Inserting mechanical drugs into living cells (w/video)
(Nanowerk Spotlight) HeLa cells (a lineage of continually dividing cells that have become the most widely used human cell line in biological research) have an average diameter of 20-21 µm. In nanotechnology terms this is quite large and so it is no surprise that researchers have been using microelectronic fabrication techniques – the same technologies used for the fabrication of memory and microprocessor chips of our mobile phones and laptops – to make tiny devices that can be internalized inside living cells.
"Scientists have begun to realize that a cell's physical structure – composed of microtubules, actin filaments, and intermediate filaments – is as important as the biochemical part of the cell," Prof. José A. Plaza, who leads the Micro- & NanoTools Group at Instituto de Microelectrónica de Barcelona, IMB-CNM (CSIC), explains to Nanowerk. "In this sense, the determination of the intracellular mechanical properties and forces encompass crucial information that explain the function or malfunction of living cells."
Whereas in previous work, Plaza and his team designed structures to fit inside a cell, in their new work reported in Advanced Materials ("Intracellular Mechanical Drugs Induce Cell-Cycle Altering and Cell Death") they designed devices that, at 23.5 µm diameter, are actually slightly longer than the diameter of a mitotic cell to study cell mechanics during the cell cycle and to alter the global mechanical equilibrium of the cells. This work was done in collaboration with the Margarita Salas Center for Biological Research (CIB-CSIC) and the Institute of Nanoscience and Nanotechnology of the University of Barcelona (IN2UB).
A star shaped silicon chip enters a living HeLa cell. (Video: CIB, CSIC)
Historically, the studies of disease and therapeutics have usually resulted in biochemical drugs intended to deal with diseases in which the mechanical performance of the cell is decisive. For instance, anticancer drugs are designed to destroy the mechanical intracellular structures of cancer cells, which play a role in the tumorous cells' increased process of division.
Over the years, scientists realized that cell mechanics, the mechanical properties of the cells and their intracellular mechanical loads, are as relevant for a cell's accurate functioning as the biochemical portion of the cell.
In their recent work, Plaza and his group present a novel perspective that addresses the study of cell mechanics and even the alteration of the mechanical equilibrium of the cells by using silicon chips as an intracellular intervention mechanism.
The scientists hypothesized that, what they termed 'mechanical drugs', would destabilize cell mechanics during mitosis (cell division), allowing them to study intracellular reinforcements and constraints.
Design and fabrication of intracellular mechanical drugs. a) Schematic of the 8-pronged star showing the definition of main geometrical parameters (t = thickness, w and L = cantilever’s width and length, Φ = device diameter, ΦHeLa = mitotic diameter of a HeLa cell. b) Schematic of the technology. i) A 1 µm-thick silicon oxide layer (green) is grown on a silicon substrate. ii) A polysilicon layer is deposited as a structural layer. iii) A photolithographic process defines the shape of the chips. iv) Photoresist is striped and polysilicon dry etched to pattern the device. v) Photoresist is removed. vi) Silicon oxide sacrificial etching to release the chips. vii) Released chips are immersed on ethanol and collected in an Eppendorf. (Reprinted with permission by Wiley-VCH Verlag)
To test their hypothesis, the team designed and fabricated devices with varying shapes (star and disk) and and diameters (13.7 µm and 23.5 µm), and with varying thicknesses (50, 110, 230, and 500 nm).
"Our results show that HeLa cells favored the internalization of large devices with features akin to pseudo-1D structures, as with our star design," says Plaza. "This suggests that particle shape plays a dominant role in internalization by HeLa cells, a phenomenon also reported in professional phagocytes."
"Moreover" he continues, "the use of these mechanical drugs has allowed us to directly determine the forces associated with the spindle, the apparatus responsible to split the chromosomes correctly during cell division, which play a relevant role in development and disease studies."
These 'mechanical drugs' open the possibility to determine and measure intracellular forces and internalization routes (which is an important aspect in nanotoxicology studies). They can also produce intracellular mechanical perturbations by altering the cell cycle and even, if they are designed accordingly, destroy the cells.
The fabrication technology used by the researchers offers the possibility to fabricate devices with different shapes and dimensions in order to better understand cell internalization processes, the basis of mechanical affectation on cell cycle, and even cell death and to avoid or promote devices as drug delivery carriers.
"Conducting cell biology research with intracellular chips is in its infancy," Plaza concludes. "However, ongoing advances in the fabrication and miniaturization of chips open avenues for numerous applications of silicon chips inside living cells."