Scientists from the University of Cambridge have developed a highly porous type of nanoparticle known as a metal-organic framework (MOF) that can deliver disease gene targeting small interfering RNAs (siRNAs) directly to cells. There is considerable interest in using siRNAs to knock down disease-related genes as an approach to cancer therapy, but delivering the molecules to cells intact is challenging. The Cambridge University-led researchers, headed by David Fairen-Jimenez, PhD, at the department of chemical engineering and biotechnology, used computational simulations to find a MOF with the ideal pore size, which could encapsulate and carry siRNA molecules to target cells, and then break down and release its siRNA cargo once inside the cells.

“People that have done this before have used MOFs that don’t have a porosity that’s big enough to encapsulate the siRNA, so a lot of it is likely just stuck on the outside,” commented Michelle Teplensky, a former PhD student in Fairen-Jimenez’s group, and first author of the team’s paper, which is published in Chem. “We used a MOF that could encapsulate the siRNA and when it’s encapsulated you offer more protection.” The researchers’ studies are reported in a paper titled, “A Highly Porous Metal-Organic Framework System to Deliver Payloads for Gene Knockdown.”

siRNAs are small, double-stranded RNA fragments, typically 21 to 23 nucleotides in length, which can be designed to block the expression of specific genes and so prevent their proteins from being produced. The technology holds great potential as a therapeutic approach against hard-to-treat cancers, as well as other neurological disorders and viral infections, the authors noted. siRNAs are particularly attractive as a strategy for blocking over-expressed cancer-driving genes because the molecules are highly efficient at gene knockdown, but they are also nontoxic, highly specific—which minimizes the risk of off-target effects—and don’t trigger immune responses. All that is needed to design a siRNA is the sequence of the gene that is targeted for knockdown. And, unlike traditional drug design strategies that are based on expensive and time-consuming rounds of synthesizing and testing new drugs for each condition, redesigning an siRNA to target a different gene is relatively simple.

One of the problems with using siRNAs to treat disease is that the molecules are very unstable and are often broken down before they can reach their targets. “Although siRNA therapy has the potential to benefit patients with cancer, the main limitation is its lack of stability and ease of degradation by native biological enzymes,” the investigators wrote. Chemically modifying the siRNA molecules can make them more stable, but this can compromise their ability to knock down the target genes. It’s also difficult to get the siRNA molecules into cells. They need to be transported by another vehicle acting as a delivery agent.

Fairen-Jimenez leads research into advanced materials, and in particular MOFs, which are self-assembling 3D compounds made of metallic and organic building blocks connected together. “ … MOFs, a class of porous self-assembled materials composed of metal ions or clusters connected by organic linkers, are one of the most promising materials for biomedicine,” the team stated. There are thousands of different types of MOFs that researchers can synthesize, including more than 84,000 MOF structures currently in the Cambridge Structural Database—to which 1,000 new structures are published each month. The properties of MOFs can also be fine-tuned for specific purposes. By changing different components of the MOF structure, researchers can create molecules with different pore sizes, stabilities, and toxicities, including structures that can carry molecules such as siRNAs into cells without harmful side effects.

“With traditional cancer therapy if you’re designing new drugs to treat the system, these can have different behaviors, geometries, sizes, and so you’d need a MOF that is optimal for each of these individual drugs,” said Fairen-Jimenez. “But for siRNA, once you develop one MOF that is useful, you can in principle use this for a range of different siRNA sequences, treating different diseases.”

For siRNA delivery, the MOF carrier needs to be biodegradable so that it doesn’t then build up in cells. The MOF that Teplensky and team selected breaks down into harmless components that are easily recycled by the cell without toxic side effects. “The MOF we chose is made of a zirconium-based metal node and we’ve done a lot of studies that show zirconium is quite inert and it doesn’t cause any toxicity issues,” Teplensky commented. The large pore size of the MOF designed by the team also means that a significant amount of siRNA can be loaded into each MOF carrier, so the dosage needed can be kept very low.

“One of the benefits of using a MOF with such large pores is that we can get a much more localized, higher dose than other systems would require,” said Teplensky. “siRNA is very powerful, you don’t need a huge amount of it to get good functionality. The dose needed is less than 5% of the porosity of the MOF.”

One potential problem with using MOFs or other vehicles to carry small molecules into cells is that they can be halted by endosomal entrapment, a cellular defense mechanism that prevents unwanted components from entering the cell. Fairen-Jimenez’s team added extra components to their MOF to stop them from being trapped during transport into the cell, and so help to ensure that the siRNA reached its target. “… through combined encapsulation of siRNA in the MOF with various cofactors (proton sponge, KALA peptide, and NH4Cl), we show that endosomal retention can be evaded and ensure that gene knockdown is efficacious.”

The team used the system to knock down a gene that produces fluorescent proteins in a test cell line. Working in collaboration with super-resolution microscopy specialists Clemens Kaminski, PhD, and Gabi Kaminski-Schierle, PhD, who lead research in the department of chemical engineering and biotechnology, the investigators applied microscopy imaging techniques to measure and compare the fluorescence emitted by the proteins in cells that were, or were not treated using the siRNA-loaded MOFs. Their experiments showed that by using the MOF platform they could consistently prevent gene expression by 27%, a level that is compatible with the potential use of the system for knocking down cancer genes.

“To the best of our knowledge, this work is the first to utilize a large porous network to internally encapsulate siRNAs in sufficient quantities to achieve gene knockdown,” the authors concluded. “The stability of the MOF material offers future advantages in long-term storage, while the tunability of the MOFs can allow further modifications to improve efficacy. Through this work, we show how the efficacy and efficiency of gene therapy can be improved with the implementation of this highly porous material.”

Fairen-Jimenez believes that it will also be possible to further increase the efficacy of the system. The next steps will be to apply the platform to genes involved in driving hard-to-treat cancers. “One of the questions we get asked a lot is ‘why do you want to use a metal-organic framework for healthcare?’ because there are metals involved that might sound harmful to the body,” Fairen-Jimenez acknowledged. “But we focus on difficult diseases such as hard-to-treat cancers for which there has been no improvement in treatment in the last 20 years. We need to have something that can offer a solution; just extra years of life will be very welcome.

Cells with MOFs carrying siRNA. [University of Cambridge]

The versatility of the system will enable the team to use the same adapted MOF to deliver different siRNA sequences and target different genes. Because of its large pore size, the MOF also has the potential to deliver multiple drugs at once, which could lead to the development of combination therapies.

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