Development of nanodroplets for histotripsy-mediated cell ablation

Article abstract of DOI:10.1021/mp500419w

This report describes the synthesis of amphiphilic copolymers (ABC-1 and ABC-2) composed of a hydrophilic poly(ethylene glycol) (PEG) block, a central poly(acrylic acid) (PAA) block, and a random copolymer of heptadecafluorodecyl methacrylate (HDFMA) and methyl methacrylate (MMA) forming the hydrophobic block, which are used to form nanodroplets for ultrasound-mediated cell ablation. Specifically, the effect of molecular weight of PEG and P(HDFMA-co-MMA) blocks on polymers ability to self-assemble around a variable amount (0%, 1%, and 2% v/v) of perfluoropentane (PFP) forming nanodroplets is investigated. The ability of different nanodroplets formulations embedded with a monolayer of red blood cells (RBCs) in tissue-mimicking agarose phantoms to initiate and sustain a bubble cloud in response to ultrasound treatments with different acoustic pressures and the associated ablation of RBCs were also investigated. Results show that ABC-1 polymer composed of a 2 kDa PEG block and a 6.7 kDa P(HDFMA-co-MMA) block better encapsulate the PFP core compared to ABC-2 polymer composed of a 5 kDa PEG block and 11.4 kDa P(HDFMA-co-MMA) block. Further, the ablative capacity indicated by the damage area in the RBCs monolayer increased with the increase in PFP content and reached its maximum with the nanodroplets formulated using ABC-1 polymer and encapsulating 2% v/v PFP. The nanodroplets formulated using ABC-1 polymer and loaded with 2% PFP produced the cavitation cloud and exhibited their ablative effect at an acoustic pressure that is 2.5-fold lower than the acoustic pressure needed to generate the same effect using a histotripsy (ultrasound) pulse alone, which indicates the ability of these nanodroplets to achieve targeted and self-limiting fractionation of disease cells while sparing neighboring healthy ones. Results also show that effective nanodroplets maintained their size and concentration upon incubation with bovine serum albumin at 37 °C for 24 h, which indicates their stability in physiologic conditions and their promise for in vivo cancer cell ablation.

Full text of DOI:10.1021/mp500419w

Article
pubs.acs.org/molecularpharmaceutics
Development of Nanodroplets for Histotripsy-Mediated Cell Ablation
Yasemin Yuksel Durmaz,† Eli Vlaisavljevich,† Zhen Xu,*,† and Mohamed ElSayed*,†,‡
†Department of Biomedical Engineering, College of Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States
‡Macromolecular Science and Engineering Program, University of Michigan, Ann Arbor, Michigan 48109, United States
S
* Supporting Information
ABSTRACT: This report describes the synthesis of amphi-
philic copolymers (ABC-1 and ABC-2) composed of a
hydrophilic poly(ethylene glycol) (PEG) block, a central
poly(acrylic acid) (PAA) block, and a random copolymer of
heptadecafluorodecyl methacrylate (HDFMA) and methyl
methacrylate (MMA) forming the hydrophobic block, which
are used to form nanodroplets for ultrasound-mediated cell
ablation. Specifically, the effect of molecular weight of PEG
and P(HDFMA-co-MMA) blocks on polymer’s ability to self-
assemble around a variable amount (0%, 1%, and 2% v/v) of
perfluoropentane (PFP) forming nanodroplets is investigated.
The ability of different nanodroplets formulations embedded
with a monolayer of red blood cells (RBCs) in tissue-
mimicking agarose phantoms to initiate and sustain a bubble cloud in response to ultrasound treatments with different acoustic
pressures and the associated ablation of RBCs were also investigated. Results show that ABC-1 polymer composed of a 2 kDa
PEG block and a 6.7 kDa P(HDFMA-co-MMA) block better encapsulate the PFP core compared to ABC-2 polymer composed
of a 5 kDa PEG block and 11.4 kDa P(HDFMA-co-MMA) block. Further, the ablative capacity indicated by the damage area in
the RBCs monolayer increased with the increase in PFP content and reached its maximum with the nanodroplets formulated
using ABC-1 polymer and encapsulating 2% v/v PFP. The nanodroplets formulated using ABC-1 polymer and loaded with 2%
PFP produced the cavitation cloud and exhibited their ablative effect at an acoustic pressure that is 2.5-fold lower than the
acoustic pressure needed to generate the same effect using a histotripsy (ultrasound) pulse alone, which indicates the ability of
these nanodroplets to achieve targeted and self-limiting fractionation of disease cells while sparing neighboring healthy ones.
Results also show that effective nanodroplets maintained their size and concentration upon incubation with bovine serum
albumin at 37 °C for 24 h, which indicates their stability in physiologic conditions and their promise for in vivo cancer cell
ablation.
KEYWORDS: amphiphilic polymer, perfluorocarbon, nanodroplets, therapeutic ultrasound, cell ablation
ultrasound-mediated cell ablation. Therefore, it would be of
great interest to develop well-defined nanodroplets that can
withstand the shear forces in the blood circulation, resist the
adsorption of serum proteins that will trigger their phagocytosis
by macrophages (e.g., Kupffer cells), and allow the covalent
attachment of targeting ligands to be used for mechanical
ablation of cancer cells.
To address this need, we designed and synthesized the
amphiphilic poly[(ethylene glycol-b-(acrylic acid)-b-(heptade-
cafluorodecyl methacrylate-co-methyl methacrylate)] [PEG-b-
(PAA)-b-P(HDFMA-co-MMA)] triblock copolymer, which
encapsulates perfluoropentane (PFP, an US contrast agent)
in the core forming nanodroplets with an average diameter of
100−350 nm (Table 1 and Figure 1). The hydrophilic PEG
INTRODUCTION
■
Interest in ultrasound (US) imaging and delivery of therapeutic
agents to the extravascular space particularly into solid tumors
has motivated the development of nanosized contrast
agents.1−7 Lipids,8−10 fluoroalkyl surfactant,11 and amphiphilic
block copolymers13 have been used to encapsulate perfluor-
ocarbon (PFC) forming nanodroplets. The potential of
nanodroplets for US contrast imaging,14,15 local delivery of
chemotherapeutic agents,14,16 and nontargeted/targeted high
intensity focus ultrasound (HIFU)17−20 has been extensively
investigated. For example, Dayton et al. used oil to solubilize
paclitaxel and incorporate it with the PFC core to develop
therapeutic and imaging nanodroplets.21 Recently, lipid-coated
nanodroplets encapsulating perfluoropentane (PFP) were
developed for cell-specific imaging and therapy.17 Further,
attachment of targeting ligands to nanodroplets increased their
accumulation in tumor tissue, which enhanced contrast
resolution in US imaging.22,23 Despite their promise in US
imaging and drug delivery, nanodroplets have not been used for
Received: June 15, 2014
Revised: August 10, 2014
Accepted: August 19, 2014
Published: August 19, 2014
© 2014 American Chemical Society
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Table 1. Composition of Amphiphilic PEG-b-PAA-b-P(HDFMA-co-MMA) Copolymers
a
,
b
a
a
e
Mn,PEG (g/mol)
(no. of units)
Mn,PtBA (g/mol)
Mn,P(MMA‑co‑HDFMA) (g/mol)
no. of HP/no. of
Mn,ABC
Mn,ABC
c
d
e
polymer
(no. of units)
(no. of units)
M
nHP/MnHF
HF units
(g/mol)
(g/mol)
PD
ABC-1
ABC-2
2000 (45)
1640 (10)
6720 (29)
1.84
1.71
0.53
0.42
10370
18040
9613
1.11
1.12
5000 (113)
1640 (10)
11390 (52.5)
17510
a
b
c
Calculated from 1H NMR spectra. tert-Butyl groups were efficiently hydrolyzed into acid group to obtain the final ABC block copolymer. MnHP
/
d
MnHF: molecular weight of hydrophobic block (HP)/molecular weight of hydrophilic blocks (HF). no. of HP/no. of HF units: The number of
hydrophobic units (HP)/the number of hydrophilic units. Determined by gel permeation chromatography (GPC) using Styragel HR 4E column
e
compared to a series of PMMA standards (PolyAnalitik Inc., Canada) using THF as a mobile phase at a flow rate of 1 mL/min at 35 °C. Data were
analyzed using Viscotek OmniSEC Omni-01 software.
block is used to inhibit the adsorption of serum proteins and
inhibit the uptake by Kupffer cells,24 which will consequently
increase droplets residence time in the systemic circulation.
Further, the free end of the PEG block can be used to conjugate
different types of targeting ligands prior to the formulation of
the nanodroplets, which will allow the presentation of a well-
defined and tunable number of targeting ligands on the
droplets’ surface to mediate selective binding to cancer cells.24
The carboxylic acid groups in the central PAA block are used
for covalent cross-linkage of the polymer chains to form a
flexible shell that stabilizes the nanodroplets. The fluorinated
and hydrophobic P(HDFMA-co-MMA) block is designed to
facilitate the encapsulation of PFP in the droplets’ core.11,25
Histotripsy is a noninvasive, nonthermal, image-guided,
ultrasound ablation method that uses extremely high acoustic
pressure and microseconds long pulses to generate a cluster of
microbubbles (bubble cloud) from pre-existing gas pockets in
tissue.12,26−29 Rapid expansion and energetic collapse (i.e.,
cavitation) of the formed microbubbles, each with an average
diameter >50 μm, mechanically fragment the adjacent cells into
subcellular debris.30−33 Histotripsy has been used to ablate
tumor cells in a canine prostate cancer model34 and dissolve
deep vein thrombosis in a porcine model.35 However, the
required high acoustic pressure and the inability to detect/
image small metastatic tumor foci are some of histotripsy
limitations, which can be overcome by combining histotripsy
with our nanodroplets
We hypothesize that PFP-loaded nanodroplets with an
average diameter <500 nm can diffuse across tumor’s leaky
vasculature and accumulate in the cancer lesion when
administered into the systemic circulation.36−38 Applying
specific US pulse sequences to the tumor lesion will deliver
the acoustic energy necessary to convert the PFP core from the
liquid to the gaseous phase in a process known as acoustic
droplet vaporization (ADV),36,39,40 which will allow real time
US imaging of tumor tissue. Unlike histotripsy that relies on
cavitation nuclei derived from rare gas pockets in the tissue and
extremely high acoustic pressures to initiate the cavitation
process, the gas bubbles formed by ADV can act as cavitation
nuclei to generate and maintain the cavitation bubble cloud at a
significantly reduced pressure. The significantly reduced
cavitation threshold will allow us to deliver histotripsy to the
tumor tissue “tagged“ with the nanodroplets, resulting in
selective fractionation of cancer cells while sparing the
surrounding normal tissue.
assembly of amphiphilic ABC-1 and ABC-2 polymers around
the PFP core forming nanodroplets with variable PFP content
(0%, 1%, and 2% v/v), the chemical cross-linkage of the
polymer shell to stabilize the droplets, and their character-
ization in terms of size, shape, and intrinsic cytotoxicity toward
mammalian cells. We investigated the relationship between
polymer composition (ABC-1 versus ABC-2) and PFP content
(0%, 1%, and 2% v/v) on the ability of the formulated
nanodroplets to initiate a bubble cloud in agarose gel phantoms
with similar mechanical and acoustic properties to tumor tissue
in response to a 2 cycles long histotripsy pulse. Further, we
correlated the behavior of the bubble cloud to the observed
damage of a layer of red blood cells (RBCs) embedded with
different nanodroplets in the agarose gel phantom to identify
the ablative capacity of each formulation. In addition, we
showed effective hemolysis of RBCs using the combination of
most effective composition and histotripsy under the
physiological conditions. Futher, we examined the change in
nanodroplets size and concentration upon incubation with
bovine serum albumin (BSA) as a model serum protein for 24 h
at 37 °C while stirring to determine the stability of different
nanodroplets formulations in a condition that resembles the
systemic circulation in vivo. Results establish the potential of
combining these nanodroplets with histotripsy to achieve
ultrasound-mediated cell ablation.
EXPERIMENTAL SECTION
■
Materials. Methyl methacrylate (MMA, Sigma-Aldrich,
99%), 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyl
methacrylate (HDFMA, Sigma-Aldrich, 97%), tert-butyl acryl-
ate (tBA, Sigma-Aldrich, 98%), and N,N,N′,N″,N″-pentam-
ethyldiethylenetriamine (PMDETA, Sigma-Aldrich, 99%,) were
passed through basic alumina column to remove the inhibitor.
N-(n-Pentyl)-2-pyridylmethanimine was synthesized according
to the literature procedure.41 Copper(I) bromide (CuBr,
Sigma-Aldrich, 99.9%), 2-bromoisobutyryl bromide (Fluka,
>97%), tetrahydrofuran anhydrous (THF, Sigma-Aldrich,
>99.9%), N,N′-dicyclohexylcarbodiimide (DCC, Sigma-Aldrich,
99%), dimethylaminopyridine (DMAP, Acros, 99%), 4-
pentynoic acid (Sigma-Aldrich, 99%), furan (Sigma-Aldrich,
≥99%), maleic anhydride (Fluka, ≥99%), 9-anthracene
methanol (Aldrich, ≥ 99%), perfluoropentane (PFC, Alfa
Aesar, 97% ca. 85% n-isomer), N-hydroxy succinimide (NHS,
Fluka, 97%), N-(3-(dimethylamino)propyl)-N′-ethylcarbodii-
mide hydrochloride (EDC, Fluka >98%), poly(ethylene glycol)
monomethyl ether (Me-PEG, Mn 2000 and 5000, Sigma-
Aldrich), sodium azide (NaN3, Acros, 99%), 2-(N-
morpholino)ethanesulfonic acid monohydrate (MES, Acros,
99%), triethylamine (TEA, Sigma-Aldrich, ≥99%), trifluoro-
acetic acid (TFA, Acros, 99%), ethylene carbonate (Sigma-
Aldrich, 98%), 2,2′-(ethylenedioxy)-bis(ethylamine) (Sigma-
This article summarizes the first phase toward confirming our
hypothesis. Specifically, we describe the synthesis of two
triblock copolymers (ABC-1 and ABC-2) where the molecular
weight of the P(HDFMA-co-MMA) (6.5 and 11.5 kDa) and
PEG (2 and 5 kDa) blocks was varied to examine their effect on
encapsulation efficiency of different amounts of PFP (1% and
2% v/v) and overall droplets stability. We describe the self-
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Figure 1. (A) Scheme for synthesis of amphiphilic PEG-b-(PAA)-b-P(HDFMA-co-MMA) copolymers using one-pot “click” reaction. (B) Schematic
presentation showing the formulation of PFP-loaded nanodroplets using amphiphilic PEG-b-(PAA)-b-P(HDFMA-co-MMA) copolymers.
Aldrich, 98%) agarose powder (Type VII; Sigma-Aldrich), and
citratephosphate-dextrose (CPD, Sigma-Aldrich) were used as
received. RPMI medium 1640, fetal bovine serum (FBS), 0.25%
trypsin/0.20% ethylene diamine teraacetic acid (EDTA),
phosphate buffered saline (PBS), penicillin/streptomycin/
amphotericin, sodium pyruvate, and nonessential amino acid
solutions were purchased from Invitrogen Corporation
(Carlsbad, CA). MTT Assay Kit was purchased from American
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Type Culture Collection (Manassas, VA). T-75 flasks, Costar
96-well plates, and cell culture supplies were purchased from
Corning Inc. (Corning, NY).
analyzing the data. The samples were measured for 60 s with
manual shutter and gain adjustments. The error bars were
obtained by the standard deviation of the different measure-
ments of each sample. The mean size and standard deviation
values obtained by the NTA software correspond to arithmetic
values calculated with the sizes of all particles analyzed by the
software. The error bars displayed on the graphs were obtained
by the standard deviation of a minimum three different samples.
All statistical evaluations were carried out with unpaired two-
tailed Student’s t tests. A p-value of less than 0.05 (p < 0.05)
was considered significant.
Ablation of Red Blood Cells in Tissue Phantoms.
Agarose gel phantoms with an embedded red blood cell
(RBCs) layer were used to demonstrate the ability of using
nanodroplets to mechanically ablate tissue.44 This phantom
provides a thin, translucent red layer of RBCs as a visual
indicator between the two transparent agorose gel layers that
locally appears more transparent when RBCs layer is damaged.
Tissue phantoms were prepared using a mixture of agarose
powder (type VII; Sigma-Aldrich Co., St. Louis, Missouri,
USA) and canine RBCs in 0.9% isotonic saline. Fresh canine
blood was obtained from adult research subjects in an unrelated
study and added to an anticoagulant solution of citratephos-
phate-dextrose (CPD, Sigma-Aldrich Co.) with a CPD-to-
blood ratio of 1:9 mL. Whole blood was separated in a
centrifuge at 3000 rpm for 10 min. The plasma and white buffy
coat were removed, and the RBCs were saved for addition to
the phantom.
Agarose was slowly combined with saline while stirring at 20
°C (1.5% w/v agarose/saline), forming a translucent solution.
The solution was heated in a microwave oven for 30 s and then
stirred. Heating at 30 s intervals and stirring was repeated until
the solution turned entirely transparent. The solution was then
placed under a partial vacuum of 20.5 psi for 30 min to degas
the mixture. After removing the mixture from the vacuum,
mixture was cooled to 37 °C and PFP encapsulated micelles
were added to experimental phantom for comparison with 1 or
2% PFP encapsulated nanodroplets and micelles phantoms. A
layer of these phantom mixtures was then poured into a
rectangular polycarbonate housing to fill half of it. The housing
was placed in a refrigerator at 4 °C to allow the agarose to cool
and solidify. The remaining agarose solution was kept at 37 °C.
A small amount of agarose solution was mixed with the RBCs
(5% RBCs v/v). The frame with solidified agarose was removed
from refrigeration, and a thin layer of the RBC−agarose
solution was poured onto the gel surface to allow the entire
surface to coat in a thin layer. After 5 min, the RBC−agarose
layer was solidified, and the remaining agarose solution without
RBCs was poured to completely fill the frame. This procedure
created a thin layer of RBCs suspended in the center of the
agarose phantom.
The focus of a 32 element 500 kHz transducer was aligned
with the center of the red blood cell phantom layer. Histotripsy
pulses were applied at a pulse repetition frequency of 10 Hz at
peak negative pressures of 11.0 and 20.7 MPa to the center of
phantoms containing red blood cell layer embedded with PFP
encapsulated micelles and empty micelles. Red blood cell
fractionation was monitored using a high-speed, 1 megapixel
CCD camera (Phantom V210, Vision Research) capable of a
maximum frame rate of 2000 fps. The camera was focused to
the red blood cell layer and backlit by a continuous light source.
The camera was triggered to record two images for each
applied pulse, one 10 μs after the pulse reached the focus to
Formulation of PFP-Loaded Nanodroplets. Copolymers
were synthesized via a one-pot “click” reaction42,43 of PEG,
poly(tert-butyl acrylate) (PtBA), and P(HDFMA-co-MMA)
blocks following the synthesis scheme outlined in Figure 1A.
Detailed description of the synthesis procedure and supporting
spectra are available in the Supporting Information (Figure S1−
S11). Self-assembly of amphiphilic fluorinated triblock
copolymers in the presence of PFP was resulted in PFP
encapsulated nanodroplets because hydrophobic PFP prefers to
stay in a hydrophobic core of nanosized self-assembly. PFP is
most commonly used PFC with boiling point of 29 °C, which is
close to room temperature. To minimize PFP evaporation, each
step of nanodroplets preparation was performed in an ice bath
(Figure 1B). PEG-b-(PAA)-b-P(HDFMA-co-MMA) copoly-
mers were dissolved in tetrahydrofuran (THF) (0.2% w/v)
and cooled down to 0 °C before addition of PFP (1 or 2% v/v).
An equal amount of water was slowly added to this solution
mixture to trigger micelle formation, and mixture was stirred for
1 h in ice bath. The solution was transferred into a dialysis bag
(MWCO of 1 kDa) and dialyzed against ice-cold MES solution
or water to remove THF. After 12 h of dialysis, a slightly milky
solution of PFP encapsulated non-cross-linked nanodroplets
was obtained. These nanodroplets were further reacted with
2,2′-(ethylenedioxy)-bis(ethylamine) via NHS/EDC coupling
(2/4 equiv of COOH groups) to obtain a cross-linked shell.
Briefly, 5 mL of this nanodroplet solution (3.18 × 10−4 mmol
of ABC-1, 3.18 × 10−3 mmol of carboxylic acid groups) was
transferred into round-bottom flask. Then 127.2 μL of NHS
solution (6.36 × 10−3 mmol, 50 mM solution in 100 mM MES
buffer pH: 5.5) and 254.4 μL of EDC solution (1.27 × 10−2
mmol, 50 mM solution in 100 mM MES buffer pH: 5.5) were
added to nanodroplets solution and stirred for 45 min followed
by addition 159 μL of 2,2′-(ethylenedioxy)-bis(ethylamine) as a
cross-linker (1.59 × 10−3 mmol, 10 mM solution in PBS buffer
pH: 7.4). After 6 h stirring in ice bath, the nanodroplet solution
was transferred into a dialysis bag to remove buffer impurities.
Nanodroplets size was monitored using DLS before and after
cross-linking.
Nanodroplets Characterization. Self-assembly of amphi-
philic triblock copolymers without PFP form nanosized
micelles. In the presence of PFP, these micelles have
hydrophobic liquid (PFP) in their core, which makes them
nanodroplets. Micelles and nanodroplets size were measured
using both dynamic light scattering (DLS) 90Plus particle size
analyzer with ZetaPALS capability (Brookhaven Instruments
Corporation, Holtsville, NY) and nanoparticle tracking analyzer
(NTA) methods at 22 and 37 °C. DLS was used as most
common size measurement techniques, which allowed us to
monitor our preparation process before and after critical points
such as PFP encapsulation and shell cross-linked. NTA was
used not only for size measurement but also for determination
of particle concentration. The technique combines light
scattering microscopy with a camera, which enables visual-
ization and recording of Brownian motion of nanoparticles in
solution, which allows later evaluation of their size and
concentration. NTA measurements were performed with
Nanosight NS500 (Nanosight, Amesbury, United Kingdom)
equipped with a sample chamber with a 640 nm laser and a
fluoroelastomer O-ring at 22 °C. Each sample was analyzed six
times. NTA 2.3 Build 127 was used as software to capture and
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visualized the bubble activity, and another frame was captured
between pulses to assess tissue damage. The camera exposure
time was 10 μs. The bubbles appeared as black regions in the
shadow graphic image, while red blood cell fractionation was
visualized as red blood cells turning transparent after
fractionation. Tissue fraction was compared between RBC
phantoms with nanodroplets and micelles as controls.
copolymer were placed in contact with the model proteins at
concentrations of 20 μg/mL of BSA (100 μL). Nanodroplets
size and concentration were measured before and after (24 h
later) incubation using NTA.
RESULTS AND DISCUSSION
■
Synthesis of Amphiphilic Fluorinated Triblock Co-
polymers. Two PEG-b-(PAA)-b-P(HDFMA-co-MMA) poly-
mers were synthesized via a one-pot “click” reaction of PEG,
poly(tert-butyl acrylate) (PtBA), and P(HDFMA-co-MMA)
blocks following the synthesis scheme outlined in Figure1A.
Detailed description of the synthesis procedure and supporting
spectra are available in the Supporting Information (Figure S1−
S11). Briefly, commercial PEG (2 and 5 kDa) was reacted with
4-pentynoic acid to introduce a terminal alkyne group forming
PEG-alkyne (block A). The PtBA block was synthesized via
atom transfer radical polymerization (ATRP) using anthracene-
functional initiator followed by azidation of the terminal Br
group to yield the Anth-PtBA-N3 block (block B) (Figure 1A).
Earlier reports show that the percentage of fluorinated
monomers play a critical role in the amount of encapsulated
PFC and the stability of the formed droplets.11 Therefore, we
synthesized two different P(HDFMA-co-MMA) copolymers
(block C) that incorporate a different number of fluorinated
HDFMA units (8 and 13.5 units) and different molecular
weights (6.7 and 11.4 kDa) (Supporting Information, Table
S1) via ATRP using a maleimide-functional initiator (Figure
1A). Pure A, B, and C blocks were coupled via a one-pot
alkyne−azide and Diels−Alder “click” reactions in the presence
of a copper catalyst (Figure1A) to yield two ABC triblock
copolymers (ABC-1 and ABC-2) that vary in the length of the
PEG and P(HDFMA-co-MMA) blocks but have similar number
of PtBA units (Table 1). The synthesized polymers (ABC-1
and ABC-2) were purified and characterized (Supporting
Information, Figures S8−S11) before acid hydrolysis of the tert-
butyl groups to obtain the corresponding acrylic acid.
Hemolysis of Red Blood Cells Using Nanodroplets-
Mediated Histotripsy. The plasma supernatant of bovine
blood (10 mL) was discarded after centrifuge (3500g for 5
min), and the RBCs were washed three times using a 150 mM
saline solution. After the third wash, the RBCs solution was
resuspended in 100 mM PBS (pH:7.4), followed with 10-fold
dilution using PBS. Desired nanodroplets solutions ((8.45 μL)
ND-ABC-1−2% PFP and (3.68 μL) ND-ABC-1−0% PFP) to
obtain 2.36 × 108 particles/mL as a final nanodroplets
concentration were mixed with 400 μL of RBCs solution and
PBS to reach 2 mL of final volume. Each solution was prepared
as triplicate as well as control solutions and kept at 4 °C until
they were treated with histotripsy pulses. The focus of a 32
element 500 kHz transducer was aligned with the RBCs filled
rubber tube, and histotripsy pulses were applied at a pulse
repetition frequency of 50 Hz at peak negative pressures of 20.7
MPa by scanning the rubber tube inside the water tank at 37
°C. At the end of 10 min of treatment time, RBCs solutions
were centrifuged at 14000g for 5 min, resulting in intact and
ruptured RBCs to pellet out, leaving the hemoglobin in the
supernatant solution in which its absorbance was measured at
541 nm as an indication of hemolysis. The observed hemolysis
of RBCs in PBS solutions without histotripsy and in 0.1% v/v
Triton X-100 solution were used as negative and positive
controls, respectively. The observed hemolytic activity was
normalized to 0.1% v/v Triton X-100 solution.
Cytotoxicity of Micelles Prepared Using ABC-1 and
ABC-2 Copolymer. We investigated the effect of the micelles
prepared using ABC-1 and ABC-2 copolymers on the viability
of PC-3 (human prostate cancer) cells using the MTT assay
(American Type Culture Collection, Manassas, VA) following
manufacturer’s specifications. Briefly, human prostate cancer
cells (PC-3) were seeded in 96-well plates at a seeding density
of 1 × 105 cells/well and allowed to adhere overnight before
replacing the culture medium with RPMI medium 1640
solution (without phenol red) containing different concen-
tration of micelles and incubating for 24 h under normal culture
conditions. The cells were then incubated with 10 μL of the
yellow tetrazolium dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide) for 2 h, which was reduced to a purple
formazan precipitate that was dissolved by mixing with 100 μL
of the detergent, incubating for 3 h in the dark at room
temperature, followed by measuring the absorbance of this
solution at 570 nm using a Multiskan microplate reader
(Thermo Fisher Scientific Inc., Waltham, MA). Contribution of
free culture medium was eliminated by subtracting the
absorbance of equal volume of culture medium at this
wavelength. PC-3 cells with RPMI1640 culture medium
(untreated cells) and 5% v/v Triton X-100 solution were
used as negative and positive controls, respectively. The
micelles concentration that result in statistically significant
reduction in cell viability was considered cytotoxic.
Formulation of Nanodroplets. ABC-1 and ABC-2
copolymers were dissolved in tetrahydrofuran (THF) followed
by cooling down the polymer solution in an ice bath and adding
different volumes of PFP (0%, 1%, or 2% v/v) while mixing. An
equal amount of water was slowly added to the polymer/PFP
mixture to initiate polymers self-assembly into nanosized
micelles that will encapsulate the hydrophobic PFP solution
forming nanodroplets (Figure 1B). This solution mixture was
kept stirring on an ice bath for 1 h before transferring to a
dialysis bag and dialyzing against ice-cold water for 12 h to yield
a homogeneous milky solution indicating nanodroplets
formation. We used 2,2′-(ethylenedioxy)-bis(ethylamine) link-
er to cross-link the polymer chains via standard NHS/EDC
coupling reactions with the central PAA block.45,46 Cross-linked
nanodroplets were further purified by dialysis against ice cold
water before their characterization and use in ablation studies.
We confirmed the cross-linkage of the central PAA block by
examining the thermal behavior of the amphiphilic polymer
(e.g., ABC-1), the hydrophobic P(HDFMA-co-MMA) block,
cross-linked droplets, and non-cross-linked droplets when
heated between 20 and 120 °C using a differential scanning
calorimeter. The P(HDFMA-co-MMA) block with an average
molecular weight of 6.7 kDa has a glass transition temperature
(Tg) of 61.4 °C, which increased to 76.5 °C with the increase in
molecular weight to 11.4 kDa (Figure 2 and Supporting
Information, Table S2). Because the PEG and P(HDFMA-co-
MMA) blocks are not miscible, the thermograph of ABC-1
Stability of Nanodroplets. The stability of the nano-
droplets and its interaction with bovine serum albumin (BSA)
have been investigated by incubating them in BSA solution at
37 °C. Typically, 900 μL of nanodroplets solution of each block
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°C) and body (37 °C) temperatures. We relied on the
Nanoparticle Tracking Analysis 2.3 software to capture and
analyze images of different nanodroplets solutions and calculate
the average size of each formulation (Figure 3). Results show
that nanodroplets formulated using ABC-1 polymer have an
average diameter of 192 4.7 to 218 2.1 nm at 22 °C, which
slightly increased to 201
2.61 to 211
4.7 nm at 37 °C
(Figure 3). Nanodroplets formulated using ABC-2 polymer
have average diameter of 202 2.7 to 236 2.6 nm at 22 °C,
which also slightly increased to 206 2.9 to 246 3.4 nm at
37 °C. Results show that the increase in PFP loading from 0%−
2% v/v did not influence the average size of all the formulated
nanodroplets. It is important to note that NTA software
identified the major fraction in each formulation and calculated
its average mode size and concentration (Table 2). Results
show that the average mode size for ND-ABC-1−1% and ND-
ABC-1−2% droplets at 37 °C is 111.9 6.8 and 135.3 7.1
nm, respectively. Similarly, the average mode size for ND-ABC-
2−1% and ND-ABC-2−2% droplets at 37 °C is 167.0 9.0
Figure 2. Thermal properties of P(HDFMA-co-MMA), PEG-b-PAA-b-
P(HDFMA-co-MMA) (ABC-1), non-cross-linked micelles, and cross-
linked micelles made out of ABC-1 block copolymer.
and 166.8
4.3 nm, respectively (Table 2 and Supporting
Information, Figure S12). These results clearly show that the
average size of cross-linked (CL) nanodroplets prepared using
ABC-1 and ABC-2 polymers with different PFP content is
much smaller than the size cutoff (∼500 nm) of the tumor
vasculature.13,48 Furthermore, the concentration of cross-linked
ND-ABC-1 and ND-ABC-2 nanodroplets did not decrease
upon increasing the solution temperature from 22 to 37 °C,
which indicates droplets resistance to dissolution and stability
at body temperature (Table 2). These results collectively
indicate the promise of these nanodroplets in future in vivo
evaluation.
We visualized different nanodroplets formulations using a
FEI Quanta 3D dual-beam environmental scanning electron
microscope (FEI Co., Oregon, USA) under low pressure (5.2
Torr) and temperature (1.3 °C) to examine droplets
morphology. Images of all PFP-loaded formulations show
spherical nanodroplets (Supporting Information, Figure S13)
polymer shows two transitions at 50.8 and 60.9 °C,
corresponding to the PEG melting point and Tg of the
P(HDFMA-co-MMA) block, respectively (Figure 2). Increasing
the molecular weight of the PEG and P(HDFMA-co-MMA)
blocks increased their melting point and Tg, respectively
(Supporting Information, Table S2). The restricted mobility of
the polymer chains in cross-linked droplets prepared using
ABC-1 polymer caused an increase in the Tg compared to the
parent polymer and non-cross-linked droplets (Figure 2 and
Supporting Information, Table S2). However, the insignificant
difference in the Tg between cross-linked and non-cross-linked
droplets prepared using ABC-2 is due to the increase in PEG
content (38%) compared to ABC-1 (24%), which acts as a
plasticizer and reduces the observed Tg as shown in previous
reports.47
Characterization of Nanodroplets. We used Nanosight
NS500 (NanoSight Limited, Amesbury, United Kingdom)
equipped with a sample chamber with a 640 nm laser and a
fluoroelastomer O-ring to measure the size and concentration
(i.e., number of nanodroplets/mL) of cross-linked (CL)
nanodroplets formulated using ABC-1 and ABC-2 polymers
as a function of PFP loading (0%, 1%, and 2% v/v) at room (22
with average size of 316 49, 342 11, 310 10, and 333
9
nm for ND-ABC-1−1% PFP, ND-ABC-1−2% PFP, ND-ABC-
2−1% PFP, and ND-ABC-2−2% PFP, respectively. The higher
average size observed with scanning electron microscopy could
possibly be as a result of the smaller number of droplets
manually analyzed in each field compared to NTA. We used
Malvern ZetasizerNano ZSP (Malvern, United Kingdom) to
Figure 3. Size of cross-linked nanodroplets prepared using ABC-1 and ABC-2 polymers with different PFP content (0%, 1%, and 2% v/v) at (A) 22
°C and (B) 37 °C calculated using the Nanoparticle Tracking Analysis 2.3 software. Results are reported as the average of six different measurements
collected from three independent solutions + standard error of the mean.
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Table 2. Properties of the Nanodroplets Prepared Using ABC-1 and ABC-2 Polymers
mode size at 22 °C
concentration at 22 °C (1010
mode size at 37 °C
concentration at 37 °C (1010
ζ potential
a
a
a
a
b
nanodroplets code
(nm)
particles/mL)
(nm)
particles/mL)
(mV)
ND-ABC-1−0% PFP
ND-ABC-1−1% PFP
ND-ABC-1−2% PFP
ND-ABC-1−0% PFP
ND-ABC-2−1% PFP
ND-ABC-2−2% PFP
164.5 8.5
117.5 9.7
126.3 8.0
178.9 7.4
163.0 11.1
159.1 6.2
2.31 0.08
2.50 0.05
3.07 0.11
2.98 0.10
3.59 0.09
4.41 0.11
162.0 6.5
111.9 6.8
135.3 7.1
187.3 10.8
167.0 9.0
166.8 4.3
2.96 0.07
2.58 0.04
3.00 0.12
2.98 0.09
4.09 0.11
4.85 0.12
−40.1 7.16
−39.9 7.55
−37.0 24.8
−24.5 6.71
−26.7 7.32
−38.8 13.6
a
Mode size represents the most abundant fraction in the nanodroplets solution identified and calculated using Nanoparticles Tracking Analysis and
NTA 2.3 build 127 software. Results are the average of six measurements of a minimum of three independent experiments standard error of the
b
mean. The average size of the nanodroplets is presented in Figure 3. Measured using 90Plus particle size analyzer with Zeta PALS capability at
room temperature.
measure the ζ potential of nanodroplets formulations, which
was in the range of −40.1 7.16 and −24.5 6.71 mV,
Vision Research) was positioned to image the plane of the
RBCs layer throughout the histotripsy treatment using a flash
lamp as a backlight for the phantom to record the bubble cloud
formed from the expansion of the embedded nanodroplets into
microbubbles as well as the resulting damage to the RBC tissue
phantom.
showing similar surface charge with most of the PEG
containing micelles49 and liposome surfaces50,51 (Table 2).
Ablation of Red Blood Cells in Tissue Phantoms.
Agarose tissue phantoms (1% w/v) with an embedded layer of
red blood cells (RBCs) have been used as model tissue
phantoms to investigate the ablative effects of histotripsy
therapy.44 To compare the effects of our nanodroplet-mediated
ablation to histotripsy alone at higher pressure, we treated RBC
agarose tissue phantoms (1% w/v) containing each of our
nanodroplet formulations (Figure 4). Briefly, the agarose
phantom was placed in the focal zone of a 32 element, 500
kHz transducer inside a degassed water tank at 37 °C (Figure
4A). A high-speed, 1 megapixel CCD camera (Phantom V210,
We hypothesized that ultrasound treatment of the embedded
nanodroplets will trigger acoustic droplet vaporization forming
microbubbles, which will expand reaching an average size >50
μm before they violently collapse, causing disruption of the
neighboring RBCs (Figure 4B). Disruption of the translucent
RBCs layer renders the agarose gel more transparent, which
provides visual evidence of cell ablation. Previous work has
demonstrated that damage to the RBC tissue phantoms directly
correlates to the tissue damage formed by histotripsy therapy.44
We tested our hypothesis by applying histotripsy pulses to the
center of the agarose gels at the RBC layer using a pulse
repetition frequency of 10 Hz at a peak negative pressure of
20.7 MPa. We embedded an equal number of nanodroplets
(2.36 × 108 droplets/mL) in the agarose gel to eliminate the
effect of droplets concentration on the observed ablation
behavior but rather focus on investigating the effect of polymer
composition (ABC-1 versus ABC-2) and PFP content (0%, 1%,
and 2% v/v) on ablation capacity to identify the most effective
formulation.
Results show that histotripsy application could not initiate a
bubble cloud from empty nanodroplets (i.e., 0% PFP loading)
prepared using ABC-1 and ABC-2 polymers at the applied
acoustic pressure (20.7 MPa), and no damage was observed in
the RBCs layer (Figure 5A). A similar histotripsy application to
tissue phantoms containing nanodroplets prepared using ABC-
1 polymer loaded with 1% and 2% v/v PFP, resulted in the
formation of a bubble cloud in the center of the gel and
formation of a corresponding transparent zone, confirming the
ablation of the RBCs (Figure 5A). The nanodroplets prepared
using ABC-2 polymer and loaded with 1% and 2% v/v PFP also
resulted in cavitation and lesion formation in response to the
histotripsy treatment (Figure 5A). We measured the surface
area of the lesions formed in each tissue phantom and plotted it
against nanodroplets composition and PFP content (Figure
5B). Results show that empty nanodroplets (i.e., 0% PFP
loading) caused no ablation to RBCs regardless of the polymer
composition used to formulate the nanodroplets.
Figure 4. (A) Schematic drawing of the experimental setup
incorporating a 500 kHz transducer attached to a motorized 3D
positioning system controlled using a PC console. The transducer
focus was aligned with the center of the red blood cell (RBC) layer
embedded in the agarose gel. Formation of a bubble cloud in response
to histotripsy treatment was monitored using high-speed optical
imaging (Phantom V210, Vision Research). (B) Schematic drawing
showing the expansion of the nanodroplets (100−350 nm) forming
microbubbles (>500 μm) before energetically collapsing and
mechanically fractionating neighboring cells.
This demonstrates that the loading of PFP in the droplets’
core is critical for bubble formation in response to histotripsy
treatment. Increasing PFP content from 1% to 2% v/v caused
an increase in the surface area of the ablation lesion from 0.67
0.23 to 5.60
0.52 mm2 (8.4-fold) for nanodroplets
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Figure 5. (A) Effect of 2-cycle histotripsy pulse (20.7 MPa) with a pulse repetition frequency of 10 MHz on the nanodroplets prepared using ABC-1
and ABC-2 polymers with different PFP loading and embedded with RBCs in agarose gels. Images were captured using high-speed, 1 megapixel
CCD camera (Phantom V210, Vision Research) capable of a maximum frame rate of 2000 fps and show the generated bubble cloud and the
corresponding transparent ablation zone. (B) The area of the ablation zone (mm2) observed after treatment of agarose gels using different
nanodroplet formulations calculated using MATLAB (The Math-Works, Natick, MA, USA). (C) Hemolysis of RBCs upon incubation with ND-
ABC-1−2% PFP and treatment with histotripsy pulse (20.7 MPa, pulse repetition frequency = 50 MHz) for 10 min. Statistical difference between
the area of the ablation zone for different nanodroplets was evaluated using student’s t test where * denotes p ≤ 0.05, ** denotes p ≤ 0.01, and ***
denotes p ≤ 0.001.
prepared using ABC-1 polymer. Similarly, increasing PFP
content in the nanodroplets prepared using ABC-2 polymer
from 1% to 2% v/v increased the surface area of the ablation
lesion from 0.37 0.17 to 1.59 1.86 mm2 (4.3-fold). It is
interesting to note that nanodroplets prepared using ABC-1
polymer exhibit higher ablation capacity compared to those
prepared using ABC-2 polymer, which can be attributed to
difference in the polymer’s capacity to encapsulate and retain
the loaded PFP. Specifically, ABC-2 polymer has an average of
13.5 units of the fluorinated monomer with the heptadeca-
fluorodecyl groups distributed along a long hydrophobic
backbone, which results in strong interaction of the hydro-
phobic block in the droplet’s core that limits PFP loading and
encapsulation.
Our results are supported by previously published reports
showing that the number of fluorinated monomers plays a
critical role in PFC loading and encapsulation by a polymeric
shell.11,25 For example, Yokoyama and co-workers synthesized a
series of amphiphilic PEG-b-poly(fluoroheptyl aspartate)
copolymers with a variable number of fluorinated alkyl chains
and investigated their ability to encapsulate PFC forming
nanodroplets.11,25 Results showed that introducing 10% of
fluoroheptyl units in the polymer backbone was sufficient to
encapsulate the loaded PFP (45.5 mg/mL) forming nano-
droplets. Incorporation of higher or lower percentages of
fluoroheptyl units in the polymer backbone reduced PFP
encapsulation by the polymer and negatively affected droplets
stability. Further, the increase in the number of fluorinated
carbon atoms in the fluoroalkyl chain above 9 decreased the
encapsulation of PFP. These results collectively show that the
nanodroplets formulated using ABC-1 polymer and loaded by
2% PFP exhibit the highest ablation capacity among all
formulations.
We also examined the effect of PFP-loaded nanodroplets
coupled with ultrasound on hemolysis of RBCs solution.
Specifically, the hemolysis ability of nanodroplets (ND-ABC-
1−2% PFP) at the same concentration used for phantom
experiment was evaluated as combined with histotripsy pulse at
a pulse repetition frequency of 50 Hz and a peak negative
pressure of 20.7 MPa (Figure 5C). The results showed that
PFP-loaded nanodroplets combined with ultrasound hemo-
lyzed 60.9% of RBCs in the solution while empty micelles
combined with ultrasound and ultrasound alone caused <1%
hemolysis of the RBCs.
Using ND-ABC-1−2% PFP nanodroplets, we investigated
the ability to generate a bubble cloud and ablate the RBCs in
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Figure 6. Effect of 2-cycle histotripsy pulse (11.0 MPa) with a pulse repetition frequency of 10 MHz on ND-ABC-1−2% PFP embedded with RBCs
in agarose gels. Images were captured using high-speed camera (Phantom V210, Vision Research) and show the generated bubble cloud and the
corresponding transparent ablation zone.
Table 3. Effect of Incubating Nanodroplets at 37°C for 24 h in Presence and Absence of Bovine Serum Albumin on Droplets
Size and Concentration
a,
b
a
a,c
nanodroplets code
mode size (nm)
mean size (nm)
concentration (1010 particles/mL)
ND-ABC-1−2% PFP (0 h)
ND-ABC-1−2% PFP (24 h)
ND-ABC-1−2% PFP+BSA (24 h)
ND-ABC-2−2% PFP (0 h)
ND-ABC-2−2% PFP (24 h)
ND-ABC-2−2% PFP+BSA (24 h)
131.3 10.9
142.0 10.5
144.5 13.9
160.0 4.4
152.6 4.3
159.0 7.8
237.35 6.1
243.9 6.0
242.6 4.1
207.6 3.2
207.0 3.3
209.6 2.5
1.98 0.09
1.81 0.06
1.93 0.07
3.87 0.17
3.45 0.16
2.94 0.12
a
b
Results are the average of six measurements of a minimum of three independent experiments standard error of the mean. Mode size represents
the most abundant fraction in the nanodroplets solution. Nanoparticles concentration is calculated using Nanoparticles Tracking Analysis and NTA
2.3 build 127 software.
c
Figure 7. Effect of incubating (A) ND-ABC-1−2% PFP and (B) ND-ABC-2−2% PFP nanodroplets for 24 h at 37 °C in the presence and absence of
bovine serum albumin (BSA) as a model serum protein on droplets size and concentration.
agarose gels at a lower acoustic pressure. We applied histotripsy
pulses at a pulse repetition frequency of 10 Hz and a peak
negative pressure of 11.0 MPa, which is 2.5-fold lower than the
intrinsic threshold of ∼26−30 MPa required to generate
cavitation using 2-cycle histotripsy pulses alone.52 Results show
the generation of a bubble cloud and a clear ablation zone with
sharp margins of the RBCs at this reduced acoustic pressure
(Figure 6). This clearly shows that PFP-loaded nanodroplets
can reduce the histotripsy cavitation threshold and be used to
generate lesions at significantly lower pressure than histotripsy
alone. Previous in vivo histotripsy study shows that tissue
fractionation is always formed when a cavitation bubble cloud is
generated and maintained in various organs such as
prostate,53,54 heart,32 kidney,55−57 and liver58 in large animals.
The observed reduction in the acoustic pressure necessary to
generate and maintain a bubble cloud coupled with the sharp
demarcation of the ablation zone exhibited by these nano-
droplets are critical properties that indicate their potential for
future in vivo use to allow selective ablation of specific lesions
(e.g., solid tumors) where the nanodroplets will accumulate
while neighboring healthy tissue will be preserved.
Stability of Nanodroplets. We investigated the stability of
nanodroplets prepared using ABC-1 and ABC-2 polymers and
loaded with 2% v/v PFP upon incubation for 24 h at 37 °C in
the presence and absence of bovine serum albumin (20 μg/
mL) as a model serum protein. Using NTA software, we
measured the mode and mean size for each formulation at the
beginning of the incubation time (0 h) and after 24 h along
with droplet concentration. Results show that the nanodroplets
prepared using ABC-1 polymer have an initial mode and mean
size of 131.3 10.9 and 237.35 6.1 nm, respectively (Table 3
and Figure 7A). Incubating the droplets at 37 °C for 24 h
caused a slight shift in mode size and mean size to 142.0 10.5
and 243.9 6.0 nm, respectively. Incubating the nanodroplets
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with BSA did not affect nanodroplets mode size (144.5 13.9
proved to generate a cavitation bubble cloud that ablates
neighboring RBCs at reduced acoustic pressure compared to
histotripsy alone. The increase in PFP content from 1% to 2%
v/v proved to increase the ablation lesion induced by the
nanodroplets. Further, the nanodroplets formulated using
ABC-1 appear to better encapsulate the loaded PFP and
exhibit higher ablation capacity compared to those formulated
by ABC-2 polymer at equal PFP loading. On the basis of the
surface area of the ablation zone, ND-ABC-1−2% PFP
nanodroplets exhibit effective ablation of RBCs at 2.5-fold
lower acoustic pressure than the intrinsic threshold required to
initiate the bubble cloud using the same 2-cycle histotripsy
pulses alone. Furthermore, ND-ABC-1−2% PFP nanodroplets
caused 60.9% of RBCs hemolysis comparing empty nano-
droplets and PBS solution under the same histotripsy and
physiological conditions. Further, these nanodroplets main-
tained their average size and concentration upon incubation
with BSA for 24 h at 37 °C, which prove their promise for
cancer cell ablation and warrant their future testing in vivo.
Moreover, the viability of PC-3 cells was not affected at any of
the tested concentration of nanodroplets, indicating their
biocompatibility.
nm) and mean size (242.6
4.1 nm), which indicates the
success of the PEG brush displayed on droplets surface in
suppressing BSA adsorption. Results also show that nano-
droplets concentration did not change throughout the 24 h
incubation time in presence or absence of BSA, which indicates
their stability against dissolution and aggregation. Analysis of
mode and mean size of the nanodroplets prepared using ABC-2
and encapsulated 2% v/v PFP show that they maintained their
size throughout the incubation time and in the presence of BSA
(Table 3 and Figure 7B). However, there was a 25% reduction
in droplet concentration upon incubation with BSA for 24 h,
which is probably due to droplet dissolution or poor PFP
encapsulation indicated by the low ablation capacity of these
droplets shown in Figure 5. These results show that ND-ABC-
1−2% PFP nanodroplets resist the adsorption of serum
proteins and are suited for in vivo testing.
Toxicity of Nanodroplets. Finally, we investigated the
toxicity of ABC-1 and ABC-2 polymers toward PC-3 prostate
cancer cells as a function of polymer concentration using the
MTT assay (ATCC, Manassas, VA) following manufacturer’s
specifications. Briefly, we incubated PC-3 cells with ABC-1 and
ABC-2 polymers formulated as empty nanodroplets (i.e., 0%
loaded PFP) for 24 h under normal culture conditions and
compared the percentage of viable cells at each polymer
concentration (0.3−75 μg/mL) to that observed for PC-3
cultured in normal culture medium (0 μg/mL polymer).
Results show that the nanodroplets did not affect the viability of
PC-3 cells at any of the tested concentrations (Figure 8),
ASSOCIATED CONTENT
■
S
* Supporting Information
Materials, characterization, synthesis of amphiphilic block
copolymer, preparation of precursors, thermal properties of
1
nanodroplets and its precursors, H and 13C NMR spectra,
GPC results, size and concentration changes, and ESEM
images. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*For M.E.S.: phone, 1-734-615-9404; fax, 1-734-647-4834; E-
mail, melsayed@umich.edu; website, www.bme.umich.edu/
centlab.php; address, University of Michigan, Department of
Biomedical Engineering, 2150 Lurie Biomedical Engineering
Building, 1101 Beal Avenue, Ann Arbor, Michigan 48109,
United States.
*For Z.X.: phone, 1-734-647-4961; fax, 1-734-939-1905; E-
mail, zhenx@umich.edu; website, www.bme.umich.edu/labs/
xulab; address, University of Michigan, Department of
Biomedical Engineering, 2107 Carl A. Gerstacker Building,
2200 Bonisteel Boulevard, Ann Arbor, Michigan 48109, United
States.
Figure 8. Evaluation of in vitro cytotoxicity of nanodroplets by MTT
assay. PC-3 prostate cancer cells were incubated with different
concentration of nanodroplets for 24 h.
Notes
The authors declare no competing financial interest.
indicating their biocompatibility. We did not evaluate the
toxicity of the PFP given that it is commonly used in FDA-
approved ultrasound contrast agents and its concentration in
the blood circulation drops by 90% within 10 min of its
administration.25
ACKNOWLEDGMENTS
■
This research is funded by grants from University of Michigan-
Prostate Cancer SPORE and Department of Defense-Prostate
Cancer Research Program (W81XWH-11-PCRP-ID) to Dr.
Mohamed ElSayed. Dr. Yasemin Yuksel Durmaz recognizes the
financial support of Susan G. Komen Breast Cancer
Foundation. Mr. Eli Vlaisavljevich recognizes the financial
support of the National Science Foundation Graduate Research
Fellowship.
CONCLUSIONS
■
To summarize, we have designed and synthesized two
amphiphilic PEG-b-(PAA)-b-P(HDFMA-co-MMA) polymers
(ABC-1 and ABC-2) that proved to encapsulate 1%−2% v/v
PFP, forming nanodroplets. Combining histotripsy pulses with
nanodroplets formulated using ABC-1 and ABC-2 polymers
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