Injection of Propidium Iodide into HeLa Cells Using a Silicon Nanoinjection Lance Array

Abstract

Delivering foreign molecules into human cells is a wide and ongoing area of research. Gene therapy, or delivering nucleic acids into cells via nonviral or viral pathways, is an especially promising area for pharmaceutics. All gene therapy methods have their respective advantages and disadvantages, including limited delivery efficiency and low viability. We present an electromechanical method for delivering foreign molecules into human cells. Nanoinjection, or delivering molecules into cells using a solid lance, has proven to be highly efficient while maintaining high viability levels. This paper describes an array of solid silicon microlances that was tested to determine efficiency and viability when nanoinjecting tens of thousands of HeLa cells simultaneously. Propidium iodide (PI), a dye that fluoresces when bound to nucleic acids and does not fluoresce when unbound, was delivered into cells using the lance array. Results show that the lance array delivers PI into up to 78% of a nanoinjected HeLa cell culture, while maintaining 78-91 % viability. With these results, we submit the nanoinjection method using a silicon lance array as another promising particle delivery method for mammalian culture cells.

The effect of injection speed and serial injection on propidium iodide entry into cultured HeLa and primary neonatal fibroblast cells using lance array nanoinjection

“…Second, because the lances are 1–2.5 μm in diameter, the resulting pores created during the injection event are relatively large, making it possible for large molecules to transiently pass into the cell. Even though the pores are relatively large, the trauma induced during the process is relatively minimal, as evidenced by high cell viability rates (78–91 %) previously noted (Lindstrom et al 2014 ). This latter feature of cell viability is an issue in some instrumentation based transfection methods, such as electroporation (Barsoum 1995 ) and microinjection (Aten et al 2012 ).…”

“…The parameters for treatments were selected based on data from previous work (Lindstrom et al 2014 ) with the exception of changing the initial voltage control setting to be a current control setting. This modification allows the molecular load of interest to be electrically attracted to the lance array in greater quantities because there is current flowing continuously in the solution as opposed to the voltage control setting that sees an exponential decay in current flow in the solution.…”

“…One non-viral transfection biotechnology, known as lance array nanoinjection (LAN), has been created with these design requirements in mind. LAN works by using a combination of physical penetration of target cell membranes and electrical delivery of molecular loads using a microfabricated silicon etched array of lances (Lindstrom et al 2014 ; Teichert et al 2013 ). Figure 1 shows an SEM image of a lance array which contains 10 μm length lances spaced 10 μm from center to center in a grid pattern, ultimately forming 4 million lances on a 2 by 2 cm chip.…”

A review of genetic engineering biotechnologies for enhanced chronic wound healing

“…This same technology has been modified in a second generation to consist of a microfabricated silicon wafer that has a grid of four million 10‐micron‐length lances that allow for effective delivery of genetic loads to hundreds of thousands of cells simultaneously . As an emerging method, nanoinjection shows promise in regard to in vitro CRISPR‐Cas9 chronic wound applications, given the high efficiency of transfection and high cell survival rates as well as the lack of cytotoxic effects.…”

CRISPR-Cas9 directed knock-out of a constitutively expressed gene using lance array nanoinjection

“…LAN uses a microfabricated silicon etched array of lances to physically penetrate hundreds of thousands of cells simultaneously and electrically deliver attached molecular loads (Lindstrom et al 2014; Sessions et al 2014; Teichert et al 2013) (see Fig. 1).…”