Bioorthogonal Uncaging of Cytotoxic Paclitaxel through Pd Nanosheet-Hydrogel Frameworks

Article abstract of DOI:10.1021/acs.jmedchem.0c00781

The promising potential of bioorthogonal catalysis in biomedicine is inspiring incremental efforts to design strategies that regulate drug activity in living systems. To achieve this, it is not only essential to develop customized inactive prodrugs and biocompatible metal catalysts but also the right physical environment for them to interact and enable drug production under spatial and/or temporal control. Toward this goal, here, we report the first inactive precursor of the potent broad-spectrum anticancer drug paclitaxel (a.k.a. Taxol) that is stable in cell culture and labile to Pd catalysts. This new prodrug is effectively uncaged in cancer cell culture by Pd nanosheets captured within agarose and alginate hydrogels, providing a biodegradable catalytic framework to achieve controlled release of one of the most important chemotherapy drugs in medical practice. The compatibility of bioorthogonal catalysis and physical hydrogels opens up new opportunities to administer and modulate the mobility of transition metal catalysts in living environs.

Full text of DOI:10.1021/acs.jmedchem.0c00781

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Article
Bioorthogonal Uncaging of Cytotoxic Paclitaxel through Pd
Nanosheet−Hydrogel Frameworks
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Ana M. Perez-Lopez, Belen Rubio-Ruiz, Teresa Valero, Rafael Contreras-Montoya,
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Luis Alvarez de Cienfuegos, Víctor Sebastian, Jesus Santamaría, and Asier Unciti-Broceta*
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ABSTRACT: The promising potential of bioorthogonal catalysis in
biomedicine is inspiring incremental efforts to design strategies that
regulate drug activity in living systems. To achieve this, it is not only
essential to develop customized inactive prodrugs and biocompatible
metal catalysts but also the right physical environment for them to
interact and enable drug production under spatial and/or temporal
control. Toward this goal, here, we report the first inactive precursor of
the potent broad-spectrum anticancer drug paclitaxel (a.k.a. Taxol) that
is stable in cell culture and labile to Pd catalysts. This new prodrug is
effectively uncaged in cancer cell culture by Pd nanosheets captured
within agarose and alginate hydrogels, providing a biodegradable
catalytic framework to achieve controlled release of one of the most
important chemotherapy drugs in medical practice. The compatibility of
bioorthogonal catalysis and physical hydrogels opens up new opportunities to administer and modulate the mobility of transition
metal catalysts in living environs.
round-bottom flasks to cell culture plates and animals.
Ruthenium-,12−17 palladium-,15−31 gold-,32−34 copper-,35,36 or
iron-based37,38 catalysts have demonstrated their compatibility
with saline aqueous solutions and their capacity to mediate
chemo-specific processes in complex biological systems. The
emerging field of bioorthogonal catalysis has enriched the
wealth of methods available to label biomolecules,20,21 release
cytotoxic drugs,14,17,24,26,38 or modulate biological func-
tions.15,16,23,31 In this context, our lab has contributed to the
use of palladium (Pd) as a heterogeneous catalytic system to
generate clinically approved anticancer drugs in living
environs.24,25,39−43 Such an approach aims to achieve increased
control over when and where prodrugs are converted into
active drugs by exploiting activation strategies that do not rely
on metabolic reactions.44,45 To date, nondegradable materials
have been used to capture Pd nanoparticles and protect them
from the biological milieu, meaning that the devices will stay at
the affected tissue even after completing their catalytic
function.41 This situation is certainly useful for cancers that
tend to relapse at the same location, but not quite as much for
INTRODUCTION
■
Despite the growing impact of molecularly targeted therapies
in cancer, chemotherapy still stands as the main pharmaco-
therapeutic choice to treat most kinds of solid malignancies.1−3
The third-generation cytotoxic drug paclitaxel (PTX) is one of
the most widely used chemotherapeutics4,5 and the anticancer
drug currently enrolled in more active clinical trials (>1100).6
First isolated from the bark of the Pacific yew tree, the
initiation of PTX’s phase 2 trials in the 1980s raised ecological
concerns over the impact on yew populations and sparked the
renowned race to complete its total synthesis.5,7 Nowadays,
PTX is produced by a semisynthetic protocol from sustainable
sources7,8 and globally used, alone or in combination, in the
treatment of many of the most prevalent types of cancers,
including breast, ovarian, and lung cancers.4,5 There are,
however, important dose-limiting adverse effects associated
with PTX therapy (myelosuppression, peripheral neuropathy,
and cardiac toxicity) that restrains its systemic anticancer
efficacy. For this reason, over the last decades, much research
on PTX has been steered toward developing efficient drug
delivery systems that improve its clinical tolerability8,9 and low-
toxicity prodrugs.10,11 Innovative methods to localize drug
activity at the tumor site are still essential to maximize the
clinical potential of PTX, not least because of its ever-growing
role in chemotherapy regimens.
Received: May 8, 2020
Published: August 5, 2020
The catalytic scope of some of the transition metals most
commonly used in chemistry labs has recently expanded from
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a
Scheme 1. Proposed Pd-Mediated Activation Strategy in Cell Culture (Left) and Synthesis of Pro-PTX (Right)
a
Cell culture: Pd-mediated O-depropargylation of Pro-PTX is thermodynamically favored by the generation of CO2 and designed to trigger an
intramolecular cyclization event that releases PTX and 1,3-dimethyl-2-imidazolidinone. The dealkylation step in water is expected to afford
nontoxic 1-hydroxyacetone.24 Reaction byproducts could not be confirmed by LCMS. Chemistry lab: Carbamate-protected Pro-PTX is
semisynthetically prepared from PTX in two steps by successive reactions with p-nitrophenyl chloroformate and N-(propargyloxycarbonyl)-N,N′-
dimethylethylenediamine, 4.
short-lived neoadjuvant therapies or patients with multiple
tumor foci. Hence, it would also be desirable to develop
implantable systems that securely host Pd nanoparticles to
induce local prodrug activation for a limited period of time
and, afterward, degrade and naturally eliminate from the
organism.
Here, we report the development of a novel PTX precursor
that exhibits up to 700-fold lower activity than the parent drug
and incorporates a Pd-sensitive masking group (Scheme 1). In
the presence of Pd catalysts, a rapid depropargylation reaction
takes place to trigger the release of PTX. In addition, we report
the first investigation on the use of physical hydrogels to
capture Pd catalysts and their capacity to carry out
bioorthogonal uncaging reactions under physiological con-
ditions.
mate, followed by Boc deprotection under acidic conditions
provided compound 4 in a high yield (90%, two steps). Finally,
coupling of 1 and 4 in the presence of DIPEA generated Pro-
PTX in a moderate yield (36%).
Cytotoxicity Study: PTX vs Pro-PTX. To evaluate the
inactivation strategy, viability assays were performed in three
cell lines: non-small-cell lung carcinoma A549 cells (cell model
of human cancer clinically treated with PTX),4,5 human
glioblastoma U87 cells (model of aggressive brain cancer),49
and human brain vascular pericytes (HBVPs, noncancerous
cells that are critical components of the blood−brain barrier49
and similar to those found in peripheral nerves). Cells were
treated with PTX and Pro-PTX at a range of concentrations
(up to 10 μM) and viability measurements were carried out
after 5 days of treatment. Results were normalized to the
untreated cell control (1% dimethyl sulfoxide (DMSO)) and
half-maximal effective concentration (EC50) values were
calculated from the generated dose−response curves (see
Figure 1). As expected, PTX treatment induced a very potent
cytotoxic effect against the two cancer cell lines, with EC50
values in the range of 2−3 nM. In contrast, Pro-PTX did not
show activity at most concentrations tested in these cell lines,
with reduced cell viability only seen at doses ≥1 μM and
exhibiting an EC50 value of 1.75 μM for A549 and 0.54 μM for
U87 cells (Figure 1a,b). This vast drop of anticancer activity
(200- to 700-fold lower than PTX) highlights the essential role
of the C2′-OH of PTX for binding its target (lumenal side of
microtubules)4,5 and the stability of the masking group to
enzymatic cleavage and nucleophilic attacks by intracellular
biomolecules. As expected, PTX and Pro-PTX displayed much
lower cytotoxicity in low-proliferating HBVPs (Figure 1c).
Under treatment with PTX, cell proliferation was reduced to
75% at concentrations ≥1 μM, with an EC50 value of 0.047
μM. The antiproliferative effect of Pro-PTX in HBVPs was
negligible at most concentrations (EC50 = 4.4 μM), which is in
agreement with an increased safety profile relative to its parent
drug.
RESULTS AND DISCUSSION
■
Design and Synthesis of a Biochemically Stable Pd-
Sensitive Prodrug of PTX. The structure−activity relation-
ships of PTX and its analogues have been the subject of
intensive research.46,10 It is well established that one of the
essential groups for their bioactivity is the secondary alcohol at
the C2′ position of the side chain. Masking of this aliphatic
OH with various chemical groups (carbonates, esters,
carbamates, etc.) has been used to develop PTX prodrugs
for different purposes.47,10 In light of previous studies25
showing that carbamate-based masking groups exhibit
significantly higher stability in cell culture than carbonate
ones and the need to generate a prodrug that endures the cell
metabolism, a biscarbamate group connected through an N,N′-
dimethylethylenediamine spacer48 was used to mask the C2′-
OH of PTX (Scheme 1). To provide sensitivity to Pd catalysis,
an O-propargyl moiety was incorporated at the terminal
carbamate that, after cleavage, triggers a self-immolative
cascade resulting in the release of PTX.
Pro-PTX was prepared following the semisynthetic route
described in Scheme 1 (see the synthetic procedure in the
Experimental Section and NMR spectra in the Supporting
Information). PTX was reacted with p-nitrophenyl chlor-
oformate in the presence of pyridine to incorporate a reactive
carbonate group at its C2′-OH position. The reaction of N-
Boc-protected ethylenediamine 2 with propargyl chlorofor-
Prodrug-into-Drug Conversion Studies. Next, the
sensitivity of the prodrug to Pd under biologically relevant
conditions was tested by incubating Pro-PTX at 37 °C in PBS
(pH = 7.4, isotonicity) with or without a polymer-supported
Pd catalyst, 30 μm of Pd devices41 (which can be easily filtered
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bioorthogonal reactions when they are properly protected
inside exosomes.30 Because of their minute size (<2 nm in
thickness), these ultrathin metallic structures could be
precisely injected at desired anatomical sites (e.g., inside
small tumor lesions) and, in time, be eliminated from the
organism by the renal and/or hepatobiliary routes,51 features
that are optimal for short-lived therapeutic applications.
However, such small dimensions also make them capable of
permeating through most biological barriers, which would
defeat the purpose of catalyzing bioorthogonal reactions
exclusively at the point of implantation.
Hydrogels are insoluble porous networks with high water
contents (up to 95%) that have been extensively used as drug
reservoirs in controlled-release systems.52 However, the
potential use of physical hydrogels as biocompatible scaffolds
for bioorthogonal catalysts has never been tested before.
Among the broad variety of gelling agents available, the FDA-
approved natural polysaccharides agarose and alginate are
especially attractive for medical and pharmaceutical applica-
tions because of their biodegradable and nonthrombogenic
nature.53,54 Driven by the idea of creating implantable catalytic
devices capable of uncaging Pro-PTX and feature the potential
to gradually biodegrade over time, we decided to investigate
the use of agarose- and alginate-based hydrogels as scaffolds for
the immobilization of Pd nanosheets.
Pd nanosheets were prepared using a new method (adapted
from previous work30,55,56) consisting of treating an aqueous
solution of Na2PdCl4, poly(vinyl pyrrolidone), and KBr to a
CO atmosphere in a high-pressure reactor (6 bar) at 80 °C for
40 min (see the full protocol in the Experimental Section).
Low-toxicity reagents were used to increase the biocompati-
bility of the metallic nanostructures, whereas CO was used as a
reductant and capping agent to control the shape and size of
the nanoparticles.55,56 TEM analysis showed the planar and
homogeneous shape of the resulting structures (Figure 3a,b),
which displayed an average width and thickness of 12 2.2
Figure 1. Nine-point semilog dose−response curves and calculated
EC50 values for (a) A549 cells, (b) U87 cells, and (c) HBVPs after 5
days of treatment with PTX and Pro-PTX at concentrations ranging
from 0.001 to 10 μM. Error bars: SEM from n = 3.
off to facilitate the analysis of the reaction mixture). Reactions
were monitored by MS. While the prodrug remained intact in
the absence of Pd (Figure 2a), reaction analysis showed the
complete disappearance of the prodrug after 10 h incubation
with Pd devices and the formation of major mass peaks
corresponding to PTX (Figure 2b). The observation of the
[M2 + H]+ peak in the mass spectrum, which corresponds to
the amino derivative 5 (highly ionizable pre-immolation
intermediate), is in agreement with the proposed prodrug
activation mechanism.
Biodegradable Solid Supports for Pd Catalysts.
Bidimensional Pd nanostructuresalso called Pd nano-
sheetshave been reported to display good biocompatibility
in culture and in vivo50 and, more recently, shown to catalyze
and 1.4
0.1 nm, respectively, making them a priori good
candidates to become trapped in polymeric fibrillar networks.
Their planar morphology (which corresponds to less than nine
atomic layers) further agrees with the capacity of the Pd
nanosheets to absorb in the near-infrared range (Figure 3c).50
A fluorogenic test was first used to determine the catalyst
compatibility with physiological conditions. The reaction was
performed by incubation in PBS at 37 °C with the Pd-labile
off/on probe O-propargyl-resorufin (Pro-Res)30 at a range of
concentrations of Pd nanosheets (5−40 μg/mL in Pd).
Figure 2. Pd-mediated conversion of Pro-PTX into PTX under biologically relevant conditions. (a) MS spectrum of Pro-PTX (100 μM) after 10 h
incubation in PBS at 37 °C. (b) MS spectrum of Pro-PTX (100 μM) after 10 h incubation with Pd devices (2 mg/mL) in PBS at 37 °C.
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in PBS at 37 °C during 24 h. The mixture was filtered off using
StageTips to eliminate the solid nanostructures and the
quantity of soluble Pd in the filtered solution was determined
by ICP-OES. Analysis detected 8.337 ( 3.049) and 16.675
ng/mL ( 6.090) of Pd in the samples, respectively, indicating
that just a very small fraction of Pd (approx. 0.042%) leaches
from the freestanding Pd nanosheets over this period. Next, to
test the inherent cytotoxicity of the nanodevices,57 A549 cells
were treated with Pd nanosheets for 3 days at the previously
tested range of concentrations. Cell viability results showed no
signs of toxicity at 5 μg/mL of Pd nanosheets and good cell
tolerability at 10 μg/mL (Figure S4, Supporting Information).
Finally, a prodrug activation study was performed to
corroborate the capacity of the nanodevices to dealkylate
Pro-PTX. The reaction was performed as described above but
using Pd nanosheets instead of Pd-functionalized microdevices.
The Pd nanodevices were filtered off using StageTips before
analyzing the samples by LCMS. Analysis showed the time-
dependent disappearance of the prodrug and the formation of
the peaks corresponding to PTX and the highly ionizable
amino derivative 5 (Figure S5, Supporting Information).
Once the catalytic activity and the biocompatible dose range
of the Pd nanosheets were confirmed, the use of hydrogels to
immobilize these functional nanodevices was investigated. Two
biopolymers, agarose and alginate, were tested to take
advantage of their different gelling properties and to assess
the scope of the capture strategy. Hydrogel formation in the
presence of catalysts was first studied by dissolving agarose in
warm biological-grade water at a range of concentrations,
followed by mixing with a prewarmed suspension of Pd
nanosheets. The mixture (60 μL) was then transferred to tissue
culture inserts (0.4−8 μm pore) and allowed to cool down to
room temperature to generate dark-colored hydrogel disks (Pd
Agarose) ready to be tested in cell culture (Figure 4a). Optimal
catalyst capture was obtained using 5 mg/mL of agarose and
0.4 mg/mL of Pd nanosheets.
Figure 3. (a, b) TEM images of freestanding Pd nanosheets at two
magnifications. (c) UV−Vis absorption spectrum of Pd nanosheets.
Next, the uncaging capabilities of the hydrogel-entrapped
catalysts from the tissue culture inserts were evaluated by
incubation with the off/on probe Pro-Res under physiological
conditions. Notably, treatment of the probe with Pd Agarose
(inserts of 1−8 μm pore size) resulted in an increase in the
fluorescence intensity equivalent to that obtained by free-
standing Pd nanosheets at the same concentration (Figure 5a).
In agreement with the preservation of the catalytic activity,
analysis of the hydrogel by SEM shows the apparent capture
and homogeneous distribution of the metallic Pd onto the
Changes in fluorescence intensity were measured at 24 h with a
spectrofluorometer (Ex/Em: 540/590 nm) and conversion
rates were calculated using a standard curve of resorufin.
Analysis revealed dose-dependent fluorescence increases, with
conversion rates ranging from 46 to 94% (Figure S3,
Supporting Information). To determine the fraction of Pd
atoms that leaches from the Pd nanosheets under the reaction
conditions, 20 and 40 μg/mL of Pd nanosheets were incubated
Figure 4. (a) Snapshots of the preparation of Pd Agarose and Pd Alginate catalytic hydrogels. (b) Representative SEM images of agarose hydrogel
(secondary electron detector) and Pd Agarose (backscatter electron detector). Bright points observed in the Pd Agarose image indicate the
presence of metallic Pd.
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Figure 5. (a, b) Conversion of Pro-Res (100 μM) into resorufin by (a) Pd Agarose (40 μg/mL of Pd nanosheets placed in 0.4−8 μm pore size
inserts) or (b) Pd Alginate (5−20 μg/mL of Pd nanosheets) after incubation in DMEM culture media (600 or 500 μL, respectively) for 16 h at 37
°C in a tissue culture incubator. Pro-Res (100 μM) with or without Pd nanosheets were used as negative and positive controls, respectively. The
fluorescence signal was measured at λex/em 540/590 nm. Error bars: SEM from n = 3. (c, d) A549 cell viability study after treatment with (a) Pd
Agarose and (b) Pd Alginate hydrogels at different concentrations. Pd nanosheets were included as controls. Cell viability was measured at day 3.
Error bars: SEM from n = 3.
biopolymer fibers (Figure 4b). This indicates that gelification
in the presence of a suspension of Pd nanosheets favors their
trapping and distribution throughout the fibrillar network with
little agglomeration,58,59 thus increasing the catalytic exposition
area while minimizing the escape of the metal. The presence of
Pd was further corroborated by energy-dispersive X-ray
(Figure S6, Supporting Information).
S7, Supporting Information). This study demonstrates that,
despite its structural complexity, Pro-PTX is able to efficiently
diffuse across the porous hydrogel network and react with Pd
nanosheets to be converted into PTX, which then diffuses back
through the hydrogel mesh into the culture media.
Finally, to corroborate that the mechanism of cell death
mediated by the Pro-PTX/Pd hydrogel combination was
analogous to that mediated by PTX, immunofluorescence
studies were performed to image microtubule stabilization.4,5
Cells were fixed after 2 days treatment and incubated with an
anti-α-tubulin IgG, cell nuclei DAPI stain, and TRITC-
Phalloidin, and imaged by confocal microscopy (Olympus
FV1000). As observed in Figure 7 (see additional control
experiments in Figure S8, Supporting Information), whereas
controls showed the tubulin signal (green channel) spread
throughout the cytoplasm (panels a and b), microtubule
accumulation was patently visible after PTX treatment (panel
d). Importantly, microtubule stabilization was equally evident
in cells treated with the Pro-PTX/Pd hydrogel combination
(panel c), thereby proving that the anticancer effect mediated
by that combination is a result of the in situ formation of PTX.
Next, because of the capacity of alginate to spontaneously
form hydrogels in Ca2+ solutions and inspired by previous
work by Fortin and co-workers60 on the “injection & capture”
strategy to immobilize radioactive metal nanoparticles in
tumors, the entrapment of Pd nanosheets was also tested in
alginate hydrogels. A suspension of sodium alginate (20 mg/
mL) and Pd nanosheets (0.3 mg/mL) was first prepared in
warm biological-grade water. Then, hydrogels were simply
made by dropwise addition of the mixture into PBS or culture
media supplemented with 100 mM CaCl2, to generate
spherical dark-colored hydrogels (Figure 4a and Movie S1).
As before, treatment of the off−on Pd-labile probe Pro-Res
with Pd Alginate spheres demonstrated the functional
operativity of the catalytic hydrogel (Figure 5b). Remarkably,
cell vitality studies showed that both Pd Agarose and Pd
Alginate hydrogels were better tolerated by A549 cells than
freestanding Pd nanosheets at equivalent metal concentrations
(Figure 5c,d).
Encouraged by the catalytic properties and biocompatibility
of the catalyst-loaded hydrogels, the capacity of Pro-PTX to
enter and be activated by these novel catalytic systems was
tested in A549 cells by incubation with Pro-PTX in the
presence of Pd- or catalyst-free hydrogels. As shown in Figure
6, no reduction of cell proliferation was observed after
separately treating A549 cells with either Pro-PTX or the
catalytic hydrogels. In contrast, the combination of Pro-PTX
and Pd Agarose disk/Pd Alginate spheres potently inhibited
cancer cell proliferation, resulting in an anticancer activity
equivalent to the direct treatment with the parent drug PTX.
Equivalent results were obtained in the U87 cell line (Figure
CONCLUSIONS
■
In the exploration of new ways to improve the safety and
efficacy of one of the most important anticancer drugs in
clinical use, a new prodrug of PTX has been developed. This
inactive drug precursor features excellent stability in cancer cell
culture and high sensitivity to metallic Pd, thus enabling
selective drug release in the presence of Pd catalysts. The
biscarbamate chemical mask used to block the 2′-OH position
of PTX provides optimal protection for aliphatic alcohols and
metal sensitivity, representing a novel and useful addition to
the current toolbox of bioorthogonal uncaging strategies.
Moreover, a biocompatible catalytic system based on the
immobilization of Pd catalysts in physical hydrogels has been
developed for the first time. Entrapment of Pd nanosheets in
agarose and alginate hydrogels exhibited equivalent catalytic
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Figure 6. Prodrug activation assays with (a) Pd Agarose and (b) Pd
Alginate. Experiments: 0.1% DMSO (untreated control, light gray),
Pd Agarose or Pd Alginate (−ve control, dark gray), 0.75 μM of Pro-
PTX (−ve control, blue), Pd Agarose or Pd Alginate + 0.75 μM of
Pro-PTX (activation assay, pink), and 0.75 μM of PTX (+ve control,
black). Cell viability was measured on day 3. Error bars: SEM, n = 3.
properties as the free catalysts, and displayed the capacity to
convert the inactive prodrug Pro-PTX into PTX in cancer cell
culture. By capturing transition metal catalysts within an
inherently biodegradable scaffold, this investigation provides a
strategy to temporarily elicit localized biorthogonal uncaging of
chemotherapeutics. As exemplified with alginate, the in situ
gelling strategy in which a liquid suspension containing
catalytic nanodevices solidifies in contact with environs rich
in bivalent cations could offer a minimally invasive solution to
locally administer and immobilize the catalyst at the disease
site. Subsequent studies will focus on progressing these
innovations into preclinical animal models.
Figure 7. Immunofluorescence study. Experiments: (a) Pd Alginate,
(b) 0.75 μM of Pro-PTX + Alginate (no Pd), (c) Pd Alginate + 0.75
μM of Pro-PTX (prodrug activation experiment), and (d) 0.75 μM of
PTX. Forty-eight hours after treatment, cells were fixed and stained
with anti-α-tubulin IgG, TRITC-Phalloidin, and DAPI. Left panels: α-
tubulin stain (green). Right panels: nuclei (cyan) and phalloidin
(red). Scale bar = 20 μm.
plates. High-resolution mass spectrometry was performed in a Bruker
MicrOTOF II. The purity of PTX and Pro-PTX was >95% for cell
studies, as measured by HPLC using an Agilent 1200 system. HPLC
method: eluent A: water and formic acid (0.1%); eluent B: methanol
and formic acid (0.1%); and A/B = 95:5 to 5:95 in 4 min and isocratic
2 min (flow = 1 mL/min). Prodrug-into-drug conversion experiments
were conducted in an LCMS (Agilent 1200) using a micrOTOF II
detector. Method A: eluent A: water and formic acid (0.1%); eluent
B: acetonitrile and formic acid (0.1%); and A/B = 95:5 isocratic 0.5
min, 95:5 to 0:100 in 4.5 min, isocratic 2 min, 0:100 to 95:5 in 0.5
min, and isocratic 2.5 min (flow = 0.2 mL/min). Method B: A/B =
80:20 to 0:100 in 10 min, isocratic 3 min, and 0:100 to 80:20 in 5 min
(flow = 0.3 mL/min). Stock solutions (100 mM) were prepared in
DMSO. SEM images were obtained using an FEI Quanta 400 ESEM
equipped with an EDX analytical system. A portion of hydrogel was
EXPERIMENTAL SECTION
■
General Methods. Chemicals and solvents were purchased from
Fisher Scientific, Sigma-Aldrich, VWR International Ltd., or TCI UK
Ltd. PTX was purchased from Fluorochem UK. NMR spectra were
recorded at ambient temperature on a 500 MHz Bruker Avance III
spectrometer. Chemical shifts are reported in parts per million (ppm)
relative to the solvent peak. Rf values were determined on Merck TLC
Silica gel 60 F254 plates under a 254 nm UV source. Purifications
were carried out by flash column chromatography using commercially
available silica gel (220−440 mesh, Sigma-Aldrich) or via semi-
preparative TLC chromatography on Merck TLC Silica gel 60 F254
F
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supercritically dried and coated with a fine carbon layer. Afterward,
the samples were examined by SEM using secondary and backscatter
electron detectors. ICP-OES measurements were carried out in a
Varian 715 ICP optical emission spectrometer.
Synthesis of 2′-(4-Nitrophenoxycarbonyl)paclitaxel (1). 2′-(4-
Nitrophenoxycarbonyl)paclitaxel, 1, was synthesized according to the
literature procedure.11
replaced with fresh media, containing compounds PTX and Pro-PTX
(0.001−10 μM). Untreated cells were incubated with DMSO (0.1%
v/v). After 3 days of incubation, PrestoBlue cell viability reagent (10%
v/v) was added to each well and the plate was incubated for 90 min.
Fluorescence emission was detected using a PerkinElmer EnVision
2101 multilabel reader (Ex/Em: 540/590 nm). All conditions were
normalized to the untreated cells (100%) and curves were fitted using
GraphPad Prism using a sigmoidal variable slope curve. Experiments
were performed in triplicate.
Prodrug-into-Drug Conversion Studies. Pro-PTX (100 μM) was
dissolved in PBS (1 mL) with 2 mg of Pd devices (Pd-resin diameter
size 30 μm, prepared as previously reported41) and incubated at 37 °C
in a Thermomixer at 1200 rpm for 10 h. Reaction crudes were
centrifuged (13 000 rpm, 5 min) to sediment the Pd devices and
supernatants were analyzed by LCMS/MS (Agilent 1200) using a
micrOTOF II detector. PTX (100 μM) was dissolved in PBS (1 mL)
for 10 h and analyzed as a positive control.
Synthesis of N-(Propargyloxycarbonyl)-N,N′-dimethylethylene-
diamine (4). Tert-butyl methyl[2-(methylamino)ethyl]carbamate, 2
(284 mg, 1.5 mmol), and pyridine (275 μL, 3.4 mmol) were dissolved
in a mix of H2O:1,4-dioxane (3:6, 9 mL). A solution of propargyl
chloroformate (221 μL, 2.3 mmol) in 1,4-dioxane (1 mL) was then
added dropwise to the mixture at room temperature and the reaction
was stirred overnight. Subsequently, solvents were removed by rotary
evaporation, the crude was dissolved in CH2Cl2 (20 mL), and the
mixture was washed with an aqueous solution of 1 N HCl (2 × 15
mL) and water (2 × 15 mL). The organic phase was dried over
anhydrous MgSO4 and concentrated by rotary evaporation. The crude
residue was purified by flash chromatography with 2.5% MeOH in
CH2Cl2 to give compound 3 as a pale oil. Rf = 0.48 (5% MeOH in
CH2Cl2). Without further characterization, compound 3 was then
dissolved in a 9:1 (v/v) mixture of TFA/water (5 mL) and the
mixture was stirred at room temperature for 2 h. Solvents were
removed by rotary evaporation and the addition of cold diethyl ether
(10 mL) afforded pure compound 4 as a yellow solid (90% yield, two
steps). Rf = 0.15 (5% MeOH in CH2Cl2). 1H NMR (500 MHz,
DMSO-d6) δ 8.48 (s, 1H), 4.68 (s, 2H), 3.55 (s, 1H), 3.50 (t, J = 6.0
Hz, 2H), 3.08 (t, J = 6.0 Hz, 2H), 2.87 (s, 3H), 2.58 (s, 3H). 13C
NMR (126 MHz, DMSO-d6) δ 155.8 (C), 79.5 (C), 78.0 (CH), 53.4
(CH2), 46.5 (CH2), 45.5 (CH2), 34.4 (CH3), 33.2 (CH3). HRMS
(ESI) m/z [M + H]+ calcd for C8H15N2O2, 171.11280; found,
171.11360.
Synthesis of Pro-PTX. Compound 1 (14 mg, 14 μmol) was
dissolved in dry DMF (1 mL) under a N2 atmosphere and cooled
down to 0 °C. Compound 4 (7 mg, 42 μmol) and DIPEA (12 μL, 70
μmol) were dissolved in dry DMF (0.5 mL) and added dropwise to
the solution and the mixture was allowed to reach room temperature
and stirred overnight. The solvent was removed by rotary evaporation
and the crude was purified via semipreparative TLC chromatography
(2.5% MeOH in CH2Cl2) to yield a white solid (36% yield). Rf = 0.40
(5% MeOH in CH2Cl2). 1H NMR (500 MHz, DMSO-d6) δ 9.17 (d, J
= 8.9 Hz, 1H), 7.98−7.94 (m, 2H), 7.87−7.81 (m, 2H), 7.73 (t, J =
7.5 Hz, 1H), 7.65 (t, J = 7.5 Hz, 2H), 7.59−7.53 (m, 1H), 7.49 (t, J =
7.3 Hz, 2H), 7.47−7.43 (m, 4H), 7.19−7.17 (m, 1H), 6.29 (s, 1H),
5.87−5.81 (m, 1H), 5.66−5.54 (m, 1H), 5.41 (d, J = 7.2 Hz, 1H),
5.30−5.12 (m, 1H), 4.88 (dd, J = 14.5, 8.5 Hz, 2H), 4.62−4.52 (m,
2H), 4.13−4.08 (m, 1H), 4.03−3.97 (m, 2H), 3.58 (d, J = 7.1 Hz,
1H), 3.46 (d, J = 6.6 Hz, 1H), 3.40 (s, 1H), 2.88 (s, 1H), 2.85 (s,
1H), 2.81 (s, 1H), 2.77 (d, J = 6.9 Hz, 1H), 2.72 (d, J = 0.6 Hz, 2H),
2.35−2.20 (m, 4H), 2.10 (d, J = 1.5 Hz, 3H), 1.81−1.78 (m, 4H),
1.62 (t, J = 13.0 Hz, 2H), 1.49 (s, 4H), 1.34 (s, 1H), 1.22 (s, 2H),
1.01 (d, J = 7.4 Hz, 6H). 13C NMR (126 MHz, DMSO-d6) δ 202.9,
170.1, 169.2, 167.0, 165.7, 155.1, 140.9, 140.4, 137.8, 134.7, 134.0,
131.9, 130.5, 130.1, 129.2, 128.8, 128.6, 128.2, 127.8, 126.8, 116.6,
84.1, 80.7, 79.6, 77.6, 77.2, 76.5, 75.8, 75.2, 75.0, 71.1, 70.9, 66.1,
65.5, 60.2, 57.8, 55.4, 54.5, 52.9, 46.6, 43.4, 37.0, 34.9, 33.7, 32.9,
26.8, 23.0, 21.9, 21.2, 21.1, 20.8, 19.3, 15.6, 14.6, 14.4, 10.2. HRMS
(ESI) m/z [M + H]+ calcd for C56H64N3O17, 1050.42302; found,
1050.42120. Purity as measured by HPLC was >99%.
Preparation of Pd Nanosheets. The synthesis of Pd nanosheets
was based on previously reported protocols,30,55,56 but with
modifications to avoid the use of toxic quaternary ammonium salts.
The palladium growth solution was prepared by mixing 11 mg of
Na2PdCl4, 30 mg of poly(vinyl pyrrolidone) (MW = 55 000), and 130
mg of KBr in Milli-Q water (400 μL). The resulting homogeneous red
solution was mixed with 4 mL of DMF. The Pd nanosheet precursor
solution was introduced into a high-pressure stainless-steel Teflon-
lined reactor. CO gas was introduced into the high-pressure reactor to
reduce the Pd precursor and control the anisotropic shape of the Pd
nanostructures.55 The pressure inside the reactor was maintained at 6
bar and the reactor was placed in a heated water bath (80 °C for 40
min). The solution was gently stirred with a magnetic flea located on
the high-pressure reactor. A dark blue colloid was obtained after the
CO treatment. Pd nanosheets were collected by centrifugation
(10 000 rpm, 10 min) by mixing the dark blue colloid and acetone in
a volume ratio of 1 to 3. Finally, Pd nanosheets were dispersed in
Milli-Q water and kept at 5 °C for further use. The Pd concentration
was determined by MP-AES (Microwave Plasma-Atomic Emission
Spectrometer 4100 Agilent Technologies) and the optical properties
were analyzed by UV−VIS spectrophotometry (Jasco V-670). TEM
(Tecnai FEI T20) was used to study the Pd nanosheet morphology by
operating at an acceleration voltage of 200 kV with a LaB6 electron
source fitted with a SuperTwin objective lens allowing a point-to-
point resolution of 2.4 Å. TEM images showed that the Pd nanosheets
tend to stack during the drying process of the TEM grid preparation.
This effect enabled us to observe the ultrathin sheetlike morphology
and made their direct thickness measurement easy. Leaching of Pd
nanosheets after incubation for 24 h in PBS was studied by ICP-OES
using a Varian 715 ICP optical emission spectrometer.
Preparation of Pd Agarose. Pd nanosheets were embedded in
agarose hydrogels (5 mg/mL) with a Pd concentration of 0.4 mg/mL.
Agarose powder (UltraPure Agarose, Thermo Fisher Scientific) was
weighted in an Eppendorf tube and sterilized by UV radiation. Milli-Q
water was filtered through a 0.22 μm mesh filter and was added to the
agarose powder. The mixture was shaken and warmed at 80 °C for 5
min to achieve a complete solution. The solution obtained was added
on a prewarmed Pd nanosheet suspension and was homogenized by
pipetting up and down several times. Immediately after, 60 μL of the
suspension was pipetted into tissue culture inserts (Corning
Transwell), placed in a 24-well plate, and allowed to cool at room
temperature for 1 h. Afterward, the 24-well plate was sterilized under
UV radiation for 30 min before characterization and use in cell assays.
Preparation of Pd Alginate. Pd nanosheets were embedded in
alginate hydrogels with a Pd nanosheet concentration of 0.3 mg/mL.
Sodium alginate powder (Sigma Aldrich, W201502) was dissolved in
sterile water and Pd nanosheets were added and vortexed until
homogenization. The mixture was sterilized under UV for 15 min.
The suspension was added dropwise into standard cell culture
medium (DMEM, 10% FBS, 2 mM L-glutamine) supplemented with
100 mM CaCl2 dihydrate in 24-well plates to form dark alginate
beads. Beads were washed twice with DMEM and transferred to the
Synthesis of O-Propargyl-resorufin. Pro-Res was synthesized
according to the literature procedure.30
Cell Viability Study: PTX vs Pro-PTX. A549 cells (a kind gift from
Dr. Wilkinson) and U87 cells (a kind gift from Dr. Gammoh) were
seeded in a 96-well plate format (at 1500 and 2000 cells/well,
respectively) in DMEM supplemented with 10% of FBS and L-
glutamine (2 mM) and incubated in a tissue culture incubator at 37
°C and 5% CO2 for 24 h before treatment. HBVPs (a kind gift from
Dr. Caporali) were seeded at 10 000 cells (to reach confluency) in
pericyte media (ScienCell Research Laboratories, Inc.) on gelatin
(0.1%)/fibronectin (10 μg/mL)-precoated dishes. Each well was then
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Article
experimental wells (3 beads/500 μL, final concentration of Pd of
approx. 20 μg/mL).
immersion objective. The images were acquired using the FV10-
ASW program in a sequential mode using software preconfigured
settings for Alexa Fluor 488 and TIRTC and analyzed with Image-J
software to obtain maximal projections.
Cell Toxicity Studies: Pd Nanosheets vs Pd Hydrogels. A549 cells
were cultured in DMEM supplemented with 10% of FBS and L-
glutamine (2 mM) and incubated in a tissue culture incubator at 37
°C and 5% CO2. A549 cells were seeded in a 24-well plate format (at
9000 cells/well) and incubated for 24 h before treatment. For Pd
nanosheets, each well was replaced with a suspension of Pd
nanosheets in fresh culture media at 5, 10, 20, and 40 μg/mL of
metal (600 μL final volume). For Pd Agarose, each well was replaced
with fresh media (540 μL). Agarose hydrogels containing Pd
nanosheets were prepared following the procedure previously
described and placed in cell culture inserts with 1 μm pore size (60
μL, 5−40 μg/mL of metal concentration). For Pd Alginate, each well
was replaced with fresh media (500 μL). Pd Alginate hydrogels were
prepared as described above and added at 1−3 beads/well (5−20 μg/
mL of metal concentration). After 3 days of incubation, PrestoBlue
cell viability reagent (10% v/v) was added to each well and the plate
was incubated for 180 min. Fluorescence emission was detected using
a PerkinElmer EnVision 2101 multilabel reader (Ex/Em: 540/590
nm). Experiments were performed in triplicate.
Fluorogenic Assays: Pd Nanosheets vs Pd Hydrogels. Pro-Res
(100 μM) was dissolved in DMEM culture media (in 540 μL for Pd
Agarose hydrogels or in 500 μL for Pd Alginate hydrogels) in a 24-
well plate format. For Pd Agarose, hydrogels containing Pd
nanosheets at 40 μg/mL (60 μL) were prepared following the
procedure described above and placed in cell culture inserts (0.4−8
μm pore size). For Pd Alginate, hydrogels containing Pd nanosheets
at approximately 5−20 μg/mL (1−3 beads/well) were prepared
following the procedure previously described. Pd nanosheets (20 and
40 μg/mL) were used as controls. Samples were incubated at 37 °C in
a tissue culture incubator. Pd nanosheets were removed by
centrifugation (13 000 rpm, 30 min). The fluorescence intensity of
100 μL of supernatants transferred to a 96-well plate format was
measured in a PerkinElmer EnVision 2101 multilabel reader (Ex/Em:
540/590 nm). Samples were repeated in triplicate. The percentage of
conversion was calculated based on the fluorescence signal of the
positive control Resorufin at 100 μM.
Prodrug Activation Assays with Pd Hydrogels. A549 were seeded
in a 24-well plate format (at 9000 cells/well) and incubated for 24 h
before treatment. Each well was replaced with fresh culture media
containing Pro-PTX or PTX (0.75 μM). Untreated cells were
incubated with DMSO (0.1% v/v). Pd Agarose and Alginate
hydrogels (40 μg/mL of metal and 20 μg/mL, respectively) were
prepared following the procedure previously described. Pd Agarose
hydrogels were placed in cell culture inserts with 1 μm pore size and
Pd Alginate hydrogels were added at 3 beads/well. After 3 days of
incubation, cell viability was determined as described above.
Experiments were performed in triplicate.
Immunofluorescence Studies. A549 cells were seeded on 10 mm
poly(L-lysine)-precoated coverslips in 24-well plates (5000 cells/well).
After 24 h, cells were incubated in the presence or in the absence of
alginate gels and/or Pro-PTX/PTX (0.75 μM) in 500 μL of DMEM
in triplicate. After 48 h, cells were fixed with paraformaldehyde (4% v/
v) for 10 min and washed three times with PBS every 5 min. Cells
were permeabilized for 15 min with 0.3% Tween in PBS and washed
three times with PBS every 5 min. Coverslips were incubated in
blocking buffer (1× PBS, 5% goat serum, 0.3% Triton X-100) for 60
min. Anti-α-tubulin Rabbit mAb (Cell Signaling Technology) was
incubated overnight at 4 °C in antibody dilution buffer (1× PBS, 1%
BSA, 0.3% Triton X-100) at a dilution of 1:25. After washing three
times with PBS, coverslips were incubated for 30 min in antibody
dilution buffer (1× PBS, 1% BSA, 0.3% Triton X-100) with Alexa
Fluor 488 Goat anti-Rabbit IgG (H + L) secondary antibody
(Invitrogen) at a dilution of 1:400. Coverslips were washed three
times with PBS and mounted on a slide with a 1:1 mixture of
VECTASHIELD Hardset Antifade Mounting Medium with Phalloi-
din: VECTASHIELD Antifade Mounting Medium with DAPI. Cells
were imaged using a scanning confocal inverted microscope Olympus
FluoView FV1000 (Olympus, Tokyo, Japan) with a 60× oil
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00781.
■
sı
NMR for 4 and Pro-PTX, Figures S1−S8 (PDF)
Movie S1: Preparation of Pd Alginate (MP4)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
■
Asier Unciti-Broceta − Cancer Cancer Research UK Edinburgh
Centre, Institute of Genetics and Molecular Medicine, University
of Edinburgh, Edinburgh EH4 2XR, U.K.; orcid.org/0000-
0003-1029-2855; Phone: 0044-(0)-1316518500;
Email: Asier.Unciti-Broceta@igmm.ed.ac.uk
Authors
́
́
Ana M. Perez-Lopez − Cancer Cancer Research UK Edinburgh
Centre, Institute of Genetics and Molecular Medicine, University
of Edinburgh, Edinburgh EH4 2XR, U.K.; orcid.org/0000-
0002-3900-3335
́
Belen Rubio-Ruiz − Cancer Cancer Research UK Edinburgh
Centre, Institute of Genetics and Molecular Medicine, University
of Edinburgh, Edinburgh EH4 2XR, U.K.; orcid.org/0000-
0003-4720-6578
Teresa Valero − Cancer Cancer Research UK Edinburgh Centre,
Institute of Genetics and Molecular Medicine, University of
Edinburgh, Edinburgh EH4 2XR, U.K.
́
Rafael Contreras-Montoya − Departamento de Quımica
́
Organica, Facultad de Ciencias, Universidad de Granada,
Granada 18002, Spain; orcid.org/0000-0002-0766-004X
Luis Alvarez de Cienfuegos − Departamento de Quımica
Organica, Facultad de Ciencias, Universidad de Granada,
́
́
́
Granada 18002, Spain; orcid.org/0000-0001-8910-4241
́
Víctor Sebastian − Department of Chemical Engineering and
Environmental Technology; Instituto de Nanociencia y
́
Materiales de Aragon (INMA), CSIC-Universidad de
Zaragoza, Zaragoza 50009, Spain; Networking Research
Center on Bioengineering Biomaterials and Nanomedicine
(CIBER- BBN), Madrid 28029, Spain; orcid.org/0000-
0002-6873-5244
́
Jesus Santamaría − Department of Chemical Engineering and
Environmental Technology; Instituto de Nanociencia y
́
Materiales de Aragon (INMA), CSIC-Universidad de
Zaragoza, Zaragoza 50009, Spain; Networking Research
Center on Bioengineering Biomaterials and Nanomedicine
(CIBER- BBN), Madrid 28029, Spain; orcid.org/0000-
0002-8701-9745
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c00781
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank EPSRC (Healthcare Technology Challenge Award,
no. EP/N021134/1) and ERC (Advanced Grant CADENCE,
■
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
́
́
(13) Sanchez, M. I.; Penas, C.; Vazquez, M. E.; Mascarenas, J. L.
̃
no. ERC-2016-ADG-742684) for funding. B.R.-R. and T.V.
thank the EC for an MSC Fellowship (H2020-MSCA-IF-2014-
658833 and H2020-MSCA-IF-2016-749299, respectively).
R.C.M. is grateful for the support of the Vicerrectorate of
Research of University of Granada. R.C.-M. and L.A.C. thank
Metal-catalyzed uncaging of DNA-binding agents in living cells. Chem.
Sci. 2014, 5, 1901−1907.
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Junta de Andalucıa (Spain) for funding project P12-FQM-790.
́
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L.A.C. thanks the Ministerio de Ciencia, Innovacion y
Universidades, for a Salvador de Madariaga visiting professor
fellowship (PRX18/00017) and the Agencia Estatal de
́
Investigacion (Ministerio de Economia y Competitividad)
and the European Regional Development Fund for cofunding
project FIS2017-85954-R. V.S. acknowledges the financial
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LIST OF ABBREVIATIONS
chemistry. Nat. Chem. 2011, 3, 239−243.
■
́
(19) Unciti-Broceta, A.; Johansson, E. M. V.; Yusop, R. M.; Sanchez-
Boc, tert-butyloxycarbonyl; DAPI, 4′,6-diamidino-2-phenyl-
indole; DIPEA, N,N-diisopropylethylamine; DMF, dimethyl-
formamide; DMEM, Dulbecco’s modified Eagle’s medium;
DMSO, dimethylsulfoxide; EC50, half-maximal effective con-
centration; FDA, Food and Drug Administration; ICP-OES,
inductively coupled plasma atomic emission spectroscopy;
IgG, immunoglobulin G; MP-AES, microwave plasma-atomic
emission spectrometer; MS, mass spectrometry; NSCLC, non-
small-cell lung carcinoma; PBS, phosphate-buffered saline; Pd,
palladium; ppm, parts per million; rt, room temperature; SEM,
scanning electron microscopy; SEM, standard error of mean;
TEM, transmission electron microscopy; TFA, trifluoroacetic
acid; TLC, thin-layer chromatography
Martín, R. M.; Bradley, M. Synthesis of polystyrene microspheres and
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