Desilylation Induced by Metal Fluoride Nanocrystals Enables Cleavage Chemistry in Vivo

Article abstract of DOI:10.1021/jacs.0c10399

Metal fluoride nanocrystals are widely used in biomedical studies owing to their unique physicochemical properties. The release of metal ions and fluorides from nanocrystals is intrinsic due to the solubility equilibrium. It used to be considered as a drawback because it is related to the decomposition and defunction of metal fluoride nanocrystals. Many strategies have been developed to stabilize the nanocrystals, and the equilibrium concentrations of fluoride are often <1 mM. Here we make good use of this minimum amount of fluoride and unveil that metal fluoride nanocrystals could effectively induce desilylation cleavage chemistry, enabling controlled release of fluorophores and drug molecules in test tubes, living cells, and tumor-bearing mice. Biocompatible PEG (polyethylene glycol)-coated CaF2 nanocrystals have been prepared to assay the efficiency of desilylation-induced controlled release of functional molecules. We apply the strategy to a prodrug activation of monomethyl auristatin E (MMAE), showing a remarkable anticancer effect, while side effects are almost negligible. In conclusion, this desilylation-induced cleavage chemistry avails the drawback on empowering metal fluoride nanocrystals with a new function of perturbing or activating for further biological applications.

Full text of DOI:10.1021/jacs.0c10399

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Article
Desilylation Induced by Metal Fluoride Nanocrystals Enables
Cleavage Chemistry In Vivo
Dongban Duan, Hao Dong, Zhiyu Tu, Chunhong Wang, Qunfeng Fu, Junyi Chen, Haipeng Zhong,
Ping Du, Ling-Dong Sun, and Zhibo Liu*
Cite This: J. Am. Chem. Soc. 2021, 143, 2250−2255
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sı Supporting Information
ABSTRACT: Metal fluoride nanocrystals are widely used in
biomedical studies owing to their unique physicochemical
properties. The release of metal ions and fluorides from
nanocrystals is intrinsic due to the solubility equilibrium. It used
to be considered as a drawback because it is related to the
decomposition and defunction of metal fluoride nanocrystals.
Many strategies have been developed to stabilize the nanocrystals,
and the equilibrium concentrations of fluoride are often <1 mM.
Here we make good use of this minimum amount of fluoride and
unveil that metal fluoride nanocrystals could effectively induce
desilylation cleavage chemistry, enabling controlled release of
fluorophores and drug molecules in test tubes, living cells, and
tumor-bearing mice. Biocompatible PEG (polyethylene glycol)-coated CaF nanocrystals have been prepared to assay the efficiency
2
of desilylation-induced controlled release of functional molecules. We apply the strategy to a prodrug activation of monomethyl
auristatin E (MMAE), showing a remarkable anticancer effect, while side effects are almost negligible. In conclusion, this
desilylation-induced cleavage chemistry avails the drawback on empowering metal fluoride nanocrystals with a new function of
perturbing or activating for further biological applications.
19,20
INTRODUCTION
and NaF under aqueous conditions.
Recently, our group
■
constructed a bioorthogonal system based on organotrifluor-
oborate-mediated desilylation and achieved tumor-selective
targeting for controlled release of a client protein from the
Metal fluoride nanocrystals (NCs) such as alkaline earth
fluorides and rare earth fluorides have been widely used in
1
−4
5,6
7−9
bioimaging,
drug delivery, and biosensing,
owing to
21
their unique and advantageous optical, electrical, and magnetic
endocytosed nanoparticles. We wondered whether metal
fluoride NCs could also trigger efficient desilylation and if so
whether the reaction could be developed into a useful
controlled release system, therefore making metal fluoride
NCs a perturbing matrix for controlled release of functional
1
0
properties. The tunable size and morphology, controllable
excitation and emission behaviors, and varied surface
modifications make metal fluorides of high relevance in
biomedical applications.
be chemically stable in water to maintain their structural
integrity and optical properties. However, the release of
metal ions and fluoride is intrinsic due to solubility
equilibrium, especially under highly diluted or acidic
11,12
Ideally, metal fluoride NCs should
−
13
molecules. Besides, compared to small molecules such as F or
a trifluoroborate-derived amino acid for desilylation, metal
fluoride NCs may benefit from the enhanced permeability and
retention (EPR) effect for tumor targeting and give sustained
1
4,15
conditions.
To protect the metal fluoride NCs from
disintegration, various surface coatings have been successfully
developed. The decompositions of metal fluoride NCs are
often <5% while the equilibrium fluoride concentrations are <1
−
F release for the localized desilylation reaction, empowering
metal fluoride NCs to rescue the “function-caged” biomole-
cules in a tumor-selective manner.
1
6,17
mM.
Although the dissolution of metal fluoride NCs is undesired,
the resulting product fluoride (F ) could trigger a rapid and
−
Received: September 29, 2020
Published: February 1, 2021
efficient desilylation reaction and is commonly used to remove
18
the protecting silyl ether group in organic synthesis. Various
silyl ether “caged” fluorescent probes have been successfully
developed as fluoride sensors, which could be readily activated
by tetrabutylammonium fluoride (TBAF), hydrofluoric acid,
©
2021 American Chemical Society
https://dx.doi.org/10.1021/jacs.0c10399
J. Am. Chem. Soc. 2021, 143, 2250−2255
2
250

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Article
As shown in Figure 1c, the equilibrium concentrations of F−
were determined by a fluoride electrode and are 0.45, 1.9, 7.4,
RESULTS AND DISCUSSION
■
To pursue this idea, we have developed six model metal
fluoride NCs to evaluate their effectiveness for desilylation
reaction. Figure 1a shows the transmission electron microscope
0
.059, 0.13, and 0.44 mM from 1 mg/mL CaF , SrF , BaF ,
2 2 2
LaF , NaYF , and NaYF @CaF NCs, respectively. These
3
4
4
2
results corroborate with the solubility of different metal
fluoride NCs and are slightly higher than the equilibrium
concentrations calculated from Ksp (solubility product
constant) that was measured when they are not nanoscale
−
(
Table S1). The greater F release from nanosized metal
−
fluoride NCs was further verified by comparing the F release
kinetics of CaF of different sizes (Figure S3), which could be
2
explained by the additional surface energy as the particle sizes
2
5,26
decrease.
It is worth noting that the equilibrium
−
concentrations of F from CaF , SrF , BaF , and NaYF @
2
2
2
4
CaF NCs are >0.44 mmol/L, which are abundant for driving
2
the desilylation reaction. To evaluate the effectiveness of metal
fluoride NC induced desilylation, we developed an ortho-
carbamoylmethylene silyl-phenolic ether system, in which the
carbamate carbon is linked to the client molecule MeRho
(
Figure 1b). Desilylation of this system will trigger the
elimination of the carbamate, leading to the breakdown of the
amide bond and therefore release the highly fluorescent
MeRho. As opposed to the amine-blocked MeRho in the silyl−
phenolic ether system, the bright fluorescence of MeRho thus
provides a simple and quantitative assay for metal fluoride NC
induced desilylation.
The desilylation assays with different metal fluoride NCs
were performed under physiological conditions. The increased
fluorescence signal from decaged TBS−MeRho was recorded
to obtain the kinetic curve for each NC (Figure 1d). As
−
expected, the desilylation reaction rates are highly F -
dependent in the order BaF > SrF > CaF ≈ NaYF @CaF
2
2
2
4
2
>
NaYF > LaF . In addition, the reaction selectivity of
4 3
desilylation has been investigated by treating TBSO−MeRho
with various biologically relevant or corresponding metal ions
Figure 1. Metal fluoride NCs induced desilylation reaction enabled
controlled release of fluorophore under aqueous solution. (a)
Representative TEM images of CaF , SrF , BaF , LaF , NaYF , and
−
−
−
−
−
−
2+
(
1 mM), including Cl , Br , I , BF , NO , H PO , Ca ,
4 3 2 4
2+
2+
3+
3+
−
Sr , Ba , La , and Y ; only the F ion can efficiently trigger
the release of free MeRho (Figure S4), indicating the
possibility of using metal fluoride NCs for desilylation
reactions in living systems. For the subsequent biological
assays, CaF2 NCs were chosen due to their sufficient
desilylation rate and the low toxicity of metal ions in contrast
2
2
2
3
4
NaYF @CaF NCs. (b) Desilylation-to-release of fluorescent MeRho
4
2
−
from TBS-MeRho as a model reaction. (c) F concentrations of metal
fluoride NCs in aqueous suspensions (1 mg/mL) measured by a
fluoride-ion-selective electrode at 25 °C. (d) Kinetic profile of the
desilylation reaction for different metal fluoride NCs (1 mg/mL) via
fluorogenic uncaging of TBS-MeRho (20 μM) in HEPES buffer (10
mM, pH 7.4) solution at 37 °C.
27,28
to SrF NCs and BaF NCs.
2
2
To achieve better colloidal stability and biocompatibility,
CaF NCs were surface-coated by PEG, and the coating was
2
confirmed by FTIR spectroscopy and thermogravimetric
(
TEM) images of CaF , SrF , BaF , LaF , NaYF , and NaYF @
2
2
2
3
4
4
CaF NCs synthesized with thermal decomposition combined
with a seed-mediated shell growth strategy.
2
16,22,23
All of the
metal fluoride NCs exhibit uniform morphology and good
monodispersity. To confirm the core−shell structure of
NaYF @CaF NCs, high-angle annular dark-field scanning
transmission electron microscopy and dispersive X-ray spec-
troscopy line scanning were used, and no separate CaF2
nanoparticles were observed (Figure S1). The X-ray diffraction
4
2
(
XRD) patterns (Figure S2) suggest that CaF , SrF , BaF ,
2
2
2
NaYF , and NaYF @CaF NCs exhibit a face-centered-cubic
In fact, PEG coatings will slightly slow down the dissolution,
but show a limited impact on the dissolution equilibrium
(Figure S9), which corroborates the previous report. We
4
4
2
phase and LaF NCs exhibit a hexagonal phase. These metal
3
25
fluoride NCs were coated with oleic acid, which are rather
lipophilic and easy to aggregate under aqueous conditions. We
removed this lipophilic coating by using a ligand exchange
have also recorded the dissolution procedure of CaF −PEG
2
NCs by TEM. As shown in Figure 2a, after treating CaF −PEG
2
2
4
method, and the resulting metal fluoride NCs are hydrophilic
NCs (0.1 mg/mL) with acetate buffers (10 mM, pH = 7.4) at
37 °C for 6 h, the cubic morphology gradually transformed
and well-dispersed in water.
2
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desilylation reaction was then investigated in a cell-based
assay. Cells were incubated with TBS−MeRho and CaF −PEG
2
NCs and analyzed by live-cell fluorescence microscopy (Figure
2
b). U78MG cells showed a 5.6-fold increase in fluorescence of
the entire cell population compared to untreated cells (Figure
c). The similar experimental results were obtained in
2
BGC823 cells and B16F10 cells (Figure S14). This suggests
that CaF −PEG NC induced cleavage chemistry remains
2
functional in vitro.
Peptide−drug conjugates (PDCs) hold promise in anti-
cancer therapy, but suffer from uncontrolled and inefficient
3
0
release of the drug. We applied CaF −PEG NC desilylation
2
to the PDC system. Specifically, the TBS ether-containing
carbamate was used to conjugate monomethyl auristatin E
(
3
MMAE) to RGD (α β integrin targeting peptide) (Figure
v 3
a). CaF −PEG NC incubation caused about 90% MMAE
2
Figure 2. Biocompatible CaF −PEG NCs are prepared for
2
desilylation in vitro. (a) TEM images to record the morphology
changes of CaF −PEG NCs (0.1 mg/mL) in acetate buffers (10 mM,
2
pH = 6.0 or 7.4) at 37 °C. (b) Representative confocal images of
U87MG cells after treatment with 0.2 mg/mL CaF −PEG NCs, 20
2
μM TBS−MeRho, or the combination for 4 h. Scale bars, 20 μm. (c)
Quantification of the cellular fluorescence intensities with the
treatment of CaF −PEG NCs or CaF −PEG NCs + TBS−MeRho,
2
2
respectively. The data were collected from the ROIs (n = 20) within
the cells.
into a sphere and the size of the nanoparticles showed a slight
decrease from 23.1 ± 3.2 nm to 22.3 ± 3.3 nm. It was
noteworthy that the dissolution was more obvious in acid
conditions (pH = 6.0). Almost all cubic NCs transformed into
spheres, and the particle size decreased from 22.9 ± 3.1 nm to
2
0.3 ± 3.6 nm after 6 h of treatment. Moreover, during the
degradation process, small-sized residues could be observed in
Figure 3. CaF −PEG NC desilylation triggered controlled release of
2
the carbon film and the surface of nanoparticles, which was
drugs and its application to a peptide−drug conjugate. (a) Scheme
showing the RGD−MMAE being converted by fluoride into uncaged
MMAE. (b) Quantification of the released MMAE from RGD−
also observed for CaF NCs without a PEG coating (Figure
2
S10). Given the dissolution kinetics results as mentioned
above, we believe that the small-sized residues could be the
MMAE (1 μM) after incubation with CaF −PEG NCs (0.2 mg/mL)
2
for the indicated time points. (c) Cell viability assays of U87MG cells
recrystallized CaF nanoparticles formed after the solution
2
treated with MMAE, RGD−MMAE, and RGD−MMAE + CaF −
2
becomes saturated. Collectively, we identified CaF −PEG NCs
2
PEG NCs, respectively (n = 5).
−
as a sustained F -releasing source which is promising to power
the desilylation reaction in a living system.
Before testing the desilylation reactivity of CaF −PEG NCs
release from the RGD−MMAE conjugate (Figure 3b and
Figure S15). By treating U87MG cells with RGD−MMAE and
CaF −PEG NCs, the IC of the prodrug showed a 22-fold
2
in vitro, the cytotoxicity of CaF −PEG NCs has been
2
evaluated systematically. The cell viability was greater than
2
50
8
7
0% after incubation with 400 μg/mL of CaF −PEG NCs for
decrease from 59.6 nM to 2.7 nM, which is close to the
2
31
2 h (Figure S11), proving that CaF −PEG NCs were
potency of MMAE (IC , 1.3 nM to U87MG cells). An
2
50
biocompatible in live cells. The cellular uptake of CaF −PEG
NCs was evaluated by ICP-OES and confocal microscopy
inhibition assay of tubulin polymerization has been performed
that further validated the generation of MMAE from the
2
32
(Figure S12). The concentration of calcium in U87MG cells
prodrug (Figure S16). By treating U87MG cells with RGD−
increased over time and reached 12.8 pg/cell after 6 h of
MMAE only (10 nM) or CaF −PEG NCs only (0.2 mg/mL),
2
incubation. Interestingly, CaF −PEG NCs within cells are
no significant change of microtubule network was observed,
2
spherical but not cubic (Figure S13), which could be attributed
while the treatment of RGD−MMAE+CaF −PEG NCs caused
2
2
9
to the dissolution of CaF −PEG NCs in cells. The
substantial disruption to the cellular microtubule network,
2
2
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causing them to round up into clusters that then underwent
mice, the volume of U87MG tumors increased by about 20-
apoptosis. Together, these results indicate that RGD−MMAE
fold in 20 days (Figure 4d). Upon three rounds of RGD−
could be activated effectively by CaF −PEG NCs in vitro.
MMAE + CaF −PEG NC treatments, remarkable tumor
2
2
To accomplish desilylation-mediated controlled release of
drugs in the tumor, a selective, intense, and sustainable tumor
accumulation of F− source is essential. [ F]NaF positron
emission tomography (PET) indicates that blood clearance of
shrinkage occurred (Figure 4d). Injection of RGD−MMAE or
CaF −PEG NCs alone or RGD−MMAE + NaF did not cause
2
18
tumor shrinkage. Tumor weight measurements confirmed the
antitumor effect caused by desilylation-released MMAE
−
free F is rapid and mainly through the renal system (Figure
(Figure 4e, Figure S20).
Considering that the release F from CaF −PEG NCs might
diffuse and liberate prodrugs out of the tumor cells, the
−
S17). The bone uptake is highly intense, which has been well
2
3
3,34
89
known from the previous reports.
In contrast, [ Zr]CaF −
2
PEG NC PET imaging shows elongated blood circulation and
notable tumor accumulation (Figure 4a and Figures S18 and
biosafety of the treatment has been further evaluated. Mice
treated with RGD−MMAE + CaF −PEG NCs did not show
2
significant changes in body weight during the treatment, and
almost no tissue damage in the primary organs was observed
from histology analysis (Figures S21 and 22). In order to assess
whether CaF −PEG NCs would cause any long-term toxicity,
2
a series of experiments including blood chemistry and bone
microstructure analysis have been performed (Figures S23 and
S24). The parameters of alkaline phosphatase, calcium, and the
skeletons of mice treated with CaF −PEG NCs did not
2
significantly change compared with untreated mice. Collec-
tively, these data suggested the promising potential of the
CaF −PEG NCs in desilylation-mediated prodrug activation
2
without causing long-term side effects.
CONCLUSIONS
■
In conclusion, we take CaF NCs as an example to show that
the metal fluoride NCs could release adequate F to trigger the
2
−
desilylation chemistry both in vitro and in vivo. The nanometer
dimension of metal fluoride NCs enables elongated blood
circulation and enhanced tumor permeability compared with
small molecules, benefiting persistent tumor treatment. The
fluoride NCs should be versatile for all fluoride-containing
compounds, especially the less stable fluoride NCs such as
biodegradable ones. More importantly, the fluoride NCs can
further be functionalized with optical and magnetic properties
through doping techniques for imaging-guided theranostics.
This work offers exciting opportunities for the design of more
intriguing and valuable inorganic−organic prodrugs for on-
demand biological applications.
Figure 4. CaF −PEG NCs + RGD−MMAE treatment causes tumor
2
regression in mice. (a) Representative whole-body PET/CT imaging
of U87MG tumor-bearing mice at the indicated time points after i.v.
1
8
89
administration of 200 μCi of [ F]NaF or [ Zr]CaF −PEG NCs. t,
2
8
9
tumor; l, liver; b, bone. (b) Time−activity curve of [ Zr]CaF −PEG
2
NCs in the liver, blood, tumor, and muscle in mice. (c) Treatment
scheme. (d) Tumor growth curves of tumor-bearing mice after the
indicated treatments (n = 6). (e) Average tumor weight at 20 days
after the first treatment (mean ± s.e.m., n = 6).
METHODS
■
Measurement of Fluoride Ion Concentration of Nanosized
Metal Fluorides. The metal fluoride NC dispersion (20 mg, 1 mL)
was first prepared by sonication for 5 min. The particles were
collected from the solution by centrifugation (18000g, 20 min) and
dispersed in 20 mL of water. The fluoride-ion-selective electrode
S19). According to the 3D region-of-interest (ROI) analysis,
8
9
tumor uptake of [ Zr]CaF −PEG NCs increases until
2
(
PXSJ-226, Shanghai Precision & Scientific Instrument Inc., Shanghai,
reaching a plateau of 7.57 ± 0.71% injected dose per gram
%ID/g), which is >0.3 mg/mL within the tumor at 6 h after
China) was immersed in a metal fluoride NC solutions directly. The
fluoride ion concentration was obtained by the standardized curve
method.
(
tail-vein injection (Figure 4b). As a comparison, 0.1 mg/mL
CaF −PEG NCs were used for the aforementioned fluoride
2
Fluoride Ion Release Kinetics Measured by a Fluoride-Ion-
Selective Electrode. The ion release experiments were conducted in
a 125 mL plastic beaker containing 100 mL of a 10 mM acetate buffer
release assay (Figure S8), and the fluoride concentration would
reach 0.1 mM within 1 min. Although the local fluoride
concentration in the tumor might be decreased due to the
diffusion, it should be enough to desilylate and activate the
at 25 °C. A 0.5 mL amount of CaF NCs (20 mg/mL calculated by
2
CaF ) was added to the medium with stirring, and the fluoride ion
2
−
8
9
concentration (displayed by −log[F ]) was measured in situ by the
fluoride-ion-selective electrode. The final concentration of CaF was
MMAE prodrug. Noteworthily, [ Zr]CaF −PEG NCs exhibit
2
2
sustained retention in tumors up to 24 h, and the results from
an ex vivo biodistribution assay (Figure S18) corroborate with
0
.1 mg/mL.
Visual Detection of Disintegration with TEM. The 0.1 mg/mL
the PET imaging, showing that CaF −PEG NCs could be a
2
dilutions of CaF NCs were made with 0.01 M acetate buffer (pH = 6
−
2
sustainable F source for desilylation-mediated drug release in
or pH = 7.4) and incubated with slow shaking at 37 °C. At
appropriate time points, a 2 μL droplet of the samples was placed on a
lacey carbon film supported on a TEM copper grid, dried at 37 °C,
and analyzed with a JEOL JEM-2100 TEM operated at 200 kV.
tumors.
The treatment efficacy of this prodrug activation system was
evaluated on U87MG tumor-bearing mice. In PBS-treated
2
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Prodrug Activation In Vitro. Cells were plated on flat-bottom
and Molecular Engineering, Peking University, Beijing
00871, China
Haipeng Zhong − Beijing National Laboratory for Molecular
Sciences, Radiochemistry and Radiation Chemistry Key
Laboratory of Fundamental Science, College of Chemistry
and Molecular Engineering, Peking University, Beijing
3
9
6-well plates at a density of 3 × 10 per well. After 12 h of
1
attachment, cells were treated with CaF −PEG NCs (0.2 mg/mL),
2
RGD−MMAE (10 nmol/L), or a combination for 72 h before
measuring the viability by the CCK8 assay.
Antitumor Efficacy Study. In vivo therapy experiments were
3
performed when the tumor volume reached 50 mm in average
100871, China
diameter (7 days after implant). The mice were divided into five
groups (n = 6). Mice were intravenously injected with CaF −PEG
Ping Du − Beijing National Laboratory for Molecular Sciences,
State Key Laboratory of Rare Earth Materials Chemistry and
Applications, PKU-HKU Joint Laboratory in Rare Earth
Materials and Bioinorganic Chemistry, College of Chemistry
and Molecular Engineering, Peking University, Beijing
2
NCs (100 mg/kg) or NaF (5 mg/kg) on day 0, 4, and 8, and RGD−
MMAE (1 mg/kg) was injected intravenously 8 h later. The mice
were monitored for 20 days after treatment. The average and
distribution of the weights of the tumors collected from animals in
each treatment group were also recorded at the time of sacrifice.
100871, China
Ling-Dong Sun − Beijing National Laboratory for Molecular
Sciences, State Key Laboratory of Rare Earth Materials
Chemistry and Applications, PKU-HKU Joint Laboratory in
Rare Earth Materials and Bioinorganic Chemistry, College of
Chemistry and Molecular Engineering, Peking University,
Beijing 100871, China
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c10399.
■
*
Experimental details, characterization of organic mole-
cules, supplementary figures (PDF)
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c10399
AUTHOR INFORMATION
Corresponding Author
■
Notes
The authors declare no competing financial interest.
Zhibo Liu − Beijing National Laboratory for Molecular
Sciences, Radiochemistry and Radiation Chemistry Key
Laboratory of Fundamental Science, College of Chemistry
and Molecular Engineering and Peking-Tsinghua Center for
Life Sciences, Peking University, Beijing 100871, China;
Beijing Tiantan Hospital, Advanced Innovation Center for
Human Brain Protection, Capital Medical University, Beijing
ACKNOWLEDGMENTS
■
This work was financially supported by National Natural
Science Foundation of China (no. NSFC U1867209 and no.
NSFC 21778003) and the Ministry of Science and Technology
of the People’s Republic of China (2017YFA0506300).
1
00070, China; orcid.org/0000-0002-5587-4165;
Email: zbliu@pku.edu.cn
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