CN113599572B

CN113599572B - Freeze-dried powder and application thereof

Info

Publication number: CN113599572B
Application number: CN202110938410.0A
Authority: CN
Inventors: 袁暾; 倪潘显志; 叶胜; 李任鹏; 熊书婷
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2022-09-09
Anticipated expiration: 2041-08-16
Also published as: CN113599572A

Abstract

The invention provides freeze-dried powder and application thereof, wherein the freeze-dried powder can be chitosan-beta-sodium glycerophosphate freeze-dried powder, lactobionic acid modified chitosan-beta-sodium glycerophosphate freeze-dried powder or lactobionic acid modified chitosan/chitosan-beta-sodium glycerophosphate freeze-dried powder. The freeze-dried powder hydrogel precursor solution formed by mixing the freeze-dried powder provided by the invention and deionized water can form the freeze-dried powder hydrogel in a short time at 37 ℃, the hydrogel has good injection performance, better mechanical strength and tissue adhesion, and good cell compatibility, and has an obvious protection effect on cells in an acid environment. In addition, the freeze-dried powder hydrogel provided by the invention is stable in storage and convenient to use, and can still meet most requirements of ESD (electro-static discharge) operations. Importantly, the freeze-dried powder can effectively solve the problems that the transportation and storage of hydrogel in an ESD operation are difficult, temporary preparation is needed during use, and the whole operation is complicated, and has certain production potential.

Description

Freeze-dried powder and application thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to freeze-dried powder and application thereof.

Background

Endoscopic Submucosal Dissection (ESD) is a common treatment for early stage gastric cancer in recent years and has been shown to be effective in terms of overall resection rate and recurrence rate. However, due to the deep and submucosal depth of lesion excision, bleeding, perforation and other complications are common during and after the operation. The chitosan temperature-sensitive hydrogel system has good cell compatibility, has obvious protection effect on cells in an acidic environment, has good performance as an intraoperative biomaterial, and is proved to be an effective strategy for reducing the occurrence of complications in ESD operations.

However, the performance of the temperature-sensitive hydrogel of chitosan is susceptible to temperature (the gel is easily formed by the increase of temperature), so that the hydrogel must be stored under refrigeration. The temperature must be monitored throughout the course of transport and storage, and the associated costs are high. In addition, studies have reported that the temperature-sensitive hydrogel of chitosan lacks long-term stability even under refrigerated conditions (4 ℃), and a precursor solution of the hydrogel forms a gel or undergoes modification over a period of time. On the other hand, the temporary preparation of hydrogels prior to ESD surgery can introduce complications to the overall surgical procedure and new problems for the clinician. These problems have hindered the large-scale production and use of such intraoperative biomaterials.

Disclosure of Invention

Based on the problems of the chitosan temperature-sensitive hydrogel in the prior art, the invention aims to develop the intraoperative biomaterial which is stable in storage, convenient to use and effective in clinical application. Therefore, the invention provides freeze-dried powder and application thereof, which are used for effectively solving the problems of transportation and storage of biological materials used in the existing ESD and the complexity of the whole operation caused by the temporary preparation required during use.

In a first aspect, the invention provides a freeze-dried powder, wherein the freeze-dried powder is chitosan-beta-sodium glycerophosphate freeze-dried powder, lactobionic acid modified chitosan/chitosan-beta-sodium glycerophosphate freeze-dried powder or lactobionic acid modified chitosan-beta-sodium glycerophosphate freeze-dried powder.

Preferably, the freeze-dried powder is mixed with deionized water to form a freeze-dried powder hydrogel precursor solution, wherein the proportion of the deionized water to the freeze-dried powder in the freeze-dried powder hydrogel precursor solution is 1: 20-1: 100.

Preferably, the freeze-dried powder hydrogel precursor solution can form hydrogel at 37 ℃, and the hydrogel has a porous network and a layered structure.

Preferably, when the freeze-dried powder is chitosan-beta-sodium glycerophosphate freeze-dried powder, the preparation method of the freeze-dried powder comprises the following steps:

step 1: dissolving chitosan in 1% acetic acid solution, stirring at room temperature to dissolve completely to obtain 3.33% solution A, and storing at 4 deg.C; dissolving beta-sodium glycerophosphate in sodium bicarbonate and sodium carbonate buffer solution to obtain 0.15g/mL solution B; wherein the sodium bicarbonate concentration is 0.1g/mL, the sodium carbonate concentration is 5mg/mL, and the mixture is stored at 4 ℃;

step 2: mixing the solution A and the solution B prepared in the step 1 under the condition of ice-water bath to prepare a first group of freeze-dried powder precursor solution;

and step 3: and (3) pre-freezing the first group of freeze-dried powder precursor solution prepared in the step (2), transferring to a freeze dryer for freeze drying, grinding and screening to obtain freeze-dried powder.

Preferably, in the step 2, the first group of lyophilized powder precursor solutions is prepared by mixing 15ml of solution A and 10ml of solution B; in the first group of freeze-dried powder precursor solution, the mass concentration of the solution A is 2%, and the mass concentration of the solution B is 6%; in the step 3, the pre-freezing conditions include: the temperature is minus 18 ℃ and the time is 8-10 h; the freeze-drying time in the freeze dryer was 48 h.

Preferably, when the freeze-dried powder is lactobionic acid modified chitosan-beta-sodium glycerophosphate freeze-dried powder, the preparation method of the freeze-dried powder comprises the following steps:

step 1': dissolving chitosan modified by lactobionic acid in deionized water to prepare a solution C with the mass concentration of 3.33%; storing at 4 deg.C; dissolving beta-sodium glycerophosphate in sodium bicarbonate and sodium carbonate buffer solution to obtain 0.15g/mL solution B; wherein the sodium bicarbonate concentration is 0.1g/mL, the sodium carbonate concentration is 5mg/mL, and the sodium bicarbonate is stored at the temperature of 4 ℃;

step 2': mixing the solution C and the solution B prepared in the step 1' under the condition of ice-water bath to prepare a second group of freeze-dried powder precursor solution;

step 3': and (3) pre-freezing the second group of freeze-dried powder precursor solution prepared in the step 2', transferring to a freeze dryer for freeze drying, grinding and screening to obtain freeze-dried powder.

Preferably, in the step 2', the second group of lyophilized powder precursor solutions is prepared by mixing 15ml of solution C with 10ml of solution B; in the second group of freeze-dried powder precursor solution, the mass concentration of the solution C is 2%, and the mass concentration of the solution B is 6%; in said step 3', said pre-freezing conditions comprise: the temperature is-18 ℃ and the time is 8-10 h; the freeze-drying time in the freeze dryer was 48 h.

Preferably, when the freeze-dried powder is lactobionic acid modified chitosan/chitosan-beta-sodium glycerophosphate freeze-dried powder, the preparation method of the freeze-dried powder comprises the following steps:

step 1': dissolving chitosan in acetic acid solution with mass concentration, stirring at room temperature until the chitosan is completely dissolved to obtain solution A with mass concentration of 3.33%, and storing at 4 ℃; and dissolving the lactobionic acid modified chitosan in deionized water to prepare a solution C with the mass concentration of 3.33%. Storing at 4 deg.C; dissolving beta-sodium glycerophosphate in sodium bicarbonate and sodium carbonate buffer solution to obtain 0.15g/mL solution B; wherein the sodium bicarbonate concentration is 0.1g/mL, the sodium carbonate concentration is 5mg/mL, and the mixture is stored at 4 ℃;

step 2': mixing the solution A, the solution B and the solution C prepared in the step 1' under the condition of ice-water bath to prepare a third group of freeze-dried powder precursor solution;

step 3': pre-freezing the third group of freeze-dried powder precursor solution prepared in the step 2', transferring to a freeze dryer for freeze drying, grinding and screening to obtain freeze-dried powder.

Preferably, in the step 2 ", the third group of lyophilized powder precursor solutions is prepared by mixing 6ml of solution a, 9ml of solution C and 10ml of solution B; in the third group of freeze-dried powder precursor solution, the mass concentration of the solution A/the solution C is 2%, and the mass concentration of the solution B is 6%; in said step 3 ", said pre-freezing conditions comprise: the temperature is-18 ℃ and the time is 8-10 h; the freeze-drying time in the freeze dryer was 48 h.

In a second aspect, the invention provides an application of a freeze-dried powder, which specifically comprises the following steps: the freeze-dried powder of the first aspect is used as a biological material for endoscopic mucosal dissection.

Compared with the prior art, the invention has the following advantages:

(1) the freeze-dried powder provided by the invention can be chitosan-beta-sodium glycerophosphate freeze-dried powder, lactobionic acid modified chitosan-beta-sodium glycerophosphate freeze-dried powder or lactobionic acid modified chitosan/chitosan-beta-sodium glycerophosphate freeze-dried powder, and can be reconstructed into hydrogel for ESD surgery under the action of deionized water. The hydrogel is formed by a lyophilized powder hydrogel precursor solution prepared from the lyophilized powder provided by the invention and deionized water at 35-38 ℃, has good injection performance, good mechanical strength and tissue adhesion, and good cell compatibility, and has an obvious protective effect on cells in an acidic environment. Therefore, the freeze-dried powder hydrogel prepared from the freeze-dried powder provided by the invention still has good physicochemical and biological properties when being used as an intraoperative biological material of ESD.

(2) The raw materials of the freeze-dried powder provided by the invention comprise chitosan, lactobionic acid modified chitosan and sodium glycerophosphate, wherein the total concentration of lactobionic acid modified chitosan and chitosan is 2%, and the concentration of sodium glycerophosphate is 6%.

(3) The freeze-dried powder provided by the invention is solid powder, so that the freeze-dried powder has good long-term stability and can be stored at normal temperature. Therefore, the temperature control in the whole process is not needed in the transportation and storage processes, and the related transportation and storage cost is reduced. And when in use, the corresponding hydrogel can be prepared only by directly mixing the powder with deionized water, so the use is very convenient, and the trouble that a doctor needs to prepare a hydrogel precursor solution on site according to different proportions of raw materials before an ESD operation is solved.

Drawings

FIG. 1 shows the storage modulus (G ') and loss modulus (G') over time for various sets of lyophilized powder hydrogel precursor solutions of the examples of the present application at 37 ℃;

FIG. 2 shows an infrared spectrum of each set of lyophilized powders in the examples of the present application;

FIG. 3 shows a microstructure diagram of each group of lyophilized powder in the embodiment of the present application;

fig. 4 shows gel formation times for various sets of lyophilized powder hydrogels in the examples of the present application;

FIG. 5 shows pH values of various groups of lyophilized powder hydrogels in an example of the present invention;

FIG. 6 shows the energy storage modulus values of the groups of lyophilized powder hydrogels with frequencies of 1Hz, 2Hz, and 5Hz in the examples of the present application;

FIG. 7 shows the degradation rate of each group of lyophilized powder hydrogel in acidic PBS solution containing pepsin in the examples of the present application;

FIG. 8 shows the microstructure of various groups of lyophilized powder hydrogels in the examples of the present application;

FIG. 9 shows the viscosities of various sets of lyophilized powder hydrogel precursor solutions at 4 ℃ in the examples of the present application;

FIG. 10 shows the injection force at 4 ℃ for each set of lyophilized powder hydrogel precursor solutions in the examples of the present application;

FIG. 11 shows the percent hydrogel residues that were adhered to the tissue surface after rinsing for each set of lyophilized powder hydrogels in the examples of the present application;

FIG. 12 shows the adhesion of various groups of lyophilized powder hydrogels to tissue after gelation of the tissue surface in the examples of the present application;

FIG. 13 shows the relative proliferation rate of L929 cells after culturing L929 cells in each set of lyophilized powder hydrogel extract solution for 24h in the present application example;

FIG. 14 shows the cell morphology of GES-1 cells in each set of lyophilized powder hydrogels in the examples of the present application;

FIG. 15 shows the proliferation of GES-1 cells on the surface of each group of lyophilized powder hydrogels in the examples of the present application;

FIG. 16 shows the relative proliferation rates of GES-1 cells on the hydrogel surfaces of various groups of lyophilized powders under acidic conditions in the examples of the present application;

FIG. 17 shows the adhesion capacity of GES-1 cells on the surface of each set of lyophilized powder hydrogels in the examples of the present application.

Detailed Description

The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.

The specific experimental procedures or conditions are not indicated in the examples and can be performed according to the procedures or conditions of the conventional experimental procedures described in the prior art in this field. The reagents and other instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

First, the inventors found that chemical or physical degradation reactions are inhibited or sufficiently slowed down in the lyophilized state of the object, thereby improving long-term stability. The freeze-dried powder has better stability and is easy to operate in the processes of transportation, storage and use. However, lyophilization of a precursor solution of a hydrogel into a powder may also present problems: the freeze-drying process can affect the key properties of the chitosan temperature-sensitive hydrogel, such as mechanical strength, injectability and the like, and the properties are very important for ESD operation. The injectability of a hydrogel is related to the homogeneity of the hydrogel precursor solution. It has been reported that the introduction of lactose fragments into chitosan molecular chains can significantly improve water solubility at neutral pH. Lactic Acid (LA) has antioxidant, biodegradable, biocompatible and chelating properties that make it attractive for potential medical applications. Therefore, by introducing lactose, the molecular chain of the chitosan is prolonged, the number of H bonds is increased, an H bond network is formed, and the mechanical strength of the chitosan hydrogel can be improved.

According to the research, the chitosan hydrogel is prepared into freeze-dried powder by adopting a freeze-drying method, and the injectable temperature-sensitive hydrogel is reconstructed on the basis of the freeze-dried powder hydrogel.

In a first aspect, embodiments of the present invention provide a lyophilized powder, which may be a chitosan- β -sodium glycerophosphate lyophilized powder, a lactobionic acid modified chitosan- β -sodium glycerophosphate lyophilized powder, or a lactobionic acid modified chitosan/chitosan- β -sodium glycerophosphate lyophilized powder.

In the embodiment of the present invention, preferably, the lyophilized powder is mixed with deionized water to form a lyophilized powder hydrogel precursor solution, and in the lyophilized powder hydrogel precursor solution, the ratio of the deionized water to the lyophilized powder is 1: 20-1: 100.

In the embodiment of the present invention, preferably, the lyophilized powder hydrogel precursor solution can form a lyophilized powder hydrogel at 37 ℃, and the lyophilized powder hydrogel has a porous network and a layered structure.

In the embodiment of the present invention, preferably, when the lyophilized powder is chitosan- β -sodium glycerophosphate lyophilized powder, the preparation method thereof comprises:

step 1: dissolving chitosan in acetic acid solution with mass concentration, stirring at room temperature until the chitosan is completely dissolved to obtain solution A with mass concentration of 3.33%, and storing at 4 ℃; dissolving beta-sodium glycerophosphate in sodium bicarbonate and sodium carbonate buffer solution to obtain 0.15g/mL solution B; wherein the sodium bicarbonate concentration is 0.1g/mL, the sodium carbonate concentration is 5mg/mL, and the mixture is stored at 4 ℃;

and 2, step: mixing the solution A and the solution B prepared in the step 1 under the ice-water bath condition to prepare a first group of freeze-dried powder precursor solution;

In the embodiment of the present invention, preferably, in step 2, the first group of lyophilized powder precursor solutions is prepared by mixing 15ml of solution a and 10ml of solution B; in the first group of freeze-dried powder precursor solution, the mass concentration of the solution A is 2%, and the mass concentration of the solution B is 6%; in step 3, the pre-freezing conditions include: the temperature is minus 18 ℃ and the time is 8-10 h; the freeze-drying time in the freeze-dryer was 48 h.

In the embodiment of the present invention, preferably, when the lyophilized powder is lactobionic acid modified sodium chitosan- β -glycerophosphate lyophilized powder, the preparation method of the lyophilized powder comprises:

step 1': dissolving chitosan modified by lactobionic acid in deionized water to prepare a solution C with the mass concentration of 3.33%; storing at 4 deg.C; dissolving beta-sodium glycerophosphate in sodium bicarbonate and sodium carbonate buffer solution to obtain 0.15g/mL solution B; wherein the sodium bicarbonate concentration is 0.1g/mL, the sodium carbonate concentration is 5mg/mL, and the mixture is stored at 4 ℃;

in specific implementation, the process for obtaining lactobionic acid modified chitosan is as follows: firstly, respectively preparing 2% (w/v) chitosan solution and 2.22% (w/v) lactobionic acid solution; then EDS and NHS are added into 2.22% lactobionic acid solution, and the pH value is adjusted to any value between 4 and 6 to carry out an amide reaction, so as to realize the activation of carboxyl in lactobionic acid; and finally, mixing the activated lactobionic acid solution with a 2% chitosan solution, reacting at room temperature for 24 hours, after the reaction is finished, adding ethanol into the reaction system to precipitate the materials in the reaction system for 24 hours, filtering to obtain a precipitate, re-dissolving the precipitate in deionized water to obtain a dissolution system, dialyzing the dissolution system for 48 hours, and freezing and drying the dialyzed dissolution system to obtain the lactobionic acid modified Chitosan (CSLA) similar to a sponge.

Step 2': mixing the solution C and the solution B prepared in the step 1' under the ice-water bath condition to prepare a second group of freeze-dried powder precursor solution;

In the embodiment of the present invention, preferably, in step 2', the second set of lyophilized powder precursor solutions is prepared by mixing 15ml of solution C with 10ml of solution B; in the second group of freeze-dried powder precursor solution, the mass concentration of the solution C is 2%, and the mass concentration of the solution B is 6%; in step 3', the pre-freezing conditions include: the temperature is-18 ℃ and the time is 8-10 h; the freeze-drying time in the freeze-dryer was 48 h.

In the embodiment of the present invention, preferably, when the lyophilized powder is lactobionic acid modified chitosan/chitosan- β -sodium glycerophosphate lyophilized powder, the preparation method of the lyophilized powder comprises:

step 1': dissolving chitosan in acetic acid solution with mass concentration, stirring at room temperature until the chitosan is completely dissolved to obtain solution A with mass concentration of 3.33%, and storing at 4 ℃; and dissolving the lactobionic acid modified chitosan in deionized water to prepare a solution C with the mass concentration of 3.33%. Storing at 4 deg.C; dissolving beta-sodium glycerophosphate in sodium bicarbonate and sodium carbonate buffer solution to obtain 0.15g/mL solution B; wherein the sodium bicarbonate concentration is 0.1g/mL, the sodium carbonate concentration is 5mg/mL, and the sodium bicarbonate is stored at the temperature of 4 ℃;

step 3': and (3) pre-freezing the third group of freeze-dried powder precursor solution prepared in the step 2', transferring the third group of freeze-dried powder precursor solution to a freeze dryer for freeze drying, grinding and screening to obtain freeze-dried powder.

In the embodiment of the present invention, preferably, in step 2 ″, the third group of lyophilized powder precursor solutions is prepared by mixing 6ml of solution a, 9ml of solution C and 10ml of solution B; in the third group of freeze-dried powder precursor solution, the mass concentration of the solution A/the solution C is 2%, and the mass concentration of the solution B is 6%; in step 3 ", the pre-freezing conditions include: the temperature is-18 ℃ and the time is 8-10 h; the freeze-drying time in the freeze-dryer was 48 h.

The preparation method of the lyophilized powder is described in detail by the following examples.

Example 1:

first, Chitosan (CS) (molecular weight 180kDa, degree of deacetylation 84%), sodium beta-Glycerophosphate (GP) (C) ₃ H ₇ Na ₂ O ₆ P· ₅ H ₂ O), Fluorescein Diacetate (FDA), Propidium Iodide (PI) and pepsin (from porcine gastric mucosa) were purchased from Sigma-Aldrich, usa. Lactobionic Acid (LA) (C) ₁₂ H ₂₂ O ₁₂ ) N-hydroxysuccinimide C ₄ H ₅ NO ₃ (NHS), carbodiimide C ₈ H ₁₇ N ₃ HCl (EDC) from Shanghai Allatin Biochemical technology, Inc. DMEM high-sugar medium and RPMI-1640 medium were purchased from Thermo Fisher Scientific Corporation, USA. L929 cells (mouse fibroblasts) and GES-1 cells (human gastric epithelial cells) were purchased from Kunming cell Bank, Chinese academy of sciences.All other chemicals used were reagent grade and were used without further purification.

Step 1: three solutions were prepared:

dissolving Chitosan (CS) in acetic acid solution with mass concentration of 1%, stirring at room temperature until completely dissolving, wherein the mass concentration of the obtained solution is 3.33%, and storing at 4 ℃;

preparing a buffer solution with the concentration of 0.1g/mL of sodium bicarbonate and the concentration of 5mg/mL of sodium carbonate, and dissolving beta-sodium glycerophosphate in the buffer solution to obtain a solution with the mass concentration of 0.15g/mL of beta-sodium Glycerophosphate (GP); storing at 4 deg.C;

dissolving lactobionic acid modified Chitosan (CSLA) in deionized water to obtain solution C with the mass concentration of 3.33%; storing at 4 deg.C;

step 2: preparing a lyophilized powder precursor solution

Mixing the prepared 3.33% (w/v) chitosan solution, 3.33% (w/v) lactobionic acid modified chitosan solution and beta-sodium glycerophosphate solution with the concentration of 0.15g/ml under the condition of ice-water bath according to the volume given in the following table to prepare freeze-dried powder precursor solutions with different components.

In 3 groups of hydrogel precursor solutions prepared according to Table 1, the final concentrations of CS, CSLA/CS and CSLA were each 2% and the final concentration of GP was 6%. Each group was prepared under ice bath conditions and mixed with stirring until complete mixing.

And step 3: freeze-drying

And (3) placing the 3 groups of freeze-dried powder precursor solutions prepared in the step (2) in a culture dish, refrigerating for 8-10 nights in a refrigerator at the temperature of-20 ℃, then transferring to a freeze dryer for freeze drying for 48 hours, finally grinding the freeze-dried gel into freeze-dried powder, and sieving once by using a 100-mesh sieve. 100mg of hydrogel freeze-dried powder is added into a 5ml Eppendorf (EP) tube, 2ml of deionized water is added, the water and the freeze-dried powder are uniformly mixed under the ice bath condition, and the mixture is stored at 4 ℃. The hydrogel based on lyophilized powder is defined as lyophilized powder hydrogel.

And 4, step 4: characterization of gel lyophilized powder

(1) Temperature sensitive gelling property

The hydrogel based on the gel freeze-dried powder can form gel at 37 ℃ (physiological temperature), which is the key of whether the hydrogel can be used as biological material in ESD operation. Adding the lyophilized powder hydrogel precursor solution into an EP tube, and placing in an incubator at 37 ℃. After a period of time, the flow of the solution can be observed to determine if the solution has coagulated. The rheological properties of the hydrogels prepared were measured with a rotational rheometer (Anto Paar MCR-302, Austria) with a sweep of oscillation time, a shear strain of 1%, a frequency of 1Hz, a sweep time of 320s and a temperature of 37 ℃ which was maintained. The test geometry was 40mm diameter with a 1.0mm gap. All experiments were repeated three times.

In this example, fig. 1 shows the change of storage modulus (G') and loss modulus (G ") at 37 ℃ over time for each set of lyophilized powder hydrogel precursor solutions of the examples of the present application. As shown in figure 1, the storage modulus (G') of the 3 groups of lyophilized powder hydrogel is always greater than the loss modulus (G "), which also proves that the lyophilized powder hydrogel precursor solution of the 3 groups of lyophilized powder hydrogel can form gel at 37 ℃, i.e. the lyophilized powder hydrogel maintains the temperature-sensitive property of the original hydrogel.

Here, it should be noted that, in the present embodiment, the gel temperature is set to 37 ℃, and it is understood from the experimental results of this embodiment that the gel is automatically formed at 37 ℃. In order to further confirm the temperature-sensitive gelling property of the lyophilized powder prepared in this example, the inventor continuously performed the same experimental operations as in the above step 4(1) at 35 ℃, 36 ℃ and 38 ℃, respectively, and the results show that the three lyophilized powders prepared in this example can form gel at 35 ℃, 36 ℃ and 38 ℃ when prepared into their respective lyophilized powder hydrogel precursor solutions. Therefore, the three kinds of freeze-dried powder prepared by the embodiment have good temperature-sensitive gelling characteristics when being prepared into the freeze-dried powder hydrogel precursor solution.

(2) Fourier transform Infrared Spectroscopy (FTIR) analysis

Respectively weighing 1mg of CS-GP, CSLA/CS-GP and CSLA-GP freeze-dried powder samples, mixing the samples with 50mg of KBr, grinding the mixture into powder, pressing the powder into tablets, and performing temperature change at the room temperature of 4000 cm to 400cm ^-1 In range, FTIR spectra were recorded using a KBr disk on an FTIR spectrometer (Nicolet FTIR 6700, USA).

Fig. 2 shows an infrared spectrum of each group of lyophilized powders in the example of the present application. As shown in FIG. 3, 3400cm ^-1 The peaks at (a) correspond to the free hydroxyl groups in CS and CSLA. 1660cm ^-1 And 1290cm ^-1 The peak at (A) corresponds to amide I and amide III, respectively, and the peak value is 1290cm along with the increase of the CSLA concentration ^-1 The strength is enhanced.

(3) Micro-morphology of freeze-dried powder

A layer of ultrathin gold is plated on the surfaces of CS-GP, CSLA/CS-GP and CSLA-GP freeze-dried powder samples respectively by an ion sputtering method, and then the microstructure of the freeze-dried powder is observed by a scanning electron microscope (SEM, Hitachi S-4800, Japan).

Fig. 3 shows a microstructure diagram of each group of lyophilized powders in the embodiment of the present application. As shown in fig. 3, the microstructures of the three groups of lyophilized powders were different. The CS-GP freeze-dried hydrogel powder is granular and has the grain diameter of about 3-6 mu m. The hydrogel freeze-dried powder added with CSLA is more uniform and has a regular needle-shaped structure. The needle structure of the CSLA-GP hydrogel freeze-dried powder is denser and thinner.

And 5: characterization of lyophilized powder hydrogels

(1) Gel formation time and pH

Under the ice-water bath condition, CS-GP, CSLA/CS-GP and CSLA-GP freeze-dried powders are respectively prepared into freeze-dried powder hydrogel precursor solutions, the prepared hydrogels are used as a control group, and the gelling time of the hydrogels is measured by an inverted test tube method. Adding 2ml of hydrogel precursor solution prepared by freeze-dried powder into a 5ml EP tube, and quickly transferring the hydrogel precursor solution into an incubator at 37 ℃. Each set of tubes was observed every 1 minute to assess the color change of the solution in the tubes, while the tubes were inverted to observe the flow of the solution to determine if the solution had gelled. The final gel time was recorded and all experiments were repeated 5 times.

The pH values of the 3 lyophilized powder hydrogels were measured using a pH meter (BPH-303). The experiments were all repeated three times.

Fig. 4 shows gel formation times for various sets of lyophilized powder hydrogels in the examples of the present application. As shown in fig. 4, the gel formation time of 6 experimental groups is within 5min, and the gel formation time of the freeze-dried powder hydrogel is longer than that of the corresponding existing hydrogel.

Fig. 5 shows pH values of various groups of lyophilized powder hydrogels in the examples of the present invention. As can be seen from FIG. 5, the pH values of the 6 experimental groups were all around 7 and were close to neutral. This indicates that the freeze-drying process has little effect on the pH. Wherein, the pH values of the CS-GP and CSLA/CS-GP freeze-dried powder hydrogel are slightly higher than those of the existing prepared hydrogel, and the pH values of the CSLA-GP hydrogel before and after freeze-drying are not obviously different.

(2) Dynamic Mechanical Analysis (DMA)

The hydrogel was characterized at room temperature using a dynamic mechanical analyzer (DMA, TAQ800, USA)

Storage modulus of (2). The hydrogel reached a swelling equilibrium in Phosphate Buffered Saline (PBS) and the storage modulus was measured at an amplitude of 80mm, a pre-stress of 1mN and a frequency of 1Hz, 2Hz, 5 Hz. Three parallel samples were measured to find the average.

FIG. 6 shows the energy storage modulus values of the groups of lyophilized powder hydrogels with frequencies of 1Hz, 2Hz, and 5Hz in the examples of the present application. The results are shown in the figure. Under three different frequencies, the storage modulus of the freeze-dried powder based on CS-GP, CSLA/CS-GP and CSLA-GP hydrogels is increased compared with that of the corresponding existing hydrogels, which indicates that the strength of the freeze-dried powder hydrogel is enhanced.

(3) Rate of degradation

To characterize the degradability of the hydrogel in the gastric acid environment, the lyophilized hydrogel samples were weighed

(W _o ) Then incubated in 5mL of PBS containing pepsin (Sigma-Aldrich, USA) at pH 4 at 37 ℃. The hydrogel was removed at a predetermined time point and weighed. Three replicates of hydrogels were measured to obtain an average. Finally, the degradation rate of each sample was calculated according to equation (1):

percent degradation [ (% W) ₀ -W _d )/W ₀ ]×100％

W _d Mass of the sample after degradation.

W ₀ Mass of the initial sample.

Fig. 7 shows the degradation rate of each set of lyophilized powder hydrogels in acidic PBS solution containing pepsin in the examples of the present application. As shown in fig. 7, in the first three days, the hydrogel of the lyophilized powder was maintained at 70% of the original state, and by the fifth day, the degradation rate of the hydrogel decreased by about half. After one week, the degradation rate was about 80%. The degradation rate of the freeze-dried powder hydrogel is higher than that of three types of in-situ prepared hydrogels.

(4) Microcosmic appearance of freeze-dried powder hydrogel

In order to understand the effect of the freeze-drying process on the microstructure of the chitosan hydrogel, the cross-sectional microstructure of each group of hydrogels was observed by scanning electron microscopy. 100 mu LCS-GP, CSLA/CS-GP and CSLA-GP freeze-dried powders are respectively prepared into freeze-dried powder hydrogel precursor solution, the freeze-dried powder hydrogel precursor solution is added into a silica gel mold with the diameter of 8mm and the height of 2mm, and the silica gel mold is placed in a constant temperature oven at 37 ℃ for 30min, so that a freeze-dried powder hydrogel precursor solution sample is completely gelatinized. Taking out each group of hydrogel samples, fixing the shape by using liquid nitrogen, breaking the hydrogel samples by using forceps, putting the hydrogel samples into a freeze dryer for freeze drying for 48 hours, removing the samples, spraying gold to prepare samples, and observing the microstructure of the cross section of the hydrogel by using a scanning electron microscope.

Fig. 8 shows the microstructure of each set of lyophilized powder hydrogels in the examples of the present application. As shown in fig. 8. The 6 groups of hydrogels all had porous network, layered structure. After the CS-GP, CSLA/CS-GP and CSLA-GP aquagels are freeze-dried and ground, the freeze-dried aquagels basically keep the original structures.

(5) Low temperature fluidity and feasibility of injection

The ESD intraoperative biomaterial is injected through a catheter. Therefore, it is necessary to evaluate its low temperature fluidity and the feasibility of injection. The viscosity of the lyophilized powder hydrogel precursor solution at 4 ℃ indicates the low temperature fluidity of the hydrogel. Thus, the viscosity, shear rate and shear stress of the lyophilized powder hydrogel precursor solutions of each set of lyophilized powder formulations were measured at 4 ℃ with a rotational rheometer (austria Anto Paar MCR-302) maintained at constant values. Each sample was tested in triplicate.

The feasibility of hydrogel precursor solution injection was studied using an endoscopic injection needle (25 gauge needle (0.23mm), channel diameter 2.8mm, length 180cm) and an endoscopic spray tube (diameter 1.8mm, channel diameter 2.2mm, length 180cm) provided in the third national hospital of metropolis. Meanwhile, the freshly prepared hydrogel samples were stored in a refrigerator at 4 ℃ for 10 days as a control. Then, the injection pushing force of the hydrogel precursor solution was evaluated using an electromechanical universal tester (Shimadzu Autograph AGS-X, Japan), the pushing speed was 30mm/min, and the maximum load was 500N. 7 mm in diameter and 146 mm in length, and is equipped with a cannula having an inner diameter of 5 mm and a length of 75 mm. And 3 replicates per sample were tested using saline as a control.

Fig. 9 shows the viscosity of each set of lyophilized powder hydrogel precursor solutions at 4 ℃ in the examples of the present application. As shown in FIG. 9, the viscosities of the CS-GP, CSLA/CS-GP and CSLA-GP lyophilized powder hydrogel precursor solutions at 4 ℃ were significantly reduced relative to their freshly prepared hydrogels. This indicates that the low temperature fluidity of the lyophilized powder hydrogel precursor solution is significantly improved.

Fig. 10 shows injection forces for various sets of lyophilized powder hydrogel precursor solutions in the examples of the present application. As can be seen from fig. 10, the injection force of the lyophilized powder hydrogel precursor solution was significantly reduced compared to the corresponding ready-made hydrogel. The injection force of the three lyophilized powder hydrogels is similar to that of physiological saline.

(6) Tissue adhesion of lyophilized powder hydrogel

The tissue adhesiveness of the hydrogel prepared was measured by a tissue retention method. 3 fresh pig stomachs were purchased from a slaughterhouse and rinsed with 0.1mol/L hydrochloric acid solution. Cutting pig stomach tissue (3cm × 6cm) of a certain area, and absorbing the tissue surface with filter paperWater is fixed on a self-made chute. Then slowly and uniformly pouring 2ml (V) from the upper edge of the stomach tissue of the pig ₁ ) A hydrogel solution. After 5min, use 10ml (V) ₂ ) Distilled water was used to flush the tissue at a rate of 1ml/s, the fluid was collected and the volume (V) was measured ₃ ). Finally, the percentage of hydrogel samples of each group adhering to the stomach tissue was calculated:

adhesion Rate [ (% V) ₁ +V ₂ -V ₃ )/(V ₁ +V ₂ )]×100％

Briefly, a certain area of pig stomach tissue is cut, filter paper is used for absorbing moisture on the surface of the tissue, and then a layer of pig stomach tissue with the height of 1mm and the area of 30 multiplied by 20mm is paved on the pig stomach tissue ² Another piece of tissue was gently pressed against the hydrogel surface and then placed in an incubator at 37 ℃ for 30 minutes to ensure complete gel formation. The tension meter was pulled away from one end of the upper tissue and the maximum force required to pull away was recorded. Three replicates of each hydrogel were tested.

All animal subjects were used according to the university of Sichuan "guidelines for care and use of laboratory animals" and ISO10993-2:2006 standard.

The tissue adherence of the hydrogel is characterized by measuring the amount of hydrogel adhering to the tissue surface after rinsing. Figure 11 shows the percent hydrogel residue that remained adhered to the tissue surface after rinsing for each set of lyophilized powder hydrogels in the examples of the present application. From the results in FIG. 11, it can be seen that all six groups of hydrogels had some tissue adhesion. The tissue adhesivity of the three prepared aquagels is slightly stronger than that of the corresponding freeze-dried powder aquagel.

Fig. 12 shows the adhesion of each set of lyophilized powder hydrogels to the tissue after gelation on the tissue surface in the examples of the present application. FIG. 12 shows the results of adhesion testing of hydrogels to tissue surfaces. The CS-GP hydrogel has no obvious difference with the CS-GP freeze-dried powder hydrogel in tissue adhesion force. Generally speaking, the tissue adhesion of CSLA/CS-GP and CSLA-GP hydrogels was slightly greater than that of their lyophilized powder hydrogels.

And 6: biological experiments

(1) Cytotoxicity test

CCK-8 experiment evaluation of hydrogels on mouse fibroblasts (L)929 cells). And (3) leaching the freeze-dried powder hydrogel in a DMEM high-sugar complete culture medium with the leaching concentration of 0.1g/mL for 24 hours, and diluting the leaching liquor to 50% and 25% in the complete culture medium. Cells were cultured in the extract at a cell concentration of L929 of 20000cells/mL for 24 hours, and then the culture was taken out and incubated with fresh serum-free medium (containing 10% CCK-8) for 3 hours (37 ℃, 5% CO) ₂ ). Finally, the absorbance was measured at 450nm with a microplate reader (Multiskan FC, USA).

FIG. 13 shows the relative proliferation rate of L929 cells after culturing L929 cells in each set of lyophilized powder hydrogel extract solution for 24h in the present application example. As shown in FIG. 13, the hydrogels tested in the six groups exhibited a relative proliferation rate of L929 cells of 70% or more even when the concentration of the extract solution was 100%. These groups of hydrogels were non-toxic according to GB/T16886.5-2017 (evaluation of medical device biology part 5: in vitro cytotoxicity test).

(2) Proliferation and morphology of hydrogel three-dimensionally encapsulated cells

Cells three-dimensionally encapsulated in hydrogel were stained with fluorescein diacetate/propidium iodide (FDA/PI) for viability and visualized with a laser confocal microscope (CLSM, Leica-TCS-SP5, Germany). Specifically, L929 cells were encapsulated in hydrogel at a concentration of 1X 106cells/mL, and soaked in DMEM high-sugar complete medium. The hydrogel encapsulating the cells was cultured in vitro for 1, 3 or 5 days. The gels were removed at different time points, washed 3 times with PBS, then soaked for 2min in PBS solution containing 5. mu.g/mL FDA and 5. mu.g/mL PI, and the viability and distribution of cells in the hydrogel were observed with a confocal laser microscope.

FIG. 14 shows the cell morphology of GES-1 cells in each group of lyophilized powder hydrogels in the examples of the present application. The activity of hydrogel three-dimensionally encapsulated L929 cells was investigated by FDA/PI staining. After 1, 3, and 5 days of culture, the cells were observed using a confocal laser microscope, and the results are shown in fig. 5. Viable cells were stained green by FDA and dead cells were stained red by PI. During the culture, most cells were stained green, and only a few cells were stained red. On the first day, cells were evenly distributed throughout the hydrogel. As the culture time is prolonged, cells proliferate and aggregate in the hydrogel, and the fluorescence intensity continuously increases. The lyophilized powder hydrogel and the corresponding in-situ prepared hydrogel (not lyophilized) show good cell compatibility.

(3) Ges-1 cell proliferation on hydrogel surface

GES-1 cells (human gastric epithelial cells) were used to study cell proliferation on the hydrogel surface. Cells were seeded on the surface of lyophilized powder hydrogel at a cell concentration of 20000cells/mL, and proliferation of cell growth was measured on

days

1, 3, and 5 using CCK-8.

FIG. 15 shows the proliferation of GES-1 cells on the surface of each group of lyophilized powder hydrogels in the examples of the present application. It can be seen that as the culture time was extended, the Optical Density (OD) increased, reflecting good cell proliferation on the hydrogel surface. The cell growth rate of the surface of the prepared hydrogel is superior to that of the surface of the freeze-dried powder gel.

(4) Protective effect of hydrogel on cells in acidic environment

The protective effect of the freeze-dried powder hydrogel on GES-1 cells in an acidic environment is researched by a Transwell culture mode. 10000 cells were seeded in the upper chamber of each well of a Transwell 12-well plate and 1ml of RPMI-1640 complete medium was added to the lower chamber. After the cells are attached to the wall, 400ul of freeze-dried powder hydrogel precursor solution is added to the upper chamber to cover the cell surface. After the formation of the hydrogel of the lyophilized powder, the complete medium is added and the pH is adjusted to 2 or 4 with hydrochloric acid. After 24h of culture, the cell proliferation rate was measured using CCK-8.

FIG. 16 shows the relative proliferation rate of GES-1 cells on the surface of each set of lyophilized powder hydrogels under acidic conditions in the examples of the present application. As can be seen from FIG. 16, under acidic conditions, the hydrogel has a significant protective effect on GES-1 cells. The cells covered with hydrogel grew significantly better than those without hydrogel, indicating that the six hydrogels had significant protective effects on cells at low pH.

(5) Adhesion of GES-1 cells to the gel surface

200. mu.L of the hydrogel precursor was added to a 24-well plate and gelled in a 37 ℃ incubator. 20000 GES-1 cells were seeded on the hydrogel surface and cultured for 12h to allow the cells to adhere. Then theWashing the hydrogel surface with 1ml/s flow rate of medium for 20s, sucking away the poorly adhered or non-adhered cells and medium, adding fresh serum-free medium containing 10% CCK-8, and incubating at 37 deg.C for 3h (37 deg.C, 5% CO) ₂ ). Finally, the absorbance was measured at 450nm with a microplate reader (Multiskan FC, USA). The cell adhesion of the hydrogel was evaluated based on the number of cells still adhering to the hydrogel surface.

FIG. 17 shows the adhesion capacity of GES-1 cells on the surface of each set of lyophilized powder hydrogels in the examples of the present application. FIG. 17 shows the adhesion capacity of GES-1 cells on the hydrogel surface. All six hydrogels showed some cell adhesion. The cell adhesion of the CS-GP hydrogel and the CS-GP freeze-dried powder hydrogel prepared in the prior art has no obvious difference, but the cell adhesion capability of the CSLA/CS-GP hydrogel and the CSLA-GP hydrogel is slightly larger than that of the corresponding freeze-dried powder hydrogel.

The data obtained from the above examples are expressed as mean ± Standard Deviation (SD) and analyzed using SPSS 11.0(SPSS, Chicago, IL, USA). Statistical significance was determined for significant differences between groups using analysis of variance (ANOVA). P <0.05 is statistically significant for differences between groups, and p <0.01 is very significant.

In a second aspect, an embodiment of the present invention provides an application of a lyophilized powder, in which the lyophilized powder provided in the present invention is used as a biomaterial for endoscopic mucosal dissection.

Based on the characterization and detection results in the above examples, the inventors concluded that:

endoscopic submucosal dissection is an effective method for early treatment of gastric cancer, but intraoperative and postoperative complications such as bleeding, perforation and the like limit the wide application of the endoscopic submucosal dissection. In recent years, new intraoperative biomaterials have been developed to reduce the incidence of these complications in ESD. Previous studies have demonstrated that chitosan temperature sensitive hydrogels can be used as intraoperative biomaterials for ESD. However, the poor long-term stability of such hydrogels, coupled with the complexity of extemporaneous preparation, presents challenges for large-scale production and application. In order to solve the problems, the research introduces lactose groups into chitosan molecular chains, and reconstructs injectable temperature-sensitive hydrogel based on chitosan hydrogel freeze-dried powder. We conducted intensive studies on the influence of the lyophilization process on the physicochemical and biological properties of hydrogels.

Based on the research of the above embodiments, the temperature-sensitive gelling property, gelling time, mechanical strength, injectability, tissue adhesion and other physicochemical properties of the chitosan temperature-sensitive hydrogel as a biomaterial in ESD surgery are of great importance. The lyophilized powder hydrogel precursor solutions evaluated in this study can form gels at 37 deg.C, depending on the measured rheological properties (FIG. 1). Researches show that amino and hydroxyl on a chitosan side chain play an important role in regulating and controlling the temperature-sensitive gelling property of the chitosan. Physical methods such as freeze drying and grinding have no great influence on amino on chitosan and hydroxyl on a side chain of chitosan, which is the reason why the freeze-dried powder hydrogel still can keep the temperature-sensitive property. Although the gel forming time of the CSLA/CS/GP freeze-dried powder hydrogel is longer than that of the corresponding existing prepared hydrogel, the gel forming time is within 5min (figure 4), and the time requirement of ESD minimally invasive surgery is met. The DMA results (figure 6) show that the mechanical strength of the lyophilized powder hydrogel is improved relative to the three currently formulated hydrogels, which may be related to the concentration of the lyophilized powder. Among the three lyophilized powder hydrogels, the hydrogel based on CSLA-GP gel powder has the best mechanical strength. This is probably because the introduction of lactose groups lengthens the chitosan molecular chain, increasing the number of H bonds, leading to the formation of H-bond networks. Partial flocculation appears after the prepared hydrogel precursor solution is stored for 10 days at 4 ℃, which reflects the poor long-term stable storage of the chitosan temperature-sensitive hydrogel to a certain extent. The results of low-temperature fluidity and injection force tests (fig. 9-10) show that the injection feasibility of the freeze-dried powder hydrogel is obviously improved compared with that of the corresponding existing hydrogel. In fact, the injection force is similar to that of physiological saline, which means that the hydrogel can be injected through an endoscopic spray tube. Furthermore, the CSLA-GP lyophilized powder hydrogel showed the best injectability (fig. 10), which is related to its CSLA content. According to FTIR Spectroscopy (FIG. 2), 1290cm ^-1 The peak at (a) corresponds to amide bond III, which increases in intensity with increasing CSLA concentration. The hydrogel lyophilized powder added with CSLA is more uniform and has regular needle-like structure(fig. 3), which may help to improve injectability. The lyophilized powder hydrogel also exhibited some tissue adhesion, although the tissue adhesion was slightly lower than that of the freshly prepared hydrogel (FIGS. 11-12). Other relevant properties of lyophilized powder hydrogels were also investigated. The final pH value of the gel system is not greatly influenced by the freeze-drying process, and the pH value of the freeze-dried powder hydrogel is close to neutral (figure 5). The in vitro degradation assay results (figure 7) show that the hydrogel can be maintained in acidic PBS solution with pepsin for several days. From the SEM image, the hydrogel of the lyophilized powder basically maintains the original porous network structure (FIG. 8). In conclusion, the CSLA/CS/GP freeze-dried powder hydrogel basically keeps the original good performance, and compared with the existing prepared hydrogel, the injectability and the mechanical strength of the hydrogel are improved.

Biological experiment results show that the CSLA/CS/GP freeze-dried powder hydrogel has no cytotoxicity, and shows good cell proliferation both in the hydrogel interior and on the hydrogel surface, thereby confirming that the hydrogels have good cell compatibility (FIGS. 13-15). More importantly, the lyophilized powder hydrogel had a significant protective effect on GES-1 cells in an acidic environment, and cells covered with hydrogel grew significantly better than those not covered (fig. 16). This is probably because the strength of the hydrogel increases, and the hydrogel hinders the flow of the acidic solution. In addition, the hydroxyl and amino groups in the hydrogel may ensure a less acidic environment around the cell. The CSLA/CS/GP lyophilized powder hydrogel also exhibits a certain cell adhesion, probably because the hydrogel has a porous network structure, provides a large number of cell attachment sites, and promotes the diffusion and further proliferation of cells. The experimental results prove that the freeze-dried powder provided by the invention can be stably stored, is convenient to use, meets most requirements of an ESD operation, and can be used as a biological material in the ESD operation.

The solid adhesive provided by the invention, the preparation method and the application thereof are described in detail, the principle and the implementation mode of the invention are explained by applying specific examples, and the description of the examples is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.

Claims

1. The freeze-dried powder is characterized by being chitosan-beta-sodium glycerophosphate freeze-dried powder, lactobionic acid modified chitosan-beta-sodium glycerophosphate freeze-dried powder or lactobionic acid modified chitosan/chitosan-beta-sodium glycerophosphate freeze-dried powder;

the freeze-dried powder is used for reconstructing injectable temperature-sensitive hydrogel used in endoscopic submucosal dissection, and comprises the following components: the freeze-dried powder is mixed with deionized water to form a freeze-dried powder hydrogel precursor solution, wherein the proportion of the deionized water to the freeze-dried powder is 1: 20-1: 100, respectively;

the freeze-dried powder hydrogel precursor solution can form hydrogel at 37 ℃, and the hydrogel has a porous network and a layered structure;

the chitosan-beta-sodium glycerophosphate freeze-dried powder is obtained by freeze drying a mixed solution consisting of a chitosan solution with the mass concentration of 2% and a beta-sodium glycerophosphate solution with the mass concentration of 6%;

the lactobionic acid modified chitosan-beta-sodium glycerophosphate freeze-dried powder is obtained by freeze drying a mixed solution consisting of a lactobionic acid modified chitosan solution with the mass concentration of 2% and a beta-sodium glycerophosphate solution with the mass concentration of 6%;

the lactobionic acid modified chitosan/chitosan-beta-sodium glycerophosphate freeze-dried powder is obtained by freeze-drying a mixed solution consisting of a lactobionic acid modified chitosan/chitosan solution with the mass concentration of 2% and a beta-sodium glycerophosphate solution with the mass concentration of 6%.

2. The lyophilized powder of claim 1, wherein when the lyophilized powder is chitosan- β -sodium glycerophosphate lyophilized powder, the method of preparing the lyophilized powder comprises:

step 1: dissolving chitosan in 1% acetic acid solution, stirring at room temperature to dissolve completely to obtain 3.33% solution A, and storing at 4 deg.C;

dissolving beta-sodium glycerophosphate in sodium bicarbonate and sodium carbonate buffer solution to obtain 0.15g/mL solution B;

wherein the sodium bicarbonate concentration is 0.1g/mL, the sodium carbonate concentration is 5mg/mL, and the sodium bicarbonate is stored at the temperature of 4 ℃;

3. Lyophilized powder according to claim 2, wherein in step 2, the first set of lyophilized powder precursor solutions is prepared by mixing 15ml of solution A with 10ml of solution B; in the first group of freeze-dried powder precursor solution, the mass concentration of the solution A is 2%, and the mass concentration of the solution B is 6%;

in the step 3, the pre-freezing conditions include: the temperature is-18 ℃ and the time is 8-10 h; the freeze-drying time in the freeze dryer was 48 h.

4. The lyophilized powder of claim 1, wherein when the lyophilized powder is lactobionic acid modified sodium chitosan- β -glycerophosphate lyophilized powder, the method of preparing the lyophilized powder comprises:

step 3': and (3) pre-freezing the second group of freeze-dried powder precursor solution prepared in the step 2', transferring to a freeze dryer for freeze drying, grinding and screening to obtain freeze-dried powder with uniform particles.

5. Lyophilized powder according to claim 4, wherein in step 2', the second set of lyophilized powder precursor solutions is prepared by mixing 15ml of solution C with 10ml of solution B; in the second group of freeze-dried powder precursor solution, the mass concentration of the solution C is 2%, and the mass concentration of the solution B is 6%;

in said step 3', said pre-freezing conditions comprise: the temperature is minus 18 ℃ and the time is 8-10 h; the freeze-drying time in the freeze dryer was 48 h.

6. The lyophilized powder of claim 1, wherein when the lyophilized powder is lactobionic acid modified chitosan/chitosan-beta-sodium glycerophosphate lyophilized powder, the method of preparing the lyophilized powder comprises:

step 1': dissolving chitosan in acetic acid solution with mass concentration, stirring at room temperature until the chitosan is completely dissolved to obtain solution A with mass concentration of 3.33%, and storing at 4 ℃; dissolving chitosan modified by lactobionic acid in deionized water to prepare a solution C with the mass concentration of 3.33%, and storing at 4 ℃; dissolving beta-sodium glycerophosphate in sodium bicarbonate and sodium carbonate buffer solution to obtain 0.15g/mL solution B; wherein the sodium bicarbonate concentration is 0.1g/mL, the sodium carbonate concentration is 5mg/mL, and the mixture is stored at 4 ℃;

step 2': mixing the solution A, the solution B and the solution C prepared in the step 1 '' under the condition of ice-water bath to prepare a third group of freeze-dried powder precursor solution;

step 3': pre-freezing the third group of lyophilized powder precursor solution prepared in the step 2 '', transferring to a lyophilizer for freeze-drying, grinding and sieving to obtain lyophilized powder.

7. Lyophilized powder according to claim 6, wherein in step 2 ", the third set of lyophilized powder precursor solutions is prepared by mixing 6ml of solution A, 9ml of solution C and 10ml of solution B; in the third group of freeze-dried powder precursor solution, the mass concentration of the solution A/the solution C is 2%, and the mass concentration of the solution B is 6%;

in said step 3 ", said pre-freezing conditions comprise: the temperature is-18 ℃ and the time is 8-10 h; the freeze-drying time in the freeze dryer was 48 h.

8. Use of a lyophilized powder according to any of claims 1-7 for the preparation of a medical device for endoscopic mucosal dissection.