CN114984322B

CN114984322B - Double-layer hydrogel material, preparation method and application thereof

Info

Publication number: CN114984322B
Application number: CN202210657427.3A
Authority: CN
Inventors: 黄炜; 乔铭薪; 赵志河; 吴晓悦; 朱舟; 刘家伶; 张博
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-06-10
Filing date: 2022-06-10
Publication date: 2022-12-20
Anticipated expiration: 2042-06-10
Also published as: CN114984322A

Abstract

The invention discloses a double-layer hydrogel material, a preparation method and application thereof, and relates to the technical field of biological medicines. The inner hydrogel is formed by methacrylated hyaluronic acid (HAMA) and F127, outer hydrogel coated on the inner hydrogel is formed by methacrylated gelatin (GelMA), melatonin is loaded on the inner hydrogel and the outer hydrogel, high-concentration melatonin on the outer hydrogel has the functions of inhibiting tumor proliferation, differentiation and metastasis, and low-concentration melatonin on the inner hydrogel has the capacity of promoting osteogenic differentiation of stem cells. By constructing an MT @ GelMA/HAMA/F127 double-layer hydrogel drug-loading system, the drug is released in a programmed way, the tumor cells are quickly killed in the early stage after the operation, the melatonin is slowly released in the later stage, and a support is provided for osteoblasts to promote bone repair, so that the aims of inhibiting postoperative recurrence of osteosarcoma and promoting bone repair of a lesion area are fulfilled.

Description

Double-layer hydrogel material, preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a double-layer hydrogel material, and a preparation method and application thereof.

Background

Osteosarcoma can greatly influence the life quality of a patient, the bone defect recovery caused by surgical resection of osteosarcoma still faces a plurality of challenges, and generally, the postoperative recovery treatment of osteosarcoma has the problems of high tumor recurrence rate, slow bone growth, poor hard tissue function recovery in a damaged area, poor life quality of the patient, low survival rate and the like.

In the early postoperative stage of osteosarcoma treatment, residual tumor cells need to be killed, stem cells need to be recruited for bone repair after the operation area is cleaned, and an urgent need exists for exploring a treatment method which can prevent tumor recurrence and promote bone regeneration. However, the existing hydrogel materials for bone tissue engineering still have the following defects:

(1) Failure to mimic the course of osteosarcoma treatment;

(2) Cannot inhibit tumor cells and promote bone regeneration;

(3) The mechanical strength is not ideal, and the scaffold for repairing bone tissues cannot be provided.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a double-layer hydrogel material and a preparation method thereof, which can inhibit tumor cells and promote bone regeneration.

The invention also aims to provide application of the double-layer hydrogel material in the field of drug delivery.

The third purpose of the present invention is to provide the application of the above-mentioned double-layer hydrogel material in bone tissue engineering.

The invention is realized by the following steps:

in a first aspect, the invention provides a double-layer hydrogel material, which comprises an inner-layer hydrogel and an outer-layer hydrogel coated on the inner-layer hydrogel, wherein the inner-layer hydrogel is formed by mixing methacrylated hyaluronic acid and F127 and then photocuring, the outer-layer hydrogel is formed by photocuring methacrylated gelatin, melatonin is loaded on both the inner-layer hydrogel and the outer-layer hydrogel, and the loading amount of the melatonin on the inner-layer hydrogel is smaller than that on the outer-layer hydrogel.

In alternative embodiments, the mass ratio of methacrylated hyaluronic acid to F127 in the inner layer hydrogel is 1:1-2.0; preferably 1:1;

preferably, the mass volume ratio of the methacrylated hyaluronic acid to the solution of the photoinitiator LAP is 5-10g:2-5g, namely 100mL, wherein the mass fraction of the photoinitiator LAP solution is 0.2-0.3%;

more preferably, the mass-to-volume ratio between the methacrylated hyaluronic acid, F127 and the photoinitiator LAP solution is 5 g.

In a second aspect, the present invention provides a method of preparing a bi-layer hydrogel material according to any one of the preceding embodiments, comprising: preparing the inner-layer hydrogel by taking methacrylated hyaluronic acid, F127 and melatonin as raw materials, and then preparing the outer-layer hydrogel on the inner-layer hydrogel by taking methacrylated hyaluronic acid and melatonin as raw materials;

preferably, the mass volume ratio between the outer layer of methacrylated gelatin and the photoinitiator LAP solution is 5-10g.

In an alternative embodiment, the method comprises the following steps: mixing methacryloylated hyaluronic acid, F127 and a photoinitiator LAP to obtain a first mixed solution, mixing the first mixed solution with melatonin to obtain an inner-layer mixed solution, and curing and forming the inner-layer mixed solution under the illumination condition to obtain inner-layer drug-loaded hydrogel;

mixing methacrylated gelatin and photoinitiator LAP to obtain a second mixed solution, uniformly mixing the second mixed solution with melatonin to obtain an outer drug-loaded hydrogel mixed solution, and uniformly wrapping the cured inner hydrogel with the outer drug-loaded hydrogel mixed solution to perform light curing so as to form the outer drug-loaded hydrogel on the inner drug-loaded hydrogel.

In an alternative embodiment, the mass fraction of the methacrylated hyaluronic acid in the first mixed solution is 0.05-0.1g/mL, the mass fraction of the F127 in the first mixed solution is 0.02-0.05g/mL, and the amount of melatonin used per mL of the first mixed solution is 0.23-2.32mg;

preferably, the mass fraction of the methacrylated hyaluronic acid in the first mixed solution is 0.05-0.10g/mL, the mass fraction of F127 in the first mixed solution is 0.02-0.05g/mL, and the dosage of melatonin corresponding to each milliliter of the first mixed solution is 1.1-1.3mg.

In an alternative embodiment, the mass fraction of the methacrylated gelatin in the second mixed solution is 0.05-0.10g/mL, and the amount of melatonin per mL of the second mixed solution is 2.90-92.91mg;

preferably, the mass fraction of the methacrylated gelatin in the second mixed solution is 0.05-0.10g/mL, and the dosage of melatonin corresponding to each mL of the second mixed solution is 20-30mg;

preferably, the preparation process of the second mixed solution comprises: the methacrylated gelatin and photoinitiator LAP were mixed and then treated at 50-60 ℃ to melt the starting materials.

In an alternative embodiment, the inner hydrogel and the outer hydrogel are cured and formed for 0.5-2min under the irradiation of a light curing lamp;

preferably, the light curing lamp emits light with a wavelength of 400nm to 480nm; preferably 405nm.

In a third aspect, the present invention provides a double-layer hydrogel material according to any one of the preceding embodiments or a double-layer hydrogel material prepared by the method according to any one of the preceding embodiments, for use in the field of drug delivery.

In a fourth aspect, the present invention provides a use of the bilayer hydrogel material of any one of the preceding embodiments or prepared by the method of any one of the preceding embodiments in bone tissue engineering.

The invention has the following beneficial effects: the inner hydrogel is formed by methacrylated hyaluronic acid (HAMA) and F127, outer hydrogel coated on the inner hydrogel is formed by methacrylated gelatin (GelMA), melatonin is loaded on the inner hydrogel and the outer hydrogel, high-concentration melatonin on the outer hydrogel has the functions of inhibiting tumor proliferation, differentiation and metastasis, and low-concentration melatonin on the inner hydrogel has the capacity of inducing stem cell osteogenic differentiation and mineralization.

In addition, gelMA and HAMA/F127 have a portable forming mode and good biocompatibility, and can also provide a good mechanical microenvironment for bone repair, and meanwhile, the inner layer hydrogel can stably and slowly release drugs and can promote stem cells to form bone for a long time, so that the bone repair is promoted, and therefore, the drug delivery system can be applied to postoperative recovery treatment of osteosarcoma patients and has a good application prospect.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.

Fig. 1 is a diagram of a matter of a constructed melatonin-loaded double-layer hydrogel drug-loading system provided in an embodiment of the present invention;

FIG. 2 shows the effect of leachate obtained by varying the amounts of F127 and HAMA in the inner layer structure on the proliferation of BMSCs;

fig. 3 is an outer layer GelMA scanning electron microscope of the double-layer hydrogel drug-loading system provided by the embodiment of the invention;

FIG. 4 is a scanning electron micrograph of inner layer 5% HAMA +5% of the bilayer hydrogel drug delivery system provided by an embodiment of the present invention;

fig. 5 is a scanning electron microscope image of a prepared double-layer hydrogel drug-loaded system provided by an embodiment of the invention;

fig. 6 is a CCK8 test of the proliferation effect of the leachate after the double-layer hydrogel drug-loading system provided by the embodiment of the invention loads melatonin at various concentrations on the outer layer on osteosarcoma cells MG63 and U2 OS;

fig. 7 is a CCK-8 detection result of the influence of leachate after the double-layer hydrogel drug-loaded system carries melatonin at various concentrations on the proliferation of rat mesenchymal stem cells according to the embodiment of the present invention;

FIG. 8 is a colony assay performed on the outer drug-loaded hydrogel leachate of the double-layer hydrogel drug-loaded system according to the experimental example of the present invention, wherein the colony assay is performed by uniformly seeding 1000 cells/well in a 6-well plate, then adding complete medium, 5% GelMA leachate, and 5% GelMA + melatonin (100 mM) leachate, respectively, to culture the cells, and performing crystal violet staining on day 10 to detect the number of colonies;

fig. 9 is an ALP quantitative determination of the influence of the leaching solution after melatonin is loaded in the inner layer of the double-layer hydrogel drug-loaded system provided by the embodiment of the invention on osteogenic differentiation of rat bone marrow mesenchymal stem cells;

FIG. 10 is a diagram of a procedure for performing an in vivo degradation experiment of a material, including shaving, disinfecting, incising the skin, placing a double hydrogel system, and suturing for disinfection;

FIG. 11 is a state diagram of a hydrogel material before and after a degradation test; FIG. 11 (a) is a diagram showing a real object of the hydrogel of the inner layer, the outer layer and the double layer in the synthesized product; (b) - (d) is a schematic diagram showing the bilayer hydrogel taken out after 1 week of the degradation test.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The embodiment of the invention provides a preparation method of a double-layer hydrogel drug-loading system, which comprises the following steps:

s1, construction of inner layer hydrogel drug-loading system

The inner hydrogel is prepared from raw materials of methacryloylated hyaluronic acid (HAMA), F127 and melatonin, and can be prepared in a photocuring mode in the actual operation process, and the preparation method comprises the following specific steps: mixing methacrylated hyaluronic acid, F127 and a photoinitiator LAP solution to obtain a first mixed solution, mixing the first mixed solution with melatonin to obtain an inner-layer mixed solution, and curing and molding the inner-layer mixed solution under the illumination condition to obtain the inner-layer hydrogel.

Specifically, HAMA is a biological hydrogel material with photosensitivity by introducing a methacrylic group into a hyaluronic acid molecular chain, is an existing raw material, and has a portable forming mode and good biocompatibility. F127 is also an existing raw material, F127 has excellent thermal gelation characteristics and good biological safety, a material system based on F127 is widely applied to the field of biomedicine, for example, a drug carrier, a wound auxiliary material and the like, F127 is matched with HAMA, not only is the inner layer hydrogel have larger material strength, but also the degradation time can be prolonged, a good slow release effect is achieved, a scaffold is provided for bone repair, and meanwhile, osteogenic differentiation of stem cells is promoted.

The study of Melatonin (MT) in osteosarcoma opens a new field of vision for the treatment of osteosarcoma in the clinic. MT is a neuroendocrine hormone in the human body, is an indole hormone, has a chemical name of N-acetyl-5-methoxytryptamine, and is a precursor of L-tryptophan.

In order to further improve the capability of promoting bone regeneration of the hydrogel of the inner layer, the dosage of the raw materials is optimized:

the mass fraction of the methacrylated hyaluronic acid in the first mixed solution is 0.05-0.1g/mL, the mass fraction of F127 in the first mixed solution is 0.02-0.05g/mL, and the dosage of melatonin corresponding to each milliliter of the first mixed solution is 1.1-1.3mg. The amount of the raw materials is preferably controlled within the above range to ensure better bone regeneration promoting performance.

Specifically, the mass fraction of the methacrylated hyaluronic acid in the first mixed solution is 0.05g/mL, 0.06g/mL, 0.07g/mL, 0.08g/mL, 0.09g/mL, 0.1g/mL, or the like, and may be any value between the above adjacent concentration values; the mass fraction of F127 in the first mixed solution is 0.02g/mL, 0.03g/mL, 0.04g/mL, 0.05g/mL or the like, and may be any value between the above adjacent concentration values; the amount of melatonin per ml of the first mixed solution is any of 1.1-1.3mg.

In the actual operation process, the curing molding under the illumination condition is to cure for 0.5-2min (such as 0.5min, 1.0min, 1.5min, 2.0min, etc.) under the illumination of a light curing lamp, and the wavelength of light emitted by the light curing lamp is 400nm-480nm.

Specifically, the photoinitiator used in the preparation of the inner layer hydrogel may be a conventional photoinitiator LAP, and the required use concentration is 0.25% (mass to volume ratio of photoinitiator LAP (as powder)/PBS).

Construction of S2, gelMA/HAMA/F127 double-layer hydrogel drug-loading system

And (3) preparing an outer hydrogel on the inner hydrogel by using methacrylic acid gelatin (GelMA) as a raw material to construct a GelMA/HAMA/F127 double-layer hydrogel drug-loading system.

In particular, gelMA is an existing photosensitive biological hydrogel material, which has excellent biocompatibility and can be excited by ultraviolet light or visible light to perform a curing reaction to form a three-dimensional structure with certain strength suitable for cell growth and differentiation. The biocompatibility of the collagen is far superior to that of matrigel and fibrin glue, the biocompatibility of the collagen is close to that of collagen, and the forming performance of the collagen is far superior to that of collagen.

In some embodiments, the outer hydrogel can also be formed by light curing, which comprises the following steps: mixing the methacrylated gelatin and the photoinitiator LAP solution to obtain a second mixed solution, mixing the second mixed solution with melatonin to obtain an outer-layer hydrogel and melatonin mixed solution, and uniformly wrapping the photocured inner-layer melatonin-loaded hydrogel with the outer-layer hydrogel and melatonin mixed solution. The specific shaping procedure allows the preparation of two layers of hydrogel by means of different size molds.

In order to improve the performance of the outer hydrogel in inhibiting tumor cells, the inventor optimizes the dosage of the raw materials: the mass volume ratio of the outer layer of methacrylated gelatin to the photoinitiator LAP solution is 5-10g. The mass fraction of the methacrylated gelatin in the second mixed solution is 0.05-0.10g/mL, and the dosage of melatonin corresponding to each milliliter of the second mixed solution is 2.90-92.91mg; preferably, the amount of melatonin used per ml of the second mixed solution is 20-30mg.

Specifically, by controlling the amount of the photoinitiator, the mass fraction of the methacrylated gelatin in the second mixed solution can be 0.05-0.10g/mL or the like, or can be any value between the adjacent concentration values; the dosage of the melatonin corresponding to each milliliter of the second mixed solution is any value between 2.90 and 92.91.

In some embodiments, the preparing of the second mixed solution comprises: mixing methacrylated gelatin and photoinitiator, treating at 50-60 deg.c to melt the material and mix it with melatonin, and subsequent light curing.

In an alternative embodiment, the outer hydrogel is cured and formed under the irradiation of a light curing lamp for 0.5 to 2min (e.g., 0.5min, 1.0min, 1.5min, 2.0min, etc.); the wavelength of light emitted by the light curing lamp is 400-480nm.

Specifically, the photoinitiator used in the preparation of the outer hydrogel may be a conventional photoinitiator LAP, and the required concentration used is 0.25% (mass to volume ratio of photoinitiator LAP (as powder)/PBS).

The embodiment of the invention also provides a double-layer hydrogel material which can be prepared by the preparation method. Specifically, the hydrogel comprises an inner layer hydrogel and an outer layer hydrogel coated on the inner layer hydrogel, wherein the inner layer hydrogel is formed by mixing methacrylated hyaluronic acid and F127 and then photocuring, the outer layer hydrogel is formed by photocuring methacrylated gelatin, melatonin is loaded on both the inner layer hydrogel and the outer layer hydrogel, and the loading amount of the melatonin on the inner layer hydrogel is smaller than that on the outer layer hydrogel.

When the hydrogel is used, the outer GelMA layer carries high-concentration melatonin to inhibit the growth of tumor cells, the low-concentration GelMA layer can degrade and release high-concentration melatonin in a period of time, and the hydrogel formed by mixing the inner HAMA layer and the inner HAF 127 layer carries low-concentration melatonin to promote osteogenesis. The melatonin carried in the inner-outer layer structure is released in order to kill tumor cells at a high concentration in the initial stage, and the melatonin at a low concentration is released continuously in the later stage to promote bone repair. The MT @ GelMA drug-carrying system is a core component for inhibiting tumor proliferation, differentiation and metastasis, and the HAMA/F127 mixed hydrogel is porous and has a multi-network structure which is a main part of secondary drug release and also provides a good support for bone repair.

It should be noted that, in order to achieve the effects of inhibiting tumor and promoting bone formation, the embodiment of the present invention constructs a novel double-layer hydrogel melatonin-loaded system to programmatically release melatonin with different concentrations, so as to achieve the processes of inhibiting tumor at the initial stage of tumor surgery and promoting bone repair at the later stage. The outer layer of the embodiment of the invention is formed by photocuring after loading high-concentration melatonin by GelMA, the inner layer is formed by photocuring after mixing HAMA, F127 and low-concentration melatonin, gelMA is hydrogel with good biocompatibility, and has the characteristics of high degradation speed, low strength and the like, the GelMA at the outer layer is rapidly degraded at the initial stage of operation to release the high-concentration melatonin to kill tumor cells, and meanwhile, the concentration of the hydrogel loaded at the outer layer is not so high as to kill mesenchymal stem cells in marrow. On the other hand, the HAMA/F127 hydrogel in the inner layer has higher strength after being mixed and photocured, can slowly release low-concentration melatonin to promote bone regeneration and bone defect repair, can achieve the multi-stage slow release effect of the medicine, and enables MT to play a more lasting effect.

In order to improve the inhibition effect of the outer hydrogel on tumor cells and the capability of promoting bone regeneration of the inner hydrogel, the inventor optimizes the content of each component in the product: the mass-volume ratio of the methacrylated hyaluronic acid to the solution of the photoinitiator LAP is 5-10g:2-5g, namely 100mL, wherein the mass fraction of the photoinitiator LAP solution is 0.2-0.3%; preferably, the mass-to-volume ratio of the methacrylated hyaluronic acid, the F127 and the photoinitiator LAP solution is 5 g.

It should be noted that the double-layer hydrogel provided by the embodiment of the present invention has a very wide application range, and can be widely applied in the field of biological medicine, for example, the double-layer hydrogel constructed by the embodiment of the present invention can be applied in the field of drug loading. The more important application is that the double-layer hydrogel is applied to bone tissue engineering and is used for postoperative recovery and treatment of osteosarcoma patients.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

This example provides a method for preparing a bilayer hydrogel, including the following steps:

(1) Construction of inner hydrogel drug-loading system

Dissolving 5g of HAMA, 5g of F127 and 100mL of photoinitiator LAP (the concentration is 0.25%) at normal temperature to obtain a first mixed solution, uniformly mixing each 1mL of the first mixed solution with 1.16mg of melatonin to obtain an inner layer mixed solution, injecting the inner layer mixed solution into small molds (the length, the width and the height of an inner cavity of each mold are respectively 5X 2.5, the same is applied below), wherein each small mold needs 60 mu l of the inner layer mixed solution, and then curing for 1min by adopting a light curing lamp with the wavelength of 450nm to form the inner layer hydrogel.

(2) Construction of GelMA/HAMA/F127 hydrogel

Mixing 5g GelMA with 100mL of photoinitiator LAP (with the concentration of 0.25%), heating in a water bath at 55 ℃ to melt the raw materials to obtain a second mixed solution, and uniformly mixing every 1mL of the second mixed solution with 23.23mg of melatonin to obtain an outer layer mixed solution.

And (3) injecting the outer layer mixed solution into a large-size mold (the length, the width and the height are respectively 7 × 4.45, the same below), putting the hydrogel solid obtained in the step (1) into the outer layer mixed solution, enabling the outer layer mixed solution to uniformly wrap the hydrogel solid, and then curing for 1min by adopting a light curing lamp with the wavelength of 450nm to obtain the product shown in the figure 1.

In fig. 1, the inner hydrogel is stained in order to better distinguish the inner hydrogel from the outer hydrogel.

Example 2

(1) 5g of HAMA, 2g of F127 and 100mL of photoinitiator LAP (the concentration is 0.25%) are mixed, dissolved at normal temperature to obtain a first mixed solution, every 1mL of the first mixed solution and 0.23mg of melatonin are uniformly mixed to obtain an inner layer mixed solution, the inner layer mixed solution is injected into a small-sized mold, and then a light curing lamp with the wavelength of 450nm is used for curing for 1min to form inner layer hydrogel.

(2) Construction of GelMA/HAMA/F127 hydrogel

Mixing 4g GelMA with 100mL of photoinitiator LAP (with the concentration of 0.25%), heating in a water bath at 55 ℃ to melt the raw materials to obtain a second mixed solution, and uniformly mixing every 1mL of the second mixed solution with 2.90mg of melatonin to obtain an outer layer mixed solution.

And (2) placing the hydrogel solid obtained in the step (1) in a large-size mold (the length, width and height of an inner cavity of the mold are respectively 5 × 2.5), injecting the outer-layer mixed solution into the large-size mold (the length, width and height are respectively 7 × 4.45), enabling the hydrogel solid to be in the middle position, enabling the outer-layer mixed solution to uniformly wrap the hydrogel solid, and then curing for 1min by adopting a light curing lamp with the wavelength of 450 nm.

Example 3

(1) Mixing 10g of HAMA, 5g of F127 and 100mL of photoinitiator LAP (the concentration is 0.25%), dissolving at normal temperature to obtain a first mixed solution, uniformly mixing every 1mL of the first mixed solution with 2.32mg of melatonin to obtain an inner layer mixed solution, injecting the inner layer mixed solution into a small-size mold, and curing for 1min by adopting a light curing lamp with the wavelength of 450nm to form inner layer hydrogel.

(2) Construction of GelMA/HAMA/F127 hydrogel

6g of GelMA and 100mL of photoinitiator LAP (with the concentration of 0.25%) are mixed, the mixture is heated in a water bath kettle at the temperature of 55 ℃ to melt the raw materials to obtain a second mixed solution, and every 1mL of the second mixed solution and 92.91mg of melatonin are uniformly mixed to obtain an outer layer mixed solution.

And (2) placing the hydrogel solid obtained in the step (1) in a large-size mold (the length, width and height of an inner cavity of the mold are respectively 5 x 2.5), injecting the outer-layer mixed solution into the large-size mold (the length, width and height are respectively 7 x 4.45), enabling the hydrogel solid to be in the middle position, enabling the outer-layer mixed solution to uniformly wrap the hydrogel solid, and curing for 1min by adopting a light curing lamp with the wavelength of 450 nm.

Test example 1

The effect of F127 and HAMA concentrations on BMSCs cell proliferation was investigated by CCK8 experiments, varying only F127 and HAMA concentrations on the basis of example 1, and the results are shown in fig. 2.

The results show that: 5% F127+10% HAMA has a significant inhibitory effect on the proliferation of BMSCs, considering that the increase in F127 and HAMA concentrations can increase the material hardness, provide a scaffold for bone repair, while not significantly affecting the proliferation of BMSCs, the 5% F127+5% HAMA formulation was selected to synthesize the inner layer material.

Note: 5% F127+5% by weight of HAMA means: 5gF127 and 5gHAMA were dissolved in 100mL of photoinitiator LAP.

Test example 2

The structure of GelMA as an outer layer of the product prepared in example 1 was tested, and as a result, as shown in fig. 3, it can be seen that GelMA has a large amount of relatively uniform porous structure.

The inner HAMA/F127 structure of the product prepared in example 1 was tested, and as a result, as shown in fig. 4, it can be seen that the inner pores are uniform in size and denser than the outer structure.

The combined inner and outer layer structure of the product double hydrogel system prepared in example 1 was tested and the results are shown in figure 5.

Test example 3

By using the antitumor effect of the drug-loaded outer hydrogel in the obtained bilayer hydrogel, only the concentration of melatonin was changed based on example 1, for example, when the concentration of melatonin was 12.5mM, the mass-to-volume ratio of melatonin to GelMA was 12.5mg:4.3051mLGELMA, outer layer leachate is used for treating osteosarcoma cell lines MG63 and U2OS, and then CCK-8 proliferation detection is carried out at 3d, and the result is shown in FIG. 6.

The test method comprises the following steps: the leaching solution is prepared according to the conventional report of the literature, and the leaching solution is obtained by mixing the hydrogel and the medicine and soaking the mixture in a complete culture medium for 24 hours at the temperature of 37 ℃. Obtained according to the ratio of hydrogel to drug formation volume ratio complete medium =1 before synthesis.

As can be seen from fig. 6, the outer hydrogel had a significant anti-tumor effect.

Only the melatonin concentration was changed based on example 1, rat BMSCs were treated with the outer layer leachate, and the influence of the outer layer hydrogel on BMSCs while killing osteosarcoma cells was examined, with the results shown in fig. 7. As can be seen from fig. 7, the leachate had no significant effect on the proliferation of BMSCs when the concentration of melatonin carried by the outer hydrogel was less than 100mM, whereas significant inhibition of the proliferation of BMSCs cells began to occur when the concentration of melatonin carried by the outer hydrogel was 100mM, and this inhibitory effect increased as the amount of melatonin carried by the outer layer increased.

Test example 4

The melatonin-loaded extract from the outer hydrogel obtained in example 1 was tested for its effect on the proliferation of MG63 and U2OS, and the results are shown in fig. 8.

The test method comprises the following steps: cells were seeded evenly into 6-well plates at a density of 1000 cells/well. After the cells adhere to the wall, different leachates (complete medium, gelMA leachate, gelMA + melatonin leachate) are added respectively. After further 10 days of culture, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Images of stained colonies were taken, the number of colonies was recorded and compared in different groups. Each experiment was independently repeated three times.

The results show that leachate from the GelMA + melatonin (100 mM) group had inhibitory effects on the proliferation of MG63 and U2 OS.

Note: the concentration of 100mM is the concentration of melatonin dissolved in the initial F127+ HAMA solution, and the mixture of the F127+ HAMA + melatonin is photocured after the melatonin is dissolved and uniformly mixed.

Test example 5

Only the amount of melatonin added was changed based on example 1, and whether the inner hydrogel of the two-layer hydrogel obtained by testing the mixture of melatonin at different concentrations had the effect of promoting osteogenic differentiation and the optimum concentration of promoting osteogenic differentiation of stem cells was tested.

The test method comprises the following steps: rat BMSCs were treated with the leachate, and 7 days later, the effect of the leachate on osteogenic differentiation of BMSCs was examined using a quantitative ALP assay, and the results are shown in FIG. 9.

As can be seen from fig. 9 using the ALP quantitative analysis method, differences in osteogenic differentiation abilities of BMSCs were observed by the inner layer leachate having different concentrations at the 7 th day of osteogenic induction, wherein the leachate promoted osteogenic differentiation of BMSCs most strongly when the drug loading concentration of the inner layer was 5 mM.

Test example 6

The degradability of the bilayer hydrogel obtained in example 1 was tested, and the results are shown in fig. 10, in which (a) to (e) in fig. 10 show the specific procedures of the test method.

The test method comprises the following steps: double-layered hydrogel structures were placed on the backs of 6-week-old SD rats and sutured. The material was removed after 7 days and the results showed significant degradation of the bilayer hydrogel structure, with the outer layers completely degraded, and the specific test results are shown in table 1. FIG. 11 (a) is a schematic diagram showing an inner layer, an outer layer and a double layer hydrogel in the synthesized product; (b) - (d) is a schematic diagram showing the bilayer hydrogel taken out after the degradation test.

TABLE 1 concrete test results of degradability test

In conclusion, the GelMA + HAMA/F127 double-layer hydrogel scaffold is constructed on the basis of killing residual tumor cells and promoting bone defect repair when an osteosarcoma patient recovers after a surgery; the dual-layer hydrogel drug-loaded system has the advantages that the dual effects of inhibiting tumors and promoting bone repair by focusing MT @ GelMA + HAMA/F127 are achieved, the problem that osteosarcoma tumors are easy to relapse and slow in osteogenesis is solved by constructing the MT @ GelMA/HAMA/F127 double-layer hydrogel drug-loaded system, the double-layer hydrogel system has osteoinductive property, tumor inhibition and drug-loaded slow-release potential, and important theoretical guidance is provided for postoperative recovery and treatment of osteosarcoma patients.

The inventor proves that the double-layer hydrogel prepared in the embodiment of the invention has good biocompatibility and good degradability through in-vivo experiments, and simultaneously, in-vitro experiments show that the outer-layer hydrogel leaching solution loaded with high-concentration melatonin can effectively inhibit the growth of tumor cells, and the inner-layer hydrogel loaded with low-concentration melatonin can obviously improve the osteogenic differentiation of mesenchymal stem cells. Finally, the effects of inhibiting tumor and promoting bone formation are achieved, and a new thought and direction are provided for postoperative recovery of osteosarcoma patients.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A double-layer hydrogel material, which is characterized by comprising an inner layer hydrogel and an outer layer hydrogel coated on the inner layer hydrogel, wherein the inner layer hydrogel is formed by mixing methacrylated hyaluronic acid and F127 and then photocuring the mixture, the outer layer hydrogel is formed by photocuring methacrylated gelatin, melatonin is loaded on both the inner layer hydrogel and the outer layer hydrogel, and the loading amount of the melatonin on the inner layer hydrogel is smaller than that on the outer layer hydrogel;

the preparation method comprises the following steps: mixing methacryloylated hyaluronic acid, F127 and a photoinitiator LAP solution to obtain a first mixed solution, uniformly mixing the first mixed solution and melatonin to obtain an inner layer mixed solution, and curing and molding the inner layer mixed solution under the illumination condition to obtain an inner layer drug-loaded hydrogel;

mixing methacrylated gelatin and a photoinitiator LAP solution to obtain a second mixed solution, uniformly mixing the second mixed solution with melatonin to obtain an outer-layer drug-loaded hydrogel mixed solution, uniformly wrapping the cured inner-layer hydrogel with the outer-layer hydrogel mixed solution, and then carrying out illumination curing to form the outer-layer hydrogel on the inner-layer hydrogel;

the mass-volume ratio of the methacrylated hyaluronic acid to the solution of the photoinitiator LAP is 5-10g:2-5g, namely 100mL, wherein the mass fraction of the photoinitiator LAP solution is 0.2-0.3%;

the mass volume ratio of the outer layer of methacrylated gelatin to the photoinitiator LAP solution is 5-10g;

the mass fraction of the methacrylated hyaluronic acid in the first mixed solution is 0.05-0.1g/mL, the mass fraction of F127 in the first mixed solution is 0.02-0.05g/mL, and the dosage of melatonin corresponding to each milliliter of the first mixed solution is 0.23-2.32mg;

the mass fraction of the methacrylated gelatin in the second mixed solution is 0.05-0.10g/mL, and the dosage of melatonin corresponding to each milliliter of the second mixed solution is 2.90-92.91mg.

2. The bilayer hydrogel material of claim 1, wherein the mass-to-volume ratio of methacrylated hyaluronic acid, F127 and photoinitiator LAP solution is 5 g.

3. A method for preparing the bilayer hydrogel material of any one of claims 1-2, comprising:

mixing methacryloylated hyaluronic acid, F127 and a photoinitiator LAP solution to obtain a first mixed solution, uniformly mixing the first mixed solution and melatonin to obtain an inner layer mixed solution, and curing and molding the inner layer mixed solution under the illumination condition to obtain an inner layer drug-loaded hydrogel;

mixing methacrylated gelatin and a photoinitiator LAP solution to obtain a second mixed solution, uniformly mixing the second mixed solution with melatonin to obtain an outer-layer drug-loaded hydrogel mixed solution, uniformly wrapping the cured inner-layer hydrogel with the outer-layer hydrogel mixed solution, and then carrying out light curing to form the outer-layer hydrogel on the inner-layer hydrogel;

4. The method according to claim 3, wherein the mass fraction of methacryloylated hyaluronic acid in the first mixed solution is 0.05-0.10g/mL, the mass fraction of F127 in the first mixed solution is 0.02-0.05g/mL, and the amount of melatonin per mL of the first mixed solution is 1.1-1.3mg.

5. The method according to claim 3, wherein the mass fraction of the methacrylated gelatin in the second mixed solution is 0.05-0.10g/mL, and the amount of melatonin in the second mixed solution is 20-30 mg/mL.

6. The method according to claim 5, comprising, during the preparation of the second mixed solution: the methacrylated gelatin and photoinitiator LAP were mixed and then treated at 50-60 ℃ to melt the starting material.

7. The method according to claim 3, wherein the inner layer hydrogel and the outer layer hydrogel are cured and formed for 0.5-2min under the irradiation of a light curing lamp.

8. The method of claim 7, wherein the light curing lamp emits light having a wavelength of 400nm to 480nm.

9. The method of claim 8, wherein the light emitted from the light curing lamp has a wavelength of 405nm.