Injectable hydrogel for promoting repair of defect of old bone and preparation method of injectable hydrogel

Abstract

The invention provides injectable hydrogel for regulating and controlling ROS (reactive oxygen species) levels inside and outside senescent cells to promote repair of senescent bone defects and a preparation method of the injectable hydrogel. The method comprises the following steps: 1) preparing a double-selenium block polycarbonate micelle with reactive oxygen species (ROS) responsiveness; (2) loading an autophagy accelerant rapamycin into the double-selenium block polycarbonate micelle; and (3) compounding the double-selenium block polycarbonate loaded with the rapamycin with the polyethylene glycol-polysebacic acid glyceride hydrogel. According to the injectable hydrogel for regulating and controlling ROS levels inside and outside senescent cells and promoting repair of the senescent bone defects, the ROS levels inside and outside the senescent cells can be removed, meanwhile, ROS continuously generated by the senescent cells is reduced, a senescent bone tissue regeneration microenvironment is improved, senescence of stem cells is delayed, the regeneration capacity of the senescent stem cells is recovered, and the repairing effect of the senescent bone defects is improved. And a direction is provided for clinical experiments in the future.

Description

Technical Field

[0001] The invention relates to the fields of material science and biological tissue engineering, and in particular to an injectable hydrogel for promoting the repair of aged bone defects and a preparation method thereof. Background Art

[0002] As the global population ages, age-related bone defects, as the main consequence of bone loss, have become a major challenge in the field of public health due to their high disability and mortality rates. Age-related bone repair is always hampered by the loss of bone tissue homeostasis and progressive damage to regenerative potential, and has been considered a manifestation of bone aging in recent years. Cellular senescence accompanied by deterioration of the bone microenvironment is a key factor in bone aging and decreased regenerative capacity.

[0003] Excessive accumulation of reactive oxygen species (ROS) is one of the most common causes of cellular aging. In senescent cells, damaged mitochondria produce a large amount of ROS due to insufficient autophagy. In addition, the reduced levels or inactivation of antioxidant enzymes such as SOD, CAT, and GPx's lead to a weakened ability to scavenge reactive oxygen species, thereby exacerbating the oxidative stress (OS) response of senescent cells and continuously posing a threat to components such as proteins, lipids, and DNA. It is worth noting that age-related iron accumulation increases the production of hydroxyl radicals (OH-) in the cellular microenvironment, further exacerbating the OS of senescent cells. In the bone defect microenvironment, OS-induced senescent bone marrow stem cells (BMSCs), which are critical for bone regeneration, have a decreased proliferation capacity, a decreased osteogenic differentiation capacity, and an increased lipid differentiation capacity, leading to impeded bone repair.

[0004] Eliminating ROS and improving the oxidative microenvironment are key factors in improving the regenerative capacity of aging stem cells. Existing studies have shown that reducing ROS accumulation or generation is an effective way to inhibit bone loss in aged mice or promote the repair of osteoporotic bone defects. The regulation of ROS levels is a dynamic and complex process, which is accompanied by the accumulation of intracellular and extracellular ROS and continuous ROS generation, especially in the defective aging environment.

[0005] Therefore, developing hydrogels that simultaneously remove intracellular and extracellular ROS and reduce ROS production to improve the regenerative environment of aging stem cells has significant clinical application value. Summary of the invention

[0006] The present invention aims to develop a hydrogel that can simultaneously remove intracellular and extracellular ROS and reduce ROS generation, and specifically relates to an injectable hydrogel that regulates the level of ROS inside and outside aging cells to promote the repair of aged bone defects.

[0007] In a first aspect of the present invention, an injectable hydrogel PSeR is provided for regulating the level of ROS inside and outside senescent cells to promote the repair of aged bone defects. The injectable hydrogel comprises the following components:

[0008] PEGylated polysebacic acid glycerol hydrogel PEGS, and nanodrug-loaded micelle SeR;

[0009] Wherein, the PEGS is polymerized by aminated polyethylene glycol-polyglycerol sebacate (PEG-NH2) and γ-polyglutamic acid (γ-PGA) in the presence of a condensation agent;

[0010] The nano drug-loaded micelle SeR is prepared by loading the fat-soluble drug R on the diselenide block nano micelle (Se).

[0011] In another preferred embodiment, the condensing agent is selected from the following group: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC)

[0012] In another preferred embodiment, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).

[0013] In another preferred embodiment, the PEGS accounts for 25-40% of the total mass fraction of the PSeR hydrogel, preferably 30-35%.

[0014] In another preferred embodiment, the drug-loaded nano-micelle SeR is prepared by self-assembly of amphiphilic diselenide block copolymer mPEG-bP (TMC-co-MSeSe) and drug molecules through an aqueous phase method.

[0015] In another preferred embodiment, the nano drug-loaded micelle SeR is prepared by the following method: mPEG-bP (TMC-co-MSeSe) copolymer and drug R are self-assembled in aqueous phase to obtain nano drug-loaded micelle SeR.

[0016] In another preferred embodiment, the method comprises: adding mPEG-bP (TMC-co-MSeSe) copolymer and drug R into dimethylformamide, stirring sufficiently in ultrapure water, and then dialyzing with ultrapure water to obtain nano drug-loaded micelle SeR.

[0017] In another preferred embodiment, the molecular weight of the γ-PGA is 400,000-600,000.

[0018] In another preferred embodiment, the amphiphilic diselenide block copolymer mPEG-bP (TMC-co-MSeSe) has a structural characteristic formula as shown in Formula II:

[0019]

[0020] Wherein, m represents a molecular weight of 4500-5500, x is an integer of 100-200, and y is an integer of 10-20.

[0021] In another preferred embodiment, the mPEG-bP (TMC-co-MSeSe) is prepared by the following steps:

[0022] a) mixing mPEG, trimethyl chitosan (TMC) and diethylene diselenide carbonate dimer (MSeSe) to form a reaction mixture;

[0023] b) adding lipase CA to the mixture described in a) to carry out reaction to obtain mPEG-bP(TMC-co-MSeSe).

[0024] In another preferred embodiment, the mass ratio of mPEG, TMC and diethylene diselenide carbonate dimer (MSeSe) in step a) is 1:1.5-2.5:1.5-2.5.

[0025] In another preferred embodiment, the mass ratio of mPEG, TMC and diethylene diselenide carbonate dimer (MSeSe) in step a) is 1:2:2.

[0026] In another preferred embodiment, the drug R is selected from the following group: rapamycin, resveratrol, aspirin, or a combination thereof; preferably, the drug R is rapamycin.

[0027] In another preferred embodiment, the PEG-NH2 has a structure as shown in the following formula I, wherein: n is a positive integer of 1-20

[0028]

[0029] number, m is a positive integer from 5 to 60.

[0030] In another preferred embodiment, the PEG-NH2 is prepared by the following steps:

[0031] (1) Under a nitrogen atmosphere, PEG, boc-glycine, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine are dissolved in DCM and reacted to obtain a mixed reaction solution;

[0032] (2) filtering the mixed reaction solution and deprotecting the Boc group to obtain a purified PEG-NH2 solution.

[0033] In another preferred embodiment, the PEG-NH2 solution obtained in step (2) can be purified by the following method: the crude product is dialyzed against water and ethanol in sequence to obtain a purified product.

[0034] In a second aspect of the present invention, a method for preparing the hydrogel according to the first aspect is provided, the method comprising the steps of:

[0035] (S1) providing mPEG-bP(TMC-co-MSeSe) copolymer and drug R dissolved in dimethylformamide, fully stirred in ultrapure water, and dialyzed against ultrapure water to obtain nano drug-loaded micelle SeR;

[0036] (S2) Providing PEG-NH2 and γ-PGA to be fully dissolved in SeR, polymerizing in the presence of a cross-linking agent, thereby forming the hydrogel PSeR described in the first aspect.

[0037] In another preferred embodiment, in step (S2), PEG-NH2 accounts for 15-25% of the mass fraction of PSeR hydrogel, γ-PGA accounts for 5-15% of the mass fraction of PSeR hydrogel, the effective concentration of Se in PSeR hydrogel is 50-200 μg / mL, and the concentration of R in PSeR hydrogel is 50-200 μM.

[0038] In another preferred embodiment, in step (S2), the EDC accounts for 3-5% of the mass fraction of the PSeR hydrogel.

[0039] In the third aspect of the present invention, there is provided a use of the hydrogel according to the first aspect and the second aspect, wherein the hydrogel is used to prepare a pharmaceutical composition for treating an indication selected from the following group:

[0040] (i) Regulate ROS levels inside and outside aging cells;

[0041] (ii) improving mitochondrial function in aging cells;

[0042] (iii) reduce the secretion of SASP-related factors;

[0043] (iv) reducing senescence of aged bone marrow mesenchymal stem cells (BMSCs); and

[0044] (v) Repair of aged bone tissue.

[0045] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1The hydrogen nuclear magnetic resonance spectra of (a) amino-polyethylene glycol-polysebacic acid glycerol PEG-NH2 and (b) diselenide block copolymer mPEG-bP (TMC-co-MSeSe) of the present invention are shown; (c) transmission electron microscopy characterization of SeR nano-drug loaded micelles; (d) laser confocal microscopy characterization of Se nano-micelles uniformly distributed in PEGS hydrogel.

[0047] Figure 2 The present invention shows (a) the injectability of the PSeR hydrogel; (b) the characterization of the material's ability to bind Fe2+ in solution; c) the characterization of the material's antioxidant properties; and (d) the release of drug R from the PSeR hydrogel under different concentrations of hydrogen peroxide.

[0048] Figure 3 The hydrogel of the present invention is characterized in its ability to regulate ROS levels in the internal and external environments of aging cells: (a) redox potential; (b) characterization of intracellular ROS content; (c) characterization of intracellular mitochondrial membrane potential.

[0049] Figure 4 The effect of the hydrogel of the present invention on BMSCs aging is shown: (a) SASP-related gene expression; (b) aging-related β-galactosidase (SA-β-Gal) staining

[0050] Figure 5 The effect of the hydrogel of the present invention on the differentiation ability of aged BMSCs is shown: (a) alkaline phosphatase staining; (b) alizarin red staining.

[0051] Figure 6 The effect of the hydrogel of the present invention on the regeneration capacity of aged bones is shown: (a) Micro-CT two-dimensional image and three-dimensional image of the defect; (b) bone volume fraction at the defect; (c) trabecular bone density at the defect; (d) hematoxylin-eosin staining (H&E) image of the defect. DETAILED DESCRIPTION

[0052] After extensive and in-depth research, the inventors have for the first time used materials to remove ROS inside and outside senescent cells and improve the microenvironment of aging bone regeneration. PSeR hydrogel is injected into the local defect site through minimally invasive treatment to match the defect morphology. The hydrogel contains abundant carboxyl groups that can chelate the excessive ferrous ions (Fe 2+ ), thereby maintaining the oxidative microenvironment outside the senescent cells. Subsequently, the SeR micelles are released from the hydrogel and reach the inside of the senescent cells, where the ROS-sensitive diselenide bonds are broken, and the drug R is released while clearing the excessively accumulated ROS in the senescent cells. R activates the autophagy of senescent cells, thereby clearing the damaged mitochondria in the senescent cells and reducing the continuous generation of ROS in the senescent cells. The present invention was completed on this basis.

[0053] Terminology

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0055] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0056] As used herein, the terms "reactive oxygen species," "ROS," and "ROS" are used interchangeably.

[0057] As used herein, the terms "polyethylene glycol-polyglyceryl sebacate", "PEGS" are used interchangeably.

[0058] As used herein, the terms "aminated polyethylene glycol-polyglyceryl sebacate", "PEG-NH2" are used interchangeably.

[0059] As used herein, the terms "γ-polyglutamic acid", "γ-PGA" are used interchangeably.

[0060] As used herein, the terms "1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride," "EDC," are used interchangeably.

[0061] As used herein, the terms "phosphate buffered saline," "PBS," are used interchangeably.

[0062] As used herein, the terms "bone marrow mesenchymal stem cells," "BMSCs," are used interchangeably.

[0063] As used herein, the terms "senescence-associated β-galactosidase," "SA-β-gal," are used interchangeably.

[0064] As used herein, the terms "senescence-associated secretory phenotype," "SASP," are used interchangeably.

[0065] As used herein, the terms "micro-computed tomography", "Micro CT" are used interchangeably.

[0066] As used herein, the terms "hematoxylin-eosin staining," "H&E," are used interchangeably.

[0067] Injectable hydrogels that regulate ROS levels inside and outside senescent cells to promote bone defect repair in the elderly (PSeR)

[0068] The injectable hydrogel of the present invention comprises the following components:

[0069] PEGylated polysebacic acid glycerol hydrogel (PEGS), and nanodrug-loaded micelle SeR;

[0070] The PEGS is polymerized by amino polyethylene glycol-polysebacic acid glyceride (PEG-NH2) and γ-polyglutamic acid (γ-PGA) under the action of a condensation agent.

[0071] In another preferred embodiment, the condensing agent is selected from the following group: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC).

[0072] In another preferred embodiment, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).

[0073] The injectable hydrogel material (PSeR) for regulating the ROS level inside and outside senescent cells of the present invention is composed of a PEGS material for maintaining the ROS microenvironment outside senescent cells, a Se nano-micelle for clearing ROS accumulated in senescent cells, and a drug R for reducing the continuous generation of ROS by senescent cells.

[0074] According to some embodiments of the present invention, said:

[0075] 1) PEGS accounts for 34.3% of the total mass fraction of PSeR hydrogel;

[0076] 2) The effective concentration of Se nanomicelles in PSeR hydrogels to remove ROS in senescent cells is 50-200 μg / mL;

[0077] 3) The size of SeR nanomicelles is between 30-200 nm;

[0078] 4) The effective concentration of drug R in reducing the continuous generation of ROS in PSeR hydrogel is 50-200 nM;

[0079] 5) SeR drug-loaded nanomicelles are uniformly dispersed in PEGS hydrogel;

[0080] 6) PSeR has antioxidant properties and can adapt to the concentration-responsive release of drug R in an oxidative environment.

[0081] Preparation method of hydrogel PSeR of the present invention

[0082] In some embodiments of the present invention, the hydrogel PSeR of the present invention is prepared by the following method:

[0083] (S1) Preparation of PEG-NH2:

[0084] (S1a) Under nitrogen atmosphere, PEG, boc-glycine, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine were uniformly dissolved in anhydrous DCM and stirred to mix;

[0085] (S1b) filtering the mixture, concentrating the filtrate, deprotecting the Boc group with trifluoroacetic acid and exposing the amino group under continuous stirring, dialyzing to obtain a purified PEG-NH2 solution, and drying the obtained PEG-NH2 solution in a vacuum to obtain PEG-NH2;

[0086] (S2) Preparation of mPEG-bP(TMC-co-MSeSe)

[0087] (S2a) mPEG, TMC and diethylene diselenide carbonate dimer (MSeSe) are mixed in a mass ratio of 1:1-3:1-3 under argon to form a reaction mixture,

[0088] (S2b) dissolving LipaseCA in anhydrous toluene, adding the mixture to the above reaction mixture, stirring, diluting with dichloromethane and filtering; and

[0089] (S2c) taking the residue obtained by filtration in step b) and precipitating it in cold anhydrous ether, and then further drying it in vacuum to obtain mPEG-bP(TMC-co-MSeSe);

[0090] (S3) Preparation of Nano-drug-loaded Micellar SeR

[0091] The mPEG-bP (TMC-co-MSeSe) copolymer and drug R are dissolved in dimethylformamide, fully stirred in ultrapure water, and dialyzed with ultrapure water to obtain nano drug-loaded micelle SeR;

[0092] (S4) Preparation of PEGS:

[0093] PEG-NH2 and γ-PGA are fully dissolved in SeR and polymerized under the action of crosslinking agent EDC to form the hydrogel PSeR of the present invention.

[0094] Use of the injectable hydrogel material of the present invention for regulating ROS levels inside and outside aging cells

[0095] The injectable hydrogel material (PSeR) prepared by the present invention can regulate the ROS level inside and outside senescent cells, improve the ROS microenvironment inside and outside senescent cells, reduce the aging-related phenotype of senescent bone marrow mesenchymal stem cells (BMSCs), and has a new use of improving the antioxidant capacity of senescent BMSCs and further restoring the self-renewal ability and osteogenic differentiation ability of BMSCs. According to some embodiments of the present invention, the:

[0096] 1) PEGS accounts for 34.3% of the total mass fraction of PSeR hydrogel;

[0097] 2) The improvement of the extracellular ROS microenvironment of aging cells was demonstrated by extracting serum to detect the redox potential, where young and aged serum were taken from 1-month-old SD male mice and 24-month-old SD male mice, respectively;

[0098] 3) Elimination of ROS content in senescent cells was demonstrated by ROS staining, where the larger the green-positive area was considered to be, the higher the ROS content in the cell;

[0099] 4) Improving the mitochondrial function of aging cells is demonstrated by mitochondrial membrane staining, where the red area is the distribution of mitochondria with high membrane potential, that is, the distribution of normal mitochondria, and the green area is the distribution of mitochondria with low membrane potential, that is, the distribution of damaged mitochondria;

[0100] 5) The reduction in the secretion of SASP-related factors was determined by reverse transcription-polymerase chain reaction (RT-PCR);

[0101] 6) The senescence of BMSCs was determined by senescence-associated β-galactosidase (SA-β-Gal) staining, in which green-positive cells were considered to be senescent BMSCs.

[0102] 7) The enhanced osteogenic differentiation ability of BMSCs was demonstrated by alkaline phosphatase and alizarin red staining experiments. The darker the staining, the stronger the osteogenic differentiation ability.

[0103] The novel use of the present invention for regulating the level of ROS inside and outside senescent cells to promote the repair of aging bone defects

[0104] According to some embodiments of the present invention, the hydrogel PSeR prepared by the method of the present invention has the following new uses:

[0105] 1) PEGS accounts for 34.3% of the total mass fraction of PSeR hydrogel;

[0106] 2) PSeR hydrogel was injected and filled into the bone defect of aged C57 / BL6 males;

[0107] 3) The regeneration of bone defects was demonstrated by bone parameters measured by Micro Computed Tomography (Micro CT);

[0108] 4) The regenerated bone microstructure was demonstrated by bone parameters measured by Micro-CT and hematoxylin-eosin staining (H&E).

[0109] Compared with the prior art, the present invention has the following beneficial effects:

[0110] (1) The present invention provides a method for preparing an injectable hydrogel that regulates the level of ROS inside and outside senescent cells, demonstrating a new idea of ​​PSeR hydrogel in treating bone damage in the elderly by regulating the level of ROS inside and outside senescent cells;

[0111] (2) The PSeR hydrogel provided by the present invention improves the bone tissue microenvironment by regulating the ROS levels inside and outside senescent cells, thereby effectively delaying the aging of mesenchymal stem cells and restoring their osteogenic differentiation ability;

[0112] (3) The PSeR hydrogel of the present invention improves the regeneration rate of aged bone defects, and has a new and good application value in the field of treating aged bone diseases, providing a new clinical treatment strategy for this type of bone damage repair.

[0113] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0114] Example 1: Preparation of injectable hydrogels for promoting bone defect repair in the elderly by regulating ROS levels inside and outside senescent cells Preparation

[0115] A method for preparing an injectable hydrogel for promoting the repair of aged bone defects by regulating the level of ROS inside and outside aging cells comprises the following steps:

[0116] 1) Sebacic acid (10.11 g, 1 equivalent), polyethylene glycol diglycidyl ether (25 g, 1 equivalent) and catalyst TBAS (0.21 g, 0.006 equivalent) were mixed, uniformly dissolved in 100 mL of anhydrous dimethylformamide (DMF), and placed in a glove box filled with high-purity nitrogen to prepare polyethylene glycol polysebacic acid glycerol ester (PEG).

[0117] 2) Take PEG (7.02 g) in 1) and mix it with boc-glycine (4.20 g), N,N'diisopropylcarbodiimide (DIC; 5.05 g) and 4-dimethylaminopyridine (DMAP) and uniformly dissolve it in 100 mL of anhydrous dichloromethane (DCM) in a round-bottom flask in a glove box under a high-purity nitrogen atmosphere. After sealing, the flask was removed from the glove box and stirred for 24 hours under a nitrogen atmosphere at room temperature, and then the mixture was filtered, the filtrate was concentrated, and then stirred continuously in 15 mL of trifluoroacetic acid for 30 minutes. Then, the crude product was dialyzed with water for 48 hours and ethanol for 24 hours to obtain a purified amino-polyethylene glycol-polysebacic acid glyceride (PEG-NH2) solution, and the resulting solution was dried in a vacuum for further use; the structure and hydrogen nuclear magnetic resonance spectrum of PEG-NH2 are shown as follows Figure 1 a).

[0118] 3) mPEG, N-trimethylchitosan (TMC) and diethylene diselenide carbonate dimer (MSeSe) were added to a dry 10 mL vial and detected under Ar atmosphere. 2 mL of lipase CA was added to 2 mL of anhydrous toluene and then added to the mixture. After the reaction mixture was stirred at 70°C for 48 hours, it was diluted with 2 mL of dichloromethane and filtered. The residue was precipitated in cold anhydrous ether and then further dried in a vacuum. Finally, mPEG-bP (TMC-co-MSeSe) was obtained as a yellow sticky solid, and its structure and hydrogen NMR spectrum are shown in Figure 2. Figure 1 b).

[0119] 4) The mPEG-bP (TMC-co-MSeSe) copolymer (10 mg) described in 3) was dissolved in dimethylformamide (5 mL), and an appropriate amount of drug R was added, and the mixture was dropped into ultrapure water (10 mL) and stirred thoroughly. After dialysis in ultrapure water for 48 hours, SeR micelles were obtained, and the SeR micelle solution (0.5 mg / mL) was diluted for further use. The size of the SeR micelles was 30-200 nm, and the transmission electron microscopy image thereof was as follows: Figure 1 c) as shown.

[0120] 5) Take appropriate amount of PEG-NH2 and γ-polyglutamic acid (γ-PGA) and dissolve them in SeR solution. After fully dissolved, add EDC and stir evenly. Inject and shape within one minute. Figure 2 a) as shown; Se nanomicelles need to be evenly dispersed in the hydrogel, such as Figure 1 d), Se nanomicelles carrying FITC can be uniformly dispersed in the hydrogel; wherein PEG-NH2 accounts for 20% by mass, γ-PGA accounts for 10% by mass, the effective concentration of Se nanomicelles in the SeR solution is 50-200 μg / mL, and the effective concentration of drug R in the SeR solution is 50-200 nM.

[0121] Example 2: The PSeR hydrogel of the present invention has the ability to respond to oxidative environments

[0122] 1) Hydrogels for Fe 2+ Characterization of binding capacity: Prepare 10 μM Fe 2+ Solution, place the fully swollen hydrogel in the solution, hydrogel volume: Fe 2+ Solution volume = 1:10, control group is untreated 10μM Fe 2+ After the solution was left to stand for 12 hours, Fe in the solution was detected. 2+ Concentration. The hydrogel contains a large amount of γ-PGA, and sufficient carboxyl groups can chelate Fe in the environment. 2+ , thereby reducing Fe in the environment 2+ The concentration of Se nano-micelles has little effect on the results. Figure 2 b).

[0123] 2) The antioxidant activity of the hydrogel was evaluated by scavenging hydroxyl radicals. Hydroxyl radicals were generated by Fenton reaction of 5 mM hydrogen peroxide and 1.8 mM FeSO4 at 37 °C for 10 minutes; then the hydrogels in the experimental group were fully swollen and added to a solution containing hydroxyl radicals, the volume of hydrogel: the volume of the solution of hydroxyl radicals = 1:10, and the control group was a solution of untreated hydroxyl radicals. After 30 minutes of reaction, 1.8 mM salicylic acid reacted with the residual hydroxyl radicals to produce a characteristic absorbance. The darker the color, the higher the content of the remaining hydroxyl radicals in the solution. The photo of the reaction solution was taken by a digital camera, as shown in the figure. Figure 2 c) as shown.

[0124] 3) HPLC analysis of drug R release: PSeR was dialyzed with hydrogen peroxide solution, and then the dialyzate was subjected to HPLC analysis, with mobile phase: methanol: water = 80:20, detection wavelength 278 nm, flow rate 1 mL / min, injection volume 10 μL, column temperature 45 °C; PSeR showed obvious hydrogen peroxide responsiveness to the release of drug R, indicating that PSeR hydrogel can respond to the oxidation level of the environment, such as Figure 2 d).

[0125] Example 3: The PSeR hydrogel of the present invention regulates the level of ROS inside and outside aging cells

[0126] 1) Serum extraction: Blood was collected from the young (1-month-old SD rats) and old (24-month-old SD rats) groups using a serum separation tube. The blood was allowed to stand at room temperature for 35 min and then centrifuged at 3500 rpm for 30 min to separate the clear upper serum.

[0127] 2) Redox potential detection: The redox potential of serum was measured using a microelectrode research system containing microelectrodes, and the potential was recorded after the value stabilized; the serum in the control group was young serum and aged serum, and the redox potential of the serum in the control group was the stable potential during the detection; the serum in the experimental group was aged serum after the addition of hydrogel material, the hydrogel volume: serum volume = 1:10, and the redox potential of the serum in the experimental group was the stable potential detected after the addition of hydrogel; after the addition of P group hydrogel and PSe group hydrogel, the redox potential of aged serum could be restored to a redox potential close to that of young serum, and the difference in redox potential between the P group and the PSe group was small, proving that the hydrogel obtained by crosslinking PEG-NH2 and γ-PGA during the experiment could significantly maintain the extracellular oxidation level of aged cells, such as Figure 3 a).

[0128] 3) Cell model: serum was mixed into complete culture medium at a ratio of 10% to prepare conditioned medium for the young group and the aged group; BMSCs were cultured in the conditioned medium for 3 days to obtain young BMSCs and aged BMSCs; the experimental group consisted of BMSCs co-cultured with hydrogels of different components for 3 days, and the control group consisted of young BMSCs and BMSCs without aging intervention;.

[0129] 4) Detection of intracellular ROS levels: After staining with DCFH-DA probe for 30 minutes, the cells were washed with serum-free medium, and the fluorescence intensity of DCF (Ex = 488nm, Em = 525nm) was measured using a laser confocal microscope to characterize the intracellular ROS level. The BMSCs in the experimental group needed to be removed and washed once with PBS before staining. The hydrogels in the P group, the PSe group, and the PSeR hydrogel all had an effect on the intracellular ROS level of aging BMSCs; compared with the P group, the ROS content in the aging cells of the PSe group decreased more significantly, proving that the introduction of Se nano-micelles can more effectively remove the ROS accumulated in aging cells, such as Figure 3 b).

[0130] 5) Characterization of mitochondrial membrane potential: The cells were stained using the JC-1 kit, and the fluorescence intensity of red and green light was measured using a laser confocal microscope to characterize the mitochondrial membrane function of the cells. The BMSCs in the experimental group needed to be removed and washed once with phosphate buffered saline (PBS) before staining. PSe group hydrogel and PSeR hydrogel both had an effect on the mitochondrial membrane potential of aged BMSCs cells; compared with the PSe group, the mitochondrial membrane potential of aged cells in the PSeR group was more significantly improved, proving that the introduction of drug R can more effectively improve the mitochondrial function of aged BMSCs, such as Figure 3 c) as shown.

[0131] Example 4: PSeR hydrogel of the present invention regulates BMSCs aging

[0132] 1) The senescent cell model and grouping are consistent with those described in 3) of Example 2;

[0133] 2) Aging-associated β-galactosidase (SA-β-Gal) staining: After induction culture, the BMSCs in each group were removed, rinsed with PBS, and fixed with 4% paraformaldehyde at room temperature for 20 minutes. After fixation, rinsed twice with PBS, added staining working solution, sealed, and placed in a CO2-free 37°C incubator for overnight incubation. The dye was aspirated the next day, rinsed with PBS three times, and observed under an optical microscope. Cells stained green were identified as senescent cells. Figure 4 As shown in a), PSeR hydrogel significantly reduced the number of SA-β-Gal-positive BMSCs after induction with conditioned medium.

[0134] 3) Expression of senescence-associated secretory phenotype (SASP) genes: After induction culture, the BMSCs in each group were removed, rinsed twice with PBS, lysed with Trizol cell lysis buffer, and extracted RNA by centrifugation. The RNA was then converted into cDNA using a reverse transcription kit, and the expression of SASP-related genes in each group of cells was detected by RT-qPCR. Figure 4 The gene expression matrix shown in b) indicated that PSeR treatment inhibited the expression of SASP-related genes in senescent BMSCs.

[0135] Example 5: The PSeR hydrogel of the present invention improves the osteogenic differentiation ability of BMSCs

[0136] 1) The senescent cell model and grouping are consistent with those described in 3) of Example 2;

[0137] 2) Alkaline phosphatase staining experiment: After BMSCs in each group were treated and cultured for 14 days, the materials were taken out and washed once with PBS, and the osteogenic differentiation was detected by alkaline phosphatase staining kit; Figure 5 As shown in a), PSeR hydrogel significantly increased the expression of alkaline phosphatase in aged BMSCs, proving that the osteogenic differentiation ability of aged BMSCs was improved.

[0138] 3) Alizarin red staining experiment: After BMSCs in each group were treated with osteogenic induction culture for 21 days, the materials were taken out, washed once with PBS, and the osteogenic differentiation was detected by Alizarin red kit; Figure 5 As shown in b), PSeR hydrogel significantly enhanced the osteogenic mineralization ability of aged BMSCs.

[0139] Example 6: The PSeR hydrogel of the present invention promotes the repair of bone defects in the elderly

[0140] 1) Animal model: All operations were carried out in accordance with the National Laboratory Animal Health Guidelines and approved by Shanghai Ruitaimos Biotechnology Co., Ltd. (approval number: 20230601 (02), 20230604 (03)). This study selected 20-month-old male C57 / BL6 mice. After intraperitoneal injection of pentobarbital (40 mg / kg) for anesthesia, distal femoral defect surgery was performed. After flushing the blood from the defect site with PBS, the composite hydrogel was implanted into the distal femur defect (1.2 mm in diameter) of the C57 / BL6 mouse. After suturing the skin, penicillin was injected intramuscularly. After surgery, the experimental mice were allowed to move freely and were carefully cared for, and their physiological conditions were observed. Four weeks after surgery, the experimental animals were killed by cervical dislocation, and their femurs were taken for further analysis. The control group was a group without hydrogel implantation, and the experimental group was a group with hydrogel implantation.

[0141] 2) Micro-CT: The femur was removed and fixed in 4% neutral tissue fixative. The sample was then scanned using Micro-CT technology, and the image was reconstructed in three dimensions. Bone parameters were quantitatively analyzed, including bone volume percentage (%), number of trabeculae (mm -1 );like Figure 6 As shown in a), the repair speed of the defect in the PSeR group was significantly faster than that in the other groups four weeks after surgery, proving that multi-dimensional ROS-regulated hydrogels can accelerate the repair of aged bones. The PSeR group also had the highest bone volume percentage and number of trabeculae, proving that PSeR hydrogels improved the quality and efficiency of aged bone repair.

[0142] 3) Hematoxylin-Eosin staining (H&E) (e.g. Figure 6 d): The femur specimen was fixed with 4% neutral paraformaldehyde, decalcified with ethylenediaminetetraacetic acid (0.5M), dehydrated with ethanol, and embedded in paraffin. The femur specimen was cut into 4.5 μm slices. H&E staining was performed to observe the bone repair at the defect. Figure 6 b is the percentage of bone volume fraction at the defect site. Compared with the control group, the bone repair quality of the P group, PSe group, and PSeR group was improved, and the new bone in the PSeR group was more dense ( Figure 6 c) It proves that regulating ROS inside and outside senescent cells can improve the regeneration environment of aged bone and accelerate the repair process.

[0143] All data are expressed as mean ± standard deviation. All analyses were performed by at least three independent experiments. All analyses were performed using GraphPad Prism 8 software. Unpaired two-tailed Student's t-test was used for comparison between the two groups, and one-way ANOVA with post hoc test was used for statistical analysis. *P<0.005, **P<0.01, ***P<0.005, ****P<0.001.

[0144] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An injectable hydrogel PSeR that regulates the level of ROS inside and outside senescent cells to promote the repair of aged bone defects, characterized in that: The injectable hydrogel comprises the following components: PEGylated polysebacic acid glycerol hydrogel PEGS, and nanodrug-loaded micelle SeR; Wherein, the PEGS is polymerized by aminated polyethylene glycol-polyglycerol sebacate (PEG-NH2) and γ-polyglutamic acid (γ-PGA) in the presence of a condensation agent; The nano drug-loaded micelle SeR is prepared by loading the fat-soluble drug R on the diselenide block nano micelle (Se).

2. The hydrogel according to claim 1, characterized in that The nano drug-loaded micelle SeR is prepared by self-assembly of amphiphilic diselenide block copolymer mPEG-bP (TMC-co-MSeSe) and drug molecules through an aqueous phase method.

3. The hydrogel according to claim 2, characterized in that The amphiphilic diselenide block copolymer mPEG-bP (TMC-co-MSeSe) has a structural characteristic formula as shown in Formula II: Wherein, m represents a molecular weight of 4500-5500, x is an integer of 100-200, and y is an integer of 10-20.

4. The hydrogel according to claim 3, characterized in that The mPEG-bP (TMC-co-MSeSe) was prepared by the following steps: a) mixing mPEG, trimethyl chitosan (TMC) and diethylene diselenide carbonate dimer (MSeSe) to form a reaction mixture; b) adding lipase CA to the mixture described in a) to carry out reaction to obtain mPEG-bP(TMC-co-MSeSe).

5. The hydrogel according to claim 1, characterized in that The drug R is selected from the following group: rapamycin, resveratrol, aspirin, or a combination thereof; preferably, the drug R is rapamycin.

6. The hydrogel according to claim 1, wherein The PEG-NH2 has the following formula I Structure, wherein: n is a positive integer from 1 to 20, and m is a positive integer from 5 to 60.

7. The hydrogel according to claim 1, characterized in that The PEG-NH2 is prepared by the following steps: (1) Under a nitrogen atmosphere, PEG, boc-glycine, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine are dissolved in DCM and reacted to obtain a mixed reaction solution; (2) filtering the mixed reaction solution and deprotecting the Boc group to obtain a purified PEG-NH2 solution.

8. A method for preparing the hydrogel according to claim 1, characterized in that: The method comprises the steps of: (S1) providing mPEG-bP(TMC-co-MSeSe) copolymer and drug R dissolved in dimethylformamide, fully stirred in ultrapure water, and dialyzed against ultrapure water to obtain nano drug-loaded micelle SeR; (S2) Providing PEG-NH2 and γ-PGA fully dissolved in SeR, polymerizing in the presence of a cross-linking agent, thereby forming the hydrogel PSeR described in claim 1.

9. The preparation method according to claim 8, characterized in that: In step (S2), PEG-NH2 accounts for 15-25% of the mass fraction of PSeR hydrogel, γ-PGA accounts for 5-15% of the mass fraction of PSeR hydrogel, the effective concentration of Se in PSeR hydrogel is 50-200 μg / mL, and the concentration of R in PSeR hydrogel is 50-200 μM.

10. A use of the hydrogel according to claims 1-9, characterized in that: The hydrogel is used to prepare a pharmaceutical composition for treating an indication selected from the following group: (i) Regulate ROS levels inside and outside aging cells; (ii) improving mitochondrial function in aging cells; (iii) reduce the secretion of SASP-related factors; (iv) reducing senescence of aged bone marrow mesenchymal stem cells (BMSCs); and (v) Repair of aged bone tissue.

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