A programmable cascade active targeted nucleic acid delivery system and uses thereof

Abstract

This invention discloses a programmable cascaded active targeted nucleic acid delivery system and its applications. This system overcomes the limitations of traditional "one carrier, one target" approaches by constructing a lipid nanoparticle platform comprising ionizable cationic lipids, sterols, phospholipids, and lipid complexes with at least two functional ligands. The functional ligands are selected from immune escape ligands, cell-targeting ligands, and membrane-penetrating ligands. By designing and adjusting the types and molar ratios of these functional modules, the in vivo target organs of the delivery system can be reprogrammed, achieving efficient and specific cascaded targeting of organs such as the spleen, liver, or lymph nodes. This system significantly improves the efficiency of nucleic acid drug delivery, providing a versatile and customizable solution for developing targeted nucleic acid drugs for various diseases.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and more specifically, to a programmable cascaded active targeted nucleic acid delivery system and its application. Background Technology

[0002] Nucleic acid drugs (such as mRNA, siRNA, DNA, etc.) have significant potential in disease treatment, but their large molecular weight, high hydrophilicity, and easy degradation make it difficult for them to autonomously cross the membrane and reach intracellular targets. Therefore, developing a safe and efficient targeted delivery system is a key bottleneck to their clinical translation.

[0003] Currently, the mainstream strategies for achieving organ-selective targeting mainly rely on the following technical pathways: Passive targeting strategies, which involve finely controlling the physicochemical properties of the delivery carrier, including particle size, surface charge, and lipid composition, in order to utilize the unique physiological structure of specific organs to achieve passive drug accumulation. However, this method heavily depends on the coupling between the physicochemical parameters of the carrier and the physiological structure of the body, resulting in limited targeting efficiency, poor specificity, and difficulty in achieving precise delivery to extrahepatic organs, leading to low controllability; Single active targeting strategies, which aim to modify the carrier surface with a ligand that can specifically bind to receptors on the surface of specific organs or cells, thereby improving targeting specificity through recognition mechanisms. While improving delivery specificity to some extent, the "one ligand, one target" single mode has inherent drawbacks. For example, a single ligand is difficult to overcome multiple physiological barriers in the in vivo delivery process and has poor versatility. Every time a new target is developed, a completely new carrier formulation or ligand needs to be designed and screened from scratch, resulting in a long R&D cycle and high costs. The biomimetic targeting strategy uses cell membranes to encapsulate the delivery carrier in order to utilize the biological functions of the source cell to achieve long-term circulation or homologous targeting. However, this approach faces industrialization challenges such as complex preparation processes, large batch-to-batch variations, and difficulties in quality control.

[0004] To improve delivery efficiency, extending the carrier's blood circulation time is also an important direction. Common methods include surface modification with polyethylene glycol (PEG) to enhance hydrophilicity and reduce protein adsorption, or modification with CD47-derived "self-peptides" (SPs) to inhibit phagocytosis by interacting with macrophage SIRPα, thereby prolonging in vivo circulation. Regarding promoting cellular uptake, cyclic transmembrane peptides such as W5R4K (WRK) can promote intracellular delivery through the interaction of their arginine / tryptophan residues with the cell membrane, and have been used in siRNA / DNA delivery, but there are few reports on their application in mRNA delivery.

[0005] In summary, existing delivery strategies have shortcomings in terms of targeting accuracy, versatility, and industrialization. There is an urgent need in this field to develop a modular, programmable, universal targeting platform capable of flexibly and efficiently achieving specific cascade delivery to different organs through the rational combination of different functional modules, thereby promoting the clinical translation of nucleic acid drugs. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a programmable cascaded active targeted nucleic acid delivery system and its applications, breaking away from the existing "one carrier, one target" model and offering a modular, programmable, and universal delivery platform. Its core is the construction of a stable lipid nanoparticle core framework composed of ionizable cationic lipids, sterols, and phospholipids. Based on this, a functional ligand-lipid complex combination is innovatively introduced. This complex combination contains functional modules targeting different physiological barriers during delivery. Simultaneously, by precisely optimizing the molar ratio of these modules in the carrier using experimental design software, the in vivo targeting fate of the entire delivery system can be reprogrammed without altering the core framework, achieving specific cascaded active targeting of different organs (such as the spleen, liver, and lymph nodes).

[0007] In a first aspect, the present invention provides a programmable cascaded active targeted nucleic acid delivery system, characterized in that the nucleic acid delivery system comprises at least two types of functional ligand lipid complexes; the functional ligand lipid complexes are formed by covalently linking functional ligands with cholesterol via linkers; wherein the functional ligands are selected from immune escape ligands, cell-targeting ligands, and membrane-penetrating ligands.

[0008] Preferably, the linker is a polyethylene glycol chain.

[0009] Preferably, the immune escape ligand is a self-peptide with the sequence shown in SEQ ID NO: 1.

[0010] Preferably, the cell-targeting ligand is a mannose molecule.

[0011] Preferably, the membrane-penetrating ligand is a cyclic membrane-penetrating peptide with the sequence shown in SEQ ID NO: 2.

[0012] Secondly, the present invention provides a method for preparing a programmable cascaded active targeted nucleic acid delivery system, characterized by comprising the following steps: S1: Dissolve ionizable cationic lipids, cholesterol, and the functional ligand in an organic solvent, then remove the organic solvent to form a lipid membrane; S2: Add an aqueous buffer solution to the lipid membrane, and after hydration reaction, a colloidal solution is obtained; S3: The colloidal solution is ultrasonically treated and then filtered to obtain the programmable cascaded active targeted nucleic acid delivery system; The molar percentages of each component in S1 are as follows: 35-50% ionizable cationic lipids, 29-40% cholesterol, 0.1-10% self-peptides, 0.1-10% mannose, and 0.1-10% cyclic transmembrane peptides.

[0013] Preferably, the molar percentages of each component in S1 are 50% ionizable cationic lipids, 35.96% cholesterol, 2.32% self-peptide, 2.82% mannose, and 2.34% cyclic transmembrane peptide, and the delivery system is used for spleen targeting.

[0014] Preferably, the molar percentages of each component in S1 are 50% ionizable cationic lipids, 35.96% cholesterol, 4.37% self-peptides, 6.29% mannose, and 3.38% cyclic transmembrane peptides, and the delivery system is used for liver targeting.

[0015] Preferably, when the molar percentages of each component in S1 are preferably 14.50% ionizable cationic lipids, 35.96% cholesterol, 5.00% peptides, and 5.00% mannose, the delivery system is used for lymph node targeting.

[0016] Thirdly, the present invention provides an application of a programmable cascaded active targeted nucleic acid delivery system in the preparation of nucleic acid targeted drugs.

[0017] In summary, the present invention has at least one of the following beneficial technical effects: 1. This invention is the first to propose and experimentally verify the core idea that "the in vivo targeting of a delivery system can be reprogrammed by changing and adjusting the ligand combination." The delivery system, as a general platform, does not have its targeting specificity determined by a fixed formulation, but rather by selecting functional modules and optimizing their molar ratios. Subsequent systematic adjustments allow for flexible and efficient switching of the target from the spleen to other organs such as the liver and lymph nodes, greatly expanding the application scope of the technology and avoiding the enormous workload and time cost of developing new targets from scratch.

[0018] 2. This invention achieves a synergistic amplification effect by synergistically combining functional ligands targeting different physiological barriers, overcoming multiple physiological barriers and realizing more efficient gene delivery and protein expression to target cells.

[0019] 3. Taking the spleen-targeted mRNA delivery system (Os-LLNs) constructed using the platform of this invention as an example, in vitro and in vivo experimental data fully demonstrate that this system can effectively evade phagocytosis and clearance by macrophages, specifically accumulate in the spleen, and promote the uptake of antigens by dendritic cells. In two different tumor-bearing animal models, this embodiment effectively activated the body's immune system, exhibiting a significant tumor-suppressive effect, and the experimental animals maintained stable body weight, demonstrating good safety. This proves the feasibility and enormous therapeutic application potential of the platform technology of this invention. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The diagram shows the ligand, cholesterol, and ionizable cationic lipid structures of the targeted delivery vector provided in this application embodiment; Figure 2 The particle size of the SMW-LLNs / mRNA formulation provided in the embodiments of this application is shown. Figure 2 A and potential Figure 2 B; Figure 3 This paper illustrates the effect of SP on reducing the uptake of SMW-LLNs / Cy5 by macrophages in vitro, as provided in an embodiment of this application; wherein... Figure 3 A is a confocal image of Raw264.7 cells incubated with PBS, Cy5, MW-LLNs / Cy5, and SMW-LLNs / Cy5 for 2 h; Figure 3 B is Cy5 + Flow cytometry analysis results of MFI in Raw264.7 cells Figure 4 The diagram illustrates the effect of WRK and Mannose on increasing the uptake of SMW-LLNs / Cy5 by DC2.4 cells, as provided in the embodiments of this application; wherein... Figure 4 A is a flow cytometry image captured by SMW-LLNs / Cy5; Figure 4 B is Cy5 + Flow cytometry analysis results of cell MFI; Figure 5 A schematic diagram of the CCD-optimized SMW-LLNs / mRNA formulation provided in an embodiment of this application is shown; Figure 6The diagram illustrates the expression of luciferase in mice by two formulations, before and after optimization, provided in the embodiments of this application. Figure 7 The tumor growth curves and body weight changes of Os-LLNs / OVA-immunized mice provided in the embodiments of this application are shown. Figure 8 The tumor growth curves of Os-LLNs / LMP2 immunized mice provided in the embodiments of this application are shown. Figure 8 A and weight changes Figure 8 C; Figure 9 The diagram shows the expression and distribution of tdTomato in Ai9 mice after intravenous administration of Ol-LLNs / Cre, as provided in the embodiments of this application. Figure 9 A is a fluorescence image showing the expression and distribution of tdTomato; Figure 9 B is a statistical result graph of the fluorescence intensity of tdTomato; Figure 10 The diagram illustrates the luciferase expression of luc-mRNA delivered in lymph nodes via vectors of the ligand-free, single-ligand-modified, and optimized multi-ligand-modified formulations provided in this application. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention. Furthermore, all other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of the present invention.

[0023] Specific experimental steps or conditions are not specified in the embodiments; they can be performed according to the conventional experimental steps or conditions described in the prior art. Reagents and other instruments used, unless otherwise specified, are all commercially available conventional reagent products. Furthermore, the accompanying drawings are merely illustrative diagrams of the embodiments of the present invention and are not necessarily drawn to scale. The same reference numerals in the drawings denote the same or similar parts, and therefore, repeated descriptions of them will be omitted. Some block diagrams shown in the drawings are functional entities and do not necessarily correspond to physically or logically independent entities.

[0024] Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of this specification.

[0025] In the description of this invention, it should be understood that the use of terms such as "first" and "second" to define components is merely for the purpose of distinguishing the corresponding components. Unless otherwise stated, the above terms have no special meaning and therefore should not be construed as limiting the scope of protection of this invention.

[0026] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0027] To enable those skilled in the art to better understand this application, the following embodiments will be used to provide a detailed description of a programmable cascaded active targeted nucleic acid delivery system and its applications.

[0028] Example Example 1: Preparation and characterization of multi-ligand modified cascaded active targeted delivery systems (LLNs) This embodiment presents a multi-ligand modified cascade active targeted delivery system based on lipid nanoparticles. Its core design involves co-encapsulating three functional ligand compounds within a lipid nanoparticle framework composed of ionizable cationic lipids (GO-C14), auxiliary phospholipids, distearate phosphatidylcholine (DSPC), and structural lipids, cholesterol (Chol), via a thin-film dispersion method. This allows the ligands to integrate in situ into the lipid structure during nanoparticle self-assembly, thereby constructing a multifunctional delivery system.

[0029] The specific preparation steps are as follows: S1: Thin-film dispersion method is used. Precisely weigh the prescribed amounts of G0-C14, DSPC, Chol, and three functional ligand compounds according to the predetermined molar ratio: SP (a self-peptide), as an immune escape ligand used to send a "don't eat me" signal to macrophages; Mannose, as a cell-targeting ligand used to target mannose receptors on the surface of dendritic cells; and W5R4K (WRK), a cyclic transmembrane ligand used to promote cellular memory function.

[0030] The above components were placed in a 100 mL round-bottom flask, and anhydrous ethanol / chloroform mixed solution (volume ratio 1:1) was added to bring the total volume to 2 mL. The mixture was shaken thoroughly to dissolve all components. Subsequently, the organic solvent was removed by rotary evaporation at 37°C under vacuum, forming a uniform lipid film on the flask wall.

[0031] S2: Add 2 mL of RNase-free water to the lipid membrane and hydrate the lipid membrane at 60°C to obtain a colloidal solution.

[0032] S3: The colloidal solution was sonicated for 3 minutes using a probe (power 100 W, cycle: 3 seconds of sonication, 3 seconds of intermittent sonication), and then filtered through a 0.22 μm sterile filter membrane to obtain a series of multi-ligand modified lipid nanoparticles (LLNs) with a final G0-C14 concentration of 2 mg / mL. Figure 1 As shown.

[0033] Key physical properties of the prepared LLNs were characterized. The results are as follows: Figure 2 As shown, the average particle size ranges from 125 to 140 nm, the polydispersity index (PDI) is less than 0.3, and the zeta potential ranges from +30 to +45 mV. This indicates that the formulation prepared by this invention has uniform particle size, narrow distribution, and stable system, meeting the basic requirements for injectable nanomedicines.

[0034] Example 2: Verification of the inhibitory effect of immune escape ligand SP on macrophage uptake. This embodiment verifies the inhibitory effect of immune escape ligand self-peptide on macrophage uptake in the delivery system of the present invention, thereby proving the effectiveness of its "don't eat me" signaling function.

[0035] RAW264.7 mouse macrophage cells in logarithmic growth phase were collected and the cell density was adjusted to 8 × 10⁻⁶ cells / year. 4 Cells were seeded at a rate of 1 mL / well in 24-well plates and incubated overnight at 37°C with 5% CO2. The next day, SP-modified SMW-LLNs / Cy5 (experimental group) and unmodified MW-LLNs / Cy5 (control group) were added to the cell culture medium at a dose of 0.5 μg Cy5-mRNA per well and incubated for another 2 hours. After incubation, the following assays were performed: Cells were collected from 24-well plates, washed twice with pre-cooled phosphate-buffered saline (PBS), resuspended, and then the mean fluorescence intensity (MFI) of Cy5-positive cells was detected by flow cytometry to quantitatively analyze the uptake level of nanoparticles by cells.

[0036] Cells in confocal dishes were washed with pre-cooled PBS, then incubated with Hoechst 33258 (1 µg / mL) staining solution for 15 minutes to label the cell nuclei. After washing again, the cells were observed and photographed using a confocal microscope to visually compare the fluorescence distribution and intensity within the cells.

[0037] like Figure 3As shown, flow cytometry and confocal microscopy results consistently indicate that the uptake of SP-modified SMW-LLNs / Cy5 in RAW264.7 cells was significantly lower than that of unmodified MW-LLNs / Cy5. These results demonstrate that SP modification effectively inhibits macrophage phagocytosis of the delivery system and significantly enhances the system's immune escape capability, providing crucial experimental evidence for achieving the "circulatory escape" cascade targeting step in this invention.

[0038] Example 3: Validation of the synergistic effect of cell-targeting ligand Mannose and transmembrane ligand WRK in promoting dendritic cell uptake. This embodiment verifies the synergistic targeting and uptake-promoting effects of the cell-targeting ligand Mannose and the transmembrane ligand WRK on dendritic cells in the delivery system of the present invention.

[0039] Mouse dendritic cell line (DC2.4) in logarithmic growth phase was collected, and the cell density was adjusted to 8 × 10⁻⁶. 4 Cells / mL were seeded at 1 mL / well in 24-well plates and incubated overnight at 37°C in a 5% CO2 incubator for cell adhesion.

[0040] The following day, different groups of formulations were added to the cell culture medium at a dose of 0.5 μg Cy5-mRNA per well. The formulation groups included: a complete system that simultaneously modifies SP, Mannose and WRK (SMW-LLNs / Cy5); SP and Mannose only, without WRK (SM-LLNs / Cy5); and SP and WRK only, without Mannose (SW-LLNs / Cy5).

[0041] After incubating the cells and the formulation in a 37°C incubator for 2 h, the cells were collected, washed twice with pre-cooled PBS, resuspended, and then the MFI of Cy5 positive cells was detected by flow cytometry to quantitatively analyze the uptake level of different nanoparticles by the cells.

[0042] like Figure 4 As shown, flow cytometry analysis revealed that the uptake of SMW-LLNs / Cy5 in DC2.4 cells was significantly higher than that of SM-LLNs / Cy5, demonstrating that WRK modification effectively increases the internalization of nanoparticles by cells. The uptake of SMW-LLNs / Cy5 in DC2.4 cells was also significantly higher than that of SW-LLNs / Cy5, indicating that mannose modification can significantly promote active uptake by targeting mannose receptors on the surface of dendritic cells. Furthermore, the SMW-LLNs / Cy5 group exhibited the highest uptake level.

[0043] The above results demonstrate that co-modification with Mannose and WRK can synergistically improve the uptake efficiency of the delivery system in dendritic cells through active targeting and membrane penetration promotion mechanisms, respectively.

[0044] Example 4: Application of Cascaded Active Targeting System in Spleen Delivery This embodiment optimizes the ratio of three functional ligands, using the independent variables of the peptide (SP, X1), mannose (X2), and cyclic transmembrane peptide W5R4K (WRK, X3) and the dependent variable of liver bioluminescence intensity (Y1), employing a central composite design. A 3-factor, 5-level experimental design was conducted in Design-Expert software to predict the optimal prescription for spleen targeting. Figure 5 As shown.

[0045] The optimal spleen-targeting mRNA delivery system was determined through software analysis and prediction, and named Os-LLNs. Its final formulation composition (molar percentage) is: G0-C14 50%, Chol 35.96%, SP 2.32%, Mannose 2.82%, WRK 2.34%.

[0046] To verify the above optimization results, an Os-LLNs formulation loaded with luc-mRNA was prepared. C57BL / 6 mice were administered the drug via tail vein injection. Six hours after administration, the expression of luciferase in the mice was detected using a small animal in vivo imaging system. The in vivo imaging results are as follows: Figure 6 As shown, compared with the original formulation, the optimized Os-LLNs / luc-mRNA significantly increased the expression level of luciferase protein in the spleen, while significantly decreasing the expression level in the liver.

[0047] This embodiment demonstrates that by adjusting the composition ratio of functional ligands, the targeting fate of the programmable delivery platform described in this invention can be successfully reprogrammed to spleen-specific targeting. The obtained Os-LLNs system exhibits highly efficient spleen-targeting mRNA delivery capability, fully demonstrating the "programmable" characteristics and cascaded active targeting advantages of the platform of this invention.

[0048] Example 5: Application and efficacy validation of a programmable cascaded active targeting system in mRNA tumor vaccines. Lymphoma cells expressing ovalbumin model antigen (E.G7-OVA cells) in the logarithmic growth phase were collected, resuspended in sterile PBS, and the cell density was adjusted to 1 × 10⁻⁶. 7 Cells / mL. 100 μL of cell suspension was subcutaneously injected into the right rib area of ​​6-8 week old male C57BL / 6 mice. After 5 days of visible tumor formation, the mice were randomly assigned to groups and intravenous administration began.

[0049] The experimental groups included: Control group (blank control), Os-LLNs / GFP group (spleen-targeting system encapsulates GFP mRNA, which is an unrelated gene and serves as an antigen negative control), Or-LLNs / OVA group (ordinary non-targeting system encapsulates OVA antigen mRNA) and Os-LLNs / OVA group (spleen-targeting system encapsulates OVA antigen mRNA).

[0050] Nasopharyngeal carcinoma cells (TC-1-LMP2 cells) expressing the HPV LMP2 model antigen in the logarithmic growth phase were collected, mixed with an equal volume of matrix gel, and the cell density was adjusted to 1 × 10⁻⁶. 7 Cells / mL. 150 μL of cell mixture was subcutaneously injected into the right rib area of ​​6-8 week old male C57BL / 6 mice. After tumor formation 7 days after inoculation, mice were randomly assigned to groups and began intravenous drug administration. The experimental group was configured as above, except that the antigen was LMP2.

[0051] like Figure 7 , 8 As shown, in the E.G7-OVA model, tumors grew rapidly in the Control group and the Os-LLNs / GFP group; tumor growth slowed in the Or-LLNs / OVA group; while the tumor volume in the Os-LLNs / OVA group initially increased slowly and then decreased significantly. At day 18, the tumor volume in the Os-LLNs / OVA group was significantly smaller than in all other groups. Consistent with the above trends in the TC-1-LMP2 model, the Os-LLNs / LMP2 group exhibited the best tumor suppression effect, with its tumor volume significantly smaller than other control groups.

[0052] This embodiment fully demonstrates that the spleen-targeting mRNA vaccine (Os-LLNs) constructed using the programmable cascade active targeting system described in this invention can stimulate a strong anti-tumor immune response in different tumor models by delivering specific tumor antigens, thereby achieving significant therapeutic effects, and the systemic administration shows good safety. Example 6: Construction and Validation of Liver Targeting Program for Programmable Delivery System This embodiment uses a central composite design with hepatic bioluminescence intensity (Y1) as the dependent variable, and the peptides SP (X1), mannose (X2), and the cyclic transmembrane peptide W5R4K (WRK, X3) as independent variables. A three-factor, five-level experimental design was performed using Design-Expert software to predict the optimal liver-targeting formulation. Through software analysis and prediction, the optimal liver-targeting mRNA delivery system was obtained and named Ol-LLNs. Its final formulation composition (molar percentage) is: G0-C1 450%, Chol 35.96%, SP 4.37%, Mannose 6.29%, and WRK 3.38%.

[0053] To verify the above optimization results, an Ol-LLNs formulation loaded with Cre-mRNA was prepared. Ai9 reporter mice were administered the drug via tail vein injection. Three days after injection, in vivo imaging was performed on the mice, and major organs were dissected for in vitro fluorescence imaging analysis.

[0054] In vivo and in vitro imaging results as follows Figure 9 As shown, Cre mRNA was successfully translated into a functional Cre recombinase in hepatocytes, effectively deleting the stop sequence before the tdTomato gene, thereby activating the expression of red fluorescent protein. The tdTomato fluorescent signal was strongly expressed mainly in liver tissue, with small amounts expressed in the spleen and lungs, and very little expressed in the heart and kidneys.

[0055] Example 7: Construction and Validation of Lymph Node Targeting Program for Programmable Delivery System This embodiment uses the self-peptide (SP, X1), mannose (X2), and the cyclic transmembrane peptide W5R4K (WRK, X3) as independent variables, and the lymph node luciferase expression intensity (Y1) as the dependent variable. A central composite design was employed, and a 3-factor, 5-level experimental design was performed using Design-Expert software to predict the optimal formulation for lymph node targeting. Through software analysis and prediction, the optimal lymph node-targeting mRNA delivery system was obtained and named SM-LLNs. Its final formulation composition (molar percentage) is: G0-C1 4.50%, Chol 35.96%, SP 5.00%, Mannose 5.00%, WRK 0%.

[0056] To verify the above optimization results, SM-LLNs formulations loaded with luc-mRNA were prepared. C57BL / 6 mice were administered the drug via tail vein injection. Six hours after administration, the expression of luciferase in vivo and in isolated organs of the mice was detected using a small animal in vivo imaging system.

[0057] In vivo and in vitro imaging results as follows Figure 10 As shown, compared with the unoptimized formulation and the non-targeted control group, the expression level of luciferase protein in lymph nodes was significantly increased by SM-LLNs / luc-mRNA, while the expression level in other major organs such as the liver was relatively low. This embodiment demonstrates that by rationally adjusting the composition and ratio of functional ligands, the targeting fate of the programmable delivery platform described in this invention can be successfully reprogrammed to lymph node-specific targeting.

[0058] The embodiments of this invention systematically demonstrate the construction and verification process of a programmable cascaded active targeting platform. Lipid nanoparticles composed of GO-C14, cholesterol, and a lipid complex with multiple functional ligands (self-peptide SP, mannose, and membrane-penetrating peptide WRK) were successfully prepared by thin-film dispersion, exhibiting uniform particle size and stable potential.

[0059] In vitro experiments validated the synergistic effects of the various functional ligands: SP effectively inhibited macrophage phagocytosis of the carrier, demonstrating its immune escape function; Mannose and WRK synergistically promoted the active uptake and internalization of the carrier by dendritic cells. Using central combinatorial design (CCD) software, we precisely optimized the ligand ratios based on protein expression in different organs, successfully "reprogramming" the in vivo targeting fate of this platform: obtaining spleen-targeting Os-LLNs, liver-targeting Ol-LLNs, and lymph node-targeting SM-LLNs. In vivo pharmacodynamic experiments showed that the spleen-targeting system Os-LLNs, as an example, efficiently delivered antigen mRNA in two tumor-bearing models, activated specific immune responses, produced significant tumor suppression effects, and demonstrated good safety.

[0060] In summary, these embodiments collectively demonstrate that by rationally adjusting the combination and ratio of ligands, the same platform can be flexibly and efficiently customized for different target purposes without altering the core framework.

[0061] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0062] Although preferred embodiments of the present application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present application.

[0063] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0064] The above provides a detailed description of a programmable cascaded active targeted nucleic acid delivery system and its application. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A programmable cascaded active targeted nucleic acid delivery system, characterized in that, The nucleic acid delivery system comprises at least two types of functional ligand lipid complexes; the functional ligand lipid complexes are formed by covalently linking functional ligands with cholesterol via linkers; wherein the functional ligands are selected from immune escape ligands, cell-targeting ligands, and membrane-penetrating ligands.

2. The programmable cascaded active targeted nucleic acid delivery system according to claim 1, characterized in that, The linker is a polyethylene glycol chain.

3. The programmable cascaded active targeted nucleic acid delivery system according to claim 1, characterized in that, The immune escape ligand is a self-peptide with the sequence shown in SEQ ID NO:

1.

4. The programmable cascaded active targeted nucleic acid delivery system according to claim 1, characterized in that, The cell-targeting ligand is a mannose molecule.

5. A programmable cascaded active targeted nucleic acid delivery system according to claim 1, characterized in that, The membrane-penetrating ligand is a cyclic membrane-penetrating peptide with the sequence shown in SEQ ID NO:

2.

6. A method for preparing a programmable cascaded active targeted nucleic acid delivery system as described in claims 1-5, characterized in that, Includes the following steps: S1: Dissolve ionizable cationic lipids, cholesterol, and the functional ligand in an organic solvent, then remove the organic solvent to form a lipid membrane; S2: Add an aqueous buffer solution to the lipid membrane, and after hydration reaction, a colloidal solution is obtained; S3: The colloidal solution is ultrasonically treated and then filtered to obtain the programmable cascaded active targeted nucleic acid delivery system; The molar percentages of each component in S1 are as follows: 35-50% ionizable cationic lipids, 29-40% cholesterol, 0.1-10% self-peptides, 0.1-10% mannose, and 0.1-10% cyclic transmembrane peptides.

7. The method for preparing a programmable cascaded active targeted nucleic acid delivery system according to claim 6, characterized in that, The preferred molar percentages of each component in S1 are 50% ionizable cationic lipids, 35.96% cholesterol, 2.32% self-peptide, 2.82% mannose, and 2.34% cyclic transmembrane peptide. The delivery system is used for spleen targeting.

8. The method for preparing a programmable cascaded active targeted nucleic acid delivery system according to claim 6, characterized in that, The preferred molar percentages of each component in S1 are 50% ionizable cationic lipids, 35.96% cholesterol, 4.37% self-peptides, 6.29% mannose, and 3.38% cyclic transmembrane peptides. The delivery system is used for liver targeting.

9. A method for preparing a programmable cascaded active targeted nucleic acid delivery system according to claim 6, characterized in that, The preferred molar percentages of each component in S1 are 14.50% ionizable cationic lipids, 35.96% cholesterol, 5.00% peptides, and 5.00% mannose. The delivery system is used for lymph node targeting.

10. The application of the programmable cascaded active targeted nucleic acid delivery system as described in any one of claims 1 to 5 in the preparation of nucleic acid targeted drugs.

Images