assembly-operation-an-acoustofluidic-device-for-enhanced-delivery

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

AS1411 is a 26-mer G-rich DNA oligonucleotide that forms a variety of G-quadruplex structures. It was identified based on its cancer-selective antiproliferative activity and subsequently determined to be an aptamer to nucleolin, a multifunctional protein that preferentially binds quadruplex nucleic acids and which is present at high levels on the surface of cancer cells. AS1411 has exceptionally efficient cellular internalization compared to non-quadruplex DNA sequences.

G-quadruplex oligonucleotide AS1411 as a cancer-targeting agent: Uses and mechanisms.

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.

Systemic delivery of conventional chemotherapies can cause negative systemic toxicity, including reduced immunity and damage to organs such as the heart and kidneys-limiting the maximum dose that can be administered. Targeted therapies appear to address this problem by having a specific target while mitigating off-target effects. Biocompatible perfluorocarbon-based nanodroplet emulsions encapsulated by a phospholipid shell are in development for delivery of molecular compounds and hold promise as vehicles for targeted delivery of chemotherapeutics to tumors. When ultrasound is applied, perfluorocarbon will undergo a phase change-ultimately inducing transient perforation of the cell membrane when in close proximity, which is more commonly known as "sonoporation." Sonoporation allows enhanced intracellular delivery of molecular compounds and will reseal to encapsulate the molecular compound intracellularly. In this study, we investigated delivery of thymoquinone (TQ), a natural hydrophobic phytochemical compound with bioactivity in cancer cells. In addition, we conjugated a G-quadruplex aptamer, 'AS1411', to TQ-loaded nanodroplets and explored their effects on multiple human cancer cell lines. AS1411 binds nucleolin, which is over-expressed on the surface of cancer cells, and in addition to its tumor-targeting properties AS1411 has also been shown to induce anti-cancer effects. Thymoquinone was loaded onto AS1411-conjugated nanodroplet emulsion to assess activity against cancer cells. Confocal microscopy indicated uptake of AS1411-conjugated nanodroplets by cancer cells. Furthermore, AS1411-conjugated nanoemulsions loaded with TQ significantly enhanced cytotoxicity in cancer cells compared to free compound. These results demonstrate that AS1411 can be conjugated onto nanodroplet emulsions for targeted delivery to human cancer cells. This novel formulation offers significant potential for targeted delivery of hydrophobic chemotherapeutics to tumors for cancer treatment.

Article	Year
G-quadruplex oligonucleotide AS1411 as a cancer-targeting agent: Uses and mechanisms. Biochimica et biophysica acta. General subjects\| PubMed ID: 28007579	2017
Effect of Molecular Weight on Sonoporation-Mediated Uptake in Human Cells. Ultrasound in medicine & biology\| PubMed ID: 30274682	2018
Ultrasound-induced molecular delivery to erythrocytes using a microfluidic system. Biomicrofluidics\| PubMed ID: 32341725	2020
Delivery of thymoquinone to cancer cells with as1411-conjugated nanodroplets. PloS one\| PubMed ID: 32437399	2020

Emily M. Murphy

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