Perfluorinated modified PEI carrier, carrier / siRNA compound and preparation method thereof

Abstract

The invention discloses a perfluorinated modified PEI carrier, a carrier / siRNA compound and a preparation method of the carrier / siRNA compound, and belongs to the technical field of gene delivery systems. The polyethyleneimine carrier is prepared from PF-DBCO (Polypropylene Factor-Diphenyl Carbonate) and PF-N3; wherein the PF-DBCO has a structure as shown in a formula I, and the PF-N3 has a structure as shown in a formula II; formula I; formula II; through combination of perfluorinated modification, click crosslinking and disulfide bond controllable release, the problems that an existing siRNA carrier is insufficient in stability, uncontrollable in release, high in cytotoxicity and the like are solved, and a novel siRNA delivery system with the triple advantages of efficient delivery, stable protection and accurate release is constructed.

Description

Technical Field

[0001] This invention belongs to the field of gene delivery system technology, and particularly relates to a perfluorinated modified PEI vector, a vector / siRNA complex, and a method for preparing the same. Background Technology

[0002] Small interfering RNA (siRNA) is a highly specific gene silencing molecule that can regulate disease-related genes by binding to specific mRNA sequences and blocking their translation. Therefore, siRNA shows great promise for applications in gene therapy, antiviral therapy, and tumor treatment. However, significant obstacles exist in the in vivo application of siRNA. Due to its large molecular weight and strong negative charge, siRNA is easily degraded by nucleases in the bloodstream and has difficulty effectively crossing the cell membrane into the cytoplasm, resulting in low delivery efficiency. Therefore, improving its transmembrane delivery and cytoplasmic release efficiency while protecting its stability is a pressing issue that needs to be addressed.

[0003] To this end, researchers have developed a variety of delivery systems. Among them, polycationic macromolecular carriers (such as polyethyleneimine, PEI) can form stable complexes with siRNA due to their positive charge, thereby protecting siRNA to a certain extent and enabling cellular uptake.

[0004] However, traditional PEI vectors suffer from several drawbacks, including high toxicity, limited delivery efficiency (siRNA delivery primarily relies on endocytosis for entry into cells, resulting in insufficient endosome escape efficiency and low cytoplasmic release efficiency), insufficient complex stability, and difficulty in achieving precise release at specific intracellular locations. These issues severely restrict their clinical application. Therefore, structural optimization of PEI is urgently needed to simultaneously improve delivery efficiency, reduce toxicity, and achieve effective regulation of siRNA release. Summary of the Invention

[0005] The perfluorinated PEI delivery system proposed in this invention combines a direct transmembrane delivery mechanism with a controllable cross-linking release strategy, which can effectively solve the shortcomings of existing vectors in terms of stability, delivery efficiency and toxicity control, and provides a new solution for the efficient delivery of siRNA and the clinical translation of gene therapy.

[0006] This invention proposes a perfluorinated modified polyethyleneimine support, comprising PF-DBCO and PF-N3; wherein PF-DBCO has the structure shown in Formula I, and PF-N3 has the structure shown in Formula II; Formula I; Formula II.

[0007] Furthermore, the polyethyleneimine is a branched polyethyleneimine, and the weight-average molecular weight of the branched polyethyleneimine is 2000-40000 Daltons; Preferably, the branched polyethyleneimine has a weight-average molecular weight of 20,000-30,000 Daltons; More preferably, the branched polyethyleneimine has a weight-average molecular weight of 25,000 Daltons.

[0008] The present invention also proposes a method for preparing the perfluorinated modified polyethyleneimine carrier described above. (A) Methods for preparing PF-DBCO include: Dibenzocyclooctyne-disulfide-carboxylic acid was dissolved in N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added. The first reaction was carried out to obtain a reaction solution with carboxyl activated group. The above carboxyl-activated reaction solution was added to PF solution for the second reaction. After dialyzing, the resulting reaction solution was freeze-dried to obtain PF-DBCO. (B) The preparation methods of PF-N3 include: NHS-PEG4-N3 was added to the PF solution, and a third reaction was carried out. The resulting reaction solution was dialyzed and then freeze-dried to obtain PF-N3. PF has the structure shown in Formula III, and NHS-PEG4-N3 has the structure shown in Formula IV. Formula III; Formula IV. Furthermore, (A) satisfies at least one of the following conditions: (1) The first reaction is specifically: stirring at 25-30°C for 3-5 hours; (2) The second reaction is specifically carried out by stirring at 25-30°C for 12-24 hours; (3) The molar ratio of the dibenzocyclooctyn-disulfide-carboxylic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:(1.2-1.5):(1.2-1.5); (4) The ratio of dibenzocyclooctyne-disulfide-carboxylic acid to N,N-dimethylformamide is 35~50 µmol: 10mL; (5) The concentration of the PF solution is 4.0-5.0 mg / mL; (6) The molar ratio of the dibenzocyclooctyn-disulfide-carboxylic acid to PF is 4~10:1; And / or, said (B) satisfies at least one of the following conditions: (7) The third reaction is specifically: stirring at 25-30°C for 4-12 hours; (8) The molar ratio of NHS-PEG4-N3 to PF is 3~10:1.

[0009] Furthermore, the PF is prepared by the following steps: Branched polyethyleneimine was dissolved in methanol, pentafluoropropionic anhydride was added, and the reaction was carried out in the fourth step. The resulting reaction solution was dialyzed and then freeze-dried to obtain solid PF.

[0010] Furthermore, at least one of the following conditions must be met: (1) The fourth reaction is specifically: react at 25-30°C for 24-48 hours; (2) The molar ratio of the branched polyethyleneimine to pentafluoropropionic anhydride is 1:(80-160); (3) The concentration of the branched polyethyleneimine solution obtained by dissolving branched polyethyleneimine in methanol is 2-20 mg / mL. Furthermore, dialysis specifically involves transferring the obtained reaction solution into a dialysis bag for dialysis. Preferably, the molecular weight cutoff of the dialysis bag is 3000-5000 Da; More preferably, the dialysis is performed by first dialysis in an HCl solution and then dialysis in ultrapure water; More preferably, the concentration of the HCl solution is 0.01 mol / L.

[0011] The present invention also proposes a carrier / siRNA complex, which is obtained by combining a perfluorinated polyethyleneimine carrier with siRNA prepared by any of the above-mentioned perfluorinated polyethyleneimine carriers or by any of the above-mentioned preparation methods.

[0012] This invention also proposes a method for preparing any of the above-mentioned vector / siRNA complexes, comprising the following steps: S1. Dissolve the carrier PF-DBCO and the carrier PF-N3 in PBS buffer to obtain PF-DBCO solution and PF-N3 solution, respectively. S2. Mix the above PF-DBCO solution, the above PF-N3 solution and the siRNA solution and let stand to obtain the PFND / siRNA complex.

[0013] Furthermore, at least one of the following conditions must be met: (1) In S1, the concentrations of PF-DBCO solution and PF-N3 solution are both 0.1~0.5 mg / mL; (2) In S2, the concentration of siRNA is 10 pmol / uL; (3) In S2, the volume ratio of PF-DBCO solution, PF-N3 solution and siRNA solution is (1-2):(1-2):1; (4) In S2, the mixing and standing process is as follows: after mixing, let stand for 3-5 minutes.

[0014] This invention has the following advantages: This invention proposes a perfluorinated PEI vector, which, through a specific perfluorination modification, alters the physicochemical properties of the vector, allowing it to bypass the traditional endosome pathway and directly cross the cell membrane into the cytoplasm, thereby improving the cytoplasmic delivery efficiency and transfection effect of siRNA. Furthermore, a controllable cross-linking system is introduced, utilizing click chemistry between -DBCO and -N3 to achieve cross-linking, enhancing the stability of the vector / siRNA complex in the extracellular environment. Simultaneously, the disulfide bonds introduced in the cross-linking molecules can break under the reducing environment of the cytoplasm, triggering the targeted release of siRNA. In short, this invention, through the combination of "perfluorination modification + click cross-linking + controllable release via disulfide bonds," solves the problems of insufficient stability, uncontrollable release, and high cytotoxicity of existing siRNA vectors, constructing a novel siRNA delivery system with the triple advantages of high-efficiency delivery, stable protection, and precise release. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The 1H NMR spectrum of PF in Test Example 1 of this invention; Figure 2 The 1H NMR spectrum of PF-DBCO in Test Example 1 of this invention; Figure 3 The 1H NMR spectrum of PF-N3 in Test Example 1 of this invention; Figure 4 The particle size distribution of PFND / siRNA in Test Example 1 of this invention; Figure 5 This invention provides an example of testing the hemolysis of erythrocytes in the PFND / siRNA complex in Example 2. Figure 6 The cytotoxicity of the PFND / siRNA complex in Test Example 2 of this invention; Figure 7 The images show the CLSM and FCM quantification results of NCI-H460 cells taking up Cy5-siRNA in Test Example 3 of this invention. Figure 8The images show the CLSM and FCM quantification results of Hep-G2 cells taking up Cy5-siRNA in Test Example 3 of this invention. Figure 9 The images show the CLSM and FCM quantification results of MDA-MB-468 cells taking up Cy5-siRNA in Test Example 3 of this invention. Figure 10 The uptake efficiency of HeLa cells to PFND / siRNA or PFND / pDNA under 37°C, 4°C and NaN3 conditions in Test Example 4 of this invention; Figure 11 The test example 5 of this invention tested the level of GAPDH mRNA in NCI-H460 cells; Figure 12 The test example 5 of this invention tested the level of GAPDH mRNA in Hep-G2 cells; Figure 13 The test results were used to measure the level of GAPDH mRNA in MDA-MB-468 cells in Example 5 of this invention. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0018] In a first aspect, embodiments of the present invention provide a perfluorinated modified polyethyleneimine (PEI) support, comprising PF-DBCO and PF-N3; wherein PF-DBCO has the structure shown in Formula I, and PF-N3 has the structure shown in Formula II; Formula I; Formula II; In one embodiment of the present invention, the perfluorinated polyethyleneimine is branched polyethyleneimine (bPEI), and the weight-average molecular weight of the branched polyethyleneimine is 2000-40000 Daltons.

[0019] Preferably, the branched polyethyleneimine has a weight-average molecular weight of 20,000-30,000 Daltons. More preferably, the branched polyethyleneimine has a weight-average molecular weight of 25,000 Daltons.

[0020] This invention proposes to perfluorinate polyethyleneimine. Perfluorination gives the vector the ability to bypass the endosome and directly enter the cytoplasm without energy dependence, which is particularly suitable for improving the delivery and transfection efficiency of siRNA, and can achieve efficient, stable and controllable siRNA delivery. Secondly, embodiments of the present invention also propose a method for preparing a perfluorinated modified PEI support. The preparation methods of PF-DBCO include: Dibenzocyclooctyn-disulfide-carboxylic acid (DBCO-ss-COOH) was dissolved in N,N-dimethylformamide (DMF), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were added. The first reaction was carried out to obtain a reaction solution with carboxyl activation. The above carboxyl-activated reaction solution was added to PF solution for the second reaction. After dialyzing, the resulting reaction solution was freeze-dried to obtain PF-DBCO. The preparation methods of PF-N3 include: NHS-PEG4-N3 was added to the PF solution, and the third reaction was carried out. The resulting reaction solution was dialyzed and then freeze-dried to obtain PF-N3. Among them, PF has the structure shown in Formula III; NHS-PEG4-N3 has the structure shown in Formula IV; Formula III; Formula IV. In the preparation method of PF-DBCO in this invention, a large number of amino groups are retained on the PF molecular chain, while DBCO-ss-COOH contains terminal carboxyl groups (-COOH). After activation by EDC / NHS, a stable active ester intermediate can be generated, which is convenient to react with the amino groups on the PF molecular chain to form PF-DBCO which simultaneously has amino cations (binding to siRNA), fluoroalkyl (membrane-penetrating function) and DBCO (binding to N3).

[0021] In one embodiment of the present invention, the first reaction is specifically: stirring at 25-30°C for 3-5 hours.

[0022] In one embodiment of the present invention, the second reaction is specifically: a stirring reaction at 25-30°C for 12-24 hours. In one embodiment of the present invention, the molar ratio of the dibenzocyclooctyn-disulfide-carboxylic acid (DBCO-ss-COOH) to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) is 1:(1.2-1.5):(1.2-1.5). In one embodiment of the present invention, the ratio of dibenzocyclooctylene-disulfide-carboxylic acid (DBCO-ss-COOH) to N,N-dimethylformamide (DMF) is 35~50 µmol:10 mL.

[0023] In one embodiment of the present invention, the concentration of the PF solution is 4.0-5.0 mg / mL.

[0024] In one embodiment of the present invention, the molar ratio of dibenzocyclooctyn-disulfide-carboxylic acid (DBCO-ss-COOH) to PF is 4~10:1. It should be noted that once the weight-average molecular weight of branched polyethyleneimine is determined, the molecular weight of PF can be determined accordingly. In one embodiment of the present invention, dialysis specifically involves transferring the obtained reaction solution to a dialysis bag for dialysis. Preferably, the molecular weight cutoff (MWCO) of the dialysis bag is 3000-5000 Da; more preferably, the molecular weight cutoff of the dialysis bag is 3500 Da.

[0025] In one embodiment of the present invention, dialysis is performed first in an HCl solution and then in ultrapure water. Preferably, the dialysis time is 18-72 hours, wherein dialysis in the HCl solution is performed for 9-36 hours and dialysis in ultrapure water is performed for 9-36 hours. More preferably, the concentration of the HCl solution is 0.01 mol / L. For example, dialysis is performed three times in 0.01 M HCl solution and ultrapure water, with each dialysis session lasting 3-12 hours.

[0026] In one embodiment of the present invention, the freeze-drying is specifically vacuum freeze-drying, with drying conditions of -50°C and 0.1 Pa, and a drying time of 24 hours. In the preparation method of PF-N3 in this invention: NHS-PEG4-N3 is a pre-activated functional molecule with an NHS active ester group at its end, which can directly react with the amino group of PF to obtain stable PF-N3, which is convenient for subsequent binding with PF-DBCO and siRNA.

[0027] In one embodiment of the present invention, the third reaction is specifically: a stirring reaction at 25-30°C for 4-12 hours.

[0028] In one embodiment of the present invention, the molar ratio of NHS-PEG4-N3 to PF is 3~10:1.

[0029] In one embodiment of the present invention, the concentration of the PF solution is 4-5 mg / mL. Preferably, the concentration of the PF solution is 4.5 mg / mL. In one embodiment of the present invention, dialysis specifically involves transferring the obtained reaction solution to a dialysis bag for dialysis. Preferably, the molecular weight cutoff (MWCO) of the dialysis bag is 3000-5000 Da. More preferably, the molecular weight cutoff of the dialysis bag is 3500 Da. In one embodiment of the present invention, dialysis is performed first in an HCl solution and then in ultrapure water. Preferably, the dialysis time is 18-72 hours, wherein dialysis in the HCl solution is performed for 9-36 hours and dialysis in ultrapure water is performed for 9-36 hours. More preferably, the concentration of the HCl solution is 0.01 mol / L. For example, dialysis is performed three times in 0.01 M HCl solution and ultrapure water, with each dialysis session lasting 3-12 hours.

[0030] In one embodiment of the present invention, the freeze-drying is specifically vacuum freeze-drying, with drying conditions of -50°C and 0.1 Pa, and a drying time of 24 hours. In one embodiment of the present invention, PF is prepared by the following steps: Branched polyethyleneimine (bPEI) was dissolved in methanol, pentafluoropropionic anhydride was added, and the reaction was carried out in the fourth step. The resulting reaction solution was dialyzed and then freeze-dried to obtain solid PF.

[0031] In one embodiment of the present invention, the branched polyethyleneimine has a weight-average molecular weight of 2,000-40,000 Daltons. Preferably, the branched polyethyleneimine has a weight-average molecular weight of 20,000-30,000 Daltons; more preferably, the branched polyethyleneimine has a weight-average molecular weight of 25,000 Daltons.

[0032] In one embodiment of the present invention, the fourth reaction is specifically: reacting at 25-30°C for 24-48 hours.

[0033] In one embodiment of the present invention, the molar ratio of branched polyethyleneimine (bPEI) to pentafluoropropionic anhydride is 1:(80-160).

[0034] In one embodiment of the present invention, the concentration of the branched polyethyleneimine solution obtained by dissolving branched polyethyleneimine in methanol is 2-20 mg / mL. Preferably, the concentration of the branched polyethyleneimine solution is 10 mg / mL. In one embodiment of the present invention, dialysis specifically involves transferring the obtained reaction solution to a dialysis bag for dialysis. Preferably, the molecular weight cutoff (MWCO) of the dialysis bag is 3000-5000 Da. More preferably, the molecular weight cutoff of the dialysis bag is 3500 Da. In one embodiment of the present invention, the dialysis is performed first in an HCl solution and then in ultrapure water. Preferably, the dialysis time is 18-72 hours, wherein dialysis in HCl solution is performed for 9-36 hours and dialysis in ultrapure water is performed for 9-36 hours. More preferably, the concentration of the HCl solution is 0.01 mol / L. For example, dialysis is performed three times in a 0.01 M HCl solution and then three times in ultrapure water, with each dialysis session lasting 3-12 hours.

[0035] In one embodiment of the present invention, the freeze-drying is specifically vacuum freeze-drying, with drying conditions of -50°C and 0.1 Pa, and a drying time of 24 hours. Thirdly, embodiments of the present invention also propose a vector / siRNA complex obtained by combining a perfluorinated PEI vector with siRNA. The vector / siRNA complex obtained by combining a perfluorinated PEI vector with siRNA proposed in this invention allows the perfluorinated vector to effectively deliver siRNA using a non-endocytic mechanism, and it even maintains its activity under energy-inhibiting conditions.

[0036] Fourthly, embodiments of the present invention also provide a method for preparing a vector / siRNA complex, comprising the following steps: S1. Dissolve the carrier PF-DBCO and the carrier PF-N3 in PBS buffer to obtain PF-DBCO solution and PF-N3 solution, respectively. S2. Mix the above PF-DBCO solution, the above PF-N3 solution and the siRNA solution and let stand to obtain the PFND / siRNA complex.

[0037] This invention introduces perfluorinated PEI into a controllable cross-linking system. The cross-linked structure constructed by the click chemical reaction (-DBCO / -N3) effectively enhances the stability of the vector / siRNA complex, preventing premature release of siRNA during transport. Furthermore, the disulfide bonds introduced into the cross-linking molecules can break under the reducing environment of the cytoplasm, achieving controllable release of siRNA and thus significantly improving gene silencing effects. In this way, this method effectively overcomes the problems of insufficient stability, lack of controllable release site, and high cytotoxicity of existing siRNA delivery vectors.

[0038] In one embodiment of the present invention, in S1, the concentrations of both the PF-DBCO solution and the PF-N3 solution are 0.1~0.5 mg / mL. The PBS buffer is specifically phosphate-buffered saline.

[0039] In one embodiment of the present invention, in S2, the concentration of siRNA is 10 pmol / uL.

[0040] In one embodiment of the present invention, in S2, the volume ratio of PF-DBCO solution, PF-N3 solution, and siRNA solution is (1-2):(1-2):1. Preferably, the volume ratio of PF-DBCO solution, PF-N3 solution, and siRNA solution is 1.5:1.5:1.

[0041] In one embodiment of the present invention, in step S2, the mixing and standing process specifically involves mixing and then standing for 3-5 minutes.

[0042] Example 1Preparation of perfluorinated PEI supports 1) Synthesis of PF 200 mg bPEI (25 kDa) was dissolved in 20 mL methanol (MeOH), and 292 mg pentafluoropropionic anhydride was added. The reaction system was then stirred in a 25°C oil bath for 24 hours. The reaction solution was transferred to a 3500 Da dialysis bag and dialyzed in 0.01 M HCl aqueous solution and pure water, respectively (dialyzed three times in HCl solution and then three times in ultrapure water, each dialysis time was 5 hours). The dialyzed sample was then freeze-dried under vacuum (-50°C, 0.1 Pa, 24 hours) to obtain PEI-F solid (abbreviated as PF, molecular weight 28796 Da).

[0043]

[0044] 2) Synthesis of PF-DBCO 19.5 mg of dibenzocyclooctyne-disulfide-carboxylic acid (DBCO-ss-COOH) was dissolved in 10 mL of DMF, and 10.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 6.2 mg of N-hydroxysuccinimide (NHS) were added. The reaction system was then placed in an oil bath at 25°C and stirred for 4 hours to activate the carboxyl group, resulting in a carboxyl-activated reaction solution.

[0045] 230 mg of PF was dissolved in 50 mL of DMF. The carboxyl-activated reaction solution was added to the PF solution. The reaction system was then stirred in a 25°C oil bath for 12 hours. The reaction solution was then transferred to a 3500 Da dialysis bag and dialyzed in 0.01 M HCl aqueous solution and pure water, respectively (dialyzed three times in HCl solution and then three times in ultrapure water, each dialysis time was 5 hours). The dialyzed sample was then freeze-dried under vacuum (-50°C, 0.1 Pa, 24 hours) to obtain PF-DBCO solid.

[0046] 3) Synthesis of PF-N3 225 mg of PF was dissolved in 50 mL of DMF, and 15.8 mg of NHS-PEG4-N3 was added. The reaction system was stirred in an oil bath at 25°C for 8 hours. The reaction solution was then transferred to a 3500 Da dialysis bag and dialyzed in pure water and 0.01 M HCl aqueous solution (dialyzed three times in HCl solution and then three times in ultrapure water, each dialysis time was 5 hours). The dialyzed sample was then freeze-dried under vacuum (-50°C, 0.1 Pa, 24 hours) to obtain solid PF-N3.

[0047] Example 2 Preparation of vector / siRNA complex The PFND / siRNA complex was prepared as follows: 0.5 μg of PF-DBCO and 0.5 μg of PF-N3 were dissolved in 1.5 μL of PBS buffer, respectively. Then, the 1.5 μL PF-DBCO solution and 1.5 μL PF-N3 solution were mixed with 1 μL of siRNA solution (10 pmol), gently mixed, and allowed to stand for 3-5 minutes to obtain the PFND / siRNA complex.

[0048] Test Example 1 Characterization of vectors and vector / siRNA complexes (1) Structural characterization of the carrier and its intermediates 1) PF's proton NMR spectrum results pass 1 H-NMR was used to verify the PF structure. For example... Figure 1 As shown, based on the integrated area of ​​the methylene (-CH2-) proton peak (chemical shift: 2.2-3.4 ppm) in bPEI and the methylene (-CH2-) proton peak (chemical shift: 3.5 ppm) at the fluorine modification site, it is calculated that approximately 26 mol of perfluorinated molecules are linked to each mol of bPEI molecule.

[0049] 2) 1H NMR spectrum of PF-DBCO pass 1 The PF-DBCO structure was verified by H-NMR. For example... Figure 2 As shown, the number of DBCO grafts was calculated to be 7.4 based on the integrated area of ​​the proton peak of diphenylcyclooctyne (chemical shift: 7.0-7.5 ppm) and the methylene (-CH2-) proton peak in bPEI.

[0050] 3) 1H NMR spectrum results of PF-N3 pass 1 The PF-N3 structure was verified by H-NMR. For example... Figure 3 As shown, the area change of the methylene (-CH2-) proton peak (chemical shift: 3.5 ppm) at the ether bond and the integral area of ​​the methylene (-CH2-) proton peak in bPEI were used to calculate the grafting number of NHS-PEG4-N3 as 7.5.

[0051] (2) Characterization of the vector / siRNA complex The particle size distribution of the PFND / siRNA complex was detected by dynamic light scattering (DLS), and the results are as follows: Figure 4 As shown (the horizontal axis represents particle size, and the vertical axis represents intensity percentage), the particle size of the PFND / siRNA complex is 67.6 ± 3.9 nm, and the PDI is 0.22 ± 0.02.

[0052] Test Example 2 Biocompatibility of PFND / siRNA complex (1) Red blood cell hemolysis rate The PFND / siRNA complex solution was prepared according to the method described in Example 2, and bPEI / siRNA was prepared as a control as follows: 1 μg of the vector bPEI was dissolved in 3 μL of PBS buffer. Then, the above 3 μL of bPEI solution was mixed with 1 μL of siRNA solution (10 pmol), gently mixed, and allowed to stand for 3-5 minutes to obtain the bPEI / siRNA complex.

[0053] Take 10 mL of sterile defibrinated sheep blood and place it in a 50 mL sterile enzyme-free centrifuge tube. Centrifuge at 1000 g for 3 min and discard the supernatant. Gently resuspend the red blood cells in 20 mL of PBS, and wash repeatedly until the supernatant is clear and colorless. Collect the precipitated red blood cells. Dilute the red blood cells to 2% (v / v) with PBS and mix gently. PBS was set as a blank control, and Tween 80 (1 mg / mL) and pure water were used as positive controls. Take 100 μL of the red blood cell suspension and mix it with an equal volume of bPEI / siRNA (final siRNA concentration 10 nM), PFND / siRNA complex solution (final siRNA concentration 10 nM), PBS, Tween 80, or pure water. Incubate at 37℃ for 2 h or 4 h. Take 200 μL of the sample, centrifuge at 1000 g at room temperature for 3 min, and add 100 μL of the supernatant to a 96-well plate. Measure the absorbance at 540 nm. Each group was tested three times. The hemolysis rate (%) was calculated using the following formula: Hemolysis rate (%) = (OD200%) / (OD200%) 样品 -OD 阴性对照 ) / (OD 阳性对照 -OD 阴性对照 )×100%.

[0054] The results are as follows Figure 5 As shown (the horizontal axis represents time, and the vertical axis represents hemolysis rate), the hemolysis rate of erythrocytes after PFND / siRNA incubation with erythrocytes for 2 h and 4 h remained below 5%, which was significantly lower than that of Tween 80 (58.8% and 82.0%) and bPEI / siRNA (5.6% and 8.7%).

[0055] (2) Cytotoxicity The cytotoxicity of PFND / siRNA to HeLa cells was evaluated using the 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide colorimetric method (MTT method).

[0056] HeLa cells were divided into 1×10 4 Seeds were planted at a density of [number] cells / well in 96-well plates and incubated for 24 h at 37°C and 5% CO2 in Dalberg modified Eagle medium (DMEM medium containing 10% FBS) containing 10% fetal bovine serum. The medium was then replaced with 100 μL of fresh medium containing Lipo 3000 / siRNA (commercially available product, prepared according to the Lipo 3000 / siRNA product instructions), bPEI / siRNA, and PFND / siRNA complex (siRNA concentration 10 nM), and incubated for another 48 h. The medium was discarded, and the plates were washed twice with PBS. 100 μL of medium containing MTT (0.5 mg / mL) was added to each well, and the plates were incubated for another 4 h. After discarding the supernatant, 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals, and the mixture was shaken to mix. The absorbance was measured at 570 nm using a microplate reader, with a background correction of 630 nm. The results are shown below. Figure 6 As shown (the vertical axis represents cell viability), the cell viability remained at around 95% after 48 hours of incubation with the PFND / siRNA complex, demonstrating good cell compatibility.

[0057] Test Example 3 Cellular uptake capacity of PFND / siRNA Cy5-labeled siRNA was used, and the uptake of siRNA by NCI-H460, Hep-G2, and MDA-MB-468 cells was observed using laser confocal microscopy. NCI-H460, Hep-G2, or MDA-MB-468 cells were labeled with 1×10⁻⁶ cells, respectively. 6Cells were seeded at a density of [number] cells / well in 6-well plates and incubated in DMEM medium containing 10% FBS at 37°C and 5% CO2 for 24 h. PFND / Cy5-siRNA complexes were prepared according to the method in Example 2, bPEI / Cy5-siRNA was prepared according to the method in Test Example 2, and Lipo 3000 / Cy5-siRNA was prepared according to the manufacturer's instructions. These were then added to 2 mL of fresh medium in each well (siRNA concentration of 10 nM per well), and incubated for another 24 h. Cy5-siRNA uptake was observed under a laser confocal microscope (CLSM). The uptake efficiency of Cy5-siRNA in each group of cells was quantified by flow cytometry (FCM).

[0058] The results are as follows Figure 7 , Figure 8 , Figure 9 As shown in the figure (the vertical axis represents cell uptake, RFU, and relative fluorescence units), in the three cell types, the uptake effect of the PFND / Cy5-siRNA experimental group was significantly better than that of the unmodified bPEI, and also better than the best-selling Lipo 3000 transfection reagent on the market.

[0059] Test Example 4 Non-energy-dependent endocytosis efficiency of PFND / siRNA To definitively confirm the unique mechanism by which PFND delivers siRNA, the cell’s energy-dependent endocytosis process was disrupted using a metabolic inhibitor (take-up in serum-free medium containing 10 mM sodium azide for 2 hours) and cryotherapy (take-up at 4°C for 2 hours).

[0060] In conventional delivery systems, since network protein-mediated endocytosis and other forms of endocytosis are mostly energy-dependent processes, metabolic inhibitors or cryotherapy significantly reduce intracellular nucleic acid uptake. However, as Figure 10 As shown, the PFND / siRNA complex of this invention exhibits significant cellular uptake even in the presence of sodium azide or at 4°C, demonstrating its energy-independent uptake pathway. This characteristic allows it to avoid endocytosis-related degradation pathways. In contrast, when PFND delivers plasmid DNA (pDNA), a sharp decrease in uptake efficiency is observed under the same inhibitory conditions. This differential behavior strongly suggests that the delivery mechanism of fluorinated compound vectors depends on the physicochemical properties of the delivered nucleic acid, such as its size. Therefore, the PFND vector constructed in this invention is particularly suitable for the delivery of siRNAs among nucleic acid types.

[0061] Test Example 5 Gene silencing efficiency of PFND / siRNA In NCI-H460, Hep-G2, and MDA-MB-468 cells, siRNA (GAPDH) was used as an siRNA model (working concentration of 10 nM) to detect the gene silencing efficiency of Lipo 3000 / siRNA, bPEI / siRNA, and PFND / siRNA. NCI-H460, Hep-G2, or MDA-MB-468 cells were treated with 1×10⁻⁶ cells per cell line. 6 Cells were seeded at a density of [number] cells / well in 6-well plates and incubated in DMEM medium containing 10% FBS at 37°C and 5% CO2 for 24 h. bPEI / siRNA was prepared as described in Test Example 2, PFND / siRNA complex was prepared as described in Example 2, and Lipo 3000 / siRNA was prepared according to the manufacturer's instructions. These were then added to 2 mL of fresh medium in each well (siRNA concentration of 10 nM per well), and incubated for another 24 h. The silencing efficiency of GAPDH was then detected using quantitative polymerase chain reaction (qPCR). Results are as follows: Figure 11 , Figure 12 , Figure 13 As shown (vertical axis represents GAPDH mRNA level), in all three cell types, the gene silencing efficiency of the PFND / siRNA experimental group was above 90%, which was significantly better than that of bPEI and Lipo 3000 transfection reagents.

[0062] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A perfluorinated modified polyethyleneimine carrier, characterized in that, Including PF-DBCO and PF-N3; Among them, PF-DBCO has the structure shown in Formula I, and PF-N3 has the structure shown in Formula II; Equation I; Formula II.

2. The preparation method according to claim 1, characterized in that, The polyethyleneimine is a branched polyethyleneimine, and the weight-average molecular weight of the branched polyethyleneimine is 2000-40000 Daltons. Preferably, the branched polyethyleneimine has a weight-average molecular weight of 20,000-30,000 Daltons; More preferably, the branched polyethyleneimine has a weight-average molecular weight of 25,000 Daltons.

3. The method for preparing the perfluorinated modified polyethyleneimine carrier according to claim 1 or 2, characterized in that, (A) Methods for preparing PF-DBCO include: Dibenzocyclooctyne-disulfide-carboxylic acid was dissolved in N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added. The first reaction was carried out to obtain a reaction solution with carboxyl activated group. The above carboxyl-activated reaction solution was added to PF solution for the second reaction. After dialyzing, the resulting reaction solution was freeze-dried to obtain PF-DBCO. (B) Methods for preparing PF-N3 include: NHS-PEG4-N3 was added to the PF solution, and a third reaction was carried out. The resulting reaction solution was dialyzed and then freeze-dried to obtain PF-N3. PF has the structure shown in Formula III, and NHS-PEG4-N3 has the structure shown in Formula IV. Formula III; Formula IV.

4. The preparation method according to claim 3, characterized in that, The (A) satisfies at least one of the following conditions: (1) The first reaction is specifically: stirring at 25-30°C for 3-5 hours; (2) The second reaction is specifically carried out by stirring at 25-30°C for 12-24 hours; (3) The molar ratio of the dibenzocyclooctyn-disulfide-carboxylic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:(1.2-1.5):(1.2-1.5); (4) The ratio of dibenzocyclooctyne-disulfide-carboxylic acid to N,N-dimethylformamide is 35~50 µmol: 10 mL; (5) The concentration of the PF solution is 4.0-5.0 mg / mL; (6) The molar ratio of the dibenzocyclooctyn-disulfide-carboxylic acid to PF is 4~10:1; And / or, said (B) satisfies at least one of the following conditions: (7) The third reaction is specifically: stirring at 25-30°C for 4-12 hours; (8) The molar ratio of NHS-PEG4-N3 to PF is 3~10:

1.

5. The preparation method according to claim 3, characterized in that, The PF is prepared by the following steps: Branched polyethyleneimine was dissolved in methanol, pentafluoropropionic anhydride was added, and the reaction was carried out in the fourth step. The resulting reaction solution was dialyzed and then freeze-dried to obtain solid PF.

6. The preparation method according to claim 5, characterized in that, At least one of the following conditions must be met: (1) The fourth reaction is specifically: react at 25-30°C for 24-48 hours; (2) The molar ratio of the branched polyethyleneimine to pentafluoropropionic anhydride is 1:(80-160); (3) The concentration of the branched polyethyleneimine solution obtained by dissolving branched polyethyleneimine in methanol is 2-20 mg / mL.

7. The preparation method according to claim 2 or 5, characterized in that, Dialysis specifically involves transferring the resulting reaction solution into a dialysis bag for dialysis. Preferably, the molecular weight cutoff of the dialysis bag is 3000-5000 Da; More preferably, the dialysis is performed by first dialysis in an HCl solution and then dialysis in ultrapure water; More preferably, the concentration of the HCl solution is 0.01 mol / L.

8. A carrier / siRNA complex, obtained by combining a perfluorinated polyethyleneimine carrier with siRNA prepared using the perfluorinated polyethyleneimine carrier of claim 1 or the preparation method of any one of claims 2 to 7.

9. The method for preparing the vector / siRNA complex according to claim 8, comprising the following steps: S1. Dissolve the carrier PF-DBCO and the carrier PF-N3 in PBS buffer to obtain PF-DBCO solution and PF-N3 solution, respectively. S2. Mix the above PF-DBCO solution, the above PF-N3 solution and the siRNA solution and let stand to obtain the PFND / siRNA complex.

10. The preparation method according to claim 9, characterized in that, At least one of the following conditions must be met: (1) In S1, the concentrations of PF-DBCO solution and PF-N3 solution are both 0.1~0.5 mg / mL; (2) In S2, the concentration of siRNA is 10 pmol / uL; (3) In S2, the volume ratio of PF-DBCO solution, PF-N3 solution and siRNA solution is (1-2):(1-2):1; (4) In S2, the mixing and standing process is as follows: after mixing, let stand for 3-5 minutes.

Images