Near-Infrared Light Activated Formulation for the Spatially Controlled Release of CRISPR-Cas9 Ribonucleoprotein for Brain Gene Editing.

The CRISPR/Cas9 system has emerged as a promising platform for gene editing; however, the lack of an efficient and safe delivery system to introduce it into cells continues to hinder clinical translation. Here, we report a rationally designed gene-editing nanoparticle (NP) formulation for brain applications: an sgRNA:Cas9 ribonucleoprotein complex is immobilized on the NP surface by oligonucleotides that are complementary to the sgRNA. Irradiation of the formulation with a near-infrared (NIR) laser generates heat in the NP, leading to the release of the ribonucleoprotein complex. The gene-editing potential of the formulation was demonstrated in vitro at the single-cell level. The safety and gene editing of the formulation were also demonstrated in the brains of reporter mice, specifically in the subventricular zone after intracerebral administration and in the olfactory bulb after intranasal administration. The formulation presented here offers a new strategy for the spatially controlled delivery of the CRISPR system to the brain.

ENPP1 induces blood-brain barrier dysfunction and promotes brain metastasis formation in HER2-positive breast cancer.

BACKGROUND: Brain metastasis (BrM) is a devastating end-stage neurological complication that occurs in up to 50% of HER2+ breast cancer patients. Understanding how disseminating tumor cells manage to cross the blood-brain barrier (BBB) is essential for developing effective preventive strategies. We identified the ecto-nucleotidase ENPP1 as specifically enriched in the secretome of HER2+ brain metastatic cells, prompting us to explore its impact on BBB dysfunction and BrM formation. METHODS: We used in vitro BBB and in vivo premetastatic mouse models to evaluate the effect of tumor-secreted ENPP1 on brain vascular permeability. BBB integrity was analyzed by real-time fluorescence imaging of 20 kDa Cy7.5-dextran extravasation and immunofluorescence staining of adherens and tight junction proteins. Pro-metastatic effects of ENPP1 were evaluated in an experimental brain metastatic model. RESULTS: Systemically secreted ENPP1 from primary breast tumors impaired the integrity of BBB with loss of tight and adherens junction proteins early before the onset of BrM. Mechanistically, ENPP1 induced endothelial cell dysfunction by impairing insulin signaling and its downstream AKT/GSK3ß/ß-catenin pathway. Genetic ablation of ENPP1 from HER2+ brain metastatic cells prevented endothelial cell dysfunction and reduced metastatic burden while prolonging the overall and metastasis-free survival of mice. Furthermore, plasmatic ENPP1 levels correlate with brain metastatic burden and inversely with overall survival. CONCLUSIONS: We demonstrated that metastatic breast cancer cells exploit the ENPP1 signaling for cell transmigration across the BBB and brain colonization. Our data implicate ENPP1 as a potential biomarker for poor prognosis and early detection of BrM in HER2+ breast cancer.

Engineered extracellular vesicles as brain therapeutics.

Extracellular vesicles (EVs) are communication channels between different cell types in the brain, between the brain and the periphery and vice-versa, playing a fundamental role in physiology and pathology. The evidence that EVs might be able to cross the blood-brain barrier (BBB) make them very promising candidates as nanocarriers to treat brain pathologies. EVs contain a cocktail of bioactive factors, yet their content and surface can be further engineered to enhance their biological activity, stability and targeting ability. Native and engineered EVs have been reported for the treatment of different brain pathologies, although issues related to their modest accumulation and limited local therapeutic effect in the brain still need to be addressed. In this review, we cover the therapeutic applications of native and bioengineered EVs for brain diseases. We also review recent data about the interaction between EVs and the BBB and discuss the challenges and opportunities in clinical translation of EVs as brain therapeutics.

Therapeutic Nanoparticles for the Different Phases of Ischemic Stroke.

Stroke represents the second leading cause of mortality and morbidity worldwide. Ischemic strokes are the most prevalent type of stroke, and they are characterized by a series of pathological events prompted by an arterial occlusion that leads to a heterogeneous pathophysiological response through different hemodynamic phases, namely the hyperacute, acute, subacute, and chronic phases. Stroke treatment is highly reliant on recanalization therapies, which are limited to only a subset of patients due to their narrow therapeutic window; hence, there is a huge need for new stroke treatments. Nonetheless, the vast majority of promising treatments are not effective in the clinical setting due to their inability to cross the blood-brain barrier and reach the brain. In this context, nanotechnology-based approaches such as nanoparticle drug delivery emerge as the most promising option. In this review, we will discuss the current status of nanotechnology in the setting of stroke, focusing on the diverse available nanoparticle approaches targeted to the different pathological and physiological repair mechanisms involved in each of the stroke phases.

Cell Membrane-Coated Magnetic Nanocubes with a Homotypic Targeting Ability Increase Intracellular Temperature due to ROS Scavenging and Act as a Versatile Theranostic System for Glioblastoma Multiforme.

In this study, hybrid nanocubes composed of magnetite (Fe3 O4 ) and manganese dioxide (MnO2 ), coated with U-251 MG cell-derived membranes (CM-NCubes) are synthesized. The CM-NCubes demonstrate a concentration-dependent oxygen generation (up to 15%), and, for the first time in the literature, an intracellular increase of temperature (6 °C) due to the exothermic scavenging reaction of hydrogen peroxide (H2 O2 ) is showed. Internalization studies demonstrate that the CM-NCubes are internalized much faster and at a higher extent by the homotypic U-251 MG cell line compared to other cerebral cell lines. The ability of the CM-NCubes to cross an in vitro model of the blood-brain barrier is also assessed. The CM-NCubes show the ability to respond to a static magnet and to accumulate in cells even under flowing conditions. Moreover, it is demonstrated that 500 µg mL-1 of sorafenib-loaded or unloaded CM-NCubes are able to induce cell death by apoptosis in U-251 MG spheroids that are used as a tumor model, after their exposure to an alternating magnetic field (AMF). Finally, it is shown that the combination of sorafenib and AMF induces a higher enzymatic activity of caspase 3 and caspase 9, probably due to an increment in reactive oxygen species by means of hyperthermia.