Injectable oxygen-carrying and drug-carrying microbubble composite hydrogel with immunoregulation function and preparation method of injectable oxygen-carrying and drug-carrying microbubble composite hydrogel

Abstract

The invention discloses injectable oxygen-carrying and medicine-carrying microbubble composite hydrogel with immunoregulation performance and a preparation method of the injectable oxygen-carrying and medicine-carrying microbubble composite hydrogel, and belongs to the field of tissue damage treatment. The preparation method comprises the following steps: carrying out a reaction on hyaluronic acid HA and dipalmitoyl phosphatidyl ethanolamine DPPE to construct a microbubble monomer DPPE-HA; the preparation method comprises the following steps: fully dissolving DPPC (dipalmitoyl phosphatidylcholine), DPPE-HA (dipalmitoyl phosphatidylcholine) and a therapeutic drug into a solvent, introducing oxygen and carrying out mechanical oscillation to obtain oxygen-carrying and drug-carrying microbubbles; the oxygen-carrying and drug-carrying microbubbles are uniformly doped into a vinyl modified natural polymer solution, and the oxygen-carrying and drug-carrying microbubble composite hydrogel is formed through photo-crosslinking. The obtained injectable microbubble hydrogel can be used for treatment of different tissue injuries, including wound repair and burn treatment, osteochondral injury repair, cardiovascular disease treatment and nerve injury repair, in addition, the injectable microbubble hydrogel can also be applied to the fields of cosmetic plastic surgery, skin regeneration and the like, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of tissue damage treatment, specifically relating to an injectable oxygen- and drug-carrying microbubble composite hydrogel with immunomodulatory properties and its preparation method. Background Technology

[0002] The inflammatory microenvironment plays a crucial role in the occurrence and development of many diseases, especially those related to immune system dysregulation, such as rheumatoid arthritis (RA). RA is a chronic, systemic autoimmune disease. During the development of RA, the immune system mistakenly attacks the synovial tissue of the joints, leading to synovitis. A large number of inflammatory factors secreted by immune cells in the synovial tissue further erode the articular cartilage and bone tissue, resulting in joint damage and loss of joint function. Currently, drug therapy is the main clinical treatment for RA and its complications, primarily including nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, antirheumatic drugs (ARDDs), and targeted biologics. However, adverse reactions from long-term administration, such as osteonecrosis, infection, and liver and kidney dysfunction, make these drugs particularly cautious in clinical treatment selection. Simultaneously, the rapid metabolism of drugs in the body significantly reduces drug efficiency, thus requiring increased dosing frequency, which further increases the physical and economic burden on patients. To reduce the side effects of long-term administration and address the drawbacks of rapid drug metabolism, microbubble drug delivery technology, which loads drugs into microbubbles to improve drug targeting, stability, and efficacy, shows great potential in areas such as RA treatment.

[0003] Microbubbles are tiny gaseous structures, typically between 1 and 10 micrometers in diameter, usually composed of biocompatible materials (such as phospholipids, lipids, and polymers). Drugs can be loaded into microbubbles through various methods, including adsorption, encapsulation, or copolymerization. Microbubble drug delivery technology offers several advantages: 1) Drug release can be controlled by stimulating microbubbles physically or chemically; 2) Microbubbles can be surface-modified to bind specific target molecules (such as antibodies, peptides, and glycosyl groups), delivering drugs directly to diseased tissues or cells, thereby improving therapeutic efficacy and reducing side effects on healthy tissues; 3) The microbubble structure protects drugs, preventing premature degradation or inactivation in vivo, thus improving drug stability and bioavailability; 4) Microbubble delivery systems can optimize drug penetration and distribution in vivo by adjusting parameters such as microbubble size, surface properties, and drug loading, thereby improving therapeutic efficacy. However, existing microbubble drug delivery technologies also have intractable drawbacks, such as the fragility of the microbubble structure, making it prone to rupture or degradation in vivo or during preparation. Furthermore, although microbubble drug delivery systems employ various methods to control drug release (such as external stimuli like ultrasound, temperature, and pH), precisely controlling the release rate remains a challenge. Excessively rapid drug release may accelerate drug metabolism, ultimately impacting therapeutic efficacy.

[0004] Therefore, the key to material design is to leverage the advantages of microbubble drug delivery technology while addressing the problems that microbubbles present in drug delivery. Summary of the Invention

[0005] To address the above issues, this invention provides an injectable oxygen- and drug-carrying microbubble composite hydrogel with immunomodulatory properties and its preparation method. By controlling the microbubbles and hydrogel components of the microbubble hydrogel, immunomodulatory properties are achieved, ultimately leading to the repair of damaged tissues. Simultaneously, this material also possesses injectability, ultrasound-responsive drug release properties, and controllable mechanical properties. Specifically, the technical solution of this invention addresses the shortcomings of microbubbles in drug delivery by constructing microbubble hydrogels. Immunomodulatory drug molecules are loaded into oxygen-carrying microbubbles, and then the microbubbles are incorporated into the hydrogel, endowing the microbubble hydrogel with immunomodulatory properties, ultimately achieving tissue repair. The microbubble hydrogel combines the characteristics of microbubbles with the biocompatibility, drug loading capacity, and long-lasting release advantages of hydrogels, giving it several significant advantages in the field of drug delivery. Especially in terms of increasing drug loading, prolonging drug release time, improving biocompatibility, and addressing complex treatment needs, the microbubble hydrogel shows superior potential compared to traditional microbubble delivery systems. Furthermore, the microbubble hydrogel developed in this invention is injectable and can be flexibly processed to meet the specific needs of different disease treatments.

[0006] According to a first aspect of the present invention, a method for preparing an injectable oxygen- and drug-carrying microbubble composite hydrogel with immunomodulatory properties is provided, the specific process of which is as follows:

[0007] Step 1: Construct the microbubble monomer DPPE-HA by reacting hyaluronic acid (HA) and dipalmitoylphosphatidylethanolamine (DPPE);

[0008] Step 2: Obtain oxygen-carrying drug-carrying microbubbles by fully dissolving dipalmitoylphosphatidylcholine (DPPC), DPPE-HA, and the therapeutic drug in a solvent, introducing oxygen, and accompanied by mechanical vibration.

[0009] Step 3: The oxygen-carrying and drug-carrying microbubbles are uniformly incorporated into a vinyl-modified natural polymer solution, and an injectable oxygen-carrying and drug-carrying microbubble composite hydrogel is formed through photocrosslinking.

[0010] Furthermore, step 1 specifically includes:

[0011] Hyaluronic acid (HA) was weighed and dissolved in ultrapure water, and activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to adjust the pH of the solution. Dipalmitoylphosphatidylethanolamine (DPPE) was added to the activated hyaluronic acid solution to carry out the reaction. After the reaction was completed, the unreacted reagents were removed, and the microbubble monomer DPPE-HA was obtained by freeze drying.

[0012] Further, in step 1, after activation with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), the pH of the solution is adjusted to 4-6;

[0013] Dipalmitoylphosphatidylethanolamine (DPPE) is added to an activated hyaluronic acid solution for reaction. The pH of the reaction system is controlled at 8-10, the temperature at 30-50℃, and the reaction time at 6-24h. After the reaction is completed, the unreacted reagent is removed by dialysis for 3-5 days.

[0014] Further, in step 1, the mass ratio of hyaluronic acid (HA) to dipalmitoylphosphatidylethanolamine (DPPE) is 14:1, the pH of the hyaluronic acid solution is 4, the pH of the reaction system is 8.6, the temperature is 37°C, and the reaction time is 24 hours.

[0015] Furthermore, step 2 specifically includes:

[0016] Dipalmitoylphosphatidylcholine (DPPC), the microbubble monomer DPPE-HA, and the therapeutic drug are dissolved in a solvent, stirred in a water bath, and oxygen is introduced until the emulsion bubbles. Then, mechanical vibration is used to promote microbubble formation. Finally, the emulsion is diluted with PBS to obtain the oxygen-carrying and drug-carrying microbubbles.

[0017] Furthermore, in step 2, the therapeutic drug is methotrexate (MTX).

[0018] Furthermore, step 2 also includes a component that promotes the fluidity of the microbubble membrane, such as glycerol, cholesterol, or polyethylene glycol 200.

[0019] Furthermore, in step 2, the reaction system temperature is controlled at 25-50℃, the stirring time is 10-60 min, the frequency of mechanical vibration is set to 60-100 Hz, and the time is 1-5 min.

[0020] Further, in step 2, the mass ratio of dipalmitoylphosphatidylcholine (DPPC), the microbubble monomer DPPE-HA, and methotrexate is 2:5:1, the reaction system temperature is controlled at 40℃, the stirring time is 30 min, and the mechanical vibration frequency is set to 60 Hz for 1 min.

[0021] Furthermore, step 3 specifically includes:

[0022] Step 31: Dissolve gelatin in deionized water, then add methacrylic anhydride to the gelatin solution, adjust the pH of the solution, collect the reaction product after a certain reaction time, dialyze, filter and freeze dry to obtain GelMA;

[0023] Step 32: GelMA is dissolved in PBS buffer containing Irgacure2959 to obtain a hydrogel. Then, the oxygen-carrying drug-carrying microbubbles are uniformly incorporated into the hydrogel and transferred to a mold. After photocuring under ultraviolet light, the mold is removed to obtain an injectable oxygen-carrying drug-carrying microbubble composite hydrogel.

[0024] Furthermore, in step 31, the solution pH is adjusted to 8-10, the reaction temperature is set to 40-60℃, and the reaction product is collected after 3-12 hours of reaction.

[0025] Furthermore, in step 31, the solution pH is adjusted to 8.0, the reaction temperature is set to 50°C, and the reaction time is 3 hours.

[0026] Further, the reaction conditions in step 32 are as follows: the concentration of GelMA dissolved in PBS buffer containing Irgacure2959 is 1-10 w / v, the content of Irgacure2959 is 0.1-0.5 w / v, the volume ratio of the oxygen-carrying drug-carrying microbubbles to the volume of the GelMA solution is 0-1 / 4, and the UV irradiation time is 30-180 s.

[0027] Further, the reaction conditions in step 32 are as follows: the concentration of GelMA dissolved in PBS buffer containing Irgacure2959 is 10 w / v, the content of Irgacure2959 is 0.5 w / v, the volume ratio of the oxygen-carrying drug-carrying microbubbles to the volume of the GelMA solution is 1 / 6, and the irradiation time of the ultraviolet light source is 30 s.

[0028] According to a second aspect of the present invention, an injectable oxygen- and drug-carrying microbubble composite hydrogel prepared by the preparation method described in any of the above aspects is provided.

[0029] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0030] 1. This invention provides a method for preparing an oxygen- and drug-carrying microbubble hydrogel with immunomodulatory properties. This method is universally applicable; customized treatments can be achieved by adjusting the preparation process parameters according to the specific tissue damage.

[0031] 2. This invention provides a method for preparing an oxygen-carrying and drug-carrying microbubble hydrogel with immunomodulatory properties. Compared with traditional microbubbles, this composite hydrogel has better biocompatibility. As a carrier for microbubbles, the hydrogel can protect the microbubbles and ensure their long-term stability, thus prolonging the drug release time and achieving long-acting drug release.

[0032] 3. The microbubble hydrogel prepared by this invention can slow down the release rate of the drug loaded in the microbubbles, avoid drug burst release, increase drug utilization, reduce the frequency of administration, and thus reduce the toxic side effects of the drug.

[0033] 4. The microbubble hydrogel prepared by this invention has good injectability, can realize minimally invasive treatment, and can adapt to various tissue damage shapes.

[0034] 5. The microbubble hydrogel prepared by this invention has ultraviolet light crosslinking characteristics, and the mechanical properties of the microbubble hydrogel can be controlled by adjusting the ultraviolet light irradiation time.

[0035] 6. The drug release of the microbubble hydrogel prepared by this invention is ultrasonically responsive, and the drug release can be controlled by adjusting the ultrasonic parameters. Attached Figure Description

[0036] Figure 1 The Fourier transform infrared spectrum of DPPE-HA synthesized according to an embodiment of the present invention is shown.

[0037] Figure 2 These are optical microscope images of oxygen-carrying drug-loaded microbubbles according to an embodiment of the present invention;

[0038] Figure 3 This is a particle size distribution image of microbubbles according to an embodiment of the present invention;

[0039] Figure 4 This is a Zeta potential diagram of microbubbles according to an embodiment of the present invention;

[0040] Figure 5 This is a scanning electron microscope image of a GelMA hydrogel and a microbubble composite hydrogel according to an embodiment of the present invention;

[0041] Figure 6 The Fourier transform infrared spectrum of a microbubble hydrogel according to an embodiment of the present invention;

[0042] Figure 7 These are images showing the release rate of MTX, a drug loaded in a microbubble hydrogel according to an embodiment of the present invention, under different conditions.

[0043] Figure 8 These are images showing the release rate of oxygen carried by a microbubble hydrogel under different ultrasonic conditions according to an embodiment of the present invention.

[0044] Figure 9 This study measured the proliferation of RAW264.7 macrophages after culture in different hydrogels.

[0045] Figure 10 The determination of intracellular reactive oxygen species (ROS) in RAW264.7 macrophages after culture in different hydrogels;

[0046] Figure 11 This study measured the expression levels of intracellular pro-inflammatory cytokines (CD86) and anti-inflammatory cytokines (CD206) in RAW264.7 macrophages after culture in different hydrogels. Detailed Implementation

[0047] The following specific implementation examples further illustrate the embodiments of the present invention. These implementation examples are merely illustrative and not intended to limit the scope of the invention. Furthermore, various equivalent modifications made to the present invention by those skilled in the art after reading the teachings of this invention also fall within the scope claimed in the claims of this application.

[0048] This invention provides an injectable oxygen- and drug-carrying microbubble composite hydrogel with immunomodulatory properties and its preparation method. The method involves constructing a microbubble precursor DPPE-HA by reacting HA and dipalmitoylphosphatidylethanolamine (DPPE); then, by fully dissolving dipalmitoylphosphatidylcholine (DPPC), DPPE-HA, and a therapeutic drug in a solvent, introducing oxygen, and accompanied by mechanical vibration to obtain oxygen- and drug-carrying microbubbles; finally, uniformly incorporating the microbubbles into a vinyl-modified natural polymer solution, and forming a microbubble-loaded composite hydrogel through photocrosslinking. This preparation method first prepares oxygen- and drug-carrying microbubbles, then uniformly loads them onto a modified natural hydrogel, and finally prepares the oxygen- and drug-carrying microbubble composite hydrogel through photocrosslinking.

[0049] HA can target macrophages to regulate immune function. Additionally, HA is an important component of synovial fluid, playing a role in joint lubrication. Therefore, it is introduced into the microbubble precursor DPPE-HA through a chemical reaction. The introduced oxygen serves as a template for microbubble formation, and the oxygen released during application also improves the cellular microenvironment and exerts its immunomodulatory effect. Furthermore, vinyl groups impart photocurable properties to the hydrogel, and the mechanical properties of the hydrogel can be controlled by adjusting the UV irradiation time.

[0050] The structural features of the injectable oxygen- and drug-carrying microbubble composite hydrogel are as follows: using an injectable natural hydrogel as the matrix, the microbubbles are dispersed in the network structure of the matrix gel through electrostatic attraction, van der Waals forces, and other interactions. The collective gel polysaccharide molecules form electrostatic adsorption with the groups on the surface of the microbubbles, thus encapsulating the microbubbles on the surface of the microbubbles, thereby enabling the microbubbles to exist stably in the matrix gel and ensuring the stability of the microbubbles during injection and cross-linking. At the same time, the microbubble acts as a gel core, promoting the aggregation of gel matrix molecules on the surface of the microbubble, enhancing the effect of the gel matrix molecules, increasing the number of cross-linking points, and thus regulating the mechanical properties and flowability of the hydrogel.

[0051] The aforementioned microbubble hydrogel is characterized by the following: HA-modified microbubbles exhibit good biocompatibility; the oxygen and drugs loaded within the microbubbles can regulate the immune microenvironment of damaged tissues, improving the immune microenvironment and achieving effective tissue repair. Furthermore, due to the ultrasonic cavitation effect of the microbubbles, the controlled release of the microbubble-loaded components can be achieved by controlling the ultrasonic conditions. The presence of the injectable hydrogel not only protects the fragile microbubbles but also further slows down the rapid short-term release, thereby improving the utilization rate of the loaded drugs and effectively reducing systemic toxicity caused by long-term, high-dose administration. Simultaneously, the preparation process is simple, low-cost, suitable for mass production, and has promising clinical application prospects. The biocompatible injectable oxygen- and drug-loaded microbubble hydrogel prepared using the above method possesses controllable mechanical properties, good in vivo enzymatic hydrolysis performance, and immunomodulatory properties, making it suitable for repairing various tissue injuries.

[0052] Specifically, the present invention first provides a method for preparing an injectable oxygen- and drug-carrying microbubble composite hydrogel with immunomodulatory properties. By designing a microbubble hydrogel by incorporating microbubbles into a modified natural hydrogel, the precise and controllable release of the drug carried by the microbubbles can be achieved, exhibiting good biocompatibility and immunomodulatory capabilities for the treatment of tissue damage. The specific process is as follows:

[0053] Step 1: Synthesize oxygen- and drug-carrying microbubbles.

[0054] (1) First, DPPE-HA was synthesized. HA was weighed and dissolved in ultrapure water, and activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to adjust the pH of the solution to 4-6. Subsequently, DPPE was added to the pre-activated HA solution to carry out the reaction. The pH of the reaction system was controlled at 8-10, the temperature at 30-50℃, and the reaction time at 6-24h. After the reaction was completed, the unreacted reagents were removed by dialyzing for 3-5 days, and DPPE-HA was obtained by freeze-drying.

[0055] (2) Add the microbubble monomer DPPE-HA obtained in (1) to a solution containing DPPC and methotrexate (MTX), gently stir in a water bath, and introduce oxygen until the emulsion bubbles. Then promote the formation of microbubbles by mechanical vibration. The temperature of the reaction system is controlled at 25-50℃, the stirring time is 10-60 min, the frequency of mechanical vibration is set to 60-100 Hz, and the time is 1-5 min.

[0056] The emulsion was then diluted with PBS to obtain drug-loaded and oxygen-carrying microbubbles.

[0057] Step 2: Synthesize photocrosslinked methacrylamide gelatin (GelMA).

[0058] (1) Dissolve gelatin in deionized water, then add methacrylic anhydride to the gelatin solution, adjust the pH of the solution to 8-10, and the reaction temperature to 40-60℃. After reacting for 3-12 hours, collect the reaction product, dialyze, filter and freeze dry to obtain GelMA.

[0059] Step 3: Prepare microbubble hydrogels.

[0060] (1) Dissolve the GelMA from step two in a PBS buffer containing a photoinitiator, and then uniformly incorporate a certain amount of oxygen-carrying drug-carrying microbubbles into the hydrogel by mechanical stirring. Transfer the mixed solution to a mold and irradiate it with an ultraviolet light source for a certain period of time. After the hydrogel is photocured, it is demolded to obtain a microbubble hydrogel.

[0061] (2) The reaction conditions in (1) above are as follows: the concentration of GelMA in PBS buffer is 1-10 w / v, the content of Irgacure2959 is 0.1-0.5 w / v, the volume ratio of microbubble emulsion to GelMA solution is 0-1 / 4, and the UV irradiation time is 30-180 s.

[0062] Preferably, the reaction conditions for step one are: the mass ratio of HA to DPPE is 14:1, the pH value of the HA solution is 4, the pH value of the reaction system is 8.6, the temperature is 37°C, and the reaction time is 24h.

[0063] Preferably, the monomers used in step one for preparing oxygen- and drug-carrying microbubbles are typically materials used to construct microbubble membranes. These materials can be surfactants, lipids, polymers, or natural materials, and one or more can be used simultaneously to form a stable membrane structure and generate microbubbles in a liquid. These materials include, but are not limited to, lecithin, fatty acid derivatives, dioleoylglycerol, phosphatidylcholine, polylactic-co-hydroxyacetic acid copolymer, polydimethylsiloxane, gelatin, chitosan, and other materials.

[0064] Preferably, the drug loaded in the microbubbles in step one has good immunomodulatory ability and can effectively inhibit cellular inflammation, etc. One or more drugs can be used simultaneously, including but not limited to glucocorticoids such as prednisone, nonsteroidal anti-inflammatory drugs such as aspirin, anti-tumor necrosis factor drugs such as infliximab, and immunosuppressants such as methotrexate.

[0065] Preferably, the reaction conditions for step two are as follows: the mass ratio of DPPC, DPPE-HA and MTX is 2:5:1, the reaction system temperature is controlled at 40°C, the stirring time is 30 min, the mechanical vibration frequency is set to 60 Hz, and the time is 1 min.

[0066] Preferably, in step two, the monomers used to prepare the photocrosslinked hydrogel primarily possess good biocompatibility and photoreactivity, and one or more of these materials can be used simultaneously, including but not limited to acrylamide, acrylic acid, 2-hydroxyethyl methacrylate, glycerol dimethacrylate, etc. The hydrogel used exhibits good biocompatibility, bioactivity, and stability, and is mainly composed of natural polymer compounds, including but not limited to gelatin, chitosan, hyaluronic acid, chondroitin sulfate, sodium alginate, and collagen, etc.

[0067] Preferably, the reaction conditions for step three are: adjusting the pH of the reaction system to 8.0, the temperature to 50°C, and the reaction time to 3 hours.

[0068] Preferably, the photoinitiator used in step three is biocompatible, non-toxic, and has highly efficient photoinitiation activity. One or more of these photoinitiators can be used simultaneously, including but not limited to IrGacure 2959, water-soluble benzoyl peroxide, camphorquinone, and other materials.

[0069] Preferably, the reaction conditions for step four are as follows: the concentration of GelMA in PBS buffer is 10 w / v, the content of Irgacure2959 is 0.5 w / v, the volume ratio of microbubble emulsion to GelMA solution is 1 / 6, and the irradiation time of ultraviolet light source is 30 s.

[0070] The present invention also provides an injectable oxygen- and drug-carrying microbubble composite hydrogel prepared by the preparation method described in any of the above aspects.

[0071] The oxygen- and drug-carrying microbubble hydrogel designed in this invention possesses injectability, photocrosslinking properties, and tunable mechanical properties. The preparation method is simple and easy to operate, using inexpensive and widely available raw materials with broad applicability. Furthermore, it exhibits good biocompatibility and immunomodulatory properties, and is suitable for treating various types of tissue injuries. This microbubble hydrogel can be used to treat tissue injuries including RA and osteoarthritis-induced joint injuries, skin wounds, and osteoarthritis, achieving close contact with damaged tissues and meeting the material shape design requirements for different shaped damaged tissues.

[0072] Example 1

[0073] Step 1: Weigh 0.5 g of HA and dissolve it in 100 mL of ultrapure water. Activate the carboxyl group of HA with 0.1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and adjust the pH of the solution to 4. Then, add 0.036 g of DPPE to the pre-activated HA solution to react, adjust the pH of the solution to 8.6, and react at 37 °C for 24 h. After the reaction, dialyze the solution through a 14 kDa dialysis bag for 3 days to remove unreacted reagents. After freeze-drying, obtain HA-modified DPPE (DPPE-HA).

[0074] Figure 1 In the Fourier transform infrared (FTIR) curves, compared to the DPPE group, the DPPE-HA group showed higher wavelengths at 1742 cm⁻¹. -1 The presence of a distinct absorption peak indicates that the reaction between DPPE and HA has successfully undergone an amide reaction to generate DPPE-HA.

[0075] Step 2: Dissolve 5 mg DPPC, 10 mg DPPE-HA, 2 mg MTX, and 50 μl glycerol in 450 μl PBS buffer and gently stir in a 40°C water bath for 30 min, continuously aerating until the emulsion bubbles. During this process, use ultrasonic mechanical vibration at a frequency of 60 Hz for 1 min. Then dilute the emulsion with PBS to obtain oxygen-loaded drug-carrying liposome microvesicles (O2+MTX@DPPC / DPPE-HA).

[0076] Figure 2 This indicates that the microbubbles are uniformly spherical. Figure 3 The microbubbles were observed to have a diameter of approximately 10 μm. Additionally, Figure 4 The zeta potentials of different microbubble surfaces are shown, indicating that the surface of O2+MTX@DPPC / DPPE-HA microspheres is negatively charged, which is beneficial to their uniform dispersion.

[0077] Step 3: Dissolve 10g of gelatin in 100ml of deionized water, then add 1ml of methacrylic anhydride to the solution, adjust the pH of the solution to 8, react at 50℃ for 3h, collect the reaction product, dialyze it using a 14kDa dialysis bag, filter and freeze dry to obtain methacrylic acylated gelatin (GelMA).

[0078] Step 4: Dissolve 1g GelMA and 50mg IrGacure2959 in 10ml PBS buffer, then uniformly incorporate 1.67mL of oxygen- and drug-carrying microbubbles into the hydrogel using mechanical stirring. Transfer the mixture to a mold and irradiate with a UV light source for 30s. After photocuring, demold to obtain the microbubble hydrogel (MBs / GelMA).

[0079] like Figure 5 As shown, compared to the uniform and smooth pore structure of GelMA hydrogel, smaller pores and spherical microbubble structures can be observed in the pores of microbubble hydrogel. Figure 6 Fourier transform infrared spectroscopy indicates that microbubbles were successfully loaded into the GelMA hydrogel. Figure 7 The results of MTX release from the microbubble hydrogel showed that ultrasound could significantly enhance the release of MTX from the microbubble hydrogel, and the release rate of MTX decreased with the increase of GelMA content. Figure 8 The O2 release experiment also showed that ultrasonic treatment is beneficial for the release of O2 from the microbubble hydrogel. Figure 9 This indicates that the microbubble hydrogel has good biocompatibility. Figure 10 The results showed that microbubble hydrogels could significantly reduce oxidative stress in macrophages. Correspondingly, Figure 11 This indicates that microbubble hydrogels can significantly inhibit M1 polarization of macrophages, while promoting M2 polarization.

[0080] Example 2

[0081] In Example 2, the other steps and parameters are the same as in Example 1, except that step 2 is adjusted to dissolve 5 mg DPPC, 10 mg DPPE-HA, 2 mg MTX and 50 μl cholesterol in 450 μl PBS buffer.

[0082] Experimental results show that the microbubble hydrogel in Example 2 also has good biocompatibility, can significantly reduce oxidative stress of macrophages, and significantly inhibit M1 polarization of macrophages, while promoting macrophages to M2 polarization.

[0083] Example 3

[0084] In Example 3, the other steps and parameters are the same as in Example 1, except that step 2 is adjusted to dissolve 5 mg DPPC, 10 mg DPPE-HA, 2 mg MTX and 50 μl polyethylene glycol 200 in 450 μl PBS buffer.

[0085] Experimental results show that the microbubble hydrogel in Example 3 also has good biocompatibility, can significantly reduce oxidative stress of macrophages, significantly inhibit M1 polarization of macrophages, and promote macrophages to M2 polarization.

[0086] In summary, this invention provides an injectable oxygen- and drug-carrying microbubble composite hydrogel with immunomodulatory properties and its preparation method. By combining the advantages of microbubbles loaded with immunomodulatory drug molecules and bioactive natural polymers, the microbubble hydrogel exhibits stable drug loading capacity and can achieve long-term stable and controllable release through ultrasonic treatment, thus possessing good biocompatibility and tissue repair capabilities, effectively reducing the systemic toxicity caused by long-term, high-dose administration in current clinical drug therapy and microbubble therapy. Furthermore, the microbubble hydrogel possesses excellent injectability, photocrosslinking properties, and controllable mechanical properties, enabling it to address complex treatment needs with different shapes, mechanical properties, and tissue injuries, demonstrating superior potential compared to traditional microbubble delivery systems. The microbubble hydrogel preparation process is simple, low-cost, and suitable for mass production and industrialization. The obtained microbubble hydrogel can be used to treat various tissue injuries, including wound repair and burn treatment, osteochondral injury repair, cardiovascular disease treatment, and nerve injury repair. It can also be applied in cosmetic surgery and skin regeneration, showing broad clinical application prospects.

[0087] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing an injectable oxygen- and drug-carrying microbubble composite hydrogel with immunomodulatory properties, characterized in that, The specific process is as follows: Step 1: Construct the microbubble monomer DPPE-HA by reacting hyaluronic acid (HA) and dipalmitoylphosphatidylethanolamine (DPPE); Step 2: Obtain oxygen-carrying and drug-carrying microbubbles by fully dissolving dipalmitoylphosphatidylcholine, DPPE-HA and the therapeutic drug in a solvent, introducing oxygen and accompanied by mechanical vibration. Step 3: The oxygen-carrying and drug-carrying microbubbles are uniformly incorporated into a vinyl-modified natural polymer solution, and an injectable oxygen-carrying and drug-carrying microbubble composite hydrogel is formed through photocrosslinking.

2. The preparation method according to claim 1, characterized in that, Step 1 specifically includes: Hyaluronic acid was weighed and dissolved in ultrapure water, and activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to adjust the pH of the solution. Dipalmitoylphosphatidylethanolamine was added to the activated hyaluronic acid solution to carry out the reaction. After the reaction was completed, the unreacted reagents were removed, and the microbubble monomer DPPE-HA was obtained by freeze drying.

3. The preparation method according to claim 2, characterized in that, In step 1, after activation with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the pH of the solution is adjusted to 4-6. During the reaction process, dipalmitoylphosphatidylethanolamine is added to an activated hyaluronic acid solution. The pH of the reaction system is controlled at 8-10, the temperature at 30-50℃, and the reaction time at 6-24 hours. After the reaction is completed, the unreacted reagents are removed by dialysis for 3-5 days.

4. The preparation method according to claim 1, characterized in that, Step 2 specifically includes: Dipalmitoylphosphatidylcholine, the microbubble monomer DPPE-HA, and the therapeutic drug are dissolved in a solvent, stirred in a water bath, and oxygen is introduced until the emulsion bubbles. Then, mechanical vibration is used to promote microbubble formation. Finally, the emulsion is diluted with PBS to obtain the oxygen-carrying and drug-carrying microbubbles.

5. The preparation method according to claim 1, characterized in that, In step 2, the therapeutic drug is methotrexate.

6. The preparation method according to claim 4, characterized in that, In step 2, the reaction system temperature is controlled at 25-50℃, the stirring time is 10-60 min, the mechanical vibration frequency is set to 60-100 Hz, and the time is 1-5 min.

7. The preparation method according to claim 1, characterized in that, Step 3 specifically includes: Step 31: Dissolve gelatin in deionized water, then add methacrylic anhydride to the gelatin solution, adjust the pH of the solution, collect the reaction product after a certain reaction time, dialyze, filter and freeze dry to obtain GelMA; Step 32: GelMA is dissolved in PBS buffer containing Irgacure2959 to obtain a hydrogel. Then, the oxygen-carrying drug-carrying microbubbles are uniformly incorporated into the hydrogel and transferred to a mold. After photocuring under ultraviolet light, the mold is removed to obtain an injectable oxygen-carrying drug-carrying microbubble composite hydrogel.

8. The preparation method according to claim 7, characterized in that, In step 31, the solution pH is adjusted to 8-10, the reaction temperature is 40-60℃, and the reaction product is collected after 3-12 hours of reaction.

9. The preparation method according to claim 7, characterized in that, The reaction conditions for step 32 are as follows: the concentration of GelMA dissolved in PBS buffer containing Irgacure2959 is 1-10 w / v, the content of Irgacure2959 is 0.1-0.5 w / v, the volume ratio of the oxygen-carrying drug-carrying microbubbles to the volume of the GelMA solution is 0-1 / 4, and the UV irradiation time is 30-180 s.

10. An injectable oxygen- and drug-carrying microbubble composite hydrogel prepared by the preparation method according to any one of claims 1 to 9.

Images