Multicompartment Theranostic Nanoemulsions Stabilized by a Triphilic Semifluorinated Block Copolymer

Article abstract of DOI:10.1021/acs.molpharmaceut.7b00624

The presence of a perfluorocarbon block in a multiblock polymer has been shown to be an additional driving force toward nanoparticle assembly. In the preparation of nanoemulsions, this perfluorocarbon block also provides enhanced particle stability. Herei

Full text of DOI:10.1021/acs.molpharmaceut.7b00624

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Article
Multicompartment Theranostic Nanoemulsions Stabilized
by a Triphilic Semifluorinated Block Copolymer
Alexa R. Barres, Megan R. Wimmer, and Sandro Mecozzi
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00624 • Publication Date (Web): 25 Sep 2017
Downloaded from http://pubs.acs.org on September 26, 2017
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Multicompartment Theranostic Nanoemulsions
Stabilized by a Triphilic Semifluorinated Block
Copolymer
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Alexa R. Barres a, Megan R. Wimmerb, Sandro Mecozzia,b*
aDepartment of Chemistry and bSchool of Pharmacy, University of WisconsinꢀMadison,
Madison, WI 53705, United States
KEYWORDS
*School of Pharmacy, Pharmaceutical Sciences Division, University of WisconsinꢀMadison, 777
Highland Avenue, Madison, WI 53705, United States. Tel: (608) 262ꢀ7810 Fax: (608) 262ꢀ5345
Eꢀmail: sandro.mecozzi@wisc.edu
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Semifluorinated polymers, block copolymers, nanoemulsions, drug delivery, hydrophobic drugs,
paclitaxel, 19FꢀMRI, theranostics
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ABSTRACT
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The presence of a perfluorocarbon block in a multiblock polymer has been shown to be
an additional driving force towards nanoparticle assembly. In the preparation of nanoemulsions,
this perfluorocarbon block also provides enhanced particle stability. Herein, the synthesis of a
new triphilic, semifluorinated copolymer, M2F8H18, is introduced. This ABC type block
copolymer can be used to formulate extremely stable nanoemulsions, assembled around a
lipophilic droplet, with lifetimes of one year or more. The central oil droplet can stably
solubilize high concentrations of hydrophobic drugs making this system an ideal drug delivery
vehicle. The incorporation of the perfluorocarbon block modulates drug release from the
lipophilic core via the surrounding fluorous shell. Fluorous imaging agents incorporated into the
fluorous shell prolong drug release even further as well as provide potent 19FꢀMRI contrast
ability. In vitro studies show that these nanoemulsions efficiently inhibit cancer cell growth, thus
providing a theranostic drug delivery system.
INTRODUCTION
Copolymers that can form nanoscale assemblies in aqueous solution have vast potential
in the field of drug delivery. Different polymer designs result in varied nanoparticle sizes,
shapes, and properties, as desired by the specific application. Incorporation of a fluorous block
into the polymer provides an enhanced driving force towards assembly and particle stability
overall, in comparison to typical hydrocarbon polymers. This is a result of the unique properties
of fluorous blocks which include both hydrophobicity and lipophobicity, high thermal and
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chemical stability, high surface tension, and biological inertness. In addition, due to the
extremely strong carbonꢀfluorine bond (472 kJ/mol) and to the size of a carbonꢀbound fluorine
atom (vdW radius = 1.47 Å) the fluorous blocks have very low polarizability and rigid, helical
structures.1–3 These properties result in the formation of an energetically favored fluorous phase
in solution and with it, increased particle stability. For these reasons, semifluorinated polymers
are highly valuable for the design of drug delivery systems.
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Colloidal nanoemulsions are kinetically stable nanoparticles in the biomedically useful
size range of 100ꢀ400 nm.4 These particles can be formulated by the input of a high amount of
energy into a solution of two immiscible liquids and an appropriate surfactant. The
nanoemulsions described here are oilꢀinꢀwater emulsions, in which a drugꢀcontaining oil droplet
is stabilized in an aqueous environment by a shell of M2F8H18 surfactant. Nanoemulsions of
this design are especially advantageous over other nanoparticle systems due to their large oil
droplet core and their resulting ability to solubilize and deliver copious amounts of hydrophobic
drugs. Furthermore, due to its particle size, this nanoscale system is able to exploit the enhanced
permeability and retention (EPR) effect to preferentially accumulate within tumor tissue over
healthy tissue due to leaky tumor vasculature and a poorly functioning tumor lymphatic system.5–
7
This type of selective delivery to tumor tissue, as well as the high drug payload afforded by
nanoemulsions, makes them a potential therapeutic tool for use in cancer treatment.
Previous work in our laboratory with nanoemulsions has primarily focused on
semifluorinated diblock copolymerꢀbased nanoemulsions for successful emulsification of
fluorous anesthetics. Here, a new semifluorinated, triblock ABC copolymer is introduced
consisting of hydrophilic, fluorophilic, and lipophilic segments that can form triphilic
nanoemulsions8–12. This innovative triphilic design is not only ideal for the delivery of solid
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chemotherapeutic drugs dissolved in the lipophilic core, but also for use as a theranostic system
by the addition of a fluorous imaging agent present in the intermediary fluorous shell. The
introduction of the fluorocarbon block also allows for the development of nanoparticles that can
modulate drug retention and exhibit enhanced kinetic stability. The unique properties of the
triphilic polymer M2F8H18 (Figure 1) allow for high loading and retention of hydrophobic drugs
within the oil droplet core. In the emulsion nanoparticles, the lipophilic block of the polymer
penetrates the oil core, stabilizing the particles and interacting nonꢀcovalently with the drug and
oil. Drug release from the core is then modulated by the thickness of the nanoparticle
intermediate fluorous shell. Particle waterꢀsolubility and biocompatibility is achieved through the
methoxy capped poly(ethylene glycol) (mPEG) hydrophilic block comprising the particle
corona. The addition of a fluorous imaging agent provides further particle stability, enhanced
drug retention, as well as potent 19Fꢀmagnetic resonance imaging (MRI) signal and imaging
possibilities.
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Figure 1. Hypothesized organization of the ABC polymer M2F8H18 into therapeutic
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nanoemulsion particles.
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Drugꢀencapsulation and release properties of the nanoemulsions were tested using the
chemotherapeutic paclitaxel. This antimitotic drug was selected for its high potency against
many cancer types including breast, ovarian, and lung cancer as well as Kaposi’s carcinoma.
Paclitaxel functions by the promotion of tubulin polymerization and equimolar binding to tubulin
dimers, resulting in mitotic arrest.13,14 Paclitaxel is highly hydrophobic with a water solubility <
1 µg/mL and exhibits poor in vivo pharmacokinetics. Due to these challenging characteristics,
paclitaxel was first administered intravenously as Taxol® in combination with Cremophor EL®,
a nonionic surfactant with polyoxyethylated castor oil, and ethanol. However, Taxol® leads to
widespread hypersensitivity reactions, plasticizer leaching, and even neuropathy due to the
Cremophor EL® surfactant and other solvents.15. An additional paclitaxel formulation,
Abraxane®, was approved in 2005 involving albuminꢀbound paclitaxel nanoparticles.16 Despite
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dramatic improvements in side effect toxicity, this formulation still lacks high potency due to the
discrepancy between injected drug and drug that actually reaches the tumor.17,18 Thus, an
improved delivery method for this highly cytotoxic drug is still needed. Due to the extremely
poor solubility of paclitaxel in water and other solvents, an optimization was performed to
determine what lipophilic medium could best carry this chemotherapeutic payload. Common oils
were investigated including 2ꢀoctylꢀ1ꢀdodecanol, soybean oil, glycerol, and their combinations.
These media resulted in many issues including very low paclitaxel solubility, high oil viscosity,
and bacterial growth within the oil. Further investigation lead to the use of medium chain
triglycerides (MCT) an FDA approved lipophilic media mainly derived from coconut oil.19
Similar to soybean oil, MCT contains a triglyceride backbone but in contrast to soybean oil that
contains both saturated and unsaturated fatty acids, MCT only contains fully saturated fatty
acids. The MCT grade used here (Neobee® Mꢀ5) contains a range of eight to ten carbon units
per chain. Furthermore, MCT has been used widely in nutritional supplements and drug
formulations since this oil is easily hydrolyzed, metabolized, and absorbed, which results in its
fast conversion to energy.20,21 Paclitaxel interacts favorably with MCT with formulations up to
15 mg paclitaxel per mL MCT reported herein. Due to its many advantageous properties, MCT
was selected as the medium of choice to compose the lipophilic core of the M2F8H18
nanoemulsions.
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Theranostics is a term describing dual therapeutic and diagnostic character. Theranostic
nanomedicine is an expanding field geared towards personalized medicine that combines
therapeutic and imaging treatments for a clear picture of drug distribution and efficacy. As such,
theranostic agents frequently involve administration of a chemotherapy, gene therapy, or
radiation therapy with an imaging agent.22 The described M2F8H18 nanoemulsions are
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formulated into a theranostic system upon combination of paclitaxel, the therapeutic portion,
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with an FꢀMRI contrast agent, the diagnostic portion. Perfluoroꢀ15ꢀcrownꢀ5ꢀether (PFCE) was
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selected as the fluorous contrast agent to be used in these nanoemulsions. This clear, organic oil
is very stable under physiological conditions with a high boiling point. The structure of the
macrocycle is thought to tend toward a puckered ring with high flexibility in solution leading to
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all twenty fluorine atoms exhibiting a single peak in FꢀNMR.23 The presence of just a single
resonance signal makes this system highly sensitive in 19FꢀNMR and therefore in 19FꢀMRI
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applications. Previously, PFCE has found FꢀMRI uses in labeling and tracking dendritic cells,
stem cells, plaques, and even imaging sites of inflammation.24–30 The use of our semifluorinated
M2F8H18 copolymer allows for the binding of very high concentrations of fluorophilic PFCE in
the intermediate fluorous shell (Figure 1) thereby providing a sensitive imaging system with
greater stability than a traditional surfactant.
The use of MRI for diagnostic purposes continues to expand due to its noninvasive
nature, deep tissue penetration, excellent sensitivity, and widespread application. Most
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frequently, H nuclei are utilized due to their high sensitivity of 1.0, natural abundance of 99%,
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and the large amount of water present in anatomical systems. Though less common, F nuclei
can be used easily for MRI with similar sensitivity to proton, 0.83, and an even higher natural
abundance of 100%.29 Furthermore, fluorine is not a naturally occurring biological element and
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this leads to a nearꢀzero background in FꢀMRI as well as an excellent signalꢀtoꢀnoise ratio.30,31
In order to clearly visualize tissues in traditional 1H MRI, a contrast agent is commonly required;
two of the most common include chelated gadolinium and superparamagnetic iron oxide (SPIO).
Both metallic compounds are very efficient at indirect imaging but have exhibited several
toxicity concerns including oxidative stress, altered gene expression, and in extreme cases,
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kidney failure.32 In an analogous manner, fluorocarbons with high signal intensity can be used to
image anatomical systems. In this case, however, the imaging can be performed directly. This
work utilizes the intrinsic properties of 19F nuclei via the incorporation of perfluoroꢀ15ꢀcrownꢀ5ꢀ
ether (PFCE). This excipient was selected due to its high signal intensity and reportedly low
toxicity compared to other fluorinated crown ethers.33
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Described in this article are the synthetic methods and physiochemical characteristics of
the M2F8H18 copolymer. The formulation of nanoemulsions stabilized by M2F8H18 will be
discussed herein including their unprecedented longꢀterm stability, in vitro drug release profiles,
and in vitro and in vivo toxicity profiles. The theranostic potential of these nanoemulsions will
also be presented through preliminary 19FꢀMRI work.
EXPERIMENTAL SECTION
Materials and Methods
1H,1H,10H,10Hꢀperfluorodecaneꢀ1,10ꢀdiol was purchased from SynQuest Laboratories Inc.
(Alachua, FL) and perfluoroꢀ15ꢀcrownꢀ5ꢀether was purchased from Oakwood Chemical (Estill,
SC). Normal saline (AirLife sterile 0.9% sodium chloride for irrigation USP) was purchased
from the University of Wisconsin Hospital Pharmacy and paclitaxel from LC Laboratories
(Woburn, MA). Solvents and all other reagents were purchased form Sigma Aldrich Co.
(Milwaukee, WI) and Spectrum (Gardena, CA) and used as received, unless otherwise
mentioned. Small molecular and polymer chromatography was accomplished with Silicycle 60
Å SiO2 or using a Teledyne CombiFlash® Rf 4x (Lincoln, NE) equipped with an ELSD for
visualization and REDIꢀsep Rf high performance silica or C18 columns.
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1Hꢀ and 19FꢀNMR spectra were obtained on a Varian UnityꢀInova 400 or Varian UnityꢀInova 500
spectrometer using CDCl3 as the solvent (unless otherwise specified) and TMS as the internal
reference. Polymer purity was confirmed by HPLC with a Gilson 321 Pump (Middleton, WI)
equipped with a Jordi Gel DVB 500 Å (Bellingham, MA) column and a Gilson PrepꢀELS
detector and by MALDIꢀMS on a Bruker Ultraflex III MALDI TOF/TOF using αꢀcyanoꢀ4ꢀ
hydroxycinnamic acid matrix unless otherwise specified.
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Critical micelle concentration (CMC) – surface tensiometry
Polymer was dissolved in Millipore MilliꢀQ water in a 20 mL disposable scintillation vial to the
desired maximum concentrations (1.0 and 0.32 mM). The solutions were shaken and sonicated
for 3 h. Solutions were then allowed to equilibrate for 24 h. Serial dilutions were then prepared
from these two stock solutions to achieve the desired concentrations. Each serial dilution was
also sonicated 3 h and allowed to equilibrate prior to the next dilution. Once all solutions were
prepared, they were allowed to equilibrate for an additional 24 h. Surface tensions were
measured on a KSV sigma 701 tensiometer (KSV Instruments, Helsinki, Finland). A custom
round platinum rod, with a diameter of 1.034 mm and a wetted length of 3.248 mm from KSV
Instruments (Helsinki, Finland), was initially cleaned with ethanol and dried in a Bunsen burner
flame. Before running the experimental samples, the surface tension of Millipore MilliꢀQ water
was measured as control to confirm the rod was fully cleaned and surface tension was within ± 2
mN mꢀ1 of the literature value, 78.2 mN mꢀ1. The surface tension of each sample was then
measured using the Wilhelmy method, beginning with the least concentrated solution and
proceeding to successively more concentrated solutions. The surface tension at each
concentration was measured in quadruplet and the average recorded. The CMC value was
determined from the intersection of the slope at the crossover point of two lines: the baseline of
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minimal surface tension and the slope where surface tension showed linear decline. Error was
determined by weighted least squares analysis.
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Nanoemulsion formulation
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Aqueous polymer solutions were prepared freshly (20 mM) in sterile, normal saline and
sonicated at room temperature until fully dissolved. Saline was composed of 0.9% (w/w) sodium
chloride.
Paclitaxel/medium chain triglycerides (MCT) solutions were prepared freshly. Two mL of
paclitaxel solution (prepared at a concentration of 7.5 mg/mL in 50:50, acetonitrile:ethanol) was
dissolved in MCT followed by heating and stirring until fully solubilized. All traces of
acetonitrile and ethanol were removed by vacuum.
Paclitaxel/MCT solution and PFCE were added to the polymer solution. The homogenizer and
microfluidizer were first cleaned with 100% and 70% ethanol followed by 100% and 70%
methanol and finally three rinses with Millipore MilliꢀQ water to remove all traces of any
previous nanoemulsions. The prepared mixture was then homogenized with the highꢀspeed
homogenizer (Power Gen 500, Fisher Scientific, Hampton, NH) for 1 min at 21,000 rpm at room
temperature. The resulting crude emulsion was then further mixed with the microfluidizer
(model Mꢀ110S, Microfluidics Corp., Newton, MA) for 1 min under 5,000 psi with the cooling
bath kept at 0°C. The final emulsion was then filtered with a 0.45µm nylon filter and stored in a
sterile, plastic centrifuge tube (Corning Inc., Corning, NY) at 4°C.
Particle size determination via dynamic light scattering (DLS)
Longꢀterm nanoemulsion size and stability was monitored via dynamic light scattering
(NICOMP 380ZLS, Particle Sizing Systems, Santa Barbara, CA). The nanoemulsions were
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diluted at the intensity factor of 500 by adding 5 µL of the nanoemulsion to 3.0 mL of Millipore
MilliꢀQ water. Each particle size analysis was run for 5 min at room temperature in a quartz
cuvette and repeated three times. The data was analyzed using Gaussian analysis and reported as
intensity weighted average diameters.
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Micellar aggregate size, distribution, and histogram data were determined via dynamic light
scattering (Zetasizer NanoꢀZS, Malvern Instruments, Worcestershire, UK). The nanoemulsions
were diluted at the same ratio as above, 5 µL nanoemulsion to 3.0 mL of Millipore MilliꢀQ
water. Each particle analysis was run as a set of 10 scans in a semiꢀmicro polystyrene cuvette at
room temperature and repeated three times. The data was analyzed using Malvern software
analysis and reported as volume weighted average diameters.
In vitro drug release
The nanoemulsion was initially diluted by a factor of 20 (0.125 mL nanoemulsion plus 2.375 mL
Millipore MilliꢀQ water). A timeꢀzero time point was established by diluting 100 µL diluted
nanoemulsion mixture above in 900 µL acetonitrile (ACN). A 3 mL capacity SlideꢀAꢀLyzer
Dialysis cassette (G2 2,000 MWCO from Thermo Fisher Scientific Inc., Fitchburg, WI) was
hydrated by stirring for 12 h in a 3 L PBS bath (300 mL 10x PBS and 2,700 mL Millipore Milliꢀ
Q water) at 37 ºC. After this time, the remaining diluted nanoemulsion solution and 100 µL PBS
(1x) was added to the cassette which was then returned to the PBS bath and allowed to stir for 1
week at 37 ºC; this was performed in triplicate. Time points were taken at 0.5, 2, 3, 6, 9, 12, 24,
36, 48, 72, 96, 120, 144, and 168 h. At each time point a longꢀstemmed glass pipette was used to
mix the contents of the cassette three times. Then a 100 µL aliquot of nanoemulsion was
removed from the cassette and diluted with 900 µL ACN. The nanoemulsion aliquot was then
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replaced in the cassette by 100 µL of fresh PBS (1x). Sink conditions were maintained by
replacing the 3 L PBS baths at the 3, 6, 9, and 12 h time points and every 12 h thereafter.
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The paclitaxel concentration remaining in the nanoemulsion at each time point was quantified by
reverse phase HPLC. The HPLC system used was a Shimadzu prominence HPLC system
(Shimadzu, Japan) equipped with an LCꢀ20AT pump, SILꢀ20 AC HT autosampler, CTOꢀ20 AC
column oven, and an SPDꢀM20A diode array detector. For each time point sample, 20 µL was
injected into a C18 column (Agilent XDBꢀC18, 4.6 Å x 150 mm) and eluted with an isocratic
mixture of 25% water and 75% ACN. The run time was 7 min, the flow rate was 1.0 mL minꢀ1,
and the detection was set at 227 nm. Paclitaxel eluted at 4.15 min. Concentration of paclitaxel
was determined by integrating the area of the peak and extrapolation from a standard calibration
curve (500, 100, 50, 25, 10, 5, 2.5 µg mLꢀ1).
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In vitro cytotoxicity
A549, a human nonꢀsmall cell lung carcinoma cell line, was used to perform threeꢀday
cytotoxicity studies. A549 cells were cultured in RPMIꢀ1640 media containing 10% FBS. The
cells were plated in 96ꢀwell plates, 5,000 cells/well, and incubated at 37 ºC for 24 h. After
incubation, the cells were treated with 10 µL of each solution: emulsion solutions containing
paclitaxel (0.04, 0.4, 4, 400, 4,000, 40,000 nM in relation to paclitaxel concentration), standard
paclitaxel solutions (0.04, 0.4, 4, 40, 400, 4,000 nM in relation to paclitaxel concentration), and
emulsion solutions with no paclitaxel (17 nM, 1,700 nM, 17 µM in relation to polymer
concentration) diluted in 90 µL of fresh media and allowed to incubate for 24 h at 37 ºC.
Paclitaxel standard concentrations were prepared at least three magnitudes above and two below
its reported IC50 for the A549 cell line, 4 nM.34 On the third day, all liquid was removed from
each well and 100 µL of CellTiterꢀBlue® reagent (CellTiterꢀBlue® cell viability assay,
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Promega) was added to each well.35 The cells were incubated for another three hours at 37 ºC.
The fluorescence intensity at 560 nm was analyzed using a plate reader. The cell viability in
each well was calculated relative to the untreated control wells and each type of well was
averaged (n = 6).
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In vivo developmental toxicity study
Due to the novel nature of the M2F8H18 copolymer, there is no preceding toxicity data. As a
result, a thorough study of developmental effects related to M2F8H18 was performed using an
embryoꢀlarval zebrafish model. This animal model was selected because zebrafish eggs remain
transparent from fertilization until the tissues become dense as a mature adult. Several
developmental endpoints can be simultaneously monitored providing valuable developmental
toxicity data for a variety of chemicals.36 Following shortꢀterm exposure, survival, and hatching,
nonꢀlethal malformations were monitored including curved body axis and pericardia edemas.
Mortality rate was also monitored.
Zebrafish (Danio rerio) of the AB strain were obtained from Dr. Michael Taylor at the
University of WisconsinꢀMadison School of Pharmacy, where the fish were cultured until sexual
maturation for crossing. The fish were maintained in a light/dark cycle of 14:10 h at 28.5 ºC in
egg water (0.03% Instant Ocean, Blacksburg, VA, USA). Zebrafish were fed live brine shrimp
(Artemia nauplii) twice a day. Embryos were obtained from healthy adult fish with a ratio of 1:2
for female to male. Six breeding groups were placed in separate spawning aquariums, equipped
with a mesh bottom to prevent the eggs from being cannibalized. Crossing was induced in the
morning when the light was turned on. One hour later, eggs free of macroscopically discernable
symptoms of infection and disease were collected, rinsed with egg water, and transferred into
Petri dishes until chemical exposure.
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The embryo–larvae toxicity assay was carried out according to standard protocols. Briefly, 8
fertilized eggs of 2 hours post fertilization (hpf) stage were placed into each well of a 24ꢀwell
plate and each filled with 600 µL egg water. Six concentrations (1 mM, 333 µM, 111 µM, 37
µM, 12.3 µM, and 4.1 µM) plus two controls were plated. The plate was covered and incubated
at 28.5 ºC in a light/dark cycle of 14:10 throughout the 96 hpf exposure period. The observations
of zebrafish development were made directly in the well using a stereomicroscope (Nikon
SMZ18) every 24 h. Endpoints including mortality, spontaneous movement, hatching success,
pericardial edema, and curved body axis, were selected to monitor the effects of M2F8H18.
Embryos and larvae were considered dead when no heartbeat was observed. The number of
hatched embryos and a cumulative mortality tally was recorded and every 24 hr from 2 hpf. The
number of larvae displaying pericardial edemas or curved body axes were also recorded every 24
hr from 2 hpf. At 96 hpf, following the final observations, representative larvae were
anesthetized with 0.4% tricaine mesylate solution and mounted on petri dishes using low melting
point agarose. The larvae were then photographed using a highꢀdefinition color microscope
camera (Nikon DSꢀFi2) and finally, euthanized.
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Magnetic resonance imaging (MRI)
Samples were prepared fully concentrated, without any dilution. The nuclear magnetic resonance
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(NMR) internal temperature was maintained at 25 °C. The F relaxation parameters T1 and T2,
of the perfluoroꢀ15ꢀcrownꢀ5ꢀether present in the nanoemulsions, were measured on a Varian
UnityꢀInova 500 MHz (11.7 T) NMR spectrometer. The T1 was determined using an inversion
recovery experiment acquired with 12 independent, quadratically spaced variable (tau) values
covering a range up to 10 times the estimated T1, 0.75 s. The T2 was determined using a Carr–
Purcell–Meiboom–Gill (CPMG) pulse sequence experiment acquired with 12 independent,
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quadratically spaced variable (tau) values covering a range up to 10 times the estimated T2, 0.32
s. For T1 and T2 measurements: 90° pulse = 17.1 µs, nt = 16, spectral width (T1) = 46948.4 Hz
and spectral window (T2) = 46948.4 Hz.
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MR images were acquired using an Agilent 4.7 T small animal horizontal bore scanner using a
homeꢀbuilt 19F quadrature volume coil with a 1.5″ diameter and a 3″ length. The temperature was
maintained at 25 °C. A nanoemulsion stock of M2F8H18 was prepared by direct dilution of the
nanoemulsion to the highest phantom concentration in sterile, normal saline (0.9% (w/w) sodium
chloride). Lower phantom concentrations were made as serial dilutions from the stock solution in
sterile, normal saline (0.9% (w/w) sodium chloride).
Nanoemulsion formation was confirmed by DLS. Phantom nanoemulsion solutions at PFCE
concentrations of 154 mM, 61 mM, 31 mM, 3 mM, and 0 mM were transferred to polystyrene
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microꢀcentrifuge tubes. H images of the phantoms were acquired using a gradient echo pulse
sequence with 0.19 x 0.19 x 2 mm3 spatial resolution, 48 x48 mm2 field of view (FOV), 8.82 ms
TR, 4.43 ms TE, 20° flip angle, 195.3 Hz/voxel, 16 averages and 36.2 s imaging time. 19F
images of the phantoms were acquired using a fast spin echo pulse sequence with a 0.25 × 0.25 ×
2.0 mm3 spatial resolution, 48 × 48 mm2 FOV, 0.500 s TR, 16.08 ms TE, echo train length of 8
echoes, 104.2 Hz/voxel, 50 averages, and 10 min 1 s imaging time. Image background threshold
was adjusted to only view above 5% voxels.
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Synthesis
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“BnOF8OH” 1H, 1H, 10H, 10H-monobenzyl-perfluorodecanol (1). To a dry roundbottom
flask was added 1H,1H,10H,10Hꢀperfluorodecaneꢀ1,10ꢀdiol (8.5 mmol, 4 g) and anhydrous
dimethylformamide (40 mL). This solution was allowed to stir under argon for 10 min followed
by the addition of sodium hydride (8.5 mmol, 0.20 g). This mixture was sonicated, under argon,
at room temperature for an additional 10 min then benzyl bromide (8.5 mmol, 1 mL) was added
dropwise. The reaction was sonicated for 3 h under argon. The reaction was then neutralized
with 5% HCl and extracted with ethyl acetate, saturated aqueous NaHCO3 solution, and brine.
The organics were dried with MgSO4, concentrated in vacuo, and purified via silica gel column
chromatography. A gradient of 0ꢀ15% ethyl acetateꢀhexanes was used. The separation was
monitored with thin layer chromatography (TLC) and KMnO4 staining. Isolated product resulted
in 1.77 grams (37.5% yield). 1H NMR (500 MHz, CDCl3) δ 7.35 (m, 5H), 4.68 (s, 2H), 4.10 (td,
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J = 15.2, 13.0, 7.6 Hz, 2H), 3.94 (t, J =13.8 Hz, 2H), 1.95 (t, J = 7.5 Hz, 1H). F NMR (470
MHz, CDCl3) δ ꢀ119.36 (t, J = 13.7 Hz), ꢀ121.92 (m), ꢀ122.42 (t, J = 13.6 Hz), ꢀ123.31 (bs), ꢀ
123.57 (bs).
“H18OMs” octadecyl methanesulfonate (2). To a dry roundbottom flask was added anhydrous
dichloromethane (100 mL), 1ꢀoctadecanol (21 mmol, 5.6808 g), and triethyl amine (51 mmol,
7.10 mL). This mixture was stirred and gently heated until all solid 1ꢀoctadecanol was dissolved.
Then the flask was put in an ice bath under argon. This mixture was allowed to react for 30 min
before methanesulfonyl chloride (27 mmol, 2.10 mL) was added dropwise. The solution turned
slightly cloudy. After running overnight, the solution was diluted with more DCM and extracted
with saturated aqueous NH4Cl solution. The crude mixture was then dried with MgSO4 and
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concentrated in vacuo. Isolated product resulted in 7.15 grams (97.7% yield). H NMR (400
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MHz, CDCl3) δ 4.22 (t, J = 6.6 Hz, 2H), 3.00 (s, 3H), 1.75 (p, J = 6.9 Hz, 2H), 1.39 (m, 3H) 1.26
(bs, 31H), 0.88 (t, J = 6.6 Hz, 3H).
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“BnOF8OH18” 1H, 1H, 10H, 10H-monobenzyl-octadecyl-perfluorodecane (3). To a dry
roundbottom flask was added anhydrous benzotrifluoride (50 mL) and 1 (3.4 mmol, 1.90 g). This
mixture was flushed with argon before adding sodium hydride (14 mmol, 0.3418 g). The mixture
was allowed to stir for one hour before 2 was added (4.4 mmol, 1.5611 g). The mixture was then
heated to reflux for 24 h. After running overnight, the solution was diluted with DCM and
extracted with saturated aqueous NH4Cl solution. The crude mixture was then dried with MgSO4
and concentrated in vacuo. Purification was performed via silica gel chromatography using a
gradient of 0ꢀ5% ethyl acetateꢀhexanes. The separation was monitored with thin layer
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chromatography and KMnO4 staining. Isolated product resulted in 2.53 grams (91.3% yield). H
NMR (500 MHz, CDCl3) δ 7.35 (m, 5H), 4.68 (s, 2H), 3.93 (q, J = 13.6 Hz, 4H), 3.59 (t, J = 6.6
Hz, 2H), 1.60 (p, J = 6.7 Hz, 2H), 1.25 (m, 32H), 0.88 (t, J = 6.8 Hz, 3H). 19F NMR (470 MHz,
CDCl3) δ ꢀ119.75 (t, J = 15.7 Hz), ꢀ120.03 (t, J = 15.7 Hz), ꢀ121.96 (m), ꢀ123.40 (d, J = 78.8
Hz).
“HOF8OH18” 1H, 1H, 10H, 10H-octadecyl-perfluorodecanol (4).
Route 1) To a dry roundbottom flask was added reagent grade methanol (100 mL), anhydrous
tetrahydrofuran (50 mL), and 3 (2.90 mmol, 2.34 g). This mixture was stirred and flushed with
argon for one hour then palladium on carbon catalyst (3.9 mmol, 0.4100 g) was added. This
mixture was flushed with argon for one more hour then a balloon of hydrogen gas was dispelled
into the flask with a bubbler attached. A second balloon of hydrogen gas was then put on the
reaction. The reaction was allowed to stir and run for 24 h under static hydrogen atmosphere. To
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workup the reaction, the balloon was removed and the flask flushed with argon for one hour
again. The mixture was then filtered through celite with copious rinsing with DCM. Solvent was
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removed in vacuo. Isolated product resulted in 2.25 grams (96.2 % recovered yield). H NMR
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(500 MHz, CDCl3) δ 4.10 (td, J = 14.0, 7.5 Hz, 2H), 3.92 (t, J = 14.0 Hz, 2H), 3.59 (t, J = 6.6
Hz, 2H), 1.93 (t, J = 7.6 Hz, 1H), 1.60 (p, J = 6.5 Hz, 2H), 1.25 (m, 24H), 0.88 (t, J = 6.9 Hz,
3H). 19F NMR (470 MHz, CDCl3) δ ꢀ119.65 (m), ꢀ121.96 (m), ꢀ122.42 (t, J = 12.6 Hz), ꢀ123.51
(d, J = 51.2 Hz).
Route 2) To a dry roundbottom flask was added 1H, 1H, 10H, 10Hꢀperfluorodecaneꢀ1,10ꢀdiol
(14.4 mmol, 6.6557 g) and anhydrous dimethylformamide (220 mL). The mixture was stirred
and flushed with argon until fully dissolved then sodium hydride (14.5 mmol, 0.3482 g) was
added. This was allowed to sonicate for 30 min, under argon, before 2 (14.3 mmol, 5.0 g) was
added. The reaction was sonicated for 21 h under argon. The reaction was then neutralized with
5% HCl and extracted with ethyl acetate, saturated aqueous NaHCO3 solution, and brine. The
crude mixture was dried with MgSO4 and concentrated in vacuo. Purification was performed
using automated column chromatography, CombiFlash®, using a silica gel column. A gradient
of 0ꢀ20% ethyl acetateꢀhexanes was used. The separation was monitored using an evaporative
light scattering detector (ELSD). Isolated product resulted in 1.84 grams (17.9% yield). 1H NMR
(500 MHz, CDCl3) δ 4.10 (td, J = 15.5, 13.1, 7.6 Hz, 2H), 3.92 (t, J = 13.9 Hz, 2H), 3.59 (t, J =
6.6 Hz, 2H), 1.96 (t, J = 7.5 Hz, 1H), 1.60 (p, J = 7.5 Hz, 2H), 1.25 (m, 33H), 0.88 (t, J = 6.9 Hz,
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3H). F NMR (470 MHz, CDCl3) δ ꢀ119.63 (t, J = 13.7 Hz), ꢀ121.95 (m), ꢀ122.43 (t, J = 13.5
Hz), ꢀ123.53 (d, J = 46.5 Hz).
“M2OMs” monomethyl poly(ethylene glycol) methanesulfonate (5). To a dry roundbottom
flask, in an ice bath and under argon, was added anhydrous dichloromethane (50 mL),
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poly(ethylene glycol) monomethyl ether 2,000 (2.5 mmol, 5.0195 g), and triethyl amine (7.5
mmol, 1.50 mL). This was allowed to react for 30 min before methanesulfonyl chloride (6.25
mmol, 0.50 mL) was added dropwise. After running overnight, the reaction was diluted with
more DCM and extracted with saturated aqueous NH4Cl solution. The crude mixture was dried
with MgSO4 and concentrated in vacuo. Purification was performed on the crude mixture
dissolved in minimal DCM via precipitation of pure 5 with cold ether in a dry ice/acetone bath.
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Isolated product resulted in 4.93 grams (94.1% yield). H NMR (400 MHz, CDCl3) δ 4.38 (m,
2H), 3.64 (m, 182H), 3.38 (s, 3H), 3.09 (s, 3H).
M2F8H18 (6). To a dry roundbottom flask was added 4 (3.7 mmol, 2.63 g) and anhydrous
benzotrifluoride (150 mL). This mixture was cooled over ice, flushed with argon, and then
sodium hydride was added (7.4 mmol, 0.1831 g). This reaction was stirred for 30 min then 5 was
added (1.9 mmol, 3.4526 g). The flask was heated to reflux and allowed to stir at reflux for 5
days. Upon completion, the reaction was cooled, diluted with DCM, and extracted with saturated
aqueous NH4Cl solution. The crude mixture was dried with MgSO4 and concentrated in vacuo.
Purification was performed via automated column chromatography, CombiFlash®, using a C18
column and reverse phase conditions of a 90ꢀ0% waterꢀmethanol (0.1% formic acid) to 0ꢀ100%
dichloromethaneꢀmethanol gradient. Polymer purity was confirmed via NMR, HPLC, and
MALDIꢀMS. Isolated product resulted in 2.57 grams (83.9% yield). 1H NMR (400 MHz, CDCl3)
δ 4.04 (t, J = 14.1 Hz, 2H), 3.92 (t, J = 14.0 Hz, 2H), 3.79 (m, 3H), 3.64 (m, 174 H), 3.47 (t, J =
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5.0 Hz, 1H), 3.38 (s, 3H), 1.60 (p, J = 7.0 Hz, 3H), 1.26 (m, 36H), 0.88 (t, J = 6.7 Hz, 4H). F
NMR (376 MHz, CDCl3) δ ꢀ119.73 (dt, J = 58.9, 11.9 Hz), ꢀ121.97 (m), ꢀ123.47 (bs). MALDIꢀ
MS [M + Na]+ calculated for C113H212F16O44Na+: 2600.40 m/z; found: 2600.584 m/z. [M + K]+
calculated for C113H212F16O44K+: 2616.37 m/z; found: 2616.556 m/z.
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RESULTS AND DISCUSSION
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Synthesis of M2F8H18 semifluorinated polymer
The synthesis of M2F8H18 was performed using two different routes adapted from previous
work in the Mecozzi group37,38 as well as other published works.39 The nomenclature for the
polymer is based on the three different blocks: Mx represents methoxy capped poly(ethylene
glycol) where x is the average molecular weight in the thousands, Fy represents the fluorocarbon
block where y is the number of carbon atoms substituted with fluorine atoms, and Hz represents
the hydrocarbon block where z is the number of carbon atoms substituted with hydrogens. Route
1 (see Scheme 1) begins with the monoꢀbenzylation of 1H,1H,10H,10Hꢀperfluorodecaneꢀ1,10ꢀ
diol under basic conditions and sonication to yield the protected fluorous alcohol, 1. Next, 1ꢀ
octadecanol was mesylated under basic conditions to isolate 2. Compounds 1 and 2 were
coupled through a reflux reaction under basic conditions to yield the benzylated diblock
intermediate, 3. Hydrogenation of 3 catalyzed by palladium on carbon resulted in the alcohol
diblock intermediate, 4. Then, methoxy capped poly(ethylene glycol) (mPEG) with average
molecular weight of 2,000 was mesylated under basic conditions, 5. Finally, the alcohol diblock
intermediate 4 was coupled to mesylated mPEG 5 through a reflux reaction under basic
conditions to afford the final, desired M2F8H18 polymer, 6. Compound 6 was purified using an
automated CombiFlash® system resulting in very high yields of the isolated polymer.
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Scheme 1. Synthesis of M2F8H18 copolymer.
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1) NaH, DMF
O
C
OH
8
HO
C
OH
8
F2
F2
2) BnBr, sonication
3 hr.
1
, 38%
1) TEA, DCM
1
, NaH, BTF
OH
16
O
O
C
O
8
S
16
F2
2) MsCl, ice bath
24 hr.
reflux
24 hr.
16
O O
2
, 98%
3
, 91%
MeOH/THF
O
O
C
O
HO
C
O
8
F2
16
F2
16
H2, Pd/C
24 hr.
8
4, 96%
1) TEA, DCM
4, NaH, BTF
O
O
O
O
O
S
O O
H
O
m
C
O
O
m
reflux
5 days
F2
2) MsCl, ice bath
24 hr.
m
16
8
5, 94%
6, 84%
M2F8H18
m = average molecular weight of 2,000 Daltons
Alternatively, synthetic route 2 (Scheme 2) begins with the mesylation of 1ꢀoctadecanol under
basic conditions to produce compound 2. Next, 1H,1H,10H,10Hꢀperfluorodecaneꢀ1,10ꢀdiol was
coupled directly to 2 without prior protection steps (Scheme 1). This coupling reaction occurred
under basic conditions and sonication to directly afford 4 after purification. The final two steps
mimic what was accomplished in Scheme 1 including mesylation of mPEG 5, and the final
coupling of 4 and 5 under basic conditions and reflux. The final M2F8H18 polymer 6, was once
again purified using an automated CombiFlash® system.
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Scheme 2. Alternative, shortened synthesis of M2F8H18 copolymer.
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1) TEA, DCM
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O
OH
16
S
16
2) MsCl, ice bath
24 hr.
O O
2, 98%
2, NaH, DMF
HO
C
O
8
HO
C
F2
OH
8
16
F2
sonication
21 hr.
4, 18%
1) TEA, DCM
4
, NaH, BTF
O
O
O
O
O
O
H
O
m
C
O
O
S
O O
reflux
5 days
m
m
F2
2) MsCl, ice bath
24 hr.
16
8
5, 94%
6, 84%
M2F8H18
m = average molecular weight of 2,000 Daltons
Synthetic route 1 proved to be reliable and high yielding, overall, though it does involve a more
extensive set of reactions including a protection and deprotection sequence. On the other hand,
synthetic route 2 significantly shorted the overall reaction scheme by two steps but with a
moderate loss of yield in the initial reactions.
Physicochemical characterization of polymer aggregates
The CMC of the M2F8H18 micellar aggregates was measured using surface tensiometry. The
concentration that induces aggregation for M2F8H18, 2.2 µM, is an intermediate value compared
to the related polymers M1F13 and M2H18 (Figure 2). A specific focus was placed on M2H18
due to its similarity in structure to the new M2F8H18 polymer, minus the fluorocarbon block.
The
standard
polymer,
1,2ꢀdistearoylꢀsnꢀglyceroꢀ3ꢀphosphoethanolamineꢀNꢀ
[methoxy(polyethylene glycol)ꢀ2000], M2DSPE was also measured for reference and found to
aggregate at a concentration of 12.6 µM. The size of the micellar aggregates was also analyzed
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using DLS (Table 1). Based on aggregate size (Table 1) it can be concluded that M2F8H18
forms compact, stable particles.
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Figure 2. Chemical structure of M2F8H18 and related polymers.
F2
C
F2
C
F2 F2
O
C
C
O
M2F8H18
O
C
C
C
C
F2
F2
F2
F2
m
F2
C
F2
C
F2 F2
C C
F2
C
F2
C
O
M1F13
CF3
O
C
C
C
C
C
C
F2
F2
F2
F2
F2
F2
p
O
O
M2H18
O
m
O
O
O
O
P
M2DSPE
O
O
O
O
m
N
H
O
O
m = average molecular weight of 2,000 Daltons
p = average molecular weight of 1,000 Daltons
Table 1. Physicochemical characteristics of aggregates.
CMC
log(M)
ꢀ5.7 ± 0.2
Micellar
Particle Size (nm)
16.3 ± 4.5
Polymer
M2F8H18
M1F13
ꢀ6.1 ± 0.1
ꢀ5.1 ± 0.1
ꢀ4.9 ± 0.2
17.2 ± 1.9
12.2 ± 3.1
13.9 ± 1.6
M2H18
M2DSPE
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Nanoemulsion preparation and stability
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Formulation of M2F8H18 nanoemulsions with MCT or an MCT/PTX combination was very
successful resulting in opaque, milky colloidal solutions with particleꢀsizes centered around 230
nm initially. High concentrations of paclitaxel, from 1ꢀ4 mg drug per mL nanoemulsion, were
loaded into the core of the particles. Nanoemulsions containing the highest concentrations of
paclitaxel were stable for over one year, showing no signs of phase separation. The M2F8H18ꢀ
based nanoemulsions also exhibited very minimal Ostwald ripening (Figure 3), one of the most
common mechanisms leading to the growth and destabilization of emulsion particles, resulting in
consistent nanoparticle size over time. The nanoemulsion sizes observed for M2F8H18 are
appropriate to take advantage of the EPR effect and significantly below the FDA standard size
cutoff of 500 nm for intravenous delivery of nanoparticles.40,41 The addition of the PFCE
imaging agent resulted in increased colloidal stability and better drug retention, as hypothesized
by the design of the M2F8H18 copolymer. This added stability from the fluorous excipient
further supports the idea that the more dense the fluorous shell surrounding the oil droplet, the
slower the drug release will be, which is critical for reducing systemic toxicity during the
administration of chemotherapeutics.
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Figure 3. Change in particle size of M2F8H18 nanoemulsions over time as measured by DLS:
(red) 4 mL MCT only, (orange) 2 mL MCT and 1 mg/mL paclitaxel, (blue) 4 mL MCT and 4
mg/mL paclitaxel, (green) 2 mL MCT and 10% v/v PFCE, (purple) MCT, 1 mg/mL paclitaxel,
and 23.5% v/v PFCE. All nanoemulsions were prepared in 0.9% w/w NaCl. Note: Error bars are
removed here for clarity. They are available in the Supporting Information, see Figure S2 and
Table S1.
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In vitro drug release profile
The in vitro paclitaxel release profile for M2F8H18 showed slow, steady release with highly
sustained halfꢀlives. Burst release is a common issue exhibited by many nanoparticles but no
trace of that is seen here. The linear M2F8H18 nanoemulsion without the imaging agent showed
a promising halfꢀlife (t ) of 69.5 h (Figure 4). This is an improvement compared to other drug
½
delivery nanocarriers. For instance, reported in vitro paclitaxel release halfꢀlives for micellar
delivery systems range from 2ꢀ25 h.42–46 The observed controlled release of the M2F8H18
nanoemulsions can be attributed to both the penetration of the long hydrocarbon chains of
M2F8H18 into the MCT oil droplet where the drug is solubilized, and to the perfluorocarbon
shell surrounding the oil droplet. The fluorous shell provides a barrier to modulate drug release
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from the internal oil core while at the same time providing enhanced stability to the
nanoparticles. The effect of the fluorous shell can be appreciated by comparing drug release halfꢀ
lives from fluorous M2F8H18ꢀbased nanoemulsions to the M2H18ꢀbased nanoemulsions devoid
of a fluorous block: 69.5 and 52.9 h respectively. Upon addition of the PFCE imaging agent to
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the M2F8H18 nanoemulsions, up to a 23.5% v/v, the drug release is even further modulated (t
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of 92.4 h, Figure 4). Thus, not only does the PFCE add imaging potential to the nanoparticles but
it also affords enhanced modulation of drug release.
Figure 4. In vitro nanoemulsion drug release profiles monitored for 168 h: (blue) M2F8H18 with
MCT only, (green) M2F8H18 with MCT and PFCE.
In vitro and in vivo toxicity profile
Cellular cytotoxicity was examined in vitro using A549 nonꢀsmall cell lung carcinoma cells. The
A549 cells were selected due to their robust nature and frequent use in the field. The cells were
treated with three solutions including a M2F8H18 nanoemulsion containing 4 mg/mL paclitaxel
with 4 mL MCT, a M2F8H18 nanoemulsion containing no paclitaxel with 4 mL MCT, and
paclitaxel standard solutions prepared with low concentrations of dimethyl sulfoxide (DMSO).
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Because DMSO has been shown to be toxic to A549 cells at high concentrations DMSO
cytotoxicity was also monitored in this study.47 Paclitaxel standard concentrations were centered
around the reported IC50 for the A549 cell line, 4 nM.34 As seen below (Figure 5), the media
containing 0.05% DMSO, which is the highest concentration used in this study, showed little to
no cell inhibition confirming that it was appropriate to use low concentrations of DMSO in
solution preparation without any cytotoxic effects. The wells treated with the M2F8H18
nanoemulsion devoid of drug exhibited very high cell viability, confirming that both the polymer
and MCT oil do not inhibit cell growth. The M2F8H18 nanoemulsion loaded with paclitaxel did
inhibit cell growth at similar concentrations as the standard paclitaxel solutions. In fact, the
paclitaxel delivered via the nanoemulsion inhibited cell growth at an even lower concentration
compared to the standards. This data suggests that the chemotherapeutic containing
nanoemulsions can successfully inhibit cancer cell growth, as they were designed to do.
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Figure 5. A549 cell viability studies: (blue) nonꢀtreated control, (orange) 0.05% DMSO control,
(purple) M2F8H18 nanoemulsion with MCT but no drug, (green) M2F8H18 nanoemulsion
loaded with 4 mg/mL paclitaxel and MCT, (red) paclitaxel standard solutions in DMSO.
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The M2F8H18 polymer was also screened for potential intrinsic toxicity due to the novel
nature of the polymer using a zebrafish model, the primary model used to study nanotoxicity.48–50
A thorough study of developmental effects related to M2F8H18 was performed using an
embryoꢀlarval zebrafish model. This animal model was selected because zebrafish eggs remain
transparent from fertilization until the tissues become dense as a mature adult. Several
developmental endpoints can be simultaneously monitored providing valuable developmental
toxicity data for new chemicals. After crossing the fish and fertilization of the eggs had occurred,
the embryos were collected and plated. The embryos were then treated with M2F8H18 and
positive control solutions of the following concentrations: 1 mM, 333 µM, 111 µM, 37 µM, 12.3
µM, and 4.1 µM. The embryos were then incubated, monitored, and photographed over a 96 h
experiment period. Survival rates for the nonꢀtreated control fish (Figure 6) were very high, as
would be expected from healthy zebrafish at 96 hpf. The positive control for the experiment,
perfluorooctanoic acid (PFOA), was selected for its documented toxicity to zebrafish and the
contrasting difference between toxic perfluoro acids and benign perfluorocarbonꢀcontaining
copolymers.51,52 The PFOA caused mortality in all fish at the highest concentration of 1 mM
resulting in 0% survival at 96 hpf. At PFOA concentrations of 333 µM, 67% of fish survived
and some developmental malformations were observed including curved body axis and mild
pericardia edemas. The PFOA results are varied at concentrations at and below 111 µM
indicating subꢀlethal concentrations may have been reached. No other malformations were noted
at the lower PFOA concentrations. In great contrast to PFOA, the novel M2F8H18 polymer
induced 100% survival in the fish of this study at the highest concentration of 1 mM. The small
decrease in survival rate, 78%, seen at 111 µM is thought to be a random anomaly based on the
collective zebrafish data. None of the fish exposed to M2F8H18 in this study exhibited
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malformations such as curved body axis or pericardia edemas, as well. This data suggests there
are no developmental toxicity concerns involving 1 mM and below concentrations of the newly
synthesized M2F8H18 polymer. Research in this area may progress to further in vivo stages.
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Figure 6. Zebrafish survival rates after exposure for 96 hpf: (blue) nonꢀtreated control, (red)
positive control of perfluorooctanoic acid (PFOA), (green) M2F8H18 polymer.
19F-MR imaging characterization
M2F8H18ꢀbased nanoemulsions can be loaded with high concentrations of PFCE. The addition
of this fluorous imaging agent enhances the drug release profile of the nanoemulsions, slowly
drug release, and provides further stability to the colloidal particles. Next, the imaging potential
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of this molecule was investigated. Preliminary FꢀNMR studies of PFCE at 11.7 T show an
intense, single peak at δ ꢀ91.8 ppm with CFCl3 as an internal refernce.23 This pattern is due to the
twenty magnetically equivalent fluorine atoms and ring flexibility in solution of the PFCE. The
19F relaxation parameters T1, longitudinal relaxation or delay between pulses, and T2, transverse
relaxation or signal decay over time, were also measured to monitor potential PFCE use as an
efficient contrast agent. The T1 was determined using an inversion recovery experiment while T2
was determined using a Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence experiment. The
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