Predicting the size and morphology of nanoparticle clusters driven by biomolecular recognition.

Nanoparticle aggregation is a driving principle of innovative materials and biosensing methodologies, improving transduction capabilities displayed by optical, electrical or magnetic measurements. This aggregation can be driven by the biomolecular recognition between target biomolecules (analytes) and receptors bound onto nanoparticle surface. Despite theoretical advances on modelling the entropic interaction in similar systems, predictions of the fractal morphologies of the nanoclusters of bioconjugated nanoparticles are lacking. The morphology of resulting nanoclusters is sensitive to the location, size, flexibility, average number of receptors per particle f̄, and the analyte-particle concentration ratio. Here we considered bioconjugated iron oxide nanoparticles (IONPs) where bonds are mediated by a divalent protein that binds two receptors attached onto different IONPs. We developed a protocol combining analytical expressions for receptors and linker distributions, and Brownian dynamics simulations for bond formation, and validated it against experiments. As more bonds become available (e.g., by adding analytes), the aggregation deviates from the ideal Bethe's lattice scenario due to multivalence, loop formation, and steric hindrance. Generalizing Bethe's lattice theory with a (not-integer) effective functionality feff leads to analytical expressions for the cluster size distributions in excellent agreement with simulations. At high analyte concentration steric impediment imposes an accessible limit value facc to feff, which is bounded by facc < feff < f̄. A transition to gel phase, is correctly captured by the derived theory. Our findings offer new insights into quantifying analyte amounts by assessing nanocluster size, and predicting nanoassembly morphologies accurately is a first step towards understanding variations of physical properties in clusters formed after biomolecular recognition.

Correlative 3D cryo X-ray imaging reveals intracellular location and effect of designed antifibrotic protein-nanomaterial hybrids.

Revealing the intracellular location of novel therapeutic agents is paramount for the understanding of their effect at the cell ultrastructure level. Here, we apply a novel correlative cryo 3D imaging approach to determine the intracellular fate of a designed protein-nanomaterial hybrid with antifibrotic properties that shows great promise in mitigating myocardial fibrosis. Cryo 3D structured illumination microscopy (cryo-3D-SIM) pinpoints the location and cryo soft X-ray tomography (cryo-SXT) reveals the ultrastructural environment and subcellular localization of this nanomaterial with spatial correlation accuracy down to 70 nm in whole cells. This novel high resolution 3D cryo correlative approach unambiguously locates the nanomaterial after overnight treatment within multivesicular bodies which have been associated with endosomal trafficking events by confocal microscopy. Moreover, this approach allows assessing the cellular response towards the treatment by evaluating the morphological changes induced. This is especially relevant for the future usage of nanoformulations in clinical practices. This correlative super-resolution and X-ray imaging strategy joins high specificity, by the use of fluorescence, with high spatial resolution at 30 nm (half pitch) provided by cryo-SXT in whole cells, without the need of staining or fixation, and can be of particular benefit to locate specific molecules in the native cellular environment in bio-nanomedicine.

Reduction of cardiac TGFß-mediated profibrotic events by inhibition of Hsp90 with engineered protein.

Myocardial fibroblast activation coupled with extracellular matrix production is a pathological signature of myocardial fibrosis and is governed mainly by transforming growth factor TGFß-Smad2/3 signaling. Targeting the ubiquitous TGFß leads to cellular homeostasis deregulation with adverse consequences. We previously showed the anti-fibrotic effects upon downregulation of 90-kDa heat shock protein (Hsp90), a chaperone that associates to the TGFß signaling cascade. In the present study, we use a fluorescent-labeled Hsp90 protein inhibitor (CTPR390-488) with specific Hsp90 binding properties to reduce myocardial pro-fibrotic events in vitro and in vivo. The mechanism of action involves the disruption of TGFßRI-Hsp90 complex, resulting in a decrease in TGFß signaling and reduction in extracellular matrix collagen. In vivo, decreased myocardial collagen deposition was observed upon CTPR390-488 treatment in a pro-fibrotic mouse model. This is the first study demonstrating the ability of an engineered Hsp90 protein inhibitor to block collagen expression, reduce the motility of myocardial TGFß-activated fibroblasts and ameliorate angiotensin-II induced cardiac myocardial fibrosis in vivo.

An experimental and computational framework for engineering multifunctional nanoparticles: designing selective anticancer therapies.

A key challenge in the treatment of cancer with nanomedicine is to engineer and select nanoparticle formulations that lead to the desired selectivity between tumorigenic and non-tumorigenic cells. To this aim, novel designed nanomaterials, deep biochemical understanding of the mechanisms of interaction between nanomaterials and cells, and computational models are emerging as very useful tools to guide the design of efficient and selective nanotherapies. This works shows, using a combination of detailed experimental approaches and simulations, that the specific targeting of cancer cells in comparison to non-tumorigenic cells can be achieved through the custom design of multivalent nanoparticles. A theoretical model that provides simple yet quantitative predictions to tune the nanoparticles targeting and cytotoxic properties by their degree of functionalization is developed. As a case study, a system that included a targeting agent and a drug and is amenable to controlled experimental manipulation and theoretical analysis is used. This study shows how at defined functionalization levels multivalent nanoparticles can selectively kill tumor cells, while barely affecting non-tumorigenic cells. This work opens a way to the rational design of multifunctionalized nanoparticles with defined targeting and cytotoxic properties for practical applications.

Glycophorin as a receptor for Escherichia coli alpha-hemolysin in erythrocytes.

Escherichia coli alpha-hemolysin (HlyA) can lyse both red blood cells (RBC) and liposomes. However, the cells are lysed at HlyA concentrations 1-2 orders of magnitude lower than liposomes (large unilamellar vesicles). Treatment of RBC with trypsin, but not with chymotrypsin, reduces the sensitivity of RBC toward HlyA to the level of the liposomes. Since glycophorin, one of the main proteins in the RBC surface, can be hydrolyzed by trypsin much more readily than by chymotrypsin, the possibility was tested of a specific binding of HlyA to glycophorin. With this purpose, a number of experiments were performed. (a) HlyA was preincubated with purified glycophorin, after which it was found to be inactive against both RBC and liposomes. (b) Treatment of RBC with an anti-glycophorin antibody protected the cells against HlyA lysis. (c) Immobilized HlyA was able to bind glycophorin present in a detergent lysate of RBC ghosts. (d) Incorporation of glycophorin into pure phosphatidylcholine liposomes increased notoriously the sensitivity of the vesicles toward HlyA. (e) Treatment of the glycophorin-containing liposomes with trypsin reverted the vesicles to their original low sensitivity. The above results are interpreted in terms of glycophorin acting as a receptor for HlyA in RBC. The binding constant of HlyA for glycophorin was estimated, in RBC at sublytic HlyA concentrations, to be 1.5 x 10(-9) m.