Spleen-targeted membrane fusion type delivery carrier as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a spleen-targeted membrane fusion type delivery carrier as well as a preparation method and application thereof. The delivery carrier comprises a dye and lipid nanoparticles, wherein the lipid nanoparticles comprise an ionizable cationic lipid, an auxiliary lipid and a PEG lipid; the dye is selected from one or more of CyBI7, ICG, Nile Red, IR780, IR820, ICG COOH, Nile Blue, ROX alkynine, ROX amine and BODIPY (Boron Dipyrromethene). The delivery carrier has spleen targeting and membrane fusion characteristics, and the utilization rate and expression efficiency of nucleic acid drugs are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a spleen-targeting membrane fusion delivery carrier, its preparation method, and its application. Background Technology

[0002] From mRNA, siRNA, and antisense oligonucleotides to plasmid DNA, therapeutic nucleic acids have become a core tool for programmable drugs. Compared to traditional small protein molecules, nucleic acid drugs have inherent advantages such as unlimited target sites, rapid action, and easy large-scale synthesis, which are particularly well-suited to the rapid iteration needs of personalized tumor treatment, rare disease gene compensation, and infectious disease vaccines. However, nucleic acid molecules have large molecular weights, high negative charge density, are easily degraded by nucleases, and have difficulty crossing cell membranes to enter the cytoplasm to exert their functions. Therefore, they must rely on safe and efficient in vivo delivery systems to realize their clinical potential.

[0003] Lipid nanoparticles (LNPs) are currently the only approved systemic mRNA delivery platform, but they passively accumulate in the liver after intravenous injection, making effective transfection of immune organs such as the spleen difficult. Meanwhile, the classical endocytosis pathway causes most nucleic acids to remain in endosomes and lysosomes and be degraded, resulting in extremely low cytoplasmic release efficiency. Compared to endocytosis, membrane fusion pathways can promote lysosomal escape, exhibiting superior release efficiency and improving the utilization and efficacy of nucleic acid molecules. However, there are currently few reports on delivery systems targeting membrane fusion mechanisms, and research in this area is still in its early stages.

[0004] The spleen is the largest peripheral immune organ in the human body, rich in dendritic cells, macrophages, and T / B lymphocytes. Approximately 90% of circulating blood is filtered through the spleen, making it a natural "reactor" for inducing rapid anti-tumor or antiviral cellular immune responses. Precisely delivering mRNA encoding tumor neoantigens, cytokines, or immune checkpoint antibodies to the spleen allows for efficient expression within local antigen-presenting cells, directly activating and expanding cytotoxic T cells while avoiding first-pass metabolism in the liver and systemic toxicity. However, clinically approved liver nuclei (LNPs) lack an active spleen-targeting mechanism, and traditional passive particle size / charge regulation strategies struggle to overcome hepatic sinusoidal blockage. Therefore, developing a novel nucleic acid delivery system that simultaneously possesses a high spleen / liver distribution ratio, active membrane fusion into cells, and high cytoplasmic release is crucial for advancing mRNA-based tumor immunotherapy, infectious disease vaccines, and personalized gene therapy. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems existing in the prior art and provide a spleen-targeting membrane fusion delivery vector, its preparation method, and its application. This delivery vector combines spleen targeting and membrane fusion properties, significantly improving the utilization rate and expression efficiency of nucleic acid drugs.

[0006] To achieve the above objectives, the present invention provides a spleen-targeted membrane-fusion delivery carrier comprising a dye and lipid nanoparticles, wherein the lipid nanoparticles comprise ionizable cationic lipids, auxiliary lipids, and PEG lipids. The dye is selected from one or more of CyBI7, ICG, Nile Red, IR780, IR820, ICG COOH, Nile Blue, ROXalkyne, ROX amine, and BODIPY.

[0007] A second aspect of the present invention provides a method for preparing the above-mentioned delivery carrier, the method comprising: contacting an organic phase containing dye and lipid nanoparticles with an aqueous phase to obtain a delivery carrier.

[0008] A third aspect of the present invention provides a delivery system comprising the above-described delivery carrier and a nucleic acid drug.

[0009] A fourth aspect of the present invention provides a method for preparing the above-described delivery system, the method comprising: contacting an organic phase containing dyes and lipid nanoparticles with an aqueous phase containing nucleic acid drugs to obtain a delivery system, wherein the organic phase, the aqueous phase, and the contacting treatment are as defined in the second aspect above.

[0010] The fifth aspect of the present invention provides an application of the above-described delivery vector or delivery system in live gene delivery.

[0011] Traditionally, dyes have been used only as visual markers or passive tracers. However, the inventors of this invention have discovered that certain dyes, when combined with specific lipid nanoparticles, can endow delivery carriers with membrane fusion properties, thereby avoiding the drawbacks of traditional endocytosis pathways such as easy drug degradation, release, and low expression efficiency. The introduction of these specific dyes also further enhances the spleen-targeting performance of the delivery carrier, achieving highly efficient expression in the cytoplasm. Attached Figure Description

[0012] Figure 1 This is a schematic diagram illustrating the delivery system of the present invention for delivering nucleic acid drugs via a membrane fusion pathway; Figure 2 Particle size and polydispersity index of each delivery carrier obtained in Test Example 1; Figure 3 Potential diagrams of each delivery carrier obtained in Test Example 1; Figure 4 The UV absorption spectrum of LNP21 obtained in Test Example 1; Figure 5 The UV absorption spectrum of LNP22 obtained in Test Example 1; Figure 6The UV absorption spectrum of LNP28 obtained in Test Example 1; Figure 7 The graph shows the change in cellular uptake fluorescence intensity over time for each delivery vector obtained in Test Example 2. Figure 8 The graph shows the membrane fusion efficiency of each delivery carrier obtained in Test Example 3 as a function of time. Figure 9 The bar chart shows the uptake fluorescence intensity of LNP20 obtained in test example 4; Figure 10 The bar chart shows the uptake fluorescence intensity of LNP21 obtained in test example 4; Figure 11 The bar chart shows the uptake fluorescence intensity of LNP28 obtained in test example 4; Figure 12 Particle size and polydispersity index of each delivery system obtained in Test Example 5; Figure 13 Potential diagrams of each delivery system obtained in Test Example 5; Figure 14 The encapsulation efficiency characterization diagrams for each delivery system obtained from Test Example 6 are shown below. Figure 15 The graph shows the change in transfection efficiency of each delivery system after 2 days of storage, obtained from Test Example 7. Figure 16 The graph shows the change in transfection efficiency of each delivery system after 23 days of storage, as obtained in Test Example 7. Figure 17 Bar charts showing cell viability at different transfection doses for each delivery system obtained in Test Example 8; Figure 18 The transfection efficiency of each delivery system obtained in Test Example 9 when transfecting EGFP expression is shown in the graph. Figure 19 The graph shows the average fluorescence intensity of positive cells transfected with EGFP by each delivery system obtained in Test Example 9. Figure 20 Bioluminescent imaging of major organs of mice obtained in Test Example 10 after intravenous injection of various delivery systems; Figure 21 The graph shows the expression of LNP20@mFluc in major organs of mice obtained from Test Example 10 after intravenous injection. Figure 22 This figure shows the expression of LNP28@mFluc in major organs of mice obtained from Test Example 10 after intravenous injection. Detailed Implementation

[0013] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0014] In one aspect, the present invention provides a spleen-targeted membrane-fusion delivery carrier comprising a dye and lipid nanoparticles, wherein the lipid nanoparticles comprise ionizable cationic lipids, auxiliary lipids, and PEG lipids. The dye is selected from one or more of CyBI7, ICG, Nile Red, IR780, IR820, ICG COOH, Nile Blue, ROXalkyne, ROX amine, and BODIPY.

[0015] According to the present invention, by combining specific dyes and lipid nanoparticles, the resulting delivery carrier can be made to have both spleen targeting and membrane fusion properties by utilizing the mutual influence and synergistic effect between the two. In order to further improve the performance of the delivery carrier, the dye is preferably selected from one or more of CyBI7, ICG and Nile Red.

[0016] CyBI7 is a cyanine dye, which can be obtained by referring to the method in Nano Research, 2021, 14(7), 2432-2440. The CAS number of ICG is 3599-32-4. The CAS number of Nile Red is 7385-67-3. The CAS number of IR780 is 207399-07-3. The CAS number of IR820 is 172616-80-7. The CAS number of ICG COOH is 181934-09-8. The CAS number of Nile Blue is 2381-85-3. The CAS number of ROX alkyne is 2264016-88-6. The CAS number of ROX amine is 53170-19-7. The CAS number of BODIPY is 947328-71-4.

[0017] According to the present invention, in order to enable the delivery carrier to better achieve both spleen targeting and membrane fusion functions, it is also necessary to regulate the ratio of dye to lipid nanoparticles. Preferably, the molar ratio of the dye to the lipid nanoparticles is 1:10-50, more preferably 1:20-40, for example, it can be 1:20, 1:25, 1:33 and 1:40 and any range between these values.

[0018] According to the present invention, by adjusting the proportion of various lipids in the lipid nanoparticles, the dye can be better coordinated, thereby improving the spleen-targeting effect. Preferably, the molar ratio of the ionizable cationic lipid, the auxiliary lipid, and the PEG lipid is 1:0.1-1.2:0.005-0.08, more preferably 1:0.2-1:0.01-0.05, for example, it can be 1:0.97:0.03, 1:0.2:0.03, 1:0.65:0.02, and 1:0.85:0.05, or any value between these values.

[0019] According to the present invention, the delivery carrier has suitable particle size, polydispersity index, and electrical potential, thereby exhibiting good dispersibility and stability. Preferably, the particle size of the delivery carrier is 50-200 nm, more preferably 70-150 nm, for example, it can be 70 nm, 105 nm, 120 nm, 135 nm, and 150 nm, or any range between these values.

[0020] Preferably, the polydispersity coefficient of the delivery carrier is 0.05-0.4, more preferably 0.1-0.3, and can be, for example, values ​​such as 0.12, 0.18, 0.22, 0.26 and 0.3, or any value between them.

[0021] Preferably, the potential of the delivery carrier is 2-30mV, more preferably 5-20mV, for example, it can be 7mV, 8.6mV, 12mV, 15mV and 20mV and any range between these values.

[0022] According to the present invention, in order to better cooperate with the dye to achieve spleen targeting and membrane fusion, various lipids can be further selected. Preferably, the ionizable cationic lipids are selected from one or more of SM-102, DODMA, ALC-0315, Dlin-MC3-DMA, C12-200 and DlinDMA, preferably SM-102 and / or DODMA.

[0023] Preferably, the auxiliary lipid is selected from one or more of DOPE, DOTAP, DOPC, DOPG and DOPS, and more preferably DOPE and / or DOPC.

[0024] Preferably, the PEG lipid is selected from one or more of DMG-PEG2000, DSPE-PEG2000, ALC-0159 and TPGS, and is more preferably DMG-PEG2000 and / or DSPE-PEG2000.

[0025] The CAS numbers for the following are listed: SM-102 (2089251-47-6), DODMA (104162-47-21), ALC-0315 (2036272-55-4), Dlin-MC3-DMA (1224606-06-7), C12-200 (1220890-25-4), DlinDMA (871258-12-7), DOPE (4004-05-1), DOTAP (144189-73-1), DOPC (4235-95-4), DOPG (62700-69-0), and DOPS (6811-55-8). The CAS number for DMG-PEG2000 is 160743-62-4. The CAS number for DSPE-PEG2000 is 147867-65-0. The CAS number for ALC-0159 is 1849616-42-7. The CAS number for TPGS is 9002-96-4.

[0026] A second aspect of the present invention provides a method for preparing the above-mentioned delivery carrier, the method comprising: contacting an organic phase containing dye and lipid nanoparticles with an aqueous phase to obtain a delivery carrier.

[0027] According to the present invention, in order to obtain a delivery carrier with better properties and performance, preferably, the total concentration of the dye and the lipid nanoparticles in the organic phase is 1-15 mg / mL, more preferably 4-8 mg / mL, for example, it can be 4 mg / mL, 5 mg / mL, 6 mg / mL and 8 mg / mL and any range between these values.

[0028] Preferably, the solvent of the organic phase is selected from one or more of ethanol, methanol and dimethyl sulfoxide, and more preferably ethanol and / or methanol.

[0029] Preferably, the aqueous phase is selected from one or more acidic buffer solutions, preferably citric acid-citrate buffer solution and / or acetate-acetate buffer solution, for example, citric acid-sodium citrate buffer solution.

[0030] Preferably, the pH of the aqueous phase is 3-6, more preferably 4-5, for example, it can be 4, 4.5 and 5 or any range between these values.

[0031] Preferably, the volume ratio of the organic phase to the aqueous phase is 1:1-6, more preferably 1:2-4, and can be, for example, 1:2, 1:3, 1:3.5 and 1:4, or any range between these values.

[0032] According to the present invention, the contact treatment method can be selected from a wide range, such as using dual-channel microfluidic technology, microfluidic technology, vortexing and blowing. In order to obtain a delivery carrier with better properties, the contact treatment preferably includes: obtaining a nano-suspension by using dual-channel microfluidic technology to obtain the organic phase and the aqueous phase, and then performing dialysis treatment to obtain the delivery carrier.

[0033] Preferably, the conditions for the dual-channel microfluidic technology include: a total flow rate of 0.2-5 mL / min, an organic phase to aqueous phase flow rate ratio of 1:1-6, and a temperature of 10-40℃. More preferably, the conditions for the dual-channel microfluidic technology include: a total flow rate of 0.5-2 mL / min (e.g., values ​​such as 0.5 mL / min, 0.8 mL / min, 1.5 mL / min, and 2 mL / min, or any range thereof), an organic phase to aqueous phase flow rate ratio of 1:2-4 (e.g., values ​​such as 1:2, 1:3, 1:3.5, and 1:4, or any range thereof), and a temperature of 20-30℃ (e.g., values ​​such as 20℃, 25℃, 28℃, and 30℃, or any range thereof). Wherein, the total flow rate refers to the sum of the organic phase flow rate and the aqueous phase flow rate.

[0034] Preferably, the dialysis conditions include a temperature of -5°C to 15°C and a time of 2-10 hours. More preferably, the dialysis conditions include a temperature of 0-8°C (e.g., values ​​such as 0°C, 4°C, 6°C, and 8°C, or any range thereof) and a time of 4-8 hours (e.g., values ​​such as 4 hours, 6 hours, 7 hours, and 8 hours, or any range thereof). The dialysis treatment may, for example, use a dialysis bag with a molecular weight cutoff of 3500 Da, and the external aqueous phase may be, for example, double-distilled water, PBS buffer, or physiological saline, and the external aqueous phase must be sterile and enzyme-free.

[0035] A third aspect of the present invention provides a delivery system comprising the above-described delivery carrier and a nucleic acid drug.

[0036] According to the present invention, the nucleic acid drug can be selected from a wide range of sources. Preferably, the nucleic acid drug is selected from one or more of mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA, lncRNA and DNA, and more preferably from one or more of mRNA.

[0037] According to the present invention, in order to obtain better loading capacity and delivery efficiency, preferably, the amount of the nucleic acid drug and the delivery carrier is such that the nitrogen / phosphorus molar ratio in the delivery system is 1-10:1, preferably 2-6:1, for example, it can be 2:1, 4:1, 5:1 and 6:1 and any range between these values.

[0038] According to the present invention, the delivery system has suitable particle size, polydispersity index, and potential, thereby exhibiting good dispersibility and stability. Preferably, the particle size of the delivery system is 50-200 nm, more preferably 70-150 nm, and can be, for example, values ​​such as 80 nm, 105 nm, 128 nm, 139 nm, and 150 nm, or any range between these values.

[0039] Preferably, the polydispersity coefficient of the delivery system is 0.05-0.4, more preferably 0.08-0.3, and can be, for example, values ​​such as 0.098, 0.155, 0.194, 0.253 and 0.3, or any value between them.

[0040] Preferably, the potential of the delivery system is 1-30mV, more preferably 2-10mV, for example, it can be 2mV, 3mV, 3.56mV, 5.14mV and 10mV and any range between these values.

[0041] According to the present invention, a schematic diagram of the delivery system for delivering nucleic acid drugs (mRNA) via a membrane fusion pathway is shown below. Figure 1 As shown, the delivery system of the present invention can enter the cytoplasm and release nucleic acid drugs more quickly, while the traditional endocytosis route requires multiple steps and is extremely inefficient.

[0042] A fourth aspect of the present invention provides a method for preparing the above-described delivery system, the method comprising: contacting an organic phase containing dyes and lipid nanoparticles with an aqueous phase containing nucleic acid drugs to obtain a delivery system, wherein the organic phase, the aqueous phase, and the contacting treatment are as defined in the second aspect above.

[0043] According to the present invention, preferably, the concentration of the nucleic acid drug in the aqueous phase is 0.05-1 mg / mL, more preferably 0.1-0.3 mg / mL, for example, it can be 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL and 0.3 mg / mL and any range between these values.

[0044] The fifth aspect of the present invention provides an application of the above-described delivery vector or delivery system in live gene delivery.

[0045] Traditional dyes generally only have staining properties, serving as visual markers or passive tracers. However, the inventors of this invention discovered that certain dyes, when combined with specific lipid nanoparticles, can endow delivery carriers with membrane fusion properties, thereby avoiding the drawbacks of traditional endocytosis pathways such as easy drug degradation, release, and low expression efficiency. The introduction of these specific dyes also further enhances the spleen-targeting performance of the delivery carrier, enabling it to possess a high spleen / liver distribution ratio, active membrane fusion into cells, and high cytoplasmic release—a triple function that significantly improves the utilization and expression efficiency of nucleic acid drugs. The delivery carrier and delivery system of this invention are simple to prepare, exhibit excellent results, and show promising application prospects in gene therapy.

[0046] The present invention will be described in detail below through embodiments.

[0047] In the following examples, all the apparatus used are conventional in the field, all the operations performed are conventional in the field, and all the raw materials and reagents used are commercially available. CyBI7 was prepared using the method provided in Nano Research, 2021, 14(7), 2432-2440. SM-102 was purchased from Shanghai Shunna Biotechnology Co., Ltd. DOPC, DOPE, DMG-PEG2000, cholesterol, NBD-PE (lipid fluorescent probe), and Rho-PE (lipid fluorescent probe) were purchased from Avanti PolarLipids. ICG and DiR (CAS No. 100068-60-8) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Nile Red, MTT (thiazolyl blue tetrazolium bromide), and D-Luciferin (luciferase substrate) were purchased from Adamas-beta (Chongqing) Pharmaceutical Technology Co., Ltd. mFluc and mEGFP were purchased from APExBIO Technology LLC, USA, with product numbers R1013 and R1016, respectively. PBS dry powder was purchased from Jiangsu Zhonghui Hecai Group Co., Ltd. Triton X-100 was purchased from Beijing Bailingwei Technology Co., Ltd. MeβCD, CPZ, and DEPC (diethyl pyrocarbonate) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Genistein was purchased from MedChemExpress (MCE). 50×TAE electrophoresis buffer was purchased from Jizhi Biochemical Technology Co., Ltd. GeneGreen nucleic acid dye was purchased from Beijing Tiangen Biotech Co., Ltd. 2×Triton-TE buffer was purchased from Shanghai Maokang Biotechnology Co., Ltd.

[0048] Example 1 This embodiment illustrates the preparation process of LNP21, a spleen-targeting membrane fusion delivery vector.

[0049] (1) Add dye CyBI7, ionizable cationic lipid SM-102, auxiliary lipid DOPE and PEG lipid DMG-PEG2000 to ethanol to obtain an organic phase.

[0050] The total concentration of CyBI7, SM-102, DOPE and DMG-PEG2000 in the organic phase was 6.2 mg / mL; the molar ratio of CyBI7 to the total molar ratio of SM-102, DOPE and DMG-PEG2000 was 1:33.3; and the molar ratio of SM-102, DOPE and DMG-PEG2000 was 1:0.97:0.03.

[0051] (2) Use a citric acid-sodium citrate buffer solution with a pH of 4 as the aqueous phase.

[0052] (3) A nano-suspension was prepared by using a dual-channel microfluidic technology (total flow rate of 0.8 mL / min, flow rate ratio of organic phase to aqueous phase of 1:3, temperature of 25℃) to prepare the organic phase and aqueous phase. The nano-suspension was added to a dialysis bag (specification of molecular weight cutoff of 3500 Da), with double-distilled water as the external aqueous phase, and dialyzed at 4℃ for 6 h to obtain the spleen-targeted membrane fusion delivery carrier LNP21.

[0053] Example 2-3 This embodiment illustrates the preparation process of spleen-targeting membrane fusion delivery vectors LNP22 and LNP28.

[0054] Example 2: The method of Example 1 is followed, except that in step (1), CyBI7 is replaced with an equimolar amount of ICG. The spleen-targeting membrane fusion delivery vector LNP22 is finally obtained.

[0055] Example 3: The method of Example 1 is followed, except that in step (1), CyBI7 is replaced with an equimolar amount of Nile Red. The spleen-targeting membrane fusion delivery vector LNP28 is finally obtained.

[0056] Examples 4-6 This embodiment illustrates the preparation process of spleen-targeted membrane fusion delivery systems LNP21@mFluc, LNP28@mFluc, and LNP28@mEGFP.

[0057] Example 4: The method of Example 1 is followed, except that step (2) is as follows: mFluc is added to a citrate-sodium citrate buffer solution with a pH of 4 to obtain an aqueous phase, wherein the concentration of mFluc in the aqueous phase is 0.109 mg / mL. Finally, a spleen-targeted membrane fusion delivery system LNP21@mFluc with a nitrogen / phosphorus molar ratio of 4:1 is obtained.

[0058] Example 5: The method of Example 3 is followed, except that step (2) is as follows: mFluc is added to a citrate-sodium citrate buffer solution with a pH of 4 to obtain an aqueous phase, wherein the concentration of mFluc in the aqueous phase is 0.109 mg / mL. Finally, a spleen-targeted membrane fusion delivery system LNP28@mFluc with a nitrogen / phosphorus molar ratio of 4:1 is obtained.

[0059] Example 6: The method of Example 3 is followed, except that step (2) is as follows: mEGFP is added to a citrate-sodium citrate buffer solution at pH 4 to obtain an aqueous phase, wherein the concentration of mEGFP in the aqueous phase is 0.109 mg / mL. Finally, a spleen-targeted membrane fusion delivery system LNP28@mEGFP with a nitrogen / phosphorus molar ratio of 4:1 is obtained.

[0060] Comparative Example 1 The method is the same as in Example 1, except that the dye CyBI7 was not added in step (1). The delivery carrier LNP20 was finally obtained.

[0061] Comparative Example 2 The method is based on Example 6, except that the dye CyBI7 was not added in step (1). The final delivery system LNP20@mFluc is obtained.

[0062] Comparative Example 3 The method of Example 6 is different in that, in step (1), the dye CyBI7 is not added; and in step (2), mFluc is replaced with an equal mass of mEGFP. The final delivery system LNP20@mEGFP is obtained.

[0063] Test Example 1 LNP20, LNP21, LNP22, and LNP28 were selected, and their particle size, polydispersity index, and potential were measured using a dynamic light scattering particle size analyzer (detection medium: 5wt% glucose solution). The results are shown in Table 1 and [Table data missing]. Figure 2-3 As shown, LNP21, LNP22, and LNP28, which employ the technical solution of this invention, exhibit uniform size and good dispersion.

[0064] LNP21, LNP22, and LNP28 were selected, and their UV absorption spectra in water and methanol were measured using a UV-Vis spectrophotometer. Aqueous solutions (lipid concentration 0.2 mM) and methanol solutions (lipid concentration 0.2 mM) of each LNP were sequentially added to the sample cell. The scanning program was started, and the absorption curves were recorded. The results are shown below. Figure 4-6As shown, the maximum absorption peaks of LNP21 and LNP22 in water are red-shifted by 20 nm compared to the absorption peaks of the corresponding free dyes (in methanol), while LNP28 is blue-shifted by 29 nm, indicating that CyBI7, ICG, and Nile Red are in an aggregated state in LNPs.

[0065] Table 1

[0066] Test Example 2 LNP20, LNP21, LNP22, and LNP28 were selected, and their uptake kinetics on DC2.4 cells were studied using the following method: DC2.4 cells were injected at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of 1 cell / well in 24-well plates and cultured until the cell density reached approximately 70%. Seven time points were set: 0.5h, 1h, 2h, 4h, 8h, 12h, and 24h. The culture medium was aspirated, and the cells were washed twice with PBS buffer. Then, medium containing 12.33 μL of LNP solution (lipid concentration 24 μM, with LNP20 pre-labeled with DiR) was added to each well, and the cells were cultured for another 24 hours. After reaching the predetermined time points (0.5h, 1h, 2h, 4h, 8h, 12h, and 24h), the cells were quickly washed three times with pre-cooled PBS buffer (4°C) to remove untaken LNPs. The cells were then resuspended by pipetting with PBS buffer, and the resulting cell suspension was transferred to flow cytometry tubes. The fluorescence intensity of intracellular LNPs was detected using flow cytometry. Three replicates were set up for each time point to ensure data reliability. Finally, by analyzing the changes in cell fluorescence intensity at different time points, uptake curves were plotted. The results are shown below. Figure 7 As shown in the figure, LNP20 is taken up by cells at a slower rate, while LNP21, LNP22, and LNP28 are all taken up by cells rapidly, with LNP21 and LNP28 showing more significant effects.

[0067] Test Example 3 LNP20, LNP21, and LNP28 were selected, and their membrane fusion efficiency was determined using the following method: (1) Preparation of cell membrane-mimicking liposomes FRET-L (composed of DOPC, cholesterol, NBD-PE and Rho-PE in a molar ratio of 10:1:0.1:0.05). LNP20 or LNP21 solution (2 mM, 90 μL) was mixed with FRET-L solution (2 mM, 10 μL) and diluted with PBS buffer (10 mM, pH=7.4, 500 μL). Using a fluorescence spectrometer (HITACHI, F-7000), the emission spectra were continuously acquired from 500 to 700 nm (slit width 10 nm / 10 nm, voltage 670 V) at excitation of 475 nm. The FRET signals of NBD-PE and Rho-PE were monitored at 0.5, 5, 10, 15, and 20 min. The acceptor / donor fluorescence intensity ratio (592 nm / 516 nm) was denoted as R. t After 20 minutes, 66.66 μL of 1 wt% Triton X-100 was added to completely dissolve the liposomes, and the ratio at this point was recorded as R. 100 The ratio of FRET-L (2 mM, 10 μL) in 590 μL PBS buffer was used as R0. Membrane fusion efficiency was calculated using the following formula: =(R t R0) / (R 100 R0)×100%.

[0068] (2) Prepare cell membrane-mimicking liposome NBD-L (composed of DOPC, cholesterol, and NBD-PE in a molar ratio of 10:1:0.033) and liposome IL100 (composed of DOPC, cholesterol, NBD-PE, and Nile Red in a molar ratio of 10:1:0.01:2.95) to simulate 100% fusion efficiency of NBD-L and LNP28. Mix LNP28 solution (2 mM, 90 μL) with NBD-L (2 mM, 10 μL) and dilute with PBS buffer (10 mM, pH=7.4, 500 μL). The emission spectra of NBD-PE and Nile Red were continuously acquired from 500 to 700 nm using a fluorescence spectrometer (model HITACHI, F-7000) with excitation at 420 nm (slit width 10 nm / 10 nm, voltage 670 V). The FRET signals of NBD-PE and Nile Red were monitored at 0.5, 5, 10, 15, and 20 min. The acceptor / donor fluorescence intensity ratio (625 nm / 535 nm) was denoted as R. t The ratio of NBD-L (2 mM, 10 μL) in 590 μL PBS buffer was used as R0. The ratio of IL100 (2 mM, 100 μL) in 500 μL PBS buffer was used as R0. 100 The membrane fusion efficiency is calculated using the following formula: =(R t R0) / (R 100 R0)×100%.

[0069] The membrane fusion efficiency variation curves of LNP20, LNP21, and LNP28 were obtained using the above method, as follows: Figure 8 As shown, after mixing for 10 minutes, the membrane fusion efficiency of LNP21 and LNP28 can reach over 80%, while that of LNP20 is only around 30%.

[0070] Test Example 4 LNP20, LNP21, and LNP28 were selected, and their uptake mechanism on DC2.4 cells was studied using the following method: DC2.4 cells were inoculated at a concentration of 5 × 10⁻⁶ cells / cells. 4 Cells were seeded at a density of 1 mL / well in 24-well plates and cultured until the cell density reached approximately 80%. Cells were then divided into an endocytosis inhibitor group, a cryogenic group, and a control group. The endocytosis inhibitor group was incubated for 1.5 h at 37°C and 5% CO2 with fresh culture medium containing CPZ (chlorpromazine, 10 μg / mL), Genistein (genistein, 150 μM), and MeβCD (methyl-β-cyclodextrin, 5 mg / mL), respectively. LNP20, LNP21, and LNP28 were then added (to achieve a lipid concentration of 24 μM in the medium, with LNP20 pre-labeled with DiR) and incubated for another h. The cryogenic group was incubated for 1 h at 4°C after adding LNP20, LNP21, and LNP28 (to achieve a lipid concentration of 24 μM in the medium, with LNP20 pre-labeled with DiR). The control group was incubated with LNP20, LNP21, and LNP28 (addition amounts to achieve a lipid concentration of 24 μM in the culture medium, with LNP20 pre-labeled with DiR) at 37°C for 1.5 h. After washing with PBS buffer and replacing the culture medium with fresh medium, intracellular fluorescence intensity was measured by flow cytometry. The results are as follows: Figure 9-11 As shown, LNP20 is significantly affected by endocytosis inhibitors, while LNP21 and LNP28 are almost unaffected by endocytosis inhibitors and enter cells entirely via membrane fusion.

[0071] Test Example 5 LNP20@mFluc, LNP21@mFluc, LNP28@mFluc, LNP20@mEGFP, and LNP28@mEGFP were selected, and their particle size, polydispersity index, and potential were measured using a dynamic light scattering particle size analyzer (detection medium: 5wt% glucose solution). The results are shown in Table 2 and... Figure 12-13 As shown, the delivery systems are uniform in size and well-distributed.

[0072] Table 2

[0073] Test Example 6 LNP20@mFluc, LNP21@mFluc, LNP28@mFluc, LNP20@mEGFP, and LNP28@mEGFP were selected, and their encapsulation efficiency was determined using the following method: DEPC water was pre-cooled to 4°C for later use. 20 μL of 50×TAE electrophoresis buffer was diluted to 1 L with pre-cooled DEPC water to obtain 1×TAE electrophoresis buffer. 0.25 μg agarose was dissolved in 20 μL of 1×TAE electrophoresis buffer and heated to boiling in a microwave oven. After removing the microwave oven and allowing the temperature to drop to approximately 70°C, 5 μL of GeneGreen nucleic acid dye was added, and the mixture was shaken well before being added to the electrophoresis tank. For sample preparation, 8 μL of the aforementioned LNP@mRNA was added to each sample. After membrane perforation, 8 μL of 2×Triton-TE buffer was added to the buffer, and the samples were incubated at 37°C for 5 min. An appropriate amount of nucleic acid loading buffer was added to each sample. The electrophoresis apparatus voltage was set to 150V for 30 min, and finally, the samples were photographed under UV light. The encapsulation efficiency of different LNP@mRNAs was obtained by quantifying the bands on agarose gels using ImageJ image processing software, such as... Figure 14 As shown, the encapsulation efficiency of the above LNP@mRNAs is all above 80%, demonstrating excellent encapsulation efficiency.

[0074] Test Example 7 LNP20@mFluc, LNP21@mFluc, and LNP28@mFluc were selected, and their transfection stability in DC2.4 cells after long-term storage was determined using the following method: The LNP@mFluc samples were stored at 4°C in the dark, and samples were taken on days 1, 2, 3, and 23. DC2.4 cells were transfected at a rate of 1×102... 4 LNP@mFluc was seeded at a density of 10 cells / well in 96-well plates and cultured until the cell density reached approximately 80%. The culture medium was replaced with fresh medium, and LNP@mFluc was added to each well (to achieve a final mFluc concentration of 0.2 μg / well). After incubation at 37°C for 24 h, the supernatant was removed, and the cells were washed with PBS buffer. 100 μL of luciferase substrate (2 mg / mL) was added to each well. After 5 min, the luminescence value was measured using a small animal in vivo imaging system (IVIS Lumina II) to evaluate the transfection stability after long-term storage. The results are as follows: Figure 15-16 As shown in the figure, LNP21@mFluc and LNP28@mFluc exhibit good transfection stability. Among them, LNP21@mFluc is slightly worse than LNP20@mFluc, while LNP28@mFluc is better than LNP20@mFluc.

[0075] Test Example 8 LNP20@mFluc and LNP28@mFluc were selected, and their effects on the in vitro survival rate of DC2.4 cells were determined using the following method: DC2.4 cells were inoculated at 1×10⁻⁶ cells per cell line. 4 LNP@mFluc was seeded at a density of 10 cells / well in 96-well plates and cultured until the cell density reached approximately 80%. The medium was replaced with fresh medium, and LNP@mFluc was added to each well (to achieve final mFluc concentrations of 0, 0.05, 0.1, and 0.2 μg / well). After incubation at 37°C for 24 h, the supernatant was removed, and the cells were washed with PBS buffer. 10 μL of MTT solution (5 mg / mL) was added, and the cells were incubated at 37°C for 4 h. After incubation, the medium was removed, 150 μL of dimethyl sulfoxide was added, and the cells were shaken to dissolve. The absorbance was measured at 490 nm using a microplate reader, and cell viability was calculated (compared to untreated control cells). The results are shown below. Figure 17 As shown in the figure, LNP20@mFluc and LNP28@mFluc showed no significant cytotoxicity at mRNA final concentrations of 0-0.2 μg / well, and the survival rate of all cells was higher than 85%.

[0076] Test Example 9 LNP20@mEGFP and LNP28@mEGFP were selected, and their transfection efficiency in DC2.4 cells was determined using the following method: DC2.4 cells were transfected at a rate of 2 × 10⁶ cells / year. 5 Cells were seeded at a density of 10 cells / well in 24-well plates and cultured until the cell density reached approximately 80%. The medium was then replaced with fresh medium, and LNP@mEGFP was added to each well (to achieve a final mEGFP concentration of 0.2 μg / well). The plates were incubated at 37°C for 24 h. After incubation, cells were directly detached from the plates, collected, and centrifuged at 800 rpm for 5 min. The supernatant was removed, and the cells were resuspended in 200 μL of PBS. Transfection efficiency and the mean fluorescence intensity of positive cells were measured using flow cytometry. The results are shown below. Figure 18-19 As shown, the transfection efficiency of LNP28@mEGFP in DC2.4 cells and the average fluorescence intensity of positive cells are significantly better than those of LNP20@mEGFP.

[0077] Test Case 10 LNP20@mFluc and LNP28@mFluc were selected, and their transfection efficiency of luciferase mRNA in mice was determined by the following method: Seven-week-old female Balb / c mice (weighing approximately 20g) were injected intravenously with the above-mentioned LNP@mFluc (mFluc dose: 0.25mg / kg). Six hours after injection, 200μL of D-Luciferin (10mg / mL) was injected intraperitoneally. Five minutes later, the mice were sacrificed, and the heart, liver, spleen, lung, and kidney were placed in a small animal in vivo imaging system (model IVIS Lumina II). Bioluminescence mode was selected, automatic exposure was performed, and the expression of luciferase in each organ was monitored. The results are as follows: Figure 20-22 As shown, LNP28@mFluc achieves a spleen targeting efficiency of over 90%, which is far superior to LNP20@mFluc.

[0078] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A spleen-targeted membrane-fusion type delivery vehicle, characterized by, The delivery carrier comprises a dye and a lipid nanoparticle, the lipid nanoparticle comprising an ionizable cationic lipid, a helper lipid and a PEG lipid; the dye is selected from one or more of CyBI7, ICG, Nile Red, IR780, IR820, ICG COOH, Nile Blue, ROX alkyne, ROX amine and BODIPY.

2. The delivery vehicle of claim 1, wherein, the dye is selected from one or more of CyBI7, ICG and Nile Red; and / or, the molar ratio of the dye to the lipid nanoparticle is 1:10-50, preferably 1:20-40; and / or, the molar ratio of the ionizable cationic lipid, the helper lipid and the PEG lipid is 1:0.1-1.2:0.005-0.08, preferably 1:0.2-1:0.01-0.05; and / or, the particle size of the delivery carrier is 50-200 nm, preferably 70-150 nm; and / or, the polydispersity index of the delivery carrier is 0.05-0.4, preferably 0.1-0.3; and / or, the zeta potential of the delivery carrier is 2-30 mV, preferably 5-20 mV.

3. The delivery vector of claim 1 or 2, wherein, the ionizable cationic lipid is selected from one or more of SM-102, DODMA, ALC-0315, Dlin-MC3-DMA, C12-200 and DlinDMA, preferably SM-102 and / or DODMA; and / or, the helper lipid is selected from one or more of DOPE, DOTAP, DOPC, DOPG and DOPS, preferably DOPE and / or DOPC; and / or, the PEG lipid is selected from one or more of DMG-PEG2000, DSPE-PEG2000, ALC-0159 and TPGS, preferably DMG-PEG2000 and / or DSPE-PEG2000.

4. A method for the preparation of the delivery vehicle according to any one of claims 1 to 3, characterized in that, The method comprises: contacting an organic phase comprising a dye and a lipid nanoparticle with an aqueous phase to obtain a delivery carrier.

5. The method of claim 4, wherein, the total concentration of the dye and the lipid nanoparticle in the organic phase is 1-15 mg / mL, preferably 4-8 mg / mL; and / or, the solvent of the organic phase is selected from one or more of ethanol, methanol and dimethyl sulfoxide, preferably ethanol and / or methanol; and / or, the aqueous phase is selected from one or more of acidic buffer solutions, preferably citric acid-citrate buffer solution and / or acetic acid-acetate buffer solution; and / or, the pH of the aqueous phase is 3-6, preferably 4-5; and / or, the volume ratio of the organic phase to the aqueous phase is 1:1-6, preferably 1:2-4.

6. A delivery system comprising the delivery carrier of any one of claims 1-3 and a nucleic acid drug.

7. The delivery system of claim 6, wherein, the nucleic acid drug is selected from one or more of mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA, lncRNA and DNA, preferably one or more of mRNA; the nucleic acid drug is selected from one or more of mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA, lncRNA and DNA, preferably one or more of mRNA; and / or the amount of the nucleic acid drug and the delivery carrier is such that the nitrogen / phosphorus molar ratio in the delivery system is 1-10:1, preferably 2-6:1; and / or the particle size of the delivery system is 50-200 nm, preferably 70-150 nm; and / or the polydispersity index of the delivery system is 0.05-0.4, preferably 0.08-0.3; and / or the zeta potential of the delivery system is 1-30 mV, preferably 2-10 mV.

8. A method of making the delivery system of claim 6 or 7, characterized in that, The method comprises: contacting an organic phase comprising a dye and a lipid nanoparticle with an aqueous phase comprising a nucleic acid drug to obtain a delivery system, wherein the organic phase, the aqueous phase and the contacting are as defined in claim 4 or 5.

9. The method of claim 8, wherein, The concentration of the nucleic acid drug in the aqueous phase is 0.05-1 mg / mL, preferably 0.1-0.3 mg / mL.

10. Use of the delivery carrier of any one of claims 1-3 or the delivery system of any one of claims 6-7 in in vivo gene delivery.

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