Polymer-Based Bioorthogonal Nanocatalysts for the Treatment of Bacterial Biofilms

Article abstract of DOI:10.1021/jacs.0c01758

Bioorthogonal catalysis offers a unique strategy to modulate biological processes through the in situ generation of therapeutic agents. However, the direct application of bioorthogonal transition metal catalysts (TMCs) in complex media poses numerous challenges due to issues of limited biocompatibility, poor water solubility, and catalyst deactivation in biological environments. We report here the creation of catalytic "polyzymes", comprised of self-assembled polymer nanoparticles engineered to encapsulate lipophilic TMCs. The incorporation of catalysts into these nanoparticle scaffolds creates water-soluble constructs that provide a protective environment for the catalyst. The potential therapeutic utility of these nanozymes was demonstrated through antimicrobial studies in which a cationic nanozyme was able to penetrate into biofilms and eradicate embedded bacteria through the bioorthogonal activation of a pro-antibiotic.

Full text of DOI:10.1021/jacs.0c01758

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Article
Polymer-Based Bioorthogonal Nanocatalyst
for the Treatment of Bacterial Biofilms
Rui Huang, Cheng-Hsuan Li, Roberto Cao-Milán, Luke D He, Jessa
Marie Makabenta, Xianzhi Zhang, Erlei Yu, and Vincent M. Rotello
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c01758 • Publication Date (Web): 28 May 2020
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Polymer-Based Bioorthogonal Nanocatalysts for the Treatment of
Bacterial Biofilms
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†
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Rui Huang , Cheng-Hsuan Li , Roberto Cao-Milán , Luke D. He , Jessa Marie Makabenta , Xianzhi
†
†, §
, †
Zhang , Erlei Yu and Vincent M. Rotello*
†
0
Department of Chemistry, University of Massachusetts Amherst, 710 North Pleasant Street, Amherst, Massachusetts
1003, United States
§
Key Laboratory for Green Process of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical
Engineering, Shihezi University, Shihezi 832003, P. R. China
KEYWORDS. Bioorthogonal chemistry, Transition Metal Catalysts (TMCs), nanozymes, polymer nanoparticles, biofilms.
ABSTRACT: Bioorthogonal catalysis offers a unique strategy to modulate biological processes through the in situ generation of
therapeutic agents. However, the direct application of bioorthogonal transition metal catalysts (TMCs) in complex media poses
numerous challenges due to issues of limited biocompatibility, poor water solubility, and catalyst deactivation in biological
environments. We report here the creation of catalytic “polyzymes”, comprised of self-assembled polymer nanoparticles engineered
to encapsulate lipophilic TMCs. The incorporation of catalysts into these nanoparticle scaffolds creates water-soluble constructs that
provide a protective environment for the catalyst. The potential therapeutic utility of these nanozymes was demonstrated through
antimicrobial studies in which a cationic nanozyme was able to penetrate into biofilms and eradicate embedded bacteria through the
bioorthogonal activation of a pro-antibiotic.
Introduction
which bacterial cells are embedded in a self-produced
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extracellular polymeric substance (EPS).
This polymeric
Bioorthogonal chemistry has emerged as a promising
strategy for modulating bioprocesses using reactions that
cannot be performed by natural enzymes.
unmasking reactions mediated by transition metal catalysts
TMCs) present a powerful tool for biomedical applications
matrix provides protection to bacterial cells and severely
hinders the penetration of most hydrophobic molecules, making
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Bioorthogonal
40,41
treatment particularly challenging.
(
Here, we report a self-assembled polymeric nanoparticle that
encapsulates TMCs into a hydrophobic pocket, providing a
bioorthogonal “polyzyme”. We hypothesized that beyond
solubilizing and stabilizing the catalyst molecules, the cationic
nanoparticle would act as a vehicle to facilitate the penetration
arising from their ability to generate therapeutic agents in situ,
minimizing off-target effects. In this prodrug strategy, a drug
6-15
is first modified with a masking group, rendering it inactive
(i.e., a prodrug); TMCs then remove the protecting group at the
targeted location to restore activity, providing on-site
therapeutic action. However, the direct application of TMC-
mediated reactions in biological environment is challenging due
4
2-44
of TMCs into biofilms.
To this end, we synthesized a
quaternary ammonium polymer possessing hydrophobic alkyl
side chains (Figure 1a). This polymer self-assembles into
nanoparticles, encapsulating TMCs within a protective
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to their limited water solubility and biocompatibility.
hydrophobic environment. Moreover, the cationic nature of the
polymer facilitates penetration of the particle into biofilms. The
penetration ability of our polyzyme was demonstrated by
performing in vitro activation of the non-fluorescent pro-dye
The incorporation of TMCs into nanomaterial hosts
solubilizes and stabilizes hydrophobic catalyst molecules while
preserving catalytic activity and stability in complex biological
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media.
In addition, nanomaterial scaffolds can be
(Figure 1a) using either polyzyme or free catalyst molecules.
engineered to localize within therapeutically important targets,
such as bacterial biofilms, tissues and cellular environments,
providing a targeting strategy for bioorthogonal activation of
Biofilms treated with polyzyme exhibited bright fluorescence
whereas biofilms incubated with free catalyst molecule showed
negligible activation. The penetration ability of the polyzymes
was further evidenced by monitoring a fluorescent metal-free
analog. The therapeutic potential of the bioorthogonal
polyzyme platform was demonstrated through the activation of
a pro-antibiotic to effectively eradicate bacterial biofilms.
24-34
prodrugs.
Bacterial biofilms are important potential targets for
biorthogonal therapeutics as their formation results in persistent
infections on wounds and indwelling medical devices,
including urinary catheters, arthro-prostheses and dental
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implants.
Biofilms are aggregates of micro-organisms in
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volume of THF. The solutions were mixed, and then stirred
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Results and Discussion
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overnight to allow the encapsulation of the catalyst into the
hydrophobic pockets of polymeric nanoparticles. Next, the
sample was dried under flowing nitrogen, and then redissolved
in water. The polyzyme solution was subsequently filtered 5
times to remove the precipitated catalyst and dialyzed against
water to remove any unbound catalyst. Dynamic light scattering
Design and synthesis of the polyzyme. The polyzyme
features two main components: a self-assembled polymer
nanoparticle and the encapsulated transition metal catalyst
(TMC). The hosting polymer was designed with monomers
featuring two crucial components: i) an alkyl chain that fosters
formation of a self-assembled nanoparticle to provide a
hydrophobic inner shell to encapsulate and protect TMCs, and
ii) a cationic terminal groups for maintaining water solubility
(DLS, Figure 1b) measurements on the polyzymes showed the
formation of ~50 nm nanocatalysts with no aggregation
observed after the encapsulation of [Fe(TPP)]Cl (Nanozyme
PDI = 0.147). The particle structure was also confirmed by
using transmission electron microscopy (TEM). As shown in
Figure 1b, the [Fe(TPP)]Cl (black dots) readily self-assembled
through stacking and were encapsulated into polymer
nanoparticles. The amount of [Fe(TPP)]Cl encapsulated in
polymer nanoparticles was determined quantitively using UV-
Vis (0.59 mg per mg of polyzyme, Figure S2, Supporting
Information).
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and penetrating the biofilm. For the polymer, we chose a
quaternary ammonium poly(oxanorborneneimide) containing a
C11-bridged alkyl chain that we have previously shown can
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penetrate into biofilms.
We chose 5,10,15,20-tetraphenyl-21H,23H-porphine (TPP)-
containing complex, [Fe(TPP)]Cl as the catalyst due to its high
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catalytic efficiency (Figure 1a). This iron-based complex
catalyzes the reduction of aryl azides to their respective amines
(Figure 2a) in the presence of biogenic thiols. In this catalytic
cycle, we used glutathione (GSH) as the reducing agent to
convert intermediate Fe -nitrene to the reduced amine.
IV
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To fabricate the polyzyme (Figure 1a), the polymer was
dissolved in water while [Fe(TPP)]Cl was dissolved in equal
Figure 1. a) Structures of [Fe(TPP)]Cl and polymeric nanoparticle. b) Transmission electron microscopy (TEM) imaging and dynamic light
scattering measurement (DLS). Scale bar is 100 nm. c) Schematic representation of the bioorthogonal activation for imaging and therapeutics.
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Figure 2. a) Schematic representation of the bioorthogonal activation of aryl azide-protected resorufin. b) Catalytic activity of polyzyme
(100 nM) was tracked by measuring changes in fluorescence (Ex. 570nm, Em. 590nm) of pro-resorufin solutions at different concentrations.
The curve is fit with a non-linear exponential equation. 1 mM GSH was used as the cofactor. c) Polyzyme kinetics is shown as a function of
substrate concentration; line is the regression curve corresponding to Michaelis-Menten kinetics.
Figure 3. Polyzyme analog (200 nM) diffusion into GFP - expressing E. coli (CD-2) biofilms after incubation for 1hr in M9 media, as
measured by confocal microscopy images. (a) free TPP, (b) encapsulated TPP. Scale bars are 50 μm. 1 mM GSH was used as the cofactor.
Catalytic efficacy of polyzymes in solution. Nanozyme
activity was quantified through activation of a resorufin-based
pro-fluorophore (Pro-Res, Figure 2a). This pro-dye was masked
with an aryl azide carbonate unit bound to the phenolate of
resorufin to render it non-fluorescent. The catalytic reduction of
azide group mediated by [Fe(TPP)]Cl results in fragmentation,
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releasing the fluorescent resorufin molecule.
The
fluorogenesis of this dye provides a direct way for studying
catalytic kinetics.
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Resorufin fluorescence was monitored in M9 media at 37 °C
penetration of the hydrophobic TMCs into biofilms
(Quantitative enhancement of fluorescence intensity is in
Figure S4, Supporting Information).
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to quantify the catalytic properties of the polyzyme (Figure 2b).
Fluorescence increased immediately after mixing the substrate
and polyzyme (100 nM), with no significant change in
fluorescence observed for the pro-dye only. Polyzyme activity
followed a standard Michaelis-Menten kinetic model (Figure
Catalytic efficacy of polyzymes in biofilms. Having
established biofilm penetration, the catalytic activity of
polyzymes was studied in biofilms through pro-dye activation.
GFP - expressing E. coli biofilms were first incubated with the
polyzyme for 1 hr in M9 media to internalize the catalyst.
Bacterial biofilms were then washed with PBS three times to
remove any polyzyme from the surface of the biofilm. Next,
fresh M9 media containing the pro-resorufin was added,
followed by a 1-hour incubation. Biofilms incubated with
substrate alone served as the negative control (Figure 4). By
analyzing the red channel, it can be observed that in the
presence of polyzyme pro-resorufin was activated in biofilms
and showed bright fluorescence. (Quantitative enhancement of
fluorescence intensity is in Figure S4, Supporting Information).
Biofilms first treated with pro-dye or pro-dye and free TPP
catalyst only exhibited negligible fluorescence. In merged
channels, the colocalization of the generated red fluorophore
and GFP - expressing E. coli biofilms demonstrates that the
activated resorufin was distributed within the biofilm,
consistent with the catalyst distribution observed in the biofilm.
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c), allowing facile quantification of the catalytic process.
Penetration of polyzymes into biofilms. Having
characterized the catalytic activity of the polyzyme in solution,
we next probed bioorthogonal catalysis in biofilms. As the
ability of the catalyst to accumulate inside of biofilms is crucial
for in situ activation, the penetration ability of polyzymes was
determined using confocal microscopy. To monitor the non-
fluorescent [Fe(TPP)]Cl, we replaced the encapsulated catalyst
with the metal-free analog, tetraphenyl porphyrin, TPP, which
has red fluorescence. A GFP (green fluorescent protein) -
expressing E. coli biofilm was chosen to facilitate microscopy
and the biofilm formation was confirmed by staining assay
using crystal violet (Figure S7, Supporting Information).
Biofilms were incubated with polyzyme analog and free TPP
for 1 hr in M9 media and then washed with PBS buffer three
times. The experiment showed that encapsulated TPP readily
penetrated biofilms whereas free porphyrin had significant
difficulty in penetration (Figure 3). This indicates that polymer-
based nanometric scaffolds act as a vehicle to facilitate the
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Figure 4. Confocal microscopy images of biofilms treated with polyzyme (1 hr, 200 nM) followed by incubation with pro-dye (1 hr, 4 μM);
negative controls are biofilms incubated with (a) pro-dye only and (b) free catalyst followed by incubation with pro-dye. Scale bars are 50
μm. 1 mM GSH was used as the cofactor.
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Antimicrobial activity through activation of prodrugs binding to their two target enzymes, DNA gyrase and
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using polyzymes. We next carried out prodrug activation using
the polyzyme. Two fluoroquinolones, moxifloxacin and
ciprofloxacin, were chosen as the antimicrobial agents owing to
topoisomerase IV. A bulky aryl-azide carbamate moiety was
introduced as a caging group to turn moxifloxacin or
ciprofloxacin into
a prodrug, reducing both of their
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their broad-spectrum activities. These drugs was converted
into prodrugs via functionalization on their secondary amino
groups (Figure 5a, 5b), as modification at this site blocks the
antimicrobial activities by more than two orders of magnitude
(see Table S1 for planktonic bacteria and Figure S6 for
biofilms,).
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Figure 5. Schematic representation of the activation of (a) pro-Moxifloxacin and (b) pro-Ciprofloxacin. 1 mM GSH was used as the cofactor.
c)Viability of E. coli (CD-2) biofilms incubated with polyzyme (2 hrs, 200 nM) followed by treatment with prodrugs (6 hrs). (d) Viability
of P. aeruginosa (ATCC-27853) biofilms incubated with polyzyme (2 hrs, 1 μM) followed by treatment with prodrugs (6 hrs). Biofilms
treated with prodrug, drug and prodrug + polymers were used as controls. Each experiment was performed in triplicate. Error bars represent
standard deviation.
(
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To demonstrate the generality of this prodrug strategy, pro-
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Fibroblast cells were incubated with polyzymes and cell
viability quantified using an Alamar Blue assay. As shown in
Figure 6, no acute cytotoxicity of polyzymes was observed at
any concentrations used for biofilm treatment. This result
suggests that our polyzyme is a promising candidate to
eliminate pathogenic biofilms in the presence of mammalian
cells, important for wound therapy.
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moxifloxacin was used to treat a uropathogenic clinical isolate,
E. coli (CD-2) while pro-ciprofloxacin was applied to treat P.
aeruginosa (ATCC-27853). Biofilms were incubated in M9
media for 2 hrs with polyzyme at concentrations of 200 nM and
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μM, respectively. After washing three times using PBS,
biofilms were then incubated with different concentrations of
prodrugs. In these experiments, biofilms treated with prodrug
only and the combination of prodrug and polymer were used as
negative controls while those incubated with drugs were used
as positive controls. After incubating for 6 hrs, biofilm viability
was determined using an Alamar Blue assay.
Conclusion
In summary, we have described a new platform for the
application of bioorthogonal transition metal catalysts (TMCs)
in complex biological milieus. The catalytic polyzyme is
comprised of self-assembled polymer nanoparticles
functionalized with hydrophobic side chains to encapsulate
TMCs. Loading TMCs into the polymeric nanoparticles
enhances their stability and water solubility. The potential
therapeutic application of these polyzymes was demonstrated
via antimicrobial studies. Polyzymes featuring cationic surface
functionality provides the ability to penetrate into bacterial
biofilms. Significantly, polyzymes exhibit excellent catalytic
efficiency for drug activation and demonstrate high
antimicrobial activity within the biofilms at concentrations that
do not compromise the fibroblast cell viability. Taken together,
polyzyme-polyzymes provide a versatile strategy with potential
in antimicrobial therapies as well as broader biomedical
applications where in situ drug uncaging can be utilized.
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Figure 5c and 5d show the results obtained from bacterial
biofilms exposed to each treatment. As expected, in the
presence of the polyzyme, biofilms treated with prodrug
exhibited significant decreased viability with efficacy
essentially identical to moxifloxacin. In contrast, biofilms
treated with prodrug alone demonstrated negligible toxicity at
any concentration studied. Similarly, the combination of
prodrug and the polymer did not show toxicity to biofilms,
indicating that prodrug activation was mediated by catalyst
loaded in the polymer rather than polymer itself. The
antimicrobial efficacy of this strategy was also validated against
planktonic bacteria (Figure S5, Supporting Information).
Cytotoxicity assessment of polyzymes with mammalian
cells. We chose fibroblast cells for these studies due to their
51
importance in the wound healing process.
investigated the
ASSOCIATED CONTENT
Supporting Information. Materials; Synthesis of the
oxanorbornene Polymer; Synthesis of the polyzyme; Methods of
DLS and TEM; Zeta potential of the polyzyme; Quantification of
cytotoxicity of polyzymes to mammalian NIH 3T3 (ATCC
CRL-1658) fibroblast cells.
[Fe(TPP)]Cl loaded in polyzyme; Catalytic activity of Nanozymes;
Minimum inhibitory concentration (MIC) and Minimum biofilm
inhibitory concentration (MBIC) of Mox, Cip, pro-Mox and pro-
Cip; Quantitative enhancement of fluorescence intensity in
confocal study; Antimicrobial activity test with planktonic bacterial
cells; Activation of pro-dye and prodrug in bacterial biofilms;
Crystal violet assay; Synthesis of Pro-Resorufin and the
intermediates; Synthesis of Pro-Mox and Pro-Cip;
AUTHOR INFORMATION
Corresponding Author
*
E-mail: rotello@chem.umass.edu
Figure 6. Viability of 3T3 fibroblast cells after treating with
polyzyme for overnight. Each result is an average of three
experiments, and the error bars designate the standard deviations.
Notes
The authors declare no competing final interest.
ACKNOWLEDGMENT
This research was supported by NIH EB022641 and AI134770.
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