A USF Health preclinical study indicates nanoparticles containing a micro-RNA switch offers promising biotechnology to advance the fight against atherosclerotic cardiovascular disease

Illustration of angioplasty with a stent

Tampa, FL (Feb. 9, 2021) – Percutaneous coronary intervention (PCI), commonly known as angioplasty with a stent, opens clogged arteries and saves lives. Despite its benefit in treating atherosclerosis that causes coronary artery disease, this common minimally invasive procedure still poses severe complications for some patients.

Angioplasty involves inflating a balloon at the tip of a catheter to compress fatty deposits (plaques) against the artery wall, thereby restoring blood flow to the narrowed or blocked vessels. The image-guided procedure is often combined with the placement of either uncoated stents — tiny expandable mesh devices– or stents coated with slowly-released antiproliferative drugs. The drug-eluting stents help avert the growth of scar tissue (smooth muscle cell proliferation) in the artery so that the vessel does not eventually close again, known as restenosis.

However, current antiproliferative drugs indiscriminately inhibit the growth of all nearby cells, including the layer of endothelial cells lining the blood vessels. These endothelial cells prevent blood clots (thrombosis) within the stent and the formation of more plaques (neoatherosclerosis), which can trigger a heart attack or sudden cardiac death.

Focused on tackling this treatment complication, University of South Florida Health (USF Health) Morsani College of Medicine researchers recently developed a next-generation nanotherapy. Their preclinical findings are detailed in a study published Feb. 2 in Molecular Therapy.

Hana Totary-Jain, PhD, USF Health associate professor of molecular pharmacology and physiology, was principal investigator for the nanotherapy study.

The nanotherapy comprised of a nontoxic peptide known as p5RHH and a synthetic messenger RNA (mRNA) that carries the genetic instructions, or code, needed by cells to make proteins. By simply mixing up the p5RHH with the mRNA, they spontaneously self assemble into compacted nanoparticles that specifically target the injured regions of the arteries in mouse models mimicking angioplasty. The nanoparticles contain an microRNA switch added to the mRNA.

“One of the main challenges of cardiovascular disease remains the delivery of targeted therapies specifically to the plaque regions and the cells that form plaques, including the smooth muscle cells and inflammatory cells — without affecting the endothelial cells or the healthy regions,” said the study’s principal investigator Hana Totary-Jain, PhD, an associate professor of molecular pharmacology physiology at USF Health Morsani College of Medicine.

To do this, the researchers used mRNA that encodes for p27 protein, which blocks cell growth, and added to the mRNA an endothelial cell-specific microRNA to generate a microRNA switch. The design of this microRNA switch allowed the researchers to turn on the mRNA in smooth muscle cells to inhibit their growth and the formation of restenosis. It also enabled them to turn off the mRNA in endothelial cells so these cells could grow uninhibited and quickly heal the damaged blood vessel.

John Lockhart, PhD, was the paper’s lead author.

“If we can come up with an antiproliferative therapy that specifically targets the cardiovascular smooth muscles cells and the infiltrating inflammatory cells but spares the endothelial cells – which we’ve done with the design of our microRNA switches – then we should be able to achieve the therapeutic effects of drug-eluting stents without the downside of thrombosis and neoatherosclerosis,” said the paper’s lead author John Lockhart, PhD, who worked on the study as a doctoral student at USF Health Molecular Pharmacology and Physiology. Dr. Lockhart is continuing his postdoctoral training at Moffitt Cancer Center.

The latest study builds upon previous research by Dr. Totary-Jain, indicating that a microRNA-based therapy worked better than drug-eluting stents in a rat model of angioplasty. That work used an adenovirus vector to carry the cell-selective therapy to injured arteries. In this study the viral vector was replaced with a nanoparticle alternative – a change needed to avoid safety concerns and advance the therapy toward use in patients.

The investigational nanoparticles were injected into mice with arteries mimicking post-angioplasty vessel injury every three days for two weeks (5 doses total). Mice treated with the nanoparticles containing the miRNA switch had significantly reduced restenosis and completely restored endothelial cell growth in the injured artery, compared to animals treated with nanoparticles containing mRNA without the miRNA switch, the researchers report.

Above: Injured control artery treated with near infrared florescent protein, depicts restenosis in center. Below: Injured artery treated with the microRNA switch nanotherapy shows open artery (no restenosis) and clear endothelial cell layer marked in green. | Images courtesy of Hana Totary-Jain, USF Health

In addition, the nanoparticles efficiently delivered its mRNA cargo, without degradation, solely to regions of the artery where endothelial cells were damaged. The particles did not toxically accumulate either in the cells of healthy organs (the liver, spleen. lungs or kidneys), or in uninjured arteries adjacent to those requiring treatment. The researchers observed no adverse reactions or outcomes in mice treated with the nanoparticles.

Overall, the findings suggest that the miRNA-switch nanoparticles could be applied clinically to selectively prevent restenosis after PCI by specifically targeting areas of endothelial cell damage to allow quicker cell regrowth and repair of injured arteries.

The USF Health researchers next plan to investigate the potential of the microRNA-switch nanoparticles to directly treat atherosclerotic plaques, thereby eliminating the need for PCI.

“Cardiovascular disease is still the number one cause of death,” said Dr. Totary-Jain, a member of the USF Health Heart Institute. “This research offers promise for the development of novel biomolecular therapies to advance the fight against coronary artery disease and peripheral artery disease,”

One person dies of cardiovascular disease every 36 seconds in the U.S., according to the Centers for Disease Control and Prevention.

The USF Health research was supported by grants from the National Institutes of Health. Samuel Wickline, MD, director of the USF Health Heart Institute, and Hua Pan, PhD, assistant professor at the Heart Institute, collaborated on the study.

A USF Health-Johns Hopkins Medicine team shows that nanoparticles targeting the blood-clotting protein reduces necrotizing enterocolitis-like injury in neonatal mice

.

TAMPA, Fla. (May 4, 2020) — Necrotizing enterocolitis (NEC), a rare inflammatory bowel disease, primarily affects premature infants and is a leading cause of death in the smallest and sickest of these patients. The exact cause remains unclear, and there is no effective treatment. Since no test can definitively diagnose the devastating condition early, infants with suspected NEC are carefully monitored and administered supportive care, such as IV fluids and nutrition, and antibiotics to fight infection caused by bacteria invading the gut wall. Surgery must be done to excise damaged intestinal tissue if the condition worsens.

A new preclinical study by researchers at the University of South Florida Health (USF Health) Morsani College of Medicine and Johns Hopkins University School of Medicine offers promise of a specific treatment for NEC, one of the most challenging diseases confronting neonatologists and pediatric surgeons.  The team found that inhibiting the inflammatory and blood-clotting molecule thrombin with targeted nanotherapy can protect against NEC-like injury in newborn mice.

Their findings were reported May 4 in the Proceedings of the National Academy of Sciences.

“Our data identified the inflammatory molecule thrombin, which plays a critical role in platelet-activated blood clotting, as a potential new therapeutic target for NEC,” said coauthor Samuel Wickline, MD, professor of cardiovascular sciences at Morsani College of Medicine and director of the USF Health Heart Institute. “We showed that anti-thrombin nanoparticles can find, capture and inactivate all the active thrombin in the gut, thereby preventing or reducing the small blood vessel damage and clotting that accelerates NEC.”

The PNAS paper’s senior author is Akhil Maheshwari, MD, professor of pediatrics and director of neonatology at the Johns Hopkins University School of Medicine. Before joining Johns Hopkins Medicine (Baltimore) in 2018, Dr. Maheshwari’s group at USF Health was the first to demonstrate that platelet activation is an early, critical event in causing NEC, and therapeutic measures to block these platelets might be a new way to prevent or reduce intestinal injury in NEC.

The nanotherapy platform created by Dr. Wickline and USF Health biomedical engineer Hua Pan, PhD, delivers high drug concentrations that specifically inhibit thrombin from forming blood clots on the intestinal blood vessel wall without suppressing the (clotting) activity needed to prevent bleeding elsewhere in the body. This localized treatment is particularly important for premature infants, Dr. Wickline said, because the underdeveloped blood vessels in their brains and other vital organs are still fragile and susceptible to rupture and bleeding.

Samuel Wickline, MD, and Hua Pan, PhD, of the USF Health Heart Institute, created the treatment platform used to deliver anti-thrombin nanoparticles to the site of gut damage.

For this study the researchers used a model they created — infant mice, or pups, induced to develop digestive tract damage resembling human NEC, including the thrombocytopenia commonly experienced by premature infants with NEC. Thrombocytopenia is characterized by low counts of blood cell fragments known as platelets, or thrombocytes, which normally stop bleeding from a cut or wound by clumping together to plug breaks in injured blood vessels.

The molecule thrombin plays a key role in the bowel inflammation driven by overactive platelets. While investigating role of platelet depletion in NEC-related thrombocytopenia, the USF-Johns Hopkins researchers were surprised to find that thrombin mediates platelet-activated blood clotting early in the pathology of NEC-like injury – before bacteria leaks from inside the gut to circulating blood or other organs. This clotting clogs small blood vessels and restricts blood flow to the inflamed bowel. Eventually, the lining of the damaged intestinal wall can begin to die off.

The investigative therapy essentially works “like a thrombin sponge” that is exponentially more potent than current agents used to inhibit clotting, Dr. Wickline explained. “It literally puts trillions of nanoparticles at that damaged (intestinal wall) site to sponge up all the overactive thrombin, which tones down the clotting and inflammation processes promoted by thrombin.”

“We are so excited about finding this new way to attenuate intestinal injury in NEC,” Dr. Maheshwari said.

The same approach has also been shown in preclinical studies to inhibit the growth of atherosclerotic plaques and certain kidney injuries without causing systemic bleeding problems, Dr. Wickline added. “The nanoparticles can be tailored to other inflammatory diseases highly dependent on thrombin for their progression.”

The study authors conclude that their experimental targeted treatment for NEC merits further evaluation in clinical trials. Grants from the National Institutes of Health supported the project.

A Washington University-USF Health preclinical study finds that the novel nanoparticle delivers gene-level treatment to suppress KRAS-driven cancer without adverse effects

Despite advances in cancer survival, more than 90 percent of people with pancreatic cancer die within five years. Most patients with pancreatic tumors (and half of those with colorectal cancers) carry a mutation in the KRAS gene, which normally controls cell growth and death.

The KRAS oncogene was discovered more than 35 years ago and is considered one of the most desirable targets in cancer biology — particularly for cancers (like pancreatic) often diagnosed late and in desperate need of improved therapies to prolong survival. Yet KRAS has earned a reputation as being “undruggable” by researchers who continue searching for effective ways to inhibit the mutated form of RAS proteins driving the growth of deadly tumors.

Now a preclinical study led by Washington University School of Medicine in St. Louis, including co-investigators at the University of South Florida Health (USF Health) Morsani College of Medicine, Tampa, Fla., has demonstrated that specially designed peptide-based nanoparticles can suppress pancreatic cancer growth without the toxic side effects and therapeutic resistance seen in drug trials. The findings were published July 30 in Oncotarget.

The nontoxic, peptide-based p5RHH nanoparticles designed by USF Health researchers Samuel Wickline, MD, and Hua Pan, PhD, MBA, deliver an RNA molecule known as small interfering RNA, or siRNA (also known as silencing RNA). The molecules silence the chemical signal (message) telling the KRAS oncogene to make more mutated KRAS proteins that cause pancreatic cells to grow uncontrollably and largely resist existing cancer-killing drugs.

Confocal microscopy image shows fluorescent-tagged nanoparticles (pink) carrying siRNA diffusely taken up by mouse colorectal cancer cells.| Photo courtesy of Washington University School of Medicine in St. Louis

“We’ve developed a nanoparticle system that gets enough of the therapeutic molecule from the bloodstream to the tumor cell without (the molecule) being metabolized or excreted,” said coauthor Dr. Wickline, professor of cardiovascular sciences and director of the USF Health Heart Institute. “The nanoparticle is actively taken up by the targeted tumor cells and then the molecule escapes and does its job to prevent production of mutated KRAS proteins.”

“Approaches that precisely target tumors with various therapies are the future of cancer care. Nanoparticle delivery allows higher concentrations of drugs to reach their target while sparing normal tissues any side effects,” said senior author Ryan Fields, MD, professor of surgery and chief of surgical oncology at Washington University School of Medicine. “In pancreatic cancer, where breaking through the resistant tumor microenvironment is a current unmet need, this approach has the potential to improve therapeutics and patient outcomes.”

Compared to control cells, nanoparticle treatment of both pancreatic and colorectal cells was shown to deliver KRAS-specific siRNA, decrease KRAS RNA expression and lead to increased tumor cell death. Using a genetically engineered mouse model for spontaneously arising pancreatic cancer, the researchers also demonstrated that the intravenously administered nanoparticles could selectively target silencing RNA against the KRAS oncogene to ultimately slow KRAS-driven pancreatic cancer growth.  Their system effectively delivered this nanotherapy even in the “stroma-rich” environment of pancreatic tumors.

3D reconstruction of a single pancreatic cancer cell treated with siRNA nanoparticles. Confocal microscopy depicts accumulation of strong fluorescent silencing RNA signaling (pink) within the cell membrane (cyan) boundaries, but separate from lysosomes (yellow) that can degrade the nano-delivered molecules. | Photo courtesy of Washington University School of Medicine in St. Louis

Pancreatic cancer is so difficult to treat in part because fibrous tissue, or stroma, that surrounds the solid tumor “like the shell of a clam” is much denser than the stroma surrounding other more treatable tumors, Dr. Wickline explained. This protective stromal barrier can complicate nanoparticle-delivered treatment.

“Our p5RHH peptide nanoparticle is relatively small and it can squeeze in and out of tight spaces to get (treatment) safely into tumor-specific cells while staying out of normal tissue,” he said.  “It avoids adverse ‘off-target’ effects.”

Cancer is very efficient at evading treatments that target one, or even a few mutated genes or proteins, Dr. Wickline said. “The advantage of this nanoplatform carrying siRNA is that it’s easy to change out the target you want silenced, or add many more targets for simultaneous treatment in the same tumor cell.”

The nanoparticles formulated by Dr. Wickline and Dr. Pan have also shown promise for siRNA treatment in mouse models of atherosclerosis and arthritis.

The Oncotarget reported study was supported in part by grants from the National Cancer Institute.

Samuel Wickline, MD, and Hua Pan, PhD, researchers at the USF Health Heart Institute, designed the siRNA nanoparticle system tested in the study using tumor cells and a genetically engineered mouse model for pancreatic cancer. | Photo by Eric Younghans

The peptide-siRNA nanoparticle technology applied in the preclinical study was developed by USF Health Heart Institute researchers

Advanced ovarian and uterine cancers are deadly diseases. Ovarian cancers, in particular, present with vague symptoms common to other diseases, and often are not diagnosed until a late stage when cancer has spread throughout the abdomen.  More options are needed to effectively treat these metastasized gynecological cancers and improve patient survival rates.

A preclinical study published recently in Scientific Reports demonstrates that nanoparticle-delivered small interfering RNA (siRNA) targeting production of the protein AXL (AXL siRNA) inhibits metastasis of ovarian and uterine cancer cells.  The study was conducted by researchers at Washington University School of Medicine, St. Louis, Mo., and the USF Health Heart Institute, University of South Florida Morsani College of Medicine, Tampa, Fla.

3D illustration of ovarian cancer

The research team used a new nanoparticle system developed by USF Health co-investigators Samuel Wickline, MD, and Hua Pan, MBA, PhD, to test the experimental nanotherapy in human uterine and ovarian tumor cells and in immunodeficient mice implanted with these cancer cells.

“We’ve figured how to package in a simple peptide all the critical steps needed to efficiently get this particular small interfering RNA (also known as silencing RNA) into tumor cells and then release the siRNA so it can do its job,” said Dr. Wickline, a professor of cardiovascular sciences who direct the USF Health Heart Institute. “The nanoparticle basically hijacks the tumor cells’ biological machinery to get the siRNA where it needs to go – without being destroyed along the way, or creating harmful side effects.”

The nanoparticle combines two components in one delivery package 100 times smaller than a red blood cell: AXL siRNA and the peptide p5RHH.  AXL siRNA is designed to target and silence the expression of AXL, a key molecule that drives uterine and ovarian cancers. The p5RHH nanoparticles are derived from a major substance of bee venom called melittin, detoxified and selectively modified to facilitate timely escape of AXL siRNA from the nanostructure once the silencing RNA is delivered inside the targeted tumor cells.

Among the findings of the Washington University-USF Health study:

• In cell culture, treatment with p5RHH-siAXL nanoparticles decreased the ability of uterine and ovarian cancer cells to migrate and invade neighboring normal tissues.
• Mice with established uterine and ovarian tumors were intravenously and abdominally (intraperitoneally) injected with nanoparticles containing p5RHH and fluorescent control siRNA. The peptide nanoparticles localized to and released their contents into both tumor cell types regardless of the injection route, but fluorescent imaging showed that intraperitoneal administration was more effective than IV administration.
• In the mouse models, p5RHH-siALX treatment significantly reduced metastasis of both uterine and ovarian cancer without toxic effects.

USF Health Heart Institute Director Samuel Wickline, MD, and biomedical engineer Hua Pan, PhD, build nanoparticles to safely and efficiently deliver drugs or other therapeutic agents to specific cell types.

Overall, the study demonstrates this nanoparticle approach shows promise for treating patients with ovarian or uterine cancers, the authors conclude.

Challenges in translating preclinical successes into patient care remain, but Dr. Wickline believes nanoparticle-mediated delivery of siRNA has applications beyond just suppressing one target (AXL) implicated in other cancers in addition to uterine and ovarian.

The power of harnessing tiny nanotechnology for gene therapies lies in its flexibility, he said.

“As we identify new disease-modifying targets, it offers the potential to attack multiple different targets at the same time.  So, one nanoparticle could deliver a whole host of genetic materials – a combination of RNA interference drugs, or other types of synthetic RNA or DNA-based drugs —  to hit any specific cell types where treatment is needed.”