CN113171464B - Graphene-reinforced hydrogel, graphene-reinforced hydrogel bacterial vector, and preparation method and application thereof - Google Patents

Graphene-reinforced hydrogel, graphene-reinforced hydrogel bacterial vector, and preparation method and application thereof Download PDF

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CN113171464B
CN113171464B CN202110416280.4A CN202110416280A CN113171464B CN 113171464 B CN113171464 B CN 113171464B CN 202110416280 A CN202110416280 A CN 202110416280A CN 113171464 B CN113171464 B CN 113171464B
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graphene
reinforced hydrogel
hydrogel
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刘梓瑞
王晶
张玉珊
李嫄渊
焦旸
曹建平
祁小飞
张琦
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Suzhou University
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Abstract

The invention provides a graphene-reinforced hydrogel, a graphene-reinforced hydrogel bacterial vector, a preparation method and an application thereof, and relates to the technical field of biomedical materials, wherein the graphene-reinforced hydrogel comprises the following components in percentage by mass: 0.1-5% of amphiphilic graphene, 0.25-3% of sodium lactate, 0.5-5% of calcium alginate and the balance of water. The graphene-reinforced hydrogel provided by the invention has excellent biocompatibility, excellent mechanical properties and water retention stability, can block the permeability of oxygen, prolongs the in-vitro preservation time of anaerobic flora, can be used as a carrier of bacterial drugs, and can effectively expand the application range of the hydrogel.

Description

Graphene-reinforced hydrogel, graphene-reinforced hydrogel bacterial vector, and preparation method and application thereof
Technical Field
The invention relates to the technical field of biomedical materials, in particular to a graphene-reinforced hydrogel, a graphene-reinforced hydrogel bacterial vector, a preparation method and an application.
Background
A hydrogel is a hydrophilic polymeric material with a network structure, with hydrophilic polymer chains forming a three-dimensional solid by crosslinking, and water serving as a dispersion medium. Hydrogel materials have excellent biocompatibility and ion transport capacity, so that the hydrogel materials have the potential of being widely applied to the medical fields of biomimetic materials, artificial tissues and the like.
Sodium alginate is a naturally occurring copolymer with which Ca can be reacted2+The gelling properties of such polyvalent metal ions are well known. It is a natural, low-cost, readily available polysaccharide with good biocompatibility, has been widely used in the food industry and has received extensive attention. The hydrogel system formed by crosslinking sodium alginate and divalent cations has the advantages of water absorption, water retention, slow release, body temperature resistance and the like. However, the application of sodium alginate in the field of biomedical materials is limited due to the poor mechanical and thermal properties of sodium alginate.
In view of the above, the invention is particularly provided.
Disclosure of Invention
One of the purposes of the invention is to provide a graphene reinforced hydrogel, so as to solve the technical problems that the mechanical property and the thermal property of a hydrogel system formed by crosslinking the existing sodium alginate and divalent cations are poor, and the application of the hydrogel system in the field of biological materials is limited.
The graphene reinforced hydrogel provided by the invention comprises the following components in percentage by mass: 0.1-5% of amphiphilic graphene, 0.25-3% of sodium lactate, 0.5-5% of calcium alginate and the balance of water.
Further, the amphiphilic graphene is mainly prepared by connecting a carboxyl-containing hydrophilic polymer to the surface of graphene oxide through diisocyanate;
preferably, the amphiphilic graphene is prepared according to the following steps:
modifying diisocyanate to the surface of graphene oxide, and then adding a carboxyl-containing hydrophilic polymer to enable the carboxyl of the carboxyl-containing hydrophilic polymer to react with the diisocyanate on the surface of the graphene oxide to obtain the amphiphilic graphene.
The carboxyl-containing polymer comprises at least one of polyacrylic acid, polyacrylic acid-polyethylene glycol block copolymer or polyacrylic acid-polyethylene glycol monomethyl ether block copolymer;
preferably, the molar mass of the polyacrylic acid is 2000-50000g/mol, preferably 4000-6000;
preferably, in the polyacrylic acid-polyethylene glycol block copolymer, the molar mass of the polyethylene glycol block is 5000-10000 g/mol and the molar mass of the polyacrylic acid block is 1000-5000-mol;
preferably, in the polyacrylic acid-polyethylene glycol monomethyl ether block copolymer, the molar mass of the polyethylene glycol monomethyl ether block is 2000-5000g/mol, and the molar mass of the polyacrylic acid block is 1000-10000 g/mol.
The invention also aims to provide a preparation method of the graphene reinforced hydrogel, which comprises the following steps:
adding the amphiphilic graphene into a sodium alginate solution, and then adding a calcium lactate solution to obtain the graphene reinforced hydrogel.
The invention also aims to provide application of the graphene reinforced hydrogel in preparation of bacteria, medicines or gene vectors.
The fourth purpose of the present invention is to provide a graphene-reinforced hydrogel bacterial vector, which comprises the graphene-reinforced hydrogel provided by one of the purposes of the present invention and bacteria, wherein the bacteria are loaded in the graphene-reinforced hydrogel.
Further, the bacteria include at least one of lactobacillus casei, lactobacillus acidophilus, lactobacillus, mucinous-Ackermansia type bacteria or coprophilous fungi;
preferably, the fecal bacteria is an intestinal fecal bacteria extract;
preferably, the activity of the bacterium in the graphene-enhanced hydrogel is 1-10U.
The fifth purpose of the invention is to provide a preparation method of the graphene reinforced hydrogel bacterial vector, which comprises the following steps:
adding the amphiphilic graphene into a sodium alginate solution, adding a bacteria solution, uniformly mixing, and finally adding a calcium lactate solution to obtain the graphene reinforced hydrogel bacteria carrier.
Further, inert gas is adopted for protection in the process of adding and mixing the bacterial solution;
preferably, the graphene-enhanced hydrogel bacterial vector is stored at 4 ℃.
The invention also aims to provide the application of the graphene-reinforced hydrogel bacterial vector in preparing a medicament for treating chronic diarrhea, depression or radioactive intestinal injury.
The invention provides a graphene reinforced hydrogel, which at least has the following beneficial effects:
(1) the biocompatibility is good, and the organism anaphylactic reaction is not easy to cause;
(2) the water-retaining agent has excellent mechanical property and water-retaining stability, and can effectively slow down the rate of water loss;
(3) can block the permeability of oxygen and prolong the in vitro preservation time of anaerobic flora;
(4) has controllable release characteristic, regulates and controls the release and metabolic behavior of the drug in vivo;
(5) can promote the stable establishment and balance of normal flora and relieve the symptoms of patients.
The graphene-reinforced hydrogel bacterial vector provided by the invention has excellent biocompatibility, can adjust the release and metabolism behaviors of flora in vivo and directionally release the flora to a controllable intestinal environment, is suitable for the preservation of normal anaerobic flora in intestinal tract due to the anoxic environment in the hydrogel, can block the permeability of oxygen and prolong the in vitro preservation time of the anaerobic flora, and meanwhile, the amphiphilic-reinforced hydrogel can promote the establishment and balance of the steady state of the normal flora and relieve the symptoms of patients.
Drawings
Figure 1 is a time-mass plot of hydrogels provided in comparative example 1 and example 5 and drug-loaded hydrogels provided in example 7 at 4 ℃ and room temperature;
figure 2 is a time-quality plot of hydrogels provided in comparative example 1 and example 5 and drug-loaded hydrogels provided in example 7;
fig. 3 is a graph of time versus cumulative release of curcumin from the drug-loaded graphene-enhanced hydrogels provided in examples 10-12;
FIG. 4 is a graph showing a diarrhea behavior of mice fed with the fecal bacteria-loaded graphene reinforced hydrogel feed provided in example 13 and with a general feed;
FIG. 5 is a graph of the bacterial effects in patients with chronic diarrhea who took the graphene enhanced hydrogel provided in example 5;
fig. 6 is a graph showing activity of bacteria in the hydrogel at different times in the graphene reinforced hydrogel loaded with lactobacillus acidophilus provided in example 14;
FIG. 7 is a graph showing the effect of administering the Lactobacillus casei-loaded graphene-reinforced hydrogel provided in example 15 and the effect of not administering the hydrogel on depressed mice.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, 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.
According to one aspect of the present invention, the present invention provides a graphene reinforced hydrogel, comprising the following components by mass:
0.1-5% of amphiphilic graphene, 0.25-3% of sodium lactate, 0.5-5% of calcium alginate and the balance of water.
In the invention, the amphiphilic graphene is abbreviated as amphiphilic reduced graphene oxide.
Typically, but not by way of limitation, in the graphene reinforced hydrogel provided by the present invention, the content of amphiphilic graphene is, for example, 0.1%, 0.2%, 0.3%, 0.5%, 0.8%, 1%, 1.5%, 2%, 3%, 4%, or 5%; sodium lactate content of 0.25%, 0.3%, 0.4%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5% or 3%; the content of calcium alginate is 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.
According to the graphene-reinforced hydrogel provided by the invention, the amphiphilic graphene, the sodium lactate and the calcium alginate are mutually cooperated by adding the amphiphilic graphene into the raw materials, so that the graphene-reinforced hydrogel has excellent mechanical properties and water retention stability, effectively slows down the water loss efficiency, can block the permeability of oxygen, prolongs the in-vitro preservation time of anaerobic flora, has excellent biocompatibility and controllable release characteristics, can regulate and control the release and metabolism behaviors in vivo, can promote the establishment and balance of normal flora, and can more effectively relieve the symptoms of patients.
In a preferred embodiment of the present invention, the amphiphilic graphene is mainly prepared by connecting a carboxyl-containing hydrophilic polymer to the surface of graphene oxide through diisocyanate.
Preferably, the amphiphilic graphene is prepared according to the following steps:
modifying diisocyanate to the surface of graphene oxide, and then adding a carboxyl-containing hydrophilic polymer to enable the carboxyl of the carboxyl-containing hydrophilic polymer to react with the diisocyanate on the surface of the graphene oxide to obtain the amphiphilic graphene.
Preferably, the diisocyanate is hexamethylene diisocyanate. Isocyanate at one end of diisocyanate reacts with carboxyl or hydroxyl on the surface of graphene oxide to be connected to the surface of graphene oxide.
Preferably, the polymer containing carboxyl is any one or a mixture of more than two of polyacrylic acid, polyacrylic acid-polyethylene glycol block copolymer or polyacrylic acid-polyethylene glycol monomethyl ether block copolymer. The carboxyl-containing polymer modifies graphene oxide with a plurality of carboxyl groups to functionalize the graphene oxide.
Preferably, the amphiphilic graphene prepared by using the polyacrylic acid with the molar mass of 2000-50000g/mol, preferably 4000-6000, and more preferably 5000 has more excellent hydrophilic performance.
Typically, but not limitatively, the polyacrylic acid has a molar mass of, for example, 2000, 3000, 4000, 5000, 8000, 10000, 20000, 30000, 40000 or 50000 g/mol.
Preferably, in the polyacrylic acid-polyethylene glycol block copolymer, the molar mass of the polyethylene glycol block is 5000-10000 g/mol and the molar mass of the polyacrylic acid block is 1000-5000-mol, so that the amphiphilic graphene prepared by the method has more excellent hydrophilicity.
Typically, but not by way of limitation, in the polyacrylic acid-polyethylene glycol block copolymer, the polyethylene glycol block has a molar mass of, for example, 2000, 2500, 3000, 3500, 4000, 4500, or 5000g/mol, and the polyacrylic acid block has a molar mass of, for example, 1000, 2000, 3000, 4000, 5000, 8000, or 10000 g/mol.
Preferably, in the polyacrylic acid-polyethylene glycol monomethyl ether block copolymer, the molar mass of the polyethylene glycol monomethyl ether block is 2000-5000g/mol, and the molar mass of the polyacrylic acid block is 1000-10000g/mol, so that the amphiphilic graphene prepared by the method has excellent hydrophilicity.
Typically, but not by way of limitation, in the polyacrylic acid-polyethylene glycol block copolymer, the polyethylene glycol monomethyl ether block has a molar mass of, for example, 2000, 2500, 3000, 3500, 4000, 4500, or 5000g/mol, and the polyacrylic acid block has a molar mass of, for example, 1000, 2000, 3000, 4000, 5000, 8000, or 10000 g/mol.
In a preferred embodiment of the present invention, in the preparation method of the graphene reinforced hydrogel, diisocyanate is modified on the surface of graphene oxide, and the reaction is performed at 80-160 ℃, and then a polymer containing carboxyl is added, and the reaction is performed at 80-160 ℃ to react the carboxyl of the polymer with the diisocyanate on the surface of the graphene oxide, so as to obtain the amphiphilic graphene.
Preferably, the mass ratio of the diisocyanate to the graphene oxide is 10:1, and the mass ratio of the graphene oxide to the carboxyl group-containing polymer is 1: 10.
According to a second aspect of the present invention, the present invention provides a method for preparing a graphene-reinforced hydrogel, comprising the following steps:
adding the amphiphilic graphene into a sodium alginate solution, then adding a calcium lactate solution, and carrying out a cross-linking reaction on the sodium alginate and calcium ions to obtain the graphene reinforced hydrogel.
According to a third aspect of the present invention, there is provided the use of a graphene-reinforced hydrogel in the preparation of a bacterial, pharmaceutical or genetic vector.
Preferably, the bacteria comprise fecal bacteria, which refers to intestinal fecal bacterial extracts.
The graphene-reinforced hydrogel provided by the invention can also be used for preparing a medicament for treating chronic diarrhea.
According to a fourth aspect of the present invention, there is provided a graphene reinforced hydrogel bacterial vector comprising a bacterium and the graphene reinforced hydrogel provided in the first aspect of the present invention, wherein the bacterium is supported on the graphene reinforced hydrogel.
The graphene-reinforced hydrogel bacterial carrier provided by the invention loads bacteria in the graphene-reinforced hydrogel, so that the graphene-reinforced hydrogel bacterial carrier has excellent biocompatibility, can adjust the release and metabolism behaviors of flora in vivo and directionally release the flora to a controllable intestinal environment, is suitable for the storage of normal anaerobic flora in intestinal tract, can block the permeability of oxygen and prolong the in-vitro storage time of the anaerobic flora, and meanwhile, the amphiphilicity-reinforced hydrogel can promote the establishment and balance of the steady state of the normal flora and relieve the symptoms of patients.
Preferably, the activity of the bacteria in the graphene-reinforced hydrogel is 1-10U.
Typically, but not by way of limitation, the activity of the bacteria in the graphene-enhanced hydrogel is, for example, 1, 2, 3, 5, 8, or 10U.
Preferably, the bacteria are selected from any one of lactobacillus casei, lactobacillus acidophilus, lactobacillus, muciniphilic-akkermansia or coprophila or a mixture of at least two flora.
Preferably, the fecal bacteria is an extract of human or mouse intestinal fecal bacteria.
Preferably, the graphene reinforced hydrogel is digested with EDTA (ethylene diamine tetraacetic acid) and the bacterial flora and the turbidity of the fecal bacteria are measured.
Preferably, 16rRNA is used to measure the gene expression level of bacteria, coprophila or coprophila extract in the graphene-enhanced hydrogel, and the abundance and content of bacteria in different species are compared.
According to a fifth aspect of the present invention, the present invention provides a preparation method of the above graphene-reinforced hydrogel bacterial vector, comprising the following steps:
adding the amphiphilic graphene into a sodium alginate solution, adding a bacteria solution, uniformly mixing, and finally adding a calcium lactate solution to obtain the graphene reinforced hydrogel bacteria carrier.
Preferably, inert gas is used for protection during the mixing process of adding the bacteria solution, so as to avoid the bacteria from being oxidized and deteriorated.
The graphene-reinforced hydrogel bacterial vector provided by the invention can be stored at 4 ℃ for 1-30 days, so that the in-vitro storage time of flora is effectively prolonged.
According to a sixth aspect of the present invention, there is provided the use of a graphene-reinforced hydrogel bacterial vector in the manufacture of a medicament for the treatment of chronic diarrhea, depression or radiation-induced bowel injury.
In order to facilitate the understanding of those skilled in the art, the technical solutions provided by the present invention will be further described below with reference to examples and comparative examples.
Example 1
The embodiment provides a graphene reinforced hydrogel, which is prepared according to the following steps:
dissolving 2.54g of sodium alginate and 0.1g of amphiphilic graphene in 80.96g of deionized water, dissolving 1.4g of calcium lactate in 15g of deionized water, and dripping the calcium lactate solution into the sodium alginate-amphiphilic graphene mixed solution to obtain the graphene reinforced hydrogel, wherein the mass ratio of the calcium alginate is 2.5%, the mass ratio of the sodium lactate is 1.5%, the mass ratio of the amphiphilic graphene is 0.1%, and the mass ratio of the water is 95.9%.
The amphiphilic graphene is prepared by the following steps:
mixing 10g of hexamethylene diisocyanate (sufficient excess) and 1g of graphene oxide in anhydrous N, N-Dimethylformamide (DMF), reacting at 80 ℃ for 12h, reacting isocyanate at one end of the hexamethylene diisocyanate with carboxyl or hydroxyl on the surface of the graphene oxide to be connected to the surface of the graphene oxide, washing the product with anhydrous DMF for several times to remove unreacted diisocyanate, adding 5g of polyacrylic acid (preferably with the number average molecular weight of 5000) into the reaction solution, and reacting at 80 ℃ for 12h to react carboxyl in the polyacrylic acid with isocyanate at the other end of the diisocyanate to obtain the amphiphilic reduced graphene oxide (referred to as amphiphilic graphene).
Example 2
The present example provides a graphene-reinforced hydrogel, which is different from example 1 in that 2.5g of amphiphilic graphene is added, and in the obtained graphene-reinforced hydrogel, the mass ratio of calcium alginate is 0.5%, the mass ratio of sodium lactate is 0.3%, the mass ratio of amphiphilic graphene is 2.5%, and the mass ratio of water is 96.7%.
Example 3
The present example provides a graphene-reinforced hydrogel, which is different from example 1 in that 5g of amphiphilic graphene is added, and in the obtained graphene-reinforced hydrogel, the mass ratio of calcium alginate is 0.5%, the mass ratio of sodium lactate is 0.3%, the mass ratio of amphiphilic graphene is 5%, and the mass ratio of water is 94.2%.
Example 4
The embodiment provides a graphene reinforced hydrogel which is prepared according to the following steps:
dissolving 5.08g of sodium alginate and 0.1g of amphiphilic graphene in 80g of deionized water, adding 2.79g of calcium lactate solution in 25g of deionized water, and dripping the calcium lactate solution into the sodium alginate-amphiphilic graphene mixed solution to obtain the graphene reinforced hydrogel, wherein the mass ratio of the calcium alginate is 5%, the mass ratio of the sodium lactate is 3%, the mass ratio of the amphiphilic graphene is 0.1%, and the mass ratio of the water is 91.9%.
Example 5
The present example provides a graphene-reinforced hydrogel, which is different from example 4 in that 2.5g of amphiphilic graphene is added, and in the obtained graphene-reinforced hydrogel, the mass ratio of calcium alginate is 5%, the mass ratio of sodium lactate is 3%, the mass ratio of amphiphilic graphene is 2.5%, and the mass ratio of water is 89.5%.
Example 6
The embodiment provides a graphene reinforced hydrogel, which is different from embodiment 4 in that 5g of amphiphilic graphene is added, and in the graphene reinforced hydrogel, the mass ratio of calcium alginate is 5%, the mass ratio of sodium lactate is 3%, the mass ratio of amphiphilic graphene is 5%, and the mass ratio of water is 87%.
Example 7
The embodiment provides a drug-loaded graphene reinforced hydrogel, which is prepared according to the following steps:
(1) carrying out ultrasonic treatment on 10mg of amphiphilic graphene in a water bath ultrasonic instrument for 30min to uniformly disperse the amphiphilic graphene; dissolving 80mg of curcumin in sufficient DMSO (dimethyl sulfoxide), dropwise adding the curcumin into the amphiphilic graphene aqueous solution subjected to ultrasonic dispersion, stirring overnight, centrifuging for 0.5h (r is 15000r/min), removing supernatant, rinsing, and drying at room temperature to obtain amphiphilic graphene-curcumin particles with the mass ratio of 1: 4;
(2) taking 0.5g of amphiphilic graphene-curcumin particles, adding 0.51g of sodium alginate, dissolving in 80g of deionized water, dissolving 0.28g of calcium lactate in 11.71g of deionized water, and dripping a calcium lactate solution into a mixed solution of graphene-curcumin and sodium alginate to obtain the curcumin-loaded graphene reinforced hydrogel, wherein the mass ratio of calcium alginate is 0.5%, the mass ratio of sodium lactate is 0.3%, the mass ratio of amphiphilic graphene is 0.1%, the mass ratio of curcumin is 0.4%, and the mass ratio of water is 98.7%.
Example 8
The embodiment provides a drug-loaded graphene reinforced hydrogel, which is prepared according to the following steps:
(1) carrying out ultrasonic treatment on 10mg of amphiphilic graphene in a water bath ultrasonic instrument for 30min to uniformly disperse the amphiphilic graphene; dissolving 40mg of curcumin in sufficient DMSO (dimethyl sulfoxide), dropwise adding the curcumin into the amphiphilic graphene aqueous solution subjected to ultrasonic dispersion, stirring overnight, centrifuging for 0.5h (r is 15000r/min), removing supernatant, rinsing, and drying at room temperature to obtain amphiphilic graphene-curcumin particles with the mass ratio of 1: 2;
(2) taking 3g of amphiphilic graphene-curcumin particles, adding 0.51g of sodium alginate, dissolving the sodium alginate in 80g of deionized water, dissolving 0.28g of calcium lactate in 16.21g of deionized water, and dripping a calcium lactate solution into a mixed solution of graphene-curcumin and sodium alginate to obtain the curcumin-loaded graphene reinforced hydrogel, wherein the mass ratio of calcium alginate is 0.5%, the mass ratio of sodium lactate is 0.3%, the mass ratio of amphiphilic graphene is 1%, the mass ratio of curcumin is 2%, and the mass ratio of water is 96.2%.
Example 9
The embodiment provides a drug-loaded graphene reinforced hydrogel, which is prepared according to the following steps:
(1) carrying out ultrasonic treatment on 10mg of amphiphilic graphene in a water bath ultrasonic instrument for 30min to uniformly disperse the amphiphilic graphene; dissolving 10mg of curcumin in sufficient DMSO (dimethyl sulfoxide), dropwise adding the curcumin into the amphiphilic graphene aqueous solution subjected to ultrasonic dispersion, stirring overnight, centrifuging for 0.5h (r is 15000r/min), removing supernatant, rinsing, and drying at room temperature to obtain amphiphilic graphene-curcumin particles with the mass ratio of 1: 0.5;
(2) taking 7.5g of amphiphilic graphene-curcumin particles, adding 5.08g of sodium alginate, dissolving in 75g of deionized water, dissolving 2.79g of calcium lactate in 9.63g of deionized water, and dripping a calcium lactate solution into a mixed solution of graphene curcumin and sodium alginate to obtain the curcumin-loaded graphene reinforced hydrogel, wherein the mass ratio of calcium alginate to sodium lactate is 5%, the mass ratio of amphiphilic graphene to curcumin is 5%, the mass ratio of curcumin to sodium alginate is 2.5%, and the mass ratio of water to water is 84.5%.
Example 10
The embodiment provides a drug-loaded graphene reinforced hydrogel, wherein the mass ratio of amphiphilic graphene is 1%, the mass ratio of curcumin is 2%, the mass ratio of calcium alginate is 2.5%, the mass ratio of sodium lactate is 1.5%, and the balance is water.
Example 11
The embodiment provides a drug-loaded graphene reinforced hydrogel, wherein the mass ratio of amphiphilic graphene is 3%, the mass ratio of curcumin is 2%, the mass ratio of calcium alginate is 2.5%, the mass ratio of sodium lactate is 1.5%, and the balance is water.
Example 12
The embodiment provides a drug-loaded graphene reinforced hydrogel, wherein the mass ratio of amphiphilic graphene is 5%, the mass ratio of curcumin is 2%, the mass ratio of calcium alginate is 2.5%, the mass ratio of sodium lactate is 1.5%, and the balance is water.
Example 13
The embodiment provides a coprophila-loaded graphene reinforced hydrogel, which is prepared according to the following steps:
(1) dissolving 5.08g of sodium alginate and 2.5g of amphiphilic graphene in 80g of deionized water to obtain a sodium alginate-amphiphilic graphene mixed solution;
(2) adding the excrement filtrate into the sodium alginate-amphiphilic graphene mixed solution, uniformly mixing, and blowing nitrogen for 5-60 minutes; wherein, the fecal filtrate is extracted from 10 normal mice feces and is prepared according to the following steps:
(s1) collecting feces by promoting defecation of normal control group mice by anus stimulation method at 8:00 in the morning, collecting 2 fresh-formed feces of each mouse, mixing 20 feces uniformly, and weighing 5g feces;
(s2) preparation of fecal suspension: dissolving mouse excrement in physiological saline according to the mass ratio of 1:10, and stirring in a stirrer to obtain excrement suspension;
(s3) filtration: filtering the excrement suspension by using double-layer sterile gauze, and collecting the filtered excrement suspension;
(s4) centrifugation: the filtered fecal suspension was centrifuged at 6000r/min for 20min (centrifugation radius 10 cm).
(s5) resuspension, namely centrifuging, then discarding the supernatant, suspending the precipitate in 50mL of normal saline, and turning upside down and mixing uniformly to obtain the fecal filtrate. The fecal filtrate was prepared for use the day.
(3) Dissolving 2.79g of calcium lactate in 25g of deionized water to obtain a calcium lactate solution, adding the calcium lactate solution into the mixed solution provided in the step (2), and uniformly mixing to obtain the coprophilous-bacterium-loaded graphene reinforced hydrogel.
Example 14
The embodiment provides a lactobacillus acidophilus-loaded graphene-reinforced hydrogel bacterial vector, which is prepared according to the following steps:
(1) dissolving 2.5g of amphiphilic graphene and 2.54g of sodium alginate in 80g of deionized water;
(2) adding 50 mu L of lactobacillus acidophilus liquid in logarithmic phase into the mixed solution of the amphiphilic graphene and the sodium alginate, and blowing nitrogen for 5-60 minutes;
(3) and (3) dissolving 1.4g of calcium lactate in 15g of deionized water, and dripping the calcium lactate solution into the mixed solution obtained in the step (2) to obtain the lactobacillus acidophilus-loaded graphene reinforced hydrogel.
Example 15
This example provides a cheese lactic acid bacteria-loaded graphene reinforced hydrogel, which is prepared according to the following steps:
(1) dissolving 2.5g of amphiphilic graphene and 2.54g of sodium alginate in 80g of deionized water;
(2) adding 50 mu L of cheese lactobacillus bacteria liquid in logarithmic phase into the mixed solution of the amphiphilic graphene and the sodium alginate, and blowing nitrogen for 5-60 minutes;
(3) and (3) dissolving 1.4g of calcium lactate in 15g of deionized water, and dripping the calcium lactate solution into the mixed solution obtained in the step (2) to obtain the cheese lactic acid bacteria-loaded graphene reinforced hydrogel.
Example 16
The embodiment provides a graphene-reinforced hydrogel bacterial vector loaded with mucinous-Ackermanella, which is prepared according to the following steps:
(1) dissolving 2.5g of amphiphilic graphene and 2.54g of sodium alginate in 80g of deionized water;
(2) adding 50 mu L of the mucinous-Ackermanella bacterium liquid in the logarithmic phase into the mixed solution of the amphiphilic graphene and the sodium alginate, and blowing nitrogen for 5-60 minutes;
(3) and (3) dissolving 1.4g of calcium lactate in 15g of deionized water, and dripping the calcium lactate solution into the mixed solution obtained in the step (2) to obtain the graphene reinforced hydrogel loaded with the mucinous-Ackermanella.
Comparative example 1
The comparative example provides a hydrogel prepared according to the following steps:
0.51g of sodium alginate is dissolved in 89.21g of deionized water, 0.28g of calcium lactate is dissolved in 10g of deionized water, and the calcium lactate solution is dripped into the sodium alginate solution to obtain the hydrogel, wherein the mass ratio of the calcium alginate is 0.5%, the mass ratio of the sodium lactate is 0.3%, and the mass ratio of the water is 99.2%.
Test example 1
The hydrogels provided in examples 1 to 9 and comparative example 1 were respectively subjected to tensile strength test using a universal material testing machine, and the prepared hydrogel sheets were cut into a standard dumbbell shape (dumbbell portion 2 mm. times.12 mm) by a cutter, and then subjected to mechanical test on the testing machine with the tensile rate set at 50 mm/min. Initial parameters of the hydrogel disks were measured by a vernier caliper. In order to prevent the water evaporation in the stretching process from influencing the mechanical property of the hydrogel, the surface of the hydrogel is coated with a layer of silicone oil. Each set of data was measured at least three times. The results are shown in table 1 below. As can be seen from the data, the tensile strength of the material gradually increases with the addition of the amphiphilic graphene.
TABLE 1 hydrogel tensile Strength Properties data sheet
Group of Tensile Strength (kPa)
Example 1 354±24
Example 2 361±33
Example 3 430±31
Example 4 423±35
Example 5 725±24
Example 6 1203±36
Example 7 292±32
Example 8 336±35
Example 9 1233±27
Comparative example 1 44±18
Test example 2
The hydrogels provided in comparative example 1 and example 5 and the drug-loaded hydrogels provided in example 7 were subjected to equilibrium water loss rate testing at 4 ℃ and room temperature (20 ℃), respectively, and the results are shown in fig. 1.
The specific test method comprises the following steps: starting with hydrochloric acid and pepsin to simulate an artificial gastric fluid environment at pH 1.35, the initial weight of the sample (m) was first recorded0) Placing in a sealed container, taking out every 2h, wiping with filter paper to record mass, reaching water loss balance when the mass is not changed, and measuring balance mass m1Equilibrium water loss rate (m)1/m0)*100％。
As can be seen from FIG. 1, in the environment of 4 ℃ or room temperature (20 ℃), the hydrogel water retention performance is obviously improved after the amphiphilic graphene or the amphiphilic graphene-curcumin are added, but the influence of drug loading on the hydrogel water retention performance is not great.
Test example 3
The hydrogels provided in comparative example 1 and example 5 and the drug-loaded hydrogels provided in example 7 were subjected to swelling performance tests, and the results are shown in fig. 2.
The swelling properties were carried out as follows:
hydrochloric acid and pepsin are used as raw materials to simulate an artificial gastric juice environment with the pH value of 1.35, the artificial gastric juice is used, the hydrogel provided in comparative example 1 and example 5 and the drug-loaded hydrogel provided in example 7 are respectively placed in a constant temperature shaking table (r is 200rad/min) environment at 37 ℃, the artificial gastric juice is taken out every 2 hours, the hydrogel and the drug-loaded hydrogel are weighed and recorded by an electronic balance after the samples are wiped by filter paper, and the swelling balance is achieved when the mass is not changed, as can be seen from fig. 2, the hydrogel swelling performance is obviously improved after the amphiphilic graphene or the amphiphilic graphene-curcumin is added, and the hydrogel swelling performance is not greatly influenced when the curcumin is not added.
Test example 4
The drug-loaded graphene-reinforced hydrogels provided in examples 10-12 were cut to about 2mm3The pieces were weighed to about 1g and placed in 10mL Erlenmeyer flasks, and 10mL of PBS was added to each flask. Placing the culture dish with hydrogel in a constant temperature shaking table (r is 150rad/min) at 37 ℃, taking out 3mL of curcumin release solution every 1 hour after placing, measuring the ultraviolet absorbance value, then continuing to measure the absorbance values of 24 hours, 48 hours and 72 hours, respectively, adding 3mL of fresh PBS into the original culture dish after each measurement, and the measurement result is shown in figure 3.
As can be seen from fig. 3, the drug-loaded graphene reinforced hydrogel provided in examples 10 to 12 can slowly release curcumin, and as the mass ratio of amphiphilic graphene increases, the cumulative release rate and release rate of curcumin also increase, which proves that the graphene reinforced hydrogel has a good drug-loading function.
Test example 5
10 mice with chronic diarrhea were randomly divided into a control group and an example group, each group was divided into 5 mice, the graphene reinforced hydrogel loaded with fecal bacteria provided in example 13 was fed to the mice in the experimental group, the mice in the control group were fed with a common feed, and the mice in the experimental group were fed with a feed containing the graphene reinforced hydrogel loaded with fecal bacteria provided in example 13, wherein the amount of the graphene reinforced hydrogel loaded with fecal bacteria added to the feed was 25% of the total mass of the feed, and the number of diarrhea in the mice in 6 weeks was continuously observed, and the results are shown in fig. 4.
As can be seen from the comparison of the experimental group and the control group in FIG. 4, the diarrhea of the mice in the experimental group is significantly improved.
Test example 6
10 healthy rats were selected and randomly divided into control groups and example groups, with 5 rats per group. The experimental group was administered the graphene-reinforced hydrogel provided in example 5 stored at 4 ℃ for 1 day. The control group was administered placebo with nausea, vomiting, abdominal pain as adverse outcome events. The number of occurrences of adverse outcome events within 3 days was investigated. 1 adverse outcome event occurs in the experimental group and the control group, and no significant difference exists, which indicates that the graphene reinforced hydrogel can be orally taken.
Test example 7
10g of each of the hydrogels provided in example 5 and comparative example 1 were taken, and the hydrogels provided in example 5 and comparative example 1 were taken out from the nitrogen atmosphere, exposed to air for 1 hour, and then soaked in 50mL ddH2And O, standing for 30min, and sealing. The dissolved oxygen content in water was measured in two groups separately by fluorescence spectrophotometry, and the results are shown in Table 2.
TABLE 2 hydrogel dissolved oxygen content data sheet
Dissolved oxygen content (mg)
Example 5 0.038
Comparative example 1 0.070
As can be seen from Table 2, the content of dissolved oxygen in the hydrogel water provided in example 5 was reduced by 54.3%.
Test example 8
8 patients with refractory diarrhea older than 50 years after informed consent were administered the graphene-reinforced hydrogel provided in example 5 three times a day, 5g each time. Taking a feces specimen every day three days before taking, taking a feces specimen every day after taking, storing at-80 ℃, and carrying out microbial flora analysis by 16SrRNA sequencing. Phyla and family levels were used to analyze changes in the composition of fecal bacteria, as shown in figure 5. As can be seen from FIG. 5, the number of harmful bacteria such as Escherichia coli in the intestinal tract of the patient decreased after administration, while the number of beneficial bacteria such as Lactobacillus acidophilus was relatively increased after administration, as compared to before administration. The graphene reinforced hydrogel can also generate a certain inhibition effect on harmful bacteria in the intestinal tract under the condition of not loading the bacteria, and has little influence on normal flora. After the graphene reinforced hydrogel is taken, clinical symptoms of 8 patients are remarkably improved (P is less than 0.01) compared with the prior clinical symptoms evaluated by doctors.
Test example 9
Taking 100g of lactobacillus acidophilus-loaded graphene reinforced hydrogel provided in example 14, 1d, 3d, 5d, 7d, 9d, 12d and 15d after the lactobacillus acidophilus-loaded graphene reinforced hydrogel is formed are dissolved in EDTA, and the turbidity and the gene expression level of lactobacillus acidophilus are measured, so as to judge the activity of lactobacillus acidophilus. The final activity was defined as 5U as the lowest limit, as shown in FIG. 6.
As can be seen from fig. 6, the activity of lactobacillus acidophilus was lower than 5U after 15d, indicating an optimal consumption period within 15 d.
Test example 10
14 patients over 50 years old suffering from chronic diarrhea were selected and randomly divided into an experimental group and a control group, each group consisting of 7 patients, the lactobacillus acidophilus-loaded graphene-reinforced hydrogel provided in example 14, which was stored at 4 ℃ for 1 day, was administered to the experimental group at a dose of 20mL, wherein the lactobacillus acidophilus-loaded graphene-reinforced hydrogel provided in example 14 was 1g/mL, and the lactobacillus acidophilus-loaded graphene-reinforced hydrogel provided in example 5, which was stored at 4 ℃ for 1 day, was administered to the control group as a placebo at a dose of 20mL, wherein the density of the graphene-reinforced hydrogel provided in example 5 was 1g/mL, the average number of stools per week before and after administration was used as statistical data, and the reduction of the number of stools was used as an effective criterion, and the results are shown in table 3.
TABLE 3 average stool number table for chronic diarrhea patients
Figure RE-GDA0003384354220000181
As can be seen from table 3, the lactobacillus acidophilus-loaded graphene reinforced hydrogel has a certain improvement effect on the symptoms of the elderly patients with chronic diarrhea.
Test example 11
The method comprises the steps of selecting 20 mice, establishing a mouse depression model by CUMS combined isolated culture, randomly dividing the mice into an intervention group and a control group, selecting 10 mice in each group, using the graphene reinforced hydrogel loaded with lactobacillus casei provided in the embodiment 15 as an intervener, intervening the mice in the intervention group, and not intervening the control group.
And observing the curiosity degree of the mouse to the new environment and the exploration capacity of the mouse in the new environment by adopting an open field experiment, and recording the activity condition of the mouse in a quiet environment within 5min, including the total distance and the activity rate. The results showed that the distance traveled by each mouse in the open box was significantly reduced after molding (P <0.05), while the results after intervention showed that the distance traveled by the intervention group was significantly increased compared to the control group (P <0.05), as shown in fig. 7. This demonstrates that lactobacillus casei loaded graphene reinforced hydrogel can improve symptoms in depressed mice.
Test example 12
Taking 30 adult mice, establishing a radioactive intestinal injury mouse model, randomly dividing the 30 adult mice into 3 groups, namely an experiment 1 group, an experiment 2 group and a control group, wherein the control group eats common feed, the experiment group 1 eats feed mixed with the graphene reinforced hydrogel provided by the embodiment 5, and the addition amount of the graphene reinforced hydrogel in the feed is 25% of the total mass. Experimental group 2 was fed with coprophil-loaded graphene-reinforced hydrogel provided in example 13, and the amount of coprophil-loaded graphene-reinforced hydrogel added to the feed was 25% of the total mass, and the inflammatory response of the intestinal tract of mice at weeks 3, 5, and 7 was recorded. The results are shown in Table 4.
TABLE 4
Figure RE-GDA0003384354220000191
As can be seen from Table 4, the inflammatory response of the intestinal tract was better and the recovery time was faster (P <0.05) in the experimental group 1 than in the control group, and the inflammatory response of the intestinal tract was better and the recovery time was faster (P <0.05) in the experimental group 2 than in the experimental group 1. This indicates that the graphene-reinforced hydrogel can improve the recovery ability of mice after radioactive intestinal injury, and the further recovery of mice is promoted by normal flora transplantation.
Test example 13
Taking 30 healthy adult rats, irradiating the rats by using 5Gy rays, establishing a radioactive intestinal injury model, randomly dividing the 30 adult rats into 3 groups, namely an experiment 1 group, an experiment 2 group and a control group, wherein the control group eats common feed, the experiment 1 group eats feed mixed with the graphene reinforced hydrogel loaded with lactobacillus acidophilus provided in the embodiment 14, and the addition amount of the graphene reinforced hydrogel loaded with lactobacillus acidophilus in the feed is 25% of the total mass. Experiment 2 groups consumed the mucin-akkermansia-loaded graphene-reinforced hydrogel provided in example 16, and the amount of the mucin-akkermansia-loaded graphene-reinforced hydrogel added to the feed was 25% of the total mass, and the number of defecation and fecal status in 7 days were recorded in 3 groups of rats, and blood was detected in the feces by using a fecal occult blood test card, and the results are shown in table 5.
TABLE 5
Figure RE-GDA0003384354220000192
Figure RE-GDA0003384354220000201
Note: (-) does not change into blue within 2 min; after 30-60s, the color turns blue; (++) is immediately blue-green; (+++) immediately turns dark green.
As can be seen from Table 5, compared with the control group, the rats in the experiment 1 group have obviously improved gastrointestinal tract state, and intestinal peristalsis and defecation times are increased; the intestinal absorption and metabolism effects of the rats in the experiment 2 group are obviously improved compared with those of the control group. The pathological histological examination shows that the recovery of rats in the experimental group 1 and the experimental group 2 after the damage of the intestinal mucosa epithelium is obviously improved compared with that in the control group (P < 0.05).
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (11)

1. The graphene reinforced hydrogel is characterized by comprising the following components in percentage by mass: 0.1-5% of amphiphilic graphene, 0.25-3% of sodium lactate, 0.5-5% of calcium alginate and the balance of water;
the amphiphilic graphene is prepared according to the following steps:
modifying diisocyanate to the surface of graphene oxide, and then adding a carboxyl-containing hydrophilic polymer to enable the carboxyl of the carboxyl-containing hydrophilic polymer to react with the diisocyanate on the surface of the graphene oxide to obtain amphiphilic graphene;
wherein the carboxyl-containing polymer is polyacrylic acid, and the molar mass of the polyacrylic acid is 4000-6000 g/mol.
2. The method for preparing the graphene-reinforced hydrogel according to claim 1, comprising the steps of:
adding the amphiphilic graphene into a sodium alginate solution, and then adding a calcium lactate solution to obtain the graphene reinforced hydrogel.
3. Use of the graphene-reinforced hydrogel according to claim 2 for the preparation of a bacterial, pharmaceutical or genetic vector.
4. A graphene-reinforced hydrogel bacterial vector comprising the graphene-reinforced hydrogel of claim 1 and a bacterium, wherein the bacterium is loaded into the graphene-reinforced hydrogel.
5. The graphene-reinforced hydrogel bacterial vector of claim 4, wherein the bacteria comprise at least one of Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus, Ackermansia muciniphila, or fecal bacteria.
6. The graphene-reinforced hydrogel bacterial vector of claim 5, wherein the fecal bacteria is an intestinal fecal bacterial extract.
7. The graphene-reinforced hydrogel bacterial vector according to claim 5, wherein the activity of the bacteria in the graphene-reinforced hydrogel is 1-10U.
8. The method for preparing the graphene-reinforced hydrogel bacterial vector according to any one of claims 4 to 7, comprising the following steps:
adding the amphiphilic graphene into a sodium alginate solution, adding a bacteria solution, uniformly mixing, and finally adding a calcium lactate solution to obtain the graphene reinforced hydrogel bacteria carrier.
9. The method for preparing the graphene-reinforced hydrogel bacterial vector as claimed in claim 8, wherein the inert gas is used for protection during the process of adding the bacterial solution and mixing.
10. The method for preparing a graphene-reinforced hydrogel bacterial vector according to claim 8,
the graphene-enhanced hydrogel bacterial vector is stored at 4 ℃.
11. Use of the graphene-reinforced hydrogel bacterial vector according to any one of claims 4 to 7 for the preparation of a medicament for the treatment of chronic diarrhea, depression or radiation-induced bowel injury.
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