Temperature-sensitive injectable pedunculoside hydrogel as well as preparation method and application of temperature-sensitive injectable pedunculoside hydrogel

Abstract

The invention relates to the technical field of medicines, and discloses pedunculoside temperature-sensitive injectable hydrogel as well as a preparation method and application thereof. Specifically, the invention provides application of the temperature-sensitive injectable hydrogel with the pedunculoside in preparation of the medicine for treating the cardiovascular diseases, and the temperature-sensitive injectable hydrogel with the pedunculoside comprises the pedunculoside, a lactic acid-glycolic acid copolymer, polyvinyl alcohol and dipolylactic acid-glycolic acid copolymer polyethylene glycol. The microsphere-gel combined delivery preparation PE-M / Ge prepared by the preparation method disclosed by the invention is good in injectability and has a good temperature-sensitive gelling characteristic in vitro; the obvious gel property is realized, and the microspheres are uniformly distributed in the gel; the release of the drug can be effectively reduced, the release curve is closer to zero-order release, the dual controlled release system can more stably release the drug, the toxic and side effects caused by non-characteristic distribution of PE are reduced, and the bioavailability is improved. And the PE-M / Ge has no cytotoxicity to H9c2 cells and HUVECs cells, so that a foundation is laid for in-vivo pharmacodynamic experiments of rats.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to a thermosensitive injectable hydrogel of irisin and a preparation method and application thereof. Background Art

[0002] Cardiovascular disease is currently the leading cause of death in humans, and most of them are related to myocardial ischemia. Myocardial infarction (MI) refers to long-term local myocardial ischemia caused by coronary artery sclerosis and (or) blockage, which leads to irreversible diffuse myocardial fibrosis and necrosis.

[0003] Jiubiying, also known as Bear Gallwood, White Wood, and Sheep Does Not Eat, was first recorded in Lingnan Collection of Medicinal Herbs. It is the dried bark or root bark of the Ilex rotunda Thunb. of the Aquifoliaceae family. It tastes bitter and is cold in nature. It enters the lung, stomach, large intestine, and liver meridians. It has the effects of clearing away heat and detoxifying, promoting dampness and relieving pain. It is mainly used to treat summer heat fever, sore throat, damp-heat diarrhea, abdominal distension and pain, rheumatic arthralgia, eczema, sores, and injuries from falls. Main pharmacological effects: (1) Antibacterial effect; (2) Analgesic and anti-inflammatory effects; (3) Hepatoprotective effect; (4) Anti-tumor effect; (5) Cardiovascular protection effect.

[0004] Pedunculoside (PE) is a triterpenoid saponin component extracted from the herb Psoralea corylifolia and is one of its main active ingredients. The current literature reports that the pharmacological effects of PE include: hepatoprotective effect and cardiovascular protection effect. However, the oral bioavailability of PE is low. The hydrogel can provide mechanical support for weak myocardial tissue, alleviate the biological and mechanical pathophysiological vicious cycle of tissues after myocardial infarction, and at the same time has strong plasticity and good tissue compatibility, and can release solvent drugs for a long time. Therefore, the present invention proposes to improve its bioavailability by preparing a PE hydrogel preparation.

[0005] The structural formula of hollyglucoside is as follows:

[0006]

[0007] At present, the research on thermosensitive hydrogels is quite extensive, but the delivery of a single preparation often results in burst release. The combination of different preparations for multiple controlled release can make the drug release more uniform and reduce the toxic and side effects caused by burst release. Microsphere preparations are widely used local in situ drug delivery dosage forms in high-efficiency preparations, that is, the drug is dispersed in microspheres formed by polymer materials. In clinical practice, in situ administration or subcutaneous injection is mainly used to form a drug reservoir at the lesion site, improve the targeting of the drug, reduce the dosage and frequency of administration, prolong the drug's action time, and effectively avoid the peak and valley phenomenon of the drug in the body caused by the frequent administration of conventional preparations, thereby reducing toxic and side effects and improving efficacy.

[0008] Therefore, the present invention adopts an emulsified solvent evaporation method to prepare pyrifos glycoside microspheres (PE-M), and then embeds the microspheres in PLGA-PEG-PLGA hydrogel to prepare pyrifos glycoside microsphere hydrogel (PE-M / Gel), and systematically characterizes and investigates the biocompatibility in vitro to lay a foundation for its further in vivo pharmacodynamics. Summary of the invention

[0009] The purpose of the present invention is to provide a thermosensitive injectable hydrogel of hollyhock glycoside with high bioavailability, and a preparation method and application thereof. By preparing hollyhock glycoside microspheres by an emulsified solvent volatilization method, and embedding the microspheres in a PLGA-PEG-PLGA hydrogel to prepare the hollyhock glycoside microsphere hydrogel, more uniform drug release can be achieved, toxic side effects caused by sudden release can be avoided, and the therapeutic effect of the drug in treating cardiovascular diseases can be improved.

[0010] In order to achieve the above object, the technical solution of the present invention is as follows:

[0011] The invention provides a thermosensitive injectable hydrogel of hollyhock glycoside. The thermosensitive injectable hydrogel of hollyhock glycoside comprises hollyhock glycoside, lactic acid-glycolic acid copolymer, polyvinyl alcohol and dipolylactic acid-glycolic acid copolymer polyethylene glycol.

[0012] Furthermore, the drug loading amount of the hollygold glycoside is 0.50-0.72%.

[0013] The present invention also provides a method for preparing a thermosensitive injectable hydrogel of hollyglucoside, comprising the following steps:

[0014] S1, preparing polyvinyl alcohol (PVA) solution and NaCl solution;

[0015] S2, preparing pyrifos glycoside microspheres PE-M by emulsification solvent evaporation method;

[0016] S3. Preparation of phytosylvatica glycoside hydrogel PE-M / Gel.

[0017] Preferably, in step S1, the polyvinyl alcohol (PVA) solution is a PVA solution with a mass fraction of 1%, and the NaCl solution is a NaCl solution with a molar concentration of 0.5 mol / L.

[0018] Preferably, in step S2, the method for preparing the pyrifos glycoside microspheres PE-M comprises the following steps:

[0019] S21, dissolving: firstly, adding an appropriate amount of hollyglucoside to an organic reagent and dissolving it by ultrasonication, adding lactic acid-glycolic acid copolymer PLGA to the organic reagent solution, and heating and dissolving in a 40° C. water bath to obtain an organic solution of hollyglucoside;

[0020] S22, emulsification: adding 2 ml of the polyvinyl alcohol (PVA) solution prepared in step S1 to the organic solution of hollygol prepared in step S21, and centrifuging;

[0021] S23, solidification: pour the centrifuged solution obtained in step S22 into 10 ml of the NaCl solution prepared in step S1, stir for 4 hours and then evaporate the solvent to obtain a solid;

[0022] S24, washing: the solid obtained in step S23 is washed three times with ultrapure water.

[0023] S25, freeze-drying: the washed product obtained in step S24 is placed in a -80°C refrigerator for pre-freezing overnight, and then freeze-dried to obtain PE-M.

[0024] Preferably, the organic reagent is ethyl acetate, the mass volume ratio of the euphorbia glabra glycoside to ethyl acetate is 1 mg:1 ml, and the mass ratio of the euphorbia glabra glycoside to the lactic acid-glycolic acid copolymer PLGA is 1:200.

[0025] Preferably, in step S22, the specific parameters of the centrifugal treatment are a temperature of 4°C, a rotation speed of 16000 rpm, and a centrifugal time of not less than 60 s.

[0026] Preferably, in step S24, each cleaning step needs to be centrifuged at 4500 rpm for at least 10 minutes.

[0027] 11. Preferably, in step S3, the preparation method of the pyrifos glycoside hydrogel PE-M / Gel is as follows: weigh PE-M with a mass fraction of M1 (0%<M1≤25%), add it to a thermosensitive blank hydrogel solution in an ice bath, and stir evenly to form a suspension solution to obtain the pyrifos glycoside microsphere hydrogel. The thermosensitive blank hydrogel is prepared by adding a certain amount of PLGA-PEG-PLGA carrier to 4°C deionized water to prepare a gel solution with a concentration of 20%.

[0028] The present invention also provides an application of the thermosensitive injectable hydrogel of hollyhock glycoside in the preparation of cardiovascular treatment drugs.

[0029] In summary, the present invention has the following beneficial effects:

[0030] 1. The present invention adopts the O / W single emulsion solvent evaporation method to prepare PE-M microspheres with good dispersibility, a particle size range of 18.33±1.63μm, an encapsulation rate of 95.5%, a yield of 85%, uniform microsphere size, uniform distribution of drugs in the microspheres, and good stability.

[0031] 2. The microsphere-gel combined delivery preparation PE-M / Gel prepared by the present invention has good thermosensitive gelling properties in vitro; it has significant gel properties and good injectability, and the microspheres are evenly distributed in the gel.

[0032] 3. The microsphere-gel combined delivery preparation PE-M / Gel prepared by the present invention overcomes the technical problem that the single PE preparation has a certain burst release phenomenon in vitro, and can effectively reduce the release of drugs, making its release curve closer to zero-order release. In addition, PE-M / Gel has a dual controlled release system that can more stably control the release of drugs, which is beneficial to reduce the toxic and side effects caused by the non-characteristic distribution of PE and improve its bioavailability. At the same time, PE-M / Gel has no cytotoxicity to H9c2 cells and HUVECs cells, laying the foundation for further in vivo pharmacodynamic experiments in rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a diagram of the preparation process of the thermosensitive injectable hydrogel of pyrifos glycoside (PE-M / Gel);

[0034] Figure 2 is a morphological characteristic diagram of PE-M in Example 1 (A is a morphological characteristic diagram of PE-M in liquid state, and B is a morphological characteristic diagram of PE-M in solid state);

[0035] Figure 3 is the in vitro release curve of PE-M in Example 1;

[0036] Figure 4 The states of the blank hydrogel and PE-M / Gel in Example 2 at different temperatures (A is a liquid sol image of the blank hydrogel Gel at 4°C, B is a gel image of the blank hydrogel Gel at 37°C, C is a liquid sol image of the PE-M / Gel containing 5% PE-M at 4°C, and D is a gel image of the PE-M / Gel containing 5% PE-M at 37°C);

[0037] Figure 5 is the modulus change of the blank hydrogel and PE-M / Gel in Example 2 for frequency and shear stress scanning (A is the modulus change for frequency scanning, B is the modulus change for shear stress scanning);

[0038] Figure 6 The side view and top view of the PE-M / Gel injected into 37°C water and its gelled state in Example 2 (A is the side view, B is the top view);

[0039] Figure 7 The effect of the PE-M content in PE-M / Gel in Example 2 on its water uptake and gel time (A is the effect on water uptake, B is the effect on gel time);

[0040] Figure 8 This is a graph showing the gelling properties of PE-M / Gel in Example 2 (containing a small amount of Evans Blue, where A is a liquid hydrogel and B is a semi-solid hydrogel);

[0041] Fig. 9 is the in vitro release curve of PE-M / Gel in Example 2;

[0042] Fig.10 The cytotoxicity of Gel and PE-M / Gel in Example 2 on H9C2 cells and HUVECs cells and their effects on cell proliferation. DETAILED DESCRIPTION

[0043] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0044] like Figure 1 As shown, the present invention provides a method for preparing a thermosensitive injectable hydrogel of hollyglucoside, comprising the following steps:

[0045] S1. Prepare a polyvinyl alcohol (PVA) solution with a mass fraction of 1% and a NaCl solution with a molar concentration of 0.5 mol / L.

[0046] S2, preparing pyrifos glycoside microspheres PE-M by an emulsified solvent volatilization method, comprising the following steps:

[0047] S21, dissolving: firstly, adding an appropriate amount of pyrifos glycoside to an ethyl acetate solution and ultrasonicating for 30 minutes to fully dissolve it, adding lactic acid-glycolic acid copolymer PLGA to the above ethyl acetate solution, heating in a 40° C. water bath to dissolve it, and obtaining an ethyl acetate solution of pyrifos glycoside;

[0048] The mass volume ratio of the euphorbia glabra glycoside to ethyl acetate is 1 mg:1 ml, and the mass ratio of the euphorbia glabra glycoside to the lactic acid-glycolic acid copolymer PLGA is 1:200.

[0049] S22, emulsification: add 2 ml of the 1% polyvinyl alcohol (PVA) solution prepared in step S1 to the ethyl acetate solution of euphorbia glabra glycoside prepared in step S21, and centrifuge at 4°C and 16000 rpm for 60 seconds.

[0050] S23, solidification: the centrifuged solution obtained in step S22 is quickly poured into 10 ml of the 0.5 mol / L NaCl solution prepared in step S1, and the solvent is evaporated after stirring for 4 hours to obtain a solid;

[0051] S24, washing: the solid obtained in step S23 was washed with ultrapure water for 3 times, and each time the solid was centrifuged at 4500 rpm for 10 min.

[0052] S25, freeze-drying: the washed product obtained in step S24 is placed in a -80°C refrigerator for pre-freezing overnight, and then freeze-dried to obtain PE-M.

[0053] S3. Preparation of pyrifos glycoside hydrogel PE-M / Gel, the method is as follows:

[0054] First, a certain amount of PLGA-PEG-PLGA carrier was weighed, and deionized water at 4°C was added to prepare a 20% concentration gel solution to obtain a thermosensitive blank hydrogel. Then, PE-M with a mass fraction of M1 (0% < M1 ≤ 25%) was weighed, and added to the blank thermosensitive hydrogel solution in an ice bath. The solution was stirred evenly to form a suspension solution, and the hollyglucoside microsphere hydrogel was obtained.

[0055] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0056] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0057] Embodiment 1:

[0058] Preparation of phytochrome glycoside microspheres (PE-M)

[0059] 1 Experimental methods

[0060] 1.1 Solution preparation

[0061] 1% polyvinyl alcohol (PVA) solution: weigh 10.08 g PVA and add it into 1000 mL ultrapure water, stirring until it is completely dissolved;

[0062] 0.5Mol / L NaCl solution: weigh 29.22g NaCl powder and add it into 1000mL ultrapure water, stirring until it is completely dissolved.

[0063] 1.2 Preparation of PE-M microspheres

[0064] PE-M was prepared by using the emulsified solvent evaporation method and using the biodegradable material poly(latic-co-glycolic acid), PLGA as a carrier. The preparation method is as follows:

[0065] (1) Weighing: ① Weigh 1 mg of pyrifos glycoside, add 1 mL of ethyl acetate, and sonicate for 30 min to fully dissolve. ② Weigh 200 mg of PLGA (PLGA needs to be taken out of the -20°C refrigerator and placed at room temperature for 30 min to avoid moisture absorption), add it to the above ethyl acetate solution, and heat in a 40°C water bath to dissolve.

[0066] (2) Emulsification: Add the above ethyl acetate solution into 2 mL of 1% PVA solution and centrifuge at 4°C, 16,000 rpm for 60 s.

[0067] (3) Solidification: The above solutions were quickly poured into 10 mL of 0.5 M NaCl solution, stirred at room temperature for 4 h, and the solvent was evaporated to form PE-M doped products.

[0068] (4) Washing: The PE-M doped product was washed three times with ultrapure water (centrifugation at 4500 rpm for 10 min).

[0069] (5) Freeze-drying: Pre-freeze in a -80°C refrigerator overnight, and then freeze-dry to obtain PE-M.

[0070] 1.3 Characterization of PE-M

[0071] 1.3.1 Observation of PE-M appearance

[0072] A certain amount of PE-M freeze-dried powder was placed on a glass slide, and the freeze-dried powder was dispersed with an appropriate amount of ultrapure water. The surface morphology, actual size, and structural shape of PE-M were observed under an optical microscope, and photos were taken.

[0073] 1.2.1 PE-M particle size determination

[0074] Weigh an appropriate amount of PE-M and dissolve it in a 5g / L Tween 80 aqueous solution to make the PE-M concentration 1mg / mL. Disperse it by ultrasonication for 10 minutes, and then immediately measure the particle size with a laser particle size analyzer. Repeat the measurement 3 times for each sample and take the average value.

[0075] 1.3.3 PE-M encapsulation efficiency determination

[0076] Sample treatment: 20 mg of PE-M was accurately weighed, 500 μL of methanol was added, ultrasonicated for 10 min, centrifuged at 12000 rpm for 10 min, the supernatant was taken, and the PE content was determined by HPLC.

[0077] HPLC chromatographic conditions: Kromasil C 18Column (4.6×250mm, 5.0μm), with 0.1% formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B for gradient elution (0-10min, 30-75% B; 10-11min, 75%-95% B; 11-14min, 95% B; 14-15min, 95-30% B; 15-18min, 70% B), total running time: 18min. UV detection wavelength: 215nm, flow rate: 0.8mL / min, column temperature: 40℃, injection volume: 20μL.

[0078] Encapsulation efficiency calculation formula:

[0079]

[0080] 1.3.4 Determination of drug loading of PE-M

[0081] Drug loading calculation formula:

[0082]

[0083] 1.3.5 PE-M yield determination

[0084] The total weight of the obtained PE-M is: m1, the weight of the PE feed is: m2, the weight of the PLGA feed is: m3, and the PE-M yield calculation formula is as follows:

[0085]

[0086] 1.3.6 PE-M in vitro drug release assay

[0087] Weigh 20 mg of PE-M accurately and place it in a 50 mL round bottom centrifuge tube, add 45 mL of pH 7.4 PBS buffer solution, place it in a 37 ° C constant temperature oscillator and oscillate at a speed of 100 rpm, and centrifuge it at 12000 rpm for 10 minutes at a predetermined time point, take 5 mL of the supernatant, sample it every 24 hours, and add an equal amount of release medium, continue constant temperature oscillation, and continuously sample for 14 days. The obtained samples are stored at 4 ° C. The same pH 7.4 PBS buffer solution is used as a blank control, and the absorbance value is measured using a UV spectrophotometer with a fixed wavelength (215nm), repeated 3 times, and the results are averaged. The concentration of PE in the sample, the cumulative release percentage, and the total amount of PE released are obtained by the standard curve equation, and the PE-M in vitro release curve is drawn with time as the horizontal axis and the cumulative release percentage as the vertical axis.

[0088] 1.3.7 PE-M body stability study

[0089] Weigh 30 mg of PE-M, divide it into 2 mL centrifuge tubes, and store them under different conditions (room temperature, 4°C, -20°C, -80°C). Sampling is performed at 0, 7, 14, 21, and 28 days to determine the PE-M particle size and PE content.

[0090] 2. Experimental results

[0091] 2.1 PE-M Characterization

[0092] 2.1.1 Observation of PE-M appearance

[0093] The prepared PE-M was observed to be fine white powder by naked eyes. PE-M was observed to be regular spherical with slightly rough surface and uniform size under optical microscope. Figure 2 shown.

[0094] 3.1.2 Determination of PE-M particle size, encapsulation efficiency, drug loading and yield

[0095] As shown in Table 1, the particle size of PE-M was (18.33±1.63) μm, the encapsulation efficiency was (95.17±12.09)%, the drug loading was (0.61±0.11)%, and the yield was (85.50±4.51)%.

[0096] Table 1 PE-M particle size, encapsulation efficiency, drug loading test results

[0097]

[0098] 1.1.3 In vitro drug release of PE-M

[0099] The cumulative drug release of PE-M in 14 days reached (94.05±5.80)%. Figure 3 .

[0100] 1.1.4PE-M stability

[0101] The PE-M particle size and PE content at 0, 7, and 14 days were investigated under storage conditions of room temperature, 4°C, -20°C, and -80°C. The results are shown in Table 2. PE-M showed good stability.

[0102] Table 2 PE-M stability investigation results

[0103]

[0104] In summary, the microspheres prepared by the O / W single emulsion solvent evaporation method in the present invention have good dispersibility, a particle size range of 18.33±1.63 μm, an encapsulation rate of 95.5%, a yield of 85%, uniform size of the microspheres, uniform distribution of the drugs in the microspheres, and good stability.

[0105] Embodiment 2:

[0106] Preparation of phytochrome hydrogel (PE-M / Gel)

[0107] 1. Experimental methods

[0108] 1.1.1 Preparation of thermosensitive blank hydrogel

[0109] A certain amount of PLGA-PEG-PLGA carrier was weighed and added to 4° C. deionized water to prepare a 20% concentration gel solution to obtain a thermosensitive blank hydrogel.

[0110] 1.1.2 Preparation of PE-M / Gel

[0111] A certain amount of PE-M prepared in Example 1 was weighed and added to the blank hydrogel solution in an ice bath, and stirred evenly to form a suspension solution to obtain the hollygold glycoside microsphere hydrogel, which was stored in a refrigerator at 4°C for later use.

[0112] 1.1 Characterization of PE-M / Gel

[0113] 1.1.1 PE-M / Gel appearance and morphology

[0114] Weigh a certain amount of hollygold microsphere hydrogel and add it to 4℃ water, place it in a 4℃ refrigerator to swell to obtain low-temperature liquid hydrogel, keep taking pictures at low temperature, put the hydrogel in a 37℃ water bath, wait for it to gel to form a solid hydrogel, and then continue to take pictures with a camera to record its appearance changes. Take pictures of the appearance of blank hydrogel and PE-M / Gel respectively.

[0115] 1.1.2 Rheological characterization of PE-M / Gel

[0116] The mechanical characteristics of PE-M / Gel were analyzed and characterized by rotational rheometer.

[0117] (1) Stress scanning: strain amplitude 0.1%-10%, angular frequency 1 rad / s, temperature 25°C, and obtain the graphs of elastic modulus G' and viscous modulus G" versus stress. Scans were performed on 20% Gel, 20% Gel containing 10% PE-M, and 20% Gel containing 1% PE-M, respectively.

[0118] (2) Frequency sweep: strain amplitude 1%, angular frequency 0.1-100 rad / s, temperature 25°C, and obtain the graph of elastic modulus G' and viscous modulus G" versus frequency.

[0119] 1.1.3 Characterization of PE-M / Gel Injectability

[0120] Prepare a set of 50 mL small beakers, add about 40 mL of 37 °C water, use a 1 mL syringe to draw 4 °C liquid PE-M / Gel and inject it into the water, observe the gelation of the hydrogel and take photos to record.

[0121] 1.1.4 Effect of PE-M content on water content of PE-M / Gel

[0122] Prepare six groups of 5mL vials, weigh m0 first after marking, add a certain amount of completely swollen blank hydrogel and different amounts of PE-M (PE-M accounts for 0, 5%, 10%, 15%, 20% and 25%) to each bottle, mix evenly to form PE-M / Gel, let it stand at room temperature for 5 minutes, weigh m1 again after it is fully gelled, put it in a -80℃ refrigerator for pre-freezing overnight, freeze-dry it in a freeze dryer for 48 hours the next day, weigh m2 again after freeze-drying, and calculate the effect of changes in PE-M content on the water content of PE-M / Gel according to the following formula.

[0123]

[0124] 1.1.5 Effect of PE-M content on gelation time of PE-M / Gel

[0125] Prepare six groups of 5mL glass tubes, place them in 4℃ water, add 1mL of blank hydrogel (20%, W / V) and different amounts of PE-M (PE-M accounts for 0, 5%, 10%, 15%, 20%, 25%), mix well, take out and quickly wipe off the water on the wall of the glass tube, place it at room temperature and start timing, and gently invert the test tube after 60s to observe whether the hydrogel is gelled. According to the preliminary experiment, the hydrogel gelation in the time period of 60-90s is concentrated. When the hydrogel is viscous and does not flow down, it is considered that a solid hydrogel is formed, and this period of time is recorded. Each group contains at least 3 replicates.

[0126] 1.1.6 Investigation of PE-M / Gel gelling properties

[0127] Prepare a circular mold, add 200 μL of PE-M / Gel into the circular groove, and observe and record the distribution of hydrogel in the groove and its gelation at room temperature to investigate the gelation characteristics of PE-M / Gel in the mold groove.

[0128] 1.1.7 In vitro release study of PE-M / Gel

[0129] First, PE-M and Gel were mixed to form PE-M / Gel, and then placed in a 50mL round-bottom centrifuge tube, placed in a 37°C constant temperature box for 5 minutes, and after it fully formed a solid hydrogel, added to 45mL of pH 7.4 PBS solution, placed in a 37°C constant temperature oscillator and oscillated at 100rpm, and centrifuged at 12000rpm for 10 minutes at a predetermined time point, and 5mL of supernatant was taken. Samples were taken every 24 hours, and an equal amount of release medium was added at the same time. Constant temperature oscillation was continued, and samples were taken continuously for 14 days. The samples obtained were stored at 4°C. The determination method is the same as 1.1.6 PE-M in vitro drug release determination, and the cumulative release of the drug over time is plotted.

[0130] 1.1.8 PE-M / Gel Cell Biocompatibility Test

[0131] The specific experimental operation is as follows: (1) Preparation of cell suspension: After the cultured H9c2 cells were washed once with 5 mL PBS, 1 mL of trypsin was added. When most of the cells became round and fell off, 5 mL of complete culture medium was added to terminate the digestion. After gently mixing, the cells were aspirated, centrifuged at 1200 rpm for 3 min, the supernatant was discarded, and 1 mL of culture medium was added to prepare a cell suspension. (2) PE-M / Gel was spread on the bottom of the 96-well plate: After adding the following different concentrations of PE-M / Gel to the cultured 96-well plate, it was placed in a 37°C incubator for 10 min to make it gel. (3) H9c2 cells were spread on the top of the hydrogel: 100 μL of cell suspension was added to each well of the 96-well plate, and the cells were cultured in the incubator for 24 h. (4) Addition of CCK-8 solution: 10 μL of CCK-8 solution was added to each well, and the culture plate was incubated in the incubator for 2 h. (5) Determination: The absorbance at 450 nm was measured using an ELISA reader. The cell viability calculation formula is as follows:

[0132] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0133] As: Experimental well (containing cell culture medium, CCK-8, test substance)

[0134] Ac: control well (containing cell culture medium, CCK-8, no test substance)

[0135] Ab: Blank well (medium without cells and test substance, CCK-8)

[0136] 2 Experimental results

[0137] 2.1 PE-M / Gel Characterization

[0138] 2.1.1 Characterization of PE-M / Gel morphology

[0139] The obtained thermosensitive hydrogel PE-M / Gel of hollyhock glycosides is in a white hydrogel state. Figure 4 It is a flowable liquid at 4°C and quickly forms a semi-solid hydrogel at 37°C, with obvious hydrogel properties.

[0140] 2.1.2 Rheological characterization of PE-M / Gel

[0141] This experiment investigated the mechanical strength and viscoelasticity of the hydrogel. Figure 5 As shown in Figure A, the frequency scanning results show that as the shear frequency increases, the elastic modulus G' of the hydrogel system is greater than the viscous modulus G", and it also increases accordingly, indicating that the hydrogel is frequency-dependent. The addition of PE-M will reduce the elastic modulus G' and the viscous modulus G" to a certain extent. This is because the addition of PE-M occupies a certain space, which makes the cross-linking degree between the hydrogel materials lower. Figure 5 As shown in B, the stress scanning results show that Gel or PE-M / Gel decreases slowly with the increase of shear force, among which PE-M / Gel changes very little and remains basically unchanged with only slight fluctuations, indicating that the system can maintain a good gelling system.

[0142] 2.1.3 Characterization of PE-M / Gel Injectability

[0143] In order to investigate the injectability of PE-M / Gel and simulate its gelation in vivo, the experiment slowly injected the cryopreserved PE-M / Gel into 37°C water. The injection process was relatively smooth without obvious resistance, and the injectability was good. The PE-M / Gel injected into the water quickly formed a gel, causing the gelation speed to be very fast, so a clear linear gel was formed, such as Figure 6 The experimental results show that PE-M / Gel can also gel quickly in vivo and has good gelling properties.

[0144] 2.1.4 Characterization of water uptake of PE-M / Gel

[0145] In order to investigate the changes in water uptake of PE-M / Gel, the gel was freeze-dried and weighed to calculate the water uptake. Figure 7 As shown in A, the experimental results show that the water uptake of PE-M / Gel accounts for 70-82% of the total preparation, and the addition of PE-M has no significant effect on the water uptake of the gel.

[0146] 2.1.5 Effect of PE-M content on gelation time of PE-M / Gel

[0147] The gelation time of Gel and PE-M / Gel at 37℃ was 78-88s. Figure 7As shown in B. With the increase of PE-M content, the gelation time of PE-M / Gel was slightly prolonged, but there was no significant change compared with the blank gel. This shows that 0-20% PE-M has no significant effect on the gelation properties and gelation time of Gel, and PE-M / Gel still has good temperature sensitivity.

[0148] 2.1.6 Investigation of PE-M / Gel Gelation Properties

[0149] The PE-M / Gel stored at 4°C was injected into the circular mold. The gel preparation was quickly and evenly dispersed and spread inside the mold. After it gelled, the gel was peeled off to form a regular circle. Figure 8 As shown, it can be seen that PE-M / Gel has good gelling properties.

[0150] 2.1.7 In vitro release study of PE-M and PE-M / Gel

[0151] Drug burst release is a common problem. If the drug release reaches 10-80% of the total amount within 24 hours of administration, it is considered as burst release. The cumulative release curve of PE-M / Gel within 14 days is closer to zero-order release, such as Fig. 9 This indicates that the combination of microspheres and gels can effectively control the sudden release of drugs, effectively reduce drug toxicity and side effects, and improve bioavailability.

[0152] 2.1.8 PE-M / Gel Cytotoxicity Study

[0153] The cytotoxic effects of 0-25% Gel on H9c2 cells and HUVECs cells were investigated. Fig.10 The results showed that the concentration of Gel within 20% had no effect on the viability of H9C2 cells and HUVECs cells, while the concentration of Gel at 25% inhibited cell growth. At the same time, PE-M / Gel loaded with 2.5-10μM PE significantly promoted the proliferation of H9c2 cells, and 10μM PE-M / Gel promoted the proliferation of HUVECs cells.

[0154] In summary, the PE-M microspheres prepared in Example 1 are used as raw materials to prepare the microsphere-gel combined delivery preparation PE-M / Gel that meets the requirements. The above experimental results systematically characterize that PE-M / Gel has good thermosensitive gelling properties in vitro. Rheology proves that the microsphere gel preparation has significant gel properties, good injectability, and the microspheres are evenly distributed in the gel. The release of a single PE preparation in vitro has a certain burst release phenomenon, while PE-M / Gel can effectively reduce the release of the drug, making its release curve closer to zero-order release, which shows that the PE-M / Gel dual controlled release system can release the drug more stably, which is conducive to reducing the toxic and side effects caused by the non-characteristic distribution of PE and improving its bioavailability. At the same time, PE-M / Gel has no cytotoxicity to H9c2 cells and HUVECs cells. This experimental result lays the foundation for further in vivo pharmacodynamic experiments in rats.

[0155] The above-described embodiments only express the preferred implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A thermosensitive injectable hydrogel of hollyhock glycoside, characterized in that: The thermosensitive injectable hydrogel of hollyhock glycoside comprises hollyhock glycoside, lactic acid-glycolic acid copolymer, polyvinyl alcohol and di-lactic acid-glycolic acid copolymer polyethylene glycol.

2. The thermosensitive injectable hydrogel of irisin according to claim 1, characterized in that: The drug loading amount of silene glycoside in the silene glycoside microspheres in the thermosensitive hydrogel is 0.50-0.72%.

3. A method for preparing the thermosensitive injectable hydrogel of irisin according to claim 1 or claim 2, characterized in that: The steps include: S1, preparing polyvinyl alcohol (PVA) solution and NaCl solution; S2, preparing pyrifos glycoside microspheres PE-M by emulsification solvent evaporation method; S3. Preparation of phytosylvatica glycoside hydrogel PE-M / Gel.

4. The method for preparing the thermosensitive injectable hydrogel of irisin according to claim 3, characterized in that: In step S1, the polyvinyl alcohol (PVA) solution is a PVA solution with a mass fraction of 1%, and the NaCl solution is a NaCl solution with a molar concentration of 0.5 mol / L.

5. The method for preparing a thermosensitive injectable hydrogel of irisin according to claim 4, characterized in that: In step S2, the preparation method of the pyrifos glycoside microspheres PE-M comprises the following steps: S21, dissolving: firstly, adding an appropriate amount of hollyglucoside to an organic reagent and dissolving it by ultrasonication, adding lactic acid-glycolic acid copolymer PLGA to the organic reagent solution, and heating and dissolving in a 40° C. water bath to obtain an organic solution of hollyglucoside; S22, emulsification: adding 2 ml of the polyvinyl alcohol (PVA) solution prepared in step S1 to the organic solution of hollygol prepared in step S21, and centrifuging; S23, solidification: pour the centrifuged solution obtained in step S22 into 10 ml of the NaCl solution prepared in step S1, stir for 4 hours and then evaporate the solvent to obtain a solid; S24, washing: the solid obtained in step S23 is washed three times with ultrapure water. S25, freeze-drying: the washed product obtained in step S24 is placed in a -80°C refrigerator for pre-freezing overnight, and then freeze-dried to obtain PE-M.

6. The method for preparing the thermosensitive injectable hydrogel of irisin according to claim 5, characterized in that: The organic reagent is ethyl acetate, the mass volume ratio of the euphorbia glabra glycoside to ethyl acetate is 1 mg:1 ml, and the mass ratio of the euphorbia glabra glycoside to the lactic acid-glycolic acid copolymer PLGA is 1:

200.

7. The method for preparing the thermosensitive injectable hydrogel of irisin according to claim 5, characterized in that: In step S22, the specific parameters of the centrifugal treatment are a temperature of 4°C, a rotation speed of 16000 rpm, and a centrifugal time of not less than 60 seconds.

8. The method for preparing the thermosensitive injectable hydrogel of irisin according to claim 5, characterized in that: In step S24, each cleaning step needs to be centrifuged at 4500 rpm for at least 10 minutes.

9. The method for preparing the thermosensitive injectable hydrogel of irisin according to claim 3, characterized in that: In step S3, the preparation method of the pyrifos glycoside hydrogel PE-M / Gel is as follows: weigh PE-M with a mass fraction of M1, 0%<M1≤25%, add it to the thermosensitive blank hydrogel solution in an ice bath, and stir evenly to form a suspension solution to obtain the pyrifos glycoside microsphere hydrogel. The thermosensitive blank hydrogel is prepared by adding a certain amount of PLGA-PEG-PLGA carrier to 4°C deionized water to make a 20% concentration gel solution.

10. Use of the thermosensitive injectable hydrogel of irisin according to claim 1 or claim 2 in the preparation of cardiovascular drugs.

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