Figure 3
Figure 3. Pharmacokinetics and siglec specificity of BPCNeuAc liposomes. (A) BPCNeuAc liposomes selectively bind to Daudi cells in mouse blood. In vitro (top panels): Daudi cells were spiked into an aliquot of mouse whole blood followed by addition of fluorescent naked or BPCNeuAc liposomes. Cells were stained with anti–human CD19 to distinguish Daudi cells from other cells in the mouse blood. In vivo (bottom panels): after intravenous injection of Daudi cells, mice were injected with fluorescent naked or BPCNeuAc liposomes. After 2 hours, a blood sample was drawn and the binding of liposomes to Daudi cells was analyzed by FACS. The numbers in the quadrants represent percentages of CD19 Daudi cells that bound or did not bind to liposomes. Shown are data from 1 of 3 independent experiments. (B-C) Dox-loaded liposomes were injected intravenously to the tumor-free SCID mice (3 mice per group) without or with pretreatment of clodronate to deplete tissue macrophages. A sample of blood was withdrawn at 0.5, 2, and 25 hours after liposome injections. The plasma concentration of dox was measured, and data are presented as percentage remaining of the initial injected drug plus or minus SD. (D) FACS analysis for binding of naked or BPCNeuAc liposomes to siglec-expressing CHO lines and Daudi, A20, and TSn cell lines that express hCD22, mCD22 and hSn, respectively. Binding is shown as MCF plus or minus SD (n = 3). Binding degree of BPCNeuAc liposomes to CHO-mSn, CHO-hCD22, Daudi, and TSn cell lines was significant in comparison to the same cell line that was treated with the naked liposomes (*P < .01). (E) Comparison of BPCNeuAc or BPANeuAc liposomes in binding to cell lines expressing hCD22, mCD22, hSn, and mSn. Binding of liposomes is expressed as MCF plus or minus SD (n = 3). (F) Top panel shows that the structures of the trisaccharide ligands designed to be specific for human CD22 are based on the parent compound NeuAcα2-6Galβ1-4GlcNAc, varying the biphenyl substituent at C-9 (R1). Bottom panel shows that BPANeuAc liposomes exhibit a long circulation time in vivo. A sample of blood was withdrawn from mice (n = 3) that received dox-loaded naked, BPCNeuAc, or BPANeuAc liposomes at 0.5, 2, and 25 hours after liposome injections. The plasma concentration of dox was detected using a fluorometer. Data are presented as percentage remaining of the initial dose ± SD. (G) Pharmacokinetics analysis for naked and BPCNeuAc liposomes in wild-type C57BL/6 and Sn knockout mice. Data are presented as percentage remaining of the initial dose ± SD (n = 3).

Pharmacokinetics and siglec specificity of BPCNeuAc liposomes. (A) BPCNeuAc liposomes selectively bind to Daudi cells in mouse blood. In vitro (top panels): Daudi cells were spiked into an aliquot of mouse whole blood followed by addition of fluorescent naked or BPCNeuAc liposomes. Cells were stained with anti–human CD19 to distinguish Daudi cells from other cells in the mouse blood. In vivo (bottom panels): after intravenous injection of Daudi cells, mice were injected with fluorescent naked or BPCNeuAc liposomes. After 2 hours, a blood sample was drawn and the binding of liposomes to Daudi cells was analyzed by FACS. The numbers in the quadrants represent percentages of CD19 Daudi cells that bound or did not bind to liposomes. Shown are data from 1 of 3 independent experiments. (B-C) Dox-loaded liposomes were injected intravenously to the tumor-free SCID mice (3 mice per group) without or with pretreatment of clodronate to deplete tissue macrophages. A sample of blood was withdrawn at 0.5, 2, and 25 hours after liposome injections. The plasma concentration of dox was measured, and data are presented as percentage remaining of the initial injected drug plus or minus SD. (D) FACS analysis for binding of naked or BPCNeuAc liposomes to siglec-expressing CHO lines and Daudi, A20, and TSn cell lines that express hCD22, mCD22 and hSn, respectively. Binding is shown as MCF plus or minus SD (n = 3). Binding degree of BPCNeuAc liposomes to CHO-mSn, CHO-hCD22, Daudi, and TSn cell lines was significant in comparison to the same cell line that was treated with the naked liposomes (*P < .01). (E) Comparison of BPCNeuAc or BPANeuAc liposomes in binding to cell lines expressing hCD22, mCD22, hSn, and mSn. Binding of liposomes is expressed as MCF plus or minus SD (n = 3). (F) Top panel shows that the structures of the trisaccharide ligands designed to be specific for human CD22 are based on the parent compound NeuAcα2-6Galβ1-4GlcNAc, varying the biphenyl substituent at C-9 (R1). Bottom panel shows that BPANeuAc liposomes exhibit a long circulation time in vivo. A sample of blood was withdrawn from mice (n = 3) that received dox-loaded naked, BPCNeuAc, or BPANeuAc liposomes at 0.5, 2, and 25 hours after liposome injections. The plasma concentration of dox was detected using a fluorometer. Data are presented as percentage remaining of the initial dose ± SD. (G) Pharmacokinetics analysis for naked and BPCNeuAc liposomes in wild-type C57BL/6 and Sn knockout mice. Data are presented as percentage remaining of the initial dose ± SD (n = 3).

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