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Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. ...

University of Louisville

Ultrasound-induced microbubble destruction can enhance drug delivery to cells. The molecular weight of therapeutic compounds varies significantly (from <1 kDa for small molecule drugs, to 7-15 kDa for siRNAs/miRNAs, to >1000 kDa for DNA plasmids). Therefore, the objective of this study was to determine the relationship between uptake efficiency and molecular weight using equal molar concentrations. Uptake efficiency of fluorescent compounds with different molecular weights (0.3, 10 and 2000 kDa) was explored in vitro using human cardiac mesenchymal cells and breast cancer cells exposed to microbubbles and 2.5-MHz ultrasound pulses. Uptake by viable cells was quantified using flow cytometry. After correction for the fluorescence yield of each compound, there was a significant size-dependent difference in fluorescence intensity, indicating an inverse relationship between size and uptake efficiency. These results suggest that diffusion of therapeutic compounds across permeabilized cell membranes may be an important mechanism for ultrasound-mediated drug delivery.

Effect of Molecular Weight on Sonoporation-Mediated Uptake in Human Cells.

Preservation of erythrocytes in a desiccated state for storage at ambient temperature could simplify blood transfusions in austere environments, such as rural clinics, far-forward military operations, and during space travel. Currently, storage of erythrocytes is limited by a short shelf-life of 42 days at 4 °C, and long-term preservation requires a complex process that involves the addition and removal of glycerol from erythrocytes before and after storage at -80 °C, respectively. Natural compounds, such as trehalose, can protect cells in a desiccated state if they are present at sufficient levels inside the cell, but mammalian cell membranes lack transporters for this compound. To facilitate compound loading across the plasma membrane via ultrasound and microbubbles (sonoporation), a polydimethylsiloxane-based microfluidic device was developed. Delivery of fluorescein into erythrocytes was tested at various conditions to assess the effects of parameters such as ultrasound pressure, ultrasound pulse interval, microbubble dose, and flow rate. Changes in ultrasound pressure and mean flow rate caused statistically significant increases in fluorescein delivery of up to 73 ± 37% (< 0.05) and 44 ± 33% (< 0.01), respectively, compared to control groups, but no statistically significant differences were detected with changes in ultrasound pulse intervals. Following freeze-drying and rehydration, recovery of viable erythrocytes increased by up to 128 ± 32% after ultrasound-mediated loading of trehalose compared to control groups (< 0.05). These results suggest that ultrasound-mediated molecular delivery in microfluidic channels may be a viable approach to process erythrocytes for long-term storage in a desiccated state at ambient temperatures.

Ultrasound-induced molecular delivery to erythrocytes using a microfluidic system.

Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was classified as a pandemic by the World Health Organization and has caused over 550,000 deaths worldwide as of July 2020. Accurate and scalable point-of-care devices would increase screening, diagnosis, and monitoring of COVID-19 patients. Here, we demonstrate rapid label-free electrochemical detection of SARS-CoV-2 antibodies using a commercially available impedance sensing platform. A 16-well plate containing sensing electrodes was pre-coated with receptor binding domain (RBD) of SARS-CoV-2 spike protein, and subsequently tested with samples of anti-SARS-CoV-2 monoclonal antibody CR3022 (0.1 μg/ml, 1.0 μg/ml, 10 μg/ml). Subsequent blinded testing was performed on six serum specimens taken from COVID-19 and non-COVID-19 patients (1:100 dilution factor). The platform was able to differentiate spikes in impedance measurements from a negative control (1% milk solution) for all CR3022 samples. Further, successful differentiation and detection of all positive clinical samples from negative control was achieved. Measured impedance values were consistent when compared to standard ELISA test results showing a strong correlation between them (R=0.9). Detection occurs in less than five minutes and the well-based platform provides a simplified and familiar testing interface that can be readily adaptable for use in clinical settings.

Rapid detection of SARS-CoV-2 antibodies using electrochemical impedance-based detector.

Respiratory droplets emitted during speech can transmit oral bacteria and infectious viruses to others, including COVID-19. Loud speech can generate significantly higher numbers of potentially infectious respiratory droplets. This study assessed the effect of speech volume on respiratory emission of oral bacteria as an indicator of potential pathogen transmission risk. Loud speech (average 83 dBA, peak 94 dBA) caused significantly higher emission of oral bacteria (p = 0.004 compared to no speech) within 1 ft from the speaker. N99 respirators and simple cloth masks both significantly reduced emission of oral bacteria. This study demonstrates that loud speech without face coverings increases emission of respiratory droplets that carry oral bacteria and may also carry other pathogens such as COVID-19.

Effect of speech volume on respiratory emission of oral bacteria as a potential indicator of pathogen transmissibility risk.

Despite recent advances in biostabilization, clinical blood supplies still experience shortages and storage limitations for red blood cells (RBCs) have not yet been sufficiently addressed. Storing RBCs in a frozen or dried state is an appealing solution to address storage limitations, but many promising cryoprotectants, including the non-reducing sugar trehalose, are impermeant to mammalian cell membranes and cannot be utilized effectively using currently available compound-loading methods. We found that transient pore formation induced by ultrasound and microbubbles (sonoporation) offers an effective means of loading trehalose into RBCs to facilitate long-term storage in a frozen or desiccated state. The protective potential of trehalose loading was demonstrated by freezing processed RBCs at -1 °C/min to -80 °C, then either storing the cells at -80 °C or lyophilizing them. RBCs were either thawed or rehydrated after 42 days of storage and evaluated for membrane integrity and esterase activity to estimate recovery and cell viability. The intracellular concentration of trehalose reached 40 mM after sonoporation and over 95% of treated RBCs were recovered after loading. Loading of trehalose was sufficient to maintain RBC morphology and esterase activity in most cells during freezing (>90% RBC recovery) and to a lower degree after lyophilization and rehydration (>20% recovery). Combining sonoporation with an integrated fluidics device allowed for rapid loading of up to 70 mM trehalose into RBCs. These results demonstrate the potential of sonoporation-mediated trehalose loading to increase recovery of viable RBCs, which could lead to effective methods for long-term stabilization of RBCs.

Mariah C. Priddy

Department of Bioengineering

University of Louisville

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Mariah C. Priddy

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Bioengineering

University of Louisville

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Department of Bioengineering