Polyethyleneimine derivatives and their use as gene drug carriers

Abstract

The application discloses a polyethylene imine derivative and application of the polyethylene imine derivative as a gene medicine carrier, and the polyethylene imine derivative is obtained by connecting an iron chelating agent to a side chain of polyethylene imine through an amide bond. The application takes polyethylene imine as a basic carrier material, connects the iron metabolism regulating medicine through an amide bond to synthesize a polyethylene imine derivative, and then combines with a nucleic acid medicine through self-assembly to form a nanoparticle core, and connects a PEG end to form a final preparation. The application first proposes a combined drug mode targeting epigenetics. Different medicines are respectively directed against tumor iron metabolism and RNA methylation, which can effectively enhance the anti-tumor effect, different from a traditional drug administration mode.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polyethyleneimine derivative and its application as a gene drug carrier. Background Technology

[0002] Epigenetics refers to the biological phenomenon where gene function undergoes heritable variations without altering the DNA sequence, leading to changes in an organism's phenotype. Cancer is an epigenetic disease, and its development is closely related to abnormal gene expression. Abnormal modifications of DNA, RNA, and histones can all affect oncogene expression. With the development of cancer treatment, various anti-tumor drugs have emerged. However, due to tumor heterogeneity and the unique microenvironment, many drugs fail to produce good therapeutic effects. Therefore, targeting epigenetic anti-tumor targets holds promise for providing new directions in cancer treatment. Targets for epigenetics mainly include the inhibition of core enzymes, the inhibition of histone modification recognition, and the inhibition of allosteric organs. Currently, some anti-epigenetic anti-tumor drugs have been approved for marketing, but the development of such drugs still faces many challenges. Most drugs have a single action, weak response in solid tumors, and limited therapeutic effects on tumors. Therefore, drug combination therapy to overcome the limitations of indications will become a new breakthrough.

[0003] mRNA N6-adenosine methylation (m 6 A) is the most common type of RNA modification, when m 6 Abnormalities in A modification can lead to disease, most commonly tumors. Studies have shown that YTHDF1 overexpression is present in many cancers, and YTHDF1 acts as an m... 6 A reading protein can accelerate mRNA translation, promoting tumor growth; it also restricts MHC-I expression, degrades antigens, and disables tumor immunity. Iron metabolism is also closely related to epigenetic regulation; some key epigenetic enzymes, such as DNA demethylases, require iron ions to function. Therefore, linking iron metabolism regulation with mRNA expression is crucial for tumor growth. 6 Combining the inhibition of A-related enzymes with dual regulation of epigenetics could be an effective strategy for developing anti-tumor drugs. Summary of the Invention

[0004] One objective of this invention is to provide a polyethyleneimine derivative obtained by modifying the side chain of polyethyleneimine with an iron chelating agent, wherein the iron chelating agent is derarosiform, cholic acid, drosidopa, or carbidopa.

[0005] Further, the polyethyleneimine is a low molecular weight polyethyleneimine (<1000), a medium molecular weight polyethyleneimine (1000-10000), or a high molecular weight polyethyleneimine (>10000).

[0006] In this invention, NHS and EDC are first used to activate the side chains of polyethyleneimine, and then an iron chelating agent is added to react and the two are linked by amide bonds.

[0007] A second objective of this invention is to provide the application of the above-mentioned polyethyleneimine derivative in the preparation of nucleic acid drug delivery carriers.

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

[0009] A third objective of this invention is to provide a nucleic acid drug delivery carrier, comprising a nanoparticle core formed by the self-assembly of the aforementioned polyethyleneimine derivative and a nucleic acid drug, and PEG ends attached to the surface of the nanoparticle core. The PEG ends can be linked to the nanoparticle core via active groups such as -CHO, -COOH, or -COX (X = F, Cl, Br).

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

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

[0012] Step 1: Dissolve 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 react for 30-60 min.

[0013] Step 2: Dissolve PEI in the reaction system of Step 1, with a molar ratio of PEI to DFX of 1:10 to 1:40, and react for 12 to 36 hours.

[0014] Step 3: Dialyze, freeze-dry and store the solution obtained in Step 2 to obtain PEI-DFX;

[0015] The reaction equation is as follows:

[0016]

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

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

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

[0020] Step 1: Dissolve benzoic acid derivative, EDC, and DAMP in CH2Cl2 and react for 30-60 min. The molar ratio of benzoic acid derivative to EDC is 1:1 to 1:2, and the molar ratio of DAMP to EDC is 1:5 to 1:10.

[0021] Step 2: Dissolve OH-PEG-OH in the reaction system of Step 1 and react for 12-36 h. The molar ratio of OH-PEG-OH to benzoic acid derivative is 1:10.

[0022] Step 3: Dialyze, freeze-dry and store the solution obtained in Step 2 to obtain the terminal-modified PEG.

[0023] The reaction equation is as follows:

[0024]

[0025] This invention uses polyethyleneimine (PEI) as the basic carrier material, linking it to an iron chelating agent (deferrasirox DFX) via amide bonds to synthesize a polyethyleneimine derivative (PEI-DFX). This derivative is then self-assembled with a nucleic acid drug (which downregulates YTHDF1 expression) and attached to a PEG terminus to form the final formulation (PPD / siYTHDF1). By using PEG to form the terminus or shell on PD / siYTHDF, and with deferrasirox acting as an iron chelating agent to disrupt iron metabolism in tumor cells, the nucleic acid drug inhibits YTHDF1 expression, leading to a decrease in tumor DNA synthesis. The combined use of these two drugs causes cell cycle arrest in tumor cells, thereby inhibiting tumor growth.

[0026] Beneficial effects:

[0027] 1. This invention is the first to propose a combination drug therapy targeting epigenetics. Unlike traditional drug administration methods, 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 this invention is simple to synthesize, has strong universality, good reproducibility of the preparation process, good carrier stability, and is easy to translate into clinical applications.

[0029] 3. The formulation prepared by this invention can improve the bioavailability of drugs, enhance the stability of nucleic acid drug delivery, and has good prospects for the development of drug combination strategies, thus possessing high clinical value. Attached Figure Description

[0030] Figure 1 The image shows the proton NMR spectrum of the prepared PEI-DFX.

[0031] Figure 2 The results show the gene inclusion detection results for PD / siYTHDF1.

[0032] Figure 3 The particle size potential characterization results for PD / siYTHDF1 and PPD / siYTHDF1 are shown.

[0033] Figure 4 The results show the RNase stability of PPD / siYTHDF1.

[0034] Figure 5 The results show the cellular uptake of PPD / FAM-siYTHDF1.

[0035] Figure 6 The results show the lysosomal escape of PPD / FAM-siYTHDF1.

[0036] Figure 7 This refers to the silencing effect of the YTHDF1 gene. Detailed Implementation

[0037] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

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

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] Example 1

[0041] 1. Synthesis of polyethyleneimine derivatives

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

[0043] Using NHS and EDC as catalysts, derafloxacin (DFX) was modified onto polyethyleneimine (PEI) via an amide reaction to synthesize PEI-DFX. The specific procedure was as follows: 0.4 mmol DFX, 1 mmol NHS, and 1 mmol EDC were dissolved in approximately 10 mL of DMSO and activated for 30-60 min; then, 0.02 mmol PEI was added to the activated system to initiate the amide reaction, and the mixture was stirred for 24 h. The molar ratio of PEI to DFX was 1:20. After the reaction was complete, the reaction solution was dialyzed through a 10 kDa dialysis bag for 48 h, then lyophilized, and subjected to further processing. 1 H-NMR analysis.

[0044] like Figure 1 As shown, according to 1 The H-NMR results showed that the proton peak of PEI appeared at 2.0-2.9 ppm, and the proton peak of the 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] Carbidopa was modified onto polyethyleneimine (PEI) via an amide reaction using NHS and EDC as catalysts to synthesize PEI-Carbidopa (PC). The specific procedure was as follows: 0.4 mmol DFX, 1 mmol NHS, and 1 mmol EDC were dissolved in approximately 10 mL of DMSO and activated for 30–60 min; then 0.02 mmol PEI was added to the activated system to initiate the amide reaction, and the mixture was stirred for 24 h. The molar ratio of PEI to Carbidopa was 1:20. After the reaction was complete, the reaction solution was dialyzed through a 10 kDa dialysis bag for 48 h, and then lyophilized.

[0047] 2. Preparation of nucleic acid drugs encapsulated in polyethyleneimine derivatives

[0048] 2.1 Preparation of PD-encapsulated siYTHDF1

[0049] W was prepared by self-assembly method. PD / W siYTHDF1 PD / siYTHDF1 ratios of 0, 0.5, 1, 2, 4, 6, and 8 were obtained. Specifically, a 50 μg / mL siYTHDF1 solution was prepared, and then PD solutions of different concentrations were prepared using a serial dilution method. Equal volumes of both solutions were mixed to obtain different PD / siYTHDF1 ratios. Agarose gel electrophoresis was then performed at 100V for 15 min, and the exposed siYTHDF1 bands were observed and photographed using a gel imaging system.

[0050] like Figure 2 As shown, in W PD / W siYTHDF1 When the value is 6 or higher, 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 PD / CRISPR ratios of 0, 0.5, 1, 2, 4, 6, and 8 were obtained. Specifically, a 50 μg / mL CRISPR solution was prepared, and then PD solutions of different concentrations were prepared using a gradient dilution method. Equal volumes of the two solutions were mixed to obtain PD / CRISPR ratios of different proportions.

[0053] 2.3 Preparation of PD-coated saRNA

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

[0055] 3. Preparation of polyvinylimine derivatives to encapsulate nucleic acid drugs and ligate them with PEG.

[0056] 3.1 Preparation of terminally modified PEG

[0057] 3.1.1 Preparation of PEG with terminal aldehyde groups

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

[0059] 3.1.2 Preparation of PEG with terminal carboxyl groups

[0060] 0.25 mmol of 4-(carboxymethyl)benzoic acid, 0.5 mmol of EDC, and 0.05 mmol of DMAP were dissolved in CH2Cl2 and reacted for 30–60 min. The molar ratio of 4-(carboxymethyl)benzoic acid to EDC was 1:2, and the molar ratio of DAMP to EDC was 1:10. Then, 0.025 mmol of OH-PEG-OH was dissolved in the above reaction system, and the reaction was carried out for 12–36 h. The molar ratio of OH-PEG-OH to 4-(carboxymethyl)benzoic acid was 1:10. After the reaction was complete, the reaction solution was dialyzed through a 10 kDa dialysis bag for 48 h, and then lyophilized to obtain terminally modified carboxyl PEG.

[0061] 3.1.3 Preparation of terminally modified acyl halide PEG

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

[0063] 3.2 Connection 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 Mix the terminal-modified aldehyde PEG solution with PD / siYTHDF1 in equal volume 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, terminally modified carboxyl PEG was linked to PD / siYTHDF1 via an amide reaction. Specifically, 0.4 mmol of terminally modified carboxyl PEG, 1 mmol of NHS, and 1 mmol of EDC were dissolved in approximately 10 mL of DMSO and activated for 30-60 min. Then, 0.02 mmol of PD / siYTHDF1 was added to the activated system to initiate the amide reaction, and the mixture was stirred for 24 h.

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

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

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

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

[0072] Test Example 1

[0073] PPD / siYTHDF1(W) was prepared using the method described in Example 1. CHO-PEG-CHO / W PD / W FAM-siYTHDF1The nanoparticles were incubated with RNase at a ratio of 6:6:1 for different time periods. SDS was then used to replace the siYTHDF1 within the nanoparticles, and the results were analyzed by agarose gel electrophoresis. The extent of RNase degradation of siYTHDF1 in the formulation was evaluated by detecting the replaced siYTHDF1, thus assessing the RNase stability of PPD / siYTHDF1. The nanoparticles were placed in an environment containing RNase (10 mg / mL) and incubated at 37°C. Samples were taken at different time points (0, 2, 4, 8, 12, 24 h), and 2% SDS was added to replace the siYTHDF1 encapsulated in the nanoparticles. Agarose gel electrophoresis was used to observe and photograph the exposed siYTHDF1 bands to evaluate the RNase stability of the formulation.

[0074] like Figure 4 As shown, after incubation with RNase for 24 hours, siYTHDF1 showed no significant leakage, demonstrating the good RNase stability of the formulation.

[0075] Test Example 2

[0076] Quantitative cellular uptake of nanoparticles was assessed using flow cytometry. 4T1 cells were cultured at a concentration of 1 × 10⁻⁶. 4 The culture medium was evenly seeded into 24-well plates at a uniform density and incubated for 24 hours. The supernatant was removed, and the plates were washed twice with sterile PBS preheated to 37°C. PBS, free FAM-siYTHDF1, and PPD / FAM-siYTHDF1 (FAM-siYTHDF1 1.50 μg / mL, W) were then diluted with blank culture medium. CHO-PEG-CHO / W PD / W FAM-siYTHDF1 The ratio of the cultures was 6:6:1 (preparation process as in Example 1), and the cells were cultured for 4 hours. After incubation, the cells were washed twice with PBS, digested with trypsin to collect the cells, and then analyzed by flow cytometry.

[0077] like Figure 5 As shown, PPD / FAM-siYTHDF1 exhibited higher cellular uptake compared to free FAM-siYTHDF1.

[0078] Test Example 3

[0079] Lysosomal escape of the formulation was examined using laser confocal microscopy. 4T1 cells were used at a concentration of 2 × 10⁻⁶. 4 / wells were seeded in confocal culture dishes and cultured at 37°C for 24 h. The PPD / FAM-siYTHDF1 nanocomposite (FAM-siYTHDF1 1.50 μg / mL, W) was diluted with blank medium. CHO-PEG-CHO / W PD / W FAM-siYTHDF1=6:6:1, the preparation process is as in Example 1), co-culture with cells for 2h, then wash the cells twice with PBS, add fresh blank culture medium and continue incubation for 0 or 4h, then add lysosomal red fluorescent probe LysoTracker Red and incubate at 37℃ for 40min, then wash the cells twice with PBS, and observe and photograph them using a laser confocal microscope.

[0080] like Figure 6 As shown, after 2 hours of incubation, the green fluorescence of FAM-siYTHDF1 and the red fluorescence of the lysosomal probe were detected to colocalize. After 6 hours of incubation, a clear separation of the fluorescence signals of FAM-siYTHDF1 and LysoTracker was observed, indicating that siYTHDF1 successfully escaped.

[0081] Test Example 4

[0082] The silencing effect of the formulation on the YTHDF1 gene was investigated by Western blotting. 4T1 cells were seeded in 12-well plates at a density of 1 × 10⁻⁶ cells / well. 5 / well. After culturing for 24 hours, PBS, DFX, and PPD / siNC (siNC concentration of 100 nM, W) were added. CHO-PEG-CHO / W PD / W FAM-siNC =6:6:1) and PPD / siYTHDF1 (siYTHDF1 concentration is 100 nM, W CHO-PEG-CHO / W PD / W FAM-siNC YTHDF1 protein was incubated with 4T1 cells in a 6:6:1 ratio for 4 h, then incubated with blank medium for another 48 h. After washing twice with ice-cold PBS, the cells were lysed with RIPA lysis buffer, the lysis buffer was collected, centrifuged at 12000 rpm for 10 min, and the supernatant was collected to collect the proteins of each group. The expression of YTHDF1 protein was detected by Western Blot.

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

Claims

1. A nucleic acid drug delivery vector, characterized in that, It includes a nanoparticle core formed by the self-assembly of a polyethyleneimine derivative and a nucleic acid drug, and PEG ends attached to the surface of the nanoparticle core; The polyethyleneimine derivative was obtained by modifying the side chain of polyethyleneimine with an iron chelating agent, wherein the iron chelating agent is derarosilicate, which is linked to the side chain of polyethyleneimine via an amide bond. The nucleic acid drug is siRNA; The PEG terminus is a terminally modified aldehyde PEG, prepared from 4-formylbenzoic acid and OH-PEG-OH, and its reaction formula is as follows: ， The molecular weight of the PEG at the PEG end is 200-20000; The mass ratio of the polyethyleneimine derivative to siRNA is 6–12:1; the mass ratio of the polyethyleneimine derivative to the terminally modified aldehyde PEG is 1:1.

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