Fig. 2.

Fabricating CMNDDs by covalent interactions in vitro. (A) Flow cytometry analysis of cell surface thiols on mouse splenocytes detected by fluorophore-conjugated malemide co-staining with lineage-specific surface markers for erythrocytes (Ter-119), T cells (CD3), B cells (B220) and hematopoietic stem cells (c-Kit). (B) Schematic of maleimide-based conjugation to cell surface thiols. MPB-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide]. (C) Confocal microscopy images of CD8⁺ effector T cells and lineage − Sca-1⁺c-Kit⁺ HSCs immediately after conjugation with fluorescent DiD-labeled multilamellar lipid nanoparticles. (D) Flow cytometry analysis of CD8⁺ T cells after incubation with DiD-labeled multilamellar lipid nanoparticles synthesized with or without maleimide-headgroup lipids. (E) Quantification of nanoparticle internalization. Immature dendritic cells (DCs), effector CD8⁺ T cells or HSCs were conjugated with carboxyfluorescein (CFSE)-tagged maleimide-bearing liposomes. A-E are preprinted with the permission from Springer Nature America, Inc., Ref [52]. (F) The schematic of metabolic labeling and cytokine conjugation of T cells with azido-sugar G400 NPs. Azido-sugar nanoparticles are directly added to T cell culture, enter T cells via endocytosis, and lead to presentation of azide group on T cell surfaces. After T cells are metabolically labeled, T cells are washed and DBCO-labeled proteins (e.g., cytokines) are directly added to produce conjugated T cells for downstream use. (G) The percentage of T cells with positive azide signal over time. T cells were treated with 200 µM G400 NP for 3 d, after which G400 NP in the medium was removed (day 0) and T cells were subsequently cultured free of G400 NP. (H) The enzyme-linked immunosorbent assay quantification of the amount of DBCO-IL-12 conjugated onto one million T cells at various DBCO-IL-12 concentrations. F-H are preprinted with the permission from PNAS [53]