Journal: Scientific Reports

Article Title: Antibody-targeted paclitaxel loaded nanoparticles for the treatment of CD20 + B-cell lymphoma

doi: 10.1038/srep45682

Figure Lengend Snippet: ( a ) A schematic representation of AR160. ( b ) Rituximab was tagged with AlexaFluor 488 and co-incubated with ABX for visualization. ImageStream reveals fluorescently labeled nanoparticles of approximately 0.1 uM. ( c ) ABX was tagged with AlexaFluor 488 and co-incubated with rituximab and Daudi cells were stained with either PE anti-human CD19, fluorescent AR160, or both. Cells were analyzed by Guava flow cytometery. Daudi cells were about 75% positive for CD19, AR160 or both. ( d ) The labeled Daudi cells were also run by ImageStream and an image of a doubly stained Daudi cell is shown. ( e ) AR160 was separated into 3 fractions: the particulate, and proteins greater than and less than 100 kD. Paclitaxel concentration in each fraction was determined by HPLC and showed about 69.2% of paclitaxel is in the particulate and the remaining paclitaxel is among proteins greater the 100 kD. Paclitaxel was measured in AR160 fractions after 24 ( f ), 48 hours ( g ), and 60 minutes in AB serum ( h ). Data shows a shift of the majority of paclitaxel from the particulate to the proteins >100 kD. ( i ) Western blot was performed on the greater than 100 kD fraction and rituximab, paclitaxel and albumin co-localized in a band of approximately 200 kD. Biacore screening of an albumin peptide library for binding to riruximab reveals 3 binding peptides ( j ) HSA peptide 4 binds to rituximab with a Kd of 5.7 × 10 −8 . ( k ) HSA peptide 13 binds to rituximab with a Kd of 4.0 × 10 −7 . ( l ) HSA peptide 40 binds to rituximab with a Kd of 5.2 × 10 −10 . ( m ) ABX was incubated for 30 minutes with 4 mg/ml rituximab and either no peptide (AR160), 10x molar excess, relative to antibody, of control peptide (ABX+Rit+control) or HSA peptide 40 (ABX+Rit+HSA peptide 40). Particle sizes were determined by nanoparticle tracking analysis utilizing the NS300. HSA peptide 40 was shown to block the formation of AR160 suggesting the peptide blocks rituximab from binding ABX. Results are representative of 3 experiments.

Article Snippet: Five million Daudi cells were implanted into the right flank of athymic nude mice (Harlan Sprague Dawley, Indianapolis, IN).

Techniques: Incubation, Labeling, Staining, Concentration Assay, Western Blot, Binding Assay, Blocking Assay

Journal: Scientific Reports

Article Title: Antibody-targeted paclitaxel loaded nanoparticles for the treatment of CD20 + B-cell lymphoma

doi: 10.1038/srep45682

Figure Lengend Snippet: ( a ) ABX and AR160 was prepared and incubated for 24 hours in saline at room temperature and size and particle number was measured at 0, 1, 2, 4, 6, and 24 hours by NS300 nanoparticle tracking system. AR160 remained stable over 24 hours as particle size and number remained unchanged during that time. The particle size distributions and particle size and numbers are shown. ( b , c ) ABX and AR160 were prepared, and 30 × 108 particles were added to human AB serum and incubated for 60 minutes in human AB serum. Particle size and numbers were determined at 5, 15, 30, and 60 minutes after being added to the AB serum. Particle size distributions for ABX ( b ) and AR160 ( c ) are shown. ( d ) Graphical representation of the particle number of ABX relative to AR160 after incubation in AB serum. ( e ) To verify toxicity of AR160, we treated CD20+ Daudi cells with AR160, ABX and rituximab. Cells were treated overnight with the drugs at concentrations from 0–200 ug/ml with the addition of EdU, a thymidine analog. The level of proliferation was determined by staining cells with FITC labeled anti-EdU. The proliferation index was calculated by normalization to the untreated positive control. ( f ) Rituximab ligand binding was confirmed by flow cytometry in which the Daudi cells were pretreated with rituximab, ABX, and AR160 and then stained with PE anti-human CD20. The drug-pretreated samples were compared to isotype control and PE anti-human CD20 alone serving as negative and positive controls, respectively. Rituximab and AR160, but not ABX alone blocked subsequent PE anti-human CD20 binding. Data is representative of 3 experiments.

Article Snippet: Five million Daudi cells were implanted into the right flank of athymic nude mice (Harlan Sprague Dawley, Indianapolis, IN).

Techniques: Incubation, Staining, Labeling, Positive Control, Ligand Binding Assay, Flow Cytometry, Binding Assay