阅读说明：本技术 一种多功能复合材料及其制备方法和应用 (Multifunctional composite material and preparation method and application thereof ) 是由 李明强 陶玉 王海霞 金圆圆 于 2021-05-25 设计创作，主要内容包括：本发明涉及一种多功能复合材料及其制备方法和应用,所述复合材料包括交替层叠设置的复合海绵层、负载抗肿瘤药物的电纺纤维层,且所述复合材料的上、下表层均为复合海绵层。本发明所涉及的多功能复合材料集止血功能、抗菌功能和化疗功能于一体,多维度地针对引起肿瘤复发的原因,更加高效地抑制术后肿瘤的复发。本发明所涉及的多功能复合材料将复合海绵层与负载抗肿瘤药物的电纺纤维层交替层叠,其中复合海绵层可以快速有效地吸收流出的血液,进一步将其凝结在内部；同时,抗肿瘤药物从电纺纤维中持续释放,从而杀死残留的肿瘤细胞。综上,本发明所涉及的复合材料是一种多功能的抑制肿瘤复发的材料。(The invention relates to a multifunctional composite material and a preparation method and application thereof. The multifunctional composite material provided by the invention integrates the hemostatic function, the antibacterial function and the chemotherapy function, and can be used for inhibiting the recurrence of the postoperative tumor more efficiently aiming at the cause of tumor recurrence in a multi-dimensional manner. The multifunctional composite material alternately laminates the composite sponge layer and the electrospun fiber layer loaded with the anti-tumor drug, wherein the composite sponge layer can quickly and effectively absorb the outflowing blood and further coagulate the outflowing blood inside; meanwhile, the anti-tumor drug is continuously released from the electrospun fiber, so that residual tumor cells are killed. In conclusion, the composite material of the present invention is a multifunctional material for inhibiting tumor recurrence.)

1. The multifunctional composite material is characterized by comprising composite sponge layers and electrospun fiber layers loaded with anti-tumor drugs, which are alternately stacked, wherein the upper surface layer and the lower surface layer of the composite material are both the composite sponge layers.

2. The multifunctional composite of claim 1 wherein said composite sponge layer is provided in 2-5 layers and said anti-tumor drug loaded electrospun fiber layer is provided in 1-4 layers;

preferably, the thickness of the composite sponge layers is 1-5mm, and the thickness of the electrospun fiber layers loaded with the antitumor drugs is 10-1000 μm.

3. The multifunctional composite material according to claim 1 or 2, wherein said antineoplastic drug comprises any one or a combination of at least two of doxorubicin hydrochloride, triptolide, paclitaxel, docetaxel, camptothecin, gemcitabine, or oxaliplatin;

preferably, the anti-tumor drug is a combination of doxorubicin hydrochloride and triptolide;

preferably, the mass ratio of the doxorubicin hydrochloride to the triptolide is (5-15): 1.

4. The multifunctional composite of any one of claims 1 to 3 wherein said electrospun fibers are of a single layer structure or a coaxial multilayer structure;

preferably, the electrospun fiber is a coaxial three-layer structure comprising an inner layer, a middle layer and an outer layer;

preferably, the inner layer matrix material is selected from any one or a combination of at least two of glycerol, water, ethylene glycol, propylene glycol or butanetriol; preferably glycerol;

preferably, the middle layer and the outer layer matrix material are independently selected from any one or a combination of at least two of polylactic acid, polycaprolactone or poly (glycolide-co-lactide); preferably, the middle layer matrix material is polylactic acid, and the outer layer matrix material is polycaprolactone;

preferably, the electrospun fiber is of a coaxial three-layer structure and comprises a glycerol inner layer loaded with doxorubicin hydrochloride, a polylactic acid middle layer and a polycaprolactone outer layer loaded with triptolide.

5. The multifunctional composite of any one of claims 1 to 4 wherein said composite sponge layer is made by cross-linking protein and cationic polymer;

preferably, the mass ratio of the protein to the cationic polymer is (1-8): 1;

preferably, the protein comprises any one of gelatin, bovine serum albumin, ovalbumin or human serum albumin or a combination of at least two thereof;

preferably, the cationic polymer comprises any one of chitosan, chitosan quaternary ammonium salt, polylysine or polyethyleneimine, or a combination of at least two of them.

6. The method of preparing the multifunctional composite of any one of claims 1 to 5, wherein said method of preparing comprises: and (3) sequentially and alternately laminating the composite sponge layer and the electrospun fiber layer loaded with the antitumor drug for compounding.

7. The method of claim 6, wherein the method of preparing the electrospun fiber layer loaded with the anti-tumor drug comprises the following steps:

preparing an inner layer solution, a middle layer solution and an outer layer solution, then respectively filling the solutions into different injectors, communicating three-axis needles according to the inner layer, the middle layer and the outer layer, and injecting into the three-axis needles for electrospinning to obtain the composite material.

8. The method of claim 7, wherein the injection rates of the inner, intermediate and outer layer solutions are set to 0.2-0.5mL/h, 0.7-0.9mL/h, 0.8-1.0mL/h, respectively;

preferably, the voltage of the electrospinning is 10-20 kV;

preferably, the rotating speed of the receiving roller of the electrospinning is 300-900 rpm;

preferably, the distance from the needle of the electrospinning needle to the receiving roller is 5-20 cm.

9. The production method according to claim 7 or 8, wherein the inner layer solution is an aqueous glycerol solution in which doxorubicin hydrochloride is dissolved;

preferably, the intermediate layer solution is an organic solution dissolved with polylactic acid, and the mass concentration of the polylactic acid is 5-10%;

preferably, the outer layer solution is an organic solution dissolved with polycaprolactone and triptolide, and the mass concentration of the polycaprolactone is 7-13%.

10. Use of the multifunctional composite material according to any one of claims 1 to 5 for the preparation of a medical material for inhibiting tumor recurrence.

Technical Field

The invention belongs to the technical field of drug delivery systems, relates to a composite material, a preparation method and application thereof, and particularly relates to a multifunctional composite material, a preparation method thereof and application thereof in preparation of medical materials for inhibiting tumor recurrence.

Background

Surgical removal of solid tumors is an important method for early treatment of cancer with high efficiency. However, postoperative patients are at high risk for tumor recurrence and metastasis. Tumor cells dispersed in blood and carried in blood circulation, and remaining at the edge of tumor resection are major factors causing tumor recurrence during surgical bleeding. In addition, infection of the surgical wound may also lead to serious postoperative complications. Therefore, a safe treatment method is urgently needed to effectively inhibit postoperative recurrence and metastasis and achieve the purpose of postoperative comprehensive treatment.

CN109528736A discloses a preparation method and application of a nanocomposite for inhibiting postoperative tumor recurrence. The invention takes polycaprolactone as a bulk material, mesoporous silica nanoparticles as a drug carrier, adriamycin as a chemotherapeutic drug, and prepares the biodegradable nano composite material doped with the inorganic nano drug by a solvent-free low-temperature ball milling method, so that the doped nano particles are uniformly distributed in the bulk polymer material, and the biodegradable nano composite material has the mechanical properties of the bulk polymer material. However, the nanocomposite material only starts from the chemotherapy point of view, and does not consider the cause of tumor recurrence in a multidimensional way, so that the capacity of inhibiting tumor recurrence is relatively limited.

CN111097070A discloses an injectable bioactive hydrogel for inhibiting tumor recurrence after resection and promoting tissue repair, a preparation method and application thereof. The injectable bioactive hydrogel contains a high molecular material, multifunctional active peptide and water, wherein the high molecular material is grafted with the multifunctional active peptide through a grafting agent, and the multifunctional active peptide is subjected to photo-crosslinking and curing under ultraviolet light through a photoinitiator to form the injectable bioactive hydrogel. The multifunctional active peptide grafted on the hydrogel polymer material can be continuously and slowly released in the whole stage from skin tumor excision to wound repair, is implemented by liquid and photo-crosslinking curing, can fill defected parts with different shapes, and better realizes the combined treatment effect of inhibiting tumor recurrence and promoting tissue repair. However, the hydrogel material does not take account of the cause of tumor recurrence in a multidimensional manner, and thus the ability to suppress tumor recurrence is relatively limited.

In recent years, bleeding during clinical surgery has been prevented by applying conventional electrocoagulation and mechanical methods, but the above methods cannot be applied to certain marginal areas of the resection site. Currently, some topical hemostatic materials have been developed, but efficacy remains to be improved. To achieve rapid, economical, efficient and safe hemostasis, it remains challenging to overcome toxicity and complex synthetic processes.

However, the prevention of bleeding by local implantation of hemostatic materials is not sufficient to completely inhibit tumor growth and recurrence, and the combination of hemostasis and chemotherapy is more effective in post-operative treatment. Systemic chemotherapy causes multiple organ toxic side effects and few drugs reach the tumor site. Topical chemotherapy can overcome the above disadvantages. By designing a local drug delivery system, the anti-tumor drug is slowly and controllably released at a target part in a long-acting manner, so that the drug delivery efficiency can be improved, and accurate and efficient chemotherapy is hopefully realized.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a composite material and a preparation method and application thereof, in particular to a multifunctional composite material and a preparation method and application thereof in preparing a medical material for inhibiting tumor recurrence.

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

in a first aspect, the invention provides a multifunctional composite material, which comprises composite sponge layers and electrospun fiber layers loaded with anti-tumor drugs, wherein the composite sponge layers and the electrospun fiber layers are alternately stacked, and the upper surface layer and the lower surface layer of the composite material are both composite sponge layers.

The multifunctional composite material provided by the invention integrates the hemostatic function, the antibacterial function and the chemotherapy function, and can be used for inhibiting the recurrence of the postoperative tumor more efficiently aiming at the cause of tumor recurrence in a multi-dimensional manner. The multifunctional composite material alternately laminates the composite sponge layer and the electrospun fiber layer loaded with the anti-tumor drug, wherein the composite sponge layer can quickly and effectively absorb the outflowing blood and further coagulate the outflowing blood inside; meanwhile, the anti-tumor drug is continuously released from the electrospun fiber, so that residual tumor cells are killed. In conclusion, the fiber/sponge composite material related by the invention is a multifunctional material for inhibiting tumor recurrence.

Preferably, the composite sponge layer is arranged into 2-5 layers, such as 2 layers, 3 layers, 4 layers and 5 layers; the electrospun fiber layer loaded with the antitumor drug is arranged into 1-4 layers, such as 1 layer, 2 layers, 3 layers and 4 layers.

Under the condition of maintaining the total load amount of the antitumor drugs and the total thickness of the sponge layer unchanged, the invention creatively discovers that when the composite sponge layer is provided with 3 layers and the electrospun fiber layer loaded with the antitumor drugs is provided with 2 layers, the comprehensive effect of the product on hemostasis, antibiosis and chemotherapy is better than that when the composite sponge layer is provided with 2 layers and the electrospun fiber layer loaded with the antitumor drugs is provided with 1 layer. Increasing the number of layers will greatly increase the difficulty of the manufacturing process, and the range of values is considered to be the most reasonable based on cost and practical considerations.

Preferably, the thickness of the composite sponge layers is each independently 1-5mm, such as 1mm, 2mm, 3mm, 4mm, 5mm, etc.; the thickness of the electrospun fiber layer loaded with the anti-tumor drug is independently 100-1000 μm, such as 100 μm, 200 μm, 300 μm, 500 μm, 600 μm, 700 μm, 800 μm, 1000 μm, and the like, and other specific values within the above numerical range can be selected, which is not described herein again.

The thicknesses of the composite sponge layer and the electrospun fiber layer are specifically limited within the numerical range, so that the better hemostatic and antibacterial effects can be fully exerted, the sustained and sufficient release of the antitumor drug can be ensured, and the comprehensive effect of inhibiting tumor recurrence is maximized.

Preferably, the antitumor drug comprises any one or a combination of at least two of doxorubicin hydrochloride, triptolide, paclitaxel, docetaxel, camptothecin, gemcitabine or oxaliplatin.

The combination of at least two of the compounds, such as the combination of doxorubicin hydrochloride and triptolide, the combination of paclitaxel and docetaxel, the combination of camptothecin and gemcitabine, and the like, can be selected in any combination manner, and are not repeated herein.

Preferably, the anti-tumor drug is a combination of doxorubicin hydrochloride and triptolide.

The anti-tumor drug related to the invention is preferably the combination of doxorubicin hydrochloride and triptolide, because the triptolide can improve the cellular uptake level of the doxorubicin and the inherent anti-tumor capacity of the doxorubicin, the doxorubicin hydrochloride and the triptolide have effective synergistic anti-cancer effect.

Preferably, the mass ratio of the doxorubicin hydrochloride to the triptolide is (5-15):1, for example, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 15:1, and the like, and other specific point values in the numerical value range can be selected, which is not described herein again.

For the product related to the invention, the mass ratio of the doxorubicin hydrochloride to the triptolide is specifically selected to be (5-15):1, because the matching relationship is satisfied, the synergistic anticancer effect of the doxorubicin and the triptolide can be maximized.

Preferably, the electrospun fibers are of a single layer structure or a coaxial multilayer structure.

Designing electrospun fibers as a coaxial multilayer structure enables co-loading of hydrophilic and hydrophobic drugs, for example, loading a hydrophilic drug in an inner layer, a hydrophobic drug in an outer layer, or also providing an intermediate layer to further separate the inner and outer layers.

Preferably, the electrospun fiber is a coaxial three-layer structure comprising an inner layer, a middle layer and an outer layer.

Preferably, the inner matrix material is selected from any one of glycerol, water, ethylene glycol, propylene glycol or butanetriol or a combination of at least two such as a glycerol and ethylene glycol mixture, a glycerol and propylene glycol mixture or a glycerol and butanetriol mixture. Further preferred is glycerin.

The inner matrix material is preferably glycerol because glycerol has good biocompatibility, good hygroscopicity, and is less volatile than other materials.

Preferably, the middle layer and the outer layer matrix material are independently selected from any one or a combination of at least two of polylactic acid, polycaprolactone or poly (glycolide-lactide). Preferably, the middle layer matrix material is polylactic acid, and the outer layer matrix material is polycaprolactone.

Polylactic acid is preferred as the intermediate matrix material herein because it allows better control of the release of the inner drug compared to other materials; polycaprolactone is preferred here as the outer matrix material because, compared to other materials, polycaprolactone degrades slowly to achieve a slow release of the outer triptolide.

As a preferred technical scheme of the invention, the electrospun fiber is of a coaxial three-layer structure and comprises a glycerol inner layer loaded with doxorubicin hydrochloride, a polylactic acid middle layer and a polycaprolactone outer layer loaded with triptolide.

Doxorubicin hydrochloride as hydrophilic medicine can be dissolved in glycerin inner layer, triptolide as hydrophobic medicine is dissolved in organic solvent containing polycaprolactone, when electrospinning is carried out, inner layer solution can form elongated ellipsoid (similar to small liquid drop) in obtained fiber, polylactic acid middle layer wraps glycerin ellipsoid dissolved with doxorubicin hydrochloride, inner and outer phases are separated, co-entrapment of doxorubicin hydrochloride and triptolide is finally realized, and synergistic anticancer effect of the doxorubicin hydrochloride and the triptolide is exerted.

Preferably, the composite sponge layer is made by crosslinking proteins and cationic polymers.

Preferably, the mass ratio of the protein to the cationic polymer is (1-8):1, such as 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, and the like, and other specific values in the numerical range can be selected, which is not described in detail herein.

The mass ratio of the protein to the cationic polymer is specifically selected to be (1-8):1 because if the relative mass of the cationic polymer is further increased, certain cytotoxicity is generated on normal tissue cells, and if the relative mass of the cationic polymer is further decreased, the blood-sucking and hemostatic efficiency and the antibacterial effect of the sponge layer are reduced.

Preferably, the protein comprises any one of gelatin, bovine serum albumin, ovalbumin or human serum albumin or a combination of at least two thereof.

Preferably, the cationic polymer comprises any one of chitosan, chitosan quaternary ammonium salt, polylysine or polyethyleneimine, or a combination of at least two of them.

In a second aspect, the present invention provides a method of preparing the multifunctional composite material according to the first aspect, the method comprising: and (3) sequentially and alternately laminating the composite sponge layer and the electrospun fiber layer loaded with the antitumor drug for compounding.

Preferably, the preparation method of the electrospun fiber layer loaded with the antitumor drug comprises the following steps:

preparing an inner layer solution, a middle layer solution and an outer layer solution, then respectively filling the solutions into different injectors, communicating three-axis needles according to the inner layer, the middle layer and the outer layer, and injecting into the three-axis needles for electrospinning to obtain the composite material.

Preferably, the injection rates of the inner, middle and outer layer solutions are set to 0.2-0.5mL/h, 0.7-0.9mL/h, 0.8-1.0mL/h, respectively.

The injection rate of the inner layer solution can be 0.2mL/h, 0.25mL/h, 0.3mL/h, 0.35mL/h, 0.4mL/h, 0.45mL/h or 0.5mL/h, and the like, and other specific values in the numerical range can be selected, so that the repeated description is omitted.

The injection rate of the interlayer solution can be 0.7mL/h, 0.75mL/h, 0.8mL/h, 0.85mL/h or 0.9mL/h, and other specific values in the numerical range can be selected, and are not described in detail herein.

The injection rate of the outer layer solution can be 0.8mL/h, 0.85mL/h, 0.9mL/h, 0.95mL/h or 1.0mL/h, and other specific values in the numerical range can be selected, and are not repeated herein.

Preferably, the voltage of the electrospinning is 10-20kV, such as 10kV, 12kV, 15kV, 18kV or 20kV, and other specific values within the numerical range can be selected, and are not described herein.

Preferably, the rotation speed of the receiving drum for electrospinning is 300-900rpm, such as 300rpm, 400rpm, 500rpm, 600rpm, 700rpm, 800rpm or 900rpm, and other specific values within the numerical range can be selected, and thus detailed description is omitted here.

Preferably, the distance from the needle of the electrospinning needle to the receiving roller is 10-20cm, such as 10cm, 12cm, 15cm, 18cm or 20cm, and other specific point values in the numerical range can be selected, and are not described in detail herein.

Under the common coordination of the spinning parameters, the electrospun fiber can present an inner, middle and outer three-layer structure and is an excellent carrier for coating the medicine.

Preferably, the inner layer solution is glycerol aqueous solution dissolved with doxorubicin hydrochloride.

Preferably, the intermediate layer solution is an organic solution in which polylactic acid is dissolved, the mass concentration of the polylactic acid is 5-10%, for example, 5%, 6%, 7%, 8%, 9%, 10%, etc., and other specific values within the numerical range can be selected, which is not described herein again.

Preferably, the outer layer solution is an organic solution in which polycaprolactone and triptolide are dissolved, the mass concentration of the polycaprolactone is 7-13%, for example, 7%, 8%, 9%, 10%, 11%, 12%, 13%, and the like, and other specific points within the numerical range can be selected, and are not repeated herein.

In the present invention, the composite sponge layer may exemplarily include the steps of:

(1) mixing and stirring a cationic polymer and a protein solution;

(2) then mixing and stirring the mixture with a glycerol aqueous solution;

(3) then mixing and stirring with calcium chloride solution;

(4) finally, mixing and stirring the mixture with a cross-linking agent for cross-linking.

In a third aspect, the present invention provides a use of the multifunctional composite material according to the first aspect in the preparation of a medical material for inhibiting tumor recurrence.

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

the multifunctional composite material provided by the invention integrates the hemostatic function, the antibacterial function and the chemotherapy function, and can be used for inhibiting the recurrence of the postoperative tumor more efficiently aiming at the cause of tumor recurrence in a multi-dimensional manner. The multifunctional composite material alternately laminates the composite sponge layer and the electrospun fiber layer loaded with the anti-tumor drug, wherein the cationic polymer and the protein in the composite sponge layer can activate the aggregation of red blood cells and platelets and limit the red blood cells and the platelets in the sponge, namely the composite sponge layer can quickly and effectively absorb the outflowing blood and further coagulate the blood in the sponge; meanwhile, the cationic polymer has excellent antibacterial performance; in addition, the anti-tumor drug is released from the electrospun fiber continuously, thereby killing residual tumor cells. In conclusion, the composite material of the present invention is a multifunctional material for inhibiting tumor recurrence.

Drawings

FIG. 1 is an SEM image of (1) a blank electrospun fiber layer of a coaxial three-layer structure in example 1;

FIG. 2 is an enlarged fragmentary view of the labeled area of FIG. 1;

FIG. 3 is an SEM photograph of (1) a composite sponge layer in example 2;

FIG. 4 is an enlarged fragmentary view of the marked area in FIG. 3;

FIG. 5 is an SEM photograph of the fiber/sponge composite layer of (1) in example 3;

FIG. 6 is an enlarged fragmentary view of the marked area in FIG. 5;

FIG. 7 is a confocal laser observation of (2) the electrospun fiber layer of the dye-loaded coaxial three-layer structure prepared in example 1 (wherein A-D represent rhodamine B, coumarin 120, FITC, overlay, respectively);

FIG. 8 is a graph of fluorescence intensity at the label in D of FIG. 7;

FIG. 9 is a graph showing the release profile of doxorubicin hydrochloride from (4) the fiber/sponge composite of 3-layer structure supporting doxorubicin hydrochloride and triptolide prepared in example 3;

FIG. 10 is a graph showing the degradation of (1) a chitosan quaternary ammonium salt and gelatin composite sponge layer prepared in example 2 in a PBS solution (pH7.4) and a PBS solution (pH7.4) containing 8. mu.g/mL of proteinase K;

FIG. 11 is a graph showing the degradation of (1) a blank electrospun fiber layer of a coaxial three-layer structure prepared in example 1 in a PBS solution (pH7.4) and a PBS solution containing 20U/mL of lipase (pH 7.4);

FIG. 12 is a statistical chart of the toxicity test results of various groups of products on H22 liver cancer cells;

FIG. 13 is a statistical chart of the toxicity test results of the products on Hepa 1-6 liver cancer cells;

FIG. 14 is a graph showing the results of H & E staining of tumor tissues of various groups of mice;

FIG. 15 is a graph showing Ki-67 immunohistochemical staining results for tumor tissues from various groups of mice;

FIG. 16 is a graph showing the result of H & E staining of organ tissues of each group of mice.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

This example prepares several electrospun fiber layers (170 μm in thickness each):

(1) the preparation method of the blank electrospun fiber layer with the coaxial three-layer structure comprises the following steps:

respectively preparing an inner layer solution, namely an aqueous solution (600 mu L of ultrapure water, 2.4mL of glycerol) containing 80% of glycerol, an intermediate layer solution, namely a dimethyl carbonate solution of polylactic acid (PDLLA) (8%, wt%), and an outer layer solution, namely a dichloromethane solution of Polycaprolactone (PCL) (10%, wt%); then the solutions are respectively filled into different syringes, the inner layer, the middle layer and the outer layer are communicated with a three-axis needle, and the injection rates of the inner layer, the middle layer and the outer layer are respectively set to be 0.36 mL/h, 0.8mL/h and 0.9 mL/h; electrospinning was carried out at a spinning voltage of 15kV, a spindle speed of 650rpm, and a needle to receiving drum distance of 15 cm. In the high-voltage electrospinning process, the spinning fiber with a three-layer structure is obtained after the organic solvent and most of water are evaporated. After preparation, the spun fibers were stored at-20 ℃.

(2) The preparation method of the electrospinning fiber layer of the coaxial three-layer structure loaded with the dye comprises the following steps:

the difference from (1) is only that rhodamine B, coumarin 120 and FITC are respectively dissolved in an inner layer solution (with the concentration of 10.0mg/mL), an intermediate layer solution (with the concentration of 3.3mg/mL) and an outer layer solution (with the concentration of 1.3mg/mL), glycerol, PDLLA and PCL are respectively labeled, and the other operation is referred to (1).

(3) The preparation method of the coaxial three-layer structure electro-spinning fiber layer loaded with the adriamycin hydrochloride comprises the following steps:

the only difference from (1) is the preparation of the inner layer solution: doxorubicin hydrochloride was dissolved in an aqueous solution containing 80% glycerol (600 μ L of ultrapure water, 2.4mL of glycerol) as an inner layer solution, and the amount of doxorubicin hydrochloride supported was 36.2 μ g/mg as measured by absorption spectroscopy. Other operations are as described in (1).

(4) The preparation method of the electro-spinning fiber layer of the coaxial three-layer structure loaded with triptolide comprises the following steps:

the only difference from (1) is the preparation of the outer layer solution: triptolide was dissolved in the outer layer solution, Polycaprolactone (PCL) (10%, wt%) in dichloromethane, and its loading was 1.8 μ g/mg as determined by HPLC. Other operations are as described in (1).

(5) The preparation method of the electrospinning fiber layer I with the coaxial three-layer structure loaded with the adriamycin hydrochloride and the triptolide comprises the following steps:

the only difference from (1) is the preparation of the inner and outer layer solutions: doxorubicin hydrochloride was dissolved in an inner layer solution, which was an aqueous solution containing 80% glycerol (600 μ L of ultrapure water, 2.4mL of glycerol), and the amount of doxorubicin hydrochloride supported was 36.2 μ g/mg as measured by absorption spectroscopy; triptolide was dissolved in the outer layer solution, Polycaprolactone (PCL) (10%, wt%) in dichloromethane, and its loading was 1.8 μ g/mg as determined by HPLC. Other operations are as described in (1).

(6) The preparation method of the electrospinning fiber layer II with the coaxial three-layer structure loaded with the adriamycin hydrochloride and the triptolide comprises the following steps:

the difference from (5) is only that the loading amounts of the doxorubicin hydrochloride and the triptolide are different: the load capacity of the doxorubicin hydrochloride is determined to be 18.1 mug/mg by absorption spectroscopy; the loading of triptolide was determined by HPLC to be 0.9. mu.g/mg. Other operations are as described in (1).

(7) The preparation method of the electrospinning fiber layer III with the coaxial three-layer structure loaded with the adriamycin hydrochloride and the triptolide comprises the following steps:

the only difference from (5) is the replacement of glycerol with propylene glycol. The other operations may be referred to in (5).

(8) The preparation method of the electrospinning fiber layer IV of the coaxial three-layer structure loaded with the adriamycin hydrochloride and the triptolide comprises the following steps:

the only difference from (5) is the replacement of polylactic acid with polyglycolide. The other operations may be referred to in (5).

(9) The preparation method of the coaxial three-layer structure electro-spinning fiber layer V loaded with doxorubicin hydrochloride and triptolide comprises the following steps:

the only difference from (5) is the replacement of polycaprolactone with polyglycolide. The other operations may be referred to in (5).

(10) The preparation method of the coaxial three-layer structure electro-spinning fiber layer loaded with the adriamycin hydrochloride and the paclitaxel comprises the following steps:

the only difference from (5) is the substitution of triptolide for paclitaxel. The other operations may be referred to in (5).

(11) The preparation method of the electrospinning fiber layer with the coaxial three-layer structure for loading doxorubicin hydrochloride and docetaxel comprises the following steps:

the only difference from (5) is that triptolide was replaced with docetaxel. The other operations may be referred to in (5).

(12) The preparation method of the electrospinning fiber layer of the coaxial three-layer structure loaded with gemcitabine and paclitaxel comprises the following steps:

the only difference from (5) is that doxorubicin hydrochloride is replaced by gemcitabine and triptolide is replaced by paclitaxel. The other operations may be referred to in (5).

Example 2

This example prepares several sponge layers as follows:

(1) compounding chitosan quaternary ammonium salt and gelatin:

preparing 2% chitosan quaternary ammonium salt (mass volume ratio, purchased from Macklin company, product number 850125, degree of substitution 95%), and stirring at 20 ℃ for 1 h; heating and stirring 4% gelatin solution (mass volume ratio) at 50 deg.C for 1 hr; stirring a mixed solution of chitosan quaternary ammonium salt and gelatin in a volume ratio of 1:2 at 20 ℃ for 1 h; adding 2% glycerol (volume ratio) and magnetically stirring for 1 h; adding 1% calcium chloride aqueous solution (100mg/mL) (volume ratio) and magnetically stirring for 1 h; 8% genipin solution (2mg/mL) (volume ratio) was added and heated at 37 ℃ with stirring for 1 h. Then, 500 mu L of the mixed solution is added into a 24-pore plate per hole, and the mixed solution is kept stand and crosslinked for 16h at the temperature of 20 ℃; standing at 4 deg.C for gelation for 6 h; pre-freezing at-80 deg.C, and freeze-drying.

(2) Compounding chitosan and gelatin:

the only difference from (1) was that the chitosan quaternary ammonium salt was replaced with chitosan (from Macklin, model C804729), and the other operations remained unchanged.

(3) Compounding chitosan quaternary ammonium salt and bovine serum albumin:

the only difference from (1) was that gelatin was replaced with bovine serum albumin (purchased from Sigma Aldrich under the trade designation A1933), and all other manipulations were kept unchanged.

(4) Compounding chitosan quaternary ammonium salt and egg albumin:

the only difference from (1) was that the chitosan quaternary ammonium salt was replaced with ovalbumin (purchased from a source leaf organism, cat # S12016), and the other procedures were kept unchanged.

Example 3

This example prepares several fiber-sponge composites as follows:

(1) fiber/sponge composite of 3-layer structure without drug loading:

preparing 2% chitosan quaternary ammonium salt (mass volume ratio), and stirring for 1h at 20 ℃; heating and stirring 4% gelatin solution (mass volume ratio) at 50 deg.C for 1 hr; stirring a mixed solution of chitosan quaternary ammonium salt and gelatin in a volume ratio of 1:2 at 20 ℃ for 1 h; adding 2% glycerol (volume ratio) and magnetically stirring for 1 h; adding 1% calcium chloride aqueous solution (100mg/mL) (volume ratio) and magnetically stirring for 1 h; 8% genipin solution (2mg/mL) (volume ratio) was added and heated at 37 ℃ with stirring for 1 h. Then, 500. mu.L of the mixed solution is added into a 24-pore plate per pore, and after standing and crosslinking are carried out for 60min at 20 ℃, the electrospun fiber layer (1) prepared in the embodiment 1 is added, and then 500. mu.L of the mixed solution is added, and standing and crosslinking are carried out for 16h at 20 ℃; standing at 4 deg.C for gelation for 6 h; pre-freezing at-80 deg.C, and freeze-drying.

(2) Fiber/sponge composite material of 3-layer structure loaded with doxorubicin hydrochloride:

the only difference from (1) is that the electrospun fiber layer (1) prepared in addition to example 1 was replaced by (3).

(3) The fiber/sponge composite material of the 3-layer structure loading triptolide:

the only difference from (1) is that the electrospun fiber layer (1) prepared in addition to example 1 was replaced by (4).

(4) The fiber/sponge composite material of 3-layer structure loaded with adriamycin hydrochloride and triptolide:

the only difference from (1) is that the electrospun fiber layer (1) prepared in addition to example 1 was replaced by (5).

(5) Fiber/sponge composite material loaded with doxorubicin hydrochloride and triptolide and having 3-layer structure

The only difference from (1) is that the electrospun fiber layer (1) obtained in addition to example 1 was replaced by (7).

(6) Fiber/sponge composite material loaded with doxorubicin hydrochloride and triptolide and having 3-layer structure

The only difference from (1) is that the electrospun fiber layer (1) obtained in addition to example 1 was replaced by (8).

(7) Fiber/sponge composite material loaded with doxorubicin hydrochloride and triptolide and having 3-layer structure

The only difference from (1) is that the electrospun fiber layer (1) obtained in addition to example 1 was replaced with (9).

(8) The fiber/sponge composite material of the 5-layer structure loading the adriamycin hydrochloride and the triptolide:

preparing 2% chitosan quaternary ammonium salt (mass volume ratio), and stirring for 1h at 20 ℃; heating and stirring 4% gelatin solution (mass volume ratio) at 50 deg.C for 1 hr; stirring a mixed solution of chitosan quaternary ammonium salt and gelatin in a volume ratio of 1:2 at 20 ℃ for 1 h; adding 2% glycerol (volume ratio) and magnetically stirring for 1 h; adding 1% calcium chloride aqueous solution (100mg/mL) (volume ratio) and magnetically stirring for 1 h; 8% genipin solution (2mg/mL) (volume ratio) was added and heated at 37 ℃ with stirring for 1 h. Then, adding 400 μ L of the mixed solution into a 24-pore plate per pore, standing and crosslinking at 20 ℃ for 60min, adding the electrospun fiber layer (6) prepared in the example 1, then adding 200 μ L of the mixed solution, standing and crosslinking at 20 ℃ for 60min, then adding the electrospun fiber layer (6) prepared in the example 1, then adding 400 μ L of the mixed solution, and standing and crosslinking at 20 ℃ for 16 h; standing at 4 deg.C for gelation for 6 h; pre-freezing at-80 deg.C, and freeze-drying.

(9) Fiber/sponge composite material of 3-layer structure loaded with adriamycin hydrochloride and paclitaxel:

the only difference from (1) is that the electrospun fiber layer (1) prepared in example 1 was replaced by (10).

(10) The fiber/sponge composite material of a 3-layer structure loaded with doxorubicin hydrochloride and docetaxel:

the only difference from (1) is that the electrospun fiber layer (1) prepared in addition to example 1 was replaced with (11).

(11) Gemcitabine and paclitaxel loaded fiber/sponge composite of 3-layer structure:

the only difference from (1) is that the electrospun fiber layer (1) prepared in example 1 was replaced with (12).

Evaluation test:

(I) SEM topography Observation

SEM morphology observation of the blank electrospun fiber layer of (1) coaxial three-layer structure in example 1 shows that as shown in FIGS. 1-6 (wherein FIGS. 2, 4 and 6 are enlarged views of marked areas of FIGS. 1, 3 and 5 respectively), as shown in FIGS. 1-2: the electrospinning structure is a bead structure, and the size of liquid beads formed by glycerol at the inner layer is about 3 mu m; FIGS. 3-4 show that: the gelatin chitosan sponge has rich porosity, and the size of pores is more than 50 mu m; FIGS. 5-6 show that: the electrospun fiber layer is positioned between the two layers of sponge and is in a sandwich shape.

(II) confocal laser observation

The electrospun fiber layer of (2) the dye-loaded coaxial three-layer structure prepared in example 1 was observed by confocal laser observation, and the results are shown in fig. 7 (wherein a to D represent rhodamine B, coumarin 120, FITC, and superimposed fluorescence) and fig. 8 (which is a fluorescence intensity map at the mark in fig. 7D). As can be seen from FIGS. 7-8: the rhodamine B-labeled glycerol layer is located on the inner layer, the coumarin 120-labeled polylactic acid layer is located on the middle layer, and the FITC-labeled polycaprolactone layer is located on the outer layer.

(III) drug Release test

The fiber/sponge composite material (4) loaded with doxorubicin hydrochloride and triptolide and having a 3-layer structure prepared in example 3 was placed in a dialysis bag of Mw3500, and placed in PBS solutions of pH7.4 and 5.0 and PBS solution containing 2U/mL lipase and 8 μ g/mL proteinase K (pH7.4), respectively, and continuously released in a constant temperature shaking chamber (37 ℃, 40rpm) for 48 hours, and 100 μ L of sample was taken at 1, 6, 24, and 48 hours to detect the release ratio of doxorubicin hydrochloride (absorbance at 480nm by a microplate reader).

The results are shown in FIG. 9, which shows that: the release rate of doxorubicin in PBS pH 5.0 was faster than that in PBS pH7.4, and in the presence of both enzymes, the release rate of the drug was faster than that in PBS pH7.4 without enzyme.

(IV) degradation experiments

The chitosan quaternary ammonium salt and gelatin composite sponge layer (1) prepared in example 2 was placed in a PBS solution (pH7.4) and a PBS solution (pH7.4) containing 8. mu.g/mL of proteinase K, shaken at a constant temperature (37 ℃, 40rpm), and the degradation of the sponge was continuously observed, observed and evaluated by photographing, and the results are shown in FIG. 10, from which: the sponge layer will be slowly degraded in PBS solution, most of the sponge is degraded in 72h without enzyme, and the sponge is completely degraded after 168 h. In the presence of proteinase K, the degradation rate of the sponge increases, and after 24h, most of the sponge is dissolved in the solution.

The blank electrospun fiber layer of (1) coaxial three-layer structure prepared in example 1 was placed in a PBS solution (pH7.4) and a PBS solution (pH7.4) containing 20U/mL of lipase, shaken at a constant temperature (37 ℃, 40rpm), and the degradation of the fiber was continuously observed, observed and evaluated by photographing, and the result is shown in FIG. 11, which shows that: the degradation speed of the electrospun fiber layer is slower than that of the sponge layer, and the significant degradation of the electrospun fiber layer begins to occur when the electrospun fiber layer begins to shrink in enzyme-free PBS solution for 168 hours. In the PBS solution of lipase, the degradation speed of the electrospun fiber layer is accelerated, and at 24h, the electrospun fiber layer starts to degrade after obvious shrinkage phenomenon occurs, but the complete macroscopic electrospun fiber layer form is still kept.

(V) cytotoxicity test

Cytotoxicity of drug-loaded electrospun was assessed using a cell proliferation and toxicity detection kit (CCK-8):

(1) at 37 ℃ 5% CO₂Culturing and amplifying H22 liver cancer cell (1640 culture medium + 10% serum + 1% double antibody) in cell culture box until cell density reaches 80-90% and 6 × 10⁴The number of cells per well was evenly plated in 48-well plates, and 500. mu.L of culture medium was added to each well. Grouping experiments: control, empty fiber (example 1 (1)), triptolide single-drug-spun fiber (example 1 (4)), doxorubicin hydrochloride single-drug-spun fiber (example 1 (3)), dual-drug-spun fiber (example 1 (5)), each set having 3 replicate wells. According to grouping, adding 2mg of different types of spinning into each hole, respectively processing for 12h and 24h, adding 50 μ L of CCK-8 detection solution into each hole, incubating in an incubator for 1.5h, and detecting 450nm position with an enzyme-labeling instrumentAbsorbance of (b). The results are shown in FIG. 12, which shows that: the hollow fiber has no toxicity to H22 cells, and in the culture experiment of 12H or 24H and cells, the double-drug spinning fiber group has obviously enhanced cytotoxicity to H22 compared with the single-drug spinning fiber group.

(2) At 37 ℃ 5% CO₂Culturing and amplifying Hepa 1-6 hepatocarcinoma cell (DMEM high-sugar medium + 10% serum + 1% double antibody) in cell culture box until cell density reaches 80-90% and 6 × 10⁴The number of cells per well was evenly plated on 48-well plates, and 500. mu.L of culture medium was added to each well for 12 hours of adherence. Grouping experiments: a control group, an empty fiber group, a triptolide single-drug spinning fiber group, an doxorubicin hydrochloride single-drug spinning fiber group and a dual-drug spinning fiber group, wherein each group is provided with 3 repeated holes. According to grouping, adding 2mg of different types of spinning into each hole, processing for 12h and 24h respectively, discarding the culture solution, washing with PBS for 1 time, adding 250 mu L of culture solution and 25 mu L of LCCK-8 detection solution into each hole, incubating in an incubator for 1.5h, and detecting the absorbance at 450nm by using an enzyme-labeling instrument. The results are shown in FIG. 13, which shows that: the hollow fiber has no toxicity to the Hepa 1-6 cells, and the cytotoxicity of the double-drug spinning fiber group is obviously enhanced compared with that of the single-drug spinning fiber group in the culture experiment of the cells in 12h or 24 h.

(VI) hemostasis test

(1) Blood coagulation index determination:

the four sponge products (1), (2), (3) and (4) prepared in example 2 and commercially available gelatin sponges were placed in beakers, respectively; then, whole blood (100. mu.L) from the mice was carefully dropped onto the corresponding sponges or fibers, respectively, and 100. mu.L of whole blood was directly dropped into an unaddressed beaker as a control. Each set of beakers was incubated in an incubator at 37 ℃ for 5 minutes, 50mL of distilled water was carefully added after 5 minutes (care was taken not to break the clot), red blood cells which failed to form a clot were hemolyzed in the water, and the absorbance (Ai) at 540nm of the liquid in the beakers to which the sponge or fiber was added was measured by a microplate reader, and the absorbance at 540nm of distilled water (50mL) and whole blood (100. mu.L) was assumed to be 100, which was used as a reference value (A0); the Blood Coagulation Index (BCI) of different materials can be quantified by the following formula:

BCI-Ai/A0 x 100 represents the blood coagulation index of hemostatic ability.

The results are shown in Table 1.

(2) Liquid absorption rate determination:

the four sponge products (1) (2) (3) (4) prepared in example 2, and a commercially available gelatin sponge were immersed in 5mL of a diluted blood solution (physiological saline: blood (v/v) ═ 49: 1). The dry weights (W) of the sponges were weighed separately_d) And wet weight of sponge after absorption of liquid (W)_w) Calculating the liquid absorption rate (V) by formula_dVolume of sponge) r ═ W_w-W_d)/V_d。

The results are shown in Table 1.

(3) Evaluation of in vivo hemostatic ability:

the method is divided into 6 groups: 1) control group, no treatment; 2-5) sponge product groups (1) (2) (3) (4) prepared in example 2; 6) the commercially available gelatin sponge group. BALB/c mice (4 per group) were anesthetized with 1% sodium pentobarbital, the livers were exposed by ventral midline incision, transverse incisions were made in the liver surface, which was seen to bleed profusely, and each group of products was immediately placed on the incision surface of the livers, covered for 5 minutes. The hemostatic effect of each group was evaluated by direct observation.

The results are shown in Table 1.

TABLE 1

From the results in Table 1, it can be seen that: example 2(1) sponge, i.e., the sponge compounded by chitosan quaternary ammonium salt and gelatin, has faster blood absorption speed, higher blood coagulation capacity and more prominent in vivo hemostatic capacity.

(VII) antibacterial experiments

The four sponge products (1), (2), (3) and (4) prepared in example 2 and commercially available gelatin sponge were UV-sterilized for half an hour, and added to the mixture containing 1X 10⁴In a number of E.coli cultures, incubated together (37 ℃, 200rpm), OD600 absorptions were measured at 0, 4, 6, and 8h using a microspectrophotometer to assess changes in E.coli concentration. The results are shown in Table 2Shown in the figure.

TABLE 2

Group of	0h	4h	6h	8h
					Example 2(1) sponge	0.01	0.1	0.72	1.2
Example 2(2) sponge	0.01	0.15	0.97	1.63
					Example 2(3) sponge	0.01	0.16	1.09	1.77
Example 2(4) sponge	0.01	0.14	1.05	1.64
					Commercially available sponge	0.01	0.19	1.14	2.18

From the results of table 2, it can be seen that: example 2(1) the sponge, i.e. the sponge compounded by chitosan quaternary ammonium salt and gelatin, has the most remarkable bacteriostatic ability.

(VIII) experiment for suppressing tumor recurrence

(1) Establishment of H22 model of subcutaneous tumor of liver cancer cell

(1.1) ascites culture of H22 hepatoma cells

Culturing and amplifying H22 liver cancer cells, centrifuging to collect cells when the density reaches 80-90%, making into cell suspension, detecting viable cell number with trypan blue, counting viable cells with optical phase contrast microscope and calculating viable cell number per ml cell suspension to make the viable cell number of cell suspension reach 2.5 × 10⁷and/mL. Pre-collected H22 cells were injected into the peritoneal cavity (200. mu.L, 5X 10) of BALB/c mice as soon as possible⁶One/only), the general health condition and ascites generation were observed every other day.

(1.2) Collection of H22 cells in ascites

Ascites was collected by performing abdominal cavity aspiration about 7 days after the injection of H22 cells. The neck and back of the mouse are fixed, the abdomen is disinfected, a needle is inserted by a No. 18 needle and the right 45-degree angle of the midline of the abdomen of the lower abdomen, and a 15mL sterile centrifuge tube is prepared in advance to receive ascites. 4-10mL of ascites can be harvested once per mouse. After the ascites is collected, the cells are precipitated by centrifugation at 1500g for 10min, the supernatant is discarded, PBS is added for washing twice, and trypan blue staining is carried out to judge the cell activity and count.

(1.3) subcutaneous inoculation of H22 cells BALB/c mice

Resuspend the above-collected H22 cells with PBS to achieve a cell density of 5X 10⁷/mL, inoculated in miceSubcutaneous, 100 μ L, 5X 10, dorsal rat⁶Subcutaneous tumorous tissue was visible 5-7 days after inoculation.

(2) Experiment for inhibiting tumor recurrence

(2.1) establishment of tumor recurrence model

After 1 week of subcutaneous inoculation of H22 cells, the molded mice were subjected to tumor mass resection, and one third of the tumor volume remained, to establish a tumor recurrence model.

(2.2) treatment and monitoring of efficacy

The mice were randomly divided into 12 groups of 6 mice each:

example 2(1) composite sponge of chitosan quaternary ammonium salt and gelatin,

Example 3(2) Adriamycin hydrochloride-supporting 3-layer-structured fiber/sponge composite,

Example 3(3) A fiber/sponge composite supporting triptolide in a 3-layer structure,

Example 3(4) 3-layer-structured fiber/sponge composite supporting Adriamycin hydrochloride and triptolide,

Example 3 (5) 3-layer-structured fiber/sponge composite supporting Adriamycin hydrochloride and triptolide,

Example 3 (6) A fiber/sponge composite supporting a 3-layer structure of doxorubicin hydrochloride and triptolide,

Example 3 (7) 3-layer-structured fiber/sponge composite supporting Adriamycin hydrochloride and triptolide,

Example 3 (8) A5-layer-structured fiber/sponge composite supporting Adriamycin hydrochloride and triptolide,

Example 3 (9) A3-layer structured fiber/sponge composite supporting Adriamycin hydrochloride and paclitaxel,

Example 3 (10) A fiber/sponge composite of 3-layer structure supporting Adriamycin hydrochloride and docetaxel,

Example 3 (11) Gemcitabine-and paclitaxel-loaded fiber/sponge composite of 3-layer Structure,

Control group (no material implanted).

The corresponding material was implanted at the position after the majority of the tumor was excised according to the group, and the incision was carefully sutured. Infection was prevented by daily intraperitoneal injections of antibiotics (0.1mL/20g) after surgery, and the volume of recurrent tumors and the body weight of mice were monitored and recorded daily, and the results are shown in table 3.

TABLE 3

From the results in Table 3, it can be seen that: the (8) of example 3, i.e., the 5-layer structure fiber/sponge complex supporting doxorubicin hydrochloride and triptolide, had the best effect of inhibiting tumor recurrence, and the (4) of example 3, i.e., the 3-layer structure fiber/sponge complex supporting doxorubicin hydrochloride and triptolide, had the next lower effect of inhibiting tumor. And the tumor inhibition effect of the compound loaded with the double drugs is more obvious than that of the compound loaded with the single drug.

Mice were sacrificed on day 17, major organs (heart, liver, spleen, lung and kidney) and tumors were collected, and H & E staining was used to visualize tumor morphology, as shown in fig. 14; h & E staining assessed organ toxicity, as shown in fig. 16; ki-67 immunohistochemical staining assessed inhibition of tumor tissue proliferation as shown in FIG. 15. In the figure, a-e are control (no treatment), blank sponge (example 2 (1)) sponge, doxorubicin-loaded spinning sponge complex (example 3(2)), triptolide-loaded spinning sponge complex (example 3(3)), and triptolide and tripterygium loaded spinning sponge complex (example 3(4)) in that order. A-E are heart, liver, spleen, lung and kidney in sequence. The scale in the figure is 500. mu.m. As can be seen from the figure: the double-drug loaded compound has the strongest inhibition effect on the proliferation of tumor cells and has no influence on other main organs.

The applicant states that the present invention is illustrated by the above examples of a multifunctional fiber-sponge composite material and a preparation method and application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.