Journal: Pharmaceutics

Article Title: Targeted Therapy of Hepatocellular Carcinoma Using Gemcitabine-Incorporated GPC3 Aptamer

doi: 10.3390/pharmaceutics12100985

Figure Lengend Snippet: In vitro affinity and specificity of G12msi aptamers. ( a ) The specific binding capacity of G12msi to HCC cells was assessed by using flow cytometry. GPC3-positive HepG2, Hep3B, and Huh7 cell lines, and GPC3-negative A431 cells were stained with Cy5-labeled G12msi or scrambled G12msi. ( b ) Confocal microscopy analysis with Cy5-labeled G12msi or scrambled G12msi aptamers (Red) on GPC3 positive HepG2, Hep3B, Huh7 cells, and GPC3 negative A431 cells at 4 °C. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bars, 20 μm.

Article Snippet: G12msi, scrambled G12msi, and Cy5-labeled G12msi aptamers were provided by Aptamer Sciences.

Techniques: In Vitro, Binding Assay, Flow Cytometry, Staining, Labeling, Confocal Microscopy

Journal: Pharmaceutics

Article Title: Targeted Therapy of Hepatocellular Carcinoma Using Gemcitabine-Incorporated GPC3 Aptamer

doi: 10.3390/pharmaceutics12100985

Figure Lengend Snippet: Internalization and intracellular localization of G12msi aptamers. ( a ) Representative z-stack images with orthogonal side-views of HepG2, Hep3B, Huh7, and A431 cells treated with Cy5-labeled G12msi or scrambled G12msi aptamers (red) at 37 °C. Nuclei were stained with DAPI (blue). ( b ) The cellular trafficking of G12ms and G12msi aptamers in HepG2 cells. Representative images showing the intracellular colocalization of the Cy5-labeled G12ms and G12msi aptamers (red) with a lysosomal marker (LysoTracker™ Green DND-26; green) by using confocal microscopy. The nuclei were counterstained with DAPI (blue). Scale bars, 20 μm.

Article Snippet: G12msi, scrambled G12msi, and Cy5-labeled G12msi aptamers were provided by Aptamer Sciences.

Techniques: Labeling, Staining, Marker, Confocal Microscopy

Journal: Advanced Healthcare Materials

Article Title: Urine‐Stable Aptamer‐Conjugated Gold Nanorods for the Early Detection of High‐Grade Bladder Cancer Residual Disease

doi: 10.1002/adhm.202403314

Figure Lengend Snippet: Features of the aptamer and tested bladder cancer cell lines. A) The secondary structure of H02 full‐length and Apt‐Itg (nucleotides from 18 to 51, boxed); structures of the H02 full‐length and Apt‐Itg were predicted by using Mfold web server, and the calculated change in Gibbs free energy (ΔG) values are reported. B) AlphaFold 3 molecular docking result of integrin α5β1‐Apt‐Itg binding complex; Apt‐Itg, integrin α5 and integrin β1 predicted structures are shown in yellow, light blue and red, respectively. The predicted interaction of Apt‐Itg with integrin α5, involving D154 and Y208 residues, is framed on the side. C) The expression of integrin α5β1 by human and murine bladder cancer cell lines evaluated by western blot analysis of the α5 and β1 chain of the integrin. The expression of the housekeeping protein α‐tubulin was used to estimate the relative fold of expression of integrin chains D). MB49‐Luc; murine bladder cancer cell line.

Article Snippet: The SH‐terminated 2′Fluoro‐Pyrimidine (2′F‐Py)‐RNA aptamer targeting integrin α5β1 (Apt‐Itg) and unrelated aptamer were purchased from Metabion (Martinsried, Germany).

Techniques: Binding Assay, Expressing, Western Blot

Journal: Advanced Healthcare Materials

Article Title: Urine‐Stable Aptamer‐Conjugated Gold Nanorods for the Early Detection of High‐Grade Bladder Cancer Residual Disease

doi: 10.1002/adhm.202403314

Figure Lengend Snippet: Binding assay of Alexa 647‐Apt‐Itg to human and murine bladder cancer cells using flow cytometry. A–C) Representative binding profiles of increasing concentrations of Alexa 647‐Apt‐Itg and Unrelated aptamer incubated with T24 (A), RT112 (B) and MB49‐Luc (C) cells. Apparent dissociation curves of aptamer‐cell interaction and Kd value of Apt‐Itg is shown. Data are presented as the mean ± SD. D) Competition binding assay by flow cytometry (left) and quantification of the gMFI (right) using 1 µ m Alexa 647‐Apt‐Itg in the absence (blue) or in the presence (red) of 30‐fold excess of unlabeled unrelated aptamer.

Article Snippet: The SH‐terminated 2′Fluoro‐Pyrimidine (2′F‐Py)‐RNA aptamer targeting integrin α5β1 (Apt‐Itg) and unrelated aptamer were purchased from Metabion (Martinsried, Germany).

Techniques: Binding Assay, Flow Cytometry, Incubation

Journal: Advanced Healthcare Materials

Article Title: Urine‐Stable Aptamer‐Conjugated Gold Nanorods for the Early Detection of High‐Grade Bladder Cancer Residual Disease

doi: 10.1002/adhm.202403314

Figure Lengend Snippet: Apt‐Itg actively internalizes into T24 bladder cancer cells. A) Representative confocal images of T24 cells incubated for indicated times at 4 °C or 37 °C with 4 µm Alexa 647‐Apt‐Itg or Alexa 647‐Unrelated aptamer as a negative control. Aptamer, WGA‐488 (cell surface), LysoTracker (lysosomes), and DAPI (nuclei) are visualized in red, green, blue, and gray, respectively. All digital images were captured at the same setting to allow direct comparison of staining patterns. Magnification 63x, 1.0x digital zoom, scale bar = 10 µm. B) Plot of MFI of Alexa 647‐Apt‐Itg normalized to cell number. Bars depict mean ± SD. *** p < 0.001.

Article Snippet: The SH‐terminated 2′Fluoro‐Pyrimidine (2′F‐Py)‐RNA aptamer targeting integrin α5β1 (Apt‐Itg) and unrelated aptamer were purchased from Metabion (Martinsried, Germany).

Techniques: Incubation, Negative Control, Comparison, Staining

Journal: Advanced Healthcare Materials

Article Title: Urine‐Stable Aptamer‐Conjugated Gold Nanorods for the Early Detection of High‐Grade Bladder Cancer Residual Disease

doi: 10.1002/adhm.202403314

Figure Lengend Snippet: Integrin α5 silencing results in reduced Apt‐Itg binding. A) T24, RT112, and MB49‐Luc cells were left untreated (NT) or transfected with the indicated Integrin α5 siRNAs or siRNA ctrl. At 24 h post‐transfection, cells were harvested, and cell lysates prepared and immunoblotted with anti‐integrin α5 or anti‐integrin β1 antibody. Equal loading was confirmed by immunoblot with anti‐α‐tubulin antibody. The histograms indicate the integrin chains/α‐tubulin ratio of the densitometric signals. Values are shown relative to siRNA ctrl, arbitrarily set to 1. B) Binding of 1 µ m Alexa647‐Apt‐Itg to human and murine bladder cancer cells following 24 h‐transfection with si‐ITGα5 (blue) and siRNA ctrl (red). C) Binding of Alexa488‐Ab‐Itg to T24 cells 24 h post‐transfection. (B, C) The histogram indicates gMFI of aptamer‐ or antibody‐treated cells normalized to the gMFI of siRNA ctrl untreated cells (black), arbitrarily set to 1. Bars depict mean ± SD of two independent experiments. * p < 0.005, ** p < 0.001, and *** p < 0.0001.

Article Snippet: The SH‐terminated 2′Fluoro‐Pyrimidine (2′F‐Py)‐RNA aptamer targeting integrin α5β1 (Apt‐Itg) and unrelated aptamer were purchased from Metabion (Martinsried, Germany).

Techniques: Binding Assay, Transfection, Western Blot

Journal: Advanced Healthcare Materials

Article Title: Urine‐Stable Aptamer‐Conjugated Gold Nanorods for the Early Detection of High‐Grade Bladder Cancer Residual Disease

doi: 10.1002/adhm.202403314

Figure Lengend Snippet: Characterization of the final nanosystem, GNRs@Chit‐Apt‐Itg. A) Scheme representative of the aptamer Apt‐Itg conjugated to the GNRs@Chit via PEG 12 linker. B) TEM images of nanoparticles. C) Left , representative PCR calibration curve with unconjugated Apt‐Itg (red); samples from TOP, 1/2 and 1/10 GNRs@Chit‐Apt‐Itg preparations are shown in blue, green, and black, respectively. Right , quantification of the Apt‐Itg and unrelated aptamer loaded onto GNRs@Chitosan; data shown mean ± SD of three independent experiments. D) UV–vis spectra of GNRs@CTAB, GNRs@Chit, and GNRs@Chit‐Apt‐Itg/SCR showing no significant differences in the absorption spectrum of GNRs after surface modification; FAAS analysis of GNRs@Chit‐Apt‐Itg with calibration line; thermogravimetric analysis of GNRs@Chit in N 2 and air atmosphere (above 600 °C). GNRs@Chit‐Apt‐Itg stability in human urine was investigated by E) VIS‐NIR spectra of GNRs@Chit‐Apt‐Itg compared to the VIS‐NIR spectra of GNRs@CTAB and GNRs@Chit, and F) VIS‐NIR spectra and absorption intensity of GNRs@Chit‐Apt‐Itg in the presence of human urine over time. G) Binding of GNRs@Chit‐Apt‐Itg to MB49 cells after 15 min of incubation, followed by cell internalization of GNRs and accumulation into lysosome after 4 h. SCR: unrelated aptamer.

Article Snippet: The SH‐terminated 2′Fluoro‐Pyrimidine (2′F‐Py)‐RNA aptamer targeting integrin α5β1 (Apt‐Itg) and unrelated aptamer were purchased from Metabion (Martinsried, Germany).

Techniques: Modification, Binding Assay, Incubation

Journal: Cancers

Article Title: Are Integrins Still Practicable Targets for Anti-Cancer Therapy?

doi: 10.3390/cancers11070978

Figure Lengend Snippet: Selected clinical trials of agents targeting integrins. Non-exhaustive listing of the recent most important clinical studies with integrin inhibitors and their salient features and results).

Article Snippet: Volociximab (MEDI-522, anti α5β1 integrin antibody) (AbbVie): total trials 7 (+ 3 discontinued early) , , , , , , .

Techniques: Clinical Proteomics, Control, Activity Assay, Comparison

Journal: The Febs Journal

Article Title: Neuropilin‐1 is required for endothelial cell adhesion to soluble vascular endothelial growth factor receptor 1

doi: 10.1111/febs.16119

Figure Lengend Snippet: Flt 2‐11 peptide does not interfere with NRP‐1 binding to either α5β1 integrin or semaphorin 3A. SPR analysis of NRP‐1 interaction with α5β1 integrin or semaphorin 3A, in the absence or presence of Flt 2‐11 peptide or ‘scrambled’ Flt 2‐11 . NRP‐1 is immobilized on sensor chip. (A) α5β1 integrin is injected at different concentrations (from bottom to top: 20 n m , cyan; 40 n m , red; 100 n m , green; 200 n m , blue; and 500 n m , black). Kinetic and thermodynamic parameters are: k on = 2.1 × 10 4 M −1 ·s −1 ; k off = 2.9 × 10 −4 s −1 ; K D = 14 ± 4 n m . (B) α5β1 integrin is injected at 200 n m concentration together with either Flt 2‐11 peptide or scrambled Flt 2‐11 peptide at different concentrations (no peptides, black; Flt 2‐11 peptide 2 M μ and 20 μ m , blue and cyan, respectively; scrambled Flt 2‐11 peptide at 2 μ m and 20 μ m , red and purple, respectively). (C) Semaphorin 3A is injected at different concentrations (from bottom to top: 20 n m , cyan; 30 n m , red; 50 n m , green; 200 n m , blue; and 500 n m , black). Interaction parameters: k on = 3.7 × 10 3 M −1 ·s −1 ; k off = 9 × 10 −5 s −1 ; K D = 24 ± 6 n m . (D) Semaphorin 3A is injected at 200 n m , and peptide Flt 2‐11 at different concentrations (no peptides, black; Flt 2‐11 peptide 1 μ m and 10 μ m blue and cyan, respectively; scrambled Flt 2‐11 peptide 50 μ m , red).

Article Snippet: Purified α5β1 integrin (CC1052), octyl‐β‐ d ‐glucopyranoside formulation, was from Merck Millipore (Billerica, MA, USA).

Techniques: Binding Assay, Injection, Concentration Assay

Journal: The Febs Journal

Article Title: Neuropilin‐1 is required for endothelial cell adhesion to soluble vascular endothelial growth factor receptor 1

doi: 10.1111/febs.16119

Figure Lengend Snippet: Evaluation of RNA interference for NRP‐1 in HUVECs. (A) EC adhesion to either sVEGFR‐1 or fibronectin (FN) in the presence of Flt 2‐11 peptide or scrambled (Scr) Flt 2‐11 peptide or in the absence of any peptide (No add). Results are expressed as percentage of basal EC adhesion to each protein with respect to the control (No add). Student’s t ‐test: ** P < 0.01. (B) Real‐time RT‐PCR on total RNA extracted from ECs, either interfered for NRP‐1 expression (shRNA NRP‐1) or transfected with a shRNA for an unrelated, not‐targeted gene (shRNA NT). Results are reported as relative fold expression with respect to shRNA NT‐transfected cells and represent the mean ± SE of experiments performed in triplicate out of three independent determinations. (C) FACS analysis was performed to determine integrin α5β1 and NRP‐1 levels in ECs interfered for NRP‐1 expression (shRNA NRP‐1) and transfected with a control shRNA (shRNA NT). Panels show the results of a representative experiment, and histograms indicate the quantification of α5β1 and NRP‐1 expression, evaluated as variations in the geometric mean fluorescence intensity. Results represent the mean values ± SE of four independent determinations. In B and C, Student’s t ‐test: * P ≤ 0.05, ** P ≤ 0.01.

Article Snippet: Purified α5β1 integrin (CC1052), octyl‐β‐ d ‐glucopyranoside formulation, was from Merck Millipore (Billerica, MA, USA).

Techniques: Control, Quantitative RT-PCR, Expressing, shRNA, Transfection, Fluorescence

Journal: The Febs Journal

Article Title: Neuropilin‐1 is required for endothelial cell adhesion to soluble vascular endothelial growth factor receptor 1

doi: 10.1111/febs.16119

Figure Lengend Snippet: Availability of peptide 12 residues for interaction. SASA (Å 2 ) of peptide 12 residues in the isolated peptide 12 and in the context of the 3D structure of the second Ig‐like domain of VEGFR‐1 (PDB identifier: 1FLT, Resolution: 1.7 Å). a.a.: amino acid type, three‐letter code; Nb: residue number in the PDB file; chain x, chain y: name of the chains of the two sVEGFR‐1 Ig‐like domain 2 copies present in the experimental structure; Peptide, Domain and Diff: SASA of each residue and of the whole peptide 12 in the free state and in the context of the whole domain, and difference between these values, respectively. Residues previously shown to play an essential role in EC adhesion and/or α5β1 integrin binding to sVEGFR‐1 [ 34 ] are bold.

Article Snippet: Purified α5β1 integrin (CC1052), octyl‐β‐ d ‐glucopyranoside formulation, was from Merck Millipore (Billerica, MA, USA).

Techniques: Isolation, Residue, Binding Assay

Journal: The Febs Journal

Article Title: Neuropilin‐1 is required for endothelial cell adhesion to soluble vascular endothelial growth factor receptor 1

doi: 10.1111/febs.16119

Figure Lengend Snippet: sVEGFR‐1/α5β1 integrin interaction. (A) SPR analysis of the interaction between sVEGFR‐1 and α5β1 integrin. sVEGFR‐1 is immobilized on sensor chips and α5β1 integrin is injected at different concentrations (from bottom to top: 80 n m , cyan; 160 n m , red; 350 n m , green; 600 n m , blue; and 1 μ m , black). Interaction parameters: k on = 1.5 × 10 3 M −1 ·s −1 ; k off = 2.9 × 10 −4 s −1 ; K D = 195 ± 40 n m . (B) Secondary structure representation of the 3D structure of VEGFR‐1 Ig‐like domain II. β‐strands are represented by arrows and labelled from A to H; loops are shown as tubular ribbons; the N‐ and C‐terminal ends of the domain are indicated by labels. Colour coding: peptide 12 (interacting with α5β1 integrin) is blue; Flt 2‐11 peptide (interacting with NRP‐1) is yellow in the five‐residue NITVT region corresponding to the Flt 2‐5 peptide (see text), and green in the remaining six‐residues region; the rest of the domain is magenta. (C) Proposed mechanism of action of Flt 2‐11 peptide. Left: Light blue arrows indicate the high‐affinity interactions: (i) between NRP‐1 (yellow) and α5β1 integrin (black) on the EC membrane; and (ii) between NRP‐1 on the EC membrane and the sVEGFR‐1 substrate (green). The dark blue arrow indicates the low‐affinity interaction between α5β1 integrin on the EC membrane and the sVEGFR‐1 substrate. The low‐affinity interaction is stabilized by the two high‐affinity interactions, and the angiogenic stimulus is triggered. Centre: The Flt 2‐11 peptide inhibits the high‐affinity interaction between NRP‐1 on the EC membrane and the sVEGFR‐1 substrate. The low‐affinity interaction between α5β1 integrin on ECs and the sVEGFR‐1 substrate is not sufficiently stable to trigger the angiogenic stimulus that starts upon integrin engagement. Right: The affinity of the interaction between α5β1 integrin (black) on the EC membrane and the P12 peptide substrate (light blue arrow) is high enough that additional stabilizing interactions are not required. Consequently, the α5β1 integrin/P12 peptide interaction takes place both in the presence and in the absence of the Flt 2‐11 peptide.

Article Snippet: Purified α5β1 integrin (CC1052), octyl‐β‐ d ‐glucopyranoside formulation, was from Merck Millipore (Billerica, MA, USA).

Techniques: Injection, Residue, Membrane

Journal: Acta biomaterialia

Article Title: Interplay Between Degradability and Integrin Signaling on Mesenchymal Stem Cell Function within Poly(ethylene glycol) Based Microporous Annealed Particle Hydrogels

doi: 10.1016/j.actbio.2019.11.009

Figure Lengend Snippet: Design and characterization of MAP hydrogels with varying degradability. a) A schematic of MAP hydrogels assembled from off-stoichiometric PEG microgels via a secondary thiol-ene photopolymerization. b) Peptide sequences designed as crosslinkers and cell-adhesive ligands to achieve varying degradability and integrin binding. c) A representative Z-stack projection image of MAP hydrogels labeled with Alexa Fluor 488- succinimidyl ester illustrating the internal porous structure. Scale bar is 100 μm. d) Degradation curves of non-deg, slow-deg and fast-deg MAP hydrogels in a 0.2 mg/mL collagenase solution at 37 °C. e) Storage modulus of slow-deg and fast deg MAP hydrogels after degradation with 0%, 33%, and 67% mass loss.

Article Snippet: The α5β1 integrin targeting peptide c(RRETAWA) was purchased from AAPPtec and was synthesized via an on-resin cyclization reaction of Ac-CAhxK(Alloc)RRETAWAE(ODmab), as previously described by Gandavarapu et al. [ 30 ].

Techniques: Binding Assay, Labeling

Journal: Acta biomaterialia

Article Title: Interplay Between Degradability and Integrin Signaling on Mesenchymal Stem Cell Function within Poly(ethylene glycol) Based Microporous Annealed Particle Hydrogels

doi: 10.1016/j.actbio.2019.11.009

Figure Lengend Snippet: The effect of degradability on hMSC spreading and proliferation in c(RRETAWA)-functionalized MAP hydrogels. a) Maximum intensity Z- projection of cytoskeleton staining of hMSCs cultured in MAP hydrogels after 2 and 8 days. Green represents F- actin and blue represents nuclei. Scale bars are 100 μm. b) Quantification of cell number. c) Quantification of cell volume. * comparison factor: degradability; # comparison factor: time. ** indicates p < 0.01, *** and ### indicate p < 0.0001, Two-way ANOVA and Tukey’s multiple comparisons test.

Article Snippet: The α5β1 integrin targeting peptide c(RRETAWA) was purchased from AAPPtec and was synthesized via an on-resin cyclization reaction of Ac-CAhxK(Alloc)RRETAWAE(ODmab), as previously described by Gandavarapu et al. [ 30 ].

Techniques: Staining, Cell Culture

Journal: Acta biomaterialia

Article Title: Interplay Between Degradability and Integrin Signaling on Mesenchymal Stem Cell Function within Poly(ethylene glycol) Based Microporous Annealed Particle Hydrogels

doi: 10.1016/j.actbio.2019.11.009

Figure Lengend Snippet: ECM proteins, collagen type I and fibronectin synthesized by hMSCs in MAP hydrogels with varying degradability and integrin-binding peptides after 8 days of culture. a) Z-projection images from confocal microscopy. Scale bars are 100 μm. b) Quantification of total ECM protein per 50 μL MAP hydrogels. * comparison factor: degradability; # comparison factor: integrin-binding peptide. * indicates p < 0.05, *** and ### indicate p < 0.0001, Two-way ANOVA by Tukey’s multiple comparisons test.

Article Snippet: The α5β1 integrin targeting peptide c(RRETAWA) was purchased from AAPPtec and was synthesized via an on-resin cyclization reaction of Ac-CAhxK(Alloc)RRETAWAE(ODmab), as previously described by Gandavarapu et al. [ 30 ].

Techniques: Synthesized, Binding Assay, Confocal Microscopy

Journal: Acta biomaterialia

Article Title: Interplay Between Degradability and Integrin Signaling on Mesenchymal Stem Cell Function within Poly(ethylene glycol) Based Microporous Annealed Particle Hydrogels

doi: 10.1016/j.actbio.2019.11.009

Figure Lengend Snippet: hMSC expression of OPG in RGDS and c(RRETAWA)-functionalized MAP hydrogels with varying degradability after 2, 5, and 8 days of culture. Three-way ANOVA results: time (p < 0.0001), time×degradability (p < 0.0001), integrin-binding peptide×degradability (p < 0.05).

Article Snippet: The α5β1 integrin targeting peptide c(RRETAWA) was purchased from AAPPtec and was synthesized via an on-resin cyclization reaction of Ac-CAhxK(Alloc)RRETAWAE(ODmab), as previously described by Gandavarapu et al. [ 30 ].

Techniques: Expressing, Binding Assay

Journal: Acta biomaterialia

Article Title: Interplay Between Degradability and Integrin Signaling on Mesenchymal Stem Cell Function within Poly(ethylene glycol) Based Microporous Annealed Particle Hydrogels

doi: 10.1016/j.actbio.2019.11.009

Figure Lengend Snippet: hMSC secretion of a, b) VEGF and c, d) BMP2 in MAP hydrogels functionalized with RGDS and c(RRETAWA) and with varying degradability after 8 days of culture. Data presented are both before and after normalization. * comparison factor: degradability; # comparison factor: integrin-binding peptide. * and # indicate p < 0.05, ## indicates p < 0.01, *** indicates p < 0.0001, Two-way ANOVA by Tukey’s multiple comparisons test.

Article Snippet: The α5β1 integrin targeting peptide c(RRETAWA) was purchased from AAPPtec and was synthesized via an on-resin cyclization reaction of Ac-CAhxK(Alloc)RRETAWAE(ODmab), as previously described by Gandavarapu et al. [ 30 ].

Techniques: Binding Assay