(M1430-07-37) Intracellular Trafficking of Cationic Bicelles and siRNA Cargo in an In Vitro BBB Model

Purpose: Drug delivery to the endothelium is critical for addressing blood-brain barrier (BBB) dysfunction, which is believed to be a significant contributor to the progression of Alzheimer’s disease (AD). It is widely acknowledged that proinflammatory cytokines can induce BBB dysfunction. Studies have shown that TNF-α promotes the expression of GATA-6, which in turn stimulates the expression of vascular adhesion molecule-1 (VCAM-1). Circulating leukocytes adhere to VCAM-1 and disrupt the tight junctions, resulting in a leaky BBB. Reinstating BBB integrity is hypothesized to slow, and even reverse, neurodegeneration.⁴ GATA-6 has no known pharmacological inhibitors, but can be modulated through RNA-based therapeutics such as GATA-6 siRNA. Nucleic acids have been successfully delivered in vivo through lipid nanoparticles (LNPs) to various cellular targets such as the liver and muscle,⁵ but not the BBB endothelium. To optimize LNPs for endothelial delivery, we modulated their shape and surface characteristics. Discoidal particles exhibit enhanced vascular margination, increased adhesion to cell surface receptors, and faster endocytosis than spherical particles.^6–9 Indeed, the cationic bicelles formulated in our lab—DPPC/DC₇PC/DOTAP at a molar ratio of 63.8/25.0/11.2¹⁰ and nitrogen-to-phosphate (N/P) ratio of 4:1¹¹—demonstrated greater transfection efficiency of FITC-labeled siRNA than commercially available Lipofectamine RNAiMAX in an in vitro blood-brain barrier (BBB) model.¹¹ Our current study is focused on the cellular uptake and trafficking of the siRNA-bicelle complex in an in vitro BBB model. We aim to elucidate the mechanisms of cellular uptake, track the intracellular movement of the bicelles and siRNA with time, and model the kinetics of their uptake and clearance. Elucidating these pathways will allow us to evaluate the cellular pharmacokinetics and make informed decisions about modifying the lipid composition, such as incorporating PEGylated lipids or targeting ligands, to optimize cellular delivery and transfection.

Methods: Chloroform solutions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DC7PC), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were added at a molar ratio of 63.8/25.0/11.2. The solvent was removed through rotary evaporation, and the resultant thin lipid film was hydrated with an aqueous solution of siRNA at an N/P ratio of 4:1. Particle size was controlled through sonication in a water bath at room temperature.Polarized human cerebral microvascular endothelial cells (hCMEC/D3) monolayers were used as an in vitro BBB model. The mechanism of uptake was determined through pharmacological inhibition and siRNA knockdown approaches. Cells were pretreated with 80 μM Dynasore, 10 μM cytochalasin A (CytoA), or at 4 °C for 30 min, followed by DiI-laced bicelles for 1 h. The DiI fluorescence associated with bicelles was then measured through flow cytometry. Clathrin/caveolin-1 was also knocked down in cells using siRNA, and the cells were treated with DiI-bicelles for 1 h. Uptake was observed through fluorescent microscopy. Nuclei were stained with DAPI and clathrin/caveolin-1 was labeled with Alexa Fluor 647. Intracellular trafficking following endocytosis was observed through fluorescent microscopy. Bicelles were laced with DiI/DiD and siRNA was labeled with FITC. Accumulation in lysosomes was determined by labeling acidic organelles with Lysotracker Green or Deep Red. Accumulation in early endosomes, late endosomes, and recycling endosomes was determined by transfecting cells with m-CFP/Rab5, GFP/Rab7, and GFP/Rab11 respectively. Colocalization was determined based on the Pearson correlation coefficient (PCC). Image analysis was done using ImageJ, and parameters for compartmental models were estimated using SAAM II.

Results: Pretreatment with 80 μM Dynasore, 10 μM CytoA, or at 4 °C reduced the cellular uptake of bicelles by 50%, 40%, and 90%, respectively, compared to the positive control. For clathrin knockdown cells, protein expression was reduced by 69% and bicelle uptake was reduced by 62%. For caveolin-1 knockdown cells, protein expression was reduced by 58% and bicelle uptake was reduced by 40%. PCC values greater than 0.6 are indicative of significant colocalization. The PCC values between bicelles and siRNA were 0.854, 0.782, and 0.557 at 1h, 2h, and 3h respectively. The PCC values between lysosomes and bicelles/siRNA generally did not exceed 0.2 at those same timepoints. Bicelles also exhibited poor colocalization with Rab11, but strong colocalization with Rab5 and Rab7. Meanwhile, siRNA exhibited poor colocalization with Rab5. The mean fluorescence of bicelles was normalized to the mean fluorescence of Rab5 or Rab7, and a three-compartment model was built based on these data points. DiD fluorescence intensity peaked at 4 h and dropped off over the course of 12 h.

Conclusion: DPPC/DC₇PC/DOTAP bicelles undergo multiple routes of endocytosis, including clathrin-mediated, caveolin-mediated, and macropinocytosis. This is consistent with other findings in the literature.^12–14 Following endocytosis, the cationic bicelles are hypothesized to facilitate the endosomal escape of their cargo by fusing with the endosomal membrane.¹⁵ Our findings support this hypothesis. Bicelles and siRNA appeared to be trafficked separately over time, with bicelles being primarily located in early/late endosomes and siRNA being primarily located in the cytosol.

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(Left) Micrographs of polarized hCMEC/D3 monolayers after clathrin or caveolin-1 knockdown and treatment with cationic bicelles. Nuclei were stained with DAPI (blue), bicelles were laced with DiI (yellow), and clathrin or caveolin-1 were tagged with Alexa Fluor 647 (red). The scale bar is 20 μm. (Right) Protein expression and bicelle uptake in clathrin or caveolin-1 knockdown hCMEC/D3 cells expressed as a percentage relative to the wild-type. Values were quantified based on mean fluorescence and normalized to mean DAPI fluorescence. Mean ± SD (n = 6).

Micrographs of polarized hCMEC/D3 monolayers after treatment with siRNA-bicelle complexes at an N/P ratio of 4:1. Nuclei were stained with DAPI (blue), bicelles were laced with DiI or DiD (red), siRNA was labeled with FITC (green), and lysosomes were tagged with Lysotracker Green/Deep Red (green/red). The scale bar is 20 μm. Pearson correlation coefficients (PCC) were calculated between bicelles and siRNA, bicelles and Lysotracker, or siRNA and Lysotracker. Mean ± SD (n ≥ 6).

Pearson correlation coefficients (PCC) between bicelles and early endosomes (Rab5), bicelles and late endosomes (Rab7), bicelles and recycling endosomes (Rab11), and siRNA and early endosomes (Rab5) at different time points (n = 12). Values above the red dotted line indicate significant colocalization.