A pH reversibly activatable NIR photothermal/photodynamic-in-one agent integrated with renewable nanoimplants for image-guided precision phototherapy

Article abstract of DOI:10.1039/d0sc04408c

Phototherapy has great potential to revolutionize conventional therapeutic modalities. However, most phototherapeutic strategies based on multicomponent therapeutic agents generally lack tumor-specificity, resulting in asynchronous therapy and superimposed side-effects. Severe heat damage is also inevitable because of the necessity of continuous external irradiation. Here we show the design of an acid-activated and continuous external irradiation-free photothermal and photodynamic (PTT/PDT) synchronous theranostic nanoplatform for precision tumor-targeting near-infrared (NIR) image-guided therapy. pH-reversibly responsive brominated asymmetric cyanine is designed as the tumor-specific NIR PTT/PDT-in-one agent to enhance anticancer efficiency and reduce side-effects. Ultra-small NIR persistent luminescence nanoparticles are prepared as both the imaging unit and renewable nanoimplant. Biotin functionalized polyethylene glycol is introduced to endow active tumor-targeting ability and prolong blood-circulation. The developed smart platform offers merits of reversible activation, PTT/PDT synergetic enhancement, tumor targetability and continuous external irradiation-free properties, allowing autofluorescence-free image-guided phototherapy only in tumor sites. This work paves the way to developing smart theranostic nanoplatforms for precision medicine.

Full text of DOI:10.1039/d0sc04408c

Chemical
Science
View Article Online
View Journal
EDGE ARTICLE
A pH reversibly activatable NIR photothermal/
photodynamic-in-one agent integrated with
renewable nanoimplants for image-guided
precision phototherapy†
Cite this: DOI: 10.1039/d0sc04408c
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Xu Zhao, *abc Kai-Chao Zhao, Li-Jian Chen,
cd
abc
c
cd
Yu-Shi Liu, Jia-Lin Liu
abcd
and Xiu-Ping Yan
*
Phototherapy has great potential to revolutionize conventional therapeutic modalities. However, most
phototherapeutic strategies based on multicomponent therapeutic agents generally lack tumor-
specificity, resulting in asynchronous therapy and superimposed side-effects. Severe heat damage is also
inevitable because of the necessity of continuous external irradiation. Here we show the design of an
acid-activated and continuous external irradiation-free photothermal and photodynamic (PTT/PDT)
synchronous theranostic nanoplatform for precision tumor-targeting near-infrared (NIR) image-guided
therapy. pH-reversibly responsive brominated asymmetric cyanine is designed as the tumor-specific NIR
PTT/PDT-in-one agent to enhance anticancer efficiency and reduce side-effects. Ultra-small NIR
persistent luminescence nanoparticles are prepared as both the imaging unit and renewable
nanoimplant. Biotin functionalized polyethylene glycol is introduced to endow active tumor-targeting
ability and prolong blood-circulation. The developed smart platform offers merits of reversible activation,
PTT/PDT synergetic enhancement, tumor targetability and continuous external irradiation-free
properties, allowing autofluorescence-free image-guided phototherapy only in tumor sites. This work
paves the way to developing smart theranostic nanoplatforms for precision medicine.
Received 11th August 2020
Accepted 29th October 2020
DOI: 10.1039/d0sc04408c
rsc.li/chemical-science
Although considerable efforts have been made to integrate PTT
and PDT into one platform to improve therapeutic efficacy,
Introduction
8–11
Phototherapy, a localized, spatiotemporally controllable and most PTT and PDT synergistic platforms are based on multi-
1
1,12
less invasive therapy tool, has great potential to revolutionize component PTT and PDT agents,
conventional therapeutic modalities for precision therapy.
resulting in asynchronous
1
therapy and superimposed side-effects. Besides, conventional
Photothermal therapy (PTT) and photodynamic therapy (PDT), phototherapeutic agents are mostly in the “always on” state and
two modalities of phototherapy, produce local hyperthermia lack tumor specicity, leading to severe nonspecic damage to
3,13,14
and generate cytotoxic reactive oxygen species (ROS), respec- normal tissue.
The therapeutic effect of conventional pho-
tively, to induce cell apoptosis or even necrosis upon light totherapy also strongly depends on continuous external irradi-
2–4
excitation. However, PTT and PDT are oen plagued by the ation, inevitably causing severe overheating damage to normal
1
5,16
thermoresistance of cancer cells and inadequate oxygen supply tissue.
Hence, an enormous challenge remains in devel-
5
–7
in a hypoxic tumor microenvironment, respectively. Thus, oping innovative smart activatable PTT and PDT integrated
using PTT or PDT alone is difficult for efficient therapy. agents with precision tumor-targeting and efficient therapy. In
this sense, exploring ‘clean’ excitation sources without
nonspecic thermal damage is of particular signicance.
Persistent luminescence nanoparticles (PLNPs) are magical
optical materials that can store excitation energy and emit
a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi
2
b
14122, China. E-mail: zhaoxu2017@jiangnan.edu.cn; xpyan@jiangnan.edu.cn
International Joint Laboratory on Food Safety, Jiangnan University, Wuxi 214122,
17–19
persistent luminescence aer the cessation of excitation.
China
Besides, the luminescence of near-infrared (NIR) PLNPs can be
reactivated with deep tissue penetrable NIR light, enabling
detection and imaging without the need for continuous in situ
excitation, thereby eliminating autouorescence interfer-
c
Institute of Analytical Food Safety, School of Food Science and Technology, Jiangnan
University, Wuxi 214122, China
d
Key Laboratory of Synthetic and Biological Colloids, Ministry of Education, School of
Chemical and Material Engineering, Jiangnan University, Wuxi, 214122, China
2
0–22
†
Electronic supplementary information (ESI) available: Chemicals and materials, ence
and avoiding phototoxicity and tissue thermal damage
1
13
instrumentation, synthetic procedures, experimental details, H and C NMR
23–26
from long-time continuous excitation.
PLNPs, therefore, are
data, mass spectra and other supplementary gures. See DOI: 10.1039/d0sc04408c
This journal is © The Royal Society of Chemistry 2020
Chem. Sci.

View Article Online
Chemical Science
Edge Article
Fig. 1 Design of the PLNP-BAC-PEGBT theranostic platform. (a) Design strategy and synthetic route for BAC and PLNP-BAC-PEGBT. (b)
Illustration of PLNP-BAC-PEGBT as a pH-reversibly activatable PDT/PTT synchronous theranostic platform for autofluorescence-free image-
guided precision phototherapy without continuous external irradiation.
not only competent for long-term in vivo autouorescence-free develop high-performance PLNPs with excellent persistent
imaging, but also show tremendous promise in continuous luminescence and controllable morphology. In particular,
external irradiation-free phototherapy. The great potential of intelligent integration of high-performance PLNPs with a smart
PLNPs as a built-in light source to reduce the side-effects of PDT activatable PTT/PDT-in-one agent is of great signicance for
associated with continuous external irradiation has been precision therapy.
revealed, but exclusively restricted to ‘always on’ PDT
Herein, we report a rational design of an NIR-emitting ultra-
23–27
systems.
Besides, most PLNPs suffer from serious agglom- small PLNP sensitized pH-reversibly activatable PTT/PDT-in-
22,26,28–31
eration or poor luminescence performance,
impeding one agent for precision tumor-targeting and autouorescence-
further biomedical applications. So, it is still challenging to free image-guided synchronous phototherapy without
Chem. Sci.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Edge Article
Chemical Science
continuous external irradiation. pH reversibly responsive the indole ring N atoms leads to the transition of BAC from its
brominated asymmetric cyanine (BAC) is designed as an acti- base form to its acid form and vice versa (Fig. S15†). The
vatable NIR PTT/PDT-in-one agent for precision tumor-targeting reversible characteristic absorption change of BAC with pH lays
and high-efficiency therapy. Ultra small-sized monodispersed a solid foundation for pH-reversibly activatable PDT/PTT.
PLNPs with excellent NIR persistent luminescence are prepared Density functional theory (DFT) calculation shows that the
as both the autouorescence-free imaging unit and built-in protonation of the indole ring N atoms narrows the gap (E
light source to guide and trigger PDT/PTT. Meanwhile, biotin between the highest occupied molecular orbital (HOMO) and
BT) functionalized polyethylene glycol (PEGBT) is introduced the lowest unoccupied molecular orbital (LUMO) of BAC,
g
)
(
into the PLNP sensitized pH reversibly activatable PTT/PDT-in- leading to a distinct redshi of the absorption peak (Fig. 2b).
one nanoplatform (PLNP-BAC-PEGBT) to endow active tumor- Besides, the acidic environment enables the activation of the
32,33
targeting ability and prolong blood-circulation.
The as- NIR uorescence of BAC with a characteristic emission peak at
prepared PLNP-BAC-PEGBT theranostic nanoplatform not only 830 nm (Fig. 2a and S16a†) due to the protonation of the indole
exhibits precision tumor-targeted imaging without auto- ring N atoms accompanied by the intramolecular change

uorescence interference, but also shows an ultra-strong PTT/ transfer process. The pK
a
and the pH sensitive intervals of the
PDT effect with excellent tumor-specicity and ignorable side- synthesized BAC determined by means of uorescence titration
effects, holding great potential for precision medicine.
are 5.2 and 4.5–7.0, respectively (Fig. S16†). The results indicate
that the as-synthesized BAC has pH reversible response features
and tumor acidic microenvironment-matched pH sensitive
intervals, providing great potential for tumor microenviron-
ment specic activation.
Results and discussion
Design of the PLNP-BAC-PEGBT theranostic nanoplatform
We then investigated the photothermal and photodynamic
performance of BAC. DFT calculation shows that the protonated
BAC can be excited to the S1 state under 808 nm irradiation in
the acidic microenvironment (Fig. 2c). The S1 state consumes
its energy not only through a nonradiative decay pathway
accompanied by heat release due to the strong ICT effect of the
protonated BAC, but also via an enhanced ISC from the S1 state
to the T1 state owing to the heavy atom effect. Meanwhile,
Fig. 1 shows the design strategy of the PLNP-BAC-PEGBT
theranostic platform for autouorescence-free image-guided
precision tumor-targeting phototherapy without continuous
external irradiation. To realize precision tumor-targeting and
PTT/PDT synchronous efficient therapy, we design a non-N-
alkylated BAC molecule as the pH-reversibly activatable PTT/
PDT-in-one agent. Unsubstituted N atoms in the indole ring
of BAC are employed as the specic recognition sites for the acid
microenvironment to realize tumor-specic activation. The
heavy atom Br is introduced to improve intersystem crossing
energy transfer from the T1 state to triplet oxygen further occurs
1
to yield singlet oxygen ( O
2
) (Fig. 2c). Hence, BAC has great
potential for tumor acidic microenvironment specically acti-
vated photothermal and photodynamic performance. The acid-
activated photothermal effect of BAC at various power densities
of 808 nm laser irradiation was evaluated rst. The BAC solution
exhibited an obvious increase of temperature at pH 6.0 and
displayed power density and exposure time-dependent hyper-
thermia (Fig. 2d). The photothermal conversion performance of
indocyanine green (ICG), approved by the United States Food
(
ISC) from the lowest excited singlet (S1) state to the lowest
34,35
excited triplet (T1) state to enhance the ROS generation.
Meanwhile, a chemically active ester is introduced into BAC to
integrate NIR-emitting PLNPs as a built-in excitation source for
continuous external irradiation-free imaging and phototherapy.
PEGBT is further introduced on the surface of the PLNP sensi-
tized BAC theranostic platform (PLNP-BAC) with chloroacetyl
chloride as bridges to endow active tumor-targeting ability and
prolong blood-circulation (Fig. 1a). As such, the designed PLNP-
BAC-PEGBT enables autouorescence-free NIR imaging-guided
precision tumor-targeting phototherapy with no need for
continuous external irradiation.
4,38–40
and Drug Administration,
was also measured as a refer-
ence. Importantly, BAC showed a more obvious increase of
temperature than ICG (with the same concentration and power
density) (Fig. 2d), indicating that the as-prepared BAC at pH 6.0
has a superior photothermal effect. The photothermal conver-
ꢀ
2
sion efficiency of BAC at 0.6 W cm of 808 nm irradiation was
calculated to be 37.9% (Fig. S17†). In comparison, the control
Preparation and characterization of BAC
The synthetic route for the synthesis of BAC is illustrated in groups, phosphate buffer solution (PBS, pH 7.4) and BAC at pH
Fig. 1a while the synthesis procedures are detailed in the ESI. 7.4, did not trigger any increase of temperature even if the
ꢀ
2
The structures of the synthesized BAC and the important power density and exposure time were up to 0.6 W cm and
intermediates were conrmed from the nuclear magnetic 10 min, respectively (Fig. 2d). These results clearly show that the
resonance, mass spectra and FT-IR spectra (Fig. S1–S14†). BAC photothermal effect of BAC could be switched on only in an
presents a characteristic absorption peak at ca. 506 nm in acidic microenvironment, making it qualied for tumor acidic
a weakly basic medium (ethanol/water, 3/7 v/v; pH 7.4, simu- microenvironment specically activated PTT. Besides, BAC has
lated normal physiological pH), but shows a new shoulder band much higher photostability than ICG, especially at pH 7.4
at ca. 783 nm with an evident decreased absorption band at (Fig. 2e, S18 and S19†), ensuring strong stability in blood
5
06 nm in a weakly acidic medium (pH 6.0, simulated tumor circulation and therapy. Considering both photothermal effi-
36,37
ꢀ2
acidic microenvironment ) (Fig. 2a). Meanwhile, the BAC ciency and the photobleaching effect, 0.6 W cm was taken as
solution turns from pink to green because the protonation of the irradiating power in the follow-up study.
This journal is © The Royal Society of Chemistry 2020
Chem. Sci.

View Article Online
Chemical Science
Edge Article
Fig. 2 Characterization of BAC. (a) UV-vis-NIR absorption (dotted lines) and fluorescence (solid lines) spectra of BAC at pH 6.0 (blue lines) and
.4 (magenta lines). (b) Optimized structures and frontier molecular orbital (HOMO and LUMO) energies from DFT calculations (Gaussian 09/6-
7
1
3
1G (d)) of BAC in its ground state (S0). (c) Illustration for photothermal and O
.0 and 7.4 and ICG at pH 7.4 under continuous 808 nm laser irradiation with different power densities. (e) Effect of irradiation time on the
absorbance of BAC at pH 7.4 or 6.0 and ICG at pH 7.4 at the corresponding maximum absorption wavelength. (f) Time-dependent absorbance of
DPBF (A/A ) at 410 nm without and with BAC or ICG at pH 6.0 and 7.4 under continuous 808 nm laser irradiation. A is the initial absorbance of
DPBF, while A is the absorbance of DPBF at a certain irradiation time.
2
generation of BAC. (d) Time dependent temperature of BAC at pH
6
0
0
The acid-activated photodynamic effect of BAC was evaluated PLNPs need UV light excitation to produce persistent lumines-
further under various power densities of 808 nm laser irradia- cence and have limited ability to reactivate the persistent
1
tion. The O
8
(
2
generation efficiency of BAC at pH 7.4 or 6.0 under luminescence with visible and NIR light, which is unfavourable
26
08 nm laser was measured with 1,4-diphenyl-2,3-benzofuran for long-term in vivo application. In a previous report, we
DPBF) as the O probe. The decrease in the absorbance of proposed a surfactant-aided hydrothermal method in combi-
DPBF due to O oxidation was used to evaluate the relative nation with a short term calcination method to prepare mon-
generation efficiency of O . DBPF alone is stable during 5 min odispersed triple-doped NIR PLNPs (ca. 40 nm) with bright
of continuous irradiation using an 808 nm laser (0.6 W cm
Fig. 2F and S20†). The negligible reduction of the absorbance feature,
of DPBF indicates that BAC at pH 7.4 under the same laser obtain the small-sized nanoparticles, leading to low yield. Here
1
2
1
2
1
2
ꢀ
2
)
luminescence and red LED light or 808 nm laser renewable
26,28
(
but gradient centrifugation was still inevitable to
1
2
irradiation did not produce O (Fig. 2f and S21a†). However, we show the preparation of ultra-small size, ultra-bright and
BAC at pH 6.0 under the same laser irradiation generated NIR light renewable PLNPs with no need for gradient centrifu-
1
signicant O2 as conrmed by the rapid decrease of the gation via careful control of the hydrothermal and calcination
absorbance of DPBF (Fig. 2f and S21b†). In contrast, ICG conditions (Fig. S23†). The doping elements and doping ratio
1
enabled O generation at pH 7.4 under the same irradiation are consistent with the stoichiometry of the chemical formula
2
3
+
3+
3+
(
Fig. 2f and S22†). The distinct feature of BAC for the efficient (Zn1.25Ga1.5Ge0.25
O
4
:0.5%Cr ,2.5%Yb ,0.25%Er ) in our
1
26,28
generation of O
2
only in an acidic environment indicates that previous work.
The solvothermal method with a high-
BAC is also promising for tumor-specic PDT. The unique pH temperature and long time (220 C, > 24 h) in combination
reversible response activation and weak acidity specically with low-temperature calcination (800 C, 1 h) signicantly
ꢁ
ꢁ
activated PTT/PDT effect provide BAC with great potential as improved the optical performance of the PLNPs with no obvious
a smart activatable PTT/PDT-in-one agent for tumor-targeting agglomeration (Fig. S23†). Our improved method yielded ultra-
precision phototherapy.
small sized, mono-dispersed and NIR light renewable PLNPs
with an average diameter of 12.8 ꢂ 1.1 nm (calculated from 100
randomly selected particles) and a uniform lattice spacing of
Preparation and characterization of PLNPs
0
.24 nm without further gradient centrifugation (Fig. S24a and
b†).
The as-prepared PLNPs gave an NIR E / A
Ultra-small sized PLNPs with excellent NIR persistent lumi-
nescence and super-long aerglow are the fundamental
requirements of in vivo imaging and the built-in excitation
source. However, traditional preparation methods hardly
provide a compromise between the small size and excellent
luminescence performance. Besides, most of the available
2
4
2
luminescence
emission peak from 650 to 900 nm (Fig. S24c†). The overlap
between the luminescence emission peak of the as-prepared
PLNPs and the absorption peak of the synthesized BAC in its
Chem. Sci.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Edge Article
Chemical Science
acid form enables the PLNPs to sensitize BAC based on F ¨o rster of PLNPs (Fig. S26†). The content of BAC conjugated onto the
ꢀ
4
ꢀ1
resonance energy transfer (FRET). Besides, the PLNPs exhibited surface of PLNPs was measured to be 1.3 ꢃ 10 mol g via UV-
super long-lasting and repeatable NIR aerglow upon pre- vis-NIR absorption spectrometry. In addition, the prepared
excitation with 254 nm UV light (Fig. S24d and g†). Interest- PLNP-BAC-PEGBT gave a pure spinel phase of ZnGa O4 and
2
2 4
ingly, the persistent luminescence of the PLNPs can be reac- Zn GeO (JCPDS le number 38-1240 and 25-1018, respectively)
tivated with 650 nm red LED light and 808 nm laser irradiation like PLNPs (Fig. 3e), indicating no crystal phase change during
26,41
(Fig. S24e and f†) via a photo-assisted excitation process.
the whole functionalization process.
These unique optical properties and ultra-small size make the
Conjugation of BAC and PEGBT to PLNPs had an insigni-
as-prepared PLNPs qualied for long-term autouorescence- cant effect on the inherent luminescence intensity and the
free in vivo imaging and as a renewable built-in light source persistent luminescence of PLNPs at pH 7.4 (Fig. 3f, S26 and
for continuous external irradiation-free PTT and PDT.
S27b†). The change of pH from 7.4 to 6.0 slightly decreased the
luminescence intensity and persistent luminescence (Fig. 3f
and S27†) owing to the FRET effect from PLNPs to BAC origi-
nating from the overlap between the absorption peak of BAC in
its acid form and the emission peak of PLNPs. Even so, the
Preparation and characterization of the PLNP-BAC-PEGBT
theranostic nanoplatform
The above results encouraged us to employ the prepared BAC luminescence of PLNP-BAC-PEGBT was still clearly observed
and PLNPs to construct a PLNP sensitized BAC theranostic using the in vivo imaging system (Fig. 3g). These results also
platform for autouorescence-free image-guided precision show that the FRET process between PLNPs and BAC occurs if
tumor-targeting phototherapy without continuous external and only if a tumor acidic microenvironment is present, making
irradiation. To enhance its reaction activity, BAC was rst smart tumor-specic PDT/PTT therapy via PLNPs as the built-in
hydrolyzed
and
then
reacted
with
1-ethyl-3-(3- excitation light source possible.
dimethylaminopropyl) carbodiimide hydrochloride (EDC$HCl)
and N-hydroxysuccinimide (NHS) via amide condensation to
yield the active succinimide ester of BAC (BAC-NHS) (Fig. 1a).
In vitro PTT/PDT performance of PLNP-BAC-PEGBT
Amino functionalized PLNPs (PLNP-NH
2
) were then obtained To reveal the synergistic acid-activated and continuous
(Fig. S25†) to covalently bond with BAC-NHS via ester-amide external irradiation-free photothermal effect of PLNP-BAC-
condensation to form the PLNP sensitized activatable PTT/ PEGBT, continuous 808 nm laser irradiation as well as frac-
PDT-in-one theranostic platform (PLNP-BAC). To endow active tionated 808 nm laser irradiation (repeated cycles for 1 min
ꢀ
2
tumor-targeting ability and prolong blood-circulation, PEGBT laser on/1 min laser off) was carried out at 0.6 W cm . An
was further introduced onto the surface of PLNP-BAC with obvious temperature increase of PLNP-BAC-PEGBT was
chloroacetyl chloride as a bridge. For this purpose, PLNP-BAC observed under both continuous and fractionated irradiation
was modied with chloroacetyl chloride to yield PLNP-BAC-Cl at pH 6.0 (Fig. 3h). In comparison, a slight temperature uc-
2
via an acylation reaction between the –NH group of PLNP- tuation was noted for BAC at pH 6.0 under fractionated
BAC and the –COCl group of chloroacetyl chloride with potas- 808 nm laser irradiation (Fig. 3h). Besides, naked PLNPs
sium carbonate as the deacid reagent. The obtained PLNP-BAC- (either at pH 6.0 or pH 7.4) or PLNP-BAC-PEGBT (pH 7.4) only
2
Cl had a terminal –CH Cl group to further react with PEGBT via did not trigger any notable temperature increase upon 808 nm
nucleophilic substitution to produce PEGBT modied PLNP- laser irradiation up to 10 min (Fig. 3h and S28†). The above
BAC (PLNP-BAC-PEGBT) (Fig. 1a). As such, a smart theranostic results show that the as-prepared PLNP-BAC-PEGBT has great
platform PLNP-BAC-PEGBT was successfully constructed.
Conjugation of BAC and PEGBT did not lead to obvious in light source for repeatable PTT due to their long NIR
morphological change or signicant aggregation of PLNP-NH aerglow.
potential for tumor-specic PTT and PLNPs can act as a built-
2
1
(Fig. 3a vs. Fig. S24a†), but remarkably increased the hydrody-
The O
2
generation performance of PLNP-BAC-PEGBT at pH
namic size of PLNP-NH
2
from 58.9 ꢂ 2.3 nm to 91.6 ꢂ 3.2 nm 7.4 or 6.0 under continuous and fractionated 808 nm laser
1
and 122.2 ꢂ 3.7 nm, respectively, owing to the conjugated rigid irradiation was then tested with DPBF as the probe for
2
O .
1
plane structure of BAC and the large hydrodynamic volume of PLNP-BAC-PEGBT at pH 6.0 generated signicant
PEGBT (Fig. 3b). Meanwhile, the zeta potential turned from continuous irradiation as conrmed by signicant and rapid
2.67 ꢂ 4.38 mV to 11.63 ꢂ 3.48 and ꢀ16.97 ꢂ 3.24 mV, reduction of the absorbance of DPBF (Fig. 3i and S29b†).
respectively (Fig. 3c). The disappearance of the characteristic Interestingly, fractionated 808 nm laser irradiation of PLNP-
2
O under
1
ꢀ
1
1
2 2
bands at 3241 cm (–NH stretching vibration of PLNPs) and BAC-PEGBT (pH 6.0) gave an O generation efficiency similar
ꢀ1
1
703 cm (–COOH stretching vibration of BAC) in conjunction to continuous irradiation in a period of 10 min (Fig. 3i). In
ꢀ
ꢀ
1
with the appearance of the characteristic peaks at 1650 cm
contrast, PLNP-BAC-PEGBT at pH 7.4 under the same contin-
uous or fractionated irradiation did not produce any O as
2
1
1
(
(
stretching vibration of –CONH–) and 1600–1450 cm
stretching vibration of the benzene skeleton) in the FT-IR veried by negligible reduction of the absorbance of DPBF
spectrum of PLNP-BAC-PEGBT also conrm the successful (Fig. 3i and S29a†). The above results clearly indicate that the as-
conjugation of BAC and PEGBT onto the PLNPs (Fig. 3d). The prepared PLNP-BAC-PEGBT has excellent potential for tumor-
color of PLNP-BAC turned from white to pink (color of BAC in its specic PDT, and PLNPs are promising as the built-in light
base form) further indicates the presence of BAC on the surface source for assisted enhanced PDT.
This journal is © The Royal Society of Chemistry 2020
Chem. Sci.

View Article Online
Chemical Science
Edge Article
Fig. 3 Characterization of PLNP-BAC-PEGBT. (a) TEM images of PLNP-BAC-PEGBT. (b) Hydrodynamic diameter distribution of PLNPs, PLNP-
OH, PLNP-NH , PLNP-BAC and PLNP-BAC-PEGBT. (c) Zeta potentials of PLNP-OH, PLNP-NH , PLNP-BAC and PLNP-BAC-PEGBT. (d) FT-IR
spectra of PLNP-NH , BAC, PEGBT and PLNP-BAC-PEGBT. (e) XRD patterns of PLNPs, PLNP-BAC-PEGBT, ZnGa and Zn GeO . (f) Photo-
luminescence spectra of PLNPs and PLNP-BAC-PEGBT at pH 6.0 and 7.4. (g) Reactivated NIR persistent luminescence images of PLNPs, PLNP-
NH and PLNP-BAC-PEGBT at pH 7.4 and 6.0 recorded on a PerkinElmer IVIS Lumina III imaging system. (h) Irradiation time dependent
temperature change curves of BAC and PLNP-BAC-PEGBT at pH 6.0 with continuous or fractionated 808 nm laser irradiation. (i) Time-
dependent absorbance of DPBF (A/A ) at 410 nm with PLNP-BAC-PEGBT at pH 6.0 and 7.4 under continuous or fractionated 808 nm laser
irradiation. A is the initial absorbance of DPBF, while A is the absorbance of DPBF at a certain irradiation time.
2
2
2
2
O
4
2
4
2
0
0
Cell targeting NIR imaging and PTT/PDT
used (Fig. 4a), indicating that the BT on the surface of PLNP-
BAC-PEGBT exhibits active targeting specicity and enhances
the internalization of the nanoprobe. Besides, the BT mediated
active tumor-targeting effect of PLNP-BAC-PEGBT was veried
and quantied by ow cytometry analysis (Fig. S32†). The mean
uorescence intensity (MFI) of the cells incubated with PLNP-
BAC-PEGBT exhibited about a 1.4-fold increase compared with
that of the control groups. Furthermore, the CLSM and ow
cytometry analysis of HeLa cells gave the same results (Fig. S33
and S34†). However, no noticeable difference in the results of
CLSM and ow cytometry was observed in 3T3 cells treated with
PLNPs or PLNP-BAC-PEGBT (Fig. S35 and S36†). These results
clearly indicate that the internalization of PLNP-BAC-PEGBT by
tumor cells was signicantly improved by BT due to the
enhanced permeation and retention effect and BT receptor
mediated active tumor-targeting effect.
The potential of PLNP-BAC-PEGBT for in vitro cell targeting
luminescence imaging and PTT/PDT was demonstrated with
HeLa and A549 cells, two kinds of biotin overexpressed cancer
4
2–44
cells,
as model cells and biotin receptor-negative 3T3 cells as
44
the negative control. The cytotoxicity of PLNP-BAC-PEGBT
towards the above cells was rst evaluated via the MTT assay.
The cell viability of these cells remained over 95% aer 24 h of
incubation even if the test concentration was up to 500 mg
ꢀ
1
mL , demonstrating negligible cytotoxicity and good biocom-
patibility of PLNP-BAC-PEGBT (Fig. S30 and S31†).
Cell internalization and imaging of PLNP-BAC-PEGBT were
then investigated to show its targeting capability. To this point,
ꢀ
1
7
5 mg mL PLNP-BAC-PEGBT was incubated with HeLa, A549
and 3T3 cells, respectively, and cell internalization was moni-
tored using a confocal laser scanning microscope (CLSM). The
luminescence signal in A549 cells incubated with PLNP-BAC-
PEGBT was clearly brighter than that in BT pre-treated A549
cells or the A549 cells treated with PLNPs (control groups)
although the same amount of PLNP-BAC-PEGBT (as PLNPs) was
The photothermal and photodynamic cytotoxicity of PLNP-
BAC-PEGBT was tested on tumor cells (A549 and HeLa, pH
6.0) and normal cells (3T3, pH 7.4). PLNP-BAC-PEGBT irradi-
ated with an 808 nm laser exhibited obvious cytotoxicity against
the tumor cells in an irradiation time dependent manner
Chem. Sci.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Edge Article
Chemical Science
Fig. 4 Cell cytotoxicity and internalization of PLNP-BAC-PEGBT. (a) Cell internalization and cell imaging of PLNP-BAC-PEGBT toward A549 cells
scale bar, 30 mm). (b) Cell viability of PLNP-BAC-PEGBT with continuous or fractionated 808 nm laser irradiation against A549, HeLa and 3T3
(
ꢀ2
cells. Control refers to continuous 808 nm laser irradiation (0.6 W cm , 10 min); 2 min and 10 min refer to 2 min and 10 min continuous 808 nm
laser irradiation (0.6 W cm ) with PLNP-BAC-PEGBT, respectively; 10 min (1/1 min) represents fractionated 808 nm laser irradiation (repeated
cycles for 1 min laser on/1 min laser off, 0.6 W cm ) with PLNP-BAC-PEGBT for a total time of 10 min. (c) CLSM imaging of A549 cells incubated
with or without PLNP-BAC-PEGBT under 10 min of 808 nm laser irradiation (0.6 W cm ) (Calcein-Am and PI staining).
ꢀ2
ꢀ2
ꢀ2
(Fig. 4b, c and S37†). An extremely low cell viability (ca. 5%) was guided precision phototherapy. The in vivo precision tumor-
observed for A549 and HeLa cells treated with PLNP-BAC- targeting imaging performance of PLNP-BAC-PEGBT was
PEGBT under continuous or fractionated 808 nm laser irradia- demonstrated through intravenously injecting PLNP-BAC-
tion for a total time of 10 min (Fig. 4b, c, and S37†). In contrast, PEGBT (experimental group) or PLNP-BAC (control group) into
PLNP-BAC-PEGBT did not induce discernible cytotoxicity HeLa tumor-bearing mice (Fig. 5a). The luminescence signal
against 3T3 cells (cell viability ca. 90%) under all the above appeared at the tumor site of the experimental group at ca. 4 h,
irradiation conditions, because the PTT/PDT effect of PLNP- gradually increased to the maximum at ca. 6 h, and remained
BAC-PEGBT was inactive in normal cells (Fig. 4b and S38†). clear up to at least 12 h, indicating that the maximum amount
These results show that PLNPs can act as an efficient built-in of PLNP-BAC-PEGBT was accumulated in the tumor site at 6 h
light source for triggering PTT/PDT with no need for contin- aer injection. Hence, 6 h post-injection was chosen as the most
uous external light irradiation and the PTT/PDT effect of PLNP- appropriate time point for follow-up therapy. In contrast,
BAC-PEGBT could merely be activated in an acidic microenvi- almost no luminescence signal appeared at the tumor site of the
ronment, ensuring synergistic efficient therapy without side- control group, while the luminescence signal in the liver and
effects to adjacent normal tissues.
lungs in the control group was slightly weaker than that in the
experimental group, conrming that the introduction of PEGBT
is indeed helpful in enhancing the tumor-targeting accumula-
tion and prolonging the blood circulation time. Besides, auto-
uorescence background was effectively avoided during the
imaging process because the imaging signal was acquired with
no need for in situ excitation by taking advantage of the
persistent luminescence feature. The above results conrm that
PLNP-BAC-PEGBT has great potential for in vivo
In vivo autouorescence-free tumor-targeting NIR imaging
and precision therapy
The excellent biocompatibility, cell-targeting imaging and high-
efficiency in vitro anti-tumor effect of PLNP-BAC-PEGBT
encouraged us to explore whether it is suitable for in vivo
autouorescence-free tumor-targeting NIR imaging and image-
This journal is © The Royal Society of Chemistry 2020
Chem. Sci.

View Article Online
Chemical Science
Edge Article
Fig. 5 PLNP-BAC-PEGBT-mediated in vivo luminescence images in HeLa tumor-bearing mice. (a) In vivo persistent luminescence imaging of
HeLa tumor-bearing mice after intravenous injection with PLNP-BAC-PEGBT or PLNP-BAC pre-irradiated with a 254 nm UV light for 10 min
before injection and irradiated with 650 nm LED light for 2 min before acquisition. (b) Ex vivo persistent luminescence images of the major organs
and the tumor dissected from the mice at 6 h post-injection with PLNP-BAC-PEGBT (1–8: tumor, heart, liver, spleen, lungs, kidneys, stomach and
intestines, respectively). (c) Luminescence intensity of the major organs and the tumor dissected from mice at 6 h after intravenous injection with
PLNP-BAC-PEGBT or PLNP-BAC. (d) Biodistribution of PLNP-BAC-PEGBT or PLNP-BAC in the main organs and the tumor of the mice by
measuring the content of the Zn element from PLNPs at 6 h after injection. The corresponding main organs and tumor tissue of HeLa tumor-
bearing mice without any treatment were collected and measured as blank. Center values and error bars are defined as mean and s.d.,
respectively (n ¼ 3).
autouorescence-free tumor-targeting imaging and guiding the 6 h post-injection as the treatment groups, while other tumor-
stimulus in the best time and at the accurate location.
bearing mice without any treatment or treated with PBS in
The major organs and the tumor tissues were then collected combination with continuous/fractionated laser irradiation or
at 6 h post intravenous injection for ex vivo imaging to further PLNP-BAC-PEGBT only set as the control groups. The treatment
investigate the tumor-targeting accumulation and bio- group under continuous 808 nm laser exposure shows an
distribution (Fig. 5b and c). Although the luminescence signal obvious temperature increase in tumor sites (from the initial
ꢁ
ꢁ
in the lungs and liver and a slight luminescence signal in the 32.5 C to 49 C) (Fig. S39†). Importantly, the treatment group
spleen were inevitable due to the strong phagocytosis of retic- under fractionated irradiation exhibits almost the same
uloendothelial system organs, a noticeable luminescence signal thermal increase (Fig. S39†), suggesting that the PLNP core can
existed at tumor sites only in the PLNP-BAC-PEGBT group, act as an effective built-in light for tumor PTT/PDT. In contrast,
further showing that the tumor-targeting accumulation was all control groups did not induce any increase of temperature
specically improved by PEGBT. Moreover, the tumor-targeting (Fig. S39†), indicating that the photothermal effect of PLNP-
accumulation of PLNP-BAC-PEGBT was conrmed by induc- BAC-PEGBT was specically governed by the tumor acidic
ꢀ2
tively coupled plasma mass spectrometry for the determination microenvironment and the 0.6 W cm laser irradiation power
of Zn (originating from the PLNP core) in major organs and (continuous or fractionated exposure) was acceptable with no
tumor tissues 6 h aer the injection as well (Fig. 5d). The obvious photothermal side-effects itself. Further experiments
quantitative results further indicate that the PLNP-BAC-PEGBT with a high power laser demonstrate that fractionated laser
group has higher tumor-targeting accumulation than PLNP- irradiation can effectively reduce damage of the laser itself
BAC, making PLNP-BAC-PEGBT promising for tumor-targeting compared with the traditional continuous irradiation
accumulation and autouorescence-free tumor-specic (Fig. S40†).
imaging.
A time-dependent change in tumor volume indicates that
We then evaluate the in vivo precision PTT/PDT of PLNP- tumor tissues are obviously suppressed and disappear in the
BAC-PEGBT. Tumor-bearing mice were intravenously injected late-stage for PLNP-BAC-PEGBT with continuous or fractionated
with PLNP-BAC-PEGBT and subjected to continuous or frac- laser irradiation treatment groups, whereas the tumor tissues
tionated (repeated cycles for 1 min laser on/1 min laser off) still increased signicantly in the above control groups (Fig. 6a
ꢀ2
8
08 nm laser exposure (0.6 W cm for a total time of 10 min) at and b). To further investigate the in vivo therapeutic effect and
Chem. Sci.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Edge Article
Chemical Science
mechanism of PLNP-BAC-PEGBT under laser irradiation, (Fig. S41†). Meanwhile, the treatment groups showed obvious
histological and immunohistochemical (IHC) analyses inhibition of cell proliferation and signicant promotion of
(
including Ki67 and cleaved caspase 3 for cell proliferation and apoptosis as conrmed by the obvious decrease of the expres-
apoptosis, respectively) of tumor tissue slices were carried out at sion of Ki67 and the increase of caspase 3 (Fig. S42†), which is
4 h post treatment. The hematoxylin and eosin (H&E) staining consistent with the observed tumor tissue inhibition or even
2
results show that the tumor tissues in treatment groups were disappearance. Furthermore, the systemic toxicity of the PLNP-
seriously damaged with obvious karyopyknosis and many void BAC-PEGBT platform was also evaluated by histology analysis
spaces of tumor cells in comparison with the control groups aer the treatment process, where no obvious organ damage
Fig. 6 PLNP-BAC-PEGBT-mediated in vivo PTT/PDT in HeLa tumor-bearing mice models. (a) Representative photos for different groups of
mice during the PTT/PDT therapy process. (b) Tumor volume change in mice during the PTT/PDT process relative to initial tumor volumes. (c)
Body weight change of mice during the PTT/PDT process. (d) H&E staining of the main organs and tumor tissues of mice after the treatment
(
scale bar, 100 mm). Center values and error bars are defined as mean and s.d., respectively (n ¼ 3).
This journal is © The Royal Society of Chemistry 2020
Chem. Sci.

View Article Online
Chemical Science
Edge Article
and inammation were observed (Fig. 6d). Besides, neither
remarkable body weight loss nor abnormal behavior occurred
in the treatment groups compared to the controls (Fig. 6c). All
the above results consistently conrm that the developed PLNP-
BAC-PEGBT platform is competent for tumor-specic ablation
by a single intravenous injection and one-time continuous or
even fractionated laser irradiation with good therapeutic effects
and no obvious side-effects.
Notes and references
1 W. Xiao, P. Wang, C. Ou, X. Huang, Y. Tang, M. Wu, W. Si,
J. Shao, W. Huang and X. Dong, Biomaterials, 2018, 183, 1–9.
2 G. Lan, K. Ni and W. Lin, Coord. Chem. Rev., 2019, 379, 65–81.
3 J. F. Lovell, T. W. B. Liu, J. Chen and G. Zheng, Chem. Rev.,
2010, 110, 2839–2857.
4 L. Y. Li, Y. M. Zhou, R. Y. Gao, X. C. Liu, H. H. Du, J. L. Zhang,
X. C. Ai, J. P. Zhang, L. M. Fu and L. H. Skibsted, Biomaterials,
2
019, 190–191, 86–96.
Conclusions
5
6
7
8
C. Wang, B. Huang, G. Yang, Y. Ouyang, J. Tian and
W. Zhang, Biomacromolecules, 2019, 20, 4218–4229.
W. Fan, W. Bu, B. Shen, Q. He, Z. Cui, Y. Liu, X. Zheng,
K. Zhao and J. Shi, Adv. Mater., 2015, 27, 4155–4161.
Y. Cheng, H. Cheng, C. Jiang, X. Qiu, K. Wang, W. Huan,
A. Yuan, J. Wu and Y. Hu, Nat. Commun., 2015, 6, 8785.
P. Li, L. Liu, Q. Lu, S. Yang, L. Yang, Y. Cheng, Y. Wang,
S. Wang, Y. Song, F. Tan and N. Li, ACS Appl. Mater.
Interfaces, 2019, 11, 5771–5781.
In summary, we have reported a PLNP-BAC-PEGBT theranostic
nanoplatform based on pH-reversibly activatable NIR PTT/PDT-
in-one agent BAC and NIR renewable nanoimplant PLNPs for
precision tumor-targeting and autouorescence-free image-
guided efficient phototherapy without the need for contin-
uous external irradiation. The reversibly activatable PTT/PDT
synergetic enhancement effect of BAC in combination with
the super-long NIR aerglow feature of PLNPs makes the PLNP-
BAC-PEGBT effectively overcome the shortcomings of limited
therapeutic efficiency and non-specic damage in the previous
9
M. Sun, L. Xu, W. Ma, X. Wu, H. Kuang, L. Wang and C. Xu,
Adv. Mater., 2016, 28, 898–904.
1
1
1
1
1
1
0 B. Tian, C. Wang, S. Zhang, L. Feng and Z. Liu, ACS Nano,
011, 5, 7000–7009.
1 X. Hu, H. Tian, W. Jiang, A. Song, Z. Li and Y. Luan, Small,
018, 14, 1802994.
‘always on’ or irreversible ‘turn-on’ PTT/PDT platforms with the
2
need for continuous and long term in situ irradiation. Such
a pH-reversibly activatable PTT/PDT synergetic effect, needless
continuous external laser irradiation and BT-mediated active
targeting combined with autouorescence-free NIR image-
guiding endow the smart theranostic nanoplatform with great
potential for precision tumor-targeting and excellent thera-
peutic effects without nonspecic damage. This work furnishes
a prospective strategy to design an intelligent theranostic plat-
form for precision medicine.
2
2 Y. K. Kim, H. K. Na, S. Kim, H. Jang, S. J. Chang and
D. H. Min, Small, 2015, 11, 2527–2535.
3 X. Li, S. Kolemen, J. Yoon and E. U. Akkaya, Adv. Funct.
Mater., 2017, 27, 1604053.
4 X. Li, S. Lee and J. Yoon, Chem. Soc. Rev., 2018, 47, 1174–
1
188.
5 Q. Zhan, J. Qian, H. Liang, G. Somesfalean, D. Wang, S. He,
Z. Zhang and S. Andersson-Engels, ACS Nano, 2011, 5, 3744–
3
757.
Ethical statement
1
6 G. Yang, D. Yang, P. Yang, R. Lv, C. Li, C. Zhong, F. He, S. Gai
All experiments were performed in strict accordance with the
and J. Lin, Chem. Mater., 2015, 27, 7957–7968.
Chinese National Standard Laboratory animal Guideline for the 17 S. K. Sun, H. F. Wang and X. P. Yan, Acc. Chem. Res., 2018, 51,
ethical review of animal welfare (GB/T 35892-2018) and were
1131–1143.
approved by the Institutional Animal Care and Use Committee 18 Y. Li, M. Gecevicius and J. Qiu, Chem. Soc. Rev., 2016, 45,
of Jiangnan University (Wuxi, China).
2090–2136.
1
9 T. Maldiney, A. Lecointre, B. Viana, A. Bessi `e re, M. Bessodes,
D. Gourier, C. Richard and D. Scherman, J. Am. Chem. Soc.,
Conflicts of interest
2011, 133, 11810–11815.
2
2
0 Z. Pan, Y. Y. Lu and F. Liu, Nat. Mater., 2011, 11, 58–63.
1 Z. Li, Y. Zhang, X. Wu, X. Wu, R. Maudgal, H. Zhang and
G. Han, Adv. Sci., 2015, 2, 1500001.
There are no conicts to declare.
2
2 Z. Li, Y. Zhang, X. Wu, L. Huang, D. Li, W. Fan and G. Han, J.
Am. Chem. Soc., 2015, 137, 5304–5307.
Acknowledgements
The authors appreciate the support from the National Natural 23 W. Fan, N. Lu, C. Xu, Y. Liu, J. Lin, S. Wang, Z. Shen, Z. Yang,
Science Foundation of China (No. 21934002, 21804056 and
1804057), the Natural Science Foundation of Jiangsu Province,
J. Qu, T. Wang, S. Chen, P. Huang and X. Chen, ACS Nano,
2017, 11, 5864–5872.
2
China (No. BK20180581 and BK20180584), the China Post- 24 R. Abdurahman, C. X. Yang and X. P. Yan, Chem. Commun.,
doctoral Science Foundation (No. 2018M630511 and
2016, 52, 13303–13306.
018M630509), the National First-class Discipline Program of 25 J. Wang, Y. Li, R. Mao, Y. Wang, X. Yan and J. Liu, J. Mater.
2
Food Science and Technology (No. JUFSTR20180301), and the
Collaborative Innovation Center of Food Safety and Quality
Control in Jiangsu Province.
Chem. B, 2017, 5, 5793–5805.
Chem. Sci.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Edge Article
Chemical Science
26 Y. J. Li, C. X. Yang and X. P. Yan, Anal. Chem., 2018, 90, 4188– 37 B. P. Lin, H. T. Chen, D. Y. Liang, W. Lin, X. Y. Qi, H. P. Liu
4195.
and X. Y. Deng, ACS Appl. Mater. Interfaces, 2019, 11, 11157–
27 L. Hu, P. Wang, M. Zhao, L. Liu, L. Zhou, B. Li,
11166.
F. H. Albaqami, A. M. El-Toni, X. Li, Y. Xie, X. Sun and 38 Z. Sheng, D. Hu, M. Zheng, P. Zhao, H. Liu, D. Gao, P. Gong,
F. Zhang, Biomaterials, 2018, 163, 154–162.
8 Y. J. Li and X. P. Yan, Nanoscale, 2016, 8, 14965–14970.
G. Gao, P. Zhang, Y. Ma and L. Cai, ACS Nano, 2014, 8,
12310–12322.
2
2
9 J. Wang, Q. Ma, X. X. Hu, H. Liu, W. Zheng, X. Chen, Q. Yuan 39 M. Zheng, P. Zhao, Z. Luo, P. Gong, C. Zheng, P. Zhang,
and W. Tan, ACS Nano, 2017, 11, 8010–8017.
0 B. B. Srivastava, A. Kuang and Y. Mao, Chem. Commun., 2015,
C. Yue, D. Gao, Y. Ma and L. Cai, ACS Appl. Mater.
Interfaces, 2014, 6, 6709–6716.
3
3
5
1, 7372–7375.
40 W. S. Kuo, Y. T. Chang, K. C. Cho, K. C. Chiu, C. H. Lien,
C. S. Yeh and S. J. Chen, Biomaterials, 2012, 33, 3270–3278.
1 E. Teston, S. Richard, T. Maldiney, N. Li `e vre, G. Y. Wang,
L. Motte, C. Richard and Y. Lalatonne, Chem.-Eur.J., 2015, 41 F. Liu, Y. Chen, Y. Liang and Z. Pan, Opt. Lett., 2016, 41, 954–
1, 7350–7354. 957.
2 J. Jiang, Z. Zhao, Z. Hai, H. Wang and G. Liang, Anal. Chem., 42 R. Kumar, E. J. Kim, J. Han, H. Lee, W. S. Shin, H. M. Kim,
2
3
3
2017, 89, 9625–9628.
S. Bhuniya, J. S. Kim and K. S. Hong, Biomaterials, 2016,
104, 119–128.
3 X. Li, C. Y. Kim, S. Lee, D. Lee, H. M. Chung, G. Kim,
S. H. Heo, C. Kim, K. S. Hong and J. Yoon, J. Am. Chem. 43 X. Li, C. Y. Kim, S. Lee, D. Lee, H. M. Chung, G. Kim,
Soc., 2017, 139, 10880–10886.
4 F. Hu, S. Xu and B. Liu, Adv. Mater., 2018, 30, 1801350.
S. H. Heo, C. Kim, K. S. Hong and J. Yoon, J. Am. Chem.
Soc., 2017, 31, 10880–10886.
3
3
5 J. Cao, J. Chi, J. Xia, Y. Zhang, S. Han and Y. Sun, ACS Appl. 44 D. N. Heo, D. H. Yang, H. Moon, J. B. Lee, M. S. Bae, S. C. Lee,
Mater. Interfaces, 2019, 11, 25720–25729.
6 E. Boedtkjer and S. F. Pedersen, Annu. Rev. Physiol., 2020, 82,
W. J. Lee, I. C. Sun and I. K. Kwon, Biomaterials, 2012, 33,
856–866.
3
103–126.
This journal is © The Royal Society of Chemistry 2020
Chem. Sci.