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

Abstract

Lipid nanoparticles have shown success in targeting major organs such as the liver, spleen, and lungs, but crossing the blood-brain barrier (BBB) remains a major challenge. Effective brain-targeted delivery systems are essential for advancing gene therapy for neurological diseases but remain limited by low transport efficiency and poor nucleic acid stability. Here, we report a library of ionizable lipids based on the tetrahydroisoquinoline structure of protoberberine alkaloids, designed to improve BBB penetration via dopamine D3 receptor-mediated endocytosis. These nanoparticles offer three key advantages: enhanced brain uptake, improved nucleic acid stability through poly(A) self-assembly, and minimal immunogenicity with inherent neuroprotective properties. In murine models, they demonstrate therapeutic potential in Alzheimer's disease, glioma, and cryptococcal meningitis. This berberine-inspired delivery system integrates precise receptor targeting with nucleic acid stabilization, offering a promising platform for brain-targeted therapeutics.

Fig. 1. Design and mechanism of berberine-inspired lipidoid nanoparticles (BE) for targeted brain delivery.

a Construction of a three-dimensional combinatorial synthesis library and formation of BE@RNA complexes through berberine derivative-poly(A) affinity and self-assembly properties. b Blood-brain barrier penetration mechanism through dopamine D3 receptor (D3R)-mediated transport, enabling targeted therapeutic delivery for neurodegenerative disorders, including Alzheimer’s disease.

Fig. 2. Design and characterization of tetrahydroisoquinoline alkaloid-based ionizable lipids.

a Chemical structures of lipid building blocks comprising five amine heads and 15 alkylated tails. b Optimization workflow for BE lipidoid molecule development, involving 27 formulations derived from 75 novel ionizable compounds. (Created with BioRender.com) c Affinities of various ionizable lipidoid molecules for poly(A) measured using fluorescence spectroscopy. d, e Cellular uptake of LNPs assessed by fluorescence co-localization studies and flow cytometry analysis. LNPs were labeled DiD (red). Nuclei were stained with Hoechst 33342 (blue). Scale bar, 20 µm. f In vivo imaging of mice after injection of LNP@DiR at 0.5, 1, 2, 4, and 8 h. g Ex vivo fluorescence distribution showing brain-to-liver ratio at 1 h. Data are presented as mean ± SD (n = 3 independent experiments). Statistical significance was determined by two-tailed unpaired Student’s t test (two groups) or one-way ANOVA with Dunnett’s multiple comparison tests (multiple groups); **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 3. In vitro characterization of BE-based LNPs containing ionizable lipid A2-B13.

a Gel retardation assay of BE@siRNA at LNP/siRNA weight ratios from 0.2 to 20 to evaluate complex stability. b Size distribution analysis by dynamic light scattering. c Transmission electron microscopy (TEM) image showing particle morphology of BE@siRNA. Scale bar, 100 nm. d Cellular uptake comparison between BE@FAM-siRNA and MC3@FAM-siRNA by fluorescence microscopy. Scale bar, 25 µm. e High-magnification imaging of subcellular localization of BE@siRNA and MC3@siRNA. FAM channel shows BE@FAM-siRNAs (green). LysoTracker channel shows lysosome (red). Nuclei were counter stained with Hoechst 33342 (blue). Scale bar, 5 µm. f Analysis of BE-mediated endocytosis mechanisms. g, h LNP@DiD penetration across an in vitro blood-brain barrier (BBB) model using transwell assays. LNPs were labeled DiD (red). Nuclei were counter stained with Hoechst 33342 (blue). Scale bar, 1000 µm. Data are presented as mean ± SD (n = 3 independent experiments). Statistical significance was determined by two-tailed unpaired Student’s t test (two groups) or one-way ANOVA with Dunnett’s multiple comparison tests (multiple groups); ***P < 0.001; NS not significant. Data are representative of three (c, d) and two (e) independent experiments with similar results. Source data are provided as a Source Data file.

Fig. 4. Brain-targeting evaluation of BE-ST nanoparticles.

a Cellular uptake analysis by flow cytometry following DRD3 knockdown with siRNA. b Zeta potential measurements of BE complexed with different organic acids. c Brain-to-liver fluorescence distribution ratio of BE formulated with various organic acids by ex vivo imaging. d In vivo and ex vivo imaging of LNP@DiR distribution 1 h post-injection of BE-organic acid complexes. e Plasma concentration-time profiles of BE and BE-ST (1:5 w/w) in SD rats following intravenous administration. f Time-course in vivo imaging of LNP@DiR distribution at 0.5, 1, 2, 4, and 8 h post-injection. g Ex vivo brain imaging at 1, 2, 4, and 8 h post-administration. h Quantitative analysis of brain fluorescence intensity over time. Data are presented as mean ± SD (n = 3 independent experiments). Statistical significance was determined by two-tailed unpaired Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS not significant. Source data are provided as a Source Data file.

Fig. 5. BE(-ST)@siBACE1 therapy improves cognitive function in APP/PS1 mice.

a Experimental timeline showing treatment regimen of APP/PS1 and wild-type (WT) mice receiving tail vein injections of LNP@siBACE1 or PBS every 2 days for seven cycles, followed by Morris water maze (MWM) testing and tissue collection. b–e MWM probe test results: b Representative swimming trajectories during platform search. c Time spent in target quadrant as percentage of total time. d Platform location crossing frequency. e Mean swimming speed across groups. Data are presented as mean ± SD (n = 6 biologically independent animals). Statistical significance was determined by two-tailed unpaired Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 6. Therapeutic synergy of BE(-ST) and siBACE1 in modulating Alzheimer’s disease (AD) hallmarks in APP/PS1 mice.

a Representative western blot images showing BACE1, phosphorylated tau (p-tau), and GSK3β levels in the hippocampus and cortex of BE(-ST)@siBACE1-treated APP/PS1 mice, control APP/PS1 groups, and wild-type (WT) mice. b Quantification of western blot for BACE1, p-tau, and phosphorylated GSK3β (p-GSK3β), normalized to GAPDH. The samples derive from the same experiment and that blots were processed in parallel. c Representative confocal laser scanning microscopy images showing amyloid-β (Aβ) plaque burden in the hippocampus and cortex of APP/PS1 and WT mice. Aβ plaques (green) and nuclei (Hoechst 33342, blue). Scale bars: top row, 500 µm; bottom row, 250 µm. d Nissl staining of brain sections 14 days post-treatment. Scale bar, 50 µm. ELISA results for complement activation-related pseudoallergy (CARPA) markers: e C5b9, f C3a, and g monocyte chemoattractant protein (MCP-1) in serum. Data are means ± SD (n = 3 biologically independent samples). Statistical significance was determined by two-tailed unpaired Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data in c, d are representative of two independent experiments with similar results. Source data are provided as a Source Data file.

Fig. 7. BE-ST enables efficient delivery of small-molecule and macromolecular drugs in vivo.

a Experimental timeline for cryptococcal meningitis treatment in mice using amphotericin B (AMB). b, c In vivo fluorescence imaging and quantification. Data are presented as mean ± SD (n = 5 biologically independent animals). d Brain tissue fungal colony burden quantification. Data are presented as mean ± SD (n = 6 biologically independent animals). e Kaplan-Meier survival analysis (n = 10 biologically independent animals). f Fluorescence microscopy of eGFP mRNA in vitro transfection. Scale bar, 25 µm. g Three-dimensional visualization of polyA-A2-B13 binding site interactions (PDB ID: 3GIB). h Agarose gel electrophoresis showing BE-loaded mRNA stability. i Three-dimensional hyalinization microscopy of brain tissue following 10 µg eGFP mRNA administration. Scale bar, 1500 µm. Statistical significance was determined by two-tailed unpaired Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are representative of three (f, h) and two (i) independent experiments with similar results. Source data are provided as a Source Data file.