Targeted drug-loaded nanoparticles, their preparation methods and applications, and a targeted drug delivery system based on the CRISPR cleavage system

Abstract

The present invention belongs to the technical field of anti-tumor drugs, and particularly relates to a targeted drug-loaded nanoparticle, a preparation method and application thereof, and a targeted drug delivery system based on a CRISPR cleavage system. The targeted drug-loaded nanoparticle provided by the present invention comprises a hollow silica nanoparticle, a telomerase inhibitor loaded in the hollow structure; and a targeting material and a pore-sealing material chemically modified on the outer surface of the shell structure of the hollow carrier. The targeted drug delivery system based on the CRISPR cleavage system provided by the present invention comprises a targeted drug-loaded nanoparticle; a DNA substrate; a crRNA and an Lba Cas12a (Cpf 1). The targeted drug delivery system provided by the present invention releases a telomerase inhibitor by means of the highly expressed telomerase in tumor cells, and acts on the inhibition of the activity of telomerase in tumor cells, providing a unique and novel tumor telomerase "suicide" behavior mode, and realizing the effective killing of tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drugs, and specifically relates to targeted drug-loaded nanoparticles, a preparation method and application thereof, and a targeted drug delivery system based on the CRISPR cleavage system. Background Art

[0002] Cancer is a rapidly growing abnormal cell that grows beyond its normal boundaries and can invade and spread to other organs. Globally, nearly one-sixth of deaths are caused by cancer. Worldwide, cancer is a major cause of morbidity and mortality. According to WTO statistics, there were approximately 14 million new cancer cases in 2012.

[0003] Among the various treatment methods for cancer, chemotherapy, as one of the most important methods, can be used to treat various types of tumors. Chemotherapy drugs are characterized by systemic administration and are usually administered at the maximum tolerated dose. However, the current chemotherapy effect is still poor, and only less than 1% of the anti-cancer drugs can reach the target cancer cells. The remaining drugs will attack the normal cells of the body in vivo, which may cause serious toxic side effects. On the other hand, the high incidence of tumor recurrence and the easy generation of drug resistance in patients lead to poor anti-cancer effects.

[0004] Therefore, there is an urgent need to develop new effective drugs and treatment systems for cancer treatment. Summary of the Invention

[0005] The object of the present invention is to provide targeted drug-loaded nanoparticles, a preparation method and application thereof, and a targeted drug delivery system based on the CRISPR cleavage system. The targeted drug delivery system provided by the present invention can release telomerase inhibitors by means of the highly expressed telomerase in tumor cells, which in turn inhibits the activity of telomerase, providing a unique and effective telomerase "suicide" behavior pattern, thereby promoting tumor cell senescence and achieving the purpose of damaging tumor cells.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a targeted drug-loaded nanoparticle, comprising a hollow carrier, the hollow carrier is composed of a shell structure with nanoscale pores and a hollow structure; a drug loaded in the hollow structure of the hollow carrier; and a targeting material and a pore-sealing material chemically modified on the outer surface of the shell structure of the hollow carrier, the pore-sealing material is used to block the nanoscale pores of the shell structure of the hollow carrier;

[0008] The hollow carrier is a hollow silica nanoparticle;

[0009] The drug is a telomerase inhibitor;

[0010] The sealing material is obtained by hybridizing FQssDNA with ssDNA1 and ssDNA2 simultaneously; the nucleotide sequence of ssDNA1 is shown as SEQ ID NO.1, the nucleotide sequence of ssDNA2 is shown as SEQ ID NO.2, and the nucleotide sequence of FQssDNA is shown as SEQ ID NO.3.

[0011] Preferably, the targeting material is the MUC1 nucleic acid aptamer capable of targeting and recognizing MCF-7 breast cancer cells.

[0012] Preferably, the diameter of the hollow carrier is 150 - 200 nm.

[0013] Preferably, the thickness of the shell structure with nanoscale pores is 30 - 40 nm, and the size of the nanoscale pores is ~3.8 nm.

[0014] The present invention provides a method for preparing the targeted drug-loaded nanoparticles described in the above technical solution, comprising the following steps:

[0015] Mix the carboxylated hollow carrier and the telomerase inhibitor for loading to obtain drug-loaded particles;

[0016] Activate the drug-loaded particles with a carboxyl activator to obtain activated drug-loaded particles;

[0017] Mix the activated drug-loaded particles, amino-modified ssDNA1, amino-modified ssDNA2, and amino-modified targeting material for the first co-incubation, and mix the obtained modified particles with FQssDNA for the second co-incubation to obtain the targeted drug-loaded nanoparticles.

[0018] Preferably, the method for preparing the carboxylated hollow carrier comprises the following steps:

[0019] Carboxylate the hollow carrier by mixing it with a strong acid solution and 3-(triethoxysilyl)propyl succinic anhydride to obtain the carboxylated hollow carrier.

[0020] Preferably, the mass ratio of the carboxylated hollow carrier to the telomerase inhibitor is 10:1.

[0021] Preferably, the molar ratio of the amino-modified ssDNA1, amino-modified ssDNA2, amino-modified targeting material, and FQssDNA is 1:1:1:2.

[0022] The present invention provides the use of the targeted drug-loaded nanoparticles described in the above technical solution or the targeted drug-loaded nanoparticles prepared by the preparation method described in the above technical solution in the preparation of anti-tumor drugs.

[0023] The present invention provides a targeted drug delivery system based on the CRISPR cleavage system, comprising the targeted drug-loaded nanoparticles described in the above technical solution or the targeted drug-loaded nanoparticles prepared by the preparation method described in the above technical solution;

[0024] A DNA substrate, which is formed by hybridization of a truncated sequence and its complementary sequence. The nucleotide sequence of the truncated sequence is as shown in SEQ ID NO.4, and the nucleotide sequence of the complementary sequence of the truncated sequence is as shown in SEQ ID NO.5;

[0025] A crRNA, the nucleotide sequence of which is as shown in SEQ ID NO.6;

[0026] Lba Cas12a (Cpf 1).

[0027] The present invention provides a targeted drug-loaded nanoparticle, comprising a hollow carrier, which is composed of a shell structure with nanoscale pores and a hollow structure; a drug loaded in the hollow structure of the hollow carrier; and a targeting material and a pore-blocking material chemically modified on the outer surface of the shell structure of the hollow carrier. The pore-blocking material is used to block the nanoscale pores of the shell structure of the hollow carrier; the hollow carrier is a hollow silica nanoparticle; the drug is a telomerase inhibitor; the pore-blocking material is obtained by hybridizing FQssDNA with ssDNA1 and ssDNA2 simultaneously; the nucleotide sequence of ssDNA1 is as shown in SEQ ID NO.1, the nucleotide sequence of ssDNA2 is as shown in SEQ ID NO.2, and the nucleotide sequence of FQssDNA is as shown in SEQ ID NO.3. For the targeted drug-loaded nanoparticle provided by the present invention, by modifying the targeting material on the surface of the shell structure, the nanoparticle can specifically target tumor cells. By modifying the pore-blocking material on the surface of the shell structure, the pore-blocking material obtained by hybridizing FQssDNA with ssDNA1 and ssDNA2 simultaneously can effectively block the nanoscale pores on the shell structure, thereby blocking the telomerase inhibitor (active drug) located in the hollow structure. When the targeted drug-loaded nanoparticle reaches the target tumor cell, after FQssDNA is cleaved, the telomerase inhibitor is released from the hollow structure, thereby effectively inhibiting the activity of telomerase in the tumor cell and promoting the senescence of the tumor cell, achieving the purpose of damaging the tumor cell. The targeted drug-loaded nanoparticle provided by the present invention only uses a telomerase inhibitor as the active drug, and blocks and releases the active drug by hybridizing FQssDNA with ssDNA1 and ssDNA2 simultaneously, realizing the killing effect on tumor cells, with a simple structure and good effect.

[0028] The present invention provides a targeted drug delivery system based on the CRISPR cleavage system, including the targeted drug-loaded nanoparticles described in the above technical solution or the targeted drug-loaded nanoparticles prepared by the preparation method described in the above technical solution; a DNA substrate, which is formed by hybridization of a truncated sequence and its complementary sequence, the nucleotide sequence of the truncated sequence is as shown in SEQ ID NO.4, and the nucleotide sequence of the complementary sequence of the truncated sequence is as shown in SEQ ID NO.5; a crRNA, the nucleotide sequence of which is as shown in SEQ ID NO.6; Lba Cas12a (Cpf1). In the targeted drug delivery system provided by the present invention, the DNA substrate in the targeted drug delivery system is recognized and extended by telomerase highly expressed in tumor cells so as to be complementary paired with the crRNA, thereby effectively activating Cas12a (Lba Cas12a (Cpf1)). The activated CRISPR-Cas12a precisely cleaves FQdsDNA (FQdsDNA is a sealing material obtained by hybridizing FQssDNA with ssDNA1 and ssDNA2 simultaneously), releasing the telomerase inhibitor (BIBR1532) in the hollow structure of the hollow carrier, thereby inhibiting the activity of telomerase in tumor cells and promoting the senescence of tumor cells, achieving the purpose of damaging tumor cells. The targeted drug delivery system provided by the present invention releases the telomerase inhibitor by means of telomerase highly expressed in tumor cells, and acts on inhibiting the activity of telomerase in tumor cells, providing a unique and novel "suicide" behavior mode of tumor telomerase, and realizing the effective killing of tumor cells. Description of the Drawings

[0029] Figure 1 is the TEM image of HSN in the example ( Figure 1 Figure A in Figure 1 ), and the DLS image (

[0030] Figure 2 is the nitrogen adsorption-desorption isotherm ( Figure 2 Figure A in Figure 2 ), and the pore size distribution diagram (

[0031] Figure 3 of hollow silica and carboxylated hollow silica in the example;

[0032] Figure 4 is the result of agarose gel electrophoresis test;

[0033] Figure 5 is the zeta potential characterization result of HSN, HSN-COOH, BIBR1532@HSN, Apts, FQssDNA, ssDNA and BIBR1532@HSN / FQdsDNA / Apts in the example;

[0034] Figure 6 are the fluorescence spectra ( Figure 6 Figure A in Figure 6 ) and fluorescence intensity graphs ( Figure 6 Figure B in Figure 6 ) for each experimental group. In Figure 6 , 1 represents Cas12a + crRNA, DNA substrate, telomerase, BIBR1532@HSN / FQdsDNA / Apts; Figure 6 in Figure 6 , 2 represents DNA substrate, telomerase, BIBR1532@HSN / FQdsDNA / Apts;

[0035] Figure 7 are the cell fluorescence imaging graphs ( Figure 7 Figure A in Figure 7 ) and flow cytometry analysis of FAM content under different conditions ( Figure 7 Figure B in Figure 7 ). In Figure 7 , (1) represents PBS; Figure 7 in Figure 7 , (2) represents Cas12a, crRNA; Figure 7 in

[0036] Figure 8 are the fluorescence images after MCF-7 breast cancer cells were co-incubated with BIBR1532@HSN / FQdsDNA / Apts nanoparticles for 1 h, 2 h, and 3 h respectively ( Figure 8 Figure A in Figure 8 ) and the fluorescence images after MDA-MB-231 breast cancer cells were co-incubated with BIBR1532@HSN / FQdsDNA / Apts nanoparticles for 1 h, 3 h, and 5 h respectively ( Figure 8 Figure B in

[0037] The scale bar in each is: 20 μm;Figure 9 The senescence of MCF-7 breast cancer cells was detected by a β-galactosidase kit under the conditions of PBS; HSN; BIBR1532@HSN / FQdsDNA / Apts; and BIBR1532@HSN / FQdsDNA / Apts / Cas, Figure 9 The scale bar for the images is: 1 μm;

[0038] Figure 10 The cell viability of MCF-7 cells after co-incubation with different concentrations of HSN ( Figure 10 Figure A in), the cell viability of MCF-7 cells after co-incubation with different concentrations of BIBR1532@HSN / FQdsDNA / Apts ( Figure 10 Figure B in), and the cell viability of MCF-7 cells after co-incubation with different concentrations of BIBR1532@HSN / FQdsDNA / Apts while adding the CRISPR system (Cas12a + crRNA, DNA substrate) ( Figure 10 Figure C in). Detailed implementation method

[0039] The present invention provides a drug-loaded targeted nanoparticle, which includes a hollow carrier composed of a shell structure with nanoscale pores and a hollow structure; a drug loaded in the hollow structure of the hollow carrier; and a targeting material and a pore-blocking material chemically modified on the outer surface of the shell structure of the hollow carrier, where the pore-blocking material is used to block the nanoscale pores of the shell structure of the hollow carrier;

[0040] The hollow carrier is a hollow silica nanoparticle;

[0041] The drug is a telomerase inhibitor;

[0042] The pore-blocking material is obtained by hybridizing FQssDNA with ssDNA1 and ssDNA2 simultaneously; the nucleotide sequence of ssDNA1 is as shown in SEQ ID NO.1, the nucleotide sequence of ssDNA2 is as shown in SEQ ID NO.2, and the nucleotide sequence of FQssDNA is as shown in SEQ ID NO.3.

[0043] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well-known to those skilled in the art.

[0044] The targeted drug-loaded nanoparticles provided by the present invention comprise a hollow carrier, which consists of a shell structure with nanoscale pores and a hollow structure. In the present invention, the hollow carrier is preferably a hollow silica nanoparticle. The diameter of the hollow carrier is preferably 150 - 200 nm, more preferably 190 nm. The thickness of the shell structure with nanoscale pores is preferably 30 - 40 nm, and the size of the nanoscale pores is preferably ~3.8 nm.

[0045] The targeted drug-loaded nanoparticles provided by the present invention comprise a drug loaded in the hollow structure of the hollow carrier. In the present invention, the drug is a telomerase inhibitor (BIBR1532).

[0046] The targeted drug-loaded nanoparticles provided by the present invention comprise a targeting material and a pore-blocking material chemically modified on the outer surface of the shell structure of the hollow carrier, and the pore-blocking material is used to block the nanoscale pores of the shell structure of the hollow carrier. In the present invention, the targeting material is a MUC1 nucleic acid aptamer capable of targeting and recognizing MCF-7 breast cancer cells. The nucleotide sequence of the MUC1 nucleic acid aptamer capable of targeting and recognizing MCF-7 breast cancer cells is shown as SEQ ID NO.7. The SEQ ID NO.7 is: 5’-GCAGTTGATCCTTTGGATACCCTGG-3’. In the present invention, the pore-blocking material is obtained by hybridizing FQssDNA with ssDNA1 and ssDNA2 simultaneously; the pore-blocking material obtained by hybridizing FQssDNA with ssDNA1 and ssDNA2 simultaneously is named FQdsDNA.

[0047] In the present invention, the nucleotide sequence of ssDNA1 is shown as SEQ ID NO.1, the nucleotide sequence of ssDNA2 is shown as SEQ ID NO.2, and the nucleotide sequence of FQssDNA is shown as SEQ ID NO.3. The SEQ ID NO.1 is: 5’-GCGCGGCGCG-3’. The SEQ ID NO.2 is: 5’-GCGCGGCGCG-3’. The SEQ ID NO.3 is:

[0048] 5’-CGCGCCGCGCTTATTCGCGCCGCGC-3’

[0049] In a specific embodiment of the present invention, in order to prove by fluorescence color reaction that the activated CRISPR-Cas12a can cleave the "TTATT" site of the nucleotide sequence of FQdsDNA and realize the release of the drug in the hollow structure of the hollow carrier, the present invention preferably uses fluorescence labeling to modify the nucleotide sequence of FQssDNA, and the nucleotide sequence of the fluorescence-labeled FQssDNA is preferably:

[0050] 5’-CGCGCCGCGC-FAM-TTATT-BHQ1-CGCGCCGCGC-3’.

[0051] When the "TTATT" site of the nucleotide sequence of the accurately cleaved FQdsDNA by the activated CRISPR-Cas12a is cleaved, the green fluorescence of FAM is generated.

[0052] In the embodiments of the present invention, the purpose of adding the fluorescent group is to verify the cleavage performance of Cas12a.

[0053] The present invention provides a method for preparing the targeted drug-loaded nanoparticles described in the above technical solution, including the following steps:

[0054] Mix the carboxylated hollow carrier and the telomerase inhibitor for loading to obtain drug-loaded particles;

[0055] Activate the drug-loaded particles with a carboxyl activator to obtain activated drug-loaded particles;

[0056] Mix the activated drug-loaded particles, amino-functionalized ssDNA1, amino-functionalized ssDNA2, and amino-functionalized targeting material for the first co-incubation, and mix the obtained modified particles with FQssDNA for the second co-incubation to obtain the targeted drug-loaded nanoparticles.

[0057] In the present invention, the carboxylated hollow carrier and the telomerase inhibitor are mixed for loading to obtain drug-loaded particles.

[0058] In the present invention, the preparation method of the carboxylated hollow carrier includes the following steps:

[0059] Mix the hollow carrier, a strong acid solution, and 3-(triethoxysilyl)propyl succinic anhydride for carboxylation to obtain the carboxylated hollow carrier.

[0060] In the present invention, the preparation method of the hollow carrier preferably includes the following steps: First, mix ethanol, water, ammonia water, and tetraethyl orthosilicate (TEOS) for hydrolysis reaction to obtain solid silica nanoparticles; Second, mix the solid silica nanoparticles, water, cetyltrimethylammonium bromide (CTAB), ethanol, and tetraethyl orthosilicate (TEOS) to obtain core-shell structured nanoparticles; Third, mix the core-shell structured nanoparticles and an aqueous solution of carbonate for etching to obtain the hollow carrier.

[0061] In the present invention, ethanol, water, ammonia water and tetraethyl orthosilicate (TEOS) are first mixed for a hydrolysis reaction to obtain solid silica nanoparticles. In the present invention, the mass percentage content of the ammonia water is preferably 25-28%. The volume ratio of ethanol, water, ammonia water and tetraethyl orthosilicate (TEOS) used in the first mixing is preferably 100:8:4:3. The first mixing preferably includes the following steps: stirring and mixing the ethanol, water and ammonia water to obtain an alkaline solution; mixing the alkaline solution and the tetraethyl orthosilicate (TEOS). The temperature of the stirring and mixing is preferably 30°C, and the time is preferably 10 min. The temperature of the hydrolysis reaction is preferably 30°C, and the time is preferably 6 h.

[0062] After obtaining the solid silica nanoparticles, the present invention secondarily mixes the solid silica nanoparticles, water, cetyltrimethylammonium bromide (CTAB), ethanol and tetraethyl orthosilicate (TEOS) to obtain core-shell structured nanoparticles. In the present invention, the dosage ratio of water, cetyltrimethylammonium bromide (CTAB), ethanol and tetraethyl orthosilicate (TEOS) used in the second mixing is preferably 220 mL: 1200 mg: 10 mL: 1.075 mL. The dosage ratio of the solid silica nanoparticles to the tetraethyl orthosilicate (TEOS) is preferably 150 mg: 1.075 mL. The second mixing preferably includes the following steps: stirring and mixing the solid silica nanoparticles, water, cetyltrimethylammonium bromide (CTAB) and ethanol to obtain a mixed material liquid and mixing it with the tetraethyl orthosilicate (TEOS). The time of the stirring and mixing is preferably 30 min. The time of the second mixing is preferably 15 h.

[0063] After obtaining the core-shell structured nanoparticles, the present invention mixes the core-shell structured nanoparticles and an aqueous solution of carbonate for etching to obtain the hollow carrier. In the present invention, the carbonate is preferably sodium carbonate. The mass concentration of the aqueous solution of carbonate is preferably 0.4 mol / L. The present invention has no special requirement for the dosage of the aqueous solution of carbonate, and it is sufficient to immerse the core-shell structured nanoparticles. The etching is preferably carried out under an oil bath condition, the temperature of the etching is preferably 50°C, the etching is carried out under stirring, and the time of the stirring is preferably 2.5 h. After the etching is completed, the present invention preferably washes and dries the solid product obtained by the etching in sequence to obtain the hollow carrier. The reagent used for washing is preferably a mixed solution of concentrated hydrochloric acid and ethanol, and the volume ratio of the concentrated hydrochloric acid to the ethanol is preferably 1:10. The number of washing times is preferably 3 times. The drying is preferably freeze-drying.

[0064] In the present invention, the strong acid solution is preferably a hydrochloric acid solution, and the molar concentration of the hydrochloric acid solution is preferably 0.1 mol / L. The dosage ratio of the hollow carrier, the strong acid solution and 3-(triethoxysilyl)propyl succinic anhydride (TESPSA) is preferably 50 mg: 50 mL: 0.1 mL. The carboxylation temperature is preferably 50 °C, and the carboxylation is carried out under stirring conditions, and the stirring time is preferably 5 h. In the present invention, during the carboxylation process, under the acidic conditions formed by the strong acid solution, the anhydride structure in the TESPSA is converted into a carboxyl group, thereby forming a carboxyl group-modified hollow carrier (i.e., a carboxylated hollow carrier). After the carboxylation is completed, the present invention preferably separates the obtained reaction material liquid into solid and liquid, and the obtained solid product is washed and dried in sequence to obtain the carboxylated hollow carrier. The solid-liquid separation is preferably centrifugation. The specific implementation manner of the washing is preferably centrifugal washing. The drying is preferably freeze-drying.

[0065] In the present invention, during the loading process, the carboxylated hollow carrier is preferably mixed in the form of an aqueous solution of the carboxylated hollow carrier, and the mass concentration of the aqueous solution of the carboxylated hollow carrier is preferably 1 mg / mL. The telomerase inhibitor is preferably mixed in the form of a telomerase inhibitor solution, and the mass concentration of the telomerase inhibitor solution is preferably 1 mg / mL. The mass ratio of the carboxylated hollow carrier to the telomerase inhibitor is 10:1. The loading is preferably carried out under light-shielded conditions, preferably at room temperature, and preferably under stirring conditions, and the stirring time is preferably 24 h. After the loading is completed, the present invention preferably separates the loading material liquid into solid and liquid to obtain drug-loaded particles. The solid-liquid separation is preferably centrifugal separation, the rotation speed of the centrifugal separation is preferably 10,000 rpm, and the centrifugal separation time is preferably 10 min. The drug-loaded particles are preferably dissolved in dimethyl sulfoxide (DMSO) for storage.

[0066] After obtaining the drug-loaded particles, the present invention activates the drug-loaded particles with a carboxyl activator to obtain activated drug-loaded particles. In the present invention, the activation is preferably carried out using an activation reagent, and the activation reagent is preferably N-hydroxysulfosuccinimide sodium salt (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). The activation preferably includes the following steps: mixing the activation reagent and the drug-loaded particles for activation. The activation reagent is preferably NHS and EDC. The NHS is preferably used in the form of an NHS solution, and the molar concentration of the NHS solution is preferably 0.5 mM. The EDC is preferably used in the form of an EDC solution, and the molar concentration of the EDC solution is preferably 0.5 mM. The drug-loaded particles are preferably used in the form of a drug-loaded particle solution, and the mass concentration of the drug-loaded particle solution is preferably 1 mg / mL. The volume ratio of the NHS solution, the EDC solution, and the drug-loaded particle solution is preferably 1:1:50. The activation is preferably carried out at room temperature and preferably under shaking conditions, and the activation time is preferably 1 h. After the activation is completed, the present invention preferably washes the solid product obtained by activation, and the reagent used for washing is preferably PBS. The washing is preferably centrifugal washing, and the number of washing times is preferably 3 times.

[0067] After obtaining the activated drug-loaded particles, the present invention mixes the activated drug-loaded particles, amino-functionalized ssDNA1, amino-functionalized ssDNA2, and amino-functionalized targeting material for the first co-incubation to obtain modified particles, and then mixes the obtained modified particles with FQssDNA for the second co-incubation to obtain the targeted drug-loaded nanoparticles. In the present invention, the amino group in the amino-functionalized ssDNA1 is preferably modified at the 5'-end of the nucleotide sequence shown in SEQ ID NO.1, and the nucleotide sequence of the amino-functionalized ssDNA1 is: 5'-NH2-GCGCGGCGCG-3'. The amino group in the amino-functionalized ssDNA2 is preferably modified at the 3'-end of the nucleotide sequence shown in SEQ ID NO.2, and the nucleotide sequence of the amino-functionalized ssDNA2 is: 5'-GCGCGGCGCG-NH2-3'. The amino-functionalized targeting material is preferably an amino-functionalized MUC1 nucleic acid aptamer capable of targeting and recognizing MCF-7 breast cancer cells, and the amino group in the amino-functionalized targeting material is preferably modified at the 5'-end of the nucleotide sequence shown in SEQ ID NO.7. The nucleotide sequence of the amino-functionalized MUC1 nucleic acid aptamer capable of targeting and recognizing MCF-7 breast cancer cells is:

[0068] 5'-NH2-GCAGTTGATCCTTTGGATACCCTGG-3'.

[0069] The molar ratio of the aminated ssDNA1, aminated ssDNA2, aminated targeting material, and FQssDNA is preferably 1:1:1:2. The ssDNA1 is preferably used in the form of an ssDNA1 solution, and the molar concentration of the ssDNA1 solution is preferably 2 μM. The ssDNA2 is preferably used in the form of an ssDNA2 solution, and the molar concentration of the ssDNA2 solution is preferably 2 μM. The aminated targeting material is preferably used in the form of an aminated targeting material solution, and the molar concentration of the aminated targeting material solution is preferably 2 μM. The temperature of the first incubation is preferably 37 °C, and the time is preferably 2 h. The solid product obtained from the first incubation is preferably washed to obtain the modified particles. The reagent used for washing is preferably PBS. The washing is preferably centrifugal washing, and the number of washing times is preferably 3 times.

[0070] During the second incubation, the FQssDNA is preferably used in the form of an FQssDNA solution, and the molar concentration of the FQssDNA solution is preferably 2 μM. The temperature of the second incubation is preferably 37 °C, and the time is preferably 2 h. The solid product obtained from the second incubation is preferably washed to obtain the targeted drug-loaded nanoparticles. The reagent used for washing is preferably PBS. The washing is preferably centrifugal washing, and the number of washing times is preferably 3 times. The targeted drug-loaded nanoparticles are preferably dispersed in a PBS buffer solution and stored at 4 °C.

[0071] The present invention provides the use of the targeted drug-loaded nanoparticles described in the above technical solution or the targeted drug-loaded nanoparticles prepared by the preparation method described in the above technical solution in the preparation of anti-tumor drugs.

[0072] The present invention provides a targeted drug delivery system based on the CRISPR cleavage system, including the targeted drug-loaded nanoparticles described in the above technical solution or the targeted drug-loaded nanoparticles prepared by the preparation method described in the above technical solution;

[0073] A DNA substrate, which is formed by hybridization of a truncated sequence and its complementary sequence. The nucleotide sequence of the truncated sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the complementary sequence of the truncated sequence is shown in SEQ ID NO.5;

[0074] A crRNA, the nucleotide sequence of which is shown in SEQ ID NO.6;

[0075] Lba Cas12a (Cpf 1).

[0076] In the targeted drug delivery system based on the CRISPR cleavage system provided by the present invention, telomerase is a unique ribonucleoprotein that can prevent telomere shortening. In normal cells, telomeres gradually shorten after each replication cycle, causing the cells to gradually age and apoptose. In tumor cells, due to the overexpression of telomerase, telomeres do not shorten, enabling tumor cells to proliferate infinitely and obtain "immortality". Therefore, telomerase has always been a recognized tumor marker. The significant difference in telomerase activity between normal cells and tumor cells provides a biological basis for the design of telomerase-responsive drug carriers. Recently, the CRISPR-associated proteins (CRISPR-Cas) system has become an effective tool for genetic engineering and biosensing. Generally, in the CRISPR-Cas detection system, the Cas effector protein is deployed to a specific sequence target through a programmable guide RNA (gRNA or crRNA), and then uses its inherent cis-cleavage property to cleave the target, laying the foundation for precise gene editing. CRISPR-Cas12a in its CRISPR family simultaneously exhibits a unique target-activated trans-cleavage ability, which can significantly cleave single-stranded DNA around the target nucleic acid. The present invention designs a nano-drug delivery platform based on the telomerase self-extension response in the presence of the CRISPR-Cas12a system. The system provided by the present invention has the following advantages: 1): The nano-carrier can specifically target MCF-7 breast cancer cells; 2): The nano-carrier will only release BIBR1532 after being cleaved by CRISPR-Cas12a in response to telomerase, inhibiting telomerase activity, which is a novel "suicide" mode.

[0077] In the targeted drug delivery system based on the CRISPR cleavage system provided by the present invention: the crRNA and the LbaCas12a (Cpf 1) are preferably used in the form of a crRNA and Lba Cas12a (Cpf 1) complex.

[0078] In the present invention, the preparation method of the crRNA and Lba Cas12a (Cpf 1) complex comprises the following steps:

[0079] The solutions of crRNA and Lba Cas12a (Cpf 1) are blended and incubated to obtain a crRNA and Lba Cas12a (Cpf 1) complex. When blending and incubating, the molar concentration of the crRNA solution is preferably 0.2 μM; the molar concentration of the Lba Cas12a (Cpf 1) solution is preferably 0.2 μM. The volume ratio of the crRNA solution to the Lba Cas12a (Cpf 1) solution is 1:1.

[0080] The targeted drug delivery system based on the CRISPR cleavage system provided by the present invention includes the targeted drug-loaded nanoparticles described in the above technical solution or the targeted drug-loaded nanoparticles prepared by the preparation method described in the above technical solution. The targeted drug-loaded nanoparticles can release the telomerase inhibitor (BIBR1532), thereby inhibiting the activity of telomerase in tumor cells, promoting the senescence of tumor cells, and achieving the purpose of damaging tumor cells.

[0081] The targeted drug delivery system based on the CRISPR cleavage system provided by the present invention includes a DNA substrate, which is formed by hybridization of a truncated sequence and its complementary sequence. The nucleotide sequence of the truncated sequence is as shown in SEQ ID NO.4, and the nucleotide sequence of the complementary sequence of the truncated sequence is as shown in SEQ ID NO.5. The SEQ ID NO.4 is: 5’-GCAATCCGTCGAGCAGAGTT-3’. The SEQ ID NO.5 is 5’-AACCCTAACCCTTCGAGAGCTCGACGGATTGC-3’.

[0082] The targeted drug delivery system based on the CRISPR cleavage system provided by the present invention includes a crRNA, and the nucleotide sequence of the crRNA is as shown in SEQ ID NO.6. The SEQ ID NO.6 is: 5’-UAAUUUCUACUAAGUGUAGAUAACUCUGCUCGACGGAUUGC-3’.

[0083] The targeted drug delivery system based on the CRISPR cleavage system provided by the present invention includes Lba Cas12a (Cpf1).

[0084] The DNA substrate in the targeted drug delivery system based on the CRISPR cleavage system provided by the present invention is recognized and extended by telomerase highly expressed in tumor cells so as to be complementary paired with the crRNA, and then effectively activate Cas12a. The activated CRISPR-Cas12a precisely cleaves FQdsDNA, releases BIBR1532 in the hollow structure of the hollow vector, thereby inhibiting the activity of telomerase in tumor cells, promoting the senescence of tumor cells, and achieving the purpose of damaging tumor cells.

[0085] The present invention provides targeted drug-loaded nanoparticles (BIBR1532@HSN / FQdsDNA / Apts). The present invention provides a targeted drug delivery system based on the CRISPR cleavage system. FQdsDNA and the MUC1 aptamer capable of specifically recognizing MCF-7 breast cancer cells are covalently immobilized on the surface of carboxyl-functionalized HSN. Modifying HSN with the MUC1 aptamer enables the nanoparticles to specifically target MCF-7 breast cancer cells, and FQdsDNA is used to block the telomerase inhibitor (BIBR1532) in HSN. The DNA substrate in the CRISPR system is recognized and extended by the highly expressed telomerase in MCF-7 breast cancer cells to complementarily pair with crRNA, thereby effectively activating Cas12a. The activated CRISPR-Cas12a precisely cleaves FQdsDNA, releasing BIBR1532 in HSN, thus inhibiting the activity of telomerase, promoting the senescence of MCF-7 breast cancer cells, and achieving the purpose of damaging tumor cells. The present invention utilizes the highly expressed telomerase in MCF-7 breast cancer cells to release the telomerase inhibitor, which in turn inhibits the activity of telomerase, providing a unique and novel "suicide" behavior pattern of telomerase.

[0086] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0087] The sources of materials and reagents used in the following examples are as follows:

[0088] Hexadecyltrimethylammonium bromide (CTAB), N-hydroxysulfosuccinimide sodium salt (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), MTT cell proliferation kit, cytotoxicity assay kit, amino-modified MUC1 aptamer (5'-NH2-GCAGTTGATCCTTTGGATACCCTGG-3'), crRNA (5'-UAAUUUCUACUAAGUGUAGAUAACUCUGCUCGACGGAUUGC-3'), FQssDNA (5'-CG CGCCGCGC-FAM-TTATT-BHQ1-CGCGCCGCGC-3'), aminated ssDNA1 (5'-NH2-GCGCGGCGCG-3'), aminated ssDNA2 (5'-GCGCGGCGCG-NH2-3'), truncated sequence (5'-GCAATCCGTCGAGCAGAGTT-3') and its complementary sequence (5'-AACCCTAACCCTTCGAGAGCTCGACGGATTGC-3') were all purchased from Shanghai Biotech Co., Ltd. Among them, the DNA substrate is formed by hybridization of the truncated sequence and its complementary sequence. Lba Cas12a (Cpf 1) was purchased from Beijing New England Biolabs. Sodium carbonate (Na2CO3), tetraethyl orthosilicate (TEOS), anhydrous ethanol, ammonia solution, and TESPSA were purchased from Sinopharm. DAPI and cell senescence β-galactosidase staining kits were purchased from Biotech. Dimethyl sulfoxide (DMSO) was purchased from Macklin. 6× DNA loading buffer, nucleic acid dye Super Red, and DNA molecular weight standard marker (50-500bp) were purchased from Solebol. Telomerase inhibitor (BIBR1532) was purchased from Aladdin. Lipofectamine 3000 transfection reagent was purchased from Thermo Fisher. Fetal bovine serum was provided by Solebol. MCF-7 breast cancer cells were from Wuhan Pronocell Life Science Co., Ltd.

[0089] The instruments used in the following examples include:

[0090] Laser particle size analyzer (ZetasizerNano ZS90, Malvern Instrument Ltd., UK). Transmission electron microscope (JEM2100, Japan). Three-dimensional imaging luminescence spectrometer (FL-4700, Hitachi). Flow cytometer (CytoFLEX, Beckman). Microplate reader (K3 touch, Thermo Fisher). Fluorescence microscope (Ti2-E, Nikon, Japan).

[0091] The cell culture method used in the following examples is:

[0092] MCF-7 breast cancer cells were cultured in a constant temperature and humidity incubator at 37 °C with a humidity of 95% and a CO2 concentration of 5%. The culture medium used for the cells was DMEM containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin).

[0093] Example 1

[0094] (1) Preparation of carboxyl-modified hollow silica nanoparticles (HSN)

[0095] First, 8 mL of water and 4 mL of ammonia water (the mass percentage of ammonia water is 28%) were added to 100 mL of ethanol, stirred at 30 °C for 10 min, and then 3 mL of tetraethyl orthosilicate (TEOS) solution was added and stirred at 30 °C for 6 h to obtain solid silica nanoparticles.

[0096] 150 mg of the solid silica nanoparticle suspension was added with 220 mL of water, 1200 mg of cetyltrimethylammonium bromide (CTAB), and 10 mL of ethanol, stirred for 30 min, and then 1.075 mL of tetraethyl orthosilicate (TEOS) was added and stirred overnight to obtain core-shell nanoparticles.

[0097] The obtained core-shell nanoparticles were redispersed in 0.4 mol / L aqueous Na2CO3 solution, heated and stirred in an oil bath at 50 °C for 2.5 h. Finally, the obtained product was washed 3 times with a mixture of concentrated hydrochloric acid and ethanol (V:V = 1:10). Hollow silica nanoparticles were obtained by freeze-drying, denoted as HSN.

[0098] The hollow silica nanoparticles were dissolved in 50 mL of hydrochloric acid (0.1 mol / L) and 0.1 mL of TESPSA, stirred at 50 °C for 5 h. Under acidic conditions, the anhydride groups were converted into carboxyl groups to form carboxyl-modified HSN. The precipitate was collected by centrifugation and washing (10000 rpm, 10 min), and carboxylated HSN was obtained by freeze-drying.

[0099] (2) Preparation of BIBR1532@HSN / FQdsDNA / Apts

[0100] Under light-shielded conditions, 500 μL of carboxylated HSN (1 mg / mL) and 50 μL of BIBR1532 (1 mg / mL) were stirred for 24 h. After centrifugation (10000 rpm, 10 min) to remove the supernatant, BIBR1532@HSN was obtained and dissolved in 1 mL of DMSO for standby.

[0101] 10 μL of EDC (0.5 mM) and 10 μL of NHS (0.5 mM) were added to 500 μL of BIBR1532@HSN (1 mg / mL) and shaken for 1 hour to activate the surface carboxyl groups of BIBR1532@HSN. The mixture was washed three times by centrifugation with PBS, and 200 μL of ssDNA1 (2 μM), 200 μL of ssDNA2 (2 μM), and 200 μL of MUC1 (2 μM) were added and incubated at 37°C for 2 hours. The mixture was washed three times by centrifugation with PBS, and then 400 μL of FQssDNA (2 μM) was added and incubated at 37°C for 2 hours. After centrifugation and washing, BIBR1532@HSN / FQdsDNA / Apts was obtained. The product was dispersed in PBS buffer and stored at 4°C.

[0102] Test Case

[0103] (1) CRISPR-Cas12a system cleavage experiment

[0104] Collect 1×10 6 Cells were washed twice with ice-cold PBS (pH 7.4) and centrifuged at 4°C (2000 rpm, 5 min). The cells were resuspended in 200 μL of ice-cold CHAPS lysis buffer, incubated on ice for 30 min, and centrifuged at 4°C (2000 rpm, 20 min). The supernatant was finally collected and stored at -80°C for further experiments. 1 μL of telomerase extract was added to the telomerase reaction buffer to make the final volume 50 μL, which contained the truncated sequence (1 μM) and its complementary sequence (100 μM). The mixture was incubated at 37°C for 2 h, and then the mixture was heated to 95°C for 5 min to inactivate the telomerase activity. The extension product was finally obtained. The obtained extension product was mixed with 20 μL crRNA (0.2 μM), 20 μL Cas12a (0.2 μM), 12 μL 10×NEBuffer 2.1, and 20 μL BIBR1532@HSN / FQdsDNA / Apts. Incubate at 37°C for 2 h for cleavage reaction. Finally, 100 μL PBS was added to the reaction solution, and the fluorescence spectrum was recorded with a three-dimensional imaging luminescence spectrometer to observe the fluorescence intensity in the range of 510 nm to 650 nm (excitation wavelength was 490 nm).

[0105] (2) Intracellular cleavage experiments with the CRISPR-Cas12a system

[0106] MCF-7 breast cancer cells were inoculated in 12-well plates and cultured for 24 h to obtain a cell concentration of 1×10 6cells / mL. First, 100 nM DNA substrate was transfected in the medium with lipofectamine 3000. After 3 h, BIBR1532@HSN / FQdsDNA / Apts nanoparticles were added to MCF-7 breast cancer cells. Subsequently, 50 nM Cas12a crRNA complex was transfected with lipofectamine 3000, and the cells were co-transfected for 3 h. Finally, DAPI staining was performed, and the fluorescence phenomenon was analyzed by fluorescence microscopy and flow cytometry. The Cas12a crRNA complex was obtained by mixing and co-incubating 20 μL crRNA (0.2 μM) with 20 μL Cas12a (0.2 μM).

[0107] (4) Detection of the targeting property of the nanoparticles

[0108] MCF-7 breast cancer cells and MDA-MB-231 breast cancer cells were seeded in a 12-well plate and cultured for 24 h to obtain a cell concentration of 1×10 6 cells / mL for targeting analysis. First, 100 nM DNA substrate was transfected in the medium with lipofectamine 3000. After 3 h, BIBR1532@HSN / FQdsDNA / Apts nanoparticles were added to the two cell lines respectively. Subsequently, 50 nM Cas12a / crRNA complex was transfected with lipofectamine 3000, and the cells were co-transfected for 3 h. Finally, DAPI staining was performed, and the fluorescence phenomenon was analyzed by fluorescence microscopy.

[0109] (5) Determination of cell senescence

[0110] After MCF-7 breast cancer cells were seeded and cultured for 24 h, a cell concentration of 1×10 6cells / mL. Four groups of experiments were set up, namely: PBS (the first group), HSN (the second group), BIBR1532@HSN / FQdsDNA / Apts (the third group), and BIBR1532@HSN / FQdsDNA / Apts (the fourth group). In the fourth group, 100 nM DNA substrate was transfected in the medium by lipofectamine 3000. After 3 h, 100 μg / mL HSN was added to the second group, and 100 μg / mL BIBR1532@HSN / FQdsDNA / Apts was added to the third and fourth groups. In addition, the CRISPR system (Cas12a + crRNA) was additionally transfected into the fourth group. After the above 4 groups were co-cultured for 24 h, they were treated with a β-galactosidase staining kit. First, the cell culture medium was aspirated, washed once with PBS or HBSS, 1 mL of β-galactosidase staining fixative was added, and fixed at room temperature for 15 min. The cell fixative was aspirated, and the cells were washed 3 times with PBS or HBSS, 3 minutes each time. The PBS or HBSS was aspirated, and 1 mL of staining working solution was added to each well. Incubate overnight at 37 °C. Finally, the cell status was observed under an optical microscope to analyze cell senescence.

[0111] (6) Determination of cytotoxicity

[0112] After MCF-7 breast cancer cells were inoculated and cultured for 24 h, the cell concentration was 1×10 6 cells / mL. Four groups of experiments were set up, namely: PBS (the first group), HSN (the second group), BIBR1532@HSN / FQdsDNA / Apts (the third group), and BIBR1532@HSN / FQdsDNA / Apts (the fourth group). In the fourth group, 100 nM DNA substrate was transfected in the medium by lipofectamine 3000. After 3 h, 100 μg / mL HSN was added to the second group, and 100 μg / mL BIBR1532@HSN / FQdsDNA / Apts was added to the third and fourth groups. In addition, the CRISPR system (Cas12a + crRNA) was additionally transfected into the fourth group. After the above 4 groups were co-cultured for 24 h, the supernatant in each well was carefully aspirated, then 10 μL of MTT and 90 μL of fresh medium were added to each well, and kept in the dark at 37 °C for 4 h. After 4 h, the supernatant was aspirated, 100 μL of DMSO was added to each well, shaken at low speed on an oscillator for 10 min, detected at 490 nm by an enzyme-linked immunosorbent assay, and the cell survival rate was calculated according to the detection data.

[0113] Results and Discussion

[0114] In Example 1 of the present invention, HSN was first synthesized. TESPSA was used to successfully conjugate carboxyl groups on the surface of HSN. The carboxyl groups on the surface of HSN were activated by EDC and NHS, enabling the nanoparticles to be firmly linked to amino-functionalized FQssDNA, more effectively blocking BIBR1532 in HSN. Only when the DNA substrate in the CRISPR system is extended by telomerase can it precisely pair with crRNA, thereby effectively activating Cas12a, cleaving FQdsDNA, restoring fluorescence while releasing BIBR1532, inhibiting telomerase activity, and the decrease in telomerase activity will promote cell senescence and further damage tumor cells.

[0115] (1) Characterization of BIBR1532@HSNs / FQdsDNA / Apts

[0116] Figure 1 In which, A is the TEM image of HSN; Figure 1 In which, B is the DLS image of HSN. In the present invention, the morphology of HSN was characterized by TEM ( Figure 1 A image in which). It was found that HSN had uniform size, the shell thickness was 30 - 40 nm, and it had good dispersibility. DLS ( Figure 1 B image in which) analysis showed that the hydrodynamic diameter of HSN was 190 nm, which was basically consistent with the data in Figure 1 A image in which. Figure 2 is the nitrogen adsorption - desorption curve of HSN ( Figure 2 A image in which) and the pore size distribution diagram ( Figure 2 B image in which). Through the nitrogen adsorption - desorption experiment ( Figure 2 ), the pore size of HSN was investigated, which was about 3.8 nm. Figure 3 is the Fourier transform infrared spectroscopy (FTIR) of hollow silica and carboxylated hollow silica. In the FTIR spectrum ( Figure 3 ), HSN showed Si - O absorption bands at 1040, 800, and 470 cm -1 . For carboxyl - modified HSN, an additional peak was observed at 1720 cm -1 , which corresponded to the C = O vibration of - COOH, proving the successful synthesis of carboxylated HSN. Figure 4 is the agarose gel electrophoresis. According to the agarose gel electrophoresis ( Figure 4 ), it can be seen that FQdsDNA, FQssDNA, ssDNA, and MUC1 can freely pass through the gel pores (bands 5, 6, 7, 8), while when they are modified on the surface of HSN, they cannot pass through the gel and stay in the loading wells (bands 2, 3, 4). This is because after DNA is linked to HSN, the molecular weight becomes larger and it cannot move in the gel. Figure 5The zeta potentials of HSN, HSN-COOH, BIBR1532@HSN, Apts(MUC1), FQssDNA, ssDNA, and BIBR1532@HSN / FQdsDNA / Apts. Figure 5 The zeta potential results in Figure 5 show that the zeta potential of hollow silica in aqueous solution is -15 eV. After carboxyl modification, the zeta potential becomes -19 eV. After loading BIBR1532, the potential remains almost unchanged. The zeta potential of BIBR1532@HSN / FQdsDNA / Apts is -23 eV, which is due to the electronegativity of the nucleic acids on the surface.

[0117] (2) Cleavage effect of the CRISPR-Cas12a system

[0118] In this invention, the fluorescence intensities under five different conditions were measured by a three-dimensional imaging luminescence spectrometer, namely Cas12a + crRNA, DNA substrate, telomerase, BIBR1532@HSN / FQdsDNA / Apts (the first group); DNA substrate, telomerase, BIBR1532@HSN / FQdsDNA / Apts (the second group); Cas12a + crRNA, telomerase, BIBR1532@HSN / FQdsDNA / Apts (the third group); Cas12a + crRNA, DNA substrate, BIBR1532@HSN / FQdsDNA / Apts (the fourth group); Cas12a + crRNA, DNA substrate, telomerase (the fifth group). Figure 6 A in Figure 6 is the fluorescence spectrogram. Figure 6 B in Figure 6 is the fluorescence intensity diagram. As can be seen from Figure 6 Figure A and Figure B in Figure 6 , no fluorescence peak appears in the second, third, fourth, and fifth groups, indicating that the lack of any component in the Cas12a + crRNA, DNA substrate, telomerase, BIBR1532@HSN / FQdsDNA system does not meet the cleavage conditions of Cas12a. A distinct fluorescence peak appears in the first group because the presence of telomerase causes the extension of the DNA substrate, and the extended product pairs specifically with crRNA, effectively activating Cas12a to cleave FQdsDNA, resulting in the recovery of fluorescence. This demonstrates the good feasibility of this invention.

[0119] (3) Exploration of the intracellular cleavage effect of the CRISPR-Cas12a system

[0120] After co-incubating the five groups of solutions with cells, the cleavage ability of CRISPR-Cas12a in MCF-7 breast cancer cells was detected by a fluorescence microscope and a flow cytometer. According to Figure 7In Panel A, there are PBS (Group 1); Cas12a, crRNA (Group 2); Cas12a, crRNA, DNA substrate (Group 3); Cas12a, crRNA, BIBR1532@HSN / FQdsDNA / Apts (Group 4); Cas12a, crRNA, DNA substrate, BIBR1532@HSN / FQdsDNA / Apts (Group 5). It can be seen that Groups 2, 3, and 4 are the same as PBS, and no green fluorescence is produced. This is because the lack of any one of Cas12a, crRNA, DNA substrate, and BIBR1532@HSN / FQdsDNA / Apts results in the ineffective activation of CRISPR-Cas12a and the inability to cleave FQdsDNA, so the fluorescence is not restored. In Group 5, obvious green fluorescence appears. This is because there are a large number of telomerases in MCF-7 breast cancer cells, which cause the extension of the DNA substrate and its specific pairing with crRNA, effectively activating the trans-cleavage ability of Cas12a, forming a complete CRISPR-Cas12a system, cleaving FQdsDNA, and restoring the fluorescence. The above data are consistent with Figure 7 the results of the flow cytometry experiment in Panel B of

[0121] (4) Targeting study of BIBR1532@HSN / FQdsDNA / Apts

[0122] To study the targeting of BIBR1532@HSN / FQdsDNA / Apts nanoparticles to MCF-7 breast cancer cells, MDA-MB-231 breast cancer cells were used as a control experimental group. Figure 8 Panel A in shows the fluorescence images of MCF-7 breast cancer cells co-incubated with BIBR1532@HSN / FQdsDNA / Apts nanoparticles for 1 h, 2 h, and 3 h respectively. Figure 8 Panel B in shows the fluorescence images of MDA-MB-231 breast cancer cells co-incubated with BIBR1532@HSN / FQdsDNA / Apts nanoparticles for 1 h, 3 h, and 5 h respectively. Figure 8 The scale bar in is: 20 μm for all. After BIBR1532@HSN / FQdsDNA / Apts nanoparticles were co-incubated with MCF-7 breast cancer cells for 1 h, green fluorescence gradually appeared ( Figure 8Figure A in [reference] shows that BIBR1532@HSN / FQdsDNA / Apts nanoparticles can rapidly enter MCF-7 breast cancer cells. After 2 hours of incubation, the green fluorescence significantly increases, indicating that a large amount of BIBR1532@HSN / FQdsDNA / Apts enters MCF-7 breast cancer cells. The change in green fluorescence is not obvious after 3 hours, indicating that between 2 and 3 hours, BIBR1532@HSN / FQdsDNA / Apts nanoparticles have completely entered the cells. Compared with Figure 8 Figure B in [reference], the green fluorescence of MDA-MB-231 breast cancer cells is significantly less. When co-incubated for 1 hour, there is no obvious green fluorescence, indicating that the nanoparticles have not entered MDA-MB-231 breast cancer cells. Green fluorescence gradually appears from 3 to 5 hours, indicating that the nanoparticles gradually enter MDA-MB-231 breast cancer cells, but the amount of nanoparticles is small. The presence of MUC1 aptamer is beneficial for the nanoparticles to specifically recognize and rapidly enter MCF-7 breast cancer cells, and BIBR1532@HSN / FQdsDNA / Apts nanoparticles have good targeting to MCF-7 breast cancer cells.

[0123] (5) Determination of cell senescence by β-galactosidase

[0124] The cell senescence situation was studied using a cell senescence β-galactosidase staining kit. Figure 9 The β-galactosidase kit was used to detect the senescence of MCF-7 breast cancer cells under the conditions of (1) PBS; (2) HSN; (3) BIBR1532@HSN / FQdsDNA / Apts; (4) BIBR1532@HSN / FQdsDNA / Apts / Cas, respectively. Figure 9 The scale bar in [reference] is: 1 μm. Using X-Gal in the β-galactosidase staining kit as a substrate, a dark blue product will be generated under the catalysis of senescence-specific β-galactosidase. As Figure 9 shown, compared with the PBS group, there is no obvious change in the HSN group and the BIBR1532@HSN / FQdsDNA / Apts group, and the cells do not show obvious dark blue, indicating that the HSN group and the BIBR1532@HSN / FQdsDNA / Apts group do not promote the senescence of MCF-7 breast cancer cells. The cells in the BIBR1532@HSN / FQdsDNA / Apts / Cas12a group show more dark blue, indicating severe cell senescence. This is because the CRISPR system (Cas12a + crRNA, DNA substrate) cleaves FQdsDNA, releasing BIBR1532, inhibiting telomerase activity, and promoting the senescence of MCF-7 breast cancer cells.

[0125] (6) MTT assay for cytotoxicity

[0126] To further study the cytotoxic effect of nanomaterials on cancer cells, the present invention used an MTT detection kit to detect the cytotoxicity of HSN, BIBR1532@HSN / FQdsDNA / Apts, and BIBR1532@HSN / FQdsDNA / Apts / Cas12a. Figure 10 In [A], the cell survival rate is the result of co-incubating MCF-7 cells with different concentrations of HSN. Figure 10 In [B], the cell survival rate is the result of co-incubating MCF-7 cells with different concentrations of BIBR1532@HSN / FQdsDNA / Apts. Figure 10 In [C], the cell survival rate is the result of co-incubating MCF-7 cells with different concentrations of BIBR1532@HSN / FQdsDNA / Apts while adding the CRISPR system (Cas12a + crRNA, DNA substrate). As Figure 10 can be seen from the data in [A], after adding different concentrations of HSN, as the concentration of HSN increases, the cell viability decreases slightly, indicating that HSN has good biosafety. Through Figure 10 the data in [B], it can be seen that as the concentration of BIBR1532@HSN / FQdsDNA / Apts increases, the cell viability is high and the change is not obvious, indicating that BIBR1532@HSN / FQdsDNA / Apts does not damage the cells, and at the same time, it also indicates that the encapsulation effect of FQdsDNA is good. As Figure 10 shown in [C], as the concentration of BIBR1532@HSN / FQdsDNA / Apts / Cas12a increases, the cell viability decreases significantly. This is because after the DNA substrate is extended by the highly expressed telomerase in MCF-7 breast cancer cells, crRNA hybridizes with it complementarily, effectively activating the cleavage ability of Cas12a, resulting in the cleavage of FQdsDNA and the release of BIBR1532, damaging the cells.

[0127] As can be seen from the above embodiments, the present invention provides a targeted drug delivery system (BIBR1532@HSN / FQdsDNA / Apts) based on hollow silica nanoparticles (HSN) modified with FQdsDNA and MUC1 aptamer. The MUC1 aptamer facilitates the specific and rapid targeting of MCF-7 breast cancer cells by the nanoparticles, and FQdsDNA is used to block BIBR1532 in the HSN. At the same time, the DNA substrate in the CRISPR system is recognized by the highly expressed telomerase in MCF-7 breast cancer cells and the corresponding sequence is extended. The extended sequence is complementary paired with the crRNA, effectively activating Cas12a. The activated CRISPR-Cas12a precisely cleaves FQdsDNA, releasing BIBR1532 in the HSN, inhibiting telomerase activity, promoting cell senescence, and damaging tumor cells. This article provides a unique "suicide" new method that relies on "telomerase eliminating telomerase".

[0128] In addition, due to the presence of the MUC1 aptamer, this article is only applicable to MCF-7 breast cancer cells. (MUC1 only targets MCF-7), and the present invention is applicable to tumor cells with overexpressed telomerase. By selecting nucleic acid aptamers that specifically target tumor cells, corresponding tumor cells can be targeted. In the targeting study of BIBR1532@HSN / FQdsDNA / Apts prepared in Example 1 of the present invention, a control experiment was conducted with MDA-MB-231 breast cancer cells, demonstrating that BIBR1532@HSN / FQdsDNA / Apts prepared in Example 1 of the present invention has good targeting to MCF-7.

[0129] The research work of the present invention is funded by the Natural Science Foundation of Shandong Province (Project No.: ZR2023JQ004), and the project name is: Nanoprobe and Cell Imaging.

[0130] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments can also be obtained based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A targeted drug-loaded nanoparticle, characterized in that, Comprising a carboxylated hollow carrier, which consists of a shell structure with nanoscale pores and a hollow structure; a drug loaded in the hollow structure of the carboxylated hollow carrier; and a targeting material and a pore-blocking material chemically modified on the outer surface of the shell structure of the carboxylated hollow carrier, wherein the pore-blocking material is used to block the nanoscale pores of the shell structure of the carboxylated hollow carrier; The carboxylated hollow carrier is carboxyl-modified hollow silica nanoparticles; The drug is a telomerase inhibitor, and the telomerase inhibitor is BIBR1532; The targeting material is a MUC1 nucleic acid aptamer capable of targeting and recognizing MCF-7 breast cancer cells, and the nucleotide sequence of the MUC1 nucleic acid aptamer capable of targeting and recognizing MCF-7 breast cancer cells is shown in SEQ ID NO.7; The pore-blocking material is obtained by hybridizing FQssDNA with ssDNA1 and ssDNA2 at the same time; the nucleotide sequence of ssDNA1 is shown in SEQ ID NO.1, the nucleotide sequence of ssDNA2 is shown in SEQ ID NO.2, and the nucleotide sequence of FQssDNA is shown in SEQ ID NO.

3.

2. The targeted drug-loaded nanoparticles according to claim 1, characterized in that, The diameter of the carboxylated hollow carrier is 150-200 nm.

3. The targeted drug-loaded nanoparticles according to claim 1 or 2, characterized in that, The thickness of the shell structure with nanoscale pores is 30-40 nm, and the size of the nanoscale pores is 3.8 nm.

4. The preparation method of the targeted drug-loaded nanoparticles according to any one of claims 1 to 3, characterized in that, Comprising the following steps: Mixing the carboxylated hollow carrier and the telomerase inhibitor for loading to obtain drug-loaded particles; Activating the drug-loaded particles with a carboxyl activator to obtain activated drug-loaded particles; Mixing the activated drug-loaded particles, amino-modified ssDNA1, amino-modified ssDNA2 and amino-modified targeting material for the first co-incubation to obtain modified particles, and mixing the obtained modified particles with FQssDNA for the second co-incubation to obtain the targeted drug-loaded nanoparticles.

5. The preparation method according to claim 4, wherein The preparation method of the carboxylated hollow carrier comprises the following steps: Mixing the hollow carrier, a strong acid solution and 3-(triethoxysilyl)propyl succinic anhydride for carboxylation to obtain the carboxylated hollow carrier.

6. The preparation method according to claim 4, characterized in that, The mass ratio of the carboxylated hollow carrier to the telomerase inhibitor is 10:

1.

7. The preparation method according to claim 4, characterized in that The molar ratio of the amino-modified ssDNA1, amino-modified ssDNA2, amino-modified targeting material and FQssDNA is 1:1:1:

2.

8. Use of the targeted drug-loaded nanoparticles according to any one of claims 1 to 3 or the targeted drug-loaded nanoparticles prepared by the preparation method according to any one of claims 4 to 7 in the preparation of anti-tumor drugs.

9. A targeted drug delivery system based on the CRISPR cleavage system, characterized in that, Comprising the targeted drug-loaded nanoparticles according to any one of claims 1 to 3 or the targeted drug-loaded nanoparticles prepared by the preparation method according to any one of claims 4 to 7; A DNA substrate, which is formed by hybridizing a truncated sequence and its complementary sequence, the nucleotide sequence of the truncated sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the complementary sequence of the truncated sequence is shown in SEQ ID NO.5; crRNA, the nucleotide sequence of the crRNA is as shown in SEQ ID NO.6; Lba Cas12a (Cpf 1).

Images