NIRF turn-on nanoparticles based on the tumor microenvironment for monitoring intracellular protein delivery

Article abstract of DOI:10.1039/c9cc07768e

A dual responsive NIRF turn-on protein delivery system incorporating an NIRF turn-on probe and protein into one single nanoparticle has been constructed. It can be taken up efficiently by A549 cells, where protein release and NIRF recovery happen simultaneously in response to low pH and excessive H₂O₂. This work provides a novel system for monitoring intracellular protein delivery.

Full text of DOI:10.1039/c9cc07768e

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NIRF turn-on nanoparticles based on the tumor
microenvironment for monitoring intracellular
protein delivery†
Cite this: Chem. Commun., 2019,
55, 14930
Received 3rd October 2019,
Accepted 18th November 2019
Huaibao Xu,‡ Yi Wang,‡ Zhichao Pei,
Wei Ji and Yuxin Pei
*
DOI: 10.1039/c9cc07768e
rsc.li/chemcomm
A dual responsive NIRF turn-on protein delivery system incorporating allows for non-invasive deep tissue penetration with minimal
an NIRF turn-on probe and protein into one single nanoparticle has interference.8 For example, active fluorophore QCy7 (quinone-
been constructed. It can be taken up efficiently by A549 cells, where cyanine) can be released from QCy7-PBA (QCy7 conjugated with
protein release and NIRF recovery happen simultaneously in response phenylboronic acid) in the presence of H2O2,9 which makes
to low pH and excessive H2O2. This work provides a novel system for QCy7-PBA a terrific H2O2-responsive NIRF-probe to tumor cells
monitoring intracellular protein delivery.
which overproduce H2O2. Unfortunately, due to the fragile
nature of proteins and the complexity of the incorporation of
Protein therapy holds appealing promise for cancer treatment due the turn-on probe into one single nanocarrier, the design and
to its potentially high potency.1 However, its clinical application fabrication of stimuli-responsive systems for monitoring intra-
is severely limited due to the poor membrane permeability of cellular protein delivery in a safe and efficient way remains a
proteins as well as lack of efficient delivery vehicles and indica- tremendous challenge.10
tive methods of protein release. To solve these problems, numer-
Previously, PBA has been successfully used for the modification
ous strategies and various nanocarriers2 have been developed for of enzymes (including trypsin, papain, DNase I) and ‘‘click’’ with
intracellular protein delivery, which are able to respond to the salicyl hydroxamates (SHA) conjugated on a dendron of PAMAM to
tumor cell microenvironment (such as low pH, high concentration form a stable boronate complex for the intracellular release of
of glutathione, or overproduced H2O2) to achieve spatiotemporal enzymes at low pH with a preserved activity.11 Inspired by this
protein delivery and controlled release at sites of interest.2,3 Never- work, we envision that a NIRF turn-on nanoparticle responsive to
theless, few of them are capable of protein-tracking or diagnostic pH and H2O2 can be constructed by conjugating QCy7-PBA and
imaging.4
protein-PBA (in this work, trypsin was used as a model protein)
Lately, a protein delivery platform doped with a fluorescent to SHA functionalized nanoparticles (NPs) via the interaction
dye has been explored for monitoring intracellular protein between PBA and SHA. Such a protein delivery system with a
delivery where the protein was released while the fluorescence protein and a turn-on NIRF probe incorporated into one single
was turned on based on the hypoxic microenvironment of most nanoparticle can first lead to protein-PBA and QCy7-PBA release
solid tumors,5 which displayed a higher selectivity toward the at low pH, and then QCy7-PBA underwent oxidation on the
tumor microenvironment in comparison with conventional PBA moiety, followed by hydrolysis and 1,6-elimination of
always-on probes that heavily relied on their concentration the p-quinone-methide to release an active fluorophore QCy7
and resulted in inevitable signal interference.6 Thus, this kind in the presence of H2O2, which can be considered as a signal of
of platform can better achieve intracellular monitoring of protein protein release. Thus, this NIRF turn-on protein delivery system
delivery. Recently, near-infrared fluorescence (NIRF) turn-on probes provides a new strategy for monitoring intracellular protein release.
have attracted considerable attention for in vivo imaging7 since they To the best of our knowledge, there is no such activatable NIRF
not only undergo intrinsic signal evolution upon the intended imaging-guided protein delivery system reported to date.
stimuli, but also have an emission range of 650–900 nm, which
We first studied the activity of Try-PBA (trypsin after the
modification of PBA).11 The modification was carried out via
condensation of its surface lysine residues with pre-activated
4-carboxyphenylboronic acid (Scheme S1, ESI†).11 The charac-
terization and the analysis of the biological activity of Try-PBA
revealed 23.5 equivalents of PBA per trypsin molecule and
negligible loss of biological activity (Fig. S1–S5, ESI†). This is
Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of
Chemistry & Pharmacy, Northwest A&F University, Yangling, Shaanxi 712100,
P. R. China. E-mail: peiyx@nwafu.edu.cn
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9cc07768e
‡ These authors contributed equally.
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in agreement with that reported.11 Therefore, Try-PBA was used
as a model protein for the construction of protein delivery
systems.
Then, the NIRF recovery of QCy7-PBA (for synthesis and
characterization details, see the ESI†) upon incubation with
H2O2 (PBS 7.4) was investigated using UV-Vis and fluorescence
spectroscopies. As illustrated in Fig. S6 (ESI†), in the absence of
H2O2, QCy7-PBA in a PBS/DMSO buffer solution (95/5, v/v, pH
7.4, 10 mM) showed a maximum absorption peak at 411 nm
and negligible fluorescence (Ex/Em: 600/670 nm); whereas upon
incubation with H2O2, a strong bathochromic shift was observed,
accompanied by the color change from yellow to purple in day-
light (inset in Fig. S6, ESI†). In addition, the absorption displayed
a broad band with two maximum absorption peaks at 474 and
529 nm; while a strong NIRF emission peak at 670 nm with
B12.5-fold enhanced intensity was observed when excited at
600 nm. The H2O2-responsive property of QCy7-PBA was further
evaluated by varying the concentration of H2O2 and duration of
incubation. The results showed a significant increasing trend in
NIRF intensity with the concentration of H2O2 (Fig. S7, ESI†) or
incubation duration (reaching an apex at 4.5 min), in contrast to
minimal changes in the absence of H2O2 (Fig. S8, ESI†). A H2O2
selectivity test for QCy7-PBA was conducted which showed that
QCy7-PBA is highly selective towards H2O2 over other interfering
substances present in cells (Fig. S9, ESI†). This indicated that
QCy7-PBA was an ideal turn-on probe which had the potential for
NIRF imaging in H2O2 overproduced cancer cells.
Based on the above results, our target protein delivery system of
MNP@PDA@PEG/SHA-Q/T was built by following the procedure
depicted in Scheme 1. MNP@PDA was used as scaffolds, where the
magnetic nanoparticles (MNP) core makes the separation of the
NPs in the following preparation steps convenient; while poly-
dopamine (PDA) serves as a reactive surface for the conjugation
of SHA and PEG due to the reductive nature of the o-quinone units
in PDA, which allows reactivity towards amino groups and thiol
groups. Thus, by mixing MNP@PDA with SHA-NH2, MNP@PDA@-
SHA was prepared. To improve the dispersity of the NPs in aqueous
solution and provide a protective layer for the protein, a PEG layer
was introduced to the NPs by mixing MNP@PDA@SHA with
PEG-SH to obtain MNP@PDA@PEG/SHA.12 Finally, via complexa-
tion between PBA and SHA,11 the turn-on probes of QCy7-PBA and
Try-PBA were covalently conjugated to MNP@PDA@PEG/SHA, which
led to the successful fabrication of MNP@PDA@PEG/SHA-Q/T
(for the detailed assembly process and details of characteriza-
tion, see the ESI,† Fig. S10–S16 and Tables S1, S2) with a loading
capacity of Try-PBA and QCy7-PBA on MNP@PDA@PEG/SHA-Q/T
as 6.45% and 4.95%, respectively (cf. standard curve in Fig. S17
and S18, ESI†).
Scheme 1 Illustration of the fabrication procedure for MNP@PDA@PEG/
SHA-Q/T, pH-triggered Try-PBA and QCy7-PBA release, and H2O2-induced
fluorescence turn-on in the tumor microenvironment.
the cumulative release amount of the protein increased rapidly
to 53.5% by 30 min, and reached a plateau of 77.1% by 105 min.
In contrast, protein release plateaued at 20.0% at pH 7.4. Such a
pH-responsive behaviour was attributed to the cleavage of the
complex formed between PBA and SHA under acidic conditions.
In addition, the activity of Try-PBA released from MNP@PDA@-
PEG/SHA-Q/T at pH 5.5 was measured to be 264.7 U mgÀ1
(Fig. S5, ESI†), very similar to native trypsin (269.1 U mgÀ1). This
further confirmed that the method designed in this work allows
the constructed NPs to effectively release protein in some acidic
microenvironment of cancer cells with preserved activity.14
It is known that the antitumor potential of protein drugs is
closely dependent on their cellular uptake capability. The cellular
uptake of MNP@PDA@PEG/SHA-TF was studied with A549 cells
using flow cytometry. As shown in Fig. 1b, the fluorescence
intensity of A549 cells incubated with MNP@PDA@PEG/SHA-TF
To study the release profile of protein,13 a fluorescent protein
loaded nanoparticle (MNP@PDA@PEG/SHA-TF) was synthesized
by mixing fluorescein isothiocyanate (FITC) labelled Try-PBA
(FITC-Try-PBA) with MNP@PDA@PEG/SHA. The release of the
protein was carried out at pH 5.5 in order to simulate the unique
acidic environment of cancer cells, and was determined by
measuring the fluorescence intensity of FITC-Try-PBA at 517 nm
(cf. standard curve in Fig. S19, ESI†). As shown in Fig. 1a, at pH 5.5,
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up to 120 min, which was attributed to the weakly acidic micro-
environment and high concentration of H2O2 in A549 cells
(Fig. 1c(a–e)). These results demonstrate that QCy7-PBA possesses
the capability of NIRF imaging.
Having proved intracellular protein release and NIRF recovery,
we investigated the cytotoxicity of MNP@PDA@PEG/SHA-Q/T. A549
cells were incubated with MNP@PDA@PEG/SHA-Q/T for 3 h at
37 1C and 5% CO2 for MTT analysis. The cells treated under the
same conditions but incubated with PBS (blank), trypsin, and
Try-PBA, respectively, were used as the controls (Fig. 2a). The
results showed that MNP@PDA@PEG/SHA-Q/T displayed strong
cytotoxicity and resulted in nearly 90% cell death. In contrast, the
cell death of the groups from the controls, including trypsin and
Try-PBA, was merely 12.0% and 16.6%, respectively, indicating
the low efficacy of the two proteins alone due to the inefficient
internalization by A549 cells. Notably, cells incubated with
MNP@PDA@PEG/SHA-Q did not show obvious cytotoxicity, as
the cell viability was determined to be 85.8%, indicating that the
system is moderately biocompatible (Fig. S21 and S22, ESI†), and
cell death observed with MNP@PDA@PEG/SHA-Q/T was indeed
mainly caused by Try-PBA released from the protein loaded NPs.
The cytotoxicity of MNP@PDA@PEG/SHA-Q/T was further eval-
uated using the annexin V conjugate staining assay. The cell
death and apoptosis of A549 cells incubated with MNP@PDA@-
PEG/SHA-Q/T, as shown in Fig. 2b, reached 37.0% compared to
merely 9.3% with Try-PBA (Fig. 2c).
In order to verify the ability of monitoring protein release of
MNP@PDA@PEG/SHA-Q/T, a confocal laser scanning microscope
Fig. 1 (a) FITC-Try-PBA release profiles at different pH values; (b) flow
cytometry analysis of A549 cells after incubation with MNP@PDA@PEG/
SHA-TF (0.25 mg mLÀ1) for 15, 30, and 60 min. A549 cells incubated with
PBS and FITC-Try-PBA (16.1 mg mLÀ1) for 60 min were used as the control;
(c) CLSM images of A549 cells after incubation with MNP@PDA@PEG/
SHA-Q (0.25 mg mLÀ1) at different durations. The lysosomes were stained
with LysoTracker Green. The last photo in each line is an enlarged view of
the marked area in the overlay. QCy7-PBA: red signal, lex = 561 nm;
LysoTracker Green: green signal, lex = 488 nm.
clearly increased with incubation duration. In contrast, the fluores-
cence intensity of A549 cells incubated with free FITC-Try-PBA was
only slightly higher than that of the blank (cells incubated with
PBS) even after 60 min incubation. This result indicated that the
synthesized NPs can be internalized much more efficiently by A549
cells in comparison with free FITC-Try-PBA. Furthermore, confocal
laser scanning microscopy (CLSM) imaging studies indicated a
successful endolysomal escape of released protein (Fig. S23, ESI†),
which was attributed to the proton sponge effect caused by the
high amount of amino groups on the PDA shell.15 To test the
intracellular NIRF recovery of the turn-on probe QCy7-PBA, CLSM
was used to study A549 cells incubated with MNP@PDA@PEG/
SHA-Q for different durations and dyed with LysoTracker Green
(Fig. 1c). As expected, NPs were internalized by A549 cells efficiently
and the intensity of NIRF emitted by QCy7 increased with time,
Fig. 2 (a) Cell viability of A549 cells incubated with PBS, trypsin, Try-PBA,
MNP@PDA@PEG/SHA-Q and MNP@PDA@PEG/SHA-Q/T for 3 h, respectively;
(b and c) flow cytometry studies of A549 cells after 3 h of treatment with
MNP@PDA@PEG/SHA-Q/T (b, PBS as blank) and Try-PBA (c). The cells were
stained using the Annex V-FITC/PI apoptosis kit. The concentration of trypsin
and Try-PBA used was 16.1 mg mLÀ1; the concentration of the NPs used was
0.25 mg mLÀ1, which corresponds to 16.1 mg mLÀ1 Try-PBA for MNP@PDA@-
PEG/SHA-Q/T.
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our work provides a new approach to fabricate biocompatible and
efficient stimuli-responsive turn-on systems for monitoring intra-
cellular protein delivery. We believe that the strategy developed in
this work may find broad applications in drug release monitoring
and cancer cell imaging.
This work was financially supported by the National Natural
Science Foundation of China (21772157 and 21572181).
Conflicts of interest
There are no conflicts to declare.
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