Journal: Advanced Science

Article Title: Screening and Identification of Novel DNA Aptamer for Targeted Delivery to Injured Podocytes in Glomerular Diseases

doi: 10.1002/advs.202412356

Figure Lengend Snippet: Identification and Characterization of Aptamers Targeting Injured Podocytes Using Cell‐SELEX. A) Schematic representation of the Cell‐SELEX process outlining the methodology for obtaining aptamers that specifically bind to injured podocytes. B) Analysis of the binding abilities of different aptamer pools to podocytes via flow cytometry. MPC5 cells were incubated with 250 nM of the amplified FAM‐labeled products at 4 °C. FAM‐labeled library was used as controls. Confocal images showing f‐actin in MPC5 cells after 48 h of treatment with 0.3 µg mL −1 adriamycin (ADR), 30 µg mL −1 puromycin aminonucleoside (PAN), or 50 m m high glucose medium (HG). Untreated MPC5 cells (NC) served as the control. Actin filaments were stained with FITC‐phalloidin (green), and nuclei were stained with DAPI (blue). Scale bars, 25 µm. C) Composition of the candidate aptamers, excluding the primer region. Candidate aptamers are named based on the type of injury, with numbers indicating their abundance rank. D) Phylogenetic tree showing the relationship among candidate aptamers. E) Flow cytometry analysis of the binding of seven candidate aptamers (S1‐S7).

Article Snippet: The immortalized mouse podocyte cell line (MPC5) was donated by Professor Peter Mundel from Goldfinch Bio.

Techniques: Binding Assay, Flow Cytometry, Incubation, Amplification, Labeling, Control, Staining

Journal: Advanced Science

Article Title: Screening and Identification of Novel DNA Aptamer for Targeted Delivery to Injured Podocytes in Glomerular Diseases

doi: 10.1002/advs.202412356

Figure Lengend Snippet: Optimization and Characterization of S7 Variants and RLS‐2 for Podocyte Targeting. A) Predicted secondary structures of S7 (S7‐A, S7‐B, S7‐C) and its derived variants (S7‐1, S7‐2, S7‐3). The primer region, indicated by nucleotides with green borders, is shown separately. Mismatched nucleotides in S7 (highlighted in blue) were modified to create the variants. RLS‐2 was derived from S7‐2 by truncation, as denoted by the dashed circle. B) Binding ability of S7, its variants, and RLS‐2 to podocytes, as determined by flow cytometry. C) Binding ability of RLS‐2 and its phosphorothioate‐modified variant (PS‐RLS‐2) to injured podocytes, as analyzed by flow cytometry. D‐E) Evaluation of the serum stability of RLS‐2 and PS‐RLS‐2 in a 10% serum incubation medium (D), and the corresponding quantitative analysis (E) with data presented as mean ± standard deviation. n = 3. ** p < 0.01. F) The dissociation constant (K d ) curves and statistical analysis for S7 and RLS‐2, with data presented as mean ± standard deviation. n = 3.

Article Snippet: The immortalized mouse podocyte cell line (MPC5) was donated by Professor Peter Mundel from Goldfinch Bio.

Techniques: Derivative Assay, Modification, Binding Assay, Flow Cytometry, Variant Assay, Incubation, Standard Deviation

Journal: Advanced Science

Article Title: Screening and Identification of Novel DNA Aptamer for Targeted Delivery to Injured Podocytes in Glomerular Diseases

doi: 10.1002/advs.202412356

Figure Lengend Snippet: Binding of RLS‐2 to Injured Podocytes Analyzed by Confocal Microscopy. Binding ability of RLS‐2 to injured podocytes analyzed by confocal microscopy. Hoechst 33 342 staining (blue) indicates cell nuclei, while RLS‐2 is represented by green signals. Scale bars, 10 µm.

Article Snippet: The immortalized mouse podocyte cell line (MPC5) was donated by Professor Peter Mundel from Goldfinch Bio.

Techniques: Binding Assay, Confocal Microscopy, Staining

Journal: Advanced Science

Article Title: Screening and Identification of Novel DNA Aptamer for Targeted Delivery to Injured Podocytes in Glomerular Diseases

doi: 10.1002/advs.202412356

Figure Lengend Snippet: Characterization of RLS‐2: Binding Efficiency, Serum Stability, and Safety in Kidney Cell Lines. A) Binding assays of RLS‐2 with various human‐derived kidney cell lines, including human mesangial cells (HMC), human proximal tubular epithelial cells (HK‐2), human renal glomerular endothelial cells (HGREC), and human podocytes (HPC). HPC‐ADR, HPC‐PAN, and HPC‐HG represent injured human podocytes induced by different methods. B) Quantification of RLS‐2‐specific binding after subtraction of library fluorescence intensity. Error bars indicate mean ± standard deviation. n = 3. ** p < 0.01; *** p < 0.001. C) Cell viability of podocytes after 24 h treatment with scrambled RLS‐2 and RLS‐2, evaluated using the CCK‐8 assay. Error bars indicate mean ± standard deviation. D) Urinary albumin‐to‐creatinine ratio (UACR) in BALB/c mice following five consecutive days of aptamer administration (0.16 nmol g −1 body weight per day) via tail vein injection. Error bars indicate mean ± standard deviation. n = 3. ns, not significant.

Article Snippet: The immortalized mouse podocyte cell line (MPC5) was donated by Professor Peter Mundel from Goldfinch Bio.

Techniques: Binding Assay, Derivative Assay, Fluorescence, Standard Deviation, CCK-8 Assay, Injection

Journal: Advanced Science

Article Title: Screening and Identification of Novel DNA Aptamer for Targeted Delivery to Injured Podocytes in Glomerular Diseases

doi: 10.1002/advs.202412356

Figure Lengend Snippet: Cellular Uptake and Intracellular Trafficking of RLS‐2 in Podocytes: Effects of Time, Concentration, and Endocytic Pathways. A,B) Time‐dependent (A) and concentration‐dependent (B) cellular uptake of scrambled RLS‐2 and RLS‐2 in podocytes analyzed by flow cytometry. Data are presented as mean ± standard deviation. n = 3. C) Percentage of RLS‐2 uptake by injured podocytes at 4 and 37 °C following a 30 min pre‐treatment with endocytosis inhibitors (AML: amiloride, GEN: genistein, CPZ: chlorpromazine). Uptake in cells treated with DMSO was considered 100%. Error bars indicate mean ± standard deviation. n = 3. * , p < 0.05; ns, not significant. D) Representative confocal microscopy images showing the distribution of Cy5‐labeled aptamers (red) and their co‐localization with a lysosome marker (LysoTracker Green DND‐26, green) in injured MPC5 cells after a 60‐min incubation at 37 °C. Nuclei were counterstained with Hoechst 33 342 (blue). Scale bars, 10 µm.

Article Snippet: The immortalized mouse podocyte cell line (MPC5) was donated by Professor Peter Mundel from Goldfinch Bio.

Techniques: Concentration Assay, Flow Cytometry, Standard Deviation, Confocal Microscopy, Labeling, Marker, Incubation

Journal: Advanced Science

Article Title: Screening and Identification of Novel DNA Aptamer for Targeted Delivery to Injured Podocytes in Glomerular Diseases

doi: 10.1002/advs.202412356

Figure Lengend Snippet: Verification of RLS‐2 Distribution in Podocytes. A,B) Representative confocal images showing the co‐localization of RLS‐2 (red) with the podocyte marker synaptopodin (green). Nuclei were stained with DAPI (blue). Scale bars, 50 µm. C,D) Representative flow cytometry results showing the mean fluorescence intensity in podocytes and non‐podocytes from mice after tail vein injection of FAM‐labeled aptamer. E–F). Quantitative analysis of the mean fluorescence intensity ratio of podocyte to non‐podocyte. Error bars indicate mean ± standard deviation. n = 3. *** p < 0.001; ns, not significant. G,H) Representative flow cytometry results showing the mean fluorescence intensity in podocyte and non‐podocyte from kidney cell suspensions of different mouse groups after in vitro incubation with the FAM‐labeled aptamer. I,J) Quantitative analysis of the mean fluorescence intensity ratio of podocyte to non‐podocyte. Error bars indicate mean ± standard deviation. n = 3. *** p < 0.001; ns, not significant.

Article Snippet: The immortalized mouse podocyte cell line (MPC5) was donated by Professor Peter Mundel from Goldfinch Bio.

Techniques: Marker, Staining, Flow Cytometry, Fluorescence, Injection, Labeling, Standard Deviation, In Vitro, Incubation

Journal: Advanced Science

Article Title: Screening and Identification of Novel DNA Aptamer for Targeted Delivery to Injured Podocytes in Glomerular Diseases

doi: 10.1002/advs.202412356

Figure Lengend Snippet: Identification of RLS‐2 Target Protein and Knockdown efficiency of RLS‐2–siRNA Chimeras. A) Coomassie Brilliant Blue staining was used to analyze proteins separated by SDS‐PAGE, including input protein (Input), beads pull‐down protein (Beads), biotin‐conjugated scrambled RLS‐2 pull‐down protein (bio‐Scrambled RLS‐2), and biotin‐conjugated RLS‐2 pull‐down protein (bio‐RLS‐2). B) The spectrum score of the candidate proteins in the RLS‐2 pulldown group (Score (RLS‐2)) and the ratio of spectrum score (RLS‐2) to spectrum score (Scrambled‐RLS‐2). C) Representative images showing the co‐localization of RLS‐2 (green) and EPB41L5 (red). Nuclei were stained with DAPI (blue). Scale bars, 10 µm. D) Schematic representation of RLS‐2‐si Epb41l5 and RLS‐2‐si Mmp10 chimeras. E) Quantitative analysis of qRT‐PCR results showing the knockdown efficiency of the RLS‐2‐si Epb41l5 chimera (800 nM) in normal MPC5 cells after 24 h incubation. Saline, RLS‐2 (800 nM), and scrambled RLS‐2‐si Epb41l5 chimera (800 nM) were used as controls. Error bars indicate mean ± standard deviation. n = 3. * p < 0.05; ns, not significant. F) Quantitative analysis of qRT‐PCR results showing the knockdown efficiency at different concentrations of the RLS‐2‐si Epb41l5 chimera. 800 nM RLS was used as the control. Error bars indicate mean ± standard deviation. n = 3. *** p < 0.001. G) Quantitative analysis of qRT‐PCR results showing the knockdown efficiency of the RLS‐2‐si Mmp10 chimera (800 nM) in normal MPC5 cells. Saline, RLS‐2 (800 nM), and scrambled RLS‐2‐si Mmp10 chimera (800 nM) were used as controls. Error bars indicate mean ± standard deviation. n = 3. *** p < 0.001; ns, not significant.

Article Snippet: The immortalized mouse podocyte cell line (MPC5) was donated by Professor Peter Mundel from Goldfinch Bio.

Techniques: Knockdown, Staining, SDS Page, Quantitative RT-PCR, Incubation, Saline, Standard Deviation, Control