WO2022007153A1 - 一种同时包载光敏剂和蛋白质的肿瘤靶向纳米药物及其应用 - Google Patents

一种同时包载光敏剂和蛋白质的肿瘤靶向纳米药物及其应用 Download PDF

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WO2022007153A1
WO2022007153A1 PCT/CN2020/112583 CN2020112583W WO2022007153A1 WO 2022007153 A1 WO2022007153 A1 WO 2022007153A1 CN 2020112583 W CN2020112583 W CN 2020112583W WO 2022007153 A1 WO2022007153 A1 WO 2022007153A1
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tumor
protein
nanomedicine
zif
targeting
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French (fr)
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张晓龙
曾永毅
刘小龙
丁磊
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福建医科大学孟超肝胆医院(福州市传染病医院)
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A61K38/415—Cytochromes
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    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
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    • A61K47/6927—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
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    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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    • A61K47/6929—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
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    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0036—Porphyrins
    • A—HUMAN NECESSITIES
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    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0045—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo
    • A—HUMAN NECESSITIES
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • 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

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to a multifunctional nanomedicine, which has active targeting of tumor cells, responsiveness to tumor microenvironment, can be used for fluorescence imaging and can realize the combination of photodynamic therapy and protein therapy, and a synthesis method and application thereof. .
  • Cancer is one of the leading causes of morbidity and mortality worldwide, and the number of deaths due to cancer has increased rapidly over the past few decades.
  • Small molecule anticancer drugs are a mainstream cancer treatment and one of the most commonly used methods.
  • free forms of anticancer drugs may develop multidrug resistance. Therefore, scientists have been looking for other therapeutic agents to circumvent these limitations.
  • Photodynamic therapy with its noninvasive clinical therapeutic efficacy and inherent fluorescence imaging properties, has been a promising cancer treatment approach since the early 2000s.
  • PDT has the advantages of reproducible administration, controllable light dose, rapid efficacy, and site-specific treatment through localized irradiation.
  • PDT uses specific wavelengths of light to excite photosensitizers (PSs), transfer energy to molecular oxygen (O 2 ), and generate cytotoxic singlet oxygen ( 1 O 2 ), which in turn leads to tumor cell apoptosis and/or necrosis.
  • PSs photosensitizers
  • O 2 transfer energy to molecular oxygen
  • 1 O 2 cytotoxic singlet oxygen
  • PSs are usually hydrophobic, which leads to their easy self-aggregation in aqueous solution, which not only reduces their PDT effect, but also results in the ineffective delivery of PSs molecules to tumor sites.
  • molecular aggregates PSs greatly reduce the chance of contact between 2 PSs molecules and O, is not conducive to play the efficacy of PDT.
  • Protein therapy is an emerging cancer therapy that has shown promise for highly effective treatment in preclinical and clinical trials. Compared to traditional chemotherapeutics, protein therapy employs proteins that may activate tumor cell apoptosis or block tumor growth signals. For example, studies have found that cytochrome c (Cyt c) is involved in the initiation of the mitochondrial apoptotic pathway. In particular, high levels of Cyt c, which activate caspase proteases and induce DNA breaks in subsequent nuclei, can also be used to circumvent chemoresistance in tumors and improve therapeutic efficacy. However, the clinical translation of protein-based anticancer drugs has long been hindered by a series of problems.
  • Cyt c cytochrome c
  • Free proteins are known to be highly unstable in biologically relevant environments due to their sensitivity to chemicals, pH changes, temperature changes and enzymatic degradation, while denatured proteins can trigger immune responses and cause adverse health effects. Furthermore, the cellular uptake efficiency of the protein is unsatisfactory due to its size and surface charge.
  • the single treatment method often has certain deficiencies in the therapeutic effect, and cannot achieve the complete treatment of the tumor.
  • the advent of nanotechnology has opened up new avenues for the development of combined therapeutic modalities of photodynamic therapy and protein therapy.
  • nanocarriers can efficiently deliver hydrophobic photosensitizers and proteins, improve tumor uptake of photosensitizers and protect the functional integrity of protein drugs during delivery, while surface engineering techniques can be used to increase material stability and target cells. uptake capacity. Therefore, the development of a multifunctional nanomedicine with active tumor cell targeting, tumor microenvironment responsiveness, fluorescence imaging, and combination of photodynamic therapy and protein therapy has great application prospects in improving tumor therapy.
  • the purpose of the present invention is to provide a tumor-targeted nanomedicine that simultaneously encapsulates photosensitizers and proteins and its application.
  • the present invention adopts the following technical solutions:
  • One of the protection purposes of the present invention is a tumor-targeted nanomedicine that simultaneously encapsulates photosensitizers and proteins, which uses metal-organic framework material ZIF-8 as a carrier, simultaneously encapsulates hydrophobic photosensitizers and protein drugs, and adopts Nanoparticles formed by modifying the modifier; the particle size of the nanoparticles is 20-400nm.
  • the hydrophobic photosensitizer is one of chlorin e6 (Ce6), curcumin, hypericin, protoporphyrin (IX), tetraphenyl porphyrin, zinc phthalocyanine, and rhodochrousin B or more.
  • the protein drug is cytochrome c (Cyt c), carboxypeptidase G2, interferon alpha-2b, ribonuclease, interleukin-10, PD-1 antibody, and granzyme B.
  • the modifier is one of hyaluronic acid (HA), polyethyleneimine, dextran, chitosan, fucose, albumin, gelatin, liposome, polyvinylpyrrolidone or variety.
  • the second protection purpose of the present invention is the preparation method of the tumor-targeted nanomedicine, which is to self-assemble into metal-organic framework material ZIF-8 by dimethylimidazole (2-MIM) and Zn 2+, and simultaneously synthesize and encapsulate it in one step.
  • Hydrophobic photosensitizers and protein drugs are used to prepare pre-nano-drugs, and the tumor-targeting nano-drugs are synthesized by the coordination of carboxyl groups on the modifier and Zn 2+.
  • the specific steps are as follows:
  • the third protection purpose of the present invention is the application of the tumor-targeting nanomedicine in the preparation of anti-tumor drugs.
  • the tumor-targeting nanomedicine can be targeted to tumor cells and undergo pH-sensitive response degradation in the tumor microenvironment. , release the drug, and realize the synergistic treatment of photodynamic therapy and protein therapy at the same time of fluorescence imaging.
  • the synthesis steps of the tumor-targeted nanomedicine of the present invention are simple, the conditions are mild, and the activity of the protein is not affected;
  • the tumor-targeting nanomedicine of the present invention efficiently encapsulates hydrophobic photosensitizers and protein drugs through non-covalent actions such as self-assembly and electrostatic interaction, thereby avoiding the destruction of the molecular structure of the drug by covalent modification;
  • the tumor-targeting nanomedicine of the present invention is modified by a modifier, so that it can be degraded in response to the micro-acid microenvironment of the tumor, so as to realize the stability of the drug in the physiological environment and the efficient targeted release in the tumor;
  • the tumor-targeted nanomedicine of the present invention has both photodynamic therapy ability and protein therapy ability, and can realize the synergistic therapy of photodynamic therapy and protein therapy through the catalytic and/or therapeutic effect of protein, and enhance the combined killing ability of tumor.
  • the nanomedicine can not only perform fluorescence imaging at the tumor site, but also can be used as a combined therapeutic agent for cancer.
  • Figure 1 shows the nanomedicine Ce6/Cyt TEM image of c@ZIF-8/HA (A), particle size distribution of different nanomedicines (B), Zeta potential map (C), UV-Vis absorption spectrum (D), fluorescence excited by Ce6 excitation wavelength Spectrogram (E), fluorescence spectrogram (F) excited at the excitation wavelength of FITC.
  • Figure 2 shows the variation of the absorption value of DPBF in the range of 300-600 nm with irradiation time under 670 nm laser irradiation (where A is Cyt c@ZIF-8/HA, B is Ce6/Cyt c@ZIF-8, C is Ce6/Cyt c@ZIF-8/HA) and the normalized change in absorbance at 415 nm (D).
  • Figure 3 shows the nanomedicine Ce6/Cyt Time-dependent release of Ce6 (A) and Cyt c (B) from c@ZIF-8/HA at different pH.
  • Figure 4 shows the nanomedicine Ce6/Cyt The time-varying diagram of the decomposition of c@ZIF-8/HA in the presence of hydrogen peroxide to produce oxygen (A) and the comparison of reactive oxygen species produced under normoxic and hypoxic conditions (B).
  • Figure 5 shows the nanomedicine Ce6/Cyt Fluorescence imaging comparison of c@ZIF-8/HA uptake by cells.
  • Figure 6 is a comparison of fluorescence imaging of the intracellular reactive oxygen species generated by different nanomedicines under illumination and non-illumination conditions.
  • Figure 7 is a comparison diagram of PI staining fluorescence imaging of cells treated with different nanomedicines under illumination and non-illumination conditions.
  • Figure 8 shows the quantitative detection results of cell viability after treatment of cells with different nanomedicines under illumination and non-illumination conditions.
  • Figure 9 shows the detection results of apoptosis of cells treated with different nanomedicines under illumination and non-illumination conditions.
  • ZIF-8/HA without loading photosensitizer and protein drug
  • Ce6@ZIF-8/HA without loading protein drug
  • Cyt c@ZIF-8/HA without loading photosensitizer
  • Ce6/Cyt c@ZIF-8 no modifier was used
  • Ce6@ZIF-8 no protein drug loaded and no modifier was used
  • together with Ce6, Cyt c, and ZIF-8 were used as reference samples for comparative studies.
  • Example 1 The nanomedicine synthesized in Example 1 was characterized by tests such as transmission electron microscope (TEM), nanoparticle size and Zeta potential analyzer (DLS, Zeta potential), ultraviolet-visible spectrophotometer (UV-Vis), and fluorescence spectrometer. , the results are shown in Figure 1.
  • TEM transmission electron microscope
  • DLS nanoparticle size and Zeta potential analyzer
  • UV-Vis ultraviolet-visible spectrophotometer
  • fluorescence spectrometer fluorescence spectrometer
  • Example 2 To investigate the ability of the nano-drug samples synthesized in Example 1 to generate reactive oxygen species under 670 nm laser irradiation, that is, using 1,3-diphenylisobenzofuran (DPBF) as the reactive oxygen indicator probe, different nanometer After the drug sample is mixed with DPBF, the laser is irradiated for different times, and the test is 300-600 The variation of the absorbance in the nm range was normalized by taking the absorbance at 415 nm to prove the reactive oxygen generation ability of the metal-organic framework nanomedicine. The results are shown in Fig. 2.
  • DPBF 1,3-diphenylisobenzofuran
  • Example 4 The ability of the nanomedicine synthesized in Example 1 to catalyze the generation of oxygen from hydrogen peroxide and improve the photodynamic effect was investigated, that is, the nanomedicine was reacted with 500 ⁇ M hydrogen peroxide, and the change of dissolved oxygen was measured by the oxygen electrode. At the same time, using DPBF as a probe, the ability of nanomedicines to generate reactive oxygen species under normoxic and hypoxic conditions in the presence of hydrogen peroxide was compared. The results are shown in Figure 4.
  • nano-drugs were incubated with HeLa cells for 4 hours, the nano-drugs were washed away, and then each was divided into two groups, one of which was subjected to 670 24 hours later, cells were stained with PI dye for 20 minutes, and then detected by fluorescence microscope. The results are shown in Figure 7.
  • c@ZIF-8/HA also has a certain improvement, which is because the effective uptake of nano-drugs by cells decreases after HA blocking treatment, thus reducing the effect of combined treatment.
  • c@ZIF-8/HA also has a certain improvement, which is because the effective uptake of nano-drugs by cells decreases after HA blocking treatment, thus reducing the effect of combined treatment.
  • apoptosis kit (AnnexinV-FITC/PI) quantitatively test the therapeutic effect of nanomedicines under light and no light conditions by flow cytometry. Specifically, HeLa cells were cultured in a 6-well plate, and different nano-drugs were added for co-incubation for 4 hours, and the nano-drugs were washed away, and then divided into two groups, one of which was exposed to 670 nm light for 5 min, and the other was Without illumination as a comparison, after 24 hours, according to the operation method of the apoptosis kit (AnnexinV-FITC/PI), flow cytometry was used for detection. The results are shown in Figure 9.
  • c@ZIF-8/HA also has a certain improvement, which is because the effective uptake of nano-drugs by cells decreases after HA blocking treatment, thus reducing the effect of combined treatment.

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Abstract

一种同时包载光敏剂和蛋白质的肿瘤靶向纳米药物及其在制备抗肿瘤药物中的应用，该肿瘤靶向纳米药物是利用金属有机框架包载疏水性光敏剂和蛋白质药物，并采用改性剂进行修饰而构成。所述肿瘤靶向纳米药物具有肿瘤细胞主动靶向及肿瘤微环境响应释放药物的特性，并能在荧光成像的同时实现光动力治疗和蛋白质治疗的协同治疗，从而达到良好的诊断与治疗效果。

Description

一种同时包载光敏剂和蛋白质的肿瘤靶向纳米药物及其应用 技术领域
本发明属于生物医药领域，具体涉及一种具有肿瘤细胞主动靶向性、肿瘤微环境响应性、可用于荧光成像并可实现光动力治疗和蛋白质治疗联合的多功能纳米药物及其合成方法与应用。
 背景技术
癌症是全球发病率和死亡率的主要原因之一，在过去的几十年中，由于癌症导致的死亡人数迅速增加。小分子抗癌药物是一种主流的癌症治疗方法，也是最常用的方法之一。然而，游离形式的抗癌药物都可能产生多药耐药性。因此，科学家们一直在寻找其他治疗剂来规避上述局限性。
自21世纪初以来，具有无创临床治疗效果和固有荧光成像特性的光动力疗法（PDT）一直是一种有前景的癌症治疗方法。与放疗、外科手术和化学疗法等传统疗法相比，PDT具有可重复给药、可控制的光剂量、快速的疗效以及通过局部照射进行定点治疗的优势。PDT是利用特定波长光照射激发光敏剂（PSs），将能量转移到分子氧（O 2），产生具有细胞毒性的单线态氧（ 1O 2），进而导致肿瘤细胞凋亡和/或坏死。但是，常用的高效PSs通常是疏水性的，导致其易在水溶液中自聚集，这不仅降低了其PDT的效果，而且导致PSs分子无法有效递送到肿瘤部位。另外，PSs分子的聚集大大减少了PSs分子与O 2之间接触的机会，也不利于PDT功效的发挥。
技术问题
蛋白治疗是一种新兴的癌症治疗方法，已经在临床前和临床试验中显示出了高效治疗的希望。与传统的化学治疗剂相比，蛋白治疗采用的蛋白可能会激活肿瘤细胞的凋亡或阻断肿瘤的生长信号。例如，研究发现细胞色素c（Cyt c）参与线粒体凋亡途径的启动。特别是，高水平的Cyt c可以激活半胱天冬氨酸蛋白酶，并在随后的细胞核中诱导DNA断裂，也可用于规避肿瘤的化疗耐药性，并提高治疗效果。然而，蛋白质类抗癌药物的临床转化长期以来一直受到一系列问题的阻碍。众所周知，游离蛋白质因为对化学物质、pH变化、温度变化和酶降解等因素非常敏感，使其在生物相关的环境中高度不稳定，而变性蛋白质能够引发免疫反应并造成不利的健康影响。此外，由于蛋白质的大小和表面电荷，使其细胞摄取效率不令人满意。
同时，单一治疗方法在治疗效果方面往往存在一定的不足，无法实现肿瘤的完全治疗。纳米技术的出现为开发光动力治疗和蛋白质治疗的联合治疗方式开辟了新途径。研究表明，纳米载体可以高效递送疏水性光敏剂和蛋白质，提高肿瘤对光敏剂的摄取并保护蛋白质药物在递送过程中功能的完整性，同时能使用表面工程技术来增加材料稳定性和靶细胞的摄取能力。因此，开发一种具有肿瘤细胞主动靶向性、肿瘤微环境响应性、可荧光成像并可实现光动力治疗和蛋白质治疗联合的多功能纳米药物，在提高肿瘤治疗方面具有极大的应用前景。
 技术解决方案
本发明的目的是提供一种同时包载光敏剂和蛋白质的肿瘤靶向纳米药物及其应用。
为实现上述目的，本发明采用如下技术方案：
本发明的保护目的之一是一种同时包载光敏剂和蛋白质的肿瘤靶向纳米药物，其是以金属有机框架材料ZIF-8为载体，同时包载疏水性光敏剂和蛋白质药物，并采用改性剂进行修饰而构成的纳米颗粒；该纳米颗粒的粒径为20-400nm。
所述疏水性光敏剂为二氢卟吩e6（Ce6）、姜黄素、金丝桃素、原卟啉（IX）、四苯基卟啉、锌酞菁、竹红菌素B中的一种或多种。
所述蛋白质药物为细胞色素c（Cyt c）、羧肽酶G2、干扰素α-2b、核糖核酸酶、白介素-10、PD-1抗体、颗粒酶B。
所述改性剂（SA）为透明质酸（HA）、聚乙烯亚胺、葡聚糖、壳聚糖、岩藻糖、白蛋白、明胶、脂质体、聚乙烯吡咯烷酮中的一种或多种。
本发明的保护目的之二是所述肿瘤靶向纳米药物的制备方法，是以二甲基咪唑（2-MIM）和Zn 2+自组装成金属有机框架材料ZIF-8，同时一步合成包载疏水性光敏剂和蛋白质药物，制得前纳米药物，再利用改性剂上的羧基和Zn 2+的配位作用，合成该肿瘤靶向纳米药物。其具体步骤如下：
1）将2-MIM水溶液、蛋白质药物水溶液与疏水性光敏剂溶液（将疏水性光敏剂溶于N,N-二甲基甲酰胺中）混合均匀并搅拌分散5 min，然后快速滴加含醋酸锌或硝酸锌的水溶液，300~600 r/min（优选为400 r/min）搅拌5~60 min（优选为10 min）后，于4℃、8000~14800 r/min离心5~60 min，收集下层沉淀，用水离心洗涤多次，得到金属有机框架包载的前纳米药物PS/Protein@ZIF-8；其中，所用疏水性光敏剂、蛋白质药物与醋酸锌或硝酸锌的重量比为1~20：1~20：100（优选为7.5：8：100），所用醋酸锌或硝酸锌与2-MIM的摩尔比为1：1~200（优选为1:70）；
2）将所得PS/Protein@ZIF-8重新分散在水中，在超声条件下缓慢滴加改性剂溶液，然后避光搅拌2～6 h后，加入反应体系体积1/10的无水乙醇，于8000~14800 r/min离心洗涤20～40分钟，再用水离心洗涤多次，得到靶向纳米药物PS/Protein@ZIF-8/SA，于2~8℃避光保存；其中，所述反应体系中PS/Protein@ZIF-8和改性剂的质量比为1：0.1~10（优选为1：1）。
本发明的保护目的之三是所述肿瘤靶向纳米药物在制备抗肿瘤药物中的应用，所述肿瘤靶向纳米药物可以靶向至肿瘤细胞，并在肿瘤微环境下发生pH敏感的响应降解、释放药物，还能在荧光成像的同时实现光动力治疗和蛋白质治疗的协同治疗。
 有益效果
本发明的有益效果主要体现在：
（1）本发明肿瘤靶向纳米药物的合成步骤简单、条件温和，不影响蛋白质的活性；
（2）本发明肿瘤靶向纳米药物通过自组装、静电作用等非共价作用高效包载疏水性光敏剂和蛋白质药物，避免了共价修饰对药物分子结构的破坏；
（3）本发明肿瘤靶向纳米药物采用改性剂修饰，使其可响应肿瘤微酸微环境而降解，实现药物在生理环境中的稳定及肿瘤中的高效靶向释放；
（4）本发明肿瘤靶向纳米药物兼具光动力治疗能力和蛋白质治疗能力，通过蛋白质的催化和/或治疗效果可实现光动力治疗和蛋白质治疗的协同治疗，增强对肿瘤的联合杀伤能力。
综上所述，该纳米药物不仅可以在肿瘤部位进行荧光成像，还可作为癌症的联合治疗剂应用。
 附图说明
图1为纳米药物Ce6/Cyt c@ZIF-8/HA的透射电镜图（A），不同纳米药物的粒径分布图（B）、Zeta电位图（C）、紫外可见光吸收光谱图（D）、以Ce6激发波长激发的荧光光谱图（E）、以FITC激发波长激发的荧光光谱图（F）。
图2为在670 nm激光照射下，DPBF在300-600 nm范围内的吸收值随照射时间的变化情况（其中，A为Cyt c@ZIF-8/HA、B为Ce6/Cyt c@ZIF-8、C为Ce6/Cyt c@ZIF-8/HA）及在415 nm处的吸收值归一化变化图（D）。
图3为纳米药物Ce6/Cyt c@ZIF-8/HA在不同pH下Ce6（A）和Cyt c（B）的释放随时间的变化情况。
图4为纳米药物Ce6/Cyt c@ZIF-8/HA在过氧化氢存在下分解产生氧气的时间变化图（A）和在常氧和乏氧条件下产生的活性氧对比图（B）。
图5为纳米药物Ce6/Cyt c@ZIF-8/HA被细胞摄取的荧光成像对比图。
图6为不同纳米药物在光照及未光照条件下在细胞内产生活性氧的荧光成像对比图。
图7为不同纳米药物在光照及未光照条件下对细胞治疗后的PI染色荧光成像对比图。
图8为不同纳米药物在光照及未光照条件下对细胞治疗后的细胞活力定量检测结果。
图9为不同纳米药物在光照及未光照条件下对细胞治疗后的细胞凋亡检测结果。
 本发明的实施方式
为了使本发明所述的内容更加便于理解，下面结合具体实施方式对本发明所述的技术方案做进一步的说明，但是本发明不仅限于此。
实施例1 肿瘤靶向纳米药物的制备
分别称取2.27 g的2-MIM、8 mg的Cyt c（作为本实施中的蛋白质药物）、6 mg的Ce6（作为本实施中的光敏剂），将其分别溶于8 mL蒸馏水，0.8 mL蒸馏水以及0.6 mL N,N-二甲基甲酰胺（DMF）中，然后以400 r/min避光搅拌5 min使三者充分混匀；另称取86 mg的醋酸锌溶于0.8 mL的蒸馏水中，并将所得醋酸锌溶液快速滴加入上述反应液中，400 r/min避光搅拌10 min后，收集反应液，于4℃、13300 r/min离心30 min，收集下层沉淀，用蒸馏水离心洗涤三次，然后重悬在蒸馏水中配成浓度为2 mg/mL的前纳米药物Ce6/Cyt c@ZIF-8溶液。
向2 mg/mL的前纳米药物溶液中加入等体积2 mg/mL的HA（作为本实施中的改性剂）水溶液，超声充分混匀，然后以500 r/min避光振荡3 h后，在反应体系中加入其体积1/10的无水乙醇，于4℃、10000 r/min离心20 min，再用蒸馏水离心洗涤三次，即为Ce6/Cyt c@ZIF-8/HA，重新分散在蒸馏水中，置于4℃冰箱避光保存。
另外，参照上述方法，分别合成ZIF-8/HA（未负载光敏剂和蛋白质药物）、Ce6@ZIF-8/HA（未负载蛋白质药物）、Cyt c@ZIF-8/HA（未负载光敏剂）、Ce6/Cyt c@ZIF-8（未使用改性剂）、Ce6@ZIF-8（未负载蛋白质药物且未使用改性剂），并与Ce6、Cyt c、ZIF-8共同作为参照样品用于比较研究。
实施例2：
1. 对实施例1中合成的纳米药物通过透射电镜(TEM)、纳米粒度及Zeta电位分析仪(DLS，Zeta电位)、紫外-可见分光光度计(UV-Vis)、荧光光谱仪等测试进行表征，结果如图1所示。
由图1可见，合成的纳米药物Ce6/Cyt c@ZIF-8/HA尺寸约110 nm（A）；且不同纳米药物样品的尺寸分布集中，说明合成的样品较为均匀（B）；且通过Zeta电位图（C）和紫外可见光吸收光谱图（D）可见，蛋白质药物Cyt c和光敏剂Ce6成功的包载在金属有机框架中；由荧光光谱图（E）、（F）可见，有包裹光敏剂和荧光标记的蛋白质药物时，纳米药物Ce6/Cyt c@ZIF-8/HA的谱线中分别出现了Ce6在660 nm处的荧光峰和FITC在520 nm处的荧光峰，进一步证明了光敏剂和蛋白质成功包裹进金属有机框架中。
2. 考察实施例1中合成的纳米药物样品在670 nm激光照射下产生活性氧的能力，即以1,3-二苯基异苯并呋喃(DPBF)为活性氧指示探针，将不同纳米药物样品与DPBF混合后，激光照射不同时间，测试300-600 nm范围内吸收值的变化，并取415 nm处的吸收值进行归一化处理，以证明金属有机框架纳米药物的活性氧产生能力，结果如图2所示。
由图2可见，有包载Ce6的纳米药物样品在415 nm处的吸收值随光照时间延长而降低，而未包载Ce6的样品没有明显变化，说明包载有Ce6的纳米药物可以在光照条件下产生活性氧。
3. 考察实施例1中合成的纳米药物响应释放的能力，即将纳米药物分别溶于pH=5.0和pH=7.4的PBS缓冲溶液中，通过测定吸收，来研究纳米药物释放Ce6和Cyt c的情况，结果如图3所示。
由图3可见，在pH=5.0的PBS缓冲液中时，纳米药物Ce6/Cyt c@ZIF-8/HA发生明显降解，几乎完全释放包载的Ce6和Cyt c，证明了其具有酸响应性释放能力。
4. 考察实施例1中合成的纳米药物催化过氧化氢产生氧气和提高光动力作用的能力，即将纳米药物与500 μM 过氧化氢反应，通过氧电极测定其溶解氧的变化。同时以DPBF为探针，比较常氧和乏氧条件下，在过氧化氢存在时光照纳米药物产生活性氧的能力。结果如图4所示。
由图4可见，纳米药物中存在Cyt c可以有效催化过氧化氢分解成氧气（A），提高活性氧产生能力（B），从而有利于提高光动力治疗效果。
实施例3：
1. 以宫颈癌HeLa细胞为模型，利用共聚焦荧光显微镜测试纳米药物Ce6/Cyt c@ZIF-8/HA在细胞层面运载Ce6和Cyt c的能力及HA的靶向肿瘤细胞能力。其具体是将纳米药物分别与L929细胞（小鼠成纤维细胞）、HeLa细胞及HA封闭处理的HeLa细胞共孵育2小时后，利用共聚焦荧光显微镜分别检测Ce6和FITC-Cyt c的荧光，结果如图5所示。
由图5可见，HeLa细胞中出现了Ce6和FITC的荧光图像，证明了该纳米药物可成功将Ce6和Cyt c递送进细胞。
2. 结合活性氧荧光指示剂2',7'-二氯荧光黄双乙酸盐（DCFH-DA）考察不同纳米药物在细胞内产生活性氧的能力。其具体是将纳米药物分别与HeLa细胞共孵育4小时后，洗去纳米药物后，加入荧光指示剂DCFH-DA孵育20分钟，然后各分为两组，其中一组对细胞进行670 nm光照5 min，另外一组不进行光照作为对比，光照后用荧光显微镜检测，结果如图6所示。
由图6可见，含有Ce6的纳米药物组(Ce6@ZIF-8/HA、Ce6/Cyt c@ZIF-8/HA、HA+Ce6/Cyt c@ZIF-8/HA)出现了DCFH明显的荧光，证明含有Ce6的纳米药物可以在光照下产生活性氧，故可用于细胞光动力治疗。
3. 结合死细胞荧光指示剂（碘化丙啶，PI）考察不同纳米药物在光照和无光照条件下的治疗效果。其具体是将纳米药物分别与HeLa细胞共孵育4小时后，洗去纳米药物，然后各分为两组，其中一组对细胞进行670 nm光照5 min，另外一组不进行光照组作为对比，24小时后，用PI染料对细胞染色20分钟，之后用荧光显微镜检测，结果如图7所示。
由图7可见，无光照时，各纳米药物对细胞的杀伤作用较低；而光照后，相对于不含有Ce6的试验组（Control和Cyt c），含有Ce6的纳米药物组（Ce6@ZIF-8/HA、Ce6/Cyt c@ZIF-8/HA、HA+Ce6/Cyt c@ZIF-8/HA）对癌细胞的杀伤能力显著提高，说明其具有良好的光动力治疗效果。另外，Ce6/Cyt c@ZIF-8/HA相比于HA+Ce6/Cyt c@ZIF-8/HA也有一定提高，这是由于HA封闭处理后，纳米药物被细胞有效摄取变少，从而降低了联合治疗效果。
4. 利用细胞活力定量检测试剂盒(CCK-8试剂盒)定量测试纳米药物在在光照和无光照条件下的治疗效果。其具体是将HeLa细胞培养于96孔板中，分别加入不同纳米药物共孵育4小时后，洗去纳米药物，然后各分为两组，其中一组对细胞进行670 nm光照5 min，另外一组不进行光照作为对比，24小时后，按照细胞活力定量检测试剂盒操作方法进行检测，结果如图8所示。
由图8可见，无光照时，各纳米药物对细胞的杀伤作用较低；而光照后，相对于不含有Ce6组（Control和Cyt c），含有Ce6的纳米药物组（Ce6@ZIF-8/HA、Ce6/Cyt c@ZIF-8/HA、HA+Ce6/Cyt c@ZIF-8/HA）对癌细胞的杀伤能力显著提高。另外，Ce6/Cyt c@ZIF-8/HA相比于HA+Ce6/Cyt c@ZIF-8/HA也有一定提高，这是由于HA封闭处理后，纳米药物被细胞有效摄取变少，从而降低了联合治疗效果。
5. 利用凋亡试剂盒（AnnexinV-FITC/PI），通过流式细胞术定量测试纳米药物在在光照和无光照条件下的治疗效果。其具体是将HeLa细胞培养于6孔板，分别加入不同纳米药物共孵育4小时后，洗去纳米药物，然后各分为两组，其中一组对细胞进行670 nm光照5 min，另外一组不进行光照作为对比，24小时后，按照凋亡试剂盒（AnnexinV-FITC/PI）操作方法，通过流式细胞术进行检测，结果如图9所示。
由图9可见，无光照时，各纳米药物对细胞的杀伤作用较低；而光照后，相对于不含有Ce6组（Control和Cyt c），含有Ce6的纳米药物组（Ce6@ZIF-8/HA、Ce6/Cyt c@ZIF-8/HA、HA+Ce6/Cyt c@ZIF-8/HA）对癌细胞的杀伤能力显著提高。另外，Ce6/Cyt c@ZIF-8/HA相比于HA+Ce6/Cyt c@ZIF-8/HA也有一定提高，这是由于HA封闭处理后，纳米药物被细胞有效摄取变少，从而降低了联合治疗效果。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。

Claims (9)

  1. 一种同时包载光敏剂和蛋白质的肿瘤靶向纳米药物，其特征在于：所述肿瘤靶向纳米药物是以二甲基咪唑和Zn 2+自组装成的金属有机框架材料ZIF-8为载体，同时包载疏水性光敏剂和蛋白质药物，并采用改性剂进行修饰而构成的纳米颗粒。
  2. 根据权利要求1所述的同时包载光敏剂和蛋白质的肿瘤靶向纳米药物，其特征在于：所述疏水性光敏剂为二氢卟吩e6、姜黄素、金丝桃素、原卟啉、四苯基卟啉、锌酞菁、竹红菌素B中的一种或多种。
  3. 根据权利要求1所述的同时包载光敏剂和蛋白质的肿瘤靶向纳米药物，其特征在于：所述蛋白质药物为细胞色素c、羧肽酶G2、干扰素α-2b、核糖核酸酶、白介素-10、PD-1抗体、颗粒酶B。
  4. 根据权利要求1所述的同时包载光敏剂和蛋白质的肿瘤靶向纳米药物，其特征在于：所述改性剂为透明质酸、聚乙烯亚胺、葡聚糖、壳聚糖、岩藻糖、白蛋白、明胶、脂质体、聚乙烯吡咯烷酮中的一种或多种。
  5. 根据权利要求1所述的同时包载光敏剂和蛋白质的肿瘤靶向纳米药物，其特征在于：所述纳米颗粒的粒径为20-400nm。
  6. 一种如权利要求1-5所述的肿瘤靶向纳米药物的制备方法，其特征在于：具体步骤如下：
    1）将2-甲基咪唑、蛋白质药物与疏水性光敏剂混合均匀并搅拌分散5 min，然后快速滴加含醋酸锌或硝酸锌的水溶液，搅拌5~60min后，于4℃、8000~14800 r/min离心5~60min，收集下层沉淀，用水离心洗涤多次，得到金属有机框架包载的前纳米药物PS/Protein@ZIF-8；
    2）将所得PS/Protein@ZIF-8重新分散在水中，在超声条件下缓慢滴加改性剂溶液，然后避光搅拌2～6 h后，加入反应体系总体积1/10的无水乙醇，于8000~14800 r/min离心洗涤20～40分钟，再用水离心洗涤多次，得到靶向纳米药物PS/Protein@ZIF-8/SA。
  7. 根据权利要求6所述的肿瘤靶向纳米药物的制备方法，其特征在于：步骤1）中所用疏水性光敏剂、蛋白质药物与醋酸锌或硝酸锌的重量比为1~20：1~20：100；所用醋酸锌或硝酸锌与2-甲基咪唑的摩尔比为1：1~200。
  8. 根据权利要求6所述的肿瘤靶向纳米药物的制备方法，其特征在于：步骤2）所述反应体系中PS/Protein@ZIF-8和改性剂的质量比为1：0.1~10。
  9. 一种如权利要求1所述的肿瘤靶向纳米药物在制备抗肿瘤药物中的应用，其特征在于：所述肿瘤靶向纳米药物具有肿瘤细胞主动靶向及肿瘤微环境响应释放药物的特性，并能在荧光成像的同时实现光动力治疗和蛋白质治疗的协同治疗。
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