M.Sc Thesis


M.Sc StudentKeren David
SubjectUltrasound for the Delivery of Genes to Suspended Cells:
Design and Characterization
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professors Machluf Marcelle


Abstract

The main problem associated with delivery of genes to suspended cells is the low transfection level. The present research is aimed at optimizing the therapeutic ultrasound (TUS) system for delivery of genes to suspended cells.


To find the optimal parameters and configuration of the TUS system in transfecting the cells (T lymphocytes) we examined different frequencies, intensities and time of application. Moreover, two synthetic molecules with positive charge, AMD-3100 and T-134, were used to mediate between the plasmid DNA (pDNA) and the cells prior to TUS application.

Using the AMD-3100 and T-134 in combination with TUS (1 MHz, 2 W/cm2, 30 and 50 % DC and 30 min), we were able to increase the level of transfection by 5 and 20 fold respectively when compared to the transfection using pDNA alone.

A 24 hrs follow-up on membrane permeability using FACS revealed that 5hrs after the TUS the membrane was highly permeable. Twenty-four hrs after the TUS, the membrane returned to its original condition.

Using the optimized TUS parameters and mediators, we were able to deliver Interleukin 4, one of the most important cytokines in the suppression of inflammatory autoimmune diseases to the T cells. Its secretion was confirmed using Western-Blot and RT-PCR and biological test confirmed its activity.

To conclude, this research emphasizes the advantages in the use of TUS to deliver genes to suspended cells.