Double-selenium lipid nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a double-selenium lipid nanoparticle as well as a preparation method and application thereof. According to the nanoparticle, a skeleton is composed of cationic lipid, neutral phospholipid, cholesterol and functionalized PEG lipid, meanwhile, mRNA and a TLR agonist for coding therapeutic protein are carried, and the nanoparticle is synthesized through an ethanol injection method; the nanoparticle has both active targeting and strong transfection capabilities, the lipid nanoparticle is actively targeted to a tumor microenvironment and is taken by tumor cells by using biotin modified PEG lipid, and rapid'inclusion-lysosome 'escape is realized in the tumor cells by using trifluorogroup modified PEG lipid. Meanwhile, the nanoparticles are degraded in a glutathione / hydrogen peroxide dual-mode, so that the entrapped medicine is quickly and effectively released; the drug delivery system provided by the invention is simple in synthesis method, high in gene transfection efficiency and good in biological safety, and has potential value in anti-tumor research and clinical application.

Description

Technical Field

[0001] The invention relates to diselenide lipid nanoparticles, and also relates to a preparation method and application of the nanoparticles. Background Art

[0002] Malignant tumors, as one of the leading causes of death worldwide, have become a major disease that seriously endangers human life and health. In the continuous exploration and evolution of anti-tumor treatment, gene therapy, as an emerging and promising treatment method, is gradually showing its unique advantages over traditional anti-tumor methods. The core of gene therapy is to directly intervene in the genetic basis of tumor occurrence and development, using ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) as a template to achieve the continuous production of therapeutic target proteins, blocking the growth and spread of tumors at the source, which not only improves the accuracy of treatment, but also can achieve individualized treatment at the molecular level, significantly improving the effectiveness and safety of treatment, and opening up new avenues for cancer treatment. Among them, messenger RNA (mRNA), as a single-stranded RNA, carries protein coding information and has several advantages over DNA: first, mRNA does not need to enter the cell nucleus, but directly initiates protein translation in the cytoplasm. This process is more efficient than DNA first entering the cell nucleus and then being transcribed into mRNA; second, mRNA will not be integrated into the host genome, avoiding the risk of genome contamination; finally, mRNA can be synthesized through in vitro transcription (IVT) technology, which is a low-cost and fast process. However, as a large, negatively charged and unstable molecule, mRNA is easily degraded by nucleases in the body. Traditional nanoparticles are difficult to effectively load and protect mRNA. Therefore, the development of high-performance carrier systems to accurately and stably deliver mRNA to the target site has become an urgent need for current research.

[0003] Vectors for delivering mRNA are divided into two categories: viral vectors (mainly including lentivirus, adenovirus, retrovirus, adeno-associated virus, etc.) and non-viral vectors (mainly including liposome complexes, cationic polymers, chitosan polymers, etc.). In clinical gene therapy, viral vectors have potential safety issues and limited capacity. These shortcomings have promoted the development of non-viral vector systems. Non-viral vectors have the advantages of low cost, simple preparation, easy large-scale production, high safety, and no restrictions on the length of exogenous genes.

[0004] Among them, lipid nanoparticles (LNPs) are ideal nucleic acid delivery vectors for clinical applications because of their good safety, large-scale production capabilities, and versatility in effectively delivering mRNA. However, the escape efficiency of LNP-mRNA complexes in endosomes is low, and only about 1% to 4% of mRNA can be successfully transferred to the cytoplasm. This limitation significantly hinders the production of sufficient protein. In addition, LNPs usually require a high N / P ratio to ensure complete encapsulation of mRNA, thereby giving it high stability. However, there is an inherent contradiction between this high stability and the ideal release performance-high in vivo stability often means that the drugs carried by lipid nanoparticles are difficult to fully release in cells, thereby weakening the overall delivery efficiency. In view of this, in order to improve the effect of gene therapy in anti-tumor treatment, it is urgent to develop new lipid nanoparticles with excellent endosomal escape ability and spatiotemporally controllable degradation characteristics. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a diselenide lipid nanoparticle with both active targeting and strong transfection ability, and also to provide a preparation method of the above diselenide lipid nanoparticle and its application in the preparation of anti-tumor drugs.

[0006] Technical solution: The present invention discloses a diselenide lipid nanoparticle, which has a skeleton composed of cationic lipids, neutral phospholipids, cholesterol, and functionalized PEG lipids. The positively charged cationic lipids and the negatively charged nucleic acid molecules form a multilayer core structure inside the lipid layer through electrostatic action, and are loaded with spherical vesicles composed of mRNA encoding therapeutic proteins and TLR agonists.

[0007] The cationic lipid is N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), the neutral phospholipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), and the functionalized PEG lipid is DMG-PEG 2000 -Biotin(BPD) and DSPE-Se-Se-PEG 2000 -CF3(FPSeD), the mRNA encoding the therapeutic protein is IL-15mRNA, and the TLR agonist is resiquimod (R848).

[0008] The preparation method of the above-mentioned diselenide lipid nanoparticles comprises the following steps:

[0009] (1) Dissolve 4,4,4-trifluorobutyric acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS), and triethylamine in dichloromethane (DCM), stir under ice bath, and then add DSPE-Se-Se-PEG 2000-NH2 was added to the above system, stirred at room temperature, dichloromethane was removed by rotary evaporation, dialyzed, and freeze-dried to obtain DSPE-Se-Se-PEG 2000 -CF3(FPSeD);

[0010] (2) N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), cholesterol (CHOL), 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), DMG-PEG 2000 -Biotin(BFD), DSPE-Se-Se-PEG 2000 -CF3(FPSeD) and TLR agonist are dissolved in ethanol to form an ethanol phase, mRNA encoding therapeutic protein is dissolved in an aqueous phase, the ethanol phase is dripped into the aqueous phase with rapid stirring, dialyzed, and filtered to obtain diselenide lipid nanoparticles.

[0011] Wherein, in step (1), 4,4,4-trifluorobutyric acid, EDCI, NHS, triethylamine, DSPE-PEG 2000 The molar ratio of -NH2 is 1-2:1-2:1-2:2-4:1.

[0012] Wherein, in step (1), the stirring speed is 450-550 rpm, preferably 500 rpm; the ice bath, the stirring time is 30-60 minutes; the room temperature stirring time is 12-24 hours; the rotary evaporation conditions are 90 rpm, 37°C, 10 minutes; the dialysis medium is ultrapure water, the time is 1-3 days, and the molecular weight of the dialysis bag is 500Da.

[0013] Wherein, in step (2), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), cholesterol (CHOL), 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), DMG-PEG 2000 -Biotin(BFD), DSPE-Se-Se-PEG 2000 The molar ratio of -CF3(FPSeD) is 50:36-40:10:1-3:1-3.

[0014] Wherein, in step (2), the mass ratio of resiquimod (R848) to total lipids is 1-2:10, and the mass ratio of IL-15mRNA to cationic lipids is 1:6-8; the total lipids are cationic lipids, neutral phospholipids, cholesterol and functionalized PEG lipids; and the volume ratio of the ethanol phase to the aqueous phase is 1:3.

[0015] Wherein, in step (2), the aqueous phase is a citrate buffer solution, preferably a citrate buffer solution with a pH of 4.0 and 10 mM.

[0016] Wherein, in step (2), the stirring speed is 900-1100 rpm, the stirring time is 25-35 minutes, preferably 30 minutes; the dialysis medium is PBS buffer, preferably PBS buffer with pH 7.4, the dialysis time is 2 hours, and the molecular weight of the dialysis bag is 3500Da.

[0017] The above-mentioned diselenide lipid nanoparticles can also be used in the preparation of anti-tumor drugs.

[0018] Invention principle: The diselenide lipid nanoparticles of the present invention have both active targeting and strong transfection ability, and their main components include: cationic lipids, neutral phospholipids, cholesterol, and functionalized PEG lipids; they are also loaded with mRNA encoding therapeutic proteins and TLR agonists, and are synthesized by ethanol injection. The nanoparticles have strong targeting and cell transfection ability; the functionalized PEG lipid is biotin-modified PEG lipid (DMG-PEG 2000 -Biotin, BPD) and trifluoro group-modified PEG lipids (DSPE-Se-Se-PEG 2000 -CF3, FPSeD), biotin and trifluoro groups are functionalized parts.

[0019] Specifically, on the one hand, biotin-modified PEG lipids are used to actively target lipid nanoparticles to the tumor microenvironment and be taken up by tumor cells. On the other hand, within tumor cells, trifluoro-modified PEG lipids are used to achieve rapid "endosome-lysosome" escape. At the same time, the nanoparticles are degraded in a dual mode of glutathione / hydrogen peroxide (2), achieving rapid and effective release of the encapsulated drug. The degradation diagram is shown in the figure below. Figure 3As shown in the figure, after the nanoparticles are taken up into tumor cells, the high concentration of glutathione and hydrogen peroxide in the cells causes the diselenide bond to break, which accelerates the rapid release of IL-15mRNA and R848 molecules. IL-15mRNA is rapidly translated in the tumor cytoplasm and continuously produces IL-15 protein, activating innate immunity and adaptive immunity. R848, as a small molecule, is passively discharged from tumor cells and returns to the tumor microenvironment, binding to TLR7 / 8 on the surface of M2-like TAMs, prompting M2 TAMs to repolarize to M1 phenotypes, eliminating cancer cells' resistance to IL-15 while reshaping the tumor microenvironment. These effects work sequentially and synergize with each other to effectively inhibit tumor growth. The drug delivery system provided by the present invention has a simple synthesis method, high gene transfection efficiency, good biosafety, and high stability under physiological conditions. In tumor tissue, the trifluoromethyl group on the surface of the material promotes endosomal-lysosomal escape. In addition, the nanomaterial degrades and rapidly releases the drug under dual-response stimulation of GSH and H2O2, thereby improving the gene transfection efficiency while protecting the activity of mRNA, and has potential value in anti-tumor research and clinical application.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The diselenide lipid nanoparticles of the present invention can degrade and quickly release drugs under the dual response stimulation of GSH and H2O2, the effective release rate of TLR agonists is higher than 80%, and the mRNA transfection efficiency is as high as 90%. The gene transfection efficiency is improved while protecting the mRNA activity, and the stability is high. It has good biosafety and can be completely degraded in the body and can be excreted through normal metabolic pathways; (2) The synthesis method has the characteristics of simple process, low cost, mild requirements, stable products, etc., and is suitable for industrial production; (3) As a carrier of drugs with poor stability, it has universality and promotion, has good conversion value, and can cooperate with multiple treatment methods according to different needs to achieve synergistic tumor treatment and achieve the best tumor treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The synthetic route of FPSeD;

[0022] Figure 2 Schematic diagram of the synthesis of the diselenide lipid nanoparticles RIL@BFSeP of the present invention;

[0023] Figure 3 It is a schematic diagram of the degradation of the diselenide lipid nanoparticles RIL@BFSeP of the present invention;

[0024] Figure 4 The transmission electron microscope image (a) and particle size distribution diagram (b) of L1 prepared in Example 1;

[0025] Figure 5The transmission electron microscope image (a) and particle size distribution diagram (b) of L2 prepared in Example 2;

[0026] Figure 6 The transmission electron microscope image (a) and particle size distribution diagram (b) of L3 prepared in Example 2;

[0027] Figure 7 The transmission electron microscope image (a) and particle size distribution diagram (b) of L4 prepared in Example 2;

[0028] Figure 8 is the gene transfection efficiency of GL0, GL1, GL2, GL3, and GL4 prepared in Example 3;

[0029] Fig. 9 : is a stability curve diagram of RIL@BFSeP in Example 3 of the present invention, wherein a is a stability image of RIL@BFSeP in PBS, and b is a stability image of RIL@BFSeP in RPMI 1640 medium;

[0030] Fig.10 Agarose gel electrophoresis images of RIL@BFSeP with different nitrogen-phosphorus ratios in Example 4;

[0031] Fig.11 This is the degradation image of RIL@BFSeP in Example 5;

[0032] Fig.12 is the release curve of TLR agonist R848 in Example 6, wherein a is the effect of GSH on the release rate of R848, and b is the effect of H2O2 on the release rate of R848;

[0033] Fig.13 The cytotoxicity bar graph of the toxicity test in Example 7;

[0034] Fig.14 is the absorbance curve of R848 outside the tumor cells after being treated with RIL@BFSeP in Example 8;

[0035] Fig.15 This is a graph showing the body weight of mice in different treatment groups in Example 9;

[0036] Fig.16 This is a curve chart of tumor volume of mice in different treatment groups in Example 9. DETAILED DESCRIPTION

[0037] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0038] Example 1

[0039] The preparation method of the diselenide lipid nanoparticle RIL@BFSeP with active targeting and high transfection performance of the present invention comprises the following steps. The synthesis schematic diagram is shown in FIG. Figure 2 As shown:

[0040] (1) 1.5 equivalents of 4,4,4-trifluorobutyric acid, 1.5 equivalents of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1.5 equivalents of N-hydroxysuccinimide (NHS), and 3 equivalents of triethylamine were dissolved in 10 ml of dichloromethane (DCM) in a 25 ml eggplant-shaped bottle and stirred for 30 minutes under ice bath; then 1 equivalent of DSPE-PEG 2000 -NH2 was added to the above system and stirred at room temperature for 12 hours. DCM was removed by rotary evaporator. Ultrapure water was added to transfer the product to a dialysis bag (500Da), dialyzed for 2 days, and freeze-dried for 2 days to obtain FPSeD. The synthetic route is as follows: Figure 1 As shown;

[0041] (2) 5 mg of DOTMA, 3.75 mg of CHOL, 1 mg of DOPE, 0.075 mg of BFD, 0.175 mg of FPSeD, and 2 mg of TLR agonist were dissolved in 1 ml of ethanol, and 300 μg of mRNA encoding the therapeutic protein was dissolved in 3 ml of pH 4.0 10 mM citrate buffer. The ethanol phase was dripped into the aqueous phase under rapid stirring at 1000 rpm and stirring was continued for 30 minutes; dialyzed with pH 7.4 10 mM PBS for 2 hours and filtered through a 0.22 μm filter membrane to obtain the diselenide lipid nanoparticles of the present invention, which were recorded as L1.

[0042] Example 2

[0043] According to the method of Example 1, the material ratio was changed to prepare diselenide lipid nanoparticles, which were marked as L2, L3 and L4;

[0044] Among them, compared with L1, the mass ratio of FSePD:BPD in L2 is 1:1;

[0045] Compared with L1, the mass ratio of FSePD:BPD in L3 is 1.5:1;

[0046] Compared with L1, the mass ratio of FSePD:BPD in L4 is 3:1.

[0047] like Figures 4 to 7As shown, the transmission electron micrographs and particle size distribution diagrams of L1 to L4 are all spherical in shape and the PDI is less than 0.2. The hydration kinetic diameter of L1 is about 127.84nm, the hydration kinetic diameter of L2 is about 121.99nm, the hydration kinetic diameter of L3 is about 118.82nm, and the hydration kinetic diameter of L4 is about 123.52nm. Among them, the particle size and PDI of L3 are the smallest, so L3 is selected for subsequent experiments.

[0048] Example 3

[0049] Investigation of gene transfection efficiency of RIL@BFSeP

[0050] (1) According to the method of Example 2, IL-15 mRNA was replaced by GFP mRNA to prepare diselenide lipid nanoparticles, which were marked as GL1, GL2, GL3 and GL4, and a control group GL0 without FSePD;

[0051] (2) One day in advance, 4T1 cells were plated on a 96-well plate and incubated in a cell culture incubator at 37°C overnight.

[0052] (3) GL0, GL1, GL2, GL3 and GL4 were diluted to the same concentration with blank RPMI 1640 medium and incubated with the cells for 6 h. Then, the drug-containing medium was discarded and fresh complete RPMI 1640 medium was added and incubated for another 18 h.

[0053] (4) Flow cytometry was used to count the percentage of GFP-positive cells.

[0054] The results are as follows Figure 8 As shown, the gene transfection efficiency of GL0~GL4, among which the transfection efficiency of GL3 is as high as 90%. Combined with the hydration dynamic diameter and PDI, the proportion of each component in GL3 was used in subsequent experiments.

[0055] Example 4

[0056] Stability investigation of RIL@BFSeP

[0057] (1) To investigate the stability of the particles, the RIL@BFSeP solution was diluted with PBS and RPMI 1640 medium and placed at 37°C;

[0058] (2) Take points on the 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th days;

[0059] (3) The changes in the hydration kinetic diameter and PDI of RIL@BFSeP were investigated using a Malvern laser particle size analyzer.

[0060] The results are as follows Fig. 9As shown, this is the stability curve of RIL@BFSeP. Within seven days, the hydration kinetic diameter and PDI of RIL@BFSeP fluctuated slightly within a certain range, indicating that the tight binding of cationic lipids and mRNA makes the nanoparticles have good stability under normal physiological conditions.

[0061] Example 5

[0062] Encapsulation of mRNA by RIL@BFSeP with different nitrogen-phosphorus ratios.

[0063] (1) RIL@BFSeP with N / P of 0, 2, 4, 6, and 8 were synthesized respectively.

[0064] (2) Weigh 0.4 mg of agarose powder into a conical flask containing 40 mL of 1× TAE solution and heat until the powder dissolves.

[0065] (3) After boiling for 5 minutes, add 4 μL of Gel-Red nucleic acid dye, shake well and cool to room temperature, then pour it into the mold and let it stand for 30 minutes until the agarose is completely solidified.

[0066] (4) Add 10 μL of 1× loading buffer to the channels at both ends of the gel, and add maker and 10 μL of RIL@BFSeP with different nitrogen-phosphorus ratios to the remaining channels. Start running the gel using a gel electrophoresis instrument and expose it with a fluorescent imager after completion.

[0067] (5) Gel electrophoresis conditions: voltage 180 V, time 20-25 minutes.

[0068] The results are as follows Fig.10 The figure shows the agarose gel electrophoresis images of RIL@BFSeP with different nitrogen-phosphorus ratios. When the nitrogen-phosphorus ratio is lower than 6, the mRNA band can be observed in the lane of gel electrophoresis, indicating that the mRNA cannot be completely encapsulated by the cationic liposomes. When N / P≥6, no mRNA band can be observed in the lane of gel electrophoresis, indicating that when N / P≥6, the cationic liposomes can stably load the mRNA.

[0069] Example 6

[0070] Degradation of RIL@BFSeP

[0071] (1) Take 1 mL of 1 mg / mL RIL@BFSeP and place it in a 3500 Da dialysis bag, and then place the dialysis bag in 19 mL of buffered saline solution.

[0072] (2) The conditions of the buffered saline solutions were pH 7.4 PBS, pH 7.4 PBS + 5 mM GSH, and pH 7.4 PBS + 100 μM H2O2.

[0073] (3) Pipette 1 mL of solution at 4, 24, and 72 h, respectively.

[0074] (4) The sample was dropped onto a copper grid and degradation photos were taken using a transmission electron microscope.

[0075] The results are as follows Fig.11 As shown, it is the degradation image of RIL@BFSeP. In the presence of GSH or H2O2, RIL@BFSeP at 4h, 24h, and 72h are degraded to varying degrees. Among them, RIL@BFSeP is almost completely degraded at 72h, indicating that due to the presence of diselenide bonds, RIL@BFSeP has good GSH / H2O2 dual response ability.

[0076] Example 7

[0077] Release profile of TLR agonist R848 in RIL@BFSeP

[0078] (1) 1 mL of the previously prepared RIL@BFSeP was placed in a 3500 Da dialysis bag, which was then placed in a centrifuge tube containing 19 mL of different buffer solutions.

[0079] (2) The conditions of the buffered saline solutions were pH 7.4 PBS, pH 7.4 PBS + 5 mM GSH, and pH 7.4 PBS + 100 μM H2O2.

[0080] (3) At a specific time point, collect 1 mL of sample solution and add 1 mL of fresh buffer.

[0081] (4) The concentration of released drug was measured using a UV-visible spectrophotometer.

[0082] The results are as follows Fig.12 The figure shows the release curve of TLR agonist R848. In the absence of GSH or H2O2, the cumulative release rate of R848 within 72 hours is less than 20%, indicating that the nanoparticles do not have drug leakage problems. In the presence of GSH or H2O2, the cumulative release rate of R848 within 72 hours is above 80%, indicating that the presence of diselenide bonds makes RIL@BFSeP have good responsive release ability.

[0083] Example 8

[0084] The toxicity of the nanoparticles to 4T1 tumor cells was verified by MTT assay.

[0085] The verification method includes the following steps:

[0086] S1. 4T1 cells were cultured in 96-well plates at a density of 5 × 103 cells per well and incubated at 37°C overnight;

[0087] S2. Add different concentrations of materials and incubate with cells for 24 h, add 20 μL of 5 mg·mL-1 MTT to each well and incubate for 4 h;

[0088] S3. Remove the medium containing MTT and add 150 μL DMSO. Detect the optical density (OD) at 490 nm using a microplate reader, and calculate the cell viability according to the following formula: cell viability (%) = OD (sample) / OD (control) × 100%.

[0089] The results are as follows Fig.13 The figure shows the cytotoxicity bar graph of toxicity detection, and the groups are PBS (Control), lipid nanoparticles co-loaded with R848 and IL-15mRNA (RIL@BFSeP), lipid nanoparticles loaded with IL-15mRNA only (IL@BFSeP), cationic liposomes loaded with R848 only (R@BFSeP), and blank cationic carrier (BFSeP). As can be seen from the figure, the cell survival rates of the Control, RIL@BFSeP, and IL@BFSeP treated groups are all above 90%, while the survival rates of cells treated with R@BFSeP and BFSeP are far below 70%, indicating that the cationic lipids and mRNA combined through electrostatic interaction greatly reduce the cytotoxicity of lipid nanoparticles, and RIL@BFSeP has good biocompatibility.

[0090] Example 9

[0091] The absorbance curve of R848 outside tumor cells after being treated with RIL@BFSeP includes the following steps:

[0092] S1. 4T1 cells were seeded in a 96-well plate one day in advance.

[0093] S2. RIL@BFSeP was diluted to a safe concentration using blank culture medium and co-incubated with 4T1 cells.

[0094] S3. Take out 1 ml of culture medium at the set time, centrifuge at 1800 rpm for 5 minutes, and keep 500 μl of supernatant.

[0095] S4. Measure the absorbance in the range of 300-350nm using an enzyme reader.

[0096] The results are as follows Fig.14As shown in the figure, the absorbance curve of R848 outside the tumor cells after RIL@BFSeP treatment. R848 does not directly interact with tumor cells, but is excreted to the extracellular space through passive transport and indirectly exerts anti-tumor effects by binding to antigen-presenting cells. The R848 content in the supernatant was the lowest at 4h, which was the peak of uptake. Soon after, the R848 content in the supernatant gradually increased to almost unchanged, indicating that RIL@BFSeP can achieve rapid "endosome-lysosome" escape through trifluoromethyl, and at the same time, the diselenide bond responds to GSH and H2O2 rupture, and the two work synergistically to achieve rapid and effective release of the encapsulated drug.

[0097] Example 10

[0098] Verifying the in vivo anti-tumor effect of the nanoparticles of the present invention comprises the following steps:

[0099] S1. 4T1 cells were inoculated into the right legs of BALB / c mice to establish a subcutaneous tumor model;

[0100] S2. The tumor-bearing mice for treatment were randomly divided into 6 groups (5 mice in each group) and intravenously injected with 100 μL of normal saline, BFSeP, R@BFSeP, IL@BFSeP, and RIL@BFSeP, once every three days for a total of 3 times;

[0101] S3. The tumor size and mouse weight were measured every other day, and the tumor volume (V) was calculated using the formula V=W2×L / 2 (W and L are the shortest and longest diameters of the tumor).

[0102] The results are as follows Fig.15 and 16 The figure shows the weight and tumor volume curves of mice in different treatment groups. There is no significant difference in the weight of mice treated with different preparations, indicating that all preparations are safe. The tumor volume of mice treated with Control and BFSeP preparations increased significantly, indicating that the blank liposomes themselves have no anti-tumor ability. The tumor volume of mice treated with R@BFSeP and IL@BFSeP was significantly smaller than that of the Control and BFSeP groups, but it also exceeded 500 cubic millimeters, indicating that IL-15mRNA and R848 alone have anti-tumor ability but poor efficacy and cannot completely inhibit tumor growth. On the contrary, the RIL@BFSeP group significantly inhibited tumor growth, which shows that IL-15 and R848 formed a combined treatment and improved the effect.

[0103] Therefore, the diselenide lipid nanoparticles of the present invention have both active targeting and strong transfection capabilities. The biotin-modified PEG lipids are used to enable the lipid nanoparticles to actively target the tumor microenvironment and be taken up by tumor cells. The trifluoro group-modified PEG lipids are used in tumor cells to achieve rapid "endosome-lysosome" escape. At the same time, due to the presence of diselenide bonds, the nanoparticles exhibit glutathione / hydrogen peroxide dual-mode degradation, thereby achieving rapid and effective release of the encapsulated drugs. The nanoparticles have high gene transfection efficiency and good biosafety, and have potential value in anti-tumor research and clinical applications.

Claims

1. A diselenide lipid nanoparticle, characterized in that: The nanoparticles are composed of a cationic lipid, a neutral phospholipid, cholesterol, and a functionalized PEG lipid as the skeleton; the positively charged cationic lipid and the negatively charged nucleic acid molecule form a multilayer core structure inside the lipid layer through electrostatic interaction, and are loaded with spherical vesicles composed of mRNA encoding therapeutic proteins and TLR agonists.

2. according to claim 1, diselenide lipid nanoparticles, characterised in that, The cationic lipid is N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, the neutral phospholipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine, and the functionalized PEG lipid is DMG-PEG 2000 -Biotin and DSPE-Se-Se-PEG 2000 -CF3, the mRNA encoding the therapeutic protein is IL-15mRNA, and the TLR agonist is resiquimod.

3. A method for preparing diselenide lipid nanoparticles according to claim 1, characterized in that: The following steps are involved: (1) Dissolve 4,4,4-trifluorobutyric acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS), and triethylamine in dichloromethane (DCM), stir under ice bath, and then add DSPE-Se-Se-PEG 2000 -NH2 was added to the above system, stirred at room temperature, dichloromethane was removed by rotary evaporation, dialyzed, and freeze-dried to obtain DSPE-Se-Se-PEG 2000 -CF3; (2) N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, cholesterol, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine, DMG-PEG 2000 -Biotin, DSPE-Se-Se-PEG 2000 -CF3 and TLR agonist are dissolved in ethanol to form an ethanol phase, and mRNA encoding therapeutic protein is dissolved in the water phase. The ethanol phase is dripped into the water phase with rapid stirring, dialyzed, and filtered to obtain diselenide lipid nanoparticles.

4. The preparation method according to claim 3, characterized in that: In step (1), 4,4,4-trifluorobutyric acid, EDCI, NHS, triethylamine, DSPE-PEG 2000 The molar ratio of -NH2 is 1-2:1-2:1-2:2-4:

1.

5. The preparation method according to claim 3, characterized in that: In step (1), the stirring speed is 450-550 rpm; the stirring time in the ice bath is 30-60 minutes; the stirring time at room temperature is 12-24 hours; the dialysis medium is ultrapure water, and the time is 1-3 days.

6. The preparation method according to claim 3, characterized in that: In step (2), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, cholesterol, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine, DMG-PEG 2000 -Biotin, DSPE-Se-Se-PEG 2000 -CF3 mass ratio is 50:36-40: 10：1-3：1-3。 7. The preparation method according to claim 3, characterized in that: In step (2), the mass ratio of TLR agonist to total lipid is 1-2:10, and the mass ratio of IL-15 mRNA to cationic lipid is 1:6-8.

8. The preparation method according to claim 3, characterized in that: In step (2), the aqueous phase is a citrate buffer solution.

9. The preparation method according to claim 3, characterized in that: In step (2), the stirring speed is 900-1100 rpm, and the stirring time is 25-35 minutes; and the dialysis medium is PBS buffer.

10. Use of the diselenide lipid nanoparticles according to claim 1 in the preparation of anti-tumor drugs.

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