Thermally Gated Bio-orthogonal Nanozymes with Supramolecularly Confined Porphyrin Catalysts for Antimicrobial Uses

Full text of DOI:10.1016/j.chempr.2020.01.015

Article
Thermally Gated Bio-orthogonal Nanozymes
with Supramolecularly Confined Porphyrin
Catalysts for Antimicrobial Uses
Roberto Cao-Mil a´ n, Sanjana
Gopalakrishnan, Luke D. He, ...,
Federica Scaletti, Richard W.
Vachet, Vincent M. Rotello
rotello@chem.umass.edu
HIGHLIGHTS
Development of
thermoresponsive bio-orthogonal
nanocatalysts, i.e., nanozymes
Confined dynamics of iron catalyst
assemblies in gold nanoparticle
monolayers
Catalytic and thermoresponsive
activity in complex biological
systems
Eradication of biofilms through
controlled activation of antibiotics
using nanozymes
This paper describes the fabrication of thermoresponsive bio-orthogonal catalytic
systems though the integration of transition metal catalysts into gold
nanoparticles. The confined assemblies of the catalysts provide a temperature-
regulated system able to controllably activate antibiotics within biofilms. This work
presents a blueprint for synthesizing a family of reversible thermoresponsive
nanozymes with tailored activation temperatures and preserved bio-orthogonal
activity in complex biological environments.
Cao-Mil a´ n et al., Chem 6, 1–12
April 9, 2020 ª 2020 Elsevier Inc.
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alysts for Antimicrobial Uses, Chem (2020), https://doi.org/10.1016/j.chempr.2020.01.015
Article
Thermally Gated Bio-orthogonal Nanozymes
with Supramolecularly Confined Porphyrin
Catalysts for Antimicrobial Uses
1
1
1
1
1
Roberto Cao-Mil a´ n, Sanjana Gopalakrishnan, Luke D. He, Rui Huang, Li-Sheng Wang,
1
1
1
1
1
Laura Castellanos, David C. Luther, Ryan F. Landis, Jessa Marie V. Makabenta, Cheng-Hsuan Li,
Xianzhi Zhang, Federica Scaletti, Richard W. Vachet, and Vincent M. Rotello1,2,*
1
1
1
SUMMARY
The Bigger Picture
Bio-orthogonal catalysis has the capability of localized generation of imaging
and therapeutic molecules in vitro and in vivo. The integration of these catalysts
into thermoresponsive nanoparticle platforms would generate bio-orthogonal
Bio-orthogonal chemistry is a
medical tool that uses abiotic
chemical reactions to generate
diagnostic and therapeutic agents
in the body without interfering
with endogenous processes. The
integration of catalytic systems,
such as transition metal catalysts
(TMCs) into nanoparticle scaffolds
provides nanocatalysts that bio-
orthogonally generate imaging
and therapeutic agents through
the enzyme-like activation of
profluorophores and prodrugs.
This study describes the
‘‘nanozymes’’ that could be controlled through endogenous or exogenous
thermal control. We have fabricated thermoresponsive nanozymes by confining
supramolecular assemblies of porphyrins into the monolayer of gold nanopar-
ticles. The resulting nanodevices feature an on-off gated thermal response
ꢀ
occurring over a 3 C range with commensurate tunability of activation temper-
ꢀ
ꢀ
ature from 25 C to 37 C. Reversible activation of catalysis was demonstrated
in complex biological environments, and the efficacy of bi-stable thermorespon-
sive nanozymes demonstrated through thermal activation of antibiotic-based
prodrugs to effectively treat bacterial biofilms.
INTRODUCTION
Bio-orthogonal chemistry provides a tool for biological and biomedical science that
uses abiotic chemical reactions that do not interfere with endogenous enzymatic
fabrication of thermoresponsive
nanozymes through the
1
,2
processes. Bio-orthogonal catalysis extends this capability to generate imaging
supramolecular confinement of
TMC assemblies into
and therapeutic agents on demand through enzyme-like activation of prodrugs
3
–5
and profluorophores.
biocompatible gold
nanoparticles. The confined
assemblies provide a means of
regulating catalytic behavior
through temperature. The
potential application of this
platform for the temperature-
regulated generation of
The integration of bio-orthogonal catalytic systems such as transition-metal catalysts
(
TMCs) into nanoparticle hosts provides catalysts that replicate key features of en-
6
–8
zymes.
The nanoparticle scaffolds of these nanozymes solubilize and stabilize
1
catalysts in complex biological milieus. Additionally, these scaffolds can be used
to regulate the activity of the catalytic center, by using supramolecular interactions
to provide exogenous activation of catalysis in cells, as well as providing complex
9
–11
kinetic behavior replicating enzymatic catalysis.
These bioinspired nanodevices
therapeutics was demonstrated
by utilizing these
with programable catalytic properties are especially appealing for biomedical
applications where repetitive localized therapy is required such as biofilm infec-
1
2,13
thermoresponsive nanozymes for
controlled activation of
tions.
Spatiotemporally controlled generation of antibiotics could facilitate
1
2
generation of therapeutic strategies with reduced side effects.
proantibiotics for the eradication
of biofilms.
Thermally responsive systems provide a means of controlling catalysis that
would allow the regulation of bioorthogonal processes by using endogenous
1
4
or exogenous temperature gradients. Most existing methods for creating ther-
1
5–18
moresponsive catalysts involve the insertion of either heterogeneous
or
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alysts for Antimicrobial Uses, Chem (2020), https://doi.org/10.1016/j.chempr.2020.01.015
Figure 1. Thermoresponsive Nanozyme Design
(
(
(
(
A) Structure of FeTPP.
B) Conversion of profluorophore pro-Res through the catalytic reduction of aryl azides.
C) Structure of AuTTMA.
D) Confined dynamics of FeTPP assemblies within AuTTMA monolayer; FeTPP forms a packed
structure with ligands preventing substrates from reaching active sites. Increasing temperature
triggers the dissociation of FeTPP assemblies and redistribution of FeTPP molecules within the
monolayer, forming structures that allow substrates to access active centers, enabling catalysis.
This process is reversed by cooling down the system.
1
9,20
molecular
catalytic species into temperature-sensitive scaffolds such as poly-
22–27
6
–10,21
mers
or gels.
These systems modulate their catalytic activity through
2
8
temperature-driven changes in volume and/or hydrophobicity of the scaffold,
7
,16
thereby controlling the access of substrates to active sites.
As a result, these
systems generally exhibit attenuation in their catalytic properties with increasing
7
,15
temperature,
providing turn-off-based regulation that is challenging to translate
to many biomedical applications.
In our previous studies, we incorporated hydrophobic TMCs into the monolayer of
6
,29–31
2
-nm gold nanoparticles (NPs),
in complex biological environments.
lizing and stabilizing the TMCs, we could use the confinement provided by the
to generate nanozymes that were functional
1
9,20,22,23
We hypothesized that beyond solubi-
3
2
monolayer to regulate intercatalyst supramolecular assemblies, providing a new
approach to the thermoregulation of catalysis (Figure 1; see also Data S1–S3). For
these studies we employed iron (III) tetraphenyl porphyrin (FeTPP), a system readily
3
3,34
capable of self-assembly through stacking (Figure 1A).
At low temperatures, the
confined FeTPP assemblies display cooperative interactions with the ligands of the
NP scaffold (Figure 1C), forming a compact structure that blocks substrate access
to the active centers, thus deactivating the catalytic process.
An increase in temperature triggers the disassembly and redistribution of TMCs
within the monolayer of NPs, enabling substrates to access the active centers and
1Department of Chemistry, University of
Massachusetts Amherst, 710 North Pleasant
Street, Amherst, MA 01003, USA
ꢀ
reactivating catalysis (Figure 1D). Activation occurred over a very narrow (3 C)
range, providing bistable off-on control. Changing the FeTPP:NP stoichiometry
2Lead Contact
enabled fine control of the gating temperature over the physiologically relevant
ꢀ
ꢀ
ꢀ
range of 25 C–37 C, with a resolution of 3 C. The inherent biostability of the
AuNP platform yielded nanozymes that retain their thermal gating properties in
*Correspondence: rotello@chem.umass.edu
https://doi.org/10.1016/j.chempr.2020.01.015
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Figure 2. Demonstrating the Tunability and Reversibility of the Catalytic Activity of
Thermoresponsive Nanozymes
(
A) The thermoresponsive behavior of Fe-NZ_25, Fe-NZ_28, Fe-NZ_31, Fe-NZ_34, and Fe-NZ_37
was monitored by measuring the catalytic activity of each nanozyme (at equal concentrations) in the
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
temperature range of 25 C–37 C. Nanozymes were exposed to 25 C, 28 C, 31 C, 34 C, and 37 C
ꢀ
for a fixed time interval (60 min each except at 25 C), represented by the appropriately colored
columns.
ꢀ
(
B) Thermal activation of the catalytic properties of Fe-NZ_37 (red) at 37 C compared with free
FeTPP (blue). Each column represents a 60-min period (except for the red column) of exposure to a
ꢀ
given temperature. Fe-NZ _37 is only active at 37 C (red column) while free FeTPP (blue plot)
ꢀ
displays catalytic activity even at 25 C.
(
C) Reversibility of catalytic properties of Fe-NZ_37 was monitored through changes in catalytic
ꢀ
ꢀ
activity because of alternate heating (to 37 C) and cooling (to 25 C) of Fe-NZ_37. Increase in
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Figure 2. Continued
fluorescence intensity is observed upon heating. Exposure to both temperatures was limited to a
duration of 20 min each as denoted by the alternately colored columns. Experiments were
conducted in PBS (pH 7.4) solutions containing glutathione 5 mM as a cofactor for the catalytic
process. Each experiment was replicated thrice, and error bars represent standard deviations of
these measurements.
complex biological environments. The potential of this platform for bio-orthogonal
therapy was demonstrated through a thermally regulated treatment of bacterial
biofilms via the catalytic uncaging of antibiotic-based prodrugs. On the whole,
this platform presents a new tool for the gated thermal regulation of biosystems
through bioorthogonal catalysis.
RESULTS AND DISCUSSION
Design and Synthesis of a Thermoresponsive Nanozyme
Nanozymes (NZs) were prepared by incorporating FeTPP into AuTTMA scaffolds.
The FeTPP molecule catalyzes the reduction of aryl azides to the corresponding
3
5
amines providing a robust and efficient bio-orthogonal catalyst that self-assem-
bles into stacked aggregates (see Data S9 for substrate design strategies). The
host NPs use the biocompatible scaffold used in our prior nanozyme systems
6
that provides a hydrophobic environment for catalyst encapsulation.
Nanozymes with different activation temperatures (Figure 2) were fabricated by
mixing AuTTMA aqueous solutions (10 mM) with FeTPP solutions (THF) of concentra-
tions ranging from 0.28 mM to 2.84 mM (see Data S5). Dynamic light-scattering
measurements showed no change in size of the AuNPs after encapsulation of the
FeTPP assemblies (see Data S1), indicating retention of nanopartricle architecture
and absence of aggregation. The incorporation of FeTPP assemblies of different
sizes into NPs produced a family of thermoresponsive nanozymes, Fe-NZ_25, Fe-
NZ_28, Fe-NZ_31, Fe-NZ_34, and Fe-NZ_37, which had different activation temper-
ꢀ
ꢀ
atures ranging from 25 C to 37 C (see Data S2 and Data S5).
The catalytic properties of the nanozymes were studied in PBS buffer through the
activation of a nonfluorescent resorufin-based profluorophore (pro-Res, Figure 1B),
where reduction of the azide results in fragmentation and release of the fluorescent
resorufin molecule (Figure 1B).
Demonstrating the Reversibility and Tunability of Thermoresponsive Behavior
Catalytic activity for each nanozyme was measured by monitoring changes in the
fluorescence intensity of pro-Res solutions at different temperatures (Ex. 568 nm
and Em. 588 nm). The FeTPP:AuTTMA stoichiometry was quantified using induc-
tively coupled plasma mass spectrometry (ICP-MS). Table 1 summarizes the ratio
of FeTPP:AuTTMA in each nanozyme and their corresponding rates for activation
of substrates at their respective temperatures of activation. Figure 2A shows
turn-on of catalysis for each nanozyme at their respective temperatures of activation.
Similar catalytic activity across different nanozymes was observed at their activation
temperature (Figure 2A and Table 1) consistent with the nanozymes having a similar
ꢀ
number of accessible FeTPP when they activate. In contrast, at 45 C, the catalytic
efficiency of nanozymes is proportional to the amount of encapsulated FeTPP,
indicating that at such temperatures most of encapsulated FeTPP are dissociated
and catalytically active (see Data S10).
The mixture of FeTPP solutions in THF with water instantly produced assemblies of
different sizes on the basis of the concentration of FeTPP solution, as inferred from
4
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Table 1. Number of FeTPP Molecules Encapsulated in Each Fe-NZ and the Rate of Substrate
Activation of Each Nanozyme at Its Activation TemperatureCatalytic activity was tracked by
monitoring changes in fluorescence of pro-Res solutions (Ex. 568 nm, em. 588 nm).
À1
À1
)
Fe-NZ
FeTPP:NP (Molecules per
Particle)
V
TA (Mol [NZ(Mol)] min
Fe-NZ_25
Fe-NZ_28
Fe-NZ_31
Fe-NZ_34
Fe-NZ_37
4 G 1
8 G 1
9 G 1
14 G 2
28 G 3
98.17 G 8.448
100.2 G 30.11
86.74 G 4.7008
123.2 G 20.90
113.1 G 14.19
UV-vis analyses. These FeTPP assemblies are confined in the AuTTMA scaffold to
obtain nanozymes. It is expected that FeTPP assemblies of smaller sizes would
have lower contact with the ligands of the AuTTMA scaffold, thereby reducing the
strength of hydrophobic interactions. As a result, the assemblies are expected to
disaggregate into their open conformations at lower temperatures, thereby
providing access of active centers to the substrates. Conversely, FeTPP assemblies
of larger sizes would require higher temperatures to generate their open conforma-
tions because of stronger hydrophobic interactions with the ligands of the AuTTMA
scaffold. Therefore, it was concluded that the transformation of substrates takes
place only after the dissociation of the FeTPP assemblies within the AuTTMA
scaffold.
ꢀ
Further studies were performed using Fe-NZ_37 as 37 C is the ideal temperature
for most bacterial and cell-culture studies. This would allow us to elucidate the ther-
moresponsive behavior in biological environments. Temperature-controlled activa-
tion of the nanozymes was verified by comparing the catalytic activity of Fe-NZ_37
with that of free FeTPP assemblies. As shown in Figure 2B, Fe-NZ_37 was activated
ꢀ
ꢀ
at 37 C, whereas free FeTPP solution was activated at 25 C. Panels correspond to
0-min exposure periods for each temperature. We also investigated the revers-
6
ibility of thermoresponsive behavior by using Fe-NZ_37. Figure 2C demonstrates
the reversible nature of thermal activation of Fe-NZ_37, wherein the nanozyme
can be returned to its dormant state by simply cooling the system.
We therefore conclude that confining FeTPP assemblies in the monolayer of our NPs
produces nanozymes that respond to subtle changes in temperature by reversibly
gating the access of substrates to active centers.
Elucidation of the Thermal Gating Mechanism of Thermoresponsive
Nanozymes
We studied the UV-vis spectra of the nanozyme at different temperatures to validate
that the mechanism of Fe-NZ_37 activation is based on the dynamics of the confined
FeTPP assemblies. Iron porphyrin aggregates present a characteristic absorption
band (Soret band) located at ꢁ410 nm, which tends to increase in intensity and
3
6,37
blue shift when such assemblies dissociate into smaller structures.
The spectra
of Fe-NZ_37 show the electronic transitions of both chromophores, the gold core,
and the confined FeTPP assemblies (Data S4). The absorption bands of the NP scaf-
folds show minimal changes within the studied range of temperatures (Data S3),
which allows their subtraction from the Fe-NZ_37 spectra to provide more insight
into the behavior of the confined FeTPP assemblies at different temperatures. The
subtracted spectra of Fe-NZ_37 are presented in Figure 3B. At higher temperatures,
the increased intensity of the Soret band is accompanied by a small blue shift. This
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Figure 3. Thermally Triggered Reversible Dissociation of FeTPP Assemblies in the Monolayer
of NPs
ꢀ
(
(
A) Scheme of Fe-NZ_37 at temperatures below (blue panel) and above (above) 37 C.
B) Subtracted UV-vis spectra of Fe-NZ_37 at different temperatures. The inset shows intensity of
4
12-nm band with increments of temperature. Significant variations in A412 are observed for Fe-
ꢀ
NZ_37 at temperatures above 37 C (red column).
ꢀ
ꢀ
(
(
C) Intensity of the band at 412 nm during cycles of heating at 37 C (red circles) and cooling at 25 C
ꢀ
blue circles). Fe:Au ratio of Fe_NZ-37 solutions subjected to 37 C (for different lapse of time) and
ꢀ
to 25 C. Measurements were conducted by ICP-MS. Each experiment was made in triplicate. Error
bars represent standard deviations of these measurements.
behavior indicates that confined FeTPP assemblies dissociate at increased
temperatures. Analysis of the variations of intensity of the Soret band (412 nm)
with temperature shows pronounced changes only when the temperature reaches
ꢀ
the 37 C turn-on temperature, consistent with porphyrin catalyst dissociation at
the activation temperature (Figure 3D). Next, we analyzed variations in the
Soret band of confined FeTPP assemblies in Fe-NZ_37 when exposed to different
ꢀ
ꢀ
cycles of heating (at 37 C) and cooling (at 25 C) (see Data S4). Figure 3C shows
ꢀ
that the intensity of the Soret band reversibly increases at 37 C and decreases at
room temperature. These results are consistent with the observed reversible
6
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thermoresponsive behavior of the catalytic properties of Fe-NZ_37 (Figure 2D).
Resorufin showed no change in fluorescence due to changes in temperature
(
Data S6).
We next conducted ICP-MS experiments to detect whether the dissociation pro-
cesses of the confined FeTPP assemblies occurred solely within the monolayer of
the Fe-NZ_37, or if the catalysts could escape to solution. For this, Fe-NZ_37 sam-
ꢀ
ples were incubated at 37 C for 4 and 24 h, then immediately filtered (Amicon ultra
4
, 10K). Samples remaining in the filters were then collected, digested, and analyzed
by ICP. Figure 3D shows that the heated Fe-NZ_37 samples display the same
Fe:Au ratio (mol:mol) as the samples kept at RT. These results confirm that all dy-
namic processes detected by UV-vis only occur within the confining environment
of the monolayer of Fe-NZ_37 as represented in Figure 1D. Furthermore, we demon-
strated that only dissociated FeTPP contribute to catalysis. This can be seen in
ꢀ
Data S10 where upon heating all Fe-NZs to 45 C, the rate of catalysis is directly
proportional to the amount of catalyst encapsulated in each of the Fe-NZs.
Thermoresponsive Behavior of Fe-NZ_37 in Biological Systems
Having characterized our thermoresponsive nanozymes in solution, we subse-
quently studied their potential for the eradication of bacterial biofilms. The temper-
ature-dependent properties of Fe-NZ_37 in the biofilm were evaluated through
the catalytic deprotection of pro-Res as determined through confocal microscopy.
Biofilms comprising of green fluorescent protein-expressing E. coli (GFP-E. coli)
were incubated with Fe-NZ_37 in M9 media for 1 h to allow internalization of
2
0
nanozymes within the biofilms. Bacterial biofilms were then washed three times
with lead sulfide (PBS) to remove excess nanozyme. The thermoresponsive behavior
of the internalized nanozymes was then tested by incubating the biofilms in M9
ꢀ
ꢀ
media containing pro-Res (10 mM) at 25 C and 37 C for ꢁ1 h before obtaining the
images shown in Figure 4. Biofilms incubated only with pro-Res served as the nega-
tive control.
Figure 4 shows confocal studies that examine the penetration of nanozymes into
bacterial biofilms. By analyzing the green channel, the GFP-E. coli biofilms can be
visualized (Figures 4B-i, 4C-i, and 4D-i). Notably, the biofilms treated with Fe-
ꢀ
NZ_37 at 37 C (Figure 4D-i) resulted in higher bacterial density, presumably
due to a combination of growth-stimulating effects (hormesis) of the cationic
2
3
nanoparticles at low concentrations. Analysis of the red channel illustrates negli-
ꢀ
gible fluorescence in biofilms treated with pro-Res and Fe-NZ_37 at 25 C (Fig-
ꢀ
ure 4C-i) and also those treated with only pro-Res at 37 C (Figure 4B-i). These results
ꢀ
indicate that pro-Res deprotection occurs only in the presence of Fe-NZ_37 at 37 C.
The merged channel shows that the generated red fluorophore co-localizes with
GFP-E. coli, implying that it is being activated within the biofilm. This suggests
that the nanozymes successfully penetrated the bacterial biofilms and were able
to retain thermoresponsive catalytic activity within the biofilms.
From an antimicrobial perspective, a strategy that relies on proantibiotic activation
through bio-orthogonal thermoresponsive nanozymes can provide spatiotempo-
rally controlled generation of antibiotics. As a result, these systems could
facilitate the development of antimicrobial therapies with reduced side effects for
mammalian tissues, organs, and beneficial microorganisms. We demonstrated
this by using Fe-NZ_37 for the activation of pro-Mox (a protected moxifloxacin de-
rivative) (Figure 5 and Data S8).
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Figure 4. Thermoresponsive Behavior of Nanozymes in Biofilms
(
A–D) Shown in (A) is the internalization of Fe-NZ_37 in biofilms and temperature-controlled activation of pro-Res. Confocal microscopy images of
ꢀ
ꢀ
biofilms treated (B) with pro-Res at 37 C, (C) with a combination of Fe-NZ_37, and (D) with pro-Res at 25 C, and with a combination of Fe-NZ_37 and
ꢀ
pro-Res at 37 C. Each channel is represented with sub index -i (green), -ii (red), and -iii (merged). Scale bars, 50 mM.
E. coli biofilms, treated with Fe-NZ_37 and different concentrations of pro-Mox,
ꢀ
ꢀ
were incubated at 25 C and 37 C for 6 h. The samples were then washed with
PBS, and their viability was analyzed through an alamarBlue assay. Biofilms treated
with varying concentrations of pro-Mox only and Fe-NZ_37 only were tested as
negative controls and varying concentrations of Mox + Fe-NZ_37 were used as pos-
itive controls.
Figures 5B and 5C show the results obtained from bacterial biofilms exposed to each
ꢀ
ꢀ
treatment described above at 25 C and 37 C, respectively. As expected, samples
treated with different concentrations of pro-Mox displayed negligible variations in
8
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Figure 5. Deprotection of Antimicrobials in Biofilms Using Thermoresponsive Nanozymes
Fe-NZ _37 was used for the controlled activation of antimicrobial prodrugs that modulate biofilms
viability.
ꢀ
(
(
A) Scheme of activation of pro-Mox by p-p aggregates At 37 C.
ꢀ
ꢀ
B and C) E. coli biofilms treated with pro-Mox and Fe-NZ_37 (red bars) at (B) 25 C and at (C) 37 C.
Biofilms treated only with pro-Mox (blue bars) or with a combination of moxifloxacin (Mox) and Fe-
NZ_37 (pink bars) were used in all experiments as negative and positive controls, respectively. Each
experiment was replicated five times. Error bars represent standard deviations of these
measurements. The thermoresponsive properties of Fe-NZ_37 combined with its ability to
penetrate biofilms is ideal for controlled deprotection of pro-Mox within biofilms. Mox was chosen
3
8
as the antibacterial agent (Figure 5A, right) because of its broad-spectrum activity. The secondary
2
4
amino group is crucial for binding two target enzymes of the drug. Masking this group with the
bulky aryl-azide carbamate (Figure 5A, left) reduces the antibiotic activity of this drug by more than
two orders of magnitude, as can be seen in SI-7. This large reduction in the bactericidal activity of
pro-Mox (with respect to Mox) makes it ideal in combination with Fe-NZ_37 to thermally control the
viability of bacterial biofilms.
bacterial viability at both temperatures. On the contrary, biofilms responded in a
dose-dependent manner to treatments containing Fe-NZ_37 and different concen-
trations of active Mox, regardless of the temperature used. Biofilms treated with
ꢀ
pro-Mox and Fe-NZ_37 exhibited biofilm inhibitory effects at 37 C in a dose-depen-
ꢀ
dent manner, whereas biofilms exposed to pro-Mox and Fe-NZ_37 at 25 C (Fig-
ure 5B, red bars) displayed no significant variations in viability. Notable bactericidal
effect was registered with pro-Mox concentrations of above 2 mM. These results
show that Fe-NZ_37 can thermally regulate bioprocesses via the uncaging of modu-
latory drugs even in the presence of complex biological environments.
Conclusions
In this report, we highlight a strategy for fabricating thermoresponsive nanocata-
lysts that reversibly switch between dormancy and high rates of substrate process-
ing over slight variations in temperature. This strategy is based on the encapsula-
tion of transition metal catalysts, in the form of supramolecular assemblies, into
the monolayer of gold nanoparticles. We demonstrated that by confining supra-
molecular structures (p-p aggregates, in our case) into semi-rigid biocompatible
scaffolds, the stimuli-responsive behavior of these supramolecular complexes is
preserved in biological environments and can be employed in applications
that include localized therapy and imaging. This enables the TMC assemblies to
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reversibly dissociate within the hosting scaffold upon heating, consequently
resuming catalytic activity. This gating mechanism is based on the dynamics of
confined TMC assemblies and allows the nanozymes to display thermoresponsive
behavior in complex biological systems. Together with high rates of substrate pro-
cessing, these properties make our nanozymes well-suited for biomedical applica-
tions that include not only antibacterial therapies, but also the self-modulation of
inflammatory processes, which typically produce local heating. This strategy can
be adapted to integrate other self-assembled chromophores and/or fluorescent
3
7,38
molecules that can be used in the fabrication of nanometric sensors,
which
detect subtle changes in cellular microenvironments, and programable nanobots
that exert preventive regulatory functions in biosystems. This work presents a blue-
print for synthesizing a family of reversible thermoresponsive nanozymes with
tailored activation temperatures and preserved biorthogonal activity in complex
biological environments.
EXPERIMENTAL PROCEDURES
Synthesis and characterization of ligands, NPs (Figure 1D), nanozymes, and sub-
strates are described in the Supplemental Information. Detailed sample preparation
for ICP-MS, UV-vis characterization of nanozymes and FeTPP assemblies, and
additional bacteria culture studies can also be found in the Supplemental
Information.
Fabrication of Thermoresponsive Nanozymes
0
.5 mL of the FeTPP solution in THF was mixed with an equal volume of aqueous
solution of NPs (10 mM) (see Data S11 and Data S12 for AuTTMA synthesis), and
stirred for 10 min. THF was then slowly removed by evaporation to induce the encap-
sulation of FeTPP assemblies into the NP monolayer, with excess catalyst precipi-
tating. Fe-NZ solutions were then purified by centrifugation at 14,000 rpm to
eliminate precipitated FeTPP and filtered (Amicon ultra 4, 10K) six times to remove
the remaining free catalyst. For synthesizing Fe-NZ_37, Fe-NZ_34, Fe-NZ_31, Fe-
NZ_28, and Fe-NZ_25, 1 mL of FeTPP solutions with concentrations of 2.84 mM,
2
84 mM, 28.4 mM, 2.84 mM, and 0.28 mM, respectively, were mixed with equal
volumes of NP solutions at 10 mM (Data S13). The resulting solutions were then
concentrated by roto-evaporation and purified through multiple sessions of centri-
fugation and filtration (Amicon ultra 4, 10K). For long term preservation of Fe-NZ
solutions (1–2 months), solid ascorbic acid was added to stock solutions at concen-
trations ranging from 1 to 6 mg/mL.
Determination of Temperature of Activation of Thermoresponsive
Nanozymes
Each kinetic experiment was performed in a 96-well black plate with 100 mL of PBS
solution containing the corresponding Fe-NZ (50 nm), pro-Res (20 mM), and 5 mM
of glutathione (See Data S14 for pro-Res synthesis). Samples were then immediately
ꢀ
inserted into the plate reader at 25 C to analyze the evolution of fluorescence
(
Ex. 568 nm, Em. 588 nm). At the end of each 60-min interval, the incubation temper-
ꢀ
ature was increased by 3 C, and fluorescence was analyzed for another 60 min.
When nanozymes were observed to become thermally active, fluorescence was
monitored until reaching a maximum value (typically around 2,000 a.u.) representing
pro-Res total transformation.
Thermoresponsive Activation of Prodrug in Bacterial Biofilms
E. coli strains CD 2 and GFP-E. coli were inoculated in 3 mL LB broth and grown to
ꢀ
the stationary phase at 37 C. The cultures were then washed thrice in 0.85% sodium
10
Chem 6, 1–12, April 9, 2020

Please cite this article in press as: Cao-Mil a´ n et al., Thermally Gated Bio-orthogonal Nanozymes with Supramolecularly Confined Porphyrin Cat-
alysts for Antimicrobial Uses, Chem (2020), https://doi.org/10.1016/j.chempr.2020.01.015
chloride solution through centrifugation. The concentration of the resuspended
bacteria solutions was estimated by measuring the optical density at 600 nm. Seed-
8
ing solutions for both strains were prepared by diluting to 0.1 OD600 (ꢁ10 colony
forming units/ml) in M9 minimal media.
For the confocal studies, GFP-E. coli seeding solutions were spiked with IPTG
(
Isopropyl b-D-1-thiogalactopyranoside) so that the final solution contained 1mM
of IPTG. The seeding solution (1 ml) was placed in confocal dishes and incubated
at room temperature for ꢁ3 days to grow biofilms. M9 minimal media was replaced
each day. Biofilms were washed with PBS three times on day 3 and incubated with
Fe-NZ_37 in M9 media for 1 h. Bacterial biofilms were next washed three times
ꢀ
with PBS to remove excess nanozyme and incubated pro-Res (100 mM) at 25 C
ꢀ
and 37 C for ꢁ1 h before obtaining images.
For the biofilm viability studies, 100 mL of the E. coli (CD 2) seeding solution was
added into each well of a 96 well-plate and incubated overnight at room tempera-
ture to grow the biofilms. The biofilms were then washed thrice with PBS and treated
with nanozymes (100 nM) and/or pro-Mox/Mox (see Data S15 for pro-Mox synthesis)
at varied concentrations prepared in M9 media. One set of plates was incubated
ꢀ
ꢀ
at 37 C for 5 h while the other was kept at 25 C. After this treatment, the biofilms
were washed with PBS three times and their viability was determined using the ala-
marBlue assay according to the protocol established by the manufacturer
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.chempr.
2
020.01.015.
ACKNOWLEDGMENTS
This work was supported by the NIH EB022641 and GM008515, to D.C.L., Research
Corporation for a TREE award to V.M.R., and CHE-1808199 to V.M.R. and R.W.V.
AUTHOR CONTRIBUTIONS
R.C.-M. and V.M.R. conceived the idea. R.C.-M., L.D.H., R.H., R.F.L., and X.Z. syn-
thesized nanozymes and substrates. R.C.-M., L.D.H., and L.S.W. designed and per-
formed the experiments in solutions. R.C.-M., S.G., R.H., J.M.M., and C.H.L.
conceived and designed bacteria experiments. R.C.-M., L.C., D.C.L., and R.W.V.
conceived and performed ICP-MS analyses. All authors analyzed and discussed
the data. R.C.-M., L.D.H., S.G., and V.M.R. co-wrote the manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: August 8, 2019
Revised: September 16, 2019
Accepted: January 20, 2020
Published: February 13, 2020
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