Polyethyleneimine derivative and application thereof as gene drug carrier

Abstract

The invention discloses a polyethyleneimine derivative and application thereof as a gene drug carrier. The polyethyleneimine derivative is obtained by connecting an iron chelating agent to a side chain of polyethyleneimine through an amido bond. Polyethyleneimine is used as a basic carrier material and is connected with an iron metabolism regulating drug through an amido bond to synthesize a polyethyleneimine derivative, then the polyethyleneimine derivative is combined with a nucleic acid drug through self-assembly to form a nanoparticle core, and the nanoparticle core is connected with a PEG terminal to form a final preparation. The invention provides a targeted epigenetic combined medication mode for the first time. Different from a traditional administration mode, different drugs respectively aim at tumor iron metabolism and RNA methylation, and the anti-tumor curative effect can be effectively enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a polyethyleneimine derivative and its application as a gene drug carrier. Background Art

[0002] Epigenetics refers to the biological phenomenon that gene function produces heritable variations without changing the DNA sequence, leading to changes in the biological phenotype. Tumors are an epigenetic disease, and their development is closely related to abnormal gene expression. Abnormal modifications of DNA, RNA and histones can affect the expression of oncogenes. With the development of tumor treatment, various anti-tumor drug research and development have emerged, but due to problems such as tumor heterogeneity and special microenvironment, many drugs cannot produce good therapeutic effects. Therefore, anti-tumor targets targeting epigenetics are expected to bring new directions to tumor treatment. The targets targeting epigenetics mainly include inhibition of core enzymes, inhibition of histone modification recognition and inhibition of allosteric devices. At present, some anti-epigenetic anti-tumor drugs have been approved for marketing, but there are still many problems in the development of anti-epigenetic anti-tumor drugs. Most drugs have a single effect, weak responsiveness to solid tumors, and limited therapeutic effects on tumors. Therefore, drug combination to break through the limitations of indications will become a new breakthrough.

[0003] mRNA N6-adenylate methylation (m 6 A) is the most common type of RNA modification. 6 When the modification of mA is abnormal, it will cause diseases, the most common of which is tumors. Studies have shown that YTHDF1 is overexpressed in many cancers. 6 A reading protein can accelerate the translation of mRNA and promote tumor growth; at the same time, it will limit the expression of MHC-I, degrade antigens, and make tumor immunity ineffective. Iron metabolism is also closely related to epigenetic regulation. Some key epigenetic enzymes, such as DNA demethylase, require the participation of iron ions to function. Therefore, it is necessary to link iron metabolism regulation with m 6 Combining the inhibition of A-related enzymes with dual regulation of epigenetics will be an effective strategy for developing anti-tumor drugs. Summary of the invention

[0004] One of the purposes of the present invention is to provide a polyethyleneimine derivative obtained by modifying an iron chelator with a polyethyleneimine side chain, wherein the iron chelator is deferasirox, cholic acid, droxidopa or carbidopa.

[0005] Furthermore, the polyethyleneimine is low molecular weight polyethyleneimine (<1000), medium molecular weight polyethyleneimine (1000-10 000) or high molecular weight polyethyleneimine (>10 000).

[0006] In the present invention, NHS and EDC are first used to activate the side chain of polyethyleneimine, and then an iron chelating agent is added to react, and the two are connected through an amide bond.

[0007] The second object of the present invention is to provide the use of the above polyethyleneimine derivatives in the preparation of nucleic acid drug delivery vectors.

[0008] Furthermore, the nucleic acid drug is DNA, mRNA, siRNA, microRNA or saRNA.

[0009] The third object of the present invention is a nucleic acid drug delivery carrier, comprising a nanoparticle core formed by self-assembly of the above-mentioned polyethyleneimine derivative and the nucleic acid drug, and a PEG end connected to the surface of the nanoparticle core. The PEG end can be connected to the nanoparticle core through an active group such as -CHO, -COOH or -COX (X=F, Cl, Br).

[0010] Furthermore, the molecular weight of the PEG in the PEG shell and / or the end is 200-20000.

[0011] In one embodiment of the present invention, the iron chelator uses deferasirox (DFX) as a model drug, which is connected to the side chain of polyethyleneimine through an amide bond to obtain a derivative PEI-DFX. The specific preparation method includes the following steps:

[0012] Step 1, dissolving DFX, NHS and EDC in DMSO, with the molar ratio of NHS to EDC being 1:1, and the molar ratio of DFX to NHS being 1:1 to 1:3, and reacting for 30-60 minutes;

[0013] Step 2, dissolving PEI in the reaction system of step 1, the molar ratio of PEI to DFX is 1:10 to 1:40, and reacting for 12 to 36 hours;

[0014] Step 3, dialyzing, freeze-drying and storing the solution obtained in step 2 to obtain PEI-DFX;

[0015] The reaction formula is as follows:

[0016]

[0017] Afterwards, siRNA siYTHDF1 was used as a nucleic acid drug, which was combined with PEI-DFX through self-assembly to form the nanoparticle core PD / siYTHDF1, and the PEG end was connected to form the final preparation PPD / siYTHDF1.

[0018] In one embodiment of the present invention, the mass ratio of the polyethyleneimine derivative PEI-DFX to siRNA is 6-12:1; the mass ratio of the polyethyleneimine derivative to the terminal modified PEG is 1:1.

[0019] In one embodiment of the present invention, the terminal modified PEG is prepared by the following steps:

[0020] Step 1, dissolving a benzoic acid derivative, EDC, and DMAP in CH2Cl2, reacting for 30-60 min, wherein the molar ratio of the benzoic acid derivative to EDC is 1:1-1:2, and the molar ratio of DAMP to EDC is 1:5-1:10;

[0021] Step 2, dissolving OH-PEG-OH in the reaction system of step 1, reacting for 12 to 36 hours, wherein the molar ratio of OH-PEG-OH to the benzoic acid derivative is 1:10;

[0022] Step 3, dialyzing, freeze-drying and storing the solution obtained in step 2 to obtain the terminal-modified PEG.

[0023] The reaction formula is as follows:

[0024]

[0025] The present invention uses polyethyleneimine (PEI) as a basic carrier material, connects with an iron chelator (deferasirox DFX) through an amide bond, synthesizes a polyethyleneimine derivative (PEI-DFX), and then combines with a nucleic acid drug (nucleic acid drug downregulates YTHDF1 expression) through self-assembly, and connects the PEG end to form a final preparation (PPD / siYTHDF1). PEG is connected to PD / siYTHDF to form an end or shell. Deferasirox, as an iron chelator, can destroy the iron metabolism level of tumor cells; nucleic acid drugs inhibit the expression of YTHDF1, resulting in reduced tumor DNA synthesis; the combination of the two drugs causes cell cycle arrest in tumor cells, thereby inhibiting tumor growth.

[0026] Beneficial effects:

[0027] 1. This invention proposes for the first time a combined drug therapy method targeting epigenetics. Different from the traditional drug administration method, different drugs target tumor iron metabolism and RNA methylation respectively, which can effectively enhance the anti-tumor efficacy.

[0028] 2. The polyethyleneimine derivative carrier prepared by the present invention has simple synthesis, strong universality, good reproducibility of the preparation process, good carrier stability, and is easy for clinical transformation.

[0029] 3. The preparation prepared by the present invention can improve the bioavailability of drugs, enhance the delivery stability of nucleic acid drugs, and has good development prospects for drug combination strategies and high clinical use value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the prepared PEI-DFX.

[0031] Figure 2 This is the gene packaging detection result of PD / siYTHDF1.

[0032] Figure 3 These are the particle size potential characterization results of PD / siYTHDF1 and PPD / siYTHDF1.

[0033] Figure 4 This is the RNase stability result of PPD / siYTHDF1.

[0034] Figure 5 The results of cellular uptake of PPD / FAM-siYTHDF1 are shown in Figure 2.

[0035] Figure 6 This is the lysosomal escape result of PPD / FAM-siYTHDF1.

[0036] Figure 7 This is the silencing effect of the YTHDF1 gene. DETAILED DESCRIPTION

[0037] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] Example 1

[0041] 1. Synthesis of polyethyleneimine derivatives

[0042] 1.1 Synthesis of PEI-DFX(PD)

[0043] Using NHS and EDC as catalysts, the deferasirox DFX was modified onto polyethyleneimine PEI through an amide reaction to synthesize PEI-DFX. The specific process is: 0.4mmol DFX, 1mmol NHS and 1mmol EDC were dissolved in about 10mL DMSO and activated for 30-60min; then 0.02mmol PEI was added to the above activation system to initiate the amide reaction, stirred for 24h, and the molar ratio of PEI to DFX was 1:20. After the reaction was completed, the reaction solution was dialyzed for 48h using a 10kDa dialysis bag, then freeze-dried and 1 H-NMR analysis.

[0044] like Figure 1 As shown, according to 1 H-NMR results showed that the proton peak of PEI appeared at 2.0-2.9 ppm, and the proton peak of DFX benzene ring appeared at 6.5-8.0 ppm, proving that DFX was successfully grafted onto PEI.

[0045] 1.2 Synthesis of PEI-Carbidopa (PC)

[0046] Using NHS and EDC as catalysts, carbidopa was modified onto polyethyleneimine PEI through an amide reaction to synthesize PEI-Carbidopa (PC). The specific process is as follows: 0.4mmol DFX, 1mmol NHS and 1mmol EDC were dissolved in about 10mL DMSO and activated for 30-60min; then 0.02mmol PEI was added to the above activation system to initiate an amide reaction, stirred for 24h, and the molar ratio of PEI to Carbidopa was 1:20. After the reaction was completed, the reaction solution was dialyzed for 48h using a 10kDa dialysis bag and then freeze-dried.

[0047] 2. Preparation of Polyethyleneimine Derivatives Encapsulating Nucleic Acid Drugs

[0048] 2.1 Preparation of PD-loaded siYTHDF1

[0049] W was prepared by self-assembly method PD / W siYTHDF1 The ratio of PD / siYTHDF1 was 0, 0.5, 1, 2, 4, 6, and 8. Specifically, a 50 μg / mL siYTHDF1 solution was prepared, and then PD solutions of different concentrations were prepared by gradient dilution method, and the two were mixed in equal volumes to obtain different ratios of PD / siYTHDF1. Then, an agarose gel electrophoresis experiment was performed at 100 V for 15 min, and the exposed siYTHDF1 bands were observed and photographed using a gel imager.

[0050] like Figure 2 As shown, in W PD / W siYTHDF1 When it is above 6, PD can completely encapsulate siYTHDF1.

[0051] 2.2 Preparation of PD-encapsulated CRISPR / Cas

[0052] W was prepared by self-assembly method PD / W CRISPR The PD / CRISPR ratios are 0, 0.5, 1, 2, 4, 6, and 8. Specifically, a 50 μg / mL CRISPR solution is prepared, and then PD solutions of different concentrations are prepared by gradient dilution method, and equal volumes of the two are mixed to obtain PD / CRISPR solutions of different ratios.

[0053] 2.3 Preparation of PD-encapsulated saRNA

[0054] W was prepared by self-assembly method PD / W saRNA The PD / saRNA ratios are 0, 0.5, 1, 2, 4, 6, and 8. Specifically, a 50 μg / mL saRNA solution is prepared, and then PD solutions of different concentrations are prepared by gradient dilution method, and equal volumes of the two are mixed to obtain PD / saRNA of different ratios.

[0055] 3. Preparation of polyethyleneimine derivatives to encapsulate nucleic acid drugs and connect PEG

[0056] 3.1 Preparation of terminally modified PEG

[0057] 3.1.1 Preparation of terminally modified aldehyde PEG

[0058] 0.25mmol 4-formylbenzoic acid, 0.5mmol EDC, and 0.05mmol DMAP were dissolved in CH2Cl2 and reacted for 30-60min. The molar ratio of 4-formylbenzoic acid to EDC was 1:2, and the molar ratio of DAMP to EDC was 1:10. Then 0.025mmol OH-PEG 2000 -OH is dissolved in the above reaction system and reacted for 12 to 36 hours. 2000 The molar ratio of -OH to 4-formylbenzoic acid was 1: 10. After the reaction was completed, the reaction solution was dialyzed for 48 h using a 10 kDa dialysis bag and then freeze-dried to obtain terminally modified aldehyde PEG (CHO-PEG-CHO).

[0059] 3.1.2 Preparation of terminally modified carboxyl PEG

[0060] Dissolve 0.25mmol 4-(carboxymethyl)benzoic acid, 0.5mmol EDC, and 0.05mmol DMAP in CH2Cl2 and react for 30-60min. The molar ratio of 4-(carboxymethyl)benzoic acid to EDC is 1:2, and the molar ratio of DAMP to EDC is 1:10. Then, dissolve 0.025mmol OH-PEG-OH in the above reaction system and react for 12-36h. The molar ratio of OH-PEG-OH to 4-(carboxymethyl)benzoic acid is 1:10. After the reaction is completed, the reaction solution is dialyzed for 48h using a 10kDa dialysis bag, and then freeze-dried to obtain terminally modified carboxyl PEG.

[0061] 3.1.3 Preparation of terminally modified acyl halide PEG

[0062] 0.25mmol 4-(carboxymethyl)benzoyl chloride, 0.5mmol EDC, and 0.05mmol DMAP were dissolved in CH2Cl2 and reacted for 30-60min. The molar ratio of 4-(carboxymethyl)benzoyl chloride to EDC was 1:2, and the molar ratio of DAMP to EDC was 1:10. Then 0.025mmol OH-PEG-OH was dissolved in the above reaction system and reacted for 12-36h. The molar ratio of OH-PEG-OH to 4-(carboxymethyl)benzoyl chloride was 1:10. After the reaction was completed, the reaction solution was dialyzed for 48h using a 10kDa dialysis bag, and then freeze-dried to obtain terminally modified acyl halide PEG.

[0063] 3.2 Ligation of PEG and PD / siYTHDF1

[0064] 3.2.1 Ligation of terminally modified aldehyde PEG and PD / siYTHDF1

[0065] Preparation of W PD / W siYTHDF1 = 6:1 PD / siYTHDF1, then press W CHO-PEG-CHO / W PD / W FAM-siYTHDF1 =6:6:1 The terminally modified aldehyde PEG solution and PD / siYTHDF1 were mixed in equal volumes to obtain PPD / siYTHDF1 connected to the PEG end or shell.

[0066] 3.2.2 Ligation of terminally modified carboxyl PEG and PD / siYTHDF1

[0067] Using NHS and EDC as catalysts, the terminally modified carboxyl PEG was connected to PD / siYTHDF1 through an amide reaction. The specific process is: 0.4mmol of terminally modified carboxyl PEG, 1mmol NHS and 1mmol EDC were dissolved in about 10mL DMSO and activated for 30-60min; then 0.02mmol PD / siYTHDF1 was added to the above activation system to initiate the amide reaction and stirred for 24h.

[0068] 3.2.3 Ligation of terminally modified acyl halide PEG and PD / siYTHDF1

[0069] The reaction was carried out under alkaline conditions. Commonly used bases include triethylamine, pyridine, sodium hydroxide, etc. The benzoyl halide reacted with the amino group to connect the terminal modified acyl halide PEG to PD / siYTHDF1. 100 mg of PD / siYTHDF1 was dissolved in dichloromethane, and an appropriate amount of NaOH was added. Then, an equal volume of COCl-PEG-COCl of different concentrations was added dropwise under ice bath conditions while stirring. The reaction was carried out for 24 hours to obtain the product.

[0070] The particle size and zeta potential of the siRNA nanocomplexes were then measured using a dynamic light scattering (DLS) analyzer (Brookhaven) and a ZetaPlus-zeta potential analyzer (Brookhaven).

[0071] like Figure 3 As shown, compared with PD / siYTHDF1, the particle size of PPD / siYTHDF1 increased from 113nm to 126nm after connecting the terminal modified aldehyde PEG, and the potential decreased from +36.3mV to +20.0mV.

[0072] Test Example 1

[0073] PPD / siYTHDF1 (W) was prepared by the method of Example 1 CHO-PEG-CHO / W PD / W FAM-siYTHDF1=6:6:1), then the nanoparticles were incubated under RNase conditions for different periods of time, SDS was used to displace the siYTHDF1 therein for agarose gel electrophoresis detection, and the displaced siYTHDF1 was detected to evaluate the degree of degradation of siYTHDF1 in the preparation by RNase, and then the RNase stability of PPD / siYTHDF1 was evaluated. The nanoparticles were placed in an environment containing RNase (10 mg / mL) and incubated in a 37°C water bath, and samples were taken at different time points (0, 2, 4, 8, 12, 24h), 2% SDS was added to displace the siYTHDF1 encapsulated in the nanoparticles, and the exposed siYTHDF1 bands were observed and photographed using agarose gel electrophoresis to evaluate the RNase stability of the preparation.

[0074] like Figure 4 As shown, after incubation with RNase for 24 h, there was no obvious leakage of siYTHDF1, demonstrating the good RNase stability of the preparation.

[0075] Test Example 2

[0076] Flow cytometry was used to quantitatively investigate the cellular uptake of nanoparticles. 4 The cells were evenly inoculated at a density of 100 μg / well in a 24-well plate and cultured for 24 h. The upper layer of waste culture medium was removed by aspiration, and the cells were washed twice with sterile PBS preheated to 37°C. PBS, free FAM-siYTHDF1, PPD / FAM-siYTHDF1 (FAM-siYTHDF1 1.50 μg / mL, W CHO-PEG-CHO / W PD / W FAM-siYTHDF1 =6:6:1, the preparation process is the same as in Example 1), and the cells are cultured for 4 hours. After the incubation, the cells are washed twice with PBS, and the cells are collected by trypsin digestion and then detected by flow cytometry.

[0077] like Figure 5 As shown, PPD / FAM-siYTHDF1 showed higher cellular uptake than free FAM-siYTHDF1.

[0078] Test Example 3

[0079] The lysosomal escape of the preparations was investigated by laser confocal microscopy. 4 The cells were inoculated into the confocal culture dish and cultured at 37°C for 24 h. The PPD / FAM-siYTHDF1 nanocomplex (FAM-siYTHDF1 1.50 μg / mL, W CHO-PEG-CHO / W PD / W FAM-siYTHDF1=6:6:1, the preparation process is the same as in Example 1), co-cultured with cells for 2 h, then washed the cells twice with PBS, added fresh blank culture medium and continued to incubate for 0 or 4 h, then added lysosomal red fluorescent probe LysoTracker Red and incubated at 37°C for 40 min, then washed the cells twice with PBS, and observed and photographed under a laser confocal microscope.

[0080] like Figure 6 As shown in the figure, after 2 h of incubation, the green fluorescence of FAM-siYTHDF1 was detected to be co-localized with the red fluorescence of the lysosomal probe. After 6 h of incubation, a clear separation of the FAM-siYTHDF1 and LysoTracker fluorescence signals was observed, indicating that siYTHDF1 successfully escaped.

[0081] Test Example 4

[0082] Western Blot was used to investigate the silencing effect of the preparation on the YTHDF1 gene. 4T1 cells were seeded in a 12-well plate at a density of 1×10 5 After culturing for 24 h, PBS, DFX, PPD / siNC (siNC concentration was 100 nM, W CHO-PEG-CHO / W PD / W FAM-siNC =6:6:1) and PPD / siYTHDF1 (siYTHDF1 concentration was 100 nM, W CHO-PEG-CHO / W PD / W FAM-siNC =6:6:1) were incubated with 4T1 cells for 4 h, then replaced with blank medium for another 48 h, washed twice with ice-cold PBS, lysed with RIPA lysis buffer, the lysate was collected, centrifuged at 12000 rpm for 10 min, the supernatant was collected to collect various histones, and the expression of YTHDF1 protein was detected by Western Blot.

[0083] like Figure 7 As shown, the YTHDF1 protein level in the PPD / siYTHDF1 group was significantly inhibited, proving that the preparation has a good gene silencing effect.

Claims

1. A polyethyleneimine derivative obtained by modifying an iron chelator with a polyethyleneimine side chain, wherein the iron chelator is deferasirox, cholic acid, droxidopa or carbidopa.

2. The polyethyleneimine derivative according to claim 1, characterized in that The polyethyleneimine is low molecular weight polyethyleneimine, medium molecular weight polyethyleneimine or high molecular weight polyethyleneimine.

3. Use of the polyethyleneimine derivative according to claim 1 in preparing a nucleic acid drug delivery vector.

4. The use according to claim 3, characterized in that: The nucleic acid drug is DNA, mRNA, siRNA, microRNA or saRNA.

5. A nucleic acid drug delivery vector, characterized in that: The nanoparticle comprises a nanoparticle core formed by self-assembly of the polyethyleneimine derivative according to claim 1 and a nucleic acid drug, and a PEG terminal connected to the surface of the nanoparticle core.

6. The nucleic acid drug delivery vector according to claim 5, characterized in that: The nucleic acid drug is DNA, mRNA, siRNA, microRNA or saRNA.

7. The nucleic acid drug delivery vector according to claim 6, characterized in that: The molecular weight of the PEG in the PEG terminal is 200-20000.

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