CN101062017B - Hyaluronic acid and preparation process thereof - Google Patents
Hyaluronic acid and preparation process thereof Download PDFInfo
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- CN101062017B CN101062017B CN200610078103A CN200610078103A CN101062017B CN 101062017 B CN101062017 B CN 101062017B CN 200610078103 A CN200610078103 A CN 200610078103A CN 200610078103 A CN200610078103 A CN 200610078103A CN 101062017 B CN101062017 B CN 101062017B
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Abstract
The invention discloses a hyaluronic acid particle, which is characterized by the following: comprising a polymer; reacting hyaluronic acid and alkylate with aldolisation; getting the polymer; comprising at least one hydroxide radical in the hyaluronic acid; comprising at least dialdehyde in the alkylate; proceeding aldolisation with the hydroxide radical and aldehyde group; generating crosslinking bridge formation among the hyaluronic acid; crosslinking the hyaluronic acid and alkylate in emulsion; getting the product. This product can be used to hypodermic injection and biological medical treatment.
Description
Technical field
The present invention relates to a kind of hyaluronic acid, particularly a kind of hyaluronic acid particle.
The invention still further relates to the above-mentioned hyaluronic method of preparation, refer to a kind of method for preparing the hyaluronic acid particle that is applicable to especially.
Background technology
By tanning by the sun or cutis laxa and wrinkle that other factors causes of aging, sunlight, often can be improved by subcutaneous filling injection (soft tissue augment) mode, its benefit is simply, accurate and do not have a wound.The material that is usually used in subcutaneous filling injection at present has silica gel, Teflon glue, collagen protein and hyaluronic acid ... or the like.Yet subcutaneous filling injection can be subjected to its injection needle restriction, makes its implant must be limited to the material that can be injected by pin hole smoothly.
Hyaluronic acid (Hyaluronic Acid, claim hyaluronic acid again) be a kind of natural polysaccharides, be present in crystalline tissue of vertebrate connective tissue, mucous tissue and eyeball or the like at occurring in nature widely, have quite good bio-compatibility and decomposability.In addition, hyaluronic acid also has powerful filling and moisture-keeping functions.Therefore, now be widely used in subcutaneous filling injection, other is used and also comprises pharmaceutical material and cosmetics etc.
Therefore, in the technique known, have the people that single candy or few candy are made cross-linking agent with multi-functional acyl group, it is crosslinked at room temperature to utilize emulsion reaction to carry out.Make single candy or few candy through being cross-linked to form micron or nanoparticle; And the molecular weight of its carbohydrate is limited in below 5000.Someone is that cross-linking agent is prepared into viscoelastic colloidal with the macromolecule polysaccharides of bio-compatibility to go back the oxygen base, and this colloid composition is water miscible polysaccharides glue.The people is also arranged with the hyaluronic acid or derivatives thereof, as cross-linking agent, produce the hyaluronic acid colloid with the reagent of phosphorus element-containing.
Yet hyaluronic molecular weight can directly influence its soaking effect.Just do not fill and moistening effect if the hyaluronic acid molecular weight is too low, loosely organized.So the hyaluronan molecule amount is bigger, network structure is more complete, its suction and filling effect are just better.Unfortunately, hyaluronic molecular weight also can influence its viscosity.Under finite concentration, hyaluronic molecular weight is bigger, and viscosity is just bigger, makes high-molecular weight hyaluronic acid can't pass the injection orifice of syringe cylinder smoothly.Therefore, present hyaluronic application is restricted to lower concentration and molecular weight.
Summary of the invention
The purpose of this invention is to provide a kind of hyaluronic acid.
Another purpose of the present invention provides the above-mentioned hyaluronic method of preparation.
For achieving the above object, hyaluronic acid provided by the invention is the hyaluronic acid particle of a kind of particle diameter less than 1 μ m and tool bio-compatibility, and its viscosity under identical concentration is hyaluronic 75% less than straight chain, and wherein this concentration is greater than 1wt%.
Described hyaluronic acid particle, wherein the viscosity of this hyaluronic acid particle is to measure with rotary viscosimeter.
Described hyaluronic acid particle, wherein this hyaluronic acid particle is used in the human body implantation.
Particle diameter of the present invention is less than the hyaluronic acid particle of 1 μ m, comprise a polymer by at least one hyaluronic acid and one and the alkyl compound reaction that produces aldolisation of this hyaluronic acid form, wherein this hyaluronic acid includes at least one hydroxy, this alkyl compound includes dialdehyde-based at least, this hydroxy and this aldehyde radical produce crosslinked bridge formation via aldolisation between this hyaluronic acid, and this hyaluronic acid and this alkyl compound be in emulsion, carry out crosslinked.
Described hyaluronic acid particle, wherein this hyaluronic acid particle is used in the human body implantation.
Described hyaluronic acid particle, wherein this hyaluronic molecular weight is between 30,000 to 5000,000.
Described hyaluronic acid particle, the wherein preparation of this emulsion, be with an organic solvent and an emulsifying agent according to the mixed of weight ratio 1: 0.01~0.3 to form this emulsion.
Described hyaluronic acid particle, wherein this organic solvent is olive oil, mineral oil, glycerol, toluene or isobutyltrimethylmethane..
Described hyaluronic acid particle, wherein this emulsifying agent is ionic emulsifying agent, nonionic emulsifier or amphoteric emulsifying agent.
Described hyaluronic acid particle, wherein this alkyl compound has the structure as shown in the formula (I):
R-(CHO)
n(I)
Wherein n is 2 to 4 integer, and R is C
3To C
15Alkyl, C
3To C
15Cycloalkyl, six carbon candy rings or five carbon candy rings.
Described hyaluronic acid particle, wherein this alkyl compound is glutaraldehyde (Glutaraldehyde).
The method of the above-mentioned hyaluronic acid particle of preparation provided by the invention may further comprise the steps:
(A) with an organic solvent and an emulsifier, form one first mixed solution, wherein the weight ratio of this organic solvent and this emulsifying agent be 1: 0.01 to 0.3;
(B) with a hyaluronic acid and water according to mixing, form one second mixed solution, wherein the weight ratio of this hyaluronic acid and this water is 0.001 to 0.08: 1, the weight ratio of this organic solvent and this water is at least greater than 5: 1;
(C) this second mixed solution is added formation one the 3rd mixed solution in this first mixed solution;
(D) with a chemical compound as shown in the formula (I)
R-(CHO)
n(I)
Add in the 3rd mixed solution, form one the 4th mixed solution, and carry out cross-linking reaction, wherein n is 2 to 4 integer, and R is C
3To C
15Alkyl, C
3To C
15Cycloalkyl, six carbon candy rings or five carbon candy rings;
(E) stir the 4th mixed solution; And
(F) filter the 4th mixed solution, to obtain a precipitate.
Described preparation method, wherein this organic solvent is olive oil, mineral oil, glycerol, toluene or isobutyltrimethylmethane..
Described preparation method, wherein this emulsifying agent is a nonionic emulsifier.
Described preparation method, wherein this emulsifying agent is Polyethylene Glycol 8 monostearates (PEG (8) monostearate), Polyethylene Glycol 8 Laurel ethers (PEG (8) laurylether), sorbitan monostearate (Sorbitan monosterate), sorbitan mono-oleic acid ester (this Pan 80) (Sorbitan monooleate (Span80)), Polyethylene Glycol 20 sorbitol anhydride monopalmitates (PEG (20) Sorbitanmonolaurate), Polyethylene Glycol 20 sorbitan mono-oleic acid esters (PEG (20) Sorbitanmonooleate), Polyethylene Glycol 40 sorbitan monostearates (PEG (40) Sorbitanmonostearte), nonyl phenol 10 (Nonylphenol (10)), nonyl phenol 20 (Nonylphenol (20)), nonyl phenol 7.5 (Nonylphenol (7.5)), octyl phenol 10 (Octylphenol (10)), monopalmitin (Glycerinemonolaurate), monoglyceride (Monoglyceride), fatty glyceride (mono and diglyceride), or leaf fat monoglyceride (Lard monoglyceride), propylene glycol ethylene oxide copolymer (Pluronic F68).
Described preparation method, wherein this emulsifying agent is an anionic emulsifier.
Described preparation method, wherein this emulsifying agent is sodium alkyl benzene sulfonate (Alkyl benzenesulfonic acid sodium), sulphuric acid ten diester sodium (C
12H
25OSO
3Na (SDS)), sodium hexadecyl sulfate (C
16H
33OSO
3Na), sodium stearate (C
17H
35COON), succinic acid monooctyl ester sulfonate (Dioctylsulfosuccinate), alkylaryl hydroxide ethylsulfonic acid ester (Alkyl hyroxyethane sulfonate) or polyoxyethylene alkylphenol sulphonic acid ester (Polyoxy ethylene alkyl phenol sulfonate).
Described preparation method, wherein this emulsifying agent is a cationic emulsifier.
Described preparation method, wherein this emulsifying agent is hexadecyltrimethylammonium chloride (Cetyltrimethyl ammonium chloride), cetyl trimethyl ammonium bromide (Cetyltrimethylammonium bromide), hexadecyldimethyl benzyl ammonium ammonium bromide (Cetyldimethyl ammoniumbromide), chlorination Petiolus Trachycarpi hexadecyldimethyl benzyl ammonium amine (Palmiticalkyl dimethyl benzylammonium chloride), cetyl chloride arsenic pyridine (Cetylpyridinium chloride), zephiran (Alkyl dimethyl chlorobenzyl ammonium chloride), or Fluhyzon chlorination arsenic pyridine (Alkyl naphthalene pyridinium chloride).
Described preparation method, wherein this emulsifying agent is the amphoteric emulsifying agent.
Described preparation method, wherein this emulsifying agent lauryl amine base ethyl Ammoniom-Acetate (Dodecyl aminoethyl glycine), oleamide (Oleic acid amide), lauric acid diethyl amide (Lauricdiethanol amide) or diethanolamine (Diethanol amine).
Described preparation method, the chemical compound of its Chinese style (I) are glutaraldehyde (Glutaraldehyde).
Described preparation method, wherein the rotating speed of this step (E) stirring is 5,000 to 25, between the 000rpm.
Described preparation method, wherein this step (E) stirs with homogenizer.
Described preparation method, wherein this step (F) is finished with centrifuge.
Described preparation method comprises also that wherein a step (G) cleans this precipitate.
Described preparation method, wherein this step (G) is finished with organic solvent, evacuation, super dialysis or its combination.
Furtherly, particle diameter of the present invention is less than the hyaluronic acid particle of 1 μ m, and (greater than 1wt%) viscosity is less than straight chain hyaluronic 75% under identical concentration.The molecular weight of this hyaluronic acid particle can be preferably 30,000 to 5000,000 between more than 30,000, is more preferred from 150,000 to 2500,000.The hyaluronic molecular weight of this straight chain can be preferably between 30,000 to 5000,000 between more than 30,000.The viscosity of this hyaluronic acid particle can any known mode be measured, and preferablely measures with rotary viscosimeter.And, because hyaluronic acid particle of the present invention does not have bio-toxicity,, especially be applicable to the implantation of human body therefore applicable to any known application.
Therefore, even hyaluronic acid particle molecular weight of the present invention is very high, its viscosity still can significantly reduce.That is to say that under identical concentration (greater than 1wt%), the viscosity of hyaluronic acid particle of the present invention is less than straight chain hyaluronic 75%.Therefore, hyaluronic acid particle of the present invention can solve high-molecular weight hyaluronic acid because viscosity is too high wayward when injection, and is not suitable for the problem of subcutaneous filling injection.
In addition, the organic solvent that the preparation method of hyaluronic acid particle of the present invention is used can be any known and immiscible solvent of water, is preferably olive oil (oliver oil), mineral oil (Mineral oil), glycerol (Glycerol), isobutyltrimethylmethane. (Isooctane), toluene (Toluene) etc.The emulsifying agent that preparation method of the present invention is used can be any public nonionic emulsifier, anionic emulsifier, amphoteric emulsifying agent, or cationic emulsifier.Nonionic emulsifier is preferably Polyethylene Glycol 8 monostearates (PEG (8) monostearate), Polyethylene Glycol 8 Laurel ethers (PEG (8) laurylether), Sorbitanmonostearte, sorbitan mono-oleic acid ester (this Pan 80) (Sorbitan monooleate (Span80)), PEG (20) Sorbitan monolaurate, PEG (20) Sorbitan monooleate, Polyethylene Glycol 40 sorbitan monostearates (PEG (40) Sorbitan monostearte), nonyl phenol 10 (Nonylphenol (10)), nonyl phenol 20 (Nonylphenol (20)), nonyl phenol 7.5 (Nonylphenol (7.5)), octyl phenol 10 (Octylphenol (10)), monopalmitin (Glycerinemonolaurate), monoglyceride (Monoglyceride), fatty glyceride (monoand diglyceride), or leaf fat monoglyceride (Lard monoglyceride), propylene glycol ethylene oxide copolymer (Pluronic F68).In the anionic emulsifier, be preferably sodium alkyl benzene sulfonate (Alkylbenzene sulfonic acid sodium), sulphuric acid ten diester sodium (C
12H
25OSO
3Na (SDS)), sodium hexadecyl sulfate (C
16H
33OSO
3Na), sodium stearate (C
17H
35COON), succinic acid monooctyl ester sulfonate (Dioctyl sulfosuccinate), alkylaryl hydroxide ethylsulfonic acid ester (Alkyl hyroxyethanesulfonate) or polyoxyethylene alkylphenol sulphonic acid ester (Polyoxy ethylene alkyl phenolsulfonate).Be preferably hexadecyltrimethylammonium chloride (Cetyltrimethylammonium chloride) in the cationic emulsifier, cetyl trimethyl ammonium bromide (Cetyltrimethyl ammoniumbromide), hexadecyldimethyl benzyl ammonium ammonium bromide (Cetyldimethyl ammonium bromide), chlorination Petiolus Trachycarpi hexadecyldimethyl benzyl ammonium amine (Palmiticalkyl dimethyl benzyl ammoniumchloride), cetyl chloride arsenic pyridine (Cetylpyridinium chloride), zephiran (Alkyl dimethyl chlorobenzyl ammonium chloride), or Fluhyzon chlorination arsenic pyridine (Alkyl naphthalene pyridinium chloride).Be preferably lauryl amine base ethyl Ammoniom-Acetate (Dodecyl amino ethyl glycine), oleamide (Oleic acidamide), lauric acid diethyl amide (Lauric diethanol amide) or diethanolamine (Diethanolamine) in the amphoteric emulsifying agent.In the preparation method of the present invention, the alkyl of the chemical compound of formula (I) can be C
3To C
15Alkyl is preferably C
3To C
10Alkyl is more preferred from amyl group (that is the chemical compound of formula (I) is glutaraldehyde (Glutaraldehyde)).
In the preparation method of the present invention, the rotating speed that step (E) stirs can be any rotating speed, is preferably between 5000~25000rpm.Preparation method of the present invention, wherein step (E) can any known mode be finished, and is preferably with homogenizer and stirs.Preparation method of the present invention, wherein step (F) can any known mode be finished, and is preferably with centrifuge and finishes.Preparation method of the present invention can comprise more that a step (G) cleans this precipitate, this step (G) can any known mode or solvent finish, be preferably with normal hexane (n-hexane), acetone (acetone), evacuation, super dialysis or its combination and finished.
Description of drawings
Fig. 1 is the particle size distribution figure of one embodiment of the invention.
Fig. 2 is the bio-toxicity comparison diagram of one embodiment of the invention.
Fig. 3 is the particle size distribution figure of another embodiment of the present invention.
Fig. 4 is the bio-toxicity comparison diagram of another embodiment of the present invention.
The specific embodiment
Embodiment one: 1,730K level hyaluronic acid emulsifying processing procedure experiment: oil/water 40/1, rotating speed 8,000rpm
(Oliver oil) inserts in the reaction bulb with the 500g olive oil, getting 50g Si Pan 80 (span80) pours in the olive oil and stirs. get the hyaluronic acid (SciVision of 0.094g, MW:1730K) be dissolved in the 12.5ml secondary water, 0.125g glutaraldehyde (Glutaraldehyde) be dissolved in the 10ml secondary water, place respectively in two isobaric funnels, earlier hyaluronic acid aqueous solution is delayed and splash in the olive oil, again glutaraldehyde water solution is splashed into, with homogenizer 8,000rpm stirs, temperature is 0 ℃, reaction mixing time 4 hours. the product that reaction is finished separates the water oil reservoir with centrifuge earlier, collect the water layer product, clean for several times with normal hexane (n-Hexane) and acetone (Acetone) again, collect product and dry, place vacuum equipment that solvent is drained product, utilizing water dialysis that residue is removed, obtaining end product in the lyophilization mode at last and its collection.
Test result
Hyaluronic acid nanometer microgranule poured in the secondary water stir, (DynamicLight Scattering, DLS, Malvern Zeta 1000HS) measures its size, mean diameter (Z with dynamic light scattering
Ave) be 637.2nm, as shown in Figure 1.
In addition, hyaluronic bio-toxicity also is one of the index of clinical practice.Therefore, with MTT tetramethyl fixed nitrogen azoles salt (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) (ISO 10993-5 (E)) cytotoxicity experiment carries out the bio-toxicity analysis of product, can find that by Fig. 2 its cell survival rate is close with primary hyaluronic acid raw material.Be with, the hyaluronic acid nanometer microgranule of present embodiment is the subcutaneous filling injection that is applicable to human body, medical and cosmetic material.
Embodiment two: 1730K level hyaluronic acid emulsifying processing procedure experiment: oil/water 20/1, rotating speed 8000rpm
(Oliver oil) inserts in the reaction bulb with the 500g olive oil, gets 50g Si Pan 80 (span80) and pours in the olive oil and stir.Get the hyaluronic acid (SciVision of 0.188g, MW:1730K) be dissolved in the 25ml secondary water, 0.125g glutaraldehyde (Glutaraldehyde) be dissolved in the 10ml secondary water, place respectively in two isobaric funnels, first hyaluronic acid aqueous solution is delayed splashes in the olive oil, glutaraldehyde water solution splashed into again, with homogenizer 8,000rpm stirs, and temperature is 0 ℃, reaction mixing time 4 hours.The product that reaction is finished separates the water oil reservoir with centrifuge earlier, collect the water layer product, clean for several times with normal hexane (n-Hexane) and acetone (Acetone) again, collect product and dry, place vacuum equipment that solvent is drained product, utilizing water dialysis that residue is removed, obtaining end product in the lyophilization mode at last and its collection.
Hyaluronic acid nanometer microgranule poured in the secondary water stir, (DynamicLight Scattering, DLS, Malvern Zeta 1000HS) measures its size, mean diameter (Z with dynamic light scattering
Ave) be 770.4nm.
Embodiment three: 1730K level hyaluronic acid emulsifying processing procedure experiment: oil/water 20/1, rotating speed 11000rpm
(Oliver oil) inserts in the reaction bulb with the 500g olive oil, gets 50g Si Pan 80 (span80) and pours in the olive oil and stir.Get the hyaluronic acid (SciVision of 0.75g, MW:1730K) be dissolved in the 25ml secondary water, 0.125g glutaraldehyde (Glutaraldehyde) be dissolved in the 10ml secondary water, place respectively in two isobaric funnels, first hyaluronic acid aqueous solution is delayed splashes in the olive oil, glutaraldehyde water solution splashed into again, with homogenizer 11,000rpm stirs, and temperature is 0 ℃, reaction mixing time 4 hours.The product that reaction is finished separates the water oil reservoir with centrifuge earlier, collect the water layer product, clean for several times with normal hexane (n-Hexane) and acetone (Acetone) again, collect product and dry, place vacuum equipment that solvent is drained product, utilizing water dialysis that residue is removed, obtaining end product in the lyophilization mode at last and its collection.
Hyaluronic acid nanometer microgranule poured in the secondary water stir, (DynamicLight Scattering, DLS, Malvern Zeta 1000HS) measures its size, mean diameter (Z with dynamic light scattering
Ave) be 459nm.
Embodiment four: 1,730K level hyaluronic acid emulsifying processing procedure experiment: oil/water 20/1, rotating speed 16,000rpm
(Oliver oil) inserts in the reaction bulb with the 500g olive oil, getting 50g Si Pan 80 (span80) pours in the olive oil and stirs. get the hyaluronic acid (SciVision of 0.75g, MW:1,730K) be dissolved in the 25ml secondary water, 0.125g glutaraldehyde (Glutaraldehyde) be dissolved in the 10ml secondary water, place respectively in two isobaric funnels, earlier hyaluronic acid aqueous solution is delayed and splash in the olive oil, again glutaraldehyde water solution is splashed into, with homogenizer 16,000rpm stirs, temperature is 0 ℃, reaction mixing time 4 hours. the product that reaction is finished separates the water oil reservoir with centrifuge earlier, collects the water layer product, cleans for several times with normal hexane (n-Hexane) and acetone (Acetone) again, collect product and dry, place vacuum equipment that solvent is drained product, utilizing water dialysis that residue is removed, obtain end product in the lyophilization mode at last and its collection.
Hyaluronic acid nanometer microgranule poured in the secondary water stir, (DynamicLight Scattering, DLS, Malvern Zeta 1000HS) measures its size, mean diameter (Z with dynamic light scattering
Ave) be 358nm.
Embodiment five: 1,730K level hyaluronic acid emulsifying processing procedure experiment: oil/water 20/1, rotating speed 22,000rpm
(Oliver oil) inserts in the reaction bulb with the 500g olive oil, gets 50g Si Pan 80 (span80) and pours in the olive oil and stir.Get the hyaluronic acid (SciVision of 0.75g, MW:1730K) be dissolved in the 25ml secondary water, 0.125g glutaraldehyde (Glutaraldehyde) be dissolved in the 10ml secondary water, place respectively in two isobaric funnels, first hyaluronic acid aqueous solution is delayed splashes in the olive oil, glutaraldehyde water solution splashed into again, and stirs with homogenizer 22000rpm, temperature is 0 ℃, reaction mixing time 4 hours.The product that reaction is finished separates the water oil reservoir with centrifuge earlier, collect the water layer product, clean for several times with normal hexane (n-Hexane) and acetone (Acetone) again, collect product and dry, place vacuum equipment that solvent is drained product, utilizing the water dialysis with the residue removing, at last with lyophilization mode Unwinding end product and with its collection.
Hyaluronic acid nanometer microgranule poured in the secondary water stir, (DynamicLight Scattering, DLS, Malvern Zeta 1000HS) measures its size, mean diameter (Z with dynamic light scattering
Ave) be 322nm.
Table one is the particle diameter interpretation of result of 1730K level hyaluronic acid emulsifying processing procedure.
Table one:
Embodiment six: 220K level hyaluronic acid emulsifying processing procedure experiment: oil/water 20/1, rotating speed 8000rpm
(Oliver oil) inserts in the reaction bulb with the 500g olive oil, gets 50g Si Pan 80 (span80) and pours in the olive oil and stir.Get the hyaluronic acid (Lifecore of 0.375g, MW:220K) be dissolved in the 25m1 secondary water, 0.125g glutaraldehyde (Glutaraldehyde) be dissolved in the 10ml secondary water, place respectively in two isobaric funnels, first hyaluronic acid aqueous solution is delayed splashes in the olive oil, glutaraldehyde water solution splashed into again, with homogenizer 8,000rpm stirs, and temperature is 0 ℃, reaction mixing time 4 hours.
Hyaluronic acid nanometer microgranule poured in the secondary water stir, (DynamicLight Scattering, DLS, Malvern Zeta 1000HS) measures its size, mean diameter (Z with dynamic light scattering
Ave) be 270nm, as shown in Figure 3.
In addition, also carry out the bio-toxicity analysis of product with the MTT cytotoxicity experiment, MTT is a kind of cytotoxicity experiment of giving birth to doctor's material that is commonly used to analyze.MTT tetramethyl fixed nitrogen azoles salt (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is a water miscible salt, under the interference of no phenol red (phenol red), be yellow, can be the cell active absorption enters in the cell, under the effect of the dehydrogenation ferment (dehydrogenase) of living cells grain line body, cut MTT and go up tetrazole ring (tetrazolium ring), reduction produces the water-fast Jia Za of navy blue (formazan), and be deposited in the cell, after adding dimethyl sulfoxide (DMSO) dissolving, can measure also quantitatively by the extinction of wavelength 560nm.In cell suspending liquid, cell is spheroidal, is called round cell, on a biological compatible good base material, cell should be able to be very fast attaching (attach) and utilize its pseudopodium to extend to be tiled on the base material, to move (migration) and hypertrophy (proliferation) again.Utilize the ability of cell reduction MTT to can be applicable to experiments such as analysis of cells survival rate, estimation cell number, this patent promptly utilizes the method to come propagation situation of analysis of cells (cell proliferation) and relative activity.Can find the hyaluronic acid nanometer microgranule of present embodiment by Fig. 4, its cell survival rate is close with primary hyaluronic acid raw material.Be with, hyaluronic acid nanometer microgranule of the present invention is the packing material that is applicable to human body.
Table two is the particle diameter interpretation of result of 220K level hyaluronic acid emulsifying processing procedure
In addition, hyaluronic acid also needs low viscosity to be beneficial to subcutaneous filling injection except having good bio-compatibility, is also to carry out viscosity test with present embodiment.The classification of viscometer is a lot, and commonly used have capillary type, a rotary and vibration type, and present embodiment system carries out hyaluronic viscosity measurement with rotary viscosimeter.Following table three be the straight chain type hyaluronic acid of general high molecular (MW=220K) and present embodiment high molecular (MW=220K) microparticles of hyaluronic acid ratio of viscosities.
Table three
Can find out obviously that by table three under 4.0wt% concentration, high-molecular weight its viscosity of straight chain type hyaluronic acid is much larger than the microparticles of hyaluronic acid of present embodiment.The more important thing is that when increasing along with concentration, the viscosity differences of the viscosity of microparticles of hyaluronic acid and straight chain type hyaluronic acid solution is bigger, demonstrate the restriction of straight chain type hyaluronic acid on the injection product is used.Relative, the hyaluronic acid nanoparticle of present embodiment then demonstrates high concentration and low viscous characteristic.
The hyaluronic viscosity of known straight chain type can improve along with the increase of molecular weight, makes high-molecular weight straight chain type hyaluronic acid because of the difficult control when injecting of viscosity height.Therefore, the employed hyaluronic acid of present subcutaneous filling injection is limited in lower molecular weight and concentration.Yet hyaluronic molecular weight and concentration are lower, and its filling and suction moistening effect are just poorer, and degradation in vivo speed is also bigger simultaneously.In other words, though the straight chain type hyaluronic acid of high molecular or high concentration filling moistening effect is preferable, viscosity is too high, is unfavorable for subcutaneous filling injection; Though and but the straight chain type hyaluronic acid of low-molecular-weight or low concentration is beneficial to subcutaneous injection moistening effect variation.Be that with the filling moistening effect of high molecular weight hyaluronic acid, and viscosity is very low, has quite good progressive with, microparticles of hyaluronic acid of the present invention.
The foregoing description only is to give an example for convenience of description, and the interest field that the present invention advocated should be as the criterion so that claim is described certainly, but not only limits to the foregoing description.
Claims (26)
1. a particle diameter is less than the hyaluronic acid particle of 1 μ m and tool bio-compatibility, and its viscosity under identical concentration is hyaluronic 75% less than straight chain, and wherein this concentration is greater than 1wt%; Prepare by following method:
(A) with an organic solvent and an emulsifier, form one first mixed solution, wherein the weight ratio of this organic solvent and this emulsifying agent be 1: 0.01 to 0.3;
(B) with a hyaluronic acid and water according to mixing, form one second mixed solution, wherein the weight ratio of this hyaluronic acid and this water is 0.001 to 0.08: 1, the weight ratio of this organic solvent and this water is at least greater than 5: 1;
(C) this second mixed solution is added formation one the 3rd mixed solution in this first mixed solution;
(D) with a chemical compound as shown in the formula (I)
R-(CHO)
n(I)
Add in the 3rd mixed solution, form one the 4th mixed solution, and carry out cross-linking reaction, wherein n is 2 to 4 integer, and R is C
3To C
15Alkyl, C
3To C
15Cycloalkyl, six carbon candy rings or five carbon candy rings;
(E) stir the 4th mixed solution; And
(F) filter the 4th mixed solution, to obtain the hyaluronic acid particle.
2. hyaluronic acid particle as claimed in claim 1, wherein the viscosity of this hyaluronic acid particle is to measure with Rotary Viscosimeter.
3. hyaluronic acid particle as claimed in claim 1, wherein this hyaluronic acid particle is used in the human body implantation.
4. a particle diameter is less than the hyaluronic acid particle of 1 μ m, comprise a polymer by at least one hyaluronic acid and one and the alkyl compound reaction that produces aldolisation of this hyaluronic acid form, wherein this hyaluronic acid includes at least one hydroxy, this alkyl compound includes dialdehyde-based at least, this hydroxy and this aldehyde radical produce crosslinked bridge formation via aldolisation between this hyaluronic acid, and this hyaluronic acid and this alkyl compound be in emulsion, carry out crosslinked.
5. hyaluronic acid particle as claimed in claim 4, wherein this hyaluronic acid particle is used in the human body implantation.
6. hyaluronic acid particle as claimed in claim 4, wherein this hyaluronic molecular weight is between 30,000 to 5000,000 dalton.
7. hyaluronic acid particle as claimed in claim 4, the wherein preparation of this emulsion, be with an organic solvent and an emulsifying agent according to the mixed of weight ratio 1: 0.01~0.3 to form this emulsion.
8. hyaluronic acid particle as claimed in claim 7, wherein this organic solvent is olive oil, mineral oil, glycerol, toluene or isobutyltrimethylmethane..
9. hyaluronic acid particle as claimed in claim 7, wherein this emulsifying agent is ionic emulsifying agent, nonionic emulsifier or amphoteric emulsifying agent.
10. hyaluronic acid particle as claimed in claim 4, wherein this alkyl compound has the structure as shown in the formula (I):
R-(CHO)
n(I)
Wherein n is 2 to 4 integer, and R is C
3To C
15Alkyl, C
3To C
15Cycloalkyl, six carbon candy rings or five carbon candy rings.
11. hyaluronic acid particle as claimed in claim 10, wherein this alkyl compound is a glutaraldehyde.
12. prepare the method for the described hyaluronic acid particle of claim 1, may further comprise the steps:
(A) with an organic solvent and an emulsifier, form one first mixed solution, wherein the weight ratio of this organic solvent and this emulsifying agent be 1: 0.01 to 0.3;
(B) hyaluronic acid and water are mixed, form one second mixed solution, wherein the weight ratio of this hyaluronic acid and this water is 0.001 to 0.08: 1, and the weight ratio of this organic solvent and this water is at least greater than 5: 1;
(C) this second mixed solution is added formation one the 3rd mixed solution in this first mixed solution;
(D) with a chemical compound as shown in the formula (I)
R-(CHO)
n(I)
Add in the 3rd mixed solution, form one the 4th mixed solution, and carry out cross-linking reaction, wherein n is 2 to 4 integer, and R is C
3To C
15Alkyl, C
3To C
15Cycloalkyl, six carbon candy rings or five carbon candy rings;
(E) stir the 4th mixed solution; And
(F) filter the 4th mixed solution, to obtain a precipitate;
Wherein this organic solvent is olive oil, mineral oil, glycerol, toluene or isobutyltrimethylmethane..
13. the preparation method of hyaluronic acid particle as claimed in claim 12, wherein this emulsifying agent is a nonionic emulsifier.
14. the preparation method of hyaluronic acid particle as claimed in claim 13, wherein this emulsifying agent is Polyethylene Glycol 8 monostearates, Polyethylene Glycol 8 Laurel ethers, sorbitan monostearate, sorbitan mono-oleic acid ester, Polyethylene Glycol 20 sorbitol anhydride monopalmitates, Polyethylene Glycol 20 sorbitan mono-oleic acid esters, Polyethylene Glycol 40 sorbitan monostearates, nonyl phenol 10, nonyl phenol 20, nonyl phenol 7.5, octyl phenol 10, monopalmitin, monoglyceride, fatty glyceride, leaf fat monoglyceride or propylene glycol ethylene oxide copolymer.
16. the preparation method of hyaluronic acid particle as claimed in claim 12, wherein this emulsifying agent is an anionic emulsifier.
17. the preparation method of hyaluronic acid particle as claimed in claim 16, wherein this emulsifying agent is sodium alkyl benzene sulfonate, sulphuric acid ten diester sodium, sodium hexadecyl sulfate, sodium stearate, succinic acid monooctyl ester sulfonate, alkylaryl hydroxide ethylsulfonic acid ester or polyoxyethylene alkylphenol sulphonic acid ester.
18. the preparation method of hyaluronic acid particle as claimed in claim 12, wherein this emulsifying agent is a cationic emulsifier.
19. the preparation method of hyaluronic acid particle as claimed in claim 18, wherein this emulsifying agent is hexadecyltrimethylammonium chloride, cetyl trimethyl ammonium bromide, hexadecyldimethyl benzyl ammonium ammonium bromide, chlorination Petiolus Trachycarpi hexadecyldimethyl benzyl ammonium amine, the pyridine of cetyl chloride arsenic, zephiran or the pyridine of Fluhyzon chlorination arsenic.
20. the preparation method of hyaluronic acid particle as claimed in claim 12, wherein this emulsifying agent is the amphoteric emulsifying agent.
21. the preparation method of hyaluronic acid particle as claimed in claim 20, wherein this emulsifying agent lauryl amine base ethyl Ammoniom-Acetate, oleamide, lauric acid diethyl amide or diethanolamine.
22. the preparation method of hyaluronic acid particle as claimed in claim 12, the chemical compound of its Chinese style (I) is a glutaraldehyde.
23. the preparation method of hyaluronic acid particle as claimed in claim 12, wherein the rotating speed of this step (E) stirring is 5,000 to 25, between the 000rpm.
24. the preparation method of hyaluronic acid particle as claimed in claim 12, wherein this step (E) stirs with homogenizer.
25. the preparation method of hyaluronic acid particle as claimed in claim 12, wherein this step (F) is finished with centrifuge.
26. the preparation method of hyaluronic acid particle as claimed in claim 12 comprises also that wherein a step (G) cleans this precipitate.
27. the preparation method of hyaluronic acid particle as claimed in claim 26, wherein this step (G) is finished with organic solvent, evacuation, super dialysis or its combination.
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