Learn to understand how nanomaterials enter cells and how cells react

June 20, 2022

(Spotlight on Nanowerk) The formation of supramolecular complexes between DNA and carbon nanotubes (CNTs) – single-walled and multi-walled – has attracted the attention of researchers since the synthesis of the first DNA-wrapped CNTs in 2003. These hybrid structures can take advantage of the unique mechanical, electrical, thermal and optical properties of nanotubes and combine them with the remarkable bio-recognition capabilities of DNA. Supramolecular DNA-CNT complexes have been proposed for applications such as chemical biosensors, cellular transporters that target structures such as mRNA inside cells, fibers for artificial muscles, and bioelectrodes for battery cells. combustible.

Although scientists know that CNTs can penetrate almost any cell (wall), the detailed mechanism of uptake is still unclear and most likely depends on the state of the cell cycle. Equally important is the elucidation of potential cytotoxicity and cellular damage caused by variations in CNT size, purity, concentration, and functionalization.

“Each cell relies on the uptake (endocytosis) of materials like proteins, cytokines, and even synthetic carbon nanomaterials, to perform its required cell fate functions,” Sabrina Jedlickaassociate professor of bioengineering and materials science and engineering and associate dean of Rossin College of Engineering at Lehigh University, tells Nanowerk.

“Studying this process in detail is an extremely difficult and therefore extremely interesting goal in biophysics,” adds Slava V. Rotkin, Frontier Professor of Engineering Science and Mechanics with an appointment at the Materials Research Institute at Penn State. “Therefore, endocytosis is of interest for bringing therapeutic targets into cells. Studying the pathways by which materials enter the cell can help unravel the trafficking to design more effective targeted drug and gene therapies.

Identifying new ways to differentiate and maintain healthy cell populations could lead to new therapies in regenerative medicine. Nanomaterials could hold the key, but researchers still know too little about the detailed mechanisms underlying nanomaterial-cell interactions to safely design treatment protocols.

In a further step towards uncovering exactly how synthetic nanomaterials interact with cells, Jedlicka from Lehigh University, Rotkin from Penn State and Prof. Tetyana Ignatova at the University of North Carolina at Greensboro and their teams, conducted a series of experiments that allowed them to determine the mechanisms of cellular uptake of single-walled DNA-wrapped carbon nanotubes (SWCNTs), and how they depend on the cell cycle (ie the state of the cell during its division).

They published their findings in Biophysical reports (“Cell cycle-dependent endocytosis of DNA-wrapped single-walled carbon nanotubes (DNA-SWCNTs) by neural progenitor cells”). Raman microscopy image of a fixed neural stem cell (right) superimposed on the Raman image showing three SWCNTs (left, highlighted by boxes around each SWCNT location). (Image courtesy of the researchers)

The material used in this study has already been characterized in depth: SWCNTs synthesized by CoMoCat enveloped with (GT)20 single-stranded DNA oligomers. DNA conjugation serves as a biomolecule mask for cells – even though this artificial strand would not be recognized by the cell – also greatly increasing the solubility of nanomaterials in aqueous buffer and inhibiting nanotube coalescence.

“We hypothesized that the presence of the nanotubes, in optimized concentrations and external conditions, may upregulate aspects of natural cell fate processes that can be exploited to better understand the system, which can be used to the future for developing improved nanosensors and therapeutic delivery,” Jedlicka and Rotkin explain the background to this work. “It requires cellular and molecular scale analysis of the nanotube-cell system to understand the modification of developmental behavior cells in response to potential nanomaterial-based therapies as well as the downstream implications of cell-material interactions on these therapies.”

The team’s study focused primarily on the modes of entry of SWCNTs into cells and how internalization changes depending on the phase state of the cell. Despite all the research that has been done in this area, the detailed aspects of endocytic mechanisms and pathways are still not fully understood. Since scientists are familiar with SWCNT-cell interactions, this information has primarily been averaged over whole cell populations where the cells are of different age (i.e. what stage each cell is in the cycle cell) or taken from a single cell with random age.

“These input mechanisms are accompanied by respective stimuli that trigger various downstream cellular responses and signal transduction changes in the cell as a result,” Jedlicka points out. “Unsurprisingly, this results in biochemical changes in the cell, vital for its growth and development, in addition to the innate dynamic reorganization of the internal components of the cell.”

The researchers’ overall results in this study correlate strongly with the hypothesis that nanomaterials enter the cell by more than one method and that multiple methods of endocytosis may be responsible for the uptake of a single cargo.

Going forward, the team’s next study will focus on the mechanisms by which CNTs influence cell differentiation. This study will examine traditional means of cell differentiation in vitro compared to CNT-based differentiation. This will assess whether the mechanisms by which nanotubes interact with cells are similar to the natural internal drivers of cell differentiation, allowing for mapping of signaling pathways and assessment of any potential challenges around gene regulation.

“Extending our approach to other cell types and other types of nanomaterials is definitely the right way to move forward towards these goals,” Rotkin concludes. “It’s a very big volume of work, which our groups cannot do alone. It’s up to the scientific community to contribute in this area.”

“As far as we are concerned,” he adds, “we would like to deepen our knowledge of the processes inside the cell, potentially using high-resolution, non-destructive optical characterization techniques such as Raman microscopy ( see for example: “Micro-Raman spectroscopy as a tool enabling long-term intracellular studies of nanomaterials at nanomolar concentration levels”) and Scattering Scanning Near-field Optical Microscopy (sSNOM) (see for example: “Multidimensional Imaging Reveals Mechanisms Controlling Label-Free Multimodal Biosensing in 2DM Vertical Heterostructures”).” By

Michael is the author of three Royal Society of Chemistry books:
Nano-society: pushing the limits of technology,
Nanotechnology: the future is tinyand
Nanoengineering: the skills and tools that make technology invisible
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