The vehicles look nothing like delivery trucks, thoughthat is their function once inside the body. Instead, these so-callednanoparticles, which are assembled from three short pieces ofribonucleic acid, resemble miniature triangles. The microscopicparticles possess both the right size to gain entry into cells and alsothe right structure to carry other therapeutic strands of RNA insidewith them, where they are able to halt viral growth or cancer'sprogress. The team has already tested the nanoparticles successfullyagainst cancer growth in mice and lab-grown human cells.
"RNA hasimmense promise as a therapeutic agent against cancer, but until now wehave not had an efficient system to bring multiple therapeutic agentsdirectly into specific cancer cells where they can perform differenttasks," said research team leader Peixuan Guo, who is a professor ofmolecular virology at Purdue with joint appointments in Purdue's CancerResearch Center, School of Veterinary Medicine and Weldon School ofBiomedical Engineering. "Physicians have hoped that nanotechnologymight provide a solution to the problem, and it's possible that theapplication of these tiny triangles could lead to the solution."
"Withthese devices, Dr. Guo was able to deliver three different therapeuticagents into a cell at the same time," said Jean Chin, a scientist atthe National Institute of General Medical Sciences, which is part ofthe National Institutes of Health. "This is an incredibleaccomplishment that points to the versatility and potential medicalvalue of these nanoparticles."
The research appears in tworelated papers being published in the scientific journals Nano Lettersand Human Gene Therapy. Members of Guo's research team are from Purdue,the University of Central Florida and the University of California,Riverside, including Songchuan Guo, Annette Khaled, Feng Li, SulmaMohammed and Nuska Tschammer.
Guo's team created theirnanoparticles by linking together different kinds of RNA, a task thattheir previous research has given them ample opportunities to practice.Several years after building a tiny "motor" from several strands of RNAthat mimic those in a bacteria-killing virus called phi29, the teamlearned how to manipulate these stringy molecules into differentshapes, including rods, triangles and arrays.
"We speculated atthat time that these shapes would be useful purely as physicalscaffolding on which more sophisticated nanodevices could beconstructed," Guo said. "But RNA, which carries genetic messages withincells, also has many therapeutic functions. We realized that if webuilt different kinds of therapeutic RNA onto the RNA scaffolding andcreated a single structure, we might be able to respond to severalchallenges that have confronted the medical field."
RNA moleculescome in many variant forms, and the sort that the team mimicked fromthe phi29 virus – called pRNA – also can be linked to other types ofRNA to form longer, hybrid strands with properties the researcherscould assign.
"We looked around for RNA strands that would behavein certain ways when they encounter a cancer cell because each of themneeds to perform one step of the therapy," Guo explained. "An effectiveagent against cancer needs to accomplish several tasks. It needs firstto recognize the cancer cell and gain access to its interior, and thenit needs to destroy it. But we'd also like the agent to leave a trailfor us, to mark the path the molecule has taken somehow. That way, wecan pinpoint the location of the cancer and trace the outcome after thetreatment."
To accomplish these tasks, the team turned to otherforms of RNA that can interfere with the goings-on inside cells. Theteam sorted through a variety of RNA forms that have shown promise fordisease treatment and found three that could perform each of thedesired tasks. One example is "small interfering RNA," or siRNA, whichdeactivates certain genes in cells. The others are RNA aptamers, whichbind to cancer cell surface markers, and ribozymes, which can bedesigned to degrade specific RNA in cancer cells or viruses.
"Welinked each of the three therapeutic strands with a piece of pRNA,forming three hybrid strands," Guo said. "Then, using techniques welearned from our earlier work, we were able to combine all three intotriangles that are between 25 and 40 nanometers wide. This is theGoldilocks size for any nanoparticle that is to be used in the body –not too big, not too small."
Particles larger than about 100nanometers are generally too large to pass through cell membranes intothe cell's interior, Guo said, and the body has a hard time retainingparticles smaller than 10 nanometers. But the tiny triangles fit, andthey worked well enough to interrupt the growth of human breast cancercells and leukemia model lymphocytes in laboratory experiments.
"Onecharacteristic of cancer cells is that they do not stop growing, whichis one reason tumors develop," Guo said. "Once inside, the siRNAessentially instructs the cells to 'stop not stopping.' Thenanoparticles had done their work on the breast cancer cell cultureswithin a few days."
Additionally, the team found that thenanoparticles completely block cancer development in living mice. Agroup of mice that were in the process of developing cancer were testedwith the nanoparticles, and they did not develop the disease. A secondgroup that was tested with mutated inactive RNA all developed tumors.
"Theresults are very promising, but we still have several hurdles to jumpbefore we can test this therapy on people," Guo said. "First andforemost, we must ensure that it is as safe as we think it is. Some RNAcan be toxic to noncancerous cells as well, and though ournanoparticles appear to go straight to the cancer cells where we wantthem to go, we have to be sure they do not go anywhere else before wecan inject them into a living person."
Stability of the RNA alsois a factor the team must consider. Although they previously publisheddata indicating that phi29 RNA nanoparticles are more stable than otherRNA, Guo said the team still needs to find better ways to protect theRNA from degradation by enzymes in the body.
Although the groupstill needs to prove the safety of their tiny creations, Guo said, theyremain confident that their work is a milestone for medicalnanotechnology. The team has already obtained further results thatcould help create safer RNA nanoparticles.
"Many studies haveshown that therapeutic forms of RNA, such as siRNA or ribozymes, couldbe put together to kill cancer, but the main obstacle has been findingthe delivery method that can bring them to specific cellssimultaneously," Guo said. "Nanotechnology is beginning to pay off herein that it may have provided us with a solution to the problem. We hopeto enhance the work we have done so far and refine it for human trials."
The team's work is supported in part by grants from the National Institutes of Health and the Department of Defense.
Guo is affiliated with Purdue's Cancer Center and Birck Nanotechnology Center.
TheCancer Center, one of just eight National Cancer Institute-designatedbasic-research facilities in the United States, attempts to help cancerpatients by identifying new molecular targets and designing futureagents and drugs for effectively detecting and treating cancer.
TheBirck Nanotechnology Center is located in Purdue's new Discovery Park,located on the southwestern edge of campus. Programs includeundergraduate teaching, graduate research and technology-transferinitiatives with industry partners. Scientists in biology, chemistry,physics and several engineering disciplines participate in the research.
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