Nuclear targeting peptide modified DNA nano-particles based on microfluidic technology and preparation method and application of nuclear targeting peptide modified DNA nano-particles

Abstract

The invention discloses a nuclear targeting peptide modified DNA nano-particle based on a microfluidic technology and a preparation method and application thereof, and relates to the technical field of biological medicine, a poly (beta-amino ester) cationic polymer is used as a gene delivery carrier, the gene delivery carrier comprises a microtubule related sequence MTAS and a nuclear localization signal NLS oligopeptide, the gene delivery carrier and the plasmid DNA carrying the target gene are mixed to prepare nano-particles, and the plasmid DNA carrying the target gene is uniformly dispersed in the nano-particles by adopting a microfluidic mixing process; the nuclear targeting peptide modified DNA nano-particle is ingenious in structural design, the mixing uniformity of a nano-carrier and plasmid DNA is improved by adopting a micro-fluidic mixing process, the performance of the nano-particle is further improved by optimizing dialysis conditions, potential cytotoxicity and adverse effects are reduced, and the nuclear targeting peptide modified DNA nano-particle is suitable for large-scale production. And after transduction of non-activated T cells, strong tumor killing ability and cytokine secretion can be generated.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and specifically to a nuclear targeting peptide-modified DNA nanoparticle based on microfluidic technology, a preparation method thereof, and an application thereof. Background Art

[0002] Chimeric antigen receptor T cells (CAR-T cells) have played a significant role in the research of hematological malignancies by infusing gene-reprogrammed immune cells. When preparing gene-reprogrammed immune cells for infusion, an ideal gene delivery vector is required. An ideal gene delivery vector should have cell adsorption ability, be able to effectively penetrate the cell membrane, achieve endosome / lysosome escape and nuclear localization. Therefore, designing an ideal gene delivery vector is of great significance. On the other hand, the preparation process of CAR-T cells still faces the following problems: First, the current preparation of CAR-T cells relies on viral vectors for gene transduction, and steps such as virus packaging, purification, in vitro activation, and amplification of T cells need to be completed in a GMP-grade clean workshop, resulting in high production costs and limiting its clinical accessibility; second, in vitro-activated T cells are prone to differentiate into terminal effector cells during rapid proliferation, losing their proliferative potential and undergoing functional exhaustion, resulting in the difficulty of maintaining long-term anti-tumor activity in vivo and affecting the durability of the therapeutic effect; third, non-activated T cells (such as naive T cells) have stronger self-renewal ability and anti-exhaustion characteristics and are ideal target cells for gene modification. However, the current gene delivery vectors (including viral vectors and non-viral vectors) have extremely low transduction efficiency for non-activated T cells and cannot be directly used for gene reprogramming.

[0003] Therefore, there is an urgent need to develop a delivery vector that can directly transport nucleic acids into non-activated T cells to achieve effective gene delivery. Summary of the Invention

[0004] To solve the above problems, the purpose of the present invention is to provide a nuclear targeting peptide-modified DNA nanoparticle based on microfluidic technology, a preparation method thereof, and an application thereof, which can directly transport nucleic acids into non-activated T cells to achieve effective gene delivery.

[0005] To achieve the above object, the present invention is realized through the following technical solutions: A nuclear targeting peptide-modified DNA nanoparticle based on microfluidic technology, including using a poly(β-amino ester) cationic polymer as a gene delivery vector, on which a microtubule-associated sequence MTAS and a nuclear localization signal NLS short peptide are included. The gene delivery vector is mixed with plasmid DNA carrying a target gene to prepare nanoparticles, and the plasmid DNA carrying the target gene is uniformly dispersed in the nanoparticles by a microfluidic mixing process.

[0006] Preferably, the target gene is a CAR gene.

[0007] Preferably, the target gene is a CAR gene targeting CD19, CD38, B7H3 or CS1.

[0008] The present invention also includes a preparation method of a nuclear targeting peptide-modified DNA nanoparticle based on microfluidic technology, comprising the following steps: First, prepare a poly(β-amino ester) cationic polymer as a gene delivery vector. Second, add a microtubule-associated sequence MTAS and a nuclear localization signal NLS short peptide to the polymer vector. Finally, use a microfluidic mixing process to uniformly mix the polymer vector with plasmid DNA carrying the target gene to prepare nanoparticles, and uniformly disperse the plasmid DNA carrying the target gene in the nanoparticles.

[0009] Preferably, the preparation method comprises the following steps: ① Prepare a poly(β-amino ester) cationic polymer as a gene delivery vector: Synthesize an acrylate-capped polymer by Michael addition reaction of 1,4-butanediol diacrylate and 4-amino-1-butanol, and then react with 1-(3-aminopropyl)-4-methylpiperazine to obtain poly(β-amino ester). Dissolve the obtained poly(β-amino ester) in DMSO to obtain a PBAE-447 solution, and store it at -20 °C for later use; ② Add a microtubule-associated sequence MTAS and a nuclear localization signal NLS short peptide to the nanoparticles: Dissolve the crosslinker N-(p-maleimidophenyl) isocyanate in DMSO, add it to the PBAE-447 solution obtained in step ①, and react at 20-35 °C for 3 h to obtain an activated PBAE-447-maleimide derivative. After mixing and reacting the obtained activated PBAE-447-maleimide derivative with the microtubule-associated sequence MTAS and the nuclear localization signal NLS short peptide for 3 h, a mixed solution is obtained. The obtained mixed solution is filtered and vacuum dried to obtain conjugated PBAE-447-MTAS-NLS; dissolve the obtained conjugated PBAE-447-MTAS-NLS in DMSO and store it at -20 °C for later use; ③ Prepare plasmid DNA: Use two plasmids. Plasmid 1 expresses PiggyBac transposase; Plasmid 2 drives the expression of the target gene by the hPGK promoter; The construction method of plasmid 2: Connect the target gene by hPGK, and then insert it into the EcoRI / NotI of the PiggyBac-CMV-MCS-Fluc-EF1a-copGFP-T2A-Puro plasmid to obtain; ④ Preparation of peptide-modified DNA nanoparticles: The conjugated PBAE-447-MTAS-NLS dissolved in DMSO is diluted with an organic solvent to serve as the organic phase; plasmid 1 and plasmid 2 are mixed to obtain plasmid DNA, and the plasmid DNA is dissolved in a NaAc / HAc buffer solution with a pH of 5.0 to serve as the acidic aqueous phase. The obtained organic phase and acidic aqueous phase are mixed through a microfluidic device, and excess reagents are removed by dialysis to obtain nuclear targeting peptide-modified DNA nanoparticles; When the obtained organic phase and acidic aqueous phase are mixed through a microfluidic device, the mass ratio of conjugated PBAE-447-MTAS-NLS to plasmid 2 is 60:1; the ratio of plasmid 1 to plasmid 2 is 1:3; Preferably, the organic solvent is obtained by mixing ethanol and DMSO in a volume ratio of 1:1.

[0010] The present invention also includes the use of nuclear targeting peptide-modified DNA nanoparticles based on microfluidic technology for the transduction of non-activated T cells.

[0011] The present invention has the following advantages compared with the prior art: The nuclear targeting peptide-modified DNA nanoparticles based on microfluidic technology of the present invention are ideal gene delivery vectors. They can not only penetrate cell membranes, achieve endosome / lysosome escape and nuclear localization, but also rapidly reprogram non-activated T cells. There is no need for in vitro activation and amplification culture, and the freshly isolated T cells can be intravenously infused in vivo 2 - 6 hours after transduction to obtain CAR-T cells with strong anti-tumor ability.

[0012] The nuclear targeting peptide-modified DNA nanoparticles based on microfluidic technology of the present invention have a clever structural design. The microfluidic mixing process is used to uniformly mix the nanoparticles with plasmid DNA carrying the target gene, improving the uniformity of the mixing of the nanocarrier and plasmid DNA. The optimization of the dialysis conditions further improves the performance of the nanoparticles, reduces potential cytotoxicity and adverse effects, and can produce strong tumor killing ability and cytokine secretion after transducing non-activated T cells.

[0013] In addition, the PBAE-447 polymer of the present invention can electrostatically bind to nucleic acids to form nanoparticles, enabling them to enter cells through endocytosis and prevent degradation by nucleases. Subsequently, protonation occurs at the low pH value of lysosomes to achieve endosome escape. Its special chemical structure and functional design can overcome the biological barriers of non-activated T cells and achieve efficient gene delivery. In addition, the nuclear localization signal peptide (MTAS-NLS) with a microtubule-related sequence is introduced into the nanoparticles, which helps the nanoparticles to accurately target the nucleus inside the cell. Description of the Drawings

[0014] Figure 1 It is a synthetic route diagram of the PBAE-447 nanocarrier; Figure 2 It is the map of plasmid 2; Figure 3 It is the schematic diagram of the preparation process of peptide-modified DNA nanoparticles; Figure 4 It is the comparison result diagram of the transduction efficiency of non-peptide-modified DNA nanoparticles in transducing and activating T cells and non-activated T cells; Figure 5 It is the schematic diagram of the influence of non-peptide-modified DNA nanoparticles on the transduction efficiency of non-activated T cells under different conditions; among them Figure 5 a in it is the schematic diagram of the influence of nanoparticles with different doses on the transduction efficiency of non-activated T cells, Figure 5 b in it is the schematic diagram of the influence of transduction time on the transduction efficiency of non-activated T cells, Figure 5 c in it is the schematic diagram of the influence of the dose of cytokine IL2 on the transduction efficiency of non-activated T cells, Figure 5 d in it is the schematic diagram of the influence of the specific surface area of transduction on the transduction efficiency of non-activated T cells; Figure 6 It is the influence of adding MTAS and NLS peptides to the nanoparticles on the transduction efficiency; Figure 7 It is the schematic diagram of the influence of peptide-modified nanoparticles on the transduction efficiency of non-activated T cells under different conditions; among them Figure 7 a in it is the schematic diagram of the influence of nanoparticles with different doses on the transduction efficiency of non-activated T cells, Figure 7 b in it is the schematic diagram of the influence of transduction time on the transduction efficiency of non-activated T cells, Figure 7 c in it is the schematic diagram of the influence of the dose of cytokine IL2 on the transduction efficiency of non-activated T cells, Figure 7 d in it is the schematic diagram of the influence of the specific surface area of transduction on the transduction efficiency of non-activated T cells; Figure 8 It is the schematic diagram of the killing effect of different T cells on tumor cells. Detailed implementation method

[0015] The purpose of the present invention is to provide a nuclear targeting peptide-modified DNA nanoparticle based on microfluidic technology, its preparation method and application. The following will further describe the present invention in combination with specific embodiments.

[0016] Plasmid 1 was provided by Wuhan Miaoling Biotechnology Co., Ltd.

[0017] PiggyBac-CMV-MCS-Fluc-EF1a-copGFP-T2A-Puro (P27701) plasmid was provided by Wuhan Miaoling Biotechnology Co., Ltd. Example 1

[0018] Preparation method of nuclear targeting peptide-modified DNA nanoparticles based on microfluidic technology, comprising the following steps: ① Synthesis of PBAE-447 nanocarrier: The basic monomer molecule 1,4-butanediol diacrylate and the side chain monomer molecule 4-amino-1-butanol are subjected to a Michael addition reaction to synthesize a polymer capped with acrylate. The obtained polymer after the reaction is reacted with 1-(3-aminopropyl)-4-methylpiperazine to obtain an amino-capped PBAE-447 nanopolymer, namely poly(β-amino ester). This polymer is dissolved in DMSO and stored at -20 °C for later use; ② Coupling of PBAE-447 nanocarrier and MTAS-NLS peptide: First, the crosslinking agent N-(p-maleimidophenyl) isocyanate (PMPI) is dissolved in DMSO and added to the PBAE-447 solution. After reacting at room temperature for 3 h, the activated PBAE-447-maleimide derivative and MTAS-NLS are mixed and reacted for 3 h. Then, the mixed solution is filtered and vacuum dried to obtain PBAE-447-MTAS-NLS; The MTAS-NLS peptide sequence is SEQ ID No.1, and the specific content is: GRYLTQETNKVETYKEQPLKTPGKKKKGKPGKRKEQEKKKRRTR, with a cysteine added at the N-terminus.

[0019] ③ Preparation of plasmid DNA: Two plasmids are used. Plasmid 1 expresses PiggyBac transposase; Plasmid 2 drives the expression of the target gene by the hPGK promoter, and the target gene is preferably the CAR gene; Construction method of Plasmid 2: The synthetic gene (hPGK-CAR) is inserted into the EcoRI / NotI site of the PiggyBac-CMV-MCS-Fluc-EF1a-copGFP-T2A-Puro (P27701) plasmid by a conventional method, and the constructed plasmid is named PiggyBac-cmv-PGK-BBZ19-copGFP-Puro; The CAR gene is a common CAR structure. Taking the CAR targeting CD19 (CD19 CAR) as an example, the gene coding sequence of CD19 CAR is an existing gene sequence, as shown in the paper "A safe and potent anti-CD19 CAR T cell therapy, Nature Medicine 2019; 25: 947–953"; ④ Preparation of peptide-modified DNA nanoparticles: Plasmid 1 and plasmid 2 were mixed at a mass ratio of 1:3, and the PBAE nanocarrier was mixed with plasmid 1 at a mass ratio of 60:1; PBAE-447 was used as the organic phase, and the plasmid DNA obtained by mixing plasmid 1 and plasmid 2 was used as the acidic aqueous phase and directly mixed through a microfluidic device. During this process, DNA molecules were uniformly dispersed in the polymer network, and the mixed solution was dialyzed at room temperature to remove excess reagents; the final DNA nanocarrier had a size of 145 ± 40 nm and a zeta potential of 15.5 ± 2.1 mV. Example 2

[0020] A method for preparing nuclear-targeting peptide-modified DNA nanoparticles based on microfluidic technology, comprising the following steps: ① Synthesis of PBAE-447 nanocarrier: As Figure 1 shown, accurately weigh 100 g of 1,4-butanediol diacrylate and 2.56 g of 4-amino-1-butanol, add them to a round-bottom flask, synthesize the acrylate-capped basic polymer poly(1,4-butanediol diacrylate-co-4-amino-1-butanol), and stir at 70 °C for 24 hours. Take 2.30 g of the reacted polymer and dissolve it in 2 mL of tetrahydrofuran (THF), add it to 13 mL of tetrahydrofuran containing 786 mg of 1-(3-aminopropyl)-4-methylpiperazine, and stir at 8000 r / min at room temperature for 2 h for capping. The final PBAE-447 polymer was dissolved in 75 mL of ether for washing, allowed to stand for 0.5 h to remove unreacted small molecules, the polymer was collected, and the supernatant was removed. The polymer was repeatedly washed and purified with ether twice. After completion, the collected polymer was vacuum dried at room temperature for 48 h, and then the obtained PBAE-447 polymer was dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 100 mg / ml, aliquoted into small tubes, and frozen at -20 °C to avoid repeated freezing and thawing.

[0021] ② Coupling of PBAE-447 nanocarrier and MTAS-NLS peptide: Dissolve 6 mg of N-(p-maleimidophenyl)-isocyanate (PMPI) in DMSO to a concentration of 20 mg / ml, and then add it to 50 mg of PBAE-447 (100 mg / ml). After shaking and mixing at room temperature for 3 h, PBAE-447-maleimide derivative was obtained. The obtained PBAE-447-maleimide derivative was added to a 5.3 ml DMSO solution containing 50 mg of MTAS-NLS, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP•HCl; 3 mg / ml) was added to the solution. After mixing at room temperature for 3 h, it was filtered using a 7k Zeba spin column. The coupled PBAE-447-MTAS-NLS was redissolved in DMSO to a concentration of 100 mg / ml and stored at -20 °C.

[0022] ③ Prepare plasmid DNA: Two plasmids are used. Plasmid 1 expresses PiggyBac transposase; plasmid 2 drives the expression of the target gene by the hPGK promoter, and the target gene is preferably the CAR gene; Construction method of plasmid 2: The synthetic gene (hPGK-CAR) is inserted into the EcoRI / NotI of the PiggyBac-CMV-MCS-Fluc-EF1a-copGFP-T2A-Puro (P27701) plasmid by a conventional method, and the constructed plasmid is named PiggyBac-cmv-PGK-BBZ19-copGFP-Puro; the CAR gene is a common CAR structure. Taking the CAR targeting CD19 (CD19 CAR) as an example, the map of plasmid 2 is as Figure 2 shown; ④ Preparation of peptide-modified DNA nanoparticles: Prepare a NaAc / HAc buffer solution with pH = 5.0 as the acidic aqueous phase, and prepare an organic phase with a volume ratio of ethanol to DMSO of 1:1 for standby; Dilute the conjugated PBAE-447-MTAS-NLS in 100 mg / ml DMSO to 18 mg / ml in the organic phase; plasmid 1 and plasmid 2 are mixed at a mass ratio of 1:3 to obtain plasmid DNA, and dissolved in the acidic aqueous phase according to the ratio of conjugated PBAE-447-MTAS-NLS to plasmid DNA = 240:4 (w / w), as Figure 3 shown. Mix the organic phase and the aqueous phase directly at a volume ratio of 2:1 through a microfluidic device. During this process, plasmid DNA molecules are evenly dispersed in the polymer network. The prepared nanoparticles are first dialyzed in ammonia water with pH = 10 for 1 h, and then dialyzed in PBS for 1 h to remove organic solvents. The dialysis temperature is controlled between 4 - 8 °C to obtain nuclear-targeting peptide-modified DNA nanoparticles.

[0023] Prepare non-peptide-modified DNA nanoparticles. The steps are as shown in ①③④ above. Compared with the steps of Example 2, there is no step ②, and the steps ①③ are exactly the same. The operation of step ④ is slightly different, as follows: ④ Preparation of non-peptide-modified DNA nanoparticles: Prepare a NaAc / HAc buffer solution with pH = 5.0 as the acidic aqueous phase, and prepare an organic phase with a volume ratio of ethanol to DMSO of 1:1 for standby; Dilute PBAE-447 in 100 mg / ml DMSO to 18 mg / ml in the organic phase; mix plasmid 1 and plasmid 2 at a mass ratio of 1:3 to obtain plasmid DNA, dissolve it in the acidic aqueous phase at a ratio of PBAE-447 polymer to plasmid 1 = 60:1 (w / w), and directly mix the organic phase and the aqueous phase at a volume ratio of 2:1 through a microfluidic device. During this process, plasmid DNA molecules are uniformly dispersed in the polymer network. The prepared nanoparticles are first dialyzed in ammonia water with pH = 10 for 1 h, and then dialyzed in PBS for 1 h to remove organic solvents. The dialysis temperature is controlled between 4 - 8 °C to obtain non-peptide modified DNA nanoparticles.

[0024] Experiment 1: Comparison of transduction efficiency between non-peptide modified DNA nanoparticles transducing activated T cells and non-activated T cells Take 100 μl each of activated T cells and non-activated T cells transduced with non-peptide modified DNA nanoparticles respectively.

[0025] Process of nanoparticles transducing non-activated T cells: Directly add the above-prepared nanoparticles containing 1.5 μg plasmid DNA to 3×10 5 freshly isolated T cells, mix well, incubate at 37 °C and 5% CO2 for 2 - 6 h, wash thoroughly and then continue to culture for 48 h.

[0026] Preparation process of nanoparticles transducing activated T cells: After activating T cells with anti-CD3 / CD28 magnetic beads for 24 h, remove the magnetic beads, add the above-prepared 1.5 μg DNA nanoparticles to the cells, mix well, incubate at 37 °C and 5% CO2 for 2 - 6 h, wash thoroughly and then continue to culture for 48 h. After 48 h, detect the fluorescence of nanoparticles on a flow cytometer (Flow Cytometry), collect and analyze the data to obtain a flow cytometry graph. As Figure 4 shown, although it is proved that nanoparticles have advantages in transducing non-activated T cells, the transduction efficiency of about 3% is still not sufficient to generate potent CAR-T cells.

[0027] Experiment 2: Investigated the transduction efficiency of non-peptide modified DNA nanoparticles on non-activated T cells under different conditions Change the culture conditions, and investigate the transduction efficiency of non-peptide modified DNA nanoparticles on non-activated T cells by adjusting the dose of nanoparticles, changing the transduction time, adding cytokine IL-2, and increasing the transduction specific surface area. The results are as Figure 5 shown.

[0028] As Figure 5 seen from a in, although the transduction efficiency can be improved by increasing the dose of nanoparticles, when the maximum dose of 2.0 μg is used, the transduction efficiency is still lower than 5%; as Figure 5It can be seen from Fig. b that increasing the transduction time can improve the transduction efficiency, but the increasing rate becomes smaller. Using the longest transduction time of 6 h, the transduction efficiency is also lower than 5%. From Figure 5 It can be seen from Fig. c that increasing the cytokine IL-2 has little effect on the transduction efficiency. From Figure 5 It can be seen from Fig. d that increasing the specific surface area of transduction has little effect on the transduction efficiency. Therefore, it is difficult to improve the transduction efficiency of non-peptide-modified DNA nanoparticles on non-activated T cells by only changing the culture conditions.

[0029] Experiment 3: Add MTAS and NLS to the nanoparticles to explore their effects on the transduction efficiency Transduce non-activated T cells with MTAS-NLS peptide-modified and non-peptide-modified DNA nanoparticles respectively. Specific steps: Directly add 1.5 μg of nuclear targeting peptide-modified DNA nanoparticles to freshly isolated human T cells, mix well, incubate at 37 °C and 5% CO2 for 2 - 6 h, wash thoroughly and then continue to culture for 48 h. Directly add 1.5 μg of non-peptide-modified DNA nanoparticles to freshly isolated human T cells, mix well, incubate at 37 °C and 5% CO2 for 2 - 6 h, wash thoroughly and then continue to culture for 48 h. After 48 h, detect the fluorescence of the nanoparticles on a flow cytometer, collect and analyze the data to obtain a flow cytometry graph. As Figure 6 shown, compared with non-peptide-modified nanoparticles, the transduction rate of peptide-modified DNA nanoparticles on non-activated T cells is increased by about 5 times.

[0030] Experiment 4: Explore the transduction efficiency of peptide-modified DNA nanoparticles on non-activated T cells under different conditions Change the culture conditions, and investigate the transduction efficiency of peptide-modified DNA nanoparticles on non-activated T cells by adjusting the dose of the nanoparticles, changing the transduction time, adding the cytokine IL-2 and increasing the specific surface area of transduction. The results are as Figure 7 shown. It can be seen that the transduction efficiency of peptide-modified nanoparticles on non-activated T cells is affected by the DNA dose and transduction time to a certain extent. 1.5 μg of DNA at a transduction time of 4 h is used as the dose for subsequent experiments; Adding the cytokine IL-2 and increasing the transfection specific surface area instead reduce the transduction efficiency of peptide-modified nanoparticles on non-activated T cells.

[0031] Experiment 5: Verify the killing effect of T cells transfected with peptide-modified DNA nanoparticles on tumor cells Co-culture the non-activated T cells transfected with the nanoparticles and RS411 lymphoma cells at a cell ratio of 3:1 at 37 °C and 5% CO2, and detect the apoptosis of tumor cells with an enzyme-labeled instrument after 4 days.

[0032] As Figure 8As shown, T cells transduced with peptide-modified DNA nanoparticles can kill lymphoma cells. In contrast, untransduced T cells and T cells transduced with non-peptide-modified nanoparticles not only have no killing effect on lymphoma cells, but instead stimulate the growth of tumor cells.

Claims

1. A nuclear-targeting peptide-modified DNA nanoparticle based on microfluidic technology, characterized in that: Using poly(β - amino ester) cationic polymer as a gene delivery vector, which contains a microtubule - associated sequence MTAS and a nuclear localization signal NLS short peptide. The gene delivery vector is mixed with plasmid DNA carrying a target gene to prepare nanoparticles, and the plasmid DNA carrying the target gene is uniformly dispersed in the nanoparticles by a microfluidic mixing process.

2. The DNA nanoparticles modified with nuclear targeting peptides based on microfluidic technology according to claim 1, wherein: The target gene is a CAR gene.

3. The DNA nanoparticles modified with nuclear targeting peptides based on microfluidic technology according to claim 1, wherein: The target gene is a CAR gene targeting CD19, CD38, B7H3 or CS1.

4. The preparation method of the nuclear targeting peptide-modified DNA nanoparticles based on microfluidic technology according to claim 1, characterized in that: It includes the following steps: Firstly, prepare poly(β - amino ester) polymer as a gene delivery vector. Secondly, add a microtubule - associated sequence MTAS and a nuclear localization signal NLS short peptide to the polymer vector. Finally, use a microfluidic mixing process to uniformly mix the polymer vector with plasmid DNA carrying the target gene to prepare nanoparticles, and the plasmid DNA carrying the target gene is uniformly dispersed in the nanoparticles.

5. The preparation method of the nuclear targeting peptide-modified DNA nanoparticles based on microfluidic technology according to claim 4, wherein: It includes the following steps: ① Prepare poly(β - amino ester) cationic polymer as a gene delivery vector: Synthesize acrylate - terminated polymer by Michael addition reaction of 1,4 - butanediol diacrylate and 4 - amino - 1 - butanol, and then react with 1 - (3 - aminopropyl) - 4 - methylpiperazine to obtain poly(β - amino ester). Dissolve the obtained poly(β - amino ester) in DMSO to get PBAE - 447 solution, and store it at - 20 °C for later use. ② Add a microtubule - associated sequence MTAS and a nuclear localization signal NLS short peptide to the PBAE - 447 polymer: Dissolve the cross - linker N - (p - maleimidophenyl) isocyanate in DMSO and add it to the PBAE - 447 solution obtained in step ①. React at 20 - 35 °C for 3 h to obtain the activated PBAE - 447 - maleimide derivative. After mixing and reacting the obtained activated PBAE - 447 - maleimide derivative with the microtubule - associated sequence MTAS and the nuclear localization signal NLS short peptide for 3 h, a mixed solution is obtained. The obtained mixed solution is filtered and vacuum - dried to obtain conjugated PBAE - 447 - MTAS - NLS. Dissolve the obtained conjugated PBAE - 447 - MTAS - NLS in DMSO and store it at - 20 °C for later use. ③ Prepare plasmid DNA: Use two plasmids. Plasmid 1 expresses PiggyBac transposase; Plasmid 2 drives the expression of the target gene by the hPGK promoter. The construction method of plasmid 2: Connect the target gene with hPGK, and then insert it into the EcoRI / NotI site of the PiggyBac - CMV - MCS - Fluc - EF1a - copGFP - T2A - Puro plasmid. ④ Preparation of peptide - modified DNA nanoparticles: The conjugated PBAE-447-MTAS-NLS dissolved in DMSO is diluted with an organic solvent and used as the organic phase; plasmid 1 and plasmid 2 are mixed to obtain plasmid DNA, and the plasmid DNA is dissolved in a NaAc / HAc buffer solution with pH = 5.0 as the acidic aqueous phase. The obtained organic phase and acidic aqueous phase are mixed through a microfluidic device, and excess reagents are removed by dialysis to obtain nuclear-targeting peptide-modified DNA nanoparticles; When the obtained organic phase and acidic aqueous phase are mixed through a microfluidic device, the mass ratio of PBAE-447-MTAS-NLS to plasmid 2 is 60:1; plasmid 1 and plasmid 2 are mixed at a mass ratio of 1:

3.

6. The preparation method of the nuclear targeting peptide-modified DNA nanoparticles based on the microfluidic technology according to claim 5, characterized in that: The organic solvent is obtained by mixing ethanol and DMSO at a volume ratio of 1:

1.

7. Use of a nuclear targeting peptide-modified DNA nanoparticle based on microfluidic technology according to claim 1, characterized in that: It is used for the transduction of non-activated T cells.

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