Preparation method and application of injectable hydrogel microspheres

Abstract

This application discloses a preparation method and application of injectable hydrogel microspheres. By integrating the hydrogel drug delivery system with liposome microparticles obtained by microfluidic technology, NMN is encapsulated by liposome material, and the mitochondrial targeting peptide SS-31 is introduced into the liposome to achieve a more sustained and stable release, thereby enabling NMN to selectively target cell mitochondria and improve the application effect.

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 a 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 nanosystems that have been most studied in clinical applications to date. Liposomes are composed of micron- or nano-sized phospholipid vesicles whose membranes consist of a phospholipid bilayer that isolates their internal aqueous cavity from the external aqueous environment. Because their bilayer phospholipids can form a lubricating layer formed by the phosphocholine head, they have porosity and injectability, thus being used in pharmaceuticals and medicine. Because they exhibit a lipid bilayer and allow the encapsulation of various biomolecules within 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 will 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, centered around energy metabolism disturbances caused by decreased NAD+ levels, is the key driver of skeletal muscle aging. Nicotinamide mononucleotide (NMN) supplementation can increase NAD+ levels and improve energy metabolism. However, even with the aforementioned administration methods, there is currently a lack of effective and long-lasting supplementation strategies. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of this application is to provide a preparation method and application of injectable hydrogel microspheres to solve the problems of short action time and low supplementation effect in the prior art administration method.

[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 The lipid phase flow rate is 200-300 μL·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; the oxidized hyaluronic acid is then modified with methacrylic anhydride in a buffer solution, reacted in the dark at room temperature for 0.5 to 2 hours, and filtered and dried to obtain a hydrogel microsphere precursor material 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 an initiator is then added thereto to obtain a mixture, which is passed into a microfluidic device to allow the AHM and NMN@Lipo-s to be mixed and assembled, and irradiated with 365 nm or 405 nm ultraviolet light at the mixing position in the microfluidic device to cause a polymerization reaction to obtain the hydrogel microspheres NMN@Lipo-s@AHM;

[0014] 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. This application integrates the hydrogel drug delivery system with liposome microparticles obtained by microfluidic technology, uses liposome materials to encapsulate NMN, and introduces the mitochondrial targeting peptide SS-31 into the liposomes to achieve 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 this 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 targetedly delivered to mitochondria, 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 Figure 3 is a graph showing the expression of proteins and mRNA related to the inhibition of muscle decomposition 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; F is the mRNA expression level of Trim63 in muscle cells.

[0025] Figure 4 The results of the biosafety experiment of NMN@Lipo-s@AHM hydrogel microspheres are shown in Figure 1. A is the staining results of live and dead cells in different treatment groups on the 1st, 2nd and 3rd days, 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 figure shows the therapeutic effect of NMN@Lipo-s@AHM hydrogel microspheres on myositis model mice.

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

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

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

[0030] This application will provide a clear and complete description of the technical solutions in the embodiments of this application in conjunction with the accompanying drawings. 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 are within 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 The lipid phase flow rate is 200-300 μL·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; the oxidized hyaluronic acid is then modified with methacrylic anhydride in a buffer solution, reacted in the dark at room temperature for 0.5 to 2 hours, and filtered and dried to obtain a hydrogel microsphere precursor material 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 an initiator is then added thereto to obtain a mixture, which is passed into a microfluidic device to allow the AHM and NMN@Lipo-s to be mixed and assembled, and irradiated with 365 nm or 405 nm ultraviolet light at the mixing position in the microfluidic device to cause a polymerization reaction to obtain the hydrogel microspheres NMN@Lipo-s@AHM;

[0039] 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 the use of hydrogels for administration will increase the local concentration of the drug, 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 structurally, changing the mechanical stability and membrane integrity of encapsulated liposomes by hydrogels. Based on this, the present application adopts 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, in combination with the use of linear stereo targeting peptides, NMN is delivered to the mitochondria, achieving accurate and continuous improvement of mitochondrial energy metabolism, thereby delaying the aging of skeletal muscle cells. The hydrogel microspheres prepared in the present application are connected to the mitochondria targeting peptide to continuously supply NMN to the mitochondria, which can effectively delay skeletal muscle aging, provide a new treatment option for treating aging-related diseases (such as sarcopenia), and are a biomaterial with great potential.

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

[0042] In some embodiments, 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). The present application selects cholesterol, neutral lipids and cationic lipids as ingredients to prepare liposomes to encapsulate 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, as cationic lipids, effectively encapsulate negatively charged NMN through electrostatic interactions, significantly improving the encapsulation rate and cellular uptake efficiency; SS31, as an antioxidant peptide targeting mitochondria, further enhances the efficiency of liposomes in targeting mitochondria. The formula and ratio of the present application, while ensuring the drug loading efficiency, effectively avoid the inefficient encapsulation problem caused by the lack of cationic lipids in other liposomes, 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 this 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 this application.

[0043] In some embodiments, SS31 is processed 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 extending 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 this application.

[0045] In some embodiments, in step 2, hyaluronic acid is oxidized with sodium periodate at room temperature in the dark to obtain oxidized hyaluronic acid. After dialysis to remove impurities, the oxidized hyaluronic acid is modified with methacrylic anhydride in a PBS buffer at pH 7.0-9.0. The reaction is carried out at room temperature in the dark for 0.5-2 hours, and the hydrogel microsphere precursor AHM is obtained after filtration and drying. 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 at a pH of 7-9. Methacryl is introduced into the oxidized hyaluronic acid to enable cross-linking via a photopolymerization reaction. The purpose of this chemical modification is to impart specific controllable cross-linking properties to the oxidized hyaluronic acid for subsequent coupling with a photosensitizer to the microspheres. If the methacrylation substitution degree of the 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 too high a substitution degree may cause the natural biocompatibility of the 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 the oxidized hyaluronic acid, thereby achieving 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 the 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 this application.

[0046] 2. Application of an injectable hydrogel microsphere

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

[0048] 3. Examples and Comparative Examples

[0049] Example 1:

[0050] The preparation method of this embodiment of the invention 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 aqueous phase and lipid phase were assembled into micron-scale liposome particles NMN@Lipo-s using a microfluidic device. The main parameters during the assembly process were: aqueous phase flow rate 200 μl·min -1 , lipid phase flow rate 800 μl·min -1 The aqueous phase contained the drug NMN at a concentration of 3 mg / ml, and the lipid phase contained lipids at a concentration of 1 mg / ml, which were mixed under a microfluidic chip. The lipids included cholesterol, DOTAP, DLin-K-C3-DMA, SM102, DODAC, and SS31; the mass ratios of cholesterol, DOTAP, DLin-K-C3-DMA, SM102, DODAC, and SS31 were 1:1.8:0.5:0.95:2:0.12. The collected liposomes were diluted 5-fold with water and then ultrafiltered and centrifuged, leaving 0.5 ml of solution for subsequent use.

[0055] Step 3: Preparation of methacryloyl hyaluronic acid

[0056] A hyaluronic acid aqueous solution was oxidized with sodium periodate at 25°C for 1 hour to obtain oxidized hyaluronic acid. The molar ratio of sodium periodate to hyaluronic acid was 1:1. The oxidized hyaluronic acid was modified with methacrylic anhydride in a weakly alkaline environment to obtain a hydrogel microsphere precursor. The molar ratio of methacrylic anhydride to oxidized hyaluronic acid was 1:2, and the buffer solution was PBS at a pH of 7-9. In a weakly alkaline environment, the lone pair electrons of the hydroxyl oxygen atoms of the oxidized hyaluronic acid are more nucleophilic, increasing the reaction rate and hindering the self-polymerization of the methacrylic anhydride's own double bonds. The weakly alkaline environment makes the hydroxyl groups in the hyaluronic acid molecules more nucleophilic, favoring the attack of the anhydride molecules, thereby promoting the acylation reaction.

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

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

[0059] Specifically, the methacryloyl-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. They were 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] Example 2:

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

[0062] Example 3:

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

[0064] Example 4:

[0065] The method was modified 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 in Example 1.

[0066] Comparative Example 1

[0067] Adjustments were made based on Example 1, with the difference that the cationic lipid was removed and the release effects of the drug with and without the addition of cationic lipid were compared. Figure 6 .

[0068] Comparative Example 2

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

[0070] Comparative Example 3

[0071] Adjustments were made based on Example 1, except that SS31 was removed, and the liposomes were labeled green with FITC to compare their co-localization with myocyte mitochondria.

[0072] 4. Effect Analysis

[0073] The microparticles prepared in the examples and comparative examples (using hyaluronic acid to directly load NMN) were added, and 0.20 g of the microspheres prepared in the examples and comparative examples were added to 50 mL of phosphate buffer solution (pH 7.0), stirred at a speed of 100 r / min, and 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 using a high performance liquid chromatograph, 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 Shown, what comparative example 2 adopts is the hyaluronic acid hydrogel without any treatment, and it can be seen that it just releases medicine completely in a short time (5 hours).And comparative example 1 does not add cationic lipid, and drug release gradually reaches steady after 15 hours, which also illustrates that in the early stage, the rate of release of comparative example 1 is obviously faster.The embodiment of the present application then continues to release medicine slowly and steadily, even after 35 hours, still in slow-release medicine, and this also proves that the sustained-release effect of embodiment is obviously better than comparative example 1 and comparative example 2.

[0076] (2) Figure 3The hydrogel microspheres prepared in this application are shown in the table below in inhibiting the expression of muscle breakdown-related proteins and mRNA. 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, and the Lipo-s@AHM obtained by combining ordinary liposome particles with AHM carriers is used to test its effect on the target indicators. NMN@Lipo-s@AHM prepared in this application is another experimental group for comparison. The above substances were injected into muscle cells to observe the expression of the above substances in inhibiting muscle breakdown-related proteins and mRNA. In 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 DAPI-stained nuclei, and green represents F-actin expression. Figure 3 C and 3D show the relative expression intensities of FBXO32 and Trim63 proteins in the different treatment groups. It can be seen that the Blank and Lipo-s@AHM groups showed increased expression of FBXO32 and Trim63 compared to the Control group, while the expression in the NMN@Lipo-s@AHM group was significantly lower than that in the 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 from this 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 three-day period, the number of viable cells and optical density values ​​of each treatment group remained relatively stable, indicating that the hydrogel microspheres described in this application did 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 application 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 muscle fiber diameter is shown. As can be seen from the figure, there are significant differences in the muscle fiber diameters of the different treatment groups: the muscle fiber diameter of the control group is about 80μm, the highest among all groups. The muscle fiber diameter of the Blank group dropped significantly to about 20μm, indicating that the blank control group has severe muscle fiber atrophy. The muscle fiber diameter of the Lipo-s@AHM group recovered to about 40μm. Although lower than the Control group, it is significantly higher than 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 treatment of 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 showed 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 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 capabilities and can more effectively deliver drugs or other bioactive substances to target cells. The enhancement of this targeting capability 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. This higher uptake efficiency can make the concentration of drugs in cells higher, thereby producing a stronger physiological effect, 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 and can more accurately deliver drugs to target cells, reducing damage to other healthy cells. They also have a longer half-life and can stay in the body longer, 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 capability and cellular uptake efficiency as carriers. They 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 solutions of the present application and not to limit the technical solutions. Those skilled in the art should understand that modifications or equivalent replacements of the technical solutions of the present application that do not depart from the purpose and scope of the technical solutions of the present application should be included in the scope of the claims of the present application.

Claims

1. An application of injectable hydrogel microspheres, characterized in that: The hydrogel microspheres are used to prepare a drug for treating sarcopenia; the hydrogel microspheres are prepared by the following steps: 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 microparticles NMN@Lipo-s. During the assembly process, the aqueous phase flow rate was 700-800 μL·min -1 The 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; 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. The oxidized hyaluronic acid is then modified with methacrylic anhydride in a buffer solution, reacted in the dark at room temperature for 0.5 to 2 hours, and filtered and dried to obtain a hydrogel microsphere precursor material AHM. The molar ratio of sodium periodate to hyaluronic acid is (1:1) to (5:1), and the molar ratio of methacrylic anhydride to oxidized hyaluronic acid is (1:2) to (1:5). 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 an initiator is then added thereto to obtain a mixture, which is passed into a microfluidic device to allow the AHM and NMN@Lipo-s to be mixed and assembled, and irradiated with 365 nm or 405 nm ultraviolet light at the mixing position in the microfluidic device to cause a polymerization reaction to obtain the hydrogel microspheres NMN@Lipo-s@AHM; Calculated by mass percentage, the amount of AHM in the mixture 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 application 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 application 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.

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