Intracellular proteomics and extracellular vesiculomics as a metric of disease recapitulation in 3D-bioprinted aortic valve arrays.

In calcific aortic valve disease (CAVD), mechanosensitive valvular cells respond to fibrosis- and calcification-induced tissue stiffening, further driving pathophysiology. No pharmacotherapeutics are available to treat CAVD because of the paucity of (i) appropriate experimental models that recapitulate this complex environment and (ii) benchmarking novel engineered aortic valve (AV)-model performance. We established a biomaterial-based CAVD model mimicking the biomechanics of the human AV disease-prone fibrosa layer, three-dimensional (3D)-bioprinted into 96-well arrays. Liquid chromatography-tandem mass spectrometry analyses probed the cellular proteome and vesiculome to compare the 3D-bioprinted model versus traditional 2D monoculture, against human CAVD tissue. The 3D-bioprinted model highly recapitulated the CAVD cellular proteome (94% versus 70% of 2D proteins). Integration of cellular and vesicular datasets identified known and unknown proteins ubiquitous to AV calcification. This study explores how 2D versus 3D-bioengineered systems recapitulate unique aspects of human disease, positions multiomics as a technique for the evaluation of high throughput-based bioengineered model systems, and potentiates future drug discovery.

Nanoparticle-Mediated Delivery of Anti-PU.1 siRNA via Localized Intracisternal Administration Reduces Neuroinflammation.

Neuroinflammation is a hallmark of neurodegenerative disorders including Alzheimer's disease (AD). Microglia, the brain's immune cells, express many of the AD-risk loci identified in genome wide association studies and present a promising target for anti-inflammatory RNA therapeutics but are difficult to transfect with current methods. Here, several lipid nanoparticle (LNP) formulations are examined, and a lead candidate that supports efficient RNA delivery in cultures of human stem cell-derived microglia-like cells (iMGLs) and animal models of neuroinflammation is identified. The lead microglia LNP (MG-LNP) formulation shows minimal toxicity and improves delivery efficiency to inflammatory iMGLs, suggesting a preference for delivery into activated microglia. Intraperitoneal injection of the MG-LNP formulation generates widespread expression of the delivered reporter construct in all organs, whereas local intracisternal injection directly into the cerebrospinal fluid leads to preferential expression in the brain. It is shown that LNP-mediated delivery of siRNA targeting the PU.1 transcription factor, a known AD-risk locus, successfully reduces PU.1 levels in iMGLs and reduces neuroinflammation in mice injected with LPS and in CK-p25 mice that mimic the chronic neuroinflammation seen in AD patients. The LNP formulation represents an effective RNA delivery vehicle when applied intrathecally and can be broadly utilized to test potential neuroinflammation-directed gene therapies.

Nucleic acid delivery for therapeutic applications.

Recent medical advances have exploited the ability to address a given disease at the underlying level of transcription and translation. These treatment paradigms utilize nucleic acids - including short interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), and messenger RNA (mRNA) - to achieve a desired outcome ranging from gene knockdown to induced expression of a selected target protein. Towards this end, numerous strategies for encapsulation or stabilization of various nucleic acid structures have been developed in order to achieve intracellular delivery. In this review, we discuss several therapeutic applications of nucleic acids directed towards specific diseases and tissues of interest, in particular highlighting recent technologies which have reached late-stage clinical trials and received FDA approval.

Nucleic acid delivery and nanoparticle design for COVID vaccines.

ABSTRACT: Nucleic acid therapeutics offer a new paradigm to rapidly respond to global health problems. The versatility of nucleic acids, especially in RNA therapies, provides the ability to tune levels of specific protein expression, achieving downregulation through short interfering RNA (siRNA) or upregulation by messenger RNA (mRNA) administration. Recent advances in the development of delivery vehicles, including nonviral nanoparticles are crucial to overcome the innate barriers to nucleic acid delivery. Toward this end, current clinical approaches have utilized mRNA and lipid nanoparticles (LNPs) to address the COVID-19 pandemic through novel vaccine strategies, producing efficacious vaccines within one year of sequencing the SARS-CoV-2 genome. Here, we review fundamental concepts required to achieve successful nucleic acid delivery, including the design of LNP systems optimized for mRNA vaccine applications.

Outlooks on Three-Dimensional Printing for Ocular Biomaterials Research.

Given its potential for high-resolution, customizable, and waste-free fabrication of medical devices and in vitro biological models, 3-dimensional (3D) bioprinting has broad utility within the biomaterials field. Indeed, 3D bioprinting has to date been successfully used for the development of drug delivery systems, the recapitulation of hard biological tissues, and the fabrication of cellularized organ and tissue-mimics, among other applications. In this study, we highlight convergent efforts within engineering, cell biology, soft matter, and chemistry in an overview of the 3D bioprinting field, and we then conclude our work with outlooks toward the application of 3D bioprinting for ocular research in vitro and in vivo.

Polymers for extended-release administration.

Developing strategies to deliver the required dose of therapeutics into target tissues and cell populations within the body is a principal aim of controlled release and drug delivery. Specifically, there is an interest in developing formulations that can achieve drug concentrations within the therapeutic window, for extended periods of time, with tunable release profiles, and with minimal complication and distress for the patient. To date, drug delivery systems have been developed to serve as depots, triggers, and carriers for therapeutics including small molecules, biologics, and cell-based therapies. Notably, the efficacy of these systems is intricately tied to the manner in which they are administered. For example, systemic and oral routes of administration are common, but both can result in rapid clearance from the organism. Towards this end, what formulation and administration route strategies are available to prolong the bioavailability of therapeutics? Here, we discuss historical and modern drug delivery systems, with the intention of exploring how properties including formulation, administration route and chemical structure influence the ability to achieve extended-release drug release profiles within the body.

ß-Aminoacrylate Synthetic Hydrogels: Easily Accessible and Operationally Simple Biomaterials Networks.

The development of new material platforms can improve our ability to study biological processes. Here, we developed a water-compatible variant of a click-like polymerization between alkynoates and secondary amines to form ß-aminoacrylate synthetic polyethylene glycol (PEG) based hydrogels. These materials are easy to access-PEG alkynoate was synthesized on a 100 gram scale and the amines were available commercially; these materials are also operationally simple to formulate-gel formation occurred upon simple mixing of precursor solutions without the need for initiators, catalysts, nor specialized equipment. Three-dimensional cell culture experiments also indicated cytocompatibility of these gels with >90 % viability retained in THP-1 and NIH/3T3 cells after 72 hours in culture. This hydrogel system therefore represents an alternative platform to other click and click-like hydrogels with improved accessibility and user-friendliness for biomaterials application.

Customizable Lipid Nanoparticle Materials for the Delivery of siRNAs and mRNAs.

RNAs are a promising class of therapeutics given their ability to regulate protein concentrations at the cellular level. Developing safe and effective strategies to deliver RNAs remains important for realizing their full clinical potential. Here, we develop lipid nanoparticle formulations that can deliver short interfering RNAs (for gene silencing) or messenger RNAs (for gene upregulation). Specifically, we study how the tail length, tail geometry, and linker spacing in diketopiperazine lipid materials influences LNP potency with siRNAs and mRNAs. Eight lipid materials are synthesized, and 16 total formulations are screened for activity in vitro; the lead material is evaluated with mRNA for in vivo use and demonstrates luciferase protein expression in the spleen. In undertaking this approach, not only do we develop synthetic routes to delivery materials, but we also reveal structural criteria that could be useful for developing next-generation delivery materials for RNA therapeutics.

Engineering a 3D-Bioprinted Model of Human Heart Valve Disease Using Nanoindentation-Based Biomechanics.

In calcific aortic valve disease (CAVD), microcalcifications originating from nanoscale calcifying vesicles disrupt the aortic valve (AV) leaflets, which consist of three (biomechanically) distinct layers: the fibrosa, spongiosa, and ventricularis. CAVD has no pharmacotherapy and lacks in vitro models as a result of complex valvular biomechanical features surrounding resident mechanosensitive valvular interstitial cells (VICs). We measured layer-specific mechanical properties of the human AV and engineered a three-dimensional (3D)-bioprinted CAVD model that recapitulates leaflet layer biomechanics for the first time. Human AV leaflet layers were separated by microdissection, and nanoindentation determined layer-specific Young’s moduli. Methacrylated gelatin (GelMA)/methacrylated hyaluronic acid (HAMA) hydrogels were tuned to duplicate layer-specific mechanical characteristics, followed by 3D-printing with encapsulated human VICs. Hydrogels were exposed to osteogenic media (OM) to induce microcalcification, and VIC pathogenesis was assessed by near infrared or immunofluorescence microscopy. Median Young’s moduli of the AV layers were 37.1, 15.4, and 26.9 kPa (fibrosa/spongiosa/ventricularis, respectively). The fibrosa and spongiosa Young’s moduli matched the 3D 5% GelMa/1% HAMA UV-crosslinked hydrogels. OM stimulation of VIC-laden bioprinted hydrogels induced microcalcification without apoptosis. We report the first layer-specific measurements of human AV moduli and a novel 3D-bioprinted CAVD model that potentiates microcalcification by mimicking the native AV mechanical environment. This work sheds light on valvular mechanobiology and could facilitate high-throughput drug-screening in CAVD.