WO2023240505A1 - 一种l-4-二羟基硼苯丙氨酸-n-羧酸内酸酐单体和聚氨基酸及其制备方法和应用 - Google Patents

一种l-4-二羟基硼苯丙氨酸-n-羧酸内酸酐单体和聚氨基酸及其制备方法和应用 Download PDF

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WO2023240505A1
WO2023240505A1 PCT/CN2022/099010 CN2022099010W WO2023240505A1 WO 2023240505 A1 WO2023240505 A1 WO 2023240505A1 CN 2022099010 W CN2022099010 W CN 2022099010W WO 2023240505 A1 WO2023240505 A1 WO 2023240505A1
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block copolymer
dihydroxyborophenylalanine
cur
carboxylic acid
sor
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PCT/CN2022/099010
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English (en)
French (fr)
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邓超
章强
刘媛媛
谢吉国
钟志远
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苏州大学
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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
    • A61K47/30—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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • 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
    • C08G69/42—Polyamides containing atoms other than carbon, hydrogen, oxygen, and nitrogen

Definitions

  • the invention constructs a polypeptide nanomaterial with bioresponsiveness and good biocompatibility, specifically involving the synthesis of L-4-dihydroxyborophenylalanine N -carboxylic acid anhydride and its ring opening Polymerization prepares a range of polymers and has applications in drug delivery.
  • Polypeptides are widely used in drug delivery, tissue engineering and other fields due to their unique secondary structure and biocompatibility.
  • existing polypeptide materials usually have problems such as insufficient functionality, complicated introduction of functional groups, and harsh preparation conditions.
  • polypeptide-based nanomedicines can improve drug solubility and pharmacokinetics to a certain extent, reduce toxicity to normal cells, and broaden the therapeutic window, they still suffer from poor stability and uncontrollable drug release (i.e., drug release during circulation). limitations such as premature release and slow drug release from tumor sites). Therefore, the construction of polypeptide nanomedicine carriers with good biocompatibility and bioresponsiveness (reduction responsiveness, pH responsiveness, ROS responsiveness or enzyme responsiveness, etc.) for release has become a research hotspot.
  • the present invention designs and synthesizes an L-4-dihydroxyborophenylalanine N -carboxylic acid anhydride monomer, and uses polyethylene glycol to initiate the L-4-dihydroxyborophenylalanine N -carboxylic acid anhydride monomer.
  • polyethylene glycol to initiate the L-4-dihydroxyborophenylalanine N -carboxylic acid anhydride monomer.
  • other types of N -carboxylic acid anhydride monomer ring-opening polymerization to prepare a series of block copolymers, these block polymers can self-assemble to form polymer micelles for drug delivery.
  • the technical solution adopted by the present invention is: a kind of L-4-dihydroxyborophenylalanine N -carboxylic acid anhydride with the structure of formula I: .
  • R 1 comes from the initiator polyethylene glycol and is its terminal functional group, preferably , , , or etc.;
  • R2 comes from other types of amino acids, preferably different types of amino acids are L-tyrosine, N ⁇ -Boc-L-lysine, ⁇ -benzyl-L-aspartic acid;
  • m is 70 to 210 , x ranges from 5 to 30, y ranges from 0 to 15, and n ranges from 5 to 45.
  • linear polyethylene glycol initiates the ring-opening polymerization of L-4-dihydroxyborophenylalanine N -carboxylic acid anhydride to form polyethylene glycol.
  • Glycol-poly(L-4-dihydroxyborophenylalanine) copolymer when y is not 0, it is linear polyethylene glycol initiated L-4-dihydroxyborophenylalanine N -carboxylic internal anhydride Ring-opening polymerization with other types of N -carboxylic acid anhydrides to form polyethylene glycol-poly(L-4-dihydroxyborophenylalanine)-poly other types of amino acid copolymers; other types of amino acids refer to copolymers with poly(L-4 -dihydroxyborophenylalanine) different amino acids, such as L-tyrosine, N ⁇ -Boc-L-lysine, ⁇ -benzyl-L-aspartic acid, etc.
  • the polyethylene glycol-poly(L-4-dihydroxyborophenylalanine) copolymer is as formula III: .
  • R 3 comes from the initiator polyethylene glycol and is its terminal functional group, preferably , , , or etc.; m is 70-210, n is 5-70; preferably m is 90-150, n is 10-30.
  • a branched block copolymer with a formula IV structure is .
  • m is 20 to 150
  • n is 2 to 20
  • x is 2 to 8; preferably m is 50 to 120, n is 4 to 8, x is 4 or 8;
  • R 4 is initiator branched polyethylene glycol
  • four-arm or eight-arm polyethylene glycol ammonia is preferred as the initiator in the present invention.
  • the invention discloses a preparation method of the above-mentioned L-4-dihydroxyborophenylalanine- N -carboxylic acid anhydride, which uses L-4-dihydroxyborophenylalanine, ⁇ -pinene and triphosgene as reactants, React in anhydrous tetrahydrofuran to prepare the L-4-dihydroxyborophenylalanine- N -carboxylic acid anhydride.
  • the specific reaction process is as follows: add L-4-dihydroxyborophenylalanine and ⁇ -pinene to anhydrous tetrahydrofuran solution under nitrogen protection environment, then dropwise add triphosgene tetrahydrofuran solution at 50-60°C The reaction lasted for 3 to 7 hours. After the reaction was completed, the reactants that did not participate in the reaction were filtered out, and the reaction liquid was concentrated by rotary evaporation and then precipitated in ice petroleum ether to obtain a crude product.
  • the molar ratio of L-4-dihydroxyborophenylalanine, ⁇ -pinene and triphosgene is 2:4-6:1-2, preferably 2:5:1.
  • the present invention uses linear polyethylene glycol as the initiator, L-4-dihydroxyborophenylalanine N -carboxylic acid anhydride as the monomer, or L-4-dihydroxyborophenylalanine N -carboxylic acid anhydride and Other types of N -carboxylic acid anhydrides are monomers that undergo ring-opening polymerization to obtain linear block copolymers.
  • branched polyethylene glycol as the initiator and L-4-dihydroxyborophenylalanine N -carboxylic acid anhydride as the monomer
  • ring-opening polymerization is performed to obtain a branched block copolymer.
  • organic solvents such as N , N -dimethylformamide (DMF), dichloromethane (DCM), chloroform, and tetrahydrofuran (THF).
  • N , N -dimethylformamide (DMF) is preferred as the solvent;
  • the ring-opening polymerization temperature is 30 to 90°C, and the preferred polymerization temperature in this invention is 80°C;
  • the polymerization reaction time is 1 to 5 days, and the preferred reaction time in this invention is for 3 days.
  • the invention discloses a targeting block copolymer, which is obtained by coupling a linear block copolymer with a formula II structure to a targeting molecule, or a branched block copolymer with a formula IV structure coupled with a targeting molecule.
  • a targeting block copolymer which is obtained by coupling a linear block copolymer with a formula II structure to a targeting molecule, or a branched block copolymer with a formula IV structure coupled with a targeting molecule.
  • either the PEG end or the amino acid end can be chemically coupled to specific targeting molecules, including short peptides (ApoE, Angiopep-2, cRGD, cNGQ, etc.), small molecule targeting molecules (glucose, folic acid, etc.) etc.), antibodies and antibody fragments, etc.
  • the invention further discloses the application of the above-mentioned block copolymer, especially the self-assembly of the amphiphilic polymer polyethylene glycol-poly(L-4-dihydroxyborophenylalanine) into nanomicelles in drug delivery;
  • the drug is a hydrophobic chemical drug, a drug containing cis-1,2 or 1,3-diol, a peptide drug, a protein drug or a nucleic acid drug.
  • the drug is curcumin (Cur) and/or sorafil tosylate. Sor.
  • the above-mentioned block copolymer especially the application of the amphiphilic polymer polyethylene glycol-poly(L-4-dihydroxyborophenylalanine) in the preparation of drug carriers.
  • the polymer of the present invention has a suitable hydrophilic/hydrophobic ratio
  • a series of polymer micelles with controllable sizes can be prepared by a solvent replacement method.
  • the polymer micelles are prepared by a solvent replacement method, that is, block copolymerization.
  • the material is dissolved in dimethyl sulfoxide (DMSO) or N , N -dimethylformamide (DMF) to obtain a polymer solution, and then the polymer solution is added drop by drop to a buffer solution such as HEPES or PB, and finally The organic solvent was removed by dialysis using a dialysis bag with a molecular weight cutoff (MWCO) of 7000 to obtain polymer micelles with a particle size of 18 to 200 nm.
  • MWCO molecular weight cutoff
  • the present invention can prepare specifically targeted polymer micelles by adjusting the ratio of targeting block copolymers and non-targeting block copolymers.
  • targeting molecules can also be introduced to the surface of prepared non-targeting polymer micelles through post-modification, such as introducing short peptides (ApoE, Angiopep-2, cRGD, cNGQ, etc.) at the PEG end of the micelles.
  • Small molecule targeting molecules glucose, folic acid, etc.
  • antibodies and antibody fragments etc.
  • the present invention has the following advantages compared with the prior art: the ⁇ -amino acid- N -carboxylic acid anhydride prepared by the invention is a new type of internal acid anhydride monomer, which can be obtained by ring-opening polymerization with controllable properties. Polypeptides, this type of polyamino acids have better biological responsiveness and wider applicability than other polyamino acids.
  • the present invention uses polyethylene glycol ammonia as the initiator and obtains polymers with controllable molecular weight and narrow molecular weight distribution through ring-opening polymerization, which greatly broadens the types of amphiphilic polypeptides.
  • the polymer disclosed in the invention has excellent biocompatibility, can prepare (tumor targeting) polymer micelles, and can be suitable for hydrophilic and hydrophobic chemical drugs, cis-1,2 or 1,3-diol-containing drugs, and polypeptides Efficient loading and delivery of drugs, protein drugs, nucleic acid drugs, etc.
  • Boron-10-rich L-4-dihydroxyborophenylalanine is an important boron carrier for boron neutron capture therapy.
  • the synthesis process disclosed in the present invention is also applicable to the synthesis of boron-10-rich polypeptides.
  • the obtained polypeptide is also suitable for the above-mentioned micelle preparation process, which will broaden the application scope of L-4-dihydroxyborophenylalanine and is expected to be used as a new boron carrier for boron neutron capture therapy.
  • the preparation method of the invention is simple and the raw materials used come from a wide range of sources, thereby having good application prospects.
  • Figure 1 is a schematic diagram of the reactions of Example 2, Example 3 and Example 4.
  • Figure 2 is the hydrogen nuclear magnetic spectrum (A) and carbon nuclear magnetic spectrum (B) of BPA-NCA in Example 1, and the infrared monitoring chart (C) and PEG 5k - during the synthesis of PEG 5k -PBPA 4k in Example 2.
  • Figure 3 is the hydrogen nuclear magnetic spectrum (A) of PEG 5k -P (Tyr 1k -BPA 4k ), the hydrogen nuclear magnetic spectrum (B) of PEG 5k -P (BLA 1k -BPA 4k ), and PEG 5k - The hydrogen NMR spectrum of P(Lys 1k -BPA 4k ) (C) and the hydrogen NMR spectrum of 4-arm-PEG 20k -PBPA 3k (D).
  • Figure 4 is the hydrogen nuclear magnetic spectrum (A) of ApoE-PEG 5k -PBPA 4k and the hydrogen nuclear magnetic spectrum (B) of 4-arm-PEG 20k -PBPA 1.5k -Glucose in Example 4.
  • Figure 5 is the particle size distribution (embedded TEM image) of the micelle-encapsulated drugs Cur and Sor formed by the self-assembly of the polymer PEG-PBPA in Example 5 (A), dilution, serum and long-term storage stability (B) and In vitro responsiveness (C) and in vitro release (D).
  • Figure 6 is a graph showing the cytotoxicity results (A), free Cur and Sor of Example 6 hollow polymer micelles on L929 mouse fibroblasts, U87 MG-luc human brain glioma cells and B16F10 mouse melanoma cells.
  • A free Cur and Sor of Example 6 hollow polymer micelles on L929 mouse fibroblasts, U87 MG-luc human brain glioma cells and B16F10 mouse melanoma cells.
  • B the toxicity of single-loaded drug micelles PBN-Cur and PBN-Sor to U87 MG-luc cells
  • C curcumin and sorafenib tosylate on U87 Cytotoxicity
  • D long-term cell inhibition effect of MG-luc mouse melanoma cells
  • Figure 7 shows the hemolysis experiment (A) and hemolysis rate (B) of red blood cells treated with Cur, Sor, PBN-Cur, PBN-Sor, PBN-Cur/Sor (low) and PBN-Cur/Sor (high).
  • Figure 8 shows the apoptosis (A) of U87 MG-luc cells treated with Cur, Sor, PBN-Cur, PBN-Sor and PBN-Cur/Sor (Cur: 1.5 ⁇ g/mL, Sor: 1.5 ⁇ g/mL) and treated with Cur and cell cycle arrest of U87 MG-luc cells treated with PBN-Cur (Cur: 1.5 ⁇ g/mL) (B).
  • Figure 9 is the NMR spectrum of 10 BPA-NCA (A) and mPEG-P 10 BPA rich in boron-10 (B).
  • the present invention discloses for the first time L-4-dihydroxyborophenylalanine N -carboxylic acid anhydride, which complexes with cis 1,2 or 1,3-dihydroxy compounds as well as monosaccharides, polysaccharides, nucleic acids, etc. to form reversible
  • Five-membered or six-membered ring lactones or boron-nitrogen coordination are combined to construct carriers such as nanomedicines or hydrogels, which can achieve stable and efficient encapsulation of various drugs.
  • the formed borate ester bonds and boron-nitrogen coordination is a dynamic effect that can dissociate and release the entrapped drug under certain conditions (such as ROS, low pH), and can also interact with sialic acid highly expressed on the surface of tumor cells. Therefore, the present invention can be used in many fields such as biosensing and drug delivery.
  • BPA-NCA L-4-dihydroxyborophenylalanine- N -carboxylic acid anhydride
  • the invention also discloses a block copolymer ( Polyethylene glycol-poly(L-4-dihydroxyborophenylalanine) copolymer), in which the molecular weight of polyethylene glycol is 2000 to 40000, and the poly(L-4-dihydroxyborophenylalanine) The molecular weight is 1000 ⁇ 50000.
  • the linear block copolymer is prepared by linear polyethylene glycol initiating the ring-opening polymerization of the above-mentioned L-4-dihydroxyborophenylalanine- N -carboxylic acid anhydride monomer, and the chemical structural formula is as follows: .
  • m is 70 to 210, n is 5 to 70; preferably m is 90 to 150, n is 10 to 30; R 3 is the terminal functional group of the polyethylene glycol initiator, and polyethylene glycol is preferred in the present invention.
  • Ammonia initiator the molecular formula is as follows: .
  • R 3 is preferably , , , or wait.
  • the branched polymer is prepared by ring-opening polymerization of the above-mentioned L-4-dihydroxyborophenylalanine- N -carboxylic acid anhydride monomer initiated by branched polyethylene glycol.
  • the chemical structural formula is as follows: .
  • m is 20 to 150
  • n is 2 to 20
  • x is 2 to 8; preferably m is 50 to 120, n is 4 to 8, x is 4 or 8;
  • R 2 is a branched polyethylene glycol initiator
  • the branching center of the agent, the present invention preferably uses the existing four-arm or eight-arm polyethylene glycol ammonia as the initiator.
  • Eight-arm polyethylene glycol ammonia .
  • the invention also discloses the method of using linear polyethylene glycol as an initiator to initiate the random copolymerization of the above-mentioned L-4-dihydroxyborophenylalanine- N -carboxylic acid anhydride monomer and other types of N -carboxylic acid anhydride monomers.
  • a block copolymer in which the molecular weight of polyethylene glycol is 2000 to 20000, and the molecular weight of poly(amino acid- co -L-4-dihydroxyborophenylalanine) is 1000 to 50000.
  • the amino acids here refer to other types Product obtained by polymerization of N -carboxylic acid anhydride monomer.
  • the block copolymer is prepared by random copolymerization of the above-mentioned L-4-dihydroxyborophenylalanine- N -carboxylic acid anhydride monomer and other types of N -carboxylic acid anhydride monomers using linear polyethylene glycol as an initiator.
  • the chemical structural formula is as follows: .
  • m is 70 to 210
  • x is 5 to 30, y is 0 to 15, n is 5 to 45
  • R 1 is the terminal functional group of the polyethylene glycol initiator, and ⁇ -methoxy- is preferred in the present invention.
  • ⁇ -amino-polyethylene glycol and ⁇ -maleimide- ⁇ -amino-polyethylene glycol are initiators;
  • R 2 is different types of amino acids, and L-tyrosine, N ⁇ -Boc- are preferred in the present invention.
  • N , N -dimethyl is preferred.
  • Formamide (DMF) is used as the solvent; the ring-opening polymerization temperature is 30 to 90°C, and the preferred polymerization temperature in the present invention is 80°C; the polymerization reaction time is 1 to 5 days, and the preferred reaction time in the present invention is 3 days.
  • Example 1 Synthesis of L-4-dihydroxyborophenylalanine- N -carboxylic acid anhydride (BPA-NCA): In a nitrogen atmosphere, L-4-dihydroxyborophenylalanine (500 mg, 2.39 mmol) and ⁇ -pinene (948 ⁇ L, 5.97 mmol) were added to a dry three-neck round-bottom flask, followed by anhydrous tetrahydrofuran (THF, 150 mL), and then the THF solution of triphosgene was added dropwise to the above In the suspension, the reaction system was placed in an oil bath at 55°C and stirred evenly.
  • BPA-NCA L-4-dihydroxyborophenylalanine- N -carboxylic acid anhydride
  • Example Preparation of diblock copolymer PEG-PBPA uses polyethylene glycol with an amino terminal as an initiator to initiate the ring-opening polymerization of L-4-dihydroxyborophenylalanine- N -carboxylic acid anhydride.
  • Polymers PEG-PBPA with different chain lengths can be prepared by adjusting the molar ratio of polyethylene glycol and L-4-dihydroxyborophenylalanine- N -carboxylic acid anhydride (Table 1).
  • BPA-based branched polymers were synthesized using 4/8-arm PEG- NH as initiator.
  • four-arm PEG-NH 2 (1.32 g, 0.066 mmol) dissolved in anhydrous DMF was added to a BPA-NCA (0.250 g, 1.06 mmol) solution under a nitrogen atmosphere, and the reaction was carried out at 80 °C for 24 h. .
  • PEG-P(Lys(Boc)- co -BPA), PEG-P(BLA- co -BPA) and 4-arm-PEG-PBPA also follows the above operation process.
  • PEG-P(Lys(Boc)- co -BPA) was deprotected in trifluoroacetic acid to obtain PEG-P (Lys- co -BPA).
  • the present invention uses maleimide-modified polyethylene glycol as an initiator to initiate the above-mentioned L-4-dihydroxyborane- N -carboxylic acid anhydride monomer and/or other types of N -carboxyl groups.
  • Internal anhydride monomers are used to obtain block polymers such as Mal-PEG-PBA or Mal-PEG-P (BPA- co -Tyr), and then their PEG terminals can be chemically coupled to specific targets such as ApoE, Angiopep-2, cRGD, etc. Molecule.
  • the NMR characterization of the ApoE-PEG-PBPA block copolymer is shown in Figure 4A.
  • the grafting efficiency of ApoE was measured as 92% by the 9,10-phenanthrenequinone method.
  • glucose molecules can be modified at the end of the polymer.
  • Figure 4B shows the NMR spectrum of the polymer modified with glucose. The yield is: 82%. Through NMR, it can be calculated that approximately 3.6 glucose molecules are modified into the polymer 4-arm-PEG- on PBPA.
  • the actual drug loading of Cur and Sor measured by HPLC reached 7.8 wt% and 9.8 wt%, respectively.
  • the polymer micelles have good stability under high dilution (50 times), 10% fetal bovine serum (FBS), and long-term storage at 4°C.
  • Example 6 MTT method to test the cytotoxicity of empty polymer micelles and drug-loaded micelles: The MTT method was used to evaluate the effects of empty polymer micelles PBN on tumor cells (B16F10 cells, U87 MG-luc cells) and normal mouse fibroblasts ( L929 cells) toxicity. Specifically: spread the cells into a 96-well plate (80 ⁇ L, 5 ⁇ 10 3 cells/well), incubate in a 37°C incubator for 24 hours, and then add 20 ⁇ L of PBN micelles with different concentration gradients to each well. solutions to give final concentrations in the wells of 0.05, 0.1, 0.2, 0.5, 0.75, and 1.0 mg/mL.
  • the cells were then digested with trypsin, and the cells of each sample group (300 cells/well) were re-seeded into a 6-well plate and placed in a cell culture incubator for 9 days, during which the culture medium was changed every three days. Finally, the culture medium was aspirated, washed twice with PBS, fixed with 4% paraformaldehyde solution for 15 minutes, and then stained with crystal violet staining solution. Finally, the crystal violet was slowly washed away and air dried.
  • both PBN-Cur (Cur: 1.5 ⁇ g/mL) and PBN-Sor (Sor: 1.5 ⁇ g/mL) have a certain long-term inhibitory effect on tumor cell proliferation, and the inhibitory effect of the PBN-Sor group is greater than Slightly better than the PBN-Cur group, the double-drug combination PBN-Cur/Sor group has the best inhibitory effect. This result is consistent with the MTT experimental results.
  • Example 7 Hemolysis results of drug-loaded micelles: In this experiment, deionized water was used as a positive control, 0.9% NaCl was used as a negative control, and Cur, Sor, PBN-Cur, PBN-Sor, and PBN-Cur/Sor (low) were studied. and hemolysis of PBN-Cur/Sor (high) (Fig. 7A). Drug concentration was set to 50 ⁇ g/mL, PBN-Cur/Sor (high; Cur: 110 ⁇ g/mL, Sor: 110 ⁇ g/mL) group.
  • Example 8 Apoptosis and cell cloning experiments of drug-loaded micelles:
  • the present invention uses Annexin V-FITC/Propidium iodine (PI) double-staining technology and uses flow cytometry to study Cur, Sor, PBN-Cur, and PBN-Sor. and the apoptotic effect of PBN-Cur/Sor on U87 MG-luc cells.
  • U87 MG-luc cells (2 ⁇ 10 5 cells/well) in logarithmic growth phase were inoculated into a 6-well plate and cultured in an incubator overnight.
  • Cur, Sor, PBN-Cur, PBN-Sor or PBN-Cur/Sor (Cur: 1.5 ⁇ g/mL, Sor: 1.5 ⁇ g/mL, Cur/Sor: 1.5/1.5 ⁇ g/mL) was incubated for 48 hours, then the medium was discarded and washed twice with PBS , add trypsin (without EDTA) to digest the cells, collect by centrifugation, wash twice with pre-cooled PBS at 4°C, then add 100 ⁇ L binding buffer to resuspend the cells (1 ⁇ 10 6 cells/mL) , transfer 100 ⁇ L of cell suspension into the flow tube, and add 5 ⁇ L of Annexin V-FITC staining solution and 10 ⁇ L of PI staining solution in sequence, mix evenly, and then stain at room temperature in the dark for 15 minutes, and finally add 400 ⁇ L of PBS.
  • Example 8 Synthesis of boron-10-rich 10 BPA-NCA and mPEG-P 10 BPA: The synthesis of 10 BPA-NCA and mPEG-P 10 BPA follows Example 1 and Example 2 respectively. The only difference is that during the monomer synthesis process, boron-10-rich BPA is used, which is characterized in Figure 9A&B.

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Abstract

本发明公开了一种L-4-二羟基硼苯丙氨酸-N-羧酸内酸酐单体和聚氨基酸及其制备方法和应用，构建了一种具有生物响应性和良好生物相容性的聚多肽纳米材料，具体涉及一种L-4-二羟基硼苯丙氨酸N-羧基内酸酐的合成，以及由其开环聚合制备的一系列聚合物和药物递送方面的应用。本发明公开的聚合物具有优异的生物相容性，可以制备（肿瘤靶向）聚合物胶束，可适用于亲疏水性化药、含顺式1,2或1,3-二醇药物、多肽药物、蛋白药物以及核酸药物等的高效装载与递送。

Description

一种L-4-二羟基硼苯丙氨酸-N-羧酸内酸酐单体和聚氨基酸及其制备方法和应用 技术领域
本发明构建了一种具有生物响应性和良好生物相容性的聚多肽纳米材料，具体涉及一种L-4-二羟基硼苯丙氨酸 N-羧基内酸酐的合成，以及由其开环聚合制备的一系列聚合物和药物递送方面的应用。
背景技术
聚多肽由于其独特的二级结构和生物相容性等特性使其在药物递送、组织工程等领域得到广泛应用。但现有的聚多肽材料通常存在功能性不足、功能性基团引入复杂繁琐、制备条件苛刻等问题。另外，基于聚多肽的纳米药物虽然在一定程度上可改善药物溶解度和药代动力学，降低对正常细胞的毒性，扩宽治疗窗口，但依然存在稳定性差和药物释放不可控（即循环期间药物过早释放和肿瘤部位药物释放缓慢）等局限性。因此，构建出具有良好的生物相容性和生物响应性（还原响应性、pH响应性、ROS响应性或酶响应性等）释放的聚多肽纳米药物载体成为研究的热点。
 技术问题
本发明设计合成了一种L-4-二羟基硼苯丙氨酸 N-羧基内酸酐单体，并用聚乙二醇引发L-4-二羟基硼苯丙氨酸 N-羧基内酸酐单体或/和其他类型 N-羧基内酸酐单体开环聚合制备系列嵌段共聚物，这些嵌段聚合物能自组装形成聚合物胶束用于药物递送。
技术解决方案
为达到上述目的，本发明采用的技术方案是：一种具有式I结构的L-4-二羟基硼苯丙氨酸 N-羧基内酸酐： 。
一种具有式II结构的线型嵌段共聚物： 。
其中，R 1来自引发剂聚乙二醇，为其末端官能基团，优选为 ， ， ， 或 等；R 2来自其他类型的氨基酸，优选不同类型的氨基酸为L-酪氨酸、 N ε-Boc-L-赖氨酸、 β-苄基-L-天冬氨酸；m为70～210，x为5～30，y为0～15，n为5～45。
本发明具有式II结构的线型嵌段共聚物中，y为0时，为线型聚乙二醇引发L-4-二羟基硼苯丙氨酸 N-羧基内酸酐开环聚合形成聚乙二醇-聚（L-4-二羟基硼苯丙氨酸）共聚物；y不为0时，为线型聚乙二醇引发L-4-二羟基硼苯丙氨酸 N-羧基内酸酐与其他类型 N-羧基内酸酐开环聚合形成聚乙二醇-聚（L-4-二羟基硼苯丙氨酸）-聚其他类型氨基酸共聚物；其他类型氨基酸是指与聚（L-4-二羟基硼苯丙氨酸）不同的氨基酸，比如L-酪氨酸、 N ε-Boc-L-赖氨酸、 β-苄基-L-天冬氨酸等。
本发明中，y为0时聚乙二醇-聚（L-4-二羟基硼苯丙氨酸）共聚物如式Ⅲ： 。
其中，R 3来自引发剂聚乙二醇，为其末端官能基团，优选为 ， ， ， 或 等；m为70～210，n为5～70；优选的m为90～150，n为10～30。
一种具有式Ⅳ结构的支化嵌段共聚物： 。
其中，m为20～150，n为2～20，x为2～8；优选的m为50～120，n为4～8，x为4或8；R 4为引发剂支化聚乙二醇的支化中心，本发明优选四臂或八臂聚乙二醇氨为引发剂。
本发明公开了上述L-4-二羟基硼苯丙氨酸- N-羧基内酸酐的制备方法，以L-4-二羟基硼苯丙氨酸、 α-蒎烯、三光气为反应物，在无水四氢呋喃中反应，制备得到所述的L-4-二羟基硼苯丙氨酸- N-羧基内酸酐。具体反应过程如下：在氮气保护环境下将L-4-二羟基硼苯丙氨酸和 α-蒎烯加入到无水四氢呋喃溶液中，然后滴加三光气的四氢呋喃溶液，在50～60℃下反应3～7小时，反应结束后滤掉没有参与反应的反应物，再将反应液旋蒸浓缩后在冰石油醚中沉淀得到粗产物。粗产品复溶于四氢呋喃，再次沉淀，重复进行2～3次，得到最终的白色粉末固体，即为L-4-二羟基硼苯丙氨酸- N-羧基内酸酐（BPA-NCA）。
上述技术方案中，L-4-二羟基硼苯丙氨酸、 α-蒎烯和三光气的摩尔比为2：4～6：1～2，优选2：5：1。
本发明以线性聚乙二醇为引发剂，L-4-二羟基硼苯丙氨酸 N-羧基内酸酐为单体，或者L-4-二羟基硼苯丙氨酸 N-羧基内酸酐与其他类型 N-羧基内酸酐为单体，开环聚合，得到线性嵌段共聚物。以支化聚乙二醇为引发剂，L-4-二羟基硼苯丙氨酸 N-羧基内酸酐为单体，开环聚合，得到支化嵌段共聚物。
上述线型或支化型嵌段共聚物的制备在有机溶剂如 N, N-二甲基甲酰胺（DMF）、二氯甲烷（DCM）、三氯甲烷、四氢呋喃（THF）中进行，本发明优选 N, N-二甲基甲酰胺（DMF）作溶剂；开环聚合的温度为30～90℃，本发明优选聚合温度为80℃；聚合反应时间为1～5天，本发明优选反应时间为3天。
本发明公开了一种靶向嵌段共聚物，由具有式Ⅱ结构的线型嵌段共聚物偶联靶向分子得到，或者由式Ⅳ结构的支化嵌段共聚物偶联靶向分子得到。本发明的嵌段聚合物中，PEG末端或氨基酸末端均可以化学偶联特异性靶向分子，包括短肽（ApoE、Angiopep-2、cRGD、cNGQ等）、小分子靶向分子（葡萄糖、叶酸等）、抗体及抗体片段等。
本发明进一步公开了上述嵌段共聚物，尤其是两亲性聚合物聚乙二醇-聚（L-4-二羟基硼苯丙氨酸）自组装成纳米胶束在药物递送方面的应用；药物为亲疏水性化药、含顺式1,2或1,3-二醇药物、多肽药物、蛋白药物或者核酸药物，优选的，药物为姜黄素（Cur）和/或甲苯磺酸索拉非尼（Sor）。具体地，上述嵌段共聚物，尤其是两亲性聚合物聚乙二醇-聚（L-4-二羟基硼苯丙氨酸）在制备药物载体中的应用。由于本发明聚合物具有合适的亲疏水比例，因此可以通过溶剂置换法制备得到一系列尺寸可控的聚合物胶束，具体的，聚合物胶束是通过溶剂置换法制备得到，即将嵌段共聚物溶于二甲基亚砜（DMSO）或 N, N-二甲基甲酰胺（DMF）中，得到聚合物溶液，然后将聚合物溶液逐滴滴加到HEPES或PB等缓冲溶液中，最后使用截留分子量（MWCO）为7000的透析袋透析除去有机溶剂得到粒径在18～200 nm的聚合物胶束。本发明可以通过调节靶向嵌段共聚物和非靶向嵌段共聚物的比例制备得到特异性靶向的聚合物胶束。此外，还可通过后修饰的方式将靶向分子引入到制备好的非靶向聚合物胶束表面，比如在胶束的PEG端引入短肽（ApoE、Angiopep-2、cRGD、cNGQ等），小分子靶向分子（葡萄糖、叶酸等），抗体及抗体片段等。
有益效果
由于上述技术方案的应用，本发明与现有技术相比具有下列优点：本发明制备的 α-氨基酸- N-羧基内酸酐为一种新型内酸酐单体，可以通过开环聚合得到性能可控的聚多肽，该类聚氨基酸相较于其他聚氨基酸具有更优异的生物响应性和更宽泛的适用性。
本发明利用聚乙二醇氨为引发剂、通过开环聚合得到分子量可控、分子量分布较窄聚合物，极大拓宽了两亲性聚多肽的种类。
本发明公开的聚合物具有优异的生物相容性，可以制备（肿瘤靶向）聚合物胶束，可适用于亲疏水性化药、含顺式1,2或1,3-二醇药物、多肽药物、蛋白药物以及核酸药物等的高效装载与递送。
富含硼-10的L-4-二羟基硼苯丙氨酸是硼中子俘获治疗重要的的硼携带剂，本发明公开的合成工艺同样适用于富含硼-10的聚多肽的合成，得到的聚多肽也同样适用于上述胶束的制备工艺，这将会拓宽L-4-二羟基硼苯丙氨酸的应用范围，有望用作硼中子俘获治疗的新型硼携带剂。
本发明制备方法简单，所用原料来源广泛，从而具有良好的应用前景。
 附图说明
图1是实施例二、实施例三、实施例四的反应示意图。
图2是实施例一中BPA-NCA的核磁氢谱图（A）、核磁碳谱图（B）、以及实施例二中PEG 5k-PBPA 4k合成时的红外监测图（C）和PEG 5k-PBPA 4k的核磁氢谱图（D）。
图3是实施例三中PEG 5k-P(Tyr 1k-BPA 4k)的核磁氢谱图（A）、PEG 5k-P(BLA 1k-BPA 4k)的核磁氢谱图（B）、PEG 5k-P(Lys 1k-BPA 4k)的核磁氢谱图（C）和4-arm-PEG 20k-PBPA 3k的核磁氢谱图（D）。
图4是实施例四中ApoE-PEG 5k-PBPA 4k的核磁氢谱图（A）、4-arm-PEG 20k-PBPA 1.5k-Glucose的核磁氢谱图（B）。
图5是实施例五中聚合物PEG-PBPA自组装形成的胶束包载药物Cur和Sor的粒径分布（内嵌TEM图）（A），稀释、血清以及长期存放稳定性（B）和体外响应性（C）和体外释放（D）。
图6是实施例六中空聚合物胶束对L929小鼠成纤维细胞、U87 MG-luc人脑胶质瘤细胞和B16F10小鼠黑色素瘤细胞和的细胞毒性结果图（A）、游离Cur和Sor以及单载药物胶束PBN-Cur和PBN-Sor对U87 MG-luc细胞的毒性（B）、聚合物胶束装载姜黄素和甲苯磺酸索拉非尼对U87 MG-luc小鼠黑色素瘤细胞的细胞毒性（C）和细胞长期抑制效果图（D）。
图7为Cur、Sor、PBN-Cur、PBN-Sor、PBN-Cur/Sor（低）和PBN-Cur/Sor（高）等处理红细胞的溶血实验（A）和溶血率（B）。
图8是Cur、Sor、PBN-Cur、PBN-Sor和PBN-Cur/Sor（Cur: 1.5μg/mL, Sor: 1.5μg/mL）处理的U87 MG-luc细胞凋亡（A）和用Cur和PBN-Cur（Cur: 1.5μg/mL）处理的U87 MG-luc细胞的细胞周期阻滞（B）。
图9是富含硼-10的 10BPA-NCA（A）和mPEG-P 10BPA核磁谱图（B）。
本发明的实施方式
本发明首次公开了L-4-二羟基硼苯丙氨酸 N-羧基内酸酐，其与顺式1,2或1,3-双羟基化合物以及单糖、多糖、核酸等络合形成可逆的五元或六元环内酯或硼氮配位作用结合，从而构建得到纳米药物或水凝胶等载体，能实现对各种药物的稳定高效包载，形成的硼酸酯键和硼氮配位作用是一种动态作用，在一定条件下（如ROS、低pH）能解离释放出包载的药物，还能与肿瘤细胞表面高表达的唾液酸作用。因此，本发明用于生物传感及药物递送等众多领域。
上述L-4-二羟基硼苯丙氨酸- N-羧基内酸酐（BPA-NCA）的制备方案可表示如下： 。
本发明还公开了用线型或支化型聚乙二醇为引发剂引发上述L-4-二羟基硼苯丙氨酸- N-羧基内酸酐单体开环聚合制备的嵌段共聚物（聚乙二醇-聚（L-4-二羟基硼苯丙氨酸）共聚物），其中聚乙二醇的分子量为2000～40000，聚（L-4-二羟基硼苯丙氨酸）的分子量为1000～50000。
线型嵌段共聚物通过线型聚乙二醇引发上述L-4-二羟基硼苯丙氨酸- N-羧基内酸酐单体开环聚合制备得到化学结构式如下所示： 。
其中，m为70～210，n为5～70；优选的m为90～150，n为10～30；R 3为聚乙二醇引发剂的末端官能基团，本发明优选聚乙二醇氨引发剂，分子式如下： 。
其中，R 3优选为 ， ， ， 或 等。
支化型聚合物是通过支化型聚乙二醇引发上述L-4-二羟基硼苯丙氨酸- N-羧基内酸酐单体开环聚合制备得到，化学结构式如下所示： 。
其中，m为20～150，n为2～20，x为2～8；优选的m为50～120，n为4～8，x为4或8；R 2为支化聚乙二醇引发剂的支化中心，本发明优选现有四臂或八臂聚乙二醇氨为引发剂。
四臂聚乙二醇氨： 。
八臂聚乙二醇氨： 。
本发明还公开了用线型聚乙二醇为引发剂引发上述L-4-二羟基硼苯丙氨酸- N-羧基内酸酐单体和其他类型 N-羧基内酸酐单体无规共聚制备的嵌段共聚物，其中聚乙二醇的分子量为2000～20000，聚（氨基酸- co-L-4-二羟基硼苯丙氨酸）的分子量为1000～50000，此处的氨基酸指其他类型 N-羧基内酸酐单体聚合得到的产物。由线型聚乙二醇为引发剂引发上述L-4-二羟基硼苯丙氨酸- N-羧基内酸酐单体和其他类型 N-羧基内酸酐单体无规共聚制备得到嵌段共聚物，化学结构式如下所示： 。
其中，m为70～210，x为5～30，y为0～15，n为5～45；R 1为聚乙二醇引发剂的末端官能基团，本发明优选 α-甲氧基- ω-氨基-聚乙二醇和 α-马来酰亚胺- ω-氨基-聚乙二醇为引发剂；R 2为不同类型的氨基酸，本发明优选L-酪氨酸、 N ε-Boc-L-赖氨酸、 β-苄基-L-天冬氨酸。
上述聚合物的制备过程在有机溶剂如 N, N-二甲基甲酰胺（DMF）、二氯甲烷（DCM）、三氯甲烷、四氢呋喃（THF）中进行，本发明优选 N, N-二甲基甲酰胺（DMF）作溶剂；开环聚合的温度为30～90℃，本发明优选聚合温度为80℃；聚合反应时间为1～5天，本发明优选反应时间为3天。
本发明的反应，参见图1，采用的原料都是市售产品，具体制备方法以及测试方法都为常规技术，下面结合附图以及实施例对本发明作进一步描述。
实施例一L-4-二羟基硼苯丙氨酸- N-羧基内酸酐（BPA-NCA）的合成：在氮气气体环境下，将L-4-二羟基硼苯丙氨酸（500 mg，2.39 mmol）和 α-蒎烯（948 μL，5.97 mmol）加入干燥处理的三颈圆底烧瓶中，随后加入无水四氢呋喃（THF，150 mL），然后再将三光气的THF溶液滴加到上述悬浊液中，反应体系置于55℃的油浴中搅拌均匀，反应5小时后将反应液自然冷却至室温，用砂芯漏斗过滤，再将澄清的下清反应液旋蒸浓缩至10 mL，随后在预冷的石油醚中沉淀得到粗产物；再将粗产物复溶于四氢呋喃，再次沉淀，重复进行3次，得到白色粉末固体，即为L-4-二羟基硼苯丙氨酸- N-羧基内酸酐单体（BPA-NCA，产率: 60%）。BPA-NCA核磁表征见附图2， 1H NMR (400 MHz, DMSO- d 6 , δ): 9.09 (s, 1H, -CON H-), 8.03 (s, 2H, -B(O H) 2), 7.71 and 7.14 (d, J = 7.6 Hz, 4H, -C 6 H 4 -), 4.79 (t, J = 5.2 Hz, 1H, -COC HNH-), 3.03 (d, J = 5.2 Hz, 2H, -C 6H 4C H 2 -); 13C NMR (100 MHz, DMSO- d 6, δ): 171.27, 152.09, 137.05, 134.67, 133.31, 129.19, 58.62, 36.81. BPA-NCA的元素分析为: C, 53.19; H, 4.85; N, 5.53（理论: C, 51.11; H, 4.29; N, 5.96）；质谱: MS (m/z): 235.1（理论: 235.1）。
实施例二 嵌段共聚物PEG-PBPA的制备：本发明以末端为氨基的聚乙二醇为引发剂引发L-4-二羟基硼苯丙氨酸- N-羧基内酸酐开环聚合，通过调节聚乙二醇与L-4-二羟基硼苯丙氨酸- N-羧基内酸酐的摩尔比可制备得到不同链长的聚合物PEG-PBPA（表1）。以合成PEG-PBPA（ M n = 5.0-4.0 kg/mol）为例：在氮气氛围下，将PEG-NH 2（0.25 g, 0.05 mmol）的DMF溶液加入到密闭反应器中，在搅拌的条件下将BPA-NCA（0.25 g, 1.06 mmol）的DMF溶液加入密闭反应器，在80℃的恒温油浴中反应3天（红外谱图中对应的BPA-NCA单体的羰基伸缩峰1770 cm -1和1845 cm -1完全消失，表明聚合完全）。反应结束后，聚合物溶液用冰的无水***沉淀、离心，接着用甲醇复溶，重复沉淀3次，收集固体沉淀并真空干燥48小时，得到白色固体产物，产率：81%。PEG-PBPA嵌段共聚物的核磁表征见附图2。 1H NMR (DMSO- d 6/CD 3OD (v/v = 2/1), 400 MHz, δ): 7.68 and 7.22 (- C 6H 4 B(OH) 2), 4.49 (-CO CHNH-), 3.51 (-O CH 2CH 2 O-), 2.96-2.78 (-C 6H 4 CH 2 -)。
同样，以4/8臂PEG-NH 2为引发剂合成了基于BPA的支化型聚合物。例如，将溶解在无水DMF中的四臂PEG-NH 2（1.32 g，0.066 mmol）在氮气气氛下添加到BPA-NCA（0.250 g，1.06 mmol）溶液中，反应在80℃下进行24小时。反应结束后，聚合物溶液用冰的无水***沉淀、离心，接着用甲醇复溶，重复沉淀3次，收集固体沉淀并真空干燥48小时，得到4-arm-PEG-PBPA ( M n = 20.0-3.0 kg/mol). 1H NMR (400 MHz, DMSO- d 6/ CD 3OD(v/v = 2/1), δ):7.68 and 7.22 (-C 6 H 4 B(OH) 2), 4.50 (-COC HNH-), 3.51 (-OC H 2C H 2O-), 2.98-2.74 (-C 6H 4 CH 2 -)；同样方法，采用八臂PEG-NH 2得到8-arm-PEG-PBPA ( M n = 20.0-12.0 kg/mol), 1H NMR (400 MHz, DMSO- d 6/ CD 3OD (v/v = 2/1), δ):7.68 and 7.22 (-C 6 H 4 B(OH) 2), 4.50 (-COC HNH-), 3.51 (-OC H 2C H 2O-), 2.98-2.74 (-C 6H 4 CH 2 -)。
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实施例三嵌段共聚物PEG-P(Tyr- co-BPA)、PEG-P(Lys- co-BPA)以及PEG-P(BLA- co-BPA)等的合成：本发明以线型聚乙二醇为引发剂引发上述L-4-二羟基硼苯丙氨酸- N-羧基内酸酐单体和其他类型 N-羧基内酸酐单体（如Tyr-NCA、Lys(Boc)-NCA以及BLA-NCA等）无规共聚得到具有不同分子结构、不同亲疏水性、不同电荷和不同功能基团的苯硼酸基聚多肽材料（表3）。以合成PEG-P(Tyr- co-BPA)（ M n = 5.0-1.0-4.0 kg/mol）为例：用甲氧基聚乙二醇氨为引发剂，在氮气环境下将PEG-NH 2（0.25 g, 1.06 mmol）的DMF溶液加到Tyr-NCA（0.121 g, 0.396 mmol）和BPA-NCA（0.25 g, 1.06 mmol）的DMF溶液中，然后在80℃下反应3天。反应结束后，聚合物溶液用冰的无水***沉淀、离心，接着用甲醇复溶，重复沉淀3次，收集固体沉淀并真空干燥48小时，得到灰白色产物，产率：78%。PEG-P(Tyr- co-BPA)嵌段共聚物的核磁表征见附图3。 1H NMR (DMSO- d 6/CD 3OD (v/v = 2/1), 400 MHz, δ): 7.68 and 7.22 (-C 6 H 4 B(OH) 2), 6.95 and 6.61 (- C 6H 4 OH), 4.47 and 4.37 (-CO CHNH-), 3.52 (-O CH 2CH 2 O-), 2.82-2.63 (-C 6H 4 CH 2 -)。
PEG-P(Lys(Boc)- co-BPA)、PEG-P(BLA- co-BPA)以及4-arm-PEG-PBPA的合成同样遵循上述操作流程，PEG-P(Lys(Boc)- co-BPA)在三氟乙酸中脱保护后得到PEG-P(Lys- co-BPA)。
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实施例四 ApoE修饰的PEG-PBPA聚合物（ApoE-PEG-PBPA）的合成。
参照上述方法，本发明以马来酰亚胺修饰的聚乙二醇为引发剂引发上述L-4-二羟基硼苯丙氨酸- N-羧基内酸酐单体和/或其他类型 N-羧基内酸酐单体得到（Mal-PEG-PBA或Mal-PEG-P(BPA- co-Tyr)等嵌段聚合物，然后其PEG末端可化学偶联ApoE、Angiopep-2、cRGD等特异性靶向分子。以合成ApoE-PEG-PBPA为例：在氮气氛围下，将Mal-PEG-PBPA（100 mg, 0.011 mmol）和ApoE肽（30.7 mg, 0.013 mmol）溶解在除氧的DMSO/CH 3OH（v/v = 9/1, 0.1 mL）中，在37℃下反应24小时，得到聚多肽溶液。反应结束后，将聚多肽溶液置于DMSO/CH 3OH中透析24小时，接着置于二次水中透析以置换有机溶剂，最后冻干后得到白色固体产物ApoE-PEG-PBPA，产率：78%。ApoE-PEG-PBPA嵌段共聚物的核磁表征见附图4A。 1H NMR (400 MHz, DMSO- d 6/ CD 3OD(v/v = 2/1), δ): 7.68 and 7.22 (-C 6 H 4 B(OH) 2), 4.49 (-CO CHNH-), 3.51 (-O CH 2CH 2 O-), 2.98-2.74 (-C H 2 NH-), 0.78-2.41 (ApoE)。通过9,10-菲醌法测得ApoE的接枝效率为92%。同样地，在聚合物末端可修饰葡萄糖分子，附图4B为修饰了葡萄糖的聚合物核磁谱图，产率：82%。通过核磁可以算出大约有3.6个葡萄糖修饰到聚合物4-arm-PEG-PBPA上。
实施例五 PEG-PBPA聚合物胶束（PBN）装载姜黄素和甲苯磺酸索拉非尼及体外释放：将聚合物PEG-PBPA（ M n = 5.0-4.0 kg/mol）、药物姜黄素（Cur）、甲苯磺酸索拉非尼（Sor）分别溶解在DMSO溶液中，并按一定的量混合后滴加到搅拌状态的HEPES、PB缓冲溶液或超纯水中，然后用截留分子量为7000的透析袋透析除去有机溶剂和游离药物，透析过程在pH = 7.4的HEPES、PB缓冲液或纯水中进行（表4）。以共包载Cur和Sor（理论载药量均为10 wt%）为例：取10 μL溶解好的PEG-PBPA聚合物溶液（100 mg/mL）、5.55 μL的Cur溶液和5.55 μL的Sor溶液充分混合后，滴加到979 μL的HEPES缓冲溶液中，并在HEPES缓冲溶液中透析以除去有机溶剂和未包载的药物。最终，通过动态光散射粒度分析仪（DLS）测得聚合物胶束的尺寸为95 nm，粒径分布较窄（< 0.2）。通过HPLC测得Cur和Sor的实际载药量分别达到7.8 wt%和9.8 wt%。而且，该聚合物胶束在高倍稀释（50倍）、10%的胎牛血清（FBS）以及4℃长期放置条件下均具有良好的稳定性。
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Cur和Sor的体外释放实验是在37℃，200 rpm的恒温摇床中进行的。具体地，将1.0 mL的PBN-Cur/Sor（Cur和Sor载药量为10 wt%）胶束溶液转移到释放袋（MWCO = 12-14 KD）中，并浸没在25.0 mL不同条件的释放介质（pH 7.4、100 μM H 2O 2）中，在预定时间点吸取5.0 mL释放介质并补充等体积的新鲜释放介质，待所有样品冷冻冻干后用0.3 mL的乙腈复溶，通过HPLC测定Cur和Sor的含量。附图5D为Cur和Sor累积释放量与时间的关系，从图中可以看出载药胶束能通过扩散作用释放药物。
实施例六 MTT法测试空聚合物胶束和载药胶束的细胞毒性：通过MTT法评价空聚合物胶束PBN对肿瘤细胞（B16F10细胞、U87 MG-luc细胞）和正常鼠成纤维细胞（L929细胞）的毒性。具体为：将细胞铺到96孔板（80 μL，5×10 3个细胞/孔）中，在37℃培养箱中孵育24小时，随后向每个孔加入20 μL不同浓度梯度的PBN胶束溶液，使孔内最终浓度为0.05、0.1、0.2、0.5、0.75和1.0 mg/mL。共孵育48小时后，向每孔再加入10 μL浓度为5.0 mg/mL的MTT溶液继续培养4小时，除去孔内培养基并加入150 μL的DMSO溶解MTT与活细胞作用产生的紫色甲瓒结晶，最后通过多功能酶标仪测定孔板中每孔在570 nm处的吸光值。细胞存活率通过各样品组吸光值与空白对照组的比值测定，其中空白对照组是加入等体积的PBS。每组设定5个平行孔。附图6A为聚合物PEG-PBPA（ M n = 5.0-4.0 kg/mol）自组装形成的胶束对B16F10、U87 MG-luc和L929和的细胞毒性结果，可看出，当聚合物胶束的浓度从0.1增到1.0 mg/mL时，所有组别细胞的存活率仍高于80%，说明该聚合物胶束具有良好的生物相容性。
研究实施例五的载药胶束（PBN-Cur、PBN-Sor和PBN-Cur/Sor）对U87 MG-luc细胞的毒性，细胞培养操作同上述一致。当加入不同梯度浓度的PBN-Cur、PBN-Sor（Cur或Sor终浓度为0.01、0.1、0.5、1、2、4、6、8和10 μg/mL）以及不同药物比的PBN-Cur/Sor胶束溶液与细胞共孵育48小时后加入MTT，处理和测定吸光度同上述一致。实验结果参见附图6B&C，图中结果显示出Cur和Sor均表现出一定的杀伤效果，PBN-Cur和PBN-Sor相比于自由药，杀伤效果有提升。对于两种药物共同作用于U87 MG-luc，当两种药物的质量比为1/1时，对肿瘤细胞的协同效果（CI = C CA/C A+C CB/C B；C CA：联用药物A、B时A的IC 50浓度、C CB：联用药物A、B时B的IC 50浓度、C A和C B分别为A和B的IC 50浓度）最为显著，其半数致死浓度（IC 50）为1.47 μg/mL。说明本发明的胶束具有很好的递送Cur和Sor的能力，并实现有效的释放，最终杀死肿瘤细胞。
此外，细胞克隆形成实验对PBN-Cur、PBN-Sor以及PBN-Cur/Sor的细胞毒性进行验证。具体步骤为：将U87 MG-luc细胞（2×10 5个细胞/孔）接种于6孔板内，培养24小时后分别加入Cur、Sor、PBN-Cur、PBN-Sor以及PBN-Cur/Sor（Cur: 1.5 μg/mL, Sor: 1.5 μg/mL, Cur/Sor: 1.5/1.5 μg/mL）共孵育48小时。随后用胰酶消化细胞，将各样品组细胞（300个细胞/孔）重新接种于6孔板内，放置在细胞培养箱中培养9天，期间每隔三天更换一次培养基。最后吸去培养基，PBS浸洗2次后用4%多聚甲醛溶液固定细胞15分钟，接着用结晶紫染色液对孔内细胞进行染色，最后缓慢洗去结晶紫并空气干燥。从图6D可以看出，PBN-Cur（Cur: 1.5 μg/mL）和PBN-Sor（Sor: 1.5 μg/mL）均具有一定的长期抑制肿瘤细胞增殖效果，且PBN-Sor组的抑制效果要略微优于PBN-Cur组，双药联用的PBN-Cur/Sor组具有最优异的抑制效果，这一结果与MTT实验结果具有一致性。
实施例七载药胶束的溶血结果：本实验以去离子水作阳性对照，0.9% NaCl为阴性对照，研究了Cur、Sor、PBN-Cur、PBN-Sor、PBN-Cur/Sor（低）和PBN-Cur/Sor（高）的溶血情况（图7A）。药物浓度设置为50 μg/mL，PBN-Cur/Sor（高；Cur: 110 μg/mL，Sor: 110 μg/mL）组除外。图7B的结果显示出，游离Sor具有出明显的血液毒性，在50 μg/mL的浓度下诱导约45%的溶血率，而相同浓度下的纳米药物PBN-Sor仅表现出轻微的溶血（HR为4.3%）。此外，低浓度（Cur: 50 μg/mL；Sor: 50 μg/mL）和高浓度的PBN-Cur/Sor也显示出优越的血液相容性，HR约为2.3%，可为同时静脉注射Cur和Sor提供有效策略。
实施例八 载药胶束的细胞凋亡和细胞克隆实验：本发明采用Annexin V-FITC/Propidium iodine（PI）双染技术，利用流式细胞仪研究Cur、Sor、PBN-Cur、PBN-Sor以及PBN-Cur/Sor对U87 MG-luc细胞的凋亡效果。具体地，取对数生长期的U87 MG-luc细胞（2×10 5个细胞/孔）接种于6孔板中，置于培养箱中培养过夜，每孔加入Cur、Sor、PBN-Cur、PBN-Sor或PBN-Cur/Sor（Cur: 1.5 μg/mL, Sor: 1.5 μg/mL, Cur/Sor: 1.5/1.5 μg/mL）共孵育48小时，随后弃去培养基并用PBS洗涤2次，加入胰酶（不含EDTA）消化细胞后离心收集，用4℃预冷的PBS洗涤2次后加入100 μL结合缓冲液（binding buffer）进行细胞重悬（1×10 6个细胞/mL），取100 μL细胞悬液转入流式管，并依次加入5 μL Annexin V-FITC染液和10 μL PI染液，混合均匀后于室温下避光染色15分钟，最后补加400 μL PBS，混合均匀后用流式细胞仪检测细胞处于各个凋亡阶段的百分数。对照组的双染样品处理步骤同样品组。另外，早凋样品的制备是将细胞放入50℃水浴中处理5分钟，晚凋样品的制备是加入4%多聚甲醛处理5分钟。所有数据均使用Flowjo软件进行分析。附图8A可知，PBN-Cur/Sor处理引起了超过50%的凋亡，明显高于PBN-Cur（7.1%）、PBN-Sor（23.4%）、Cur（6.8%）和Sor（21.5%）。
此外，据文献报道Cur具有抑制细胞周期的能力，因此PBN-Cur对于U87 MG-luc细胞的细胞周期阻滞能力也进行了相应地测试。具体地，将U87 MG-luc细胞（2×10 5个细胞/孔）接种于6孔板内，孵育24小时后将Cur或PBN-Cur（Cur: 1.5 μg/mL）加入再孵育24小时。接着用胰酶消化并收集细胞，用4℃预冷的PBS洗涤细胞3次，最终使细胞分散在1.0 mL的PBS中，将细胞悬液滴加到4.0 mL匀速震荡的95%乙醇中，4℃固定24小时，随后用细胞周期试剂盒染色30分钟，并通过流式细胞仪进行细胞周期检测。从图8B中可知，PBS组多数处于G1期（67.28%），S期细胞只有23.98%，G2/M期含量仅为8.74%，而Cur和PBN-Cur对U87 MG-luc细胞的周期阻滞情况优于PBS组，且PBN-Cur组较Cur组有略微提升。
实施例八富含硼-10的 10BPA-NCA和mPEG-P 10BPA的合成： 10BPA-NCA的合成和mPEG-P 10BPA的合成分别遵循实施例一和实施例二。唯一不同是在单体合成过程中，使用的是富含硼-10的BPA，图9A&B为其表征。 10BPA-NCA的 1H NMR (400 MHz, DMSO- d 6, δ): 9.09 (s, 1H), 8.03 (s, 2H), 7.71 (d, J = 7.6 Hz, 2H), 7.14 (d, J = 7.6 Hz, 2H), 4.79 (t, J = 5.2 Hz, 1H), 3.03 (d, J = 5.2 Hz, 2H)。mPEG-P 10BPA的 1H NMR (DMSO- d 6/CD 3OD (v/v = 2/1), 400 MHz, δ): 7.68 (- C 6H 2 B(OH) 2), 7.22 (- C 6H 2 CH 2-), 4.49 (-CO CHNH-), 3.51 (-O CH 2CH 2 O-), 3.26-2.85 (-C 6H 4 CH 2 -)。

Claims (10)

  1. 一种具有式I结构的L-4-二羟基硼苯丙氨酸 N-羧基内酸酐：
    。
  2. 权利要求1所述L-4-二羟基硼苯丙氨酸 N-羧基内酸酐的制备方法，其特征在于，以L-4-二羟基硼苯丙氨酸、 α-蒎烯、三光气为反应物，在溶剂中反应，制备得到所述L-4-二羟基硼苯丙氨酸- N-羧基内酸酐。
  3. 一种具有式II结构的线型嵌段共聚物：
    其中，R 1来自引发剂聚乙二醇；R 2来自其他类型的氨基酸；m为70～210，x为5～30，y为0～15，n为5～45。
  4. 权利要求3所述线型嵌段共聚物的制备方法，其特征在于，以线型聚乙二醇为引发剂，L-4-二羟基硼苯丙氨酸为单体或者L-4-二羟基硼苯丙氨酸与其他类型 N-羧基内酸酐为单体，开环聚合，得到线性嵌段共聚物。
  5. 一种具有式Ⅳ结构的支化嵌段共聚物：
    其中，m为20～150，n为2～20，x为2～8；R 4为引发剂支化聚乙二醇的支化中心。
  6. 权利要求5所述支化嵌段共聚物的制备方法，其特征在于，以支化聚乙二醇为引发剂，L-4-二羟基硼苯丙氨酸为单体，开环聚合，得到支化嵌段共聚物。
  7. 一种靶向嵌段共聚物，其特征在于，由权利要求3所述线型嵌段共聚物偶联靶向分子得到，或者由权利要求5所述支化嵌段共聚物偶联靶向分子得到。
  8. 一种药物递送体系，其特征在于，由权利要求3所述线型嵌段共聚物或者权利要求5所述支化嵌段共聚物装载药物得到；或者由权利要求3所述线型嵌段共聚物或者权利要求5所述支化嵌段共聚物装载药物后偶联靶向分子得到；或者由权利要求3所述线型嵌段共聚物与权利要求7所述靶向嵌段共聚物共装载药物得到；或者由权利要求5所述支化嵌段共聚物与权利要求7所述靶向嵌段共聚物共装载药物得到。
  9. 权利要求1所述L-4-二羟基硼苯丙氨酸 N-羧基内酸酐、权利要求3所述线型嵌段共聚物、权利要求5所述支化嵌段共聚物、权利要求7所述靶向嵌段共聚物或者权利要求8所述药物递送体系在制备药物中的应用。
  10. 权利要求1所述L-4-二羟基硼苯丙氨酸 N-羧基内酸酐、权利要求3所述线型嵌段共聚物、权利要求5所述支化嵌段共聚物、权利要求7所述靶向嵌段共聚物在制备药物载体中的应用。
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