Spleen-targeted nano platform construction method suitable for tumor vaccination
By optimizing the combination of nanocarriers and adjuvants, spleen-targeted delivery of tumor vaccines is achieved, which solves the problem of insufficient targeting efficiency of traditional vaccines, improves the activation efficiency of splenic red pulp cells and tumor inhibition effect, and avoids systemic inflammation and organ damage.
Patent Information
- Application Number
- CN202510654439.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-21
- Publication Date
- 2025-09-19
AI Technical Summary
Traditional vaccines have insufficient targeting efficiency during delivery, making it difficult to activate myeloid cells in the red pulp of the spleen, resulting in low efficiency of cellular immune activation and potentially causing systemic inflammation or organ damage.
CL15H6-DOPSLNPs with a particle size greater than 100 nm and a negatively charged surface, spherical polymer vesicles and red blood cell membrane-coated nanoparticles were used as spleen-targeted nanocarriers. Mn2+-doped LNPs, ultrasound-responsive liposomes and CD3 antibody coupling were combined to optimize the liposome chain length and surface topology to achieve precise delivery of tumor vaccines and immune activation.
Significantly improve the spleen enrichment rate, enhance the uptake efficiency of red pulp myeloid cells, activate the STING pathway, promote the proportion of antigen-specific CD8+T cells, reduce non-target organ exposure, avoid spleen pathological damage, and achieve efficient tumor suppression effect.
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Figure CN120661470A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of vaccine technology, and in particular to a method for constructing a spleen-targeted nanoplatform suitable for tumor vaccination. Background Art
[0002] Tumor vaccines are biological agents that activate the patient's own immune system by delivering tumor antigens (such as tumor cells, related proteins / peptides, and antigen-encoding genes), inducing specific cellular immunity (such as CD8+ T cells) and humoral immune responses, thereby achieving tumor control or clearance. The spleen-targeted nanoplatform is a delivery system that uses nanocarrier technology to precisely deliver tumor vaccines (such as mRNA, antigenic proteins, or immune adjuvants) to splenic immune cells (such as dendritic cells and myeloid cells).
[0003] Traditional vaccines have some shortcomings when used, such as insufficient targeting efficiency of traditional vaccines, and non-targeted delivery systems are easily intercepted by organs such as the liver and lungs, resulting in low spleen enrichment rate and difficulty in activating myeloid cells in the spleen red pulp. Antigens and adjuvants are difficult to deliver simultaneously to spleen immune cells, resulting in low efficiency of cellular immune activation, and traditional delivery systems cause systemic inflammation or organ damage due to nonspecific immune activation.
[0004] Therefore, we made improvements to this and proposed a method to construct a spleen-targeted nanoplatform suitable for tumor vaccination. Summary of the Invention
[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:
[0006] The present invention provides a method for constructing a spleen-targeted nanoplatform suitable for tumor vaccination, comprising the following steps:
[0007] S1. Selection of spleen-targeted nanocarriers. CL15H6-DOPSLNPs, spherical polymersomes, and erythrocyte membrane-coated nanoparticles were selected as spleen-targeted nanocarriers based on the particle size range and surface charge conditions.
[0008] S2, integrated immune adjuvant: Mn2+-doped LNPs, ultrasound-responsive liposomes, and CD3 antibody coupling were selected as integrated materials, where the adjuvants were manganese ions, TLR agonists, and CD3 antibodies, respectively;
[0009] S3. In vivo delivery: Animal models were selected for in vivo delivery and efficacy verification. The animal models were B16F10 melanoma mice, MC38 colon cancer mice, and non-human primates.
[0010] As a preferred technical solution of the present invention, the spleen-targeted nanocarrier needs to be a material with a particle size greater than 100 nm and a negatively charged surface, and the surface charge of the material is -10 mV to -25 mV.
[0011] As a preferred technical solution of the present invention, CL15H6 combined with DOPS LNPs achieves efficient transfection of splenic dendritic cells with a transfection rate greater than 32%. DOPS selects a 30% ratio and optimizes targeting through SORT technology.
[0012] As a preferred technical solution of the present invention, the particle size distribution and storage stability were detected by dynamic light scattering, and the carrier was kept at 4° C. and had no aggregation for 21 days.
[0013] As a preferred technical solution of the present invention, Mn2+ increases the DC maturation rate by activating the STING pathway, and the Mn2+ concentration is 0.5-2mM.
[0014] As a preferred technical solution of the present invention, the liposome chain length is C14-C18, and combined with ultrasound parameter optimization, the ultrasound parameter is 1-3 MHz; the CD3 antibody coupling density is 1-2 μg / mg lipid.
[0015] As a preferred technical solution of the present invention, mRNA is delivered in vivo at a dose of 0.1-0.5 mg / kg; after in vivo delivery, spleen enrichment is confirmed by PET imaging to avoid exceeding 25% ID / g.
[0016] As a preferred technical solution of the present invention, serum and immune tolerance are monitored after in vivo delivery.
[0017] The beneficial effects of the present invention are:
[0018] By optimizing liposome chain length (C14-C18) and surface topology (e.g., spherical polymersomes), the present invention significantly improves spleen enrichment and enhances red pulp myeloid cell uptake. Combining this with a manganese adjuvant for co-delivery activates the STING pathway and promotes type I interferon secretion, increasing the proportion of antigen-specific CD8+ T cells and tumor inhibition rates.
[0019] The present invention uses redox-responsive liposomes to reduce exposure to non-target organs (liver and kidneys) and avoid pathological damage to the spleen. Stable spleen targeting was verified in a non-human primate model, providing reliable data support for clinical translation. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:
[0021] Figure 1 It is a schematic flow chart of a method for constructing a spleen-targeted nanoplatform suitable for tumor vaccination according to the present invention. DETAILED DESCRIPTION
[0022] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.
[0023] Example: Figure 1 As shown, the present invention provides a method for constructing a spleen-targeted nanoplatform suitable for tumor vaccination, comprising the following steps:
[0024] S1. Selection of spleen-targeted nanocarriers: CL15H6-DOPSLNPs, spherical polymer vesicles, and erythrocyte membrane-coated nanoparticles were selected as spleen-targeted nanocarriers based on the particle size range and surface charge conditions. A comparison of the various carriers can be found in Table 1 below.
[0025]
[0026] In Table 1 , CL15H6-DOPS LNPs, spherical polymersomes, and erythrocyte membrane-coated nanoparticles were compared in terms of particle size, surface charge, spleen uptake rate, and antigen loading type. When the particle size was greater than 100 nm and the surface charge was between -10 and -25 mV, myeloid cell uptake was enhanced.
[0027] The lipid structure of CL15H6 was optimized through SORT technology, enabling specific targeting of splenic antigen-presenting cells (APCs). The mRNA transfection rate was >35%, significantly higher than that of traditional LNPs. The anionic properties of DOPS can stably load manganese ions (Mn2+), activate the STING pathway, and increase the dendritic cell (DC) maturation rate to 85%.
[0028] Spherical polymer vesicles (such as polyester copolymers) with a particle size of 150–200 nm are preferentially taken up by splenic red pulp myeloid cells, with an uptake rate of >25% ID / g. Non-human primate experiments showed that their spleen enrichment characteristics are consistent with those of the mouse model, making them suitable for clinical translation.
[0029] Erythrocyte membrane-coated nanoparticles (such as PEI25000-C14 / PLGA complexes) can reduce reticuloendothelial clearance, prolong in vivo circulation time, and utilize tumor-associated antigens derived from induced pluripotent stem cells (iPSCs) to cover multiple cancer types.
[0030] In summary, CL15H6-DOPS LNPs are suitable for mRNA vaccine delivery due to their efficient transfection and adjuvant compatibility; spherical polymer vesicles have become the first choice for β-glucan delivery due to their cross-species consistency; and red blood cell membrane-coated nanoparticles achieve broad-spectrum antigen delivery through immune escape properties, so the three are selected as carriers.
[0031] S2. Integrated immune adjuvant: Mn2+-doped LNPs, ultrasound-responsive liposomes, and CD3 antibody coupling were selected as integrated materials, wherein the adjuvants were manganese ions, TLR agonists, and CD3 antibodies, respectively. For details, please refer to Table 2 below:
[0032]
[0033] Mn2+-doped LNPs can enhance the efficiency of STING pathway activation while also exhibiting spleen targeting and adjuvant synergy. By enhancing the binding affinity of cGAMP for STING, Mn2+ significantly activates the cGAS-STING pathway, promoting dendritic cell (DC) maturation (maturation rate >85%) and type I interferon secretion. Mn2+-doped LNPs at concentrations of 0.5–1 mM can both activate the immune response and prevent systemic inflammation. Mn@mRNA-LNPs constructed using SORT technology can achieve splenic DC-specific targeting (splenic enrichment >25% ID / g), simultaneously delivering mRNA antigens and Mn2+ adjuvants, enhancing antigen presentation efficiency.
[0034] Ultrasound-responsive liposomes can precisely regulate drug release, improve the immunosuppressive microenvironment, and enhance splenic accumulation. High-fluidity liposomes (C14-C18 chain length) combined with ultrasound triggering (1–3 MHz) enable spatiotemporally controlled antigen / adjuvant release at the tumor site, enhancing the efficiency of immune activation in the tumor microenvironment. Ultrasound-responsive liposomes recruit T cells to the tumor site by releasing chemokines (such as CCL5), reducing interstitial pressure and enhancing T cell infiltration (>30%).
[0035] CD3 antibody conjugates can directly activate T cell immunity and synergistically amplify the immune response. CD3 antibody conjugates (density 1–2 μg / mg lipid) can target and bind to T cell surface receptors, promote CAR-T cell generation (generation rate > 30%) and tumor-specific killing activity (inhibition rate > 65%). When used in combination with Mn2+ adjuvant, CD3 antibody-conjugated nanocarriers can simultaneously activate innate immunity (DC maturation) and adaptive immunity (T cell expansion), forming a "dual-signal" immune enhancement mechanism.
[0036] CD3 antibody conjugation requires optimization of coupling density and verification of antibody activity retention (>90%). mRNA and adjuvant are co-encapsulated using microfluidic technology with an encapsulation efficiency of >90% to ensure simultaneous delivery to splenic APCs.
[0037] The synergy and adaptation scenarios of integrated materials are shown in Table 3 below:
[0038]
[0039] S3. In vivo delivery: Animal models were selected for in vivo delivery and efficacy verification. The animal models were B16F10 melanoma mice, MC38 colon cancer mice, and non-human primates. For details, please refer to Table 4 below:
[0040]
[0041] The B16F10 melanoma mouse model has high invasiveness and immune escape characteristics, as well as immunotherapy responsiveness. B16F10 melanoma cells are highly metastatic and can simulate the immunosuppressive microenvironment of advanced tumors. It is suitable for evaluating the efficacy of delivery systems to overcome drug resistance (such as PD-1 / PD-L1 blockade). The subcutaneous tumor-bearing model has a short tumor formation time (about 7 days), which facilitates rapid verification of anti-tumor effects (tumor volume calculation standardization: V = ab 2 / 2). This model is sensitive to CAR-T cell therapy and immune checkpoint inhibitors, and can quantitatively analyze T cell infiltration rate and tumor-specific killing activity.
[0042] The MC38 colon cancer mouse model has moderate immunogenicity and tumor transformation potential, as well as suitability for combined chemotherapy and immunotherapy. The MC38 colon cancer model has moderate immunogenicity and is suitable for validating the ability of delivery systems to convert "cold tumors" into "hot tumors" (e.g., via STING agonists or ferroptosis inducers). It is sensitive to immune cycle activation (e.g., enhanced antigen presentation efficiency) and can evaluate the synergistic effects of adaptive immune responses. The MC38 model is often used to validate chemotherapy-immunity combination strategies (e.g., oxaliplatin combined with PD-1 antibodies) and supports quantitative analysis of multi-mechanism synergistic anti-tumor effects.
[0043] Non-human primate models have cross-species translational validation capabilities and can simulate complex immune systems. The physiological structure of non-human primates (such as macaques) is highly similar to that of humans, allowing verification of the spleen-enrichment characteristics (>20% ID / g) and safety (ALT / AST <50U / L) of nanocarriers (such as spleen-targeted polymer vesicles), supporting preclinical pharmacokinetic (PK) and toxicology studies (such as systemic toxicity assessment of IL-12 self-replicating RNA drugs). Non-human primate models can simulate human T cell differentiation, antibody-dependent cellular cytotoxicity (ADCC) and other mechanisms, and are suitable for evaluating CAR-T cell generation rate (>30%) and long-lasting immune memory effects.
[0044] Model selection and scene adaptation are shown in Table 5 below:
[0045]
[0046] Spleen-targeted nanocarriers require materials with a particle size greater than 100 nm and a negative surface charge ranging from -10 mV to -25 mV. This negative surface charge reduces nonspecific adsorption of plasma proteins (such as albumin and fibrinogen), preventing the formation of a "protein corona" that interferes with targeting. Surface charge stability (fluctuations <5 mV over 24 hours) can be verified in vitro using simulated body fluids (such as PBS containing 10% fetal bovine serum) to prevent charge reversal caused by the in vivo environment (pH and ionic strength).
[0047] CL15H6-DOPS LNPs achieved efficient transfection of splenic dendritic cells, with a transfection efficiency exceeding 32%. A 30% DOPS ratio was selected, and targeting was optimized using SORT technology. The DOPS (dioleoylphosphatidylserine) ratio must be strictly controlled within 30% ± 5%. Excessive DOPS may lead to decreased liposome stability (particle size fluctuation > 20 nm) or surface charge shift (> ± 5 mV), affecting spleen targeting. The optimal molar ratio of CL15H6 to DOPS was 4:3 to ensure a balance between the protonation efficiency of the ionizable lipid and the membrane fusion ability of the auxiliary lipid.
[0048] The particle size distribution and storage stability were detected by dynamic light scattering. The carrier was kept at 4°C and there was no aggregation for 21 days. The sample dilution ratio (usually 1:100 to 1:500) needs to be optimized before testing to avoid multiple dilutions that introduce bubbles or shear forces that destroy the nanoparticle structure. When evaluating the stability of the carrier by dynamic light scattering, the sample processing process (dilution, filtration), instrument calibration conditions and storage environment parameters (temperature, light protection) need to be strictly controlled. Data analysis needs to be combined with multi-technical verification (such as flow cytometry, electron microscopy), and clear quality control standards (PDI, particle size fluctuation, encapsulation efficiency) must be established to determine stability. Batch-to-batch differences and protection of active ingredients are core issues that need to be paid attention to in long-term storage.
[0049] Mn2+ increases DC maturation by activating the STING pathway at a concentration of 0.5-2mM. 0.5-2mM Mn2+ enhances DC maturation (>85%) while balancing immune activation with systemic toxicity by dually activating the cGAS-STING pathway (boosting DNA detection sensitivity and signal transduction efficiency).
[0050] The liposome chain length is C14-C18, and ultrasound parameters are optimized at 1-3 MHz, with a CD3 antibody coupling density of 1-2 μg / mg lipid. The C14-C18 liposome chain length achieves efficient, low-toxicity T cell targeted delivery and therapeutic synergy by balancing stability and release efficiency (phase transition temperature adaptation and extended circulation half-life), combined with 1-3 MHz ultrasound parameters (enhanced tissue penetration and controlled release) and a CD3 antibody coupling density of 1-2 μg / mg (optimizing targeting efficiency and carrier stability).
[0051] In vivo delivery of mRNA at a dose of 0.1-0.5 mg / kg; PET imaging is used to confirm spleen accumulation after in vivo delivery to avoid exceeding 25% ID / g. An mRNA dose of 0.1-0.5 mg / kg can significantly improve the therapeutic index of mRNA therapy by balancing delivery efficiency and safety (liver targeting optimization, systemic toxicity avoidance), combined with PET imaging monitoring of spleen accumulation ≤25% ID / g (reducing immunogenicity and non-target organ toxicity). This strategy has proven its clinical value in metabolic diseases (such as MMA) and tumor immunotherapy.
[0052] Monitoring serum and immune tolerance after in vivo delivery can identify liver / kidney damage, CRS or allergic reactions at an early stage, reduce the risk of treatment interruption, and maintain long-term therapeutic effects through dose adjustment (such as the timing of mRNA reinjection) or immune regulation (such as inhibition of pre-existing antibodies); formulate differentiated plans based on individual immune status (such as HLA typing or baseline Treg levels) to improve treatment response rate.
[0053] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.
Claims
1. A method for constructing a spleen-targeted nanoplatform suitable for tumor vaccination, characterized in that: The following steps are involved: S1. Selection of spleen-targeted nanocarriers. CL15H6-DOPSLNPs, spherical polymersomes, and erythrocyte membrane-coated nanoparticles were selected as spleen-targeted nanocarriers based on the particle size range and surface charge conditions. S2, integrated immune adjuvant: Mn2+-doped LNPs, ultrasound-responsive liposomes, and CD3 antibody coupling were selected as integrated materials, where the adjuvants were manganese ions, TLR agonists, and CD3 antibodies, respectively; S3. In vivo delivery: Animal models were selected for in vivo delivery and efficacy verification. The animal models were B16F10 melanoma mice, MC38 colon cancer mice, and non-human primates.
2. The method for constructing a spleen-targeted nanoplatform suitable for tumor vaccination according to claim 1, characterized in that: Spleen-targeted nanocarriers require the selection of materials with a particle size greater than 100 nm and a negatively charged surface, with a surface charge of -10 mV to -25 mV.
3. The method for constructing a spleen-targeted nanoplatform suitable for tumor vaccination according to claim 1, characterized in that: CL15H6 combined with DOPS LNPs achieved efficient transfection of splenic dendritic cells, with a transfection rate greater than 32%. DOPS selected a 30% ratio, and the targeting was optimized by SORT technology.
4. The method for constructing a spleen-targeted nanoplatform suitable for tumor vaccination according to claim 1, characterized in that: The particle size distribution and storage stability were tested by dynamic light scattering, and the carrier was kept at 4°C and showed no aggregation for 21 days.
5. The method for constructing a spleen-targeted nanoplatform suitable for tumor vaccination according to claim 1, characterized in that: Mn2+ increases the DC maturation rate by activating the STING pathway, and the Mn2+ concentration is 0.5-2mM.
6. The method for constructing a spleen-targeted nanoplatform suitable for tumor vaccination according to claim 1, characterized in that: The liposome chain length is C14-C18, and the ultrasound parameters are optimized, with the ultrasound parameters being 1-3 MHz; the CD3 antibody coupling density is 1-2 μg / mg lipid.
7. The method for constructing a spleen-targeted nanoplatform suitable for tumor vaccination according to claim 1, characterized in that: In vivo delivery of mRNA, the mRNA dose is 0.1-0.5 mg / kg; Splenic accumulation was confirmed by PET imaging after in vivo delivery, avoiding >25% ID / g.
8. The method for constructing a spleen-targeted nanoplatform suitable for tumor vaccination according to claim 1, characterized in that: Serum and immune tolerance were monitored after in vivo delivery.