CN1186371C - Porous biodegradable polymer crosslinking method - Google Patents
Porous biodegradable polymer crosslinking method Download PDFInfo
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- CN1186371C CN1186371C CNB011417684A CN01141768A CN1186371C CN 1186371 C CN1186371 C CN 1186371C CN B011417684 A CNB011417684 A CN B011417684A CN 01141768 A CN01141768 A CN 01141768A CN 1186371 C CN1186371 C CN 1186371C
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Abstract
The present invention relates to a porous biodegradable polymer crosslinking method. The method comprises the following procedures: (a) a porous biodegradable polymer is put in a device; (b) supercritical fluid containing a crosslinking agent is transferred into the device for carrying out a porous biodegradable polymer crosslinking reaction; in addition, after procedure (b), the method further comprises the procedure (c) as required: pure supercritical fluid is transferred into the device for cleaning the crosslinked polymer and removing the unreacted crosslinking agent. The method of the present invention can effectively solve the problems in the traditional crosslinking method that the crosslinking extent is not enough, the operating time is longer, the procedures of chemical residue and secondary cleaning are complicated, the pore shape is altered even reduced, etc. The porous biodegradable polymer prepared by the method of the present invention can be applied to drug release, cell growth mould plates, wound dressing, tissue material filling, and the decomposition inside or outside the biologic body of decomposable daily tools.
Description
Invention field
But the present invention relates to a kind of preparation method of porous biological decomposing macromolecular, but and be particularly related to a kind of new cross-linking method of porous biological decomposing macromolecular.
Background technology
The application that the decomposing macromolecular material can be widely used in drug release control, artificial skin, benefit bone material, burn and scald material, hemostasis plate and various organizational projects can fall in porousness.The macromolecular material of decomposability can decompose in atmosphere or in vivo gradually, its natural hydrolysis or decomposed by biological intravital enzyme system or absorb.For but the implantation that makes this porousness decomposing macromolecular material have more effective and long period is used to avoid in vivo quick decomposition and to reduce immunological rejection, and can when practical application, need according to actual clinical, reach the rate of decomposition of desired macromolecular material, but therefore prepared decomposing macromolecular material often need further carry out crosslinking reaction to control its rate of decomposition.Another important problem then is that this decomposable polymer is prepared into the structure with hole kenel, for example open hole (being suitable as the carrier of organizational project cell growth) or closed hole (the Chang Zuowei medicine coats the base material that discharges), how successfully preparation has suitable hole kenel and can meet the requirements of crosslinking degree with the intensity that improves this material and reduce cracking, the important research field that just becomes medical material exploitation in recent years and use.
It is many with freeze-drying (freeze drying) preparation that but general natural decomposing macromolecular is prepared into the method for porous substrate, and minority is with critical-point drying method (critical point drying) or natural seasoning (air drying) preparation.But, the porous biological decomposing macromolecular must carry out chemically crosslinked again when preparing, the process chemically crosslinked was handled and just can be increased high molecular mechanical property and increase high molecular antienzyme capacity of decomposition, otherwise, will be decomposed in the porousness polymer short period of time of preparing, can't reach due effect.
The patent documentation report, the biopolymer that contains the volume hydrogen bond for the tool protein structure carries out crosslinking reaction generally physics and chemical process dual mode.Wherein physical method has thermal dehydration [with reference to people such as Wang M-C, 1994] and irradiation UV[with reference to people such as Weadock K.S., 1995] modes such as light or gamma-radiation, its result often have crosslinking degree not high and uneven resemble existing, therefore suitable limited on using.Can be divided into liquid phase and gas phase dual mode [with reference to people such as Yannas, 1980] again as for chemical process.
1. solution phase crosslinking method:
The porousness polymer is soaked in the solution that contains chemical cross-linking agent, after the after chemical reaction, utilizes other solution to clean, residual unreacted linking agent is washed, utilize cryodesiccated mode again, prepare porous substrate.
2. gas phase crosslinking method:
The porousness polymer is positioned over the top that contains the chemically crosslinked agent solution, to contain the solution heating of chemical cross-linking agent, make the porousness polymer in being full of the vapour pressure of chemical cross-linking agent, carry out chemical reaction, when chemical reaction finishes, utilize air gas to clean the porousness polymer, unreacted chemical cross-linking agent is cleaned up.
Solution and crosslinked this dual mode of vapor phase are the crosslinking methods of the most normal application, but its defective is all arranged.The mutually crosslinked defective of solution is, needs the lyophilize through again, and is consuming time and step is numerous and diverse, and destroyed the pore space structure and the kenel of original base material.The crosslinked defective of gas phase is, regular meeting causes the inhomogeneous of crosslinking degree, and some places are excessively crosslinked, and the some places crosslinking degree is not enough, and, usually can there be the residue problem of linking agent, be difficult to remove.It is crosslinked that for example commercial glutaraldehyde commonly used is made gas phase, yet glutaraldehyde easily has residual and entraps globule easily causes the local organization calcify reaction and cause cell-cytotoxic reaction etc. [with reference to people such as Trowbridge E.A., 1988 and Speer, people such as D.P., 1980].
Comprehensive above method utilizes traditional way to finish crosslinking reaction as can be known, and existing is not that crosslinking degree is not enough, is exactly to cross chemical residues, operating time the permanent problems such as step is miscellaneous that reach.Therefore this patent proposes with supercutical fluid (scCO for example
2) technology carries out open type stephanoporate biodegradable polymer crosslinking reaction, to address the above problem.
It is as follows as the prior art of reference file to introduce the present invention:
1. U.S. Patent number 5,629,353, and it utilizes aromatic monomer to mix with vinylbenzene, and adding linking agent (cross-linking agent) carries out polyreaction.Linking agent is the acyl halide (acyl halides) and benzene halide formic acid (benzylic halids) of multiple functional radical.Co-polymer colloid (copolymer gel) soaked place in the solvent, unreacted monomer and linking agent are removed, utilize Co 2 supercritical fluid and solvent exchange to come out again, be heated to critical drying temperature (critical point temperature) and open pore (opencellednanopore) can occur;
2. U.S. Patent number 5,710,187, and it mixes multiple aromatic monomer and huge monomer (comprising 2,6-dimethyl phenylene oxide, vinylbenzene, methylphenyl siloxane), and add catalyzer and linking agent carries out polyreaction.The co-polymer colloid soaked place in the solvent, unreacted monomer and linking agent are removed, utilize Co 2 supercritical fluid and solvent exchange to come out again, be heated to critical drying temperature and open pore can occur.The small holes polymkeric substance of the crosslinked mistake of this kind can be applicable to separating film (separation membrane);
3. U.S. Patent number 4,873,218, and it utilizes the poly-hydroxybenzene (polyhydroxybenzene) of different ratios and formaldehyde (formaldehyde) to add catalyzer, and catalyzer is a yellow soda ash.Become aerogel (aerogel) after the compound, being heated to high temperature can become low density porous property colloid;
4. U.S. Patent number 4,997,804, and it adds Resorcinol-formaldehyde aerogel becomes aerogel after yellow soda ash mixes, and being heated to high temperature can become low density porous property colloid, and density range is 30～100mg/cc.
5. the reported in literature volume hydrogen bond of biopolymer contain to(for) the tool protein structure carries out crosslinking reaction physics and chemical process dual mode.Wherein physical method has thermal dehydration [with reference to people such as WangM-C, Biomaterials, 15,507,1994.] and irradiation UV light [with reference to people such as Weadock K.S., J.Biomed.Mater.Res., 29,1373,1995.] or mode such as gamma-radiation, its result often have crosslinking degree not high and uneven resemble existing, therefore suitable limited on using;
6. reported in literature utilizes supercritical carbon dioxide fluid (scCO
2) next dry porousness collagen protein base material, it mainly is more different drying step, freeze-drying, critical-point drying method, natural seasoning are arranged, produce porousness collagen protein base material, and utilize liquid phase and two kinds of crosslinked porousness collagen proteins of Chemical Crosslinking Methods of gas phase base material.[with reference to people such as Dagalakis N., Mater.Res., 14,511,1980.];
7. the animal pericardium [people such as Trowbridge E.A. of Trowbridge to soak glutaraldehyde solution in 1988, Cardiovasc.Surg., 95,577-585,1988.], the heart valve that replaces patient, they find can produce really up to the mark and phenomenon calcification through the biological tissue that glutaraldehyde cross-linking was handled.And when the concentration of glutaraldehyde solution is higher than 3 to 25ppm, can pair cell toxigenous influence, therefore the biological tissue that handles with glutaraldehyde cross-linking may be to the human body cell toxigenicity behind implant into body;
8.West with Mangan once respectively at [West in 1970, J.and Mangan, J.L., Nature, 228,466-468,1970.], with lipase (lipase) and trypsin trypsin) decompose the chloroplastin of crossing through glutaraldehyde (glutaraldehyde) crosslinking Treatment (chloroplast), they find that decomposing the green proteic ability of crosslinking Treatment posterior lobe by these enzymes has all reduced significantly.
Summary of the invention
One of purpose of the present invention is exactly in order to improve the defective of solution and vapor phase crosslinking method, but and a kind of new cross-linking method of porous biological decomposing macromolecular is provided;
Two of purpose of the present invention just provides a kind of porous biodegradable polymer crosslinking method, and it can not destroy the pore space structure and the kenel of original base material;
Three of purpose of the present invention just provides a kind of porous biodegradable polymer crosslinking method, and it can obtain crosslinked uniform porousness polymer;
Four of purpose of the present invention just provides a kind of porous biodegradable polymer crosslinking method, and it can solve the residual problem of linking agent;
Five of purpose of the present invention just provides a kind of porous biodegradable polymer crosslinking method, and it can use various linking agents, can not be subjected to the insufficient restriction of linking agent vapour pressure;
Six of purpose of the present invention just provides a kind of simple to operate, quick and porous biodegradable polymer crosslinking method that control easily.
For reaching above-mentioned purpose, feature of the present invention is to adopt supercutical fluid to carry linking agent to carry out crosslinking reaction, and utilizes supercutical fluid effectively to remove unreacted linking agent.Yi Yan's, method of the present invention comprises following key step: but (a) a porous biological decomposing macromolecular is placed a device; And (b) supercutical fluid that contains linking agent is fed this device, to carry out the porous biodegradable polymer crosslinking reaction.In addition, (b) can optionally further comprise afterwards in step: (c) a pure supercutical fluid is fed this device, to clean above-mentioned crosslinked polymer.Wherein the supercutical fluid of step (b) further comprises a cosolvent.
The present invention relates to a kind of porous biodegradable polymer crosslinking method, it comprises the following steps:
(a) but a porous biological decomposing macromolecular is placed a device;
(b) supercritical co that contains linking agent is fed this device, to carry out this porous biodegradable polymer crosslinking reaction; And
(c) a pure supercritical co is fed this device, to clean this crosslinked polymer, till this linking agent removes in fact from this polymer.
Method of the present invention can solve effectively in traditional cross-linking method that crosslinking degree is not enough, the operating time permanent, chemical residues and the secondary cleaning step is numerous and diverse and may cause the hole external form to change even problem such as dwindle.And this Biodegradable porousness polymer crosslinking degree, abscess kenel also can be borrowed operational condition and be made variation and change, and reach its decomposition rate of may command with as different application.
Supercutical fluid is the gas or the liquid of postcritical.Under stagnation point, gas has identical physical properties (particularly density) with liquid, and for a particular fluid, the temperature of its stagnation point and pressure are certain value.Can use various supercutical fluids in the method for the invention, for example carbonic acid gas, ammonia (NH
3), rare gas element (as Ar), refrigerant and low-carbon (LC) alkanes (as propane) and composition thereof, but wherein supercritical co is preferable.
Method of the present invention is specially adapted to the decomposability polymer crosslinking, for example the wetting ability biopolymer is protein-based as collagen protein, gelatin and other animality, vegetable protein, polyose such as hyaluronic acid, chitin, chitosan and other polysaccharide body, the synthetic decomposable polymer of class, as PVA (polyvinyl alcohol), PLGA (polyglycolic acid-altogether-lactic acid), PLA (poly(lactic acid)), PGA (polyglycolic acid), PCL (polycaprolactone) but and other decomposing macromolecular, and above-mentioned high molecular mixture or multipolymer etc.In addition, method of the present invention also can be used for general polymer crosslinking.
But there is no particular restriction for the hole pattern of porous biological decomposing macromolecular, can be the open communication hole of runner type, open cell type, or dead front type closed hole, or having open one closed hole simultaneously, its hole scope can be between 500 μ m～0.01 μ m; Its porosity can be between 0.99～0.05.
The linking agent that is used to crosslinked biological tissue at present includes: aldehydes, as formaldehyde, glutaraldehyde [Nimni, M.E., Cheung, D., Strates, B., Kodama, M., Sheikh, K., " Bioprosthesis derived from cross-linked and chemically modifiedcollagenous tissue, " in Collagen Vol.III, Florida, M.E.Nimni (ed.), CRC Press, Boca Raton, 1-38,1988], dialdehyde starch (dialdehydestarch) [Rosenberg, D., " Dialdehyde starch tanned bovineheterografts:Develoment, " New York, in Vascular Grafts, P.N.Sawyer and M.J.Kaplitt (eds.), Appleton-Century-Crofts, 261-270,1978] and epoxide [with reference to Tu, R., et al, 1993] etc.And glutaraldehyde is to use maximum biological tissue's linking agents at present clinically.
It should be noted, because the present invention utilizes supercutical fluid to carry linking agent, but not utilize the steam of linking agent to carry out crosslinking reaction, therefore linking agent used in the present invention can not be subjected to the too low restriction of vapour pressure, can be extensive use of the linking agent of various types.Except above-mentioned mentioned linking agent, other as epoxide, carbodiimide (carbodiimide), isocyanic ester (isocyanates), metal crosslinking agent, ion crosslinking agent, organic heterocyclic molecule (as genipin, it is a gardenin, a kind of natural goods that extract by the fruit of flower of Capa Jasmine) and acrylic acid series derivative and composition thereof etc., all applicable to the present invention.
In addition, in order to promote the solubleness of linking agent at supercutical fluid, enter linking agent bucket 7 before, can install another and contain the cosolvent bucket (cosolvent definition: the mixing liquid that comprises two or more composition), described cosolvent is aqueous phase solution, alcohol solution etc. for example.
According to one of feature of the present invention, but can control the crosslinking degree of porous biological decomposing macromolecular material simply by the variation of operating time, crosslinker concentration, pressure, temperature and flow, wherein the concentration of linking agent can be any concentration, need decide on indivedual linking agent kinds, but be good generally with the minimum concentration dosage that can reach cross-linking effect; Pressure range is greater than a normal atmosphere, need decide on indivedual linking agent kinds; Temperature range is a room temperature to 150 ℃, and is preferable between 20 ℃～50 ℃; The flow range of supercutical fluid is 0.1～10L/min; Reaction times is at least greater than 1 minute, usually between 30 minutes～8 hours.
According to another characteristic of the invention, crosslinking reaction finish after, but can utilize supercutical fluid to clean crosslinked porousness decomposing macromolecular, to remove unreacted linking agent.The supercutical fluid that is used for cleaning usefulness herein can be identical or different with the supercutical fluid of previous use, and it can be selected from carbonic acid gas, nitrogen, rare gas element, refrigerant, low-carbon (LC) alkanes and composition thereof.Till the operating time of cleaning can continue until linking agent removed from polymer in fact fully.So can avoid the residual toxicity or the other problem of causing of linking agent.
According to the porousness polymer of aforesaid method gained can be applicable to drug release, cell growth template, wound dressings, organize foam that material for repairing, decomposable daily apparatus can decompose etc. can be in vivo or external decomposition use.
In sum, the present invention utilizes supercritical fluid technology, carries out porous biodegradable polymer crosslinking reaction, and defective such as go to pot to overcome cellular structure, crosslinking degree is inhomogeneous, chemical residues and operating time are long.But this processing procedure can be controlled the crosslinking degree of porous biological decomposing macromolecular material simply by the variation of operating time, crosslinker concentration, pressure, temperature and flow in addition, and borrow supercutical fluid to flush out residual linking agent and simple to operate quick again, make this processing procedure have more the progressive of innovation and have the benefit that in fact can commercially produce based on these advantages.
Description of drawings
For above and other objects of the present invention, feature and advantage can be become apparent, conjunction with figs. hereinafter is described in detail below:
Fig. 1 is the cross-linking method schema of a preferred embodiment of the present invention;
Fig. 2 is the employed device synoptic diagram of a preferred embodiment of the present invention;
Fig. 3 is gelatin porous property base material sweep electron microscope (SEM) figure, 100 times;
Fig. 4 is with the porousness gelatin polymer (embodiment 1 gelatin) of Non-crosslinked processing and the porousness gelatin polymer of handling through linking agent (embodiment 2 sample Exp1 and embodiment 3 sample Exp2), soak at once and place phosphate buffer soln (PBS), the figure of photograph;
Fig. 5 is porousness gelatin polymer (embodiment 1 gelatin) that Non-crosslinked is handled and the porousness gelatin polymer of handling through linking agent (embodiment 2 sample Exp1 and embodiment 3 sample Exp2), soaks to place PBS, through the figure of 2 minutes back photographs;
Fig. 6 is with the porousness gelatin polymer (embodiment 1 gelatin) of Non-crosslinked processing and the porousness gelatin polymer of handling through linking agent (embodiment 2 sample Exp1 and embodiment 3 sample Exp2), soak and place PBS, through after 51 days the figure of photograph, can find out obviously that the method via this patent can reach effectively crosslinked purpose really;
Fig. 7 is the result of swelling test.Exp1 is the sample of embodiment 2, and Exp2 is the sample of embodiment 3;
Fig. 8 is that porousness gelatin base material decomposes experimental result.Exp1 is the sample of embodiment 2, and Exp2 is the sample of embodiment 3, " G " expression " gelatin " among the figure;
Fig. 9 is the test result that porousness gelatin base material adopts " ASTM F-895 Agar DiffusionCytotoxicity " working specification.
Embodiment
To be example below, cooperate the schema of following Fig. 1 and the device synoptic diagram of Fig. 2, operation steps of the present invention will be described with the supercritical co.Nomenclature among Fig. 2:
1～carbon dioxide steel cylinder; 2～setting device; 3～pump;
4～back pressure regulator device; 5～metering valve; 6～3-way valve;
7～linking agent bucket; 8～high pressure vessel; 9～thermostatic bath;
10～anemometer measuring device.
At first, but a porousness decomposing macromolecular is placed high pressure vessel 8, the setting operation temperature is also opened CO
2Pump makes CO
2Pressure and flow reach setting point.Then, rotate 3-way valve 6-1,6-2 makes CO
2Flow through in the linking agent bucket 7, rotate 3-way valve 6-3 after about 1～2 hour, 6-4 makes the CO that contains cosolvent and linking agent
2But enter in the porousness decomposing macromolecular to a certain set time.When experiment is finished, rotate 3-way valve 6-1,6-2 utilizes pure high pressure CO
2But clean crosslinked porousness decomposing macromolecular, to remove unreacted linking agent, after cleaning is finished, removal pressure, but from high pressure vessel, taking out the porous biological decomposing macromolecular, can finish.
Embodiment 1: the high molecular preparation of porous biological
Deionized water added be made into 1wt% solution in the gelatin, stir fully the back put freeze drier (VIS, USA) in.Temperature and vacuum tightness are made as-80 ℃ and 100 millitorrs respectively carry out 24 hours freezing dryings that vacuumize, solvent is removed, under solid-state to make the open type stephanoporate bioabsorbable polymer material.Its porosity of the resulting porous material of operational condition＞99% thus.The porosity of product is determined by following equation:
P＝[1-(D
1/D
2)]×100
D wherein
1Be foam density, D
2Be real density, density by densometer (Electricdencimeter, MP-200S) measured.The hole form of this porous material, by the figure of sweep electron microscope (SEM) as can be known, its hole size is an open type stephanoporate structure (as shown in Figure 3) between 50 to 200 μ m.
Embodiment 2: the reaction of porous biological polymer crosslinking
Porous biological polymer by embodiment 1 gained experimentizes, and at first the porous biological polymer is placed high pressure vessel, sets CO
2Fluid is respectively 1000psi, 30 ℃ and 1L/min (outside the system) to carry out 1 hour crosslinking reaction through pressure, temperature and the flow of linking agent (glutaraldehyde) groove.After question response finishes with CO
2Pressure is adjusted to 3000psi and cleans crosslinked porous biological polymer 1 hour (Exp 1), to remove unreacted linking agent glutaraldehyde.
Embodiment 3: the reaction of porous biological polymer crosslinking
Porous biological polymer by embodiment 1 gained experimentizes, and at first the porous biological polymer is placed high pressure vessel, sets CO
2Fluid is respectively 1000psi, 30 ℃ and 1L/min (outside the system) to carry out 1 hour crosslinking reaction through pressure, temperature and the flow of linking agent (glutaraldehyde) groove.After question response finishes with CO
2Pressure is adjusted to 3000psi and cleans crosslinked porous biological polymer 3 hours (Exp 2), to remove unreacted linking agent glutaraldehyde.
The porousness gelatin polymer of embodiment 1 gained that Non-crosslinked is handled places phosphate buffer soln (Phosphate Buffer Solution; PBS), can hole be subsided and dissolve.But, then can in same solvent, maintain its former hole kenel for some time and can not subside (as Fig. 4, Fig. 5, shown in Figure 6) is enough to show that cross-linking effect is fairly good and uniformity coefficient is preferable through the porousness gelatin of this crosslinking reaction gained.
Swelling test (Swelling Test)
Experimental procedure:
1. the different condition sample is cut into homalographic (about 1 * 1.3cm), place 6-well tray (6-well dish) respectively, each condition repeats to do 3 times;
2. measure the length of all samples and wide in advance, and it is placed in 30 ℃ of baking ovens, oven dry is more than 3 hours;
3. measure its weight behind the taking-up sample at once, and record;
4. sample is immersed in the phosphate buffer soln of equivalent about 8ml;
5.1 hour, 6 hours, 24 hours back taking-up, blot gently with filter paper, weighing is also measured its area;
6. the sample after will measuring places 30 ℃ of baking ovens to dry spend the night (over night);
7. after the sample of oven dry takes out, measure its weight immediately, and record;
8. calculation formula:
Swelling rate (swelling ratio) %=(Ww-Wd)/Wd * 100%
Ww wherein: the weight in wet base of sample; Wd: the dry weight of sample
Experimental result as shown in Figure 7, the porousness gelatin base material that Non-crosslinked is handled, immersion through PBS, just dissolving in one minute, therefore there is not experimental data, but about 13 times of the Exp1 swelling of crossing via glutaraldehyde cross-linking, but about 11 times of Exp2 swelling, and do not have the dispersion phenomenon that dissociates, its demonstration truly has crosslinked effect.
Permeability gelatin base material decomposes experiment (Degradation Test)
Experimental procedure:
1. the different condition sample is cut into homalographic (about 1 * 1.3cm), place 6-well tray respectively, each condition repeats to do 3 times;
2. measure the length of all samples and wide in advance, and it is placed in 30 ℃ of baking ovens, oven dry is more than 3 hours;
3. measure its weight behind the taking-up sample at once, and record;
4. sample is immersed among the PBS of equivalent about 8ml;
5.1 hour, 6 hours, 24 hours back taking-up, sample places 30 ℃ of baking ovens oven dry to spend the night;
6. after the sample of oven dry takes out, measure its weight immediately, and record;
7. calculation formula:
Keep weight ratio (retention weight) %=100-[(Wd1-Wd2)/Wd1 * 100%]
Wd1 wherein: the dry weight of sample; Wd2: the dry weight of sample behind the soaking solution
Experimental result as shown in Figure 8, the porousness gelatin base material that Non-crosslinked is handled, immersion through PBS, just dissolving in one minute, so there is not experimental data, be immersed among the PBS, Exp1 and Exp2 still can keep 98% weight after six hours, and Exp1 and Exp2 still can keep 90% weight after 24 hours.Can be by finding significantly in the experimental result that the gelatin base material of the crosslinked mistake of process linking agent soaks and can also keep 90% weight in 24 hours.
The cytotoxicity test
Present cell culture test program mainly contains three kinds of methods and more often is used promptly: directly contact (Direct Contact), agar diffusion (Agar Diffusion) extraction method (Extraction).What cytotoxicity test of the present invention was taked is ASTM F-895 " Standard Test Method forAgar Diffusion Cell Culture for Cytotoxicity " working specification.This test method is applicable to the material of many kinds of shapes and the material of nonessential sterilization; also be applicable to the material quantity condition of limited; for example; little assembly or powder can place going up of agar and hinder the situation of cell growth also can be inspected; and agar layer can act as buffer layer is avoided sample with the protection cell weight; hinder diffusion or cause the injury of cell mechanicalness yet understand weight agar owing to some material is quite heavy, for this this test method of class material and inapplicable.This test method(s) is also inapplicable when extract can't spread via agar or gel (Agarose).
Experimental result
Utilizing the toluylene red stain to dye viable cell, is red by the visible viable cell of photo, and is and spins the Duo type and stretch.
Index of Response=area indices/dissolving index
(Response?index＝Zone?index/Lysis?index)
Critical region is described (Zone Description) as table 1, differentiates dissolving and describes (LysisDescription) as table 2, experimental result such as table 3 and Fig. 9.Can find significantly that the toxicity of Exp2 is lower, prove that supercritical carbon dioxide fluid can carry away unreacted linking agent.
Table 1: region description
The area indices region description
0 near sample or sample following do not have detectable zone
The area of 1 region limits under sample
2 zones are above sample but less than 0.5 centimeter
3 zones surpass 0.5～1.0 centimeter on sample
4 zones surpass sample more than 1.0 centimeters but do not betide in the whole dish
5 zones betide in the whole dish
Table 2: dissolving is described
The dissolving exponential region is described
0 cytotoxicity that not can observe
1 is affected less than 20% zone
2 20%～39% zones are affected
3 20%～59% zones are affected
4 20%～80% zones are affected
5 are affected greater than 80% zone
Table 3: cytotoxicity experiment result
The example reaction index
Negative control group (latex rubbers) 4/4
Positive regulation group (tetrafluoroethylene) 0/0
Gelatin 0/0
Exp1 2/3
Exp2 1D/2
Though the present invention discloses as above with preferred embodiment, so it is not in order to restriction the present invention, any those of ordinary skills, without departing from the spirit and scope of the present invention, when the change that can do some permission and retouching.
Claims (12)
1. porous biodegradable polymer crosslinking method, it comprises the following steps:
(a) but a porous biological decomposing macromolecular is placed a device; Wherein but this porous biological decomposing macromolecular is selected from protein-based, polyose, synthetic class and composition thereof or multipolymer; And
(b) supercritical co that contains linking agent is fed this device, to carry out this porous biodegradable polymer crosslinking reaction, wherein this linking agent is selected from aldehydes, epoxide, carbodiimide, isocyanic ester, metal crosslinking agent, ion crosslinking agent, organic heterocyclic molecule, acrylic acid series derivative and composition thereof.
2. the method for claim 1, but wherein this porous biological decomposing macromolecular has open hole, closed hole or has open-closed hole simultaneously.
3. the method for claim 1, wherein the supercritical co of step (b) further comprises a cosolvent.
4. the method for claim 1 wherein further comprises afterwards in step (b):
(c) a pure supercritical co is fed this device, to clean this crosslinked polymer.
5. method as claimed in claim 4 is till wherein step (c) lasts till that this linking agent removes in fact from this polymer.
6. porous biodegradable polymer crosslinking method, it comprises the following steps:
(a) but a porous biological decomposing macromolecular is placed a device, but wherein this porous biological decomposing macromolecular is selected from protein-based, polyose, synthetic class and composition thereof or multipolymer;
(b) supercritical co that contains linking agent is fed this device, to carry out this porous biodegradable polymer crosslinking reaction, wherein this linking agent is selected from aldehydes, epoxide, carbodiimide, isocyanic ester, metal crosslinking agent, ion crosslinking agent, organic heterocyclic molecule, acrylic acid series derivative and composition thereof; And
(c) a pure supercritical co is fed this device, to clean this crosslinked polymer, till this linking agent removes in fact from this polymer.
7. method as claimed in claim 6, but wherein this porous biological decomposing macromolecular has open hole, closed hole or has open-closed hole simultaneously.
8. method as claimed in claim 6, wherein the supercritical co of step (b) further comprises a cosolvent.
9. method as claimed in claim 6, wherein the flow range of the supercritical co of step (b) is 0.1～10L/min.
10. method as claimed in claim 6, wherein the operating time of step (b) was greater than 1 minute.
11. method as claimed in claim 6, wherein the operating temperature range of step (b) is a room temperature to 150 ℃.
12. method as claimed in claim 6, wherein the working pressure scope of step (b) is greater than a normal atmosphere.
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