Figure 2
Figure 2. BPCNeuAc liposomes are bound and internalized by CD22-expressing cells. (A) FACS analysis for binding of fluorescently (NBD) labeled liposomes to wild-type CHO cells or CHO cells expressing recombinant human CD22. Cells were incubated with the naked (blue) or BPCNeuAc (green) liposomes or left untreated (gray) at 37°C for 1.5 hours before analysis. (B) CHO cells expressing CD22 were compared for their binding to the fluorescent (green) naked or BPCNeuAc liposomes. CD22 was detected with anti–human CD22 (red), and the nuclei were visualized by staining with DAPI. (C) Fluorescence microscopy analysis of the colocalization of BPCNeuAc liposomes with early endosomes. CHO-CD22 cells were incubated with fluorescent liposomes (green) as described. Early endosomes were visualized by staining with an Alexa Fluor 555–labeled anti-EEA1 (red). (D) Binding of BPCNeuAc liposomes to Daudi human B lymphoma cells. Daudi cells were incubated in mouse serum with liposomes containing 0% to 5% BPCNeuAc lipids or without liposomes (gray) before FACS analysis. (E) BPCNeuAc liposomes rapidly bind to Daudi cells. Fluorescent naked or BPCNeuAc liposomes were added to an aliquot of Daudi cells in mouse serum and incubated at 37°C for the indicated time before FACS analysis. Data are presented as mean channel of fluorescence (MCF) plus or minus SD. (n = 3). (F) Competitive binding of BPCNeuAc liposomes to Daudi cells in the presence of the free BPCNeuAc ligands. Fluorescent naked or BPCNeuAc liposomes were incubated with Daudi cells with the presence of the monovalent BPCNeuAc ligands at indicated concentration. Data were analyzed by FACS and shown as normalized MCF plus or minus SD. (n = 3). (G) Cytotoxicity of dox-loaded liposomes toward Daudi B cells. Cells were subjected to free dox, dox-loaded naked liposomes, or BPCNeuAc liposomes for 1 hour at 37°C. Cells were washed and incubated at 37°C for an additional 48 hours before measuring cell viability. Data shown are means of triplicate plus or minus SD. Representative data from 1 of 3 independent experiments are shown. Data were fitted using the Prism nonlinear regression software.

BPCNeuAc liposomes are bound and internalized by CD22-expressing cells. (A) FACS analysis for binding of fluorescently (NBD) labeled liposomes to wild-type CHO cells or CHO cells expressing recombinant human CD22. Cells were incubated with the naked (blue) or BPCNeuAc (green) liposomes or left untreated (gray) at 37°C for 1.5 hours before analysis. (B) CHO cells expressing CD22 were compared for their binding to the fluorescent (green) naked or BPCNeuAc liposomes. CD22 was detected with anti–human CD22 (red), and the nuclei were visualized by staining with DAPI. (C) Fluorescence microscopy analysis of the colocalization of BPCNeuAc liposomes with early endosomes. CHO-CD22 cells were incubated with fluorescent liposomes (green) as described. Early endosomes were visualized by staining with an Alexa Fluor 555–labeled anti-EEA1 (red). (D) Binding of BPCNeuAc liposomes to Daudi human B lymphoma cells. Daudi cells were incubated in mouse serum with liposomes containing 0% to 5% BPCNeuAc lipids or without liposomes (gray) before FACS analysis. (E) BPCNeuAc liposomes rapidly bind to Daudi cells. Fluorescent naked or BPCNeuAc liposomes were added to an aliquot of Daudi cells in mouse serum and incubated at 37°C for the indicated time before FACS analysis. Data are presented as mean channel of fluorescence (MCF) plus or minus SD. (n = 3). (F) Competitive binding of BPCNeuAc liposomes to Daudi cells in the presence of the free BPCNeuAc ligands. Fluorescent naked or BPCNeuAc liposomes were incubated with Daudi cells with the presence of the monovalent BPCNeuAc ligands at indicated concentration. Data were analyzed by FACS and shown as normalized MCF plus or minus SD. (n = 3). (G) Cytotoxicity of dox-loaded liposomes toward Daudi B cells. Cells were subjected to free dox, dox-loaded naked liposomes, or BPCNeuAc liposomes for 1 hour at 37°C. Cells were washed and incubated at 37°C for an additional 48 hours before measuring cell viability. Data shown are means of triplicate plus or minus SD. Representative data from 1 of 3 independent experiments are shown. Data were fitted using the Prism nonlinear regression software.

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