JP5997902B2 - Multiple stimulus responsive polymer and method for producing the same - Google Patents

Description

  The present invention relates to a multi-stimulus responsive polymer that reversibly exhibits a phase transition in response to pH and temperature, and a method for producing the same.

Stimulation-responsive polymers exhibit phase transitions when stimulated from the outside. Therefore, use of these properties as biomaterials such as DDS (drug delivery system) and cell scaffolds has been studied.
Poly (N-isopropylacrylamide) (PNIPAAm) is known as such a stimulus-responsive polymer, and this reversibly exhibits a phase transition at 32 ° C. (for example, Non-Patent Document 1). , 2). That is, when the solution temperature exceeds 32 ° C., PNIPAAm aggregates and loses solubility (in this way, the temperature at which a polymer dissolved at a low temperature causes phase separation due to an increase in temperature is referred to as LCST or lower critical solution temperature). ). However, the PNIPAAm LCST is constant and cannot be changed.
Therefore, it has been studied to control LCST by pH by introducing N-isopropylacrylamide (NIPAAm) as a graft chain into pH-responsive polyacrylic acid (PAA) or the like (for example, see Non-Patent Document 3). ). According to this method, it is possible to control the LCST with pH, but the change in LCST with respect to the change in pH is slight and not sufficient.
Furthermore, since PNIPAAm is not biodegradable, its application as a biomaterial has been limited.

Y. G. Takei et al., Bioconjugate Chem. , 4, 341, 1993 H. Feil et al., Macromolecules, 26, 2496, 1993. Allan S.M. Hoffman, Nature. , 373, 49, 1995

  Accordingly, an object of the present invention is to provide a multi-stimulation response polymer exhibiting excellent biocompatibility and exhibiting highly sensitive pH and temperature responsiveness and a method for producing the same.

In order to solve the above problems, the present inventor has intensively studied.
In the process, first, it was considered to develop pH responsiveness by introducing an ionic functional group (defined as “functional group capable of being ionized in an appropriate external pH range”) as a side chain. In addition, further studies were conducted to introduce a highly sensitive temperature response to this pH response. In consideration of biocompatibility, focusing on elastin protein derived from a living body, the VPGVG (V represents a valine residue, P represents a proline residue, and G represents a glycine residue. In the present invention, Val, Elastin-like peptides are systematically synthesized using the characteristic amino acid sequence of “Pro, Gly” in some cases, and in addition to the ionic functional group as a polymer side chain, the elastin By introducing a similar peptide, highly sensitive pH and temperature responsiveness is expressed, and when a valine residue in the amino acid sequence is replaced with another α-amino acid residue, excellent multi-stimulus responsiveness is obtained. It has also been found that the response pH range and the response temperature range can be changed without loss.

  Furthermore, it has also been found that such a multi-stimulus responsive polymer can be easily produced by grafting a specific peptide onto a part of the ionic functional group of the polymer having an ionic functional group in the side chain.

The present invention has been completed based on the above findings.
That is, the multiple stimulus responsive polymer according to the present invention includes a first side chain containing a carboxyl group as an ionic functional group and a peptide structure having the amino acid sequence represented by the following formula (1) as a structural unit. It is a multi-stimulus responsive polymer that has at least a second side chain and reversibly exhibits a phase transition in response to pH and temperature, which is represented by the following formula (2).

In the method for producing a multi-stimulation responsive polymer according to the present invention, the N-terminus of the peptide represented by the following formula (3) is inserted into a part of the carboxyl group of the polyacrylic acid with respect to polyacrylic acid via a spacer. In this way, a multi-stimulation responsive polymer represented by the above formula (2) and reversibly showing a phase transition in response to pH and temperature is produced by grafting the peptide chain.

(In the above formula (3), m is in the range of 1 to 8, and R ¹ and R ² are α-amino acid side chains corresponding to the types of α-amino acid residues selected from hydrophobic amino acid residues, respectively. .)

  In the present invention, the term “residue” with respect to an amino acid refers to a structural portion in which at least one of the OH of the carboxyl group of the amino acid and the hydrogen atom of the amino group is removed by a peptide bond.

  According to the present invention, since an elastin-like peptide having biocompatibility is used, it can be expected to be widely used as a biomaterial, and is excellent in that it exhibits a highly sensitive pH and temperature responsiveness. Since the structure is used, there is an advantage that the response region can be controlled with a high degree of freedom by a simple method of changing the amino acid sequence.

It is a graph which shows the temperature responsiveness under constant pH of the multistimulation response type polymer in Example 1 and a prior art. It is the graph which plotted the measurement result of LCST for every pH about the multiple stimulus responsive type polymer of Examples 1-5. 2 is a graph showing the pH responsiveness of the multi-stimulus response polymer of Example 1 at a constant temperature. It is a graph which shows the time-dependent change of the turbidity after adding an enzyme to the sample exceeding LCST about the multi-stimulation response type polymer of Example 1. It is a graph which shows the temperature dependence of the turbidity after adding an enzyme to the sample below LCST about the multi stimulus response type | mold polymer | macromolecule of Example 1. FIG. 2 is a CD spectrum for the multi-stimulus response polymer of Example 1. FIG. It is the graph which plotted the measurement result of CD spectrum in wavelength 198nm for every temperature about the multiple stimulus responsive type polymer of Examples 1-5.

  Hereinafter, preferred embodiments of the multi-stimulus responsive polymer and the production method thereof according to the present invention will be described in detail. However, the scope of the present invention is not limited to these descriptions, and the present invention is not limited to the following examples. Modifications can be made as appropriate without departing from the spirit of the invention.

[Multiple stimulus responsive polymer]
The multi-stimulation responsive polymer according to the present invention has at least a first side chain and a second side chain described in detail below.

<First side chain>
The first side chain contains an ionic functional group. This ionic functional group ionizes reversibly when the external pH fluctuates, but when ionized, repulsion due to charge occurs between the ions. The polymer is given pH responsiveness by the presence or absence of repulsion between ions depending on the external pH.

  Examples of the ionic functional group include an anionic functional group such as a carboxyl group, a phosphoric acid group, and a sulfonic acid group, and a cationic functional group such as an amino group and a quaternary ammonium base. Of these, a carboxyl group or an amino group is preferred.

<Second side chain>
The second side chain includes a peptide structure having an amino acid sequence represented by the following (1) as a structural unit. This second side chain includes the specific peptide structure similar to elastin, thereby showing a structural transition between a random coil and a β-turn structure depending on the external temperature, thereby making the polymer temperature-responsive. give.

(In the above, X ¹ and X ² are each independently any α-amino acid residue.)

Here, if the second side chain has an ionic functional group, an interaction due to electric charge occurs with the first side chain, which may complicate the control of multiple stimulus responsiveness. Therefore, in order to avoid such complicated control, it is preferable to select nonionic amino acid residues as X ¹ and X ^2. For example, valine residue, alanine residue, isoleucine residue, leucine Hydrophobic amino acid residues such as residues and phenylalanine residues are preferred. Of these, a valine residue, an alanine residue, and an isoleucine residue are preferable.
In the above (1), for convenience, X ¹ is displayed at the left end and Gly is displayed at the right end, but this does not mean that the N-terminal side must be X ¹ and the C-terminal side must be Gly. That is, any of the α-amino acid units constituting the amino acid sequence may be on the N-terminal side. For example, Gly is on the N-terminal side, and X ² , Gly, X ^{1 in} that order toward the C-terminal side. , Pro is also included in (1) above.

The peptide structure may contain only one structural unit consisting of the amino acid sequence represented by (1) above, or may contain two or more. Especially, what contains 1-8 of the said structural unit is preferable, and what contains 3-5 is more preferable. When two or more are included, X ¹ and X ² in each structural unit may be the same or different.

  Further, the peptide structure may be directly bonded to the main chain, or may be bonded via an appropriate spacer. Such a spacer is not particularly limited as long as it has a function of linking the main chain and the peptide structure without harming the multi-stimulus responsiveness of the present invention, and specifically, it reacts at both ends. An organic chain obtained by using a bifunctional compound having a functional functional group, such as an amino acid, a diol, a diamine, or a dicarboxylic acid, is easy to introduce and is preferable. Two or more amino acids can be used in combination (as a peptide chain).

<Multiple stimulus responsive polymer>
The multi-stimulus responsive polymer according to the present invention only needs to have at least a first side chain and a second side chain, and the first side chain and the second side chain, as long as the effects of the present invention are not impaired. You may have side chains other than a 2nd side chain. The ratio of the first side chain and the second side chain is not particularly limited. For example, in the molar ratio, when the sum of both is 100, the ratio of the second side chain is 15 or more and less than 100. It is preferable that If either of them is excessive, there is a possibility that multiple stimulus responsiveness cannot be sufficiently obtained.

  The side chain referred to here includes not only a side chain bonded to a linear main chain but also a side chain in a multi-branched polymer such as a dendron in a dendrimer. As the dendrimer, those having a polyamidoamine structure are widely known and not particularly limited, but those having this polyamidoamine structure can also be suitably employed in the present invention. As will be described later, the polyamidoamine dendrimer can relatively easily obtain the multi-stimulation responsive polymer of the present invention by introducing a peptide chain serving as the second side chain into a part of the amine.

  Although it does not necessarily limit as what has the structure which the said 1st side chain and 2nd side chain couple | bonded with the linear main chain, For example, the multiple stimulation represented by the following Formula (2) Responsive polymers are preferred.

In the above formula (2), x is equal to the molar ratio of the first side chain and the second side chain, and is not particularly limited, but as described above, it may be in the range of 15 or more and less than 100. preferable.
n represents the degree of polymerization of the polymer and is not particularly limited, but is preferably in the range of 50 to 15000.
In the above formula (2), the specific amino acid sequence in parentheses in the second side chain has X ¹ on the N-terminal side and is directed toward the C-terminal side in the expression described in (1) above. In this order, Pro, Gly, X ² and Gly are combined. R ¹ and R ² are α-amino acid side chains that are automatically determined depending on the type of α-amino acid residues X ¹ and X ² in (1), and are, for example, isopropyl groups for valine residues. If it is an alanine residue, it is a methyl group, and if it is an isoleucine residue, it is a sec-butyl group.
m is the number of repetitions of the specific amino acid sequence and is not particularly limited, but is preferably in the range of 1 to 8, more preferably in the range of 3 to 5, as described above.
In the multiple stimulus responsive polymer represented by (2) above, amino acids are used as spacers. That is, Y is a structural portion derived from an amino acid residue of an amino acid serving as a spacer, and is not particularly limited as such an arbitrary amino acid residue. For example, a β-alanine residue (-Y-=-CH ₂ -CH ₂ -), and the like preferably.

[Method for producing multi-stimulus responsive polymer]
The above-described multiple stimulus responsive polymer of the present invention can be easily produced by, for example, the production method of the present invention described in detail below.

  That is, the above-described multi-stimulation responsive polymer of the present invention has a peptide represented by the following formula (3) as a part of the ionic functional group compared to the polymer having an ionic functional group in the side chain. The peptide chain can be produced by grafting the peptide chain by binding the N-terminus or the C-terminus of the peptide represented by the following formula (4) directly or via a spacer.

As described above for the multi-stimulus responsive polymer, m is preferably in the range of 1 to 8, and more preferably in the range of 3 to 5.
R ¹ and R ² are α-amino acid side chains that are automatically determined according to the type of α-amino acid residue.

  Examples of the ionic functional group of the polymer include an anionic functional group such as a carboxyl group, a phosphoric acid group, and a sulfonic acid group, an amino group, a quaternary ammonium base, etc. And a cationic functional group. Of these, a carboxyl group or an amino group is preferred.

  The polymer having a carboxyl group in the side chain is not particularly limited, and examples thereof include carboxyl group-containing vinyl monomers such as acrylic acid, methacrylic acid, itaconic acid, maleic acid, crotonic acid, and maleic anhydride. And a polypeptide having an acidic amino acid such as glutamic acid or aspartic acid as a main constituent amino acid.

  The polymer having an amino group in the side chain is not particularly limited. For example, a homopolymer or copolymer of an amino group-containing vinyl monomer such as allylamine, or a basic polymer such as lysine or chitosan. Examples include polypeptides having amino acids as the main constituent amino acids. Further, a multi-branched polymer having an amino group in the side chain, such as a polyamide amine dendrimer, is also preferable.

  In the above, as an example in which the N-terminal of the peptide represented by the formula (3) or the C-terminal of the peptide represented by the formula (4) is “directly” bonded to a part of the ionic functional group of the polymer Is, for example, a case where an N-terminal of the peptide represented by the formula (3) is amide-bonded to a part of the carboxyl group of the polymer having a carboxyl group in the side chain, or a polymer having an amino group in the side chain. Preferred examples include a case where the C-terminal of the peptide represented by the formula (4) is amide-bonded to a part of the amino group.

In the case where the N-terminus of the peptide represented by the formula (3) or the C-terminus of the peptide represented by the formula (4) is bonded to a part of the ionic functional group of the polymer “via a spacer” As an example, for example, if the spacer is one or more amino acids, the carboxyl group of the polymer having a carboxyl group in the side chain and the N-terminus of the peptide, or the amino group in the side chain via this spacer The amino group of the polymer having the peptide can be linked to the C-terminal of the peptide.
If the spacer is a diol, diamine or the like, the carboxyl group of the polymer having a carboxyl group in the side chain can be linked to the C-terminal of the peptide via this.
Furthermore, if the spacer is a dicarboxylic acid or the like, the amino group of the polymer having an amino group in the side chain and the N-terminal of the peptide can be linked via this spacer.
Thus, there can be various modes depending on the type of ionic functional group possessed by the polymer and the type of spacer used.
Of these, amino acids are used as spacers, through which the carboxyl group of the polymer having a carboxyl group in the side chain and the N-terminus of the peptide, or the amino group of the polymer having an amino group in the side chain and the peptide It is preferable to link the C-terminal of the polymer, or to link the carboxyl group of the polymer having a carboxyl group in the side chain with the C-terminal of the peptide via diethylene glycol as a spacer.

  Of the various embodiments described above, polyacrylic acid is used as the polymer having the ionic functional group in the side chain, and the peptide represented by the above formula (3) is part of the carboxyl group of the polyacrylic acid. Particularly preferred is a method of grafting a peptide chain by binding the N-terminus of each of them directly or via one or more arbitrary amino acids (for example, β-alanine acid).

The peptide represented by the above formula (3) or the above formula (4) is a solid phase using a protective group such as an Fmoc group (9-fluorenylmethyloxycarbonyl group) or a Boc group (tert-butoxycarbonyl group). It can be synthesized by a conventionally known method such as a peptide synthesis method.
However, if the second side chain has an ionic functional group, interaction with the first side chain due to charge may occur, and control of multiple stimulus responsiveness may be complicated. The peptide represented by (3) is amidated at the C-terminus, and the peptide represented by the above formula (4) is acetylated at the N-terminus.
The C-terminal amidation in the peptide represented by the above formula (3) can be obtained, for example, by using a peptide amine resin as a carrier. As such a resin for peptide amines, for example, Fmoc-NH-SAL-MBHA resin, CLEAR-Amide resin and the like are known.
The N-terminal acetylation in the peptide represented by the above formula (4) can be easily performed by, for example, acting acetic anhydride on the N-terminal. For example, a method of adding an acetic anhydride solution, a DIPC (diisopropylcarbodiimide) solution, and a HOAt (1-hydroxybenzotriazole) solution to the peptide solution, followed by stirring and mixing may be mentioned. In this case, as the solvent, for example, N, N-dimethylformamide (DMF) is suitable.

  Next, a condensation reaction between a carboxyl group and an amino group in the polymer and the peptide can also be performed by a conventionally known method. Usually, the reaction is carried out in a solvent in the presence of a condensing agent.

  Examples of the solvent include N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile, organic solvents such as ethanol, water, and the like. A solvent can be used individually by 1 type or in mixture of 2 or more types.

When a condensing agent is used, for example, 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholine salt (DMT-MM), dicyclohexylcarbodiimide (DCC), 1-ethyl -3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC or WSC) and the like. Further, activators such as N-methylmorpholine (NMP) and NHS can be used in combination with the condensing agent.
Although the said condensation reaction is not necessarily limited, For example, it can carry out at the temperature of the range of 0-60 degreeC for about 1 to 48 hours.

In addition, the multiple stimulus responsive polymer of the present invention can be obtained by a copolymerization technique other than the above production method using a graft technique.
For example, it can be obtained by separately preparing a monomer having an ionic functional group such as a carboxyl group or an amino group and a monomer having the predetermined peptide chain and copolymerizing them. In this case, the copolymerization may be random copolymerization or block copolymerization, but random copolymerization is more preferable. In the case of block copolymerization, the resulting multi-stimulation responsive polymer is such that the first side chains having an ionic functional group are close to each other and the second side chains having a peptide structure are close to each other. As a result, the behavior of both side chains may appear individually, and the desired multiple stimulus responsiveness based on these synergistic effects may not be obtained.

[Applications of multi-stimulus responsive polymers]
The multi-stimulation response type polymer of the present invention exhibits a phase transition according to pH and temperature, and therefore can be suitably used as a DDS, a cell scaffold material, an actuator, a separation material, or the like.

  Hereinafter, although the multistimulation response type | mold polymer concerning this invention and its manufacturing method are demonstrated using an Example, this invention is not limited to these Examples.

(Synthesis of elastin-like peptide)
According to the following synthetic pathway 1, the elastin-like peptide (ELP) concerning the synthesis examples 1-3 was synthesize | combined.

<Synthesis Example 1>
As described in detail below, the ELP of Synthesis Example 1 in which X = Val in the final product described in Synthesis Route 1 was obtained.
That is, first, 1.5 g (0.67 mmol / g) of Fmoc-NH-SAL-MBHA resin was swollen overnight with 15 mL of DMF. The Fmoc-NH-SAL-MBHA resin was sequentially subjected to the following (1) deprotection group and (2) amino acid chain extension.

(1) Deprotecting group (De-Fmoc group)
In order to remove the Fmoc group, 15 mL of piperidine / DMF (volume ratio = 1/4) was added and stirred. This operation was performed 3 minutes × 2 times and 20 minutes × 1 time. Thereafter, washing was repeated with DMF until the solution became neutral.

(2) Amino acid chain elongation (condensation reaction)
Fmoc-amino acid in DMF solution (7 mL) according to the amino acid chain to be extended, DIPC: 0.38 g (3.02 mmol) in DMF solution (3 mL), HOAt: 0.42 g (3.02 mmol) in DMF solution (5 mL) And the desired Fmoc-amino acid chain was condensed and bonded by stirring for 2 hours. After the condensation, washing with DMF was performed to remove unreacted Fmoc-amino acid. The amount of Fmoc-amino acid used was as follows: Fmoc-Gly-OH: 0.9 g (3.02 mmol), Fmoc-Val-OH: 1.02 g (3.02 mmol), Fmoc-Pro-OH: 1.01 g ( 3.02 mmol).

Fmoc- (Val-Pro-Gly-Val-Gly) ₄ -NH-SAL-MBHA resin was obtained by repeating the above procedures (1) and (2).

Thereafter, Fmoc-β-Ala-OH: 0.936 g (3.02 mmol) is introduced as a spacer, and the Fmoc group is removed to remove NH ₂ -β-Ala- (Val-Pro-Gly-Val-Gly) _4. -NH-SAL-MBHA resin was synthesized.

Next, the synthesized NH ₂ -β-Ala- (Val-Pro-Gly-Val-Gly) ₄ -NH-SAL-MBHA resin was washed with DMF and DCM, and then dried under reduced pressure in a desiccator. After drying, the peptide was cleaved from the resin by stirring with 30 mL of TFA / DCM (volume ratio = 9/1) for 2 hours × 2 times. Thereafter, methanol and TFA were azeotropically concentrated under reduced pressure. Finally, it was dissolved in water and freeze-dried to obtain the target elastin-like peptide NH ₂ -β-Ala- (Val-Pro-Gly-Val-Gly) ₄ -NH ₂ .

<Synthesis Example 2>
In the final product described in Synthesis Route 1, X = Ile except that Fmoc-Ile-OH: 1.07 g (3.02 mmol) was used as one of Fmoc-amino acids in the same manner as in Synthesis Example 1. The ELP of Synthesis Example 2 was obtained.

<Synthesis Example 3>
In the final product described in Synthesis Route 1 above, X = Ala except that Fmoc-Ala-OH: 0.99 g (3.02 mmol) was used as one of the Fmoc-amino acids in the same manner as in Synthesis Example 1. The ELP of Synthesis Example 3 was obtained.

[Synthesis of multi-stimulus responsive polymers]
According to the following synthesis route 2, the multi-stimulus responsive polymers according to Examples 1 to 5 were synthesized.

<Example 1>
As described in detail below, the multi-stimulus responsive polymer of Example 1 in which x = 36 and X = Val in the final product described in Synthesis Route 2 was obtained.
That is, first, 0.80 g (0.46 mmol) of the elastin-like peptide of Synthesis Example 1 was dissolved in DMF in an eggplant flask, and 77 μl (0.576 mmol) of triethylamine was added to remove the trifluoro salt. .
Here, 0.083 g (0.00035 mmol) of polyacrylic acid (PAA) having a weight average molecular weight of 250,000 was dissolved. After dissolution, condensation was performed in 30 ml of DMF using 0.45 g (1.44 mmol) of DMT-MM as a condensing agent. The condensation was performed at room temperature for 24 hours. After condensation, DMF is removed under reduced pressure, dissolved in ultrapure water, and then dialyzed using a dialysis membrane with a molecular weight cut off of 12,000 to 14000 at 4 ° C. to remove unreacted peptide and condensing agent. Purification was performed at Purification was confirmed by UV-vis spectrum. Specifically, when exchanging distilled water, the external solution after dialysis was measured and confirmed from the disappearance of the peak near 198 nm based on the π-π ^* transition of the carbonyl group of the peptide. After dialysis, the multi-stimulus responsive polymer of Example 1 was obtained by freeze-drying the target aqueous solution. The structure of this multiple stimulus-responsive polymer was confirmed by ¹ H-NMR spectrum, and the graft ratio x = 36 (%) was calculated.

<Examples 2 to 5>
In Example 1, in place of the elastin-like peptide of Synthesis Example 1, the elastin-like peptide of Synthesis Examples 2 and 3 was used, and each charge was changed in the same manner except that the amounts shown in Table 1 were used. Multiple stimuli-responsive polymers according to Examples 2 and 3 having different constituent amino acid residues and graft rates were obtained. Moreover, in Example 1, the multistimulus response type polymer according to Examples 4 and 5 having different graft ratios was obtained in the same manner except that the amounts charged were as shown in Table 1.

[Evaluation of physical properties and performance]
<Evaluation of multiple stimulus responsiveness by turbidity measurement>
(Measurement method of turbidity)
Multiple stimulus responsiveness was evaluated by measuring turbidity, and this turbidity measurement was performed using J-820 type manufactured by JASCO Corporation. A quartz cell having an optical path length of 1 cm was used, and the temperature change was performed using a PTC-423L type Peltier thermostatic device manufactured by JASCO Corporation and E200 manufactured by LAUDA at a temperature gradient of 1 ° C./min. Turbidity at each temperature was determined from absorbance at 700 nm, and the temperature at which the turbidity changed by 50% was defined as LCST.

(Temperature response under constant pH)
A sample (polymer concentration = 1 wt%) was prepared by using the multiple stimulus-responsive polymer of Example 1 and dissolving it in a 50 mM Na ₂ HPO ₄ / citrate buffer solution having a predetermined pH. According to the above method, the temperature dependence of turbidity at a constant pH (pH 3.3) was measured. Similarly, each of pH 3.9, pH 4.2, pH 4.9, pH 5.4, pH 5.9, and pH 6.4 was measured.
The results are summarized in FIG. 1 (the lower diagram in FIG. 1).
For comparison, the temperature responsiveness of a conventional multi-stimulus responsive polymer is also shown in FIG. 1 (upper figure in FIG. 1. Quoted from Allan S. Hoffman, Nature. 373, 49 (1995)).
This conventional multi-stimulation responsive polymer has a structure represented by the following formula, and is a polyacrylic acid (weight average molecular weight 250,000) grafted with NIPAAm (weight average molecular weight 2200).

  As shown in FIG. 1, the multi-stimulus responsive polymer of the present invention has a temperature responsiveness sensitivity to pH change as compared with the conventional multi-stimulus responsive polymer (ΔLCST = 18 ° C. with respect to ΔpH 2.5). Is very big.

  Next, multiple stimuli responsive polymers of Examples 2 and 3 having different constituent amino acid residues (and graft ratios) in the peptide structure of the second side chain, and multiple stimulus responsive types of Examples 4 and 5 having different graft ratios A sample (polymer concentration = 1 wt%) was similarly prepared for each of the polymers, and the temperature responsiveness under a constant pH was measured and the LCST was derived in accordance with the above-mentioned method while varying the pH. These results are summarized in FIG. 2 together with the results of Example 1.

  As shown in FIG. 2, it can be seen that by changing the constituent amino acid residues and the graft ratio in the peptide structure of the second side chain, the response region can be changed without impairing the multi-stimulus responsiveness.

(PH responsiveness at a constant temperature)
Using the multiple stimulus-responsive polymer of Example 1, sample preparation and turbidity measurement were performed in the same manner as described above, and pH responsiveness at a constant temperature (= 35 ° C.) was measured.
The results are shown in FIG.

  From FIG. 3, it was found that the solubility characteristics can be changed even when the pH is changed with the temperature kept constant. A slight difference was observed when the pH was changed from 3.5 to 6.0 and when the pH was changed from 6.0 to 3.5.

(Stimulus response behavior when enzyme is added)
The second side chain of the multi-stimulation responsive polymer of the present invention contains an elastin-like peptide structure. The unique behavior based on this characteristic configuration was observed.
That is, first, papain was dissolved in 50 mM, pH 4.2, citric acid / Na ₂ HPO ₄ buffer solution in which the multi-stimulation response type polymer of Example 1 was dissolved, and the sample (polymer concentration = 1 wt%, enzyme concentration = 1 mg / mL) was obtained. The turbidity of this sample was measured according to the above method, and the change with time was measured at 50 ° C. (temperature exceeding LCST).
The results are shown in FIG.

From the obtained results, it was found that the polymer in the aggregated state was dissolved in about 5 minutes after adding the enzyme (papain) under the temperature condition of 50 ° C. or higher than LCST.
In the same manner as described above, a sample (polymer concentration = 1 wt%, enzyme concentration = 1 mg / mL) at 5 ° C. (temperature lower than LCST) was prepared, and this was heated to 50 ° C. (temperature exceeding LCST). As a result, as shown in FIG. 5, aggregation no longer occurred.
From the above results, it was confirmed that the multi-stimulus responsive polymer of the present invention has a biodegradability characteristic not found in conventional multi-stimulus responsive polymers. It can be seen that by having such characteristics, the cohesiveness can be forcibly and irreversibly lost regardless of temperature or pH, for example.

<Temperature dependence of conformation>
The circular dichroism (CD) spectrum measurement shows that the characteristic behavior in the multiple stimulus-responsive polymer of the present invention as described above is brought about by the characteristic peptide structure in the second side chain. This was confirmed by measuring the temperature dependence of the conformation.

This CD spectrum was measured using a J-820 type manufactured by JASCO Corporation, using a quartz cell with an optical path length of 0.1 cm, and the number of integrations was 4, and the scanning speed was 100 nm / min.
Specifically, each polymer to be measured was dissolved in 5 mM, pH 6.4, Na ₂ HPO ₄ / citrate buffer solution to obtain a stock solution. A sample solution having a pH of 6.4 was prepared by diluting the stock solution with a buffer solution. About these sample solutions, UV-vis spectrum measurement was performed in a quartz cell having an optical path length of 1 mm using a UV-2100 spectrophotometer manufactured by Shimadzu Corporation, and the concentration was determined by measuring absorption based on peptide bonds near 198 nm. (Polymer concentration = 40 μM). When measuring the CD spectrum at different temperatures, using a PTC-423L type Peltier thermostatic device manufactured by JASCO Corporation and E200 manufactured by LAUDA, the temperature was increased at a temperature gradient of 1 ° C./min and reached that temperature. Measurement was performed 50 seconds after the test.

FIG. 6 shows the CD spectrum of the multiple stimulus-responsive polymer of Example 1 measured as described above.
Moreover, the graph which plotted the measured value for every temperature in wavelength 198nm about the multi stimulus response type | mold polymer of Examples 1-5 is shown in FIG.

  From the results shown in FIGS. 6 and 7, it can be seen that the multi-stimulus responsive polymer of the present invention reversibly adopts two conformations of a random coil and a β-turn depending on the external temperature. It turns out that characteristic multiple stimulus responsiveness is expressed in combination with pH responsiveness based on the first side chain. Since the reversible change in the conformation in Examples 1 to 5 above is due to the interaction between the valine residue of the peptide structure in the second side chain and each amino acid side chain of X, these valine residues It was demonstrated that multiple stimulus responsiveness can be controlled with a high degree of freedom by changing the types of X and X in various ways.

  The multi-stimulation responsive polymer and the method for producing the same according to the present invention can be suitably used particularly for biomaterials such as DDS and cell scaffolds.

Claims (2)

It has at least a first side chain containing a carboxyl group as an ionic functional group and a second side chain containing a peptide structure having the amino acid sequence represented by the following formula (1) as a structural unit. 2) A multi-stimulus responsive polymer that reversibly exhibits a phase transition in response to pH and temperature.

(In the above formula (1), X ¹ and X ² are each independently any α-amino acid residue selected from hydrophobic amino acid residues . In the above formula (2), x is 15 The range is less than 100, n is in the range of 50 to 15000, m is in the range of 1 to 8, and Y is derived from any amino acid residue linking the main chain and the second side chain. And R ¹ and R ² are α-amino acid side chains corresponding to the types of the α-amino acid residues X ¹ and X ² , respectively. By grafting the peptide chain to the polyacrylic acid by bonding the N-terminus of the peptide represented by the following formula (3) to a part of the carboxyl group of the polyacrylic acid via a spacer, the following formula ( A method for producing a multi-stimulus responsive polymer, which is represented by 4) and which produces a multi-stimulus responsive polymer reversibly in response to pH and temperature.

(In the above formulas (3) and (4), m is in the range of 1 to 8, and R ¹ and R ² are each an α-amino acid according to the type of α-amino acid residue selected from hydrophobic amino acid residues. In the above formula (4), x is in the range of 15 or more and less than 100, n is in the range of 50 to 15000, and Y is an arbitrary connecting the main chain and the N-terminus of the peptide. The structural part derived from the amino acid residue of

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