WO2023070868A1 - Oxygen self-supply photosensitizer, and preparation method therefor and application thereof - Google Patents

Oxygen self-supply photosensitizer, and preparation method therefor and application thereof Download PDF

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WO2023070868A1
WO2023070868A1 PCT/CN2021/137758 CN2021137758W WO2023070868A1 WO 2023070868 A1 WO2023070868 A1 WO 2023070868A1 CN 2021137758 W CN2021137758 W CN 2021137758W WO 2023070868 A1 WO2023070868 A1 WO 2023070868A1
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photosensitizer
self
vermiculite
dcpy
oxygenated
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PCT/CN2021/137758
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French (fr)
Chinese (zh)
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张鹏飞
于兴华
孟文婧
蔡林涛
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深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • 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
    • 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
    • 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
    • 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 technical field of photosensitizers, and in particular relates to a preparation method and application of a self-oxygenated photosensitizer.
  • photosensitizers have been extensively improved, they still have the disadvantages of dark toxicity and poor photostability under complex physiological conditions and high reactive oxygen species environment. Due to their inherent rigid planar and hydrophobic structures, most photosensitizers often suffer from aggregation-induced fluorescence quenching and a sharp drop in the production of reactive oxygen species in the aggregated state. To some extent, it limits the anti-tumor application of PDT. Moreover, the prior art has problems such as complex preparation of the required nanocomposite material, low oxygen loading rate, and uncontrolled oxygen release.
  • the present invention proposes a near-infrared light-controlled on-demand oxygen-supplying vermiculite nanosheet diagnostic agent, which can not only improve the biocompatibility of the near-infrared photosensitizer, but also make the nano-diagnostic agent Oxygen on demand improves tissue hypoxia and enables deep photodynamic therapy through the effective delivery of near-infrared photosensitizers.
  • a self-oxygenated photosensitizer the structure of the self-oxygenated photosensitizer includes vermiculite nanosheets and an aggregation-induced luminescent luminescent agent loaded on the surface of the vermiculite nanosheets.
  • the aggregation-induced luminescent luminescent agent is one or more of DCPy, DCMa, DCIs, DCFu,
  • the aggregation-induced luminescence luminescent agent is DCPy.
  • vermiculite nanosheets have a single-layer structure or a multi-layer structure.
  • the loading amount of the aggregation-induced luminescent luminescent agent is 10-15% of the weight of the vermiculite nanosheets. w/w%.
  • the present invention also provides a preparation method of the above-mentioned self-oxygenated photosensitizer, comprising the following steps:
  • step S1 includes: adding vermiculite to the lithium salt solution modifier to obtain vermiculite nanosheets.
  • the alkali metal salt solution modifier is one or more of lithium chloride solution, lithium ethylenediaminetetraacetate solution or lithium citrate solution.
  • step S2 the ratio of the concentration of the vermiculite nanosheets to the concentration of the aggregation-induced luminescence luminescent agent is 2: (0.1-100).
  • step S2 the aggregation-induced luminescence luminescent agent is loaded onto the surface of the vermiculite nanosheets by electrostatic adsorption.
  • the present invention also provides the application of the above self-oxygenated photosensitizer in the preparation of photodynamic therapy drugs.
  • the self-oxygenated photosensitizer provided by the present invention can not only improve the effect of photodynamic therapy, but more importantly, the self-oxygenated photosensitizer controls the fixed-point and timed release of oxygen through vermiculite nanomaterials, and can be effectively used for cancer cells and In the diagnosis and treatment of tumors, it has a good prospect of in vivo biomedical application;
  • the self-oxygenated photosensitizer provided by the present invention has good biocompatibility and high specific surface area, and has good application prospects in visual photodynamic therapy;
  • the oxygen-supplied photosensitizer provided by the present invention has rich raw materials, is easy to prepare, has a wide range of applications, and can be used in industrial production.
  • Fig. 1 is the structural representation of the self-supply oxygen photosensitizer prepared by embodiment 1;
  • Fig. 2 is the transmission electron micrograph of the self-supply oxygen photosensitizer prepared in embodiment 1;
  • Fig. 3 is the atomic force microscope figure of the self-supply oxygen photosensitizer prepared by embodiment 1;
  • Fig. 4 is the average particle diameter and the average height of the self-oxygenated photosensitizer prepared by embodiment 1;
  • Fig. 5 is the visible-near-infrared spectrum and the fluorescent spectrum of the self-supplied oxygen photosensitizer prepared in embodiment 1;
  • Fig. 6 is the X-ray photoelectron energy spectrogram of the self-supplied oxygen photosensitizer prepared in embodiment 1;
  • Fig. 7 is the Zeta potential diagram of the self-supplied oxygen photosensitizer prepared in embodiment 1;
  • Fig. 8 is the oxygen production concentration of the self-supplying photosensitizer prepared in embodiment 1;
  • Figure 9 is the 1 O electron spin resonance amplitude of the self-supplied oxygen photosensitizer prepared in Example 1;
  • Figure 10 is the OH electron spin resonance amplitude of the self-supplied oxygen photosensitizer prepared in Example 1;
  • Figure 11 is a confocal laser scanning microscope (CLSM) image of Experimental Example 3;
  • Figure 12 is a time-lapse biological imaging of Balb/c mice
  • Figure 13 is the biological imaging of major organs and tumors after 24 hours of intravenous injection
  • Figure 14 is the measurement results of the average fluorescence intensity of DCPy and NSs@DPCy on the main organs and tumors of mice;
  • Fig. 15 is a graph of tumor growth curves after systemic administration of PBS, NSs, DCPy and NSs-DCPy to living mice without light irradiation or with light irradiation for 5 minutes;
  • Figure 16 is the results of immunohistochemical analysis of Caspase3 expression (scale bar is 100 ⁇ m);
  • Figure 17 is the results of immunohistochemical analysis of GPX4 expression (scale bar is 100 ⁇ m);
  • Figure 18 is the hematoxylin and eosin staining images obtained from the heart, liver, spleen, lung and kidney of living mice in different groups (scale bar is 1000 ⁇ m);
  • Figure 19 is the measurement result of mean corpuscular hemoglobin concentration
  • Figure 20 is the mean platelet volume measurement result
  • Figure 21 is the hemoglobin concentration measurement result
  • Figure 22 is the measurement result of white blood cell count
  • Figure 23 is the mean corpuscular hemoglobin measurement result
  • Figure 24 is the analysis result of coefficient of variation of red blood cell distribution width
  • Figure 25 is the red blood cell assay result
  • Fig. 26 is platelet measurement result
  • Figure 27 is the measurement result of urea nitrogen
  • Figure 28 is the assay result of aspartate aminotransferase
  • Figure 29 is alanine aminotransferase assay result
  • Figure 30 shows the results of albumin measurement.
  • the present invention proposes a novel oxygen self-sufficient photodynamic cancer therapy strategy.
  • Vermiculite nanosheets were synthesized by a simple Li-ion intercalation method, and then the aggregation-induced luminescence luminescent agent (DCPy) was loaded onto the surface of the nanosheets by electrostatic adsorption.
  • DCPy aggregation-induced luminescence luminescent agent
  • NSs@DPCy was taken up by hypoxic tumor cells and exposed to visible light, NSs not only catalyzed the decomposition of hydrogen peroxide to generate oxygen, but also simultaneously catalyzed hydrogen peroxide and oxygen to generate multiple reactive oxygen species ROS ( ⁇ OH and 1 O 2 ).
  • ROS reactive oxygen species
  • NSs continuously produce oxygen and relieve hypoxia, which greatly enhances the efficacy of PDT.
  • NSs are able to regulate the tumor microenvironment (TME) by depleting glutathione that causes ferroptosis in tumor cells. Therefore, the present invention provides a new method for the synthesis of vermiculite nanosheet NSs, and develops a smart therapeutic platform based on vermiculite nanosheet NSs for iron poisoning assisted oxygen self-sufficient photodynamic cancer therapy.
  • a self-oxygenated photosensitizer the structure of the self-oxygenated photosensitizer includes vermiculite nanosheets and an aggregation-induced luminescent luminescent agent DCPy loaded on the surface of the vermiculite nanosheets, and the structural formula of DCPy is
  • the number of layers of the vermiculite nanosheets is 1 layer, and the loading amount of the aggregation-induced luminescence luminescent agent is 10.1w/w% of the weight of the vermiculite nanosheets.
  • the preparation method comprises the following steps:
  • the concentration of DCPy was determined by subtracting the corresponding NSs peak from the UV-Vis absorption spectrum at 452 nm.
  • the present invention also provides the application of the above-mentioned oxygen-supplying photosensitizer in the preparation of photodynamic therapy drugs.
  • a self-oxygenated photosensitizer includes vermiculite nanosheets and an aggregation-induced luminescent luminescent agent DCPy loaded on the surface of the vermiculite nanosheets.
  • DCPy The structural formula of DCPy is
  • the number of layers of the vermiculite nanosheets is 2 layers, and the loading amount of the aggregation-induced luminescence luminescent agent is 12w/w% of the weight of the vermiculite nanosheets.
  • the preparation method comprises the following steps:
  • the concentration of DCPy was determined by subtracting the corresponding NSs peak from the UV-Vis absorption spectrum at 452 nm.
  • the present invention also provides the application of the above-mentioned oxygen-supplying photosensitizer in the preparation of photodynamic therapy drugs.
  • a self-oxygenated photosensitizer includes vermiculite nanosheets and an aggregation-induced luminescent luminescent agent DCPy loaded on the surface of the vermiculite nanosheets.
  • DCPy The structural formula of DCPy is
  • the number of layers of the vermiculite nanosheets is 3 layers, and the loading amount of the aggregation-induced luminescence luminescent agent is 15w/w% of the weight of the vermiculite nanosheets.
  • the preparation method comprises the following steps:
  • the concentration of DCPy was determined by subtracting the corresponding NSs peak from the UV-Vis absorption spectrum at 452 nm.
  • the present invention also provides the application of the above-mentioned oxygen-supplying photosensitizer in the preparation of photodynamic therapy drugs.
  • a self-oxygenated photosensitizer includes vermiculite nanosheets and an aggregation-induced luminescent luminescent agent DCPy loaded on the surface of the vermiculite nanosheets.
  • DCPy The structural formula of DCPy is
  • the number of layers of the vermiculite nanosheets is 1 layer, and the loading amount of the aggregation-induced luminescence luminescent agent is 10w/w% of the weight of the vermiculite nanosheets.
  • the preparation method comprises the following steps:
  • the concentration of DCPy was determined by subtracting the corresponding NSs peak from the UV-Vis absorption spectrum at 452 nm.
  • the present invention also provides the application of the above-mentioned oxygen-supplying photosensitizer in the preparation of photodynamic therapy drugs.
  • Extracellular O 2 production test NSs@DCPy, DCPy, and NSs at a final concentration of 0.2 mg/mL were added to a H 2 O 2 solution with a final concentration of 10 mM, and the O 2 produced by the solution was measured with a dissolved oxygen meter.
  • the results are shown in Figure 8. It can be seen that the oxygen production of NSs@DCPy group is much larger than that of DCPy group and NSs group. It can be proved that the self-oxygenation photosensitizer provided by the present invention has excellent oxygen supply capacity.
  • the AIE photosensitizer DCPy can be attracted by static electricity and adsorbed on the negatively charged surface of NSs, as shown in Figure 1 shown.
  • Figure 2 and Figure 3 are the characterization of the surface morphology of NSs@DCPy by transmission electron microscope (TEM) and atomic force microscope (AFM), respectively. It can be seen from Figure 4 that the average width of NSs@DCPy is 320nm, and the average height of NSs is about 1.2nm.
  • the reason for the slight thickening of NSs is that there is a small amount of DCPy coating on the surface of NSs.
  • the coating amount of DCPy on the NSs surface was ⁇ 10.1% (w/w %) of NSs@DCPy, which was determined by the absorbance of NSs@DCPy.
  • NSs@DCPy The optical properties of NSs@DCPy were evaluated by ultraviolet-visible-near-infrared spectroscopy and fluorescence spectroscopy, as shown in Figure 5, where labels 1-3 represent the absorbance of NSs, DCPy, and NSs@DCPy, and labels 4-6 The distributions represent the fluorescence emission intensities of NSs, DCPy, and NSs@DCPy, and it can be seen from the results that NSs@DCPy exhibits a broad absorption band from ultraviolet to near-infrared.
  • NSs@DCPy has a broad luminescence band at 672 nm, and the fluorescence of the loaded DCPy is partially quenched, which may be caused by the strong interaction between DCPy and vermiculite NSs.
  • the chemical composition and crystal structure of NSs@DCPy were further confirmed by X-ray photoelectron spectroscopy (XPS), as shown in Figure 6, 1 represents the X-ray photoelectron spectrum of NSs, and 2 represents the X-ray photoelectron spectrum of NSs@DCPy It can be seen from Figure 6 that the peak positions of NSs@DCPy and NSs are consistent, indicating that the main elements of the two materials are the same.
  • XPS X-ray photoelectron spectroscopy
  • the zeta potential of DCPy in phosphate-buffered saline is +12.8 mV, and it can be speculated that its positive charge characteristics will promote the adsorption of negatively charged NSs materials through electrostatic interaction.
  • the zeta potential of NSs was measured ( Figure 7), and the surface charge of NSs@DCPy was observed to increase compared with the previous NSs ( ⁇ 40.3 mV vs ⁇ 45.4 mV), indicating that NSs were successfully modified with DCPy.
  • the ordinate represents the oxygen content, indicating that the redox reaction between Fe 3+ and H 2 O 2 can continuously generate O 2 , alleviating the hypoxia in the tumor microenvironment.
  • the very reliable electron paramagnetic resonance (EPR) technique was used to further detect the species of ROS. Both ROS ( ⁇ OH and 1 O 2 ) exhibited obvious EPR signals, further illustrating that NSs@DCPy has a strong ROS generating ability (Fig. 9, 10).
  • this example uses NSs@DCPy and different commercial organelle-specific fluorescent probes (Mito Tracker Green for mitochondrial staining, ER Tracker Green for MC38 cells were co-stained for endoplasmic reticulum staining and Lyso Tracker Green for lysosome staining).
  • the results showed that the red fluorescence of NSs@DCPy had high co-localization with the green fluorescence of Mito Tracker green, ER Tracker green and Lyso Tracker green (Pearson correlation coefficient of mitochondria was 0.900, Pearson correlation coefficient of endoplasmic reticulum was 0.906, The Pearson correlation coefficient of the enzyme body is 0.846).
  • Colocalization experiments indicated that NSs@DCPy could enter cancer cells and accumulate widely in multiple organelles.
  • mice Divide MC38 tumor-bearing BALB/c mice into 8 groups (5 mice in each group): (1) PBS (20 ⁇ L); (2) PBS (20 ⁇ L) + (white light 90 mW/cm2, 5 min);(3) NSs (20 mg/kg, 20 ⁇ L);(4) NSs (20 mg/kg, 20 ⁇ L) +(white light 90 mW/cm2, 5 min);(5) DCPy (20 mg/ kg, 20 ⁇ L); (6) DCPy (20 mg/kg, 20 ⁇ L) +(white light 90 mW/cm2, 5 min);(7) NSs@DCPy (20 mg/kg, 20 ⁇ L);(8) NSs@DCPy (20 mg/kg, 20 ⁇ L) +(white light 90mw/cm2, 5 min).
  • the mice received intravenous injections of different solutions. After 12 h, the tumor area was irradiated with white light. Mouse tumor volume and weight were recorded every 2 days. The mice were sacrificed on day 14 to obtain major organs, blood and tumors
  • Figure 12 is the time-lapse bioimaging of BALB/c mice, in which the tumor site is marked with a white dotted line
  • Figure 13 is the bioimaging of the main organs and tumors of the mice after 24 hours of intravenous injection, wherein H represents the heart; Li Denotes liver; S, spleen; Lu, lung; K, kidney; T, tumor.
  • H represents the heart
  • Li Denotes liver
  • S spleen
  • Lu lung
  • K kidney
  • T tumor.
  • the average fluorescence intensity of DCPy and NSs@DPCy measured in Figures 12 and 13 in major organs and tumor sites was processed into a data map, and Figure 14 was obtained.
  • tumor-bearing mice were randomly divided into the following treatment groups: (1) PBS (20 ⁇ L); (2) PBS (20 ⁇ L) + (white light 90 mW/ cm 2 , 5 min); (3) NSs (20 mg/kg, 20 ⁇ L); (4) NSs (20 mg/kg, 20 ⁇ L) + (white light 900 mW/cm 2 , 5 min); (5) DCPy (20 mg/kg, 20 ⁇ L) ;(6) DCPy (20 mg/kg, 20 ⁇ L) +(white light 900 mW/cm 2 , 5 min);(7) NSs@DCPy (20 mg/kg, 20 ⁇ L);(8) NSs@DCPy ( 20 mg/kg, 20 ⁇ L) + (white light 900 mW/cm 2 , 5 min).
  • mice in groups (4), (6), and (8) irradiated the tumor area with white light (90 mW/cm 2 ) for 5 min 12 hours after injection of the therapeutic drug, and groups (1) and (7) did not receive light. irradiated.
  • Tumor volume and body weight were recorded every other day during the 14 days of treatment. The results of tumor volume recording are shown in Figure 15, where dark means no light irradiation, and light means light irradiation.
  • the tumor volumes of the PBS group, the NSs+light group and the NSs@DCPy(dark) group increased rapidly with almost the same increase.
  • mice in the NSs@DCPy + light treatment group were sacrificed on the first day after complete tumor ablation.
  • Hematoxylin-eosin staining (H&E) and immunohistochemical staining were used to detect tumor proliferation activity.
  • FIG. 18 The H&E staining images of major organs (heart, liver, spleen, lung, and kidney) are shown in Figure 18. It can be seen from Figure 18 that no matter whether NSs, DCPy or NSs@DCPy are treated under the condition of no light or light, the The H&E staining results of the main organs of the mouse, heart, liver, spleen, lung, and kidney, were similar to those of the PBS group, and the morphology was intact, which further confirmed the good biocompatibility of NSs@DCPy ( Figure 18).
  • the self-supplying photosensitizer provided by the present invention can not only improve the effect of photodynamic therapy, but more importantly, the self-supplying photosensitizer controls the fixed-point and timed release of oxygen through vermiculite nanomaterials, and the self-supplying photosensitizer
  • the agent will automatically gather at the tumor site, can be effectively used in the diagnosis and treatment of cancer cells and tumors, and has a good prospect for in vivo biomedical application;
  • the self-oxygenated photosensitizer provided by the present invention has good biocompatibility and high specific surface area, and can be used in Visual photodynamic therapy has a good application prospect;
  • the self-oxygenated photosensitizer provided by the invention has rich raw materials, is easy to prepare, has a wide range of applications, and can be used in industrial production.

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Abstract

Provided are an oxygen self-supply photosensitizer, and a preparation method therefor and an application thereof. The structure of the oxygen self-supply photosensitizer comprises a vermiculite nano-sheet and an aggregation-induced emission luminogen loaded on the surface of the vermiculite nano-sheet. According to the prepared oxygen self-supply photosensitizer, the biocompatibility of a near-infrared photosensitizer can be improved, oxygen can be supplied as required to alleviate the problem of tissue hypoxia, and deep photodynamic therapy can also be implemented by means of effective delivery of the near-infrared photosensitizer.

Description

一种自供氧光敏剂及其制备方法和应用A self-oxygenated photosensitizer and its preparation method and application 技术领域technical field
本发明属于光敏剂技术领域，具体涉及一种自供氧光敏剂的制备方法及应用。The invention belongs to the technical field of photosensitizers, and in particular relates to a preparation method and application of a self-oxygenated photosensitizer.
背景技术Background technique
癌症一直是危害人类健康的疾病之一,近年来光动力治疗(PDT)由于微创、副作用小、空间选择性强等优点而广受关注。实体瘤缺氧、药物在肿瘤处富集效果差、肿瘤内药物渗透性差以及激发光敏剂(PS)的光源穿透深度有限等问题成为限制PDT发展的最重要因素。目前,利用肿瘤微环境(TME)触发或光触发释放药物、光敏剂和氧气等的纳米复合诊疗材料成为研究热点。Cancer has always been one of the diseases that endanger human health. In recent years, photodynamic therapy (PDT) has attracted widespread attention due to its advantages of minimal invasiveness, small side effects, and strong space selectivity. Hypoxia in solid tumors, poor accumulation of drugs in tumors, poor drug penetration in tumors, and limited penetration depth of light sources that excite photosensitizers (PS) have become the most important factors that limit the development of PDT. At present, the use of tumor microenvironment (TME)-triggered or light-triggered release of drugs, photosensitizers and oxygen nanocomposite therapeutic materials has become a research hotspot.
现阶段，光敏剂虽然得到了广泛的改进，但在复杂的生理条件和高活性氧环境下仍存在暗毒性和光稳定性差等缺点。由于其固有的刚性平面和疏水结构，大多数的光敏剂经常遭受聚集诱导的荧光猝灭和聚集态的活性氧产量急剧下降。在一定程度上限制了PDT的抗肿瘤应用。并且，现有技术存在所需的纳米复合材料制备复杂，载氧率低，氧释放不受控制等问题。At present, although photosensitizers have been extensively improved, they still have the disadvantages of dark toxicity and poor photostability under complex physiological conditions and high reactive oxygen species environment. Due to their inherent rigid planar and hydrophobic structures, most photosensitizers often suffer from aggregation-induced fluorescence quenching and a sharp drop in the production of reactive oxygen species in the aggregated state. To some extent, it limits the anti-tumor application of PDT. Moreover, the prior art has problems such as complex preparation of the required nanocomposite material, low oxygen loading rate, and uncontrolled oxygen release.
技术问题technical problem
为了克服上述现有技术的缺陷，本发明提出了一种近红外光控制按需供氧蛭石纳米片诊疗剂，既能改善近红外光敏剂的生物相容性，又能使该纳米诊疗剂按需供氧改善组织乏氧问题，也能够通过近红外光敏剂的有效递送实现深层光动力治疗。In order to overcome the defects of the above-mentioned prior art, the present invention proposes a near-infrared light-controlled on-demand oxygen-supplying vermiculite nanosheet diagnostic agent, which can not only improve the biocompatibility of the near-infrared photosensitizer, but also make the nano-diagnostic agent Oxygen on demand improves tissue hypoxia and enables deep photodynamic therapy through the effective delivery of near-infrared photosensitizers.
技术解决方案technical solution
一种自供氧光敏剂，所述自供氧光敏剂的结构包括蛭石纳米片以及负载于所述蛭石纳米片表面的聚集诱导发光发光剂。A self-oxygenated photosensitizer, the structure of the self-oxygenated photosensitizer includes vermiculite nanosheets and an aggregation-induced luminescent luminescent agent loaded on the surface of the vermiculite nanosheets.
进一步地，所述聚集诱导发光发光剂为DCPy、DCMa、DCIs、DCFu中的一种或多种，Further, the aggregation-induced luminescent luminescent agent is one or more of DCPy, DCMa, DCIs, DCFu,
其中，所述DCPy的结构式为Wherein, the structural formula of the DCPy is
Figure dest_path_image001
，
Figure dest_path_image001
,
所述DCMa的结构式为The structural formula of the DCMa is
Figure dest_path_image002
，
Figure dest_path_image002
,
所述DCIs的结构式为The structural formula of the DCIs is
Figure dest_path_image003
，
Figure dest_path_image003
,
所述DCFu的结构式为The structural formula of the DCFu is
Figure dest_path_image004
。
Figure dest_path_image004
.
优选地，所述聚集诱导发光发光剂为DCPy。Preferably, the aggregation-induced luminescence luminescent agent is DCPy.
进一步地，所述蛭石纳米片为单层结构或多层结构。Further, the vermiculite nanosheets have a single-layer structure or a multi-layer structure.
进一步地，所述聚集诱导发光发光剂的负载量为所述蛭石纳米片重量的10-15 w/w%。Further, the loading amount of the aggregation-induced luminescent luminescent agent is 10-15% of the weight of the vermiculite nanosheets. w/w%.
本发明还提供上述自供氧光敏剂的制备方法，包括以下步骤：The present invention also provides a preparation method of the above-mentioned self-oxygenated photosensitizer, comprising the following steps:
S1：通过锂离子插层法合成蛭石纳米片；S1: Synthesis of vermiculite nanosheets by lithium ion intercalation;
S2：将聚集诱导发光发光剂负载到所述蛭石纳米片表面，得到自供氧光敏剂。S2: loading the aggregation-induced luminescence luminescence agent on the surface of the vermiculite nanosheets to obtain a self-oxygenation photosensitizer.
进一步地，步骤S1包括：将蛭石加入到锂盐溶液改性剂中得到蛭石纳米片。Further, step S1 includes: adding vermiculite to the lithium salt solution modifier to obtain vermiculite nanosheets.
优选地，所述碱金属盐溶液改性剂为氯化锂溶液、乙二胺四乙酸锂溶液或柠檬酸锂溶液中的一种或多种。Preferably, the alkali metal salt solution modifier is one or more of lithium chloride solution, lithium ethylenediaminetetraacetate solution or lithium citrate solution.
进一步地，步骤S2中，蛭石纳米片的浓度与聚集诱导发光发光剂的浓度比为2：（0.1-100）。Further, in step S2, the ratio of the concentration of the vermiculite nanosheets to the concentration of the aggregation-induced luminescence luminescent agent is 2: (0.1-100).
进一步地，步骤S2中，通过静电吸附将聚集诱导发光发光剂负载到所述蛭石纳米片表面。Further, in step S2, the aggregation-induced luminescence luminescent agent is loaded onto the surface of the vermiculite nanosheets by electrostatic adsorption.
本发明还提供上述自供氧光敏剂在制备光动力治疗药物上的应用。The present invention also provides the application of the above self-oxygenated photosensitizer in the preparation of photodynamic therapy drugs.
有益效果Beneficial effect
本发明的有益效果包括以下几个方面：The beneficial effects of the present invention include the following aspects:
1.本发明提供的自供氧光敏剂不仅能够提高光动力治疗的效果，更重要的是该自供氧光敏剂是通过蛭石纳米材料控制氧气的定点定时释放，能够有效用于癌细胞和肿瘤的诊疗中，具有较好的体内生物医学应用前景；1. The self-oxygenated photosensitizer provided by the present invention can not only improve the effect of photodynamic therapy, but more importantly, the self-oxygenated photosensitizer controls the fixed-point and timed release of oxygen through vermiculite nanomaterials, and can be effectively used for cancer cells and In the diagnosis and treatment of tumors, it has a good prospect of in vivo biomedical application;
2.本发明提供的自供氧光敏剂生物相容性好，比表面积高，在可视化光动力治疗方面具有良好的应用前景；2. The self-oxygenated photosensitizer provided by the present invention has good biocompatibility and high specific surface area, and has good application prospects in visual photodynamic therapy;
3.本发明提供的自供氧光敏剂原料丰富，易于制备，适用范围广，可用于工业化生产。3. The oxygen-supplied photosensitizer provided by the present invention has rich raw materials, is easy to prepare, has a wide range of applications, and can be used in industrial production.
附图说明Description of drawings
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，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的附图。In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application. For those skilled in the art, other drawings can also be obtained based on these drawings without creative effort.
图1为实施例1制备的自供氧光敏剂的结构示意图；Fig. 1 is the structural representation of the self-supply oxygen photosensitizer prepared by embodiment 1;
图2为实施例1制备的自供氧光敏剂的透射电子显微镜图；Fig. 2 is the transmission electron micrograph of the self-supply oxygen photosensitizer prepared in embodiment 1;
图3为实施例1制备的自供氧光敏剂的原子力显微镜图；Fig. 3 is the atomic force microscope figure of the self-supply oxygen photosensitizer prepared by embodiment 1;
图4为实施例1制备的自供氧光敏剂的平均粒径和平均高度；Fig. 4 is the average particle diameter and the average height of the self-oxygenated photosensitizer prepared by embodiment 1;
图5为实施例1制备的自供氧光敏剂的可见-近红外光谱和荧光光谱；Fig. 5 is the visible-near-infrared spectrum and the fluorescent spectrum of the self-supplied oxygen photosensitizer prepared in embodiment 1;
图6为实施例1制备的自供氧光敏剂的X射线光电子能谱图；Fig. 6 is the X-ray photoelectron energy spectrogram of the self-supplied oxygen photosensitizer prepared in embodiment 1;
图7为实施例1制备的自供氧光敏剂的Zeta电位图；Fig. 7 is the Zeta potential diagram of the self-supplied oxygen photosensitizer prepared in embodiment 1;
图8为实施例1制备的自供氧光敏剂的氧气生产浓度；Fig. 8 is the oxygen production concentration of the self-supplying photosensitizer prepared in embodiment 1;
图9为实施例1制备的自供氧光敏剂的 1O 2电子自旋共振振幅； Figure 9 is the 1 O electron spin resonance amplitude of the self-supplied oxygen photosensitizer prepared in Example 1;
图10为实施例1制备的自供氧光敏剂的·OH电子自旋共振振幅;Figure 10 is the OH electron spin resonance amplitude of the self-supplied oxygen photosensitizer prepared in Example 1;
图11为实验例3的激光共聚焦扫描显微镜（CLSM）图；Figure 11 is a confocal laser scanning microscope (CLSM) image of Experimental Example 3;
图12为Balb/c小鼠的延时生物成像；Figure 12 is a time-lapse biological imaging of Balb/c mice;
图13为24小时静脉注射后主要器官和肿瘤的生物成像；Figure 13 is the biological imaging of major organs and tumors after 24 hours of intravenous injection;
图14为DCPy和NSs@DPCy在小鼠的主要器官和肿瘤上的平均荧光强度测量结果；Figure 14 is the measurement results of the average fluorescence intensity of DCPy and NSs@DPCy on the main organs and tumors of mice;
图15为对活体小鼠全身给药PBS、NSs、DCPy和NSs-DCPy在不用光照射或用光照射5分钟后的肿瘤生长曲线图；Fig. 15 is a graph of tumor growth curves after systemic administration of PBS, NSs, DCPy and NSs-DCPy to living mice without light irradiation or with light irradiation for 5 minutes;
图16为Caspase3表达的免疫组化分析结果图（比例尺为100μm）；Figure 16 is the results of immunohistochemical analysis of Caspase3 expression (scale bar is 100 μm);
图17为GPX4表达的免疫组织化学分析结果图（比例尺为100μm）；Figure 17 is the results of immunohistochemical analysis of GPX4 expression (scale bar is 100 μm);
图18为从不同组的活体小鼠的心脏、肝脏、脾脏、肺和肾脏获得的苏木精和伊红染色图像（比例尺为1000μm）；Figure 18 is the hematoxylin and eosin staining images obtained from the heart, liver, spleen, lung and kidney of living mice in different groups (scale bar is 1000 μm);
图19为平均红细胞血红蛋白浓度测定结果；Figure 19 is the measurement result of mean corpuscular hemoglobin concentration;
图20为平均血小板体积测定结果；Figure 20 is the mean platelet volume measurement result;
图21为血红蛋白浓度测定结果；Figure 21 is the hemoglobin concentration measurement result;
图22为白细胞计数测定结果；Figure 22 is the measurement result of white blood cell count;
图23为平均红细胞血红蛋白测定结果；Figure 23 is the mean corpuscular hemoglobin measurement result;
图24为红细胞分布宽度变异系数分析结果；Figure 24 is the analysis result of coefficient of variation of red blood cell distribution width;
图25为红细胞测定结果；Figure 25 is the red blood cell assay result;
图26为血小板测定结果；Fig. 26 is platelet measurement result;
图27为尿素氮测定结果；Figure 27 is the measurement result of urea nitrogen;
图28为天冬氨酸转氨酶测定结果；Figure 28 is the assay result of aspartate aminotransferase;
图29为谷丙转氨酶测定结果；Figure 29 is alanine aminotransferase assay result;
图30为白蛋白测定结果。Figure 30 shows the results of albumin measurement.
本发明的实施方式Embodiments of the present invention
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，所描述的实施例仅是本发明的一部分实施例，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前提下所获得的所有其他实施例，都属于本发明保护的范围。The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
由于肿瘤的光动力治疗(PDT)的效率依赖于氧气浓度，所以PDT治疗技术受到缺氧的限制。为了克服肿瘤缺氧诱导的PDT耐药，本发明提出了一种新的氧自给光动力癌症治疗策略。采用简单的锂离子插层法合成了蛭石纳米片(NSs)，然后通过静电吸附将聚集诱导发光发光剂(DCPy)负载到纳米片表面。NSs@DPCy一旦被缺氧的肿瘤细胞吸收，暴露在可见光下，NSs不仅催化过氧化氢分解生成氧气，还同时催化过氧化氢和氧气生成多个活性氧ROS(·OH和 1O 2)。此外，NSs持续产生氧气，缓解缺氧，极大地提高了PDT的疗效。值得关注的是，NSs能够通过消耗引起肿瘤细胞铁下垂的谷胱甘肽来调节肿瘤微环境(TME)。因此，本发明为蛭石纳米片NSs的合成提供了一种新的方法，而且开发了一种基于蛭石纳米片NSs的智能治疗平台，用于铁中毒辅助氧自给光动力癌症治疗。 Since the efficiency of photodynamic therapy (PDT) of tumors is dependent on oxygen concentration, PDT treatment techniques are limited by hypoxia. To overcome tumor hypoxia-induced PDT resistance, the present invention proposes a novel oxygen self-sufficient photodynamic cancer therapy strategy. Vermiculite nanosheets (NSs) were synthesized by a simple Li-ion intercalation method, and then the aggregation-induced luminescence luminescent agent (DCPy) was loaded onto the surface of the nanosheets by electrostatic adsorption. Once NSs@DPCy was taken up by hypoxic tumor cells and exposed to visible light, NSs not only catalyzed the decomposition of hydrogen peroxide to generate oxygen, but also simultaneously catalyzed hydrogen peroxide and oxygen to generate multiple reactive oxygen species ROS (·OH and 1 O 2 ). In addition, NSs continuously produce oxygen and relieve hypoxia, which greatly enhances the efficacy of PDT. Interestingly, NSs are able to regulate the tumor microenvironment (TME) by depleting glutathione that causes ferroptosis in tumor cells. Therefore, the present invention provides a new method for the synthesis of vermiculite nanosheet NSs, and develops a smart therapeutic platform based on vermiculite nanosheet NSs for iron poisoning assisted oxygen self-sufficient photodynamic cancer therapy.
实施例1Example 1
一种自供氧光敏剂，自供氧光敏剂的结构包括蛭石纳米片以及负载于蛭石纳米片表面的聚集诱导发光发光剂DCPy，DCPy的结构式为A self-oxygenated photosensitizer, the structure of the self-oxygenated photosensitizer includes vermiculite nanosheets and an aggregation-induced luminescent luminescent agent DCPy loaded on the surface of the vermiculite nanosheets, and the structural formula of DCPy is
Figure dest_path_image005
，
Figure dest_path_image005
,
蛭石纳米片的层数为1层，聚集诱导发光发光剂的负载量为蛭石纳米片重量的10.1w/w%。The number of layers of the vermiculite nanosheets is 1 layer, and the loading amount of the aggregation-induced luminescence luminescent agent is 10.1w/w% of the weight of the vermiculite nanosheets.
制备方法包括以下步骤：The preparation method comprises the following steps:
S1、通过锂离子插层法合成蛭石纳米片：S1. Synthesis of vermiculite nanosheets by lithium ion intercalation method:
S2、将聚集诱导发光发光剂负载到蛭石纳米片表面：将200μg/mL蛭石纳米片(NSs)分散体与浓度为1.0mg/mL的DCPy在PBS中混合，超声处理30 min，室温搅拌过夜。多余的卸料在Amicon管(MWCO 100kDa;Millipore)， 3500 rpm, 30分钟，然后用PBS洗三次，得到自供氧光敏剂。S2. Load the aggregation-induced luminescence luminescent agent on the surface of vermiculite nanosheets: mix 200 μg/mL vermiculite nanosheets (NSs) dispersion with DCPy at a concentration of 1.0 mg/mL in PBS, sonicate for 30 min, and stir at room temperature overnight. The excess unloaded in an Amicon tube (MWCO 100kDa; Millipore), 3500 rpm, 30 minutes, and then washed three times with PBS to obtain a self-oxygenated photosensitizer.
DCPy的浓度是通过在452 nm处的紫外-可见吸收光谱减去相应的NSs峰值测定。The concentration of DCPy was determined by subtracting the corresponding NSs peak from the UV-Vis absorption spectrum at 452 nm.
本发明还提供上述供氧光敏剂在制备光动力治疗药物上的应用。The present invention also provides the application of the above-mentioned oxygen-supplying photosensitizer in the preparation of photodynamic therapy drugs.
实施例2Example 2
一种自供氧光敏剂，自供氧光敏剂的结构包括蛭石纳米片以及负载于蛭石纳米片表面的聚集诱导发光发光剂DCPy，DCPy 的结构式为A self-oxygenated photosensitizer. The structure of the self-oxygenated photosensitizer includes vermiculite nanosheets and an aggregation-induced luminescent luminescent agent DCPy loaded on the surface of the vermiculite nanosheets. The structural formula of DCPy is
Figure dest_path_image006
，
Figure dest_path_image006
,
蛭石纳米片的层数为2层，聚集诱导发光发光剂的负载量为蛭石纳米片重量的12w/w%。The number of layers of the vermiculite nanosheets is 2 layers, and the loading amount of the aggregation-induced luminescence luminescent agent is 12w/w% of the weight of the vermiculite nanosheets.
制备方法包括以下步骤：The preparation method comprises the following steps:
S1、通过锂离子插层法合成蛭石纳米片：S1. Synthesis of vermiculite nanosheets by lithium ion intercalation method:
S2、将聚集诱导发光发光剂负载到蛭石纳米片表面：将200μg/mL蛭石纳米片(NSs)分散体与浓度为2.0 mg/mL的DCPy 在PBS中混合，超声处理30 min，室温搅拌过夜。多余的卸料在Amicon管(MWCO 100kDa;Millipore)， 3500 rpm, 30分钟，然后用PBS洗三次，得到自供氧光敏剂。S2. Load the aggregation-induced luminescence luminescent agent on the surface of vermiculite nanosheets: mix 200 μg/mL vermiculite nanosheets (NSs) dispersion with DCPy at a concentration of 2.0 mg/mL in PBS, sonicate for 30 min, and stir at room temperature overnight. The excess unloaded in an Amicon tube (MWCO 100kDa; Millipore), 3500 rpm, 30 minutes, and then washed three times with PBS to obtain a self-oxygenated photosensitizer.
DCPy的浓度是通过在452 nm处的紫外-可见吸收光谱减去相应的NSs峰值测定。The concentration of DCPy was determined by subtracting the corresponding NSs peak from the UV-Vis absorption spectrum at 452 nm.
本发明还提供上述供氧光敏剂在制备光动力治疗药物上的应用。The present invention also provides the application of the above-mentioned oxygen-supplying photosensitizer in the preparation of photodynamic therapy drugs.
实施例3Example 3
一种自供氧光敏剂，自供氧光敏剂的结构包括蛭石纳米片以及负载于蛭石纳米片表面的聚集诱导发光发光剂DCPy，DCPy 的结构式为A self-oxygenated photosensitizer. The structure of the self-oxygenated photosensitizer includes vermiculite nanosheets and an aggregation-induced luminescent luminescent agent DCPy loaded on the surface of the vermiculite nanosheets. The structural formula of DCPy is
Figure dest_path_image007
，
Figure dest_path_image007
,
蛭石纳米片的层数为3层，聚集诱导发光发光剂的负载量为蛭石纳米片重量的15w/w%。The number of layers of the vermiculite nanosheets is 3 layers, and the loading amount of the aggregation-induced luminescence luminescent agent is 15w/w% of the weight of the vermiculite nanosheets.
制备方法包括以下步骤：The preparation method comprises the following steps:
S1、通过锂离子插层法合成蛭石纳米片：S1. Synthesis of vermiculite nanosheets by lithium ion intercalation method:
S2、将聚集诱导发光发光剂负载到蛭石纳米片表面：将200μg/mL蛭石纳米片(NSs)分散体与浓度为10 mg/mL的DCPy在PBS中混合，超声处理20 min，室温搅拌过夜。多余的卸料在Amicon管(MWCO 100kDa;Millipore)， 3500 rpm, 30分钟，然后用PBS洗三次，得到自供氧光敏剂。S2. Load the aggregation-induced luminescence luminescent agent on the surface of vermiculite nanosheets: mix 200 μg/mL vermiculite nanosheets (NSs) dispersion with DCPy at a concentration of 10 mg/mL in PBS, sonicate for 20 min, and stir at room temperature overnight. The excess unloaded in an Amicon tube (MWCO 100kDa; Millipore), 3500 rpm, 30 minutes, and then washed three times with PBS to obtain a self-oxygenated photosensitizer.
DCPy的浓度是通过在452 nm处的紫外-可见吸收光谱减去相应的NSs峰值测定。The concentration of DCPy was determined by subtracting the corresponding NSs peak from the UV-Vis absorption spectrum at 452 nm.
本发明还提供上述供氧光敏剂在制备光动力治疗药物上的应用。The present invention also provides the application of the above-mentioned oxygen-supplying photosensitizer in the preparation of photodynamic therapy drugs.
实施例4Example 4
一种自供氧光敏剂，自供氧光敏剂的结构包括蛭石纳米片以及负载于蛭石纳米片表面的聚集诱导发光发光剂DCPy，DCPy 的结构式为A self-oxygenated photosensitizer. The structure of the self-oxygenated photosensitizer includes vermiculite nanosheets and an aggregation-induced luminescent luminescent agent DCPy loaded on the surface of the vermiculite nanosheets. The structural formula of DCPy is
Figure dest_path_image008
，
Figure dest_path_image008
,
蛭石纳米片的层数为1层，聚集诱导发光发光剂的负载量为蛭石纳米片重量的10w/w%。The number of layers of the vermiculite nanosheets is 1 layer, and the loading amount of the aggregation-induced luminescence luminescent agent is 10w/w% of the weight of the vermiculite nanosheets.
制备方法包括以下步骤：The preparation method comprises the following steps:
S1、通过锂离子插层法合成蛭石纳米片：S1. Synthesis of vermiculite nanosheets by lithium ion intercalation method:
S2、将聚集诱导发光发光剂负载到蛭石纳米片表面：将200μg/mL蛭石纳米片(NSs)分散体与浓度为0.01 mg/mL的DCPy在PBS中混合，超声处理30 min，室温搅拌过夜。多余的卸料在Amicon管(MWCO 100kDa;Millipore)， 3500 rpm, 30分钟，然后用PBS洗三次，得到自供氧光敏剂。S2. Load the aggregation-induced luminescence luminescent agent on the surface of vermiculite nanosheets: mix 200 μg/mL vermiculite nanosheets (NSs) dispersion with DCPy at a concentration of 0.01 mg/mL in PBS, sonicate for 30 min, and stir at room temperature overnight. The excess unloaded in an Amicon tube (MWCO 100kDa; Millipore), 3500 rpm, 30 minutes, and then washed three times with PBS to obtain a self-oxygenated photosensitizer.
DCPy的浓度是通过在452 nm处的紫外-可见吸收光谱减去相应的NSs峰值测定。The concentration of DCPy was determined by subtracting the corresponding NSs peak from the UV-Vis absorption spectrum at 452 nm.
本发明还提供上述供氧光敏剂在制备光动力治疗药物上的应用。The present invention also provides the application of the above-mentioned oxygen-supplying photosensitizer in the preparation of photodynamic therapy drugs.
实验例1Experimental example 1
细胞外O 2生产试验：在终浓度为10 mM的H 2O 2溶液中加入终浓度为0.2 mg/mL的NSs@DCPy、DCPy以及NSs，用溶解氧计测量溶液产生的O 2。结果参见图8，可见，NSs@DCPy组的产氧量远大于DCPy组和NSs组。可以证明，本发明提供的自供氧光敏剂具有优秀的供氧能力。 Extracellular O 2 production test: NSs@DCPy, DCPy, and NSs at a final concentration of 0.2 mg/mL were added to a H 2 O 2 solution with a final concentration of 10 mM, and the O 2 produced by the solution was measured with a dissolved oxygen meter. The results are shown in Figure 8. It can be seen that the oxygen production of NSs@DCPy group is much larger than that of DCPy group and NSs group. It can be proved that the self-oxygenation photosensitizer provided by the present invention has excellent oxygen supply capacity.
实验例2Experimental example 2
实施例1中提供的纳米片中，由于Al 3+杂质的取代引起NSs层上的负电荷，而由于静电作用，AIE光敏剂DCPy能够被静电吸引，吸附在NSs的负电荷表面，如图1所示。图2、图3分别是通过透射电子显微镜(TEM)和原子力显微镜(AFM)对NSs@DCPy的表面形貌进行表征。从图4可以看出NSs@DCPy的平均宽度为320nm，NSs的平均高度约为1.2nm。NSs微增厚的原因是NS表面有少量的DCPy包覆。NSs表面的DCPy包覆量为NSs@DCPy的≈10.1% (w/w %)，该包覆量由NSs@DCPy的吸光度测定。通过紫外-可见-近红外光谱和荧光光谱对NSs@DCPy的光学性能进行了评价，如图5所示，其中，标号1-3分别表示NSs、DCPy、NSs@DCPy的吸光度，标号4-6分布表示NSs、DCPy、NSs@DCPy的荧光发射强度，由结果可以看出，NSs@DCPy表现出从紫外到近红外的宽吸收带。此外，NSs@DCPy在672 nm处有一个较宽的发光带，并且负载的DCPy荧光部分猝灭，这可能是由于DCPy与蛭石NSs之间的强相互作用造成的。通过X射线光电子能谱(XPS)进一步证实了NSs@DCPy的化学组成和晶体结构，如图6所示，1表示NSs的X射线光电子能谱图，2表示NSs@DCPy的X射线光电子能谱图，从图6可以看出，NSs@DCPy与NSs一致的峰位，表示两种材料主要元素相同。如图7所示，DCPy在磷酸盐缓冲盐水(PBS)中的zeta电位为+12.8 mV，可以推测其正电荷特征会通过静电作用促进负电荷NSs材料的吸附。测定了NSs的zeta电位(图7)，观察到NSs@DCPy的表面电荷相对先前NSs的增加(−40.3 mV vs−45.4 mV)，说明NSs成功修饰上了DCPy。此外，如图8所示，纵坐标表示氧含量，说明Fe 3+与H 2O 2之间的氧化还原反应可以持续产生O 2，缓解了肿瘤微环境中的缺氧。考虑到·OH和 1O 2的生命周期短、化学活性高，采用非常可靠的电子顺磁共振(EPR)技术进一步检测ROS的种类。两种ROS(·OH和 1O 2)均表现出明显的EPR信号，进一步说明NSs@DCPy具有较强的ROS生成能力(图9, 10)。 In the nanosheets provided in Example 1, due to the substitution of Al 3+ impurities, the negative charge on the NSs layer is caused, and due to the electrostatic interaction, the AIE photosensitizer DCPy can be attracted by static electricity and adsorbed on the negatively charged surface of NSs, as shown in Figure 1 shown. Figure 2 and Figure 3 are the characterization of the surface morphology of NSs@DCPy by transmission electron microscope (TEM) and atomic force microscope (AFM), respectively. It can be seen from Figure 4 that the average width of NSs@DCPy is 320nm, and the average height of NSs is about 1.2nm. The reason for the slight thickening of NSs is that there is a small amount of DCPy coating on the surface of NSs. The coating amount of DCPy on the NSs surface was ≈10.1% (w/w %) of NSs@DCPy, which was determined by the absorbance of NSs@DCPy. The optical properties of NSs@DCPy were evaluated by ultraviolet-visible-near-infrared spectroscopy and fluorescence spectroscopy, as shown in Figure 5, where labels 1-3 represent the absorbance of NSs, DCPy, and NSs@DCPy, and labels 4-6 The distributions represent the fluorescence emission intensities of NSs, DCPy, and NSs@DCPy, and it can be seen from the results that NSs@DCPy exhibits a broad absorption band from ultraviolet to near-infrared. In addition, NSs@DCPy has a broad luminescence band at 672 nm, and the fluorescence of the loaded DCPy is partially quenched, which may be caused by the strong interaction between DCPy and vermiculite NSs. The chemical composition and crystal structure of NSs@DCPy were further confirmed by X-ray photoelectron spectroscopy (XPS), as shown in Figure 6, 1 represents the X-ray photoelectron spectrum of NSs, and 2 represents the X-ray photoelectron spectrum of NSs@DCPy It can be seen from Figure 6 that the peak positions of NSs@DCPy and NSs are consistent, indicating that the main elements of the two materials are the same. As shown in Figure 7, the zeta potential of DCPy in phosphate-buffered saline (PBS) is +12.8 mV, and it can be speculated that its positive charge characteristics will promote the adsorption of negatively charged NSs materials through electrostatic interaction. The zeta potential of NSs was measured (Figure 7), and the surface charge of NSs@DCPy was observed to increase compared with the previous NSs (−40.3 mV vs −45.4 mV), indicating that NSs were successfully modified with DCPy. In addition, as shown in Figure 8, the ordinate represents the oxygen content, indicating that the redox reaction between Fe 3+ and H 2 O 2 can continuously generate O 2 , alleviating the hypoxia in the tumor microenvironment. Considering the short lifetime and high chemical activity of OH and 1O2 , the very reliable electron paramagnetic resonance (EPR) technique was used to further detect the species of ROS. Both ROS (·OH and 1 O 2 ) exhibited obvious EPR signals, further illustrating that NSs@DCPy has a strong ROS generating ability (Fig. 9, 10).
实验例3Experimental example 3
为了探索制备的NSs@DCPy在活细胞中的亚细胞位置(图11)，本实施例用NSs@DCPy和不同的商业细胞器特异性荧光探针(Mito Tracker Green用于线粒体染色，ER Tracker Green用于内质网染色，Lyso Tracker Green用于溶酶体染色)对MC38细胞进行了共染色。结果表明，NSs@DCPy的红色荧光与Mito Tracker green、ER Tracker green和Lyso Tracker green的绿色荧光具有较高的共定位(线粒体的Pearson相关系数为0.900，内质网的Pearson相关系数为0.906，溶酶体的Pearson相关系数为0.846)。共定位实验表明NSs@DCPy能进入癌细胞并在多个细胞器中广泛积累。In order to explore the subcellular location of the prepared NSs@DCPy in living cells (Figure 11), this example uses NSs@DCPy and different commercial organelle-specific fluorescent probes (Mito Tracker Green for mitochondrial staining, ER Tracker Green for MC38 cells were co-stained for endoplasmic reticulum staining and Lyso Tracker Green for lysosome staining). The results showed that the red fluorescence of NSs@DCPy had high co-localization with the green fluorescence of Mito Tracker green, ER Tracker green and Lyso Tracker green (Pearson correlation coefficient of mitochondria was 0.900, Pearson correlation coefficient of endoplasmic reticulum was 0.906, The Pearson correlation coefficient of the enzyme body is 0.846). Colocalization experiments indicated that NSs@DCPy could enter cancer cells and accumulate widely in multiple organelles.
实验例4Experimental example 4
将MC38荷瘤BALB/c小鼠分为8组(每组5只):(1)PBS (20 μL);(2) PBS (20 μL) +(白光90 mW/cm2, 5 min);(3) NSs (20 mg/kg, 20 μL);(4) NSs (20 mg/kg, 20 μL) +(白光90 mW/cm2, 5 min);(5) DCPy (20 mg/kg, 20 μL);(6) DCPy (20 mg/kg, 20 μL) +(白光90 mW/cm2, 5 min);(7) NSs@DCPy (20 mg/kg, 20 μL);(8) NSs@DCPy (20 mg/kg, 20 μL) +(白光90mw /cm2, 5 min)。这些小鼠接受了不同溶液的静脉注射。12 h后，用白光照射肿瘤区域。每2天记录小鼠肿瘤体积和重量。这些小鼠在第14天被处死，以获取主要器官、血液和肿瘤进行检查。Divide MC38 tumor-bearing BALB/c mice into 8 groups (5 mice in each group): (1) PBS (20 μL); (2) PBS (20 μL) + (white light 90 mW/cm2, 5 min);(3) NSs (20 mg/kg, 20 μL);(4) NSs (20 mg/kg, 20 μL) +(white light 90 mW/cm2, 5 min);(5) DCPy (20 mg/ kg, 20 μL); (6) DCPy (20 mg/kg, 20 μL) +(white light 90 mW/cm2, 5 min);(7) NSs@DCPy (20 mg/kg, 20 μL);(8) NSs@DCPy (20 mg/kg, 20 μL) +(white light 90mw/cm2, 5 min). The mice received intravenous injections of different solutions. After 12 h, the tumor area was irradiated with white light. Mouse tumor volume and weight were recorded every 2 days. The mice were sacrificed on day 14 to obtain major organs, blood and tumors for examination.
评估DCPy和NSs@DCPy在MC38荷瘤小鼠(n=3/组)中的生物分布。在注射后1、12和24小时，一个带有Cy5通道的IVIS小动物成像***来可视化这些动物的生物分布概况。24 h后处死另一组小鼠，采集肿瘤及主要器官标本进行组织特异性成像(图12-14)。图12为BALB/c小鼠的延时生物成像，其中，肿瘤部位用白色虚线标出；图13是24小时静脉注射后小鼠的主要器官和肿瘤的生物成像，其中，H表示心脏；Li表示肝脏；S表示脾脏；Lu表示肺；K表示肾；T表示肿瘤。将图12、13测量得到的DCPy和NSs@DPCy在主要器官和肿瘤部位的平均荧光强度处理成数据图，得图14，由图14可以看出，NSs@DCPy组比DCPy组的信号更强，进一步证明NSs@DCPy通过血液循环的体内靶向作用和药物载体NSs的增强渗透性保留(EPR)作用。The biodistribution of DCPy and NSs@DCPy in MC38 tumor-bearing mice (n=3/group) was evaluated. An IVIS small animal imaging system with a Cy5 channel was used to visualize the biodistribution profile of these animals at 1, 12 and 24 hours post-injection. Another group of mice was sacrificed 24 hours later, and tumor and major organ samples were collected for tissue-specific imaging (Fig. 12-14). Figure 12 is the time-lapse bioimaging of BALB/c mice, in which the tumor site is marked with a white dotted line; Figure 13 is the bioimaging of the main organs and tumors of the mice after 24 hours of intravenous injection, wherein H represents the heart; Li Denotes liver; S, spleen; Lu, lung; K, kidney; T, tumor. The average fluorescence intensity of DCPy and NSs@DPCy measured in Figures 12 and 13 in major organs and tumor sites was processed into a data map, and Figure 14 was obtained. It can be seen from Figure 14 that the signal of the NSs@DCPy group is stronger than that of the DCPy group , further demonstrating the in vivo targeting effect of NSs@DCPy through blood circulation and the enhanced permeability retention (EPR) effect of drug carrier NSs.
为进一步评价NSs@DCPy抗肿瘤作用，将荷瘤小鼠随机分为以下治疗组:(1)PBS (20μL);(2) PBS (20μL) +(白光90 mW/ cm 2, 5 min);(3) NSs (20 mg/kg, 20μL);(4) NSs (20 mg/kg, 20μL) +(白光900 mW/cm 2, 5 min);(5) DCPy (20 mg/kg, 20μL);(6) DCPy (20 mg/kg, 20 μL) +(白光900 mW/cm 2, 5 min);(7) NSs@DCPy (20 mg/kg, 20 μL);(8) NSs@DCPy (20 mg/kg, 20 μL) +(白光900 mW/cm 2, 5 min)。 To further evaluate the anti-tumor effect of NSs@DCPy, tumor-bearing mice were randomly divided into the following treatment groups: (1) PBS (20 μL); (2) PBS (20 μL) + (white light 90 mW/ cm 2 , 5 min); (3) NSs (20 mg/kg, 20μL); (4) NSs (20 mg/kg, 20μL) + (white light 900 mW/cm 2 , 5 min); (5) DCPy (20 mg/kg, 20μL) ;(6) DCPy (20 mg/kg, 20 μL) +(white light 900 mW/cm 2 , 5 min);(7) NSs@DCPy (20 mg/kg, 20 μL);(8) NSs@DCPy ( 20 mg/kg, 20 μL) + (white light 900 mW/cm 2 , 5 min).
（4）、（6）、（8）组小鼠在注射治疗药物12 h后，将白光(90 mW/cm 2)照射小鼠肿瘤区域5 min，（1）和（7）组不进行光照射。在14天的治疗过程中，每隔一天记录肿瘤体积和体重。肿瘤体积记录结果见图15所示，其中，dark表示无光照射，light表示有光照射。PBS组、NSs + light组和NSs@DCPy（dark）组的肿瘤体积迅速增加，增幅几乎相同。说明与对照组（PBS组）相比，光单独照射NSs或不照射NSs@DCPy均不能抑制肿瘤生长。DCPy + light组(仅PDT)的治疗效果优于NSs+ light组(仅铁下垂)，这可能与NSs浓度有限有关。NSs@DCPy + light组(在PDT和铁下垂双重协同效果下)表现出了最佳的肿瘤抑制效果。 The mice in groups (4), (6), and (8) irradiated the tumor area with white light (90 mW/cm 2 ) for 5 min 12 hours after injection of the therapeutic drug, and groups (1) and (7) did not receive light. irradiated. Tumor volume and body weight were recorded every other day during the 14 days of treatment. The results of tumor volume recording are shown in Figure 15, where dark means no light irradiation, and light means light irradiation. The tumor volumes of the PBS group, the NSs+light group and the NSs@DCPy(dark) group increased rapidly with almost the same increase. It shows that compared with the control group (PBS group), neither irradiation of NSs alone nor irradiation of NSs@DCPy can inhibit tumor growth. The treatment effect of the DCPy + light group (only PDT) was better than that of the NSs+ light group (only ferroptosis), which may be related to the limited concentration of NSs. The NSs@DCPy + light group (under the dual synergistic effects of PDT and ferroptosis) showed the best tumor suppressive effect.
为了进一步验证PDT联合铁下垂治疗对肿瘤的抑制作用，NSs@DCPy + light治疗组小鼠于肿瘤完全消融后第1天处死。采用苏木精-伊红染色(H&E)和免疫组化染色检测肿瘤的增殖活性。In order to further verify the inhibitory effect of PDT combined with ferroptosis therapy on tumors, mice in the NSs@DCPy + light treatment group were sacrificed on the first day after complete tumor ablation. Hematoxylin-eosin staining (H&E) and immunohistochemical staining were used to detect tumor proliferation activity.
小鼠解剖肿瘤组织上的半胱天冬酶-3（Caspase 3）和GPX4的免疫组化分析结果如图16、17所示，与其他组相比，NSs@DCPy + light组肿瘤细胞凋亡最为显著。肿瘤组织切片免疫组化染色结果显示NSs@DCPy +light光照射下，GPX4明显下调（褐色部分减少），说明治疗效果显著归因于铁下垂。NSs@DCPy +light组在PDT辅助下表现出明显的肿瘤生长抑制作用。这些结果有力地证实了NSs@DCPy通过PDT和铁下垂途径的协同治疗，能够抑制癌细胞的增殖和损伤肿瘤血管组织。此外，各组小鼠体重均缓慢增加，治疗期间无小鼠死亡，证实所有治疗均无明显副作用。The results of immunohistochemical analysis of Caspase-3 (Caspase 3) and GPX4 on dissected tumor tissues of mice are shown in Figures 16 and 17. Compared with other groups, NSs@DCPy The apoptosis of tumor cells in the + light group was the most significant. The results of immunohistochemical staining of tumor tissue sections showed that under NSs@DCPy + light light irradiation, GPX4 was significantly down-regulated (the brown part was reduced), indicating that the therapeutic effect was significantly attributed to ferroptosis. NSs@DCPy The +light group showed significant tumor growth inhibition with the assistance of PDT. These results strongly confirm that NSs@DCPy can inhibit the proliferation of cancer cells and damage tumor vascular tissue through the synergistic treatment of PDT and ferroptosis pathway. In addition, the body weight of mice in each group increased slowly, and no mice died during the treatment, confirming that all treatments had no obvious side effects.
主要器官(心、肝、脾、肺、肾)的H&E染色图像见图18，由图18可以看出，不论是用NSs、DCPy还是NSs@DCPy在无光照或有光照条件下处理，各组小鼠主要器官心、肝、脾、肺、肾的H&E染色结果均与PBS组相近，形态都是完好的，这进一步证实了NSs@DCPy具有良好的生物相容性(图18)。此外，通过血常规和血液生化分析（包括检测分析平均红细胞血红蛋白浓度、平均血小板体积、血红蛋白浓度、白细胞计数、平均红细胞血红蛋白浓度、红细胞分布宽度变异系数、红细胞、血小板、尿素氮、天冬氨酸转氨酶、谷丙转氨酶、白蛋白这些项目），由结果可以看出所有治疗方法的毒性均可忽略不计，各组的数据几乎都在正常范围内(图19-30)，进一步说明NSs@DCPy的治疗安全、无毒性。The H&E staining images of major organs (heart, liver, spleen, lung, and kidney) are shown in Figure 18. It can be seen from Figure 18 that no matter whether NSs, DCPy or NSs@DCPy are treated under the condition of no light or light, the The H&E staining results of the main organs of the mouse, heart, liver, spleen, lung, and kidney, were similar to those of the PBS group, and the morphology was intact, which further confirmed the good biocompatibility of NSs@DCPy (Figure 18). In addition, through blood routine and blood biochemical analysis (including detection and analysis of mean corpuscular hemoglobin concentration, mean platelet volume, hemoglobin concentration, white blood cell count, mean corpuscular hemoglobin concentration, coefficient of variation of red blood cell distribution width, red blood cells, platelets, blood urea nitrogen, aspartic acid Aminotransferase, alanine aminotransferase, albumin, etc.), the results show that the toxicity of all treatment methods can be ignored, and the data of each group are almost within the normal range (Figure 19-30), which further shows that NSs@DCPy The treatment is safe and non-toxic.
综上所述，本发明提供的自供氧光敏剂不仅能够提高光动力治疗的效果，更重要的是该自供氧光敏剂是通过蛭石纳米材料控制氧气的定点定时释放，自供氧光敏剂会自动聚集在肿瘤部位，能够有效用于癌细胞和肿瘤的诊疗中，具有较好的体内生物医学应用前景；本发明提供的自供氧光敏剂生物相容性好，比表面积高，在可视化光动力治疗方面具有良好的应用前景；此外，本发明提供的自供氧光敏剂原料丰富，易于制备，适用范围广，可用于工业化生产。In summary, the self-supplying photosensitizer provided by the present invention can not only improve the effect of photodynamic therapy, but more importantly, the self-supplying photosensitizer controls the fixed-point and timed release of oxygen through vermiculite nanomaterials, and the self-supplying photosensitizer The agent will automatically gather at the tumor site, can be effectively used in the diagnosis and treatment of cancer cells and tumors, and has a good prospect for in vivo biomedical application; the self-oxygenated photosensitizer provided by the present invention has good biocompatibility and high specific surface area, and can be used in Visual photodynamic therapy has a good application prospect; in addition, the self-oxygenated photosensitizer provided by the invention has rich raw materials, is easy to prepare, has a wide range of applications, and can be used in industrial production.
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。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

Claims (10)

  1. 一种自供氧光敏剂，其特征在于，所述自供氧光敏剂的结构包括蛭石纳米片以及负载于所述蛭石纳米片表面的聚集诱导发光发光剂。A self-oxygenated photosensitizer, characterized in that the structure of the self-oxygenated photosensitizer includes vermiculite nanosheets and an aggregation-induced luminescent luminescent agent loaded on the surface of the vermiculite nanosheets.
  2. 根据权利要求1所述的自供氧光敏剂，其特征在于，所述聚集诱导发光发光剂为DCPy、DCMa、DCIs、DCFu中的一种或多种；The self-oxygenated photosensitizer according to claim 1, wherein the aggregation-induced luminescent luminescent agent is one or more of DCPy, DCMa, DCIs, DCFu;
    其中，所述DCPy的结构式为Wherein, the structural formula of the DCPy is
    Figure dest_path_image001
    ；
    Figure dest_path_image001
    ;
    所述DCMa的结构式为The structural formula of the DCMa is
    Figure dest_path_image002
    ；
    Figure dest_path_image002
    ;
    所述DCIs的结构式为The structural formula of the DCIs is
    Figure dest_path_image003
    ；
    Figure dest_path_image003
    ;
    所述DCFu的结构式为The structural formula of the DCFu is
    Figure dest_path_image004
    。
    Figure dest_path_image004
    .
  3. 根据权利要求2所述的自供氧光敏剂，其特征在于，所述聚集诱导发光发光剂为DCPy。The self-oxygenated photosensitizer according to claim 2, wherein the aggregation-induced luminescence luminescent agent is DCPy.
  4. 根据权利要求1所述的自供氧光敏剂，其特征在于，所述蛭石纳米片为单层结构或多层结构。The self-oxygenated photosensitizer according to claim 1, characterized in that, the vermiculite nanosheets have a single-layer structure or a multi-layer structure.
  5. 根据权利要求1所述的自供氧光敏剂，其特征在于，所述聚集诱导发光发光剂的负载量为所述蛭石纳米片重量的5-55w/w%。The self-oxygenated photosensitizer according to claim 1, wherein the loading of the aggregation-induced luminescence luminescent agent is 5-55w/w% of the weight of the vermiculite nanosheets.
  6. 一种根据权利要求1-5任一项所述的自供氧光敏剂的制备方法，其特征在于，包括以下步骤：A preparation method of the self-oxygenated photosensitizer according to any one of claims 1-5, characterized in that it comprises the following steps:
    S1：通过锂离子插层法合成蛭石纳米片；S1: Synthesis of vermiculite nanosheets by lithium ion intercalation;
    S2：将聚集诱导发光发光剂负载到所述蛭石纳米片表面，得到自供氧光敏剂。S2: loading the aggregation-induced luminescence luminescence agent on the surface of the vermiculite nanosheets to obtain a self-oxygenation photosensitizer.
  7. 根据权利要求6所述的自供氧光敏剂的制备方法，其特征在于，步骤S1包括：将蛭石加入到锂盐溶液改性剂中得到蛭石纳米片。The preparation method of self-oxygenated photosensitizer according to claim 6, characterized in that step S1 comprises: adding vermiculite to lithium salt solution modifier to obtain vermiculite nanosheets.
  8. 根据权利要求6所述的自供氧光敏剂的制备方法，其特征在于，步骤S2中，蛭石纳米片的浓度与聚集诱导发光发光剂的浓度比为2：（0.1-100）。The method for preparing a self-oxygenated photosensitizer according to claim 6, characterized in that, in step S2, the ratio of the concentration of the vermiculite nanosheets to the concentration of the aggregation-induced luminescent luminescent agent is 2: (0.1-100).
  9. 根据权利要求6所述的自供氧光敏剂的制备方法，其特征在于，步骤S2中，通过静电吸附将聚集诱导发光发光剂负载到所述蛭石纳米片表面。The method for preparing a self-oxygenated photosensitizer according to claim 6, characterized in that, in step S2, the aggregation-induced luminescence luminescent agent is loaded onto the surface of the vermiculite nanosheets by electrostatic adsorption.
  10. 根据权利要求1-5任一项所述的自供氧光敏剂或根据权利要求6-9任一项所述方法制备得到的自供氧光敏剂在制备光动力治疗药物上的应用。Application of the self-oxygenated photosensitizer according to any one of claims 1-5 or the self-oxygenated photosensitizer prepared according to the method according to any one of claims 6-9 in the preparation of photodynamic therapy drugs.
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