Berberine-inspired ionizable lipid for self-structure stabilization and potent brain targeting delivery of nucleic acid therapeutics

Berberine-inspired ionizable lipid for self-structure stabilization and potent brain targeting delivery of nucleic acid therapeutics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Berberine-inspired ionizable lipid for self-structure stabilization and potent brain targeting delivery of nucleic acid therapeutics Zhi-Hong Jiang, Chong Li, Xufei Bian, Qian Guo, Ling Yang, Xiaoyou Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4626003/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Mar, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Despite advancements in targeting organs such as the liver, spleen, and lungs with lipid nanoparticles (LNPs), the challenge of traversing the blood-brain barrier (BBB) significantly impedes the progress of gene therapies for neurological disorders. Motivated by the structural and functional characteristics of alkaloids, we developed a novel library of ionizable lipid molecules based on the tetrahydroisoquinoline structure characteristic of the protoberberine family. Our findings reveal that: (i) LNPs incorporating berberine-derived ionizable lipids notably enhance the ability to cross the BBB, increasing in vitro endocytosis efficiency by up to 65-fold and achieving an in vivo brain-to-liver distribution ratio of approaching 20%; (ii) these lipids form stable self-assemblies with polyA, enhancing nucleic acid stability through mechanisms beyond conventional electrostatic interactions, thus providing effective RNA protection without the need for additional modifications; (iii) the lipids inherit the diverse brain-protective properties of protoberberine-type alkaloids, including anti-inflammatory and antioxidant effects, thereby synergistically enhancing the therapeutic management of brain diseases while exhibiting minimal immunogenicity. Biological sciences/Drug discovery/Drug delivery Biological sciences/Neuroscience/Blood–brain barrier Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In recent years, small-molecule natural products have gained prominence not only as lead compounds in drug development but also as facilitators of drug delivery. Illustrative examples include the complexation of monoclonal antibodies, such as Herceptin, with flavonoids like (−)-epigallocatechin-3- O -gallate (EGCG) 1 to counteract cancer, and the TANNylation of therapeutic proteins or peptides with phenols such as tannic acid (TA) 2 for specific targeting of elastins and collagens, thereby augmenting the treatment of heart diseases. Additionally, the substitution of cholesterol with terpenes, exemplified by ginsenoside Rg 3 in the lipid bilayer of liposomes, has demonstrated the ability to target GLUT1 receptors, thereby enhancing cancer therapy. While various classes of natural small molecules 4 , including flavonoids, terpenes, and others like coumarins, quinones, and steroids, have been explored for drug delivery, the application of alkaloids as auxiliary materials in this field is still in its nascent stage. Alkaloids, with their unique structural features of tertiary and quaternary amines, show potential for robust interactions with nucleic acid drugs, positioning them as promising carriers in nucleic acid therapeutics. Their distinctive biomembrane penetration capabilities can enhance targeted delivery, as evidenced by their direct effects on bacterial biofilms 5 , 6 , cell membranes 7 , 8 , and organelle membranes 9 , 10 . Berberine (BE) 11 , a prominent alkaloid in the isoquinoline series, and its analogs such as jateorrhizine, African tetrandrine, and palmatine are naturally occurring protoberberine alkaloids found in various plant species. Tetrahydroberberine-type alkaloids 12 , derived from plants or synthesized through hydrogenation of berberine-type alkaloids, have garnered attention due to their increased stability, bioavailability, safety, and potential therapeutic efficacy. These protoberberine alkaloids, with their isoquinoline or tetrahydroisoquinoline skeletons, display a versatile pharmacological profile 13 , 14 , possessing antimicrobial, anti-inflammatory, antioxidant, and antitumor properties. Since the 1960s, the nucleic acid interactions of protoberberine, particularly its ability to bind DNA 15 with a preference for thymidine-adenine pairs, have been extensively studied. Recent studies have highlighted the ability of isoquinoline alkaloids to bind mRNA 16 through partial intercalation, specifically targeting the poly(A) tails of mRNA, leading to self-structure formation via hydrogen bonding and π-π stacking 17 . These interactions help maintain the structural integrity of polyA. Further studies have revealed specific interactions between protoberberine 18 – 20 and receptor proteins on cell surfaces, notably exhibiting high affinity for dopamine receptors, particularly the dopamine D3 receptor (D3R) 21 , which is abundant in brain regions associated with Alzheimer's disease (AD), such as the hippocampus and cortex 22 , 23 , as well as the pituitary gland region 24 – 26 which are prone to brain infections and tumors. This suggests that the tetrahydroisoquinoline ring-fused skeleton of protoberberine-type alkaloids, when complexed with nucleic acid drugs, may offer unique advantages for brain-targeted delivery. In response to these findings, we have designed a series of innovative ionizable lipids inspired by the protoberberine alkaloid structure. These lipids, focusing on the stable and safe tetrahydroberberines, have undergone extensive validation from molecular investigations to animal studies. Our findings reveal the following multifaceted advantages: (i) a robust capacity for nucleic acid loading and stability; (ii) enhanced endosomal escape of nucleic acid drugs and improved intracellular transport; (iii) significantly improved brain targeting characteristics in vitro , corroborated by in vivo studies; and (iv) the inheritance of pharmacological activity from protoberberine alkaloids, enabling synergistic treatment with loaded nucleic acid drugs. Results Design and screening of BE lipidoids Inspired by the structural frameworks of tetrahydroberberine-type alkaloids and their analogs, we conducted preliminary screenings of five head groups. The tail chains were designed to incorporate diverse, representative structural features such as unsaturated bonds, ester groups, and alcoholic hydroxyl groups, which are conducive to gene delivery (Fig. 1 a). For nucleic acid delivery, we employed a combination of helper lipids (DSPC, DOPC, and DOPE), DMG-PEG 2000 , cholesterol (Chol), and newly proposed ionizable lipidoids. The lipid compositions in the BE formulations were meticulously optimized with the following relative molar percentages: ionizable lipids (29–39%), helper lipids (10–30%), Chol (20–59%), and DMG-PEG 2000 (1–2%), ensuring the sum of molar ratios reached 100% for each formulation (Supplementary Table S1 ). Multiple screening rounds using blank lipid nanoparticles (LNPs) were performed (Fig. 1 b). Initially, optimal formulation 10 was identified based on the size of various preparations (Supplementary Table S2). Subsequently, BEs were prepared using 27 candidate compounds, each with a size smaller than 100 nm (Supplementary Table S3). The second screening phase measured the pKa of 10 eligible compounds, revealing values within the optimal range for effective RNA delivery (6.0–7.0) (Supplementary Fig. S1 ). In the third phase, we assessed the binding interactions of these derivatives with the poly(A) tail which is crucial for RNA stabilization 27 – 29 , resulting in the identification of five candidate compounds (Fig. 1 c). In vitro targeting experiments demonstrated that the LNP containing these compounds exhibited higher uptake rates than the control group (NP) (Fig. 1 d-e). This trend was consistently observed in vivo , with compounds A2-B13 emerging as the most promising lipidoid (Fig. 1 f-g and Supplementary Fig. S2). Representative ionizable lipids (A2-B13) were synthesized as outlined in the Supplementary Information, and their structural integrity was confirmed by 1 H nuclear magnetic resonance (NMR) analysis (Supplementary Fig. S3). Preparation and characterization of BE@siRNA The lead structure (A2-B13) and its formulation composition (molar ratio A2-B13:DOPC:Chol:PEG-DMG = 39:20:30:1) were thoyoughly screened and selected. Gel retardation assay analysis confirmed that BE effectively entrapped siRNA at mass ratios exceeding 1:2 (Fig. 2 a). The BE and BE@siRNA complexes exhibited rounded morphologies with hydrodynamic sizes of approximately 70 nm and 90 nm, respectively (Fig. 2 b-c). To evaluate the transfection efficiency, FAM-labeled siRNA was encapsulated in BE, demonstrating robust transfection efficacy comparable to the commercial lipidoid DLin-MC3-DMA, as evidenced by equivalent intracellular fluorescence intensity (Fig. 2 d). Additionallye, BE facilitated siRNA escape from endosomes/lysosomes (Fig. 2 e). We further investigated the endocytic pathways by comparing the uptake efficiency of BE@DiD in the presence of various inhibitors against BE@DiD uptake without inhibitors. Cells pretreated with inhibitors exhibited significant variations in endocytosis efficiency, with chlorpromazine notably reducing clathrin-mediated cellular entry of BE@DiD (Fig. 2 f). The capability of BE to traverse the blood-brain barrier (BBB) in vitro was confirmed using a transwell model, illustrating significantly enhanced penetration of BE-treated conditions compared to fluorescein DiD control nanoparticles (NP) and MC3 (Fig. 2 g-h), highlighting BE’s potential to endocytose endothelial cells efficiently. Brain targeting of BE-ST (BE + Scutellarin) Our in vitro quantitative flow cytometry analysis indicated an obviously enhanced uptake of BE, showing 65.84 times the uptake compared to NP (Fig. 1 e). This uptake was markedly higher than that observed with nanoparticles modified with classical ligands, which typically showed enhancements ranging from 0.2-fold to 1.6-fold 30 – 34 . To elucidate the underlying mechanisms, we downregulated the expression of the DRD3 protein using siRNA. Protein expression was reduced by approximately 50% after 72 h post-transfection with siDRD3 compared to the negative control (Supplementary Fig. S4). The decreased uptake of BE in cells with reduced DRD3 expression suggests an interaction between BE and this protein, corroborating literature reports 21 and our findings (Fig. 3 a, Supplementary Table S4, Fig. S5). For in vivo brain targeting, we implemented an acid-base pair strategy to neutralize charges, aiming to mitigate the impact of the positive charge on in vivo metabolism and distribution. After charge neutralization with organic acids (Fig. 3 b) such as scutellarin (ST) 35 , EGCG 36 , and citric acid (CA) 37 , we observed a reduced distribution in the liver. Notably, the combination with ST resulted in a brain-to-liver distribution ratio approaching 20% (Fig. 3 c-d). This charge neutralization also enhanced pharmacokinetics, including a higher peak plasma concentration and a 1.73-fold increase in half-life for the BE-ST group compared to the BE group.(Fig. 3 e). To evaluate the brain-targeting efficiency of nanoparticles in vivo , different LNPs labeled with DiR were administered to normal mice. The BE-ST nanoparticles showed significantly stronger fluorescence intensity in the brain compared to those without BE-ST (Fig. 3 f). Ex vivo imaging conducted at 0.5, 1, 2, 4, and 8 h post-injection further demonstrated that the BE-ST group exhibited stronger fluorescence signals in the brain compared to the positive control, wherein glutathione (GSH) served as the target head group (GSH-NP) and is currently in clinical trials for brain-targeted delivery 38 (Fig. 3 g, Supplementary Fig. S6). Quantitative analysis reinforced these observations, indicating that the fluorescence intensity in the brain for the BE-ST group was approximately 167% of that observed in the positive control group (Fig. 3 h). Brain-targeted delivery of siBACE1 by BE-ST for comprehensive treatment of AD The efficacy of the BE-ST delivery system was initially assessed using AD as a model, targeting the beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) 39 as a crucial pharmacological intervention point. BE-ST@siBACE1 or control BE-ST@siScr (siRNA, 1 mg/kg) were administered bilaterally via caudal vein injections to APP/PS1 mice, as illustrated in Fig. 4 a, with MC3@siBACE1 serving as a comparative control. Additionally, PBS was injected into both APP/PS1 and wild-type (WT) mice to establish baseline AD-related deficits. Spatial learning and memory were assessed using the Morris water maze (MWM). Mice treated with PBS and MC3@siBACE1 exhibited an undirected search pattern, suggesting no improvement in cognitive abilities, as depicted in Fig. 4 b. There were no significant differences in swimming speed (Fig. 4 e). Conversely, APP/PS1 mice treated with BE-ST@siBACE1 demonstrated marked improvement, spending 1.95 times longer in the target quadrant and crossing it more frequently compared to PBS-treated controls (Fig. 4 c, d). These results strongly support the effectiveness of BE-ST@siBACE1 in enhancing cognitive performance in APP/PS1 mice. After the behavioral evaluations, mice were euthanized, and brain tissues were harvested to assess the levels of BACE1 and its impact on amyloid-β (Aβ) and tau pathology accumulation. Our results showed that BE-ST@siBACE1 treatment led to significant reductions in BACE1 levels in the hippocampus and cortex (Fig. 5 a, b; Supplementary Fig. S7 a ). Moreover, there was a noticeable decrease in both the number and size of amyloid plaques, a critical neuropathological feature of AD resulting from the cleavage of amyloid precursor protein (APP) by BACE1, within the hippocampus and cortex of BE-ST@siBACE1-treated mice (Fig. 5 c). Additionally, a reduction in the levels of hyperphosphorylated tau protein (p-tau), a key marker of advanced AD’s pathology, was observed in the hippocampus and cortex of the BE-ST@siBACE1-treated mice compared to those receiving PBS (Fig. 5 a, b; Supplementary Fig. S7 b ). Protoberberine and its derivatives are known for their synergistic 40 , 41 effects on p-tau and Aβ through the inhibition of glycogen synthase kinase-3β (GSK-3β). This interaction regulates Ser9 41 phosphorylation and affects BACE1 activity. To explore the potential of a tetrahydroberberine derivative in this context, we conducted a detailed study. Our findings revealed that phosphorylation at Ser9 of GSK3β (p-GSK3β) was significantly reduced in both the hippocampus and cortex of APP/PS1 mice treated with BE-ST@siBACE1 and BE-ST@siScr, compared to the untreated AD group (Fig. 5 a, b; Supplementary Fig. S7 c ). This reduction suggests that BE-ST maintains its inhibitory effect on GSK-3β and BACE1, leading to a synergistic therapeutic effect against dementia, corroborating the behavioral improvements observed in BE-ST@siScr-treated APP/PS1 mice. Oxidative stress is a crucial factor in the development and progression of AD. In this study, we observed that treatment with BE-ST@siScr and BE-ST@siBACE1 resulted in diminished levels of superoxide dismutase (SOD) and malondialdehyde (MDA), along with core proinflammatory cytokines in APP/PS1 mice. This led to a notable alleviation of oxidative stress (Supplementary Fig. S8 a - b ) and inflammation (Supplementary Fig. S8 c - f ) within the cerebral context, shedding light on potential mechanisms for the observed reductions in Aβ and p-tau levels. Furthermore, the application of BE-ST and BACE1 significantly preserved neuronal integrity and morphology, preventing substantial neuronal shrinkage (Fig. 5 d). Routine blood biochemical analyses and histological examinations of tissues samples collected two weeks post-injection revealed no significant differences between the PBS-treated and BE(-ST)@siRNA-treated groups, confirming the excellent biocompatibility of BE(-ST) (Supplementary Figs. S9, S10). Additionally, there was no discernible increase in immunogenicity markers such as complement components and cytokines. Specifically, levels of C5b9, C3a, and monocyte chemoattractant protein (MCP-1) remained stable two weeks after BE(-ST) treatment (Fig. 5 e-g). In contrast, the MC3-treated group displayed a significant deviations from baseline, aligning with previous reports that LNPs containing commercially available ionizable lipids like DLin-MC3-DMA can trigger immune system activation 42 , potentially resulting in CARPA, a severe immunological reaction similar to anaphylactic shock. Furthermore, we utilized a real-time quantitative single-cell detection technique to investigate reactive oxygen species (ROS) generation. BE induced ROS at significantly lower levels over time compared to commercial MC3 (Supplementary Fig. S11), further highlighting the favorable safety profile of BE(-ST). BE-ST as a versatile platform for brain disease drug delivery Exploring the versatility of BE-ST, we examined its efficacy across various pathological models by administering different therapeutic agents. Initially, we utilized a brain tumor model with GL261-Luc cells to evaluate the delivery and therapeutic potential of siVEGF (Supplementary Fig. S12 a ). Live imaging revealed a noticeable decrease in luminescence, indicating tumor activity reduction post-treatment variation with BE-ST@siVEGF. Quantitative analysis demonstrated a significant reduction in the mean fluorescence intensity (MFI) of brain tumors treated with BE-ST, with reductions of 27.96% at 3 days and 38.82% at 6 days post-administration, in comparison to the MC3-treated group (Supplementary Fig. S12 b , c ). The ex vivo imaging results highlighted BE-ST’s enhanced precision in targeting the lesion site (Supplementary Fig. S12 d ), suggesting its potential as a robust delivery platform for siRNA therapeutics in brain tumor treatment. Expanding our investigation to include small-molecule drugs and mRNA, we tested BE-ST in a broader range of animal models, covering both cancer and neurodegenerative diseases. Specifically, we addressed the challenge of treating escalating fungal meningitis, a condition with increasing incidence and high mortality, aggravated by the ineffective BBB penetration of conventional treatments like amphotericin B (AmB). We developed BE-ST@AmB for use in a cryptococcal meningitis model (Fig. 6 a). The administration of BE-ST@AmB significantly enhanced the delivery and efficacy of AmB, as evidenced by live imaging which showed a drastic reduction in luminescence variation. Quantitative assessments confirmed a reduction in the MFI of the brain by 94.15% at 3 days and 99.28% at 6 days post-treatment compared to the commercially available AmBisome-treated group (Fig. 6 b, c). Examination of the brain fungal burden indicated recovery in BE-ST@AmB-treated mice, in stark contrast to significant cryptococcal infiltration in untreated or AmBisome-treated mice (Fig. 6 d). Long-term therapeutic studies further demonstrated a superior survival rate in mice treated with BE-ST compared to other treatment groups (Fig. 6 e). In vitro studies on mRNA transfection capabilities involved encapsulating eGFP mRNA into BE, revealing superior transfection efficiency compared to the commercial lipid nanoparticle DLin-MC3-DMA. This was evidenced by elevated intracellular fluorescence levels (Fig. 6 f), likely due to the strong affinity between BE and polyA, a relationship supported by literature 43 , 44 and molecular docking studies (Fig. 6 g-h and Supplementary Fig. S13). The ionizable lipid derivative of tetrahydroberberine incorporated into BE not only preserved this affinity but also enhanced the structural stability of mRNA, eliminating the need for chemical modifications typically required by other nucleic acid carriers. Intravenous administration of BE-ST@eGFP mRNA effectively delivered mRNA to the brain, achieving deeper penetration and higher fluorescence in brain tissue compared to MC3@eGFP mRNA (Fig. 6 i). Discussion In the evolving landscape of ionizable lipid designs 45 , conventional strategies have predominantly favored simpler linear tertiary amine groups to enhance pH sensitivity due to reduced steric hindrance. However, leveraging the complexation capabilities of the protoberberine family with nucleic acids, as documented in several studies 15 , 43 , 46 – 49 , we adopted a novel approach in ionizable lipid design. Our design features a dual tetrahydroisoquinoline fused-ring skeleton, which uniquely binds to the polyA tail of RNA, forming stable self-structures. This innovative approach moves beyond traditional electrostatic interactions used to compact RNA 50 , enabling efficient nucleic acid loading and lysosomal escape while preserving nucleic acid integrity through partial intercalation without necessitating complex chemical modifications. Moreover, the use of synthetic lipidoids often falls short in biological performance, with associated cellular toxicity and immunogenicity 42 posing significant drawbacks, particularly in repeated administration scenarios. In contrast, tetrahydroisoquinoline derivatives contribute inherent pharmacological benefits, such as antioxidant and anti-inflammatory properties, potentially reducing the acute immune response typically associated with RNA therapeutics. These derivatives also serve as synergistic therapeutic adjuvants, enhancing the overall therapeutic efficacy. Despite advancements in organ-specific targeting, the BBB continues to present a formidable challenge. While various lipid nanoparticle platforms have demonstrated promise in targeting organs like the liver 51 – 54 , spleen 53 , 55 , and lungs 56 , 57 , brain targeting remains particularly complex. Traditional strategies often rely on ligand-receptor interactions using ligands such as transferrin 58 and peptides like apamin 31 , glutathione 38 , angiopep-2 59 , and RVG peptide 60 . These modifications generally improve uptake efficiency by 0.2-fold to 1.6-fold 30 – 34 in vitro in primary or immortalized brain microvascular endothelial cells, yet they typically achieve brain-to-liver distribution ratio below 10%. In stark contrast, our novel ionizable lipids derived from tetrahydroisoquinoline showcase a unique capability as small molecular ligands that efficiently penetrate the BBB. These lipids demonstrate superior in vitro uptake and exceptional in vivo performance, especially when combined with organic acids, such as ST 35 , EGCG, and citric acid for charge neutralization. In conclusion, the berberine-inspired LNPs not only effectively deliver siRNA into the brain but also show potential for the delivery of small molecules and biological macromolecules like mRNA. This opens new avenues for the application of these innovative lipids in neurological therapies, promising enhanced treatment modalities for a range of brain disorders. Methods 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-PEG 2000 (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% CO 2 . DRD3 −/− bEnd.3 cells were transfected with siDRD3 using Lipofectamine 2000 (Invitrogen). Cryptococcus neoformans ( C. neoformans ) s trains 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 previously 61 . 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 tetrahydroberberine derivatives N 6 , N 6 -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 1 H nuclear magnetic resonance (NMR, Bruker, USA) with the following shifts (400 MHz, DMSO- d 6 +C 2 D 4 O 2 ): 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-DMG 2000 , 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) b inding e xperiments 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 Na 2 HPO 4 , 0.5 mM NaH 2 PO 4 , 0.25 mM EDTA, 6 mM NaCl) or citrate-phosphate (CP) buffer (5 mM Na 2 HPO 4 ) with pH adjusted to 7.1 and 4.5 using citric acid, following established protocols 49 . 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% CO 2 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 es c ape bEnd.3 cells were seeded and incubated for 12 h at 37°C with 5% CO 2 . 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 2.5.4 64 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 1.0 65 for compatibility with AutoDock Vina. Docking was conducted using AutoDock Vina 1.1.2 66 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 protocol 67 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% CO 2 . (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 software 68 . 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 I n V ivo 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. I n 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 I n V ivo 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 methodologies 69 . 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 steps 70 . 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 M n 500 (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. Declarations Acknowledgements This work was supported by the National Key Research and Development Program of China (Grant No. 2023YFF0724200), the National Natural Science Foundation of China (NSFC Grant Nos. 82373808, 82073789), and the Chongqing Science Fund for Distinguished Young Scholars (Grant No. CSTB2023NSCQ-JQX0021) awarded to C.L. We extend our gratitude to Dr. Huan Zhao and Xiaogang Wang from Revvity for their invaluable advice on experimental design and for engaging in productive discussions that enhanced this research. Additionally, we are thankful for the support provided by the Academy of Agricultural Sciences at Southwest University, which included access to essential instrumentation and technical expertise. We also appreciate the equipment support received from Rayme Biotechnology and Revvity. Author contributions Chong Li, Zhi-Hong Jiang, Xufei Bian, Qian Guo, Xiaoyou Wang and Xurong Qin conceived the project, designed all the experiments, analyzed the data, and wrote the manuscript. Ling Yang, Shikang Zhao, and Shiqiong Wu conducted the experiments and analyzed the data. All authors edited the manuscript. Declaration of interests The authors declare no conflict of interest. References Chung, J.E. et al. Self-assembled micellar nanocomplexes comprising green tea catechin derivatives and protein drugs for cancer therapy. Nature nanotechnology 9 , 907-912 (2014). Shin, M. et al. Targeting protein and peptide therapeutics to the heart via tannic acid modification. Nature biomedical engineering 2 , 304-317 (2018). Wang, X. et al. Identification and construction of a novel biomimetic delivery system of paclitaxel and its targeting therapy for cancer. Signal transduction and targeted therapy 6 , 33 (2021). Fu, S. & Yang, X. Recent advances in natural small molecules as drug delivery systems. J Mater Chem B 11 , 4584-4599 (2023). Huigens, R.W., 3rd et al. Control of bacterial biofilms with marine alkaloid derivatives. Mol Biosyst 4 , 614-621 (2008). Yan, Y. et al. Research Progress on Antibacterial Activities and Mechanisms of Natural Alkaloids: A Review. Antibiotics (Basel) 10 (2021). Lebecque, S. et al. Interaction between the barley allelochemical compounds gramine and hordenine and artificial lipid bilayers mimicking the plant plasma membrane. Sci Rep 8 , 9784 (2018). Efimova, S.S., Zakharova, A.A. & Ostroumova, O.S. Alkaloids Modulate the Functioning of Ion Channels Produced by Antimicrobial Agents via an Influence on the Lipid Host. Front Cell Dev Biol 8 , 537 (2020). Patalas-Krawczyk, P. et al. Effects of plant alkaloids on mitochondrial bioenergetic parameters. Food Chem Toxicol 154 , 112316 (2021). Shitan, N. & Yazaki, K. Accumulation and membrane transport of plant alkaloids. Curr Pharm Biotechnol 8 , 244-252 (2007). Singh, S., Pathak, N., Fatima, E. & Negi, A.S. Plant isoquinoline alkaloids: Advances in the chemistry and biology of berberine. European journal of medicinal chemistry 226 , 113839 (2021). Kim, A.N., Ngamnithiporn, A., Du, E. & Stoltz, B.M. Recent Advances in the Total Synthesis of the Tetrahydroisoquinoline Alkaloids (2002-2020). Chem Rev 123 , 9447-9496 (2023). Feng, J.H. et al. The composition, pharmacological effects, related mechanisms and drug delivery of alkaloids from Corydalis yanhusuo. Biomed Pharmacother 167 , 115511 (2023). Shang, X.F. et al. Biologically active isoquinoline alkaloids covering 2014-2018. Med Res Rev 40 , 2212-2289 (2020). Krey, A.K. & Hahn, F.E. Berberine: complex with DNA. Science 166 , 755-757 (1969). Giri, P. & Kumar, G.S. Molecular aspects of small molecules-poly(A) interaction: an approach to RNA based drug design. Curr Med Chem 16 , 965-987 (2009). Chatterjee, S. & Suresh Kumar, G. Small molecule induced poly(A) single strand to self-structure conformational switching: evidence for the prominent role of H-bonding interactions. Mol Biosyst 13 , 1000-1009 (2017). Kawano, M., Takagi, R., Kaneko, A. & Matsushita, S. Berberine is a dopamine D1- and D2-like receptor antagonist and ameliorates experimentally induced colitis by suppressing innate and adaptive immune responses. J Neuroimmunol 289 , 43-55 (2015). Niwa, M., Mibu, H., Nozaki, M., Tsurumi, K. & Fujimura, H. Dopaminergic unique affinity of tetrahydroberberine and l-tetrahydroberberine-d-camphor sulfonate. Pharmacology 43 , 329-336 (1991). Wang, J.B. & Mantsch, J.R. l-tetrahydropalamatine: a potential new medication for the treatment of cocaine addiction. Future Med Chem 4 , 177-186 (2012). Xu, P. et al. Structures of the human dopamine D3 receptor-G(i) complexes. Mol Cell 81 , 1147-1159.e1144 (2021). Crist, A.M. et al. Transcriptomic analysis to identify genes associated with selective hippocampal vulnerability in Alzheimer's disease. Nature communications 12 , 2311 (2021). Rao, Y.L. et al. Hippocampus and its involvement in Alzheimer's disease: a review. 3 Biotech 12 , 55 (2022). Zhu, H. et al. The clinical characteristics and molecular mechanism of pituitary adenoma associated with meningioma. J Transl Med 17 , 354 (2019). Pekic, S., Miljic, D. & Popovic, V. in Endotext. (eds. K.R. Feingold et al.) (MDText.com, Inc. Copyright © 2000-2023, MDText.com, Inc., South Dartmouth (MA); 2000). Yildirim, M., Cengil, E., Eroglu, Y. & Cinar, A. Detection and classification of glioma, meningioma, pituitary tumor, and normal in brain magnetic resonance imaging using deep learning-based hybrid model. Iran Journal of Computer Science 6 , 455-464 (2023). Torabi, S.F. et al. RNA stabilization by a poly(A) tail 3'-end binding pocket and other modes of poly(A)-RNA interaction. Science 371 (2021). Li, J. et al. 3'-Poly(A) tail enhances siRNA activity against exogenous reporter genes in MCF-7 cells. J RNAi Gene Silencing 2 , 195-204 (2006). Mitton-Fry, R.M., DeGregorio, S.J., Wang, J., Steitz, T.A. & Steitz, J.A. Poly(A) tail recognition by a viral RNA element through assembly of a triple helix. Science 330 , 1244-1247 (2010). van Rooy, I., Mastrobattista, E., Storm, G., Hennink, W.E. & Schiffelers, R.M. Comparison of five different targeting ligands to enhance accumulation of liposomes into the brain. Journal of controlled release : official journal of the Controlled Release Society 150 , 30-36 (2011). Oller-Salvia, B. et al. MiniAp-4: A Venom-Inspired Peptidomimetic for Brain Delivery. Angewandte Chemie (International ed. in English) 55 , 572-575 (2016). Huile, G. et al. A cascade targeting strategy for brain neuroglial cells employing nanoparticles modified with angiopep-2 peptide and EGFP-EGF1 protein. Biomaterials 32 , 8669-8675 (2011). Dos Santos Rodrigues, B., Arora, S., Kanekiyo, T. & Singh, J. Efficient neuronal targeting and transfection using RVG and transferrin-conjugated liposomes. Brain Res 1734 , 146738 (2020). Shen, J. et al. Poly(ethylene glycol)-block-poly(D,L-lactide acid) micelles anchored with angiopep-2 for brain-targeting delivery. J Drug Target 19 , 197-203 (2011). Wang, L. & Ma, Q. Clinical benefits and pharmacology of scutellarin: A comprehensive review. Pharmacol Ther 190 , 105-127 (2018). Bakun, P. et al. Tea-break with epigallocatechin gallate derivatives - Powerful polyphenols of great potential for medicine. European journal of medicinal chemistry 261 , 115820 (2023). Habteyes, T.G. et al. Hierarchical Self-Assembly of Carbon Dots into High-Aspect-Ratio Nanowires. Nano Lett 23 , 9474-9481 (2023). Gaillard, P.J. et al. Pharmacokinetics, brain delivery, and efficacy in brain tumor-bearing mice of glutathione pegylated liposomal doxorubicin (2B3-101). PloS one 9 , e82331 (2014). Ohno, M. et al. BACE1 gene deletion prevents neuron loss and memory deficits in 5XFAD APP/PS1 transgenic mice. Neurobiology of disease 26 , 134-145 (2007). Gao, D. & Liu, Y. Berberine regulates endocrine function in mice with polycystic ovary syndrome through PI3K/Akt/GSK-3β insulin signaling pathway. Tropical Journal of Pharmaceutical Research (2022). Bajad, N.G. et al. Combined structure and ligand-based design of dual BACE-1/GSK-3β inhibitors for Alzheimer’s disease. Chemical Papers 76 , 7507-7524 (2022). Au, L. et al. Cytokine release syndrome in a patient with colorectal cancer after vaccination with BNT162b2. Nature medicine 27 , 1362-1366 (2021). Yuan, Z.Y. et al. TATA boxes in gene transcription and poly (A) tails in mRNA stability: New perspective on the effects of berberine. Sci Rep 5 , 18326 (2015). Yuan, Z. et al. Berberine inhibits mRNA degradation by promoting the interaction between the poly A tail and its binding protein PABP. Journal of Chinese Pharmaceutical Sciences 26 , 53-62 (2017). Hou, X., Zaks, T., Langer, R. & Dong, Y. Lipid nanoparticles for mRNA delivery. Nature reviews. Materials 6 , 1078-1094 (2021). Debnath, D., Kumar, G.S. & Maiti, M. Circular dichroism studies of the structure of DNA complex with berberine. J Biomol Struct Dyn 9 , 61-79 (1991). Saran, A., Srivastava, S., Coutinho, E. & Maiti, M. 1H NMR investigation of the interaction of berberine and sanguinarine with DNA. Indian J Biochem Biophys 32 , 74-77 (1995). Nandi, R., Debnath, D. & Maiti, M. Interactions of berberine with poly(A) and tRNA. Biochim Biophys Acta 1049 , 339-342 (1990). Yadav, R.C. et al. Berberine, a strong polyriboadenylic acid binding plant alkaloid: spectroscopic, viscometric, and thermodynamic study. Bioorg Med Chem 13 , 165-174 (2005). Han, X. et al. An ionizable lipid toolbox for RNA delivery. Nature communications 12 , 7233 (2021). Han, X. et al. Ligand-tethered lipid nanoparticles for targeted RNA delivery to treat liver fibrosis. Nature communications 14 , 75 (2023). Pattipeiluhu, R. et al. Anionic Lipid Nanoparticles Preferentially Deliver mRNA to the Hepatic Reticuloendothelial System. Adv Mater 34 , e2201095 (2022). Kim, M. et al. Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver. Science advances 7 (2021). Böttger, R. et al. Lipid-based nanoparticle technologies for liver targeting. Adv Drug Deliv Rev 154-155 , 79-101 (2020). Sinegra, A.J., Evangelopoulos, M., Park, J., Huang, Z. & Mirkin, C.A. Lipid Nanoparticle Spherical Nucleic Acids for Intracellular DNA and RNA Delivery. Nano Lett 21 , 6584-6591 (2021). Lokugamage, M.P. et al. Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs. Nature biomedical engineering 5 , 1059-1068 (2021). Radmand, A. et al. The Transcriptional Response to Lung-Targeting Lipid Nanoparticles in Vivo. Nano Lett 23 , 993-1002 (2023). Okuyama, T. et al. Iduronate-2-Sulfatase with Anti-human Transferrin Receptor Antibody for Neuropathic Mucopolysaccharidosis II: A Phase 1/2 Trial. Molecular therapy : the journal of the American Society of Gene Therapy 27 , 456-464 (2019). Beola, L., Iturrioz-Rodríguez, N., Pucci, C., Bertorelli, R. & Ciofani, G. Drug-Loaded Lipid Magnetic Nanoparticles for Combined Local Hyperthermia and Chemotherapy against Glioblastoma Multiforme. ACS nano 17 , 18441-18455 (2023). Villa-Cedillo, S.A. et al. CDNF overexpression prevents motor-cognitive dysfunction by intrastriatal CPP-based delivery system in a Parkinson's disease animal model. Neuropeptides 102 , 102385 (2023). Davidson, R.C. et al. Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans. Fungal Genet Biol 29 , 38-48 (2000). Chien, E.Y. et al. Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist. Science 330 , 1091-1095 (2010). Link, T.M., Valentin-Hansen, P. & Brennan, R.G. Structure of Escherichia coli Hfq bound to polyriboadenylate RNA. Proceedings of the National Academy of Sciences of the United States of America 106 , 19292-19297 (2009). Delano, W.L. PyMOL: An Open-Source Molecular Graphics Tool. (2002). Ravindranath, P.A., Forli, S., Goodsell, D.S., Olson, A.J. & Sanner, M.F. AutoDockFR: Advances in Protein-Ligand Docking with Explicitly Specified Binding Site Flexibility. PLoS Comput Biol 11 , e1004586 (2015). Trott, O. & Olson, A.J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31 , 455-461 (2010). Assmann, J.C. et al. Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice. Bio-protocol 7 (2017). Zhang, Y., Huo, M., Zhou, J. & Xie, S. PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Comput Methods Programs Biomed 99 , 306-314 (2010). Puzzo, D., Lee, L., Palmeri, A., Calabrese, G. & Arancio, O. Behavioral assays with mouse models of Alzheimer's disease: practical considerations and guidelines. Biochemical pharmacology 88 , 450-467 (2014). Jing, D. et al. Tissue clearing of both hard and soft tissue organs with the PEGASOS method. Cell research 28 , 803-818 (2018). Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files Supportinginformation.docx Scheme1.docx Cite Share Download PDF Status: Published Journal Publication published 10 Mar, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4626003","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":324620293,"identity":"137fba33-e69e-4e7a-acf1-f746cd1f4296","order_by":0,"name":"Zhi-Hong Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYDACCYaEA0BKDsplJl6LMRAzNhCrBQwSG4jWIj+74eGBnztq09fOSH/+gKHCOrFB7PABvFoY5xxIONh75njuths5hg0MZ9ITG6TTEvBqYZZISDjA23YMpIWxgbHtMFBLjgFeLWxALQf/th1LN7uR/rCB8R8RWniAWg7zttUkmN1IMGxgbCBCiwRIi2zbAcNtZ94Yzkg4lm7cRsgv8jNykj++bauTNzue/uDDhxpr2X7p5AN4tQCdBjLzMIQNYrIRUA8E7CAz6wirGwWjYBSMgpELABFVTprl2IzdAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-7956-2481","institution":"Macau University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Zhi-Hong","middleName":"","lastName":"Jiang","suffix":""},{"id":324620294,"identity":"de3df1f2-dc9a-4215-8404-1dd770042b08","order_by":1,"name":"Chong Li","email":"","orcid":"https://orcid.org/0000-0002-6049-7675","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Chong","middleName":"","lastName":"Li","suffix":""},{"id":324620295,"identity":"94fef644-d8f7-4299-b15e-f17d97aeace4","order_by":2,"name":"Xufei Bian","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Xufei","middleName":"","lastName":"Bian","suffix":""},{"id":324620296,"identity":"df9ceaf2-9469-4060-90c7-8cf88524c2e2","order_by":3,"name":"Qian Guo","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Guo","suffix":""},{"id":324620297,"identity":"2fa84e3f-9c37-4f64-b569-aa002c30c5c7","order_by":4,"name":"Ling Yang","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Yang","suffix":""},{"id":324620298,"identity":"c68bcf1a-80e5-4216-82d6-723e3bbd0dae","order_by":5,"name":"Xiaoyou Wang","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyou","middleName":"","lastName":"Wang","suffix":""},{"id":324620299,"identity":"fc1c2127-14ce-4c2b-9961-18f7cc33a28f","order_by":6,"name":"Shikang Zhao","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Shikang","middleName":"","lastName":"Zhao","suffix":""},{"id":324620300,"identity":"2501b2dd-5632-49bc-9471-98cbd153d621","order_by":7,"name":"Shiqiong Wu","email":"","orcid":"","institution":"School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, P. R. China","correspondingAuthor":false,"prefix":"","firstName":"Shiqiong","middleName":"","lastName":"Wu","suffix":""},{"id":324620301,"identity":"b136e6f5-420d-4c41-9eab-47de804cf343","order_by":8,"name":"Xurong Qin","email":"","orcid":"https://orcid.org/0000-0002-9290-1943","institution":"Engineering Research Center of Coptis Development and Utilization, Ministry of Education, College of Pharmaceutical Sciences, Southwest University.","correspondingAuthor":false,"prefix":"","firstName":"Xurong","middleName":"","lastName":"Qin","suffix":""},{"id":324620302,"identity":"c7d28df7-2124-4a56-ac0a-dd81cb352783","order_by":9,"name":"Lee-Fong Yau","email":"","orcid":"https://orcid.org/0000-0001-9627-0088","institution":"Macau University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Lee-Fong","middleName":"","lastName":"Yau","suffix":""}],"badges":[],"createdAt":"2024-06-23 16:30:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4626003/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4626003/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-57488-0","type":"published","date":"2025-03-10T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59982878,"identity":"e17a2e24-070b-40a4-8ddd-f6c1337889fe","added_by":"auto","created_at":"2024-07-10 06:34:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":282480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening and optimization of a novel ionizable lipidoid based on \u0026nbsp;tetrahydroisoquinine alkaloids (tetrahydroberberine)\u003c/strong\u003e. (\u003cstrong\u003ea\u003c/strong\u003e) Chemical structures of the lipid building blocks, which include five amine head groups and fifteen alkylated tails. (\u003cstrong\u003eb\u003c/strong\u003e) Optimization scheme for the lipidoid molecule BE; this phase involved the preparation and evaluation of 27 different formulations derived from 75 novel ionizable compounds. (\u003cstrong\u003ec\u003c/strong\u003e) The affinitiesof variousionizable lipidoid molecules for poly(A) measured using fluorescence spectroscopy. (\u003cstrong\u003ed-e\u003c/strong\u003e) The cellular uptake of the LNP via fluorescence co-localization studies and flow cytometry analysis (Scale bar: 20 µm). Data are presented as means ± SD (\u003cem\u003en\u003c/em\u003e = 3 biologically independent samples).(\u003cstrong\u003ef\u003c/strong\u003e) \u003cem\u003eIn vivo\u003c/em\u003e imaging of mice after injection of LNP@DiR at time intervals of 0.5, 1, 2, 4, and 8h (\u003cstrong\u003eg\u003c/strong\u003e) \u003cem\u003eEx vivo\u003c/em\u003e fluorescence distribution showing thebrain-liver ratio after 1 h. Statistical significane is denoted by **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001, based on two-tailed unpaired \u003cem\u003et\u003c/em\u003e-tests. Data are presented as means ± SD from three biologically independent samples.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4626003/v1/0c43366b9ece6420b9e49944.png"},{"id":59982294,"identity":"774ef268-dc07-4f67-9c2d-917567ab3f4f","added_by":"auto","created_at":"2024-07-10 06:26:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":220997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e \u003cstrong\u003echaracterization of\u003c/strong\u003e \u003cstrong\u003eLNP (BE) composed of ionizable lipid A2-B13\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Gel retardation assay of BE@siRNA acrossLNP/siRNA weight ratios of 0.2, 0.5, 1, 2, 5, 10, 15, and 20, evaluating complex stability. (\u003cstrong\u003eb\u003c/strong\u003e) Dynamic light scattering measurement of the size distribution and (\u003cstrong\u003ec\u003c/strong\u003e) transmission electron microscopy (TEM) image of BE@siRNA, indicating particle morphology (Scale bar: 100 nm). (\u003cstrong\u003ed\u003c/strong\u003e) Comparison of cellular transfection efficiency between BE@siRNA with MC3@siRNA using a fluorescence microscope (Scale bar: 25 µm). (\u003cstrong\u003ee\u003c/strong\u003e) Subcellular localization studies of BE@siRNA or MC3@siRNA observed under higher magnification (Scale bar: 5 µm). (\u003cstrong\u003ef\u003c/strong\u003e) Investigation of the endocytosis mechanism mediated by BE, with statistical significance denoted as ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; \u003cem\u003eNS\u003c/em\u003e indicates no significant difference. Data are presented as means ± SD from three biologically independent samples, analyzed using two-tailed unpaired \u003cem\u003et\u003c/em\u003e-tests. (\u003cstrong\u003eg\u003c/strong\u003e-\u003cstrong\u003eh\u003c/strong\u003e) Evaluation of LNP@DiD permeability using an \u003cem\u003ein vitro\u003c/em\u003e blood-brain barrier (BBB) model implemented with a transwell assay (Scale bar: 1000 µm).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4626003/v1/5b603c3107cc73d19daed6d8.png"},{"id":59981045,"identity":"3b15b71e-ebf4-4c38-9f71-a1ecd2e0d6d6","added_by":"auto","created_at":"2024-07-10 06:10:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":322273,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of brain targeting of BE-ST. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Flow cytometry analysis of cellular uptake of BE-ST in the presence and absence of DRD3, whose expression was downregulated using siRNA.(\u003cstrong\u003eb\u003c/strong\u003e) Measurement of the zeta potential for BE when paired with different organic acids. (\u003cstrong\u003ec\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eEx vivo\u003c/em\u003e analysis showing the fluorescence distribution of the brain-liver ratio after administration of BE paired with different organic acids. (\u003cstrong\u003ed\u003c/strong\u003e) Combined \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e imaging of mice at 1 h post-injection of LNP@DiR, following administration of BE paired with different organic acids. (\u003cstrong\u003ee\u003c/strong\u003e) Plasma concentration of BE and BE-ST (BE paired with Scutellarin at a weight ratio of 1:5) in SD rats after intravenous administration. (\u003cstrong\u003ef\u003c/strong\u003e) \u003cem\u003eIn vivo \u003c/em\u003eimaging time-course of mice after administration of LNP, captured at 0.5, 1, 2, 4, and 8 h post-injection. (\u003cstrong\u003eg\u003c/strong\u003e) E\u003cem\u003ex vivo \u003c/em\u003eimaging of mouse brains at 1, 2, 4, and 8 h post-administration of LNP. (\u003cstrong\u003eh\u003c/strong\u003e) Semi‒quantitative analysis of fluorescence intensity across the time points. Data are presented as means ± SD for three biologically independent samples. Statistical significance is indicated by *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001; \u003cem\u003eNS\u003c/em\u003e denotes no significant difference. Statistical analyses were performed using two-tailed unpaired \u003cem\u003et\u003c/em\u003e-tests.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4626003/v1/d15693457afaa4855957dcda.png"},{"id":59981047,"identity":"f5e482b5-014d-48b6-bfe2-044113528e79","added_by":"auto","created_at":"2024-07-10 06:10:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":218407,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBehavioral evaluation of BE(-ST)@siBACE1 therapy in APP/PS1 mice\u003c/strong\u003e.(\u003cstrong\u003ea\u003c/strong\u003e) Schematic of the experimental design: APP/PS1 and wild-type (WT) mice received tail vein injections of LNP@siBACE1 or PBS every 2 days over seven cycles. Following treatments, mice underwent the Morris water maze (MWM) tests to evaluate memoryfunctions, and tissue samples were collected for molecular pathological assessments.(\u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003ee\u003c/strong\u003e) Results from the probe tests in the MWM: (\u003cstrong\u003eb\u003c/strong\u003e) Representative swimming paths of mice searching for the submerged platform;(\u003cstrong\u003ec\u003c/strong\u003e) ratio of time spent in the target quadrant where the platform was previously located; (\u003cstrong\u003ed\u003c/strong\u003e) number of times each group crossed the former platform location during the probe test day, (\u003cstrong\u003ee\u003c/strong\u003e) average swimming speeds across all groups. Behavioral test results are presented as means ± SD from six biologically independent samples. Statistical significance is denoted by *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, determined using two-tailed unpaired \u003cem\u003et\u003c/em\u003e-tests.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4626003/v1/8c083c21aa0a31f86e5881ad.png"},{"id":59981052,"identity":"531d9272-e53a-4c04-8113-c4abb2813ebe","added_by":"auto","created_at":"2024-07-10 06:10:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":508244,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTherapeutic\u003c/strong\u003e \u003cstrong\u003esynergy of BE(-ST) and siBACE1 to modulating Alzheimer’s disease (AD) hallmarks in APP/PS1 mice\u003c/strong\u003e.(\u003cstrong\u003ea\u003c/strong\u003e) Representative western blot images showing levels of BACE1, phosphorylated tau (p-tau), and GSK3β in the hippocampus and cortex of BE(-ST)@siBACE1-treated APP/PS1 mice, control APP/PS1 groups, and wild-type (WT) mice. (\u003cstrong\u003eb\u003c/strong\u003e) Quantitative analysis of western blot for BACE1, p-tau, and phosphorylated GSK3β (p-GSK3β), normalized to GAPDH. (\u003cstrong\u003ec\u003c/strong\u003e) Representative confocal laser scanning microscopy images depicting amyloid (Aβ) plaque burden in the hippocampus and cortex of APP/PS1 and WT mice, with Aβ plaques stained green and nuclei stained with DAPI (blue). (Top row scale bar = 500 µm, bottom row scale bar = 250 µm) (\u003cstrong\u003ed\u003c/strong\u003e) Nissl staining of brain sections taken at 14 days post-treatment from different experimental groups (Scale bar: 50 µm). (\u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e) ELISA results for the evaluation of complement activation-related pseudoallergy (CARPA) markers: C5b9, C3a, and monocyte chemoattractant protein (MCP-1) in serum. Data are presented as means ± SD for three biologically independent samples. Statistical significance is indicated by *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. Analyses were performed using two-tailed unpaired \u003cem\u003et\u003c/em\u003e-tests.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4626003/v1/11ad810dc84b969e94bc63fb.png"},{"id":59981873,"identity":"73b29db3-45d3-44c0-9c81-0f0d0a919c2d","added_by":"auto","created_at":"2024-07-10 06:18:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":400451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e evaluation of the efficacy of BE-ST for the delivery of small-molecule and macromolecule drugs\u003c/strong\u003e. (\u003cstrong\u003ea\u003c/strong\u003e) Schematic of the experimental timeline for treating a mouse model of cryptococcal meningitis. AMB denotes amphotericin B. (\u003cstrong\u003eb-c\u003c/strong\u003e) Fluorescence imaging and quantitative live imaging analysis of mice. Data are presented as means ± SD for five biologically independent samples; *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01. Statistical significances were calculated using multiple \u003cem\u003et\u003c/em\u003e-tests.(\u003cstrong\u003ed\u003c/strong\u003e) Quantification of fungal colony burdens in brain tissues of treated mice. Data are presented as means ± SD for six biologically independent samples; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. Analysis was performedwith two-tailed unpaired \u003cem\u003et\u003c/em\u003e-tests. (\u003cstrong\u003ee\u003c/strong\u003e) Kaplan-Meier survival curves for each treatment group for ten biologically independent samples. (\u003cstrong\u003ef\u003c/strong\u003e) Fluorescence microscope images showing \u003cem\u003ein vitro\u003c/em\u003e transfection of eGFP mRNA (Scale bar: 25 µm). (\u003cstrong\u003eg\u003c/strong\u003e) Three-dimensional (3D) ligand-RNA interaction visualization for the binding site of polyA with the leadcompound A2-B13. (\u003cstrong\u003eh\u003c/strong\u003e) Analysis of the stability of BE-loaded mRNA usingagarose gel electrophoresis. (\u003cstrong\u003ei\u003c/strong\u003e) 3D hyalinization microscopy images of brain tissue after administration of 10 mg eGFP mRNA (Scale bar: 1500 µm).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4626003/v1/456d6dfbb9bf6a7dcba4ebf5.png"},{"id":78227060,"identity":"ab35b831-b2a8-4b44-8539-fa3dbfe8e31f","added_by":"auto","created_at":"2025-03-11 07:07:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3648389,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4626003/v1/961c829b-9690-4e88-b32f-f1ff9e984dad.pdf"},{"id":59981874,"identity":"f56169a2-0403-43c2-b31e-99f11dc753e0","added_by":"auto","created_at":"2024-07-10 06:18:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2119237,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4626003/v1/93e2625be00a91395ed872b0.docx"},{"id":59981870,"identity":"6c426e16-7f7e-4643-be5b-449c9e1443bd","added_by":"auto","created_at":"2024-07-10 06:18:58","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":862151,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4626003/v1/2c092ebe052506862cc9e2a5.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Berberine-inspired ionizable lipid for self-structure stabilization and potent brain targeting delivery of nucleic acid therapeutics","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, small-molecule natural products have gained prominence not only as lead compounds in drug development but also as facilitators of drug delivery. Illustrative examples include the complexation of monoclonal antibodies, such as Herceptin, with flavonoids like (\u0026minus;)-epigallocatechin-3-\u003cem\u003eO\u003c/em\u003e-gallate (EGCG)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e to counteract cancer, and the TANNylation of therapeutic proteins or peptides with phenols such as tannic acid (TA)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e for specific targeting of elastins and collagens, thereby augmenting the treatment of heart diseases. Additionally, the substitution of cholesterol with terpenes, exemplified by ginsenoside Rg\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e in the lipid bilayer of liposomes, has demonstrated the ability to target GLUT1 receptors, thereby enhancing cancer therapy. While various classes of natural small molecules\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, including flavonoids, terpenes, and others like coumarins, quinones, and steroids, have been explored for drug delivery, the application of alkaloids as auxiliary materials in this field is still in its nascent stage. Alkaloids, with their unique structural features of tertiary and quaternary amines, show potential for robust interactions with nucleic acid drugs, positioning them as promising carriers in nucleic acid therapeutics. Their distinctive biomembrane penetration capabilities can enhance targeted delivery, as evidenced by their direct effects on bacterial biofilms\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, cell membranes\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and organelle membranes\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBerberine (BE)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, a prominent alkaloid in the isoquinoline series, and its analogs such as jateorrhizine, African tetrandrine, and palmatine are naturally occurring protoberberine alkaloids found in various plant species. Tetrahydroberberine-type alkaloids\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, derived from plants or synthesized through hydrogenation of berberine-type alkaloids, have garnered attention due to their increased stability, bioavailability, safety, and potential therapeutic efficacy. These protoberberine alkaloids, with their isoquinoline or tetrahydroisoquinoline skeletons, display a versatile pharmacological profile\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, possessing antimicrobial, anti-inflammatory, antioxidant, and antitumor properties. Since the 1960s, the nucleic acid interactions of protoberberine, particularly its ability to bind DNA\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e with a preference for thymidine-adenine pairs, have been extensively studied. Recent studies have highlighted the ability of isoquinoline alkaloids to bind mRNA\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e through partial intercalation, specifically targeting the poly(A) tails of mRNA, leading to self-structure formation via hydrogen bonding and π-π stacking\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These interactions help maintain the structural integrity of polyA. Further studies have revealed specific interactions between protoberberine\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and receptor proteins on cell surfaces, notably exhibiting high affinity for dopamine receptors, particularly the dopamine D3 receptor (D3R)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, which is abundant in brain regions associated with Alzheimer's disease (AD), such as the hippocampus and cortex\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, as well as the pituitary gland region\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e which are prone to brain infections and tumors. This suggests that the tetrahydroisoquinoline ring-fused skeleton of protoberberine-type alkaloids, when complexed with nucleic acid drugs, may offer unique advantages for brain-targeted delivery.\u003c/p\u003e \u003cp\u003eIn response to these findings, we have designed a series of innovative ionizable lipids inspired by the protoberberine alkaloid structure. These lipids, focusing on the stable and safe tetrahydroberberines, have undergone extensive validation from molecular investigations to animal studies. Our findings reveal the following multifaceted advantages: (i) a robust capacity for nucleic acid loading and stability; (ii) enhanced endosomal escape of nucleic acid drugs and improved intracellular transport; (iii) significantly improved brain targeting characteristics \u003cem\u003ein vitro\u003c/em\u003e, corroborated by \u003cem\u003ein vivo\u003c/em\u003e studies; and (iv) the inheritance of pharmacological activity from protoberberine alkaloids, enabling synergistic treatment with loaded nucleic acid drugs.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDesign and screening of BE lipidoids\u003c/h2\u003e \u003cp\u003eInspired by the structural frameworks of tetrahydroberberine-type alkaloids and their analogs, we conducted preliminary screenings of five head groups. The tail chains were designed to incorporate diverse, representative structural features such as unsaturated bonds, ester groups, and alcoholic hydroxyl groups, which are conducive to gene delivery (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). For nucleic acid delivery, we employed a combination of helper lipids (DSPC, DOPC, and DOPE), DMG-PEG\u003csub\u003e2000\u003c/sub\u003e, cholesterol (Chol), and newly proposed ionizable lipidoids. The lipid compositions in the BE formulations were meticulously optimized with the following relative molar percentages: ionizable lipids (29\u0026ndash;39%), helper lipids (10\u0026ndash;30%), Chol (20\u0026ndash;59%), and DMG-PEG\u003csub\u003e2000\u003c/sub\u003e (1\u0026ndash;2%), ensuring the sum of molar ratios reached 100% for each formulation (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMultiple screening rounds using blank lipid nanoparticles (LNPs) were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Initially, optimal formulation 10 was identified based on the size of various preparations (Supplementary Table S2). Subsequently, BEs were prepared using 27 candidate compounds, each with a size smaller than 100 nm (Supplementary Table S3). The second screening phase measured the pKa of 10 eligible compounds, revealing values within the optimal range for effective RNA delivery (6.0\u0026ndash;7.0) (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In the third phase, we assessed the binding interactions of these derivatives with the poly(A) tail which is crucial for RNA stabilization\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, resulting in the identification of five candidate compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). \u003cem\u003eIn vitro\u003c/em\u003e targeting experiments demonstrated that the LNP containing these compounds exhibited higher uptake rates than the control group (NP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-e). This trend was consistently observed \u003cem\u003ein vivo\u003c/em\u003e, with compounds A2-B13 emerging as the most promising lipidoid (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-g and Supplementary Fig. S2).\u003c/p\u003e \u003cp\u003eRepresentative ionizable lipids (A2-B13) were synthesized as outlined in the Supplementary Information, and their structural integrity was confirmed by \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH nuclear magnetic resonance (NMR) analysis (Supplementary Fig. S3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and characterization of BE@siRNA\u003c/h2\u003e \u003cp\u003eThe lead structure (A2-B13) and its formulation composition (molar ratio A2-B13:DOPC:Chol:PEG-DMG\u0026thinsp;=\u0026thinsp;39:20:30:1) were thoyoughly screened and selected. Gel retardation assay analysis confirmed that BE effectively entrapped siRNA at mass ratios exceeding 1:2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The BE and BE@siRNA complexes exhibited rounded morphologies with hydrodynamic sizes of approximately 70 nm and 90 nm, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-c). To evaluate the transfection efficiency, FAM-labeled siRNA was encapsulated in BE, demonstrating robust transfection efficacy comparable to the commercial lipidoid DLin-MC3-DMA, as evidenced by equivalent intracellular fluorescence intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Additionallye, BE facilitated siRNA escape from endosomes/lysosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further investigated the endocytic pathways by comparing the uptake efficiency of BE@DiD in the presence of various inhibitors against BE@DiD uptake without inhibitors. Cells pretreated with inhibitors exhibited significant variations in endocytosis efficiency, with chlorpromazine notably reducing clathrin-mediated cellular entry of BE@DiD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). The capability of BE to traverse the blood-brain barrier (BBB) \u003cem\u003ein vitro\u003c/em\u003e was confirmed using a transwell model, illustrating significantly enhanced penetration of BE-treated conditions compared to fluorescein DiD control nanoparticles (NP) and MC3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg-h), highlighting BE\u0026rsquo;s potential to endocytose endothelial cells efficiently.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBrain targeting of BE-ST\u003c/b\u003e (BE\u0026thinsp;+\u0026thinsp;Scutellarin)\u003c/p\u003e \u003cp\u003eOur \u003cem\u003ein vitro\u003c/em\u003e quantitative flow cytometry analysis indicated an obviously enhanced uptake of BE, showing 65.84 times the uptake compared to NP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). This uptake was markedly higher than that observed with nanoparticles modified with classical ligands, which typically showed enhancements ranging from 0.2-fold to 1.6-fold\u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32 CR33\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. To elucidate the underlying mechanisms, we downregulated the expression of the DRD3 protein using siRNA. Protein expression was reduced by approximately 50% after 72 h post-transfection with siDRD3 compared to the negative control (Supplementary Fig. S4). The decreased uptake of BE in cells with reduced DRD3 expression suggests an interaction between BE and this protein, corroborating literature reports\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and our findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, Supplementary Table S4, Fig. S5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e brain targeting, we implemented an acid-base pair strategy to neutralize charges, aiming to mitigate the impact of the positive charge on \u003cem\u003ein vivo\u003c/em\u003e metabolism and distribution. After charge neutralization with organic acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) such as scutellarin (ST)\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, EGCG\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, and citric acid (CA)\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, we observed a reduced distribution in the liver. Notably, the combination with ST resulted in a brain-to-liver distribution ratio approaching 20% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d). This charge neutralization also enhanced pharmacokinetics, including a higher peak plasma concentration and a 1.73-fold increase in half-life for the BE-ST group compared to the BE group.(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eTo evaluate the brain-targeting efficiency of nanoparticles \u003cem\u003ein vivo\u003c/em\u003e, different LNPs labeled with DiR were administered to normal mice. The BE-ST nanoparticles showed significantly stronger fluorescence intensity in the brain compared to those without BE-ST (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). \u003cem\u003eEx vivo\u003c/em\u003e imaging conducted at 0.5, 1, 2, 4, and 8 h post-injection further demonstrated that the BE-ST group exhibited stronger fluorescence signals in the brain compared to the positive control, wherein glutathione (GSH) served as the target head group (GSH-NP) and is currently in clinical trials for brain-targeted delivery\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, Supplementary Fig. S6). Quantitative analysis reinforced these observations, indicating that the fluorescence intensity in the brain for the BE-ST group was approximately 167% of that observed in the positive control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBrain-targeted delivery of siBACE1 by BE-ST for comprehensive treatment of AD\u003c/h2\u003e \u003cp\u003eThe efficacy of the BE-ST delivery system was initially assessed using AD as a model, targeting the beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1)\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e as a crucial pharmacological intervention point. BE-ST@siBACE1 or control BE-ST@siScr (siRNA, 1 mg/kg) were administered bilaterally via caudal vein injections to APP/PS1 mice, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, with MC3@siBACE1 serving as a comparative control. Additionally, PBS was injected into both APP/PS1 and wild-type (WT) mice to establish baseline AD-related deficits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSpatial learning and memory were assessed using the Morris water maze (MWM). Mice treated with PBS and MC3@siBACE1 exhibited an undirected search pattern, suggesting no improvement in cognitive abilities, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb. There were no significant differences in swimming speed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Conversely, APP/PS1 mice treated with BE-ST@siBACE1 demonstrated marked improvement, spending 1.95 times longer in the target quadrant and crossing it more frequently compared to PBS-treated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). These results strongly support the effectiveness of BE-ST@siBACE1 in enhancing cognitive performance in APP/PS1 mice.\u003c/p\u003e \u003cp\u003eAfter the behavioral evaluations, mice were euthanized, and brain tissues were harvested to assess the levels of BACE1 and its impact on amyloid-β (Aβ) and tau pathology accumulation. Our results showed that BE-ST@siBACE1 treatment led to significant reductions in BACE1 levels in the hippocampus and cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b; Supplementary Fig. S7\u003cb\u003ea\u003c/b\u003e). Moreover, there was a noticeable decrease in both the number and size of amyloid plaques, a critical neuropathological feature of AD resulting from the cleavage of amyloid precursor protein (APP) by BACE1, within the hippocampus and cortex of BE-ST@siBACE1-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Additionally, a reduction in the levels of hyperphosphorylated tau protein (p-tau), a key marker of advanced AD\u0026rsquo;s pathology, was observed in the hippocampus and cortex of the BE-ST@siBACE1-treated mice compared to those receiving PBS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b; Supplementary Fig. S7\u003cb\u003eb\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProtoberberine and its derivatives are known for their synergistic\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e effects on p-tau and Aβ through the inhibition of glycogen synthase kinase-3β (GSK-3β). This interaction regulates Ser9\u003csup\u003e41\u003c/sup\u003e phosphorylation and affects BACE1 activity. To explore the potential of a tetrahydroberberine derivative in this context, we conducted a detailed study. Our findings revealed that phosphorylation at Ser9 of GSK3β (p-GSK3β) was significantly reduced in both the hippocampus and cortex of APP/PS1 mice treated with BE-ST@siBACE1 and BE-ST@siScr, compared to the untreated AD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b; Supplementary Fig. S7\u003cb\u003ec\u003c/b\u003e). This reduction suggests that BE-ST maintains its inhibitory effect on GSK-3β and BACE1, leading to a synergistic therapeutic effect against dementia, corroborating the behavioral improvements observed in BE-ST@siScr-treated APP/PS1 mice.\u003c/p\u003e \u003cp\u003eOxidative stress is a crucial factor in the development and progression of AD. In this study, we observed that treatment with BE-ST@siScr and BE-ST@siBACE1 resulted in diminished levels of superoxide dismutase (SOD) and malondialdehyde (MDA), along with core proinflammatory cytokines in APP/PS1 mice. This led to a notable alleviation of oxidative stress (Supplementary Fig. S8\u003cb\u003ea\u003c/b\u003e-\u003cb\u003eb\u003c/b\u003e) and inflammation (Supplementary Fig. S8\u003cb\u003ec\u003c/b\u003e-\u003cb\u003ef\u003c/b\u003e) within the cerebral context, shedding light on potential mechanisms for the observed reductions in Aβ and p-tau levels. Furthermore, the application of BE-ST and BACE1 significantly preserved neuronal integrity and morphology, preventing substantial neuronal shrinkage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eRoutine blood biochemical analyses and histological examinations of tissues samples collected two weeks post-injection revealed no significant differences between the PBS-treated and BE(-ST)@siRNA-treated groups, confirming the excellent biocompatibility of BE(-ST) (Supplementary Figs. S9, S10). Additionally, there was no discernible increase in immunogenicity markers such as complement components and cytokines. Specifically, levels of C5b9, C3a, and monocyte chemoattractant protein (MCP-1) remained stable two weeks after BE(-ST) treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-g). In contrast, the MC3-treated group displayed a significant deviations from baseline, aligning with previous reports that LNPs containing commercially available ionizable lipids like DLin-MC3-DMA can trigger immune system activation\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, potentially resulting in CARPA, a severe immunological reaction similar to anaphylactic shock. Furthermore, we utilized a real-time quantitative single-cell detection technique to investigate reactive oxygen species (ROS) generation. BE induced ROS at significantly lower levels over time compared to commercial MC3 (Supplementary Fig. S11), further highlighting the favorable safety profile of BE(-ST).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBE-ST as a versatile platform for brain disease drug delivery\u003c/h2\u003e \u003cp\u003eExploring the versatility of BE-ST, we examined its efficacy across various pathological models by administering different therapeutic agents. Initially, we utilized a brain tumor model with GL261-Luc cells to evaluate the delivery and therapeutic potential of siVEGF (Supplementary Fig. S12\u003cb\u003ea\u003c/b\u003e). Live imaging revealed a noticeable decrease in luminescence, indicating tumor activity reduction post-treatment variation with BE-ST@siVEGF. Quantitative analysis demonstrated a significant reduction in the mean fluorescence intensity (MFI) of brain tumors treated with BE-ST, with reductions of 27.96% at 3 days and 38.82% at 6 days post-administration, in comparison to the MC3-treated group (Supplementary Fig. S12\u003cb\u003eb\u003c/b\u003e, \u003cb\u003ec\u003c/b\u003e). The \u003cem\u003eex vivo\u003c/em\u003e imaging results highlighted BE-ST\u0026rsquo;s enhanced precision in targeting the lesion site (Supplementary Fig. S12\u003cb\u003ed\u003c/b\u003e), suggesting its potential as a robust delivery platform for siRNA therapeutics in brain tumor treatment.\u003c/p\u003e \u003cp\u003eExpanding our investigation to include small-molecule drugs and mRNA, we tested BE-ST in a broader range of animal models, covering both cancer and neurodegenerative diseases. Specifically, we addressed the challenge of treating escalating fungal meningitis, a condition with increasing incidence and high mortality, aggravated by the ineffective BBB penetration of conventional treatments like amphotericin B (AmB). We developed BE-ST@AmB for use in a cryptococcal meningitis model (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The administration of BE-ST@AmB significantly enhanced the delivery and efficacy of AmB, as evidenced by live imaging which showed a drastic reduction in luminescence variation. Quantitative assessments confirmed a reduction in the MFI of the brain by 94.15% at 3 days and 99.28% at 6 days post-treatment compared to the commercially available AmBisome-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c). Examination of the brain fungal burden indicated recovery in BE-ST@AmB-treated mice, in stark contrast to significant cryptococcal infiltration in untreated or AmBisome-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Long-term therapeutic studies further demonstrated a superior survival rate in mice treated with BE-ST compared to other treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e studies on mRNA transfection capabilities involved encapsulating eGFP mRNA into BE, revealing superior transfection efficiency compared to the commercial lipid nanoparticle DLin-MC3-DMA. This was evidenced by elevated intracellular fluorescence levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), likely due to the strong affinity between BE and polyA, a relationship supported by literature\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e and molecular docking studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg-h and Supplementary Fig. S13). The ionizable lipid derivative of tetrahydroberberine incorporated into BE not only preserved this affinity but also enhanced the structural stability of mRNA, eliminating the need for chemical modifications typically required by other nucleic acid carriers. Intravenous administration of BE-ST@eGFP mRNA effectively delivered mRNA to the brain, achieving deeper penetration and higher fluorescence in brain tissue compared to MC3@eGFP mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the evolving landscape of ionizable lipid designs\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, conventional strategies have predominantly favored simpler linear tertiary amine groups to enhance pH sensitivity due to reduced steric hindrance. However, leveraging the complexation capabilities of the protoberberine family with nucleic acids, as documented in several studies\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, we adopted a novel approach in ionizable lipid design. Our design features a dual tetrahydroisoquinoline fused-ring skeleton, which uniquely binds to the polyA tail of RNA, forming stable self-structures. This innovative approach moves beyond traditional electrostatic interactions used to compact RNA\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, enabling efficient nucleic acid loading and lysosomal escape while preserving nucleic acid integrity through partial intercalation without necessitating complex chemical modifications.\u003c/p\u003e \u003cp\u003eMoreover, the use of synthetic lipidoids often falls short in biological performance, with associated cellular toxicity and immunogenicity\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e posing significant drawbacks, particularly in repeated administration scenarios. In contrast, tetrahydroisoquinoline derivatives contribute inherent pharmacological benefits, such as antioxidant and anti-inflammatory properties, potentially reducing the acute immune response typically associated with RNA therapeutics. These derivatives also serve as synergistic therapeutic adjuvants, enhancing the overall therapeutic efficacy.\u003c/p\u003e \u003cp\u003eDespite advancements in organ-specific targeting, the BBB continues to present a formidable challenge. While various lipid nanoparticle platforms have demonstrated promise in targeting organs like the liver\u003csup\u003e\u003cspan additionalcitationids=\"CR52 CR53\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, spleen\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, and lungs\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, brain targeting remains particularly complex. Traditional strategies often rely on ligand-receptor interactions using ligands such as transferrin\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e and peptides like apamin\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, glutathione\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, angiopep-2\u003csup\u003e59\u003c/sup\u003e, and RVG peptide\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. These modifications generally improve uptake efficiency by 0.2-fold to 1.6-fold\u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32 CR33\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e \u003cem\u003ein vitro\u003c/em\u003e in primary or immortalized brain microvascular endothelial cells, yet they typically achieve brain-to-liver distribution ratio below 10%.\u003c/p\u003e \u003cp\u003eIn stark contrast, our novel ionizable lipids derived from tetrahydroisoquinoline showcase a unique capability as small molecular ligands that efficiently penetrate the BBB. These lipids demonstrate superior \u003cem\u003ein vitro\u003c/em\u003e uptake and exceptional \u003cem\u003ein vivo\u003c/em\u003e performance, especially when combined with organic acids, such as ST\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, EGCG, and citric acid for charge neutralization. In conclusion, the berberine-inspired LNPs not only effectively deliver siRNA into the brain but also show potential for the delivery of small molecules and biological macromolecules like mRNA. This opens new avenues for the application of these innovative lipids in neurological therapies, promising enhanced treatment modalities for a range of brain disorders.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical approval for animal\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll\u0026nbsp;procedures involving\u0026nbsp;animal were approved by the Institutional Animal Care and Use Committee (IACUC) of Southwest University Laboratory Animal Center (Approval\u0026nbsp;No. IACUC-20230529-02).\u0026nbsp;The\u0026nbsp;experiments were conducted\u0026nbsp;in accordance with\u0026nbsp;the guidelines of the Ethical Review Committee\u0026nbsp;for\u0026nbsp;Experimental Animals at Southwest University,\u0026nbsp;China.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemicals and reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLipids including\u0026nbsp;DOPC (S01007), DSPC (S01005), DOPE (S03005), DMG-PEG\u003csub\u003e2000\u003c/sub\u003e (O02005), cholesterol (O01001) and Dlin-MC3-DMA (AVT0003) were\u0026nbsp;obtained from\u0026nbsp;A.V.T. Pharmaceutical Ltd. (Shanghai, China)\u0026nbsp;with\u0026nbsp;a\u0026nbsp;purity\u0026nbsp;of over\u0026nbsp;98%.\u0026nbsp;siRNA sequences targeting\u0026nbsp;DRD3 (siDRD3: GGUGGAGUCUGGAAUUUCA), BACE1 (siBACE1: GAACCUAUGCGAUGCGAAUTT), and VEGF (siVEGF: GGAGUACCCUGAUGAGAUCTT) were purchased from Shanghai Sangon Biotechnology Co., Ltd.\u0026nbsp;Antibodies were acquired as follows: anti-BACE1 (ab183612, 1:1000) from Abcam (Cambridge, MA, USA),\u0026nbsp;phospho-GSK3β (Ser9) (PA5-97339, 1:1000) and phospho-Tau (Ser396) (44-752G, 1:1000) from Invitrogen (Carlsbad, CA, USA), and\u0026nbsp;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\u0026nbsp;(H+L) (SA00003-2, 1:100),\u0026nbsp;and horseradish peroxidas (HRP)-conjugated Affinipure Goat Anti-Rabbit IgG\u0026nbsp;(H+L) (SA00001-2, 1:5000) were purchased from Proteintech Group, Inc. (Wuhan, China). D-Luciferin was\u0026nbsp;sourced\u0026nbsp;from Gold Biotechnology Co., Ltd. (St Louis, USA), and\u0026nbsp;LysoRed (KGMP006) from KeyGEN Biotechnology Co., Ltd. (Jiangsu, China). eGFP mRNA (05297410) was\u0026nbsp;supplied by\u0026nbsp;Novoprotein Technology Co., Ltd. (Suzhou, China). ELISA kits\u0026nbsp;and biochemical criterion kits were\u0026nbsp;obtained\u0026nbsp;from Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China)\u0026nbsp;and\u0026nbsp;Grace Biotechnology Co., Ltd. (Suzhou, China), respectively. All other reagents\u0026nbsp;used\u0026nbsp;were\u0026nbsp;of\u0026nbsp;analytical grade.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eculture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ebEnd.3\u0026nbsp;and GL261-Luc cell\u0026nbsp;lines\u0026nbsp;were\u0026nbsp;acquired\u0026nbsp;from KeyGen Biotech (Jiangsu, China).\u0026nbsp;Cells\u0026nbsp;were\u0026nbsp;cultured in DMEM (KeyGen)\u0026nbsp;supplemented with fetal bovine serum (FBS, Gibco) and maintained\u0026nbsp;at 37°C\u0026nbsp;in\u0026nbsp;a humidified atmosphere\u0026nbsp;containing\u0026nbsp;5% CO\u003csub\u003e2\u003c/sub\u003e. DRD3\u003csup\u003e−/−\u003c/sup\u003e bEnd.3 cells were transfected with siDRD3 using Lipofectamine 2000 (Invitrogen).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCryptococcus neoformans (\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eC. neoformans\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e)\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003cstrong\u003etrains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo\u0026nbsp;create\u0026nbsp;luciferase-expressing strains of \u003cem\u003eC. neoformans\u0026nbsp;\u003c/em\u003e(wild-type H99), the luciferase gene LUC1 was amplified\u0026nbsp;using\u0026nbsp;primers TL1943/1944 from plasmid pCDW104-Luciferase. The amplified product was\u0026nbsp;cloned into\u0026nbsp;vector\u0026nbsp;pTBL6 to\u0026nbsp;construct\u0026nbsp;pTBL402 (P_ACTIN-LUC1). The resulting vector\u0026nbsp;was\u0026nbsp;linearized with XbaI,\u0026nbsp;precipitated onto gold microcarrier beads (0.6 μm, Bio-Rad) and biolistically transformed into\u0026nbsp;the\u0026nbsp;H99 ura5\u0026nbsp;strain\u0026nbsp;as described previously\u003csup\u003e61\u003c/sup\u003e.\u0026nbsp;Transformants were selected\u0026nbsp;for stability\u0026nbsp;on SD-URA medium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal housing conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale and female\u0026nbsp;Balb/c mice (5 weeks, 18–22 g), C57BL/6 mice (8 months, 28–32 g), and Sprague Dawley\u0026nbsp;(SD)\u0026nbsp;rats (180–220 g) were obtained from Chongqing Academy of Chinese Materia Medica (Chongqing, China).\u0026nbsp;Additionally,\u0026nbsp;APP-PS-1 mice (8 months, 28–32 g) were\u0026nbsp;procured\u0026nbsp;from Viewsolid Biotechnology Co., Ltd. (Beijing, China). All animals were\u0026nbsp;maintained\u0026nbsp;in a pathogen-free environment\u0026nbsp;under\u0026nbsp;a 12 h light/dark cycle at 22–24°C and 30–50% humidity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of tetrahydroberberine\u003c/strong\u003e\u003cstrong\u003ederivatives\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003eN\u003c/em\u003e\u003csup\u003e6\u003c/sup\u003e, \u003cem\u003eN\u003c/em\u003e\u003csup\u003e6\u003c/sup\u003e-bis (2-hydroxy-dodecyl) lysine (180 mg, 0.35 mmol) was dissolved in dry dichloromethane (30 mL). \u003cem\u003eN\u003c/em\u003e, \u003cem\u003eN\u003c/em\u003e-dicyclohexyl carbodiimide (DCC, 124 mg, 0.6 mmol) and dimethylaminopyridine p-toluene sulfonate (DPTS, 177 mg, 0.6 mmol) were added and stirred\u0026nbsp;under\u0026nbsp;nitrogen at room temperature for 30 min.\u0026nbsp;Tetrahydrotetrandrine (34 mg, 0.1 mmol) in \u003cem\u003eN\u003c/em\u003e, \u003cem\u003eN\u003c/em\u003e-dimethylformamide (DMF, 2 mL)\u0026nbsp;was then added.\u0026nbsp;The reaction was stirred\u0026nbsp;under\u0026nbsp;nitrogen at room temperature for 24 h, monitored by thin layer chromatography.\u0026nbsp;The final product\u0026nbsp;was filtered,\u0026nbsp;concentrated under vacuum,\u0026nbsp;and purified by column chromatography. The structures were confirmed by \u003csup\u003e1\u003c/sup\u003eH nuclear magnetic resonance (NMR, Bruker, USA)\u0026nbsp;with the following shifts\u0026nbsp;(400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e+C\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e):\u0026nbsp;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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of BE-LNP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTetrahydroberberine-derivative lipid nanoparticles (BE-LNP) were\u0026nbsp;synthesized\u0026nbsp;using a microfluidic\u0026nbsp;approach. The\u0026nbsp;lipid\u0026nbsp;components,\u0026nbsp;comprising\u0026nbsp;ionizable lipids, helper lipids, cholesterol, and PEG-DMG\u003csub\u003e2000\u003c/sub\u003e, were dissolved in ethanol at\u0026nbsp;specific\u0026nbsp;ratios\u0026nbsp;detailed\u0026nbsp;in\u0026nbsp;Supplementary\u0026nbsp;Table S1. The ionizable lipid\u0026nbsp;component\u0026nbsp;included\u0026nbsp;various tetrahydroberberine derivatives selected from our molecular library, while\u0026nbsp;helper lipids\u0026nbsp;involved\u0026nbsp;DOPE, DOPC, and DSPC, aligned\u0026nbsp;with best practices in nanoparticle formulation. This\u0026nbsp;lipid mixture was subsequently combined with a 6.25 mM sodium acetate buffer (pH\u0026nbsp;5.0), containing siRNA, at a\u0026nbsp;water-to-ethanol\u0026nbsp;ratio of 3:1.\u0026nbsp;Homogeneous mixing was achieved using a\u0026nbsp;microfluidic mixer (AITESEN, China).\u0026nbsp;Following\u0026nbsp;mixing, the preparations\u0026nbsp;underwent overnight\u0026nbsp;dialysis\u0026nbsp;against PBS (pH 7.4) to remove\u0026nbsp;residual ethanol and unincorporated components.\u0026nbsp;Concentration of the nanoparticles was performed\u0026nbsp;using an ultracentrifugal filter\u0026nbsp;unit\u0026nbsp;(Millipore, Billerica, MA), optimizing\u0026nbsp;the formulation for further\u0026nbsp;\u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e testing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGel retardation assay\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;for siRNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the siRNA gel retardation assay, siRNA (1\u0026nbsp;OD) was dissolved in 125 μL of diethyl pyrocarbonate (DEPC)-treated water\u0026nbsp;and mixed\u0026nbsp;with varying weight ratios of siRNA to LNP. After 30\u0026nbsp;min\u0026nbsp;at room temperature, the complexes\u0026nbsp;containing 500 ng of siRNA were mixed with 6× loading buffer, and then\u0026nbsp;were electrophoresed\u0026nbsp;on\u0026nbsp;a 2%\u0026nbsp;agarose gel\u0026nbsp;containing\u0026nbsp;0.02% Goldview gel stain. Electrophoresis was conducted\u0026nbsp;at\u0026nbsp;180 V for 20 min\u0026nbsp;in\u0026nbsp;1× tris-acetate-EDTA (TAE)\u0026nbsp;buffer.\u0026nbsp;Gel analysis\u0026nbsp;was performed\u0026nbsp;using a gel image analysis system (Tanon, China).\u003c/p\u003e\n\u003cp\u003eFor mRNA studies,\u0026nbsp;mRNA (1 mg/mL) was\u0026nbsp;prepared in\u0026nbsp;DEPC-treated water\u0026nbsp;and\u0026nbsp;mixed with LNPs at a\u0026nbsp;fixed weight ratio (1:5).\u0026nbsp;Samples were\u0026nbsp;incubated at room temperature for varying durations\u0026nbsp;(0, 0.25, 0.5, 1, 2, 4 h).\u0026nbsp;Complexes containing 1.5 ug of mRNA were\u0026nbsp;analyzed using a similar gel electrophoresis procedure as described for siRNA, but\u0026nbsp;with 0.02% GelRed gel stain. After electrophoresis, gels\u0026nbsp;was analyzed\u0026nbsp;with the same\u0026nbsp;gel image analysis system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysiochemical characterization of BE-LNP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe physicochemical\u0026nbsp;properties\u0026nbsp;of BE-LNP were\u0026nbsp;thoroughly assessed. Particle size and zeta potential\u0026nbsp;were determined\u0026nbsp;using dynamic light scattering (DLS)\u0026nbsp;with a\u0026nbsp;Zetasizer\u0026nbsp;(Malvern, England).\u0026nbsp;The structural morphology of\u0026nbsp;the\u0026nbsp;LNPs was\u0026nbsp;examined\u0026nbsp;using\u0026nbsp;transmission electron microscopy (TEM) (HITACHI, Japan).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of the acid dissociation constant (pKa)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor pKa determination, BE-LNP was\u0026nbsp;first\u0026nbsp;prepared and diluted to a concentration of approximately 10 mM in phosphate-buffered saline (PBS).\u0026nbsp;A\u0026nbsp;\u0026nbsp;specialized\u0026nbsp;buffer solution was\u0026nbsp;then prepared,\u0026nbsp;containing\u0026nbsp;130 mM NaCl, 100 mM ammonium acetate, 10 mM HEPES, and 10 mM MES. This\u0026nbsp;solution was\u0026nbsp;pH-adjusted using 0.1\u0026nbsp;M NaOH or HCl to\u0026nbsp;span\u0026nbsp;the desired pH range.\u0026nbsp;In a black 96-well plate, 95 μL of\u0026nbsp;each\u0026nbsp;pH-adjusted\u0026nbsp;buffer, 5 μL of BE-LNP,\u0026nbsp;and 1μL of TNS solution (100 μM) were\u0026nbsp;combined.\u0026nbsp;Fluorescence spectra were\u0026nbsp;recorded\u0026nbsp;at room temperature using an excitation wavelength of 321 nm and an emission wavelength of 445 nm. The pKa was\u0026nbsp;accurately\u0026nbsp;determined by\u0026nbsp;identifying\u0026nbsp;the pH\u0026nbsp;corresponding\u0026nbsp;to half of the maximum fluorescence\u0026nbsp;intensity, a\u0026nbsp;recognized method for\u0026nbsp;pKa determination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePoly(A)\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003einding\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003cstrong\u003experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescence\u0026nbsp;measurements\u0026nbsp;were\u0026nbsp;conducted using a black microplate (96-well, Corning, USA) on a Multifunctional Microplate Reader\u0026nbsp;at an\u0026nbsp;excitation wavelength\u0026nbsp;of\u0026nbsp;350 nm.\u0026nbsp;Reactions were\u0026nbsp;carried out\u0026nbsp;in BPES buffer (1.5\u0026nbsp;mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 0.5\u0026nbsp;mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.25\u0026nbsp;mM EDTA, 6\u0026nbsp;mM NaCl) or citrate-phosphate (CP) buffer (5\u0026nbsp;mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e) with pH adjusted to 7.1 and 4.5 using citric acid, following established\u0026nbsp;protocols\u003csup\u003e49\u003c/sup\u003e.\u0026nbsp;A\u0026nbsp;blank buffer\u0026nbsp;and a control solution containing only\u0026nbsp;polyA\u0026nbsp;were\u0026nbsp;measured to\u0026nbsp;account for background signals. Fresh solutions of polyA and the alkaloid were prepared daily at a concentration of 10 μM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCellular uptake and endocytosis mechanism analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCultured bEnd.3 and DRD3\u003csup\u003e−\u003c/sup\u003e\u003csup\u003e/\u003c/sup\u003e\u003csup\u003e−\u003c/sup\u003e bEnd.3 cells were seeded in culture plates and incubated at 37°C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 12 h.\u0026nbsp;Cellular uptake\u0026nbsp;was assessed following the introduction of\u0026nbsp;LNP@DiD formulations to the cells for a 2\u0026nbsp;h\u0026nbsp;duration.\u0026nbsp;To investigate\u0026nbsp;endocytotic pathways, bEnd.3 cells\u0026nbsp;underwent treatment with\u0026nbsp;chlorpromazine (12.5 μM), filipin (12.5 μM), monensin (20 μM), brefeldin A (40 μM), colchicine (25 μM), and fresh DMEM for 30 min.\u0026nbsp;Post-treatment, LNP@DiD was\u0026nbsp;added\u0026nbsp;to the corresponding wells, and cells were incubated for an additional 2 h at 37°C.\u0026nbsp;Post-incubation,\u0026nbsp;cellular\u0026nbsp;imaging\u0026nbsp;was\u0026nbsp;performed\u0026nbsp;using a high-content analysis system (Operetta CLS, PerkinElmer, USA). Cells\u0026nbsp;were then subjected to\u0026nbsp;trypsinization\u0026nbsp;(0.25%), followed by rigorous PBS washes,\u0026nbsp;and resuspended in 300 μL of PBS\u0026nbsp;for flow cytometric analysis\u0026nbsp;using a flow cytometer (FACSverse, BD) with FlowJo 7.6 software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntracellular transfection and endosomal es\u003c/strong\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003cstrong\u003eape\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ebEnd.3 cells were seeded\u0026nbsp;and incubated for 12 h\u0026nbsp;at 37°C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u0026nbsp;For\u0026nbsp;transfection,\u0026nbsp;cells were exposed to various\u0026nbsp;LNP@FAM-siRNA formulations\u0026nbsp;in serum-free medium.\u0026nbsp;Following\u0026nbsp;the experiment, cells\u0026nbsp;were trypsinized\u0026nbsp;(0.25%), washed\u0026nbsp;thrice\u0026nbsp;with PBS,\u0026nbsp;and resuspended\u0026nbsp;in 300\u0026nbsp;μL\u0026nbsp;of PBS.\u0026nbsp;Transfection efficiency was quantified using flow cytometry\u0026nbsp;with FlowJo 7.6 software.\u003c/p\u003e\n\u003cp\u003eFor\u0026nbsp;detailed\u0026nbsp;intracellular\u0026nbsp;analysis, cells were stained with Lysotracker Red (KeyGEN BioTECH, China) at 37°C for 2 h, followed by\u0026nbsp;exposure to LNP@FAM-siRNA formulations\u0026nbsp;in serum-free medium for\u0026nbsp;4\u0026nbsp;h.\u0026nbsp;Post-incubation, cells\u0026nbsp;were washed thrice\u0026nbsp;with PBS\u0026nbsp;and\u0026nbsp;stained\u0026nbsp;with Hoechst 33342 (Beyotime, China) for 10 min at room temperature.\u0026nbsp;Images were captured using a high-content analysis system (Operetta CLS, PerkinElmer, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCrystal structures of the dopamine D3 receptor (PDB ID: 3PBL)\u003csup\u003e62\u003c/sup\u003e and\u0026nbsp;poly(A) (PDB ID: 3GIB)\u003csup\u003e63\u003c/sup\u003e were\u0026nbsp;retrieved\u0026nbsp;from the RCSB Protein Data Bank.\u0026nbsp;Using Chem3D 14.0, three-dimensional structures of the small molecule compounds were constructed\u0026nbsp;and minimized using\u0026nbsp;the MMFF94 force field. The protein structures\u0026nbsp;were\u0026nbsp;prepared\u0026nbsp;with\u0026nbsp;PyMol 2.5.4\u003csup\u003e64\u003c/sup\u003e by\u0026nbsp;removing hydrogen atoms, water molecules, and other non-ligand molecules.\u0026nbsp;A\u0026nbsp;bounding box,\u0026nbsp;or\u0026nbsp;\"butt box\" was\u0026nbsp;created\u0026nbsp;around the\u0026nbsp;active\u0026nbsp;protein pocket. Both small compounds and\u0026nbsp;receptor proteins\u0026nbsp;were converted to PDBQT format using\u0026nbsp;ADFRsuite 1.0\u003csup\u003e65\u003c/sup\u003e for\u0026nbsp;compatibility\u0026nbsp;with\u0026nbsp;AutoDock Vina.\u0026nbsp;Docking was conducted using AutoDock Vina 1.1.2\u003csup\u003e66\u003c/sup\u003e with\u0026nbsp;a conformational search detail of 32,\u0026nbsp;with\u0026nbsp;other parameters\u0026nbsp;at\u0026nbsp;default settings. The conformation exhibiting the highest affinity score\u0026nbsp;was chosen for further analysis and\u0026nbsp;visualized\u0026nbsp;using\u0026nbsp;PyMol version 2.5.4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and cultivation of primary brain microvascular endothelial cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe isolation\u0026nbsp;and cultivation\u0026nbsp;of\u0026nbsp;primary Brain Microvascular Endothelial Cells (BMECs)\u0026nbsp;were conducted using\u0026nbsp;a\u0026nbsp;detailed\u0026nbsp;protocol\u003csup\u003e67\u003c/sup\u003e outlined\u0026nbsp;in the following steps: (i) Brain tissue dissection: Fresh brain tissue from 4-6 week-old SD rats was\u0026nbsp;processed by\u0026nbsp;first removing\u0026nbsp;the meninges and choroid plexus through dissection.\u0026nbsp;The\u0026nbsp;cleaned\u0026nbsp;brain tissue was\u0026nbsp;then\u0026nbsp;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\u0026nbsp;37°C\u0026nbsp;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\u0026nbsp;with 5% CO\u003csub\u003e2\u003c/sub\u003e. (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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;blood-brain barrier penetration assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMECs 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\u0026nbsp;Ω·cm²\u0026nbsp;indicating suitable bilayer integrity.\u003c/p\u003e\n\u003cp\u003eFollowing 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\u0026nbsp;measuring\u0026nbsp;its fluorescence intensity using a Multimode Microplate Reader (BioTek Synergy H1, USA)\u0026nbsp;at\u0026nbsp;excitation wavelength of 485 nm and emission wavelength of 535 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePharmacokinetic Studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSD rats\u0026nbsp;were administered\u0026nbsp;BE and BE-ST\u0026nbsp;via\u0026nbsp;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,\u0026nbsp;24 and 48 h).\u0026nbsp;Each sample\u0026nbsp;(~200 μL)\u0026nbsp;was centrifuged at 4°C for 10 min\u0026nbsp;at\u0026nbsp;11000 × \u003cem\u003eg\u003c/em\u003e).\u0026nbsp;Samples were then\u0026nbsp;processed by mixing\u0026nbsp;50 µL\u0026nbsp;of\u0026nbsp;serum\u0026nbsp;with\u0026nbsp;150 µL\u0026nbsp;of\u0026nbsp;methanol, followed\u0026nbsp;by ultrasonication for 10 min\u0026nbsp;and a second centrifugation under the same conditions.\u0026nbsp;The\u0026nbsp;supernatant\u0026nbsp;(20 µL)\u0026nbsp;was analyzed using HPLC (SHIMADZU, Japan).\u0026nbsp;Pharmacokinetic parameters were calculated using PKsolver 2.0.10 software\u003csup\u003e68\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn Vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate\u0026nbsp;brain targeting, healthy mice were intravenously injected with LNP@DiR via the tail vein. Fluorescence images were captured at\u0026nbsp;predefined\u0026nbsp;time points (0.5, 1, 2, 4, and 8 h)\u0026nbsp;using the VISQUE\u0026nbsp;\u003cem\u003eI\u003c/em\u003e\u003cem\u003en\u0026nbsp;\u003c/em\u003e\u003cem\u003eV\u003c/em\u003e\u003cem\u003eivo\u003c/em\u003e Smart-LF System (Vieworks, Korea). After imaging\u0026nbsp;sessions, mice were euthanized at predetermined intervals and their major organs were\u0026nbsp;harvested\u0026nbsp;for \u003cem\u003eex vivo\u003c/em\u003e imaging analysis.\u003c/p\u003e\n\u003cp\u003eFor the establishment of a brain tumor model, 5\u0026nbsp;μL\u0026nbsp;of\u0026nbsp;a\u0026nbsp;GL261-Luc\u0026nbsp;cell suspension\u0026nbsp;(10,000 cells/μL) was carefully injected into the right\u0026nbsp;hemisphere of the\u0026nbsp;brain over a 3\u0026nbsp;min period.\u0026nbsp;Mice were anesthetized and secured\u0026nbsp;in a stereotactic apparatus to ensure precise administration.\u0026nbsp;The needle was slowly\u0026nbsp;withdrawn post-injection.\u0026nbsp;\u003cem\u003eI\u003c/em\u003e\u003cem\u003en vivo\u003c/em\u003e imaging\u0026nbsp;of the\u0026nbsp;pathological model\u0026nbsp;was performed\u0026nbsp;after a 10-day post-operation period,\u0026nbsp;with\u0026nbsp;mice receiving\u0026nbsp;daily intravenous injections of LNP@siVEGF (1 mg/kg)\u0026nbsp;over five\u0026nbsp;consecutive days.\u0026nbsp;Imaging was conducted using the Lumina III Imaging System (PerkinElmer, USA)\u0026nbsp;on days\u0026nbsp;0, 3, and 6 post-injection. For precision targeting \u003cem\u003eex vivo\u003c/em\u003e imaging\u0026nbsp;of the\u0026nbsp;pathological model, mice were intravenously injected with LNP@DiR. Their brains were\u0026nbsp;harvested\u0026nbsp;for \u003cem\u003eex vivo\u003c/em\u003e imaging at\u0026nbsp;8 h post-injection\u0026nbsp;using the VISQUE\u0026nbsp;\u003cem\u003eI\u003c/em\u003e\u003cem\u003en\u0026nbsp;\u003c/em\u003e\u003cem\u003eV\u003c/em\u003e\u003cem\u003eivo\u003c/em\u003e Smart-LF System.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn\u0026nbsp;the construction of a meningitis model, a fungal suspension\u0026nbsp;containing\u0026nbsp;2500 CFU of a luciferase-expressing strain of \u003cem\u003eC. neoformans\u003c/em\u003e was\u0026nbsp;injected into the\u0026nbsp;mouse\u0026nbsp;brain\u0026nbsp;using the same method as the tumor model.\u0026nbsp;After a 48 h post-operation period, mice\u0026nbsp;received\u0026nbsp;daily intravenous injections of LNP@AmB, consisting of either\u0026nbsp;1 mg/kg of amphotericin B or 25 mg/kg of flucytosine,\u0026nbsp;for a total of five days.\u0026nbsp;Imaging was\u0026nbsp;subsequently\u0026nbsp;performed using the Lumina III Imaging System at 0, 3, and 6 days post-surgery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorris water maze\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo\u0026nbsp;assess\u0026nbsp;spatial learning and memory, the Morris water maze (MWM) paradigm was\u0026nbsp;employed\u0026nbsp;following established methodologies\u003csup\u003e69\u003c/sup\u003e. The\u0026nbsp;maze consisted of a\u0026nbsp;water pool divided into four quadrants, each\u0026nbsp;marked\u0026nbsp;by a unique symbol (pentagram, square, triangle, and circle)\u0026nbsp;on\u0026nbsp;the corresponding quadrant wall\u0026nbsp;to serve as\u0026nbsp;spatial cues. The water maintained at 22 ± 1°C, and food-grade titanium dioxide was\u0026nbsp;used\u0026nbsp;to\u0026nbsp;obscure the water and facilitate\u0026nbsp;tracking of mouse movements.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll trials were conducted in the afternoon in a controlled environment\u0026nbsp;devoid of\u0026nbsp;extraneous noise\u0026nbsp;and\u0026nbsp;intense light.\u0026nbsp;Mice were\u0026nbsp;acclimatized\u0026nbsp;to the test room for 2 h\u0026nbsp;prior to the start of the experiments. The training phase\u0026nbsp;lasted for\u0026nbsp;five consecutive days, with each mouse undergoing four trials per day and a\u0026nbsp;20-30 min\u0026nbsp;inter-trial interval. Mice were placed facing the\u0026nbsp;pool\u0026nbsp;wall\u0026nbsp;and tasked with locating a\u0026nbsp;hidden\u0026nbsp;platform. The time\u0026nbsp;to locate the\u0026nbsp;platform was recorded.\u0026nbsp;If a mouse failed to find the platform within\u0026nbsp;60 s,\u0026nbsp;it was\u0026nbsp;guided to the platform and allowed to\u0026nbsp;remains there for 10 s.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing a 24\u0026nbsp;h interval post-training, the platform was removed\u0026nbsp;for\u0026nbsp;a 60\u0026nbsp;s probe test.\u0026nbsp;Mice\u0026nbsp;were\u0026nbsp;placed\u0026nbsp;in the water facing the quadrant opposite to the target quadrant.\u0026nbsp;Performance\u0026nbsp;metrics, such as time spent in the target quadrant and the number of crossings at the\u0026nbsp;former\u0026nbsp;platform location, were recorded using EthoVision XT8.5 tracking software, indicating spatial memory retention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the assessment of targeted molecular mechanisms, bEnd.3 and DRD3\u003csup\u003e−/−\u003c/sup\u003e bEnd.3 cells were collected. Following behavioral assessments, murine brain tissues were obtained for therapeutic efficacy\u0026nbsp;evaluation.\u0026nbsp;Mice\u0026nbsp;were ethically and humanely euthanized, and transcardial perfusion with saline was performed\u0026nbsp;prior to tissue\u0026nbsp;extraction, including the entire hippocampus and cortex. These\u0026nbsp;samples\u0026nbsp;were\u0026nbsp;homogenized\u0026nbsp;in lysis buffer\u0026nbsp;containing\u0026nbsp;1% phosphatase inhibitors and 1% PMSF (Beyotime, China). After homogenization, the mixture was centrifugated at\u0026nbsp;12,000 rpm\u0026nbsp;for 15 min\u0026nbsp;at\u0026nbsp;4°C. The protein concentration\u0026nbsp;in\u0026nbsp;the supernatant was determined using\u0026nbsp;a\u0026nbsp;BCA Protein Assay Kit (Beyotime, China). Approximately 20 μg of protein was\u0026nbsp;subjected\u0026nbsp;to\u0026nbsp;SDS-PAGE on\u0026nbsp;a 10% gel\u0026nbsp;and\u0026nbsp;transferred\u0026nbsp;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.\u0026nbsp;Overnight incubation at 4°C with primary antibodies, including BACE1, p-tau, p-GSK3β, DRD3, or GAPDH,\u0026nbsp;was followed by incubation with\u0026nbsp;HRP-conjugated IgG rabbit secondary antibodies for 1 h at 37°C.\u0026nbsp;Blots were visualized using ECL (Beyotime, China), with GAPDH\u0026nbsp;serving\u0026nbsp;as a\u0026nbsp;loading control.\u0026nbsp;Quantification was performed using ImageJ, and results were recorded\u0026nbsp;with\u0026nbsp;a gel image analysis system (Tanon, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-Linked Immunosorbent Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum 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\u0026nbsp;at 2000 rpm for 10 min at 4°C\u0026nbsp;to\u0026nbsp;isolate\u0026nbsp;plasma. To\u0026nbsp;assess\u0026nbsp;potential immune response and toxicity of LNP formulations, ELISA kits measures\u0026nbsp;complement activation-related pseudoallergy (CARPA) indicators\u0026nbsp;(complement\u0026nbsp;C5b-9, C3a, MCP-1).\u0026nbsp;Additionally, levels of key\u0026nbsp;inflammatory cytokines\u0026nbsp;(IL-1β, IL-6, IFNγ, and TNFα)\u0026nbsp;were\u0026nbsp;evaluated\u0026nbsp;using specific ELISA kits to understand\u0026nbsp;the synergistic impact of LNP@siBACE1 on AD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood Biochemical Profiling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the potential hepatotoxic and nephrotoxic effects of LNP formulations, serum biochemical examinations were performed\u0026nbsp;every other day for\u0026nbsp;two weeks. Blood was\u0026nbsp;obtained from the retro-orbital plexus and centrifuged\u0026nbsp;at 2000 rpm for 10 min at 4°C. The plasma was analyzed for biochemical parameters including\u0026nbsp;alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acid (UA), urea (UREA), and creatinine (CREA),\u0026nbsp;providing a comprehensive\u0026nbsp;evaluation of hepatic and renal function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing\u0026nbsp;the treatment regimen, humane euthanasia was performed on APP/PS-1 mice, followed by transcardial perfusion. The brains were\u0026nbsp;fixed\u0026nbsp;in 4% paraformaldehyde for 24 h,\u0026nbsp;then underwent\u0026nbsp;dehydration, embedding in OCT, and freezing.\u0026nbsp;Frozen sections of 20 μm\u0026nbsp;thickness\u0026nbsp;were obtained using a high-precision freezing microtome (Leica, Germany). For immunofluorescence, sections\u0026nbsp;were treated with\u0026nbsp;4% paraformaldehyde\u0026nbsp;for 30 min, rinsed\u0026nbsp;with PBS,\u0026nbsp;permeabilized with\u0026nbsp;0.1% Triton X-100\u0026nbsp;for 15 min,\u0026nbsp;and\u0026nbsp;blocked\u0026nbsp;with 5% BSA for 2 h.\u0026nbsp;Primary anti-β-amyloid antibody\u0026nbsp;incubation was performed overnight\u0026nbsp;at 4°C.\u0026nbsp;After\u0026nbsp;three PBS washes, sections were incubated with FITC-conjugated goat anti-rabbit IgG for 2 h at room temperature. Nuclei were\u0026nbsp;stained with\u0026nbsp;Hoechst 33342 for 10 min.\u0026nbsp;Confocal microscopy (OLYMPUS FV3000, Japan)\u0026nbsp;acquired high-resolution fluorescence images. Nissl staining identified\u0026nbsp;potential\u0026nbsp;neuronal damage. Furthermore,\u0026nbsp;vital organs like\u0026nbsp;hearts, lungs, livers, spleens, and kidneys,\u0026nbsp;were\u0026nbsp;fixed\u0026nbsp;in a 4% paraformaldehyde,\u0026nbsp;sectioned, and\u0026nbsp;stained with\u0026nbsp;hematoxylin and eosin (HE). Images were captured using an inverted fluorescence microscope (DMi8, Leica, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of reactive oxygen species levels in single cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess oxidative stress and intracellular levels of ROS, a\u0026nbsp;ROS\u0026nbsp;assay kit\u0026nbsp;(Beyotime, China)\u0026nbsp;was utilized.\u0026nbsp;bEnd.3 cells from each treatment group\u0026nbsp;were incubated\u0026nbsp;at 37°C for 30 min in\u0026nbsp;serum-free\u0026nbsp;medium containing 10 μM DCHF- diacetate\u0026nbsp;(DA).\u0026nbsp;Post-incubation, the medium was\u0026nbsp;replaced with fresh medium\u0026nbsp;containing\u0026nbsp;ionizable molecules at\u0026nbsp;a concentration\u0026nbsp;of 5 mg/mL,\u0026nbsp;and\u0026nbsp;the cells were further\u0026nbsp;incubated for 2, 12,\u0026nbsp;or 24 h.\u0026nbsp;Following this,\u0026nbsp;cells were washed three times in PBS containing 0.1% BSA\u0026nbsp;and\u0026nbsp;mounted on glass slides with PBS for microscopy\u0026nbsp;analysis.\u0026nbsp;The fluorescence intensity\u0026nbsp;of each\u0026nbsp;cell,\u0026nbsp;indicating ROS levels,\u0026nbsp;was\u0026nbsp;quantified\u0026nbsp;using a real-time single-cell multimode analyzer equipped with optical fiber probes (Rayme, China).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue burden assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were\u0026nbsp;anesthetized and securely positioned using a stereotactic apparatus\u0026nbsp;before inoculating\u0026nbsp;2500 CFU of \u003cem\u003eC. neoformans\u003c/em\u003e into the right cerebral hemisphere over a meticulous 3\u0026nbsp;min period. This\u0026nbsp;procedure\u0026nbsp;was\u0026nbsp;carefully\u0026nbsp;conducted with precision and\u0026nbsp;a\u0026nbsp;gradual withdrawal\u0026nbsp;of the injection needle.\u0026nbsp;After\u0026nbsp;a 48\u0026nbsp;h interval, mice were subjected to intravenous administration of LNP@AmB. The\u0026nbsp;treatment\u0026nbsp;regimen\u0026nbsp;included daily doses\u0026nbsp;of\u0026nbsp;either\u0026nbsp;1 mg/kg of amphotericin B or 25 mg/kg of flucytosine, administered over a span of\u0026nbsp;five\u0026nbsp;consecutive days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the sixth day\u0026nbsp;post-inoculation, all mice\u0026nbsp;were\u0026nbsp;humanely\u0026nbsp;euthanized, and\u0026nbsp;their\u0026nbsp;brain were harvested. The\u0026nbsp;brain\u0026nbsp;tissues were weighed and homogenized\u0026nbsp;in\u0026nbsp;sterile saline at a ratio of 1 g of tissue to 3 mL of saline. The homogenates\u0026nbsp;were\u0026nbsp;subjected to sequential dilutions\u0026nbsp;in\u0026nbsp;sterile saline.\u0026nbsp;A\u0026nbsp;30 μL\u0026nbsp;aliquot of each dilution\u0026nbsp;was carefully plated onto YPD\u0026nbsp;agar\u0026nbsp;and incubated at 30°C for 48 h to determine\u0026nbsp;the\u0026nbsp;colony-forming units (CFU) per gram of brain tissue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurvival rates study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter\u0026nbsp;a 48 h incubation period after inoculating the mice with\u0026nbsp;\u003cem\u003eC. neoformans\u003c/em\u003e to\u0026nbsp;establish\u0026nbsp;a\u0026nbsp;brain infection\u0026nbsp;model, the mice were randomly divided into five groups. Each group received\u0026nbsp;daily\u0026nbsp;intravenous\u0026nbsp;treatments for five days. The treatments\u0026nbsp;consisted\u0026nbsp;of either saline or different\u0026nbsp;formations of\u0026nbsp;LNP@AmB, with doses set\u0026nbsp;at\u0026nbsp;1 mg/kg of amphotericin B or 25 mg/kg of flucytosine. The survival of the mice was\u0026nbsp;monitored\u0026nbsp;for a duration\u0026nbsp;of 45 days\u0026nbsp;following the conclusion of\u0026nbsp;the treatment\u0026nbsp;regimen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eeGFP-mRNA transfection \u003cem\u003ein vitro\u003c/em\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCultured cells were seeded in a 12-well culture dish and allowed to reach 60 to 70%\u0026nbsp;confluence.\u0026nbsp;Transfection was conducted using eGFP mRNA complexed with distinct LNP\u0026nbsp;formulations\u0026nbsp;in\u0026nbsp;serum-free DMEM.\u0026nbsp;The cells were incubated\u0026nbsp;at 37°C for 4 h\u0026nbsp;to facilitate mRNA uptake.\u0026nbsp;Following the initial transfection period,\u0026nbsp;FBS was reintroduced\u0026nbsp;to the medium, and\u0026nbsp;the\u0026nbsp;cells were incubated\u0026nbsp;for an additional 20 h.\u0026nbsp;The expression of eGFP was then\u0026nbsp;assessed using a fluorescence microscope (DMi8, Leica, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eeGFP-mRNA transfection \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the intravenous administration of LNP@eGFP mRNA at a dosage\u0026nbsp;of 1 mg/kg, a 24\u0026nbsp;h\u0026nbsp;monitoring\u0026nbsp;period\u0026nbsp;ensured. After this period, all mice were humanely euthanized, and\u0026nbsp;their\u0026nbsp;brain tissues were\u0026nbsp;harvested and fixed\u0026nbsp;in a 4% paraformaldehyde solution for 24 h.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe preparation of\u0026nbsp;brain tissues for imaging involved several carefully controlled steps\u003csup\u003e70\u003c/sup\u003e.\u0026nbsp;Initially, tissues underwent decolorization by immersion in a solution containing 25% v/v\u0026nbsp;Quadrol\u0026nbsp;and 5% v/v ammonium in water at 37°C\u0026nbsp;for two days. This was followed by a gradient\u0026nbsp;delipidation\u0026nbsp;process using\u0026nbsp;tert-butanol (tB)\u0026nbsp;solutions\u0026nbsp;at concentrations of 30%, 50%, and 70% v/v, with\u0026nbsp;pH adjustments\u0026nbsp;above 9.5\u0026nbsp;using\u0026nbsp;3% w/v Quadrol. Subsequently, the tissues were\u0026nbsp;dehydrated in a\u0026nbsp;solution comprised\u0026nbsp;of 70% v/v tB, 27% v/v PEG methacrylate M\u003csub\u003en\u003c/sub\u003e500 (PEGMMA500), and 3% w/v Quadrol\u0026nbsp;for two days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe final clearing step involved submerging the samples in\u0026nbsp;BB-PEG Clearing Medium, which consists of\u0026nbsp;75% v/v benzyl benzoate (BB),\u0026nbsp;25% v/v PEGMMA500,\u0026nbsp;and\u0026nbsp;3% w/v Quadrol, achieving a refractive index of 1.543. This medium was used for 507 days until the tissues reached\u0026nbsp;optical transparency.\u0026nbsp;The cleared samples were then preserved at room temperature in the\u0026nbsp;same\u0026nbsp;clearing medium.\u003c/p\u003e\n\u003cp\u003eFor imaging,\u0026nbsp;cleared brain\u0026nbsp;samples were analyzed using\u0026nbsp;a light sheet microscope (LiToneXL, Light Innovation Technology, China),\u0026nbsp;equipped with a 43×\u0026nbsp;objective lens (NA = 0.28, working distance = 20 mm).\u0026nbsp;The imaging process utilized thin light sheets\u0026nbsp;to\u0026nbsp;illuminate the samples from all four sides,\u0026nbsp;capturing\u0026nbsp;and merging\u0026nbsp;images\u0026nbsp;to visualize the expression and distribution of eGFP effectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative data\u0026nbsp;from the experiments\u0026nbsp;are presented as means ± SD.\u0026nbsp;Statistical significance was determined using two-tailed unpaired \u003cem\u003et\u003c/em\u003e-tests and multiple \u003cem\u003et\u003c/em\u003e-tests\u0026nbsp;with\u0026nbsp;GraphPad Prism software\u0026nbsp;(version\u0026nbsp;8). The threshold for statistical significance was set at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;with 95% confidence intervals.\u0026nbsp;Notably, in instances\u0026nbsp;where \u003cem\u003ep\u003c/em\u003e-values fell below 0.0001, the software was unable to provide an exact value, indicating extremely significant differences.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (Grant No.\u0026nbsp;2023YFF0724200),\u0026nbsp;the\u0026nbsp;National Natural Science Foundation of China (NSFC\u0026nbsp;Grant\u0026nbsp;Nos. 82373808, 82073789),\u0026nbsp;and the\u0026nbsp;Chongqing Science Fund for Distinguished Young Scholars (Grant No.\u0026nbsp;CSTB2023NSCQ-JQX0021)\u0026nbsp;awarded\u0026nbsp;to C.L.\u0026nbsp;We extend our gratitude\u0026nbsp;to Dr. Huan Zhao and Xiaogang Wang\u0026nbsp;from\u0026nbsp;Revvity for their\u0026nbsp;invaluable\u0026nbsp;advice on experimental design and\u0026nbsp;for engaging in productive discussions that enhanced this research.\u0026nbsp;Additionally, we are thankful for the support provided by\u0026nbsp;the Academy of Agricultural Sciences\u0026nbsp;at\u0026nbsp;Southwest University,\u0026nbsp;which included\u0026nbsp;access to\u0026nbsp;essential\u0026nbsp;instrumentation and technical\u0026nbsp;expertise. We also appreciate the equipment\u0026nbsp;support\u0026nbsp;received from\u0026nbsp;Rayme Biotechnology and Revvity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChong Li, Zhi-Hong Jiang, Xufei Bian, Qian Guo, Xiaoyou Wang and Xurong Qin conceived the project, designed all the experiments, analyzed the data, and wrote the manuscript. Ling Yang, Shikang Zhao, and Shiqiong Wu conducted the experiments and analyzed the data. All authors edited the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChung, J.E. et al. Self-assembled micellar nanocomplexes comprising green tea catechin derivatives and protein drugs for cancer therapy. \u003cem\u003eNature nanotechnology\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 907-912 (2014).\u003c/li\u003e\n\u003cli\u003eShin, M. et al. Targeting protein and peptide therapeutics to the heart via tannic acid modification. \u003cem\u003eNature biomedical engineering\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 304-317 (2018).\u003c/li\u003e\n\u003cli\u003eWang, X. et al. Identification and construction of a novel biomimetic delivery system of paclitaxel and its targeting therapy for cancer. \u003cem\u003eSignal transduction and targeted therapy\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 33 (2021).\u003c/li\u003e\n\u003cli\u003eFu, S. \u0026amp; Yang, X. Recent advances in natural small molecules as drug delivery systems. \u003cem\u003eJ Mater Chem B\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 4584-4599 (2023).\u003c/li\u003e\n\u003cli\u003eHuigens, R.W., 3rd et al. Control of bacterial biofilms with marine alkaloid derivatives. \u003cem\u003eMol Biosyst\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 614-621 (2008).\u003c/li\u003e\n\u003cli\u003eYan, Y. et al. Research Progress on Antibacterial Activities and Mechanisms of Natural Alkaloids: A Review. \u003cem\u003eAntibiotics (Basel)\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e (2021).\u003c/li\u003e\n\u003cli\u003eLebecque, S. et al. Interaction between the barley allelochemical compounds gramine and hordenine and artificial lipid bilayers mimicking the plant plasma membrane. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 9784 (2018).\u003c/li\u003e\n\u003cli\u003eEfimova, S.S., Zakharova, A.A. \u0026amp; Ostroumova, O.S. Alkaloids Modulate the Functioning of Ion Channels Produced by Antimicrobial Agents via an Influence on the Lipid Host. \u003cem\u003eFront Cell Dev Biol\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 537 (2020).\u003c/li\u003e\n\u003cli\u003ePatalas-Krawczyk, P. et al. Effects of plant alkaloids on mitochondrial bioenergetic parameters. \u003cem\u003eFood Chem Toxicol\u003c/em\u003e \u003cstrong\u003e154\u003c/strong\u003e, 112316 (2021).\u003c/li\u003e\n\u003cli\u003eShitan, N. \u0026amp; Yazaki, K. Accumulation and membrane transport of plant alkaloids. \u003cem\u003eCurr Pharm Biotechnol\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 244-252 (2007).\u003c/li\u003e\n\u003cli\u003eSingh, S., Pathak, N., Fatima, E. \u0026amp; Negi, A.S. Plant isoquinoline alkaloids: Advances in the chemistry and biology of berberine. \u003cem\u003eEuropean journal of medicinal chemistry\u003c/em\u003e \u003cstrong\u003e226\u003c/strong\u003e, 113839 (2021).\u003c/li\u003e\n\u003cli\u003eKim, A.N., Ngamnithiporn, A., Du, E. \u0026amp; Stoltz, B.M. Recent Advances in the Total Synthesis of the Tetrahydroisoquinoline Alkaloids (2002-2020). \u003cem\u003eChem Rev\u003c/em\u003e \u003cstrong\u003e123\u003c/strong\u003e, 9447-9496 (2023).\u003c/li\u003e\n\u003cli\u003eFeng, J.H. et al. The composition, pharmacological effects, related mechanisms and drug delivery of alkaloids from Corydalis yanhusuo. \u003cem\u003eBiomed Pharmacother\u003c/em\u003e \u003cstrong\u003e167\u003c/strong\u003e, 115511 (2023).\u003c/li\u003e\n\u003cli\u003eShang, X.F. et al. Biologically active isoquinoline alkaloids covering 2014-2018. \u003cem\u003eMed Res Rev\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 2212-2289 (2020).\u003c/li\u003e\n\u003cli\u003eKrey, A.K. \u0026amp; Hahn, F.E. Berberine: complex with DNA. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e166\u003c/strong\u003e, 755-757 (1969).\u003c/li\u003e\n\u003cli\u003eGiri, P. \u0026amp; Kumar, G.S. Molecular aspects of small molecules-poly(A) interaction: an approach to RNA based drug design. \u003cem\u003eCurr Med Chem\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 965-987 (2009).\u003c/li\u003e\n\u003cli\u003eChatterjee, S. \u0026amp; Suresh Kumar, G. Small molecule induced poly(A) single strand to self-structure conformational switching: evidence for the prominent role of H-bonding interactions. \u003cem\u003eMol Biosyst\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1000-1009 (2017).\u003c/li\u003e\n\u003cli\u003eKawano, M., Takagi, R., Kaneko, A. \u0026amp; Matsushita, S. Berberine is a dopamine D1- and D2-like receptor antagonist and ameliorates experimentally induced colitis by suppressing innate and adaptive immune responses. \u003cem\u003eJ Neuroimmunol\u003c/em\u003e \u003cstrong\u003e289\u003c/strong\u003e, 43-55 (2015).\u003c/li\u003e\n\u003cli\u003eNiwa, M., Mibu, H., Nozaki, M., Tsurumi, K. \u0026amp; Fujimura, H. Dopaminergic unique affinity of tetrahydroberberine and l-tetrahydroberberine-d-camphor sulfonate. \u003cem\u003ePharmacology\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 329-336 (1991).\u003c/li\u003e\n\u003cli\u003eWang, J.B. \u0026amp; Mantsch, J.R. l-tetrahydropalamatine: a potential new medication for the treatment of cocaine addiction. \u003cem\u003eFuture Med Chem\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 177-186 (2012).\u003c/li\u003e\n\u003cli\u003eXu, P. et al. Structures of the human dopamine D3 receptor-G(i) complexes. \u003cem\u003eMol Cell\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 1147-1159.e1144 (2021).\u003c/li\u003e\n\u003cli\u003eCrist, A.M. et al. Transcriptomic analysis to identify genes associated with selective hippocampal vulnerability in Alzheimer\u0026apos;s disease. \u003cem\u003eNature communications\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 2311 (2021).\u003c/li\u003e\n\u003cli\u003eRao, Y.L. et al. Hippocampus and its involvement in Alzheimer\u0026apos;s disease: a review. \u003cem\u003e3 Biotech\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 55 (2022).\u003c/li\u003e\n\u003cli\u003eZhu, H. et al. The clinical characteristics and molecular mechanism of pituitary adenoma associated with meningioma. \u003cem\u003eJ Transl Med\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 354 (2019).\u003c/li\u003e\n\u003cli\u003ePekic, S., Miljic, D. \u0026amp; Popovic, V. in Endotext. (eds. K.R. Feingold et al.) (MDText.com, Inc. Copyright \u0026copy; 2000-2023, MDText.com, Inc., South Dartmouth (MA); 2000).\u003c/li\u003e\n\u003cli\u003eYildirim, M., Cengil, E., Eroglu, Y. \u0026amp; Cinar, A. Detection and classification of glioma, meningioma, pituitary tumor, and normal in brain magnetic resonance imaging using deep learning-based hybrid model. \u003cem\u003eIran Journal of Computer Science\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 455-464 (2023).\u003c/li\u003e\n\u003cli\u003eTorabi, S.F. et al. RNA stabilization by a poly(A) tail 3\u0026apos;-end binding pocket and other modes of poly(A)-RNA interaction. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e371\u003c/strong\u003e (2021).\u003c/li\u003e\n\u003cli\u003eLi, J. et al. 3\u0026apos;-Poly(A) tail enhances siRNA activity against exogenous reporter genes in MCF-7 cells. \u003cem\u003eJ RNAi Gene Silencing\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 195-204 (2006).\u003c/li\u003e\n\u003cli\u003eMitton-Fry, R.M., DeGregorio, S.J., Wang, J., Steitz, T.A. \u0026amp; Steitz, J.A. Poly(A) tail recognition by a viral RNA element through assembly of a triple helix. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e330\u003c/strong\u003e, 1244-1247 (2010).\u003c/li\u003e\n\u003cli\u003evan Rooy, I., Mastrobattista, E., Storm, G., Hennink, W.E. \u0026amp; Schiffelers, R.M. Comparison of five different targeting ligands to enhance accumulation of liposomes into the brain. \u003cem\u003eJournal of controlled release : official journal of the Controlled Release Society\u003c/em\u003e \u003cstrong\u003e150\u003c/strong\u003e, 30-36 (2011).\u003c/li\u003e\n\u003cli\u003eOller-Salvia, B. et al. MiniAp-4: A Venom-Inspired Peptidomimetic for Brain Delivery. \u003cem\u003eAngewandte Chemie (International ed. in English)\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 572-575 (2016).\u003c/li\u003e\n\u003cli\u003eHuile, G. et al. A cascade targeting strategy for brain neuroglial cells employing nanoparticles modified with angiopep-2 peptide and EGFP-EGF1 protein. \u003cem\u003eBiomaterials\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 8669-8675 (2011).\u003c/li\u003e\n\u003cli\u003eDos Santos Rodrigues, B., Arora, S., Kanekiyo, T. \u0026amp; Singh, J. Efficient neuronal targeting and transfection using RVG and transferrin-conjugated liposomes. \u003cem\u003eBrain Res\u003c/em\u003e \u003cstrong\u003e1734\u003c/strong\u003e, 146738 (2020).\u003c/li\u003e\n\u003cli\u003eShen, J. et al. Poly(ethylene glycol)-block-poly(D,L-lactide acid) micelles anchored with angiopep-2 for brain-targeting delivery. \u003cem\u003eJ Drug Target\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 197-203 (2011).\u003c/li\u003e\n\u003cli\u003eWang, L. \u0026amp; Ma, Q. Clinical benefits and pharmacology of scutellarin: A comprehensive review. \u003cem\u003ePharmacol Ther\u003c/em\u003e \u003cstrong\u003e190\u003c/strong\u003e, 105-127 (2018).\u003c/li\u003e\n\u003cli\u003eBakun, P. et al. Tea-break with epigallocatechin gallate derivatives - Powerful polyphenols of great potential for medicine. \u003cem\u003eEuropean journal of medicinal chemistry\u003c/em\u003e \u003cstrong\u003e261\u003c/strong\u003e, 115820 (2023).\u003c/li\u003e\n\u003cli\u003eHabteyes, T.G. et al. Hierarchical Self-Assembly of Carbon Dots into High-Aspect-Ratio Nanowires. \u003cem\u003eNano Lett\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 9474-9481 (2023).\u003c/li\u003e\n\u003cli\u003eGaillard, P.J. et al. Pharmacokinetics, brain delivery, and efficacy in brain tumor-bearing mice of glutathione pegylated liposomal doxorubicin (2B3-101). \u003cem\u003ePloS one\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, e82331 (2014).\u003c/li\u003e\n\u003cli\u003eOhno, M. et al. BACE1 gene deletion prevents neuron loss and memory deficits in 5XFAD APP/PS1 transgenic mice. \u003cem\u003eNeurobiology of disease\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 134-145 (2007).\u003c/li\u003e\n\u003cli\u003eGao, D. \u0026amp; Liu, Y. Berberine regulates endocrine function in mice with polycystic ovary syndrome through PI3K/Akt/GSK-3\u0026beta; insulin signaling pathway. \u003cem\u003eTropical Journal of Pharmaceutical Research\u003c/em\u003e (2022).\u003c/li\u003e\n\u003cli\u003eBajad, N.G. et al. Combined structure and ligand-based design of dual BACE-1/GSK-3\u0026beta; inhibitors for Alzheimer\u0026rsquo;s disease. \u003cem\u003eChemical Papers\u003c/em\u003e \u003cstrong\u003e76\u003c/strong\u003e, 7507-7524 (2022).\u003c/li\u003e\n\u003cli\u003eAu, L. et al. Cytokine release syndrome in a patient with colorectal cancer after vaccination with BNT162b2. \u003cem\u003eNature medicine\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 1362-1366 (2021).\u003c/li\u003e\n\u003cli\u003eYuan, Z.Y. et al. TATA boxes in gene transcription and poly (A) tails in mRNA stability: New perspective on the effects of berberine. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 18326 (2015).\u003c/li\u003e\n\u003cli\u003eYuan, Z. et al. Berberine inhibits mRNA degradation by promoting the interaction between the poly A tail and its binding protein PABP. \u003cem\u003eJournal of Chinese Pharmaceutical Sciences\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 53-62 (2017).\u003c/li\u003e\n\u003cli\u003eHou, X., Zaks, T., Langer, R. \u0026amp; Dong, Y. Lipid nanoparticles for mRNA delivery. \u003cem\u003eNature reviews. Materials\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 1078-1094 (2021).\u003c/li\u003e\n\u003cli\u003eDebnath, D., Kumar, G.S. \u0026amp; Maiti, M. Circular dichroism studies of the structure of DNA complex with berberine. \u003cem\u003eJ Biomol Struct Dyn\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 61-79 (1991).\u003c/li\u003e\n\u003cli\u003eSaran, A., Srivastava, S., Coutinho, E. \u0026amp; Maiti, M. 1H NMR investigation of the interaction of berberine and sanguinarine with DNA. \u003cem\u003eIndian J Biochem Biophys\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 74-77 (1995).\u003c/li\u003e\n\u003cli\u003eNandi, R., Debnath, D. \u0026amp; Maiti, M. Interactions of berberine with poly(A) and tRNA. \u003cem\u003eBiochim Biophys Acta\u003c/em\u003e \u003cstrong\u003e1049\u003c/strong\u003e, 339-342 (1990).\u003c/li\u003e\n\u003cli\u003eYadav, R.C. et al. Berberine, a strong polyriboadenylic acid binding plant alkaloid: spectroscopic, viscometric, and thermodynamic study. \u003cem\u003eBioorg Med Chem\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 165-174 (2005).\u003c/li\u003e\n\u003cli\u003eHan, X. et al. An ionizable lipid toolbox for RNA delivery. \u003cem\u003eNature communications\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 7233 (2021).\u003c/li\u003e\n\u003cli\u003eHan, X. et al. Ligand-tethered lipid nanoparticles for targeted RNA delivery to treat liver fibrosis. \u003cem\u003eNature communications\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 75 (2023).\u003c/li\u003e\n\u003cli\u003ePattipeiluhu, R. et al. Anionic Lipid Nanoparticles Preferentially Deliver mRNA to the Hepatic Reticuloendothelial System. \u003cem\u003eAdv Mater\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, e2201095 (2022).\u003c/li\u003e\n\u003cli\u003eKim, M. et al. Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver. \u003cem\u003eScience advances\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e (2021).\u003c/li\u003e\n\u003cli\u003eB\u0026ouml;ttger, R. et al. Lipid-based nanoparticle technologies for liver targeting. \u003cem\u003eAdv Drug Deliv Rev\u003c/em\u003e \u003cstrong\u003e154-155\u003c/strong\u003e, 79-101 (2020).\u003c/li\u003e\n\u003cli\u003eSinegra, A.J., Evangelopoulos, M., Park, J., Huang, Z. \u0026amp; Mirkin, C.A. Lipid Nanoparticle Spherical Nucleic Acids for Intracellular DNA and RNA Delivery. \u003cem\u003eNano Lett\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 6584-6591 (2021).\u003c/li\u003e\n\u003cli\u003eLokugamage, M.P. et al. Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs. \u003cem\u003eNature biomedical engineering\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 1059-1068 (2021).\u003c/li\u003e\n\u003cli\u003eRadmand, A. et al. The Transcriptional Response to Lung-Targeting Lipid Nanoparticles in Vivo. \u003cem\u003eNano Lett\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 993-1002 (2023).\u003c/li\u003e\n\u003cli\u003eOkuyama, T. et al. Iduronate-2-Sulfatase with Anti-human Transferrin Receptor Antibody for Neuropathic Mucopolysaccharidosis II: A Phase 1/2 Trial. \u003cem\u003eMolecular therapy : the journal of the American Society of Gene Therapy\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 456-464 (2019).\u003c/li\u003e\n\u003cli\u003eBeola, L., Iturrioz-Rodr\u0026iacute;guez, N., Pucci, C., Bertorelli, R. \u0026amp; Ciofani, G. Drug-Loaded Lipid Magnetic Nanoparticles for Combined Local Hyperthermia and Chemotherapy against Glioblastoma Multiforme. \u003cem\u003eACS nano\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 18441-18455 (2023).\u003c/li\u003e\n\u003cli\u003eVilla-Cedillo, S.A. et al. CDNF overexpression prevents motor-cognitive dysfunction by intrastriatal CPP-based delivery system in a Parkinson\u0026apos;s disease animal model. \u003cem\u003eNeuropeptides\u003c/em\u003e \u003cstrong\u003e102\u003c/strong\u003e, 102385 (2023).\u003c/li\u003e\n\u003cli\u003eDavidson, R.C. et al. Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans. \u003cem\u003eFungal Genet Biol\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 38-48 (2000).\u003c/li\u003e\n\u003cli\u003eChien, E.Y. et al. Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e330\u003c/strong\u003e, 1091-1095 (2010).\u003c/li\u003e\n\u003cli\u003eLink, T.M., Valentin-Hansen, P. \u0026amp; Brennan, R.G. Structure of Escherichia coli Hfq bound to polyriboadenylate RNA. \u003cem\u003eProceedings of the National Academy of Sciences of the United States of America\u003c/em\u003e \u003cstrong\u003e106\u003c/strong\u003e, 19292-19297 (2009).\u003c/li\u003e\n\u003cli\u003eDelano, W.L. PyMOL: An Open-Source Molecular Graphics Tool. (2002).\u003c/li\u003e\n\u003cli\u003eRavindranath, P.A., Forli, S., Goodsell, D.S., Olson, A.J. \u0026amp; Sanner, M.F. AutoDockFR: Advances in Protein-Ligand Docking with Explicitly Specified Binding Site Flexibility. \u003cem\u003ePLoS Comput Biol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e1004586 (2015).\u003c/li\u003e\n\u003cli\u003eTrott, O. \u0026amp; Olson, A.J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. \u003cem\u003eJ Comput Chem\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 455-461 (2010).\u003c/li\u003e\n\u003cli\u003eAssmann, J.C. et al. Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice. \u003cem\u003eBio-protocol\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e (2017).\u003c/li\u003e\n\u003cli\u003eZhang, Y., Huo, M., Zhou, J. \u0026amp; Xie, S. PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. \u003cem\u003eComput Methods Programs Biomed\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 306-314 (2010).\u003c/li\u003e\n\u003cli\u003ePuzzo, D., Lee, L., Palmeri, A., Calabrese, G. \u0026amp; Arancio, O. Behavioral assays with mouse models of Alzheimer\u0026apos;s disease: practical considerations and guidelines. \u003cem\u003eBiochemical pharmacology\u003c/em\u003e \u003cstrong\u003e88\u003c/strong\u003e, 450-467 (2014).\u003c/li\u003e\n\u003cli\u003eJing, D. et al. Tissue clearing of both hard and soft tissue organs with the PEGASOS method. \u003cem\u003eCell research\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 803-818 (2018).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4626003/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4626003/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDespite advancements in targeting organs such as the liver, spleen, and lungs with lipid nanoparticles (LNPs), the challenge of traversing the blood-brain barrier (BBB) significantly impedes the progress of gene therapies for neurological disorders. Motivated by the structural and functional characteristics of alkaloids, we developed a novel library of ionizable lipid molecules based on the tetrahydroisoquinoline structure characteristic of the protoberberine family. Our findings reveal that: (i) LNPs incorporating berberine-derived ionizable lipids notably enhance the ability to cross the BBB, increasing \u003cem\u003ein vitro\u003c/em\u003e endocytosis efficiency by up to 65-fold and achieving an \u003cem\u003ein vivo\u003c/em\u003e brain-to-liver distribution ratio of approaching 20%; (ii) these lipids form stable self-assemblies with polyA, enhancing nucleic acid stability through mechanisms beyond conventional electrostatic interactions, thus providing effective RNA protection without the need for additional modifications; (iii) the lipids inherit the diverse brain-protective properties of protoberberine-type alkaloids, including anti-inflammatory and antioxidant effects, thereby synergistically enhancing the therapeutic management of brain diseases while exhibiting minimal immunogenicity.\u003c/p\u003e","manuscriptTitle":"Berberine-inspired ionizable lipid for self-structure stabilization and potent brain targeting delivery of nucleic acid therapeutics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-10 06:10:53","doi":"10.21203/rs.3.rs-4626003/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4e972774-cd6b-4663-a575-512fe351b113","owner":[],"postedDate":"July 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34334665,"name":"Biological sciences/Drug discovery/Drug delivery"},{"id":34334666,"name":"Biological sciences/Neuroscience/Blood\u0026#x2013;brain barrier"}],"tags":[],"updatedAt":"2025-03-11T07:07:05+00:00","versionOfRecord":{"articleIdentity":"rs-4626003","link":"https://doi.org/10.1038/s41467-025-57488-0","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-03-10 04:00:00","publishedOnDateReadable":"March 10th, 2025"},"versionCreatedAt":"2024-07-10 06:10:53","video":"","vorDoi":"10.1038/s41467-025-57488-0","vorDoiUrl":"https://doi.org/10.1038/s41467-025-57488-0","workflowStages":[]},"version":"v1","identity":"rs-4626003","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4626003","identity":"rs-4626003","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}