A polypeptide-modified cationic polymeric nanoparticle and a preparation method and application thereof

Abstract

The application discloses a polypeptide modified cationic polymer nanoparticle and a preparation method and application thereof, relates to the technical field of genetic engineering drugs and vaccine manufacturing, and the polypeptide modified cationic polymer nanoparticle comprises a cationic polymer nanoparticle PBAE-536, plasmid DNA which is electrostatically combined with the PBAE-536 and tightly wound, a cell penetrating peptide HIV-TAt which is indirectly coupled to the surface of the PBAE-536, an auxiliary peptide HA2-TAt which is mixed with the PBAE-536, and a short peptide comprising a microtubule associated sequence MTAS and a nuclear localization signal NLS which is coupled with the PBAE-536 carrier. The polypeptide modified cationic polymer nanoparticle has low cytotoxicity, can enter cells through endocytosis after being incubated with T lymphocytes from peripheral blood for 30 min, and realizes rapid and efficient gene delivery.

Description

Technical Field

[0001] This invention relates to the field of gene-engineered drugs and vaccine manufacturing technology, specifically to a polypeptide-modified cationic polymer nanoparticle, its preparation method, and its application. Background Technology

[0002] Chimeric antigen receptor T cells (Chimeric antigen receptor T cells) can be used in adoptive cell immunotherapy with durable efficacy. However, existing Chimeric antigen receptor T cell protocols require in vitro T cell activation, viral vector transfection, and an expansion process that takes 2-3 weeks, resulting in high costs and a high risk of T cell exhaustion and differentiation. For patients with rapidly progressing hematologic malignancies or solid tumors, the rapid preparation of suitable Chimeric antigen receptor T cells can significantly improve survival rates. Therefore, developing a gene delivery system capable of transfecting inactive T cells, bypassing the in vitro activation step, and shortening the expansion time is of paramount importance.

[0003] Poly(β-amino esters) (PBAEs), as non-viral carriers, electrostatically assemble nucleic acid nanoparticles and enter inactive T cells via endocytosis. Protonation in the acidic endosome environment allows for lysosomal escape, theoretically providing a possibility for direct transfection of resting T cells to prepare CAR-T cells. However, three problems limit the application of PBAEs in practice: First, the cytoplasmic DNA of PBAE polymers as carriers is difficult to efficiently enter the cell nucleus, currently relying mainly on nuclear-targeting peptide (NLS) conjugation; second, the high positive charge of PBAE polymers is difficult to balance with cytotoxicity; and finally, existing PBAE polymers also suffer from insufficient cell transmembrane efficiency and low endosome escape rate, affecting the basic delivery efficiency of inactive T cells.

[0004] To improve transmembrane efficiency, nuclear localization ability, and endosome escape ability, measures such as optimizing PBAE structure and regulating pH responsiveness can be taken. However, strategies based on optimizing the material's inherent properties have significant limitations in terms of efficiency improvement. Therefore, new approaches are needed to enhance the transmembrane efficiency of PBAE structures. In principle, introducing exogenous active peptides with different functions can enhance the transmembrane efficiency of PBAE structures. However, due to the limitations of the biochemical and biophysical properties of the exogenous active peptides themselves, direct introduction also faces application bottlenecks. For example, coupled transmembrane peptides (such as HIV-TAt) often cause nanoparticle aggregation due to the high positive charge of TAT, leading to inactivation by protein corona encapsulation in serum; while fusion peptides (such as HA2-TAt) may disrupt the active conformation of HA2 through chemical coupling, and the high density of positive charge may cause membrane damage. Although these exogenous peptide modifications can improve efficiency in localized areas, their individual application bottlenecks and the unpredictable synergistic toxicity risks that may arise in combined applications severely limit their practical application potential.

[0005] Therefore, there is an urgent need for PBAE nanoparticles with excellent gene delivery capabilities to achieve a synergistic increase in membrane penetration efficiency, escape efficiency, and nuclear localization efficiency, without a significant decrease in cell viability, thereby greatly shortening the preparation cycle of CAR-T cells and significantly reducing production costs. Summary of the Invention

[0006] To address the aforementioned issues, the present invention aims to provide a polypeptide-modified cationic polymer nanoparticle, its preparation method, and its applications. Based on the core molecular mechanisms of nanoparticle transmembrane penetration, endosome escape, and nuclear localization, the invention innovatively introduces three types of bioactive substances simultaneously: transmembrane peptides, accessory peptides, and nuclear localization peptides, directly intervening in and enhancing the efficiency of these three key delivery steps.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] A peptide-modified cationic polymer nanoparticle includes cationic polymer nanoparticle PBAE-536, plasmid DNA tightly entangled with PBAE-536 by electrostatic binding; cell-penetrating peptide HIV-TAt indirectly coupled to the surface of PBAE-536, helper peptide HA2-TAt mixed with PBAE-536, and a short peptide containing microtubule-associated sequence MTAS and nuclear localization signal NLS coupled to the PBAE-536 vector.

[0009] The cationic polymer nanoparticles PBAE-536 were obtained by polymerization of 1,5-pentanediol diacrylate and 3-amino-1-propanol, followed by end-capping with 2-(3-aminopropylamino)ethanol.

[0010] The plasmid DNA comprises two plasmids: plasmid 1 expresses PiggyBac transposase; plasmid 2 is driven by the hPGK promoter to express the target gene; plasmid 2 is obtained by linking the CD19 target gene with hPGK and then inserting it into the EcoRI / NotI site of the PiggyBac-CMV-MCS-Fluc-EF1a-CopGFP-T2A-Puro plasmid.

[0011] The short peptide sequences of the microtubule-associated sequence MTAS and the nuclear localization signal NLS are SEQ ID NO.1.

[0012] This invention also includes a method for preparing peptide-modified cationic polymer nanoparticles, comprising the following steps:

[0013] S1. Preparation of PBAE-536 polymer carrier material;

[0014] S2. Couple the cell-penetrating peptide HIV-TAt with polyglutamate PGA to obtain PGA-HIV-TAt for later use;

[0015] S3. Add a short peptide containing microtubule-associated sequence MTAS and nuclear localization signal NLS to the PBAE-536 polymer carrier material to obtain PBAE-536-NLS-MTAS.

[0016] S4. Using two-phase microfluidic technology, PBAE-536-NLS-MTAS obtained in step S3 and plasmid DNA are tightly wrapped together, and after dialysis, PBAE-536-NLS-MTAS-DNA nanoparticles are obtained.

[0017] S5. Add the PGA-HIV-TAt obtained in step S2 to the PBAE-536-NLS-MTAS-DNA nanoparticles obtained in step S4, and then add the auxiliary peptide HA2-TAt to obtain peptide-modified cationic polymer nanoparticles.

[0018] Preferably, step S1, which prepares the PBAE-536 polymer carrier material, includes the following steps:

[0019] 1,5-Pentanediol diacrylate and 3-amino-1-propanol were mixed and stirred at 85-95°C for 23-25 ​​hours to obtain an acrylate-terminated polymer. This polymer was dissolved in tetrahydrofuran and then added to a mixture of 2-(3-aminopropylamino)ethanol and tetrahydrofuran. The end-capping reaction was stirred for 2-3 hours to obtain a crude PBAE-536 polymer product. After washing and drying, PBAE-536 polymer was obtained. The obtained PBAE-536 polymer was dissolved in dimethyl sulfoxide for later use.

[0020] Preferably, step S2 involves conjugating the cell-penetrating peptide HIV-TAt with polyglutamate (PGA) to obtain PGA-HIV-TAt, specifically including the following steps:

[0021] Polyglutamic acid was dissolved in water, and N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide·HCl was added. The mixture was vortexed for 10-15 seconds, allowed to stand for 5-10 minutes, and then mixed with the cell-penetrating peptide HIV-TAt. The mixture was allowed to stand at 20-30°C for 4-6 hours, and the solvent was removed by centrifugation to obtain PGA-HIV-TAt.

[0022] Preferably, step S3 specifically includes the following steps:

[0023] First, PBAE-536 was reacted with N-(p-maleimide phenyl) isocyanate at 20-30°C for 2-4 hours to obtain the activated PBAE536-maleimide derivative. This was then mixed with a short peptide containing microtubule-associated sequence (MTAS) and nuclear localization signal (NLS) and reacted for 2-4 hours. After filtration, PBAE-536-NLS-MTAS was obtained.

[0024] Preferably, in step S4, the mass ratio of PBAE-536-NLS-MTAS obtained in step S3 to plasmid DNA is 30~90:1. During dialysis, ammonia water is used for dialysis for 45 minutes to 75 minutes, followed by dialysis with PBS solution for 45 minutes to 75 minutes. The ammonia water is obtained by diluting 10 ml of concentrated ammonia water with 480~520 ml of water.

[0025] Preferably, the mass-to-volume ratio of 1,5-pentanediol diacrylate to 3-amino-1-propanol is 78-80:212;

[0026] The 2-(3-aminopropylamino)ethanol-tetrahydrofuran mixture is composed of 2-(3-aminopropylamino)ethanol and tetrahydrofuran at a mass-volume ratio of 30~35mg:13~15ml;

[0027] The mass ratio of 2-(3-aminopropylamino)ethanol in the mixture of 3-amino-1-propanol and 2-(3-aminopropylamino)ethanol-tetrahydrofuran is 212:2.77~2.80.

[0028] The present invention also includes the use of peptide-modified cationic polymer nanoparticles in the preparation of CAR-T cell, CAR-NK cell or CAR-γδT cell drugs.

[0029] The present invention has the following advantages over the prior art:

[0030] The peptide-modified cationic polymer nanoparticles of this invention utilize a cell-penetrating peptide HIV-TAt, a helper peptide HA2-TAt, and a short peptide containing microtubule-associated sequences (MTAS) and nuclear localization signals (NLS) to co-modify PBAE-536 nanoparticles. This allows for rapid in vitro transfection of inactive T cells, NK cells, macrophages, and other immune cells. The transfected immune cells function normally and kill specific tumor cells. The peptide-modified cationic polymer nanoparticles of this invention exhibit low cytotoxicity. The peptide-modified nanoparticle carrier improves cell transfection efficiency through cell membrane penetration, endosome escape, and nuclear localization. After incubation with peripheral blood T lymphocytes for 30 minutes, the nanoparticles enter the cells via endocytosis, achieving rapid and efficient gene delivery.

[0031] The peptide-modified cationic polymer nanoparticles of the present invention, wherein the HIV-TAt membrane-penetrating peptide can improve the membrane-penetrating efficiency of the nanoparticles, are first coupled with the anionic polymer PGA to obtain a negatively charged polymer, and then electrostatically bound to PBAE nanoparticles. This can effectively avoid the particle aggregation problem caused by the positive charge of TAT, and at the same time, it can be adsorbed onto the surface of PBAE nanoparticles through electrostatic interaction, shielding the excessively high positive charge on the PBAE surface and reducing cytotoxicity. It can also induce the particles to quickly cross the cell membrane and enter the cell through the TAT portion.

[0032] The peptide-modified cationic polymer nanoparticles of the present invention incorporate a short peptide containing the microtubule-associated sequence MTAS and the nuclear localization signal NLS, thereby enabling DNA to rapidly reach the cell nucleus via the microtubule transport mechanism and improving gene transduction efficiency. The helper peptide HA2-TAt is directly mixed with the nanoparticles, avoiding the problem of HA2 conformational inactivation caused by coupling, thus enabling the nanoparticles to rapidly achieve endosome escape. Plasmid DNA encoding hematologic malignancy-specific CD19-CAR is mounted on the nanoparticles, thereby giving the delivered nanoparticles anti-tumor programming capabilities. Attached Figure Description

[0033] Figure 1 Synthesis route diagram for PBAE-536 polymer carrier material;

[0034] Figure 2 The effect of the mixing ratio of PBAE-536 and DNA and dialysis conditions on the efficiency of transfection of 293T cells with non-peptide-modified DNA nanoparticles is shown in the figure.

[0035] Figure 3 Figure 1 shows the effect of the mixing ratio of PBAE-536 and DNA and dialysis conditions on the transfection efficiency of non-peptide-modified DNA nanoparticles into Jurkat cells.

[0036] Figure 4 The image shows the gel electrophoresis results of nanoparticles containing PBAE-536 mixed with DNA at different mass ratios and dialyzed in ammonia and PBS.

[0037] Figure 5 A schematic diagram illustrating the effect of different DNA doses and transfection times on the transfection efficiency of non-activated T cells.

[0038] Figure 6 A schematic diagram illustrating the effect of different DNA doses on the transfection efficiency of MTAS-NLS-modified nanoparticles on non-activated T cells.

[0039] Figure 7A schematic diagram illustrating the effect of MTAS-NLS-modified nanoparticles on the transfection efficiency of non-activated T cells at different transfection times.

[0040] Figure 8 A schematic diagram illustrating the effect of nanoparticles co-modified with cell-penetrating peptide HIV-TAt and nuclear localization peptide MTAS-NLS on the transfection efficiency of non-activated T cells at different mixing ratios of HIV-TAt and DNA.

[0041] Figure 9 A schematic diagram illustrating the effect of different HA2-TAt concentrations on the transfection efficiency of nanoparticles co-modified with the releasing peptide HA2-TAt and the nuclear localization peptide MTAS-NLS on non-activated T cells.

[0042] Figure 10 A schematic diagram illustrating the time-dynamic effects of transfection of non-activated T cells with nanoparticles co-modified by HIV-TAt, HA2-TAt, and MTAS-NLS.

[0043] Figure 11 Representative flow cytometry results at different times;

[0044] Figure 12 A schematic diagram illustrating the effects of PBAE-536 and its peptides on cell viability;

[0045] Figure 13 This diagram illustrates the effect of non-activated T cells and Raij lymphoma cells transfected with different proportions of nanoparticles on tumor killing. Detailed Implementation

[0046] The purpose of this invention is to provide a polypeptide-modified cationic polymer nanoparticle, its preparation method, and its application. The invention will be further described below with reference to specific embodiments.

[0047] Plasmid 1 was provided by Wuhan Miaoling Biotechnology Co., Ltd.

[0048] The PiggyBac-CMV-MCS-Fluc-EF1a-CopGFP-T2A-Puro (P27701) plasmid was provided by Wuhan Miaoling Biotechnology Co., Ltd.

[0049] Construction method of plasmid 2: The synthetic gene (hPGK-CAR) was inserted into the EcoRI / NotI region of the PiggyBac-CMV-MCS-Fluc-EF1a-CopGFP-T2A-Puro (P27701) plasmid using conventional methods. The constructed plasmid was named PiggyBac-cmv-PGK-BBZ19-CopEGFP-Puro. The CAR gene is a commonly used CAR structure. Taking the CD19-targeting CAR (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".

[0050] The cell-penetrating peptide HIV-TAt was provided by Nanjing Genscript Biotech Co., Ltd.

[0051] The helper peptide HA2-TAt was provided by Nanjing Genscript Biotech Co., Ltd.

[0052] The short peptide containing the microtubule-associated sequence MTAS and the nuclear localization signal NLS was provided by Apson;

[0053] The short peptide sequences of the microtubule-associated sequence MTAS and the nuclear localization signal NLS are SEQ ID NO.1:

[0054] GRYLTQETNKVETYKEQPLKTPGKKKKGKPGKRKEQEKKKRRTR. Example 1

[0055] The preparation method and synthetic route of PBAE-536 polymer carrier material are as follows: Figure 1 As shown, it includes the following steps:

[0056] 212 g of 1,5-pentanediol diacrylate and 78 g of 3-amino-1-propanol were mixed and stirred at 85 °C for 25 hours to obtain an acrylate-terminated polymer. This polymer was dissolved in 25 ml of tetrahydrofuran and then added to a mixture of 2-(3-aminopropylamino)ethanol and tetrahydrofuran. The end-capping reaction was carried out for 2 hours to obtain a crude PBAE-536 polymer product. After washing and drying, PBAE-536 polymer was obtained. The obtained PBAE-536 polymer was dissolved in dimethyl sulfoxide to a concentration of 100 mg / ml. The solution was dispensed into small tubes and frozen at -20 °C to avoid repeated freeze-thaw cycles for later use.

[0057] The 2-(3-aminopropylamino)ethanol-tetrahydrofuran mixture consists of 2.77 g of 2-(3-aminopropylamino)ethanol and 1.2 L of tetrahydrofuran. Example 2

[0058] The preparation method and design route of PBAE-536 polymer carrier material are as follows: Figure 1 As shown, it includes the following steps:

[0059] 212 g of 1,5-pentanediol diacrylate and 80 g of 3-amino-1-propanol were mixed and stirred at 95 °C for 23 hours to obtain an acrylate-terminated polymer. This polymer was dissolved in 30 ml of tetrahydrofuran and then added to a mixture of 2-(3-aminopropylamino)ethanol and tetrahydrofuran. The end-capping reaction was carried out for 3 hours to obtain a crude PBAE-536 polymer product. After washing and drying, PBAE-536 polymer was obtained. The obtained PBAE-536 polymer was dissolved in dimethyl sulfoxide to a concentration of 100 mg / ml. The solution was dispensed into small tubes and frozen at -20 °C to avoid repeated freeze-thaw cycles for later use.

[0060] The 2-(3-aminopropylamino)ethanol-tetrahydrofuran mixture consists of 2.80 g of 2-(3-aminopropylamino)ethanol and 1.2 L of tetrahydrofuran. Example 3

[0061] The preparation method and design route of PBAE-536 polymer carrier material are as follows: Figure 1 As shown, it includes the following steps:

[0062] 6.58 g of 1,5-pentanediol diacrylate was added to 2.56 g of 3-amino-1-propanol. The two monomers were mixed in a round-bottom flask and stirred at 90 °C for 24 h to synthesize the acrylate-terminated base polymer. The reacted polymer was dissolved in 8 mL of tetrahydrofuran and added to 16 mL of tetrahydrofuran containing 1.43 g of 2-(3-aminopropylamino)ethanol. The mixture was stirred at 25 °C for 2 h to achieve end-capping. The final PBAE-536 polymer was dissolved in dimethyl sulfoxide for washing, allowed to stand for 0.5 h to remove unreacted monomer molecules, and the polymer was collected. The polymer was purified by washing with dimethyl sulfoxide twice. After purification, the collected polymer was vacuum dried at room temperature for 48 h. The obtained PBAE-536 polymer was then dissolved in dimethyl sulfoxide to a final concentration of 100 mg / mL. The polymer was aliquoted into small tubes and frozen at -20 °C to avoid repeated freeze-thaw cycles. Example 4

[0063] A method for preparing peptide-modified cationic polymer nanoparticles includes the following steps:

[0064] S1. Preparation of PBAE-536 polymer carrier material. In this example, the PBAE-536 polymer carrier material obtained in Example 3 is used.

[0065] S2. Couple the cell-penetrating peptide HIV-TAt with polyglutamic acid to obtain PGA-HIV-TAt, for later use;

[0066] The cationic transmembrane peptide HIV-TAt was coupled with negatively charged PGA to reduce its high positive charge. The specific procedure was as follows: Equal volumes of polyglutamic acid (PGA) and N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide·HCl (EDC•HCl) were mixed at room temperature for 5 min. The initial PGA concentration was 20 mg / ml. The mixture was placed on a magnetic stirrer and stirred continuously at 500 rpm until it was completely dissolved and formed a homogeneous and transparent solution. The initial EDC•HCl concentration was 4 mg / ml. After mixing with PGA, the mixture was vortexed for 10-15 seconds and reacted at room temperature for 5 min. The carboxyl group of PGA was activated by EDC to form an O-acylisourea intermediate, which was then immediately mixed with HIV-TAt at a 1:1 molar ratio and reacted at room temperature for 5 h. The mixture was then centrifuged through a 50 K MWCO ultrafiltration centrifuge tube to remove excess reagents.

[0067] S3. Add a short peptide containing microtubule-associated sequence MTAS and nuclear localization signal NLS to the PBAE-536 polymer carrier material to obtain PBAE-536-NLS-MTAS.

[0068] 12 mg of N-(p-maleimide-phenyl)-isocyanate (PMPI) was dissolved in 0.6 mL of DMSO and then added to 86 mg of PBAE-536 (100 mg / mL). After shaking at room temperature for 3 h, the 536-maleimide derivative was added to a 5.3 mL DMSO solution containing 100 mg of MTAS-NLS, with tris(2-carbonylethyl)phosphohydrochloride (TCEP•HCl; 15.9 mg) added. After shaking and mixing at room temperature for 3 h, desalting was performed using a 7 kWh MWCO column. The coupled 536-NLS-MTAS was redissolved in DMSO to a concentration of 100 mg / mL and stored at -20 °C.

[0069] S4. Using two-phase microfluidic technology, PBAE-536-NLS-MTAS obtained in step S3 and plasmid DNA are tightly wrapped together, and after dialysis, PBAE-536-NLS-MTAS-DNA nanoparticles are obtained.

[0070] The nanoparticles were prepared using a two-phase microfluidic strategy. The organic phase was a mixture of ethanol and DMSO at a volume ratio of 1.5:1; the acidic aqueous phase was a sodium acetate buffer solution with a pH of 5.0. 536-MTAS-NLS was dissolved in the organic phase. Plasmid 1 and plasmid 2 were mixed at a mass ratio of 1:3 to obtain plasmid DNA, which was then dissolved in the aqueous phase at PBAE:DNA ratios of 30:1, 60:1, or 90:1 (w / w), respectively. The two phases were mixed at a volume ratio of 1:2 using a microfluidic device. In this process, the DNA molecules in the nanoparticles formed were tightly entangled with PBAE, unlike in traditional methods where DNA molecules are adsorbed onto the surface of PBAE particles. The prepared nanoparticles were dialyzed in PBS for 2 hours, or in ammonia solution (pH 10) and PBS for 1 hour each.

[0071] S5. Add the PGA-HIV-TAt obtained in step S2 to the PBAE-536-NLS-MTAS-DNA nanoparticles obtained in step S4, and then add the auxiliary peptide HA2-TAt to obtain peptide-modified cationic polymer nanoparticles.

[0072] The prepared PGA-HIV-TAt was added to the dialyzed nanoparticles at HIV-TAt:DNA mass ratios of 0.25:1, 0.5:1, 0.75:1, 1:1, and 1.5:1. The mixture was vortexed for 10 s and then incubated at room temperature for 5 min. Then, the peptide-based delivery agent HA2-TAt was added directly. 1 mM HA2-TAt, frozen at -80 ℃, was diluted to 15 μM, 25 μM, and 35 μM in PBS, and added to the nanoparticle solution at a volume ratio of 1:3 (prepared fresh for each use).

[0073] Experiment 1: Screening of the mixing ratio of PBAE and plasmid DNA and dialysis conditions;

[0074] Non-peptide-modified DNA nanoparticles were transfected into 293T and Jurkat cells;

[0075] PBAE-536 and DNA were mixed via a microfluidic device at ratios of 30:1, 60:1, and 90:1 (w / w). One group was dialyzed twice in 500 ml PBS for 1 h each time, while the other group was dialyzed for 1 h each in ammonia water and PBS at pH 10. The process of transfecting 293T cells and Jurkat cells with DNA nanoparticles was described; 293T cells were transfected at 5.0 × 10⁻⁶ ml. 4 / well, Jurkat cells 2.5 × 10⁶ 4The nanoparticles were seeded in 96-well plates and incubated overnight for 24 h. Nanoparticles containing 1.0 μg and 1.5 μg of DNA, after dialyzing, were added to 293T cells and Jurkat cells, respectively, mixed, and incubated at 37°C with 5% CO2 for 48 h. The fluorescence of the nanoparticles was detected using flow cytometry, and the data were acquired, analyzed, and used to obtain flow cytometry plots. Figure 2 and Figure 3 The results showed that the transfection rates of nanoparticles dialyzed in PBS were significantly lower for 293T and Jurkat cells than those dialyzed in ammonia and PBS, with the highest efficiency observed at a PBAE:DNA ratio of 60:1. Among these, for example... Figure 2 As shown, nanoparticles can achieve high transfection efficiency in 293T cells at a mass ratio of 30, while Jurkat requires a mass ratio of 60 to achieve good transfection results (e.g., ...). Figure 3 (As shown). Gel electrophoresis experiments revealed, as... Figure 4 As shown, when the mass ratio was 30, free DNA bands were observed, indicating that PBAE could not completely encapsulate the plasmid DNA at this ratio. As the mass ratio increased, the DNA bands disappeared, indicating that the DNA was completely compressed. Therefore, we chose a dialysis method of 1 hour of ammonia followed by 1 hour of PBS, with a PBAE to plasmid DNA mass ratio of 60:1.

[0076] Experiment 2: Investigating the transfection efficiency of non-peptide-modified DNA nanoparticles on inactive T cells;

[0077] Peripheral blood was collected from healthy individuals, and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation. High-purity T cell populations were isolated from PBMCs through positive sorting and using the T cell-specific surface marker (CD3). The T cells were kept in an inactive state for subsequent transfection analysis. The inactive T cells were then centrifuged at 3 × 10⁻⁶ cells / day. 5 The cells were seeded in 96-well plates, and nanoparticles with different DNA concentrations were immediately added. One group was incubated for 2 hours, 4 hours, and 6 hours, respectively, followed by thorough washing and replacement with fresh culture medium. Another group was incubated directly without medium change. After 48 hours, the transfection efficiency was measured by flow cytometry. Figure 5 As shown, non-peptide-modified nanoparticles exhibit low transfection efficiency in non-activated T cells, and increasing the transfection dose and incubation time does not significantly improve transfection efficiency. The transfection results indicate that, at the same incubation time, further increasing the DNA transfection dose of 1.5 μg does not improve transfection efficiency. At the same transfection dose, efficiency reaches a steady state after 4 hours of incubation.

[0078] Experiment 3: Investigating the transfection efficiency of nanoparticles modified with different peptides on non-activated T cells;

[0079] (1) Transfection of non-activated T cells with nanoparticles modified with microtubule-associated sequences and nuclear localization signal peptide MTAS-NLS;

[0080] Freshly isolated non-activated T cells were used at 3 × 10⁻⁶ 5 Cells were seeded in 96-well plates at doses of 0.5, 1.0, 1.5, and 2.0 μg DNA per well. After incubation for 4 hours, cells were thoroughly washed and replaced with fresh culture medium for further culture. Transfection efficiency at different concentrations was analyzed by flow cytometry after 48 hours. Figure 6 As shown, MTAS-NLS modification significantly improved the transfection efficiency of nanoparticles on inactive T cells, and the efficiency increased significantly with increasing transfection dose, reaching a stable level at a DNA dose of 1.5 μg. When the DNA dose was fixed at 1.5 μg, the incubation time between the nanoparticles and T cells was varied, as shown... Figure 7 As shown, the transfection efficiency tends to stabilize when the incubation time is more than 3 hours.

[0081] (2) Transfection of non-activated T cells by nanoparticles co-modified with cell-penetrating peptide HIV-TAt and nuclear localization peptide MTAS-NLS;

[0082] The prepared PGA-HIV-TAt was added to dialysis-treated MTAS-NLS-modified nanoparticles at HIV-TAt:DNA mass ratios of 0.25:1, 0.5:1, 0.75:1, 1:1, and 1.5:1. The mixture was vortexed for 10 s and then incubated at room temperature for 5 min. Freshly isolated inactive T cells were cultured at 3 × 10⁻⁶ cells / mL. 5 Cells were seeded in 96-well plates at a dose of 1.5 μg DNA per well, incubated for 0.5 h, washed thoroughly, and cultured in fresh medium. Figure 8 As shown, co-modification with HIV-TAt and MTAS-NLS significantly reduced the transfection time of nanoparticles to non-activated T cells, and the transfection efficiency tended to stabilize when the HIV-TAt:DNA mass ratio was 0.75:1. This transfection efficiency was close to that of nanoparticles modified with MTAS-NLS alone at 4 h.

[0083] (3) Transfection of non-activated T cells by nanoparticles co-modified with releasing peptide HA2-TAt and nuclear localization peptide MTAS-NLS;

[0084] Freshly isolated non-activated T cells were used at 3 × 10⁻⁶ 5 The nanoparticles, containing 1.5 μg of DNA and modified with nuclear-targeting peptides, were seeded into each well of a 96-well plate. Immediately afterward, HA2-TAt solutions diluted to different concentrations were added. After incubation for 0.5 h, the nanoparticles were thoroughly washed and replaced with fresh culture medium for further culturing. Transfection efficiency at different quality levels was analyzed by flow cytometry after 48 h. Figure 9 As shown, compared with nanoparticles modified with the single nuclear localization peptide MTAS-NLS, when the HA2-TAt concentration is 25 μM, the transfection efficiency can be increased by about 5 times with a DNA dose of 1.5 μg and an incubation time of 0.5 h.

[0085] The above experiments demonstrate that the nuclear localization peptide MTAS-NLS significantly improves the transfection efficiency of nanoparticles on inactive T cells. However, this modification has certain limitations: at a DNA dose of 1.5 μg, the transfection efficiency only increases to over 20% after incubation of MTAS-NLS-modified nanoparticles with inactive T cells for more than 3 hours. The experiments also show that synergistic modification can significantly shorten the incubation time of nanoparticles on inactive T cells. Adding an appropriate proportion of the cell-penetrating peptide HIV-TAt to the MTAS-NLS modification reduces the transfection efficiency to the higher 3-6 hours achieved by MTAS-NLS alone after only 0.5 hours of incubation. Adding an appropriate proportion of the releasing peptide HA2-TAt to the MTAS-NLS modification, while not as significant as HIV-TAt, still increases the transfection efficiency by approximately 5-fold with a 0.5-hour incubation time.

[0086] Experiment 4: Investigating the transfection efficiency of DNA nanoparticles co-modified with peptides on inactive T cells;

[0087] Non-activated T cells were transfected with nanoparticles co-modified with three peptides.

[0088] The prepared PGA-HIV-TAt was added to dialysis-modified MTAS-NLS nanoparticles at an HIV-TAt:DNA mass ratio of 0.75:1. The mixture was vortexed for 10 s and then incubated at room temperature for 5 min. Freshly isolated inactive T cells were cultured at 3 × 10⁻⁶ cells / mL. 5 Cells were seeded in 96-well plates at a dose of 1.5 μg DNA per well, and immediately incubated with 25 μM HA2-TAt solution for 0.5 h. After incubation, the cells were thoroughly washed and replaced with fresh culture medium for further culture. Transfection efficiency at different time points was analyzed by flow cytometry. Figure 10 As shown, within an incubation period of 0.5 h, the co-modification of MTAS-NLS with HIV-TAt and HA2-TAt significantly improved the transfection efficiency of nanoparticles on non-activated T cells, and the transfection efficiency remained stable for a certain period of time. Figure 11 These are several representative flow cytometry results. Cell viability was immediately determined using AOPI staining after washing; the control group consisted of untransfected T cells. Results are as follows: Figure 12 As shown, although the addition of PBAE-536 and the peptide reduced cell viability, this reduction was considered acceptable compared to the significant increase in transfection efficiency.

[0089] Non-activated T cells transfected with nanoparticles were co-cultured with Raij lymphoma cells at cell ratios of 1:1, 3:1, and 5:1 at 37°C and 5% CO2. Tumor cell apoptosis was detected by ELISA after 5 days. Figure 13 As shown, T cells transfected with nanoparticles co-modified with peptides exhibited significant tumor-killing effects.

Claims

1. A method for preparing polypeptide-modified cationic polymer nanoparticles, characterized in that: Includes the following steps: S1. Preparation of PBAE-536 polymer carrier material; 1,5-pentanediol diacrylate and 3-amino-1-propanol were mixed and stirred at 85-95°C for 23-25 ​​hours to obtain an acrylate-terminated polymer. This polymer was dissolved in tetrahydrofuran and then added to a mixture of 2-(3-aminopropylamino)ethanol and tetrahydrofuran. The end-capping reaction was stirred for 2-3 hours to obtain a crude PBAE-536 polymer. After washing and drying, PBAE-536 polymer was obtained. The obtained PBAE-536 polymer was dissolved in dimethyl sulfoxide for later use. S2. Couple the cell-penetrating peptide HIV-TAt with polyglutamate PGA to obtain PGA-HIV-TAt for later use; S3. Add a short peptide containing microtubule-associated sequence MTAS and nuclear localization signal NLS to the PBAE-536 polymer carrier material to obtain PBAE-536-NLS-MTAS. S4. Using two-phase microfluidic technology, PBAE-536-NLS-MTAS obtained in step S3 and plasmid DNA are tightly wrapped together, and after dialysis, PBAE-536-NLS-MTAS-DNA nanoparticles are obtained. The mass ratio of PBAE-536-NLS-MTAS and plasmid DNA obtained in step S3 is 30~90:

1. During dialysis, ammonia water is used for 60 minutes, followed by PBS solution for 60 minutes. S5. Add the PGA-HIV-TAt obtained in step S2 to the PBAE-536-NLS-MTAS-DNA nanoparticles obtained in step S4, and then add the auxiliary peptide HA2-TAt to obtain peptide-modified cationic polymer nanoparticles. The MTAS and NLS short peptide sequences are SEQ ID NO.1: GRYLTQETNKVETYKEQPLKTPGKKKKGKPGKRKEQEKKKRRTR.

2. The method for preparing polypeptide-modified cationic polymer nanoparticles according to claim 1, characterized in that: Step S2 involves conjugating the cell-penetrating peptide HIV-TAt with polyglutamate (PGA) to obtain PGA-HIV-TAt, specifically including the following steps: Polyglutamic acid was dissolved in water, and N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide·HCl was added. The mixture was vortexed for 10-15 seconds, allowed to stand for 5-10 minutes, and then mixed with the cell-penetrating peptide HIV-TAt. The mixture was allowed to stand at 20-30°C for 4-6 hours, and the solvent was removed by centrifugation to obtain PGA-HIV-TAt.

3. The method for preparing polypeptide-modified cationic polymer nanoparticles according to claim 1, characterized in that: Step S3 specifically includes the following steps: First, PBAE-536 was reacted with N-(p-maleimide phenyl) isocyanate at 20-30°C for 2-4 hours to obtain the activated PBAE536-maleimide derivative. This was then mixed with a short peptide containing microtubule-associated sequence (MTAS) and nuclear localization signal (NLS) and reacted for 2-4 hours. After filtration, PBAE-536-NLS-MTAS was obtained.

4. A polypeptide-modified cationic polymer nanoparticle obtained by any of the preparation methods of claims 1 to 3, characterized in that: Includes cationic polymer nanoparticles PBAE-536, plasmid DNA tightly entangled with PBAE-536 by electrostatic binding; cell-penetrating peptide HIV-TAt indirectly coupled to the surface of PBAE-536, helper peptide HA2-TAt mixed with PBAE-536, and short peptides containing microtubule-associated sequence MTAS and nuclear localization signal NLS coupled to the PBAE-536 vector. The cationic polymer nanoparticles PBAE-536 were obtained by polymerization of 1,5-pentanediol diacrylate and 3-amino-1-propanol, followed by end-capping with 2-(3-aminopropylamino)ethanol. The plasmid DNA comprises two plasmids: plasmid 1 expresses PiggyBac transposase; plasmid 2 is driven by the hPGK promoter to express the target gene; plasmid 2 is obtained by linking the CD19 target gene with hPGK and then inserting it into the EcoRI / NotI region of the PiggyBac-CMV-MCS-Fluc-EF1a-CopGFP-T2A-Puro plasmid.

5. The application of the polypeptide-modified cationic polymer nanoparticles according to claim 4, characterized in that: Application in the preparation of CAR-T cell drugs.

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