TWI358415B - Method for applying chondroitin sulfate or the lik - Google Patents

Description

1358415 IX. Description of the invention: [Technical field to which the invention pertains] The technology of the present invention relates to the use of pirimidin or the like for cell culture, coating, and the like. [Prior Art] - A material that is also sufficient to contain aqueous substances (typical representative example: an object commonly known as a water gel), which is known in the relevant industry. Its properties are between liquid and @body, and when it is manufactured, it contains The amount of water can vary from several times to thousands of times of its own weight, and the volume increases accordingly, and it is also released according to different environmental stimuli (such as temperature, pH, etc.). Because of the amount of water that can be used in the water, the water gel is similar to the physiological condition, soft and has appropriate mechanical properties. Therefore, it has good biocompatibility and is not easy to produce inflammation in the living body. Therefore, it is in the field of biomedicine. It is often used in drug release, molecular separation procedures, cell and enzyme immobilization, and the like. Its t drug release is the most commonly used field, using the moisturizing properties of water gel, embedding the drug in the water gel, and after the water gel reaches the fixed point, it is stimulated by different environments (such as temperature and PH value). , ionic strength, etc.), release the embedded drug. Designing different types of water gels can make them respond differently to different environmental stimuli and can cater for a wide range of applications. A typical temperature-sensitive water gel - a representative molecular copolymer of N-isopropylacrylamide. The structure of such a molecular copolymer must have a hydrophobic functional group such as methyl (ethyl) or ethyl (Ethyl), and its phase critical temperature (LCST) is between about 31 and 34 °C. If the N-isopropylacrylamide monomer is copolymerized with other hydrophilic monomers, the phase transfer temperature of the copolymer can be increased, such as N-isopropylacrylamide (Molecular Copolymer of Acrylic Acid). 'The phase transition temperature can be gradually increased with the addition of different proportions of Acrylic acid. In addition to the addition of different hydrophilic monomers, the polymer structure design will also have a considerable impact on the material response, designed to be different. The structure, its response to the environment 4 1358415 shape also changes, such as the design of the county is the comb-like structure of its response =. Among these temperature-sensitive materials, there have been many patents published, such as % = not US patent No. 6,296,83 describes the use of radioactive method er et al. to synthesize a copolymer of amine and propionic acid {that is, Poly (leg Am, AAc)} to obtain acrylic acid, and its phase transition temperature varies with different groups. The situation 'understand the phase transfer of materials: household = ^ = in the carrier as a radiopharmaceutical drug' as a drug for the treatment of cancer on the tumor:: carrier. In the flattened special towel, the secret of the compound, the county plus When the field amine is biased, η sulfate) or a monomer such as an amine analog. In addition, Bae et al. applied 6% of the US patents (it is difficult to use isopropyl acrylic acid _ with acrylic acid synthesis) to apply to the intubation material to solve the intubation process, patients often have The problem of feeding and vomiting, the intubation material can be stimulated by the temperature of various parts of the body, and the configuration of the material can be changed, and the intubation device can be easily taken out from the patient. It can reduce the pain of patients and achieve the purpose of medical treatment. In recent years, the application of water gel has expanded to the field of tissue engineering, replacing the materials used for cell culture and in vitro cell interstitial with water gel. For example, Gevaert et al. In patent No. 6 525, a biocompatible and biodegradable material (in the case of acid) is crosslinked with high molecular weight polydextrose to obtain biocompatibility and biodegradability. Hydrocolloid polymer. Because polydextrose has hydrophilic properties, and polylactic acid has hydrophobic properties, the cross-linked hydrogel polymer also has the characteristics of hydrophilicity and hydrophobicity, which is generated by environmental stimulation. The contractile response is therefore applied by Gevaert et al. in drug release as a drug carrier. On the other hand, since this material is also biocompatible and biodegradable, it can be made into a porous structure. Tissue filling, tissue coating and extracellular interstitial applications. Although the polymers thus obtained by Gevaert et al. are biocompatible and biodegradable, and similar to natural materials, their material processes require high temperatures and catalysts. In the process of production and purification, there are considerable difficulties, and the yield is not large, so there are still many problems to be solved in order to commercialize this material. In view of this, if the case is synthesized in a novel way A kind of "filament material" with both water-gel property, biocompatibility and temperature sensitivity, 5 1358415 can be used as a material, because the process is simple and economical, and the finished product has better characteristics, the application benefit will be very = body specific = material (four) biological hybrid treatment, can make it closer to the original cell growth of the ring brewing I grow with the help of phase # so there have been many research attempts in recent years Glue add 2 aging age, so that the transfer has more tangible for biocompatibility, and can promote cell growth ^ affixed 'for example · oxime (chondroitinsulfate, abbreviated as cs) for tissue / soft moon The important structure of the connection (4) is mainly located on the surface of the cell or the surface of the extracellular matrix. The biological characteristics of the cell are combined with growth factors to regulate cell division and inhibit protein secretion. The chondroitin sulfate is added to the water gel. Hydrogel has an environment similar to the growth of chondrocytes. When cell culture is carried out, chondrocytes are easier to grow. This not only promotes cell growth, but also helps cells to adhere to the surface of the material. Saki et al., U.S. Patent No. 6,620,927 In the case, first with cystamine and

Ethyl isothiocyanate human L σ into 2'2-dithiobis (N-ethyl (N, ethylcarbodiimide)) cross-linking agent, followed by addition of hyaluronic acid dissolved in deionized water for cross-linking reaction, the reaction is carried out in acidic % The acidification reaction of the hyaluronic acid salt is first carried out, and several hyaluronic acid molecules are cross-linked together, and the structure has a disulfide bond, and the t-form formed by the method has a rather viscous viscosity and has Very high cell-compatible activity, so it can be applied to materials that avoid tissue sticking after surgery, or substrates for tissue engineering in vitro cell culture or cell regeneration in vivo, but the materials are expensive, so At present, the general applicability is not great. According to the literature published by G. masci et al. (p〇iymer 43, 2002, 5587-5593), pullulian polymer and glycidyl methacrylate, 4-(N, N-dimethylamino)pyridine are dissolved in DMS0 solvent and added to HC1. Thereafter, the reaction was stirred at room temperature for 48 hours, and nitrogen was passed through, and then the product was taken out by freeze-drying to obtain a polymer having a double bond on Pullulan, and then subjected to radical polymerization with NIPAAm (decylamine isopropylamine). The reaction is carried out to obtain a water-sensitive polymer having temperature sensitivity, which is applied to the field of drug release. 6 1358415 Other documents related to the technical field of the present invention: US Patent Nos. 6, 602, 975, 6, 562, 362, 6, 525, 145 ' 6, 486, 213, 6, 451, 429, 6, 306, 922, 6, 296, 83, 6, 277, 768, 6, 060, 582, 5, 986, 043, 5, 969, 052, 5, 626, 863, 5, 567, 435, 5, 410, 016, and the like. SUMMARY OF THE INVENTION One object of the present invention is to provide a cell culture substrate which has at least one of various characteristics such as temperature sensitivity, temperature stability, hydrophilicity, biocompatibility and the like. The second object of the present invention is to provide a substance which can contain, absorb or extract water-containing substances and has better temperature sensitivity. The third object of the present invention is to provide a substance which can contain, or absorb, and release an aqueous substance, and has a stable temperature stability (that is, a large temperature is fine). The fourth object of the present invention is to provide a material that can contain, absorb or release an aqueous substance, and has a difficult biological complexity, a warm temperature, a temperature stability, or preferably has the object of the present invention. As a substance for culturing a cell substrate. The sixth object of the present invention is to provide a substance as a drug carrier. And the seventh is to provide a substance that can contain, or absorb, and release aqueous substances, :' /, with better biocompatibility. Features, advantages, application of a temperature sensitivity, temperature stability, affinity for cell culture, overlay. </ br> A simple and economical acquisition of a cell culture with superior characteristics The invention is characterized by a water-based, biological phase, and the like. The main advantage of the invention is the material to be raised and applied. 7 Other features and advantages of the technology of the present invention can be further clarified by the following description and drawings. The technique of the present invention is to make isopropyl amide (N-iS〇pr_aer fine ide) molecule and ehQndrQitin The molecule of Liejia (_su or its congener) undergoes a copolymerization reaction, and the double bond between the isopropyl propyl __ molecular towel and another carbon-carbon (as shown in Figure la) is converted into One-button connection also allows one carbon in the "molecular molecule" to be connected to the double bond between the other carbon (as shown in item 1c), and the conversion is a single-key connection. Thus, the isopropyl riding _ molecule is __ The carbon and the other carbon each have a bond (as shown in FIG. 1b) for respectively connecting other molecules, and the one carbon of the ch〇ndr〇itin sulfate molecule and the other carbon also have a bond (see FIG. Shown) can be used to connect other molecules separately. When the number of interconnected molecules reaches a certain amount, a molecule is obtained, which encapsulates or absorbs the released aqueous matter. Since ch〇ndr〇itin suifate itself has more -OH functional groups, the hydrophilicity of the material is improved, and the synthesized ruthenium molecule has a ring-like structure in the main chain, so the temperature is stable. Sexuality can greatly improve the stability of the substrate to temperature. Since ch〇ndr〇itin sulfate is an important structural substance for the connection between tissue and chondrocytes, it is added to the synthesized polymer, which makes the structure of the polymer more similar to the environment of cell growth, and is beneficial for cell culture. Substrate. Compared with the conventional water gel, the synthesized molecular copolymer has better biocompatibility, temperature sensitivity and temperature stability, and can effectively increase the amount of water contained. The molecular structure of the glycosaminol (or the like) used in the above copolymerization reaction contains at least one carbon-carbon double bond. The glycosaminol (or the like) may be a mixture of any one or more of a disaccharide, a tetrasaccharide, a hexasaccharide, an octasaccharide or the like. The glycosaminol (or its analog) is chondroitin sulfate, hyaluronic acid, heparin, heparin sulfate, dextran sulfate, Among the pentosan sulfates, dermatan sulfate, etc., to one of 1358415. The glycosaminol (or the like) used in the above copolymerization reaction is preferably a small molecule ch〇ndr〇itin sulfate obtained by cutting a polymer of chondroitin sulfate by an enzyme, and the small molecule obtained by the cleavage The 〇ndr〇itin button "closes at the end of its ring structure will produce double bonds, which can be utilized for free radical polymerization with other temperature-sensitive monomers" to obtain the novel molecular copolymer of the present invention. The above copolymerization can be Further adding acrylic acid in which a double bond between one carbon in the acrylic molecule and another carbon (as shown in FIG. 8) is also converted into a single bond (as shown in FIG. The acrylic acid molecule has two bonds for respectively connecting the other two molecules. When the interconnected molecules reach a certain amount, a molecular copolymer is obtained, which can contain, or absorb, and release the aqueous substance. The purpose of adding acrylic acid is Acrylic acid itself also has biocompatible properties, and it also increases the hydrophilicity of the material. For temperature-sensitive polymers, it can increase the phase transition temperature. The cell is not dissolved by the temperature. Compared with the conventional water gel, the molecular copolymer obtained by the copolymerization of acrylic acid has better temperature sensitivity (for example, increased phase transition temperature) and temperature stability. In addition to hydrophilicity, it also has better biocompatibility. A representative example of the invention is a molecular copolymer, the molecule of which contains at least two structures, and the main component of the first structure is a pseudo-different The main component of the second structure of the propyl acrylate amide molecule is a pseudo-glycosamine molecule. The difference between the isopropylidene amide amine molecule and the isopropyl isopropyl amide molecule is: The (i.e., all) carbon of the imidazolylamide molecule is linked to the carbon by a single bond (as shown in Figure lb). The difference between the molecule of the pseudoglucosamine molecule and the glycosamine congener is: All (i.e., all) carbon-to-carbon single-link bonds of the pseudo-glycosin molecule (as shown in Figure Id) 'the first structure and the second structure Key (e.g., a chemical reaction between the key procured elements to each other) is connected to a first male "Structure of the percentage of at least 80 up to 95 'mole percentage of the second structure is at least 1 up 2〇. The preferred molecular weight range of the above molecular copolymer is at least 20,000 up to 250,000. 9 1358415 The molecular copolymer, wherein the molecule of the glycosamine congener comprises a molecular structure of a carbon-carbon double bond. The molecular copolymer described above may further comprise a third structure, and the main component of the third structure is A pseudo-acrylic acid molecule's difference between a pseudo-acrylic acid molecule and an acrylic acid molecule is that all (ie, all) of the pseudo-acrylic molecules are linked to a single bond between carbon and carbon (as shown in Figure Show), the percentage of the mole number of the third structure is at most 15. In the above molecular copolymer, the chemical bond is a single bond between carbon and carbon, that is, the chemical bond between one molecule and another molecule, which is between one carbon of the molecule and one carbon of the other molecule. single bond. Another representative example of the invention of the present invention is an object comprising moisture and the above-mentioned molecular copolymer, that is, the above-mentioned molecular copolymer has absorbed an aqueous substance and contains or forms an object when the aqueous substance is contained. The moisture is at least 80 to 650 by weight relative to the molecular copolymer. Obviously, the above-mentioned molecular copolymer is suitable as a material which can absorb an aqueous substance, and is also suitable as a material which can contain an aqueous substance, and is also suitable as a material which can release an aqueous substance. The molecular copolymer described above has a temperature sensitivity of: phase transition temperature (10) τ) of between about 35 and 4 °C. The knife copolymer of 3 contains the third structure (the main component is the pseudo-acrylic acid molecule), which is a pro-. There are at least one of the following characteristics: temperature sensitive, biocompatible, and acid sensitive: the sub-copolymer 'through-series of cells# test found that it can also be used as a base for cell culture The coating is more dense, so it can be tested _ engineering, in the material used to culture the cells, which contains the above-mentioned points of the use of cross-linking agents (such as EGDMA, that is, Ethylene 10! The material for culturing the cell contains a small amount of the cross-linking agent and the photoinitiator in addition to the molecular copolymer phase disc described above. The amount of the photoinitiator used in the process is ί. The percentage of the zipper copolymer is at least 0 01 to 0. The percentage of mGle used in the process of the copolymer is up to 1. 1 to 2 / t. The material used to culture the cells can be of various types (for example, in the form of a rod or a disk). 20=, and comprising a plurality of pores, the diameter of which is usually at least 5[deg.] micrometers to the representative of the invention of the present invention, which is a pharmaceutical carrier comprising the above-mentioned molecular type of polymer. The drug carrier has been contained in the aqueous substance. (such as syrup), the generation of the invention in this case _ A music carrier comprising the above-mentioned molecular copolymer and an aqueous substance (for example, a syrup). A further representative example of the invention is a molecular copolymerization process comprising: a formation-mixture comprising at least an isopropyl group醯 醯 a a a y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y The percentage of the number of walls is at least 1 to 20; and heated to the hull, the absorption peak of the Wei Lai lying on the mixture of the straight dragon mixture disappears, while the other chemical _ read _ shift, _ read peak refers to the infrared filament spectrum ( Ir: Infrared spect (clear spect) or NMR spectroscopy (NMR: (5) coffee leg priest e spect dirty opy) shows the most absorption of light (that is, the light penetration {transmittance 丨 minimum ). In the above molecular copolymerization process, the mixture may further comprise a kind of acrylic acid (the acid), and the mole percentage of the acrylic acid is at most 15. The above-mentioned knife path + U system dissolves the isopropyl propyl __ with the glycosamine congener and the like in the ageing agent', and the pure scale is heated and the silk is heated to make the temperature of the mixture reach 70°. C. Another representative example of the invention of the present invention is a cell culture substrate process, which uses the molecular copolymer of the present invention to prepare a cell culture substrate, which is used for culturing cells, and the cell culture substrate process Including: promoting a crosslinking reaction of the molecular copolymer with a photoinitiator (such as Benzoin) and a crosslinking agent (such as EGDMA, that is, Ethylene glycol dimethacrylate), the photoinitiator relative to the molecular copolymer The percentage of 〇le is at least 〇.01 up to 0.5, and the percentage of the crosslinking agent relative to the molecular copolymer is at least 〇1 to 2. In the above process of the cell culture substrate, the crosslinking reaction is a photopolymerization crosslinking reaction, and the photopolymerization crosslinking reaction uses an ultraviolet light source of about 100 watts, and the thickness of the molecular copolymer is at least 公 1 cm at most 0· 4 cm, light time at least 1 hour up to 4 hours. After the crosslinking reaction of the molecular copolymer, it is moistened. After the molecular copolymer is wetted, a plurality of pores are formed in the molecular copolymer by salting out and freeze-drying. The salt in the interior can be washed with water to remove it. In order to form a pore structure in the cell culture substrate, the cell culture substrate process further comprises: immersing the molecular copolymer after the crosslinking reaction with water to remove the solvent and sodium chloride before the molecular copolymer is wetted. So that the molecular copolymer is wetted and has a pore structure therein. The above process of the cell culture substrate may further comprise: freezing the molecular copolymer of the condensed body with liquid nitrogen to maintain the shape of the original turbidity, and then freeze-drying, the freezing temperature is below zero零5〇β (: between. [Embodiment] preparation of chondroitin sulfate monomer will buy Chondroitin sulfate A (Sigma), in the environment of the aqueous solution, add chondroitin hydrolase ABC In order to carry out the dissociation reaction, after the dissociation, an active double bond is generated on the ring, and the surface is to be partially secreted. The riding procedure is a liquid chromatography method using eight sieves, and the cracked ch〇ndroitin can be touched. A, the integer length of the long-term sugar production '故_Molecular_phase chromatograph, the non-chain length of the oligo-segment ς 12 1358415 line separation, and get a specific length of sugar (including disaccharide, tetrasaccharide, hexasaccharide , octasaccharide, etc.), and then purified by a strong anion parent for liquid chromatography, the amount of sulfation using chondroitin sulfate A and its position have an absolute influence on the performance of different electronegativity, so the strong anion is further pure The disaccharide molecule obtained by the purification step is identified by infrared light spectrometer, mass spectrometer, nuclear magnetic resonance instrument. In the infrared spectrometer, the sulfate of chondroitin sulfate is in the spectrometer and other kinds of functional groups. Significantly different, so the absorption intensity can be used for quantification. In the mass spectrometer, the sulfate of chondroitin sulfate is very easy to break during the ionization process, so the appropriate ion pairing reagent should be used to protect the sulfate. The quality of the complex, and then from the data obtained to reverse the quality of sugar (including disaccharide, tetrasaccharide, hexose, octasaccharide, etc.). In the aspect of the use of nuclear magnetic resonance, using one-dimensional selective stimulation Sorting the oligosaccharides of different fragments to obtain the desired disaccharide molecule. The ch〇ndr〇itin sulfate disaccharide used in the present invention has the structure shown in Figure ic, and its IR absorption peak: in the IR spectrum. The characteristic absorption peak (or simply the absorption peak) of ch〇ndr〇itin sulfate includes—the large and strong absorption peak near the 34〇〇cnrl C00 is 1633 cm 1 The absorption is called symmetry absorption at 14〇1(10)-1, the asymmetric absorption at s=〇 is 1425 αιΓ1, the symmetric absorption is at 1229 cnfl, (^ has a special absorption peak at 112〇cnfl, and the most important carbon-carbon double bond absorption The peak is at 1595 cnfl, and the age of the double bond disappears after participating in the copolymerization reaction, but other characteristic absorption peaks (or simply absorption peaks) still exist and are slightly offset (as shown in Fig. 2). A representative example of a molecular copolymerization process mainly comprises: forming a mixture comprising N-1SOpr〇pylacrylamide and a sugar amine (or its ugly substance), or more comprising Uetylie aeid) The percentage of m〇le (mole) of acrylamide is at least 刖 at most 95 '% of the mole of the glycosaminol (or its analog) is at least! Up to 2〇, the percentage of the acrylic acid is at most 151 and less than zero (depending on the application requirements, it is decided whether to add ^ 13 1358415 to heat the mixture until the absorption peak corresponding to the double bond of the molecule in the mixture disappears, and other The absorption peak of the chemical bond is shifted. An implementation example of the representative example of the molecular copolymerization process of the above invention is as follows: 95 m〇le (mole) % N-isopropylacrylamide and 0 to 5 mole % acryllc acid ' And the chondroitin sulfate with a mole percentage of 0.1 to 1% relative to the first two monomers is first dissolved in DMS0, and after being completely dissolved, it is placed in a nitrogen atmosphere, a total reflux condenser and a three neck reaction. In the bottle, first freeze with liquid nitrogen for 2 minutes' then vacuum, until the solids gradually dissolve, 'pass with nitrogen (this action is repeated four times), remove the oxygen of the reactor towel by pain; afterwards, dissolve The temperature is gradually heated to 4 ° C under the reduced cover, and when the temperature reaches 4 ° C &gt; C, the cesium (O.Olwt (% by weight) AIBN in 5 ml DMS0) previously dissolved in DMS0 is injected into the syringe. In the reaction bottle, and Continue heating to scratch, and let it react for 24 hours to obtain a non-volatile material leg c (abbreviation of molecular copolymer of N_is〇pr〇pylacr careful ac1d, chondroitin suifate, etc.), conversion percentage (by PNAC weight divided by monomer The total weight percentage is measured. Up to 8 times or more. After the reaction is completed, the solution is poured into a ten-fold volume (10), and the polymer is precipitated, and then placed in a box at a temperature of 35 C. In the aspect of structure identification, after the reaction, the absorption front of the double bond position disappears, but the characteristic absorption peaks of other positions are still there, but the financial offset, such as (10). There is asymmetric absorption in the secret (10), S=G The asymmetric absorption is at 1458(10)-丨, the symmetric absorption is at 1387(10)-, so, so has a special absorption peak at mw, and the absorption peak at c=0 is at (10)^ (as shown in Figure 3), except for the enemy, after the reaction is completed, In the ugly map, there is a characteristic peak of chQndr〇itin :te = in the vicinity of 2·9 Qing. There is a main characteristic peak of NIPAAm in the special 1PPm position of the yang ndr〇itin between the 2 qingqing, and the area ratio is about: 6 (As shown in Figure 4, where A, 5, β = M4〇T, ·;, 1. It can be confirmed that (10) ttr polymer, its molecular weight can be between 1〇~~2 faces. In, = square_determination, TGA and DSC as measuring tools, test its cracking at tga&gt; dish degree (five) around the boot 'So its stability is quite good at room temperature and its glass transition temperature (Tg) A is found at about 13 1358415. It is determined that the material has a considerable amount of the invention of the molecular silk process, the percentage of the coffee, the result Roughly the same. The molecular copolymerization process of the white knives of the white knives (or _) is not included in the molecular copolymerization process of glycosaminoglycans (or their analogs). It is indicated that isopropyl acrylate acid amine (N-1S) 〇 pr_aerylamide) and propylene _ kind of molecular copolymerization process is as follows: 95% by weight of Ni Ning opylacrylamide and 5% by weight of (four) _, placed in the neck of the anti-wrinkle 'additional amount _ turn, the financial simplified after the domain There are lion equipment ^ = environment, total reflux condenser and three-neck reaction bottle, with nitrogen, the solution is Γτ ^ιΤ / Γ slowly heated to 40c, when the temperature reaches 4 (rc, will be dissolved beforehand) _ medium (0.01wt% calendar in 5 ml _) is injected into the reaction bottle with a syringe and continuously added 24 PNA ^ (measured by dividing the total weight percentage of the monomer) up to 9〇% or more to be reacted After that, the liquid was poured into ten volumes of acetonitrile, and the polymer was precipitated, and then the solvent was drained under vacuum at a temperature of 35 C. In the structural identification, the characteristic of the biliary in the IR was absorbed. The peak, in 3200~340 (there is also a strong absorption between W, and, as shown in Figure 5). And in the ugly figure (3) (four), where (four) (4) ^ (four) is a ritual... (four) =-24·22, Η The characteristic absorption peak of bit = 12.45), the absorption bee of Nip Shi (abbreviation of N-qing ropylacr fine ide) at 4 _ and 1 ppm, the area ratio is about i to 6, and the characteristic absorption peak of the two places is NIPAAm. On the main absorption, it was thus possible to identify a synthetic PolKNIPM-AAe polymer (Nu is a molecular copolymer of N-tank and a mixture of acrylic acid) for comparison. Other thermal properties were analyzed using TGA and dsc as thermal analysis tools. At the TGA test, the material cleavage temperature (Τ5χ) was approximately 16Q&lt;&gt;c, while the DSC test, its glass transition temperature α) was approximately 11 〇β (:, by these thermal properties analysis, the material was at 37° The cell culture incubator in C has a fairly stable nature, but the pyrolysis temperature (Tsx) and glass of PNA (abbreviation of molecular copolymerization of N-isopr〇pylacryiamide, acrylic acid, etc. 15 1358415) compared with the polymer of Example j. The transfer temperature (Tg) is lower than that of pNAC. It may be due to the addition of chondroitin sulfate to the polymer of Example 1, because of its cyclic structure, the polymer backbone is less mobile. Therefore, the pyrolysis temperature and glass transition temperature of PNAC are higher. High cross-linking reaction of isopropylamine acrylate, acrylic acid, glycosamine (or its analogs), etc., and preparation of cell culture substrate. The reaction and preparation of cell culture substrate are mainly light. The initiator (for example, Benz〇in) and a crosslinking agent (such as EGDMA, that is, Ethylene glyC〇l dimethacrylate) promote the crosslinking reaction of the molecular copolymer, and one of the implementation details is explained. As follows: Take appropriate amount of sulfate) synthesized from Example 1 and add 1 wt% of N-isopropylacrylamide and 0.1 m〇% of Benzoin as photoinitiator (the total amount relative to other monomers is 〇〇1~) 〇5 m〇1%), 〇1齢1%

Ethylene glycol dimethacrylate is used as a cross-linking agent (the content of cross-linking agent is between (1) and mol%), and then DMS is added as a solvent to make the solid content of 6〇%, and it is placed in an ultrasonic oscillator and uniformly mixed. And rushed to the bubbles, after which the sodium chloride salt was added, and the added sodium chloride was used as a solid for the pores. After mixing uniformly, it was placed in a Teflon mold and placed in a UV lamp of 1 〇〇w. The crosslinking reaction was carried out by irradiating for 4 hours. After the reaction is completed, it is cooled at room temperature, and then the sample is taken out, placed in deionized water to moisten the material, and the vaporized sodium salt in the material is removed. In the process of moistening, the deionized water is replaced multiple times to confirm After removing the salt in the sample, it takes about 72 hours, and then the sample is freeze-dried for 24 hours to remove the moisture in the sample to obtain a three-dimensional three-dimensional porous material (for example, 1 cm in diameter, thickness). 〇_2 cm of the original disc type material) 'porosity can reach more than 70%, the pore diameter is between i 〇 ~ 10 〇 / zm. The molecular copolymer phase transfer temperature (LCST) test

The copolymer of isopropyl methacrylate, glycosamine (or its analogy), acrylic acid, and the like, and a copolymer of isopropylamine acrylate, acrylic acid, etc., are respectively dissolved in PBS 16 1358415 buffer / In combination, the towel g has been set to a concentration of 5 wt%, which is measured by an ultraviolet/visible spectrophotometer to observe the change in the degree of Wei as a function of temperature. When the absorbance is suddenly increased, the temperature is its phase transfer temperature. When the temperature continues to increase, the solution has a white colloid, Poly 〇 έ 4 Poly(N-isopropylacrylamide)^-^#^·®^^^ 31~33Ί' while adding other hydrophilic monomers, the copolymerized polymer will have a phase transition temperature of @'如如: Add Aerylie aeid (AAe) to the copolymer of NIpAAm (Morbi ΝΙΡΑΑπκΑΑϋ) The phase transfer temperature can be increased to about 36. π, and the temperature is increased again. The polymer can form a jelly-like water gel and tightly enclose the solvent. When adding 〇~ c^ntoiti(iv) lfate to polymerize the copolymer polymer (10) c) its phase transfer temperature is about k to 36 38C (shown in Table 1), and as the amount of ch〇ndr〇jtin suifate increases, its phase The transfer temperature also has a slight increase, but the change is not large. Above the phase transition temperature, it can break the test between the polymer and the right, so that the polymer molecules can be polymerized, and the polymer can be produced at a temperature lower than the phase transition temperature. In the above-mentioned characteristics, the molecular copolymer of the present invention is very suitable as a drug release carrier, a protein separation procedure, and the like. Polymer Moisture Test Water Gel Concentration Test The pore water gel prepared by the copolymerization process of isopropyl acrylamide, acrylic acid, glycosaminol (or its analogs, etc.) was subjected to the Lai test. The degree _ represents the degree of water content of the polymer. The experimental method is that after the water gel is vacuum dried under the machine, the weight obtained is weighed to dry weight (5). The water gel was placed in the sample crucible, 2〇8 pure water or buffer solution was added and the experimental temperature was controlled with a water bath. After the passage of the section, the sample is taken out to remove the moisture from the surface of the lens and the weight is weighed. This step is reversed until the value is no longer changed to achieve a more balanced balance. The weight obtained is the wet weight (W·). The degree of sweUing is calculated as: 17 1358415 脉 χ 100% The meaning is the weight of water absorbed by the polymer per unit weight. In terms of the test results of the balance, PNA (NIPAAm: AAc=95: 5) The wetness is about 250% (the above NIPAAm is the abbreviation of isopropyl acrylate, aac is the abbreviation of acrylic acid), and the polymer PNAC (NIPAAm: AAc: CS=) is added with 1% chondroitin sulfate. 95:5:l), its equilibrium moisture can reach about 600% (the above CS is short for glycosamine or its kind), compared with PNA polymer, its water content is about 3 times larger, it may be Since ch()ndr〇itin sulfate itself has a negative charge, it has a large water content due to the appearance of charge repulsion during hydration. Because of the abundant water content, imipenyl isopropyl acrylate, acrylic acid, and glycosamine are used in this case. Porous water gel prepared by copolymerization reaction process (one of its uses: as cell culture medium #) is also close to physiological conditions, so it is suitable for application in the field of biomedicine. Water gel response reversibility test ...will not reach the (five) city of the water county to reach Pinglin Job, at its critical temperature _ back and forth change / phoenix to change the polymer type to understand whether its response during the shrinkage and swelling process is fast enough. Put the sputum weight after placing it in 25. 〇 pure water towel to wait for it After the swell is balanced, the temperature is adjusted to 37C, and the amount is turned off every other time. After the balance is reached, the temperature is changed.

2. This is done several times to observe the situation of swelling and contraction. Experimental results found that PNA

At-95 · ruthenium polymer has less obvious response characteristics, and its phenomenon with temperature changes is slower. It takes about three hours. The demanding PNAC deletes Am:AAc.CS=95.5^t to another temperature. Pingna Run, also observed ^ 0.5.1) With the temperature response, after changing the temperature, about '卩 can reach another new balance _, faster response than m polymer special 咐州(4) (4) The tenderness __ toxicity test was raised at 37t'5 %. __Box (-(10), inverted to see its growth situation 1 cell length 18 1358415 trypsin-EDTA wash the cells in the culture dish, mix trypan blue, calculate the cell number under the microscope with a cell counter ixl 〇 5celis / Cc· was placed in each culture dish and placed in a quantitative polymer. The culture was continued for another three days, and then the growth of the cells around the substrate in the culture dish and the change in the actual number of cells under the microscope were observed. System) contains only cells and medium. From the experimental results, it was observed that the number of cells originally grown at a concentration of 1*105 cells/cc (cc is cc) was cultured, and then PNAC (NIPAAm: AAc: CS=95: 5: l%) The number of cells increased to 3.25*105 cells/cc. Compared with the control group (completely cultured only in the empty culture), the control group increased the cell number to 4_31*1 after the same conditions. The cell concentration of 5 cells/cc, this data shows that the substrate of the present invention has reached an acceptable cytotoxicity test range (the results are shown in Table 2). In addition, the substrate is directly observed by a microscope. In the case of cell growth, it was found that the cells in the control group could be completely attached to the surface of the culture dish and closely arranged; while around the substrate of the present invention, the cell growth was slightly sparse on the surface of the control dish, and the rest was It is still the same as the growth of cells in the control group. [Table of brief description] Table 1 shows the different ratios of isopropyl acrylate, acrylic acid 'glycosamine or its symmetry to the molecule formed by the (4) reaction The phase transition temperature of the copolymer changes. Table 2 shows the results of the cytotoxicity test of the molecular copolymer formed by the polymerization reaction in this case as a cell culture substrate. ° LCST (°C) 32.5 36.5 37.14 Relative to other orders 95 5 〇95 5 0.5 19 1358415 37.5 37.64 Cell number (cells/cc) 4. 31 Wood 105 2.76 wood 105 0. 94氺10s .25 Wood 105 95 5 1 95 5 2 NIPAAm Representative: isopropyl acrylate Amine AAC stands for: Acrylic acid CS stands for: Glycosamine or its analogue

Sample

Control system P(NIPAAm) PNACCNIPAAin: AAc: CS=95:5:1%) P(NIPAAm) stands for: Polymer PNA formed only with isopropyl acrylamide (NIPAAm: AAc=95: 5) PNA stands for: The molecular copolymer PNAC formed by the polymerization of isopropylamine amide with acrylic acid represents: a molecular copolymer formed by polymerization of isopropyl acrylate, acrylic acid, glycosaminol or the like. The above description is intended to provide a better or more practical embodiment of the present invention. The spirit and scope of the present invention is not limited to the embodiments disclosed above, but rather, various modifications or the like may be included. ^ [Simple description of the diagram] Figure 1 is used to illustrate the phenomenon in which the double bond between carbon and carbon is converted into a single bond in each monomer molecule of the copolymerization reaction. Figure 2 is an infrared spectroscopy (infrared spectroscopy) of the modified ch〇ndroitin sulfate [where the horizontal axis represents the wave number per centimeter (wave number port), and the vertical axis represents the penetration rate ( Transmittance). Fig. 3 is an infrared spectrum diagram of a molecular copolymer formed by the polymerization reaction of isopropylamine acrylate, acrylic acid, glycosamine or the like, wherein the horizontal axis represents the wave number peron. The vertical axis represents the transmission rate. 20 1358415 Figure 4 is the isopropyl acrylamide, acrylic acid, tyrosine or its ugly substance, etc. The molecules formed by the polymerization reaction in this case are (4) 暇 NMR 丝 丨 丨 丨 丨 已 已 已

Resonance Spectroscopy). Fig. 5 is an infrared spectrum of a molecular copolymer formed by the polymerization reaction of isopropyl acrylic acid, propylene acid, etc. in the present case @ 'the towel _ represents the wave number per square centimeter (wave_berper cm), and the vertical axis represents wearing Overshoot (transmittance). Fig. 6 is a nuclear magnetic resonance spectrum of the reddish molecular copolymer of the present invention in the polymerization of isopropylpropanoid, propylene, and the like. [Main component symbol description] C carbon 〇 oxygen N氤 Η hydrogen S sulfur ― single bond = double bond 21

Claims (1)

X. The scope of application for patents: A method for cultivating cells by using sulphamine or a congener thereof, comprising: converting a double bond connection between two carbons in a hetero bis amide amide molecule into a single bond a molecule of guanidinium isopropyl acrylate having two carbons which can be linked to each other by a single bond; two of the glycosaminoglycans or their congeners (glycosaminoglycans) The double bond between carbons is converted to a single bond to form a pseudo-glycosin molecule, which has two carbons that can be linked to other molecules by a single bond; let a first amount of the The guanidinium isopropylamine molecule is chemically bonded to a second amount of the tyrosine molecule to obtain a molecular copolymer: and the molecular copolymer is used as a substrate for culturing cells. 2. The method of using a glycosamine or a congener thereof for culturing cells as described in claim 1 wherein the molecular copolymer has a molecular weight ranging from at least 20,000 to 250,000. 3_ A method for using a glycosamine or a congener thereof for culturing cells as described in claim 1 wherein the first amount is at least 8 〇 at most 95, and the second amount is mole. The percentage is at least 1 to 2 〇. 4. The method of using a glycosaminol or a congener thereof for culturing a cell as described in claim 1, wherein the molecule of the glycosaminol or the like contains a molecular structure of a carbon-carbon double bond. 5_ The method for using a glycosamine or a congener thereof for culturing a cell according to the above-mentioned claim, wherein the glycosaminol or the like is a disaccharide, a tetrasaccharide, a hexasaccharide, an octasaccharide or the like. At least one of them. 6. A method for using a glycosamine or a congener thereof for culturing a cell as described in claim 1, wherein the glycosaminoglycan or its congener is chondroitin sulfate, hyaluronic acid (hyaluronic) Acid), heparin, heparin sulfate, dextran sulfate, pentosan sulfate, dermatan sulfate, etc.丄 Zero 15 less one. 7. A method of using a glycosaminoglycan or a congener thereof for culturing a cell as described in claim 1, wherein the chemical bond is a single bond between carbon and carbon. 8. The method of using a glycosaminoglycan or a congener thereof for culturing a cell as described in claim 1, wherein the imidazomethic acid molecule and the pseudo-glycosin molecule are similar in a ring shape. The chain structure is connected in a way. 9_ The method for using a glycosamine or a congener thereof for culturing cells according to claim 1, further comprising: promoting a crosslinking reaction of the copolymer by a photoinitiator and a crosslinking agent; . 10. The method of using a glycosaminoglycan or a congener thereof for culturing a cell according to the scope of the patent application, further comprising: converting a double bond connection between two carbon atoms in an acrylic molecule into a single bond connection, A pseudo-propionic acid molecule is formed. The pseudo-acrylic molecule has two single bonds for connecting the respective ages, and the mole percentage of the pseudo-propane ship molecule is at most 15. U. The method for using a glycosamine or a congener thereof for culturing cells as described in claim 10, wherein the pseudo-(10) acid molecule and the iridino-propionylamine are in the middle of riding At least - the boots are connected, drop out _ pin ^ = single button. 12. A method for using a glycosaminoglycan or a congener thereof for culturing a cell, comprising: forming a first carbon-containing molecule, wherein two single bonds are respectively connectable to another molecule; The homologue (glyeQSaminQglycans) is formed—the second carbon-containing molecule' also has two single bonds which can be respectively linked to other molecules; 3 the first carbon-containing molecule and the second carbon-containing molecule are made by the monomorphic The bond structure is linked into a molecular copolymer: and a bond, and the molecular copolymer is used as a substrate for the cultured cells. 13. A method for using a glycosaminoglycan or a congener thereof for culturing a cell, comprising: a double bond between two carbons in a molecule of isopropyl propyl decanoate 23 'Formation - bribe isopropyl propyl ship secret, isopropyl isopropyl surface amine two carbon can be connected to other molecules by a single bond; let a sugar amine or its ugly (glycosaminoglycans) two The double bond connection between carbons is converted into a single bond to form a pentaamine molecule, and the two molecules of the fructosamine can be linked to each other by a single bond; let a first amount of the pseudo-isopropyl a guanamine acrylate molecule is chemically bonded to a second amount of the sucralose molecule to obtain a molecular copolymer; a photoinitiator and a crosslinking agent are used to facilitate crosslinking reaction of the molecular copolymer; The molecular copolymer having a crosslinking reaction is used as a substrate for culturing cells. 14. A method of using a glycosaminoglycan or a congener thereof for culturing a cell, comprising: converting a double bond between two carbon atoms in a molecule of isopropyl acrylate to a single bond, forming a pseudo-differentiation a propyl acrylate amine molecule having two carbons which can be linked to each other by a single bond; a double of a carbon of a glycosaminol or its analogous (giycosaminoglycans) The bond linkage is converted to a single bond to form a pseudo-glycosin molecule having two carbons which can be linked to each other by a single bond; the first number of the pseudo-isopropyl propylene The acid amine molecule is linked to a second amount of the fructosamine molecule in a ring-like chain structure to form a molecular copolymer; and the molecular copolymer is used as a substrate for the cultured cells. 15. A method of using a glycosaminoglycan or a congener thereof for culturing a cell, comprising: forming a first carbon-containing molecule having two single bonds to link to other molecules; using a glycosaminol or the like (glyCOSamin〇glycans) form a second carbon-containing molecule, and two single bonds can be respectively connected to other molecules; let the first carbon-containing molecule and the second carbon-containing molecule pass the single bonds a cyclic chain-like structure connected to form a molecular copolymer; a photoinitiator and a crosslinking agent to facilitate crosslinking reaction of the molecular copolymer; and the molecular copolymer having a crosslinking reaction as a cultured cell The substrate. twenty four