A basic amino acid-modified 3S-PCL, its preparation method and uses

Abstract

This invention discloses a basic amino acid-modified 3S-PCL, its preparation method, and its applications, relating to the field of biomedical technology. The structural formula of the basic amino acid-modified 3S-PCL is as follows: [Structure formula would be inserted here]; where R is a basic amino acid; m, m1, and m2 are all positive integers, and m, m1, and m2 ≥ 1. This invention designs and synthesizes a basic amino acid-modified three-armed polycaprolactone (3S-PCL) material, which exhibits good biocompatibility and can replace cationic liposomes for nucleic acid delivery via lipid nanoparticles. The preparation method of this basic amino acid-modified 3S-PCL material is simple and rapid, particularly suitable for industrial production. Lipid nanoparticles prepared using this basic amino acid-modified 3S-PCL material as a gene carrier can achieve highly efficient delivery of nucleic acid drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a basic amino acid-modified 3S-PCL, its preparation method, and its uses. Background Technology

[0002] With the rapid development of nanotechnology and improvements in nucleic acid chemical modification, gene therapy has achieved certain results in treating ischemic injuries, including peripheral artery disease and severe limb ischemia. Gene therapy refers to the introduction of exogenous normal genes into target cells to correct or compensate for diseases caused by gene defects and abnormalities, thereby achieving therapeutic goals. Upregulation of gene expression can be achieved by delivering plasmids (pDNA) expressing specific genes, chemically modified mRNA, circular RNA, and other nucleic acid drugs. Downregulation of gene expression can be achieved by delivering molecules such as small interfering RNA, microRNA, and antisense oligonucleotides. Messenger RNA (mRNA) is a direct template for protein biosynthesis. Compared to DNA, which needs to enter the core, mRNA does not pose a risk of integration into the genome. However, the unstable structure of mRNA is easily affected by external factors such as acidity, alkalinity, high temperature, and nucleases, leading to inactivation. Therefore, nucleic acid drugs need to be loaded onto gene vectors to enhance transmembrane transport and improve nucleic acid stability. Viral vectors can deliver exogenous genes into cells using the viral genome, but they have drawbacks such as low packaging capacity, high immunogenicity, and potential biological risks. Non-viral nanocarriers, such as cationic polymers, lipid complexes, natural biomacromolecules, and DNA frameworks, can also protect nucleic acids, preventing their degradation by nucleases, promoting their absorption by cells, and allowing for the controlled release of encapsulated therapeutic drugs. Based on this, this invention aims to develop a novel biodegradable gene vector to achieve efficient delivery of nucleic acid drugs. Summary of the Invention

[0003] The purpose of this invention is to provide a basic amino acid-modified 3S-PCL, its preparation method, and its uses, to solve the problems existing in the prior art. Lipid nanoparticles prepared using this basic amino acid-modified 3S-PCL material as a gene carrier can achieve highly efficient delivery of nucleic acid drugs.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides a basic amino acid-modified 3S-PCL, the structural formula of which is as follows:

[0006] ;

[0007] Where R is a basic amino acid; m, m1 and m2 are all positive integers, and m, m1 and m2 ≥ 1.

[0008] Furthermore, the basic amino acid is lysine, arginine, or histidine.

[0009] The present invention also provides a method for preparing the above-mentioned basic amino acid-modified 3S-PCL, comprising the following steps:

[0010] 3S-PCL and BOC-protected basic amino acids were dissolved in a solvent, and then 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide were added to carry out an amino acid modification reaction. After purification, the basic amino acid-modified 3S-PCL was obtained.

[0011] The 3S-PCL is obtained by mixing and reacting caprolactone, stannous octoate, and triethanolamine, followed by purification.

[0012] Furthermore, the BOC-protected basic amino acid is Boc-Lys(Boc)-OH, Boc-Arg(Boc)2-OH, or Boc-His-OH.

[0013] Further, the solvent is at least one selected from dichloromethane, trichloromethane, tetrahydrofuran, N,N-dimethylformamide, and dimethyl sulfoxide.

[0014] Further, the molar ratio of the 3S-PCL to the BOC-protected basic amino acid is 1:(3-5); and / or

[0015] The molar ratio of the BOC-protected basic amino acid, the 4-dimethylaminopyridine, and the N,N'-dicyclohexylcarbodiimide is 1:(0.1-1.5):(1-5); and / or

[0016] The molar ratio of caprolactone, stannous octoate, and triethanolamine is (10-50):(0.01-0.03):2.

[0017] Furthermore, the amino acid modification reaction is carried out at a temperature of 0-60℃ for a time of 12-24h.

[0018] Furthermore, the temperature of the mixing reaction is 120-160℃, and the time is 6-12h.

[0019] Furthermore, the purification process in the preparation of the 3S-PCL includes reprecipitation and dialysis.

[0020] The solvent used for the reprecipitation is at least one of methanol, diethyl ether, petroleum ether, methyl tert-butyl ether, n-hexane, and n-heptane.

[0021] The present invention also provides the application of the above-mentioned basic amino acid-modified 3S-PCL as a substitute for cationic lipids in the preparation of lipid nanoparticle nucleic acid drugs.

[0022] The present invention discloses the following technical effects:

[0023] This invention designs and synthesizes a basic amino acid-modified three-armed polycaprolactone (3S-PCL) material with good biocompatibility, which can replace cationic liposomes for the delivery of nucleic acids via lipid nanoparticles. The preparation method of this basic amino acid-modified 3S-PCL material is simple and rapid, making it particularly suitable for industrial production. Lipid nanoparticles prepared using this basic amino acid-modified 3S-PCL material as a gene carrier can achieve highly efficient delivery of nucleic acid drugs.

[0024] This invention synthesizes a novel polyester biomaterial capable of efficiently delivering nucleic acid drugs and scavenging reactive oxygen species by modifying 3S-PCL material with basic amino acids. In the treatment of peripheral vascular diseases, this novel polyester biomaterial encapsulates and delivers VEGF-mRNA, upregulates VEGF expression at the damaged site, promotes angiogenesis at ischemic sites, and scavenges reactive oxygen species at inflammatory sites, thereby achieving tissue repair and regeneration. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Infrared spectra of 3S-PCL, 3SPL, and 3SPA;

[0027] Figure 2 The hydrogen NMR spectra of 3S-PCL, 3SPL, and 3SPA are shown.

[0028] Figure 3 Figure showing the biocompatibility results of 3SPL and 3SPA on HUVEC cells;

[0029] Figure 4 The graph shows the hydrogen peroxide removal results for 3S-PCL, 3SPL, and 3SPA.

[0030] Figure 5 Figure showing the total nitric oxide formation results for 3S-PCL, 3SPL, and 3SPA;

[0031] Figure 6 Particle size distribution of 3SPL-Lipo / VEGF mRNA lipid nanoparticles;

[0032] Figure 7Zeta potential diagram of 3SPL-Lipo / VEGF mRNA lipid nanoparticles;

[0033] Figure 8 Particle size distribution of 3SPA-Lipo / VEGF mRNA lipid nanoparticles;

[0034] Figure 9 Zeta potential diagram of 3SPA-Lipo / VEGF mRNA lipid nanoparticles;

[0035] Figure 10 Figure 1 shows the ELISA results of VEGF content in the culture supernatant of HUVEC cells after treatment with 3SPL-Lipo / VEGF mRNA and 3SPA-Lipo / VEGF mRNA lipid nanoparticles.

[0036] Figure 11 Western blot results of HUVEC cells after treatment with 3SPL-Lipo / VEGF mRNA and 3SPA-Lipo / VEGF mRNA lipid nanoparticles. Detailed Implementation

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0040] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0041] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0042] Example 1

[0043] The preparation method of three-armed polycaprolactone 3S-PCL is as follows:

[0044] Caprolactone (3.42 g, 30 mmol), stannous octoate (12.2 mg, 0.03 mmol), and triethanolamine (298.4 mg, 2 mmol) were mixed and placed in a dry, flask-equipped with supports. The mixture was thoroughly shaken to ensure proper mixing. The flask was evacuated for 10 minutes using an oil pump, then purged with nitrogen. This process was repeated three times, with the final evacuation time being 60 minutes at 50°C to completely remove oxygen and moisture. The flask was then placed in a 140°C oven for 6 hours, with occasional shaking to ensure proper mixing. After the reaction, the mixture was allowed to cool naturally to room temperature. The resulting product was a white, waxy substance, which was dissolved in dichloromethane. After complete dissolution, some of the dichloromethane was removed by vacuum evaporation. The concentrated solution was then added dropwise to methanol for redeposition. The precipitated product was again dissolved in dichloromethane, evaporated under reduced pressure, and redepositioned using methyl tert-butyl ether. This methanol and methyl tert-butyl ether redeposition process was repeated three times. Finally, unreacted small molecules were further removed from methanol using a dialysis bag (molecular weight cutoff MW 1000 Da). The final white solid methanol solution was dried under reduced pressure using a rotary evaporator to obtain a white waxy substance, 3S-PCL.

[0045] Example 2

[0046] The preparation method of three-armed polycaprolactone 3S-PCL is as follows:

[0047] Caprolactone (1.41 g, 10 mmol), stannous octoate (8.1 mg, 0.02 mmol), and triethanolamine (298.4 mg, 2 mmol) were mixed and placed in a dry, flask-equipped with supports. The mixture was thoroughly shaken to ensure proper mixing. The flask was evacuated for 10 minutes using an oil pump, then purged with nitrogen. This process was repeated three times, with the final evacuation time conducted at 40°C for 30 minutes to completely remove oxygen and moisture. The flask was then placed in a 120°C oven and reacted for 12 hours, with occasional shaking to ensure proper mixing. After the reaction, the mixture was allowed to cool naturally to room temperature. The resulting product was a white, waxy substance, which was dissolved in dichloromethane. After complete dissolution, some of the dichloromethane was removed by vacuum evaporation. The concentrated solution was then added dropwise to methanol for redeposition. The precipitated product was again dissolved in dichloromethane, evaporated under reduced pressure, and then redepositioned using diethyl ether. This methanol and diethyl ether redeposition process was repeated three times. Finally, unreacted small molecules were further removed from methanol using a dialysis bag (molecular weight cutoff MW 1000 Da). The final white solid methanol solution was dried under reduced pressure using a rotary evaporator to obtain a white waxy substance, 3S-PCL.

[0048] Example 3

[0049] The preparation method of three-armed polycaprolactone 3S-PCL is as follows:

[0050] Caprolactone (5.71 g, 50 mmol), stannous octoate (4.1 mg, 0.01 mmol), and triethanolamine (298.4 mg, 2 mmol) were mixed and placed in a dry, flask-equipped with supports. The mixture was thoroughly shaken to ensure proper mixing. The flask was evacuated for 10 minutes using an oil pump, then purged with nitrogen. This process was repeated three times, with the final evacuation time at 45°C for 45 minutes to completely remove oxygen and moisture. The flask was then placed in a 160°C oven for 6 hours, with occasional shaking to ensure proper mixing. After the reaction, the mixture was allowed to cool naturally to room temperature. The resulting product was a white, waxy substance, which was dissolved in dichloromethane. After complete dissolution, some of the dichloromethane was removed by vacuum evaporation. The concentrated solution was then added dropwise to methanol for redeposition. The precipitated product was again dissolved in dichloromethane, evaporated under reduced pressure, and then redepositioned using n-hexane. This methanol and n-hexane redeposition process was repeated three times. Finally, unreacted small molecules were further removed from methanol using a dialysis bag (molecular weight cutoff MW 1000 Da). The final white solid methanol solution was dried under reduced pressure using a rotary evaporator to obtain a white waxy substance, 3S-PCL.

[0051] Example 4

[0052] The preparation method of lysine-modified 3S-PCL (3SPL) is as follows:

[0053] (1) 200.0 mg (0.1 mmol) of 3S-PCL prepared in Example 1 and 173.2 mg (0.5 mmol) of 2,6-di-tert-butoxycarbonylaminohexanoic acid (Boc-Lys(Boc)-OH) were dissolved in a reaction flask containing 5 mL of dichloromethane. After complete dissolution, 6.1 mg (0.05 mmol) of 4-dimethylaminopyridine (DMAP) was added to the reaction solution and stirred to dissolve. After the reaction system was dissolved, 154 mg (0.75 mmol) of N,N'-dicyclohexylcarbodiimide (DCC) was added. After reacting at 25 °C for 12 hours, the dichloromethane solvent was evaporated under reduced pressure to obtain a white solid product.

[0054] (2) Dissolve the white solid product obtained in step (1) in 2 mL of trifluoroacetic acid dichloromethane solution in an ice bath, and continue to react in an ice bath for 1 hour. After the reaction is completed, remove dichloromethane and trifluoroacetic acid using an oil pump. Further remove unreacted small molecules in double-distilled water using a dialysis bag (molecular weight cutoff MW 2000Da). After purification, freeze dry for 48 hours to obtain lysine-modified 3S-PCL. The volume ratio of trifluoroacetic acid to dichloromethane in the trifluoroacetic acid dichloromethane solution is 1:2.

[0055] Example 5

[0056] The preparation method of lysine-modified 3S-PCL (3SPL) is as follows:

[0057] (1) 200.0 mg (0.1 mmol) of 3S-PCL prepared in Example 1 and 103.9 mg (0.3 mmol) of Boc-Lys(Boc)-OH were dissolved in a reaction flask containing 5 mL of dichloromethane. After complete dissolution, 36.7 mg (0.3 mmol) of DMAP was added to the reaction solution and stirred to dissolve. After the reaction system was dissolved, 61.9 mg (0.3 mmol) of DCC was added. After reacting at 0 °C for 24 hours, the dichloromethane solvent was removed by vacuum evaporation to obtain a white solid product.

[0058] (2) Dissolve the white solid product obtained in step (1) in 2 mL of trifluoroacetic acid dichloromethane solution in an ice bath, and continue to react in an ice bath for 2 hours. After the reaction is completed, remove dichloromethane and trifluoroacetic acid using an oil pump. Further remove unreacted small molecules in double-distilled water using a dialysis bag (molecular weight cutoff MW 2000Da). After purification, freeze dry for 48 hours to obtain lysine-modified 3S-PCL. The volume ratio of trifluoroacetic acid to dichloromethane in the trifluoroacetic acid dichloromethane solution is 1:2.

[0059] Example 6

[0060] The preparation method of lysine-modified 3S-PCL (3SPL) is as follows:

[0061] (1) 200.0 mg (0.1 mmol) of 3S-PCL prepared in Example 1 and 138.6 mg (0.4 mmol) of Boc-Lys(Boc)-OH were dissolved in a reaction flask containing 5 mL of dichloromethane. After complete dissolution, 4.9 mg (0.04 mmol) of DMAP was added to the reaction solution and stirred to dissolve. After the reaction system was dissolved, 412.7 mg (2 mmol) of DCC was added. After reacting at 60 °C for 12 hours, the dichloromethane solvent was removed by vacuum evaporation to obtain a white solid product.

[0062] (2) Dissolve the white solid product obtained in step (1) in 2 mL of trifluoroacetic acid dichloromethane solution in an ice bath, and continue to react in an ice bath for 2 hours. After the reaction is completed, remove dichloromethane and trifluoroacetic acid using an oil pump. Further remove unreacted small molecules in double-distilled water using a dialysis bag (molecular weight cutoff MW 2000Da). After purification, freeze dry for 48 hours to obtain lysine-modified 3S-PCL. The volume ratio of trifluoroacetic acid to dichloromethane in the trifluoroacetic acid dichloromethane solution is 1:2.

[0063] Example 7

[0064] The preparation method of arginine-modified 3S-PCL (3SPA) is as follows:

[0065] (1) 200.0 mg (0.1 mmol) of 3S-PCL prepared in Example 1 and 237.3 mg (0.5 mmol) of tri-tert-butoxycarbonyl arginine (Boc-Arg(Boc)2-OH) were dissolved in a reaction flask containing 5 mL of dichloromethane. After complete dissolution, 6.1 mg (0.05 mmol) of DMAP was added to the reaction solution and stirred to dissolve. After the reaction system was dissolved, 154 mg (0.75 mmol) of DCC was added. After reacting at 25 °C for 12 hours, the dichloromethane solvent was removed by vacuum evaporation to obtain a white solid product.

[0066] (2) Dissolve the white solid product obtained in step (1) in 2 mL of trifluoroacetic acid dichloromethane solution in an ice bath, and continue to react in an ice bath for 1 hour. After the reaction is completed, remove dichloromethane and trifluoroacetic acid using an oil pump. Further remove unreacted small molecules in double-distilled water using a dialysis bag (molecular weight cutoff MW 2000Da). After purification, freeze dry for 48 hours to obtain arginine-modified 3S-PCL. The volume ratio of trifluoroacetic acid to dichloromethane in the trifluoroacetic acid dichloromethane solution is 1:2.

[0067] Example 8

[0068] The preparation method of arginine-modified 3S-PCL (3SPA) is as follows:

[0069] (1) 200.0 mg (0.1 mmol) of 3S-PCL prepared in Example 1 and 142.4 mg (0.3 mmol) of tritert-butoxycarbonyl arginine (Boc-Arg(Boc)2-OH) were dissolved in a reaction flask containing 5 mL of dichloromethane. After complete dissolution, 36.7 mg (0.3 mmol) of DMAP was added to the reaction solution and stirred to dissolve. After the reaction system was dissolved, 61.9 mg (0.3 mmol) of DCC was added. After reacting at 0 °C for 24 hours, the dichloromethane solvent was removed by vacuum evaporation to obtain a white solid product.

[0070] (2) Dissolve the white solid product obtained in step (1) in 2 mL of trifluoroacetic acid dichloromethane solution in an ice bath, and continue to react in an ice bath for 2 hours. After the reaction is completed, remove dichloromethane and trifluoroacetic acid using an oil pump. Further remove unreacted small molecules in double-distilled water using a dialysis bag (molecular weight cutoff MW 2000Da). After purification, freeze dry for 48 hours to obtain arginine-modified 3S-PCL. The volume ratio of trifluoroacetic acid to dichloromethane in the trifluoroacetic acid dichloromethane solution is 1:2.

[0071] Example 9

[0072] The preparation method of arginine-modified 3S-PCL (3SPA) is as follows:

[0073] (1) 200.0 mg (0.1 mmol) of 3S-PCL prepared in Example 1 and 189.8 mg (0.4 mmol) of tri-tert-butoxycarbonyl arginine (Boc-Arg(Boc)2-OH) were dissolved in a reaction flask containing 5 mL of dichloromethane. After complete dissolution, 4.9 mg (0.04 mmol) of DMAP was added to the reaction solution and stirred to dissolve. After the reaction system was dissolved, 412.7 mg (2 mmol) of DCC was added. After reacting at 60 °C for 12 hours, the dichloromethane solvent was removed by vacuum evaporation to obtain a white solid product.

[0074] (2) Dissolve the white solid product obtained in step (1) in 2 mL of trifluoroacetic acid dichloromethane solution in an ice bath, and continue to react in an ice bath for 2 hours. After the reaction is completed, remove dichloromethane and trifluoroacetic acid using an oil pump. Further remove unreacted small molecules in double-distilled water using a dialysis bag (molecular weight cutoff MW 2000Da). After purification, freeze dry for 48 hours to obtain arginine-modified 3S-PCL. The volume ratio of trifluoroacetic acid to dichloromethane in the trifluoroacetic acid dichloromethane solution is 1:2.

[0075] The following uses Examples 1, 4, and 7 as examples to characterize and perform performance tests on 3S-PCL, 3SPL, and 3SPA:

[0076] Example 10

[0077] 3S-PCL, lysine-modified 3S-PCL (3SPL), and arginine-modified 3S-PCL (3SPA) were characterized by proton NMR spectroscopy and infrared spectroscopy, respectively. The infrared spectra are shown below. Figure 1 As shown, the proton NMR spectrum is as follows: Figure 2 As shown, this confirms that all materials were successfully synthesized.

[0078] Example 11

[0079] Safety testing of lysine-modified 3S-PCL and arginine-modified 3S-PCL in human umbilical vein endothelial cells (HUVECs):

[0080] Dilute 3SPL (or 3SPA) with culture medium to the predetermined concentration. Six replicates were prepared for each concentration, and control and blank control groups were included. Human umbilical vein endothelial cells (HUVECs) were seeded at a density of 5000 cells / well in 96-well plates and cultured for 24 h. The cell culture medium was then replaced with medium containing 1, 3, 10, 30, 100, and 300 μg / mL 3SPL (or 3SPA), and cultured for another 20 h each time. 20 μL of CCK-8 reagent was added to each well. Incubation was continued for 4 h, and the absorbance was measured at 450 nm, with 650 nm as the reference wavelength. Cell viability was calculated using the following formula:

[0081] Cell viability (%) = [(OD) 实验 -OD 空白 ) / (OD 对照 -OD 空白 )]×100%.

[0082] The results of cell viability testing are shown below. Figure 3 The results showed that within the measured concentration range, both 3SPL and 3SPA maintained HUVEC cell viability above 85%, indicating good biocompatibility of the materials. This demonstrates that the basic amino acid-modified 3S-PCL provided by this invention has advantages in terms of safety.

[0083] Example 12

[0084] Hydrogen peroxide (H2O2) scavenging experiments of 3S-PCL, lysine-modified 3S-PCL, and arginine-modified 3S-PCL:

[0085] The materials to be tested (3S-PCL, 3SPL, or 3SPA) were diluted with PBS to prepare solutions of 0, 5, 10, 20, 50, 100, and 200 μg / mL, respectively. An equal volume of 200 μM H2O2 solution was added to each concentration solution, and the solutions were incubated at 37°C for 12 h. An appropriate volume of Amplexred and horseradish peroxidase (HRP) solution was added to each group to bring the final concentrations of Amplexred and HRP to 1 mmol / L and 1 mU / mL, respectively, and the solutions were incubated at room temperature for 20 min. The H2O2 content of each group was detected using fluorescence spectrophotometry (excitation and emission wavelengths were approximately 571 nm and 585 nm, respectively).

[0086] The results of the hydrogen peroxide removal rate test are shown in [the table below]. Figure 4 The results showed that, compared with 3S-PCL, both 3SPL and 3SPA exhibited higher hydrogen peroxide scavenging capabilities. 3SPA showed a more significant hydrogen peroxide scavenging ability at a concentration of 100 µg / mL, which may be due to the presence of a large number of guanidine groups in its structure. This indicates that the basic amino acid-modified 3S-PCL provided by this invention can scavenge hydrogen peroxide and has anti-ROS and anti-inflammatory effects.

[0087] Example 13

[0088] Nitric oxide release experiments of 3S-PCL, lysine-modified 3S-PCL, and arginine-modified 3S-PCL:

[0089] The materials to be tested (3S-PCL, 3SPL, or 3SPA) were diluted with PBS to prepare solutions of 0, 5, 10, 20, 50, 100, and 200 μg / mL, respectively. An equal volume of 200 μM H2O2 solution was added to each concentration solution, and the solutions were incubated at 37°C for 12 h. Following the instructions of the total nitric oxide assay kit (S0023, Beyotime), the absorbance at 540 nm was measured using a microplate reader, and the total nitric oxide content of each group was calculated.

[0090] The results of the total nitric oxide content test are shown below. Figure 5 The results showed that 3SPL and 3SPA could significantly generate nitric oxide, with 3SPA producing the most. This is likely due to the presence of numerous guanidinium functional groups in the 3SPA structure that promote nitric oxide production. This indicates that the basic amino acid-modified 3S-PCL provided in this invention can release nitric oxide during hydrogen peroxide scavenging.

[0091] Example 14

[0092] The preparation method of lysine-modified 3S-PCL (3SPL)-loaded VEGF mRNA lipid nanoparticles (abbreviated as 3SPL-Lipo / VEGF mRNA) includes the following steps:

[0093] The nucleotide sequence of VEGF mRNA is shown in SEQ ID NO.1.

[0094] SEQ ID NO.1:

[0095] .

[0096] 10.0 mg of 3SPL (prepared in Example 4) was dissolved in 1 mL of acetonitrile until completely dissolved. The solution was then filtered through a 0.22 μm filter to remove insoluble matter before use. 16.0 mg of distearate phosphatidylcholine (DSPC), 4.0 mg of cholesterol, and 3.3 mg of DMG-PEG2000 were dissolved in 2.3 mL of anhydrous ethanol until completely dissolved. After filtering through a 0.22 μm filter to remove insoluble matter, the solution was rapidly injected into 7.0 mL of aqueous solution (containing 1.0 mg of VEGF mRNA). The solution gradually changed from clear and transparent to clear milky white. After injection, the solution was stirred for 20 min. The organic solvent in the system was removed by centrifugation (4000 rpm, 4 °C) using an ultrafiltration tube to obtain 3SPL-Lipo / VEGF mRNA lipid nanoparticles (the composition of the raw materials is shown in Table 1).

[0097] Table 1. Composition of raw materials for the preparation of 3SPL-Lipo / VEGF mRNA

[0098]

[0099] The particle size and zeta potential of the 3SPL-Lipo / VEGF mRNA lipid nanoparticles prepared in this example were determined using a Malvern particle size analyzer. The particle size was 113.8 nm, and the PDI was 0.067. Figure 6 As shown; the potential is +1.91 mV, as... Figure 7 As shown in the figure, the encapsulation efficiency and drug loading of the lipid nanoparticles were determined by the RiboGreen method, with an encapsulation efficiency of 86.5% and a drug loading of 2.5%.

[0100] Example 15

[0101] The preparation method of arginine-modified 3S-PCL-loaded VEGF mRNA lipid nanoparticles (abbreviated as 3SPA-Lipo / VEGFmRNA) includes the following steps:

[0102] 10.0 mg of 3SPA (prepared in Example 7) was dissolved in 1 mL of acetonitrile until completely dissolved. The solution was then filtered through a 0.22 μm filter to remove insoluble matter before use. 16.0 mg of distearate phosphatidylcholine, 4.0 mg of cholesterol, and 3.3 mg of DMG-PEG2000 were dissolved in 2.3 mL of anhydrous ethanol until completely dissolved. After filtering through a 0.22 μm filter to remove insoluble matter, the solution was rapidly injected into 7.0 mL of aqueous solution (containing 1.0 mg of VEGF mRNA). The solution gradually changed from clear and transparent to clear milky white. After injection, the solution was stirred for 20 min. The organic solvent in the system was removed by centrifugation (4000 rpm, 4 °C) using an ultrafiltration tube to obtain 3SPL-Lipo / VEGF mRNA lipid nanoparticles (the composition of the raw materials is shown in Table 2).

[0103] Table 2. Composition of raw materials for the preparation of 3SPA-Lipo / VEGF mRNA

[0104]

[0105] The particle size and zeta potential of the 3SPA-Lipo / VEGF mRNA lipid nanoparticles prepared in this example were determined using a Malvern particle size analyzer. The particle size was 123.5 nm, and the PDI was 0.119. Figure 8 As shown; the potential is +10.3 mV, as Figure 9 As shown in the figure, the encapsulation efficiency and drug loading of the lipid nanoparticles were determined by the RiboGreen method, with an encapsulation efficiency of 94.7% and a drug loading of 2.7%.

[0106] Example 16

[0107] Detection of gene transfection efficiency of 3SPL-Lipo / VEGF mRNA and 3SPA-Lipo / VEGF mRNA lipid nanoparticles in HUVEC cells in vitro:

[0108] HUVEC cells were loaded at a rate of 2 × 10 5 HUVEC cells were seeded at a density of 10 cells / well and cultured for 24 h. 3SPL-Lipo / VEGF mRNA lipid nanoparticles prepared in Example 14, 3SPA-Lipo / VEGF mRNA lipid nanoparticles prepared in Example 15 (based on VEGF mRNA 1 µg / well), and free VEGF mRNA (1 µg / well) were added to each well and incubated for 24 h. These three groups were named 3SPL-Lipo, 3SPA-Lipo, and Free mRNA, respectively. The negative control (NC) was PBS with an equal volume of lipid nanoparticles; the positive control was 1,2-dioleoyl-3-trimethylammonium chloride propyl ester (DOTAP) liposomes (group named DOTAP-Lipo; formulation mass ratio DSPC:cholesterol:DOTAP:DMG-PEG). 2000 = 48:12:30:10) and Dlin-MC3-DMA liposomes (group name MC3-Lipo; formulation mass ratio DSPC: cholesterol: Dlin-MC3-DMA: DMG-PEG) 2000 = 13.5:25:55:6.5), the N / P ratio of the above preparations was 10:1. After incubation, the VEGF content in the cell culture medium was quantitatively analyzed by ELISA, and the VEGF content in HUVEC cells was detected by Western blotting.

[0109] Depend on Figure 10 as well as Figure 11 As shown, both 3SPL-Lipo / VEGF mRNA and 3SPA-Lipo / VEGF mRNA lipid nanoparticles upregulated VEGF expression, and the expression levels were higher than those of free mRNA and DOTAP liposomes. The transfection efficiency of 3SPL-Lipo / VEGF mRNA lipid nanoparticles was comparable to that of Dlin-MC3-DMA liposomes, while 3SPA-Lipo / VEGF mRNA showed higher VEGF expression levels in both culture supernatant and intracellular samples, and the transfection efficiency of 3SPA-Lipo / VEGF mRNA lipid nanoparticles was significantly higher than that of 3SPL-Lipo / VEGF mRNA and Dlin-MC3-DMA liposomes.

[0110] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A basic amino acid-modified three-armed polycaprolactone 3S-PCL, characterized in that, Its structural formula is as follows: ； Where R is a basic amino acid; m, m1, and m2 are all positive integers, and m, m1, and m2 ≥ 1; The basic amino acid is lysine, arginine, or histidine.

2. A method for preparing the basic amino acid-modified three-arm polycaprolactone 3S-PCL as described in claim 1, characterized in that, Includes the following steps: 3S-PCL and BOC-protected basic amino acids were dissolved in a solvent, and then 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide were added to carry out an amino acid modification reaction. After purification, the basic amino acid-modified 3S-PCL was obtained. The 3S-PCL is obtained by mixing and reacting caprolactone, stannous octoate, and triethanolamine, followed by purification.

3. The preparation method according to claim 2, characterized in that, The BOC-protected basic amino acid is Boc-Lys(Boc)-OH, Boc-Arg(Boc)2-OH, or Boc-His-OH.

4. The preparation method according to claim 2, characterized in that, The solvent is at least one selected from dichloromethane, trichloromethane, tetrahydrofuran, N,N-dimethylformamide, and dimethyl sulfoxide.

5. The preparation method according to claim 2, characterized in that, The molar ratio of 3S-PCL to the BOC-protected basic amino acid is 1:(3-5); and / or The molar ratio of the BOC-protected basic amino acid, the 4-dimethylaminopyridine, and the N,N'-dicyclohexylcarbodiimide is 1:(0.1-1.5):(1-5); and / or The molar ratio of caprolactone, stannous octoate, and triethanolamine is (10-50):(0.01-0.03):

2.

6. The preparation method according to claim 2, characterized in that, The amino acid modification reaction is carried out at a temperature of 0-60℃ for 12-24 hours.

7. The preparation method according to claim 2, characterized in that, The mixing reaction is carried out at a temperature of 120-160℃ for 6-12 hours.

8. The preparation method according to claim 2, characterized in that, The purification process in the preparation of 3S-PCL includes reprecipitation and dialysis. The solvent used for the reprecipitation is at least one of methanol, diethyl ether, petroleum ether, methyl tert-butyl ether, n-hexane, and n-heptane.

9. The application of the basic amino acid-modified three-armed polycaprolactone 3S-PCL as described in claim 1 as a substitute for cationic lipids in the preparation of lipid nanoparticle nucleic acid drugs.

Images