CN113754829A - Dendrimer/modified gelatin composite microgel, preparation method and application thereof - Google Patents
Dendrimer/modified gelatin composite microgel, preparation method and application thereof Download PDFInfo
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- CN113754829A CN113754829A CN202110674703.2A CN202110674703A CN113754829A CN 113754829 A CN113754829 A CN 113754829A CN 202110674703 A CN202110674703 A CN 202110674703A CN 113754829 A CN113754829 A CN 113754829A
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- C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
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- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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Abstract
The invention discloses a dendrimer/modified gelatin composite microgel, a preparation method and application thereof. Based on the capability of effective envelope and environmental shielding of guest molecules provided by the unique topological structure of the dendriform alkoxy ether element, the composite microgel can be used as an intelligent carrier of active biomolecules to realize the effective envelope of the guest molecules and can maintain higher catalytic activity under an acidic condition or a high-temperature environment. In addition, the invention combines the biological characteristics of natural macromolecules, and the composite microgel can be used as a cell culture matrix material to promote the adhesion and proliferation of cells. Based on the excellent properties of the composite microgel, the composite microgel has very wide application prospect in the fields of biological medicine, cell culture and tissue engineering.
Description
Technical Field
The invention belongs to the field of biomedical materials and intelligent composite materials, and particularly relates to a dendrimer/modified gelatin composite gel, a preparation method thereof and application thereof in enzyme activity protection.
Background
The protease is a substance with high activity, strong specificity, low toxicity and definite biological function, and has important application value, for example, as a medicament, the protease can be endowed with high-efficiency anti-tumor and anti-virus functions; as an antibody, can act as a vaccine against infection; the catalyst can realize high-efficiency, mild and green key chemical reaction. The function of protein depends on its biological activity, but the protein is very sensitive to the environment, such as sensitive to the environmental parameters of pH, temperature, ion species and concentration, etc., and can easily lose activity under uncomfortable conditions. The activity of the protein can be irreversibly damaged during the transportation, storage and even use processes, so that the use effect is reduced, and the use cost is increased. Especially as pharmaceuticals and vaccines, also pose an unpredictable risk. It is therefore important to provide a suitable carrier for proteins to overcome the impairment of their biological activity by adverse environmental factors.
Microgels stand out in a wide variety of protein carriers due to their good biocompatibility and biodegradability, as well as flexibility in size, morphology and surface function. The protease is limited in the three-dimensional network of the microgel, so that the stability of the protease structure is favorably maintained. The dendronized polymer is a highly branched polymer and is constructed by taking a linear polymer as a main chain and taking a high-density tree-shaped element as a side group. They have several characteristics different from the conventional linear polymers, including monomolecular large scale, high rigidity, and multidentate cooperativity. More importantly, the alkoxy ether branched polymer has excellent temperature-sensitive behavior, and can realize effective envelope on guest molecules through the synergistic effect given by high-density stacking of the tree-shaped side chains, so that a limited microenvironment different from the outside is provided for the guest molecules. This property will bring new properties to the microgel. The alkoxy ether element with temperature responsiveness is combined with the natural polymer gelatin, so that the composite microgel with intelligent characteristics and biological functions can be prepared, and the composite microgel has wide application prospect in the fields of biological medicines and the like.
The microgel is prepared in a controllable way, and has important significance for the development of the performance and the application expansion. Conventional methods for preparing microgel include emulsion polymerization, precipitation polymerization, spray drying, and the like. These methods are suitable for the preparation of microgels in submicron size for industrial applications, but still have certain challenges in precisely controlling the size, composition and characteristics of the microgels prepared.
Disclosure of Invention
In order to solve the problems of the prior art, the invention aims to overcome the defects in the prior art and provide a tree polymer/modified gelatin composite microgel, a preparation method and application thereof. The intelligent composite microgel of the invention shows excellent temperature-sensitive performance and good biocompatibility and biodegradability, can be used as a protease carrier, realizes the high-efficiency protection of protease activity, and can also be used as a cell culture matrix material.
In order to achieve the purpose, the invention adopts the following technical scheme:
a composite microgel of tree-shaped polymer/modified gelatin is prepared from the tree-shaped alkoxy ether element with temp-sensitive performance and the gelatin with biocompatibility and biodegradability through dripping and microflow control.
Preferably, the composite microgel with different components and different sizes is obtained by regulating and controlling the feed ratio of the dendritic alkoxy ether to the modified gelatin and the flow rate of the dispersed phase and the continuous phase.
Preferably, the dendrimer/modified gelatin composite microgel of the present invention has an average diameter of 115 μm.
The preparation method of the dendrimer/modified gelatin composite microgel comprises the following steps:
a. mixing a dendritic alkoxy ether monomer and modified gelatin according to the ratio of (5-1): 1, dissolving in water, adding a photoinitiator which is not less than 1 wt% of the mixed solution to obtain an aqueous phase solution with the total solid content of not less than 10 wt% as a disperse phase, and taking HFE7500 containing not less than 1 wt% of a surfactant as a continuous phase;
b. by means of the liquid drop micro flow control method, the dispersed phase forms micro liquid drops with uniform size under the high speed shearing action of the continuous phase, and the micro liquid drops are irradiated by ultraviolet light in the collecting pipe to generate cross-linking polymerization reaction to form the polymer/modified gelatin composite microgel.
Preferably, in step a, the adopted dendritic alkoxy ether monomer has the structural formula:
wherein, X is methoxy, ethoxy or hydroxyl; n is 1 to 5.
Preferably, in the step b, the flow rate ratio of the dispersed phase to the continuous phase is 1/10-1/5; the light source wavelength of the selected ultraviolet light is 254-415 nm.
Preferably, in the step b, the flow rate of the dispersed phase is set to be not higher than 40 μ L/h and the flow rate of the continuous phase is set to be not higher than 300 μ L/h by a peristaltic pump, and the polymer/modified gelatin composite microgel is prepared by a droplet microfluidic method.
The invention relates to an application of a dendrimer/modified gelatin composite microgel, which utilizes a mechanism that a limited microenvironment constructed by the dendrimer/modified gelatin composite microgel has enveloping and shielding effects on guest molecules and is used for a biological macromolecular activity protection material.
Preferably, the biomacromolecule comprises at least one of a protein, a nucleic acid, and a polysaccharide.
Preferably, the dendrimer/modified gelatin composite microgel is used as a drug carrier material or a cell culture matrix material.
Preferably, the dendrimer/modified gelatin composite microgel can promote cell adhesion and proliferation when being used as a cell culture matrix material.
Compared with the prior art, the invention has the following obvious and prominent substantive characteristics and remarkable advantages:
1. the invention combines the temperature-responsive dendriform alkoxy ether and the natural gelatin with biocompatibility and biodegradability into a whole, and prepares an intelligent composite microgel with uniform size by a droplet microfluidic technology; the microgel not only shows good temperature-sensitive performance, but also has good biocompatibility and biodegradability, can be used as an intelligent carrier of enzyme protein or nucleic acid bioactive molecules to realize high-efficiency protection of object molecules, and can also be used as a cell culture matrix material to promote adhesion and proliferation of cells;
2. the novel intelligent composite microgel obtained by the method can be used as a protein carrier material and a cell culture matrix material; when the complex is used as a protein carrier material, the complex can carry out effective envelope and activity protection on guest molecules; when used as a cell culture matrix material, the material can promote cell adhesion and proliferation;
3. the dendrimer/modified gelatin composite microgel provided by the invention combines the dendrimer alkoxy ether with excellent temperature-sensitive property with the modified gelatin with good biocompatibility and biodegradability, and a novel intelligent composite microgel with the characteristics of both is prepared by a droplet microfluidic technology, has the properties of controllable components, structure and size, and has the phenomenon of reversible shrinkage of surface phase temperature induced volume in an aqueous solution; in addition, the limited microenvironment constructed by the composite microgel can provide effective enveloping and shielding effects for guest molecules, and is expected to be applied to the field of biological medicines.
Drawings
FIG. 1 is an optical microscopic view and a particle size distribution of a composite microgel prepared in example two of the present invention.
FIG. 2 is a graph comparing temperature-sensitive properties of composite microgel according to a preferred embodiment of the present invention. Wherein FIG. 2a) is an optical microscope photograph of the microgel compounded at different temperatures in the first, second and third examples; FIG. 2b) is a graph showing the change in volume of the compounded microgel at different temperatures in the first, second and third examples; FIG. 2c) is a graph comparing the dimensional change of the composite microgel of example two during the passage of a plurality of heating/cooling cycles.
FIG. 3 is a graph comparing the protection of the microgel prepared in example two of the present invention against horseradish peroxidase (HRP) under different pH conditions.
FIG. 4 is a graph comparing the protection of the microgel prepared in example two of the present invention against HRP under different temperature conditions.
FIG. 5 is a confocal microscope photograph of the microgel prepared in example two of the present invention after 24h co-incubation with cells and double-staining by AO-EB.
FIG. 6 is a confocal image of microgel prepared in the third example of the invention after being treated with collagenase for different times.
FIG. 7 is a schematic diagram of a dendrimer/modified gelatin composite microgel and a preparation process thereof according to the present invention.
Detailed Description
The following examples combine temperature-responsive dendritic alkoxy ether with biocompatible and biodegradable natural gelatin to prepare an intelligent composite microgel with uniform size by a droplet microfluidic technology. The microgel not only shows good temperature-sensitive performance, but also has good biocompatibility and biodegradability, can be used as an intelligent carrier of bioactive molecules, realizes high-efficiency protection of object molecules, and can be used as a cell culture matrix material to promote adhesion and proliferation of cells.
Referring to fig. 7, the following examples illustrate a method for preparing an intelligent composite microgel, comprising the steps of:
a. dissolving a dendriform alkoxy ether monomer, modified gelatin and a photoinitiator in water according to different proportions to obtain an aqueous phase solution with the total solid content of 10 wt% as a dispersion phase;
b. HFE7500 containing a surfactant as a continuous phase;
c. by adopting a droplet microfluidic technology, the dispersed phase forms micro droplets with uniform size under the high-speed shearing action of the continuous phase;
d. the micro-droplets are subjected to cross-linking polymerization reaction in the collecting pipe by ultraviolet irradiation to form microgel.
The above-described scheme is further illustrated below with reference to specific embodiments, which are detailed below:
the first embodiment is as follows:
in this example, a branched polymer/modified gelatin composite microgel D5G1The preparation method comprises the following steps:
30MG of monomer MG and 6MG of modified gelatin GMA were dissolved in water at different mass ratios, and 4MG of photoinitiator 2959 was added to prepare an aqueous solution having a total solid content of 10 wt% as a dispersed phase. As the continuous phase, a fluorinated oil HFE7500 containing 1% by weight of surfactant was used. The flow rate of the dispersed phase was set to 40. mu.L/h and the flow rate of the continuous phase was set to 300. mu.L/h by means of a peristaltic pump. Preparation of composite microgel D by droplet microfluidic method5G1。
Example two:
this embodiment is substantially the same as the first embodiment, and is characterized in that:
in this example, a branched polymer/modified gelatin composite microgel D3G1The preparation method comprises the following steps:
27MG of monomer MG1 and 9MGThe gelatin GMA was dissolved in water at different mass ratios and 4mg of photoinitiator 2959 was added to prepare an aqueous solution with a total solid content of 10 wt% as a dispersed phase. As the continuous phase, a fluorinated oil HFE7500 containing 1% by weight of surfactant was used. The flow rate of the dispersed phase was set to 40. mu.L/h and the flow rate of the continuous phase was set to 300. mu.L/h by means of a peristaltic pump. Preparation of composite microgel D by droplet microfluidic method3G1。
Example three:
this embodiment is substantially the same as the previous embodiment, and is characterized in that:
in this example, a branched polymer/modified gelatin composite microgel D1G1The preparation method comprises the following steps:
18MG of monomer MG1 and 18MG of modified gelatin GMA were dissolved in water at different mass ratios, and 4MG of photoinitiator 2959 was added to prepare an aqueous solution having a total solid content of 10 wt% as a dispersed phase. As the continuous phase, a fluorinated oil HFE7500 containing 1% by weight of surfactant was used. The flow rate of the dispersed phase was set to 40. mu.L/h and the flow rate of the continuous phase was set to 300. mu.L/h by means of a peristaltic pump. Preparation of composite microgel D by droplet microfluidic technology1G1。
Example four:
this embodiment is substantially the same as the previous embodiment, and is characterized in that:
in this example, the microgel sample prepared in example two was observed by an optical microscope.
mu.L of the microgel taken from the continuous phase was placed on a glass slide, the morphology thereof was observed by means of an optical microscope, and the size of the microgel was statistically analyzed by means of a software Digimizer (image measuring software). As shown in FIG. 1, the composite microgel has a narrow size distribution and an average diameter of about 115 μm.
Example five:
this embodiment is substantially the same as the previous embodiment, and is characterized in that:
in this example, the temperature-sensitive properties of the microgels prepared in examples one, two and three were measured using an optical microscope equipped with a heating stage apparatus:
taking a proper amount of microgel on a single-concave glass slide, carrying out mounting treatment by using a cover glass, observing the appearance of the microgel under the room temperature condition under a microscope, heating by using a temperature control table, and setting the heating rate to be 5 ℃ per minute-1And maintaining for 10min after reaching the target temperature, respectively observing the size change of the microgel at 25 ℃, 37 ℃, 40 ℃ and 50 ℃, and taking the microgel GMA without alkoxy ether as a control. And observing the recovery capability of the microgel in the continuous heating and cooling processes. The test result is shown in figure 2, and the composite microgel has excellent temperature-sensitive performance.
Example six:
this embodiment is substantially the same as the previous embodiment, and is characterized in that:
in this example, the microgel prepared in example two was tested for its ability to protect HRP at different pH conditions:
phosphate buffers of different pH, pH 3.0-pH7.0, were prepared with 50mM disodium hydrogen phosphate and sodium dihydrogen phosphate solutions. 5.0mg of microgel is uniformly dispersed into 900 mu L of phosphate solution, and 100 mu L of 10 mu g/mL microgel is added-1Placing the HRP solution in a shaking table at room temperature, shaking and standing for 24h, detecting the enzyme catalytic activity of the HRP, and taking free HRP as a control group. The protective effect of the microgel on HRP under different pH conditions is shown in figure 3, and the microgel has a better protective effect on HRP in a phosphate solution with pH of 3.5-7.0. The microgel lowers the optimum pH of HRP from pH 5.0 to pH 4.0.
Example seven:
this embodiment is substantially the same as the previous embodiment, and is characterized in that:
in this example, the microgel prepared in example two was tested for its ability to protect HRP under different temperature conditions:
phosphate buffer pH4.0 was prepared with 50mM disodium hydrogen phosphate and sodium dihydrogen phosphate solutions. 5.0mg of microgel is uniformly dispersed into 900 mu L of phosphate solution, and 100 mu L of 10 mu g/mL microgel is added-1Placing the HRP solution at 40 ℃ and 65 ℃ for 12h and 30min respectively, detecting the enzyme catalytic activity of the HRP, andfree HRP was used as a control. The protection effect of the microgel on HRP under different temperature conditions is shown in figure 4, after high-temperature treatment, the activity of free HRP is almost completely lost, and the HRP in the microgel still maintains higher catalytic activity. The composite microgel can effectively envelop HRP and provide thermal protection
Example eight:
this embodiment is substantially the same as the previous embodiment, and is characterized in that:
in this example, the microgel prepared in example two was tested for biocompatibility:
50. mu.L of 10 mg. multidot.mL was added to each well of a 96-well plate -1200. mu.L of a cell (PC-12) solution was added to each well, and the seeding density of the cells was 4X 104 cells. multidot.mL-1. The microgel is suspended in cell culture fluid prior to use. After 24h, detecting cell viability by using acridine orange/ethidium bromide (AO-EB) dye, adding 10 mu L of AO-EB solution into each hole, standing for 2min, transferring the microgel onto a glass slide, observing the adhesion state and cell viability of cells on the surface of the microgel under a laser confocal microscope, wherein live cells emit green fluorescence, dead cells are red, the excitation wavelengths are 488nm and 561nm respectively, and meanwhile, the microgel GMA is used as a control. As shown in FIG. 5, after 24h, the microgel adhered to the surface and survived about 100%. The composite microgel has good biological property and can be used as a cell culture matrix.
Example nine:
this embodiment is substantially the same as the previous embodiment, and is characterized in that:
in this example, the microgel prepared in example three was tested for biodegradability:
0.1 mg/mL of PBS solution was prepared-1The collagenase solution of (1) is prepared by placing 10 mu L of microgel loaded with FITC-HRP into 200 mu L of collagenase solution, and monitoring the degradation process of the microgel in real time by using a laser confocal microscope. The result is shown in figure six, the composite microgel can be completely degraded within 50 min.
In summary, the dendrimer/modified gelatin composite microgel of the embodiment combines the temperature-responsive dendrimer alkoxy ether polymer and the biocompatible and biodegradable modified gelatin into a whole, and the synthetic macromolecule/natural macromolecule intelligent composite microgel with uniform size is prepared by the droplet microfluidic technology. Based on the capability of effective envelope and environmental shielding of guest molecules provided by the unique topological structure of the dendriform alkoxy ether element, the composite microgel can be used as an intelligent carrier of active biomolecules to realize the effective envelope of the guest molecules (enzymes), and can maintain higher catalytic activity under an acidic condition or a high-temperature environment. Especially, the high catalytic activity is maintained at pH 3.5-7.0 and 40-65 ℃. In addition, the composite microgel can be used as a cell culture matrix material to promote the adhesion and proliferation of cells by combining the biological characteristics of natural macromolecules. Based on the excellent properties of the composite microgel, the composite microgel has very wide application prospect in the fields of biological medicine, cell culture and tissue engineering.
The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made according to the purpose of the invention, and any changes, modifications, substitutions, combinations or simplifications made according to the spirit and principle of the technical solution of the present invention should be replaced with equivalents as long as the object of the present invention is met, and the technical principle and the inventive concept of the present invention are not departed from the scope of the present invention.
Claims (10)
1. A tree polymer/modified gelatin composite microgel is characterized in that: the dendritic alkoxy ether element with temperature-sensitive property and gelatin with biocompatibility and biodegradability are combined, and a droplet microflow control method is utilized to prepare the synthetic polymer/natural polymer intelligent composite microgel with uniform size.
2. The dendrimer/modified gelatin composite microgel of claim 1, wherein: the composite microgel with different components and different sizes is obtained by regulating and controlling the feed ratio of the dendritic alkoxy ether to the modified gelatin and the flow rate of the dispersed phase and the continuous phase.
3. The dendrimer/modified gelatin composite microgel of claim 1, wherein: the average diameter was 115. mu.m.
4. A method for preparing the dendrimer/modified gelatin composite microgel of claim 1, comprising the steps of:
a. mixing a dendritic alkoxy ether monomer and modified gelatin according to the ratio of (5-1): 1, dissolving in water, adding a photoinitiator which is not less than 1 wt% of the mixed solution to obtain an aqueous phase solution with the total solid content of not less than 10 wt% as a disperse phase, and taking HFE7500 containing not less than 1 wt% of a surfactant as a continuous phase;
b. by means of the liquid drop micro flow control method, the dispersed phase forms micro liquid drops with uniform size under the high speed shearing action of the continuous phase, and the micro liquid drops are irradiated by ultraviolet light in the collecting pipe to generate cross-linking polymerization reaction to form the polymer/modified gelatin composite microgel.
5. The method for preparing dendrimer/modified gelatin composite microgel according to claim 4, wherein: in the step a, the structural formula of the adopted dendric alkoxy ether monomer is as follows:
wherein, X is methoxy, ethoxy or hydroxyl; n is 1 to 5.
6. The method for preparing dendrimer/modified gelatin composite microgel according to claim 4, wherein: in the step b, the flow rate ratio of the dispersed phase to the continuous phase is 1/10-1/5; the light source wavelength of the selected ultraviolet light is 254-415 nm.
7. Use of the dendrimer/modified gelatin composite microgel of claim 1, wherein: the mechanism that the limited microenvironment constructed by the dendrimer/modified gelatin composite microgel has enveloping and shielding effects on guest molecules is used for biological macromolecular active protective materials.
8. Use of the dendrimer/modified gelatin composite microgel according to claim 7, wherein: the biological macromolecule comprises at least one of protein, nucleic acid and polysaccharide.
9. Use of the dendrimer/modified gelatin composite microgel according to claim 7, wherein: the dendrimer/modified gelatin composite microgel is used as a drug carrier material or a cell culture matrix material.
10. Use of the dendrimer/modified gelatin composite microgel according to claim 9, wherein: the dendrimer/modified gelatin composite microgel can promote cell adhesion and proliferation when being used as a cell culture matrix material.
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