A nanomaterial for relieving tendinopathy inflammation and pain and its preparation method

Abstract

This invention provides a nanomaterial for relieving inflammation and pain in tendinopathy and its preparation method, relating to the field of biomedical technology. The nanomaterial comprises decellularized tendon DTM and hybrid membrane vesicles (MSM) of macrophages and nerve cells. The aforementioned nanomaterial for relieving inflammation and pain in tendinopathy includes decellularized tendon DTM and hybrid membrane vesicles (MSM) of macrophages and nerve cells. Combining these two components yields DTM-MSM, which regulates the proliferation and differentiation of tendon stem cells, modulates the local immune microenvironment, and relieves pain. It can improve inflammation and pain in tendinopathy and accelerate the repair of damaged tendons. In other words, DTM-MSM can not only alleviate the pathophysiological changes in tendinopathy but also rapidly improve the main clinical symptoms of tendinopathy, such as pain, swelling, and functional limitation, thus overcoming the current shortcomings of tendinopathy treatments, including slow onset of action, poor compliance, significant side effects, and unclear efficacy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nanomaterial for relieving tendinopathy inflammation and pain, and its preparation method. Background Technology

[0002] Tendons connect muscles and bones, transmitting the force generated by the muscles to the bones, thus enabling joint movement. Injury and overuse can alter the microstructure of tendons, leading to tendinopathy. Since 2000, the prevalence of tendinopathy has been increasing globally, making it one of the most common orthopedic diseases. Currently, only limited conservative treatments are used clinically to relieve the pain symptoms of tendinopathy. Therefore, there is an urgent need to develop new treatment strategies for tendinopathy.

[0003] Exercise therapy, injections, shockwave therapy, and medication are currently the main conservative treatment options for patients with tendinopathy. Exercise therapy typically involves 12 weeks of exercise, performed 3-4 times per week. Clinical outcomes regarding pain and function take a considerable amount of time to become apparent, gradually improving over weeks to months; however, not all patients achieve a full recovery, and residual symptoms often persist. Therefore, exercise therapy is slow to take effect, making it difficult for patients to adhere to and resulting in poor compliance.

[0004] Commonly used injectable therapies include corticosteroid injections and platelet-rich plasma (PRP) injections. Corticosteroid injections, as a monotherapy, have shown good short-term clinical results in tendinopathy, but their long-term efficacy is poor. A recent study published in JAMA showed that PRP injections did not alleviate Achilles tendon dysfunction at 6 months. Furthermore, the therapeutic effects of these commonly used injectable therapies remain unclear.

[0005] Extracorporeal shock wave therapy (ESWT) focuses high-amplitude shock waves into the tendon to potentially induce cavitation, promoting the release of healing factors at that site by inducing micro-damage. However, the ESWT procedure is painful, and due to the lack of monitoring of the focal pressure by clinical equipment, it is unclear in clinical studies whether cavitation is achieved, or even within the target area.

[0006] The most commonly used oral medications are nonsteroidal anti-inflammatory drugs (NSAIDs). In the treatment of tendinopathy, some patients cannot tolerate the gastrointestinal reactions caused by NSAIDs. Summary of the Invention

[0007] In view of the deficiencies in the existing technology, the technical problem solved by the present invention is to provide a nanomaterial for relieving tendinopathy inflammation and pain and its preparation method, which can effectively relieve the inflammation, pain and degeneration of tendinopathy.

[0008] To achieve the above objectives, in a first aspect, the present invention provides a nanomaterial for relieving tendinopathy inflammation and pain, said nanomaterial comprising decellularized tendon (DTM), macrophage and nerve cell hybrid membrane vesicle (MSM).

[0009] Preferably, in the nanomaterial, the mass ratio of the MSM to the volume ratio of the DTM is 40-120 μg / mL.

[0010] Secondly, the present invention also provides a method for preparing nanomaterials for relieving tendinopathy inflammation and pain, the method comprising: uniformly mixing decellularized tendon DTM with hybrid membrane vesicles of macrophages and nerve cells MSM to obtain the nanomaterials.

[0011] Preferably, the method for preparing the MSM includes: extracting the cell membranes of macrophages and nerve cells, mixing the two cell membranes to obtain a mixed cell membrane solution, and obtaining the MSM by extrusion of the mixed cell membrane solution.

[0012] Preferably, the method for extracting the cell membranes of macrophages and nerve cells includes: performing a first centrifugation, ultrasonic disruption, and a second centrifugation on macrophages and nerve cells respectively, to obtain cell membrane precipitates of macrophages and nerve cells respectively, wherein the ratio of macrophages to nerve cells is 1:1.

[0013] Preferably, the specific steps of the extrusion method include: extruding the mixed cell membrane solution through a polycarbonate porous membrane to obtain MSM with an average particle size of 160-240 nm.

[0014] Preferably, the method for preparing DTM includes: sequentially freezing, grinding, and sterilizing the decellularized tendon tissue, followed by enzymatic hydrolysis and gelation to obtain gel-like DTM.

[0015] Preferably, the freeze-drying step includes: placing the decellularized tendon tissue in an environment of -22~-18℃ for 12-14 hours and then freeze-drying under vacuum;

[0016] And / or, the grinding step includes: grinding the freeze-dried tendon tissue into powder with a particle size of less than 1 mm;

[0017] And / or, the sterilization step includes: placing the ground tendon tissue and a culture dish filled with ethanol together in an incubator at 37°C for 18-24 hours.

[0018] Preferably, the enzymatic hydrolysis method specifically includes: adding the sterilized tendon tissue to a pepsin solution and stirring continuously at 22-28°C for 24-48 hours to obtain a DTM solution;

[0019] And / or the method for preparing the pepsin solution includes: dissolving pepsin in hydrochloric acid at pH=2 to obtain the pepsin solution;

[0020] And / or, the concentration of the pepsin solution is 1 mg / mL;

[0021] And / or, the mass ratio of tendon tissue to pepsin after the sterilization treatment is (5-10):1.

[0022] Preferably, the gelation method specifically includes: adjusting the pH of the DTM solution obtained after enzymatic hydrolysis to neutral, and then placing it in a 37°C water bath for 30 minutes to obtain gel-like DTM.

[0023] Compared with existing technologies, the advantages of this invention are as follows: The nanomaterial used in this invention to relieve inflammation and pain in tendinopathy includes decellularized tendon (DTM), macrophage and nerve cell hybrid membrane vesicles (MSM). Combining these two materials yields DTM-MSM, which regulates tendon stem cell proliferation and differentiation, modulates the local immune microenvironment, and relieves pain. This improves inflammation and pain in tendinopathy and accelerates the repair of damaged tendons. In other words, DTM-MSM not only alleviates the pathophysiological changes in tendinopathy but also rapidly improves the main clinical symptoms of tendinopathy, such as pain, swelling, and limited function, overcoming the current shortcomings of tendinopathy treatments, including slow onset of action, poor compliance, significant side effects, and unclear efficacy. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 A process flow diagram for preparing hybrid membrane vesicle-decellularized tendon (MSM-DTM) according to an embodiment of the present invention;

[0026] Figure 2 This is a diagram illustrating the mechanism of action of MSM-DTM prepared in this embodiment of the invention in relieving tendinopathy inflammation and pain.

[0027] Figure 3 The image shows a transmission electron microscope (TEM) image of the MSM prepared in Example 1 of this invention.

[0028] Figure 4 This is a characteristic protein expression map of the MSM prepared in Example 1 of the present invention;

[0029] Figure 5 This is a flow cytometry result of the MSM prepared in Example 1 of the present invention;

[0030] Figure 6 The Zeta potential diagram of the MSM prepared in Example 1 of this invention;

[0031] Figure 7 This is a particle size distribution diagram of the MSM prepared in Example 1 of the present invention;

[0032] Figure 8 This is a graph showing the effect of MSM concentration obtained in Example 1 of the present invention on cell viability;

[0033] Figure 9 H&E staining and DAPI staining images of fresh tissue and DTM prepared in Example 1 of this invention;

[0034] Figure 10 Scanning electron microscope (SEM) images of fresh tissue and DTM prepared in Example 1 of this invention;

[0035] Figure 11 The graph shows the content analysis of DNA, collagen, and GAG in fresh tissue and DTM prepared in Example 1 of this invention.

[0036] Figure 12 This is a DTM gel image prepared in Example 1 of the present invention;

[0037] Figure 13 The rheological diagram is shown for the DTM gel prepared in Example 1 of this invention.

[0038] Figure 14 This is a diagram showing the release of MSM prepared in MSM-DTM according to Example 1 of the present invention;

[0039] Figure 15 This is a graph showing the adsorption capacity of the MSM prepared in Example 1 of the present invention for various cytokines and neuropeptides.

[0040] Figure 16 Bar chart showing the relative mRNA expression of inflammatory markers IL-6 (left) and CCL-2 (right) after treatment in each group;

[0041] Figure 17 Western blot plots showing the expression levels of COL1A2, MMP3, and GAPDH proteins after treatment in each group;

[0042] Figure 18 Western blot plots showing the expression levels of p16, p21, and GAPDH proteins after treatment in each group;

[0043] Figure 19 Image showing cell senescence as detected by β-galactosidase staining;

[0044] Figure 20 The images show the immunofluorescence results of treating TSPCs with DTM and MSM-DTM prepared in Example 1 of this invention for 21 days.

[0045] Figure 21 Immunofluorescence quantitative analysis of differentiation indicators TNMD (left) and TNC (right);

[0046] Figure 22 The figures show the RT-qPCT results of TSPCs treated for 21 days using the DTM and MSM-DTM prepared in Example 1 of this invention, respectively.

[0047] Figure 23 The Transwell migration result diagram;

[0048] Figure 24 Schematic diagram of establishing a rat tendinopathy model by injecting collagenase I;

[0049] Figure 25 This is a gross morphological image of the tendon recorded 28 days after injection.

[0050] Figure 26 This is a schematic diagram of HE staining of the tendon 4 weeks after injection.

[0051] Figure 27 This is a schematic diagram of Masson staining of the tendon 4 weeks after injection.

[0052] Figure 28 The image shows the results of immunofluorescence staining of the tendon 4 weeks after injection.

[0053] Figure 29 Statistical graph of mechanical pain threshold in rats after local injection of DTM and MSM-DTM;

[0054] Figure 30 Statistical graph of thermal pain threshold in rats after local injection of DTM and MSM-DTM;

[0055] Figure 31 Footprints of rats after local injection of DTM and MSM-DTM;

[0056] Figure 32 This is a graph showing the assessment of Achilles tendon function index in rats after local injection of DTM and MSM-DTM. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] This invention provides a nanomaterial for relieving inflammation and pain in tendinopathy and its preparation method. The nanomaterial includes decellularized tendon (DTM), macrophage and nerve cell hybrid membrane vesicles (MSM). This nanomaterial simultaneously regulates the proliferation and differentiation of tendon stem cells, modulates the local immune microenvironment, and relieves pain. It can not only alleviate the pathophysiological changes of tendinopathy, but also rapidly improve the main clinical symptoms of tendinopathy, such as pain, swelling, and functional limitation. This solves a series of defects in current tendinopathy treatments, such as slow onset of action, poor compliance, large side effects, and unclear efficacy.

[0059] To achieve the above-mentioned technical effects, the overall concept of this invention is as follows:

[0060] In a first aspect, the present invention provides a nanomaterial for relieving tendinopathy inflammation and pain, said nanomaterial comprising decellularized tendon (DTM), macrophage and nerve cell hybrid membrane vesicles (MSM).

[0061] This invention discovered that macrophage-neuron hybrid membrane vesicles (MSMs) can bind to various inflammatory factors, trapping them and regulating the immune microenvironment around the tendon. A long-term inflammatory microenvironment leads to tendon aging, which MSMs significantly alleviate. Furthermore, MSMs can capture neuropeptides and neurotransmitters around the tendon, relieving tendinopathy pain. Therefore, in this invention, MSMs act as "traps" for inflammatory factors, adsorbing them and alleviating tendon inflammation; simultaneously, they capture neurotrophic factors in the microenvironment, preventing them from binding to receptors on normal cells and relieving tendinopathy pain. In addition, this invention also found that decellularized tendon (DTM) not only has low immunogenicity, effectively avoiding the side effects of immune rejection, but also promotes tendon cell migration and differentiation, accelerating tendon repair. In addition, cell membrane vesicles can highly express integrins. Specific integrins (such as α5β1, α2β1 and α10β1) can interact with collagen and other extracellular matrix proteins. Since DTM contains a large amount of extracellular matrix components, MSM and DTM can be anchored and slowly released in DTM through the binding mechanism of integrins and extracellular matrix proteins.

[0062] This invention combines DTM and MSM to obtain DTM-MSM, which can exert the synergistic effect of both. Its mechanism of action on tendons is as follows: Figure 2 As shown, DTM-MSM can regulate tendon stem cell proliferation and differentiation, modulate the local immune microenvironment, and relieve pain, thus improving inflammation and pain in tendinopathy and accelerating the repair of damaged tendons. In other words, the nanomaterial (DTM-MSM) can not only alleviate the pathophysiological changes of tendinopathy but also rapidly improve the main clinical symptoms of tendinopathy, such as pain, swelling, and limited function, overcoming the current shortcomings of tendinopathy treatments, including slow onset of action, poor compliance, significant side effects, and unclear efficacy.

[0063] Preferably, in the above-mentioned nanomaterials, the mass ratio of MSM to DTM is 40-120 μg / mL. Experiments show that the higher the MSM concentration, the better the therapeutic effect on tendinopathy; however, experiments also show that... Figure 8 As shown, when the mass of MSM in 1 mL of DTM exceeds 120 μg, negative effects such as inhibition of cell growth occur. Therefore, the mass ratio of MSM to the volume of DTM is 40-120 μg / mL, which can achieve a good therapeutic effect on tendinopathy while ensuring biosafety. In a more preferred embodiment, the dosage ratio of MSM to DTM is 120 μg: 1 mL, which is the optimal usage condition under the condition of ensuring biosafety.

[0064] Preferably, the decellularized tendon (DTM) raw material of the present invention is tendon, taken from animal tendons, such as those of cattle, sheep, horses, pigs, etc. Since the DTM raw material of the present invention is taken from animal tendons, it has the advantages of low price and wide availability, thus making the preparation of the nanomaterials of the present invention easier and lower in cost.

[0065] Secondly, such as Figure 1 As shown, the present invention also provides a method for preparing nanomaterials for relieving tendinopathy inflammation and pain, the method comprising: uniformly mixing decellularized tendon (DTM) with hybrid membrane vesicles (MSM) of macrophages and nerve cells to obtain the nanomaterials.

[0066] This invention obtains DTM-MSM nanomaterials by uniformly mixing decellularized tendon (DTM) with macrophage and nerve cell hybrid membrane vesicles (MSM). In this invention, DTM is a decellularized matrix in a gel form, such as... Figure 12As shown, cell membrane vesicles can highly express integrins. Specific integrins (such as α5β1, α2β1, and α10β1) can interact with collagen and other extracellular matrix proteins. Since DTM contains a large amount of extracellular matrix components, MSM and DTM can achieve anchoring and slow release of MSM in DTM through the binding mechanism of integrins and extracellular matrix proteins.

[0067] Extensive research has demonstrated that DTM-MSM nanomaterials can regulate tendon stem cell proliferation and differentiation, modulate the local immune microenvironment, and alleviate pain. They can improve inflammation and pain associated with tendinopathy and accelerate the repair of damaged tendons. In other words, the nanomaterial (DTM-MSM) can not only alleviate the pathophysiological changes of tendinopathy but also rapidly improve the main clinical symptoms of tendinopathy, such as pain, swelling, and functional limitations. This addresses the shortcomings of current tendinopathy treatments, including slow onset of action, poor patient compliance, significant side effects, and unclear efficacy.

[0068] Preferably, the method for preparing the MSM includes: extracting the cell membranes of macrophages and nerve cells respectively; mixing the macrophage cell membranes and nerve cell membranes to obtain a mixed cell membrane solution; and obtaining the MSM by extrusion of the mixed cell membrane solution. The morphology of the MSM obtained by the above preparation method is as follows: Figure 3 As shown, its particle size distribution is as follows: Figure 7 As shown, the average particle size of MSM is 201.9 nm, and the particle size distribution is between 50 and 600 nm.

[0069] Preferably, the method for extracting the cell membranes of macrophages and nerve cells includes: sequentially centrifuging macrophages and nerve cells, followed by ultrasonic disruption, and then centrifuging again to obtain the cell membranes of macrophages and nerve cells, respectively, wherein the ratio of macrophages to nerve cells is 1:1. Hybrid membrane vesicles obtained by mixing the cell membranes extracted from macrophages and nerve cells at a 1:1 ratio can simultaneously achieve immunomodulation and pain relief, exhibiting good effects.

[0070] The specific steps of the first centrifugation include: collecting macrophages and nerve cells separately into centrifuge tubes, centrifuging at 1500 r / min for 5 minutes, and discarding the supernatant; washing each tube once with PBS; centrifuging at 1500 r / min for 5 minutes and discarding the supernatant; resuspending the cells in PBS and mixing the cells in both tubes by inverting them; centrifuging at 1500 r / min for 5 minutes and discarding the supernatant to obtain macrophage and nerve cell pellets, respectively.

[0071] Macrophage and nerve cell precipitates were disrupted by ultrasound. The specific steps of ultrasound disruption included: placing the cell precipitate in an ice bath, performing ultrasound for 3 seconds, pausing for 3 seconds, and repeating the above steps 5 times. The ultrasound power was 100-120W, resulting in broken macrophage cells and broken nerve cells.

[0072] The fragmented macrophage cells and the fragmented nerve cells were subjected to a second centrifugation. The specific steps of the second centrifugation included: taking the supernatant of the fragmented cells and centrifuging it at 3000×g for 5 minutes, then taking the supernatant after centrifugation and centrifuging it at 20000×g for 30 minutes, and then taking the supernatant after centrifugation and centrifuging it at 100000×g for 40 minutes. The resulting precipitates were the cell membranes of macrophages and nerve cells, respectively.

[0073] Preferably, in this invention, the cell membrane deposits of macrophages and nerve cells are resuspended in equal volumes of PBS and then mixed in equal volumes to obtain a mixed cell membrane solution.

[0074] Preferably, the specific steps of the extrusion method include: extruding the mixed cell membrane solution through a polycarbonate porous membrane with a pore size of 200 nm to obtain MSM with an average particle size of 160-240 nm. The MSM obtained by extrusion through the polycarbonate porous membrane has a uniform particle size.

[0075] Preferably, the concentration of MSM obtained by extrusion in this invention is 600 μg / mL.

[0076] Preferably, the method for preparing DTM includes: sequentially freezing, grinding, and sterilizing the decellularized tendon tissue, followed by enzymatic hydrolysis and gelation to obtain DTM.

[0077] Preferably, the freeze-drying step includes: placing the decellularized tendon tissue in an environment of -22~-18℃ for 12-14 hours and then freeze-drying under vacuum.

[0078] Preferably, the grinding step includes grinding the freeze-dried tendon tissue into powder with a particle size of less than 1 mm.

[0079] Preferably, the sterilization step includes: placing the ground tendon tissue and a culture dish filled with ethanol together in an incubator at 37°C for 24 hours.

[0080] Preferably, the enzymatic hydrolysis method specifically includes: adding the sterilized tendon tissue to a pepsin solution and stirring continuously at 25°C for 24-48 hours to obtain a DTM solution.

[0081] Preferably, the method for preparing the pepsin solution includes: dissolving pepsin in hydrochloric acid at pH=2 to obtain the pepsin solution.

[0082] Preferably, the concentration of the pepsin solution is 1 mg / mL.

[0083] Preferably, the mass ratio of the sterilized tendon tissue to pepsin is (5-10):1. This concentration range ensures sufficient contact between the tendon and pepsin, thereby improving the enzymatic hydrolysis effect. In a more preferred embodiment, the mass ratio of the sterilized tendon tissue to pepsin is 10:1.

[0084] Preferably, the gelation method specifically includes: adjusting the pH of the DTM solution obtained after enzymatic hydrolysis to neutral, and then placing it in a 37°C water bath for 25-35 minutes to obtain gelled DTM. Since the DTM solution obtained after enzymatic hydrolysis is acidic and cannot be used directly, it needs to be adjusted to neutral before use. The DTM obtained after adjusting the pH of the DTM solution to neutral will be in a gel state.

[0085] Preferably, the preparation method of the present invention further includes a decellularization process of the tendon: after repeatedly freezing and thawing the cleaned tendon tissue several times, deoxyribonuclease (DNase) is added to obtain the decellularized tendon tissue.

[0086] Preferably, the tendon tissue of the present invention is derived from animal tendons, such as those of cattle, sheep, horses, and pigs. Since the DTM raw material of the present invention is derived from animal tendons, it has the advantages of low price and wide availability, thus making the preparation of the nanomaterials of the present invention easier and less costly.

[0087] The following specific embodiments illustrate a nanomaterial for relieving tendinopathy inflammation and pain, and its preparation method. All raw materials used in the embodiments of this invention were purchased commercially. Deoxyribonuclease (DNase) was purchased from Beyotime.

[0088] Example 1

[0089] Example 1 of this invention provides a method for preparing nanomaterials for relieving tendinopathy inflammation and pain, comprising the following steps:

[0090] (1) Preparation of decellularized tendon (DTM):

[0091] The tendons were purchased from the market as bovine tendons. The tendons were cut into 5mm x 5mm pieces and placed in centrifuge tubes. 2% Triton X-100 nonionic surfactant was added to the centrifuge tubes, ensuring the Triton X-100 completely covered the tendons. The mixture was shaken to ensure thorough contact between the tendons and the reagent. The tendons were subjected to five freeze-thaw cycles using liquid nitrogen and a 37°C water bath. After thawing, the centrifuge tubes were filled with Triton X-100 reagent and then placed in a shaker at 18°C ​​for 48 hours. After shaking, the tubes were centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, and the tubes were filled with 1% SDS (sodium dodecyl sulfate). The mixture was shaken and incubated for 24 hours. The liquid in the tubes was discarded, and the tubes were filled with 750 mU / mL DNase and incubated at 37°C for 48 hours. After shaking well, centrifuge at 1200 r / min for 5 minutes, discard the supernatant, fill the centrifuge tube with water, shake well, centrifuge again and discard the supernatant (1200 r / min, 5 minutes), repeat 3 times to obtain the decellularized tendon;

[0092] The decellularized tendon was placed in a culture dish and incubated overnight at -20°C, followed by vacuum freeze-drying. The freeze-dried tendon was then ground into powder with a particle size less than 1 mm using a grinding rod. This powder was then placed in a culture dish along with two other culture dishes filled with 95% ethanol, and placed in an iron box. The mixture was then incubated at 37°C for 24 hours for sterilization. 30 mg of the sterilized decellularized tendon tissue was weighed for enzymatic digestion: 3 mg of pepsin was dissolved in 3 ml of hydrochloric acid (pH=2) to obtain a pepsin solution. The 30 mg of sterilized decellularized tendon was added to the filtered pepsin solution for digestion, and the mixture was stirred continuously at 25°C for 48 hours to obtain the enzymatically digested decellularized tendon tissue. The pH of the enzymatically digested decellularized tendon tissue solution was then adjusted to neutral, and the solution was placed in a 37°C water bath for 25-35 minutes to obtain a gel-like decellularized tendon (DTM).

[0093] (2) Preparation of hybrid membrane vesicles (MSM) of macrophages and nerve cells:

[0094] Macrophages and nerve cells (in a 1:1 ratio) were collected separately into centrifuge tubes and centrifuged at 1500 rpm for 5 minutes, discarding the supernatant. Each tube was washed once with PBS. The cells were then resuspended in PBS, and the two tubes were inverted to mix. The cells were centrifuged at 1500 rpm for 5 minutes, discarding the supernatant. The cells were then sonicated for 30 seconds (100W power, 3 seconds sonication, 3 seconds pause, 5 cycles, sonication performed on ice), and the supernatant was collected. The supernatant from the sonicated cells was centrifuged at 3000×g for 5 minutes, and then centrifuged at 20000×g for 30 minutes. Finally, the supernatant was centrifuged again at 100000×g for 40 minutes, and the cell membrane pellet was collected for subsequent experiments.

[0095] Hybrid membrane vesicles were generated using an extruder (Genizer HandExtruder - 1 mL, USA): The two cell membrane precipitates were resuspended in equal volumes of PBS, mixed in equal volumes to obtain a mixed cell membrane solution, and slowly extruded through an extruder using a 200 nm pore size polycarbonate porous membrane at 25°C to obtain vesicles of relatively uniform size. Each extrusion process was repeated 50 times, and the final MSM concentration was 600 μg / mL.

[0096] (3) Preparation of MSM-DTM:

[0097] 120 μg of MSM was incorporated into 1 mL of DTM at 4 °C and mixed uniformly to obtain MSM-DTM.

[0098] Example 2

[0099] The MSM, DTM, and MSM-DTM nanomaterials obtained in Example 1 of this invention were characterized structurally and their effects were verified.

[0100] (1) The structure of the MSM prepared in Example 1 was characterized by transmission electron microscopy (TEM). Figure 3 The image shows a transmission electron microscope (TEM) image of the MSM prepared in Example 1 of this invention. As can be seen from the image, the MSM is oval or round, consistent with the typical morphology of extracellular vesicles. Simultaneously, this invention also performed a Western blot assay, and the results are as follows: Figure 4 As shown, the experimental results demonstrate that MSM simultaneously expresses macrophage membrane characteristic proteins F4 / 80 and CD14, and nerve cell surface characteristic proteins TRPV1 and TrkA, proving that the hybrid membrane vesicles prepared in this invention were obtained.

[0101] Furthermore, before preparing MSM, this invention labels macrophages and nerve cells with cell membrane dyes DiO and DiI, respectively, and then detects the obtained MSM by flow cytometry. The results of flow cytometry are as follows: Figure 5 As shown, Figure 5 The fourth image shows that 80.8% of the vesicles are double positive for DiO and DiI, confirming the successful fusion of the cell membranes of macrophages and nerve cells.

[0102] (2) The zeta potential and particle size distribution of the MSM prepared in Example 1 were tested using a nanoparticle size and zeta potential analyzer. Figure 6 This is a Zeta potential map of the MSM prepared in Example 1 of the present invention. Figure 7 This is a particle size distribution diagram of the MSM prepared in Example 1 of the invention. Figure 6 and Figure 7 It can be seen that the zeta potential of MSM is approximately -24.3 mV, the average diameter is approximately 201.9 nm, and the particle size ranges from 50 to 600 nm.

[0103] (3) The CCK8 assay was used to detect the biosafety of MSM. The specific steps of the CCK8 assay are as follows: MSM at concentrations of 20 μg / mL, 40 μg / mL, 80 μg / mL, 120 μg / mL, and 160 μg / mL was co-cultured with tendon stem cells (TSPCs). The effects of different concentrations of MSM on the cell viability of TSPCs were investigated. Figure 8 As shown. Figure 8 The experimental results showed that low concentrations of MSM did not cause a significant decrease in cell viability. However, when the concentration of MSM reached 160 μg / mL, cell viability decreased. Therefore, the present invention preferably uses 120 μg / mL as the optimal therapeutic concentration of MSM.

[0104] (4) The DTM obtained in Example 1 of this invention was subjected to H&E staining and DAPI staining to evaluate the decellularization effect. The results showed that... Figure 9 As shown, Figure 9 The images on the left and right sides show the results of H&E staining and DAPI staining of fresh tissue (tendon tissue); Figure 9 The images on the right and left sides show the H&E staining and DAPI staining results of DTM in Example 1 of this invention, respectively. A comparison of the left and right images shows that no visible cell nuclei were found in the H&E and DAPI stained tissue sections obtained from DTM in Example 1 of this invention.

[0105] (5) The structure of the DTM obtained in Example 1 of this invention was characterized using a scanning electron microscope (SEM). Figure 10 This is a scanning electron microscope (SEM) image of the DTM prepared in Example 1 of the present invention. Figure 10 This indicates that the DTM obtained in Example 1 of the present invention still maintains the fibrous structure of the tendon, can effectively support tendon cells, and facilitates tendon cell adhesion.

[0106] (6) Figure 11 The DNA, collagen, and glycosaminoglycan (GAG) contents of DTM obtained in Example 1 of this invention and fresh tissue (tendon tissue) are shown. Compared with fresh tissue, the DNA content in DTM is significantly reduced, while the collagen and GAG contents are only slightly reduced.

[0107] Figure 9 , Figure 10 , Figure 11The results showed that cellular components were removed after the decellularization process, while the main components of the ECM (extracellular matrix) were retained. Since cells and nucleic acids are crucial components that trigger immune responses, effectively removing these immune-inducing components through the decellularization process enhances biocompatibility and is more conducive to the clinical translation of the nanomaterials of this invention.

[0108] (7) Figure 12 The image shows the appearance of the DTM obtained in Embodiment 1 of the present invention. Figure 12 It can be seen that the DTM obtained in Example 1 of the present invention is a stable gel.

[0109] (8) Rheological tests were performed on the DTM, and the results are as follows: Figure 13 As shown in the figure. Rheological measurements indicate that the storage modulus (G') of the DTM gel is significantly higher than its loss modulus (G''), suggesting that it primarily exhibits elastic properties, which is beneficial for DTM to maintain structural stability under physiological conditions.

[0110] (9) MSM was labeled with green fluorescent DiO, and the labeled MSM was mixed into DTM. The supernatant was collected at intervals and the absorbance was measured. The results are as follows: Figure 14 As shown, Figure 14 The results showed that MSM release reached 70% on the first day and was almost completely released within seven days.

[0111] (10) The cell membranes of macrophages and sensory neurons (nerve cells) are rich in receptors for inflammatory cytokines and neuropeptides, respectively. MSM, formed by the fusion of these two membranes, possesses surface properties, thus giving it the dual ability to adsorb pro-inflammatory cytokines and neuropeptides. To verify this, the present invention incubated MSM with TNF-α, IL-1β, IL-6, and LPS (500 pg / mL each) and IFN-γ and NGF (100 pg / mL each) at 37°C for 1 hour. After incubation, the MSM was removed by centrifugation, and the residual cytokine levels in the supernatant were measured using an enzyme-linked immunosorbent assay (ELISA). The results are as follows: Figure 15 As shown, Figure 15 The values ​​represent the remaining amounts of TNF-α, IL-1β, IL-6, LPS, IFN-γ, and NGF in the system after adding different concentrations of MSM. The experimental results show that 120 μg of MSM can adsorb 253.96 pg of TNF-α, 398.87 pg of IL-1β, 264.10 pg of IL-6, 48.77 pg of IFN-γ, 253.70 pg of LPS, and 41.53 pg of NGF, confirming that MSM has a strong adsorption capacity for various cytokines and neuropeptides.

[0112] (11) In this invention, interleukin-1β (IL-1β) was used to treat TSPCs for 24 hours to induce inflammation in TSPCs, while the following four different treatments were given.

[0113] The NC group received no additional treatment, the IL-1 group received only IL-1β, the DTM group received IL-1β followed by DTM, and the MSM-DTM group received IL-1β followed by MSM-DTM. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed, and the relative mRNA expression levels of IL-6 and CCL-2 were then measured. The results are as follows: Figure 16 As shown, Figure 16 The results showed that inflammatory markers IL-6 and CCL-2 decreased significantly after MSM-DTM treatment.

[0114] (12) This invention also demonstrated through Western blot experiments that MSM-DTM can alleviate tendon inflammation. The results of the Western blot experiments are as follows: Figure 17 As shown, the results indicate that MSM-DTM treatment significantly increased COL1A2 (type I collagen α2 chain), a key marker protein for tendon healing. Simultaneously, MMP3 (matrix metalloproteinase 3) decreased, indicating that MSM-DTM alleviated the inflammatory state. DTM alone could not achieve the same effects.

[0115] Figure 16 and Figure 17 The results shown indicate that the MSM-DTM obtained in Example 1 of this invention can alleviate inflammation in tendinopathy.

[0116] (13) This invention found that TSPCs underwent significant senescence after 7 days of treatment with IL-1β. To investigate the effect of MSM-DTM in alleviating TSPC senescence, this invention added MSM-DTM to the IL-1β treatment (MSM-DTM group). Western blot results are as follows: Figure 18 As shown, the results indicated that, compared to the IL-1β group (i.e., treatment with only IL-1β), the p16 and p21 proteins in the MSM-DTM group were significantly downregulated. Figure 18 In this study, the NC group served as the control group without any treatment. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is widely used as an internal reference in biochemical and molecular biology experiments.

[0117] Simultaneously, β-galactosidase staining was performed on each of the above groups, and the results are as follows: Figure 19As shown, the blue color in the MSM-DTM group was significantly reduced compared to other groups, indicating a significant decrease in the positive rate of the MSM-DTM group. This suggests that MSM-DTM can significantly alleviate cellular senescence caused by long-term inflammation.

[0118] (14) The migration and differentiation ability of TSPCs is crucial for tendon repair. This study found that DTM and MSM-DTM can promote the migration and differentiation of TSPCs.

[0119] During tendon aging, osteogenic differentiation capacity increases while tendon-forming capacity decreases. This invention treats TSPCs with DTM and MSM-DTM for 21 days, respectively. Immunofluorescence results showed that the tendon differentiation markers TNMD (tendinogen monoxide) and TNC (tendinogen monoxide-C) were significantly increased in both the DTM and MSM-DTM groups (e.g., ...). Figure 20 , 21 (As shown). Meanwhile, as... Figure 22 As shown, RT-qPCR results also demonstrated that treatment with DTM and MSM-DTM significantly increased the tendon differentiation indices TNC and TNMD.

[0120] This invention conducted Transwell migration experiments, and the experimental results are as follows: Figure 23 As shown in the figure, the results indicate that the migration ability of TSPCs is significantly increased after DTM and MSM-DTM treatment, which demonstrates that DTM and MSM-DTM improve the migration ability of TSPCs.

[0121] (15) This invention further investigated the therapeutic effect of MSM-DTM on the Achilles tendon in vivo. After establishing a rat tendinopathy model by injecting collagenase I, phosphate-buffered saline (PBS), DTM, and MSM-DTM were locally injected into the Achilles tendon, corresponding to the COL group, DTM group, and MSM-DTM group, respectively. The injection process is as follows: Figure 24 As shown. Healthy rats were used as a negative control, and the gross morphology of the tendon was recorded 28 days after injection. Figure 25 As shown, the tendons of healthy rats (NC group) appeared white with no synovial hyperplasia, while those of rats with tendinopathy (COL group) exhibited hyperplastic synovium and yellow tendons. After 4 weeks of local injection, the tendinopathy in the MSM-DTM group was significantly alleviated, showing a white appearance and less synovial hyperplasia, with a more significant improvement than that in the DTM group.

[0122] (16) To further evaluate the efficacy of MSM-DTM, the microstructure of the tendon was also investigated. Tendons from all groups were collected after 4 weeks and subjected to HE staining and Masson staining, as shown below. Figure 26 , 27As shown, after 4 weeks of treatment, the tendons in the COL group exhibited disordered collagen, abundant round nuclei, and uneven ECM (extracellular matrix); however, these abnormal structures of the tendons were slightly improved when treated with DTM, and significantly improved when treated with MSM-DTM. Figure 28 As shown, immunofluorescence staining results indicate that, compared with the COL group and the DTM group, the MSM-DTM group showed a significant increase in type I collagen (COL I), a key marker of tendon healing.

[0123] Figures 25-28 The results showed that MSM-DTM can effectively relieve local inflammation in tendinopathy in vivo.

[0124] (17) Chronic pain is one of the main symptoms of tendinopathy. Repetitive stimulation induces tendon cells to release neurotrophic factors. Neurotrophic factors, such as nerve growth factor (NGF), nourish nerve endings, leading to nerve budding and inward growth of nerve fibers into the tendon. In vitro studies have shown that MSM-DTM has the effect of adsorbing nerve growth factor. Next, this invention investigated the effect of local injection of MSM-DTM in relieving tendinopathy pain in rats. After establishing tendinopathy in rats as described above, the mechanical pain threshold and thermal pain threshold of rats were tested before collagenase I injection, one day before local injection of nanomaterials, and on days 3, 6, 12, 18, 24, and 28 after injection. Figure 29 , 30 The results showed that the thermal and mechanical pain thresholds of rats with tendinopathy were significantly decreased. On days 3, 6, and 12 after local injection, the mechanical and thermal pain thresholds of rats in the MSM-DTM group were significantly higher than those in the COL group. After 18 days of treatment, the mechanical and thermal pain thresholds in the MSM-DTM group essentially returned to normal, while those in the COL group took 28 days to return to normal. A low pain threshold indicates sensitivity to pain. MSM-DTM increased the pain threshold in the experimental animals, indicating relief of pain sensitivity, which indirectly explains the relief of tendinopathy pain. Therefore, the test results demonstrate that MSM-DTM accelerates the recovery of the pain threshold in tendinopathy and alleviates tendinopathy pain.

[0125] Meanwhile, we conducted Achilles tendon function tests on the animals 4 weeks after modeling. The Achilles tendon function test method was as follows: the rats walked freely on a path covered with white paper, which was 80cm long and 15cm wide (the rats' hind paws were coated with black ink, and they left footprints on the white paper after walking) (rat footprints are shown in the figure). Figure 31 As shown), the Achilles tendon function index (AFI) was calculated by analyzing the rat footprints. Figure 32 (As shown). The NC group consisted of normal rats, the COL group consisted of rats with tendinopathy, the DTM group consisted of rats with tendinopathy that received local injections of DTM, and the MSM-DTM group consisted of rats with tendinopathy that received local injections of MSM-DTM. Figure 31 and 32The results showed that the Achilles tendon function of rats was significantly restored 28 days after local injection of MSM-DTM.

[0126] It should be noted that, in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0127] This invention is not limited to the embodiments described above. Those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications are also considered within the scope of protection of this invention. Contents not described in detail in this specification are prior art known to those skilled in the art.

Claims

1. A method for preparing nanomaterials for relieving tendinopathy inflammation and pain, characterized in that, The preparation method includes: uniformly mixing decellularized tendon DTM with hybrid membrane vesicles of macrophages and nerve cells (MSM) to obtain the nanomaterial; The method for preparing the MSM includes: extracting the cell membranes of macrophages and nerve cells, mixing the two cell membranes to obtain a mixed cell membrane solution, and obtaining the MSM by extrusion of the mixed cell membrane solution; The method for preparing DTM includes: freeze-drying, grinding, and sterilizing the decellularized tendon tissue in sequence, followed by enzymatic hydrolysis and gelation to obtain gel-like DTM.

2. The method for preparing nanomaterials for relieving tendinopathy inflammation and pain as described in claim 1, characterized in that, The method for extracting the cell membranes of macrophages and nerve cells includes: performing a first centrifugation, ultrasonic disruption, and a second centrifugation on macrophages and nerve cells respectively, to obtain cell membrane precipitates of macrophages and nerve cells respectively, wherein the ratio of macrophages to nerve cells is 1:

1.

3. The method for preparing nanomaterials for relieving tendinopathy inflammation and pain as described in claim 1, characterized in that, The specific steps of the extrusion method include: extruding the mixed cell membrane solution through a polycarbonate porous membrane to obtain MSM with an average particle size of 160-240 nm.

4. The method for preparing nanomaterials for relieving tendinopathy inflammation and pain as described in claim 1, characterized in that, The freeze-drying step includes: placing the decellularized tendon tissue in an environment of -22~-18℃ for 12-14 hours and then freeze-drying it under vacuum; And / or, the grinding step includes: grinding the freeze-dried tendon tissue into powder with a particle size of less than 1 mm; And / or, the sterilization step includes: placing the ground tendon tissue and a culture dish filled with ethanol together in an incubator at 37°C for 18-24 hours.

5. The method for preparing nanomaterials for relieving tendinopathy inflammation and pain as described in claim 1, characterized in that, The enzymatic hydrolysis method specifically includes: adding the sterilized tendon tissue into a pepsin solution and stirring continuously at 22-28°C for 24-48 hours to obtain a DTM solution; And / or the method for preparing the pepsin solution includes: dissolving pepsin in hydrochloric acid at pH=2 to obtain the pepsin solution; And / or, the concentration of the pepsin solution is 1 mg / mL; And / or, the mass ratio of tendon tissue to pepsin after the sterilization treatment is (5-10):

1.

6. The method for preparing nanomaterials for relieving tendinopathy inflammation and pain as described in claim 1, characterized in that, The gelation method specifically includes: adjusting the pH of the DTM solution obtained after enzymatic hydrolysis to neutral, and then placing it in a 37°C water bath for 30 minutes to obtain gel-like DTM.

7. A nanomaterial for relieving tendinopathy inflammation and pain, prepared by the method described in any one of claims 1 to 6, characterized in that, The nanomaterials include decellularized tendon DTM, macrophage and nerve cell hybrid membrane vesicles MSM.

8. The nanomaterial for relieving tendinopathy inflammation and pain as described in claim 7, characterized in that, In the nanomaterial, the mass ratio of the MSM to the volume ratio of the DTM is 40-120 μg / mL.

Images