Art by Ellie Gabriel.
Searching through Nanoparticle Libraries
In the wake of the COVID-19 pandemic, clinical trials for potential drugs and treatments have never seemed more paramount. Scientists must carefully evaluate a drug’s interactions within the body and observe possible side effects. Particularly for drugs that must be administered intravenously, it is critical for researchers to determine the circulation half-life, or time it takes for a drug’s concentration to be halved, to gain insight into how long a drug remains in the body.
One useful technology to visualize and measure concentration is fluorescence microscopy. The principles of fluorescence rely on excitation of fluorophore molecules and emission of light. With fluorescent dyes or probes to track a target molecule, fluorescence microscopes allow biomedical researchers to perform experiments in vivo, or directly in the organism, providing strong spatiotemporal resolution to visualize physiological systems in normal and diseased states.
This is a primary research focus of the laboratory of Mark Saltzman, professor of Biomedical Engineering at Yale University. From generating polymeric nanoparticles that aid drugs in targeting brain tumors to producing bioadhesive biodegradable nanoparticles to be used in sunscreen, the Saltzman research group has aimed to devise safer and more effective technologies to prevent disease. For the past fifteen years, the Saltzman laboratory has also generated libraries of nanoparticles to determine how their chemical composition may affect the behavior of and interaction with biological specimens, including impacts to circulation half-life. “We use nanoparticles to facilitate the delivery of therapeutic molecules, protecting them from things in the blood like a protective shell, until they arrive at the target destination. Depending on the properties of the nanoparticles, you can deliver a package of molecules all at once or gradually,” said Laura Bracaglia, a postdoctoral researcher who has worked on developing these nanoparticle libraries. Evaluating the efficacies of these nanoparticles relies on an accurate method of measuring concentration over time, such as via correlating fluorescence intensity of fluorescently tagged injected agents. In a recent paper published in PNAS, co-first authors Bracaglia and postdoctoral colleague Alexandra Piotrowski-Daspit designed a quantitative microscopy approach to efficiently measure the circulation half-lives of fluorescently tagged agents, such as nanoparticles encapsulating fluorescent dye or fluorescently labeled antibodies.
Limitations of Traditional Methods
A commonly used protocol for determining concentration of fluorescently dyed nanoparticles after administration involves three steps: collecting at least twenty microliters of blood from experimental animals, separating dyed nanoparticles from the blood samples, and measuring the dye’s concentration by dissolving the nanoparticles to create a uniform solution. The process, however, can be laborious, expensive, and error-prone.
One of the greatest challenges of the traditional method is the volume of blood needed for a plate reader to detect even trace amounts of fluorescent dye within the sample. The catch-22 is that removing too much blood from an experimental animal can interfere with studying how injected drugs affect disease outcomes, since circulating drug molecules can be removed during blood collection.
Revamped Microscopy
The researchers realized that the plate reader machine typically used to measure fluorescent dye concentrations was ineffective. “You need a uniform amount of blood in the plate reader, but the measurement tends to be inaccurate depending on where in the solution you are measuring,” Piotrowski-Daspit said. To address the limitations of using large blood volumes, the researchers decided to switch to quantitative microscopy, which requires only a drop of blood on a microscope slide. “Depending on the strength of the microscope, you can see in the sub-micron level, so you don’t need that much blood to see everything,” she said. With their revamped method, only two microliters of blood, compared to the twenty microliters needed for the existing protocol, are needed to accurately measure circulation half-life.
The concentration of a drug in circulation decreases exponentially until it approaches zero, when it has been mostly eliminated from the body. A drug’s half-life is a useful measurement in understanding circulation time, and the goal of this quantitative microscopy method is to understand how the drug is transported and reacts within the body. “You can make design changes to a molecule or drug via physical or chemical methods to make it less likely to be degraded or phagocytosed in order to be circulated for a longer time in the blood,” Bracaglia said. “Sometimes, it’s also beneficial for a drug to have extended circulation to allow more time to reach a target,” Piotrowski-Daspit added.
Where to Inject?
In their study, the researchers initially focused on quantifying rodent drug delivery. Because there are two standard ways of intravenously injecting drugs to rodents—retro-orbital (RO, or behind the eye) and tail-vein (TV, or in the tail) administration—the researchers tested both routes of administration to better understand possible changes in circulation half-life. “RO is easier for some people, so we were thinking if one experimenter injects RO and another does TV, then does that matter?” Bracaglia explained. Whereas the previous protocol might not have had the resolution to accurately measure differences in half-lives between RO and TV routes, the researchers detected subtle differences in nanoparticle concentrations measured within the first thirty minutes of blood collection—a testament to the powerful resolution of their method. TV injection had higher measured concentrations, but these concentrations equalized after one hour. This initial variability was not too concerning, because “we’re sampling blood from the tail, so it makes sense that the TV concentration was higher at first than the RO, which needed more time to pass through circulation,” Piotrowski-Daspit said. Bracaglia pointed out that detecting changes in circulating concentration based on the route of administration may also be relevant for humans, since drugs are also administered using various methods.
Expanding the Data
To determine whether this improved method of measuring fluorescence concentration could be applied to molecules of different sizes, the research team also successfully tested fluorescent antibodies. “Whereas nanoparticles are usually sized between 180-250 nm, antibodies are smaller at around 10 nm. We wanted to see if we can detect a wide range of agents that might be injected into an animal model,” Piotrowski-Daspit said. Because their circulation measurements of these antibodies matched the decay profiles gathered from literature, the researchers were confident that their method could even detect small antibodies in the blood.
The data from the quantitative microscopy method can also be combined with further multivariable analyses. Saltzman emphasized the importance of observing biodistributions from these experiments—understanding what kind of tissues and what types of cells the nanoparticles are found in over time. “By coupling with other methods, you end up with a powerful high-throughput, comprehensive look at how long these particles circulated and where they end up,” he said. Furthermore, because only a small amount of blood is needed for each sample, more data can be collected from a single experiment and animal. “Using different nanoparticles each with separate dyes, you can track these nanoparticles in one animal. Because this can also introduce differences in half-life and biodistribution than when injected alone, it’s an interesting way to see what happens when you administer more than one drug at once,” Piotrowski-Daspit explained. This method could provide researchers opportunities to better understand combination therapies in humans as well.
Overcoming Obstacles
The researchers faced a few challenges on their path to developing this improved microscopy method. First, a major concern with nanoparticle research is the possibility that the fluorescent dye (which is visualized) and the nanoparticle itself have unexpectedly separated, so the dye is no longer indicating where the nanoparticle is. To address this, the team ordered a commercially available polymer that is chemically linked to a fluorescent dye, and then imaged both the polymer and a separate encapsulated dye. “The observation that they colocalized served as evidence that going forward, if we look only for the encapsulated dye, we can be confident that it is also with the nanoparticle of interest,” Bracaglia said. Another concern was that measuring fluorescent agents might not be as accurate as measuring radiolabeled agents, so the team carefully compared their experimental half-lives with examples from literature. Not only did they confirm similar half-life values, but their method was also less complicated and more accessible for the average lab, which may not have equipment for measuring radioactivity.
Future Projects
Armed with a more effective method to measure circulation half-lives of drugs, the Saltzman research group plans to rapidly screen through their nanoparticle libraries. “We are excited to see where these new nanoparticles go and how long they stay in the blood, and to learn more about how changes to physical and chemical properties can affect drug delivery success,” Bracaglia said.
An upcoming challenge for these researchers involves what happens after nanoparticles are delivered into circulation. Because the liver functions to detoxify drugs from the blood, nanoparticles often accumulate in the liver instead of the desired target organ. The researchers hope to discover ways to bypass the liver, using “decoy” nanoparticles. “These molecules potentially may be used to pre-treat and take up residence in the liver, such that anything that comes afterwards can remain in circulation longer and reach other organs,” Piotrowski-Daspit explained.
The main advantage of this novel protocol is that the improved quantitative fluorescent microscopy has drastically reduced sample blood volume. Previous limitations from sample blood volume often prevented experiments involving essential animals with rare tumors or diseases. “You normally don’t want to waste these animals doing a half-life experiment. If you’re treating the tumor, you want to save these animals to see if the treatment worked,” Bracaglia said. Drug circulation, however, might significantly differ between non-experimental and diseased animals. With this new time-and-cost effective, accessible microscopy method, scientists may soon be able to screen a wide range of therapeutic agents and provide more accurate measurements for preclinical studies, enabling researchers everywhere to answer the growing need for innovative drugs.
Acknowledgements: The author would like to thank Laura Bracaglia, Alexandra Piotrowski-Daspit, and Mark Saltzman for their time and thoughtful discussions about their research.
About the Author: Anna Sun is a junior in Jonathan Edwards College majoring in Molecular, Cellular and Developmental Biology. She currently serves as Managing Editor for the Yale Scientific Magazine. Outside of YSM, she studies riboswitches, volunteers in the hospital, and reads with New Haven youth. She also enjoys dancing and exploring the food scene in New Haven with her friends.
Sources:
- Bracaglia, L. G., Piotrowski-Daspit, A. S., Lin, C., Moscato, Z. M., Wang, W., Tietjen, G. T., & Saltzman, W. M. (2020). High-throughput quantitative microscopy-based half-life measurements of intravenously injected agents. PNAS, 117(7), 3502-3508.
- Bracaglia, L. G., Piotrowski-Daspit, A. S., & Saltzman, W. M. (Personal interview, March 4, 2020).
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