WO2023202090A1 - 一种响应释放一氧化碳的羰基铁载体及其制备方法和应用 - Google Patents

一种响应释放一氧化碳的羰基铁载体及其制备方法和应用 Download PDF

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WO2023202090A1
WO2023202090A1 PCT/CN2022/137364 CN2022137364W WO2023202090A1 WO 2023202090 A1 WO2023202090 A1 WO 2023202090A1 CN 2022137364 W CN2022137364 W CN 2022137364W WO 2023202090 A1 WO2023202090 A1 WO 2023202090A1
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carbonyl
siderophore
thiol
preparation
polymer
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龚萍
马功成
张鹏飞
蔡林涛
刘中轲
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Shenzhen Institute of Advanced Technology of CAS
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    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/001—Macromolecular compounds containing organic and inorganic sequences, e.g. organic polymers grafted onto silica
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
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    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
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    • 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
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
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    • 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
    • A—HUMAN NECESSITIES
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    • A61P35/00—Antineoplastic agents
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    • B82—NANOTECHNOLOGY
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    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters

Definitions

  • the present invention relates to the field of nanomedicine, and in particular to a carbonyl siderophore that responds to the release of carbon monoxide and its preparation method and application.
  • the normal body produces CO under conditions of increased pressure through the expression of the heme oxygenase-1 (HO-1) gene, however, this gene is inactive in cancer cells.
  • CO can reverse the Warburg effect, selectively induce cell apoptosis, and inhibit the metastasis of cancer cells, while normal cells are induced to enter a dormant state and are protected from cytotoxicity.
  • the significant anti-cancer selectivity of CO is superior to traditional chemotherapy/radiotherapy drugs. Therefore, combination therapy is emerging as a promising therapeutic strategy.
  • CO is small in size, has high transmembrane diffusivity and has no purpose. High blood drug concentrations and low intratumoral accumulation lead to the risk of CO poisoning and limited combined therapeutic effects, respectively.
  • Previous CO treatment research mainly focused on how to effectively deliver CO to tumor tissues.
  • the existing means of achieving CO gas therapy mainly involve the use of other complex drug carriers to deliver CO from the blood to the tumor site.
  • the current supply of CO gas mainly relies on the delivery of carriers such as mesoporous silicon and metal nanocages.
  • Traditional delivery methods require additional drug carriers and encapsulation methods, resulting in extremely complex design of nanotherapeutic agents, difficult preparation, and cumbersome processes, which are not conducive to industrial production or clinical experiments. And too many materials add additional uncontrollable factors and physiological toxicity.
  • the biosafety of carrier materials such as metallic nanocages and mesoporous silicon is questionable.
  • the light-controlled CO release also means that the material is unstable under light conditions.
  • CO concentration in tumor tissue is extremely difficult to control, and the preparation of complex nanosystems will also increase the uncertainty of pharmacokinetics and operational difficulty, reducing the reproducibility of clinical translation. Therefore, there is an urgent need for a drug carrier that can intelligently respond to the tumor microenvironment and accurately release CO.
  • the present invention proposes a carbonyl siderophore that responds to the release of carbon monoxide and its preparation method and application.
  • the present invention uses carbonyl iron compounds and thiol end-group polymers to prepare an intelligent responsive carrier capable of supplying CO.
  • the present invention provides a carbonyl siderophore that responds to the release of carbon monoxide, including a carbonyl iron compound and a thiol terminal polymer, and the carbonyl iron compound and the thiol terminal polymer are connected through a coordination reaction.
  • the carbonyl iron compound is selected from any one of tricarbonyl iron, pentacarbonyl iron, nonacarbonyl iron, and dodecacarbonyl iron.
  • the thiol-terminated polymer is selected from any one of polyethylene glycol polymers, polypropylene polymers, polystyrene polymers, and polypropylene ester polymers.
  • the molecular weight of the thiol-terminated polymer is 1000 ⁇ 8000. As the molecular weight of the thiol-terminated polymer increases, the loading of CO in the carrier decreases, and the reverse is true as the molecular weight of the thiol-terminated polymer decreases.
  • the relative ratio between thiol-terminated polymer and CO loading is set to X. As the molecular weight and CO loading of the thiol-terminated polymer change, the amphiphilicity of the carrier changes. When X increases, the carrier will become more hydrophilic, otherwise it will become more hydrophobic. Both states are not conducive to the use of the carrier.
  • the carrier When the molecular weight of the thiol-terminated polymer is less than 1,000, the carrier has poor water solubility and is difficult to dissolve. When the molecular weight of the thiol-terminated polymer is greater than 8,000, the proportion of hydrophobic ends of the carrier is too small and the drug cannot be effectively loaded. Therefore, the molecular weight of the thiol-terminated polymer needs to be between 1000-8000.
  • the invention also provides a method for preparing the carbonyl iron carrier, which includes the following steps: dissolve the carbonyl iron compound and the thiol-terminated polymer in tetrahydrofuran, and stir under a nitrogen flow; at the end of the reaction, the solution changes from dark blue to Brownish yellow; cool to room temperature, add liquid alkane to obtain brown precipitate, wash with organic solvent and dry to obtain the carbonyl siderophore.
  • the preparation step also includes a purification step, specifically as follows: redissolve the carbonyl siderophore prepared in tetrahydrofuran, freeze it in a -20°C environment for 5-24 h, and filter the precipitated crystals to obtain Purified carbonyl siderophores.
  • the mass ratio of the carbonyl iron compound and the thiol-terminated polymer is 1:(4 ⁇ 8).
  • the nitrogen flow temperature is 50-120°C
  • the stirring time is 1-12 h.
  • liquid alkane is n-hexane or n-pentane.
  • the organic solvent is diethyl ether or n-butyl ether.
  • the present invention also provides the use of the carbonyl siderophore in the preparation of anti-tumor drugs.
  • the current supply technology for CO relies on the design of small molecule CO prodrugs, but the present invention directly designs the CO prodrug as a polymer carrier, which can not only be released as a responsive prodrug, but also encapsulate other drugs, contrast agents or Nanosystems are more conducive to imaging monitoring and collaborative treatment of CO gas therapy.
  • the carbonyl siderophores prepared by the present invention have high yield and short preparation time.
  • the nanoparticles prepared by the present invention can exist stably, and no sedimentation or flocculation occurs after 15 days.
  • the preparation method is simple and easy to operate and promote.
  • This carbonyl siderophore responds to ROS in the tumor microenvironment, releases CO, and can be successfully enriched in the tumor site to achieve long-term treatment.
  • Figure 1 is a schematic diagram of the process of using PG-CO to encapsulate dyes in the present invention
  • Figure 2 shows the results of the change in absorption of the nanoparticles with concentration after using PG-CO to encapsulate the fluorescent dye IR-813 in Example 3;
  • Figure 3 is a verification experiment of the response to active oxygen after the PG-CO carrier of Example 4 is loaded with Bodipy dye;
  • Figure 4 shows the gradual enrichment and precipitation of PG-CO in Example 5 after responding to hydrogen peroxide
  • Figure 5 is a picture of the aggregation of PG-CO nanoparticles in Example 6 in response to ROS under a high-resolution transmission electron microscope;
  • Figure 6 shows the HE stained sections of Example 7, showing that PG-CO has no obvious effect on mouse organs
  • Figure 7 is a picture of the PG-CO nanoparticles of Comparative Example 1 under a high-resolution transmission electron microscope.
  • the present invention constructs a polymer carrier based on metal carbonyl through coordination reaction.
  • the carrier can simultaneously store and release CO in response to reactive oxygen species.
  • the carbonyl iron compound at the end of the polymer was modified, and a new amphiphilic polymer PG-CO was obtained. Due to the strong hydrophobicity of the metal complex, PG-CO disperses into a molecular state in chloroform and aggregates into nanoparticles in water.
  • ROS reactive oxygen species
  • PG-CO will release enough CO gas through a Fenton-like reaction, and the carbonyl iron side will be oxidized to iron oxide, leading to particle deposition.
  • PG-CO Due to the lack of ROS, PG-CO does not release CO in normal cell tissues, but in the tumor microenvironment with overexpression of ROS, PG-CO will gradually release CO and deposit it in the tumor site for long-term treatment.
  • PG-CO simultaneously meets the following requirements: 1. As a drug carrier; 2. Reactive oxygen species respond to CO gas release system; 3. Accumulate in tumor tissue. 4. Simple structure and convenient synthesis; 5. Stable and safe.
  • the preparation method of the carbonyl siderophore of the present invention has the following steps:
  • the preparation step also includes a purification step, specifically as follows: redissolve the prepared carbonyl siderophore in tetrahydrofuran, freeze it in a -20°C environment for 5-24 h, and filter the precipitated crystals to obtain the purified carbonyl siderophore. Siderophores.
  • the carbonyl iron compound is selected from any one of tricarbonyl iron, pentacarbonyl iron, nonacarbonyl iron, and dodecacarbonyl triiron.
  • the following examples take iron dodecacarbonyl as an example.
  • Iron tricarbonyl, iron pentacarbonyl, and iron nonacarbonyl all have similar physical and chemical properties. Therefore, those skilled in the art can know that iron tricarbonyl, iron pentacarbonyl, and iron nonacarbonyl can be used.
  • the technical solution of the present invention can be realized.
  • the thiol-terminated polymer is selected from any one of mercapto polyethylene glycol, mercapto polypropylene, mercapto polystyrene, and mercapto polypropylene ethyl ester.
  • the following examples take mercapto polyethylene glycol as an example.
  • Mercapto polypropylene, mercapto polystyrene, and mercapto polypropylene ethyl ester all have similar physical and chemical properties. Therefore, those skilled in the art can know that mercapto polypropylene, mercapto polystyrene, Both mercapto polypropylene ethyl esters can realize the technical solution of the present invention.
  • Iron dodecacarbonyl due to its strong hydrophobicity, is generally coated on other carriers for transportation.
  • This example uses the substitution reaction of ferric dodecacarbonyl and thiol to modify one end of the methyl polyethylene glycol polymer methoxypolyethylene glycol thiol (mPEG-SH) as reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • Responsive amphiphilic polymer carrier PG-CO The specific preparation process is as follows. Iron dodecacarbonyl (5-100 mg) and mPEG-SH (M.W. ⁇ 2000) (100-600 mg) are dissolved in tetrahydrofuran (THF) and stirred at 50-120°C under nitrogen flow. 1-12 hours.
  • the solution changed from dark blue to brown. Cool to room temperature, add n-hexane to obtain brown precipitate, wash with diethyl ether and dry to obtain PG-CO. Redissolve PG-CO in THF, freeze it in a -20°C environment for 5-24 hours, and filter the precipitated crystals to obtain a new smart carbonyl siderophore PG-CO.
  • PG-CO is a brown solid that is soluble in water and organic solutions.
  • PG-CO can be used to coat various small molecule drugs, polymer drugs, fluorescent probes, nanoparticles, etc.
  • FPG-CO is used to coat polymer fluorescent probes TPB, MCH-PPV, Bodipy, etc.
  • THF polymer fluorescent probes
  • MCH-PPV polymer fluorescent probes
  • Bodipy a polymer fluorescent probes
  • Example 3 Results of changes in the absorption of nanoparticles with concentration after using PG-CO to encapsulate the fluorescent dye IR-813
  • the experimental steps of using PG-CO to encapsulate the fluorescent dye IR-813 are as in Example 2.
  • the concentration of the obtained nanoparticles is 100-120mM. Dilute the nanoparticle solution and take 5mM, 10mM, 20mM, 40mM, 50mM and 60mM concentrations for UV measurement. Absorption, use a UV spectrophotometer to measure the UV absorption of the above-mentioned concentration nanoparticles in the wavelength range of 500-1000nm. The results are shown in Figure 2.
  • the concentrations from bottom to top are 1uM, 2uM, 3uM, 4uM, 5uM and 6uM. It shows that its UV absorption increases with the increase of nanoparticle concentration.
  • the experimental steps for using PG-CO to encapsulate Bodipy dye are as in Example 2. Dilute the concentration of the obtained nanoparticles to 20-30mM, take 1mL and put it into a cuvette, add 1-2 drops of 0.3 ⁇ hydrogen peroxide solution, and then Record the color change of the solution every hour from 0 to 4 hours.
  • the Fenton reaction between carbonyl iron and hydrogen peroxide gradually produces ROS.
  • the specific phenomenon is that the nanoparticle solution changes from light green to light pink over time, as shown in Figure 3 (the solution gradually becomes brighter). It shows that after adding hydrogen peroxide solution, the nanoparticles gradually release ROS.
  • Example 5 PG-CO gradually enriches and precipitates in response to hydrogen peroxide
  • Example 6 Pictures of PG-CO nanoparticles under high-resolution transmission electron microscopy in response to aggregation of ROS
  • Example 7 HE stained sections show that PG-CO has no obvious effect on mouse organs
  • Iron dodecacarbonyl (5-100 mg) and mPEG-SH (M.W. ⁇ 1w-1.2w) (100-600 mg) were dissolved in tetrahydrofuran (THF) and stirred at 50-120°C under nitrogen flow 1-12 h. At the end of the reaction, the solution changed from dark blue to brown. Cool to room temperature, add n-hexane to obtain brown precipitate, wash with diethyl ether and dry to obtain PG-CO. Redissolve PG-CO in THF, freeze it in a -20°C environment for 5-24 hours, and filter the precipitated crystals to obtain a new smart carbonyl siderophore PG-CO.
  • THF tetrahydrofuran
  • the characterization and detection results of the carbonyl siderophore synthesized by the present invention prove its ability as a carrier and the ability to release CO in response to ROS, and the carbonyl siderophore is used to successfully encapsulate berberine drugs, such as TPE, TPA, MCH-PPV, Bodipy fluorescent molecules and PBPTV fluorescent polymers, etc.; proved the biosafety of the vector, and had no effect on mouse organs after tail vein injection; proved the enrichment of the vector in response to ROS ability.
  • the present invention uses carbonyl iron compounds and thiol-terminated polymers to prepare an intelligent responsive carrier capable of supplying CO, which greatly simplifies the delivery process and improves delivery efficiency.
  • the carrier can load other drugs or contrast agents to achieve Collaborative treatment of disease.

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Abstract

一种响应释放一氧化碳的羰基铁载体及其制备方法和应用，所述羰基铁载体包括羰基铁类化合物和硫醇端基聚合物，所述羰基铁类化合物和所述硫醇端基聚合物通过配位反应连接。利用羰基铁类化合物和硫醇端基聚合物制备出能够供应CO的响应型载体，极大简化了递送的流程，提高递送效率，并且该载体能够负载其它的药物或者造影剂，实现疾病的协同治疗。

Description

一种响应释放一氧化碳的羰基铁载体及其制备方法和应用 技术领域
本发明涉及纳米医学领域，特别涉及一种响应释放一氧化碳的羰基铁载体及其制备方法和应用。
 背景技术
世界卫生组织估计，2021年中有8400万人在没有干预的情况下死于癌症。癌细胞不受控制的增殖和扩散，几乎可以影响人体的任何部位。肿瘤的复杂性、多样性和异质性严重损害了治疗的潜力。气体疗法作为一种新的、有前途的治疗方法，利用治疗/治疗辅助气体（NO、CO、H 2S、H 2、O 2、SO 2和CO 2）及其前体药物来抑制癌细胞的增殖和转移，在治疗中越来越受到重视。CO作为一种信号分子，在血红蛋白降解过程中产生，并在应激和炎症中触发一系列细胞保护机制。正常机体通过血红素加氧酶-1（HO-1）基因的表达在压力增加的情况下产生CO，然而，该基因在癌细胞中无效。在合适的浓度范围内，CO可以逆转Warburg效应，选择性诱导细胞凋亡，抑制癌细胞的转移，而正常细胞则被诱导进入休眠状态，免受细胞毒性的影响。CO的显著抗癌选择性优于传统的化疗/放疗药物。因此，联合治疗正在成为一种有前途的治疗策略。
CO体积小，跨膜扩散率高且无目的。高血药浓度和低肿瘤内蓄积分别导致CO中毒风险和有限的联合治疗效果。以往的CO治疗研究主要集中在如何有效地将CO输送到肿瘤组织。现有的实现CO气体治疗的手段，主要体现在使用其它复杂的药物载体，实现CO从血液到肿瘤部位的递送。当前CO气体的供应主要依赖于介孔硅、金属纳米笼等载体的递送。传统的递送方法需要额外使用药物载体和封装手段，导致纳米诊疗剂的设计极其复杂，制备困难，流程繁琐，不利于工业化生成或临床实验。而且过多的材料增加了额外的不可控因素和生理毒性。金属纳米笼和介孔硅等载体材料的生物安全性存疑。光控制的CO释放方式同时也意味着材料在光条件下的不稳定。肿瘤组织中CO浓度极难调控，复杂纳米系统的制备也会增加药代动力学的不确定性和操作难度，降低临床转化的可重复性。因此目前亟需一种能够智能响应肿瘤微环境并且精准释放CO的药物载体。
 技术问题
针对现有技术中的缺陷，本发明提出了一种响应释放一氧化碳的羰基铁载体及其制备方法和应用。本发明利用羰基铁类化合物和硫醇端基聚合物，制备出能够供应CO的智能响应型载体。
 技术解决方案
本发明提供一种响应释放一氧化碳的羰基铁载体，包括羰基铁类化合物和硫醇端基聚合物，所述羰基铁类化合物和所述硫醇端基聚合物通过配位反应连接。
进一步的，所述羰基铁类化合物选自三羰基铁、五羰基铁、九羰基铁、十二羰基三铁中的任意一种。
进一步的，所述硫醇端基聚合物选自聚乙二醇类聚合物、聚丙烯类聚合物、聚苯乙烯类聚合物、聚丙烯乙酯类聚合物中的任意一种。
进一步的，所述硫醇端基聚合物的分子量为1000~8000。随着硫醇端基聚合物分子量的增加，载体中CO的负载量降低，随着硫醇端基聚合物的降低，则相反。硫醇端基聚合物和CO负载量相对比设值为X。随着硫醇端基聚合物分子量和CO负载量变化时，载体的两亲性会发生变化。当X增加时，载体会更加亲水，反之更加疏水，两种状态都不利于载体的使用。硫醇端基聚合物分子量小于1000时，载体水溶性不好，不易溶解。硫醇端基聚合物分子量大于8000时，载体的疏水端占比太小，不能有效载药。所以硫醇端基聚合物的分子量需要在1000-8000之间。
本发明还提供所述的羰基铁载体的制备方法，包括如下步骤：羰基铁类化合物和硫醇端基聚合物溶于四氢呋喃中，并在氮气流下搅拌；反应结束时，溶液由深蓝色变为棕黄色；冷却至室温，加入液态烷烃获得棕色沉淀，用有机溶剂洗涤并干燥后获得所述的羰基铁载体。
进一步的，所述制备步骤后还包括纯化的步骤，具体如下：将制备得到羰基铁载体的复溶在四氢呋喃中，在-20℃环境中冻存5-24 h，将析出的结晶过滤后得到纯化的羰基铁载体。
进一步的，所述羰基铁类化合物和所述硫醇端基聚合物的质量比为1：（4~8）。
进一步的，所述氮气流温度为50-120℃，搅拌时间为1-12 h。
进一步的，所述液态烷烃为正已烷或正戊烷。
进一步的，所述有机溶剂为乙醚或正丁醚。
本发明还提供所述的羰基铁载体在制备抗肿瘤药物中的应用。
 有益效果
综上，与现有技术相比，本发明达到了以下技术效果：
1. 利用羰基铁类化合物和硫醇端基聚合物，直接制备出能够供应CO的智能响应型载体，大大简化了递送的流程，提高递送效率，并且该载体能够负载其它的药物或者造影剂，实现疾病的协同治疗。同时聚乙二醇类聚合物已经通过美国联邦食品药物和化妆品法规的食品添加剂增补条例，批准把食物化学品药典级的聚乙二醇直接或间接地用作食品添加剂，材料的安全性具有良好的保障。
2. 当前对于CO的供应技术依赖于小分子CO前药的设计，而本发明直接将CO前药设计为聚合物载体，不仅可以作为前药响应型释放，而且可以封装其它药物、造影剂或者纳米系统，更利于实现CO气体治疗的成像监控和协同治疗。
3.本发明制备的羰基铁载体产率高，制备时间短。
4.本发明制备的纳米颗粒能够稳定存在，15天后未出现沉降、凝絮现象。
5. 该制备方法简便易行，便于操作推广。
6. 该羰基铁载体在肿瘤微环境中响应ROS，释放CO，并能够成功在肿瘤部位富集实现长时间治疗。
 附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明使用PG-CO包载染料的过程示意图；
图2为实施例3利用PG-CO包载荧光染料IR-813后纳米颗粒的吸收随浓度的变化的结果；
图3为实施例4的PG-CO载体包载Bodipy染料后对活性氧的响应验证实验；
图4为实施例5的PG-CO在响应双氧水后逐渐富集沉淀；
图5为实施例6的PG-CO纳米颗粒在高分辨透射电镜下的响应ROS后的聚集图片；
图6为实施例7的HE染色切片表明PG-CO对小鼠脏器无明显影响；
图7为对比例1的PG-CO纳米颗粒在高分辨透射电镜下的图片。
 本发明的实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
本发明通过配位反应构建了基于羰基金属的聚合物载体。该载体能够同时存储和响应活性氧簇释放CO。通过硫醇与羰基金属络合物的配位反应，对聚合物末端的羰基铁化合物进行改性，得到了一种新型两亲性聚合物PG-CO。由于金属络合物的强疏水性，PG-CO在氯仿中分散成分子状态，而在水中聚集成纳米颗粒。在活性氧（ROS）存在的情况下，PG-CO会通过类Fenton反应释放出足够的CO气体，羰基铁侧会氧化为氧化铁，导致颗粒沉积。由于缺乏ROS，PG-CO在正常细胞组织中不会释放CO，而在ROS过度表达的肿瘤微环境中，PG-CO会逐渐释放CO并沉积在肿瘤部位进行长期治疗。作为一种多功能智能纳米载体，PG-CO同时满足以下要求：1。作为药物载体；2.活性氧响应CO气体释放系统；3.在肿瘤组织中积聚。4.结构简单，合成方便；5.稳定与安全。
本发明的羰基铁载体的制备方法，步骤如下：
羰基铁类化合物和硫醇端基聚合物溶于四氢呋喃中，并在氮气流下搅拌；反应结束时，溶液由深蓝色变为棕黄色；冷却至室温，加入液态烷烃获得棕色沉淀，用有机溶剂洗涤并干燥后获得所述的羰基铁载体。
所述制备步骤后还包括纯化的步骤，具体如下：将制备得到羰基铁载体的复溶在四氢呋喃中，在-20℃环境中冻存5-24 h，将析出的结晶过滤后得到纯化的羰基铁载体。
其中，羰基铁类化合物选自三羰基铁、五羰基铁、九羰基铁、十二羰基三铁中的任意一种。以下实施例以十二羰基三铁为例，三羰基铁、五羰基铁、九羰基铁均具有相似的理化性质，因此本领域技术人员可以知晓采用三羰基铁、五羰基铁、九羰基铁均能实现本发明的技术方案。
硫醇端基聚合物选自巯基聚乙二醇、巯基聚丙烯、巯基聚苯乙烯、巯基聚丙烯乙酯中的任意一种。以下实施例以巯基聚乙二醇为例，巯基聚丙烯、巯基聚苯乙烯、巯基聚丙烯乙酯均具有相似的理化性质，因此本领域技术人员可以知晓采用巯基聚丙烯、巯基聚苯乙烯、巯基聚丙烯乙酯均能实现本发明的技术方案。
实施例 1 本发明的羰基铁载体的制备和纯化
十二羰基三铁，由于其强疏水性，一般被涂覆在其他载体上用于输送。本实施例使用十二羰基三铁与硫醇的取代反应来修饰甲基聚乙二醇类聚合物甲氧基聚乙二醇硫醇（mPEG-SH）的一端，来作为活性氧（ROS）响应性两亲性聚合物载体PG-CO。具体的准备过程如下，十二羰基三铁（5-100 mg）和mPEG-SH（M.W.≈2000）（100-600 mg）溶于四氢呋喃（THF）中，并在50-120℃和氮气流下搅拌1-12 h。反应结束时，溶液由深蓝色变为棕黄色。冷却至室温，加入正己烷以获得棕色沉淀，用乙醚洗涤并干燥后获得PG-CO。将PG-CO复溶在THF中，在-20℃环境中冻存5-24 h，将析出的结晶过滤后得到新型智能羰基铁载体PG-CO。PG-CO是一种棕色固体，可溶解于水和有机溶液中。
实施例 2  利用本发明的羰基铁载体合成智能纳米制剂
PG-CO可以用于包覆各种小分子药物、聚合物药物、荧光探针、纳米颗粒等。例如利用FPG-CO包覆聚合物荧光探针TPB、MCH-PPV，Bodipy等。首先将1-50 mg PG-CO和 5-10 mg MCH-PPV染料溶解于THF中。超声5-10 min后，加入10 ml去离子水，用氮气吹出THF，共沉淀。用超滤管离心5分钟并重复洗涤3次，采用共沉淀来形成稳定且均匀的纳米颗粒，为粒径50-200 nm的颗粒。
实施例 3  利用PG-CO包载荧光染料IR-813后纳米颗粒的吸收随浓度的变化的结果
利用PG-CO包载荧光染料IR-813实验步骤如实施例2，得到的纳米颗粒浓度为100-120mM，将纳米颗粒溶液稀释，分别取5mM，10mM，20mM，40mM，50mM，60mM浓度测量紫外吸收，利用紫外分光光度计分别测量上述浓度纳米颗粒在500-1000nm波长范围内紫外吸收，结果见附图2，浓度由下往上分别为1uM，2uM，3uM，4uM，5uM和6uM。说明其紫外吸收随纳米颗粒浓度增大而增加。
实施例 4  PG-CO载体包载Bodipy染料后对活性氧的响应验证实验
利用PG-CO包载Bodipy染料实验步骤如实施例2，将得到的纳米颗粒浓度稀释至20-30mM，取1mL放入比色皿，滴加1-2滴0.3‰的过氧化氢溶液，之后0-4小时每隔1小时记录溶液颜色变化。羰基铁和双氧水发生芬顿反应逐渐产生ROS，具体现象表现为纳米颗粒溶液随时间从淡绿色转变为淡粉色，如附图3（溶液逐渐变明亮）。表明加入过氧化氢溶液后，纳米颗粒逐渐释放ROS。
实施例 5  PG-CO在响应双氧水后逐渐富集沉淀
取制备的PG-CO溶于水制得40-50mM溶液，各取0.5-1mL于两个离心管，往其中一个滴加1-2滴0.3‰的过氧化氢溶液，另一个不做处理。等待1-2小时后观察离心管中溶液，可以看到未处理的PG-CO溶液中无沉淀，而加入过氧化氢溶液后的PG-CO溶液则有沉淀于离心管壁，如附图4。表明PG-CO在响应双氧水后逐渐富集沉淀。
实施例 6  PG-CO纳米颗粒在高分辨透射电镜下的图片响应ROS后的聚集
取实施例4中加入过氧化氢溶液后的PG-CO溶液，制备电镜样品，寄出去请专业机构拍摄高分辨透射电镜图片，如附图5。可以看到PG-CO纳米颗粒在响应ROS后发生了聚集现象。
实施例 7  HE染色切片表明PG-CO对小鼠脏器无明显影响
取制备的PG-CO溶于水制得40-50mM溶液，取0.5-1mL通过尾静脉注射的方法注入小鼠体内，正常饲养1-2天后将小鼠解剖，取出脏器，寄出去请专业机构做HE染色切片，如附图6。可以看到小鼠脏器正常，表明PG-CO对小鼠脏器无明显不良影响。
对比例 1  使用分子量大于8000的硫醇端基聚合物制备羰基铁载体
十二羰基三铁（5-100 mg）和mPEG-SH（M.W.≈1w-1.2w）（100-600 mg）溶于四氢呋喃（THF）中，并在50-120℃和氮气流下搅拌1-12 h。反应结束时，溶液由深蓝色变为棕黄色。冷却至室温，加入正己烷以获得棕色沉淀，用乙醚洗涤并干燥后获得PG-CO。将PG-CO复溶在THF中，在-20℃环境中冻存5-24 h，将析出的结晶过滤后得到新型智能羰基铁载体PG-CO。
将1-50 mg PG-CO和 5-10 mg Bodipy染料溶解于THF中。超声5-10 min后，加入10 ml去离子水，用氮气吹出THF，共沉淀。用超滤管离心5分钟并重复洗涤3次，采用共沉淀来形成稳定且均匀的纳米颗粒。将得到的纳米颗粒浓度稀释至20-30mM，取1mL放入比色皿，滴加1-2滴0.3‰的过氧化氢溶液，制备电镜样品，寄出去请专业机构拍摄高分辨透射电镜图片，如附图7。可以看到PG-CO纳米颗粒与图5相比在响应ROS后并没有发生聚集现象，说明使用分子量大于8000的硫醇端基聚合物制备羰基铁载体不能有效制成均一稳定的纳米颗粒。
综合以上实施例和对比例，本发明合成的羰基铁载体的表征和检测结果证明了其作为载体的能力和对ROS响应释放CO的能力，并利用该羰基铁载体成功包载黄连素药物，如TPE、TPA、MCH-PPV、Bodipy荧光分子和PBPTV荧光聚合物等；证明了该载体的生物安全性，尾静脉注射后对小鼠脏器没有影响；证明了该载体对ROS响应后的富集能力。本发明利用羰基铁类化合物和硫醇端基聚合物制备出能够供应CO的智能响应型载体，极大简化了递送的流程，提高递送效率，并且该载体能够负载其它的药物或者造影剂，实现疾病的协同治疗。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种响应释放一氧化碳的羰基铁载体，其特征在于，包括羰基铁类化合物和硫醇端基聚合物，所述羰基铁类化合物和所述硫醇端基聚合物通过配位反应连接。
  2. 根据权利要求1所述的羰基铁载体，其特征在于，所述羰基铁类化合物选自三羰基铁、五羰基铁、九羰基铁、十二羰基三铁中的任意一种。
  3. 根据权利要求1所述的羰基铁载体，其特征在于，所述硫醇端基聚合物选自聚乙二醇类聚合物、聚丙烯类聚合物、聚苯乙烯类聚合物、聚丙烯乙酯类聚合物中的任意一种。
  4. 根据权利要求1所述的羰基铁载体，其特征在于，所述硫醇端基聚合物的分子量为1000~8000。
  5. 权利要求1-4任一项所述的羰基铁载体的制备方法，其特征在于，包括如下步骤：
    羰基铁类化合物和硫醇端基聚合物溶于四氢呋喃中，并在氮气流下搅拌；反应结束时，溶液由深蓝色变为棕黄色；冷却至室温，加入液态烷烃获得棕色沉淀，用有机溶剂洗涤并干燥后获得所述的羰基铁载体。
  6. 根据权利要求5所述的制备方法，其特征在于，所述制备步骤后还包括纯化的步骤，具体如下：
    将制备得到羰基铁载体的复溶在四氢呋喃中，在-20℃环境中冻存5-24 h，将析出的结晶过滤后得到纯化的羰基铁载体。
  7. 根据权利要求5所述的制备方法，其特征在于，所述羰基铁类化合物和所述硫醇端基聚合物的质量比为1：（4~8）。
  8. 根据权利要求5所述的制备方法，其特征在于，所述氮气流温度为50-120℃，搅拌时间为1-12 h。
  9. 根据权利要求5所述的制备方法，其特征在于，所述液态烷烃为正已烷或正戊烷；所述有机溶剂为乙醚或正丁醚。
  10. 权利要求1-4任一项所述的羰基铁载体在制备抗肿瘤药物中的应用。
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CN115010939A (zh) * 2022-04-22 2022-09-06 深圳先进技术研究院 一种响应释放一氧化碳的羰基铁载体及其制备方法和应用

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WO2025107185A1 (zh) * 2023-11-22 2025-05-30 中国科学院深圳先进技术研究院 一种载体、靶向纳米颗粒及其制备方法和应用

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