Comparison of the DNA concentrations.

<p>Comparison of the DNA concentration in the tissues form the intact (Control) and denuded HAMs with TrypLE Express (TrypLE), trypsin/EDTA and thermolysin treatment directly after de-epithelialization. Each bar represents mean ± SD from 3 determinations (***<i>P</i> < 0.001).</p

Immunostaining of BM.

<p>Distribution of BM collagen type IV α2 chain (green; A) or laminin α5 (green; B) in intact (Control) and denuded HAM: TrypLE Express, trypsin/EDTA, thermolysin treatment. Intact HAM (primary antibody omitted), was used as negative control. Cell nuclei were stained with the propidium iodide (red). Scale bar represents 100 μm.</p

The RT-PCR analysis of hAECs.

<p>The RT-PCR analysis of hAECs after de-epithelialization and each passage (P0-P5). The iPS cells were used as a positive (iPS) and corneal fibroblasts as negative control (CF). Sample without cDNA (NTC) was used as non-template control. One representative experiment of 3 (with identical results) is shown.</p

The viability of hAECs.

<p>Comparison of the hAECs viability after TrypLE Express, trypsin/EDTA and thermolysin treatment. Cells were stained with trypan blue and counted via hemocytometer. Each bar represents mean ± SD from 15 determinations (***<i>P</i> < 0.001).</p

Comparison of the intact and denuded HAMs and HAM cryosections.

<p>Comparison of the intact (Control) and denuded HAMs (A) and HAM cryosections (B) after TrypLE Express, trypsin/EDTA and thermolysin treatment stained with H/E for light microscopy. Scale bar represents 100 μm.</p

The morphology of hAECs.

<p>The comparison of morphology of cultured hAECs after trypsin/EDTA treatment in complete DMEM medium. The cells for the light microscopy were photographed before each passage (after de-epithelialization, before 1^st, 2^nd, 3^rd, 4^th and 5^th passage). Results of one out of 3 identical experiment is shown. Scale bars represent 100 μm.</p

The metabolic activity of hAECs.

<p>Comparison of metabolic activity of the epithelial cells unstimulated (Uns) and stimulated with EGF (EGF) after each passage. WST-1 reagent was added to the cell cultures for 4 h to form formazan. The absorbance was measured at a wave-length of 450 nm. Each bar represents mean ± SD from 3 determinations.</p

Primer sequences used for real-time PCR.

<p>Primer sequences used for real-time PCR.</p

Effect of Polycation Structure on Interaction with Lipid Membranes

Interaction of polycations with lipid membranes is a very important issue in many biological and medical applications such as gene delivery or antibacterial usage. In this work, we address the influence of hydrophobic substitution of strong polycations containing quaternary ammonium groups on the polymer–zwitterionic membrane interactions. In particular, we focus on the polymer tendency to adsorb on or/and incorporate into the membrane. We used complementary experimental and computational methods to enhance our understanding of the mechanism of the polycation–membrane interactions. Polycation adsorption on liposomes was assessed using dynamic light scattering (DLS) and zeta potential measurements. The ability of the polymers to form hydrophilic pores in the membrane was evaluated using a calcein-release method. The polymer–membrane interaction at the molecular scale was explored by performing atomistic molecular dynamics (MD) simulations. Our results show that the length of the alkyl side groups plays an essential role in the polycation adhesion on the zwitterionic surface, while the degree of substitution affects the polycation ability to incorporate into the membrane. Both the experimental and computational results show that the membrane permeability can be dramatically affected by the amount of alkyl side groups attached to the polycation main chain