WO2022257855A1 - 一种可实现靶向光热和化学协同治疗的h-BN/MoS2纳米探针及其制备方法和应用 - Google Patents

一种可实现靶向光热和化学协同治疗的h-BN/MoS2纳米探针及其制备方法和应用 Download PDF

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WO2022257855A1
WO2022257855A1 PCT/CN2022/096890 CN2022096890W WO2022257855A1 WO 2022257855 A1 WO2022257855 A1 WO 2022257855A1 CN 2022096890 W CN2022096890 W CN 2022096890W WO 2022257855 A1 WO2022257855 A1 WO 2022257855A1
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mos
mos2
quantum dots
dna
nanoprobe
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何鹏
赵林
李甜甜
隋永鹍
韩文豪
李东祥
徐景坤
毕成
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青岛科技大学
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Definitions

  • the invention relates to the technical field of nanoprobes, in particular to an h - BN/MoS2 nanoprobe capable of realizing targeted photothermal and chemical synergistic therapy, a preparation method and application thereof.
  • 2D nanocomposites are interesting nanomaterials for biomedical applications due to their excellent physicochemical properties and high diversity in morphology, size, and biodegradability, and are widely used in multimodal state-of-the-art imaging, biosensors, drug/gene delivery, and cancer therapy.
  • two-dimensional nanocomposites can effectively adsorb various types of molecules, such as drugs, fluorescent probes, nucleic acids, proteins, and other molecules, through covalent or non-covalent interactions, and stimulate , to achieve controlled release.
  • a series of functional nanoparticles including Fe3O4 nanoparticles, TiO2 nanoparticles, Au nanoparticles can be effectively adsorbed to the surface of 2D nanocomposites, endowing them with corresponding additional functions, such as magnetism, imaging, near-infrared Absorbs and mimics properties such as enzymes.
  • 2D nanocomposites have proven to be a promising functional material, which is of great significance for constructing superior nanoplatforms for the integration of diagnosis and treatment.
  • Liu Jia et al. designed a multifunctional therapeutic platform CuPc@HG@BN consisting of hexagonal boron nitride nanosheets, DNA oligonucleotides, and copper(II) phthalocyanine.
  • the detection of intracellular miRNA-21 and the photodynamic therapy of tumor cells were realized by surface-enhanced Raman spectroscopy and photodynamic therapy of CuPc molecules.
  • this method has a single treatment and detection method, which is limited in terms of treatment effect and detection accuracy.
  • Qunhong Weng et al. used thermal substitution method to prepare water-soluble boron nitride materials with high degree of hydroxylation. Although hydroxylated boron nitride has high biocompatibility, it can be loaded with anticancer drugs for chemotherapy of cancer cells. However, the effect of this single mode of chemotherapy has certain limitations. On the other hand, the drug does not have the ability to specifically recognize cancer cells, and it is also lethal to normal cells, so it cannot achieve accurate detection and treatment of cancer cells.
  • h-BN nanosheets are only used for Raman signal detection, with a single detection standard, high background signal and limited sensitivity;
  • h-BN is used as a carrier to adsorb a single therapeutic drug, and chemotherapy and photodynamic therapy are performed separately, the treatment method is single, and the treatment effect is limited;
  • the present invention provides a targeted, low background signal, compatible with cells h-BN/MoS 2 nanoprobes with better capacitive properties and capable of targeted photothermal and chemical synergistic therapy and their preparation methods and applications, broaden the scope of 2D nanosheets by combining two-dimensional nanosheets with quantum dots
  • the application of fluorescence Raman dual-mode detection and fluorescence in situ imaging of miRNA can be realized, and it can be used as a nano-preparation for cancer chemotherapy and photothermal therapy.
  • An all-in-one platform lays the groundwork.
  • the present invention provides a method for preparing h - BN/MoS nanoprobes capable of targeted photothermal and chemical synergistic therapy, comprising the following steps:
  • DNA hairpin H1-Rox (rhodamine) and DNA strand H2-Rox were annealed in PBS buffer solution and cooled to room temperature to obtain DNA hairpin H1 and DNA hairpin H2, and h-BN/MoS 2 composite material , DNA hairpin H1 and DNA hairpin H2 were shaken and incubated in PBS buffer solution for 2 hours, so that the h-BN/MoS 2 composite material adsorbed DNA strands, and centrifuged to remove unadsorbed DNA hairpins, and h-BN/MoS 2 nanometer Probes, redisperse h - BN/MoS2 nanoprobes in PBS buffer solution, store at 4°C for later use; the sequences of DNA chain H1-Rox and DNA chain H2-Rox are shown in SEQ ID No.1 and SEQ ID No. .2 shown.
  • h-BN nanosheets are prepared according to the following method:
  • the mass volume ratio of hexagonal boron nitride powder to concentrated sulfuric acid is 1g:50mL
  • the mass ratio of hexagonal boron nitride powder to KMnO 4 is 1:2
  • the mass volume ratio of hexagonal boron nitride powder to H 2 O 2 The ratio is 1g:10mL
  • the H2O2 concentration is 30wt %.
  • the FA - PEI modified MoS2 quantum dots are prepared according to the following method:
  • the MoS 2 quantum dots were prepared by using the solvent stripping method, and the MoS 2 quantum dots and L-cysteine were dispersed in water to obtain a mixed dispersion; then EDC (1-(3-dimethylaminopropyl base)-3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide), to obtain a reaction solution, adjust the pH of the reaction solution to 6.8, and continue stirring; add PEI ethanol solution to the reaction solution, Stir to form PEI-MoS 2 quantum dots, centrifuge to separate, retain the centrifuged black powder, wash to remove unreacted chemical substances, freeze-dry and store;
  • Folic acid, EDC and NHS were dispersed in secondary water to obtain a mixed dispersion, adjust the pH of the mixed dispersion to 6.8, stir for 4 hours at 37°C in the dark, then disperse the PEI-MoS 2 quantum dot powder in the mixed dispersion, and continue stirring, FA-PEI modified MoS 2 quantum dots were formed, washed by centrifugation to remove unreacted chemical substances, and stored after freeze-drying.
  • the mass ratio of MoS 2 quantum dots to L-cysteine is 1:0.125
  • the mass ratio of MoS 2 quantum dots to EDC is 1:2.5
  • the mass ratio of MoS 2 quantum dots to NHS is 1:1.5
  • the mass volume ratio of MoS 2 quantum dots to PEI ethanol solution is 1g:10mL, and the concentration of PEI ethanol solution is 1wt%;
  • the mass ratio of PEI-MoS2 quantum dots to EDC was 1 :0.2
  • the mass ratio of PEI-MoS2 quantum dots to NHS was 1 :0.1
  • the mass ratio of PEI - MoS2 quantum dots to folic acid was 1:0.2.
  • MoS 2 quantum dots are prepared according to the following method:
  • the mass volume ratio of MoS 2 to DMF is 1g:100mL.
  • the present invention provides a h-BN/MoS 2 nanoprobe prepared according to the above preparation method that can realize targeted photothermal and chemical synergistic therapy.
  • the present invention provides an application of the above-mentioned h-BN/MoS 2 nanoprobe in miRNA fluorescence and Raman dual-mode detection and/or fluorescence in situ imaging.
  • the BN/MoS 2 composite is used as a carrier, enters the cell through endocytosis, and meets intracellular miRNA-21. Based on the foothold effect, miRNA-21 will first open the hairpin H1 from the 3' end, and the exposed 5' of H1 The end will open H2 again, thereby releasing miRNA-21, and the release of miRNA-21 will continue to open H1 and H2, forming a CHA cycle amplification.
  • the DNA double strand will leave from h-BN/MoS 2 , based on the Raman enhancement and fluorescence quenching of h-BN/MoS 2 , the Rox-modified After the DNA is detached from h-BN/MoS 2 , the fluorescence of Rox is restored and the Raman signal is weakened, thereby realizing the dual-mode detection and/or fluorescence in situ imaging of intracellular miRNA fluorescence and Raman.
  • the present invention provides an application of the aforementioned h-BN/MoS 2 nanoprobe as a nano-preparation.
  • the nano-preparation is a photothermal preparation and/or a drug carrier.
  • the nano-preparation enters the cancer cells through endocytosis, and the chemical drugs (such as doxorubicin, etc.) loaded on the h-BN/MoS 2 nano-probe will be slowly released from the surface under the action of the acidic tumor microenvironment. DNA in the nucleus, preventing the transcription process of cancer cells, and causing cancer cell apoptosis.
  • 808nm laser irradiation can produce photothermal effect and promote the release of chemical drugs, so the synergistic treatment of cancer by chemotherapy and photothermal therapy can be realized.
  • the beneficial effect of the present invention is that,
  • the h-BN/MoS2 nanoprobe provided by the present invention is a novel two -dimensional nanocomposite material with DNA and drug loading functions, photothermal properties and fluorescence quenching properties.
  • the MoS 2 quantum dots used in the present invention have high near-infrared absorption rate, are suitable for photothermal conversion, and have superior optical properties, and can be used as a fluorescence quencher.
  • PEI modification can improve the stability and water solubility of MoS 2 quantum dots ;
  • the positive charge is conducive to the adsorption on the surface of h-BN nanosheets through electrostatic interaction to form a two-dimensional nanocomposite material.
  • further modification of folic acid (FA) outside PEI-MoS 2 quantum dots can increase the targeting of nanomaterials, target folic acid receptors on the surface of cancer cells, and improve the accuracy and targeting of cell imaging and treatment.
  • the present invention uses FA-PEI to modify the positive charge of MoS 2 quantum dots and the negative charge of h-BN nanosheets, mix the two, and obtain h-BN/MoS 2 composite material based on electrostatic interaction.
  • the composite material can utilize The unique two-dimensional structure, large surface area and strong conjugation effect of h-BN nanosheets combine with single-stranded DNA through ⁇ - ⁇ interaction, while taking advantage of the superior optical properties of MoS2 quantum dots and can be used as a fluorescence quencher
  • h-BN nanosheets can also adsorb the chemical drug doxorubicin, and MoS 2 quantum dots, as a transition metal dichalcogenide, also have relatively strong Strong near-infrared absorption ability can be used as a photothermal material, so the composite material can also be used to synthesize nano-preparations with targeted chemotherapy and photothermal therapy.
  • Figure 1 is a TEM image of h-BN nanosheets, MoS 2 quantum dots and h-BN/MoS 2 composites;
  • Figure 2 is the elemental mapping of the h-BN/MoS composite
  • Fig. 3 is the temperature change curve diagram of h - BN/MoS composite material
  • Figure 4 is the UV-vis-NIR spectrum of the h - BN/MoS nanoprobe
  • Fig. 5 is the Rox fluorescence signal map of different materials
  • Fig. 6 is the electrophoresis figure of CHA polyacrylamide
  • Figure 7 is a Rox Raman signal map of different materials
  • Figure 8 is a Rox fluorescence signal map of miRNA-21
  • Figure 9 is a Rox signal diagram of different miRNAs or base-mismatched RNAs.
  • Figure 10 is a Rox Raman signal map of miRNA-21
  • Figure 11 is a Rox Raman signal map of different miRNAs or base-mismatched RNAs
  • Figure 12 is a fluorescence imaging diagram of miRNA-21 in cells
  • Figure 13 is a diagram of the hydration kinetic particle size and Zeta potential of the h - BN/MoS nanoprobe
  • Figure 14 is the Rox signal diagram of the h - BN/MoS nanoprobe
  • Figure 15 is an imaging diagram of DOX in HEK293 and HeLa cells
  • Figure 16 is a histogram of cell viability.
  • the deionized water used in the present invention is produced by a water purification system with a resistance of 18.2 M ⁇ /cm.
  • the DNA and RNA sequences used in the present invention were purchased from Shanghai Sangong Co., Ltd., as shown in Table 1 below.
  • the h - BN/MoS2 nanoprobe was prepared according to the following preparation method:
  • molybdenum disulfide powder 0.5 g was dissolved in 50 mL of DMF, and subjected to ultrasonic treatment at room temperature for 4 h (ultrasonic power 360 W). Then the mixed solution was put into a 100mL flask, heated under reflux in an oil bath at 140°C, and stirred for 6h. Finally, centrifuge at 8000rpm for 10min to remove the precipitate, collect the supernatant, and centrifuge at 12000rpm for 10min to obtain black powder MoS2 quantum dots. Washed three times with ethanol, freeze-dried and stored for future use.
  • 0.2g of MoS 2 quantum dots and 25mg of L-cysteine were dispersed in 25mL of secondary water, ultrasonically dispersed for 3min, and stirred at 37°C for 4h. Then, 0.5 g of EDC and 0.3 g of NHS were added in sequence to adjust the pH of the reaction solution to 6.8. After continuing to stir for 2 h, 2 mL of 1% PEI ethanol solution was added dropwise, and stirred for 4 h to form PEI-MoS 2 quantum dots. After the reaction, the final product was centrifuged (12000 rpm, 10 min), washed three times with secondary water and ethanol to remove unreacted chemical substances, and the black precipitate was freeze-dried and stored.
  • the DNA strands (H1-Rox, H2-Rox) were annealed in PBS buffer solution (1 ⁇ 0.0067M, pH7.4) and cooled to room temperature, so that the DNA strands perfectly formed the hairpin structure (H1, H2). Subsequently, 200 ⁇ L of PBS buffer solution containing h-BN/MoS 2 composite material (100 ⁇ g/mL), annealed hairpins H1 (1 ⁇ M) and H2 (1 ⁇ M), incubated at 37 ° C for 2 h with shaking to make h-BN/MoS 2 The composite material fully adsorbs DNA strands.
  • Test example 1 TEM characterization of h-BN nanosheets, MoS 2 quantum dots, and h-BN/MoS 2 composites
  • the h-BN nanosheets prepared in step (1) of Example 1, the MoS quantum dots prepared in step ( 2 ) and the h-BN/MoS composite material prepared in step ( 3 ) were detected by TEM and characterized respectively Microstructure and morphology of three nanoparticles.
  • a in Figure 1 shows the TEM image of the two-dimensional structure h-BN nanosheets. From the TEM image, it can be seen that the size of the h-BN nanosheets is about 100nm, and there is a partial stacking phenomenon; B in Figure 1 is MoS 2 quantum The TEM image of the dots shows that the size of the MoS 2 quantum dots is uniformly dispersed in the figure, and the particle size is 3nm; h-BN nanosheets and FA-PEI modified MoS 2 quantum dots are electrostatically interacted to obtain h-BN/MoS 2 composites such as As shown in C in Figure 1, it can be clearly found that the FA-PEI modified MoS 2 quantum dots can be perfectly combined with h-BN nanosheets and uniformly dispersed on the surface of h-BN nanosheets, and the h-BN/MoS 2 composites The size is basically the same as that of h-BN nanosheets (100nm).
  • the elemental composition of the as-synthesized h - BN/MoS2 composite was determined by elemental mapping image analysis. The results are shown in Fig. 2, and the Mo, S distributions match those of B, N, confirming the uniform distribution of MoS2 quantum dots on the surface of h-BN nanosheets.
  • Test Example 4 Fluorescence Quenching Ability of h-BN/MoS 2 Composite Materials and Response of h-BN/MoS 2 Nanoprobes to miRNA-21
  • the fluorescence of the Rox-labeled DNA hairpin H1/H2 adsorbed by the ⁇ - ⁇ interaction will be quenched, which indicates that h- Preliminary synthesis of BN/MoS nanoprobes.
  • the CHA cycle reaction of the hairpin H1/H2 starts, and the rigid double-stranded DNA will leave the surface of the h-BN/MoS 2 composite to realize the recovery of Rox fluorescence, and then realize the detection of miRNA-21. detection.
  • H1-Rox+H2-Rox a
  • h-BN 50 ⁇ g/mL
  • target miRNA-21 5nM
  • h-BN in the absence of target miRNA-21 b
  • h-BN/MoS 2 nanoprobe 50 ⁇ g/mL
  • target miRNA-21 5nM
  • the h-BN/MoS 2 nanoprobe exhibited a better Raman signal due to the Raman-enhancing effect of the h-BN/MoS 2 composite; the hairpin H1
  • the CHA cycle reaction of /H2 starts, the rigid double-stranded DNA will leave the h-BN/MoS 2 surface, and the Raman signal decreases, thereby realizing the Raman detection of miRNA-21.
  • h-BN/MoS 2 nanoprobe can have different signal responses to different concentrations of miRNA-21
  • miRNA-21 in vitro experiments were performed, and the miRNA-21(D) sequence with the same sequence as miRNA-21 was used for experiments.
  • the h-BN/MoS 2 nanoprobe (50 ⁇ g/mL) was reacted with different concentrations of target miRNA-21 (D) at 37 °C for 2 h.
  • Adopt F4600 fluorescence spectrophotometer to measure the emission peak of fluorescein Rox at 610nm under the excitation of 543nm, the results are shown in A in Figure 8, curves aj in the figure respectively represent 0nM, 0.2nM, 0.5nM, 1.0nM, 2.0nM, Fluorescence signal curves corresponding to 5.0nM, 8.0nM, 10nM, 20nM, 50nM concentrations of miRNA-21, as the target concentration of miRNA-21 (D) increases, the Rox of the h-BN/MoS 2 nanoprobe is at 610nm The fluorescence signal intensity at 610nm is gradually increased; in addition, as shown in Figure 8 B and 8 C, with miRNA-21 (D) concentration C as the abscissa, the fluorescence signal intensity of Rox at 610nm is fitted.
  • h-BN/MoS 2 nanoprobes The selectivity of h-BN/MoS 2 nanoprobes to miRNA-21 was also studied. Different miRNAs or alkaline reagent mismatched RNAs were used to react with h-BN/MoS 2 nanoprobes. The RNA concentration was 5nM, and the reaction conditions were all It was reacted at 37°C for 2 hours, and the emission peak of fluorescein Rox at 610nm under 543nm excitation was measured by F4600 fluorescence spectrophotometer. The results are shown in Figure 9. Compared with the blank control, only the presence of target miRNA can induce high intensity Fluorescent response. Therefore, the h - BN/MoS nanoprobe has excellent selectivity.
  • the Raman signal intensity of the Rox of the h-BN/MoS 2 nanoprobe at 1644 cm -1 gradually decreases;
  • miRNA-21(D) concentration C as the abscissa
  • h-BN/MoS 2 nanoprobes The selectivity of h-BN/MoS 2 nanoprobes to miRNA-21 was also studied. Different miRNAs or alkaline reagent mismatched RNAs were used to react with h-BN/MoS 2 nanoprobes. The RNA concentration was 1pM and the reaction conditions were all After reacting at 37°C for 2 hours, the Raman signal value of Rox was measured, and the results are shown in Figure 11. Compared with the blank control, only the presence of the target miRNA can produce a weakened Raman signal. Therefore, the h - BN/MoS nanoprobe has excellent selectivity.
  • HeLa cells were seeded in 96-well plates (1 ⁇ 10 4 cells per well) by trypsinization, and cultured for 12 hours. When the cells reached an appropriate number, incubate the cells with DMEM medium (100 ⁇ L) containing h-BN/MoS nanoprobes (100 ⁇ g/mL) for 6 h at 37 °C, 5% CO 2 , and perform confocal laser scanning microscopy. For cell imaging, the imaging uses a 543nm laser to excite Rox fluorescence, and collects fluorescence emission spectra in the range of 600-650nm. The results are shown in A in Figure 12. The Rox fluorescence signal in HeLa cells increases with time, and a bright fluorescent signal can be observed after 6 hours of incubation, so 6 hours is selected as the optimal incubation time between cells and probes.
  • the release ability of the DOX@h-BN/MoS 2 nano-preparation was further investigated by the fluorescence in the supernatant in different pH buffer media.
  • the fluorescence intensity transformation is shown in Fig. 14.
  • Curves ae are the supernatant fluorescence, DOX release at pH 7.4, DOX release at pH 6.5, DOX release at pH 5.5, and h - Fluorescence curve of supernatant of BN/MoS 2 unloaded with DOX.
  • DOX release is slow, which can avoid the premature release of DOX@h-BN/MoS2 nanoformulations when the internal environment circulates.
  • the release of DOX is larger. Therefore, the h - BN/MoS2 nanoprobe can be used as a drug carrier to transport DOX to the tumor site and release it rapidly to realize the drug treatment of tumor cells.
  • HeLa cells cervical cancer cells, tumor cells
  • HEK293 cells human embryonic kidney cells, normal cells
  • the cells were incubated with DMEM medium (100 ⁇ L) containing DOX@h-BN/MoS2 nano - formulation (100 ⁇ g/mL) at 37 °C, 5% CO2 for different time, and scanned by confocal laser
  • the microscope is used for cell imaging, and the fluorescence imaging uses a 488nm laser to excite the fluorescence of DOX, and collects the fluorescence emission spectrum in the range of 550-620nm.
  • HEK293 cells were treated in the same way for fluorescence imaging analysis.
  • HeLa cells were used as a model to evaluate the synergistic therapeutic effect.
  • HeLa cells (1 ⁇ 10 4 cells per well) were seeded in a 96-well plate by trypsinization, and cultured for 12 hours to obtain an appropriate number of cells.
  • DOX@h-BN/MoS 2 nano-formulations exhibited excellent synergistic therapeutic effects of chemotherapy and photothermal therapy under the stimulation of near-infrared 808 nm laser.

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Abstract

一种可实现靶向光热和化学协同治疗的h-BN/MoS 2纳米探针及其制备方法和应用，制备方法包括：h-BN纳米片分散液和FA-PEI修饰的MoS 2量子点分散液等体积混合得到h-BN/MoS 2复合材料；制备DNA发夹，h-BN/MoS 2复合材料吸附DNA链，得到h-BN/MoS 2纳米探针；h-BN/MoS 2纳米探针可用于miRNA荧光和拉曼双模式检测和/或荧光原位成像及制备纳米制剂。

Description

一种可实现靶向光热和化学协同治疗的h-BN/MoS 2纳米探针及其制备方法和应用
本申请要求于2021年06月07日提交中国专利局、申请号为202110631331.5、发明名称为“一种可实现靶向光热和化学协同治疗的h-BN/MoS 2纳米探针及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及纳米探针技术领域，具体涉及一种可实现靶向光热和化学协同治疗的h-BN/MoS 2纳米探针及其制备方法和应用。
背景技术
二维纳米复合材料因其优异的物理化学性能以及形态、尺寸、生物降解性方面的高度多样性，这些性质和特性使其成为有意义的生物医学应用的纳米材料，并被广泛用于多模态成像、生物传感器、药物/基因传递和癌症治疗。
因具有较大的比表面积，二维纳米复合材料可以通过共价或非共价相互作用有效吸附各种类型的分子，如药物、荧光探针、核酸、蛋白质和其他分子，并通过靶向刺激，实现可控释放。包括Fe 3O 4纳米颗粒、TiO 2纳米粒子、Au纳米颗粒在内的一系列功能纳米粒子可以有效地吸附到二维纳米复合材料表面，赋予其相应的附加功能，如磁性、成像、近红外吸收和模拟酶等特性。基于这些优势，二维纳米复合材料已被证明是一种有前途的功能材料，对构建诊疗一体化的优越纳米平台具有重要意义。
Liu Jia等人设计了一种由六方氮化硼纳米片、DNA寡核苷酸和酞菁铜(Ⅱ)组成的多功能治疗平台CuPc@HG@BN。利用CuPc分子的表面增强拉曼光谱和光动力治疗实现对细胞内miRNA-21的检测及肿瘤细胞的光动力治疗。然而，该方法治疗和检测手段单一，在治疗效果和检测的精准度方面受到一定限制。
Qunhong Weng等人采用热取代方法，制备了具有高羟基化度的水溶性氮化硼材料。虽然羟基化的氮化硼具有高生物相容性，可负载抗癌药物用于癌细胞的化学治疗。但是这种单一模式的化学治疗效果具有一定的局 限性，另一方面该药物没有特异性识别癌细胞的能力，对正常细胞也具有杀伤性，不能实现对癌细胞的精准检测与治疗。
综上所述，现有技术存在的问题是：
(1)h-BN纳米片在细胞检测过程中，仅用于拉曼信号检测，检测标准单一，背景信号较高且灵敏度有限；
(2)现有探针只是进行荧光或者拉曼检测，没有进行荧光和拉曼双模式检测分析；
(3)现有技术以h-BN为载体吸附单一治疗药物，分别进行化学治疗和光动力治疗，治疗手段单一，治疗效果有限；
(4)现阶段，使用探针进行检测与治疗时，手段单一，不能实现拉曼和荧光双模态检测，以及化学治疗和光热治疗协同治疗，在诊疗一体化平台的构建上具有一定的局限性。
发明内容
为扩展二维纳米材料在生物学检测和治疗方面的应用，实现表面增强拉曼和荧光双模式检测及肿瘤的多模式治疗，本发明提供一种具有靶向性、背景信号低、与细胞相容性较好、并且可实现靶向光热和化学协同治疗的h-BN/MoS 2纳米探针及其制备方法和应用，通过将二维纳米片与量子点相结合，拓宽了2D纳米片的应用，可实现对miRNA的荧光拉曼双模式检测和荧光原位成像，并能够作为纳米制剂用于癌症的化学治疗和光热治疗，为进一步开发用于癌症标志物检测和协同治疗的诊疗一体化平台奠定了基础。
第一方面，本发明提供一种可实现靶向光热和化学协同治疗的h-BN/MoS 2纳米探针的制备方法，包括如下步骤：
(1)将相同浓度的h-BN纳米片分散液和FA(叶酸)-PEI(聚乙烯亚胺)修饰的MoS 2量子点分散液等体积混合，匀速搅拌组装，离心洗涤，冷冻干燥并保存，得到h-BN/MoS 2复合材料；
(2)DNA链H1-Rox(罗丹明)及DNA链H2-Rox在PBS缓冲溶液中分别退火并冷却至室温，得到DNA发夹H1及DNA发夹H2，将h-BN/MoS 2复合材料、DNA发夹H1及DNA发夹H2在PBS缓冲溶液中震荡孵育2h，使h-BN/MoS 2复合材料吸附DNA链，离心洗涤去除未吸附的DNA发夹，得到h-BN/MoS 2纳米探针，将h-BN/MoS 2纳米探针重 新分散在PBS缓冲溶液中，4℃储存备用；DNA链H1-Rox及DNA链H2-Rox的序列分别如SEQ ID No.1、SEQ ID No.2所示。
进一步的，所述h-BN纳米片按照如下方法制备：
将六方氮化硼粉溶解分散于浓硫酸中并搅拌，加入KMnO 4，继续搅拌；然后向混合溶液中加入H 2O 2，待溶液冷却至室温，超声得到悬浮液；离心悬浮液，保留离心白色固体粉末，除去粒径过大或过小的h-BN纳米片；真空干燥白色粉末，得到h-BN纳米片。
更进一步的，六方氮化硼粉与浓硫酸的质量体积比为1g：50mL，六方氮化硼粉与KMnO 4的质量比为1：2，六方氮化硼粉与H 2O 2的质量体积比为1g：10mL，H 2O 2浓度为30wt％。
进一步的，所述FA-PEI修饰的MoS 2量子点按照如下方法制备：
使用溶剂剥离法制得MoS 2量子点，将MoS 2量子点与L-半胱氨酸分散于水中，得混合分散液；然后依次向混合分散液中加入EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)、NHS(N-羟基琥珀酰亚胺)，得到反应液，调整反应液pH为6.8，继续搅拌；向反应液中加入PEI乙醇溶液，搅拌形成PEI-MoS 2量子点，离心分离，保留离心黑色粉末，洗涤去除未反应的化学物质，冻干保存；
叶酸、EDC和NHS分散在二次水中得到混合分散液，调节混合分散液pH为6.8，37℃黑暗条件下搅拌4h，然后将PEI-MoS 2量子点粉末分散在混合分散液中，继续搅拌，形成FA-PEI修饰的MoS 2量子点，离心洗涤，去除未反应的化学物质，冻干后保存备用。
进一步的，MoS 2量子点与L-半胱氨酸的质量比为1：0.125，MoS 2量子点与EDC的质量比为1：2.5，MoS 2量子点与NHS的质量比为1：1.5，MoS 2量子点与PEI乙醇溶液的质量体积比为1g：10mL，PEI乙醇溶液的浓度为1wt％；
PEI-MoS 2量子点与EDC的质量比为1：0.2，PEI-MoS 2量子点与NHS的质量比为1：0.1，PEI-MoS 2量子点与叶酸的质量比为1：0.2。
进一步的，所述MoS 2量子点按照如下方法制备：
将MoS 2溶解在DMF(N,N-二甲基甲酰胺)中，超声得到混合溶液；将混合溶液回流加热、搅拌；8000rpm下离心去除沉淀，收集上清液，12000rpm下离心，保留离心黑色粉末，得到MoS 2量子点，洗涤去除未 反应的化学物质，冻干后保存备用。
更进一步的，MoS 2与DMF的质量体积比为1g：100mL。
第二方面，本发明提供一种按照上述制备方法制得的可实现靶向光热和化学协同治疗的h-BN/MoS 2纳米探针。
第三方面，本发明提供一种上述h-BN/MoS 2纳米探针在miRNA荧光和拉曼双模式检测和/或荧光原位成像中的应用，具体应用原理为：DNA发夹以h-BN/MoS 2复合材料为载体，通过细胞胞吞作用进入细胞，与细胞内miRNA-21相遇，基于立足点效应，miRNA-21会率先从3’端打开发夹H1，裸露的H1的5’端又会打开H2，从而释放miRNA-21，释放miRNA-21会继续打开H1、H2，形成CHA循环放大。由于形成的刚性DNA双链结构降低π-π相互作用，DNA双链会从h-BN/MoS 2上离去，基于h-BN/MoS 2的拉曼增强和荧光猝灭作用，Rox修饰的DNA从h-BN/MoS 2上脱离后，实现Rox的荧光恢复和拉曼信号减弱，进而实现对细胞内miRNA荧光和拉曼双模式检测和/或荧光原位成像。
第四方面，本发明提供一种上述h-BN/MoS 2纳米探针作为纳米制剂的应用。
进一步的，所述纳米制剂为光热制剂和/或药物载体。该纳米制剂通过胞吞作用进入癌细胞，在偏酸肿瘤微环境作用下，负载在h-BN/MoS 2纳米探针上的化学药物(如阿霉素等)会从表面缓慢释放出来，作用于细胞核内DNA，阻止癌细胞的转录过程，进而引起癌细胞凋亡。另外，基于MoS 2量子点较强的NIR吸收能力，808nm激光照射下可以产生光热效应，促进化学药物的释放，因此可以实现化学治疗和光热治疗对癌症的协同治疗。
本发明的有益效果在于，
本发明提供的h-BN/MoS 2纳米探针是一种具有DNA和药物负载功能、光热性能与荧光猝灭性能的新型二维纳米复合材料。
本发明使用的MoS 2量子点近红外吸收率高，适合于光热转换，且具有优越的光学性质，可作为荧光猝灭剂。
使用FA-PEI修饰MoS 2量子点，一方面PEI修饰能够提高MoS 2量子点的稳定性、水溶性作用；另一方面PEI修饰能够改变MoS 2量子点的电性，使其由负电性转变为正电性，有利于通过静电作用吸附在h-BN纳米 片表面，形成二维纳米复合材料。同时，在PEI-MoS 2量子点外进一步修饰叶酸(FA)，能够增加纳米材料的靶向性，靶向癌细胞表面的叶酸受体，提高细胞成像与治疗的准确度与靶向性。
本发明利用FA-PEI修饰MoS 2量子点的正电性和h-BN纳米片的负电性，将两者混合，基于静电作用得到h-BN/MoS 2复合材料，该复合材料一方面能利用h-BN纳米片独特的二维结构、大的表面积和强的共轭效应，通过π-π相互作用与单链DNA相结合，同时利用MoS 2量子点优越的光学性能和可作为荧光猝灭剂使用的特性，实现了对细胞内miRNA的检测；另一方面，h-BN纳米片还可以吸附化学药物阿霉素，而MoS 2量子点作为一种过渡金属二硫族化合物，也具备较强的近红外吸收能力，可作为光热材料使用，因此该复合材料还可用于合成具有靶向性化学治疗和光热治疗的纳米制剂。
说明书附图
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，对于本领域普通技术人员而言，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。
图1为h-BN纳米片、MoS 2量子点及h-BN/MoS 2复合材料的TEM图；
图2是h-BN/MoS 2复合材料的元素映射图；
图3是h-BN/MoS 2复合材料的温度变化曲线图；
图4是h-BN/MoS 2纳米探针的UV-vis-NIR光谱图；
图5是不同材料的Rox荧光信号图；
图6是CHA聚丙烯酰胺电泳图；
图7是不同材料的Rox拉曼信号图；
图8是miRNA-21的Rox荧光信号图；
图9是不同miRNA或碱基错配RNA的Rox信号图；
图10是miRNA-21的Rox拉曼信号图；
图11是不同miRNA或碱基错配RNA的Rox拉曼信号图；
图12是细胞中miRNA-21的荧光成像图；
图13是h-BN/MoS 2纳米探针水合动力粒径及Zeta电位图；
图14是h-BN/MoS 2纳米探针Rox信号图；
图15是HEK293和HeLa细胞中DOX的成像图；
图16是细胞存活率柱状图。
具体实施方式
为了使本技术领域的人员更好地理解本发明中的技术方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，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，都应当属于本发明保护的范围。
本发明所使用的DMEM培养液、PBS缓冲溶液(1×0.0067M)、胰蛋白酶购自通用电气中国医疗有限公司；Hela细胞(宫颈癌细胞)和HEK293细胞(人胚肾细胞)购自北京银紫晶生物医学技术有限公司；L-半胱氨酸、EDC、NHS、PEI(M w＝10000)、DOX购自上海阿拉丁化学试剂公司；MoS 2纳米片(<2μm，99.5％)、六方氮化硼(99.9％，1-2μm)购自上海麦克林公司；浓硫酸、DMF、乙醇(99.7％)和H 2O 2(30％)购自天津天力化学试剂有限公司；CCK-8试剂盒购自上海碧云天生物技术有限公司。
本发明所使用的去离子水由电阻为18.2MΩ/cm的净水系统制得。
本发明所使用的DNA、RNA序列购自上海生工有限公司，具体如下表1所示。
表1本发明所使用的DNA、RNA序列
Figure PCTCN2022096890-appb-000001
Figure PCTCN2022096890-appb-000002
实施例1
h-BN/MoS 2纳米探针，按照如下制备方法制得：
(1)h-BN纳米片的合成
将1g六方氮化硼粉溶解分散在50mL浓硫酸中，并使用磁力搅拌，缓慢加入0.5gKMnO 4，溶液由白色转变为绿色，继续搅拌12h。然后，向混合溶液中加入10mL 30％的H 2O 2。待溶液冷却到室温，超声处理1h得到悬浮液，将悬浮液离心10min，保留8000-12000rpm的离心白色固体粉末，以除去粒径过大或过小的h-BN纳米片。用去离子水反复清洗离心白色固体粉末，至溶液pH值为中性。最后，白色固体粉末在60℃下真空干燥12h，得到h-BN纳米片。
(2)MoS 2量子点的合成与修饰
0.5g的二硫化钼粉末溶解在50mLDMF中，室温超声处理4h(超声功率360W)。然后将混合溶液放入100mL烧瓶中，140℃油浴回流加热、搅拌6h。最后，8000rpm离心10min去除沉淀，收集上清液，12000rpm离心10min得到黑色粉末MoS 2量子点。乙醇洗涤三次，冻干后保存备用。
0.2g MoS 2量子点和25mg L-半胱氨酸分散在25mL二次水中，超声分散3min，37℃搅拌4h。然后依次加入0.5gEDC和0.3g NHS，调整反应液pH为6.8。继续搅拌2h后，滴加2mL 1％PEI乙醇溶液，搅拌4h形成PEI-MoS 2量子点。反应后，离心分离(12000rpm，10min)最终产物，用二次水和乙醇洗涤三次，去除未反应的化学物质，黑色沉淀冻干保存。
20mg叶酸、20mg EDC和10mg NHS分散在10mL二次水中得到混合分散液，调节混合分散液pH为6.8，37℃黑暗条件下搅拌4h，然后将100mg粉末状PEI-MoS 2量子点超声溶解在混合分散液中，继续搅拌12h。反应后，离心洗涤三次，去除未反应的化学物质，得到FA-PEI修饰的 MoS 2量子点，冷冻干燥并储存。
(3)h-BN/MoS 2复合材料的合成
用去离子水分别分散h-BN纳米片和FA-PEI修饰的MoS 2量子点，得到相同浓度的h-BN纳米片分散液和FA-PEI修饰的MoS 2量子点分散液，等体积混合，室温下快速搅拌1h，得到最终浓度为1mg/mL的混合液，分散均匀的混合液室温下匀速搅拌6h。然后离心洗涤(10000rpm，10min)，除去未吸附的PEI修饰MoS 2量子点。最后，沉淀物冻干保存，得到h-BN/MoS 2复合材料。
(4)h-BN/MoS 2纳米探针的组装
将DNA链(H1-Rox、H2-Rox)在PBS缓冲溶液(1×0.0067M，pH7.4)中分别退火并冷却到室温，使DNA链完美形成发夹结构(H1、H2)。随后，200μLPBS缓冲溶液包含h-BN/MoS 2复合材料(100μg/mL)、退火后的发夹H1(1μM)和H2(1μM)，在37℃下震荡孵育2h，使h-BN/MoS 2复合材料充分吸附DNA链。然后，离心洗涤(10000rpm，10min，4℃)去除未吸附的DNA发夹，将得到的h-BN/MoS 2纳米探针重新分散在200μL PBS缓冲溶液，4℃储存备用。
测试例1 h-BN纳米片、MoS 2量子点、h-BN/MoS 2复合材料的TEM表征
对实施例1步骤(1)制得的h-BN纳米片、步骤(2)制得的MoS 2量子点及步骤(3)制得的h-BN/MoS 2复合材料进行TEM检测，分别表征三种纳米粒子的微观结构和形貌。
图1中的A显示了二维结构h-BN纳米片的TEM图像，从TEM图中看出h-BN纳米片的大小为100nm左右，存在部分堆叠现象；图1中的B为MoS 2量子点的TEM图像，图中MoS 2量子点的大小分散均匀，粒径大小在3nm；h-BN纳米片与FA-PEI修饰的MoS 2量子点通过静电作用得到h-BN/MoS 2复合材料如图1中的C所示，可以清楚发现FA-PEI修饰的MoS 2量子点能够与h-BN纳米片完美结合并均匀分散在h-BN纳米片表面，且h-BN/MoS 2复合材料的大小与h-BN纳米片基本相同(100nm)。
为了进一步确定MoS 2量子点吸附在h-BN纳米片上，通过对合成的h-BN/MoS 2复合材料进行元素映射图像分析，确定其元素组成。结果如图 2所示，Mo、S分布与B、N的分布相匹配，证实了MoS 2量子点在h-BN纳米片表面均匀分布。
测试例2 h-BN/MoS 2复合材料的光热性能分析
分别配制浓度为200μg/mL的PBS、h-BN纳米片、MoS 2量子点和h-BN/MoS 2复合材料溶液，各取1mL置于不同石英皿中，利用808nm近红外激光器(1.2W/cm 2)照射溶液10min，同时用手持式热敏成像仪器每隔1min拍照记录温度变化，并绘制温度变化曲线。
结果如图3中的A所示，在近红外808nm激光照射下，功率为1.2W/cm 2下比较相同浓度(200μg/mL)的四种溶液(PBS、h-BN纳米片、MoS 2量子点和h-BN/MoS 2复合材料)照射10min的温度变化情况，在室温为23℃下，h-BN/MoS 2复合材料溶液在10min内温度可达到50.2℃，MoS 2量子点可达51.4℃。而在相同条件下，经过连续光照，PBS和h-BN纳米片的温度分别仅为25.2℃和27.6℃，表明h-BN/MoS 2复合材料的光热效应来自MoS 2量子点。
按照相同的步骤，改变h-BN/MoS 2复合材料溶液浓度(0、50、100、200、400μg/mL)、光照功率(0.5、0.8、1.2、1.5W/cm 2)，记录温度变化数据。如图3中的B、3中的C所示，h-BN/MoS 2复合材料的光热行为对材料浓度和光照功率均表现出较强的依赖性，随材料浓度、光照功率的增加，h-BN/MoS 2复合材料溶液温度都有升高。
测试例3 h-BN/MoS 2纳米探针紫外表征
分别检测DNA、h-BN/MoS 2复合材料、h-BN/MoS 2纳米探针在200-1000nm波长范围内的吸收峰，图4曲线a、b、c分别为DNA、h-BN/MoS 2复合材料、h-BN/MoS 2纳米探针紫外曲线图，由于h-BN/MoS 2纳米探针曲线既有DNA在260nm处的吸收峰，又具有材料本身近红外吸收峰，可以说明h-BN/MoS 2纳米探针的成功制备。
测试例4 h-BN/MoS 2复合材料的荧光猝灭能力及h-BN/MoS 2纳米探针对miRNA-21的响应
分别检测无靶miRNA-21存在时h-BN/MoS 2纳米探针(a)以及h-BN/MoS 2纳米探针(50μg/mL)与靶miRNA-21(5nM)37℃下反应2h(b)、H1-Rox+H2-Rox(c)的荧光(543nm激发下610nm处的发射峰)。 结果如图5所示，由于h-BN/MoS 2复合材料具有荧光猝灭作用，会将因π-π相互作用吸附的Rox标记的DNA发夹H1/H2的荧光猝灭，这表明h-BN/MoS 2纳米探针的初步合成。然后，由于miRNA-21的存在，发夹H1/H2的CHA循环反应启动，刚性双链DNA会从h-BN/MoS 2复合材料表面离开，实现Rox荧光的恢复，进而实现对miRNA-21的检测。
同时，通过12％聚丙烯酰胺凝胶电泳分析确定CHA反应的结构形成和可行性。如图6所示，在没有目标miRNA-21存在下，发夹H1、H2不会发生CHA；与5nM的miRNA-21孵育后，H1、H2之间发生CHA，形成分子量较大的长链双链DNA，故电泳迁移率降低，电泳条带较H1、H2上移，出现新的明亮条带。
测试例5 h-BN/MoS 2复合材料对miRNA-21的SERS响应
分别检测H1-Rox+H2-Rox(a)、h-BN(50μg/mL)与靶miRNA-21(5nM)37℃下反应2h(b)、无靶miRNA-21存在时h-BN(c)、h-BN/MoS 2纳米探针(50μg/mL)与靶miRNA-21(5nM)37℃下反应2h(d)、无靶miRNA-21存在时h-BN/MoS 2纳米探针(e)的拉曼信号。如图7所示，由于h-BN/MoS 2复合材料具有增强拉曼的作用，h-BN/MoS 2纳米探针表现出较好的拉曼信号；由于miRNA-21的存在，发夹H1/H2的CHA循环反应启动，刚性双链DNA会从h-BN/MoS 2表面离开，拉曼信号降低，进而实现对miRNA-21的拉曼检测。
测试例6体外miRNA-21荧光线性和选择性实验
为证明h-BN/MoS 2纳米探针能够对不同浓度miRNA-21有不同信号响应，进行miRNA-21体外实验，使用与miRNA-21相同序列的miRNA-21(D)序列进行实验。将h-BN/MoS 2纳米探针(50μg/mL)与不同浓度的目标miRNA-21(D)在37℃下反应2h。采用F4600荧光分光光度计测定荧光素Rox在543nm激发下610nm处的发射峰，结果如图8中的A所示，图中曲线a-j分别表示0nM、0.2nM、0.5nM、1.0nM、2.0nM、5.0nM、8.0nM、10nM、20nM、50nM浓度的miRNA-21所对应的荧光信号曲线图，随着目标靶浓度miRNA-21(D)增加，h-BN/MoS 2纳米探针的Rox在610nm处的荧光信号强度逐渐增加；另外，如图8中的B、8中的C所示，以miRNA-21(D)浓度C为横坐标，Rox在610nm处的荧光信号强度进行 拟合，在0.2-10nM范围内呈线性工作曲线，方程为y＝56.368C+30.181，检测限为62.3pM。
还研究了h-BN/MoS 2纳米探针对miRNA-21的选择性，使用不同miRNA或碱剂错配RNA与h-BN/MoS 2纳米探针反应，RNA浓度均为5nM，反应条件均为37℃下反应2h，采用F4600荧光分光光度计测定荧光素Rox在543nm激发下610nm处的发射峰，结果如图9所示，与空白对照相比，只有靶miRNA存在时才能诱导高强度的荧光响应。因此，h-BN/MoS 2纳米探针具有优异的选择性。
测试例7体外miRNA-21 SERS线性和选择性实验
为证明DNA@h-BN/MoS 2纳米探针能够对不同浓度miRNA-21有不同信号响应，进行miRNA-21体外实验，使用与miRNA-21相同序列的miRNA-21(D)序列进行实验。采用表面增强测定拉曼分子罗丹明(Rox)在633nm激光下测定1640cm -1为Rox的拉曼特征吸收峰。结果如图10中的A所示，由曲线a到曲线i分别为0fM、5.0fM、10fM、50fM、0.1pM、0.5pM、1.0pM、10pM、100pM浓度的miRNA-21所对应的拉曼信号曲线图。随着目标靶浓度miRNA-21(D)增加，h-BN/MoS 2纳米探针的Rox在1644cm -1处的拉曼信号强度逐渐降低；另外，如图10中的B、10中的C所示，以miRNA-21(D)浓度C为横坐标，Rox在1644cm -1处的拉曼信号强度进行拟合，在5.0fM-1pM范围内呈线性工作曲线，方程为y＝-859.77lg C-9767.35，检测限为2.43fM。
还研究了h-BN/MoS 2纳米探针对miRNA-21的选择性，使用不同miRNA或碱剂错配RNA与h-BN/MoS 2纳米探针反应，RNA浓度均为1pM，反应条件均为37℃下反应2h，测定Rox的拉曼信号值，结果如图11所示，与空白对照相比，只有靶miRNA存在时才能产生拉曼信号减弱。因此，h-BN/MoS 2纳米探针具有优异的选择性。
体外miRNA-21线性和选择性实验证明h-BN/MoS 2纳米探针具有稳定的荧光信号、高选择性和高的灵敏度。
测试例8细胞中miRNA-21荧光成像
将HeLa细胞通过胰蛋白酶消化接种于96孔板内(1×10 4cells每孔)，培养12h。当细胞达到适当数量，用含有h-BN/MoS 2纳米探针(100μg/mL) 的DMEM培养液(100μL)在37℃、5％CO 2条件下孵育细胞6h，通过共聚焦激光扫描显微镜进行细胞成像，成像使用543nm激光激发Rox荧光，收集600-650nm范围的荧光发射光谱。结果如图12中的A所示，HeLa细胞内的Rox荧光信号随时间增加而增加，孵育时间6h可观察到明亮的荧光信号，因此选择6h作为细胞与探针之间的最佳孵育时间。
此外，还研究了h-BN/MoS 2纳米探针对不同细胞内的成像。不同种类的细胞内miRNA-21的表达不同。将相同浓度的h-BN/MoS 2纳米探针与HeLa、HEK293两种细胞分别孵育6h，然后进行共聚焦荧光成像。结果如图12中的B所示，通过比较Rox荧光信号强度可知，ROX荧光在HeLa细胞中的强度显著而HEK293细胞荧光信号微弱。这一结果与miRNA-21在HeLa细胞中的表达高于HEK293细胞一致。
实施例2 DOX@h-BN/MoS 2纳米制剂的构建与应用
①DOX的加载与释放
将h-BN/MoS 2纳米探针(0.2mL，1.0mg/mL)和DOX溶液(0.2mL，1.0mg/mL)用PBS缓冲液稀释至1mL，室温下震荡24h，离心分离DOX@h-BN/MoS 2纳米制剂和未加载的DOX溶液。收集上清溶液，采用日立F-4600荧光分光光度计(E X＝488nm)测定上清液的荧光。并通过水合动力粒径和Zeta电位实验比较h-BN/MoS 2纳米探针加载DOX前后变化以及不同pH的PBS缓冲液(pH＝5.5、6.5和7.4)，室温下震荡12h变化后的DOX荧光。
进一步通过不同pH缓冲液介质下上清液中的荧光考察DOX@h-BN/MoS 2纳米制剂的释放能力。将DOX@h-BN/MoS 2纳米制剂(200μg)分散于1mL不同pH的PBS缓冲液(pH＝5.5、6.5和7.4)，室温下震荡12h。离心取200μL上清液，测定上清液的荧光。
h-BN/MoS 2纳米探针加载DOX前后的水合动力粒径和Zeta电位变化如图13所示，结果表明，DOX的负载对h-BN/MoS 2纳米探针的粒径大小几乎没有影响，但会使其电位发生变化。
荧光强度变换如图14所示，曲线a-e分别为h-BN/MoS 2纳米探针加载DOX后的上清液荧光、pH7.4 DOX释放、pH6.5 DOX释放、pH5.5 DOX释放和h-BN/MoS 2未加载DOX的上清液荧光曲线图。在生理条件下 (pH7.4)，DOX释放缓慢，可以避免DOX@h-BN/MoS 2纳米制剂在内部环境循环时过早释放。相反，在肿瘤微环境(pH6.5-6.8)以及肿瘤细胞器(pH5.0-6.0)中，DOX释放量较大。因此，h-BN/MoS 2纳米探针可作为药物载体，运输DOX到肿瘤部位，并快速释放，实现肿瘤细胞的药物治疗。
②DOX细胞成像
以HeLa细胞(宫颈癌细胞，肿瘤细胞)和HEK293细胞(人胚肾细胞，正常细胞)为模型，研究DOX@h-BN/MoS 2在细胞中释放过程及靶向识别。首先，将HeLa细胞通过胰蛋白酶消化传代接种于96孔板内(1×10 4cells每孔)，培养12h。当细胞达到适当数量，用含有DOX@h-BN/MoS 2纳米制剂(100μg/mL)的DMEM培养液(100μL)在37℃、5％CO 2条件下孵育细胞不同时间，通过共聚焦激光扫描显微镜进行细胞成像，荧光成像使用488nm激光激发DOX的荧光，收集550-620nm范围的荧光发射光谱。采用相同的方法处理HEK293细胞，进行荧光成像分析。
结果如图15所示，孵育6h后HeLa细胞核有明亮的DOX的荧光，而HEK293细胞核内仅呈现极其微弱的DOX荧光。这是由于与正常细胞相比，在肿瘤细胞表面有过表达的叶酸受体，制备的DOX@h-BN/MoS 2纳米制剂由于标记了叶酸，能够对肿瘤细胞特异性识别，提高了肿瘤细胞对探针的摄取效率，增大了阿霉素的摄取量，而正常细胞表面叶酸受体的缺乏，降低了正常细胞对探针的摄取率，减少了阿霉素进入正常细胞的量，可以有效避免药物对正常细胞的损伤，实现靶向治疗。
③DOX@h-BN/MoS 2纳米制剂的协同治疗效果
以HeLa细胞为模型，用CCK-8细胞毒性实验评估协同治疗效果。首先，将HeLa细胞(1×10 4cells每孔)通过胰蛋白酶消化接种在96孔板中，培养12h得到数量合适的细胞。PBS清洗两次后，分为h-BN/MoS 2纳米探针、h-BN/MoS 2纳米探针+NIR(近红外光，808nm，10min)，DOX@h-BN/MoS 2纳米制剂、DOX@h-BN/MoS 2纳米制剂+NIR(808nm，10min)四组处理细胞，然后用含有10μL CCK-8的DMEM培养液(100μL)继续孵育细胞3h。最后，测定每孔460nm处的吸光度，计算细胞存活率。 结果如图16所示，照射808nm近红外光10min，100μg/mL DOX@h-BN/MoS 2纳米制剂处理的HeLa细胞存活率在40％左右。与对照组和其他不光照组相比，该方法明显降低了细胞存活率，从而证实了DOX@h-BN/MoS 2纳米制剂+NIR具有更好的治疗效果。因此，在近红外808nm激光的刺激下，DOX@h-BN/MoS 2纳米制剂表现出优异的化学治疗与光热治疗协同治疗效果。
尽管通过参考附图并结合优选实施例的方式对本发明进行了详细描述，但本发明并不限于此。在不脱离本发明的精神和实质的前提下，本领域普通技术人员可以对本发明的实施例进行各种等效的修改或替换，而这些修改或替换都应在本发明的涵盖范围内/任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。

Claims (16)

  1. 一种可实现靶向光热和化学协同治疗的h-BN/MoS2纳米探针的制备方法，其特征在于，包括如下步骤：
    (1)将相同浓度的h-BN纳米片分散液和FA-PEI修饰的MoS2量子点分散液等体积混合，匀速搅拌组装，离心洗涤，冷冻干燥并保存，得到h-BN/MoS2复合材料；
    (2)DNA链H1-Rox及DNA链H2-Rox在PBS缓冲溶液中分别退火并冷却至室温，得到DNA发夹H1及DNA发夹H2，将所述h-BN/MoS2复合材料、DNA发夹H1及DNA发夹H2在PBS缓冲溶液中震荡孵育2h，使h-BN/MoS2复合材料吸附DNA链，离心洗涤去除未吸附的DNA发夹，得到h-BN/MoS2纳米探针；DNA链H1-Rox及DNA链H2-Rox的序列分别如SEQ ID No.1、SEQ ID No.2所示。
  2. 一种可实现靶向光热和化学协同治疗的h-BN/MoS 2纳米探针的制备方法，其特征在于，包括如下步骤：
    (1)将相同浓度的h-BN纳米片分散液和FA-PEI修饰的MoS 2量子点分散液等体积混合，匀速搅拌组装，离心洗涤，冷冻干燥并保存，得到h-BN/MoS 2复合材料；
    (2)DNA链H1-Rox及DNA链H2-Rox在PBS缓冲溶液中分别退火并冷却至室温，得到DNA发夹H1及DNA发夹H2，将所述h-BN/MoS 2复合材料、DNA发夹H1及DNA发夹H2在PBS缓冲溶液中震荡孵育2h，使h-BN/MoS 2复合材料吸附DNA链，离心洗涤去除未吸附的DNA发夹，得到h-BN/MoS 2纳米探针，将所述h-BN/MoS 2纳米探针重新分散在PBS缓冲溶液中，4℃储存备用；DNA链H1-Rox及DNA链H2-Rox的序列分别如SEQ ID No.1、SEQ ID No.2所示。
  3. 如权利要求1或2所述的制备方法，其特征在于，所述h-BN纳米片按照如下方法制备：
    将六方氮化硼粉溶解分散于浓硫酸中并搅拌，加入KMnO 4，继续搅拌；然后向混合溶液中加入H 2O 2，待溶液冷却至室温，超声得到悬浮液；离心悬浮液，保留离心白色固体粉末，除去粒径过大或过小的h-BN纳米 片；真空干燥白色粉末，得到h-BN纳米片。
  4. 如权利要求3所述的制备方法，其特征在于，六方氮化硼粉与浓硫酸的质量体积比为1g：50mL，六方氮化硼粉与KMnO 4的质量比为1：2，六方氮化硼粉与H 2O 2的质量体积比为1g：10mL。
  5. 如权利要求3所述的制备方法，其特征在于，所述保留离心白色固体粉末为保留8000-12000rpm的离心白色固体粉末。
  6. 如权利要求1或2所述的制备方法，其特征在于，所述FA-PEI修饰的MoS 2量子点按照如下方法制备：
    使用溶剂剥离法制得MoS 2量子点，将MoS 2量子点与L-半胱氨酸分散于水中，得混合分散液；然后依次向混合分散液中加入EDC、NHS，得到反应液，调整反应液pH值为6.8，继续搅拌；向反应液中加入PEI乙醇溶液，搅拌形成PEI-MoS 2量子点，离心分离，保留离心黑色粉末，洗涤去除未反应的化学物质，冻干保存；
    将PEI-MoS 2量子点粉末分散在EDC、NHS、叶酸混合分散液中，搅拌形成FA-PEI修饰的MoS 2量子点，离心洗涤，去除未反应的化学物质，冻干后保存备用。
  7. 如权利要求6所述的制备方法，其特征在于，MoS 2量子点与L-半胱氨酸的质量比为1：0.125，MoS 2量子点与EDC的质量比为1：2.5，MoS 2量子点与NHS的质量比为1：1.5，MoS 2量子点与PEI乙醇溶液的质量体积比为1g：10mL；
    PEI-MoS 2量子点与EDC的质量比为1：0.2，PEI-MoS 2量子点与NHS的质量比为1：0.1，PEI-MoS 2量子点与叶酸的质量比为1：0.2。
  8. 如权利要求6所述的制备方法，其特征在于，所述MoS 2量子点按照如下方法制备：
    将MoS 2溶解在DMF中，超声得到混合溶液；将混合溶液回流加热、搅拌；8000rpm离心去除沉淀，收集上清液，12000rpm离心，保留离心黑色粉末，得到MoS 2量子点，洗涤去除未反应的化学物质，冻干后保存备用。
  9. 一种可实现靶向光热和化学协同治疗的h-BN/MoS 2纳米探针，其特征在于，所述h-BN/MoS 2纳米探针采用如权利要求1-8任一项所述的制 备方法制得。
  10. 一种如权利要求9所述的h-BN/MoS 2纳米探针在miRNA荧光和拉曼双模式检测和/或荧光原位成像中的应用。
  11. 一种如权利要求9所述的h-BN/MoS 2纳米探针作为纳米制剂的应用。
  12. 如权利要求11所述的应用，其特征在于，所述纳米制剂为光热制剂和/或药物载体。
  13. 权利要求9所述h-BN/MoS 2纳米探针在靶向光热和化学协同治疗癌症中的应用。
  14. 一种加载阿霉素的h-BN/MoS 2纳米探针，其特征在于，所述加载阿霉素的h-BN/MoS 2纳米探针的制备方法包括以下步骤：
    将0.2mL 1.0mg/mL的h-BN/MoS 2纳米探针和0.2mL 1.0mg/mL的DOX溶液用PBS缓冲液稀释至1mL，室温下震荡24h，离心分离DOX@h-BN/MoS 2纳米制剂。
  15. 权利要求14所述加载阿霉素的h-BN/MoS 2纳米探针在靶向光热和化学协同治疗癌症中的应用。
  16. 根据权利要求15所述的应用，其特征在于，所述癌症包括宫颈癌。
PCT/CN2022/096890 2021-06-07 2022-06-02 一种可实现靶向光热和化学协同治疗的h-BN/MoS2纳米探针及其制备方法和应用 WO2022257855A1 (zh)

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