CN114395164A - Polysaccharide composite gel and preparation method and application thereof - Google Patents
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Abstract
The invention provides a polysaccharide composite gel, which is prepared from the following substances: polysaccharide substance, biodegradable high molecular material, acidic aqueous solution containing cross-linking agent, polypeptide, dispersing assistant, and its preparation method and application in preparing filler for injection are provided. Compared with the prior art, the polysaccharide composite gel, the preparation method and the application thereof provided by the invention have the following advantages: the polycaprolactone PCL is dispersed more uniformly and is firmly fixed in the gel; the dosage of the cross-linking agent is less; the situations of needle blockage, component separation and the like are avoided; reduce the adverse reaction after operation such as red swelling, induration, etc.; the cell quality of the tissue of the operation area is enhanced, and the tissue and the skin of the operation area have the functions of resisting aging and rejuvenating; the absorption and metabolism time of the cross-linked hyaluronic acid is prolonged; the filling and shaping are good, and the collapse and deformation are avoided; effectively improving the satisfaction and comfort after the operation.
Description
Technical Field
The invention relates to the technical field of gels for injection, in particular to a polysaccharide composite gel and a preparation method and application thereof.
Background
Hyaluronic acid, also called hyaluronic acid, hyaluronic acid (HA for short) is a natural polysaccharide structure, is composed of a disaccharide structure formed by repeated D-glucuronic acid and N-acetyl glucose through beta-1, 3-glycosidic bonds, widely exists in animals and microorganisms, HAs no species difference and immunogenicity, HAs high biological safety, is a novel concerned biomedical material, and HAs high medical value. The hyaluronic acid can improve the nutrition metabolism of the skin, makes the skin tender and smooth, removes wrinkles, increases elasticity and prevents aging, is a good transdermal absorption promoter while preserving moisture, and can play a more ideal effect of promoting nutrition absorption when being matched with other nutrient components. The injectable gel has good embedding property with the repaired tissue in vivo, realizes in-situ injection to achieve the aim of minimally invasive, and becomes a hot spot for repairing, reconstructing and filling soft tissue. The structure is folded to form a three-dimensional network, and physiological effects are generated. Including generating fluid resistance, maintaining water balance and homeostasis in the body, influencing the solubility, spatial configuration, chemical balance and systemic osmotic pressure of biological macromolecules, preventing pathogen from playing, and guiding the deposition of collagen fiber secretory substances. It is connected with protein which can not be separated to form polymer, and can maintain the shape and volume of chemical space structure of tissue and ensure reversible compressive resistance of tissue. It has certain effect on macrophage, adherent cell, lymphocyte and natural killer cell.
Polycaprolactone, namely PCL, has good biodegradability, biocompatibility and nontoxicity, is widely used as a medical biodegradable material and a drug controlled release system, and is used as a drug sustained release system in tissue engineering. At present, the product is also used as a filling product in the medical beauty industry. PCL is a few of high polymer material degradable artificial synthetic biomaterials which are approved by the US FDA and are safe and have excellent filling effect, has the advantages of good biocompatibility, low toxicity, easy modification and the like, and can cause giant cell reaction of a foreign body, so that collagen is gradually generated. The corium layer is thickened along with the increase of time, the filling position is completely replaced by the new autogenous tissue, and the filling position can be maintained in a human body for more than 2 years, so that a permanent beautifying effect is obtained.
In recent years, soft tissue augmentation surgery has become popular in micro plastic surgery, mainly for filling wrinkles, filling depressions, and correcting facial contours. Among the used soft tissue fillers, hyaluronic acid is the most commonly used soft tissue filler at home and abroad due to the advantages of high safety, good biocompatibility, less adverse reaction after operation, immediate display of the filling effect after operation and the like. However, the hyaluronic acid-based filler is limited by itself, and the maintenance time in vivo is short, and is only 6 to 12 months.
Oligopeptides, also called small peptides, are polypeptides, generally having a molecular weight of less than 1000 daltons, and are compounds of small active peptides formed by the condensation of 2 to 10 amino acids. Oligopeptides can be further divided into: the oligopeptide-1, the oligopeptide-3, the oligopeptide-5, the oligopeptide-6 and the like act on skin through exogenous action, can change the metabolism of skin cell tissues after being combined with skin corium cells, improve microcirculation, promote proliferation and differentiation of corium fibroblasts, effectively recover the growth of subcutaneous connective tissues, enable the skin to be fine and smooth and have luster, increase elasticity and eliminate wrinkles, have deep repair effect, and can be used for repairing newly generated pits, laser, spots, nevus and acnes after removing the acnes and repairing the red blood silk instruments after operation. Can stimulate and improve the cell synthesis ability, repair the damaged elastic fibers, ensure that each cell of the skin is full of youthful activity, obviously improve the aging phenomena of dry skin, loose skin, fine wrinkles and the like, and ensure that the skin is compact and young.
In the prior art, patent with publication number CN 104258470 a provides a preparation method of polylactic acid microsphere and cross-linked hyaluronic acid mixed gel for injection, which comprises the steps of firstly preparing cross-linked sodium hyaluronate gel, then adding polylactic acid microsphere and uniformly mixing to obtain mixed gel. The disadvantages are: 1. the storage life of the filler is short, and the PLLA molecules collapse more than one month. 2. The polylactic acid microspheres are easily separated from the gel, and the components are not uniform. 3. The occurrence probability of the blockage of the syringe needle, 4. subcutaneous nodule and granuloma is increased by several times during the injection process.
Patent publication No. CN 105879124A provides a preparation method of L-polylactic acid filler for injection, which is a freeze-dried product prepared by compounding L-polylactic acid, honeycomb HA and a synergist, and HAs the following defects: 1. ready to use and ready to be prepared. 2. Honeycomb HAs low HA content, 3. the effect is poor immediately after injection. 4. The final effect cannot be accurately evaluated. 5. The effect is slow.
Patent publication No. CN 102911380A provides a method for preparing a modified material of hyaluronic acid and biodegradable polymer, which comprises dissolving polylactic acid and hyaluronic acid in organic phase dichloromethane, activating carbodiimide (EDC) under acidic condition, and crosslinking with Adipic Dihydrazide (ADH) to obtain water-insoluble solid particles. The disadvantages are: 1. the final product contains toxic substances (EDC toxic). 2. The purification difficulty in the production process of the product is high. 3. The resulting product is not a gel. 4. The side effects of the product are unacceptable or unusable.
Disclosure of Invention
Aiming at the existing restriction limitation, the invention provides a polysaccharide composite gel and a preparation method and application thereof, and overcomes the defects in the background technology.
In order to achieve the purpose, the invention adopts the following technical scheme:
1. the invention provides a polysaccharide composite gel, which is prepared from the following substances in parts by weight: 0.5-1.5 parts of polysaccharide substance, 0.1-10 parts of biodegradable high polymer material, 3-10 parts of acidic aqueous solution containing cross-linking agent, 0.2-6 parts of polypeptide and 0.5-3 parts of dispersing auxiliary agent.
The polysaccharide composite gel is prepared by carrying out composite crosslinking and grafting on polysaccharide and derivatives thereof and a biodegradable high polymer material with an active functional group in an acidic environment through a crosslinking agent, and polypeptide and a dispersing aid are added into the composite gel, wherein the polypeptide can effectively assist the combination of the gel and skin cell tissues, improve the differentiation capacity and healing capacity of the skin cell tissues, and have a deep repair effect on skin.
The amount of polysaccharide substance can be selected from 0.5 parts, 0.7 parts, 1 part, 1.2 parts and 1.5 parts, preferably 1 part;
the usage amount of the biodegradable high polymer material can be selected from 0.1 part, 0.5 part, 1 part, 3 parts, 5 parts, 7 parts, 9 parts and 10 parts, and 5 parts is preferred;
the amount of the acidic aqueous solution containing the crosslinking agent can be selected from 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts and 10 parts, and preferably 6 parts;
the amount of the polypeptide used can be selected from 0.2 parts, 0.5 parts, 1 part, 2 parts, 3 parts, 4 parts, 5 parts and 6 parts, and preferably 3 parts;
the amount of the dispersion aid used may be selected from 0.5 parts, 1 part, 1.5 parts, 2 parts, 2.5 parts and 3 parts, and preferably 1.5 parts.
Further, in the polysaccharide composite gel, the concentration of acid in the acidic aqueous solution containing the cross-linking agent is 0.1-1 mmol/L.
In the acidic aqueous solution containing the crosslinking agent, the concentration of the acid is preferably 0.5 to 1mmol/L, and most preferably 0.8 mmol/L.
The acid concentration is selected from 0.1mmol/L, 0.2 mmol/L, 0.3 mmol/L, 0.4 mmol/L, 0.5 mmol/L, 0.6 mmol/L, 0.7 mmol/L, 0.8mmol/L, 0.9 mmol/L, and 1 mmol/L.
Further, in the polysaccharide composite gel, the acidic aqueous solution containing the cross-linking agent is one or more of hydrochloric acid aqueous solution, acetic acid aqueous solution and lactic acid aqueous solution.
In the acidic aqueous solution containing the cross-linking agent, the acidic aqueous solution can be independently selected from hydrochloric acid aqueous solution, acetic acid aqueous solution or lactic acid aqueous solution, or any two or all three of the hydrochloric acid aqueous solution, the acetic acid aqueous solution or the lactic acid aqueous solution can be compounded according to the equal volume ratio and then used together, and most preferably the lactic acid aqueous solution.
Further, in the polysaccharide composite gel, in the acidic aqueous solution containing the cross-linking agent, the cross-linking agent is any one or more of modified polyethylene glycol and 1, 4-butanediol diglycidyl ether.
The cross-linking agent can be used singly or by compounding a plurality of modified polyethylene glycols.
Preferably a modified polyethylene glycol, more preferably a polyethylene glycol diglycidyl ether or a four-arm star polyethylene glycol epoxide.
The ethylene glycol diglycidyl ether is epoxy resin containing an ethylene glycol chain, belongs to low-viscosity water-soluble epoxy resin or aliphatic epoxy resin, and has the molecular weight of 400-5000, and can be selected from 400, 1000, 2000, 3000, 4000 and 5000;
the molecular structural formula of the polyethylene glycol diglycidyl ether is shown as the following formula I:
The four-arm star polyethylene glycol epoxide (four-arm-polyethylene glycol-epoxide) is a multi-arm polyethylene glycol derivative having an epoxy or epoxy group at each end of the four arms attached to a pentaerythritol core, and the molecular weight of the four-arm star polyethylene glycol epoxide provided in the present invention is 200-40000, which can be selected from 200, 400, 600, 800, 1000, 2000, 3400, 5000, 10000, 20000, 30000, 40000;
the molecular structural formula of the four-arm star polyethylene glycol epoxide is shown as the following formula II:
Further, in the polysaccharide complex gel, the polysaccharide substance is one or more of hyaluronic acid and hyaluronate.
Hyaluronic acid is a main component constituting connective tissues such as human intercellular substance, ocular vitreous body, joint synovial fluid, etc., and plays important physiological functions of retaining water, maintaining extracellular space, regulating osmotic pressure, lubricating, and promoting cell repair in vivo. The hyaluronic acid molecule contains a large amount of carboxyl and hydroxyl, and is in water; hydrogen bonds in molecules and among molecules are formed in the solution, so that the solution has a strong water retention effect and can combine water which is 400 times more than that of the solution per se; at higher concentrations, aqueous solutions have significant viscoelastic properties due to the complex tertiary network structure formed by intermolecular interactions. Hyaluronic acid, as a main component of an intercellular matrix, directly participates in the regulation of the communication of electrolytes inside and outside cells, and plays a role of a filter for physical and molecular information. Hyaluronic acid has unique physicochemical properties and physiological functions, and has been widely used in medicine.
Hyaluronic acid can be used as a viscoelastic agent for ophthalmologic intraocular lens implantation surgery, a filler for joint surgery such as osteoarthritis and rheumatoid arthritis, is widely applied in eye drops as a medium, and is also used for preventing postoperative adhesion and promoting healing of skin wounds. The compound formed by the reaction of hyaluronic acid and other medicines has a slow release effect on the medicines, and can achieve the purpose of directional and timed release.
The hyaluronate is preferably hyaluronic acid sodium salt, hyaluronic acid potassium salt and/or hyaluronic acid calcium salt, and can be used alone or mixed at equal mass, and has molecular weight of 200-3000kDa, preferably 200kDa, 500 kDa, 1000kDa, 2000 kDa, 3000kDa, and preferably 1000 kDa.
Further, in the above polysaccharide composite gel, the biodegradable polymer material is a polymer with a molecular weight of 5-9kDa, preferably any one or more of polylactic acid, polyglycolic acid, polyglycolide, polycaprolactone, polyamino acid, polyethylene glycol and polypropylene oxide.
The molecular weight of the biodegradable high molecular material can be selected from 5 kDa, 6 kDa, 7kDa, 8 kDa and 9kDa, and is preferably 7 kDa; the molecular particle diameter is 5-80 μm, and can be selected from 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, preferably 50 μm;
specifically, any one of the substances listed above can be used alone or any combination of a plurality of the substances, the total molecular weight of the substances reaches 5-9kDa, the particle size of the molecules is 5-80 μm, and the substances are preferably used alone.
Further, in the polysaccharide composite gel, the polypeptide is a small molecular polypeptide which is obtained by condensing 2-10 amino acids and has a molecular weight of less than 1 kDa; preferably any one or more of oligopeptide-1, oligopeptide-3, oligopeptide-5 and oligopeptide-6.
The polypeptide is selected to be small molecular polypeptide, preferably oligopeptide, the molecular weight can be selected to be 100Da, 200 Da, 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1000 Da, oligopeptide-1, oligopeptide-3, oligopeptide-5 and oligopeptide-6, and the total molecular weight is less than 1000 Da, preferably oligopeptide 1, oligopeptide 3 or oligopeptide 5, and most preferably oligopeptide 5.
Further, in the polysaccharide composite gel, the dispersing auxiliary agent is hesperetin.
The hesperetin has the effect of enhancing the dispersion effect, and can also effectively inhibit inflammation generated in the use process of the gel, and after the hesperetin is added, the dispersion effect of the polysaccharide substance and the biodegradable high polymer material can be effectively promoted in the dispersion process, so that the polysaccharide substance and the biodegradable high polymer material can be more tightly compounded.
Another objective of the present invention is to provide a method for preparing a polysaccharide complex gel, wherein the polysaccharide complex gel is the above polysaccharide complex gel, and the preparation method comprises: dispersing and compounding polysaccharide and biodegradable polymer material, adding into acid water solution containing cross-linking agent, cross-linking at least twice, and adding polypeptide.
The important invention of the invention is that not only the dispersing assistant is adopted to improve the dispersing effect of the polysaccharide substance, but also the times of crosslinking and the external environment of crosslinking are adjusted, at least two times of crosslinking can effectively improve the crosslinking degree of the polysaccharide substance and the biodegradable high molecular material, and simultaneously, the external environment of the crosslinking process adopts an acid environment, which is beneficial to further improving the crosslinking degree of the polysaccharide substance and the biodegradable high molecular material, and simultaneously, the crosslinked product in the acid environment can be more effectively adapted to the subsequent secondary crosslinking.
Further, the preparation method of the polysaccharide composite gel specifically comprises the following steps:
s1, adding the biodegradable high molecular material and the dispersing assistant into the aqueous solution of the polysaccharide substance, performing ultrasonic dispersion, uniformly mixing, and performing vacuum freeze drying to obtain a compound;
s2, adding the compound obtained in the step S1 into an acidic aqueous solution containing a cross-linking agent, uniformly mixing, adjusting the temperature to perform cross-linking at least twice, changing the cross-linking temperature after each cross-linking, and performing next cross-linking to obtain a cross-linked product;
s3, adjusting the pH of the cross-linked product obtained in the step S2 to be neutral, dialyzing to obtain composite gel, and performing vacuum freeze drying for decrement again to obtain decrement composite gel;
and S4, when the reduced composite gel obtained in the step S3 is reduced to 90-99.5 percent of the original weight, dripping equivalent polypeptide, and uniformly mixing to obtain the polysaccharide composite gel.
Further, in the above preparation method of the polysaccharide composite gel, in the step S1, the ultrasonic dispersion condition is 100-200W, 30-40 ℃, 15-35 min; the temperature of the vacuum freeze drying is-10 ℃ to-50 ℃, and the drying time is 24-60 h.
The ultrasonic dispersion can be selected from 100W, 125W, 150W, 175W, and 200W, 30 deg.C, 32 deg.C, 34 deg.C, 35 deg.C, 36 deg.C, 38 deg.C, 40 deg.C, 15min, 20 min, 25min, 30 min, and 35 min; preferably 150W, 35 ℃ and 25 min.
The vacuum freeze drying temperature can be selected from-10 deg.C, -20 deg.C, -30 deg.C, -40 deg.C, -50 deg.C, preferably-30 deg.C, and the drying time is 24h, 30 h, 40h, 50 h, 60h, preferably 40 h.
Further, in the above method for preparing the polysaccharide complex gel, in step S2, the temperature of the present crosslinking is higher than the temperature of the next crosslinking.
The process that the cross-linking itself is for line type or branch type polymer chain with covalent bond linkage network or size polymer between the polymer chain, when twice cross-linking, the temperature of first cross-linking need be greater than the cross-linking temperature of second time, its reason mainly lies in, first cross-linking need be to carry out covalent bond linkage earlier with the big particle size polymer in polyoses material and the biodegradable macromolecular material, the structure of connecting this moment is network structure mostly, the secondary cross-linking needs the lowering temperature, make the two small particle size polymer carry out covalent bond linkage once more, form inseparabler more firm stable size structure, can effectively improve the cross-linking degree.
Further, in the preparation method of the polysaccharide composite gel, the crosslinking frequency is two times, the temperature of the first crosslinking is 40-60 ℃, and the time is 2-10 hours; the temperature of the second cross-linking is 20-50 ℃ and the time is 2-24 h.
The first crosslinking can be selected at 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C for 2h, 4h, 6h, 8 h, 10h, preferably 50 deg.C, 6 h;
the second crosslinking can be selected at 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C for 2h, 5h, 10h, 15h, 20 h, and 24h, preferably 30 deg.C and 15 h;
further, in the above preparation method of the polysaccharide complex gel, in the step S3, a weak alkaline solution is used to adjust the pH value of the cross-linked product to 6.8-7.3, the dialysis method is to use a phosphate buffer solution to dialyze and purify the cross-linked product until the osmotic pressure is 270-330mmol/L, the conditions of vacuum freeze drying decrement are that the temperature is-10 ℃ to-50 ℃, the drying time is 24-60h, and the decrement is 20-30% of the original volume; the weakly alkaline solution is preferably sodium bicarbonate or disodium bicarbonate.
The adjusted pH value can be selected to be 6.8, 6.9, 7.0, 7.1, 7.2 and 7.3;
dialyzing until osmotic pressure is 270 mmol/L, 280 mmol/L, 290 mmol/L, 300mmol/L, 310 mmol/L, 320 mmol/L, 330 mmol/L;
the freeze drying temperature can be selected from-10 deg.C, -20 deg.C, -30 deg.C, -40 deg.C and-50 deg.C;
the drying time can be selected from 24h, 30 h, 40h, 50 h and 60 h;
preferably, the pH value is 7.0, the osmotic pressure is 300mmol/L, the freeze-drying temperature is-40 ℃, and the drying time is 40 h.
The third purpose of the invention is to provide an application of polysaccharide composite gel in preparing a filling agent for injection, wherein the polysaccharide composite gel is the polysaccharide composite gel or the polysaccharide composite gel prepared by the preparation method.
Compared with the prior art, the polysaccharide composite gel, the preparation method and the application thereof provided by the invention have the following advantages:
1. the polycaprolactone PCL is dispersed more uniformly and is firmly fixed in the gel;
2. the dosage of the cross-linking agent is less;
3. the situations of needle blockage, component separation and the like are avoided;
4. reduce the adverse reaction after operation such as red swelling, induration, etc.;
5. the cell quality of the tissue of the operation area is enhanced, and the tissue and the skin of the operation area have the functions of resisting aging and rejuvenating;
6. the absorption and metabolism time of the cross-linked hyaluronic acid is prolonged;
7. the filling and shaping are good, and the collapse and deformation are avoided;
8. effectively improving the satisfaction and comfort after the operation.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below. It is to be understood that the description herein is only illustrative of the present invention and is not intended to limit the scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in the specification of the present invention are for the purpose of describing particular embodiments only and are not intended to limit the present invention. The reagents and instruments used in the present invention are commercially available, and the characterization means involved can be referred to the description in the prior art, which is not repeated herein.
For a further understanding of the present invention, reference will now be made in detail to the preferred embodiments of the present invention.
Example 1
The polysaccharide composite gel is prepared from the following substances in parts by weight: 0.5-1.5 parts of polysaccharide substance, 0.1-10 parts of biodegradable high polymer material, 3-10 parts of acidic aqueous solution containing cross-linking agent, 0.2-6 parts of polypeptide and 0.5-3 parts of dispersing auxiliary agent.
The amount of polysaccharide substance can be selected from 0.5 parts, 0.7 parts, 1 part, 1.2 parts and 1.5 parts, preferably 1 part;
the usage amount of the biodegradable high polymer material can be selected from 0.1 part, 0.5 part, 1 part, 3 parts, 5 parts, 7 parts, 9 parts and 10 parts, and 5 parts is preferred;
the amount of the acidic aqueous solution containing the crosslinking agent can be selected from 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts and 10 parts, and preferably 6 parts;
the amount of the polypeptide used can be selected from 0.2 parts, 0.5 parts, 1 part, 2 parts, 3 parts, 4 parts, 5 parts and 6 parts, and preferably 3 parts;
the amount of the dispersion aid used may be selected from 0.5 parts, 1 part, 1.5 parts, 2 parts, 2.5 parts and 3 parts, and preferably 1.5 parts.
The concentration of the acid in the acidic aqueous solution containing the crosslinking agent is 0.1-1 mmol/L.
In the acidic aqueous solution containing the crosslinking agent, the concentration of the acid is preferably 0.5 to 1mmol/L, and most preferably 0.8 mmol/L.
The acid concentration is selected from 0.1mmol/L, 0.2 mmol/L, 0.3 mmol/L, 0.4 mmol/L, 0.5 mmol/L, 0.6 mmol/L, 0.7 mmol/L, 0.8mmol/L, 0.9 mmol/L, and 1 mmol/L.
In the acidic aqueous solution containing the cross-linking agent, the acidic aqueous solution is one or more of hydrochloric acid aqueous solution, acetic acid aqueous solution and lactic acid aqueous solution.
In the acidic aqueous solution containing the cross-linking agent, the acidic aqueous solution can be independently selected from hydrochloric acid aqueous solution, acetic acid aqueous solution or lactic acid aqueous solution, or any two or all three of the hydrochloric acid aqueous solution, the acetic acid aqueous solution or the lactic acid aqueous solution can be compounded according to the equal volume ratio and then used together, and most preferably the lactic acid aqueous solution.
In the acidic aqueous solution containing the cross-linking agent, the cross-linking agent is any one or more of modified polyethylene glycol and 1, 4 butanediol diglycidyl ether.
The cross-linking agent can be used singly or by compounding a plurality of modified polyethylene glycols.
Preferably a modified polyethylene glycol, more preferably a polyethylene glycol diglycidyl ether or a four-arm star polyethylene glycol epoxide.
The molecular weight of the polyethylene glycol diglycidyl ether is 400-;
the molecular weight of the four-arm star-shaped polyethylene glycol epoxide is 200-40000, which can be selected from 200, 400, 600, 800, 1000, 2000, 3400, 5000, 10000, 20000, 30000 and 40000;
the polysaccharide substance is one or more of hyaluronic acid and hyaluronate.
The hyaluronate is preferably hyaluronic acid sodium salt, hyaluronic acid potassium salt and/or hyaluronic acid calcium salt, and can be used alone or mixed at equal mass, and has molecular weight of 200-3000kDa, preferably 200kDa, 500 kDa, 1000kDa, 2000 kDa, 3000kDa, and preferably 1000 kDa.
The biodegradable high molecular material is a polymer with a molecular weight of 5-9kDa, preferably one or more of polylactic acid, polyglycolic acid, poly (glycolide-co-lactide), polycaprolactone, polyamino acid, polyethylene glycol and polypropylene oxide.
The molecular weight of the biodegradable high molecular material can be selected from 5 kDa, 6 kDa, 7kDa, 8 kDa and 9kDa, and is preferably 7 kDa; the molecular particle diameter is 5-80 μm, and can be selected from 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, preferably 50 μm;
specifically, any one of the substances listed above can be used alone or any combination of a plurality of the substances, the total molecular weight of the substances reaches 5-9kDa, the particle size of the molecules is 5-80 μm, and the substances are preferably used alone.
The polypeptide is a micromolecular polypeptide which is obtained by condensing 2-10 amino acids and has a molecular weight of less than 1 kDa; preferably any one or more of oligopeptide-1, oligopeptide-3, oligopeptide-5 and oligopeptide-6.
The polypeptide is selected to be small molecular polypeptide, preferably oligopeptide, the molecular weight can be selected to be 100Da, 200 Da, 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1000 Da, oligopeptide-1, oligopeptide-3, oligopeptide-5 and oligopeptide-6, and the total molecular weight is less than 1000 Da, preferably oligopeptide 1, oligopeptide 3 or oligopeptide 5, and most preferably oligopeptide 5.
The dispersing auxiliary agent is hesperetin.
The preparation method of polysaccharide composite gel comprises dispersing and compounding polysaccharide substance and biodegradable high molecular material, adding into acidic aqueous solution containing cross-linking agent, cross-linking at least twice, and adding polypeptide.
The method specifically comprises the following steps:
s1, adding the biodegradable high molecular material and the dispersing assistant into the aqueous solution of the polysaccharide substance, performing ultrasonic dispersion, uniformly mixing, and performing vacuum freeze drying to obtain a spongy compound;
s2, adding the spongy compound obtained in the step S1 into an acidic aqueous solution containing a cross-linking agent, uniformly mixing, adjusting the temperature to perform cross-linking at least twice, changing the cross-linking temperature after each cross-linking, and performing next cross-linking to obtain a cross-linked product;
s3, adjusting the pH of the cross-linked product obtained in the step S2 to be neutral, dialyzing to obtain composite gel, and performing vacuum freeze drying for decrement again to obtain decrement composite gel;
and S4, when the reduced composite gel obtained in the step S3 is reduced to 90-99.5% of the original reduced composite gel, dripping equal amount of polypeptide (small molecular polypeptide, namely oligopeptide 1, oligopeptide 3, oligopeptide 4, oligopeptide 5 and oligopeptide 6) and uniformly mixing to obtain the polysaccharide composite gel.
In step S1, the ultrasonic dispersion condition is 100 and 200W, 30-40 ℃, 15-35 min; the temperature of the vacuum freeze drying is-10 ℃ to-50 ℃, and the drying time is 24-60 h.
The ultrasonic dispersion can be selected from 100W, 125W, 150W, 175W, and 200W, 30 deg.C, 32 deg.C, 34 deg.C, 35 deg.C, 36 deg.C, 38 deg.C, 40 deg.C, 15min, 20 min, 25min, 30 min, and 35 min; preferably 150W, 35 ℃ and 25 min.
The vacuum freeze drying temperature can be selected from-10 deg.C, -20 deg.C, -30 deg.C, -40 deg.C, -50 deg.C, preferably-30 deg.C, and the drying time is 24h, 30 h, 40h, 50 h, 60h, preferably 40 h.
In step S2, the temperature of the present crosslinking is higher than the temperature of the next crosslinking.
The number of crosslinking times is two, the temperature of the first crosslinking is 40-60 ℃, and the time is 2-10 h; the temperature of the second cross-linking is 20-50 ℃ and the time is 2-24 h.
The first crosslinking can be selected at 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C for 2h, 4h, 6h, 8 h, 10h, preferably 50 deg.C, 6 h;
the second crosslinking can be selected at 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C for 2h, 5h, 10h, 15h, 20 h, and 24h, preferably 30 deg.C and 15 h;
in step S3, a weakly alkaline solution is used to adjust the pH value of the cross-linked product to 6.8-7.3, the dialysis method is to use a phosphate buffer solution to dialyze and purify the product until the osmotic pressure is 270-330mmol/L, the conditions of vacuum freeze drying decrement are that the temperature is minus 10 ℃ to minus 50 ℃, the drying time is 24-60h, and the decrement is 20-30% of the original volume; the weakly alkaline solution is preferably sodium bicarbonate or disodium bicarbonate.
The adjusted pH value can be selected to be 6.8, 6.9, 7.0, 7.1, 7.2 and 7.3;
dialyzing until osmotic pressure is 270 mmol/L, 280 mmol/L, 290 mmol/L, 300mmol/L, 310 mmol/L, 320 mmol/L, 330 mmol/L;
the freeze drying temperature can be selected from-10 deg.C, -20 deg.C, -30 deg.C, -40 deg.C and-50 deg.C;
the drying time can be selected from 24h, 30 h, 40h, 50 h and 60 h;
preferably, the pH value is 7.0, the osmotic pressure is 300mmol/L, the freeze-drying temperature is-40 ℃, and the drying time is 40 h.
The polysaccharide composite gel is mainly used for preparing fillers for injection.
Example 2
The preparation of the polysaccharide composite gel comprises the following steps:
s1, adding the biodegradable high molecular material and the dispersing assistant into the aqueous solution of the polysaccharide substance, performing ultrasonic dispersion, uniformly mixing, and performing vacuum freeze drying to obtain a spongy compound;
s2, adding the spongy compound obtained in the step S1 into an acidic aqueous solution containing a cross-linking agent, uniformly mixing, adjusting the temperature to perform cross-linking at least twice, changing the cross-linking temperature after each cross-linking, and performing next cross-linking to obtain a cross-linked product;
s3, adjusting the pH of the cross-linked product obtained in the step S2 to be neutral, dialyzing to obtain composite gel, and performing vacuum freeze drying for decrement again to obtain decrement composite gel;
and S4, when the reduced composite gel obtained in the step S3 is reduced to 90-99.5% of the original reduced composite gel, dripping equal amount of polypeptide (small molecular polypeptide, namely oligopeptide 1, oligopeptide 3, oligopeptide 4, oligopeptide 5 and oligopeptide 6) and uniformly mixing to obtain the polysaccharide composite gel.
In the step S1, 0.5-1.5g of polysaccharide substance, 0.1-10g of biodegradable polymer material and 0.5-3g of dispersing aid are added, and the ultrasonic dispersion condition is 100-200W, 30-40 ℃ and 15-35 min; the temperature of the vacuum freeze drying is-10 ℃ to-50 ℃, and the drying time is 24-60 h;
the ultrasonic dispersion can be selected from 100W, 125W, 150W, 175W, and 200W, 30 deg.C, 32 deg.C, 34 deg.C, 35 deg.C, 36 deg.C, 38 deg.C, 40 deg.C, 15min, 20 min, 25min, 30 min, and 35 min; preferably 150W, 35 ℃ and 25 min.
The vacuum freeze drying temperature can be selected from-10 deg.C, -20 deg.C, -30 deg.C, -40 deg.C, -50 deg.C, preferably-30 deg.C, and the drying time is 24h, 30 h, 40h, 50 h, 60h, preferably 40 h.
The polysaccharide substance is one or more of hyaluronic acid and hyaluronate.
The hyaluronate is preferably hyaluronic acid sodium salt, hyaluronic acid potassium salt and/or hyaluronic acid calcium salt, and can be used alone or mixed at equal mass, and has molecular weight of 200-3000kDa, preferably 200kDa, 500 kDa, 1000kDa, 2000 kDa, 3000kDa, and preferably 1000 kDa.
The biodegradable high molecular material is a polymer with a molecular weight of 5-9kDa, preferably one or more of polylactic acid, polyglycolic acid, poly (glycolide-co-lactide), polycaprolactone, polyamino acid, polyethylene glycol and polypropylene oxide.
The molecular weight of the biodegradable high molecular material can be selected from 5 kDa, 6 kDa, 7kDa, 8 kDa and 9kDa, and is preferably 7 kDa; the molecular particle diameter is 5-80 μm, and can be selected from 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, preferably 50 μm;
specifically, any one of the substances listed above can be used alone or any combination of a plurality of the substances, the total molecular weight of the substances reaches 5-9kDa, the particle size of the molecules is 5-80 μm, and the substances are preferably used alone.
The dispersing auxiliary agent is hesperetin.
In step S2, 3-10g of an acidic aqueous solution containing a crosslinking agent,
the concentration of the acid in the acidic aqueous solution containing the crosslinking agent is 0.1-1 mmol/L.
In the acidic aqueous solution containing the crosslinking agent, the concentration of the acid is preferably 0.5 to 1mmol/L, and most preferably 0.8 mmol/L.
The acid concentration is selected from 0.1mmol/L, 0.2 mmol/L, 0.3 mmol/L, 0.4 mmol/L, 0.5 mmol/L, 0.6 mmol/L, 0.7 mmol/L, 0.8mmol/L, 0.9 mmol/L, and 1 mmol/L.
In the acidic aqueous solution containing the cross-linking agent, the acidic aqueous solution is one or more of hydrochloric acid aqueous solution, acetic acid aqueous solution and lactic acid aqueous solution.
In the acidic aqueous solution containing the cross-linking agent, the acidic aqueous solution can be independently selected from hydrochloric acid aqueous solution, acetic acid aqueous solution or lactic acid aqueous solution, or any two or all three of the hydrochloric acid aqueous solution, the acetic acid aqueous solution or the lactic acid aqueous solution can be compounded according to the equal volume ratio and then used together, and most preferably the lactic acid aqueous solution.
In the acidic aqueous solution containing the cross-linking agent, the cross-linking agent is any one or more of modified polyethylene glycol and 1, 4 butanediol diglycidyl ether.
The cross-linking agent can be used singly or by compounding a plurality of modified polyethylene glycols.
Preferably a modified polyethylene glycol, more preferably a polyethylene glycol diglycidyl ether or a four-arm star polyethylene glycol epoxide.
The molecular weight of the polyethylene glycol diglycidyl ether is 400-;
the molecular weight of the four-arm star-shaped polyethylene glycol epoxide is 200-40000, which can be selected from 200, 400, 600, 800, 1000, 2000, 3400, 5000, 10000, 20000, 30000 and 40000;
the number of crosslinking times is two, the temperature of the first crosslinking is 40-60 ℃, and the time is 2-10 h; the temperature of the second cross-linking is 20-50 ℃ and the time is 2-24 h.
The first crosslinking can be selected at 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C for 2h, 4h, 6h, 8 h, 10h, preferably 50 deg.C, 6 h;
the second crosslinking can be selected at 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C for 2h, 5h, 10h, 15h, 20 h, and 24h, preferably 30 deg.C and 15 h;
in step S3, a weakly alkaline solution is used to adjust the pH value of the cross-linked product to 6.8-7.3, the dialysis method is to use a phosphate buffer solution to dialyze and purify the product until the osmotic pressure is 270-330mmol/L, the conditions of vacuum freeze drying decrement are that the temperature is minus 10 ℃ to minus 50 ℃, the drying time is 24-60h, and the decrement is 20-30% of the original volume; the weakly alkaline solution is preferably sodium bicarbonate or disodium bicarbonate.
The adjusted pH value can be selected to be 6.8, 6.9, 7.0, 7.1, 7.2 and 7.3;
dialyzing until osmotic pressure is 270 mmol/L, 280 mmol/L, 290 mmol/L, 300mmol/L, 310 mmol/L, 320 mmol/L, 330 mmol/L;
the freeze drying temperature can be selected from-10 deg.C, -20 deg.C, -30 deg.C, -40 deg.C and-50 deg.C;
the drying time can be selected from 24h, 30 h, 40h, 50 h and 60 h;
preferably, the pH value is 7.0, the osmotic pressure is 300mmol/L, the freeze-drying temperature is-40 ℃, and the drying time is 40 h.
Example 3
The preparation of the polysaccharide composite gel comprises the following steps:
s1, adding the biodegradable high molecular material and the dispersing assistant into the aqueous solution of the polysaccharide substance, performing ultrasonic dispersion, uniformly mixing, and performing vacuum freeze drying to obtain a spongy compound;
s2, adding the spongy compound obtained in the step S1 into an acidic aqueous solution containing a cross-linking agent, uniformly mixing, adjusting the temperature to perform cross-linking at least twice, changing the cross-linking temperature after each cross-linking, and performing next cross-linking to obtain a cross-linked product;
s3, adjusting the pH of the cross-linked product obtained in the step S2 to be neutral, dialyzing to obtain composite gel, and performing vacuum freeze drying for decrement again to obtain decrement composite gel;
and S4, when the reduced composite gel obtained in the step S3 is reduced to 95 percent of the original reduced composite gel, dripping equal amount of polypeptide (small molecular polypeptide, namely oligopeptide 1, oligopeptide 3, oligopeptide 4, oligopeptide 5 and oligopeptide 6), and uniformly mixing to obtain the polysaccharide composite gel.
In step S1, 1g of polysaccharide substance, 5g of biodegradable high polymer material and 2g of dispersing aid are subjected to ultrasonic dispersion under the conditions of 150W, 35 ℃ and 25 min; the temperature of vacuum freeze drying is-30 ℃, and the drying time is 40 h;
the polysaccharide is hyaluronic acid sodium salt, and has molecular weight of 1000 kDa.
The biodegradable high molecular material is polycaprolactone which is used independently.
The molecular weight of the biodegradable high polymer material is 7 kDa; the molecular particle size is 50 μm;
the dispersing auxiliary agent is hesperetin.
In step S2, 6.5g of an acidic aqueous solution containing a crosslinking agent,
the concentration of the acid in the acidic aqueous solution containing the crosslinking agent was 0.8 mmol/L.
In the acidic aqueous solution containing the crosslinking agent, the acidic aqueous solution is a lactic acid aqueous solution.
In an acidic aqueous solution containing a cross-linking agent, the cross-linking agent is a four-arm star polyethylene glycol epoxide.
The molecular weight of the four-arm star-shaped polyethylene glycol epoxide is 20000;
the number of crosslinking times is two, the temperature of the first crosslinking is 50 ℃, and the time is 6 hours; the temperature of the second crosslinking was 30 ℃ and the time was 15 hours.
In step S3, adjusting the pH value of the cross-linked product to 7.0 by using weak alkaline solution, wherein the dialysis method comprises the steps of dialyzing and purifying by using phosphate buffer until the osmotic pressure is 300mmol/L, and reducing the volume by vacuum freeze drying at-40 ℃ for 40h to 25% of the original volume; the weakly alkaline solution is preferably sodium bicarbonate or disodium bicarbonate.
The embodiment has the following effects:
1. in order to verify the effect of the technical scheme of the invention, the inventor independently performs experiments on key technical points in the technology, and the experiments are specifically grouped as follows according to various choices of the key technical points:
1) the polysaccharide substances are respectively selected as follows: hyaluronic acid sodium salt alone (example 3), hyaluronic acid potassium salt alone (example 4), hyaluronic acid calcium salt alone (example 5), hyaluronic acid sodium salt + hyaluronic acid potassium salt (example 6), hyaluronic acid sodium salt + hyaluronic acid calcium salt (example 7), hyaluronic acid potassium salt + hyaluronic acid calcium salt (example 8), hyaluronic acid sodium salt + hyaluronic acid potassium salt + hyaluronic acid calcium salt (example 9);
2) the biodegradable high polymer materials are respectively selected as follows: polyglycolic acid alone (example 10), polycaprolactone alone (example 11), poly (lactide-co-glycolide) alone (example 12), polypropylene oxide alone (example 13), polycaprolactone + polyglycolic acid (example 14), polycaprolactone + poly (lactide-co-glycolide) (example 15), polycaprolactone + polypropylene oxide (example 16);
3) the dispersing auxiliary agent is selected from: hesperetin (example 17), no dispersing aid (example 18);
4) the acidic aqueous solution containing the cross-linking agent is selected from: aqueous hydrochloric acid (example 19), aqueous lactic acid (example 20), aqueous acetic acid + lactic acid (example 21), polyethylene glycol diglycidyl ether (example 22), four-armed star polyethylene glycol epoxide (example 23), polyethylene glycol diglycidyl ether + four-armed star polyethylene glycol epoxide (example 24);
5) the polypeptides were selected as: oligopeptide 1 (example 25), oligopeptide 3 (example 26), oligopeptide 5 (example 27), oligopeptide 6 (example 28);
6) the specific conditions of the crosslinking process are as follows: first 40 ℃/10h, second 20 ℃/24h (example 29), first 60 ℃/2h, second 50 ℃/2h (example 30), first 50 ℃/6h, second 30 ℃/15h (example 31).
2. The effect data is embodied from the following aspects:
1) dynamic viscosity test: measuring by using a rotational viscometer according to a VIG second method recorded in pharmacopoeia of the people's republic of China, and testing at the conditions that the shear rate is not less than 0.25Hz and the temperature is 25 ℃;
2) testing osmotic pressure;
3) the dosage of the cross-linking agent is as follows: compared with three prior art documents given in the background art, the prior art documents are respectively patent documents with publication numbers of CN 104258470A, CN 105879124A and CN 102911380A;
4) evaluating the effect of inhibiting inflammation; the grade is divided into A-inflammation recovery, B-inflammation partial recovery and C-inflammation no change;
5) verifying the absorption and metabolism time of the polysaccharide composite gel by a mouse experiment;
6) adverse reactions were determined in mouse experiments.
The mouse experiment of 5) and 6) is specifically carried out as follows:
evaluation of local injection reaction: SPF-grade Wistar rats were selected for testing, half male and female, weighing approximately 180-230 g. 45 rats were divided into 9 groups on average, and each group was injected with 5 gels or fillers as given in examples 3 to 31 and comparative examples 1 to 3 (i.e., patent documents with publication numbers CN 104258470 a, CN 105879124 a and CN 102911380 a given in the background art). The injection method comprises the following steps: 0.5ml of gel or filler is respectively implanted into the back subcutaneous part of a rat in an injection mode, the pushing difficulty degree of the injector in the injection process is sensed, and whether the adverse reactions such as red swelling, subcutaneous nodules and the like occur or not is observed after one week.
3. The results of the effect verification and the statistics of the effect data are shown in table 1 below.
As can be seen from Table 1, the kinematic viscosity of comparative examples 1-3 is significantly higher than that of examples 3-31, the amount of the crosslinking agent is also significantly higher than that of examples 3-31, and the inflammation inhibitory effect is poor and the adverse reaction is severe.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. The polysaccharide composite gel is characterized by being prepared from the following substances in parts by weight: 0.5-1.5 parts of polysaccharide substance, 0.1-10 parts of biodegradable high polymer material, 3-10 parts of acidic aqueous solution containing cross-linking agent, 0.2-6 parts of polypeptide and 0.5-3 parts of dispersing auxiliary agent.
2. The polysaccharide composite gel according to claim 1, wherein the concentration of acid in the acidic aqueous solution containing the cross-linking agent is 0.1-1 mmol/L; the acidic aqueous solution is one or more of hydrochloric acid aqueous solution, acetic acid aqueous solution and lactic acid aqueous solution; the cross-linking agent is any one or more of modified polyethylene glycol and 1, 4 butanediol diglycidyl ether.
3. The polysaccharide composite gel according to claim 1, wherein the polysaccharide substance is one or more of hyaluronic acid and a hyaluronate salt.
4. The polysaccharide composite gel according to claim 1, wherein the biodegradable polymer material is a polymer having a molecular weight of 5-9 kDa.
5. The polysaccharide composite gel according to claim 1, wherein the polypeptide is a small-molecule polypeptide with a molecular weight of less than 1kDa, which is obtained by condensation of 2-10 amino acids.
6. The polysaccharide composite gel according to claim 1, wherein the dispersion aid is hesperetin.
7. A method for preparing a polysaccharide complex gel, wherein the polysaccharide complex gel is the polysaccharide complex gel according to any one of claims 1 to 6, and the method comprises the following steps: dispersing and compounding polysaccharide and biodegradable polymer material, adding into acid water solution containing cross-linking agent, cross-linking at least twice, and adding polypeptide.
8. The preparation method of the polysaccharide composite gel according to claim 7, which is characterized by comprising the following steps:
s1, adding a biodegradable high polymer material and a dispersing aid into an aqueous solution of a polysaccharide substance, performing ultrasonic dispersion, uniformly mixing, and performing vacuum freeze drying to obtain a compound;
s2, adding the compound obtained in the step S1 into an acidic aqueous solution containing a cross-linking agent, uniformly mixing, adjusting the temperature to perform cross-linking at least twice, changing the cross-linking temperature after each cross-linking, and performing next cross-linking to obtain a cross-linked product;
s3, adjusting the pH value of the cross-linked product obtained in the step S2 to be neutral, dialyzing to obtain composite gel, and performing vacuum freeze drying for decrement again to obtain decrement composite gel;
s4, when the reduced composite gel obtained in the step S3 is reduced to 90-99.5%, adding polypeptide with the same amount dropwise, and mixing uniformly to obtain the polysaccharide composite gel.
9. The method as claimed in claim 8, wherein the ultrasonic dispersion conditions in step S1 are 100-200W, 30-40 ℃, 15-35 min; the temperature of the vacuum freeze drying is-10 ℃ to-50 ℃, and the drying time is 24-60 h; in the step S2, the temperature of each cross-linking is higher than the temperature of the next cross-linking, the number of cross-linking is two, the temperature of the first cross-linking is 40-60 ℃, and the time is 2-10 h; the temperature of the second crosslinking is 20-50 ℃, and the time is 2-24 h; in the step S3, a weak alkaline solution is used to adjust the pH value of the cross-linked product to 6.8-7.3, the dialysis method is to use a phosphate buffer solution to dialyze and purify the cross-linked product until the osmotic pressure is 270-330mmol/L, the conditions of vacuum freeze drying decrement are that the temperature is minus 10 ℃ to minus 50 ℃, the drying time is 24-60h, and the decrement is 20-30% of the original volume; the weak alkaline solution is sodium bicarbonate or disodium bicarbonate.
10. The application of the polysaccharide composite gel in preparing the filling agent for injection is characterized in that the polysaccharide composite gel is the polysaccharide composite gel of claims 1 to 6 or the polysaccharide composite gel prepared by the preparation method of claims 7 to 9.
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