mRNA vaccine designed to target glioma by sequentially reaching specific organs and cells

Cell membrane and lysosome escape are key barriers to intracellular mRNA delivery⁴². The positional isomers of tLNP resulted in different mRNA delivery efficiencies, prompting us to investigate their cellular uptake and endosomal escape characteristics. Confocal laser scanning microscopy (CLSM) images showed no significant difference in lysosome escape among meta-, ortho- and para-tLNP (Figs. S7–S11). However, the cellular uptake of meta-tLNP was significantly higher than that of ortho-/para-tLNP, suggesting that the higher mRNA delivery efficiency of meta-tLNP was due to their superior cellular uptake (Fig. S12). Next, we studied the pathway of cellular uptake of tLNP by using inhibitors to block different cellular uptake pathways of nanoparticles. These results showed that the cellular uptake efficiency of mXO10 tLNP decreased by 63% after the addition of chloroquine, which altered the pH on the cell membrane surface and inhibited endocytosis⁴³. This indicated that endocytosis was the main pathway for the cellular uptake of mXO10 tLNP (Fig. 2c). On the other hand, MβCD reduced the cellular uptake efficiency of oXO10 tLNP and pXO10 tLNP by 75% and 78%, respectively, suggesting that the primary pathway for their uptake by cells was clathrin-dependent endocytosis (Fig. 2d, e). Therefore, meta-tLNP were more efficient in delivering mRNA than ortho-/para-tLNP due to their higher cellular uptake. To understand why positional isomers lead to differences in the cellular uptake of tLNP, we conducted an in-depth study on the relationship between the geometry of ionizable lipidoids and their mRNA delivery efficiency. We calculated the P values of three ionizable lipidoids (mXO10, oXO10, and pXO10). As an important parameter, the P value can provide key clues for us to predict the nanostructures that ionizable lipidoids may form under specific conditions. P = v/(ℓa), a represents the head area of the ionizable lipidoids molecule, which reflects the size and shape of the lipidoids molecule head; v and ℓ represent the tail volume and length of the ionizable lipidoids molecule, respectively. These physical properties of the tail have an important influence on the overall structure and function of the lipidoid molecule. We can calculate the P value of different ionizable lipidoids to predict the nanostructures they may form. When the P value is less than 1/3, these ionizable lipidoids molecules tend to form spherical structures during aggregation. This structure has relatively stable physical and chemical properties, but it is not as conducive to vector delivery as other structures; when 1/3 ≤ P ≤ 1/2, ionizable lipidoids will be arranged into hexagonal phase (HI phase) nanostructures; and when 1/2 < P ≤ 1, planar nanostructures will be formed. Hexagonal phase (HI phase) and planar nanostructures have different characteristics and mechanisms of action in the process of lipid nanoparticle delivery. Compared with spherical structures, they may be more conducive to vector delivery in some aspects. When P is greater than 1, this type of ionizable lipidoids has the typical structural characteristics of “small head and large tail”. This special structure makes them easier to form HII phase or reverse spherical nanostructures, and the larger the P value, the easier it is to form this structure. The interaction between HII phase and reverse spherical nanostructure and endosomal membrane in biological function effectively improves the delivery efficiency of carriers. After calculation, we found that the P value of mXO10 was 2.1, which was significantly higher than the P value of oXO10 (P = 1.6) and the P value of pXO10 (P = 1.7) (Fig. 2f, g). mXO10 was more likely to assemble into HII phase nanostructures, which provided more favorable conditions for intracellular delivery of mRNA. These experimental results indicated that, compared with oXO10 and pXO10, mXO10 was more readily assembled into HII phase nanostructures, leading to higher cellular uptake efficiency of meta-tLNP and consequently resulting in improved mRNA delivery efficiency. Thus, we selected the meta-tLNP (mXO10 tLNP and mXE14 tLNP) with the best mRNA delivery efficiency for further experiments. In the following text, mXO10 tLNP@mRNA/mXE14 tLNP@mRNA refered to the three-component lipid nanoparticles prepared using mXO10/mXE14, DOPE, and cholesterol in a 9:6:4 molar ratio, with mRNA-loaded nanoparticles prepared by microfluidics.

Cellular uptake and endosomal escape of lipid nanoparticles are critical for the intracellular delivery of mRNA. We next investigated the efficiency of mRNA delivery by mXO10 tLNP and mXE14 tLNP in cellular uptake. Cy5-labeled firefly luciferase mRNA (Cy5-mLuc) was delivered to DC2.4 and GL261 cells using mXO10 tLNP and mXE14 tLNP. CLSM images showed that both mXO10 tLNP and mXE14 tLNP effectively delivered mLuc into DC2.4 and GL261 cells compared to the control group (Figs. 4f and S23). Flow cytometry results indicated that mXO10 tLNP delivered mLuc to DC2.4 cells with 4 times the efficiency of the free mRNA group, while mXE14 tLNP was 7.7 times more efficient, demonstrating that tLNP significantly enhanced mRNA uptake and intracellular delivery efficiency (Figs. 4g and S24). The acidic environment of lysosomes protonates the ionizable lipidiods nanoparticles, allowing them to escape from lysosomes and ensure that the mRNA reaches the cytoplasm. Therefore, lysosomal escape of LNP is a critical factor in mRNA delivery. We studied the lysosomal escape of tLNP using CLSM. The result showed that, compared to the control group, naked mRNA could barely enter DC2.4 cells, whereas mXO10 tLNP@mRNA and mXE14 tLNP@mRNA showed significant lysosomal escape and a substantial amount of mRNA entering the cytoplasm, providing a foundation for efficient protein expression. The result indicated that mXO10 tLNP and mXE14 tLNP significantly improved the efficiency of mRNA uptake and lysosomal escape, enhancing the delivery and translation of mRNA into proteins. Previous data have shown that mXO10 tLNP and mXE14 tLNP effectively deliver luciferase mRNA (mLuc) into cells and efficiently translate it into luciferase. Next, we explored whether these tLNP could deliver other mRNA and efficiently translate them into proteins. mCherry mRNA (mCherry) encodes a red fluorescent protein. We used mXO10 tLNP and mXE14 tLNP to deliver mCherry mRNA into cells and observed the expression of mCherry protein in DC2.4 and GL261 cells at 3 h, 6 h, 18 h, 24 h, and 36 h using CLSM. The results showed very strong red fluorescent signals in both DC2.4 and GL261 cells, with the intensity of the red fluorescence increasing over time, peaking at around 24 h (Figs. 4h and S25). The result demonstrated that mXO10 tLNP and mXE14 tLNP effectively delivered mCherry mRNA into cells and efficiently expressed mCherry protein. Additionally, we investigated whether mXO10 tLNP and mXE14 tLNP could efficiently deliver glioma-associated antigen IDH1R132H mRNA (mIDH1). Western blotting results showed that mXO10 tLNP@mIDH1 and mXE14 tLNP@mIDH1 successfully expressed the IDH1 R132H peptide in cells (Fig. 4i). These findings indicated that mXO10 tLNP and mXE14 tLNP were capable of effectively delivering different functional mRNA and translating them efficiently into protein. Therefore, tLNP could serve as a versatile mRNA delivery platform for developing mRNA therapies for various diseases.

In addition, we evaluated the biological safety of the mXO10 tLNP@mIDH1 mRNA vaccine in vivo. As shown in Fig., no abnormal histologic changes were observed in the heart, liver, spleen, or kidney in all groups, suggesting the good biocompatibility of mXO10 tLNP@mIDH1 vaccine and mXO10 tLNP@mIDH1 vaccine plus Anti-PD-1. Furthermore, serum biochemistry parameters, including albumin (ALB), alanine aminotransferase (ALT), creatinine (CREA), and glucose (GLU), were detected, and the results showed that the blood biochemistry parameters in the control group, the mXO10 tLNP@mIDH1 vaccine group, and the mXO10 tLNP@mIDH1 vaccine plus Anti-PD-1 group were all within the safe range, indicating the good biosafety of this mRNA vaccine and its potential for future clinical application (Fig.). S31 S32

HEK293T cells, DC2.4 cells, and GL261 cells were obtained from the American Type Culture Collection (ATCC). All eukaryotic cell lines used were authenticated by short tandem repeat (STR) analysis and routinely screened for mycoplasma contamination via loop-mediated isothermal amplification (LAMP) to ensure the absence of mycoplasma. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with GlutaMAX, 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin. The lentiviral plasmids were constructed and produced by Getein Biotech, Inc.

Male BALB/c mice (6–8 weeks old, 20 g) and male C57 mice (6–8 weeks old, 20 g) were bred in a temperature- and humidity-controlled SPF condition (20–25 °C and 45–55% humidity) under 12/12 h light/dark cycles with free access to food and water. The maximum tumor volume/load approved by the Sun Yat-sen University Ethics Committee is that tumor growth must not exceed 10% of the animal’s original body weight and that the average tumor diameter in mice must not exceed 20 mm; we confirm that these limits were not exceeded during the experiment. All animal husbandry and experimental procedures were approved by the Animal Ethics Committee of the Laboratory Animal Research Center of Sun Yat-sen University.

Meta/ortho/para-xylylenediamine (Sigma-Aldrich) was individually reacted with 1,2-epoxydecane, 1,2-epoxydodecane, 1,2-epoxytetradecane, 1,2-epoxyhexadecane, or 1,2-epoxyoctadecane (Sigma–Aldrich) at a molar ratio of 1:2.2 in 95% ethanol at 85 °C for 48 h. This series of reactions resulted in the crude products mXO8, mXO10, mXO12, mXO14, mXO16, oXO8, oXO10, oXO12, oXO14, oXO16, pXO8, pXO10, pXO12, pXO14, and pXO16, respectively. Similarly, meta/ortho/para-xylylenediamine was reacted with decyl acrylate, dodecyl acrylate, tetradecyl acrylate, hexadecyl acrylate, octadecyl acrylate, or docosyl acrylate at a molar ratio of 1:2.2 in isopropanol at 85 °C for 48 h. This process yielded the crude products mXE10, mXE12, mXE14, mXE16, mXE18, oXE10, oXE12, oXE14, oXE16, oXE18, pXE10, pXE12, pXE14, pXE16, and pXE18, respectively. The m/o/pXO10 and m/o/pXE14 compounds obtained from the above reactions were purified using silica gel column chromatography. Subsequently, the purified compounds were subjected to structural verification through Nuclear Magnetic Resonance (¹H-NMR) spectroscopy.

Preparation of m/o/pXO(n + 1) tLNP@mLuc (n = 7, 9, 11, 13, 15): The m/o/pXO(n + 1) ionizable lipidoids, DOPE (Avanti Polar Lipids), and cholesterol (Sigma–Aldrich) were mixed in ethanol at a molar ratio of 9:6:4. Luciferase mRNA (mLuc) was dissolved in PBS buffer. The ethanol phase and aqueous phase were mixed at a ratio of 1:9 to obtain m/o/pXO(n + 1) tLNP@mLuc with varying N:P ratios. Similarly, the preparation of m/o/pXE(n + 3) tLNP@mLuc (n = 7, 9, 11, 13, 15) was conducted, but the mLuc was dissolved in a pH 4 citrate-sodium citrate buffer instead of PBS. This variation in pH conditions affected the interaction of tLNP with mRNA. Preparation of mXO10 tLNP@OligoT: mXO10, DOPE, and cholesterol were dissolved in ethanol at a molar ratio of 9:6:4. OligoT (a single-stranded nucleic acid sequence of 50 Ts) was dissolved in PBS buffer. The ethanol and aqueous phases were mixed at a 1:9 ratio to obtain mXO10 tLNP@OligoT. Preparation of mXO10 tLNP@mIDH1 mRNA vaccine: mXO10, DOPE, and cholesterol were dissolved in ethanol at a molar ratio of 9:6:4, and the IDH1(R132H) mRNA was prepared in PBS. The two phases were mixed at a 1:9 ratio, specifically targeting an N:P ratio of 3, to obtain the mXO10 tLNP@mIDH1 mRNA vaccine. Preparation of mXO10/mXE14 tLNP@Cy5-labeled mRNA: First, Cy5-labeled OligoT-18 (a single-stranded sequence of 18 Ts) was hybridized with mLuc by incubating them together at 37 °C for 15 min, allowing the OligoT-18 to bind to the poly-A tail of the mRNA. The tLNP preparation was then carried out following either the (1) or (2) protocol, incorporating the Cy5-labeled mLuc. Screening of tLNP in HEK-293 Cells: HEK-293 cells were seeded in a white-bottomed 96-well cell culture plate at a density of 20,000 cells per well. After 24 h of incubation, the prepared tLNP was added to the wells and co-incubated for another 24 h. Then, 100 µL of One-umi™ firefly luciferase reporter gene assay reagent (Beyotime Biotechnology) was added to each well, and the plate was gently mixed on a shaker for 10 min. The bioluminescence intensity of the cells was measured using a multimode microplate reader to evaluate the efficiency of tLNP delivery of mRNA.

A series of buffer systems with pH values ranging from 3 to 12 were prepared, and a 300 µM solution of TNS (2-(p-toluidino) naphthalene-6-sulfonic acid) (Sigma-Aldrich) was freshly prepared in DMSO (Sigma–Aldrich). Additionally, mXO10 tLNP@mLuc and mXE14 tLNP@mLuc were prepared. Different pH buffer solutions were dispensed into a black-bottomed 96-well plate (90 µL per well), followed by the addition of 10 µL of tLNP solution to each well, and the mixtures were thoroughly mixed. Subsequently, 2 µL of TNS was added to each well. The fluorescence intensity of each well was measured at an excitation wavelength of 325 nm and an emission wavelength of 435 nm using a microplate reader. The obtained fluorescence values were then plotted against the pH of the buffer solutions to generate an S-shaped curve. The logarithm of the pH value at the inflection points of this curve corresponded to the pKa value of tLNP.

An agarose gel was prepared with the addition of GEL-RED nucleic acid stain. The samples were mixed with loading buffer, and the following were loaded into separate wells: 200 ng of free luciferase mRNA (mLuc), mXE14 tLNP@mLuc + 200 ng/μL RNase, mXO10 tLNP@mLu + 200 ng/μL RNase, mXE14 tLNP@mLuc, and mXO10 tLNP@mLuc (tLNP containing 200 ng of mLuc). Electrophoresis was performed at a constant voltage of 110 V until the bromophenol blue dye migrated to approximately 1 cm from the bottom of the gel. A gel documentation system was used to image the gel.

DC2.4 cells and GL261 cells (8000 cells per well) were seeded into an 8-well chamber (Sigma-Aldrich). After 24 h of incubation, mXO10 tLNP@Cy5-labeled mRNA, mXE14 tLNP@Cy5-labeled mRNA, and free mRNA (each containing 400 ng of mRNA) were added to the respective wells and incubated for 1.5 h. The media were aspirated, and the cells were washed with PBS. The cells were then stained with Hoechst 33342 for 15 min, followed by aspiration and three washes with PBS. Next, 100 μL per well of Lysotracker Green (Thermo Fisher Scientific) staining solution (diluted 10,000-fold) was added and incubated for 30 min, followed by aspiration and further PBS washes. The prepared samples were imaged using a confocal laser scanning microscope.

BALB/c mice were injected with mXO10 tLNP@mLuc, mXE14 tLNP@mLuc, and free mLuc (at a dose of 0.5 mg/kg) via the tail vein. Bioluminescence imaging was performed at 1, 3, 6, 12, 24 h post-injection to assess the efficiency of mRNA delivery by tLNP. Additionally, BALB/c mice were injected with mXO10 tLNP@Cy5-labeled mRNA, mXE14 tLNP@Cy5-labeled mRNA, and free Cy5-labeled mRNA (at a dose of 0.1 mg/kg) via the tail vein. Fluorescence imaging was conducted using a small animal live imaging instrument at 1, 3, 6, 12, 24 h post-administration to examine the biodistribution of the tLNP.

(a) 293 T cell seeding: Seed 293T cells in a 6-well plate and culture overnight until the confluence reaches 70–90%; (b) Transfection mixture preparation: (1) Solution A: Mix the packaging plasmid, envelope plasmid, and transfer plasmid (containing the target gene) in a ratio of 0.8 μg: 0.4 μg: 1.2 μg. Add them to 100 μL of opti-MEM (serum-free and antibiotic-free), mix gently 20 times, and let it stand at room temperature for 5 min; (2) Solution B: Add 7.2 μg of PEI reagent to 100 μL of opti-MEM, mix gently 20 times, and let it stand at room temperature for 5 min; (3) Mixing and transfection: Add solution B dropwise to solution A, mixing gently after each addition. Let it stand at room temperature for 30 min. Discard the old culture medium from the 6-well plate, rinse with PBS, and add 2 mL of fresh complete culture medium. Gently add 200 μL of the transfection mixture to the cells, and gently shake the culture plate to evenly distribute the transfection mixture. Continue culturing the cells; (4) Virus collection: After 24 h, observe the expression of fluorescent protein. Collect the cell supernatant at 48 h and 72 h. Centrifuge at 4 °C and 845 × g for 10 min to remove cell debris, and filter through a 0.22 μm filter membrane; (5) Target cell seeding and infection: Seed GL261 cells in a culture plate. When the confluence reaches 30–40%, infect the cells with the virus, and add polybrene to a final concentration of 5 μg/mL. After 24 h, replace with fresh culture medium containing 10% FBS; (6) Stable cell line screening: After observing the expression of fluorescent protein, add puromycin to a final concentration of 10 μg/mL for selection. Repeated selection yields the GL261-IDH1 stable cell line.

Male C57 mice were anesthetized via intraperitoneal injection of 1% sodium pentobarbital at a dose of 50 mg/kg and then fixed onto the base of a brain stereotaxic apparatus. The head epidermis was disinfected 2–3 times with iodine and alcohol, followed by a 1.5 cm skin incision. After gently wiping the skull surface with hydrogen peroxide, the positioning needle was placed at the bregma point, designated as X = 0, Y = 0, Z = 0. At coordinates X = −1.6, Y = −4.6, a 1.5 mL syringe was used to puncture the dura mater. The positioning needle aspirated approximately 500,000 GL261 cells and was moved to X = −1.6, Y = −4.6, Z = −3. The GL261 cells, labeled with firefly luciferase, were implanted intracranially at a rate of 5 μL every 15 s. After the injection, the positioning needle was left in place for 3 min before being withdrawn at a speed of 0.01 mm/s. The skull surface was disinfected, and the skin was sutured.

On the 8th day after modeling, bioluminescence imaging was performed using a small animal live imaging system. C57 mice with bioluminescence intensity greater than 5.0 × 10⁷ and less than 5.0 × 10⁸ were selected and randomly divided into the following groups: PBS group, Free mIDH1 group, mXO10 tLNP@irrelevant group, mXO10 tLNP@mIDH1 mRNA vaccine group, Anti-PD-1 group, and mXO10 tLNP@mIDH1+Anti-PD-1 combination group. The mXO10 tLNP@mIDH1 mRNA vaccine was administered intravenously at a dose of 0.05 mg/kg on days 8, 12, and 16. Anti-PD-1 was administered intraperitoneally on days 7, 11, 14, and 18. Bioluminescence imaging was used to monitor glioma cells growth, mouse survival time, and other indicators to evaluate the efficacy of glioma mRNA vaccine therapy and its combination with Anti-PD-1 therapy.

mXO10 tLNP encapsulated with tdTomato mRNA was prepared using mXO10 as the ionizable lipid at an N:P ratio of 3. C57 mice were intravenously injected with the prepared mXO10 tLNP@ mtdTomato at a dose of 0.2 mg/kg. After 24 h, the spleen was harvested, minced, and passed through a 70 μm filter to create a cell suspension. The cell suspension was then lysed with red blood cell lysis buffer for 10 min on ice, followed by washing with PBS. Different types of immune cells were labeled using specific antibodies: DC cells were labeled with CD11c antibody, macrophages were labeled with CD11b and F4/80 antibodies, NK cells were labeled with NK1.1 antibody, and T cells were labeled with CD3 antibody. The staining process was performed on ice for 15 min, followed by centrifugation at 135 × g for 5 min to remove the supernatant. The cells were then washed once with PBS and filtered through a 300-mesh sieve before being analyzed on a flow cytometer. The flow cytometer was used to detect the expression of tdTomato mRNA in different types of immune cells isolated from the spleen. This analysis allowed us to investigate the delivery and expression of the mRNA by tLNP in specific immune cell populations.

The mXO10 tLNP@mCre was prepared using the aforementioned method with mXO10 as the ionizable lipidoids and encapsulating Cre mRNA (mCre) (N:P ratio = 3). Ai9 mice were intravenously injected with mXO10 tLNP@mCre at doses of 0.5 mg/kg and 0.2 mg/kg, respectively. After 48 h, the spleens were harvested, homogenized, and filtered through a 70-μm cell strainer to prepare a single-cell suspension. The cell suspension was treated with red blood cell lysis buffer for 3 min on ice. Subsequently, Fc receptor blocking solution was added and incubated on ice in the dark for 10 min. Different types of immune cells were labeled as follows: DC cells were labeled with CD11c antibody, macrophages were labeled with CD11b and F4/80 antibodies, NK cells were labeled with NK1.1 antibody, B cells were labeled with CD19 antibody, and T cells were labeled with CD3 antibody. During the staining process, the cells were kept on ice. After 15 min, the supernatant was removed by centrifugation at 135 × g for 5 min. The cells were washed once with PBS and then filtered through a 300-mesh sieve. The expression of tdTomato in different types of immune cells in the spleen was detected by flow cytometry.

On the 21st day after modeling, brain and spleen tissues were harvested from mice in different treatment groups. The tissues were minced and passed through a 70 μm filter to create cell suspensions. For spleen tissue, the cell suspension was treated with red blood cell lysis buffer on ice for 10 min before washing with PBS. The cells were then stained simultaneously with CD80 and CD86 antibodies on ice for 15 min. After staining, the cells were centrifuged at 135 × g for 5 min to remove the supernatant. The cells were washed once with PBS and filtered through a 300-mesh sieve before being analyzed on a flow cytometer. The number of CD80⁺CD86⁺ cells in the spleen and brain tissue samples from different treatment groups of C57 mice was quantified by flow cytometry.

On the 21st day after modeling, spleen tissues were harvested from C57 mice in different treatment groups. The tissues were homogenized using a high-speed tissue grinder in PBS, with a tissue weight to volume ratio of 1:9, and protease inhibitors were added. This process resulted in tissue homogenates. ELISA kits were used to quantify the levels of tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-12 (IL-12), and Interferon-γ (IFN-γ) in the spleen tissue homogenates from the various groups.

C57 mice administered with different treatments were sacrificed on the 21st day after modeling, and their brain tissues were harvested. The tissues were fixed in 4% paraformaldehyde for 24 h. Subsequently, the tissue samples underwent gradual dehydration using varying concentrations of ethanol (70%, 80%, 95%, and 100%) before being cleared in xylene. The dehydrated tissues were then infiltrated with molten paraffin at 60–65 °C, embedded in paraffin blocks, and sectioned using a rotary microtome. The sections were mounted on glass slides, dewaxed with a clearing agent, and rehydrated. The paraffin sections were subjected to antigen retrieval by immersing them in 0.01 M citrate buffer (pH 6.0) and heating in a microwave oven at high power until boiling, followed by 15 min at medium power. The sections were then blocked with TBS containing 10% normal serum and 1% BSA for 2 h at room temperature. Overnight incubation with specific antibodies against T cell markers CD4 and CD8 was performed at 4 °C. The slides were washed twice in TBS containing 0.025% Triton X-100 for 5 min, followed by incubation with a fluorescently labeled secondary antibody diluted in TBS with 1% BSA for 1 h. After secondary antibody incubation, the slides were washed three times with TBS for 5 min. Nuclear staining was performed using DAPI for 15 min, followed by three washes with PBS. Finally, the slides were mounted with a coverslip using a mounting medium and observed under a fluorescence microscope for imaging.

On the 21st day after modeling, spleen tissues were harvested from C57 mice in different treatment groups. The tissues were minced and passed through a 70 μm filter to create a cell suspension. Erythrocytes were lysed, and the lysing solution was removed. The cells were resuspended in PBS at a tissue weight to volume ratio of 1:19. Stimulatory agents and protein transport inhibitors, including phorbol 12-myristate 13-acetate (PMA), ionomycin, monensin, and Brefeldin A (BFA), were added to the cell suspension. The cells were incubated at 37 °C for 5 h to stimulate intracellular cytokine production. After incubation, the cells were centrifuged at 135 × g for 5 min to remove the supernatant. Fc receptor (FcR) block (1 μL per tube) was added to the cell pellet and incubated at room temperature for 10 min to block non-specific binding. CD3 and CD8 antibodies were then added to the samples and incubated on ice for 15 min to label the specific T cell subsets. The cells were centrifuged at 135 × g for 5 min, and the supernatant was discarded. To prepare the cells for intracellular staining, 300 μL of fixation buffer was added, and the cells were incubated in the dark at room temperature for 20 min. After fixation, the cells were washed and permeabilized using 100 μL of a 1:10 dilution of intracellular staining perm wash buffer. Next, 1 μL of IFN-γ antibody was added, and the cells were incubated on ice in the dark for 50 min to stain for intracellular IFN-γ. Following antibody incubation, the cells were centrifuged to remove the supernatant and washed once with PBS. The cells were resuspended in 300 μL of PBS and analyzed using a flow cytometer to quantify the number of CD3⁺CD8⁺IFN-γ⁺ cells within the spleen tissue.

On the 21st day post-modeling, blood samples were collected from the orbital sinus of C57 mice in different experimental groups. The whole blood samples were allowed to stand at 4 °C for 8 h before being centrifuged at 845 × g and 4 °C to separate the serum. The obtained serum was then stored at −80 °C to prevent repeated freeze-thaw cycles. Parameters were set on a fully automated biochemistry analyzer, and the samples were loaded for automatic measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), urea (UREA), creatinine (CREA), and uric acid (UA) levels in the serum.

On the 21st day post-modeling, mice were euthanized, and their hearts, livers, spleens, lungs, and kidneys were harvested. The organs were promptly fixed in 4% paraformaldehyde for 24 h. After fixation, the tissues were processed to create paraffin blocks. Using a microtome, sagittal sections of 5–8 μm thickness were cut from each organ block. The paraffin was removed from the sections with xylene, and the sections were then passed through a series of alcohol solutions from high to low concentrations, ending in distilled water. The deparaffinized and rehydrated tissue sections were immersed in hematoxylin solution for 5 min to stain the nuclei. Following this, the slides were briefly differentiated in acid alcohol and ammonia water for approximately 10 s each to enhance the nuclear staining contrast. The sections were rinsed under running tap water for 1 h and then briefly dipped in distilled water. To dehydrate the tissue, the slides were placed in 70% and 90% alcohol solutions for 10 min each. Finally, the sections were stained with eosin solution for 3 min to impart a pinkish-red color to the cytoplasm and other non-nucleated structures.

The analysis tools used included one-way ANOVA (Analysis of Variance) and two-way ANOVA (analysis of variance). Results are presented as mean ± SEM (standard error of the mean) to provide a comprehensive understanding of data distribution and variability. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

This study complies with all relevant ethical regulations at Sun Yat-sen University and was approved by the Institutional Review Board under ethical permit AEDGJ202301. All experimental animals were purchased from Zhuhai BesTest Biotechnology Co., Ltd.

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