Gene therapy, where genetic material is added directly to patients’ cells to compensate for abnormal or missing genes, promises to be a boon to medical care, helping to fight cancer and treat genetic diseases. But current gene therapy techniques, such as those that use viral vectors, are either costly, unsafe, or inefficient. 

In a step forward for the field, researchers at the University of California, Los Angeles (UCLA) have now developed biodegradable, magnetically guided “nanospears” (nanoscale, splinter-like structures) that can pierce cells and deliver genetic material. In experiments published recently in ACS Nano, the scientists tested their nanospears as vehicles to deliver genes that encode fluorescent proteins—90% of the targeted cells survived the mild trauma and 80% subsequently glowed fluorescent. 

“We can efficiently deliver a payload to cells and transfect cells in a way that keeps most of the cells alive,” says UCLA nanoscientist Paul S. Weiss, who was a co-lead author of the new study along with pediatric oncologist Steven Jonas. “We showed that we could do it with an individual cell, as well as up to a couple hundred thousand cells in one batch.”

Current gene therapy approaches penetrate cell membranes to deliver genes using modified viruses, external electrical fields (electroporation), or chemical reagents (such as lipofection, which transfects cells using liposomes, or lipid-based vesicles). While they show great promise in treating a range of diseases, from hemophilia to muscular dystrophy to certain types of cancer, these approaches all have their own issues, Weiss says. 

Viral methods, for example, can accidentally turn on cancer genes, and the treatments are expensive. In March 2018, US doctors used a new gene therapy to treat a child with a rare genetic disorder that caused vision loss—the virus-based treatment costed $425,000 per eye. On the other hand, electroporation, which opens pores in the cell membrane using electric fields, often destroys cells, and lipofection delivers a variable amount of DNA, making the therapy inefficient. 

Recently, researchers investigated whether “nanoneedles” could be used for gene therapy. Experiments suggest these nanoscale structures can successfully deliver genetic material into cells, but the movement of the nanoneedles developed thus far cannot be controlled. Additionally, the needles are not individual and are instead comprised of arrays on a substrate–this arrangement makes it difficult to separate punctured cells from the nanostructured substrate for further study. 

Weiss, Jonas, and their colleagues sought to come up with a gentler way to penetrate cells with nanostructures that can be precisely controlled. To fabricate their nanospears, they used an affordable and scalable approach called nanosphere lithography. They allowed polystyrene nanospheres (2 μm in diameter) to self-assemble into dense monolayers on a silicon wafer substrate, and then reduced the size of the nanospheres to 1.4 μm by exposing them to oxygen plasma. Next, to create sharp silicon needles with high aspect ratios, the team used reactive ion etching to etch the silicon substrates vertically with the polystyrene spheres serving as a template and simultaneously etch the nanospheres down to less than 50 nm. 

The researchers then evaporated nickel and gold thin films on to the silicon nanospear arrays. The nickel film allows the nanospears to be controlled with magnetic fields, while the gold film is biocompatible and can be tailored to load biomolecules. They used a multi-step, layer-by-layer process to modify the surface of the nanospear arrays and coat them with encapsulated nucleic acid molecules (plasmids). Finally, they released the plasmid-encapsulated nanospears from the silicon substrate through gentle mechanical scraping with a razor blade.

The scientists tested their 5-μm-long nanospears in vitro on U87 glioblastoma cells (malignant brain tumor cells). In their first tests, they showed that they could precisely guide a nanospear loaded with plasmids that express enhanced green fluorescent protein into a glioblastoma cell using a magnetic field produced by a small disk magnet. After 24 hours, the cell produced green fluorescence, showing that it was successfully transfected, while non-target cells remained unchanged.

In another experiment, they added approximately 1 million nanospears dispersed in deionized water into a single well containing about 200,000 U87 cells. With magnets, they guided the nanospears toward the cells, with scanning electron micrographs showing that multiple cells can dock at individual cells. Overall, 80% of the target cell population subsequently glowed green and 90% of the population remained viable. By two weeks, many of the nanospears were completely dissolved. 

In other tests, the researchers showed that they could selectively guide nanospears carrying different expression plasmids to neighboring cells. They loaded some nanospears with plasmids that express green fluorescent protein and others that express red fluorescent protein. They guided the green group to U87 cells to the left-hand portion of a cell culture well and then guided the red group to U87 to the right-hand portion. After 24 hours, the cells on the left-hand and right-hand sides of the dish glowed green and red, respectively. 

“The work is a very interesting evolution of the first initial concept of nanoneedles, in that it adds a component of control of the orientation that was not present in previous platforms,” says Ennio Tasciotti, a molecular biologist at Houston Methodist Research Institute who has worked on nanoneedles and was not involved in the current study. This level of control will allow scientists to learn more about “the inner workings of cells and how the cell interfaces with [penetrative] technologies,” he says. But the technology still has some challenges, he notes, including employing it in vivo and scaling it up to transfect millions to billions of cells (as would be required for clinical use). 

Weiss, Jonas, and their team will be optimizing the nanospears and comparing the technology head-to-head with other transfecting approaches. They are also collecting a variety of cells that are models of those needed for different diseases to see which applications appear most promising for this new gene therapy platform. Weiss says, “We are looking into a smorgasbord of different diseases and testing if this is the right approach for particular types of cells.”

Read the abstract in ACS Nano.