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Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...

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Fabrication and Characterization of Layer-By-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo ...

University of Connecticut

Chondrosarcoma is a highly aggressive primary malignant bone tumor mostly occurring in adults. There are no effective systemic treatments, and patients with this disease have poor survival. miR-181a is an oncomiR that is overexpressed in high-grade chondrosarcoma and promotes tumor progression. Regulator of G-protein signaling 16 (RGS16) is a target of miR-181a. Inhibition of RGS16 expression by miR-181a enhances CXC chemokine receptor 4 signaling, which in turn increases MMP1 and VEGF expression, angiogenesis, and metastasis. Here, we report the results of systemic treatment with anti-miRNA oligonucleotides (AMO) directed against miR-181a utilizing a nanopiece delivery platform (NPs). NPs were combined with a molecular beacon or anti-miR-181a oligonucleotides and are shown to transfect chondrosarcoma cells  and  Intratumoral injection and systemic delivery had similar effects on miR-181a expression in nude mice bearing chondrosarcoma xenografts. Systemic delivery of NPs carrying anti-miR-181a also restored  expression, decreased expression of VEGF and MMP1, MMP activity, and tumor volume by 32% at day 38, and prolonged survival from 23% to 45%. In conclusion, these data support that systemic delivery of AMO shows promise for chondrosarcoma treatment.

Anti-miRNA Oligonucleotide Therapy for Chondrosarcoma.

Mesenchymal stem cells (MSCs) have been widely applied in biomedicine due to their ability to differentiate into many different cell types and their ability to synthesize a broad spectrum of growth factors and cytokines that directly and indirectly influence other cells in their vicinity. To guide MSC infiltration to a bone fracture site, we developed a novel self-assembled Nano-Matrix which can be used as an injectable scaffold to repair bone fractures. The Nano-Matrix is formed by Janus base nanotubes (JBNTs) and fibronectin (FN). JBNTs are nucleobase-derived nanotubes mimicking collagen fibers, and FN is one of the cell adhesive glycoproteins which is responsible for cell-extracellular matrix interactions and guides stem cell migration and differentiation to desired cells types. Here, we demonstrated the successful fabrication and characterization of the JBNT/FN Nano-Matrix as well as its excellent bioactivity that encouraged human MSC migration and adhesion. This work lays a solid foundation for using the Nano-Matrix as an injectable approach to improve MSC retention and function during bone fracture healing.

Self-assembled biomimetic Nano-Matrix for stem cell anchorage.

RNA delivery into deep tissues with dense extracellular matrix (ECM) has been challenging. For example, cartilage is a major barrier for RNA and drug delivery due to its avascular structure, low cell density and strong negative surface charge. Cartilage ECM is comprised of collagens, proteoglycans, and various other noncollagneous proteins with a spacing of 20nm. Conventional nanoparticles are usually spherical with a diameter larger than 50-60nm (after cargo loading). Therefore, they presented limited success for RNA delivery into cartilage. Here, we developed Janus base nanotubes (JBNTs, self-assembled nanotubes inspired from DNA base pairs) to assemble with small RNAs to form nano-rod delivery vehicles (termed as "Nanopieces"). Nanopieces have a diameter of ~20nm (smallest delivery vehicles after cargo loading) and a length of ~100nm. They present a novel breakthrough in ECM penetration due to the reduced size and adjustable characteristics to encourage ECM and intracellular penetration.

RNA Delivery via DNA-Inspired Janus Base Nanotubes for Extracellular Matrix Penetration.

A variety of engineered nanoparticles, including lipid nanoparticles, polymer nanoparticles, gold nanoparticles, and biomimetic nanoparticles, have been studied as delivery vehicles for biomedical applications. When assessing the efficacy of a nanoparticle-based delivery system,  testing with a model delivery system is crucial because it allows for real-time,  quantitative transport analysis, which is often difficult with  animal models. The advent of tissue engineering has offered methods to create experimental models that can closely mimic the 3D microenvironment in the human body. This review paper overviews the types of nanoparticle vehicles, their application areas, and the design strategies to improve delivery efficiency, followed by the uses of engineered microtissues and methods of analysis. In particular, this review highlights studies on multicellular spheroids and other 3D tissue engineering approaches for cancer drug development. The use of bio-engineered tissues can potentially provide low-cost, high-throughput, and quantitative experimental platforms for the development of nanoparticle-based delivery systems.

Studies of nanoparticle delivery with  bio-engineered microtissues.

Nanotechnology platforms, such as nanoparticles, liposomes, dendrimers, and micelles have been studied extensively for various drug deliveries, to treat or prevent diseases by modulating physiological or pathological processes. The delivery drug molecules range from traditional small molecules to recently developed biologics, such as proteins, peptides, and nucleic acids. Among them, proteins have shown a series of advantages and potential in various therapeutic applications, such as introducing therapeutic proteins due to genetic defects, or used as nanocarriers for anticancer agents to decelerate tumor growth or control metastasis. This review discusses the existing nanoparticle delivery systems, introducing design strategies, advantages of using each system, and possible limitations. Moreover, we will examine the intracellular delivery of different protein therapeutics, such as antibodies, antigens, and gene editing proteins into the host cells to achieve anticancer effects and cancer vaccines. Finally, we explore the current applications of protein delivery in anticancer treatments.

Nanomaterials for Protein Delivery in Anticancer Applications.

Degenerative Disc Disease (DDD) causes a nagging to severe back pain as well as numbing sensation to the extremities leading to loss of overall patients' height and weakness to leg muscles. Degenerative disc disease is often observed in aging patients as well as patients who have suffered from a back injury. Cervical Degenerative Disc Disease (CDDD) is a progressive condition that leads to the degeneration of the intervertebral discs supporting the cervical vertebral column. Anterior Cervical Interbody Fusion (ACIF) has been the longstanding treatment option for severe degenerative disc disease; however, ACIF presents various novel complications, necessitating numerous comparative device studies to reduce the negative effects of spinal fusion. Cervical disc arthroplasty, the recent focus of clinical attention, was one of the alternatives studied to mitigate the complications associated with vertebral fusion but presents its own disadvantages. These complications prompted further investigation and modifications that can be introduced into these devices. We will be discussing the nano-scale interactions between the implant and extracellular matrix play a crucial role in device integration and efficacy, providing an additional approach towards a device's overall success.

Nano-Scale Surface Modifications to Advance Current Treatment Options for Cervical Degenerative Disc Disease (CDDD).

Molecular Engineering of DNA-inspired Janus base nanomaterials.

mRNA Detection with Fluorescence-base Imaging Techniques for Arthritis Diagnosis.

To realize RNA interference (RNAi) therapeutics, it is necessary to deliver therapeutic RNAs (such as small interfering RNA or siRNA) into cell cytoplasm. A major challenge of RNAi therapeutics is the endosomal entrapment of the delivered siRNA. In this study, we developed a family of delivery vehicles called Janus base nanopieces (NPs). They are rod-shaped nanoparticles formed by bundles of Janus base nanotubes (JBNTs) with RNA cargoes incorporated inside via charge interactions. JBNTs are formed by noncovalent interactions of small molecules consisting of a base component mimicking DNA bases and an amino acid side chain. NPs presented many advantages over conventional delivery materials. NPs efficiently entered cells via macropinocytosis similar to lipid nanoparticles while presenting much better endosomal escape ability than lipid nanoparticles; NPs escaped from endosomes via a "proton sponge" effect similar to cationic polymers while presenting significant lower cytotoxicity compared to polymers and lipids due to their noncovalent structures and DNA-mimicking chemistry. In a proof-of-concept experiment, we have shown that NPs are promising candidates for antiviral delivery applications, which may be used for conditions such as COVID-19 in the future.

DNA-inspired nanomaterials for enhanced endosomal escape.

Articular cartilage injury, as a hallmark of arthritic diseases, is difficult to repair and causes joint pain, stiffness, and loss of mobility. Over the years, the most significant problems for the drug-based treatment of arthritis have been related to drug administration and delivery. In recent years, much research has been devoted to developing new strategies for repairing or regenerating the damaged osteoarticular tissue. The RNA interference (RNAi) has been suggested to have the potential for implementation in targeted therapy in which the faulty gene can be edited by delivering its complementary Short Interfering RNA (siRNA) at the post-transcriptional stage. The successful editing of a specific gene by the delivered siRNA might slow or halt osteoarthritic diseases without side effects caused by chemical inhibitors. However, cartilage siRNA delivery remains a challenging objective because cartilage is an avascular and very dense tissue with very low permeability. Furthermore, RNA is prone to degradation by serum nucleases (such as RNase H and RNase A) due to an extra hydroxyl group in its phosphodiester backbone. Therefore, successful delivery is the first and most crucial requirement for efficient RNAi therapy. Nanomaterials have emerged as highly advantage tools for these studies, as they can be engineered to protect siRNA from degrading, address barriers in siRNA delivery to joints, and target specific cells. This review will discuss recent breakthroughs of different siRNA delivery technologies for cartilage diseases.

Short interfering RNA (siRNA)-Based Therapeutics for Cartilage Diseases.

Various biomaterial scaffolds have been developed for improving stem cell anchorage and function in tissue constructs for in vitro and in vivo uses. Growth factors are typically applied to scaffolds to mediate cell differentiation. Conventionally, growth factors are not strictly localized in the scaffolds; thus, they may leak into the surrounding environment, causing undesired side effects on tissues or cells. Hence, there is a need for improved tissue construct strategies based on highly localized drug delivery and a homeostatic microenvironment. This study developed an injectable nanomatrix (NM) scaffold with a layer-by-layer structure inside each nanosized fiber of the scaffold based on controlled self-assembly at the molecular level. The NM was hierarchically assembled from Janus base nanotubes (JBNTs), matrilin-3, and transforming growth factor β-1 (TGF-β1) via bioaffinity. JBNTs, which form the NM backbone, are novel DNA-inspired nanomaterials that mimic the natural helical nanostructures of collagens. The chondrogenic factor, TGF-β1, was enveloped in the inner layer inside the NM fibers to prevent its release. Matrilin-3 was incorporated into the outer layer to create a cartilage-mimicking microenvironment and to maintain tissue homeostasis. Interestingly, human mesenchymal stem cells (hMSCs) had a strong preference to anchor along the NM fibers and formed a localized homeostatic microenvironment. Therefore, this NM has successfully generated highly organized structures via molecular self-assembly and achieved localized drug delivery and stem cell anchorage for homeostatic tissue constructs.

Controlled Self-Assembly of DNA-Mimicking Nanotubes to Form a Layer-by-Layer Scaffold for Homeostatic Tissue Constructs.

Research interest in nanoscale biomaterials has continued to grow in the past few decades, driving the need to form families of nanomaterials grouped by similar physical or chemical properties. Nanotubes have occupied a unique space in this field, primarily due to their high versatility in a wide range of biomedical applications. Although similar in morphology, members of this nanomaterial family widely differ in synthesis methods, mechanical and physiochemical properties, and therapeutic applications. As this field continues to develop, it is important to provide insight into novel biomaterial developments and their overall impact on current technology and therapeutics. In this review, we aim to characterize and compare two members of the nanotube family: carbon nanotubes (CNTs) and janus-base nanotubes (JBNts). While CNTs have been extensively studied for decades, JBNts provide a fresh perspective on many therapeutic modalities bound by the limitations of carbon-based nanomaterials. Herein, we characterize the morphology, synthesis, and applications of CNTs and JBNts to provide a comprehensive comparison between these nanomaterial technologies.

Comparison between Janus-Base Nanotubes and Carbon Nanotubes: A Review on Synthesis, Physicochemical Properties, and Applications.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have recently received notable attention for their applications in nucleic acid detection. Despite many attempts, the majority of current CRISPR-based biosensors in infectious respiratory disease diagnostic applications still require target preamplifications. This study reports a new biosensor for amplification-free nucleic acid detection via harnessing the trans-cleavage mechanism of Cas13a and ultrasensitive graphene field-effect transistors (gFETs). CRISPR Cas13a-gFET achieves the detection of SARS-CoV-2 and respiratory syncytial virus (RSV) genome down to 1 attomolar without target preamplifications. Additionally, we validate the detection performance using clinical SARS-CoV-2 samples, including those with low viral loads (Ct value >30). Overall, these findings establish our CRISPR Cas13a-gFET among the most sensitive amplification-free nucleic acid diagnostic platforms to date.

Amplification-Free Detection of SARS-CoV-2 and Respiratory Syncytial Virus Using CRISPR Cas13a and Graphene Field-Effect Transistors.

The blood-brain barrier (BBB) is the most specialized biological barrier in the body. This configuration of specialized cells protects the brain from invasion of molecules and particles through formation of tight junctions. To learn more about transport to the brain, in vitro modeling of the BBB is continuously advanced. The types of models and cells selected vary with the goal of each individual study, but the same validation methods, quantification of tight junctions, and permeability assays are often used. With Transwells and microfluidic devices, more information regarding formation of the BBB has been observed. Disease models have been developed to examine the effects on BBB integrity. The goal of modeling is not only to understand normal BBB physiology, but also to create treatments for diseases. This review will highlight several recent studies to show the diversity in model selection and the many applications of BBB models in in vitro research.

Yupeng Chen

Department of Biomedical Engineering

University of Connecticut

Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

Fabrication and Characterization of Layer-By-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration