Binbin Deng

An Orthogonal Array Optimization of Lipid-like Nanoparticles for mRNA Delivery in Vivo.

Systemic delivery of mRNA-based therapeutics remains a challenging issue for preclinical and clinical studies. Here, we describe new lipid-like nanoparticles (TT-LLNs) developed through an orthogonal array design, which demonstrates improved delivery efficiency of mRNA encoding luciferase in vitro by over 350-fold with significantly reduced experimental workload. One optimized TT3 LLN, termed O-TT3 LLNs, was able to restore the human factor IX (hFIX) level to normal physiological values in FIX-knockout mice. Consequently, these mRNA based nanomaterials merit further development for therapeutic applications.

Effects of local structural transformation of lipid-like compounds on delivery of messenger RNA.

Lipid-like nanoparticles (LLNs) have shown great potential for RNA delivery. Lipid-like compounds are key components in LLNs. In this study, we investigated the effects of local structural transformation of lipid-like compounds on delivery of messenger RNA. Our results showed that position change of functional groups on lipid-like compounds can dramatically improve delivery efficiency. We then optimized formulation ratios of TNT-b10 LLNs, a lead material, increasing delivery efficiency over 2-fold. More importantly, pegylated TNT-b10 LLNs is stable for over four weeks and is over 10-fold more efficient than that of its counterpart TNT-a10 LLNs. Additionally, the optimal formulation O-TNT-b10 LLNs is capable of delivering mRNA encoding luciferase in vivo. These results provide useful insights into the design of next generation LLNs for mRNA delivery.

Silver-Zinc Redox-Coupled Electroceutical Wound Dressing Disrupts Bacterial Biofilm

Pseudomonas aeruginosa biofilm is commonly associated with chronic wound infection. A FDA approved wireless electroceutical dressing (WED), which in the presence of conductive wound exudate gets activated to generate electric field (0.3–0.9V), was investigated for its anti-biofilm properties. Growth of pathogenic P. aeruginosa strain PAO1 in LB media was markedly arrested in the presence of the WED. Scanning electron microscopy demonstrated that WED markedly disrupted biofilm integrity in a setting where silver dressing was ineffective. Biofilm thickness and number of live bacterial cells were decreased in the presence of WED. Quorum sensing genes lasR and rhlR and activity of electric field sensitive enzyme, glycerol-3-phosphate dehydrogenase was also repressed by WED. This work provides first electron paramagnetic resonance spectroscopy evidence demonstrating that WED serves as a spontaneous source of reactive oxygen species. Redox-sensitive multidrug efflux systems mexAB and mexEF were repressed by WED. Taken together, these observations provide first evidence supporting the anti-biofilm properties of WED.

Co-delivery of mRNA and SPIONs through amino-ester nanomaterials

Nanoparticles have been widely explored for combined therapeutic and diagnostic applications. For example, lipid-based nanoparticles have been used to encapsulate multiple types of agents and achieve multi-functions. Herein, we enabled a co-delivery of mRNA molecules and superparamagnetic iron oxide nanoparticles (SPIONs) by using an amino-ester lipid-like nanomaterial. An orthogonal experimental design was used to identify the optimal formulation. The optimal formulation, MPA-Ab-8 LLNs, not only showed high encapsulation of both mRNA and SPIONs, but also increased the r2 relaxivity of SPIONs by more than 1.5-fold in vitro. MPA-Ab-8 LLNs effectively delivered mRNA and SPIONs into cells, and consequently induced high protein expression as well as strong MRI contrast. Consistent herewith, we observed both mRNA-mediated protein expression and an evident negative contrast enhancement of MRI signal in mice. In conclusion, amino-ester nanomaterials demonstrate great potential as delivery vehicles for theranostic applications.

Sub-cellular In-situ Characterization of Ferritin(iron) in a Rodent Model of Spinal Cord Injury

Iron (Fe) is an essential metal involved in a wide spectrum of physiological functions. Sub-cellular characterization of the size, composition, and distribution of ferritin(iron) can provide valuable information on iron storage and transport in health and disease. In this study we employ magnetic force microscopy (MFM), transmission electron microscopy (TEM), and electron energy loss spectroscopy (EELS) to characterize differences in ferritin(iron) distribution and composition across injured and non-injured tissues by employing a rodent model of spinal cord injury (SCI). Our biophysical and ultrastructural analyses provide novel insights into iron distribution which are not obtained by routine biochemical stains. In particular, ferritin(iron) rich lysosomes revealed increased heterogeneity in MFM signal from tissues of SCI animals. Ultrastructural analysis using TEM elucidated that both cytosolic and lysosomal ferritin(iron) density was increased in the injured (spinal cord) and non-injured (spleen) tissues of SCI as compared to naïve animals. In-situ EELs analysis revealed that ferritin(iron) was primarily in Fe3+ oxidation state in both naïve and SCI animal tissues. The insights provided by this study and the approaches utilized here can be applied broadly to other systemic problems involving iron regulation or to understand the fate of exogenously delivered iron-oxide nanoparticles.

Correlative Imaging of Murine Pulmonary Valve Extracellular Matrix

Heart valves have purely biomechanical structures that respond to the fluid dynamic environment caused by heart palpitations. The pulmonary valve is responsible for unidirectional blood flow from the right ventricle of the heart to the pulmonary arteries, leading to the respiratory system. Healthy pulmonary valves consist of three leaflets, each compositionally and organizationally distinct [1]. Collagen is a fibrillar extracellular matrix (ECM) protein is that responsible for the mechanical strength of heart valves. Malformations and disorganization of collagen leads to biomechanical inefficiencies in the heart valve, which manifest as physiological conditions such as stenosis or regurgitation [2]. In order to comprehensively study the organization of the ECM, the physiological conditions of the valve must be controlled and related to the microstructure of the ECM. We are developing a correlative workflow in order to characterize the hierarchical organization of the collagen ECM and relate this to the biomechanical properties of the valve.

Cell interactions in Wound Biofilm and in vitro Biofilm Revealed by Electron Microscopy

Biofilms are aggregation of bacteria which are encapsulated in extracellular polymeric substance (EPS) and form complex structures. Biofilm associated chronic wound is a significant burden for patient and healthcare system [1-2]. Treatment of biofilm infection designed based on in vitro culture rarely promote the chronic wound healing successfully. The principal limitation of applying results from in vitro biofilm to clinical wound biofilm treatment is that the controlled culture condition cannot represent the real environment of wound biofilm. To understand the structural details of how bacteria survive and thrive in wound, and also compare the structural differences of wound biofilm and in vitro biofilm, we applied Electron Microscopy (EM) techniques in structural study of porcine wound biofilm and in vitro biofilm.

Correlative Microscopy Application in Spinal Cord Injury Research

Spinal cord injury (SCI) affects approximately 250,000 people in the United States, with about 11,000 new injuries each year. Neuron regeneration, a critical process to restore the communication between the body and the brain after SCI, is an import topic in both basic science and clinical research. After a severe SCI, surviving neurons repair and develop functional synapses to re-establish axonal pathways. Glia cells are believed to regulate many signaling mechanisms that control the neural repair ability before and after SCI [1, 2]. Increased numbers of glia cells gather around motor neurons after SCI. The interaction between motor neurons and surrounding glia cells may play critical roles in reconnecting neural circuits. Three-dimensional (3D) structural information about the interaction between motor neuron and surrounding glia cells at synapses would help researchers understand the process of neuron repair. However, such 3D structures are currently unavailable.

Ferritin Mineral Core Composition in Health and Disease

1. The Dorothy M. Davis Heart and Lung Research Institute, the Ohio State University, Columbus, OH 2. Biomedical Engineering Department, the Ohio State University, Columbus, OH 3. Center for Electron Microscopy and Analysis, the Ohio State University, Columbus, OH 4. Department of Pathology, the Ohio State University, Columbus, OH 5. Department of Neuroscience, the Ohio State University, Columbus, OH 6. Department of Material Science and Engineering, the Ohio State University, Columbus, OH

FIB/SEM Tomography of Wound Biofilm

Biofilm has a complex architecture and show high tolerance to host immune responses and antimicrobial agents [1]. Biofilm infection of chronic wounds often makes them very resistant to treatment, and can be a severe health problem in patients with compromised immune systems. Despite the extensive study of biofilm in the past 15 years [2], the pathogenic biofilm structures remain to be characterized. DualBeam FIB/SEM tomography is a suitable tool to investigate three-dimensional (3D) structures of biological materials at intermediate to high resolution. In order to understand the biofilm architecture in animal model and clinical chronic wound, we have studied a porcine pre-clinical wound model involving single-species and multi-species infection as well as chronic wound from a patient using FIB/SEM tomography.