WO2020057086A1 - 一种Fe 3+/2+-NO供体混价配位聚合物及其应用 - Google Patents

一种Fe 3+/2+-NO供体混价配位聚合物及其应用 Download PDF

Info

Publication number
WO2020057086A1
WO2020057086A1 PCT/CN2019/079877 CN2019079877W WO2020057086A1 WO 2020057086 A1 WO2020057086 A1 WO 2020057086A1 CN 2019079877 W CN2019079877 W CN 2019079877W WO 2020057086 A1 WO2020057086 A1 WO 2020057086A1
Authority
WO
WIPO (PCT)
Prior art keywords
donor
coordination polymer
bpdb
mixed
nanoparticles
Prior art date
Application number
PCT/CN2019/079877
Other languages
English (en)
French (fr)
Inventor
丁娅
黄张建
Original Assignee
中国药科大学
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 中国药科大学 filed Critical 中国药科大学
Publication of WO2020057086A1 publication Critical patent/WO2020057086A1/zh

Links

Images

Classifications

    • 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
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/008—Supramolecular polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/80—Polymers containing hetero atoms not provided for in groups A61K31/755 - A61K31/795
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the fields of biomedicine technology and nanotechnology, and particularly relates to a Fe 3 + / 2 + -NO donor mixed-valence coordination polymer and its antitumor application.
  • Nitric oxide is an important endogenous messenger substance and / or effector molecule and is involved in many physiological and pathological processes in the body.
  • NO is an important endogenous messenger substance and / or effector molecule and is involved in many physiological and pathological processes in the body.
  • the relationship between NO and tumorigenesis, development, apoptosis, invasion and metastasis has aroused widespread concern and has become a new direction for tumor prevention and treatment.
  • Numerous studies have shown that NO is a "double-edged sword" and has dual effects on tumors.
  • Low concentrations of NO promote tumor development, angiogenesis, and metastasis, while high concentrations of NO can inhibit tumor growth, accelerate tumor cell apoptosis, and exhibit antitumor effects. Therefore, releasing a high concentration of NO at the tumor site can effectively achieve anti-tumor therapy.
  • NO donor prodrugs have improved the anti-tumor efficacy of NO to a certain extent, most of these NO donors have low molecular weight, are easily removed by the reticuloendothelial system, and have limited stability. Therefore, it is still difficult to achieve the desired therapeutic effect with NO donor treatment alone.
  • nano-drugs have the dual identities of drugs and nano-materials. Compared with traditional drugs, nano-drugs have their unique advantages, such as improving the stability of the drug; high specific surface area, capable of carrying a large number of drugs; and high-efficiency solid tumor Qualcomm Permeability and Retention Effect (EPR) can passively target tumors; by controlling the optical signal, temperature, magnetic field of the nanomaterials, or using the tumor microenvironment response, the controlled release of drugs at the tumor site can be achieved Wait.
  • EPR Qualcomm Permeability and Retention Effect
  • NO donor compound BPDB containing two free carboxyl groups. It can be activated by over-expressed GSH in tumor tissues or cells, releasing 4 molecules of NO, and exhibiting a certain proliferation inhibitory activity on HL-60 cells.
  • this NO donor molecule similar to the previously reported NO donor molecule JS-K, relies on GSH activation, and normal tissues and plasma also contain a certain amount of GSH, resulting in poor plasma stability of these compounds. Short half-life.
  • the present invention considers from three aspects: 1 improving the plasma stability of BPDB, extending its antitumor effect time, and making it have the potential for in vivo application; 2 BPDB is single-use, the activity is not ideal enough, in order to design new Molecules, which can simultaneously produce NO and other cytotoxic drugs, thereby exerting synergistic anti-tumor effects and improving anti-tumor activity, making them have practical value in preclinical or clinical applications; 3 reducing the toxic and side effects of BPDB on normal tissues or cells to achieve The purpose of synergistic and attenuating is to overcome the existing problems of BPDB, improve its drug-making properties, and make it have value and potential for further clinical application.
  • the present invention uses metal iron ions to have strong coordination ability with carboxyl group (-COOH), and can form coordination polymeric nanomaterials with organic ligands with -COOH, especially multiple -COOH under milder conditions.
  • -COOH carboxyl group
  • using Fe 2+ as a Fenton reagent can effectively catalyze the reaction of H 2 O 2 to generate more cytotoxic reactive oxygen species (ROS) in order to achieve Chemodynamic Therapy (CDT).
  • ROS cytotoxic reactive oxygen species
  • CDT Chemodynamic Therapy
  • BPDB cytotoxic reactive oxygen species
  • BPDB N-proline azo-O 2 --2,4-dinitrobenzene
  • the proportions of Fe 3+ and Fe 2+ in the Fe 3 + / 2 + -NO donor-complexed coordination polymer change due to different preparation conditions.
  • the molecular formula of the Fe 3 + / 2 + -NO donor mixed coordination polymer is Fe x + 4 (BPDB) (H 2 O) 2 , where Fe x + represents Fe 3+ and Fe 2+ .
  • Fe 3+ has an Fe 2+ ratio of 2.3: 1.7
  • one molecule of BPDB can coordinate with 4 Fe x + ions, and Fe x + is a mixture of Fe 3+ and Fe 2+ . This is because Fe 3+ -BPDB is prepared in the preparation method, and then Fe 2+ is used. Ion exchange, when the exchange reaches equilibrium, a mixed-valence coordination polymer having both Fe 3+ and Fe 2+ and BPDB coordination is obtained.
  • the above coordination polymers can form polymeric nanoparticles under specific conditions, with a particle size of 270-530 nm, preferably 270 nm.
  • Fe 3 + / 2 + -NO donor mixed-valence coordination polymer nanoparticles have better water solubility and stability in vivo than NO donor alone, and are suitable for in vivo antitumor treatment.
  • Another object of the present invention is to provide a method for preparing the above-mentioned Fe 3 + / 2 + -NO donor mixed coordination polymer.
  • the NO donor 1,5-bis (L-proline azo-O-O 2 -)-2,4-dinitrobenzene and Fe 3+ coordinate to form a polymer, which is further prepared by ion exchange with Fe 2+ .
  • the preparation method of the Fe 3 + / 2 + -NO donor mixed-valence coordination polymer nanoparticles is as follows:
  • NO donor 1,5-bis (L-proline azo-O 2 -)-2,4-dinitrobenzene and Fe 3+ water-soluble trivalent iron salts can be used, such as FeCl 3 .6H 2 O) is dispersed in an aqueous solution, and the pH value of the solution is adjusted to weakly acidic;
  • Fe 3+ -BPDB-NCP exchange Fe 3+ -BPDB-NCP with Fe 2+ (water-soluble divalent iron salts such as FeCl 2 .4H 2 O can be used) in an aqueous solution. After the exchange reaches equilibrium, centrifuge, wash, and collect the product To obtain Fe 3 + / 2 + -NO donor mixed-valence coordination polymer nanoparticles (Fe 3 + / 2 + -BPDB-NCP).
  • the molar ratio of the NO donor and Fe 3+ is 1:15 to 1:10, and preferably 2:15.
  • the poor solvent is selected from one or more of alcohol solvents such as anhydrous methanol and absolute ethanol, and is preferably anhydrous methanol.
  • alcohol solvents such as anhydrous methanol and absolute ethanol
  • the molar ratio of Fe 3+ -BPDB-NCP to Fe 2+ is 2: 1 to 1: 2, preferably 1: 1.
  • the NO donor according to the present invention can be prepared by a method disclosed in a document published in the Journal of Medicinal Chemistry entitled Aryl Bis (diazeniumdiolates): Potential Inducers of S-Glutathionylation of Cellular Proteins and Their in Vitro Antiproliferative Activities. The reaction route is shown in Figure 1A.
  • Another object of the present invention is to provide an application of the Fe 3 + / 2 + -NO donor covalent coordination polymer in the preparation of an antitumor drug. Further, the Fe 3 + / 2 + -NO donor mixed coordination polymer can be used as a prodrug in response to high concentrations of GSH and H 2 O 2 in the tumor microenvironment to release NO and generate ROS, respectively.
  • Another object of the present invention is to provide a pharmaceutical preparation containing the Fe 3 + / 2 + -NO donor mixed coordination polymer according to the present invention.
  • the Fe 3 + / 2 + -NO donor mixed coordination polymer according to the present invention passively targets tumors through the high permeability and retention effect (EPR effect) of solid tumors; high concentrations in the tumor microenvironment In response to GSH and high concentration of H 2 O 2 , it releases NO and generates ROS, realizing high-efficiency, low-toxicity, and synergistic treatment of tumors.
  • EPR effect high permeability and retention effect
  • FIG. 1 (A) is a synthetic roadmap of a NO donor in the present invention
  • (B) is a synthetic roadmap of a Fe 3 + / 2 + -NO donor mixed-valence coordination polymerized nanoparticle according to the present invention.
  • FIG. 2 is (A) TEM, (B) EDS, and (C) XPS images of each coordination polymerized nanoparticle in Example 1 of the present invention.
  • FIG. 3 is a DLS characterization result of the Fe 2+ -BPDB coordination polymer in Example 2 of the present invention.
  • FIG. 4 is a result of examining the water solubility (A) and stability (B) of Fe 3 + / 2 + -BPDB-NCP and NO donors prepared in Embodiment 1 of the present invention.
  • Fig. 5 is the in vitro (A) relationship between GSH concentration and NO release, (B) fluorescence curves generated by hydroxyl radicals and superoxide anions, (C) active oxygen cluster generation (D) H 2 O 2 concentration and the relationship between active oxygen clusters.
  • FIG. 6 is a confocal imaging diagram of (A) NO release and (B) ROS confocal imaging diagram of each coordinated polymeric nanoparticle HepG2 cell in Example 6 of the present invention.
  • FIG. 7 is a pharmacodynamic result of (a) physiological saline, (b) NO donor, (c) Fe 3 + / 2 + -BPDB-NCP nanoparticle preparation in Example 7 of the present invention: (A) relative tumor volume , (B) mouse weight, (C) survival and (D) tumor weight.
  • the invention constructs a Fe 3 + / 2 + -NO donor mixed-valence coordination polymerized nanoparticle, which has better water solubility and stability in vivo than a single NO donor, and is more suitable for application in vivo antitumor therapy.
  • the in vitro characterization, in vitro NO release and ROS production, cell-level NO release and ROS production, and animal pharmacodynamics were studied.
  • the NO donor 1,5-bis (L-proline azo-O 2 -)-2,4-dinitrobenzene (BPDB) and FeCl 3 .6H 2 O were dispersed at a molar ratio of 2:15 In aqueous solution and adjust pH to 5.5 with dilute NaOH solution. Then, add anhydrous methanol under vigorous stirring to induce the formation of nanoparticles, and continue stirring for a period of time until the particle growth is complete. After centrifugation and washing, Fe 3+ -BPDB-NCP can be obtained.
  • the method can regulate the synthesis of nanoparticles of different sizes by adjusting the rate of adding anhydrous methanol.
  • Fe 3+ -BPDB-NCP nanoparticles are exchanged with FeCl 2 .4H 2 O in an equimolar ratio in an aqueous solution. After the exchange reaches equilibrium, the product is centrifuged, washed, and collected to obtain Fe 3 + / 2 + -BPDB-NCP.
  • the NO donor 1,5-bis (L-proline azo-O 2 -)-2,4-dinitrobenzene (BPDB) and FeCl 3 .6H 2 O were dispersed at a molar ratio of 2:15 In DMF, then heated to 90 ° C for 8h. It was found during the experiment that during the heating process, the NO donor was thermally decomposed to generate NO, which could not effectively form the required coordination polymer.
  • the above experiments show that the synthesis of coordination polymers by NO donors requires mild reaction conditions, such as the poor solvent method in the present invention.
  • the NO donor 1,5-bis (L-proline azo-O 2 -)-2,4-dinitrobenzene (BPDB) and FeCl 2 .4H 2 O were dispersed at a molar ratio of 2:15 In aqueous solution and adjust pH to 5.5 with dilute NaOH solution. Then, the anhydrous methanol was added under vigorous stirring to induce the formation of Fe 2+ -BPDB particles, and stirring was continued for a period of time until the particle growth was completed. After centrifugation and washing, Fe 2+ -BPDB particles were obtained. Repeated three times, the DLS characterization results of the three samples are shown in Figure 2. The results show that the hydrated particle size of Fe 2+ -BPDB is larger than 1 ⁇ m, and the required nano-sized particles cannot be further prepared.
  • the Fe 3+ -BPDB-NCP and Fe 3 + / 2 + -BPDB-NCP freshly prepared in the poor solvent method of Example 1 were taken, diluted to a certain multiple and dropped on a copper mesh. After air-drying, the materials were characterized on a transmission electron microscope Morphology, size (TEM), and elemental composition (EDS).
  • FIG. 3 is (A) TEM, (B) EDS, and (C) XPS diagrams of each coordination polymerized nanoparticle prepared in Example 1 of the present invention. The results show that the coordination polymer nanoparticles with NO donor BPDB as the ligand and Fe 3 + / 2 + mixed valence as the metal coordination ions were successfully synthesized.
  • the molecular formula of Fe 3 + / 2 + -NO donor mixed coordination polymer is calculated as Fe x + 4 (BPDB) (H 2 O) 2 , and Fe x + is Fe 3+ and The ratio of Fe 2+ , Fe 3+ and Fe 2+ is 2.3: 1.7.
  • Water solubility Disperse the NO donor 1,5-bis (L-proline azo-O 2 -)-2,4-dinitrobenzene (BPDB) in anhydrous methanol and water, respectively, Fe 3+ / 2 + -BPDB-NCP was dispersed in water to prepare a 1 mg / mL solution. Their dispersibility and solubility in various solvents were recorded by taking pictures. As shown in FIG. 4A, BPDB can only be dissolved in a methanol solution and has poor dispersibility in water, and Fe 3 + / 2 + -BPDB-NCP can be well dissolved in water.
  • the method for detecting in vitro NO release and active oxygen cluster generation of Fe 3 + / 2 + -BPDB-NCP according to the present invention is as follows:
  • the multifunctional NO donor is GSH-responsive
  • the release of NO by the coordination polymer nanoparticles is also GSH-triggered.
  • Fe 3 + / 2 + -BPDB-NCP was incubated with GSH at different concentrations for 2 h, and the amount of NO released was measured with Griess reagent.
  • Fe 2+ in NO donor coordination polymer nanoparticles is an effective Fenton reagent, which can catalyze H 2 O 2 to produce active oxygen clusters including hydroxyl radicals and superoxide anions.
  • the Fe 3 + / 2 + -BPDB- NCP and H 2 O 2 were incubated 30min, respectively terephthalic acid, and ethidium dihydro DCFH-DA detection OH, O 2 -.. And generation of ROS.
  • the results are shown in FIG. 5, which indicate that the Fe 3 + / 2 + -NO donor mixed coordination polymer nanoparticles synthesized by the present invention can release NO and generate ROS under the response of GSH and H 2 O 2 , respectively.
  • DMEM Discard DMEM, wash 3 times with PBS solution, and add 5% serum-DMEM solution, 5% serum-DMEM solution containing 20 ⁇ g / mL Fe 3+ -BPDB-NCP, and 20 ⁇ g / mL Fe 3 + / 2 + - A 5% serum-DMEM solution of BPDB-NCP.
  • the incubation was continued for 3 hours, the supernatant was discarded and washed three times with PBS, and 1 ⁇ g / mL Hoechst 33342 was added for nuclear staining. After nuclear staining for 15 min, washing with PBS three times, the cells were fixed with 4% paraformaldehyde for confocal imaging observation.
  • FIG. 6 shows that the Fe 3 + / 2 + -NO donor mixed coordination polymer nanoparticles synthesized by the present invention can effectively release NO and generate ROS at the cell level, and the amount of released NO is more than that of NO alone.
  • the body has more BPDB and more ROS than Fe 3+ -BPDB-NCP, which provides a basis for NO-CDT cooperative therapy in vivo.

Landscapes

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

Abstract

本发明公开了一种Fe 3+/2+-NO供体混价配位聚合物Fe x+ 4(BPDB)(H 2O) 2，所述分子式中Fe x+代表Fe 3+和Fe 2+，BPDB为NO供体：1,5-二(L-脯氨酸偶氮翁-O 2-)-2,4-二硝基苯。本发明还提供了上述Fe 3+/2+-NO供体混价配位聚合物的纳米颗粒，该纳米颗粒具有更高的水溶性和稳定性，能够通过实体瘤的高通透性和滞留效应(EPR效应)被动靶向肿瘤；在肿瘤微环境高浓度GSH和高浓度H 2O 2响应下，释放NO以及产生ROS，实现肿瘤的高效、低毒、协同治疗。

Description

一种Fe 3+/2+-NO供体混价配位聚合物及其应用 技术领域
本发明属于生物医药技术和纳米科技领域，具体涉及一种Fe 3+/2+-NO供体混价配位聚合物及其抗肿瘤应用。
背景技术
一氧化氮(NO)是一种重要的内源性信使物质和(或)效应分子，参与体内众多的生理和病理过程。近年来，NO与肿瘤的发生、发展、凋亡、侵袭和转移的关系引起了人们的广泛关注，成为肿瘤预防和治疗的新方向。已有大量研究表明，NO是一把“双刃剑”，对肿瘤具有双重作用。低浓度的NO促进肿瘤的发展、血管生成和转移，而高浓度的NO则可以抑制肿瘤的生长，加速肿瘤细胞凋亡，表现出抗瘤作用。因此，在肿瘤部位释放高浓度的NO，能够有效实现抗肿瘤治疗。
然而NO气体的半衰期极短、稳定性差，将体外NO直接输送至体内肿瘤部位非常困难，尤其是高浓度的NO。NO供体，即能够在体内产生NO的化合物，备受关注，已成为NO用于肿瘤治疗的重要化合物。尽管NO供体前药在一定程度上提高了NO抗肿瘤的疗效，但这些NO供体大部分分子质量较低，容易被网状内皮系统清除且本身稳定性有限等。因而，单独的NO供体治疗仍然难以达到预期的治疗效果。
随着纳米科技的飞速发展，纳米技术和纳米材料被广泛应用于疾病的诊疗。其中，纳米药物兼具药物和纳米材料的双重身份，与传统药物相比有其独特的优点，如能提高药物的稳定性；高比表面积，能够负载大量的药物；具有高效的实体瘤高通透性和滞留效应(Enhanced Permeability and Retention Effect，EPR)，能够被动靶向肿瘤；可通过调控纳米材料的光信号、温度、磁场或利用肿瘤微环境响应等，实现在肿瘤部位药物的可控释放等。
2008年发表于Journal of Medicinal Chemistry杂志题为Aryl Bis(diazeniumdiolates):Potent Inducers of S-Glutathionylation of Cellular Proteins and Their in Vitro Antiproliferative Activities的文献公开了一种含有2个游离羧基的NO供体化合物BPDB，其可以被肿瘤组织或细胞中过表达的GSH激活，释放出4分子的NO，并对HL-60细胞表现出一定的的增殖抑制活性。但是，此NO供体分子(BPDB)，与前期报道的NO供 体分子JS-K类似，均依靠GSH激活，而正常组织和血浆中也含有一定量的GSH，造成该类化合物血浆稳定性差，半衰期短。即化合物在未到达肿瘤组织或进入肿瘤细胞时，绝大部分已被降解并产生NO，造成抗肿瘤活性大大降低，同时在非肿瘤部位产生的NO，会对正常组织或细胞产生较大的毒副作用。因此，上述问题限制了该NO供体作为抗肿瘤药物的进一步研发及临床应用。
发明内容
基于上述研究背景，本发明从3个方面考虑，①提高BPDB的血浆稳定性，延长其发挥抗肿瘤作用的时间，使其具有体内应用的潜力；②BPDB单一适用，活性不够理想，以期设计新的分子，其同时可产生NO及其他细胞毒药物，从而发挥协同抗肿瘤作用，提高抗肿瘤活性，使其具有临床前或临床应用的实际价值；③降低BPDB对正常组织或细胞的毒副作用，实现其增效减毒的目的，以期克服目前BPDB存在的问题，提高其成药性，使其具有进一步临床应用的价值和潜力。
本发明一方面利用金属铁离子与羧基(-COOH)有较强的配位能力，在较温和的条件下能与带有-COOH尤其是多个-COOH的有机配体形成配位聚合纳米材料；另一方面，利用Fe 2+作为芬顿试剂，能有效催化H 2O 2反应生成细胞毒性更大的活性氧簇(ROS)，以期实现化学动力学治疗(Chemodynamic Therapy,CDT)，设计了一种基于NO供体(BPDB)的Fe 3+/2+-NO供体混价配位聚合物纳米颗粒，能在肿瘤组织部位累积，实现肿瘤微环境GSH和H 2O 2响应的NO和CDT的协同抗肿瘤治疗。
本发明具体技术方案如下：
一种Fe 3+/2+-NO供体混价配位聚合物，NO供体1,5-二(L-脯氨酸偶氮翁-O 2-)-2,4-二硝基苯(简称为BPDB)与Fe 3+和Fe 2+形成混价配位聚合物。BPDB结构如下：
Figure PCTCN2019079877-appb-000001
上述Fe 3+/2+-NO供体混价配位聚合物中Fe 3+和Fe 2+的比例因制备条件不同而发生变化。本发明一个具体的技术方案中，Fe 3+/2+-NO供体混价配位聚合物的分子式为Fe x+ 4(BPDB)(H 2O) 2，所述Fe x+代表Fe 3+和Fe 2+。优选Fe 3+的Fe 2+比例为2.3:1.7
上述分子式中一分子BPDB可以与4个Fe x+离子形成配位，Fe x+为Fe 3+和Fe 2+的混合，这是因为制备方法中先制备Fe 3+-BPDB，再采用Fe 2+进行离子交换，交换达到平衡时，得到的是既有Fe 3+也有Fe 2+与BPDB配位的混价配位聚合物。
上述配位聚合物可以在特定条件下形成聚合纳米颗粒，粒径为270–530nm，优选270nm。Fe 3+/2+-NO供体混价配位聚合物纳米颗粒的水溶性和体内稳定性优于单独的NO供体，适合用于体内抗肿瘤治疗。
本发明另一目的在于提供上述Fe 3+/2+-NO供体混价配位聚合物的制备方法，由所述NO供体1,5-二(L-脯氨酸偶氮翁-O 2-)-2,4-二硝基苯和Fe 3+配位形成聚合物，再进一步与Fe 2+经离子交换制得。
上述Fe 3+/2+-NO供体混价配位聚合物纳米颗粒的制备方法如下：
(1)将NO供体1,5-二(L-脯氨酸偶氮翁-O 2-)-2,4-二硝基苯和Fe 3+(可使用水溶性三价铁盐，如FeCl 3.6H 2O)分散于水溶液中，调节溶液pH值至弱酸性；
(2)在剧烈搅拌下加入不良溶剂诱导纳米颗粒的形成，继续搅拌直至颗粒生长完成、离心、洗涤得到Fe 3+-NO供体混价配位聚合物纳米颗粒(Fe 3+-BPDB-NCP)；
(3)将Fe 3+-BPDB-NCP与Fe 2+(可使用水溶性二价铁盐，如FeCl 2.4H 2O)在水溶液中进行交换，交换达到平衡后，离心、洗涤、收集产品，即可得到Fe 3+/2+-NO供体混价配位聚合物纳米颗粒(Fe 3+/2+-BPDB-NCP)。优选的，所述NO供体和Fe 3+的摩尔比为1:15～1:10，优选为2:15。
上述不良溶剂选自无水甲醇、无水乙醇等醇类溶剂中的一种或几种，优选无水甲醇。优选的，所述Fe 3+-BPDB-NCP与Fe 2+摩尔比为2:1～1:2，优选为1:1。
本发明所述NO供体可采用发表于Journal of Medicinal Chemistry杂志题为Aryl Bis(diazeniumdiolates):Potent Inducers of S-Glutathionylation of Cellular Proteins and Their in Vitro Antiproliferative Activities的文献中公开的方法制备而成。反应路线如图1A所示。本发明的另一目的在于提供所述的Fe 3+/2+-NO供体混价配位聚合物在制备抗肿瘤药物中的应用。进一步的，所述Fe 3+/2+-NO供体混价配位聚合物可以作为前药受到肿瘤微环境高浓度GSH和H 2O 2响应分别释放NO和产生ROS。
本发明的另一目的在于提供一种药物制剂，含有本发明所述的Fe 3+/2+-NO供体混价配位聚合物。
本发明优点：
(1)本发明所述的Fe 3+/2+-NO供体混价配位聚合物通过实体瘤的高通透性和滞留效应(EPR效应)被动靶向肿瘤；在肿瘤微环境高浓度GSH和高浓度H 2O 2响应下，释放NO以及产生ROS，实现肿瘤的高效、低毒、协同治疗。
(2)本发明研究中发现，直接使用NO供体和Fe 2+制备Fe 2+-BPDB配位聚合物无法制成纳米颗粒，因此巧妙地先将NO供体和Fe 3+配位形成络合物，进一步与Fe 2+经离子交换后制得Fe 3+/2+-NO供体混价配位纳米颗粒。制得的纳米颗粒相对于单独的NO供体具有更高的水溶性和稳定性，适合应用于体内治疗。
附图说明
图1(A)为本发明中NO供体的合成路线图；(B)为本发明所述Fe 3+/2+-NO供体混价配位聚合纳米颗粒的合成路线图。
图2为本发明实施例1中各配位聚合纳米颗粒的(A)TEM，(B)EDS和(C)XPS图。图3为本发明实施例2中Fe 2+-BPDB配位聚合物的DLS表征结果。
图4为本发明实施1制得的Fe 3+/2+-BPDB-NCP和NO供体的水溶性(A)和稳定性(B)考察结果。
图5为本发明实施例5中各配位聚合纳米颗粒体外(A)GSH浓度与NO释放的关系曲线，(B)羟自由基和超氧阴离子产生的荧光曲线，(C)活性氧簇产生的荧光曲线和(D)H 2O 2浓度与活性氧簇产生的关系曲线。
图6为本发明实施例6中各配位聚合纳米颗粒HepG2细胞内(A)NO释放的共聚焦成像图和(B)ROS产生的共聚焦成像图。
图7为本发明实施例7中(a)生理盐水，(b)NO供体，(c)Fe 3+/2+-BPDB-NCP纳米颗粒制剂的药效学结果：(A)相对肿瘤体积，(B)小鼠体重，(C)生存期和(D)肿瘤重量。
具体实施方式
本发明构建了一种Fe 3+/2+-NO供体混价配位聚合纳米颗粒，其水溶性和体内稳定性优于单独NO供体，更适合应用于体内抗肿瘤治疗。并对其体外表征，体外NO的释放和ROS的产生，细胞水平NO的释放和ROS的产生以及动物药效学进行了研究。
实施例1 Fe 3+/2+-NO供体混价配位聚合纳米颗粒(Fe 3+/2+-BPDB-NCP)的构建
一、NO供体1,5-二(L-脯氨酸偶氮翁-O 2-)-2,4-二硝基苯的合成，合成路线如图1A所示。
(a)
Figure PCTCN2019079877-appb-000002
：将L-(+)-脯氨醇溶于无水乙醚，加入NaOMe/MeOH，置于高压反应釜中，通入氮气(N 2)置换空气，再通入NO气体，将反应釜密封反应36h。反应结束后，向反应液中加入无水乙醚，搅拌析出白色固体，抽滤，用无水乙醚洗涤，干燥得到L-(+)-脯氨醇偶氮鎓钠盐，即化合物2。
(b)
Figure PCTCN2019079877-appb-000003
：称取一定量化合物2溶于H 2O中，反应置于冰浴中搅拌，在N 2保护下，缓缓地加入1,5-二氟-2,4-二硝基苯的四氢呋喃溶液，保温0.5h，加入叔丁醇，继续保温0.5h。而后将反应移至室温，搅拌过夜。待反应结束后，向反应液中加入5％的碳酸氢钠水溶液，出现黄色浑浊，过滤收集固体，干燥，进一步柱层析得到浅黄色固体，即化合物3。
(c)
Figure PCTCN2019079877-appb-000004
：将化合物3溶于丙酮，加入高碘酸钠(NaIO 4)水溶液，搅拌至澄清。将反应瓶置于冰浴中，缓慢加入三氯化钌(RuCl 3)，而后移至室温反应4h。反应结束后，过滤、浓缩、洗涤得到淡黄色固体，即为目标化合物4。
二、Fe 3+/2+-NO供体混价配位聚合纳米颗粒(Fe 3+/2+-BPDB-NCP)的制备
分别采用不良溶剂法、溶剂热法对Fe 3+/2+-NO供体混价配位聚合纳米颗粒的制备进行了研究。
1.不良溶剂法
Fe 3+/2+-BPDB-NCP的合成路线如图1B所示：
将NO供体1,5-二(L-脯氨酸偶氮翁-O 2-)-2,4-二硝基苯(BPDB)和FeCl 3.6H 2O以摩尔比为2:15分散于水溶液中，并用稀NaOH溶液调节pH值至5.5。然后，在剧烈搅拌下加入无水甲醇诱导纳米颗粒的形成，继续搅拌一段时间，直至颗粒生长完成。离心、洗涤即可得到Fe 3+-BPDB-NCP。该方法可通过调节加入无水甲醇的速度，调控合成不同尺寸的纳米颗粒。
基于离子交换的方法，将Fe 3+-BPDB-NCP纳米颗粒与等摩尔比的FeCl 2.4H 2O在水溶液中进行交换，交换达到平衡后，离心、洗涤、收集产品，即可得到Fe 3+/2+-BPDB-NCP。
2.溶剂热法
将NO供体1,5-二(L-脯氨酸偶氮翁-O 2-)-2,4-二硝基苯(BPDB)和FeCl 3.6H 2O以摩 尔比为2:15分散于DMF中，然后加热至90℃保持8h。实验过程中发现，加热过程中NO供体受热分解产生了NO，不能有效形成所需的配位聚合物。上述实验说明，NO供体合成配位聚合物需要温和的反应条件，如本发明中的不良溶剂法。
实施例2 Fe 2+-BPDB配位聚合物的制备
将NO供体1,5-二(L-脯氨酸偶氮翁-O 2-)-2,4-二硝基苯(BPDB)和FeCl 2.4H 2O以摩尔比为2:15分散于水溶液中，并用稀NaOH溶液调节pH值至5.5。然后，在剧烈搅拌下加入无水甲醇诱导Fe 2+-BPDB颗粒的形成，继续搅拌一段时间，直至颗粒生长完成。离心、洗涤得到Fe 2+-BPDB颗粒。重复三次，三次样品的DLS表征结果如图2所示。结果表明Fe 2+-BPDB的水合粒径大于1μm，无法进一步制备得到所需纳米级的颗粒。
DLS表征：
取新鲜制备的Fe 2+-BPDB配位聚合物200μL，蒸馏水稀释至3mL，在粒径电位仪上对脂质体制剂进行其水合粒径的测定。
实施例3 Fe 3+/2+-NO供体混价配位聚合物纳米颗粒(Fe 3+/2+-BPDB-NCP)的表征
TEM和EDS测定：
取实施例1不良溶剂法新鲜制备的Fe 3+-BPDB-NCP和Fe 3+/2+-BPDB-NCP，稀释一定倍数后滴到铜网上，待自然风干后，于透射电子显微镜上表征材料的形貌、尺寸(TEM)以及元素组成(EDS)。
XPS测定：
制备好的Fe 3+-BPDB-NCP和Fe 3+/2+-BPDB-NCP冷冻干燥后，收集粉末，用X射线光电子能谱分析仪表征材料表面铁离子，并通过软件分峰计算得到Fe 3+和Fe 2+的比例。图3为本发明实施例1制得的各配位聚合纳米颗粒的(A)TEM，(B)EDS和(C)XPS图。表明成功合成了以NO供体BPDB为配体，以Fe 3+/2+混价为金属配位离子的配位聚合纳米颗粒。
根据上述TEM，EDS和XPS结果，计算出Fe 3+/2+-NO供体混价配位聚合物的分子式为Fe x+ 4(BPDB)(H 2O) 2，Fe x+为Fe 3+和Fe 2+，Fe 3+和Fe 2+比例为2.3:1.7。
实施例4 NO供体和Fe 3+/2+-NO供体混价配位聚合纳米颗粒(Fe 3+/2+-BPDB-NCP)的水溶性和稳定性考察
水溶性：将NO供体1,5-二(L-脯氨酸偶氮翁-O 2-)-2,4-二硝基苯(BPDB)分别分散 于无水甲醇和水中，Fe 3+/2+-BPDB-NCP分散于水中，制备得到1mg/mL的溶液。通过拍照记录它们在各溶剂中的分散性和溶解性。如图4A所示，BPDB只能溶解于甲醇溶液中，在水中分散性差，而Fe 3+/2+-BPDB-NCP能很好的溶解在水中。
稳定性：15mg/kg的BPDB和Fe 3+/2+-BPDB-NCP通过尾静脉注射入大鼠内体，通过血压计测量大鼠尾巴的舒张压来表征NO在体内循环中的稳定性，如图4B所示。其结果表明Fe 3+/2+-BPDB-NCP在体内有较高的稳定性，而单独的NO供体BPDB体内稳定性差，在未到达肿瘤部位就开始释放NO了。
实施例5 Fe 3+/2+-NO供体混价配位聚合物纳米颗粒(Fe 3+/2+-BPDB-NCP)的体外释放行为研究
本发明的Fe 3+/2+-BPDB-NCP体外NO释放和活性氧簇产生的检测方法如下：
一方面，由于多功能NO供体是GSH响应的，因而配位聚合物纳米颗粒释放NO也是GSH触发的。将Fe 3+/2+-BPDB-NCP与不同浓度的GSH共孵育2h，用Griess试剂检测NO的释放量。另一方面，NO供体配位聚合物纳米颗粒中Fe 2+是有效的Fenton试剂，能够催化H 2O 2产生活性氧簇包括羟自由基、超氧阴离子。将Fe 3+/2+-BPDB-NCP与H 2O 2共孵育30min，分别用对苯二甲酸、二氢乙锭和DCFH-DA检测 .OH、 .O 2 -和ROS的产生。结果如图5所示，表明本发明合成的Fe 3+/2+-NO供体混价配位聚合纳米颗粒在GSH和H 2O 2响应下能分别释放出NO和产生ROS。
实施例6 Fe 3+/2+-NO供体混价配位聚合物纳米颗粒(Fe 3+/2+-BPDB-NCP)的细胞成像研究
(a)NO成像：将HepG2细胞接种于共聚焦培养皿内，接种密度为1×10 5个/孔，并在5％CO 2无菌培养箱(37℃，饱和湿度)内孵育过夜。24h后，将原培养液吸出，加入无血清含有5μM DAF-FM DA NO检测探针的DMEM，于培养箱中培育20min。将DMEM弃掉，用PBS溶液洗涤3次，分别加入5％血清-DMEM溶液、含有20μg/mL BPDB的5％血清-DMEM溶液和20μg/mL Fe 3+/2+-BPDB-NCP的5％血清-DMEM溶液。继续孵育3h，弃掉上清用PBS洗涤3次，加入1μg/mL Hoechst 33342进行核染。核染15min，PBS洗涤3次后用4％的多聚甲醛固定细胞用于共聚焦成像观察。
(b)ROS成像：将HepG2细胞接种于共聚焦培养皿内，接种密度为1×10 5个/孔，并在5％CO 2无菌培养箱(37℃，饱和湿度)内孵育过夜。24h后，将原培养液吸出， 加入无血清含有10μM DCFH-DA ROS检测探针的DMEM，于培养箱中培育20min。将DMEM弃掉，用PBS溶液洗涤3次，分别加入5％血清-DMEM溶液、含有20μg/mL Fe 3+-BPDB-NCP的5％血清-DMEM溶液和20μg/mL Fe 3+/2+-BPDB-NCP的5％血清-DMEM溶液。继续孵育3h，弃掉上清用PBS洗涤3次，加入1μg/mL Hoechst 33342进行核染。核染15min，PBS洗涤3次后用4％的多聚甲醛固定细胞用于共聚焦成像观察。
结果如图6所示，表明本发明合成的Fe 3+/2+-NO供体混价配位聚合纳米颗粒在细胞水平上能有效释放NO和产生ROS，且释放NO的量较单独NO供体BPDB多，产生ROS的量也多于Fe 3+-BPDB-NCP，为体内NO-CDT协同治疗提供依据。
实施例7 Fe 3+/2+-NO供体混价配位聚合物纳米颗粒(Fe 3+/2+-BPDB-NCP)的药效学研究
对健康ICR小鼠进行腋下植瘤，每只小鼠注射Heps瘤溶液(1×10 7个/mL)100μL，当瘤体积至约100～200mm 3时，将移植瘤小鼠随机分成3组(n＝8)，将给药的第一天设为第0天，则分别于第0、2、4、6、8、10天注射生理盐水、NO供体和Fe 3+/2+-BPDB-NCP制剂，剂量均为15mg/kg。同时隔天记录小鼠的体重和肿瘤的大小，最后一次给药后，将小鼠处死，剥离肿瘤，并称重。瘤体积的计算方法采用以下公式：Volume＝(W 2×L)/2其中W为肿瘤最短的半径，L为肿瘤最长半径。
结果如图7所示，证明本发明合成的Fe 3+/2+-NO供体混价配位聚合纳米颗粒在老鼠体内的抗肿瘤治疗效果明显优于单独的NO供体BPDB，且没有明显的毒副作用。

Claims (10)

  1. 一种Fe 3+/2+-NO供体混价配位聚合物，其特征在于NO供体1,5-二(L-脯氨酸偶氮翁-O 2-)-2,4-二硝基苯与Fe 3+和Fe 2+形成混价配位聚合物。
  2. 如权利要求1所述的Fe 3+/2+-NO供体混价配位聚合物，其特征在于分子式为Fe x+ 4(BPDB)(H 2O) 2，所述Fe x+代表Fe 3+和Fe 2+，BPDB为NO供体1,5-二(L-脯氨酸偶氮翁-O 2-)-2,4-二硝基苯。
  3. 如权利要求1或2所述的Fe 3+/2+-NO供体混价配位聚合物，其特征在于所述配位聚合物为纳米颗粒。
  4. 如权利要求1或2所述的Fe 3+/2+-NO供体混价配位聚合物的制备方法，其特征在于所述NO供体和Fe 3+配位形成聚合物，再进一步与Fe 2+经离子交换制得。
  5. 如权利要求3所述的Fe 3+/2+-NO供体混价配位聚合物的制备方法，其特征在于包括如下步骤：
    (1)将NO供体1,5-二(L-脯氨酸偶氮翁-O 2-)-2,4-二硝基苯和Fe 3+分散于水溶液中，调节溶液pH值至弱酸性；
    (2)在剧烈搅拌下加入不良溶剂诱导纳米颗粒的形成，继续搅拌直至颗粒生长完成、离心、洗涤得到Fe 3+-NO供体混价配位聚合物纳米颗粒；
    (3)将Fe 3+-NO供体混价配位聚合物纳米颗粒与Fe 2+在水溶液中进行交换，交换达到平衡后，离心、洗涤、收集产品，即可得到Fe 3+/2+-NO供体混价配位聚合物纳米颗粒。
  6. 如权利要求5所述的方法，其特征在于所述NO供体和Fe 3+的摩尔比为1:15～1:10。
  7. 如权利要求5所述方法，其特征在于所述不良溶剂为无水醇类溶剂。
  8. 如权利要求5所述的方法，其特征在于Fe 3+-NO供体混价配位聚合物纳米颗粒与Fe 2+摩尔比为2:1～1:2。
  9. 如权利要求1-3任一项所述的Fe 3+/2+-NO供体混价配位聚合物在制备抗肿瘤药物中的应用。
  10. 如权利要求8所述的应用，其特征在于所述Fe 3+/2+-NO供体混价配位聚合物作为前药受到肿瘤微环境高浓度GSH和H 2O 2响应分别释放NO和产生ROS。
PCT/CN2019/079877 2018-09-20 2019-03-27 一种Fe 3+/2+-NO供体混价配位聚合物及其应用 WO2020057086A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811102995.7 2018-09-20
CN201811102995.7A CN109369923B (zh) 2018-09-20 2018-09-20 一种Fe3+/2+-NO供体混价配位聚合物及其应用

Publications (1)

Publication Number Publication Date
WO2020057086A1 true WO2020057086A1 (zh) 2020-03-26

Family

ID=65401610

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/079877 WO2020057086A1 (zh) 2018-09-20 2019-03-27 一种Fe 3+/2+-NO供体混价配位聚合物及其应用

Country Status (2)

Country Link
CN (1) CN109369923B (zh)
WO (1) WO2020057086A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109369923B (zh) * 2018-09-20 2020-06-02 中国药科大学 一种Fe3+/2+-NO供体混价配位聚合物及其应用
CN112516308B (zh) * 2020-11-19 2023-01-17 暨南大学 一种近红外ii区激光控释药物纳米脂质体及其制备方法与应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105622607A (zh) * 2016-01-12 2016-06-01 沈阳药科大学 一类具有抗肿瘤活性的呋咱类no供体型吴茱萸碱衍生物
CN109369923A (zh) * 2018-09-20 2019-02-22 中国药科大学 一种Fe3+/2+-NO供体混价配位聚合物及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105030674A (zh) * 2015-07-17 2015-11-11 中国药科大学 子母弹型抗肿瘤金纳米结合物杂合脂质体制备方法和应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105622607A (zh) * 2016-01-12 2016-06-01 沈阳药科大学 一类具有抗肿瘤活性的呋咱类no供体型吴茱萸碱衍生物
CN109369923A (zh) * 2018-09-20 2019-02-22 中国药科大学 一种Fe3+/2+-NO供体混价配位聚合物及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANDREI, DANIELA ET AL.: "Aryl Bis(diazeniumdillates):Potent Inducers of S-Glutathionylation of Cellular Proteins and Their in Vitro Antiproliferative Activities", J. MED. CHEM, vol. 51, no. 24, 18 November 2008 (2008-11-18), pages 7944 - 7952, XP055696816, DOI: 10.1021/jm800831y *
WILLIAM J. RIETER ET AL.: "Nanoscale coordination polymers for platinium-based anticancer drug dilivery", J. AM. CHEM. SOC, vol. 130, 8 August 2008 (2008-08-08), pages 11584 - 11585, XP055164871 *

Also Published As

Publication number Publication date
CN109369923B (zh) 2020-06-02
CN109369923A (zh) 2019-02-22

Similar Documents

Publication Publication Date Title
Della Rocca et al. Polysilsesquioxane nanoparticles for triggered release of cisplatin and effective cancer chemoradiotherapy
Liu et al. Photosensitizer loaded PEG-MoS 2–Au hybrids for CT/NIRF imaging-guided stepwise photothermal and photodynamic therapy
Pan et al. A combination of glioma in vivo imaging and in vivo drug delivery by metal–organic framework based composite nanoparticles
Li et al. Core-satellite metal-organic framework@ upconversion nanoparticle superstructures via electrostatic self-assembly for efficient photodynamic theranostics
CN110787146B (zh) 氧化还原响应性肿瘤靶向顺铂纳米递药系统的制备方法及其应用
Cao et al. Surface PEGylation of MIL-101 (Fe) nanoparticles for co-delivery of radioprotective agents
WO2022067885A1 (zh) 掺杂型二氧化钛在制备声敏剂中的应用
WO2020057086A1 (zh) 一种Fe 3+/2+-NO供体混价配位聚合物及其应用
CN108727353A (zh) 联合光热治疗和化疗的ir820-ptx两亲性小分子前药及其纳米粒制备方法和应用
Pang et al. Aptamer modified MoS2 nanosheets application in targeted photothermal therapy for breast cancer
Xie et al. Modification of magnetic molybdenum disulfide by chitosan/carboxymethylcellulose with enhanced dispersibility for targeted photothermal-/chemotherapy of cancer
Yang et al. Ferrocene-based multifunctional nanoparticles for combined chemo/chemodynamic/photothermal therapy
CN111298140B (zh) 还原响应的t1/t2切换型mri造影剂、其制备方法及应用
Yu et al. Nanozyme-nanoclusters in metal–organic framework: GSH triggered Fenton reaction for imaging guided synergistic chemodynamic-photothermal therapy
Cao et al. Ultrasound-assisted continuous-flow synthesis of PEGylated MIL-101 (Cr) nanoparticles for hematopoietic radioprotection
CN110917349B (zh) 一种碗状isp复合功能性纳米粒子及其制备方法和应用
CN113750252A (zh) 一种钴掺杂的金属有机框架的纳米颗粒的制备方法及其应用
CN111848658A (zh) 一种靶向线粒体的氟硼二吡咯类化合物及其脂质体包裹纳米粒子的制备方法和用途
Wu et al. Engineering phosphopeptide-decorated magnetic nanoparticles as efficient photothermal agents for solid tumor therapy
CN117085147B (zh) 一种负载阿霉素的碳基给药系统及其制备方法和应用
CN116271097B (zh) 一种基于金属有机骨架的工程化外泌体及其制备方法和应用
CN114767868B (zh) Cox-2抑制剂与化疗药物在制备抗肿瘤药物中的应用
CN113368235B (zh) 一种Gd-NGQDs/BTS@PLGA-PEG纳米材料及其制备方法和应用
CN118203671A (zh) 一种基于电荷组装并具有响应性的纳米药物组合物及其制备方法和应用
Jiang et al. A metal–organic framework complex for enhancing tumor treatments through synergistic effect of chemotherapy and photodynamic therapy

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: 19862794

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: 19862794

Country of ref document: EP

Kind code of ref document: A1