JP2008267822A - Cd44 recognition property coating agent and porous body - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a CD44 recognition property coating agent that has temperature responsiveness where it is hydrophilic at a temperature lower than a predetermined temperature (T) lower than a living body temperature and it is hydrophobic at a temperature higher than the predetermined temperature, and contains hyaluronic acid or its derivative. <P>SOLUTION: This CD44 recognition property coating agent is made of polymer material carrying hyaluronic acid or its derivative. The polymer material is hydrophilic at a temperature lower than the predetermined temperature (T) lower than the living body temperature, and is hydrophobic at a temperature higher than the predetermined temperature (T). The polymer material is hydrophilic at a temperature lower than the predetermined temperature (T) lower than the living body temperature, and is hydrophobic at a temperature higher than the predetermined temperature (T). The polymer material is preferably obtained by block-polymerizing N-isopropylacrylamide and 3-N, N-dimethylamino propyl acrylamide to a compound which has three or more N,N-dialkyl-dithiocarbamyl methyl molecule groups in the same molecule, which serves as an iniferter. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a CD44-recognizing coating agent having a property of recognizing CD44-positive cells and a porous body coated with this CD44-recognizing coating agent.

  A technique using a monoclonal antibody as a ligand for CD44, which is highly expressed in cancer cells, is known. Monoclonal antibodies have very high selective specificity, and only CD44 can be recognized unless the surface is coated with non-specific adsorption such as heterogeneous proteins. However, monoclonal antibodies are very expensive, and are practical if they are experimental systems that can obtain the maximum effect with a minute amount such as immunostaining. If necessary, it is disadvantageous in terms of cost.

  Hyaluronic acid is known as a ligand that recognizes CD44 at low cost (for example, the column of the conventional technique in Patent Document 1 below). Hyaluronic acid is a mucopolysaccharide composed of β (1-3) glucuronic acid and β (1-4) glocosamine having a molecular weight of about 50,000 to 8 million, and is a polymer having a carboxyl group in the molecule.

  The cell separation column allows cells to be separated by the affinity for the ligand immobilized on the column by passing the cell suspension through the cell separation column. Is fixed in water-insoluble.

When fixing hyaluronic acid as a ligand to a porous material in a water-insoluble manner, a method of introducing a chemically active functional group such as an amino group on the surface of the porous material and fixing it using hyaluronic acid and a chemical cross-linking agent is considered. The organic solvent used for the surface treatment infiltrates into the micropores inside the porous body and has a problem that it remains difficult to remove. It is also difficult to remove the solvent molecules existing between the polymer molecules forming the porous body. In the case of a chemical crosslinking agent, since it is a highly active compound compared with a solvent, the problem of residual is larger. In addition, there are porous substrates that are violated by organic solvents, and the types of porous substrates are also restricted.
JP 9-25299 A

  The present invention solves the above-mentioned conventional problems, is water-soluble (hydrophilic) at a temperature lower than a predetermined temperature (T) lower than the living body temperature, and water-insoluble (at a temperature higher than the predetermined temperature ( It is an object of the present invention to provide a hyaluronic acid or its derivative-containing CD44 recognizing coating agent and a porous body coated with this CD44 recognizing coating agent, which have a temperature responsiveness that is hydrophobic.

  The CD44 recognizing coating agent of the present invention is a CD44 recognizing coating agent comprising a polymer material supporting hyaluronic acid or a derivative thereof, and the polymer material is at a temperature lower than a predetermined temperature (T) lower than the living body temperature. It is hydrophilic and is hydrophobic at a temperature higher than the predetermined temperature (T).

  The predetermined temperature (T) is preferably a temperature between 25 and 35 ° C.

  The polymer material preferably has a block copolymer chain of a cationic monomer and N-isopropylacrylamide or a derivative thereof.

  The polymer material uses a compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule as an iniferter, and a light-irradiation living polymerization of a vinyl monomer and N-isopropylacrylamide or a derivative thereof. It is preferable to be a branched polymer.

  The compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus and three or more N, N-dialkyl-dithio as a branched chain in this nucleus. Those having a carbamylmethyl molecular group bonded thereto are preferred. For this iniferter, either a cationic monomer and N-isopropylacrylamide or a derivative thereof may be polymerized first.

  The porous body of the present invention is coated with the CD44 recognizing coating agent of the present invention.

  The CD44 recognizing coating agent of the present invention is hydrophilic and water-soluble at a temperature lower than the predetermined temperature. Therefore, the CD44-recognizing coating agent is dissolved in water and applied to a living body or a porous body, and then becomes hydrophobic (water insoluble) by setting the temperature higher than a predetermined temperature. Adhere to. The attached CD44 recognizing coating agent exhibits a CD44 recognizing action by the supported hyaluronic acid or its derivative. Therefore, according to the coating agent of the present invention, hyaluronic acid is fixed from the aqueous solution to the material surface without using an organic solvent, a chemical cross-linking agent, or the like, thereby imparting selective affinity to CD44-positive cells and making it insoluble in water. Can be fixed.

  This CD44 recognizing coating agent can be stably stored for a long time as an aqueous solution, and can also be applied to a hybrid material or a living body.

  When the polymer material constituting the CD44 recognizing coating agent has a cationic polymer block, hyaluronic acid or a derivative thereof has a negative charge because it has a carboxyl group having a high pKa. It is supported so as to form an ionic complex.

  When this polymer material has a plurality of branched chains made of a block copolymer of a cationic monomer and N-isopropylacrylamide or a derivative thereof, the adhesion of the CD44-recognizing coating agent to the porous body is high. That is, in the CD44-recognizing coating agent having a plurality of branched chains, if all of the plurality of branched chains in one molecule that are hydrophobically bonded to the surface of a porous body or the like do not peel at the same time, the molecule (polymer) Can not be peeled off from the material surface, and even if one piece is peeled off, if the remaining branched chain is bonded, the once peeled branched chain can be rebonded to the material surface. On the other hand, in the case of a linear block polymer having only one polymer chain in one molecule, if this one polymer chain is peeled off, the molecule is peeled off as it is, so that the adhesion is weak. Conceivable.

  The CD44 recognizing coating agent of the present invention comprises a polymer material supporting hyaluronic acid or a derivative thereof, and the polymer material is preferably a block having a cationic polymer block and a block of N-isopropylacrylamide or a derivative thereof. Has copolymer chains.

  As the polymer material, a compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule is used as an iniferter, and this is irradiated with a cationic monomer and N-isopropylacrylamide or a derivative thereof. A branched polymer polymerized by living polymerization is preferred.

  In this specification, the iniferter is a polymerization initiator that generates radicals upon irradiation with light, a function as a chain transfer agent, a function that bonds with the growth terminal to stop the growth, and a polymerization when light irradiation stops. It is a molecule that functions as a polymerization initiator / termination agent for stopping.

  As a compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule, three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups are bonded as a branched chain to the benzene ring. These are preferred, and specific examples are as follows. That is, as a 3-branched chain, 1,3,5-tri (bromomethyl) benzene and sodium N, N-dithiocarbamate (sodium N, N-dithiocarbamate) can be obtained by addition reaction in ethanol. , 3,5-tri (N, N-dithiocarbamylmethyl) benzene, and four branched chains include 1,2,4,5-tetrakis (bromomethyl) benzene and sodium N, N-dithiocarbamylate ( 1,2,4,5-tetrakis (N, N-dithiocarbamylmethyl) benzene obtained by addition reaction with sodium N, N-dithiocarbamate) in ethanol. Hexakis (N, N-dithio) obtained by addition reaction of bromomethyl) benzene and sodium N, N-dithiocarbamate in ethanol Rubamirumechiru) is a benzene.

  The above iniferter is almost insoluble in polar solvents such as alcohol, but is easily soluble in nonpolar solvents. The nonpolar solvent is preferably a hydrocarbon, an alkyl halide or an alkylene halide, and particularly preferably benzene, toluene, chloroform or methylene chloride, particularly toluene and chloroform.

  Monomers to be polymerized to this iniferter are a cationic monomer and N-isopropylacrylamide, and any of them may be polymerized first. In addition, (meth) acrylate represents an acrylate and / or a methacrylate.

  As the cationic monomer, vinyl monomers, particularly 3-N, N-dimethylaminopropylacrylamide, are suitable.

  To react the iniferter with the above monomer, prepare a raw material solution containing the iniferter and one monomer, and irradiate it with light, whereby the first step reaction product in which the one monomer is bonded to the iniferter Is generated. As the solvent in this case, the solvent of the above iniferter may be used. Next, the other monomer is block copolymerized with each branched chain of the first step reaction product.

  The concentration of the cationic monomer in the raw material solution is preferably 0.1 M or more, for example, 0.1 to 2.5 M. The concentration of N-isopropylacrylamide or a derivative thereof is preferably 0.1M or more, for example, about 0.1 to 2.5M.

  The concentration of the iniferter is preferably about 0.1 to 100 mM.

200-400 nm is suitable for the wavelength of the light to irradiate. The irradiation time of the light depends on the irradiation intensity, about 1 to 60 minutes are preferred, about 1 to 30 minutes at a low irradiation intensity of about 1μW ^/ cm ² ~10mW ^/ cm 2 is particularly preferred.

  The reaction product produced in the light irradiation step (first light irradiation step) using one monomer is preferably purified, and then preferably dissolved in alcohol and the second light irradiation using the other monomer. Perform the process. As the alcohol, methanol or ethanol, particularly methanol is preferable. The concentration of the first step reaction product in the alcohol solution is preferably about 0.01 mM to 100 mM.

  The polymerization degree of the cationic monomer is particularly preferably 10 to 6000, and the number average molecular weight of the polymer block of N-isopropylacrylamide or a derivative thereof is about 2,000 to 500,000, particularly about 3,000 to 150,000. Is preferred.

  Since the target branched polymer is produced in the reaction solution by this light irradiation, the CD44 recognizing coating agent comprising the branched polymer can be obtained by purification as necessary.

  The molecular weight of this branched polymer depends on the number of branched chains, but is preferably about 3,000 to 600,000, particularly 3,000 to 150,000, and more preferably about 3,000 to 100,000.

  The branched polymer thus produced has a polymer block of N-isopropylacrylamide or a derivative thereof. This polymer chain of N-isopropylacrylamide or a derivative thereof has a temperature dependency that is hydrophilic at a temperature lower than 30 ° C. and hydrophobic at a temperature higher than 30 ° C., whereby the CD44 recognizing coating agent is about It is water insoluble at temperatures above 30 ° C. and water soluble at temperatures below about 30 ° C.

  Accordingly, an aqueous solution of a CD44 recognizing coating agent having a temperature lower than about 30 ° C., for example, about 10 to 25 ° C. (concentration is preferably about 3 to 150 mg / mL) is attached to a living body or a porous body by coating or the like. By raising the temperature to a higher temperature and drying as necessary, a water-insoluble CD44 recognizing coating having a cationic polymer block is formed. In the case of a living body, the temperature increase at this time is performed by the body temperature of the living body. The formed CD44 recognizing coating has a plurality of branched chains containing a polymer block of N-isopropylacrylamide or a derivative thereof, and thus has high adhesion to a living body or a porous body and is difficult to peel off.

  Moreover, the branched polymer produced | generated in this way has a cationic polymer block. Hyaluronic acid or a derivative thereof is supported on the cationic polymer block so as to form an ionic complex. In order to support hyaluronic acid or a derivative thereof on this polymer material, it is preferable to add an aqueous solution of hyaluronic acid or a derivative thereof to a low temperature aqueous solution of the polymer material and mix them.

As this porous body,
Columns packed with polystyrene beads and chitosan beads,
Porous structures such as polyolefin sponge and polyurethane sponge,
porous films such as e-PTFE membrane and polypropylene membrane,
Porous hollow fibers such as polysulfone and regenerated cellulose,
Nonwoven fabrics and laminates of woven fabrics are exemplified.

  In order to attach the CD44 recognizing coating agent to the surface of the porous body, the porous body may be brought into contact with a 0.01 to 10% by weight aqueous solution of the coating agent and dried at a temperature equal to or higher than the predetermined temperature.

  This porous body can be used as follows, for example. That is, a CD44-recognizing coating agent is attached to a polyurethane sponge having an average pore size of 200 μm, and a cell suspension is passed through the cells to collect cells that show a delay tendency in passage time, thereby concentrating CD44 positive cells. It can also be used for cytodiagnosis for determining the presence or absence of CD44-positive cells, or for selecting CD44-positive cells in the process of differentiation induction.

Example 1
i) Synthesis of iniferter According to the following reaction formula, 1,2,4,5-tetrakis (NN-diethyldithiocarbamylmethyl) benzene was synthesized as follows.

  2.0 g of 1,2,4,5-tetrakis (bromomethylbenzene) and 12.0 g of sodium N, N-diethyldithiocarbamate were added to 10 mL of ethanol and stirred at room temperature for 4 days under light shielding. The precipitate was filtered, dried under reduced pressure, dissolved in 40 mL of chloroform, 50 mL of water was added, liquid-liquid extraction separation was performed, and sodium bromide was removed. After repeating this operation three times, the chloroform layer was dried with about 10 g of magnesium sulfate for 24 hours, filtered, added with n-hexane, purified by recrystallization, and a slightly light blue-white white Needle-like crystals of 1,2,4,5-tetrasakis (N, N-diethyldithiocarbamylmethyl) benzene were obtained (yield 90%). The starting material peak disappeared by high performance liquid chromatography, and it was confirmed that the purified product was a single substance.

The measurement results of ¹ H NMR (in CDCl ₃ ) are σ7.48 (s, 2H, Ar—H), σ4.57 (s, 2H × 4, Ar—CH ₂ S—), σ4.03 (q, 2H). × 4, N—CH ₂ −, J = 10.7 Hz), σ 3.72 (q, 2H × 4, N—CH ₂ −, J = 10.7 Hz), σ 1.26 to 1.31 (t, 3H) × _8, became _{-CH 2 -CH 3, J = 6.6Hz} ).

ii) Synthesis of cationic homopolymer comprising 4-branched star polymer by photopolymerization According to the following reaction formula, 1,2,4,5-tetrakis [(N, N-diethyldithiocarbamyl) A cationic homopolymer composed of (poly (3-N, N-dimethylaminopropylacrylamide) -methyl] benzene (hereinafter sometimes referred to as pDMAPAAm) was synthesized.

That is, 0.043 g of 1,2,4,5-tetrasakis (N, N-diethyldithiocarbamylmethyl) benzene synthesized by i) above was dissolved in 10 mL of toluene, and 3-N, N-dimethylaminopropylacrylamide was dissolved. 5.2 g of (3-N, N-DMAPAAm) was added and mixed, and the total amount was adjusted to 20 mL with toluene. After purging with high-purity nitrogen gas for 5 minutes with vigorous stirring in a quartz cell, ultraviolet light was irradiated for 10 minutes with a 200 W high-pressure mercury lamp. The irradiation intensity was adjusted to 1 mW / cm ² (250 nm) using an illuminometer (UVR-1, TOPCON, Tokyo, Japan). The polymerization solution is concentrated by an evaporator, and the polymer is purified by reprecipitation with diethyl ether, dissolved in a small amount of water, filtered through a 0.2 μm filter, and lyophilized to obtain a 4-branched star homopolymer 1, A cationic polymer composed of 2,4,5-tetrakis [(N, N-diethyldithiocarbamyl (poly (3-N, N-dimethylaminopropylacrylamide) -methyl) benzene (pDMAPAAm) was obtained (polymerization rate 40 The molecular weight was determined to be 3700 by GPC.

The measurement results of ¹ H NMR (in CDCl ₃ ) are as follows: σ 3.10-3.39 (br, —NH—CH ₂ —), σ 2.25-2.42 (br, —CH ₂ —N (CH ₃ ) _{2), σ2.21 (br, -N} (CH 3) 2), σ1.48-1.76 (br, -CH 2 -CH -, - CH 2 -CH 2 -CH 2 -) became.

iii) Synthesis of polymer material (4-branched pDMAPAAm-b-pNIPAM) by block copolymerization of N-isopropylacrylamide with cationic homopolymer According to the following reaction formula, tetrakis [(N, N-diethyl) Synthesis of dithiocarbamyl (poly (3-N, N-dimethylaminopropylacrylamide) -block-poly- (N-isopropylacrylamide) -methyl] benzene (hereinafter sometimes referred to as pDMAPAAm-b-pNIPAM). went.

  That is, 645 mg of the 4-branched pDMAPAAm homopolymer synthesized in the above ii) was dissolved in 10 mL of methanol, and 1.2 g of N-isopropylacrylamide (NIPAM) was mixed to adjust the total amount to 20 mL with methanol. Polymerization material pDMAPAAm-b-pNIPAM consisting of a block polymer of 4-branched pDMAPAAm and poly N-isopropylacrylamide (pNIPAM) after light irradiation polymerization under the same conditions as in ii) and purification in methanol / diethyl ether system (Polymerization rate 80%) was obtained. The molecular weight was measured to be 9,300 by GPC.

The measurement result of ¹ H NMR (in CDCl ₃ ) is as follows: σ 3.10-3.39 (br, —NH—CH ₂ —), σ 2.25-2.42 (br, —CH ₂ —N (CH ₃ ) ₂ ), σ 2.21 (br, —N (CH ₃ ) ₂ ), σ 1.48-1.76 (br, —CH ₂ —CH—, —CH ₂ —CH ₂ —CH ₂ —), σ 1.08 _{-1.23 (br, -CH- (CH 3} ) 2) became.

iv) Ion complexation with hyaluronic acid Ion complexation was possible by mixing the polymer material synthesized in (iii) above (4-branched pDMAPAAm-b-pNIPAM) and hyaluronic acid in an aqueous solution at about 20 ° C. .

  In order to make the CD44 recognizing treatment agent insoluble or hardly soluble in water, the mixing ratio with hyaluronic acid was determined as follows. The relationship between the absorbance and temperature of a solution in which 20 mg of 4-branched pDMAPAAm-b-pNIPAM and hyaluronic acid were dissolved in 2 mL of water in the range of 0 mg to 50 mg was plotted, and the cloud point was measured. It was confirmed that the cloud point shifted to a low temperature region when the amount of hyaluronic acid introduced was large. In order to be insoluble in water near the human body temperature, the amount of hyaluronic acid had to be about 20 mg or less.

v) Coating treatment 20 mg of the above polymer material (4-branched pDMAPAAm-b-pNIPAM block copolymer) and 15 mg of hyaluronic acid were dissolved in water to adjust the total amount to 2 mL. 10 μL of this solution was homogeneously cast onto a 2 cm × 3 cm square PET film and dried by a dryer to carry out coating treatment. The film was placed on a plate temperature-controlled at 37 ° C., and the contact angle was measured. The contact angle in this state was 15 °, and the hydrophilization phenomenon of the film surface presumed to be caused by hyaluronic acid was confirmed. When this film was continuously washed with warm water at 37 ° C., the contact angle of the surface gradually increased with the washing time and reached a plateau at about 44 °. Although the contact angle of the PET film approached 74 °, it was confirmed that hyaluronic acid was insolubilized and fixed.

Comparative Example 1
(N, N-diethyldithiocarbamylmethyl) benzene was prepared in the same manner as i) of Example 1 except that (bromomethyl) benzene was used instead of 1,2,4,5-tetrakis (bromomethylbenzene). Synthesized. The purified product was a colorless liquid, and the yield was about 93%.

The measurement results of ¹ H NMR (in CDCl ₃ ) are σ7.41 to 7.27 ppm (m, 5H, Ar—H), σ4.54 ppm (s, 2H, Ar—CH ₂ —S—), σ4.08 to 4.01 ppm (q, 2H, N—CH ₂ —), σ 3.72 ppm (q, 2H, N—CH ₂ —), σ 1.25-1.31 ppm (m, 6H, —CH ₂ —CH ₃ ) became.

  Next, a linear block polymer pDMAPAAm-b-pNIPAM having only one linear chain was synthesized according to the above methods ii) and iii), and the coating treatment and water washing on the PET film of (v) above The experiment was conducted. The contact angle increased from 15 ° to 74 ° just by washing lightly at 37 ° C., and it was confirmed that the coated polymer was quickly peeled off from the PET film.

  From the above, the superiority of the branched structure of Example 1 was confirmed. That is, in the CD44-recognizing coating agent of Example 1, the molecules are peeled from the material surface unless all of the branched pNIPAM blocks in one molecule that are hydrophobically bonded to the material surface are peeled from the material surface at the same time. If the remaining branched chain is bonded even if one peels, the branched chain once peeled can be recombined, so it is thought that it is firmly fixed to the material surface. . On the other hand, the linear block polymer synthesized in Comparative Example 1 has only one p-NIPAM polymer chain in one molecule. If this P-NIPAM polymer chain peels from the material surface, the molecule remains as it is. Since it peels from the surface of the material, it is considered weak against the cleaning treatment.

Claims (9)

In a CD44 recognizing coating agent comprising a polymer material supporting hyaluronic acid or a derivative thereof,
The CD44 recognizing coating, wherein the polymer material is hydrophilic at a temperature lower than a predetermined temperature (T) lower than a living body temperature and is hydrophobic at a temperature higher than the predetermined temperature (T). Agent.   2. The CD44 recognizing coating agent according to claim 1, wherein the polymer material has a block copolymer chain of a cationic monomer and N-isopropylacrylamide or a derivative thereof.   3. The CD44 recognizing coating agent according to claim 2, wherein the cationic polymer block has a molecular weight of 2,000 to 500,000.   4. The CD44 recognizing coating agent according to claim 3, wherein the block copolymer has a molecular weight of 3,000 to 600,000.   5. The CD44 recognizing coating agent according to claim 1, wherein the predetermined temperature (T) is a temperature of 25 to 35 ° C. 6.   6. The cationic homopolymer according to claim 1, wherein the cationic homopolymer is an iniferter having a compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule. A CD44 recognizing coating agent, characterized in that it is a branched polymer obtained by light-irradiating living polymerization.   7. The compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule as defined in claim 6, wherein the benzene ring is a nucleus and three or more N, N- A CD44-recognizing coating agent, wherein a dialkyl-dithiocarbamylmethyl molecular group is bonded.   8. The CD44 recognizing coating agent according to claim 6, wherein the vinyl monomer is 3-N, N-dimethylaminopropylacrylamide.   A porous body coated with the CD44 recognizing coating agent according to any one of claims 1 to 8.