WO2019071945A1 - 一种杂化水凝胶材料的制备方法及应用 - Google Patents

一种杂化水凝胶材料的制备方法及应用 Download PDF

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WO2019071945A1
WO2019071945A1 PCT/CN2018/086241 CN2018086241W WO2019071945A1 WO 2019071945 A1 WO2019071945 A1 WO 2019071945A1 CN 2018086241 W CN2018086241 W CN 2018086241W WO 2019071945 A1 WO2019071945 A1 WO 2019071945A1
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polyethylene glycol
polylysine
lys
nca
product
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French (fr)
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陈平
王翔
行岳真
支三军
胡华友
仲慧
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淮阴师范学院
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    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • 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
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • 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
    • C08G69/40—Polyamides containing oxygen in the form of ether groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • 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
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L77/00—Compositions of polyamides obtained by reactions forming a carboxylic amide link in the main chain; Compositions of derivatives of such polymers
    • C08L77/04—Polyamides derived from alpha-amino carboxylic acids

Definitions

  • the invention relates to the technical field of new materials, in particular to a preparation method and application of a hybrid hydrogel material constructed based on the electrostatic action of a polypeptide-polyethylene glycol-polypeptide triblock copolymer and DNA.
  • the hydrogel material is a gel material in which water is a dispersion medium, and a water-soluble polymer having a network cross-linked structure introduces a part of a hydrophobic group and a hydrophilic residue, and the hydrophilic residue is combined with water molecules to water
  • Patent No. CN104780909A discloses a hydrogel forming material which can be formed by dissolving and dispersing a gelling agent in a medium, dissolving it, cooling it, and then allowing it to stand at room temperature while stirring. And a hydrogel material capable of re-forming a gel even after the hydrogel is sol-geled by shaking and then allowed to stand at room temperature.
  • Patent CN106397793A discloses an L-alanine, L-glutamic acid-5-benzyl ester polypeptide copolymer hydrogel, which is a nano-silver-loaded polyethylene glycol and a poly(L-alanine-co-L - an aqueous solution of a block copolymer of - glutamic acid-5-benzyl ester), which is simple in loading, high in loading, and has good in vitro antibacterial effect.
  • this is based on the transformation of the secondary structure between amphiphilic polypeptides or polypeptides to regulate the structure and properties of the hydrogel. This gel network formation mechanism may exist in certain environmental applications. Limitations.
  • Bio macromolecular DNA in addition to its function as a gene carrier, is also an important class of natural polyelectrolyte-based polymer materials. Due to the order and controllability of its structural units, it is considered to be a supermolecular self-assembled structure. Ideal raw material.
  • Patent CN103910893A discloses a polypeptide-DNA hydrogel and a preparation method thereof, comprising a polypeptide covalently bound to a single-stranded DNA molecule and a double-stranded DNA molecule having two cohesive ends, through a single-stranded DNA molecule and double-stranded DNA
  • the complementary molecules form a crosslinked structure, and the hydrogel has mechanical strength and biocompatibility.
  • the operating conditions are complex and irreversible, and a method of constructing a hybrid supramolecular hydrogel material by electrostatic action is not disclosed.
  • this patent utilizes two kinds of degradable biopolymers with excellent properties, such as polypeptide and DNA molecule, as the main building block component to construct hybrid supramolecular hydrogel materials, which can fully utilize the advantages of the above two structures in structure and properties. , giving hybrid materials more performance.
  • the invention provides a method for coupling a polyethylene glycol (PEG) end group by a coupling reaction of phenylalanine with a polyethylene glycol terminal hydroxyl group to obtain a macroinitiator-phenylalanine end group.
  • Polyethylene glycol (NH 2 -Phe-PEG 227 -Phe-NH 2 ) and then the ring-opening polymerization of Lys(Z)-NCA is initiated by its terminal amino group, and then successfully synthesized by deprotecting the group t-butoxycarbonyl group.
  • a triblock copolymer PLL-b-PEG-b-PLL was obtained.
  • a first aspect of the invention relates to a method for preparing a hybrid hydrogel material, which comprises a charge of 1:(10-20) from a polypeptide-polyethylene glycol-polypeptide triblock copolymer and fish sperm DNA.
  • the molar ratio is prepared by electrostatic bonding.
  • the charge molar ratio is 1: (14-18); further preferably, the charge molar ratio is 1:16.
  • the polypeptide-polyethylene glycol-polypeptide triblock copolymer is polylysine-polyethylene glycol-polylysine;
  • the dissociation constant of the amino group on the polylysine side chain It is about 10.5, so it can exist in a positively charged dissociated state in an aqueous solution of a pH neutral neutral system;
  • DNA acts as a natural anionic polyelectrolyte, and its phosphate structure in the phosphodiester bond is in the aqueous solution. It is usually present in a negatively charged dissociated state, thereby enabling it to electrostatically interact with the previously synthesized positively charged polylysine side chain to form a coupled binding site.
  • the positively charged pendant group of the polylysine block in the triblock copolymer can form a stable riveting site with the negatively charged phosphate group in the DNA backbone, while the copolymer midblock is electrically neutral and flexible PEG.
  • the segment acts as a crosslinked network connection structure, which allows the formed hydrogel material to have more structural controllability.
  • step 2) Mixing the polypeptide-polyethylene glycol-polypeptide triblock copolymer dissolved in step 1) with the dissolved fish sperm DNA.
  • step 2) according to the negatively charged phosphate group in the DNA phosphodiester bond and the positive charge of the protonated protonated side chain in the triblock copolymer, the charge molar relationship between the two The ratio is (10-20):1 mixed, and the mixture is allowed to stand overnight at normal temperature, and the minimum gelation mass concentration for forming the hybrid hydrogel structure is determined by inverting the sample tube; further preferably, between the two The molar ratio of charge is (14-18):1; most preferably, the molar ratio of charge between the two is 16:1; wherein, the positively charged amino group of polylysine and the negatively charged phosphoric acid in the DNA structure The charge concentration of the groups was controlled at 20 mM, respectively.
  • the synthetic steps of the polylysine-polyethylene glycol-polylysine triblock copolymer of the present invention are as follows:
  • step 2) transferring the dissolved Lys(Z)-NCA in step 2) to the product of step 1) to obtain a mixed solution
  • the degree of polymerization was calculated.
  • the mixture is precipitated with diethyl ether, and the hydrolysis is hydrolyzed by hydrobromic acid and glacial acetic acid. More preferably, the step 4) is a mixture of diethyl ether precipitated and trifluoroacetic acid (TFA) dissolved and precipitated.
  • TFA trifluoroacetic acid
  • step 5 After hydrolysis of 5 times equivalent of hydrobromic acid and glacial acetic acid water bath, diethyl ether precipitation; in step 5), the pH is adjusted with dilute HCl, and the precipitate is dissolved in water for dialysis, and the drying is dialysate Freeze drying to give a polylysine-polyethylene glycol-polylysine product.
  • the structural formula of the polylysine-polyethylene glycol-polylysine is as follows:
  • n is 3 to 6 in the structural formula; preferably, n is 4 to 5 in the structural formula; and further preferably, n is 4 in the structural formula.
  • step 2) removing the crude product in step 2) from the protective group t-butoxycarbonyl, washing to obtain phenylalanine-terminated polyethylene glycol, and the phenylalanine-terminated polyethylene glycol is two A polyethylene glycol having a macroinitiator modified with a primary amino group.
  • the coupling reaction is dissolved in dichloromethane in an ice water bath of N-tert-butoxycarbonyl-L-phenylalanine, and DCC and DMAP are added thereto at 0 ° C.
  • the reaction in the ice water bath is 2 h, and then at room temperature overnight;
  • the filtration in the step 2) includes room temperature filtration and cooling filtration;
  • the protecting group in the step 3) is tert-butoxycarbonyl (Boc), deprotection
  • the method of the group tert-butoxycarbonyl is to add 5 times equivalent of trifluoroacetic acid, and the washing liquid used for washing is a saturated NaHCO 3 , H 2 O, HCl and saturated NaCl washing liquid.
  • the Lys(Z)-NCA synthesis step of the present invention is as follows:
  • step 2) After the reaction in step 1) is completed, a crude product of ⁇ -lysyl-N-carboxy-cyclic internal anhydride is obtained;
  • H-Lys(Z)-OH is 0.8 g, 2.85 mmol-1.5 g, 5.35 mmol, corresponding to the triphosgene is 0.339 g, 1.14 mmol-0.635 g, 2.14 mmol, further Preferably, the H-Lys(Z)-OH is 1.00 g, 3.57 mmol, the triphosgene is 0.42 g, 1.43 mmol, the solvent is anhydrous tetrahydrofuran (THF); the reaction temperature in the step 2) 45-55 ° C, preferably 50 ° C, to clarify and continue to react for 0.5-1.5 h, preferably 1 h, the crude product is ⁇ -lysyl-N-carboxy-cyclic internal acid anhydride; in step 3)
  • the solvent used for the recrystallization is a mixed solvent of anhydrous tetrahydrofuran and n-hexane, and the number of times of recrystallization is preferably
  • a second aspect of the invention relates to a hybrid hydrogel material obtained according to the method described above.
  • the hybrid hydrogel material preparation materials of the present invention include polypeptide-polyethylene glycol-polypeptide and fish sperm DNA.
  • the polypeptide-polyethylene glycol-polypeptide is polylysine-polyethylene glycol-polylysine; the polylysine-polyethylene glycol-polylysine and fish sperm
  • the charge molar ratio of DNA is 1: (10-20); further preferably, the charge molar ratio of the polylysine-polyethylene glycol-polylysine to the fish sperm DNA is 1: (14-18) Most preferably, the charge molar ratio of the polylysine-polyethylene glycol-polylysine to the fish sperm DNA is 1:16.
  • the preparation raw material of polylysine-polyethylene glycol-polylysine according to the present invention includes an initiator and Lys(Z)-NCA.
  • the mass ratio of the initiator to Lys(Z)-NCA in the raw material is 1: (3-20); further preferably, the mass ratio of the initiator to Lys(Z)-NCA in the raw material is 1 : (8-20); Most preferably, the mass ratio of the initiator to Lys(Z)-NCA in the raw material is 1:10.
  • the initiator is a phenylalanine-terminated polyethylene glycol.
  • the raw material for preparing the hybrid hydrogel material further comprises 3-7 equivalents of trifluoroacetic acid; further preferably 5 equivalents of trifluoroacetic acid.
  • the preparation raw materials of the initiator of the present invention include the following components and mass contents:
  • the starting materials for the preparation of the initiator include the following components and mass contents:
  • the starting material for the initiator comprises the following components and mass content:
  • the Lys(Z)-NCA of the present invention is prepared by reacting L-lysine (H-Lys(Z)-OH) with triphosgene, wherein:
  • it is prepared by reacting 1.00 g, 3.57 mmol of H-Lys(Z)-OH with 0.42 g of 1.43 mmol of triphosgene.
  • a third aspect of the invention relates to the use of the method described above or the hybrid hydrogel material described above for tissue engineering, medical materials and drug loading.
  • the dichloromethane (H 2 CCl 2 ), tetrahydrofuran (THF) and n-hexane according to the present invention are used after bubbling oxygen removal by high-purity nitrogen and drying using an activated alumina column.
  • the hybrid supramolecular hydrogel material of the invention not only has the biodegradability of the copolymer and the structural unit of the DNA is ordered, controllable, and has the characteristics of a transient network structure, and simultaneously solves the traditional use of photoinitiators and The toxicity caused by the chemical cross-linking agent; at the same time, the method for preparing the hybrid supramolecular hydrogel material by the method of the invention has the advantages of low cost, simplicity, high efficiency, mild formation condition and responsiveness, reversibility and self-healing property, etc.
  • the strategy of constructing supramolecular network structures by electrostatic interaction has potential application value in the preparation of more complex functional materials.
  • Figure 1 Synthesis of NH 2 -Phe-PEG 227 -Phe-NH 2 ;
  • Figure 3 Synthesis of a polylysine-polyethylene glycol-polylysine (PLL-b-PEG-b-PLL) triblock copolymer
  • Figure 4 Nuclear magnetic hydrogen spectroscopy, wherein A is a nuclear magnetic hydrogen spectrum of PZLL-Phe-PEG-Phe-PZLL, and B is a nuclear magnetic hydrogen spectrum of PLL-Phe-PEG-Phe-PLL;
  • Figure 5 Characterization of NH 2 -Phe-PEG 227 -Phe-NH 2 with the initiated polymerization product PZLL-Phe-PEG 227 -Phe-PZLL size exclusion chromatography (SEC);
  • Figure 6 Schematic diagram of the formation of a supramolecular gel structure of triblock copolymer PLL-b-PEG-b-PLL and fish sperm DNA, wherein A: triblock copolymer and anion DNA form electrostatic coupling interaction by electrostatic interaction Point; B: a process of forming a hybrid network hydrogel material; C: a stable hydrogel structure.
  • N-tert-Butoxycarbonyl-L-phenylalanine Boc-Phe-OH, Shanghai Jill Biochemical, 99%;
  • N,N'-dicyclohexylcarbodiimide DCC, Shanghai Jill Biochemistry, 99%;
  • Trifluoroacetic acid TFA, Aladdin, 99.5%;
  • Deionized water (resistivity > 18 M ⁇ cm -1 ): Millipore Milli-Q purified.
  • the charge concentration of the positively charged amino group of polylysine and the negatively charged phosphate group in the DNA structure was controlled at 20 mM, respectively, and the charge molar ratio of the triblock copolymer to the fish sperm DNA was 1:16.
  • the first step coupling reaction of polyethylene glycol (HO-PEG 227- OH) with N-tert-butoxycarbonyl-L-phenylalanine (Boc-Phe-OH) to obtain a conjugate;
  • the HO-PEG 227- OH and Boc-Phe-OH were dissolved in 200 mL of dichloromethane, and DCC and DMAP were added thereto to react in an ice water bath at 0 ° C for 2 h, and the reaction system was further heated to room temperature overnight. ;
  • the second step filtering and removing impurities to obtain a crude product
  • the resulting insoluble by-product N,N'-dicycloethylurea was removed by filtration, and the residual DCU was further removed by a cooling filtration method to obtain a crude product.
  • the third step the crude product is deprotected from the tert-butoxycarbonyl group, washed to obtain a phenylalanine-terminated polyethylene glycol, and the phenylalanine-terminated polyethylene glycol is modified at both ends.
  • a polyethylene glycol of a primary amine initiator A polyethylene glycol of a primary amine initiator.
  • the raw material H-Lys(Z)-OH and triphosgene are reacted in anhydrous tetrahydrofuran (THF) solvent at 50 ° C until clarification, and the reaction is continued for one hour; after completion of the reaction, ⁇ -lysine-N-carboxy-ring is obtained.
  • THF tetrahydrofuran
  • the crude product of the internal anhydride (NCA) was placed in a glove box and recrystallized three times with a mixed solvent of THF and n-hexane to obtain the final Lys(Z)-NCA product.
  • NH 2 -Phe-PEG 227 -Phe-NH 2 was vacuum dried in an oil bath at 50 ° C for 4 h, then cooled to room temperature, and Lys (Z)-NCA was dissolved in dry DMF in a glove box, then extracted by syringe and transferred.
  • a mixed liquid was obtained; the mixed solution was nitrogen-protected, and reacted at 40 ° C for three days, and the reaction was confirmed to be completely obtained by FT-IR spectroscopy, and a small amount of the crude product was prepared to be 5 mg/mL for SEC/LLS test.
  • the precipitated product was adjusted to a suitable pH with dilute HCl, dissolved in water for dialysis, and the dialyzate was freeze-dried to obtain the product polylysine-polyethylene glycol-polylysine.
  • the deprotection group t-butoxycarbonyl reaction was confirmed to be complete by 1 H-NMR, and finally the product PLL 4 -b-PEG 227 -b-PLL 4 was obtained .
  • Volume exclusion chromatography/laser scattering (SEC/LLS) was connected to Wyatt Optilab DSP and Wyatt DAWN EOS laser scatter detector using SSI pump with 0.02 M LiBr in DMF as the mobile phase at a flow rate of 1.0 mL/min. The sample concentration was approximately 5 mg/mL at 50 °C.
  • the fish sperm DNA and the triblock copolymer PLL 4 -b-PEG 45 -b-PLL 4 prepared in 2.3 are respectively dissolved in water according to the negatively charged phosphate group and the triblock copolymer in the DNA phosphodiester bond.
  • the strategy for modifying the terminal hydroxyl group of PEG not only has the characteristics of simple and efficient synthesis, but also the characteristic chemical shift of the benzyl group on the pendant phenylalanine side group in 1 H-NMR, so that the end The modified product is readily characterized.
  • the yield of the crude product was 80%, and the NH 2 -Phe-PEG 227 -Phe-NH 2 product obtained after removal of the protective group t-butoxycarbonyl group was obtained. The yield was 75%.
  • the triblock copolymer PZLL-Phe-PEG-Phe-PZLL was synthesized by a ring-opening polymerization method using NH 2 -Phe-PEG 227 -Phe-NH 2 as a macroinitiator under certain conditions. Determination of the PEG methylene unit with a chemical shift of 3.3-3.8 and the chemical shift of 4.9-5.1 polylysine block side chain benzyl Central Asia in the 1 H-NMR spectrum (Fig.
  • the obtained triblock copolymer PLL-b-PEG 227- b-PLL has the following characteristics: first, the polymer middle block is a water-soluble and biocompatible polyethylene glycol segment, and the PEG The long segment (227 units) makes it flexible in constructing hybrid materials; in addition, the triblock copolymer is biodegradable polylysine block at both ends, which is beneficial to the organism. Medical materials applications.
  • the Zeta potential was measured by the Zetron potential particle size analyzer of the Malvern Company in the United States. The results showed that the zeta potential of the above aqueous solution of the triblock copolymer of 20 mg/mL was about +10 mV, which proved that the triblock copolymer was concentrated on both sides.
  • the lysine block carries a certain positive charge at the appropriate pH.
  • the triblock copolymer and the anionic DNA form a coupling binding site by electrostatic interaction as shown in FIG. 6A, and the PEG 227 which is flexible and has a long segment length in the triblock copolymer can serve as a bridge structure and will be aggregated.
  • the amino acid is linked to the coupling sites formed by DNA to form a hybrid hydrogel material with a spatial network structure (Fig. 6B), and the stability of the formation is achieved when the total mass concentration of the two components is about 3%.
  • the hydrogel structure is shown in Figure 6C.
  • the charge concentration of the positively charged amino group of polylysine and the negatively charged phosphate group in the DNA structure was controlled at 20 mM, respectively, and the charge molar ratio of the triblock copolymer to the fish sperm DNA was 1:10.
  • NH 2 -Phe-PEG 227 -Phe-NH 2 was vacuum dried in an oil bath at 55 ° C for 4 h, then cooled to room temperature, and Lys (Z)-NCA was dissolved in dry DMF in a glove box, then extracted by syringe and transferred.
  • a mixed liquid was obtained; the mixed solution was nitrogen-protected, and reacted at 45 ° C for three days, and the reaction was confirmed to be completely obtained by FT-IR spectroscopy, and a small amount of crude product was prepared to prepare 5 mg/mL for SEC/LLS test.
  • the precipitated product was adjusted to a suitable pH with dilute HCl, dissolved in water for dialysis, and the dialyzate was freeze-dried to obtain the product polylysine-polyethylene glycol-polylysine. It was confirmed by 1 H-NMR that the deprotection reaction was completed to give the product PLL 6 -b-PEG 227 -b-PLL 6 .
  • the stable hydrogel structure is formed in the case where the total mass concentration of the two components is about 2.5%.
  • the charge concentration of the positively charged amino group of polylysine and the negatively charged phosphate group in the DNA structure was controlled at 20 mM, respectively, and the charge molar ratio of the triblock copolymer to the fish sperm DNA was 1:20.
  • the precipitated product was adjusted to a suitable pH with dilute HCl, dissolved in water for dialysis, and the dialyzate was freeze-dried to obtain the product polylysine-polyethylene glycol-polylysine. It was confirmed by 1 H-NMR that the deprotection group t-butoxycarbonyl reaction was completed to finally obtain the product PLL 3 -b-PEG 227 -b-PLL 3 .
  • the stable hydrogel structure is formed in the case where the total mass concentration of the two components is about 4%.

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Abstract

提供了一种杂化水凝胶材料及其制备方法，以及在组织工程、医用材料及药物负载方面的应用。该杂化水凝胶材料由聚多肽-聚乙二醇-聚多肽三嵌段共聚物与鱼精DNA静电结合制备而成。该制备方法成本低、形成条件温和，并表现出响应性、可逆性和自愈性等特点。采用该方法制备的杂化水凝胶材料，既具有共聚物的生物降解性和DNA的结构单元有序性、可控性，又具有瞬态网络结构的特征，同时解决了使用光引发剂和化学交联剂而带来的毒性。

Description

一种杂化水凝胶材料的制备方法及应用 技术领域
本发明涉及新材料技术领域，具体涉及一种基于聚多肽-聚乙二醇-聚多肽三嵌段共聚物与DNA静电作用构建的杂化水凝胶材料的制备方法及应用。
背景技术
水凝胶材料是以水为分散介质的凝胶材料，是具有网状交联结构的水溶性高分子中引入一部分疏水基团和亲水残基，亲水残基与水分子结合，将水分子连接在网状内部，而疏水残基遇水膨胀的交联聚合物。由于其良好的性能，在组织工程和药物负载方面都具有广泛地应用前景，特别是基于聚多肽的水凝胶材料。
聚多肽的水凝胶材料具有生物可降解性与生物相容性，该类材料在生物医学材料领域应用时拥有独特的优势。专利CN104780909A公开了一种经由将胶凝剂在介质中加热搅拌使其溶解、分散后，一边搅拌一边放冷后，在室温下静置的工序的情况下，也能够形成水凝胶形成用材料，以及即使将水凝胶通过振荡溶胶化后，然后通过在室温下静置也能够再形成凝胶的水凝胶材料。专利CN106397793A公开了一种L-丙氨酸、L-谷氨酸-5-苄酯多肽共聚物水凝胶，为负载纳米银的聚乙二醇与聚(L-丙氨酸-co-L-谷氨酸-5-苄酯)的嵌段共聚物水溶液，该纳米银的水凝胶剂负载简便、负载量高并具有良好的体外抗菌效果。但是这种基于两亲性多肽之间或聚多肽之间二级结构的转变，来对水凝胶的结构与性能进行调控，这种凝胶网络形成机理在某些环境的应用过程中可能存在一定的局限性。
生物大分子DNA，除了能够作为基因载体的功能外，还是一类重要的天然聚电解质类高分子材料，由于其结构单元的有序性与可控性，被认为是构建超分子自组装结构的理想原材料。
由两种或两种以上不同类型高分子复合而成的杂化材料，由于可以同时兼具不同种类单体或聚合物的性质，使该类材料拥有独特的结构和功能。专利CN103910893A公开了一种聚多肽-DNA水凝胶及其制备方法，包括共价结合有单链DNA分子的多肽和具有两个粘性末端的双链DNA分子，通过单链DNA分子与双链DNA分子的互补形成交联结构，该 水凝胶机械强度可调节、生物相容性良好。但是通过共价结合的方式，操作条件复杂、不可逆，同时也未公开通过静电作用构筑杂化超分子水凝胶材料的方法。
因此，本专利利用聚多肽与DNA分子这两类性能优良的可降解生物高分子作为主要构筑单元组分构建杂化超分子水凝胶材料，能够充分利用上述两者在结构与性质上的优势，赋予杂化材料更多的性能。
发明内容
本发明提供了一种通过苯丙氨酸与聚乙二醇末端羟基进行偶联反应的方法对聚乙二醇(PEG)端基进行改性，获得大分子引发剂-苯丙氨酸端基化聚乙二醇(NH 2-Phe-PEG 227-Phe-NH 2)，然后利用其末端氨基引发Lys(Z)-NCA的开环聚合，再经脱保护基团叔丁氧羰基后成功合成得到三嵌段共聚物PLL-b-PEG-b-PLL。进一步利用该三嵌段聚合物中两端聚赖氨酸中的氨基与DNA分子磷酸二酯键骨架中的磷酸基团，在一定的pH条件下，通过正负电荷之间的静电相互作用，构建杂化交联网络结构，从而建立一种构筑杂化超分子水凝胶材料，该杂化超分子水凝胶材料既具有共聚物的生物降解性和DNA的结构单元有序、可控，又由于是通过静电作用方式结合的杂化超分子而具有瞬态网络结构的特征，同时解决了传统使用光引发剂和化学交联剂带来的毒性。
本发明的第一方面，涉及一种杂化水凝胶材料的制备方法，由聚多肽-聚乙二醇-聚多肽三嵌段共聚物与鱼精DNA以1:(10-20)的电荷摩尔比通过静电结合制备而成。
优选的，所述电荷摩尔比为1:(14-18)；进一步优选的，所述电荷摩尔比为1:16。
优选的，所述的聚多肽-聚乙二醇-聚多肽三嵌段共聚物为聚赖氨酸-聚乙二醇-聚赖氨酸；聚赖氨酸侧链上的氨基的解离常数为10.5左右，因此在通常为pH中性的生物体系水溶液中，能够以正电荷的解离状态存在；DNA作为天然阴离子性聚电解质，其骨架结构磷酸二酯键中的磷酸基团，在水溶液中通常以负电荷的解离状态存在，从而使其能够与上述合成得到的带正电荷的聚赖氨酸侧链利用静电相互作用形成偶联结合位点。三嵌段共聚物中聚赖氨酸嵌段带正电荷的侧基可以与DNA骨架中带负电荷的磷酸基团形成稳定的铆合位点，同时共聚物中间嵌段电中性的柔性PEG链段作为交联网络连接结构，使形成的水凝 胶材料具有更多地结构可控性。
本发明所述的杂化水凝胶材料的制备方法步骤如下：
1)将聚多肽-聚乙二醇-聚多肽三嵌段共聚物与鱼精DNA分别溶解于水；
2)将步骤1)中溶解的聚多肽-聚乙二醇-聚多肽三嵌段共聚物与溶解的鱼精DNA混合。
优选的，所述步骤2)中按照DNA磷酸二酯键中带负电荷的磷酸基团与三嵌段共聚物中聚赖氨酸侧链质子化的氨基正电荷，两者之间的电荷摩尔比例为(10-20):1混合，常温下，静置过夜，利用翻转样品管的方法，确定形成杂化水凝胶结构的最低成胶质量浓度；进一步优选的，所述两者之间的电荷摩尔比例为(14-18):1；最优选的，两者之间的电荷摩尔比例为16:1；其中，聚赖氨酸带正电荷的氨基与DNA结构中带负电荷的磷酸基团的电荷浓度分别控制在20mM。
本发明所述的聚赖氨酸-聚乙二醇-聚赖氨酸三嵌段共聚物合成步骤如下：
1)将苯丙氨酸端基化聚乙二醇油浴、干燥、冷却；
2)将Lys(Z)-NCA溶解于DMF中；
3)将步骤2)中溶解的Lys(Z)-NCA转移至步骤1)产物中，获得混合液；
4)确认步骤3)中的混合液反应完全后，沉淀，得到初步处理物，将初步处理物水解；
5)调节pH值、透析、干燥得到聚赖氨酸-聚乙二醇-聚赖氨酸产物。
优选的，所述步骤1)中油浴温度为45-55℃，优选为50℃，所述干燥为真空干燥，干燥约4小时，冷却至室温；所述步骤2)中将Lys(Z)-NCA在手套箱中溶解于干燥DMF中；所述步骤3)中用注射器抽取后转移到上述反应体系中，其中Lys(Z)-NCA：苯丙氨酸端基化聚乙二醇＝(3-20):1，优选的，Lys(Z)-NCA：苯丙氨酸端基化聚乙二醇＝(8-20):1，最优选的，Lys(Z)-NCA/苯丙氨酸端基化聚乙二醇＝10:1，并将获得的混合液氮气保护、35-45℃，优选为40℃，反应三天；所述步骤4)中确认反应完全利用FT-IR光谱，并利用产物的 1H-NMR相应特征吸收峰面积比例关系，计算得聚合度。所述步骤4)中混合液沉淀采用乙醚，所述的水解采用氢溴酸和冰醋酸水解，更优选的，所述的步骤4)为混合液采用乙醚沉淀、三氟乙酸(TFA)溶解沉淀物、5倍当量的氢溴酸和冰醋酸水浴水解后， 再进行乙醚沉淀；所述步骤5)中用稀HCl调节pH值，使沉淀物溶解于水中进行透析，所述干燥为将透析液冷冻干燥，得到聚赖氨酸-聚乙二醇-聚赖氨酸产物。
在本发明的一个实施例中，所述聚赖氨酸-聚乙二醇-聚赖氨酸的结构式如下：
Figure PCTCN2018086241-appb-000001
其中，所述结构式中n为3～6；优选的，所述结构式中n为4～5；进一步优选的，所述结构式中n为4。
本发明所述的初步处理物的结构式如下：
Figure PCTCN2018086241-appb-000002
本发明所述的苯丙氨酸端基化聚乙二醇合成步骤如下：
1)聚乙二醇与N-叔丁氧羰基-L-苯丙氨酸进行偶联反应，得到偶联物；
2)所述步骤1)中反应完全后，过滤除杂，得到粗产物；
3)将步骤2)中的粗产物脱去保护基团叔丁氧羰基，洗涤，得到苯丙氨酸端基化聚乙二醇，所述苯丙氨酸端基化聚乙二醇为两端修饰有伯胺基的大分子引发剂的聚乙二醇。
其中，所述苯丙氨酸端基化聚乙二醇的结构式如下：
Figure PCTCN2018086241-appb-000003
优选的，所述步骤1)中偶联反应为聚乙二醇与N-叔丁氧羰基-L-苯丙氨酸冰水浴中溶解于二氯甲烷中，向其中加入DCC和DMAP后0℃的冰水浴中反应2h，然后室温过夜；所述步骤2)中所述过滤包括常温过滤和冷却过滤；所述步骤3)中所述保护基团为叔丁氧羰基(Boc)，脱去保护基团叔丁氧羰基的方法为加入5倍当量的三氟乙酸，所述洗涤所用洗涤液为饱和NaHCO 3、H 2O、HCl和饱和NaCl洗涤分液。
本发明所述的Lys(Z)-NCA合成步骤如下：
1)将原料H-Lys(Z)-OH和三光气溶解于溶剂中；
2)步骤1)中反应完全后得到α-赖基酸-N-羧基-环内酸酐粗产品；
3)将步骤2)中获得的粗产品置于手套箱中，重结晶得到Lys(Z)-NCA产物。
其中，所述Lys(Z)-NCA的结构式如下：
Figure PCTCN2018086241-appb-000004
优选的，所述步骤1)中H-Lys(Z)-OH为0.8g，2.85mmol-1.5g，5.35mmol，对应的所述三光气为0.339g，1.14mmol-0.635g，2.14mmol，进一步优选的，所述H-Lys(Z)-OH为1.00g，3.57mmol，所述三光气为0.42g，1.43mmol，所述溶剂为无水四氢呋喃(THF)；所述步骤2)中反应温度为45-55℃，优选为50℃，至澄清后继续反应0.5-1.5h，优选为1h，所述粗产品为α-赖基酸-N-羧基-环内酸酐；所述步骤3)中重结晶所用溶剂为无水四氢呋喃和正己烷混合溶剂，所述重结晶次数优选为3次。
本发明的第二方面，涉及根据上述所述的方法获得的杂化水凝胶材料。
本发明所述的杂化水凝胶材料制备原料包括聚多肽-聚乙二醇-聚多肽和鱼精DNA。
优选的，所述聚多肽-聚乙二醇-聚多肽为聚赖氨酸-聚乙二醇-聚赖氨酸；所述聚赖氨酸-聚乙二醇-聚赖氨酸与鱼精DNA的电荷摩尔比例为1:(10-20)；进一步优选的，所述聚赖氨酸-聚乙二醇-聚赖氨酸与鱼精DNA的电荷摩尔比例为1:(14-18)；最优选的，所述聚赖氨酸-聚乙二醇-聚赖氨酸与鱼精DNA的电荷摩尔比例为1:16。
本发明所述的聚赖氨酸-聚乙二醇-聚赖氨酸的制备原料包括引发剂和Lys(Z)-NCA。
优选的，所述原料中引发剂与Lys(Z)-NCA的质量比为1:(3-20)；进一步优选的，所述原料中引发剂与Lys(Z)-NCA的质量比为1:(8-20)；最优选的，所述原料中引发剂与 Lys(Z)-NCA的质量比为1:10。
在本发明的一个实施例中，所述引发剂为苯丙氨酸端基化聚乙二醇。
优选的，所述杂化水凝胶材料的制备原料中还包括3-7倍当量的三氟乙酸；进一步优选为5倍当量的三氟乙酸。
本发明所述引发剂的制备原料包括以下组分和质量含量：
Figure PCTCN2018086241-appb-000005
优选的，所述引发剂的制备原料包括以下组分和质量含量：
Figure PCTCN2018086241-appb-000006
在本发明的一个实施例中，所述引发剂的制备原料包括以下组分和质量含量：
Figure PCTCN2018086241-appb-000007
本发明所述Lys(Z)-NCA由L-赖氨酸(H-Lys(Z)-OH)与三光气反应制得，其中：
H-Lys(Z)-OH                  0.8g，2.85mmol-1.5g，5.35mmol
三光气                       0.339g，1.14mmol-0.635g，2.14mmol
优选的，由1.00g，3.57mmol的H-Lys(Z)-OH与0.42g，1.43mmol的三光气反应制得。
本发明的第三方面，涉及来源于上述所述的方法或所述的杂化水凝胶材料在组织工程、医用材料及药物负载方面的应用。
本发明所述的二氯甲烷(H 2CCl 2)、四氢呋喃(THF)和正己烷经高纯氮鼓泡除氧再利用活性氧化铝柱干燥后使用。
本发明所述的杂化超分子水凝胶材料既具有共聚物的生物降解性和DNA的结构单元有序、可控，又具有瞬态网络结构的特征，同时解决了传统使用光引发剂和化学交联剂带来的毒性；同时采用本发明的方法制备杂化超分子水凝胶材料，成本低、简便、高效、形成条件温和并表现出响应性、可逆性和自愈性等特点，利用静电作用构建超分子网络结构的策略，在制备更加复杂的功能化材料方面具有潜在的应用价值。
附图说明
以下，结合附图来详细说明本发明的实施例，其中：
图1：NH 2-Phe-PEG 227-Phe-NH 2的合成过程；
图2：Lys(Z)-NCA的合成过程；
图3：聚赖氨酸-聚乙二醇-聚赖氨酸(PLL-b-PEG-b-PLL)三嵌段共聚物的合成过程；
图4：核磁氢谱图，其中，A为PZLL-Phe-PEG-Phe-PZLL的核磁氢谱图，B为PLL-Phe-PEG-Phe-PLL的核磁氢谱图；
图5：NH 2-Phe-PEG 227-Phe-NH 2与引发聚合产物PZLL-Phe-PEG 227-Phe-PZLL体积排阻色谱(SEC)的表征；
图6：三嵌段共聚物PLL-b-PEG-b-PLL与鱼精DNA形成超分子凝胶结构示意图，其中，A：三嵌段共聚物与阴离子DNA利用静电相互作用形成偶联结合位点；B：形成空间网络结构的杂化水凝胶材料的过程；C：稳定的水凝胶结构。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本申请实施例所用的材料、仪器：
L-赖氨酸：H-Lys(Z)-OH上海吉尔生化，99％；
N-叔丁氧羰基-L-苯丙氨酸：Boc-Phe-OH，上海吉尔生化，99％；
N,N'-二环己基碳二亚胺：DCC，上海吉尔生化，99％；
三光气：上海吉尔生化，99％；
4-二甲氨基吡啶：DMAP，阿拉丁，99％；
三氟乙酸：TFA，阿拉丁，99.5％；
鱼精DNA(2000bp)、聚乙二醇(HO-PEG-OH，M n＝10 000g/mol)：Sigma-Aldrich；
去离子水(电阻率＞18MΩ·cm -1)：Millipore Milli-Q纯化。
实施例1
1、原料
1.1合成大分子引发剂NH 2-Phe-PEG 227-Phe-NH 2的原料
Figure PCTCN2018086241-appb-000008
1.2合成Lys(Z)-NCA的原料
H-Lys(Z)-OH                        1g，3.57mmol
三光气                             0.42g，1.43mmol
1.3合成聚赖氨酸-聚乙二醇-聚赖氨酸的原料
NH 2-Phe-PEG 227-Phe-NH 2              1g，0.1mmol
Lys(Z)-NCA                         0.31g，1mmol
1.4合成杂化水凝胶的原料
聚赖氨酸带正电荷的氨基与DNA结构中带负电荷的磷酸基团的电荷浓度分别控制在20mM，三嵌段共聚物与鱼精DNA的电荷摩尔比例1:16。
2、制备方法
2.1合成苯丙氨酸端基化聚乙二醇(NH 2-Phe-PEG 227-Phe-NH 2)
苯丙氨酸端基化聚乙二醇(NH 2-Phe-PEG 227-Phe-NH 2)的合成过程如图1所示，具体步骤如下：
第一步：聚乙二醇(HO-PEG 227-OH)与N-叔丁氧羰基-L-苯丙氨酸(Boc-Phe-OH)进行偶联反应，得到偶联物；
将HO-PEG 227-OH与Boc-Phe-OH冰水浴中溶解于200mL二氯甲烷中，向其中加入DCC和DMAP后0℃的冰水浴中反应2h，进一步将反应体系升温到室温下反应过夜；
第二步：过滤除杂，得到粗产物；
通过过滤的方法除去产生的不溶性副产物N,N'-二环乙基脲(DCU)，并利用冷却过滤的方法再进一步除去残余的DCU后得到粗产物。
第三步：将粗产物脱去保护基团叔丁氧羰基，洗涤，得到苯丙氨酸端基化聚乙二醇，所述苯丙氨酸端基化聚乙二醇为两端修饰有伯胺基的大分子引发剂的聚乙二醇。
在粗产物中加入5倍当量的三氟乙酸脱去末端的保护基团(Boc)，再依次用饱和NaHCO 3、H 2O、HCl和饱和NaCl洗涤分液，得到NH 2-Phe-PEG 227-Phe-NH 2。
2.2合成Lys(Z)-NCA
Lys(Z)-NCA的合成过程如图2所示，具体步骤如下：
将原料H-Lys(Z)-OH和三光气于无水四氢呋喃(THF)溶剂中、50℃反应至澄清后再继续反应一个小时；反应完全后得到α-赖基酸-N-羧基-环内酸酐(NCA)粗产品；将获得的NCA单体粗产物置于手套箱中，经THF与正己烷混合溶剂重结晶纯化三次后得最终的Lys(Z)-NCA产物。
2.3合成聚赖氨酸-聚乙二醇-聚赖氨酸(PLL-b-PEG-b-PLL)三嵌段共聚物
聚赖氨酸-聚乙二醇-聚赖氨酸的合成过程如图3所示，具体步骤如下：
将NH 2-Phe-PEG 227-Phe-NH 2在50℃油浴真空干燥4h后冷却到室温，并将Lys(Z)-NCA在手套箱中溶解于干燥DMF中，然后用注射器抽取后转移到上述反应体系中，得混合液；将混合液氮气保护、40℃下反应三天，利用FT-IR光谱确认反应完全得到粗产物，取少量 粗产物配制成5mg/mL进行SEC/LLS测试，确定多分散性(PDI)与分子量，并利用产物的 1H-NMR相应特征吸收峰面积比例关系，计算得到引发聚合的两端聚氨基酸嵌段的聚合度DP n为4；加入大量乙醚沉淀后，用少量三氟乙酸(TFA)溶解粗产物，加入5倍当量的氢溴酸/冰醋酸(HBr/CH 3COOH)于冰水浴中反应4h(图3)，后加入过量的乙醚沉淀。最后将沉淀产物用稀HCl调节适当pH值，使其溶解于水中进行透析，透析液冷冻干燥后得到产物聚赖氨酸-聚乙二醇-聚赖氨酸。通过 1H-NMR确认脱保护基团叔丁氧羰基反应进行完全，最终得到产物PLL 4-b-PEG 227-b-PLL 4。其中体积排除色谱/激光散射(SEC/LLS)使用SSI泵连接到Wyatt Optilab DSP和Wyatt DAWN EOS激光散射检测器，以0.02M LiBr的DMF溶液作为流动相，流速为1.0mL/min，测量温度为50℃，样品浓度大约为5mg/mL。
2.4合成杂化水凝胶
将鱼精DNA和2.3制备的三嵌段共聚物PLL 4-b-PEG 45-b-PLL 4分别溶解于水中，按照DNA磷酸二酯键中带负电荷的磷酸基团与三嵌段共聚物中聚赖氨酸侧链质子化的氨基正电荷，按两者之间的电荷摩尔比例混合进行混合后，常温下静置过夜，利用翻转样品管的方法，确定形成杂化水凝胶结构的最低成胶质量浓度。
3、结果
关于对PEG末端羟基进行改性的策略，不仅具有合成方法简单高效的特点，而且改性后末端苯丙氨酸侧基上的苄基在 1H-NMR中具有特征的化学位移，从而使端基改性后的产物易于进行表征。在NH 2-Phe-PEG 227-Phe-NH 2的合成过程中，得到粗产物产率为80％，去除保护基叔丁氧羰基后得到的NH 2-Phe-PEG 227-Phe-NH 2产物产率为75％。
通过开环聚合的方法，以NH 2-Phe-PEG 227-Phe-NH 2作为大分子引发剂，在一定条件下，合成出三嵌段共聚物PZLL-Phe-PEG-Phe-PZLL。利用Bruker AV400 FT-NMR核磁共振仪测定 1H-NMR谱图(图4A)中化学位移3.3-3.8的PEG亚甲基单元与化学位移4.9-5.1聚赖氨酸嵌段侧链苄基中亚甲基吸收峰面积的比例，估算PZLL嵌段的聚合度，并由于PEG两端的伯胺基引发NCA开环聚合的几率相等，因此可以推断合成的PZLL-b-PEG-b-PZLL三嵌段共聚物中两端PZLL嵌段的聚合度为4个单元左右。从 1H-NMR(图4B)表征结果 可以看出，PZLL侧链的苄氧羰基中亚甲基的吸收峰(化学位移4.9-5.1)，在上述脱保护反应后完全消失，最终确定了我们成功合成得到两端各含有4个赖氨酸嵌段的三嵌段共聚物PLL 4-b-PEG 227-b-PLL 4。通过以0.02mol/L LiBr DMF溶液为流动相的SEC/LLS获得的分子量分布PDI＝1.08(如图5)，证明上述合成得到的聚合物具有较好的多分散性。
得到的三嵌段共聚物PLL-b-PEG 227-b-PLL具有如下特点：首先该聚合物中间嵌段为水溶性且生物相容性较好的聚乙二醇链段，并且该PEG的链段较长(227个单元)，使其在构筑杂化材料时具有良好的柔性；另外该三嵌段共聚物两端为生物可降解性的聚赖氨酸嵌段，有利于其在生物医用材料方面的应用。
使用英国Malvern公司Nano ZS ZEN3600型Zeta电位粒度仪侧量Zeta电位，结果证明，20mg/mL的上述三嵌段共聚物水溶液的Zeta电位为+10mV左右，证明该三嵌段共聚物中两侧聚赖氨酸嵌段在适当的pH的条件下带有一定的正电荷。该三嵌段共聚物与阴离子DNA利用静电相互作用形成偶联结合位点如图6A所示，同时三嵌段共聚物中柔性且链段长度较长的PEG 227能够作为桥链结构，将聚氨基酸与DNA形成的偶联位点连接起来，最终形成具有空间网络结构的杂化水凝胶材料(图6B)，同时两种组分总的质量浓度为3％左右的情况下，形成的稳定水凝胶结构如图6C所示。
实施例2
1、原料
1.1合成大分子引发剂NH 2-Phe-PEG 227-Phe-NH 2的原料
Figure PCTCN2018086241-appb-000009
1.2合成Lys(Z)-NCA的原料
H-Lys(Z)-OH                       0.8g,2.85mmol
三光气                            0.339g,1.14mmol
1.3合成聚赖氨酸-聚乙二醇-聚赖氨酸的原料
NH 2-Phe-PEG 227-Phe-NH 2             0.8g,0.08mmol
Lys(Z)-NCA                        0.49g,1.6mmol
1.4合成杂化水凝胶的原料
聚赖氨酸带正电荷的氨基与DNA结构中带负电荷的磷酸基团的电荷浓度分别控制在20mM，三嵌段共聚物与鱼精DNA的电荷摩尔比例1:10。
2、制备方法
2.1合成苯丙氨酸端基化聚乙二醇(NH 2-Phe-PEG 227-Phe-NH 2)的方法同实施例1。
2.2合成Lys(Z)-NCA
Lys(Z)-NCA的合成过程如图2所示，具体步骤如下：
将原料H-Lys(Z)-OH和三光气于无水四氢呋喃(THF)溶剂中、45℃反应至澄清后再继续反应1.5小时；反应完全后得到α-赖基酸-N-羧基-环内酸酐(NCA)粗产品；将获得的NCA单体粗产物置于手套箱中，经THF与正己烷混合溶剂重结晶纯化三次后得最终的Lys(Z)-NCA产物。
2.3合成聚赖氨酸-聚乙二醇-聚赖氨酸(PLL-b-PEG-b-PLL)三嵌段共聚物
聚赖氨酸-聚乙二醇-聚赖氨酸的合成过程如图3所示，具体步骤如下：
将NH 2-Phe-PEG 227-Phe-NH 2在55℃油浴真空干燥4h后冷却到室温，并将Lys(Z)-NCA在手套箱中溶解于干燥DMF中，然后用注射器抽取后转移到上述反应体系中，得混合液；将混合液氮气保护、45℃下反应三天，利用FT-IR光谱确认反应完全得到粗产物，取少量粗产物配制成5mg/mL进行SEC/LLS测试，确定多分散性(PDI)与分子量，并利用产物的 1H-NMR相应特征吸收峰面积比例关系，计算得到引发聚合的两端聚氨基酸嵌段的聚合度DP n为6；加入大量乙醚沉淀后，用少量三氟乙酸(TFA)溶解粗产物，加入5倍当量的氢溴酸/冰醋酸(HBr/CH 3COOH)于冰水浴中反应4h(图3)，后加入过量的乙醚沉淀。最后将沉淀产物用稀HCl调节适当pH值，使其溶解于水中进行透析，透析液冷冻干燥后得到产物聚赖氨酸-聚乙二醇-聚赖氨酸。通过 1H-NMR确认脱保护反应进行完全，最终得 到产物PLL 6-b-PEG 227-b-PLL 6。
2.4合成杂化水凝胶的方法同实施例1。
3、结果
在NH 2-Phe-PEG 227-Phe-NH 2的合成过程中，得到粗产物产率为62％，去除保护基叔丁氧羰基后得到的NH 2-Phe-PEG 227-Phe-NH 2产物产率为57％。
应用 1H-NMR表征确定了我们成功合成得到两端各含有6个赖氨酸嵌段的三嵌段共聚物PLL 6-b-PEG 227-b-PLL 6，并具有很好的多分散性。
在通过三嵌段共聚物与DNA静电合成水凝胶材料时，两种组分总的质量浓度为2.5％左右的情况下，形成的稳定水凝胶结构。
实施例3
1、原料
1.1合成大分子引发剂NH 2-Phe-PEG 227-Phe-NH 2的原料
Figure PCTCN2018086241-appb-000010
1.2合成Lys(Z)-NCA的原料
H-Lys(Z)-OH                       1.5g,5.35mmol
三光气                            0.635g,2.14mmol
1.3合成聚赖氨酸-聚乙二醇-聚赖氨酸的原料
NH 2-Phe-PEG 227-Phe-NH 2             1.2g,0.12mmol
Lys(Z)-NCA                        0.294g,0.96mmol
1.4合成杂化水凝胶的原料
聚赖氨酸带正电荷的氨基与DNA结构中带负电荷的磷酸基团的电荷浓度分别控制在20mM，三嵌段共聚物与鱼精DNA的电荷摩尔比例1:20。
2、制备方法
2.1合成苯丙氨酸端基化聚乙二醇(NH 2-Phe-PEG 227-Phe-NH 2)的方法同实施例1。
2.2合成Lys(Z)-NCA
Lys(Z)-NCA的合成过程如图2所示，具体步骤如下：
将原料H-Lys(Z)-OH和三光气于无水四氢呋喃(THF)溶剂中、55℃反应至澄清后再继续反应0.5小时；反应完全后得到α-赖基酸-N-羧基-环内酸酐(NCA)粗产品；将获得的NCA单体粗产物置于手套箱中，经THF与正己烷混合溶剂重结晶纯化三次后得最终的Lys(Z)-NCA产物。
2.3合成聚赖氨酸-聚乙二醇-聚赖氨酸(PLL-b-PEG-b-PLL)三嵌段共聚物
聚赖氨酸-聚乙二醇-聚赖氨酸的合成过程如图3所示，具体步骤如下：
将NH 2-Phe-PEG 227-Phe-NH 2在45℃油浴真空干燥4h后冷却到室温，并将Lys(Z)-NCA在手套箱中溶解于干燥DMF中，然后用注射器抽取后转移到上述反应体系中，得混合液；将混合液氮气保护、35℃下反应三天，利用FT-IR光谱确认反应完全得到粗产物，取少量粗产物配制成5mg/mL进行SEC/LLS测试，确定多分散性(PDI)与分子量，并利用产物的 1H-NMR相应特征吸收峰面积比例关系，计算得到引发聚合的两端聚氨基酸嵌段的聚合度DP n为3；加入大量乙醚沉淀后，用少量三氟乙酸(TFA)溶解粗产物，加入5倍当量的氢溴酸/冰醋酸(HBr/CH 3COOH)于冰水浴中反应4h(图3)，后加入过量的乙醚沉淀。最后将沉淀产物用稀HCl调节适当pH值，使其溶解于水中进行透析，透析液冷冻干燥后得到产物聚赖氨酸-聚乙二醇-聚赖氨酸。通过 1H-NMR确认脱保护基团叔丁氧羰基反应进行完全，最终得到产物PLL 3-b-PEG 227-b-PLL 3。
2.4合成杂化水凝胶的方法同实施例1。
3、结果
在NH 2-Phe-PEG 227-Phe-NH 2的合成过程中，得到粗产物产率为73％，去除保护基叔丁氧羰基后得到的NH 2-Phe-PEG 227-Phe-NH 2产物产率为64％。
应用 1H-NMR表征确定了我们成功合成得到两端各含有3个赖氨酸嵌段的三嵌段共聚 物PLL 3-b-PEG 227-b-PLL 3，并具有很好的多分散性。
在通过三嵌段共聚物与DNA静电合成水凝胶材料时，两种组分总的质量浓度为4％左右的情况下，形成的稳定水凝胶结构。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (10)

  1. 一种杂化水凝胶材料的制备方法，其特征在于，由聚多肽-聚乙二醇-聚多肽三嵌段共聚物与鱼精DNA以1:(10-20)的电荷摩尔比通过静电结合制备而成。
  2. 根据权利要求1所述的方法，其特征在于，所述的聚多肽-聚乙二醇-聚多肽三嵌段共聚物为聚赖氨酸-聚乙二醇-聚赖氨酸。
  3. 根据权利要求2所述的方法，其特征在于，所述聚赖氨酸-聚乙二醇-聚赖氨酸三嵌段共聚物合成步骤如下：
    1)将苯丙氨酸端基化聚乙二醇油浴、干燥、冷却；
    2)将Lys(Z)-NCA溶解于DMF中；
    3)将步骤2)中溶解的Lys(Z)-NCA转移至步骤1)中的产物中，获得混合液；
    4)确认步骤3)中的混合液反应完全后，沉淀，获得初步处理物，将初步处理物水解；
    5)调节pH值、透析、干燥得到聚赖氨酸-聚乙二醇-聚赖氨酸产物。
  4. 根据权利要求3所述的方法，其特征在于，所述聚赖氨酸-聚乙二醇-聚赖氨酸的结构式如下：
    Figure PCTCN2018086241-appb-100001
    其中，所述结构式中n为3～6。
  5. 根据权利要求3所述的方法，其特征在于，所述苯丙氨酸端基化聚乙二醇合成步骤如下：
    1)聚乙二醇与N-叔丁氧羰基-L-苯丙氨酸进行偶联反应，得到偶联物；
    2)所述步骤1)中反应完全后，过滤除杂，得到粗产物；
    3)将步骤2)中的粗产物脱去保护基团叔丁氧羰基，洗涤，得到苯丙氨酸端基化聚乙二醇。
  6. 根据权利要求5所述的方法，其特征在于，所述苯丙氨酸端基化聚乙二醇的结构式如下：
    Figure PCTCN2018086241-appb-100002
  7. 根据权利要求3所述的方法，其特征在于，所述Lys(Z)-NCA合成步骤如下：
    1)将原料H-Lys(Z)-OH和三光气溶解于溶剂中；
    2)步骤1)中反应完全后得到α-赖基酸-N-羧基-环内酸酐粗产品；
    3)将步骤2)中获得的粗产品重结晶得到Lys(Z)-NCA产物。
  8. 根据权利要求7所述的方法，其特征在于，所述Lys(Z)-NCA的结构式如下：
    Figure PCTCN2018086241-appb-100003
  9. 根据权利要求1-7任一项所述的方法获得的杂化水凝胶材料。
  10. 权利要求9所述的杂化水凝胶材料在组织工程、医用材料及药物负载方面的应用。
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