Specific targeting to B cells by lipid-based nanoparticles conjugated with a novel CD22-ScFv

Abstract

The CD22 antigen is a viable target for therapeutic intervention for B-cell lymphomas. Several therapeutic anti-CD22 antibodies as well as an anti-CD22-based immunotoxin (HA22) are currently under investigation in clinical settings. Coupling of anti-CD22 reagents with a nano-drug delivery vehicle is projected to significantly improve treatment efficacies. Therefore, we generated a mutant of the targeting segment of HA22 (a CD22 scFv) to increase its soluble expression (mut-HA22), and conjugated it to the surface of sonicated liposomes to generate immunoliposomes (mut-HA22-liposomes). We examined liposome binding and uptake by CD22(+) B-lymphocytes (BJAB) by using calcein and/or rhodamine PE-labeled liposomes. We also tested the effect of targeting on cellular toxicity with doxorubicin-loaded liposomes. We report that: (i) Binding of mut-HA22-liposomes to BJAB cells was significantly greater than liposomes not conjugated with mut-HA22 (control liposomes), and mut-HA22-liposomes bind to and are taken in by BJAB cells in a dose and temperature-dependent manner, respectively; (ii) This binding occurred via the interaction with the cellular CD22 as pre-incubation of the cells with mut-HA22 blocked subsequent liposome binding; (iii) Intracellular localization of mut-HA22-liposomes at 37 degrees C but not at 4 degrees C indicated that our targeted liposomes were taken up through an energy dependent process via receptor-mediated endocytosis; and (iv) Mut-HA22-liposomes loaded with doxorubicin exhibited at least 2-3 fold more accumulation of doxorubicin in BJAB cells as compared to control liposomes. Moreover, these liposomes showed at least a 2-4 fold enhanced killing of BJAB or Raji cells (CD22(+)), but not SUP-T1 cells (CD22(-)). Taken together these data suggest that these 2nd-generation liposomes may serve as promising carriers for targeted drug delivery to treat patients suffering from B-cell lymphoma.

Figure 1. Characterization of mut-HA22

Figure 1A: Analysis of mut-HA22. Mut-HA22 was analyzed by gel electrophoresis. (Left Panel), mutations to the CDR3 region of HA22 increased soluble expression of the scFv. (Right Panel), verification of mut-HA22 reduction by gel electrophoresis: (a) mut-HA22, shown as a monomer and dimer, (b) mut-HA22 reduced with tributylphosphine (TBP), and (c) mut-HA22 reduced with dithiothreitol (DTT). Figure 1B: Mut-HA22 binds to CD22-expressing cells. To analyze specific binding of mut-HA22 with CD22, BJAB cells were incubated with 2μg mut-HA22 followed by sequential incubations with mouse anti-histidine IgG, and FITC-conjugated goat anti-mouse IgG. A commercial anti-CD22 IgG MYG13 was used as positive control, and was stained with FITC-conjugated goat anti-mouse IgG. SUP-T1 and Jurkat cells were used as negative controls. Graphs representing corresponding cell types are indicated. Fluorescence distributions of stained cells are: solid black curves, cells alone; black lines, immunostaining of mut-HA22; grey lines, immunostaining of MYG13; black dotted lines, mut-HA22's secondary Abs (mouse anti-his IgG and FITC conjugated goat anti-mouse IgG; dotted grey lines, MYG13's secondary Abs (FITC-conjugated goat anti-mouse IgG). These results were reproducible from at least three independent experiments. Bkg, background binding.

Figure 1. Characterization of mut-HA22

Figure 1A: Analysis of mut-HA22. Mut-HA22 was analyzed by gel electrophoresis. (Left Panel), mutations to the CDR3 region of HA22 increased soluble expression of the scFv. (Right Panel), verification of mut-HA22 reduction by gel electrophoresis: (a) mut-HA22, shown as a monomer and dimer, (b) mut-HA22 reduced with tributylphosphine (TBP), and (c) mut-HA22 reduced with dithiothreitol (DTT). Figure 1B: Mut-HA22 binds to CD22-expressing cells. To analyze specific binding of mut-HA22 with CD22, BJAB cells were incubated with 2μg mut-HA22 followed by sequential incubations with mouse anti-histidine IgG, and FITC-conjugated goat anti-mouse IgG. A commercial anti-CD22 IgG MYG13 was used as positive control, and was stained with FITC-conjugated goat anti-mouse IgG. SUP-T1 and Jurkat cells were used as negative controls. Graphs representing corresponding cell types are indicated. Fluorescence distributions of stained cells are: solid black curves, cells alone; black lines, immunostaining of mut-HA22; grey lines, immunostaining of MYG13; black dotted lines, mut-HA22's secondary Abs (mouse anti-his IgG and FITC conjugated goat anti-mouse IgG; dotted grey lines, MYG13's secondary Abs (FITC-conjugated goat anti-mouse IgG). These results were reproducible from at least three independent experiments. Bkg, background binding.

Figure 2. Characterization of mut-HA22-liposomes

Figure 2A: Determination of mut-HA22 conjugation to liposomes. Concentrated liposomes were prepared in sample buffer with or without DTT, and were loaded on a 4-12% Bis-Tris gel (under non-reducing conditions). The gel was run in MES-SDS running buffer at 200V for 35 minutes and proteins were stained with Microwave Blue. Conjugation of mut-HA22 to DSPE-PEG2000-Maleimide is verified by the molecular weight increase of free mut-HA22 to liposomal mut-HA22 (indicated by the arrow). Figure 2B: Hydrodynamic size distribution of liposomes before and after conjugation with mut-HA22. Hydrodynamic size was measured by backlight scattering in phosphate buffered saline with a Malvern Zetasizer Nano ZS instrument. The data are plotted as volume (top panel) and intensity (bottom panel) weighted distributions. Figure 2C: Stability of mut-HA22-liposomes in the presence of serum at 37°C. Calcein release from liposomes was measured after liposome incubation at 37°C in PBS with 0, 10, or 50% FBS for 0, 1, 4, or 24 hours before and after addition of Triton X-100 (TX100). Leakage is expressed as a percent of total, where 100% calcein leakage was obtained by addition of 20 μl Triton-X100 (10 % w/v) and 0% calcein leakage was the background fluorescence value before liposome incubation at 37°C. Error bars represent ± SD from at least three samples within a single experiment. Figure 2D: Elution profile of DOX-loaded, mut-HA22 liposomes on Sepharose CL-6B column Top panel: mut-HA22 conjugated liposomes were separated from free, unconjugated mut-HA22 scFv through size exclusion chromatography on a Sepharose CL-6B column (1x40cm). 1 mL fractions were collected at an elution rate of 0.37 mL/min. Absorbance at 280nm (as measured by UV detector) is shown. Peak I: DOX-loaded, mut-HA22 conjugated liposomes; Peak II: unconjugated mut-HA22 scFv; Peak III: Free DOX. Bottom panel: Samples in peak I were analyzed (see Fig 2A) to confirm mut-HA22 conjugation to liposomes. (a): mut-HA22 scFv standard; (b): DOX-loaded, mut-HA22 conjugated liposomes before and after Sepharose CL-6B column. The fractions in peak II were analyzed for the presence of mut-HA22 by gel electrophoresis as described (see Methods section, data not shown). Similarly, all fractions were also analyzed for DOX and peak III was assigned accordingly (data not shown).

Figure 2. Characterization of mut-HA22-liposomes

Figure 2A: Determination of mut-HA22 conjugation to liposomes. Concentrated liposomes were prepared in sample buffer with or without DTT, and were loaded on a 4-12% Bis-Tris gel (under non-reducing conditions). The gel was run in MES-SDS running buffer at 200V for 35 minutes and proteins were stained with Microwave Blue. Conjugation of mut-HA22 to DSPE-PEG2000-Maleimide is verified by the molecular weight increase of free mut-HA22 to liposomal mut-HA22 (indicated by the arrow). Figure 2B: Hydrodynamic size distribution of liposomes before and after conjugation with mut-HA22. Hydrodynamic size was measured by backlight scattering in phosphate buffered saline with a Malvern Zetasizer Nano ZS instrument. The data are plotted as volume (top panel) and intensity (bottom panel) weighted distributions. Figure 2C: Stability of mut-HA22-liposomes in the presence of serum at 37°C. Calcein release from liposomes was measured after liposome incubation at 37°C in PBS with 0, 10, or 50% FBS for 0, 1, 4, or 24 hours before and after addition of Triton X-100 (TX100). Leakage is expressed as a percent of total, where 100% calcein leakage was obtained by addition of 20 μl Triton-X100 (10 % w/v) and 0% calcein leakage was the background fluorescence value before liposome incubation at 37°C. Error bars represent ± SD from at least three samples within a single experiment. Figure 2D: Elution profile of DOX-loaded, mut-HA22 liposomes on Sepharose CL-6B column Top panel: mut-HA22 conjugated liposomes were separated from free, unconjugated mut-HA22 scFv through size exclusion chromatography on a Sepharose CL-6B column (1x40cm). 1 mL fractions were collected at an elution rate of 0.37 mL/min. Absorbance at 280nm (as measured by UV detector) is shown. Peak I: DOX-loaded, mut-HA22 conjugated liposomes; Peak II: unconjugated mut-HA22 scFv; Peak III: Free DOX. Bottom panel: Samples in peak I were analyzed (see Fig 2A) to confirm mut-HA22 conjugation to liposomes. (a): mut-HA22 scFv standard; (b): DOX-loaded, mut-HA22 conjugated liposomes before and after Sepharose CL-6B column. The fractions in peak II were analyzed for the presence of mut-HA22 by gel electrophoresis as described (see Methods section, data not shown). Similarly, all fractions were also analyzed for DOX and peak III was assigned accordingly (data not shown).

Figure 2. Characterization of mut-HA22-liposomes

Figure 2A: Determination of mut-HA22 conjugation to liposomes. Concentrated liposomes were prepared in sample buffer with or without DTT, and were loaded on a 4-12% Bis-Tris gel (under non-reducing conditions). The gel was run in MES-SDS running buffer at 200V for 35 minutes and proteins were stained with Microwave Blue. Conjugation of mut-HA22 to DSPE-PEG2000-Maleimide is verified by the molecular weight increase of free mut-HA22 to liposomal mut-HA22 (indicated by the arrow). Figure 2B: Hydrodynamic size distribution of liposomes before and after conjugation with mut-HA22. Hydrodynamic size was measured by backlight scattering in phosphate buffered saline with a Malvern Zetasizer Nano ZS instrument. The data are plotted as volume (top panel) and intensity (bottom panel) weighted distributions. Figure 2C: Stability of mut-HA22-liposomes in the presence of serum at 37°C. Calcein release from liposomes was measured after liposome incubation at 37°C in PBS with 0, 10, or 50% FBS for 0, 1, 4, or 24 hours before and after addition of Triton X-100 (TX100). Leakage is expressed as a percent of total, where 100% calcein leakage was obtained by addition of 20 μl Triton-X100 (10 % w/v) and 0% calcein leakage was the background fluorescence value before liposome incubation at 37°C. Error bars represent ± SD from at least three samples within a single experiment. Figure 2D: Elution profile of DOX-loaded, mut-HA22 liposomes on Sepharose CL-6B column Top panel: mut-HA22 conjugated liposomes were separated from free, unconjugated mut-HA22 scFv through size exclusion chromatography on a Sepharose CL-6B column (1x40cm). 1 mL fractions were collected at an elution rate of 0.37 mL/min. Absorbance at 280nm (as measured by UV detector) is shown. Peak I: DOX-loaded, mut-HA22 conjugated liposomes; Peak II: unconjugated mut-HA22 scFv; Peak III: Free DOX. Bottom panel: Samples in peak I were analyzed (see Fig 2A) to confirm mut-HA22 conjugation to liposomes. (a): mut-HA22 scFv standard; (b): DOX-loaded, mut-HA22 conjugated liposomes before and after Sepharose CL-6B column. The fractions in peak II were analyzed for the presence of mut-HA22 by gel electrophoresis as described (see Methods section, data not shown). Similarly, all fractions were also analyzed for DOX and peak III was assigned accordingly (data not shown).

Figure 2. Characterization of mut-HA22-liposomes

Figure 2A: Determination of mut-HA22 conjugation to liposomes. Concentrated liposomes were prepared in sample buffer with or without DTT, and were loaded on a 4-12% Bis-Tris gel (under non-reducing conditions). The gel was run in MES-SDS running buffer at 200V for 35 minutes and proteins were stained with Microwave Blue. Conjugation of mut-HA22 to DSPE-PEG2000-Maleimide is verified by the molecular weight increase of free mut-HA22 to liposomal mut-HA22 (indicated by the arrow). Figure 2B: Hydrodynamic size distribution of liposomes before and after conjugation with mut-HA22. Hydrodynamic size was measured by backlight scattering in phosphate buffered saline with a Malvern Zetasizer Nano ZS instrument. The data are plotted as volume (top panel) and intensity (bottom panel) weighted distributions. Figure 2C: Stability of mut-HA22-liposomes in the presence of serum at 37°C. Calcein release from liposomes was measured after liposome incubation at 37°C in PBS with 0, 10, or 50% FBS for 0, 1, 4, or 24 hours before and after addition of Triton X-100 (TX100). Leakage is expressed as a percent of total, where 100% calcein leakage was obtained by addition of 20 μl Triton-X100 (10 % w/v) and 0% calcein leakage was the background fluorescence value before liposome incubation at 37°C. Error bars represent ± SD from at least three samples within a single experiment. Figure 2D: Elution profile of DOX-loaded, mut-HA22 liposomes on Sepharose CL-6B column Top panel: mut-HA22 conjugated liposomes were separated from free, unconjugated mut-HA22 scFv through size exclusion chromatography on a Sepharose CL-6B column (1x40cm). 1 mL fractions were collected at an elution rate of 0.37 mL/min. Absorbance at 280nm (as measured by UV detector) is shown. Peak I: DOX-loaded, mut-HA22 conjugated liposomes; Peak II: unconjugated mut-HA22 scFv; Peak III: Free DOX. Bottom panel: Samples in peak I were analyzed (see Fig 2A) to confirm mut-HA22 conjugation to liposomes. (a): mut-HA22 scFv standard; (b): DOX-loaded, mut-HA22 conjugated liposomes before and after Sepharose CL-6B column. The fractions in peak II were analyzed for the presence of mut-HA22 by gel electrophoresis as described (see Methods section, data not shown). Similarly, all fractions were also analyzed for DOX and peak III was assigned accordingly (data not shown).

Figure 3. Binding of mut-HA22-liposomes to CD22-expressing cells

Cells (10⁷/mL in RPMI-1640, 10% serum) were incubated with fluorescently labeled mut-HA22-liposomes (2 μg of mut-HA22/46μg of phospholipids) or control liposomes (0 μg of mut-HA22/46 μg of phospholipids) for 40 minutes at 37°C. At the end of incubations, cells were centrifuged and washed twice with PBS-BSA to remove unbound liposomes; cell-bound fluorescence was examined by microscopy and FACS. Figure 3A: Binding of mut-HA22-liposomes to CD22-receptor expressing cells. Fluorescence images were captured using the Nikon Eclipse TE200 inverted microscope with a 40X oil objective (N.A. 1.30). Images shown are for calcein and rhodamine fluorescence as indicated ((a-d), BJAB cells, (e-h) SUP-T1 cells). Figure 3B: Flow cytometry analysis of incubated cells. The data shown are for calcein fluorescence. Figure 3C: Pre-incubation of cells with free mut-HA22 inhibits subsequent binding of liposomes. BJAB cells were incubated with (b) 8, (c) 2, or (d) 0 μg of free mut-HA22 at 4°C for 30 minutes in the dark, washed twice with cold PBS-BSA, and then incubated with fluorescently labeled mut-HA22-liposomes (1μg/10⁶ cells) at 4°C for 30 minutes ((a) cells only)).

Figure 3. Binding of mut-HA22-liposomes to CD22-expressing cells

Cells (10⁷/mL in RPMI-1640, 10% serum) were incubated with fluorescently labeled mut-HA22-liposomes (2 μg of mut-HA22/46μg of phospholipids) or control liposomes (0 μg of mut-HA22/46 μg of phospholipids) for 40 minutes at 37°C. At the end of incubations, cells were centrifuged and washed twice with PBS-BSA to remove unbound liposomes; cell-bound fluorescence was examined by microscopy and FACS. Figure 3A: Binding of mut-HA22-liposomes to CD22-receptor expressing cells. Fluorescence images were captured using the Nikon Eclipse TE200 inverted microscope with a 40X oil objective (N.A. 1.30). Images shown are for calcein and rhodamine fluorescence as indicated ((a-d), BJAB cells, (e-h) SUP-T1 cells). Figure 3B: Flow cytometry analysis of incubated cells. The data shown are for calcein fluorescence. Figure 3C: Pre-incubation of cells with free mut-HA22 inhibits subsequent binding of liposomes. BJAB cells were incubated with (b) 8, (c) 2, or (d) 0 μg of free mut-HA22 at 4°C for 30 minutes in the dark, washed twice with cold PBS-BSA, and then incubated with fluorescently labeled mut-HA22-liposomes (1μg/10⁶ cells) at 4°C for 30 minutes ((a) cells only)).

Figure 3. Binding of mut-HA22-liposomes to CD22-expressing cells

Cells (10⁷/mL in RPMI-1640, 10% serum) were incubated with fluorescently labeled mut-HA22-liposomes (2 μg of mut-HA22/46μg of phospholipids) or control liposomes (0 μg of mut-HA22/46 μg of phospholipids) for 40 minutes at 37°C. At the end of incubations, cells were centrifuged and washed twice with PBS-BSA to remove unbound liposomes; cell-bound fluorescence was examined by microscopy and FACS. Figure 3A: Binding of mut-HA22-liposomes to CD22-receptor expressing cells. Fluorescence images were captured using the Nikon Eclipse TE200 inverted microscope with a 40X oil objective (N.A. 1.30). Images shown are for calcein and rhodamine fluorescence as indicated ((a-d), BJAB cells, (e-h) SUP-T1 cells). Figure 3B: Flow cytometry analysis of incubated cells. The data shown are for calcein fluorescence. Figure 3C: Pre-incubation of cells with free mut-HA22 inhibits subsequent binding of liposomes. BJAB cells were incubated with (b) 8, (c) 2, or (d) 0 μg of free mut-HA22 at 4°C for 30 minutes in the dark, washed twice with cold PBS-BSA, and then incubated with fluorescently labeled mut-HA22-liposomes (1μg/10⁶ cells) at 4°C for 30 minutes ((a) cells only)).

Figure 3. Binding of mut-HA22-liposomes to CD22-expressing cells

Cells (10⁷/mL in RPMI-1640, 10% serum) were incubated with fluorescently labeled mut-HA22-liposomes (2 μg of mut-HA22/46μg of phospholipids) or control liposomes (0 μg of mut-HA22/46 μg of phospholipids) for 40 minutes at 37°C. At the end of incubations, cells were centrifuged and washed twice with PBS-BSA to remove unbound liposomes; cell-bound fluorescence was examined by microscopy and FACS. Figure 3A: Binding of mut-HA22-liposomes to CD22-receptor expressing cells. Fluorescence images were captured using the Nikon Eclipse TE200 inverted microscope with a 40X oil objective (N.A. 1.30). Images shown are for calcein and rhodamine fluorescence as indicated ((a-d), BJAB cells, (e-h) SUP-T1 cells). Figure 3B: Flow cytometry analysis of incubated cells. The data shown are for calcein fluorescence. Figure 3C: Pre-incubation of cells with free mut-HA22 inhibits subsequent binding of liposomes. BJAB cells were incubated with (b) 8, (c) 2, or (d) 0 μg of free mut-HA22 at 4°C for 30 minutes in the dark, washed twice with cold PBS-BSA, and then incubated with fluorescently labeled mut-HA22-liposomes (1μg/10⁶ cells) at 4°C for 30 minutes ((a) cells only)).

Figure 4

A: Binding of mut-HA22--liposomes with 1% or 4% DSPE-PEG2000-Maleimide to CD22- expressing cells. Cells (10⁷/mL in RPMI-1640, 10% serum) were incubated with various concentrations of calcein-loaded mut-HA22-liposomes for 40 minutes at 37°C. At the end of incubations, cells were centrifuged and washed twice with PBS-BSA to remove unbound liposomes; cell-bound fluorescence was examined by FACS. The graph represents mean fluorescence intensity values (y-axis). Three liposome preparations (see Table 1) corresponding to either 0 mol% (=0 mut-HA22/liposome), 1 mol% (=47 mut-HA22/liposome) or 4 mol% (=90 mut-HA22/liposome) were used for incubations with either BJAB or SUP-T1 cells. The amounts of liposomal mut-HA22 added are given in x-axis. Cell types are indicated at the end of each curve. The numbers in parentheses indicate the average number of mut-HA22/liposome. Figure 4B: Dose-dependent binding of mut-HA22-liposomes to BJAB cells. BJAB cells were incubated with various concentrations of calcein-loaded liposomes for 40 minutes at 37°C. At the end of incubations, cells were washed and analyzed for calcein fluorescence by FACS. Concentrations of liposome-conjugated mut-HA22 added to cells: (a) cells alone, (b) 0.01 μg mut-HA22, (c) 0.05 μg mut-HA22 (d), 0.2 μg mut-HA22, (e) 0.5 μg mut-HA22, and (f) 2 μg mut-HA22. Control liposomes contained the corresponding phospholipid quantities without any mut-HA22.

Figure 4

A: Binding of mut-HA22--liposomes with 1% or 4% DSPE-PEG2000-Maleimide to CD22- expressing cells. Cells (10⁷/mL in RPMI-1640, 10% serum) were incubated with various concentrations of calcein-loaded mut-HA22-liposomes for 40 minutes at 37°C. At the end of incubations, cells were centrifuged and washed twice with PBS-BSA to remove unbound liposomes; cell-bound fluorescence was examined by FACS. The graph represents mean fluorescence intensity values (y-axis). Three liposome preparations (see Table 1) corresponding to either 0 mol% (=0 mut-HA22/liposome), 1 mol% (=47 mut-HA22/liposome) or 4 mol% (=90 mut-HA22/liposome) were used for incubations with either BJAB or SUP-T1 cells. The amounts of liposomal mut-HA22 added are given in x-axis. Cell types are indicated at the end of each curve. The numbers in parentheses indicate the average number of mut-HA22/liposome. Figure 4B: Dose-dependent binding of mut-HA22-liposomes to BJAB cells. BJAB cells were incubated with various concentrations of calcein-loaded liposomes for 40 minutes at 37°C. At the end of incubations, cells were washed and analyzed for calcein fluorescence by FACS. Concentrations of liposome-conjugated mut-HA22 added to cells: (a) cells alone, (b) 0.01 μg mut-HA22, (c) 0.05 μg mut-HA22 (d), 0.2 μg mut-HA22, (e) 0.5 μg mut-HA22, and (f) 2 μg mut-HA22. Control liposomes contained the corresponding phospholipid quantities without any mut-HA22.

Figure 5. Uptake of mut-HA22-liposomes by BJAB cells at 37°C

BJAB cells (10⁷/mL RPMI-1640) were incubated with liposomes at 37°C or 4°C. Cells were then washed twice with cold PBS-BSA and kept on ice until analyzed. Figure 5A: Temperature Dependent Binding/Uptake of mut-HA22-liposomes by BJAB cells. Cells were incubated with 4 μg of liposome-conjugated mut-HA22 (or equivalent amount of phospholipids for control liposomes) for 40 minutes at 37°C or 4°C (see methods section). Cells were then washed and rhodamine fluorescence of cells was analyzed by FACS. Figure 5B: Intracellular Localization of mut-HA22-scFv liposomes by BJAB Cells. Cells were incubated with 0.2 μg of liposome-conjugated mut-HA22 (or equivalent amount of phospholipids for control liposomes) for 20 or 40 minutes at 37 or 4°C. Z-stacks of 14-16 optical slices at 1.7 micron intervals were acquired. Calcein fluorescence of cells is shown in a single slice of the acquired images.

Figure 5. Uptake of mut-HA22-liposomes by BJAB cells at 37°C

BJAB cells (10⁷/mL RPMI-1640) were incubated with liposomes at 37°C or 4°C. Cells were then washed twice with cold PBS-BSA and kept on ice until analyzed. Figure 5A: Temperature Dependent Binding/Uptake of mut-HA22-liposomes by BJAB cells. Cells were incubated with 4 μg of liposome-conjugated mut-HA22 (or equivalent amount of phospholipids for control liposomes) for 40 minutes at 37°C or 4°C (see methods section). Cells were then washed and rhodamine fluorescence of cells was analyzed by FACS. Figure 5B: Intracellular Localization of mut-HA22-scFv liposomes by BJAB Cells. Cells were incubated with 0.2 μg of liposome-conjugated mut-HA22 (or equivalent amount of phospholipids for control liposomes) for 20 or 40 minutes at 37 or 4°C. Z-stacks of 14-16 optical slices at 1.7 micron intervals were acquired. Calcein fluorescence of cells is shown in a single slice of the acquired images.

Figure 6

A: Cellular accumulation of liposomal DOX by Raji cells Cells (3×10⁵ cells per sample) were incubated with 0.5 – 2 ug of liposomal DOX for one hour at 37°C. The cells were then washed with phenol-red free culture medium (0.5mL×3) and resuspended in 0.3 mL of PBS and transferred to a 96-well plate (0.1mL×3). Cellular accumulation of DOX was determined using a standard curve of known DOX concentrations in the presence of cell lysates without incubation with liposomes (left panel). Right panel: accumulation of DOX in nanograms. Solid gray bars, control liposomes; black diagonal line bars, mut-HA22 liposomes. Error bars represent ± SD of triplicate samples within a single experiment. Figure 6B: Cell viability in the continuous presence of liposomal DOX Cells were plated in duplicates in a 96-well plate (10⁵/0.1mL per well) and incubated with 0-7.5 ug of liposomal or free DOX for 72 hours at 37°C. Cell viability was calculated taking control sample (without DOX) as 100%. Values represent an average of duplicate samples. (a) and (b) Cytotoxicity by liposomal DOX: (a) BJAB (CD22⁺), (b) SUP-T1 (CD22^-) mut-HA22 liposomes (◆) and control liposomes (●). (c) Cytotoxicity by free DOX: SUP-T1 (X), BJAB (▲), and Raji (■). Figure 6C: Cell viability following pre-incubation with liposomes Cells were incubated in triplicates with liposomal DOX for 1 hour, and any unbound liposomes were washed off. Following an additional 72-hour incubation, cell viability was determined (see Figure 6B). (a) Raji (CD22⁺), (b) BJAB (CD22⁺), and (c) SUP-T1 (CD22^-) mut-HA22 liposomes (◆) and control liposomes (●) (d) A snapshot of cell viability at a concentration of 10 ug/mL liposomal DOX is shown (Data from the respective curves in Figure 6C (a-c)). In this graph, the total number of cells remaining viable in the presence of control liposomes were taken as 100%. mut-HA22 liposomes (diagonal line bars), control liposomes (gray bars)

Figure 6

A: Cellular accumulation of liposomal DOX by Raji cells Cells (3×10⁵ cells per sample) were incubated with 0.5 – 2 ug of liposomal DOX for one hour at 37°C. The cells were then washed with phenol-red free culture medium (0.5mL×3) and resuspended in 0.3 mL of PBS and transferred to a 96-well plate (0.1mL×3). Cellular accumulation of DOX was determined using a standard curve of known DOX concentrations in the presence of cell lysates without incubation with liposomes (left panel). Right panel: accumulation of DOX in nanograms. Solid gray bars, control liposomes; black diagonal line bars, mut-HA22 liposomes. Error bars represent ± SD of triplicate samples within a single experiment. Figure 6B: Cell viability in the continuous presence of liposomal DOX Cells were plated in duplicates in a 96-well plate (10⁵/0.1mL per well) and incubated with 0-7.5 ug of liposomal or free DOX for 72 hours at 37°C. Cell viability was calculated taking control sample (without DOX) as 100%. Values represent an average of duplicate samples. (a) and (b) Cytotoxicity by liposomal DOX: (a) BJAB (CD22⁺), (b) SUP-T1 (CD22^-) mut-HA22 liposomes (◆) and control liposomes (●). (c) Cytotoxicity by free DOX: SUP-T1 (X), BJAB (▲), and Raji (■). Figure 6C: Cell viability following pre-incubation with liposomes Cells were incubated in triplicates with liposomal DOX for 1 hour, and any unbound liposomes were washed off. Following an additional 72-hour incubation, cell viability was determined (see Figure 6B). (a) Raji (CD22⁺), (b) BJAB (CD22⁺), and (c) SUP-T1 (CD22^-) mut-HA22 liposomes (◆) and control liposomes (●) (d) A snapshot of cell viability at a concentration of 10 ug/mL liposomal DOX is shown (Data from the respective curves in Figure 6C (a-c)). In this graph, the total number of cells remaining viable in the presence of control liposomes were taken as 100%. mut-HA22 liposomes (diagonal line bars), control liposomes (gray bars)

Figure 6

A: Cellular accumulation of liposomal DOX by Raji cells Cells (3×10⁵ cells per sample) were incubated with 0.5 – 2 ug of liposomal DOX for one hour at 37°C. The cells were then washed with phenol-red free culture medium (0.5mL×3) and resuspended in 0.3 mL of PBS and transferred to a 96-well plate (0.1mL×3). Cellular accumulation of DOX was determined using a standard curve of known DOX concentrations in the presence of cell lysates without incubation with liposomes (left panel). Right panel: accumulation of DOX in nanograms. Solid gray bars, control liposomes; black diagonal line bars, mut-HA22 liposomes. Error bars represent ± SD of triplicate samples within a single experiment. Figure 6B: Cell viability in the continuous presence of liposomal DOX Cells were plated in duplicates in a 96-well plate (10⁵/0.1mL per well) and incubated with 0-7.5 ug of liposomal or free DOX for 72 hours at 37°C. Cell viability was calculated taking control sample (without DOX) as 100%. Values represent an average of duplicate samples. (a) and (b) Cytotoxicity by liposomal DOX: (a) BJAB (CD22⁺), (b) SUP-T1 (CD22^-) mut-HA22 liposomes (◆) and control liposomes (●). (c) Cytotoxicity by free DOX: SUP-T1 (X), BJAB (▲), and Raji (■). Figure 6C: Cell viability following pre-incubation with liposomes Cells were incubated in triplicates with liposomal DOX for 1 hour, and any unbound liposomes were washed off. Following an additional 72-hour incubation, cell viability was determined (see Figure 6B). (a) Raji (CD22⁺), (b) BJAB (CD22⁺), and (c) SUP-T1 (CD22^-) mut-HA22 liposomes (◆) and control liposomes (●) (d) A snapshot of cell viability at a concentration of 10 ug/mL liposomal DOX is shown (Data from the respective curves in Figure 6C (a-c)). In this graph, the total number of cells remaining viable in the presence of control liposomes were taken as 100%. mut-HA22 liposomes (diagonal line bars), control liposomes (gray bars)