Multilayered polyelectrolyte microcapsules: interaction with the enzyme cytochrome C oxidase

Cell-sized polyelectrolyte capsules functionalized with a redox-driven proton pump protein were assembled for the first time. The interaction of polyelectrolyte microcapsules, fabricated by electrostatic layer-by-layer assembly, with cytochrome c oxidase molecules was investigated. We found that the cytochrome c oxidase retained its functionality, that the functionalized microcapsules interacting with cytochrome c oxidase were permeable and that the permeability characteristics of the microcapsule shell depend on the shell components. This work provides a significant input towards the fabrication of an integrated device made of biological components and based on specific biomolecular functions and properties

Cyt<i>c</i>O redox activity measured polarographically.

<p>The O₂-consumption as a function of time for reaction mixture without Cyt<i>c</i>O, Cyt<i>c</i>O in solution, PSS/PAH)₄/Cyt<i>c</i>O NPCs and PSS/PAH)₄ NPCs. 100% indicate the initial amount of O₂ in all the different samples.</p

QCM-D result showing the in situ build-up of the multilayer PAH-(PSS/PAH)₄/Cyt<i>c</i>O films at a pH 8.5.

<p>Frequency changes are recorded as a function of time for the 3^rd, 5^th and 7^th armonics.</p

(CHI/PEC)₄/Cyt<i>c</i>O NPCs sequence of images before (Panel A) and after addition of dye (B–D).

<p>The dashed line indicates the border of one (CHI/PEC)₄/Cyt<i>c</i>O NPCs which upon addition of the dye swells and finally explodes. Scale bar is 5 µm.</p

Confocal images of NPCs in the presence of rhodamine.

<p>Forward (a, d), confocal (b, e) and merged (c, f) images of empty capsules (top) can be compared with the corresponding images of (PSS/PAH)₄/Cyt<i>c</i>O NPCs (bottom). The scale bar is equivalent to 5 µm.</p

Surface coverage grow-up of the multilayer (PAH/PSS)₄/PAH/Cyt<i>c</i>O at pH 8.5 as a function of the number of layers, calculated with the Voigt model by QCM-D.

<p>Step number 11 correspond to surface coverage value of the multilayer after the addition of Cyt<i>c</i>O.</p

Image-based tracking of anticancer drug-loaded nanoengineered polyelectrolyte capsules in cellular environments using a fast Benchtop Mid-Infrared (MIR) microscope

Drug delivery monitoring and tracking in the human body are two of the biggest challenges in targeted therapy to be addressed by nanomedicine. The ability of imaging drugs and micro-/nanoengineered drug carriers and of visualizing their interactions at the cellular interface in a labelfree manner is crucial in providing the ability of tracking their cellular pathways and will help understand their biological impact, allowing thus to improve the therapeutic efficacy. We present a fast, label-free technique to achieve high-resolution imaging at the mid-infrared (MIR) spectrum that provides chemical information. Using our custom-made benchtop infrared microscope using a high-repetition-rate pulsed laser (80 MHz, 40 ps), we were able to acquire images with subwavelength resolution (0.8 × λ) at very high speeds. As a proof-of-concept, we embarked on the investigation of nanoengineered polyelectrolyte capsules (NPCs) containing the anticancer drug, docetaxel. These NPCs were synthesized using a layer-by-layer approach built upon a calcium carbonate (CaCO3) core, which was then removed away with ethylenediaminetetraacetic acid. The obtained MIR images show that NPCs are attached to the cell membrane, which is a good step toward an efficient drug delivery. This has been confirmed by both three-dimensional confocal fluorescence and stimulated emission depletion microscopy. Coupled with additional instrumentation and data processing advancements, this setup is capable of video-rate imaging speeds and will be significantly complementing current super-resolution microscopy techniques while providing an unperturbed view into living cells