Protection and Isolation of Bioorthogonal Metal Catalysts by Using Monolayer-Coated Nanozymes

Article abstract of DOI:10.1002/cbic.202000207

We demonstrate here the protection of biorthogonal transition metal catalysts (TMCs) in biological environments by using self-assembled monolayers on gold nanoparticles (AuNPs). Encapsulation of TMCs in this hydrophobic environment preserves catalytic act

Full text of DOI:10.1002/cbic.202000207

Combining Chemistry and Biology
Accepted Article
Title: Protection and isolation of bioorthogonal metal catalysts using
monolayer-coated nanoparticle nanozymes
Authors: Xianzhi Zhang, Stefano Fedeli, Sanjana Gopalakrishnan, Rui
Huang, Aarohi Gupta, David C. Luther, and Vincent Rotello
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: ChemBioChem 10.1002/cbic.202000207
Link to VoR: https://doi.org/10.1002/cbic.202000207
01/2020

10.1002/cbic.202000207
ChemBioChem
COMMUNICATION
Protection and isolation of bioorthogonal metal catalystsusing
monolayer-coated nanoparticle nanozymes
Xianzhi Zhang, Stefano Fedeli, Sanjana Gopalakrishnan, Rui Huang, Aarohi Gupta, David C. Luther,
and Vincent M. Rotello*
E-mail: rotello@chem.umass.edu
*
Department of Chemistry
University of Massachusetts Amherst
710 North Pleasant Street, Amherst, Massachusetts 01003, United States
Supporting information for this article is given via a link at the end of the document.
Abstract: We present here demonstration of the protection of
biorthogonal transition metal catalysts (TMCs) in biological
environments using self-assembled monolayers on gold
nanoparticles (AuNPs). Encapsulation of transition metal catalysts
(TMCs) into this hydrophobic environment preserves catalytic activity
in presence of pH conditions and complex biological media that
deactivates free catalyst. Significantly, the protection afforded by
these ‘nanozymes’ extends to isolation of the catalyst ‘active site’, as
demonstrated by the independence of rate over a wide pH range, in
strong contrast to the behavior of the free catalyst.
Introduction
Bioorthogonal chemistry provides a tool for performing reactions
in biological system without interfering with natural processes.1-3
Bioorthogonal processes can be used to generate imaging and
therapeutic agents in biosystems with high specificity for
reactants. Bioorthogonal catalysis is a particularly attractive
strategy, providing access to enzyme-like systems.4-7 Transition
metal catalysts (TMCs) are excellent candidates for bioorthogonal
processes, featuring high reactivity, selectivity and ready access
to non-biological processes. 8 , 9 Researchers have developed
ruthenium-10-12 palladium-9,13-19 and iron-20 based catalysts for
generation of fluorophores and therapeutics as well as for in situ
protein modification. However, the direct use of TMCs is
challenging due to poor water solubility, low stability and limited
biocompatibility of these reactive systems.21 In particular, these
TMCs can be very sensitive to physiological pH variations, limiting
their applicability in biological systems.22
Incorporating TMCs in nanomaterials can enhance both
their solubility and stability in biological environments.8,23,24 As an
example, previous reports have demonstrated the ability to attach
Figure 1. Schematic representation of the insulation properties of the
nanozyme. (a) Structure of AuNP-TTMA containing the hydrophobic aliphatic
chain (black), the hydrophobic segment (green) and the cationic head group
(blue). (b) Structure of TMCs and encapsulation of TMCs within AuNP-TTMA.
(c) Catalytic deprotection of non-fluorescent pro-Rho into fluorescent Rho
though free TMCs or nanozymes. (d) Retention of catalytic activity in
nanozyme in different environments due to supramolecular interactions with
monolayer.
palladium catalyst on micron-size polystyrene scaffold for
25
therapeutic drug activation
, or into mesoporous silica
nanoparticles for controlled catalysis. 26
Recently, we have developed nano-sized reactors that
show enzymatic kinetic behavior (i.e. nanozymes) by
encapsulating TMCs into the self-assembled monolayers of 2 nm
AuNPs.23 Through appropriate design, these NZs are able to
respond to exogenous stimuli such as supramolecular
processes,8, 27 and localize at desired intracellular/extracellular,28
and biofilm locations. 29
1
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Figure 2. Characterization of AuNP-TTMA and NZ-TTMA. (a) and (b) TEM image of AuNP-TTMA (2.4±1.0 nm) and NZ-TTMA (1.8±1.1 nm) confirmed that no
aggregation occur after encapsulation (c) The DLS result showed similar hydrodynamic diameter of AuNP-TTMA and NZ-TTMA (d) Zeta potential of AuNP-TTMA
and NZ-TTMA indicated no significant change of surface charge. Each experiment had 3 replicates, and the error bars represent the standard deviation.
Here we demonstrate the ability of the particle monolayer to
protect the TMCs, preserving activity in complex biological media
as well as over the full range of physiological pH. Going well
beyond protection of the TMCs activity, the monolayer isolates the
catalyst from the outside environment, as demonstrated by
minimal changes in catalyst efficiency with pH, in stark contrast to
the free catalyst that showed strong pH dependence.
unbound TMCs (details in experimental section). The diameter of
the overall particle is ~7 nm in water with 2 nm core which is
verified by DLS and TEM measurements respectively and is not
affected by the encapsulation of the TMCs (Figure 2,
experimental details in Supporting information). The TMCs
loading was obtained by ICP-MS analysis, revealing ~20 TMCs
molecule per nanoparticle (Table S1).
The ability of the AuNP-TTMA scaffold to protect the TMCs
was evaluated first by measuring the catalytic activity in aqueous
media (pH = 7.4). The catalytic activity was evaluated by
measuring the increase in fluorescence intensity due to the
activation of pro-Rho. (Figure 1c) and compared with free TMCs.
Previous studies demonstrated that catalyst is retained in the
monolayer,27 so pro-dye activation occurred in situ. According to
the calibration curve of Rhodamine 110 (Figure S1), the rate of
activation was calculated in Figure 3. The results show a
remarkable difference in the behavior of the two systems. It is
seen in Figure 3a and Figure 3b that the free TMC is completely
deactivated within 4 h of incubation. On the contrary, ~60% of the
catalytic activity of the TMCs was retained even after 4 h with NZ-
TTMA. Clearly, the AuNP-TTMA monolayer can stabilize the TMC
over a prolonged duration in aqueous environments.
Result and discussion
AuNP (2 nm AuNP core) scaffolds (AuNP-TTMA) were designed
with three structural features (shown in Figure 1a) – (1) a
hydrophobic aliphatic monolayer (2) a hydrophilic tetra ethylene
glycol spacer to enhance biocompatibility (3)
a terminal
quaternary ammonium to improve solubility in aqueous
environment. The hydrophobic monolayer has been used
previously to encapsulate hydrophobic drugs30 as well as TMCs.8
Figure 1b shows the TMCs utilized in this study Cp*Ru(cod)Cl
[(Cp*
=
pentamethylcyclopentadienyl
cod
=
1,5-
cyclooctadiene)].10 This TMC is able to perform the uncaging of
the allyoxycarbonyl group on substrates. The substrate utilized is
the non-fluorescent pro-Rhodamine 110 (pro-Rho) (Figure 1c)
which is converted into the fluorescent Rhodamine 110 (Rho)
upon catalysis, providing straightforward determination of activity.
The nanozyme (NZ-TTMA) was synthesized from
pentanethiol capped core AuNP-TTMA using a place exchange
reaction.23 Then the TMCs were encapsulated into the monolayer
through nanoprecipitation followed by ultrafiltration to remove the
We next evaluated the stability of the NZ-TTMA in simulated
biological environments. For this, the catalytic activity of the NZ-
TTMA as well as the free TMCs were measured in 1% serum
concentration. As can be seen in Figure 4, the catalytic activity of
Figure 3. Stability of nanozyme in aqueous media. (a) Catalytic activity of free TMCs with time. For the free TMCs, catalytic activity drastically reduced within 4 h.
(b) Catalytic activity of nanozyme with time. For nanozyme no significant change in activity was observed. (c) Comparison of rates of activation of TMC and nanozyme
at 0,1,2 and 4h. Each experiment had 3 replicates, and the error bars represent the standard deviation.
2
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Figure 4. Stability of NZ-TTMA in serum. (a) Catalytic activity of free TMCs with time. The activity of the free TMCs was drastically reduced in serum. (b) Catalytic
activity of NZ-TTMA with time. For nanozyme no significant change in activity is observed between 0.5 and 1h. (c) Comparison of rates of activation of TMCs and
NZ-TTMA at 0, 0.5 and 1 h. Each experiment had 3 replicates, and the error bars represent the standard deviation.
the NZ-TTMA remains unchanged while the activity of the free
TMCs drops significantly with over 30 min. The catalytic activity of
the NZ-TTMA is slightly reduced with respect to aqueous media,
attributed to the formation of a protein corona31 around cationic
nanoparticles that modestly decreases activity in NZ-TTMA
featuring surfaces such as NZ-TTMA that resist irreversible
protein binding.32
Having established protection of the catalyst, we next
explored the ability of the monolayer to isolate the TMC from the
external environment. To test for isolation, we measured catalytic
activity at different pH. According to literature, the product
Rhodamine is not sensitive to pH change.33, 34 Figure 5 shows a
dramatic difference between the activity of the two systems at
physiological and acidic pH. Free TMCs have excellent catalytic
activity at pH = 4.1, but over 99% of the activity was lost at pH =
5.5 and 7.4. In contrast, the NZ-TTMA retained comparable
activity over the range of pH 4.1-7.4. This result indicates that the
AuNP monolayer is able to shield the encapsulated TMCs from
the external environment, including acids. We hypothesize that
the hydrophobic pocket generated by strong supramolecular
interactions between the TMCs and the aliphatic monolayer is
responsible for the insulating property of the AuNP scaffold.
In summary, we have demonstrated that the monolayer of AuNP-
TTMA protects and isolates biorthogonal TMC catalysts - through
supramolecular interactions. Encapsulation of catalyst into AuNP-
TTMA hydrophobic pockets eliminates environmental effects,
preserving high catalytic activity. This NZ-TTMA strategy provides
highly promising strategy for the application of bioorthogonal
catalysis in a wide range of fundamental, imaging, and diagnostic
uses.
Experimental section
Encapsulation of ruthenium catalyst into AuNP-TTMA: 2.0 mg
of ruthenium catalyst were dissolved in 1 mL of acetone and
added dropwise to 1 mL of 15 µM of AuNP-TTMA. The resulting
solution was stirred at room temperature for half an hour followed
by slowly evaporating acetone by Rotavapor. Excess of TMC was
removed by 0.22 µm PES membrane filter. Then the dispersion
was washed with ultra-centrifugal filters (MWCO = 10 KDa),
washing with Milli-Q water three more times after no color
observed in the flow through. The concentration of NZ-TTMA was
measured by the absorption at 506 nm and the TMC amount was
measured by ICP-MS by tracking 101Ru and 197Au.
Synthesis of pro-Rhodamine: Rhodamine 110 (1 eq, 100mg)
and pyridine (70µL, 3.2 eq) was dissolved in 2mL
dimethylformamide (DMF) in ice bath. Allyl chloroformate (87 µL,
Conclusion
Figure 5. Stability of nanozyme in acidic and physiological pH. (a) Catalytic activity of free TMCs at pH = 4.1, 5.5 and 7.4. It was observed that activity
drastically reduced within at pH = 5.5 as compared to pH = 4.1(b) Catalytic activity of nanozyme with time. No significant change in activity was observed (c)
Comparison of rates of activation of TMCs and nanozymes at pH = 4.1, 5.5 and 7.4. Each experiment had 3 replicates, and the error bars represent the standard
deviation.
3
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3 eq) was added to the solution by dropwise. The resulting
solution was stirred in ice bath for two hours and warmed up to
room temperature overnight. The product was purified by a
column with 2:1 hexane to ethyl acetate as eluent. Pro-
Rhodamine 110 was obtained as a pinkish white powder. 1H-NMR
(400 MHz, DMSO-d6) 10.05 (s, 2H), 8.0 (d, 1H), 7.77 (t, 1H) 7.7
(t, 1H), 7.55 (s, 2H), 7.24 (d, 1H), 7.14 (d, 2H), 6.69 (d, 2H), 5.8
(m, 2H), 5.35 (d, 2H), 5.22 (d, 2H), 4.61 (d, 4H).
Protection from NZ-TTMA: NZ-TTMA and free TMC were
incubated in PBS (pH = 7.4) for 4h, 2h, 1h and 0h or PBS
containing 1% serum for 0h, 0.5h and 1h at room temperature
prior kinetic study. After incubation, a solution containing 10 µM
of pro-Rho and 200 nM NZ-TTMA or 4 µM free TMC was prepared
in 96 well black plate. The kinetics study was done by detecting
the increase of fluorescence of Rhodamine (λex = 488 nm, λem
=
521 nm) by Molecular Devices SpectraMax M2 microplate reader
at 37 °C. Each experiment comprised three replicates
Kinetic study of insulation: 10 µL of pro-Rho in DMSO was
added into 96 well black plate, followed by adding 100 µL of NZ-
TTMA or free TMC solution in PBS buffer (pH = 7.4, 5.5 and 4.1),
obtaining the final concentration of 10 µM of pro-Rho and 200 nM
of NZ-TTMA or 4 µM free TMC. The kinetic study was based on
tracking the increase of fluorescence intensity of Rhodamine (λex
= 488 nm, λem = 521 nm) by Molecular Devices SpectraMax M2
microplate reader at 37 °C. Each experiment comprised three
replicates.
Acknowledgements
This research was supported by NIH EB022641
Keywords: Bioorthogonal catalysis • nanozymes • protection •
insulation • monolayer
4
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Entry for the Table of Contents
We demonstrate the retained catalytic activity of bioorthogonal transition metal catalysts through encapsulation within the
hydrophobic monolayer of gold nanoparticles. This encapsulation strategy stabilizes the catalyst in environmental conditions like
serum, prevents degradation with time and insulates the catalyst to changes in pH. This allows for in situ generation of imaging and
therapeutic agents in biological systems.
Institute and/or researcher Twitter usernames:
References
1
C. R. Bertozzi, Acc. Chem. Res. 2011, 44, 651–653.
2.
J. A. Prescher, C. R. Bertozzi, Nat. Chem. Biol. 2005, 1, 13–21.
3.
4
E. M. Sletten, C. R. Bertozzi, Angew. Chemie - Int. Ed. 2009, 48, 6974–6998.
R. Cao-Milan, L. D. He, S. Shorkey, G. Y. Tonga, L.-S. Wang, X. Zhang, I. Uddin, R. Das, M. Sulak, V. R. Rotello, Mol. Syst. Des. Eng., 2017,2, 624-628
5
6
7
J. Li, P. R. Chen, Nat. Chem. Biol. 2016, 12, 129–137.
J. Wu, X. Wang, Q. Wang, Z. Luo, S. Li, Y. Zhu, L. Qin, H. Wei, Chem. Soc. Rev. 2019, 48, 1004-1076.
Y. Huang, J. Ren, X. Qu, Chem. Rev. 2019, 119, 4357-4412
8
G. Y. Tonga, Y. Jeong, B. Duncan, T. Mizuhara, R. Mout, R. Das, S. T. Kim, Y. C. Yeh, B. Yan, S. Hou, et al., Nat. Chem. 2015, 7, 597–603.
F. Wang, Y. Zhang, Z. Du, J. Ren, X. Qu, Nat. Commun. 2018, 9, 1209.
9
10
11
12
13
14
15
16
C. Streu, E. Meggers, Angew. Chemie Int. Ed. 2006, 45, 5645–5648.
C. Vidal, M. Tomástomás-Gamasa, A. Gutiérrezgutiérrez-Gonzaíez, J. J. Mascareñ, J. Am. Chem. Soc 2019, 141, 5129.
T. Völker, E. Meggers, Curr. Opin. Chem. Biol. 2015, 25, 48–54.
J. M. Chalker, C. S. C. Wood, B. G. Davis, J. Am. Chem. Soc. 2009, 131, 16346–16347.
J. Li, J. Yu, J. Zhao, J. Wang, S. Zheng, S. Lin, L. Chen, M. Yang, S. Jia, X. Zhang, et al., Nat. Chem. 2014, 6, 352–361.
R. M. Yusop, A. Unciti-Broceta, E. M. V. Johansson, R. M. Sánchez-Martín, M. Bradley, Nat. Chem. 2011, 3, 239–243.
M. Sancho-Albero, B. Rubio-Ruiz, A. M. Pérez-López, V. Sebastián, P. Martín-Duque, M. Arruebo, J. Santamaría, A. Unciti-Broceta, Nat. Catal. 2019, 2,
864–872.
17
18
19
20
M. A. Miller, B. Askevold, H. Mikula, R. H. Kohler, D. Pirovich, R. Weissleder, Nat. Commun. 2017, 8, 15906.
P. Destito, A. Sousa-Castillo, J. R. Couceiro, F. López, M. A. Correa-Duarte, J. L. Mascareñas, Chem. Sci. 2019, 10, 2598–2603.
M. Martínez-Calvo, J. R. Couceiro, P. Destito, J. Rodríguez, J. Mosquera, J. L. Mascareñas, ACS Catal. 2018, 8, 6055–6061.
P. K. Sasmal, S. Carregal-Romero, A. A. Han, C. N. Streu, Z. Lin, K. Namikawa, S. L. Elliott, R. W. Köster, W. J. Parak, E. Meggers, ChemBioChem 2012,
13, 1116–1120.
21
22
23
24
25
M. Martínez-Calvo, J. L. Mascareñas, Coord. Chem. Rev. 2018, 359, 57–79.
Y. Bai, J. Chen, S. C. Zimmerman, Chem. Soc. Rev. 2018, 47, 1811–1821.
X. Zhang, R. Huang, S. Gopalakrishnan, R. Cao-Milán, V. M. Rotello, Trends Chem. 2019, 1, 90–98.
Y. Jeong, G. Y. Tonga, B. Duncan, B. Yan, R. Das, C. Sahub, V. M. Rotello, Small 2018, 14, 1702198.
A. Unciti-Broceta, E. M. V. Johansson, R. M. Yusop, R. M. Sánchez-Martín, M. Bradley, Nat. Protoc. 2012, 7, 1207–1218.
26
27
P. Destito, A. Sousa-Castillo, J. R. Couceiro, F. López, M. A. Correa-Duarte, J. L. Mascareñas, Chem. Sci. 2019, 10, 2598–2603.
R. Cao-milan, S. Gopalakrishnan, L. D. He, R. Huang, L. Wang, L. Castellanos, D. C. Luther, R. F. Landis, J. M. V Makabenta, C. Li, et al., Chem 2020,
https://doi.org/10.1016/j.chempr.2020.01.015
28
R. Das, R. F. Landis, G. Y. Tonga, R. Cao-Milán, D. C. Luther, V. M. Rotello, ACS Nano 2019, 13, 229–235.
29
30
A. Gupta, R. Das, G. Yesilbag Tonga, T. Mizuhara, V. M. Rotello, ACS Nano 2018, 12, 89–94.
C. K. Kim, P. Ghosh, C. Pagliuca, Z. Zhu, S. Menichetti, V. M. Rotello, J. Am. Chem. Soc. 2009, 131, 1360–1361.
31
32
K. Saha, M. Rahimi, M. Yazdani, S. T. Kim, D. F. Moyano, S. Hou, R. Das, R. Mout, F. Rezaee, M. Mahmoudi, et al., ACS Nano 2016, 10, 4421–4430.
X. Zhang, Y. Liu, S. Gopalakrishnan, L. Castellanos-Garcia, G. Li, M. Malassine, I. Uddin, R. Huang, D. C. Luther, R. W. Vachet, et al. ACS Nano 2020,
https://doi.org/10.1021/acsnano.0c00629
33
S. Brauch, M. Henze, B. Osswald, K. Naumann, L. A. Wessjohann, S. S. Van Berkel, B. Westermann, Org. Biomol. Chem., 2012, 10, 958-965
5
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COMMUNICATION
34
J. Gooch, V. Abbate, B. Daniel, N. Frascione, Analyst, 2016,141, 2392-2395
6
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