CN113041216B

CN113041216B - Multifunctional liver extracellular matrix composite hydrogel for liver cancer treatment and repair integration and preparation method thereof

Info

Publication number: CN113041216B
Application number: CN202110328033.9A
Authority: CN
Inventors: 郑辉东; 何晶晶; 张进; 林哲; 阮任杰; 林延带
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2021-03-26
Filing date: 2021-03-26
Publication date: 2022-07-08
Anticipated expiration: 2041-03-26
Also published as: CN113041216A

Abstract

The invention discloses a multifunctional liver extracellular matrix composite hydrogel for liver cancer treatment and repair integration and a preparation method thereof. When the liposome is injected into a human body, carboxyl on the liposome and amino on ECM are bonded at the body temperature to form amido bond, the liposome has the advantages of minimal invasion, safety, strong controllability and the like, and the hydrogel contains various bioactive molecules of extracellular matrix, is favorable for adhesion and growth of cells, and has great benefits for repairing inflammation and reconstructing tissues after treatment. Therefore, the multifunctional liver extracellular matrix composite hydrogel is expected to be applied to the integration of clinical treatment and repair of liver cancer.

Description

Multifunctional liver extracellular matrix composite hydrogel for liver cancer treatment and repair integration and preparation method thereof

Technical Field

The invention belongs to the field of biomaterial preparation and biomedical application, and particularly relates to multifunctional liver extracellular matrix composite hydrogel for liver cancer treatment and repair integration and a preparation method thereof.

Background

Liver is an important tissue organ of human body, and liver diseases are always puzzled to human beings, so that effective drug therapy is one of the most effective methods for treating liver diseases. To date, a series of chemotherapeutic drugs have been developed by the scientific community to treat patients, but the methods of directly using drugs have some similar disadvantages: the toxicity to cells and tissues in vivo is high, the affected area cannot be accurately positioned, the absorption rate is low, and the like, so that the patient is treated and suffers from great side effects.

Liposomes have been recognized as an effective drug delivery system due to their simple preparation and unique properties. However, conventional liposomes do not meet the need for on-demand release of the content, which limits their therapeutic utility. The gold nanoparticles are Nano-sized substances with good optical properties, have unique local surface plasmon resonance effect and good photo-thermal effect, the resonance wavelength of the gold nanoparticles is matched with the wavelength of incident light through the adjustment of the shape and the size, and the gold nanoparticles can convert absorbed light energy into heat to kill cancer cells after laser irradiation (Zhang D, Wu T, et al, Nano Letters, 2019, 19(9): 6635-. The invention provides a bilayer liposome vesicle containing gold nanoparticles with antibacterial effect and an anticancer drug. The near infrared light can penetrate through the skin to stimulate the gold nanoparticles to have GSH response with hydrogen peroxide in vivo, hydroxyl free radicals are generated, and a liposome membrane is destabilized, so that a large amount of medicines and the gold nanoparticles are released from the liposome cavity, and controllable release is realized. The continuous illumination can achieve the purpose of the synergistic treatment of photothermal treatment, chemotherapy and chemokinetic treatment. However, in a deeply buried cancer site, the laser energy required to kill cancer cells is relatively large, and thermal damage is likely to occur to the epidermis. Moreover, excessive local temperature, due to tissue heat diffusion, inevitably causes damage to surrounding normal tissue and inflammation, which leads to delayed healing and scar hyperplasia, while normal scar tissue formation completely restores the skin barrier function, but the important structure, appearance and function of the tissue are not completely restored.

Extracellular matrix (ECM) is a biological network structure composed of macromolecules, and is a natural biologically-derived material from which cellular components in biological tissues are removed by physicochemical methods, and the main components include collagen, non-collagen, elastin, proteoglycan, aminoglycan, and bioactive substances in parts of natural tissues, and can provide physical support and a suitable site for cell growth, promote cell adhesion, growth, proliferation and differentiation, and regenerate damaged tissues and organs (Donald, Bejleri, et al. Advanced Healthcare Materials, 2019, 8(5): 1801217). The hydrogel formed by the extracellular matrix is rich in a large number of extracellular matrix components, can provide a large number of nutrient substances for tissue repair, has a porous reticular structure presented inside, is favorable for the attachment and migration of regenerative cells, has the temperature-sensitive characteristic, can be formed by gel under the action of body temperature, is favorable for the targeted delivery and the controlled release of the loaded drug, and is easy to degrade in vivo without causing strong immunological rejection. However, the decellularization process of organs such as kidney and bladder reported at present is complex, a perfusion method is mostly adopted, the whole complete organ is difficult to obtain except hospitals and common laboratories, and liver decellularization is not reported in a patent at present. Therefore, in order to promote the wide application of the liver extracellular matrix in scientific research, an experimental method which is simple and convenient, can remove the ECM completely and can keep the ECM effective components intact should be developed. However, in the conventional process of injecting ECM hydrogel for repair after treatment, secondary damage to the diseased part is easily caused, the gelling time is too long, the mechanical property is poor, the function of supporting cells is difficult to play, and the whole recovery period is increased.

Therefore, a simple and convenient method for preparing a multifunctional composite hydrogel which has the combined treatment performance of PTT, CDT and chemotherapy and can perform quick crosslinking for resisting bacteria, repairing and promoting tissue regeneration on an injured part after operation is needed.

Disclosure of Invention

The invention aims to provide a multifunctional liver extracellular matrix composite hydrogel for liver cancer treatment and repair integration and a preparation method thereof, the composite hydrogel not only has good photo-thermal, chemical kinetics and antibacterial performance, but also is beneficial to cell adhesion and growth due to containing various bioactive molecules of the extracellular matrix, and has great benefits for repair of inflammation and reconstruction of tissues after treatment, namely the composite hydrogel has dual effects of tumor treatment and tissue repair, and is expected to be applied to clinical treatment and repair integration of liver cancer.

In order to realize the purpose, the invention adopts the following technical scheme:

a multifunctional liver extracellular matrix composite hydrogel for liver cancer treatment and repair integration is prepared by the following steps:

(1) repeatedly freezing and thawing fresh animal livers, slicing, repeatedly washing with deionized water, then carrying out acellular treatment with a mixed solution of ammonia water and Triton X-100, magnetically stirring and washing with deionized water until the mixture is neutral, and then soaking with a PBS (phosphate buffer solution) solution with pH =7.4 overnight to obtain an acellular scaffold precursor P-DECM; soaking in peroxyacetic acid for disinfection, and washing with PBS and water in sequence;

(2) freeze-drying the P-DECM cleaned in the step (1), shearing into pieces, and grinding at a low temperature of-20-4 ℃ to obtain extracellular matrix powder F-DECM;

(3) adding the F-DECM obtained in the step (2) into hydrochloric acid solution containing pepsin, stirring at a proper temperature until the F-DECM is completely dissolved, then adding sodium hydroxide solution to adjust the pH value to be neutral, adding 10 XPBS to adjust the osmotic pressure to be balanced, and obtaining a liver extracellular matrix hydrogel precursor H-DECM;

(4) mixing and stirring a chloroauric acid solution and sodium citrate uniformly in a conical flask, then adding an ice-cold sodium borohydride solution into the obtained solution, stirring and reacting to obtain a nano-gold solution, centrifuging, and freeze-drying to obtain gold nano-particles GNPs;

(5) dissolving dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), azido diphenyl phosphate (DPPA), phospholipid modified polyethylene glycol (DSPE-PEG 2000-COOH) with carboxyl at the tail end and GNPs in a mixed solution of methanol and chloroform, uniformly mixing by ultrasound, and performing rotary evaporation to form a membrane to obtain a nano material precursor P-GNBs wrapped by liposome vesicles;

(6) mixing glycerol, 1 XPBS and block polyether F-68 to obtain a hydration liquid;

(7) adding the hydration liquid obtained in the step (6) into the P-GNBs obtained in the step (5), and dissolving a liposome membrane by ultrasonic waves to obtain a nano particle GNBs solution coated by liposome vesicles (NBs); adding an anti-cancer drug (ACD) into the GNBs solution, carrying out oscillation reaction in a perfluoropropane atmosphere under the condition of oil bath, centrifuging and washing, drying at 37 ℃, and weighing to obtain a drug-loaded nano material GNBs/ACD;

(8) and (3) rapidly stirring and uniformly mixing the H-DECM obtained in the step (3) and the medicine-carrying nano material GNBs/ACD obtained in the step (7) on an ice bath, then performing temperature programming, and finally maintaining the self-crosslinking at 37 ℃ to obtain the multifunctional liver extracellular matrix composite hydrogel GNBs/ACD/H-DECM, and sterilizing under the irradiation of an ultraviolet lamp of a biological super clean bench for later use.

Freezing and thawing fresh animal livers for 1-10 times; in the mixed solution of the ammonia water and the Triton X-100, the volume concentration of the ammonia water is 0.01-20%, and the volume concentration of the Triton X-100 is 0.1-30%.

And (3) cutting the P-DECM into particles with the diameter of 1 mu m-3 cm in the step (2), and grinding the particles into powder with the diameter of 10 nm-1 mm.

The concentration of hydrochloric acid in the hydrochloric acid solution containing the pepsin in the step (3) is 0.00001-10M, and the concentration of the pepsin is 0.01-1000 mg/mL; the addition amount of the F-DECM is 0.01-1000 mg/mL; the stirring temperature is 0-60 ℃, and the stirring time is 2 h-7 d; the concentration of the sodium hydroxide solution is 0.0001-100M.

The concentration of chloroauric acid in the gold solution obtained in the step (4) is 0.001-250 mM, and the concentration of sodium citrate is 0.001-250 mM; the concentration of the sodium borohydride solution is 0.1M, the volume ratio of the amount of the sodium borohydride solution to the gold solution is (0.001-15): 20, and the stirring reaction time is 10 min-4 h; the rotation speed of the nano gold solution centrifugation is 5000-30000 rpm, and the particle size of the obtained gold nano particles GNPs is 1-100 nm.

The volume ratio of methanol to chloroform in the mixed solution of methanol and chloroform in the step (5) is (0.1-200): 1, the addition amount of DPPC is 0.002-10 mg/mL, the addition amount of DPPE is 0.002-10 mg/mL, the addition amount of DPPA is 0.002-10 mg/mL, the addition amount of DSPE-PEG2000-COOH is 0.002-10 mg/mL, and the addition amount of GNPs is 0.002-100 mg/mL; the ultrasonic time is 5 min-1 h; the time for the spin-evaporation film forming is 5 min-3 h.

The volume ratio of glycerol to 1 multiplied by PBS in the hydration liquid obtained in the step (6) is (0.00001-100) to 1, and the content of F-68 is 0.005-50 mg/mL.

In the step (7), the ultrasonic dissolution time of the liposome membrane is 10 s-30 min; after the anti-cancer drugs are added, the concentration of the anti-cancer drugs in the GNBs solution is 0.001-10 mg/mL, and the anti-cancer drugs comprise any one of adriamycin, paclitaxel and docetaxel; the temperature of the oscillation reaction is 20-120 ℃, the time is 10 min-8 h, and the rotating speed is 100-1000 rpm; the centrifugation time is 30 s-1 h, and the rotating speed is 100-10000 rpm; the washing is carried out for 1-10 times by adopting PBS solution for centrifugal cleaning, the centrifugal speed is 1000-30000 rpm, and the time is 30 s-30 min.

The mass-volume ratio of GNBs/ACD and H-DECM used in the step (8) is 5-850 microgram/mL, and the temperature programming speed is 0.1-10 ℃/s.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a multifunctional liver extracellular matrix composite hydrogel for liver cancer treatment and repair integration, which is characterized in that liposome nano vesicles are used as a carrier, and nano gold particles and anti-cancer drugs are loaded, so that the hydrogel has good photo-thermal, chemical kinetics and antibacterial properties, and can reach 42 ℃ under the irradiation of near infrared light of 808 nm, while anti-tumor drugs are slowly released, and the nano gold and in-vivo H are mixed₂O₂The effect being produced^.OH can achieve the effect of killing the tumor in a synergistic targeting way. Meanwhile, the invention grinds the animal liver after decellularization into powder, and then prepares the hydrogel through enzymolysis and digestion, and the hydrogel has good biocompatibility; after pH regulation, the composite nano vesicles are wrapped in the composite nano vesicles, and the liposome and the ECM are kept independent at low temperature. During the temperature programming, the carboxyl groups on the liposome can form amide bonds with amino groups in the ECM, and in addition, the ECM and the ECM are re-crosslinked, so that the gel is formed more quickly and the crosslinking is firmer. The multifunctional liver extracellular matrix composite hydrogel can be formed into gel at the temperature of a human body by injecting the multifunctional liver extracellular matrix composite hydrogel into the human body, has the advantages of micro-wound, safety, strong controllability and the like, contains various bioactive molecules of the extracellular matrix, is favorable for the adhesion and growth of cells, and has great benefits for the repair of inflammation and the reconstruction of tissues after treatment. Meanwhile, the raw material animal liver is a natural source, so that rejection risk is reduced, and the raw material animal liver can be degraded automatically finally. Therefore, the multifunctional liver extracellular matrix composite hydrogel has double effects of tumor treatment and tissue repair, is expected to be applied to the integration of clinical treatment and repair of liver cancer, and avoids secondary damage.

Drawings

FIG. 1 shows Transmission Electron Microscopy (TEM), hydrodynamic particle size in aqueous phase (DLS), Zeta potential diagram, and ultraviolet absorption spectrum (UV-vis) of GNPs (A), NBs (B), and GNBs nanomaterial (C) prepared in example 1;

FIG. 2 is a staining chart of a tissue section of the protohepatic and DECM scaffolds of example 1;

FIG. 3 is a graph comparing the DNA content of the protohepatic and DECM scaffolds in example 1;

FIG. 4 is a Scanning Electron Micrograph (SEM) of the H-DECM hydrogel precursor prepared in example 1;

FIG. 5 is a graph of the porosity and pore size results for the H-DECM hydrogel precursor prepared in example 1;

FIG. 6 is an SEM image of GNBs/DOX/H-DECM composite material prepared in example 1;

FIG. 7 is a graph showing the injection properties of GNBs/DOX/H-DECM composite hydrogel precursors prepared in example 1;

FIG. 8 is a diagram of the cytotoxicity test of GNBs nanomaterial prepared in example 1;

FIG. 9 is a cell proliferation assay chart of H-DECM prepared in example 1;

FIG. 10 is a graph of photothermal temperature increase (A), photothermal temperature difference (B) and photothermal imaging (C) of NBs, GNBs and GNBs/DOX of the NBs and GNBs nanomaterial prepared in example 1;

FIG. 11 is a result chart of the apoptosis rate of the flow cytometer detection of hepatoma cell HepG2 by the GNBs/DOX composite material prepared by different ratios of GNBs/DOX and H-DECM;

FIG. 12 is a graph of the ability of GNBs/DOX/H-DECM composite material with a mixture ratio of 80 μ g/mL to treat tumors;

FIG. 13 shows the measurement results of the contents of proinflammatory factors IL-1 β, IL-6, TNF- α and anti-inflammatory factor IL-10 in animals after animal experiments with GNBs/DOX/H-DECM composite materials prepared from GNBs/DOX and H-DECM according to different ratios;

FIG. 14 shows the storage modulus (solids-like) of hydrogels prepared with different concentrations of H-DECM and different ratios of GNBs/DOX at the same concentration.

Detailed Description

The invention discloses a multifunctional liver extracellular matrix composite hydrogel for liver cancer treatment and repair integration, which is prepared by the following steps:

(3) adding the F-DECM obtained in the step (2) into hydrochloric acid solution containing pepsin, stirring at a proper temperature until the F-DECM is completely dissolved, then adding sodium hydroxide solution to adjust the pH value to be neutral, adding 10 x PBS to adjust the osmotic pressure to be balanced, and obtaining liver extracellular matrix hydrogel precursor H-DECM;

(4) mixing and stirring a chloroauric acid solution and sodium citrate uniformly in a conical flask, then adding an ice-cold sodium borohydride solution into the obtained gold solution, stirring and reacting to obtain a nano gold solution, centrifuging, and freeze-drying to obtain gold nano-particles GNPs;

(7) adding the hydrated liquid obtained in the step (6) into the P-GNBs obtained in the step (5), and dissolving a liposome membrane by ultrasonic waves to obtain a nano particle GNBs solution coated by liposome vesicles (NBs); adding an anti-cancer drug (ACD) into the GNBs solution, carrying out oscillation reaction in a perfluoropropane atmosphere under the condition of oil bath, centrifuging and washing, drying at 37 ℃, and weighing to obtain a drug-loaded nano material GNBs/ACD;

The concentration of hydrochloric acid in the hydrochloric acid solution containing the pepsin in the step (3) is 0.00001-10M, and the concentration of the pepsin is 0.01-1000 mg/mL; the adding amount of the F-DECM is 0.01-1000 mg/mL; the stirring temperature is 0-60 ℃, and the stirring time is 2 h-7 d; the concentration of the sodium hydroxide solution is 0.0001-100M.

The concentration of chloroauric acid in the gold solution obtained in the step (4) is 0.001-250 mM, and the concentration of sodium citrate is 0.001-250 mM; the concentration of the sodium borohydride solution is 0.1M, the volume ratio of the amount of the sodium borohydride solution to the gold solution is (0.001-15): 20, and the stirring reaction time is 10 min-4 h; the rotation speed of the nano gold solution centrifugation is 5000-30000 rpm, and the particle size of the obtained gold nano-particle GNPs is 1-100 nm.

The volume ratio of glycerol to 1 XPBS in the hydration solution obtained in the step (6) is (0.00001-100) to 1, and the content of F-68 is 0.005-50 mg/mL.

In the step (7), the ultrasonic dissolution time of the liposome membrane is 10 s-30 min; after the anti-cancer drugs are added, the concentration of the anti-cancer drugs in the GNBs solution is 0.001-10 mg/mL, and the anti-cancer drugs comprise any one of adriamycin (DOX), paclitaxel and docetaxel; the temperature of the oscillation reaction is 20-120 ℃, the time is 10 min-8 h, and the rotating speed is 100-1000 rpm; the centrifugation time is 30 s-1 h, and the rotating speed is 100-10000 rpm; the washing is carried out for 1-10 times by adopting PBS solution for centrifugal cleaning, the centrifugal speed is 1000-30000 rpm, and the time is 30 s-30 min.

The invention takes fresh animal liver as raw material, slices, and carries out enzymolysis and digestion after decellularization to prepare hydrogel, and the hydrogel is assembled with added liposome nano vesicles wrapping nano-gold and anti-cancer drugs to prepare the multifunctional injectable composite hydrogel with photo-thermal and chemical dynamic properties, antibiosis, promotion of cell proliferation, damaged tissue repair and the like, and can simultaneously solve the problems of poor tumor treatment effect and timely repair of postoperative damaged parts.

For further understanding of the present invention, the following examples are provided to illustrate the preparation method of the multifunctional drug testing template provided by the present invention, and the scope of the present invention is not limited by the following examples.

Example 1

(1) Repeatedly freezing and thawing fresh animal livers for 3 times, slicing, repeatedly washing with deionized water, then carrying out acellular treatment with a mixed solution containing 0.1vol% ammonia water and 10 vol% Triton X-100, washing with deionized water to neutrality, and soaking in a PBS (phosphate buffer solution) with pH =7.4 overnight to obtain P-DECM; soaking in peroxyacetic acid for disinfection, and washing with PBS and water in sequence;

(2) cutting the prepared P-DECM after freeze drying into particles with the diameter of 1 mu m, and then grinding at the low temperature of-20-4 ℃ to obtain F-DECM with the diameter of 100 nm;

(3) adding F-DECM into 0.01M hydrochloric acid solution containing 1 mg/mL pepsin to enable the concentration of the solution to be 10 mg/mL, stirring for 72 hours at 25 ℃ until the solution is completely dissolved, then adding 0.1M sodium hydroxide solution to adjust the pH value to be neutral, adding 1 mL 10 XPBS to adjust the osmotic pressure to be balanced, and obtaining H-DECM;

(4) mixing and stirring chloroauric acid solution and sodium citrate uniformly in a conical flask, wherein the total volume of the obtained gold solution is 20 mL, the concentration of chloroauric acid is 1 mM, the concentration of sodium citrate is 1 mM, then stirring at 550 rpm at normal temperature, adding 0.6 mL of refrigerated sodium borohydride solution with the concentration of 0.1M, stirring for 30 min to obtain solution nanogold solution, then centrifuging at 25000 rpm, and freeze-drying to obtain GNPs with the average particle size of 3.49 nm;

(5) dissolving DPPC, DPPE, DPPA, DSPE-PEG2000-COOH and GNPs in a mixed solution prepared from 10 mL of methanol and 5 mL of chloroform, so that the concentration of each substance is 2 mg/mL of DPPC, 6 mg/mL of DPPE, 6 mg/mL of DPPA, 6 mg/mL of DSPE-PEG2000-COOH and 7.68 mg/mL of GNPs; performing ultrasonic treatment for 5 min, and performing rotary evaporation at 55 ℃ for 1 h to form a film to obtain P-GNBs;

(6) mixing 500 muL of glycerol, 5 mL of PBS and 3 mg of F-68 to obtain a hydration solution;

(7) adding 5.5 mL of hydration liquid into the P-GNBs prepared in the step 5), and ultrasonically dissolving a liposome membrane for 5 min to obtain a GNBs solution; adding DOX into the obtained GNBs solution to enable the concentration to be 3.64 mg/mL, carrying out oil bath for 1 h at 75 ℃, then oscillating for 2 h at the rotating speed of 500 rpm in the perfluoropropane atmosphere, centrifuging for 10 min at 800 rpm, washing for 3 times by PBS, wherein the centrifuging speed is 20000 rpm, and the time is 5 min to obtain the drug-loaded nano material GNBs/DOX;

(8) and rapidly stirring the obtained H-DECM and the medicine-carrying nano material GNBs/DOX in a mass-volume ratio of 20 mug/mL under ice bath, uniformly mixing, then carrying out temperature programming at a speed of 1.0 ℃/s, and finally carrying out autonomous crosslinking at 37 ℃ to obtain the GNBs/DOX/H-DECM.

FIG. 1 shows Transmission Electron Microscopy (TEM), hydrodynamic particle size in aqueous phase (DLS), Zeta potential diagram and ultraviolet absorption spectrum (UV-vis) of the GNPs (A), NBs (B) and GNBs nanomaterial (C) prepared in this example. As can be seen from FIG. 1, the average particle size of the GNPs prepared in this example was 3.49 nm, and the average potential was-35 mV. NBs successfully encapsulated GNPs, and the resulting GNBs had an average particle size of 450 nm and an average potential of-1.10 mV.

FIG. 2 is a staining chart of tissue sections of the protoliver and the DECM scaffold prepared in this example (DAPI, H & E, Masson and Sirius red, scale 1000 μm). As can be seen from the DAPI staining pattern, the liver scaffolds after decellularization had almost no nuclei compared to the protoliver, indicating that they were completely removed. As can be seen from H & E staining diagram, compared with the original liver, the decellularized liver scaffold retains the original extracellular matrix components such as elastic fibers and collagen fibers. As can be seen from the Masson staining pattern, the liver scaffold after decellularization has almost no tissues such as muscle and the like compared with the primitive liver, and the collagen fiber is well preserved. From the Sirius red staining pattern, it can be seen that the liver scaffolds after decellularization almost preserved type i collagen fibers, compared to the primitive liver.

FIG. 3 is a graph comparing the DNA content of the protoliver and the DECM scaffold prepared in this example. As can be seen from the figure, the DNA content in DECM was very low compared to that of the original liver, indicating that the decellularized scaffold was indeed completely removed.

FIG. 4 is an SEM image of the H-DECM hydrogel precursor prepared in this example. As can be seen from the figure, H-DECM is in a porous structure, and preserves original fibrin, thereby being beneficial to the growth and adhesion of cells.

FIG. 5 is a graph of the porosity and pore size results for the H-DECM hydrogel precursor prepared in this example. As can be seen from the figure, the average porosity thereof is 62% and the pore size thereof is 95 μm.

FIG. 6 is an SEM image of GNBs/DOX/H-DECM composite material prepared in this example. As can be seen from the figure, the GNBs/DOX nanoparticles are uniformly loaded on the surface of the H-DECM.

FIG. 7 is a graph showing the injection properties of the GNBs/DOX/H-DECM composite hydrogel precursors prepared in this example. As can be seen from the figure, after the pH of the composite hydrogel is adjusted to be neutral, it is in a flowing state, and has injectability, and can be solidified into a gel at 37 ℃.

Fig. 8 is a graph of cytotoxicity experiments of GNBs nanomaterials prepared in this example on hepatocytes LO2 at different concentrations and times. As can be seen from the figure, even if the material concentration reaches 200 mug/mL and the culture is carried out for 48 hours, the cells still maintain high survival rate, which indicates that the biocompatibility of the material is good.

FIG. 9 is a graph showing the cell proliferation assay of H-DECM prepared in this example. As can be seen from the figure, after the liver cells LO2 are cultured on H-DECM for 1, 3, 5 and 7 days, the survival rate of LO2 cells is basically 100%, which indicates that the hydrogel has good cell proliferation promoting performance and has great benefits for repairing inflammation and reconstructing tissues after treatment.

FIG. 10 is a graph (A) showing the photothermal temperature rise and the photothermal temperature difference of the NBs and GNBs nanomaterial prepared in this example, and a graph (C) showing photothermal imaging of NBs, GNBs, and GNBs/DOX. As can be seen from the figure, the GNBs prepared in this example are at 808 nm (2W/cm)²) After the laser irradiates for 10 min, the temperature difference of the temperature rise is about 25 ℃, and the temperature required by killing the tumor is completely met. And after three cycles of temperature rise and drop, the material still keeps good temperature rise effect, which shows that the material can continuously carry out photothermal therapy for a long time.

Example 2

(1) Repeatedly freezing and thawing fresh animal livers for 3 times, slicing, repeatedly washing with deionized water, then performing acellular treatment with a mixed solution containing 0.1vol% ammonia water and 3.33 vol% Triton X-100, washing with deionized water to neutrality, and soaking in a PBS (phosphate buffer solution) with pH =7.4 overnight to obtain P-DECM; soaking in peroxyacetic acid for disinfection, and washing with PBS and water in sequence;

(2) cutting the prepared P-DECM after freeze drying into particles with the diameter of 1 mu m, and then grinding at low temperature of-20-4 ℃ to obtain F-DECM with the diameter of 100 nm;

(4) mixing and stirring chloroauric acid solution and sodium citrate uniformly in a conical flask, wherein the total volume of the obtained gold solution is 20 mL, the concentration of chloroauric acid is 0.025 mM, the concentration of sodium citrate is 0.025 mM, then stirring at the normal temperature of 550 rpm, adding 5 mL of refrigerated sodium borohydride solution with the concentration of 0.1M, stirring for 2 h to obtain solution nanogold solution, then centrifuging at the speed of 10000 rpm, and freeze-drying to obtain GNPs with the average particle size of 10 nm;

(5) dissolving DPPC, DPPE, DPPA, DSPE-PEG2000-COOH and GNPs in a mixed solution prepared from 10 mL of methanol and 5 mL of chloroform, so that the concentration of each substance is 20 mg/mL of DPPC, 6 mg/mL of DPPE, 6 mg/mL of DPPA, 6 mg/mL of DSPE-PEG2000-COOH and 5 mg/mL of GNPs; performing ultrasonic treatment for 5 min, and performing rotary evaporation at 55 ℃ for 1 h to form a film to obtain P-GNBs;

(8) and rapidly stirring the obtained H-DECM and the medicine-carrying nano material GNBs/DOX in an ice bath according to the mass-volume ratio of 20 mug/mL, uniformly mixing, performing temperature programming at the speed of 1.0 ℃/s, and finally performing self-crosslinking at the temperature of 37 ℃ to obtain the GNBs/DOX/H-DECM.

Example 3

(1) Repeatedly freezing and thawing fresh animal livers for 3 times, slicing, repeatedly washing with deionized water, then carrying out cell removal treatment with a mixed solution containing 1vol% ammonia water and 7 vol% Triton X-100, washing with deionized water to neutrality, and soaking in PBS (phosphate buffer solution) with pH =7.4 overnight to obtain P-DECM; soaking in peroxyacetic acid for disinfection, and washing with PBS and water in sequence;

(3) adding F-DECM into 0.001M hydrochloric acid solution containing 0.1 mg/mL pepsin to make the concentration of the solution be 20 mg/mL, stirring at 25 ℃ for 12H until the solution is completely dissolved, then adding 0.1M sodium hydroxide solution to adjust the pH value to be neutral, adding 1 mL 10 XPBS to adjust the osmotic pressure to be balanced, and obtaining H-DECM;

(4) mixing and stirring chloroauric acid solution and sodium citrate uniformly in a conical flask, wherein the total volume of the obtained gold solution is 20 mL, the concentration of chloroauric acid is 0.025 mM, the concentration of sodium citrate is 0.025 mM, then stirring at 550 rpm at normal temperature, adding 0.6 mL of refrigerated sodium borohydride solution with the concentration of 0.1M, stirring for 30 min to obtain solution nanogold solution, then centrifuging at the speed of 12000 rpm, and freeze-drying to obtain GNPs with the average particle size of 15 nm;

(5) dissolving DPPC, DPPE, DPPA, DSPE-PEG2000-COOH and GNPs in a mixed solution prepared from 10 mL of methanol and 5 mL of chloroform, so that the concentration of each substance is 2 mg/mL of DPPC, 4 mg/mL of DPPE, 4 mg/mL of DPPA, 4 mg/mL of DSPE-PEG2000-COOH and 7.68 mg/mL of GNPs; performing ultrasonic treatment for 5 min, and performing rotary evaporation at 60 ℃ for 1 h to form a film to obtain the P-GNBs.

(8) rapidly stirring the obtained P-H-DECM and the drug-loaded nano material GNBs/DOX in a mass-volume ratio of 20 microgram/mL under ice bath, uniformly mixing, then carrying out temperature programming at a speed of 1.0 ℃/s, and finally carrying out self-crosslinking at 37 ℃ to finally obtain the GNBs/DOX/H-DECM.

Example 4

(1) Repeatedly freezing and thawing fresh animal livers for 3 times, slicing, repeatedly washing with deionized water, then performing acellular treatment with a mixed solution containing 0.05 vol% ammonia water and 7.5 vol% Triton X-100, washing with deionized water to neutrality, and soaking in a PBS (phosphate buffer solution) with pH =7.4 overnight to obtain P-DECM; soaking in peroxyacetic acid for disinfection, and washing with PBS and water in sequence;

(2) cutting the prepared P-DECM after freeze drying into particles with the diameter of 5 mu m, and then grinding at low temperature of-20-4 ℃ to obtain F-DECM with the diameter of 270 nm;

(3) adding F-DECM into 0.1M hydrochloric acid solution containing 1 mg/mL pepsin to enable the concentration of the solution to be 10 mg/mL, stirring for 72 hours at 25 ℃ until the solution is completely dissolved, then adding 0.1M sodium hydroxide solution to adjust the pH value to be neutral, adding 4 mL 10 XPBS to adjust the osmotic pressure to be balanced, and obtaining H-DECM;

(4) mixing and stirring chloroauric acid solution and sodium citrate uniformly in a conical flask, wherein the total volume of the obtained gold solution is 20 mL, the concentration of chloroauric acid is 5 mM, the concentration of sodium citrate is 5 mM, then stirring at 550 rpm at normal temperature, adding 0.6 mL of refrigerated sodium borohydride solution with the concentration of 0.1M, stirring for 30 min to obtain solution nanogold solution, then centrifuging at 25000 rpm, and freeze-drying to obtain GNPs with the average particle size of 3.49 nm;

(5) dissolving DPPC, DPPE, DPPA, DSPE-PEG2000-COOH and GNPs in a mixed solution prepared from 10 mL of methanol and 5 mL of chloroform, so that the concentration of each substance is 10 mg/mL of DPPC, 10 mg/mL of DPPE, 11 mg/mL of DPPA, 25 mg/mL of DSPE-PEG2000-COOH and 7.68 mg/mL of GNPs; performing ultrasonic treatment for 5 min, and performing rotary evaporation at 55 ℃ for 1 h to form a film to obtain P-GNBs;

(7) adding 5.5 mL of hydration solution into the P-GNBs prepared in the step 5), and ultrasonically dissolving a liposome membrane for 5 min to obtain a GNBs solution; adding DOX into the obtained GNBs solution to enable the concentration to be 3.64 mg/mL, carrying out oil bath for 1 h at 80 ℃, then oscillating for 2 h at the rotating speed of 500 rpm in the perfluoropropane atmosphere, centrifuging for 10 min at 800 rpm, washing for 3 times by PBS, wherein the centrifuging speed is 15000 rpm, and the time is 10 min to obtain the drug-loaded nano material GNBs/DOX;

(8) and rapidly stirring the obtained H-DECM and the medicine-carrying nano material GNBs/DOX in an ice bath at a mass-volume ratio of 20 mug/mL, uniformly mixing, performing temperature programming at a speed of 1.0 ℃/s, and finally performing self-crosslinking at 37 ℃ to finally obtain the GNBs/DOX/H-DECM.

Example 5

(1) Repeatedly freezing and thawing fresh animal livers for 3 times, repeatedly washing the livers with deionized water after slicing, then carrying out decellularization treatment on the livers with a mixed solution containing 0.04 vol% ammonia water and 6.9 vol% Triton X-100, washing the livers with deionized water until the mixture is neutral, and soaking the livers in a PBS (phosphate buffer solution) solution with the pH =7.4 overnight to obtain P-DECM; soaking in peroxyacetic acid for disinfection, and washing with PBS and water in sequence;

(3) adding F-DECM into 0.1M hydrochloric acid solution containing 1 mg/mL pepsin to enable the concentration of the solution to be 10 mg/mL, stirring for 48 hours at 30 ℃ until the solution is completely dissolved, then adding 10M sodium hydroxide solution to adjust the pH value to be neutral, adding 4 mL 10 x PBS to adjust the osmotic pressure to be balanced, and obtaining H-DECM;

(4) mixing and stirring chloroauric acid solution and sodium citrate uniformly in a conical flask, wherein the total volume of the obtained gold solution is 20 mL, the concentration of chloroauric acid is 5 mM, the concentration of sodium citrate is 5 mM, then stirring at 550 rpm at normal temperature, adding 0.6 mL of refrigerated sodium borohydride solution with the concentration of 0.1M, stirring for 30 min to obtain solution nanogold solution, then centrifuging at the speed of 21000 rpm, and freeze-drying to obtain GNPs with the average particle size of 39 nm;

(5) dissolving DPPC, DPPE, DPPA, DSPE-PEG2000-COOH and GNPs in a mixed solution prepared from 10 mL of methanol and 5 mL of chloroform, so that the concentration of each substance is 10 mg/mL of DPPC, 10 mg/mL of DPPE, 11 mg/mL of DPPA, 25 mg/mL of DSPE-PEG2000-COOH and 7.68 mg/mL of GNPs; performing ultrasonic treatment for 5 min, and performing rotary evaporation at 68 ℃ for 1 h to form a film to obtain P-GNBs;

(6) mixing 500 muL of glycerol, 5 mL of PBS and 1 mg of F-68 to obtain a hydration solution;

Example 6

(1) Repeatedly freezing and thawing fresh animal livers for 3 times, slicing, repeatedly washing with deionized water, then carrying out acellular treatment with a mixed solution containing 0.01 vol% ammonia water and 6.3 vol% Triton X-100, washing with deionized water to neutrality, and soaking in a PBS (phosphate buffer solution) with pH =7.4 overnight to obtain P-DECM; soaking in peroxyacetic acid for disinfection, and washing with PBS and water in sequence;

(4) mixing and stirring chloroauric acid solution and sodium citrate uniformly in a conical flask, wherein the total volume of the obtained gold solution is 20 mL, the concentration of chloroauric acid is 16 mM, the concentration of sodium citrate is 16 mM, then stirring at 550 rpm at normal temperature, adding 5 mL of refrigerated sodium borohydride solution with the concentration of 0.1M, stirring for 1 h to obtain solution nanogold solution, then centrifuging at 25000 rpm, and freeze-drying to obtain GNPs with the average particle size of 40 nm;

(7) adding 6mL of hydration liquid into the P-GNBs prepared in the step 5), and ultrasonically dissolving a liposome membrane for 5 min to obtain a GNBs solution; adding DOX into the obtained GNBs solution to enable the concentration to be 1 mg/mL, carrying out oil bath at 50 ℃ for 1 h, then oscillating at 500 rpm for 2 h in perfluoropropane atmosphere, centrifuging at 800 rpm for 20 min, washing for 3 times by PBS, wherein the centrifugation speed is 20000 rpm, and the time is 5 min to obtain the drug-loaded nano material GNBs/DOX;

Example 7

(1) Repeatedly freezing and thawing fresh animal livers for 3 times, repeatedly washing the livers with deionized water after slicing, then carrying out decellularization treatment on the livers with a mixed solution containing 0.5 vol% ammonia water and 4.44 vol% Triton X-100, washing the livers with deionized water until the mixture is neutral, and soaking the livers in a PBS (phosphate buffer solution) solution with the pH =7.4 overnight to obtain P-DECM; soaking in peroxyacetic acid for disinfection, and washing with PBS and water in sequence;

(2) cutting the prepared P-DECM after freeze drying into particles with the diameter of 100 mu m, and then grinding at low temperature of-20-4 ℃ to obtain F-DECM with the diameter of 400 nm;

(3) adding F-DECM into 0.01M hydrochloric acid solution containing 1 mg/mL pepsin to enable the concentration to be 1 mg/mL, stirring for 50H at 25 ℃ until the solution is completely dissolved, then adding 0.5M sodium hydroxide solution to adjust the pH value to be neutral, adding 7 mL 10 XPBS to adjust the osmotic pressure to be balanced, and obtaining H-DECM;

(4) mixing and stirring chloroauric acid solution and sodium citrate uniformly in a conical flask, wherein the total volume of the obtained gold solution is 20 mL, the concentration of chloroauric acid is 0.025 mM, the concentration of sodium citrate is 0.025 mM, then stirring at 550 rpm at normal temperature, adding 0.6 mL of refrigerated sodium borohydride solution with the concentration of 0.1M, stirring for 30 min to obtain solution nanogold solution, then centrifuging at the speed of 23000 rpm, and freeze-drying to obtain GNPs with the average particle size of 370 nm;

(5) dissolving DPPC, DPPE, DPPA, DSPE-PEG2000-COOH and GNPs in a mixed solution prepared from 10 mL of methanol and 5 mL of chloroform, so that the concentration of each substance is 20 mg/mL of DPPC, 5 mg/mL of DPPE, 6 mg/mL of DPPA, 6 mg/mL of DSPE-PEG2000-COOH and 3 mg/mL of GNPs; performing ultrasonic treatment for 5 min, and performing rotary evaporation at 66 ℃ for 1 h to form a film to obtain P-GNBs;

(6) mixing 400 mu L of glycerol, 8 mL of PBS and 3 mg of F-68 to obtain a hydration solution;

(7) adding 5.5 mL of hydration liquid into the P-GNBs prepared in the step 5), and ultrasonically dissolving a liposome membrane for 5 min to obtain a GNBs solution; adding DOX into the obtained GNBs solution to enable the concentration of the DOX to be 3.64 mg/mL, carrying out oil bath for 1 h at 90 ℃, then oscillating for 4 h at the rotating speed of 500 rpm in the perfluoropropane atmosphere, centrifuging for 10 min at 800 rpm, washing for 2 times by PBS, wherein the centrifuging speed is 16000 rpm, and the time is 3 min to obtain the drug-loaded nano material GNBs/DOX;

(8) and rapidly stirring the obtained H-DECM and the medicine-carrying nano material GNBs/DOX in an ice bath at a mass-volume ratio of 100 mug/mL, uniformly mixing, performing temperature programming at a speed of 1.0 ℃/s, and finally performing self-crosslinking at 37 ℃ to finally obtain the GNBs/DOX/H-DECM.

Example 8

(1) Repeatedly freezing and thawing fresh animal livers for 3 times, slicing, repeatedly washing with deionized water, then performing acellular treatment with a mixed solution containing 1.37 vol% ammonia water and 1.45 vol% Triton X-100, washing with deionized water to neutrality, and soaking in a PBS (phosphate buffer solution) with pH =7.4 overnight to obtain P-DECM; soaking in peroxyacetic acid for disinfection, and washing with PBS and water in sequence;

(4) mixing and stirring chloroauric acid solution and sodium citrate uniformly in a conical flask, wherein the total volume of the obtained gold solution is 20 mL, the concentration of chloroauric acid is 27 mM, the concentration of sodium citrate is 68 mM, then stirring at 550 rpm at normal temperature, adding 5.93 mL of refrigerated sodium borohydride solution with the concentration of 0.1M, stirring for 30 min to obtain solution nanogold solution, then centrifuging at 25000 rpm, and freeze-drying to obtain GNPs with the average particle size of 16 nm;

(5) dissolving DPPC, DPPE, DPPA, DSPE-PEG2000-COOH and GNPs in a mixed solution prepared from 10 mL of methanol and 5 mL of chloroform, so that the concentration of each substance is 21 mg/mL of DPPC, 3.6 mg/mL of DPPE, 6.8 mg/mL of DPPA, 7.5 mg/mL of DSPE-PEG2000-COOH and 7.68 mg/mL of GNPs; performing ultrasonic treatment for 5 min, and performing rotary evaporation at 55 ℃ for 1 h to form a film to obtain P-GNBs;

(7) adding 5.5 mL of hydration liquid into the P-GNBs prepared in the step 5), and ultrasonically dissolving a liposome membrane for 5 min to obtain a GNBs solution; adding DOX into the obtained GNBs solution to enable the concentration of the DOX to be 3.64 mg/mL, carrying out oil bath for 1 h at 75 ℃, then oscillating for 2 h at the rotating speed of 500 rpm in the perfluoropropane atmosphere, centrifuging for 10 min at 800 rpm, washing for 3 times with PBS (phosphate buffer solution) at the centrifuging speed of 20000 rpm for 5 min, and obtaining the drug-loaded nano material GNBs/DOX;

Example 9

(1) Repeatedly freezing and thawing fresh animal livers for 3 times, slicing, repeatedly washing with deionized water, then carrying out acellular treatment with a mixed solution containing 1.5 vol% ammonia water and 7.53 vol% Triton X-100, washing with deionized water to neutrality, and soaking in a PBS (phosphate buffer solution) with pH =7.4 overnight to obtain P-DECM; soaking in peroxyacetic acid for disinfection, and washing with PBS and water in sequence;

(2) cutting the prepared P-DECM after freeze drying into particles with the diameter of 478 mu m, and then grinding at low temperature of-20-4 ℃ to obtain F-DECM with the diameter of 196 nm;

(3) adding F-DECM into 0.01M hydrochloric acid solution containing 17 mg/mL pepsin to enable the concentration to be 16 mg/mL, stirring for 72 hours at 25 ℃ until the solution is completely dissolved, then adding 0.1M sodium hydroxide solution to adjust the pH value to be neutral, adding 1 mL 10 XPBS to adjust the osmotic pressure to be balanced, and obtaining H-DECM;

(4) mixing and stirring chloroauric acid solution and sodium citrate uniformly in a conical flask, wherein the total volume of the obtained gold solution is 20 mL, the concentration of chloroauric acid is 0.025 mM, the concentration of sodium citrate is 0.025 mM, then stirring at 550 rpm at normal temperature, adding 0.6 mL of refrigerated sodium borohydride solution with the concentration of 0.1M, stirring for 30 min to obtain solution nanogold solution, then centrifuging at the speed of 25000 rpm, and freeze-drying to obtain GNPs with the average particle size of 3.49 nm;

(5) dissolving DPPC, DPPE, DPPA, DSPE-PEG2000-COOH and GNPs in a mixed solution prepared from 10 mL of methanol and 5 mL of chloroform, so that the concentration of each substance is 20 mg/mL of DPPC, 6 mg/mL of DPPE, 6 mg/mL of DPPA, 6 mg/mL of DSPE-PEG2000-COOH and 7.68 mg/mL of GNPs; performing ultrasonic treatment for 5 min, and performing rotary evaporation at 55 ℃ for 1 h to form a film to obtain P-GNBs;

(7) adding 5.5 mL of hydration liquid into the P-GNBs prepared in the step 5), and ultrasonically dissolving a liposome membrane for 5 min to obtain a GNBs solution; adding DOX into the obtained GNBs solution to enable the concentration of the DOX to be 6 mg/mL, carrying out oil bath at 75 ℃ for 1 h, then oscillating at 500 rpm for 2 h in perfluoropropane atmosphere, centrifuging at 800 rpm for 10 min, washing for 3 times by using PBS, wherein the centrifugation speed is 20000 rpm, and the time is 5 min to obtain the drug-loaded nano material GNBs/DOX;

(8) rapidly stirring the obtained P-H-DECM and the drug-loaded nano material GNBs/DOX in a mass-volume ratio of 20 microgram/mL in an ice bath, uniformly mixing, performing temperature programming at a speed of 1.0 ℃/s, and finally performing self-crosslinking at 37 ℃ to finally obtain the GNBs/DOX/H-DECM.

Comparative example 1

(1) Repeatedly freezing and thawing fresh animal livers for 3 times, slicing, repeatedly washing with deionized water, then performing acellular treatment with a mixed solution containing 0.1vol% ammonia water and 0.05 vol% Triton X-100, washing with deionized water to neutrality, and soaking in a PBS (phosphate buffer solution) with pH =7.4 overnight to obtain P-DECM; soaking in peroxyacetic acid for disinfection, and washing with PBS and water in sequence;

Compared with example 1, under the same conditions, the concentration of Triton X-100 used in the comparative example is reduced, the corresponding degree of decellularization is more incomplete, and the biocompatibility of the hydrogel and the proliferation capacity of cells are greatly reduced.

Comparative example 2

(7) adding 5.5 mL of hydration liquid into the P-GNBs prepared in the step 5), and ultrasonically dissolving a liposome membrane for 5 min to obtain a GNBs solution; adding DOX into the obtained GNBs solution to enable the concentration of the DOX to be 3.64 mg/mL, carrying out oil bath for 1 h at 75 ℃, then oscillating for 2 h at the rotating speed of 500 rpm in the perfluoropropane atmosphere, centrifuging for 10 min at 70 rpm, washing for 3 times by PBS, wherein the centrifuging speed is 20000 rpm, and the time is 5 min to obtain the drug-loaded nano material GNBs/DOX;

(8) and rapidly stirring the obtained P-H-DECM and the drug-loaded nano material GNBs/DOX respectively at mass-volume ratios of 20, 40, 80, 160 and 320 mug/mL in an ice bath, uniformly mixing, then carrying out temperature programming at a speed of 1.0 ℃/s, and finally carrying out self-crosslinking at 37 ℃ to obtain the GNBs/DOX/H-DECM with different ratios.

TABLE 1 comparison of gelling times for GNBs/DOX/H-DECM composites prepared in different ratios

FIG. 11 is a result chart of the apoptosis rate of the GNBs/DOX/H-DECM composite material prepared by different ratios of GNBs/DOX and H-DECM on hepatoma cell HepG2 detected by a flow cytometer. As can be seen from the figure, the composite material prepared in the embodiment has a very strong cancer cell killing function, and compared with the control group PBS, the higher the ratio of GNBs/DOX is, the higher the apoptosis rate is.

As can be seen from table 1 and fig. 11, under the same conditions, the larger the GNBs/DOX ratio, the better the crosslinking degree, and the shorter the gelling time, the better the photothermal treatment effect.

FIG. 12 is a graph showing the capacity of the GNBs/DOX/H-DECM composite material to treat tumors at a ratio of 80 μ g/mL. Tumor inoculation is carried out on nude mice by using liver cancer cells HepG2, PBS is injected into a control group two weeks after tumor inoculation, GNBs/DOX/H-DECM with the concentration of 80 mug/mL is injected into an experimental group, the experimental group is periodically irradiated by a 808 nm laser for treatment, and tumor masses are taken out on the tenth day. It is evident from the figure that the tumor mass volume of the treated group is smaller than that of the control group, indicating the capacity of the GNBs/DOX/H-DECM composite material to treat tumors in vivo.

FIG. 13 shows the measurement results of the contents of proinflammatory factors IL-1 beta, IL-6, TNF-alpha and anti-inflammatory factor IL-10 in animals after animal experiments on GNBs/DOX/H-DECM composite materials prepared by different ratios of GNBs/DOX and H-DECM. As can be seen from the figure, the higher the content of added H-DECM, the more anti-inflammatory factors are produced and the lower the content of pro-inflammatory factors is, compared with the control group GNBs/DOX, indicating that GNBs/DOX/H-DECM has the functions of treating inflammation and repairing.

FIG. 14 shows the storage modulus (solids-like) of hydrogels prepared with different concentrations of H-DECM and different ratios of GNBs/DOX at the same concentration. When the concentration of H-DECM is increased from 10 mg/mL to 20 mg/mL, the storage modulus is increased, which shows that increasing the concentration of H-DECM can increase the stress for elastic deformation. When the concentration of the H-DECM is kept to be 20 mg/mL, and 100 mug/mL and 200 mug/mL of GNBs/DOX are added to be compounded with the H-DECM, the storage modulus is sequentially increased, which shows that the internal structure is changed and the stress required by elastic deformation is further increased by adding the GNBs/DOX, namely, the mechanical property is enhanced.

The above embodiments are merely provided to aid understanding of the method of the present invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of multifunctional liver extracellular matrix composite hydrogel for liver cancer treatment and repair integration is characterized by comprising the following steps: the method comprises the following steps:

(1) repeatedly freezing and thawing fresh animal livers, slicing, repeatedly washing with deionized water, then carrying out acellular treatment with a mixed solution of ammonia water and Triton X-100, magnetically stirring and washing with deionized water until the mixture is neutral, and then soaking with a PBS (phosphate buffer solution) solution with pH =7.4 overnight to obtain an acellular scaffold precursor P-DECM;

(2) freeze-drying the P-DECM prepared in the step (1), shearing into pieces, and grinding at a low temperature of-20-4 ℃ to obtain extracellular matrix powder F-DECM;

(5) dissolving dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, diphenylphosphoryl azide, DSPE-PEG2000-COOH and GNPs in a mixed solution of methanol and chloroform, ultrasonically mixing uniformly, and performing rotary evaporation to form a membrane to obtain a nano material precursor P-GNBs wrapped by liposome vesicles;

(7) adding the hydrated liquid obtained in the step (6) into the P-GNBs obtained in the step (5), and dissolving a liposome membrane by ultrasonic waves to obtain a nano particle GNBs solution coated by the liposome vesicle; adding an anti-cancer drug into the GNBs solution, carrying out oscillation reaction in perfluoropropane atmosphere under the condition of oil bath, centrifuging, washing, drying at 37 ℃, and weighing to obtain a drug-loaded nano material GNBs/ACD;

(8) and (4) rapidly stirring and uniformly mixing the H-DECM obtained in the step (3) and the medicine-carrying nano material GNBs/ACD obtained in the step (7) on an ice bath, and finally, maintaining the self-crosslinking at 37 ℃ after temperature programming to obtain the multifunctional liver extracellular matrix composite hydrogel GNBs/ACD/H-DECM.

2. The preparation method of the multifunctional liver extracellular matrix composite hydrogel according to claim 1, wherein the preparation method comprises the following steps: in the mixed solution of the ammonia water and the Triton X-100 in the step (1), the volume concentration of the ammonia water is 0.01-20%, and the volume concentration of the Triton X-100 is 0.1-30%.

3. The preparation method of the multifunctional liver extracellular matrix composite hydrogel according to claim 1, wherein the preparation method comprises the following steps: and (3) cutting the P-DECM into particles with the diameter of 1 mu m-3 cm in the step (2), and grinding the particles into powder with the diameter of 10 nm-1 mm.

4. The preparation method of the multifunctional liver extracellular matrix composite hydrogel according to claim 1, wherein the preparation method comprises the following steps: the hydrochloric acid concentration of the hydrochloric acid solution containing the pepsin in the step (3) is 0.00001-10M, and the pepsin concentration is 0.01-1000 mg/mL; the addition amount of the F-DECM is 0.01-1000 mg/mL; the stirring temperature is 0-60 ℃, and the stirring time is 2 h-7 d; the concentration of the sodium hydroxide solution is 0.0001-100M.

5. The preparation method of the multifunctional liver extracellular matrix composite hydrogel according to claim 1, wherein the preparation method comprises the following steps: the concentration of chloroauric acid in the gold solution obtained in the step (4) is 0.001-250 mM, and the concentration of sodium citrate is 0.001-250 mM; the concentration of the sodium borohydride solution is 0.1M, the volume ratio of the amount of the sodium borohydride solution to the gold solution is (0.001-15): 20, and the stirring reaction time is 10 min-4 h; the particle size of the obtained GNPs is 1-100 nm.

6. The preparation method of the multifunctional liver extracellular matrix composite hydrogel according to claim 1, wherein the preparation method comprises the following steps: in the step (5), the volume ratio of methanol to chloroform in the mixed solution of methanol and chloroform is (0.1-200) to 1; the addition amount of dipalmitoyl phosphatidylcholine is 0.002-10 mg/mL, the addition amount of dipalmitoyl phosphatidylethanolamine is 0.002-10 mg/mL, the addition amount of diphenylphosphorylazide is 0.002-10 mg/mL, the addition amount of DSPE-PEG2000-COOH is 0.002-10 mg/mL, and the addition amount of GNPs is 0.002-100 mg/mL.

7. The preparation method of the multifunctional liver extracellular matrix composite hydrogel according to claim 1, wherein the preparation method comprises the following steps: the volume ratio of glycerol to 1 multiplied by PBS in the hydration liquid obtained in the step (6) is (0.00001-100) to 1, and the content of F-68 is 0.005-50 mg/mL.

8. The preparation method of the multifunctional liver extracellular matrix composite hydrogel according to claim 1, wherein the preparation method comprises the following steps: after the anti-cancer drugs are added in the step (7), the concentration of the anti-cancer drugs in the GNBs solution is 0.001-10 mg/mL, and the anti-cancer drugs comprise any one of adriamycin, paclitaxel and docetaxel; the temperature of the oscillation reaction is 20-120 ℃, the time is 10 min-8 h, and the rotating speed is 100-1000 rpm.

9. The preparation method of the multifunctional liver extracellular matrix composite hydrogel according to claim 1, wherein the preparation method comprises the following steps: the mass-volume ratio of GNBs/ACD and H-DECM used in the step (8) is 5-850 microgram/mL, and the temperature programming speed is 0.1-10 ℃/s.

10. A multifunctional liver extracellular matrix composite hydrogel for liver cancer treatment and repair integration prepared by the method of claims 1-9.