Thioketal Nanoparticles Targeting Inflammation

The article Orally delivered thioketal nanoparticles loaded with TNF-alpha-siRNA target inflammation and inhibit gene expression in the intestines published in Nature Materials by Wilson, Dalmasso, Wang, Sitaraman, Merlin, and Murthy was one of the most dense articles we discussed this week, but arguably, one of the most well-thought out articles too in its processes. I'm going to do my best to summarize the logic that each experiment in the article was founded on. The researchers aimed to design a novel way to prevent the excess inflammation in inflammatory bowel disease and target the macrophages in the mucosal lining to downregulate pro-inflammatory cytokines that worsen symptoms. They developed a novel method where siRNAs, or small interrupting RNAs, are specifically delivered to areas of inflammation and reduce pro-inflammatory cytokine production locally.

Challenge 1: How do you target only diseased intestinal tissue in the release of siRNAs/drug delivery?

Researchers identified high levels of reactive oxygen species (ROS) that are released from sites of inflammation through studying ROS concentrations in biopsies from ulcerative colitis, colon cancer, and Helicobacter pylori infection patients. They developed the ROS-sensitive polymer thioketal nanoparticles (TKN) to encapsulate the siRNAs until they are released in the intestine at sites with high ROS, or inflammation sites, but still stay resistant to pH changes in the digestional tract. 

Challenge 2: Wait, how do we really know TKN degrades due to ROS and not due to pH changes? And how exactly do we know it can carry a drug/siRNAs?

Researchers took the polymer material and exposed it to three different solutions in an experiment: one solution was acidic, one was basic, and the final was concentrated with ROS. The polymer's weight reduced to less than a tenth of its original size in the superoxide solution, but stayed the same in the basic and acidic environments. They also tested delivery of an agent by making TKNs carrying a fluorescent dye and releasing them around activated macrophages. As such, the activated macrophages had higher count of fluorescent markers than inactive macrophages, indicating a release by TKN.

Challenge 3: So, now that we know the delivery method "works" releasing the material, how do we make sure the siRNA/drug gets into the cell and where we want it to go (ie. the bowels)?

Researchers added DOTAP, a positively charged lipid, to make sure the TNF-alpha siRNA particles in the TKNs (hereby known as "TNF-a-TKN"s). They also made the size roughly around ~600 nm that altered which cells would take them and where they would bind in the mucosa. 

Challenge 4: Remind me again, how do we know this "TNF-a-TKN" actually works to silence the TNF-alpha (pro-inflammatory) production in these immune cells?

Macrophages were 'activated' and then scientists studied how these cells reacted to TNF-a-TKN and controls (random siRNA fragments or placebo). TNF-a-TKN treated cells exhibited a significant reduction in TNF-alpha production.

Challenge 5: Can we get these siRNAs into the gut successfully of an IBD patient where it is supposed to work?

Mice models finally come into play here. To model an IBD patients, researchers induced an IBD-like reaction in the GI tract by tainting the experimental mice's water with DSS. These DSS mice and control mice were both given TKNs with fluorescence-tagged, random siRNAs in them to prove that the TKNs "can localize orally delivered siRNA to sites of intestinal inflammation." Researchers found three times the fluorescent siRNAs at inflammation sites compared to non-inflammatory sites.

Challenge 6: So, the entire capsule of TKNs can get into the body, through the digestive tract, and release its siRNAs directly to sites of inflammation instead of random sites in the GI tract. Does this actually work as we want now in mouse model to reduce the production of pro-inflammatory factors locally?
Once again, researchers induced IBD in mice by tainting their water. This time they administered TNF-a-TNKs (remember, TNF-a is a proinflammatory agent) to mice along with appropriate control groups. They calculated the levels of individual proinflammatory factors (the targeted TNF-alpha along with IL-6, IL-1, and IFN-gamma) in each of the groups of mice. Mice with TNF-a-TNKs exhibited a 10-fold decrease in colonic TNF-a mRNA (which produces this factor); these same mice also had inhibited levels of IL-6, IL-1, and IFN-gamma. Other experiments included in the paper came to similar conclusions, supporting the idea that TNF-a-TKNs did allow the delivery of this agent (siRNAs against TNF-alpha) to help treat local inflammation involved with IBD.


Researchers did continue to test for other factors during the study including the relative toxicity in comparison to the existing FDA-approved PGLA for drug delivery and a few tests that were aimed to discover the correct dosage level. However, this highlighted was the general logical pathway I imagined the researchers following, as conveyed by the logical progression of the article. As always there are some lose ends left by the researchers, but I believe they conveyed their main aim of the project (the development of the novel method for drug delivery specifically with IBD) and achieved that goal, despite occasionally missing some more medicinal or pharmacological areas of focus. As always with novel developments, the topic needs to be repeated and studied more thoroughly. However, I am very excited to hear of such an invention.

For more information about the tests undergone, the results recorded, and overall more information than the summary, please feel free to read the article for yourself.

Citation
Wilson, S., Dalmasso, G., Wang, L., Sitaraman, S.V., Merlin, D., and Murthy, N. (2010) Orally delivered thioketal nanoparticles loaded with TNF-a-siRNA target inflammation and inhibit gene expression in the intestines. Nature Materials.