Journal: Biomacromolecules
Article Title: Development of CD33-Targeted Dual Drug-Loaded Nanoparticles for the Treatment of Pediatric Acute Myeloid Leukemia
doi: 10.1021/acs.biomac.4c00672
Figure Lengend Snippet: Binding of nanoformulations to recombinant CD33-Fc and CD33-expressing cells. (A–D) Binding of rhodamine 6G-loaded NPs to recombinant CD33-Fc in FLISA assays: (A) dose-dependent binding, (B) binding of NPs (50 μg polymer/mL) ± preincubation with CD33-Fc (10 μg/mL), (C) binding of NPs (500 μg polymer/mL) ± preblock with CD33 mAb (40 μg/mL), (D) binding of NPs (500 μg polymer/mL) in competition with varying concentrations of gemtuzumab (0.00256–40 μg/mL). Data are presented as mean ± SD, n = 3. (E) Binding of nonfluorescent nanoformulations to CD33-Fc and IgG-Fc evaluated by SPR: (i) binding of the NPs at 4 mg/mL to CD33-Fc and IgG-Fc, (ii) binding of CD33 NPs to CD33-Fc at varying concentrations, with linear regression and corresponding goodness of fit ( R 2 ). Binding is presented as response relative to baseline observed 5 s before the end of the injection period. Data are presented as mean ± SD, n = 2. (F) Cells were treated with blank CD33 or nude NPs (750 μg polymer/mL) for 1 h at 4 °C. Then, cells were washed, stained with PE-labeled anti-CD33 antibody or isotype control antibody, and PE-fluorescence was analyzed by flow cytometry. Representative histograms are shown for each condition tested, (i), as well as the corresponding reduction of fluorescence compared with the positive stained control observed after treatment with the NPs for each cell line (ii). (G) Confocal microscopy images of MOLM-13 cells treated with 500 μg polymer/mL rhodamine 6G-loaded NPs for 1 h at 4 °C, followed by a washing step and a further 2 h-incubation at 37 °C. Scale bar is 50 μm, and blue and red staining denote cell nuclei and nanoparticles, respectively. Representative data from n = 2.
Article Snippet: FLISA studies were performed as previously described, using recombinant human CD33-Fc (Sino Biological) at 1 μg/mL to coat the plate wells.
Techniques: Binding Assay, Recombinant, Expressing, Fluorophore-linked Immunoabsorbent Assay, Polymer, Injection, Staining, Labeling, Control, Fluorescence, Flow Cytometry, Confocal Microscopy, Incubation
![Identification of <t>CD33-specific</t> SdAbs (A) CD33 indirect ELISA curves of llama plasma at different stages during immunization (percentage of signal vs. plasma dilution factor [DF]). (B) Output/input (O/I) phage ratio for each panning round. (C) Phage ELISA of the original library and the final output AO3. (D) Preliminary ELISA screening of isolated E. coli clones infected with bacteriophages. As positive control, one positive clone from a previous round output (C+) was offered. (E) Final ELISA screening of isolated E. coli clones expressing the SdAbs from the expression vector pETMod. The positive control (C+) was C4 from the preliminary screening, and the negative control was an irrelevant SdAb-expressing clone culture supernatant from a panning round against another target. (F) Sequence alignment of the five candidate SdAbs selected for further characterization. (G) Phylogenetic tree of the identified SdAb sequences against CD33, clustered into five families based on half distance. (H) Coomassie blue-stained SDS-PAGE and anti-HA Western blot of the IMAC and IEC purified SdAbs. (I) Cross reactivity test (ELISA) of the five candidate SdAbs against antigens from the same llama library.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4528/pmc11904528/pmc11904528__gr1.jpg)
