CN107513145A - The synthesis of biodegradable amphiphilic block class antibacterial peptide and the preparation method and application of class antibacterial peptide vesica - Google Patents

The synthesis of biodegradable amphiphilic block class antibacterial peptide and the preparation method and application of class antibacterial peptide vesica Download PDF

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CN107513145A
CN107513145A CN201710728104.8A CN201710728104A CN107513145A CN 107513145 A CN107513145 A CN 107513145A CN 201710728104 A CN201710728104 A CN 201710728104A CN 107513145 A CN107513145 A CN 107513145A
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antibacterial peptide
reaction
vesica
hydrophobic polymer
amino acid
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周春才
周欣宇
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Tongji University
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Tongji University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/28—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the compounds used containing active hydrogen
    • C08G18/40—High-molecular-weight compounds
    • C08G18/42—Polycondensates having carboxylic or carbonic ester groups in the main chain
    • C08G18/4266—Polycondensates having carboxylic or carbonic ester groups in the main chain prepared from hydroxycarboxylic acids and/or lactones
    • C08G18/4269—Lactones
    • C08G18/4277—Caprolactone and/or substituted caprolactone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/28—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the compounds used containing active hydrogen
    • C08G18/40—High-molecular-weight compounds
    • C08G18/60—Polyamides or polyester-amides
    • C08G18/603—Polyamides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
    • C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
    • C08G63/08—Lactones or lactides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/68—Polyesters containing atoms other than carbon, hydrogen and oxygen
    • C08G63/685—Polyesters containing atoms other than carbon, hydrogen and oxygen containing nitrogen
    • C08G63/6852—Polyesters containing atoms other than carbon, hydrogen and oxygen containing nitrogen derived from hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/10—Alpha-amino-carboxylic acids
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    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/07—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media from polymer solutions
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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    • C08J2387/00—Characterised by the use of unspecified macromolecular compounds, obtained otherwise than by polymerisation reactions only involving unsaturated carbon-to-carbon bonds

Abstract

The invention provides a kind of synthesis of biodegradable amphiphilic block class antibacterial peptide and the preparation method and application of class antibacterial peptide vesica.The synthesis of such antibacterial peptide is connected by the amino-terminated hydrophobic polymer segment in the hydrophilic polypeptides segment of the group containing amido protecting, one end with the isocyano of diisocyanate unit by covalent bond to be made；Class antibacterial peptide vesica is formed by such antibacterial self-assembling peptide, and class antibacterial peptide vesica can be applied as antiseptic or pharmaceutical carrier in terms of critical anti-inflammatory, cancer therapy drug Targeting delivery or nanosecond medical science.The class antibacterial peptide vesica of the present invention has selected biodegradable hydrophobic polymer segment to instead of the hydrophobic amino acid segment in antibacterial peptide, therefore does not have cytotoxicity, and has excellent biocompatibility and biodegradability；In addition, it has excellent broad spectrum antibacterial performance, the film similar with natural antibacterial peptide destruction Antibacterial Mechanism is identical, is not easy Induction of bacterial and produces drug resistance.

Description

The synthesis of biodegradable amphiphilic block class antibacterial peptide and class antibacterial peptide capsule The preparation method and application of bubble
Technical field
The invention belongs to field of biomedical polymer materials, and in particular to a kind of biodegradable amphipathic block is total to Cluster the synthesis of antibacterial peptide and the preparation method and application of antibacterial peptide vesica.
Background technology
Abuse of antibiotics causes the generation of drug-fast bacteria, and the antiseptic found different from conventional antibiotic Antibacterial Mechanism is compeled in eyebrow Eyelash.Natural antibacterial peptide has broad-spectrum high efficacy bactericidal activity, and its film failure mechanism is different from conventional antibiotic, that is, passes through electrostatic interaction The surface of bacterial cell membrane is adsorbed onto, then hydrophobic amino acid residue is inserted into bacterial cell membrane, causes to produce on its cell membrane Raw hole.This special Antibacterial Mechanism makes bacteria peptide be difficult to produce drug resistance.But the extraction of natural antibacterial peptide Journey is complicated, yields poorly and cost is high, therefore the mode of chemical synthesis synthesizes the antiseptic similar to natural antibacterial peptide structure and had Far-reaching Research Significance.At the same time, the hydrophobic amino acid residues of part antibacterial peptide such as phenyl ring etc. has to human normal cell Larger toxicity, constrain development of the antibacterial peptide in fields such as clinical treatments, synthesis no cytotoxicity, the antibacterial of good biocompatibility Agent is always the common objective of anti-biotic material researcher.
The content of the invention
In view of the shortcomings of the prior art, primary and foremost purpose is to provide a kind of biodegradable amphiphilic block to the present invention Class antibacterial peptide.
Second object of the present invention is to provide a kind of conjunction of biodegradable amphiphilic block class antibacterial peptide Into.
Third object of the present invention is the preparation method and application for providing a species antibacterial peptide vesica.
To reach above-mentioned purpose, solution of the invention is：
A kind of biodegradable amphiphilic block class antibacterial peptide, its structural formula are as follows：
Wherein, integers of the x in 1-100, integers of the y in 1-100, x and y represent the polymerization of each segment respectively Degree, R1Represent the hydrophilic radical of hydrophilic amino acid, R2Represent the alkane segment of diisocyanate, R3Represent biodegradable The repeat unit of hydrophobic polymer, A represent the initiator of hydrophilic amino acid polymerization, and A ' represents hydrophobic polymer polymerization Initiator.
Preferably, any one of hydrophilic amino acid in lysine, arginine and histidine.
Preferably, diisocyanate is selected from hexamethylene diisocyanate, MDI, to the methylene of benzene two Any one in group diisocyanate and PPDI.
Preferably, hydrophobic polymer is in polycaprolactone, PLA, polyglycolic acid and PTMC Any one or a few.
A kind of preparation method of above-mentioned biodegradable amphiphilic block class antibacterial peptide, it includes following step Suddenly：
(1), the preparation of the hydrophilic polypeptides segment of the group containing amido protecting：
(1-1), the hydrophilic amino acid and triphosgene of the group containing amido protecting reacted in organic solvent, obtain containing ammonia The hydrophilic amino acid of base blocking group-N carboxy α amino acid anhydride monomer, its structural formula are：
Wherein, R1The hydrophilic radical of hydrophilic amino acid is represented, Z ' represents amido protecting group；
(1-2), by the hydrophilic amino acid of the group containing amido protecting-N carboxy α amino acid anhydride monomer and hydrophilic ammonia The initiator of base acid polymerization is dissolved in organic solvent, and is reacted under vacuo, obtains the hydrophilic polypeptides of the group containing amido protecting Segment, its structural formula are：
Wherein, A represents the initiator of hydrophilic amino acid polymerization；
(2), the preparation of the amino-terminated hydrophobic polymer segment in one end：
(2-1), the hydrophobic polymer of the cyclic monomer of hydrophobic polymer, catalyst, the group containing amido protecting gathered The initiator of conjunction is dissolved in organic solvent, and is reacted under nitrogen protection, obtains the hydrophobic polymer of the group containing amido protecting Segment, its structural formula are：
Wherein, R3The repeat unit of biodegradable hydrophobic polymer is represented, A ' represents hydrophobic polymer polymerization Initiator, Z ' represent amido protecting group；
(2-2), add deprotection agent in the hydrophobic polymer segment of the group containing amido protecting and reacted, obtain the One reaction solution；Precipitating reagent is added in the first reaction solution, taking precipitate is as the first crude product；By the washing of the first crude product, do It is dry, the amino-terminated hydrophobic polymer segment in one end is obtained, its structural formula is：
(3), the preparation of biodegradable amphiphilic block class antibacterial peptide：
(3-1), by the hydrophilic polypeptides segment and diisocyanate of the group containing amido protecting of step (1) in organic solvent Middle reaction, obtain the second reaction solution；N-hexane is added in the second reaction solution, purifying obtains：
Wherein, R2Represent the alkane segment of diisocyanate；
(3-2), the amino-terminated hydrophobic polymer segment in one end of step (2) is dissolved in organic solvent, and added Reacted in step (3-1), obtain the block polymer of the group containing amido protecting, its structural formula is：
(3-3), addition deprotection agent is reacted in step (3-2), obtains the second crude product；Second crude product is saturating Analysis, dry, obtain biodegradable amphiphilic block class antibacterial peptide, its structural formula is：
Preferably, any one of amido protecting group in benzyloxycarbonyl and tert-butoxycarbonyl.
Preferably, the cyclic monomer of hydrophobic polymer is selected from caprolactone, lactide, glycolide and trimethylene carbonate In any one or a few.
Preferably, the initiator of hydrophilic amino acid polymerization is in isobutyl amine, n-butylamine, isopropylamine, propylamine and aniline Any one.
Preferably, the initiator of the hydrophobic polymer polymerization of the group containing amido protecting is selected from tert-butoxycarbonyl-amino Any one in ethanol and tert-butoxycarbonyl-amino propyl alcohol.
Preferably, deprotection agent be selected from the methanol solution of the hydrogen chloride containing 25wt%, the hydrobromic acid containing 33wt% ethanol solution, Any one in the acetic acid solution of trifluoroacetic acid and the hydrobromic acid containing 30wt%.
Preferably, any one or a few in water, n-hexane, acetone, methanol and ethanol of precipitating reagent.
Preferably, organic solvent is selected from tetrahydrofuran, DMF, dichloromethane, toluene, acetone and diformazan Any one or a few in base sulfoxide.
Preferably, the mol ratio of hydrophilic amino acid and triphosgene is 2-2.3:1.
Preferably, the initiator and the hydrophilic amino acid-N- carboxyls of the group containing amido protecting that hydrophilic amino acid polymerize- The mol ratio of a-amino acid anhydride monomer is 1:1‐100.
Preferably, the initiator of hydrophobic polymer polymerization and the ring-type list of hydrophobic polymer of the group containing amido protecting The mol ratio of body is 1:1‐100.
Preferably, the temperature of the reaction of step (1-1) is 40-60 DEG C, and the time of reaction is 3-6h.
Preferably, the temperature of the reaction of step (1-2) is 20-35 DEG C, and the time of reaction is 10-16h.
Preferably, the temperature of the reaction of step (2-1) is 90-120 DEG C, and the time of reaction is 32-48h.
Preferably, the temperature of the reaction of step (2-2) is 20-35 DEG C, and the time of reaction is 2-6h.
Preferably, the temperature of the reaction of step (3-1) is 10-30 DEG C, and the time of reaction is 0.5-1h.
Preferably, the temperature of the reaction of step (3-2) is 10-30 DEG C, and the time of reaction is 0.5-1h.
Preferably, the temperature of the reaction of step (3-3) is 20-35 DEG C, and the time of reaction is 2-6h.
One species antibacterial peptide vesica, its by above-mentioned biodegradable amphiphilic block class antibacterial self-assembling peptide and Into.
A kind of preparation method of above-mentioned class antibacterial peptide vesica, it comprises the following steps：
(a), by biodegradable amphiphilic block class antibacterial peptide
It is dissolved in organic solvent, obtains suspension；
(b) deionized water, is added dropwise in suspension, stirs, dialysis, obtains class antibacterial peptide vesica.
Preferably, in step (a), organic solvent is selected from tetrahydrofuran, dimethyl sulfoxide (DMSO), DMF, two Any one or a few in chloromethanes and acetone.
Preferably, in step (a), the concentration of suspension is 1-100mg/mL.
Preferably, in step (b), the time of dialysis is 12-24h.
A kind of above-mentioned class antibacterial peptide vesica is released as antiseptic or pharmaceutical carrier in critical anti-inflammatory, cancer therapy drug targeting Put or nanosecond medical science in terms of in application.
Due to using such scheme, the beneficial effects of the invention are as follows：
Firstth, class antibacterial peptide vesica prepared by the present invention has selected biodegradable hydrophobic polymer segment instead of Hydrophobic amino acid segment in antibacterial peptide, therefore there is no cytotoxicity, and can be dropped with excellent biocompatibility and biology Xie Xing.
Secondth, class antibacterial peptide vesica prepared by the present invention has excellent broad spectrum antibacterial performance, similar with natural antibacterial peptide Film destroy Antibacterial Mechanism it is identical, be not easy Induction of bacterial produce drug resistance.
3rd, class antibacterial peptide vesica prepared by the present invention can form stable assembly in water, can be used as antibacterial Agent or pharmaceutical carrier are applied in terms of critical anti-inflammatory, cancer therapy drug Targeting delivery or nanosecond medical science.
4th, the structure-controllable of class antibacterial peptide vesica prepared by the present invention, synthesis step are easy, cost is cheap, and can Realize industrialization production.
Brief description of the drawings
Fig. 1 is the class antibacterial peptide vesica (Polylys of the present invention20‐HDI‐PCL16And Polylys11‐HDI‐PCL16) grain Footpath and particle polydispersity index (PDI) figure, transverse axis are Hydrodynamic diameter (Hydrodynamic Diameter, nm).
Fig. 2 is the class antibacterial peptide vesica Polylys of embodiments of the invention 120‐HDI‐PCL16Transmission electron microscope (TEM) Figure.
Fig. 3 is the class antibacterial peptide vesica Polylys of embodiments of the invention 211‐HDI‐PCL16Transmission electron microscope (TEM) Figure.
Embodiment
The invention provides a kind of synthesis of biodegradable amphiphilic block class antibacterial peptide and class antibacterial peptide capsule The preparation method and application of bubble.
<Biodegradable amphiphilic block class antibacterial peptide>
A kind of biodegradable amphiphilic block class antibacterial peptide, its by the group containing amido protecting hydrophilic polypeptides The amino-terminated hydrophobic polymer segment of segment and one end with the isocyano of diisocyanate unit by forming covalent bond It is connected and is made, its structural formula is as follows：
Wherein, integers of the x in 1-100, integers of the y in 1-100, x and y represent the polymerization of each segment respectively Degree, R1Represent the hydrophilic radical of hydrophilic amino acid, R2Represent the alkane segment of diisocyanate, R3Represent biodegradable The repeat unit of hydrophobic polymer, A represent the initiator of hydrophilic amino acid polymerization, and A ' represents hydrophobic polymer polymerization Initiator.
(hydrophilic amino acid)
Hydrophilic amino acid can be selected from any one in lysine (Lys), arginine and histidine, hydrophilic amino The hydrophilic radical of acid can be the imidazole radicals of arginic guanidine radicals, the amino of lysine and histidine.
Wherein, the hydrophilic amino acid of the group of the invention containing amido protecting is preferably benzyloxycarbonyl-lysine.
(diisocyanate)
Diisocyanate can be selected from hexamethylene diisocyanate (HDI), MDI (MDI), right Any one in XDI (XDI) and PPDI (PPDI).
(hydrophobic polymer)
Hydrophobic polymer can be selected from polycaprolactone (PCL), PLA (PLA), polyglycolic acid (PGA) and poly- Sanya Any one or a few in methyl carbonic (PTMC).
In fact, above-mentioned class antibacterial peptide is similar to the structure of natural cationic type antibacterial peptide, thus its Antibacterial Mechanism also with day Right cationic antibacterial peptide is similar, is film damage type Antibacterial Mechanism.Positive charge on the hydrophilic segment of above-mentioned class antibacterial peptide with Bacterial cell membrane with negative electrical charge produces electrostatic adsorption, and hydrophobic segment is inserted inside cell membrane, destroys the integrality of film, Cause the cellular content in cell membrane to leak, finally cause bacterial death.When above-mentioned class antibacterial peptide is used as antiseptic, it is not easy to Induction of bacterial produces drug resistance.
<The preparation method of biodegradable amphiphilic block class antibacterial peptide>
A kind of preparation method of above-mentioned biodegradable amphiphilic block class antibacterial peptide, it includes following step Suddenly：
(1), the preparation of the hydrophilic polypeptides segment of the group containing amido protecting：
(1-1), the hydrophilic amino acid and triphosgene of the group containing amido protecting reacted in organic solvent, obtain containing ammonia The hydrophilic amino acid of base blocking group-N carboxy α amino acid anhydride monomer, its structural formula are：
Wherein, R1The hydrophilic radical of hydrophilic amino acid is represented, Z ' represents amido protecting group；
(1-2), by the hydrophilic amino acid of the group containing amido protecting-N carboxy α amino acid anhydride monomer and hydrophilic ammonia The initiator of base acid polymerization is dissolved in organic solvent, and is reacted under vacuo, obtains the hydrophilic polypeptides of the group containing amido protecting Segment, its structural formula are：
Wherein, A represents the initiator of hydrophilic amino acid polymerization；
(2), the preparation of the amino-terminated hydrophobic polymer segment in one end：
(2-1), the hydrophobic polymer of the cyclic monomer of hydrophobic polymer, catalyst, the group containing amido protecting gathered The initiator of conjunction is dissolved in organic solvent, and is reacted under nitrogen protection, obtains the hydrophobic polymer of the group containing amido protecting Segment, its structural formula are：
Wherein, R3The repeat unit of biodegradable hydrophobic polymer is represented, A ' represents hydrophobic polymer polymerization Initiator, Z ' represent amido protecting group；
(2-2), add deprotection agent in the hydrophobic polymer segment of the group containing amido protecting and reacted, obtain the One reaction solution；Precipitating reagent is added in the first reaction solution, taking precipitate is as the first crude product；By the washing of the first crude product, do It is dry, the amino-terminated hydrophobic polymer segment in one end is obtained, its structural formula is：
(3), the preparation of biodegradable amphiphilic block class antibacterial peptide：
(3-1), by the hydrophilic polypeptides segment of the group containing amido protecting of step (1) and it is excessive (be above-mentioned hydrophily 4-10 times of the amount of polypeptide chain material) diisocyanate react in organic solvent, obtain the second reaction solution；It is anti-second Answer and n-hexane is added in liquid, remove excessive unreacted diisocyanate, purifying obtains：
Wherein, R2Represent the alkane segment of diisocyanate；
(3-2), the amino-terminated hydrophobic polymer segment in one end of step (2) is dissolved in organic solvent, and added Reacted in step (3-1), obtain the block polymer of the group containing amido protecting, its structural formula is：
(3-3), addition deprotection agent is reacted in step (3-2), obtains the second crude product；Second crude product is saturating Analysis, dry, obtain biodegradable amphiphilic block class antibacterial peptide, its structural formula is：
Wherein, in step (1-1), the temperature of reaction can be 40-60 DEG C, preferably 50 DEG C；The time of reaction can be with For 3-6h, preferably 5h.
In step (1-2), the temperature of reaction can be 20-35 DEG C, preferably 25 DEG C；The time of reaction can be 10- 16h, preferably 12h.
In step (2-1), the temperature of reaction can be 90-120 DEG C, preferably 110 DEG C；The time of reaction can be 32-48h, preferably 48h.
In step (2-2), the temperature of reaction can be 20-35 DEG C, preferably 25 DEG C；The time of reaction can be 2- 6h, preferably 3h.
In step (3-1), the temperature of reaction can be 10-30 DEG C, preferably 20 DEG C；The time of reaction can be 0.5- 1h, preferably 40min.
In step (3-2), the temperature of reaction can be 10-30 DEG C, preferably 20 DEG C；The time of reaction can be 0.5- 1h, preferably 40min.
In step (3-3), the temperature of reaction can be 20-35 DEG C, preferably 25 DEG C；The time of reaction can be 2- 6h, preferably 4h.
(amido protecting group)
Amido protecting group can be selected from any one in benzyloxycarbonyl (Cbz) and tert-butoxycarbonyl (Boc).
(cyclic monomer of hydrophobic polymer)
The cyclic monomer of hydrophobic polymer can be in caprolactone, lactide, glycolide and trimethylene carbonate Any one or a few.
(initiator of hydrophilic amino acid polymerization)
Hydrophilic amino acid polymerization initiator can be in isobutyl amine, n-butylamine, isopropylamine, propylamine and aniline appoint Meaning is a kind of.
(initiator of the hydrophobic polymer polymerization of the group containing amido protecting)
The initiator of the hydrophobic polymer polymerization of the group containing amido protecting can be selected from tert-butoxycarbonyl (Boc)-ammonia Any one in base ethanol and tert-butoxycarbonyl (Boc)-aminopropanol.
(deprotection agent)
Deprotection agent can be selected from methanol solution, the second of hydrobromic acid containing 33wt% (HBr) of hydrogen chloride containing 25wt% (HCl) Any one in the acetic acid solution of alcoholic solution, trifluoroacetic acid and hydrobromic acid containing 30wt% (HBr).
(precipitating reagent)
Precipitating reagent can be selected from any one or a few in water, n-hexane, acetone, methanol and ethanol.
(organic solvent)
Organic solvent can be selected from tetrahydrofuran (THF), N,N-dimethylformamide (DMF), dichloromethane (DCM), first Any one or a few in benzene, acetone and dimethyl sulfoxide (DMSO) (DMSO).
The mol ratio of hydrophilic amino acid and triphosgene can be 2-2.3:1, preferably 2:1.
Hydrophilic amino acid-N- carboxyls-alpha-amido of the initiator and the group containing amido protecting of hydrophilic amino acid polymerization The mol ratio of anhydride monomers can be 1:1-100, preferably 1:23.
The initiator of hydrophobic polymer polymerization and the cyclic monomer of hydrophobic polymer of the group containing amido protecting rub Your ratio can be 1:1-100, preferably 1:20.
<Class antibacterial peptide vesica>
One species antibacterial peptide vesica, its by above-mentioned biodegradable amphiphilic block class antibacterial self-assembling peptide and Into.
<The preparation method of class antibacterial peptide vesica>
A kind of preparation method of above-mentioned class antibacterial peptide vesica, it comprises the following steps：
(a), by biodegradable amphiphilic block class antibacterial peptide
It is dissolved in organic solvent, obtains suspension；
(b) deionized water, is added dropwise in suspension, is stirred overnight, dialyses, obtains class antibacterial peptide vesica.
Wherein, in step (a), organic solvent can be selected from tetrahydrofuran (THF), dimethyl sulfoxide (DMSO) (DMSO), N, N- Any one or a few in dimethylformamide (DMF), dichloromethane (DCM) and acetone.
The concentration of suspension can be 1-100mg/mL, preferably 5mg/mL.
In step (b), the time of dialysis can be 12-24h, preferably 24h.
<The application of class antibacterial peptide vesica>
A kind of above-mentioned class antibacterial peptide vesica in critical anti-inflammatory, cancer therapy drug Targeting delivery or can be received as antiseptic Rice medical science etc. is applied.
A kind of above-mentioned class antibacterial peptide vesica can also be used as pharmaceutical carrier in critical anti-inflammatory, cancer therapy drug Targeting delivery Or nanosecond medical science etc. is applied.
Gathered in fact, the present invention selects by N carboxy α amino acid anhydride (N-carboxy- α-anhydride, NCA) open loop It is anti-by being coupled to close lysine (Lysine), Amino End Group polycaprolactone (PCL) and the hexamethylene diisocyanate (HDI) formed Above-mentioned amphiphilic block class antibacterial peptide should be prepared.Wherein, hydrophilic amino acid (lysine) segment carries a large amount of positive electricity Lotus, produce electrostatic interaction with the bacterial phospholipid bilayer of negative electrical charge and adsorb on cell membrane, while hydrophobic polymer is (poly- Caprolactone) in segment insertion phospholipid bilayer, the integrality of cell membrane is destroyed so as to reach the purpose of bactericidal, due to poly- Caprolactone is widely used biodegradable and the excellent macromolecule of biocompatibility, and it is isostructural more hydrophobic than containing phenyl ring Acidic amino acid segment toxicity is low.Therefore, above-mentioned amphiphilic block class antibacterial peptide no cytotoxicity, the biology that prepared by the present invention Assembly that is degradable and can forming stabilization, that is, be assembled into class antibacterial peptide vesica, anti-in clinic as antiseptic or pharmaceutical carrier Inflammation, cancer therapy drug Targeting delivery or nanosecond medical science etc. are all with a wide range of applications.
The present invention will be further described with reference to the accompanying drawings.
Embodiment 1：
The synthesis of the biodegradable amphiphilic block class antibacterial peptide of the present embodiment and the system of class antibacterial peptide vesica Preparation Method, it comprises the following steps：
(1) 20.000g (71.428mmol) benzyloxycarbonyl group-lysine and 10.598g (35.714mmol) triphosgene are added Enter in 200mL tetrahydrofurans, reaction temperature is 50 DEG C, and reacts 5h, obtains benzyloxycarbonyl group-lysine-N- carboxyls-a-amino acid Anhydride monomer, its structural formula are：
(2) using 30.475mg (0.417mmol) isobutyl amine (as initiator), 3.000g (9.804mmol) benzyloxycarbonyl group- Lysine-N carboxy α amino acid anhydride monomer is dissolved in 20mL dimethyl sulfoxide (DMSO)s, and reaction 12h is vacuumized at 25 DEG C, is obtained Poly- (benzyloxycarbonyl group-lysine), its structural formula are：
(3) using 2.121g (13.158mmol) tertbutyloxycarbonyl (Boc)-ethylaminoethanol (as initiator), 30.00g (263.16mmol) caprolactone is added in 100mL toluene, adds 2 drop stannous octoates (as catalyst), and be passed through in system Nitrogen is protected, and is reacted 48h at 110 DEG C, is obtained mixed liquor；1000mL methanol extractions are instilled in mixed liquor and obtain crude product, By filtration of crude product, washing, drying, the Amino End Group polycaprolactone of tertbutyloxycarbonyl protection is obtained, its structural formula is：
(4) the Amino End Group polycaprolactone that tertbutyloxycarbonyl is protected is dissolved in 20mL acetone, adds 50mL chlorine containing 25wt% Change the methanol solution (as deprotection agent) of hydrogen (HCl), stirring reaction 3h at 25 DEG C；It is molten in rotary evaporation reaction solution under vacuum Agent, the crude product gone out with water sedimentation after deprotection, and repeatedly wash to crude product in neutrality, filter, be dried to obtain one end ammonia The polycaprolactone of base end-blocking, its structural formula are：
(5) poly- (benzyloxycarbonyl group-lysine) obtained by 2.73g steps (2) is dissolved in 20mL dimethyl sulfoxide (DMSO)s, it is completely molten The hexamethylene diisocyanate (0.86g) of the amount of 10 times of poly- (benzyloxycarbonyl group-lysine) materials is added after solution, it is anti-at 20 DEG C Answer 40min；Then a large amount of (about 100mL) n-hexanes (as extractant) are added and extract the unreacted isocyanic acid of hexa-methylene two Ester, upper strata n-hexane is toppled over after standing, be repeated 5 times extracting operation；30min is rotated at 30 DEG C and removes residual minim n-hexane, Pure reaction solution is obtained, its structural formula is：
(6) the amino-terminated polycaprolactone in one end obtained by 1.40g steps (4) is dissolved in dimethyl sulfoxide (DMSO), be completely dissolved Add afterwards in reaction solution obtained by step (5), react 40min at 20 DEG C, obtain mixed liquor；1000mL is added in gained mixed liquor Water sedimentation obtains crude product, by filtration of crude product, washs, is dried to obtain the poly- isocyanide of (benzyloxycarbonyl group-lysine)-hexa-methylene two Acid esters-polycaprolactone block polymer, its structural formula are：
(7) gather to poly- (benzyloxycarbonyl group-lysine)-hexamethylene diisocyanate-polycaprolactone block obtained by step (6) The acetic acid solution (as deprotection agent) containing 30wt%HBr is added in compound, stirring reaction 4h at 25 DEG C；Then lower 50 DEG C of vacuum Rotary evaporation removes the dimethyl sulfoxide (DMSO) in reaction system, then adjusts pH value to faintly acid with sodium acid carbonate, finally by crude product Dialysis 48h in bag filter (molecular cut off 3500) is placed in, is freezed in freeze drier, obtains the methylene of polylysine-six Group diisocyanate-poly caprolactone antibacterial peptide (Polylys20‐HDI‐PCL16), its structural formula is：
(8) by polylysine-hexamethylene diisocyanate-poly caprolactone antibacterial peptide obtained by 5.5mg steps (7) (Polylys20‐HDI‐PCL16) be dissolved in 1mL tetrahydrofurans, being slowly added to 5.5mg dropwise in suspension after being completely dissolved goes Ionized water, it is added dropwise in 10min, is stirred overnight after being added dropwise, is then dialysed with the bag filter that molecular cut off is 3500 24h removes tetrahydrofuran, during which changes a water per 2h, obtains class antibacterial peptide vesica Polylys20‐HDI‐PCL16。
In fact, in step (1-1), the temperature of reaction is within 40-60 DEG C, the time of reaction is within 3-6h Can with.
In step (1-2), the temperature of reaction is within 20-35 DEG C, the time of reaction is within 10-16h can be with 's.
In step (2-1), the temperature of reaction is within 90-120 DEG C, the time of reaction is within 32-48h can be with 's.
In step (2-2), the temperature of reaction is within 20-35 DEG C, the time of reaction is possible within 2-6h.
In step (3-1), the temperature of reaction is within 10-30 DEG C, the time of reaction is within 0.5-1h can be with 's.
In step (3-2), the temperature of reaction is within 10-30 DEG C, the time of reaction is within 0.5-1h can be with 's.
In step (3-3), the temperature of reaction is within 20-35 DEG C, the time of reaction is possible within 2-6h.
Embodiment 2：
The synthesis of the biodegradable amphiphilic block class antibacterial peptide of the present embodiment and the system of class antibacterial peptide vesica Preparation Method, it comprises the following steps：
(1) 10.000g (35.714mmol) benzyloxycarbonyl group-lysine and 5.299g (17.857mmol) triphosgene are added In 100mL tetrahydrofurans, reaction temperature is 50 DEG C, and reacts 5h, obtains benzyloxycarbonyl group-lysine-N carboxy α amino acid anhydride Monomer, its structural formula are：
(2) using 92.348mg (1.263mmol) isobutyl amine (as initiator), 5.000g (16.340mmol) benzyloxy carbonyl Base-lysine-N carboxy α amino acid anhydride monomer is dissolved in 20mL dimethyl sulfoxide (DMSO)s, and reaction 12h is vacuumized at 20 DEG C, Poly- (benzyloxycarbonyl group-lysine) is obtained, its structural formula is：
(3) using 2.121g (13.158mmol) tertbutyloxycarbonyl (Boc)-ethylaminoethanol (as initiator), 30.00g (263.16mmol) caprolactone is added in 100mL toluene, adds 2 drop stannous octoates (as catalyst), and be passed through in system Nitrogen is protected, and is reacted 48h at 110 DEG C, is obtained mixed liquor；1000mL methanol extractions are instilled in mixed liquor and obtain crude product, By filtration of crude product, washing, drying, the Amino End Group polycaprolactone of tertbutyloxycarbonyl protection is obtained, its structural formula is：
(4) the Amino End Group polycaprolactone that tertbutyloxycarbonyl is protected is dissolved in 20mL acetone, adds 50mL chlorine containing 25wt% Change the methanol solution (as deprotection agent) of hydrogen (HCl), stirring reaction 3h at 20 DEG C；It is molten in rotary evaporation reaction solution under vacuum Agent, the crude product gone out with water sedimentation after deprotection, and repeatedly wash to crude product in neutrality, filter, be dried to obtain one end ammonia The polycaprolactone of base end-blocking, its structural formula are：
(5) poly- (benzyloxycarbonyl group-lysine) obtained by 3.20g steps (2) is dissolved in 20mL dimethyl sulfoxide (DMSO)s, it is completely molten The hexamethylene diisocyanate (1.82g) of the amount of 10 times of poly- (benzyloxycarbonyl group-lysine) materials is added after solution, it is anti-at 30 DEG C Answer 40min；Then a large amount of (about 100mL) n-hexanes (as extractant) are added and extract the unreacted isocyanic acid of hexa-methylene two Ester, upper strata n-hexane is toppled over after standing, be repeated 5 times extracting operation；30min is rotated at 30 DEG C and removes residual minim n-hexane, Pure reaction solution is obtained, its structural formula is：
(6) the amino-terminated polycaprolactone in one end obtained by 2.10g steps (4) is dissolved in dimethyl sulfoxide (DMSO), be completely dissolved Add afterwards in reaction solution obtained by step (5), react 40min at 25 DEG C, obtain mixed liquor；1000mL is added in gained mixed liquor Water sedimentation obtains crude product, by filtration of crude product, washs, is dried to obtain the poly- isocyanide of (benzyloxycarbonyl group-lysine)-hexa-methylene two Acid esters-polycaprolactone block polymer, its structural formula are：
(7) gather to poly- (benzyloxycarbonyl group-lysine)-hexamethylene diisocyanate-polycaprolactone block obtained by step (6) The acetic acid solution (as deprotection agent) containing 30wt%HBr is added in compound, stirring reaction 5h at 20 DEG C；Then lower 50 DEG C of vacuum Rotary evaporation removes the dimethyl sulfoxide (DMSO) in reaction system, then adjusts pH value to faintly acid with sodium acid carbonate, finally by crude product Dialysis 48h in bag filter (molecular cut off 3500) is placed in, is freezed in freeze drier, obtains the methylene of polylysine-six Group diisocyanate-poly caprolactone antibacterial peptide (Polylys11‐HDI‐PCL16), its structural formula is：
(8) by polylysine-hexamethylene diisocyanate-poly caprolactone antibacterial peptide obtained by 5.5mg steps (7) (Polylys11‐HDI‐PCL16) be dissolved in 1mL tetrahydrofurans, being slowly added to 5.5mg dropwise in suspension after being completely dissolved goes Ionized water, it is added dropwise in 10min, is stirred overnight after being added dropwise, is then dialysed with the bag filter that molecular cut off is 3500 24h removes tetrahydrofuran, during which changes a water per 2h, obtains class antibacterial peptide vesica Polylys11‐HDI‐PCL16。
Embodiment 3：
The synthesis of the biodegradable amphiphilic block class antibacterial peptide of the present embodiment and the system of class antibacterial peptide vesica Preparation Method, it comprises the following steps：
(1) 10.000g (35.714mmol) benzyloxycarbonyl group-lysine and 5.299g (17.857mmol) triphosgene are added In 100mL tetrahydrofurans, reaction temperature is 50 DEG C, and reacts 5h, obtains benzyloxycarbonyl group-lysine-N carboxy α amino acid anhydride Monomer, its structural formula are：
(2) using 20.317mg (0.278mmol) isobutyl amine (as initiator), 3.000g (9.804mmol) benzyloxycarbonyl group- Lysine-N carboxy α amino acid anhydride monomer is dissolved in 20mL dimethyl sulfoxide (DMSO)s, and reaction 12h is vacuumized at 30 DEG C, is obtained Poly- (benzyloxycarbonyl group-lysine), its structural formula are：
(3) using 2.121g (13.158mmol) tertbutyloxycarbonyl (Boc)-ethylaminoethanol (as initiator), 30.00g (263.16mmol) caprolactone is added in 100mL toluene, adds 2 drop stannous octoates (as catalyst), and be passed through in system Nitrogen is protected, and is reacted 48h at 110 DEG C, is obtained mixed liquor；1000mL methanol extractions are instilled in mixed liquor and obtain crude product, By filtration of crude product, washing, drying, the Amino End Group polycaprolactone of tertbutyloxycarbonyl protection is obtained, its structural formula is：
(4) the Amino End Group polycaprolactone that tertbutyloxycarbonyl is protected is dissolved in 20mL acetone, adds 50mL chlorine containing 25wt% Change the methanol solution (as deprotection agent) of hydrogen (HCl), stirring reaction 3h at 30 DEG C；It is molten in rotary evaporation reaction solution under vacuum Agent, the crude product gone out with water sedimentation after deprotection, and repeatedly wash to crude product in neutrality, filter, be dried to obtain one end ammonia The polycaprolactone of base end-blocking, its structural formula are：
(5) poly- (benzyloxycarbonyl group-lysine) obtained by 3.71g steps (2) is dissolved in 20mL dimethyl sulfoxide (DMSO)s, it is completely molten The hexamethylene diisocyanate (0.79g) of the amount of 10 times of poly- (benzyloxycarbonyl group-lysine) materials is added after solution, it is anti-at 25 DEG C Answer 40min；Then a large amount of (about 100mL) n-hexanes (as extractant) are added and extract the unreacted isocyanic acid of hexa-methylene two Ester, upper strata n-hexane is toppled over after standing, be repeated 5 times extracting operation；30min is rotated at 30 DEG C and removes residual minim n-hexane, Pure reaction solution is obtained, its structural formula is：
(6) the amino-terminated polycaprolactone in one end obtained by 0.89g steps (4) is dissolved in dimethyl sulfoxide (DMSO), be completely dissolved Add afterwards in reaction solution obtained by step (5), react 40min at 25 DEG C, obtain mixed liquor；1000mL is added in gained mixed liquor Water sedimentation obtains crude product, by filtration of crude product, washs, is dried to obtain the poly- isocyanide of (benzyloxycarbonyl group-lysine)-hexa-methylene two Acid esters-polycaprolactone block polymer, its structural formula are：
(7) gather to poly- (benzyloxycarbonyl group-lysine)-hexamethylene diisocyanate-polycaprolactone block obtained by step (6) The acetic acid solution (as deprotection agent) containing 30wt%HBr is added in compound, stirring reaction 5h at 20 DEG C；Then lower 50 DEG C of vacuum Rotary evaporation removes the dimethyl sulfoxide (DMSO) in reaction system, then adjusts pH value to faintly acid with sodium acid carbonate, finally by crude product Dialysis 48h in bag filter (molecular cut off 3500) is placed in, is freezed in freeze drier, obtains the methylene of polylysine-six Group diisocyanate-poly caprolactone antibacterial peptide (Polylys30‐HDI‐PCL16), its structural formula is：
(8) by polylysine-hexamethylene diisocyanate-poly caprolactone antibacterial peptide obtained by 5.5mg steps (7) (Polylys30‐HDI‐PCL16) be dissolved in 1mL tetrahydrofurans, being slowly added to 5.5mg dropwise in suspension after being completely dissolved goes Ionized water, it is added dropwise in 10min, is stirred overnight after being added dropwise, is then dialysed with the bag filter that molecular cut off is 3500 24h removes tetrahydrofuran, during which changes a water per 2h, obtains class antibacterial peptide vesica Polylys30‐HDI‐PCL16。
<Experiment>
Production is used as using above-described embodiment 1 and the biodegradable amphiphilic block class antibacterial peptide vesica of embodiment 2 Product are tested as follows respectively.
<Experiment 1>
This experiment is to verify biodegradable amphiphilic block class antibacterial peptide vesica (Polylys20‐HDI‐ PCL16With Polylys11‐HDI‐PCL16) in gram-positive bacteria (staphylococcus aureus) and Gram-negative bacteria (large intestine bar Bacterium) in anti-microbial property.
Wherein, minimal inhibitory concentration (MIC) is to assess the important parameter of antibacterial antiplaque agent performance.This experiment is respectively using leather Lan Shi negative bacteriums (Escherichia coli) and gram-positive bacteria (staphylococcus aureus) are biodegradable amphipathic embedding to determine The anti-microbial property of section Copolymer antibacterial peptide vesica.
Experimental procedure is as follows：
(1) Escherichia coli and staphylococcus aureus for taking 1mL to activate respectively, LB meat bones are removed with physiological saline Soup, and the strain of the strain of Escherichia coli and staphylococcus aureus is separately added into and is well mixed in 1mL physiological saline, it is Facilitate bacterium colony during coated plate to count, bacterium liquid is diluted 10 with decimal dilution method5Times；
(2) configuration concentration is 1000 μ g/mL class antibacterial peptide solution, and the class antibacterial of various concentrations is obtained with double of dilution method Peptide solution, concentration be followed successively by 1000 μ g/mL, 500 μ g/mL, 250 μ g/mL, 125 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ g/m, 8μg/mL、4μg/mL；100 μ L are respectively taken to be placed in 1mL centrifuge tubes；
(3) bacterium liquid (Escherichia coli or the golden yellow added into step (2) in each centrifuge tube after 100 μ L dilutions Staphylococcus), class antibacterial peptide concentration be changed into 500 μ g/mL, 250 μ g/mL, 125 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL, cultivate 2h under 37 DEG C of constant temperature, and similarity condition culture does not add the pure of class antibacterial peptide Bacterium is as control sample；
(4) mixed liquor of 100 μ L various concentrations is added in culture dish, adds the 10mL agar solutions of warm, is rocked Agar solidification is treated after even by culture dish back-off, is placed under 37 DEG C of constant temperatures after cultivating 18h and carries out bacterium colony counting, observation suppresses thin The minimum class antibacterial peptide concentration of bacteria growing.
By above-mentioned experiment, following experimental result is obtained：
Class antibacterial peptide vesica Polylys20‐HDI‐PCL16After acting on (experimental group) with Escherichia coli, compared to not adding class Antibacterial peptide vesica Polylys20‐HDI‐PCL16The bacterium colony dense degrees of pure Escherichia coli (control group) be reduced, illustrate class Antibacterial peptide vesica Polylys20‐HDI‐PCL16The growth of Escherichia coli can effectively be suppressed, under 4 μ g/mL concentration, experiment Half of the clump count of group still less than control group clump count.In the range of effective Mlc, with class antibacterial peptide vesica Polylys20‐HDI‐PCL16Concentration increase, experimental group culture dish colony number significantly reduces, especially in 125-500 μ g/mL Concentration under the colony growth without Escherichia coli.
Class antibacterial peptide vesica Polylys20‐HDI‐PCL16After acting on (experimental group) with staphylococcus aureus, compared to not Add class antibacterial peptide vesica Polylys20‐HDI‐PCL16The bacterium colony dense degrees of proof gold staphylococcus aureus (control group) obtain To reduction, illustrate class antibacterial peptide vesica Polylys20‐HDI‐PCL16The growth of staphylococcus aureus can effectively be suppressed, 8 Under μ g/mL concentration, the half of the clump count of experimental group still less than control group clump count.In the range of effective Mlc, With class antibacterial peptide vesica Polylys20‐HDI‐PCL16Concentration increase, experimental group culture dish colony number significantly reduces, special Grown not under 250-500 μ g/mL concentration without S. aureus colonies.
Class antibacterial peptide vesica Polylys11‐HDI‐PCL16After acting on (experimental group) with Escherichia coli, compared to not adding class Antibacterial peptide vesica Polylys11‐HDI‐PCL16The bacterium colony dense degrees of pure Escherichia coli (control group) be reduced, illustrate class Antibacterial peptide vesica Polylys11‐HDI‐PCL16The growth of Escherichia coli can effectively be suppressed, under 32 μ g/mL concentration, experiment Half of the clump count of group still less than control group clump count.In the range of effective Mlc, with class antibacterial peptide vesica Polylys11‐HDI‐PCL16Concentration increase, experimental group culture dish colony number significantly reduces, and concentration increases to 500 μ g/mL When without E. coli clones grow.
Class antibacterial peptide vesica Polylys11‐HDI‐PCL16After acting on (experimental group) with staphylococcus aureus, compared to not Add class antibacterial peptide vesica Polylys11‐HDI‐PCL16The bacterium colony dense degrees of proof gold staphylococcus aureus (control group) obtain To reduction, illustrate class antibacterial peptide vesica Polylys11‐HDI‐PCL16The growth of staphylococcus aureus can effectively be suppressed, Under 32 μ g/mL concentration, half of the experimental group clump count still less than control group clump count.In the range of effective Mlc, With class antibacterial peptide vesica Polylys11‐HDI‐PCL16Concentration increase, experimental group culture dish colony number significantly reduces, dense Degree grows when increasing to 500 μ g/mL without S. aureus colonies.
According to above-mentioned experimental result, class antibacterial peptide vesica Polylys is calculated respectively20‐HDI‐PCL16With class antibacterial peptide vesica Polylys11‐HDI‐PCL16Minimum inhibitory concentration (MIC) value in Escherichia coli, staphylococcus aureus, in this experiment most Small Mlc refers to the minimum addition concentration of class antibacterial peptide that clump count in culture dish is less than control group clump count half, above-mentioned MIC Value is as shown in table 1：
Table 1：Polylys20‐HDI‐PCL16With Polylys11‐HDI‐PCL16Minimum inhibitory concentration (MIC)
From table 1 it follows that biodegradable amphiphilic block class antibacterial peptide vesica prepared by the present invention (Polylys20‐HDI‐PCL16With Polylys11‐HDI‐PCL16) there is excellent restraining and sterilizing bacteria effect, and to Escherichia coli (leather Lan Shi negative bacteriums) and staphylococcus aureus (gram-positive bacteria) be respectively provided with good antibacterial effect, therefore, the present invention system Standby biodegradable amphiphilic block class antibacterial peptide vesica has excellent broad spectrum antibacterial performance.
<Experiment 2>
This experiment is to verify biodegradable amphiphilic block class antibacterial peptide vesica (Polylys20‐HDI‐ PCL16With Polylys11‐HDI‐PCL16) assembling situation.
The particle diameter and particle diameter distribution situation of vesica are the important parameters of assembly performance, more points of particle diameter distribution situation particle Dissipate sex index PDI (Polydispersed index) to represent, when PDI values are less than 0.3, then it represents that the particle size dispersion of assembly Uniformly.Using dynamic light scattering test come the particle diameter of test class antibacterial peptide vesica and particle polydispersity index PDI.
Experimental procedure is as follows：The class antibacterial peptide vesica (Polylys that will have been assembled20‐HDI‐PCL16With Polylys11‐ HDI‐PCL16) solution respectively takes 1.5mL to be put into glass dish respectively, be then placed in Zetasizer Nano serial nanos granularities and Dynamic light scattering test is carried out in zeta potential instrument.
Class antibacterial peptide vesica Polylysx‐HDI‐PCLyThe particle diameter and particle polydispersity index of (x=20,11, y=16) PDI value is as shown in figure 1, wherein (a) x=20, y=16；(b) x=11, y=16.
It will be seen from figure 1 that the particle diameter of class antibacterial peptide vesica, between 80-400nm, PDI values are respectively less than 0.3, illustrate this Invent the uniform particle sizes of the class antibacterial peptide vesica prepared, favorable dispersibility.
The transmission electron microscope shape appearance figure of class antibacterial peptide vesica further verifies the assembling situation of above-mentioned vesica, and its experimental procedure is such as Under：
Class antibacterial peptide vesicle solution is diluted to 150 μ g/mL by researcher at room temperature, takes 3 μ L to drop on copper mesh, in room Dyed after being dried under temperature with phosphotungstic acid (PTA, 1.0wt%) solution.A drop PTA solution is taken to drop in hydrophobic membrane, by copper mesh The one side for having loaded sample is placed on PTA drops top, infiltrates 1min, after the unnecessary PTA solution of Adsorption of Filter Paper, copper mesh is existed It is dried overnight at room temperature, you can to carry out transmission electron microscope test.
Class antibacterial peptide vesica Polylys20‐HDI‐PCL16Transmission electron microscope picture as shown in Fig. 2 class antibacterial peptide vesica Polylys11‐HDI‐PCL16Transmission electron microscope picture as shown in figure 3, and from Fig. 2 and Fig. 3, class antibacterial peptide vesica (Polylys20‐HDI‐PCL16With Polylys11‐HDI‐PCL16) vesica is successfully assembled into aqueous.
The above-mentioned description to embodiment is that this hair is understood that and used for the ease of those skilled in the art It is bright.Those skilled in the art obviously can readily make various modifications to these embodiments, and described herein one As principle be applied in other embodiment, without by performing creative labour.Therefore, the invention is not restricted to above-described embodiment. Those skilled in the art do not depart from improvement that scope of the invention made and modification all should be at these according to the principle of the present invention Within the protection domain of invention.

Claims (10)

  1. A kind of 1. biodegradable amphiphilic block class antibacterial peptide, it is characterised in that：Its structural formula is as follows：
    Wherein, integers of the x in 1-100, integers of the y in 1-100, x and y represent the degree of polymerization of each segment, R respectively1Table Show the hydrophilic radical of hydrophilic amino acid, R2Represent the alkane segment of diisocyanate, R3Represent that biodegradable hydrophobicity is high The repeat unit of molecule, A represent the initiator of hydrophilic amino acid polymerization, and A ' represents the initiator of hydrophobic polymer polymerization.
  2. 2. biodegradable amphiphilic block class antibacterial peptide according to claim 1, it is characterised in that：The parent Any one of water-based amino acid in lysine, arginine and histidine；And/or
    It is different that the diisocyanate is selected from hexamethylene diisocyanate, MDI, terephthalylidene two Any one in cyanate and PPDI；And/or
    The hydrophobic polymer is any in polycaprolactone, PLA, polyglycolic acid and PTMC It is one or more of.
  3. 3. a kind of preparation method of biodegradable amphiphilic block class antibacterial peptide as claimed in claim 1 or 2, its It is characterised by：It comprises the following steps：
    (1), the preparation of the hydrophilic polypeptides segment of the group containing amido protecting：
    (1-1), the hydrophilic amino acid and triphosgene of the group containing amido protecting reacted in organic solvent, obtain protecting containing amino Hydrophilic amino acid-N carboxy α amino acid anhydride monomer of group is protected, its structural formula is：
    Wherein, R1The hydrophilic radical of hydrophilic amino acid is represented, Z ' represents amido protecting group；
    (1-2), by the hydrophilic amino acid of the group containing amido protecting-N carboxy α amino acid anhydride monomer and hydrophilic ammonia The initiator of base acid polymerization is dissolved in organic solvent, and is reacted under vacuo, obtains the hydrophilic polypeptides of the group containing amido protecting Segment, its structural formula are：
    Wherein, A represents the initiator of hydrophilic amino acid polymerization；
    (2), the preparation of the amino-terminated hydrophobic polymer segment in one end：
    (2-1), the hydrophobic polymer of the cyclic monomer of hydrophobic polymer, catalyst, the group containing amido protecting polymerize Initiator is dissolved in organic solvent, and is reacted under nitrogen protection, obtains the hydrophobic polymer segment of the group containing amido protecting, Its structural formula is：
    Wherein, R3The repeat unit of biodegradable hydrophobic polymer is represented, A ' represents the initiation of hydrophobic polymer polymerization Agent, Z ' represent amido protecting group；
    (2-2), add deprotection agent in the hydrophobic polymer segment of the group containing amido protecting and reacted, obtain the One reaction solution；Precipitating reagent is added in first reaction solution, taking precipitate is as the first crude product；By first crude product Washing, dry, obtain the amino-terminated hydrophobic polymer segment in one end, its structural formula is：
    (3), the preparation of biodegradable amphiphilic block class antibacterial peptide：
    (3-1), by the hydrophilic polypeptides segment and diisocyanate of the group containing amido protecting of step (1) in organic solvent Middle reaction, obtain the second reaction solution；N-hexane is added in second reaction solution, purifying obtains：
    Wherein, R2Represent the alkane segment of diisocyanate；
    (3-2), the amino-terminated hydrophobic polymer segment in described one end of step (2) is dissolved in organic solvent, and added Reacted in the step (3-1), obtain the block polymer of the group containing amido protecting, its structural formula is：
    (3-3), addition deprotection agent is reacted in the step (3-2), obtains the second crude product；By the described second thick production Thing dialysis, dry, obtain the biodegradable amphiphilic block class antibacterial peptide, its structural formula is：
  4. 4. preparation method according to claim 3, it is characterised in that：The amido protecting group be selected from benzyloxycarbonyl and Any one in tert-butoxycarbonyl；And/or
    The cyclic monomer of the hydrophobic polymer appointing in caprolactone, lactide, glycolide and trimethylene carbonate Meaning is one or more of；And/or
    The initiator of the hydrophilic amino acid polymerization is any one in isobutyl amine, n-butylamine, isopropylamine, propylamine and aniline Kind；And/or
    The initiator of the hydrophobic polymer polymerization of the group containing amido protecting is selected from tert-butoxycarbonyl-amino ethanol and uncle Any one in butoxycarbonyl-amino propyl alcohol；And/or
    The deprotection agent is selected from the methanol solution of the hydrogen chloride containing 25wt%, the ethanol solution of the hydrobromic acid containing 33wt%, trifluoro second Acid and the hydrobromic acid containing 30wt% acetic acid solution in any one；And/or
    Any one or a few in water, n-hexane, acetone, methanol and ethanol of the precipitating reagent；And/or
    The organic solvent is selected from tetrahydrofuran, N,N-dimethylformamide, dichloromethane, toluene, acetone and dimethyl sulfoxide (DMSO) In any one or a few.
  5. 5. preparation method according to claim 3, it is characterised in that：The hydrophilic amino acid rubs with the triphosgene Your ratio is 2-2.3:1；And/or
    The initiator and the hydrophilic amino acid-N- carboxyls-α of the group containing amido protecting that the hydrophilic amino acid polymerize- The mol ratio of amino anhydride monomers is 1:1‐100；And/or
    The cyclic monomer of the initiator and the hydrophobic polymer of the hydrophobic polymer polymerization of the group containing amido protecting Mol ratio be 1:1‐100.
  6. 6. preparation method according to claim 3, it is characterised in that：The temperature of the reaction of the step (1-1) is 40-60 DEG C, the time of reaction is 3-6h；And/or
    The temperature of the reaction of the step (1-2) is 20-35 DEG C, and the time of reaction is 10-16h；And/or
    The temperature of the reaction of the step (2-1) is 90-120 DEG C, and the time of reaction is 32-48h；And/or
    The temperature of the reaction of the step (2-2) is 20-35 DEG C, and the time of reaction is 2-6h；And/or
    The temperature of the reaction of the step (3-1) is 10-30 DEG C, and the time of reaction is 0.5-1h；And/or
    The temperature of the reaction of the step (3-2) is 10-30 DEG C, and the time of reaction is 0.5-1h；And/or
    The temperature of the reaction of the step (3-3) is 20-35 DEG C, and the time of reaction is 2-6h.
  7. A 7. species antibacterial peptide vesica, it is characterised in that：By as claimed in claim 1 or 2 biodegradable amphipathic embedding Section Copolymer antibacterial self-assembling peptide forms.
  8. A kind of 8. preparation method of class antibacterial peptide vesica as claimed in claim 7, it is characterised in that：It comprises the following steps：
    (a), by biodegradable amphiphilic block class antibacterial peptide
    It is dissolved in organic solvent, obtains suspension；
    (b) deionized water, is added dropwise in the suspension, stirs, dialysis, obtains the class antibacterial peptide vesica.
  9. 9. the preparation method of class antibacterial peptide vesica according to claim 8, it is characterised in that：It is described organic in step (a) Solvent in tetrahydrofuran, dimethyl sulfoxide (DMSO), N,N-dimethylformamide, dichloromethane and acetone any one or it is several Kind；And/or
    The concentration of the suspension is 1-100mg/mL；And/or
    In step (b), the time of the dialysis is 12-24h.
  10. 10. a kind of class antibacterial peptide vesica as claimed in claim 7 is as antiseptic or pharmaceutical carrier in critical anti-inflammatory, anticancer Application in terms of drug targeting release or nanosecond medical science.
CN201710728104.8A 2017-08-23 2017-08-23 The synthesis of biodegradable amphiphilic block class antibacterial peptide and the preparation method and application of class antibacterial peptide vesica Pending CN107513145A (en)

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CN110054713A (en) * 2019-04-19 2019-07-26 同济大学 Amphipathic graft copolymer and its preparation method and application containing antibacterial peptide
CN111228223A (en) * 2020-03-06 2020-06-05 同济大学 Polymer vesicle for promoting wound healing and preparation method and application thereof
CN111228223B (en) * 2020-03-06 2021-06-18 同济大学 Polymer vesicle for promoting wound healing and preparation method and application thereof
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CN113694027A (en) * 2021-08-27 2021-11-26 中国科学技术大学 Polymer vesicle and preparation method thereof, and drug-loaded vesicle
CN113694027B (en) * 2021-08-27 2022-12-30 中国科学技术大学 Polymer vesicle and preparation method thereof, and drug-loaded vesicle

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