A microfluidic technology-based nuclear-targeting peptide-modified DNA nanoparticle, a preparation method and application thereof

Abstract

The application discloses a kind of nuclear targeting peptide modified DNA nanoparticles based on microfluidic technology and its preparation method and application, it is related to biological medicine technical field, including poly (β- amino ester) cationic polymer as gene delivery carrier, microtubule associated sequence MTAS and nuclear localization signal NLS short peptide are included on the gene delivery carrier, the gene delivery carrier is mixed with plasmid DNA carrying target gene to prepare into nanoparticle, the plasmid DNA carrying target gene is uniformly dispersed in nanoparticle using microfluidic mixing process;The nuclear targeting peptide modified DNA nanoparticles of the application structure design is ingenious, using microfluidic mixing process improves the uniformity of nanoparticle carrier and plasmid DNA mixing, the performance of nanoparticle is further improved by optimizing dialysis condition, reduces potential cytotoxicity and adverse effect, can produce strong tumor killing capacity and cytokine secretion after transducing non-activated T cell.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a nucleic acid targeting peptide modified DNA nanoparticle based on microfluidic technology and a preparation method and application thereof. BACKGROUND

[0002] Chimeric antigen receptor T cells (CAR-T cells) are immune cells reprogrammed by gene infusion, which have made remarkable achievements in the research of hematological malignancies. In the preparation of immune cells reprogrammed by gene infusion, an ideal gene delivery carrier is needed, which should have cell adsorption capacity, be able to effectively penetrate the cell membrane, achieve endosome / lysosome escape and nuclear localization, so it is of great significance to design an ideal gene delivery carrier. On the other hand, the preparation process of CAR-T cells still faces the following problems:

[0003] Firstly, the current CAR-T cell preparation relies on viral vectors for gene transduction, which requires the completion of virus packaging, purification, T cell in vitro activation, expansion and other steps in a GMP-level clean room, resulting in high production cost and limiting its clinical accessibility. Secondly, in vitro activated T cells are prone to differentiate into terminal effector cells during rapid proliferation, which lose proliferative potential and undergo functional exhaustion, making it difficult to maintain long-term antitumor activity in vivo and affecting the durability of efficacy. Thirdly, non-activated T cells (such as naive T cells) have stronger self-renewal ability and anti-exhaustion properties, making them ideal target cells for gene modification. However, the transduction efficiency of existing gene delivery carriers (including viral vectors and non-viral vectors) on non-activated T cells is extremely low, which cannot be directly used for gene reprogramming.

[0004] Therefore, there is an urgent need to develop a delivery carrier that can directly transport nucleic acids into non-activated T cells to achieve effective gene delivery. SUMMARY

[0005] To solve the above problems, the purpose of the present application is to provide a nucleic acid targeting peptide modified DNA nanoparticle based on microfluidic technology and a preparation method and application thereof, which can directly transport nucleic acids into non-activated T cells to achieve effective gene delivery.

[0006] To achieve the above purpose, the present application realizes the following technical solutions:

[0007] A nucleic acid targeting peptide modified DNA nanoparticle based on microfluidic technology, comprising a poly(beta-amino ester) cationic polymer as a gene delivery carrier, wherein a microtubule-associated sequence MTAS and a nuclear localization signal NLS short peptide are contained on the gene delivery carrier, the gene delivery carrier is mixed with plasmid DNA carrying a target gene to prepare a nanoparticle, and the plasmid DNA carrying the target gene is uniformly dispersed in the nanoparticle by using a microfluidic mixing process.

[0008] Preferably, the target gene is a CAR gene.

[0009] Preferably, the target gene is a CAR gene targeting CD19, CD38, B7H3 or CS1.

[0010] The present application also includes a preparation method of a nucleic acid targeting peptide modified DNA nanoparticle based on microfluidic technology, comprising the following steps:

[0011] First, a poly (beta-amino ester) cationic polymer is prepared as a gene delivery carrier, second, a microtubule associated sequence MTAS and a nuclear localization signal NLS short peptide are added to the polymer carrier, and finally, the polymer carrier and plasmid DNA carrying the target gene are uniformly mixed to prepare nanoparticles by using a microfluidic mixing process, so that the plasmid DNA carrying the target gene is uniformly dispersed in the nanoparticles.

[0012] Preferably, the preparation method comprises the following steps:

[0013] ①Preparation of poly (beta-amino ester) cationic polymer as a gene delivery carrier:

[0014] 1,4-butanediol diacrylate and 4-amino-1-butanol are subjected to a Michael addition reaction to synthesize an acrylate-terminated polymer, which is then reacted with 1-(3-aminopropyl)-4-methylpiperazine to obtain poly (beta-amino ester), and the obtained poly (beta-amino ester) is dissolved in DMSO to obtain a PBAE-447 solution, which is stored at -20℃ for standby;

[0015] ②Adding a microtubule associated sequence MTAS and a nuclear localization signal NLS short peptide to the nanoparticles:

[0016] The crosslinking agent N-(p-maleimide phenyl) isocyanate is dissolved in DMSO and added to the PBAE-447 solution obtained in step ①, and reacted at 20~35℃ for 3h to obtain an activated PBAE-447-maleimide derivative, which is mixed with the microtubule associated sequence MTAS and the nuclear localization signal NLS short peptide and reacted for 3h to obtain a mixed solution, and the mixed solution is filtered and vacuum dried to obtain coupled PBAE-447-MTAS-NLS; the obtained coupled PBAE-447-MTAS-NLS is dissolved in DMSO and stored at -20℃ for standby;

[0017] ③Preparation of plasmid DNA: two plasmids are used, plasmid 1 expresses PiggyBac transposase; and plasmid 2 expresses the target gene driven by the hPGK promoter;

[0018] The construction method of the plasmid 2: connecting the target gene from hPGK, and then inserting into the EcoRI / NotI of the PiggyBac-CMV-MCS-Fluc-EF1a-copGFP-T2A-Puro plasmid to obtain;

[0019] IV. Preparation of the peptide-modified DNA nanoparticle:

[0020] The coupled PBAE-447-MTAS-NLS dissolved in DMSO is diluted as an organic phase by an organic solvent; the plasmid 1 and the plasmid 2 are mixed to obtain plasmid DNA, the plasmid DNA is dissolved in a NaAc / HAc buffer with pH=5.0 as an acidic aqueous phase, the obtained organic phase and the acidic aqueous phase are mixed by a microfluidic device, and the excess reagent is removed by dialysis to obtain the nucleus-targeting peptide-modified DNA nanoparticle;

[0021] The mass ratio of the coupled PBAE-447-MTAS-NLS and the plasmid 2 is 60:1 when the obtained organic phase and the acidic aqueous phase are mixed by the microfluidic device; the ratio of the plasmid 1 and the plasmid 2 is 1:3.

[0022] Preferably, the organic solvent is obtained by mixing ethanol and DMSO according to a volume ratio of 1:1.

[0023] The application also includes the nucleus-targeting peptide-modified DNA nanoparticle based on the microfluidic technology for the transduction of non-activated T cells.

[0024] Compared with the prior art, the application has the following advantages:

[0025] The nucleus-targeting peptide-modified DNA nanoparticle based on the microfluidic technology is an ideal gene delivery carrier, which can penetrate the cell membrane, realize the endosome / lysosome escape and the nuclear localization, and quickly reprogram the non-activated T cells without in-vitro activation and expansion culture.

[0026] The nucleus-targeting peptide-modified DNA nanoparticle based on the microfluidic technology has a clever structure design, the nanoparticle and the plasmid DNA carrying the target gene are uniformly mixed by the microfluidic mixing process, the uniformity of the mixing of the nanoparticle carrier and the plasmid DNA is improved, the performance of the nanoparticle is further improved by the optimization of the dialysis condition, the potential cytotoxicity and adverse effects are reduced, and the strong tumor killing ability and cytokine secretion can be generated after the transduction of the non-activated T cells.

[0027] In addition, the PBAE-447 polymer of the present application can be electrostatically combined with nucleic acid to form nanoparticles, so as to enter cells through endocytosis and prevent degradation by nucleases. Subsequently, protonation occurs at the low pH of lysosomes, realizing endosome escape. The special chemical structure and functional design can overcome the biological obstacles of non-activated T cells, and realize 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 be precisely targeted to the nucleus in cells. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Synthesis flow chart of PBAE-447 nanoparticle carrier;

[0029] Figure 2 Map of plasmid 2;

[0030] Figure 3 Preparation flow chart of peptide-modified DNA nanoparticles;

[0031] Figure 4 Comparison results of transduction efficiency of non-activated T cells and activated T cells by non-peptide-modified DNA nanoparticles;

[0032] Figure 5 Influence diagram of transduction efficiency of non-activated T cells by non-peptide-modified DNA nanoparticles under different conditions; wherein Figure 5 a is an influence diagram of transduction efficiency of non-activated T cells by nanoparticles of different doses, Figure 5 b is an influence diagram of transduction efficiency of non-activated T cells by transduction time, Figure 5 c is an influence diagram of transduction efficiency of non-activated T cells by cytokine IL2 dose, Figure 5 d is an influence diagram of transduction efficiency of non-activated T cells by specific surface area of transduction;

[0033] Figure 6 Influence of adding MTAS and NLS peptides on transduction efficiency of nanoparticles;

[0034] Figure 7 Influence diagram of transduction efficiency of non-activated T cells by peptide-modified nanoparticles under different conditions; wherein Figure 7 a is an influence diagram of transduction efficiency of non-activated T cells by nanoparticles of different doses, Figure 7 b is an influence diagram of transduction efficiency of non-activated T cells by transduction time, Figure 7 c is an influence diagram of transduction efficiency of non-activated T cells by cytokine IL2 dose, Figure 7 d is an influence diagram of transduction efficiency of non-activated T cells by specific surface area of transduction;

[0035] Figure 8 Schematic diagram of killing effect of different T cells on tumor cells. DETAILED DESCRIPTION

[0036] The purpose of the present application is to provide a nucleic acid targeting peptide modified DNA nanoparticle based on microfluidic technology, as well as a preparation method and application thereof, which will be further described below in combination with specific embodiments.

[0037] Plasmid 1 is provided by Wuhan Moli Life Science Co., Ltd.

[0038] PiggyBac-CMV-MCS-Fluc-EF1a-copGFP-T2A-Puro (P27701) plasmid is provided by Wuhan Moli Life Science Co., Ltd. Example 1

[0039] The preparation method of the nucleic acid targeting peptide modified DNA nanoparticle based on microfluidic technology comprises the following steps:

[0040] ① Synthesis of PBAE-447 nanocarrier: the base monomer molecule 1,4-butanediol diacrylate and the side chain monomer molecule 4-amino-1-butanol are subjected to Michael addition reaction to synthesize an acrylate-terminated polymer. The polymer after reaction is reacted with amino 1-(3-aminopropyl)-4-methylpiperazine to obtain an amino-terminated PBAE-447 nanopolymer, i.e., poly(β-amino ester), which is dissolved in DMSO and stored at -20℃ for standby;

[0041] ② Coupling of PBAE-447 nanocarrier and MTAS-NLS peptide: first, the crosslinking agent N-(p-maleimide phenyl) isocyanate (PMPI) is dissolved in DMSO and added to the PBAE-447 solution, which is reacted at room temperature for 3 h. After the activated PBAE-447-maleimide derivative is mixed with MTAS-NLS for 3 h, the mixed solution is filtered and vacuum dried to obtain PBAE-447-MTAS-NLS;

[0042] The MTAS-NLS short peptide sequence is SEQ ID No. 1, and the specific content is:

[0043] GRYLTQETNKVETYKEQPLKTPGKKKKGKPGKRKEQEKKKRRTR, with a cysteine added at the N-terminus.

[0044] ③ Preparation of plasmid DNA: two plasmids are used, plasmid 1 expresses PiggyBac transposase; and plasmid 2 expresses a target gene driven by an hPGK promoter, wherein the target gene is preferably a CAR gene;

[0045] Construction method of plasmid 2: the synthetic gene (hPGK-CAR) is inserted into the EcoRI / NotI of PiggyBac-CMV-MCS-Fluc-EF1a-copGFP-T2A-Puro (P27701) plasmid by conventional method, and the constructed plasmid is named PiggyBac-cmv-PGK-BBZ19-copGFP-Puro; the CAR gene is a commonly used CAR structure, and the CD19 CAR targeting CD19 is taken as an example, and the gene coding sequence of the 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";

[0046] ④Preparation of peptide-modified DNA nanoparticles: plasmid 1 and plasmid 2 are mixed at a mass ratio of 1:3, and PBAE nanoparticle carriers are mixed with plasmid 1 at a mass ratio of 60:1; PBAE-447 is used as an organic phase, and the plasmid DNA obtained by mixing plasmid 1 and plasmid 2 is used as an acidic aqueous phase, which is directly mixed by a microfluidic device, in the process, the DNA molecules are uniformly dispersed in the polymer network, and the mixed solution is dialyzed at room temperature to remove excess reagents; the final DNA nanoparticle carrier has a size of 145±40nm and a zeta potential of 15.5±2.1mV. Example 2

[0047] The preparation method of the nuclear-targeting peptide-modified DNA nanoparticle based on the microfluidic technology comprises the following steps:

[0048] ①Synthesis of PBAE-447 nanoparticle carrier: as shown in Figure 1 , 100g of 1,4-butanediol diacrylate and 2.56g of 4-amino-1-butanol are accurately weighed and added to a round-bottom flask to synthesize the acrylate-terminated base polymer poly(1,4-butanediol diacrylate-co-4-amino-1-butanol), and stirring is performed at 70°C for 24 hours, 2.30g of the polymer after reaction is dissolved in 2-mL of tetrahydrofuran (THF), and then added to 13mL of tetrahydrofuran containing 786mg of 1-(3-aminopropyl)-4-methylpiperazine, stirring is performed at 8000r / min and room temperature for 2h to perform termination. The final PBAE-447 polymer is dissolved in 75mL of ether for cleaning, and then placed for 0.5h to remove unreacted small molecules, and the polymer is collected and the supernatant is removed. The purification of the polymer is repeated twice by using ether, and after the end, the collected polymer is vacuum dried at room temperature for 48h, and then the obtained PBAE-447 polymer is dissolved in dimethyl sulfoxide (DMSO) to obtain a final concentration of 100mg / ml, and then divided into small tubes and frozen at-20°C to avoid repeated freezing and thawing.

[0049] (2) Coupling of PBAE-447 nanocarrier with MTAS-NLS peptide: 6 mg of N-(p-maleimidophenyl) isocyanate (PMPI) was dissolved in DMSO to a concentration of 20 mg / ml, and then added to 50 mg of PBAE-447 (100 mg / ml). After 3 hours of shaking mixing at room temperature, PBAE-447-maleimide derivative was obtained, and 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 3 hours of mixing at room temperature, a 7k Zeba spin column was used for filtration. The coupled PBAE-447-MTAS-NLS was again dissolved in DMSO to a concentration of 100 mg / ml, and stored at -20°C.

[0050] (3) Preparation of plasmid DNA: two plasmids were used, plasmid 1 expresses PiggyBac transposase; and plasmid 2 expresses a target gene driven by the hPGK promoter, wherein the target gene is preferably a CAR gene;

[0051] Construction method of plasmid 2: the synthesized gene (hPGK-CAR) was inserted into the EcoRI / NotI site of the PiggyBac-CMV-MCS-Fluc-EF1a-copGFP-T2A-Puro (P27701) plasmid by a conventional method to construct a plasmid named PiggyBac-cmv-PGK-BBZ19-copGFP-Puro; the CAR gene is a commonly used CAR structure, and a CAR (CD19 CAR) targeting CD19 is taken as an example, and the map of plasmid 2 is as shown in Figure 2 ;

[0052] (4) Preparation of DNA nanoparticles modified with peptides:

[0053] Prepare NaAc / HAc buffer with pH=5.0 as the acidic aqueous phase, prepare ethanol and DMSO with a volume ratio of 1:1 as the organic phase, and reserve;

[0054] The coupled PBAE-447-MTAS-NLS in 100 mg / ml DMSO was diluted to 18 mg / ml in the organic phase; plasmid 1 and plasmid 2 were mixed according to a mass ratio of 1:3 to obtain plasmid DNA, and were dissolved in the acidic aqueous phase according to a ratio of coupled PBAE-447-MTAS-NLS, plasmid DNA = 240:4 (w / w), as shown in Figure 3As shown, the organic phase and the aqueous phase are directly mixed through the microfluidic device at a volume ratio of 2:1, in the process, the plasmid DNA molecules are uniformly dispersed in the polymer network, the prepared nanoparticles are first dialyzed in ammonia water with pH=10 for 1h, and then dialyzed in PBS for 1h to remove the organic solvent, the dialysis temperature is controlled between 4-8℃, and the core-targeting peptide modified DNA nanoparticles are obtained.

[0055] The non-peptide modified DNA nanoparticles are prepared, the steps are shown as above ① ③ ④, compared with the steps of Example 2, there is no step ②, steps ① ③ are completely the same, and the operation of step ④ is slightly different, as shown below:

[0056] ④ Preparation of non-peptide modified DNA nanoparticles:

[0057] Prepare NaAc / HAc buffer with pH=5.0 as the acidic aqueous phase, prepare ethanol and DMSO with a volume ratio of 1:1 as the organic phase, and prepare them for use;

[0058] Dilute PBAE-447 in DMSO with 100mg / ml to 18mg / ml in the organic phase; mix plasmid 1 and plasmid 2 according to the mass ratio of 1:3 to obtain plasmid DNA, and dissolve them in the acidic aqueous phase according to the ratio of PBAE-447 polymer to plasmid 1=60:1(w / w), directly mix the organic phase and the aqueous phase through the microfluidic device at a volume ratio of 2:1, in the process, the plasmid DNA molecules are uniformly dispersed in the polymer network, the prepared nanoparticles are first dialyzed in ammonia water with pH=10 for 1h, and then dialyzed in PBS for 1h to remove the organic solvent, the dialysis temperature is controlled between 4-8℃, and the non-peptide modified DNA nanoparticles are obtained.

[0059] Experiment one, comparison of the transduction efficiency of non-peptide modified DNA nanoparticles transduced activated T cells and non-activated T cells

[0060] Take 100ul of activated T cells and non-activated T cells transduced by non-peptide modified DNA nanoparticles respectively.

[0061] Process of nanoparticle transduction of non-activated T cells: directly add the above prepared nanoparticles containing 1.5ug plasmid DNA into 3*10 5 T cells freshly isolated, mix well, incubate at 37℃, 5% CO2 for 2~6h, and continue to culture for 48h after washing.

[0062] Nanoparticle transduction of activated T cell preparation process: after T cells were activated with anti-CD3 / CD28 magnetic beads for 24 hours, the magnetic beads were removed, 1.5 ug of DNA nanoparticles prepared above were added to the cells, mixed, incubated at 37°C for 2-6 hours in 5% CO2, and then washed thoroughly and cultured for 48 hours. After 48 hours, the nanoparticle fluorescence was detected on the flow cytometry, the data was collected and analyzed, and the flow chart was obtained. As shown in Figure 4 , although the nanoparticles have advantages in transducing non-activated T cells, the transduction efficiency of about 3% is still insufficient to generate potent CAR-T cells.

[0063] Experiment two, the transduction efficiency of non-peptide modified DNA nanoparticles on non-activated T cells under different conditions was explored

[0064] By adjusting the dose of nanoparticles, changing the transduction time, adding cytokine IL-2 and increasing the specific surface area of transduction, the transduction efficiency of non-peptide modified DNA nanoparticles on non-activated T cells was investigated, and the results are shown in Figure 5 .

[0065] As shown in Figure 5 a, although the transduction efficiency can be improved by increasing the dose of nanoparticles, the transduction efficiency is still less than 5% when the maximum dose of 2.0 ug is used; as shown in Figure 5 b, increasing the transduction time can improve the transduction efficiency, but the increasing rate becomes smaller, and the transduction efficiency is still less than 5% when the longest transduction time of 6 hours is used; as shown in Figure 5 c, the addition of cytokine IL-2 has little effect on the transduction efficiency; as shown in Figure 5 d, increasing the specific surface area of transduction has little effect on the transduction efficiency, so it is difficult to improve the transduction efficiency of non-peptide modified DNA nanoparticles on non-activated T cells by changing the culture conditions only.

[0066] Experiment three, MTAS and NLS were added to the nanoparticles to explore their effect on the transduction efficiency

[0067] Non-activated T cells were transduced with MTAS-NLS peptide modified and non-peptide modified DNA nanoparticles, respectively. The specific steps are as follows: 1.5 ug of DNA nanoparticles modified with nuclear targeting peptide were directly added to freshly isolated human T cells, mixed, incubated at 37°C for 2-6 hours in 5% CO2, and then washed thoroughly and cultured for 48 hours. 1.5 ug of non-peptide modified DNA nanoparticles were directly added to freshly isolated human T cells, mixed, incubated at 37°C for 2-6 hours in 5% CO2, and then washed thoroughly and cultured for 48 hours. After 48 hours, the nanoparticle fluorescence was detected on the flow cytometry, the data was collected and analyzed, and the flow chart was obtained. As shown in Figure 6As shown, the transduction efficiency of the peptide-modified DNA nanoparticles on non-activated T cells was about 5 times higher than that of the non-peptide-modified nanoparticles.

[0068] Experiment Four: The transduction efficiency of the peptide-modified DNA nanoparticles on non-activated T cells under different conditions was explored.

[0069] By changing the culture conditions, adjusting the dose of nanoparticles, changing the transduction time, adding cytokine IL-2, and increasing the specific surface area of transduction, the transduction efficiency of the peptide-modified DNA nanoparticles on non-activated T cells was investigated, and the results are shown in Figure 7 As shown, the transduction efficiency of the peptide-modified nanoparticles on non-activated T cells was affected to some extent by the DNA dose and the transduction time. The 1.5 ug dose of DNA transduction for 4 hours was used as the subsequent experimental dose. The addition of cytokine IL-2 and the increase of the specific surface area of transfection actually reduced the transduction efficiency of the peptide-modified nanoparticles on non-activated T cells.

[0070] Experiment Five: Verification of the killing effect of T cells transfected by peptide-modified DNA nanoparticles on tumor cells

[0071] The non-activated T cells transfected by the nanoparticles were co-cultured with RS411 lymphoma cells at a cell ratio of 3:1 at 37°C and 5% CO2. After 4 days, the apoptosis of the tumor cells was detected by the enzyme label instrument.

[0072] As shown in Figure 8 , the T cells transfected by the peptide-modified DNA nanoparticles could kill the lymphoma cells. On the contrary, the non-transfected T cells and the T cells transfected by the non-peptide-modified nanoparticles not only had no killing effect on the lymphoma cells, but also stimulated the growth of the tumor cells.

Claims

1. A nuclear-targeted peptide-modified DNA nanoparticle based on microfluidic technology, characterized in that: Using a poly(β-amino ester) cationic polymer as a gene delivery vector, the gene delivery vector contains a microtubule-associated sequence (MTAS) and a nuclear localization signal (NLS) short peptide. The gene delivery vector is mixed with plasmid DNA carrying the target gene to prepare nanoparticles. The plasmid DNA carrying the target gene is uniformly dispersed in the nanoparticles using a microfluidic mixing process. The target gene is a CAR gene. The nuclear-targeted peptide-modified DNA nanoparticles based on microfluidics technology were prepared through the following steps: ① Preparation of poly(β-amino ester) cationic polymers as gene delivery vectors: An acrylate-terminated polymer was synthesized by Michael addition reaction of 1,4-butanediol diacrylate and 4-amino-1-butanol. Then, it was reacted with 1-(3-aminopropyl)-4-methylpiperazine to obtain poly(β-amino ester). The obtained poly(β-amino ester) was dissolved in DMSO to obtain PBAE-447 solution, which was stored at -20°C for later use. ② Add a short peptide containing the microtubule-associated sequence MTAS and the nuclear localization signal NLS to the PBAE-447 polymer: The cross-linking agent N-(p-maleimide phenyl) isocyanate was dissolved in DMSO and added to the PBAE-447 solution obtained in step ①. The reaction was carried out at 20~35℃ for 3h to obtain the activated PBAE-447-maleimide derivative. The obtained activated PBAE-447-maleimide derivative was mixed with the microtubule-associated sequence MTAS and the nuclear localization signal NLS short peptide and reacted for 3h to obtain a mixed solution. The obtained mixed solution was filtered and vacuum dried to obtain the coupled PBAE-447-MTAS-NLS. The obtained coupled PBAE-447-NLS-MTAS was dissolved in DMSO and stored at -20℃ for later use. ③Prepare plasmid DNA: Use two plasmids, plasmid 1 expresses PiggyBac transposase; plasmid 2 is driven by the hPGK promoter to express the target gene; Construction method of plasmid 2: The target gene is ligated by hPGK and then inserted into EcoRI / NotI of the PiggyBac-CMV-MCS-Fluc-EF1a-copGFP-T2A-Puro plasmid; ④ Preparation of peptide-modified DNA nanoparticles: The PBAE-447-NLS-MTAS, after being coupled and dissolved in DMSO, was diluted with an organic solvent to form the organic phase. Plasmid 1 and plasmid 2 were mixed to obtain plasmid DNA. The plasmid DNA was dissolved in NaAc / HAc buffer at pH 5.0 to form the acidic aqueous phase. The resulting organic phase and acidic aqueous phase were mixed using a microfluidic device, and excess reagents were removed by dialysis to obtain nuclear-targeted peptide-modified DNA nanoparticles. When the obtained organic phase and acidic aqueous phase were mixed by a microfluidic device, the mass ratio of PBAE-447-NLS-MTAS to plasmid 2 was 60:1; plasmid 1 and plasmid 2 were mixed at a mass ratio of 1:

3. During dialysis, the patient was first dialyzed in ammonia water at pH 10 for 1 hour, and then dialyzed in PBS for 1 hour. The dialysis temperature was controlled at 4-8℃. The organic solvent is obtained by mixing ethanol and DMSO in a volume ratio of 1:

1.

2. The nuclear-targeted peptide-modified DNA nanoparticles based on microfluidic technology according to claim 1, characterized in that: The target gene is a CAR gene that targets CD19, CD38, B7H3, or CS1.

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