Preparation method and application of lipid nanoparticles based on phenolic hydroxyl lipids for peptide antigen / manganese adjuvant co-delivery.

Abstract

This invention discloses a lipid nanoparticle co-delivery system based on phenolic hydroxyl lipids and a manganese adjuvant. Addressing the shortcomings of traditional HPV therapeutic peptide vaccines—weak immunogenicity, easy degradation and inactivation in vivo, low bioavailability of manganese ion adjuvants making spatiotemporal co-delivery with antigens, and poor encapsulation efficiency and insufficient biosafety of conventional lipid nanocarriers—this invention constructs an integrated nanovaccine delivery system by embedding phenolic hydroxyl functional lipids into a nanocarrier framework, synergistically loading HPV E6 / E7 specific antigen peptides and manganese ion immune adjuvants. This system achieves efficient delivery, immune activation, and anti-tumor efficacy, integrating the functions of efficient antigen delivery, potent immune activation, and precise anti-tumor action. The preparation process is simple and exhibits excellent stability, overcoming the limitations of traditional HPV peptide vaccines with poor efficacy when used alone. It has broad research value and clinical translation prospects in the field of precision immunotherapy for HPV-related cervical cancer, head and neck squamous cell carcinoma, and other malignant tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical and tumor immunotherapy technology, specifically relating to a lipid nanoparticle based on HPV E6 / E7 polypeptide antigen / manganese adjuvant co-delivery with phenolic hydroxyl lipids, its preparation method, and the application of the nanoparticle in the preparation of HPV-related tumor therapeutic drugs. Background Technology

[0002] Persistent infection with high-risk human papillomavirus (HPV), especially HPV types 16 and 18, is a core pathogenic factor in the development and progression of malignant tumors such as cervical cancer, anal cancer, and head and neck squamous cell carcinoma. HPV-encoded E6 and E7 oncoproteins can disrupt cell cycle regulation by degrading tumor suppressor proteins such as p53 and pRb, inducing cell immortalization and malignant transformation. Furthermore, these oncoproteins are persistently and stably expressed in HPV-positive tumor cells and show no homology to normal tissues, making them specific targets for HPV-related tumor immunotherapy.

[0003] Therapeutic vaccines based on E6 / E7 peptides have become an important research direction for precision immunotherapy of HPV-related tumors due to their advantages such as simple preparation process, high safety, and strong targeting. However, free E6 / E7 peptides have inherent defects such as weak immunogenicity, easy degradation by proteases in vivo, poor cell membrane penetration, and low intracellular delivery efficiency. They are difficult to achieve ideal anti-tumor effects when used alone, and there is an urgent need for efficient delivery systems for transport.

[0004] Manganese ions (Mn) 2+ As a novel and highly effective immune adjuvant, it can specifically activate the cyclic GMP-AMP synthase-stimulating factor interferon gene (cGAS-STING) pathway, promote dendritic cell (DC) maturation, enhance antigen presentation capacity, tandem innate and adaptive immunity, and significantly improve the immune activation effect of peptide vaccines. However, Mn 2+ It is easily metabolized and cleared in vivo, has low bioavailability, and cannot achieve spatiotemporal co-delivery with E6 / E7 peptides, making it difficult to exert synergistic effects in antigen-presenting cells. At the same time, it has potential toxicity at high concentrations, which seriously restricts its clinical application.

[0005] Lipid nanoparticles (LNPs), as ideal carriers for the delivery of biomolecules, have been widely used in vaccine delivery. They can achieve the co-delivery of antigens and adjuvants. However, traditional LNPs rely on the electrostatic interaction between ionizable lipids and drugs for encapsulation, requiring the use of excessive cationic lipids, which can easily trigger inflammatory responses and cytotoxicity. Furthermore, they exhibit low encapsulation efficiency and poor stability for substances with insignificant charge properties, such as peptides and metal ions, making them unsuitable for encapsulating E6 / E7 peptides and Mn. 2+ The need for efficient and secure co-delivery.

[0006] Phenolic hydroxyl-containing lipids, as a novel type of functional lipid, possess phenolic hydroxyl groups in their molecular structure that can form stable bonds with peptides and metal ions through hydrogen bonding and coordination, offering a new approach to address the shortcomings of traditional LNP encapsulation, such as low efficiency and high toxicity. Currently, there are no studies on the application of phenolic hydroxyl-containing lipids in the encapsulation of HPV E6 / E7 peptides and Mn... 2+ There are reports on adjuvant co-delivery and its application in the treatment of HPV-related tumors. Therefore, this study aims to develop a method based on phenolic hydroxyl lipids for the efficient co-delivery of E6 / E7 peptides and Mn. 2+ Lipid nanoparticles containing adjuvants overcome the bottlenecks of existing delivery systems and have significant clinical application value and research significance. Summary of the Invention

[0007] This invention addresses the shortcomings of existing technologies, such as weak immunogenicity and low delivery efficiency of HPV E6 / E7 peptides, low bioavailability of Mn²⁺ adjuvants and difficulty in spatiotemporal co-delivery with peptides, and low encapsulation efficiency and high toxicity of traditional LNPs. It provides a lipid nanoparticle (E6 / E7 / Mn²⁺) for co-delivering HPV E6 / E7 peptide antigen / manganese adjuvant based on phenolic hydroxyl lipids. 2+ / TA@LNP (EMT@LNP for short) is a nanoparticle that enables efficient and stable co-delivery of E6 / E7 peptides and Mn²⁺ adjuvant, enhancing immune activation and reducing toxicity.

[0008] The present invention also provides a method for preparing the above-mentioned lipid nanoparticles based on phenolic hydroxyl lipids co-delivered by peptide antigen / manganese adjuvant and their applications.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing lipid nanoparticles based on phenolic hydroxyl lipids co-delivered with peptide antigens / manganese adjuvants, comprising the following steps: 1) Synthesis of phenolic hydroxyl lipids: Aldehydes and amines were added to organic solvent one and reacted at 30±5 ℃ for 5-7 h. Then, carboxylic acid was added and the reaction continued for 1-2 h. After that, isonitriles were added and the reaction was carried out at 40±5 ℃ for 20-28 h. The product was purified by column chromatography to obtain phenolic hydroxyl lipids. 2) Preparation of lipid nanoparticles: Organic phase preparation: Phenolic hydroxyl lipids, auxiliary lipids, cholesterol, and PEG-lipids were mixed and dissolved in organic solvent II to obtain the organic phase; Aqueous phase preparation: The peptides HPV E6, HPV E7, and Mn were prepared... 2+ Tannic acid is dissolved in pure water to obtain an aqueous phase; Nanoparticle assembly: The organic phase is added to the aqueous phase, mixed evenly, and purified by dialysis to obtain the final product.

[0010] Specifically, in step 1), the molar ratio of the aldehyde, amine, carboxylic acid, and isonitrile is 1-1.2:1:1:1, and the amount of amine used is 1 mmol.

[0011] Furthermore, in step 1), the aldehyde includes 2-ethylhexanal, etc.; the amine includes dopamine, etc.; the carboxylic acid is stearic acid, etc.; the isonitrile is octadecyl isocyanate, etc.; the eluent used for column chromatography purification is a mixture of petroleum ether and ethyl acetate, preferably in a volume ratio of 10:1.

[0012] More preferably, in step 1), the organic solvent is methanol.

[0013] Specifically, in step 2), the auxiliary lipid is phospholipid, and the molar ratio of the phenolic hydroxyl lipid, phospholipid, cholesterol, and PEG-lipid is 50:10:38-39:1-2; the amount of the phenolic hydroxyl lipid is 50-60 μg, and the second organic solvent is ethanol, with an ethanol volume of 10-40 μL.

[0014] Furthermore, in step 2), the dosage of both HPV E6 and HPV E7 is 4-6 μg; Mn 2+ The molar ratio with tannic acid is 1:1.

[0015] In a further preferred embodiment, in step 2), dialysis is performed using a dialysis bag with a molecular weight cutoff of 1000 Da in pure water for 2-3 hours.

[0016] This invention provides lipid nanoparticles based on phenolic hydroxyl lipids and co-delivered by a peptide antigen / manganese adjuvant, prepared by the above method.

[0017] The present invention also provides the application of the above-mentioned peptide antigen / manganese adjuvant co-delivery lipid nanoparticles based on phenolic hydroxyl lipids in the preparation of antitumor drugs.

[0018] As a preferred technical solution, the above-mentioned method for preparing lipid nanoparticles based on phenolic hydroxyl lipid-containing peptide antigen / manganese adjuvant co-delivery includes the following steps: 1) Synthesis of phenolic hydroxyl-containing lipids: The Ugi four-component reaction requires one molecule each of aldehyde, amine, carboxylic acid, and isonitrile. 1 mmol of aldehyde and 1 mmol of amine were added to a glass vial containing 1-2 mL of methanol, an organic solvent, and reacted at 30 °C for 6 h. Then, 1 mmol of carboxylic acid was added and reacted at 30 °C for 1 h. Finally, 1 mmol of octadecyl isocyanate was added, and the reaction was carried out at 40 °C for 24 h. The resulting product was purified by column chromatography. The accuracy of the structure was verified by 1H NMR spectroscopy. 2) Preparation of lipid nanoparticles: Organic phase preparation: Phenolic hydroxyl lipids, auxiliary lipids, cholesterol, and PEG-lipids were mixed in a molar ratio of phenolic hydroxyl lipids: phospholipids: cholesterol: PEG-lipids = 50:10:38.5:1.5 (the amount of phenolic hydroxyl lipids was 55 μg), and dissolved in 15-30 μL of organic solvent ethanol to obtain the organic phase; Aqueous phase preparation: The peptides HPV E6, HPV E7, and Mn were prepared... 2+ Dissolve tannic acid in 40-80 μL of pure water to obtain an aqueous phase; wherein, HPV E6 and HPV E7 are each 5 μg, and the molar ratio of Mn is... 2+ Tannic acid = 1:1, the Mn 2+ MnCl2·4H2O was selected, with a tannic acid concentration of 1 mg / mL; Nanoparticle assembly: The organic phase is added to the aqueous phase and mixed evenly. The mixture is then dialyzed in 2 L of pure water for 2 h using a dialysis bag with a molecular weight cutoff of 1000 Da.

[0019] This invention delivers functionalized lipid nanoparticles containing HPV E6 / E7 peptides and manganese adjuvant. The carrier backbone consists of phenolic hydroxyl lipids, auxiliary lipids, cholesterol, PEG-lipids, and tannic acid, internally encapsulating HPV16 type E6 / E7 antigenic peptides and manganese adjuvant. 2+ Immunoadjuvant; the phenolic hydroxyl-containing lipid is the core carrier material, the auxiliary lipid is distearylphosphatidylcholine (phospholipid, DSPC), and the PEGylated lipid (PEG-lipid) is distearylglycerol-polyethylene glycol 2000 (DSG-PEG2000). The prepared lipid nanoparticles have an average particle size of approximately 150 nm and a dispersion index (PDI) ≤ 0.2; they are regularly spherical in appearance and exhibit excellent in vitro stability; they can be efficiently taken up by bone marrow-derived dendritic cells, achieving lysosomal escape, effectively activating the cGAS-STING immune pathway, and significantly promoting dendritic cell maturation; in TC-1 tumor-bearing mice, they can target and accumulate in draining lymph nodes, inducing antigen-specific CD8. + It exhibits T-cell response, inhibits tumor growth, and shows no significant tissue damage to major organs such as the heart, liver, spleen, lungs, and kidneys in mice, demonstrating good biocompatibility. The applicable animal model is a subcutaneous TC-1 tumor-bearing model constructed from 6-8 week old female C57BL / 6 mice. The administration method is intramuscular injection at a dose of 100 μL per mouse, administered once every 7 days for 3 consecutive times. Before application, TC-1 tumor cells must be aseptically cultured and the cell concentration adjusted to 1×10⁻⁶. 6 The tumor was inoculated at a rate of 100 μL on the back of mice near the hind limbs to establish the tumor model. Once the tumor volume reached the target level, drug treatment and subsequent testing were carried out.

[0020] This invention uses phenolic hydroxyl-containing lipids as the core carrier material, utilizing the hydrogen bonding and coordination dual effects mediated by phenolic hydroxyl groups to achieve the interaction between HPV E6 / E7 peptides and Mn. 2+ The efficient and stable co-encapsulation of adjuvants overcomes the limitations of traditional LNP encapsulation relying on electrostatic interactions, solves the technical problems of low encapsulation efficiency and poor stability of traditional LNPs for peptides and metal ions, and avoids the use of excessive cationic lipids, significantly reducing the cytotoxicity of the carrier and improving biosafety.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1) The lipid nanoparticles constructed in this invention have uniform particle size (approximately 150 nm) and good dispersibility, allowing for efficient uptake by dendritic cells and lysosomal escape, effectively protecting E6 / E7 peptides from degradation by proteases in vivo. Simultaneously, the carrier-loaded Mn... 2+ It can precisely activate the cGAS-STING pathway, link innate and adaptive immunity, promote DC cell maturation and antigen cross-presentation, significantly enhance antigen-specific cellular immune responses, effectively break HPV-related tumor immune tolerance, and has a significantly better anti-tumor effect than free peptides, single adjuvants or traditional LNP delivery systems.

[0022] 2) The preparation method of the present invention is simple, convenient to operate, and has good reproducibility. It does not require complex equipment and can achieve large-scale production. Moreover, the prepared lipid nanoparticles have excellent stability and can be stably stored for more than one month at 4 ℃, which is convenient for clinical translation and application.

[0023] 3) The lipid nanoparticles of the present invention have strong targeting and can preferentially accumulate in tumor tissues and draining lymph nodes, reducing off-target delivery. While improving the anti-tumor effect, they reduce damage to normal tissues and have good in vivo safety and clinical translation potential. They provide a new feasible solution for the precision immunotherapy of HPV-related tumors and also provide experimental basis and technical support for the design and development of a co-delivery system of peptide antigen and metal ion adjuvant. Attached Figure Description

[0024] Figure 1 The structural formulas of the aldehydes, acids, ammonia, and isocyanates selected for the preparation of phenolic hydroxyl lipids in this invention (the structural formulas of the compounds selected for the Ugi reaction); Figure 2 The images show the particle size, transmission electron microscopy (TEM) image, and zeta potential of the lipid nanoparticles of this invention: A) Particle size of EMT@LNP nanoparticles; B) TEM image of EMT@LNP nanoparticles; C) Zeta potentials of T@LNP, ET@LNP, MT@LNP, and EMT@LNP. The images show that the lipid nanoparticles have a negatively charged surface, uniform particle size, spherical shape, and good dispersibility. Figure 3The following are the infrared, Raman, and XPS spectra of the lipid nanoparticles of this invention: A) Infrared absorption spectra of T@LNP, MT@LNP, ET@LNP, and EMT@LNP; B) Infrared absorption spectrum of EMT@LNP; C) Raman spectra of MnO2 and EMT@LNP; D) XPS spectra of Mn in MnO2 and EMT@LNP; F) Changes in particle size and PDI of EMT@LNP nanoparticles over one week. Figure 4 The images show the uptake of lipid nanoparticles by Raw Blue and BMDC cells; (A) Confocal microscopy image of nanoparticles uptake by Raw Blue; (B) Flow cytometry analysis of uptake of Cy5-labeled nanoparticles by BMDCs after 4 h of incubation; and (C) Cy5 fluorescence intensity statistics. Figure 5 The activation effect of nanoparticles on BMDCs; (A) Flow cytometry detection of the expression level of the activation marker (CD80+CD86+) after nanoparticles were incubated with BMDCs for 24 h; (B) Statistical graph of the activation marker (CD80+CD86+) after nanoparticles were incubated with BMDCs for 24 h (n=3). **p<0.01, ***p<0.001, ****p<0.0001; Figure 6 Western blots of sting, p-TBK1, p-sting, and p-IRF3 proteins in TC-1 cells after different treatments; Figure 7 In the images, (A) in vivo imaging of mice immediately after intramuscular injection of Cy5-labeled E6 / E7 or EMT@LNP; (B) ex vivo imaging of axillary and inguinal lymph nodes 24 h after intramuscular injection of Cy5-labeled E6 / E7 or EMT@LNP; (C) quantitative analysis of fluorescence intensity of axillary lymph nodes; and (D) quantitative analysis of fluorescence intensity of inguinal lymph nodes (n=3). Figure 8 Tumor growth in each group of mice over 21 days; Figure 9 Nanoparticles were used to activate the innate immunity of mice. (A) Flow cytometry of CD86 expression on CD11C and (B) Statistical analysis of the data; (C) CD4 + CD8 + (D) Flow cytometry analysis of CD103 expression on DCs; (E) Flow cytometry analysis of CD103 expression on DCs and (F) Statistical analysis of CD103 expression on DCs; Figure 10 For flow cytometry analysis of immune cells within tumors. (AB)CD4 + CD8 +Representative flow cytometry plots and statistical analysis; (CD) CD3 - NK1.1 + Representative flow cytometry plots and statistical analysis; (EF) CD25 + FoxP3 + Representative flow cytometry plots and statistical analysis; (GH) CD11b + Gr-1 + Representative flow cytometry plots and statistical analysis; Figure 11 Representative hematoxylin-eosin (H&E) staining images of major organs (heart, liver, kidney, lung and spleen) of mice treated with various nanovaccines show that the organs are morphologically normal and without obvious damage. Detailed Implementation

[0025] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0026] In the following examples, all raw materials used were commercially available products that could be directly purchased or prepared using conventional techniques in the art. For example, phospholipids, cholesterol, and PEGylated lipids (PEG-lipids, distearylglycerol-polyethylene glycol 2000, DSG-PEG2000) were all purchased from Avitol, among which HPV16 E7 49-57 ( 49 RAHYNIVTF 57 HPV E7) and HPV16 E6 49-57 ( 49 VYDFAFRDL 57 HPV E6 was purchased from Shanghai Jier Biochemical Biotechnology Co., Ltd. Example 1

[0027] Preparation of lipid nanoparticles co-delivered with HPV E6 / E7 peptides and manganese adjuvant: 1) Synthesis of phenolic hydroxyl lipids: The Ugi four-component reaction requires one mole each of aldehyde, amine, carboxylic acid, and isonitrile, with the following structure: Figure 1 As shown, 1 mmol of 2-ethylhexanal and 1 mmol of dopamine were added to a glass bottle containing 1 mL of methanol and reacted at 30 °C for 6 h. Then, 1 mmol of stearic acid was added and reacted at 30 °C for 1 h. Finally, 1 mmol of octadecyl isocyanate was added and reacted at 40 °C for 24 h. The product was purified by column chromatography using petroleum ether and ethyl acetate (v / v, 10:1) to obtain the phenolic hydroxyl-containing lipid. The accuracy of the structure was verified by 1H NMR spectroscopy.

[0028] 2) Preparation of lipid nanoparticles: Organic phase preparation: Phenolic hydroxyl lipids, phospholipids, cholesterol, and PEG-lipids were mixed in a molar ratio of phenolic hydroxyl lipids: phospholipids: cholesterol: PEG-lipids = 50:10:38.5:1.5, wherein the amount of phenolic hydroxyl lipids was 55 μg. The mixture was dissolved in 20 μL of ethanol organic solvent to obtain the organic phase.

[0029] Aqueous phase preparation: HPV peptides, Mn 2+ The tannic acid was dissolved in 60 μL of pure water to obtain an aqueous phase; 5 μg each of HPV E6 and HPV E7 were present; the molar ratio of Mn was... 2+ Tannic acid = 1:1, Mn 2+ The concentration of tannic acid is MnCl2·4H2O, and the concentration of tannic acid is 1 mg / mL.

[0030] Nanoparticle assembly: The organic phase was added to the aqueous phase and mixed thoroughly. The mixture was then dialyzed in 2 L of pure water for 2 h using a dialysis bag with a molecular weight cutoff of 1000 Da to obtain the desired lipid nanoparticles E6 / E7 / Mn. 2+ / TA@LNP (abbreviated as EMT@LNP).

[0031] 3) Preparation of LNPs: Organic phase preparation: Phenolic hydroxyl lipids, auxiliary lipids phospholipids, cholesterol, and PEG-lipids were mixed at a molar ratio of phenolic hydroxyl lipids:phospholipids:cholesterol:PEG-lipids = 50:10:38.5:1.5, with 55 μg of phenolic hydroxyl lipids. The mixture was dissolved in 20 μL of ethanol organic solvent to obtain the organic phase. Nanoparticle assembly: The organic phase was added to 60 μL of pure water and mixed evenly. The mixture was dialyzed in 2 L of pure water for 2 h using a dialysis bag with a molecular weight cutoff of 1000 Da to obtain the desired lipid nanoparticles (LNPs).

[0032] 4) Preparation of TA@LNP: Organic phase preparation: Phenolic hydroxyl lipids, auxiliary lipids, cholesterol, and PEGylated lipids were mixed at a molar ratio of phenolic hydroxyl lipids: phospholipids: cholesterol: PEG-lipids = 50:10:38.5:1.5, with 55 μg of phenolic hydroxyl lipids. The mixture was dissolved in 20 μL of ethanol to obtain the organic phase. Aqueous phase preparation: Tannic acid (TA) was dissolved in 60 μL of pure water to obtain the aqueous phase; the TA concentration was 1 mg / mL. Nanoparticle assembly: The organic phase was added to the aqueous phase and mixed thoroughly. The mixture was dialyzed in 2 L of pure water for 2 h using a dialysis bag with a molecular weight cutoff of 1000 Da to obtain the desired lipid nanoparticles TA@LNP (abbreviated as T@LNP).

[0033] 5) Mn 2+Preparation of / TA@LNP: Organic phase preparation: Phenolic hydroxyl lipids, auxiliary lipids, cholesterol, and PEG-lipids were mixed in a molar ratio of phenolic hydroxyl lipids: phospholipids: cholesterol: PEG-lipids = 50:10:38.5:1.5, with 55 μg of phenolic hydroxyl lipids. The mixture was dissolved in 20 μL of ethanol to obtain the organic phase. Aqueous phase preparation: Mn... 2+ The tannic acid was dissolved in 60 μL of pure water to obtain an aqueous phase; wherein, the molar ratio of Mn was... 2+ Tannic acid = 1:1, Mn 2+ The tannin concentration was 1 mg / mL, and the organic phase was added to the aqueous phase and mixed thoroughly. The mixture was then dialyzed in 2 L of pure water for 2 h using a dialysis bag with a molecular weight cutoff of 1000 Da to obtain the desired lipid nanoparticles (MnCl2·4H2O). 2+ / TA@LNP (abbreviated as MT@LNP).

[0034] 6) Preparation of E6 / E7 / TA@LNP: Organic phase preparation: Phenolic hydroxyl lipids, auxiliary lipids, cholesterol, and PEGylated lipids were mixed at a molar ratio of phenolic hydroxyl lipids: phospholipids: cholesterol: PEG-lipids = 50:10:38.5:1.5, with 55 μg of phenolic hydroxyl lipids. The mixture was dissolved in 20 μL of ethanol to obtain the organic phase. Aqueous phase preparation: HPV peptides and tannic acid were dissolved in 60 μL of pure water to obtain the aqueous phase; 5 μg each of HPV E6 and E7 were present, and the TA concentration was 1 mg / mL. Nanoparticle assembly: The organic phase was added to the aqueous phase and mixed thoroughly. The mixture was dialyzed in 2 L of pure water for 2 h using a dialysis bag with a molecular weight cutoff of 1000 Da to obtain the desired lipid nanoparticles E6 / E7 / TA@LNP (abbreviated as ET@LNP).

[0035] 7) Preparation of Cy5-labeled E6 / E7 (Cy5-E6 / E7): Weigh 0.021 g of sodium bicarbonate and 0.0265 g of sodium carbonate, dissolve them in 200 mL of deionized water, stir to dissolve, adjust the pH to 8.5-9.0, and bring the volume to 250 mL using a volumetric flask. Filter 1 mL of the solution through a 0.22 µm filter membrane. Weigh 1 mg of each of the peptides HPV E6 and HPV E7, and dissolve them in 2 mL of sodium bicarbonate buffer at pH 8.5. Dissolve the dye: Dissolve 0.62 mg of the dye NHS-Cy5 in 80 μL of anhydrous DMSO to prepare a solution (vortex to mix, store in the dark). Mix the protein and dye: Transfer 2 mL of the E6 solution and E7 solution to 1.5 mL of brown, light-protected centrifuge tubes and place them in an ice bath. Add dye dropwise: Slowly add 80 μL of NHS-Cy5 DMSO solution using a pipette, while gently mixing with a pipette. Incubate in the dark, wrap the centrifuge tube with aluminum foil and let it stand at room temperature for 1 hour, then place it on a shaker at 25 ℃ (150 rpm) for 1 hour. Terminate the reaction: Add 50 μL of 50 mM glycine (pH 7.4), mix gently, and incubate at room temperature in the dark for 15 minutes. Purify by dialysis in the dark: Pre-treat the dialysis bag, cut an appropriate length (capable of holding 25 mL of liquid), and soak it in deionized water for 5 minutes. Immerse the dialysis bag in deionized water and boil for 1 hour, then wash it three times with deionized water at 60 ℃. Temporarily soak it in dialysis buffer (PBS) for later use. Dialysis: Immerse the dialysis bag in 2 L of pure water (pH=7.4) and dialyze at 4 ℃ with vigorous stirring for 6-8 hours. Change the buffer: Replace with 2 L of pure water and continue dialysis for 8 hours. Final dialysis: Repeat the buffer replacement 2-3 times, with a total dialysis time of 24-48 hours (ensuring complete removal of free dye). Sample collection: Open the dialysis bag and transfer the purified Cy5-E6 / E7 solution to a light-protected centrifuge tube for subsequent experiments.

[0036] 8) Preparation of Cy5-labeled EMT@LNP lipid nanoparticles: Replace E6 / E7 with Cy5-E6 / E7, and follow the same steps as described in the preparation of lipid nanoparticles (EMT@LNP). The prepared sample is referred to as Cy5-EMT@LNP. Example 2

[0037] 1. Characterization of lipid nanoparticles.

[0038] 80 μL of the prepared lipid nanoparticles were mixed thoroughly in 900 μL of deionized water and then added to a particle size analyzer or a zeta potential analyzer. The particle size and zeta potential of each nanoparticle were measured using a Malvern laser particle size analyzer. The morphology of the EMT@LNP lipid nanoparticles was characterized using transmission electron microscopy. The results are shown below. Figure 2 .

[0039] Figure 2 It can be seen that the prepared lipid nanoparticles EMT@LNP nanoparticles are a complex with a diameter of about 150 nm, a dense spherical morphology, and a negatively charged surface.

[0040] 2. E6 / E7 peptides and Mn 2+ An investigation into the interaction forces between lipid nanoparticles.

[0041] Further analysis of E6 / E7 and Mn was conducted using infrared spectroscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy. 2+ The interaction force between TA@LNP. See the results. Figure 3 , Figure 3 It can be seen that in Fourier transform infrared (FTIR) spectroscopy, the absorption peak of the hydroxyl stretching vibration of the nanoparticles is from 3397.76 cm⁻¹. −1 Moved to 3424.13 cm −1 ( Figure 3 A), at the same time Figure 3 In B, it can be seen at 420 cm -1 and 523 cm -1 There is a significant Mn-O stretching vibration. To further prove that the Mn-O stretching vibration is due to the interaction between the phenolic hydroxyl functional groups in LNP and TA and Mn... 2+ The coordination effect, rather than Mn 2+ It was oxidized to MnO2, and then further characterized using Raman spectroscopy, such as... Figure 3 As shown in C, MnO2 at 662.19 cm⁻¹ -1 There is a distinct Mn-O octahedral-enhanced symmetric stretching vibration at this location, while in EMT@LNP, at 507.46 cm⁻¹... -1 The location is Mn 2+ Coordination with phenolic hydroxyl groups, and XPS results indicate that Mn is involved in the coordination of Mn. 2+ The form of existence ( Figure 3 DE). These results all indicate that E6 / E7, Mn 2+ It binds to TA and LNP through hydrogen bonding and coordination. The results are as follows: Figure 3 As shown in Figure F, EMT@LNP exhibits only negligible size changes in particle size and PDI after one week of storage, indicating its good stability. Example 3

[0042] Evaluation of the in vitro bioactivity of lipid nanoparticles.

[0043] 1. Cellular Uptake Assay: Using bone marrow-derived dendritic cells (BMDCs) as a model, lipid nanoparticles (fluorescently labeled) prepared in Example 1 were co-cultured with BMDCs in 1640 complete medium at 37 °C and 5% CO2 for 4 h. Cellular uptake efficiency was detected by flow cytometry. The uptake of Cy5-labeled lipid nanoparticles by cells was further investigated using laser confocal microscopy. Using Raw Blue cells as a model, Cy5-labeled E6 / E7 and EMT@LNP were added to confocal dishes and incubated in a cell culture incubator in the dark for another 4 h. Then, 50 μL of nuclear dye containing 4,6-diamidinyl-2-phenylindole (DAPI) was added, and the uptake of lipid nanoparticles by cells was observed under a confocal microscope. Results are shown below. Figure 4 .

[0044] Figure 4 The results show that: Figure 4 As shown in Figure A, a clear separation between the red signal from Cy5-E6 / E7 and the green signal from Lysotracker Green was observed within the cells, indicating that EMT@LNP possesses strong lysosomal escape capabilities. To further demonstrate this result, we investigated the uptake of nanoparticles by BMDC cells using flow cytometry, such as... Figure 4 As shown in the flow cytometry in BC, after incubation with cells, both the E6 / E7 and EMT@LNP groups showed a certain degree of rightward shift in the Cy5 channel compared to the PBS group, indicating that both groups could be taken up by BMDCs. Furthermore, the rightward shift was more pronounced in the EMT@LNP group than in the E6 / E7 group. Quantitative analysis of Cy5 fluorescence intensity revealed that the fluorescence intensity of the EMT@LNP group was significantly higher than that of the E6 / E7 group, indicating that the nanoparticle delivery carrier enhanced the cell's uptake capacity.

[0045] 2. BMDC cell maturation assay: Lipid nanoparticles and BMDCs were co-cultured in 1640 complete medium at 37 ℃ and 5% CO2 for 24 h. The expression of DC cell maturation markers (CD80, CD86) was detected by flow cytometry. Results are shown below. Figure 5 .

[0046] Figure 5 The results showed that, compared with the control group (free polypeptide + Mn²⁺), the lipid nanoparticles of the present invention could significantly upregulate the expression levels of CD80 and CD86 (P<0.01), indicating that they can effectively promote the maturation of DC cells.

[0047] 3. Nanoparticle activation of the cGAS-STING pathway in TC-1 tumor cells: TC-1 tumor cells were incubated with PBS, free E6 / E7, E6 / E7@LNP (ET@LNP), and EMT@LNP in 1640 complete medium at 37 °C and 5% CO2 for 4 h. Proteins were lysed and extracted, and protein concentrations were determined using a BCA protein assay kit. Equal amounts of protein were then separated by electrophoresis on a 10% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked in a protein-free rapid blocking buffer for 30 min. After blocking, the membrane was incubated overnight at 4 °C with primary antibodies against p-STING (72971, CST), p-TBK1 (5483, CST), p-IRF-3 (29047, CST), and STING (13647, CST). After washing, the membrane was incubated with secondary antibodies at room temperature for 1.5 h, and then imaged using a gel imaging system. Results are shown below. Figure 6 .

[0048] Figure 6 The results of Western blotting show that EMT@LNP increased the expression of p-TBK1, p-sting, and p-IRF3, while the overall levels of these proteins did not change significantly. These results further demonstrate that Mn 2+ The introduction of nanoparticles enabled the successful activation of the cGAS-STING pathway in TC-1 tumor cells.

[0049] 4. Enrichment of nanoparticles in mouse lymph nodes: Six- to eight-week-old female C57 BL / 6 mice were intramuscularly injected with lipid nanoparticles or the corresponding free forms of E6 / E7 at a dose of 0.1 mL / mouse, equivalent to 5 μg of Cy5-labeled E6 / E7. Mice were euthanized 24 hours post-injection (n=3), and inguinal and axillary lymph nodes were excised. Small animal in vivo imaging was used for imaging. Results are shown below. Figure 7 .

[0050] like Figure 7 As shown, both Cy5-E6 / E7 and Cy5-EMT@LNP showed significant enrichment in inguinal lymph nodes, while Cy5-EMT@LNP also showed significant enrichment in axillary lymph nodes. Furthermore, quantitative analysis revealed that the fluorescence intensity of both inguinal and axillary lymph nodes in the Cy5-EMT@LNP group was higher than that in the Cy5-E6 / E7 group. Example 4

[0051] Evaluation of the in vivo antitumor effect of lipid nanoparticles.

[0052] 1. Tumor-bearing model establishment: TC-1 tumor cells (HPV16 positive) were subcutaneously injected into the back of 6-8 week old female C57BL / 6 mice, with each mouse receiving 100 μL (1×10⁻⁶) of the tumor-bearing cell line. 6 (1 cell) to construct a TC-1 tumor-bearing mouse model.

[0053] 2. Grouping and administration: Tumor-bearing mice were randomly divided into 3 groups of 6 mice each: control group (PBS buffer), free polypeptide group (E6 / E7 polypeptide), and lipid nanoparticle group of the present invention. Administration was started on the 7th day after tumor inoculation, by intramuscular injection, with a dose of 0.1 mL / mouse each time, once every 7 days, for a total of 3 times.

[0054] 3. Tumor growth monitoring: Mouse body weight and tumor volume were measured every 3 days, and mouse survival time was recorded. Results are shown below. Figure 8 .

[0055] Figure 8 The results showed that, compared with the other two groups, the tumor growth rate of mice in the lipid nanoparticle group of this invention was significantly slower, indicating that the EMT@LNP vaccine exhibited stronger anti-tumor activity during treatment.

[0056] 4. Immune Response Detection: After sacrifice, the proportion of E6 / E7 antigen-specific CD8⁺ T cells in lymph nodes and tumor tissues was detected by flow cytometry. Results are shown below. Figure 9 and Figure 10 .

[0057] Figure 9 The results showed that both E6 / E7 and EMT@LNP significantly upregulated the activation marker CD11C in DCs. + CD86 + The expression levels of Mn were significantly increased, with EMT@LNP showing the strongest upregulation, indicating that EMT@LNP can activate antigen-presenting cells in lymph nodes, particularly Mn. 2+ It can further enhance the activation effect. Simultaneously, it expands T cells, replenishing the immune "force," and EMT@LNP can significantly upregulate CD4 in DCs. + CD8 + and CD11C + CD103 + The expression levels of these substances were observed. These results collectively confirm that EMT@LNP can effectively drive the proliferation and activation of CD8⁺ T cells in lymph nodes by promoting dendritic cell maturation and enhancing antigen cross-presentation, thereby further translating into anti-tumor effects at the tumor site. The proportions of antigen-specific CD86, CD8, and CD103 cells in the spleen of mice in the lipid nanoparticle group of this invention were significantly higher than in the other two groups (P<0.001), indicating that it can effectively induce antigen-specific cellular immune responses.

[0058] Figure 10 The results showed that EMT@LNP effectively drove the proliferation and activation of CD8⁺ T cells in lymph nodes by promoting dendritic cell maturation and enhancing antigen cross-presentation. Subsequently, these activated immune cells were effectively recruited to the tumor microenvironment. Experimental results indicated that CD8⁺ T cells in the tumor sites of mice in the EMT@LNP group increased significantly. + T cells ( Figure 10 AB), CD3⁺NK1.1⁺ co-killing cells ( Figure 10 The ratio of CD25⁺Foxp3⁺ regulatory T cells (CD25⁺Foxp3⁺ T cells) showed a significantly increased proportion compared to other groups, forming a synergistic killing effect of adaptive and innate immunity; at the same time, EMT@LNP also significantly reduced the proportion of immunosuppressive cells in the tumor microenvironment, including CD25⁺Foxp3⁺ regulatory T cells (CD25⁺Foxp3⁺ T cells). Figure 10 EF), CD11b⁺Gr-1⁺myeloid-derived suppressor cells ( ...) Figure 10 The proportion of GH was significantly reduced, thereby breaking the immunosuppression. In summary, the anti-tumor mechanism is the presence of Mn in EMT@LNP. 2+ The adjuvant activates the sting pathway, which then triggers an effective and specific immune response against the antigen. The activated T cells then migrate from the lymph nodes to the tumor site, thereby transforming the TME from suppression to killing.

[0059] 5. In vivo safety evaluation: After sacrifice, the mice were dissected and the main organs, including the heart, liver, spleen, lungs, and kidneys, were separated. Paraffin sections were prepared and stained with hematoxylin and eosin (HE). The organ morphology was observed under a microscope. Results are shown below. Figure 11 .

[0060] Figure 11 The results showed that there were no obvious tissue damage or inflammatory infiltration in the heart, liver, kidneys, lungs and spleen, indicating that the lipid nanoparticles of the present invention have good in vivo safety.

[0061] In summary, addressing the shortcomings of traditional HPV therapeutic peptide vaccines, such as weak immunogenicity, easy degradation and inactivation in vivo, low bioavailability of manganese ion adjuvants and difficulty in achieving spatiotemporal co-delivery with antigens, and poor encapsulation efficiency and insufficient biosafety of conventional lipid nanocarriers, this invention constructs an integrated nanovaccine delivery system that combines efficient delivery, immune activation, and anti-tumor efficacy by embedding phenolic hydroxyl functional lipids into the nanocarrier framework and synergistically loading HPVE6 / E7 specific antigen peptides and manganese ion immune adjuvants. A series of characterization tests, including dynamic light scattering, transmission electron microscopy, infrared spectroscopy, Raman spectroscopy, XPS, and flow cytometry, confirmed that the functionalized lipid nanoparticles have regular morphology, good dispersibility, and excellent antigen and adjuvant encapsulation efficiency. In vitro cell experiments showed that the nanocarrier could be efficiently taken up by dendritic cells and stably released intracellularly through multiple interactions mediated by phenolic hydroxyl groups, effectively activating the cGAS-STING immune pathway. In vivo anti-tumor experiments showed that the delivery system could significantly induce antigen-specific immune responses and inhibit tumor proliferation and growth. Histopathological and safety tests proved that the carrier did not cause significant damage to the major organs of mice and had good in vivo biocompatibility. The HPV peptide-manganese adjuvant co-delivery nanovaccine constructed in this invention achieves the functional integration of efficient antigen delivery, potent immune activation, and precise anti-tumor action. The preparation process is simple and the stability is excellent, overcoming the limitations of traditional HPV peptide vaccines with poor efficacy when used alone. It has broad scientific research value and clinical translation prospects in the field of precision immunotherapy for HPV-related cervical cancer, head and neck squamous cell carcinoma, and other malignant tumors.

Claims

1. A method of preparing a polypeptide antigen / manganese adjuvant co-delivery lipid nanoparticle based on a phenolic hydroxyl group-containing lipid, characterized by, Includes the following steps: 1) Synthesis of phenolic hydroxyl lipids: Aldehydes and amines were added to organic solvent one and reacted at 30±5 ℃ for 5-7 h. Then, carboxylic acid was added and the reaction continued for 1-2 h. After that, isonitriles were added and the reaction was carried out at 40±5 ℃ for 20-28 h. The product was purified by column chromatography to obtain phenolic hydroxyl lipids. 2) Preparation of lipid nanoparticles: Organic phase preparation: Phenolic hydroxyl lipids, auxiliary lipids, cholesterol, and PEG-lipids were mixed and dissolved in organic solvent II to obtain the organic phase; Aqueous phase preparation: polypeptides HPV E6, HPV E7, Mn 2+ and tannic acid were dissolved in pure water to obtain the aqueous phase; Nanoparticle assembly: The organic phase is added to the aqueous phase, mixed evenly, and purified by dialysis to obtain the final product.

2. The method of claim 1, wherein the method of preparing the polypeptide antigen / manganese adjuvant co-delivery lipid nanoparticle based on phenolic hydroxyl group-containing lipid is characterized by, In step 1), the molar ratio of the aldehyde, amine, carboxylic acid, and isonitrile is 1-1.2:1:1:1, and the amount of amine used is 1 mmol.

3. The method of claim 2, wherein the method of preparing the polypeptide antigen / manganese adjuvant co-delivery lipid nanoparticle based on phenolic hydroxyl group-containing lipid is characterized by, The aldehyde includes 2-ethylhexanal; the amine includes dopamine.

4. The method of claim 2, wherein the preparation of the polypeptide antigen / manganese adjuvant co-delivery lipid nanoparticle based on phenolic hydroxyl group-containing lipid is characterized by, The carboxylic acid is stearic acid; the isonitrile is octadecyl isocyanate; and the eluent used for column chromatography purification is a mixture of petroleum ether and ethyl acetate.

5. The method for preparing peptide antigen / manganese adjuvant co-delivery lipid nanoparticles based on phenolic hydroxyl lipids as described in claim 1, characterized in that, In step 1), the organic solvent is methanol.

6. The method for preparing peptide antigen / manganese adjuvant co-delivery lipid nanoparticles based on phenolic hydroxyl lipids as described in claim 1, characterized in that, In step 2), the auxiliary lipid is phospholipid, and the molar ratio of the phenolic hydroxyl lipid, phospholipid, cholesterol, and PEG-lipid is 50:10:38-39:1-2; the amount of the phenolic hydroxyl lipid is 50-60 μg, and the second organic solvent is ethanol, with an ethanol volume of 10-40 μL.

7. The method for preparing polypeptide antigen / manganese adjuvant co-delivery lipid nanoparticles based on phenolic hydroxyl lipids as described in claim 6, characterized in that, In step 2). The amount of HPV E6, HPV E7 was 4-6 μg; Mn 2+ The molar ratio with tannic acid was 1:

1.

8. The method for preparing polypeptide antigen / manganese adjuvant co-delivery lipid nanoparticles based on phenolic hydroxyl lipids as described in claim 6, characterized in that, In step 2), dialysis is performed using a dialysis bag with a molecular weight cutoff of 1000 Da in pure water for 2-3 hours.

9. Peptide antigen / manganese adjuvant co-delivery lipid nanoparticles based on phenolic hydroxyl lipids prepared by any one of the methods described in claims 1 to 8.

10. The use of the polypeptide antigen / manganese adjuvant co-delivery lipid nanoparticles based on phenolic hydroxyl lipids as described in claim 9 in the preparation of antitumor drugs.

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