YSM Issue 93.2

Nanoscience
FOCUS
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 halflife
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
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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
PHOTOGRAPH COURTESY OF KATE KELLY
A photograph of Dr. Piotrowski-Daspit looking
through a microscope, accompanied by Dr.
Laura Bracaglia. These research scientists
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
September 2020 Yale Scientific Magazine 11