decellularized adipose tissue in deionized water to obtain a decellularized adipose tissue solution; Fully dissolving polydopamine in the decellularized adipose tissue solution to obtain a mixed solution, wherein the concentration of polydopamine in the mixed solution is 0.5 mg / ml to 4 mg / ml; The pH of the mixed solution was adjusted to 7-9 using Tris buffer and stirred vigorously at room temperature for 1 h; (III) Preparation of photothermal antibacterial materials The mixed solution was centrifuged and washed three times with ultrapure water to obtain the photothermal antibacterial material.

8. The preparation method according to claim 6, It is characterized in that Also includes: (IV) Preparation of photothermal antibacterial scaffolds Dissolving the photothermal antibacterial material in deionized water to obtain a photothermal antibacterial material solution, wherein the concentration of the photothermal antibacterial material solution is 0.5 mg / ml to 4 mg / ml; The photothermal antibacterial material solution is freeze-dried to obtain a photothermal antibacterial scaffold.

9. A photothermal antibacterial material based on decellularized adipose tissue, prepared by the preparation method according to any one of claims 1 to 7. It is characterized in that Includes decellularized adipose tissue and polydopamine.

10. The photothermal antibacterial material according to claim 8, It is characterized in that The mass ratio of polydopamine to decellularized adipose tissue is 0.05-1:

1.

11. Use of the photothermal antibacterial material prepared by the preparation method according to any one of claims 1 to 7 or the photothermal antibacterial material according to any one of claims 8 to 9 in the preparation of bioremediation materials.

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