Nested lipid nanoparticles for immunotherapy synergy as well as preparation method and application of nested lipid nanoparticles

Abstract

The invention relates to the technical field of drug delivery and tumor immunotherapy, and discloses nested lipid nanoparticles for immunotherapy synergy as well as a preparation method and application of the nested lipid nanoparticles. The nLNP has a double-layer nested structure with an inner layer carrying Hsc70 mRNA and an outer layer carrying an STING agonist MSA-2 prodrug, and an inner layer nitrogen stacking group and an outer layer DBCO group are connected through a click reaction to realize tumor pre-targeting enrichment; the pH response lipid on the outer layer is dissociated preferentially in a slightly acidic environment, firstly releases an MSA-2 prodrug to activate innate immunity, and then releases Hsc70 mRNA to reverse PD-L1 related drug resistance, so that the curative effect of the immune checkpoint inhibitor is synergistically enhanced. The preparation provided by the invention has the advantages of strong targeting property, controllable release and good safety, and is suitable for drugs or pharmaceutical compositions for tumor immunotherapy synergy.

Description

Technical Field

[0001] This invention relates to the fields of drug delivery and tumor immunotherapy, specifically to a nested lipid nanoparticle (nLNP) with a "click-target-step release" function, its preparation method, and its application in reversing immune checkpoint tolerance and enhancing tumor treatment efficacy. Background Technology

[0002] Malignant tumors pose a serious threat to human health. Tumor immunotherapy, by activating the body's immune system to recognize and eliminate tumor cells, has shown clinical benefits in various tumor types. However, in solid tumors, the overall response rate of immunotherapy remains limited, and inherent or acquired tolerance is easily developed. The main limiting factors typically include: the tumor's immunosuppressive microenvironment leading to restricted maturation of antigen-presenting cells (APCs) and activation of effector T cells; insufficient local immune activation signals and low antigen presentation efficiency in the tumor; and uneven distribution of immunomodulatory factors in the body and systemic toxicity caused by non-specific uptake, thus limiting the therapeutic window and sustainable response of immunotherapy.

[0003] Lipid nanoparticles (LNPs) have attracted attention due to their biocompatibility and ability to deliver nucleic acid therapeutics, aiming to improve the delivery efficiency of immunomodulatory drugs and expand the application of nucleic acid therapy in tumor immunotherapy. Patent document CN113271926A discloses various LNP compositions and preparation strategies to enhance nucleic acid delivery and cellular uptake. However, existing LNP systems generally face the following shortcomings in tumor immunotherapy: First, their in vivo distribution is easily affected by reticuloendothelial system clearance, with significant accumulation in non-target organs such as the liver, limiting tumor targeting efficiency. Second, it is difficult to achieve stable co-loading and mutual isolation of immune agonists and nucleic acid regulatory factors within the same nanosystem. Third, and more importantly, existing systems often struggle to achieve programmable responses and stepwise release to the tumor microenvironment, thus failing to meet the temporal synergistic requirement of "first enhancing innate immune activation—then reshaping the tumor cell immune phenotype and drug resistance-related checkpoint axis."

[0004] On the other hand, to improve the localization and enrichment efficiency of drugs in tumors, the "pre-targeting" strategy represented by bioorthogonal click reactions provides a new technical route for precise tumor delivery. Patent document CN107496937A / B discloses a pre-targeted drug delivery system based on the reaction of DBCO with azide groups, which can achieve enhanced target enrichment through the introduction of reactive groups followed by click coupling. However, existing click-based pre-targeting technologies mostly focus on the single enrichment of drugs or carriers or their integration with chemotherapy, imaging, and other scenarios. They typically lack deep integration with nucleic acid delivery systems and are difficult to further achieve: the stepwise release of different immunomodulatory components triggered by the tumor microenvironment, and the "temporal-spatial" coupling between immune activation and immune escape axis regulation. This limits their systematic application in enhancing the efficacy of immune checkpoint therapy and reversing drug resistance.

[0005] In summary, while existing technologies have made progress in both nucleic acid delivery LNP and pre-targeted click localization, they still struggle to simultaneously meet the following key requirements: (1) Achieve highly specific and precise localization of tumor tissue in vivo and reduce non-target accumulation; (2) Utilizing the characteristics of the tumor microenvironment to achieve responsive dissociation and programmed stepwise release of nanosystems; (3) The collaborative delivery and timing matching of “first activating the innate immune pathway and then delivering nucleic acid regulatory factors to reshape the tumor cell immune phenotype and drug resistance-related checkpoint axis” can be completed within the same platform; thereby reducing systemic toxicity and improving the overall benefit of solid tumors such as TNBC to immunotherapy (including ICIs).

[0006] Therefore, there is an urgent need to construct a novel nanodelivery system that can organically unify pre-targeting precision positioning, microenvironment response dissociation, and the stepwise release of multiple immune regulatory factors, in order to meet the practical needs of enhancing the efficacy of tumor immunotherapy and overcoming drug resistance. Summary of the Invention

[0007] 1. Technical problems to be solved This invention aims to address the problems of insufficient tumor enrichment, non-specific distribution leading to toxicity risks, and lack of controllable sequential release in existing immunomodulatory drugs and nucleic acid delivery systems, which make it difficult to achieve synergistic immune enhancement. Based on the above objectives, this invention provides a "click-target-step-release" nested lipid nanoparticle for enhancing immunotherapy efficacy, its preparation method, and its applications. Technical solution

[0008] This invention provides a nested lipid nanoparticle (nLNP) with "click-targeting-stepwise release" function, its preparation method, and its application in enhancing tumor immunotherapy. The nLNP comprises an inner lipid nanoparticle and an outer lipid shell: the inner lipid nanoparticle encapsulates mRNA encoding heat shock homolog 70 (Hsc70), and its surface is incorporating an azide group (N3); the outer lipid shell contains strained alkyne group lipids (DBCO) and pH-responsive lipid components, and can be loaded with the STING agonist MSA-2 prodrug. Through a copper-free bioorthogonal click reaction (SPAAC) between the inner N3 and outer DBCO layers, an nLNP with "click-targeting-stepwise release" function is formed. Simultaneously, selective covalent coupling of the nLNP with azide-labeled tumor cells is achieved, thereby obtaining in-situ targeted enrichment of tumor cells. The outer shell preferentially dissociates and releases MSA-2 prodrug in the tumor's microacidic environment to initiate innate immune activation; subsequently, the inner layer releases Hsc70 mRNA and upregulates Hsc70 expression in tumor cells, promoting the reversal of the PD-L1-related drug resistance axis, thereby enhancing the efficacy of immune checkpoint blockade therapy. This invention features a mild and reproducible preparation process, yielding nLNPs with controllable particle size, strong drug loading capacity, good targeting, and high biosafety, making it suitable for immunomodulatory therapy and industrialization of solid tumors such as triple-negative breast cancer.

[0009] To achieve the above objectives, this invention provides an nLNP with "click-targeting-gradual release" function, its preparation method, and its application: S1. By introducing DSPE-PEG-DBCO into the outer shell and DMG-PEG-N3 into the inner shell, the inner and outer layers can form a stable nested structure through a copper-free SPAAC click reaction; at the same time, the outer DBCO can react with azide-labeled tumor cells to achieve in situ targeted enrichment of tumor cells. S2. By introducing the pH-responsive lipid component DSPE-PEOz into the outer layer, the outer shell is preferentially dissociated in the tumor microacidic environment, releasing the MSA-2 prodrug first to initiate innate immune activation; S3. Subsequently, the inner layer releases Hsc70 mRNA and upregulates Hsc70 expression, which helps to promote the reversal of PD-L1-related tolerance, thereby enhancing the efficacy of ICIs.

[0010] In a first aspect, the present invention provides nested lipid nanoparticles, comprising: Inner lipid nanoparticles: contain ionized lipids, auxiliary lipids, cholesterol and PEG lipids with azide groups. The inner lipid nanoparticles may or may not contain Hsc70 mRNA, and their surface contains azide groups (N3). Outer lipid shell: Contains dibenzocyclooctylene functional group lipids and pH-responsive lipids, and may or may not be loaded with the STING agonist MSA-2 prodrug; In this process, the azide groups on the surface of the inner lipid nanoparticles and the dibenzocyclooctyn functional groups on the surface of the outer lipid shell are covalently linked through a copper-free bioorthogonal click reaction (SPAAC), enabling the nested lipid nanoparticles to click-target azide-labeled tumor cells; the outer lipid shell preferentially dissociates in the acidic tumor microenvironment and achieves stepwise release.

[0011] Furthermore, depending on the active ingredient contained therein, the nested lipid nanoparticles include at least the following formulations: (1) nLNP empty vector: The inner layer does not carry Hsc70 mRNA and the outer layer does not carry the STING agonist MSA-2 prodrug; (2) nLNM: The outer layer carries the STING agonist MSA-2 prodrug and the inner layer does not carry Hsc70 mRNA; (3) nLNH: The inner layer carries Hsc70 mRNA and the outer layer does not carry the STING agonist MSA-2 prodrug; (4) nLNMH: The outer layer carries the STING agonist MSA-2 prodrug and the inner layer carries Hsc70 mRNA.

[0012] Further, the inner lipid nanoparticles comprise ionized lipid SM102, cofactor lipid DOPE, cholesterol, and DMG-PEG-N3, wherein the molar ratio of ionized lipid SM102, cofactor lipid DOPE, cholesterol, and DMG-PEG-N3 is (40–60):(5–20):(30–50):(0.5–5). Preferably, it is 48:10:40:2. Furthermore, the nitrogen and phosphorus equivalence ratio of the ionized lipids to Hsc70 mRNA is 4–10:1, preferably 6:1.

[0013] Further, the outer lipid shell layer comprises egg yolk lecithin, cholesterol, DSPE-PEG-DBCO, and DSPE-PEOz; wherein the molar ratio of egg yolk lecithin, cholesterol, DSPE-PEG-DBCO, and DSPE-PEOz is (20–50):(1–15):(1–15):(1–15). Preferably, it is 35:5:8:6. Further, the mass ratio of the STING agonist MSA-2 prodrug to the outer lipid shell is 1:(10–25). Preferably, it is 1:14. Furthermore, the molar ratio of the inner lipid nanoparticles to the outer lipid shell is 1:(1–3). Preferably, it is 1:1.5. Secondly, the present invention provides a method for preparing the aforementioned nested lipid nanoparticles, comprising the following steps: S1. Preparation of inner lipid nanoparticles: Ionized lipids, auxiliary lipids, cholesterol and PEG lipids with azide groups were dissolved in ethanol to form an alcohol phase; Hsc70 mRNA or empty vector buffer was dissolved in acidic buffer to form an aqueous phase; the aqueous phase was added to the alcohol phase and mixed to self-assemble, resulting in a dispersion of inner lipid nanoparticles with azide groups on the surface. S2. Preparation of outer lipid alcohol phase: Dissolve the outer lipid material containing dibenzocyclooctylene functional group lipid and pH-responsive lipid in ethanol to form the outer lipid alcohol phase, and add the STING agonist MSA-2 prodrug as needed; S3. Constructing a nested structure: Using the inner lipid nanoparticle dispersion as the aqueous phase, it is added to the outer lipid alcohol phase and mixed to complete the self-assembly of the outer lipid shell. At the same time, the SPAAC click reaction occurs to form nested lipid nanoparticles. S4. Purification: Remove ethanol and free components by dialysis or equivalent methods to finally obtain nested lipid nanoparticles.

[0014] Further, the acidic buffer in step S1 is a 10–50 mM citrate buffer with a pH of 3.5–4.5.

[0015] Furthermore, in step S3, after mixing, the mixture is allowed to stand at room temperature for 5–20 min to complete the self-assembly and SPAAC click reaction.

[0016] Further, in step S4, a dialysis bag with a molecular weight cutoff of 10–30 kDa is used to dialyze in PBS to remove ethanol, free lipids and / or free drugs.

[0017] Thirdly, the present invention provides the use of the aforementioned nested lipid nanoparticles in the preparation of medicaments or pharmaceutical compositions for enhancing the efficacy of tumor immunotherapy.

[0018] Preferably, the nested lipid nanoparticles are used to promote the activation of the cGAS-STING pathway and promote the reversal of the PD-L1-related tolerance / resistance axis by upregulating Hsc70 mRNA expression, thereby enhancing the efficacy of immune checkpoint blockade therapy.

[0019] 3. The beneficial effects of the present invention: Compared with the prior art, the present invention has at least the following advantages: 1. Strong targeting and reduced risk of non-targeted accumulation: Selective covalent coupling with azide-labeled tumor-related structures is achieved through click chemistry, improving the enrichment and retention capacity at the tumor site; 2. Clear step-by-step release and well-defined synergistic pathway: The outer layer releases immune agonist factors first, followed by the inner layer releasing nucleic acid regulatory factors, achieving a temporal synergy of "activation first, remodeling later"; 3. Helps reverse immune tolerance: By regulating Hsc70 mRNA expression, it intervenes in the formation of the PD-L1-related immune escape axis, thereby enhancing the sensitivity of ICIs; 4. Mild process, reproducible and with scale-up potential: The preparation conditions are mild and the parameters are controllable, which facilitates batch-to-batch consistency and large-scale production; 5. Adjustable materials and wide applicability: Lipid materials and functional components can be replaced and optimized within a pharmaceutically acceptable range, facilitating the expansion of combination therapies. Attached Figure Description

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

[0021] Figure 1 This is a schematic diagram of the structure and synthesis of the click-targeted stepwise release nested lipid nanoparticles (nLNP) of the present invention (with the click-targeted release of inner and outer N3-DBCO layers in response to pH as the core).

[0022] Figure 2 MSA-2, the STING agonist in Example 1 1 H NMR structural characterization.

[0023] Figure 3 For the MSA-2 prodrug in Example 2 1 H NMR structural characterization and mass spectrometry (MS) confirmation results.

[0024] Figure 4 The images show a TEM image (a) and a particle size distribution (DLS) image (b) of the inner mRNA-LNP in Example 5.

[0025] Figure 5 The images show TEM images (a) and particle size distribution (DLS) images (b) of nLNMH in Examples 6 and 7.

[0026] Figure 6 The results of the click reaction feasibility verification in Example 8 (in vitro click kinetics-fluorescence change curve of 3-azido-7-hydroxycoumarin and nLNP).

[0027] Figure 7 This is a graph showing the MSA-2 prodrug release curves of nLNMH under different pH conditions in Example 9.

[0028] Figure 8 This is a line graph showing the serum stability of nLNMH in Example 10.

[0029] Figure 9 Results of the in vitro click-targeted uptake experiment in Example 11: confocal microscopy and quantitative analysis of intracellular fluorescence intensity; data are expressed as mean ± standard deviation (n=3). Statistical analysis was performed on differences between groups, with *** indicating... p <0.001.

[0030] Figure 10 Flow cytometry analysis results of DC maturation-related indicators in Example 12: Flow cytometry detection and quantitative statistical analysis of maturation indicators (CD80 / CD86); data are expressed as mean ± standard deviation (n=3), and differences between groups were analyzed using statistical tests. **** indicates... p <0.0001.

[0031] Figure 11 This is a Western blot analysis of intracellular Hsc70 protein expression after treatment with different doses of Hsc70 mRNA in Example 13. Figure 12 The following are the flow cytometry analysis results of PD-L1 expression in Example 14: PD-L1 expression was detected by flow cytometry, and the positive rate and MFI were quantitatively analyzed. Data are expressed as mean ± standard deviation (n=3). Differences between groups were analyzed using statistical tests. ** indicates... p <0.01, *** indicates p <0.001, **** indicates p <0.0001.

[0032] Figure 13 The results of the in vitro safety evaluation (cytotoxicity) in Example 15 are shown.

[0033] Figure 14 The results of in vivo biodistribution and tumor targeting ability evaluation in Example 16 (in vivo imaging and ex vivo organ imaging).

[0034] Figure 15 The figures show the tumor volume change curves and statistical analysis of mice in different treatment groups in Example 17. Data are expressed as mean ± standard deviation (n=5). Statistical analysis was performed on differences between groups. **** indicates... p <0.0001. Detailed Implementation

[0035] 1. Standardized Explanation of Terminology and Group Naming To avoid ambiguity, this instruction manual adopts the following consistent naming convention: PBS: Phosphate-buffered saline (PBS) control.

[0036] nLNP (empty payload): Inner empty payload N3-LNP + outer empty payload shell (containing DBCO and pH-responsive lipids, but not MSA-2 prodrug or Hsc70 mRNA).

[0037] nLNM: Inner layer empty N3-LNP + outer layer containing MSA-2 prodrug shell.

[0038] nLNH: Inner layer carrying Hsc70 mRNA N3-mRNA-LNP + outer empty shell.

[0039] nLNMH: Inner layer contains N3-mRNA-LNP carrying Hsc70 mRNA, outer layer contains MSA-2 prodrug shell.

[0040] The above formulations are identical in key structural features (N3 / DBCO click site and outer pH-responsive lipid) except for whether they contain MSA-2 prodrug and whether they contain Hsc70 mRNA, to ensure comparability with the control.

[0041] 2. Sources of raw materials and reagents: SM-102 (ionizable lipid, CAS 2089251-47-6) was purchased from Cayman Chemical (USA, Catalog No. 33474).

[0042] DOPE [1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, CAS 4004-05-1] was purchased from Aladdin (AL-D155634).

[0043] Cholesterol (CAS 57-88-5) was purchased from Aladdin (AL-C104036).

[0044] DMG-PEG(2000)-Azide (DMG-PEG2000-N3, CAS 2389048-44-4) was purchased from Avanti PolarLipids (Catalog No. 880237).

[0045] Egg yolk lecithin / phosphatidylcholine (CAS93685-90-6) was purchased from Aladdin (L305002).

[0046] DSPE-PEG(2000)-DBCO (copper-free click DBCO functionalized PEG lipid) was purchased from Avanti PolarLipids (Catalog No. 880226).

[0047] DSPE-PEOz (mPEOz-DSPE, CAS: N / A) was purchased from Shenzhen MeloPEG Technology Co., Ltd. (item number 131331).

[0048] The chemically synthesized reagents used in Examples 1–2 (such as succinic anhydride, aluminum trichloride, dichloromethane, 4-morpholinobutanol, TBTU, DIEA, anhydrous sodium sulfate, silica gel and TLC plates, etc.), the molecular biology reagents used in Examples 3–4 (such as PCR kits, T7 in vitro transcription and purification reagents, etc.), and routine reagents for cell / animal experiments (such as culture media, serum, PBS, etc.) were all purchased from commercial suppliers such as Aladdin, Sinopharm Group, Thermo Fisher, Takara, Vazyme, Beyotime, and Gibco.

[0049] Unless otherwise stated, all reagents used in this invention are commercially available analytical grade / biochemical grade. The listed catalog numbers are only used to indicate the source of materials used in the implementation of this invention. Commercial materials of equivalent purity and specifications may be used as substitutes without affecting the technical effect of this invention.

[0050] 3. Overview of the general preparation process for nLNP (1) Preparation of inner layer dispersion: SM102, DOPE, cholesterol, and DMG-PEG-N3 were dissolved in ethanol to form an alcohol phase; Hsc70 mRNA (or empty vector buffer) was dissolved in citrate buffer to form an aqueous phase; the aqueous phase was added to the alcohol phase, mixed well, self-assembled, and dialyzed to obtain the inner layer dispersion.

[0051] (2) Outer shell construction: The outer lipid material (e.g., egg yolk lecithin, cholesterol, DSPE-PEG-DBCO, DSPE-PEOz) is dissolved in ethanol to form the outer alcohol phase; MSA-2 prodrug is added as needed; the inner dispersion is added to the outer alcohol phase as an aqueous phase and mixed to complete the outer self-assembly and SPAAC click. The ethanol and free components are removed by dialysis to obtain the target nLNP formulation.

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0053] Note: The following embodiments provide specific proportions and operating conditions, which are preferred implementation methods and do not constitute a limitation on the scope of protection.

[0054] Example 1: Synthesis and characterization of the STING agonist MSA-2, including the following steps: S1. Succinic anhydride (3.1 g, 30.9 mmol) and aluminum trichloride (2.7 g, 20.6 mmol) were dissolved in dichloromethane (DCM, 10 mL) and stirred at 0 °C for 1 h to obtain reaction solution A; S2. Dissolve 5,6-dimethoxybenzo[b]thiophene (2.0 g, 10.3 mmol) in DCM (40 mL) and add it dropwise to reaction solution A (dropwise addition for about 30 min). After the addition is complete, continue stirring at 43 °C overnight. S3. After the reaction was completed by TLC monitoring, the reaction solution was poured into ice water, the pH was adjusted to about 10 with sodium hydroxide solution, and the solution was filtered to obtain the filtrate; then the solution was acidified with concentrated hydrochloric acid to about 2 to precipitate the solid. S4. Filter and collect the solid, wash with water and DCM in sequence, and dry to obtain MSA-2 solid (2.6 g, yield about 85.6%). S5. Adopt 1 H NMR (400 MHz, DMSO-d6) characterization confirmed (see [link to H NMR characterization]). Figure 2 ).

[0055] like Figure 2 As shown, MSA-2's 1 The H NMR spectrum showed characteristic chemical shifts and peak distribution consistent with the target structure, with no obvious impurity peaks, indicating that the synthesized product has the correct structure and the purity meets the requirements for subsequent prodrug synthesis and formulation construction.

[0056] Example 2: Synthesis and characterization of MSA-2 prodrug, including the following steps: S1. Dissolve 4-morpholinobutanol (140 mg, 0.88 mmol) in anhydrous DCM (3 mL), then add MSA-2 (200 mg, 0.68 mmol), TBTU (284 mg, 0.88 mmol) and DIEA (114 mg, 0.88 mmol) in sequence, and stir overnight at 43 °C. S2. Remove solvent by rotary evaporation, dissolve the residue in DCM, and wash successively with 5% citric acid solution, saturated NaHCO3 solution and saturated saline solution; S3. The organic phase was dried over anhydrous Na2SO4, filtered, and then rotary evaporated to obtain the crude product; purified by silica gel column chromatography to obtain a pink solid MSA-2 prodrug (approximately 258 mg, yield approximately 85%). S4. Adopt 1 H NMR (400 MHz, CDCl3) and MS-confirmed structures (see [link to H NMR]) Figure 3 ).

[0057] like Figure 3 As shown, the MSA-2 prodrug 1 The H NMR spectrum and mass spectrometry confirmation results are consistent with the target molecule structure, proving that the prodrug was successfully synthesized; the structural confirmation provides a basis for subsequent loading and release evaluation.

[0058] Example 3: PCR amplification and linearization quantification of Hsc70 coding sequence template, including the following steps: S0. Sequence origin and template plasmid description: The Hsc70 (also known as Hspa8) coding sequence is derived from publicly available nucleotide sequence information in public databases, which can be obtained by those skilled in the art through public channels such as NCBI GenBank. In this embodiment, the coding region sequence corresponding to the published sequence of Mus musculus Hspa8 mRNA (Accession No. NM_031165.5; Gene ID: 15481) is used as the basis for the in vitro transcription template sequence, and is cloned into the in vitro transcription vector pGP-T7-MCS-polyA (the vector contains a T7 promoter and a polyA structure) to obtain a template plasmid containing the T7 promoter-Hsc70 coding sequence-polyA, which is used for subsequent PCR amplification to obtain a linearized transcription template.

[0059] Because the target sequence contains a HindIII restriction endonuclease recognition site, it is not suitable to linearize it using HindIII single enzyme digestion. Therefore, universal vector primers were used to amplify the template plasmid by PCR to obtain linear DNA as an in vitro transcription template.

[0060] S1. Using a plasmid containing the T7 promoter sequence and the Hsc70 coding sequence as a template, take 20 ng of the template plasmid; S2. Prepare the PCR reaction system (total volume 50 μL): add ddH2O to 50 μL, 2×Proofast® MasterMix 25 μL, primer 1 (T7, 10 μM) 2 μL, primer 2 (polyAR, 10 μM) 2 μL, template DNA 20 ng; S3. PCR cycling conditions: 94 ℃ pre-denaturation for 3 min; followed by 36 cycles: 94 ℃ for 30 s, 60 ℃ for 50 s, 72 ℃ for 30 s; and finally 72 ℃ extension for 5 min. S4. Fragment size of PCR products was verified by agarose gel electrophoresis; S5. The concentration of linearized DNA was determined using a NanoDrop micro-spectrophotometer and used as a template for subsequent in vitro transcription of mRNA.

[0061] Example 4: In vitro transcription of Hsc70 mRNA, including the following steps: S1. Using the linearized DNA described above as a template, Hsc70 mRNA was prepared using the T7 RNA polymerase in vitro transcription system; S2. After transcription, the mRNA is deDNA removed and purified to obtain high-purity mRNA; S3. The mRNA concentration and the A260 / A280 ratio were determined using NanoDrop. S4. Aliquot the mRNA and store it at -80 ℃ for later use.

[0062] Example 5: Preparation of inner layer mRNA-LNP: Taking the preparation of a batch of inner layer mRNA-LNPs as an example, the steps include: S1. Alcohol phase (1 volume): Dissolve SM102, DOPE, cholesterol, and DMG-PEG-N3 in anhydrous ethanol at a molar ratio of 48:10:40:2 to prepare an inner lipid alcohol phase solution. S2. Aqueous phase (3 volumes): Dissolve Hsc70 mRNA in 25 mM sodium citrate buffer (pH=4.0) at a nitrogen / phosphorus (N / P) equivalent ratio of 6 to obtain an aqueous mRNA solution; S3. Quickly add the aqueous phase to the alcohol phase and vortex mix for 60 s, then let stand at room temperature for 10 min to self assemble into mRNA-LNP; S4. Place the obtained solution in a 20 kDa dialysis bag and dialyze with PBS to remove acidic buffer and free lipids, to obtain the inner mRNA-LNP dispersion and store it quantitatively. S5. Particle size and PDI were determined by dynamic light scattering (DLS); morphology was observed by transmission electron microscopy (TEM).

[0063] Note: For the inner empty N3-LNP (used for nLNP empty or nLNM), the Hsc70 mRNA in step S2 can be replaced with an equal volume of acidic buffer, with the other conditions remaining the same.

[0064] Example 6: Construction of nLNMH (outer layer containing MSA-2 prodrug, inner layer carrying Hsc70 mRNA) via click reaction, including the following steps: S1. Outer lipid alcohol phase (1 volume): Egg yolk lecithin, cholesterol, DSPE-PEG-DBCO, and DSPE-PEOz are dissolved in anhydrous ethanol at a molar ratio of 35:5:8:6 to obtain the outer lipid alcohol phase; S2. Prepare a 100 mg / mL solution of MSA-2 prodrug and add the outer lipid alcohol phase according to the formula "drug: outer lipid = 1:(10–25) (mass ratio), for example 1:10, 1:14 or 1:20, preferably 1:14", and mix well to obtain the drug-containing outer lipid alcohol phase. S3. Aqueous phase (3 volumes): Take the inner mRNA-LNP dispersion obtained in Example 5 as the aqueous phase, and control the ratio of "inner lipid:outer lipid = 1:(1–3) (molar ratio), preferably inner lipid:outer lipid = 1:1.5"; S4. The aqueous phase is rapidly added to the drug-containing outer alcohol phase and vortexed for 120 s. After standing at room temperature for 10 min, the outer shell self-assembles to cover the inner layer. At the same time, the inner N3 and the outer DBCO undergo a SPAAC click reaction to form a stable nested structure. S5. Place the system in a 20 kDa dialysis bag and dialyze with PBS to remove ethanol and free components, obtaining nLNMH, which is then quantitatively stored.

[0065] Note: nLNH can be obtained by removing only the MSA-2 prodrug as described in Example 6.

[0066] Example 7: Characterization of particle size, dispersibility, and morphology of the final formulation nLNMH and inner layer mRNA-LNP, including the following steps: S1. Take inner layer mRNA-LNP and nLNMH samples respectively, and measure the average particle size and PDI by DLS; S2. The sample was negatively stained with copper mesh, and the morphology and nested structure differences were observed by TEM. S3. Organize the DLS curves and TEM images (see...) Figures 4-5 ).

[0067] like Figure 5 As shown, the nested lipid nanoparticle system of the present invention exhibits regular morphology and good particle size distribution after the formation of the inner carrier and the final formulation assembly.

[0068] like Figure 4 As shown, the inner mRNA-LNP exhibits a uniform spherical structure and good dispersibility under TEM; its DLS particle size distribution is concentrated and its PDI is low, indicating that the inner vector can be stably formed and meet the basic conditions for subsequent outer layer assembly.

[0069] like Figure 5 As shown, the final formulation nLNMH also maintained a near-spherical shape and good dispersibility. Compared with the inner mRNA-LNP, the particle size was larger and tended to be more concentrated, indicating that the outer shell was successfully assembled and formed a nested structure. The DLS results further showed that the system had good homogeneity and stability.

[0070] Example 8: Feasibility verification of click reaction (in vitro click kinetics), including the following steps: S1. Set the molar percentage gradient of DMG-PEG-N3 in the inner layer (0–5 mol% of the inner layer lipids (SM102, DOPE, cholesterol, DMG-PEG-N3)) and prepare nLNP samples with different N3 percentages according to Examples 5–6; S2. Add 3-azido-7-hydroxycoumarin probe to each sample to make the final concentration 6.15 μM; S3. Continuously monitor changes in fluorescence intensity using a fluorescence microplate reader; S4. Compare the fluorescence signals of samples with different N3 proportions to assess the degree of click consumption of DBCO functional groups and verify that the nested structure is formed by N3-DBCO click (see...). Figure 6 ).

[0071] Supplement: Excessive design of outer DBCO and control of the accessible amount of residual DBCO exposure: To simultaneously meet the dual requirements of "inner N3-outer DBCO click forming a stable nested structure" and "the outer layer retaining sufficient DBCO for subsequent targeted click conjugation to the tumor N3 site," this invention employs a significantly excessive design of the outer DSPE-PEG-DBCO layer relative to the inner DMG-PEG-N3 layer. Specifically, the amount of outer DSPE-PEG-DBCO added should be significantly higher than the surface-accessible sites of the inner DMG-PEG-N3 layer, such that: a portion of the DBCO undergoes a SPAAC reaction with the inner N3 layer to construct the nested structure, while the other portion of the DBCO remains exposed as PEG end groups and remains accessible for subsequent targeted click conjugation to azide-labeled tumors.

[0072] 1. Total molar ratio control: In the formulation design, the DBCO:N3 (molar ratio) ≈ 3:1 (under preferred conditions) can ensure the click consumption required for shell fixation while retaining sufficient exposed DBCO for subsequent tumor pre-targeting clicks.

[0073] 2. Threshold for the amount of residual DBCO exposed: The amount of "residual accessible DBCO" in the finished nLNP should reach ≥30% of the initial amount of outer DBCO introduced (under preferred conditions) to ensure efficient subsequent click with the tumor N3 site.

[0074] 3. Verification method: Fluorescent azide probe titration method: Take a certain amount of nLNP sample, add excess fluorescent azide probe (3-azido-7-hydroxycoumarin used in Example 8 can be used), record the fluorescence rise over time and fit the endpoint value; convert the endpoint fluorescence signal into "reactive DBCO amount" to obtain the residual DBCO accessible amount. like Figure 6As shown, the 0% group exhibited the fastest fluorescence signal increase and the highest intensity, indicating the presence of numerous free DBCO functional groups capable of reacting with the probe. As the molar percentage of the inner DMG-PEG-N3 layer increased from 0% to 5%, the fluorescence growth rate and endpoint intensity of each group showed a gradual decreasing trend, indicating that the outer DBCO functional groups were preferentially consumed by the inner N3 layer through click chemistry, leading to a reduction in the number of DBCO groups capable of reacting with the probe. This result, from a kinetic perspective, proves that the nested structure of this invention is indeed formed by the click chemistry interaction between the inner DMG-PEG-N3 layer and the outer DSPE-PEG-DBCO layer.

[0075] Considering both the degree of click consumption and the controllability of the system, a DMG-PEG-N3 content of 2 mol% (corresponding to the preferred molar ratio of 48:10:40:2) can achieve a stable and repeatable click assembly effect, and is therefore determined to be the preferred N3 content of this invention.

[0076] Example 9: Outer shell dissociation and MSA-2 prodrug release under different pH conditions, including the following steps: S1. Take nLNMH and place it in a dialysis bag (e.g., 3.5 kDa), and place it in a buffer system of pH 7.4, pH 6.5, and pH 5.5 respectively, and release it on a shaker at 37 ℃; S2. Collect external fluid at 0.5, 1, 2, 4, 6, 8, 12, 24, 36, and 48 h and replenish with an equal volume of fresh buffer solution; S3. HPLC quantifies MSA-2 prodrug in the external solution, calculates cumulative release and plots curves (see...). Figure 7 ).

[0077] Figure 7 The cumulative release curves of nLNMH under different acid and alkaline environments (pH 7.4, pH 6.5, pH 5.5) are shown. The horizontal axis represents the release time (h), and the vertical axis represents the cumulative release percentage of MSA-2 prodrug (%). Different curves correspond to different pH conditions.

[0078] like Figure 7 As shown, under physiological conditions at pH 7.4, the release rate of MSA-2 prodrug was generally slow, indicating that the outer shell is structurally stable in a neutral environment, which helps reduce premature leakage in systemic circulation. When the ambient pH dropped to tumor microacidity (pH 6.5) and stronger acidity (pH 5.5, simulating the acidification environment of endocytosis / lysosomes), the release curve shifted upward and showed faster release kinetics, indicating that the outer shell underwent more significant responsive dissociation, thereby promoting the release of the loaded MSA-2 prodrug.

[0079] This result supports the "stepwise release" design of the nLNP in this invention from the perspective of "the outer layer responding first to the acidic microenvironment and preferentially releasing the immune agonist": the pH-responsive lipid components of the outer layer trigger structural loosening / dissociation in the microacidic environment, so that the STING agonist can be preferentially released in the tumor-associated acidic microenvironment, laying the foundation for subsequent immune activation.

[0080] Example 10: Evaluation of serum stability of nLNMH, including the following steps: S1. Disperse nLNMH in PBS and serum-containing medium (e.g., 10% FBS) respectively, and incubate at 37 °C; S2. Samples were taken at 0, 7, 14, and 21 days. S3. DLS was used to determine the changes in particle size and PDI over time to evaluate the stability of nLNMH in the serum environment. (See...) Figure 8 ) like Figure 8 As shown, the particle size and PDI of nLNMH changed little over time during incubation in PBS and serum-containing conditions, indicating that the nested lipid nanoparticles have good colloidal stability in the serum environment and can meet the stability requirements of in vivo administration and circulation.

[0081] Example 11: Confocal validation of in vitro click-targeted uptake, including the following steps: S0. Setting up tumor cell azide metabolism markers (N3+) and unlabeled controls (N3-): S0-1. Cell plating: Select tumor cells such as 4T1, plate them in a confocal glass dish, and incubate overnight to ensure that the cell confluence is about 50-70% the next day.

[0082] S0-2. Azide metabolism marker (N3+): Ac4ManNAz (tetraacetyl N-azidoacetylmannosamine) was added to the complete culture medium at a final concentration of 50 μM and incubated at 37 °C and 5% CO2 for 24 h.

[0083] S0-3. Washing: After incubation, discard the sugar-containing medium, gently wash 2–3 times with PBS, and replace with fresh complete medium. To reduce the impact of residual free sugar, incubate for another 1 hour after replacing with fresh medium before proceeding to the subsequent click-targeted uptake experiment.

[0084] S0-4. Unlabeled control (N3-) setting: No Ac4ManNAz was added, only an equal volume of solvent (PBS) was added and incubated for the same amount of time; S1. Azide-based metabolic labeling of tumor cells: A sugar precursor that can be metabolized to introduce azide groups is added to the culture medium, and incubation is carried out to form N3 groups on the cell surface; S2. Set up an unlabeled group (N3-) and a labeled group (N3+), and add fluorescently labeled nLNMH containing DSPE-PEG-DBCO. To ensure fairness of comparison, the N3+ and N3− groups were completely identical in terms of cell density, incubation time, number of washes, and amount of fluorescently labeled nLNMH. S3. After incubation, wash and observe the distribution of fluorescence in the cell membrane and intracellular cells using confocal microscopy. S4. Comparing the differences in uptake under N3+ and N3- conditions demonstrates "DBCO-N3 click-mediated targeted adhesion and uptake" (see...). Figure 9 ).

[0085] Figure 9 a (Confocal imaging): Comparison of N3- (unlabeled by azide metabolism) and N3+ (expressed by azide groups via Ac4ManNAz, etc.) tumor cells, showing the difference in cell membrane / intracellular fluorescence distribution after the addition of nLNMH with DBCO functional group and fluorescent label.

[0086] Figure 9 b (Quantitative fluorescence intensity): For Figure 9 The intracellular fluorescence signal of a was statistically quantified (mean ± standard deviation, n=3), *** indicates p <0.001.

[0087] like Figure 9 As shown, the N3- cell group showed only weak non-specific background fluorescence, while the N3+ cell group showed significantly enhanced fluorescence signal, with the signal more concentrated at the cell membrane periphery and obvious intracellular distribution; quantitative results ( Figure 9 b) The intracellular fluorescence intensity of the N3+ group was significantly higher than that of the N3− group.

[0088] The above results indicate that the DBCO functional group introduced into the outer layer of nLNMH can undergo a copper-free SPAAC click reaction with the N3 group on the cell surface, thereby achieving click-mediated adhesion enrichment and enhanced uptake. This "pre-targeting-click coupling" mechanism can significantly improve the uptake efficiency of tumor cells, providing in vitro evidence for subsequent in vivo tumor enrichment and efficacy enhancement.

[0089] Example 12: Flow cytometry detection of DC maturation, including the following steps: S1. Obtain bone marrow-derived dendritic cell (BMDC) lines and treat them with PBS control, nLNP, nLNM, nLNH, nLNMH, etc., according to the experimental groups; S2. After processing for a certain period of time, cells were collected and antibody staining was used to detect DC maturation markers; S3. Flow cytometry was used to acquire data and analyze the expression levels of maturation-related molecules such as CD80 / CD86 to evaluate the APC activation and immune-enhancing capabilities of each group. (See...) Figure 10 ) Figure 10 a (flow cytometry): Maturation markers (CD80, CD86) are detected in BMDC, and the expression distribution of CD80 / CD86 in different treatment groups is displayed as a scatter plot (commonly a four-quadrant or two-parameter plot of CD80 vs CD86).

[0090] Figure 10 b (Quantitative Statistics): Quantifying maturity-related indicators (e.g., CD80) + CD86 + The proportion of double positives (or MFI of CD80 / CD86) was compared, and the differences between PBS, nLNP, nLNM, nLNH, and nLNMH were compared.

[0091] like Figure 10 As shown: the overall maturation level of the PBS and nLNP (empty vector) groups was low, reflecting the limited stimulation of the basal state or the vector itself; the nLNM group containing MSA-2 prodrug showed an increase in maturation-related indicators, suggesting that the release of the STING agonist promoted DC maturation; the nLNH group (containing only Hsc70 mRNA) showed some changes, but the overall maturation range was generally weaker than that of the group containing immune agonists; the nLNMH group showed the most significant upregulation of maturation indicators (reflected in the figure by a higher double-positive rate / MFI and significance markings), indicating that the combined delivery of "immune agonist + nucleic acid regulatory factor" is more conducive to APC activation and immune enhancement.

[0092] The results demonstrate that the formulation of this invention can promote the maturation of dendritic cells (DCs) and enhance antigen presentation-related functions, thus supporting its application in "enhancing the efficacy of immunotherapy" from an immune mechanism perspective; it also aligns with the design goal of this invention of "releasing an immune agonist from the outer layer first to initiate innate immunity".

[0093] Example 13: Western blot detection of intracellular Hsc70 protein expression after treatment with different doses of Hsc70 mRNA, including the following steps: S1. Obtain the cell lines to be tested and seed them for culture. The control group and different doses of Hsc70 mRNA treatment groups (e.g., 0.25 μg, 0.5 μg, 1 μg, 2 μg, 4 μg) were given according to the experimental groups. S2. After processing for a certain period of time, cells were collected, lysed, and total protein was extracted, followed by SDS-PAGE electrophoresis and membrane transfer. S3. Immunoblot detection was performed using anti-Hsc70 primary antibody and anti-β-Actin primary antibody. The band grayscale was analyzed, and the β-Actin-normalized Hsc70 expression level was used to evaluate the protein expression effect of different doses of Hsc70 mRNA. (See...) Figure 11 ) like Figure 11 As shown, the intensity of the Hsc70 protein band in cells changed accordingly after treatment with different doses of Hsc70 mRNA, suggesting that mRNA delivery can effectively upregulate Hsc70 expression; this result was used to determine the effective dose range of mRNA required for subsequent cell and animal experiments.

[0094] Example 14: Detection of PD-L1 expression by flow cytometry, including the following steps: S1. Obtain the cell lines to be tested, and treat them with PBS control, nLNP, nLNM, nLNH, nLNMH, etc. according to the experimental groups; S2. After processing for a certain period of time, cells were collected and stained with anti-mouse PD-L1-PE by flow cytometry. S3. Flow cytometry was used to acquire and analyze PD-L1 expression levels (positive rate and / or MFI) to evaluate the regulatory effects of each group on PD-L1, a molecule related to tumor cell immune escape. (See...) Figure 12 ) Figure 12 a (PD-L1 flow cytometry results): Displays the changes in the proportion of PD-L1 positive groups in each group.

[0095] Figure 12 b (Quantitative statistics): Intergroup comparisons were performed for PBS, nLNP, nLNM, nLNH, and nLNMH.

[0096] like Figure 12 As shown, the positive rate of PD-L1 after treatment with different formulations showed differences between groups: the group containing MSA-2 prodrug (nLNM) showed an upregulation trend of PD-L1, which is more common when immune activation is accompanied by enhanced type I interferon signaling, and can be regarded as an indirect manifestation of "innate immunity being activated"; the group containing Hsc70 mRNA (nLNH) showed an inhibitory trend of PD-L1, suggesting that nucleic acid regulatory factors have an intervention effect on the immune escape-related axis; the PD-L1 expression level of the nLNMH group showed a comprehensive effect: while preserving immune activation, it regulated the PD-L1-related drug resistance axis, making the overall expression level different from that of a single group.

[0097] This result serves to demonstrate that the present invention can not only "initiate immunity" but also further regulate the "immune escape axis (PD-L1)," thereby providing key cellular evidence for its synergistic effect and tolerance reversal in immune checkpoint blockade (ICIs) therapy.

[0098] Example 15: In vitro safety evaluation of cytotoxicity, including the following steps: S1. Tumor cells were seeded into 96-well plates, and after adhesion, different concentrations of nLNP (empty vector), nLNM, nLNH, and nLNMH were added and incubated for 24–48 h. S2. MTT assay for cell viability, calculation of survival rate, and assessment of biocompatibility (see...) Figure 13 ).

[0099] like Figure 13 As shown, within the set concentration range, the changes in cell viability after treatment with each formulation were used to comprehensively evaluate the in vitro safety and biocompatibility of the system; the results indicate that the nested lipid nanoparticles of the present invention have a good safety window.

[0100] Example 16: Evaluation of in vivo biodistribution and tumor targeting ability (with / without glucose labeling), including the following steps: S1.4T1 cells were inoculated into the right leg of BALB / c mice to establish a subcutaneous tumor model. The mice were randomly divided into two groups (n=3 per group). S1-1. Establish a tumor model and begin azide metabolism labeling when the tumor volume reaches a preset range (80–120 mm³).

[0101] S1-2. Preparation of azidosaccharide for drug delivery: Weigh Ac4ManNAz, dissolve it with a small amount of DMSO, and then dilute it with sterile PBS to the working solution; control the volume fraction of DMSO in the final preparation to ≤5% to reduce local irritation.

[0102] S1-3. Route of administration and dosage (intratumoral injection regimen, preferred): Metabolic markers are performed by intratumoral (it) injection; single injection volume is 50 μL / tumor; the dosage of Ac4ManNAz can be set to 1 mg / tumor.

[0103] S1-4. Dosage frequency and time interval: once daily for 2 consecutive days; each injection is approximately 24 hours apart.

[0104] S1-5. Time interval with nLNP injection: Cy7-nLNMH was injected intravenously 12 hours after the last Ac4ManNAz intratumoral injection for in vivo click pre-targeting enrichment and imaging evaluation.

[0105] S1-6. Unlabeled control (N3-) setup: Mice in the unlabeled control group were treated with the same injection frequency and volume, but the injection solution was a carrier solution without Ac4ManNAz (DMSO+PBS in the same ratio); this was used to exclude differences caused by intratumoral injection operation and solvent.

[0106] S2. Cy7-labeled nLNMH was used to set up two groups: a sugar-free labeling group (direct injection of Cy7-nLNMH) and a sugar-labeled group (intratumoral injection of Ac4ManNAz metabolic marker to form N3 label before injection of Cy7-nLNMH); 100 μL was injected intravenously into each animal. S3. Live imaging was performed at 0, 2, 4, 6, 8, 10, 12, 24, and 48 hours. S4. 48 h of in vitro imaging evaluation of tumor and major organs to assess distribution (see...) Figure 14 ).

[0107] Figure 14 a / 14b (In vivo imaging and quantification): Fluorescence distribution of Cy7-labeled nLNMH in mice at different time points (0–48h); comparison of the sugar-free group (without Ac4ManNAz metabolic labeling) and the sugar-labeled group (Ac4ManNAz pretreatment), and statistical analysis of tumor fluorescence intensity. Figure 14 c / 14d (Ex vivo organ imaging and quantification): 48 h. Tumors and major organs (heart, liver, spleen, lung, kidney, etc.) were collected for ex vivo imaging, and the fluorescence intensity of tumors and each organ was statistically analyzed to show the contrast between "tumor enrichment" and "non-target distribution".

[0108] like Figure 14 As shown, compared with the sugar-labeled group, the sugar-labeled group (Ac4ManNAz metabolic label) showed stronger fluorescence signals in the tumor region and maintained for a longer time; in vitro imaging and quantitative results further showed enhanced fluorescence signals in tumor tissue and increased signal-to-weight ratios in tumors and major organs.

[0109] These results demonstrate that a two-step strategy of "Ac4ManNAz pre-labeling tumor + nLNMH intravenous injection" can achieve click-mediated pre-targeted enrichment in vivo, thereby improving the localization and retention of nLNPs at the tumor site and providing a basis for reducing the risk of off-target accumulation and expanding the therapeutic window. This is consistent with the "click-targeting-gradual release" integrated strategy proposed in this invention. Example 17: In vivo antitumor effects of nanomaterials, including the following steps: S1. 4T1 cells were inoculated into the right leg of BALB / c mice to establish a subcutaneous tumor model; S2. The tumor mice used for treatment were randomly divided into 5 groups (n=5 per group) and injected intravenously with 100 μL of physiological saline, nLNP, nLNM, nLNH and nLNMH respectively, once every other day for a total of 3 times. S3. Measure the tumor size every other day. The formula for calculating tumor volume (V) is V=W 2 ×L / 2 (W and L are the shortest and longest diameters of the tumor). (See...) Figure 15 ) like Figure 15 As shown, the tumor volume of mice in different treatment groups changed differently over time. The nLNMH group showed a more significant tumor inhibition trend compared with the control and single-component formulation groups, indicating that the stepwise synergistic strategy of "releasing immune agonists first and then releasing mRNA regulatory factors" of the present invention can be used to improve the overall anti-tumor efficacy.

[0110] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

[0111] This invention is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A nested lipid nanoparticle, characterized in that, include: Inner lipid nanoparticles: contain ionized lipids, auxiliary lipids, cholesterol and PEG lipids with azide groups. The inner lipid nanoparticles may or may not contain Hsc70 mRNA, and their surface contains azide groups. Outer lipid shell: Contains dibenzocyclooctylene functional group lipids and pH-responsive lipids, and may or may not be loaded with the STING agonist MSA-2 prodrug; In this process, the azide groups on the surface of the inner lipid nanoparticles and the dibenzocyclooctyn functional groups on the surface of the outer lipid shell are covalently linked through a copper-free bioorthogonal click reaction, enabling the nested lipid nanoparticles to have the ability to click-target azide-labeled tumor cells; the outer lipid shell preferentially dissociates in the acidic tumor microenvironment and achieves stepwise release.

2. The nested lipid nanoparticles according to claim 1, characterized in that, Depending on the active ingredient contained therein, the nested lipid nanoparticles include at least the following formulations: (1) nLNP empty vector: The inner layer does not carry Hsc70 mRNA and the outer layer does not carry the STING agonist MSA-2 prodrug; (2) nLNM: The outer layer carries the STING agonist MSA-2 prodrug and the inner layer does not carry Hsc70 mRNA; (3) nLNH: The inner layer carries Hsc70 mRNA and the outer layer does not carry the STING agonist MSA-2 prodrug; (4) nLNMH: The outer layer carries the STING agonist MSA-2 prodrug and the inner layer carries Hsc70 mRNA.

3. The nested lipid nanoparticles according to claim 1 or 2, characterized in that, The inner lipid nanoparticles comprise ionized lipid SM102, auxiliary lipid DOPE, cholesterol, and DMG-PEG-N3, with a molar ratio of (40–60):(5–20):(30–50):(0.5–5).

4. The nested lipid nanoparticles according to claim 1 or 2, characterized in that, The nitrogen and phosphorus equivalent ratio of the ionized lipids to Hsc70 mRNA is 4–10:

1.

5. The nested lipid nanoparticles according to claim 1 or 2, characterized in that, The outer lipid shell contains egg yolk lecithin, cholesterol, DSPE-PEG-DBCO and DSPE-PEOz; wherein the molar ratio of egg yolk lecithin, cholesterol, DSPE-PEG-DBCO and DSPE-PEOz is (20–50):(1–15):(1–15):(1–15).

6. The nested lipid nanoparticles according to claim 1 or 2, characterized in that, The mass ratio of the STING agonist MSA-2 prodrug to the outer lipid shell is 1:(10–25).

7. The nested lipid nanoparticles according to claim 1 or 2, characterized in that, The molar ratio of the inner lipid nanoparticles to the outer lipid shell is 1:(1–3).

8. A method for preparing nested lipid nanoparticles according to claim 1, characterized in that, Includes the following steps: S1. Preparation of inner lipid nanoparticles: Ionized lipids, auxiliary lipids, cholesterol and PEG lipids with azide groups were dissolved in ethanol to form an alcohol phase; Hsc70 mRNA or empty vector buffer was dissolved in acidic buffer to form an aqueous phase; the aqueous phase was added to the alcohol phase and mixed to self-assemble, resulting in a dispersion of inner lipid nanoparticles with azide groups on the surface. S2. Preparation of outer lipid alcohol phase: Dissolve the outer lipid material containing dibenzocyclooctylene functional group lipid and pH-responsive lipid in ethanol to form the outer lipid alcohol phase, and add the STING agonist MSA-2 prodrug as needed; S3. Constructing a nested structure: Using the inner lipid nanoparticle dispersion as the aqueous phase, it is added to the outer lipid alcohol phase and mixed to complete the self-assembly of the outer lipid shell. At the same time, the SPAAC click reaction occurs to form nested lipid nanoparticles. S4. Purification: Remove ethanol and free components by dialysis or equivalent methods to finally obtain nested lipid nanoparticles.

9. The preparation method according to claim 8, characterized in that, The acidic buffer solution mentioned in step S1 is a 10–50 mM citrate buffer solution with a pH of 3.5–4.

5.

10. The use of the nested lipid nanoparticles as described in claim 1 or the nested lipid nanoparticles prepared by the preparation method of claim 8 in the preparation of a drug or pharmaceutical composition for enhancing the efficacy of tumor immunotherapy.

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