WO2023237060A1 - 一种两亲嵌段聚合物和放化疗纳米增敏剂及其制备方法 - Google Patents

一种两亲嵌段聚合物和放化疗纳米增敏剂及其制备方法 Download PDF

Info

Publication number
WO2023237060A1
WO2023237060A1 PCT/CN2023/099185 CN2023099185W WO2023237060A1 WO 2023237060 A1 WO2023237060 A1 WO 2023237060A1 CN 2023099185 W CN2023099185 W CN 2023099185W WO 2023237060 A1 WO2023237060 A1 WO 2023237060A1
Authority
WO
WIPO (PCT)
Prior art keywords
chemotherapy
block polymer
amphiphilic block
group
derivatives
Prior art date
Application number
PCT/CN2023/099185
Other languages
English (en)
French (fr)
Inventor
喻青松
甘志华
唐小虎
俞艳婷
Original Assignee
北京化工大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京化工大学 filed Critical 北京化工大学
Publication of WO2023237060A1 publication Critical patent/WO2023237060A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
    • C08F8/32—Introducing nitrogen atoms or nitrogen-containing groups by reaction with amines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • 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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/54—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 non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2/00—Processes of polymerisation
    • C08F2/38—Polymerisation using regulators, e.g. chain terminating agents, e.g. telomerisation
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
    • C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2438/00—Living radical polymerisation
    • C08F2438/03—Use of a di- or tri-thiocarbonylthio compound, e.g. di- or tri-thioester, di- or tri-thiocarbamate, or a xanthate as chain transfer agent, e.g . Reversible Addition Fragmentation chain Transfer [RAFT] or Macromolecular Design via Interchange of Xanthates [MADIX]

Definitions

  • the invention belongs to the technical field of biomedical polymer materials, and specifically relates to an amphiphilic block polymer, a nano-sensitizer for radiotherapy and chemotherapy and a preparation method thereof.
  • the clearance system of the human blood system which limits the effective concentration of drugs reaching the tumor tissue
  • the second reason is the presence of dense fibrosis in the cancer tissue Factors such as excessive internal pressure generated by the intercellular matrix and interstitial fluid and lack of capillary vascular network hinder the diffusion of drugs into tumor tissues, especially the diffusion of macromolecules.
  • traditional PEG water-soluble nanomedicines or liposome nanomedicines can effectively avoid the clearance system in the body, on the one hand, their penetrating ability is very weak, and on the other hand, they are difficult to be engulfed by cancer cells, so to a large extent limit their efficacy.
  • radiosensitizers for adjuvant radiotherapy, such as gemcitabine, capecitabine or fluorouracil.
  • Individualized chemoradiotherapy that simultaneously combines multiple targeted drugs and radiosensitizers has also been shown to prolong patient survival and increase treatment variability for patients with different cancers.
  • nanotechnology is also used in the research of personalized radiotherapy and chemotherapy with radiosensitizers.
  • the present invention provides an amphiphilic block polymer and a nanosensitizer for radiotherapy and chemotherapy and a preparation method thereof.
  • Nanosensitizers for radiotherapy and chemotherapy are used to solve the biological barriers and microenvironmental limitations faced by personalized tumor treatment.
  • the amphiphilic block polymer prepared through chemical modification has the characteristics of deep tumor penetration, enzyme-responsive drug release/activation and hypoxia-responsiveness.
  • the hypoxia-responsive chemotherapy prodrug and amphiphilic block polymer are combined
  • the block polymer self-assembles through physical encapsulation to obtain a stable and high drug-carrying rate chemoradiotherapy nanosensitizer, which has charge reversal and cascade response characteristics.
  • the chemotherapeutic prodrugs and amphiphilic block polymers released by the chemoradiotherapy nanosensitizer provided by the invention can effectively make tumor cells resistant to chemoradiotherapy. sensitive.
  • the radiochemotherapy nanosensitizer can achieve significant The tumor suppressive effect shows great application potential in the field of combined radiotherapy and chemotherapy with personalized treatment.
  • a first aspect of the present invention provides an amphiphilic block polymer, wherein the amphiphilic block polymer has a structure represented by formula (1):
  • R 1 is a living free radical polymerization initiating group
  • R 10 is a H atom or a C 1 -C 5 alkyl group
  • R 11 is an O atom or an N atom
  • R 2 is an enzyme response molecule
  • R 3 is an anoxic response.
  • R 30 is a redox-sensitive group
  • R 4 is an initiator connecting group
  • X is an enzyme response molecule bonding group, 10 ⁇ m ⁇ 100, 20 ⁇ n ⁇ 200, m and n are both integers.
  • the living radical polymerization initiating group R 1 is dithioester and its derivatives, trithioester and its derivatives, dithiocarbonate and its derivatives, dithiocarbonate and its derivatives, Thiocarbamates and their derivatives or halogen groups and their derivatives.
  • hypoxia response group R 3 is 2-nitroimidazole and its derivatives, 4-nitroimidazole and its derivatives, or 5-nitroimidazole and its derivatives.
  • the redox-sensitive group R 30 is a disulfide ketal bond, a monosulfide bond, a monoselenium bond, a disulfide bond, a trisulfide bond or a diselenide bond.
  • the enzyme response molecule R 2 is a ⁇ -glutamyl transpeptidase response molecule, a fibroblast protease response molecule, Metalloproteinase-responsive molecules or cathepsin-responsive molecules.
  • the enzyme-responsive molecule bonding group X has the structure shown in formula (3):
  • the second aspect of the present invention provides a nanosensitizer for chemoradiotherapy, wherein the nanosensitizer for chemoradiotherapy contains the above-mentioned amphiphilic block polymer and chemotherapeutic prodrug, and the amphiphilic block polymer and chemotherapeutic prodrug are
  • the mass ratio of prodrug is 100:(0 ⁇ 100);
  • the chemotherapy prodrug has the structure shown in formula (4):
  • R 5 is an anti-cancer chemotherapy drug molecule.
  • the anti-cancer chemotherapeutic drug molecule R 5 is paclitaxel, paclitaxel derivatives, doxorubicin, epirubicin, camptothecin, camptothecin derivatives, cisplatin drugs, Changchun alkali, vincristine, docetaxel, gemcitabine, curcumin, or salvianolic acid.
  • a third aspect of the present invention provides a method for preparing a nanosensitizer for chemoradiotherapy, wherein the method includes the following steps:
  • R 5 is an anti-cancer chemotherapy drug molecule
  • step S2 Add the solution containing the amphiphilic block polymer and chemotherapy prodrug obtained in step S1 to the organic solvent II-pure water mixed phase and stir at high speed to obtain a solution containing the nanosensitizer for radiotherapy and chemotherapy;
  • step S3 Dialyze the solution containing the nanosensitizer for chemoradiotherapy obtained in step S2 into pure water to remove organic solvent I and organic solvent II to obtain the nanosensitizer for chemoradiotherapy.
  • the fourth aspect of the present invention provides a nanosensitizer for radiotherapy and chemotherapy prepared by the above method.
  • the present invention constructs a nanosensitizer for radiotherapy and chemotherapy with high drug loading efficiency, high stability and cascade response characteristics.
  • enzyme-responsive molecules, hypoxia-responsive groups and redox-sensitive groups are simultaneously introduced into the amphiphilic block polymer.
  • the polymer can achieve charge conversion after enzyme activation, achieving effective endocytosis and transcytosis, thereby further driving deep tumor penetration.
  • Chemotherapy prodrugs loaded on amphiphilic block polymers are chemically modified with metronidazole, so their drug loading rate and encapsulation rate are relatively high, and they can be selectively activated by excess enzymes in cancer cells.
  • the released chemotherapeutic drugs and metronidazole residues can effectively sensitize tumor cells to radiochemotherapy.
  • Figure 1 shows the NMR spectrum of N 3 -PEG-CTA
  • Figure 2 shows the NMR spectrum of MASSCOOH
  • FIG. 3 shows the NMR spectrum of MASSMI
  • FIG. 4 shows the NMR spectrum of PSSM
  • FIG. 5 shows the NMR spectrum of BAP
  • Figure 6 shows the NMR spectrum of GBAP
  • FIG. 7 shows the NMR spectrum of PSSMG
  • Figure 8 shows the CM release curve of chemoradiotherapy nanosensitizer nanoparticles in response to GSH (10 ⁇ M, 5mM and 10mM);
  • Figure 9 shows the DLS particle size change curve of nanosensitizer nanoparticles for radiotherapy and chemotherapy in response to 10mM GSH;
  • Figure 10 shows the CM release curve of chemoradiotherapy nanosensitizer nanoparticles under hypoxic response
  • Figure 11 shows the DLS particle size change curve of nanosensitizer nanoparticles for radiotherapy and chemotherapy under hypoxic response
  • Figure 12 is the Zeta potential charge reversal curve of nanosensitizer nanoparticles for radiotherapy and chemotherapy catalyzed by GGT enzyme;
  • Figure 13 is a histogram of intracellular endocytosis of chemoradiotherapy nanosensitizer nanoparticles catalyzed by GGT enzyme;
  • Figure 14 shows the MTT cell survival curve of different drug administration experimental groups under normoxia and (B) the effect of different drug administration experimental groups on BxPC-3 cells under hypoxia;
  • Figure 15 is a picture of a representative colony of cells sensitized by radiotherapy under hypoxia under different drug administration experimental groups and BxPC-3 cells;
  • Figure 16 is a confocal microscope picture of the transcytosis of PSSMG nanoparticles catalyzed by GGT enzyme
  • Figure 17 is a confocal microscopy picture of 3D tumor spheres penetrated into tumors by PSSM nanoparticles and PSSMG nanoparticles;
  • Figures 18 and 19 show the combined synergistic tumor inhibition curves of different administration experimental groups on subcutaneous BxPC-3 pancreatic cancer subcutaneous tumor model mice.
  • a first aspect of the present invention provides an amphiphilic block polymer, wherein the amphiphilic block polymer has a structure represented by formula (1):
  • R 1 is a living free radical polymerization initiating group
  • R 10 is a H atom or a C 1 -C 5 alkyl group
  • R 11 is an O atom or an N atom
  • R 2 is an enzyme response molecule
  • R 3 is an anoxic response.
  • R 30 is a redox-sensitive group
  • R 4 is an initiator connecting group
  • X is an enzyme response molecule bonding group, 10 ⁇ m ⁇ 100, 20 ⁇ n ⁇ 200, m and n are both integers.
  • the living radical polymerization initiating group R 1 can be a reversible addition-fragmentation chain transfer polymerization (RAFT) chain transfer group, an atom transfer radical polymerization (ATRP) chain transfer group or other non-free radicals At least one of the polymeric chain transfer groups.
  • the living radical polymerization initiating group R 1 can be dithioester and its derivatives, trithioester and its derivatives, dithiocarbonate and its derivatives, dithiocarbamate Esters and their derivatives or halogen groups and their derivatives.
  • the living radical polymerization initiating group R 1 is trithioester and its derivatives, and its specific structure is particularly preferably shown in formula (1-1):
  • R 1-1 can be a C 1 -C 20 alkyl group, preferably a C 10 -C 20 alkyl group, and more preferably an n-dodecyl group.
  • the enzyme cleavage site connecting the enzyme response molecule R 2 to the amphiphilic block polymer is the amino group in the structure shown in the above formula (1). After the enzyme cleavage, the amino group is exposed to polymerize the amphiphilic block. Objects have a positive charge.
  • the enzyme responsive molecule R 2 is preferably a ⁇ -glutamyl transpeptidase responsive molecule, a fibroblast protease responsive molecule, a metalloproteinase responsive molecule or a cathepsin responsive molecule, and more preferably a ⁇ -glutamyl transpeptidase responsive molecule.
  • formula (1) contains a stimulus-responsive chemical bond with the structure shown in formula (2):
  • R 10 may be a H atom or a C 1 -C 5 alkyl group, and R 11 may be an O atom or an N atom.
  • the hypoxia response group R 3 is preferably 2-nitroimidazole and its derivatives, 4-nitroimidazole and its derivatives or 5-nitroimidazole and its derivatives, in a specific
  • the hypoxia-responsive group R 3 is metronidazole, which can be reduced in a hypoxic environment, thereby having hypoxia responsiveness.
  • the redox-sensitive group R 30 is preferably a disulfide ketal bond, a monosulfide bond, a monoselenium bond, a disulfide bond, a trisulfide bond or a disulfide bond, and is more preferably a disulfide bond, which can Reduced by glutathione, making it redox responsive.
  • amphiphilic block polymer provided by the present invention has dual response properties of hypoxia response (metronidazole) and reduction response (disulfide bond).
  • the initiator linking group R 4 is not particularly limited, as long as it can connect the amphiphilic block polymer.
  • the initiator linking group R 4 has the structure shown in formula (1-2):
  • the enzyme-responsive molecule bonding group X is introduced by introducing the enzyme-responsive molecule R 2 into two The bonding group formed on the block polymer, the enzyme response molecule bonding group X has the structure shown in formula (3):
  • the enzyme-responsive molecule bonding group X is a bonding group formed by introducing the enzyme-responsive molecule R 2 onto the amphiphilic block polymer through (b) click chemistry.
  • the bonding group X of the enzyme response molecule is triazole.
  • the structure of the triazole is shown in formula (3b-1):
  • amphiphilic block polymer prepared by chemical modification in the present invention has hypoxia responsiveness, redox responsiveness and enzyme responsiveness, and thus has the characteristics of deep tumor penetration and enzyme-reactive drug release/activation.
  • the second aspect of the present invention provides a nanosensitizer for radiotherapy and chemotherapy, which contains the above-mentioned amphiphilic block polymer and chemotherapy prodrug, and the mass ratio of the amphiphilic block polymer and chemotherapy prodrug is 100: (0 ⁇ 100), such as 100:0, 100:0.1, 100:1, 100:5, 100:10, 100:15, 100:20, 100:30, 100:40, 100:50, 100:60 , 100:70, 100:80, 100:90, 100:100, etc.
  • the chemotherapy prodrug has the structure shown in formula (4):
  • R 5 is an anti-cancer chemotherapy drug molecule.
  • the anti-cancer chemotherapy drug molecule R 5 is paclitaxel, paclitaxel derivatives, doxorubicin, epirubicin, camptothecin, camptothecin derivatives, cisplatin drugs, vinblastine, vincristine, At least one of docetaxel, gemcitabine, curcumin, and salvianolic acid.
  • specific examples of chemotherapy prodrugs include but are not limited to cisplatin-based drugs.
  • the structure of cisplatin-based chemotherapy prodrugs is shown in formula (4-1):
  • a third aspect of the present invention provides a method for preparing a nanosensitizer for chemoradiotherapy, wherein the preparation method includes the following steps:
  • R 5 is an anti-cancer chemotherapy drug molecule
  • step S2 Add the solution containing the amphiphilic block polymer and chemotherapy prodrug obtained in step S1 to the organic solvent II-pure water mixed phase and stir at high speed to obtain a solution containing the radiochemotherapy nanosensitizer;
  • step S3 Dialyze the solution containing the nanosensitizer for chemoradiotherapy obtained in step S2 into pure water to remove organic solvents I and II to obtain the nanosensitizer for chemoradiotherapy.
  • organic solvent I the two organic solvents required in the preparation process are called “organic solvent I” and “organic solvent II” only for the purpose of distinguishing different objects and cannot be understood. To indicate or imply their relative importance.
  • the present invention has no special restrictions on the organic solvent I used in step S1, as long as it can fully dissolve the amphiphilic block polymer and chemotherapeutic prodrugs.
  • specific examples of the organic solvent I include but are not limited to: DMF and/or DMSO.
  • the present invention has no special restrictions on the organic solvent II used in step S2, as long as the mixed phase with pure water can prepare a physically embedded self-assembled nanosensitizer for radiotherapy and chemotherapy.
  • organic solvent II and organic solvent I are the same organic solvent.
  • the present invention has no particular limitation on the high-speed stirring conditions used in step S2. It is preferable to use a stirring rate of 400 to 600 rpm, such as 400 rpm, 600 rpm, and 600 rpm.
  • the stirring time is 40 to 80 minutes, such as 40 minutes, 50 minutes, 60 minutes, 70 minutes, or 80 minutes.
  • the present invention has no special restrictions on the dialysis conditions in step S3, as long as the solvent-containing machines I and II can be removed.
  • the fourth aspect of the present invention provides a nanosensitizer for radiotherapy and chemotherapy prepared by the above method.
  • PSSMG was synthesized via CuAAC click chemistry from alkynyl-terminated GBAP and azido-terminated PSSM.
  • PMDETA pentamethyldiethylenetriamine
  • PSSMG solid product
  • Its NMR spectrum is shown in Figure 7.
  • the obtained product is PSSMG.
  • PSSMG polymer is an amphiphilic block polymer with dual response characteristics of enzyme response and redox response.
  • Test Example 1 Test of the performance of the nanosensitizer for chemoradiotherapy obtained in Preparation Example 1
  • the experimental results are shown in Figure 8.
  • the particle size distribution of chemoradiotherapy nanosensitizer nanoparticles in a high GSH concentration (10mM) environment was monitored through DLS characterization.
  • the experimental results are shown in Figure 9.
  • Each set of experiments is set up with three sets of parallel experiments. It can be seen from the experimental results in Figures 8 and 9 that the chemoradiotherapy nanosensitizer solution provided by the present invention has GSH-responsive drug release/activation characteristics.
  • the specific operation is as follows: In order to simulate the hypoxic environment in the body, a three-neck flask is evacuated, and 100 ⁇ M sodium bisulfite solution is added while argon gas is continuously flowing.
  • the MWCO3000KD dialysis bag loaded with 3 mL of chemoradiotherapy nanosensitizer solution obtained in Preparation Example 1 and loaded with cisplatin chemotherapy prodrug CM was placed in a three-neck flask, and sodium bisulfite was used to simulate the hypoxic reducing environment in the body.
  • the three-necked flask was wrapped in tin foil to avoid light, and placed in a constant temperature shaker at 37°C and 100r/min for 24h. Sampling was carried out at 1h, 2h, 9h, 12h, 24h, 48h and 96h respectively, and the content of CM released outside the dialysis bag was monitored by HPLC.
  • the experimental results are shown in Figure 10.
  • chemoradiotherapy nanosensitizer nanoparticles in a hypoxic environment was monitored through DLS characterization.
  • the experimental results are shown in Figure 11. Each set of experiments is set up with three sets of parallel experiments. It can be seen from the experimental results in Figures 10 and 11 that the chemoradiotherapy nanosensitizer solution provided by the present invention has the characteristics of CM drug release/activation under hypoxic conditions.
  • Human pancreatic cancer cells (BxPC-3) were evenly spread in a six-well plate at 10 5 cells per well, and a control group (NPs) and a GGTi group were set up (the GGTi group added 1.0 ⁇ 10 cells to the cells 24 h before co-culture).
  • NPs control group
  • GGTi group added 1.0 ⁇ 10 cells to the cells 24 h before co-culture.
  • PSSM nanoparticles containing 5 ⁇ g/mL Nile Red and PSSMG nanoparticles containing 5 ⁇ g/mL Nile Red were added to each group respectively, and the cells were incubated. After incubation for 6 h, they were digested with trypsin and digested with trypsin.
  • Drug nanoparticles PSSMCM (referring to the PSSM synthesized in Preparation Example 1 (2) for CM drug loading, that is, no GGT enzyme responsive molecule modification is performed, and the preparation method is the same as Preparation Example 1 (5)) and drug-loaded nanoparticles PSSMGCM (referring to Preparation Example 1 (5)).
  • the PSSMG synthesized in Example 1 (3a) was loaded with CM drugs, that is, the chemoradiotherapy nanosensitizer synthesized in Preparation Example 1 (5)), and the concentration gradient was 100 ⁇ M, 64 ⁇ M, 32 ⁇ M, 16 ⁇ M, 8 ⁇ M, 4 ⁇ M, 2 ⁇ M, 1 ⁇ M and 0.5 ⁇ M, three parallel groups were set up for each concentration, and then the two sets of well plates of BXPC-3 cells were incubated in normoxic and hypoxic incubators for 24 h respectively, and the NIH-3T3 cell well plates were placed in a conventional Incubate in the incubator for 24h.
  • CM drugs that is, the chemoradiotherapy nanosensitizer synthesized in Preparation Example 1 (5)
  • the concentration gradient was 100 ⁇ M, 64 ⁇ M, 32 ⁇ M, 16 ⁇ M, 8 ⁇ M, 4 ⁇ M, 2 ⁇ M, 1 ⁇ M and 0.5 ⁇ M
  • Relative cell survival rate (%) (A x -A 0 )/(A c -A 0 ) ⁇ 100..................(1)
  • Ax , A0 and Ac are the absorbance values of the experimental group, zeroing group and negative control group respectively.
  • the MTT cell survival curve of the experimental group administered CM, CDDP, PSSMCM and PSSMGCM under normoxia and hypoxia and the effect of BxPC-3 cells is shown in Figure 14. It can be seen from the experimental results in Figure 14 that the nanosensitizer for chemoradiotherapy provided by the present invention has a high cell growth inhibitory effect in a hypoxic environment.
  • Its electron linear accelerator 23Ex, Varian, USA was used as the irradiation source.
  • the dose rate was set to 2Gy/min, the handpiece was rotated 180°, the fixed irradiation area was 15 ⁇ 15cm, and a 1.5cm dose compensator was placed on the culture plate to eliminate the dose accumulation effect.
  • the cells After being placed at low temperature for 0.5 h, the cells were digested with trypsin, seeded on a six-well plate at 1,000 cells per well, and then added with fresh RPMI-1640 medium to continue culturing; 14 days later, glutaraldehyde (6.0% v/v) was used to The cell groups visible to the naked eye were fixed, stained with crystal violet (0.5% w/v) at room temperature for 1 hour, and clones of ⁇ 50 cells were counted.
  • the experimental results are shown in Figure 15. It can be seen from the experimental results in Figure 15 that the nanosensitizer for radiotherapy and chemotherapy provided by the present invention has good radiotherapy sensitivity in a hypoxic environment.
  • BxPC-3 cells were seeded on coverslips (1)-(3) and incubated overnight. Use two parallel coverslips for each step: one for CLSM observation after 15 minutes of Hoechst 33342 staining; the other for co-incubation in the next step.
  • the cells on the coverslip (1) were incubated with Nile Red-labeled PSSM (1.0 ⁇ 10 -5 M) and Nile Red-labeled PSSMG (1.0 ⁇ 10 -5 M) for 6 hours respectively.
  • 1.0 ⁇ 10 -5 M GGsTop was added to the cells in the GGTi group containing PSSMG 24 hours before co-culture.
  • the cells on the coverslip (1) were washed three times with PBS buffer and then incubated with the fresh cells on the coverslip (2) in fresh RAPI-1640 medium for 10 h. Then wash the cells on the coverslip (2) with PBS buffer and incubate them with the fresh cells on the coverslip (3) in fresh culture medium for another 10 hours.
  • the experimental results are shown in Figure 16. It can be seen from the experimental results in Figure 16 that the nanosensitizer for chemoradiotherapy provided by the present invention has an effective sub-encapsulation effect.
  • a 3D tumor sphere experiment was used to verify the tumor penetration effect, using 3D tumor spheroids were obtained in 96-well hanging drop plates (3D Biomatrix, Michigan, USA). The specific steps are as follows: 40 ⁇ L of human pancreatic cancer cells (BxPC-3) in logarithmic phase with good growth status are seeded in the plate at 3 ⁇ 10 cells per well, and incubated at 37°C with 5% CO for 14 days. , replace part of the fresh RPMI-1640 culture medium every day. When the tumor sphere reaches the ideal size, add 1.0 ⁇ 10 -5 M Nile Red equivalent of PSSM and PSSMG to each well and incubate for 4 hours.
  • the subcutaneous pancreatic cancer model mice constructed by the model were divided into 8 groups (5 mice/group).
  • the designed experimental groups are: saline Control group, radiotherapy RT group (that is, no injection of any drugs or nanoparticles), metronidazole MI group, free drug cisplatin CDDP group, cisplatin chemotherapy prodrug CM group, non-drug-loaded PSSMG group, drug-loaded nanoparticle PSSMCM group and drug-loaded nanoparticle PSSMGCM group.
  • Administration via tail vein injection The total concentration of the drug is cisplatin equivalent 4 mg/kg, 100 ⁇ L/animal, administered once every 3 days. Radiotherapy with a dose of 3 Gy was performed 24 hours after the injection, administered 4 times. Measure and record the length (L) and width (W) of the tumor every 3 days, and weigh and record the weight of the mouse.
  • Vn is the tumor volume on the nth day.
  • V n is the tumor volume on the nth day
  • V 0 is the tumor volume of the initial administration mouse
  • the macromolecular chain transfer agent N 3 -PEG-CTA is replaced with the same molar amount of macromolecular chain transfer agent N 3 -PEG-S/S, and the monomer MASSMI is replaced with the same molar amount of a monomer containing a single selenium bond.
  • the remaining conditions were the same as Preparation Example 1 to obtain PSeM.
  • PSSM was replaced with the same molar amount of PSeM, and the remaining conditions were the same as Preparation Example 1 to obtain PSeMG.
  • PSSMG was replaced with the same weight part of PSeMG, and the other conditions were the same as Preparation Example 1 to obtain the radiochemotherapy nanosensitizer PSeMGCM, which was stored in a 4°C refrigerator.
  • the monomer MASSMI is replaced with the same molar amount of monosulfide bond-containing monomer, and the N 3 -PEG-CTA is replaced with the same molar amount of dithiocarbonate macromolecular chain transfer agent.
  • the remaining conditions are the same as Preparation Example 1. Get PSMG.
  • PSSM was replaced with the same molar amount of PSM, and the remaining conditions were the same as Preparation Example 1 to obtain PSMG.
  • PSSMG was replaced with the same weight part of PSMG, and the remaining conditions were the same as Preparation Example 1 to obtain the chemoradiotherapy nanosensitizer PSMGCM, which was stored in a 4°C refrigerator.
  • the monomer MASSMI is replaced with the same molar amount of trisulfide bond-containing monomer, and the N 3 -PEG-CTA is replaced with the same molar amount of dithiocarbamate macromolecular chain transfer agent.
  • the remaining conditions are the same as in Preparation Example 1 Same, get PSSSM.
  • PSSM was replaced with the same molar amount of PSSSM, and the remaining conditions were the same as Preparation Example 1 to obtain PSSSMG.
  • PSSMG was replaced with the same weight part of PSSSMG, and the other conditions were the same as Preparation Example 1 to obtain the radiochemotherapy nanosensitizer PSSSMGCM, which was stored in a 4°C refrigerator.
  • the reaction is quenched by liquid nitrogen, and neutral alumina is used to remove the copper salt.
  • pure water is used for dialysis to remove unreacted monomers and impurities, and the product is freeze-dried to obtain the product, which is recorded as PSCSM.
  • PSSM was replaced with the same molar amount of PSCSM, and the remaining conditions were the same as Preparation Example 1 to obtain PSCSMG.
  • PSSMG was replaced with the same weight part of PSCSMG, and the other conditions were the same as Preparation Example 1 to obtain the radiochemotherapy nanosensitizer PSCSMGCM, which was stored in a 4°C refrigerator.
  • the monomer MASSMI is replaced with the same molar amount of a diselenide bond-containing monomer, and the remaining conditions are the same as Preparation Example 1 to obtain PSeSeM.
  • PSSM was replaced with the same molar amount of PSeSeM, and the remaining conditions were the same as Preparation Example 1 to obtain PSeSeMG.
  • PSSMG was replaced with the same weight part of PSeSeMG, and the other conditions were the same as Preparation Example 1 to obtain the radiochemotherapy nanosensitizer PSeSeMGCM, which was stored in a 4°C refrigerator.
  • GBAP was replaced with the same weight part of fibroblast-responsive molecules, and the other conditions were the same as Preparation Example 1 to obtain PSSMF.
  • PSSMG was replaced with the same weight part of PSSMF, and the other conditions were the same as Preparation Example 1 to obtain the radiochemotherapy nanosensitizer PSSMFCM, which was stored in a 4°C refrigerator.
  • GBAP was replaced with the same weight part of metalloprotease-responsive molecules, and the remaining conditions were the same as Preparation Example 1 to obtain PSSMM.
  • PSSMG was replaced with the same weight part of PSSMM, and the other conditions were the same as Preparation Example 1 to obtain the radiochemotherapy nanosensitizer PSSMMCM, which was stored in a 4°C refrigerator.
  • GBAP was replaced with the same weight part of cathepsin-responsive molecules, and the other conditions were the same as Preparation Example 1 to obtain PSSMC.
  • PSSMG was replaced with the same weight part of PSSMC, and the other conditions were the same as Preparation Example 1 to obtain the radiochemotherapy nanosensitizer PSSMCCM, which was stored in a 4°C refrigerator.
  • the subcutaneous pancreatic cancer model mice constructed by the model were divided into 16 groups (5 mice/group).
  • the designed experimental groups are: saline Control group, radiotherapy RT group (that is, no injection of any drugs or nanoparticles), metronidazole MI group, free drug cisplatin CDDP group, cisplatin chemotherapy prodrug CM group, drug-free PSSMG group, drug-loaded nanoparticles (monosulfide bond + GGT enzyme response) PSMGCM ( S CM G ) group, drug-loaded nanoparticles ( Trisulfide bond + GGT enzyme response) PSSSMGCM ( SSS CM G ) group, drug-loaded nanoparticles (disulfide ketal bond + GGT enzyme response) PSCSMGCM ( SCS CM G ) group, drug-loaded nanoparticles (single selenium bond + GGT enzyme Response) PSeMGCM ( Se CM G ) group, drug-loaded nanoparticles (diselenium bond + GGT enzyme
  • the total concentration of the drug is cisplatin equivalent 4 mg/kg, 100 ⁇ L/animal, administered once every 3 days. Radiotherapy with a dose of 3 Gy was performed 24 hours after the injection, administered 4 times. Measure and record the length (L) and width (W) of the tumor every 3 days, and weigh and record the weight of the mouse.
  • Vn is the tumor volume on the nth day.
  • V n is the tumor volume on the nth day
  • V 0 is the tumor volume of the initial administration mouse

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Inorganic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

生物医用高分子材料领域的一种两亲嵌段聚合物和放化疗纳米增敏剂及其制备方法。经过化学改性，使得制备得到的两亲嵌段聚合物具有深度肿瘤穿透、酶响应性药物释放/激活的特性，同时将具有乏氧响应性的化疗前药与两亲嵌段聚合物通过物理包埋自组装得到稳定的高载药率的放化疗纳米增敏剂，其具有电荷反转以及级联响应特性。在影响大多数常规肿瘤个体化疗效的缺氧肿瘤微环境中，所述放化疗纳米增敏剂所释放的化疗前药和两亲嵌段聚合物可以有效地使肿瘤细胞对放化疗敏感，可以同时解决药物深度渗透以及克服缺氧脱敏的问题。

Description

一种两亲嵌段聚合物和放化疗纳米增敏剂及其制备方法
相关申请的交叉引用
本申请要求于2022年06月09日提交至中国专利局、申请号为2022106508360、申请名称为“一种两亲嵌段聚合物和放化疗纳米增敏剂及其制备方法”的中国专利申请的优先权，将其全部内容通过引用结合至本申请中。
技术领域
本发明属于生物医用高分子材料技术领域，具体涉及一种两亲嵌段聚合物和放化疗纳米增敏剂及其制备方法。
背景技术
随着现代社会的人类寿命增长以及环境因素影响，癌症正成为患病率最高以及致死率最高的疾病之一。据权威机构发布的论文显示，仅2020年一年共新增癌症患者就达1929万，死亡人数竟高达996万。目前临床常用的治疗方法是手术切除辅助化学治疗，这是唯一可以治愈的治疗方法。但是并不是所有患者都可以进行手术切除，另外手术切除存在术后并发症、手术切除不彻底以及切除过多造成的创伤性等缺点。对于无法进行手术切除的患者，则只能选择化学治疗和放射性疗法进行治疗。尽管目前临床上多种药物的综合化疗方法和不断研发的新型化疗药物延长了患者的生存期并提高了生存率，但是存在着产生毒副作用以及治疗效果并不显著等缺点，这不仅会减低患者的生存质量同时也限制了临床药物的治疗连续性，最终导致治疗效果有限、副作用过大等现状。
导致大多数癌症患者化疗效果有限的主要原因有两个：第一个原因是人体血液系统的清除系统，其限制了药物到达肿瘤组织的有效浓度；第二个原因是癌症组织存在致密的纤维化细胞间质、间质液产生的内压过高以及毛细管血管网缺失等因素，这阻碍了药物向肿瘤组织的扩散，尤其是大分子的扩散。传统的PEG水溶性纳米药物或者脂质体纳米药物尽管可以有效地避免身体中的清除系统，但是一方面其穿透能力很弱，另外一方面其又很难被癌症细胞吞噬，因此很大程度上限制了它们的疗效。
目前，具有促进渗透特性的智能纳米药物被认为可以克服这些生物屏障，这些药物具有靶向性、响应性并且可以随着环境改变自身理化性质，可以激活药物在肿瘤组织内部的有效被动扩散。这种理化性质的改变不仅可以提高纳米药物在体内的运输效率，同时本身阳离子的特性可以促进其被细胞吞噬以及增加其对肿瘤的渗透。因此，利用理化性质转变同时改变运输效率和渗透作用的新策略被认为是克服化疗治疗有限的解决方案。
另外，癌症内部血管缺失和致密间质导致内部产生乏氧区，乏氧会阻碍放疗治疗的效果，导致癌症患者表现出放疗抗性。目前临床上的解决方法是使用放射增敏剂进行辅助放疗，如吉西他滨、卡培他滨或氟尿嘧啶等。同时结合多个靶向药物和放射增敏剂的个体化放化疗也显示可以延长患者的生存期，增加对不同癌症患者的治疗差异性。此外，纳米技术也应用于放射增敏剂的个体化放化疗研究中，鉴于临 床上常用的小分子放射增敏剂药物具有系统毒性，具有更好安全性的纳米放射增敏剂不可避免地成为更具竞争力的选择。然而，目前研究报道的纳米技术无法同时解决药物深度渗透以及能够克服缺氧脱敏的问题。因此，亟需设计一种可以同时解决药物深度渗透以及能够克服缺氧脱敏问题的纳米技术。
发明内容
针对现有技术中的纳米技术无法同时解决药物深度渗透以及能够克服缺氧脱敏的问题，本发明提供了一种两亲嵌段聚合物和放化疗纳米增敏剂及其制备方法，将该放化疗纳米增敏剂用于解决肿瘤个体化治疗所面临的生物屏障和微环境限制。本发明经过化学改性制备得到的两亲嵌段聚合物具有深度肿瘤穿透、酶响应性药物释放/激活的特性以及乏氧响应性，同时将具有乏氧响应性的化疗前药与两亲嵌段聚合物通过物理包埋自组装得到稳定的高载药率放化疗纳米增敏剂，其具有电荷反转以及级联响应特性。在影响大多数常规肿瘤个体化疗效的缺氧肿瘤微环境中，本发明所提供的放化疗纳米增敏剂所释放的化疗前药和两亲嵌段聚合物可以有效地使肿瘤细胞对放化疗敏感。体外细胞实验验证了其在乏氧环境中具有电荷反转、高内吞速率、高细胞生长抑制效果和放疗敏感性，同时在构建的皮下肿瘤模型中，该放化疗纳米增敏剂可以实现显著的肿瘤抑制作用，展示了在放化疗联合个体化治疗领域中的极大应用潜力。
为了实现上述发明目的，本发明的第一方面提供了一种两亲嵌段聚合物，其中，所述两亲嵌段聚合物具有式(1)所示的结构：
其中，R1为活性自由基聚合引发基团，R10为H原子或C1-C5的烷基，R11为O原子或N原子，R2为酶响应分子，R3为乏氧响应基团，R30为氧化还原敏感基团，R4为引发剂连接基团，X为酶响应分子键合基团，10≤m≤100，20≤n≤200，m、n均为整数。
在一种优选的实施方式中，所述活性自由基聚合引发基团R1为二硫代酯及其衍生物、三硫代酯及其衍生物、二硫代碳酸酯及其衍生物、二硫代氨基甲酸酯及其衍生物或卤素基团及其衍生物。
在一种优选的实施方式中，所述乏氧响应基团R3为2-硝基咪唑及其衍生物、4-硝基咪唑及其衍生物或5-硝基咪唑及其衍生物。
在一种优选的实施方式中，所述氧化还原敏感基团R30为二硫缩酮键、单硫键、单硒键、二硫键、三硫键或二硒键。
在一种优选的实施方式中，所述酶响应分子R2为γ-谷氨酰转肽酶响应分子、成纤维蛋白酶响应分子、 金属蛋白酶响应分子或组织蛋白酶响应分子。
在一种优选的实施方式中，所述酶响应分子键合基团X具有式(3)所示的结构：
本发明的第二方面提供了一种放化疗纳米增敏剂，其中，所述放化疗纳米增敏剂含有上述两亲嵌段聚合物和化疗前药，所述两亲嵌段聚合物和化疗前药的质量比为100:(0～100)；所述化疗前药具有式(4)所示的结构：
其中，R5为抗癌化疗药物分子。
在一种优选的实施方式中，所述抗癌化疗药物分子R5为紫杉醇、紫杉醇衍生物、阿霉素、表阿霉素、喜树碱、喜树碱衍生物、顺铂类药物、长春碱、长春新碱、多西他赛、吉西他滨、姜黄素或丹酚酸。
本发明的第三方面提供了一种放化疗纳米增敏剂的制备方法，其中，该方法包括以下步骤：
S1：于避光条件下，将上述两亲嵌段聚合物以及化疗前药溶解于有机溶剂I中，得到含两亲嵌段聚合物和化疗前药的溶液；所述化疗前药具有式(4)所示的结构：
其中，R5为抗癌化疗药物分子；
S2：将步骤S1所得含两亲嵌段聚合物和化疗前药的溶液加入到有机溶剂II-纯水混合相中进行高速搅拌，得到含放化疗纳米增敏剂的溶液；
S3：将步骤S2所得含放化疗纳米增敏剂的溶液于纯水中进行透析以除去有机溶剂I和有机溶剂II，得到放化疗纳米增敏剂。
本发明的第四方面提供了由上述方法制备得到的放化疗纳米增敏剂。
本发明为了克服肿瘤放化疗个体化治疗过程中的药物递送障碍和放射抗性，构建了一种具有高载药效率、高稳定性和级联响应特性的放化疗纳米增敏剂。通过化学修饰，在两亲嵌段聚合物上同时引入酶响应分子、乏氧响应基团以及氧化还原敏感基团，该聚合物在酶激活过后可以实现电荷转换，实现了有效的内吞作用和转胞吞作用，从而进一步驱动肿瘤深度穿透。在两亲嵌段聚合物上负载的化疗前药因为有甲硝唑的化学修饰，因此其载药率和包封率都比较高，并且可以在癌细胞内被过量酶选择性地激活。此外，在影响大多数常规个体化放化疗效的缺氧肿瘤微环境中，释放的化疗药物和甲硝唑残基可以有效地使肿瘤细胞对放化疗敏感。
附图说明
图1为N3-PEG-CTA的核磁谱图；
图2为MASSCOOH的核磁谱图；
图3为MASSMI的核磁谱图；
图4为PSSM的核磁谱图；
图5为BAP的核磁谱图；
图6为GBAP的核磁图谱；
图7为PSSMG的核磁图谱；
图8为放化疗纳米增敏剂纳米粒子在GSH(10μM、5mM和10mM)响应下CM释放曲线；
图9为放化疗纳米增敏剂纳米粒子在10mM的GSH响应下DLS粒径变化曲线；
图10为放化疗纳米增敏剂纳米粒子在乏氧响应下CM释放曲线；
图11为放化疗纳米增敏剂纳米粒子在乏氧响应下DLS粒径变化曲线；
图12为放化疗纳米增敏剂纳米粒子在GGT酶催化下Zeta电位电荷反转曲线；
图13为放化疗纳米增敏剂纳米粒子在GGT酶催化下细胞内吞量柱状图；
图14(A)为常氧下不同给药实验组和(B)为乏氧下不同给药实验组与BxPC-3细胞作用的MTT细胞生存曲线；
图15为乏氧下不同给药实验组与BxPC-3细胞作用的细胞放疗增敏代表性集落图片；
图16为PSSMG纳米粒子在GGT酶催化下转胞吞作用的共聚焦显微镜图片；
图17为PSSM纳米粒子和PSSMG纳米粒子对肿瘤渗透的3D肿瘤球的共聚焦显微镜图片；
图18和图19为不同给药实验组对皮下BxPC-3胰腺癌皮下肿瘤模型小鼠的联合协同抑瘤曲线。
具体实施方式
以下将通过实施例对本发明进行详细描述。
本发明的第一方面提供了一种两亲嵌段聚合物，其中，所述两亲嵌段聚合物具有式(1)所示的结构：
其中，R1为活性自由基聚合引发基团，R10为H原子或C1-C5的烷基，R11为O原子或N原子，R2为酶响应分子，R3为乏氧响应基团，R30为氧化还原敏感基团，R4为引发剂连接基团，X为酶响应分子键合基团，10≤m≤100，20≤n≤200，m、n均为整数。
本发明中，所述活性自由基聚合引发基团R1可以为可逆加成-断裂链转移聚合(RAFT)链转移基团、原子转移自由基聚合(ATRP)链转移基团或其他非自由基聚合的链转移基团中的至少一种。优选地，所述活性自由基聚合引发基团R1可以为二硫代酯及其衍生物、三硫代酯及其衍生物、二硫代碳酸酯及其衍生物、二硫代氨基甲酸酯及其衍生物或卤素基团及其衍生物。在一种具体的实施方式中，所述活性自由基聚合引发基团R1为三硫代酯及其衍生物，其具体结构特别优选如式(1-1)所示：
其中，R1-1可以为C1-C20的烷基，优选为C10-C20的烷基，更优选为正十二烷基。
本发明中，所述酶响应分子R2与两亲嵌段聚合物连接的酶切作用位点为上述式(1)所示结构中的氨基，酶切作用过后暴露氨基使两亲嵌段聚合物带正电荷。所述酶响应分子R2优选为γ-谷氨酰转肽酶响应分子、成纤维蛋白酶响应分子、金属蛋白酶响应分子或组织蛋白酶响应分子，更优选为γ-谷氨酰转肽酶响应分子。
本发明中，其中，式(1)中含有具有式(2)所示结构的刺激响应性化学键：
式(2)中，R10可以为H原子或C1-C5的烷基，R11可以为O原子或N原子。
本发明中，所述乏氧响应基团R3优选为2-硝基咪唑及其衍生物、4-硝基咪唑及其衍生物或5-硝基咪唑及其衍生物，在一种具体的实施方式中，所述乏氧响应基团R3为甲硝唑，其在乏氧环境下可以被还原，从而具有乏氧响应性。
本发明中，所述氧化还原敏感基团R30优选为二硫缩酮键、单硫键、单硒键、二硫键、三硫键或二硒键，更优选为二硫键，其能够被谷胱甘肽还原，从而具有氧化还原响应性。
在一种具体的实施方式中，本发明所提供的两亲嵌段聚合物具有乏氧响应(甲硝唑)和还原响应(二硫键)的双响应性质。
本发明对所述引发剂连接基团R4为没有特别的限定，只要能够起到连接两亲嵌段聚合物即可。在一种具体的实施方式中，所述引发剂连接基团R4具有式(1-2)所示的结构：
本发明中，所述酶响应分子键合基团X为通过(a)酰胺化反应、(b)点击化学和(c)迈克尔加成反应中的至少一种将酶响应分子R2引入到两亲嵌段聚合物上所形成的键合基团，所述酶响应分子键合基团X具有式(3)所示的结构：
在一种具体的实施方式中，所述酶响应分子键合基团X为通过(b)点击化学将酶响应分子R2引入到两亲嵌段聚合物上所形成的键合基团。考虑到合成的难易程度，酶响应分子键合基团X特别优选为三氮唑。所述三氮唑的结构如式(3b-1)所示：
本发明通过化学改性制备得到的两亲嵌段聚合物具有乏氧响应性、氧化还原响应性以及酶响应性，从而具有深度肿瘤穿透、酶反应性药物释放/激活的特性。
本发明的第二方面提供了一种放化疗纳米增敏剂，其中，含有上述两亲嵌段聚合物和化疗前药，所述两亲嵌段聚合物和化疗前药的质量比为100:(0～100)，如100:0、100:0.1、100:1、100:5、100:10、100:15、100:20、100:30、100:40、100:50、100:60、100:70、100:80、100:90、100:100等。其中，所述化疗前药具有式(4)所示的结构：
其中，R5为抗癌化疗药物分子。
优选地，所述抗癌化疗药物分子R5为紫杉醇、紫杉醇衍生物、阿霉素、表阿霉素、喜树碱、喜树碱衍生物、顺铂类药物、长春碱、长春新碱、多西他赛、吉西他滨、姜黄素和丹酚酸中的至少一种。考虑到合成的难易程度，化疗前药的具体实施例包括但不限于为顺铂类药物，顺铂类化疗前药的结构如式(4-1)所示：
本发明的第三方面提供了一种放化疗纳米增敏剂的制备方法，其中，该制备方法包括以下步骤：
S1：于避光条件下，将上述两亲嵌段聚合物以及化疗前药溶解于有机溶剂I中，得到含两亲嵌段聚合物和化疗前药的溶液；所述化疗前药具有式(4)所示的结构：
其中，R5为抗癌化疗药物分子
S2：将步骤S1中所得含两亲嵌段聚合物和化疗前药的溶液加入到有机溶剂II-纯水混合相中进行高速搅拌，得到含放化疗纳米增敏剂的溶液；
S3：将步骤S2中所得含放化疗纳米增敏剂的溶液于纯水中进行透析以除去有机溶剂I、II，得到放化疗纳米增敏剂。
需要说明的是，在本发明中，为了便于描述，将制备过程中所需的两次有机溶剂称为“有机溶剂I”和“有机溶剂II”的目的仅是为了区别不同对象，而不能理解为指示或暗示其的相对重要性。
本发明对步骤S1中所采用的有机溶剂I没有特别的限制，只要是能够将两亲嵌段聚合物以及化疗前药充分溶解即可，优选地，有机溶剂I的具体实例包括但不限于：DMF和/或DMSO。
本发明对步骤S2中所采用的有机溶剂II没有特别的限制，只要是其与纯水的混合相能够制备得到物理包埋自组装的放化疗纳米增敏剂即可。优选地，有机溶剂II与有机溶剂I为相同的有机溶剂。本发明对步骤S2中所采用的高速搅拌的条件没有特别的限定，优选采用搅拌速率为400～600rpm，例如：400rpm、600rpm、600rpm。搅拌时间为40～80min，例如40min、50min、60min、70min、80min。
本发明对步骤S3中的透析条件没有特别的限制，只要是能够将有溶剂机I、II除净即可。
本发明的第四方面提供了一种由上述方法制备得到的放化疗纳米增敏剂。
制备例1
(1)合成叠氮端甲氧基聚乙二醇二硫代正十二烷酯(N3-PEG-CTA)链转移剂
(1a)合成小分子引发剂(CTA-COOH)
准确称取(7.3g,130mmol)KOH固体于三颈烧瓶中并加入220mL去离子水溶解，缓慢滴加(24mL,95mmol)正十二硫醇并高速搅拌形成水乳浊液。再准确称取(0.4g,1mmol)Aliqut336(甲基三辛基氯化铵)和(6mL,100mmol)CS2，混匀后缓慢滴加至上述水乳浊液中，滴加完毕后于室温下反应1h。接着于冰盐浴-5℃下缓慢加入(10g,52.45mmol)对甲苯磺酰氯反应2h，之后再于冰浴0℃下反应1h。反应结束后采用布氏漏斗进行过滤，冰水洗涤得黄色固体，进一步采用丙酮进行重结晶，过滤，所得晶体即为中间产物，记为1-a。
准确称取(3.25g)中间产物1-a和(3.25g,11.60mmol)引发剂V501(4,4'-偶氮双(4-氰基戊酸))，加入乙酸乙酯溶解并于90℃下避光加热回流24h。反应结束后对所得溶液进行旋蒸浓缩得到浓缩液，浓缩液以纯乙酸乙酯作为流动相进行硅胶柱过柱纯化，收集合并流动相并进行旋蒸得到黄色固体，记为CTA-COOH。
(1b)合成大分子链转移剂N3-PEG-CTA：
准确称取(2.25g,0.45mmol)叠氮端甲氧基聚乙二醇mPEG5K-N3以及(1.612g,4mmol)CTA-COOH并溶于50mL新鲜除水的二氯甲烷中，依次加入4-二甲氨基吡啶(DMAP)、乙酸乙酯和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)，于室温条件下反应3d。反应结束后采用布氏漏斗进行过滤，收集滤液并旋干溶剂后采用无水乙醚进行重结晶并过滤，将固体产物置于真空烘箱中进行干燥12h得到所需产物，记为N3-PEG-CTA，其核磁谱图见图1。从图1可以看出，得到的产物即为N3-PEG-CTA。
(2)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇二硫代酯(PSSM)
(2a)合成单体MASSMI
准确称取(1g,7.68mmol)甲基丙烯酸羟乙酯HEMA、(6.45g,30.72mmol)3,3'-二硫代二丙酸DTDPA和(85.29mg,0.7mmol)DMAP进行混合，加入60mL新鲜除水的THF后并于室温下反应12h，得到反应混合物。接着在0℃冰浴条件下冷却反应混合物1h。将(1.74g,8.44mmol)二环己基碳二亚胺(DCC)溶于20mL新鲜除水的THF后滴入到反应混合物中进行剧烈搅拌，在室温下反应24h。反应结束后采用布氏漏斗进行过滤，收集滤液并进行旋蒸，加入50mL CHCl3溶解后过滤，并采用盐水洗涤三次后，旋转蒸发即得产物，记为MASSCOOH，其核磁谱图见图2。从图2可以看出，得到的产物即为MASSCOOH。
准确称取(7.431g,23mmol)MASSCOOH、(3.944g,23mmol)甲硝唑(MI)和(0.25g,2.3mmol)DMAP进行混合，加入100mL新鲜除水的THF后在室温下反应12h，得到反应混合物。接着在0℃冰浴条件下冷却反应混合物1h。将(5.22g,25.32mmol)DCC溶于20mL新鲜除水的THF后滴入到反应混合物中进行剧烈搅拌，在室温下反应24h。反应结束后采用布氏漏斗进行过滤，收集滤液并进行旋蒸浓缩后进行硅胶柱过柱纯化，收集合并流动相并进行旋蒸得到产物，记为MASSMI，其核磁谱图见图3。从图3可以看出，得到的产物即为MASSMI。
(2b)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇二硫代酯(PSSM)
取一干燥洁净的Schlenk瓶，进行抽真空-充氩气三次，在氩气环境中依次加入准确称量好的(0.2g,0.036mmol)大分子链转移剂N3-PEG-CTA、2mg引发剂V501和(0.6g,1.26mmol)单体MASSMI，用5mL超干DMSO溶剂进行搅拌溶解，反应体系冷冻抽排循环三次，然后在70℃条件下反应24h。反应结束后液氮猝灭反应，采用纯水进行透析除去未反应的单体及杂质，冻干得到产物，记为PSSM，其核磁谱图见图4。从图4可以看出，得到的产物即为PSSM。
(3)合成具有酶响应和氧化还原响应的双响应叠氮端甲氧基聚乙二醇二硫代酯(PSSMG)
(3a)合成GGT酶响应缀合物(GBAP)
准确称取(2g,9.85mmol)Boc-L-2-氨基丁酸和(1.78g,10.98mmol)N,N-碳酰二咪唑(CDI)溶解在20mL新鲜除水的二氯甲烷中，并于室温下搅拌2h后加入(0.4g,15mmol)炔丙胺和(3.5mL,24.7mmol)三乙胺，在室温下反应24h。反应结束后分别采用1M HCl(50mL)和饱和NaHCO3水溶液(50mL)进行洗涤三次，收集有机层进行无水Na2SO4干燥后真空蒸发，将所得产物记为BTP。之后将所得产物BTP溶于(v:v＝1:1,8mL)CH2Cl2/TFA(三氟乙酸)溶液中，室温下搅拌过夜后进行旋蒸得到中间产物，记为BAP，其核磁谱图见图5。从图5可以看出，得到的产物即为BAP。
准确称取(0.42g,3.72mmol)BAP和(0.67g,4.14mmol)CDI溶解在20mL新鲜除水的二氯甲烷中，并于室温下搅拌2h后加入(1.3mL,9.32mmol)三乙胺和(1.13g，3.72mmol)boc-Glu-otbu(Boc-L-谷氨酸-1-叔丁酯)，在室温下反应24h，接着分别采用1M HCl(50mL)以及饱和NaHCO3水溶液(50mL)进行洗涤三次，收集有机层进行无水Na2SO4干燥后真空蒸发，将所得产物记为GBTP。之后将所得产物BTP溶于(v:v＝1:1,8mL)CH2Cl2/TFA(三氟乙酸)溶液中，室温下搅拌过夜后进行旋蒸得到中间产物，记为GBAP，其核磁谱图见图6。从图6可以看出，得到的产物即为GBAP。
(3b)合成具有酶响应和氧化还原响应的双响应叠氮端甲氧基聚乙二醇二硫代酯(PSSMG)
PSSMG是通过炔基封端的GBAP和叠氮基封端的PSSM经CuAAC点击化学合成。将(0.02g,0.28mmol)GBAP、(0.2g,0.028mmol)PSSM和(80mg,0.028mmol)五甲基二乙烯三胺(PMDETA)以及5mL水加入到25mL的schlenk瓶中，在对体系进行冷冻抽排解冻循环三次之后，在氩气保护下添加(0.055g,0.28mmol)抗坏血酸钠和(0.035g,0.14mmol)硫酸铜五水化物，在室温下反应24h后结束反应，之后将所得溶液用PBS缓冲液透析24h，冻干得到固体产物，记为PSSMG，其核磁谱图见图7。从图7可以看出，得到的产物即为PSSMG。PSSMG聚合物为两亲嵌段聚合物，同时具有酶响应和氧化还原响应的双响应特性。
(4)合成顺铂化疗前药(CM)
(4a)合成氧化顺铂(Pt-OH)
准确称取(1g,3.33mmol)顺铂并溶于25mL水中，然后滴加35mL双氧水溶液，在70℃条件下避光反应5h。反应结束后于4℃条件下结晶，采用布氏漏斗进行过滤，所得产物用冰水和冰乙醇分别洗涤，干燥后得到氧化顺铂产物，记为Pt-OH。
(4b)合成羧酸化顺铂(Pt-COOH)
准确称取(0.8g,2.4mmol)Pt-OH并溶于30mL新鲜除水的DMF中，加入(0.24g,2.4mmol)丁二酸酐，在75℃条件下反应12h。反应结束后采用布氏漏斗进行过滤，收集滤液进行旋蒸浓缩后于冰乙醚和丙酮中沉淀重结晶，真空干燥得到羧酸化顺铂，记为Pt-COOH。
(4c)合成顺铂化疗前药(CM)
准确称取(0.5g,1.15mmol)Pt-COOH、(0.197g,1.15mmol)甲硝唑、(0.22g，1.15mmol)EDC和(0.014 g，0.015mmol)DMAP并溶于20mL新鲜除水的DMF中，室温下反应24h。反应结束后采用布氏漏斗进行过滤，收集滤液进行旋蒸浓缩得到浓缩液，浓缩液以乙酸乙酯作为流动相进行硅胶柱过柱纯化，收集合并流动相并进行旋蒸得到顺铂化疗前药，记为CM。
(5)合成放化疗纳米增敏剂
准确称取10mg具有酶响应和氧化还原响应的双响应叠氮端甲氧基聚乙二醇二硫代酯PSSMG和5mg顺铂化疗前药CM，将这两者加入1mLDMF中进行充分溶解，得到含两亲嵌段聚合物PSSMG和顺铂化疗前药CM的溶液。随后用注射器吸取1mL含两亲嵌段聚合物PSSMG和顺铂化疗前药CM的溶液，安置在注射泵上，以1mL/h流速注入到9mL混合相(由DMF和十倍体积的纯水组成)当中以500rpm的搅拌速率进行高速搅拌60min得到含物理包埋自组装的放化疗纳米增敏剂的溶液。组装完成后继续搅拌24h，随后将含放化疗纳米增敏剂的溶液转移至MWCO3000KD的透析袋中，在超纯水中透析以除去DMF有机溶剂。透析结束后，用0.45μm滤膜进行过滤得到纯化的放化疗纳米增敏剂，并放入4℃冰箱冷藏保存。
测试例1制备例1所得放化疗纳米增敏剂性能的测试
(1)GSH响应体外释放
采用渗析法研究了在10μM、5mM和10mM谷胱甘肽(GSH)的浓度下，放化疗纳米增敏剂在20mL(pH＝7.4,0.01M)的PBS缓冲液中的药物释放行为。具体操作步骤如下：将制备例1所得负载有顺铂化疗前药CM的3mL放化疗纳米增敏剂溶液和GSH的MWCO3000KD渗析袋置于20mL(pH＝7.4,0.01M)的PBS缓冲液(吐温80体积浓度为0.5％)中，离心管用锡纸包裹进行避光处理，放入37℃、100r/min恒温摇床中培养24h。在1h、2h、9h、12h、24h、48h和96h时分别进行取样，释放至透析袋外的CM的含量通过HPLC监测，实验结果见图8。通过DLS表征监测高GSH浓度(10mM)环境下放化疗纳米增敏剂纳米粒子的粒径分布，实验结果见图9。每组实验均设定三组平行实验。从图8和图9实验结果可知，本发明所提供的放化疗纳米增敏剂溶液具有GSH反应性药物释放/激活的特性。
(2)乏氧响应释放
采用渗析法研究了在乏氧环境中，制备例1所得放化疗纳米增敏剂在20mL(pH＝7.4,0.01M)的PBS缓冲液中的药物释放行为。具体操作如下：为了模拟体内缺氧环境，对一个三颈烧瓶进行抽真空，并在持续通入氩气的情况下，加入100μM亚硫酸氢钠溶液。将制备例1所得负载有顺铂化疗前药CM的3mL的放化疗纳米增敏剂溶液的MWCO3000KD渗析袋放入三颈烧瓶当中，使用亚硫酸氢钠来模拟体内的缺氧还原环境。三颈烧瓶用锡纸包裹进行避光处理，放入37℃、100r/min恒温摇床中培养24h。在1h、2h、9h、12h、24h、48h和96h时分别进行取样，释放至透析袋外的CM的含量通过HPLC监测，实验结果见图10。通过DLS表征监测乏氧环境下放化疗纳米增敏剂纳米粒子的粒径分布，实验结果见图11。每组实验均设定三组平行实验。从图10和图11实验结果可知，本发明所提供的放化疗纳米增敏剂溶液在乏氧条件下具有CM药物释放/激活的特性。
(3)GGT酶响应电荷反转测定
研究了制备例1所得放化疗纳米增敏剂在谷酰胺转肽酶(GGT酶)的催化下的电荷反转过程。具体 操作如下：准确称取2mg放化疗纳米增敏剂溶于1mL(pH＝7.4,10mM)的Hepes缓冲液中，加入10U的GGT酶，放入37℃、100r/min恒温摇床中培养24h。在0h、2h、4h、8h、16h和24h时分别进行取样，通过粒径分析仪表征监测在GGT酶催化环境下放化疗纳米增敏剂纳米粒子的Zeta电位，实验结果见图12。每组实验均设定三组平行实验。从图12实验结果可知，本发明所提供的放化疗纳米增敏剂具有电荷反转特性。
(4)细胞内吞实验
将人胰腺癌细胞(BxPC-3)以每孔105个细胞数均匀铺在六孔板中，设置对照组(NPs)和GGTi组(GGTi组在共培养前24h向细胞中加入1.0×10-5M的GGsTop)，于各组中分别加入含5μg/mL尼罗红的PSSM纳米粒子和5μg/mL尼罗红的PSSMG纳米粒子，对细胞进行孵育，孵育6h后用胰酶消化，用PBS缓冲液洗涤离心两次后再加入500μLPBS缓冲液制备成细胞悬浮液，用细胞流式仪检测其细胞内荧光值，实验结果见图13。从图13实验结果可知，本发明所提供的放化疗纳米增敏剂具有高内吞速率。
(5)MTT细胞毒性实验
将生长状态良好处于对数期的人胰腺癌细胞(BxPC-3)和小鼠胚胎成纤维细胞(NIH-3T3)以5000个/孔的细胞密度加入至96孔板中，弃用孔板外圈孔，改加PBS缓冲液。待观察细胞已经贴壁生长，长满整个培养皿的70％左右，吸走旧培养基，加入100μL稀释好的顺铂化疗前药CM、游离药物顺铂CDDP(指化疗药物顺铂)、载药纳米粒子PSSMCM(指制备例1(2)中合成的PSSM进行CM药物负载，即未进行GGT酶响应分子修饰，其制备方法同制备例1(5))和载药纳米粒子PSSMGCM(指制备例1(3a)合成的PSSMG进行CM药物负载，即制备例1(5)中合成得到的放化疗纳米增敏剂)，浓度梯度依次为100μM、64μM、32μM、16μM、8μM、4μM、2μM、1μM和0.5μM，每个浓度均设3个平行组，而后分别将BXPC-3细胞的两组孔板分别在常氧及乏氧培养箱中孵育24h，将NIH-3T3细胞孔板置于常规培养箱中孵育24h。一段时间后，96孔板替换为每孔加入10μL MTT溶液和90μL RPMI-1640培养基，继续孵育4h。之后在每孔加入100μL的Formazan溶解液，适当混匀，在细胞培养箱内再继续孵育待Formazan全部溶解。最后在570nm处测定吸光度，计算细胞存活率，利用公式(1)计算细胞相对存活率：
细胞相对存活率(％)＝(Ax-A0)/(Ac-A0)×100…………………(1)
其中Ax、A0以及Ac分别为实验组、调零组以及阴性对照组吸光度值。
常氧和乏氧下CM、CDDP、PSSMCM和PSSMGCM给药实验组与BxPC-3细胞作用的MTT细胞生存曲线见图14。从图14实验结果可知，本发明所提供的放化疗纳米增敏剂在乏氧环境中具有高细胞生长抑制效果。
(6)细胞放疗增敏实验
(6a)放疗照射参数
采用其电子直线加速器(23Ex,Varian,USA)为照射源。剂量率设定为2Gy/min，机头旋转180°，固定照射面积是15×15cm，将1.5cm剂量补偿器放置在培养板上以消除剂量累积效应。
(6b)具体实验流程
将生长状态良好处于对数期的人胰腺癌细胞(BxPC-3)接种到6孔板中，并分别在乏氧条件下孵育。 设置对照组(Ctrl组)、甲硝唑(MI组)以及各实验组中分别加入1.0×10-5M Pt当量的顺铂化疗前药CM、游离药物顺铂CDDP、纳米粒子PSSMG和载药纳米粒子PSSMGCM，孵育48h，然后用0Gy、2Gy和4Gy的X射线按照上述条件照射。照射完成后，将六孔板置于冰上，抑制细胞的自我修复功能。低温放置0.5h后，用胰蛋白酶消化细胞，按照每孔1000个细胞接种在六孔板上，然后加入新鲜RPMI-1640培养基继续培养；14天后，用戊二醛(6.0％v/v)固定肉眼可见的细胞群，室温结晶紫(0.5％w/v)染色1h，计数克隆≥50个细胞，实验结果见图15。从图15实验结果可知，本发明所提供的放化疗纳米增敏剂在乏氧环境中具有良好的放疗敏感性。
(7)转胞吞实验
将BxPC-3细胞接种在盖玻片(1)-(3)上并孵育过夜。每个步骤使用两张平行的盖玻片：一张用于Hoechst 33342染色15分钟后的CLSM观察；另一个用于下一步的共同孵化。首先将盖玻片(1)上的细胞分别与尼罗红标记的PSSM(1.0×10-5M)和尼罗红标记的PSSMG(1.0×10-5M)一起孵育6h。此外，含有PSSMG的GGTi组在共培养前24小时向细胞中加入1.0×10-5M的GGsTop。盖玻片(1)上的细胞用PBS缓冲液洗涤3次，然后与盖玻片(2)上的新鲜细胞在新鲜RAPI-1640培养基中孵育10h。然后用PBS缓冲液冲洗盖玻片(2)上的细胞，并与盖玻片(3)上的新鲜细胞在新鲜培养基中再孵育10h，实验结果见图16。从图16实验结果可知，本发明所提供的放化疗纳米增敏剂具有有效的转包吞作用。
(8)肿瘤穿透实验
采用3D肿瘤球实验来验证肿瘤穿透效果，使用96孔悬滴板(3D Biomatrix,Michigan,USA)获得3D肿瘤球。具体操作步骤如下：将40μL生长状态良好处于对数期的人胰腺癌细胞(BxPC-3)以每孔3×105个细胞接种在板中，在37℃下用5％CO2孵育14天，每天更换部分新鲜RPMI-1640培养基。当肿瘤球达到理想大小时，每孔加入1.0×10-5M尼罗红当量的PSSM和PSSMG后孵育4h。孵育后，使用激光共聚焦显微镜在z堆栈扫描模式下拍摄图像。在抑制实验中，3D球体在实验前24h用1.0×10-5M的GGTi预处理，然后在每孔中加入等量的尼罗红标记的PSSMG纳米颗粒，再孵育4h。之后，使用相同的条件在激光共聚焦显微镜下观察3D肿瘤球，实验结果见图17。从图17实验结果可知，本发明所提供的放化疗纳米增敏剂具有深度肿瘤穿透能力。
(9)抑瘤实验
将模型构建好的皮下胰腺癌模型小鼠分为8组(5只/组)，设计实验组分别为：生理盐水Control组、放疗RT组(即不注射任何药物和纳米粒子)、甲硝唑MI组、游离药物顺铂CDDP组、顺铂化疗前药CM组、无载药PSSMG组、载药纳米粒子PSSMCM组和载药纳米粒子PSSMGCM组。通过尾静脉注射给药：药物总浓度为顺铂当量4mg/kg，100μL/只，每隔3天给一次药，注射完24h后进行剂量为3Gy的放疗，给药4次。每3天量取并记录肿瘤的长(L)和宽(W)，以及称量记录小鼠体重，小鼠肿瘤体积按公式(2)计算：
Vn＝1/2LW2…….………………(2)
其中Vn为第n天肿瘤体积。相对肿瘤体积比按公式(3)为：
A＝Vn/V0……….………………(3)
其中Vn为第n天肿瘤体积，V0为起始给药小鼠肿瘤体积。
从图18实验结果可以看出，本发明所提供的放化疗纳米增敏剂可以实现显著的肿瘤抑制作用，展 示了其在放化疗联合个体化治疗领域中的极大应用潜力。
制备例2
(1)合成二硫代酯大分子链转移剂
准确称取(2.25g,0.45mmol)叠氮端甲氧基聚乙二醇mPEG5K-N3以及(0.849g,4mmol)S-(硫代苯甲酰基)硫代乙酸并溶于50mL新鲜除水的二氯甲烷中，依次加入4-二甲氨基吡啶(DMAP)、乙酸乙酯和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)，于室温条件下反应3d。反应结束后采用布氏漏斗进行过滤，收集滤液并旋干溶剂后采用无水乙醚进行重结晶并过滤，将固体产物置于真空烘箱中干燥12h，得到二硫代酯大分子链转移剂N3-PEG-S/S。
(2)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇二硫代酯(PSeM)
(2a)合成含单硒键单体
准确称取(1g,7.68mmol)甲基丙烯酸羟乙酯HEMA、(9.34g,30.72mmol)3,3'-硫代二丙酸和(85.29mg,0.7mmol)DMAP进行混合，加入60mL新鲜除水的THF后并于室温下反应12h，得到反应混合物。接着在0℃冰浴条件下冷却反应混合物1h。将(1.74g,8.44mmol)二环己基碳二亚胺(DCC)溶于20mL新鲜除水的THF后滴入到反应混合物中进行剧烈搅拌，在室温下反应24h。反应结束后采用布氏漏斗进行过滤，收集滤液并进行旋蒸，加入50mL CHCl3溶解后过滤，收集滤液并进行旋蒸浓缩后进行硅胶柱过柱纯化，流动相为(V乙酸乙酯:V正己烷＝1:2.5)，收集合并流动相并进行旋转蒸发即得产物含单硒键单体。
(2b)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇二硫代酯(PSeM)
将大分子链转移剂N3-PEG-CTA采用相同摩尔量的大分子链转移剂N3-PEG-S/S替代，并将单体MASSMI采用相同摩尔量的含单硒键单体替代，其余条件与制备例1相同，得到PSeM。
(3)合成具有酶响应和氧化还原响应的双响应叠氮端甲氧基聚乙二醇二硫代酯(PSeMG)
将PSSM采用相同摩尔量的PSeM替代，其余条件与制备例1相同，得到PSeMG。
(4)合成顺铂化疗前药(CM)
与制备例1相同。
(5)合成放化疗纳米增敏剂
将PSSMG采用相同重量份的PSeMG替代，其余条件与制备例1相同，得到放化疗纳米增敏剂PSeMGCM，并放入4℃冰箱冷藏保存。
制备例3
(1)合成二硫代碳酸酯大分子链转移剂
(1a)合成二硫代碳酸酯小分子引发剂
准确称取(16.3g，0.1mol)乙基黄原酸钾、(17.91g，0.15mol)氯仿、(88.1g，1.0mol)环己酮溶于150mL甲苯中，混合15min后滴加(1.36g，4mmol)硫酸氢四丁基铵，混合完毕后将反应体系在氮气下冷却至15℃，并搅拌30min。搅拌完毕后分批加入(20g，0.5mol)氢氧化钠，加入冰块使温度始终保持在25℃以下。加完后将反应物搅拌过夜。待反应完毕，往混合物中加入100mL水，用浓盐酸酸化水层，酸化完毕后使用乙酸乙酯萃取产物三次，合并所有的乙酸乙酯层，并用无水硫酸镁干燥过夜。第二天减压蒸去溶剂在干燥后收集固体，将其从环己烷中重结晶，得到二硫代碳酸酯小分子引发剂。
(1b)合成二硫代碳酸酯大分子链转移剂：
将CTA-COOH采用相同摩尔量的二硫代碳酸酯小分子引发剂替代，其余条件与制备例1相同，得到二硫代碳酸酯大分子链转移剂。
(2)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇二硫代酯(PSM)
(2a)合成含单硫键单体
准确称取(1g,7.68mmol)甲基丙烯酸羟乙酯HEMA、(5.47g,30.72mmol)3,3'-硫代二丙酸和(85.29mg,0.7mmol)DMAP进行混合，加入60mL新鲜除水的THF后并于室温下反应12h，得到反应混合物。接着在0℃冰浴条件下冷却反应混合物1h。将(1.74g,8.44mmol)二环己基碳二亚胺(DCC)溶于20mL新鲜除水的THF后滴入到反应混合物中进行剧烈搅拌，在室温下反应24h。反应结束后采用布氏漏斗进行过滤，收集滤液并进行旋蒸，加入50mL CHCl3溶解后过滤，收集滤液并进行旋蒸浓缩后进行硅胶柱过柱纯化，流动相为(V乙酸乙酯:V正己烷＝1:3)，收集合并流动相并进行旋转蒸发即得产物含单硫键单体。
(2b)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇二硫代酯(PSM)
将单体MASSMI采用相同摩尔量的含单硫键单体替代，并将N3-PEG-CTA采用相同摩尔量的二硫代碳酸酯大分子链转移剂替代，其余条件与制备例1相同，得到PSMG。
(3)合成具有酶响应和氧化还原响应的双响应叠氮端甲氧基聚乙二醇二硫代酯(PSMG)
将PSSM采用相同摩尔量的PSM替代，其余条件与制备例1相同，得到PSMG。
(4)合成顺铂化疗前药(CM)
与制备例1相同。
(5)合成放化疗纳米增敏剂
将PSSMG采用相同重量份的PSMG替代，其余条件与制备例1相同，得到放化疗纳米增敏剂PSMGCM，并放入4℃冰箱冷藏保存。
制备例4
(1)合成二硫代氨基甲酸酯大分子链转移剂
(1a)合成二硫代氨基甲酸酯小分子引发剂
准确称取(2.88g，0.12mol)镁条于三口烧瓶中并加入催化量的碘和90mLTHF，再准确称取(20.7g，0.1mol)a-溴萘在冰浴中缓慢滴加至上述溶液，滴加完毕后在45℃反应3h。反应结束后冷却至室温，在室温条件下滴加(7.6g，0.10mol)二硫化碳，滴完后回流反应8小时，反应结束后加入少量水终结反应。将反应物倒入100mL冷水中，过滤除去镁盐，用2M盐酸酸化，使用乙酸乙酯萃取直到水层无色。合并红色的乙酸乙酯层，并用无水硫酸镁干燥过夜。第二天减压蒸去溶剂，残留物转入250mL三口瓶中，加入50mL乙酸乙酯，通氮气30min后加入催化剂量的碘，常温下滴加(2.53g，0.032mol)DMSO，滴完后避光过夜，然后在三颈瓶中再加入(10.67g，0.065mol)V501和50mL苯，通N230分钟后升温至65℃反应15小时，反应结束后旋蒸去除大部分溶剂，剩余物质通过硅胶柱进行分离。浓缩液以流动相(V乙酸乙酯:V石醚＝1:10)进行硅胶柱过柱纯化，并进行旋蒸得到二硫代氨基甲酸酯小分子引发剂。
(1b)合成二硫代氨基甲酸酯大分子链转移剂：
将CTA-COOH采用相同摩尔量的二硫代氨基甲酸酯小分子引发剂替代，其余条件与制备例1相同，得到二硫代碳酸酯大分子链转移剂。
(2)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇二硫代酯(PSSSM)
(2a)合成含三硫键单体
准确称取(0.76g，5mmol)3-溴丙酸和(1.61g，6.5mmol)硫代硫酸钠五水合物并溶于20ml水中，在60℃下反应5h。当溶液变清时，将反应混合物冷却至室温，然后在室温下反应过夜。然后准确称取(0.14，1.5mmol)硫化钠水合物溶液滴加至在上述混合物中，继续反应12h。待反应完毕后使用HCl溶液将反应混合物的pH调节至4.0至5.0。然后用乙酸乙酯提取上清液三次以获得目标粗产物，并通过制备液相色谱法纯化，得到3,3'-三硫代二丙酸。
准确称取(1g,7.68mmol)甲基丙烯酸羟乙酯HEMA、(7.43g,30.72mmol)3,3'-三硫代二丙酸和(85.29mg,0.7mmol)DMAP进行混合，加入60mL新鲜除水的THF后并于室温下反应12h，得到反应混合物。接着在0℃冰浴条件下冷却反应混合物1h。将(1.74g,8.44mmol)二环己基碳二亚胺(DCC)溶于20mL新鲜除水的THF后滴入到反应混合物中进行剧烈搅拌，在室温下反应24h。反应结束后采用布氏漏斗进行过滤，收集滤液并进行旋蒸，加入50mL CHCl3溶解后过滤，收集滤液并进行旋蒸浓缩后进行硅胶柱过柱纯化，流动相为(V乙酸乙酯:V正己烷＝1:1)，旋转蒸发即得产物含三硫键单体。
(2b)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇二硫代酯(PSSSM)
将单体MASSMI采用相同摩尔量的含三硫键单体替代，并将N3-PEG-CTA采用相同摩尔量的二硫代氨基甲酸酯大分子链转移剂替代，其余条件与制备例1相同，得到PSSSM。
(3)合成具有酶响应和氧化还原响应的双响应叠氮端甲氧基聚乙二醇二硫代酯(PSSSMG)
将PSSM采用相同摩尔量的PSSSM替代，其余条件与制备例1相同，得到PSSSMG。
(4)合成顺铂化疗前药(CM)
与制备例1相同。
(5)合成放化疗纳米增敏剂
将PSSMG采用相同重量份的PSSSMG替代，其余条件与制备例1相同，得到放化疗纳米增敏剂PSSSMGCM，并放入4℃冰箱冷藏保存。
制备例5
(1)合成叠氮端PEG卤素大分子引发物
准确称取(2.25g,0.45mmol)叠氮端羟基聚乙二醇mPEG5K-N3、(0.52g，2.25mmol)2-溴异丁酰溴(BIBB)、(0.14g，1.35mmol)三乙胺(TEA)并加入DCM中，室温反应48h。反应完成后过滤除盐，分别用0.1M盐酸、饱和NaHCO3溶液、去离子水洗涤，保留有机相，无水硫酸钠干燥除水。浓缩有机相后在乙醚中沉淀，真空烘干得到初产物。初产物使用乙醇重结晶，收集白色固体,于真空烘箱中充分干燥后得到卤素大分子引发物。
(2)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇溴基(PSCSM)
(2a)合成含二硫缩酮键单体
准确称取丙酮(7.42g，127.9mmol)和巯基乙酸(2.36g，255.9mmol)溶解在50mL新鲜除水的THF中，在冰浴中连续搅拌15min后，加入(2.98g，12.80mmol)氯化锆。混合物在室温下搅拌2小时后，旋蒸除去溶剂。通过硅胶色谱法纯化粗产物，得到无色油状的中间产物1。
准确称取(1g,7.68mmol)甲基丙烯酸羟乙酯HEMA、(7.68g,30.72mmol)中间产物1和(85.29mg,0.7mmol)DMAP进行混合，加入60mL新鲜除水的THF后并于室温下反应12h，得到反应混合物。接着在0℃冰浴条件下冷却反应混合物1h。将(1.74g,8.44mmol)二环己基碳二亚胺(DCC)溶于20mL新鲜除水的THF后滴入到反应混合物中进行剧烈搅拌，在室温下反应24h。反应结束后采用布氏漏斗进行过滤，收集滤液并进行旋蒸，加入50mL CHCl3溶解后过滤，收集滤液并进行旋蒸浓缩后进行硅胶柱过柱纯化，流动相为(V乙酸乙酯:V正己烷＝1:2)，收集合并流动相并进行旋蒸得到产物含二硫缩酮键单体MASCSMI。
(2b)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇溴基(PSCSM)
将单体MASSMI采用相同摩尔量的含二硫缩酮键单体MASCSMI替代，取一干燥洁净的Schlenk瓶，进行抽真空-充氩气三次，在氩气环境中依次加入准确称量好的(0.2g,0.036mmol)大分子链转移剂N3-PEG-Br、20μL的PMEDTA和(0.6g,1.26mmol)单体MASCSMI，用5mL超干DMSO溶剂进行搅拌溶解，反应体系冷冻抽排循环两次，称取(5mg，0.035mmol)溴化亚铜加入到体系中，然后冷冻抽排循环两次，最后在45℃条件下反应24h。反应结束后液氮猝灭反应，使用中性氧化铝除掉铜盐，旋蒸浓缩过后采用纯水进行透析除去未反应的单体及杂质，冻干得到产物，记为PSCSM。
(3)合成具有酶响应和氧化还原响应的双响应叠氮端甲氧基聚乙二醇溴基(PSCSMG)
将PSSM采用相同摩尔量的PSCSM替代，其余条件与制备例1相同，得到PSCSMG。
(4)合成顺铂化疗前药(CM)
与制备例1相同。
(5)合成放化疗纳米增敏剂
将PSSMG采用相同重量份的PSCSMG替代，其余条件与制备例1相同，得到放化疗纳米增敏剂PSCSMGCM，并放入4℃冰箱冷藏保存。
制备例6
(1)合成叠氮端甲氧基聚乙二醇二硫代正十二烷酯(N3-PEG-CTA)链转移剂
与制备例1相同。
(2)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇二硫代酯(PSeSeM)
(2a)合成含二硒键单体
准确称取(1g,7.68mmol)甲基丙烯酸羟乙酯HEMA、(9.34g,30.72mmol)3,3'-二硒代二丙酸和(85.29mg,0.7mmol)DMAP进行混合，加入60mL新鲜除水的THF后并于室温下反应12h，得到反应混合物。接着在0℃冰浴条件下冷却反应混合物1h。将(1.74g,8.44mmol)二环己基碳二亚胺(DCC)溶于20mL新鲜除水的THF后滴入到反应混合物中进行剧烈搅拌，在室温下反应24h。反应结束后采用布氏漏斗进行过滤，收集滤液并进行旋蒸，加入50mL CHCl3溶解后过滤，收集滤液并进行旋蒸浓缩后进行硅胶柱过柱纯化，流动相为(V乙酸乙酯:V正己烷＝1:2)，旋转蒸发即得产物含二硒键单体。
(2b)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇二硫代酯(PSeSeM)
将单体MASSMI采用相同摩尔量的含二硒键单体替代，其余条件与制备例1相同，得到PSeSeM。
(3)合成具有酶响应和氧化还原响应的双响应叠氮端甲氧基聚乙二醇二硫代酯(PSeSeMG)
将PSSM采用相同摩尔量的PSeSeM替代，其余条件与制备例1相同，得到PSeSeMG。
(4)合成顺铂化疗前药(CM)
与制备例1相同。
(5)合成放化疗纳米增敏剂
将PSSMG采用相同重量份的PSeSeMG替代，其余条件与制备例1相同，得到放化疗纳米增敏剂PSeSeMGCM，并放入4℃冰箱冷藏保存。
制备例7
(1)合成叠氮端甲氧基聚乙二醇二硫代正十二烷酯(N3-PEG-CTA)链转移剂
与制备例1相同。
(2)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇二硫代酯(PSSM)
与制备例1相同。
(3)合成具有酶响应和氧化还原响应的双响应叠氮端甲氧基聚乙二醇二硫代酯(PSSMF)
(3a)合成成纤维蛋白响应分子
准确称取(2g,9.85mmol)Boc-L-2-氨基丁酸和(1.78g,10.98mmol)N,N-碳酰二咪唑(CDI)溶解在20mL新鲜除水的二氯甲烷中，并于室温下搅拌2h后加入(0.4g,15mmol)炔丙胺和(3.5mL,24.7mmol)三乙胺，在室温下反应24h。反应结束后分别采用1M HCl(50mL)和饱和NaHCO3水溶液(50mL)进行洗 涤三次，收集有机层进行无水Na2SO4干燥后真空蒸发，将所得产物记为BTP。之后将所得产物BTP溶于(v:v＝1:1,8mL)CH2Cl2/TFA(三氟乙酸)溶液中，室温下搅拌过夜后进行旋蒸得到中间产物1。
准确称取(0.42g,3.72mmol)中间产物1和(0.67g,4.14mmol)CDI溶解在20mL新鲜除水的二氯甲烷中，并于室温下搅拌2h后加入(1.3mL,9.32mmol)三乙胺和(1.29g，3.72mmol)N-Boc-4-氧代-脯氨酸，在室温下反应24h，接着分别采用1M HCl(50mL)以及饱和NaHCO3水溶液(50mL)进行洗涤三次，收集有机层进行无水Na2SO4干燥后真空蒸发，将所得产物记为GBTP。之后将所得产物BTP溶于(v:v＝1:1,8mL)CH2Cl2/TFA(三氟乙酸)溶液中，室温下搅拌过夜后进行旋蒸得到中间产物2。
准确称取(1.71g，3.72mmol)中间产物2和(0.68g，5.58mmol)1，3-丙磺酸内脂溶解在20mL的甲醇中，并于室温下搅拌30min后加入碳酸钾，在70℃条件下回流反应3h，接着分别采用1M HCl(50mL)以及饱和NaHCO3水溶液(50mL)进行洗涤三次，收集有机层进行无水Na2SO4干燥后真空蒸发，得到成纤维蛋白响应分子。
(3b)合成具有酶响应和氧化还原响应的双响应叠氮端甲氧基聚乙二醇二硫代酯(PSSMF)
将GBAP采用相同重量份的成纤维蛋白响应分子替代，其余条件与制备例1相同，得到PSSMF。
(4)合成顺铂化疗前药(CM)
与制备例1相同。
(5)合成放化疗纳米增敏剂
将PSSMG采用相同重量份的PSSMF替代，其余条件与制备例1相同，得到放化疗纳米增敏剂PSSMFCM，并放入4℃冰箱冷藏保存。
制备例8
(1)合成叠氮端甲氧基聚乙二醇二硫代正十二烷酯(N3-PEG-CTA)链转移剂
与制备例1相同。
(2)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇二硫代酯(PSSM)
与制备例1相同。
(3)合成具有酶响应和氧化还原响应的双响应叠氮端甲氧基聚乙二醇二硫代酯(PSSMM)
(3a)合成金属蛋白酶响应分子
准确称取(0.42g,3.72mmol)中间产物1和(0.67g,4.14mmol)CDI溶解在20mL新鲜除水的二氯甲烷中，并于室温下搅拌2h后加入(1.3mL,9.32mmol)三乙胺和(2.06g，3.72mmol)MMP-2响应的多肽CPLGLAGG，在室温下反应24h，接着分别采用1M HCl(50mL)以及饱和NaHCO3水溶液(50mL)进行洗涤三次，收集有机层进行无水Na2SO4干燥后真空蒸发，将所得产物记为GBTP。之后将所得产物BTP溶于(v:v＝1:1,8mL)CH2Cl2/TFA(三氟乙酸)溶液中，室温下搅拌过夜后进行旋蒸得到金属蛋白酶响应分子。
(3b)合成具有酶响应和氧化还原响应的双响应叠氮端甲氧基聚乙二醇二硫代酯(PSSMM)
将GBAP采用相同重量份的金属蛋白酶响应分子替代，其余条件与制备例1相同，得到PSSMM。
(4)合成顺铂化疗前药(CM)
与制备例1相同。
(5)合成放化疗纳米增敏剂
将PSSMG采用相同重量份的PSSMM替代，其余条件与制备例1相同，得到放化疗纳米增敏剂PSSMMCM，并放入4℃冰箱冷藏保存。
制备例9
(1)合成叠氮端甲氧基聚乙二醇二硫代正十二烷酯(N3-PEG-CTA)链转移剂
与制备例1相同。
(2)合成具有氧化还原响应的叠氮端甲氧基聚乙二醇二硫代酯(PSSM)
与制备例1相同。
(3)合成具有酶响应和氧化还原响应的双响应叠氮端甲氧基聚乙二醇二硫代酯(PSSMC)
(3a)组织蛋白酶响应分子
准确称取(0.42g,3.72mmol)中间产物1和(0.67g,4.14mmol)CDI溶解在20mL新鲜除水的二氯甲烷中，并于室温下搅拌2h后加入(1.3mL,9.32mmol)三乙胺和(1.98g，3.72mmol)组织蛋白酶响应的多肽FRRG(Phe-Arg-Arg-Gly)，在室温下反应24h，接着分别采用1M HCl(50mL)以及饱和NaHCO3水溶液(50mL)进行洗涤三次，收集有机层进行无水Na2SO4干燥后真空蒸发，将所得产物记为GBTP。之后将所得产物BTP溶于(v:v＝1:1，8mL)CH2Cl2/TFA(三氟乙酸)溶液中，室温下搅拌过夜后进行旋蒸得到组织蛋白酶响应分子。
(3b)合成具有酶响应和氧化还原响应的双响应叠氮端甲氧基聚乙二醇二硫代酯(PSSMC)
将GBAP采用相同重量份的组织蛋白酶响应分子替代，其余条件与制备例1相同，得到PSSMC。
(4)合成顺铂化疗前药(CM)
与制备例1相同。
(5)合成放化疗纳米增敏剂
将PSSMG采用相同重量份的PSSMC替代，其余条件与制备例1相同，得到放化疗纳米增敏剂PSSMCCM，并放入4℃冰箱冷藏保存。
测试例2
将模型构建好的皮下胰腺癌模型小鼠分为16组(5只/组)，设计实验组分别为：生理盐水Control组、放疗RT组(即不注射任何药物和纳米粒子)、甲硝唑MI组、游离药物顺铂CDDP组、顺铂化疗前药CM组、无载药PSSMG组、载药纳米粒子(单硫键+GGT酶响应)PSMGCM(SCMG)组、载药纳米粒子(三硫键+GGT酶响应)PSSSMGCM(SSSCMG)组、载药纳米粒子(二硫缩酮键+GGT酶响应)PSCSMGCM(SCSCMG)组、载药纳米粒子(单硒键+GGT酶响应)PSeMGCM(SeCMG)组、载药纳米粒子(二硒键+GGT酶响应)PSeSeMGCM(SeSeCMG)组、载药纳米粒子(二硫键+组织蛋白酶酶响应)PSSMCCM(SSCMC)组、载药纳米粒子(二硫键+金属蛋白酶酶响应)PSSMMCM(SSCMM)组、载药纳米粒子(二硫键+成纤维细胞蛋白酶酶响应)PSSMFCM(SSCMF)组。通过尾静脉注射给药：药物总浓度为顺铂当量4mg/kg，100μL/只，每隔3天给一次药，注射完24h后进行剂量为3Gy的放疗，给药4次。每3天量取并记录肿瘤的长(L)和宽(W)，以及称量记录小鼠体重，小鼠肿瘤体积按公式(2)计算：
Vn＝1/2LW2…….………………(2)
其中Vn为第n天肿瘤体积。相对肿瘤体积比按公式(3)为：
A＝Vn/V0……….………………(3)
其中Vn为第n天肿瘤体积，V0为起始给药小鼠肿瘤体积。
所得结果见图19。从图19中可以看出，本发明所提供的放化疗纳米增敏剂可以实现显著的肿瘤抑制作用，展示了其在放化疗联合个体化治疗领域中的极大应用潜力。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (10)

  1. 一种两亲嵌段聚合物，其特征在于，所述两亲嵌段聚合物具有式(1)所示的结构：
    其中，R1为活性自由基聚合引发基团，R10为H原子或C1-C5的烷基，R11为O原子或N原子，R2为酶响应分子，R3为乏氧响应基团，R30为氧化还原敏感基团，R4为引发剂连接基团，X为酶响应分子键合基团，10≤m≤100，20≤n≤200，m、n均为整数。
  2. 根据权利要求1所述的两亲嵌段聚合物，其特征在于，所述活性自由基聚合引发基团R1为二硫代酯及其衍生物、三硫代酯及其衍生物、二硫代碳酸酯及其衍生物、二硫代氨基甲酸酯及其衍生物或卤素基团及其衍生物。
  3. 根据权利要求1所述的两亲嵌段聚合物，其特征在于，所述乏氧响应基团R3为2-硝基咪唑及其衍生物、4-硝基咪唑及其衍生物或5-硝基咪唑及其衍生物。
  4. 根据权利要求1所述的两亲嵌段聚合物，其特征在于，所述氧化还原敏感基团R30为二硫缩酮键、单硫键、单硒键、二硫键、三硫键或二硒键。
  5. 根据权利要求1所述的两亲嵌段聚合物，其特征在于，所述酶响应分子R2为γ-谷氨酰转肽酶响应分子、成纤维蛋白酶响应分子、金属蛋白酶响应分子或组织蛋白酶响应分子。
  6. 根据权利要求1所述的两亲嵌段聚合物，其特征在于，所述酶响应分子键合基团X具有式(3)所示的结构：
  7. 一种放化疗纳米增敏剂，其特征在于，所述放化疗纳米增敏剂含有权利要求1所述的两亲嵌段聚合物和化疗前药，所述两亲嵌段聚合物和化疗前药的质量比为100:(0～100)；所述化疗前药具有式(4)所示的结构：
    其中，R5为抗癌化疗药物分子。
  8. 根据权利要求7所述的放化疗纳米增敏剂，其特征在于，所述抗癌化疗药物分子R5为紫杉醇、紫杉醇衍生物、阿霉素、表阿霉素、喜树碱、喜树碱衍生物、顺铂类药物、长春碱、长春新碱、多西他赛、吉西他滨、姜黄素或丹酚酸。
  9. 一种放化疗纳米增敏剂的制备方法，其特征在于，该方法包括以下步骤：
    S1：于避光条件下，将权利要求1所述的两亲嵌段聚合物以及化疗前药溶解于有机溶剂I中，得到含两亲嵌段聚合物和化疗前药的溶液；所述化疗前药具有式(4)所示的结构：
    其中，R5为抗癌化疗药物分子；
    S2：将步骤S1所得含两亲嵌段聚合物和化疗前药的溶液加入到有机溶剂II-纯水混合相中进行高速搅拌，得到含放化疗纳米增敏剂的溶液；
    S3：将步骤S2所得含放化疗纳米增敏剂的溶液于纯水中进行透析以除去有机溶剂I和有机溶剂II，得到放化疗纳米增敏剂。
  10. 由权利要求9所述的方法制备得到的放化疗纳米增敏剂。
PCT/CN2023/099185 2022-06-09 2023-06-08 一种两亲嵌段聚合物和放化疗纳米增敏剂及其制备方法 WO2023237060A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210650836.0 2022-06-09
CN202210650836.0A CN114989375B (zh) 2022-06-09 2022-06-09 一种两亲嵌段聚合物和放化疗纳米增敏剂及其制备方法

Publications (1)

Publication Number Publication Date
WO2023237060A1 true WO2023237060A1 (zh) 2023-12-14

Family

ID=83033484

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/099185 WO2023237060A1 (zh) 2022-06-09 2023-06-08 一种两亲嵌段聚合物和放化疗纳米增敏剂及其制备方法

Country Status (2)

Country Link
CN (1) CN114989375B (zh)
WO (1) WO2023237060A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114989375B (zh) * 2022-06-09 2023-05-26 北京化工大学 一种两亲嵌段聚合物和放化疗纳米增敏剂及其制备方法
CN115806566A (zh) * 2022-12-08 2023-03-17 福州大学 一种用于克服肿瘤氧异质性分布的硝基还原酶激活的多功能分子前药的制备方法及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110694076A (zh) * 2019-09-09 2020-01-17 浙江大学 一种羟基氯喹两亲性聚合物药物前体、制备方法及其应用
US20200215197A1 (en) * 2017-09-30 2020-07-09 Zhejiang University Polymer of gamma-glutamyl transpeptidase catalyzing hydrolysis-induced charge reversal and its application in the field of drug delivery
CN113801276A (zh) * 2020-06-12 2021-12-17 北京化工大学 一种用于肿瘤放化疗协同治疗的纳米药物及其制备方法和应用
CN114989375A (zh) * 2022-06-09 2022-09-02 北京化工大学 一种两亲嵌段聚合物和放化疗纳米增敏剂及其制备方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110123785B (zh) * 2019-05-29 2021-05-11 山东大学 一种荷载化疗药物的双敏感型靶向纳米粒制剂及制备方法
KR20220074757A (ko) * 2020-11-27 2022-06-03 주식회사 에스엔바이오사이언스 난용성 캄토테신 화합물을 포함하는 나노 입자를 포함하는 암 치료용 약제학적 조성물 및 이의 병용요법

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200215197A1 (en) * 2017-09-30 2020-07-09 Zhejiang University Polymer of gamma-glutamyl transpeptidase catalyzing hydrolysis-induced charge reversal and its application in the field of drug delivery
CN110694076A (zh) * 2019-09-09 2020-01-17 浙江大学 一种羟基氯喹两亲性聚合物药物前体、制备方法及其应用
CN113801276A (zh) * 2020-06-12 2021-12-17 北京化工大学 一种用于肿瘤放化疗协同治疗的纳米药物及其制备方法和应用
CN114989375A (zh) * 2022-06-09 2022-09-02 北京化工大学 一种两亲嵌段聚合物和放化疗纳米增敏剂及其制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GLASS EVAN B., MASJEDI SHIRIN, DUDZINSKI STEPHANIE O., WILSON ANDREW J., DUVALL CRAIG L., YULL FIONA E., GIORGIO TODD D.: "Optimizing Mannose "Click" Conjugation to Polymeric Nanoparticles for Targeted siRNA Delivery to Human and Murine Macrophages", ACS OMEGA, ACS PUBLICATIONS, vol. 4, no. 16, 15 October 2019 (2019-10-15), US , pages 16756 - 16767, XP093113943, ISSN: 2470-1343, DOI: 10.1021/acsomega.9b01465 *
TANG XIAOHU, DU XIAOMENG, YU YANTING, QIN MENG, QIAN LILI, ZHANG MENG, YANG YAN, YU QINGSONG, GAN ZHIHUA: "Deep‐Penetrating Triple‐Responsive Prodrug Nanosensitizer Actuates Efficient Chemoradiotherapy in Pancreatic Ductal Adenocarcinoma Models", SMALL, WILEY, HOBOKEN, USA, vol. 18, no. 31, 1 August 2022 (2022-08-01), Hoboken, USA, XP093113942, ISSN: 1613-6810, DOI: 10.1002/smll.202202834 *
ZHOU QUAN; SHAO SHIQUN; WANG JINQIANG; XU CHANGHUO; XIANG JIAJIA; PIAO YING; ZHOU ZHUXIAN; YU QINGSONG; TANG JIANBIN; LIU XIANGRUI: "Enzyme-activatable polymer–drug conjugate augments tumour penetration and treatment efficacy", NATURE NANOTECHNOLOGY, NATURE PUB. GROUP, INC., LONDON, vol. 14, no. 8, 1 July 2019 (2019-07-01), London , pages 799 - 809, XP036851969, ISSN: 1748-3387, DOI: 10.1038/s41565-019-0485-z *

Also Published As

Publication number Publication date
CN114989375B (zh) 2023-05-26
CN114989375A (zh) 2022-09-02

Similar Documents

Publication Publication Date Title
WO2023237060A1 (zh) 一种两亲嵌段聚合物和放化疗纳米增敏剂及其制备方法
US11534435B2 (en) Drug carrier and preparation method thereof
CA3009252C (en) Biodegradable amphiphilic polymer, polymeric vesicles prepared therefrom, and application of biodegradable amphiphilic polymer in preparation of medicines for targeted therapy of lung cancer
JP6677914B2 (ja) 卵巣癌用の特異的に標的化された生分解性両親媒性ポリマー、それから製造されたポリマーベシクル及びその使用
KR102237234B1 (ko) 간 표적성을 갖는 유전자/약물을 공동으로 운반하는 통합식 나노 시스템 및 이의 제조 방법
CN107298741B (zh) 一种嵌段聚合物、含有其的药物载体及其制备方法和应用
CN106995516B (zh) 肿瘤特异性富集的纳米载药体系及其制备方法
CN109966507A (zh) 一种肿瘤靶向的pH和氧化还原双重响应的大分子纳米前药及其制备方法与应用
Chen et al. Real-time monitoring of a controlled drug delivery system in vivo: construction of a near infrared fluorescence monomer conjugated with pH-responsive polymeric micelles
CN111000823A (zh) 一种同时载有siRNA和顺铂前药的新型酸敏纳米载体及其制备方法与应用
CN104877092A (zh) 含有缩醛键的双重靶向的两亲共聚物及其制备和作为抗肿瘤药物载体的应用
CN111333786B (zh) 基于两性离子及叶酸靶向的酸敏感性阿霉素前药的制备方法
CN104784700B (zh) 一种药物共载复合物、胶束及胶束的制备方法
CN108774301A (zh) 一类基于葡聚糖的酸响应聚合物药物的制备方法及其应用
CN104650307B (zh) 基于PDEAEMA的pH敏感五嵌段线性聚合物及胶束
CN113041355B (zh) 一种可精准调控联用药物比率的共递送纳米药物及应用
CN104415339A (zh) 一种自组装靶向纳米药物载体胶束
CN108815536B (zh) 一种具有pH和双重氧化还原响应性的药物递送材料及其制备方法和应用
CN117777378A (zh) 一种具有pH和氧化还原双重刺激响应型聚合物载体的制备方法及应用
CN105169405A (zh) 阿霉素类高分子药物的制备方法
CN111840228A (zh) 肿瘤免疫脂质体，其制备方法和用途
CN104414999A (zh) 抗肿瘤靶向纳米载药微胶囊的制备方法
CN113402661B (zh) 一种两性离子聚合物基一氧化氮驱动纳米马达及其制备方法和应用
CN113603811B (zh) 一种pH敏感和氧增敏的透明质酸氟化聚合物及其合成方法与应用
WO2023155755A1 (zh) 一种交联聚合物结构及其制备方法与应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23819224

Country of ref document: EP

Kind code of ref document: A1