Abstract
Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150 MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca2+ and propidium iodide (PI) and the delivery of 3 kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3 kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6 hr) cell viability test and long-term (40 hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity.
Highlights
Cells by the generated ultrasound field is usually quite large because the focal area of low frequency ultrasound is in the millimeter range
The intracellular delivery of macromolecules, i.e., 3 kDa dextran labeled with Alexa 488, was investigated with two different input parameters that did not induce cell death
We found that conditions I (Vpp = 22 V, tp = 30 μ s, pulse repetition frequency (PRF) = 0, and number of electric pulse (NP) = 1) and II (Vpp = 43V, tp = 10 μ s, PRF = 0, and NP = 1) were two possible upper limits of input parameters right before inducing cell death
Summary
Cells by the generated ultrasound field is usually quite large because the focal area of low frequency ultrasound is in the millimeter range. Binding of Ca2+ between CaM and M13 results in the FRET ratio increase, which indicates the Ca2+ influx into cell cytoplasm (Supplementary Figure 3) This genetically encoded molecular biosensor can target subcellular regions to visualize more accurate Ca2+ concentration than fluorescence dye. After an acoustic pulse was applied to a targeted single-cell to induce the intracellular delivery of molecules, i.e., acoustic-transfection, the cell was monitored by an epi-fluorescence microscope[34,35,36]. Direct and sustained intracellular delivery of macromolecules, which utilizes a high frequency ultrasound-induced perturbation on the cell membrane, was introduced and single-cell level targeting capability and low cytotoxicity of acoustic-transfection was established
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