Preparation method of binary guanidyl block copolymer for human vascular endothelial cell nucleic acid delivery

Abstract

The invention relates to a preparation method of a binary guanidyl block copolymer (Poly-PEG-GUA) for nucleic acid transfection of human vascular endothelial cells (HUVEC). According to the invention, N-(2-(3-carbamyl guanidyl) ethyl) methacrylamide with different proportions is subjected to reversible addition-fragmentation chain transfer (RAFT) polymerization to polyethylene glycol modified 4-cyano-4-(phenyl thioformyl sulfenyl) valeric acid (PEG-CPADN), so that five kinds of binary guanidyl block copolymers with different molecular weights are constructed; the HUVEC gene and messenger ribonucleotide (EGFP-mRNA) of green fluorescent protein are assembled in the HUVEC for screening of transfection efficiency, and the optimal molecular weight for HUVEC transfection is optimized. Experimental results show that the Poly-PEG-GUA with the molecular weight of 53575 Daltons can be used for effectively delivering green fluorescent protein (EGFP) mRNA (messenger ribonucleic acid) to HUVEC (human umbilical vein endothelial cells), and the highest transfection efficiency is shown. The optimized Poly-PEG-GUA in the invention has a certain reference value for the research and development of subsequent cardiovascular transfection products.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and specifically relates to the preparation and application of a binary guanidine-based block copolymer capable of transfecting human vascular endothelial cells. Background Art

[0002] Endothelial cells (HUVEC) are a single layer of flat epithelial cells that are crucial in the cardiovascular system. They not only form a barrier for the vascular wall, but also participate in a variety of physiological processes, including regulating vascular tension, maintaining the balance between anticoagulation and procoagulant, regulating inflammatory response, promoting angiogenesis and repair, and metabolic function. These functions are extremely important for maintaining the normal functioning of the cardiovascular system. When endothelial cells are damaged or dysfunctional, such as in disease states such as hypertension and atherosclerosis, a series of cardiovascular complications may occur.

[0003] Although significant progress has been made in the study of the function of endothelial cells, attempts to directly intervene in endothelial cells to treat related diseases face many challenges. In particular, the process of efficiently and safely delivering exogenous nucleic acids (such as DNA or RNA) into endothelial cells is particularly difficult. This is mainly due to the following reasons: First, vascular endothelial cells are located at the interface of blood flow, making it difficult to effectively implement traditional transfection methods; second, endothelial cells have highly selective permeability barrier properties and have low efficiency in the uptake of macromolecules; third, even after successfully entering the cells, exogenous nucleic acids are easily degraded by intracellular nucleases. Therefore, the development of efficient targeted delivery systems has become one of the focuses of current research.

[0004] In recent years, with the development of nanotechnology, non-viral vectors based on block copolymers have shown great potential in the field of nucleic acid delivery due to their unique advantages. This type of material is usually composed of monomer units with two different properties, hydrophilicity and hydrophobicity, and can form polymer chains with specific structural characteristics. By adjusting the proportion and arrangement of each component, researchers can precisely control key parameters such as the size, shape and surface charge of the final nanoparticles, thereby optimizing the efficiency of their interaction with target cells. In addition, block copolymers also exhibit good biocompatibility and low cytotoxicity, laying the foundation for safe and effective gene delivery.

[0005] Guanidinium is a functional group with a strong positive charge. When designing block copolymers for nucleic acid delivery, the introduction of guanidinium can bring multiple advantages. The positive charge carried by the guanidinium band can be tightly bound to the negatively charged phosphate backbone in the nucleic acid molecule through electrostatic interaction, thereby improving the encapsulation efficiency and stability of the nucleic acid. At the same time, the guanidinium group can also enhance the interaction between the carrier and the negatively charged glycoproteins on the cell surface, thereby promoting the entry of nanoparticles into cells through endocytosis. Inside the cell, the guanidinium group can help escape the lysosomal pathway by destroying the lysosomal membrane, which helps to increase the proportion of nucleic acids released from the lysosome to the cytoplasm, thereby improving the transfection efficiency. In addition, polymers containing guanidinium groups generally show lower cytotoxicity and higher biocompatibility. These advantages make block copolymers containing guanidinium groups one of the important materials for the development of new, efficient and low-toxic gene delivery systems.

[0006] To this end, the present invention uses two components, N-(2-(3-carbamoylguanidinyl)ethyl)methacryloyl and polyethylene glycol-modified 4-cyano-4-(phenylthiocarbamoylthio)pentanoic acid (PEG-CPADN), and prepares five binary guanidinyl block copolymers with different molecular weights by adjusting the mass ratio of the two components. Based on the material characterization and the verification of the transfection efficiency, a binary guanidinyl block copolymer with the optimal molecular weight is screened out for transfection of HUVEC. Summary of the invention

[0007] The purpose of the present invention is to prepare a green fluorescent protein mRNA nano drug delivery system with HUVEC as the editing object, which is used for HUVEC intracellular delivery and provides a new method for gene therapy of cardiovascular diseases. The specific steps are as follows:

[0008] Step 1: In the presence of initiator AIBN and inert gas, N-(2-(3-carbamoylguanidinyl)ethyl)methacrylamide of different weights was polymerized onto polyethylene glycol-modified 4-cyano-4-(phenylthiocarbamoylthio)pentanoic acid (PEG-CPADN) molecules by reversible addition-fragmentation chain transfer polymerization (RAFT), and the reaction was carried out at high temperature overnight. The reaction solution was dialyzed in pure water for 1-2 days, and the dialyzate was lyophilized to obtain binary guanidine block copolymers of different molecular weights.

[0009] Step 2: Add EGFP-mRNA to the above polymer at a certain mass ratio, and incubate at room temperature for a period of time to obtain the EGFP-mRNA nano drug delivery system of the present invention.

[0010] Preferably, in the step 1, the mass ratios of N-(2-(3-carbamoylguanidinyl)ethyl)methacrylamide to N-(2-(3-carbamoylguanidinyl)ethyl)methacrylamide are 1.6:1, 3.2:1, 4.8:1, 6.4:1, and 8:1, respectively, and they are named Poly-PEG-GUA1 (43409 Daltons), Poly-PEG-GUA2 (44995 Daltons), Poly-PEG-GUA3 (48176 Daltons), Poly-PEG-GUA4 (49893 Daltons), and Poly-PEG-GUA5 (53575 Daltons), respectively.

[0011] Preferably, in step 2, the mass ratio of EGFP-mRNA to the binary guanidine block copolymer carrier is 1:4.

[0012] Preferably, in step 2, the incubation time of EGFP-mRNA and the binary guanidine block copolymer carrier at room temperature is 30 minutes.

[0013] The beneficial effects of the present invention are:

[0014] The binary guanidine-based block copolymer of the present invention can be used as a new nano-carrier material to improve transfection efficiency.

[0015] The delivery of mRNA by the binary guanidine-based block copolymer of the present invention can be used as a gene therapy strategy for cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a binary guanidine block copolymer carrier structure.

[0017] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the binary guanidine block copolymer.

[0018] Figure 3 Molecular weight detection of five binary guanidine block copolymers.

[0019] Figure 4 Transmission electron microscopy images of nanoparticles assembled from Poly-PEG-GUA with five different molecular weights and EGFP-mRNA.

[0020] Figure 5 The hydrated particle sizes after assembly of five different molecular weight Poly-PEG-GUA and nucleic acid.

[0021] Figure 6 The potentials of five different molecular weight Poly-PEG-GUA after assembly with nucleic acid.

[0022] Figure 7 The encapsulation efficiency of five different molecular weight Poly-PEG-GUA assembled with nucleic acid.

[0023] Figure 8 The efficiency of five different molecular weight Poly-PEG-GUA in transfecting EGFP-mRNA was screened for high content.

[0024] Fig. 9 The figure shows the cell viability results after HUVEC was treated with five different molecular weight Poly-PEG-GUA for 24 hours as determined by the CCK-8 method. DETAILED DESCRIPTION

[0025] The following will be combined with the accompanying drawings in the embodiments of the present invention to further fully explain the technical solutions in the embodiments of the present invention. The described embodiments are only some embodiments of the present invention. After reading the content of the present invention, researchers in the field can understand the present invention more comprehensively. The experimental methods used in the present invention are all conventional methods in the field, and the reagents used are all commercially available.

[0026] Implementation Example 1: Synthesis and verification of binary guanidine-based block copolymers.

[0027] Synthesis of Poly-PEG-GUA. Take 50 mg of PEG-CPADN and mix with 79.55 mg, 159.1 mg, 238.65 mg, 318.2 mg, and 379.75 mg of N-(2-(3-carbamoylguanidinyl)ethyl)methacrylamide, respectively, add 0.85 mg of initiator azobisisobutyronitrile, and react overnight at high temperature under the protection of inert gas. The reaction solution is dialyzed in pure water for 1-2 days and freeze-dried to obtain a binary guanidinyl block copolymer Poly-PEG-GUA. The structural formula of the polymer is as follows Figure 1 shown.

[0028] Structural verification of Poly-PEG-GUA. The structure of the binary guanidine block copolymer was analyzed by nuclear magnetic resonance. The specific steps are as follows: take an appropriate amount of Poly-PEG-GUA freeze-dried powder, vacuum dry the powder, dissolve the powder in deuterated dimethyl sulfoxide, place it in a nuclear magnetic resonance tube, and detect the chemical structure by nuclear magnetic resonance hydrogen spectrum. The results are shown in Figure 2.

[0029] Poly-PEG-GUA molecular weight test. Take five appropriate amounts of Poly-PEG-GUA and dissolve them to a concentration of about 0.1%-5%. Filter with a 0.45μm filter membrane and let the polymer solution reach room temperature before testing. The results are as follows Figure 3 shown.

[0030] Embodiment 2: Assembly performance of binary guanidine-based block copolymer and EGFP-mRNA system.

[0031] Methods: The assembly performance of polymer and EGFP-mRNA was investigated by transmission electron microscopy, Malvern particle size analyzer, Ribogreen and other methods.

[0032] Characterization by transmission electron microscopy: Five kinds of lyophilized powders of binary guanidine block copolymers Poly-PEG-GUA with different molecular weights were resuspended in an appropriate amount of pure water. After being evenly dispersed, the Poly-PEG-GUA suspension was dripped onto the copper mesh respectively, air-dried at room temperature, and the morphology and dispersibility of the particles were observed by transmission electron microscopy. Figure 4 It can be seen that after the two are assembled, a nanoparticle structure is formed, with an average particle size of about 30 nm, uniform size and good dispersion.

[0033] Malvern particle sizer characterization: Five different molecular weight binary guanidine block copolymers Poly-PEG-GUA were assembled with EGFP-mRNA, and after uniform dispersion, the hydrated particle size and potential change after assembly were detected in a Malvern particle sizer. Figure 5 It can be seen that after Poly-PEG-GUA with a molecular weight of 48176 Daltons is assembled with nucleic acid, its hydrated particle size is the smallest.

[0034] Ribogreen detection of encapsulation efficiency. ① Establishment of the standard curve for determining nucleic acid encapsulation efficiency by Ribogreen method. ② Determination of sample encapsulation efficiency. Take five different molecular weights of binary guanidine-based block copolymers Poly-PEG-GUA, assemble them with EGFP-mRNA, and after uniform dispersion, use an enzyme marker to determine their encapsulation efficiency. Figure 7 It can be seen that the encapsulation rate of nucleic acid by Poly-PEG-GUA with a molecular weight of 53575 Daltons is about 87%, which can efficiently encapsulate nucleic acid.

[0035] Example 3: Transfection efficiency of nano-delivery system.

[0036] This example uses high content to detect the transfection efficiency of five different molecular weight Poly-PEG-GUA in delivering EGFP-mRNA in HUVEC. The method is as follows:

[0037] Cell culture: Human vascular smooth muscle cells (HUVEC) were cultured in 1640 liquid culture medium containing 10% fetal bovine serum in an incubator at 37° C. and 5% carbon dioxide.

[0038] Transfection: Add cell slides to a 24-well plate and inoculate at a density of 40,000 cells / well, with a total of 18 wells inoculated. After the cells adhere to the wall, divide the cells into 6 groups, with 3 wells in each group. Incubate EGFP-mRNA and Poly-PEG-GUA of different molecular weights at room temperature for 30 minutes to obtain a transfection complex solution. Add the transfection complex solution to HUVEC, incubate for 48 hours, discard the culture medium, wash with sterile PBS 3 times, add 1 mL of Hochest staining solution, and incubate at 37°C for 20 minutes. Use high content to detect the expression of green fluorescent protein. Figure 8 It can be seen that Poly-PEG-GUA with a molecular weight of 53575 Daltons has the highest transfection efficiency.

[0039] Implementation Example 4: This example uses CCK8 colorimetry to investigate the effects of five different molecular weight Poly-PEG-GUA concentrations on HUVEC cell viability, as follows:

[0040] Cell culture: The method is the same as Example 3.

[0041] Cell viability assay: cells were seeded in 96-well plates at a density of 8,000 cells / well, with a total of 48 wells. When the cells adhered to the wall and grew to 60% of the wells, the cells were divided into 16 groups, with 3 wells in each group. 0 μg / mL (control group), 2 μg / mL Poly-PEG-GUA with five different molecular weights, 10 μg / mL Poly-PEG-GUA with five different molecular weights, and 50 μg / mL Poly-PEG-GUA with five different molecular weights were added to each group. After 24 hours of drug treatment, the culture medium was removed, and after rinsing 3 times with sterile PBS, 100 μL of 1640 culture medium containing 10% (volume ratio) CCK8 was added to each well. After incubation in the incubator for 2 hours, the absorbance at 450 nm was measured on an enzyme reader. The absorbance value of each well divided by the absorbance value of the control group was taken as the cell viability value, and then graphed and analyzed using Graphpad. Fig. 9 As shown, Poly-PEG-GUA has no obvious toxicity to cell viability within a certain concentration range.

Claims

1. A binary guanidine-based block copolymer, characterized in that Successful delivery of green fluorescent protein (EGFP) mRNA in HUVECs was achieved.

2. A binary guanidine-based block copolymer as claimed in claim 1, characterized in that According to the different feed ratios of polyethylene glycol-modified 4-cyano-4-(phenylthioformylthio) valeric acid and N-(2-(3-carbamoylguanidinyl)ethyl) methacrylamide, the invention synthesizes polymers with different molecular weights.

3. A method for preparing a binary guanidine-based block copolymer as described in claims 1-2, characterized in that: The specific synthetic route is as follows: Take an appropriate amount of polyethylene glycol-modified 4-cyano-4-(phenylthioformylthio) pentanoic acid and an appropriate amount of N-(2-(3-carbamoylguanidino)ethyl) methacrylamide, add initiator azobisisobutyronitrile (AIBN), and react at high temperature overnight under the protection of inert gas. The reaction solution is dialyzed in pure water for 1-2 days, and the dialyzate is freeze-dried to obtain a binary guanidine block copolymer.

4. A method for preparing a binary guanidine-based block copolymer as claimed in claims 1 to 3, characterized in that: The mass ratios of guanidine side chain to PEG-CPADN were 1.6:1, 3.2:1, 4.8:1, 6.4:1, and 8:1, respectively, and five binary guanidine block copolymers with molecular weights of 43409 Daltons, 44995 Daltons, 48176 Daltons, 49893 Daltons, and 53575 Daltons were synthesized.

5. The use according to any one of claims 1 to 4, characterized in that: The preparation method of the green fluorescent protein (EGFP) mRNA delivery system comprises mixing five guanidine block copolymer carriers, binary guanidine block copolymers and green fluorescent protein (EGFP) mRNA in the presence of an aqueous solution. Preferably, the mass ratio of the green fluorescent protein (EGFP) mRNA to the binary guanidine block copolymer carrier is 1:4; preferably, the mixing time is 30 minutes.

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