CN110812529A

CN110812529A - Injectable hydrogel and preparation method thereof

Info

Publication number: CN110812529A
Application number: CN201910987307.8A
Authority: CN
Inventors: 易小玉
Original assignee: Individual
Current assignee: Hunan Meibai Biomedical Co ltd
Priority date: 2019-10-17
Filing date: 2019-10-17
Publication date: 2020-02-21
Anticipated expiration: 2039-10-17
Also published as: CN110812529B

Abstract

The invention discloses an injectable hydrogel and a preparation method thereof, wherein the preparation method comprises the following steps: sequentially carrying out cell disruption, primary proteolysis, cleaning, degreasing and decellularization on biological tissues to obtain a decellularized matrix, and freeze-drying the decellularized matrix for later use; carrying out second proteolysis on the freeze-dried acellular matrix to obtain 6-40mg/mL water-soluble extracellular matrix protein solution; crosslinking the extracellular matrix protein solution with 10-100mg/ml multi-arm modified polyethylene glycol containing succinimide group, and/or sulfhydryl group, and/or maleimide group, and/or amino group to obtain the injectable hydrogel. Injectable hydrogels are made by the above method. The preparation method provided by the invention is simple, and the prepared hydrogel not only has the biological inductivity of collagen, but also has the property of controllable degradation rate of a high polymer material.

Description

Injectable hydrogel and preparation method thereof

Technical Field

The invention belongs to the technical field of biomedical materials and tissue engineering, and particularly relates to an injectable hydrogel used as an in vivo biological scaffold and a preparation method thereof.

Background

Tissue engineering (tissue engineering) is an emerging discipline for the in vitro or in vivo construction of tissues or organs by the combination of cell biology and material science. Classical tissue engineering methods require the use of basic scaffolds of living cells and cell cultures to mimic and replace the natural structure of the tissue, while providing temporary functional support for the cells.

It is well known that there exists a complex extracellular matrix (ECM) among most mammalian cells, which is a macromolecule synthesized and secreted extracellularly by animal cells and distributed on the cell surface or between cells, and the composition of the ECM can be divided into three major groups, ① glycosaminoglycans (glycosaminoglycans), proteoglycans (proteoglycans), which are capable of forming an aqueous jelly in which many other matrix components are embedded, ② structural proteins, such as collagen and elastin, which impart strength and toughness to the extracellular matrix, ③ adhesion proteins (adhesin), also known as fibronectin, which promote cell homomatrix binding, ECM forms a fibroreticular complex on the cell surface using collagen and proteoglycans as the basic scaffold, which complex is directly linked to cell surface receptors via fibronectin or laminin and other linking molecules, or attached to receptors, forms a microenvironment for three-dimensional growth of cells, supports tissue architecture, regulates tissue generation and cell physiological activities, and connective tissue survival, and provides a number of connective tissue-embedding characteristics, which are important for the physical tissue-mediated by the extracellular matrix, and provides a balance of cell growth, such as well as cell organization, cell migration, cell morphology, cell differentiation, and cell morphology.

Therefore, ECM or ECM-derived collagen or the like is often used as a bioscaffold material in the field of medical regeneration, but in actual practice, when ECM protein is used as an injectable dosage form, it is often necessary to dissolve ECM protein in advance and then form a hydrogel by self-carrying functional groups on the collagen. However, during the dissolution process, the three-dimensional matrix cross-linked network of ECM proteins is destroyed, and various active enzyme components capable of degrading ECM protein components, such as matrix metalloprotease and cathepsin, are also present in the implant, and thus, pure extracellular matrix injected into the implant is rapidly degraded in a short time and its degradation rate is not controlled, so that its function as an in vivo bioscaffold is limited.

In order to overcome these limitations, a new method for manufacturing an internal vascular structure by using a synthetic material (e.g., polymer) having a solid free form has been developed, polyethylene glycol (PEG) is a polymeric compound with good biosolubility, and is widely used in the fields of medicine, food and industry, PEG has good hydrophilicity, and in the field of medicine, pegylation of proteins can increase the water solubility of antibodies or recombinant proteins, and simultaneously reduce the clearance rate of these protein drugs in blood, and a linear structure is synthesized.

A series of tissue engineering products based on collagen scaffolds are being developed, i.e. collagen-based biomaterials forming dispersions of fibers, films, sheets, sponges and fibrils, which can potentially be used as tissue engineering scaffolds for repairing or enhancing body tissues, but in many practical tissue engineering applications the structure and proteolytic susceptibility of reconstituted protein hydrogels in a bioscaffold system are still much less than those of natural tissues, besides the prior art methods for improving the physical properties of collagen gels based on covalent cross-linking are complicated to operate, require prior thiolation of natural proteins and also require uv irradiation or additional initiators and photoinitiators for induction during cross-linking.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, and provides an injectable hydrogel and a preparation method thereof.

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

a method of preparing an injectable hydrogel comprising the steps of:

sequentially carrying out cell disruption, primary proteolysis, cleaning, degreasing and decellularization on biological tissues to obtain a decellularized matrix, and freeze-drying the decellularized matrix for later use;

carrying out second proteolysis on the freeze-dried acellular matrix to obtain 6-40mg/mL water-soluble extracellular matrix protein solution;

and (2) crosslinking the extracellular matrix protein solution with 10-100mg/ml of multi-arm modified polyethylene glycol containing succinimide groups, sulfydryl groups, maleimide groups and/or amino groups to obtain the injectable hydrogel, wherein the molecular weight of the multi-arm modified polyethylene glycol is 10-30KD, and the number of the multi-arms is 3-8.

In one embodiment, the biological tissue is adipose tissue.

In one embodiment, the cell disruption is: the biological tissue is repeatedly frozen and thawed, and then is placed into hypertonic liquid for swelling and crushing.

In one embodiment, the number of times of freeze thawing is 3-6, and the swelling and crushing is to soak the mixture for more than 4 hours with 0.4-0.6mol/L NaCl and then soak the mixture for more than 4 hours with 0.9-1.1mol/L NaCl.

In one embodiment, the first proteolysis is a digestion with 0.24-0.26 wt% trypsin solution at 35-38 ℃ for 3-10 hours and the defatting treatment is a treatment with isopropanol for 12-24 hours.

In one embodiment, the decellularization treatment is carried out by treating with 0.9-1.1 wt% of sodium dodecyl sulfate for 2-24h, and then treating with 0.9-1.1 wt% of polyethylene glycol octyl phenyl ether for more than 48 h.

In one embodiment, the lyophilization is at a temperature of-80 deg.C, a pressure of 0.038-0.042mbar, and for a time of 36h or more.

In one embodiment, the second proteolysis is a proteolysis of 0.5-5mg/mL pepsin in an acidic environment for 12-24 hours, and then the pepsin is inactivated to obtain an extracellular matrix protein solution.

In one embodiment, the crosslinking is at a temperature of 25-38 ℃ for 20-60 min.

An injectable hydrogel prepared by the method.

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

aiming at the defects of the prior art, the multi-arm modified polyethylene glycol containing a succinimide group, and/or a sulfhydryl group, and/or a maleimide group, and/or an amino group is spontaneously combined with functional groups of an extracellular matrix protein, such as amino groups, to form a novel injectable three-dimensional hydrogel material containing a high molecular material and an extracellular matrix dissolving protein. The preparation method can re-crosslink the extracellular matrix soluble protein to form a three-dimensional matrix structure, and particularly obtains a tissue acellular matrix after acellular treatment is carried out on biological tissues such as fat and the like, then obtains the soluble extracellular matrix protein through enzymolysis on the extracellular matrix, and the extracellular matrix protein obtained through the operation mainly takes collagen as a main component and contains a large amount of cysteine (tissue specificity), so that a large amount of amino and sulfydryl can be provided to be further spontaneously combined with functional groups of multi-arm modified polyethylene glycol. The method not only enables the prepared polymer hydrogel to have the biological inductivity of collagen, but also has the property of controllable degradation rate of polymer materials.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic representation of the covalent cross-linking of ECM-solubilized collagen with PEG having a reactive functional group in examples 1-3 of the present invention;

FIG. 2 is a schematic representation of covalent cross-linking of ECM-solubilized collagen to PEG having various reactive functional groups in examples 4-6 of the present invention;

FIG. 3 is an infrared spectrum of each detection object in the experimental results of the present invention;

fig. 4 is a scanning electron microscope image of an injectable hydrogel provided in example 5 of the present invention;

FIG. 5 is a graph comparing the degradation rates of example 1 and example 5 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example 1

A method of preparing an injectable hydrogel comprising the steps of:

(1) taking adipose tissues for repeated freeze thawing for 3 times, then soaking in 0.4mol/L NaCl hypertonic liquid for 4h, and then soaking in 0.9mol/L NaCl for 4h to break fat cells; digesting the mixture for 3 hours at the temperature of 35 ℃ by using 0.24 wt% trypsin solution, washing the mixture for 3 hours by using deionized water, treating the washed mixture for 12 hours by using isopropanol to remove fat, then sequentially treating the washed mixture for 2 hours by using 0.9 wt% sodium dodecyl sulfate and treating the treated mixture for 48 hours by using 0.9% polyethylene glycol octyl phenyl ether to perform acellular treatment to obtain an acellular matrix, and freeze-drying the acellular matrix for 36 hours at the temperature of 80 ℃ below zero and under the condition of 0.038mbar for later use;

(2) carrying out enzymolysis on the freeze-dried acellular matrix for 12 hours in an acid environment by adopting 0.5mg/mL pepsin, and then inactivating the pepsin to obtain a 6mg/mL extracellular matrix protein solution for later use;

(3) and (2) carrying out crosslinking reaction on the extracellular matrix protein solution and 10mg/ml multi-arm modified polyethylene glycol (PEG-NHS) containing succinimide groups at the temperature of 25 ℃ for 20min, wherein the molecular weight of the multi-arm modified polyethylene glycol is 10KD, and the number of the multi-arms is 3, so as to obtain the injectable hydrogel.

Example 2

As shown in fig. 1, a method for preparing an injectable hydrogel includes the steps of:

(1) taking adipose tissues for repeated freeze thawing for 4 times, then soaking in 0.5mol/L NaCl hypertonic liquid for 4 hours, and then soaking in 1mol/L NaCl for 4 hours to break the fat cells; digesting for 6 hours at the temperature of 37 ℃ by using 0.25 t% trypsin solution, washing for 4 hours by using deionized water, treating for 18 hours by using isopropanol after washing to remove fat, then sequentially treating for 18 hours by using 1 wt% of sodium dodecyl sulfate and for 48 hours by using 1% of polyethylene glycol octyl phenyl ether to carry out acellular treatment to obtain an acellular matrix, and freeze-drying the acellular matrix for 36 hours at the temperature of 80 ℃ below zero and 0.04mbar for later use;

(2) carrying out enzymolysis on the freeze-dried acellular matrix for 18h in an acid environment by adopting 3mg/mL pepsin, and then inactivating the pepsin to obtain a 20mg/mL extracellular matrix protein solution for later use;

(3) and (2) carrying out crosslinking reaction on the extracellular matrix protein solution and 60mg/ml of multi-arm modified polyethylene glycol (PEG-SH) containing sulfhydryl groups at the temperature of 37 ℃ for 40min, wherein the molecular weight of the multi-arm modified polyethylene glycol is 20KD, and the number of the multi-arms is 6, so as to obtain the injectable hydrogel.

Example 3

A method of preparing an injectable hydrogel comprising the steps of:

(1) taking adipose tissues for repeated freeze thawing for 6 times, then soaking in 0.6mol/L NaCl hypertonic liquid for more than 4h, and then soaking in 1.1mol/L NaCl for 5h to break fat cells; then digesting the mixture for 10 hours at the temperature of 38 ℃ by using 0.26 wt% trypsin solution, washing the mixture for 4 hours by using deionized water, treating the washed mixture for 24 hours by using isopropanol to remove fat, then sequentially treating the washed mixture for 24 hours by using 1.1 wt% sodium dodecyl sulfate and treating the treated mixture for 50 hours by using 1.1% polyethylene glycol octyl phenyl ether to perform acellular treatment to obtain an acellular matrix, and freeze-drying the acellular matrix for 38 hours at the temperature of 80 ℃ below zero and under the condition of 0.042mbar for later use;

(2) carrying out enzymolysis on the freeze-dried acellular matrix for 24 hours in an acid environment by adopting 5mg/mL pepsin, and then inactivating the pepsin to obtain a 40mg/mL extracellular matrix protein solution for later use;

(3) crosslinking and reacting the extracellular matrix protein solution with 100mg/ml multi-arm modified polyethylene glycol (PEG-Mal) containing maleimide groups at 38 ℃ for 60min, wherein the molecular weight of the multi-arm modified polyethylene glycol is 30KD, and the number of the multi-arms is 8, so as to obtain the injectable hydrogel.

Example 4

A method of preparing an injectable hydrogel, different from example 2:

mixing the extracellular matrix protein solution with a solution containing succinimide group (NHS), sulfhydryl group (SH), amino group (NH)₂) And crosslinking the multi-arm modified polyethylene glycol with four groups of maleimide group (Mal) to obtain the injectable hydrogel.

Example 5

A method of preparing an injectable hydrogel, different from example 2:

contacting an extracellular matrix protein solution with a solution containing succinimide groups (NHS) and amino groups (NH)₂) Crosslinking reaction is carried out on the multi-arm modified polyethylene glycol of the two groups to obtain the injectable hydrogel.

Example 6

As shown in fig. 2, a method for preparing an injectable hydrogel, which is different from example 2, is:

carrying out crosslinking reaction on the extracellular matrix protein solution and multi-arm modified polyethylene glycol containing a sulfhydryl group (SH) and a maleimide group (Mal) to obtain injectable hydrogel;

the experimental results are as follows:

the comparison example, the example 2 and the example 6 are respectively tested by taking pure PEG high molecular material gel and collagen gel as comparison examples, and the example 1 is designated as the strategy one and the example 5 is designated as the strategy two, so that as shown in an infrared spectrogram in figure 3, compared with the pure PEG high molecular material gel or collagen gel in the comparison example, the strategy one and the strategy two form more obvious chemical bond combination at the positions indicated by arrows.

As shown in the sem of fig. 4, it can be seen that the polymer hydrogel of strategy two can form a distinct porous material structure. As shown in fig. 5, the injectable hydrogel synthesized by strategy one was found to degrade significantly to the liquid state while the injectable hydrogel synthesized by strategy two degraded more slowly when soaked for 24 hours in the presence of serum.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. A method of preparing an injectable hydrogel, comprising the steps of:

2. The method of claim 1, wherein the biological tissue is adipose tissue.

3. The method of claim 1 or 2, wherein the cell disruption is: the biological tissue is repeatedly frozen and thawed, and then is placed into hypertonic liquid for swelling and crushing.

4. The method according to claim 1 or 2, wherein the number of times of freeze thawing is 3 to 6, and the swelling and crushing is performed by soaking in 0.4 to 0.6mol/L NaCl for 4 hours or more, and then soaking in 0.9 to 1.1mol/L NaCl for 4 hours or more.

5. The method according to claim 1 or 2, wherein the first proteolysis is a digestion with a 0.24-0.26 wt% trypsin solution at 35-38 ℃ for 3-10 hours, and the defatting treatment is a treatment with isopropanol for 12-24 hours.

6. The method according to claim 1 or 2, wherein the decellularization is carried out by treating with 0.9-1.1 wt% sodium dodecyl sulfate for 2-24h, and then treating with 0.9-1.1 wt% octyl phenyl ether of polyethylene glycol for more than 48 h.

7. The method of claim 1 or 2, wherein the lyophilization is at a temperature of-80 ℃, a pressure of 0.038 to 0.042mbar, and a time of 36h or more.

8. The preparation method according to claim 1 or 2, wherein the second proteolysis is a proteolysis with pepsin of 0.5-5mg/mL for 12-24h in an acidic environment, and then the pepsin is inactivated to obtain an extracellular matrix protein solution.

9. The method of claim 1 or 2, wherein the crosslinking is carried out at a temperature of 25 to 38 ℃ for 20 to 60 min.

10. An injectable hydrogel produced by the production method according to any one of claims 1 to 9.