Cytoplasmic Molecular Delivery with Shock Waves:Importance of Impulse

Abstract

Cell permeabilization using shock waves may be a way of introducing macromolecules and small polar molecules into the cytoplasm, and may have applications in gene therapy and anticancer drug delivery. The pressure profile of a shock wave indicates its energy content, and shock-wave propagation in tissue is associated with cellular displacement, leading to the development of cell deformation. In the present study, three different shock-wave sources were investigated; argon fluoride excimer laser, ruby laser, and shock tube. The duration of the pressure pulse of the shock tube was 100 times longer than the lasers. The uptake of two fluorophores, calcein (molecular weight: 622) and fluorescein isothiocyanate-dextran (molecular weight: 71,600), into HL-60 human promyelocytic leukemia cells was investigated. The intracellular fluorescence was measured by a spectrofluorometer, and the cells were examined by confocal fluorescence microscopy. A single shock wave generated by the shock tube delivered both fluorophores into approximately 50% of the cells (p < 0.01), whereas shock waves from the lasers did not. The cell survival fraction was >0.95. Confocal microscopy showed that, in the case of calcein, there was a uniform fluorescence throughout the cell, whereas, in the case of FITC-dextran, the fluorescence was sometimes in the nucleus and at other times not. We conclude that the impulse of the shock wave (i.e., the pressure integrated over time), rather than the peak pressure, was a dominant factor for causing fluorophore uptake into living cells, and that shock waves might have changed the permeability of the nuclear membrane and transferred molecules directly into the nucleus.

Delivery of ribosome-inactivating protein toxin into cancer cells with shock waves

“…In contrast with our previous studies [7,8] and our present results with OVCAR-5 cells, when we measured the cell viability of HT29 cells after shock waves alone using a clonogenic assay, we found a significant loss of viability using shock waves alone and saporin alone, not seen with the MTT assay. Although many workers have shown that the MTT assay correlates well with clonogenic assays for measuring loss of viability in vitro after several toxic treatments [16][17][18], others have found discrepancies (reviewed in Ref.…”

“…We went on to demonstrate that shock waves generated by a shock tube could also accomplish this permeabilization with no toxicity to the cells as determined by the MTT assay. Molecules as large as 2 MDa fluoresceinlabeled dextran were delivered into the cytoplasm [8], and it was proposed that the shock tube-generated shock waves had the correct parameters (pressure, rise-time, and duration) to give a threshold of impulse (defined as the integral of pressure with duration) for molecular delivery into cells [7]. Cellular deformation and membrane effects due to varying shear stresses produced by the impulse of the shock wave on the relative motion of cells and fluid phase was proposed to be responsible for the permeabilization observed.…”

“…Shock waves were generated with a double diaphragm shock tube that was obtained from Pharma Wave (Boston, MA) and is described in detail elsewhere [7,8]. The shock wave was delivered into the tube containing the cell pellet through the covering of parafilm.…”

“…We have shown that a single shock wave generated by a shock tube has the correct parameters to deliver fluorescent dextran molecules into the cytoplasm of cells without toxicity [7,8]. In this report we show that from one to five shock waves from a shock tube potentiates the cytotoxicity of saporin towards human cancer cells as determined by two different assays for cell viability, chosen to provide measures of loss of viability in both the short term (24 h) and the long term (2 weeks).…”

Noninvasive micromanipulation of live HIV-1 infected cells via laser light

“…Some applications involve introduction of potentially therapeutic material, including proteins, oligonucleotides, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) [1]. There are numerous means of promoting the inclusion of foreign genes and other macromolecules into mammalian cells.…”