Mechanical oscillations enhance gene delivery into suspended cells

Abstract

Suspended cells are difficult to be transfected by common biochemical methods which require cell attachment to a substrate. Mechanical oscillations of suspended cells at certain frequencies are found to result in significant increase in membrane permeability and potency for delivery of nano-particles and genetic materials into the cells. Nanomaterials including siRNAs are found to penetrate into suspended cells after subjecting to short-time mechanical oscillations, which would otherwise not affect the viability of the cells. Theoretical analysis indicates significant deformation of the actin-filament network in the cytoskeleton cortex during mechanical oscillations at the experimental frequency, which is likely to rupture the soft phospholipid bilayer leading to increased membrane permeability. The results here indicate a new method for enhancing cell transfection.

Figure 1

(a) Death rate of K562 cells exposed to mechanical oscillations at different frequencies (0, 10, 100, 500 and 800 Hz) and 10 volt amplitude for 0, 1, 2, 4, 8, 12, 16 mins; (b) Death rate of K562 cells exposed to oscillations at 100 Hz and 10 volt amplitude for 4 mins following by culture for 1, 3 and 7 days, measured using hemocytometry.

Figure 2. MTT measurements of K562 cells oscillated with nano-particles of different sizes, showing smaller particles producing higher cytotoxicity after oscillations at 100 Hz and 10 volt amplitude for 4 mins.

Figure 3. Flow-cytometry results of actin-siRNAs transfection, showing that fluorescence labelled siRNAs penetrated into the cells after mechanical oscillations.

(a) Flow cytometry graph of FITC-A vs PE-A, indicating the actin-siRNAs transfection efficiency and death rate of K562 cells with and without subjecting to mechanical oscillations; (b) Flow cytometry graph of the siRNAs transfection efficiency of K562 cells with and without subjecting to mechanical oscillations; (c) Histogram of actin-siRNAs transfection efficiency of K562 cells with and without subjecting to mechanical oscillations; the error bars indicate standard deviations of three measurements; (d) Optical image showing FITC labelled actin-siRNAs transfected in the K562 cells.

Figure 4

(a) Western Blot results showing designed SiR-actin inhibits the β-actin protein expression significantly. Control: K562 cells without exposing to mechanical oscillations; SiR-control: K562 cells mixed with the negative siRNAs sequence and exposed to mechanical oscillations; SiR-Actin: K562 cells mixed with the actin-siRNAs sequence and exposed to mechanical oscillations; (b) Optical images of indentation by optical tweezers manipulation, captured at 488 nm excitation wavelength in a dim condition. The white arrows indicate the 2.5 μm polystyrene indenter beads trapped by laser; (c) Histogram showing the measured elastic moduli of K562 cells transfected with negative control of siRNAs and FITC labeled actin siRNAs. The error bars indicate standard deviations of 7 measurements.

Figure 5. Transfection efficiency of leukemia cell lines THP-1, OCI-AML3 and trypsinized adherent cell line Hela measured by flow cytometey.

(a) Optical images of the three non-adherent and adherent cell types. Scale bars indicate 50 μm for all images. (b) Histogram of actin-siRNAs transfection efficiency of the three cell types with and without subjecting to mechanical oscillations; the error bars indicate standard deviations of three measurements. (c) Flow cytometry graph of the siRNAs transfection efficiency of the three cell types with and without subjecting to mechanical oscillations.

Figure 6. Finite-element simulation of the cytoskeleton cortex.

(a) Typical simulated configurations of the cortex structure before (left) and after (right) 5 cycles of vibration. An attached movie shows the simulated oscillations. (b) Frequency plots of the pore areas on the cortex layer, and fitted Gaussian distributions, before and after 5 cycles of vibration.