Inefficient drug delivery across the blood–brain barrier (BBB) and into target cells in the brain hinders the treatment of neurological diseases. One strategy to increase the brain penetration of drugs is to use vesicular nanoparticles functionalized with multiple ligands of BBB transporters as vehicles. Once within the brain, however, drugs must also be able to reach their therapeutic targets in the different cell types. It is, therefore, favorable if such nanocarriers are designed that can deliver their cargo not only to brain endothelial cells, but to other cell types as well. Here, we show that alanine-glutathione dual-targeting of niosomes enhances the delivery of a large protein cargo into cultured cells of the neurovascular unit, namely brain endothelial cells, pericytes, astrocytes and neurons. Furthermore, using metabolic and endocytic inhibitors, we show that the cellular uptake of niosomes is energy-dependent and is partially mediated by endocytosis. Finally, we demonstate the ability of our targeted nanovesicles to deliver their cargo into astroglial cells after crossing the BBB in vitro. These data indicate that dual-labeling of nanoparticles with alanine and glutathione can potentially be exploited to deliver drugs, even biopharmacons, across the BBB and into multiple cell types in the brain.

Nanosized drug delivery systems targeting transporters of the blood-brain barrier (BBB) are promising carriers to enhance the penetration of therapeutics into the brain. The expression of solute carriers (SLC) is high and shows a specific pattern at the BBB. Here we show that targeting ligands ascorbic acid, leucine and glutathione on nanoparticles elevated the uptake of albumin cargo in cultured primary rat brain endothelial cells. Moreover, we demonstrated the ability of the triple-targeted nanovesicles to deliver their cargo into midbrain organoids after crossing the BBB model. The cellular uptake was temperature- and energy-dependent based on metabolic inhibition. The process was decreased by filipin and cytochalasin D, indicating that the cellular uptake of nanoparticles was partially mediated by endocytosis. The uptake of the cargo encapsulated in triple-targeted nanoparticles increased after modification of the negative zeta potential of endothelial cells by treatment with a cationic lipid or after cleaving the glycocalyx with an enzyme. We revealed that targeted nanoparticles elevated plasma membrane fluidity, indicating the fusion of nanovesicles with endothelial cell membranes. Our data indicate that labeling nanoparticles with three different ligands of multiple transporters of brain endothelial cells can promote the transfer and delivery of molecules across the BBB.

In vitro regeneration of Arabidopsis from roots is generally achieved via indirect organogenesis. First, transdifferentiation of lateral root primordia to calli is achieved by a balanced auxin-to-cytokinin ratio that is followed by the induction of shoot meristem formation using a high cytokinin level. Here we demonstrate that if the root explants were transferred onto a hormone-free medium after a transient (4-days) cytokinin treatment, embryogenic marker genes (LEC1, LEC2, FUS3) started to be expressed. App. 50% of the regeneration foci developed into plantlets with trichome-less cotyledon-like leaves. Moreover, the somatic embryogenesis defective lec1 mutant could regenerate only shoots with trichome-bearing leaves under this condition. Based on these observations, the mixed accomplishment of shoot organogenesis and somatic embryogenesis is hypothesized in the Arabidopsis root explants cultured under hormone-free conditions following cytokinin induction. Using whole seedlings instead of root explants in the same experimental set up, no regenerates were formed on the roots. Applying the auxin transport inhibitor TIBA to the root-to-shoot junction of the seedlings, the regeneration ability of the root could be restored. The observations indicate that shoot-derived endogenous auxin blocks the cytokinin-induced regeneration process in the roots of whole seedlings. The expression of the wound-induced transcription factor WIND1 could be detected in the roots of unwounded seedlings if the shoot-to-root auxin transport was inhibited. Manipulating the exogenous cytokinin level together with the endogenous shoot-to-root auxin transport therefore could mimic the effect of wounding (removal of shoot) on plant regeneration from roots.

Microfluidics-based protoplast isolation, which enabled intracellular uptake of singlet oxygen sensor fluorophore and DNA oligonucleotides in Symbiodinium spp.

Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., “polyplexes” that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.

Plasmonically enhanced fluorescence is a widely studied and applied phenomenon, however, only a comparative theoretical and experimental analysis of coupled fluorophores and plasmonic nanoresonators makes it possible to uncover how this phenomenon can be controlled. A numerical optimization method was applied to design configurations that are capable of resulting in an enhancement of excitation and emission, moreover, of both phenomena simultaneously in coupled Cy5 dye molecule and gold nanorod systems. Parametric sensitivity studies revealed how the fluorescence enhancement depends on the molecule’s location, distance and orientation. Coupled systems designed for simultaneous improvement exhibited the highest (intermediate directional) total fluorescence enhancement, which is accompanied by intermediate sensitivity to the molecule’s parameters, except the location and orientation sensitivity at the excitation wavelength. Gold nanorods with a geometry corresponding to the predicted optimal configurations were synthesized, and DNA strands were used to control the Cy5 dye molecule distance from the nanorod surface via hybridization of the Cy5-labelled oligonucleotide. State-of-the-art dSTORM microscopy was used to accomplish a proof-of-concept experimental demonstration of the theoretically predicted (directional) total fluorescence enhancement. The measured fluorescence enhancement was in good agreement with theoretical predictions, thus providing a complete kit to design and prepare coupled nanosystems exhibiting plasmonically enhanced fluorescence.