Regulation of cerebral blood flow boosts precise brain targeting of vinpocetine-derived ionizable-lipidoid nanoparticles

Abstract

Despite advances in active drug targeting for blood-brain barrier penetration, two key challenges persist: first, attachment of a targeting ligand to the drug or drug carrier does not enhance its brain biodistribution; and second, many brain diseases are intricately linked to microcirculation disorders that significantly impede drug accumulation within brain lesions even after they cross the barrier. Inspired by the neuroprotective properties of vinpocetine, which regulates cerebral blood flow, we propose a molecular library design centered on this class of cyclic tertiary amine compounds and develop a self-enhanced brain-targeted nucleic acid delivery system. Our findings reveal that: (i) vinpocetine-derived ionizable-lipidoid nanoparticles efficiently breach the blood-brain barrier; (ii) they have high gene-loading capacity, facilitating endosomal escape and intracellular transport; (iii) their administration is safe with minimal immunogenicity even with prolonged use; and (iv) they have potent pharmacologic brain-protective activity and may synergize with treatments for brain disorders as demonstrated in male APP/PS1 mice.

Fig. 1. Screening and component optimization of the ionizable-lipidoid molecule based on the skeleton of vinca alkaloids (vinpocetine).

a, b Chemical structures of lipid building blocks, including 5 amine heads and 15 alkylated tails. c Optimization scheme for VIP; 27 formulations were prepared and evaluated in this study based on 75 ionizable compounds. (Created with BioRender.com). d Schematic diagram of laser speckle flowmetry for detecting cerebral microcirculation blood perfusion in mice. (Created with BioRender.com). e Quantitative curve of blood flow changes of 9 candidate compounds and vinpocetine (Vin) within 60 min after administration. Data are presented as means ± SD, were obtained by measuring the blood perfusion unit every 10 min. f Laser speckle flowgraphy images of 3 candidate compounds and vinpocetine (Vin). g Molecular interaction between the lipidoid molecule (A5-B1-C4.2) and PDE1 protein investigated through surface plasmon resonance (SPR). h Three-dimensional ligand-protein interaction mode for the binding site of PDE1 (Uniport ID: Q14123) with the leading compound A5-B1-C4.2. Data in (e, f) are representative of two independent experiments with similar results. Source data are provided as a Source Data file.

Fig. 2. Biophysical characterization and in vitro studies of VIP@siRNA.

a Physicochemical properties of VIP before and after siRNA encapsulation, including size, polydispersity index (PDI), zeta potential, and encapsulation efficiency (E.E.). b Transmission electron microscopy image of VIP@siRNA; scale bar = 50 nm. c Gel retardation assay of VIP@siRNA at siRNA/LNP weight ratios of 0.2, 1, 2, 5, 10, 15, and 20. d Detection of cellular transfection by flow cytometry and comparison of mean fluorescence intensity of VIP@siRNA and MC3@siRNA. Data are presented as means ± SD (n = 3 biologically independent samples). e Subcellular localization of VIP@siRNA or MC3@siRNA; scale bar = 5 µm. f Exploration of mechanisms underlying VIP-mediated siRNA transfection. Data are presented as means ± SD (n = 3 biologically independent samples). g Schematic diagram of a real-time single-cell multimodal analyzer combined with a fluorescent probe to detect reactive oxygen species levels in a single-cell. (Created with BioRender.com). h Reactive oxygen species production in bEnd.3 cells after administration of high doses (0.25 mg/mL) for 2 h. Data are presented as means ± SD (n = 6 biologically independent samples). i Reactive oxygen species production in bEnd.3 cells after administration of low doses (0.05 mg/mL) for 1 h, 4 h, 12 h, 24 h.Data are presented as means ± SD (n = 10 biologically independent samples). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS means no significance. Statistical significance was calculated with two-tailed unpaired t-tests. Data are representative of three (b, c) and two (e) independent experiments with similar results. Source data are provided as a Source Data file.

Fig. 3. Validation of brain targeting and related mechanism.

a Flow cytometry detection of cellular uptake of LNP. Data are presented as means ± SD (n = 3 biologically independent samples). b, c Flow cytometry detection of downregulated expression of PDE1 with siRNA and corresponding cellular uptake. Data are presented as means ± SD (n = 3 biologically independent samples). d, e An in vitro blood-brain barrier (BBB) model was established with a Transwell assay to verify the permeability of LNP through the barrier; scale bar = 1000 µm. f Plasma DiD concentration in SD rats after intravenous administration of NP@DiD, MC3-siNC@DiD, or VIP-siNC@DiD. Data are presented as means ± SD (n = 3 biologically independent samples). g, h In vivo imaging of mice at 0.5 h, 1 h, 2 h, 4 h, and 8 h after administration of LNP. i Blood flow enhancement effect of VIP on brain microvessels detected by using laser speckle flowgraphy in three classical models of brain disease. Data are presented as means ± SD, were obtained by measuring the blood perfusion unit every 10 min. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS means no significance. Statistical significance was calculated with a, b two-tailed unpaired t-tests and c multiple t-tests. Data in (d, i) are representative of two independent experiments with similar results. Source data are provided as a Source Data file.

Fig. 4. Behavioral evaluation of VIP@siBACE1 therapy in APP/PS1 mice.

a Schematic of the experimental timeline; APP/PS1 and wild-type (WT) mice were treated with LNP@siBACE1 or PBS via tail vein injection every 2 d (7 cycles). Mice were then subjected to the novel object recognition (NOR) and Morris water maze (MWM) tests to evaluate memory, and samples were collected for molecular pathological assessments. (Created with BioRender.com). b Setup for the NOR test. c, d Results from the NOR test. c Discrimination index and d preference index of each group after LNP@siBACE1 treatment. e–i Data in the MWM. e The 5-day learning curve for the MWM experiment. f Representative swimming track, g swimming speed, h ratio of time spent in the target quadrant, and i numbers of crossing the platform location of each group on the probe test day. Data are presented as means ± SD (n = 6 biologically independent samples). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical significance was calculated with c, e, h, i two-tailed unpaired t-tests and d multiple t-tests. Source data are provided as a Source Data file.

Fig. 5. Therapeutic synergy of VIP and siBACE1 to modulate AD hallmarks in APP/PS1 mice.

a–c Representative western blot data for BACE1, p-tau, and p-GSK3β protein expression in hippocampus and cortex from VIP@siBACE1-treated APP/PS1 mice, control APP/PS1 groups, and WT mice. Quantification of western blotting analysis of BACE1, p-tau, and p-GSK3β protein expression is shown relative to GAPDH. Data are presented as means ± SD (n = 3 biologically independent experiments). d Confocal laser scanning microscopy images of amyloid plaque burden; immunofluorescence of Aβ plaques (green) in hippocampus and cortex from APP/PS1 transgenic and WT mice. Nuclei were stained with DAPI (blue). Scale bar in top row = 500 µm, scale bar in bottom row = 250 µm. e ELISA evaluation of amyloid plaques in serum. Data are presented as means ± SD (n = 3 biologically independent samples). f BACE1 relative mRNA expression level in cortex was quantified by qPCR. Data are presented as means ± SD (n = 3 biologically independent samples). g, h Oxidative stress markers (such as SOD and MDA) and proinflammatory cytokines (such as IL-1β, IL-6, IFN-γ, and TNF-α) in serum were assessed. Data are presented as means ± SD (n = 3 biologically independent samples). i Nissl staining of representative brain sections at 14 d after treatment with different formulations; scale bar = 40 µm. j ELISA evaluation of complement activation-related pseudoallergy (CARPA) markers such as C5b9, C3a, and monocyte chemoattractant protein-1 (MCP-1) in serum. Data are presented as means ± SD (n = 3 biologically independent samples). All samples were collected after seven injections of LNP. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS means no significance. Statistical significance was calculated with two-tailed unpaired t-tests. Data in (d, i) are representative of two independent experiments with similar results. Source data are provided as a Source Data file.

Fig. 6. In vivo evaluation of the efficacy of VIP-NP for the delivery of small-molecule and macro-molecule drugs.

a Schematic of the experimental timeline for treating a mouse model of cryptococcal meningitis. AMB, amphotericin B. (Created with BioRender.com). b, c Qualitative and quantitative analysis of live imaging of C. neoformans-Luc infected mice. Data are presented as means ± SD (n = 5 biologically independent samples). d Blood flow enhancement effect on brain microvessels detected by using laser speckle flowmetry in brain C. neoformans infected model on the 3, 5, 7 day after infection treated with PBS or VIP@AMB as above. Data are presented as means ± SD, were obtained by measuring the blood perfusion unit every 10 min. e Fungal colony burdens in the brain tissue of C. neoformans infected mice in each group after treatment. Data are presented as means ± SD (n = 6 biologically independent samples). f Survival curves of each treatment group. (n = 10 biologically independent animals). **P < 0.01, ***P < 0.001, ****P < 0.0001, NS means no significance. Statistical significance was calculated with e two-tailed unpaired t-tests and c multiple t-tests. g, i VIP effectively delivers eGFP mRNA in vitro and in vivo. g Fluorescence microscope images of eGFP mRNA transfected in vitro, scale bar = 50 µm. h Fluorescence imaging of brain tissue sections after administration of 10 μg eGFP mRNA, scale bar = 100 µm. i 3D hyalinization microscope images of brain tissue processed by PEGASOS tissue-clearing protocols after administration of 10 μg eGFP mRNA, scale bar = 1500 µm. Data are representative of three (g, h) and two (d, i) independent experiments with similar results. Source data are provided as a Source Data file.