A BP-PEI@RNA nano-drug complex, preparation method and application thereof in treating cancer

Abstract

The present invention discloses a BP-PEI@RNA nano-drug complex, a preparation method and its application in treating cancer. The BP-PEI@RNA nano-drug complex is composed of RNA and BP modified by PEI. The nano-drug complex can achieve efficient and stable delivery of RNA, transport exogenous gene drugs (RNA) into tumor cells, and inhibit the progression of prostate cancer by increasing the expression level of tumor suppressor genes in tumor cells, and has good clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a BP-PEI@RNA nano drug complex, a preparation method and an application thereof in treating cancer. Background Art

[0002] The occurrence and development of malignant tumors is a complex biological process. A large number of studies have shown that it is regulated by a variety of gene network patterns. Among them, the endogenous competing endogenous RNA (ceRNA) network regulation pattern has been confirmed in a variety of malignant tumors. In this process, short-chain RNA (miRNA) with a length of about 22 nucleotides can bind to the 3'-UTR of the corresponding messenger RNA (mRNA), inhibit its transcription and translation process by cleaving the target gene, and play a role in reverse regulation of the expression of the target gene. Long non-coding RNA (lncRNA) is a non-coding RNA with a length of more than 200 nucleotides. As an important member of the ceRNA network regulation pattern, it can competitively bind to miRNA, thereby blocking the binding inhibitory effect of miRNA on its target gene, thereby upregulating the expression level of the target gene. Therefore, lncRNA is expected to be used as a gene drug for the treatment of malignant tumors. However, RNA itself is unstable and easily degraded. At the same time, it needs a carrier to transport it into tumor cells to play a role. This study uses the cell internalization effect of nanomaterials to efficiently deliver RNA into tumor cells, while improving the stability of RNA in vivo and in vitro environments. Nano-black phosphorus (BP) sheets are considered to be an ideal new type of nano-drug carrier due to their advantages such as degradability, good biocompatibility and large specific surface area. Summary of the invention

[0003] The present invention aims to use RNA sequences as gene drugs, adopt PEI-modified nano BP, and use positively charged PEI to load negatively charged gene drugs onto nano BP carriers through the attraction of positive and negative charges to construct BP-PEI@RNA nano drug complexes. BP-PEI@RNA can efficiently deliver RNA to tumor tissues and cells, while also protecting RNA and reducing the degradation of nucleases in vivo and in vitro; further, by means of the competitive binding of the gene drug with specific miRNA, the degradation of target gene mRNA by miRNA is reduced, thereby increasing the expression level of the target gene in tumor cells, and ultimately playing a role in inhibiting the progression of prostate cancer.

[0004] The invention provides a BP-PEI@RNA nano drug complex, which comprises a positively charged nano material and a negatively charged nucleic acid.

[0005] Furthermore, the nucleic acid is RNA.

[0006] Furthermore, the RNA includes lncRNA and circRNA.

[0007] In a specific embodiment of the present invention, the RNA sequence is selected from any one or more of SEQ ID NO.1-4.

[0008] Furthermore, the positively charged nanomaterial is a nanomaterial modified by PEI.

[0009] Furthermore, the nanomaterials include nano black phosphorus sheets, liposomes, graphene oxide, nano gold, gelatin, silk fibroin, collagen, chitosan, and nano polyester materials.

[0010] In a specific embodiment of the present invention, the nanomaterial is nano black phosphorus sheet.

[0011] The present invention also provides a BP-PEI@RNA nano-drug complex labeled with a fluorescent substance, wherein the BP-PEI@RNA nano-drug complex labeled with a fluorescent substance comprises the aforementioned BP-PEI@RNA nano-drug complex and a fluorescent substance connected to RNA.

[0012] Furthermore, the fluorescent substance is 6-FAM or Cy3.

[0013] The present invention also provides a method for preparing the above-mentioned BP-PEI@RNA nano-drug complex, the preparation method comprising the following steps:

[0014] 1) Prepare RNA;

[0015] 2) Preparation of negatively charged nanomaterials;

[0016] 3) Co-incubating the RNA base with the negatively charged nanomaterial.

[0017] Further, the specific preparation process of step 2) is as follows: take a nano black phosphorus sheet solution, remove the supernatant after centrifugation, resuspend in enzyme-free water, mix and then sonicate, repeat the centrifugation-resuspend in enzyme-free water-sonication steps to obtain a nano black phosphorus sheet aqueous phase dispersion solution. Then add PEI solution to the above nano black phosphorus sheet aqueous phase dispersion solution and sonicate. After centrifugation and washing to remove excess PEI, resuspend.

[0018] Furthermore, the volume ratio of the nano black phosphorus sheet aqueous dispersion solution to the PEI solution is 1:1.5.

[0019] Furthermore, the ultrasound is ice water bath ultrasound.

[0020] Further, the specific preparation process of step 2) is as follows: take 1mL of nano black phosphorus sheet solution (0.1mg / mL) in an EP tube, centrifuge at 4°C (16000rpm, 20min) and remove the supernatant, resuspend in an equal amount of enzyme-free water, mix by blowing and ice-water bath ultrasound (40kHz, 100W, 20min), repeat the centrifugation-enzyme-free water resuspension-ice-water bath ultrasound steps 2 times to obtain a well-dispersed nano black phosphorus sheet aqueous phase dispersion solution. Subsequently, an appropriate amount of PEI (polyetherimide, PEI) (2.5k, 10mg / mL, nano black phosphorus sheet and PEI volume ratio of 1:1.5) was added to the above-mentioned nano black phosphorus sheet aqueous solution and ultrasonicated in an ice-water bath for 3-4h. Resuspend after centrifugal washing to remove excess PEI.

[0021] Furthermore, the specific preparation process of step 3) is as follows: obtain a PEI-modified nano black phosphorus sheet solution through step 2), add RNA, and incubate them together, while continuously rotating and mixing, to obtain a BP-PEI@RNA nano drug complex.

[0022] Furthermore, the mass ratio of nano black phosphorus sheets to RNA is 1:0.4.

[0023] Furthermore, the specific preparation process of step 3) is as follows: RNA (BP to RNA mass ratio is 1:0.4) is added and incubated at 4°C for 10 minutes, during which continuous rotation and mixing are performed to obtain a BP-PEI@RNA nanodrug complex.

[0024] The present invention also provides a method for preparing the aforementioned fluorescently labeled BP-PEI@RNA nano-drug complex, the preparation method comprising the following steps:

[0025] a) preparing RNA labeled with a fluorescent substance;

[0026] b) preparing nanomaterials with negative charges;

[0027] c) Incubating RNA with negatively charged nanomaterials.

[0028] Furthermore, the specific preparation process of step a) is as follows: using the linearized DNA of the RNA gene drug as a template and the ribonucleotide labeled with a fluorescent substance as a fluorescent substrate, the RNA gene drug labeled with a fluorescent substance is obtained by in vitro transcription.

[0029] Further, the specific preparation process of step b) is as follows: take a nano black phosphorus sheet solution, remove the supernatant after centrifugation, resuspend in enzyme-free water, mix and then sonicate, repeat the centrifugation-resuspend in enzyme-free water-sonication steps to obtain a nano black phosphorus sheet aqueous phase dispersion solution. Then add PEI solution to the above nano black phosphorus sheet aqueous phase dispersion solution and sonicate. After centrifugation and washing to remove excess PEI, resuspend.

[0030] Furthermore, the volume ratio of the nano black phosphorus sheet aqueous dispersion solution to the PEI solution is 1:1.5.

[0031] Furthermore, the ultrasound is ice water bath ultrasound.

[0032] Further, the specific preparation process of step b) is as follows: take 1mL of nano black phosphorus sheet solution (0.1mg / mL) in an EP tube, centrifuge at 4°C (16000rpm, 20min) and remove the supernatant, resuspend in an equal amount of enzyme-free water, mix by blowing and ice-water bath ultrasound (40kHz, 100W, 20min), repeat the centrifugation-enzyme-free water resuspension-ice-water bath ultrasound steps 2 times to obtain a well-dispersed nano black phosphorus sheet aqueous phase dispersion solution. Subsequently, an appropriate amount of PEI (polyetherimide, PEI) (2.5k, 10mg / mL, nano black phosphorus sheet and PEI volume ratio of 1:1.5) is added to the above-mentioned nano black phosphorus sheet aqueous solution and ultrasonicated in an ice-water bath for 3-4h. Resuspend after centrifugal washing to remove excess PEI.

[0033] Furthermore, the specific preparation process of step c) is as follows: the PEI-modified nano black phosphorus sheet solution obtained in step b) is added with the RNA labeled with a fluorescent substance obtained in step a), and the mixture is incubated with continuous rotation and mixing to obtain a BP-PEI@RNA nanodrug complex labeled with a fluorescent substance.

[0034] Furthermore, the mass ratio of nano black phosphorus sheets to RNA is 1:0.4.

[0035] Furthermore, the specific preparation process of step c) is as follows: RNA labeled with fluorescent substance (BP to RNA mass ratio is 1:0.4) is added and incubated at 4° C. for 10 minutes, during which the mixture is continuously rotated and mixed to obtain BP-PEI@RNA nanodrug complex.

[0036] The present invention also provides an application of the above-mentioned BP-PEI@RNA nano-drug complex in the preparation of disease treatment.

[0037] Furthermore, the disease is a tumor.

[0038] Furthermore, the tumor is prostate cancer.

[0039] The present invention also provides an application of the above-mentioned BP-PEI@RNA nano-drug complex in the preparation of disease treatment.

[0040] Furthermore, the tumor is cancer.

[0041] Furthermore, the tumor is prostate cancer.

[0042] Advantages and beneficial effects of the present invention:

[0043] The present invention utilizes nano-BP to load specific RNA sequences to construct a BP-PEI@RNA nano-drug complex, which can achieve efficient and stable delivery of RNA and transport exogenous gene drugs (RNA) into tumor cells; by increasing the expression level of tumor suppressor genes in tumor cells, it plays a role in inhibiting the progression of prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The graphs show the morphology and structure characterization results of BP and BP-PEI@RNA nanodrug complexes, including (a) transmission electron microscopy observation results of BP-PEI@RNA; (b) atomic force microscopy observation results of BP-PEI@RNA; (c) Zeta potential results of BP-PEI@RNA; (d) hydration particle size results of BP-PEI@RNA; (e) Raman spectroscopy results;

[0045] Figure 2 Figures showing the results of cellular uptake and subcellular localization of BP-PEI@RNA, including (a) cellular uptake of BP-PEI@RNA at different time points; (b) subcellular localization of BP-PEI@RNA after cellular uptake at different time points;

[0046] Figure 3 Results of the effect of BP-PEI@RNA on the viability of prostate cancer cells, including (a) CCK8 results; (b) cell images of live / dead cell staining method; (c) cell survival rate statistics of live / dead cell staining method;

[0047] Figure 4 The results of the effect of BP-PEI@RNA on PC3 cell cycle are shown in Figure 1, including (a) flow cytometry graph; (b) statistical graph;

[0048] Figure 5 Figures showing the effect of BP-PEI@RNA on cell apoptosis, including (a) flow cytometry graph; (b) statistical graph;

[0049] Figure 6 Result graphs showing the in vivo anti-tumor effect of BP-PEI@RNA, including (a) tumor growth curve graph; (b) tumor size measurement graph; (c) tumor weight result graph; (d) cell apoptosis result graph. DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0051] Example

[0052] 1. Preparation of Nano-Black Phosphorus Sheets (BP)

[0053] Nano-black phosphorus flakes were purchased from Zhongke Mophosphorus Technology Co., Ltd. (0.1 mg / mL) and had a size of approximately 200-300 nm.

[0054] 2. Preparation of RNA gene drugs

[0055] The four RNA sequences are shown in SEQ ID NO: 1-4.

[0056] RNA-1:

[0057]

[0058] RNA-2:

[0059]

[0060] RNA-3:

[0061] (SEQ ID NO:3)

[0062] RNA-4:

[0063] (SEQ ID NO:4)

[0064] Prostate cancer PC3 cell mRNA was extracted and reverse transcribed to obtain cDNA. PCR amplification of 4 segments of RNA was performed using cDNA as a template. The resulting gene products were subjected to agarose gel electrophoresis and gel excision recovery. Subsequently, a T vector was constructed, and linearized DNA was obtained through experiments such as transformation, shake amplification, plasmid extraction, sequencing, gene sequence verification, linearized plasmid, agarose gel electrophoresis, gel excision recovery, and finally 4 segments of RNA gene drugs were obtained by in vitro transcription.

[0065] 3. Construction of Nanodrug Complex (BP-PEI@RNA)

[0066] Take 1mL BP solution (0.1mg / mL) in an EP tube, centrifuge at 4℃ (16000rpm, 20min), remove the supernatant, resuspend in an equal amount of enzyme-free water, mix by blowing, and then ultrasonicate in an ice-water bath (40kHz, 100W, 20min). Repeat the centrifugation-resuspend in enzyme-free water-ultrasonication in an ice-water bath for 2 times to obtain a well-dispersed BP aqueous phase dispersion solution. Then, add an appropriate amount of polyetherimide (PEI) (2.5k, 10mg / mL, BP to PEI volume ratio of 1:1.5) to the above BP aqueous solution and ultrasonicate in an ice-water bath for 3-4h. After centrifugation and washing to remove excess PEI, resuspend in enzyme-free water, add an appropriate amount of RNA-2 (BP to RNA-2 mass ratio of 1:0.4) and incubate at 4℃ for 10 minutes, during which continuous rotation and mixing are obtained to obtain BP-PEI@RNA nanodrug complex.

[0067] The morphology and structure characterization results of BP and BP-PEI@RNA nanodrug complexes are shown in the figure. Figure 1 As shown: Figure 1 (a) is the transmission electron microscopy observation result of BP-PEI@RNA. As shown in the figure, BP is an ultra-thin nanosheet with an average lateral size mainly between 200-300nm. After PEI modification and RNA-2 loading, the BP morphology did not change significantly. Figure 1 (b) is the result of atomic force microscopy observation of BP-PEI@RNA. The result is consistent with TEM. The original BP material is an ultra-thin nanosheet with an average lateral size of 200-300nm. After PEI modification and RNA loading, its particle size and morphology change significantly. Figure 1 (c) is the Zeta potential result of BP-PEI@RNA. The average Zeta potentials of BP, BP-PEI, and BP-PEI@RNA were -22.93±0.58, 36.47±1.94, and 22.1±0.56 mV, respectively. Figure 1(d) is the BP-PEI@RNA hydrated particle size results. The average particle sizes of BP, BP-PEI, and BP-PEI@RNA in their aqueous dispersion systems are 178.67±7.00, 318.10±2.18, and 592.7±6.45 nm, respectively. Figure 1 (e) is the Raman spectrum result. The three typical Raman spectrum peaks of BP appear at 360.8 cm –1 、438.7cm –1 and 465.8cm –1 .

[0068] 4. RNA Fluorescence Labeling

[0069] Method 1: 6-carboxyfluorescein (6-FAM) labeling of BP-PEI@RNA

[0070] In the preparation process of RNA gene drugs, linearized DNA is obtained through relevant steps, and 6-FAM-labeled ribonucleotides are used as fluorescent substrates to obtain 6-FAM-labeled RNA gene drugs through in vitro transcription, and then step 3 is repeated to obtain BP-PEI@RNA-6FAM nanodrug complexes.

[0071] Method 2: Cy3 fluorescent dye labeling RNA

[0072] In the preparation process of RNA gene drugs, linearized DNA is obtained through relevant steps, and Cy3-labeled ribonucleotides are used as fluorescent substrates to obtain Cy3-labeled RNA gene drugs through in vitro transcription, and then step 3 is repeated to obtain BP-PEI@RNA-Cy3 nanodrug complexes.

[0073] 5. Cellular uptake and subcellular localization of BP-PEI@RNA

[0074] When PC3 cells were expanded in 1640 medium to a cell density of 50%-60%, the cells were exposed to BP-PEI@RNA labeled with 6-FAM and Cy3 fluorescent dyes (BP concentration was 1 μg / mL; RNA concentration was 0.4 μg / mL). The uptake and subcellular localization of BP-PEI@RNA in PC3 cells were observed by laser confocal microscopy at different time points after exposure. The cellular uptake and subcellular localization of BP-PEI@RNA are shown in Figure 2. Figure 2 ( Figure 2 The RNA in the RNA is RNA-2): Figure 2(a) BP-PEI@RNA uptake by cells at different time points. RNA was labeled with 6-FAM and BP-PEI@RNA-6FAM nanodrug complex was constructed. PC3 cells were exposed to the nanodrug complex and laser confocal observation was performed. In the figure, the red fluorescence is the cytoskeleton, the green is BP-PEI@RNA-6FAM labeled with 6-FAM, and the blue is the cell nucleus. The BP-PEI@RNA-6FAM nanodrug complex was taken up by cells. After exposure for 3h and 6h, the cell uptake of the BP-PEI@RNA-6FAM nanodrug complex gradually increased. Figure 2 (b) Subcellular localization of BP-PEI@RNA after being taken up by cells at different time points. The red color in the figure is Cy3-labeled RNA or BP-PEI@RNA, and the green color is lysosomes. After the free RNA exposed cells, no exogenous RNA was detected in the cells. However, 6 hours after the BP-PEI@RNA exposed cells, a large amount of exogenous RNA was taken up by the cells, and the taken-up RNA was mainly distributed in the lysosomes (the orange-yellow fluorescence in the figure indicates the co-localization of RNA and lysosomes). When the exposure time was extended to 24 hours, the RNA taken up into the lysosomes was released into the cytoplasm. This indicates that BP-PEI@RNA enters the lysosomes after being taken up by the cells, and then the lysosomes lyse and release BP-PEI@RNA into the cytoplasm. The above data show that after the free RNA is exposed to the cells, the RNA may not be able to enter the cells due to its own instability or weak ability to penetrate the cell membrane. When loaded into BP-PEI, RNA can be efficiently taken up by cells. After uptake, the BP-PEI@RNA nanodrug complex is mainly distributed in lysosomes, and then the lysosomes lyse and release BP-PEI@RNA into the cytoplasm to exert its effect.

[0075] 6. Evaluation of BP-PEI@RNA's anti-tumor function in vitro

[0076] When PC3 cells were grown in 1640 medium to a density of 50%-60%, they were exposed to BP-PEI@RNA (BP concentration of 1 μg / mL; RNA concentration of 0.4 μg / mL) for 24 h, and then cell function was evaluated.

[0077] BP-PEI loaded with four different RNA sequences were exposed to PC3 cells respectively, and the effect of BP-PEI@RNA on cell viability was evaluated by CCK8 and cell live / dead staining. The experimental results are shown in Figure 3 Shown by: Figure 3 (a) It can be seen that the cell viability of the BP-PEI@RNA group was significantly weakened compared with the BP-PEI group and the control group. Figure 3 In (b), green fluorescence indicates living cells, and red fluorescence indicates dead cells. The number of cell deaths in the BP-PEI@RNA exposure group was significantly higher than that in the BP-PEI group and the control group; Figure 3 (c) is the survival rate of cells in the experimental group. It can be seen that the cell survival rate in the BP-PEI@RNA group is significantly lower than that in the BP-PEI group and the control group.

[0078] BP-PEI loaded with four different RNA sequences were exposed to PC3 cells respectively, and the effect of BP-PEI@RNA on the cell cycle was evaluated by detecting the cell cycle. Figure 4 As shown: Figure 4 (a) and Figure 4 (b) is the result of flow cytometry detection of cell cycle. It can be seen that after BP-PEI@RNA exposed cells, the proportion of cells arrested in the G0 / G1 phase (pre-DNA synthesis phase) increased significantly, while the proportion of cells entering the G2 / M phase (late DNA synthesis / mitosis phase) decreased significantly, indicating that BP-PEI@RNA blocked the cell cycle process and thus inhibited cell proliferation.

[0079] BP-PEI loaded with four different RNA sequences were exposed to PC3 cells respectively, and the effect of BP-PEI@RNA on cell apoptosis was evaluated by detecting cell apoptosis. Figure 5 As shown: Figure 5 (a) and Figure 5 In (b), after BP-PEI@RNA exposure, the apoptotic cell ratio was significantly increased compared with that of the BP-PEI group and the control group. This indicates that BP-PEI@RNA exposure of PC3 cells can promote cell apoptosis.

[0080] 7. Evaluation of the anti-tumor effect of BP-PEI@RNA in vivo

[0081] 2×10 6 The tumor-bearing mouse model was established by using PC3 cells. Two weeks later, the BP-PEI@RNA nanodrug complex was injected into the tumor (injected once every four days, for a total of 4 injections). The tumor size was measured every 3 days after the injection of the drug, and the tumor growth curve was drawn. The mice were killed on the 5th day after the injection, and the tumor tissue was removed and photographed and weighed. At the same time, the Tunel experiment was used to detect the level of cell apoptosis in the tumor tissue. The experimental results are shown in Figure 2. Figure 6 As shown ( Figure 6 RNA in the tumor is RNA-2): Figure 6 In (a), the tumor volume of the BP+RNA group was significantly lower than that of the Cont., BP, and RNA groups; Figure 6 In (b) and (c), the size and weight of the excised tumors in the BP+RNA group were significantly lower than those in the other experimental groups (Cont., BP, and RNA groups). Figure 6As shown in (f), the apoptosis expression level of tumor tissue in the BP+RNA group was significantly higher than that in the control group. The above data show that BP-PEI@RNA has a strong anti-tumor effect in vivo.

[0082] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A BP-PEI@RNA nanodrug complex, characterized in that: The BP-PEI@RNA nano drug complex comprises a positively charged nano material and a negatively charged RNA; the RNA sequence is shown in SEQ ID NO.2, the nano material is a nano black phosphorus sheet, and the PEI is polyetherimide.

2. A BP-PEI@RNA nanodrug complex labeled with a fluorescent substance, characterized in that: The BP-PEI@RNA nano-drug complex labeled with a fluorescent substance comprises the BP-PEI@RNA nano-drug complex according to claim 1 and a fluorescent substance connected to RNA.

3. The BP-PEI@RNA nanodrug complex labeled with a fluorescent substance according to claim 2, characterized in that: The fluorescent substance is 6-FAM or Cy3.

4. A method for preparing the BP-PEI@RNA nanodrug complex according to claim 1, characterized in that: The preparation method comprises the following steps: 1) Prepare RNA; 2) Preparation of negatively charged nanomaterials; 3) co-incubating RNA with negatively charged nanomaterials; The specific preparation process of step 2) is as follows: take a nano black phosphorus sheet solution, remove the supernatant after centrifugation, resuspend in enzyme-free water, mix and then sonicate, repeat the centrifugation-resuspend in enzyme-free water-sonication steps to obtain a nano black phosphorus sheet aqueous phase dispersion solution; then add PEI solution to the above nano black phosphorus sheet aqueous phase dispersion solution and sonicate; remove excess PEI by centrifugation and washing, and then resuspend; The volume ratio of the aqueous dispersion solution of nano-black phosphorus sheets to the PEI solution is 1:1.5; The ultrasound is ice water bath ultrasound; The specific preparation process of step 2) is as follows: take 1 mL of a 0.1 mg / mL nano black phosphorus sheet solution in an EP tube, centrifuge at 4°C 16000 rpm for 20 min, remove the supernatant, resuspend in an equal amount of enzyme-free water, mix by blowing, and then ultrasonicate in an ice-water bath at 40kHz and 100W for 20 min, repeat the centrifugation-enzyme-free water resuspension-ice-water bath ultrasonication steps twice to obtain a well-dispersed nano black phosphorus sheet aqueous phase dispersion solution; then add 2.5k PEI with a concentration of 10 mg / mL to the above nano black phosphorus sheet aqueous solution, the volume ratio of nano black phosphorus sheet to PEI is 1:1.5, and ultrasonicate in an ice-water bath for 3-4 h; remove excess PEI by centrifugal washing and resuspend; The specific preparation process of step 3) is as follows: obtain a PEI-modified nano black phosphorus sheet solution through step 2), add RNA, and co-incubate, and continuously rotate and mix during the process to obtain a BP-PEI@RNA nano drug complex; The mass ratio of nano-black phosphorus sheets to RNA is 1:0.4; The specific preparation process of step 3) is as follows: RNA is added so that the mass ratio of the nano black phosphorus sheet to the RNA is 1:0.4, and the mixture is incubated at 4° C. for 10 minutes, during which the mixture is continuously rotated and mixed to obtain a BP-PEI@RNA nanodrug complex; The PEI is polyetherimide.

5. A method for preparing the BP-PEI@RNA nano-drug complex labeled with a fluorescent substance according to claim 2, the method comprising the following steps: a) preparing RNA labeled with a fluorescent substance; b) preparing nanomaterials with negative charges; c) co-incubating RNA with negatively charged nanomaterials; The specific preparation process of step a) is as follows: using RNA linearized DNA as a template and ribonucleotides labeled with fluorescent substances as fluorescent substrates, obtaining RNA labeled with fluorescent substances by in vitro transcription; The specific preparation process of step b) is as follows: taking a nano black phosphorus sheet solution, removing the supernatant after centrifugation, resuspending in enzyme-free water, mixing and ultrasonicating, repeating the centrifugation-resuspending in enzyme-free water-ultrasonicating steps to obtain a nano black phosphorus sheet aqueous phase dispersion solution; then adding a PEI solution to the above nano black phosphorus sheet aqueous phase dispersion solution and ultrasonicating; centrifuging and washing to remove excess polyetherimide and then resuspending; The volume ratio of the aqueous dispersion solution of nano-black phosphorus sheets to the PEI solution is 1:1.5; The ultrasound is ice water bath ultrasound; The specific preparation process of step b) is as follows: take 1 mL of a 0.1 mg / mL nano black phosphorus sheet solution in an EP tube, centrifuge at 4°C and 16000 rpm for 20 min, remove the supernatant, resuspend in an equal amount of enzyme-free water, mix by blowing, and then ultrasonicate in an ice-water bath at 40kHz and 100W for 20 min, repeat the centrifugation-resuspend in enzyme-free water-ultrasonication in an ice-water bath for 2 times to obtain a well-dispersed nano black phosphorus sheet aqueous phase dispersion solution; then add 2.5k PEI with a concentration of 10 mg / mL to the above nano black phosphorus sheet aqueous solution, the volume ratio of nano black phosphorus sheet to PEI is 1:1.5, and ultrasonicate in an ice-water bath for 3-4 h; remove excess PEI by centrifugal washing and resuspend; The specific preparation process of step c) is as follows: the PEI-modified nano black phosphorus sheet solution obtained in step b) is added with the RNA labeled with a fluorescent substance obtained in step a), and the mixture is incubated with continuous rotation and mixing to obtain a BP-PEI@RNA nano drug complex labeled with a fluorescent substance; The mass ratio of nano black phosphorus sheets to RNA labeled with fluorescent substances is 1:0.4; The specific preparation process of step c) is as follows: adding RNA labeled with a fluorescent substance so that the mass ratio of the nano black phosphorus sheet to the fluorescent substance label is 1:0.4, incubating at 4° C. for 10 minutes, and continuously rotating and mixing during the period to obtain a BP-PEI@RNA nanodrug complex; The PEI is polyetherimide.

6. Use of the BP-PEI@RNA nano-drug complex according to claim 1 in the preparation of a drug for treating prostate cancer.

7. Use of the BP-PEI@RNA nano-drug complex labeled with a fluorescent substance as claimed in claim 2 in the preparation of a drug for treating prostate cancer.

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