CN112933286B

CN112933286B - Crystal gel for stopping bleeding and bearing anticancer drugs and preparation method thereof

Info

Publication number: CN112933286B
Application number: CN202110189338.6A
Authority: CN
Inventors: 郭保林; 梁永平; 乔李鹏; 李勐; 黄颖
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2021-02-19
Filing date: 2021-02-19
Publication date: 2022-07-12
Anticipated expiration: 2041-02-19
Also published as: CN112933286A

Abstract

The invention discloses a crystal gel for hemostasis and bearing anticancer drugs and a preparation method thereof²⁺And (3) preparing the dopamine modified ZIF-8 by the coordination of ions. The components are respectively prepared into aqueous solution or dispersion, and then under the action of an initiator, the multifunctional water/blood triggered shape memory crystal gel is formed at low temperature through double bond polymerization. The invention takes polysaccharide and other biological macromolecules with good biocompatibility and degradation performance as the basis, and has the process of chemically grafting double bonds or polyphenol compounds, and also has the characteristics of low toxicity and no pollution.

Description

Crystal gel for stopping bleeding and bearing anticancer drugs and preparation method thereof

[ technical field ] A method for producing a semiconductor device

The invention belongs to the technical field of biomedical materials, and particularly relates to a crystal gel for stopping bleeding and bearing anticancer drugs and a preparation method thereof.

[ background of the invention ]

Surgical resection remains the first and most effective method of cancer treatment during tumor therapy. However, bleeding is inevitably encountered during tumor resection, especially from tumors with abundant blood vessels, such as liver cancer, so that hemostasis becomes particularly important. Despite the many reported or commercially available hemostatic agents, such as tissue adhesives, glutaraldehyde cross-linked hemostatic agents or gelatin hemostatic agents, that have a high hemostatic effect on surface bleeding wounds, there is still a lack of effective strategies for complex in vivo bleeding. For example, currently, the clinical treatment of bleeding in resection of malignant liver tumor is mainly performed by simple gauze packing, and the common in vivo hemostatic agents such as gelatin sponge have different degrees of problems such as difficult degradation and poor hemostatic effect. This greatly increases the risk of death during surgery and also places a greater burden on the blood supply of the medical system.

On the other hand, inevitable tissue damage and bleeding during tumor resection will greatly increase the risk of tumor recurrence due to tumor metastasis with blood flow. Therefore, it is very important to prepare a substance which can stop bleeding during tumor resection and help prevent postoperative recurrence of tumor.

[ summary of the invention ]

The invention aims to overcome the defects of the prior art and provide a crystal gel for stopping bleeding and carrying an anti-cancer drug and a preparation method thereof, so as to solve the problems that a hemostatic article capable of inhibiting internal bleeding and an article capable of inhibiting postoperative transfer along with blood flow are lacked in the prior art.

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

a preparation method of crystal gel for hemostasis and bearing anticancer drugs comprises the following steps:

step 1, synthesizing a compound A by a double-bond functionalized reagent and a first biological macromolecule;

step 2, synthesizing a compound B through a polyphenol compound and a second biological macromolecule;

step 3, synthesizing nanoparticles C by using an anti-cancer substance, dopamine and ZIF-8;

and 4, preparing the crystal gel through the compound A, the compound B and the nano particles C.

The invention is further improved in that:

preferably, in step 1, the first biological macromolecule is chitosan or a derivative thereof, gelatin or a derivative thereof, hyaluronic acid or a derivative thereof, alginate or a derivative thereof, dextran or a derivative thereof, cellulose or a derivative thereof, polyvinyl alcohol or a derivative thereof, polyethylene glycol or a derivative thereof, or pluronic F127 or a derivative thereof; the first biological macromolecule is used in one or more matching ways;

the double-bond functionalized reagent is acryloyl chloride, methacryloyl chloride, acrylic anhydride, methacrylic anhydride or glycidyl methacrylate.

Preferably, in step 2, the second biomacromolecule is hyaluronic acid or a derivative thereof, gelatin or a derivative thereof, alginate or a derivative thereof, dextran or a derivative thereof, cellulose or a derivative thereof, polyvinyl alcohol or a derivative thereof, polyethylene glycol or a derivative thereof, or pluronic F127 or a derivative thereof; the second biological macromolecule is used in one or more matching ways;

the polyphenol compound is a compound containing an o-phenylphenol structure.

Preferably, the compound containing the o-phenylphenol structure is dopamine or a derivative thereof, levodopa or a derivative thereof, dihydrocaffeic acid or a derivative thereof, tannic acid or a derivative thereof, 3, 4-dihydroxybenzaldehyde or a derivative thereof, or gallic acid or a derivative thereof; when the compounds with the structure of the o-phenylphenol are used, one or more compounds are used in a matching way.

Preferably, the anti-cancer substance is an anti-cancer drug, a photosensitizer in photodynamic therapy of cancer or a sonosensitizer in sonodynamic therapy of cancer.

Preferably, the specific process of step 4 is:

(A) respectively preparing an aqueous solution of the compound A, an aqueous solution of the compound B and an aqueous dispersion of the nano particles C, and respectively carrying out ice bath treatment on each aqueous solution and each aqueous dispersion;

(B) preparing an initiator solution, and carrying out ice-bath treatment on the initiator solution;

(C) uniformly mixing the solutions prepared in the step (A) and the step (B), inverting the mixture in a mold, and standing the mixture at a temperature of between 20 ℃ below zero and 0 ℃ for 24 to 72 hours to form process crystal glue;

(D) and thawing the process crystal gel and freeze-drying to obtain the blood triggered shape memory crystal gel.

Preferably, in the step (A), the concentration of the aqueous solution of the compound A is 1wt% to 20wt%, and the concentration of the aqueous solution of the compound B is 1wt% to 20 wt%; the concentration of the aqueous dispersion of the nanoparticles C is 5-20 mg/mL.

Preferably, in the step (B), the initiator is hydrogen peroxide, potassium persulfate, ammonium persulfate, azo or benzoyl; the amount of the initiator is 0.5 to 5 percent of the molar weight of the double bonds in the compound A in the prepared prepolymer solution.

Preferably, in the step (C), the volume mixing volume ratio of the compound a, the compound B, the nanoparticles C and the initiator solution is: 6: 2: 1: 1.

the crystal gel for stopping bleeding and bearing anticancer drugs prepared by any one of the preparation methods is a porous reticular structure, and the porous reticular structure is formed by staggering a compound A and a compound B; the nano particles C are attached to the surface of the net structure and are nano particles.

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

the invention discloses a preparation method of a crystal gel for hemostasis and carrying anticancer drugs, which is characterized in that double bonds are modified on a first biological macromolecule to form a compound A, a polyphenol compound is grafted on a second biological macromolecule to form a compound B, and dopamine molecules and Zn are utilized²⁺The coordination of ions is used for preparing the dopamine modified ZIF-8, and meanwhile, the adsorption of the ZIF-8 and the wide chemical reactivity of dopamine are used for loading anti-cancer substances. The components are respectively prepared into aqueous solution or dispersion, and then under the action of an initiator, the multifunctional water/blood triggered shape memory crystal gel is formed at low temperature through double bond polymerization. During the preparation process, the cross-linking of the double-bond functionalized first biomacromolecule (complex a) under the action of the initiator serves as the first major network of the crystal gel. The polyphenol endows the biomacromolecule with good tissue adhesion performance for modification of the biomacromolecule, and the hemostasis performance of the crystal gel is further enhanced. Meanwhile, the polyphenol modified biological macromolecules (compound B) and the double-bond functionalized biological macromolecules (compound A) are combined through the actions of macromolecular physical winding, electrostatic interaction, hydrogen bonds and the like to form a second network, and the crystal glue network is further enhanced. Introduction of dopamine modified ZIF-8 on one hand simultaneously utilizes adsorbability of ZIF-8 and extensive chemical reactivity of dopamine to complete loading of anticancer substances, and on the other hand, dopamine on the ZIF-8Can combine with the polyphenol compound on the compound B, thereby bringing the whole nano particles C into a crystal colloid system. The non-covalent connection between the second biological macromolecule and the first re-crystallized gel network is used as a bridge to load the nano-particle C, and the nano-particle C is used for delivering the anti-cancer drug, so that the formed crystal gel network can be used as a carrier for bearing the anti-cancer drug. The preparation method is based on biomacromolecules such as polysaccharide and the like with good biocompatibility and degradation performance, and has the characteristics of low toxicity and no pollution in the process of chemically grafting double bonds or polyphenol compounds. The double-bond functionalized biomacromolecule used in the invention has simple preparation and mature synthesis technology, and the prepared product has good general type.

The invention also discloses a crystal gel for stopping bleeding and bearing anticancer drugs, wherein the crystal gel is of a porous reticular structure, the porous reticular structure forms a crystal gel body, and nanoparticles are attached to the reticular structure. The crystal gel can adjust the porosity, rheological property, mechanical property, swelling property, degradability and the like of the double-bond functionalized first biological macromolecule (compound A) and the polyphenol modified second biological macromolecule (compound B) by changing the content and the proportion of the double-bond functionalized first biological macromolecule and the polyphenol modified second biological macromolecule. The good mechanical property of the crystal gel is kept in the repeated compression process, the prepared crystal gel can quickly realize shape recovery after being compressed under the condition of triggering water or blood, coagulation is further exerted through the enrichment of blood cells and blood platelets, and quick hemostasis is realized. Molecular sieve imidazole skeleton (ZIF-8) is a novel porous metal-organic skeleton with excellent adsorption performance, and has good drug-loading performance proved by high specific surface area. More importantly, it is more easily degraded under acidic condition, and is an ideal carrier for pH-responsive release of anticancer drugs. Therefore, the dopamine modified ZIF-8 loaded with the anticancer drug is added into the crystal gel system, and the effect of preventing postoperative recurrence of cancer through slow release of the anticancer drug is also given to the crystal gel. The crystal gel prepared by the invention has good water/blood triggered shape recovery performance and the function of activating blood cells/platelets to aggregate, and simultaneously, the loaded anticancer drug can also play an anticancer role.

[ description of the drawings ]

FIG. 1(a) is a photomicrograph and TEM image of prepared ZIF-8, dopamine modified ZIF-8(ZIF-8/DA) and HMME loaded dopamine modified ZIF-8(ZIF-8/DA/HMME, ZDH); FIG. 1(b) is the ultraviolet spectra of ZIF-8, ZIF-8/DA and ZDH; FIG. 1(c) is a SEM photograph and a macro photograph showing the original and fixed morphology of QCSG/HA-DA (QH) and QH/ZDH crystal gels; FIG. 1(d) is a graph showing the swelling ratio of the crystal gel.

FIG. 2(a) is a graph of water triggered shape recovery time after compression of different gels; FIG. 2(b) is the recovery behavior of different gels after compression under wet and dry conditions; FIG. 2(c) HMME release profile.

FIG. 3 shows the results of quantitative antibacterial tests of different crystalloids against gram-positive Staphylococcus aureus, gram-negative Escherichia coli and MRSA.

FIG. 4(a) shows the blood sucking behavior and the blood clotting behavior of gelatin sponge and different gels (I-III); FIG. 4(b) is the blood coagulation index of blank, gauze, gelatin sponge, different gel groups; FIG. 4(c) is a scanning electron micrograph of platelets and erythrocytes after blood aspiration with gauze, gelatin sponge and different crystal gels.

FIGS. 5(a) and 5(b) are liver partial excision wound bleeding models in rats and rabbits, respectively. I) A schematic representation of an animal model; II) representative pictures; III) quantitatively counting the amount of bleeding; IV) different groups of hemostasis times.

FIG. 6(a) is a quantitative result and a representative photograph of a blood compatibility test of different gels; FIG. 6(b) is a result of quantification by a cytocompatibility test; FIG. 6(c) is a quantitative statistic of the ROS production in DCFH-DA fluorescence detection H22 cells; fig. 6(d) is a quantitative statistical plot of H22 cell viability.

FIG. 7(a) is a graph of quantitative statistics of bleeding volume for different treatment methods in a tumor resection bleeding model; FIG. 7(b) is a quantitative statistical plot of the mass of regenerated tumors 18 days after tumor resection; FIG. 7(c) is a quantitative analysis of the relative expression of different sets of HIF-1. alpha. and IL-6.

Fig. 8 is a schematic view of a crystal glue structure prepared by the embodiment of the invention.

[ detailed description ] embodiments

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

The invention discloses a crystal gel for stopping bleeding and bearing anticancer drugs, which comprises the following steps:

(1) synthesizing complex a by double bond functionalizing agent and the first biopolymer;

(2) synthesizing a complex B by a polyphenolic compound and a second biomacromolecule;

(3) synthesizing nanoparticles C by using an anticancer substance, dopamine and ZIF-8;

(4) and preparing the crystal gel from the compound A, the compound B and the nano particles C.

the polyphenol compound is a compound containing an o-phenylphenol structure.

Preferably, the compound containing the o-phenylphenol structure is dopamine or a derivative thereof, levodopa or a derivative thereof, dihydrocaffeic acid or a derivative thereof, tannic acid or a derivative thereof, 3, 4-dihydroxybenzaldehyde or a derivative thereof, or gallic acid or a derivative thereof; when the compounds with the o-phenylphenol structure are used, one or more compounds are used in a matching way.

Preferably, the anti-cancer substance is an anti-cancer drug, a photosensitizer in cancer photodynamic therapy or a sonosensitizer in cancer sonodynamic therapy, such as Doxorubicin (DOX), chlorin (Ce6), hematoporphyrin monomethyl ether (HMME), 4-methylphenylporphyrin (TPP), or the like.

Preferably, the specific process of step 4 is:

(C) uniformly mixing the solutions prepared in the step (A) and the step (B), inverting the mixture in a mould, and standing the mixture for 24 to 72 hours at a temperature of between 20 ℃ below zero and 0 ℃ to form process crystal gel;

(D) and unfreezing the process crystal gel and then freeze-drying to obtain the blood triggered shape memory crystal gel.

Preferably, in the step (B), the initiator is hydrogen peroxide, potassium persulfate, ammonium persulfate, azo compounds such as azodimethyl N-2-hydroxybutyl acrylamide (VA-086), benzoyl compounds such as phenyl-2, 4, 6-trimethyl benzoyl lithium phosphite (LAP), etc.; the amount of the initiator is 0.5 to 5 percent of the molar weight of the double bonds in the compound A in the prepared prepolymer solution.

The crystal gel prepared by the invention has the water/blood triggered shape recovery, and can quickly stop bleeding and slowly release anticancer drugs.

Example 1

(1) Compound A, synthesis of glycidyl methacrylate modified Quaternized Chitosan (QCSG);

in the step (1), the preparation of the glycidyl methacrylate modified quaternized chitosan comprises the following steps:

(1A) suspending 1g of chitosan in 36mL of deionized water to obtain a chitosan suspension;

(1B) adding 180 mu L of glacial acetic acid into the chitosan suspension obtained in the step (1A), and stirring for 30min at 55 ℃;

(1C) adding the chitosan-glacial acetic acid solution obtained in the step (1B) under continuous stirring, wherein the molar ratio of the chitosan-glacial acetic acid solution to the amino on the chitosan skeleton is 2: 1, stirring the reaction mixture at 55 ℃ for 15 hours;

(1D) after the reaction in the step (1C) is finished, GMA is dropwise added into the reaction mixture under the continuous stirring at 55 ℃, and the ratio of GMA to amino on the main chain of pure chitosan is fixed to be 0.5: 1.0, carrying out reaction for 15 hours at 55 ℃ under the dark condition;

(1E) after completion of the reaction, the insoluble polymer was removed by centrifuging the mixture at 4500 rpm for 20 minutes at room temperature. The supernatant was precipitated into pre-cooled acetone to give the crude product. To purify the product, the crude product was dissolved in deionized water and then dialyzed thoroughly against deionized water (MWCO 3500) under dark conditions for 3 days. The pure product was obtained by lyophilization. The degree of quaternization of the QCSG copolymer was determined by chloride titration. The GTMAC substitution was 35%.

The quaternized chitosan modified by the glycidyl methacrylate can quickly coagulate blood and exert good antibacterial activity;

(2) complex B, synthesis of dopamine-modified hyaluronic acid (HA-DA);

in the step (2), the preparation of the dopamine-modified hyaluronic acid comprises the following steps:

(2A) dissolving 0.4g hyaluronic acid in 40mL degassed deionized water and storing the solution under nitrogen;

(2B) EDC (230mg, 1.2mmol) and NHS (138mg, 1.2mmol) were slowly added to the solution and stirred for 20 min;

(2C) adding dopamine hydrochloride (227.6mg, 1.2mmol) to the solution in step (2B), monitoring the pH of the solution, and adjusting the pH between 5 and 6 by adding 0.1M hydrochloric acid or NaOH, followed by allowing the solution to react at room temperature overnight;

(2D) after the reaction, the solution was purified by dialysis (MWCO 10,000) under acidic conditions for 3 days, and lyophilized. The resulting white foam should be stored at-20 ℃ prior to use;

the dopamine-modified hyaluronic acid has good biocompatibility and water retention, and can absorb blood and promote blood coagulation, and in addition, the grafted dopamine has good wet tissue adhesion, so that the capability of the material for rapidly closing a wound is improved;

(3) synthesizing a nanoparticle C and a dopamine modified molecular sieve imidazole skeleton (ZDH) loaded by a sonosensitizer HMME;

in the step (3), the preparation of the dopamine modified molecular sieve imidazole skeleton loaded by the sound-sensitive agent HMME comprises the following steps:

(3A) 2-methylimidazole (3.280 g, 40 mmol) and Zn (NO)₃)₂·6H₂O (1.487 g, 5 mmol) was added to 100 ml of methanol. After vigorous stirring at room temperature for 1 hour, the final white product ZIF-8 was purified by centrifugation and vacuum drying;

(3B) ZIF-8(500mg), methanol (10mL) and dopamine (50mg) were mixed and refluxed at 60 ℃. After 6 hours, HMME (5mg in 5mL methanol) was added to the previous solution and stirred at room temperature for an additional 12 h;

(3C) the mixed solution obtained in (1B) was washed with ultrapure water, and then centrifuged (10000rmp, 10 minutes) to remove excess HMME. The final product was obtained by vacuum drying. The detected HMME loading was 85%, obtained by measuring HMME in the total liquid obtained by centrifugation in the uv spectrum;

(4) preparation of crystal glue

In the step (4), the preparation of the crystal glue comprises the following steps:

(4A) QCSG, HA-DA and ZDH nanoparticles were prepared in 5 wt%, 3 wt% and 1wt% aqueous solutions or dispersions, respectively. mu.L of QCSG aqueous solution was mixed with 100. mu.L of aqueous LZDH dispersion and 200. mu.L of aqueous HA-DA solution and pre-cooled in an ice bath.

(4B) A solution of 100mg/mL Ammonium Persulfate (APS) and 20. mu.L/mL Tetramethylethylenediamine (TEMED) was prepared and pre-cooled in an ice bath.

(4C) Add 50. mu.L of APS solution 50. mu.L of TEMED solution to the mixed pre-cooled solution in (4A). After fully mixing, transferring the mixture to a mold, putting the mold into ethanol precooled at the temperature of-20 ℃ in a refrigerator for 48 hours, then unfreezing the crystal gel, and according to the different mass ratio of HA-DA and QCSG in the mixture, preparing the crystal gel named as QH 20/ZDH.

Example 2

Unlike example 1, QCSG, HA-DA and ZDH nanoparticles were prepared in step (4A) after 5 wt%, 1.5 wt% and 1wt% aqueous solutions or dispersions, respectively. The 600. mu.L of QCSG aqueous solution was mixed with 100. mu.L of aqueous dispersion of LZDH and 200. mu.L of aqueous solution of HA-DA, pre-cooled in an ice bath, and the correspondingly prepared gel was named QH 10/ZDH.

Example 3

Unlike example 1, QCSG, HA-DA and ZDH nanoparticles were prepared in step (4A) after 5 wt%, 4.5 wt% and 1wt% aqueous solutions or dispersions, respectively. The 600. mu.L of QCSG aqueous solution was mixed with 100. mu.L of aqueous dispersion of LZDH and 200. mu.L of aqueous solution of HA-DA, pre-cooled in an ice bath, and the correspondingly prepared gel was named QH 30/ZDH.

Example 4

In contrast to example 1, QCSG was replaced by a quaternized carboxymethyl chitosan modified with glycidyl methacrylate (QCMCSG).

Example 5

Unlike example 1, QCSG was replaced with methacrylyl Gelatin (GMA), and GMA was prepared at a concentration of 10 wt% in step (4A).

Example 6

Unlike example 1, QCSG was replaced with polyethylene glycol diacrylate (PEGDA), and in step (4A), PEGDA was prepared at a concentration of 5 wt% to 20wt% depending on the molecular weight of polyethylene glycol PEG used.

Example 7

In contrast to example 1, HA-DA was replaced by gelatin grafted dopamine (GT-DA), while in step (4A) a GT-DA concentration of 10 wt% was provided.

Example 8

In contrast to example 1, alginate-branched dopamine (SA-DA) was used instead of HA-DA.

Example 9

Unlike example 1, ZDH was replaced with dopamine-modified ZIF-8 loaded with Ce 6.

Example 10

In contrast to example 1, ZDH was replaced with DOX-loaded dopamine-modified ZIF-8.

Example 11

In contrast to example 1, potassium persulfate was used in place of ammonium persulfate.

Example 12

In contrast to example 1, VA-086 was used in place of ammonium persulfate and TEMED.

Example 13

In contrast to example 1, LAP was used instead of ammonium persulfate and TEMED, supplemented with UV irradiation.

The following table shows the experimental parameters of example 14 to example 25, all parameters not mentioned in the table being identical to those of example 1.

TABLE 1 Experimental parameters for examples 14-19

Table 2 experimental parameters for examples 20-25

Comparative example 1

(1D) after the reaction in the step (1C) is finished, GMA is dropwise added into the reaction mixture under the continuous stirring at 55 ℃, and the ratio of GMA to amino on the main chain of pure chitosan is fixed to be 0.5: 1.0, carrying out reaction for 15 hours at 55 ℃ under dark conditions;

(2) complex B, synthesis of dopamine-modified hyaluronic acid (HA-DA);

(2D) after the reaction, the solution was purified by dialysis (MWCO 10,000) under acidic conditions for 3 days and lyophilized. The resulting white foam should be stored at-20 ℃ prior to use;

the dopamine modified hyaluronic acid has good biocompatibility and water retention, and can absorb blood and promote blood coagulation, and in addition, the grafted dopamine has good wet tissue adhesion, so that the capability of the material for rapidly closing a wound is improved;

(3) synthesizing nanoparticles C and a dopamine modified molecular sieve imidazole skeleton (ZDH) loaded by a sonosensitizer HMME;

in the step (3), the preparation method of the dopamine modified molecular sieve imidazole skeleton loaded with the sonosensitizer HMME comprises the following steps:

(3) preparation of crystal glue

In the step (3), the preparation of the crystal glue comprises the following steps:

(3A) QCSG and HA-DA were prepared in 5 wt% and 3 wt% aqueous solutions, respectively. mu.L of QCSG aqueous solution was mixed with 0. mu.L, 100. mu.L, 200. mu.L and 300. mu.L of HA-DA aqueous solution, 300. mu.L, 200. mu.L, 100. mu.L and 0. mu.L of deionized water were added to ensure a mixture of 900. mu.L, respectively, and pre-cooled in an ice bath.

(3B) A solution of 100mg/mL Ammonium Persulfate (APS) and 20. mu.L/mL Tetramethylethylenediamine (TEMED) was prepared and pre-cooled in an ice bath.

(3C) Add 50. mu.L of APS solution 50. mu.L of TEMED solution to the mixed pre-cooled solution in (4A). After thorough mixing, the mixture was transferred to a mold and placed in ethanol pre-cooled in a refrigerator at-20 ℃ for 48h, and then the gel was thawed. The prepared gels were named QH0, QH10, QH20 and QH30, depending on the volume of HA-DA added to the mixture.

In the experiment of the attached figure, QCSG crystal glue (QH0) without HA-DA, QH10, QH20 and QH30, and QH20(QH20/ZDH) crystal glue with ZDH prepared in example 1 are taken as examples to illustrate the performance of the crystal glue prepared.

FIGS. I-III of FIG. 1(a) show photomicrographs of ZIF-8, ZIF-8/DA and ZIF-8/DA/HMME (ZDH). The dopamine modified ZIF-8 exhibited a cyan color different from the white color of ZIF-8, and the loading of HMME further turned the color of the ZDH nanoparticles to light brown. It can be seen that the ZIF-8 nanoparticles have a uniform size and good morphology, with a particle size of about 100nm (fig. IV). FIGS. V and VI are TEM images of representative ZIF-8 and ZDH nanoparticles, respectively. Compared to the apparent polyhedral structure of ZIF-8 in panel V, the ZDH nanoparticles in panel VI have ambiguous edges and angles, indicating successful modification of dopamine and HMME.

The successful synthesis of ZDH is further confirmed by the UV spectrum in FIG. 1 (b). Compared with ZIF-8, dopamine modified ZIF-8 shows a characteristic dopamine absorption peak near 290nm, while ZDH nanoparticles further show a characteristic absorption peak of HMME at 410 nm.

Fig. 1(c) is an SEM image of the prepared gel. It can be seen that as the HA-DA content increases, the pore size of the crystal gel decreases, indicating that the network structure of the crystal gel is enhanced.

FIG. 1(d) shows that as the HA-DA content increases, the swelling ratio decreases, but there is no significant difference overall, and the addition of ZDH nanoparticles does not significantly decrease the swelling ratio.

After the gel prepared in example 1 was compressed to 80% of the original thickness in fig. 2(a), the gel in the dry state was recovered only to a small amount. However, after contact with water, the fixed gel rapidly returned to its original state within 5.3 ± 0.2 s.

Fig. 2(b) shows that as the HA-DA content increases, the time from compression of 80% to recovery of the gel to 100% gradually decreases.

FIG. 2(c) shows that dopamine modified ZIF-8 nanoparticles dissociate more readily under acidic conditions, and the pH-responsive release capacity of HMME from ZDH nanoparticles was tested, indicating a significant increase in HMME release at pH 6.8 compared to normal physiological pH (7.4). Furthermore, the release of HMME showed a further increase when the pH was lowered to 5.5. This ability ensures that the nanoparticles are more effective at the tumor site with less damage to normal tissues. Furthermore, the entire release process lasts for a long time, thereby ensuring a long-term effect of HMME.

Fig. 3 shows that as the HA-DA content of the gel system increases, the antibacterial rate tends to decrease, which is related to the negative charge of HA, which impairs the antibacterial ability of the quaternary ammonium cationic groups. However, when Zn is to be contained²⁺Due to quaternary ammonium cationic groups and Zn when added to the crystal system²⁺The antibacterial rate is obviously improved to more than 98 percent.

As can be seen from I-III in fig. 4(a), when a commercial gelatin sponge is placed on a certain amount of blood, the blood is not rapidly absorbed, but when a gelatin is placed on the blood, the blood is rapidly absorbed into the pores; figures IV-VI show that when a drop of blood was dropped on the blank or gelatin sponge or crystal and water was added after 5 minutes, most of the blood in the blank was dispersed in the water, indicating essentially no coagulation, a portion of the blood still in the gelatin sponge group was diffused into the water, indicating partial coagulation, while the water in the crystal was completely colorless, indicating good coagulation performance.

As can be seen in FIG. 4(b), the blood coagulation indices of all the cryogel and commercial gelatin sponges were significantly lower than those of the blank and gauze groups (P < 0.01). Furthermore, all gels, except the QH0 group, had better clotting effects than the gelatin sponge group (P < 0.05). More importantly, the QH20/ZDH gel group with ZDH nanoparticles added further showed significantly lower coagulation index than the other gel groups (P <0.05), indicating that ZDH nanoparticles promote blood coagulation.

FIG. 4(c) shows the number of platelets and red blood cells on each set of materials after blood absorption. Apparently, the number of platelets and erythrocytes on the surface of QH20/ZDH gel was much higher than in the gauze and gelatin sponge groups, indicating that the gel exerts its clotting effect by enriching platelets and erythrocytes. This is because of the positively charged quaternized chitosan and Zn²⁺Can be combined with negatively charged cell membrane, thereby promoting the aggregation of red blood cells. Furthermore, HA-Carboxyl on DA and DA stimulate blood platelet, improve the function of blood coagulation.

A bleeding model of a rat liver partial resection was established in fig. 5(a) to simulate the actual fact that a portion of the liver will be resected during tumor treatment. By comparing the blood loss of the blank group and the commercial gelatin sponge group, the gel group showed not only a significant reduction in blood loss compared to the blank group (P <0.001), but also a significant difference compared to the gelatin sponge group (P <0.05, panel III). When the hemostasis time was further calculated, a similar rule was found and the hemostasis time was significantly reduced for the gel group compared to the gelatin sponge (P <0.01, panel IV). It can also be seen from the representative photograph in fig. II that the gauze of the control group was stained with blood, and the gauze of the gelatin sponge was also partially stained with blood. In contrast, the gel does not yet penetrate into the blood.

FIG. 5(b) in a more blood loss animal model (partial hepatectomy in rabbits), again demonstrates the significant difference between the gel group and the control group (P <0.001), and the more significant difference between the gel group and the commercially available gelatin sponge (P <0.01), further confirming the great advantage of these gels in treating massive bleeding. In addition, the hemostasis time is also similar. It was also observed that the amount of blood loss was significantly less for the group of gels than for the other groups, indicating that these gels have a great potential for major bleeding hemostasis during tumor therapy.

FIG. 6(a) shows that different concentrations of different gels were tested for their hemolysis rate. The hemolysis rate gradually decreases with the increase of HA-DA content in the crystal gel, and when ZDH nano-particles are further added, the hemolysis rate of QH20/ZDH crystal gel is basically consistent with that of QH20 crystal gel. Even if the sample concentration is as high as 8mg/mL, only the QH0 crystal gel group has a hemolysis rate of more than 5%, and the hemolysis rate of less than 5% is the most common standard for good blood compatibility of materials. The inset picture shows that the solution color does not significantly turn red as the concentration of the gel increases.

After co-culturing the different materials with L929 cells for 1 day, 3 days, and 5 days in fig. 6(b), the effect of the gel on the viability of L929 cells showed a rising trend. With the increase of HA-DA content in the crystal gel, the cell activity is increased. On day 5, the cell activity was even higher than that of TCP in some groups.

The quantitative data for active oxygen in FIG. 6(c) shows that the QH20/ZDH + sonicated group produced significantly more active oxygen than the blank, doxorubicin, sonicated and QH20/ZDH groups (P < 0.001).

The H22 cell viability test in fig. 6(d) showed that the QH20/ZDH + ultrasound group had lower cell viability and the difference was statistically significant (P <0.01) compared to the blank, doxorubicin, ultrasound and QH20/ZDH groups. More importantly, the significant difference between the QH20/ZDH + ultrasound group and the doxorubicin group further demonstrates the advantage of sonodynamic anticancer therapy provided by HMME release.

FIG. 7(a) assesses the hemostatic effect of QH20/ZDH crystalloid during tumor resection. In actual surgery, it was found that tumor resection was very likely to cause bleeding, which if left untimely, further resulted in massive blood loss and even death of the mice. In contrast, QH20/ZDH crystal gel showed significant differences in blood loss from the blank group (P <0.001) and the commercially available gelatin sponge group (P <0.01), which greatly reduced not only the intraoperative bleeding risk leading to mouse death, but also the risk of cancer cell metastasis with blood.

Fig. 7(b) shows the weight of recurrent tumor tissue isolated from the liver of mice on day 18 post-surgery. The mean weights of recurrent tumors in mice treated with QH20/ZDH crystalloid combined with ultrasound were significantly lower than those in the blank, ultrasound and QH20/ZDH groups (P < 0.001).

FIG. 7(c) is a graph quantifying the relative expression of HIF-1. alpha. and IL-6. The result shows that the IL-6(P <0.05) expression of the QH20/ZDH + ultrasonic group is obviously higher than that of other groups, which indicates that the addition of the sound-sensitive agent HMME has certain effect of promoting the tumor immunotherapy. More importantly, compared with the adriamycin group, the HIF-1 alpha expression in the QH20/ZDH + ultrasonic group is also obviously reduced, which indicates that the sonodynamic anticancer therapeutic effect provided by HMME is better than that of anticancer drugs.

Referring to fig. 8, a schematic diagram of the network structure of the crystal gel formed in example 1 of the present invention, as shown in the figure, the crosslinking of the double-bond functionalized first biological macromolecule (compound a) of the present invention under the action of the initiator serves as the first major network of the crystal gel. The polyphenol modified biomacromolecule (compound B) and the double-bond functionalized biomacromolecule (compound A) are combined through the actions of macromolecular physical winding, electrostatic interaction, hydrogen bonds and the like to form a second repeating network, the crystal glue network is further enhanced, and the non-covalent connection between the second biomacromolecule and the first crystal glue network is used as a bridge to load the nano particles C.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A preparation method of crystal gel for stopping bleeding and bearing anticancer drugs is characterized by comprising the following steps:

step 3, synthesizing nanoparticles C by using an anti-cancer substance, dopamine and ZIF-8; the dopamine modified ZIF-8 is loaded with an anti-cancer substance, and the dopamine is combined with a polyphenol compound on the compound B to bring the nano particles C into a crystal gel system; the anti-cancer substance is a photosensitizer in cancer photodynamic therapy or a sonosensitizer in cancer sonodynamic therapy;

step 4, preparing crystal gel through the compound A, the compound B and the nano particles C;

the specific process of the step 4 is as follows:

2. The method for preparing crystalloid used for stopping bleeding and carrying anticancer drugs as claimed in claim 1, wherein in step 1, the first biological macromolecule is chitosan or its derivatives, gelatin or its derivatives, hyaluronic acid or its derivatives, alginate or its derivatives, dextran or its derivatives, cellulose or its derivatives, polyvinyl alcohol or its derivatives, polyethylene glycol or its derivatives, or pluronic F127 or its derivatives; the first biological macromolecule is used in one or more matching ways;

3. The method of claim 1, wherein in step 2, the second biomacromolecule is hyaluronic acid or a derivative thereof, gelatin or a derivative thereof, alginate or a derivative thereof, dextran or a derivative thereof, cellulose or a derivative thereof, polyvinyl alcohol or a derivative thereof, polyethylene glycol or a derivative thereof, or pluronic F127 or a derivative thereof; the second biological macromolecule is used in one or more matching ways;

the polyphenol compound is a compound containing an o-phenylphenol structure.

4. The method for preparing a crystalloid colloid for hemostasis and bearing anticancer drugs as claimed in claim 3, characterized in that the compound containing orthophthalic polyphenol structure is dopamine or its derivatives, levodopa or its derivatives, dihydrocaffeic acid or its derivatives, tannic acid or its derivatives, 3, 4-dihydroxybenzaldehyde or its derivatives, or gallic acid or its derivatives; when the compounds with the structure of the o-phenylphenol are used, one or more compounds are used in a matching way.

5. The method for preparing a crystalloid colloid used for hemostasis and anticancer drug bearing of claim 1, wherein in step (A), the concentration of the aqueous solution of the compound A is 1wt% -20wt%, and the concentration of the aqueous solution of the compound B is 1wt% -20 wt%; the concentration of the aqueous dispersion of the nanoparticles C is 5-20 mg/mL.

6. The method for preparing the crystalloid colloid for hemostasis and bearing anticancer drugs as claimed in claim 1, wherein in the step (B), the initiator is hydrogen peroxide, potassium persulfate, ammonium persulfate or benzoyl; the initiator amount is 0.5-5% of the double bond molar amount in the compound A in the prepared prepolymer solution.

7. The method for preparing the crystalloid colloid for hemostasis and anticancer drug bearing of claim 1, wherein in the step (C), the volume mixing volume ratio of the compound A, the compound B, the nano particles C and the initiator solution is: 6: 2: 1: 1.

8. a crystalloid colloid for hemostasis and carrying anticancer drugs prepared by the preparation method of any one of claims 1 to 7, characterized in that the crystalloid colloid is a porous reticular structure composed of compound A and compound B which are interlaced; the nano particles C are attached to the surface of the net structure, and are nano particles.