Cationic polymer, process for its preparation and use

Abstract

The application relates to the technical field of high polymer materials, in particular to a cationic polymer, a preparation method and application thereof. The application provides a cationic polymer with a structure shown in formula I or formula II: wherein 5<=x<=300, and 60<=m<=600. The linear polyethylene imine (LPEI) is technically reformed. The LPEI itself has a good DNA transfection effect, but has almost no effect on RNA transfection. By introducing a phenylboronic acid group through a simple method, the RNA transfection efficiency of the LPEI is greatly improved on the basis of retaining the DNA transfection advantage of the LPEI itself, high-efficiency DNA transfection and RNA transfection can be realized. Moreover, the phenylboronic acid group is introduced through a one-step method (bromination or epoxy ring opening), other reagents (such as an activating agent and a condensing agent) are not introduced, the complexity of synthesis is reduced, expenses are saved, and the one-step method is suitable for large-scale production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of high molecular materials, in particular to a cationic polymer, a preparation method and application thereof. BACKGROUND

[0002] Gene therapy is considered as one of the effective means to cope with various genetic and major diseases, by introducing exogenous genes into target cells to repair harmful genes of lesions or mutations, so as to achieve the purpose of treating diseases. Successful gene therapy depends on the gene carrier to deliver nucleic acid material into target cells to play a role. Common gene carriers are divided into viral and non-viral, viral carriers are troubled by safety hazards; non-viral carriers, especially cationic polymer carriers, have the advantages of easy preparation, safety and efficiency, no immunogenicity, etc., and become a research hotspot. The most common non-viral gene carriers include cationic polylysine (PLys), polyethyleneimine (PEI) and liposome carriers.

[0003] The above-mentioned cationic gene carriers mainly combine nucleic acid materials such as DNA, RNA, etc. through electrostatic interaction, and all have a large charge density. Researchers can reduce the cytotoxicity and improve the transfection efficiency to a certain extent through biocompatibility modification (such as modification of polysaccharides, polyamino acid derivatives, etc.), hydrophobic modification (introduction of benzene ring, alkyl and other hydrophobic groups) and other means. However, in most cases, the efficiency is not significantly improved, and the preparation process is complex.

[0004] Furthermore, the existing carriers generally have poor universality (for example, only DNA or RNA has good efficiency, and cannot be used for both), poor stability (for example, the transfection efficiency changes greatly after 1-2 months of preparation), complex preparation process (usually requiring multiple reactions and purification), poor biocompatibility, etc. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to provide a cationic polymer, a preparation method and application thereof. The cationic polymer provided by the present application can be used as a gene carrier and can realize high-efficiency DNA transfection and RNA transfection.

[0006] The present application provides a cationic polymer having a structure represented by Formula I or Formula II:

[0007]

[0008] wherein 5≤x≤300, 60≤m≤600.

[0009] The present application further provides a preparation method of the cationic polymer, comprising the following steps:

[0010] reacting linear polyethyleneimine with structure shown in formula III and bromomethyl phenylboronic acid in a first solvent to obtain cationic polymer with structure shown in formula I;

[0011]

[0012] or

[0013] reacting linear polyethyleneimine with structure shown in formula III and 4-(oxiran-2-ylmethoxy)phenylboronic acid pinacol ester in a second solvent to obtain cationic polymer with structure shown in formula II;

[0014]

[0015] wherein, 5≤x≤300, 60≤m≤600.

[0016] Preferably, the molar ratio of the linear polyethyleneimine and bromomethyl phenylboronic acid is 1:25-250.

[0017] Preferably, the first solvent comprises dimethyl sulfoxide, N’N dimethyl formamide or dioxane.

[0018] Preferably, the temperature for the reaction of the linear polyethyleneimine with structure shown in formula III and bromomethyl phenylboronic acid in the first solvent is 30-60℃, and the time is 90-100h.

[0019] Preferably, the molar ratio of the linear polyethyleneimine and 4-(oxiran-2-ylmethoxy)phenylboronic acid pinacol ester is 1:25-250.

[0020] Preferably, the second solvent comprises dimethyl sulfoxide, N’N dimethyl formamide or dioxane.

[0021] Preferably, the temperature for the reaction of the linear polyethyleneimine with structure shown in formula III and 4-(oxiran-2-ylmethoxy)phenylboronic acid pinacol ester in the second solvent is 60-90℃, and the time is 65-80h.

[0022] Preferably, after the reaction, the method further comprises dialysis.

[0023] The pore size of the dialysis membrane is 2000-10000Da.

[0024] After the dialysis, the method further comprises lyophilization.

[0025] The application further provides a use of the cationic polymer described above or prepared by the preparation method described above as a gene carrier.

[0026] The application provides a cationic polymer with structure shown in formula I or formula II:

[0027]

[0028] wherein 5≤x≤300, 60≤m≤600.

[0029] The present application is directed to technical modification of linear polyethyleneimine (LPEI), which has good DNA transfection effect itself, but almost no effect on transfection of RNA. By introducing phenylboronic acid group through a simple method, the RNA transfection efficiency of LPEI is greatly improved on the basis of retaining the DNA transfection advantage of LPEI itself, and high-efficiency DNA transfection and RNA transfection can be realized. Moreover, the present application introduces phenylboronic acid group through one-step method (bromination or epoxy ring-opening), without introducing other reagents (such as activating agent, condensing agent), which reduces the complexity of synthesis, saves expenses, etc., and is suitable for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 NMR spectrum of the cationic polymer (LPEIPBA-1) of Example 1 of the present application;

[0031] Figure 2 NMR spectrum of the cationic polymer (LPEIPBA-2) of Example 2 of the present application. DETAILED DESCRIPTION

[0032] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0033] The present application provides a cationic polymer having the structure shown in Formula I or Formula II:

[0034]

[0035] wherein 5≤x≤300, 60≤m≤600. Specifically, x=102, m=512.

[0036] The present application also provides a preparation method of the cationic polymer, comprising the following steps:

[0037] reacting linear polyethyleneimine having the structure shown in Formula III with bromomethylphenylboronic acid in a first solvent to obtain the cationic polymer having the structure shown in Formula I;

[0038]

[0039] or

[0040] reacting a linear polyethylenimine having a structure shown in Formula III and 4- (oxiran-2-ylmethyloxy)phenylboronic acid pinacol ester in a second solvent to obtain a cationic polymer having a structure shown in Formula II;

[0041]

[0042] wherein 5≤x≤300, 60≤m≤600. Specifically, x=102, m=512.

[0043] For the first scenario:

[0044] reacting a linear polyethylenimine having a structure shown in Formula III and 4- (oxiran-2-ylmethyloxy)phenylboronic acid pinacol ester in a second solvent to obtain a cationic polymer having a structure shown in Formula II;

[0045]

[0046] wherein 5≤x≤300, 60≤m≤600. Specifically, x=102, m=512.

[0047] Specifically, comprising:

[0048] reacting a linear polyethylenimine having a structure shown in Formula III and 4- (oxiran-2-ylmethyloxy)phenylboronic acid pinacol ester in a second solvent to obtain a cationic polymer having a structure shown in Formula II;

[0049] The solvent in the linear polyethylenimine solution having a structure shown in Formula III and the bromomethylphenylboronic acid solution is a first solvent.

[0050] The bromomethylphenylboronic acid has a structure shown in Formula IV:

[0051]

[0052] In some embodiments of the present application, the molar ratio of the linear polyethylenimine and the bromomethylphenylboronic acid is 1:25-250, such as 1:102.

[0053] In some embodiments of the present application, the first solvent comprises dimethyl sulfoxide, N’N dimethylformamide or dioxane.

[0054] In some embodiments of the present application, the concentration of the linear polyethylenimine solution having a structure shown in Formula III is 10-50 mg / mL;

[0055] The concentration of the bromomethylphenylboronic acid solution is 10-50 mg / mL.

[0056] In some embodiments of the present application, the temperature of the reaction is 30-60℃, and the time is 90-100h, such as 96h.

[0057] In some embodiments of the present application, after the reaction, the method further comprises dialysis. The pore size of the dialysis membrane is 2000-10000Da, such as 1000-7000Da. After the dialysis, the method further comprises lyophilization.

[0058] For the second scenario:

[0059] reacting a linear polyethyleneimine having a structure shown in Formula III and 4-(oxiran-2-ylmethyloxy)phenylboronic acid pinacol ester in a second solvent to obtain a cationic polymer having a structure shown in Formula II;

[0060]

[0061] wherein 5≤x≤300, 60≤m≤600. Specifically, x=102, m=512.

[0062] Specifically, the method comprises:

[0063] reacting a linear polyethyleneimine having a structure shown in Formula III and 4-(oxiran-2-ylmethyloxy)phenylboronic acid pinacol ester in a second solvent to obtain a cationic polymer having a structure shown in Formula II.

[0064] The solvents in the linear polyethyleneimine solution having a structure shown in Formula III and the 4-(oxiran-2-ylmethyloxy)phenylboronic acid pinacol ester solution are both the second solvent.

[0065] The 4-(oxiran-2-ylmethyloxy)phenylboronic acid pinacol ester bromomethylphenylboronic acid has a structure shown in Formula V:

[0066]

[0067] In some embodiments of the present application, the molar ratio of the linear polyethyleneimine and the 4-(oxiran-2-ylmethyloxy)phenylboronic acid pinacol ester is 1:25-250, such as 1:102.

[0068] In some embodiments of the present application, the second solvent comprises dimethyl sulfoxide, N’N dimethylformamide or dioxane.

[0069] In some embodiments of the present application, the concentration of the linear polyethyleneimine solution having a structure shown in Formula III is 10-50mg / mL;

[0070] The concentration of the 4-(oxiran-2-ylmethyloxy)phenylboronic acid pinacol ester solution is 10-50mg / mL.

[0071] In some embodiments of the present application, the temperature of the reaction is 60-90℃, and the time is 65-80h, such as 72h.

[0072] In some embodiments of the present application, after the reaction, the method further comprises dialysis. The pore size of the dialysis membrane is 2000-10000Da, such as 1000-7000Da. After the dialysis, the method further comprises lyophilization.

[0073] The present application also provides a use of the cationic polymer described above or prepared by the preparation method described above as a gene carrier.

[0074] The raw materials used in the present application are not particularly limited and can be commercially available.

[0075] The present application is a technical improvement of linear polyethylene imine (LPEI). LPEI itself has good DNA transfection effect, but has little effect on RNA transfection. By introducing phenylboronic acid groups through a simple method, the RNA transfection efficiency of LPEI is greatly improved while retaining the DNA transfection advantage of LPEI itself.

[0076] The present application introduces phenylboronic acid groups through a one-step method (bromination or epoxy ring opening), without introducing other reagents (such as activating agents, condensing agents), thereby reducing the complexity of synthesis, saving costs, etc. and being suitable for large-scale production.

[0077] The present application can achieve high-efficiency DNA and RNA transfection.

[0078] The present application greatly reduces the cytotoxicity of the carrier by introducing phenylboronic acid.

[0079] In order to further illustrate the present application, a cationic polymer, its preparation method and application provided by the present application are described in detail below in conjunction with examples, but it should not be understood as limiting the scope of protection of the present application.

[0080] In the examples, linear polyethylene imine having the structure shown in formula III is prepared according to the preparation method described in the literature (Histidinylated linear PEI: a new efficient non-toxic polymer for gene transfer, Chem. Commun., 2011, 47, 12547-12549.).

[0081] Example 1

[0082] Preparation of the cationic polymer having the structure shown in formula I:

[0083] 0.1 mol of linear polyethyleneimine with the structure shown in Formula III was dissolved in dimethyl sulfoxide to obtain a linear polyethyleneimine solution with the structure shown in Formula III, with a concentration of 25 mg / mL;

[0084] 10.2 mol of bromomethylphenylboronic acid with the structure shown in Formula IV was dissolved in dimethyl sulfoxide to obtain a bromomethylphenylboronic acid solution, with a concentration of 25 mg / mL;

[0085] The bromomethylphenylboronic acid solution was added to the linear polyethyleneimine solution, and after 96 h of reaction at 45°C, dialysis was performed using a dialysis membrane with a pore size of 1000-7000 Da, and lyophilization was performed to obtain a cationic polymer with the structure shown in Formula I (LPEIPBA-1). In the corresponding structure shown in Formula I, x = 102, and m = 512.

[0086] The nuclear magnetic resonance spectrum of the cationic polymer with the structure shown in Formula I (LPEIPBA-1) prepared in Example 1 is shown in Figure 1 . Figure 1 The nuclear magnetic resonance spectrum of the cationic polymer (LPEIPBA-1) of Example 1 of the present application.

[0087] Example 2

[0088] Preparation of a cationic polymer with the structure shown in Formula II:

[0089] 0.1 mol of linear polyethyleneimine with the structure shown in Formula III was dissolved in dimethyl sulfoxide to obtain a linear polyethyleneimine solution with the structure shown in Formula III, with a concentration of 25 mg / mL;

[0090] 10.2 mol of 4-(oxirane-2-ylmethoxy)phenylboronic acid pinacol ester with the structure shown in Formula V was dissolved in dimethyl sulfoxide to obtain a 4-(oxirane-2-ylmethoxy)phenylboronic acid pinacol ester solution, with a concentration of 25 mg / mL;

[0091] The 4-(oxirane-2-ylmethoxy)phenylboronic acid pinacol ester solution was added to the linear polyethyleneimine solution, and after 72 h of reaction at 75°C, dialysis was performed using a dialysis membrane with a pore size of 1000-7000 Da, and lyophilization was performed to obtain a cationic polymer with the structure shown in Formula II (LPEIPBA-2). In the corresponding structure shown in Formula II, x = 102, and m = 512.

[0092] The nuclear magnetic resonance spectrum of the cationic polymer with the structure shown in Formula II (LPEIPBA-2) prepared in Example 2 is shown in Figure 2 . Figure 2 The nuclear magnetic resonance spectrum of the cationic polymer (LPEIPBA-2) of Example 2 of the present application.

[0093] Application Example (cells used are purchased from Fuheng Biological)

[0094] 1) Particle size characterization of the complex of cationic polymer and nucleic acid, taking calf thymus DNA (Shanghai Yuanye Biotechnology Co., Ltd.) as an example.

[0095] Prepare a 0.05 mg / mL aqueous solution of calf thymus DNA, and a 0.1 mg / mL aqueous solution of cationic polymer. Mix 1 mL of the aqueous calf thymus DNA solution with 1 mL of the aqueous cationic polymer solution by equal volume and vortex, and then let stand at room temperature for 20 min. Characterize the particle size and zeta potential of the carrier / DNA complex, as shown in Table 1.

[0096] Table 1 Particle size and zeta potential of carrier / DNA complex

[0097] Nanoparticle Diameter (nm) Potential (mV) LPEI / DNA 168.5 23.3 LPEI PBA-1 / DNA 112.4 5.67 LPEI PBA-2 / DNA 106.6 3.68

[0098] wherein LPEI is a linear polyethyleneimine having a structure shown in Formula III.

[0099] As can be seen from Table 1, the particle size of the modified complex is about 110 nm, and does not exceed 120 nm, and the zeta potential is below 6 mV, which is much lower than the particle size and zeta potential of the linear polyethyleneimine LPEI / DNA complex.

[0100] 2) Transfection evaluation of cationic polymer in cells

[0101] 2-1) Cell culture

[0102] The cells were cultured in a fetal bovine serum culture solution with a volume fraction of 10%, and the culture was carried out in a constant temperature incubator at 37°C with a CO2 volume fraction of 5%.

[0103] 2-2) Cell transfection

[0104] 24 h before transfection, log phase cells were taken, trypsinized, diluted with a fetal bovine serum culture solution with a volume fraction of 10%, and plated in a 96-well cell culture plate at a density of 1 x 10 4 The cells were plated in a 96-well cell culture plate at a density of 1 x 10

[0105] The determination of cell transfection efficiency was carried out by using cationic polymer to mediate pGL3 (luciferase plasmid) and pEGFPNl (green fluorescent protein plasmid) to transfect cells.

[0106] a) Detection of luciferase activity

[0107] The cell culture plate was taken out from the incubator, the cell culture solution was removed, and the cells were washed twice with PBS. Cell lysate was added and placed in -80°C for 20 min. 50 μL of lysate was taken into a 1.5 mL centrifuge tube, and then 20 μL of luciferase substrate was added to each well and mixed uniformly. The cell transfection efficiency was determined quantitatively by using a luminometer. The results are shown in Table 2.

[0108] Table 2 Cell transfection efficiency

[0109]

[0110] As can be seen from the results shown in Table 2, the transfection efficiency of LPEI PBA carrying luciferase plasmid was greatly improved compared with LPEI itself. For example, in HeLa cells, the transfection efficiency was increased by 2-10 times, in B16F10 cells, the transfection efficiency was increased by more than 30 times, and in 293T cells, the transfection efficiency was increased by more than 8 times.

[0111] b) Expression of green fluorescent protein (GFP)

[0112] The percentage of transfected positive cells was determined by flow cytometry. The results are shown in Table 3.

[0113] Table 3 Percentage of transfected positive cells

[0114]

[0115] As can be seen from the results shown in Table 3, the transfection efficiency of LPEI PBA carrying green fluorescent protein plasmid was greatly improved compared with LPEI itself. For example, in HeLa cells and B16F10 cells, the transfection efficiency was increased by more than 30%, and in 293T cells, the transfection efficiency was increased by more than 20%.

[0116] Determination of cell transfection efficiency:

[0117] Luc siRNA was used to transfect various cell lines by using cationic polymer to mediate silencing of luciferase. The cell culture plate was taken out from the incubator, the cell culture solution was removed, and the cells were washed twice with PBS. Cell lysate was added and placed in -80°C for 20 min. 50 μL of lysate was taken into a 1.5 mL centrifuge tube, and then 20 μL of luciferase substrate was added to each well and mixed uniformly. The cell transfection efficiency was determined quantitatively by using a luminometer. The silencing efficiency of luciferase is shown in Table 4.

[0118] Table 4 Silencing efficiency of luciferase

[0119]

[0120] From the results shown in Table 4, it can be seen that the silencing efficiency of luciferase of LPEIPBA carrying Luc siRNA to silence luciferase on the cell line, HuH-7 cells, CT26 cells and HeLa cells, which express luciferase constantly, is increased by more than 75% compared with LPEI.

[0121] 3) Detection of cytotoxicity (MTT)

[0122] The cytotoxicity of the carrier / pDNA complex was evaluated by the tetrazolium salt (thiazolyl blue) colorimetric method.

[0123] 24h before transfection, logarithmic growth phase cells were taken, diluted with a volume fraction of 10% fetal bovine serum culture solution after trypsin digestion, and plated at a density of 1x10 4 The cells were plated in a 96-well cell culture plate at a density of cells / well and incubated in a constant temperature incubator at 37°C with a CO2 volume fraction of 5% until the cell confluence reached 80%-90%. Different concentrations of cationic polymers were co-cultured with cells for 24h, then 20μL thiazolyl blue solution (mass fraction 0.5%) was added to each well, and incubated at 37°C for another 4h. Then the culture solution was aspirated, 200μL DMSO was added to each well, and the absorption of each well of the culture plate was detected by an enzyme marker with a wavelength of 490nm. The survival rate of the cells was calculated according to formula (1):

[0124] Cell survival rate (%) = (Asample / Acontrol) x 100 (1)

[0125] Asample is the absorption of the cell sample well after transfection, and Acontrol is the absorption of the sample well without materials. Each group of experiments was repeated three times, and the results are shown in Table 5.

[0126] Table 5 Test results of cytotoxicity

[0127]

[0128] From the results shown in Table 5, it can be seen that the cytotoxicity of LPEIPBA is significantly reduced compared with LPEI, and the cell survival rate is more than 90% in the three cells, while only about half of the cells survive in the LPEI group.

[0129] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and that the appended claims are intended to cover all such modifications that do not depart from the true spirit and scope of the application. Therefore, the application is not limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A cationic polymer having the structure shown in Formula II: Formula II; in, 5≤x≤300, 60≤m≤600; The method for preparing the cationic polymer includes the following steps: Linear polyethyleneimine having the structure shown in Formula III and pinacol 4-(ethylene oxide-2-ylmethoxy)phenylboronic acid pinacol ester were reacted in a second solvent to obtain a cationic polymer having the structure shown in Formula II. Formula III; The molar ratio of the linear polyethyleneimine and 4-(ethylene oxide-2-ylmethoxy)phenylboronic acid pinacol ester is 1:25~250.

2. A method for preparing a cationic polymer, comprising the following steps: Linear polyethyleneimine having the structure shown in Formula III and pinacol 4-(ethylene oxide-2-ylmethoxy)phenylboronic acid pinacol ester were reacted in a second solvent to obtain a cationic polymer having the structure shown in Formula II. Formula III; Formula II; Where 5≤x≤300, 60≤m≤600.

3. The preparation method according to claim 2, characterized in that, The second solvent includes dimethyl sulfoxide, N'N dimethylformamide, or dioxane.

4. The preparation method according to claim 2, characterized in that, The linear polyethyleneimine having the structure shown in Formula III and 4-(ethylene oxide-2-ylmethoxy)phenylboronic acid pinacol ester are reacted in a second solvent at a temperature of 60-90°C for a time of 65-80 h.

5. The preparation method according to claim 2, characterized in that, The reaction is followed by: dialysis; The dialysis membrane has a pore size of 2000~10000 Da; The process after dialysis also includes: lyophilization.

6. The application of the cationic polymer of claim 1, or the cationic polymer prepared by any one of claims 2 to 5, as a gene carrier.

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