Hydrogel with oxygen carrying and photo-thermal functions and preparation method thereof

Abstract

The invention discloses a preparation method of hydrogel with oxygen carrying and photo-thermal functions. The preparation method comprises the following steps: 1, dissolving chitosan, adding dopamine and hemoglobin, continuously stirring, and freeze-drying to obtain a hydrogel precursor; 2, dissolving the hydrogel precursor in deionized water or a phosphate buffer solution, adding dialdehyde polyethylene glycol, uniformly stirring, and standing to obtain hydrogel; the invention also discloses a hydrogel with oxygen carrying and photo-thermal functions. Chitosan, dopamine and hemoglobin are adopted as raw materials to prepare a hydrogel precursor, reversible imine bonds are formed through a Schiff base reaction between polydopamine and chitosan, covalent bonds are cross-linked through a Michael addition reaction, and the hydrogel precursor is synthesized through the complexing effect of polydopamine-hemoglobin; and then mixing the hydrogel precursor with dialdehyde polyethylene glycol to form the hydrogel applied to diabetes wound treatment. The invention is applicable to the technical field of polymer materials.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of high polymer materials, and particularly relates to a hydrogel with oxygen-carrying and photothermal functions and a preparation method thereof. BACKGROUND

[0002] Diabetes has evolved into a global health crisis, and chronic hyperglycemia can cause various body injuries. Among its complications, chronic wound healing disorders are particularly serious, significantly affecting the quality of life of patients. The main obstacle to chronic wound healing is the disorder of angiogenesis, which is closely related to persistent inflammatory response. Hyperglycemia and advanced glycation end products (AGEs) maintain the pro-inflammatory microenvironment by inhibiting the polarization of M2 macrophages. A large number of studies have shown that timely promotion of the polarization of macrophages to the M2 phenotype can reduce inflammation, stimulate angiogenesis and accelerate wound healing.

[0003] Hydrogels have become promising wound repair materials due to their ability to encapsulate bioactive substances and provide a moist wound healing microenvironment. In hydrogel materials, chitosan (CS) is widely used due to its excellent biocompatibility and biodegradability.

[0004] Hemoglobin (Hb) as a natural oxygen transport protein has become an ideal oxygen supply material candidate due to its high oxygen binding affinity and excellent biocompatibility. However, there are major challenges in integrating hemoglobin into chitosan hydrogels: the weak physical interaction between chitosan and hemoglobin often leads to rapid leakage of encapsulated hemoglobin, severely weakening its oxygen-carrying capacity. Overcoming this limitation is crucial for realizing the efficient treatment of oxygen-carrying hydrogels in diabetic wound repair. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art and provide a preparation method of a hydrogel with oxygen-carrying and photothermal functions. The preparation method uses chitosan, dopamine and hemoglobin as raw materials to prepare a hydrogel precursor, utilizes the Schiff base reaction between polydopamine and chitosan to form a reversible imine bond, the Michael addition reaction to cross-link the covalent bond, and the polydopamine-hemoglobin complexation to synthesize the hydrogel precursor; then the hydrogel precursor is mixed with dialdehyde-based polyethylene glycol to form a hydrogel, solving the problem of weak physical interaction between chitosan and hemoglobin, which easily leads to rapid leakage of encapsulated hemoglobin.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is: a preparation method of a hydrogel with oxygen-carrying and photothermal functions, characterized in that the preparation method comprises the following steps:

[0007] Step one, dissolve chitosan, add dopamine and hemoglobin, and continuously stir, then freeze-dry to obtain a hydrogel precursor;

[0008] Step 2: dissolving the hydrogel precursor obtained in step 1 in deionized water or phosphate buffer, then adding dialdehyde polyethylene glycol, stirring evenly and letting it stand to obtain a hydrogel.

[0009] The present invention combines hemoglobin in the hydrogel and utilizes the high oxygen binding affinity and excellent biocompatibility of hemoglobin to enable the hydrogel to have oxygen-carrying function, thereby reducing the probability of chronic hypoxia in diabetic wounds, thereby promoting angiogenesis, increasing the angiogenic activity of macrophages, and ultimately promoting inflammation resolution and tissue regeneration. However, due to the weak physical interaction between chitosan and hemoglobin, it is easy to cause rapid leakage of encapsulated hemoglobin, seriously weakening its oxygen-carrying capacity; therefore, the present invention uses dopamine as a raw material and utilizes the powerful complexing ability of polydopamine to effectively fix hemoglobin and significantly enhance its stability in complex physiological environments. At the same time, polydopamine has excellent photothermal conversion function, which can promote endothelial cell proliferation and new blood vessel formation, further enhancing the hydrogel's promoting effect on chronic wound healing.

[0010] The above-mentioned method for preparing a hydrogel with oxygen-carrying and photothermal functions is characterized in that the deacetylation degree of the chitosan in step 1 is not less than 90%, and the viscosity is 50 mPa·s to 500 mPa·s.

[0011] The above-mentioned method for preparing a hydrogel with oxygen-carrying and photothermal functions is characterized in that the deacetylation degree of the chitosan is not less than 95% and the viscosity is 100 mPa·s to 200 mPa·s.

[0012] The invention can increase the cross-linking points by controlling the deacetylation degree of chitosan to be not less than 95%.

[0013] The above-mentioned method for preparing a hydrogel with oxygen-carrying and photothermal functions is characterized in that the mass ratio of dopamine to hemoglobin in step 1 is 1 to 2:1.

[0014] The present invention controls the mass ratio of dopamine to hemoglobin, thereby enabling hemoglobin to be loaded to the maximum extent under the premise of hydrogel formation.

[0015] The above-mentioned method for preparing a hydrogel with oxygen-carrying and photothermal functions is characterized in that the stirring temperature in step 1 is 25°C to 40°C, and the stirring time is more than 24 hours.

[0016] The present invention is used to ensure that dopamine is fully oxidized and polymerized by controlling the stirring temperature and time.

[0017] The above-mentioned method for preparing a hydrogel with oxygen-carrying and photothermal functions is characterized in that the phosphate buffer in step 2 contains probiotic exosomes, and the probiotic exosomes are lactobacillus-derived exosomes.

[0018] The present invention adds probiotic exosomes and utilizes Schiff base crosslinking to form a final probiotic exosome hydrogel, thereby achieving sustained release of probiotic exosomes and continuously releasing probiotic exosomes, promoting macrophage polarization to the M2 phenotype; at the same time, hemoglobin oxygen supply and local photothermal stimulation synergistically activate vascular endothelial cells, jointly enhance angiogenesis and accelerate wound healing, thereby achieving the treatment of diabetic wounds.

[0019] The present invention uses Lactobacillus-derived exosomes, which can enhance angiogenesis and accelerate wound healing, and eliminate inflammation by inducing macrophage M2 polarization.

[0020] The above-mentioned method for preparing a hydrogel with oxygen-carrying and photothermal functions is characterized in that the molar mass of the dialdehyde polyethylene glycol in step 2 is 1000 g / mol to 10000 g / mol.

[0021] The above-mentioned method for preparing a hydrogel with oxygen-carrying and photothermal functions is characterized in that the molar mass of the dialdehyde polyethylene glycol is 2000 g / mol.

[0022] The present invention also discloses a hydrogel with oxygen-carrying and photothermal functions, characterized in that the hydrogel is prepared using the above-mentioned preparation method.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] 1. The present invention uses chitosan, dopamine and hemoglobin as raw materials to prepare a hydrogel precursor, and utilizes the Schiff base reaction between polydopamine and chitosan to form a reversible imine bond, the Michael addition reaction to crosslink the covalent bonds, and the polydopamine-hemoglobin complexation to synthesize the hydrogel precursor; the hydrogel precursor is then mixed with dialdehyde polyethylene glycol to form a hydrogel; the hydrogel has photothermal effect, oxygen-carrying function and strong tissue adhesion, and can provide a moist wound healing microenvironment, combine with hemoglobin oxygen supply, cooperate with local photothermal stimulation to synergistically activate vascular endothelial cells, jointly enhance angiogenesis and accelerate wound healing, and achieve the treatment of diabetic wounds.

[0025] 2. The hydrogel network of the present invention has dynamic Schiff base bonds, which give it self-healing properties, allowing the hydrogel to recover its own structure after mechanical damage.

[0026] 3. The raw materials used in the present invention have good biocompatibility and are degradable, and the degradation products are non-toxic. No organic waste liquid is generated during the preparation process, which is green and environmentally friendly.

[0027] 4. The hydrogel of the present application can be used for the treatment of diabetic wounds by injection method, and can deliver probiotic exosomes and oxygen to the diabetic wounds; wherein the probiotic exosomes can be replaced by other active substances to deliver other active substances to achieve the treatment of other diseases.

[0028] The technical solutions of the present application are described in further detail below with reference to the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The microscopic image of PCPH prepared in Example 1 was taken by fluorescence microscope.

[0030] Figure 2 The microscopic morphology images of PCPH prepared in Example 1 at different magnifications.

[0031] Figure 3 The microscopic morphology images of PCPH@LExos prepared in Example 2 at different magnifications.

[0032] Figure 4 The appearance morphology image of CS-PDA-Hb prepared in Example 2.

[0033] Figure 5 The appearance morphology image of PCPH@LExos prepared in Example 2.

[0034] Figure 6 The temperature change graph of PBS and PCPH@LExos prepared in Example 2 under near-infrared light irradiation.

[0035] Figure 7 The release test result graph of LExos in PCPH@LExos prepared in Example 2.

[0036] Figure 8 The oxygen release amount change graph of Hb and PBS used in Example 1, PCPH@LExos prepared in Example 2, PCPH@LExos after near-infrared light irradiation, and PCP@LExos prepared in Comparative Example 1.

[0037] Figure 9 The microscopic image of PCPH@LExos-PKH26 prepared in Example 3 was taken by fluorescence microscope.

[0038] Figure 10 The content graph of pro-inflammatory factor TNF-α of macrophages treated with PBS used in Example 1, PCP prepared in Comparative Example 2, PCPH prepared in Example 1, PCPH@LExos prepared in Example 2, and PCPH@LExos after near-infrared light irradiation, respectively.

[0039] Figure 11 This is a graph showing the content of the anti-inflammatory factor IL-10 in macrophages after being treated with PBS used in Example 1, PCP prepared in Comparative Example 2, PCPH prepared in Example 1, PCPH@LExos prepared in Example 2, and PCPH@LExos irradiated with near-infrared light.

[0040] Figure 12 Graphs showing the healing status of full-thickness wounds in diabetic mice treated with PBS used in Example 1, PCP prepared in Comparative Example 2, PCPH prepared in Example 1, PCPH@LExos prepared in Example 2, and PCPH@LExos after near-infrared light irradiation. DETAILED DESCRIPTION

[0041] The hemoglobin used in Examples 1 to 7, Comparative Example 1 and Comparative Example 2 was all derived from bovine serum, and the pH value of the phosphate buffer used was 7.4.

[0042] Example 1

[0043] The preparation method of this embodiment comprises the following steps:

[0044] Step 1: 300 mg of chitosan (CS) and 95 μL of acetic acid were dissolved in 15 mL of deionized water under stirring, and then 125 mg of dopamine (DA) and 125 mg of hemoglobin (Hb) were added. The mixture was stirred and reacted at 40°C for 24 hours. After freeze-drying, a hydrogel precursor (CS-PDA-Hb) was obtained and stored at 4°C. The chitosan had a deacetylation degree of not less than 95% and a viscosity of 100 mPa·s to 200 mPa·s.

[0045] Step 2: Dissolve 37 mg of CS-PDA-Hb obtained in step 1 in 1 mL of deionized water, then add 100 mg of dialdehyde polyethylene glycol (CHO-PEG-CHO) with a molar mass of 2000 g / mol, stir evenly, and let stand for 5 minutes. Rinse with deionized water to obtain hydrogel PEG-CS-PDA-Hb, recorded as PCPH.

[0046] Example 2

[0047] The preparation method of this embodiment comprises the following steps:

[0048] Step 1: Dissolve 300 mg of CS and 95 μL of acetic acid in 15 mL of deionized water under stirring, then add 125 mg of DA and 125 mg of Hb, stir and react at 40 °C for 24 h, and freeze-dry to obtain Figure 4The CS-PDA-Hb is stored at 4°C; the chitosan has a deacetylation degree of not less than 95% and a viscosity of 100 mPa·s to 200 mPa·s;

[0049] Step 2: 37 mg of CS-PDA-Hb was dissolved in 1 mL of phosphate buffered saline (PBS) containing probiotic exosomes (LExos), and then 100 mg of dialdehyde polyethylene glycol (CHO-PEG-CHO) was added and stirred evenly. The mixture was allowed to stand for 5 minutes and then rinsed with PBS to obtain the following: Figure 5 The hydrogel PEG-CS-PDA-Hb@LExos shown is denoted as PCPH@LExos.

[0050] The PCPH prepared in Example 1 and the PCPH@LExos prepared in Example 2 were freeze-dried and then photographed using a scanning electron microscope. Figure 2 and Figure 3 As shown in Figure 2, both PCPH and PCPH@LExos exhibit a typical three-dimensional porous structure of hydrogels. After magnification, it can be seen that the surface of PCPH is smooth, while the surface of PCPH@LExos contains 150nm to 200nm granular substances, indicating that LExos has been successfully loaded into the hydrogel. PBS with a pH value of 7.4 and PCPH@LExos were irradiated under 808nm near-infrared light. The results are shown in Figure 2. Figure 6 As shown in the figure, the temperature of PCPH@LExos increased significantly by about 10°C after 300s of illumination, indicating that PCPH@LExos has good photothermal performance.

[0051] The PCPH@LExos prepared in Example 2 was tested for its sustained release ability. The results are as follows: Figure 7 As shown, about 80% of LExos can be released in 3 days, indicating that the PCPH@LExos prepared in the present invention can achieve long-term sustained release of LExos.

[0052] Comparative Example 1

[0053] The preparation method of this comparative example is:

[0054] Step 1: 300 mg of CS and 95 μL of acetic acid were dissolved in 15 mL of deionized water under stirring, and then 125 mg of DA was added. The mixture was stirred at 40°C for 24 hours to obtain CS-PDA, which was then stored at 4°C. The chitosan had a deacetylation degree of not less than 95% and a viscosity of 100 mPa·s to 200 mPa·s.

[0055] Step two, 37 mg of CS-PDA obtained in step one was dissolved in 1 mL of PBS containing LExos, then 100 mg of CHO-PEG-CHO with a molecular weight of 2000 g / mol was added, and after standing for 5 min, PBS was used for flushing to obtain the hydrogel PEG-CS-PDA@LExos, which was recorded as PCPH@LExos.

[0056] The oxygen release amount of Hb and PBS used in Example 1, PCPH@LExos prepared in Comparative Example 1, PCPH@LExos prepared in Example 2 and PCPH@LExos after near-infrared light irradiation (PCPH@LExos+NIR) were tested respectively, and the results are shown in Figure 8 As shown in the table, PCPH@LExos and PCPH@LExos+NIR containing Hb both continuously release oxygen, have oxygen carrying capacity, and light almost does not affect the oxygen carrying performance of the hydrogel.

[0057] Example 3

[0058] The difference between this example and Example 1 is that: step two, 37 mg of CS-PDA-Hb was dissolved in 1 mL of PBS containing PKH26 labeled LExos, then 100 mg of dialdehyde polyethylene glycol (CHO-PEG-CHO) was added and stirred uniformly, and after standing for 5 min, PBS was used for flushing to obtain the hydrogel PEG-CS-PDA-Hb@LExos-PKH26, which was recorded as PCPH@LExos-PKH26.

[0059] PCPH prepared in Example 1 and PCPH@LExos-PKH26 prepared in this example were freeze-dried and observed by fluorescence microscope, as shown in Figure 1 and Figure 9 As shown in the table, PCPH has no fluorescence, while PCPH@LExos-PKH26 has a large amount of fluorescence, indicating that LExos has been successfully loaded.

[0060] Comparative Example 2

[0061] The preparation method of this comparative example is:

[0062] Step one, 300 mg of CS and 95 μL of acetic acid were dissolved in 15 mL of deionized water under stirring, then 125 mg of DA was added, and the reaction was continuously stirred at 40℃ for 24 h to obtain CS-PDA, which was stored at 4℃ environment; the deacetylation degree of the chitosan is not less than 95%, and the viscosity is 100 mPa·s-200 mPa·s

[0063] Step 2: Dissolve 37 mg of CS-PDA obtained in step 1 in 1 mL of deionized water, then add 100 mg of CHO-PEG-CHO with a molar mass of 2000 g / mol. After standing for 5 minutes, rinse with deionized water to obtain hydrogel PEG-CS-PDA, which is recorded as PCP.

[0064] After stimulating Raw264.7 mouse macrophages with LPS (bacterial lipopolysaccharide), the macrophages were treated with PBS used in Example 1, PCP prepared in Comparative Example 2, PCPH prepared in Example 1, PCPH@LExos prepared in Example 2, and PCPH@LExos irradiated with near-infrared light (PCPH@LExos+NIR), and the changes in the levels of the pro-inflammatory factor TNF-α and the anti-inflammatory factor IL-10 were detected. The results are shown in Figure 2. Figure 10 and Figure 11 As shown in the figure, compared with PBS, the pro-inflammatory factor TNF-α in macrophages treated with PCP, PCPH, PCPH@LExos, and PCPH@LExos+NIR decreased, with PCPH@LExos and PCPH@LExos+NIR carrying LExos showing a significant decrease. Compared with PBS, the anti-inflammatory factor IL-10 in macrophages treated with PCP, PCPH, PCPH@LExos, and PCPH@LExos+NIR increased, with PCPH@LExos and PCPH@LExos+NIR carrying LExos showing a more pronounced increase. This suggests that PCPH@LExos and PCPH@LExos+NIR can significantly inhibit the pro-inflammatory factor TNF-α and promote the anti-inflammatory factor IL-10, demonstrating excellent anti-inflammatory capabilities, which are further enhanced after illumination.

[0065] A full-thickness wound model was established in diabetic mice. The wounds were treated with PBS used in Example 1, PCP prepared in Comparative Example 2, PCPH prepared in Example 1, PCPH@LExos prepared in Example 2, and PCPH@LExos irradiated with near-infrared light (PCPH@LExos+NIR). The wound healing conditions at different times were shown in Figure 2. Figure 12 As shown, compared with PBS and PCP, wound healing was significantly accelerated after treatment with PCPH, PCPH@LExos, and PCPH@LExos+NIR, especially with PCPH@LExos and PCPH@LExos+NIR loaded with LExos. This indicates that the hydrogels prepared by this invention promote the healing of diabetic wounds, and that the hydrogels loaded with LExos exhibit even better healing effects.

[0066] Example 4

[0067] The difference between this embodiment and embodiment 2 is that the temperature of the stirring reaction in step 1 is 25°C.

[0068] After testing, it was found that the PCPH@LExos prepared in this example could accelerate wound healing in diabetic mice.

[0069] Example 5

[0070] The difference between this embodiment and embodiment 2 is that the amount of DA added in step 1 is 250 mg.

[0071] After testing, it was found that the PCPH@LExos prepared in this example could accelerate wound healing in diabetic mice.

[0072] Example 6

[0073] The preparation method of this embodiment comprises the following steps:

[0074] Step 1: 300 mg of CS and 95 μL of acetic acid were dissolved in 15 mL of deionized water under stirring, and then 200 mg of DA and 125 mg of Hb were added. The mixture was stirred and reacted at 30°C for 24 hours. After freeze-drying, CS-PDA-Hb was obtained and stored at 4°C. The chitosan had a degree of deacetylation of not less than 90% and a viscosity of 50 mPa·s to 500 mPa·s.

[0075] Step 2: Dissolve 37 mg of CS-PDA-Hb obtained in step 1 in 1 mL of deionized water, then add 200 mg of CHO-PEG-CHO with a molar mass of 1000 g / mol, stir evenly, and let stand for 5 minutes. Rinse with deionized water to obtain a hydrogel PEG-CS-PDA-Hb, which is recorded as PCPH.

[0076] After testing, it was found that the PCPH prepared in this example could accelerate wound healing in diabetic mice.

[0077] Example 7

[0078] The difference between this embodiment and embodiment 6 is that: 20 mg of CHO-PEG-CHO with a molar mass of 10000 g / mol is added in step 2.

[0079] After testing, it was found that the PCPH prepared in this example could accelerate wound healing in diabetic mice.

[0080] 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 transformation made to the above embodiment based on the technical essence of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a hydrogel with oxygen-carrying and photothermal functions, characterized in that: The preparation method comprises the following steps: Step 1: dissolving chitosan, adding dopamine and hemoglobin, and continuously stirring, and freeze-drying to obtain a hydrogel precursor; Step 2: dissolving the hydrogel precursor obtained in step 1 in deionized water or phosphate buffer, then adding dialdehyde polyethylene glycol, stirring evenly and letting it stand to obtain a hydrogel.

2. The method for preparing a hydrogel with oxygen-carrying and photothermal functions according to claim 1, characterized in that: The chitosan in step 1 has a deacetylation degree of not less than 90% and a viscosity of 50 mPa·s to 500 mPa·s.

3. The method for preparing a hydrogel with oxygen-carrying and photothermal functions according to claim 2, characterized in that: The chitosan has a deacetylation degree of not less than 95% and a viscosity of 100 mPa·s to 200 mPa·s.

4. The method for preparing a hydrogel with oxygen-carrying and photothermal functions according to claim 1, characterized in that: The mass ratio of dopamine to hemoglobin in step 1 is 1 to 2:

1.

5. The method for preparing a hydrogel with oxygen-carrying and photothermal functions according to claim 1, characterized in that: The stirring temperature in step 1 is 25° C. to 40° C., and the stirring time is more than 24 hours.

6. The method for preparing a hydrogel with oxygen-carrying and photothermal functions according to claim 1, characterized in that: The phosphate buffer in step 2 contains probiotic exosomes, and the probiotic exosomes are lactobacillus-derived exosomes.

7. The method for preparing a hydrogel with oxygen-carrying and photothermal functions according to claim 1, characterized in that: The molar mass of the dialdehyde polyethylene glycol in step 2 is 1000 g / mol to 10000 g / mol.

8. The method for preparing a hydrogel with oxygen-carrying and photothermal functions according to claim 7, characterized in that: The molar mass of the bisaldehyde polyethylene glycol is 2000 g / mol.

9. A hydrogel with oxygen-carrying and photothermal functions, characterized in that: The hydrogel is prepared by the preparation method described in any one of claims 1 to 8.

Images