WO2015180655A1 - 含双硫五元环功能基团的环状碳酸酯单体及其制备方法 - Google Patents

含双硫五元环功能基团的环状碳酸酯单体及其制备方法 Download PDF

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WO2015180655A1
WO2015180655A1 PCT/CN2015/079998 CN2015079998W WO2015180655A1 WO 2015180655 A1 WO2015180655 A1 WO 2015180655A1 CN 2015079998 W CN2015079998 W CN 2015079998W WO 2015180655 A1 WO2015180655 A1 WO 2015180655A1
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functional group
membered ring
cyclic carbonate
disulfide
compound
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French (fr)
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孟凤华
邹艳
钟志远
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Soochow University
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Soochow University
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Priority to JP2017514770A priority patent/JP6296381B2/ja
Priority to ES15798938.5T priority patent/ES2642150T3/es
Priority to US15/314,207 priority patent/US9796728B2/en
Priority to EP15798938.5A priority patent/EP3150611B1/en
Priority to AU2015266505A priority patent/AU2015266505B2/en
Priority to KR1020167036688A priority patent/KR101793483B1/ko
Publication of WO2015180655A1 publication Critical patent/WO2015180655A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/10—Spiro-condensed systems
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • 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
    • C08G64/00—Macromolecular compounds obtained by reactions forming a carbonic ester link in the main chain of the macromolecule
    • C08G64/02—Aliphatic polycarbonates
    • C08G64/0208—Aliphatic polycarbonates saturated
    • C08G64/0225—Aliphatic polycarbonates saturated containing atoms other than carbon, hydrogen or oxygen
    • C08G64/025—Aliphatic polycarbonates saturated containing atoms other than carbon, hydrogen or oxygen containing sulfur
    • 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
    • C08G64/00—Macromolecular compounds obtained by reactions forming a carbonic ester link in the main chain of the macromolecule
    • C08G64/18—Block or graft polymers
    • C08G64/183—Block or graft polymers containing polyether sequences
    • 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
    • C08G64/00—Macromolecular compounds obtained by reactions forming a carbonic ester link in the main chain of the macromolecule
    • C08G64/20—General preparatory processes
    • C08G64/30—General preparatory processes using carbonates
    • C08G64/305—General preparatory processes using carbonates and alcohols
    • 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
    • C08G64/00—Macromolecular compounds obtained by reactions forming a carbonic ester link in the main chain of the macromolecule
    • C08G64/42—Chemical after-treatment
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/64—Polyesters containing both carboxylic ester groups and carbonate groups
    • 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
    • C08G64/00—Macromolecular compounds obtained by reactions forming a carbonic ester link in the main chain of the macromolecule
    • C08G64/20—General preparatory processes
    • C08G64/30—General preparatory processes using carbonates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/34—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from hydroxy compounds or their metallic derivatives
    • C08G65/48—Polymers modified by chemical after-treatment
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules

Definitions

  • the invention relates to a cyclic carbonate monomer, relating to the preparation and application of a cyclic carbonate monomer containing a disulfide five-membered ring functional group.
  • Cyclic carbonate monomers have very unique properties, for example, they can be simply synthesized to obtain high-yield, high-purity products, and biodegradable polycarbonates can be obtained by small molecules or macromolecules, and the polymers are excellent. Their properties, for example, they usually have good biocompatibility, can be degraded in the body, and the degradation products can be absorbed by the human body or excreted through the normal physiological pathways of the human body. Like aliphatic polyesters, they are widely used in various fields of biomedicine. Such as surgical sutures, bone fixation devices, biological tissue engineering scaffold materials, and drug controlled release carriers. Among them, synthetic biodegradable polymers are particularly concerned because of their low immunogenicity and their properties such as degradation properties and mechanical properties, which can be easily controlled.
  • biodegradable polymers are through cyclic carbonate monomers such as trimethylene carbonate (TMC), or cyclic ester monomers such as glycolide (GA), lactide (LA), caprolactone ( CL) and other open-loop polymerization preparations have been approved by the US Food and Drug Administration (FDA).
  • TMC trimethylene carbonate
  • cyclic ester monomers such as glycolide (GA), lactide (LA), caprolactone ( CL) and other open-loop polymerization preparations have been approved by the US Food and Drug Administration (FDA).
  • the method for preparing the above cyclic carbonate monomer comprises the steps of: reacting dibromo neopentyl glycol with sodium hydrosulfide monohydrate in a polar solvent to obtain compound A; then oxidizing compound A in air to obtain compound B; The compound B and the ethyl chloroformate are reacted in a cyclic ether solvent in a nitrogen atmosphere to obtain the above-mentioned bisulfide 5-membered ring functional group-containing cyclic carbonate monomer.
  • the molar ratio of the dibromo neopentyl glycol to the sodium hydrosulfide monohydrate is (2.5-10):1; the molar ratio of the compound B to the ethyl chloroformate is 1: (2 to 4).
  • the preparation method of the above-mentioned cyclic carbonate monomer containing a disulfide five-membered ring functional group is as follows:
  • the polar solvent is N,N-dimethylformamide (DMF); and the ether solvent is tetrahydrofuran.
  • the compound A is first dissolved in an ether solvent and then oxidized in the air to obtain the compound B; the oxidation rate of the compound A is increased.
  • the ether solvent may be tetrahydrofuran or 1,4-dioxane.
  • the solvent for the dissolution of the compound A in the step (2) is identical to the solvent for the dissolution of the compound B in the step (3).
  • the above-mentioned vacuum distillation, extraction, rotary distillation, spin concentration and recrystallization are all prior art, and those skilled in the art can select according to their needs.
  • the present invention is preferably extracted four times with ethyl acetate when the compound A is purified; and recrystallized 3-5 times with diethyl ether when the cyclic carbonate is purified.
  • the cyclic carbonate monomer containing a disulfide five-membered ring functional group may be subjected to ring-opening polymerization to obtain a polycarbonate having a double-sulfur five-membered ring in a side chain, and since the disulfide five-membered ring group does not affect ring-opening polymerization, No protection and deprotection processes are required.
  • the above cyclic carbonate monomer may be subjected to ring-opening polymerization using methylene glycol as an initiator and bis(bistrimethylsilyl)amine zinc as a catalyst in dichloromethane to form a block polymer, and the reaction formula is as follows: :
  • the above cyclic carbonate monomer may also be subjected to ring-opening copolymerization with other cyclic ester and cyclic carbonate monomers to prepare random and block copolymers;
  • the other cyclic carbonates include trimethylene cyclic carbonate (TMC)
  • TMC trimethylene cyclic carbonate
  • the other cyclic ester monomers include caprolactone ( ⁇ -CL), lactide (LA) or glycolide (GA).
  • the functional polycarbonate containing a double sulfur five-membered ring in the side chain can form a stable chemical cross-linking under the catalytic amount of a reducing agent such as dithiothreitol or glutathione, but in an intracellular reducing environment. Will quickly solve the cross-linking. Therefore, the functional polycarbonate having a double-sulfur five-membered ring in the side chain has excellent practical value, for example, it can be used for preparing a cyclically stable drug carrier, and the drug can be rapidly released in the target cell.
  • a reducing agent such as dithiothreitol or glutathione
  • the present invention has the following advantages compared with the prior art:
  • the present invention discloses for the first time a cyclic carbonate monomer containing a disulfide five-membered ring functional group, which can be conveniently and efficiently prepared in two pots (three steps) without the need for protection and deprotection in the prior art. process.
  • the cyclic carbonate monomer containing a disulfide five-membered ring functional group disclosed in the present invention since the disulfide five-membered ring group does not affect the ring-opening polymerization of the cyclic carbonate monomer, there is no need in the prior art. Protection and deprotection process , that is, ring-opening polymerization to obtain a functional polycarbonate having a side chain containing a disulfide five-membered ring;
  • the cyclic carbonate monomer disclosed in the invention is simple to prepare, and can be conveniently subjected to ring-opening polymerization to obtain a carbonate polymer having reduction-sensitive reversible crosslinking property; the polymer can be further self-assembled for controlling a drug release system.
  • tissue engineering and biochips have good application value in biomaterials.
  • Example 1 is a nuclear magnetic spectrum of a cyclic carbonate monomer containing a disulfide five-membered ring functional group in Example 1.
  • 3 is an ultraviolet absorption spectrum of a cyclic carbonate monomer containing a disulfide five-membered ring functional group in the first embodiment
  • Figure 5 is a nuclear magnetic spectrum of the block polymer PEG5k-P (CDC2.5k-co-CL3.9k) in Example 4;
  • Figure 6 is a graph showing the results of toxicity of the polymer PEG5k-b-PCDC2.8k cross-linked nanoparticles in Example 6 against Raw264.7 and MCF-7 cells;
  • Example 7 is a graph showing the in vitro release results of doxorubicin-loaded polymer PEG5k-b-PCDC2.8k crosslinked nanoparticles in Example 7;
  • Figure 8 is a polymer containing PEG5k-b loaded with doxorubicin in Example 7.
  • Figure 9 is a graph showing the results of blood circulation of doxorubicin-containing polymer PEG5k-b-PCDC2.8k crosslinked nanoparticles in mice in Example 8;
  • Figure 10 is a graph showing the results of biodistribution of doxorubicin-containing polymer PEG5k-b-PCDC2.8k cross-linked nanoparticles in Example 9 against melanoma-bearing mice;
  • FIG. 11 is a graph showing inhibition of tumor growth of a doxorubicin-containing polymer PEG5k-b-PCDC2.8k crosslinked nanoparticles in a tenth melanin-bearing tumor mouse;
  • Figure 12 is a graph showing changes in body weight of mice in Example 10.
  • Figure 13 is a graph showing survival curves of mice in Example 10.
  • Figure 1 is a nuclear magnetic spectrum of the above product CDC, 1 H NMR (400 MHz, CDCl 3 ): ⁇ 3.14 (s, 4H), 4.51 (s, 4H). Elemental analysis: C: 41.8%, H: 4.20%, O: 24.3% (theory: C: 41.67%, H: 4.17%, O: 25%, S: 33.3%), Mass spectrometry analysis of CDC monomer: MS : 192.5 (theoretical molecular weight: 192), see Figure 2.
  • Fig. 3 is a UV spectrum of a solution of the above-mentioned product monomer CDC tetrahydrofuran at different concentrations. The sulfur-sulfur five-membered ring in the monomer has absorption at 330 nm, and the absorption intensity increases as the monomer concentration increases.
  • Figure 4 is a nuclear magnetic spectrum of the above cyclic carbonate polymer: 1 H NMR (400 MHz, CDCl 3 ): 3.08 (s, -CCH 2 ), 3.30 (m, -OCH 3 ), 4.05 (s, -CH 2 OCOCHCH 2 -), 4.07 (s, -OCH 2 CCH 2 O-), 4.31 (m, -CCH 2 ).
  • Figure 5 is a nuclear magnetic spectrum of the above polymer: 1 H NMR (400 MHz, CDCl 3 ): 1.40 (m, -COCH 2 CH 2 CH 2 CH 2 CH 2 -), 1.65 (m, -COCH 2 CH 2 CH 2 CH 2 CH 2 -), 2.30 (t, -COCH 2 CH 2 CH 2 CH 2 CH 2 -), 3.08 (s, -CCH 2 ), 3.30 (m, -OCH 3 ), 4.03 (t, -COCH 2 ) CH 2 CH 2 CH 2 CH 2 O-), 4.05 (s, -CH 2 OCOCHCH 2 -), 4.07 (s, -OCH 2 CCH 2 O-), 4.31 (m, -CCH 2 ); molecular weight measured by GPC : 14.0 kDa, molecular weight distribution: 1.56.
  • Polymeric micelle nanoparticles were prepared by dialysis.
  • the polymer PEG5k-b-PCDC2.8k was dissolved in N,N-dimethylformamide (2 mg/mL), and 200 ⁇ L was added dropwise to 800 ⁇ L of phosphate buffer solution (10 mM, pH 7.4, PB) for dialysis.
  • the bag (MWCO 3500) was dialyzed overnight, replaced with five times of water, and the dialysis medium was PB (10 mM, pH 7.4).
  • the concentration of the finally obtained polymer nanoparticles was 0.2 mg/mL.
  • the crosslinking of the nanoparticles is carried out by adding a catalytic amount of dithiothreitol (DTT).
  • DTT dithiothreitol
  • the aqueous polymer nanoparticle solution was passed through a nitrogen gas for 10 minutes to remove the air as much as possible.
  • 10 ⁇ L of dithiothreitol (DTT) dissolved in secondary water (0.007 mg, 4.67 ⁇ 10) was added to the nanoparticle solution (1 mL, 0.25 mg/mL, 3.21 ⁇ 10 -5 mmol) in the closed reactor. -5 mmol, 10% by mole of lipoic acid functional group, and stirred at room temperature for 1 day.
  • the particle size was determined to be 150 nm, which is about 15% less than the particle size without cross-linking.
  • the nanoparticles have almost no change in particle size and particle size distribution after 100 times dilution; stable under physiological conditions, it can be seen that disulfide cross-linking can greatly improve the stability of nano
  • the disulfide bond can be easily cleaved by a reducing agent such as glutathione (GSH).
  • GSH glutathione
  • the particle size of the crosslinked nanoparticles was gradually destroyed over time, indicating that the disulfide ring in the polymer would break in the presence of a large amount of reducing species.
  • a high concentration of the reducing substance GSH is also present in the cytoplasm, so that the prepared nano drug carrier is stable in circulation, but can be rapidly dissociated after endocytosis, releasing the drug.
  • the cytotoxicity of the crosslinked micelle nanoparticles was tested by MTT method.
  • the cells used were MCF-7 (human breast cancer cell) cells and Raw 264.7 (mouse macrophage) cells.
  • MCF-7 cells or Raw 264.7 cells were seeded in 96-well plates at 1 ⁇ 10 4 /mL, 100 ⁇ L per well, cultured until the cells were attached, and the experimental group was added with culture medium containing different concentrations of polymer nanoparticles. Set cell blank control wells and medium blank wells, parallel to 4 replicate wells. After incubating for 24 hours in the incubator, the 96-well plate was taken out, and 10 ⁇ L of MTT (5.0 mg/mL) was added. After further incubation for 4 hours, 150 ⁇ L of DMSO was added to each well to dissolve the crystals, and the absorbance was measured at 492 nm with a microplate reader ( A) Calculate cell viability by zeroing the blank of the medium.
  • a T is the absorbance at 490 nm of the test group
  • a C is the absorbance at 492 nm of the blank control group.
  • the polymer concentrations were 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL, respectively.
  • Figure 6 is a graph showing the cytotoxicity of nanoparticles. As can be seen from Figure 6, when the concentration of polymer nanoparticles is increased from 0.1 mg/mL to 0.5 mg/mL, Raw264.7 cells and MCF-7 cells are The survival rate was still higher than 85%, indicating that the PEG5k-b-PCDC2.8k polymer nanoparticles have good biocompatibility.
  • Doxorubicin is used as a drug. Since the anticancer drug doxorubicin is a fluorescent sensitive substance, the entire operation is carried out in the dark.
  • Nanopolymer nano The granule PEG5k-b-PCDC2.8k was dissolved in N,N-dimethylformamide (DMF).
  • the doxorubicin dimethyl sulfoxide solution and the polymer nanoparticle PEG5k-b-PCDC2.8k DMF solution are uniformly mixed according to the predetermined drug to polymer mass ratio, and the mixture is slowly added four times its volume by stirring. Secondary water (15s/d), after dialysis, once water is dialyzed.
  • Crosslinking of the drug-loaded micelle nanoparticles was also carried out in accordance with the crosslinking method of Example 5.
  • 100 ⁇ L of doxorubicin-loaded polymer nanoparticle solution was freeze-dried, then dissolved in 3.0 mL of DMSO, and the encapsulation efficiency was calculated by a fluorescence spectrophotometer in combination with a standard curve of doxorubicin.
  • DLC Drug loading
  • DLE encapsulation efficiency
  • Drug loading (drug weight / polymer weight) ⁇ 100%
  • Encapsulation rate (%) (loaded drug weight / total drug input) ⁇ 100%
  • Table 1 shows the above calculation results. It can be seen that the polymer PEG5k-b-PCDC2.8k nanoparticles have an efficient embedding effect on the small molecule anticancer drug doxorubicin.
  • the release test of doxorubicin was carried out by shaking (200 rpm) in a 37 ° C constant temperature shaker. Drug release was compared using two sets of parallel samples, each with two parallel samples: the first group, cross-linked doxorubicin-loaded polymer nanoparticles were simulated by intracellular reduction with the addition of 10 mM glutathione (GSH).
  • GSH glutathione
  • DOX-containing PEG5k-b-PCDC2.8k cross-linked nanoparticles were tested for toxicity to Raw264.7 cells, MCF-7 cells, etc. by MTT assay, and the drug-free uncrosslinked nanoparticles and free drug were used as controls.
  • Raw264.7 cells were seeded at 1 ⁇ 10 4 cells/mL in 96-well plates at 100 ⁇ L per well, and cultured until the cells were attached. The experimental group was added with 0.01, 0.1, 1, and 5, respectively.
  • mice C57BL/6 black mice (Chinese Academy of Sciences Laboratory Animal Center, Chinese Academy of Sciences) weighing 18-20 grams and 4-6 weeks old. After weighing, they were evenly grouped according to body weight, and the drug-loaded nanoparticles and free drugs were passed.
  • DOX was 10 mg/kg, and about 10 ⁇ L of blood was taken at 0, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours. The blood weight was accurately weighed by the difference method. Blood was added with 100 ⁇ L of 1% Triton and 500 ⁇ L of DMF (containing 20 mM DTT, 1 M HCl); after centrifugation (20,000 rpm, 20 minutes), the supernatant was taken and each fluorescence was measured. The amount of DOX at a time.
  • Figure 9 is a graph showing the blood circulation of doxorubicin-loaded polymer PEG5k-b-PCDC2.8k cross-linked nanoparticles in mice.
  • the abscissa is the time point, and the ordinate is the total amount of DOX per gram of blood.
  • DOX injection volume ID% / g.
  • Its elimination half-life in mice can be calculated as 4.67 hours, while free DOX is only 0.21 hours, so the drug-loaded cross-linked nanoparticles are stable in mice and have a long cycle time.
  • mice C57BL/6 black mice weighing about 18-20 grams and 4-6 weeks old were injected subcutaneously with 1 ⁇ 10 6 B16 melanoma tumor cells. After about two weeks, when the tumor size was 100-200 mm 3 , The drug-loaded nanoparticles and free DOX were injected into the mice through the tail vein (DOX dose was 10 mg/kg), and the mice were sacrificed after 6, 12 and 24 hours to remove the tumor and heart, liver, spleen, lung and kidney tissues. After washing, the mixture was weighed and added with 500 ⁇ L of 1% Triton, ground by a homogenizer, and then extracted with 900 ⁇ L of DMF (containing 20 mM DTT, 1 M HCl). After centrifugation (20,000 rpm, 20 minutes), the supernatant was taken and the amount of DOX at each time point was measured by fluorescence.
  • DOX dose was 10 mg/kg
  • Figure 10 is a graph showing the biodistribution results of doxorubicin-containing polymer PEG5k-b-PCDC2.8k cross-linked nanoparticles against melanoma-bearing tumor mice.
  • the abscissa is the tissue and organs, and the ordinate is DOX per gram of tumor or tissue.
  • the amount of total DOX injection (ID% / g).
  • the tumor accumulation of drug-loaded nanoparticles at 3.12, 12, and 24 hours was 3.12, 2.93, and 2.52 ID%/g, respectively, which was 3 to 12 times higher than that of free DOX at 1.05, 0.52, and 0.29 ID%/g. It is indicated that the drug-loaded cross-linked nanoparticles accumulate more in the tumor site through the EPR effect and can last longer.
  • the experiment used C57BL/6 black mice weighing about 18-20 grams and 4-6 weeks old. After weighing, they were evenly grouped according to body weight. 1 ⁇ 10 6 B16 melanoma tumor cells were injected subcutaneously. About one week later, the tumor was tumor.
  • the drug-loaded nanoparticles and free DOX are injected into the mice through the tail vein at 0, 2 , 4, 6 and 8 days, and the amount of DOX in the drug-loaded nanoparticles is 10, 20, 30 mg. /kg, the free DOX dose is 10mg/kg, from 0 to 15 days, the body weight of each group of mice is weighed daily, and the tumor volume is accurately measured by vernier caliper.
  • Figure 11 is a graph showing the inhibition of tumor growth of doxorubicin-loaded polymer PEG5k-b-PCDC2.8k cross-linked nanoparticles on melanoma-bearing tumor mice;
  • Figure 12 is a graph showing changes in body weight of mice; Graph.
  • DOX concentration 30 mg / kg
  • DOX-loaded nanoparticles for 16 days the tumor To the obvious inhibition, while DOX can also inhibit tumor growth, but the toxic side effects on mice are very large.
  • the polymer prepared by the monomer of the invention has good biocompatibility, and when used as a drug carrier, the cycle time of the antitumor drug in the body can be increased, the enrichment rate of the drug in the tumor site can be increased, and the drug can be avoided. Damage to normal tissues can effectively kill tumor cells while having little effect on normal cells.

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Abstract

本发明公开了一种含双硫五元环功能基团的环状碳酸酯单体及其制备方法。所述环碳酸酯单体能够简单高效的合成，无需保护和脱保护过程；利用本发明所述的环碳酸酯单体可开环聚合得到分子量和分子量分布可控的聚碳酸酯，其具有生物可降解性和还原敏感可逆交联的性质。可用于药物控制释放的载体、生物组织支架或者生物芯片。

Description

含双硫五元环功能基团的环状碳酸酯单体及其制备方法 技术领域
本发明涉及一种环状碳酸酯单体，涉及一种含双硫五元环功能基团的环状碳酸酯单体的制备及应用。
背景技术
环状碳酸酯单体具有非常独特的性能，例如它们可以简单地合成得到高产量高纯度的产物，并且可以通过小分子或大分子引发得到生物可降解的聚碳酸酯，其聚合物具有非常优异的性能，例如它们通常具有良好的生物相容性，能在体内降解，降解产物可被人体吸收或通过人体正常生理途径排出体外，和脂肪族聚酯一样被广泛应用于生物医学的各个领域，如手术缝合线、骨固定器械、生物组织工程支架材料、和药物控制释放载体等。其中，合成的生物可降解聚合物由于其免疫原性较低、其性能含如降解性能和机械性能等均可方便得到控制等而尤其受到关注。常用的生物可降解聚合物是通过环状碳酸酯单体如三亚甲基环碳酸酯(TMC)，或环酯单体如乙交酯(GA)、丙交酯(LA)、己内酯(CL)等开环聚合制备得到，已获得美国食品药物管理部门(FDA)的许可。
技术问题
但是，现有的环碳酸酯或环酯单体如TMC、GA、LA和CL等结构比较单一，缺乏可用于修饰的官能团，故制备的聚合物往往难以后修饰、难以满足医学需求，例如，基于这些传统碳酸酯单体的聚合物的药物载体或是表面修饰涂层存在稳定性差的致命弱点。如何提高其体内稳定性是亟待解决的问题。
另外，现有技术中，在制备环状碳酸酯单体、和/或开环聚合过程过程中，其结构中存在易于反应的基团，多数情况都需要通过保护和脱保护步骤，这导致制备过程繁琐。
问题的解决方案
技术解决方案
本发明的目的是，提供一种含双硫五元环功能基团的环状碳酸酯单体。
为达到上述目的，本发明具体的技术方案为：一种含双硫五元环功能基团的环状碳酸酯单体，其化学结构式如下：
Figure PCTCN2015079998-appb-000001
上述环碳酸酯单体的制备方法包括以下步骤：将二溴新戊二醇与一水合硫氢化钠在极性溶剂中反应得到化合物A；然后将化合物A在空气中氧化得到化合物B；最后在氮气气氛中，在环醚类溶剂中，将化合物B与氯甲酸乙酯反应得到所述的含双硫五元环功能基团的环状碳酸酯单体。
上述技术方案中，所述二溴新戊二醇与一水合硫氢化钠的摩尔比为(2.5～10)∶1；化合物B与氯甲酸乙酯的摩尔比为1∶(2～4)。
优选的技术方案中，上述含双硫五元环功能基团的环状碳酸酯单体的制备方法为：
(1)
将一水合硫氢化钠溶解在极性溶液中，将二溴新戊二醇用恒压滴液漏斗缓慢滴加，50℃件下反应48小时，得到化合物A；
所述化合物A的化学结构式如下：
Figure PCTCN2015079998-appb-000002
化合物A在空气中氧化，得到化合物B，所述化合物B的化学结构式如下：
Figure PCTCN2015079998-appb-000003
氮气气氛中，将化合物B与氯甲酸乙酯溶解在环醚类溶剂中，然后用恒压滴液漏斗缓慢滴加三乙胺，在冰水浴中反应4小时，得到含双硫五元环功能基团的环状碳酸酯单体，所述环状碳酸酯单体的化学结构式如下：
Figure PCTCN2015079998-appb-000004
优选的技术方案中，所述极性溶剂为N，N-二甲基甲酰胺(DMF)；所述醚类溶剂为四氢呋喃。
优选的技术方案中，化合物A先溶于醚类溶剂，再在空气中氧化得到化合物B；增加了化合物A的氧化速率。醚类溶剂可以为四氢呋喃、1，4-二氧六环。为了简化反应过程、纯化反应条件，步骤(2)用于溶解化合物A的溶剂与步骤(3)用于溶解化合物B的溶剂一致。
优选的技术方案中，上述步骤(1)、步骤(3)完成后进行提纯处理，具体为：
Figure PCTCN2015079998-appb-000005
化合物A的提纯：反应结束后，反应物减压蒸馏除去溶剂，然后用蒸馏水稀释，再用乙酸乙酯萃取，最后旋蒸有机相得到黄色粘稠状化合物A；
Figure PCTCN2015079998-appb-000006
含双硫五元环功能基团的环状碳酸酯单体的提纯：反应结束后，过滤，滤液经旋转浓缩，再用乙醚重结晶，得到黄色晶体，即含双硫五元环功能基团的环状碳酸酯单体。
上述减压蒸馏、萃取、旋蒸、旋转浓缩以及重结晶都属于现有技术，本领域技术人员可以根据需要自行选择。本发明优选在化合物A提纯时，用乙酸乙酯萃取四次；在环状碳酸酯提纯时，用乙醚重结晶3-5次。
上述含双硫五元环功能基团的环状碳酸酯单体的制备过程可如下表示：
Figure PCTCN2015079998-appb-000007
上述含双硫五元环功能基团的环状碳酸酯单体可开环聚合得到侧链含双硫五元环的聚碳酸酯，由于该双硫五元环基团不影响开环聚合，无需保护和脱保护过程。例如，上述环碳酸酯单体在二氯甲烷中可以以聚乙二醇为引发剂、双(双三甲基硅基)胺锌为催化剂开环聚合，形成嵌段聚合物，其反应式如下：
Figure PCTCN2015079998-appb-000008
上述环碳酸酯单体还可以和其他环酯、环碳酸酯单体进行开环共聚反应，制备无规和嵌段共聚物；所述其他环碳酸酯包括三亚甲基环碳酸酯(TMC)，所述其他环酯单体包括己内酯(ε-CL)、丙交酯(LA)或乙交酯(GA)。
该侧链含双硫五元环的功能性聚碳酸酯可以在催化量的还原剂如二硫代苏糖醇或谷胱甘肽催化下形成稳定的化学交联，但在细胞内还原环境下会快速解交联。因此该侧链含双硫五元环的功能性聚碳酸酯具有优良的实用价值，例如可以用于制备循环稳定的药物载体，在靶细胞内可以快速释放药物。
发明的有益效果
有益效果
由于上述方案的实施，本发明与现有技术相比，具有以下优点：
1.本发明首次公开了一种含双硫五元环功能基团的环状碳酸酯单体，只需两锅(三步)即可便捷高效制备，无需现有技术中的保护和脱保护过程。
2.本发明公开的含双硫五元环功能基团的环状碳酸酯单体，由于双硫五元环基团不影响环碳酸酯单体的开环聚合，因此无需现有技术中的保护和脱保护过程 ，即可开环聚合得到侧链含双硫五元环的功能性聚碳酸酯；
3.本发明公开的环碳酸酯单体制备简单，由其可以方便的开环聚合得到具有还原敏感可逆交联特性的碳酸酯聚合物；该聚合物可进一步进行自组装用于控制药物释放体系、组织工程和生物芯片，在生物材料方面，具有良好的应用价值。
对附图的简要说明
附图说明
图1为实施例一中含双硫五元环功能基团的环状碳酸酯单体的核磁谱图；
图2为实施例一中含双硫五元环功能基团的环状碳酸酯单体的质谱图；
图3为实施例一中含双硫五元环功能基团的环状碳酸酯单体的紫外吸收图谱；
图4为实施例三中嵌段聚合物PEG5k-b-PCDC2.8k的核磁谱图；
图5为实施例四中嵌段聚合物PEG5k-P(CDC2.5k-co-CL3.9k)的核磁谱图；
图6是实施例六中聚合物PEG5k-b-PCDC2.8k交联纳米粒子对Raw264.7和MCF-7细胞的毒性结果图；
图7为实施例七中载有阿霉素的聚合物PEG5k-b-PCDC2.8k交联纳米粒子的体外释放结果图；
图8是实施例七中载有阿霉素的聚合物PEG5k-b
-PCDC2.8k交联纳米粒子对Raw264.7细胞的毒性结果图；
图9为实施例八中载阿霉素的聚合物PEG5k-b-PCDC2.8k交联纳米粒子在小鼠体内的血液循环结果图；
图10为实施例九中载阿霉素的聚合物PEG5k-b-PCDC2.8k交联纳米粒子对荷黑色素肿瘤小鼠的生物分布结果图；
图11为实施例十中载阿霉素的聚合物PEG5k-b-PCDC2.8k交联纳米粒子对荷黑色素肿瘤小鼠的抑制肿瘤生长曲线图；
图12为实施例十中小鼠体重变化曲线图；
图13为实施例十中小鼠生存曲线图。
发明实施例
本发明的实施方式
下面结合实施例和附图对本发明作进一步描述：
实施例一含双硫五元环功能基团的环状碳酸酯单体(CDC)的合成：
Figure PCTCN2015079998-appb-000009
1、一水合硫氢化钠(28.25g，381.7mmol)溶在400mL N，N-二甲基甲酰胺(DMF)中，50℃加热至完全溶解，逐滴加入二溴新戊二醇(20g，76.4mmol)，反应48小时。反应物减压蒸馏除去溶剂DMF，然后用200mL蒸馏水稀释，用250mL乙酸乙酯萃取四次，最后有机相旋蒸得到黄色粘稠状化合物A，产率70％；
2、溶解在400mL的四氢呋喃(THF)中的化合物A在空气中放置24小时，分子间巯基氧化成硫硫键，得到化合物B，产率＞98％；
3、在氮气保护下，化合物B(11.7g，70.5mmol)溶于干燥过的THF(150mL)中，搅拌至完全溶解。接着冷却到0℃，加入氯甲酸乙酯(15.65mL，119.8mmol)，然后逐滴加入Et3N(22.83mL，120.0mmol)。待滴加完毕后，该体系在冰水浴条件下继续反应4h。反应结束后，过滤掉产生的Et3N·HCl，滤液经旋转浓缩，最后用乙醚进行多次重结晶，得到黄色晶体，即含双硫五元环功能基团的环状碳酸酯单体(CDC)，产率64％。
附图1为上述产物CDC的核磁图谱，1H NMR(400MHz，CDCl3)：δ3.14(s，4H)，4.51(s，4H)。元素分析为：C：41.8％，H：4.20％，O：24.3％(理论：C：41.67％，H：4.17％，O：25％，S：33.3％)，CDC单体的质谱分析：MS：192.5(理论分子量：192)，见图2。附图3为不同浓度上述产物单体CDC四氢呋喃溶液的紫外图谱，单体中硫硫五元环在330nm处有吸收，且吸收强度随着单体浓度的增大而增强。
实施例二含双硫五元环功能基团的环状碳酸酯单体(CDC)的合成：
1、一水合硫氢化钠(28.25g，381.7mmol)溶在400mL二甲亚砜(DMSO )中，40℃加热至完全溶解，逐滴加入二溴新戊二醇(20g，76.4mmol)，反应48小时。反应物减压蒸馏除去溶剂DMSO，然后用200mL蒸馏水稀释，用250mL乙酸乙酯萃取四次，最后有机相旋蒸得到黄色粘稠状化合物A，产率42％；
2、溶解在400mL的1，4-二氧六环中的化合物A在空气中放置，分子间巯基氧化成硫硫键，得到化合物B，产率＞98％；
3、在氮气保护下，化合物B(11.7g，70.5mmol)溶于干燥过的1，4-二氧六环(150mL)中，搅拌至完全溶解。接着冷却到0℃，加入氯甲酸乙酯(15.65mL，119.8mmol)，然后逐滴加入Et3N(22.83mL，120.0mmol)。待滴加完毕后，该体系在冰水浴条件下继续反应4h。反应结束后，过滤掉产生的Et3N·HCl，滤液经旋转浓缩，最后用乙醚进行多次重结晶，得到黄色晶体，即含双硫五元环功能基团的环状碳酸酯单体(CDC)，产率32％。
实施例三两嵌段聚合物PEG5k-b-PCDC2.8k的合成
Figure PCTCN2015079998-appb-000010
式中，m＝114，n＝14.6。
在氮气环境下，将0.3g(1.56mmol)含双硫五元环功能基团的环状碳酸酯单体(CDC)、2mL二氯甲烷加入密封反应器里，然后加入分子量为5000的聚乙二醇0.5g(0.1mmol)和1mL的催化剂双(双三甲基硅基)胺锌的二氯甲烷溶液(0.1mol/L)，接着把反应器密封好，转移出手套箱，放入40℃油浴中反应1天后，用冰乙酸终止反应，在冰乙醚中沉淀，最终经过过滤、真空干燥得到产物环碳酸酯聚合物PEG5k-b-PCDC2.8k。
附图4为上述环碳酸酯聚合物的核磁图谱：1H NMR(400MHz，CDCl3)：3.08(s，-CCH2)，3.30(m，-OCH3)，4.05(s，-CH2OCOCHCH2-)，4.07(s，-OCH2CCH2O-)，4.31(m，-CCH2)。
实施例四两嵌段聚合物PEG5k-P(CDC2.5k-co-CL3.9k)的合成
Figure PCTCN2015079998-appb-000011
，式中，m＝114，x＝21.9，y＝13.0，n＝34.9。
在氮气环境下，0.28g(1.46mmol)CDC单体和0.4g(3.51mmol)的己内酯(ε-CL)溶在3mL二氯甲烷中，加入密封反应器里，然后加入分子量5000的聚乙二醇0.5g(0.1mmol)和1m的催化剂双(双三甲基硅基)胺锌的二氯甲烷溶液(0.1mol/L)，接着把反应器密封好，转移出手套箱，放入40℃油浴中反应1天后，用冰乙酸终止反应，在冰乙醚中沉淀，最终经过过滤、真空干燥得到产物环碳酸酯聚合物PEG5k-P(CDC2.5k-co-CL3.9k)。
附图5为上述聚合物的核磁图谱：1H NMR(400MHz，CDCl3)：1.40(m，-COCH2CH2CH2CH2CH2-)，1.65(m，-COCH2CH2CH2CH2CH2-)，2.30(t，-COCH2CH2CH2CH2CH2-)，3.08(s，-CCH2)，3.30(m，-OCH3)，4.03(t，-COCH2CH2CH2CH2CH2O-)，4.05(s，-CH2OCOCHCH2-)，4.07(s，-OCH2CCH2O-)，4.31(m，-CCH2)；GPC测的分子量：14.0kDa，分子量分布：1.56。
实施例五聚合物胶束纳米粒PEG5k-b-PCDC2.8k的制备
采用透析法制备聚合物胶束纳米粒。聚合物PEG5k-b-PCDC2.8k溶解在N，N-二甲基甲酰胺(2mg/mL)中，取200μL滴加到800μL磷酸盐缓冲溶液(10mM，pH 7.4，PB)中，装入透析袋(MWCO 3500)中透析过夜，换五次水，透析介质为PB(10mM，pH 7.4)。最终得到的聚合物纳米粒的浓度为0.2mg/mL。
实施例六聚合物纳米粒子PEG5k-b-PCDC2.8k的交联、解交联、细胞毒性
纳米粒的交联按加催化量的二硫代苏糖醇(DTT)进行。将聚合物纳米粒水溶液通氮气10分钟，尽量将空气赶净。然后向密闭反应器中的纳米粒溶液(1mL，0.25mg/mL，3.21×10-5mmol)中加入10μL溶解在二次水中的二硫代苏糖醇(DTT)(0.007mg，4.67×10-5mmol，硫辛酸官能团摩尔数10％)，密闭室温搅拌反应1天。测定粒子的尺寸为150纳米，和没有交联的粒径相比较少约15％。交联后的纳米粒子在浓度稀释100倍以后其 粒径和粒径分布几乎没有变化；在生理条件下稳定，由此可以看出，双硫交联可以很大程度上提高纳米粒的稳定性。
二硫键可以很容易在还原剂如谷胱甘肽(GSH)作用下断裂。在氮气保护和37℃条件下，将交联纳米粒溶液通氮气10分钟后，加入GSH使其在聚合物纳米粒子溶液中的最终浓度为10mM。交联纳米粒子粒径随着时间的推移逐步被破坏，说明聚合物中双硫环在大量还原物质存在下会断裂。在细胞质中也存在高浓度的还原物质GSH，因此制备的纳米药物载体循环稳定，但被细胞内吞后能快速解离，释放药物。
采用MTT法对交联胶束纳米粒的细胞毒性进行测试。使用到的细胞为MCF-7(人乳腺癌细胞)细胞和Raw 264.7(小鼠巨噬细胞)细胞。以1×104个/mL将MCF-7细胞或Raw 264.7细胞接种于96孔板，每孔100μL，培养至细胞贴壁后，实验组加入含有不同浓度的聚合物纳米粒的培养液，另设细胞空白对照孔和培养基空白孔，平行4个复孔。培养箱中培养24小时后取出96孔板，加入MTT(5.0mg/mL)10μL，继续培养4小时后每孔加入150μL DMSO溶解生成的结晶子，用酶标仪于492nm处测其吸光度值(A)，以培养基空白孔调零，计算细胞存活率。
Figure PCTCN2015079998-appb-000012
式中AT为试验组490nm处的吸光度，AC为空白对照组492nm处的吸光度。聚合物浓度分别为0.1，0.2，0.3，0.4，0.5mg/mL。附图6为纳米粒的细胞毒性结果，从图6中可以看出，当聚合物纳米粒的浓度从0.1mg/mL增大到0.5mg/mL时，Raw264.7细胞和MCF-7细胞的存活率仍高于85％，说明PEG5k-b-PCDC2.8k聚合物纳米粒具有良好的生物相容性。
实施例七交联胶束纳米粒PEG5k-b-PCDC2.8k的载药、体外释放及细胞毒性
以阿霉素作为药物。由于抗癌药物阿霉素是荧光敏感物质，整个操作在避光条件下进行。首先除去阿霉素的盐酸盐，其操作为：1.2mg(0.002mmol)阿霉素溶解在225μL的DMSO中，加三乙胺0.58mL(m＝0.419mg，0.004mmol)搅拌12小时，吸走上层清液。阿霉素的DMSO溶液浓度为5.0mg/mL。将纳米聚合物纳米 粒PEG5k-b-PCDC2.8k溶解在N，N-二甲基甲酰胺(DMF)中。将阿霉素的二甲亚砜溶液与聚合物纳米粒子PEG5k-b-PCDC2.8k的DMF溶液按预定的药物与聚合物质量比混合均匀，搅拌下缓慢向其中加入4倍于其体积的二次水(15s/d)，滴完后对一次水透析。
载药胶束纳米粒的交联也按实施例五的交联方法进行。将100μL交联载阿霉素的聚合物纳米粒子溶液冷冻干燥，然后溶解于3.0mL DMSO中，利用荧光分光光谱仪测试，结合阿霉素的标准曲线计算包封率。
载药量(DLC)和包封率(DLE)根据以下公式计算：
载药量(wt.％)＝(药物重量/聚合物重量)×100％
包封率(％)＝(装载药物重量/药物总投入量)×100％
表1为上述计算结果，可以看出，聚合物PEG5k-b-PCDC2.8k纳米粒子对小分子抗癌药物阿霉素具有高效的包埋作用。
表1载阿霉素交联胶束纳米粒中载药量、包封率的结果
Figure PCTCN2015079998-appb-000013
阿霉素的释放实验是在37℃恒温摇床中震荡(200rpm)进行。药物释放是用二组平行样进行对比的，每组各有二个平行样：第一组，交联载阿霉素的聚合物纳米粒子在加入10mM谷胱甘肽(GSH)模拟细胞内还原环境PB(10mM，pH 7.4)中的释放；第二组，交联载阿霉素的聚合物纳米粒子在PB(10mM，pH 7.4)中的释放；载药聚合物纳米粒子浓度为25mg/L，取0.5mL放入释放用透析袋(MWCO：12,000-14,000)中，每个试管中加入相应的透析溶剂25mL，在预定的时间间隔，取出5.0mL透析袋外部介质用作测试，同时向试管中补加5.0mL相应介质。使用EDINBURGH FLS920荧光仪测定溶液中药物浓度。附图7为阿霉素累积释放量与时间的关系。从图中可以看出，加入模拟肿瘤细胞的还原性物质谷胱甘肽(GSH)后，其释放明显要快于没有加GSH组分， 说明载药的交联纳米粒子具有10mM还原物质GSH的存在下，能有效的释放药物。
载DOX的PEG5k-b-PCDC2.8k交联纳米粒子用MTT法测试其对Raw264.7细胞、MCF-7细胞等的毒性，载药未交联纳米粒及游离药物作为对照。以Raw264.7细胞为例，将Raw264.7细胞以1×104个/mL接种于96孔板，每孔100μL，培养至细胞贴壁后，实验组分别加入含有0.01、0.1、1、5、10、50和100μg/mL的载阿霉素交联纳米粒子溶液，载阿霉素未交联纳米粒子溶液及游离阿霉素的新鲜培养液，另设细胞空白对照孔和培养基空白孔，每孔设4个复孔。培养箱中培养48小时后取出96孔板，加入MTT(5.0mg/mL)10μL，继续培养4h后每孔加入150μL DMSO溶解生成的结晶子，用酶标仪于492nm处测其吸光度值(A)，以培养基空白孔调零，计算细胞存活率。见附图8，从结果可以看出，载阿霉素的交联纳米粒子对Raw264.7细胞的半致死浓度为4.89μg/mL，所以载DOX的PEG5k-b-PCDC2.8k交联纳米粒子能有效的在细胞内释放药物并杀死癌细胞。
实施例八载药PEG5k-b-PCDC2.8k交联纳米粒在小鼠体内的血液循环测定
实验选用体重为18～20克左右，4～6周龄的C57BL/6小黑鼠(中科院上海生命科学院实验动物中心)，称重后，按体重均匀分组，将载药纳米粒和自由药物通过尾静脉注射小鼠体内，其中DOX药量为10mg/kg，在0、0.25、0.5、1、2、4、8、12和24小时定点取血约10μL，通过差量法准确称取血液重量，血液加100μL浓度为1％的曲拉通和500μL DMF(其中含有20mM的DTT，1M的HCl)萃取；离心(20000转/分钟，20分钟)后，取上层清液，通过荧光测得每个时间点DOX的量。
图9为载阿霉素的聚合物PEG5k-b-PCDC2.8k交联纳米粒子在小鼠体内的血液循环结果图，横坐标为时间点，纵坐标为每克血液中的DOX量占总的DOX注射量(ID％/g)。由图可知，自由DOX的循环时间很短2小时已很难检测到DOX，而载药交联纳米粒24小时后仍有4ID％/g。其在小鼠体内的消除半衰期可计算为4.67小时，而自由DOX仅为0.21小时，所以载药交联纳米粒在小鼠体内稳定，有较长的循环时间。
实施例二十一载药PEG5k-b-PCDC2.8k交联纳米粒对荷黑色素肿瘤小鼠的生物分布
实验选用体重为18～20克左右，4～6周龄的C57BL/6小黑鼠，在皮下注射1×106个B16黑色素肿瘤细胞，大约两周后，肿瘤大小为100～200mm3时，将载药纳米粒和自由DOX通过尾静脉注射小鼠体内(DOX药量为10mg/kg)，在6、12和24小时后处死老鼠，将肿瘤及心，肝，脾，肺和肾组织取出，清洗称重后加入500μL1％的曲拉通、通过匀浆机磨碎，再加入900μL DMF萃取(其中含有20mM的DTT，1M的HCl)。离心(20000转/分钟，20分钟)后，取上层清液，通过荧光测得每个时间点DOX的量。
图10为载阿霉素的聚合物PEG5k-b-PCDC2.8k交联纳米粒子对荷黑色素肿瘤小鼠的生物分布结果图，横坐标为组织器官，纵坐标为每克肿瘤或组织中的DOX量占总的DOX注射量(ID％/g)。载药纳米粒在6、12和24小时的肿瘤积累量分别为3.12、2.93、2.52ID％/g，相对于自由DOX的1.05、0.52和0.29ID％/g来说增加了3～12倍，说明载药交联纳米粒通过EPR效应在肿瘤部位积累较多且能持续时间较长。
实施例二十二载药PEG5k-b-PCDC2.8k交联纳米粒对荷黑色素肿瘤小鼠的治疗实验
实验选用体重为18～20克左右，4～6周龄的C57BL/6小黑鼠，称重后，按体重均匀分组，在皮下注射1×106个B16黑色素肿瘤细胞，大约一周后，肿瘤大小为30～50mm3时，将载药纳米粒和自由DOX在0、2、4、6和8天通过尾静脉注射小鼠体内，其中载药纳米粒子中DOX的量为10、20、30mg/kg，自由DOX药量为10mg/kg，从0～15天，每天称量各组小鼠的体重，通过游标卡尺准确量取肿瘤体积，其中肿瘤体积计算方法为：V＝(L×W×H)/2，(其中L为肿瘤的长度，W为肿瘤的宽度，H为肿瘤的厚度)。持续观察小鼠的生存，直到46天。
图11为载阿霉素的聚合物PEG5k-b-PCDC2.8k交联纳米粒子对荷黑色素肿瘤小鼠的抑制肿瘤生长曲线图；图12为小鼠体重变化曲线图；图13为小鼠生存曲线图。由图可知，在DOX浓度为30mg/kg，载DOX的纳米粒治疗16天后，肿瘤得 到明显抑制，而DOX虽然也能抑制肿瘤增长，但对小鼠的毒副作用很大。即使当载药纳米粒中DOX的浓度为30mg/kg时，小鼠的体重几乎没有发生改变，说明载药纳米粒对小鼠没有毒副作用，而DOX组的小鼠体重在7天时降低了23％，说明DOX对小鼠副作用很大。同样是在DOX浓度为30mg/kg，载DOX的纳米粒治疗46天后的这组老鼠全部存活，而DOX治疗10天时全部死亡，而作为对照的PBS组的老鼠在35天时也全部死亡。因此，该载药纳米粒可有效抑制肿瘤增长，并对小鼠没有毒副作用，还可延长荷瘤老鼠的生存时间。
以上结果表明本发明的单体制备得到的聚合物具有良好的生物相容性，作为药物载体应用时，可以增加抗肿瘤药物在体内的循环时间，增加药物在肿瘤部位的富集率，避免药物对正常组织的损伤，可以有效杀死肿瘤细胞，同时对正常细胞影响小。

Claims (8)

  1. 一种含双硫五元环功能基团的环状碳酸酯单体，其特征在于：所述含双硫五元环功能基团的环状碳酸酯单体的化学结构式如下：
    Figure PCTCN2015079998-appb-100001
  2. 一种权利要求1所述含双硫五元环功能基团的环状碳酸酯单体的制备方法，其特征在于，包括以下步骤：将二溴新戊二醇与一水合硫氢化钠在极性溶剂中反应得到化合物A；然后将化合物A在空气中氧化得到化合物B；最后在氮气气氛中，在环醚类溶剂中，将化合物B与氯甲酸乙酯反应得到所述的含双硫五元环功能基团的环状碳酸酯单体。
  3. 根据权利要求2所述含双硫五元环功能基团的环状碳酸酯单体的制备方法，其特征在于：所述二溴新戊二醇与一水合硫氢化钠的摩尔比为(2.5～10)∶1；化合物B与氯甲酸乙酯的摩尔比为1∶(2～4)。
  4. 根据权利要求2所述含双硫五元环功能基团的环状碳酸酯单体的制备方法，其特征在于：制备化合物A时的反应温度为50℃，反应时间为48小时；制备化合物B时化合物A的氧化时间为24小时；制备环碳酸酯单体时的反应温度为冰水浴，反应时间为4小时。
  5. 根据权利要求2所述含双硫五元环功能基团的环状碳酸酯单体的制备方法，其特征在于：所述极性溶剂为N，N-二甲基甲酰胺；环醚类溶剂为四氢呋喃或者1，4-二氧六环。
  6. 根据权利要求2所述含双硫五元环功能基团的环状碳酸酯单体的制备方法，其特征在于：化合物A先溶于醚类溶剂，再在空气中氧化得到化合物B。
  7. 根据权利要求6所述含双硫五元环功能基团的环状碳酸酯单体的制备方法，其特征在于：所述醚类溶剂为四氢呋喃或者1，4-二氧六 环。
  8. 根据权利要求2所述含双硫五元环功能基团的环状碳酸酯单体的制备方法，其特征在于，还包括提纯处理，具体为：
    Figure PCTCN2015079998-appb-100002
    化合物A的提纯：反应结束后，反应物减压蒸馏除去溶剂，然后用蒸馏水稀释，再用乙酸乙酯萃取，最后旋蒸有机相得到黄色粘稠状化合物A；
    Figure PCTCN2015079998-appb-100003
    含双硫五元环功能基团的环状碳酸酯单体的提纯：反应结束后，过滤，滤液经旋转浓缩，再用乙醚重结晶，得到黄色晶体，即含双硫五元环功能基团的环状碳酸酯单体。
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EP3150611B1 (en) 2017-08-23
CN104004001B (zh) 2016-03-30
AU2015266505A1 (en) 2017-01-12
ES2642150T3 (es) 2017-11-15
KR101793483B1 (ko) 2017-11-03
US9796728B2 (en) 2017-10-24
CN104004001A (zh) 2014-08-27
JP2017517573A (ja) 2017-06-29
EP3150611A1 (en) 2017-04-05
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