The glioblastoma (GBM) microenvironment undergoes adaptations to support tumor progression, including a dysregulated extracellular matrix, with altered heparan sulfate (HS) proteoglycans. We investigated N-deacetylase/N-sulfotransferase-1 (NDST1) because NDSTs are initial modifying enzymes of HS biosynthesis and have key roles in designing the HS sulfation pattern. This, in turn governs interactions with growth factors and other biomolecules. We report that NDST1 expression is lower in GBM than in the normal brain, and that patient-derived GBM cells, grown under neural stem cell culture conditions have lower levels of HS than normal astrocytes. Overexpression of NDST1 in GBM cells with low inherent NDST1 levels stimulates cell migration, reduce cell adhesion, induce EMT markers and increase invasion. Conversely, when NDST1 levels were reduced by shRNA in GBM cells, that had higher baseline expression, we find that invasion is reduced, and instead, self-renewal capacity increases alongside elevated stem cell marker expression. Moreover, overexpression of NDST1 changes chromatin accessibility of gene regulatory regions with the capacity to affect transcription factor expression, and pathways that favors cell motility and invasion. Furthermore, NDST1 overexpression results in increased activation of several receptor tyrosine kinases. This study shows that low NDST1 levels support GBM cell stemness, whereas high NDST1 levels endow tumor cells with a motile cell phenotype. We therefore propose that NDST1 is important for regulation of the balance between proliferation and invasive properties in GBM cells.

Topical drug delivery offers a localized and patient-friendly method for treating skin diseases and subcutaneous lesions. However, the outermost skin barrier - the stratum corneum (SC) - hinders the delivery of large molecules such as biopharmaceuticals. This study introduces rolling ultraminiaturized microneedle spheres (RUMS) as a novel solution that enables topical delivery of messenger RNA (mRNA) without the need for chemical enhancers or techniques like electroporation, iontophoresis, or microneedle patches. RUMS are engineered spherical microparticles that gently roll over the skin, creating numerous micropores while minimizing tissue damage. In ex vivo porcine skin experiments, 25 RUMS generated approximately 4,500 pores within 10 seconds, achieving penetration depths of around 20 micrometers and increasing skin permeability by up to 100-fold. In vivo studies in mice showed that combining RUMS with topical doxycycline led to a ~50% tumor size reduction within two weeks and full recovery by four weeks. In contrast, doxycycline or RUMS alone offered limited therapeutic benefit. Rapid skin healing was observed due to the small pore size. Additionally, topical delivery of lipid nanoparticle-encapsulated luciferase (luc)-encoding mRNA was successfully demonstrated in mice. Overall, use of RUMS presents a simple, painless, and potentially well-tolerated technique for enabling transdermal topical delivery of biologics.

Biocompatibility of medical implants poses a significant challenge in medical technology. Neural implants, integral to curative therapies, initially exhibit efficacy but can lead to unforeseen long-term side effects. The material composition and dimensions of implants are critical factors influencing their biocompatibility within brain tissue. Typically, neural implants are identified as foreign entities by the patient's immune system, triggering persistent inflammation and severe adverse effects. In this study, we investigate the host response in mouse brain tissue of implanted microscale constructs measuring 0.1 x 0.1 x 1 mm3 fabricated from common microfabrication materials. Magnetic Resonance Imaging (MRI) analysis reveals rapid recovery of brain parenchyma at 6 week interval post-implantation, accompanied by negligible or mild adverse immune responses during the experimental period. Histological assessments and cell marker stainings targeting astroglia, macrophages, and microglia demonstrate minimal impacts of the microconstructs on mouse brain tissue throughout the 24-week implantation period. Our findings indicate that untethered microimplants of this scale may have potential applications in medical technology and medical treatment for various brain diseases. In summary, this study supports the development of potentially biocompatible brain microimplants that could be useful for the long-term management of chronic brain disorders.

Endothelial cells play an important role in several vascular diseases, and molecular analysis of these cells could provide valuable information on underlying tissue status. However, no clinically established procedure for harvesting endothelial cells exists. A micro-3D-printed device adapted for endovascular techniques to harvest endothelial cells for transcriptomic analysis is presented. In vivo evaluation in swine (n = 6) yielded tissue samples in 60 out of 65 cases, of which 80% show a substantial amount of tissue. The cytological evaluation indicates high selectivity towards endothelial cells, and RNA-sequencing shows gene expression signatures consistent with vascular tissue. It is found that there are no short-term safety risks compared to operation with a control wire of equal dimensions and acute complications are not detected. If translated to clinical use, the device could enable increased understanding of early-stage endothelial cell-mediated disease progression and earlier diagnosis of diseases such as atherosclerosis.

Proteoglycans are key molecules in signaling, both during brain development and in malignant brain tumor formation, where cancer cells mimic, or co-opt, normal developmental programs. This chapter focuses on the role of heparan sulfate proteoglycans (HSPGs) in these processes. HSPGs are composed of a core protein with attached, heavily sulfated, polysaccharide side chains, and they are ubiquitously present on cell surfaces and in the extracellular space where they serve both as structural components and regulators of a multitude of cellular activities. HSPGs are critically involved in mammalian development, and perturbations of pathways regulated by HSPGs play major roles in human diseases. Neural stem cell programs sustain populations of stem cells that initially give rise to neural progenitors with high proliferative capacity that eventually differentiate to mature cells of the nervous system. HSPGs act as coreceptors for a wide variety of signaling pathways during these processes. Accumulated mutations in neural stem cells can cause failure to perform terminal differentiation or the inability to restrict progenitor proliferation and lead to brain tumor development. The same signaling mechanisms that promote self-renewal of neural stem cells thus also support cancer stem cells, and HSPGs are integral facilitators of brain tumor development and progression.

Pseudokinases, comprising 10% of the human kinome, are emerging as regulators of canonical kinases and their functions are starting to be defined. We previously identified the pseudokinase Nuclear Receptor Binding Protein 2 (NRBP2) in a screen for genes regulated during neural differentiation. During mouse brain development,NRBP2is expressed in the cerebellum, and in the adult brain, mainly confined to specific neuronal populations. To study the role of NRBP2 in brain tumors, we stained a brain tumor tissue array for NRPB2, and find its expression to be low, or absent, in a majority of the tumors. This includes medulloblastoma (MB), a pediatric tumor of the cerebellum. Using database mining of published MB data sets, we also find that NRBP2 is expressed at a lower level in MB than in the normal cerebellum. Recent studies indicate that MB exhibits frequent epigenetic alternations and we therefore treated MB cell lines with drugs inhibiting DNA methylation or histone deacetylation, which leads to an upregulation of NRBP2 mRNA expression, showing that it is under epigenetic regulation in cultured MB cells. Furthermore, forced overexpression of NRBP2 in MB cell lines causes a dramatic decrease in cell numbers, increased cell death, impaired cell migration and inhibited cell invasion in vitro. Taken together, our data indicate that downregulation of NRBP2 may be a feature by which MB cells escape growth regulation.

Large-molecule pharmaceuticals offer new treatment options for severe lung disease. However, delivering these drugs to the lung is challenging due to the elevated shear rates during the aerosolization process. So far, this has prevented an application in portable inhalers, holding back the portable use of biopharmaceuticals for drug delivery. We demonstrate that a micro swirl nozzle can aerosolize fragile biopharmaceuticals in an aqueous solution. Shear rate simulations of the nozzle unit indicate orders of magnitude in shear rate reduction compared with conventional aqueous aerosolization units. Catalase protein can survive the aerosolization process at up to 50 bar without significant degradation. The protein further remains enzymatically active after the spray event.

Using an in-vitro model, we present the delivery of more complex and fragile mRNA structures (Nanoluc mRNA) at high concentrations when encapsulated in solid lipid nanoparticles (LNPs) or Extracellular vesicles (EVs). These vesicles maintain their capability to pass the cell wall in in-vitro cell cultures, leading to an expression of the encapsulated protein structure within the celll. Micro swirl nozzles can enable the portable delivery of large molecule pharmaceuticals and bring new treatment options to patients who have so far had to rely on stationary devices.

Large-molecule pharmaceuticals can offer new treatment options for severe lung diseases. However, their effective delivery to the lungs is challenged by the high-shear forces generated during the aerosolization process. These forces can degrade sensitive biomolecules, limiting their compatibility with portable inhalers and, consequently, restricting the use of biopharmaceuticals in portable drug delivery systems. Here, we demonstrate that micro-swirl nozzles can effectively aerosolize fragile biopharmaceuticals in aqueous solutions. Computational shear rate simulations of the nozzle design show that it produces low shear conditions suitable for the gentle aerosolization of sensitive pharmaceuticals. We demonstrate the aerosolization of encapsulated large molecules using a swirl nozzle integrated into a portable soft-mist inhaler. Catalase protein endures the aerosolization process at pressures up to 50 bar without notable degradation, retaining enzymatic activity post-spray event. We demonstrate the successful in vitro delivery of both mRNA and proteins encapsulated in lipid nanoparticles (LNPs) and extracellular vesicles (EVs), respectively. These vesicles maintain their structural integrity and cellular uptake capabilities in vitro, facilitating intracellular expression of the delivered biomolecules. Finally, we validate the successful in vivo administration pulmonary delivery and expression of LNP-encapsulated mRNA in pigs. This study highlights the potential of micro swirl nozzles to enable portable delivery of large-molecule therapeutics, offering new treatment options for patients who previously relied on stationary, and complex delivery systems.