Photoactivatable Organic Semiconducting Pro-nanoenzymes

Article abstract of DOI:10.1021/jacs.8b13507

Therapeutic enzymes hold great promise for cancer therapy; however, in vivo remote control of enzymatic activity to improve their therapeutic specificity remains challenging. This study reports the development of an organic semiconducting pro-nanoenzyme (OSPE) with a photoactivatable feature for metastasis-inhibited cancer therapy. Upon near-infrared (NIR) light irradiation, this pro-nanoenzyme not only generates cytotoxic singlet oxygen (¹O₂) for photodynamic therapy (PDT), but also triggers a spontaneous cascade reaction to induce the degradation of ribonucleic acid (RNA) specifically in tumor microenvironment. More importantly, OSPE-mediated RNA degradation is found to downregulate the expression of metastasis-related proteins, contributing to the inhibition of metastasis after treatment. Such a photoactivated and cancer-specific synergistic therapeutic action of OSPE enables complete inhibition of tumor growth and lung metastasis in mouse xenograft model, which is not possible for the counterpart PDT nanoagent. Thus, our study proposes a phototherapeutic-proenzyme approach toward complete-remission cancer therapy.

Full text of DOI:10.1021/jacs.8b13507

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Article
Photoactivatable Organic Semiconducting Pro-nanoenzymes
Jingchao Li, Jiaguo Huang, Yan Lyu, Jingsheng Huang, Yuyan Jiang, Chen Xie, and Kanyi Pu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13507 • Publication Date (Web): 11 Feb 2019
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Photoactivatable Organic Semiconducting Pro-nanoenzymes
Jingchao Li, Jiaguo Huang, Yan Lyu, Jingsheng Huang, Yuyan Jiang, Chen Xie, and Kanyi Pu*
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School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, 637457,
Singapore
KEYWORDS: Pro-enzymes, organic nanoparticles, phototherapy, cancer therapy
ABSTRACT: Therapeutic enzymes hold great promise for cancer therapy; however, in vivo remote control of enzymatic
activity to improve their therapeutic specificity remains challenging. This study reports the development of an organic
semiconducting pro-nanoenzyme (OSPE) with a photoactivatable feature for metastasis-inhibited cancer therapy. Upon
near-infrared (NIR) light irradiation, this pro-nanoenzyme not only generates cytotoxic singlet oxygen (1O2) for
photodynamic therapy (PDT), but also triggers a spontaneous cascade reaction to induce the degradation of ribonucleic
acid (RNA) specifically in tumor microenvironment. More importantly, OSPE-mediated RNA degradation is found to
downregulate the expression of metastasis-related proteins, contributing to the inhibition of metastasis after treatment.
Such a photoactivated and cancer-specific synergistic therapeutic action of OSPE enables complete inhibition of tumor
growth and lung metastasis in mouse xenograft model, which is not possible for the counterpart PDT nanoagent. Thus,
our study proposes a phototherapeutic-proenzyme approach towards complete-remission cancer therapy.
infrared (NIR) light with deep-tissue penetrating ability
INTRODUCTION
can be used to control the enzymatic activity.20 However,
Drug therapeutics in the form of small molecules and
proteins is promising for treatments of solid tumors and
metastasis.1-4 However, most existing drugs often pose
serious threats to normal tissues owing to their “always-
on” pharmacological effect. To overcome this issue,
prodrugs that only become cytotoxic in response to
it exerts the photothermal effect of optical agents, and
thus only works for thermosensitive enzymes, which
often do not have therapeutic effect.20-22 As a result, NIR
light regulation of the activity of therapeutic proenzymes
has been barely exploited for cancer therapy.
In this study, we report the synthesis of an organic
semiconducting pro-nanoenzyme (OSPE) that can be
remotely activated with NIR photo-irradiation in tumor
microenvironment for metastasis-inhibited cancer
exogenous stimuli or endogenous cancer-specific
6
biomarkers have been proposed.5,
For example,
banoxantrone- and camptothecin-based prodrugs were
developed for cancer therapy, which were active in the
therapy. OSPE contains
a semiconducting polymer
presence of hypoxic environment and biothiols,
nanoparticle (SPN) core, which is conjugated with an
inactive proenzyme via a singlet oxygen (1O2) cleavable
linker (Figure 1a). SPNs are a new class of optical
8
respectively.7,
In addition, a dendrimer-protected
protease was used as the proenzyme with pH responsive
activity for specific induction of cancer cell apoptosis.9 All
the reported systems have shown the improved
therapeutic specificity towards diseases cells/tissues.
nanomaterials applicable for molecular imaging,23-25
30
phototherapy26-28 and biological activation.29,
The
proenzyme (EBAP) is derived from cytotoxic ribonuclease
A (RNAse A) that degrades intracellular ribonucleic acid
(RNA) to induce cell death and its activity is inhibited by
caging with a hydrogen peroxide (H2O2) responsive
phenylboronic acid pinacol (BAP) group. Upon NIR laser
Several approaches have been adapted to regulate the
activity of enzymes in living systems, including reversible
11
chemical engineering,10, conditional protein splicing12
and alternating magnetic field.13 In comparison,
photoactivation has the advantages of non-invasiveness,
simple maneuverability, flexible controllability, high
spatiotemporal resolution and minimal side-effects.14-17 In
general, the enzymatic activity is inhibited by conjugation
with certain photo-cleavable caging groups.18 However,
because majority of photo-cleavable reactions are only
responsible to ultraviolet (UV) and visible light, this
strategy has poor tissue penetration.19 In contrast, near-
1
irradiation, the SPN core generates O2 that not only
permits photodynamic therapy (PDT) but also initiates
the cleavage of the 1O2-responsible linker to release EBAP;
the released EBAP then undergoes a deprotection process
triggered by H2O2 in tumor microenvironment, restoring
the enzyme activity for cancer-specific RNA degradation.
As such, OSPE represents a smart pro-nanoenzyme,
whose full activation requires both NIR laser irradiation
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and reaction with chemical mediator in tumor
microenvironment. In association with PDT, OSPE-
mediated RNA degradation can downregulate the
expression of metastasis-related proteins, contributing to
the inhibition of metastasis after treatment. Thereby,
Supporting Information). The O2-cleavable linker, (Z)-
2,2'-(ethene-1,2-diylbis(sulfanediyl))diethanamine (BSDA),
was synthesized (Figures S3 and S4, Supporting
Information) and utilized to functionalize poly(ethylene
glycol) (PEG) chains to yield PEG-BSDA (Figure S5,
Supporting Information). The photodynamic polymer
(PCB) was synthesized via Suzuki polycondensation
reaction and then grafted with methoxy-PEG (Mw = 1000)
and PEG-BSDA (Mw = 2000) at a molar ratio of 4:1,
resulting in PCB-PEG-BSDA (Figure S6, Supporting
Information). This polymer spontaneously assembled into
the nanoparticle (termed as SPNB) in aqueous solution.
SPNB was subsequently conjugated with EBAP to afford
OSPE. To synthesize the control nanoparticles without
enzyme modification, PCB was grafted with methoxy-PEG
(Mw = 1000) and carboxyl-PEG (Mw = 2000) at a molar
ratio of 4:1 to afford PCB-PEG. PCB-PEG had the structure
similar to the previously polymer used for photothermal
therapy,20 which could also assemble into the
nanoparticle in aqueous solution, termed as SPNC (Figure
1d).
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OSPE-mediated phototherapy possesses
a
unique
synergistic pharmacological effect, demonstrating the
potential for complete-remission cancer therapy.
RESULTS AND DISCUSSION
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The organic semiconducting pro-nanoenzyme (OSPE)
was constructed through the following three main steps
(Figure 1b,c): (i) synthesis of the BAP-caged proenzyme
(EBAP); (ii) synthesis of the organic nanoparticle (SPNB)
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with the O2-cleavable linker; and (iii) conjugation of
EBAP onto the surface of SPNB. The H2O2-responsive
compound was synthesized (Figure S1, Supporting
Information) and used to cage the native RNAse A
enzyme at the lysine residues, affording EBAP. MALDI-
TOF-MASS assay confirmed the conjugation, showing
approximately 7 BAP molecules per enzyme (Figure S2,
Figure 1. Design and synthesis of OSPE for cancer therapy. (a) Illustration of the proposed mechanism for the photoactivated
synergistic therapeutic action of OSPE including PDT and intracellular RNA degradation. (b) Synthesis of O2-responsive
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polymer PCB-PEG-BSDA. (c) Synthesis of OSPE. (d) Structure of PCB-PEG and the control nanoparticle (SPNC) without enzyme
modification. (i) 4-Nitrophenyl chloroformate, triethylamine, tetrahydrofuran (THF), 25 °C, 2 h. (ii) RNAse A, 0.1 M NaHCO3
buffer (pH 8.5), dimethyl sulfoxide (DMSO), 25 °C, 12 h. (iii) Pd(PPh3)4, K2CO3, methyltrioctylammonium chloride, toluene/H2O,
100 °C, 24 h. (iv) NaN3, THF/N,N-dimethylformamide (DMF), 25 °C, 12 h. (v) Tert-butyl (2-mercaptoethyl)carbamate, NaOH,
ethanol, 25 °C, 24 h. (vi) Dichloromethane (CH2Cl2)/trifluoroacetic acid (TFA), 25 °C, 1 h. (vii) Carboxyl-PEG-alkyne, EDC/NHS,
DMSO, 25 °C, 48 h. (viii) CuBr, N,N,N’,N’’,N’’’-pentamethyldiethylenetriamine (PMDETA), methoxy-PEG-alkyne, THF, 25 °C, 48
h. (ix) EBAP, EDC/NHS, 1× PBS buffer (pH 7.4).
The hydrodynamic size of OSPE was measured to be 28
nm (Figure 2a), which was larger than SPNC (17 nm) and
SPNB (18 nm). The polydispersity indexs (PDIs) of SPNC,
SPNB and OSPE were measured to be ~0.2, indicating the
good uniformity of these nanoparticles (Figure S7a,
Supporting Information). SPNB showed a positive surface
charge (11 mV) due to the modification of BSDA, while
further conjugation of EBAP reduced the zeta potential to
-12 mV for OSPE (Figure S7b, Supporting Information).
The hydrodynamic sizes of SPNC, SPNB and OSPE did
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not significantly change after storage in PBS buffer
which was similar with our previous studies.31,
32
The
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(Figure S7c, Supporting Information), suggesting their
good colloidal stability. In agarose gel electrophoresis,
OSPE migrated a much shorter distance than SPNC and
SPNB (Figure 2b), further implying the successful
conjugation of EBAP with SPNB. The weight ratio of
enzyme to the SPN core was 84% determined by Bradford
protein assay. As indicated by transmission electron
microscopy (TEM) image, OSPE was spherical and had a
homogenous size distribution (Figure 2c).
generated O2 was able to cleave the BSDA linker to
release the proenzyme (EBAP) from OSPE (Figure 2f).
After 30 min laser irradiation, the proenzyme release
efficiency reached to 59% regardless of H2O2 treatment.
To confirm the NIR laser irradiation triggered activation
of OSPE, a RNaseAlert® Kit was used to measure the
enzymatic activity. In the presence of H2O2, the
enzymatic activity of OSPE was elevated by 3.7-, 4.6- and
6.2-fold after 10, 20 and 30 min of NIR laser irradiation,
respectively (Figure 2g). While, only slight increase in
enzymatic activity was observed for OSPE after
treatments with H2O2 or laser irradiation alone. These
results verified that the enzymatic activity of OSPE could
be specifically restored by photo-irradiation-caused
release along with H2O2 induced deprotection.
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Both absorption and fluorescence spectra of OSPE were
almost identical to those of SPNC (Figure 2d), suggesting
the surface modification had negligible influence on their
optical properties. The absorption of SPNC and OSPE
ranged from 600 to 900 nm, enabling the generation of
1O2 under NIR laser irradiation as confirmed using singlet
oxygen sensor green (SOSG) as an indicator (Figure 2e),
Figure 2. In vitro characterization of OSPE along with the control SPNC. (a) Dynamic light scattering (DLS) profiles and
(b) agarose gel electrophoresis of SPNC, SPNB and OSPE. (c) Representative TEM image of OSPE. (d) UV-Vis absorption
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and fluorescence spectra of SPNC and OSPE. (e) Generation of O2 for SPNC and OSPE under NIR laser irradiation after
different irradiating time. (f) Release of proenzyme from OSPE as a function of laser irradiating time. (g) Relative
enzymatic activity of OSPE upon NIR laser irradiation for different time in the absence and presence of H2O2. [SPN] = 40
μg/mL for SPNC, SPNB and OSPE in PBS buffer (pH = 7.4), [H2O2] = 50 μM. The laser irradiation wavelength was at 808
nm with a power density of 0.3 W/cm2. Error bars represented standard deviations of three separate measurements (n = 3).
The cellular uptake of OSPE was investigated against
4T1 cancer cells. After treatment for 24 h, the fluorescence
intensity of OSPE-treated cells was almost identical to
that of SPNC-treated cells (Figure 3a,b), suggesting
surface enzyme conjugation did not have any obvious
influence on the internalization of nanoparticles. This
should be due to the similar negative zeta potential for
SPNC and OSPE (Figure S7b, Supporting Information).
Next, the ability of OSPE for cancer therapy was evaluated.
Note that SPNC and OSPE alone caused nearly no toxicity
to 4T1 cancer cells even at a high concentration up to 50
μg/mL (Figure 3c). After treatment with SPNC or OSPE
for 24 h, 4T1 cancer cells were irradiated by NIR laser at
808 nm with a power density of 0.3 W/cm2. The laser
irradiation was controlled in a discontinuous manner to
maintain the temperature below the apoptotic threshold
(43 ºC) (Figure S8, Supporting Information). The laser
irradiation alone without nanoparticle treatment showed
negligible effect on the cell viability, while the cell
viabilities decreased with increased concentrations for
both SPNC and OSPE upon laser irradiation. At the
concentration of 40 μg/mL, the cell viability of OSPE-
treated cells was 17.9%, which was 3.4-fold lower than
that of SPNC-treated cancer cells (61.6%). These data
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verified the better therapeutic efficiency of OSPE over
SPNC.
from other treated groups. In addition, no obvious RNA
degradation occurred in OSPE-treated normal fibroblast
cells, regardless of laser irradiation. This is because the
RNAse A (EBAP) released from OSPE was in its caged and
inactive form due to the relatively low H2O2 level in the
normal cells (Figure S9a, Supporting Information). This
was again validated by the observation that free RNAse A
could induce severe RNA degradation in both 4T1 cancer
cells and normal fibroblasts with or without laser
irradiation (Figure S9b, Supporting Information).
Therefore, the synergistic therapeutic action of OSPE was
fully activated only when light irradiation and H2O2
coexisted as proposed in Figure 1a, leading to its high
specificity to cancer cells and superior anticancer efficacy
over SPNC.
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To study the therapeutic mechanism of OSPE,
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intracellular O2 generation and RNA degradation were
examined. Cell permeant 2’,7’-dichlorodihydrofluorescein
diacetate (H2DCFDA), a fluorescent turn-on indicator for
ROS, was used to detect the intracellular generation of 1O2.
Upon laser irradiation, similar fluorescence signal was
observed for SPNC- and OSPE-treated cancer cells (Figure
3d,e), while negligible fluorescence signal was detected in
the cells from other groups. Furthermore, RNA in the
treated cells was extracted and assessed using agarose gel
electrophoresis. Obvious RNA degradation was only
observed for OSPE-treated 4T1 cancer cells with laser
irradiation (Figure 3f), but RNA was intact for the cells
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Figure 3. In vitro OSPE-mediated cancer therapy. (a) Mean fluorescence intensity (MFI) of 4T1 cancer cells without treatment
(control) or after treatment with SPNC or OSPE ([SPN] = 20 μg/mL) for 24 h (N.S., no significant difference, n = 3). (b)
Representative confocal fluorescence images of 4T1 cancer cells after treatment with SPNC or OSPE ([SPN] = 20 μg/mL) for 24 h.
Blue fluorescence was from cell nucleus stained with 4’,6-diamidino-2-phenylindole (DAPI) and red fluorescence indicated the
signals from nanoparticles. (c) Cell viabilities of 4T1 cancer cells after treatments with SPNC or OSPE at different concentrations
([SPN] = 0, 10, 20, 30, 40 and 50 μg/mL) with or without 808 nm laser irradiation (0.3 W/cm2) for 10 min (*p < 0.05, n = 3). (d)
Representative confocal fluorescence images and (e) flow cytometry analysis of 4T1 cancer cells treated with SPNC or OSPE
([SPN] = 40 μg/mL) with or without 808 nm laser irradiation (0.3 W/cm2) for 10 min. The cells after different treatments were
stained with H2DCFDA. (f) Agarose gel electrophoresis of RNA exacted from 4T1 cancer cells treated with SPNC or OSPE ([SPN]
= 40 μg/mL) with or without 808 nm laser irradiation (0.3 W/cm2) for 10 min.
In vivo cancer therapy was conducted by intravenously
injecting OSPE or SPNC into 4T1 tumor-bearing mice,
followed by local NIR photoactivation (Figure 4a). Tumor
growth and metastasis were monitored after treatment.
Whole-body NIR fluorescence imaging was used to
evaluate the accumulation of nanoparticles in tumors.
The fluorescence signals in tumor areas gradually
increased and reached the similar maximum (17 times
higher than the background) at 12 h post-injection for
SPNC and OSPE (Figure 4b,c). This indicated both
nanoparticles could accumulate into tumors via the
enhanced permeability and retention (EPR) effect. Such
ideal tumor accumulation of nanoparticles should be
attributed to their high surface PEG density (0.9 PEG/nm2)
and small sizes (17 nm for SPNC and 28 nm for OSPE).
This was similarly observed in the previous reports for the
nanoparticles assembled from PEG-grafted polymers.33, 34
In addition, similar biodistribution was observed for them
(Figure S10, Supporting Information), showing the highest
accumulation in liver closely followed by tumor.
According to the imaging results, the tumors of mice
were locally irradiated with NIR laser at 808 nm at a
power density of 0.3 W/cm2 (the maximum permissible
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exposure for skin) for 10 min at 12 h post-injection of
groups did not show any damages. Thereby, the tumor
growth was completely suppressed only for OSPE-injected
mice with laser irradiation (Figure 4e), while causing no
histopathological damages to major organs including
heart, liver, spleen and kidney of living mice (Figure S14,
Supporting Information). More importantly, OSPE-
mediated cancer therapy effectively inhibited the lung
metastasis as indicated by the histological staining
(Figure 4f), but obvious tumor metastasis was detected
for other treatment groups 14 days post-treatment.
Moreover, the number of pulmonary metastatic nodules
was counted and the results showed that the number for
OSPE-injected and laser-irradiated group was reduced by
nearly 100%, as compared to the control groups (Figure
4g). Such a complete-remission cancer therapy has been
barely reported for other photodynamic agents.35
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saline, SPNC or OSPE for cancer therapy. Note that the
laser irradiation was controlled in a discontinuous
manner to maintain the tumor temperature below the
apoptotic threshold (Figure S11, Supporting Information).
Such a low laser power density and discontinuous short
irradiation time ensured the biosafety of phototherapy,
allowing for its potential use in clinics. No obvious change
in the body weights of mice for all groups was observed
(Figure S12, Supporting Information). Histological
hematoxylin and eosin (H&E) staining (Figure 4d) and
immunofluorescence caspase-3 staining (Figure S13,
Supporting Information) revealed that much severer cell
apoptosis and necrosis were observed for the tumors in
OSPE-injected mice as compared to SPNC-injected mice
after 10 min laser irradiation, but the tumors in other
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Figure 4. In vivo OSPE-mediated cancer therapy. (a) Schematic illustration of OSPE-mediated complete inhibition of tumor
growth and metastasis. (b) Fluorescence imaging of 4T1 tumor-bearing mice at representative timepoints after intravenous
injection of SPNC or OSPE (200 μL, [SPN] = 200 μg/mL). Tumors were indicated by the white dashed circles. (c) Quantification
of tumor fluorescence intensity as a function of post-injection time of SPNC or OSPE (n = 6). (d) Histological H&E staining of
tumor sections from 4T1 tumor-bearing mice after different treatments. (e) The tumor growth curves in 4T1 tumor-bearing mice
(*p < 0.05, n = 6). (f) Histological H&E staining of pulmonary metastatic tumors in 4T1 tumor-bearing mice. Tumors were
indicated by the black dashed curves. (g) Quantitative analysis of pulmonary metastatic nodules in different treatment group
(***p < 0.001, n = 6). In (d)-(g), 4T1 tumor-bearing mice were intravenously injected with saline, SPNC or OSPE (200 μL, [SPN] =
200 μg/mL) and then treated with or without 808 nm laser irradiation (0.3 W/cm2) for 10 min.
Mechanism of OSPE-mediated complete-remission
cancer therapy was studied. Generation of O2 in tumors
was examined by measuring the fluorescence signal of
SOSG. Almost no green fluorescence was detected for the
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tumors after treatments with nanoparticles or laser
degradation. Obvious RNA degradation only occurred in
the tumors of OSPE-injected mice after NIR laser
irradiation (Figure 5c). Fluorescence quantification
further revealed that RNA integrity dropped to ~60% in
OSPE-injected and laser-irradiated group, while other
groups were still close to 100 % (Figure 5d).
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irradiation alone (Figure 5a). In contrast, after laser
irradiation, obvious green fluorescence signals were
observed for the tumors of both SPNC and OSPE-injected
mice, showing ~30-fold signal increase relative to those
without laser irradiation (Figure 5b). In addition, RNA
was exacted from the tumor lysates to assess RNA
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Figure 5. Mechanism study of OSPE-mediated complete-remission cancer therapy. (a) Representative confocal fluorescence
images of tumor sections from 4T1 tumor-bearing mice after different treatment. Blue fluorescence was from cell nucleus stained
with DAPI and green fluorescence indicated the signals from SOSG. (b) Quantitative analysis of SOSG MFI in tumor sections
from 4T1 tumor-bearing mice after intravenous injection of saline, SPNC or OSPE with or without an 808 nm laser irradiation
(***p < 0.001, n = 6). (c) Agarose gel electrophoresis of RNA exacted from tumor lysates of 4T1 tumor-bearing mice. (d) RNA
integrity in the tumor tissues from 4T1 tumor-bearing mice after intravenous injection of saline, SPNC or OSPE with or without
an 808 nm laser irradiation (**p < 0.01, n = 6). (e) Representative confocal fluorescence images of VCAM-1, HGF and MTA2
expression in the tumors from 4T1 tumor-bearing mice after different treatments. Blue fluorescence was from cell nucleus
stained with DAPI and green fluorescence indicated the signals from the VCAM-1, HGF or MTA2 staining. Relative expression of
VCAM-1 (f), HGF (g) and MTA2 (h) in the tumor tissues from 4T1 tumor-bearing mice after intravenous injection of saline, SPNC
or OSPE with or without an 808 nm laser irradiation (***p < 0.001, n = 6). (i) Schematic illustration of the proposed mechanism
of OSPE-mediated inhibition of lung metastasis. In (a)-(h), 4T1 tumor-bearing mice were intravenously injected with saline,
SPNC or OSPE (200 μL, [SPN] = 200 μg/mL) and then treated with or without 808 nm laser irradiation (0.3 W/cm2) for 10 min.
To further gain insight into the underlying mechanism
of metastasis inhibition by OSPE-mediated cancer
therapy, the expression of proteins associated with
metastasis, including vascular cell adhesion molecule-1
(VCAM-1), hepatocyte growth factor (HGF) and
metastasis-associated protein 2 (MTA2) in the tumor
tissues after different treatments was studied with
invasion and metastasis.37 MTA2 inhibits the cell adhesion
molecule epithelial transmembrane glycoprotein (Ep-
CAM) and E-cadherin, and thus promotes the cell
migration and metastasis.38 Confocal fluorescence
imaging revealed that very weak green fluorescence
signals from these protein staining were observed in the
tumor slides from OSPE-injected and laser-irradiated
group (Figure 5e); in contrast, obvious green fluorescence
signals were detected in all other groups. Signal
quantification (Figure 5f-h) indicated that only OSPE-
mediated phototherapy dramatically reduced the
expression of VCAM-1 (to 22.4%), HGF (to 27.5%) and
MTA2 (to 16.7%) relative to the saline-treated control
immunofluorescence staining. VCAM-1,
a
cell–cell
adhesion protein expressed on the surface of tumor cells,
facilitates the lung metastases by binding with monocytes
through α4-inergrin mediated adhesion.36 HGF is a scatter
factor that induces tumor cells to lose their contacts with
neighbors and become highly mobile, initiating tumor
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*kypu@ntu.edu.sg
groups. As summarized in Figure 5i, these results thereby
validated that RNA degradation induced by
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Notes
photoactivation of OSPE impeded the translation of
messenger RNA and thus downregulated the expression
of metastasis-related proteins, ultimately contributing to
the complete inhibition of lung metastasis.39, 40 Note that
to fully understand the synergistic effect of the OSPE core
and EBAP in inhibiting tumor growth and metastasis,
additional in vivo experiments need to be conducted in
the future study to deconvolve their individual roles.
These can involve the evaluation of other control
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This work was supported by Nanyang Technological
University (Start-Up grant: NTU-SUG: M4081627.120) and
Singapore Ministry of Education Academic Research Fund
Tier 1 (RG133/15 M4011559, 2017-T1-002-134-RG147/17) and
Tier 2 (MOE2016-T2-1-098).
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nanoparticles, such as EBAP-conjugated SPNs via a O2-
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Corresponding Author
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