Apoe4

Cerebral cortices were harvested from newborn (P0–P2) C57BL/6 mice as previously described^{15 (link)}. Neurons were cultured with 150 nM human recombinant ApoE3 (PeproTech 350-02, NJ, USA) or ApoE4 (PeproTech 350-04) combined with 100 μM PA for 24 h. In some experiments, neurons were preincubated with 10 μM capsaicin for 30 min followed by the addition of ApoE3 or ApoE4 and PA. Human neuroblastoma-derived SH-sy5y cells were exposed to 150 nM human recombinant ApoE3 or ApoE4 combined with 100 μM PA for 24 h. Microglia-conditioned medium (MCM) was collected from the culture medium of human recombinant ApoE3- and ApoE4-treated BV2 cells. BV2 cells were preincubated with 10 μM capsaicin for 30 min, and then ApoE3 or ApoE4 was added. DIV8 neurons were incubated with 95% neuronal medium and 5% MCM at 37 °C with 5% CO₂ for 24 h.

LNPs and Apos were incubated in ddH₂O water overnight at 4 °C. LNPs equivalent of 5 µg encapsulated plasmid DNA were incubated with 3 µg of each of the different human Apos; ApoA1 (Sigma, SRP4693), ApoB (Sigma, 178456), ApoC3 (Sigma, A3106), ApoD (Sigma, SRP4326), ApoE2 (Peprotech, 350-12), ApoE4 (Peprotech, 350-04) and ApoH (Sigma, G9173), unless otherwise stated, in a final volume of 200 µl. For control experiments (i.e. LNP only), LNPs were incubated with ddH₂O water (in the absence of any apolipoprotein) overnight at 4 °C in a final volume of 200 µl. For Cryo TEM reactions were set up as described above in the absence of ddH₂O.

BLI binding experiments were performed using Octet RED96 equipment (ForteBio). The recombinant RBD (SinoBiological, 40592-V05H) and recombinant human ACE2 proteins (SinoBiological, 10108-H02H) were captured with an anti-mIgG Fc Capture (AMC) probe in PBST solution and then incubated with recombinant human ApoE2 (PeproTech, 350-12-500UG), ApoE3 (PeproTech, 350-02-500UG) and ApoE4 (PeproTech, 350-04-500UG) proteins. Then, the fully reacted solid-phase conjugates were dissociated in PBST buffer for analysis. The kinetic values were fitted to a 1:1 Langmuir binding model. The results were analysed with ForteBio Data Analysis 11.0 software to determine the association rate, dissociation rate and affinity constant.