An N, N-dimethylethylenediamine-thiodiglycolic acid monocholesteryl ester conjugate, a preparation method and application thereof
By preparing an N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate and binding it to lecithin, the particle size and potential of cationic liposomes were adjusted, solving the problems of low transfection efficiency and high cytotoxicity of cationic liposomes in gene delivery, and achieving efficient and safe gene delivery.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHANGZHOU UNIV
- Filing Date
- 2023-11-30
- Publication Date
- 2026-07-03
AI Technical Summary
Existing cationic liposomes suffer from low transfection efficiency and high cytotoxicity in gene delivery, especially due to increased cytotoxicity caused by the use of highly positively charged liposomes.
An N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate was prepared and combined with lecithin. By adjusting the ratio, the particle size and potential were adjusted to form low positively charged cationic liposomes. The high efficiency of gene transfection and low cytotoxicity were achieved by utilizing the easy hydrolysis of ester bonds and the oxidative responsiveness of sulfur atoms.
This approach achieves a combination of high transfection efficiency and low cytotoxicity. By adjusting the ratio of lecithin to conjugates, the particle size and potential of liposomes are regulated, reducing cytotoxicity and improving the safety and effectiveness of gene delivery.
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Figure CN117736254B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, and specifically relates to an N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate, its preparation method, and its application. Background Technology
[0002] Liposomes are closed vesicle-like structures composed of a phospholipid bilayer. Liposomes possess good biocompatibility and biodegradability, and have been developed for delivering various drugs, including small-molecule chemotherapy drugs, gene therapy drugs, and protein drugs. Currently, several drugs using liposomes as carriers have been approved by the FDA for clinical use, achieving good results.
[0003] Cationic liposomes hold significant value in gene drug delivery. They can bind to DNA or RNA to form complexes, facilitating the entry of these nucleic acids into cells and thus enabling gene delivery and expression. However, the intracellular gene release from currently developed cationic liposomes is uncontrollable. Achieving better transfection efficiency requires the use of cationic liposomes with higher positive charges, but this can lead to increased cytotoxicity. This technical challenge remains to be solved. Summary of the Invention
[0004] The technical problem to be solved by this invention is: how to prepare cationic liposomes with high transfection efficiency and low cytotoxicity. To solve this technical problem, this invention provides an N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate, with the following structural formula:
[0005]
[0006] The present invention also provides a method for preparing the above-mentioned N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate:
[0007] (1) Preparation of thiodiethylene glycol monocholesterol ester
[0008] Cholesterol and thiodiethylene glycol anhydride were dissolved together, and N,N-dimethylaminopyridine (DMAP) was added to the solution. The mixture was then heated under reflux. After the reaction, the solvent was evaporated to dryness, and the solution was recrystallized using ethyl acetate and ethanol to obtain purified thiodiethylene glycol monocholesterol ester. The reaction formula is as follows:
[0009]
[0010] (2) After dissolving the thiodiethylene glycol monocholesterol ester obtained in step (1) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N,N-dimethylethylenediamine was added and the mixture was stirred to react. After the reaction, the solvent was evaporated to dryness, dispersed in water, dialyzed in ultrapure water, centrifuged, and the supernatant was freeze-dried to obtain the N,N-dimethylethylenediamine-thiodiethylene glycol monocholesterol ester conjugate. The reaction formula is as follows:
[0011]
[0012] As a preferred option, in step (1), the molar ratio between cholesterol, thiodiglycolic anhydride, and N,N-dimethylaminopyridine is 1:1 to 2:0.5 to 1.
[0013] As a preferred option: in step (1), cholesterol and thiodiglycolic anhydride are dissolved together in dichloromethane.
[0014] As a preferred embodiment, in step (2), the molar ratio between thiodiglycolic acid monocholesterol ester, N,N-dimethylethylenediamine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1 to 2:2 to 10.
[0015] As a preferred embodiment, in step (2), thiodiglycolic acid monocholesterol ester and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) are dissolved together in dichloromethane.
[0016] Preferably, in step (2), the stirring reaction is carried out at room temperature (25°C).
[0017] The present invention also provides an application of N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate as a component of cationic liposomes.
[0018] Preferably, the cationic liposomes also include lecithin, and the mass ratio of lecithin and N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate is 3 to 10:1.
[0019] As a preferred method, the preparation of cationic liposomes involves dissolving lecithin and N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate together, evaporating the solvent, dispersing it with water, and then ultrasonically breaking it down to obtain cationic liposomes.
[0020] Furthermore: lecithin and N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate were co-dissolved in ethanol.
[0021] Furthermore: the ultrasonic fragmentation power is 90W, and the time is 15 minutes.
[0022] The beneficial effects of this invention are as follows: The cationic liposomes based on the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate have a low degree of positive charge (potential). The lower the positive charge, the lower the cytotoxicity and the higher the safety. Simultaneously, based on the ease with which the ester bond in the conjugate structure can be hydrolyzed by esterases and the oxidative responsiveness of the sulfur atom, the cationic liposomes can effectively release the loaded gene, achieving efficient transfection. Therefore, this cationic liposome achieves comprehensive efficacy while simplifying its composition, enabling high gene transfection efficiency and low cytotoxicity. By adjusting the ratio of lecithin to the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate, the particle size and potential of the cationic liposomes can be adjusted. Attached Figure Description
[0023] Figure 1 The NMR spectrum of the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate synthesized in Example 4 is shown in 1H NMR. Detailed Implementation
[0024] A method for preparing the above-mentioned N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate:
[0025] (1) Preparation of thiodiethylene glycol monocholesterol ester
[0026] Cholesterol and thiodiethylene glycol anhydride were dissolved together in dichloromethane, and then N,N-dimethylaminopyridine (DMAP) was added. The mixture was heated under reflux for 24 hours. The molar ratio of cholesterol, thiodiethylene glycol anhydride, and N,N-dimethylaminopyridine was 1:1–2:0.5–1. After the reaction, the solvent was evaporated to dryness, and the mixture was recrystallized using ethyl acetate and ethanol to obtain purified thiodiethylene glycol monocholesterol ester. The reaction formula is as follows:
[0027]
[0028] (2) The thiodiglycolic acid monocholesterol ester and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) obtained in step (1) were dissolved together in dichloromethane, and N,N-dimethylethylenediamine was added. The mixture was stirred at room temperature for 24 hours. The molar ratio between thiodiglycolic acid monocholesterol ester, N,N-dimethylethylenediamine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was 1:1~2:2~10. After the reaction, the solvent was evaporated to dryness, and after being dispersed in water, it was dialyzed in ultrapure water for 3 days. After centrifugation at 8000 rpm for 10 minutes, the supernatant was collected and freeze-dried to obtain the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate. The reaction formula is as follows:
[0029]
[0030] A method for preparing cationic liposomes:
[0031] Lecithin and N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate were dissolved together in ethanol at a mass ratio of 3 to 10:1. The solvent was then evaporated by heating to 50°C. After dispersion in ultrapure water, the mixture was ultrasonically broken up at 90W for 15 minutes to obtain cationic liposomes.
[0032] Example 1
[0033] Preparation of thiodiethylene glycol monocholesterol ester:
[0034] 2.0 g of cholesterol and 2.06 g of thiodiethylene glycol anhydride were dissolved together in 20 mL of dichloromethane. Then, 0.32 g of N,N-dimethylaminopyridine (DMAP) was added and stirred thoroughly to dissolve the mixture. The mixture was then heated to 43 °C and refluxed for 24 hours. After the reaction, the dichloromethane solvent was evaporated to dryness, and the mixture was recrystallized using a mixed solvent of ethyl acetate and ethanol (volume ratio 1:1) to obtain purified thiodiethylene glycol monocholesterol ester with a yield of 89% (moles of thiodiethylene glycol monocholesterol ester obtained ÷ moles of cholesterol in the reactants × 100%, the same below).
[0035] Example 2
[0036] Preparation of thiodiethylene glycol monocholesterol ester:
[0037] 2.0 g of cholesterol and 1.03 g of thiodiethylene glycol anhydride were dissolved together in 20 mL of dichloromethane. Then, 0.32 g of N,N-dimethylaminopyridine (DMAP) was added and stirred thoroughly to dissolve the mixture. The mixture was then heated to 43 °C and refluxed for 24 hours. After the reaction, the dichloromethane solvent was evaporated to dryness, and the mixture was recrystallized using a mixed solvent of ethyl acetate and ethanol (volume ratio 1:1) to obtain purified thiodiethylene glycol monocholesterol ester with a yield of 78%.
[0038] Example 3
[0039] Preparation of thiodiethylene glycol monocholesterol ester:
[0040] 2.0 g of cholesterol and 1.03 g of thiodiethylene glycol anhydride were dissolved together in 20 mL of dichloromethane. Then, 0.64 g of N,N-dimethylaminopyridine (DMAP) was added and stirred thoroughly to dissolve the mixture. The mixture was then heated to 43 °C and refluxed for 24 hours. After the reaction, the dichloromethane solvent was evaporated to dryness, and the mixture was recrystallized using a mixed solvent of ethyl acetate and ethanol (volume ratio 1:1) to obtain purified thiodiethylene glycol monocholesterol ester with a yield of 80%.
[0041] Example 4
[0042] Preparation of N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate:
[0043] 0.26 g of the thiodiethylene glycol monocholesterol ester prepared in Example 1 above and 0.96 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were dissolved together in 30 mL of dichloromethane. Then, 88 mg of N,N-dimethylethylenediamine was added, and the mixture was stirred at room temperature for 24 hours. After the reaction, the dichloromethane solvent was evaporated to dryness, and 20 mL of water was added to disperse the mixture. The mixture was then dialyzed in ultrapure water for 3 days (molecular weight cutoff 500). After centrifugation at 8000 rpm for 10 minutes, the supernatant was collected and freeze-dried to obtain the N,N-dimethylethylenediamine-thiodiethylene glycol monocholesterol ester conjugate, with a yield of 86% (moles of the obtained N,N-dimethylethylenediamine-thiodiethylene glycol monocholesterol ester conjugate ÷ moles of thiodiethylene glycol monocholesterol ester in the reactants × 100%, the same below).
[0044] The N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate synthesized in Example 4 was characterized by proton nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry, such as... Figure 1 As shown, the 1H NMR spectrum of the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate simultaneously exhibits the characteristic hydrogen signals of N,N-dimethylethylenediamine (at 2.0–3.0 ppm) and those of thiodiglycolic acid (at 3.0–4.0 ppm); simultaneously, the high-resolution mass spectra of the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate show [M+H]. + The characteristic signal (m / z 589.4410) indicates the successful preparation of the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate.
[0045] Example 5
[0046] Preparation of N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate:
[0047] 0.26 g of the thiodiethylene glycol monocholesterol ester prepared in Example 1 above and 0.48 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were dissolved together in 30 mL of dichloromethane. Then, 44 mg of N,N-dimethylethylenediamine was added, and the mixture was stirred at room temperature for 24 hours. After the reaction, the dichloromethane solvent was evaporated to dryness, and 20 mL of water was added to disperse the mixture. The mixture was then dialyzed in ultrapure water for 3 days (molecular weight cutoff 500). After centrifugation at 8000 rpm for 10 minutes, the supernatant was collected and freeze-dried to obtain the N,N-dimethylethylenediamine-thiodiethylene glycol monocholesterol ester conjugate with a yield of 79%.
[0048] Example 6
[0049] Preparation of N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate:
[0050] 0.26 g of the thiodiethylene glycol monocholesterol ester prepared in Example 1 above and 0.19 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were dissolved together in 30 mL of dichloromethane. Then, 88 mg of N,N-dimethylethylenediamine was added, and the mixture was stirred at room temperature for 24 hours. After the reaction, the dichloromethane solvent was evaporated to dryness, and 20 mL of water was added to disperse the mixture. The mixture was then dialyzed in ultrapure water for 3 days (molecular weight cutoff 500). After centrifugation at 8000 rpm for 10 minutes, the supernatant was collected and freeze-dried to obtain the N,N-dimethylethylenediamine-thiodiethylene glycol monocholesterol ester conjugate with a yield of 71%.
[0051] Example 7
[0052] Preparation of cationic liposomes:
[0053] 150 mg of lecithin and 50 mg of the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate prepared in Example 4 were added to 10 mL of ethanol and dissolved completely. The solution was then heated to 50 °C and the solvent was evaporated. 20 mL of ultrapure water was added and the solution was dispersed and then sonicated at 90 W for 15 minutes to obtain a cationic liposome dispersion with a concentration of 10 mg / mL.
[0054] Example 8
[0055] Preparation of cationic liposomes:
[0056] 350 mg of lecithin and 50 mg of the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate prepared in Example 4 were added to 20 mL of ethanol and dissolved completely. The solution was then heated to 50 °C and the solvent was evaporated. 40 mL of ultrapure water was added and the solution was dispersed and then sonicated at 90 W for 15 minutes to obtain a cationic liposome dispersion with a concentration of 10 mg / mL.
[0057] Example 9
[0058] Preparation of cationic liposomes:
[0059] 500 mg of lecithin and 50 mg of the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate prepared in Example 4 were added to 20 mL of ethanol and dissolved completely. The solution was then heated to 50 °C and the solvent was evaporated. 55 mL of ultrapure water was added and the solution was dispersed and then sonicated at 90 W for 15 minutes to obtain a cationic liposome dispersion with a concentration of 10 mg / mL.
[0060] Comparative Example 1
[0061] Preparation of 3β-[N-(dimethylaminoethyl)-carbamoyl]cholesterol conjugates and cationic liposomes:
[0062] 2.0 g of cholesterol and 0.84 g of carbonyl diimidazole were dissolved together in 50 mL of dichloromethane and reacted at room temperature for 6 hours. Then, 0.45 g of N,N-dimethylethylenediamine was added, and the reaction was continued overnight at room temperature. After the reaction, the dichloromethane solvent was evaporated to dryness, and 100 mL of ultrapure water was added to disperse the mixture. The mixture was then dialyzed against ultrapure water for 3 days (molecular weight cutoff 500). After centrifugation at 8000 rpm for 10 minutes, the supernatant was collected and freeze-dried to obtain 3β-[N-(dimethylaminoethyl)-carbamoyl]cholesterol conjugate with a yield of 64%. The structure of the product is as follows:
[0063]
[0064] 350 mg of lecithin and 50 mg of the 3β-[N-(dimethylaminoethyl)-carbamoyl]cholesterol conjugate prepared above were added to 20 mL of ethanol and dissolved completely. The solution was heated to 50 °C and the solvent was evaporated. Then, 40 mL of ultrapure water was added and dispersed. The solution was then sonicated at 90 W for 15 minutes to obtain a cationic liposome dispersion with a concentration of 10 mg / mL.
[0065] Comparative Example 2
[0066] Preparation of N,N-dimethylethylenediamine-succinic acid monocholesterol ester conjugate and cationic liposomes:
[0067] 2.0 g cholesterol and 1.56 g succinic anhydride were dissolved together in 20 mL of dichloromethane. Then, 0.32 g of N,N-dimethylaminopyridine (DMAP) was added and stirred thoroughly to dissolve the mixture. The mixture was then heated to 43 °C and refluxed for 24 hours. After the reaction, the dichloromethane solvent was evaporated to dryness, and the mixture was recrystallized using a mixed solvent of ethyl acetate and ethanol (volume ratio 1:1) to obtain purified succinic monocholesterol ester with a yield of 85%.
[0068] 0.24 g of the prepared succinic acid monocholesterol ester and 0.96 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were dissolved together in 30 mL of dichloromethane. Then, 88 mg of N,N-dimethylethylenediamine was added, and the mixture was stirred at room temperature for 24 hours. After the reaction, the dichloromethane solvent was evaporated to dryness, and 20 mL of water was added to disperse the mixture. The mixture was then dialyzed in ultrapure water for 3 days (molecular weight cutoff 500). After centrifugation at 8000 rpm for 10 minutes, the supernatant was collected and freeze-dried to obtain the N,N-dimethylethylenediamine-succinic acid monocholesterol ester conjugate with a yield of 86%. The structure of the product is as follows:
[0069]
[0070] 350 mg of lecithin and 50 mg of the N,N-dimethylethylenediamine-succinic acid monocholesterol ester conjugate prepared above were added to 20 mL of ethanol and dissolved completely. The solution was then heated to 50 °C and the solvent was evaporated. 40 mL of ultrapure water was added and the solution was dispersed and then sonicated at 90 W for 15 minutes to obtain a cationic liposome dispersion with a concentration of 10 mg / mL.
[0071] Comparative Example 3
[0072] Preparation of N,N-dimethylethylenediamine-glutaric acid monocholesterol ester conjugate and cationic liposomes:
[0073] 2.0 g cholesterol and 1.78 g glutaric anhydride were dissolved together in 20 mL of dichloromethane. Then, 0.32 g of N,N-dimethylaminopyridine (DMAP) was added and stirred thoroughly to dissolve the mixture. The mixture was then heated to 43 °C and refluxed for 24 hours. After the reaction, the dichloromethane solvent was evaporated to dryness, and the mixture was recrystallized using a mixed solvent of ethyl acetate and ethanol (volume ratio 1:1) to obtain purified glutaric acid monocholesterol ester with a yield of 82%.
[0074] 0.25 g of the prepared glutaric acid monocholesterol ester and 0.96 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were dissolved together in 30 mL of dichloromethane. Then, 88 mg of N,N-dimethylethylenediamine was added, and the mixture was stirred at room temperature for 24 hours. After the reaction, the dichloromethane solvent was evaporated to dryness, and 20 mL of water was added to disperse the mixture. The mixture was then dialyzed in ultrapure water for 3 days (molecular weight cutoff 500). After centrifugation at 8000 rpm for 10 minutes, the supernatant was collected and freeze-dried to obtain the N,N-dimethylethylenediamine-succinic acid monocholesterol ester conjugate with a yield of 82%.
[0075] 350 mg of lecithin and 50 mg of the N,N-dimethylethylenediamine-glutaric acid monocholesterol ester conjugate prepared above were added to 20 mL of ethanol and dissolved completely. The solution was then heated to 50 °C and the solvent was evaporated. 40 mL of ultrapure water was added and the solution was dispersed and then sonicated at 90 W for 15 minutes to obtain a cationic liposome dispersion with a concentration of 10 mg / mL.
[0076] Comparative Example 4
[0077] Preparation of N,N-dimethylethylenediamine-diethylene glycol monocholesterol ester conjugate and cationic liposomes:
[0078] 2.0 g cholesterol and 1.81 g diethylene glycol anhydride were dissolved together in 20 mL of dichloromethane. Then, 0.32 g of N,N-dimethylaminopyridine (DMAP) was added and stirred thoroughly to dissolve the mixture. The mixture was then heated to 43 °C and refluxed for 24 hours. After the reaction, the dichloromethane solvent was evaporated to dryness, and the mixture was recrystallized using a mixed solvent of ethyl acetate and ethanol (volume ratio 1:1) to obtain purified diethylene glycol monocholesterol ester with a yield of 85%.
[0079] 0.25 g of the prepared diethylene glycol monocholesterol ester and 0.96 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were dissolved together in 30 mL of dichloromethane. Then, 88 mg of N,N-dimethylethylenediamine was added, and the mixture was stirred at room temperature for 24 hours. After the reaction, the dichloromethane solvent was evaporated to dryness, and 20 mL of water was added to disperse the mixture. The mixture was then dialyzed in ultrapure water for 3 days (molecular weight cutoff 500). After centrifugation at 8000 rpm for 10 minutes, the supernatant was collected and freeze-dried to obtain the N,N-dimethylethylenediamine-succinic acid monocholesterol ester conjugate with a yield of 77%.
[0080] 350 mg of lecithin and 50 mg of the N,N-dimethylethylenediamine-diglycolic acid monocholesterol ester conjugate prepared above were added to 20 mL of ethanol and dissolved completely. The solution was then heated to 50 °C and the solvent was evaporated. 40 mL of ultrapure water was added and the solution was dispersed and then sonicated at 90 W for 15 minutes to obtain a cationic liposome dispersion with a concentration of 10 mg / mL.
[0081] The particle size and potential of the cationic liposomes prepared in Examples 7-9 and the comparative examples were measured using a laser particle size and zeta potential analyzer. The results are shown in Table 1.
[0082] Table 1
[0083]
[0084] As shown in Table 1, the particle size and potential of the cationic liposomes prepared in Examples 7-9 show that as the mass ratio of lecithin to N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate increases, the particle size and positive charge of the prepared cationic liposomes both show a decreasing trend. This indicates that the particle size and potential of cationic liposomes can be adjusted by adjusting the ratio of lecithin to N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate.
[0085] Example 8 maintains the same lecithin:conjugate ratio as the comparative examples. However, the liposomes in Example 8 exhibit a significantly lower positive charge (potential) compared to those in Comparative Examples 1 to 4, which were constructed using conjugates based on other structures. The applicant believes this is due to the synergistic effect between different segments of the conjugate's molecular structure in Example 8, resulting in a reduction of its own positive charge. Liposomes with lower positive charge exhibit lower cytotoxicity and higher safety in use. Gene transfection test:
[0086] Fill a 6-well plate with 2×10⁻⁶ holes per well. 5 Human liver cancer cells (HepG2), human breast cancer cells (MCF-7), or human prostate cancer cells (C4-2) were seeded at a density of 100% and cultured in a CO2 incubator at 37°C. When the cell density reached approximately 80%, the original culture medium was aspirated, and the cells were washed three times with phosphate-buffered saline. 2 mL of OPTI-MEM medium was added to each well, and the cells were then placed back into the incubator for further use.
[0087] 4 μg of green fluorescent protein particle pEGFP-C1 was gently mixed with 250 μL of OPTI-MEM medium and allowed to stand for 5 minutes. 100 μL of the cationic liposome dispersion prepared in Example 7 was gently mixed with 150 μL of OPTI-MEM medium and allowed to stand for 5 minutes. The two mixtures were then gently mixed and allowed to stand for 20 minutes (the amount used per well, the same below). The mixture was then added to the wells of the corresponding 6-well plate (n=3) and cultured in the incubator for 6 hours. The mixed culture medium in the wells was then replaced with normal culture medium (DMEM medium was used as the normal culture medium for HepG2; 1640 medium was used as the normal culture medium for MCF-7 or C4-2, the same below). After culturing for 18 hours, the expression level of green fluorescent protein in HepG2, MCF-7, and C4-2 cells was detected by flow cytometry (the average expression level of green fluorescent protein in the same cancer cell type was taken, n=3).
[0088] 4 μg of green fluorescent protein particle pEGFP-C1 was gently mixed with 250 μL of OPTI-MEM medium and allowed to stand for 5 minutes. 100 μL of the cationic liposome dispersion prepared in Example 8 was gently mixed with 150 μL of OPTI-MEM medium and allowed to stand for 5 minutes. The two mixtures were then gently mixed and allowed to stand for 20 minutes. The mixture was then added to the wells of the corresponding 6-well plates (n=3) and cultured in the incubator for 6 hours. The mixed culture medium in the wells was then replaced with normal culture medium and cultured for another 18 hours. The expression levels of green fluorescent protein in HepG2, MCF-7, and C4-2 cells were detected by flow cytometry (the average expression level of green fluorescent protein in the same cancer cell type was taken, n=3).
[0089] 4 μg of green fluorescent protein particle pEGFP-C1 was gently mixed with 250 μL of OPTI-MEM medium and allowed to stand for 5 minutes; 100 μL of the cationic liposome dispersion prepared in Example 9 was gently mixed with 150 μL of OPTI-MEM medium and allowed to stand for 5 minutes; then the two were gently mixed and allowed to stand for 20 minutes, and then added to the wells of the corresponding 6-well plates (n=3). The plates were then incubated in the incubator for 6 hours. After that, the mixed culture medium in the wells was replaced with normal culture medium and the plates were incubated for another 18 hours. The expression level of green fluorescent protein in HepG2, MCF-7 and C4-2 cells was detected by flow cytometry (the average value of the expression level of green fluorescent protein in the same cancer cell type was taken, n=3).
[0090] 4 μg of green fluorescent protein particle pEGFP-C1 was gently mixed with 250 μL of OPTI-MEM medium and allowed to stand for 5 minutes. 100 μL of the cationic liposome dispersion prepared in Comparative Example 1 was gently mixed with 150 μL of OPTI-MEM medium and allowed to stand for 5 minutes. The two mixtures were then gently mixed and allowed to stand for 20 minutes. The mixture was then added to the wells of the corresponding 6-well plates (n=3) and cultured in the incubator for 6 hours. The mixed culture medium in the wells was then replaced with normal culture medium and cultured for another 18 hours. The expression levels of green fluorescent protein in HepG2, MCF-7, and C4-2 cells were detected by flow cytometry (the average expression level of green fluorescent protein in the same cancer cell type was taken, n=3).
[0091] 4 μg of green fluorescent protein particle pEGFP-C1 was gently mixed with 250 μL of OPTI-MEM medium and allowed to stand for 5 minutes. 100 μL of the cationic liposome dispersion prepared in Comparative Example 2 was gently mixed with 150 μL of OPTI-MEM medium and allowed to stand for 5 minutes. The two mixtures were then gently mixed and allowed to stand for 20 minutes. The mixture was then added to the wells of the corresponding 6-well plates (n=3) and cultured in the incubator for 6 hours. The mixed culture medium in the wells was then replaced with normal culture medium and cultured for another 18 hours. The expression levels of green fluorescent protein in HepG2, MCF-7, and C4-2 cells were detected by flow cytometry (the average expression level of green fluorescent protein in the same cancer cell type was taken, n=3).
[0092] 4 μg of green fluorescent protein particle pEGFP-C1 was gently mixed with 250 μL of OPTI-MEM medium and allowed to stand for 5 minutes. 100 μL of the cationic liposome dispersion prepared in Comparative Example 3 was gently mixed with 150 μL of OPTI-MEM medium and allowed to stand for 5 minutes. The two mixtures were then gently mixed and allowed to stand for 20 minutes. The mixture was then added to the wells of the corresponding 6-well plates (n=3) and cultured in the incubator for 6 hours. The mixed culture medium in the wells was then replaced with normal culture medium and cultured for another 18 hours. The expression levels of green fluorescent protein in HepG2, MCF-7, and C4-2 cells were detected by flow cytometry (the average expression level of green fluorescent protein in the same cancer cell type was taken, n=3).
[0093] 4 μg of green fluorescent protein particle pEGFP-C1 was gently mixed with 250 μL of OPTI-MEM medium and allowed to stand for 5 minutes. 100 μL of the cationic liposome dispersion prepared in Comparative Example 4 was gently mixed with 150 μL of OPTI-MEM medium and allowed to stand for 5 minutes. The two mixtures were then gently mixed and allowed to stand for 20 minutes. The mixture was then added to the wells of the corresponding 6-well plates (n=3) and cultured in the incubator for 6 hours. The mixed culture medium in the wells was then replaced with normal culture medium and cultured for another 18 hours. The expression levels of green fluorescent protein in HepG2, MCF-7, and C4-2 cells were detected by flow cytometry (the average expression level of green fluorescent protein in the same cancer cell type was taken, n=3).
[0094] The results are shown in Tables 2 to 4:
[0095] Table 2. Expression levels of green fluorescent proteins in HepG2 cells
[0096] sample Average fluorescence intensity (au) Example 7 162 Example 8 142 Example 9 127 Comparative Example 1 115 Comparative Example 2 116 Comparative Example 3 118 Comparative Example 4 121
[0097] Table 3. Expression levels of green fluorescent proteins in MFC-7 cells
[0098] sample Average fluorescence intensity (au) Example 7 182 Example 8 156 Example 9 139 Comparative Example 1 130 Comparative Example 2 122 Comparative Example 3 127 Comparative Example 4 114
[0099] Table 4. Expression levels of green fluorescent proteins in C4-2 cells
[0100]
[0101]
[0102] As shown in Tables 2-4, among the cationic liposomes prepared in Examples 7-9, the cationic liposome prepared in Example 7 showed the best transfection efficiency for the green fluorescent protein gene, followed by the cationic liposome prepared in Example 8, and the cationic liposome prepared in Example 9 showed the lowest transfection efficiency. This is mainly because the cationic liposome prepared in Example 7 had the highest positive charge, followed by the cationic liposome prepared in Example 8, and the cationic liposome prepared in Example 9 had the lowest positive charge. The higher the positive charge of the cationic liposome, the stronger the attraction between the positive and negative charges of the (green fluorescent protein) gene, and the stronger the attraction with the negative charge of the cell membrane, resulting in more uptake by the cell and a better transfection effect. However, if the positive charge is too high, it will damage the cell membrane structure and produce cytotoxicity.
[0103] In Example 8, with the same lecithin:conjugate ratio as the comparative examples, the positive charge of the liposomes constructed from conjugates based on other structures in Comparative Examples 1 to 4 was significantly lower than that of the liposomes constructed from conjugates based on other structures in Comparative Examples 1 to 4. However, its transfection efficiency of the green fluorescent protein gene was higher than that of the cationic liposomes in Comparative Examples 1 to 4. The applicant believes that the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate used in the liposomes of Example 8 has an oxidative response to the sulfur atom, which can break the chain segment within the cell. Simultaneously, the ester bonds on the chain segment may be more easily hydrolyzed by esterases within the cell under the influence of the sulfur atom. Ultimately, this results in the cationic liposomes being able to more fully and effectively release the loaded gene for transfection after entering the cell.
[0104] Cytotoxicity test:
[0105] Three 96-well plates were selected. Human liver cancer cells (HepG2) were seeded into the first well at a density of 10,000 cells per well; human breast cancer cells (MCF-7) were seeded into the second well at a density of 10,000 cells per well; and human prostate cancer cells (C4-2) were seeded into the third well at a density of 10,000 cells per well. The three 96-well plates were incubated overnight at 37°C in a CO2 incubator. The original culture medium was then aspirated.
[0106] For the three 96-well plates mentioned above, add 200 μL of OPTI-MEM medium and 10 μL of the cationic liposome dispersion prepared in Example 7 to each well in some portions of the plates. Add 200 μL of OPTI-MEM medium and 10 μL of the cationic liposome dispersion prepared in Example 8 to each well in other portions of the plates. Add 200 μL of OPTI-MEM medium and 10 μL of the cationic liposome dispersion prepared in Example 9 to each well in other portions of the plates. Add 200 μL of OPTI-MEM medium and 10 μL of the cationic liposome dispersion prepared in Comparative Example 1 to each well in other portions of the plates. Add 200 μL of OPTI-MEM medium and 10 μL of the cationic liposome dispersion prepared in Comparative Example 2 to each well in other portions of the plates. Add 200 μL of OPTI-MEM medium and 10 μL of the cationic liposome dispersion prepared in Comparative Example 2 to each well in other portions of the plates. OPTI-MEM medium and 10 μL of cationic liposome dispersion prepared in Comparative Example 3 were added to another portion of the wells of each plate. 200 μL of OPTI-MEM medium and 10 μL of cationic liposome dispersion prepared in Comparative Example 4 were added to each well. The plates were then incubated in the above incubator for 6 hours. The mixed culture medium in the wells was then replaced with normal culture medium and incubated for another 18 hours. The cell viability of each group was detected by the standard MTT assay (average value, n=3). The cell viability of cells cultured in blank medium was 100% (210 μL OPTI-MEM medium for 6 hours, normal culture medium for 18 hours).
[0107] The results are shown in Tables 5 to 7:
[0108] Table 5. HepG2 cytotoxicity
[0109] sample Cell viability (%) Example 7 71 Example 8 86 Example 9 90 Comparative Example 1 63 Comparative Example 2 68 Comparative Example 3 70 Comparative Example 4 66
[0110] Table 6. MFC-7 Cytotoxicity
[0111] sample Cell viability (%) Example 7 73 Example 8 87 Example 9 89 Comparative Example 1 69 Comparative Example 2 71 Comparative Example 3 72 Comparative Example 4 69
[0112] Table 7. C4-2 Cytotoxicity
[0113]
[0114]
[0115] As shown in Tables 5-7, among the cationic liposomes prepared in Examples 7-9 of the present invention, the cationic liposome prepared in Example 7 has the highest toxicity, followed by the cationic liposome prepared in Example 8, and the cationic liposome prepared in Example 9 has the lowest cytotoxicity. This is mainly because the higher the positive charge of the cationic liposome, the stronger its destructive effect on the cell membrane and the greater its cytotoxicity. Among them, the cationic liposome prepared in Example 8 has a cell survival rate of over 80% and a better transfection effect, making it suitable for gene transfection.
[0116] When the lecithin:conjugate ratio was the same as that of the comparative examples, the positive charge of Example 8 was significantly lower than that of the liposomes constructed based on conjugates with other structures in Comparative Examples 1 to 4, and thus it also showed a significant advantage in cell safety.
[0117] In contrast, in Table 1 above, Example 7 of this application, where the positive charge of the cationic liposomes is higher than that of Comparative Examples 1 to 4, exhibits slightly lower cytotoxicity. This may be because the cationic liposomes prepared in this method are more easily hydrolyzed and broken down after entering the cell, resulting in decreased toxicity after disintegration. This indicates that the cationic liposomes prepared using the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate of this invention have advantages in both transfection efficiency and cytotoxicity.
Claims
1. An N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate, characterized in that: The structural formula of the coupling is as follows.
2. A method for preparing an N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester coupling compound, characterized in that: The preparation method is as follows: (1) Preparation of thiodiethylene glycol monocholesterol ester Cholesterol and thiodiethylene glycol anhydride were dissolved together, and N,N-dimethylaminopyridine was added to initiate a reflux reaction. After the reaction, the solvent was evaporated to dryness, and the mixture was recrystallized using ethyl acetate and ethanol to obtain purified thiodiethylene glycol monocholesterol ester. The molar ratio of cholesterol, thiodiglycolic anhydride, and N,N-dimethylaminopyridine is 1:1 to 2:0.5 to 1. (2) The thiodiethylene glycol monocholesterol ester and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride obtained in step (1) are dissolved together, and N,N-dimethylethylenediamine is added to the solution and the mixture is stirred and reacted. After the reaction, the solvent is evaporated to dryness, dispersed in water, dialyzed in ultrapure water, centrifuged, and the supernatant is freeze-dried to obtain the N,N-dimethylethylenediamine-thiodiethylene glycol monocholesterol ester conjugate. The molar ratio of the thiodiglycolic acid monocholesterol ester, the N,N-dimethylethylenediamine, and the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1 to 2:2 to 10.
3. The method for preparing the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate as described in claim 2, characterized in that: In step (1), cholesterol and thiodiglycolic anhydride are dissolved together in dichloromethane.
4. The method for preparing the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate as described in claim 2, characterized in that: In step (2), thiodiglycolic acid monocholesterol ester and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride are dissolved together in dichloromethane.
5. The method for preparing the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate as described in claim 2, characterized in that: In step (2), the stirring reaction is carried out at room temperature.
6. The use of the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate as described in claim 1 as a component of cationic liposomes.
7. The application of the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate as described in claim 6 as a component of cationic liposomes, characterized in that: The cationic liposomes also include lecithin, and the mass ratio of the lecithin to the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate is 3 to 10:
1.
8. The application of the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate as described in claim 7 as a component of cationic liposomes, characterized in that: The cationic liposomes are prepared by dissolving the lecithin and the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate together, evaporating the solvent, dispersing it with water, and then ultrasonically breaking it to obtain cationic liposomes.
9. The application of the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate as described in claim 8 as a component of cationic liposomes, characterized in that: The lecithin and the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate were dissolved together in ethanol.
10. The application of the N,N-dimethylethylenediamine-thiodiglycolic acid monocholesterol ester conjugate as described in claim 8 as a component of cationic liposomes, characterized in that: The ultrasonic fragmentation power is 90W, and the time is 15 minutes.