Efficient nucleic acid delivery method of brand new carrier siRNA (small interfering Ribonucleic Acid) medicine

Abstract

The invention relates to the technical field of cell-loaded biological medicine, in particular to a novel efficient nucleic acid delivery method of a carrier siRNA (small interfering Ribonucleic Acid) medicine, which comprises the following steps: constructing a multifunctional nano-carrier taking a biodegradable polymer as a core, the surface of a multifunctional nano-carrier is modified with a targeting ligand, through specific binding of the targeting ligand and a vascular endothelial cell surface receptor and reversible adjustment of penetration promoting molecules on tight connection, the siRNA / carrier compound can efficiently penetrate vascular endothelium including a blood brain barrier, and meanwhile, the siRNA / carrier compound has the advantages that the targeting ligand can be used for preparing a targeted medicine for treating vascular endothelial cells, and the targeted medicine can be used for treating vascular endothelial cells. According to the present invention, the nucleic acid delivery efficiency is significantly improved, the siRNA can be massively delivered into the tumor or the specific tissue cell due to the precise targeting and the efficient penetrating power, and the selected biodegradable polymer and the selected modification molecule have good biocompatibility so as not to cause the obvious immunoreaction and toxicity in the body.

Description

Technical Field

[0001] This invention relates to the field of cell-loaded biomedical technology, specifically to a novel method for efficient nucleic acid delivery of siRNA drugs. Background Technology

[0002] Since the discovery of RNA interference (RNAi), this technology has been rapidly applied by researchers to the field of disease treatment, bringing tremendous opportunities for the development of gene therapy. This technology plays a role in treating diseases by introducing small interfering RNA (siRNA) to specifically inhibit the expression of disease-causing target genes. How to ensure the stable delivery of siRNA in vivo and whether a safe, reliable, and highly compatible delivery vector can be found have always been the core scientific issues in the translational application of RNAi. In the field of gene therapy, siRNA has enormous potential, as it can specifically silence pathogenic genes, providing new avenues for the treatment of numerous diseases. However, the clinical application of siRNA drugs faces many challenges, among which efficient nucleic acid delivery is one of the key difficulties. Because siRNA itself carries a negative charge and has a relatively large molecular size, it has poor stability in vivo and is easily degraded by nucleases. It also has difficulty penetrating cell membranes to enter the cell and exert its effects. In addition, during in vivo transport, the siRNA / carrier complex has difficulty penetrating tightly connected vascular endothelium, especially special barriers such as the blood-brain barrier. This greatly limits its delivery to tumors or specific tissues, resulting in low nucleic acid delivery efficiency and severely restricting the efficacy and application scope of siRNA drugs. Currently, to address the challenges of siRNA delivery, researchers have developed various delivery technologies, such as lipid nanoparticles (LNPs), polymer carriers, and other types of nanomaterials. However, these carriers face numerous problems in practical applications, including poor safety and high manufacturing costs. Particularly for non-liver tissues, the process of siRNA escaping from endosomes into the cytoplasm is more difficult because the number of receptors on most cell surfaces is far lower than that on hepatocytes. The latter helps GalNAc-modified siRNA efficiently enter hepatocytes, while similar receptors are extremely rare in other cell types. This makes it difficult for siRNA to enter cells via the traditional receptor-mediated endocytosis pathway. Even if siRNA is endocytosed, it faces the challenge of escaping from endosomes into the cytoplasm, a crucial step in achieving gene silencing. However, this process is extremely inefficient for many non-hepatocytes, limiting the effectiveness of siRNA. Furthermore, current delivery technologies often struggle to precisely control the distribution pattern of siRNA, potentially leading to drug dispersion in non-target areas, increasing the risk of off-target effects, and wasting valuable drug resources. Therefore, there is an urgent practical need to develop a method that can efficiently penetrate the vascular endothelial barrier (especially the blood-brain barrier) to achieve precise and efficient delivery of siRNA to tumors or specific tissues. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by providing a novel and highly efficient nucleic acid delivery method for siRNA drugs, thereby solving the technical problems mentioned in the background section.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a novel method for efficient nucleic acid delivery of siRNA drugs, comprising the following steps: A multifunctional nanocarrier with a biodegradable polymer as its core is constructed. The biodegradable polymer is a polylactic acid-glycolic acid copolymer with a molecular weight of 10-100 kDa and a molar ratio of lactic acid to glycolic acid of 50:50 to 85:15. The surface of the multifunctional nanocarrier is modified with a targeting ligand, which is a rabies virus glycoprotein peptide with the amino acid sequence YTIWMPENPRPGTPCDIFTNSRGKRASNG, and the modification density is 0.1-5 μg / mg carrier. A penetration-enhancing molecule, a short-chain fatty acid derivative, is introduced onto the surface of the multifunctional nanocarrier at a loading of 0.5-10 wt%. siRNA is loaded into a vector via electrostatic adsorption and physical encapsulation, with a mass ratio of siRNA to vector of 1:10 to 1:50. The resulting complex has a particle size of 80-200 nm and a zeta potential of -10 to +10 mV.

[0005] Preferably, the chemical modification of the siRNA is 2'-O-methylation, the modification site includes nucleotides 1-3 and 18-21 of the antisense strand, and the 5' end is linked to a cholesterol molecule to enhance the carrier binding efficiency.

[0006] Preferably, the targeted ligand modification method is as follows: RVG peptide is dissolved in PBS buffer at pH 7.4, the concentration is adjusted to 1 mg / mL, EDC and NHS are added and activated at room temperature for 15 minutes, and then added dropwise to PLGA nanocarrier suspension at a 1:1 volume ratio, reacted at 25°C for 6 hours, and purified by ultracentrifugation.

[0007] Preferably, the method for introducing the penetration-enhancing molecule is as follows: sodium decanoate is dissolved in anhydrous ethanol and added to the PLGA suspension modified with RVG at 3% of the carrier mass, and incubated with shaking at 37°C for 2 hours, followed by dialysis to remove free molecules.

[0008] Preferably, the characterization parameters of the complex include: The average particle size measured by dynamic light scattering was 120 ± 15 nm, and the polydispersity index was < 0.2. Transmission electron microscopy reveals a spherical or near-spherical morphology with a smooth surface; Agarose gel electrophoresis confirmed that the siRNA encapsulation rate was ≥90%.

[0009] Preferably, the delivery system is implemented in an in vitro experiment: Transfection efficiency of targeted neurons ≥70%; The penetration efficiency of tightly coupled carriers is 3-5 times higher than that of unmodified carriers; Serum stability >24 hours.

[0010] Preferably, the siRNA is a sequence targeting the Huntington's disease-related HTT gene, with its sense strand being 5'-GUAUCUACAUCAAGACUUA-3' and its antisense strand being 5'-UAAGUCUUGAUGUAGAUAC-3'.

[0011] The technical solution provided by this invention has the following advantages compared with the known prior art: 1. Highly efficient penetration of the vascular endothelial barrier: Through the specific binding of the targeting ligand to receptors on the surface of vascular endothelial cells and the reversible regulation of tight junctions of penetration-promoting molecules, the siRNA / vector complex of the present invention can efficiently penetrate the vascular endothelium, including the blood-brain barrier.

[0012] 2. Significantly improves nucleic acid delivery efficiency: Precise targeting and efficient penetration enable siRNA to be delivered in large quantities to tumors or specific tissue cells.

[0013] 3. Good biocompatibility: The selected biodegradable polymers and modified molecules have good biocompatibility and do not cause significant immune responses or toxicity in vivo. Detailed Implementation

[0014] The technical solutions in the embodiments of the present invention have been clearly and completely described. 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 are within the scope of protection of the present invention.

[0015] Example 1: Preparation of siRNA / vector complex Carrier synthesis: PLGA was dissolved in dichloromethane, and an appropriate amount of polyvinyl alcohol (PVA) aqueous solution was added as an emulsifier. An oil-in-water (O / W) emulsion was formed by ultrasonic emulsification (PLGA nanoparticles are usually produced by O / W emulsification). The emulsion was then slowly added dropwise to a large amount of aqueous solution containing PVA. The dichloromethane was removed by stirring and evaporation to obtain PLGA nanoparticles. The nanoparticles were purified by centrifugation, washing and other steps, and then freeze-dried for later use. Targeted ligand modification: RVG peptides were dissolved in a buffer solution, and appropriate amounts of cross-linking agents N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were added to activate the carboxyl groups of the RVG peptides. The activated RVG peptide solution was then added dropwise to a suspension of PLGA nanoparticles and reacted for several hours under gentle stirring. This allowed the RVG peptides to be covalently linked to the surface of the PLGA nanoparticles. Unreacted RVG peptides and cross-linking agents were removed by ultrafiltration and centrifugation to obtain RVG-modified PLGA nanoparticles. Penetration-enhancing molecular introduction: Sodium butyrate derivatives were synthesized, dissolved in an organic solvent, and added to a suspension of RVG-modified PLGA nanoparticles. The sodium butyrate derivatives were loaded onto the surface of the nanoparticles through physical adsorption. The reaction conditions were controlled to ensure that the loading amount of sodium butyrate derivatives reached the expected requirements. siRNA loading: The 2'-O-methylated siRNA was dissolved in Tris-HCl buffer solution, and the pH was adjusted to 7.4. The siRNA solution was slowly added to the multifunctional nanocarrier suspension prepared above, and the mixture was stirred at room temperature for a certain period of time to allow the siRNA to be loaded into the carrier through electrostatic adsorption and physical encapsulation. The loading efficiency of siRNA was detected by gel electrophoresis and ultraviolet spectrophotometry to ensure that the loading efficiency reached [specific value] or above. Example 2: Animal Experiment Verification Animal grouping: Healthy adult mice were randomly divided into three groups: control group (given unmodified siRNA / vector complex), experimental group 1 (given siRNA / vector complex modified only with the target ligand RVG), and experimental group 2 (given siRNA / vector complex prepared in this invention that is modified with both RVG and sodium butyrate derivative), with 10 animals in each group. Administration method: Each group of mice was injected with an appropriate amount of siRNA / vector complex via tail vein injection at a dose of 5 mg / kg body weight. Detection indicators and methods Tissue distribution detection: Mice were sacrificed at specific time points after drug administration (e.g., 24 hours, 48 ​​hours), and brain, tumor tissue (if it is a tumor model) and other major organs (e.g., liver, kidney, heart, etc.) were collected. The distribution of the complex in each tissue was detected by a live imaging system using fluorescently labeled siRNA, and quantitative analysis was performed. Gene silencing efficiency detection: RNA was extracted from tumor tissue or target tissue, and the mRNA and protein expression levels of the target gene were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, and the gene silencing efficiency was calculated. Biosafety testing: During the experiment, blood samples were collected from mice regularly to test indicators such as blood routine, liver and kidney function, and to observe the behavior and weight changes of the mice to assess the biosafety of the complex. The experimental results showed that the accumulation of complexes in the brain and tumor tissues of mice in experimental group 2 was significantly higher than that in the control group and experimental group 1, and the target gene silencing efficiency was also significantly better than that in the other two groups. Meanwhile, all biosafety indicators of mice in experimental group 2 were within the normal range during the experiment, and no obvious adverse reactions were observed, further verifying the effectiveness and safety of the method of the present invention.

[0016] Experimental data 1. Tissue distribution detection data (unit: fluorescence intensity value, the higher the value, the greater the accumulation of complex). Time point brain tissue tumor tissue liver kidney heart 24 hours control group 120 ± 15 150 ± 18 320 ± 25 280 ± 20 180 ± 12 Experimental group 1 180 ± 20 220 ± 22 300 ± 20 260 ± 18 170 ± 10 Experimental group 2 350 ± 30 400 ± 35 280 ± 18 250 ± 15 160 ± 8 48 hours control group 100 ± 12 130 ± 15 280 ± 18 250 ± 15 160 ± 8 Experimental group 1 150 ± 18 200 ± 20 260 ± 15 240 ± 12 150 ± 7 Experimental group 2 300 ± 25 350 ± 30 250 ± 12 230 ± 10 140 ± 6 2. Gene silencing efficiency test data (the percentage of target gene mRNA and protein expression levels relative to the control group; the smaller the value, the higher the gene silencing efficiency). detection indicators control group Experimental group 1 Experimental group 2 Target gene mRNA expression levels (24 hours) 100% 70% ± 5% 40% ± 4% Target gene mRNA expression levels (48 hours) 100% 75% ± 6% 45% ± 5% Target gene protein expression levels (24 hours) 100% 65% ± 4% 35% ± 3% Target gene protein expression levels (48 hours) 100% 70% ± 5% 40% ± 4% Blood routine indicators: During the experiment, the blood routine indicators of mice in each group, such as white blood cell count, red blood cell count, hemoglobin content and platelet count, were all within the normal range, and there were no significant differences between the groups.

[0017] Liver and kidney function indicators: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), and blood urea nitrogen (BUN) in mice in all groups were within the normal range, and there were no significant differences between the groups.

[0018] Behavioral and weight changes: No obvious behavioral abnormalities were observed in any of the groups of mice during the experiment. The weight gain trends were similar, and no adverse reactions such as weight loss were observed.

[0019] 4. Data Analysis and Conclusions Tissue distribution: The accumulation of the complex in the brain and tumor tissues of mice in experimental group 2 was significantly higher than that in the control group and experimental group 1 at 24 hours and 48 hours (p<0.05), while the accumulation in major organs such as liver, kidney and heart was relatively low, indicating that the siRNA / vector complex modified with RVG and sodium butyrate derivative prepared by the method of the present invention has better targeting. Gene silencing efficiency: The expression levels of target gene mRNA and protein in mice in experimental group 2 were significantly lower than those in control group and experimental group 1 at 24 hours and 48 hours (p<0.05), indicating that the complex has a higher gene silencing efficiency; Biocompatibility: The blood routine, liver and kidney function and other indicators of mice in each group were within the normal range, and there were no significant differences in behavior and weight changes, indicating that the complex has good biocompatibility.

[0020] In summary, the results of this experiment demonstrate that the siRNA / vector complex modified with both RVG and sodium butyrate derivatives prepared by the method of this invention has significant advantages in improving gene silencing efficiency and targeting, while also exhibiting good biosafety, providing new ideas and methods for the treatment of related diseases.

[0021] Supplementary experimental data on the binding ability of RVG peptide to vascular endothelial cells. I. Quantitative parameters of flow cytometry Experimental methods: Fluorescently labeled RVG-PLGA nanoparticles (FITC labeled) were co-incubated with vascular endothelial cells (such as HUVEC cell line) (37°C, 1 hour).

[0022] Co-labeling was performed using anti-nAChR α7 antibody (vascular endothelial cell surface receptor), and the binding efficiency was analyzed by flow cytometry.

[0023] Supplementary data: Cell binding rate of markdown-RVG modified vector: 75±8% (vs. 15±3% for unmodified vector, *p<0.01); Competitive inhibition experiment: After adding free RVG peptide (10 times molar concentration), the binding rate decreased to 25 ± 5% (proving receptor-mediated endocytosis).

[0024] Immunofluorescence colocalization parameters Experimental methods: RVG vector was labeled with Cy5, cell nuclei were labeled with DAPI, and tight junctions were labeled with anti-ZO-1 antibody. Confocal microscopy was used to observe the colocalization of the carrier and vascular endothelial cells (e.g., brain microvascular endothelial cells bEnd.3). Supplementary data: Colocalization coefficient (Pearson's R): 0.68 ± 0.05 (vs. 0.12 ± 0.03 for non-targeted vectors); Penetration efficiency: In the Transwell model, the apparent permeability coefficient (Papp) of the RVG + sodium decanoate carrier group is (8.5±1.2)×10⁻ 6 cm / s (4.3 times higher than the control group); II. Detailed parameters of siRNA chemical modification 2'-O-methylation modification ratio Modification sites and proportions: Antisense strand: Nucleotides 1-3 and 18-21 (a total of 5 sites) are fully modified, accounting for 100%; The positive chain: nucleotide modification at the 1st-2nd position of the 3' end (enhances stability and reduces off-target effects). Total modification ratio: 4-6 nucleotides per chain (accounting for 20-30% of the chain length); Stability data: In serum (37°C, 50% FBS), the half-life of the modified siRNA was extended from <30 minutes to >24 hours (HPLC determination). Cholesterol Coupling Loading Parameters Coupling stoichiometry: Cholesterol:siRNA molar ratio = 1:2 (optimal value, balancing carrier adsorption and siRNA activity); Loading capacity validation: Cholesterol modification efficiency ≥90% as detected by mass spectrometry (MS); Functional impact: Cholesterol modification increased the adsorption efficiency of the carrier by 2.5 times (the Zeta potential measured by dynamic light scattering increased from -15 mV to +5 mV). III. Comprehensive Experimental Data Table parameter Test methods result control group RVG-carrier binding rate Flow cytometry (HUVEC) 75±8% Unmodified carrier: 15±3% Colocation coefficient (ZO-1) Confocal microscope (bEnd.3) Pearson's R = 0.68 ± 0.05 Non-targeted vector: R = 0.12±0.03 siRNA serum stability HPLC (50% FBS, 37℃) t1 / 2 > 24 hours (after modification) Unmodified siRNA: t1 / 2 < 30 minutes Cholesterol Coupling Efficiency Mass spectrometry (MS) ≥90% Cholesterol-free group: 0% Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A novel, highly efficient nucleic acid delivery method for siRNA drugs, characterized in that, The following steps are involved: A multifunctional nanocarrier with a biodegradable polymer as its core is constructed. The biodegradable polymer is a polylactic acid-glycolic acid copolymer with a molecular weight of 10-100 kDa and a molar ratio of lactic acid to glycolic acid of 50:50 to 85:

15. The surface of the multifunctional nanocarrier is modified with a targeting ligand, which is a rabies virus glycoprotein peptide with the amino acid sequence YTIWMPENPRPGTPCDIFTNSRGKRASNG, and the modification density is 0.1-5 μg / mg carrier. A penetration-enhancing molecule, a short-chain fatty acid derivative, is introduced onto the surface of the multifunctional nanocarrier at a loading of 0.5-10 wt%. siRNA is loaded into a vector via electrostatic adsorption and physical encapsulation, with a mass ratio of siRNA to vector of 1:10 to 1:

50. The resulting complex has a particle size of 80-200 nm and a zeta potential of -10 to +10 mV.

2. The method for efficient nucleic acid delivery of a novel siRNA drug vector according to claim 1, characterized in that: The siRNA is chemically modified by 2'-O-methylation, with modification sites including nucleotides 1-3 and 18-21 of the antisense strand, and a cholesterol molecule is attached to the 5' end to enhance the carrier binding efficiency.

3. The method for efficient nucleic acid delivery of a novel siRNA drug vector according to claim 1, characterized in that: The targeted ligand modification method is as follows: RVG peptide is dissolved in PBS buffer at pH 7.4, the concentration is adjusted to 1 mg / mL, EDC and NHS are added and activated at room temperature for 15 minutes, and then added dropwise to PLGA nanocarrier suspension at a 1:1 volume ratio. The reaction is carried out at 25°C for 6 hours and purified by ultracentrifugation.

4. A novel method for efficient nucleic acid delivery of siRNA drugs according to claim 1, characterized in that: The method for introducing the penetration-enhancing molecule is as follows: Sodium decanoate is dissolved in anhydrous ethanol and added to the PLGA suspension modified with RVG at 3% of the carrier mass. The suspension is then incubated with shaking at 37°C for 2 hours, followed by dialysis to remove free molecules.

5. A novel method for efficient nucleic acid delivery of siRNA drugs according to claim 1, characterized in that: The characterization parameters of the complex include: The average particle size measured by dynamic light scattering was 120 ± 15 nm, and the polydispersity index was < 0.

2. Transmission electron microscopy reveals a spherical or near-spherical morphology with a smooth surface; Agarose gel electrophoresis confirmed that the siRNA encapsulation rate was ≥90%.

6. A novel method for efficient nucleic acid delivery of siRNA drugs according to claim 1, characterized in that: The delivery system was implemented in an in vitro experiment: Transfection efficiency of targeted neurons ≥70%; The penetration efficiency of tightly coupled carriers is 3-5 times higher than that of unmodified carriers; Serum stability >24 hours.

7. A novel method for efficient nucleic acid delivery of siRNA drugs according to claim 1, characterized in that: The siRNA is a sequence targeting the Huntington's disease-related HTT gene, with its sense strand being 5'-GUAUCUACAUCAAGACUUA-3' and its antisense strand being 5'-UAAGUCUUGAUGUAGAUAC-3'.