Ethical approval for animal studies
All procedures involving animal were approved by the Institutional Animal Care and Use Committee (IACUC) of Southwest University Laboratory Animal Center (Approval No. IACUC-20230529-02). The experiments were conducted in accordance with the guidelines of the Ethical Review Committee for Experimental Animals at Southwest University, China.
Chemicals and reagents
Lipids including DOPC (S01007), DSPC (S01005), DOPE (S03005), DMG-PEG2000 (O02005), cholesterol (O01001) and Dlin-MC3-DMA (AVT0003) were obtained from A.V.T. Pharmaceutical Ltd. (Shanghai, China) with a purity of over 98%. siRNA sequences targeting DRD3 (siDRD3: GGUGGAGUCUGGAAUUUCA), BACE1 (siBACE1: GAACCUAUGCGAUGCGAAUTT), and VEGF (siVEGF: GGAGUACCCUGAUGAGAUCTT) were purchased from Shanghai Sangon Biotechnology Co., Ltd. Antibodies were acquired as follows: anti-BACE1 (ab183612, 1:1000) from Abcam (Cambridge, MA, USA), phospho-GSK3β (Ser9) (PA5-97339, 1:1000) and phospho-Tau (Ser396) (44-752G, 1:1000) from Invitrogen (Carlsbad, CA, USA), and anti-beta amyloid 1-16 (bs-10558R, 1:100) from Bioss Biotechnology Co., Ltd. (Beijing, China). GAPDH Polyclonal Antibody (10494-1-AP, 1:10000), Fluorescein (FITC)–conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (SA00003-2, 1:100), and horseradish peroxidas (HRP)-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (SA00001-2, 1:5000) were purchased from Proteintech Group, Inc. (Wuhan, China). D-Luciferin was sourced from Gold Biotechnology Co., Ltd. (St Louis, USA), and LysoRed (KGMP006) from KeyGEN Biotechnology Co., Ltd. (Jiangsu, China). eGFP mRNA (05297410) was supplied by Novoprotein Technology Co., Ltd. (Suzhou, China). ELISA kits and biochemical criterion kits were obtained from Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China) and Grace Biotechnology Co., Ltd. (Suzhou, China), respectively. All other reagents used were of analytical grade.
Cell culture
bEnd.3 and GL261-Luc cell lines were acquired from KeyGen Biotech (Jiangsu, China). Cells were cultured in DMEM (KeyGen) supplemented with fetal bovine serum (FBS, Gibco) and maintained at 37°C in a humidified atmosphere containing 5% CO2. DRD3−/− bEnd.3 cells were transfected with siDRD3 using Lipofectamine 2000 (Invitrogen).
Cryptococcus neoformans (C. neoformans)strains
To create luciferase-expressing strains of C. neoformans (wild-type H99), the luciferase gene LUC1 was amplified using primers TL1943/1944 from plasmid pCDW104-Luciferase. The amplified product was cloned into vector pTBL6 to construct pTBL402 (P_ACTIN-LUC1). The resulting vector was linearized with XbaI, precipitated onto gold microcarrier beads (0.6 μm, Bio-Rad) and biolistically transformed into the H99 ura5 strain as described previously61. Transformants were selected for stability on SD-URA medium.
Animal housing conditions
Male and female Balb/c mice (5 weeks, 18–22 g), C57BL/6 mice (8 months, 28–32 g), and Sprague Dawley (SD) rats (180–220 g) were obtained from Chongqing Academy of Chinese Materia Medica (Chongqing, China). Additionally, APP-PS-1 mice (8 months, 28–32 g) were procured from Viewsolid Biotechnology Co., Ltd. (Beijing, China). All animals were maintained in a pathogen-free environment under a 12 h light/dark cycle at 22–24°C and 30–50% humidity.
Synthesis of tetrahydroberberinederivatives
N6, N6-bis (2-hydroxy-dodecyl) lysine (180 mg, 0.35 mmol) was dissolved in dry dichloromethane (30 mL). N, N-dicyclohexyl carbodiimide (DCC, 124 mg, 0.6 mmol) and dimethylaminopyridine p-toluene sulfonate (DPTS, 177 mg, 0.6 mmol) were added and stirred under nitrogen at room temperature for 30 min. Tetrahydrotetrandrine (34 mg, 0.1 mmol) in N, N-dimethylformamide (DMF, 2 mL) was then added. The reaction was stirred under nitrogen at room temperature for 24 h, monitored by thin layer chromatography. The final product was filtered, concentrated under vacuum, and purified by column chromatography. The structures were confirmed by 1H nuclear magnetic resonance (NMR, Bruker, USA) with the following shifts (400 MHz, DMSO-d6+C2D4O2): δ6.98–6.90 (m, 2H), δ6.81 (s, 1H), δ6.58 (s, 1H), δ4.65–4.52 (m, 2H), δ4.32–4.22 (d, 1H), δ3.81–3.66 (m, 14H), δ3.65– 3.52 (m, 3H), δ2.01–2.81 (m, 8H), δ2.81–2.67 (m, 3H), δ1.20–1.11 (m, 42H), δ0.77–0.73 (m, 6H).
Preparation of BE-LNP
Tetrahydroberberine-derivative lipid nanoparticles (BE-LNP) were synthesized using a microfluidic approach. The lipid components, comprising ionizable lipids, helper lipids, cholesterol, and PEG-DMG2000, were dissolved in ethanol at specific ratios detailed in Supplementary Table S1. The ionizable lipid component included various tetrahydroberberine derivatives selected from our molecular library, while helper lipids involved DOPE, DOPC, and DSPC, aligned with best practices in nanoparticle formulation. This lipid mixture was subsequently combined with a 6.25 mM sodium acetate buffer (pH 5.0), containing siRNA, at a water-to-ethanol ratio of 3:1. Homogeneous mixing was achieved using a microfluidic mixer (AITESEN, China). Following mixing, the preparations underwent overnight dialysis against PBS (pH 7.4) to remove residual ethanol and unincorporated components. Concentration of the nanoparticles was performed using an ultracentrifugal filter unit (Millipore, Billerica, MA), optimizing the formulation for further in vitro and in vivo testing.
Gel retardation assay for siRNA
For the siRNA gel retardation assay, siRNA (1 OD) was dissolved in 125 μL of diethyl pyrocarbonate (DEPC)-treated water and mixed with varying weight ratios of siRNA to LNP. After 30 min at room temperature, the complexes containing 500 ng of siRNA were mixed with 6× loading buffer, and then were electrophoresed on a 2% agarose gel containing 0.02% Goldview gel stain. Electrophoresis was conducted at 180 V for 20 min in 1× tris-acetate-EDTA (TAE) buffer. Gel analysis was performed using a gel image analysis system (Tanon, China).
For mRNA studies, mRNA (1 mg/mL) was prepared in DEPC-treated water and mixed with LNPs at a fixed weight ratio (1:5). Samples were incubated at room temperature for varying durations (0, 0.25, 0.5, 1, 2, 4 h). Complexes containing 1.5 ug of mRNA were analyzed using a similar gel electrophoresis procedure as described for siRNA, but with 0.02% GelRed gel stain. After electrophoresis, gels was analyzed with the same gel image analysis system.
Physiochemical characterization of BE-LNP
The physicochemical properties of BE-LNP were thoroughly assessed. Particle size and zeta potential were determined using dynamic light scattering (DLS) with a Zetasizer (Malvern, England). The structural morphology of the LNPs was examined using transmission electron microscopy (TEM) (HITACHI, Japan).
Determination of the acid dissociation constant (pKa)
For pKa determination, BE-LNP was first prepared and diluted to a concentration of approximately 10 mM in phosphate-buffered saline (PBS). A specialized buffer solution was then prepared, containing 130 mM NaCl, 100 mM ammonium acetate, 10 mM HEPES, and 10 mM MES. This solution was pH-adjusted using 0.1 M NaOH or HCl to span the desired pH range. In a black 96-well plate, 95 μL of each pH-adjusted buffer, 5 μL of BE-LNP, and 1μL of TNS solution (100 μM) were combined. Fluorescence spectra were recorded at room temperature using an excitation wavelength of 321 nm and an emission wavelength of 445 nm. The pKa was accurately determined by identifying the pH corresponding to half of the maximum fluorescence intensity, a recognized method for pKa determination.
Poly(A) binding experiments
Fluorescence measurements were conducted using a black microplate (96-well, Corning, USA) on a Multifunctional Microplate Reader at an excitation wavelength of 350 nm. Reactions were carried out in BPES buffer (1.5 mM Na2HPO4, 0.5 mM NaH2PO4, 0.25 mM EDTA, 6 mM NaCl) or citrate-phosphate (CP) buffer (5 mM Na2HPO4) with pH adjusted to 7.1 and 4.5 using citric acid, following established protocols49. A blank buffer and a control solution containing only polyA were measured to account for background signals. Fresh solutions of polyA and the alkaloid were prepared daily at a concentration of 10 μM.
Cellular uptake and endocytosis mechanism analysis
Cultured bEnd.3 and DRD3−/− bEnd.3 cells were seeded in culture plates and incubated at 37°C with 5% CO2 for 12 h. Cellular uptake was assessed following the introduction of LNP@DiD formulations to the cells for a 2 h duration. To investigate endocytotic pathways, bEnd.3 cells underwent treatment with chlorpromazine (12.5 μM), filipin (12.5 μM), monensin (20 μM), brefeldin A (40 μM), colchicine (25 μM), and fresh DMEM for 30 min. Post-treatment, LNP@DiD was added to the corresponding wells, and cells were incubated for an additional 2 h at 37°C. Post-incubation, cellular imaging was performed using a high-content analysis system (Operetta CLS, PerkinElmer, USA). Cells were then subjected to trypsinization (0.25%), followed by rigorous PBS washes, and resuspended in 300 μL of PBS for flow cytometric analysis using a flow cytometer (FACSverse, BD) with FlowJo 7.6 software.
Intracellular transfection and endosomal escape
bEnd.3 cells were seeded and incubated for 12 h at 37°C with 5% CO2. For transfection, cells were exposed to various LNP@FAM-siRNA formulations in serum-free medium. Following the experiment, cells were trypsinized (0.25%), washed thrice with PBS, and resuspended in 300 μL of PBS. Transfection efficiency was quantified using flow cytometry with FlowJo 7.6 software.
For detailed intracellular analysis, cells were stained with Lysotracker Red (KeyGEN BioTECH, China) at 37°C for 2 h, followed by exposure to LNP@FAM-siRNA formulations in serum-free medium for 4 h. Post-incubation, cells were washed thrice with PBS and stained with Hoechst 33342 (Beyotime, China) for 10 min at room temperature. Images were captured using a high-content analysis system (Operetta CLS, PerkinElmer, USA).
Molecular docking
Crystal structures of the dopamine D3 receptor (PDB ID: 3PBL)62 and poly(A) (PDB ID: 3GIB)63 were retrieved from the RCSB Protein Data Bank. Using Chem3D 14.0, three-dimensional structures of the small molecule compounds were constructed and minimized using the MMFF94 force field. The protein structures were prepared with PyMol 2.5.464 by removing hydrogen atoms, water molecules, and other non-ligand molecules. A bounding box, or "butt box" was created around the active protein pocket. Both small compounds and receptor proteins were converted to PDBQT format using ADFRsuite 1.065 for compatibility with AutoDock Vina. Docking was conducted using AutoDock Vina 1.1.266 with a conformational search detail of 32, with other parameters at default settings. The conformation exhibiting the highest affinity score was chosen for further analysis and visualized using PyMol version 2.5.4.
Isolation and cultivation of primary brain microvascular endothelial cells
The isolation and cultivation of primary Brain Microvascular Endothelial Cells (BMECs) were conducted using a detailed protocol67 outlined in the following steps: (i) Brain tissue dissection: Fresh brain tissue from 4-6 week-old SD rats was processed by first removing the meninges and choroid plexus through dissection. The cleaned brain tissue was then fragmented and transferred into a 50 mL conical tube. (ii) Enzymatic digestion: A collagenase solution was added to the brain tissue fragments in the conical tube. This mixture was incubated at 37°C for 30 min in a constant temperature air shaker. Post-incubation, the tissue fragments were mechanically disrupted using a pipette to produce smaller fragments. These were then filtered through a cell strainer to obtain a homogeneous single-cell suspension. (iii) Cell isolation: The resulting cell suspension was centrifuged at 300 g for 5 min. The cell pellet was resuspended in DMEM/F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were then plated in culture flasks and incubated at 37°C with 5% CO2. (iv) Cell culture: The primary BMECs were meticulously monitored under a microscope and typically developed into a monolayer within approximately 7-10 days. The culture medium was replenished every 2-3 days to promote cell growth and viability.
In vitro blood-brain barrier penetration assay
BMECs were seeded at a density of 10,000 cells per well in the upper chamber of a 24-well Transwell plate (Corning, USA). Transendothelial electrical resistance (TEER) was measured using a Millicell-ERS system (Millipore, USA) at two-day intervals, with values exceeding 150 Ω·cm² indicating suitable bilayer integrity.
Following bilayer establishment, LNP@DiD formulations were introduced to the upper chambers, with incubation periods ranging from 0.5 to 8 h. Post-incubation, nuclei were stained with Hoechst 33342 and Transwell membranes were transferred onto glass microscope slides for imaging with a high-content analysis system (Operetta CLS, PerkinElmer, USA) and Imaris software. The penetration of LNPs was quantified by collecting the medium from the basolateral chamber and measuring its fluorescence intensity using a Multimode Microplate Reader (BioTek Synergy H1, USA) at excitation wavelength of 485 nm and emission wavelength of 535 nm.
Pharmacokinetic Studies
SD rats were administered BE and BE-ST via the tail vein at a dose of 5mg/kg. Blood samples were collected from the orbit at different time points (0.25, 0.5, 1, 2, 4, 8, 12, 24 and 48 h). Each sample (~200 μL) was centrifuged at 4°C for 10 min at 11000 × g). Samples were then processed by mixing 50 µL of serum with 150 µL of methanol, followed by ultrasonication for 10 min and a second centrifugation under the same conditions. The supernatant (20 µL) was analyzed using HPLC (SHIMADZU, Japan). Pharmacokinetic parameters were calculated using PKsolver 2.0.10 software68.
In Vivo Imaging
To evaluate brain targeting, healthy mice were intravenously injected with LNP@DiR via the tail vein. Fluorescence images were captured at predefined time points (0.5, 1, 2, 4, and 8 h) using the VISQUE In Vivo Smart-LF System (Vieworks, Korea). After imaging sessions, mice were euthanized at predetermined intervals and their major organs were harvested for ex vivo imaging analysis.
For the establishment of a brain tumor model, 5 μL of a GL261-Luc cell suspension (10,000 cells/μL) was carefully injected into the right hemisphere of the brain over a 3 min period. Mice were anesthetized and secured in a stereotactic apparatus to ensure precise administration. The needle was slowly withdrawn post-injection. In vivo imaging of the pathological model was performed after a 10-day post-operation period, with mice receiving daily intravenous injections of LNP@siVEGF (1 mg/kg) over five consecutive days. Imaging was conducted using the Lumina III Imaging System (PerkinElmer, USA) on days 0, 3, and 6 post-injection. For precision targeting ex vivo imaging of the pathological model, mice were intravenously injected with LNP@DiR. Their brains were harvested for ex vivo imaging at 8 h post-injection using the VISQUE In Vivo Smart-LF System.
In the construction of a meningitis model, a fungal suspension containing 2500 CFU of a luciferase-expressing strain of C. neoformans was injected into the mouse brain using the same method as the tumor model. After a 48 h post-operation period, mice received daily intravenous injections of LNP@AmB, consisting of either 1 mg/kg of amphotericin B or 25 mg/kg of flucytosine, for a total of five days. Imaging was subsequently performed using the Lumina III Imaging System at 0, 3, and 6 days post-surgery.
Morris water maze
To assess spatial learning and memory, the Morris water maze (MWM) paradigm was employed following established methodologies69. The maze consisted of a water pool divided into four quadrants, each marked by a unique symbol (pentagram, square, triangle, and circle) on the corresponding quadrant wall to serve as spatial cues. The water maintained at 22 ± 1°C, and food-grade titanium dioxide was used to obscure the water and facilitate tracking of mouse movements.
All trials were conducted in the afternoon in a controlled environment devoid of extraneous noise and intense light. Mice were acclimatized to the test room for 2 h prior to the start of the experiments. The training phase lasted for five consecutive days, with each mouse undergoing four trials per day and a 20-30 min inter-trial interval. Mice were placed facing the pool wall and tasked with locating a hidden platform. The time to locate the platform was recorded. If a mouse failed to find the platform within 60 s, it was guided to the platform and allowed to remains there for 10 s.
Following a 24 h interval post-training, the platform was removed for a 60 s probe test. Mice were placed in the water facing the quadrant opposite to the target quadrant. Performance metrics, such as time spent in the target quadrant and the number of crossings at the former platform location, were recorded using EthoVision XT8.5 tracking software, indicating spatial memory retention.
Western blot
For the assessment of targeted molecular mechanisms, bEnd.3 and DRD3−/− bEnd.3 cells were collected. Following behavioral assessments, murine brain tissues were obtained for therapeutic efficacy evaluation. Mice were ethically and humanely euthanized, and transcardial perfusion with saline was performed prior to tissue extraction, including the entire hippocampus and cortex. These samples were homogenized in lysis buffer containing 1% phosphatase inhibitors and 1% PMSF (Beyotime, China). After homogenization, the mixture was centrifugated at 12,000 rpm for 15 min at 4°C. The protein concentration in the supernatant was determined using a BCA Protein Assay Kit (Beyotime, China). Approximately 20 μg of protein was subjected to SDS-PAGE on a 10% gel and transferred onto a polyvinylidene fluoride (PVDF). To reduce nonspecific binding, the membrane was incubated at 37°C for 1 h in blocking buffer containing 5% nonfat dry milk in Tris-buffered saline. Overnight incubation at 4°C with primary antibodies, including BACE1, p-tau, p-GSK3β, DRD3, or GAPDH, was followed by incubation with HRP-conjugated IgG rabbit secondary antibodies for 1 h at 37°C. Blots were visualized using ECL (Beyotime, China), with GAPDH serving as a loading control. Quantification was performed using ImageJ, and results were recorded with a gel image analysis system (Tanon, China).
Enzyme-Linked Immunosorbent Assay
Serum analyses were performed on APP/PS-1 transgenic mice following administration every two days over a two-week period. Blood samples were collected from the retro-orbital plexus and centrifuged at 2000 rpm for 10 min at 4°C to isolate plasma. To assess potential immune response and toxicity of LNP formulations, ELISA kits measures complement activation-related pseudoallergy (CARPA) indicators (complement C5b-9, C3a, MCP-1). Additionally, levels of key inflammatory cytokines (IL-1β, IL-6, IFNγ, and TNFα) were evaluated using specific ELISA kits to understand the synergistic impact of LNP@siBACE1 on AD.
Blood Biochemical Profiling
To investigate the potential hepatotoxic and nephrotoxic effects of LNP formulations, serum biochemical examinations were performed every other day for two weeks. Blood was obtained from the retro-orbital plexus and centrifuged at 2000 rpm for 10 min at 4°C. The plasma was analyzed for biochemical parameters including alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acid (UA), urea (UREA), and creatinine (CREA), providing a comprehensive evaluation of hepatic and renal function.
Histological staining
Following the treatment regimen, humane euthanasia was performed on APP/PS-1 mice, followed by transcardial perfusion. The brains were fixed in 4% paraformaldehyde for 24 h, then underwent dehydration, embedding in OCT, and freezing. Frozen sections of 20 μm thickness were obtained using a high-precision freezing microtome (Leica, Germany). For immunofluorescence, sections were treated with 4% paraformaldehyde for 30 min, rinsed with PBS, permeabilized with 0.1% Triton X-100 for 15 min, and blocked with 5% BSA for 2 h. Primary anti-β-amyloid antibody incubation was performed overnight at 4°C. After three PBS washes, sections were incubated with FITC-conjugated goat anti-rabbit IgG for 2 h at room temperature. Nuclei were stained with Hoechst 33342 for 10 min. Confocal microscopy (OLYMPUS FV3000, Japan) acquired high-resolution fluorescence images. Nissl staining identified potential neuronal damage. Furthermore, vital organs like hearts, lungs, livers, spleens, and kidneys, were fixed in a 4% paraformaldehyde, sectioned, and stained with hematoxylin and eosin (HE). Images were captured using an inverted fluorescence microscope (DMi8, Leica, Germany).
Analysis of reactive oxygen species levels in single cells
To assess oxidative stress and intracellular levels of ROS, a ROS assay kit (Beyotime, China) was utilized. bEnd.3 cells from each treatment group were incubated at 37°C for 30 min in serum-free medium containing 10 μM DCHF- diacetate (DA). Post-incubation, the medium was replaced with fresh medium containing ionizable molecules at a concentration of 5 mg/mL, and the cells were further incubated for 2, 12, or 24 h. Following this, cells were washed three times in PBS containing 0.1% BSA and mounted on glass slides with PBS for microscopy analysis. The fluorescence intensity of each cell, indicating ROS levels, was quantified using a real-time single-cell multimode analyzer equipped with optical fiber probes (Rayme, China).
Tissue burden assessment
Mice were anesthetized and securely positioned using a stereotactic apparatus before inoculating 2500 CFU of C. neoformans into the right cerebral hemisphere over a meticulous 3 min period. This procedure was carefully conducted with precision and a gradual withdrawal of the injection needle. After a 48 h interval, mice were subjected to intravenous administration of LNP@AmB. The treatment regimen included daily doses of either 1 mg/kg of amphotericin B or 25 mg/kg of flucytosine, administered over a span of five consecutive days.
On the sixth day post-inoculation, all mice were humanely euthanized, and their brain were harvested. The brain tissues were weighed and homogenized in sterile saline at a ratio of 1 g of tissue to 3 mL of saline. The homogenates were subjected to sequential dilutions in sterile saline. A 30 μL aliquot of each dilution was carefully plated onto YPD agar and incubated at 30°C for 48 h to determine the colony-forming units (CFU) per gram of brain tissue.
Survival rates study
After a 48 h incubation period after inoculating the mice with C. neoformans to establish a brain infection model, the mice were randomly divided into five groups. Each group received daily intravenous treatments for five days. The treatments consisted of either saline or different formations of LNP@AmB, with doses set at 1 mg/kg of amphotericin B or 25 mg/kg of flucytosine. The survival of the mice was monitored for a duration of 45 days following the conclusion of the treatment regimen.
eGFP-mRNA transfection in vitro
Cultured cells were seeded in a 12-well culture dish and allowed to reach 60 to 70% confluence. Transfection was conducted using eGFP mRNA complexed with distinct LNP formulations in serum-free DMEM. The cells were incubated at 37°C for 4 h to facilitate mRNA uptake. Following the initial transfection period, FBS was reintroduced to the medium, and the cells were incubated for an additional 20 h. The expression of eGFP was then assessed using a fluorescence microscope (DMi8, Leica, Germany).
eGFP-mRNA transfection in vivo
Following the intravenous administration of LNP@eGFP mRNA at a dosage of 1 mg/kg, a 24 h monitoring period ensured. After this period, all mice were humanely euthanized, and their brain tissues were harvested and fixed in a 4% paraformaldehyde solution for 24 h.
The preparation of brain tissues for imaging involved several carefully controlled steps70. Initially, tissues underwent decolorization by immersion in a solution containing 25% v/v Quadrol and 5% v/v ammonium in water at 37°C for two days. This was followed by a gradient delipidation process using tert-butanol (tB) solutions at concentrations of 30%, 50%, and 70% v/v, with pH adjustments above 9.5 using 3% w/v Quadrol. Subsequently, the tissues were dehydrated in a solution comprised of 70% v/v tB, 27% v/v PEG methacrylate Mn500 (PEGMMA500), and 3% w/v Quadrol for two days.
The final clearing step involved submerging the samples in BB-PEG Clearing Medium, which consists of 75% v/v benzyl benzoate (BB), 25% v/v PEGMMA500, and 3% w/v Quadrol, achieving a refractive index of 1.543. This medium was used for 507 days until the tissues reached optical transparency. The cleared samples were then preserved at room temperature in the same clearing medium.
For imaging, cleared brain samples were analyzed using a light sheet microscope (LiToneXL, Light Innovation Technology, China), equipped with a 43× objective lens (NA = 0.28, working distance = 20 mm). The imaging process utilized thin light sheets to illuminate the samples from all four sides, capturing and merging images to visualize the expression and distribution of eGFP effectively.
Statistical analysis
Quantitative data from the experiments are presented as means ± SD. Statistical significance was determined using two-tailed unpaired t-tests and multiple t-tests with GraphPad Prism software (version 8). The threshold for statistical significance was set at p < 0.05, with 95% confidence intervals. Notably, in instances where p-values fell below 0.0001, the software was unable to provide an exact value, indicating extremely significant differences.