A small interfering RNA delivery system based on amphiphilic dendrimer and preparation method and use thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a fluorine-functionalized modification amphiphilic dendrimer delivery system loaded with small interfering RNA, a preparation method and application thereof. The delivery system comprises a delivery component and a therapeutic component. The delivery component is a fluorine-functionalized modification amphiphilic dendrimer, and the therapeutic component is siRNA loaded thereon and having a target gene inhibition effect. The siRNA delivery system is a positively charged nanoparticle formed by self-assembly of the fluorine-functionalized modification amphiphilic dendrimer in an aqueous solution and chemically synthesized small interfering RNA. The drug delivery system can effectively load and protect siRNA, form a stable complex, improve the stability of the complex under serum conditions in a physiological environment, safely and efficiently deliver siRNA to a target site, and has significant gene silencing activity and good biocompatibility, and has a good application prospect in the field of biological medicine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a small interfering RNA delivery system based on amphiphilic dendritic molecules, its preparation method and uses. Background Technology

[0002] Small interfering RNA (siRNA) drugs can specifically "silence" disease-causing genes, exerting therapeutic effects at the gene level and overcoming the "undruggable" nature of traditional drug targets. siRNA drugs are characterized by high specificity, abundant drug targets, long-lasting efficacy, simple design and synthesis, and high success rate in clinical translation, showing great promise for the treatment and prevention of major diseases such as malignant tumors, immune system diseases, and rare diseases.

[0003] However, siRNA itself is unstable and easily degraded by nucleases; its strong hydrophilicity and abundant negative charge make it difficult to cross cell membranes. These characteristics necessitate safe and efficient carriers to deliver siRNA to its target site. Lipid carriers and polymer carriers are among the latest and most advanced siRNA delivery technologies, effectively protecting siRNA drugs from degradation, improving their bioavailability, promoting uptake by target cells, and enhancing their therapeutic effects. Amphiphilic dendritic molecules, developed in recent years, combine the delivery advantages of lipid and polymer carriers and have significant potential for siRNA delivery. These amphiphilic dendritic molecules consist of hydrophilic dendritic structures and hydrophobic alkyl chains, and can self-assemble into dendritic molecular assemblies in aqueous solutions through non-covalent interactions (such as hydrophobic interactions, van der Waals forces, and hydrogen bonding). This allows for the specific, safe, and efficient delivery of siRNA to different cell types, difficult-to-transfect cells (such as immune cells and stem cells), and various animal models. However, the efficient in vivo delivery of siRNA by amphiphilic dendritic molecules also faces challenges related to blood circulation stability. The presence of large amounts of serum proteins and other substances in the body's blood readily adsorbs onto the surface of cationic siRNA delivery vectors, disrupting the stability of the complex and significantly reducing the siRNA delivery efficiency and in vivo gene silencing activity. In conclusion, rationally designing vectors to improve the stability of the constructed siRNA delivery system under physiological conditions, especially its stability against serum proteins, based on the in vivo delivery requirements, is a crucial issue that urgently needs to be addressed to improve delivery efficiency.

[0004] The siRNA delivery system based on fluorinated amphiphilic dendritic molecules of the present invention can stably encapsulate siRNA and has excellent anti-serum transfection ability. It solves to some extent the problem that cationic nucleic acid delivery systems are unstable under serum conditions, thus limiting the transfection efficiency. This is of great significance for the development of biological functional carrier materials with independent intellectual property rights. Summary of the Invention

[0005] The purpose of this invention is to provide an siRNA delivery system based on fluorinated amphiphilic dendritic molecules, which can effectively load and protect siRNA, form a stable complex, improve its stability under serum conditions in a physiological environment, and thus improve the delivery efficiency of siRNA.

[0006] Another objective of this invention is to provide a method for preparing an siRNA delivery system based on amphiphilic dendritic molecules.

[0007] Another object of the present invention is to provide the application of an amphiphilic dendritic siRNA delivery system in tumor therapy.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] A siRNA delivery system based on an amphiphilic dendritic molecule is disclosed. The system comprises a delivery component and a therapeutic component. The delivery component is a fluorinated amphiphilic dendritic molecule, and the therapeutic component is siRNA loaded onto the molecule with gene-targeting inhibitory effects. The fluorinated amphiphilic dendritic molecule self-assembles with the siRNA in an aqueous solution via electrostatic interactions to form positively charged nanoparticles. The fluorinated amphiphilic dendritic molecule is a compound with the structure shown in Formula I, or a pharmaceutically acceptable salt thereof.

[0010]

[0011] Fluorine-functionalized amphiphilic dendritic molecules of general formula I

[0012] in,

[0013] R1 is a biocompatible C 1-10 Fluorinated alkyl groups;

[0014] R2 is C, N, O, or S;

[0015] R3 is C 0-20 Alkylene;

[0016] R4 is an amino group or

[0017] The amphiphilic cationic dendritic lipid carrier selected in this invention has good biocompatibility. The assembly formed by this carrier can effectively load siRNA through electrostatic interactions, improve its stability under physiological conditions, and safely and efficiently deliver it to the target site. It promotes the release of siRNA through the "proton sponge effect" and exerts a gene silencing effect.

[0018] As a preferred embodiment of the present invention, R1 is -C(CF3)3;

[0019] R2 is 0;

[0020] R3 is C 15-20 Alkylene;

[0021] R4 is an amino group or

[0022] As a further preferred embodiment of the present invention, the amphiphilic dendritic molecule comprises a compound with the following structure, or a pharmaceutically acceptable salt thereof:

[0023]

[0024] As a further preferred embodiment of the present invention, the siRNA is selected from chemically modified or unmodified small nucleic acid interference molecules with symmetrical two strands, or chemically modified or unmodified small nucleic acid interference molecules with asymmetrical two-strand pairing.

[0025] As a further preferred embodiment of the present invention, the N / P ratio of the siRNA / amphiphilic dendritic molecule is 1:20 to 50:1, preferably 2:1 to 25:1; wherein the N / P ratio is the ratio of amino groups in the amphiphilic dendritic molecule to phosphate groups in the siRNA.

[0026] The method for preparing the siRNA delivery system of fluorinated amphiphilic dendritic molecules according to the present invention, wherein the fluorinated amphiphilic dendritic molecules can be formed into positively charged nanoparticles by self-assembly with siRNA through thin-film dispersion, solvent evaporation, or direct water dissolution, includes the following steps:

[0027] (1) Preparation of amphiphilic dendritic molecule solution: Under aseptic conditions, the amphiphilic dendritic molecule was dissolved in sterile water, sonicated, and allowed to stand to prepare a stock solution;

[0028] (2) Preparation of siRNA / fluorinated amphiphilic dendritic molecule delivery system: The complex solution was obtained using the following methods: ① Thin film dispersion method: A certain amount of fluorinated amphiphilic dendritic molecules were dissolved in an organic solvent and rotary evaporated to form a thin film. Then, an aqueous solution containing siRNA was added to form an aqueous solution of a nanocomplex with a certain N / P ratio (N / P is the ratio of amino groups in the dendritic molecule to phosphate groups in the nucleotide). After standing, the solution was diluted with sterile water to a certain concentration to prepare a complex solution of the required concentration. ② Solvent evaporation method: Under sterile conditions, the fluorinated amphiphilic dendritic molecules were dissolved in an appropriate amount of ethanol or other organic solvent. The solution was mixed with a certain amount of siRNA aqueous solution at a certain N / P ratio. The organic solvent was evaporated completely by sonication or stirring to obtain the complex solution. ③ Direct water dissolution method: Under sterile conditions, a certain amount of amphiphilic dendritic molecule stock solution was rapidly mixed with an aqueous solution of siRNA drug to prepare a solution with a certain N / P ratio. After mixing, the complex solution was diluted with sterile aqueous solution to a certain concentration to obtain the complex solution.

[0029] (3) Filling: Under aseptic conditions, fill and seal the single-dose complex solution or prepare it as a lyophilized powder.

[0030] The N / P ratio of the fluorinated amphiphilic dendritic molecule to siRNA is 1:20 to 50:1, and more preferably 2:1 to 25:1. N / P is the ratio of the amino group in the amphiphilic dendritic molecule to the phosphate group in the siRNA nucleotide.

[0031] The siRNA is selected from artificially synthesized, chemically modified or unmodified, double-stranded symmetrical small interfering RNA, or chemically modified or unmodified small interfering RNA with asymmetrical pairing of two strands.

[0032] The nanoparticles have a particle size distribution of 5–100 nm and a surface zeta potential distribution of 10–60 mV.

[0033] The application of the amphiphilic dendritic siRNA delivery system described in this invention in the preparation of gene therapy drugs; preferably in the preparation of anti-tumor gene therapy drugs for cancer.

[0034] Beneficial Effects: Based on the characteristics of siRNA and its in vivo delivery requirements, this invention utilizes the abundant primary amino groups on the surface of amphiphilic dendritic molecules to effectively load and protect siRNA, forming a uniform nanoscale complex. Secondly, by utilizing fluorine-functionalized amphiphilic dendritic molecules, the "fluorine-loving" effect of the fluorine functional groups is retained, enhancing the stability and anti-serum transfection performance of the carrier and the constructed siRNA delivery system under physiological conditions. The siRNA delivery system described in this invention exhibits good stability, cellular uptake, and gene silencing activity under different serum concentrations (0%-50%, especially 0-10%). Furthermore, the hydrophobic alkyl chains with different degrees of fluorine modification endow the siRNA delivery system with better safety and cellular uptake. In addition, these amphiphilic dendritic molecules can effectively enhance siRNA uptake in target cells, promoting rapid drug release from endosomes and improving the efficiency of the siRNA delivery system. This delivery system has demonstrated excellent gene silencing activity and anti-tumor therapeutic efficacy in various types of tumor cell lines and animal models. This delivery system effectively enhances the stability of siRNA delivery in vivo, exhibits good biocompatibility and excellent antiserum transfection performance, and provides a certain reference value for the development of siRNA vectors and the construction of delivery systems. Attached Figure Description

[0035] Figure 1 Screening for gene silencing activity of fluorine-functionalized amphiphilic dendritic molecules in Example 2.1

[0036] Figure 2 The preferred fluorinated amphiphilic dendritic molecule CF3-C in Example 2.2 15 -8A gene silencing activity screening, where Figure A represents siRNA / CF3-C 15 -8A complex gene silencing activity at different siRNA concentrations. Figure B represents siRNA / CF3-C 15 Gene silencing activity of the -8A complex at different N / P ratios

[0037] Figure 3 CF3-C in Example 2.3 15 -8A and C 18 8A gene silencing activity screening at different serum concentrations

[0038] Figure 4 CF3-C in Example 3.1 15 -8A binding capacity of siRNA under serum-free and serum-free conditions

[0039] Figure 5 The siRNA / CF3-C in Example 3.2 15 -8A complex particle size and potential under serum-free conditions

[0040] Figure 6 The siRNA / CF3-C in Example 4 15 Stability assessment of the -8A complex under serum conditions

[0041] Figure 7 siRNA / CF3-C in Example 5 15 Enzymatic protection of siRNA by the -8A complex under serum conditions

[0042] Figure 8 The siRNA / CF3-C in Example 6 15 Cellular uptake of the -8A complex under serum-free and serum-free conditions, where Figure A represents siRNA / CF3-C 15 The cellular uptake rate of the -8A complex, Figure B represents siRNA / CF3-C 15 The average cellular uptake fluorescence intensity of the -8A complex.

[0043] Figure 9 siRNA / CF3-C in Example 7 15 -8A complex siRNA release capacity under serum-free conditions

[0044] Figure 10 The siRNA / CF3-C in Example 8 15 Specific gene silencing activity of the -8A complex under serum-free conditions

[0045] Figure 11 siRNA / CF3-C in Example 9 15 Antitumor proliferation activity of the -8A complex under serum conditions

[0046] Figure 12 The siRNA / CF3-C in Example 10.1 15 -8A complex cytotoxicity

[0047] Figure 13 The siRNA / CF3-C in Example 10.2 15 Hemolytic toxicity of the -8A complex Detailed Implementation

[0048] The present invention will be further described below with reference to the accompanying drawings and embodiments. These embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.

[0049] Example 1: Preparation of a fluorinated amphiphilic dendritic molecular delivery system loaded with siRNA

[0050] 1.1 Preparation of siRNA / fluorinated amphiphilic dendritic molecular complex

[0051] Under aseptic conditions, a certain amount of aqueous solution of amphiphilic dendritic molecule and aqueous solution of siRNA drug are thoroughly mixed at N / P = 0.05-50 (N / P is the ratio of amino groups in dendritic molecule to phosphate groups in nucleotide), so that the final concentration of siRNA is 1-50 nM. The mixture is then incubated at room temperature for 30 min to obtain siRNA / fluorinated amphiphilic dendritic molecule complex.

[0052] Example 2: Screening for gene silencing activity using a fluorinated, siRNA-loaded amphiphilic dendritic molecular delivery system

[0053] 2.1 Screening for gene silencing activity in fluorine-functionalized amphiphilic dendritic molecules

[0054] Cells in the logarithmic growth phase were digested with 0.25% trypsin, counted, and plated. The culture medium in the wells was discarded, and the siRNA / fluorinated amphiphilic dendritic molecule complex was prepared according to the procedure described in Example 1.1. This complex was co-incubated with the cells for a certain period, then the transfection solution was aspirated, and the cells were cultured again for 72 hours with fresh medium. After 72 hours, the cells were collected, and total intracellular protein was extracted using an appropriate amount of cell lysis buffer. The siRNA transfection efficiency of the delivery system was assessed by Western blotting. The results are as follows: Figure 1 As shown, this indicates that CF3-C 15 -4A and CF3-C 15 -8A exhibits significant gene silencing activity. Gene silencing activity = 100% - (target protein expression level in the treatment group / internal reference protein expression level in the treatment group) / (target protein expression level in the control group / internal reference protein expression level in the control group) × 100%, the same below.

[0055] Table 1. Gene silencing activity of fluorine-functionalized amphiphilic dendritic molecules.

[0056]

[0057] 2.2 Screening for optimal gene silencing activity of fluorine-functionalized amphiphilic dendritic molecules

[0058] Cell transfection experiments were performed according to the procedures described in Example 2.1. Cells were then collected, and the transfection efficiency of siRNA with the preferred fluorinated amphiphilic dendritic molecule was assessed by Western blotting. Results are as follows: Figure 2As shown, under the condition of N / P = 10 and a concentration of 50 nM AKT2 siRNA, fluorinated amphiphilic dendritic molecules have significant gene silencing activity. Decreasing the siRNA concentration weakens the gene silencing activity of fluorinated amphiphilic dendritic molecules to almost nothing; decreasing the N / P ratio, fluorinated amphiphilic dendritic molecules still have some gene silencing activity at N / P = 5, but further decreasing the N / P ratio weakens the gene silencing activity to almost nothing.

[0059] Table 2. Screening for optimal fluorine-functionalized amphiphilic dendritic gene silencing activity.

[0060]

[0061] 2.3 Screening for gene silencing activity of fluorine-functionalized amphiphilic dendritic molecules under different serum conditions

[0062] Cells in the logarithmic growth phase were digested with 0.25% trypsin, counted, and plated. The culture medium in the wells was discarded. An siRNA / fluorinated amphiphilic dendritic complex was prepared under optimal transfection conditions (N / P = 10, 50 nM siRNA, hereinafter the same) in 0%–10% FBS (0%, 2%, 5%, 10% FBS). This complex was co-incubated with the cells for a certain period, then the transfection solution was aspirated, and the cells were cultured again for 72 hours with fresh culture medium. After 72 hours, cells were collected, and total intracellular protein was extracted using an appropriate amount of cell lysis buffer. The siRNA transfection efficiency of the delivery system was assessed by Western blotting.

[0063] The results are as follows Figure 3 As shown, siRNA / CF3-C 15 -8A complex and siRNA / C 18 The -8A complex exhibits comparable gene silencing activity under serum-free conditions. However, the siRNA / CF3-C complex... 15 The -8A complex still exhibits good gene silencing activity under conditions containing 0%–10% serum, while siRNA / C 18 Gene silencing activity of the -8A complex decreased with increasing FBS concentration, and was almost non-existent under 10% FBS conditions. This indicates that the siRNA drug formulation constructed based on fluorine-functionalized amphiphilic dendritic molecules exhibits excellent antiserum transfection performance.

[0064] Example 3: Characterization of a fluorinated, amphiphilic, dendritic molecular delivery system loaded with siRNA

[0065] 3.1 siRNA binding ability of fluorinated amphiphilic dendritic molecules

[0066] A siRNA / fluorinated amphiphilic dendritic molecular complex with an N / P ratio of 10 was prepared with or without FBS, with each sample containing 200 ng of siRNA. Using naked siRNA as a control, the samples were mixed with 6×RNA loading buffer, loaded into the wells of a 2% agarose gel, and electrophoresed at 100 V for 20 minutes. The gels were then detected and photographed under UV light using a gel imaging system.

[0067] The results are as follows Figure 4 As shown, in both the FBS-free and FBS-containing conditions, no siRNA bands were observed in the siRNA / fluorinated amphiphilic dendritic molecule complex groups, indicating that these fluorinated amphiphilic dendritic molecules still have good siRNA binding ability under serum conditions and will not cause siRNA leakage.

[0068] 3.2 Particle size and potential of siRNA / fluorinated amphiphilic dendritic complex

[0069] The hydrodynamic particle size distribution and zeta potential of the siRNA / fluorinated amphiphilic dendritic molecular complex were determined using a multi-angle particle size and zeta potential analyzer under serum-free conditions and under different serum concentrations.

[0070] like Figure 5 As shown, the hydrodynamic particle size distribution of the siRNA / fluorinated amphiphilic dendritic molecule complex is 102.00 ± 4.08 nm, and the Zeta potential is +23.24 ± 1.24 mV. This indicates that the fluorinated amphiphilic dendritic molecule can bind to siRNA to form a complex. After the addition of serum, the particle size of the siRNA / fluorinated amphiphilic dendritic molecule complex slightly increased, and the potential changed from +23 mV to -18 mV. This indicates that the siRNA / fluorinated amphiphilic dendritic molecule complex is stable under serum conditions, and serum proteins may adsorb onto the surface of the siRNA / fluorinated amphiphilic dendritic molecule complex through electrostatic interactions.

[0071] Table 3. Particle size and potential of siRNA / fluorinated amphiphilic dendritic complexes

[0072]

[0073] Example 4: siRNA loading stability of a fluorinated functionalized amphiphilic dendritic molecular delivery system loaded with siRNA

[0074] Two siRNA / fluorinated amphiphilic dendritic molecular complexes with an N / P ratio of 10 were prepared, with or without FBS. Each sample contained 200 ng of siRNA. In one sample, SDS was used to competitively bind to the cationic carrier, displacing the siRNA. Naked siRNA served as a control. The integrity of the siRNA loaded by the fluorinated amphiphilic dendritic molecular delivery system was assessed by comparison. After incubating the four samples at 37°C for 8 hours, they were mixed with 6×RNA loading buffer and loaded into the wells of a 2% agarose gel. Electrophoresis was performed at 100 V for 20 minutes, and the gels were then detected and photographed under UV light using a gel imaging system.

[0075] Figure 6 In the diagram, "+" indicates that the reagent was added, and "-" indicates that the reagent was not added. The results are as follows: Figure 6 As shown, under serum conditions containing FBS, the drug formulation can stably encapsulate siRNA for up to 8 hours; and after the addition of SDS, intact siRNA gel electrophoresis bands can be observed in both the serum-free and FBS-co-incubated groups. This indicates that the siRNA / fluorinated amphiphilic dendritic molecular complex can effectively load siRNA for a long time and maintain its integrity, exhibiting good antiserum encapsulation stability.

[0076] Example 5: Enzyme protection capability of fluorine-functionalized amphiphilic dendritic molecular delivery system loaded with siRNA

[0077] Under 10% FBS conditions, the siRNA / fluorinated amphiphilic dendritic molecular complex was divided into 6 equal portions. RNase A was then added to the solution at different time points (0, 30, 60, 90, 110, and 120 min), and the mixture was incubated at 37°C. Sodium dodecyl sulfate (SDS) solution was added to all groups, with naked siRNA serving as the control. The enzyme protection ability of the delivery system was observed using a fully automated chemiluminescence / fluorescence image analysis system.

[0078] like Figure 7 As shown, this delivery system can effectively protect siRNA from nuclease degradation for up to 2 hours under 10% FBS conditions. This indicates that the fluorinated amphiphilic dendritic molecule delivery system has good protective ability against siRNA and maintains good siRNA enzyme tolerance under serum conditions.

[0079] Example 6: Cellular uptake by a fluorine-functionalized amphiphilic dendritic molecular delivery system loaded with siRNA

[0080] Cells in the logarithmic growth phase were digested with 0.25% trypsin, counted, and plated. After 24 hours of cell adhesion, the culture medium in the wells was discarded. Cy5 siRNA / fluorinated amphiphilic dendritic molecule complexes were prepared under optimal transfection conditions of 0% and 10% FBS and co-incubated with the cells for 5, 10, 30, 60, 120, 180, and 240 min, respectively. The cells were then digested and washed three times with PBS at 4°C. Cy5 fluorescence signals were detected by flow cytometry.

[0081] The cell uptake rate and uptake volume of this delivery system are as follows: Figure 8 As shown, the cellular uptake of this delivery system increased over time. This indicates that the functionalized amphiphilic dendritic molecule delivery system can be effectively taken up by cells. After serum addition, the average fluorescence uptake intensity of Cy5 siRNA in cells significantly increased, and it was successfully taken up by nearly 90% of tumor cells in a very short time, with its average fluorescence intensity rapidly reaching its peak.

[0082] Example 7: siRNA release capability of a fluorinated functionalized amphiphilic dendritic molecular delivery system loaded with siRNA

[0083] A certain amount of ethidium bromide (EB)-labeled siRNA was used to prepare EB / siRNA / fluorinated amphiphilic dendritic molecular complexes under optimal transfection conditions of 0% and 10% FBS. Subsequently, heparin solutions of different concentrations (0, 1, 2, 3, 4, 15, 20, 30 U / mL) were added to the complex solutions, gently mixed, and incubated at room temperature in the dark for 30 min. The fluorescence intensity of ethidium bromide was then measured using a multi-mode microplate reader.

[0084] The siRNA release capability of this delivery system is as follows: Figure 9 As shown in the figure, the siRNA in the complex can be effectively released under the displacement of negatively charged sodium heparin. At the same sodium heparin concentration, the serum-containing siRNA / fluorinated amphiphilic dendritic molecule complex can release the loaded siRNA more quickly and effectively. This indicates that the fluorinated amphiphilic dendritic molecule siRNA drug formulation not only has excellent antiserum stability, but also has better siRNA release capability.

[0085] Example 8: Specific gene silencing activity of a fluorinated, amphiphilic, dendritic molecular delivery system loaded with siRNA under serum-free conditions.

[0086] The experimental procedure for fluorine-functionalized amphiphilic dendritic molecules is the same as in Example 2.3.

[0087] The results are as follows Figure 10As shown, compared with the control group, the single nucleic acid drug group, the single vector group, and the disordered nucleic acid vector complex group, tumor cells SKOV-3 showed improved performance in siRNA / CF3-C 15 Treatment with the -8A complex reduced the expression of the target protein. Simultaneously, the siRNA / fluorinated amphiphilic dendritic molecule complex also exhibited this specific gene silencing effect under serum-containing conditions. This result indicates that the fluorinated amphiphilic dendritic molecule CF3-C... 15 -8A can deliver siRNA into cells to exert RNAi effects and inhibit the expression of target proteins, demonstrating that the nucleic acid drug formulation constructed by fluorinated amphiphilic dendritic molecules has excellent antiserum-specific transfection performance.

[0088] Example 9: Antitumor proliferation activity of a fluorinated, amphiphilic dendritic molecular delivery system loaded with siRNA

[0089] Cells in the logarithmic growth phase were digested with 0.25% trypsin, counted, and plated. The culture medium in the wells was aspirated, and the siRNA / fluorinated amphiphilic dendritic complex was prepared under optimal transfection conditions (0% or 10% FBS). This complex was co-incubated with the cells for a certain period, then the transfection solution was aspirated, and the cells were cultured again with fresh medium for 7 days. After 7 days, MTT was added, and the cells were incubated at 37°C for 4 hours. The culture medium was then aspirated, and an equal volume of DMSO was added to each well. The plates were shaken until the formazan was completely dissolved, and the absorbance at 540 nm was measured using a multi-mode microplate reader to assess the antitumor proliferative activity of the delivery system.

[0090] The results are as follows Figure 11 As shown, this delivery system can significantly inhibit the growth of tumor cells. This indicates that the cell viability of the siRNA drug formulation group constructed from fluorinated amphiphilic dendritic molecules is significantly reduced. Furthermore, the antitumor proliferative activity of the siRNA / fluorinated amphiphilic dendritic molecule complex is comparable under serum-containing conditions to that under serum-free conditions. This demonstrates that siRNA drug formulations based on fluorinated amphiphilic dendritic molecules still possess excellent antitumor cell proliferation activity in the presence of serum.

[0091] Example 10: Safety assessment of a fluorinated, amphiphilic, dendritic molecular delivery system loaded with siRNA

[0092] 10.1 Cytotoxicity of Fluorine-Functionalized Amphiphilic Dendritic Molecule Delivery Systems Loaded with siRNA

[0093] Cells in the logarithmic growth phase were digested with 0.25% trypsin, counted, and plated. The culture medium in the wells was aspirated, and the siRNA / fluorinated amphiphilic dendritic complex was prepared under optimal transfection conditions. This complex was co-incubated with the cells for a certain period, then the transfection solution was aspirated, and the cells were cultured again with fresh medium for 48 hours. After 48 hours, MTT was added, and the cells were incubated at 37°C for 4 hours. The culture medium was then aspirated, and an equal volume of DMSO was added to each well. The plates were shaken until the formazan was completely dissolved, and the absorbance at 540 nm was measured using a multi-mode microplate reader to assess the cytotoxicity of the delivery system.

[0094] The results are as follows Figure 12 As shown, this delivery system exhibits no significant cytotoxicity in normal cell lines, with cell viability generally exceeding 90%. This indicates that the fluorinated amphiphilic dendritic molecule delivery system possesses excellent safety.

[0095] 10.2 Hemolytic toxicity of fluorine-functionalized amphiphilic dendritic molecular delivery systems loaded with siRNA

[0096] Fresh mouse blood was collected and reconstituted into a 4% erythrocyte suspension with PBS. An siRNA / fluorinated amphiphilic dendritic complex was prepared under optimal N / P ratio conditions. An equal volume of 4% erythrocyte suspension was added to the complex solution to achieve a final erythrocyte concentration of 2%. The mixture was incubated at 37°C for 2 hours. Negative control (PBS) and positive control (1% Triton X-100) were also provided. After 2 hours, the solution was centrifuged at room temperature. 150 μL of the supernatant from each group was added to a 96-well plate. The absorbance at 540 nm was measured using a multi-mode microplate reader, and the percentage of hemolysis was calculated using the following formula:

[0097] Hemolysis (%) = (A 样品 -A 阴性对照 ) / (A 阳性对照 -A 阴性对照 )×100%.

[0098] The results are as follows Figure 13 As shown, the delivery system has no obvious hemolytic toxicity, indicating that it has good biocompatibility and can be used for intravenous administration.

[0099] The dendritic molecular structures and characterization data involved in the above embodiments are as follows:

[0100]

[0101] 1H NMR(500MHz,CDCl3):δ8.03(t,J=5.7Hz,2H),7.60(t,J=5.7Hz,3H),7.50(s,1H),4.30(t,J=7.3Hz,2H),3.97(t,J=6.5Hz,2H),3.77(s,2H),3.34-3.15(m,12H),2.85-2.63(m,20H),2.49(t,J=5.9Hz,4H),2.45-2.25(m,12H),1.91-1.84(m,2H),1.66-1.59(m,2H),1.37-1.22(m,16H);

[0102] 13 C NMR(126MHz,CDCl3):δ173.0,172.5,143.5,122.9,120.6(q,J=293.6Hz),70.0,53.2,50.8,50.5,49.3,47.7,42.3,41.7,37.9,34.5,33.8,30.5,29.8,29.6,29.6,29.5,29.2,29.1,26.7,25.9;

[0103] 19 F NMR(471MHz,CDCl3):δ-70.41;

[0104] HRMS:calcd.For C 49 H 91 F9N 16 O7 2+ [M+2H] 2+ 593.3551,found 593.3550.

[0105]

[0106] 1 H NMR(500 MHz,CDCl3):δ8.03(t,J=5.7 Hz,2H),7.65(t,J=5.7 Hz,3H),7.50(s,1H),4.30(t,J=7.3 Hz,2H),3.97(t,J=6.5 Hz,2H),3.76(s,2H),3.35-3.13(m,12H),2.90-2.57(m,20H),2.49(t,J=6.0 Hz,4H),2.44-2.27(m,12H),1.92-1.82(m,2H),1.68-1.61(m,2H),1.36-1.22(m,22H);

[0107] 13 C NMR(126 MHz,CDCl3):δ173.1,172.6,122.9,120.6(q,J=293.6 Hz),70.0,53.1,50.8,50.5,49.3,47.7,42.2,41.6,37.9,34.5,33.8,30.4,29.8,29.7,29.7,29.7,29.6,29.5,29.3,29.1,26.7,25.4;

[0108] 19 F NMR(471 MHz,CDCl3):δ-70.42;

[0109] HRMS:calcd.for C 52 H 98 F9N 16 O7 3+ [M+3H] 3+ 409.9215,found 409.9213.

[0110]

[0111] 1 H NMR(400 MHz,CDCl3):δ7.92(s,1H),4.39(t,2H,J=7.0 Hz),4.07(t,2H,J=6.0 Hz),3.82(s,2H),3.29-3.27(m,28H),2.83-2.78(m,44H),2.60-2.57(m,12H),2.44-2.36(m,28H),1.94-1.89(m,2H),1.72-1.66(m,2H),1.42-1.30(m,16H);

[0112] 13 C NMR(101 MHz,CDCl3 / CD3OD=3 / 1):δ173.4,172.9,172.7,142.6,123.2,120.1(J=291.4 Hz),69.6,52.0,51.8,50.2,49.7,49.6,46.7,41.5,41.4,41.1,40.6,40.5,40.3,37.1,33.5,33.3,33.0,29.9,29.1,28.7,26.2,24.9;

[0113] 19 F NMR(376 MHz,CD3OD):δ-71.6;

[0114] HRMS:calcd.for C 89 H 172 F9N 32 O 15 3+ [M+3H] 3+ 700.1173, found 700.1171.

[0115]

[0116] 1 H NMR (500MHz, CD3OD): δ7.92 (s, 1H), 4.39 (t, 2H, J = 7.0Hz), 4.07 (t, 2H, J = 6.0Hz), 3.82 (s, 2H), 3.28-3.27 (m, 28H), 2. 83-2.78(m,44H),2.60-2.57(m,12H),2.44-2.36(m,28H),1.94-1.89(m,2H),1.72-1.66(m,2H),1.41-1.29(m,22H);

[0117] 13 C NMR (100MHz, CDCl3): δ172.9,172.5,172.3,143.1,122.9,120.3(J=291.8Hz),69.7,52.7,52.3,50.3 ,50.1,49.0,47.2,41.8,41.1,40.9,37.5,34.1,33.8,33.5,30.2,29.5,29.4,29.3,28.9,26.5,25.1;

[0118] 19 F NMR (376MHz, CDCl3): δ-70.3;

[0119] HRMS:calcd.for C 92 H 178 F9N 32 O 15 3+ [M+3H] 3+ 714.1330, found 714.1329.

[0120] The above embodiments are preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are included within the scope of protection of the present invention.

Claims

1. A fluorine-functionalized amphiphilic dendritic molecule delivery system for loading siRNA, characterized in that, The delivery system comprises a delivery component and a therapeutic component. The delivery component is a fluorinated amphiphilic dendritic molecule, and the therapeutic component is siRNA loaded onto it with gene-targeting inhibitory effects. The fluorinated amphiphilic dendritic molecule self-assembles with the siRNA in aqueous solution through electrostatic interactions to form positively charged nanoparticles. The fluorinated amphiphilic dendritic molecule is a compound with the structure shown in Formula I, or a pharmaceutically acceptable salt thereof. ， General Formula I Fluorine-functionalized amphiphilic dendritic molecules in, R1 is -C(CF3)3; R2 is 0; R3 is C 15-20 Alkylene; R4 is an amino group or .

2. The amphiphilic dendritic molecular delivery system according to claim 1, characterized in that, The fluorine-functionalized amphiphilic dendritic molecule is selected from any one of the following: ， CF3-C 15 -4A, ， CF3-C 15 -8A。 3. The amphiphilic dendritic molecule delivery system according to claim 1, characterized in that, The siRNA is selected from chemically modified or unmodified small nucleic acid interference molecules with symmetrical two strands, or chemically modified or unmodified small nucleic acid interference molecules with asymmetrical two strand pairings.

4. The amphiphilic dendritic molecule delivery system according to claim 1, characterized in that, The N / P ratio for forming the siRNA / amphiphilic dendritic molecule is 1:20 to 50:1, where N / P is the ratio of amino groups in the amphiphilic dendritic molecule to phosphate groups in the siRNA.

5. The amphiphilic dendritic molecule delivery system according to claim 1, characterized in that, The N / P ratio for forming the siRNA / amphiphilic dendritic molecule is 2:1 to 25:

1.

6. A method for preparing the amphiphilic dendritic molecular delivery system according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Preparation of amphiphilic dendritic molecule solution: Under aseptic conditions, the amphiphilic dendritic molecule was dissolved in sterile water, sonicated, and allowed to stand to prepare a stock solution; (2) Preparation of siRNA / amphiphilic dendritic molecule delivery system: Under sterile conditions, the stock solution of amphiphilic dendritic molecules and the aqueous solution of siRNA are quickly mixed to prepare a N / P solution of 1:20~50:

1. After mixing, the solution is allowed to stand to obtain the amphiphilic dendritic molecule delivery system.

7. The preparation method according to claim 6, characterized in that, The dissolution temperature of the fluorinated amphiphilic dendritic molecule described in step (1) is 10–30 °C, and the standing time is 5–30 min; the dissolution temperature of the siRNA described in step (2) is 10–30 °C, and the standing time is 5–30 min; the N / P ratio of the solution forming the siRNA / amphiphilic dendritic molecule complex is 2:1 to 25:1, and the standing time is 10–40 min.

8. The method according to claim 6, characterized in that, The method further includes (3) filling: under aseptic conditions, filling a single dose of the complex solution into an ampoule and sealing it.

9. The use of the amphiphilic dendritic molecular delivery system according to any one of claims 1-5 in the preparation of antitumor gene therapy drugs.

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