Nanoparticle-Based Artificial Mitochondrial DNA Transcription Regulator: MitoScript.

The growing knowledge of the links between aberrant mitochondrial gene transcription and human diseases necessitates both an effective and dynamic approach to control mitochondrial DNA (mtDNA) transcription. To address this challenge, we developed a nanoparticle-based synthetic mitochondrial transcription regulator (MitoScript). MitoScript provides great colloidal stability, excellent biocompatibility, efficient cell uptake, and selective mitochondria targeting and can be monitored in live cells using near-infrared fluorescence. Notably, MitoScript controlled mtDNA transcription in a human cell line in an effective and selective manner. MitoScript targeting the light strand promoter region of mtDNA resulted in the downregulation of ND6 gene silencing, which eventually affected cell redox status, with considerably increased reactive oxygen species (ROS) generation. In summary, we developed MitoScript for the efficient, nonviral modification of mitochondrial DNA transcription. Our platform technology can potentially contribute to understanding the fundamental mechanisms of mitochondrial disorders and developing effective treatments for mitochondrial diseases.

Structurally Defined Water-Soluble Metallofullerene Derivatives towards Biomedical Applications.

Endohedral metallofullerenes (EMFs) are excellent carriers of rare-earth element (REE) ions in biomedical applications because they preclude the release of toxic metal ions. However, existing approaches to synthesize water-soluble EMF derivatives yield mixtures that inhibit precise drug design. Here we report the synthesis of metallobuckytrio (MBT), a three-buckyball system, as a modular platform to develop structurally defined water-soluble EMF derivatives with ligands by choice. Demonstrated with PEG ligands, the resulting water-soluble MBTs show superb biocompatibility. The Gd MBTs exhibit superior T1 relaxivity than typical Gd complexes, potentially superseding current clinical MRI contrast agents in both safety and efficiency. The Lu MBTs generated reactive oxygen species upon light irradiation, showing promise as photosensitizers. With their modular nature to incorporate other ligands, we anticipate the MBT platform to open new paths towards bio-specific REE drugs.

High-Content Screening and Analysis of Stem Cell-Derived Neural Interfaces Using a Combinatorial Nanotechnology and Machine Learning Approach.

A systematic investigation of stem cell-derived neural interfaces can facilitate the discovery of the molecular mechanisms behind cell behavior in neurological disorders and accelerate the development of stem cell-based therapies. Nevertheless, high-throughput investigation of the cell-type-specific biophysical cues associated with stem cell-derived neural interfaces continues to be a significant obstacle to overcome. To this end, we developed a combinatorial nanoarray-based method for high-throughput investigation of neural interface micro-/nanostructures (physical cues comprising geometrical, topographical, and mechanical aspects) and the effects of these complex physical cues on stem cell fate decisions. Furthermore, by applying a machine learning (ML)-based analytical approach to a large number of stem cell-derived neural interfaces, we comprehensively mapped stem cell adhesion, differentiation, and proliferation, which allowed for the cell-type-specific design of biomaterials for neural interfacing, including both adult and human-induced pluripotent stem cells (hiPSCs) with varying genetic backgrounds. In short, we successfully demonstrated how an innovative combinatorial nanoarray and ML-based platform technology can aid with the rational design of stem cell-derived neural interfaces, potentially facilitating precision, and personalized tissue engineering applications.

Advanced Drug Delivery Modulation via Hybrid Nanofibers Enhances Stem Cell Differentiation.

Seamlessly integrating soluble factors onto biomedical scaffolds with a precisely manufactured topography for efficient cell control remains elusive since many scaffold fabrication techniques degrade payloads. Surface adsorption of payloads onto synthesized nanoscaffolds retains bioactivity by removing exposure to harsh processing conditions at the expense of inefficient drug loading and uncontrolled release. Herein, we present a nanomaterial composite scaffold paradigm to improve physicochemical surface adsorption pharmacokinetics. As a proof of concept, we integrated graphene oxide (GO) and manganese dioxide (MnO2) nanosheets onto nanofibers to increase loading capacity and tune drug release. Non-degradable GO enhances payload retention, while biodegradable MnO2 enables cell-responsive drug release. To demonstrate the utility of this hybrid nanomaterial scaffold paradigm for tissue engineering, we adsorbed payloads ranging from small molecules to proteins onto the scaffold to induce myogenesis and osteogenesis for multiple stem cell lines. Scaffolds with adsorbed payloads enabled more efficient differentiation than media supplementation using equivalent quantities of differentiation factors. We attribute this increased efficacy to a reverse uptake mechanism whereby payloads are localized around seeded cells, increasing delivery efficiency for guiding differentiation. Additionally, we demonstrate spatial control over cells since differentiation factors are delivered locally through the scaffold. When co-culturing scaffolds with and without adsorbed payloads, only cells seeded on payload-adsorbed scaffolds underwent differentiation. With this modular technology being capable of enhancing multiple differentiation fates for specific cell lines, this technology provides a promising alternative for current tissue engineering scaffolds.

Predictive Biophysical Cue Mapping for Direct Cell Reprogramming Using Combinatorial Nanoarrays.

Biophysical cues, such as nanotopographies of extracellular matrix (ECM), are key cell regulators for direct cell reprogramming. Therefore, high-throughput methods capable of systematically screening a wide range of biophysical cue-regulated cell reprogramming are increasingly needed for tissue engineering and regenerative medicine. Here, we report the development of a dynamic laser interference lithography (DIL) to generate large-scale combinatorial biophysical cue (CBC) arrays with diverse micro/nanostructures at higher complexities than most current arrays. Using CBC arrays, a high-throughput cell mapping method is further demonstrated for the systematic investigation of biophysical cue-mediated direct cell reprogramming. This CBC array-based high-throughput cell screening approach facilitates the rapid identification of unconventional hierarchical nanopatterns that induce the direct reprogramming of human fibroblasts into neurons through epigenetic modulation mechanisms. In this way, we successfully demonstrate DIL for generating highly complex CBC arrays and establish CBC array-based cell screening as a valuable strategy for systematically investigating the role of biophysical cues in cell reprogramming.

Harnessing the Therapeutic Potential of Extracellular Vesicles for Biomedical Applications Using Multifunctional Magnetic Nanomaterials.

Extracellular vesicles (e.g., exosomes) carrying various biomolecules (e.g., proteins, lipids, and nucleic acids) have rapidly emerged as promising platforms for many biomedical applications. Despite their enormous potential, their heterogeneity in surfaces and sizes, the high complexity of cargo biomolecules, and the inefficient uptake by recipient cells remain critical barriers for their theranostic applications. To address these critical issues, multifunctional nanomaterials, such as magnetic nanomaterials, with their tunable physical, chemical, and biological properties, may play crucial roles in next-generation extracellular vesicles (EV)-based disease diagnosis, drug delivery, tissue engineering, and regenerative medicine. As such, one aims to provide cutting-edge knowledge pertaining to magnetic nanomaterials-facilitated isolation, detection, and delivery of extracellular vesicles and their associated biomolecules. By engaging the fields of extracellular vesicles and magnetic nanomaterials, it is envisioned that their properties can be effectively combined for optimal outcomes in biomedical applications.

Hybrid SMART spheroids to enhance stem cell therapy for CNS injuries.

Although stem cell therapy holds enormous potential for treating debilitating injuries and diseases in the central nervous system, low survival and inefficient differentiation have restricted its clinical applications. Recently, 3D cell culture methods, such as stem cell­based spheroids and organoids, have demonstrated advantages by incorporating tissue-mimetic 3D cell-cell interactions. However, a lack of drug and nutrient diffusion, insufficient cell-matrix interactions, and tedious fabrication procedures have compromised their therapeutic effects in vivo. To address these issues, we developed a biodegradable nanomaterial-templated 3D cell assembly method that enables the formation of hybrid stem cell spheroids with deep drug delivery capabilities and homogeneous incorporation of 3D cell-matrix interactions. Hence, high survival rates, controlled differentiation, and functional recovery were demonstrated in a spinal cord injury animal model. Overall, our hybrid stem cell spheroids represent a substantial development of material-facilitated 3D cell culture systems and can pave the way for stem cell­based treatment of CNS injuries.

Bioengineering Approaches for the Advanced Organoid Research.

Recent advances in 3D cell culture technology have enabled scientists to generate stem cell derived organoids that recapitulate the structural and functional characteristics of native organs. Current organoid technologies have been striding toward identifying the essential factors for controlling the processes involved in organoid development, including physical cues and biochemical signaling. There is a growing demand for engineering dynamic niches characterized by conditions that resemble in vivo organogenesis to generate reproducible and reliable organoids for various applications. Innovative biomaterial-based and advanced engineering-based approaches have been incorporated into conventional organoid culture methods to facilitate the development of organoid research. The recent advances in organoid engineering, including extracellular matrices and genetic modulation, are comprehensively summarized to pinpoint the parameters critical for organ-specific patterning. Moreover, perspective trends in developing tunable organoids in response to exogenous and endogenous cues are discussed for next-generation developmental studies, disease modeling, and therapeutics.

Developments in Bio-Inspired Nanomaterials for Therapeutic Delivery to Treat Hearing Loss.

Sensorineural hearing loss affects millions of people worldwide and is a growing concern in the aging population. Treatment using aminoglycoside antibiotics for infection and exposure to loud sounds contribute to the degeneration of cochlear hair cells and spiral ganglion neurons. Cell loss impacts cochlear function and causes hearing loss in ∼ 15% of adult Americans (∼36 million). The number of individuals with hearing loss will likely grow with increasing lifespans. Current prosthesis such as hearing aids and cochlear implants can ameliorate hearing loss. However, hearing aids are ineffective if hair cells or spiral ganglion neurons are severely damaged, and cochlear implants are ineffective without properly functioning spiral ganglion neurons. As such, strategies that alleviate hearing loss by preventing degeneration or promoting cell replacement are urgently needed. Despite showing great promise from in vitro studies, the complexity and delicate nature of the inner ear poses a huge challenge for delivering therapeutics. To mitigate risks and complications associated with surgery, new technologies and methodologies have emerged for efficient delivery of therapeutics. We will focus on biomaterials that allow controlled and local drug delivery into the inner ear. The rapid development of microsurgical techniques in conjunction with novel bio- and nanomaterials for sustained drug delivery appears bright for hearing loss treatment.

Dual-Enhanced Raman Scattering-Based Characterization of Stem Cell Differentiation Using Graphene-Plasmonic Hybrid Nanoarray.

Surface-enhanced Raman scattering (SERS) has demonstrated great potential to analyze a variety of bio/chemical molecular interactions within cells in a highly sensitive and selective manner. Despite significant advancements, it remains a critical challenge to ensure high sensitivity and selectivity, while achieving uniform signal enhancement and high reproducibility for quantitative detection of targeted biomarkers within a complex stem cell microenvironment. Herein, we demonstrate an innovative sensing platform, using graphene-coated homogeneous plasmonic metal (Au) nanoarrays, which synergize both electromagnetic mechanism (EM)- and chemical mechanism (CM)-based enhancement. Through the homogeneous plasmonic nanostructures, generated by laser interference lithography (LIL), highly reproducible enhancement of Raman signals could be obtained via a strong and uniform EM. Additionally, the graphene-functionalized surface simultaneously amplifies the Raman signals by an optimized CM, which aligns the energy level of the graphene oxide with the target molecule by tuning its oxidation levels, consequently increasing the sensitivity and accuracy of our sensing system. Using the dual-enhanced Raman scattering from both EM from the homogeneous plasmonic Au nanoarray and CM from the graphene surface, our graphene-Au hybrid nanoarray was successfully utilized to detect as well as quantify a specific biomarker (TuJ1) gene expression levels to characterize neuronal differentiation of human neural stem cells (hNSCs). Collectively, we believe our unique graphene-plasmonic hybrid nanoarray can be extended to a wide range of applications in the development of simple, rapid, and accurate sensing platforms for screening various bio/chemical molecules.

A biodegradable hybrid inorganic nanoscaffold for advanced stem cell therapy.

Stem cell transplantation, as a promising treatment for central nervous system (CNS) diseases, has been hampered by crucial issues such as a low cell survival rate, incomplete differentiation, and limited neurite outgrowth in vivo. Addressing these hurdles, scientists have designed bioscaffolds that mimic the natural tissue microenvironment to deliver physical and soluble cues. However, several significant obstacles including burst release of drugs, insufficient cellular adhesion support, and slow scaffold degradation rate remain to be overcome before the full potential of bioscaffold-based stem-cell therapies can be realized. To this end, we developed a biodegradable nanoscaffold-based method for enhanced stem cell transplantation, differentiation, and drug delivery. These findings collectively support the therapeutic potential of our biodegradable hybrid inorganic (BHI) nanoscaffolds for advanced stem cell transplantation and neural tissue engineering.

Advanced Gene Manipulation Methods for Stem Cell Theranostics.

In the field of tissue engineering, autologous cell sources are ideal to prevent adverse immune responses; however, stable and reliable cell sources are limited. To acquire more reliable cell sources, the harvesting and differentiation of stem cells from patients is becoming more and more common. To this end, the need to control the fate of these stem cells before transplantation for therapeutic purposes is urgent. Since transcription factors orchestrate all of the gene activities inside of a cell, researchers have developed engineered and synthetic transcription factors to precisely control the fate of stem cells allowing for safer and more effective cell sources. Engineered transcription factors, mutant fusion proteins of naturally occurring proteins, comprise the three main domains of natural transcription factors including DNA binding domains, transcriptional activation domains, and a linker domain. Several key advancements of engineered zinc finger proteins, transcriptional activator-like effectors, and deficient cas9 proteins have revolutionized the field of engineered transcription factors allowing for precise control of gene regulation. Synthetic transcription factors are chemically made transcription factor mimics that use small molecule based moieties to replicate the main functions of natural transcription factors. These include hairpin polyamides, triple helix forming oligonucleotides, and nanoparticle-based methods. Synthetic transcription factors allow for non-viral delivery and greater spatiotemporal control of gene expression. The developments in engineered and synthetic transcription factors have lowered the risk of tumorigenicity and improved differentiation capability of stem cells, as well as facilitated many key discoveries in the fields of cancer and stem cell biology, thus providing a stepping stone to advance regenerative medicine in the clinic for cell replacement therapies.

NanoScript: A Versatile Nanoparticle-Based Synthetic Transcription Factor for Innovative Gene Manipulation.

Cellular reprogramming and stem cell-based therapies have shown tremendous potential in the field of regenerative medicine. To that end, developing tools to control stem cell fate is an attractive area of research for replacing damaged and diseased cells and reestablishing functional connections for tissue repair. Transcription factor (TFs) proteins are well known to regulate gene expression and direct stem cell fate. Inspired by natural TFs, NanoScript, a nanoparticle (NP)-based platform, mimics TFs to afford control over gene expression and stem cell fate for regenerative medicine. Here, we describe the construction of the NanoScript platform, which is designed with tunable properties to replicate the structure and function of TFs to bind to specific portions of the genome and regulate gene expression in a way that does not involve viral delivery.

Integrating epigenetic modulators into NanoScript for enhanced chondrogenesis of stem cells.

N-(4-Chloro-3-(trifluoromethyl)phenyl)-2-ethoxybenzamide (CTB) is a small molecule that functions by altering the chromatin architecture to modulate gene expression. We report a new CTB derivative with increased solubility and demonstrate CTB's functionality by conjugating it on the recently established NanoScript platform to enhance gene expression and induce stem cell differentiation. NanoScript is a nanoparticle-based artificial transcription factor that emulates the structure and function of transcription factor proteins (TFs) to effectively regulate endogenous gene expression. Modifying NanoScript with CTB will more closely replicate the TF structure and enhance CTB functionality and gene expression. To this end, we first conjugated CTB onto NanoScript and initiated a time-dependent increase in histone acetyltransferase activity. Next, because CTB is known to trigger the pathway involved in regulating Sox9, a master regulator of chondrogenic differentiation, we modifed a Sox9-specific NanoScript with CTB to enhance chondrogenic gene activity and differentiation. Because NanoScript is a tunable and robust platform, it has potential for various gene-regulating applications, such as stem cell differentiation.