A water-soluble pillararene-based nanodiagnosis and treatment material

Abstract

The application discloses a kind of nanodiagnosis and treatment materials based on water-soluble columnar aromatic hydrocarbon.The application designs and synthesizes a new type of columnar [5] aromatic hydrocarbon WP5, the columnar [5] aromatic hydrocarbon WP5 has water-soluble quality and contains rigid structure cavity macrocyclic molecule, can occur with porphyrin molecule host-guest complexation interaction.Using the water-soluble columnar [5] aromatic hydrocarbon WP5 and porphyrin guest molecule host-guest complexation, can realize cavity protection porphyrin compound inhibits the accumulation between porphyrin molecules, improves its stability and the ability of producing singlet oxygen, while enhancing fluorescence, and improve porphyrin compound light stability.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nanomaterial for diagnosis and treatment based on water-soluble columnar aromatic hydrocarbons [5]. Background Technology

[0002] In recent years, organic materials based on supramolecular macrocyclic compounds have developed rapidly and have been applied in fields such as biomedicine and molecular recognition. Many porphyrin compounds exist in nature, mainly composed of a large π-conjugated plane consisting of four pyrrole units and four bridging carbon atoms. Due to their unique light-harvesting ability, electrochemical properties, and energy transfer effects, they have broad application prospects in photodynamic and photothermal therapy, such as twin imaging, photoacoustic imaging, magnetic resonance imaging, and near-infrared fluorescence imaging. However, porphyrins undergo severe self-quenching in water due to hydrophobic interactions and π-π stacking, leading to fluorescence quenching and photosensitivity inhibition, which severely limits the biological applications of porphyrin compounds. Furthermore, porphyrins also exhibit poor stability under oxidative or irradiation conditions. Therefore, to be more suitable for biological applications, porphyrins urgently need to be in a non-aggregated state and possess good photostability.

[0003] Columnar aromatics, as a new generation of macrocyclic host compounds, are widely used. Compared with traditional macrocyclic compounds, columnar aromatics possess rigid structures, are easily functionalized, and can form host-guest complexes with a wider variety of guest molecules. In the research of columnar aromatics, the synthesis of water-soluble columnar aromatic structures has been a focus for many researchers. However, further research is needed to address shortcomings such as whether porphyrins can be protected by host molecules with macrocyclic cavities and to improve the photostability of porphyrin compounds. Currently, there are relatively few reported applications of complexation between porphyrin guest molecules and water-soluble columnar aromatic hosts. Summary of the Invention

[0004] The purpose of this invention is to provide a nanomaterial for diagnosis and treatment based on water-soluble columnar aromatic hydrocarbons.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Water-soluble columnar aromatic hydrocarbon WP5[5], the structural formula of which is shown below:

[0007]

[0008] The application of the above-mentioned water-soluble columnar aromatic hydrocarbon WP5 in the preparation of nano-therapeutic materials.[5]

[0009] A nanomaterial is made from the above-mentioned water-soluble columnar aromatic hydrocarbon WP5 and porphyrin.

[0010] The structural formula of the porphyrin is shown below:

[0011]

[0012] The above-mentioned nanomaterials are used in the preparation of tumor diagnostic and / or therapeutic products.

[0013] This invention designs and synthesizes a novel columnar [5]arene WP5, which is a water-soluble macrocyclic molecule containing a rigid cavity structure, capable of host-guest complexation with porphyrin molecules. By utilizing the host-guest complexation of this water-soluble columnar [5]arene WP5 with porphyrin guest molecules, the cavity can protect the porphyrin compound, inhibit the stacking between porphyrin molecules, improve its stability and ability to generate singlet oxygen, while enhancing fluorescence and improving the photostability of the porphyrin compound. Attached Figure Description

[0014] Figure 1 The image is a scanning electron microscope image of columnar aromatic hydrocarbon WP5 complexed with porphyrin molecules.[5]

[0015] Figure 2 Dynamic light scattering is the spectrum of columnar aromatic hydrocarbon WP5 complexed with porphyrin molecules [5].

[0016] Figure 3 The spectrum is used to determine the complexation constant of column[5] aromatic hydrocarbon WP5 with porphyrin molecules.

[0017] Figure 4 The calculation results are for the complexation ratio of column[5] aromatic hydrocarbon WP5 with porphyrin molecules.

[0018] Figure 5 The results of reactive oxygen species release test before and after the complexation of column[5] aromatic hydrocarbon WP5 with porphyrin molecules are shown.

[0019] Figure 6 The results of cytotoxicity tests before and after the complexation of column[5] aromatic hydrocarbon WP5 with porphyrin molecules are presented.

[0020] Figure 7 The results of cell viability detection under laser conditions after complexation of columnar aromatic hydrocarbon WP5, porphyrin molecule Por, WP5 and Por [5] are presented.

[0021] Figure 8 The results of reactive oxygen species detection under laser conditions after WP5 and Por complexation.

[0022] Figure 9 The results show the detection of singlet oxygen under laser conditions after WP5 and Por complexation.

[0023] Figure 10 The results show the detection of hydroxyl radicals under laser conditions after WP5 and Por complexation. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are within the scope of the present invention. Experimental methods and reagents not specifically described in the embodiments are performed according to conventional conditions in the art.

[0025] Example 1

[0026] Preparation of water-soluble column[5] aromatic WP5

[0027]

[0028] Ethoxy[5]arene (8.90 g, 0.10 mol) was added to 500 mL of dry chloroform, and then BBr3 (31.20 g, 124.00 mmol) was slowly added dropwise. The reaction was carried out at 0 °C, and the reaction progress was monitored by TLC. After the reaction was completed, a large amount of ice water was added, and the hydroxy[5]arene was obtained by filtration.

[0029] Hydroxyl column[5] aromatics (3.00 g, 4.80 mmol) and K2CO3 (7.20 g, 52.00 mmol) were added to 125 mL of acetonitrile and stirred for 45 min. Then KI (70.00 mg, 42.00 mmol) and methyl chloroacetate (15.00 g, 90.00 mmol) were added and the mixture was heated under nitrogen protection and refluxed for 20 h. After cooling and filtration to remove potassium carbonate and potassium iodide, the mixture was evaporated under reduced pressure and purified by column chromatography (volume ratio: dichloromethane:methanol = 100:1) to obtain the crude product. The crude product was then recrystallized from acetone to obtain ester column[5] aromatics.

[0030] Ester-based [5] aromatic hydrocarbons (0.67 g, 0.50 mmol) and 0.5 mL of NaOH aqueous solution (10.00 M) were added to 50 mL of water. The reaction was carried out at 90 °C for 24 h. After the reaction was completed, the mixture was cooled, and the pH was adjusted by adding dilute HCl. The reaction solution was extracted with ethyl acetate, and the organic layer was dried with anhydrous Na2SO4. The organic solvent was removed by rotary evaporation. The crude product was washed with a mixed solvent of n-hexane and ethyl acetate (volume ratio: n-hexane: ethyl acetate = 9:1) to obtain carboxyl-based [5] aromatic hydrocarbons.

[0031] Carboxylated columnar aromatic hydrocarbon (0.52 g, 0.50 mmol) and concentrated ammonia (0.42 g, 7.00 mM) were added to 50 mL of water and stirred at room temperature for 2 h. The solvent was removed by rotary evaporation under reduced pressure to obtain water-soluble columnar aromatic hydrocarbon WP5.

[0032] The complexation ability of water-soluble columnar aromatic hydrocarbon WP5 with porphyrin (Por) host and guest, the release of reactive oxygen species before and after complexation, and cell activity were tested below.

[0033] 1. Characterization of the host-guest complexation ability of water-soluble column[5] aromatic hydrocarbon WP5 with porphyrin (Por)

[0034] The water-soluble columnar aromatic hydrocarbon WP5 was dissolved in 1 mL of water at a concentration of 100 μM, and the porphyrin guest molecule was dissolved in 1 mL of methanol at a concentration of 50 μM. Then the aqueous solution and the methanol solution were thoroughly mixed and shaken.

[0035] The complexation of water-soluble columnar aromatic hydrocarbons with porphyrin molecules was characterized by scanning electron microscopy (SEM) and dynamic light scattering (DLS).[5] The scanning electron microscopy (SEM) images are shown below. Figure 1 As shown, the dynamic light scattering (DLS) spectrum is as follows: Figure 2 As shown.

[0036] The complexation abilities of water-soluble columnar aromatic hydrocarbons and porphyrin guest molecules were determined by ultraviolet absorption spectroscopy and fluorescence absorption spectroscopy, respectively. The procedure for determining the complexation constant was as follows: water-soluble columnar aromatic hydrocarbons were dissolved in water at a concentration of 10 μM, while porphyrin guest molecules were added dropwise to the aqueous solution of the water-soluble columnar aromatic hydrocarbons at a concentration of 2 μM each time. The results were then detected by fluorescence absorption spectroscopy. Figure 3 As shown. The determination process for the complexation ratio is as follows: water-soluble columnar aromatic hydrocarbons are dissolved in water at a concentration of 10 μM. Simultaneously, porphyrin guest molecules are added dropwise to the aqueous solution of water-soluble columnar aromatic hydrocarbons at a concentration of 2 μM each time. Then, the results are detected by ultraviolet absorption spectroscopy. Figure 4 As shown in the figure. The results show that the macrocyclic cavity of water-soluble columnar aromatic hydrocarbons [5] can form a good complex with porphyrin guest molecules. The complexation ratio of columnar aromatic hydrocarbons to porphyrin was calculated to be 2:1, which effectively improves the stability of porphyrin compounds.

[0037] 2. Test on reactive oxygen species release effect after complexation of water-soluble column[5] aromatic hydrocarbons with porphyrin (Por)

[0038] 1,3-Diphenylisobenzofuran (DPBF) was dissolved in aqueous solution at a concentration of 10 μM before and after complexation with WP5 and porphyrin guest molecules. The solution was then irradiated with a 660 nm laser at intervals under a light-shielded environment, and the results were detected by ultraviolet absorption spectroscopy. The reactive oxygen species release effect of DPBF before and after complexation with WP5 and porphyrin guest molecules in aqueous solution under 660 nm laser irradiation was compared and tested.

[0039] Figure 5 (a) is a spectrum of reactive oxygen release effect of DPBF in aqueous solution before and after being irradiated by a 660nm laser. Figure 5 (b) is a spectrum of reactive oxygen species release before and after DPBF and porphyrin are irradiated with a 660 nm laser in aqueous solution; Figure 5(c) is a spectrum of reactive oxygen species release in aqueous solution before and after being irradiated by a 660nm laser before and after DPBF complexes with WP5 and porphyrin guest molecules. Figure 5 (d) is a comparison spectrum of reactive oxygen species release efficiency before and after DPBF, DPBF with porphyrin, DPBF with WP5 and porphyrin guest molecules are irradiated with a 660nm laser in aqueous solution.

[0040] Release efficiency tests have shown that water-soluble columnar aromatic hydrocarbon WP5, due to its unique cavity structure, can interact with porphyrin molecules through host-guest complexation, thereby achieving cavity protection of porphyrin compounds and improving the photostability of porphyrin compounds, thus enabling more effective release of reactive oxygen species.

[0041] 3. Biocompatibility testing

[0042] Human cervical cancer cells (HeLa cells) were seeded into 96-well plates (1×10⁻⁶ cells per well). 4 Cells / well were incubated overnight at 37°C in a 5% CO2 incubator. Different concentrations of WP5, Por, and WP5+Por dispersions were added to the corresponding wells, with the WP5+Por dispersion mixed at a complexation ratio of 2:1. The final concentration was calculated based on the Por concentration. After culturing for another 4 hours, the cells were subjected to a 660nm laser (1W / cm²). 2 Cells were irradiated for 10 min as a control experiment and cultured at 37°C for 24 h. MTT reagent was added to each well, and after culturing for another 4 h, the culture medium was removed, and 100 μL of DMSO was added to dissolve formazan crystals. Absorbance was recorded using a microplate reader, and cytotoxicity was calculated.

[0043] like Figure 6 As shown. The nanomaterials formed by the interaction of water-soluble columnar aromatic hydrocarbon WP5 and p-diporphyrin host and guest have a significantly enhanced killing effect on tumor cells.

[0044] 4. Cellular anti-tumor research

[0045] The therapeutic effect of WP5 / Por on tumor cells (HeLa cells) was determined by in vitro cell experiments. One group of HeLa cells was cultured normally with WP5 (30 μM), Por (15 μM), or WP5 (30 μM) + Por (15 μM) dispersions, while the other group was irradiated with a 660 nm laser for 10 min and then stained with calcein / PI.

[0046] like Figure 7 As shown, without 660nm laser (1W / cm) 2 The activity of HeLa cells irradiated with WP5, Por, and WP5+Por did not cause damage to HeLa cells and had good biocompatibility; when the WP5+Por group was irradiated with 660nm laser (1W / cm²), the activity of HeLa cells did not fluctuate significantly, indicating that WP5, Por, and WP5+Por did not cause damage to HeLa cells and had good biocompatibility;2 After irradiation, most HeLa cells died; in the Por plus near-infrared laser treatment group, only some HeLa cells died, which indicates that the WP5 cavity can protect porphyrin compounds from the accumulation between porphyrin molecules, while improving their stability and effectively improving the photostability of porphyrin compounds.

[0047] 5. Reactive oxygen species release test

[0048] 2,7-Dichlorofluorescein diacetate (DCFH-DA) can be rapidly oxidized by ROS and exhibits green fluorescence. Therefore, DCFH-DA can be used as a ROS sensor to study reactive oxygen species generated in tumor cells using fluorescence microscopy.

[0049] like Figure 8 As shown, neither the control group nor HeLa cells incubated with WP5 (30 μM) + Por (15 μM) produced green fluorescence. In contrast, cells incubated with WP5 / Por and treated with a 660 nm laser (1 W / cm²) showed significant green fluorescence. 2 Irradiated HeLa cells exhibited significant fluorescence, demonstrating the large-scale production of ROS. This result indicates that WP5+Por can act as a photosensitizer to generate ROS upon near-infrared light irradiation, thereby participating in photodynamic therapy of tumors.

[0050] 6. Singlet oxygen release test

[0051] SOSG in relation to 1 Before the O2 reaction, its internal electron transfer (ET) quenches the fluorescence of the luminescent chromophore, while... 1 Following the reaction with O2, an internal peroxide is formed, which inhibits electron transfer, resulting in observable fluorescence. Therefore, SOSG can be used as... 1 The O2 sensor was used to study singlet oxygen generated in tumor cells using fluorescence microscopy.

[0052] like Figure 9 As shown, neither the control group nor HeLa cells incubated with WP5 (30 μM) + Por (15 μM) produced green fluorescence. In contrast, cells incubated with WP5 / Por and treated with a 660 nm laser (1 W / cm²) showed significant green fluorescence. 2 The irradiated HeLa cells showed obvious fluorescence, proving that... 1 The large-scale generation of O2 indicates that WP5+Por can act as a photosensitizer to generate O2 upon near-infrared light irradiation. 1 O2 can be used in photodynamic therapy for tumors.

[0053] 7. Hydroxyl radical release test

[0054] Aminophenyl fluorescein (APF) is a fluorescent probe used to detect highly reactive oxygen species (hROS) and myeloperoxidase (MPO). Its fluorescence intensity increases significantly upon reaction with hROS such as hydroxyl radicals, peroxynitrite, and hypochlorite. Therefore, APF can be used as an ·OH sensor to study hydroxyl radicals generated in tumor cells using fluorescence microscopy.

[0055] like Figure 10 As shown, neither the control group nor HeLa cells incubated with WP5 (30 μM) + Por (15 μM) produced green fluorescence. In contrast, cells incubated with WP5 / Por and treated with a 660 nm laser (1 W / cm²) showed significant green fluorescence. 2 Irradiated HeLa cells exhibited significant fluorescence, demonstrating the large-scale production of ·OH. This result indicates that WP5+Por can act as a photosensitizer to generate ·OH upon near-infrared light irradiation, thereby participating in photodynamic therapy of tumors.

Claims

1. A nanomaterial made of water-soluble columnar aromatic hydrocarbon WP5 and porphyrin; The structural formula of the water-soluble columnar aromatic hydrocarbon WP5[5] is shown below: ， The structural formula of the porphyrin is shown below: 。 2. The use of the nanomaterial according to claim 1 in the preparation of cervical cancer diagnostic and / or therapeutic drugs.

3. A diagnostic and / or therapeutic agent for cervical cancer, comprising the nanomaterial of claim 1.

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