Spatiotemporal controllable light adjuvant lipid nanoparticles and preparation and application thereof

Abstract

The application provides a spatiotemporally controllable light adjuvant lipid nanoparticle and preparation and application thereof, the light adjuvant lipid nanoparticle contains a light adjuvant molecule ICG-CHOL formed by covalent coupling of indocyanine green and cholesterol through an ester bond, and is incorporated at a specific molar ratio of 3%, while keeping uniform particle size, encapsulation efficiency > 90% and low cytotoxicity, and imparting light response function. By accurately controlling the light conditions, mild induction of active oxygen production is achieved by irradiation at a power density of 0.5 W for 1.5 minutes at 6 hours after administration, which synergistically activates the STING and TLR7 / 8 signaling pathways, promotes the maturation and activation of dendritic cells and antigen cross-presentation. The light adjuvant lipid nanoparticle combined with anti-PD-1 antibody achieves a complete remission rate of 37.5% in a melanoma model, providing a new strategy for balancing the immune activation intensity and safety of mRNA vaccines.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a spatiotemporally controllable photoadjuvant lipid nanoparticle and its preparation and application. Background Technology

[0002] mRNA vaccines, as an emerging immunotherapy strategy, have shown great potential in the field of tumor immunotherapy due to their advantages such as short development cycles, rapid personalization, and the ability to simultaneously induce humoral and cellular immunity. In recent years, breakthroughs in lipid nanoparticle (LNP) delivery technology have significantly improved the in vivo delivery efficiency and stability of mRNA vaccines, laying an important foundation for the clinical translation of tumor vaccines. However, the development of existing mRNA tumor vaccines still faces two major challenges: first, how to balance the intensity of immune activation with safety; and second, how to improve the efficiency of mRNA escape from the endosome to the cytoplasm, thereby promoting antigen cross-presentation.

[0003] Traditional mRNA vaccine delivery systems, such as the commercially available LNP, primarily rely on the protonation of ionizable lipids in the acidic environment of the endosome, leading to electrostatic interactions with the endosome membrane and inducing membrane instability, thereby releasing mRNA into the cytoplasm. However, this process is inherently passive and uncontrollable, with less than 5% of internalized mRNA successfully escaping the endosome, significantly limiting the efficiency of antigen translation and expression and the intensity of immune activation. To enhance the immune response, existing technologies typically employ a strategy of combining immune agonists (such as Toll-like receptor agonists and STING agonists). However, these immune agonists exhibit non-specific distribution in vivo, easily triggering excessive inflammatory responses in non-target tissues and organs, leading to serious safety risks such as cytokine storms. This makes striking a balance between efficacy and safety a core challenge in mRNA vaccine design.

[0004] To address the aforementioned issues, researchers have recently begun exploring strategies to regulate vaccine activity using exogenous physical stimuli (such as light and ultrasound). Photosensitive adjuvants have attracted widespread attention due to their excellent spatiotemporal controllability. Existing technologies have attempted to introduce photosensitizers into vaccine systems, for example, by inducing immunogenic cell death in tumor cells through photothermal effects, or by co-delivering photosensitizers with antigens to enhance dendritic cell activation. However, current photosensitive vaccine technologies still have the following limitations: First, at the materials design level, most existing studies simply mix photosensitizing molecules as independent adjuvant components with vaccines, or directly modify key functional components of LNPs (such as ionizable lipids). The former easily leads to separation of the photosensitizer from the delivery carrier, resulting in uncontrollable in vivo behavior; the latter, because even small changes in the structure and ratio of ionizable lipids can significantly affect the in vivo escape efficiency and targeting of LNPs, poses a high risk of delivery failure. Currently, there is a lack of an engineering strategy that can both endow LNPs with photosensitivity and retain their original high-efficiency delivery performance to the maximum extent.

[0005] Second, at the level of mechanism of action, existing research lacks sufficient understanding of the dose-response relationship of light-controlled immune activation, failing to fully explore the potential of photosensitizers as immune adjuvants rather than killing agents under mild light conditions. The relationship between light intensity and immune activation is not a simple positive correlation: excessively weak light cannot effectively stimulate an immune response, while excessively strong light may directly kill immune cells or induce excessive inflammation, which is detrimental to the establishment of specific anti-tumor immunity. Current technologies lack systematic research and parameter optimization on the finely regulated windows of mildly inducing reactive oxygen species, promoting endosome escape, and activating innate immune signaling pathways.

[0006] Third, at the in vivo application level, existing research on light-controlled vaccines largely focuses on local injection into tumors. There is a lack of systematic in vivo kinetic studies and optimization of light parameters regarding how to precisely and spatiotemporally activate the vaccine at the key immune initiation site of lymph nodes after subcutaneous immunization. Furthermore, the synergistic effect of light-controlled vaccines with immune checkpoint inhibitors (such as anti-PD-1 antibodies) and their systemic remodeling mechanisms of the tumor immune microenvironment still require further investigation.

[0007] Therefore, there is an urgent need in this field to develop a spatiotemporally controllable mRNA vaccine delivery system. This system should maintain the high efficiency of LNP delivery while precisely controlling light parameters to achieve mild and controllable immune activation in antigen-presenting cells within lymph nodes, thereby maximizing the anti-tumor immune response while ensuring safety. Summary of the Invention

[0008] To address the aforementioned issues, this invention provides spatiotemporally controllable photoadjuvant lipid nanoparticles, their preparation, and applications. These lipid nanoparticles contain the photoadjuvant molecule ICG-CHOL, formed by the covalent coupling of indocyanine green and cholesterol via ester bonds, incorporated at a specific molar ratio of 3%. This process imparts photoresponsiveness while maintaining uniform particle size, encapsulation efficiency >90%, and low cytotoxicity. By precisely controlling illumination conditions, irradiation at a power density of 0.5W for 1.5 minutes 6 hours after application can mildly induce reactive oxygen species production, synergistically activating the STING and TLR7 / 8 signaling pathways, promoting dendritic cell maturation and antigen cross-presentation. This photoadjuvant lipid nanoparticle, combined with an anti-PD-1 antibody, achieved a complete remission rate of 37.5% in a melanoma model, providing a new strategy for balancing the immune activation intensity and safety of mRNA vaccines.

[0009] On one hand, the present invention provides a photoadjuvant-modified cholesterol compound, wherein the photoadjuvant-modified cholesterol compound comprises a conjugate formed by a photosensitizer and cholesterol linked by a covalent bond; the photosensitizer comprises one or more of indocyanine green, IR-780, IR-820, dihydroporphyrin E6, pyromethesin a, zinc phthalocyanine, and methylene blue; the covalent bond comprises one or more of ester bond, amide bond, disulfide bond, thioether bond, thioketal bond, carbon ester bond, amino ester bond, hydrazone bond, and acylhydrazine bond.

[0010] This invention is based on the mechanism by which photosensitizers can generate reactive oxygen species and exert immunomodulatory effects under suitable light conditions. It covalently anchors photosensitizers, which are light-responsive substances, to the cholesterol backbone to construct novel photoadjuvant-modified cholesterol compounds. These compounds are then incorporated into LNPs to build a universal platform technology. This expands photosensitizers from traditional photodynamic tools into novel compounds with precisely tunable immune adjuvant functions, enabling spatiotemporal control of LNPs and inducing more potent immune responses.

[0011] Theoretically, any photosensitizer can be used to construct photoadjuvant-modified cholesterol compounds, thereby achieving spatiotemporal controllability of LNPs. Any covalent bond, as long as it allows the photosensitizer to couple with cholesterol, can also be used to construct photoadjuvant-modified cholesterol compounds.

[0012] Our research team previously discovered that conjugating cholesterol to drugs to replace part of the cholesterol-doped liposomes can achieve dual functions as drug delivery and vaccine adjuvants, and applied for an invention patent (CN2025112082532). Building on this, this application applies the method to prepare conjugates of photosensitizers and cholesterol, thereby replacing part of the cholesterol-doped lipid nanoparticle delivery carriers. This allows for spatiotemporal control of mRNA release from lipid nanoparticles and also helps improve therapeutic efficacy.

[0013] Furthermore, the photosensitizer is indocyanine green; the indocyanine green is linked to cholesterol via an ester bond to form a conjugate.

[0014] Indocyanine green is an FDA-approved near-infrared photosensitizer for clinical use. It has an excellent safety record and good biocompatibility. Its near-infrared fluorescence properties enable vaccines to be non-invasively traced in vivo, and can monitor the distribution dynamics of nanoparticles in lymph nodes in real time, providing a visual basis for accurately determining the 6-hour illumination window.

[0015] Ester bonds, as a type of linkage that is relatively stable under physiological conditions but can be slowly hydrolyzed by intracellular esterases, ensure the structural integrity of photoadjuvant molecules during delivery while avoiding the risk of in vivo accumulation that may be caused by non-degradable linkages. Compared to other linkage methods, ester bond synthesis offers milder conditions, higher yields, fewer side reactions, and faster metabolism, making it feasible for large-scale production. Therefore, the combination of indocyanine green and ester bonds not only meets the functional requirements of photocontrolled immune activation but also addresses the multiple requirements of clinical translation for safety, traceability, and large-scale production, making it the preferred solution of this invention.

[0016] Furthermore, the structural formula of the conjugate formed by the indocyanine green and cholesterol linked by an ester bond is shown in formula (1): Equation (1) On the other hand, the present invention provides a spatiotemporally controllable lipid nanoparticle, wherein the lipid nanoparticle comprises a photoadjuvant-modified cholesterol compound as described above.

[0017] Integrating photoadjuvant-modified cholesterol compounds into lipid nanoparticle systems can achieve compatibility and synergistic effects between photocontrol functions and LNP platform performance.

[0018] This invention employs a doping strategy, replacing a portion of ordinary cholesterol with the photoadjuvant molecule ICG-CHOL in a specific ratio, rather than directly modifying key components such as ionizable lipids in liposomes. This approach endows LNPs with photoresponsive functionality while maximally preserving their core properties such as high encapsulation efficiency, uniform particle size, low cytotoxicity, and efficient delivery.

[0019] More importantly, the intracellular transport pathway of LNP, as a delivery carrier, is highly compatible with the spatial requirements for the action of photoadjuvants. LNP precisely delivers ICG-CHOL to the lysosomes and endoplasmic reticulum of dendritic cells, where reactive oxygen species generated by light act precisely on these key signaling organelles, synergistically activating the STING and TLR7 / 8 pathways. This spatial coupling design of carrier function and light-controlled function allows the photoactivated immune enhancement effect to occur precisely within antigen-presenting cells, rather than through non-specific distribution throughout the body. This enhances efficacy while reducing systemic toxicity, laying a platform foundation for the clinical translation of light-controlled mRNA vaccines.

[0020] Furthermore, the lipid nanoparticles also contain ionizable cationic lipids, cofactor phospholipids, polyethylene glycol-modified lipids, and mRNA.

[0021] It is understandable that any type of ionizable cationic lipid, cofactor phospholipid, PEGylated lipid, and any mRNA can be used to construct spatiotemporally controllable lipid nanoparticles by doping with ICG-CHOL.

[0022] Furthermore, the molar percentage of the photoadjuvant-modified cholesterol compound in the total cholesterol of the spatiotemporally controllable lipid nanoparticles is 3%-9%.

[0023] The photoadjuvant molecule ICG-CHOL is not simply added but rather replaces part of the cholesterol. Its ratio needs to be precisely controlled within a narrow window that can impart sufficient photosensitivity without interfering with the assembly and delivery function of LNP itself.

[0024] Limiting the molar proportion of photoadjuvant-modified cholesterol compounds to 3%-9% facilitates a precise balance between photoresponsiveness and the core delivery performance of lipid nanoparticles. Studies have shown that the incorporation ratio of photoadjuvant molecules exhibits a non-linear relationship with transfection efficiency: low incorporation promotes high incorporation inhibits high incorporation. When the incorporation ratio is below 3%, the density of photosensitive molecules is insufficient to generate enough reactive oxygen species to effectively promote endosome escape and immune activation under light irradiation. Conversely, when the incorporation ratio exceeds 9%, excessive cholesterol substitution disrupts the structural integrity of the lipid bilayer, leading to decreased encapsulation efficiency, particle size inhomogeneity, and significantly increased cytotoxicity, ultimately weakening mRNA translation and expression efficiency. Therefore, the 3%-9% range falls precisely within the optimal overlap window, with 3% representing the optimal balance point after systematic screening (transfection efficiency comparable to commercially available lipid materials), while extending upwards to 9% retains flexibility for process adjustments under different application scenarios.

[0025] On the other hand, the present invention provides a spatiotemporally controllable mRNA vaccine, wherein the light-responsive mRNA vaccine comprises, as described above, a light-adjuvant modified cholesterol compound, or as described above, light-responsive lipid nanoparticles, and the spatiotemporally controllable mRNA vaccine is taken up by antigen-presenting cells and then released in a controlled manner using light.

[0026] Traditional mRNA vaccines rely on passive processes for antigen expression and immune activation (endosome escape efficiency <5% and uncontrollable), while the spatiotemporally controllable vaccine of this invention is endowed with precise spatiotemporal controllability by doping LNP with photoadjuvant-modified cholesterol compounds.

[0027] The photoadjuvant molecule provided by this invention is anchored to the LNP structure rather than being freely mixed, ensuring that the photoresponsive function is delivered synchronously to the antigen-presenting cells along with the vaccine. Through a pre-screened light window (6 hours), illumination occurs precisely at the critical stage after the nanoparticles have completed endocytosis and transport to lysosomes / endoplasmic reticulum. A mild illumination condition of 0.5W / 1.5min was selected to induce appropriate levels of reactive oxygen species to promote endosome escape and signaling pathway activation, rather than directly killing cells. This design confines the immune adjuvant effect to the antigen-presenting cells that have already taken up the vaccine, avoiding the risks of systemic immune overactivation and cytokine storms caused by non-specific drug distribution in traditional combined agonist strategies. More importantly, through the exogenous physical stimulus of light, the intensity of immune activation is controllable in terms of time (determining when to activate), space (determining activation within lymph nodes), and dosage (controlling activation intensity by adjusting light parameters), providing a precise solution for balancing the efficacy and safety of mRNA vaccines.

[0028] Furthermore, this invention provides a method for using a spatiotemporally controllable mRNA vaccine, the method comprising the following steps: (a) Delivering a spatiotemporally controlled mRNA vaccine as described above to antigen-presenting cells; (b) After the mRNA vaccine is taken up by the antigen-presenting cells, it is exposed to light.

[0029] This invention limits the use of mRNA vaccines to a two-step method: delivery first, followed by ingestion and then light exposure. The core advantage of this design is that it achieves precise control of immune activation in the time dimension, completely changing the uncontrollable mode of traditional vaccine activation upon injection.

[0030] Traditional mRNA vaccines initiate antigen expression and immune response immediately after injection. The timing, intensity, and duration of immune activation are entirely determined by the formulation itself and cannot be adjusted according to individual differences or treatment needs. The method of this invention, however, separates vaccine delivery from immune activation, providing a new regulatory dimension for clinical intervention: After step (a), the vaccine enters antigen-presenting cells but does not exert its maximum adjuvant effect immediately; once cellular uptake reaches its peak (determined to be 6 hours by kinetic studies), the photosensitive molecules have precisely located within key signaling organelles such as lysosomes and endoplasmic reticulum. Activation is then triggered by light exposure in step (b).

[0031] This sequential design of first accumulating and then activating ensures, on the one hand, that the photoadjuvant effect occurs precisely within the antigen-presenting cells where the vaccine is already in place, avoiding non-specific dissipation of the photosensitizer in systemic circulation due to premature activation; on the other hand, it provides clinicians with a decision window, allowing them to flexibly decide whether to administer light and which light parameters to select based on the patient's real-time condition (such as the presence of early adverse reactions, changes in tumor burden, etc.), thus achieving individualized regulation of immune activation intensity. This method upgrades mRNA vaccines from a fixed formulation that can be injected once to a precisely controlled system that can be activated on demand, which is the core methodological innovation that distinguishes this invention from existing technologies.

[0032] In some embodiments, the method includes the following steps: (a) Administering the spatiotemporally controlled mRNA vaccine as described above to subjects in need; (b) After administration, during the period when the mRNA vaccine is internalized by antigen-presenting cells and transported to lysosomes or endoplasmic reticulum, the lymph node region of the subject is exposed to light.

[0033] This invention precisely limits the timing of light exposure to the period when the mRNA vaccine is internalized by the antigen-presenting cell and transported to the lysosome or endoplasmic reticulum, and limits the light exposure site to the lymph node region. The core advantage of this design is that it achieves precise anchoring of immune activation in both time and space, fundamentally solving the technical problems of inaccurate and imprecise light control strategies in traditional light control strategies.

[0034] In terms of time, based on the intracellular dynamics revealed by previous confocal imaging, the nanoparticles complete the crucial transport from endocytic vesicles to lysosomes and endoplasmic reticulum exactly 6 hours after uptake. At this time, light irradiation ensures that reactive oxygen species (ROS) precisely act on signaling organelles, achieving synergistic activation of the STING pathway (located in the endoplasmic reticulum) and the TLR7 / 8 pathway (located in endosomes / lysosomes). If light irradiation is premature, the nanoparticles have not yet reached the target organelles; if light irradiation is delayed, some photosensitive molecules have already been effluxed or degraded.

[0035] In terms of spatial dimension, limiting light irradiation to the lymph node region is based on the distribution pattern verified by flow cytometry. After subcutaneous injection, the vaccine mainly accumulates in the draining lymph nodes and is selectively taken up by dendritic cells and macrophages. Local irradiation of lymph nodes can highly concentrate the immune activation effect on the sites where antigen-presenting cells are enriched, avoiding non-specific light damage and inflammatory risks to non-target tissues throughout the body (such as skin, muscles, liver, etc.).

[0036] Further, in step (b), the illumination is near-infrared light; and / or the timing of the illumination is 4-12 hours after the administration of the mRNA vaccine; and / or the parameters of the illumination are: power density 0.25-1 W, irradiation time 1-3 minutes.

[0037] By further limiting the illumination conditions to near-infrared light, a 4-12 hour window after application, a power density of 0.25-1W, and an irradiation time of 1-3 minutes, this multi-level parameter combination design makes spatiotemporal controllable a precisely executable quantitative technical solution, and achieves synergistic optimization in three dimensions: penetration depth, cell kinetic matching, and dose-effect safety window.

[0038] The near-infrared light was chosen based on the characteristic absorption peak of indocyanine green in this band (about 780 nm). Near-infrared light has the characteristics of large tissue penetration depth (up to several centimeters) and minimal damage to biological tissues. It can effectively penetrate the skin and subcutaneous tissue to act on antigen-presenting cells in deep lymph nodes, while avoiding DNA damage and phototoxicity that may be caused by ultraviolet or visible light.

[0039] The 4-12 hour light exposure window covers the complete dynamic process of nanoparticles being internalized by antigen-presenting cells and transported to lysosomes and endoplasmic reticulum (the optimal point is 6 hours). This ensures that the absorption rate differences that may exist between different individuals and injection sites still fall within the effective activation window, while also providing reasonable time flexibility for clinical operation.

[0040] The combination of 0.25-1W power density and 1-3 minutes of irradiation time was determined based on multi-gradient condition screening. Too low a power density (<0.25W or <1 minute) is insufficient to induce enough reactive oxygen species to promote endosome escape, while too high a power density (>1W or >3 minutes) will induce oxidative stress damage and apoptosis, weakening transfection efficiency. This parameter range can precisely achieve mild induction of reactive oxygen species, synergistic activation of the STING / TLR7 / 8 pathway, and promotion of antigen cross-presentation without damaging cells.

[0041] In some embodiments, in step (b), the light exposure is performed 6 hours after the application of the lipid nanoparticles.

[0042] Studies have shown that 6 hours of light exposure is the optimal time, at which point the nanoparticles have completed endocytosis, their co-localization with lysosomes reaches its peak, and they also show significant co-localization with the endoplasmic reticulum. The reactive oxygen species generated by light exposure can precisely act on key signaling nodes of the STING pathway (endoplasmic reticulum) and the TLR7 / 8 pathway (endosomes / lysosomes), maximizing the synergistic activation of the two pathways.

[0043] In some methods, the illumination parameters are: power density 0.5 W, illumination time 1.5 minutes.

[0044] The 0.5W / 1.5-minute illumination parameter was determined through a systematic screening of multiple gradient power and time combinations to find the optimal balance point. This condition can gently induce the production of reactive oxygen species, increase mRNA transfection efficiency by 3-4 times, and maintain cell viability above 90%. It avoids the shortcomings of low power in effectively promoting endosome escape, and also avoids the risks of oxidative stress damage and apoptosis caused by high power.

[0045] Furthermore, the present invention provides the use of the photoadjuvant-modified cholesterol compounds as described above, or the spatiotemporally controllable lipid nanoparticles as described above, or the mRNA vaccines as described above in the preparation of drugs for treating tumors.

[0046] Furthermore, the application includes the following steps: (a) Delivering the drug to antigen-presenting cells; (b) After the drug is taken up by the antigen-presenting cells, it is exposed to light.

[0047] Further, in step (b), the illumination is near-infrared light; and / or the timing of the illumination is 4-12 hours after the administration of the mRNA vaccine; and / or the parameters of the illumination are: power density 0.25-1 W, irradiation time 1-3 minutes.

[0048] In another aspect, the present invention provides a pharmaceutical composition comprising the mRNA vaccine and an immune checkpoint inhibitor as described above; the immune checkpoint inhibitor comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody.

[0049] This invention is based on the results validated using the B16-OVA melanoma mouse model system. Photoadjuvanted mRNA vaccine monotherapy effectively inhibits tumor growth, and when combined with anti-PD-1 antibody, it achieves a complete remission rate of 37.5%. Flow cytometry confirmed the activation of dendritic cells in lymph nodes and the presence of antigen-specific CD8 in the spleen. + Systemic immune remodeling involving T cell expansion, effector T cell infiltration in the tumor microenvironment, and M1 macrophage polarization.

[0050] In another aspect, the present invention provides the use of the spatiotemporally controllable mRNA vaccine described above, after light treatment, for preparing reagents that activate the STING and NF-κB signaling pathways and regulate dendritic cell function.

[0051] Molecular mechanism studies revealed that ICG-mVac-L (a spatiotemporally controllable mRNA vaccine after light treatment) regulates dendritic cell function by synergistically activating the STING and NF-κB signaling pathways. Western blotting results showed that under optimal light conditions, the phosphorylation levels of p-STING, p-TBK1, p-IRF3, p-NF-κB, and p-IRF7 increased, while ICG-mVac treatment alone without light treatment did not activate these pathways, confirming that the immune adjuvant effect is strictly dependent on light triggering. Transcriptomic analysis further confirmed from a global perspective that ICG-mVac-L treatment significantly enriched multiple pathways closely related to the initiation of innate and adaptive immune responses, including the NOD-like receptor signaling pathway, the Toll-like receptor signaling pathway, the cytoplasmic DNA sensing pathway, cytokine-cytokine receptor interactions, and antigen processing and presentation.

[0052] In another aspect, the present invention provides the use of the spatiotemporally controllable mRNA vaccine as described above, after light treatment, for preparing a reagent that promotes the differentiation of T cells into effector subsets, thereby enhancing the immune effect.

[0053] Furthermore, the promotion of T cell differentiation into effector subsets refers to inducing CD4+ differentiation. + or CD8 + T cells differentiate into Th1, Th17, or Tc2 cells.

[0054] Based on RNA-seq results, light treatment may have induced CD4 activation. + T cell differentiation into effector subtypes leaned towards Th1, Th2, and Th17 cell differentiation. Finally, co-incubation experiments of BMDCs and T cells showed that dendritic cells activated by ICG-mVac-L could promote CD4+ differentiation. + and CD8 + T cells differentiate into different subsets.

[0055] The present invention has the following beneficial effects: 1. A photoadjuvant molecular platform with both safety and functionality was constructed. This invention is the first to covalently couple the FDA-approved clinical photosensitizer indocyanine green (ICG-CHOL) with cholesterol via ester bonds, yielding the photoadjuvant molecule ICG-CHOL. This design offers three advantages: First, ICG-CHOL possesses excellent safety and biocompatibility, and its near-infrared fluorescence properties endow vaccines with non-invasive in vivo tracking capabilities; second, the ester bond is stable under physiological conditions and can be slowly hydrolyzed by intracellular esterases, avoiding the risk of in vivo accumulation of non-degradable linkages; third, using cholesterol as an anchoring group allows for doping modification of the four-component structure of the LNP, avoiding modification of ionizable lipid functional elements and maximizing the preservation of the LNP's efficient delivery performance.

[0056] 2. A golden ratio that balances photosensitivity and delivery efficiency has been established. By systematically investigating the effects of different incorporation ratios (1.5%-50%) on the physicochemical properties and transfection efficiency of LNP, the optimal range for the proportion of photoadjuvant molecules in total lipids was determined to be 3%-9%, with 3% being the best equilibrium point. This ratio range allows LNP to maintain excellent properties such as uniform particle size (approximately 100 nm), stable potential, encapsulation efficiency >90%, and cell viability >90%, while also endowing it with photoresponsiveness. The in vitro and in vivo transfection efficiency is comparable to that of commercial lipid material SM102.

[0057] 3. Precisely targets the treatment window for gentle activation rather than lethality. This study reveals for the first time the nonlinear relationship between light intensity and mRNA transfection efficiency, characterized by low promotion and high inhibition. It also identifies the optimal light window (a critical period for the internalization of nanoparticles by antigen-presenting cells and their transport to lysosomes / endoplasmic reticulum) and light parameters of 0.5W / 1.5 minutes. This parameter combination gently induces reactive oxygen species production, increasing transfection efficiency by 3-4 times while maintaining cell viability above 90%, achieving photocontrolled immune activation. This provides a quantifiable technical standard for the precise application of photoadjuvants in tumor immunotherapy.

[0058] 4. The molecular basis for the synergistic activation of dual signaling pathways was revealed. Through transcriptomics and Western blot validation, this study elucidates for the first time that under light-controlled activation conditions, ICG-mVac can synergistically activate the STING pathway (endoplasmic reticulum localization) and the TLR7 / 8 pathway (endosome / lysosome localization), promoting dendritic cell maturation and activation and antigen cross-presentation. This dual-pathway synergistic activation mechanism exhibits a stronger immune amplification effect compared to traditional single-pathway agonists, and its spatiotemporal control through light irradiation avoids the safety risks of non-specific systemic activation.

[0059] 5. Achieved systemic immune remodeling and significant anti-tumor effects. In a B16-OVA melanoma mouse model, the in vivo antitumor efficacy of a photoadjuvanted mRNA vaccine was validated: after subcutaneous injection, the vaccine effectively accumulated in draining lymph nodes and was selectively taken up by dendritic cells and macrophages; under optimal light conditions, the vaccine significantly inhibited tumor growth, achieving a complete remission rate of 37.5% when combined with an anti-PD-1 antibody. Mechanistic studies showed that the combination therapy reshaped antitumor immunity in multiple dimensions: early pDCs amplification and late cDC1s enrichment in lymph nodes; antigen-specific CD8 in the spleen. + T cells significantly proliferate and differentiate into effector memory phenotypes; CD8+ in the tumor microenvironment + Increased T-cell infiltration and enhanced function, macrophage polarization towards the M1 type, and cDC1s enrichment. Safety evaluation showed that this treatment regimen did not cause significant toxic damage to major organs.

[0060] In summary, this invention, through multi-dimensional synergistic innovation in material design, formulation optimization, parameter precision, mechanism elucidation, and efficacy verification, has successfully constructed a spatiotemporally controllable photoadjuvant mRNA vaccine platform. This provides a novel strategy for solving the core challenge of balancing the immune activation intensity and safety of mRNA tumor vaccines, and has significant clinical application value and commercial prospects. Attached Figure Description

[0061] Figure 1 The proton NMR spectrum of ICG-CHOL prepared in Example 1; Figure 2 The carbon spectrum of ICG-CHOL prepared in Example 1; Figure 3 This is the mass spectrum of ICG-NH-CHOL prepared in Example 1; Figure 4 This is a schematic diagram showing the particle size of the spatiotemporally controllable lipid nanoparticles prepared in Example 2; Figure 5 This is a schematic diagram of the potential of the spatiotemporally controllable lipid nanoparticles prepared in Example 2; Figure 6 This is a schematic diagram showing the nanoparticle encapsulation efficiency of the spatiotemporally controllable lipid nanoparticles prepared in Example 2; Figure 7 This is a cryo-electron microscopy image of the spatiotemporally controllable lipid nanoparticles prepared in Example 2; Figure 8 The images show fluorescence imaging of DC2.4, Hela, and 293T cells transfected with ICG-LNP at different molar ratios of EGFP mRNA prepared in Example 2 (n=3, indicating 3 replicates). Figure 9Comparison of D-fluorescein sodium fluorescence signal intensity in animals with ICG-LNP containing Luciferase mRNA at different molar ratios prepared in Example 2 (n=3). Figure 10 For the cellular toxicity assessment of ICG-LNP at different molar ratios in Example 2; Figure 11 This is a confocal imaging of ICG-mVac taken by BMDCs in Example 3; Figure 12 This is a schematic diagram of the light condition screening results in Example 3. In this diagram, a shows the comparison of EGFP protein expression after light exposure, with the control group being the light-protected group; b shows the comparison of ROS release efficiency after light exposure, with the control group being the light-protected group; c shows the effect of light exposure on cell viability; (n=3) Figure 13 This is a schematic diagram illustrating the effect of light irradiation on BMDC apoptosis in Example 3. Figure 14 This is a schematic diagram illustrating the effect of WB detection on the pyrolysis of BMDCs after illumination in Example 3; Figure 15 This is a schematic diagram of the results of photo-activated BMDCs in Example 3; Figure 16 In Example 3, ICG-mVac-L activates the immune signaling pathway, where a is the STING signaling pathway detected by Western blotting; b is the TLR7 / 8 signaling pathway detected by Western blotting. Figure 17 Example 3 shows the RNA-seq analysis of vaccine-induced anti-tumor immune pathways and gene expression characteristics. Figure a shows the KEGG pathway enrichment analysis results; the left figure compares the ICG-mVac vaccine group with the PBS control group, and the right figure compares the ICG-mVac-L group with the ICG-mVac group. The horizontal axis represents the fold enrichment. Figure b is a heatmap of differentially expressed genes, with columns representing different treatment groups (PBS, ICG-mVac, ICG-mVac-L), rows representing differentially expressed genes, and color gradients representing Z-score normalized expression levels (red for high expression, blue for low expression). Figure 18 This is a schematic diagram illustrating the effect of light-treated BMDCs on T cell subset differentiation in Example 3, where a represents the number of Th1, Th2, Th17, Tc1, Tc2, and Tc17 cells differentiated; and b represents the differentiation ratio of Th1, Th2, Th17, Tc1, Tc2, and Tc17 cells. Figure 19The distribution of ICG-mVac vaccine in lymph nodes in Example 4 (n=3) is shown in Figure a: ICG fluorescence imaging in mice after subcutaneous injection of ICG-mVac (n=3); and cell distribution in lymph nodes is shown in Figure b. Figure 20 For the screening of tumor-suppressing effects under light conditions in animals in Example 4, where a represents the drug administration and light exposure method; b represents the drug administration and light exposure regimen; c represents the tumor suppression and survival curves; and d represents the Tet ratio. + CD8 + The proportion of T cells; (n=5) Figure 21 This study evaluates the antitumor efficacy of ICG-mVac combined with αPD-1 antibody in Example 4, where a represents the administration and light exposure methods; b represents the treatment regimen; c represents the tumor suppression and survival curves; and d represents the individual tumor suppression curves (n=8). Figure 22 This section illustrates the effect of ICG-mVac on DC differentiation in lymph nodes in Example 4. Figure a shows the number of cDCs, pDCs, cDC1s, and cDC2s in the lymph nodes on day 9 of flow cytometry; figure b shows the number of cDCs, pDCs, cDC1s, and cDC2s in the lymph nodes on day 14 of flow cytometry; figure c shows a flow cytometry gating diagram of the proportion of cDC1s and cDC2s cells on day 14 of flow cytometry; and figure d shows a statistical graph of the proportion of cDC1s and cDC2s cells on day 14 of flow cytometry. (n=5) Figure 23 This section illustrates the effect of ICG-mVac on T cell subset apoptosis in lymph nodes in Example 4. Figure a shows a comparison of the proportion of apoptotic T cells in lymph nodes on day 9 of flow cytometry; figure b shows a comparison of the proportion of apoptotic T cells in lymph nodes on day 14 of flow cytometry; (n=5) Figure 24 This is a graph showing the results of T cell subset counts in the spleen in Example 4, where a represents CD3+. + T cell count; b represents CD4. + T cell count; c represents CD8. + T cell count; d represents the number of T cells. + CD8 + T cell count; e represents T cells. + CD8 + T cell percentage; f represents T cells. + CD8 + Schematic diagram of T cell proportional flow cytometry gating; flow cytometry data on day 14; (n=5). Detailed Implementation

[0062] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate understanding of the present invention and are not intended to limit it in any way. Unless otherwise stated, the experimental reagents, consumables and instruments used in the following embodiments are all commercially available products.

[0063] Example 1: Synthesis and Characterization of Photoadjuvant-Modified Cholesterol Compounds 1. Constructing a photoadjuvant-modified cholesterol compound ICG-CHOL using ester bonds 1.1 Synthesis of compound CHOL-2 The synthetic reaction formula for compound CHOL-2 is shown in formula (2): Equation (2) 1,6-hexanedithiol (130 mg, 0.87 mmol), 4-dimethylaminopyridine (DMAP, 2 equivalents), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 4 equivalents) were weighed and dissolved in 5 mL of dichloromethane (DCM), and stirred at room temperature for 15 minutes. CHOL-1 (2 equivalents) (CHOL-1 is a cholesterol succinate monoester, purchased from Bied Pharmaceuticals, CAS: 1510-21-0) was added, and the reaction was allowed to proceed overnight after the addition was complete. The reaction solution was washed three times with saturated NH4Cl solution, extracted with DCM, washed with saturated NaCl solution, dried, concentrated, and purified by an automated column chromatography system (petroleum ether:ethyl acetate = 1:1) to obtain CHOL-2 (412 mg, yield 83.3%).

[0064] Compound CHOL-2: 1H NMR (400 MHz, Chloroform- d ) δ 5.36 (dt, J = 3.5, 1.8Hz, 1H), 4.61 (tdd, J = 13.7, 7.5, 4.1 Hz, 1H), 2.87 (q, J = 7.0 Hz, 4H), 2.63 (t, J = 6.9 Hz, 2H), 2.54-2.46 (m, 2H), 2.34-2.27 (m, 2H), 1.98 (ddt, J= 20.4, 11.4, 4.3 Hz, 2H), 1.91-1.76 (m, 3H), 1.69-1.44 (m, 10H), 1.42-1.23(m, 9H), 1.18-1.06 (m, 6H), 1.04-0.89 (m, 10H), 0.86 (dd, J = 6.6, 1.8 Hz, 6H), 0.67 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 198.1, 177.9, 176.3, 174.4,171.5, 140.0, 139.7, 132.0, 131.8, 130.9, 130.3, 128.3, 127.8, 125.1, 124.9,122.8, 122.1, 77.5, 77.4, 77.2, 76.8, 74.6, 56.8, 56.3, 50.1, 42.4, 40.0,39.8, 39.6, 38.7, 38.2, 37.1, 36.9, 36.7, 36.3, 35.9, 32.0, 32.0, 31.7, 29.8, 29.8, 29.7, 29.5, 29.0, 28.9, 28.5, 28.4, 28.3, 28.1, 27.8, 27.6, 24.4, 24.0, 23.1, 23.0, 22.7, 21.1, 19.4, 18.8, 12.0. 1.2 Synthesis of compound ICG-CHOL (a conjugate of indocyanine green and cholesterol) The synthetic reaction formula for compound ICG-CHOL is shown in formula (3): Equation (3) Weigh CHOL-2 (50 mg, 0.08 mmol), add 1 mL of N-ethyldiisopropylamine (DIPEA) and 5 mL of N,N-dimethylformamide (DMF), and stir at room temperature for 15 minutes. Add indocyanine green (ICG, 0.25 equivalents, purchased from Shanghai Bid Pharmaceutical, model 2143933-81-5), and react overnight after the addition is complete. Reverse the solvent and purify by preparative thin-layer chromatography (DCM:MeOH=9:1) to obtain ICG-CHOL (18.52 mg, yield 63.6%).

[0065] Photoadjuvant molecule ICG-CHOL: 1H NMR (400 MHz, Chloroform- d ) δ 8.05 (t,J = 8.5Hz, 1H), 7.91 (dq, J = 12.7, 7.8, 6.0 Hz, 4H), 7.79 (d, J = 13.4 Hz, 1H),7.61-7.53 (m, 2H), 7.43 (dt, J = 15.1, 7.8 Hz, 4H), 6.62 (s, 2H), 6.27 (s,2H), 5.38-5.31 (m, 1H), 4.59 (ddt, J = 11.7, 7.5, 3.7 Hz, 1H), 4.26-4.10 (m,3H), 4.06 (dd, J = 9.3, 3.7 Hz, 1H), 3.80-3.60 (m, 2H), 3.48 (s, 4H), 3.01(s, 2H), 2.87-2.74 (m, 5H), 2.68 (dt, J = 12.3, 7.5 Hz, 1H), 2.59 (t, J = 7.0Hz, 2H), 2.41 (dd, J = 18.2, 3.7 Hz, 1H), 2.29 (d, J = 8.4 Hz, 4H), 2.13-1.71(m, 26H), 1.62-1.40 (m, 14H), 1.38-1.20 (m, 12H), 1.14-1.05 (m, 6H), 1.01-0.83 (m, 17H), 0.66 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 198.1, 171.5, 139.7,122.9, 77.5, 77.2, 76.8, 74.6, 56.8, 56.3, 50.1, 42.4, 39.9, 39.7, 38.7,38.2, 37.1, 36.7, 36.3, 35.9, 33.9, 32.0, 32.0, 29.8, 29.6, 28.9, 28.4, 28.3, 28.2, 27.9, 27.9, 24.7, 24.4, 24.0, 23.0, 22.7, 21.2, 19.5, 18.9, 12.0. 1.3 Structural Characterization The obtained product was analyzed by proton nuclear magnetic resonance (NMR) spectroscopy (NMR). 1 H NMR, carbon spectrum (13 Identified by C NMR, it was confirmed to be the target compound ICG-CHOL, whose structure is shown in formula (1), where the proton NMR spectrum is shown in [reference needed]. Figure 1 Carbon spectrum Figure 2 .

[0066] 2. Constructing a photoadjuvant-modified cholesterol compound ICG-NH-CHOL using amide bonds. The synthetic reaction formula for constructing ICG-NH-CHOL is shown in formula (4): Equation (4) The obtained product was analyzed by proton nuclear magnetic resonance (NMR) spectroscopy (NMR). 1 H NMR, carbon spectrum ( 13 Identified by C NMR, it was confirmed to be the target compound ICG-NH-CHOL, with the structure shown in formula (5), and the mass spectrum is shown in Figure 1. Figure 3 As shown. Equation (5) Example 2: Preparation and Screening of Lipid Nanoparticles with Different Doping Ratios of Photoadjuvants 2.1 Preparation of lipid nanoparticles Ionizable cationic lipids (SM102), distearate phosphatidylcholine (DSPC), cholesterol (CHOL), ICG-CHOL, and distearate phosphatidylethanolamine-polyethylene glycol 2000 (DMG-PEG2000) were precisely weighed according to different molar ratios (see Table 1). For example, when preparing 10 μg of mRNA-LNP with an incorporation ratio of 3%, the cationic lipid was 206.2804 μg, DSPC was 31.28994 μg, CHOL was 57.1802 μg, ICG-CHOL was 6.7271908 μg, and DMG-PEG2000 was 14.904648 μg. The nanoparticles were then dissolved in 20 μl of anhydrous ethanol as the organic phase. 10 μg of mRNA (EGFP mRNA or Luciferase mRNA; in this example, EGFP mRNA was used, purchased from Shenji Biotechnology, catalog number: 11011-CAP-1 (eGFP mRNA with N1-Me-pUTP (5'CAP))) was dissolved in 20 mM sodium citrate buffer (pH 4.0) as the aqueous phase. Under an N / P ratio of 6:1, the organic phase was rapidly added dropwise to the aqueous phase, and the mixture was vortexed for 30 seconds, resulting in self-assembly of nanoparticles. The organic solvent was removed by dialysis, and the mixture was concentrated by ultrafiltration to obtain lipid nanoparticles (ICG-LNP) with different photoadjuvant doping ratios.

[0067] Table 1. Formulations of lipid nanoparticles with different incorporation ratios (molar percentage) The incorporation ratio here refers to the molar percentage of photoadjuvant-modified cholesterol compounds in the total cholesterol of the lipid nanoparticles.

[0068] 2.2 Physicochemical property characterization For lipid nanoparticles with different photoadjuvant doping ratios, the particle size and potential were detected using dynamic light scattering, the encapsulation efficiency was determined using the Quant-iT RiboGreen kit, and the morphology was characterized by cryo-electron microscopy. Three replicates were set for each sample group. Results are as follows: Figures 4-7 As shown, where, Figure 4 This is a schematic diagram of the particle size detection results. Figure 5 This is a schematic diagram of the potential detection results. Figure 6 This is a schematic diagram of the encapsulation rate test results. Figure 7 This is a cryo-electron microscopy image of ICG-LNP.

[0069] The particle size of lipid nanoparticles (LNPs) can be used to assess vaccine manufacturing processes and influences their expression in vivo. Nanoparticles with excessively small diameters are easily cleared by the kidneys, while those with excessively large diameters tend to accumulate at the injection site and are less likely to circulate and reach the target site. Potential, on the other hand, primarily determines the stability and entry capability (binding to and escape from the cell membrane) of LNPs. These two factors are interrelated and jointly influence LNP encapsulation, stability, circulation, distribution, uptake, and ultimately, transfection efficiency. Therefore, precise control of particle size and potential is a core step in the rational design of LNPs.

[0070] Dynamic light scattering detection includes the particle size, potential, and polydispersity index (PDI) of lipid nanoparticles. Figure 4 and Figure 5 The lipid nanoparticles incorporating ICG-CHOL photoadjuvant molecules at a doping ratio of 9% or less (including 9%) have relatively uniform particle size and potential, and small potential changes, which is beneficial for cellular uptake.

[0071] The encapsulation efficiency of nanoparticles is one of the key indicators for ensuring adequate drug dosage. For example... Figure 6 As shown, most lipid nanoparticles have an encapsulation efficiency of over 80%, and lipid nanoparticles with an incorporation ratio of less than 9% have an encapsulation efficiency of over 90%, which is close to the encapsulation efficiency of the original formulation (unincorporated group).

[0072] In subsequent experimental analysis, the morphology of the cationic lipid-synthesized nanoparticles was characterized by cryo-electron microscopy (cryo-TEM) (Figure 7). The results showed that the lipid nanoparticles all exhibited a uniform size and a complete spherical morphology. Their morphological characteristics were highly consistent with the particle size distribution results measured by dynamic light scattering (DLS), further verifying the reliability of the previous experimental data.

[0073] In this embodiment, lipid nanoparticles ICG-NH-LNP were also prepared using ICG-NH-CHOL constructed with amide bonds. It was found that when the incorporation ratio was no more than 9%, it could also achieve a similar encapsulation efficiency as ICG-CHOL, and could also be used to prepare spatiotemporally controllable lipid nanoparticles.

[0074] 2.3 Screening of in vitro transfection efficiency Based on preliminary experiments, and considering both photosensitivity and physicochemical properties, three molar ratios of ICG-CHOL (3%, 6%, and 9%) were selected for subsequent transfection efficiency screening. Although a 1.5% incorporation ratio had no significant effect on LNP particle size, potential, and encapsulation efficiency, preliminary experimental results showed that its photosensitivity was weak, making it difficult to meet the requirements for subsequent photocontrolled immune activation. Therefore, this study focused on evaluating the in vitro and in vivo transfection efficiency of ICG-LNP within the 3%–9% incorporation ratio range to determine the optimal formulation that balances photosensitivity and delivery performance.

[0075] ICG-LNPs with different formulations of EGFP mRNA were transfected into DC2.4, HELA, and 293T cells, respectively, and observed under a fluorescence microscope after 24 hours. HELA, 293T, and DC2.4 cells in logarithmic growth phase were digested, counted, and then seeded at 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 μL in RPMI-1640 complete medium containing 10% fetal bovine serum into 96-well cell culture plates. Each well was incubated overnight at 37°C with 5% CO2 until cell confluence reached approximately 70%, ready for drug treatment. The LNP samples to be tested were diluted with RPMI-1640 medium to a final EGFP mRNA concentration of 1 μg / mL. The old medium in the 96-well plate was aspirated, and 100 μL of the diluted LNP solution was slowly added to each well along the well wall. After gentle vortexing, the plate was returned to the 37°C, 5% CO2 incubator for another 24 hours. The 96-well plate was then removed, and cell status and EGFP expression were observed under an inverted fluorescence microscope (excitation wavelength 488 nm, emission wavelength 509 nm). Each sample was tested in triplicate, and the experiment was repeated three times independently.

[0076] The results are as follows Figure 8The results showed that as the incorporation ratio of ICG-CHOL gradually increased from 3% to 25%, the proportion of EGFP-positive cells and the average fluorescence intensity in the three cell lines all showed a decreasing trend. Compared with the unincorporated group, 3% ICG-LNP still maintained a high transfection efficiency in all cell lines, comparable to the SM102 group; while the transfection efficiency of the 6% group decreased, and the 25% group decreased significantly. This result indicates that the incorporation of ICG-CHOL has a dose-dependent inhibitory effect on the in vitro transfection efficiency of LNP. The reasons may include: (1) the partial substitution of cholesterol by ICG-CHOL changes the composition ratio and fluidity of the lipid bilayer, which may affect the endosome escape efficiency; (2) the steric hindrance effect of ICG molecules may interfere with the interaction between ionizable lipids and endosome membranes; (3) high incorporation may affect the intracellular dissociation behavior of LNP and delay the cytoplasmic release of mRNA. Among them, the transfection efficiency of the 3% ICG-LNP group in the three cell lines was not significantly different from that of the commercial lipid material SM102-LNP group, while the 25% group was significantly reduced. Therefore, the preferred incorporation ratio is 3%.

[0077] This embodiment also investigated the in vitro transfection efficiency of ICG-NH-LNPs loaded with EGFP mRNA at different incorporation ratios. It was found that as the incorporation ratio of ICG-NH-CHOL gradually increased from 3% to 25%, the proportion of EGFP-positive cells and the average fluorescence intensity in the three cell lines also showed a decreasing trend, and effective in vitro transfection could be achieved. However, the transfection efficiency of the 3% ICG-NH-LNP group in the three cell lines was slightly weaker than that of the 3% ICG-LNP group and the unincorporated group. Therefore, it is more preferable to use ICG-CHOL to prepare lipid nanoparticles, which helps to improve the transfection efficiency.

[0078] 2.4 Screening for in vivo transfection efficiency ICG-LNPs with different formulations of Luciferase mRNA were subcutaneously injected into the groin area of ​​C57BL / 6 mice (mRNA dose 2 μg / mouse), and bioluminescent signals were detected by in vivo imaging system 6 hours later.

[0079] The results are as follows Figure 9 The results showed that the in vivo transfection results were highly consistent with the in vitro trend; the bioluminescent signal intensity at the injection site decreased sequentially with increasing ICG-CHOL incorporation. The bioluminescent signal in the 3% ICG-LNP group was closest to that in the SM102-LNP group, slightly decreased in the 6% group, and significantly weakened in the 25% group. These results confirm that the ICG-CHOL incorporation ratio is a key factor affecting the in vivo delivery efficiency of LNPs; high incorporation may ultimately weaken mRNA translation and expression by altering LNP in vivo stability, lymph node transport efficiency, or cellular uptake behavior.

[0080] In summary, the in vitro and in vivo transfection efficiency evaluation results show that the 3% ICG-CHOL incorporation ratio, while ensuring good photosensitivity, maximizes the preservation of the original LNP's high-efficiency delivery performance. Its transfection efficiency in various cell lines and bioluminescence intensity in mice are comparable to the commercial lipid material SM102-LNP formulation. Therefore, 3% ICG-LNP was selected as the optimal formulation for subsequent light condition optimization, immune regulation mechanism studies, and in vivo antitumor efficacy evaluation.

[0081] This embodiment also investigated the in vivo transfection efficiency of ICG-NH-LNPs loaded with Luciferase mRNA at different incorporation ratios. It was found that the preferred incorporation ratio was 3%, which could also achieve effective in vivo transfection. However, the transfection efficiency of the 3% ICG-NH-LNP group in mice was slightly weaker than that of the 3% ICG-LNP group and the unincorporated group. Therefore, it is more preferable to use ICG-CHOL to prepare lipid nanoparticles, which helps to improve the in vivo transfection efficiency.

[0082] 2.5 Cytotoxicity assessment The cytotoxicity of different formulations of ICG-LNP against DC2.4 cells was detected using the CCK-8 assay. The results are as follows: Figure 10 The results showed that when the ICG-CHOL incorporation ratio was ≤9%, the cell viability was above 90%, with no significant difference compared to the non-incorporated group; when the incorporation ratio reached 25%, the cell viability decreased significantly to below 80%.

[0083] This embodiment also investigated the toxicity of different formulations of ICG-NH-LNP to DC2.4 cells. When the incorporation ratio was ≤9%, the cell viability was above 90%. However, considering the transfection efficiency, ICG-LNP is preferred in subsequent embodiments.

[0084] Example 3: Optimization of Irradiation Conditions and Investigation of Mechanisms for Photoadjuvant Lipid Nanoparticles 3.1 Determining the Optimal Light Time To determine the optimal photoperiod window for the ICG-mVac vaccine, this embodiment used ICG-LNP with an incorporation ratio of 3% constructed in Example 2 as the ICG-mVac vaccine. Confocal laser scanning microscopy was used to track the uptake, transport, and subcellular distribution dynamics of ICG-mVac within bone marrow-derived dendritic cells (BMDCs). By co-incubating ICG-mVac with BMDCs for different times (3h, 6h, 12h, 24h, 36h) and co-localizing it with lysosomal trackers (LY, LysoTracker), endoplasmic reticulum trackers (ER, ER-Tracker), and mitochondrial trackers (MI, MitoTracker), the intracellular migration behavior of the nanoparticles was systematically investigated.

[0085] Experimental results are as follows Figure 11 As shown, the intracellular distribution of ICG-mVac exhibits a significant time-dependent pattern. At 3 hours of incubation, the ICG fluorescence signal was mainly distributed in the pericytoplasmic region, with low co-localization with lysosomes, suggesting that the nanoparticles were in the early endocytosis stage and had not yet entered acidic organelles in large quantities. With an incubation time extended to 6 hours, the ICG fluorescence intensity significantly increased, and co-localization with lysosomes reached its peak, while significant co-localization signals with the endoplasmic reticulum also appeared. This phenomenon indicates that at 6 hours, ICG-mVac had entered the cell via endocytosis and underwent structural dissociation in the acidic environment of endosomes / lysosomes, releasing ICG-CHOL molecules that further migrated to the endoplasmic reticulum. The endoplasmic reticulum is a key site for protein synthesis and folding, as well as for the activation of the STING signaling pathway, while mitochondria are closely related to reactive oxygen species production and apoptosis regulation, laying the spatial basis for subsequent light-induced immunogenic cell death.

[0086] After 12 hours of incubation, the intensity of the ICG signal began to decrease, and the co-localization with various organelles also weakened, suggesting that some nanoparticles may have been effluxed or degraded. By 24 and 36 hours, the intracellular ICG fluorescence signal had significantly decreased, with only a small number of punctate distributions remaining, and the co-localization with various organelles had basically disappeared, indicating that the intracellular retention of nanoparticles had reached a plateau or clearance phase.

[0087] In summary, ICG-mVac reached its peak intracellular uptake at 6 hours of incubation, exhibiting the most significant co-localization with lysosomes, followed by the endoplasmic reticulum, with almost no co-localization with mitochondria. At this point, the nanoparticles had completed their transport from endocytosis to key signaling organelles, providing the optimal opportunity for light-induced spatiotemporally controlled immune activation. Therefore, this embodiment selected 6 hours as the optimal light exposure window for subsequent experiments to ensure that the photoadjuvant LNP could act on the target organelles to the maximum extent during light exposure, achieving a highly efficient immunomodulatory effect.

[0088] 3.2 Selection of Optimal Illumination Parameters To determine the optimal illumination parameters for ICG-mVac photoadjuvant lipid nanoparticles, this embodiment set up multiple combinations of power density (0, 0.25, 0.5, 1, 2, 3 W) and irradiation time (0.5, 1, 1.5, 2, 3 min). The expression level of EGFP reporter gene in BMDCs was detected by flow cytometry and the reactive oxygen species (ROS) level was detected by DCFH-DA probe. The effects of different illumination conditions on mRNA transfection efficiency and cell state were systematically evaluated.

[0089] Experimental results are as follows Figure 12The result of α indicates a non-linear relationship between light intensity and mRNA transfection efficiency, with low light intensity promoting high transfection efficiency. Under low power density (0.25–0.5 W) and moderate irradiation time (1–1.5 min), the proportion of EGFP-positive cells and the average fluorescence intensity were significantly increased, indicating that suitable light irradiation can promote ICG-mVac-mediated intracellular release and translational expression of mRNA. Specifically, the EGFP expression level in the 0.5 W / 1.5 min treatment group reached its peak, increasing by approximately 3–4 times compared to the untreated control group, suggesting that this parameter can effectively activate the photoresponsive properties of ICG, promoting endosome escape without causing excessive cell damage.

[0090] However, as the light intensity further increased (≥1 W) or the irradiation time was prolonged (≥2 min), the EGFP expression level showed a downward trend, and the EGFP positivity rate in the high-power group (2~3W) was even lower than that in the control group without light.

[0091] Meanwhile, ROS test results showed ( Figure 12 (b) Under moderate light conditions (0.5W / 1.5min), ROS levels induced by the high-power group showed a mild increase, while the high-power group, although initially exhibiting a strong ROS burst, showed a significant decrease in cell viability, and the reduced number of live cells led to a decrease in overall EGFP expression levels. This phenomenon suggests that while excessively strong light conditions can generate a large amount of ROS instantaneously, they exceed the oxidative tolerance threshold of cells, inducing apoptosis or necrosis, and thus weakening the effective transfection and expression of mRNA.

[0092] It is worth noting that this embodiment found that the relationship between ROS levels and EGFP expression is not a simple positive correlation. For example... Figure 12 As shown in b, moderate levels of ROS can activate intracellular signaling pathways, promoting endosome escape and mRNA release; while excessive ROS directly leads to cell damage and even death. This result confirms the theory that photosensitizers induce differentiated biochemical activities under different light intensities: mild light mainly mediates controllable ROS production and activates cell function; strong light induces oxidative stress damage and loses its immune activation effect.

[0093] Based on the above results, this study determined that 0.5W-1.5min is the optimal light parameter for the ICG-mVac photoadjuvant LNP. This condition can ensure cell viability ( Figure 12 (C), cell viability >90%, induces appropriate levels of ROS production, and maximizes intracellular release and translational expression of mRNA.

[0094] 3.3 Effects of light on cell state To comprehensively evaluate the cellular safety and potential toxicity mechanisms of the ICG-mVac vaccine under different light conditions, this study used Annexin V-FITC / PI double staining combined with flow cytometry to detect apoptosis in BMDCs after treatment, and Western blotting to detect the expression levels of pyroptosis-related proteins. The experiment included an optimal light group (0.5W / 1.5 min) and a high-power light group (3W / 1.5 min), with measurements taken immediately after light exposure (early effect) and 18 hours after light exposure (recovery period), respectively.

[0095] Apoptosis detection results showed ( Figure 13 In the optimal light irradiation group, the apoptosis rate (including early and late apoptosis) after light irradiation was only slightly higher than that in the PBS control group, with the vast majority of cells remaining viable. This indicates that the 0.5 W-1.5 min light conditions have low cytotoxicity to BMDCs and a good safety window. In the high-power light irradiation group, the apoptosis rate increased significantly after light irradiation, indicating that the 3 W / 1.5 min high-power light conditions instantaneously triggered oxidative stress damage, exceeding the cell tolerance threshold. When tested again 24 h after light irradiation, the apoptosis rate in this group decreased significantly compared to 6 h, and the proportion of viable cells recovered to some extent, suggesting that some cells may recover from sublethal damage to some extent by activating endogenous antioxidant defense systems or repair mechanisms.

[0096] Western blot analysis of pyroptosis-related proteins (such as GSDMD) showed that ( Figure 14 In both the optimal light group and the high-power light group, the expression level of the pyroptosis protein activation fragment was not significantly different from that in the PBS control group and the no-light group. This result indicates that under the light conditions set in this embodiment, ICG-mVac-L did not induce significant pyroptosis in BMDCs.

[0097] In summary, the optimal illumination conditions (0.5W / 1.5min) selected in this study ensured efficient immune activation while exhibiting low cytotoxicity to BMDCs, inducing only mild and reversible apoptosis without triggering pyroptosis, thus demonstrating a good safety window. While strong illumination conditions induced a stronger initial effect, they were accompanied by a significant risk of cell damage and also did not trigger the pyroptosis pathway. This finding further validates the necessity of optimizing illumination parameters in photoadjuvant design, namely, seeking a balance between immune activation efficacy and cell safety to ensure effective activation of anti-tumor immunity while avoiding irreversible cell damage and excessive inflammatory responses.

[0098] 3.4 Mechanism of Action of Time-Controlled Lipid Nanoparticles To evaluate the activation effect of ICG-mVac-L vaccine on dendritic cells under optimal light conditions, this study used flow cytometry to detect the expression levels of co-stimulatory molecules CD80 and CD86 on the surface of BMDCs in different treatment groups. BMDCs were divided into PBS control group, SM102-mVac group, ICG-mVac group (protected from light) and ICG-mVac-L group (0.5W / 1.5 min light).

[0099] The results are as follows Figure 15 As shown, the positive rates of CD80 and CD86 in the PBS negative control group were extremely low, indicating that the untreated BMDCs were in a resting state, and the conventional mRNA lipid nanoparticles themselves only had a weak adjuvant effect. The expression of CD80 and CD86 in the SM102-mVac group and the ICG-mVac group (without light treatment) was slightly higher than that in the control group. Notably, after treatment with the ICG-mVac-L group under optimal light conditions of 0.5W / 1.5min, the expression levels of CD80 and CD86 were significantly increased, with the positive rate and average fluorescence intensity being much higher than other groups (p < 0.001), indicating that light activation activated the photosensitivity of ICG, triggering the maturation and activation of BMDCs.

[0100] To investigate the molecular mechanism by which ICG-mVac-L photoadjuvant nanoparticles regulate dendritic cell activation, this example uses Western blotting to detect the phosphorylation levels and total protein expression of key molecules in the STING and NF-κB signaling pathways in BMDCs.

[0101] The results are as follows Figure 16 As shown in Figure a, the phosphorylation levels of key STING signaling pathway molecules p-STING, p-TBK1, and p-IRF3 in the ICG-mVac-0.5W group were significantly higher than those in the PBS control group and the ICG-JW group, indicating that optimal light conditions effectively activated the photosensitivity of ICG, initiating the STING-TBK1-IRF3 cascade by inducing endoplasmic reticulum stress and cytoplasmic DNA sensor signals. Simultaneously, the expression level of the upstream aptamer protein MyD88 in the NF-κB pathway was also significantly upregulated in the ICG-mVac-0.5W group. Figure 16 The increased phosphorylation levels of p-NF-κB and p-IRF7 (b) suggest that the TLR / MyD88-dependent signaling pathway is also involved in the immune activation process after light exposure.

[0102] Notably, the levels of phosphorylated proteins in the ICG-mVac group were not significantly different from those in the PBS control group, confirming that ICG-mVac alone does not have the ability to activate the aforementioned signaling pathways, and its immune adjuvant effect is strictly dependent on light triggering. The expression of p-ERK1 / 2 was also increased in the ICG-0.5W group, suggesting that the MAPK pathway may synergistically participate in regulating the maturation and function of dendritic cells.

[0103] The above results indicate that under optimal light conditions, ICG-mVac-L promotes dendritic cell maturation and antigen presentation by synergistically activating the STING and TLR7 / 8 signaling pathways. The STING pathway is mainly responsible for the production of type I interferon, while the TLR7 / 8 pathway regulates the expression of pro-inflammatory cytokines. Their synergistic activation lays the molecular foundation for the subsequent induction of a robust adaptive immune response. This discovery reveals the deep mechanism by which the photoadjuvant LNP regulates innate immunity, providing a theoretical basis for its application in tumor immunotherapy.

[0104] To further elucidate the molecular mechanism by which ICG-mVac photoadjuvant vaccines regulate anti-tumor immune responses, this embodiment employs RNA-seq transcriptome sequencing technology to perform gene expression profiling analysis on BMDCs treated with PBS control group, ICG-mVac group, and photoadjuvant-enhanced ICG-mVac-L group. Through KEGG pathway enrichment analysis and differential gene expression heatmaps, the regulatory network was systematically analyzed.

[0105] KEGG pathway enrichment analysis results showed that ( Figure 17 (a) Compared with the PBS control group, the ICG-mVac vaccine significantly activated multiple core signaling pathways closely related to innate immune recognition and adaptive immune initiation. At the antigen processing and presentation level, the ICG-mVac group showed significant enrichment of antigen processing and presentation pathways, indicating that the vaccine can effectively promote the capture and processing of antigens by dendritic cells and MHC-mediated antigen presentation. At the pattern recognition receptor signaling level, the significant enrichment of Toll-like and NOD-like receptor signaling pathways suggests that ICG-mVac can trigger innate immune responses by activating intracellular nucleic acid sensors and inflammasome pathways, producing type I interferon and pro-inflammatory cytokines, providing necessary co-stimulatory signals for the subsequent initiation of adaptive immunity. At the T cell differentiation and effector function level, the enrichment of Th1, Th2, and Th17 cell differentiation pathways indicates that the vaccine can induce diversified CD4+ cell differentiation. + Helper T cell responses, with Th1 differentiation contributing to enhanced cytotoxic T lymphocyte function, while Th17 is involved in shaping the inflammatory microenvironment.

[0106] Furthermore, the activation of NF-κB and JAK-STAT signaling pathways further confirmed the initiation of pro-inflammatory transcriptional programs, while the enrichment of cytokine-cytokine receptor interactions and chemokine signaling pathways provided a molecular basis for immune cell recruitment and migration. In the ICG-mVac-L group, the enrichment of the above-mentioned immune-related pathways was further enhanced compared to the ICG-mVac group, indicating that the light-controlled strategy can precisely enhance the immune activation efficacy of the vaccine. Notably, the ICG-mVac-L group also activated several additional pathways with unique biological significance. The enrichment of the burial pathway suggests that light-induced local apoptosis may initiate an efficient apoptotic cell clearance mechanism. This process not only prevents secondary necrosis and excessive inflammation but also promotes antigen cross-presentation through phagocytosis of apoptotic bodies. The activation of apoptosis-related pathways is consistent with the previously observed mild apoptosis level, further confirming the controllability of cell death patterns under light-controlled conditions. Meanwhile, the enrichment of metabolic remodeling-related pathways (such as lipid metabolism and oxidative phosphorylation) suggests that photocontrolled activation may induce metabolic reprogramming in dendritic cells to meet their post-activation needs for energy and biosynthetic precursors, which is the metabolic basis for immune cells to acquire effector functions.

[0107] Gene expression heatmap ( Figure 17 b) further validated the above pathway analysis results. Compared with the PBS control group, the levels of pro-inflammatory factors (ICG-mVac and ICG-mVac-L groups) were significantly higher. Tnf, Ccl4, Cxcl10 ), co-stimulatory molecules ( Cd86 ) and signal transduction molecules ( Stat1, Jak2 The expression levels of key immune genes such as α and β were significantly upregulated. The upregulation was particularly pronounced in the ICG-mVac-L group, indicating that the light-controlled strategy can amplify immune activation signals at the transcriptional level. Chemokines Ccl4 and Cxcl10 Upregulation of these molecules helps recruit effector T cells to the tumor site, co-stimulatory molecules Cd86 High expression of this directly enhances the T-cell activation capacity of dendritic cells, while Stat1 and Jak2 As a core molecule of the JAK-STAT pathway, its upregulation further confirms the enhancement of type I interferon signaling and Th1 polarization.

[0108] In summary, the ICG-mVac vaccine constructs a robust anti-tumor immune network through multi-pathway synergy. It activates pattern recognition receptor signaling at the innate immune level, promotes T cell differentiation and effector function at the adaptive immune level, and regulates cytokine networks and chemotactic signals at the immunomodulatory level. The photoadjuvant strategy can further precisely enhance immune activation and additionally mobilize auxiliary mechanisms such as cytotoxicity and metabolic remodeling, thereby enhancing the spatiotemporal controllability of the immune response.

[0109] To investigate the effects of photoactivated dendritic cells (BMDCs) on T cell function, this study co-incubated primary mouse T cells with BMDCs treated with different methods. Flow cytometry was used to detect T cell subset differentiation and effector function-related indicators. Results are as follows: Figure 18 As shown, compared with the PBS control group, SM102-mVac group and ICG-mVac group, the ICG-mVac-L group (0.5 W-1.5 min light treatment) showed significantly enhanced ability of BMDCs to induce T cell differentiation, exhibiting a specific subset differentiation bias.

[0110] In CD4 + Among T cell subsets, the ICG-mVac-L group significantly promoted Th1 differentiation, as evidenced by increased expression levels of GATA3 (Th2 marker) and RORγT (Th17 marker), but the increase in Th1-related effector markers was more pronounced; Th17 cell differentiation was not significant in vitro. (In CD8...) + Among T cell subsets, Tc2 differentiation was more pronounced, with a slight increase in both Tc1 and Tc2 types, but no significant induction was observed in the Th17 / Tc17 subset. Furthermore, the ICG-mVac-L group induced CD4+. + and CD8 + The ability of T cells to secrete effector cytokines such as TNF-γ (a Th1 marker) was significantly enhanced, suggesting that this treatment group can effectively promote the differentiation of T cells into effector subsets and enhance their immune effector function.

[0111] In vitro experiments showed that ICG-mVac-L-activated BMDCs preferentially induced T cell differentiation into Th1 and Tc2 effector subsets, while moderately promoting Th2 and Tc1 responses, but had limited induction of Th17 / Tc17 subsets. This biased T cell differentiation pattern is conducive to establishing a cell-mediated anti-tumor immune response, providing functional mechanistic support for subsequent in vivo anti-tumor efficacy studies.

[0112] Example 4: In vivo antitumor efficacy and mechanism of runaway-controllable mRNA vaccine 4.1 Assessment of vaccine lymph node targeting Based on the inherent near-infrared fluorescence properties of ICG, this embodiment uses non-invasive in vivo tracking to study the in vivo distribution kinetics of the vaccine after injection, thereby investigating the accumulation efficiency and cellular uptake preference of ICG-mVac in draining lymph nodes. ICG-mVac was subcutaneously injected into the groin region of mice. Six hours after injection, draining lymph nodes were isolated to prepare single-cell suspensions, and the proportion of ICG-positive cells in each immune cell subset was analyzed by flow cytometry.

[0113] Experimental results are as follows Figure 19As shown, after subcutaneous injection, ICG-mVac effectively migrates to the draining lymph nodes and is selectively taken up by antigen-presenting cells within the lymph nodes. Among them, macrophages (F4 / 80) + ) are the main cell population that takes up nanoparticles, with the highest ICG positivity rate; dendritic cells (CD11c) + T cells (CD3+) also exhibited significant uptake capacity, with a positive rate second only to macrophages. In contrast, T cells (CD3+) + The absence of ICG positive signals indicates that lymphocytes themselves do not directly participate in the uptake of nanoparticles, and their activation mainly depends on the cross-presentation of antigen-presenting cells.

[0114] This distribution pattern has significant immunological implications. Macrophages and dendritic cells, as professional antigen-presenting cells, can migrate to the paracortical region of lymph nodes after vaccine uptake, presenting processed antigenic peptides to naive T cells via MHC molecules, thus initiating an adaptive immune response. ICG-mVac is mainly enriched in these cell populations in lymph nodes, providing a cellular basis for its effective induction of antigen-specific T cell activation. Simultaneously, the fluorescence tracing function of ICG facilitates precise control of subsequent illumination time: localized illumination after the nanoparticles have been efficiently uptaken by antigen-presenting cells (6 h) maximizes the immune-activating effect of the photoadjuvant while avoiding non-specific damage to non-target cells.

[0115] It is evident that ICG-mVac can effectively accumulate in draining lymph nodes after subcutaneous injection and be selectively taken up by macrophages and dendritic cells, laying the spatial and cellular foundation for subsequent light-induced activation of antigen-presenting cells and the initiation of T-cell immunity.

[0116] 4.2 Screening of animals for ICG-mVac vaccine lighting conditions Based on the established optimal in vitro light exposure parameters for the ICG-mVac vaccine, this embodiment further optimized and screened animal-level light exposure conditions in a B16-OVA subcutaneous tumor-bearing mouse model, aiming to determine the in vivo light exposure scheme that maximizes the anti-tumor effect. The experiment was conducted according to... Figure 20 The dosing regimens shown in a and b were performed: ICG-mVac vaccine (containing 20 μg OVA-mRNA) was subcutaneously administered on days 0 and 7, and near-infrared light irradiation under different conditions was applied 6 h after each vaccination. Light intensity gradients (0.25 W, 0.5 W, 0.75 W, 1 W) and irradiation time gradients (1.5 min, 3 min) were set. The undoped SM102-mVac group and the unirradiated group were used as controls. The effects of each parameter combination on tumor growth and immune response were systematically evaluated.

[0117] like Figure 20The tumor growth and survival curves shown in Figure c indicate that different light conditions significantly affect the antitumor effect of the ICG-mVac vaccine, exhibiting a non-linear relationship consistent with in vitro experimental results. The 0.5 W / 1.5 min light treatment group showed the most significant tumor-suppressing effect, with a significantly slower tumor growth rate and a significantly smaller average tumor volume at the experimental endpoint compared to other light-treated groups and the untreated control group. Survival curve analysis showed that the median survival time of mice in this group was longer than that of the untreated group and also longer than other light-treated groups to varying degrees, suggesting that this light condition most effectively activates the immunomodulatory function of photoadjuvants. Conversely, treatments with excessively strong light intensity (1 W) or excessively long duration (3 min) showed a decreased tumor-suppressing effect, with some indicators even lower than the untreated group, possibly related to local tissue damage or excessive apoptosis of immune cells caused by strong light.

[0118] To further verify the regulatory effects of different light conditions on the anti-tumor immune response, peripheral blood was collected from mice via the orbital cavity on day 14 (7 days after the second immunization). OVA tetramer staining combined with flow cytometry was used to detect antigen-specific CD8. + The proportion of T cells. Results as follows: Figure 20 As shown in d, the peripheral blood Tet in the 0.5W / 1.5min light treatment group... + CD8 + The proportion of T cells was significantly higher in this group than in other groups, consistent with the trends in tumor suppression and survival curves. This result indicates that this light exposure condition most effectively promotes the expansion and activation of antigen-specific T cells, providing a sufficient reserve of effector cells for tumor clearance. While other light exposure groups also showed some degree of T cell activity... + CD8 + T cells increased, but the increase was significantly lower than in the optimal group, further confirming the importance of optimizing light parameters.

[0119] In summary, this embodiment determined that 0.5W / 1.5min is the optimal light exposure condition for in vivo antitumor therapy with ICG-mVac vaccine. This condition, while ensuring good safety, can maximize the activation of antigen-specific T cell responses, achieving optimal tumor growth inhibition and survival prolongation effects, and providing a standardized light exposure protocol for subsequent efficacy evaluation studies of combined immune checkpoint inhibitors. Unless otherwise specified, the ICG-mVac group refers to the spatiotemporally controllable lipid nanoparticle vaccine treated with these light parameters, and the mVac group refers to the SM102-mVac (undoped) vaccine.

[0120] 4.3 Evaluation of the antitumor efficacy of ICG-mVac vaccine combined with αPD-1 Based on the optimal light conditions (0.5W / 1.5min) determined in the previous screening, this embodiment further evaluates the antitumor efficacy of ICG-mVac alone or in combination with the immune checkpoint inhibitor αPD-1 antibody in a B16-OVA subcutaneous tumor-bearing mouse model. Figure 21 The dosing regimens shown in a and b were administered three times on days 0, 7, and 14, respectively. The combination therapy group received intraperitoneal injection of αPD-1 antibody on days 1, 8, and 15. Tumor growth and mouse survival were continuously monitored.

[0121] Tumor growth curve as shown Figure 21 As shown in Figure c, compared with the PBS control group, all treatment groups exhibited varying degrees of tumor suppression. Among them, the SM102-mVac group showed limited tumor suppression, while the ICG-mVac group showed a significant slowdown in tumor growth, demonstrating a significantly better tumor suppression effect than the undoped vaccine group, indicating that the introduction of photoadjuvants effectively enhanced the anti-tumor immune response of the vaccine. Furthermore, the ICG-mVac combined with αPD-1 antibody group exhibited the strongest tumor suppression effect, the slowest tumor growth, and a 3 / 8 tumor elimination rate, with a complete tumor remission rate of 37.5%. Individual tumor growth curves ( Figure 21 (d) further confirmed the efficacy advantage of the combined treatment group.

[0122] This embodiment also investigated the anti-tumor effect of ICG-NH-mVac vaccine prepared by ICG-NH-LNP in combination with αPD-1 under the same conditions. Although the complete tumor remission rate was slightly lower than that of ICG-mVac vaccine, it still had significant anti-tumor efficacy.

[0123] In conclusion, the ICG-mVac combined with αPD-1 antibody regimen demonstrated excellent anti-tumor efficacy in a melanoma subcutaneous tumor model, providing strong evidence for further in-depth research into its immune mechanism and clinical translation.

[0124] 4.4 Changes in lymph node dendritic cell population induced by ICG-mVac vaccine Dendritic cells (DCs), as professional antigen-presenting cells, directly influence the quality of T-cell immune responses through their subset composition and functional status. In this study, draining lymph nodes were collected on days 9 and 14 of treatment in a B16-OVA-bearing mouse model, and the effects of different treatment groups on DC subset differentiation were analyzed by flow cytometry.

[0125] like Figure 22As shown in Figure a, on day 9 of treatment, there were no significant differences in the number of cDCs, cDC1s, and cDC2s among the treatment groups, but the number of pDCs was significantly increased in the ICG-mVac combined with αPD-1 group. As the main producing cells of type I interferon, the early expansion of pDCs helps to initiate the innate immune response and lays the foundation for the subsequent establishment of adaptive immunity. By day 14 of treatment (… Figure 22 (b) The total number of cDCs in the ICG-mVac combined with αPD-1 group was significantly higher than that in other groups, with a significant increase in the number of cDC1s and cDC2s, while the difference in the number of pDCs disappeared among the groups. cDC1s are cross-presented antigens to CD8. + An increase in the number of key T cell subsets predicts an enhanced antigen-specific CTL response; cDC2s are mainly involved in CD4. + T cell activation. Proportion analysis showed ( Figure 22 (c) The proportion of cDC1s in cDCs was significantly increased in the combined treatment group, suggesting that the differentiation of DC subsets is shifting in a direction that is more conducive to cross-presentation.

[0126] The above results indicate that ICG-mVac combined with αPD-1 can spatiotemporally regulate lymph node dendritic (DC) subsets: initially promoting pDC expansion to enhance innate immunity, and later driving the proliferation and differentiation of cDC1s and cDC2s, providing a cellular basis for efficient antigen presentation and T cell activation. This reveals one of the mechanisms by which combined therapy enhances anti-tumor immunity at the antigen-presenting cell level.

[0127] The degree of dendritic cell activation directly determines their antigen-presenting ability and the quality of subsequent T cell responses. This embodiment also evaluated the effect of ICG-mVac combined with αPD-1 on dendritic cell activation by detecting the expression levels of co-stimulatory molecules (CD40, CD86) and MHC-I on the surface of different dendritic cell subsets in lymph nodes. On day 9 of treatment, the expression levels of CD40, CD86, and MHC-I in cDCs, cDC1s, and cDC2s in the ICG-mVac combined with αPD-1 group were significantly higher than in other groups, indicating that the combined treatment can effectively promote the maturation and activation of traditional dendritic cell subsets in the early stages. cDC1s, as a key executor of cross-presentation, shows that increased activation levels are crucial for subsequent CD8+ expression. + The activation of T cells is crucial; the activation of cDC2s helps assist CD4. +T-cell response. Notably, there were no significant differences in the expression of pDC activation markers among the groups, suggesting that pDCs may participate in the early immune response primarily through quantitative expansion rather than increased activation levels of individual cells. By day 14 of treatment, the expression levels of CD40, CD86, and MHC-I in cDC1s remained high, while the differences in cDC2 activation markers among the groups had disappeared. This result indicates that the combination therapy has a sustained activation effect on cDC1s, which is beneficial for maintaining long-term antigen cross-presentation capacity; while the activation of cDC2s may mainly participate in the early immune initiation and gradually decline in the later stages.

[0128] In summary, ICG-mVac or ICG-mVac combined with αPD-1 can spatiotemporally regulate the activation state of dendritic (DC) subsets within lymph nodes, broadly activating cDCs and their subsets in the early stages and selectively maintaining high activation levels of cDC1s in the later stages. This precise DC activation pattern provides a cellular basis for efficient antigen presentation and sustained anti-tumor T cell responses.

[0129] 4.5 Effects of ICG-mVac vaccine on T cell apoptosis To evaluate the effect of ICG-mVac combined with αPD-1 treatment on T cell survival, the apoptosis rate of T cells in the draining lymph nodes was measured on day 9 (1 day after the second immunization phototherapy) and day 14 (7 days after the second immunization phototherapy) in this embodiment. The results are shown in Figure 23.

[0130] The test results on day 9 showed ( Figure 23 In group a), the proportion of T cell apoptosis was extremely low in the PBS control group and the αPD-1 monotherapy group, while varying degrees of T cell apoptosis were observed in all vaccine groups (including SM102-mVac, SM102-mVac combined with αPD-1, ICG-mVac, and ICG-mVac combined with αPD-1). This phenomenon may be related to activation-induced cell death after the vaccine activates a strong immune response: during the peak period of rapid T cell proliferation and activation, some activated T cells maintain immune homeostasis through apoptosis, avoiding excessive inflammatory response. It is worth noting that although the proportion of apoptosis in the combination therapy group was slightly increased, it was still within a controllable range, suggesting that the strong immune activation induced by photoadjuvant vaccines is accompanied by a certain degree of T cell turnover, which is a normal immune regulatory process. By day 14 (when tested) Figure 23 (b) The proportion of T cell apoptosis in all treatment groups returned to extremely low levels, with no significant difference between groups. This result indicates that the T cell apoptosis observed in the early stage of vaccination is temporary, and as the immune response enters the plateau phase, the T cell survival status returns to homeostasis without persistent damage or exhaustion.

[0131] Although ICG-mVac combined with αPD-1 therapy is accompanied by a certain degree of T cell apoptosis in the early stage of immune activation, this phenomenon is a normal physiological process initiating the immune response and does not have a long-term negative impact on the T cell pool. The results indicate that photoadjuvant vaccines, while potently activating anti-tumor immunity, do not induce persistent immune cell damage.

[0132] 4.6 Changes in T cells in the spleen induced by ICG-mVac vaccine The spleen, as an important organ in the systemic immune response, has a T-cell composition that reflects the level of vaccine-induced peripheral immune memory. For example... Figure 24 As shown, the flow cytometry results on day 14 indicated that CD3 levels were lower in each vaccination group. + CD4 + and CD8 + Although the total number of T cells was slightly higher in the ICG-mVac combined with αPD-1 group, the difference between the groups was not significant. It is worth noting that the OVA-specific CD8+ group showed higher levels of T cells compared to the αPD-1 group. + T cells (Tet) + CD8 + The absolute number and proportion of ) were significantly higher than those of other groups ( Figure 24 (d and e). This result indicates that although the combination therapy did not significantly expand the total T cell pool, it effectively promoted the systemic expansion of antigen-specific T cells, providing a cellular basis for long-term immune surveillance and tumor clearance.

[0133] CD8 + The activation status and memory differentiation level of T cells directly reflect the strength of vaccine-induced systemic immune memory. This study measured CD8+ levels in the spleen on day 14 of treatment. + Distribution of T cell activation markers and memory subsets. Activation marker detection showed that CD69 was present in each group. + CD8 + The number of T cells was not significantly different from that in the PBS control group, indicating that the early activation peak of T cells had passed and the cell cycle had entered the memory maintenance phase. Ki67 + CD8 + The number of T cells in all vaccine groups was lower than that in the PBS group, but the differences between groups were not significant, suggesting that each vaccine formulation induced T cell proliferation to a certain extent, while photoadjuvant combination therapy did not further amplify the proliferation signal. Memory subset analysis revealed the effect of combination therapy on CD8... + The regulatory role of T cell differentiation. Effector memory T cells (Tem, CD44) in the ICG-mVac combined with αPD-1 group. + CD62L － The absolute number and proportion of CD8 were significantly higher than those of other groups, indicating that this treatment effectively promoted CD8. +T cells differentiate into effector memory phenotypes. Tem subsets possess the ability to rapidly respond to antigen restimulation and are a key population mediating long-term anti-tumor immune protection. Simultaneously, central memory T cells (Tcm, CD44) are present in all groups receiving αPD-1 treatment (including SM102-mVac combined with αPD-1 and ICG-mVac combined with αPD-1). + CD62L + The number of αPD-1 cells was higher than that of the corresponding group that did not receive αPD-1, suggesting that immune checkpoint blockade helps maintain the long-term stability of the memory T cell pool.

[0134] In summary, ICG-mVac combined with αPD-1 induced CD8 cells biased towards Tem differentiation in the spleen. + T cell response, along with the addition of αPD-1, promotes the maintenance of Tcm. This optimized combination of memory subsets provides the body with both immediate effector function and long-term immune surveillance, which is one of the important mechanisms by which combination therapy exerts a lasting anti-tumor effect.

[0135] 4.7 ICG-mVac vaccine induces changes in immune cell subsets in the tumor microenvironment The composition and functional status of antigen-presenting cells (APCs) in the tumor microenvironment are key factors determining the intensity of the anti-tumor immune response. This study examined the number of tumor-infiltrating APC subsets and macrophage polarization status on day 14 of treatment. The number of cDCs, pDCs, cDC1s, and cDC2s in the ICG-mVac combined with αPD-1 group was significantly higher than in other groups, indicating that the combined treatment effectively promoted the recruitment and expansion of multiple APC subsets to tumor tissue. cDC1s, as the core executor of cross-presentation, was enriched with tumor antigen-specific CD8+. + The initiation and maintenance of T cells provide the cellular basis; the increase of cDC2s helps assist CD4. + T cell response; pDC infiltration provides type I interferon signaling, enhancing the tumor immune microenvironment. Tumor-associated macrophage polarization analysis showed that the M1 / M2 ratio in the ICG-mVac combined with αPD-1 group was significantly higher than in other groups, suggesting that the combined treatment effectively promoted macrophage polarization towards the anti-tumor phenotype (M1) while inhibiting the accumulation of immunosuppressive M2 macrophages. M1 macrophages participate in anti-tumor immunity through mechanisms such as secreting pro-inflammatory cytokines, enhancing antigen presentation, and directly phagocytosing tumor cells, while M2 macrophages are associated with tumor progression and immune escape. In summary, ICG-mVac combined with αPD-1 dually regulates the number and function of APCs in the tumor microenvironment: on the one hand, it increases the infiltration of cDC1s, cDC2s, and pDCs; on the other hand, it reshapes the macrophage polarization balance, creating an immune-supportive microenvironment conducive to T cell activation and effector function.

[0136] 4.8 ICG-mVac vaccine activates T cells in the tumor microenvironment The functional status of tumor-infiltrating T cells is a core factor determining the final efficacy of anti-tumor immune responses. In this embodiment, CD4+ in the tumor microenvironment was detected on day 14 of treatment. + and CD8 + The expression levels of CD69, a marker of T cell activation, Ki67, a marker of proliferation, and Granzyme B, an effector molecule, were assessed to evaluate the functional status of T cells in different treatment groups.

[0137] In the ICG-mVac combined with αPD-1 group, CD4 + CD69 T cells + Ki67 + and Granzyme B + The number of cells in all groups was significantly higher than in other groups, indicating that the combination therapy effectively promoted CD4. + Early activation, proliferation, and acquisition of effector function of helper T cells. Activated CD4+ + T cells can assist CD8 by secreting cytokines and providing co-stimulatory signals. + The maintenance and function of T cells.

[0138] In CD8 + Within the T-cell population, the combination therapy group also exhibited a significantly enhanced functional state. CD69 + CD8 + Ki67 + CD8 + and Granzyme B + CD8 + The number of T cells was significantly higher in all treatment groups than in other treatment groups, indicating that the activation, expansion, and killing functions of cytotoxic T cells were enhanced. Crucially, OVA-specific CD8+ was also observed. + T cells (Tet) + CD8 + Ki67 + and Granzyme B + The number of double-positive cells was significantly enriched in the combination therapy group, confirming that not only did the number of antigen-specific CTLs increase, but their functional activity was also enhanced simultaneously.

[0139] In summary, ICG-mVac combined with αPD-1 enhances the functional state of tumor-infiltrating T cells from multiple dimensions: promoting CD4+. + T cell helper function, enhancing CD8 + It enhances the proliferation and killing activity of T cells and specifically improves the effector function of antigen-specific CTLs.

[0140] 4.9 Analysis of in vivo immune mechanisms On days 9 and 14 of treatment, lymph nodes, spleens, and tumor tissues of mice were collected, and immune cell subsets were analyzed by multicolor flow cytometry.

[0141] Lymph nodes: Day 9, ICG-mVac-L + The number of pDCs in the aPD-1 group increased significantly; on day 14, the number of cDCs, cDC1s, and cDC2s increased significantly, and the proportion of cDC1s also increased.

[0142] Spleen: Day 14, ICG-mVac-L + aPD-1 group antigen-specific CD8 + T cells (Tet) + CD8 + The number and proportion of effector memory T cells (Tem, CD44) were significantly higher than in other groups, and the number and proportion of effector memory T cells were also significantly higher than in other groups. + CD62L - Significant enrichment.

[0143] Tumor microenvironment: Day 14, ICG-mVac-L + aPD-1 group tumor infiltration CD8 + The number of T cells, Th1 cells, and Th17 cells increased significantly; tumor-infiltrating CD8 cells were observed. + The expression levels of CD69, Ki67, and Granzyme B in T cells were significantly increased; at the same time, the number of cDC1s increased, and the M1 macrophages (M1 / M2 ratio) were significantly increased.

[0144] 4.10 Safety Evaluation After treatment, the heart, liver, spleen, lungs, and kidneys of mice in each group were subjected to H&E staining. The results showed that, except for an enlarged white pulp region in the spleen (a sign of immune activation), no obvious pathological damage was observed in the major organs, indicating that the treatment regimen has good in vivo safety.

[0145] The above embodiments illustrate the technical solution of the present invention in detail, and experimental data fully demonstrate the superiority of the spatiotemporally controllable photoadjuvant lipid nanoparticles and their mRNA vaccines constructed in this invention in terms of physicochemical properties, transfection efficiency, immune activation mechanism, antitumor efficacy and safety, providing clear guidance for those skilled in the art to implement the present invention.

[0146] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A photoadjuvant-modified cholesterol compound, characterized in that, The conjugate includes a photosensitizer and cholesterol linked by covalent bonds; the photosensitizer includes one or more of indocyanine green, IR-780, IR-820, dihydroporphyrin E6, pyromethesin a, zinc phthalocyanine, and methylene blue; the covalent bond includes one or more of ester bonds, amide bonds, disulfide bonds, thioether bonds, thioketal bonds, carbon ester bonds, amino ester bonds, hydrazone bonds, and acylhydrazide bonds.

2. The compound according to claim 1, characterized in that, The photosensitizer is indocyanine green; the indocyanine green and cholesterol are linked by an ester bond to form a conjugate.

3. The compound according to claim 2, characterized in that, The structural formula of the conjugate formed by the indocyanine green and cholesterol linked by an ester bond is shown in formula (1): Equation (1) A spatiotemporally controllable lipid nanoparticle, characterized in that the lipid nanoparticle comprises a photoadjuvant-modified cholesterol compound as described in any one of claims 1 to 3.

4. The spatiotemporally controllable lipid nanoparticles according to claim 4, characterized in that, The lipid nanoparticles also contain ionizable cationic lipids, cofactor phospholipids, polyethylene glycol-modified lipids, and mRNA.

5. The spatiotemporally controllable lipid nanoparticles according to claim 5, characterized in that, The photoadjuvant-modified cholesterol compound accounts for 3%-9% of the total cholesterol in the spatiotemporally controllable lipid nanoparticles.

6. A spatiotemporally controllable mRNA vaccine, characterized in that, The light-responsive mRNA vaccine comprises a photoadjuvant-modified cholesterol compound as described in any one of claims 1 to 3, or lipid nanoparticles as described in any one of claims 4 to 6. After the spatiotemporally controllable mRNA vaccine is taken up by antigen-presenting cells, light is used to achieve the controlled release of the mRNA vaccine.

7. The use of the photoadjuvant-modified cholesterol compound according to any one of claims 1-3, or the lipid nanoparticles according to any one of claims 4-6, or the mRNA vaccine according to claim 7 in the preparation of a medicament for treating tumors, characterized in that, The application includes the following steps: (a) Delivering the drug to antigen-presenting cells; (b) After the drug is taken up by the antigen-presenting cells, it is exposed to light.

8. The use according to claim 8, characterized in that, In step (b), the illumination is near-infrared light; and / or the timing of the illumination is 4-12 hours after the administration of the mRNA vaccine; and / or the parameters of the illumination are: power density 0.25-1 W, irradiation time 1-3 minutes.

9. A pharmaceutical composition, characterized in that, Includes the mRNA vaccine and immune checkpoint inhibitor as described in claim 7; the immune checkpoint inhibitor includes anti-PD-1 antibody and / or anti-PD-L1 antibody.

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