Design, characterization, and optimization of RIT-INPs. (A) Design of G3139-loaded RIT-INPs for CLL B cell–specific targeting to overcome immunostimulatory effects. (B) Atomic Force Microscopy images of NPs and RIT-INPs. Shown are G3139 encapsulated in NPs (1) and G3139 encapsulated in RIT-INPs (2). Particle suspensions were dried on a mica substrate. All measurements were recorded in both height and amplitude modes. Height images are presented here. (C) Confocal micrographs of uptake of cy3-G3139 (1μM) loaded in RIT-INPs under various molar ratios of RIT/total lipids (approximately 1/500-1/12 000) in Raji cells after 4 hours of incubation. DIC indicates differential interference contrast (bright-field) images. Scale bar indicates 10 μm (D) Flow cytometry analysis of binding/uptake of RIT-INP carrying FAM-G3139 in Raji cells compared with free FAM-G3139 and nontargeted NP carrying FAM-G3139. Raji cells were treated with FAM-G3139–loaded RIT-INPs with various molar ratios of RIT/total lipids. Data are presented according to mean fluorescence intensity (MFI) changes (n = 3, mean ± SD). (E) Binding of free FAM–labeled G3139 and various NP-formulated FAM-ODN to Raji (CD20⁺) and Jurkat (CD20⁻) cells. (F) Binding of free FAM-G3139 and various NP-formulated FAM-G3139 to CLL B cells. The CLL B cells were incubated with free FAM-G3139 or FAM-G3139 in HER-INP or RIT-INP with the concentration of 1μM at 37°C for 1 hour and washed twice with cold PBS. (G) Inhibition of RIT-INP binding to Raji cells by excess RIT or alemtuzumab (anti-CD52). Untreated cells (bold line), cells treated with anti-CD20 ILP (thin solid line), and cells blocked with rituximab or alemtuzumab (broken line) were assessed by flow cytometry.