A method for evaluating nanoparticle transport through the blood-brain barrier in vitro

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

Intracellular Localization during Blood–Brain Barrier Crossing Influences Extracellular Release and Uptake of Fluorescent Nanoprobes

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

Shuttle-mediated nanoparticle delivery to the blood-brain barrier.

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, we have developed a viral fusion peptide (gH625)derived from the glycoprotein gH of Herpes simplex type 1, possessing several advantages including high cell translocation potency, absence of toxicity of peptide itself and the feasibility as an efficient carrier for delivering therapeutics. Therefore, it was hypothesized that brain delivery of nanoparticles conjugated with gH625 should be efficiently enhanced. gH625 was functionalized to the surface of fluorescent aminated polystyrene nanoparticles (NPs) via a covalent binding procedure and NPuptake mechanism and permeation across in vitroBBB model were studied. At early incubation time, the uptake of NPs with gH625 by brain endothelial cells was greater than that of the NPs without the peptide and their intracellular motion was mainly characterized by a random walk behavior. Most importantly, gH625peptide decreased NP intracellular accumulation as large aggregates and enhanced the NPBBB crossing. In summary, our results establish that the 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

Transport across the cell-membrane dictates nanoparticle fate and toxicity: a new paradigm in nanotoxicology

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

Surface decoration with gH625-membranotropic peptides as a method to escape the endo-lysosomal compartment and reduce nanoparticle toxicity

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