JP5463515B2 - Material with immobilized antimicrobial peptide - Google Patents

Description

  The present invention relates to a material on which a modified peptide derived from beetle defensin is immobilized, a method for producing and using the same, and a molded article made of the material.

  An infectious disease is a disease caused by the invasion and proliferation of microorganisms such as bacteria into the human or animal body, and its onset depends on the balance between the resistance of the infecting microorganism and the infected host. ing. In particular, there are many patients with reduced resistance to infection in medical institutions, and microorganisms that do not show any pathogenicity to healthy people cause fatal symptoms for patients with reduced resistance There is. Such infections in medical institutions are often referred to as nosocomial infections, as they are different from community-acquired infections because there are many different pathogens and countermeasures from infections that occur outside hospitals.

  Since the cause of nosocomial infection is mainly contact infection, measures such as hand washing, disinfection and sterilization of medical equipment are being taken, but nosocomial infections such as MRSA are on the rise, and more effective measures Is required.

  In addition, a medical device that is placed in the body after a surgical operation such as a suture thread, an artificial blood vessel, or a catheter is likely to be a scaffold for adhesion of bacteria, and the attached bacteria grow to form a biofilm. Bacteria in biofilms formed on the surface of such medical devices increase resistance to antibacterial drugs and the immune system, often resisting treatment and causing chronic and recurrent infections, Such biofilm infections are a problem for the use of indwelling medical devices.

  The present inventors have found that beetle defensin-derived modified peptides have the activity of destroying and killing cell membranes against a wide range of bacteria including drug-resistant bacteria, pathogenic microorganisms such as trypanosoma protozoa, and cancer cells such as myeloma and leukemia. (Patent document 1). If these modified peptides derived from beetle defensin can be immobilized on the surface of a medical instrument or medical material, it is expected to be effective in preventing nosocomial infections and postoperative infections.

  Moreover, it is expected that by using an antibacterial material in which the peptide is immobilized, it is possible to provide a medical material that is not used as a scaffold for biofilm formation even if it is placed in a living body.

  However, no antibacterial material in which antimicrobial peptides are efficiently immobilized on a solid surface has been reported.

JP2007-284421A

  An object of the present invention is to provide a wide range of bacteria including drug-resistant bacteria such as MRSA, pathogenic microorganisms such as trypanosoma protozoa and modified beetle defensin-derived modified peptides having toxicity against cancers such as myeloma and leukemia. It is providing the antibacterial medical instrument and medical material which used the said material by fixing to.

  As a result of diligent efforts, the present inventors have made it possible to immobilize on the surface of a material without losing the activity of the antimicrobial peptide by using a highly flexible immobilization method, and the activity of the peptide repeatedly washed. However, the present inventors have found that it does not deteriorate, and have completed the present invention.

Based on the above findings, the present invention provides a material in which a polypeptide containing an amino acid sequence represented by the following formula is immobilized.
X1-Lue-X2-Leu-X3-Ile-X4-Arg-Arg-NH ₂
(Wherein X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, and X4 is Gly or Arg.)
Preferably, the polypeptide is Arg-Leu-Tyr-Leu-Arg-Ile-Gly-Arg-Arg-NH ₂ , Arg-Leu-Arg-Leu-Arg-Ile-Gly-Arg-Arg-NH ₂ , Ala It is selected from the group consisting of -Leu-Tyr-Leu-Ala- Ile-Arg-Arg-Arg-NH 2 or Arg-Leu-Leu-Leu- Arg-Ile-Gly-Arg-Arg-NH 2.

  In another aspect, the present invention relates to a method for producing the above material. Specifically, a material to which an antimicrobial peptide is bound is produced by the following method.

  After reacting the hydroxyl group on the substrate surface with epihalohydrin to halogenate the substrate surface, a diamino compound such as 1,3-diaminopropane or diethylene glycol bis (3-aminopropyl) ether is added to form an amino-modified group Make the material. Next, the amino group is reacted with the carboxyl group of the protected amino acid to form a peptide bond. After washing the unbound amino acid, the amino group on the substrate is deprotected with piperidine or the like, and a new protected amino acid is bound. By repeating this process, desired amino acids are sequentially bound, and finally the amino acids are deprotected to obtain a material to which the polypeptide of the present invention is bound.

  The modified peptide derived from beetle defensin used in the present invention is toxic to a wide range of bacteria including drug-resistant bacteria, pathogenic microorganisms such as Trypanosoma protozoa, and cancer cells such as myeloma and leukemia. The bonded material of the present invention stably maintains these biological activities. Since the material of the present invention does not lose its activity by washing or heat sterilization by autoclaving, a molded product having antimicrobial or anticancer activity can be obtained by using the material.

  Therefore, the present invention also relates to a molded article having antimicrobial or anticancer activity obtained by molding the base material into a desired shape.

  For example, it can be expected that bacterial infection at the wound site can be prevented by using the wound dressing material using the material of the present invention. Further, by using the material of the present invention for the dialysis membrane, the possibility of bacterial contamination of the artificial dialysis device can be reduced, and the effect of killing pathogenic microorganisms, cancer cells and the like in the blood can be expected. In addition, for example, by using clothes using the material of the present invention, it can be expected that infection by pathogenic microorganisms attached to the clothes can be prevented.

  Furthermore, since the indwelling devices such as artificial blood vessels and stents using the material of the present invention can be avoided as a scaffold for bacterial growth, biofilm infection can be prevented.

  Further, pathogenic microorganisms in the liquid can be killed by utilizing the antimicrobial activity of the material of the present invention. Therefore, this invention provides the liquid sterilization method characterized by making liquids, such as blood or plasma, contact with the raw material of this invention as one aspect | mode.

More specifically, the present invention provides the following items and methods.
(1) Amino acid sequence represented by the following formula:
X1-Lue-X2-Leu-X3-Ile-X4-Arg-Arg-NH ₂
(Wherein X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, and X4 is Gly or Arg).
(2) the polypeptide is _{Arg-Leu-Tyr-Leu-} Arg-Ile-Gly-Arg-Arg-NH 2, Arg-Leu-Arg-Leu-Arg-Ile-Gly-Arg-Arg-NH 2, Ala The material according to (1), which is Leu-Tyr-Leu-Ala-Ile-Arg-Arg-Arg-Arg-NH ₂ or Arg-Leu-Leu-Leu-Arg-Ile-Gly-Arg-Arg-NH ₂ .
(3) The material according to (1) or (2), wherein the polypeptide is bound to a substrate on the C-terminal side through a spacer.
(4) The material according to any one of (1) to (3), wherein the base material is a polymer or a metal.
(5) The material according to (4), wherein the substrate is cellulose.
(6) After reacting epihalohydrin with the substrate to halogenate the surface, 1,3-diaminopropane or diethylene glycol bis (3-aminopropyl) ether is added to perform amino modification, and protected amino acids are sequentially bonded to the amino groups. And finally removing the protective group. The method for producing a material according to any one of (1) to (5),
(7) A molded article having antimicrobial or anticancer activity, comprising the material according to any one of (1) to (5).
(8) The molded article according to (7), which is selected from the group consisting of a wound dressing, a suture, an artificial blood vessel, a catheter, a dialysis membrane, clothes, and a stent.
(9) A method for sterilizing a liquid, wherein the liquid is brought into contact with the material according to any one of (1) to (5).
(10) The sterilization method according to (9), wherein the liquid is blood or plasma.

  The present inventors have found that a modified beetle defensin-derived modified peptide can be immobilized on a solid phase while maintaining antimicrobial activity by using a highly flexible immobilization method. It was also confirmed that the immobilized peptide was very stable and maintained its activity even after repeated washing and sterilization by autoclaving.

  Therefore, by using a material in which the modified peptide derived from beetle defensin of the present invention is used, it becomes possible to provide an antimicrobial-treated medical device that can be reused for a long period of time.

  Furthermore, since antimicrobial activity can be maintained over a long period of time, it is possible to provide a medical device for in-vivo placement that is unlikely to induce a biofilm infection by using the material.

It is a schematic diagram of reaction which fix | immobilizes a peptide to a base material. The activity with respect to mouse | mouth myeloma cell P3-X63-Ag8.653 and human leukemia cell Jurkat of the cotton fabric which fix | immobilized the modified peptide derived from a beetle defensin is shown. In the figure, “no cloth” is a group containing no cloth, “untreated cloth” is a group containing a cloth on which no peptide is fixed, “peptide-fixed cotton cloth 1” and “peptide-fixed cotton cloth 2” are SEQ ID NOs: 4 shows a group containing a fabric in which four peptides are fixed via 1,3-diaminopropane or diethylene glycol bis (3-aminopropyl) ether.

The material of the present invention is an amino acid sequence X1-Lue-X2-Leu-X3-Ile-X4-Arg-Arg-NH ₂ (wherein X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, and X4 is Gly or Arg)).

As preferable examples of the polypeptide used in the present invention,
Arg-Leu-Tyr-Leu-Arg-Ile-Gly-Arg-Arg-NH ₂ (SEQ ID NO: 1),
Arg-Leu-Arg-Leu-Arg-Ile-Gly-Arg-Arg-NH ₂ (SEQ ID NO: 2),
Ala-Leu-Tyr-Leu-Ala-Ile-Arg-Arg-Arg-NH ₂ (SEQ ID NO: 3), or Arg-Leu-Leu-Leu-Arg-Ile-Gly-Arg-Arg-NH ₂ (SEQ ID NO: 3) Mention may be made of the polypeptide comprising 4).

  In the present invention, the “substrate” refers to a solid phase to which a modified peptide derived from beetle defensin is bound. The “material” refers to a base material to which the polypeptide is bound, and can be processed into a molded product such as a medical device by molding it into an artificial blood vessel or a dialysis membrane. Therefore, the shape of the base material and the material may be any shape as long as it has a function as a molded product, such as a fiber, a film, and a granule.

The substrate may be made of any material as long as it can immobilize a peptide such as a polymer such as cellulose or a metal.
In order to immobilize a peptide with a spacer by covalent bonding, it is preferable to use a material that already has an appropriate functional group that can be used for immobilization in the substrate or that can easily introduce a functional group.
Specifically, it is preferable to use a polymer such as cellulose, nylon, and polystyrene. In particular, cellulose has a hydroxyl group as a functional group, and other functional groups can be easily introduced into the hydroxyl group. Gold is particularly preferable because it itself easily reacts with a thiol group and various materials can be easily deposited on gold.

  In the present invention, the “polypeptide” may be composed of either an L-type amino acid or a D-type amino acid.

Polypeptides can be produced by sequentially binding amino acids on a solid phase via a spacer by a method according to the peptide solid phase synthesis method. Here, the “spacer” is a compound having the ability to bind a solid phase and an amino acid in the above structure, and has a functional site that can react with a functional site on the solid phase to bind the spacer on the solid phase. Is.
The solid phase and the spacer are not limited to covalent bonds, and are not limited to the type of bond as long as they can fix the solid phase and the spacer, such as ionic bond, hydrophobic bond, and antigen-antibody reaction.

  The combination of the functional site to react can be exchanged between the functional site on the solid phase and the functional site of the spacer, and which functional site is introduced is not limited.

  As a combination of functional sites to be reacted, a combination of a halogen, a carboxyl group, an aldehyde group and an amino group, particularly a diamino group such as a diaminoalkyl group or a diaminoether group is preferable. Also preferred are combinations of thioester groups, aldehydes, bromoacetyl groups and maleimide groups with thiol groups.

The spacer must have a functional site that binds to the C-terminus of the peptide in addition to the functional site that binds to the functional site on the solid phase. As the combination of the functional sites, an aldehyde, a bromoacetyl group, and a combination of a maleimide group and a thiol are preferable. Moreover, what has an amino group which can couple | bond with the C terminal of a peptide is also preferable. The bond between the spacer and the peptide is not limited to the type of bond, as with the bond between the solid phase and the spacer, and any bond may be used.
Moreover, which functional site is put in which is not limited, and two functional sites to be reacted may be introduced into either one.

  In the present invention, the “protected amino acid” refers to an amino acid whose amino group is protected with a protecting group such as a 9-fluorenylmethyloxycarbonyl group (Fmoc group) or a t-butylcarbonyl group (Boc group).

  In the present invention, the “molded product” refers to a product obtained by processing a material into a desired shape. For example, wound dressings, sutures, artificial blood vessels, catheters, dialysis membranes, stents and clothes are suitable molded articles of the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

Example 1
Immobilization of a modified peptide derived from beetle defensin at the C-terminal through a spacer 1) After epibromohydrin was reacted with cotton cloth to brominate the surface, 1,3-diaminopropane was added to synthesize an amino-modified cotton cloth. A D-type amino acid protected with a 9-fluorenylmethoxy group (Fmoc group) was bound to these amino groups, and after the protecting group was removed with piperidine, an amino acid newly protected with Fmoc was bound. In this way, a D-type amino acid was sequentially bonded to synthesize a cotton white cloth in which the D-type peptide of RLLLRIGRRR (SEQ ID NO: 4) was immobilized at the C-terminus. However, since the reaction efficiency of the first-stage amino acid was lower than that of other amino acids, the amino acids were sequentially bound after first binding glycine. The amount of peptide bond per unit area was 0.25 μg / cm ² (peptide-immobilized cotton cloth 1).

2) Using a diethylene glycol bis (3-aminopropyl) ether instead of 1,3-diaminopropane, a cotton white cloth to which RLLLRIGRRR (SEQ ID NO: 4) was bound was synthesized by the same process. The amount of peptide bond per unit area was 0.13 μg / cm ² (peptide-immobilized cotton cloth 2).

Example 2
Epibromohydrin was reacted with an immobilized cotton cloth that does not control the binding site of the modified peptide derived from beetle defensin , and the surface of the cloth was brominated. The peptide was reacted with the cotton cloth to bind any amino group in the molecule to the bromo group of the cotton cloth. Cotton fabrics on which the peptides of SEQ ID NOs: 1, 2, 3 and 4 were immobilized were designated as A, B, C and D.

Example 3
Antimicrobial activity of cotton cloth with modified peptide-immobilized peptide derived from beetle defensin i) Antimicrobial activity test of cotton cloth with peptide immobilized at the C-terminal via a spacer. Antimicrobial activity of cotton cloth with peptide immobilized indirectly in Example 1 A test was conducted. After putting the peptide-immobilized cotton cloth in the culture solution and culturing for 18 hours, no viable bacteria were observed (Experiment 1). It was found that the antibacterial activity was maintained even after the cloth used in Experiment 1 was ultrapure and was subjected to hand-washing (amplitude 30 cm × 30 times shaking × 3 times) and sterilization by autoclave four times. Experiment 2-5). In addition, after Experiment 5, the peptide-immobilized cotton cloth was washed using a household washing machine without using a surfactant (washing → rinsing → dehydration), washed again under the above conditions, and then autoclaved. , Used in Experiment 6.
This experiment showed that the antibacterial activity was stably maintained by immobilizing the peptide at the C-terminus via a spacer.

＊ 表中各数値は細菌数（ＣＦＵ）を示す。

* Each value in the table indicates the number of bacteria (CFU).

＊ 表中各数値は細菌数（ＣＦＵ）を示す。 ii) Antimicrobial activity test of cotton cloth immobilized without controlling the peptide binding site The cotton cloths A, B, C and D immobilized without controlling the peptide binding site prepared in Example 2 are in accordance with JIS standards. The antibacterial activity against Staphylococcus aureus was measured using a based antibacterial test method. After putting the peptide-immobilized cotton cloth in the culture solution and culturing for 18 hours, no viable bacteria were observed (Experiment 1). The cotton cloth used in Experiment 1 was washed by shaking under the above conditions, sterilized by an autoclave and then subjected to an antibacterial test again. As a result of repeating the washing and the antibacterial test, viable bacteria were observed in all the peptide-immobilized cotton cloths by repeating washing twice or more (Experiment 2-5).

* Each value in the table indicates the number of bacteria (CFU).

Example 4
Anticancer activity of beetle defensin-derived modified peptide-immobilized cotton cloth The activity of beetle defensin-derived modified peptide-immobilized cotton cloth on mouse myeloma cells P3-X63-Ag8.653 and human leukemia cells Jurkat was measured.

Both the cotton fabric with the peptide immobilized and the cotton fabric with no peptide immobilized were cut into a size of 5 mm × 5 mm, washed in 1 ml of ultrapure water (vortex max × 10 seconds), autoclaved and used for the test. Place a cloth on the bottom of each well of a 96-well plate, and add mouse myeloma cell P3-X63-Ag8.653 or human leukemia cell Jurkat 8 × 10 ³ cells / 100 μl / well from above, and add 5% CO _2. After culturing at 37 ° C. for 24 hours, the survival rate was measured. The medium used was RPMI1640 (Gibco11875-085: glucose 2000 mg / L, L-glutamine 300 mg / L) supplemented with bovine serum (final concentration 20%) and penicillin-streptomycin (final concentration 50 mg / L).
When the peptide-immobilized cotton cloth was placed in the medium, the survival rate of all cancer cells was greatly reduced. The peptide-immobilized cotton cloth was washed, sterilized, and tested again. The anticancer activity was maintained in repeated experiments.

Claims (12)

Amino acid sequence represented by the following formula:
X1-Lue-X2-Leu-X3-Ile-X4-Arg-Arg-NH ₂
(Wherein, X1 is Arg or Ala, X2 is Tyr, is Arg or Leu, X3 is Arg or Ala, X4 is Gly or Arg.) Spacers C-terminal side of a polypeptide comprising Material fixed to the substrate via Wherein said amino acid sequence is _{Arg-Leu-Tyr-Leu-} Arg-Ile-Gly-Arg-Arg-NH 2, Arg-Leu-Arg-Leu-Arg-Ile-Gly-Arg-Arg-NH 2, Ala-Leu- The material according to claim 1, which is Tyr-Leu-Ala-Ile-Arg-Arg-Arg-Arg-NH ₂ or Arg-Leu-Leu-Leu-Arg-Ile-Gly-Arg-Arg-NH ₂ . The amino acid sequence is Arg-Leu-Leu-Leu-Arg-Ile-Gly-Arg-Arg-NH ₂ The material according to claim 2. Spacer

Or

The raw material in any one of Claims 1-3 containing.   The material according to any one of claims 1 to 4, wherein the substrate is a polymer or a metal.   The material according to claim 5, wherein the substrate is cellulose.   The molded article which has the antimicrobial or anticancer activity which consists of the raw material in any one of Claims 1-6.   The molded article according to claim 7, wherein the molded article is selected from the group consisting of wound dressings, sutures, artificial blood vessels, catheters, dialysis membranes, clothes and stents. (I) reacting an epihalohydrin with a hydroxyl group on the substrate surface to halogenate the substrate surface;
(Ii) 1,3-diaminopropane or diethylene glycol bis (3-aminopropyl) ether is added to the substrate, and one of the amino groups of 1,3-diaminopropane or diethylene glycol bis (3-aminopropyl) ether Reacting a halogen group of epihalohydrin on the substrate surface to obtain an amino-modified substrate;
(Iii) An amino acid sequence represented by the following formula, wherein an amino acid is sequentially peptide-bonded to the other amino group of 1,3-diaminopropane or diethylene glycol bis (3-aminopropyl) ether:
X1-Lue-X2-Leu-X3-Ile-X4-Arg-Arg-NH ₂
(Wherein X1 is Arg or Ala, X2 is Tyr, Arg or Leu, X3 is Arg or Ala, and X4 is Gly or Arg.)
Immobilizing a polypeptide having a base on a substrate;
A method of manufacturing a material, including The method according to claim 9, wherein the amino acid sequence is Arg-Leu-Leu-Leu-Arg-Ile-Gly-Arg-Arg-NH ₂ . The raw material produced by the method of Claim 9 or 10. A liquid sterilization method comprising contacting a liquid (excluding body fluid) with the material according to any one of claims 1 to 6.

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