Preparation method and application of injectable hydrogel microspheres

Abstract

The invention discloses a preparation method and application of injectable hydrogel microspheres, a drug delivery system of hydrogel is integrated with liposome particles obtained by a microfluidic technology, a liposome material is utilized to wrap NMN, and meanwhile, mitochondrial targeting peptide SS-31 is introduced into liposome, so that more durable and stable release is realized, and the injectable hydrogel microspheres are prepared. Therefore, the NMN can selectively target cell mitochondria, and the application effect is improved.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a preparation method of injectable hydrogel microspheres and applications thereof. Background Art

[0002] Conventional drug delivery systems have inherent limitations, such as poor targeting and low therapeutic index, which can lead to systemic side effects and increase the cost and duration of treatment. To overcome these shortcomings, various nano-delivery systems have been developed for different therapeutic applications. Among them, liposomes (self-assembled lipid vesicles) are one of the most studied nanosystems for clinical applications to date. Liposomes are composed of micron or nano-sized phospholipid vesicles, whose membranes are composed of phospholipid bilayers, isolating their internal water cavity from the external water environment. Because their bilayer phospholipids can be porous and injectable through the lubricating layer formed by the phosphorylcholine head, they are used in pharmaceuticals and medicine. Because they exhibit a lipid bilayer and allow the encapsulation of various biomolecules in the lumen or bilayer itself and are fully biocompatible and non-toxic, they have attracted much attention in the field of biology.

[0003] Hydrogels are 3D cross-linked polymer networks with high water absorption capacity similar to body tissues, which enables them to encapsulate drugs and protect them under physiological conditions. The delivery mechanism of hydrogels is usually controlled by passive diffusion, which depends largely on the hydrogel structure (such as hydrogel pore size, cross-linking degree, stimuli-sensitive hydrogel capacity, etc.). However, due to the possibility of undesirable immediate release of drugs, this can increase the local concentration of drugs, thereby causing unexpected toxicity in vivo.

[0004] Sarcopenia is a disease that affects the quality of life of the elderly and is closely related to aging. Mitochondrial dysfunction is the key to skeletal muscle aging, with energy metabolism disorders caused by reduced NAD+ levels. Supplementation with nicotinamide mononucleotide (NMN) can increase NAD+ levels in the body and improve energy metabolism. Even with the above-mentioned administration method, there is still a lack of long-acting and efficient supplementation strategies. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a method for preparing injectable hydrogel microspheres and applications thereof, so as to solve the problems of short action time and low supplementation effect in the administration methods of the prior art.

[0006] In order to solve the above technical problems, this application adopts the following technical solutions:

[0007] A method for preparing injectable hydrogel microspheres comprises the following steps:

[0008] Step 1: Preparation of NMN liposome microparticles:

[0009] The aqueous phase and lipid phase were assembled at room temperature using a microfluidic device to obtain NMN-loaded liposome microparticles NMN@Lipo-s; during the assembly process, the aqueous phase flow rate was 700-800 μL·min -1 , lipid phase flow rate is 200~300μL·min -1 ; The aqueous phase is a β-nicotinamide mononucleotide aqueous solution with a concentration of 3 to 5 mg / L; the lipid phase is a liposome;

[0010] Step 2: Preparation of methacrylylated oxidized hyaluronic acid;

[0011] Hyaluronic acid is oxidized at room temperature using sodium periodate to obtain oxidized hyaluronic acid, and the obtained oxidized hyaluronic acid is dialyzed to remove impurities; then, the oxidized hyaluronic acid is modified using methacrylic anhydride in a buffer solution, reacted in the dark at room temperature for 0.5 to 2 hours, filtered and dried to obtain a hydrogel microsphere precursor substance AHM; wherein the molar ratio of sodium periodate to hyaluronic acid is (1:1) to (5:1), the molar ratio of methacrylic anhydride to oxidized hyaluronic acid is (1:2) to (1:5), and the buffer solution is a PBS buffer solution with a pH of 7 to 9;

[0012] Step 3: Preparation of NMN liposome-loaded hydrogel microspheres:

[0013] The AHM obtained in step 2 is mixed with the NMN@Lipo-s obtained in step 1, and then an initiator is added thereto to obtain a mixture, and the mixture is passed into a microfluidic device to mix and assemble the AHM and NMN@Lipo-s, and 365nm or 405nm ultraviolet light is irradiated at the mixing position in the microfluidic device to perform a polymerization reaction to obtain the hydrogel microspheres NMN@Lipo-s@AHM;

[0014] Among them, calculated by mass percentage, in the mixture, the amount of AHM is 1% to 12%, the amount of photoinitiator is 0.1% to 1%, and the amount of surfactant is 0.1 to 2%; in the mixture, the concentration of NMN@Lipo-s is 0.1 mg / ml to 5 mg / ml; the oil phase is paraffin oil or dimethyl silicone oil, and the surfactant is Span80.

[0015] Preferably, in step 1, the liposomes include neutral lipids, cationic lipids and SS31; the neutral lipids are cholesterol, and the cationic lipids are composed of DOTAP, DLin-K-C3-DMA, SM102, and DODAC; wherein the mass ratio of cholesterol, DOTAP, DLin-K-C3-DMA, SM102, DODAC and SS31 is 1:1.8:0.5:0.95:2:(0.12-0.4).

[0016] Preferably, SS31 is pretreated as follows:

[0017] SS31 and DSPE-PEG2K-NHS ester were mixed at a molar ratio of (1.0-1.5):1, dissolved in DMSO and stirred, and then dialyzed and freeze-dried to obtain DSPE-PEG2K-SS31 powder.

[0018] The present application also provides an application of injectable hydrogel microspheres. The hydrogel microspheres prepared by the above preparation method are used to prepare drugs for treating sarcopenia.

[0019] Compared with the prior art, this application has the following beneficial effects:

[0020] 1. The present application integrates the hydrogel drug delivery system with the liposome microparticles obtained by microfluidic technology, uses the liposome material to encapsulate NMN, and introduces the mitochondrial targeting peptide SS-31 into the liposome to achieve a more sustained and stable release, thereby enabling NMN to selectively target cell mitochondria and improve the application effect.

[0021] 2. In the hydrogel microspheres (NMN@Lipo-s@AHM) described in the present application, the hydrogel microspheres are loaded with NMN to achieve the continuous supply and sustained release of NAD+ synthetic raw materials, avoiding the rapid metabolism of NMN; at the same time, combined with the use of linear stereo targeting peptides, NMN is delivered to the mitochondria in a targeted manner, thereby achieving the purpose of accurately and continuously improving mitochondrial energy metabolism, thereby delaying the aging of skeletal muscle cells, providing a new treatment option for the treatment of aging-related diseases (such as sarcopenia), and is a highly promising biomaterial. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the preparation process of NMN@Lipo-s@AHM hydrogel microspheres prepared in this application.

[0023] Figure 2 This is a scanning electron microscope image of NMN@Lipo-s@AHM hydrogel microspheres.

[0024] Figure 3 The above is a graph showing the expression of proteins and mRNA related to the inhibition of muscle degradation by NMN@Lipo-s@AHM hydrogel microspheres; A is the immunofluorescence image of protein expression of FBXO32 and F-actin in muscle cells; B is the immunofluorescence image of protein expression of Trum63 and F-actin in muscle cells; C is the relative expression of FBXO3 protein in muscle cells; D is the relative expression of Trim63 protein in muscle cells; E is the mRNA expression level of FBXO32 in muscle cells; and F is the mRNA expression level of Trim63 in muscle cells.

[0025] Figure 4 The figure is the result of the biosafety experiment of NMN@Lipo-s@AHM hydrogel microspheres; A is the staining results of live and dead cells in different treatment groups on the 1st day, 2nd day and 3rd day, B is the number of live cells in muscle cells in different treatment groups within three days, and C is the optical density value of muscle cells in different treatment groups.

[0026] Figure 5 This is a graph showing the therapeutic effect of NMN@Lipo-s@AHM hydrogel microspheres on myositis model mice.

[0027] Figure 6 It is the sustained release effect diagram of Example 1 and Comparative Example 1.

[0028] Figure 7 In comparative example 3, the liposomes released from the liposome-loaded microspheres without SS31 modification co-localize with the muscle cells.

[0029] Figure 8 As in Example 2, the co-localization of liposomes released from SS31-modified liposome-loaded microspheres with myocytes. DETAILED DESCRIPTION

[0030] This application will combine the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on this application belong to the scope of protection of this application.

[0031] Unless otherwise indicated in specific cases in this application, the numerical ranges listed herein include the upper and lower limits, as well as all integers and fractions within the range, and are not limited to the specific values ​​listed when defining the range.

[0032] 1. A method for preparing injectable hydrogel microspheres

[0033] Step 1: Preparation of NMN liposome microparticles:

[0034] The aqueous phase and lipid phase were assembled at room temperature using a microfluidic device to obtain NMN-loaded liposome microparticles NMN@Lipo-s; during the assembly process, the aqueous phase flow rate was 700-800 μL·min -1 , lipid phase flow rate is 200~300μL·min -1 ; The aqueous phase is a β-nicotinamide mononucleotide aqueous solution with a concentration of 3 to 5 mg / L; the lipid phase is a liposome;

[0035] Step 2: Preparation of methacrylylated oxidized hyaluronic acid;

[0036] Hyaluronic acid is oxidized at room temperature using sodium periodate to obtain oxidized hyaluronic acid, and the obtained oxidized hyaluronic acid is dialyzed to remove impurities; then, the oxidized hyaluronic acid is modified using methacrylic anhydride in a buffer solution, reacted in the dark at room temperature for 0.5 to 2 hours, filtered and dried to obtain a hydrogel microsphere precursor substance AHM; wherein the molar ratio of sodium periodate to hyaluronic acid is (1:1) to (5:1), the molar ratio of methacrylic anhydride to oxidized hyaluronic acid is (1:2) to (1:5), and the buffer solution is a PBS buffer solution with a pH of 7 to 9;

[0037] Step 3: Preparation of NMN liposome-loaded hydrogel microspheres:

[0038] The AHM obtained in step 2 is mixed with the NMN@Lipo-s obtained in step 1, and then an initiator is added thereto to obtain a mixture, and the mixture is passed into a microfluidic device to mix and assemble the AHM and NMN@Lipo-s, and 365nm or 405nm ultraviolet light is irradiated at the mixing position in the microfluidic device to perform a polymerization reaction to obtain the hydrogel microspheres NMN@Lipo-s@AHM;

[0039] Among them, calculated by mass percentage, in the mixture, the amount of AHM is 1% to 12%, the amount of photoinitiator is 0.1% to 1%, and the amount of surfactant is 0.1 to 2%; in the mixture, the concentration of NMN@Lipo-s is 0.1 mg / ml to 5 mg / ml; the oil phase is paraffin oil or dimethyl silicone oil, and the surfactant is Span80.

[0040] After an in-depth study of the existing drug administration methods, the present application found that due to the possibility of undesirable immediate release of drugs, only using hydrogels for administration will increase the local concentration of drugs, thereby causing unexpected in vivo toxicity. Therefore, the present application considers integrating hydrogels and liposomes to minimize the rapid release of drugs. At the same time, hydrogels and liposomes can also improve each other in structure, and change the mechanical stability and membrane integrity of encapsulated liposomes by hydrogels. Based on this, the present application uses hydrogel microspheres to load NMN to achieve continuous supply and sustained release of NAD+ synthetic raw materials, avoiding the rapid metabolism of NMN; at the same time, combined with the use of linear stereo targeting peptides, NMN is targeted to mitochondria for delivery, so as to achieve accurate and continuous improvement of mitochondrial energy metabolism, thereby delaying the purpose of skeletal muscle cell aging. The hydrogel microspheres prepared in the present application are connected to the mitochondrial targeting peptide to continuously supply NMN to the mitochondria, which can effectively delay skeletal muscle aging, and provide a new treatment plan for the treatment of aging-related diseases (such as sarcopenia). It is a highly promising biomaterial.

[0041] In some embodiments, in step 1, during the microparticle encapsulation process, the water phase flow rate is 700-800 μL·min -1 , lipid phase flow rate is 200~300μL·min -1 Controlling the flow rates of the aqueous phase and lipid phase can ensure that the formed microparticles are loaded with enough NMN, so the flow rate of the aqueous phase can be 700 μL·min -1 , 750μL·min -1 、800μL·min -1 etc., and all ranges and subranges between the above values; lipid phase flow rate is 200 μL·min -1 , 250μL·min -1 、300μL·min -1 etc., and all ranges and sub-ranges between the above values; the aqueous phase is a β-nicotinamide mononucleotide aqueous solution, and its concentration can be 3 mg / L, 4 mg / L, 5 mg / L, etc., and all ranges and sub-ranges between the above values; the lipid phase is a liposome. It should be understood that in embodiments, any of the above ranges can be combined with any range of other reaction conditions in the present application.

[0042] In some embodiments, in step 1, the liposome includes a neutral lipid, a cationic lipid and SS31; the neutral lipid is cholesterol, and the cationic lipid is composed of DOTAP, DLin-K-C3-DMA, SM102, and DODAC; wherein the mass ratio of cholesterol, DOTAP, DLin-K-C3-DMA, SM102, DODAC and SS31 is 1:1.8:0.5:0.95:2:(0.12-0.4). The present application selects cholesterol, neutral lipids and cationic lipids as ingredients to prepare liposomes encapsulating the negatively charged water-soluble substance NMN. Cholesterol enhances the stability and rigidity of the liposome membrane and reduces drug leakage; neutral lipids are used to improve the stability of the membrane and make the charge distribution on both sides of the membrane more uniform; DOTAP, DLin-K-C3-DMA, SM102, and DODAC are cationic lipids that effectively encapsulate negatively charged NMN through electrostatic interactions, significantly improving the encapsulation rate and cellular uptake efficiency; SS31 is an antioxidant peptide targeting mitochondria, which further improves the efficiency of liposomes targeting mitochondria. The formula and ratio of the present application effectively avoid the inefficient encapsulation problem caused by the lack of cationic lipids in other liposomes while ensuring the drug loading efficiency, overcome the defects of insufficient stability and high toxicity of traditional liposomes, and achieve efficient targeted delivery of NMN. If the mass ratio of the components deviates from the range described in the present application, the stability and drug loading effect of the liposomes will be significantly reduced. Therefore, the mass ratio of cholesterol, DOTAP, DLin-K-C3-DMA, SM102, DODAC and SS31 can be 1:1.8:0.5:0.95:2:0.12, 1:1.8:0.5:0.95:2:0.2, 1:1.8:0.5:0.95:2:0.3, 1:1.8:0.5:0.95:2:0.4, etc., as well as all ranges and sub-ranges between the above values; it should be understood that in the embodiment, any of the above ranges can be combined with any range of other reaction conditions in the present application.

[0043] In some embodiments, SS31 is treated as follows:

[0044] SS31 and DSPE-PEG2K-NHS ester are mixed in a molar ratio of (1.0-1.5):1, dissolved and stirred with DMSO, dialyzed and freeze-dried to obtain SS31 powder. The water-soluble SS31 is exposed on the surface of the liposomes, and the fat-soluble DSPE can be directly inserted into the lipid layer. On the one hand, the targeting efficiency of the liposomes can be improved. On the other hand, SS31 is directly coupled to PEG to prevent it from being quickly cleared or degraded by enzymes, thereby prolonging its half-life in the body. Therefore, the molar ratio of SS31 to DSPE-PEG2K-NHS ester can be 1:1, 1.3:1, 1.5:1, etc., as well as all ranges and sub-ranges between the above values; it should be understood that in the embodiment, any of the above ranges can be combined with any range of other reaction conditions in the present application.

[0045] In some embodiments, in step 2, hyaluronic acid is oxidized by sodium periodate at room temperature in the dark to obtain oxidized hyaluronic acid, and after dialysis to remove impurities, the oxidized hyaluronic acid is modified by methacrylic anhydride in a PBS buffer of pH 7.0-9.0, and reacted at room temperature in the dark for 0.5 to 2 hours, and filtered and dried to obtain a hydrogel microsphere precursor substance AHM; wherein the molar ratio of sodium periodate to hyaluronic acid is (1:1) to (5:1), the molar ratio of methacrylic anhydride is 1:2 to 1:5, and the buffer is a PBS buffer of pH 7 to 9. Methacrylation is introduced into the oxidized hyaluronic acid so that it can be cross-linked by a photo-induced polymerization reaction. The purpose of this chemical modification is to give it a specific controllable cross-linking property for subsequent co-coupling of microspheres with a photosensitizer. If the degree of methacrylation substitution of oxidized hyaluronic acid is too low, the cross-linking density is insufficient, and the mechanical properties and structural stability of the material may not be significantly improved, resulting in easy degradation of the microspheres and poor shape retention; while excessive substitution may cause the natural biocompatibility of oxidized hyaluronic acid to decrease, reduce its performance in the biological environment, and may cause adverse biological reactions. Therefore, it is necessary to optimize the degree of substitution to ensure that it can be effectively cross-linked without affecting the biological properties of oxidized hyaluronic acid, so as to achieve the best functional effect. Therefore, the molar ratio of sodium periodate to hyaluronic acid can be 1:1, 2:1, 3:1, 4:1, 5:1, etc., and all ranges and sub-ranges between the above values; the molar ratio of methacrylic anhydride to oxidized hyaluronic acid can be 1:2, 1:3, 1:4, 1:5, etc., and all ranges and sub-ranges between the above values; it should be understood that in the embodiment, any of the above ranges can be combined with any range of other reaction conditions in the present application.

[0046] 2. Application of an injectable hydrogel microsphere

[0047] The hydrogel microspheres prepared by the preparation method described in the present application are used to prepare drugs for treating sarcopenia.

[0048] 3. Examples and Comparative Examples

[0049] Embodiment 1:

[0050] The preparation method of this embodiment is to connect the mitochondrial targeting peptide with the liposome containing NMN and then load it into the methacrylylated hyaluronic acid hydrogel microspheres, which includes the following steps:

[0051] Step 1: SS31 pretreatment

[0052] SS31 and DSPE-PEG2K-NH ester were dissolved in DMSO at a molar ratio of 1.2:1, stirred at room temperature for one day, dialyzed, and freeze-dried to obtain SS31 powder.

[0053] Step 2: Preparation of SS31-NMN liposome particles

[0054] The water phase and lipid phase were assembled into micron-scale liposome particles NMN@Lipo-s using microfluidic equipment. The main parameters during the assembly process were: water phase flow rate 200 μl·min -1 , lipid phase flow rate 800 μl·min -1 , the aqueous phase is the drug NMN, the concentration is selected as 3mg / ml, the lipid phase is selected as lipid, the concentration is selected as 1mg / ml, and they are mixed under the microfluidic chip. The lipids include cholesterol, DOTAP, DLin-K-C3-DMA, SM102, DODAC, and SS31; wherein the mass ratio of cholesterol, DOTAP, DLin-K-C3-DMA, SM102, DODAC, and SS31 is 1:1.8:0.5:0.95:2:0.12. The collected liposomes were diluted 5 times with water and then ultrafiltered and centrifuged, leaving 0.5ml of solution for subsequent use.

[0055] Step 3: Preparation of methacryloyl hyaluronic acid

[0056] The hyaluronic acid aqueous solution is oxidized by sodium periodate, the oxidation temperature is 25°C, the oxidation time is 1h, and oxidized hyaluronic acid is obtained. The molar ratio of sodium periodate to hyaluronic acid is 1:1. The oxidized hyaluronic acid is modified by methacrylic anhydride in a weakly alkaline environment to obtain a hydrogel microsphere precursor; wherein the molar ratio of methacrylic anhydride to oxidized hyaluronic acid is 1:2, and the buffer solution is a PBS buffer solution with a pH of 7 to 9. In a weakly alkaline environment, the nucleophilic ability of the lone pair of electrons of the hydroxyl oxygen atom of the oxidized hyaluronic acid is enhanced, the reaction rate is increased, and the self-polymerization of the double bond of methacrylic anhydride is hindered. The hydroxyl group in the hyaluronic acid molecule in the weakly alkaline environment is more nucleophilic, which is conducive to attacking the anhydride molecule, thereby promoting the acylation reaction.

[0057] Step 4: Preparation of hydrogel microspheres loaded with NMN liposomes

[0058] The methacryloylated oxidized hyaluronic acid is mixed with the NMN@Lipo-s prepared in step 2, and the hydrogel microspheres NMN@Lipo-s@AHM loaded with NMN liposomes are obtained by using microfluidic technology; wherein, calculated by mass percentage, in the mixture obtained after mixing, the concentration of methacryloylated oxidized hyaluronic acid is 1%, the concentration of NMN is 0.1 mg / ml, the content of photoinitiator is 0.1%, the oil phase is paraffin oil or dimethyl silicone oil, and the surfactant is Span80, and its concentration is 0.1%.

[0059] Specifically, the methacrylylated oxidized hyaluronic acid solution and the NMN liposome solution were magnetically stirred for 5 minutes to ensure uniform mixing. The mixing time was 5 minutes, and the mixed liquid was used as the aqueous phase. The aqueous phase and the oil phase were introduced into the aqueous phase channel and the oil phase channel of the microfluidic device respectively, and the flow rate ratio was adjusted to control the size of the formed microspheres. The flow rate ratio was 1:5, and an emulsion of hydrogel microspheres was generated by shear force at the outlet of the microfluidic device. The generated microspheres formed a stable dispersed phase in the oil phase. It was irradiated with 365nm ultraviolet light at the mixing position in the microfluidic device to cause a polymerization reaction. After the photopolymerization was completed, the mixed emulsion was treated with anhydrous ethanol or an appropriate amount of ether to remove the oil phase and surfactant. The microspheres were washed with water several times until the surfactant residue was completely removed.

[0060] Embodiment 2:

[0061] The method is adjusted based on Example 1, except that in step 3, the molar ratio of methacrylic anhydride to oxidized hyaluronic acid is 1:3. The other steps are the same as those in Example 1.

[0062] Embodiment 3:

[0063] The method is adjusted based on Example 1, except that in step 2, the mass ratio of DOTAP, DLin-K-C3-DMA, SM102, DODAC, and SS31 is 1:1.8:0.5:0.95:2:0.4. The other steps are the same as those in Example 1.

[0064] Embodiment 4:

[0065] The method was adjusted based on Example 1, except that the concentration of NMN used in step 2 was 5 mg / ml. The other steps were the same as those in Example 1.

[0066] Comparative Example 1

[0067] The adjustment was made on the basis of Example 1, except that the cationic lipid was removed, and the release effect of the drug with and without the addition of cationic lipid was compared. Figure 6 .

[0068] Comparative Example 2

[0069] Hyaluronic acid hydrogel is used to directly load drugs.

[0070] Comparative Example 3

[0071] The method was adjusted based on Example 1, except that SS31 was removed, and the liposomes were labeled green with FITC to compare their co-localization with muscle cell mitochondria.

[0072] 4. Effect Analysis

[0073] 0.20 g of the microspheres prepared in the embodiment and the comparative example (using hyaluronic acid to directly load NMN) were added to 50 mL of phosphate buffer solution (pH 7.0), and stirred at a speed of 100 r / min. The temperature was set to 37°C. 1.0 mL of the solution was drawn with a syringe at regular intervals, and the same volume of new buffer was immediately added. The NMN content was detected by high performance liquid chromatography, and the cumulative release rate was calculated according to the following formula.

[0074]

[0075] (1) The release test results of the comparative examples and embodiments are as follows Figure 6 As shown, Comparative Example 2 uses a hyaluronic acid hydrogel without any treatment, and it can be seen that it completely releases the drug in a short time (5 hours). Comparative Example 1 does not add cationic lipids, and the drug release gradually reaches stability after 15 hours, which also shows that in the early stage, the release rate of Comparative Example 1 is obviously faster. The embodiment of the present application then continues to release drugs slowly and steadily, even after 35 hours, it is still slowly releasing drugs, which also proves that the sustained-release effect of the embodiment is significantly better than Comparative Examples 1 and Comparative Examples 2.

[0076] (2) Figure 3The hydrogel microspheres prepared by the present application are shown in the table below in inhibiting the expression of proteins and mRNA related to muscle breakdown. Among them, Control is the control group, which is a standard control group without any special treatment and is used as a basis for comparison. Blank is a blank control group, which is a control group containing only the AHM prepared in step 2 of Example 1, and is used to exclude the influence of the carrier itself on the experimental results. Lipo-s@AHM is one of the experimental groups. Lipo-s@AHM, which is obtained by combining ordinary liposome particles with AHM carriers, is used to test its effect on the target indicators. NMN@Lipo-s@AHM prepared by the present application is another experimental group for comparison. The above substances were injected into muscle cells to observe the expression of proteins and mRNA related to muscle breakdown. Figure 3 In A and 3B, we can see the myocyte morphology and protein expression in different treatment groups (Control, Blank, Lipo-s@AHM, NMN@Lipo-s@AHM). Red represents the expression of FBXO32 or Trim63, blue represents the nucleus stained with DAPI, and green represents the expression of F-actin. Figure 3 C and 3D show the relative expression intensity of FBXO32 and Trim63 proteins in different treatment groups. It can be seen that the expression of FBXO32 and Trim63 increased in the Blank group and Lipo-s@AHM group compared with the Control group, while the expression of the NMN@Lipo-s@AHM group was significantly lower than that of other groups. Figure 3 Groups E and 3F show the relative expression levels of FBXO32 and Trim63 mRNA in different treatment groups. It can also be seen that the mRNA expression of FBXO32 and Trim63 increased in the Blank group and the Lipo-s@AHM group compared with the Control group, while the expression in the NMN@Lipo-s@AHM group was significantly lower than that in other groups. It can be seen that the protein and mRNA expression of FBXO32 and Trim63 increased in the Blank group and the Lipo-s@AHM group compared with the Control group, indicating that these two treatments may lead to upregulation of the expression of genes related to muscle breakdown. Compared with other groups, the protein and mRNA expression of FBXO32 and Trim63 in the NMN@Lipo-s@AHM group were significantly reduced, indicating that the hydrogel microspheres prepared in this application were injected into muscle cells, and this treatment method can effectively inhibit the expression of genes related to muscle breakdown. This proves that the NMN@Lipo-s@AHM hydrogel microspheres prepared in this application show good therapeutic effects, can significantly inhibit the expression of muscle breakdown-related proteins and mRNA, and are expected to become a potential muscle protection therapy.

[0077] (3) Figure 4A is the staining results of live cells / dead cells in different treatment groups on the 1st, 2nd and 3rd day. Figure 4 B is the number of living cells in muscle cells of different treatment groups within three days. Figure 4 C is the optical density value of muscle cells in different treatment groups. It can be seen that the NMN@Lipo-s@AHM prepared in this application did not significantly change the survival rate and growth state of muscle cells, indicating that the hydrogel microspheres have low toxicity to muscle cells. Over a period of three days, the number of live cells and optical density values ​​of each treatment group remained relatively stable, indicating that the hydrogel microspheres described in this application will not significantly interfere with the normal proliferation process of muscle cells. This further proves that the hydrogel microspheres described in this application have good biosafety and are suitable for use in muscle cell-related research and clinical trials.

[0078] (4) Figure 5 The effect of the NMN@Lipo-s@AHM hydrogel microspheres described in this application on the diameter of muscle fibers is shown. As can be seen from the figure, there are obvious differences in the diameter of muscle fibers in different treatment groups: the diameter of muscle fibers in the Control group is about 80μm, which is the highest among all groups. The diameter of muscle fibers in the Blank group dropped significantly to about 20μm, indicating that there is serious muscle fiber atrophy in the blank control group. The muscle fiber diameter of the Lipo-s@AHM group recovered to about 40μm, which is lower than that of the Control group, but significantly higher than that of the Blank group, indicating that the Lipo-s@AHM treatment has a certain therapeutic effect. The muscle fiber diameter of the NMN@Lipo-s@AHM group further recovered to about 70μm, close to the level of the Control group, indicating that the NMN@Lipo-s@AHM treatment has a better therapeutic effect. It can be seen that the muscle fiber diameter of the NMN@Lipo-s@AHM prepared in this application after treating the muscle fibers is significantly higher than that of the Blank group and the Lipo-s@AHM group, close to the level of the Control group, indicating that the hydrogel microspheres have a significant therapeutic effect on muscle fiber atrophy. As the treatment concentration increases (from Lipo-s@AHM to NMN@Lipo-s@AHM), the diameter of the muscle fibers gradually increases, showing a certain dose-dependent relationship. Compared with the use of Lipo-s@AHM alone, the effect of using NMN@Lipo-s@AHM is more significant, indicating that there is a synergistic effect between the two. In summary, the NMN@Lipo-s@AHM hydrogel microspheres prepared in this application show excellent therapeutic effects in the muscle fiber atrophy model, can effectively promote muscle fiber regeneration and growth, and have good application prospects.

[0079] (5) Figure 7 and Figure 8 After comparison, it can be found that the green fluorescence in the figure represents liposomes. Figure 8 In the green fluorescence ratio Figure 7 More concentrated and bright, especially in certain areas within the cell, this shows that the liposomes modified by SS31 in this application have stronger targeting ability, can more effectively deliver drugs or other bioactive substances to target cells, and the enhancement of this targeting ability can make the drug's effect better and the side effects lower. At the same time, the liposomes modified by SS31 in this application are more easily taken up by cells, and this higher uptake efficiency can make the concentration of drugs in cells higher, thereby producing stronger physiological effects, and ultimately improving the utilization rate and efficacy of drugs. This proves that after the liposomes are modified with SS31, the liposomes are more easily engulfed by cells and more easily aggregated in the mitochondrial position in the cytoplasm. Moreover, the liposomes modified by SS31 have higher precision, can more accurately deliver drugs to target cells, reduce damage to other healthy cells, and also have a longer half-life, can stay longer in the body, thereby providing a more lasting therapeutic effect. In summary, the liposomes modified by SS31 in this application have shown significant advantages in terms of targeting ability and cell uptake efficiency as carriers, and can be applied to a wider range of disease types, including those that are difficult to cure with traditional treatment methods, and are expected to become a powerful tool in the field of drug delivery in the future.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the technical solution. Ordinary technicians in this field should understand that those modifications or equivalent substitutions of the technical solution of the present application that do not depart from the purpose and scope of the technical solution of the present application should be included in the scope of the claims of the present application.

Claims

1. A method for preparing injectable hydrogel microspheres, characterized in that: The specific steps include: Step 1: Preparation of NMN liposome microparticles: The aqueous phase and lipid phase were assembled at room temperature using a microfluidic device to obtain NMN-loaded liposome particles NMN@Lipo-s; during the assembly process, the flow rate of the aqueous phase was 700-800 μL·min -1 , lipid phase flow rate is 200~300μL·min -1 ; The aqueous phase is a β-nicotinamide mononucleotide aqueous solution with a concentration of 3-5 mg / L; the lipid phase is a liposome; Step 2: Preparation of methacrylylated oxidized hyaluronic acid; Hyaluronic acid is oxidized at room temperature using sodium periodate to obtain oxidized hyaluronic acid, and the obtained oxidized hyaluronic acid is dialyzed to remove impurities; then, the oxidized hyaluronic acid is modified with methacrylic anhydride in a buffer solution, reacted at room temperature in the dark for 0.5 to 2 hours, filtered and dried to obtain a hydrogel microsphere precursor substance AHM; wherein the molar ratio of sodium periodate to hyaluronic acid is (1:1) to (5:1), the molar ratio of methacrylic anhydride to oxidized hyaluronic acid is (1:2) to (1:5), and the buffer solution is a PBS buffer solution with a pH of 7 to 9; Step 3: Preparation of NMN liposome-loaded hydrogel microspheres: The AHM obtained in step 2 is mixed with the NMN@Lipo-s obtained in step 1, and then an initiator is added thereto to obtain a mixture, and the mixture is passed into a microfluidic device to mix and assemble the AHM and NMN@Lipo-s, and 365nm or 405nm ultraviolet light is irradiated at the mixing position in the microfluidic device to perform a polymerization reaction to obtain the hydrogel microspheres NMN@Lipo-s@AHM; Among them, calculated by mass percentage, in the mixture, the amount of AHM is 1%~12%, the amount of photoinitiator is 0.1%~1%, and the amount of surfactant is 0.1~2%; the concentration of NMN@Lipo-s in the mixture is 0.1mg / ml~5mg / ml; the oil phase is paraffin oil or dimethyl silicone oil, and the surfactant is Span80.

2. The preparation method according to claim 1, characterized in that: In step 1, the liposomes include neutral lipids, cationic lipids and SS31; the neutral lipids are cholesterol, and the cationic lipids are composed of DOTAP, DLin-K-C3-DMA, SM102 and DODAC; wherein the mass ratio of cholesterol, DOTAP, DLin-K-C3-DMA, SM102, DODAC and SS31 is 1:1.8:0.5:0.95:2:(0.12~0.4).

3. The preparation method according to claim 2, characterized in that: The SS31 is pre-treated as follows: SS31 and DSPE-PEG2K-NHS ester were mixed at a molar ratio of (1.0-1.5):1, dissolved in DMSO and stirred, and then dialyzed and freeze-dried to obtain DSPE-PEG2K-SS31 powder.

4. An application of injectable hydrogel microspheres, characterized in that: The hydrogel microspheres prepared by any preparation method described in claims 1 to 3 are used to prepare a drug for treating sarcopenia.

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