Multifunctional nano-composite as well as preparation method and application thereof

Abstract

The invention discloses a multifunctional nano-composite as well as a preparation method and application thereof. The multifunctional nano-composite comprises a DNA carrier, a photothermal agent and an anti-tumor drug, the DNA carrier comprises a long DNA single chain with n repetitive units, n DNA complementary single chains, n aptamer chains with cancer cell targeting capability and n SH-DNA chains connected with photo-thermal agent nanoparticles, wherein n is an integer between 5 and 150; each repetitive unit of the long DNA single chain has 63 basic groups, and the basic groups in each repetitive unit are respectively paired and connected with the DNA complementary single chain, the aptamer chain and the SH-DNA chain. According to the invention, the aptamer with multivalent targeting capability and a large amount of photothermal agents and chemotherapeutic drugs are loaded by utilizing a multi-repetitive unit of the long DNA single-chain carrier, so that a chemotherapy / photothermal / chemical power synergistic treatment system with multivalent aptamer positioning is constructed, and cancer cells can be effectively killed.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a multifunctional nanocomposite and a preparation method and application thereof. Background Art

[0002] Malignant tumors, commonly known as cancer, are one of the most challenging diseases in the medical field. They are highly metastatic and seriously threaten human life and health. In recent years, their incidence and mortality rates have increased year by year. At present, chemotherapy (CT) and radiotherapy are the main clinical tumor treatments, but there are also inevitable limitations, such as multidrug resistance and low treatment efficiency. Therefore, other treatment methods are needed for synergistic intervention to enhance efficacy and reduce side effects.

[0003] Photothermal therapy (PTT) can not only promote the increase of blood flow and improve the absorption of chemotherapy drugs by tumor tissue, but also effectively alleviate the problems caused by tumor hypoxia. It is a more novel, safe and efficient cancer treatment method. Compared with cancer cells, normal human tissue has a relatively low absorption rate of near-infrared radiation (NIR, 700-1100 nanometers). Therefore, PTT can accurately locate the spatial targets of tumor cells and penetrate deep into the tissue, while minimizing the damage to normal biological tissues. Chemodynamic therapy (CDT) develops nanoformulations with high catalytic enzyme activity to catalyze the production of reactive oxygen species (ROS) in tumor cells, thereby breaking the oxidation-reduction balance of local tumor cells, inducing oxidative damage to biological molecules such as mitochondria, DNA and proteins, thereby leading to tumor cell death and achieving anti-tumor effects. If chemotherapy, photothermal therapy and chemodynamics can be combined for synergistic treatment, the effect of cancer treatment will be enhanced while minimizing side effects.

[0004] DNA-based multifunctional vector delivery is considered to be the best strategy for integrating various treatment methods because it uses DNA, the genetic material of organisms, as its core component and has good biocompatibility. After design and modification, the system can effectively control drug release after precise delivery to the tumor site, thereby significantly reducing multidrug resistance and maximizing efficacy. Although DNA vectors have good biocompatibility and a wide range of drug carrying capacity, the complex still cannot achieve active targeting of cancer cells without appropriate modification. Therefore, it is necessary to design aptamers that can actively target cancer cells on the multifunctional complex in order to accurately locate the target cancer cells and reduce the occurrence of side effects. Aptamers are a type of single-stranded oligonucleotide or peptide molecules that bind to target molecules with high specificity through a specific three-dimensional structure. They can bind to various types of molecular targets, including proteins, nucleic acids, small molecules, and even cells and entire organisms. Aptamers have the advantages of high specificity, high affinity, stability, designability, and easy synthesis and modification. They have been widely used in many fields such as disease diagnosis, treatment, imaging, and gene regulation. However, monovalent aptamers have problems such as weak affinity, easy clearance, potential off-target effects, and limited bioavailability. Therefore, it is of great significance to design a multi-cycle DNA vector loaded with multiple aptamers targeting cancer cells to enhance the targeting ability of its multivalent aptamers. Summary of the invention

[0005] In order to solve the above-mentioned technical problems, the present invention provides a multifunctional nanocomplex and its preparation method and application. In the present invention, a multivalent aptamer is modified on the periodic long DNA single strand (RCA strand) of the rolling circle amplification product to enhance the active targeting ability, and a photothermal agent is introduced as a carrier for photothermal therapy and chemodynamic therapy, while anti-tumor drugs are loaded. The nanocomplex organically combines chemotherapy, photothermal therapy and chemodynamic therapy to achieve a synergistic therapeutic effect. Multivalent aptamers play a key role in improving tumor targeting, preventing the nonspecific release of photothermal agents and anti-tumor drugs, and realizing CT / PTT / CDT integration. This innovative synergistic treatment platform provides a comprehensive and effective strategy for the comprehensive treatment of malignant tumors.

[0006] In a first aspect, the present invention provides a multifunctional nanocomposite, which is realized by adopting the following technical solution.

[0007] A multifunctional nanocomposite comprises a DNA vector, a photothermal agent and an anti-tumor drug; the DNA vector comprises a long DNA single strand having n repeating units, n complementary DNA single strands, n aptamer strands with the ability to target cancer cells, and n SH-DNA strands connected to photothermal agent nanoparticles, wherein n is an integer between 5 and 150, preferably, n is an integer between 8 and 100; each repeating unit of the long DNA single strand has 63 bases, and the bases in each repeating unit are paired and connected with the complementary DNA single strand, the aptamer strand, and the SH-DNA strand, respectively.

[0008] Specifically, the 63 bases in each repeating unit of the long DNA single strand are divided into three parts: L1', L2', and L3'. The complementary DNA single strand (L3) is completely paired and connected with the L3' region, the adaptor chain (L2) is paired and connected with the L2' region through the partial chain extended at the end of the adaptor sequence, and the SH-DNA chain (L1) is paired and connected with the L1' region through the partial chain extended at its 5' end.

[0009] Furthermore, the long DNA single strand is a long single strand DNA molecule produced based on the rolling circle amplification technology. The long single strand DNA molecule obtained by rolling circle amplification has periodicity and large molecular weight characteristics. This is used as a scaffold for constructing a large-capacity DNA vector, and a multifunctional DNA nanocarrier complex is constructed on this basis. In the present invention, the long DNA single strand has multi-copy properties, and the number of copies is mainly affected by the rolling circle amplification reaction time. The length ranges from 3000bp to 20000bp, preferably 5000-12000bp, and more preferably 8000bp, that is, the RCA chain has approximately 120 repeating units of 63 bases.

[0010] Furthermore, the complementary DNA strand is a short DNA strand that is complementary to the long DNA strand and forms a double helix structure with the L3' position of each repeating unit. The sequence design of the complementary DNA strand should try to avoid forming special DNA secondary structures or tertiary structures such as hairpin structures and G-quadruplexes.

[0011] Furthermore, the aptamer chain includes an aptamer sequence that specifically targets and recognizes cancer cells, and the aptamers include AS1411 aptamer (nucleolar protein-specific nucleic acid aptamer), MUC1 aptamer (targeting Mucin1 protein overexpressed in breast cancer, MUC1), Sgc8 aptamer (targeting tyrosine kinase 7 overexpressed in acute lymphoblastic leukemia, PTK7), preferably AS1411 aptamer; the 3' end of the aptamer chain is labeled with a fluorescent group.

[0012] Furthermore, the SH-DNA chain is used to connect photothermal agents / chemodynamic agents (such as inorganic nanoparticles), and its 3' end is modified with a thiol group, which can be connected to the carried inorganic nanoparticles through a semi-covalent bond. The L1 chain can be divided into two regions, one of which is completely paired and connected to the L1' region of the RCA chain, while the other end is modified with a thiol group so as to bind to the inorganic nanoparticles through a semi-covalent bond. The bases paired with the L1' region can be any complementary bases, but the design should try to avoid the formation of special secondary structures such as hairpin structures by DNA chains modified with thiol groups at the ends. In a specific embodiment, the L1 chain length ranges from 20 to 60 bp.

[0013] Furthermore, the photothermal agent is selected from inorganic photothermal materials with high-efficiency photothermal conversion performance, good biocompatibility, and easy synthesis and surface modification, including precious metal-based, semiconductor photothermal materials, and carbon-based photothermal materials; the precious metal-based includes Au, Pt and Pd; the semiconductor photothermal material includes copper sulfide compounds, and the copper sulfide compounds are selected from Cu2-xE, where E=S, Se and Te; the carbon-based photothermal materials include graphene, carbon quantum dots (CDs), nanodiamonds, carbon nanotubes (CNTs), activated carbon and fullerene. In certain embodiments, the photothermal agent can have the dual therapeutic effects of simultaneously realizing photothermal and chemodynamic therapy, preferably the semiconductor material copper sulfide compound Cu2-xE (where E=S, Se and Te, etc.), more preferably CuS nanoparticles.

[0014] By adopting the above technical scheme, the inorganic photothermal agent is first connected to the 3' end of the SH-DNA chain, and after the SH-DNA chain is completely paired with the L1' region of the RCA chain, the inorganic photothermal agent enters the cancer cell under the transportation of the DNA carrier. Under external laser irradiation and the presence of endogenous hydrogen peroxide in the cell, the photothermal agent will convert light into heat in the cell for photothermal therapy, and will be oxidized by hydrogen peroxide in the cell to produce ROS for chemodynamic therapy, thereby exerting the synergistic governance effect of photothermal / chemodynamic therapy. In the present invention, inorganic nanoparticles modified with thiol DNA chains (L1 chains) are connected to RCA chains to construct a nanocomplex with photothermal / chemodynamic combined therapy for cancer treatment. After the inorganic nanoparticles as photothermal agents are modified with L1 chains, they can not only enhance their own biocompatibility and stability, but also make them easier to load onto DNA carriers, so that they can enter the target cells in large quantities to exert therapeutic effects. In addition, the L1 chain is connected to the tandem L1' region through base complementary pairing to form a double helix structure. This process depends on the complementary bases with the RCA chain, which not only enhances the stability of the entire nanocomplex but also provides sufficient drug loading space for the subsequent loading of chemotherapeutic drugs.

[0015] Furthermore, each repeating unit of the long DNA single strand has a sequence as shown in SEQ ID NO:3; the complementary DNA single strand has a sequence as shown in SEQ ID NO:4; the adaptor chain selects one or more of the following sequences: SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8; the sequence of the SH-DNA chain is shown in SEQ ID NO:6.

[0016] Furthermore, the anti-tumor drug is a chemotherapeutic drug, which includes cyclophosphamide, paclitaxel, and doxorubicin (DOX), preferably DOX. The anti-tumor drug is connected to the DNA vector through π-π interaction.

[0017] This study demonstrated through experiments that DNA vectors can form stable anti-tumor complexes with the chemotherapy drug doxorubicin (DOX), and under the action of shear enzymes, the DOX loaded in the vector can be released to a level equivalent to the same concentration of free DOX.

[0018] In a second aspect, the present invention provides a method for preparing a multifunctional nanocomposite, which is achieved by adopting the following technical solution.

[0019] A method for preparing a multifunctional nanocomposite comprises the following steps:

[0020] S1. Preparation of circular template and purification: circularization of linear template DNA and purification using exonuclease;

[0021] S2. Preparation of long DNA single strands by rolling circle amplification technology: The purified circularized DNA template is subjected to rolling circle amplification reaction under the action of DNA polymerase to obtain long DNA single strands;

[0022] S3. Construction of DNA vector: long DNA single strand, aptamer strand and DNA complementary single strand are mixed in a molar ratio of 1:10:10 to 1:200:200 and heat treated, and then cooled to room temperature;

[0023] S4. Preparation of SH-DNA chains connected to photothermal agent nanoparticles: reducing the SH-DNA chains with a reducing agent, combining the reduced SH-DNA chains with the photothermal agent in a buffer solution to obtain a DNA-photothermal agent;

[0024] S5. Loading the photothermal agent on the DNA carrier: co-incubating the DNA carrier prepared in step S3 with the DNA-photothermal agent at a molar ratio of 1:5 to 1:100 to load the photothermal agent onto the DNA carrier;

[0025] S6. Loading the anti-tumor drug on the DNA carrier: incubating the complex prepared in step S5 with the anti-tumor drug at a molar ratio of 1:100 to 1:300 in the dark to obtain a multifunctional nanocomposite.

[0026] Specifically, a method for preparing a multifunctional nanocomposite comprises the following steps:

[0027] (1) Preparing a circular template: After mixing a linear template DNA solution with polynucleotide kinase, incubate at 10 to 60° C. for 1 to 60 minutes, and then heat at 20 to 130° C. for 1 to 60 minutes; then add primer A to the solution and allow it to cool naturally to 10 to 50° C.; then add T4 DNA ligase, react at room temperature for 1 to 3 hours, then add NaOAC and ice ethanol, precipitate in an environment of -50 to 0° C. for 6 to 20 hours, and then dry;

[0028] (2) Purifying the circular template: adding exonuclease to the dried DNA circular template, reacting at 10-60°C for 1-60 minutes, then treating at 20-130°C for 5 minutes, and finally cooling to room temperature;

[0029] (3) Preparing a long DNA single strand by rolling circle amplification technology: After the purified circularized DNA template, primer A and dNTP are evenly mixed, a DNA polymerase is added to initiate the rolling circle amplification reaction; the reaction is carried out at 10 to 60°C for 1 to 60 minutes, and then incubated at 20 to 130°C for 1 to 60 minutes to terminate the reaction, and finally the solution is cooled to room temperature to obtain a long DNA single strand;

[0030] (4) Construction of a DNA vector with large capacity and multivalent targeting ability: After mixing the long DNA single strand with the aptamer strand and the DNA complementary single strand, heat treatment is performed at 70 to 180°C, and then cooled to room temperature at a rate of 1°C / min in a PCR instrument, wherein the molar ratio of the long DNA single strand to the aptamer strand and the DNA complementary single strand is 1:10:10 to 1:200:200;

[0031] (5) Preparation of SH-DNA chains connected to photothermal agent nanoparticles: First, the SH-DNA chains are incubated with TCEP at 25°C to reduce the SH-DNA chains, and the reaction time is 0.5 to 5 hours; then, the reduced SH-DNA chains are combined with the photothermal agent under room temperature conditions containing NaCl and HEPES buffer for 6 to 24 hours to obtain a DNA-photothermal agent; finally, the obtained DNA-photothermal agent solution is stored at 4°C for subsequent use;

[0032] (6) Loading a photothermal agent on a DNA carrier: co-incubating the DNA carrier prepared in step (4) with the DNA-photothermal agent, slowly cooling from 30 to 70° C. to 20 to 28° C., repeating this process three times, and maintaining the temperature at 20 to 28° C. for 30 minutes; wherein the molar ratio of the DNA carrier to the DNA-photothermal agent is 1:5 to 1:100;

[0033] (7) Loading the anti-tumor drug on the DNA vector: incubating the complex prepared in step (6) with the anti-tumor drug in the dark at 20 to 50° C. for 10 to 60 minutes; wherein the molar ratio of the DNA vector to the anti-tumor drug is 1:100 to 1:300.

[0034] Furthermore, the sequence of the linear template DNA is shown as SEQ ID NO:1; and the sequence of primer A is shown as SEQ ID NO:2.

[0035] Further, in step (1), polynucleotide kinase can transfer the 5'-phosphate group to the 3'-OH end, thereby repairing the DNA chain. The enzyme plays an important role in DNA repair, synthesis and terminal modification, and is preferably one or more of the following: Escherichia coli polynucleotide kinase (E.coli PNK), T4 polynucleotide kinase (T4 PNK), human polynucleotide kinase (Human PNK) and recombinant polynucleotide kinase (Recombinant PNK), among which T4 PNK is particularly preferred. In addition, the DNA ligase is a key biocatalyst that catalyzes the formation of phosphodiester bonds between DNA chains, and plays a vital role in processes such as DNA repair, replication and recombination; it is preferably one or more of the following: Escherichia coli DNA ligase (E.coli DNA ligase), T4 DNA ligase (T4 DNA ligase), T7 DNA ligase (T7 DNA ligase) and recombinant DNA ligase (Recombinant DNA ligase), among which T4 DNA ligase is preferred.

[0036] Furthermore, in step (2), the exonuclease is an enzyme that can remove nucleotides one by one from the end of a polynucleotide chain, preferably one or more of the following enzymes: Escherichia coli exonuclease I (Exo I), Escherichia coli exonuclease III (ExoIII) and T4 DNA exonuclease, among which Exo I and Exo III are more recommended.

[0037] Furthermore, in step (3), the DNA polymerase is a polymerase used for biocatalytic synthesis of DNA, preferably one or more of the following polymerases are selected: Phi29 polymerase, Taq DNA polymerase, Pfu DNA polymerase, Phusion DNA polymerase and KOD Hot Start DNA polymerase, among which Phi29 polymerase is the most preferred.

[0038] In a third aspect, the present invention provides a use of a multifunctional nanocomposite, which is achieved by adopting the following technical solution.

[0039] An application of the multifunctional nanocomposite in preparing cancer therapeutic drugs.

[0040] Furthermore, the cancer refers to the uncontrolled cell growth and division of certain cells in an organism due to gene mutation or other factors, which may evolve into cancer. The cancer includes melanoma, lung cancer, liver cancer, pancreatic cancer, cervical cancer, preferably melanoma.

[0041] The multifunctional DNA nanocomplex of the present invention can be applied to mammals, including but not limited to humans, mice, monkeys, rabbits, cows, sheep, horses, etc.

[0042] The experimental results show that the multifunctional nanocomplex of the present invention exhibits high targeting and killing ability against mouse melanoma cells (B16). In addition, the chemotherapy / photothermal / chemodynamic synergistic therapy system of the present invention has the characteristics of good biocompatibility, precise specific targeting ability and powerful drug carrier function.

[0043] Beneficial Effects

[0044] (1) The present invention utilizes the multi-copy characteristics of rolling circle amplification technology and uses multiple tandemly repeated DNA units as the vector backbone. The number of repeating units can be adjusted according to specific needs. Due to the presence of a large number of repeating units, aptamers, photothermal agents and anti-tumor drugs can be efficiently loaded, thereby achieving efficient and accurate targeting of cancer cells and tumor ablation;

[0045] (2) The DNA targeting vector containing multivalent aptamers of the present invention can interact with target cancer cells through multiple binding sites, thereby enhancing their affinity and reducing the risk of being eliminated in the body. In addition, the presence of multivalent aptamers can also effectively reduce off-target effects, improve biodistribution, and increase internalization efficiency, thereby further enhancing the cancer treatment effect.

[0046] (3) The multifunctional DNA nanocomplex of the present invention can effectively carry a large amount of chemotherapeutic drugs. Due to the presence of multivalent aptamers, the DNA nanocomplex can directly enter the target cancer cells through precise positioning. After entering the target cancer cells, the inorganic nanophotothermal agent will perform photothermal / chemodynamic therapy under external near-infrared light irradiation and tumor microenvironment. At the same time, the anti-tumor drug in the carrier will be released and act on the cell nucleus to achieve chemotherapy. Therefore, the multifunctional DNA nanocomplex achieves synergistic chemotherapy / photothermal / chemodynamic therapy of cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The fluorescence curves of the targeting complexes of (A) the monovalent aptamer-modified DNA targeting vector (SOACu-FAM) of Example 4 and (B) the multivalent aptamer-modified DNA targeting vector (RMAL1Cu-FAM) of Example 7 of the present invention when they bind to mouse melanoma cells (B16 cells);

[0048] Figure 2 Cell survival rate data graphs after incubation with B16 cells of the naked CuS nanoparticles (CuS) of Example 1, the thiol DNA-modified CuS nanoparticles (L1-CuS) of Example 2, the thiol aptamer (AS1411)-modified CuS nanoparticles (Apt-CuS) of Example 3, the multivalent aptamer-modified DNA vector (RMAL1) of Example 5, the multivalent aptamer-modified DNA vector-CuS (RMAL1Cu) of Example 6, the aptamer-free DNA vector-CuS (RL1Cu) of Example 8, and the multivalent aptamer-modified DNA vector loaded with CuS and DOX (RMAL1Cu@DOX) of Example 9;

[0049] Figure 3 Cell apoptosis data after incubation with B16 of the multivalent aptamer-modified DNA vector (RMAL1) of Example 5, the multivalent aptamer-modified DNA vector-CuS (RMAL1Cu) of Example 6, the DNA vector-CuS (RL1Cu) without aptamer modification of Example 8, and the multivalent aptamer-modified DNA vector loaded with CuS and DOX (RMAL1Cu@DOX) of Example 9, respectively. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with the embodiments.

[0051] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are obtained through commercial channels unless otherwise specified.

[0052] The DNA and RNA sequences used in the present invention are all from Shanghai Sangon Biotechnology Co., Ltd. Agarose, TAE buffer solution, DNA Ladder, CCK-8 kit and Calcein / PI live / dead detection kit are all purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0053] Table 1 DNA / RNA sequences involved in the examples

[0054]

[0055] Example 1

[0056] Preparation of CuS nanoparticles (CuS NPs)

[0057] 24 mg of Na2S·9H2O was added to 100 mL of an aqueous solution containing CuCl2 (13 mg) and sodium citrate (20 mg). The solution was stirred at room temperature, and the light blue solution immediately changed and gradually turned dark brown. After 5 minutes, the mixture was heated to 90°C and kept for 15 minutes, and the resulting solution was dark green. Subsequently, the solution was transferred to ice water for a 30-minute ice bath to obtain CuS nanoparticles.

[0058] Example 2

[0059] Preparation of Thiol DNA Modified CuS Nanoparticles (L1-CuS)

[0060] First, CuS nanoparticles were prepared according to the method in Example 1, and then thiol DNA-modified CuS nanoparticles (L1-CuS) were synthesized by salt aging method, specifically, TCEP was first used to incubate thiol DNA (L1 chain) at 25° C. for 1 hour to reduce the L1 chain. L1 chain was incubated with CuS nanoparticles at room temperature for 12 hours in a buffer solution containing 100 mM NaCl and 20 mM HEPES to obtain L1-CuS nanocomplexes.

[0061] Example 3

[0062] Preparation of CuS Nanoparticles Modified with Thiol Aptamer (AS1411) (Apt-CuS)

[0063] First, CuS nanoparticles were prepared according to the method in Example 1, and then thiol aptamer DNA-modified CuS nanoparticles (Apt-CuS) were synthesized by salt aging method, specifically, TCEP was first used to incubate thiol aptamer DNA (SH-Apt, L2 chain 3' end thiol modified) for 1 hour at 25°C to achieve the reduction of thiol aptamer DNA. After SH-Apt and CuS nanoparticles were incubated at room temperature for 12 hours in a buffer solution containing 100mM NaCl and 20mM HEPES, Apt-CuS nanocomplexes were obtained.

[0064] Example 4

[0065] Preparation of monovalent aptamer-modified DNA targeting vector (SOACu-FAM)

[0066] A repeating unit sequence of the RCA chain was mixed with L2-FAM (the 3' end of the L2 chain was labeled with a fluorescent group FAM) and L3, and after heat treatment at 95°C, it was cooled to room temperature in a PCR instrument at a rate of 1°C / min. Subsequently, it was incubated with L1-CuS and slowly cooled to 25°C at 65°C. This process was cycled three times and kept at 25°C for 30 minutes to obtain a monovalent aptamer-modified DNA targeting vector (SOACu-FAM). Among them, the molar ratio between the long DNA single-strand repeat unit and L1, L2 and L3 was 1:1:1:1.

[0067] Example 5

[0068] Preparation of multivalent aptamer-modified DNA vector (RMAL1)

[0069] (1) Preparation of circular template

[0070] First, the linear template DNA (SEQ ID NO: 1) solution, T4 PNK enzyme, 10× PNK buffer and ATP were mixed, placed at 37°C for 30 minutes, and then treated at 90°C for 5 minutes. Subsequently, primer A was added and annealed to 25°C. Next, T4 DNA ligase was added and reacted at 25°C for 2 hours. Finally, sodium acetate (NaOAC) and ice ethanol were added, and the circular DNA template was obtained after precipitation at -20°C for 12 hours and centrifugation and drying.

[0071] (2) Purification of circular template

[0072] The sample was dried after high-speed centrifugation. Subsequently, 10×EXO I enzyme and 10×EXO III enzyme were added, and the reaction was carried out at 37°C for 30 minutes, and then at 90°C for 5 minutes. Next, the mixture was cooled to room temperature and electrophoresed in a 10% urea denatured polyacrylamide gel for 40 minutes, and then the gel was cut to extract the target product. Finally, NaOAC and ice ethanol were added to the extract, precipitated at -20°C for 2 hours, and the purified DNA circular template was obtained by centrifugation and drying.

[0073] (3) Preparation of RCA chains by rolling circle amplification

[0074] The circular DNA template was mixed with primer A and dNTPs in Phi29 DNA polymerase buffer, followed by the addition of Phi29 DNA polymerase for a 5-minute enzymatic reaction at 30°C. Next, a final incubation at 65°C for 10 minutes was performed to terminate the reaction. Finally, the temperature of the reaction mixture was gradually lowered to room temperature to obtain RCA chains (long single-stranded DNA).

[0075] (4) Preparation of multivalent aptamer-modified DNA vector (RMAL1)

[0076] The RCA chain was mixed with L2 and L3, and after heat treatment at 95°C, cooled to room temperature at a rate of 1°C / min in a PCR instrument to obtain a multivalent aptamer-modified DNA vector (RMAL1), wherein the molar ratio of the RCA chain to L2 and L3 was 1:60:60.

[0077] Example 6

[0078] Preparation of DNA vector modified with multivalent aptamers and CuS nanoparticles (RMAL1Cu)

[0079] (1) Preparation of circular template

[0080] First, the linear template DNA solution, T4 PNK enzyme, 10× PNK buffer and ATP were mixed and reacted at 37°C for 30 minutes, and then treated at 90°C for 5 minutes. Then, primer A was added and annealed to 25°C. T4 DNA ligase was then added, reacted at 25°C for 2 hours, and then NaOAC and ice ethanol were added. After precipitation at -20°C for 12 hours, the circular DNA template was obtained by centrifugation and drying.

[0081] (2) Purification of circular template

[0082] The sample was dried after high-speed centrifugation. Subsequently, 10×EXO I enzyme and 10×EXO III enzyme were added, and the reaction was carried out at 37°C for 30 minutes, and then at 90°C for 5 minutes. Next, the mixture was cooled to room temperature and electrophoresed in a 10% urea denatured polyacrylamide gel for 40 minutes, and then the gel was cut to extract the target product. Finally, NaOAC and ice ethanol were added to the extract, precipitated at -20°C for 2 hours, and the purified DNA circular template was obtained by centrifugation and drying.

[0083] (3) Preparation of long single-stranded DNA by rolling circle amplification

[0084] The circular DNA template was mixed with primer A and dNTPs in Phi29 DNA polymerase buffer, followed by the addition of Phi29 DNA polymerase for a 5-minute enzymatic reaction at 30°C. Next, a final incubation at 65°C for 10 minutes was performed to terminate the reaction. Finally, the temperature of the reaction mixture was gradually lowered to room temperature to obtain RCA chains (long single-stranded DNA).

[0085] (4) Preparation of DNA vector modified with multivalent aptamers and CuS nanoparticles (RMAL1Cu)

[0086] The RCA chain was mixed with L2 and L3, and after heat treatment at 95°C, it was cooled to room temperature in a PCR instrument at a rate of 1°C / min to obtain a multivalent aptamer-modified DNA carrier (RMAL1). Subsequently, the prepared RMAL1 was incubated with the L1 chain (L1-CuS) connected to CuS nanoparticles, and slowly cooled from 65°C to 25°C. The process was repeated three times and kept at 25°C for 30 minutes. Among them, the molar ratio of RCA chain to L2 and L3 was 1:60:60, and the molar ratio between DNA carrier and L1-CuS was 1:10.

[0087] Example 7

[0088] DNA vector modified with multivalent aptamer (fluorescent group (FAM) modification) and CuS nanoparticles

[0089] Preparation of (RMAL1Cu-FAM)

[0090] (1) Preparation of circular template

[0091] First, the linear template DNA solution, T4 PNK enzyme, 10× PNK buffer and ATP were mixed and reacted at 37°C for 30 minutes, and then treated at 90°C for 5 minutes. Then, primer A was added and annealed to 25°C. T4 DNA ligase was then added, reacted at 25°C for 2 hours, and then NaOAC and ice ethanol were added. After precipitation at -20°C for 12 hours, the circular DNA template was obtained by centrifugation and drying.

[0092] (2) Purification of circular template

[0093] The sample was dried after high-speed centrifugation. Subsequently, 10×EXO I enzyme and 10×EXO III enzyme were added, and the reaction was carried out at 37°C for 30 minutes, and then at 90°C for 5 minutes. Next, the mixture was cooled to room temperature and electrophoresed in a 10% urea denatured polyacrylamide gel for 40 minutes, and then the gel was cut to extract the target product. Finally, NaOAC and ice ethanol were added to the extract, precipitated at -20°C for 2 hours, and the purified DNA circular template was obtained by centrifugation and drying.

[0094] (3) Preparation of long single-stranded DNA by rolling circle amplification

[0095] The circular DNA template was mixed with primer A and dNTPs in Phi29 DNA polymerase buffer, followed by the addition of Phi29 DNA polymerase for a 5-minute enzymatic reaction at 30°C. Next, a final incubation at 65°C for 10 minutes was performed to terminate the reaction. Finally, the temperature of the reaction mixture was gradually lowered to room temperature to obtain RCA chains (long single-stranded DNA).

[0096] (4) Preparation of multivalent aptamers (fluorescent group (FAM) modified) and CuS nanoparticle-modified DNA vectors (RMAL1Cu-FAM)

[0097] The RCA chain was mixed with L2-FAM and L3, and after heat treatment at 95°C, it was cooled to room temperature at a rate of 1°C / min in a PCR instrument to obtain a multivalent aptamer-modified DNA vector (RMAL1). Subsequently, the prepared RMAL1 was incubated with the L1 chain (L1-CuS) connected to CuS nanoparticles, and slowly cooled from 65°C to 25°C. The process was repeated three times and kept at 25°C for 30 minutes. Among them, the molar ratio of RCA chain to L2-FAM and L3 was 1:60:60, and the molar ratio between DNA vector and L1-CuS was 1:10.

[0098] Example 8

[0099] Preparation of CuS nanoparticles and DNA vectors without aptamer modification (RL1Cu)

[0100] (1) Preparation of circular template

[0101] First, the linear template DNA solution, T4 PNK enzyme, 10× PNK buffer and ATP were mixed and reacted at 37°C for 30 minutes, and then treated at 90°C for 5 minutes. Then, primer A was added and annealed to 25°C. T4 DNA ligase was then added, reacted at 25°C for 2 hours, and then NaOAC and ice ethanol were added. After precipitation at -20°C for 12 hours, the circular DNA template was obtained by centrifugation and drying.

[0102] (2) Purification of circular template

[0103] The sample was dried after high-speed centrifugation. Subsequently, 10×EXO I enzyme and 10×EXO III enzyme were added, and the reaction was carried out at 37°C for 30 minutes, and then at 90°C for 5 minutes. Next, the mixture was cooled to room temperature and electrophoresed in a 10% urea denatured polyacrylamide gel for 40 minutes, and then the gel was cut to extract the target product. Finally, NaOAC and ice ethanol were added to the extract, precipitated at -20°C for 2 hours, and the purified DNA circular template was obtained by centrifugation and drying.

[0104] (3) Preparation of long single-stranded DNA by rolling circle amplification

[0105] The circular DNA template was mixed with primer A and dNTPs in Phi29 DNA polymerase buffer, followed by the addition of Phi29 DNA polymerase for a 5-minute enzymatic reaction at 30°C. Next, a final incubation at 65°C for 10 minutes was performed to terminate the reaction. Finally, the temperature of the reaction mixture was gradually lowered to room temperature to obtain RCA chains (long single-stranded DNA).

[0106] (4) Preparation of CuS nanoparticles and DNA vectors without aptamer modification (RL1Cu)

[0107] The RCA chain and the L3 chain were mixed, then heat treated at 95°C and cooled to room temperature in a PCR instrument at a rate of 1°C / min to obtain a DNA vector without aptamer modification. Next, the prepared DNA vector was incubated with L1-CuS, slowly cooled from 65°C to 25°C, and the process was repeated three times and kept at 25°C for 30 minutes. During this process, the molar ratio between the RCA chain and the L3 chain was 1:60, and the molar ratio between the DNA vector and L1-CuS was 1:10.

[0108] Example 9

[0109] Preparation of multifunctional nanocomposite (RMAL1Cu@DOX)

[0110] (1) Preparation of circular template

[0111] First, the linear template DNA solution, T4 PNK enzyme, 10× PNK buffer and ATP were mixed and reacted at 37°C for 30 minutes, and then treated at 90°C for 5 minutes. Then, primer A was added and annealed to 25°C. T4 DNA ligase was then added, reacted at 25°C for 2 hours, and then NaOAC and ice ethanol were added. After precipitation at -20°C for 12 hours, the circular DNA template was obtained by centrifugation and drying.

[0112] (2) Purification of circular template

[0113] The sample was dried after high-speed centrifugation. Subsequently, 10×EXO I enzyme and 10×EXO III enzyme were added, and the reaction was carried out at 37°C for 30 minutes, and then at 90°C for 5 minutes. Next, the mixture was cooled to room temperature and electrophoresed in a 10% urea denatured polyacrylamide gel for 40 minutes, and then the gel was cut to extract the target product. Finally, NaOAC and ice ethanol were added to the extract, precipitated at -20°C for 2 hours, and the purified DNA circular template was obtained by centrifugation and drying.

[0114] (3) Preparation of long single-stranded DNA by rolling circle amplification

[0115] The circular DNA template was mixed with primer A and dNTPs in Phi29 DNA polymerase buffer, followed by the addition of Phi29 DNA polymerase for a 5-minute enzymatic reaction at 30°C. Next, a final incubation at 65°C for 10 minutes was performed to terminate the reaction. Finally, the temperature of the reaction mixture was gradually lowered to room temperature to obtain RCA chains (long single-stranded DNA).

[0116] (4) Preparation of DNA vector modified with multivalent aptamers and CuS nanoparticles (RMAL1Cu)

[0117] The RCA chain was mixed with L2 and L3, and after heat treatment at 95°C, it was cooled to room temperature in a PCR instrument at a rate of 1°C / min to obtain a multivalent aptamer-modified DNA vector (RMAL1). Subsequently, the prepared RMAL1 was incubated with the L1 chain (L1-CuS) connected with CuS nanoparticles, and slowly cooled from 65°C to 25°C. The process was repeated three times and kept at 25°C for 30 minutes. Among them, the molar ratio of RCA chain to L2 and L3 was 1:60:60, and the molar ratio between DNA vector and L1-CuS was 1:10.

[0118] (5) Loading of chemotherapeutic drugs (such as DOX) on DNA vectors:

[0119] The complex prepared in the previous step was incubated with the chemotherapeutic drug (doxorubicin, DOX) in a dark environment at 37° C. for 30 minutes. The molar ratio of DNA vector to DOX was 1:300.

[0120] Performance Testing

[0121] In the following experiments, the tumor cells used were mouse melanoma cells (B16), and the control group was mouse fibroblasts (L929). Both cells were purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0122] The experiments for Examples 1-7 were carried out as follows:

[0123] Cell culture: The cells were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO2 humidified incubator.

[0124] Aptamer targeting experiments:

[0125] Cells in the logarithmic growth phase were prepared as a cell suspension, counted using a cell counting plate, and then inoculated into a 6-well plate so that the number of cells in each well reached 100,000. The 6-well plate was placed in a cell culture incubator and incubated for 24 hours, then all the supernatant was slowly aspirated with a pipette, and washed three times with PBS. Then, the monovalent aptamer-modified DNA targeting vector (SOACu-FAM) in Example 4 was added at a concentration of C SOACu-FAM = 0, 6, 18, 30, 60 and 90 nM, and the RMAL1Cu-FAM in Example 7 whose concentration is C RMAL1Cu-FAM = 0, 0.1, 0.3, 0.5, 1 and 1.5 nM, and then continue to culture in a 37°C incubator for 30 minutes. Use a cell scraper to separate the cells in the dark to obtain a single cell suspension. Finally, the fluorescence intensity of the nanocomposite in the cells was evaluated by flow cytometry.

[0126] CCK-8 method to determine cell viability:

[0127] Cells (B16 and L929) in the logarithmic growth phase were collected, seeded in 96-well plates and cultured overnight. Subsequently, the cells were incubated with the bare CuS nanoparticles (CuS) in Example 1, the CuS nanoparticles modified with thiol DNA in Example 2 (L1-CuS), the CuS nanoparticles modified with thiol aptamer DNA (AS1411) in Example 3 (Apt-CuS), the DNA vector modified with multivalent aptamers (RMAL1) in Example 5, the DNA vector modified with multivalent aptamers and CuS nanoparticles (RMAL1Cu) in Example 6, the DNA vector modified with CuS nanoparticles and no aptamer modification (RL1Cu) in Example 8, and the DNA vector modified with multivalent aptamers and loaded with CuS and DOX (RMAL1Cu@DOX) in Example 9 (C DOX =350nM,C CuS =100 μg / mL) at 37°C for 6 hours. Then, the incubation solution was removed, the uninternalized reagent was washed with PBS, and fresh culture medium was added. Next, each well was irradiated with 808 nm laser (power of 1.25 W / cm 2 , duration 5 minutes), and then continued to culture in the incubator for 42 hours. Finally, serum-free medium containing 10% CCK-8 solution was used for 2 hours of incubation, and the optical density (OD) at 450nm was measured by a microplate reader, and the cell viability was calculated using the following formula: Cell viability (%) = (A e -A c) / (A b -A c )×100%. Among them, A e , A b , A c Represent the average OD values ​​of the experimental group, blank group, and control group, respectively. Cells and culture medium were added to the blank cell group, while only culture medium was added to the control group.

[0128] Cell apoptosis experiment:

[0129] Cells (B16 and L929) in logarithmic growth phase were collected and seeded in 96-well plates and cultured overnight. The cells were incubated with the multivalent aptamer-modified DNA vector (RMAL1) in Example 5, the multivalent aptamer and CuS nanoparticle-modified DNA vector (RMAL1Cu) in Example 6, the CuS nanoparticle-free DNA vector (RL1Cu) in Example 8, and the multivalent aptamer-modified DNA vector loaded with CuS and DOX (RMAL1Cu@DOX) in Example 9. DOX =350nM,C CuS=100 μg / mL) at 37°C for 6 hours. Subsequently, the cells were washed three times with PBS and fresh medium was added. The cells were then heated at a power of 1.25 W / cm 2 Each well was irradiated with 808 nm laser for 5 min and then cultured for 42 h. Finally, after 15 min of Annexin V / PI reaction in the dark, apoptosis was assessed using flow cytometry (BD Biosciences AccuriC6, USA).

[0130] Experimental Results

[0131] Figure 1 The experimental results show that when B16 cells were incubated with a higher concentration of SOACu-FAM and a lower concentration of RMAL1Cu at the same time, the RMAL1Cu group showed a more significant fluorescence intensity. This indicates that the large number of aptamers on RMAL1Cu can bind to multiple receptors on the cell surface, thereby showing a stronger affinity for targeted cancer cells, further verifying the excellent ability of multivalent aptamer-modified DNA vectors in targeting cancer cell recognition.

[0132] Figure 2 The experimental results show that RMAL1Cu@DOX, which has synergistic effects of chemotherapy, photothermal and chemodynamic therapy, has a more significant inhibitory effect on cancer cells. Under the same concentration conditions, the survival rate of B16 cells treated with this system is significantly lower than that of cells treated with other loading systems. This result proves that the RMAL1Cu@DOX complex can effectively release the chemotherapy drug DOX after entering the target cells, and at the same time, the carried CuS nanoparticles react with hydrogen peroxide in the cells and perform photothermal / chemodynamic therapy under 808nm laser irradiation.

[0133] Figure 3 The experimental results show that B16 cells treated with RMAL1Cu@DOX showed a higher apoptosis rate, while RMAL1Cu with photothermal / chemodynamic therapy showed suboptimal efficacy. This result shows that the chemotherapy / photothermal / chemodynamic synergistic therapy system designed by the present invention has a far better killing effect on cancer cells than other systems and can greatly promote cancer cell apoptosis.

[0134] The embodiments of this specific implementation method are all preferred embodiments of the present application, and are not intended to limit the protection scope of the present application. Therefore, all equivalent changes made based on the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A multifunctional nanocomposite, characterized in that: It comprises a DNA vector, a photothermal agent and an anti-tumor drug; the DNA vector comprises a long DNA single strand with n repeating units, n complementary DNA single strands, n aptamer strands with the ability to target cancer cells, and n SH-DNA strands connected to photothermal agent nanoparticles, wherein n is an integer between 5 and 150; each repeating unit of the long DNA single strand has 63 bases, and the bases in each repeating unit are paired and connected with the complementary DNA single strand, the aptamer strand, and the SH-DNA strand, respectively.

2. A multifunctional nanocomposite according to claim 1, characterized in that: The long DNA single strand is a long single strand DNA molecule produced based on the rolling circle amplification technology, and the length of the long DNA single strand is 5000 bp to 20000 bp.

3. The multifunctional nanocomposite according to claim 1, characterized in that: The aptamer chain includes an aptamer sequence that specifically targets and recognizes cancer cells, and the aptamers include AS1411 aptamer, MUC1 aptamer, and Sgc8 aptamer; a partial chain is extended at the end of the aptamer sequence and paired with a long DNA single strand; the 3' end of the aptamer chain is labeled with a fluorescent group.

4. The multifunctional nanocomposite according to claim 1, characterized in that: The 3' end of the SH-DNA chain is modified with a thiol group and connected to the photothermal agent through a semi-covalent bond; the length of the SH-DNA chain ranges from 20 to 60 bp.

5. The multifunctional nanocomposite according to claim 1, characterized in that: The photothermal agent is selected from inorganic photothermal materials, including precious metal-based, semiconductor photothermal materials, and carbon-based photothermal materials; the precious metal-based includes Au, Pt and Pd; the semiconductor photothermal material includes copper chalcogenide compounds, and the copper chalcogenide compound is selected from Cu2-x E, where E=S, Se and Te; the carbon-based photothermal materials include graphene, carbon quantum dots, nanodiamonds, carbon nanotubes, activated carbon and fullerenes.

6. The multifunctional nanocomposite according to claim 1, characterized in that: Each repeating unit of the long DNA single strand has a sequence as shown in SEQ ID NO:3; the complementary DNA single strand has a sequence as shown in SEQ ID NO:4; the aptamer chain selects one or more of the following sequences: SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8; the sequence of the SH-DNA chain is shown in SEQ ID NO:

6.

7. The multifunctional nanocomposite according to claim 1, characterized in that: Anti-tumor drugs are chemotherapy drugs, including cyclophosphamide, paclitaxel, and doxorubicin.

8. A method for preparing the multifunctional nanocomposite according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. Preparation of circular template and purification: circularization of linear template DNA and purification using exonuclease; S2. Preparation of long DNA single strands by rolling circle amplification technology: The purified circularized DNA template is subjected to rolling circle amplification reaction under the action of DNA polymerase to obtain long DNA single strands; S3. Construction of DNA vector: long DNA single strand, aptamer strand and DNA complementary single strand are mixed in a molar ratio of 1:10:10 to 1:200:200 and heat treated, and then cooled to room temperature; S4. Preparation of SH-DNA chains connected to photothermal agent nanoparticles: reducing the SH-DNA chains with a reducing agent, combining the reduced SH-DNA chains with the photothermal agent in a buffer solution to obtain a DNA-photothermal agent; S5. Loading the photothermal agent on the DNA carrier: co-incubating the DNA carrier prepared in step S3 with the DNA-photothermal agent at a molar ratio of 1:5 to 1:100 to load the photothermal agent onto the DNA carrier; S6. Loading the anti-tumor drug on the DNA carrier: incubating the complex prepared in step S5 with the anti-tumor drug at a molar ratio of 1:100 to 1:300 in the dark to obtain a multifunctional nanocomposite.

9. Use of the multifunctional nanocomposite according to any one of claims 1 to 7 in the preparation of cancer therapeutic drugs.

10. The use according to claim 9, characterized in that: The cancers include melanoma, lung cancer, liver cancer, pancreatic cancer, and cervical cancer.

Images