CN102010480A - Method for preparing micro-grade polymer gel microspheres capable of loading protein - Google Patents
Method for preparing micro-grade polymer gel microspheres capable of loading protein Download PDFInfo
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Abstract
The invention discloses a method for preparing micro-grade polymer gel microspheres capable of loading protein. In the invention, with polyethyleneglycol diacrylate as monomers, the micro-class polymer gel microspheres are prepared in a polysaccharide medium containing inorganic salts by adopting a dispersion polymerization method. The microspheres have favorable pelletizing state and better grain size dispersibility. The preparation process is simple and convenient and does not use any organic solvents, thereby avoiding causing the bioactivity reduction of loaded protein-based medicines or loaded cells. The micro-grade polymer gel microspheres are expected to load chondrocyte and growth factors for accelerating the chondrocyte to grow and are used for preparing injectable tissue engineering cartilage prosthesis.
Description
Technical field
The invention belongs to the functional polymer technical field of biological materials, be specifically related to a kind of preparation method of carrying proteinic micrograde polymer gel micro-ball.
Background technology
Cytokine to chondrocyte's propagation, broken up important regulation, and the exogenous growth factor in vivo the transformation period short, injection not only increases patient's misery repeatedly, also increases economical load.Therefore the drug application slow release method can make somatomedin continue to discharge lentamente at concrete position, prolongs the action time of somatomedin.Since Yolles prepared Regular Insulin-polylactic acid microsphere first in 1971, the macromolecular microspheres drug delivery system obtained fast development and widespread use.
The traditional preparation process method of polymer gel microsphere has suspension polymerization, emulsion polymerization etc.The emulsion polymerization residual organic solvent causes that easily the biological activity of the cell of protein-based medicine or load reduces, and be difficult to prepare the micron order microballoon, as: Zhang Faai, Zhu Xinghua. the letex polymerization of hydroxyethyl methylacrylate. applied chemistry, 2008,25 (5): 592-595, this article has been studied the influence to the synthetic poly hydroxy ethyl acrylate particulate of hydroxyethyl methylacrylate emulsion polymerization of emulsification system, initiator system, temperature of reaction and ionogen, use Diisopropyl azodicarboxylate to improve monomer conversion as initiator, reduce the cohesion rate, temperature of reaction; The emulsifying agent consumption can obviously reduce the content of condensation product, but used the unavoidable residual quantities of organic emulsifier aftertreatment such as sodium lauryl sulphate and Triton X-100 to limit its application, especially as the application on the pharmaceutical carrier, and particle size dispersion is difficult to prepare the micron order particulate at 50～800nm.The microspherulite diameter scope of suspension polymerization preparation is 100～1000 μ m, but dispersed relatively poor.As: appoint beautiful jade. suspension polymerization prepares the research of poly (methyl methacrylate) micro-sphere. prints during chemical industry, 2009,23 (10): 34-36, this article is stablizer with the gelatin, and Diisopropyl azodicarboxylate is an initiator, and suspension polymerization prepares poly (methyl methacrylate) micro-sphere, microsphere features smooth surface, sphericity is better, but particle diameter approximately several microns do not wait to tens microns, size distribution is undesirable.Because dispersion copolymerization method is simple to operate, system is formed simple, and size distribution is good, and the water-soluble mono physical efficiency avoids having used organic solvent with water as solvent, becomes the main method of present preparation micron order microballoon.
Polyoxyethylene glycol (PEG) is the amphipathic nature polyalcohol of a kind of water soluble and most organic solvents, and has good biocompatibility, nontoxic, characteristics such as immunogenicity is low, nil product is tired in the body; The polyoxyethylene glycol compounds is used for clinical application by FDA (FDA) approval, is studied aspect the biologic applications widely, and it is one of the most frequently used material of organizational project.The preparation of polyoxyethylene glycol double methacrylate (PEGDA) polymer gel microsphere and micro-capsule has become the focus of research fields such as biological chemistry, pharmacy in recent years, and it has certain application prospect at biomedical sectors such as tissue engineering bracket and pharmaceutical carriers.Synthesizing monodisperse PEG hydrogel microsphere and functionalization thereof in the Huiguang Zhu. polyelectrolyte multilayer micro-capsule. chemical communication (Synthesis and functionalization of monodisperse poly (ethylene glycol) hydrogel microspheres within polyelectrolyte multilayer microcapsules.Chem.Commun.), 2006,153-155, this article forms sodium polystyrene sulfonate (PSS)/polyallylamine hydrochloride (PAH) bilayer capsule by MnCO3 particulate (about 5 μ m), MnCO3 particulate nuclear is removed the formation micro-capsule with the HCl-EDTA method then, micro-capsule wraps into polyoxyethylene glycol double methacrylate monomer and light trigger, uv photopolymerization forms microballoon, the PSS/PAH film can be sloughed by drying and obtain poly-polyoxyethylene glycol double methacrylate microballoon, the amido molecule again microballoon is carried out functionalization and be applied to biosensor and medicament slow release/rake upwards, but preparing this microballoon is by uv photopolymerization, and the forming process of microballoon is difficult to be effectively controlled.
The present invention is a monomer with the polyoxyethylene glycol double methacrylate, in containing the polysaccharide medium of inorganic salt, but the polymer gel microsphere of the water soluble protein of dispersion copolymerization method preparation load with an organic solvent not, particle diameter is about 1～10 μ m, dispersiveness is better and particle diameter is controlled, this material is expected to the load chondrocyte and promotes the somatomedin of chondrocyte's growth, is used to prepare the tissue engineering bone/cartilage dummy of syringeability.
Summary of the invention
The biological activity that the objective of the invention is to avoid with an organic solvent cause the cell of protein-based medicine or load reduces, and a kind of method that can carry proteinic micrograde polymer gel micro-ball that need not with an organic solvent to prepare is provided.
Technical scheme of the present invention is to carry out Raolical polymerizable to prepare particle size dispersion better polymerization thing gel micro-ball in containing the polysaccharide medium of inorganic salt.
The method that a kind of preparation that the present invention proposes can be carried proteinic micrograde polymer gel micro-ball is as follows:
(1) with 0.5%～2% linking agent N of the water-soluble monomer of mass concentration 5%～25%, monomer mass, 0.5%～3% initiator system of 0.5%～3% stablizer polyvinylpyrrolidone of N '-methylene-bisacrylamide, monomer mass and monomer mass is made into the aqueous solution, is referred to as monomer solution;
(2) polysaccharide is made into the aqueous solution of mass concentration 0.5%～3%, is referred to as polysaccharide solution;
(3) step (1) and (2) resulting monomer solution and polysaccharide solution are mixed after, add and press monomer mass than 10%～30% inorganic salt, formation homogeneous phase aqueous solution;
(4) with the resulting homogeneous phase aqueous solution of step (3), feed inert nitrogen gas, reacted 4～24 hours down at 60 ℃～90 ℃, obtain polymer gel microsphere, to carry out purifying and separate with centrifugal by dialysis, final drying obtains polymer gel microsphere.
Water-soluble monomer described in the step (1) is the polyoxyethylene glycol double methacrylate, and wherein the polyoxyethylene glycol weight-average molecular weight is 200～1000.
Initiator system described in the step (1) is one or more in Potassium Persulphate, ammonium persulphate, Diisopropyl azodicarboxylate, benzoyl peroxide, the hydrogen peroxide, or the mixture of Potassium Persulphate or ammonium persulphate and sulphite, the perhaps mixture of ammonium persulphate and tetramethylethylened.
Polysaccharide described in the step (2) is chitosan, glucose or dextran.
Inorganic salt described in the step (3) are one or more in Repone K, sodium-chlor, anhydrous magnesium sulfate, the anhydrous sodium sulphate.
Drying means described in the step (4) is seasoning, vacuum-drying or lyophilize.
Dialysis molecular weight cut-off described in the step (4) is 12000.
Centrifugal rotational speed described in the step (4) is 10000 rev/mins.
Adopt the inventive method, the degree of crosslinking by regulating polysaccharide concentration, monomer concentration, polymer gel microsphere etc. can prepare the controlled microballoon of particle diameter, and particle diameter can be controlled at 1 μ m～10 μ m.
Beneficial effect of the present invention and outstanding advantage: present method preparation technology is simple and convenient, avoid with an organic solvent, dispersion copolymerization method is prepared the micrograde polymer gel micro-ball in containing the polysaccharide medium of inorganic salt, this microballoon balling-up is in good condition, particle size dispersion is better, avoid making the biological activity of the cell of the protein-based medicine of its loading or load to reduce, this micrograde polymer gel micro-ball is expected to the load chondrocyte and promotes the somatomedin of chondrocyte's growth, is used to prepare the tissue engineering bone/cartilage dummy of syringeability.
Description of drawings
Fig. 1 is the sem photograph (amplifying 6000 times) that obtains polymer gel microsphere in the chitose medium.
Fig. 2 is the sem photograph (amplifying 10000 times) that obtains polymer gel microsphere in the glucose medium.
Embodiment
Embodiment 1
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifugal (10000 rev/mins) washing, lyophilize got polymer gel microsphere in 40 hours.(referring to Fig. 1)
Embodiment 2
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g glucose is dissolved in 10ml water, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.(referring to Fig. 2)
Embodiment 3
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g dextran is dissolved in 10ml water, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing, centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 4
With 1g polyoxyethylene glycol (400) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 5
With 1g polyoxyethylene glycol (1000) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 6
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g Diisopropyl azodicarboxylate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 7
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g benzoyl peroxide and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 90 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 8
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 0.005ml tetramethylethylened and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 9
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3g sodium-chlor, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 10
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add the 0.15g anhydrous sodium sulphate, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 11
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, seasoning got polymer gel microsphere in 60 hours.
Embodiment 12
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, vacuum-drying got polymer gel microsphere in 50 hours.
Embodiment 13
With 0.5g polyoxyethylene glycol (200) double methacrylate, 0.002gN, N '-methylene-bisacrylamide, 0.005g polyvinylpyrrolidone, 0.005g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 14
With 3g polyoxyethylene glycol (200) double methacrylate, 0.016gN, N '-methylene-bisacrylamide, 0.04g polyvinylpyrrolidone, 0.05g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 15
With 3g polyoxyethylene glycol (200) double methacrylate, 0.05gN, N '-methylene-bisacrylamide, 0.08g polyvinylpyrrolidone, 0.08g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.6gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 16
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.05g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.15gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 17
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.3g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.1gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 18
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 80 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 19
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 90 ℃ of reactions 6 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 20
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 16 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Embodiment 21
With 1g polyoxyethylene glycol (200) double methacrylate, 0.005gN, N '-methylene-bisacrylamide, 0.03g polyvinylpyrrolidone, 0.02g ammonium persulphate and 10ml water place the 50ml there-necked flask, and stirring and dissolving forms monomer solution; 0.2g chitosan is dissolved in the acetum of 10ml 2%, forms polysaccharide solution.Mix above-mentioned monomer solution and polysaccharide solution in there-necked flask, add 0.3gKCl, the dissolving back becomes homogeneous phase aqueous solution; After feeding nitrogen 20min, constant speed stirs down 70 ℃ of reactions 24 hours; The milky white liquid that obtains was dialysed two days at dialysis tubing (molecular weight cut-off 12000), centrifuge washing, lyophilize got polymer gel microsphere in 40 hours.
Claims (8)
1. the preparation method that can carry proteinic micrograde polymer gel micro-ball is characterized in that its step is as follows:
(1) with 0.5%～2% linking agent N of the water-soluble monomer of mass concentration 5%～25%, monomer mass, 0.5%～3% initiator system of 0.5%～3% stablizer polyvinylpyrrolidone of N '-methylene-bisacrylamide, monomer mass and monomer mass is made into the aqueous solution, is referred to as monomer solution;
(2) polysaccharide is made into the aqueous solution of mass concentration 0.5%～3%, is referred to as polysaccharide solution;
(3) step (1) and (2) resulting monomer solution and polysaccharide solution are mixed after, add and press monomer mass than 10%～30% inorganic salt, formation homogeneous phase aqueous solution;
(4) with the resulting homogeneous phase aqueous solution of step (3), feed inert nitrogen gas, reacted 4～24 hours down at 60 ℃～90 ℃, obtain polymer gel microsphere, to carry out purifying and separate with centrifugal by dialysis, final drying obtains polymer gel microsphere.
2. preparation method according to claim 1 is characterized in that: the water-soluble monomer described in the step (1) is the polyoxyethylene glycol double methacrylate, and wherein the polyoxyethylene glycol weight-average molecular weight is 200～1000.
3. preparation method according to claim 1, it is characterized in that: the initiator system described in the step (1) is one or more in Potassium Persulphate, ammonium persulphate, Diisopropyl azodicarboxylate, benzoyl peroxide, the hydrogen peroxide, or the mixture of Potassium Persulphate or ammonium persulphate and sulphite, the perhaps mixture of ammonium persulphate and tetramethylethylened.
4. preparation method according to claim 1 is characterized in that: the polysaccharide described in the step (2) is chitosan, glucose or dextran.
5. preparation method according to claim 1 is characterized in that: the inorganic salt described in the step (3) are one or more in Repone K, sodium-chlor, anhydrous magnesium sulfate, the anhydrous sodium sulphate.
6. preparation method according to claim 1 is characterized in that: the drying means described in the step (4) is seasoning, vacuum-drying or lyophilize.
7. preparation method according to claim 1 is characterized in that: the dialysis molecular weight cut-off described in the step (4) is 12000.
8. preparation method according to claim 1 is characterized in that: the centrifugal rotational speed described in the step (4) is 10000 rev/mins.
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| CN104436306A (en) * | 2014-11-11 | 2015-03-25 | 四川大学 | Cell-gel material composite microsphere as well as preparation method and application thereof |
| CN110721340A (en) * | 2019-11-28 | 2020-01-24 | 南方医科大学 | Crosslinked microsphere and preparation method and application of injectable chondrocyte complex thereof |
| CN111214706A (en) * | 2018-11-25 | 2020-06-02 | 中国科学院大连化学物理研究所 | Temperature-sensitive composite gel emulsion and application thereof |
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| CN104436306A (en) * | 2014-11-11 | 2015-03-25 | 四川大学 | Cell-gel material composite microsphere as well as preparation method and application thereof |
| CN111214706A (en) * | 2018-11-25 | 2020-06-02 | 中国科学院大连化学物理研究所 | Temperature-sensitive composite gel emulsion and application thereof |
| CN110721340A (en) * | 2019-11-28 | 2020-01-24 | 南方医科大学 | Crosslinked microsphere and preparation method and application of injectable chondrocyte complex thereof |
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