My life has much changed since leaving the world of molecular biology and into clinical medicine. I do not miss the long hours pipetting over the lab bench, frustrated over a failed 4 hour PCR. Instead, I look forward to quickly working up and treating patients. On the other hand, I have found far too many uninspired clinical publications. I miss reading an outstanding publication in the journal Nature or Science on a new second messenger system and imagining up all changes now because of this discovery.
So when the two intersect, I love it. Today, I’ll be sharing with you an example of this intersection; an example of translational medicine, the conversion of scientific discovery into what I believe will be overall health improvement…
I remember when Andrew Fire, Ph.D. and Craig C. Mello, Ph.D., two researchers who brought together the new concept of RNA interference (RNAi), were granted the 2006 Nobel Prize in Physiology & Medicine. At that time, I was graduate student enrolled in a seminar course lead by Maria Gallegos, Ph.D on RNAi. The day she shared the news that the subject of this class was acknowledged by the world is still vivid in my mind.
It was an exciting time. For months we discussed terms like miRNA, siRNA, Dicer, and RNA induced silencing complex (RISC), antisense and sense. We tried to wrap our minds around this new idea. My imagination went wild with how little oligonucleotides self-regulated their brethren.The molecular biology world changed that year with greater understandings in post-transcriptional regulation of gene expression.
Since 2006, the world of RNAi has come a long way, with a lot more resolution with the networks of molecules involved. One thing that has remained constant, however, is the idea that we can use these tiny regulatory RNA molecules to silence deleterious genes (1-6). This can be incredible treatment for some difficult diseases, like genetic disorders, cancers, and other chronic conditions.
The problem with siRNAs and miRNAs is that they last but mere seconds. Cells have counter regulatory molecules, such as ribonucleases which chop up these miRNAs and siRNAs. It is a complicated interplay of molecules. Ultimately, there have been great challenges in delivering the molecules effectively to areas of interest while maintaining its activity (7-10).
Some groups have overcome this delivery challenge by attaching inhibitory RNAs to protective chemical carriers (11-17), like steroids and other nucleotides. These body guards coat the oligonucleotides as they travel through the bloodstream to their target sites, preventing them from degradation. But local treatment trumps systemic any day, especially when sending tiny 22 nucleotide silencing RNAs all around the body that may cross react with other targets. And that is where the research I’ll be sharing today comes into play… But first some more background.
One of my surgical interests is wound healing. As a first year medical student, memorizing the steps of collagen remodeling particularly fascinated me. It was one of my favorite things to memorize, far more than the Kreb’s Cycle. As a third and fourth year medical student, I’ve wondered and how some wounds, like those of a diabetic, just don’t heal. Keloids, however, are remarkable examples of over-healing, or unchecked healing.
Three researchers from Harvard-MIT have synthesized these two worlds of mine in a new publication, “Nanolayered siRNA Dressing for Sustained Localized Knockdown,” and published it in the journal ACS Nano. Paula T. Hammond, Ph.D., is the senior author, who with her colleagues her colleagues produced a nanocoat of siRNA molecules that can be applied on bandages. As the coating slowly dissolves, it releases the RNA molecules tethered to protective nanoparticles, delivering localized, sustained treatment to a wound.
The direct release of siRNA to certain regions of the body could be of significant interest in many applications. In orthopedics and vascular surgery, siRNAs can mitigate cellular interactions with implants and stents, altering the inflammatory response to foreign bodies, allowing hardware and mesh to be incorporated into the body without rejection. In the world of surgical oncology, an excised melanoma can be ensured it won’t come back with siRNAs released slowly to degrade cancerous RNAs. Ultimately, slow and direct release of siRNAs act as a localized reservoir for sustained therapeutic benefit.
The thin films consist of two different materials. The first is a peptide called protamine sulfate (PrS), a naturally derived isolate from salmon sperm. PrS has an isoelectric point of pH 12 and stabilizes of nucleic acids because its full of arginine, which binds DNA and siRNA to protect them from nuclease degradation. The other material, calcium phosphate (CaP) nanoparticles decorate the therapeutic siRNAs with intrinsically negatively charged and have been shown to remain intact until they dissociate when the pH falls below about 6.8–6.6. Additionally, other researchers have shown that similar nanoparticles help the nucleotides evade destruction once they’re taken up by cells (J. Controlled Release 2010, DOI: 10.1016/j.jconrel.2009.11.008). The RNA and nanoparticles are negatively charged, and the peptides are positively charged. The two substances cling together due to electrostatic force, producing a film when the water dries.
To test their delivery method, the researchers coated woven nylon bandages with 80-nm-thick layer-by-layer or LbL films and applied them to Tegaderm bandages, commonly used in operating rooms. The bandages were placed on layers of human and animal cells in culture. In one experiment, a bandage loaded with 19 µg of siRNA per square centimeter released two-thirds of its load over 10 days. Other bandages made using siRNAs targeting the gene for fluorescent green protein (GFP) almost completely shut down the protein’s production in cells expressing the gene, seen below in Figure 2.
Hammond says the group is now testing bandages that knock down MMP9, a collagen-destroying protein associated with slow healing in chronic wounds. I believe we can see this discovery in clinical practice very soon because PrS and CaP are naturally derived materials, and have already been approved by the Food and Drug Administration, but the final challenge of showing that genes can turn off in animal models of wound healing remains.
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