The discovery and development of therapeutic biomolecules - such as the gene editing technology CRISPR - has rapidly evolved in recent years, however the delivery methods to target cells remain less than ideal.
Current approaches include electroporation, using an electric field to increase cell permeability, or the use of genetically modified viruses. Although regularly used in the research setting, these methods come with the undesired side effects of high levels of cell death (in the case of electroporation) and safety issues (in the case of viruses).
A collaborative study between research groups from StemTherapy and NanoLund, strategic research areas funded by the Swedish Government, has aimed to tackle this problem.
“We wanted to develop an alternative, more gentle method for the delivery of biomolecules into human hematopoietic stem cells.” explains Ludwig Schmiderer, PhD student within the Stem Cell Regulators group led by Prof. Jonas Larsson and first author of the study. “Together with Martin Hjort, a researcher at NanoLund and CTO at NAVAN Technologies, we investigated the potential of nanostraws for this purpose.”
Nanostraws are aluminium oxide tubes 100 - 200nm in diameter and 1-3 µM in length and produced locally in the Lund Nano lab, enabling fast feedback between biological experiments and material science optimization.
“In previous studies we have used these structures to repeatedly extract molecules from cells, with little effect on cell viability.” explains Martin. “In this study, we were curious if we could do the opposite and use nanostraws as a direct fluid pathway for biomolecules into human hematopoietic stem cells.”
In the study published in the journal PNAS, the research groups describe how they have developed a method termed Centrifugation enhanced Nanostraw Transfection (or CeNT for short).
During this process, stem cells are placed into a tube containing a membrane embedded with nanostraws. Below the membrane is a compartment containing the biomolecule to be transferred into the cells. The tube is gently centrifuged, bringing the cells into close contact with the nanostraws and allowing them to pierce the cell membranes. A very gentle pulsed electric field is then applied, driving the biomolecule directly into the cell cytoplasm. This whole procedure takes only ten minutes.
“Using this method we can deliver cargos ranging from the very small (including the DNA dye propidium iodide) to the very large (including dextran chains of up to 2MDa).” says Ludwig “Most importantly, we show that we are able to deliver functional genetic material to hematopoietic stem cells.”
Following CeNT of hematopoietic stem and progenitor cells with mRNA encoding green fluorescent protein, the researchers isolated green fluorescent cells after less than six hours. Not only did they observe no negative effect on cell viability using this method, they were able to verify that they had successfully targeted stem cells through extensive transplantation studies
“In addition, we could not detect any changes in gene expression associated with the nanostraws,” reflects Ludwig “the only observed differences being directly associated with the GFP mRNA transcript.” These findings are in sharp contrast to electroporation, during which the process alone alters the expression levels of over 2000 genes.
This exciting new approach offers a number of desirable qualities for the treatment of cell samples in both a research and clinical setting: the method is fast and gentle with no off-target effects with the option of keeping cells within their native medium, thus perturbing them as little as possible.
“The surprisingly gentle nature of this method encourages us to explore practical applications of nanostraws.” concludes Martin. “Moving forward, we are now using this technology to deliver gene editing agents, such as CRISPR-Cas9, which can potentially be used to correct disease causing genetic mutations.”
