Photoacoustic Imaging Quantifies Drug Release from Nanocarriers via Redox Chemistry of Dye-Labeled Cargo

Article abstract of DOI:10.1002/anie.201914120

We report a new approach to monitor drug release from nanocarriers via a paclitaxel–methylene blue conjugate (PTX-MB) with redox activity. This construct is in a photoacoustically silent reduced state inside poly(lactic-co-glycolic acid) (PLGA) nanopartic

Full text of DOI:10.1002/anie.201914120

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Accepted Article
Title: Photoacoustic Imaging Quantifies Drug Release from
Nanocarriers via Redox Chemistry of Dye-Labeled Cargo
Authors: Ananthakrishnan Soundaram Jeevarathinam, Jeanne E.
Lemaster, Fang Chen, Eric Zhao, and Jesse Jokerst
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201914120
Angew. Chem. 10.1002/ange.201914120
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10.1002/anie.201914120
Angewandte Chemie International Edition
Photoacoustic Imaging Quantifies Drug Release from Nanocarriers
via Redox Chemistry of Dye-Labeled Cargo
Ananthakrishnan Soundaram Jeevarathinam[a]‡, Jeanne E. Lemaster[a]‡, Fang Chen#[a,c], Eric
Zhao[a], and Jesse V. Jokerst[a,b,c]
*
*Corresponding Author
E-mail: jjokerst@ucsd.edu
[a].
La Jolla, CA, 92093, USA
[b]. Materials Science and Engineering Program, University of California, San Diego, 9500
Gilman Dr., La Jolla, CA 92093, USA.
[c]. Department of Radiology, University of California, San Diego, 9500 Gilman Dr., La Jolla,
Department of NanoEngineering, University of California San Diego, 9500 Gilman Drive,
CA 92093, USA.
‡ These authors contributed equally to this work.
# Current Address: Stanford University
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Abstract.
There have been remarkable advances in imaging drug nanocarriers, but there are few real-time
imaging strategies to determine if the cargo has been released from the carrier. This is important
because the pharmacokinetics and pharmacodynamics of the carrier can often be dramatically
decoupled from that of the cargo. Thus, new tools are clearly needed to image the timing and
quantity of drug release from nanocarriers. Here, we describe a simple strategy for photoacoustic
monitoring of drug release based on the redox chemistry of methylene blue, which offers
predictable redox chemistry: It can transition from the oxidized state with a bright blue color and
robust photoacoustic signal to the reduced state that the transparent with no photoacoustic signal.
We locked this drug-dye conjugate into a reduced state inside of a nanoparticle with no
photoacoustic signal. As the drug is released from the carrier, the dye is oxidized for quantification
with photoacoustic imaging. We first prepared paclitaxel-methylene blue conjugate (PTX-MB) with
strong absorbance at 640 nm and photoacoustic intensity proportional to its concentration. This
cargo was co-encapsulated in a poly(lactic-co-glycolic acid) nanoparticle with a dithiothreitol
reducing agent. The IC50 of PTX-MB-loaded NPs (PTX-MB @ PLGA NPs) was 78 µg mL-1. We
then used the redox reaction of PTX-MB to monitor its release from poly(lactic-co-glycolic acid)
(PLGA) nanoparticles (NPs). In vitro drug-release in phosphate buffer saline with 20% v/v normal
mice serum showed a 670-fold increase in photoacoustic signal. The particles showed an initial
burst release (25%) during the first 24 hours. After 24 hours, a sustained release was observed
through 120 hours leading to cumulative release of 40.6% of PTX-MB. In vivo drug release study
in mice for a duration of 12 hours showed a photoacoustic signal enhancement of up to 649%
after 10 hours. We then used this system to treat an orthotopic model of colon cancer via
luciferase-positive CT26 cells. Our data showed that tumor burden decreased by 44.7% ± 4.8%
when treated with the PTX-MB @ PLGA NPs versus the empty PLGA carrier. This work presents
a direct strategy to simultaneously monitor drug release biodistribution.
Keywords.
Photoacoustic imaging, PLGA nanoparticles, Cancer therapy, Drug-release, in vivo monitoring,
Paclitaxel, Image-guided drug delivery
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Introduction
Nano-based drug delivery can improve bio-distribution, enhance efficacy, and reduce toxicity.[1]
The therapeutic efficacy is highly dependent on the drug pharmacokinetics (PK) and
pharmacodynamics (PD), but such kinetics can be difficult to measure in vivo. Recently, the failure
of a liposomal cisplatin formulation was attributed to poor tumor penetration and insufficient free
drug release during the phase II clinical study.[2] Thus, monitoring bio-distribution of both the
nanoparticle carrier and their active pharmaceutical ingredient after systemic administration is a
top challenge in nanomedicine.[3] While periodic blood sampling coupled with LC-MS analysis[4]
will likely remain the gold standard for PK/PD, it does not offer anatomic information on the site
of release, timing, or release.[5]
Imaging is an important solution to this vexing problem. Radiolabeled analogues or fluorescent
tags can monitor bio-distribution after administration for image-guided drug delivery.[6] However,
these approaches are often “always on” and cannot answer questions about drug status, i.e.,
encapsulated or released from carrier. While FRET has been used as a strategy to monitor
release[7], fluorescence imaging suffers from poor resolution in deep tissue imaging. In contrast,
photoacoustic imaging offers high contrast imaging and can be easily co-registered to anatomic
ultrasound imaging.[8] It does not use ionizing radiation and offers video frame rates.[9] As a result,
there has been considerable interest in using photoacoustics in drug delivery. [10]Some recent
approaches using photoacoustics for drug delivery are core-satellite nanoparticle system and
polylactic acid-based particles to monitor distribution of carrier particles with photoacoustics.[11]
Here, we report a strategy to monitor drug release from a nanocarrier via photoacoustic imaging.
This strategy utilizes the popular ‘blue bottle reaction’ where the clinically approved methylene
blue (MB) switches between a colored oxidized form and a colorless reduced form.[12] Such
switching leads to “turn-on” photoacoustic signal as the MB is oxidized upon release from the
protective nanocarrier[13] even when covalently conjugated to paclitaxel. After preparing this drug-
dye conjugate, we evaluated the IC50 on CT26 cells and then used an in vivo model to validate
the imaging and therapeutic features of this construct.
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Materials and Methods
Reagents. Paclitaxel was purchased form Biotang Inc. (Massachusetts, USA). Polyvinyl alcohol
(PVA), polylactic-co-glycolic acid polymer (PLGA), succinic anhydride, ascorbic acid, and D,L-
dithiothretol (DTT) were purchased from Sigma Aldrich Inc. (Missouri, USA) and used without
further purification. Normal mouse serum was purchased from Fisher Scientific (Texas, USA). D-
Luciferin was purchased from Biosynth (Illinois, USA). RPMI 1640 media was purchased from
Sigma Aldrich Inc. (Missouri, USA). CT26-Luc cells were a kind gift from Prof. Nicole Steinmetz.
Instrumentation. Mass spectrometry of all intermediates was performed using the Micromass
Quattro Ultima Mass spectrometer using electrospray ionization mass spectrometer. Liquid
chromatography-mass spectrometry (LC-MS) analysis of paclitaxel methylene blue conjugate
(PTX-MB), 2’-succinimidylpaclitaxel (PTX-COOH), and hydroxyl-terminated methylene blue (III)
was performed using the Thermo LCQ deca-MS mass spectrometer equipped with HP1100 LC
1
station. The H NMR spectrum of PTX-MB was acquired on Bruker 300 NMR spectrometer in
deuterated chloroform medium. Transmission electron microscopy (TEM) micrographs of the
particles were recorded on a JEOL JEM 4000 transmission electron microscope. UV-Vis spectra
were recorded using Spectromax M5 plate reader using a 75 µl quartz cuvette. Photoacoustic
imaging was performed with a Vevo 2100 instrument (Visualsonics) with a 21 MHz-centered
transducer. IVIS imaging utilized a Perkin-Elmer Illumination system and LivingImage software.
The zeta potential and size of the particles was measured by dynamic light scattering (DLS)
(Zetasizer-90, Malvern Instruments).
Synthesis of precursors. PTX-COOH1 and the hydroxy-terminated dye (III), 3-(dimethylamino)-
7-((2-hydroxyethyl)(methyl)amine) phenothiazin-5-ium iodide2 were synthesized according to the
previously reported procedures.
Synthesis of paclitaxel-methylene blue conjugate (PAC-MB). In a typical reaction, hydroxy-
terminated dye (III) (M.W., 441.33 g/mol; 2.0 mg; 5.66x10-6 mol; 1.0 equiv.) and PTX-COOH
(M.W. = 953.97; 17.5 mg; 1.80x10-5 mol; 3.1 equiv.) were first dissolved in dry chloroform (4 mL)
under nitrogen atmosphere. Next, N,N’-dicyclohexylcarbodiimide (DCC) (M.W. = 206.33 g/mol;
4.5 mg ; 2.18x10-5 mol; 3.85 equiv.) dissolved in 1 mL of dry chloroform and N,N-
dimethylaminopyridine (M.W. = 122.17 g/mol; 0.4 mg; 1.63x10-6 mol; 0.57 equiv.) dissolved in dry
chloroform (400 µl) were directly injected into the reaction vessel. The mixture was stirred under
a nitrous atmosphere for 3 hours, and product formation was monitored with electrospray
ionization (ESI) mass spectrometry (MS). The crude reaction mixture was evaporated under
vacuum. Finally, the purified product was obtained by reverse phase high performance
chromatography (RP-HPLC) with a mobile phase of 30-60% acetonitrile in water with
trifluoroacetic acid (overall concentration 0.01% v/v). The fraction with product was collected and
1
lyophilized. H NMR (300 MHz, CDCl3) δ 0.5-1.3 (m, 13 H), 1.4-1.9 (m, 11H), 2.0-2.2 (m, 4 H),
2.2-2.7 (mm, 7H), 3.0-3.6 (m, 8H), 3.7-4.5 (m, 7H), 7.1-7.4 (m, 10H), 7.8-8.3 (m, 6H) ppm; MS
(LC-ESI) m/z calcd for C88H73N4O17S+ 1249.47; Found 1249.79. 1H NMR and liquid
chromatographic mass spectrometry (LC-MS) were used to analyze the yield of the reaction and
to study the purity.
Synthesis of PLGA nanoparticles with reduced PTX-MB (PTX-MB @ PLGA NPs). To prepare
the PLGA nanoparticles, poly(D,L-lactic-co-glycolic acid) (60 mg, Resomer RG 504 H; acid
terminated; MW 38000-54000; lactide:glycolide 50:50) and PTX-MB (M.W. 1377.29 g/mol; 2.0 mg;
1.46x10-6 mol) of PTX-MB were dissolved in chloroform (2 mL). Then, 1,4-dithiothreitol (DTT)
(M.W. 154.25 g/mol; 4.6 mg; 2.98x10-5 mol) was added dissolved in the mixture and
ethylenediamine (M.W. = 60.10 g/mol; 1 µL; 7.47x10-6 mol) was spiked into the solution. The deep
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10.1002/anie.201914120
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blue solution turned to colorless within 120 s. The polymer solution with PTX-MB was then mixed
with 3 mL of aqueous solution containing 2% polyvinyl alcohol (PVA) (M.W. range 31000-50000;
2 % w/v) and ascorbic acid (M.W. = 176.12 g/mol; 60 mM) by vortexing the mixture vigorously to
form an emulsion. The emulsion was then mixed with 9 mL PVA solution (2% w/v) and sonicated
with a probe sonicator for 5 minutes with 10 s bursts and a gap of 10 s between each burst. The
resulting white emulsion was mixed with 12 mL PVA solution (2% w/v) and stirred at 600 rpm
under a constant stream of nitrogen for 12 h to remove chloroform.
The PLGA NPs with PTX-LMB were isolated by centrifuging the emulsion at 14000 rpm for 10
minutes. The particles were washed twice with 60 mM aqueous solution of ascorbic acid and once
with pure Millipore water purged with nitrogen to avoid oxidation of the PTX-MB. The particles
were re-suspended in nitrogen-purged Millipore water with trehalose (7.5 mg mL-1)
(cryoprotectant) was added to the particle suspension3, 4. Finally, the suspension was frozen and
lyophilized at 0.02 mBar. The lyophilized particles with trehalose were stable for over a period of
3-4 months when stored in dark conditions at -20 °C. Experimental controls were similarly
prepared.
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In vitro study of drug release. PTX-MB @ PLGA NPs (10 mg) were suspended in deionized
water and divided into 10 equal portions in separate containers. The portions of PLGA NP
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suspensions were lyophilized and mixed with mouse serum (200 µL; 20 % v/v in PBS) in
phosphate buffer saline with sonication for 5 minutes. The particle suspensions were incubated
for 1, 3, 6, 12, 24, 48, 72, 96, or 120 hours. The particle suspensions were mixed initially for 10
minutes in a rotary mixer and then incubated at 38 °C. The samples were mixed for 10 minutes
every 24 h. The supernatants from each sample were placed in separate 0.86 mm polyethylene
tubes to acquire 3D photoacoustic image with excitation at 680 nm.
Cell culture. Colon cancer cell line CT26 labeled with luciferase were cultured using RPMI1640
media supplemented with fetal bovine serum (10%), L-glutamine (2 mM), sodium pyruvate (1mM),
and 1% antibiotic-antimycotic. Cells were incubated at 37 °C and media was replaced every 1-2
days. Cells were passaged at 80-90% confluence using Trypsin-EDTA (0.25 %)
Cytotoxicity. CT26 were plated in 96-well plates (1,250 cells/well) and incubated overnight. The
media was then replaced with 200 µl media with either PLGA or PTX-MB @ PLGA NPs at various
concentrations. The highest concentration of the nanoparticles was 864 µg mL-1 (corresponding
to 4000 nM PTX-MB released after 1 hour). The cells were incubated with the nanoparticle
suspension for 48 hours. A resazurin assay was used to determine the cell viability.
In vitro inhibitory effect of PTX-MB. The in vitro inhibitory effect of PTX-MB was tested with
CT26 colon cancer cells. The cells were plated in 96-well plates (2,500 cells/well) and incubated
overnight. PTX-MB, paclitaxel, and methylene blue were added at different concentrations to cell
media with 0.1% DMSO to assist the dissolution of the drugs. Live cells in media without DMSO
and dead cells were also included as controls. These cells were incubated 48 hours followed by
a Resazurin assay (Sigma).
Photoacoustic imaging of in vivo drug release. Mice were divided into two groups (control and
experimental groups). The PTX-MB @ PLGA NPs (25 mg mL-1) and blank PLGA NPs (25 mg mL-
1)were suspended in a mixture of matrigel, normal mice serum, and 1X PBS (50%, 10%, and 40%
respectively). The nanoparticle suspension (100 µL) was subcutaneously injected in each of the
mice of the experimental group. The photoacoustic image of the injected region on each of the
mice were obtained at 2, 4, 6, 8, 10, and 12 hours with 690 nm excitation. Mice in the control set
were injected with similarly prepared suspension containing blank PLGA NPs (100 µL, 25 mg mL-
1). The 3D photoacoustic images were analyzed on Image J to obtain the intensity. Statistical
analysis used t-tests via Microsoft Excel.
In vivo activity. Mice were divided into five groups and 250K CT26 cells in 50% matrigel and
PBS were injected in each mouse (day 1). On days 3, 5, and 7, the animals in each respective
group were injected with the following: PTX-MB @ PLGA NPs (10 mg/kg PTX equivalent), PTX
(10 mg Kg-1), PBS (100 µL), PTX (20 mg/kg), and PLGA nanoparticles (100 µL of 25 mg mL-1).
Animals were injected with luciferin (150 mg/kg in PBS) immediately prior to imaging and were
imaged on days 3, 5, 7, 11, and 14 to measure tumor volume.
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Results and Discussion.
We designed an imaging-based drug release monitoring strategy because direct monitoring of
drug release and drug distribution release remains a challenge.[7, 11a, 18] Therefore, a broadly
applicable strategy for monitoring drug release and local distribution is highly desirable. We
adopted a covalent labeling strategy where the methylene blue (MB) is linked to paclitaxel to form
a photoacoustically active conjugate (PTX-MB). Using the redox property of the MB moiety in
PTX-MB molecule, we chemically reduced it to a colorless, acoustically silent leuco form (PTX-
LMB) (no absorbance at 640 nm) and encapsulated it inside PLGA nanocarriers. Upon release
from PLGA NPs, the PTX-LMB spontaneously oxidized to acoustically active (PTX-MB with strong
absorbance at 640 nm) form.
Scheme 1. Design and synthesis of paclitaxel-methylene blue conjugate (PTX-MB). PTX-MB was
synthesized through a three step synthesis. The crude product was purified by reverse phase HPLC and
isolated as trifluoroacetate salt. The overall isolated yield after HPLC was 13.6%. Inset is a sample of PTX-
MB purified by HPLC.
Synthesis of PTX-MB.
The synthesis of PTX-MB is outlined in Scheme 1. We used the FDA-approved methylene blue
to label paclitaxel.[19] The 2’ hydroxy group on the paclitaxel molecule is reactive and is a versatile
moiety for functionalization.[14, 20] A reaction of paclitaxel with succinic anhydride in dry pyridine
resulted in the hemisuccinate form of paclitaxel (II) (see LC-MS in Figure S1 and S2) .[14] The
functionalization of either 2’ or 7 hydroxy groups in paclitaxel did not affect the cytotoxicity
significantly.[20-21] Hence, the functionalization of 2’ or/and 7 hydroxy groups in paclitaxel is used
to tag a target ligand and to make water-soluble derivatives.
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Figure 1. Structural, spectral and photoacoustic characterization of PTX-MB. (a) Mass spectrum of
HPLC purified sample of PTX-MB shows the molecular ion peak (m/z = 1249.79, C88H73N4O17S+). (b) The
photoacoustic signal intensity (ʎexc = 640 nm) shows a linear relationship with concentration of PTX-MB
(4.91x10-5 to 4.91x10-6 mil L-1 in PBS containing 20% v/v mice serum, R2 = 0.87). (c) The absorbance
spectrum of PTX-MB (black square) and PTX-LMB (red circle) (7.3x10-5 M in PBS with 20% v/v mice serum)
shows approximately 20-fold difference in absorbance at 640 nm. Inset in (c) shows photos of 7.3x10-5 M
solutions of PTX-MB (blue) and PTX-LMB (faint blue). (d) The photoacoustic spectra shows an increase in
photoacoustic intensity in the PTX-MB spectrum from 680 – 760 nm. (e) Photoacoustic imaging of PTX-MB
and PTX-LMB solutions (7.3x10-5 M in PBS with 20% mice serum) shows a 6.88-fold increase of PTX-MB
at 680 nm. The inset in (e) is the photoacoustic image of a phantom. (f) The illustration shows the redox
switching between PTX-MB and PTX-LMB leading to photoacoustic signal. Scale bars = 2 mm.
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[15]
We synthesized PTX-MB through the ester formation between compounds (II) and (III).
The
yield of crude product before HPLC purification was found to be 53.8% (Figure S5 & S6). After
reverse phase HPLC purification, a purity of 93.6% was achieved and PTX-MB was isolated as
trichloroacetate salt with isolated yield of 13.6% (see Figure S7 & S8).
1
The structure of the PTX-MB was confirmed by H NMR and ESI-MS. The ESI-MS spectrum
showed a distinct peak at 1249.79 corresponding to the cationic part of PTX-MB (corresponding
to C88H73N4O17S+) (Figure 1(a)). PTX-MB showed low solubility in pure water and PBS similar to
pure paclitaxel (4 µg mL-1).[22] Methylene blue has a higher solubility in water of 50.53 mg mL-1.
[22-23] The maximum solubility of PTX-MB in 20% v/v mice serum (in PBS) was 4.18 x 10-5 mol L-
1. This increased solubility of PTX-MB in 20% v/v mice serum (in PBS) is attributed to the 10-fold
higher solubility of paclitaxel in the presence of serum protein. [22]
A calibration of absorbance of PTX-MB (at 640 nm) versus the concentration in chloroform and
PBS with 20% v/v mouse serum was used for this purpose (Figure S9). Then we confirmed that
the photoacoustic intensity was directly proportional to the concentration of PTX-MB in 20 % v/v
mouse serum (in PBS) (Figure 1(b)). We conducted linear regression analysis and calculated a
p-value of <0.05; therefore the photoacoustic signal was dependent on the concentration. The R2
value for linear fit was calculated to be 0.87. A deviation in linear relationship of photoacoustic
signal versus the concentration is attributed to changing aggregation dynamics of PTX-MB with
concentration. Aggregation of methylene blue can cause modulation of photoacoustic signal.[25]
Optical and acoustic characterization of PTX-MB.
Methylene blue is known to undergo a redox reaction to transform between the oxidized
methylene blue (MB, ʎmax = 640 nm, photoacoustically active) form and reduced leuco methylene
blue form (LMB, ʎmax = 256 nm, photoacoustically silent) under ambient conditions.[12-13] MB
dissolved in 1x PBS at room temperature can be reduced by a wide range of reducing agents.[26]
This is attributed to the reduction of the sulfur atom in the phenothiazonium ring. A similar property
is expected in MB moiety of PTX-MB. We found that 7.3x10-5 M PTX-MB is reduced completely
by 3.2x10-2 M DTT in presence of 0.05 % v/v trimethylamine according to absorbance spectra
(Figure 1(c)). At 7.3x10-5 M concentration, the photoacoustic contrast (ʎexc = 690 nm) between
PTX-MB and PTX-LMB (formed using 3.2x10-2 M DTT) was 6.88 (Figure 1 (d) & (e)). The redox
switching of PTX-MB to PTX-LMB is illustrated in Figure 1(f).
Nanoparticle Synthesis and Characterization.
Next, we prepared and characterized PTX-MB @ PLGA NPs. Previous studies showed the leuco
form of methylene blue was stable inside a polymer matrix due to absence of oxygen within the
matrix.[27] We reacted PTX-MB with DTT in chloroform containing PLGA and encapsulated the
PTX-LMB in PLGA NPs by emulsification. DTT reduces the PTX-MB to PTX-LMB in the
chloroform medium and then is solubilized in aqueous medium. DTT can also be removed
completely after reduction and encapsulation of PTX-MB in PLGA NPs due to its higher solubility
of DTT in water (50 mg mL-1) than chloroform (10 mg mL-1).[26a] Thus, DTT can be easily removed
during particle synthesis and washing. During the particle synthesis, a 60 mM aqueous solution
of ascorbic acid (in PBS) purged with argon was used as aqueous medium to prevent
spontaneous oxidation of PTX-LMB. The particles were characterized for size distribution using
SEM and TEM. The size range of the PTX-LMB loaded particles was determined to be ~50 nm
according to TEM (Figure 2 (a)) and 100-200 nm according to SEM (Figure 2(b)). The difference
of observed particle size between TEM and SEM is attributed to the different method of sample
preparation and shrinking of pores in PLGA NPs under high vacuum in TEM.[28]
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The PLGA nanoparticles with encapsulated PTX-LMB were lyophilized and found to contain 0.018
mg (13.3 nanomol) of PTX-MB in 1.0 mg of PLGA NPs. The PTX-LMB-loaded NPs were blue in
color due to surface oxidation of PTX-LMB. However, dissolution of PTX-LMB loaded PLGA NPs
in chloroform resulted in a colorless solution (no absorbance at 640 nm). When the solution was
mixed with 100 mM potassium hexacyanoferrate (III), the solution turned dark blue with strong
absorbance at 640 nm due to oxidation. The color change was also observed even when the
solution of PTX-LMB loaded PLGA NPs (in chloroform) was exposed to air for at least 30 min.
The particles were prepared and stored as a lyophilized powder for an average of 90 days at -20
°C protected from light. Using absorbance, the percentage of conjugate existing as PTX-LMB
form inside PLGA NPs was calculated to be 94.82% (Figure 2(c)). Using absorbance, the
percentage of conjugate existing as PTX-LMB form inside PLGA NPs was calculated to be
94.82% (Figure 2(c)). The color change was also observed even when the solution of PTX-LMB
loaded PLGA NPs (in chloroform) was exposed to air for at least 30 min. Due to low redox
potential of methylene blue moiety, the PTX-LMB after release (acoustically silent) is
spontaneously oxidized to PTX-MB (acoustically active) under physiological pH and normoxic
conditions.[29]. However, when PTX-LMB is encapsulated in PLGA NPs, it is stabilized due to
absence of oxygen in the PLGA matrix.[27] Thus, the release of PTX-LMB from particles can lead
to spontaneous oxidation and turn-on photoacoustic signal. The zeta potential of the particles was
found to be 4.23 ± 0.35 mV in Millipore water. The hydrodynamic diameter of the PLGA NPs was
found to be 385.5 ± 27.5 nm.
Figure 2. Preparation and characterization of PLGA nanoparticles encapsulating PTX-LMB. (a) TEM
micrograph of PTX-LMB-loaded PLGA nanoparticles shows average diameter of 50 nm. (b) SEM
micrograph of PTX-MB @ PLGA nanoparticles indicates a diameter of 100-200 nm (scale bar is 400 nm).
The difference in size observed between TEM and SEM is due to shrinking of pores in PLGA NPs during
TEM (c) Absorbance spectra of the freshly prepared solution of PTX-MB @ PLGA NPs (15 mg mL-1) in
chloroform (black squares) shows no absorption at 640 nm and the same solution absorbs strongly at 640
nm after oxidation with potassium hexacyanoferrate (III) (red circles). Using the absorbance data from (c),
94.82% of the loaded PTX-MB existed was remained as PTX-LMB.
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Figure 3. In vitro drug release and cancer inhibitory activity of PTX-MB and PTX-MB @ PLGA NPs.
(a) The in vitro drug release profile of PTX-MB @ PLGA NPs (5.0 mg mL-1 in 20% v/v mice serum in PBS)
shows that 40.6±5.2% of PTX-LMB is released at the end of 120 h. The inset in panel (a) shows the
photograph of aliquots under visible light. (b)The photoacoustic intensity of drug release aliquots show a
669.9-fold increase in signal after 120 h. Particles isolated from these aliquots did not show significant
increase in photoacoustic signal (see Figure S3). Scale bar = 2 mm. (c) The photoacoustic signal is linearly
related to the percentage of PTX-MB (R2 = 0.92). (d) The TEM image of particles collected after 120 h of
drug release shows no change in size compared to as-prepared NPs shown in (a). (e) The in vitro activity
of PTX-MB on CT26 colon cancer cells shows that the IC50 of PTX, PTX-MB, and MB are 68, 447, and
1281 nM respectively. (f) Cancer inhibitory activity of PLGA NPs loaded with PTX-LMB shows an IC50 of
78 µg mL-1 corresponding to 362 nM PTX-MB released after 1 hour of release. Error bars are the standard
deviations of four replicates.
In vitro drug release and anticancer inhibitory activity.
Next, we studied the release of PTX-MB from the carrier and the in vitro anticancer activity against
CT26 cells. The PTX-LMB loaded PLGA NPs were studied for drug release in 20% v/v mice serum
in PBS. The release profile showed an initial burst release followed by a more gradual release of
PTX-LMB. We collected aliquots from 0-120 h of drug release; the absorbance showed an 8-fold
increase at 640 nm while the corresponding photoacoustic signal showed an increase of 669.9-
fold after 120 h corresponding to 40.6±5.2% release (Figure 3(a) & (b)). The photoacoustic signal
showed a linear increase with respect to the percentage drug released (Figure 3(c)). The
increased absorbance and photoacoustic signal are attributed to the release of PTX-LMB and its
oxidation to PTX-MB.[25, 30]
The PLGA NPs isolated from the aliquots of drug release did not show any significant change in
photoacoustic signal. Thus, the signal increase is attributed to the released conjugate (see Figure
S10). The total of 40.6±5.2% of the loaded drug was found to be released after 120 h.[31] The
aliquot collected at 120 h did not show detectable amounts of DTT according to mass
spectrometry (Figure S12). No significant change in particle size was observed in TEM after the
release time of 120 h (Figure 3(d)).
PTX-MB displayed significant anticancer activity against CT26 colon cancer cells. The IC50
values of paclitaxel (PTX) and PTX-MB were found to be 68 nM and 447 nM against CT26 cells.
The reduced cancer inhibitory activity of PTX-MB is also observed in other paclitaxel conjugates.
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Next, PTX-LMB-loaded PLGA NPs were studied for cancer inhibition against CT26 cells. The
IC50 of PTX-LMB loaded PLGA NPs was 78 µg mL-1. The IC50 of PTX-LMB loaded PLGA NPs
corresponds to 362 nM PTX-MB released after 1 hour. The difference in IC50 of released drug is
attributed to the difference in media used during the in vitro drug release study.
Figure 4. Photoacoustic in vivo drug release monitoring in mice. Images a-f show photoacoustic
images of the subcutaneous injections of mice injected with the 25 mg mL-1 dispersion of PLGA
nanoparticles loaded with PTX-LMB. The experiment was performed in triplicate. (h) The photoacoustic
signal intensity increased 168-649% from 0-8 h. Error bars represent the standard error. Scale bar = 2 mm.
The cytotoxicity of paclitaxel conjugates with 2’-succinyl ester linkage were found to be lower than
free paclitaxel.[32] We attribute this to the stability of the free PTX-MB in media and intracellular
environment throughout the 48h duration of in vitro cytotoxicity study. In addition, adding a
pendant group could obviously affect the activity of the drug. The decomposition of paclitaxel 2’-
succinyl ester were observed only after 48 hours due to acidic pH and enzyme reaction in
intracellular environment.[32] However, the dye-drug conjugate did have inherent activity against
cancer cells when studied in vitro, and thus cleavage of the dye-drug product is not a requisite for
anti-cancer activity (Fig. 3e).
In vivo drug release and anticancer activity.
Finally we studied the release of PTX-MB from carrier and antitumor activity in murine models.
We studied the PTX-MB @ PLGA NPs for in vivo release and anticancer activity. One set of the
experimental group was subcutaneously injected with PTX-MB @ PLGA NPs (25 mg mL-1) and
one control set of mice was subcutaneously injected was injected with blank PLGA NPs (25 mg
mL-1) in a similar mixture of matrigel, PBS, and mice serum. The in vivo drug release was
monitored using 3D photoacoustic images (ʎexc= 680 nm). The images acquired in experimental
and control groups (at 2, 4, 6, 8, 10 and 12 hours) showed obvious signal increase in the range
of 168-649% between 0-8 h. After 8 hours the photoacoustic signal showed irregular change due
the possible clearance of the released PTX-MB (Figure 4 (a-h) and supporting information
Figure S13).
Finally, we used a murine cancer model to determine the efficacy of PTX-MB @ PLGA-DTT NPs.
Mice with metastatic colon cancer were divided into five groups: PTX-MB @ PLGA-DTT (PTX-
MB), PTX (10 mg/kg), PBS only, PTX (high, 20 mg/kg), and PLGA particle only. Mice treated with
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PTX-MB @ PLGA NPs (10 mg/kg equivalent paclitaxel) exhibited a 44.7% ± 4.8% decrease in
tumor radiance on Day 14 compared to mice treated with the PLGA nanoparticle alone (Figure
5(a) & (b)). Additionally, mice treated with PTX-MB encapsulated in PLGA-DTT NPs exhibited no
significant weight loss or observable behavioral changes from treatment. One of the mice
receiving PTX (high) exhibited high weight loss >10%, possibly due to toxic effects from a high
dose of PTX (20 mg/kg). Fonseca et al. suggested that paclitaxel encapsulated nanoparticles
caused a reduction in side effects and toxicity due to improved tissue distribution and
pharmacokinetics[33]. Additionally, a previous study showed that a dose of 32.5 mg/kg of paclitaxel
administered in 3 consecutive days caused 100% mortality in a murine model[34] consistent with
our findings that a high dosage of paclitaxel can cause adverse side effects such as weight loss.
Figure 5. In vivo efficacy of PTX-MB @ PLGA NPs. (a) Nu/nu mice were divided into 5 groups (n=5).
250,000 luminescent CT-26 cells were injected IP on Day 1 in all mice. (a) The experimental illustration
shows the schematic. Each mouse received the following doses IP on days 3, 5, and 7: PTX-MB @ PLGA
NPs (PTX-MB) injection, PTX (10 mg/kg), PBS only, PTX (10 mg/kg), PTX (high, 20 mg/kg), and PLGA
particle only. (b) The tumor radiance plotted against time (days) shows that mice treated with PTX-MB @
PLGA NPs(10 mg/kg equivalent Paclitaxel) exhibited a 44.7% ± 4.76% decrease in tumor radiance on Day
14 compared to mice treated with the blank PLGA NPs. Error bars represent the standard error within
treatment groups.
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Conclusion.
We developed a photoacoustically-activatable probe for drug release monitoring based on the
blue-bottle reaction of methylene blue. Furthermore, we demonstrated the utility of the nanodrug
formulation in live mouse models. In contrast to the previous reports, this strategy uses a direct
photoacoustic increase from the drug tagged with an acoustically activatable chromophore.[7a, 11a,
18] The conjugated paclitaxel as well as the nanoparticle formulation with paclitaxel exhibits in vitro
and in vivo cancer inhibitory effects.
This approach is novel for two reasons. First, this approach of covalently conjugating an
activatable photoacoustic probe can be used in different drug molecules with different chemistry.
Second, the absorbance of the tagged chromophore can be changed to increase the depth of
imaging using photoacoustic technique by covalent modification of the chromophore.[35] The
phenothiazine-based chromophores have good photostability and can be dependable contrast
agents in imaging against photobleaching and loss of signal.[35b]
For PTX-MB, the depth of imaging is currently limited due to limited tissue penetration of 680 nm
laser. However methylene blue can be modified to absorb in near infrared wavelengths to improve
tissue penetration. The current approach of monitoring the in-vivo drug release can be combined
with other contrast modes such as ultrasound or magnetic resonance to locate the drug delivery
site.[36] This is a powerful tool to monitor the location and concentration of drugs as well as insight
on their status relative to nanocarriers. Our long-term vision is to use this approach for
intraperitoneal chemotherapy, which gives the drug as bolus into the peritoneum. This is a
relatively closed system and ideally suited for image-guided drug delivery.
14
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Acknowledgements.
The authors acknowledge the University of California Cancer Research Coordinating
Committee grant CRN-19-582184. J.E.L. acknowledges funding from the National Institutes of
Health (NIH) Institutional National Research Service Award T32 CA153915, Cancer Researchers
in Nanotechnology (Zhang). J.V.J. acknowledges funding from the NIH (Grants R00 HL117048
and DP2 HL137187) and infrastructure from Grants S10 OD021821 and S10 OD023555. The
transmission electron micrographs were taken in the Cellular and Molecular Medicine
Electron microscopy core facility which is supported in part by National Institutes of Health Award
number S10 OD023527. This work was performed, in part, at the San Diego Nanotechnology
Infrastructure of UCSD, a member of the National Nanotechnology Coordinated Infrastructure,
which is supported by the National Science Foundation (Grant ECCS-1542148). The in
vivo imaging studies using the IVIS system were conducted at the UCSD Screening Core. The
authors would like to thank Nicole Steinmetz, PhD and Chao Wang, PhD for their gracious supply
of CT26-Luc cells. The authors express their thanks to Erkki Ruoslahti, M.D., Ph.D and Venka
Ramana Kotamraju, Ph.D for their help in performing HPLC purifications.
15
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Table of Contents
A covalently linked paclitaxel-Methylene blue conjugate (PTX-MB) is used as a self-
indicating photoacoustic contrast agent to monitor real-time drug release using
photoacoustics. The PTX-MB reduced to acoustically silent form and encapsulated in
PLGA nanoparticles. After release, PTX-MB instantly oxidized to acoustically active form
indicating the real-time drug release and distribution.
17
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