WO2023202092A1 - 一种响应释放一氧化碳的纳米颗粒及其制备方法和应用 - Google Patents

一种响应释放一氧化碳的纳米颗粒及其制备方法和应用 Download PDF

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WO2023202092A1
WO2023202092A1 PCT/CN2022/137412 CN2022137412W WO2023202092A1 WO 2023202092 A1 WO2023202092 A1 WO 2023202092A1 CN 2022137412 W CN2022137412 W CN 2022137412W WO 2023202092 A1 WO2023202092 A1 WO 2023202092A1
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carbonyl
thiol
preparation
nanoparticle
nanoparticles
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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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Definitions

  • the present invention relates to the field of nanomedicine, and in particular to a nanoparticle that responds to the release of carbon monoxide and its preparation method and application.
  • Hyperthermia is a treatment strategy that works by raising the temperature of the area of the body affected by cancer.
  • Photothermal therapy is an optical version of hyperthermia that uses photothermal converting agents to convert light energy into heat to ablate cancer cells.
  • PTT Photothermal therapy
  • various photothermal agents with strong near-infrared absorption capabilities and high conversion efficiency have been developed.
  • Halas combines laser-excited gold-silicon nanoshells (GSN) with magnetic resonance-ultrasound fusion imaging for local excision of low- and intermediate-grade tumors within the prostate.
  • Cai Lintao's team created doxorubicin (DOX) and indocyanine green (ICG) nanoparticles (DINPs), which can simultaneously deliver DOX and ICG to the tumor area for combined chemotherapy and photothermal therapy.
  • DOX doxorubicin
  • ICG indocyanine green
  • Liu Wei's research group has successfully used the preparation of albumin-bound fluorophore nanoparticles for intraoperative NIR-II fluorescence imaging of orthotopic mouse colon tumors and metastatic lesions. Under the guidance of NIR-II fluorescence imaging, the optimized PTT can completely cure colon cancer mice.
  • high-temperature PTT poses an inevitable threat to surrounding healthy tissues and may induce undesirable inflammation due to the difficulty in preventing heat diffusion.
  • high-temperature thermal ablation can produce some adverse biological effects in cancer treatment.
  • low-temperature PTT In order to overcome these bottlenecks, low-temperature PTT was proposed, which preferentially eliminates tumors and promotes wound healing at low temperatures with little harm to normal tissues. Extensive studies have shown that cryogenic PTT can achieve excellent therapeutic properties, enabling practical biomedical applications. Although there are different temperature thresholds for hypothermic PTT, it is recognized that most hypothermic thermotherapy is performed at temperatures below 48°C. It is generally believed that the efficacy of low-temperature hyperthermia therapy may be severely affected due to the increased expression of heat shock proteins (HSPs), which can repair thermal damage and protect cells from apoptosis through multiple pathways, leading to tumor heat tolerance. stimulation related pathways.
  • HSPs heat shock proteins
  • HSPs mild PTT proteins
  • HSP-70siRNA Introduction of HSP-70siRNA at the upstream node can also block the effect of HSP.
  • Zhu Hailiang's team introduced Hsp-70siRNA to block the influence of upstream node Hsps and avoid the side effects of traditional HSPs inhibitors.
  • gas therapy utilizes therapeutic/therapeutic auxiliary gases or their prodrugs to inhibit the proliferation and metastasis of cancer cells.
  • CO carbon monoxide
  • the normal body produces CO under increased pressure through the expression of the heme oxygenase-1 (HO-1) gene.
  • HO-1 heme oxygenase-1
  • this gene is ineffective 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.
  • Gas therapy has been used previously in the treatment of cancer.
  • He Qianjun's team developed a multi-level assembly/disassembly strategy by encapsulating mitochondria-targeted and intramitochondrial microenvironment-responsive prodrugs within mesoporous silica nanoparticles, and further encapsulated them through stepwise electrostatic assembly.
  • a new smart nanomedicine can be constructed to achieve tumor tissue-cell-mitochondria-targeted multi-level delivery and controlled release of CO in a step-by-step decomposition manner.
  • gas therapy inhibiting heat shock proteins are no reports.
  • PTT photothermal therapy
  • Hypothermia therapy (below 43°C) can only temporarily inhibit tumor growth, but cannot completely ablate it. Therefore, during PTT, the tissue temperature needs to exceed 50°C to ensure complete tumor death.
  • high-temperature hyperthermia not only destroys cancer cells, but also destroys normal tissues around the lesions through heat diffusion, while the damage caused by low-temperature light and heat to cancer cells is easily repaired by stress-induced heat shock proteins (HSPs). Therefore, how to simultaneously regulate the inhibitory level of heat shock proteins in tumor tissues and provide materials that can provide hypothermic treatment has become a hot topic in PTT research. Based on this, it is imperative to develop a simple nanomaterial with photothermal conversion and heat shock protein elimination capabilities as a therapeutic agent for low-temperature PTT.
  • the present invention proposes a nanoparticle that can release CO in response to overexpressed hydrogen peroxide in the cancer environment and has high photothermal conversion efficiency and second-zone fluorescence conversion efficiency. Preparation methods and applications thereof.
  • the present invention provides a nanoparticle that responds to the release of carbon monoxide, including an outer shell and a core.
  • the outer shell includes a carbonyl iron compound and a thiol end-group polymer.
  • the carbonyl iron compound and the thiol end-group polymer are formulated by Position reaction connection; the core is a photothermal conversion agent.
  • 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 1,000 to 8,000. 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 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.
  • 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 photothermal conversion agent is any one of Bodipy type, heptamethocyanine type, AIE type or polymer type two-zone fluorescein.
  • the Bodipy class is selected from 1,3,5,7-tetramethyl-8-phenyl-4,4-difluorodiazetidine, difluoro ⁇ 2-[1-(3,5-dimethyl Base-2H-pyrrole-2-ylidene-N)ethyl]-3,5-dimethyl-1H-pyrrolo-N ⁇ boron, Fmoc-Trp-BODIPY, WinterGreen carbamoyl imidazole photocage, etc.
  • the heptamethocyanine is selected from Cy5, Cy5.5, Cy7, IR-780, IR-820, etc.
  • the AIE type is selected from MCH-PPV, PBPTB, TPA, TPE, etc.
  • the polymeric second-zone fluorescein is selected from PBPTV, PBTV, PCFDP, etc.
  • the invention also provides a method for preparing the nanoparticles, which includes the following steps:
  • Carbonyl iron compounds and thiol-terminated polymers are dissolved in tetrahydrofuran and stirred under nitrogen flow; at the end of the reaction, the solution changes from dark blue to brown; cool to room temperature, add liquid alkane to obtain a brown precipitate, use After washing with organic solvent and drying, the carbonyl siderophore is obtained;
  • step (3) Dissolve the carbonyl siderophore and photothermal conversion agent purified in step (2) in tetrahydrofuran. After sonication, add deionized water, blow out the tetrahydrofuran with nitrogen, and co-precipitate; centrifuge with an ultrafiltration tube and wash repeatedly to co-precipitate to form stable and uniform nanoparticles.
  • the mass ratio of the carbonyl iron compound and the thiol-terminated polymer in step (1) is 1: (4-8).
  • the mass ratio of the purified carbonyl siderophore and the photothermal conversion agent described in step (3) is (5-15):1.
  • the nitrogen flow temperature in step (1) is 50-120°C, and the stirring time is 1-12 hours.
  • the present invention also provides a method for inhibiting heat shock proteins, including using the nanoparticles.
  • Heat shock protein is a protective protein. Inhibiting the heat shock protein in some microorganisms or bacteria can also kill them.
  • the research field is to conduct physiological research by inhibiting heat shock proteins. For example, by inhibiting heat shock proteins, we can study the stress response of various tissue parts such as the brain and heart to external stimuli (high temperature, cold, electrical stimulation, gas, etc.). Therefore, the activation or inhibition state of heat shock proteins is not directly related to disease.
  • the invention also provides the use of the nanoparticles in preparing anti-tumor drugs.
  • the preparation method of the present invention is simple and easy to implement, and can be successfully prepared in only two steps, which is convenient for operation and promotion.
  • the nanoparticles of the present invention While killing tumor tissue, the nanoparticles of the present invention have very low toxicity to normal tissue.
  • the nanoparticles prepared by the present invention can exist stably in a normal environment, and no sedimentation or flocculation occurs after 7 days.
  • the present invention proposes for the first time a way to use carbon monoxide gas molecules to inhibit heat shock proteins and thereby improve the effect of low-temperature photothermal therapy.
  • Figure 1 is a flow chart for the preparation of PBCO in Example 1 of the present invention.
  • Figure 2 is the UV spectrum of PBCO in Example 2 of the present invention.
  • Figure 3 shows the particle size of PBCO under the hydrated particle size analyzer in Example 2 of the present invention
  • Figure 4 shows the photothermal conversion efficiency of PBCO in Example 3 of the present invention
  • Figure 5 shows the CCK-8 experiment in Example 4 of the present invention demonstrating the cytotoxicity of PBCO to cancer cells
  • Figure 6 shows the therapeutic effect of PBCO on orthotopic breast cancer in Example 5 of the present invention
  • Figure 7 shows the results of PBCO inhibiting the expression of HSP in tumors in Example 6 of the present invention.
  • the present invention first 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. 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.
  • the photothermal conversion agent (PB) and the polymer carrier PG-CO were self-assembled to construct a nanoparticle that responds to the release of carbon monoxide. It not only has near-infrared dual-region (NIR-II) fluorescence and high photothermal efficiency, but also can selectively release CO in response to overexpressed hydrogen peroxide in the tumor microenvironment.
  • the released CO can not only inhibit the proliferation and metastasis of cancer cells, but also effectively inhibit the expression of heat shock proteins (HSPs), significantly reduce the levels of heat shock proteins, destroy tumor thermal resistance during mild PTT, and induce tumor cell apoptosis.
  • HSPs heat shock proteins
  • the efficacy of low-temperature PPT is greatly improved.
  • the nanoparticles of the present invention provide an effective treatment method for low-temperature PTT. This is the first technology that uses CO gas to inhibit HSPs expressed during PTT treatment, thereby improving the therapeutic effect.
  • Carbonyl iron compounds and thiol-terminated polymers are dissolved in tetrahydrofuran and stirred under nitrogen flow; at the end of the reaction, the solution changes from dark blue to brown; cool to room temperature, add liquid alkane to obtain a brown precipitate, use The carbonyl siderophore is obtained after washing with organic solvent and drying.
  • step (3) Dissolve the carbonyl siderophore and photothermal conversion agent purified in step (2) in tetrahydrofuran. After sonication, add deionized water, blow out the tetrahydrofuran with nitrogen, and co-precipitate; centrifuge with an ultrafiltration tube and wash repeatedly to co-precipitate to form stable and uniform nanoparticles.
  • 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.
  • the photothermal conversion agent is any one of Bodipy type, heptamethocyanine type, AIE type or polymer type second zone fluorescein.
  • the following examples take PBPTV dye as an example.
  • Other above-mentioned photothermal conversion agents all have similar physical and chemical properties. Therefore, those skilled in the art can know that the technical solution of the present invention can be achieved using other above-mentioned photothermal conversion agents.
  • 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).
  • Responsive amphiphilic polymer carrier PG-CO Responsive amphiphilic polymer carrier PG-CO.
  • the specific preparation process is as follows. Iron dodecacarbonyl (5-100mg) and mPEG-SH (M.W. ⁇ 2000) (100-600mg) are dissolved in tetrahydrofuran (THF), and stirred at 50-120°C under nitrogen flow for 1- 12h. At the end of the reaction, the solution changed from dark blue to brown.
  • PG-CO is a brown solid that is soluble in water and organic solutions.
  • mPEG2000-SH can be replaced by mPEG5000-SH, mPEG8000-SH, SH-mPEG2000-SH or SH-mPEG8000-SH, etc.
  • 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, PBPTV, Bodipy, etc.
  • Dissolve the nanoparticles in pure water to a final concentration of 5-10mM Take 0.5-1mL in a cuvette and measure the average particle size with a DLS particle sizer. The results are shown in Figure 3.
  • the average particle size of the nanoparticles is about 158nm.
  • Dissolve the nanoparticles in pure water to a final concentration of 50-100mM Take 0.5-1mL in a centrifuge tube and irradiate it with an 808nm laser probe for 3 minutes. At the same time, use an infrared camera to detect the temperature change of the nanoparticle solution. After 3 minutes, turn off the laser probe, wait for it to cool naturally, and record its temperature changes at the same time. The results are shown in Figure 4. It can be seen that the solution heated up very quickly in the first 2.5 minutes, and the time required for heating was basically the same as the cooling time.
  • a 96-well plate to culture 4T1 breast cancer cells.
  • the outer ring is PBS.
  • Each row of 6 wells in the middle of the well plate is a group.
  • 10-20 ⁇ L of PBCO solution or PBPTV dye of different concentrations is added to the culture wells.
  • the concentrations are respectively It is 0,10-20,30-40,40-60,60-80,80-100 ⁇ g/mL.
  • MTT dye and use a microplate reader to detect the cell survival rate in the well plate as the first set of data.
  • the well plate was irradiated with an 808nm laser for 1-2 minutes, and the cell survival rate was also measured for 24 hours, which was used as the fourth, fifth, and sixth sets of data in sequence.
  • the abscissa is concentration, ranging from 0 to 100 from left to right, and the different colored columns in each concentration from left to right are PBCO, PBPTV, nanoparticles, PBCO plus light, PBPTV plus light, and nanoparticles. Add light. It can be seen from the figure that the dye PBPTV has no inhibitory effect on the growth of cancer cells regardless of its concentration, while the inhibitory effect of PBCO and nanoparticles on cancer cells increases as the concentration increases. At the same concentration, the inhibitory effects on cancer cells from large to small are nanoparticles plus light, carrier PBCO plus light, carrier PBCO, and nanoparticles.
  • Example 5 Therapeutic effect of nanoparticles of the present invention on in situ breast cancer tumors
  • mice Two groups of 4T1 tumor-bearing BALB/c mice were taken, with 5 mice in each group. One group of mice was only injected with 0.5-1mL of nanoparticle solution with a concentration of 20-30mM, and the other group was injected with 0.5-1mL of nanoparticle solution with a concentration of 20-30mM. , 12 hours later, the tumor site of each mouse was irradiated with 808nm NIR for 1-2 minutes. The tumor volume of the mice was recorded every 2 days thereafter until the mice were sacrificed on the 18th day. The results of tumor volume changes are shown in Figure 6. The tumor volume of mice injected with PBCO alone was inhibited but still growing, while the tumor volume of mice injected with PBCO and then treated with photothermal treatment had almost no change, which played a significant role in tumor growth. inhibitory effect.
  • Example 6 Inhibitory effect of the PBCO carrier of the present invention on heat shock proteins in tumors
  • 4T1 tumor-bearing BALB/c mice were divided into eight groups, with five mice in each group. After 12 hours, the tumor area was irradiated with 808nm NIR. The mouse tumor volume and weight were recorded every 2 days. The mice were sacrificed on day 18, and major organs and tumors were harvested for histological examination.
  • the tumors of five tumor-seeking mice at the same time were treated differently: 0.5-1 mL PBS was injected; 0.5-1 mL PBS was injected and maintained at 45°C for 5-10 min; 808nm laser with a power of 4-5W was irradiated for 3-5 min; 0.5-1 mL concentration 20- 30mM PBCO solution; inject 0.5-1mL of 20-30mM PBCO solution and irradiate with an 808nm laser with a power of 4-5W for 3-5 minutes.
  • the mouse tumor tissue was taken out, immersed in formalin solution for 40-50 hours, and embedded in wax blocks. After making paraffin sections, they are routinely dewaxed to water for antigen thermal retrieval.
  • the nanoparticles of the present invention can specifically release CO in tumor tissues to inhibit cancer proliferation, have high photothermal conversion efficiency, can provide effective photothermal treatment effects locally, and have low toxicity to normal tissues, so The provided CO gas can significantly reduce the expression of heat shock proteins during low-temperature photothermal therapy, thereby improving the therapeutic effect.

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Abstract

本发明公开了一种响应释放一氧化碳的纳米颗粒及其制备方法和应用，所述纳米颗粒包括外壳和内核，所述外壳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；所述内核为光热转化剂。本发明的制备过程简便易行，便于操作推广。该纳米颗粒在杀伤肿瘤组织的同时，对正常组织毒性很低。本发明首次提出了利用一氧化碳气体分子抑制热休克蛋白，进而提高低温光热治疗效果的途径。

Description

一种响应释放一氧化碳的纳米颗粒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纳米颗粒及其制备方法和应用。
背景技术
热疗是一种通过提高身体受癌症影响区域的温度进行治疗的策略。光热疗法(PTT)是热疗的光学版本，它利用光热转换剂将光能转换为热能以消融癌细胞。随着材料科学技术的飞速发展，各种近红外吸收能力强、转换效率高的光热剂被开发出来。例如，Halas将激光激发的金硅纳米壳(GSN)与磁共振-超声融合成像相结合，用于局部切除前列腺内的中低级别肿瘤。蔡林涛的团队制造了阿霉素(DOX)和吲哚菁绿(ICG)纳米颗粒(DINPs)，它们可以同时将DOX和ICG输送到肿瘤区域进行联合化疗光热疗法。刘伟的研究小组利用白蛋白结合荧光团纳米粒的制备已成功用于原位小鼠结肠肿瘤和转移病灶的术中NIR-II荧光成像，在NIR-II荧光成像的指导下，经优化的PTT可完全治愈结肠癌小鼠。然而，高温PTT对周围的健康组织造成不可避免的威胁，并且由于难以阻止热扩散，可能诱发不良炎症。此外，在癌症治疗中，高温热消融会产生一些不良的生物学效应。
为了克服这些瓶颈，低温PTT被提出，它优先消除肿瘤，并在低温下促进伤口愈合，对正常组织的伤害不大。大量研究表明，低温PTT可以实现出色的治疗性能，从而实现实际的生物医学应用。虽然低温PTT有不同的温度阈值，人们认识到，大多数低温热疗都是在48℃以下的温度下进行的。人们普遍认为，由于热休克蛋白(HSPs)的表达升高，能够修复热损伤并通 过多种途径保护细胞免受凋亡，因此低温热疗的疗效可能会受到严重影响导致肿瘤热耐受的应激相关途径。
抑制HSPs在肿瘤中的表达一直是温和PTT的核心问题。在以前的研究中已经开发了一些HSPs抑制剂。一些小分子热休克蛋白抑制剂，如藤黄酸、雷公藤内酯醇、瑞替宁、STA-9090和17-AAG，已被设计为PCA，以增强肿瘤细胞中温和PTT蛋白(HSPs)的治疗效果，使其对热应激更敏感，提高癌细胞的PTT效率。在上游节点引入HSP-70siRNA也可以阻断HSP的作用。最近，朱海良的团队引入了Hsp-70siRNA，以阻断上游节点Hsps的影响，避免了传统HSPs抑制剂的副作用。刘军提出了一种创新策略，首次报道了基于单原子纳米酶(SAzyme)的铁下垂促进轻度PTT，使Pd-SAzyme介导的温和PTT成为可能。蔡林涛的团队开发了智能DC(IDC)，负载有光热剂(IR-797)并涂有成熟DC膜的纳米颗粒组成。IDC进入淋巴结并刺激T细胞，活化的T细胞降低了HSPs的表达。但溶解度差、急性细胞毒性和血清不稳定性使这些抑制剂难以在复杂的肿瘤生理环境中完全沉默HSPs。
近年来，气体疗法在癌症治疗中越来越受到重视。气体疗法利用治疗/治疗辅助气体或其前体药物来抑制癌细胞的增殖和转移。作为一种信号分子，血红蛋白降解过程中产生的一氧化碳(CO)可以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。气体疗法以前曾用于癌症的治疗。例如何前军的团队开发了一种多级组装/拆卸策略，通过将线粒体靶向和线粒体内微环境-响应性前药封装在介孔二氧化硅纳米颗粒内，并通过逐步静电组装进一步包覆透明质酸，从而构建一种新的智能纳米药物，以分步分解的方式实现肿瘤组织-细胞-线粒体-靶向多 级输送和CO的控制释放。然而，还没有关于气体疗法抑制热休克蛋白的报道。
综上，光热疗法(PTT)是一种很有前途的肿瘤治疗方法。低温治疗(低于43℃)只能暂时抑制肿瘤生长，但不能完全使其消融。因此，在PTT过程中，需要组织温度超过50℃，以确保肿瘤完全死亡。然而高温热疗不仅破坏了癌细胞，而且通过热扩散破坏病变周围的正常组织，而低温光热对癌细胞的损伤很容易被应激诱导的热休克蛋白(HSPs)修复。因此，如何同时调节肿瘤组织中热休克蛋白的抑制水平，并且可以提供低温治疗的材料成为PTT研究的热点。基于此，开发一种具有光热转换和热休克蛋白消除能力的简单纳米材料作为低温PTT的治疗剂势在必行。
发明内容
针对现有技术中的缺陷，本发明提出了一种能够响应癌症环境中过表达的过氧化氢释放CO，并且同时具有高光热转化效率和二区荧光转化效率的响应释放一氧化碳的纳米颗粒及其制备方法和应用。
本发明提供一种响应释放一氧化碳的纳米颗粒，包括外壳和内核，所述外壳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；所述内核为光热转化剂。
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。
进一步的，所述光热转化剂为Bodipy类，七甲川菁类，AIE类或聚合物类的二区荧光素中的任意一种。
所述Bodipy类选自1,3,5,7-四甲基-8-苯基-4,4-二氟二氮杂丁烷，二氟{2-[1-(3,5-二甲基-2H-吡咯-2-亚基-N)乙基]-3,5-二甲基-1H-吡咯并-N}硼，Fmoc-Trp-BODIPY，WinterGreen carbamoyl imidazole photocage等。
所述七甲川菁类选自Cy5，Cy5.5，Cy7，IR-780，IR-820等。
所述AIE类选自MCH-PPV，PBPTB，TPA，TPE等。
所述聚合物类的二区荧光素选自PBPTV，PBTV，PCFDP等。
本发明还提供所述的纳米颗粒的制备方法，包括如下步骤：
(1)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羰基铁载体；
(2)将制备得到羰基铁载体的复溶在四氢呋喃中，冻存，将析出的结晶过滤后得到纯化的羰基铁载体；
(3)将步骤(2)纯化后的羰基铁载体和光热转化剂溶解于四氢呋喃中。超声后，加入去离子水，用氮气吹出四氢呋喃，共沉淀；用超滤管离心并重复洗涤，共沉淀，形成稳定且均匀的纳米颗粒。
进一步的，步骤(1)中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，步骤(3)中所述纯化后的羰基铁载体和所述光热转化剂的质量比为(5～15)：1。
进一步的，步骤(1)所述氮气流温度为50-120℃，搅拌时间为1-12h。
本发明还提供一种抑制热休克蛋白的方法，包括使用所述的纳米颗粒。热休克蛋白是一种保护蛋白，抑制一些微生物或者细菌中的热休克蛋白也能够起到杀死它们的作用。研究领域通过抑制热休克蛋白来进行生理研究，比如通过抑制热休克蛋白，研究大脑，心脏等各种组织部位对外界刺激(高温，寒冷，电刺激，气体等)的应激反应。因此，热休克蛋白的激活或者抑制状态不与疾病直接相关。
本发明还提供所述的纳米颗粒在制备抗肿瘤药物中的应用。
综上，与现有技术相比，本发明达到了以下技术效果：
1.本发明的制备方法简便易行，仅需两步便可成功制备，便于操作推广。
2.本发明的纳米颗粒在杀伤肿瘤组织的同时，对正常组织毒性很低。
3.本发明制备的纳米颗粒在正常环境中能够稳定存在，7天后未出现沉降、凝絮现象。
4.本发明首次提出了利用一氧化碳气体分子抑制热休克蛋白，进而提高低温光热治疗效果的途径。
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为本发明实施例1中PBCO的制备流程图；
图2为本发明实施例2中PBCO的紫外光谱图；
图3为本发明实施例2中水合粒径分析仪下PBCO的粒径；
图4为本发明实施例3中PBCO的光热转化效率；
图5为本发明实施例4中CCK-8实验证明PBCO对癌细胞的细胞毒性；
图6为本发明实施例5中PBCO对原位乳腺癌瘤的治疗效果；
图7本发明实施例6中PBCO抑制肿瘤中HSP的表达结果。
具体实施方式
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。
本发明首先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。
之后，将光热转化剂(PB)与聚合物载体PG-CO自组装构建了一种响应释放一氧化碳的纳米颗粒。它不仅具有近红外双区(NIR-II)荧光和高光热效率，而且能响应肿瘤微环境中过度表达的过氧化氢进而选择性地释放CO。释放的CO不仅能够抑制癌细胞的增殖和转移，还能有效抑制热休克蛋白(HSPs)表达，显著降低了热休克蛋白的水平，在温和PTT过程中破坏肿瘤热阻，诱导肿瘤细胞凋亡，大大提高了低温PPT的疗效。 本发明的纳米颗粒提供了一种有效进行低温PTT的治疗手段，这是首次利用CO气体抑制PTT治疗过程中表达的HSPs，从而提高治疗效果的技术。
本发明的纳米颗粒的制备方法，如图1所示，步骤如下：
(1)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羰基铁载体。
(2)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5-24h，将析出的结晶过滤后得到纯化的羰基铁载体。
(3)将步骤(2)纯化后的羰基铁载体和光热转化剂溶解于四氢呋喃中。超声后，加入去离子水，用氮气吹出四氢呋喃，共沉淀；用超滤管离心并重复洗涤，共沉淀，形成稳定且均匀的纳米颗粒。
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。
光热转化剂为Bodipy类，七甲川菁类，AIE类或聚合物类的二区荧光素中的任意一种。以下实施例以PBPTV染料为例，其他上述光热转化剂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其他上述光热转化剂均能实现本发明的技术方案。
实施例1 本发明的纳米颗粒的制备
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(5-100mg)和mPEG-SH(M.W.≈2000)(100-600mg)溶于四氢呋喃(THF)中，并在50-120℃和氮气流下搅拌1-12h。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5-24h，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。其中，mPEG2000-SH可以替换为mPEG5000-SH，mPEG8000-SH，SH-mPEG2000-SH或SH-mPEG8000-SH等。
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、PBPTV，Bodipy等。首先将1-50mg PG-CO和5-10mg PBPTV染料溶解于THF中。超声5-10min后，加入10ml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。
实施例2 本发明的纳米颗粒的紫外光谱和粒径
将PB-CO和PBPTV溶于纯水，终浓度5-10mM，取0.5-1mL于比色皿，用紫外分光光度计测量其从300-650nm范围内紫外吸收。结果如附图2。
将纳米颗粒溶于纯水，终浓度5-10mM，取0.5-1mL于比色皿，用DLS粒径仪测量其平均粒径。结果如图3，纳米颗粒平均粒径158nm左右。
实施例3 本发明的纳米颗粒的光热转化效率
将纳米颗粒溶于纯水，终浓度50-100mM，取0.5-1mL于离心管，用808nm激光探头对其照射3min，同时用红外摄像头检测纳米颗粒溶液的 温度变化。3min后关闭激光探头，待其自然冷却，同时记录其温度变化。结果如图4，可以看到起初2.5min溶液升温速度很快，而且升温所需时间基本与降温时间相同。
实施例4 本发明的纳米颗粒对癌细胞的细胞毒性
用96孔板培养4T1乳腺癌细胞，外圈为PBS，孔板中间每一列6个孔作为一组，在第二天向培养孔中加入10-20μL不同浓度PBCO溶液或PBPTV染料，其浓度分别为0,10-20,30-40,40-60,60-80,80-100μg/mL。然后培养箱中培养24h，然后加入MTT染料，用酶标仪检测孔板中细胞存活率，作为第一组数据。
同上，将PBCO溶液换成相同浓度的PBPTV溶液或纳米颗粒溶液，检测细胞存活率作为第二、三组数据。
在上述三组实验中，在加入载体PBCO、染料PBPTV、纳米颗粒后，用808nm激光照射孔板1-2min，同样培养24h检测细胞存活率，依次作为第四、五、六组数据。
结果如图5，横坐标是浓度，从左到右是0-100，而每个浓度中不同颜色柱子从左到右分别为PBCO、PBPTV、纳米颗粒、PBCO加光照、PBPTV加光照、纳米颗粒加光照。从图中可以看到染料PBPTV不管什么浓度对癌细胞生长都没有抑制作用，而PBCO和纳米颗粒对癌细胞的抑制作用则随浓度增加而增大。而相同浓度中对癌细胞抑制效果从大到小分别为纳米颗粒加光照、载体PBCO加光照、载体PBCO、纳米颗粒。
实施例5 本发明的纳米颗粒对原位乳腺癌瘤的治疗效果
取两组4T1荷瘤BALB/c小鼠，每组5只，一组小鼠只注射0.5-1mL浓度20-30mM的纳米颗粒溶液，另一组注射0.5-1mL浓度20-30mM的纳米颗粒溶液，12小时后，用808nm NIR照射每只小鼠的肿瘤部位1-2min。之后每2天记录小鼠肿瘤体积，直到第18天处死小鼠。肿瘤体积变化结果如图6，只注射PBCO的小鼠肿瘤体积虽受到抑制但仍然在增长，而注 射PBCO后进行光热治疗的小鼠肿瘤体积则几乎没有变化，对肿瘤的生长起到了很大的抑制作用。
实施例6 本发明的PBCO载体对肿瘤中热休克蛋白的抑制作用
4T1荷瘤BALB/c小鼠分为八组，每组五只12h后，用808nm NIR照射肿瘤区域。每2天记录小鼠肿瘤体积和重量。在第18天处死这些小鼠，获取主要器官和肿瘤进行组织学检查。
对五只同期种瘤小鼠的肿瘤作不同处理：注射0.5-1mLPBS；注射0.5-1mLPBS且45℃保持5-10min；功率4-5W的808nm激光器照射3-5min；注射0.5-1mL浓度20-30mM的PBCO溶液；注射0.5-1mL浓度20-30mM的PBCO溶液且用功率4-5W的808nm激光器照射3-5min。将小鼠肿瘤组织取出浸入福尔马林液中40-50小时，蜡块包埋。制成石蜡切片后常规脱蜡至水，进行抗原热修复。滴加一抗，37℃恒温箱孵育1小时，用PBS清洗，滴加二抗，37℃恒温箱孵育1小时，用PBS清洗，滴加DAPI染料，37℃恒温箱孵育10分钟，用PBS清洗，DBA显色，常规复染后封片。共聚焦显微镜观察，结果如附图7，表明PBCO可以抑制肿瘤中HSP的表达。
综合以上实施例，本发明的纳米颗粒在肿瘤组织中能够特异性释放CO从而抑制癌症增殖，具有高光热转化效率能够在局部提供有效的光热治疗效果，对正常组织中毒性较低，所提供的CO气体能够显著降低低温光热治疗过程中热休克蛋白的表达，从而提高治疗效果。
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 (11)

  1. 一种响应释放一氧化碳的纳米颗粒，其特征在于，包括外壳和内核，所述外壳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；所述内核为光热转化剂。
  2. 根据权利要求1所述的纳米颗粒，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所述的纳米颗粒，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所述的纳米颗粒，其特征在于，所述硫醇端基聚合物的分子量为1000～8000。
  5. 根据权利要求1所述的纳米颗粒，其特征在于，所述光热转化剂为Bodipy类，七甲川菁类，AIE类或聚合物类的二区荧光素中的任意一种。
  6. 权利要求1-5任一项所述的纳米颗粒的制备方法，其特征在于，包括如下步骤：
    (1)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羰基铁载体；
    (2)将制备得到羰基铁载体的复溶在四氢呋喃中，冻存，将析出的结晶过滤后得到纯化的羰基铁载体；
    (3)将步骤(2)纯化后的羰基铁载体和光热转化剂溶解于四氢呋喃中，超声后，加入去离子水，用氮气吹出四氢呋喃，共沉淀；用超滤管离心并重复洗涤，共沉淀，形成稳定且均匀的纳米颗粒。
  7. 根据权利要求6所述的制备方法，其特征在于，步骤(1)中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6所述的制备方法，其特征在于，步骤(3)中所述纯化后的羰基铁载体和所述光热转化剂的质量比为(5～15)：1。
  9. 根据权利要求6所述的制备方法，其特征在于，步骤(1)中所述氮气流温度为50-120℃，搅拌时间为1-12h。
  10. 一种抑制热休克蛋白的方法，其特征在于，包括使用权利要求1～5任一项所述的纳米颗粒。
  11. 权利要求1-5任一项所述的纳米颗粒在制备抗肿瘤药物中的应用。
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