CN102321248A - Injectable temperature sensitive gel used for filling and repairing damaged tissues - Google Patents
Injectable temperature sensitive gel used for filling and repairing damaged tissues Download PDFInfo
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Abstract
The invention relates to an injectable temperature sensitive gel used for filling and repairing damaged tissues. The gel is characterized in that: medical-grade hyaluronic acid and chitosan are used as gel raw materials; the gel swelling ratio is 2-500; and a temperature sensitive temperature is 32-37 DEG C. The gel provided by the invention has the advantages that the injection temperature sensitive gel has physical and chemical properties of a general gel, also has injectable and temperature sensitive properties and a good fluidity at a room temperature and is easy for injection; the formability in organisms is good and the degradation is slow so that the general gel can not be compared; a cross-linking agent is not used for the gel so that harms of the cross-linking agent to a human body are avoided; a gel system can prolong a persistence time of the gel in a body, and also can continuously stimulate the body to produce reactions so that the tissue in-situ regeneration and repair is promoted and the filling quality is increased; and the gel simultaneously has the advantages of reducing bacterial invansion, anticoagulation and low allergic reaction and the like.
Description
Technical field
The present invention relates to a kind of preparation method who is used to fill and repair the injectable thermo-sensitive gel of damaged tissue, belong to medical material method of manufacture technical field.
Background technology
Injectable filling and repair materials are a kind of intelligent macromolecule materials, are newer research fields that grows up over past ten years, are very popular research topics of intelligent macromolecule material.Temperature sensitive property injectable material is compared with conventional injectable material; Be to utilize bionic principle; Organism with occurring in nature is a source; Develop on function near in addition surpass the temperature sensitive macromolecular material of bio-tissue, thereby realize that the active biomaterial of lifeless matter is to the transition that bioactive biomaterial is arranged; Increase its response susceptibility, improve its logical-disconnected characteristic.The thermodynamics reversibility of material makes the mobile at normal temperatures enhancing of material, be beneficial to injection, and material temperature solidifies in the body, and its constancy and plasticity-strengthen.In addition injectable gel in vivo and between the repair tissue mosaic good, realize that in-situ injection reaches Wicresoft's purpose, become the focus of soft tissue repair, reconstruction, filling.Therefore, the application of the injectable gelatinous material of temperature sensitive property has significant advantage.
In recent years, the development of skin of face filling agent is very fast, and the annual growth of its produce market is then to be expected in 16.9%, 2012 year to surpass 1,600,000,000 U.S. dollars, and wherein hyaluronic acid product accounts for the largest percentage.In the technology of existing injectable weighting material and preparation method thereof, the normal injection weighting material that adopts is that crosslinked mucinase and collagen protein is main its salts solution of gel.For example: by the auspicious indigo plant of mucinase dermal filler (restvlane) of exploitation of Sweden Q-Med company and production.
Hyaluronic Acid (mucinase) is a kind of saccharan that extensively is present in biological and types of organization.The Karl Meyer of Columbia University had at first found mucinase in 1934, was one of staple of extracellular matrix, indifference between each species on the chemical structure.Mucinase is absorption and the primary structure of keeping moisture in skin, aspect storing moisture and locking water function, can absorb nearly 3000 times of water volume of storage effectively, and non-general NMF can provide.Mucinase is except making skin keep moisture, can also making eyes keep lubricated, joint maintenance elasticity.Therefore, mucinase has very good service recorder in fields such as ophthalmologic operation, arthritis treatment and Cosmetics Surgerys.Physics and biological natures such as but pure HA is soluble in water, absorb rapidly and the residence time is short in tissue limit its use as the functional component carrier, need to use chemically crosslinked to modify, and to increase its molecular weight, to prolong degradation time, improve stability.
But hyaluronicly simple crosslinkedly can not alleviate it and degrade fast.Still need repeatedly filling to reach repairing effect, therefore the patient is caused the certain economic burden.With regard to the material that makes up dermal filler and tissue repair, the thermodynamic (al) reversibility of material is most important.Though mucinase is to promote the biomaterial of tissue growth still not possess thermodynamic (al) reversibility.A kind of injectable thermo-sensitive gel of the present invention's preparation, this gelling system has the synergy of Hyaluronic Acid and chitosan, has not only prolonged the time that the injection site is repaired, and has been easier to injection, and had antibiotic, anticoagulation, character such as the interior good moldability of organism.
Summary of the invention
The invention provides a kind of preparation method of thermo-sensitive gel.Notable feature of the present invention is: chitosan is introduced temperature sensitive functional group through handling and chemical reaction, the preparation thermo-sensitive gel.Mucinase is directly handled with sodium periodate without crosslinked, obtains active aldehyde radical, reacts through the amino group on chemical reaction and the chitosan, obtains having the plural gel of temperature sensitive performance.In this method of manufacture, plural gel does not need crosslinking polymerization, has simplified production stage, has practiced thrift production cost.Temperature-sensitive makes its good fluidity at normal temperatures simultaneously, is easy to injection; Good moldability in the organism, degraded are slowly.
For realizing above-mentioned purpose, technical scheme of the present invention is following:
The purpose of this invention is to provide a kind of preparation method who prepares temperature sensitive plural gel through chemical reaction.
The contriver prepares temperature sensitive chitosan and the hyaluronic preparation of active aldehyde radical through sec.-propyl propionic acid amide in the grafting, has the gel of temperature-sensitive through the chemical reaction preparation, thereby has the injection of being prone to and persistent character.
The preparation method who is prone to the injection thermo-sensitive gel may further comprise the steps:
(1) active aldehyde radical mucinase preparation
With raw material mucinase (HA) and deionized water wiring solution-forming.Add the sodium periodate aqueous solution, stirring reaction under lucifuge, room temperature condition.Add ethylene glycol solution and stop unreacted sodium periodate.
Above-mentioned raw materials mucinase (HA) and deionized water are made into concentration for being 0.01～20g/100ml;
Above-mentioned sodium periodate concentration of aqueous solution is 0.1～10M, and volume is 1～100ml
(2) gel that step (1) is made is removed unreacted monomer and other impurity with soaked in absolute ethyl alcohol, washing, extracting, solution dialysis purifying then, and lyophilize obtains purified jel product.
(3) temperature sensitive Preparation of Chitosan
An amount of chitosan is dissolved in 1～98% the acetic acid soln, slowly adds a certain amount of N-sec.-propyl propionic acid amide (NIPAAm) and initiator, after the stirring at room dissolving, feed nitrogen stirring reaction at a certain temperature.Above-mentioned reactant is used acetone precipitation, filter.Vacuum-drying makes the jel product with temperature-sensitive;
Above-mentioned additive N-sec.-propyl propionic acid amide (NIPAAm) is 1: 90～90: 1 with chitosan by mass ratio.
Above-mentioned initiator: comprise a kind of of Potassium Persulphate (KPS), Diisopropyl azodicarboxylate, peroxo-two acyls, Lucidol or di-t-butyl peroxide.
The mass ratio of above-mentioned initiator and chitosan is 0.02%～10%.
(4) preparation of plural gel
Under the room temperature, two kinds of gels in step (2) and (3) are made into the homogeneous aqueous solution by the certain mass ratio, stirring reaction prepares plural gel under the room temperature.This gelating soln dialysis purifying then, lyophilize obtains purified jel product.With above-mentioned two kinds of gels under room temperature with the abundant swelling of damping fluid PBS, be made into injection liquid, and through needle tubing, promote homogenize with constant speed, obtain injectable hydrogel.
The proportioning of mucinase and chitosan is 1: 9～9: 1 in the above-mentioned plural gel
The concentration of above-mentioned plural gel and damping fluid PBS is 0.01～30mg/ml
(5) each factor in the above-mentioned copolymerization process (like different monomers, initiator, temperature, reaction times etc.) is influenced rule research to product property; Adopt orthogonal experiment; Each factor is estimated,, building-up process is carried out computer simulation understanding in depth on the basis that influences rule.
Beneficial effect:
1. the present invention prepares that to be prone to the injection thermo-sensitive gel be preparation and the hyaluronic preparation of active aldehyde radical through temperature sensitive chitosan; Through under the chemical reaction effect chitosan and mucinase being combined; Make gel with temperature-sensitive; The gel that makes is the liquid of normal temperature current downflow, is gel state in vivo under the temperature.Have swelling property, temperature sensitive property, be prone to promotion, characteristics such as antibiotic, persistence, good biocompatibility, be that a kind of ideal is filled and the material of repairing.The thermo-responsive hydro gel of the present invention's preparation has reduced the infringement of linking agent to human body and injection site.This gelling system can not only prolong its RT in vivo, and can constantly stimulate the body generation to reply, and promptly promotes the in-situ regeneration reparation of tissue, to improve filling quality.Simultaneously because the adding of chitosan has reduced advantages such as intrusion and the anaphylaxis of bacterium be low.The restoration, healing that can be widely used in various impaired organs are with aspects such as equipment, prosthese, implants in vivo, drug delivery system and medical and beauty treatments.
Plural gel have high purity, toxicological harmless, nonirritant, chemicalstability good, degrade, do not cause inflammation, do not have anaphylaxis, good biocompatibility, not carcinogenic, heavy metal content is low, pH value is little, does not cause performances such as haemolysis and blood coagulation.Plural gel also should have required physics, mechanical property and processing characteristics, also can stand high-temperature disinfection treatment.
3. the synergy of plural gel has certain intensity and flexural stiffness, can resist tissue repairing's ability that the damaged body of human body is pressed.In addition, the gel that simple cross-linked-hyaluronic acid obtains, because its transformation period is short, degraded fast.Therefore need multiple injection, need bigger Financial cost.But plural gel is because the bacterinertness of chitosan, the healing at the position that helps being wound and be difficult for causing the infection at position of being wound.Owing to having temperature-sensitive, it has syringeability and biocompatibility preferably simultaneously.And its long transformation period makes it have permanence, does not need multiple injection, and the economic pressures and the multiple injection that reduce the patient are painful.Because plural gel is degradable, and when the injection site deformed or be undesirable, available lyase dissolved, do not need underwent operative to remove in addition, reduce patient's misery.
Description of drawings
Fig. 1 is the injectable thermo-sensitive gel reaction synoptic diagram that the embodiment of the invention 2 is prepared.
Fig. 2 is the infrared spectrogram of the injectable thermo-sensitive gel prepared of the embodiment of the invention 2.
Fig. 3 is the phase transformation analysis of the injectable thermo-sensitive gel prepared of the embodiment of the invention 2.
Fig. 4 is the swelling kinetic curve of the injectable thermo-sensitive gel prepared of the embodiment of the invention 2.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
(1) the hyaluronic preparation of active aldehyde radical:
Raw material mucinase and deionized water are mixed by proportioning 0.01g/100ml, add 0.1M, the sodium periodate aqueous solution of 100mL at room temperature adds ethylene glycol solution behind the lucifuge stirring reaction, and stirring reaction makes the aldehyde radical mucinase at normal temperatures;
(2) with the gel that makes with soaked in absolute ethyl alcohol, washing, extracting, remove unreacted monomer and other impurity, solution dialysis purifying then, lyophilize obtains purified jel product;
(3) temperature sensitive Preparation of Chitosan:
The CS of 0.2g is dissolved in 1% the acetic acid soln, slowly adds 0.2g N-sec.-propyl propionic acid amide (NIPAAm) and 4mg Potassium Persulphate (KPS), after the stirring at room dissolving, feed the nitrogen stirring reaction.Above-mentioned reactant is used acetone precipitation, filter.Vacuum-drying makes the jel product with temperature-sensitive;
(4) preparation of plural gel:
(4.1) under the room temperature, the temperature sensitive chitosan gel rubber that hyaluronic acid derivatives that step (2) is obtained and step (3) obtain is made into the aqueous solution of 20mg/mL, stirring reaction under the room temperature, preparation plural gel with above-mentioned two kinds of gel mucinases and chitosan by mass ratio 1/9;
(4.2) with the gelating soln dialysis purifying of above-mentioned steps (4.1), lyophilize obtains purified jel product.Under room temperature, use damping fluid PBS to be mixed with concentration above-mentioned two kinds of gels and be the abundant swelling of the solution of 1mg/ml, and the needle tubing through 2.5ml, homogenize 3 times promoted with constant speed.Obtain injectable hydrogel.
Embodiment 2
(1) the hyaluronic preparation of active aldehyde radical:
Raw material mucinase and deionized water are mixed by proportioning 1g/100ml, add 0.5M, the sodium periodate aqueous solution of 5mL, at room temperature the lucifuge stirring reaction reacts the end back and adds ethylene glycol solution, stirring reaction at normal temperatures.Make hyaluronic acid derivatives;
(2) with the gel that makes with soaked in absolute ethyl alcohol, washing, extracting, remove unreacted monomer and other impurity, solution dialysis purifying then, lyophilize obtains purified jel product;
(3) temperature sensitive Preparation of Chitosan:
The chitosan of 0.2g is dissolved in 5% the acetic acid soln, slowly adds 2g N-sec.-propyl propionic acid amide (NIPAAm) and 4mg Diisopropyl azodicarboxylate, after the stirring at room dissolving, feed the nitrogen stirring reaction.Above-mentioned reactant is used acetone precipitation, filter.Vacuum-drying makes the jel product with temperature-sensitive;
(4) preparation of plural gel:
(4.1) under the room temperature, the temperature sensitive chitosan gel rubber that mucinase that step (2) is obtained and step (3) obtain is made into the aqueous solution of 20mg/ml, prepared in reaction plural gel under the room temperature with above-mentioned two kinds of gel mucinases and chitosan by mass ratio 1/9;
(4.2) with the gelating soln dialysis purifying of above-mentioned steps (4.1), lyophilize obtains purified jel product.Under room temperature, use damping fluid PBS to be mixed with concentration above-mentioned two kinds of gels and be the abundant swelling of the solution of 20mg/ml, and the needle tubing through 2.5ml, homogenize 3 times promoted with constant speed.Obtain injectable hydrogel.
Embodiment 3
(1) the hyaluronic preparation of active aldehyde radical:
Raw material mucinase and deionized water are mixed by proportioning 20g/100ml, add 10M, the sodium periodate aqueous solution of 100ml, at room temperature the lucifuge stirring reaction reacts the end back and adds ethylene glycol solution, stirring reaction at normal temperatures.Make hyaluronic acid derivatives;
(2) with the gel that makes with soaked in absolute ethyl alcohol, washing, extracting, remove unreacted monomer and other impurity, solution dialysis purifying then, lyophilize obtains purified jel product;
(3) temperature sensitive Preparation of Chitosan:
The CS of 0.2g is dissolved in 98% acetic acid soln, slowly adds 18g N-sec.-propyl propionic acid amide (NIPAAm) and 1mg Potassium Persulphate (KPS), after the stirring at room dissolving, feed the nitrogen stirring reaction.Above-mentioned reactant is used acetone precipitation, filter.Vacuum-drying makes the jel product with temperature-sensitive;
(4) preparation of plural gel:
(4.1) under the room temperature, the temperature sensitive chitosan gel rubber that mucinase that step (2) is obtained and step (3) obtain is made into the aqueous solution of 20mg/ml, stirring reaction under the room temperature, preparation plural gel with above-mentioned two kinds of gel mucinases and chitosan by mass ratio 5/5;
(4.2) with the gelating soln dialysis purifying of above-mentioned steps (4.1), lyophilize obtains purified jel product.Under room temperature, use damping fluid PBS to be mixed with concentration above-mentioned two kinds of gels and be the abundant swelling of the solution of 50mg/ml, and the needle tubing through 2.5ml, homogenize 3 times promoted with constant speed.Obtain injectable hydrogel.
Embodiment 4
(1) the hyaluronic preparation of active aldehyde radical:
Raw material mucinase and deionized water are mixed by proportioning at 10: 100, add 0.5M, the sodium periodate aqueous solution of 50ml, at room temperature the lucifuge stirring reaction reacts the end back and adds ethylene glycol solution, stirring reaction at normal temperatures.Make hyaluronic acid derivatives;
(2) with the gel that makes with soaked in absolute ethyl alcohol, washing, extracting, remove unreacted monomer and other impurity, solution dialysis purifying then, lyophilize obtains purified jel product;
(3) temperature sensitive Preparation of Chitosan:
The CS of 0.2g is dissolved in 20% the acetic acid soln, slowly adds 2g N-sec.-propyl propionic acid amide (NIPAAm) and 2g Lucidol, after the stirring at room dissolving, feed the nitrogen stirring reaction.Above-mentioned reactant is used acetone precipitation, filter.Vacuum-drying makes the jel product with temperature-sensitive;
(4) preparation of plural gel:
(4.1) under the room temperature, the temperature sensitive chitosan gel rubber that mucinase that step (2) is obtained and step (3) obtain is made into the aqueous solution of 1mg/ml, stirring reaction under the room temperature, preparation plural gel with above-mentioned two kinds of gel mucinases and chitosan by mass ratio 5/5;
(4.2) with the gelating soln dialysis purifying of above-mentioned steps (4.1), lyophilize obtains purified jel product.Under room temperature, use damping fluid PBS to be mixed with concentration above-mentioned two kinds of gels and be the abundant swelling of the solution of 0.01mg/ml, and the needle tubing through 2.5ml, homogenize 3 times promoted with constant speed.Obtain injectable hydrogel.
Embodiment 5
(1) the hyaluronic preparation of active aldehyde radical:
Raw material mucinase and deionized water are mixed by proportioning 1: 100mg/ml, add 0.5M, the sodium periodate aqueous solution of 5mL, at room temperature the lucifuge stirring reaction reacts the end back and adds ethylene glycol solution, stirring reaction at normal temperatures.Make hyaluronic acid derivatives;
(2) with the gel that makes with soaked in absolute ethyl alcohol, washing, extracting, remove unreacted monomer and other impurity, solution dialysis purifying then, lyophilize obtains purified jel product;
(3) temperature sensitive Preparation of Chitosan:
The CS of 0.2g is dissolved in 50% the acetic acid soln, slowly adds 2g N-sec.-propyl propionic acid amide (NIPAAm) and 4mg Potassium Persulphate (KPS), after the stirring at room dissolving, feed the nitrogen stirring reaction.Above-mentioned reactant is used acetone precipitation, filter.Vacuum-drying makes the jel product with temperature-sensitive;
(4) preparation of plural gel:
(4.1) under the room temperature, the temperature sensitive chitosan gel rubber that mucinase that step (2) is obtained and step (3) obtain is made into the aqueous solution of 20mg/ml, stirring reaction under the room temperature, preparation plural gel with above-mentioned two kinds of gel mucinases and chitosan by mass ratio 5/5;
(4.2) with the gelating soln dialysis purifying of above-mentioned steps (4.1), lyophilize obtains purified jel product.Under room temperature, use damping fluid PBS to be mixed with concentration above-mentioned two kinds of gels and be the abundant swelling of the solution of 50mg/ml, and the needle tubing through 2.5ml, homogenize 3 times promoted with constant speed.Obtain injectable hydrogel.
Claims (9)
1. preparation method who is used to fill and repair the injectable thermo-sensitive gel of damaged tissue is characterized in that this method may further comprise the steps:
(1) active aldehyde radical mucinase preparation: raw material mucinase (HA) and deionized water are made into certain density solution.Add the sodium periodate aqueous solution, stirring reaction under lucifuge, room temperature condition.Add ethylene glycol solution and stop unreacted sodium periodate.
(2) gel that step (1) is made is removed unreacted monomer and other impurity with soaked in absolute ethyl alcohol, washing, extracting, solution dialysis purifying then, and lyophilize obtains purified jel product.
(3) temperature sensitive Preparation of Chitosan: an amount of chitosan is dissolved in 1～98% the acetic acid soln, slowly adds a certain amount of N-sec.-propyl propionic acid amide (NIPAAm) and initiator, after the stirring at room dissolving, feed nitrogen stirring reaction at a certain temperature.Above-mentioned reactant is used acetone precipitation, filter.Vacuum-drying makes the jel product with temperature-sensitive;
(4) preparation of plural gel: under the room temperature, two kinds of gels in step (2) and (3) are made into the homogeneous aqueous solution by the certain mass ratio, stirring reaction prepares plural gel under the room temperature.This gelating soln dialysis purifying then, lyophilize obtains purified jel product.With above-mentioned two kinds of gels under room temperature with the abundant swelling of damping fluid PBS, be made into injection liquid, and through needle tubing, promote homogenize with constant speed.Obtain injectable hydrogel.
2. method according to claim 1 is characterized in that, the mucinase raw material of employing is for extracting from microbe body or obtaining through fermentation.Molecular mass is 20,000 to 3,000,000.
3. method according to claim 1 is characterized in that, raw material mucinase (HA) and deionized water are made into concentration for being 0.01～50: 100mg/ml.
4. method according to claim 1 is characterized in that, the sodium periodate concentration of aqueous solution is 0.1～10M, and volume is 1～100ml.
5. method according to claim 1 is characterized in that chitosan comes from de-acetyl chitin, and deacetylation is 15～95%.
6. method according to claim 1 is characterized in that, additive N-sec.-propyl propionic acid amide (NIPAAm) is interpolation in 1: 90～90: 1 with chitosan by mass ratio.
7. method according to claim 1 is characterized in that, initiator is a kind of of Potassium Persulphate (KPS), Diisopropyl azodicarboxylate, peroxo-two acyls, Lucidol or di-t-butyl peroxide; The mass ratio of initiator and chitosan is 0.02%～10%.
8. method according to claim 1 is characterized in that, the proportioning of mucinase and chitosan is 1: 9～9: 1 in the plural gel
9. method according to claim 1 is characterized in that, the concentration of plural gel and damping fluid is 0.01～30mg/ml.
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