YSM Issue 93.2
FOCUSNanoscienceSearching through Nanoparticle LibrariesTRACKINGDRUGSIn the wake of the COVID-19 pandemic, clinical trials forpotential drugs and treatments have never seemed moreparamount. Scientists must carefully evaluate a drug’s interactionswithin the body and observe possible side effects.Particularly for drugs that must be administered intravenously,it is critical for researchers to determine the circulation halflife,or time it takes for a drug’s concentration to be halved, togain insight into how long a drug remains in the body.One useful technology to visualize and measure concentrationis fluorescence microscopy. The principles of fluorescencerely on excitation of fluorophore molecules and emissionof light. With fluorescent dyes or probes to track a targetmolecule, fluorescence microscopes allow biomedical researchersto perform experiments in vivo, or directly in theorganism, providing strong spatiotemporal resolution to visualizephysiological systems in normal and diseased states.This is a primary research focus of the laboratory of MarkSaltzman, professor of Biomedical Engineering at Yale University.From generating polymeric nanoparticles that aiddrugs in targeting brain tumors to producing bioadhesivebiodegradable nanoparticles to be used in sunscreen, theSaltzman research group has aimed to devise safer and moreeffective technologies to prevent disease. For the past fifteenyears, the Saltzman laboratory has also generated librariesof nanoparticles to determine how their chemical compositionmay affect the behavior of and interaction with biologicalspecimens, including impacts to circulation half-life.“We use nanoparticles to facilitate the delivery of therapeuticmolecules, protecting them from things in the blood likea protective shell, until they arrive at the target destination.Depending on the properties of the nanoparticles, you candeliver a package of molecules all at once or gradually,” saidLaura Bracaglia, a postdoctoral researcher who has workedon developing these nanoparticle libraries. Evaluating the efficaciesof these nanoparticles relies on an accurate methodof measuring concentration over time, such as via correlatingfluorescence intensity of fluorescently tagged injected agents.In a recent paper published in PNAS, co-first authors Bracagliaand postdoctoral colleague Alexandra Piotrowski-Daspitdesigned a quantitative microscopy approach to efficientlymeasure the circulation half-lives of fluorescently taggedagents, such as nanoparticles encapsulating fluorescent dye orPHOTOGRAPH COURTESY OF KATE KELLYPHOTOGRAPHCOURTESY OFKATE KELLYfluorescently labeled antibodies.BY ANNA SUN10 Yale Scientific Magazine September 2020 www.yalescientific.org
NanoscienceFOCUSLimitations of Traditional MethodsA commonly used protocol for determiningconcentration of fluorescentlydyed nanoparticles after administrationinvolves three steps: collecting at leasttwenty microliters of blood from experimentalanimals, separating dyed nanoparticlesfrom the blood samples, and measuringthe dye’s concentration by dissolvingthe nanoparticles to create a uniform solution.The process, however, can be laborious,expensive, and error-prone.One of the greatest challenges of thetraditional method is the volume of bloodneeded for a plate reader to detect eventrace amounts of fluorescent dye withinthe sample. The catch-22 is that removingtoo much blood from an experimentalanimal can interfere with studying howinjected drugs affect disease outcomes,since circulating drug molecules can beremoved during blood collection.Revamped MicroscopyThe researchers realized that the platereader machine typically used to measurefluorescent dye concentrations was ineffective.“You need a uniform amount ofblood in the plate reader, but the measurementtends to be inaccurate dependingon where in the solution youare measuring,” Piotrowski-Daspit said.To address the limitations of using largeblood volumes, the researchers decidedto switch to quantitative microscopy,which requires only a drop of blood ona microscope slide. “Depending on thestrength of the microscope, you can seein the sub-micron level, so you don’t needthat much blood to see everything,” shesaid. With their revamped method, onlytwo microliters of blood, compared to thetwenty microliters needed for the existingprotocol, are needed to accurately measurecirculation half-life.The concentration of a drug in circulationdecreases exponentially until it approacheszero, when it has been mostlyeliminated from the body. A drug’s halflifeis a useful measurement in understandingcirculation time, and the goalof this quantitative microscopy methodis to understand how the drug is transportedand reacts within the body. “Youcan make design changes to a moleculewww.yalescientific.orgor drug via physical or chemical methodsto make it less likely to be degradedor phagocytosed in order to be circulatedfor a longer time in the blood,” Bracagliasaid. “Sometimes, it’s also beneficialfor a drug to have extended circulationto allow more time to reach a target,” Piotrowski-Daspitadded.Where to Inject?In their study, the researchers initiallyfocused on quantifying rodent drug delivery.Because there are two standardways of intravenously injecting drugs torodents—retro-orbital (RO, or behind theeye) and tail-vein (TV, or in the tail) administration—theresearchers tested bothroutes of administration to better understandpossible changes in circulationhalf-life. “RO is easier for some people,so we were thinking if one experimenterinjects RO and another does TV, thendoes that matter?” Bracaglia explained.Whereas the previous protocol mightnot have had the resolution to accuratelymeasure differences in half-lives betweenRO and TV routes, the researchers detectedsubtle differences in nanoparticleconcentrations measured within the firstthirty minutes of blood collection—a testamentto the powerful resolution of theirmethod. TV injection had higher measuredconcentrations, but these concentrationsequalized after one hour. Thisinitial variability was not too concerning,because “we’re sampling blood from thetail, so it makes sense that the TV concentrationwas higher at first than the RO,which needed more time to pass throughcirculation,” Piotrowski-Daspit said. Bracagliapointed out that detecting changesin circulating concentration based on theroute of administration may also be relevantfor humans, since drugs are also administeredusing various methods.Expanding the DataTo determine whether this improvedmethod of measuring fluorescence concentrationcould be applied to moleculesof different sizes, the research team alsosuccessfully tested fluorescent antibodies.“Whereas nanoparticles are usuallysized between 180-250 nm, antibodies aresmaller at around 10 nm. We wanted toPHOTOGRAPH COURTESY OF KATE KELLYA photograph of Dr. Piotrowski-Daspit lookingthrough a microscope, accompanied by Dr.Laura Bracaglia. These research scientistssee if we can detect a wide range of agentsthat might be injected into an animalmodel,” Piotrowski-Daspit said. Becausetheir circulation measurements of theseantibodies matched the decay profilesgathered from literature, the researcherswere confident that their method couldeven detect small antibodies in the blood.The data from the quantitative microscopymethod can also be combined withfurther multivariable analyses. Saltzmanemphasized the importance of observingbiodistributions from these experiments—understandingwhat kindof tissues and what types of cells thenanoparticles are found in over time. “Bycoupling with other methods, you end upwith a powerful high-throughput, comprehensivelook at how long these particlescirculated and where they end up,” hesaid. Furthermore, because only a smallamount of blood is needed for each sample,more data can be collected from asingle experiment and animal. “Usingdifferent nanoparticles each with separatedyes, you can track these nanoparticlesin one animal. Because this can alsointroduce differences in half-life and biodistributionthan when injected alone, it’san interesting way to see what happenswhen you administer more than one drugSeptember 2020 Yale Scientific Magazine 11
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FOCUS
Nanoscience
Searching through Nanoparticle Libraries
TRACKING
DRUGS
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 halflife,
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
PHOTOGRAPH COURTESY OF KATE KELLY
PHOTOGRAPH
COURTESY OF
KATE KELLY
fluorescently labeled antibodies.
BY ANNA SUN
10 Yale Scientific Magazine September 2020 www.yalescientific.org
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