O-nitrobenzyl liposomes with dual-responsive release capabilities for drug delivery

Article abstract of DOI:10.1016/j.molliq.2021.116016

To improve the therapeutic efficacy of anticancer drugs and reduce its toxic side effects, we synthesized a series of amphiphilic o-nitrobenzyl molecules 4-(4-N,N,N,N-dicarboxymethyl-diethylenetriamino)acetoxymethyl-3-nitro-N,N-dialk-ylbenzamide (N,N-NB-DTPA) with good photolysis property and acid sensitivity. Simultaneously, N, N-NB-DTPA liposomes composed of the o-nitrobenzyl molecules have good biocompatibility, low hemolysis rate and cytotoxicity, and the drug encapsulation efficiency of the liposomes exceeds 70%. N, N-NB-DTPA-DOX liposomes possess good stability and can keep uniform distribution in PBS solution for 10 days. The drug release rate of these drug-loaded liposomes reaches to the maximums under pH 5.0 and 30 min UV irradiation, revealing pH/UV dual-responsiveness of these drug-loaded liposomes. The low pH makes DOX separate from these drug-loaded liposomes, and the UV irradiation leads to o-nitrobenzyl ester bond cleave, which contribute to accelerate the release of drug from drug-loaded liposomes. Furthermore, N, N-NB-DTPA-DOX liposomes after UV irradiation have better therapeutic effect than single DOX·HCl, which may result from the production of nitrosobenzaldehyde derivatives after UV irradiation.

Full text of DOI:10.1016/j.molliq.2021.116016

Journal of Molecular Liquids 334 (2021) 116016
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
O-nitrobenzyl liposomes with dual-responsive release capabilities for
drug delivery
⇑
Weihe Yao, Chenyu Liu, Ning Wang, Hengjun Zhou, Farishta Shafiq, Simiao Yu, Weihong Qiao
State Key Laboratory of Fine Chemicals, School of Chemical Engineering, Dalian University of Technology, Dalian 116024, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
To improve the therapeutic efficacy of anticancer drugs and reduce its toxic side effects, we synthesized a
series of amphiphilic o-nitrobenzyl molecules 4-(4-N,N,N,N-dicarboxymethyl-diethylenetriamino)acetox
ymethyl-3-nitro-N,N-dialk-ylbenzamide (N,N-NB-DTPA) with good photolysis property and acid sensi-
tivity. Simultaneously, N, N-NB-DTPA liposomes composed of the o-nitrobenzyl molecules have good bio-
compatibility, low hemolysis rate and cytotoxicity, and the drug encapsulation efficiency of the
liposomes exceeds 70%. N, N-NB-DTPA-DOX liposomes possess good stability and can keep uniform dis-
tribution in PBS solution for 10 days. The drug release rate of these drug-loaded liposomes reaches to the
maximums under pH 5.0 and 30 min UV irradiation, revealing pH/UV dual-responsiveness of these drug-
loaded liposomes. The low pH makes DOX separate from these drug-loaded liposomes, and the UV irra-
diation leads to o-nitrobenzyl ester bond cleave, which contribute to accelerate the release of drug from
drug-loaded liposomes. Furthermore, N, N-NB-DTPA-DOX liposomes after UV irradiation have better
therapeutic effect than single DOXꢀHCl, which may result from the production of nitrosobenzaldehyde
derivatives after UV irradiation.
Received 7 September 2020
Revised 18 March 2021
Accepted 28 March 2021
Available online 6 April 2021
Keywords:
Anticancer
N, N-NB-DTPA
Photolysis
Dual-responsive
Nitrosobenzaldehyde
DOX
Ó 2021 Published by Elsevier B.V.
1
. Introduction
Chemotherapy, reported to be the most commonly used treat-
systems is much lower than that of multi-responsive drug delivery
systems [9–11]. In exogenous stimuli, light can provide highly
accurate external stimuli to activate DDSs composed of photo-
responsive molecules, which spatiotemporally controls the release
of the payload [12,13]. Among the photoactivable groups,
o-nitrobenzyl derivatives (ONB) are commonly used structures
for their confirmed photolysis mechanism and excellent photolysis
efficiency [13–16]. Mo et al. designed and synthesized a kind of o-
nitrobenzyl prodrug which can be degraded to 5-fluorourail by UV
irradiation [17]. Zhang et al. developed a kind of o-nitrobenzyl
nanoparticles which had high drug loading efficiency and good
UV light responsiveness [18]. They also developed a triple-
responsive drug delivery platform constructed by o-nitrobenzyl
polymer grafted HMSNs which had a better anti-cancer activity
in vitro under UV irradiation [19]. Furthermore, Pasparakis et al.
developed a drug carrier via self-assembly of o-nitrobenzyl block
copolymers, and the drug carrier was able to carry out the spa-
tiotemporally controlled drug release at the target site after activa-
tion by visible light [20].
ment for overpowering tumor cells proliferation, can not only kill
the tumor cells but also damage normal cells. Compared with the
chemotherapy, the drug delivery systems (DDS) with improved
drug efficacy and reduced toxicity have become one of the main
methods of cancer treatment [1]. DDSs have the ability to accumu-
late and penetrate the tumor by enhanced tumor permeability and
retention (EPR) effect [2]. After endogenous and exogenous stimuli,
these DDSs can be triggered to release the drug at specified loca-
tion and time [3]. Endogenous stimuli are related to tumor
microenvironment which includes the acidic pH, overexpressed
specific enzymes, overproduced glutathione (GSH) and reactive
oxygen species (ROS) [4,5], while exogenous stimuli mainly apply
external conditions, such as light, ultrasound, and magnetic fields
[
2–4].
Owing to the presence of pH gradient from the blood system
and normal tissues to tumor tissues, various pH-responsive drug
carriers have been reported [6–8]. However, current studies indi-
cates that the drug release rate of single-responsive drug delivery
The photolysis mechanism of o-nitrobenzyl derivatives is based
on the cleavage of o-nitrobenzyl ester bonds into aromatic
nitrosobenzaldehyde and carboxylic acid derivatives [21–23]. Pre-
vious reports have demonstrated that aromatic nitrosobenzalde-
hyde is harmful to cells. In brief, the released aromatic
⇑
Corresponding author.
E-mail address: qiaoweihong@dlut.edu.cn (W. Qiao).
https://doi.org/10.1016/j.molliq.2021.116016
167-7322/Ó 2021 Published by Elsevier B.V.
0

W. Yao, C. Liu, N. Wang et al.
Journal of Molecular Liquids 334 (2021) 116016
nitrosobenzaldehyde analogs could lead to significant cytotoxicity
by inhibiting the expression of poly [ADP-ribose] polymerase [24].
It has been confirmed that iniparib (a nitrobenzene derive) and its
nitroso metabolites formed non-specific protein adducts in tumor
cells and displayed a certain anticancer effect [25,26]. O’Shaugh-
nessy et al. found that iniparib plus chemotherapy technique were
able to improve the clinical benefits and survival of patients with
metastatic triple-negative breast cancer without any significant
toxic effects [27]. Therefore, the chemotherapy drug combined
with the non-toxic o-nitrobenzyl compounds under UV irradiation
can produce an enhanced anticancer effect of ‘‘0 + 1 > 1”.
2. Materials and methods
2.1. Materials
Hydrochloric acid (12 mol/L), N, N-dimethylformamide (DMF,
AR), chloroform (AR) and methanol (AR) were purchased from
Tianjin Kemiou Chemical Reagent Co., Ltd. PBS solution (0.01 M)
was prepared by sodium hydrogen phosphate, monobasic potas-
sium phosphate, potassium chloride and sodium chloride in a cer-
tain proportion. Sodium hydrogen phosphate (AR), monobasic
potassium phosphate (AR), potassium chloride (AR) and sodium
chloride (AR) were purchased from Tianjin Bodi Chemical Co.,
Ltd. Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s med-
ium (DMEM), were purchased from Thermo Fisher Scientific. Dox-
orubicin hydrochloride (DOXꢀHCl, 98%) was purchased from Dalian
Meilun Biotech Co., Ltd. MTT assays were purchased from KGI Bio-
tech. 96-well plates, 24-well plates and cell culture flasks were
purchased from Guangzhou Jet Bio-Filtration Co., Ltd.
To this end, we synthesized a series of o-nitrobenzyl amphiphi-
lic molecules (N, N-NB-DTPA) containing a hydrophilic DTPA group
(
Diethylenetriaminepentaacetic acid, a ligand of nuclear magnetic
contrast agent) and hydrophobic double long-chain o-
a
nitrobenzyl group, and these molecules were able to encapsulate
DOX to form drug-loaded liposomes (N, N-NB-DTPA-DOX lipo-
somes). We believed that the photo-responsive drug-loaded lipo-
somes would accumulate at the tumor site by EPR effect. When
N, N-NB-DTPA-DOX liposomes enter into endosomes/lysosomes
2.2. Fabrication of N, N-NB-DTPA liposomes
(
pH 4.0–6.0), these liposomes will release some anticancer drug
DOX in an acidic environment. At the same time, N, N-NB-DTPA-
DOX liposomes in endosomes/lysosomes will release more drug
molecules and photolysis products after UV irradiation. Finally,
the amount of drug will diffuse into the nucleus and inhibit the
production of DNA [28], and photolysis products will oxidize the
cysteine in ADP-nucleic acid transporter [26]. Therefore, ‘‘0 + 1 > 1”
effect triggered by UV light results in cell apoptosis (as shown in
Scheme 1).
5
mg of N, N-NB-DTPA was dissolved in 1 mL of chloroform and
methanol (1:1, v/v) mixed solution in a 25 mL round bottom flask
and the solvent was slowly removed to obtain a uniform film by
rotary evaporator. The obtained uniform film was put in a vacuum
drying oven at 45 °C for overnight to remove residual organic sol-
vent. 5 mL of PBS solution (0.01 M) was added into the uniform
film in order to hydrate it for 4 h at 37 °C, followed by sonication
of the mixed solution by an ultrasonic signal generator for
Scheme 1. N, N-NB-DTPA-DOX liposomes were fabricated by self-assembly of N, N-NB-DTPA molecules and an anti-cancer drug DOXꢀ HCl. To verify the enhanced anticancer
effect triggered by UV light, these liposomes were co-cultured with MCF-7 cells. Firstly, N, N-NB-DTPA-DOX liposomes taken in by cancer cells will enter into endosomes/
lysosomes (pH 4.0–6.0) following with the release of some DOX molecules in the acidic environment. Secondly, N, N-NB-DTPA-DOX liposomes will be broken down with the
release of more drug molecules and photolysis products after UV light illumination. Finally, photolysis products will enhance the anticancer effect of drug molecules.
2

W. Yao, C. Liu, N. Wang et al.
Journal of Molecular Liquids 334 (2021) 116016
1
0 min (ultrasound 2 s, intermittent 5 s, 100 W) to fabricate the N,
and conductivity of 10, 10-NB-DTPA was measured with the addi-
N-NB-DTPA liposomes. The prepared liposomes were used for sta-
bility study (the long-term stability and the diluted stability) of
liposomes.
tion of 0.01 M HCl solution (0.3 lL/s). The change in transmittance
of 10, 10-NB-DTPA in accordance with the change in pH was mea-
sured. In the process of acidic titration, the values of pH, conductiv-
ity and transmittance were measured by the pH meter model
(PHS-3C, Shanghai Hong Yi Instrumentation Co., Ltd), conductivity
meter (DDSJ-308A conductivity meter, Shanghai Hong Yi Instru-
ment Co., Ltd.) and a Mettler ToledoT90 instrument, respectively.
0
.2 mL of FBS was added to 1.8 mL of solutions containing N, N-
NB-DTPA liposomes to study the serum stability of N, N-NB-DTPA
liposomes. The prepared liposomes were placed in a constant tem-
perature incubator at 37 °C. The diameter and PDI of N, N-NB-DTPA
liposomes were measured by Zetasizer (Malvern Nano S90, Britain)
at different time points (0, 7, 24, 51 and 72 h).
2.5.2. Dialysis experiment
The dialysis bags (MWCO 1000) containing 1 mL of 10, 10-NB-
DTPA-DOX liposomes solution were placed in 100 mL PBS solu-
tions of different pH (pH 7.4, pH 6.8, and pH 5.0). The diameter
and the PDI of the drug-loaded liposomes were measured by Zeta-
sizer after 12 h.
2
.3. Self-assembly behavior and encapsulation efficiency of N, N-NB-
DTPA-DOX liposomes
5
mg of N, N-NB-DTPA was dissolved in 1 mL of chloroform and
methanol (1:1, v/v) mixed solution in a 25 mL round bottom flask
and the solvent was slowly removed from the mixed solution to
get a uniform film by rotary evaporator. The product was then
dried in a vacuum drying oven at 45 °C for overnight. 5 mL of
DOXꢀHCl solution (1 mg/mL and 0.5 mg/mL) was added into a
round bottom flask to hydrate for 4 h at 37 °C, and then the mixed
solution was sonicated by an ultrasonic signal generator for 10 min
2
2
.6. Photo-responsiveness of N, N-NB-DTPA
.6.1. Photolysis of N, N-NB-DTPA
1
3
mg of N, N-NB-DTPA was dissolved in 10 mL CHCl in a quartz
tube and then irradiated with UV light (365 nm, 60 W) for different
time. The distance between the light source (UV 365 nm, 60 W)
and samples was fixed at 2 cm. Changes in UV absorption curve,
FT-IR spectrum and mass spectrum were measured by UV/VIS/
NIR Spectrometer (Lambda 750 S, PerkinElmer Co., Ltd.), Fourier
transform-infrared spectrometer (NEXUS, American Thermo Nico-
let Co., Ltd.) and accurate mass liquid chromatography time-of-
flight mass spectrometer (Accurate-Mass TOF LC/MS 6224, Agilent
Technology Co., Ltd.), respectively.
(
ultrasound 2 s, intermittent 5 s, 100 W) to form a uniform solu-
tion. The resulting mixed solution was then transferred to a dialy-
sis bag (MWCO 3500), followed by immersing it in 500 mL of PBS
buffer solution (0.01 M, pH 7.4) along with stirring at a rate of
1
000 rpm at room temperature. The process of dialysis was carried
out for 8 h while changing the PBS buffer solution every 0.5 h.
L solution from every sample dialysis bag was taken and lyo-
20 l
philized. The lyophilized powder was then dissolved in 5 mL of
DMF. The absorption intensity at 486 nm of the DMF solution
was measured by ultraviolet spectrophotometer (Agilen, Cary
2
.6.2. Photolysis behavior of liposomes
Changes in diameter, PDI and Zeta potential of N, N-NB-DTPA
liposomes or N, N-NB-DTPA-DOX liposomes in quartz tube were
measured by Malvern Zetasizer Nano S90 after UV light irradiation
for different time. TEM images of N, N-NB-DTPA liposomes or N, N-
NB-DTPA-DOX liposomes before and after UV irradiation were
obtained by JOEL JEM-2000EX system. Negative staining samples
were prepared with phosphotungstic acid acetate solution (2%).
6
0), and the concentration of DOXꢀHCl was determined by the cal-
2
ibration curve (y = 0.018x ꢁ 0.001, R = 0.999, where y is the UV
absorption intensity at 486 nm, x is the concentration of DOXꢀHCl
in mg/L).
The drug encapsulation efficiency (DEE) was calculated by the
formula as follows:
Mass of loaded drug
Mass of drug in feed
2.7. Measurement of dual-responsive drug release
DEE ðwt%Þ ¼
ꢂ 100%
In order to determine the accumulative release rate of N, N-NB-
DTPA-DOX liposomes, 1 mL of N, N-NB-DTPA-DOX liposomes with
different irradiation time (15 min and 30 min) was transferred to
dialysis bags (MWCO 3500) followed by immersing them in
2
.4. Hemolysis behavior of N, N-NB-DTPA liposomes
A total of 5 mL fresh blood sample from a healthy volunteer was
1
3
00 mL PBS solution with different pH (pH 7.4, 6.8 and 5.0) at
7 °C with a stirring speed of 1000 rpm. Aliquots of 3 mL PBS solu-
taken, washed with normal saline and centrifuged at 1500 r/min
for 3 min. The above steps were repeated several times until the
supernatant was colorless. 400
2
2
tion was taken out and then replaced with 3 mL fresh PBS at pre-
determined intervals. The fluorescence intensity of these aliquots
at 590 nm was measured by a fluorescence spectrometer (F7000,
l
L of substrate was diluted to
0 mL with normal saline to form a uniform solution containing
% red blood cells. 1 mL of normal saline solution containing differ-
Hitachi). According to the doxorubicin standard curve
ent concentrations N, N-NB-DTPA and 1 mL of 2% Triton X-100 (TX-
00) solution were mixed with 1 mL of 2% red blood cells solution,
2
(
y = 133.8x-0.645, R = 0.999, y is the fluorescence emission inten-
1
sity at 590 nm, x is the concentration of DOXꢀHCl), the concentra-
tion of DOX was calculated. The cumulative release curve of
DOXꢀHCl was determined as follows:
respectively. The plates were then placed into a constant tempera-
ture incubator at 37 °C for 4 h. After 4 h incubation, the mixed red
blood cells solution in 24-well plates were centrifuged at 1500 r/
M
t
min for 3 min. The 100
lL supernatant of different samples was
Cumulative release rate of DOX ¼ M ꢂ 100%
pipetted into a 96-well plate and the absorbance of the 96-well
plate was measured by a microplate reader (Multiskan FC, Thermo,
USA) at 540 nm.
0
t
Here, M is the cumulative release mass of DOX after time t, and
M
0
is the start mass of DOX encapsulated in the liposomes.
2
2
.5. pH-responsiveness of N, N-NB-DTPA
.5.1. Acidic titration
2.8. In vitro cytotoxicity of N, N-NB-DTPA liposomes
MCF-7 and HUVEC cells in logarithmic growth stage were
1
0, 10-NB-DTPA was dissolved in 20 mL NaOH solution to pre-
seeded in 96-well plates at an approximate cell density of
5
pare 1 mM aqueous solution of 10, 10-NB-DTPA. The changes in pH
10 cells/mL, followed by placing the plates in 5% CO
2
incubator
3

W. Yao, C. Liu, N. Wang et al.
Journal of Molecular Liquids 334 (2021) 116016
at 37 °C for 24 h. The medium was removed and 100
lL of fresh
UV irradiation groups: After the cell density has reached about
medium containing N, N-NB-DTPA (0.1, 1, 10, 50, 100, 200, 250,
00 and 500 g/mL) was added. After 24 h incubation, the medium
in 96-well plates was removed and each well was washed twice by
PBS buffer solution. 150 L MTT reagent was added to 96-well
plates followed by placing the plates in 5% CO incubator at
7 °C for 4 h. The obtained formazan crystals in 96-well plates
80% confluence, the medium was then replaced with 100
DMEM medium containing N, N-NB-DTPA (0.1, 1, 10, 20, 40, 50, 60,
80 and 100 g/mL) and N, N-NB-DTPA-DOX (0.1, 1, 5, 10, 15, 20, 25,
30 and 35 g/mL). After 4 h incubation, 96-well plates were irradi-
ated with UV light (360 nm, 60 W) for 30 min (3 ꢂ 10 min). After
20 h incubation, the medium was then removed and each well was
washed twice with PBS solution.
lL fresh
4
l
l
l
l
2
3
were dissolved in 150
lL DMSO, and the absorbance at 492 nm
was measured by microplate reader after 1 min vibration. Cell via-
150
lL of MTT (0.5 mg/mL, KGI Biotech) reagent was added in
bility was determined by the formula [(Asample ꢁ A
0
)/(Acontrol ꢁ A
0
)],
the plates. The plates were then placed in CO
After 4 h incubation, the mixed solution was carefully removed
in order to obtain formazan crystals. The formazan crystals were
2
incubator at 37 °C.
where Acontrol is the average value of the absorbance from control
group, Asample is the average value of the absorbance from cells
treated with samples, A
from blank well.
0
is the average value of the absorbance
dissolved in 150
at 492 nm was measured by microplate reader. The cell viability
)], where
control is the average value of the absorbance from untreated cells,
sample is the average value of the absorbance from cells treated
is the average value of the absorbance from blank well.
lL DMSO, and the absorbance of DMSO solution
was calculated by the formula [(Asample ꢁ A
0
)/(Acontrol ꢁ A
0
A
A
2.9. Photo-induced anticancer activity of N, N-NB-DTPA liposomes and
N, N-NB-DTPA-DOX liposomes
samples, A
0
MCF-7 cells in logarithmic growth stage were seeded in 96-well
5
plates at an approximate cell density of 10 cells/mL followed by
3. Results and discussion
putting them in 5% CO
Non-UV irradiation groups: After the cell density has reached
about 80% confluence, the medium was removed and 100 L fresh
medium containing N, N-NB-DTPA and photolysis products of N, N-
NB-DTPA (0.1, 1, 10, 20, 40, 50, 60, 80 and 100
g/mL) or DOXꢀHCl
and N, N-NB-DTPA-DOX (0.1, 1, 5, 10, 15, 20, 25, 30 and 35 g/mL)
was added in each well. After 24 h incubation, the medium in each
well was removed and each well was washed twice with PBS
solution.
2
incubator at 37 °C.
3.1. Design and synthesis of photo-cleavable amphiphilic molecule N,
N-NB-DTPA
l
l
In order to achieve the drug encapsulation and preparation of
the photo-controlled release drug, we used o-nitrobenzyl group
as a linker of double long hydrophobic carbon chains and hydro-
philic DTPA group to synthesize photo-cleavable amphiphilic
molecules (N, N-NB-DTPA). The detailed synthesis route of
l
Scheme 2. Synthesis route of photo-cleavable amphiphilic molecule N, N-NB-DTPA.
Table 1
DEE of N, N-NB-DTPA at different mass ratio (drug: lipid).
Mass ratio (D:L)
8,8-NB-DTPA
10, 10-NB-DTPA
12,12-NB-DTPA
14,14-NB-DTPA
DEE (1:1)%
DEE (1:2)%
73.8 ± 4.3
81.2 ± 5.4
76.1 ± 5.1
85.2 ± 7.1
76.6 ± 4.9
86.2 ± 9.2
82.6 ± 6.3
89.7 ± 6.9
4

W. Yao, C. Liu, N. Wang et al.
Journal of Molecular Liquids 334 (2021) 116016
Fig. 1. (a) DEE of N, N-NB-DTPA at different mass ratio (drug:lipid) and (b) changes of state, diameter, Zeta potential and PDI before and after encapsulation of DOXꢀHCl.
N, N-NB-DTPA is shown in Scheme 2. The detailed synthesis and
characterization of products are recorded in supporting informa-
tion (Section 3.1).
structure of N, N-NB-DTPA increases the difficulty of purification
of the product. After a variety of attempts, we finally chose this
ratio (chloroform/methanol/water/acetic acid = 65:25:4:1 v/v) for
column chromatography separation. As shown in Fig. S12–S23,
the chemical structures of N, N-NB-DTPA were validated by MS,
4
-(Hydroxymethyl)-3-nitrobenzoic acid prepared by hydrolysis
of 4-(bromomethyl)-3-nitrobenzoic acid under reflux in alkaline
solution. 4-(hydroxymethyl)-3-nitro-N, N-benzamide (N, N-NB-
OH) was synthesized by amidation of 4-(hydroxymethyl)-3-
nitrobenzoic acid and secondary amine in presence of BOP and
DIPEA. We utilized the high reactivity of hydroxyl and anhydride
to synthesize N, N-NB-DTPA. However, the reaction was accompa-
nied by the formation of by-product N, N-NB-DTPA-NB-N, N. To
reduce the formation of this by-product, we controlled the ratio
and dripping order of hydroxyl and anhydride. The polycarboxyl
1
1
13
H nuclear magnetic resonance spectroscopy ( H NMR) and
NMR. Compared with the H NMR spectrum of N,N-NB-OH, the
C
1
6 3 2
methylene (AC H CH A) proton signal of N,N-NB-DTPA has shifted
from 5.0 ppm to 5.5 ppm. These results suggest that the esterifica-
tion reaction has occurred. In addition, MS results (shown in
Fig. S12, S15, S18 and S21) confirm that the molecular weight of
ꢁ
N, N-NB-DTPA (m/z, [MꢁH] ) is consistent with the calculated
value.
5

W. Yao, C. Liu, N. Wang et al.
Journal of Molecular Liquids 334 (2021) 116016
3.2. Characterization of N, N-NB-DTPA liposomes
Compared with the traditional surfactant, N, N-NB-DTPA mole-
cules (except for 14, 14-NB-DTPA) exhibited a comparable surface
activity (Section 3.2 in supporting information). Herein, water-
soluble cationic anticancer drug doxorubicin hydrochloride
(
DOXꢀHCl) was selected as the research object in order to study
the drug-loading properties. As shown in Table 1 and Fig. 1a, the
drug encapsulation efficiencies of N, N-NB-DTPA liposomes exceed
7
0% which is much higher than the most reported drug delivery
systems [29–32]. This higher drug encapsulation efficiencies
DEEs) may be due to the strong electrostatic interaction between
(
the polycarboxylic acid group of N, N-NB-DTPA and the amino-
group of DOXꢀHCl. The inference is further verified by the increase
of Zeta potential of DOX-loaded liposomes as shown in (Fig. 2b). In
addition, the DEEs of the N, N-NB-DTPA increase with the increase
in the length of hydrophobic chain, indicating that the longer
hydrophobic chain of N, N-NB-DTPA may form the larger liposome
hydrophilic cavity to load more DOXꢀHCl molecules. However, the
diameters of N, N-NB-DTPA liposomes decrease after the encapsu-
lation with DOXꢀHCl. We speculated that after the encapsulation
with DOXꢀHCl, the electrostatic interaction between the polycar-
boxylic acid group of N, N-NB-DTPA and the amino-group of
DOXꢀHCl have made the electrostatic repulsion between the mole-
cules in N, N-NB-DTPA liposomes decrease. Furthermore, 10, 10-
NB-DTPA liposomes and 12, 12-NB-DTPA liposomes are able to
keep uniform size after encapsulation of DOXꢀHCl by contrast of
PDI in Fig. 1b.
3.3. Hemolysis behavior of N, N-NB-DTPA liposomes
In the hemolysis test, we designed to make the solution con-
taining 2% red blood cells divided into positive control group, neg-
ative control group and test group. The positive control group was
treated with 2% TritonX-100, the negative control group was trea-
ted with normal saline and the test group was treated with N, N-
NB-DTPA at different concentrations. As seen in the Fig. 2a and b,
hemolysis rate of N, N-NB-DTPA is quite low (<5%) except 8, 8-
NB-DTPA at 400 lg/mL and 250 lg/mL. As shown in Fig. 2b, com-
pared with the negative control group and the positive control
group, the erythrocytes of N, N-NB-DTPA group have no aggrega-
tion behavior, indicating good biocompatibility of N, N-NB-DTPA.
All these results prove that N, N-NB-DTPA can be applied in drug
delivery system at certain concentration range.
3.4. Stability of N, N-NB-DTPA liposomes
Fig. 2. (a) Hemolysis rate and (b) pictures of N, N-NB-DTPA at different concen-
tration, and (c) microscope image of hemolysis result of N, N-NB-DTPA at high
concentration.
The feasibility of N, N-NB-DTPA as a drug-delivery carrier was
investigated by measuring the changes in the average diameter
of N, N-NB-DTPA liposomes and N, N-NB-DTPA-DOX liposomes at
different conditions. Serum stability test was carried out in PBS
solution containing 10% FBS at 37 °C. As shown in Fig. 3a and
Fig. S25a, N, N-NB-DTPA liposomes can keep stable in PBS solution
containing 10% FBS for 72 h. No obvious changes in the average
diameters of N, N-NB-DTPA liposomes are observed when the lipo-
somes solution is diluted to 20 times, revealing stability of these
liposomes. However, once the liposomes solution is diluted to 50
times, the average diameters and the PDIs of N, N-NB-DTPA lipo-
somes increase sharply (as shown in Fig. 3b and Fig. S25b). In
Fig. 3c, the diameters of 10, 10-NB-DTPA and 12,12-NB-DTPA basi-
cally have no change in 15 days, while the average diameters of
decrease of their average diameters. However, the average diame-
ters and the PDIs of N, N-NB-DTPA-DOX liposomes have no signif-
icant changes, which may be due to the electrostatic interaction
between DOXꢀHCl and polycarboxylic acid structure of N, N-NB-
DTPA (Fig. 3d and Fig. S25d). Especially, 10, 10-NB-DTPA-DOX lipo-
somes and 12, 12-NB-DTPA-DOX liposomes still can keep stable at
the 10th day as shown in Fig. 3d and Fig. S25d.
3.5. pH-responsiveness of N, N-NB-DTPA
8
,8-NB-DTPA increase and that of 14,14-NB-DTPA decrease. We
speculated that 8, 8-NB-DTPA liposomes aggregated to larger lipo-
somes, resulting in an increase of their average diameter. And 14,
Due to the similarity of the structure of N, N-NB-DTPA, 10,
10-NB-DTPA was chose as the study object for acidic titration.
The o-nitrobenzyl ester is prone to cleave once the pH value
exceeds 8 [33], so the pH of 1 mM 10, 10-NB-DTPA aqueous
1
4-NB-DTPA liposomes was more prone to form smaller liposomes
for low solubility of 14, 14-NB-DTPA molecules, resulting in a
6

W. Yao, C. Liu, N. Wang et al.
Journal of Molecular Liquids 334 (2021) 116016
Fig. 3. Diameter of (a) serum stability, (b) dilute stability and (c) long term stability of N, N-NB-DTPA liposomes, and (d) long term stability of N, N-NB-DTPA-DOX liposomes.
ꢁ
3
ꢁ3
Fig. 4. (a) Acidic titration curves of aqueous 10 mol/L 10, 10-NB-DTPA, and (b) turbidity of aqueous 10 mol/L 10, 10-NB-DTPA at different pH conditions.
solution is adjusted to 8, and the state of the solution is shown
in Fig. 4b. In the process of the gradual addition of 0.01 M HCl
solution to an aqueous solution of 10, 10-NB-DTPA, the changes
ment, the drug molecules are gradually separated from the surface
of the liposomes, resulting in an increase in the electrostatic
repulsion between the molecules in the liposome, which further
leads to an increase of diameter of 10, 10-NB-DTPA-DOX
liposomes.
Hence, the pH-responsiveness of 10, 10-NB-DTPA was verified
from micro and macro perspectives by combining acidic titration
with dialysis experiment.
in the pH and conductivity of the solution are shown in Fig. 4a.
ꢁ
We speculated that ANACH
2
COO was selectively protonated
with H+ to become ANH ACH
+
COO with appearance of the
‘platform period” until all ANACH
COO [34]. Subsequently, ANH ACH
transferred to poorly water-soluble ANH ACH
further addition of H , a large amount of white precipitate could
be seen in Fig. 4b.
ꢁ
2
ꢁ
+
‘
2
COO was converted to
+
ꢁ
ꢁ
ANH ACH
2
2
COO gradually
COOH with the
+
2
+
3.6. Photo-responsiveness of N, N-NB-DTPA
Compared with the diameter of 10, 10-NB-DTPA-DOX lipo-
somes at pH 7.4, the diameter of 10, 10-NB-DTPA-DOX liposomes
slightly increase at pH 6.8 and pH 5.0 (in Fig. 5). In acidic environ-
3.6.1. Photocleavage of N, N-NB-DTPA
The cleavage of o-nitrobenzyl ester bond is induced by irradia-
tion with UV light, resulting in the release of drug from the drug-
7

W. Yao, C. Liu, N. Wang et al.
Journal of Molecular Liquids 334 (2021) 116016
time as shown in Fig. 6a. As presented in Fig. 6b, the absorption
intensity at 270 nm gradually decreases and the absorption inten-
sity at 310 nm gradually increases with the increase of illumina-
tion time. Moreover, these two curves have gradually kept their
balance after 30 min, which means that the cleavage process of
the o-nitrobenzyl ester bond of 10, 10-NB-DTPA has finished after
3
0 min UV irradiation. In Fig. 6c, the infrared spectrum shows that
the ANO stretching vibration of 10, 10-NB-DTPA has disappeared,
2
which further verifies the changes in structure of 10, 10-NB-DTPA
after UV illumination. As presented in Fig. 6d, the photolysis
products of 10, 10-NB-DTPA are nitrosobenzaldehyde and DTPA,
which is consistent with the photodegradation mechanism of
o-nitrobenzyl group.
Fig. 5. The diameters and the PDIs of 10, 10-NB-DTPA-DOX liposome at pH 7.4, pH
3.6.2. Photolysis behavior of N, N-NB-DTPA liposomes and N, N-NB-
DTPA-DOX liposome
6
.8 and pH 5.0 after 12 h dialysis.
To evaluate the photolysis property of N, N-NB-DTPA and N, N-
NB-DTPA-DOX liposomes, changes in average diameter, PDI and
loaded liposomes. Taking 10, 10-NB-DTPA as an example (other N,
N-NB-DTPA compounds shown in Fig. S26), the obvious changes
can be seen from the UV–Vis spectrum after irradiation at different
Zeta potential were measured. As presented in Fig.
7 and
Fig. S27, the average diameters of N, N-NB-DTPA and N, N-NB-
DTPA-DOX liposomes increased with the increase in UV irradiation
Fig. 6. Changes in (a) UV–Vis spectrum, (b) 270 nm and 310 nm with different UV light irradiation time, changes in (c) IR spectrum and (d) MS of 10, 10-NB-DTPA before and
after UV light irradiation.
8

W. Yao, C. Liu, N. Wang et al.
Journal of Molecular Liquids 334 (2021) 116016
Fig. 7. Changes in average diameter and TEM images of (a) N, N-NB-DTPA liposomes and (b) N, N-NB-DTPA-DOX liposomes with different time UV irradiation.
Scheme 3. Mechanism of drug release in acidic environment and with UV irradiation.
9

W. Yao, C. Liu, N. Wang et al.
Journal of Molecular Liquids 334 (2021) 116016
time. TEM images in Fig. 7 shows that both 10, 10-NB-DTPA and 10,
Fig. S29, the color of PBS buffer solution deepens gradually with
prolonging UV irradiation time and with decrease in pH value, also
indicating the release rate of DOX increase. Therefore, N, N-NB-
DTPA-DOX liposomes can be used as a potential pH/photo dual-
responsive drug delivery system.
1
0-NB-DTPA-DOX liposomes have uniform spherical structure
before UV irradiation (in the top left of the Fig. 7a and b) and irreg-
ular shapes (at the bottom right of the Fig. 7a and b) after UV irra-
diation. To be precise, N, N-NB-DTPA and N, N-NB-DTPA-DOX
liposomes aggregate with each other, resulting in an increase of
their diameter. O-nitrobenzyl ester bond of N, N-NB-DTPA is
cleaved by the UV light, resulting in an increase of their Zeta poten-
tial (in Fig. S27). Furthermore, the electrostatic repulsion among
the liposomes gradually decreases, which lowers the barrier for
membrane fusion and contributes to the drug release. In Scheme 3,
the proposed mechanism explains that the increasing number of
liposoluble nitrosobenzaldehyde with the increase in UV illumina-
tion time makes the pressure change of the loose liposome inside
and outside, resulting in the drug release from liposomes [33].
3.8. In vitro cytotoxicity of N, N-NB-DTPA liposomes
In order to assess the feasibility of N, N-NB-DTPA molecules as
potential drug carriers, we performed cytotoxicity experiment
with HUVEC and MCF-7 cells treated with different concentrations
of N, N-NB-DTPA (0.1, 1, 10, 50, 100, 200, 250, 400 and 500 lg/mL).
After 24 h incubation, the cell viability was obtained by MTT
3.7. In vitro drug release of drug-loaded liposomes
In order to simulate the drug release from the drug-loaded lipo-
somes in normal and cancer cells, drug-loaded liposomes were
added into dialysis bags (MWCO 3500) to conduct experiments
of photo-triggered DOX release. The samples were exposed to UV
light for 15 and 30 min, followed by placing them in phosphate
buffer solutions having range of pH 5.0, 6.8, and 7.4 at 37 °C,
respectively. The fluorescence emission spectrometer with high
detection limit was used to detect the fluorescence intensity of
DOX released from dialysis bags immersed in different pH solu-
tions. As seen in Fig. 8, the cumulative DOX release rate of N, N-
NB-DTPA at pH 5.0 + 30 min condition is higher than the groups
of pH 5.0 + 15 min, pH 5.0, 6.8 and 7.4 after 42 h dialysis, and
the release rate gradually increases with the decrease in pH value.
The drug cumulative release rate in Fig. 8 and Fig. S28 indicates
that the acidic environment and the longer UV illumination time
can increase the release rate of DOX [29,33]. As shown in
Fig. 9. Cytotoxicity tests of N, N-NB-DTPA (n = 8, 10, 12 and 14) with HUVEC and
MCF-7 cells detected by MTT assay.
Fig. 8. Cumulative release profiles of DOX-loaded N, N-NB-DTPA liposomes (a) 8,8-NB-DTPA, (b) 10, 10-NB-DTPA, (c) 12,12-NB-DTPA and (d) 14,14-NB-DTPA in 0.01 M PBS
buffer at pH 7.4, 6.8, 5.0, pH 5.0 + 15 min UV and pH 5.0 + 30 min UV at 37 °C.
10

W. Yao, C. Liu, N. Wang et al.
Journal of Molecular Liquids 334 (2021) 116016
assays. As presented in Fig. 9, when the concentration reaches to
selected as the medium, and the illumination time was
3 ꢂ 10 min (30 min) for subsequent illumination experiments.
Due to the similar structures and the similar photolysis prod-
ucts of N, N-NB-DTPA, 10, 10-NB-DTPA liposomes were used in
study of photo-induced anticancer activity. To prove anticancer
effects of the photodegradation products of 10, 10-NB-DTPA, the
experiment was divided into three groups [35]: (1) the cells incu-
bated with 10, 10-NB-DTPA liposomes were not subjected to UV
irradiation; (2) 10, 10-NB-DTPA liposomes were irradiated by UV
light for 30 min before cell incubation; (3) the cells incubated with
2
00
with amphiphilic N, N-NB-DTPA (n = 8, 10, 12, 14) molecules is
over 90%. Moreover, when the concentration reaches to 400 g/
lg/mL, the cell viability of HUVEC and MCF-7 cells treated
l
mL, the cell viability of MCF-7 cells treated with 14, 14-NB-DTPA
is still over 80%. We speculated that this may be related to the good
biocompatibility of 14, 14-NB-DTPA molecule. In general, amphi-
philic N, N-NB-DTPA molecules could be used as drug carriers
owing to their low cytotoxicity.
1
3
0, 10-NB-DTPA liposomes for 4 h were irradiated by UV light for
0 min (3 ꢂ 10 min). The cell viability of each group was deter-
3.9. Photo-induced anticancer activity of 10, 10-NB-DTPA liposomes
mined by MTT assay. As shown in Fig. 10a, the cells treated with
1
0, 10-NB-DTPA liposomes grow well at different concentrations,
In optimization of experimental conditions (as shown Sec-
tion 3.3 and Fig. S30 in supporting information), DMEM was
and the cell viability is still above 100% at 100 g/mL. In UV pre-
l
Fig. 10. MTT assay results for 10, 10-NB-DTPA which were incubated with MCF-7 cells. (a) cytotoxicity studies, and (b) IC50 of 10, 10-NB-DTPA + 30 min UV and DOXꢀHCl.
Fig. 11. Photo and drug dual-induced anticancer activity of (a) 8, 8-NB-DTPA-DOX, (b) 10, 10-NB-DTPA-DOX, (c) 12, 12-NB-DTPA-DOX and (d) 14, 14-NB-DTPA-DOX
incubated with MCF-7 cells.
11

W. Yao, C. Liu, N. Wang et al.
Journal of Molecular Liquids 334 (2021) 116016
irradiation group, the cell viability is 90% at 100
UV irradiation group, the cell viability significantly reduces to
l
g/mL. While in
change dramatically. Moreover, acidic environment and UV irradi-
ation time can increase the cumulative release rate of the drug
from the drug-loaded liposomes over 60%. We found that the treat-
ment of N, N-NB-DTPA-DOX + UV improved the anticancer effect of
DOXꢀHCl, which may contribute to the oxidation reaction of cys-
teine and nitrosobenzaldehyde instead of the synergism caused
by the DOXꢀHCl and nitrosobenzaldehyde derivatives. In brief,
the enhanced anticancer effect of ‘‘0 + 1 > 1” is presented by UV
light triggering N, N-NB-DTPA-DOX liposomes. Hence, N, N-NB-
DTPA-DOX + UV can be used as a potential therapeutic strategy
for cancer therapy.
6
5% at 50
lg/mL. Especially, when the concentration reaches to
1
00 g/mL, the cell viability is less than 10%. In Fig. 10b, the IC50
l
ꢁ2
(
IC50 = 7.91 ꢂ 10 mM) of MCF-7 cells treated with 10,10-NB-D
ꢁ2
TPA + 30 min is close to the IC50 (IC50 = 4.77 ꢂ 10 mM) of
MCF-7 cells treated with the anticancer drug DOXꢀHCl, indicating
1
0,10-NB-DTPA + 30 min also presents a comparable anticancer
activity.
The results of above experiments indicates that the 10, 10-NB-
DTPA liposomes have low cytotoxicity, but the products formed by
photodegradation significantly inhibit the growth of cancer cells.
The cell viability of the pre-irradiation group is significantly differ-
ent from that of the irradiation group. We speculated that the
nitrosobenzene products in pre-irradiation group were either
decomposed by cancer cells or combined with extracellular serum
protein [35]. While in irradiation group, 10, 10-NB-DTPA liposomes
can produce nitrosobenzene products in cancer cells after UV irra-
diation, which may affect the cell growth. The nitrosobenzene
products produced in cancer cells by photolysis enable to oxidize
the cysteine in ADP-nucleic acid transporter, which destabilizes
the binding of ADP-nucleic acid transporter and Zn2 [26] and inhi-
bits cell proliferation.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgments
This work was supported by International Science & Technology
Cooperation Program of China No. 2015DFA41670, China Scholar-
ship Council Talent International Cooperation Project [2019]
+
1
3044, the Fundamental Research Funds for the Central Universi-
3
.10. Photo and drug dual-induced anticancer activity of N, N-NB-
ties DUT19GJ203 and China Postdoctoral Science Foundation
2020M670743.
DTPA-DOX liposomes
N, N-NB-DTPA molecules possess similarity in their structure,
so the anticancer activity of N, N-NB-DTPA (n = 8, 12 and 14) lipo-
somes after UV irradiation should be similar with 10, 10-NB-DTPA
liposomes. DOXꢀHCl was loaded in N, N-NB-DTPA liposomes to
form N, N-NB-DTPA-DOX liposomes (Drug:Lipid = 1:5). MCF-7 cells
were incubated with N, N-NB-DTPA-DOX liposomes for 4 h, which
then entered into the cytoplasm and released DOXꢀHCl molecules
into the nucleus (as shown in Fig. S31). Although anticancer activ-
ity is not good as the anticancer activity of DOXꢀHCl (in Fig. 11), N,
N-NB-DTPA-DOX liposomes produce anticancer activity when the
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molliq.2021.116016.
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