Exploiting endocytosis for transfection of mRNA for cytoplasmatic delivery using cationic gold nanoparticles

Gene therapy holds promise to cure various diseases at the fundamental level. For that, efficient carriers are needed for successful gene delivery. Synthetic 'non-viral' vectors, as cationic polymers, are quickly gaining popularity as efficient vectors for transmitting genes. However, they suffer from high toxicity associated with the permeation and poration of the cell membrane. This toxic aspect can be eliminated by nanoconjugation. Still, results suggest that optimising the oligonucleotide complexation, ultimately determined by the size and charge of the nanovector, is not the only barrier to efficient gene delivery. We herein develop a comprehensive nanovector catalogue comprising different sizes of Au NPs functionalized with two different cationic molecules and further loaded with mRNA for its delivery inside the cell. Tested nanovectors showed safe and sustained transfection efficiencies over 7 days, where 50 nm Au NPs displayed the highest transfection rates. Remarkably, protein expression was increased when nanovector transfection was performed combined with chloroquine. Cytotoxicity and risk assessment demonstrated that nanovectors are safe, ascribed to lesser cellular damage due to their internalization and delivery via endocytosis. Obtained results may pave the way to design advanced and efficient gene therapies for safely transferring oligonucleotides

Deoxygenation and elevation of intracellular magnesium induce tyrosine phosphorylation of band 3 in human erythrocytes

AbstractDeoxygenation increases the level of tyrosine phosphorylation of band 3 by ∼25% in human red blood cells (RBCs), as determined by Western blotting. The effect is much more pronounced in osmotically shrunken RBCs or in the presence of vanadate. When the rise in intracellular free Mg2+ concentration in deoxygenated RBCs is simulated via clamping of the intracellular magnesium in oxygenated RBCs by ionomycin, band 3 phosphorylation is elevated by up to 10-fold. Phosphorylated band 3 is preferentially retained by RBC skeletons, after mild extraction with Triton X-100. Elevation of intracellular free Mg2+ leads to band 3 phosphorylation and is accompanied by rigidification of the membrane skeleton as determined by analysis of RBC membrane mechanical fluctuations. These findings suggest that the visco-elastic properties of human erythrocytes may be regulated by band 3 tyrosine phosphorylation

Ca2+ promotes erythrocyte band 3 tyrosine phosphorylation via dissociation of phosphotyrosine phosphatase from band 3.

The anion-exchange band 3 protein is the main erythrocyte protein that is phosphorylated by protein tyrosine kinase (PTK). We have previously identified a band 3-associated phosphotyrosine phosphatase (PTP) that is normally highly active and prevents the accumulation of band 3 phosphotyrosine. Band 3 tyrosine phosphorylation can be induced by inhibition of PTP (vanadate, thiol oxidation), activation of PTK (hypertonic NaCl) or intracellular increased Ca(2+) (mechanism unknown). We now show that there is inhibition of dephosphorylation of band 3 in Ca(2+)/ionophore-treated erythrocytes and in membranes isolated from the treated cells. These membranes exhibit phosphatase activity upon the addition of exogenous substrate. Dephosphorylation of the endogenous substrate (band 3) can be activated in these membranes by the addition of Mg(2+). Thus the inability of PTP to dephosphorylate the band 3 phosphotyrosine is not due to inhibition of the enzyme itself. Ca(2+) rise in the erythrocyte causes dissociation of PTP from band 3, thus leaving the kinase unopposed. This is shown by a significant diminution in band 3/PTP co-precipitation. Addition of Mg(2+) to these membranes leads to reassociation of band 3 with PTP. The Ca(2+)-induced inhibition of band 3 dephosphorylation may be due to Ca(2+)-dependent alterations in membrane components and structure, affecting the interaction of band 3 with PTP. The Ca(2+)-induced tyrosine phosphorylation, involving an apparent PTP inhibition via dissociation from the substrate, may play a role in signal transduction pathways and in certain pathological disorders associated with increased cell Ca(2+)

Nanoparticle-Decorated Erythrocytes Reveal That Particle Size Controls the Extent of Adsorption, Cell Shape, and Cell Deformability

Unraveling the interaction of nanoparticles with living cells is fundamental for nanomedicine and nanotoxicology. Erythrocytes are abundant and serve as model cells with well-characterized properties. Quantitative experiments addressing the binding of carboxylated polystyrene nanoparticles to human erythrocytes reveal saturated adsorption with only sparse (∼2%) coverage of the cell membrane by partial-wrapped nanoparticles. The independence of the adsorbed area on particle size suggests a restricted number of adhesive sites on the membrane. Using a continuum membrane model combined with nanoparticle–membrane adhesion mediated by receptor–ligand bonds, we predict high bond energies and low receptor densities for partial-wrapped particles. With the help of computer simulations, we determine sets of receptor densities, receptor diffusion coefficients, minimal numbers of bound receptors required for multivalent binding, and maximal possible numbers of bound receptors that reproduce the experimental nanoparticle adsorption data. Nanoparticle decoration of erythrocytes leads to shape transformations and reduced cell deformability. We quantitatively characterize and interpret erythrocyte shape and deformability changes. The shape changes also offer insights into the modification of the mechanical properties of other mammalian cell membranes by adhered nanoparticles. A potential application of nanoparticle-loaded erythrocytes is retarded targeted drug delivery with a long lifetime of the particles in the blood circulation