Probe delivers therapeutic agent and enables monitoring of its effects
RNA interference monitored in vivo using MRI and optical imaging
David L. Shenkenberg
RNA interference can inhibit the expression of any gene that causes any disease with a genetic basis. However, experiments with it so far have monitored gene expression using bioluminescence imaging, as opposed to more clinically relevant visualization methods. Researchers from Massachusetts General Hospital’s Athinoula A. Martinos Center for Biomedical Imaging in Charlestown have developed a probe that they observed in vivo using MRI and optical imaging.
In mice bearing tumors expressing GFP and in control tumors expressing red fluorescent protein, MRI showed probe delivery. Near-infrared optical imaging confirmed delivery of the probe (left), and fluorescence optical imaging showed decreased fluorescence signal in experimental tumors. These results inspired the researchers to reduce the expression of a gene that participates in tumor cell survival. As shown in this magnetic resonance image (right), the probe was delivered, and histological results demonstrated that tumor cells died as a result of the treatment.
The scientists used the probe not only to monitor gene expression but also to transport small interfering RNAs (siRNAs), a class of molecule that can cause RNA interference. They used the probes carrying the siRNAs to kill tumor cells in mice, and they used MRI and near-infrared fluorescence optical imaging to monitor the delivery of the probes. Fluorescence optical imaging also was used to indirectly observe changes in gene expression.
Among various clinical imaging techniques, the researchers chose MRI because it is a high-resolution method with virtually unlimited depth penetration. They used optical imaging because it can rapidly and sensitively detect chemical signatures. Fluorescence optical imaging at near-infrared wavelengths enables deeper tissue penetration and causes less tissue damage than at other wavelengths.
Anna Moore, the principal investigator, said that she envisions a clinical scenario in which probes such as theirs deliver siRNA to target tissues in patients and simultaneously enable noninvasive assessment of the in vivo distribution of the siRNAs and their treatment effects. The probes likely also will advance RNA interference research. Moore said that the probes would help researchers better understand the biological fate of siRNAs and correlate it with both silencing efficiency and the resulting biological effects in an intact living organism.
To build the probes, the researchers assembled several well-characterized components. The core consisted of superparamagnetic nanoparticles of iron oxide, a contrast agent commonly used for MRI. They coated the nanoparticles with a layer of dextran, to which they easily could attach the remaining components, including a fluorophore for near-infrared optical imaging, siRNAs and peptides that enabled the probe to enter cells. For optical imaging, they used the fluorophore Cy5.5 because its structure is compatible with conjugation, and it absorbs in the near-infrared region. The siRNAs corresponded to the particular gene that they silenced in each experiment. To allow the probe to enter cells, the researchers used a myristoylated polyarginine peptide sequence. Such sequences are well-known membrane translocation agents that shuttle substances across the cell membrane and into the cytoplasm.
The scientists believe that the probe accumulates in tumors via the enhanced permeability and retention effect, a passive effect caused by the permeable vasculature of tumors. In other words, molecules tend to enter tumors faster than they do healthy tissue because tumors have more openings in their blood vessels.
Researchers developed a probe that they used both for gene therapy and for monitoring its effects by using MRI and near-infrared optical imaging.
By developing the probe, the researchers aimed to solve several problems with siRNA delivery. For example, RNA molecules of any kind — let alone siRNAs — do not last long because they rapidly pass through the liver, where they are degraded by enzymes. Although a carrier might stabilize the siRNAs, the presence of a vehicle also might hamper their ability to reduce gene expression, and the carrier may not effectively distribute the drug to the tumor. However, past research has shown that conjugation to various carriers can greatly enhance the stability of siRNAs, can improve their distribution and can increase their potency in vivo. Therefore, Moore said, they believe that their probe could improve the ability of siRNAs to effectively treat tumors.
Testing the probe
In an early in vitro experiment, the researchers determined, using GFP as a model gene in a proof of principle experiment, that the probe effectively lowers gene expression. They delivered the probes to cancer cells expressing GFP and to control cancer cells expressing red fluorescent protein. As expected, the probe reduced the fluorescence of the GFP-expressing cells in a concentration-dependent manner, but it did not alter red fluorescent protein expression in the control cells, as observed by confocal microscopy using a Carl Zeiss microscope.
In a later in vivo experiment, the researchers injected the probes into tumor-bearing mice, employing the same tumor cell lines as in the in vitro experiment —one line that expressed GFP and another line that expressed red fluorescent protein. They implanted the tumors bilaterally, just above the hind legs. They monitored the delivery of the probes using a Bruker small-animal MRI scanner and near-infrared optical imaging with a Kodak whole-animal imaging station. They used the same station for fluorescence optical imaging of GFP expression.
In vitro experiments using GFP-expressing tumor cells (top left) and control cells expressing red fluorescent protein (bottom left) showed that the probe could reduce GFP expression (top right), providing proof of principle. Nothing happened to the control cells (bottom right).
When superparamagnetic probes enter tissue, magnetic resonance images darken as they accumulate. In this study, the researchers observed a significant darkening in both tumors after probe injection, demonstrating by MRI its delivery, which was confirmed also by near-infrared optical imaging. Furthermore, optical imaging showed a decrease in GFP fluorescence in GFP-expressing tumors but no change in red fluorescent protein fluorescence in control tumors. The researchers corroborated the results of their in vivo experiment by using reverse transcriptase polymerase chain reaction (RT-PCR). Tests for toxicity and inflammation demonstrated that the probes did not hurt the mice.
After demonstrating that the probes reduced GFP expression, the researchers used a more clinically relevant siRNA. They chose to target the gene that encodes survivin, a protein that prevents apoptosis, a fail-safe mechanism that instructs cells to die when they become cancerous. Survivin was a desirable target because it is expressed in most tumors but not in healthy tissue. The researchers delivered the probes to mice bearing a colon tumor on their flank. They observed the delivery of the probes with MRI and near-infrared optical imaging and confirmed inhibition of the target gene with RT-PCR. Using histological assays, they noticed that a significant number of tumor cells died as a result of treatment with the probes. They described these results in the March 2007 issue of Nature Medicine.
For the first time, the researchers efficiently delivered siRNAs to tumors by complexing them with a nanoparticulate imaging agent, and they monitored siRNA delivery with a noninvasive and accepted clinical imaging modality, MRI. They also confirmed probe delivery and observed gene expression with fluorescence optical imaging, a noninvasive method that may one day become accepted in clinical settings. “This represents an important new step toward the application of siRNAs as cancer therapeutic agents by providing a novel strategy for the assessment of their bioavailability in tumors,” Moore said.
If RNA interference becomes an accepted clinical treatment, it will be both a type of gene therapy and a form of personalized medicine. “We believe that siRNAs will one day evolve into powerful new therapeutic agents with broad applicability to any disease amenable to genetic manipulation, yet with the exquisite specificity that will allow an individualized treatment approach,” Moore said.
All of that is in the distant future, but for now Moore said that they plan to modify the probe to specifically target cancer cells and to use the probe in combination with established therapeutic agents to kill tumors more efficiently.
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