Surface Investigation on Biomimetic Materials to Control Cell Adhesion: The Case of RGD Conjugation on PCL

The cell recognition of bioactive ligands immobilized on polymeric surfaces is strongly dependent on ligand presentation at the cell/material interface. While small peptide sequences such as Arg−Gly−Asp (RGD) are being widely used to obtain biomimetic interfaces, surface characteristics after immobilization as well as presentation of such ligands to cell receptors deserve more detailed investigation. Here, we immobilized an RGD-based sequence on poly(ε-caprolactone) (PCL), a largely widespread polymeric material used in biomedical applications, after polymer aminolysis. The surface characteristics along with the efficacy of the functionalization was monitored by surface analysis (FTIR-ATR, contact angle measurements, surface free energy determination) and spectrophotometric assays specially adapted for the analytical quantification of functional groups and/or peptides at the interface. Particular attention was paid to the evaluation of a number, morphology, and penetration depth of immobilized functional groups and/or peptides engrafted on polymeric substrates. In particular, a typical morphology in peptide distribution was evidenced on the surface raised from polymer crystallites, while a significant penetration depth of the engrafted molecules was revealed. NIH3T3 fibroblast adhesion studies verified the correct presentation of the ligand with enhanced cell attachment after peptide conjugation. Such work proposes a morphological and analytical approach in surface characterization to study the surface treatment and the distribution of ligands immobilized on polymeric substrates

Inhibition of adhesion of WM266 on different extracellular matrix coated plates (A).

<p>Cells were pre-incubated with peptides (50 µM) or anti-αvβ3 antibody (10 µg/mL) for 30 min at 4°C and then seeded on extracellular matrix pre-coated plates. Cell adhesion was evaluated after 1 h of incubation using crystal violet reagent. The results are presented as the percentage of adherent cells respect to the control (untreated cells) and are expressed as means ± SE of three independent experiments performed in triplicate. Statistical significance was analyzed using Student's t test, unpaired, two-sided (*p<0.05). RGDechi-hCit dose-effect on WM266 cell adhesion (B). Cells were pre-incubated with increasing concentrations of RGDechi-hCit for 30 min at 4°C and then seeded on vitronectin or fibronectin (10 µg/mL) pre-coated plates at 37°C. The cell adhesion was evaluated after 1 h of incubation using crystal violet reagent. The results are presented as the percentage of adherent cells respect to the control (untreated cells) and are expressed as mean ± DS from three independent experiments performed in triplicate.</p

Detachment of WM266 cells from vitronectin coated plates.

<p>Cells were plated onto vitronectin (10 µg/mL) pre-coated wells and were allowed to adhere for 1 h. Peptides (50 µM) or anti-αvβ3 antibody (10 µg/mL) were added and the incubation was continued for 2 h. Cell adhesion was determined by crystal violet assay. Results are presented as percentage of adherent cells respect to the control (untreated cells) and are expressed as means ± SE of three independent experiments performed in triplicate. Statistical significance was analyzed using Student's t test, unpaired, two-sided (* p<0.05).</p

Merge of confocal and transmitted light images of peptide cellular uptake.

<p>WM266 cells after 30 min incubation with 50 µM RGDechi-hCit (A) and scrambled peptide(B). HeLa cells after 30 minute incubation with 50 µM RGDechi-hCit (C). Green fluorescence indicates peptides. Magnification bar: 20 µm.</p

Cytotoxicity assay.

<p>WM266 were treated with starvation conditions (4% FCS) for 4 h and then peptides at different concentrations were added and incubated for 24 h. The proliferation was evaluated using crystal violet assay. The results are presented as the percentage of proliferating cells respect to the control (untreated cells) and are expressed as means ± SE of three independent experiments performed in triplicate. Statistical significance was analyzed using Student's t test, unpaired, two-sided (* p<0.05, **p<0.01).</p

RGDechi cellular uptake inhibition experiments.

<p>A–F, Confocal microscope images of WM266 cells after 30 minutes of incubation with 50 µM RGDechi. A and D control non-treated cells; B and E cells treated with 0.45 M sucrose; C and F cells treated with 5 µg/ml filipin. A–C fluorescence images; D–F merge of fluorescence and transmitted light images. Magnification bar 50 µm. G, quantitative analysis of RGDechi cellular uptake by spectrofluorimeter measurements. Data are expressed as mean ± standard deviation (* p<0.05).</p

Co-localization areas (white arrows) of RGDechi-hCit peptide (green) with αvβ3 integrin (red)(A), caveolin 1 (red)(B), clathrin (red)(C) and LAMP 2 (red)(D) in WM266 cells after 30 minute incubation.

<p>(A, C, E, G) fluorescence images; (B, D, F, H) transmitted and fluorescence image overlapping. Magnification bar: 20 µm.</p

Apoptosis analyses with annexin V-FITC/PI double staining on WM266 cells.

<p>Untreated cells (A); cilengitide treated cells (B); RGDechi-hCit treated cells (C). Upper left quadrant: necrotic cells; upper right: advanced apoptotic cells; lower left: viable cells; lower right: early apoptotic cells. These pictures are representative of three independent experiments.</p

Inter-Organelle Contact Sites Mediate the Intracellular Antioxidant Activity of Platinum Nanozymes: A New Perspective on Cell–Nanoparticle Interaction and Signaling

The catalytic and antioxidant properties of platinum nanoparticles (PtNPs) make them promising candidates for several applications in nanomedicine. However, an open issue, still shared among most nanomaterials, is the understanding on how internalized PtNPs, which are confined within endo-lysosomal compartments, can exert their activities. To address this problem, here we study the protective effect of 5 nm PtNPs on a human hepatic (HepG2) cell line exposed to dichlorodiphenylethylene (DDE) as a model of oxidative stress. Our results indicate that PtNPs are very efficient to reduce DDE-induced damage in HepG2 cells, in an extent that depends on DDE dose. PtNPs can contrast the unbalance of mitochondrial dynamics induced by DDE and increase the expression of the SOD2 mitochondrial enzyme that recovers cells from oxidative stress. Interestingly, in cells treated with PtNPsalone or in combination with DDEmitochondria form contact sites with a rough endoplasmic reticulum and endo-lysosomes containing nanoparticles. These findings indicate that the protective capability of PtNPs, through their intrinsic antioxidant properties and modulating mitochondrial functionality, is mediated by an inter-organelle crosstalk. This study sheds new light about the protective action mechanisms of PtNPs and discloses a novel nano-biointeraction mechanism at the intracellular level, modulated by inter-organelle communication and signaling