Fig. 4: In vitro uptake of NVs by M2-like macrophages via MGL-mediated endocytosis.
a–c 5 × 105 M2-like bone marrow-derived macrophages (M2-BMDMs) were seeded in 24-well plates and treated with 6 × 108 CFSE-sgPik3cg-DGA-NVs for 3 h, followed by flow cytometry analysis and fluorescent microscopy. In some cases, macrophages were pre-incubated with GlcNAc or GalNAc (100 mmol/L) for 1 h before NVs treatment. Red, F4/80; green, CFSE labeled NVs; blue, DAPI nuclear staining. Scaled bar = 50 μm. n = 3 biologically independent samples. d A total of 1 × 105 macrophages with above mentioned treatments was harvested, lysed and the fluorescence intensity was quantified using a microplate reader (Em: 488 nm; Ex: 530 nm). n = 3 biologically independent samples. e–h 5 × 105 M2-BMDMs were treated with 6 × 108 CFSE-sgPik3cg-DGA-NVs or CFSE-sgPik3cg-DHP/DGA-NVs (pre-treated with pH 6.5 or 7.4 PBS for 24 h) for 3 h and then subjected to flow cytometry analysis, microphotography and fluorescence intensity quantification. Red, F4/80; green, CFSE labeled NVs; blue, DAPI nuclear staining. Scaled bar = 50 μm. n = 3 biologically independent samples. i M2-BMDMs subjected to the aforementioned NVs treatment were fixed, stained with fluorescent-labeled F4/80 and Cas9 antibodies and imaged at indicated time points. Red, F4/80; green, Cas9; blue, DAPI nuclear staining. Scaled bar = 10 μm. j, k The levels of Cas9 and sgRNA targeting Pik3cg in 5 × 105 M2-BMDMs treated with 6 × 108 sgPik3cg-DHP/DGA-NVs (pH 6.5 PBS pre-treatment) for 3 h after which the medium was replaced with fresh medium for another 9 h. The cells were examined by western blotting and agarose gel electrophoresis. l The NVs-mediated delivery of CpG-rich genomic DNA fragments into 5 × 105 M2-BMDMs incubated with 6 × 108 NVs for 3 h was detected by PCR amplification and agarose gel electrophoresis. The experiments for (i–l) were independently repeated three times with similar results. Data are presented as the means ± SD. Statistical analyses were performed using one-way ANOVA with Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01 and ***P < 0.001, ns, no significant change. The exact P-value and source data are provided as a Source Data file.
