Layer-by-Layer Enzymatic Platform for Stretched-Induced Reactive Release

An original “all-in-one” platform combining polymers, enzymes, and enzymatic substrates in a unique film is designed. A polymeric barrier stratum prevents any contact between enzymes adsorbed on top of the film and substrates loaded in an underlying reservoir. Upon stretching of the film, a continuous diffusion of substrates through the barrier is triggered, followed by a catalytic reaction. This leads to the formation of products that are released from the film. This new platform acts as a stretch-induced reactive release system and emerges as an innovative concept in mechano-responsive materials

Mobility of Proteins in Highly Hydrated Polyelectrolyte Multilayer Films

The lateral diffusion of a protein (human serum albumin labeled with fluorescein isothiocyanate) within a highly hydrated polyelectrolyte film is studied. The film is built up with poly­(l-lysine) as polycation and hyaluronate as polyanion. Fluorescence recovery after photobleaching is used to evaluate the mobility of the labeled protein. Spatial Fourier transformation is applied to the fluorescence intensity recorded at various times after bleaching of a narrow rectangular area within an image representative of the film. This approach necessitates no hypothesis on the intensity distribution at the end of the bleaching provided that the bleach has not appreciably changed the concentration ratios of the different diffusing species. Furthermore, under the hypothesis that molecules move according to Fick’s law, we represent the Fourier transform by a weighted sum of exponentials each containing another diffusion coefficient and evaluate the proportion attached to each term of this sequence using the simulated annealing method. A criterion, combining goodness-of-fit and the entropy characterizing the diffusion coefficient spectrum, is proposed to avoid overinterpretation of the experimental data. The optimum spectrum of the diffusion coefficient is then extracted from the time evolution of the light intensity at various albumin concentrations within the films. It appears that the mobility, quantified by the amount of tracer molecules having a diffusion coefficient smaller than, e.g., 0.1 μm²/s, undergoes a transition between 20 and 2000 μg/mL of internal concentration. This suggests that the mutual interactions of the albumin molecules and the interactions between fluorescently labeled albumin and the film network become increasingly important in the reduction of the albumin mobility as the albumin concentration increases

Phenotype stability under hypoxia.

<p>After the third passage, the smooth muscle cells phenotype stability of differentiated cell cultivated under hypoxic conditions was investigated by confocal microscopy observation (A) and flow cytometry analyses (B, C). A: Confocal microscopic observations showed positive cells for contractile markers: α- Smooth Muscle Actin (α-SMA), Smooth Muscle Myosin Heavy Chain (SM-MHC) and Calponin confluence on both coated surfaces (type I collagen and Polyelectrolyte Multilayer films (PEMs)). Objective×40, NA = 0.8, scale bars 75 µm. B: Flow cytometry showed that more than 80% cells expressed SMCs markers. C: Mean fluorescence intensity analyses showed a higher SMCs contractile markers expression for differentiated cells compared to control (mature SMCs) whatever the surface coating. (§)PEMs <i>versus</i> control, (*) Collagen <i>versus</i> control, (#) PEMs <i>versus</i> collagen. (§,* and #: <i>p</i><0.05 and §§§ and ***: <i>p</i><0.001).</p

Phenotype stability under normoxia.

<p>After the third passage, the smooth muscle cells phenotype stability of differentiated cell cultivated under normoxic conditions was investigated by confocal microscopy observation (A) and flow cytometry analyses (B, C). A: Microscopical observations show positive cells for contractile markers: α- Smooth Muscle Actin (α-SMA), Smooth Muscle Myosin Heavy Chain (SM-MHC) and Calponin confluence on both coated surfaces (type I collagen and Polyelectrolyte Multilayer films (PEMs)). Objective×40, NA = 0.8, scale bars 75 µm. B: Flow cytometry showed that about 90% cells expressed SMCs markers. C: Mean fluorescence intensity analyses showed a higher SMCs contractile markers expression for differentiated cells compared to control (mature SMCs) whatever the surface coating. (§) PEMs <i>versus</i> control, (*) Collagen <i>versus</i> control. (§ and *: <i>p</i><0.05, §§ and **: <i>p</i><0.01, and *** <i>p</i><0.001).</p

Vascular cell phenotype characterization.

<p>The endothelial cell were characterized by the expression of specific markers: CD31 (A–D) and von Willebrand Factor (E–H) and the smooth muscle cells by the expression of contractile markers: α- Smooth Muscle Actin (α-SMA: E–H), Smooth Muscle Myosin Heavy Chain (SM-MHC: I–L) and Calponin (M–P). Images were obtained by confocal microscopy observation at cell confluence on both coated surfaces (type I collagen and Polyelectrolyte Multilayer films (PEMs)) and cultivated under normoxic and hypoxic conditions. Objective×40, NA = 0.8, scale bars 75 µm. The figure showed the positive expression of specific ECs markers for cell differentiated under normoxic environment and positive expression of specific contractile SMCs markers for cell differentiated under hypoxic environment.</p

Morphological aspect of differentiated cell.

<p>Optical phase contrast microscopy visualization of differentiated cells seeded on type I collagen (A, B) and polyelectrolyte multilayer films (PEMs) (C, D) until confluence under normoxic (A, C) and hypoxic (B, D) environment. Objective×20, scale bar 55 µm. The morphological examination of the confluent cells showed cobblestone shape (A, C) in normoxia and a spindle like (B, D) shape in hypoxia.</p

Stretch-Induced Biodegradation of Polyelectrolyte Multilayer Films for Drug Release

The design of stimuli-responsive polymer assemblies for the controlled release of bioactive molecules has raised considerable interest these two last decades. Herein, we report the design of mechanically responsive drug-releasing films made of polyelectrolyte multilayers. A layer-by-layer (LbL) reservoir containing biodegradable polyelectrolytes is capped with a mechanosensitive LbL barrier and responds to stretching by a total enzymatic degradation of the film. This strategy is successfully applied for the release in solution of an anticancer drug initially loaded within the architecture

SW480 cells progression through mitosis with respect to elastic moduli of the substrate.

<p>A) After seeding of synchronized SW480 cells on glass, E₅₀, E₂₀ and E₀, time-lapse image were taken every 5 min for 2h30; representative images are shown. Arrow indicates the initial position of the mitotic cell. Merged images of fluorescence (DNA in red) and phase contrast (in gray); scale bar: 20 µm. B) Time-lapse monitoring chromosome segregation in SW480 cells performed as described in A. Representative images of chromosome segregation abnormalities are displayed for E₅₀ and E₂₀; scale bar : 10 µm. C) Percentage of SW480 cells completing mitosis considered in A, determined on 2 pooled independent experiments. Fisher Exact Test shows that the proportion of cells completing mitosis on E₅₀ (46 cells on 77 cells in mitosis) is significantly smaller from the proportion on glass (185/212, <i>p</i> < 0.001). The proportion on E₂₀ (16/161) is significantly smaller than that on E₅₀ (<i>p</i> < 0.001) and the proportion on E₀ (0/146) is significantly smaller than that on E₂₀ (<i>p</i> < 0.001). D) Percentage of cells with lagging chromosomes. In C, D, the error bars represent 95% confidence intervals.</p

Percentage of SW480 cells 30 min-2h post-synchronization from fixed cells presenting abnormal chromosome morphologies on glass, E₅₀ and E₂₀, determined on 2 pooled independent experiments.

<p>Fisher Exact Test shows the proportion on E₅₀ (18 cells with chromosome anomalies on 121 cells analyzed) is significantly different than that on glass (6/153, <i>p</i> < 0.003), and the proportion on E₂₀ (14/78) is significantly different than that on glass (<i>p</i> < 0.001). The errors bars represent 95% confidence intervals.</p

DNA Replication with respect to soft substrates.

<p>A) BrdU visualized by indirect immunofluorescence of SW480 cells 4h post-synchronization on glass, E₅₀ and E₂₀; scale bar: 10 µm. B) Percentage of cells with nuclear BrdU determined on 3 pooled independent experiments. Fisher Exact Test shows that the proportion of cells with nuclear BrdU E₅₀ (108/170) is significantly smaller than that on glass (400/400, <i>p</i> < 0.001) and the proportion on E₂₀ (35/150) is significantly smaller than that on E₅₀ (<i>p</i> < 0.001). The error bars represent 95% confidence intervals.</p