A stage-specific cancer chemotherapy strategy through flexible combination of reduction-activated charge-conversional core-shell nanoparticles

Article abstract of DOI:10.7150/thno.35057

Precision medicine has increased the demand for stage-specific cancer chemotherapy. Drugs with different properties are needed for different stages of tumor development, which is, inducing rapid destruction in the early stage and facilitating deep penetration in the advanced stage. Herein, we report a novel reduction-activated charge-conversional core-shell nanoparticle (CS NP) formula based on ring-closing metathesis of the thiamine disulfide system (TDS) to deliver the chemotherapeutic agent-gambogic acid (GA). Methods: The shell consisted of hyaluronic acid-all-trans retinoid acid with a disulfide bond as the linker (HA-SS-ATRA). The core was selected from poly (γ-glutamic acid) with different grafting rates of the functional group (Fx%) of TDS. GA/C_F100%S NPs, with the strongest reduction-responsive drug release, and GA/C_F60%S NPs with the strongest penetration have been finally screened. On this basis, a stage-specific administration strategy against a two-stage hepatocellular carcinoma was proposed. Results: The developed CS NPs have been confirmed as inducing reduction-activated charge conversion from about -25 to +30 mV with up to 95% drug release within 48 h. The administration strategy, GA/C_F100%S NPs for the early-stage tumor, and sequential administration of GA/C_F60%S NPs followed by GA/C_F100%S NPs for the advanced-stage tumor, achieved excellent tumor inhibition rates of 93.86±2.94% and 90.76±6.43%, respectively. Conclusions: Our CS NPs provide a novel platform for charge conversion activated by reduction. The stage-specific administration strategy showed great promise for cancer therapy.

Full text of DOI:10.7150/thno.35057

Theranostics 2019, Vol. 9, Issue 22
6532
Ivyspring
International Publisher
Theranostics
2
019; 9(22): 6532-6549. doi: 10.7150/thno.35057
Research Paper
A stage-specific cancer chemotherapy strategy through
flexible combination of reduction-activated
charge-conversional core-shell nanoparticles
1
1
2
2
2
1
3
Lingfei Han , Yingming Wang , Xiaoxian Huang , Bowen Liu , Lejian Hu , Congyu Ma , Jun Liu , Jingwei
3
,4
2
2,3,5
2,6
1,7
Xue , Wei Qu , Fulei Liu
, Feng Feng
, Wenyuan Liu
1
2
3
4
5
6
7
.
.
.
.
.
.
.
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China
Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
The Joint Laboratory of Chinese Pharmaceutical University and Taian City Central Hospital, Taian City Central Hospital, Taian, 271000, China
Taian City institute of Digestive Disease, Taian City Central Hospital, Taian, 271000, China
Pharmaceutical Department, Taian City Central Hospital, Taian, 271000, China
Jiangsu Food and Pharmaceutical Science College, Huaian 223003, China
Hangzhou Institute of Pharmaceutical Innovation, China Pharmaceutical University, Hangzhou 310018, China

Corresponding authors: liu_fulei@126.com; fengsunlight@163.com; liuwenyuan8506@163.com.
©
The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/).
See http://ivyspring.com/terms for full terms and conditions.
Received: 2019.03.20; Accepted: 2019.06.18; Published: 2019.08.21
Abstract
Precision medicine has increased the demand for stage-specific cancer chemotherapy. Drugs with
different properties are needed for different stages of tumor development, which is, inducing rapid
destruction in the early stage and facilitating deep penetration in the advanced stage. Herein, we
report a novel reduction-activated charge-conversional core-shell nanoparticle (CS NP) formula
based on ring-closing metathesis of the thiamine disulfide system (TDS) to deliver the
chemotherapeutic agent-gambogic acid (GA).
Methods: The shell consisted of hyaluronic acid-all-trans retinoid acid with a disulfide bond as the
linker (HA-SS-ATRA). The core was selected from poly (γ-glutamic acid) with different grafting
rates of the functional group (Fx%) of TDS. GA/CF100%S NPs, with the strongest
reduction-responsive drug release, and GA/CF60%S NPs with the strongest penetration have been
finally screened. On this basis, a stage-specific administration strategy against a two-stage
hepatocellular carcinoma was proposed.
Results: The developed CS NPs have been confirmed as inducing reduction-activated charge
conversion from about -25 to +30 mV with up to 95% drug release within 48 h. The administration
strategy, GA/CF100%S NPs for the early-stage tumor, and sequential administration of GA/CF60%S NPs
followed by GA/CF100%S NPs for the advanced-stage tumor, achieved excellent tumor inhibition
rates of 93.86±2.94% and 90.76±6.43%, respectively.
Conclusions: Our CS NPs provide a novel platform for charge conversion activated by reduction.
The stage-specific administration strategy showed great promise for cancer therapy.
Key words: charge conversion, reduction-activated, tumor penetration, stage-specific chemotherapy,
hepatocellular carcinoma
Introduction
Chemotherapy still plays an important role in
cancer treatment, despite the emergence of various
promising alternatives, such as photodynamic and
photothermal therapy [1,2]. In the context of precision
medicine [3], achieving optimal efficacy at each stage
of a cancer is of prime concern, and this gives rise to
the concept of stage-specific cancer therapy [4].
Generally, an early-stage tumor develops quickly due
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to sufficient nutrition supplies from adjacent blood
vessels [5]. Hence, efficient tumor targeting and rapid
release of high doses of drugs are required to swiftly
destroy the fast-growing tumor [6], which has been
widely realized through the use of various
stimuli-responsive drug carriers [7]. For an
advanced-stage tumor, however, tumor penetration
becomes another issue of concern [8,9]. This is because
internal hypoxia becomes more prevalent with
increasing tumor volume, which could promote the
proliferation of cancer stem cells. Such cells are
particularly aggressive and can lead to metastasis and
relapses [10-12]. In addition, it is difficult for drugs to
access deep sites of an advanced-stage tumor of large
size with dense stroma due to several biological
barriers [13]. Worse still, the deep tumor cells could
gradually acquire resistance due to long-term
TDS is reduced by GSH and undergoes ring-closing to
form a thiazolium salt, which is lipophobic and
cannot cross the BBB via diffusion, thus becoming
“locked” in the brain and providing sustained release
of the drug through hydrolysis [30,31]. However, TDS
also readily enters red blood cells rich in GSH and
hemoglobin [32,33], leading to its undesirable
distribution and stability in the bloodstream. Further
research indicated that utilization of only the
4-methyl-5-hydroxyethyl thiazole derived from TDS
could overcome these drawbacks [27,34].
In view of the above findings, in this study, a
novel
reduction-activated
charge-conversional
core-shell nanoparticle (CS NP) formula based on TDS
as the functional group (FG) has been designed for
rapid drug release and deep penetration in cancer
treatment (Scheme 1). It comprised HA-SS-ATRA as
the shell (constructed as described in our previous
study [35]), poly (γ-glutamic acid) with different
grafting rates of the FG (γ-PFGA, using the grafting-to
method) as the core and GA as the chemotherapeutic
agent. The HA-coated GA/CS NPs were negatively
charged in blood circulation and accumulated in
tumor sites through an enhanced permeability and
retention (EPR) effect [36]. They were then
endocytosed through HA-CD44 receptor interaction
[37] and achieved the degradation of the shell and
core by intracellular GSH. Finally, GA-loaded γ-PFGA
exposure to
therapeutic agent and ultimately become refractory
14,15]. Thus, developing drug carrier with
a
sublethal concentration of the
[
a
improved penetration ability would be significant for
enhanced cancer treatment efficacy.
Many researchers have addressed this issue
through different strategies, including size switching
[
15,16], charge conversion [17,18], modifications with
penetration-promoting ligands [19,20] and
modulations of the tumor microenvironment [21,22].
Among these approaches, charge conversion, usually
triggered by various stimuli, such as pH, reactive
oxygen species, protease, heat or light [17], provides
an effective means of penetrating deep into tumor
cells. In general, nanoparticles with positively
charged surfaces can be effectively internalized by
cells through electrostatic interactions with the
negatively charged cell plasma membrane [23],
whereas cationic carriers in the bloodstream show
strong non-specific cellular uptake and can cause
severe serum inhibition [24,25]. Given that, it is
necessary to design charge-conversional drug carriers
to address issues of both safety and penetration.
Unfortunately, however, reduction-activated charge
conversion has rarely been reported. Glutathione
acquired
electrostatic-attraction-mediated
nuclei-targeting and deep tumor penetration abilities
owing to the positive charge generated from
ring-closing metathesis of the FG [38]. GA is a
multi-target drug, and is involved in many
mechanisms related to nuclei [39-41]. Notably,
although fast degradation of the materials would lead
to rapid drug release, less of the drugs would be left
for penetration into deep tumor cells. Therefore, four
types of GA/CS NPs (grafting rate of FG: 0%, 30%,
60% and 100%), exhibiting different properties, were
screened for the most powerful antitumor efficacy
(the rapidest drug release, GA/CF100%S NP) and the
strongest penetration ability (GA/CF60%
respectively.
S
NP)
(
GSH), as the most common biological reducing
substance, is present at a high concentration in tumor
cells (2-10 mM) [26] and has been widely exploited in
many rapid drug release carriers through cleavage of
a disulfide bond. Therefore, we sought to construct a
charge-conversional carrier induced by the cleavage
of a disulfide bond.
To further confirm the functionality of our
GA/CS NPs and embody our understanding of
precision medicine,
a
two-stage hepatocellular
carcinoma model (Heps) was established according to
the Tumor-Node-Metastasis (TNM) staging criteria
[42],
including
early
and
advanced-stages.
Fortunately, a brain-targeting strategy using
TDS termed “lock-in”, aimed at blood-brain barrier
Meanwhile, tumor biomarkers, as one of the
cornerstones for precision medicine [43], were also
utilized to assist establishing the model. On this basis,
we proposed a stage-specific administration strategy.
In brief, for early-stage tumors, GA/CF100%S NPs were
the best choice to exert a direct and strong damaging
(
BBB), gave us inspiration [27]. TDS is
a
cis-2-formylamino-ethenyl-thio derivative and has
been clinically used as a fat-soluble prodrug of
thiamine (Vitamin B1) [28,29]. On entering the BBB,
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effect due to rapid reduction-responsive drug release.
For advanced-stage tumors, sequential administration
of GA/CF60%S NPs followed by GA/CF100%S NPs,
termed the “Punch-Destruction” strategy, has been
conducted for the first time. First, GA/CF60%S NPs,
with the strongest drug-loaded penetration and
medium reduction-responsive drug release ability,
opened fissures to reach deep tumor cells, and then
GA/CF100%
S
NPs,
with
the
strongest
reduction-responsive drug release ability, killed the
remaining tumor cells along the carved-out route
(Scheme 2). All the results indicated that proper
utilization and flexible combination of these novel
reduction-activated charge-conversional CS NPs
could serve as an effective strategy for stage-specific
treatment for enhanced antitumor efficacy.
Scheme 1. The design and drug delivery of the functional GA/CS NPs for a two-stage cancer therapy. The reduction-activated charge-conversional CS NPs were composed of
HA-SS-ATRA as shell and GA-loaded γ-PFGA as core. After leakage from the tumor blood vessels, it first achieved the superficial zone. Take the advanced-stage tumor as an
example, I) HA-CD44 receptor mediated endocytosis, II) reduction-activated degradation of the shell material, III) reduction-activated degradation of the core material and
consequent charge conversion and nuclei targeting due to the ring-closing metathesis of the functional group, IV) escape of the positively charged core material with remaining
drugs from the dead tumor cells, V) electrostatic-attraction-mediated deep tumor penetration.
Scheme 2. (A) Stage-specific administration strategy for a two-stage hepatocellular carcinoma: GA/CF100%S NPs for the early-stage tumor and sequential administration of
GA/CF60%S NPs followed by GA/CF100%S NPs for the advanced-stage tumor. (B) Schematic of “Punch-Destruction” strategy for the advanced-stage tumor treatment. First, the
GA/CF60%S NPs with the strongest drug-loaded penetration ability make a hole to reach deep tumor cell; then the GA/CF100%S NPs, with the strongest destruction power due to
the rapidest reduction-responsive drug release, kill the remaining tumor cells along the carved-out route.
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2
Synthesis of 3,4-dimethyl-5-hydroxyethyl thiazolium
Methods
iodide (compound 1)
Materials
CH I (106.02 mmol) was added dropwise to
3
4
-methyl-5-hydroxyethyl thiazole (35.19 mmol) in an
Poly (L-glutamic acid) (γ-PGA-Na) (10 kDa) was
purchased from Nanjing Shineking Biotech Co., Ltd.
Jiangsu, China). Cetyltrimethylammonium bromide
CTA-Br) was purchased from Sinopharm Chemical
Reagent Co., Ltd. (Shanghai, China). Iodomethane,
-methyl-5-hydroxyethyl thiazole, 1-bromopropane,
benzyl bromide, bromomethyl benzoic acid, and
fluorescein isothiocyanate (FITC) were purchased
from Sigma-Aldrich (St. Louis, MO, USA). All of the
organic solvents used, such as dimethyl sulfoxide
autoclave at 60ºC over a period of 3 h. After
distillation under reduced pressure, the brown solid
(
(
product was washed with diethyl ether (yield 100%).
1
H NMR (300 MHz, CD
3
OD, δ): 2.54 (s, 3H, 4-CH
3
),
4
3.13 (t, 2H, J=6 Hz, -CH
-CH
2-H); MS (m/z): 158 [M+H] (Figure S1).
2
CH OH), 3.83 (t, 2H, J=6 Hz,
2
2
CH
2
3
OH), 4.16 (s, 3H, 2-CH ), 9.89 ppm (s, 1H,
+
3
Synthesis of propyl Bunte salt (compound 2)
The 1-bromopropane (49.83 mmol)
dissolved in ethanol (30 mL) containing PEG400
0.2 mL). A solution of Na ·5H O (45.21 mmol) in
was
(
DMSO),
ethanol,
ethyl
acetate,
and
N,N-dimethylformamide (DMF), were purchased
from Nanjing Chemical Reagent Co., Ltd. (Nanjing,
China). Distilled water was purified by a Millipore
Milli Q-Plus system (Millipore, Bedford, MA, USA).
(
2
S
O
2 3
2
water (10 mL) containing PEG400 (0.1 mL) was then
added dropwise and the mixture was heated under
reflux for 7 h at 69ºC. The product was obtained as a
white microcrystalline powder after distillation under
reduced pressure (yield 100%). H NMR (300 MHz,
D
Synthesis of the shell material
1
The HA-SS-ATRA conjugate was synthesized
following the methods devised in our previous
research [35]. In brief, HA-Na (14.6 kDa, 24.4 mmol)
and tetra-n-butylammonium hydroxide (TBA,
2
O, δ): 0.86 (t, 3H, J=6 Hz, -SCH
2
CH
2
CH ), 1.64 (m,
3
2H, -SCH
-SCH CH CH
2
CH
2
CH
3
), 2.95 ppm (t, 2H, J=6 Hz,
–
2
2
3
); MS (m/z): 155 [M–H] (Figure S2).
1
9.4 mmol) were combined in distilled water (60 mL),
and the solution was stirred for 1 h to generate
HA-TBA. ATRA (17.18 mmol), triethylamine
18.90 mmol), and bis(2-bromoethyl) disulfide
17.18 mmol) were mixed in THF (70 mL) by stirring
4
Synthesis of 3,4-dimethyl-5-hydroxyethyl thiazolium
iodide (ring-opening) with propyl (compound 3)
Aqueous NaOH (1 g/mL, 127.5 mmol) was
(
(
at
added to
a stirred solution of compound 2
(
21.1 mmol) in water (16 mL) under argon over a
room temperature
for 3 h
to
afford
and
period of 30 min with cooling in ice, and then ethyl
acetate (60 mL) was added over 10 min. A solution of
compound 3 (52.5 mmol) in water was added to the
above system over a period of 2 h. After separating
the layers, the ethyl acetate phase was concentrated
under reduced pressure. The concentrate was purified
ATRA-bromoethyl
disulfide.
HA-TBA
ATRA-bromoethyl disulfide (molar ratio 5:2) were
then stirred in DMSO for 48 h at 50ºC. Subsequent
dialysis with NaCl solution (5%, w/v) and distilled
water afforded HA-SS-ATRA conjugate as the shell
material. Furthermore, FITC-labeled HA-SS-ATRA
was synthesized following the reported method [44].
In brief, HA-SS-ATRA (100 mg) was dissolved in
distilled water (50 mL), and then a solution of FITC
by column chromatography on silica, eluting with
1
CH
300 MHz, CDCl
SCH CH CH ), 1.65 (m, 2H, SCH
s, 3H, 4-CH ), 2.57 (t, 2H, J=6 Hz, -CH
t, 2H, J=6 Hz, SCH CH CH ), 2.93/3.04 (s, 3H,
-CH ), 3.77 (t, 2H, J=6 Hz, -CH CH OH), 5.29 (s, 1H,
OH), 7.90/7.94 ppm (s, 1H, -CHO); MS (m/z): 272
2
Cl
2
/MeOH (50:1, v/v). Yield: 79.85%; H NMR
(
3
,
δ): 0.95 (t, 3H, J=7.5 Hz,
CH CH ), 1.95/1.99
CH OH), 2.85
(
20 mg) in ethanol (2 mL) was added dropwise. The
2
2
3
2
2
3
(
(
3
3
2
2
mixture was agitated in the dark for 3 days. The
FITC-labeled HA-SS-ATRA was recovered after
dialysis with water for 2 days and then freeze-dried.
2
2
3
3
2
2
-
[
Synthesis of the core material (Figure 1A)
+
M+Na] (Figure S3).
1
Synthesis of γ-PGA-CTA
5
Synthesis of 3,4-dimethyl-5-hydroxyethyl thiazolium
The γ-PGA-Na (10 kDa, 10 g) was dissolved in
distilled water (200 mL) under vigorous stirring at
0°C. Aqueous CTA-Br solution (3%, w/v) was then
added dropwise. The white precipitate of γ-PGA-CTA
formed was collected by filtration.
iodide (ring-opening) with propyl p-bromomethyl
benzoate (compound 4, functional group)
The 4-bromomethyl benzoic acid (32.26 mmol),
EDCI (35.75 mmol), and DMAP (16.92 mmol) were
4
mixed in CH
mixture was stirred for 30 min with cooling in ice. A
solution of compound 4 (20.05 mmol) in CH Cl
80 mL) was added over a period of 48 h at room
2
Cl
2
(120 mL) and DMF (50 mL). The
2
2
(
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Theranostics 2019, Vol. 9, Issue 22
6536
temperature. After completion of the reaction, the
were removed by threefold passage of the solution of
CS NPs through an Amicon Ultra-15 centrifugal filter
(Millipore, Billerica, MA, USA) with a molecular
weight cut-off of 100 kDa. The CS NPs were then
resuspended in water to obtain the desired final
concentration, and the suspension was stored at 4°C
prior to use. The size, polydispersity index (PDI), and
zeta potential were measured by means of a Malvern
Zetasizer Nano-ZS90 (Malvern Instruments, UK) and
a transmission electron microscope (TEM, Hitachi
HT7700). After extraction with a five-fold volume of
methanol for 5 min and filtration through a 0.22 μm
membrane, the drug encapsulation efficiency
(EE%=drug encapsulated in nanoparticles/initial
CH
2
Cl was evaporated under reduced pressure, the
2
remaining solution was poured into water (150 mL),
and the mixture was extracted with ethyl acetate
(
3×200 mL). The ethyl acetate extracts were combined,
washed with water (3×600 mL), dried over anhydrous
sodium sulfate, and concentrated. The obtained
pale-yellow solid was purified on a silica gel column,
eluting with petroleum ether/ethyl acetate (3:1, v/v) to
afford the product as a yellow oil (compound 4,
1
functional group). Yield: 22.05%; H NMR (300 MHz,
CDCl
(
(
3
, δ): 0.97 (t, 3H, J=7.5 Hz, SCH
m, 2H, SCH CH CH ), 1.96/1.98 (s, 3H, 4-CH
t, 2H, J=6 Hz, -CH CH OH), 2.93/3.02 (s, 3H, 3-CH
.09 (t, 2H, J=6 Hz, SCH CH CH ), 4.52 (t, 2H, J=6 Hz,
CH CH OH), 4.62 (s, 2H, Ph-CH Br), 7.48 (d, 2H,
J=9 Hz, Ph-3,5-H), 7.88 (s, 1H, -CHO), 8.01 ppm (d,
2
CH
2
CH
3
), 1.68
), 2.61
),
2
2
3
3
2
2
3
3
2
2
3
drug input
×
100%), drug-loading capacity
-
2
2
2
(DL%=drug encapsulated in nanoparticles/(drug +
initial material input) × 100%), drug release profile,
and in vitro stability were determined by HPLC
(Shimadzu 20AT).
+
2
H, J=9 Hz, Ph-2,6-H); MS (m/z): 446 [M+H]
(
Figure S4).
6
Synthesis of γ-PFGA with different grafting rates of
the functional group
The γ-PGA-CTA (0.962 mmol) was first
dissolved in NMP under stirring. NaHCO (0.39 g),
In vitro drug release in PBS and GSH
Aliquots (2 mL) of suspensions of different types
of GA/CS NPs (1 mg/mL) containing 0.1% (w/v)
Tween 80 were placed in dialysis bags (molecular
weight cut-off 3500 Da). They were then dialyzed
with PBS (100 mL) containing 0.1% (w/v) Tween 80
3
benzyl bromide, and compound 4 were then added to
the γ-PGA-CTA solution at four different molar ratios,
specifically 0%, 30%, 60%, and 100% (total 1 mmol).
The reaction system was stirred for 8 h at 50°C. The
mixture was then cooled to room temperature and
filtered to remove the insoluble precipitate. The
(
with or without 10 mM GSH), and the medium was
replaced every 12 h. At predetermined time points,
aliquots (50 μL) of the samples in the dialysis bags
were removed for HPLC analysis. Drug release curves
of the GA/CS NPs in PBS and GSH were plotted to
assess the release efficacy in reducing environments.
supernatant
was
poured
into
pre-cooled
methanol/water (1:1, v/v; 60 mL), containing 0.5%
HCl, and stirring was continued for 0.5 h. The
precipitate was collected by filtration and dried under
vacuum to obtain four types of core materials, namely
Reduction-responsive degradation
Aliquots (900 µL) of suspensions of different
types of CS NPs (100 μg/mL) were mixed with
aliquots (100 µL) of GSH solution (100 mM) and
incubated at 37°C. At predetermined time points, the
size distribution and morphology were measured by
dynamic light-scattering (DLS) and TEM.
0
%, 30%, 60%, and 100% γ-PFGA. The degree of
1
grafting rates was determined by
spectrometry.
H NMR
Preparation of CS NPs and GA/CS NPs with
different grafting rates of the functional group
Reduction-triggered charge conversion
The
γ-PFGA-HA-SS-ATRA
core-shell
Aliquots (900 µL) of suspensions of NPs
prepared with different core materials (100 μg/mL)
were mixed with aliquots (100 µL) of DTT solution
nanoparticles (CS NPs) were prepared by a one-step
method. Briefly, γ-PFGA (0%, 30%, 60%, 100%) was
dissolved in DMSO at 1.5 mg/mL, with or without
GA at 0.15 mg/mL, as the organic phase.
HA-SS-ATRA was dispersed in water at 0.15 mg/mL
as the aqueous phase and heated to 65°C prior to
preparation of the CS NPs. The organic phase was
then added to the preheated aqueous phase
(
100 mM) and incubated at 37°C. At predetermined
time points, the zeta potential was measured by DLS.
Further, adequate amount of different types of core
materials was treated in the same way. The reaction
was terminated by adding an equivalent of H
2
2
O at a
predetermined time point, and the mixture was
dialyzed. After work-up, the product was
freeze-dried for structure confirmation of its charge
(
0.5 mL/min)
under
vigorous
stirring.
The
temperature of the mixture was kept at 65°C during
nanoprecipitation, and the nanoparticles were
allowed to self-assemble for 2 h at room temperature.
The remaining organic solvent and free molecules
1
conversion by H NMR spectrometry.
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Theranostics 2019, Vol. 9, Issue 22
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Cell culture
group of 2 mg/mL HA was pretreated for 2 h. The
cells were then washed twice with PBS and fixed with
Human hepatocellular carcinoma cell line
HepG2, murine hepatocellular carcinoma cell line
Heps, and human hepatocyte cells (L02) were
obtained from the cell bank of the Chinese Academy
of Sciences (Shanghai, China). Cells were cultured in
4
% paraformaldehyde for 15 min. DAPI was then
added to stain the cell nuclei. Fluorescence images
were acquired by means of an LSM 800 laser confocal
scanning microscope (Zeiss, Germany). The images
were analyzed using Zeiss CLSM software. For
1
640 medium containing 10% FBS, 100 U/mL
quantitative analysis, the cells were seeded in a
penicillin, and 100 μg/mL streptomycin and
5
1
2
2-well plate (1 × 10 cells/well) and incubated for
4 h. Free C6 and C6/CS NPs were then added to each
maintained at 37°C in a humidified atmosphere
containing 5% CO
in an incubator.
2
well. For the 2 h group, another group of 2 mg/mL
HA was pretreated for 2 h. After incubation at 37°C
for 0.5 h, 2 h, or 4 h, the cells were digested with 0.25%
(w/v) trypsin, washed thrice with PBS, and then
resuspended in PBS (500 μL). Fluorescence
histograms were then recorded by FCM and analyzed
using FlowJo software.
Cytotoxicity
L02 and HepG2 cells were seeded in 96-well
plates at a density of 5×103 cells/well. After
incubation for 24 h, the cells were separately treated
with various concentrations of different types of
CS/NPs, GA/CS NPs, and GA for 24 h. Each type of
GA/CS NPs was pretreated with 50 μM BSO, 10 mM
GSH, and 2 mg/mL HA for 12 h, 2 h, and 2 h,
respectively, to investigate the enhanced cytotoxicity
due to reduction-sensitivity and HA-CD44-mediated
endocytosis. After incubation, 5 mg/mL MTT
solution (20 μL/well) was added and the mixtures
were cultured for a further 4 h. The supernatant was
discarded and DMSO was added (150 μL/well). The
absorbance at 492 nm was then measured by means of
a microplate reader (Thermo Electron Corporation).
Untreated cells were used as a 100% viability control
and triplicate determinations were performed for each
treatment.
Nuclei targeting
The method was the same as that described for
cellular uptake. For each C6/CS NP group incubated
for 4 h, samples were pretreated with 50 μM BSO and
1
0 mM GSH for 12 h and 2 h, respectively.
Intracellular shell detachment
The method was the same as that described for
cellular
uptake.
1,1-Dioctadecyl-3,3,3,3-
tetramethylindotricarbocyanine iodide (DiR,
a
near-infrared dye; Thermo Fisher Scientific, USA) was
used as a fluorescent probe. HepG2 cells were
incubated with (GA+DiR)/CF60%
S
NPs using
Cell apoptosis assay
FITC-labeled HA-SS-ATRA for 1 h or 4 h.
Apoptosis of HepG2 cells was detected using an
In vitro two-dimensional intercellular delivery
Annexin V-FITC apoptosis detection kit (BD
HepG2 cells seeded on coverslips were
4
Biosciences). The cells (5 × 10 per well) were seeded
pretreated with (GA+DiR)/CF60%
S NPs or free
in 12-well plates and cultured for 24 h. For Annexin
V-FITC assay, the cells were incubated with different
types of GA/CS NPs and GA (1 μg/mL) for 24 h. The
subsequent procedures were performed in accordance
with the manufacturer’s protocols. Finally, the cells
were analyzed by flow cytometry (Miltenyi Biotec,
Germany) and data were processed using FlowJo
software.
GA+DiR at a GA concentration of 4 μg/mL for 6 h.
The pretreated cells (A) were withdrawn and
co-incubated with fresh cells on a coverslip (B) for
1
2 h in fresh culture medium. B was then withdrawn
and co-incubated with fresh cells on another new
coverslip (C) for 12 h in fresh culture medium. The
cells were subsequently washed thrice with ice-cold
PBS and stained with propidium iodide (PI)
(20 μg/mL) for 15 min. All cells (A, B, and C) were
washed thrice with ice-cold PBS and observed by
means of CLSM.
Cellular uptake
The cellular uptake behaviors of the
nanoparticles were also studied in HepG2 cells by
loading coumarin-6 (C6; Sigma-Aldrich) instead of
GA and monitoring by flow cytometry (FCM) and
confocal laser scanning microscopy (CLSM). HepG2
cells were seeded in 35 mm confocal microscopy
In vitro tumor penetration in 3D tumor
spheroids
In vitro 3D tumor spheroids of HepG2 cells were
developed by a liquid overlay method [45]. Uniform
and compact tumor spheroids were selected for
subsequent studies. The tumor spheroids were
5
dishes at a density of 1×10 cells/well and incubated
for 24 h. The medium in each well was then replaced
with the same concentration of free C6 or C6/CS NPs
for 0.5, 2, or 4 h at 37°C. For the 4 h group, another
incubated with different types of (GA+C6)/CS
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Theranostics 2019, Vol. 9, Issue 22
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NPs and GA+C6 at a GA concentration of In vivo tumor penetration of intratumorally
injected GA/CS NPs in tumor-bearing mice
4
μg/mL, each for 6 h. The tumor spheroids were
then washed thrice with ice-cold PBS, fixed with
paraformaldehyde in PBS (4%, w/v) for 15 min, and
placed in cavity microscope slides. Images of the
tumor spheroids were acquired by Tomoscan with
Z-stack imaging at 10 μm intervals from the top of the
spheroid to the middle by CLSM. Semi-quantitative
analysis of the mean fluorescence intensity was
conducted with Image Pro Plus.
Tumor-bearing ICR normal mice were
intratumorally
injected
with
GA+DiR,
GA+DiR/CF60%S NPs, or GA+DiR/CF100%S NPs at
fixed doses of GA (6 mg/kg) and DiR (0.1 mg/kg) at a
consistent depth of needle insertion (2 mm below the
tumor surface). At 24 h post-injection, tumors were
collected, washed with PBS, and sliced into different
layers from the top to the middle by cryotome
sectioning. The nuclei of the tumor cells were stained
with Hoechst 33258 (1 μg/mL) for 30 min. The tumor
sections were observed by CLSM. Semi-quantitative
analysis of the mean fluorescence intensity was
conducted with Image Pro Plus software.
Animals and tumor xenograft models
Male ICR mice (18–22 g) were purchased from
the Medical Center of Yangzhou University
(
Yangzhou, China). All of the animals were
pathogen-free and were allowed free access to food
and water. The experiments were carried out in
compliance with the National Institute of Health
Guide for the Care and Use of Laboratory Animals of
China Pharmaceutical University and approved by
In vivo anti-tumor activity
1) Early-stage model: The
activity study was started when tumor volume in the
in vivo anti-tumor
3
mice reached around 100 mm . 2) Advanced-stage
the
Animal
Ethics
Committee
of
China
model: The
in vivo anti-tumor activity study was
Pharmaceutical University. To set up the tumor
started when tumor volume in the mice reached
6
3
xenograft
model,
1×10
Heps
cells
were
around 1000 mm . Heps xenograft tumor-bearing
subcutaneously injected into the left leg of each ICR
mouse. Tumor volume (V) was determined by
measuring length (L) and width (W), and calculated
mice were randomly divided into five groups (n=6):
saline, GA/IS (Injection solution), GA/CF60%S NPs,
GA/CF100%S NPs, and GA/CF60%S NPs + GA/CF100%
S
2
as V = 0.5×L×W .
NPs. Each group was intravenously injected with the
different formulations at a dose of 6 mg/kg every
In vivo tumor targetability
3
days. All animals were dosed four times, and the
An IVIS spectrometer (Perkin-Elmer, USA) was
used to monitor the biodistribution of CS NPs in the
tumor-bearing mice. Six tumor-bearing mice were
randomly divided into two groups, the members of
which were intravenously injected with the same dose
of DiR (0.1 mg/kg) in the form of free DiR solution,
DiR-loaded CF60%S NPs, and DiR-loaded CF100%S NPs,
respectively. Imaging was performed at 8 h after
injection to estimate the time-correlated excretion
profile. Region of interest (ROI) image analysis was
performed using Living Image 4.5 software.
first dose day was set as 0 d. The tumor volumes and
body weights of the mice were monitored every other
day. At the prescribed time (14 d), the tumors and
other immune organs (liver, spleen, and thymus)
were harvested and weighed. The tumor inhibition
rate was calculated using the formula: TIR% =
(
W
c
-W
group; W
H&E staining, the tumors were washed and fixed in
% formaldehyde solution and then embedded in
t
)/W
c
×100% (W
c
: tumor weight of the control
e
: tumor weight of the treatment group). For
4
paraffin blocks. For each type of tumor model (early
stage, advanced stage, and treatment groups), cell
proliferation was determined by EdU assay
In vivo vascular drug distribution of
intravenously injected GA/CS NPs in
tumor-bearing mice
(
BeyoClick), and the biomarker of hepatocellular
carcinoma (AFP) was determined by ELISA (EIAab)
of the tumor homogenate.
3
When the tumor volume reached 300 mm , the
tumor-bearing ICR normal mice were intravenously
injected with DiR, DiR/CF60%S NPs, and DiR/CF100%
S
Statistical analysis
NPs at the same dose of DiR (0.1 mg/kg). At 24 h
post-injection, tumors were collected, washed with
PBS, and examined by using cryotome sections.
Frozen tumor sections were stained with FITC-CD31
antibodies (Biotech Laboratories, USA) at 37ºC for
Data are given as mean ± standard deviation.
Statistical significance was tested by a two-tailed
*
Student’s t-test. Statistical significance was set at p <
**
***
0
.05, p < 0.01, and p < 0.001.
6
0 min, thrice washed with PBS, and observed by
means of CLSM.
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Theranostics 2019, Vol. 9, Issue 22
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Our prime concern was to select the best type of core
material for different goals. The shell material,
HA-SS-ATRA, was synthesized according to the
method described in our previous study [35].
Results and discussion
Synthesis and preparation of functional CS
NPs
The functional CS NPs were composed of core
and shell materials. The core material, with different
grafting rates of FG (TDS), was synthesized from
Different types of functional CS NPs were
prepared by the “one step method” optimized in our
recent work [46]. Size, polydispersity index (PDI), zeta
potential, EE% and DL% were summarized in Figure
2A and Table S2. Blank CS NPs showed a small size
(94.30±6.10 nm to 151.90±2.83 nm) and negative zeta
potential (-23.50±0.85 mV to -18.30±0.28 mV) due to
π-π stacking interactions of the core [47] and the
HA-coated shell. Notably, the CF30%S and CF60%S NPs
4
-methyl-5-hydroxyethyl thiazole, benzyl bromide
and γ-PGA (Figure 1A). The γ-PGA is a water-soluble
polymer, and so would not react readily with
water-insoluble benzyl bromide and FG. To overcome
this issue, we synthesized γ-PGA-CTA to facilitate a
homogeneous reaction. Through controlling the feed
ratio of FG and benzyl (Bz), we obtained different
were about 50 nm larger than the CF0%S and CF100%
S
grafting rates of γ-PFGA, including 0%, 30%, 60% and
NPs, which may result from the uniformity of the core
material. The more uniform the material is, the more
readily it could form a tight structure. The 30% and
1
1
00% -γ-PFGA, as characterized by H-NMR (Figure
S5). To calculate the actual grafting rate of FG,
considering 60%-γ-PFGA as an example (Figure 1B),
60%-γ-PFGA
materials
contained
different
peak 1(δ=5.08 ppm, CH
2
Ar), peak 2 (δ=5.17 ppm,
proportions of FG and Bz, leading to heterogeneity
and repulsion. In general, suitable size, negative zeta
potential, and small PDI indicated that the CS NPs
were stable and could achieve the EPR effect [36]. The
similar drug-loading capacity, nearly 100%
encapsulation efficiency (Table S2) which resulted
from π-π stacking between GA and the core material
[46] and good stability in several matrices (Figure S6),
showed the stabilization of GA by CS NPs.
PhCH ) and peak 3 (δ=8.31 ppm, NH) were used as
2
markers for Bz, FG and γ-PGA, respectively. The area
ratio of peak 2 / (peak 2 + peak 1) was calculated as
the grafting rate. The calculated grafting rate of
6
0%-γ-PFGA was 57.44%, within the margin of error.
The results of similar calculations on other types of
γ-PFGA are listed in Table S1. With the grafting rate of
FG increasing, the core material will show stronger
reduction-sensitivity and charge-conversion ability.
Figure 1. (A) Synthesis of different types of γ-PFGA. (B) H-NMR spectrum of 60%-γ-PFGA.
1
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Theranostics 2019, Vol. 9, Issue 22
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Figure 2. (A) Size, PDI and zeta potential of different types of blank and GA-loaded functional CS NPs. (B) Size distribution of different types of CS NPs incubated with 10 mM
GSH for 0, 1 and 3 h by DLS. (C) Morphology changes of different types of CS NPs incubated with 10 mM GSH for 1 and 3 h by TEM. Scale bars indicate 100 nm. (D) & (E) in
vitro drug release profiles of different types of GA/CS NPs in PBS (D) and 10 mM GSH (E). (F) Changes in the zeta potential of different types of core materials NP incubated
with 10 mM DTT over time.
with the above results. In 10 mM PBS, all kinds of
GA/CS NPs showed a similarly controlled drug
release less than 20% within 72 h. In 10 mM GSH, an
incremental increase in release was observed on going
from GA/CF0%S NPs to GA/CF100%S NPs. The t1/2 of
drug release was significantly different between
GA/CF0%S NPs and the other types (p<0.001) (Figure
S7) due to non-reduction-sensitivity of the
Reduction-activated charge conversion and
drug release
The novel functional CS NPs underwent
reduction-activated degradation followed by the
consequent charge conversion and drug release. To
assess their reduction-responsiveness, the size and
morphology changes of different types of CS NPs
incubated with 10 mM GSH (to mimic the tumor
bioreductive environment) were observed by DLS
and TEM. With the incubation time increasing, a
division of the size distribution appeared, with larger
sizes becoming more prevalent (Figure 2B). More
intuitively, in Figure 2C, the upper row shows the
initial states of the four types of CS NPs, spherical
core-shell NPs. The middle and lower rows showed
0
%-γ-PFGA.
Another important characteristic of the
functional CS NPs was their concurrent
reduction-activated charge conversion (Figure 2F). To
exclude the influences of the negatively charged
HA-coated shell and the amphiphilic ionic GSH on
the determination of zeta potential, DTT, an organic
reducing agent [48], was used instead of GSH and
incubated with the cores of CS NPs prepared from
different types of γ-PFGA. After 3 h, the higher the
grafting rate of the FG was, the more pronounced the
charge-conversion ability the core materials would
acquire (0%-γ-PFGA showed non-charge-conversion
ability). Once the FG was exposed to a reducing
environment, the disulfide bond was cleaved and the
thiazole ring was formed through ring-closing
metathesis (Scheme 1). As a consequence, positively
the morphology changes of the CF0%S and CF100%
S
NPs. For the CF0%S NPs, some degradation of the
shells could be observed after 1 h. After 3 h, the shell
was completely disassembled and only the core
structure remained. Unlike other types of γ-PFGA,
since the core of the GA/CF0%
S
NPs was
non-reduction-sensitive, the degradation rate slowed
markedly after the HA-SS-ATRA shell had been
disassembled.
C
F100%
S
NPs were taken as
representative of others owning a reduction-sensitive
core. After 1 h, more of the shell degraded and core
leakage could already be observed. Little core
structure remained after 3 h. The drug release profiles
in PBS and GSH (Figure 2D-E) were also consistent
+
charged N gradually accumulated and a stable
charged structure was formed, which was confirmed
1
by H-NMR (Figure S8). Compared to those at 0 h, the
1
H-NMR spectra of core materials incubated with
GSH for 1 h and 3 h featured a stronger new peak at
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Theranostics 2019, Vol. 9, Issue 22
6541
δ=10.00 (s, -CH=N) and weaker peaks at δ=7.75 (s,
well known that there is a high level of GSH in tumor
cells [26,51]. It serves to cleave disulfide bonds in both
the core and shell materials, thereby accelerating the
drug release.
FG-CHO) and 0.83 ppm (t, -CH
2
CH ), consistent with
3
the cleavage of the disulfide bond and the formation
+
of N .
To our knowledge, in general, more positively
charged species should exhibit stronger attraction to
negatively charged cell membranes and nuclei. Thus,
our functional CS NPs would be expected to show an
increasing ability to penetrate between cells as the
grafting rate of FG increased. This is quite important
for deep tumor treatment [8]. To think it over, in our
drug delivery system, there seems to exist a conflict
between tumor penetration and drug release. The
more positive charge CS NPs obtain, the more
promptly the material will disassemble, resulting in
rapid drug release in the superficial zone of the
tumor. Coincidently, both rapid drug release and
deep penetration are two attributes required for
different stages of tumors, laying the foundation for
our idea to design a stage-specific cancer therapy
strategy. For an early-stage tumor, rapid drug release
is beneficial for strong damage. For an
advanced-stage tumor, more of the drugs are
expected to be withheld for transport to the interior
zone. Thus, how to achieve the optimal balance and
select the best type of drug-loaded carrier for
penetration require further comparative bioactivity
tests in vitro and in vivo.
To confirm the above potential mechanism,
several control groups were examined. HepG2 cells
were pretreated with 50 mM BSO for 12 h (a GSH
biosynthesis inhibitor) [52], 10 mM GSH for 2 h (to
increase the concentration of intracellular GSH) and 2
mg/mL HA for 2 h (to saturate CD44 receptors).
Compared
to
a
blank
control
group,
concentration-dependent decreases in cytotoxicity
were observed for the groups pretreated with BSO
and HA, and an increase for that pretreated with GSH
(Figure 3C), as expected.
To assess cellular internalization of the drug, C6
was used in place of GA for fluorescence analysis.
Quantitative analysis of cellular uptake by flow
cytometry showed an obvious increase in C6/CS NPs
compared to C6 (Figure 4A). Furthermore, HA
pretreatment to saturate CD44 receptors partially
blocked the uptake of C6/CS NPs (Figure 4B). All of
the results supported the interpretation that the
enhanced cytotoxicity of GA/CS NPs could be
ascribed to more drug uptake and release.
To further investigate differences in the
cytotoxicity of the four types of GA/CS NPs, cell
apoptosis analysis using annexin V-FITC/PI staining
was performed (Figure 3D-E). With the grafting rate
of FG increasing, the total amount of necrosis and
apoptosis cells (Q2+Q3) increased (from 25.0% to
in vitro cytotoxicity, intracellular and
intercellular delivery
Cytotoxicity was evaluated by an MTT assay.
Different types of blank CS NPs at relevant doses
showed no obvious cytotoxicity towards L02 cells, a
kind of normal liver cells (Figure 3A and Table S3).
This guaranteed the biosafety of our CS NPs. For
HepG2 tumor cells, the different types of GA/CS NPs
all enhanced cell death, especially at low drug
concentrations (Figure 3B). The IC50 values of the four
types of GA/CS NPs were all less than 1.3 μg/mL,
among which GA/CF100%S NPs considerably reached
5
3
8.5%) compared to GA group (24.1%). Interestingly,
6.5% early-apoptosis cells (Q3) were observed in
GA/CF100%S NPs group. This suggested that our
GA/CS NPs might induce apoptosis through GSH
depletion as GA/CF100%
S
NPs depleted more
intracellular GSH at the same dose [53,54]. Moreover,
due to the reduction-activated charge conversion, the
nuclei-targeting abilities of different C6/CS NPs were
not identical (Figure 4C). GSH and BSO pretreatments
were also applied to validate the mechanism. No
obvious differences of the fluorescence intensity were
observed between the control, GSH and BSO
pretreatment groups for C6/CF0%S NPs due to
non-reduction-sensitivity of the core material. For the
other three types, the fluorescence intensities were in
the order of GSH (+) > control > BSO (+) due to the
decrease of reduction-activated degradation and drug
release. With C6/CF100%S NPs, numerous green
fluorescent dots were seen inside and outside of the
nuclei, indicating a superior nuclei-targeting ability
compared to the other groups. The stronger
cytotoxicity may be attributed to multiple antitumor
mechanisms involving GA being operative in the
nuclei [39-41].
0
.62±0.02 μg/mL, significantly lower than that of GA
(
2.42±0.29 μg/mL) (Table S4). The factors
underpinning this enhanced cytotoxicity may include
the following: 1) The stabilization effect of CS NPs on
GA. GA is easily metabolized by biological matrices
[
49], which caused the destruction of its 9,10
carbon-carbon double bond, a crucial site for its
antitumor activity [50]. Hence, it is important to
deliver GA into cells before it is degraded. 2)
HA-CD44 receptor mediated endocytosis. CD44
receptors are expressed on many tumor cells [37],
including HepG2 cells, and show a high affinity
towards HA (our shell material HA-SS-ATRA). 3)
GSH induced rapid intracellular drug release. It is
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Theranostics 2019, Vol. 9, Issue 22
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Figure 3. (A) Cytotoxicity of different types of CS NPs to L02 cells for 24 h. (B) Cytotoxicity of different types of GA/CS NPs to HepG2 cells for 24 h. (C) Cytotoxicity of
different types of GA/CS NPs to HepG2 cells for 24 h with BSO, GSH and HA pretreatment. (D) Flow cytometry analysis of cell apoptosis after different formulations treatment
(
GA concentration of 1 μg/mL) using annexin V-FITC/PI assay. (E) Statistical analysis of normal, necrotic and early-apoptotic cells.
To further assess the fate of our GA/CS NPs
following intracellular and intercellular delivery, we
used FITC-labeled HA-SS-ATRA to prepare
obvious in coverslip A, indicating extensive cell
apoptosis, which provided a necessary condition for
the remaining core NPs to escape and penetrate. 3)
For DiR/CF60%S NPs group, we could see that without
the cytotoxicity effect of GA, the cell apoptosis hardly
occurred, and the fluorescences of FITC and DiR were
both weak in the coverslip B and C (Figure 4G). It can
be explained that the positively charged core NPs
were mostly trapped in the live cells so that they
could not support further intercellular delivery. This
corroborated our delivery mode dependent on the
apoptosis of superficial cells. Furthermore, in vitro 3D
(
GA+DiR)/CF60%S NPs for representation of the core
and shell individually. Figure 4D shows that after
incubation for 1 h the core and shell were almost
overlapped around the nucleus, indicating uptake of
the whole CS NPs. At 4 h, the fluorescence of DiR was
released and became distributed throughout the
whole cell, indicating core–shell separation due to
intracellular reduction-activated degradation. The
core NPs, a positively charged transition state, as
confirmed in Figure S8, finally targeted the nuclei.
Notably, the cells were seen to decrease in size and
undergo apoptosis due to the effect of GA, providing
the potential for penetration of the remaining
drug-loaded core NPs.
tumor spheroid and
in vivo tumor penetration
experiments were also conducted (Figure S9). Similar
results were obtained, implying that with the depth
increasing, the fluorescence of FITC still remained in
the outer space, PI showed a strong intensity in the
superficial layer, and DiR achieved good penetration.
As far, the intracellular and intercellular
delivery mode of our GA/CS NPs could be
summarized as follows: 1) activated by intracellular
reduction environment, 2) the process depends on the
damage effect of the drug on the tumor cells; 3)
drug-loaded positively charged core NPs are
responsible for the delivery. This lays the important
foundation for our tumor penetration study.
Next, a two-dimensional intercellular delivery
experiment was conducted (Figure 4E) [9]. PI was
used to label apoptotic cells. From Figure 4F, three
conclusions can be drawn. 1) For (GA+DiR)/CF60%
S
NPs, the fluorescence of FITC decreased quickly in
coverslip B and C, while DiR achieved good
intercellular delivery (Figure 4G), meaning that the
shell was mostly detached in the coverslip A and the
core showed the penetration ability mediated by
electrostatic interactions. 2) the fluorescence of PI was
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Theranostics 2019, Vol. 9, Issue 22
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Figure 4. (A) Cellular uptake of HepG2 cells incubated with different types of C6/CS NPs for 0.5, 2 and 4 h measured by flow cytometry. (B) Cellular uptake of HepG2 cells
incubated with different types of C6/CS NPs with/without HA pretreatment for 2 h measured by flow cytometry. *p<0.05, **p<0.01 compared to without HA pretreatment. (C)
Nuclei targeting of different types of C6/CS NPs in HepG2 cells for 4 h of incubation with GSH or BSO pretreatment observed by confocal laser scanning microscopy. The nuclei
were stained by DAPI (blue) and C6 was green. Scale bars indicate 10 µm. (D) Intracellular shell detachment from the core of (GA+DiR)/CF60%S NPs incubated for 1 and 4 h. The
shell of HA-SS-ATRA was labeled with FITC (green). The nuclei were stained by DAPI (blue) and DiR was red. Scale bars indicate 10 µm. (E) Schematic of two-dimensional
intercellular delivery experiment. (I) incubate coverslip A with (GA+DiR)/CF60%S NPs for 6 h. (II) co-culture coverslip A with B for another 12 h. (III) co-culture coverslip B with
C for another 12 h. (F) Two-dimensional intercellular delivery of FITC-labeled DiR/CF60%S NPs and (GA+DiR)/CF60%S NPs. The nuclei were stained by PI (blue) and DiR was red.
Scale bars indicate 100 µm. (G) Mean fluorescence intensity of FITC or DiR in each coverslip.
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Theranostics 2019, Vol. 9, Issue 22
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intravenously injection for 24 h also told the similar
results (Figure 5B).
Tumor penetration
The above antitumor activity research from the
For
a
more direct comparison of their
two-dimensional
perspective
showed
that
penetrations in solid tumors, GA+DiR and their CS
NPs were intratumorally injected at the same depth.
After 24 h, the fluorescence at different depths below
the injection site (Figure 5C) and a semi-quantitative
analysis (Figure 5G) revealed a similar trend as in in
vitro 3D tumor spheroids. GA+DiR showed little
fluorescence owing to lack of cellular uptake and
penetration. At 200 μm, (GA+DiR)/CF100%S NPs
displayed intense and widespread fluorescence. The
fluorescence decreased sharply with increasing depth.
In contrast, (GA+DiR)/CF60%S NPs showed a slower
and smoother decrease in fluorescence intensity with
depth, indicating a superior drug-loaded penetration
ability, making it more suitable for deep tumor
treatment.
GA/CF100%S NPs had the strongest cytotoxicity due to
the rapidest drug release. Then, to further evaluate the
drug-loaded penetration abilities of different types of
GA/CS NPs, a three-dimension tumor spheroids
model was established and C6 was added with GA to
mark the location of the drug. After treatment for 6 h,
the fluorescence intensity attenuated with the
increasing distance from top (Figure 5A), which was
further analyzed semi-quantitatively (Figure 5F).
Among these, the fluorescence of GA+C6 quickly
disappeared
due
to
lack
of
charge-
conversion-induced penetration ability and lower
cellular uptake. At the same distance of 20 μm, the
strongest fluorescence intensity of (GA+C6)/CF100%
NPs was found due to their rapidest
S
Hence, two types of functional GA/CS NPs were
selected for the following pharmacodynamic research
in vivo: GA/CF60%S NPs, with the best drug-loaded
penetration ability, and GA/CF100%S NPs, with the
rapidest reduction-responsive drug release.
reduction-activated degradation of the carrier,
indicating burst-like drug release and excellent
damage potential to the superficial tumor cells.
However, it was quite obvious at the distance of 50
and 70 μm that only (GA+C6)/CF60%S NPs showed an
outstanding fluorescence, which meant that there
existed a balance point that only an appropriate
degree of degradation could allow enough drugs to
penetrate into deep cells. Otherwise, irrespective of
the charge-conversion ability, most of the drug was
released too soon. Based on the above studies, the
functional CS NPs identified as valuable for further
research were thus GA/CF60%S and GA/CF100%S NPs.
Prior to evaluation of the penetration ability in a
solid tumor, in vivo tumor-targeting ability was first
studied using DiR dye as a marker. After intravenous
injection for 8 h, free DiR was widely distributed
around the body and quickly eliminated. In
comparison, DiR/CF60%S NPs and DiR/CF100%S NPs
showed good tumor-targeting efficiency, despite
partial liver uptake (Figure 5D). The ex vivo main
organs were also imaged (Figure 5E) as well as ROI
analysis (Figure 5H). The results showed that the
Antitumor activity of the stage-specific
administration strategy
To make full use of the two types of functional
CS NPs in accordance with the concept of precision
medicine, we sought to devise a stage-specific
administration strategy for the best antitumor
efficacy. To this end, a two-stage hepatocellular
carcinoma model (murine Heps tumor), including
early and advanced stages, was established according
to the TNM staging criteria assisted with
alpha-fetoprotein (AFP) detection. Hepatocellular
carcinoma is the third most common malignancy
around the world, and the 5-year survival rate is less
than 5% [56,57]. Besides, AFP, a classic hepatocellular
carcinoma biomarker [58,59], is well defined as a
regulatory factor in the growth, recurrence and
mortality of hepatocellular carcinoma [60]. Thus, it is
essential to choose the most effective treatment
strategy for different stages of hepatocellular
carcinoma in terms of chemotherapy. After
subcutaneous injection of Heps cells, tumor volume
measurement with anatomical observation and AFP
concentration assay were conducted at the different
growth stages (Figure 6A-B). At day 7, the tumor was
fluorescence intensity of DiR/CF60%
S NPs and
DiR/CF100%S NPs were 10.0- and 10.6-fold higher,
respectively, than that of free DiR, and nearly 52%
and 57% of the available DiR was targeted to tumor
sites compared to 7% of free DiR. As is well known,
HA-related nano preparations show good active
targeting abilities to tumor, albeit with partial capture
by the reticuloendothelial system (RES), including the
liver, spleen and other immune organs [19,55]. Thus,
it was necessary to evaluate whether GA/CS NPs
would have an impact on these organs. The
drug-vascular distribution in the tumor sites after
3
small (about 100 mm ), soft, sparse and easy to
separate from other nearby tissues, and could thus be
recognized as the early stage. At day 14, the tumor
3
was large (about 1,000 mm ), hard and showed
obvious adhesions with nearby muscle tissue. At day
3
21, the tumor got even larger (about 3,000 mm ) and
vascular invasion had appeared. These tissue and
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Theranostics 2019, Vol. 9, Issue 22
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vascular invasions could be categorized as proximal
enter a dormant state as a self-protection measure due
to lack of nutrition from blood vessels [11,62,63]. After
the establishment of the two-stage hepatocellular
carcinoma, we evaluated the antitumor activity of our
functional CS NPs by applying them in the
stage-specific administration strategy (Figure 6D).
For the early-stage tumor, GA/CF100%S NPs
showed a prominent tumor inhibition ability (about
94%) compared to GA (about 54%), GA/CF60%S NPs
(about 73%), and GA/CF60%S NPs + GA/CF100%S NPs
(about 79%) (Figure 7B). These results could be easily
transfer,
corresponding
to
“regional
nodes
metastasis” of TNM. Until day 30, no distant
metastasis or ascites were seen, which might be
associated with the tumor type. Meanwhile, the AFP
concentration in tumor homogenates collected on
different days also showed a gradual increase,
reflecting the tumor progression. Therefore, through
tumor size combined with their corresponding
anatomical features and AFP levels, two stages of
Heps were established for the following research [61].
One was the early stage (day 7, tumor volume ≈ 100
understood because, as mentioned above, GA/CF100%
S
3
mm ), and the other was the advanced stage (day 14,
NPs showed the strongest antitumor efficacy, which
was sufficient for the tumor of small size in the early
stage. From the plot of the tumor volume (Figure 7A),
3
tumor volume ≈ 1,000 mm ). Besides, cell proliferation
activity at the center of the tumors was also
determined by EdU assay (Figure 6C). The green
fluorescence of Alexa488 was stronger in the early
stage, indicating that tumor cells proliferated more
quickly than those in the advanced stage. This could
be attributed to the features of cancer stem cells,
which were located more in the center of the tumor in
the advanced stage than in the early stage, and could
it can be seen that for GA/CF60%S NPs + GA/CF100%
NPs, the results were similar to that for GA/CF60%
S
S
NPs over the first 6 days; once injected with
GA/CF100%S NPs, the efficacy improved swiftly. H&E
staining also indicated that more tumor cells were
damaged by GA/CF100%S NPs (Figure 7C).
Figure 5. (A) Confocal images of 3D tumor spheroids with different treatments for 6 h at different distances from top. C6 was green. Scale bars indicate 200 µm. (B) Confocal
images of the tumor sections harvested from the mice following intravenously injection for 24 h. The blood vessels were stained by FITC-CD31 antibody (green) and DiR was
red. Scale bars indicate 100 μm. (C) in vivo penetration into the tumors of the Heps-bearing mice after intratumor injection for 24 h. The frozen tumor sections were observed
at different depths below the injection site using CLSM. The nuclei were stained by Hoechst (blue) and DiR was red. Scale bars indicate 100 μm. (D) The in vivo imaging of
tumor-bearing mice after intravenously injection for 8 h. (E) Ex vivo imaging of the major organs. (F) Mean fluorescence intensity of the 3D tumor spheroids with different
treatments at different distances from top. (G) Mean fluorescence intensity of the frozen tumor sections with different treatments at different depths below the injection site.
(
H) Average radiant efficiency of the organs measured by ex vivo ROI imaging analysis.
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Figure 6. (A) Anatomical observation of Heps tumor at different days after subcutaneous injection at the left leg. The white dotted circles indicate the features of each stage.
B) Tumor volume and AFP concentration of mice at different days after subcutaneous injection at the left leg. (C) Cell proliferation activity of the center of the tumors in
(
different stages determined by EdU assay. The EdU was labeled with Alexa488 (green), and the nuclei was labeled with Hoechst (blue). Scale bars indicates 100 μm. (D)
Administration routines of five groups for comparison. The same dose was 6 mg/kg for GA.
For the advanced-stage tumor, the excellent
tumor inhibition trend for GA/CF60% NPs
GA/CF100%S NPs (about 90%), compared to those for
GA (about 35%), GA/CF60%S NPs (about 68%) and
GA/CF100%S NPs (about 75%), was clearly evident
be viewed as a supplementary factor to the traditional
staging criteria [64]. After treatment, this index
showed a significant decrease, reminding us to pay
attention to its change as a preliminary indicator of
tumor recurrence.
S
+
(
Figure 8B). What made a difference was that
In a preliminary safety evaluation, insignificant
differences in the body weights and immune organ
coefficients of mice were found during treatment in
both two-stage models (Figure S10). H&E staining of
the normal organs after such administration revealed
no obvious toxic effects (Figure S11). Blood routine
and blood biochemistry examinations revealed no
significant differences between groups, apart from
slight increases in ALT and AST in the GA group
(Table S5), indicating slight hepatotoxicity of free GA.
Fortunately, GA/CS NPs did not elicit such damage.
Based on the above results, the potential
advantages of our functional materials may be
threefold: 1) It may avoid the risk of tolerance and
safety when increasing the dose for advanced-stage
tumors in the routine chemotherapy; 2) During
multiple dosing, similar materials could cause
receptor saturation, whether in RES or the related HA
receptors in other tissues [65]. With the sensitivity of
these receptors suppressed, subsequently introduced
similar CS NPs could escape the capture and reach the
tumor site more efficiently. 3) Due to the balance
between penetration and drug release of these types
of materials, the penetration ability of which stems
from carrier degradation, we believe that our
two-stage administration strategy is superior to those
involving only one type of particle-supported drug.
GA/CF60%S NPs + GA/CF100%S NPs showed a quite
strong efficacy when finishing the injection of
GA/CF60%S NPs and beginning to inject GA/CF100%
NPs (Figure 8A). These results demonstrated the
effectiveness of our “Punch-Damage” strategy. In
detail, GA/CF100%S NPs were initially somewhat
advantageous over GA/CF60%S NPs; the former, with
stronger antitumor efficacy, could damage the
superficial tumor cells more quickly and cause layer
S
by layer destruction. When it comes to GA/CF60%
S
NPs, despite their superior penetration ability, they
had insufficient power to clear the superficial tumor
cells completely, so the apparent tumor volume
remained larger. The most striking result was that
with GA/CF60%S NPs + GA/CF100%S NPs. A possible
explanation was that GA/CF60%S NPs first charged
ahead to punch a hole, opening the way for
GA/CF100%S NPs to destroy the remaining tumor cells
on a large scale. The same trends were also found by
H&E staining (Figure 8C). Our results implied that
this “punch-damage” strategy could reduce the tumor
size more effectively and swiftly. It also implied that
advanced-stage tumors needed drug penetration
more than early-stage tumors. Notably, the AFP
concentration in tumor homogenates, detected for
validation of our treatment efficacy (Figure 8D),
showed a stage-related trend, suggesting that it might
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Theranostics 2019, Vol. 9, Issue 22
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Figure 7. in vivo antitumor efficacy of the early-stage Heps tumor. (A) Tumor growth curves of different groups after treatment following the predetermined
administration routines every three days. *p<0.05. (B) Tumor weights and photographs of the treatment groups after sacrifice. *p<0.05, **p<0.01, ***p<0.001. (C) Histological
analyses of tumor sections after various treatments by H&E staining. Scale bars indicate 200 μm.
Figure 8. in vivo antitumor efficacy of the advanced-stage Heps tumor. (A) Tumor growth curves of different groups after treatment following the predetermined
administration routines every three days. *p<0.05. (B) Tumor weights and photographs of the treatment groups after sacrifice. *p<0.05, **p<0.01. (C) Histological analyses of
tumor sections after various treatments by H&E staining. Scale bars indicate 200 μm. (D) AFP concentration determination in tumor homogenate of different groups at different
tumor stages. *p<0.05.
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ROI:
region-of-interest;
TBA:
tetra-n-butyl
Conclusions
ammonium hydroxide; TDS: thiamine disulfide
system; TEM: transmission electron microscopy;
TNM: Tumor-Node-Metastasis.
In
summary,
a
novel
series
of
reduction-activated charge-conversional core-shell
nanoparticles with different abilities has been
developed for stage-specific cancer chemotherapy.
TDS-grafted γ-PFGA was employed as the core
material and HA-coated conjugate as the shell
material. The prepared nanoparticles showed
excellent capabilities of reduction-activated drug
release and charge-conversion-induced deep tumor
penetration. On the basis of two types of functional
CS NPs, namely GA/CF100%S NPs, with the strongest
reduction-responsive drug release ability, and
GA/CF60%S NPs, with the strongest drug-loaded
penetration ability, we proposed a stage-specific
Acknowledgements
This research work was financially supported by
the National Natural Science Foundation of China
(
Grant No. 81801819) and a program from “Double
First-Class” University project (No. CPU2018GY34).
Competing Interests
The authors have declared that no competing
interest exists.
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