WO2013181888A1 - 一种环境响应基础共聚物及其制备方法 - Google Patents

一种环境响应基础共聚物及其制备方法 Download PDF

Info

Publication number
WO2013181888A1
WO2013181888A1 PCT/CN2012/080531 CN2012080531W WO2013181888A1 WO 2013181888 A1 WO2013181888 A1 WO 2013181888A1 CN 2012080531 W CN2012080531 W CN 2012080531W WO 2013181888 A1 WO2013181888 A1 WO 2013181888A1
Authority
WO
WIPO (PCT)
Prior art keywords
tumor
copolymer
environmentally responsive
base copolymer
environmentally
Prior art date
Application number
PCT/CN2012/080531
Other languages
English (en)
French (fr)
Inventor
蓝闽波
郁荣华
赵红莉
袁慧慧
Original Assignee
华东理工大学
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 华东理工大学 filed Critical 华东理工大学
Publication of WO2013181888A1 publication Critical patent/WO2013181888A1/zh

Links

Classifications

    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • 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
    • 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

Definitions

  • the present invention relates to the field of smart pharmaceutical technology, and more particularly to an environmentally responsive base copolymer and a method of preparing the same.
  • the distribution of many drugs in the body is not selective, or the water solubility is poor, making the drug toxic to healthy tissues.
  • Targeted drug delivery and controlled release drugs can reduce the toxicity of drugs to healthy tissues while achieving therapeutic effects, and have been the focus of drug development and therapeutic research.
  • the coated micelles are used as a carrier to embed the drug to prepare targeted drug-loaded micelles for disease treatment.
  • the use of a copolymer with detectable function allows the micelle itself to be detectable or to embed diagnostic reagents to prepare a multi-functional disease micelle diagnosis platform for disease diagnosis and disease diagnosis and detection.
  • Tumor targeting is divided into active targeting and passive targeting.
  • the former is achieved by specific binding between the antibody and the ligand of the antigen or tumor-associated receptor and the receptor.
  • the latter is achieved by the EPR (enhanced penneability and retention) effect of solid tumors.
  • EPR effect Rapid and unregulated growth of tumor cells requires a large amount of blood to transport nutrients and oxygen, causing excessive and chaotic vascular growth, incomplete structure, and a large number of proliferating vascular wall endothelial cells, resulting in gaps between vascular endothelial cells. 200-600 nm ; vascular permeability factor induced by external plexus; damaged lymphatic capillaries caused by poor lymphatic excretion.
  • the EPR effect can promote the passive accumulation of macromolecules and nanoparticles in tumor tissues.
  • the tumor-targeted drug delivery system is capable of controlling the therapeutic dose to the lowest possible extent to reduce toxicity to non-targets, and to deliver a sufficient dose of the drug to the lesion for therapeutic effects.
  • An environmentally responsive base copolymer having a structure as shown in Structural Formula 1, having a well-defined triblock structure, which in turn is a hydrophobic segment, a hydrophilic environmental response segment, and a reactive poly N-methacryloyloxy group Succinimide (PNAS) chain.
  • Structural Formula 1 having a well-defined triblock structure, which in turn is a hydrophobic segment, a hydrophilic environmental response segment, and a reactive poly N-methacryloyloxy group Succinimide (PNAS) chain.
  • the hydrophobic segment material is selected from the group consisting of biodegradable hydrophobic polymers, including polyamino acids, polypeptides, polylactic acid-glycolic acid copolymers, polylactic acid, polyglycolic acid, polycaprolactone, polylactic acid polycaprolactone One or more of the mixed materials and their derivatives.
  • the environmental response of the hydrophilic environmental response segment includes temperature, pH, ultrasound, light.
  • the environmentally responsive material is selected from the group consisting of N-mercapto acrylamides, such as: N-(iso)propyl (meth) acrylamide, N-tert-butyl acrylamide, N,N-diethyl acrylamide, 2-carboxyl Isopropyl acrylamide.
  • Oleic acids and their esters such as: methacrylic acid, dimethacrylic acid, 10-undecenoic acid, [2-(dimethylamino)ethyl] methacrylate, methyl methacrylate, acrylic acid Butyl ester, diisopropylaminoethyl methacrylate, diethylaminoethyl methacrylate.
  • Others such as: methyl cellulose, carboxypropyl cellulose, vinyl alcohol-vinyl acetate copolymer, vinyl methyl ether, N-vinyl caprolactam, polyurethane.
  • the hydrophilic environmental response segment forms a micellar shell with excellent biocompatibility. It acts to stabilize and protect the micelles, and can release the drug and diagnostic reagents that are environmentally responsive to release micelles.
  • the reactive poly(N-methacryloyloxysuccinimide) (PNAS) polymer chain is polymerized by N-methacryloyloxysuccinimide.
  • the hydrophobic segment forms a hydrophobic core of micelles for entrapment of hydrophobic poorly soluble drugs and diagnostic reagents.
  • the hydrophilic environment response section forms a micelle shell with excellent biocompatibility; it functions as a stable, protective micelle, and can release the drug-embedded drug and diagnostic reagent in an environmental response;
  • PNAS chain makes the base copolymer It can be combined with different functional ligands such as folic acid, fluorescein and nitroxide to prepare various functional modules, including targeting and detection function modules.
  • Each functional module including the targeting and detection function modules, has consistent environmental response performance; each functional module can be integrated into a warehouse-based functional module library; a plurality of different functional modules are selected from the library as needed, Including the targeting and detection function modules, mixing according to the required ratio, embedding drugs or diagnostic reagents, forming multi-functional disease diagnosis and treatment micelles, achieving simultaneous targeted drug delivery and instrument detection. It provides a simple and versatile platform for laboratory research and clinical treatment of diseases (as shown in Figure 1).
  • the warehouse function module library is open; if the existing function modules in the library cannot meet the needs; the basic copolymer of the invention can be quickly and simply connected with the new functional ligand, and the new functional module can be conveniently expanded. Expanding the library of functional modules without having to design synthetic functional materials from scratch, greatly simplifies the preparation of functional materials. Achieve more and wider applications in the biomedical field.
  • An environmentally responsive base copolymer for use in the field of preparing oncology drugs is provided.
  • the invention can provide a simple and versatile platform for laboratory research and clinical treatment of diseases.
  • the multifunctional disease diagnosis and treatment micelle has a micelle diameter of less than 200 nm and an optimum value of 100 to 150 nm.
  • the multifunctional disease diagnosis and treatment micelles do not need to add other additional emulsifiers during the preparation process.
  • the most suitable preparation methods are as follows:
  • Dialysis An organic solvent that dissolves functional modules, including targeting and detection functional modules, and drugs or diagnostic reagents, while being miscible with water, such as DMF (dimethylformamide), DMSO (dimethyl sulfoxide) ), dissolving the functional module with the drug or diagnostic reagent, placing the mixed solution into the dialysis bag for dialysis against water, and self-assembling to form a micelle embedded with the drug or diagnostic reagent.
  • DMF dimethylformamide
  • DMSO dimethyl sulfoxide
  • Solvent evaporation film formation method The functional module, including the targeting and detection function module, and the drug or diagnostic reagent are simultaneously dissolved in a volatile organic solvent such as chloroform, dichloromethane, and the mixed solution is placed in a flask.
  • a volatile organic solvent such as chloroform, dichloromethane
  • the organic solvent is removed by steaming under reduced pressure to form a film on the wall of the bottle with the functional module and the drug or diagnostic reagent.
  • Water is then injected into the bottle to allow the film to be sufficiently wetted by water to self-assemble to form micelles in which the drug or diagnostic agent is embedded.
  • Final dialysis removes unembedded drugs or diagnostic reagents and other impurities.
  • a method for preparing an environmentally responsive base copolymer the specific steps of which are:
  • RAFT refers to a reversible addition-fragmentation chain transfer polymerization method
  • a second step RAFT polymerization was carried out at a ratio of 0.01 to 100:1 to prepare an environmentally responsive base copolymer.
  • the invention has the advantages that the structure of the environment-responsive base copolymer is controllable, and the hydrophobic section, the hydrophilic environment response section and the PNAS chain are independent of each other, and have a clear and clear structure, and can fully exert the performance of each segment.
  • a variety of functional modules, including targeting and detection modules, can be readily prepared from the base copolymer.
  • the corresponding targeting and detection function modules can be flexibly selected to prepare multi-functional disease-treating micelles embedding drugs or diagnostic reagents to achieve simultaneous targeted drug delivery and instrument detection.
  • FIG. 1 The functional module is used to form a warehouse function module library.
  • the corresponding functional modules are self-assembled into functional micelles as needed.
  • Figure 3 Structure (A) and 1 H NMR spectrum (B) of the base copolymer PLA-PNNUA-PNAS.
  • Figure 4 shows the pH synergy of the micelles.
  • FIG. 7 Transmission electron micrograph of multi-functional micelle (A) and particle size distribution (B)
  • FIG. 8 Multi-functional micelles were incubated with KBH cells and NIH 3T3 cells for 3 hours with laser confocal microscopy images (Fig. 8 A, B) and electron paramagnetic resonance spectra (Fig. 8 a, b).
  • Figure 9 is a transmission electron micrograph of the treatment of micelles with doxorubicin.
  • Figure 10 The particle size distribution (A) and ⁇ potential (B) of the micelles treated with doxorubicin.
  • Figure 11 The release profile of doxorubicin in micelles at 37'C in different PBS buffers.
  • FIG. 13 In vivo imaging of MCF-7 tumor-bearing mice (Fig. 13a) and tissue photographs (Fig. 13b).
  • FIG. 14 In vivo imaging of MCF-7 tumor-bearing mice (Fig. 14a) and tissue photographs (Fig. 14b).
  • FIG. 15 PLA-PNNUA-FA, PLA-PNNUA-FITC, PLA-PNNUA-TEMPO and
  • Example 1 Specific embodiments of an environmentally responsive base copolymer of the present invention and a method of preparing the same are provided below.
  • Example 1 Specific embodiments of an environmentally responsive base copolymer of the present invention and a method of preparing the same are provided below.
  • PLA polylactic acid
  • Synthesis of polylactic acid macromolecule RAFT initiator A small molecule RAFT chain transfer agent 3.6 g and a small amount of triethylamine were added to the flask, and methylene chloride was dissolved. An equimolar amount of oxalyl chloride was added and reacted for 6 hours. After filtration, the filtrate was collected, transferred to a flask containing 3.7 g of polylactic acid, and reacted at room temperature for 24 hours. Then, the reaction solution was concentrated under reduced pressure, and poured into ice-free diethyl ether to give a precipitate.
  • the precipitate was collected by centrifugation and washed, and dried in a vacuum oven at 40 'C to obtain a pale yellow powder polylactic acid macro RAFT initiator having a molecular weight of 2300 g mol. (gel permeation chromatograph, GPC).
  • PLA-PNNUA was used as the RAFT initiator, and the base copolymer PLA-PNNUA-PNAS was prepared with the monomer NAS (N-methacryloyloxysuccinimide).
  • PLA-PNNUA and NAS monomers were added to the flask, dissolved in 5 mL of DMF, and AIBN was added. Nitrogen is removed by nitrogen.
  • Mw/Mn 1.22 (condensed Glue-through chromatography, GPC).
  • the structure of PLA-PNNUA-PNAS was characterized by nuclear magnetic resonance spectroscopy (NMR, Figure 3). It indicates that the environmentally-responsive base copolymer PLA-PNNUA-PNAS was successfully prepared.
  • the flask was successively added with equimolar 2,2'-(ethylenedioxy)bis(ethylamine) and FA-NHS, dissolved in DMSO, and protected from light for 48 hours.
  • the reaction solution was poured into anhydrous diethyl ether to give an orange paddle, which was an aminated folic acid FA-NH 2 , which was collected by centrifugation and dried under vacuum.
  • PLA-PNNUA-PNAS was mixed with FA-NH 2 in DMSO and reacted in the dark for 48 hours.
  • the reaction solution was dialyzed against 1000 mL of ultrapure water for 24 hours.
  • the liquid in the dialysis bag was freeze-dried to obtain a pale yellow powder, which was a tumor targeting function module PLA-PNNUA-FA.
  • fluorescent PLA-PNNUA-FITC functional module 2,2'-(ethylenedioxy)bis(ethylamine) and FITC (fluorescein isothiocyanate) were added to the flask, and dissolved in DMSO. Protected from light for 48 hours. The reaction solution was poured into anhydrous diethyl ether to produce a yellow-green paddle at the bottom, which was aminated fluorescein isothiocyanate FITC-NH 2 , collected by centrifugation, washed, and dried at room temperature under vacuum.
  • PLA-PNNUA-PNAS and FITC-NH 2 were dissolved in DMSO and reacted in the dark for 48 hours.
  • the reaction solution was dialyzed against 1000 mL of ultrapure water for 24 hours.
  • the liquid in the dialysis bag was freeze-dried to obtain a pale yellow-green powder, which was a fluorescent functional module PLA-PNNUA-FITC.
  • TEMPO 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl radical
  • a warehouse-type functional module library is established with the three functional modules PLA-PNNUA-FA, PLA-PNNUA-FITC and PLA-PNNUA-TEMPO prepared in the embodiment 5.
  • a variety of different functional modules can be selected from the warehouse function module library, including tumor targeting and detection function modules, mixed according to any required ratio, and embedded anti-tumor drugs as needed to form a multifunctional tumor diagnosis and treatment micelle.
  • pH synergistic temperature sensitivity LCST low critical solution temperature
  • PLA-PNNUA micelles were prepared at a concentration of 500 mg/mL with PBS buffer solution (0.02 M) at pH 7.4, 6.5, and 5.0, respectively, and a curve was drawn from the light transmittance to obtain the LCST at each pH value. Figure 4). The results showed that the LCST of the micelles was 35.4 °C at pH 5.0, 37.5 °C at pH 6.5, and 39.4 °C at pH 7.4.
  • Multifunctional micelles were prepared by dialysis.
  • PLA-PNNUA-FITC and PLA-PNNUA-TEMPO were mixed in an appropriate ratio and dissolved in 1 mL of DMSO. Dialysis for 1000 mL of ultrapure water for 24 hours. At the excitation wavelength of 488 nm, the fluorescence peak at 530 nm is the fluorescence emitted by FITC in PLA-PNNUA-FITC (Fig. 5).
  • In vitro cellular uptake assay of multifunctional micelles Evaluation of the lack of expression of folate receptor overexpressing KB cells (human oral epidermoid carcinoma cells) and folate receptors by laser confocal microscopy and electron paramagnetic resonance NIH 3T3 cells (mouse embryonic fibroblasts) were examined for in vitro cellular uptake. The first was observed by laser confocal microscopy. KB cells or NIH 3T3 cells were seeded into 6-well plates with coverslips. After culturing for 24 hours, the original medium was discarded and a multi-purpose micelle culture solution of 250 mg L was added.
  • DAPI 4,',6-diamidino-2-phenylindole
  • the second part is the electron paramagnetic resonance test.
  • KB cells or NIH 3T3 cells were seeded into 6-well plates. After culturing for 24 hours, the original medium was discarded, and a multifunctional micelle culture solution having a concentration of 250 mg L was added. After continuing to culture for 3 hours, the cells were washed with PBS buffer, trypsinized, and the cells were resuspended by adding PBS buffer, and the supernatant was discarded by centrifugation to collect the cells.
  • the cells were dried by high-purity nitrogen flow, 0.2 mL of DMSO was added, and after 30 minutes of sonication, the electron paramagnetic resonance signal of the DMSO solution was detected by an electron paramagnetic resonance spectrometer X-band at 298 K.
  • Laser confocal microscopy revealed that the micelles were selectively taken up by KB cells. It can be seen from the FITC channel of Figure 8A that the fluorescence produced by PLA-PNNUA-FITC in the micelles is from the cell paddle. This is a typical feature of micelles entering cells by endocytosis of KB cells.
  • FIG. 8A and B in the lower right corner are the paramagnetic electronic signal maps of KB cells and NIH 3T3 cells, respectively. It can be found that KB cells have strong signals and NIH 3T3 cells do not detect signals. This is consistent with the results observed by laser confocal microscopy.
  • Tumor-targeted doxorubicin-treated micelles are embedded with the fluorescent anticancer drug doxorubicin (DOX), which has both tumor targeting and fluorescence functions.
  • DOX fluorescent anticancer drug doxorubicin
  • the tumor targeting function modules PLA-PNNUA-FA and PLA-PNNUA were dissolved in chloroform, and a doxorubicin chloroform solution was added. After mixing, the chloroform was removed by rotary evaporation. Ultrasound was added to 10 mL of double distilled water to obtain a tumor-targeted doxorubicin-treated micelle (sample No. FA-PN4-7).
  • the micelles were placed in a dialysis bag and dialyzed against 1000 mL of ultrapure water for 24 hours.
  • the dialysis medium was measured by ultraviolet-visible spectrophotometer at 480 nm, and the encapsulation rate and drug loading were calculated by substituting the doxorubicin standard curve.
  • the middle dark region is the hydrophobic core of the micelle embedded with doxorubicin, and the light-colored part embedded in the surrounding is dry.
  • the collapsing micelle hydrophilic segment ( Figure 9).
  • the particle size was 138.2 nm
  • the PDI disersion index
  • the monodisperse distribution was 0.160
  • the zeta potential was -33.65 mv (Fig. 10).
  • In vitro release of tumor-targeted doxorubicin-treated micelles The in vitro release of tumor-targeted doxorubicin-treated micelles (FA-PN4-7) was performed at 37 ° C with an oscillation of 100 ⁇ m. 5 mL of the freshly prepared micelle solution was placed in a dialysis bag for dialysis against the release medium. The release medium was 100 mL of PBS buffer, and the pH was 7.4, 6.8, and 5.5, respectively. The 3.0 mL release medium was taken out at regular intervals, and an equal volume of fresh release medium was immediately replenished. The concentration of doxorubicin in the release medium was determined by ultraviolet-visible spectrophotometry.
  • MCF-7 cells human breast cancer cells
  • F-PN4-7 MTT assay for tumor-targeted doxorubicin-treated micelles
  • Azamin Cytotoxicity As can be seen from Figure 12, the cytotoxicity of tumor-targeted doxorubicin-treated micelles is greater than that of free doxorubicin at the same exposure time of the same concentration. Both of them are toxic to cells in a concentration- and time-dependent manner.
  • Free doxorubicin is a small molecule state that enters cells by passive diffusion, and tumor-targeted doxorubicin-treated micelles with folic acid molecules enter the cell through receptor-mediated endocytosis and then accumulate in acidity.
  • the micelles collapse and squeeze to release the drug, and the action of entering the nucleus and DNA affects the replication of DNA and kills the cells.
  • the cytotoxicity of tumor-targeted doxorubicin micelles is related to the release of drugs. After the micelles enter the cells, the drug is released in response to the environment, and the drug concentration is greatly increased in a short period of time. The rate exceeds the passive diffusion dependence. The difference in intracellular and extracellular concentrations allows the drug to enter the nucleus faster than free doxorubicin.
  • mice Tumor-bearing mice were divided into two groups of 4 mice each. General anesthesia was administered intraperitoneally with 0.2% sodium pentobarbital 0.2 mL. Each mouse in the experimental group was administered with a single dose of 5 mg Kg body weight per vehicle intravenously with doxorubicin. The grouping is as follows:
  • mice The first group of 4 mice were treated as follows:
  • 1-A Negative control group, no administration.
  • 1-B Positive control group, free doxorubicin solution.
  • 1- C Tumor-targeted doxorubicin for the treatment of micelles.
  • 1-D Doxorubicin-loaded micelles without folate ligand.
  • the treatment of the second group of 4 mice was:
  • 2- A Negative control group, no administration.
  • 2-B The tumor site was first injected with KH 2 P0 4 solution (0.02 M, pH about 5.5) and then injected with tumor-targeted doxorubicin to treat micelles.
  • 2-C The tumor site was first injected with ⁇ 01 ⁇ 0 4 solution (0.02 M, pH about 5.5) and heated to 42 'C. Re-injection of tumor-targeted doxorubicin to treat micelles, keeping 42 ° C for half an hour.
  • 2-D The tumor site was heated to 42 °C, and then the tumor-targeted doxorubicin was used to treat the micelles and kept at 42 °C for half an hour.
  • mice were placed on the sample stage from left to right, and a 1.5 mL centrifuge tube containing doxorubicin solution was placed as a positive control.
  • Instrument excitation wavelength 470 nm
  • detect the emission wavelength 600 nm
  • the experimental results of the first group are shown in Fig. 13, and the arrow is the tumor-bearing position.
  • the fluorescence of 1-B in vivo imaging of mice showed a systemic distribution, and the overall intensity was weak, and no obvious fluorescence was observed at the tumor-bearing site, indicating that the concentration of doxorubicin in the tumor-bearing site was low.
  • the fluorescence intensity in each organ after the dissection of the mouse was weak.
  • the fluorescence intensity of the mouse 1-C tumor-bearing site is high, indicating that the concentration of doxorubicin in the tumor-bearing site is high, and the tumor-targeted doxorubicin-treated micelle has the ability to actively target the tumor. From the perspective of organ fluorescence imaging, the tumor has a higher fluorescence intensity.
  • Mouse 1-D was injected with PLA-PNNUA doxorubicin-loaded micelles without folate ligand. From the in vivo imaging, it can be seen that the distribution of doxorubicin in mice is also systemic, and the fluorescence intensity of the tumor-bearing site. Slightly stronger than other parts. The fluorescence intensity of each organ was averaged except for the higher tumor and liver.
  • the experimental results of the second group are shown in Fig. 14, and the arrow is the tumor-bearing position.
  • the tumor site of mouse 2-B was pre-injected with KH 2 P0 4 solution to cause a local weak acid environment.
  • the results showed that the tumor-bearing site of mouse 2-B showed strong fluorescence, and the tumor tissue was also strong after dissection. Fluorescence.
  • the tumor-bearing site of mouse 2-C passes through
  • the acidification of the KH 2 P0 4 solution was simultaneously heated locally to 42 ° C at the tumor-bearing site to trigger the pH synergistic temperature-sensitive response of the drug-loaded micelles.
  • the results showed extremely intense fluorescence in the tumor-bearing site of the mouse, and strong fluorescence was observed in the dissected tumor tissue.
  • Mouse 2-D was locally heated to 42 V in the tumor-bearing site, but no KH 2 P0 4 solution was injected into the tumor-bearing site.
  • the results of in vivo imaging and dissection also showed strong fluorescence in the tumor-bearing site of the mouse. .
  • the fluorescence of mouse 2-B tumor-bearing site is weaker than that of 2-C and 2-D.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Dispersion Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Dermatology (AREA)
  • Polymers & Plastics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Inorganic Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

一种环境响应基础共聚物，其具有明确的三嵌段结构，依次分别为疏水段、亲水环境响应段和具有反应活性的聚N-甲基丙烯酰氧琥珀酰亚胺（PNAS）链。该共聚物的制备方法为：将疏水段与一个小分子链转移剂连接，制备一种大分子引发剂；将环境响应单体与大分子引发剂进行第一步聚合；将N-甲基丙烯酰氧琥珀酰亚胺（NAS）单体与第一步的产物进行第二步聚合，制备出环境响应基础共聚物。所述的环境响应基础共聚物的结构是可控的，各链段之间相互独立，具有清晰明确的结构，可以充分发挥各链段的性能。

Description

一种环境响应基础共聚物及其制备方法
【技术领域】 本发明涉及智能药物技术领域， 具体地说， 是一种环境响应基础共聚物及其制备 方法。
说
【背景技术】
很多药物在体内的分布不具选择性， 或是水溶性差， 使药物对健康组织产生毒性 书
或是极易被清除。 靶向给药、 可控释药能够在取得治疗效果的同时将药物对健康组织 的毒性降低， 一直是药物开发和治疗研究的重点。 以共聚物胶束作为载体， 包埋药物 制备用于疾病治疗的靶向载药胶束。 运用具有可检测功能的共聚物， 使胶束本身具有 可检测性或包埋诊断试剂就能制备用于疾病进行诊断的同时具有疾病治疗与检测诊 断功能的多功能疾病胶束诊疗平台。
以制备一种用于肿瘤的诊疗平台为例。 肿瘤靶向分为主动靶向和被动靶向。 前者 利用抗体与抗原或肿瘤相关受体的配体与受体间的特异性结合来实现。后者通过实体 肿瘤的 EPR ( enhanced penneability and retention ) 效应实现。 EPR效应： 肿瘤细胞快 速无节制的生长需要大量血液输送营养物质、 氧气， 引发血管生长过度丰富而杂乱， 结构不完整， 存在大量的增殖性血管壁内皮细胞， 导致血管内皮细胞之间的间隙达到 了 200-600 nm; 血管通透因子引发的外湊； 损坏的淋巴毛细管引起的肿瘤淋巴排泄功 能低下。 EPR效应可以促进大分子和纳米颗粒在肿瘤组织中被动蓄积。肿瘤靶向的药 物输送系统， 能够将治疗剂量控制在尽可能低的范围内， 以降低对非靶点的毒性， 又 能够将足够剂量的药物输送到病灶取得治疗效果。
近年来， 随着纳米技术的突飞猛进， 一门新的交叉学科诊疗学（ eranostics )逐 步发展了起来。诊疗学主要综合了治疗和诊断两个概念，可以实现同步的诊断与治疗。
【发明内容】 本发明的目的在于克服现有技术的不足，提供一种环境响应基础共聚物及其制备 方法。
本发明的目的是通过以下技术方案来实现的：
一种环境响应基础共聚物， 共聚物的结构如结构式 1所示， 具有明确的三嵌段结 构， 依次分别为疏水段、 亲水环境响应段和具有反应活性的聚 N-甲基丙烯酰氧琥珀 酰亚胺 （PNAS) 链。
Figure imgf000004_0001
基础共聚物
所述的疏水段的材料选自可生物降解疏水性聚合物， 包括聚氨基酸、 多肽、 聚乳 酸 -羟基乙酸共聚物、 聚乳酸、 聚羟基乙酸、 聚己内酯、 聚乳酸聚己内酯共混材料及 其衍生物中的一种或几种。
所述的亲水环境响应段的环境响应包括温度、 pH、 超声、 光。环境响应材料选自 N-垸基丙烯酰胺类， 如： N- (异） 丙基 （甲基） 丙烯酰胺、 N-叔丁基丙烯酰胺、 N，N- 二乙基丙烯酰胺、 2-羧基异丙基丙烯酰胺。 烯酸类及其酯类， 如： 甲基丙烯酸、 双甲 基丙烯酸、 10-十一烯酸、 甲基丙烯酸 [2- (二甲基氨基） 乙基]酯、 甲基丙烯酸甲酯、 丙烯酸丁酯、 甲基丙烯酸二异丙胺基乙酯、 甲基丙烯酸二乙胺基乙酯。 其他如： 甲基 纤维素、 羧基丙基纤维素、 乙烯醇-乙酸乙烯酯共聚物、 乙烯基甲醚、 N-乙烯基己内 酰胺、 聚氨酯。 亲水环境响应段形成具有极好生物相容性的胶束外壳。 起到稳定、 保 护胶束的作用， 并可进行环境响应释放胶束包埋的药物和诊断试剂。
所述的具有反应活性的聚 N-甲基丙烯酰氧琥珀酰亚胺 （PNAS) 聚合物链为 N- 甲基丙烯酰氧琥珀酰亚胺聚合而成。
疏水段形成胶束的疏水内核， 用于包埋疏水性难溶药物和诊断试剂。
亲水环境响应段形成具有极好生物相容性的胶束外壳； 起到稳定、 保护胶束的作 用， 并可进行环境响应释放胶束包埋的药物和诊断试剂； PNAS链使基础共聚物能够 与不同的功能配体， 如叶酸、 荧光素、 氮氧自由基， 连接制备各种功能模块， 包括靶 向和检测功能模块。
各功能模块， 包括靶向和检测功能模块， 都具有一致的环境响应性能； 各功能模 块可以整合成为一个仓储式功能模块库； 根据需要从库中选取多种不同功能模块， 包 括靶向和检测功能模块， 按需要比例混合， 包埋药物或诊断试剂， 组成多功能疾病诊 疗胶束， 实现同时靶向给药和仪器的检测。 可为疾病的实验室研究与临床治疗提供一 种使用简便的多功能平台 （如附图 1所示）。
仓储式功能模块库是开放式的； 若库中现有功能模块不能满足需要； 本发明的基 础共聚物能够迅速而简单地与新的功能配体相连接， 方便地扩展出新的功能模块， 扩 充功能模块库， 而不必从头设计合成功能材料， 大大简化功能材料的制备过程。 实现 在生物医学领域更多、 更广的应用。
一种环境响应基础共聚物在制备肿瘤药物领域中的应用。
本发明可为疾病的实验室研究与临床治疗提供一种使用简便的多功能平台。 所述的多功能疾病诊疗胶束， 所述的胶束粒径小于 200 nm， 最优值为 100〜150 nm。
所述的多功能疾病诊疗胶束， 制备过程中无需添加其他额外的乳化剂。 最适合的 制备方法有如下两种：
透析法： 用可以溶解功能模块， 包括靶向和检测功能模块， 和药物或诊断试剂， 同时又与水能够互溶的有机溶剂， 如 DMF (二甲基甲酰胺）， DMSO (二甲基亚砜）， 溶解功能模块与药物或诊断试剂， 将混合溶液置入透析袋内对水透析， 自组装形成包 埋有药物或诊断试剂的胶束。
溶剂挥发成膜法制备法： 将功能模块， 包括靶向和检测功能模块， 和药物或诊断 试剂同时溶解于易挥发的有机溶剂， 如氯仿， 二氯甲垸， 将混合溶液置于烧瓶中， 减 压旋蒸除去有机溶剂， 使功能模块与药物或诊断试剂在瓶壁形成一层薄膜。 然后向瓶 中注入水， 使薄膜充分被水浸润， 自组装形成包埋有药物或诊断试剂的胶束。 最后透 析除去未包埋的药物或诊断试剂和其他杂质。
一种环境响应基础共聚物的制备方法， 其具体步骤为：
( 1 ) 将疏水段与一个小分子 RAFT链转移剂以摩尔比 1 :1的比例连接， 制备一 种大分子 RAFT引发剂； RAFT是指可逆加成-断裂链转移聚合方法；
(2)将环境响应单体与大分子 RAFT引发剂以摩尔比 100: 0.01到 100:1的比例 进行一步 RAFT聚合；
(3 )将 N-甲基丙烯酰氧琥珀酰亚胺 （NAS)单体与第一步的产物以摩尔比 100:
0.01到 100:1的比例进行第二步 RAFT聚合， 制备出环境响应基础共聚物。
与现有技术相比， 本发明的积极效果是： 本发明的优点在于环境响应基础共聚物的结构是可控的， 疏水段、 亲水环境响应 段与 PNAS链相互独立， 具有清晰明确的结构， 可以充分发挥各链段的性能。 由基础 共聚物能够简单地制备多种功能模块， 包括靶向与检测模块。 根据治疗的需要和检测 手段的不同， 灵活选取相应的靶向和检测功能模块， 制备包埋药物或诊断试剂的多功 能疾病诊疗胶束， 实现同时靶向给药和仪器的检测。
【附图说明】
附图 1 以基础共聚物开发出功能模块组成仓储式功能模块库， 根据需要选取相 应的功能模块自组装成功能胶束。
附图 2 基础共聚物 PLA-PNNUA-PNAS 的合成及其三种功能模块
PLA-PNNUA-FA, PLA-PNNUA-FITC和 PLA-PNNUA-TEMPO的合成。
附图 3 基础共聚物 PLA-PNNUA-PNAS的结构 （A) 与1 H NMR谱 （B)。 附图 4胶束的 pH协同温敏性能。
附图 5 多功能胶束的荧光图谱。
附图 6 多功能胶束的电子顺磁共振图谱。
附图 7 多功能胶束的透射电镜照片 （A) 与粒径分布 （B)
附图 8 多功能胶束对 KB细胞与 NIH 3T3细胞共同孵育 3小时的激光共聚焦显 微镜照片 （图 8 A、 B ) 与电子顺磁共振图谱 （图 8 a、 b)。
附图 9 阿霉素诊疗胶束的透射电镜照片。
附图 10 阿霉素诊疗胶束的粒度分布 （A) 和 ^^电位 （B)。
附图 11 阿霉素诊疗胶束 37'C下在不同 PBS缓冲液中的释药曲线。
附图 12游离阿霉素和阿霉素诊疗胶束对 MCF-7细胞的细胞毒性。
附图 13 MCF-7荷瘤小鼠的活体成像 （图 13 a) 与组织照片 （图 13 b)。
附图 14 MCF-7荷瘤小鼠的活体成像 （图 14 a) 与组织照片 （图 14 b)。
附图 15 PLA-PNNUA-FA， PLA-PNNUA-FITC， PLA-PNNUA-TEMPO 和
PLA-PNNUA的细胞毒性测试。
【具体实施方式】
以下提供本发明一种环境响应基础共聚物及其制备方法的具体实施方式。 实施例 1
聚乳酸 （PLA) 的制备： 在烧瓶内加入工业 DL-乳酸 22.5 g。 氮气保护下升温至 130 °C并保温 3小时。冷却至 50 °C后加入 20 mL甲苯和催化剂二水辛酸亚锡 100 mg， 160 V , 反应 24小时。 反应完后冷却至室温， 将反应液全部倒入 50 %乙醇中， 静置 产生沉淀。 过滤收集沉淀后用无水乙醇清洗， 室温真空干燥 24小时。 即为目标产物 聚乳酸 （PLA)， „ = 2 300 g mol; w/ „= 1.94 (凝胶潫透色谱仪， GPC) 。
小分子 RAFT链转移剂的合成： 烧瓶内加入正十二硫醇 20.20 g和四丁基溴化铵， 丙酮溶解。 滴加 50 %氢氧化钠水溶液 10 mL。 加入二硫化碳 7.61 g， 氯仿 20 mL和 50 %氢氧化钠溶液 20 mL， 反应过夜。 滴加入浓盐酸， 产生固体物。 过滤反应液， 收 集固体， 正己垸重结晶， 室温真空干燥后得黄色结晶小分子 RAFT链转移剂。
!H NMR (δ' ppm)： 0.88 (t， 3H， CH3)， 1.23-1.43 (m， 18H， CH2-CH2)，
1.61-1.74 (m， 8H， C-CH3和 S-CH2-CH2) ， 3.28 (t， 2H， S-CH2) 。
聚乳酸大分子 RAFT引发剂的合成：烧瓶中加入小分子 RAFT链转移剂 3.6 g和少 量三乙胺， 二氯甲垸溶解。 加入等摩尔量的草酰氯， 反应 6小时。 过滤， 收集滤液， 转移入装有聚乳酸 3.7 g的烧瓶中，室温下反应 24小时。然后将反应液减压旋蒸浓縮， 倾倒入冰无水乙醚中产生沉淀， 离心收集沉淀清洗后真空烘箱 40 'C烘干， 得到淡黄 色粉末聚乳酸大分子 RAFT引发剂， 分子量 2300 g mol (凝胶潫透色谱仪， GPC ) 。
基础共聚物 PLA-PNNUA-PNAS的合成： 第一步， 向烧瓶中加入一定比例的 N-异丙 基甲基丙烯酰胺、 N-异丙基马来酰胺酸、 10-十一烯酸， DMF (二甲基甲酰胺） 溶解。 加入 AIBN (偶氮二异丁腈） 和聚乳酸大分子 RAFT引发剂。 通氮气除氧。 80'C反应
6小时。 反应结束后将反应液倒入无水乙醚中， 将得到的沉淀离心收集， 清洗后室温 真空烘干， 得白色粉末 PLA-PNNUA, Mn = 34 400 g mol, Mw/Mn = 1.18 (凝胶潫透 色谱仪， GPC) 。 第二步， 以 PLA-PNNUA作为 RAFT引发剂， 与单体 NAS (N-甲基丙烯酰氧琥 珀酰亚胺） 制备基础共聚物 PLA-PNNUA-PNAS。 烧瓶中加入 PLA-PNNUA和 NAS 单体， 加入 5 mLDMF溶解， 加入 AIBN。 通氮气除氧。 80 °C反应 6小时。 反应结束 后将反应液倒入无水乙醚中， 将得到的沉淀离心收集， 清洗后室温真空烘干， 得白色 粉末 PLA-PNNUA-PNAS, Mn = 48 800 g mol， Mw/Mn = 1.22 (凝胶潫透色谱仪， GPC )。 核磁共振氢谱 （ NMR， 附图 3 ) 对 PLA-PNNUA-PNAS的结构经行了表征， 结果 表明成功制备了环境响应基础共聚物 PLA-PNNUA-PNAS。
实施例 2
肿瘤靶向 PLA-PNNUA-FA、 荧光 PLA-PNNUA-FITC和 TEMPO自旋标记
PLA-PNNUA-TEMPO三种不同功能模块的制备
1. 肿瘤靶向 PLA-PNNUA-FA功能模块的制备：烧瓶中加入 FA (叶酸） 0.5 g， DMSO (二甲基亚砜） 溶解。 冰水浴下加入 DCC (N,N? -二环己基碳二亚胺） 0.5 g禾 a NHS (N-羟基琥珀酰亚胺） 0.3 g。暗处反应过夜。反应完成后，过滤除去白色副产物沉淀， 将滤液倒入无水乙醚中， 产物为 NHS活化的叶酸 FA-NHS, 离心分离沉淀并清洗， 室温真空干燥。烧瓶中先后加入等摩尔 2，2'- (乙烯二氧）双（乙胺）和 FA-NHS， DMSO 溶解， 避光反应 48小时。 反应液倒入无水乙醚中， 产生橘黄色桨状物， 为氨基化的 叶酸 FA-NH2， 离心收集清洗后真空干燥。
将 PLA-PNNUA-PNAS与 FA-NH2混合溶于 DMSO， 避光反应 48小时。 反应液 对 1000 mL超纯水透析 24小时。 冻干透析袋内的液体， 得到浅黄色粉末， 为肿瘤靶 向功能模块 PLA-PNNUA-FA。
2. 荧光 PLA-PNNUA-FITC功能模块的制备： 烧瓶中加入 2，2'- (乙烯二氧） 双 （乙 胺） 和 FITC (异硫氰酸荧光素）， DMSO溶解。 避光反应 48小时。 反应液倒入无水 乙醚中， 底部产生黄绿色桨状物， 为氨基化异硫氰酸荧光素 FITC-NH2， 离心后收集 清洗后室温真空干燥。
将 PLA-PNNUA-PNAS与 FITC-NH2溶于 DMSO，避光反应 48小时。反应液对 1000 mL超纯水透析 24小时。 冻干透析袋内的液体， 得到浅黄绿色粉末， 为荧光功能模块 PLA-PNNUA-FITC。
3. TEMPO (4-氨基-2，2，6，6-四甲基哌啶 -1-氧自由基） 自旋标记 PLA-PNNUA-TEMPO 功能模块的制备：将 PLA-PNNUA-PNAS与 TEMPO溶于 DMSO，避光反应 48 小时。 反应液对 1000 mL超纯水透析 24 小时。 冻干透析袋内的液体， 得到橘红色粉末， 为
TEMPO自旋标记功能模块 PLA-PNNUA-TEMPO。
实施例 3
仓储式功能模块库的建立： 以实施例 5中制备的三种功能模块 PLA-PNNUA-FA、 PLA-PNNUA-FITC和 PLA-PNNUA-TEMPO—起建立了一个仓储式的功能模块库。 可根据需要从仓储式功能模块库中选取多种不同功能模块，包括肿瘤靶向和检测功能 模块， 按任意需要比例混合， 按需包埋抗肿瘤药物， 组成多功能肿瘤诊疗胶束。 实施例 4
pH协同温敏 LCST (低临界溶解温度） 的测定： 用可见光吸收法在波长 542 nm 处测定胶束在考察胶束的 pH协同温敏响应性能。 以超纯水作为透光率 =100 %。 将 PLA-PNNUA胶束分别用 pH值为 7.4、 6.5和 5.0的 PBS缓冲溶液 (0.02M) 配制成 浓度 500 mg/mL， 以透光率对温度绘制出曲线， 得到各 pH值下的 LCST (附图 4)。 结果显示， 胶束的 LCST在 pH值 5.0时为 35.4 °C， pH值 6.5时为 37.5 °C， pH值 7.4 时为 39.4 °C。
实施例 5
具有肿瘤靶向、 荧光和 TEMPO自旋标记功能的多功能胶束的制备与表征： 多功能 胶束由透析法制备。 将 PLA-PNNUA与三种功能模块 PLA-PNNUA-FA,
PLA-PNNUA-FITC和 PLA-PNNUA-TEMPO以适当比例混合， 溶于 1 mL DMSO中。 对超纯水 1000 mL透析 24小时。 在激发波长 488 nm下， 530 nm处的荧光峰是属于 PLA-PNNUA-FITC中 FITC所发出荧光 （附图 5 )。 附图 6为电子顺磁共振图谱， 谱 线具有典型的 14N三线态超精细裂分结构， 属于 PLA-PNNUA-TEMPO中 TEMPO的 顺磁信号。 透射电镜照片显示多功能胶束为均一分散的球形纳米颗粒， 平均水合半径 为 121.5 nm， PDI=0.19 (附图 7)。
实施例 6
多功能胶束的体外细胞摄取实验：使用激光共聚焦显微镜和电子顺磁共振方法评 价多功能胶束对叶酸受体过度表达的 KB细胞（人口腔表皮样癌细胞）和叶酸受体表 达缺乏的 NIH 3T3细胞 （小鼠胚胎成纤维细胞） 进行体外细胞摄取考察。 首先是激光共聚焦显微镜观察， 取 KB细胞或者 NIH 3T3细胞种入带有盖玻片 的 6孔板。 培养 24小时贴壁后弃去原培养基， 加入浓度 250 mg L的多功能胶束培养 液。继续培养 3小时后， PBS缓冲液洗涤， 4 %多聚甲醛， 1 % Triton (曲拉通） X-100， ^g mL DAPI (4'，6-二脒基 -2-苯基吲哚） 染色细胞核。 将盖玻片取出密封后用激光共 聚焦显微镜观察， 激发波长为 405、 488 nm, 发射波通道选择分别为 DAPI和 FITC。
第二部分是电子顺磁共振检测。 取 KB细胞或者 NIH 3T3细胞种入 6孔板。 培 养 24小时贴壁后弃去原培养基， 加入浓度 250 mg L的多功能胶束培养液。 继续培养 3小时后， PBS缓冲液洗涤， 胰酶消化， 加入 PBS缓冲液使细胞重新悬浮， 离心弃去 上清液收集细胞。 高纯氮气流吹干细胞， 加入 0.2 mL DMSO， 超声 30分钟后用电子 顺磁共振波谱仪 X-波段于 298 K下检测 DMSO溶液的电子顺磁共振信号。 激光共聚焦显微镜观察显示胶束能够选择性地被 KB细胞摄取。 从附图 8 A的 FITC通道中可以看出胶束中 PLA-PNNUA-FITC所产生的荧光发自细胞胞桨。这是胶 束通过 KB细胞的内吞作用进入细胞的典型特征。 但当多功能胶束与在同样条件下与 NIH 3T3细胞共同孵育后，由于 NIH 3T3细胞表面缺乏叶酸受体，从附图 8 B的 FITC 通道中未发现 FITC所发出的荧光。附图 8 A、B右下角小图分别是 KB细胞和 NIH 3T3 细胞的顺磁电子信号图谱，可以发现 KB细胞具有较强的信号而 NIH 3T3细胞未检测 出信号。 这与激光共聚焦显微镜观察的结果相符。
实施例 7
肿瘤靶向阿霉素诊疗胶束的制备 肿瘤靶向阿霉素诊疗胶束包埋有具有荧光的抗肿瘤药物阿霉素（DOX)， 同时具 有肿瘤靶向和荧光功能。 将肿瘤靶向功能模块 PLA-PNNUA-FA与 PLA-PNNUA溶于 氯仿， 加入阿霉素氯仿溶液。 混合后旋蒸除去氯仿。 超声下加入 10 mL双蒸水， 即得 肿瘤靶向阿霉素诊疗胶束 （样品编号 FA-PN4-7)。 将胶束置于透析袋中， 对 lOOO mL 超纯水透析 24小时。 透析介质在 480 nm波长下用紫外-可见光分光光度仪测定吸光 度， 代入阿霉素标准曲线计算包封率、 载药量。 计算公式如下： 1
Figure imgf000010_0001
载药量 (%)=¾载H药纳米i胶S束S的I质量 xi00 公式 2 经计算， 肿瘤靶向阿霉素诊疗胶束的包封率为 77.48 %， 载药量为 5.51 %。 透射 电镜照片显示胶束为均一分散的球形纳米颗粒， 而且显示出明显的核壳结构， 中间深 色区域为包埋有阿霉素的胶束疏水核心，包埋在周围的浅色部分是干燥后塌縮的胶束 亲水链段 （附图 9)。 粒径为 138.2 nm， PDI (分散指数） 为 0.160， 单分散分布， zeta 电位为 -33.65mv (附图 10)。
实施例 8
肿瘤靶向阿霉素诊疗胶束的体外释放： 肿瘤靶向阿霉素诊疗胶束 （FA-PN4-7) 的体外释放在 37°C、 100 ipm的振荡条件下进行。 取新鲜制备的胶束溶液 5 mL置透 析袋中对释放介质透析。 释放介质为 lOO mL的 PBS缓冲液， pH分别为 7.4、 6.8、 5.5。 定时取出 3.0 mL释放介质， 立即补回等体积的新鲜释放介质， 用紫外-可见光分 光光度法测定释放介质中阿霉素的浓度。 根据标准曲线计算药物的释放量， 以累计释 药百分率对时间作释药曲线。 由得到的释药曲线（附图 11 )可知， 肿瘤靶向阿霉素诊 疗胶束释药具有 pH敏感的特征，在弱酸环境下，可以较快地释放纳米胶束中阿霉素， 而在生理 pH或者是较高 pH下， 释放较为缓慢。
实施例 9
肿瘤靶向阿霉素诊疗胶束的体外抗肿瘤性能： 取 MCF-7细胞 （人乳腺癌细胞） 用 MTT法评价肿瘤靶向阿霉素诊疗胶束 （FA-PN4-7) 和游离阿霉素的细胞毒性。 从 附图 12可知， 在相同浓度相同暴露时间的条件下， 肿瘤靶向阿霉素诊疗胶束的细胞 毒性大于游离阿霉素。 且两者对细胞的毒性均具有浓度和时间依赖性。游离阿霉素是 小分子状态， 通过被动扩散进入细胞， 而表面带有叶酸分子的肿瘤靶向阿霉素诊疗胶 束是通过受体介导内吞作用的方式进入细胞内，然后积聚在酸性环境的溶酶体和内涵 体中， 在低 pH条件下胶束塌縮挤压释放出药物， 进入细胞核与 DNA作用影响 DNA 的复制从而杀死细胞。 肿瘤靶向阿霉素诊疗胶束的细胞毒性与药物的释放有关， 胶束 进入细胞后通过对环境做出响应快速释放药物， 使细胞内短时间内药物浓度大大增 加， 其速率超过被动扩散依赖细胞内外浓度差的方式， 使得药物进入细胞核的速度超 过游离阿霉素。
实施例 10
肿瘤靶向阿霉素诊疗胶束在荷 MCF-7肿瘤小鼠体内的活体成像实验：使用多功能 活体成像系统 Kodak In-vivo Imaging System FX Pro对荷 MCF-7肿瘤小鼠尾静脉单剂 量注射阿霉素或肿瘤靶向阿霉素诊疗胶束后的药物体内分布进行实时的活体成像检
将荷瘤小鼠分为两组， 每组 4头小鼠。 用 2 %戊巴比妥钠 0.2mL腹腔注射全身 麻醉。 实验组每头小鼠以阿霉素计 5 mg Kg体重单剂量尾静脉注射给药。分组情况如 下：
第一组 4头小鼠的处理方式分别为：
1-A: 阴性对照组， 不给药。 1-B: 阳性对照组， 游离阿霉素溶液。
1- C: 肿瘤靶向阿霉素诊疗胶束。 1-D: 无叶酸配体的阿霉素载药胶束。
第二组 4头小鼠的处理方式分别为：
2- A: 阴性对照组， 不给药。 2-B: 肿瘤部位先注射 KH2P04溶液 （0.02 M， pH 值约为 5.5 ) 再注射肿瘤靶向阿霉素诊疗胶束。
2-C: 肿瘤部位先注射 {01^04溶液 （0.02 M， pH值约为 5.5 ) 并加热至 42 'C， 再注射肿瘤靶向阿霉素诊疗胶束， 保持 42 °C半小时。 2-D: 肿瘤部位加热至 42 °C， 再注射肿瘤靶向阿霉素诊疗胶束， 保持 42 °C半小时。
每组注射完成后将小鼠由左至右依次摆放在样品台上， 并放置一个装有阿霉素 溶液的 1.5 mL离心管作为阳性对照。设置仪器激发波长 =470 nm，检测发射波长 =600 nm， 曝光时间 90 S。
实验结果： 第一组的实验结果如附图 13所示， 箭头处为荷瘤位置。 给药 1小时后， 小鼠 1-B活体成像的荧光呈现全身分布的特征， 而且整体强度较弱， 荷瘤部位未见明 显的荧光， 表明荷瘤部位的阿霉素浓度较低。 而且将小鼠解剖后各脏器内的荧光强度 都较弱。 小鼠 1-C荷瘤部位的荧光强度很高， 表明小鼠荷瘤部位的阿霉素浓度较高， 肿瘤靶向阿霉素诊疗胶束具有主动靶向肿瘤的能力。 从脏器荧光成像来看， 肿瘤的荧 光强度也较高。小鼠 1-D注射的是无叶酸配体的 PLA-PNNUA阿霉素载药胶束，从活 体成像上可以看出小鼠体内阿霉素的分布也是全身性的，荷瘤部位的荧光强度略强于 其他部位。 各脏器的荧光强度除了肿瘤和肝脏较高之外其余都较平均。 这是由于肿瘤 的 EPR效应使得胶束具有被动靶向肿瘤组织的能力。 本组实验结果表明与游离阿霉 素和无叶酸配体的 PLA-PNNUA阿霉素载药胶束相比，肿瘤靶向阿霉素诊疗胶束在荷 瘤小鼠体内表现出主动靶向肿瘤组织的能力，而且能够延长药物在小鼠体内的循环时 间。
第二组的实验结果如附图 14所示， 箭头处为荷瘤位置。 小鼠 2-B的肿瘤部位预 先注射 KH2P04溶液造成局部弱酸性环境， 结果显示小鼠 2-B的荷瘤部位呈现出较强 的荧光， 同时解剖后肿瘤组织也产生了较强的荧光。 小鼠 2-C 的荷瘤部位既经过
KH2P04溶液的酸化， 同时又在荷瘤部位局部加热至 42 °C， 以触发载药胶束的 pH协 同温敏响应。 结果显示在小鼠的荷瘤部位显示了极其强烈的荧光， 解剖后的肿瘤组织 也观察到了很强的荧光。 小鼠 2-D只在荷瘤部位局部加热至 42 V， 而没有在荷瘤部 位注射 KH2P04溶液， 其活体成像和解剖的结果也显示在小鼠的荷瘤部位有较强的荧 光。小鼠 2-B荷瘤部位荧光较 2-C和 2-D弱，其原因是活体成像是四头小鼠一次成像， 小鼠 2-C和 2-D的荷瘤部位荧光很强， 为了使小鼠 2-C和 2-D不过曝， 降低了整体的 荧光强度， 从而小鼠 2-B的荷瘤部位荧光就显得弱了。
实施例 11
功能模块和简单共聚物 PLA-PNNUA的细胞毒性测试 取 NIH 3T3细胞用 MTT (噻唑蓝） 法评价 PLA-PNNUA-FA、 PLA-PNNUA-FITC, PLA-PNNUA-TEMPO和 PLA-PNNUA的细胞毒性。 实验结果如附图 15所示， 各功 能模块和 PLA-PNNUA在达到较高浓度 500 mg/L, 并与细胞共同孵育 48小时之后也 未发现对细胞的存活率有明显的影响。 这表明这些共聚物材料的细胞毒性较低， 生物 相容性较好。

Claims

禾 ί l . 一种环境响应基础共聚物， 其特征在于， 共聚物的结构如结构式 1所示， 具 有明确的三嵌段结构， 依次分别为疏水段、 亲水环境响应段和具有反应活性的聚 Ν- 甲基丙烯酰氧琥珀
Figure imgf000014_0001
2. 如权利要求 1所述的一种环境响应基础共聚物，其特征在于，所述的疏水段的 材料选自生物降解疏水性聚合物。
3. 如权利要求 2所述的一种环境响应基础共聚物，其特征在于，所述的生物降解 疏水性聚合物为聚氨基酸、 多肽、 聚乳酸 -羟基乙酸共聚物、 聚乳酸、 聚羟基乙酸、 聚己内酯、 聚乳酸聚己内酯共混材料及其衍生物中的一种或几种。
4. 如权利要求 1所述的一种环境响应基础共聚物，其特征在于，所述的亲水环境 响应段的环境响应包括温度、 ρΗ、 超声、 光； 环境响应材料选自 Ν-垸基丙烯酰胺和 烯酸及烯酸酯。
5. 如权利要求 4所述的一种环境响应基础共聚物，其特征在于，所述的环境响应 材料为 Ν- (异） 丙基 （甲基） 丙烯酰胺、 Ν-叔丁基丙烯酰胺、 Ν，Ν-二乙基丙烯酰胺、 2-羧基异丙基丙烯酰胺、 甲基丙烯酸、 双甲基丙烯酸、 10-十一烯酸、 甲基丙烯酸 [2-
(二甲基氨基） 乙基]酯、 甲基丙烯酸甲酯、 丙烯酸丁酯、 甲基丙烯酸二异丙胺基乙 酯、 甲基丙烯酸二乙胺基乙酯、 甲基纤维素、 羧基丙基纤维素、 乙烯醇 -乙酸乙烯酯 共聚物、 乙烯基甲醚、 Ν-乙烯基己内酰胺、 聚氨酯中的一种或几种。
6. 如权利要求 1所述的一种环境响应基础共聚物，其特征在于，所述的具有反应 活性的聚 Ν-甲基丙烯酰氧琥珀酰亚胺（PNAS)聚合物链为 Ν-甲基丙烯酰氧琥珀酰亚 胺聚合而成。
7. 如权利要求 1所述的一种环境响应基础共聚物，其特征在于，疏水段形成胶束 的疏水内核， 用于包埋疏水性难溶药物。
8. 如权利要求 1所述的一种环境响应基础共聚物，其特征在于，亲水环境响应段 形成具有极好生物相容性的胶束外壳； 并可进行环境响应释放胶束包埋的药物和诊断 试剂。
9. 一种环境响应基础共聚物的制备方法， 其特征在于， 其具体步骤为：
( 1 ) 将疏水段与一个小分子 RAFT链转移剂以摩尔比 1 :1的比例连接， 制备一 种大分子 RAFT引发剂； RAFT是指可逆加成-断裂链转移聚合方法；
(2)将环境响应单体与大分子 RAFT引发剂以摩尔比 100: 0.01到 100:1的比例 进行第一步 RAFT聚合；
(3 )将 N-甲基丙烯酰氧琥珀酰亚胺 （NAS)单体与第一步的产物以摩尔比 100: 0.01到 100:1的比例进行第二步 RAFT聚合， 制备出环境响应基础共聚物。
10. —种环境响应基础共聚物在制备肿瘤药物领域中的应用。
PCT/CN2012/080531 2012-06-07 2012-08-24 一种环境响应基础共聚物及其制备方法 WO2013181888A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201210186758.XA CN102796235B (zh) 2012-06-07 2012-06-07 一种环境响应基础共聚物及其制备方法
CN201210186758.X 2012-06-07

Publications (1)

Publication Number Publication Date
WO2013181888A1 true WO2013181888A1 (zh) 2013-12-12

Family

ID=47195559

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2012/080531 WO2013181888A1 (zh) 2012-06-07 2012-08-24 一种环境响应基础共聚物及其制备方法

Country Status (2)

Country Link
CN (1) CN102796235B (zh)
WO (1) WO2013181888A1 (zh)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103204981B (zh) * 2013-04-23 2015-06-10 江苏大学 一种聚乳酸-嵌段-聚n-异丙基丙烯酰胺温敏材料的合成方法
CN106267223A (zh) * 2015-05-11 2017-01-04 华东理工大学 一种基于氧自由基的靶向功能材料及其应用
CN105504301B (zh) * 2015-12-07 2018-10-16 复旦大学 一种树枝大分子—共聚物细胞捕获材料及其制备方法和应用
CN108118533B (zh) * 2016-11-30 2021-05-18 北京服装学院 一种具有环境响应性的智能纤维或其制品及其制备方法
CN108721636B (zh) * 2018-06-08 2021-03-02 陕西师范大学 联硒键连接的具有双重响应性的药物递送材料及其制备方法和应用
CN113831561B (zh) * 2021-09-08 2024-05-31 长春工业大学 一种亲水可控的聚乳酸薄膜的制备方法
CN114858733A (zh) * 2022-07-07 2022-08-05 江苏满星测评信息技术有限公司 一种用于测试分析控温薄膜材料光学性能的系统及方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101045066A (zh) * 2006-03-31 2007-10-03 国家纳米科学中心 用于制备抗肿瘤血管生成的药物的温度敏感材料及其用途

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102440961B (zh) * 2011-12-08 2013-10-09 中山大学 一种具有酸敏亚表层的靶向聚合物胶束及其制备方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101045066A (zh) * 2006-03-31 2007-10-03 国家纳米科学中心 用于制备抗肿瘤血管生成的药物的温度敏感材料及其用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BERGBREITER, D.E. ET AL.: "Poly(N-isopropylacrylamide) Soluble Polymer Supports in Catalysis and Synthesis", MACROMOLECULES, vol. 31, no. IS. 18, 19 August 1998 (1998-08-19), pages 6053 - 6062 *
XU, JIANGTAO ET AL.: "Facile Access to Polymeric Vesicular Nanostructures: Remarkable m-End group Effects in Cholesterol and Pyrene Functional (Co)Polymers", vol. 44, no. IS. 2, 23 December 2010 (2010-12-23), pages 299 - 312 *

Also Published As

Publication number Publication date
CN102796235B (zh) 2014-04-23
CN102796235A (zh) 2012-11-28

Similar Documents

Publication Publication Date Title
Saadati et al. Biomedical application of hyperbranched polymers: Recent Advances and challenges
Han et al. Theranostic reduction-sensitive gemcitabine prodrug micelles for near-infrared imaging and pancreatic cancer therapy
Xing et al. Synthesis of disulfide-cross-linked polypeptide nanogel conjugated with a near-infrared fluorescence probe for direct imaging of reduction-induced drug release
Li et al. Molecular bottlebrush as a unimolecular vehicle with tunable shape for photothermal cancer therapy
Chen et al. The accumulation of dual pH and temperature responsive micelles in tumors
Yang et al. Theranostic unimolecular micelles of highly fluorescent conjugated polymer bottlebrushes for far red/near infrared bioimaging and efficient anticancer drug delivery
WO2013181888A1 (zh) 一种环境响应基础共聚物及其制备方法
US9511152B2 (en) Multicolored pH-activatable fluorescence nanoplatform
Fu et al. Galactose targeted pH-responsive copolymer conjugated with near infrared fluorescence probe for imaging of intelligent drug delivery
Yu et al. Near-infrared fluorescence imaging using organic dye nanoparticles
Shi et al. Acid-activatable theranostic unimolecular micelles composed of amphiphilic star-like polymeric prodrug with high drug loading for enhanced cancer therapy
CN106995516B (zh) 肿瘤特异性富集的纳米载药体系及其制备方法
CN104151559B (zh) 聚丙烯酰胺-丙烯腈-聚乙二醇及合成方法和应用
Xu et al. Disassembly of amphiphilic small molecular prodrug with fluorescence switch induced by pH and folic acid receptors for targeted delivery and controlled release
Peng et al. Ultrabright fluorescent cellulose acetate nanoparticles for imaging tumors through systemic and topical applications
Lin et al. Highly hemocompatible zwitterionic micelles stabilized by reversible cross-linkage for anti-cancer drug delivery
Zhuang et al. Two-photon AIE luminogen labeled multifunctional polymeric micelles for theranostics
KR102099516B1 (ko) 근적외선에 반응하는 암세포 표적 기능을 갖는 가교 마이셀, 이를 포함하는 약물 전달체 및 이의 제조방법
Chen et al. Thermal responsive micelles for dual tumor-targeting imaging and therapy
Xu et al. Design and fabrication of chitosan-based AIE active micelles for bioimaging and intelligent delivery of paclitaxel
Cheng et al. Water-soluble single-chain polymeric nanoparticles for highly selective cancer chemotherapy
Song et al. pH-Sensitive morphological transitions in polymeric tadpole assemblies for programmed tumor therapy
Tian et al. Construction of dual-functional polymer nanomaterials with near-infrared fluorescence imaging and polymer prodrug by RAFT-mediated aqueous dispersion polymerization
Shen et al. Efficient tumor accumulation, penetration and tumor growth inhibition achieved by polymer therapeutics: the effect of polymer architectures
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

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: 12878621

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12878621

Country of ref document: EP

Kind code of ref document: A1