Endoplasmic reticulum-targeted single-chain cyclic poly(β-amino esters), their preparation methods and applications

Abstract

This invention provides an endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester), its preparation method, and its applications. The structural formula of the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) is as follows. The endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) exhibits excellent affinity for DNA, and the complex nanoparticles formed with DNA are uniformly distributed, stable, biodegradable, and biocompatible. The endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) and DNA can be efficiently taken up by cells, exhibiting excellent endoplasmic reticulum targeting performance. It can overcome the limitations of gene retention in lysosomes and nuclear internalization of the complex nanoparticles, enabling efficient delivery of DNA and mRNA.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and relates to a class of endoplasmic reticulum-targeting single-chain cyclic poly(β-amino esters), their preparation methods, and gene delivery applications. Background Technology

[0002] Gene therapy refers to the use of methods to introduce genetic material with specific functions into targeted tissues or cells, replacing or inhibiting the expression of diseased genes. Compared to emerging protein and cell therapies, gene therapy can correct gene defects or mutations at their source, and has become one of the most promising treatment methods for various hereditary diseases (such as cataracts, hair loss, oral mucosal diseases, and psoriasis-like syndromes) or acquired diseases (such as Alzheimer's disease, Parkinson's disease, and rheumatoid arthritis).

[0003] However, the lack of safe and efficient gene vectors severely limits their clinical application. Currently, gene vectors are mainly divided into viral vectors and non-viral vectors. Due to their numerous advantages, such as wide availability of raw materials, flexible and diverse chemical compositions, high gene loading efficiency, low immunogenicity, and good stability with tolerable serum, cationic polymer vectors, as one of the most representative non-viral vectors, have received widespread attention. These mainly include polyethyleneimine (PEI), polydimethylaminoethyl methacrylate (PDMAEMA), and polyamide amine (PAMAM). Although cationic polymer vectors have shown some application potential, their poor degradation properties, tendency to induce high cytotoxicity, and often limited monomer types severely restrict their clinical application.

[0004] Poly(β-amino esters) have shown promise in clinical gene therapy for hereditary skin diseases and gene delivery in various cell lines due to their readily available monomers, simple and efficient synthesis, easily tunable chemical composition and structure, biodegradability, and ease of functionalization. However, for poly(β-amino ester) cationic polymer carriers, intracellular barriers such as the retention of DNA-compressed complex nanoparticles in lysosomes and the difficulty in nuclear internalization caused by excessively large particle sizes often hinder gene transfection, significantly limiting their application in clinical translational research. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a class of endoplasmic reticulum-targeted single-chain cyclic poly(β-amino esters), their preparation methods, and applications.

[0006] This invention is achieved through the following technical solution:

[0007] This invention discloses an endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester), the structural formula of which is as follows:

[0008] ;

[0009] In the formula, R1 is the spacer arm of the diacrylate monomer, R2 is a group on the organic amine, R3 is a group on the endoplasmic reticulum targeting monomer, and R4 is a group on the capping agent monomer. The organic amine is R2-NH2, the endoplasmic reticulum targeting monomer is R3-NH2, and the capping agent monomer is R4-NH2. n=5~40, m=5~60, r=3~15, p=3~20, and q=3~30.

[0010] Preferably, the molecular weight of the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) is in the range of 4000~50000 Da.

[0011] Preferably, the diacrylate monomer is 1,6-hexanediol dimethacrylate (HDDMA), 1,4-butanediol diacrylate (BDA), 2,2-dithiodiethanol diacrylate (DSDA), diethylene glycol diacrylate (DEGDA), polyethylene glycol diacrylate, tetraethylene glycol diacrylate, or bisphenol A polyoxyethylene ether diacrylate.

[0012] Preferably, the organic amine is 5-amino-1-pentanol (AP), 4-amino-1-butanol (AB), butylamine, pentanol, hexylamine, octylamine, 1-(2-aminoethyl)piperazine, N-(2-aminoethyl)morpholine, dodecylamine, or octadecylamine.

[0013] Preferably, the endoplasmic reticulum targeting monomer is the polypeptide Pardaxin or N-(2-aminoethyl)-4-methylbenzenesulfonamide (NMS).

[0014] Preferably, the end-capping agent monomer is 1-(3-aminopropyl)-4-methylpiperazine, N-(3-aminopropyl)morpholine, N-(2-aminoethyl)morpholine, 2-methyl-2-morpholin-4-ylpropane-1-amine, 1-(2-aminoethyl)piperazine, 1,4-bis(3-aminopropyl)piperazine, 1,11-diamino-3,6,9-trioxaundecanane (DATA), 1,2-ethylenediamine, or 2-methyl-1,5-pentanediamine.

[0015] This invention also discloses a method for preparing the above-mentioned endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester), comprising the following steps:

[0016] 1) A linear poly(β-amino ester) with vinyl groups at the end is obtained by Michael addition reaction of diacrylate monomers with organic amines;

[0017] 2) The linear poly(β-amino ester) with vinyl groups at the end obtained in step 1) is subjected to reversible addition-fragmentation chain transfer polymerization (RAFT) to obtain a single-chain cyclic poly(β-amino ester) containing vinyl groups.

[0018] 3) The vinyl group-containing single-chain cyclic poly(β-amino ester) obtained in step 2) is subjected to a functionalization end-capping reaction with an endoplasmic reticulum-targeting monomer to obtain an endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester) containing vinyl groups.

[0019] 4) React the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) containing vinyl groups obtained in step 3) with the end-capping agent monomer to perform functionalization end-capping treatment, and obtain endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester).

[0020] Preferably, in step 1), the molar ratio of the diacrylate monomer to the organic amine is 1:(2~4).

[0021] Preferably, step 2) specifically involves: dissolving the linear poly(β-amino ester) with vinyl groups at the end obtained in step 1) and the chain transfer agent (CTA) in a reaction solvent, and then adding the initiator azobisisobutyronitrile (AIBN) under inert gas protection to carry out a polymerization reaction to obtain a single-chain cyclic poly(β-amino ester) containing vinyl groups.

[0022] The reaction molar ratio of linear poly(β-amino ester) with vinyl groups at the end to the chain transfer agent is (2000~10):1; the reaction molar ratio of initiator azobisisobutyronitrile to the chain transfer agent is 1:(0.5~6).

[0023] Preferably, in step 3), the reaction molar ratio of the endoplasmic reticulum targeting monomer to the vinyl-containing single-chain cyclic poly(β-amino ester) is 1:(3~10).

[0024] Preferably, in step 4), the reaction molar ratio of the end-capping agent monomer to the vinyl group-containing endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) is (1~5):1.

[0025] The present invention also discloses the above-mentioned endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester) as a gene vector, wherein the gene vector is used to deliver genes to tissues or cells.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention discloses an endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester), whose backbone is composed of linear low-molecular-weight poly(β-amino ester), representing a novel type of endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester). This endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester) exhibits excellent affinity for DNA, and the complex nanoparticles formed with DNA are uniformly distributed, stable, biodegradable, and biocompatible. Mechanistic studies show that the endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester) and DNA can be efficiently taken up by cells, and laser confocal microscopy results also indicate that the complex nanoparticles entering the cell can highly overlap with the endoplasmic reticulum, confirming its excellent endoplasmic reticulum-targeting performance and overcoming the limitations of gene retention in lysosomes and the internalization of complex nanoparticles into the nucleus. The results of in vitro transfection showed that the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) can efficiently deliver DNA and mRNA in various tissue cells, which provides an application basis for the delivery of various functionalized genes. Compared with the commercial cationic polymer transfection reagents XFect and jetPEI mainly used in the current technology, the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) disclosed in this invention has greater potential for clinical application.

[0028] Furthermore, the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) of the present invention has a molecular weight in the range of 4000~50000 Da, and the resulting product exhibits a polycyclic structure and a narrow PDI.

[0029] This invention also discloses a method for synthesizing the aforementioned endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester). Using diacrylate and organic amine, a linear poly(β-amino ester) with vinyl groups at the end is first prepared via Michael addition. Then, the obtained linear poly(β-amino ester) is used in a one-pot RAFT polymerization reaction to prepare a single-chain cyclic poly(β-amino ester) with controllable structure and composition containing vinyl groups. Finally, internalization-targeted functionalization modification and functionalization end-capping treatment are performed to obtain the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester). This synthesis method is low-cost, has a simple synthesis route, and requires minimal equipment, making it particularly suitable for industrial production.

[0030] This invention also discloses the use of the above-mentioned endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester). Experiments have confirmed that it is a novel gene delivery vector with high transfection efficiency and low toxicity, which can overcome the limitations of gene retention in lysosomes and nuclear internalization of complex nanoparticles. At the same time, the accuracy and wide applicability of the invention have been verified in various tissue cells (HeLa cells, UC-3 cells, HCT 166 cells, HepG2 cells). Attached Figure Description

[0031] Figure 1A schematic diagram of the representative endoplasmic reticulum-targeted synthesis of single-chain cyclic poly(β-amino esters);

[0032] Figure 2 The image shows the gel permeation chromatography (GPC) curve of the purified linear poly(β-amino ester) from Example 1; its weight-average molecular weight is 2,000 Da.

[0033] Figure 3 For Example 1, endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) 1 The HNMR spectrum showed characteristic peaks of the benzene ring at 7.2 ppm to 7.6 ppm, confirming that the sulfonyl group was successfully grafted onto the end of the single-chain cyclic poly(β-amino ester).

[0034] Figure 4 The GPC curve of the purified endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) from Example 1 is shown, with a weight-average molecular weight of 26,000 Da.

[0035] Figure 5 This is a photograph of the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) with a weight-average molecular weight of 26,000 Da, as shown in Example 1.

[0036] Figure 6 The GPC curve of the purified linear poly(β-amino ester) in Example 2 has a weight-average molecular weight of 4,500 Da.

[0037] Figure 7 Example 2 is an endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) polymerized from linear poly(β-amino ester) with a weight-average molecular weight of 4,500 Da. 1 HNMR spectrum;

[0038] Figure 8 The image shows the GPC curve of the purified endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) from Example 2, with a weight-average molecular weight of 49,600 Da.

[0039] Figure 9 This is a physical image of the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) with a weight-average molecular weight of 49,600 Da, as shown in Example 2.

[0040] Figure 10 The affinity tests of endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) for DNA at different mass ratios confirmed its excellent DNA affinity.

[0041] Figure 11 The particle size of the complex nanoparticles formed by endoplasmic reticulum-targeted single-stranded cyclic poly(β-amino ester) compressed DNA at different mass ratios;

[0042] Figure 12Zeta potential of complex nanoparticles formed by endoplasmic reticulum-targeted single-stranded circular poly(β-amino ester) compressed DNA at different mass ratios;

[0043] Figure 13 The microstructure of the complex nanoparticles formed by endoplasmic reticulum-targeted single-stranded circular poly(β-amino ester) compressed DNA;

[0044] Figure 14 Endoplasmic reticulum targeting characterization of nanoparticles formed by endoplasmic reticulum targeting single-stranded cyclic poly(β-amino ester) and DNA labeled with AF647 fluorescent dye;

[0045] Figure 15 Fluorescence images of cells transfected with endoplasmic reticulum-targeted single-stranded circular poly(β-amino ester) encoding green fluorescent protein (GFP) DNA in different cell types, including HeLa cells, UC-3 cells, and HepG2 cells, demonstrating its broad applicability in DNA transfection;

[0046] Figure 16 To demonstrate the excellent cell activity of endoplasmic reticulum-targeted single-stranded circular poly(β-amino ester) transfected DNA in HeLa cells at different mass ratios, the study aimed to assess cell viability.

[0047] Figure 17 Fluorescence images of cells transfected with endoplasmic reticulum-targeted single-stranded circular poly(β-amino ester) encoding green fluorescent protein mRNA in different cell types, including HeLa cells, UC-3 cells, and HCT 116 cells, demonstrating its broad applicability in mRNA transfection. Detailed Implementation

[0048] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0049] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0050] The present invention will now be described in further detail with reference to the accompanying drawings:

[0051] This invention discloses an endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester), the structural formula of which is as follows:

[0052] ;

[0053] In the formula, R1 is the spacer arm of the diacrylate monomer, R2 is a group on the organic amine, R3 is a group on the endoplasmic reticulum targeting monomer, and R4 is a group on the capping agent monomer. The organic amine is R2-NH2, the endoplasmic reticulum targeting monomer is R3-NH2, the capping agent monomer is R4-NH2, n=5~40, m=5~60, r=3~15, p=3~20, and q=3~30.

[0054] This invention discloses a method for synthesizing a class of endoplasmic reticulum-targeted single-chain cyclic poly(β-amino esters), the synthetic route of which is described in [reference needed]. Figure 1 This includes the following steps:

[0055] 1) A linear poly(β-amino ester) with vinyl groups at the end is obtained by Michael addition reaction of diacrylate monomers with organic amines;

[0056] 2) The linear poly(β-amino ester) with vinyl groups at the end prepared in step 1) is subjected to a reversible addition-fragmentation chain transfer polymerization reaction to obtain a single-chain cyclic poly(β-amino ester) containing vinyl groups.

[0057] 3) The vinyl group-containing single-chain cyclic poly(β-amino ester) prepared in step 2) is added to the endoplasmic reticulum-targeting monomer for functionalization and end-capping reaction to obtain the vinyl group-containing endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester).

[0058] 4) Add a capping agent monomer to the vinyl group-containing single-chain polycyclic poly(β-amino ester) prepared in step 3) for functionalization and capping treatment to obtain endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester).

[0059] Specifically, it includes the following steps:

[0060] 1) Add a certain amount of diacrylate monomers and organic amines to a solvent (dimethyl sulfoxide or tetrahydrofuran) and stir magnetically to dissolve them completely. The molar ratio of the reaction feed of diacrylate monomers to organic amines is 1:(2~4).

[0061] The reaction is carried out under a nitrogen protective atmosphere at 60-90°C.

[0062] Furthermore, during the reaction, the polymer molecular weight was monitored using gel permeation chromatography. The reaction was terminated when the polymer molecular weight reached the preset range of 100~8000 Da. A certain amount of diacrylate monomers and solvents (dimethyl sulfoxide or tetrahydrofuran) were added to the reaction system, and the reaction was carried out at 25°C for 48 h. The product was then purified in excess diethyl ether and freeze-dried thoroughly for 72 h to obtain a low molecular weight linear poly(β-amino ester) with vinyl groups at the end of its molecular weight distribution.

[0063] 2) A certain amount of low molecular weight linear poly(β-amino ester) with vinyl groups at the end and CTA are added to the solvent and the monomers are fully dissolved by magnetic stirring. The molar ratio of linear poly(β-amino ester) with vinyl groups at the end to CTA is (2000~10):1.

[0064] Oxygen is removed by bubbling with nitrogen for 10-30 minutes. In a nitrogen atmosphere, the initiator AIBN is added rapidly, and then nitrogen is continuously introduced. The molar ratio of AIBN to CTA is 1:(0.5-6). The three-necked flask is immersed in an oil bath preheated to 40-70°C to start the reaction.

[0065] In this process, the molecular weight of the polymer was monitored using gel permeation chromatography. When the molecular weight of the polymer approached the set value of 4000~50000 Da, the reaction was stopped, and the product was purified by precipitation with excess diethyl ether and then freeze-dried.

[0066] 3) A certain amount of endoplasmic reticulum (ER)-targeting monomer is added to the reaction system and reacted with a vinyl group-containing single-chain cyclic poly(β-amino ester) to obtain an ER-targeting single-chain cyclic poly(β-amino ester) containing a vinyl group. The molar ratio of the ER-targeting monomer to the vinyl group-containing single-chain cyclic poly(β-amino ester) is 1:(3~10). The reaction is carried out for 24 hours, and the product is purified by precipitation.

[0067] 4) Add a certain amount of end-capping agent monomer to the reaction system and react with endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) containing vinyl groups for end-capping. The molar ratio of end-capping agent monomer to endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) containing vinyl groups is (1~5):1. After the reaction is completed, the product is purified by precipitation.

[0068] The diacrylate monomers used in this invention are 1,6-hexanediol diacrylate, 1,4-butanediol diacrylate, 2,2-dithiodiethanol diacrylate, diethylene glycol diacrylate, polyethylene glycol diacrylate, tetraethylene glycol diacrylate, and bisphenol A polyoxyethylene ether diacrylate, the structural formulas of which are shown below:

[0069]

[0070] Furthermore, the organic amines used in this invention are 4-amino-1-butanol, 5-amino-1-pentanol, butylamine, pentanamine, hexylamine, octylamine, 1-(2-aminoethyl)piperazine, N-(2-aminoethyl)-morpholine, dodecylamine, and octadecylamine, and their structural formulas are shown below:

[0071]

[0072] Furthermore, the endoplasmic reticulum targeting monomer used in this invention is the polypeptide Pardaxin or N-(2-aminoethyl)-4-methylbenzenesulfonamide, the structural formulas of which are shown below:

[0073]

[0074] Furthermore, the end-capping agent monomer used in this invention is 1-(3-aminopropyl)-4-methylpiperazine, N-(3-aminopropyl)morpholine, N-(2-aminoethyl)morpholine, 2-methyl-2-morpholin-4-ylpropane-1-amine, 1-(2-aminoethyl)piperazine, 1,4-bis(3-aminopropyl)piperazine, 1,11-diamino-3,6,9-trioxaundecane, 1,2-ethylenediamine, or 2-methyl-1,5-pentanediamine, and their structural formulas are shown below:

[0075]

[0076] The particle size, zeta potential, and microstructure of the complex nanoparticles formed by the endoplasmic reticulum-targeted single-stranded circular poly(β-amino ester) compressed DNA prepared in this invention were tested using the following methods:

[0077] A certain amount of endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) solution was added to the DNA solution, vortexed at high speed for 15-60 s, and then allowed to stand for 10-40 min. The mass ratio of endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) to DNA was (10-120):1. Then, 1 mL of deionized water was added, and dynamic light scattering (DLS) was used for testing. Simultaneously, composite nanoparticles were prepared using the same method, washed with deionized water to remove inorganic salt ions, freeze-dried, and finally observed using transmission electron microscopy (TEM).

[0078] The endoplasmic reticulum-targeting single-stranded circular poly(β-amino ester) prepared in this invention was transfected with DNA encoding green fluorescent protein, and cytotoxicity was tested simultaneously, as follows:

[0079] 1) Cell culture: HeLa cells, UC-3 cells, and HepG2 cells were cultured at a concentration of 1.0 × 10⁻⁶ cells / mL. 4 ~2.0×10 4 Cells were seeded at a density of 10 cells / well in 96-well plates and cultured overnight at 37°C.

[0080] 2) Mix a certain amount of endoplasmic reticulum-targeting single-stranded cyclic poly(β-amino ester) into the DNA solution encoding green fluorescent protein, let stand for 5-40 min, and then add it to the cells.

[0081] 3) 24-96 h after transfection, observe and photograph the cells transfected with green fluorescent protein DNA under a fluorescence microscope.

[0082] 4) 24-96 h after transfection, remove the cell supernatant, then add a certain amount of PBS buffer solution to wash the cells, then add Alamar Blue solution (10%), incubate in an incubator for 20-60 min, and then test the fluorescence intensity of live cells in a microplate reader. Cells without transfection treatment have 100% viability.

[0083] The endoplasmic reticulum-targeting single-stranded circular poly(β-amino ester) prepared in this invention was transfected with mRNA encoding green fluorescent protein and tested, as follows:

[0084] 1) Cell culture: HeLa cells, UC-3 cells, and HCT 166 cells were cultured at a concentration of 1.0 × 10⁻⁶ cells / mL. 4 ~2.0×10 4 Cells were seeded at a density of 10 cells / well in 96-well plates and cultured overnight at 37°C.

[0085] 2) Mix a certain amount of endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester) into the solution encoding green fluorescent protein mRNA, let stand for 5-40 min, and then add it to the cells.

[0086] 3) 24-96 h after transfection, observe and photograph the cells transfected with green fluorescent protein mRNA under a fluorescence microscope. Specific implementation examples:

[0088] Example 1

[0089] 1,4-Butanediol diacrylate (20 mmol, 3.96 g) and 5-amino-1-pentanol (20 mmol, 2.060 g) were dissolved in dimethyl sulfoxide (DMSO) (6.02 mL) to a total concentration of 100 mg / mL. The reaction was carried out at 60 °C for 20 h. Then, DMSO (10% concentration in the total reaction system) was added. After the system reached room temperature, 5 times the amount of 1,4-butanediol diacrylate (equivalent to the amount of residual primary amine at the end of the resulting polymer) was added and the reaction was carried out at room temperature for 48 h to synthesize a linear poly(β-amino ester) with vinyl groups at the end and a molecular weight of 2000 Da. Figure 2 The gel permeation chromatography (GPC) curve of the purified linear poly(β-amino ester) with a molecular weight of 2000 Da prepared for this embodiment is shown. The smooth curve confirms that it was successfully synthesized.

[0090] Then, linear poly(β-amino ester) with vinyl groups at the end (6.02 g, 20 mmol), initiator azobisisobutyronitrile (8.2 mg, 0.05 mmol), and chain transfer agent 4-cyano-4-(phenylcarbonylthio)pentanoic acid (83.7 mg, 0.3 mmol) were dissolved in the organic solvent DMSO, with the concentration of the linear poly(β-amino ester) with vinyl groups at the end being 0.1 M. The solution was bubbled with argon gas for 30 min at room temperature to remove dissolved oxygen, and then sealed to prevent oxygen from entering during the reaction. The polymerization reaction was carried out at 50°C under magnetic stirring for 48 h. Then, NMS (856 mg, 4 mmol) was added and the reaction was carried out at room temperature for 24 h. Then, excess 1,11-diamino-3,6,9-trioxaundecan (1.15 g, 6 mmol) was added for end-capping reaction. Finally, the product was precipitated in excess diethyl ether and freeze-dried to obtain endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) with a molecular weight of 26000 Da. Figure 3 The 1H NMR spectrum of the purified endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) with a molecular weight of 26,000 Da prepared for this embodiment. Figure 4 The GPC curve of the purified endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) with a molecular weight of 26,000 Da prepared in this example is shown. The characteristic peaks of the benzene ring observed in the 1H NMR spectrum from 7.2 ppm to 7.6 ppm and the integrated area of ​​the GPC curve both confirm the successful synthesis of the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester). Figure 5 This is a physical image of the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) with a weight-average molecular weight of 26,000 Da in this embodiment.

[0091] Example 2

[0092] Diethylene glycol diacrylate (20 mmol, 4.28 g) and 4-amino-1-butanol (20 mmol, 1.80 g) were dissolved in dimethyl sulfoxide (DMSO) (11.48 mL) to a total concentration of 500 mg / mL. The reaction was carried out at 60 °C for 8 h. Then, DMSO (10% concentration in the total reaction system) was added. After the system reached room temperature, 5 equivalents (compared to the equivalent of the primary amine at the end of the resulting polymer) of diethylene glycol diacrylate were added and the reaction was carried out for 36 h to synthesize a linear poly(β-amino ester) with vinyl groups at the end and a molecular weight of 4500 Da. Figure 6 The GPC curve of the purified linear poly(β-amino ester) with a molecular weight of 4500 Da prepared for this example is shown. The smooth curve confirms that it was successfully synthesized.

[0093] Then, linear poly(β-amino ester) with vinyl groups at the end (3.04 g, 10 mmol), initiator azobisisobutyronitrile (4.1 mg, 0.025 mmol), and chain transfer agent 4-cyano-4-(phenylcarbonylthio)pentanoic acid (27.9 mg, 0.1 mmol) were dissolved in the organic solvent DMSO, with the concentration of the linear poly(β-amino ester) with vinyl groups at the end being 0.1 M. The solution was bubbled with argon gas for 30 min at room temperature to remove dissolved oxygen, and then sealed to prevent oxygen from entering during the reaction. The polymerization reaction was carried out at 60°C under magnetic stirring. After 48 h of reaction, NMS (428 mg, 2 mmol) was added and the reaction was carried out at room temperature for 24 h. Then, excess 1-(3-aminopropyl)-4-methylpiperazine (1.57 g, 10 mmol) was added for end-capping reaction. Finally, the product was precipitated in excess diethyl ether and freeze-dried to obtain endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) with a molecular weight of 49600 Da. Figure 7 The purified 1H NMR spectrum of the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) with a molecular weight of 49600 Da prepared for this example showed characteristic peaks of the benzene ring at 7.2 ppm to 7.6 ppm, confirming that the sulfonyl group was successfully grafted onto the end of the single-chain cyclic poly(β-amino ester). Figure 8 The GPC curve of the purified endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) with a molecular weight of 49600 Da prepared in this example is shown. The characteristic peak of the benzene ring observed in the 1H NMR spectrum from 7.2 ppm to 7.6 ppm and the integrated area of ​​the GPC curve both confirm the successful synthesis of the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester). Figure 9 This is a physical image of the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) with a weight-average molecular weight of 49,600 Da in this embodiment.

[0094] Example 3

[0095] In Example 1, when the mass ratio of endoplasmic reticulum-targeting single-stranded cyclic poly(β-amino ester) with a molecular weight of 26000 Da to DNA was 10:1, 20:1, 30:1, and 40:1, with 1 μg of DNA used, the affinity of endoplasmic reticulum-targeting single-stranded cyclic poly(β-amino ester) to DNA was tested using PicoGreen. First, the endoplasmic reticulum-targeting single-stranded cyclic poly(β-amino ester) solution was added to the DNA solution encoding green fluorescent protein, vortexed at high speed for 30 s, and then allowed to stand for 20 min to form complex nanoparticles. Then, the complex nanoparticle solution was diluted to 100 μL with TE buffer, and 100 μL of PicoGreen working solution was added. The excitation wavelength was 480 nm, and the emission wavelength was 520 nm, and the excitation fluorescence intensity was detected. Furthermore, using a similar method, complex nanoparticles with endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) to DNA mass ratios of 10:1, 20:1, 30:1, 40:1, and 50:1 were prepared. The complex nanoparticles were diluted to 1 mL with deionized water, and their particle size and surface potential were tested using dynamic light scattering (DLS). Simultaneously, the complex nanoparticles were washed and freeze-dried, and their microstructure was characterized using TEM.

[0096] Figure 10 This example demonstrates the DNA affinity test of the 26,000 Da endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester) prepared in Example 1. The example confirms that at a mass ratio of (10~40):1, the DNA binding efficiency of the 26,000 Da endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester) prepared in Example 1 exceeds 90%, confirming its excellent DNA affinity.

[0097] Figure 11 The particle size of the complex nanoparticles formed by the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) with a molecular weight of 26,000 Da prepared in Example 1 and DNA was tested. The DLS results confirmed that the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) with a molecular weight of 26,000 Da prepared in Example 1 can effectively compress DNA, and the particle size of the complex nanoparticles is less than 150 nm.

[0098] Figure 12This refers to the surface potential test of the complex nanoparticles formed by the endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester) with a molecular weight of 26,000 Da prepared in Example 1 and DNA. DLS results confirmed that the endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester) with a molecular weight of 26,000 Da prepared in Example 1 can effectively shield the negative potential of DNA itself, and the potential of the complex nanoparticles is positive.

[0099] Figure 13 This section describes the microstructure of the nanoparticles formed by the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) with a molecular weight of 26,000 Da and DNA, prepared in Example 1. TEM results confirm that the nanoparticles formed by the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) with a molecular weight of 26,000 Da and DNA prepared in Example 1 exhibit uniform particle size distribution and relatively stable structure, demonstrating their excellent stability.

[0100] Example 4

[0101] HeLa cells at 6.0 × 10 3 Cells were seeded at a density of 10 cells / well in 96-well plates and cultured overnight at 37°C. In Example 1, endoplasmic reticulum-targeting single-stranded cyclic poly(β-amino ester) with a molecular weight of 26000 Da was used in a mass ratio of 50:1 to AF647 fluorescently labeled luciferase-encoding DNA, with 0.5 μg of AF647 fluorescently labeled luciferase-encoding DNA. This formed complex nanoparticles, which were then mixed with culture medium and added to the cells. After incubation for 4 hours, the endoplasmic reticulum of the cells was stained with Lyso-Tracker Green at 37°C for 10 minutes. The distribution of DNA in the cells was then observed using laser confocal microscopy.

[0102] Figure 14 This section describes the endoplasmic reticulum (ER) targeting characterization of the complex nanoparticles formed by the 26,000 Da ER-targeting single-stranded cyclic poly(β-amino ester) prepared in Example 1 and DNA labeled with AF647 fluorescent dye. The figure shows a clear overlap between the DNA labeled with AF647 fluorescent dye and the ER labeled with ER-tracker Green fluorescent dye. This demonstrates significant co-localization of DNA molecules within the cell and the ER, confirming that the gene vector combining the ER-targeting monomer N-(2-aminoethyl)-4-methylbenzenesulfonamide with single-stranded cyclic poly(β-amino ester) exhibits good ER targeting performance. It overcomes the retention of the complex nanoparticles in lysosomes, facilitates the entry of exogenous DNA into the cell nucleus, and increases DNA transfection efficiency.

[0103] Example 5

[0104] HeLa cells, UC-3 cells, and HepG2 cells were selected at a concentration of 2.0 × 10⁻⁶. 4 Cells were seeded at a density of 10 cells / well in 96-well plates and cultured overnight at 37°C. A sodium acetate buffer solution of endoplasmic reticulum-targeting single-stranded cyclic poly(β-amino ester) with a molecular weight of 26,000 Da prepared in Example 1 was mixed with 0.7 μg of sodium acetate buffer solution encoding green fluorescent protein DNA (the mass ratio of endoplasmic reticulum-targeting single-stranded cyclic poly(β-amino ester) to DNA was 30:1 and 60:1, respectively). After standing for 45 min, the solution was added to serum-containing culture medium, thoroughly mixed, and then slowly added to the cells. The cells were then cultured for another 48 h, and the cells transfected with green fluorescent protein DNA were observed under a fluorescence microscope.

[0105] Figure 15 This is an evaluation of the DNA transfection performance of the endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester) with a molecular weight of 26,000 Da prepared in Example 1. Figure 15 In HeLa, UC-3, and HepG2 cells, endoplasmic reticulum-targeted single-stranded cyclic poly(β-amino ester) exhibits high transfection efficiency for DNA encoding green fluorescent protein due to its advantages in overcoming intracellular barriers such as low gene lysosomal escape efficiency and nuclear internalization restrictions, as well as its compact single-stranded transformation structure. This demonstrates that it can meet the requirements for efficient DNA delivery in various tissues and cells.

[0106] Example 6

[0107] HeLa cells were used at a concentration of 2.0 × 10⁻⁶. 4 Cells were seeded at a density of 10 cells / well in 96-well plates and cultured overnight at 37°C. A sodium acetate buffer solution of endoplasmic reticulum-targeting single-stranded cyclic poly(β-amino ester) with a molecular weight of 26,000 Da prepared in Example 1 was mixed with 0.7 μg of luciferase-encoding DNA solution (the mass ratio of endoplasmic reticulum-targeting single-stranded cyclic poly(β-amino ester) to DNA was 10:1, 20:1, 30:1, and 40:1, respectively). After standing for 45 min, the mixture was slowly added to HeLa cells after being mixed with serum-containing medium. The cells were then cultured for another 48 h. The cell viability after transfection with luciferase-encoding DNA was quantitatively evaluated using 10% Alamar Blue solution.

[0108] Figure 16The cell viability evaluation following transfection with the 26,000 Da endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) prepared in Example 1 was as follows: The cell viability results show that, due to the good biocompatibility, degradability, and relatively low carrier dosage of the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester), the transfected HeLa cells maintained high cell viability. This indicates that the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) has significant advantages in DNA delivery.

[0109] Example 7

[0110] HeLa cells, UC-3 cells, and HCT 116 cells were cultured at a concentration of 2.0 × 10⁻⁶. 4 Cells were seeded at a density of 10 cells / well in 96-well plates and cultured overnight at 37°C. The endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester) sodium acetate buffer solution from Example 1 was mixed with 0.2 μg of sodium acetate buffer solution encoding green fluorescent protein mRNA, allowed to stand for 45 min, and then added to serum-containing culture medium. After thorough mixing, the mixture was slowly added to the cells. Cells were then cultured for another 48 h, and cells transfected with green fluorescent protein mRNA were observed under a fluorescence microscope.

[0111] Figure 17 This section evaluates the mRNA transfection performance of the endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester) with a molecular weight of 26,000 Da prepared in Example 1. Figure 17 The study revealed that in HeLa, UC-3, and HCT 116 cells, the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) exhibited high transfection efficiency for green fluorescent protein mRNA due to its advantages in overcoming intracellular barriers such as low gene lysosomal escape efficiency and nuclear internalization limitations, as well as its compact single-chain transformation structure. This demonstrates its ability to deliver mRNA efficiently in various tissues and cells. These results indicate that the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) possesses high structural and performance flexibility, enabling simultaneous DNA and mRNA delivery.

[0112] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. An endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester), characterized in that, The structural formula of the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) is as follows: ； In the formula, R1 is the spacer arm of the diacrylate monomer, R2 is a group on the organic amine, R3 is a group on the endoplasmic reticulum targeting monomer, and R4 is a group on the capping agent monomer. The organic amine is R2-NH2, the endoplasmic reticulum targeting monomer is R3-NH2, the capping agent monomer is R4-NH2, n=5~40, m=5~60, r=3~15, p=3~20, q=3~30; the endoplasmic reticulum targeting monomer is the polypeptide Pardaxin or N-(2-aminoethyl)-4-methylbenzenesulfonamide.

2. The endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) according to claim 1, characterized in that, The molecular weight of the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) is in the range of 4000~50000 Da.

3. The method for preparing the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) according to any one of claims 1 to 2, characterized in that, Includes the following steps: 1) A linear poly(β-amino ester) with vinyl groups at the end is obtained by Michael addition reaction of diacrylate monomers with organic amines; 2) The linear poly(β-amino ester) with vinyl groups at the end obtained in step 1) is subjected to a reversible addition-fragmentation chain transfer polymerization reaction to obtain a single-chain cyclic poly(β-amino ester) containing vinyl groups. 3) The single-chain cyclic poly(β-amino ester) containing vinyl groups obtained in step 2) is subjected to a functionalization end-capping reaction with an endoplasmic reticulum-targeting monomer to obtain an endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester) containing vinyl groups. 4) React the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) containing vinyl groups obtained in step 3) with the end-capping agent monomer to obtain the endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester).

4. The method for preparing endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) according to claim 3, characterized in that, The diacrylate monomers are 1,6-hexanediol dimethacrylate, 1,4-butanediol diacrylate, 2,2-dithiodiethanol diacrylate, diethylene glycol diacrylate, polyethylene glycol diacrylate, tetraethylene glycol diacrylate, or bisphenol A polyoxyethylene ether diacrylate. And / or, the organic amine is 5-amino-1-pentanol, 4-amino-1-butanol, butylamine, pentanol, hexylamine, octylamine, 1-(2-aminoethyl)piperazine, N-(2-aminoethyl)morpholine, dodecylamine or octadecylamine; And / or, the end-capping agent monomer is 1-(3-aminopropyl)-4-methylpiperazine, N-(3-aminopropyl)morpholine, N-(2-aminoethyl)morpholine, 2-methyl-2-morpholin-4-ylpropane-1-amine, 1-(2-aminoethyl)piperazine, 1,4-bis(3-aminopropyl)piperazine, 1,11-diamino-3,6,9-trioxaundecan, 1,2-ethylenediamine, or 2-methyl-1,5-pentanediamine.

5. The method for preparing endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) according to claim 3, characterized in that, In step 1), the molar ratio of the diacrylate monomer to the organic amine is 1:(0.5~5).

6. The method for preparing endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) according to claim 3, characterized in that, Step 2) Specifically, the linear poly(β-amino ester) with vinyl groups at the end obtained in step 1) and the chain transfer agent are added to the reaction solvent to dissolve them. Then, under the protection of an inert gas, an initiator is added to carry out the polymerization reaction to obtain a single-chain cyclic poly(β-amino ester) containing vinyl groups. Wherein, the molar ratio of the linear poly(β-amino ester) with vinyl groups at the end to the chain transfer agent is (2000~10):1; the molar ratio of the initiator to the chain transfer agent is 1:(0.5~6).

7. The method for preparing endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) according to claim 3, characterized in that, In step 3), the molar ratio of the endoplasmic reticulum targeting monomer to the single-chain cyclic poly(β-amino ester) containing vinyl groups is 1:(0.3~5).

8. The method for preparing endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) according to claim 3, characterized in that, In step 4), the molar ratio of the end-capping agent monomer to the vinyl group-containing endoplasmic reticulum-targeted single-chain cyclic poly(β-amino ester) is (1~5):

1.

9. The use of the endoplasmic reticulum-targeting single-chain cyclic poly(β-amino ester) according to any one of claims 1 to 2 in the preparation of gene vectors, characterized in that, The gene vector is used to deliver genes into tissues or cells.

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