Adeno-associated virus characterization for cargo discrimination through nanopore responsiveness.

Solid-state nanopore (SSN)-based analytical methods have found abundant use in genomics and proteomics with fledgling contributions to virology - a clinically critical field with emphasis on both infectious and designer-drug carriers. Here we demonstrate the ability of SSN to successfully discriminate adeno-associated viruses (AAVs) based on their genetic cargo [double-stranded DNA (AAVdsDNA), single-stranded DNA (AAVssDNA) or none (AAVempty)], devoid of digestion steps, through nanopore-induced electro-deformation (characterized by relative current change; ΔI/I0). The deformation order was found to be AAVempty > AAVssDNA > AAVdsDNA. A deep learning algorithm was developed by integrating support vector machine with an existing neural network, which successfully classified AAVs from SSN resistive-pulses (characteristic of genetic cargo) with >95% accuracy - a potential tool for clinical and biomedical applications. Subsequently, the presence of AAVempty in spiked AAVdsDNA was flagged using the ΔI/I0 distribution characteristics of the two types for mixtures composed of ∼75 : 25% and ∼40 : 60% (in concentration) AAVempty : AAVdsDNA.

Mechanical characterization of vesicles and cells: A review.

Vesicles perform many essential functions in all living organisms. They respond like a transducer to mechanical stress in converting the applied force into mechanical and biological responses. At the same time, both biochemical and biophysical signals influence the vesicular response in bearing mechanical loads. In recent years, liposomes, artificial lipid vesicles, have gained substantial attention from the pharmaceutical industry as a prospective drug carrier which can also serve as an artificial cell-mimetic system. The ability of these vesicles to enter through pores of even smaller size makes them ideal candidates for therapeutic agents to reach the infected sites effectively. Engineering of vesicles with desired mechanical properties that can encapsulate drugs and release as required is the prime challenge in this field. This requirement has led to the modifications of the composition of the bilayer membrane by adding cholesterol, sphingomyelin, etc. In this article, we review the manufacturing and characterization techniques of various artificial/synthetic vesicles. We particularly focus on the electric field-driven characterization techniques to determine different properties of vesicle and its membranes, such as bending rigidity, viscosity, capacitance, conductance, etc., which are indicators of their content and mobility. Similarities and differences between artificial vesicles, natural vesicles, and cells are highlighted throughout the manuscript since most of these artificial vesicles are intended for cell mimetic functions.

Electrophoretic transport and dynamic deformation of bio-vesicles.

Study of the deformation dynamics of cells and other sub-micron vesicles, such as virus and neurotransmitter vesicles are necessary to understand their functional properties. This mechanical characterization can be done by submerging the vesicle in a fluid medium and deforming it with a controlled electric field, which is known as electrodeformation. Electrodeformation of biological and artificial lipid vesicles is directly influenced by the vesicle and surrounding media properties and geometric factors. The problem is compounded when the vesicle is naturally charged, which creates electrophoretic forcing on the vesicle membrane. We studied the electrodeformation and transport of charged vesicles immersed in a fluid media under the influence of a DC electric field. The electric field and fluid-solid interactions are modeled using a hybrid immersed interface-immersed boundary technique. Model results are verified with experimental observations for electric field driven translocation of a virus through a nanopore sensor. Our modeling results show interesting changes in deformation behavior with changing electrical properties of the vesicle and the surrounding media. Vesicle movement due to electrophoresis can also be characterized by the change in local conductivity, which can serve as a potential sensing mechanism for electrodeformation experiments in solid-state nanopore setups.

Mathematical Model for Tissue-Level Hypoxic Response in Microfluidic Environment.

Availability of essential species like oxygen is critical in shaping the dynamics of tumor growth. When the intracellular oxygen level falls below normal, it initiates major cascades in cellular dynamics leading to tumor cell survival. In a cellular block with cells growing away from the blood vessel, the scenario can be aggravated for the cells further inside the block. In this study, the dynamics of intracellular species inside a colony of tumor cells are investigated by varying the cell-block thickness and cell types in a microfluidic cell culture device. The oxygen transport across the cell block is modeled through diffusion, while ascorbate (AS) transport from the extracellular medium is addressed by a concentration-dependent uptake model. The extracellular and intracellular descriptions were coupled through the consumption and traffic of species from the microchannel to the cell block. Our model shows that the onset of hypoxia is possible in HeLa cell within minutes depending on the cell location, although the nutrient supply inside the channel is maintained in normoxic levels. This eventually leads to total oxygen deprivation inside the cell block in the extreme case, representing the development of a necrotic core that maintains a dynamic balance with growing cells and scarce supply. The numerical model reveals that species concentration and hypoxic response are different for HeLa and HelaS3 cells. Results also indicate that the long-term hypoxic response from a microfluidic cellular block stays within 5% of the values of a tissue with the basal layer. The hybrid model can be very useful in designing microfluidic experiments to satisfactorily predict the tissue-level response in cancer research.

Electrodeformation of Vesicles Suspended in a Liquid Medium.

Deformation of flexible vesicles suspended in a fluid medium due to an applied electric field can provide valuable insight into deformation dynamics at a very small scale. In an electric field, the response of the vesicle membrane is strongly influenced by the conductivity of surrounding fluid, vesicle size and shape, and the magnitude of applied field. We studied the electrodeformation of vesicles immersed in a fluid media under a DC electric field. An immersed interface method is used to solve the electric field over the domain with conductive or non-conductive vesicles while an immersed boundary method is employed to solve fluid flow, fluid-solid interaction, membrane mechanics and vesicle deformation. Initial force analysis on the membrane surface reveals almost linear influence of vesicle size, but the vesicle size does not affect the long-term deformation which is consistent with experimental evidence. Highly nonlinear effect of the applied field as well as the conductivity ratios inside and outside of the vesicle are observed. Results also point towards an early linear deformation regime followed by an equilibrium stage for the membranes. Modeling results suggest that electrodeforming vesicles can create unique external flows for different conductivity ratios. Moreover, significant influence of the initial aspect ratio of the vesicle on the force distribution is observed across a range of conductivity ratios.

Hypoxic behavior in cells under controlled microfluidic environment.

BACKGROUND: Depleted oxygen levels, known as hypoxia, causes considerable changes in the cellular metabolism. Hypoxia-inducible factors (HIF) act as the major protagonist in orchestrating manifold hypoxic responses by escaping cellular degradation mechanisms. These complex and dynamic intracellular responses are significantly dependent on the extracellular environment. In this study, we present a detailed model of a hypoxic cellular microenvironment in a microfluidic setting involving HIF hydroxylation. METHODS: We have modeled the induction of hypoxia in a microfluidic chip by an unsteady permeation of oxygen from the microchannel through a porous polydimethylsiloxane channel wall. Extracellular and intracellular interactions were modeled with two different mathematical descriptions. Intracellular space is directly coupled to the extracellular environment through uptake and consumption of oxygen and ascorbate similar to cells in vivo. RESULTS: Our results indicate a sharp switch in HIF hydroxylation behavior with changing prolyl hydroxylase levels from 0.1 to 4.0µM. Furthermore, we studied the effects of extracellular ascorbate concentration, using a new model, to predict its accumulation inside the cell over a relevant physiological range. In different hypoxic conditions, the cellular environment showed a significant dependence on oxygen levels in resulting intracellular response. CONCLUSIONS: Change in hydroxylation behavior and nutrient supplementation can have significant potential in designing novel therapeutic interventions in cancer and ischemia/reperfusion injuries. GENERAL SIGNIFICANCE: The hybrid mathematical model can effectively predict intracellular behavior due to external influences providing valuable directions in designing future experiments.