Folding DNA origami for chemotherapy treatments

Chemotherapy is the primary cancer treatment used today. It involves using cytotoxic agents which attack and kill cells that are rapidly dividing, like cancer cells. However, delivering these drugs can be tricky. Usually drugs are administered into the circulatory system, exposing the whole body to their toxicity. Much of the toxicity associated with chemotherapy is due to nonspecific distribution throughout the body. Cytotoxic drugs affect more than just the tumor cells, and can lead to cell death in the immune and gastrointestinal system, along with vital organs. In an ideal world, we could develop a way of administering the drug only to the tumor, reducing the amount of toxicity to the rest of the body. Currently, there are polymers and nanoparticles that have been used to help deliver drugs to specific areas of the body. However, making a delivery system that is safer than nanoparticles and is also biocompatible has yet to be explored in great detail. Recently, a group from Xidian University in China has developed a biosynthetic method of drug delivery that uses structures made out of DNA.

To get to the bottom of the DNA structures they used, we're going to go back to 2006 when Paul Rothemund developed a way to create shapes out of DNA. He named this DNA Origami. He would take a long piece of viral,single-stranded DNA of about 7,200 basepairs. Then, using a computer algorithm, he would design small ‘staples' of 20-30 base pair long single-strandedDNA that would bind to the long single-stranded DNA in a way that would fold it into the shapeof their choice. A diagram of this is shown the right along with some examples of the shapes they made below it shown using atomic force microscopy (scale bars are 100nm). The black strand is the long single-stranded DNA strand, while the short colored portions are the small single-stranded staples. The regions the staples bind to will define how the whole structure is organized. By changing the sequence of the staples, and where they bind on the larger strand, they could design different shapes of DNA. DNA origami is an example of ‘bottom-up' fabrication. It relies on using small components to build a bigger tool. As an example, to build a house, one could take a large concrete structure, and carve a house out of it. This would be considered a top-down approach to building. On the other hand, you could use small bricks to build up the entire house, a bottom-up approach. When it comes to nanotechnology, there are advantages to both, but DNA origami relies on the bottom-up approach. It takes advantage of what we know about how DNA folds to turn it into whatever shape we want.

The researchers from Xidian University wanted to use this DNA origami to create a new way to introduce chemotherapy drugs into tumors. This method has several key characteristics that make it useful as a drug delivery vehicle. The short staple DNA sequences can be engineered to carry specific molecules. In this way, you can design them to hold a drug of interest. DNA origami has already been used to deliver different molecules, including cancer drugs, into tissue culture cells. Also, DNA origami is stable for up to 12 hours, and is degraded by cells within 72 hours. This means it has great potential for controlled drug release. Finally, because it's made out of biological material, cells may not recognize the DNA origami as a toxic substance like they do the chemotherapy drugs, so the drug delivery may be more efficient. With these advantages in mind, the researchers wanted to see if chemotherarpy drugs delivered with DNA origami would work in vivo, that is, in a living animal with a tumor. To approach this question, they designed several different sizes and shapes of the DNA origami to see which ones could be the most efficient.

The researchers used immune-compromised mice with tumors located just underneath their skin. They do this by injecting breast cancer cells they grow in culture just under the skin. The mouse will then develop a tumor. This is one of the more established ways of testing drug efficacy against tumor growth. In this scenario, the breast cancer cells they injected were also labeled with Green Fluorescent Protein (GFP), which would allow them to image the tumor and use total fluorescence as a way to measure tumor size. This would give them the opportunity to monitor the volume of the tumor easily over thecourse of the drug delivery.

They did some initial experiments using different shapes of DNA origami to see which ones would be the most efficient at targeting tumors. They added quantum dots to the DNA origami which allowed the scientists to visualize its location in the body using fluorescence imagining. They injected the DNA origami into the tail vein, and it would circulate around the mouse. There is a special property of tumor tissue that makes it easier to target with drugs. This is called the EPR effect where EPR stands for enhanced permeability and retention. Cancer cells tend to take up and retain macromolecules more readily than other tissues in the body. This meant that the DNA origami would be more likely to be targeted to and stay in the tumor. After they injected the different shapes of DNA origami, and imaged the quantum dots, they found that the DNA origami they engineered to be a triangle was far more efficient than the square or tubular ones at targeting the tumor. The triangle shape was also almost completely absent from vital organs. This is important because you don't want to introduce toxic drugs to healthy portions of the body, especially vital organs.The evidence for this is shown in the first figure above, where tumor tissue targeted by the triangle origami has very high Quantum Dot fluorescence, but nearly no fluorescence in other tissues, especially compared to the square and tube origami. From this experiment they learned they should use the triangle shaped DNA origami.

The next step was to load up the DNA origami with a chemotherapy drug named doxorubicin, referred to as DOX from here on out. The researchers had 4 conditions to test. They used 4 groups of mice and would inject either the drug, DOX, the drug plus the DNA origami, DNA/origami, the DNA origami alone, or a plain saline solution, as a control. They would inject these solutions every 3 days and measure the tumor volume and body weight every 3 days as well. Over the course of 12 days, the tumor size in the DNA/origami group decreased significantly, to about one-third of its original size. It even decreased tumor size more than injecting just DOX alone. You can see in the pictures above how the tumor size was measured. They would take fluorescent images of the entire mouse and measure how bright the region with the tumor was. Because the tumor cells expressed GFP, the amount of fluorescence represented the size of the tumor. You can see how in the DOX and DOX/origami treatments the fluorescence in the tumor is far less than in the control treatments. The quantification of tumor size is outlined in the graph to the left. Most importantly, the DNA/origami treatment exhibited nearly no toxicity on the mouse. Usually when exposed to chemotherapy drugs, there are severe immunological responses, body weight will reduce, and blood levels will drop. In the DOX/origami treated mice they had healthy body weights, especially compared to the DOX treated group, whose body weight dropped nearly 30% (shown in the graph below). The DOX/origami treated mice also showed no signs of immunological response and their blood levels, like their white and red blood cell counts, were completely normal.

Given the significant reduction in tumor size, and what appears to be a complete lack of systemic side effects, DNA origami presents a novel and effective way to target tumors. This represents a biosynthetic approach to efficiently target tumors with chemotherapeutic drugs without what seems to be any adverse side effects. Not only does DNA origami target tumors well, but because of how the DNA origami is engineered, it can be designed to contain imaging molecules so antitumor efficacy be monitored throughout treatment. It's exciting to see a technique like DNA origami that may have originally been designed for protein biochemistry experiments or to develop biological based microcircuits being used as an incredibly efficient tool in the medical field. There hasn't been any recent evidence implicating its consideration for use in primates or humans, but I would love to see this tried out in other models because it appears that DNA origami may be a groundbreaking development in drug targeting.

References:

Rothemund, P.W.K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297-302 (2006).

Zhang, Q. & Jiang, Q., et al. DNA Origami as an In Vivo Drug Delivery Vehicle for Cancer Therapy. ACS Nano 8, 6633-6643 (2014).

Image credits:

The first image is augmented from the Rothemund paper cited above. All other figures are augments from the Zhang & Jiang et al. paper cited above.