Inducers of the endothelial cell barrier identified through chemogenomic screening in genome-edited hPSC-endothelial cells.

The blood-retina barrier and blood-brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-ß pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-ß, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibiting multiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.

Modeling early pathophysiological phenotypes of diabetic retinopathy in a human inner blood-retinal barrier-on-a-chip.

Diabetic retinopathy (DR) is a microvascular disorder characterized by inner blood-retinal barrier (iBRB) breakdown and irreversible vision loss. While the symptoms of DR are known, disease mechanisms including basement membrane thickening, pericyte dropout and capillary damage remain poorly understood and interventions to repair diseased iBRB microvascular networks have not been developed. In addition, current approaches using animal models and in vitro systems lack translatability and predictivity to finding new target pathways. Here, we develop a diabetic iBRB-on-a-chip that produces pathophysiological phenotypes and disease pathways in vitro that are representative of clinical diagnoses. We show that diabetic stimulation of the iBRB-on-a-chip mirrors DR features, including pericyte loss, vascular regression, ghost vessels, and production of pro-inflammatory factors. We also report transcriptomic data from diabetic iBRB microvascular networks that may reveal drug targets, and examine pericyte-endothelial cell stabilizing strategies. In summary, our model recapitulates key features of disease, and may inform future therapies for DR.

Retinal Microvasculature-on-a-Chip for Modeling VEGF-Induced Permeability.

Relevant human in vitro models of the retinal microvasculature can be used to study the role of disease mediators on retinal barrier dysfunction and assess the efficacy of early drug candidates. This chapter describes an organ-on-a-chip model of the retinal microvasculature that allows for facile quantification of barrier permeability in response to leakage mediators, such as Vascular Endothelial Growth Factor (VEGF), and enables screening of VEGF-induced permeability inhibitors. This chapter also presents an automated confocal imaging method for the visualization of endothelial tube morphology as an additional measure of barrier integrity.

Microphysiological Neurovascular Barriers to Model the Inner Retinal Microvasculature.

Blood-neural barriers regulate nutrient supply to neuronal tissues and prevent neurotoxicity. In particular, the inner blood-retinal barrier (iBRB) and blood-brain barrier (BBB) share common origins in development, and similar morphology and function in adult tissue, while barrier breakdown and leakage of neurotoxic molecules can be accompanied by neurodegeneration. Therefore, pre-clinical research requires human in vitro models that elucidate pathophysiological mechanisms and support drug discovery, to add to animal in vivo modeling that poorly predict patient responses. Advanced cellular models such as microphysiological systems (MPS) recapitulate tissue organization and function in many organ-specific contexts, providing physiological relevance, potential for customization to different population groups, and scalability for drug screening purposes. While human-based MPS have been developed for tissues such as lung, gut, brain and tumors, few comprehensive models exist for ocular tissues and iBRB modeling. Recent BBB in vitro models using human cells of the neurovascular unit (NVU) showed physiological morphology and permeability values, and reproduced brain neurological disorder phenotypes that could be applicable to modeling the iBRB. Here, we describe similarities between iBRB and BBB properties, compare existing neurovascular barrier models, propose leverage of MPS-based strategies to develop new iBRB models, and explore potentials to personalize cellular inputs and improve pre-clinical testing.

Surface Tension-Assisted Additive Manufacturing of Tubular, Multicomponent Biomaterials.

The fabrication of functional biomaterials for organ replacement and tissue repair remains a major goal of biomedical engineering. Advances in additive manufacturing (AM) technologies and computer-aided design (CAD) are advancing the tools available for the production of these devices. Ideally, these constructs should be matched to the geometry and mechanical properties of the tissue at the needed implant site. To generate geometrically defined and structurally supported multicomponent and cell-laden biomaterials, we have developed a method to integrate hydrogels with 3D-printed lattice scaffolds leveraging surface tension-assisted AM.

Additive manufacturing in drug delivery: Innovative drug product design and opportunities for industrial application.

Additive manufacturing (AM) or 3D printing is enabling new directions in product design. The adoption of AM in various industrial sectors has led to major transformations. Similarly, AM presents new opportunities in the field of drug delivery, opening new avenues for improved patient care. In this review, we discuss AM as an innovative tool for drug product design. We provide a brief overview of the different AM processes and their respective impact on the design of drug delivery systems. We highlight several enabling features of AM, including unconventional release, customization, and miniaturization, and discuss several applications of AM for the fabrication of drug products. This includes products that have been approved or are in development. As the field matures, there are also several new challenges to broad implementation in the pharmaceutical landscape. We discuss several of these from the regulatory and industrial perspectives and provide an outlook for how these issues may be addressed. The introduction of AM into the field of drug delivery is an enabling technology and many new drug products can be created through productive collaboration of engineers, materials scientists, pharmaceutical scientists, and industrial partners.

Organ-On-A-Chip Technologies for Advanced Blood-Retinal Barrier Models.

The blood-retinal barrier (BRB) protects the retina by maintaining an adequate microenvironment for neuronal function. Alterations of the junctional complex of the BRB and consequent BRB breakdown in disease contribute to a loss of neuronal signaling and vision loss. As new therapeutics are being developed to prevent or restore barrier function, it is critical to implement physiologically relevant in vitro models that recapitulate the important features of barrier biology to improve disease modeling, target validation, and toxicity assessment. New directions in organ-on-a-chip technology are enabling more sophisticated 3-dimensional models with flow, multicellularity, and control over microenvironmental properties. By capturing additional biological complexity, organs-on-chip can help approach actual tissue organization and function and offer additional tools to model and study disease compared with traditional 2-dimensional cell culture. This review describes the current state of barrier biology and barrier function in ocular diseases, describes recent advances in organ-on-a-chip design for modeling the BRB, and discusses the potential of such models for ophthalmic drug discovery and development.

Human Retinal Microvasculature-on-a-Chip for Drug Discovery.

Retinal cells within neurovascular units generate the blood-retinal barrier (BRB) to regulate the local retinal microenvironment and to limit access to inflammatory cells. Breakdown of the endothelial junctional complexes in the BRB negatively affects neuronal signaling and ultimately causes vision loss. As new therapeutics are being developed either to prevent barrier disruption or to restore barrier function, access to physiologically relevant human in vitro tissue models that recapitulate important features of barrier biology is essential for disease modeling, target validation, and toxicity assessment. Here, a tunable organ-on-a-chip model of the retinal microvasculature using human retinal microvascular endothelial cells with integrated flow is described. Automated imaging and image analysis methods are employed for facile screening of leakage mediators and cytokine inhibitors on barrier properties. The developed retinal microvasculature-on-a-chip will enable improved understanding of BRB biology and provide an additional tool for drug discovery.

Surface tension-assisted additive manufacturing.

The proliferation of computer-aided design and additive manufacturing enables on-demand fabrication of complex, three-dimensional structures. However, combining the versatility of cell-laden hydrogels within the 3D printing process remains a challenge. Herein, we describe a facile and versatile method that integrates polymer networks (including hydrogels) with 3D-printed mechanical supports to fabricate multicomponent (bio)materials. The approach exploits surface tension to coat fenestrated surfaces with suspended liquid films that can be transformed into solid films. The operating parameters for the process are determined using a physical model, and complex geometric structures are successfully fabricated. We engineer, by tailoring the window geometry, scaffolds with anisotropic mechanical properties that compress longitudinally (~30% strain) without damaging the hydrogel coating. Finally, the process is amenable to high cell density encapsulation and co-culture. Viability (>95%) was maintained 28 days after encapsulation. This general approach can generate biocompatible, macroscale devices with structural integrity and anisotropic mechanical properties.

Nanoparticle-based drug delivery systems: a commercial and regulatory outlook as the field matures.

INTRODUCTION: Nanomedicine has emerged as a major field of academic research with direct impact on human health. While a first generation of products has been successfully commercialized and has significantly contributed to enhance patient's life, recent advances in material design and the emergence of new therapeutics are contributing to the development of more sophisticated systems. As the field matures, it is important to comprehend the challenges related to nanoparticle commercial development for a more efficient and predictable path to the clinic. Areas covered: The review provides an overview of nanoparticle-based delivery systems currently on the market and in clinical trials, and discuss the principal challenges for their commercial development, both from a manufacturing and regulatory perspective, to help gain understanding of the translational path for these systems. Expert opinion: Clinical translation of nanoparticle-based delivery systems remains challenging on account of their 3D nanostructure and requires robust nano-manufacturing process along with adequate analytical tools and methodologies. By identifying early enough in the development the product critical attributes and understanding their impact on the therapeutic performance, the developers of nanopharmaceuticals will be better equipped to develop efficient product pipelines. Second-generation products are expected to broaden nanopharmaceutical global market in the upcoming years.

Comprehensive proteomic characterization of stem cell-derived extracellular matrices.

In the stem-cell niche, the extracellular matrix (ECM) serves as a structural support that additionally provides stem cells with signals that contribute to the regulation of stem-cell function, via reciprocal interactions between cells and components of the ECM. Recently, cell-derived ECMs have emerged as in vitro cell culture substrates to better recapitulate the native stem-cell microenvironment outside the body. Significant changes in cell number, morphology and function have been observed when mesenchymal stem cells (MSC) were cultured on ECM substrates as compared to standard tissue-culture polystyrene (TCPS). As select ECM components are known to regulate specific stem-cell functions, a robust characterization of cell-derived ECM proteomic composition is critical to better comprehend the role of the ECM in directing cellular processes. Here, we characterized and compared the protein composition of ECM produced in vitro by bone marrow-derived MSC, adipose-derived MSC and neonatal fibroblasts from different donors, employing quantitative proteomic methods. Each cell-derived ECM displayed a specific and unique matrisome signature, yet they all shared a common set of proteins. We evaluated the biological response of cells cultured on the different matrices and compared them to cells on standard TCPS. The matrices lead to differential survival and gene-expression profiles among the cell types and as compared to TCPS, indicating that the cell-derived ECMs influence each cell type in a different manner. This general approach to understanding the protein composition of different tissue-specific and cell-derived ECM will inform the rational design of defined systems and biomaterials that recapitulate critical ECM signals for stem-cell culture and tissue engineering.

Chitosan Nanoparticles for SiRNA Delivery In Vitro.

RNA interference, the process in which small interfering RNAs (SiRNAs) silence a specific gene and thus inhibit the associated protein, has opened new doors for the treatment of a wide range of diseases. However, efficient delivery of SiRNAs remains a challenge, especially due to their instability in biological environments and their inability to cross cell membranes. To protect and deliver SiRNAs to mammalian cells, a variety of polymeric nanocarriers have been developed. Among them, the polysaccharide chitosan has generated great interests. This derivative of natural chitin is biodegradable and biocompatible, and can complex SiRNAs into nanoparticles on account of its positive charges. However, chitosan presents some limitations that need to be taken into account when designing chitosan/SiRNA nanoparticles. Here, we describe a method to prepare SiRNA/chitosan nanoparticles with high gene silencing efficiency and low cytotoxicity by using the ionic gelation technique.

Delivery of siRNA targeting tumor metabolism using non-covalent PEGylated chitosan nanoparticles: Identification of an optimal combination of ligand structure, linker and grafting method.

PEGylated chitosan-based nanoparticles offer attractive platforms for siRNA cocktail delivery into tumors. Still, therapeutic efficacy requires us to select a rational combination of siRNAs and an efficient tumor delivery after systemic administration. Here, we showed that non-covalent PEGylation of chitosan-based nanoparticles loaded with siRNA targeting two key transporters of energy fuels for cancer cells, namely the lactate transporter MCT1 and the glutamine transporter ASCT2, could lead to significant antitumor effects. As a ligand, we tested variations of the prototypical RGD peptidomimetic (RGDp). A higher siRNA delivery was obtained with naphthyridine-containing RGDp randomly conjugated on the PEG chain by clip photochemistry and the use of a lipophilic linker than when using traditional chain-end grafting and RGDp with a hydrophilic linker. The antiproliferative effects resulting from ASCT2 and MCT1 silencing were validated separately in vitro in conditions mimicking specific metabolic profiles of cancer cells and in vivo upon concomitant delivery. The combination of those siRNA and the selected components of targeted RGDp nanoparticles led to a dramatic tumor growth inhibition upon peri-tumoral but also systemic administration in mice. Altogether these data emphasize the convenience of using non-covalent PEGylated chitosan particles to produce sheddable stealth protection compatible with an efficient siRNA delivery in tumors.

Intracellular siRNA delivery dynamics of integrin-targeted, PEGylated chitosan-poly(ethylene imine) hybrid nanoparticles: A mechanistic insight.

Integrin-targeted nanoparticles are promising for the delivery of small interfering RNA (siRNA) to tumor cells or tumor endothelium in cancer therapy aiming at silencing genes essential for tumor growth. However, during the process of optimizing and realizing their full potential, it is pertinent to gain a basic mechanistic understanding of the bottlenecks existing for nanoparticle-mediated intracellular delivery. We designed αvß3 integrin-targeted nanoparticles by coupling arginine-glycine-aspartate (RGD) or RGD peptidomimetic (RGDp) ligands to the surface of poly(ethylene glycol) (PEG) grafted chitosan-poly(ethylene imine) hybrid nanoparticles. The amount of intracellular siRNA delivered by αvß3-targeted versus non-targeted nanoparticles was quantified in the human non-small cell lung carcinoma cell line H1299 expressing enhanced green fluorescent protein (EGFP) using a stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) approach. Data demonstrated that the internalization of αvß3-targeted nanoparticles was highly dependent on the surface concentration of the ligand. Above a certain threshold concentration, the use of targeted nanoparticles provided a two-fold increase in the number of siRNA copies/cell, subsequently resulting in as much as 90% silencing of EGFP at well-tolerated carrier concentrations. In contrast, non-targeted nanoparticles mediated low levels of gene silencing, despite relatively high intracellular siRNA concentrations, indicating that these nanoparticles might end up in late endosomes or lysosomes without releasing their cargo to the cell cytoplasm. Thus, the silencing efficiency of the chitosan-based nanoparticles is strongly dependent on the uptake and the intracellular trafficking in H1299 EGFP cells, which is critical information towards a more complete understanding of the delivery mechanism that can facilitate the future design of efficient siRNA delivery systems.

Complexity in the therapeutic delivery of RNAi medicines: an analytical challenge.

INTRODUCTION: Nucleic acids have witnessed a dramatic acceleration in their therapeutic exploitation and currently represent a growing number of applications in drug development pipelines. However, a more wide-spread development of therapeutics based on nucleic acids is restricted by their poor chemical and enzymatic stability in vivo, lack of cellular uptake and insufficient capability to reach intracellular targets. AREAS COVERED: Advanced formulation of nucleic acids in nano-sized carriers may help unlocking their potential as therapeutic agents. Nano-sized matters own specific features responsible for inducing characteristic interactions with biological molecules and tissues. These properties enable for the enhancement of the nano-formulation's therapeutic efficacy, but on the other hand, the nanomatters interactions in biological fluids are also responsible for adverse effects. The purpose of this review is to reflect on the complexity in the therapeutic delivery of RNA interference-based drugs emerging from the recent clinical experiences and report the actual technological and analytical advances introduced to solve it. EXPERT OPINION: The complexity in the therapeutic delivery of nucleic acids and the heterogeneity of side effects make the interpretation of the therapeutic outcome difficult. Hence the development of analytical approaches applicable in the field of nucleic acid delivery is becoming a major challenge.

Chitosan-based siRNA delivery systems.

Recently, chitosan has attracted significant attention in the formulation of small interfering RNA (siRNA). Because of its cationic nature, chitosan can easily complex siRNA, thus readily forming nanoparticles. Moreover, chitosan is biocompatible and biodegradable, which make it a good candidate for siRNA delivery in vivo. However, chitosan requires further development to achieve high efficiency. This review will describe the major barriers that impair the efficiency of the chitosan-based siRNA delivery systems, including the stability of the delivery system in biological fluids and endosomal escape. Several solutions to counteract these barriers have been developed and will be discussed. The parameters to consider for designing powerful delivery systems will be described, particularly the possibilities for grafting targeting ligands. Finally, optimized systems that allow in vivo therapeutic applications for both local and systemic delivery will be reviewed. This review will present recent improvements in chitosan-based siRNA delivery systems that overcome many of these system's previous pitfalls and pave the way to a new generation of siRNA delivery systems.

Nanoemulsion formulation of fisetin improves bioavailability and antitumour activity in mice.

The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) has shown antitumour activity but its administration is complicated by its low water solubility. Our aim was to incorporate fisetin into a nanoemulsion to improve its pharmacokinetics and therapeutic efficacy. Solubility and emulsification tests allowed to develop an optimal nanoemulsion composed of Miglyol 812N/Labrasol/Tween 80/Lipoid E80/water (10%/10%/2.5%/1.2%/76.3%). The nanoemulsion had an oil droplet diameter of 153 ± 2 nm, a negative zeta potential (-28.4 ± 0.6 mV) and a polydispersity index of 0.129. The nanoemulsion was stable at 4 °C for 30 days, but phase separation occurred at 20 °C. Pharmacokinetic studies in mice revealed that the fisetin nanoemulsion injected intravenously (13 mg/kg) showed no significant difference in systemic exposure compared to free fisetin. However, when the fisetin nanoemulsion was administered intraperitoneally, a 24-fold increase in fisetin relative bioavailability was noted, compared to free fisetin. Additionally, the antitumour activity of the fisetin nanoemulsion in Lewis lung carcinoma bearing mice occurred at lower doses (36.6 mg/kg) compared to free fisetin (223 mg/kg). In conclusion, we have developed a stable nanoemulsion of fisetin and have shown that it could improve its relative bioavailability and antitumour activity.