WO2021052212A1 - 基于双硒键聚合物的智能纳米粒及其制备方法与应用 - Google Patents

基于双硒键聚合物的智能纳米粒及其制备方法与应用 Download PDF

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WO2021052212A1
WO2021052212A1 PCT/CN2020/113856 CN2020113856W WO2021052212A1 WO 2021052212 A1 WO2021052212 A1 WO 2021052212A1 CN 2020113856 W CN2020113856 W CN 2020113856W WO 2021052212 A1 WO2021052212 A1 WO 2021052212A1
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selenium
double
tumor
nps
drug
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杨再兴
张连学
李友云
杨莹
段广新
徐加英
周如鸿
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苏州大学
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    • 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
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/28—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the compounds used containing active hydrogen
    • C08G18/30—Low-molecular-weight compounds
    • C08G18/38—Low-molecular-weight compounds having heteroatoms other than oxygen
    • C08G18/3897—Low-molecular-weight compounds having heteroatoms other than oxygen containing heteroatoms other than oxygen, halogens, nitrogen, sulfur, phosphorus or silicon
    • 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
    • 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
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/28—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the compounds used containing active hydrogen
    • C08G18/40—High-molecular-weight compounds
    • C08G18/48—Polyethers
    • C08G18/4833—Polyethers containing oxyethylene units
    • 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
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/28—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the compounds used containing active hydrogen
    • C08G18/65—Low-molecular-weight compounds having active hydrogen with high-molecular-weight compounds having active hydrogen
    • C08G18/66—Compounds of groups C08G18/42, C08G18/48, or C08G18/52
    • C08G18/6666—Compounds of group C08G18/48 or C08G18/52
    • 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
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/70—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the isocyanates or isothiocyanates used
    • C08G18/72—Polyisocyanates or polyisothiocyanates
    • C08G18/74—Polyisocyanates or polyisothiocyanates cyclic
    • C08G18/75—Polyisocyanates or polyisothiocyanates cyclic cycloaliphatic
    • C08G18/751—Polyisocyanates or polyisothiocyanates cyclic cycloaliphatic containing only one cycloaliphatic ring
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    • C08G18/753—Polyisocyanates or polyisothiocyanates cyclic cycloaliphatic containing only one cycloaliphatic ring containing at least one isocyanate or isothiocyanate group linked to the cycloaliphatic ring by means of an aliphatic group containing one isocyanate or isothiocyanate group linked to the cycloaliphatic ring by means of an aliphatic group having a primary carbon atom next to the isocyanate or isothiocyanate group
    • C08G18/755—Polyisocyanates or polyisothiocyanates cyclic cycloaliphatic containing only one cycloaliphatic ring containing at least one isocyanate or isothiocyanate group linked to the cycloaliphatic ring by means of an aliphatic group containing one isocyanate or isothiocyanate group linked to the cycloaliphatic ring by means of an aliphatic group having a primary carbon atom next to the isocyanate or isothiocyanate group and at least one isocyanate or isothiocyanate group linked to a secondary carbon atom of the cycloaliphatic ring, e.g. isophorone diisocyanate

Definitions

  • the invention belongs to smart medicine technology, and specifically relates to smart nanoparticles based on double selenium bond polymers and a preparation method and application thereof.
  • Radiotherapy is one of the main methods of treating cancer. More than half of cancer patients use RT alone or in combination with other radiotherapy or surgical methods in current clinical treatment.
  • High-energy radiation such as X-rays, is usually used for RT. It mainly destroys tumor tissues by interacting with the cytoplasm and nucleus of tumor cells in the irradiated area, such as the accumulation of reactive oxygen species (ROS) and DNA strand breaks. The cells are damaged.
  • ROS reactive oxygen species
  • certain tumor microenvironments make tumor cells resistant to radiation, thereby inhibiting the application potential of RT.
  • chemotherapy also has considerable toxicity, and free chemotherapeutic drugs have a low accumulation at the tumor site.
  • the invention discloses intelligent nano particles based on double selenium bonds and a preparation method and application thereof.
  • the particles realize the intelligent release of drugs in cancer cells under the synergistic triggering effect of low X-ray dose and ROS, thereby improving the synergy of RT and chemotherapy Anti-tumor effect, and reduce the adverse side effects of chemotherapy.
  • the present invention adopts the following technical solutions.
  • Polymers based on double selenium bonds have the following structural formula.
  • n 40-50, n is 8-15, and j is 6-12; preferably, m is 45, n is 11, and j is 9.
  • Smart nanoparticles based on double selenium bonds are prepared from the above-mentioned double selenium bond-based polymers.
  • Drug-loaded nanoparticles based on double selenium bonds are prepared from the above-mentioned double selenium bond-based polymers and drugs.
  • the method for preparing the polymer based on double selenium bond includes the following steps: polymerizing small molecule monomers of double selenium with IPDI to obtain a selenium-containing polymer; and then capping the selenium-containing polymer with polyethylene glycol to obtain a selenium-based polymer. Of polymers.
  • the method for preparing smart nanoparticles based on double selenium bonds includes the following steps. Small molecule monomers of double selenium are polymerized with IPDI to obtain a selenium-containing polymer; and then the selenium-containing polymer is capped with polyethylene glycol to obtain a selenium-based polymer. Bond polymer; then dissolve the polymer based on double selenium bond in a good solvent, then add water, and then pass through deionized water dialysis and ultrafiltration centrifugation to obtain smart nanoparticles based on double selenium bond.
  • the above-mentioned preparation method of drug-loaded nanoparticles based on double selenium bonds includes the following steps. Small molecule monomers of double selenium are polymerized with IPDI to obtain a selenium-containing polymer; Selenium bond polymer; dissolve the double selenium bond-based polymer and the drug in a solvent, then add water, and then pass through deionized water dialysis and ultrafiltration centrifugation to obtain drug-loaded nanoparticles based on double selenium bond.
  • the present invention discloses the application of the above double selenium bond-based polymer and double selenium bond-based smart nanoparticles in the preparation of tumor treatment drug carriers; the present invention discloses the above double selenium bond-based polymer and double selenium bond-based smart nanoparticles
  • the application of nanoparticles or drug-loaded nanoparticles based on double selenium bonds in the preparation of tumor treatment drugs preferably tumor treatment is radiotherapy and chemotherapy, that is, the drugs prepared by the present invention are suitable for simultaneous chemotherapy and radiotherapy, and have a synergistic effect.
  • the preparation of disselenium small molecule monomer is: adding bromohydrin solution to water containing sodium borohydride and selenium powder in nitrogen to react to obtain disselenium small molecule monomer; wherein, the reaction is 45-55°C.
  • the chemical structural formula of the small di-selenium monomer is as follows.
  • IPDI The chemical structure of IPDI is as follows.
  • the chemical structural formula of the selenium-containing polymer is as follows.
  • polyethylene glycol The chemical structure of polyethylene glycol is as follows: .
  • the solvent is DMF;
  • the dialysis is dialysis with deionized water in a dialysis bag with a molecular weight cut-off of 3500 Da for 24 hours; the molecular weight cut-off of ultrafiltration centrifugation is 10000 Da.
  • the drug is DOX ⁇ HCl.
  • the present invention discloses a simple amphiphilic triblock copolymer (Se-polymer) with a plurality of diselenide groups in the main chain and the production of disselenium-containing nanoparticles (Se-NP).
  • Se-polymer simple amphiphilic triblock copolymer
  • Se-NP disselenium-containing nanoparticles
  • Experimental results show that in the presence of ROS, X-ray irradiation of only 2 Gy of Se-NPs can quickly trigger the rapid disintegration and drug release of Se-NPs.
  • In vivo experiments on mice bearing 4T1 tumors have verified the significant anti-tumor effects of smart nanoparticles based on double selenium bonds as a drug delivery system. This work shows that copolymers containing di-selenium can be used in combination with RT in chemotherapy It has little toxic effect in tumor treatment and has great potential clinical application value.
  • Figure 1 is the NMR map of the product.
  • Figure 2 is a graph of the nanoparticle size test.
  • Figure 3 is a graph showing the in vitro release of NR from Nile Red-loaded nanoparticles (NR-NPs) under different conditions.
  • Figure 4 is a fluorescence image of a tumor-bearing mouse.
  • Figure 5 is an anti-tumor effect diagram.
  • Fig. 6 is a graph showing the relationship between the change in particle size distribution of D-NP and the processing time.
  • Figure 7 shows the result of live cell imaging.
  • Figure 8 shows the results of in vivo anti-tumor performance.
  • mice Female Balb/c mice (about 20 g ) were subcutaneously transplanted with 4T1 cells (2.0 ⁇ 10 6 cells/mouse) to construct tumor-bearing mice (100 mm 3 ). Mice bearing 4T1 tumors were injected with D-NP or free DOX at a dose of 7.5 mg/kg through the tail vein. Various tissues (heart, liver, spleen, lung, kidney and tumor) were extracted from mice at the designed time points (12, 24 and 48 hours) and washed with PBS. Weigh and grind parts of various tissues. At -20°C, 1 mL of methanol was used to extract DOX from the tissue. Finally, after centrifugation at 12000 rpm for 5 minutes, the fluorescence of the supernatant was measured by using a microplate reader (SpectraMax) with an excitation wavelength of 480 nm and an emission wavelength of 560 nm.
  • SpectraMax microplate reader
  • H&E hematoxylin and eosin
  • Example 1 Add 1.0 g (12.6 mmol) sodium borohydride dissolved in 10 ml of deionized water to 15 mL of deionized water containing 1.0 g (26.4 mmol) of selenium powder under stirring at room temperature , React for 10 minutes, then add 1.0 g (26.4 mmol) of selenium powder, stir for 15 minutes, then add 6.33 g (25.2 mmol) bromoundecyl alcohol dissolved in 25 ml of THF, and react in an oil bath at 50°C for 24 hours; Then filter to remove the unreacted residue, extract three times with dichloromethane, dry with anhydrous sodium sulfate for 24 hours, filter to remove sodium sulfate, and use column chromatography (volume ratio 4:1 dichloromethane/ethyl acetate as washing Removal of the agent) purify the product to obtain a yellow powder (yield 61%), which is a small molecule diselenyl monomer.
  • the above preparation method can be illustrated as follows.
  • Example 2 Add 1.0 mg DOX ⁇ HCl, 0.1 ml TEA and 10 mg Se-polymer to 2 mL DMF, stir for 2 hours, and then add 10 mL deionized water dropwise within 5 hours under stirring.
  • Deionized water dialysis MWCO 3500 Da
  • MWCO 10000 Da ultrafiltration centrifuge tube
  • D-NPs drug-loaded nanoparticles
  • DLC DOX loading
  • DLE drug loading efficiency
  • D-NPs were dried by a freeze dryer and dissolved in DMSO, and measured by a UV-vis spectrometer. The test found that the DOX loading (DLC) of the above D-NPs ) And drug loading efficiency (DLE) values are calculated to be 8.69% and 41.78%, respectively.
  • TEM Transmission electron microscopy
  • D-NP double-selenium nanoparticles loaded with DOX.
  • D-NP dynamic light scattering
  • PDI polydispersity
  • the Se-polymer was replaced with an existing polymer to obtain Nile Red nanoparticles (benzene ring-NR-NPs), as a comparison;
  • the existing polymer is as follows.
  • Example 3 Nile Red (NR) was loaded as a model compound to study drug release in vitro, because it has high stability under H 2 O 2 and X-ray conditions.
  • Figure 3 shows the in vitro release of NR from Nile Red-loaded nanoparticles (NR-NPs) under different conditions.
  • the X-ray radiation dose used at the place is comparable in the clinic, and the H 2 O 2 concentration (100 ⁇ M) is set to match the ROS level in the real tumor microenvironment; fluorescence spectrophotometer, excitation wavelength 543nm, in the wavelength of 500 to 700nm Within the range to determine the content of NR.
  • the drug release behavior was not significantly different from that of the control group.
  • the release behavior of NR was very slow.
  • the release rate of NR increased significantly compared with the previous two groups of single stimuli.
  • the benzene ring-NR-NPs nanoparticles were treated with 2Gy X-rays/100 ⁇ M H 2 O 2 for 20 hours, and the Nile Red release rate was 36%. They were treated with 5Gy X-rays/100 ⁇ M H 2 O 2 for 20 hours. After hours, the Nile Red release rate was 57%.
  • Cy-NP was injected intravenously at a dose of 5 mg/kg , And observe 48 hours through the three-dimensional imaging instrument (IVIS Spectrum) of small animals.
  • Cy-loaded Se-NP (Cy-NP) has a particle size distribution very similar to D-NP ( Figure 2B). Cy-NP was injected into tumor-bearing 4T1 mice (in accordance with the requirements of animal experiments at Soochow University) through the regular tail vein, and passed the three-dimensional imaging instrument (IVIS Spectrum) observation.
  • Example 4 The anti-tumor efficacy of D-NPs in vivo was evaluated.
  • the tumor-bearing mice were equally allocated to 6 different groups and treated with different methods: (a) PBS, (b) free DOX (F-DOX), (c) D-NP, (d) 2Gy X-ray, ( e) F-DOX combined with X-ray (F-DOX/X-ray), (f) D-NP combined with X-ray (D-NPs/X-ray).
  • F-DOX or D-NP was injected into the 4T1 tumor-bearing mice through the tail vein at the same DOX dose of 7.5 mg/kg (PBS as solvent).
  • EPR effect enriches D-NP at the tumor site and reduces the side effects of free DOX
  • X-ray + ROS precisely controls the release of DOX from D-NP at the tumor site, resulting in Enhance anti-tumor effect.
  • D the tumor volume change
  • E the image of the tumor peeled from the experimental mouse at the end of the experiment
  • Example 5 By using dynamic light scattering measurement (DLS) technology to study the change of the particle size distribution of D-NP with processing time, the drug release behavior of D-NP under different stimulus conditions was further determined from another angle. In principle, if the particle size of D-NPs is opened and the drug inside is released, its volume will become more swollen.
  • DLS dynamic light scattering measurement
  • Example 6 In order to evaluate the endocytosis of D-NP, D-NP was incubated with 4T1 cells and monitored by a live cell imaging system. The nucleus was stained with Hoechst (blue). As shown in Figure 7A, after 0.5 hours of incubation, D-NP began to be endocytosed into 4T1 cells. As the incubation time increased, the red fluorescence intensity of DOX (10 mg L ⁇ 1 ) gradually increased, indicating that more and more D-NP enters the cell. In contrast, when the cells were incubated with free DOX (F-DOX), less DOX entered the cells after 6 hours, as shown by the much weaker red fluorescence (Figure 7B).
  • DOX free DOX
  • D-NPs and F-DOX enter cells in different ways, among which D-NPs are endocytosed into cells through an endocytosis mechanism, which allows them to remain in cells for a relatively long time.
  • F-DOX enters 4T1 cells through a concentration gradient-dependent diffusion mechanism. It is also worth noting that unloaded Se-NPs (Se-copolymers) show excellent cell biocompatibility.
  • D-NP and F-DOX were compared by in vivo biodistribution analysis.
  • the mouse organs including tumor, heart, liver, spleen, lung and kidney
  • SYNERGY neo microplate reader
  • D-NPs has a good enrichment ability in tumors and shows a higher tumor enrichment rate than F-DOX.
  • the tumor uptake of DOX was about 2.1% of the injected dose per gram of tissue (%ID/g).
  • the present invention produces Se-NPs containing diselenide bonds, which are not sensitive to simple X-ray stimulation, and decompose slowly when oxidized with H 2 O 2 alone.
  • 2Gy low X-rays can trigger nanoparticles
  • the rapid disintegration of the drug is accompanied by the rapid release of the loaded drug.
  • Se-NPs develop an intelligent and rapid drug delivery system for cancer treatment.
  • In vitro cell experiments show that the toxicity of Se-NPs-loaded drugs is lower than that of free drugs at the same concentration. This means that the side effects of off-target nanomedicine on normal cells will be greatly reduced.
  • In vivo experiments on mice have shown that cancer proliferation is more effectively inhibited, while the observed side effects are significantly reduced at the same time.

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Abstract

基于双硒键聚合物的智能纳米粒及其制备方法与应用。设计了简单的两亲性含二硒的纳米颗粒(Se-NPs)。Se-NPs在X射线下可以智能的将纳米粒子中的药物释放出来。对携带4T1荷瘤小鼠的体内实验表明，由于EPR效应，装载的药物可在肿瘤部位形成富集，2Gy的低X射线剂量下(与临床单次放射治疗中采用的常规剂量相当)可触发药物快速从Se-NPs释放到肿瘤细胞中，发挥显着的联合抗肿瘤作用，而且副作用小。此外，制造的Se-NP不仅具有无毒性，而且能够减轻抗肿瘤药物对正常细胞的不良细胞毒性，显示出Se-NP具有非常好的生物相容性，为增强癌症治疗效果提供了有效的手段，具有极低的副作用。

Description

基于双硒键聚合物的智能纳米粒及其制备方法与应用 技术领域
本发明属于智能药物技术，具体涉及基于双硒键聚合物的智能纳米粒及其制备方法与应用。
 背景技术
放射治疗（RT）是治疗癌症的主要方法之一。超过一半的癌症患者在目前的临床治疗中单独使用RT或与其它放疗或者手术方法联合治疗。高能辐射，如X射线，通常用于RT，主要通过与照射区域内肿瘤细胞的细胞质和细胞核相互作用来破坏肿瘤组织，如产生活性氧（ROS）积聚和DNA链断裂，同时不可避免地对正常细胞产生损伤。但是，某些肿瘤微环境让肿瘤细胞具有抗辐射性，进而抑制了RT的应用潜力。尽管RT和抗癌药物的联合应用表现出协同抗癌作用，但化疗也具有相当大的毒性，而且游离的化疗药物在肿瘤部位具有较低累积。在过去的几十年中，人们已经付出了巨大的努力来改善这些具有不良副反应的癌症治疗药物。然而，改善治疗药物带来的毒副作用仍然是当今具有挑战性的目标。随着纳米技术和纳米医学的快速发展，近年来使用刺激响应材料来实现癌症靶向治疗引起了相当大的关注。特别地，刺激性响应性纳米材料和高能射线作为智能药物递送系统的组合已经显示出增强RT诱导的肿瘤治疗功效，具有非常大的临床应用潜力。但是现有设计的含有辐射响应的纳米结构虽然可以有效地调节药物释放。但是需要5 Gy～10 Gy的剂量照射，这与临床应用不符，无法用于临床2 Gy照射的要求。
 技术问题
本发明公开了基于双硒键的智能纳米粒子及其制备方法与应用，该粒子在低X射线剂量、ROS的协同触发效应下实现药物在癌细胞中的智能释放，从而提高RT和化学疗协同抗肿瘤效应，并减少化疗带来的不良毒副作用。
 技术解决方案
本发明采用如下技术方案。
基于双硒键的聚合物，具有以下结构式。
Figure 422752dest_path_image001
。
式中，m为40～50，n为8～15，j为6～12；优选的，m为45，n为11，j为9。
基于双硒键的智能纳米粒，由上述基于双硒键的聚合物制备得到。
基于双硒键的载药纳米粒，由上述基于双硒键的聚合物与药物制备得到。
上述基于双硒键的聚合物的制备方法，包括以下步骤，双硒小分子单体与IPDI聚合得到含硒聚合物；再将含硒聚合物经聚乙二醇封端，得到基于双硒键的聚合物。
上述基于双硒键的智能纳米粒的制备方法，包括以下步骤，双硒小分子单体与IPDI聚合得到含硒聚合物；再将含硒聚合物经聚乙二醇封端，得到基于双硒键的聚合物；再将基于双硒键的聚合物溶入良溶剂中，然后加入水，再经过去离子水透析、超滤离心，得到基于双硒键的智能纳米粒。
上述基于双硒键的载药纳米粒的制备方法，包括以下步骤，双硒小分子单体与IPDI聚合得到含硒聚合物；再将含硒聚合物经聚乙二醇封端，得到基于双硒键的聚合物；再将基于双硒键的聚合物、药物溶入溶剂中，然后加入水，再经过去离子水透析、超滤离心，得到基于双硒键的载药纳米粒。
本发明公开了上述基于双硒键的聚合物、基于双硒键的智能纳米粒在制备肿瘤治疗药物载体中的应用；本发明公开了上述基于双硒键的聚合物、基于双硒键的智能纳米粒或者基于双硒键的载药纳米粒在制备肿瘤治疗药物中的应用，优选肿瘤治疗为放化疗，即本发明制备的药物适用于化疗、放疗同时进行，具有协同作用。
本发明中，双硒小分子单体的制备为，氮气中，将溴醇溶液加入含有硼氢化钠、硒粉的水中，反应得到双硒小分子单体；其中，反应为45～55℃反应20～28小时，得到双硒小分子单体；硒粉、硼氢化钠、溴醇的摩尔比为4.1～4.2∶1∶2；溴醇溶液中溶剂为四氢呋喃；双硒小分子单体与IPDI的摩尔比为1∶1.05～1.15；聚合为45～55℃反应1.5～2.5小时；封端时的温度为45～55℃，时间为20～28小时；双硒小分子单体与IPDI聚合在有机锡化物催化条件下进行。
本发明中：溴醇的化学结构式如下。
Figure 936910dest_path_image002
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双硒小分子单体的化学结构式如下。
Figure 66540dest_path_image003
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IPDI的化学结构式如下。
Figure 615333dest_path_image004
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含硒聚合物的化学结构式如下。
Figure 172216dest_path_image005
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聚乙二醇的化学结构式如下：
Figure 173670dest_path_image006
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本发明制备纳米粒时，溶剂为DMF；透析为在截留分子量为3500Da的透析袋中，用去离子水透析24小时；超滤离心的截留分子量为10000Da。
本发明中，药物为DOX·HCl。
 有益效果
本发明公开了主链中具有多个二硒化物基团的简单两亲性三嵌段共聚物（Se-聚合物）以及制造含双硒的纳米颗粒（Se-NP）。实验结果表明，Se-NPs在ROS存在下，仅2 Gy的X射线照射可以迅速触发Se-NPs的快速解体、药物释放。通过对携带4T1肿瘤的小鼠的体内实验验证了基于双硒键的智能纳米粒作为药物递送系统的显着抗肿瘤作用，这项工作表明，含二硒的共聚物在化学疗法与RT联合用于肿瘤治疗中具有很小的毒性作用，具有很大的潜在临床应用价值。
 附图说明
图1为产物核磁图。
图2为纳米粒尺寸测试图。
图3为在不同条件下从负载尼罗红的纳米粒(NR-NPs)体外释放NR结果图。
图4为荷瘤小鼠荧光图像。
图5为抗肿瘤效果图。
图6为D-NP的粒度分布的变化与处理时间关系图。
图7为活细胞成像结果图。
图8为体内抗肿瘤性能结果。
 本发明的实施方式
硒粉、硼氢化钠、过氧化氢（H 2O 2,30％）、异佛尔酮二异氰酸酯（IPDI）、二月桂酸二丁基锡（DBTDL）、11-溴十一烷醇是购自Aladdin Reagents的分析级产品。将四氢呋喃（THF）（A.R.，Sinopharm Chemical Reagent）在KOH上干燥至少两天，然后在钠线上回流，用二苯甲酮作为指示剂直至颜色变为紫色。聚（乙二醇）单甲醚（mPEG）（Mw=2000）来自Aladdin Reagents，并通过与甲苯共沸回馏干燥。
使用氘代氯仿作为溶剂并使用四甲基硅烷（TMS）作为内标，在400MHz光谱仪（INOVA-400，Varian）上记录 1 HNMR光谱。数均分子量（M̅n），重均分子量（M̅w）和分子量分布（PDI）通过凝胶渗透色谱（GPC）仪器（HLC-8320，TOSOH）测量。使用DMF溶液作为洗脱液。使用动态光散射（DLS）仪器（Nano ZS90，Malvern）在25℃下分析纳米颗粒的尺寸和尺寸多分散指数（尺寸PDI）。透射电子显微镜（TEM，HT7700,120kV，Hitachi）用于观察纳米颗粒样品的形态。使用UV-vis光谱仪（UV2600，Shimadzu）测量DOX的含量。
体外细胞毒性试验，使用游离DOX作为对照，通过CCK-8测定单独评估纳米颗粒对4T1细胞，HUVEC细胞和MDA-MB-231细胞的细胞毒性。将细胞接种在96孔板（4×10 4细胞/孔）中，其中每孔加入100μL RPMI1640培养基，并将细胞培养12小时。除去培养基并加入含有不同浓度的药物的培养基（剂量为0.155,0.30,0.60,1.25,5.5,5.0和10.0μg/ mL DOX的游离DOX或D-NP）。进一步孵育48小时后，吸去培养液，向每个孔中加入100μL 含CCK-8的培养基。在37℃下孵育2小时后，使用酶标仪（SpectraMax，450nm）评估细胞活力，以获得光密度（OD）值。通过常规方程计算细胞存活率。
细胞摄取，通过活细胞成像系统（CELL'R，Olympus）研究游离DOX和D-NP的4T1细胞中的细胞摄取。将4T1肿瘤细胞培养在小玻璃培养皿（15×10 4个细胞/cm 2）上，并在RPMI 1640培养基中培养24小时。吸去培养基，用PBS洗涤细胞，加入1.0mL Hoechst 33342（1.0μg/mL），并在37℃下孵育30分钟。然后用PBS洗涤细胞三次。然后用含有游离DOX或D-NP（10μg/L的DOX）的样品替换培养基。每0.5小时在480nm（红色），340nm（蓝色）的激发波长下捕获图像。
活体小鼠的荧光成像。携带4T1肿瘤的小鼠通过尾静脉注射剂量为5mg/kg的Cy-NP，并通过小动物三维成像仪器（IVIS Spectrum）观察48小时。
生物分布。将雌性Balb/c小鼠（约20g）通过4T1细胞（2.0×10 6个细胞/小鼠）皮下移植以构建荷瘤小鼠（100mm 3）。携带4T1肿瘤的小鼠通过尾静脉注射剂量为7.5mg / kg的D-NP或游离DOX。在设计的时间点（12,24和48小时）从小鼠中提取各种组织（心脏，肝脏，脾脏，肺，肾和肿瘤）并用PBS洗涤。对各种组织的一部分进行称重和研磨。在-20℃条件下，用1mL甲醇来提取组织中的DOX。最后，在12000rpm离心5分钟后，通过使用激发波长为480nm且发射波长为560nm的酶标仪（SpectraMax）测量上清液的荧光。
体内抗肿瘤功效。将携带4T1肿瘤的小鼠分成不同组，并在第0天和第2天将含有7.5mg/kg剂量的各种制剂D-NP或游离DOX静脉注射到携带4T1肿瘤（100mm 3）的小鼠中。在每次静脉注射后24小时，肿瘤进行2Gy的X射线照射。每隔一天测量荷瘤小鼠的肿瘤体积（V）和体重，持续13天。根据V =（a×b 2）/2的等式计算肿瘤体积（V），其中a和b分别是肿瘤的长轴和短轴。用苏木精和伊红（H＆E）染色肿瘤制作切片，评估不同方法药物的抗肿瘤效果。
计算方法。所有密度泛函理论（DFT）模拟均使用Gaussian 09软件包执行。使用具有6-31+G(d,p)基组的M06-2x方法优化几何形状，并通过Hessian评估进一步验证。通过Grimme的经验色散（GD3）校正改进了范德华相互作用的描述，并且通过在所有计算中采用水的可极化连续模型（PCM）来考虑溶剂效应。利用6-31+G(d,p)基组，通过Berny算法定位仅具有一个虚频率的过渡态，并且执行内部反应路径（IRC）计算以验证检测到的鞍点是否对应于预期的反应物和产品路径。单点计算在M06-2x/Def2TZVP级别进行。
实施例一：室温、氮气中、搅拌下，将溶解在10毫升去离子水中的1.0 g(12.6 mmol)硼氢化钠加缓慢滴加到含1.0 g（26.4 mmol）硒粉的15 mL去离子水中，反应10分钟，随后再加入1.0 g（26.4 mmol）硒粉，搅拌15分钟，再加入溶解在25毫升THF中的6.33 g( 25.2 mmol)溴十一醇， 50℃油浴中反应24小时；然后过滤除去未反应的残渣，用二氯甲烷萃取三次，用无水硫酸钠干燥24小时，过滤除去硫酸钠，再用柱色谱法（体积比为4∶1二氯甲烷/乙酸乙酯为洗脱剂）纯化产物，得到黄色粉末（产率 61%），为双硒小分子单体。 1H-NMR (300 MHz, CDCl 3, δ) (ppm): 3.63 (4H, t, HOC H 2), 2.90 (4H, t, SeSeC H 2) 1.72-1.28(36H, m, HOCH 2 (C H 2) 9CH 2SeSe); LC-MS: 理论分子量 500.53, 测试分子量 500.16，核磁图见图1A。
室温下、氮气中， 0.2 g（0.40 mmol）的双硒单体和10 mg(0.016 mmol) DBTDL加入2mL无水THF中，再加入97.8 mg IPDI（0.44 mmol），然后于50℃油浴反应2小时，再加入溶解在1mL无水THF中的0.048g（0.44mmol）苄醇，并在50℃下进行反应2小时，然后通过旋转蒸发除去溶剂，并将残余液体用冷甲醇沉淀三次，真空干燥后得到黄色聚合物，用于测试聚合物的聚合度，核磁谱图见图1B。通过等式（1）计算聚合度（n）为11，其中Ac和Ah分别代表峰c和峰h的积分值。
Figure 356259dest_path_image007
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室温下、氮气中， 0.2 g（0.40 mmol）的双硒单体和10 mg(0.016 mmol) DBTDL加入2mL无水THF中，再加入97.8 mg IPDI（0.44 mmol），然后于50℃油浴反应2小时，再加入PEG 2000 (0.26 mmol)，然后于50℃反应12小时，再旋蒸去掉THF，用冰甲醇沉淀3次，真空干燥箱室温放置24小时，得到基于双硒键的聚合物(Se-聚合物，收率: 45%)，核磁谱图见图1C。使用1H NMR和GPC分析表征插入（Se-聚合物）的化学结构，分子量和分子量分布（图1D）。从图1C中所示的Se-聚合物的 1H NMR光谱中，可以发现所有信号都可以归因于化学结构中的质子，确定Se聚合物的分子量为11820g/mol（M + n，NMR），其中722.2是PUSeSe的每个重复单元的理论分子量。此外，GPC分析表明Se-聚合物的分子量和分子量分布分别为11950g/mol（M + n，GPC）和1.24。所有这些表明两亲性Se-聚合物的成功合成。
以上制备方法可示意如下。
Figure 759558dest_path_image008
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实施例二：将1.0 mg DOX·HCl、0.1毫升TEA和10 mg Se-聚合物加入2 mL DMF中，搅拌2h，然后在搅拌下，5小时内滴加10 mL去离子水，滴加完成用去离子水透析(MWCO 3500 Da) 24小时，再用超滤离心管 (MWCO 10000 Da)离心，得到含双硒键的载药纳米粒（D-NPs）。为了测量DOX负载量（DLC）和药物装载效率（DLE），通过冷冻干燥器干燥D-NPs并溶解在DMSO中，通过UV-vis光谱仪测量，测试发现，上述D-NPs的DOX负载量（DLC）和药物负载效率（DLE）值分别计算为8.69％和41.78％。
透射电子显微镜（TEM）表征分析如图2A所示，证明了设计的装载DOX的双硒纳米粒子（D-NPs）的成功制造。根据动态光散射（DLS）测量，D-NP具有球形形态，平均直径为83.0nm，平均动力学直径为108.0nm，多分散性（PDI）为0.24，该尺寸已被认为是纳米药物递送系统在肿瘤学中增强的渗透性和滞留（EPR）效应的有效粒径，促进肿瘤位点的NPs积累。
根据上述方法，不加入DOX·HCl，得到基于双硒键的智能纳米粒(Se-NPs)；根据上述方法，将DOX·HCl更换为Cy、尼罗红，得到负载Cy的纳米粒(Cy-NPs)、负载尼罗红的纳米粒(NR-NPs)。
将F-DOX分散在5% DMSO中作为自由药对照。
根据上述方法，将Se-聚合物更换为现有聚合物，得到载尼罗红纳米粒（苯环-NR-NPs），作为对比；现有聚合物如下。
Figure 487343dest_path_image009
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实施例三：尼罗红（NR）作为模型化合物加载，以研究体外药物释放，因为它在H 2O 2和X射线条件下具有高稳定性。图3为在不同条件下从负载尼罗红的纳米粒(NR-NPs)体外释放NR，数据表示为平均值±标准偏差，n=3，具体为PBS对照组 、单纯X射线照射组（2Gy、5Gy和10Gy）、单一H 2O 2处理组（100μM）、X射线和H 2O 2协同组（2Gy X射线、5Gy X射线、10Gy X射线分别用100μM H 2O 2共同处理）；此处使用的X射线照射剂量具有在临床中的可比性，设定H 2O 2浓度（100μM）匹配真实肿瘤微环境中的ROS水平；荧光分光光度计，激发波长543nm，在500至700nm的波长范围内进行以确定NR的含量。有趣的是，在不同剂量单独用X射线照射的所有三组中，药物释放行为与对照组没有明显差异，在H 2O 2处理组中，NR释放行为非常缓慢，令人意想不到的是，当X射线和H 2O 2共同处理时，与前两组单刺激的明显对比，NR释放速率显着升高。结果表明，在存在ROS的情况下，2Gy的X射线剂量可以足够高以有效激活NPs的药物释放。同样的方法，苯环-NR-NPs纳米粒利用2Gy X射线/100μM H 2O 2共同处理20小时后，尼罗红释放率为36%，利用5Gy X射线/100μM H 2O 2共同处理20小时后，尼罗红释放率为57%。
为了评估装载药物的二硒化物纳米颗粒的肿瘤靶向性和富集时间，通过使用Cy作为负载模型化合物的荧光成像监测Se-NP的体内分布，静脉内注射剂量为5mg/kg的Cy-NP，并通过小动物的三维成像仪器（IVIS Spectrum）观察48小时。载有Cy的Se-NP（Cy-NP）具有与D-NP非常相似的粒度分布（图2B）。将Cy-NP常规尾静脉注射到荷瘤4T1的小鼠中（符合苏州大学动物实验要求），并通过三维成像仪器（IVIS Spectrum）观察。从得到的荧光图像（图4）中可以看出在注射后的前12小时内，相当数量的Cy-NPs在肿瘤部位周围逐渐累积。24小时后，荧光主要集中在肿瘤部位，证实了由于EPR效应，Cy-NPS优选在肿瘤部位富集。
实施例四：评估了D-NPs在体内的抗肿瘤功效。将荷瘤小鼠平均分配到6个不同的组并用不同的方法处理：（a）PBS、（b）游离DOX（F-DOX）、（c）D-NP、（d）2Gy X射线、（e）F-DOX结合X射线（F-DOX/X射线）、（f）D-NP结合X射线（D-NPs/X射线）。将F-DOX或D-NP以7.5mg/kg相同DOX剂量尾静脉注射（PBS为溶剂）到携带4T1荷瘤的小鼠中。在两个X射线组中，在注射药物24小时后施加2Gy X射线照射（一共二次药物和照射）。与F-DOX相比，D-NPs显示出稍高的肿瘤抑制效率和更好的生物相容性（即体重增加），其通过EPR效应在肿瘤部位富集更多的D-NPs。从D-NP持续低释放DOX。毫不奇怪，X射线治疗抑制肿瘤生长（优于F-DOX），但导致不可忽视的毒副作用（体重减轻）。合理地，两种组合方案显示出比其他方案更好的抗肿瘤效果。F-DOX/X射线联合治疗确实大大增强了抗肿瘤作用。但是，它通过F-DOX和X射线辐射损伤的叠加毒性引起非常严重的副作用（即显着的体重减轻）。显然，这导致F-DOX/X射线组合毫无意义。更引人注目的是，D-NPs/X射线的组合不仅显着地提高了抗肿瘤效率，而且还导致相当大的体重增加，表明这种联合治疗比游离DOX和X射线的联合治疗具有明显更低的毒副作用。这意味着这种联合疗法带来更多的积极效益。这种效益可能是因为以下二种原因：1）EPR效应使D-NP在肿瘤部位富集减少了游离DOX的副作用；2）X射线+ ROS精确控制DOX从肿瘤位置的D-NP释放，导致增强抗肿瘤效果。参见附图5，其中D为肿瘤体积变化，E为在实验结束时从实验小鼠上剥离的肿瘤的图像，F为13天内不同处理组中4T1荷瘤小鼠的体重变化(n = 5, *P < 0.05, **P < 0.01, ***P < 0.001)。
实施例五：通过使用动态光散射测量（DLS）技术研究D-NP的粒度分布随处理时间的变化，从另一角度进一步测定D-NP在不同刺激条件下的药物释放行为。原则上，如果D-NPs的颗粒尺寸被打开并且内部的药物被释放，则它体积将变得更加膨胀。如图6（(A) PBS, (B) 2 Gy X-ray,(C) 100 μM H 2O 2, (D) 100 μM H 2O 2+2 Gy X-ray）显示了在t = 6,12和24小时后用PBS、X射线、H 2O 2、用X射线和H 2O 2共处理的D-NP的粒度分布曲线。在对照组中，随着处理时间的增加，纳米粒子尺寸分布变宽，峰值略有下降。但峰值位置保持不变，这表明有很少D-NP缓慢而自发地分解。在100μM H 2O 2处理组中，很长时间纳米颗粒的尺寸分布才开始略微向右移动（较大的颗粒尺寸），表明相对更多的颗粒被打开。在2 Gy X射线组中，纳米粒度分布与处理时间的变化显示出与对照组非常相似的趋势，表明单次2Gy X射线照射确实不能引发药物的额外释放。然而，在X射线和H 2O 2的共同处理下，纳米颗粒尺寸分布快速且显着地向右移动，甚至在D = 180nm的位置处出现次级峰，表明大量纳米颗粒被破坏。因此，D-NPs大小分布对四种研究刺激条件的反应强烈地证实了上述的药物释放实验。 Se-NPs快速响应X射线和H 2O 2的组合而不是它们的任何单一因子的特征的简单叠加。表明这两个因素在触发药物释放中起互补作用。
实施例六：为了评估D-NP的细胞内吞，将D-NP与4T1细胞一起孵育并通过活细胞成像系统监测。细胞核用Hoechst（蓝色）染色。如图7A所示，孵育0.5小时后，D-NP开始内吞到4T1细胞中，随着孵育时间的增加，DOX（10 mg L −1）的红色荧光强度逐渐增强，表明越来越多的D-NP进入细胞。相反，当细胞与游离DOX（F-DOX）一起孵育时，6小时后进入细胞的DOX较少，如红色荧光弱得多所示（图7B）。这些结果表明D-NPs和F-DOX通过不同方式进入细胞，其中D-NPs通过内吞作用机制胞吞到细胞中，这使得它们能够在细胞中保留相对较长的时间。另一方面，F-DOX通过浓度梯度依赖的扩散机制进入4T1细胞。另外值得注意的是，空载的Se-NPs（Se-共聚物）显示出优异的细胞生物相容性。更重要的是，当DOX包载到NP中时，它可以显着减轻其在所有四种测试细胞中的细胞毒性（图7C-F，（C）没有药物的Se-NPs对HUVEC细胞，MDA-MB-231细胞和4T1细胞的体外细胞毒性，（D）F-DOX和D-NPs对4T1细胞，（E）MDA-MB-231细胞，（F）HUVEC细胞在48小时的体外细胞毒性。数据表示为平均值±标准偏差，n = 3）。因此，可以合理地预期富含肿瘤细胞的D-NPs（而不是正常细胞）可以在其包含的药物释放的情况下精确打开，抗肿瘤效率可以显着提高。同时，大大减少F-DOX的副作用。
此外，还通过体内生物分布分析比较了D-NP和F-DOX肿瘤靶向性和富集时间。将小鼠器官（包括肿瘤，心脏，肝脏，脾脏，肺脏和肾脏）进行研磨，萃取并用酶标仪（SYNERGY neo）检测。根据公式计算不同器官中DOX的百分含量（图8A）。与其他器官相比，D-NPs在肿瘤中具有良好的富集能力，显示出比F-DOX更高的肿瘤富集率。注射后24小时，DOX的肿瘤摄取量为每克组织注射剂量的约2.1％(%ID/g)。相比之下，F-DOX摄取率收敛发生得更早（<12 h），但水平（0.8％）远低于D-NPs。因此，这些数据进一步表明D-NPs比F-DOX在肿瘤部委具有更好的积累和保留，并且再次证实24小时是实现X射线和D-NP联合照射的最佳时间。此外，还监测肿瘤重量来评估抗肿瘤效果。如图8B所示，在PBS、X射线、F-DOX、D-NPs、F-DOX/X射线、D-NPs/X射线处理14天后，平均肿瘤重量为1.22g、0.62g、0.78g、0.33g、0.18g和0.08g。
结论 ：本发明制造了含有二硒键的Se-NPs，对单纯的X射线刺激不敏感，并且当单独用H 2O 2氧化处理时也分解缓慢，然而，2Gy的低X射线可以引发纳米颗粒的快速崩解，伴随着负载药物的快速释放。在Se-NPs的基础上，开发一种智能和快速的药物输送系统用于癌症治疗。体外细胞实验表明，负载Se-NPs的药物的毒性低于相同浓度的游离药物。这意味着脱靶纳米药物对正常细胞的副作用将大大降低。对小鼠的体内实验表明，癌症增殖被更有效地抑制，而观察到的副作用同时显着减少。这主要是由于以下三个原因：（a）通过ERP效应，肿瘤部位的负载药物富集程度明显高于游离药物；（b）Se-NPs可以在很大程度上减轻抗癌药物对正常细胞的细胞毒性；（c）小剂量（2Gy）的X射线，其允许正常细胞从亚致死性损伤中快速恢复，足以显示基于Se-NP的化学疗法的协同效应。本发明的具有低剂量X射线智能响应的二硒纳米材料在癌症治疗的放疗和化疗相结合方面具有很大的临床应用前景。

Claims (10)

  1. 基于双硒键的聚合物，具有以下结构式：
    Figure 210369dest_path_image001
    式中，m为40～50，n为8～15，j为6～12。
  2. 基于双硒键的智能纳米粒，由权利要求1所述基于双硒键的聚合物制备得到。
  3. 基于双硒键的载药纳米粒，由权利要求1所述基于双硒键的聚合物与药物制备得到。
  4. 权利要求1所述基于双硒键的聚合物的制备方法，其特征在于，包括以下步骤，双硒小分子单体与IPDI聚合得到含硒聚合物；再将含硒聚合物经聚乙二醇封端，得到基于双硒键的聚合物。
  5. 根据权利要求4所述基于双硒键的智能纳米粒的制备方法，其特征在于，双硒小分子单体的制备为，氮气中，将溴醇溶液加入含有硼氢化钠、硒粉的水中，反应得到双硒小分子单体。
  6. 根据权利要求5所述基于双硒键的智能纳米粒的制备方法，其特征在于，反应为45～55℃反应20～28小时；硒粉、硼氢化钠、溴醇的摩尔比为4.1～4.2∶1∶2；溴醇溶液中溶剂为四氢呋喃。
  7. 根据权利要求4所述基于双硒键的智能纳米粒的制备方法，其特征在于，双硒小分子单体与IPDI的摩尔比为1∶1.05～1.15；聚合为45～55℃反应1.5～2.5小时。
  8. 根据权利要求4所述基于双硒键的智能纳米粒的制备方法，其特征在于，封端时的温度为45～55℃，时间为20～28小时；双硒小分子单体与IPDI聚合在有机锡化合物存在下进行。
  9. 权利要求1所述基于双硒键的聚合物、权利要求2所述基于双硒键的智能纳米粒在制备肿瘤治疗药物载体中的应用；权利要求1所述基于双硒键的聚合物、权利要求2所述基于双硒键的智能纳米粒或者权利要求3所述基于双硒键的载药纳米粒在制备肿瘤治疗药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述肿瘤治疗为放化疗。
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