Many therapeutic drugs are excluded from entering the brain due to their lack of transport through the blood-brain barrier (BBB). The development of new strategies for enhancing drug delivery to the brain is of great importance in diagnostics and therapeutics of central nervous diseases. To overcome this problem, a viral fusion peptide (gH625) derived from the glycoprotein gH of Herpes simplex virus type 1 is developed, which possesses several advantages including high cell translocation potency, absence of toxicity of the peptide itself, and the feasibility as an efficient carrier for delivering therapeutics. Therefore, it is hypothesized that brain delivery of nanoparticles conjugated with gH625 should be efficiently enhanced. The surface of fluorescent aminated polystyrene nanoparticles (NPs) is functionalized with gH625 via a covalent binding procedure, and the NP uptake mechanism and permeation across in vitro BBB models are studied. At early incubation times, the uptake of NPs with gH625 by brain endothelial cells is greater than that of the NPs without the peptide, and their intracellular motion is mainly characterized by a random walk behavior. Most importantly, gH625 peptide decreases NP intracellular accumulation as large aggregates and enhances the NP BBB crossing. In summary, these results establish that surface functionalization with gH625 may change NP fate by providing a good strategy for the design of promising carriers to deliver drugs across the BBB for the treatment of brain diseases.

The toxicity of metallic nanoparticles (MNPs) has been fully ascertained, but the mechanisms underlying their cytotoxicity remain still largely unclear. Here we demonstrate that the cytotoxicity of MNPs is strictly reliant on the pathway of cellular internalization. In particular, if otherwise toxic gold, silver, and iron oxide NPs are forced through the cell membrane bypassing any form of active mechanism (e.g., endocytosis), no significant cytotoxic effect is registered. Pneumatically driven NPs across the cell membrane show a different distribution within the cytosol compared to NPs entering the cell by active endocytosis. Specifically, they exhibit free random Brownian motions within the cytosol and do not accumulate in lysosomes. Results suggest that intracellular accumulation of metallic nanoparticles into endo-lysosomal compartments is the leading cause of nanotoxicity, due to consequent nanoparticle degradation and in situ release of metal ions.

It is possible to create sophisticated and target-specific devices for nanomedicine thanks to technological advances in the engineering of nanomaterials. When on target, these nanocarriers often have to be internalized by cells in order to accomplish their diagnostic or therapeutic function. Therefore, the control of such uptake mechanism by active targeting strategy has today become the new challenge in nanoparticle designing. It is also well-known that cells are able to sense and respond to the local physical environment and that the substrate stiffness, and not only the nanoparticle design, influences the cellular internalization mechanisms. In this frame, our work reports on the cyclic relationship among substrate stiffness, cell cytoskeleton assembly and internalization mechanism. Nanoparticles uptake has been investigated in terms of the mechanics of cell environment, the resulting cytoskeleton activity and the opportunity of activate molecular specific molecular pathways during the internalization process. To this aim, the surface of 100 nm polystyrene nanoparticles was decorated with a tripeptide (RGD and a scrambled version as a control), which was able to activate an internalization pathway directly correlated to the dynamics of the cell cytoskeleton, in turn, directly correlated to the elastic modulus of the substrates. We found that the substrate stiffness modulates the uptake of nanoparticles by regulating structural parameters of bEnd.3 cells as spreading, volume, focal adhesion, and mechanics. In fact, the nanoparticles were internalized in larger amounts both when decorated with RGD, which activated an internalization pathway directly correlated to the cell cytoskeleton, and when cells resided on stiffer material that, in turn, promoted the formation of a more structured cytoskeleton. This evidence indicates the directive role of the mechanical environment on cellular uptake of nanoparticles, contributing new insights to the rational design and development of novel nanocarrier systems.

The membranotropic peptide gH625 is able to transport different cargos (i.e., liposomes, quantum dots, polymeric nanoparticles) within and across cells in a very efficient manner. However, a clear understanding of the detailed uptake mechanism remains elusive. In this work, we investigate the journey of gH625-functionalized polystyrene nanoparticles in mouse-brain endothelial cells from their interaction with the cell membrane to their intracellular final destination. The aim is to elucidate how gH625 affects the behavior of the nanoparticles and their cytotoxic effect. The results indicate that the mechanism of translocation of gH625 dictates the fate of the nanoparticles, with a relevant impact on the nanotoxicological profile of positively charged nanoparticles.

Blood-brain barrier (BBB) represents a formidable barrier for many therapeutic drugs to enter the brain tissue. The development of new strategies for enhancing drug delivery to the brain is of great importance in diagnostics and therapeutics of central nervous system (CNS) diseases. In this context, nanoparticles are an emerging class of drug delivery systems that can be easily tailored to deliver drugs to various compartments of the body, including the brain. To identify, characterize, and validate novel nanoparticles applicable to brain delivery, in vitro BBB model systems have been developed. In this work, we describe a method to screen nanoparticles with variable size and surface functionalization in order to define the physicochemical characteristics underlying the design of nanoparticles that are able to efficiently cross the BBB.

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To improve the efficacy of nanoparticles (NPs) and boost their theragnostic potential for brain diseases, it is key to understand the mechanisms controlling blood–brain barrier (BBB) crossing. Here, the capability of 100 nm carboxylated polystyrene NPs, used as a nanoprobe model, to cross the human brain endothelial hCMEC/D3 cell layer, as well as to be consequently internalized by human brain tumor U87 cells, is investigated as a function of NPs’ different intracellular localization. We compared NPs confined in the endo-lysosomal compartment, delivered to the cells through endocytosis, with free NPs in the cytoplasm, delivered by the gene gun method. The results indicate that the intracellular behavior of NPs changed as a function of their entrance mechanism. Moreover, by bypassing endo-lysosomal accumulation, free NPs were released from cells more efficiently than endocytosed NPs. Most importantly, once excreted by the endothelial cells, free NPs were released in the cell culture medium as aggregates smaller than endocytosed NPs and, consequently, they entered the human glioblastoma U87 cells more efficiently. These findings prove that intracellular localization influences NPs’ long-term fate, improving their cellular release and consequent cellular uptake once in the brain parenchyma. This study represents a step forward in designing nanomaterials that are able to reach the brain effectively.

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