JPH06116169A - Temperature responsive physiologically active substance-oligomer complex and its production - Google Patents

Abstract

PURPOSE:To obtain the complex free from aggregation, useful for targeting system, capable of being subjected to homogeneous reaction at a lower limit codissolution temperature or lower by making a specific oligomer into an activat ed oligomer under a specific condition and reacting the oligomer with a physio logically active substance. CONSTITUTION:An N-isopropylacrylamide homooligomer containing a carboxyl group at the terminal having 1,000-80,000 molecular weight is allowed to react with N-hydroxysuccinimide in the presence of a carbodiimide to give an activated oligomer, which is reacted with a physiologically active substance containing an amino group to give the objective complex.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to N having temperature response.
-A complex of an isopropylacrylamide oligomer and a physiologically active substance, and a method for producing the same.

[0002]

N-isopropylacrylamide has a hydrophobic isopropyl group and a hydrophilic amide group in the molecule, and its homopolymer has a lower critical solution temperature in an aqueous system (M. Henskins, J. Macromol. Sci. Chem., A2,
8 , 1441 (1968)). Therefore, it exhibits water-solubility at the low temperature side at 32 ° C and almost insoluble in water at the high temperature side.

On the other hand, physiologically active substances such as enzymes and antibodies are conventionally used after being immobilized on an insoluble carrier in order to separate them from the reaction product. However, due to immobilization on the carrier,
Only about 20% of the activity of the physiologically active substance is expressed as compared with the single use under physiological conditions. Further, in such a heterogeneous system, the reaction efficiency of the entire system is not always sufficient.

As a complex of an N-isopropylacrylamide oligomer and a physiologically active substance, a complex of a random copolymer of N-isopropylacrylamide and N-acryloyloxysuccinimide and a protein is known (JP Chen, et al. ., Biomaterials, 1 1, 625
-634 (1990)). However, since this complex has many bonding points in one polymer chain, it is apt to undergo unnecessary cross-linking to easily cause aggregation, and to reduce the activity of the bonded physiologically active substance. Have.

[0005]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned drawbacks of the conventional complex of N-isopropylacrylamide oligomer and physiologically active substance, and a complex of N-isopropylacrylamide oligomer and physiologically active substance. It aims at providing the manufacturing method.

[0006]

The present invention provides a complex of a physiologically active substance and an N-isopropylacrylamide homooligomer having a functional group at the terminal and having a molecular weight of 1,000 to 80,000.
And a complex of a physiologically active substance and a co-oligomer of N-isopropylacrylamide having a functional group at the terminal with a molecular weight of 1,000 to 80,000 and a hydrophilic monomer or a hydrophobic monomer, and a method for producing these complexes.

Examples of the physiologically active substance to which the present invention is applied include enzymes, antibodies, hormones and other various proteins,
Examples include glycoproteins, nucleic acids, and glycolipids. The functional group in the oligomer used in the present invention is not particularly limited as long as it can form a covalent bond with the physiologically active substance,
For example, carboxyl group, amino group, cyano group, hydroxyl group,
Halogen atom, oxo group, formyl group, active ester,
Examples thereof include an isocyanate group and acid chloride.

Examples of the N-isopropylacrylamide homooligomer having a functional group at the terminal include those represented by the following formula (I):

[0009]

[Chemical 7]

Molecular weight composed of repeating units represented by
Examples of the oligomer include 1,000 to 80,000, one end of which is a hydrogen atom and the other end is a group represented by the following formula (II): —SCH ₂ CH ₂ COOH (II). A typical example of the homo-oligomer is the following formula (III):

[0011]

[Chemical 8]

(In the formula, n represents an integer of 10 to 700). The aforementioned physiologically active substance and N
-Complex with isopropylacrylamide homo-oligomer has a lower critical solution temperature of 32 to 34 ° C (hereinafter "LCST").
Say. ), The oligomer to be used is a co-oligomer with a hydrophilic monomer, and the LCST can be shifted to a high temperature side by increasing the ratio of the hydrophilic monomer. The LCST can be shifted to the low temperature side by using a cooligomer and increasing the ratio of the hydrophobic monomer. Examples of the hydrophilic monomer used here include N, N-dimethylacrylamide, acrylic acid, methacrylic acid, acrylamide, monomethylacrylamide, ethylacrylamide, N-vinylpyrrolidone and vinyl alcohol, and examples of the hydrophobic monomer include , The following formula:

[0013]

[Chemical 9]

(In the formula, R represents a hydrogen atom or a methyl group, and n represents an integer of 1 to 20, preferably 2 to 18) and alkyl acrylate or alkyl methacrylate, such as butyl methacrylate. Is mentioned. The N
The molar ratio of N-isopropylacrylamide and hydrophilic monomer in the co-oligomer of isopropylacrylamide and hydrophilic monomer or hydrophobic monomer is preferably 100 to 80: 0 to 20, and N-isopropylacrylamide and hydrophobic monomer are The molar ratio with is preferably
It is 100 to 90: 0 to 10.

Examples of cooligomers of N-isopropylacrylamide having a functional group at the terminal and a hydrophilic monomer include compounds represented by the following formula (I):

[0016]

[Chemical 10]

A structural unit represented by the following formula (IV):

[0018]

[Chemical 11]

The molecular weight of the structural unit represented by is linearly irregularly arranged in a molar ratio of 100 to 80: 0 to 1,000 to 80,000.
And the other is a group represented by the following formula (II): —SCH ₂ CH ₂ COOH (II).

Further, as a cooligomer of N-isopropylacrylamide having a functional group at the terminal and a hydrophobic monomer, for example, the following formula (I):

[0021]

[Chemical 12]

The structural unit represented by the following formula (V):

[0023]

[Chemical 13]

The molecular weight of the structural unit represented by is linearly irregularly arranged in a molar ratio of 100 to 90: 0 to 1,000 to 80,000.
And the other is a group represented by the following formula (II): —SCH ₂ CH ₂ COOH (II).

The oligomer used in the present invention is, for example, N
-Isopropylacrylamide (hereinafter referred to as "IPAAm"), and optionally a hydrophilic monomer or a hydrophobic monomer, and 3-mercaptopropionic acid (hereinafter referred to as "MP").
"A". It can be manufactured by reacting a) in the presence of a radical polymerization initiator. Examples of the radical polymerization initiator used here include α, α′-azobisisobutyronitrile (hereinafter referred to as “AIBN”), α,
α'-Azobis (2-methylbutyronitrile), α, α'-
Examples include a series of azo compound-based radical polymerization initiators such as azobis (2,4-dimethylvaleronitrile).

Examples of the reaction solvent include N, N-dimethylformamide (hereinafter referred to as "DMF"), dimethylsulfoxide, tetrahydrofuran and dioxane. The reaction temperature is usually 60 to 70 ° C, preferably 68 to 70
The reaction time is usually 2 to 5 hours, preferably 4
~ 5 hours.

Any molecular weight (degree of polymerization) can be obtained by changing the ratio of the monomer and MPA.
That is, the reciprocal of the number average molecular weight of the obtained oligomer is proportional to "moles of monomer / moles of MPA". The molecular weight of the oligomer used in the present invention is 1,000 to 80,000, but the molecular weight is preferably large in order that the physiologically active substance-oligomer complex is likely to precipitate due to temperature change, while the reactivity of the terminal functional group is Since the activity of the physiologically active substance decreases due to steric hindrance due to the increase in the molecular weight, the molecular weight in particular is 3,000 to 30,000.
It is preferable to use the oligomer of.

When the oligomer thus obtained is bound to a physiologically active substance having an amino group such as a protein or an enzyme, the oligomer is, if necessary, subjected to N-hydroxy in the presence of a carbodiimide such as dicyclohexylcarbodiimide. After being made into an activated oligomer by reacting with succinimide or the like, it may be reacted with a physiologically active substance having the amino group.

In the activation reaction with N-hydroxysuccinimide, examples of the reaction solvent include ester solvents such as ethyl acetate, ether solvents such as tetrahydrofuran, and halogenated hydrocarbon solvents such as dichloromethane and chloroform. The reaction temperature is usually 4 to 10
The reaction time is usually 4 to 16 hours, preferably 12
~ 16 hours.

In the reaction of the activated oligomer and the physiologically active substance, the molar ratio of the activated oligomer and the physiologically active substance is preferably 5 to 50: 1 to 5, and the reaction solvent is, for example, water, Examples include DMF and dioxane,
The reaction temperature is usually 4 to 10 ° C, and the reaction time is usually 4
~ 16 hours, preferably 12-16 hours. On the other hand, when the water-soluble carbodiimide is used for the reaction in an aqueous solution or a buffer, the oligomer can be directly reacted with the physiologically active substance without activating the oligomer. The physiologically active substance-oligomer complex of the present invention obtained as described above has temperature responsiveness, is capable of homogeneous reaction at a temperature of LCST or lower, and is insolubilized at a temperature of LCST or higher,
It can be easily separated from the reaction product. Further, it becomes possible to provide a new targeting system in which a complex of a physiologically active substance having a pharmacological effect and an oligomer is administered to a living body, and only a portion to be acted on is heated to accumulate the physiologically active substance on the portion.

[0031]

The present invention will be described in more detail with reference to Synthesis Examples and Examples, but the scope of the present invention is not limited to these. (Synthesis Example 1) Synthesis of IPAAm homooligomer (1) Reagents AIBN and DMF were purified by a conventional method. IPAAm was dissolved in toluene, poured into 20 times the amount of petroleum ether, and recrystallized. MPA by vacuum distillation at 83 ℃
A 5 mm Hg fraction was used. As other reagents, commercial products were used as they were. (2) Synthesis of IPAAm homo-oligomer having carboxyl group at one end: Collect IPAAm and MPA in the amount shown in Table 1 in a polymerization tube,
Add radical polymerization initiator AIBN 3.3g (0.4mol / l),
Diluted with 50 ml MF. After freezing and deaeration, the tube was sealed under vacuum, and telomerization was carried out while keeping the temperature at 70 ± 1 ° C. in a thermostat while shaking for 5 hours. Next, the reaction was stopped by cooling, and the obtained reaction mixture was evaporated under reduced pressure on a water bath of 40 ° C., then poured into diethyl ether to precipitate an oligomer, which was filtered off and dried under vacuum. In addition, IP in DMF
The theoretical concentration of AAm oligomer is 1.0 mol / l. The IPAAm homooligomer obtained in this example has the above formula (I
II).

Table 1 shows the amounts, yields and yields of IPAAm and MPA used.

[0033]

[Table 1]

(3) Analysis of oligomer (a) Measurement of molecular weight 0.02 g of IPAAm oligomer was added to 5 ml of tetrahydrofuran.
Gel filtration chromatography at 40 ° C (Shimadzu
C-R4AX, Shodex KF-80M × 2) weight average molecular weight (M
w) and number average molecular weight (Mn) were determined.

(B) Determination of terminal carboxyl group After dissolving 0.2 g of IPAAm oligomer in 20 ml of ultrapure water,
The mixture was allowed to stand in a thermostat at 20 ° C. for 30 minutes, and neutralization titration was performed with 0.1N sodium hydroxide aqueous solution using phenolphthalein as an indicator. (c) Measurement of change in cloud point Dissolve 0.1 g of IPAAm oligomer in 10 ml of ultrapure water, put it in a sample tube, and keep it in a constant temperature bath for 5 minutes at each temperature.
The transmittance of the IPAAm oligomer aqueous solution at 500 nm was measured with an ultraviolet spectrophotometer (Shimadzu UV-240). Separately, prepare two kinds of oligomer (Ic-50, Ic-100) aqueous solutions with different molecular weights.
Incubate at 37 ℃ for 10 minutes, then stir vigorously for 30 seconds, then reincubate
The transmittance of the supernatant of the sample, which was allowed to stand at 5 ° C for 5 minutes, was measured. In addition, the sample after the same operation was performed at 37 ℃.
After centrifuging for minutes, the transmittance of the supernatant was measured. (4) Results (a) Molecular weight etc. Table 2 shows the ratio of the molar concentration of MPA to the molar concentration of IPAAm monomer in telomerization (hereinafter referred to as S / M) and the analysis results of the molecular weight.

[0036]

[Table 2] *: Average of three times. From Table 2, the molecular weight corresponding to one carboxyl group and the number average molecular weight (Mn) by gel filtration chromatography.
It can be seen that the two match well. From this, it was considered that the reaction proceeded by telomerization as shown in the following formula, and an oligomer having a carboxyl group at one end was produced.

[0037]

[Chemical 14] (In the formula, I represents a polymerization initiator, R ^* represents a radical derived from the polymerization initiator, * represents a radical, and M represents an IPAAm monomer.) As can be seen from the results shown in Table 2, the molecular chain length is It can be controlled by changing the MPA concentration relative to the monomer. Further, an oligomer having a small molecular chain length could be obtained by using a large amount of MPA in a DMF solvent. In this telomerization, M
The chain transfer constant (Cs) of PA to IPAAm is 3.73 × 10
^-3 and MPA is methyl methacrylate (0.38 × 10 ^-4 ).
And styrene (9.4 × 10 ^-4 ) ("Polymer Handbook", J. Bra
udrup and EH Immergut Ed., John Wiley & Sons,
NY, 1975, pp.92-95).

(B) Temperature Sensitivity of Oligomer in Aqueous Solution FIG. 1 shows the change in transmittance of the obtained IPAAm oligomer aqueous solution with temperature. As is clear from FIG. 1, the transmittance of each of the oligomers having different chain lengths greatly changed around 31 to 32 ° C. This is consistent with the lower critical solution temperature of the homopolymer of IPAAm,
It was found that the oligomer also has the same temperature sensitivity as the homopolymer.

Further, the obtained IPAAm oligomer Ic-5
Fig. 2 shows the change in transmittance depending on the concentration of 0 and Ic-100 in water.
FIG. 3 shows the change in the transmittance of IPAAm oligomer Ic-50 diluted aqueous solution by centrifugation. At a concentration of 1% by weight or more, it is considered that about 90% or more of the oligomer is precipitated and settled, but at a concentration of 1% by weight or less, the oligomer having a small molecular weight did not proceed to the settling. this is,
It was considered that the number of collisions of the dehydrated oligomer was reduced in the dilute solution, and the cohesive force required for the oligomer itself to settle could not be sufficiently obtained. However, it was found that even in the state of being dispersed in an aqueous solution, if the concentration is about 0.5% by weight, it will settle out by applying a centrifugal force of 10,000 g.

Example 1 Production of Collagen-IPAAm Homo-oligomer Complex 1 g of IPAAm homo-oligomer (Ic-50) having a molecular weight of 6,100 obtained in Synthesis Example 1 was added to 0.5 g of N-hydroxysuccinimide.
And 0.5 g of dicyclohexylcarbodiimide were dissolved in 50 ml of ethyl acetate and reacted at 4 ° C. for 16 hours. The reaction solution was filtered and then poured into diethyl ether to obtain an activated oligomer. Confirmation of introduction of succinimidyl group is confirmed by IR and U
V.

500 mg of atelocollagen having a molecular weight of about 300,000 was softened with 90 ml of cold purified water. After 0.5 ml of 0.1 M hydrochloric acid was added and dissolved therein, it was diluted 30 times with cold purified water having a pH of 3.0 (about 0.3% by weight). 140 mg of activated oligomer is dried D
It was dissolved in 4 ml of MF, added to the above collagen aqueous solution 5 times every 30 minutes, and reacted at 4 ° C. for 16 hours while stirring. The total amount of activated oligomer added here is 700 mg.

After completion of the reaction, dialysis was performed using a cellulose dialysis membrane. As the external liquid, purified water (hydrochloric acid acidic pH 3.0) was used.
Then, it was freeze-dried to obtain a collagen-Ic-50 complex. A 0.3% by weight aqueous solution of collagen-Ic-50 complex was put in a sample tube and kept at the temperature for 5 minutes in a thermostat, and the transmittance at 500 nm was measured by an ultraviolet spectrophotometer (Shimadzu UV-240). The results are shown in Fig. 4.

The collagen-Ic-50 complex is soluble in water regardless of the salt environment and in polar solvents such as alcohols and DMF. (Synthesis example 2) Synthesis of IPAAm cooligomer (1) Synthesis of IPAAm-hydrophobic monomer / cooligomer Each amount of IPAAm and butyl methacrylate (hereinafter referred to as "BMA") shown in Table 3 is used as a raw material monomer. And MPA were set so that the molecular weight of the obtained cooligomer was about 6,000 (the ratio of the molar concentration of MPA to the molar concentration of all monomers of IPAAm and BMA was 0.022) and Synthesis Example 1 Similarly, IPAAm-BMA cooligomer was synthesized.

The amount of each monomer and MPA used, the yield and the yield are shown in Table 3.

[0045]

[Table 3]

Table 4 shows the results of analysis such as molecular weight.

[0047]

[Table 4] *: Average of 3 times (2) Synthesis of IPAAm-hydrophilic monomer / co-oligomer The amount of IPAAm and N, shown in Table 5, as raw material monomers,
Using N-dimethylacrylamide (hereinafter referred to as "DMAAm"), the ratio of each monomer to MPA was set so that the molecular weight of the obtained cooligomer was about 6,000 (MPA relative to the molar concentration of all monomers of IPAAm and DMAAm). The IPAAm-DMAAm cooligomer was synthesized in the same manner as in Synthesis Example 1 except that the molar concentration ratio was 0.022).

Table 5 shows the amounts, yields and yields of the respective monomers and MPA used.

[0049]

[Table 5]

Table 6 shows the results of analysis such as molecular weight.

[0051]

[Table 6] *: Average of three times (3) Sensitivity of oligomer in aqueous solution (1) Change in transmittance of IPAAm-BMA cooligomer aqueous solution obtained in (1) with temperature is shown in Fig. 5 (I).
FIG. 6 shows the change in transmittance of the PAAm-DMAAm cooligomer aqueous solution with temperature.

From FIGS. 5 and 6, the oligomer used is BM
It is possible to shift the LCST to a low temperature side by using a co-oligomer with a hydrophobic monomer such as A and increasing the ratio of the hydrophobic monomer.
It can be seen that the LCST can be shifted to a high temperature side by using a cooligomer with a hydrophilic monomer such as AAm and increasing the ratio of the hydrophilic monomer, whereby the LCST can be controlled.

Example 2 Bioactive substance-IPAAm
Production of cooligomer complex IPAAm obtained in Synthesis Example 2 (1) in the same manner as in Example 1.
-BMA cooligomer IBc-3 (LCST: 28 ° C) complex with bovine fibrinogen, and IPAAm-DMAAm cooligomer IDc-5 (LCS) obtained in Synthesis Example 2 (2).
(T: 34 ° C.) and a bovine albumin complex were prepared. The molar ratio of bovine fibrinogen and IBc-3 was 1:80, and the molar ratio of bovine albumin and IDc-5 was 1:20. Water was used as the solvent for the complex formation reaction of the activated oligomer and the physiologically active substance.

After completion of the reaction, dialysis and freeze drying were carried out in the same manner as in Example 1 to obtain a complex. 0.1 g each of the obtained bovine fibrinogen-IBc-3 complex (F- (IBc-3)) and bovine albumin-IDc-5 complex (A- (IDc-5)) were mixed in 10 ml of ultrapure water. Dissolved. This aqueous solution was placed in a sample tube and kept for 5 minutes at each temperature in a thermostat, and the transmittance at 500 nm was measured by an ultraviolet spectrophotometer (Shimadzu UV-240). The results are shown in Fig. 7.

At around 29 ° C., white turbidity / precipitation occurred. This was shaken vigorously and then centrifuged at 30 ° C. to separate and filter the supernatant. The precipitate was freeze-dried. When the filtrate was kept in the thermostat again, it became cloudy again at around 35 ° C. This precipitate was also centrifuged, filtered and freeze-dried.

It was confirmed that the first precipitate was fibrinogen by the thrombin clotting method, and that the second precipitate was albumin by the bromocresol-green method. The amount of both was negligibly small in the measurement. From the above, the protein into which IPAAm-hydrophobic monomer / co-oligomer and IPAAm-hydrophilic monomer / co-oligomer are introduced functions as a temperature-responsive physiologically active substance-oligomer complex, and the temperature of the mixture can be increased even in a mixed solution. According to the responsiveness, it will be deposited and precipitated in order,
It can be seen that recovery is possible by centrifugation and filtration.

(Example 3) Production of lipase-IPAAm cooligomer complex (1) After dissolving 50 mg of porcine pancreas-derived lipase (Wako Pure Chemical Industries) in 20 ml of 0.1M phosphate buffered saline (pH 7.4) modified with lipase , IPAAm-DMAAm cooligomer IDc-10 (LCS obtained in Synthesis Example 2 (2)).
(T: 37 ° C) was dissolved in a 10-fold or 20-fold amount by weight of lipase, and a water-soluble carbodiimide aqueous solution was added dropwise with stirring at 4 ° C, and the mixture was reacted at the same temperature for 4 hours. The obtained reaction solution was dialyzed against purified water (4 ° C, 24 hours) and then ultrafiltered (4
C.) and freeze-dried to obtain lipase-IDc-10
Complex (hereinafter, the complex obtained by using IDc-10 in a 10-fold amount by weight of lipase was used as "L- (IDc-10) A", and IDc-10 was used in a 20-fold amount by weight of lipase. The resulting complex was labeled with "L- (I
Dc-10) B ”. ) Got. 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was used as the water-soluble carbodiimide, and the concentration of the aqueous solution was 2.0 mg / ml for L- (IDc-10) A and L- (IDc- In 10) B, it was 4.0 mg / ml, and the amount used was 8 ml in all cases.

The obtained L- (IDc-10) A and L- (IDc-10)
B, raw material IDc-10 and unmodified lipase respectively
0.1 g of each was dissolved in 10 ml of ultrapure water, placed in a permeation cell and kept at a temperature of 5 minutes in a constant temperature bath, and the transmittance at 500 nm was measured by an ultraviolet spectrophotometer (Shimadzu UV-240). The results are shown in Fig. 8. From FIG. 8, it can be seen that the lipases L- (IDc-10) A and L- (IDc-10) B modified with IDc-10 are protein hybrids showing a phase transition at 37 ° C.

(2) Measurement of enzyme activity Lipases L- (IDc-10) A and L- (I modified with IDc-10
The enzyme activities of Dc-10) B and unmodified lipase as a raw material were measured as follows. ¹⁾ olive oil emulsion as substrate, after stirring for 1 hour at 32 ° C., quenched with ethanol 5 ml, was titrated with 0.001N sodium hydroxide aqueous solution of free fatty acid using phenolphthalein as an indicator. 1) Purified water 3.8 ml + enzyme solution 0.2 ml + olive oil emulsion ²⁾ 2 ml + barbital buffer ³⁾ 1 ml 2) 5 w / v% arabic gum aqueous solution 50 ml + olive oil 25 ml + 2 w
5 ml of an aqueous solution of / v% sodium benzoate + 20 ml of purified water were stirred for 20 minutes and then used. 3) Barbital sodium 1.45g ＋ Purified water 20ml ＋ 1mol / l
2.94 ml of hydrochloric acid was made up to 100 ml with purified water. The results are shown in Fig. 9. Lipase L- (I modified with IDc-10
The activities of Dc-10) A and L- (IDc-10) B and unmodified lipase were proportional to the enzyme concentration (measured by the Lowry method).

Further, the enzyme concentrations of L- (IDc-10) A and unmodified lipase were adjusted to 0.4 mg · cm ^−3, and the temperature dependence of the enzyme activity was examined. It showed the maximum enzyme activity at ℃, while L- (IDc
-10) A was insoluble at 37 ° C and showed almost no activity (Fig. 10). From the above, it can be seen that the enzyme-oligomer complex according to the present invention can be used as a system for separating a product from an enzyme by causing a phase transition after the reaction.

[0061]

INDUSTRIAL APPLICABILITY The physiologically active substance-oligomer complex of the present invention has a temperature responsiveness, is capable of a homogeneous reaction at a temperature of LCST or lower, and is insolubilized at a temperature of LCST or higher. Can be separated from. Furthermore,
Since the physiologically active substance-oligomer complex of the present invention uses an oligomer having a functional group at the terminal, unlike the conventional one using a random copolymer having many bonding points in one polymer chain, It has no drawbacks such as aggregation due to unnecessary cross-linking and reduction of the activity of the physiologically active substance, and can be quantitatively introduced into the physiologically active substance.

[Brief description of drawings]

FIG. 1 is a graph showing a change in transmittance of an IPAAm oligomer aqueous solution with temperature.

FIG. 2 is a diagram showing a change in transmittance according to an aqueous solution concentration of an IPAAm oligomer.

FIG. 3 is a view showing a change in transmittance of IPAAm oligomer dilute aqueous solution by centrifugation.

FIG. 4 is a diagram showing changes in the transmittance of a collagen-Ic-50 complex aqueous solution with temperature.

FIG. 5 is a diagram showing a change in transmittance of an IPAAm-BMA cooligomer aqueous solution with temperature.

FIG. 6 is a graph showing a change in transmittance of an IPAAm-DMAAm cooligomer aqueous solution with temperature.

FIG. 7: Bovine fibrinogen-IBc-3 complex (F- (I
Bc-3)) and bovine albumin-IDc-5 complex (A- (IDc-
5)) It is a diagram showing a change in transmittance of a mixed aqueous solution with temperature.

FIG. 8 is a graph showing changes in the transmittance of L- (IDc-10) A, L- (IDc-10) B, IDc-10, and unmodified lipase with temperature of aqueous solutions.

FIG. 9 is a diagram showing changes in activity of L- (IDc-10) A, L- (IDc-10) B and unmodified lipase depending on the enzyme concentration.

FIG. 10 is a graph showing the temperature dependence of the enzymatic activity of L- (IDc-10) A and unmodified lipase.

Claims (10)

[Claims] 1. A complex of a physiologically active substance and an N-isopropylacrylamide homooligomer having a functional group at the terminal and having a molecular weight of 1,000 to 80,000. 2. The composite according to claim 1, wherein the functional group of the N-isopropylacrylamide homooligomer having a functional group at the terminal is a carboxyl group. 3. An N-isopropylacrylamide homo-oligomer having a functional group at the terminal is represented by the following formula (I): Molecular weight consisting of repeating units shown by 1,000 to 80,000
The composite according to claim 1, wherein one of the terminals is a hydrogen atom and the other is a group represented by the following formula (II): -SCH ₂ CH ₂ COOH (II). 4. An N-isopropylacrylamide homooligomer having a functional group at the terminal is represented by the following formula (III): The composite according to claim 1, which is an oligomer represented by the formula (n represents an integer of 10 to 700). 5. A physiologically active substance and a molecular weight of 1,000 to 80,000
A complex of a co-oligomer of N-isopropylacrylamide having a functional group at the terminal and a hydrophilic monomer or a hydrophobic monomer. 6. A co-oligomer of N-isopropylacrylamide having a functional group at the terminal and a hydrophilic monomer,
The following formula (I): And a structural unit represented by the following formula (IV): An oligomer having a molecular weight of 1,000 to 80,000 in which the structural unit represented by is linearly and irregularly arranged in a molar ratio of 100 to 80: 0 to 20, one of the terminals of which is a hydrogen atom, and the other is represented by the following formula (I
I): The complex according to claim 5, which is an oligomer having a group represented by -SCH ₂ CH ₂ COOH (II). 7. A co-oligomer of N-isopropylacrylamide having a terminal functional group and a hydrophobic monomer,
Formula (I): And a structural unit represented by the following formula (V): An oligomer having a molecular weight of 1,000 to 80,000 in which the structural unit represented by is linearly and irregularly arranged at a molar ratio of 100 to 90: 0 to 10, and one of the terminals has a hydrogen atom and the other has the following formula (I
I): The complex according to claim 5, which is an oligomer having a group represented by -SCH ₂ CH ₂ COOH (II). 8. An N-isopropylacrylamide homooligomer having a carboxyl group at the terminal with a molecular weight of 1,000-80,000 is reacted with N-hydroxysuccinimide in the presence of carbodiimides to give an activated oligomer, and then a physiological group having an amino group is prepared. A method for producing a physiologically active substance-oligomer complex, which comprises reacting with an active substance. 9. A co-oligomer of N-isopropylacrylamide having a carboxyl group at a terminal with a molecular weight of 1,000 to 80,000 and a hydrophilic monomer or a hydrophobic monomer is reacted with N-hydroxysuccinimide in the presence of carbodiimides for activation. A method for producing a physiologically active substance-oligomer complex, which comprises reacting with a physiologically active substance having an amino group after forming an oligomer. 10. A cooligomer of N-isopropylacrylamide having a carboxyl group at a terminal with a molecular weight of 1,000 to 80,000 and a hydrophilic monomer or a hydrophobic monomer is used as a physiologically active substance having an amino group in the presence of water-soluble carbodiimides. Physiologically active substance characterized by reacting-
Method for producing oligomer complex.