Colloids and Surfaces B: Biointerfaces 206 (2021) 111967

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Colloids and Surfaces B: Biointerfaces

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Redox-sensitive irinotecan liposomes with active ultra-high loading and

enhanced intracellular drug release
Tao Wang, Wei He, Yawei Du, Ji Wang, Xinsong Li *
School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: In this report, a novel irinotecan (IR) encapsulated redox-responsive liposome was developed. The redox-
Disulfide phosphatidylcholine responsive liposomes were prepared based on disulfide phosphatidylcholine (SS-PC), DSPC, DSPE-PEG2000
Redox-responsive liposome and cholesterol by ethanol injection method. IR was actively loaded by triethylammonium sucrose octasulfate
Irinotecan
(TEA8-SOS) gradient method to generate IR/SS-PC liposomes (IR/SS-LP). The particle size of IR/SS-PC was
Active drug loading
characterized by using dynamic light scattering (DLS) and transmission electron microscopy (TEM). It was found
that IR/SS-LP with 30 % content of SS-PC (IR/SS30-LP) had an average size of 125.5 ± 5.8 nm with a negative
zeta potential of − 19.5 ± 0.1. The encapsulation efficiency (EE) was further determined to be 98.1 ± 0.8 % and
drug loading (DL) was 31.8 ± 0.1 %. The redox-responsiveness of IR/SS-LP was investigated by observing the
change of particle size and morphology as well as the release behavior of IR triggered by glutathione (GSH). The
data indicated GSH breaks the disulfide bonds in SS-PC and leads to the controlled release of IR. At 1 mM GSH,
60.2 % irinotecan was released from IR/SS30-LP within 24 h. Finally, the effects of IR/SS-LP in cell and animal
experiments were evaluated in detail. The results showed that IR/SS30-LP had superior pharmacokinetic and
antitumor efficacy compared to free irinotecan and traditional irinotecan liposome (ONIVYDE®-like). Taken
together, IR/SS30-LP displayed redox-responsive release of IR, ultra-high loading and enhanced anti-tumor ac­
tivity, which has great potential for clinical application as a new generation of IR liposomal formulation.

1. Introduction improve pharmacokinetics while reducing toxicity. In 2015, a liposome-

Irinotecan (IR) is a semisynthetic water-soluble camptothecin de­
rivative, targeting topoisomerase I, which is currently used for treatment
a variety of solid tumors [1,2]. IR, as a prodrug of 7-ethyl-10-hydroxy­
camptothecin (SN-38), needs to converted by nonspecific carbox­
ylesterases. Compared with IR, SN-38 shows 100- to 1000-fold more
active, which can lead to apoptosis induction in rapidly dividing tumor
cells. The first irinotecan formulation agent approved by the Food and
Drug Administration (FDA) is CAMPTO®, which serves as a first-line
drug for metastatic colorectal cancer [1,3]. However, drug’s high
toxicity, such as neutropenia (level 3–4, 47 %) and severe diarrhea (level
3–4,39 %) would lead to an ultimately fatal threat [4,5]. In addition,
active lactone rings in IR and its metabolite SN-38 are easily hydrolyzed
into an inactive carboxylate form in normal physiologic pH, causing
active ingredients’ rapid elimination and reduced circulation [6,7].
These defects of CAMPTO® contribute to the limitation of clinical
application, even though it shows outstanding efficacy [1,4,8].
Nanomedicine-based drug delivery systems (NDDSs) can be used to
encased irinotecan formulation (brand name ONIVYDE®) was approved
by the FDA as an orphan drug for metastatic pancreatic cancer [9,10].
The formulation contains main ingredients DSPC, DSPE-mPEG2000 and
cholesterol generating conventional liposomes. Improving the pharma­
cokinetics of the IR by increasing drug package, protecting active
lactone configuration, provided sustained release, prolonging circula­
tion time and increased tumor accumulation through the EPR effect [9,
10]. However, ONIVYDE® only possesses passive targeting and cannot
release IR precisely at tumor site. Actually, ONIVYDE® cannot be used
as an ideal replacement for CAMPTO® in clinical [8,11–14].
Designing active-targeted NDDSs attracted much attention in recent
years. One strategy is to decorate NDDSs surface with antibody, peptide
or aptamer [15–17]. The other is to design NDDSs that can respond to
tumor site-specific stimuli by taking advantage of significant differences
in tumor physiological microenvironment and normal tissue. [18,19].
The tumor site-specific stimuli include pH, high levels of glutathione
(GSH)/reactive oxygen species (ROS) etc [18,19]. Disulfide bonds based
polymeric redox-responsive delivery systems have been investigated

* Corresponding author.
E-mail address: lixs@seu.edu.cn (X. Li).

https://doi.org/10.1016/j.colsurfb.2021.111967
Received 31 March 2021; Received in revised form 1 July 2021; Accepted 4 July 2021
Available online 6 July 2021
0927-7765/© 2021 Elsevier B.V. All rights reserved.
T. Wang et al.
extensively in the literatures [20,21]. The disulfide bonds could be
quickly degraded in the strong reductive microenvironment of tumor
tissue, while they are stable in the extracellular space of normal tissue
with low GSH concentration. Gao et al. reported a star-like amphiphilic
copolymer (CPIO) micelles which could deliver IR in a
reduction-responsive manner [22]. Disulfide phosphatidylcholines
(SS-PCs) based redox-sensitive liposomes were first developed in our
previous work [23,24]. After passive loading with PTX, the resulting
liposomes (PTX/SS-LP) can be effectively triggered and destroyed once
exposed to reductive environment, inducing the controlled release of the
PTX, and improving anticancer activity without significant side effects
[24–26]. Compared to disulfide decorated polymeric carriers, SS-PCs
based lipid nanoparticles possesses instant reductive-responsiveness,
which might have promise in the development of liposomal formula­
tion of other drugs.
We noticed that IR has an irreplaceable role in the treatment of
gastrointestinal tumors. More importantly, IR can be encapsulated by
active loading, which brings an ultra-high drug loading (~30 %) that
PTX can hardly achieved (~5%) [27,28]. The success of PTX/SS-LP
inspired us SS-PC based liposomes might be an effective delivery sys­
tem of irinotecan to improve anti-cancer activity and reduce side effects
as an alternative of liposomal irinotecan ONIVYDE ®. In this study, a
novel redox-responsive liposome based on disulfide phosphatidylcho­
line (SS-PC, Scheme 1) for the delivery of irinotecan (IR) was developed.
The redox-responsive liposomes were prepared with different lipid
components including SS-PC, DSPC, DSPE-PEG2000 and cholesterol by
ethanol injection method. IR was actively loaded by triethylammonium
sucrose octasulfate (TEA8-SOS) gradient method to obtain IR/SS-LP. The
redox-sensitivity of IR/SS-LP was investigated by measuring the change
of the morphology and size of the liposomes under reduction condition.
In vitro cytotoxicity and in vivo anti-tumor activity were further evalu­
ated in detail. The results showed that IR/SS30-PC had superior phar­
macokinetic and antitumor efficacy compared to free irinotecan and
traditional irinotecan liposomes (ONIVYDE®-like).
2. Experimental section
2.1. Material
Disulfide phosphatidylcholine (SS-PC) with structure as shown in
Fig. 1a was synthesized in the authors’ laboratory as reported previously
Scheme 1. In vivo Mechanism of Redox-Sensitive IR-Loaded Liposomes.
2
Colloids and Surfaces B: Biointerfaces 206 (2021) 111967
[24]. Irinotecan (IR) was provided by Chunqiu Biological Engineering
Co., Ltd. (Nanjing, China). DSPE-mPEG2000 and DSPC was originated
from A.V.T. (Shanghai, China). Sodium octasulfate sucrose (SOS) was
supplied by Guokang Biotechnology Co., Ltd. (Shanxi, China). 732 ion
exchange resin was provided by Hewu Biological Co., Ltd. (Shanghai,
China). Sephadexg-50 was purchased from Jianglai Biotechnology Co.,
Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO), methylene chloride
(DCM), methanol (MT) and triethylamine (TEA) were supplied by the
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China)
The cell experiment materials were supplied by KeyGen Co., Nanjing,
China. Human breast adenocarcinoma cell line (MCF-7) and human lung
carcinoma cell line (A549) was cultured with RPMI-1640 medium which
contained 10 % fetal bovine serum. Cells were grown in a humidified
incubator at 37 ◦ C with a 5 % CO2 atmosphere. All experiments followed
strict aseptic procedures.
BALB/c mice (female, 3–8 weeks) and SD rat (female, 180–200 g)
were obtained from KeyGen BioTech Co., Ltd. (Nanjing, China).
2.2. Preparation of triethylammonium sucrose octasulfate solution
(TEA8-SOS)
1 M Sodium sucrose octasulfate (SOS) deionized aqueous solution
was prepared and Na+ and H+ were slowly exchanged through the
cation exchange resin-732. Na+ electrode was used to detect the con­
centration of cation in the exchange solution. After exchange, the con­
centration of Na+ should be reduced to at least 1 % of that before
exchange. Furthermore, the resulting solution was titrated slowly with
triethylamine (TEA) thus obtaining 250 mM TEA8-SOS. Most impor­
tantly, the pH of the solution needs to be controlled between 5.5 and 6.
[6,28].
2.3. Preparation of liposomes
All liposomes were prepared via ethanol injection method and drug-
loaded liposomes were prepared by TEA8-SOS gradient method. Briefly,
DSPC, Cholesterol, DSPE-mPEG2000 and a certain percentage of SS-PC
were dissolved in 1 mL ethanol and slowly added into 5 mL TEA8-SOS
solution followed by stirring rapidly at 65 ◦ C. Next, the ethanol was
volatilized by continuous stirring to obtain multilamellar vesicles
(MLVs). Next, the MLVs were adjusted particle size by extruding 10
times through 0.1 μm pore size polycarbonate membrane, thus forming
T. Wang et al. Colloids and Surfaces B: Biointerfaces 206 (2021) 111967

Fig. 1. (a) Structural formula of SS-PC, (b)

Schematic diagram of TEA8-SOS gradient
method, (c) Size distribution of IR/LP and IR/
SS-LP, (d) Changes of the size of both IR/LP
and IR/SS30-LP after 28 days storage, (e) Par­
ticle size distribution IR/SS30-LP, (f-g)Redox-
responsive behavior of IR/SS30-LP: TEM im­
ages of IR/SS30-LP before (f) and after (g)
treatment with 10 mM GSH, the inset in (f)
shows a cryo-TEM image. (h, j) In vitro release
of irinotecan from IR/SS-LP in PBS (pH 7.4)
containing 1 mM GSH(h) and 0.1 mM GSH(j).
(i) Image of IR/SS100-LP collapse process in
PBS (pH 7.4) containing 1 mM GSH.

generate unilamellar vesicles (ULV). Unentrapped triethylammonium IR/SS30-LP, IR/SS50-LP and IR/SS100-LP as shown in Table 1. Con­
polyanions were replaced with buffer solution (4.05 mg/mL HEPES and ventional irinotecan liposome IR/LP (ONIVYDE®-like) was prepared
8.42 mg/mL NaCl) using dialysis, and blank liposomes SS-LPs of SS15- from DSPC-LP by the same method as above.
LP, SS30-LP, SS50-LP and SS100-LP were obtained with different SS-
PC content as shown in Table 1. DSPC-LP was prepared in the same 2.4. Characterization of liposomes
manner without addition of SS-PC.
After addition of 17.5 mg irinotecan (IR), the ULV was incubated DLS (Brookhaven Instruments Co., Holtsville, NY) was used to
under stirring for 30 min at 60 ◦ C. Finally, IR-loaded liposomes were measure the size and zeta potential of liposomes. The micromorphology
quenched in ice water mixture for 15 min followed by removal of IR of liposomes was observed under TEM (JEOL, Inc., Tokyo, Japan) after
outside of liposomes via Sephadex G-50 column chromatography [6,28]. negative staining with 2 % phosphotungstic acid with a voltage of 200
The prepared irinotecan liposomes were noted as IR/SS15-LP, kV. In addition, the change in liposome particle size shown the stability
of liposomes. Storage stability of IR/SS30-LP and IR/LP was checked
after kept at 4 ◦ C for 28 days.
Table 1
Sephadex-50 was used to determine the EE of IR-loading liposome.
The formulations of liposomes.
After the removal of unentrapped IR, the IR-loading liposomes were
Formulation demulsified with methanol. And the loaded IR was measured by HPLC
liposomes DSPC SS-PC Cholesterol DSPE- irinotecan (Agilent, Palo Alto, CA). The column was Hedera ODS-2, 4.6 × 250 mm,
(mg/ (mg/ (mg/mL) mPEG2000 (mg/mL) 5 μm (HanBang, China). Column oven temperature was 35 ◦ C and the
mL) mL) (mg/mL)
detection wavelength was 368 nm. The mobile phase was methanol/
IR/LP 6.81 – 2.22 0.12 4.30 acetonitrile/water, 55/5/45 with a 1 mL/min flow rate. The DL and EE
IR/SS15- 5.79 1.22 2.22 0.12 4.30
were calculated according to the following formulas:
LP
IR/SS30- 4.68 2.44 2.22 0.12 4.30 EE(%) = Mloaded / Madded×100%
LP
IR/SS50- 3.41 4.01 2.22 0.12 4.30 DL(%) = Mloaded / (Mlipid + Mloaded) ×100%
LP
IR/SS100- – 8.13 2.22 0.12 4.30 where, Mlipid denotes the weight of the lipid materials, Madded denotes the
LP
quality of IR addition and Mloaded denotes the quality of loaded

3
T. Wang et al.
irinotecan.
2.5. Evaluation of redox-sensitivity
TEM and DLS were used to studied the redox sensitivity of IR/SS-LP.
IR/SS30-LP was exposure to reducing condition of PBS containing 10
mM glutathione (GSH) for 2 h. The changes in the morphology and size
of IR/SS-LP were measured by TEM and DLS using the method as
described in section 2.4 [25].
2.6. In vitro drug release
The In vitro release of irinotecan from IR/SS-LP was researched using
high performance liquid chromatography (HPLC). PBS solution (pH 7.4)
with 1 mM or 0.1 mM GSH was used as the release medium. Meanwhile,
free irinotecan and IR/LP (ONIVYDE®-like) were used as controls. The
release of IR/SS-LP was carried out in a regenerated cellulose dialysis
bag (MWCO 8000), which was completely immersed in 200 mL of
release medium while properly stirring at 37 ◦ C. 1 mL of the external
release medium was collected at predetermined time point while an
equal volume of fresh release medium was supplemented. The release
rate was calculated by the data which was derived from sample
measured by HPLC.
2.7. Cytotoxicity test
First, human breast adenocarcinoma cell line (MCF-7) and human
lung carcinoma cell line (A549) were used to evaluate the cytotoxicity of
SS-PC. 96-well plates were used to seed cells at density of 1 × 104 cells/
well. Then, cells were incubated in media containing control DSPC li­
posomes (DSPC-LP) or SS-LP (10 μg/mL), while control groups without
additional liposomes. The cells were cultured at 37 ◦ C with a 5 % CO2
atmosphere for 1, 4, or 7 days while culture fluid was refreshed every 2
days.
Secondly, the in vitro cytotoxicity of IR/SS-LPs was tested by using
A549 or MCF-7 cell lines. Free irinotecan and IR/LP were used as con­
trols. The cells seeded in 96-well plates were incubated in the media
containing different irinotecan liposomes. After that, the medium was
replaced by 20 μL of MTT solution with a 4 h incubation. Ultimately
MTT was replaced with 100 μL of DMSO. Spectrophotometer (Thermo
Fisher Scientific, Waltham, MA) was used to measure the optical density
(OD) of wells at 490 nm for three times. The cell viability was calculated
according to the following formula:
Cell viability(%)= (ODA-ODB) / (ODC-ODB) ×100%
where, ODA is the OD value of free irinotecan, IR/LP and IR/SS-LPs
treated cells, ODB is the OD value of DMSO while ODC is the OD value
of blank medium cultured cells without any treatment.
2.8. Cellular uptake analysis
Confocal laser scanning microscope (CLSM) was used to check
cellular uptake. Briefly, MCF-7 cells were seeded in 12-well (3 × 105
cells/well) and incubated for 24 h. After that, the cells incubated in
media containing IR, IR/LP, or IR/SS30-LP with the concentration of
irinotecan 5 mM for 6 h. The cells were fixed with 4 % para­
formaldehyde for 30 min after washed with cold PBS buffer twice. The
cells were observed by CLSM (Olympus, Japan) after washed three times
with PBS.
2.9. Apoptosis analysis
The apoptosis of MCF-7 cells was investigated by using flow
cytometry (BD Accuri C6, Bedford, MA). In brief, cells were seeded into
6-well plates at density of 2 × 105 cells/well. Next, cells incubated in
4
Colloids and Surfaces B: Biointerfaces 206 (2021) 111967
media containing IR, IR/LP, or IR/SS30-LP with the concentration of
irinotecan 50 μg/mL for 24 h. After 24 h, PBS and trypsin were used to
wash and detach cells. After centrifugation (1500 rpm, 10 min), cells
were collected and resuspended in buffer. Then, PI (2 μL) and Annexin
V-FITC (2 μL) reagents were added to stain cells. Ultimately, the samples
were measured by flow cytometer.
2.10. Pharmacokinetic study
The pharmacokinetic study of IR/SS-LP in SD rats was evaluated by
intravenous injection of IR/SS-LPs with equivalent irinotecan dose of 10
mg/kg using free irinotecan as a control. Blood samples were collected
from the orbital vein at specific time intervals and stored in heparin
sodium tubes. Blood samples were centrifuged at 3000 rpm (10 min, 4
◦
C) to obtain plasma. After that, 200 μL methanol was added to the
supernatant to extract the drug by centrifuged at 10,000 rpm (10 min, 4
◦
C). Then, the supernatant was absorbed, dried with nitrogen, and then
dissolved in methanol (100 μL). The concentration of SN38, the main
metabolite product of IR, was determined by HPLC at 368 nm. Finally,
pharmacokinetic parameters of groups were calculated by DAS software.
2.11. Anti-tumor activity
The in vivo antitumor activity was evaluated by the change of tumor
size and body weight of 4T-1 tumor-bearing BALB/c mice after intra­
venously injecting free IR, IR/LP or IR/SS30-LP [29]. BALB/c mice were
divided into 4 groups (n = 3). Free IR, IR/LP or IR/SS30-LP were
injected at an equivalent IR dose of 5 mg/kg every 3 days. Tumor vol­
ume and body weight were measured every two days. After 21 days, all
mice were sacrificed while the final tumors were weighed and
cryopreserved.
2.12. Statistical analysis
Statistical analyses were performed using SPSS (Statistics 25, IBM).
All results were presented as means ± standard deviations (SD). One-
way analysis of variance (ANOVA) was employed in data comparisons.
P < 0.05 means a statistically significant difference and P < 0.01 means
a statistically high significant difference.
3. Results and discussion
3.1. Preparation and characterization of SS-LP
Disulfide phosphatidylcholine (SS-PC) with the structure as shown in
Fig. 1a was used in the formulation of the liposomes. It’s phase transition
temperature (Tc = 58 ◦ C) is close to that of DSPC (55 ◦ C). The SS-PC
based liposomes (SS-LP) were prepared using an ethanol injection
method followed by mini extrusion. After that, irinotecan (IR) was
actively loaded into the liposomes by triethylammonium sucrose octa­
sulfate (TEA8-SOS) gradient method (Fig. 1b) [6,29,30]. The composi­
tion of the as-prepared liposomes (SS-LP) was provided in Table 1.
DLS was used to measure the size and zeta potential of the liposomes.
As listed in Table 2, the IR-loaded liposomes have an average size of
about 120 nm. The PDI is about 0.1, which confirms the liposomes are
relatively uniform with a narrow distribution (Fig. 1c.). The Zeta po­
tentials are all negative, which indicates the liposomes have strong
stability. The liposomal morphology of IR/SS30-LP was characterized by
using cryo-TEM and TEM. According to Fig. 1c, they display a uni­
lamellar vesicle structure (ULV) with a particle size of about 100 nm.
DLS was further used to demonstrate the excellent storage stability of
the liposomes since no significant change of the size was detected after
storage for 28 days at 4 ◦ C (Fig. 1d.).
The EE and DL capacity of IR/SS30-LP and IR/LP (ONIVYDE®-like)
were detected using size exclusion chromatography method. The EE and
DL of IR/LP were calculated to be 31.7 ± 0.1 % and 98.1 ± 0.8 %, while
T. Wang et al. Colloids and Surfaces B: Biointerfaces 206 (2021) 111967

Table 2 More visually, images of IR/SS100-LP collapse process in PBS (pH 7.4)
Physicochemical characteristics of liposome. containing 1 mM GSH are shown in the Fig. 1j.
sample size(nm) PDI Zeta potential DL (%) EE (%) We have supplemented the release experiments of liposomes with
(mV) different SS-PC contents at a concentration of GSH of 0.1 mM (similar to
DSPC-LP 130.1 ± 0.08 ± − 20.1 ± 0.5 – – normal intracellular concentrations). Additional experimental diagrams
4.5 0.03 are in the supporting information (Fig. 1j). The experimental results that
SS30-LP 138.3 ± 0.12 ± − 12.2 ± 1.6 – – SS-PC liposomes with content greater than 50 % are more easily
1.4 0.04 destroyed in physiological concentration of GSH, while IR/SS30-LP can
IR/LP 116.5 ± 0.14 ± − 22.4 ± 0.9 31.7 ± 98.1 ±
2.4 0.01 0.1 0.8
be more stable. It is means that IR/SS30-LP is stable under the physio­
IR/SS30- 125.5 ± 0.15 ± − 19.5 ± 0.1 32.0 ± 98.3 ± logical conditions before ingested by the tumor cells, while they could be
LP 5.8 0.02 0.2 0.7 quickly broken in a strong reductive tumor microenvironment. This trait
IR/SS15- 120.6 ± 0.11 ± − 16.9 ± 3.3 31.8 ± 97.3 ± gives IR/SS30-LP active targeting, so that it does not release drugs in
LP 8.5 0.05 0.1 1.2
normal tissues, thus achieving the effect of killing tumor cells
IR/SS50- 115.1 ± 0.12 ± − 18.2 ± 0.6 31.6 ± 96.3 ±
LP 2.4 0.06 0.1 2.2 specifically.
IR/SS100- 110.2 ± 0.12 ± − 20.1 ± 1.2 32.6 ± 98.0 ±
LP 7.2 0.10 0.1 0.5 3.4. Cellular uptake analysis

IR/SS30-LP shown similar EE and DL values of 98.3 ± 0.7 % and 32.0 ±
0.2 %, respectively. Their ultra-high drug loading and encapsulation
efficiency could be ascribed to the high negative charge density of TEA8-
SOS inside the liposomes [28]. therefore, SS-PC could sieve as a
replacement for traditional phospholipids, such as DSPC, without
negative effects on drug encapsulation and stability of liposomes.
3.2. Redox responsiveness of SS-LP
CLSM was used to investigate the uptake of free IR, IR/LP and IR/
SS30-LP into MCF-7 cells. As shown in the Fig. 2a, the cell emitted
obviously blue fluorescence due to IR. Both IR/LP and IR/SS30-LP
loaded with irinotecan showed strong blue fluorescent signals in the
MCF-7 cells, indicating high internalization efficiency. In contrast, free
irinotican as control shown much weaker fluorescence than that of the
liposomes. Based on these data, it can be inferred that the liposomes
improve the absorption efficiency of irinotecan into tumor cells and
achieve effective accumulation.

The high concentration of reductive substances in tumor sites is
mainly GSH which has been reported to be used for detecting in vitro
activation of redox-sensitive drug delivery systems [26,31,32]. In this
report, the intracellular reduction environment was simulated by 10 mM
GSH solution. And, DLS and TEM was used for checking the
redox-responsive phenomenon of IR/SS-LP.
Obviously, the evidence of the destruction of IR/SS-LP after GSH
treatment for 2 h was obtained by transmission electron microscopy
(TEM) (Fig. 1f). Compared with before treatment, IR/SS-LP lost the
spherical morphology and presented irregular black block structure
(Fig. 1g). These black blocks may be a mixture of aggregates including
lipids and residual irinotecan-SOS gels. In addition, the particle size
measured by using DLS also shown dramatically change. According to
Fig. 1e, size distribution converted from a single peak (before GSH
treatment) to multiple individual peaks after GSH treatment (Fig. 1e).
The peak of big size shown in Fig. 1e (~1000 nm) may be ascribed to the
large black lump that appears in the TEM image (Fig. 1g). The released
irinotecan gels aggregate to form larger, noncompact clusters. While the
signal peak less than 100 nm may be derived from the debris of the
destroyed liposomes. In summary, IR/SS-LP was specifically manifested
as redox-responsive release carrier of irinotecan in a reduced microen­
vironment due to the disulfide breakage of SS-PC inducing gradual
collapse followed by complete destruction of liposomal structure.
3.3. In vitro release of irinotecan
The drug release from IR/SS-LP with different SS-PC content was
monitored by dialysis. As shown in Fig. 1h, only 18.2 % irinotecan was
released from IR/SS100-LP within 24 h in PBS at pH 7.4, while 19.3 %
irinotecan was released from IR/LP. The results indicated that the sta­
bility of IR/SS-LP in PBS is comparable to that of IR/LP. Under the
condition of 1 mM GSH of PBS (pH 7.4), the final release rates of IR/SS-
LP with 0%, 15 %, 30 %, 50 % and 100 % SS-PC content were 18.2 %,
30.3 %, 60.2 %, 67.8 % and 77.1 %, respectively. Apparently, with the
increase of SS-PC content, the release rate of IR increased significantly.
Therefore, the release of irinotecan from IR/SS-LP was attributed to the
redox-reaction breakage of SS-PC inducing liposome destruction. And,
the increase of SS-PC content accelerated the drug release of IR/SS-LP in
a dose-dependent manner with an appropriate release curve at 30 %.
3.5. Analysis of cellular apoptosis
Cell apoptosis was detected by flow cytometry to investigate the
cytotoxic mechanism of the liposomes. Annexin V-FITC/PI kit was used
to stain and identify apoptotic MCF-7 cells. As shown in Fig. 2b,
apoptotic cells were found at Q3 and Q2 phases, while normal cells were
found at Q4. Accordingly, the total percentage of apoptotic cells in IR/
SS30-LP group was 49.7 %, and 38.0 % in IR/LP group, while the free
IR group has a value of 23.8 %. The results indicate that both liposomal
formulations have significantly improved cell apoptosis compared to
free IR. This finding may be explained by the fact that the liposome
structure prevents active structure of irinotecan from destroyed in the
low pH microenvironment of tumor cells. Therefore, in both the
apoptosis assays and cytotoxicity, IR/SS30-LP has displayed the best,
mainly owing to the disulfide breakage induced liposomal structure
disassociation.
3.6. In vitro cytotoxicity
Firstly, we tested the toxicity of SS-PC as a component of SS-LP li­
posomes by MTT assay. Cytotoxicity of SS30-LP was checked against
A549 cells and MCF-7 cells. As a comparison, GSH pretreated cancer
cells were used for enhancing the breakage of SS-PC induced liposomal
collapse. After incubation for 1,4 and 7 days, cell growth was deter­
mined. Fig. 3a (A549 cells) and 3b (MCF-7 cells) shown cell growth of all
samples tended to be similar without significant difference (P > 0.05).
The data proved that SS-LP as well as its degradation products had no
toxic effect on cells.
After IR loading, the in vitro cytotoxicity of IR/SS-LP with different
SS-PC content was detected against A549 and MCF-7 cell lines for 24 h.
As can be seen from the Fig. 3c-d, the cytotoxicity of the liposomes was
significantly enhanced with the increasing of SS-PC ratio. While the IR
concentration was 100 μg/mL, the cytotoxicity of IR/SS100-LP against
MCF-7 and A549 reached 69.54 % and 64.11 %, compared to 53.22 %
and 49.8 % of IR/LP. Notably, the cytotoxicity of IR/SS50-LP against
MCF-7 and A549 reached 62.1 % and 61.86 % respectively, compared to
59.36 % and 58.0 % of IR/SS30-LP. But, there was no significant dif­
ference between the latter two groups (P > 0.05). In other words, both
IR/SS50-LP and IR/SS30-LP could have similar in vitro anti-tumor

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T. Wang et al.
Fig. 2. (a) Cellular uptake of Free IR, IR/LP, and IR/SS30-LP into MCF-7 breast cancer cells at 6 h. The blue channel was IR fluorescence. scale bar: 10 μm. (b) Flow
cytometry analysis for apoptosis of MCF-7 cells induced by free irinotecan, IR/LP, and IR/SS30-LP after 24 h.
activity. Therefore, the redox-responsive liposomes might achieve a
decent improvement of cytotoxicity after incorporation of 30 % or more
SS-PC.
Furthermore, IR/SS30-LP containing 30 % SS-PC was used to
investigate the effect of additional GSH on cytotoxicity. In brief, after
cells incubation in media containing 1 mM of GSH for 12 h to take up
additional GSH, IR/SS30-LP and IR/LP were added followed by further
incubation for 24 h and 48 h [25]. Control groups of IR/SS30-LP and
IR/LP were incubated with cells without GSH pretreatment. As revealed
in Fig. 3e-h, the cytotoxicity of IR/SS30-LP was significantly higher than
that of IR/LP in both cell lines. It was noticed that after 24 h incubation
the cytotoxicity of IR/SS-LP with cells pretreated with GSH was higher
than that of IR/SS-LP without GSH, indicating that additional taken up
of GSH benefit for the killing of cancer cells. 48 h incubation leaded to
similar cytotoxicity, indicating that the effect of additional GSH taken up
was limited. Therefore, the redox response of SS-LP could be triggered
by the reducing environment within tumor cells, confirming the role of
SS-LP in enhancing anticancer efficacy.
3.7. Pharmacokinetic studies
The pharmacokinetics of IR/SS-LP and free IR in SD rats were studied
by intravenous injection of 10 mg/kg equivalent irinotecan. In this
study, the plasma concentration of 7-ethyl-10-hydroxycamptothecin
(SN38) was determined by high performance liquid chromatography,
since SN38 is a metabolite of rapid transformation of irinotecan in vivo.
The drug-time curves of free IR and IR/SS-LP were shown in Fig. 4e. DAS
software was used to calculate the pharmacokinetic parameters, which
6
Colloids and Surfaces B: Biointerfaces 206 (2021) 111967
were listed in Table 3. As is shown in Table 3, the pharmacokinetic
parameters of IR/SS-LP with different SS-PC content were significantly
different, especially reflected in the t1/2 and AUC(0-∞). Meanwhile, IR/LP
greatly improved the half-life of irinotecan, which was up to 4.89 times
that of free drug (t1/2 2.05 h).
IR/SS-LP with the SS-PC content of 15 %, 30 %, 50 % and 100 % had
t1/2 of 8.75 h, 7.16 h, 5.19 h and 3.73 h, respectively. Obviously, with
the increase of SS-PC content in IR/SS-LP, the release rate of IR was
gradually enhanced, which led to the continuous shortening of the half-
lifetime. Meanwhile, the AUC(0-∞) value firstly increased and then
decreased, and reached a maximum value of 17.68 mg h/L (IR/SS30-
LP), which was 5.05 times that of free irinotecan and 1.15 times that of
IR/LP (t1/2 10.01 h). This phenomenon might be attributed to different
half-lifetime and maximum concentration of IR/SS-LP with different SS-
PC content.
When SS-PC content was 0 % and 15 %, IR/SS-LP liposomes were
more stable in the reductive environment in vivo, resulting in lower
maximum concentration and longer drug half-life. However, when SS-
PC content exceeds 50 %, liposomes are more likely to break up and
release the drug in vivo, resulting in higher maximum concentration and
shorter half-life. In both cases of very low and high content of SS-PC, the
absorption of the drug was unsatisfactory. Particularly, SS-PC content of
30 % can maintain a delicate balance between the half-life and the
maximum concentration, reaching a higher value of AUC(0-∞). The re­
sults showed that the kinetic parameters of irinotecan metabolism in vivo
could be controlled by adjusting the content of SS-PC in the formula. The
30 % SS-PC content provided optimal pharmacokinetic parameters,
which greatly improving the half-life and AUC(0-∞) of irinotecan.
T. Wang et al. Colloids and Surfaces B: Biointerfaces 206 (2021) 111967

Fig. 3. In vitro cytotoxicity: (a, b) DSPC-LP or SS30-LP incubated with A549 (a) and MCF-7 (b) cells pretreated with or without GSH, (c, d) SS-LP with different SS-PC
content incubated with A549 (c) and MCF-7 (d) cells, (e-h) Free IR, IR/LP, and IR/SS30-LP incubated with A549 (e and g) and MCF-7 (f and h) cells pretreated with or
without GSH.

3.8. In vivo antitumor activity Negative control group was treated with normal saline. As shown in
Fig. 4a, all mice in each group gained weight during the experiment.
The in vivo antitumor activity of IR/SS30-LP versus free IR and IR/LP And, the body weight of the normal saline group was heavier than that of
was compared in 4T-1 mice with 5 mg/kg irinotecan equivalent. the two groups of liposomes, which could be attributed to the increase in

7
T. Wang et al. Colloids and Surfaces B: Biointerfaces 206 (2021) 111967

Fig. 4. (a-d) In vivo antitumor efficiency test. 4T-1 tumor-bearing mouse were treated with IR/SS30-LP versus free IR and IR/LP. (a) tumor volume, (b) body weight,
and (c) tumor weight (d) photographs of tumors (*P < 0.05 and **P < 0.01, respectively) (e) The SN38 concentration in plasma after i.v of free IR and IR/SS-LP
liposome at IR equivalent dose of 10 mg/kg.

Table 3
Pharmacokinetic parameters of free IR and IR/SS-LP after i.v injection.
Parameters (unit) t1/2(h) AUC(0–24h) (mg∙h L) AUC(0-∞) (mg h L) CL (L/h) Vd (L) MRT (h) Cmax (mg/L)

IR 2.05 3.22 3.50 0.57 1.69 2.95 1.26

IR/LP 10.01 12.57 15.32 0.13 1.89 14.45 1.04
IR/SS15-LP 8.67 14.10 16.47 0.12 1.52 12.52 1.31
IR/SS30-LP 7.16 15.49 17.68 0.11 1.17 10.33 1.95
IR/SS50-LP 5.19 12.35 12.98 0.15 1.15 7.50 1.93
IR/SS100-LP 3.73 10.06 10.21 0.20 1.05 5.38 2.03

tumor weight. The tumor weight of the IR/SS30-LP group was the lowest
(0.76 ± 0.07 g), significantly lower than that of the free IR group (2.05 ±
0.14 g), and IR/LP group (0.96 ± 0.02 g). Moreover, the tumor weight of
the three experimental groups was lower than that of the normal saline
group (2.33 ± 0.33 g). An extremely significant difference was observed
between the IR/LP group and the free IR group (P < 0.01), while there
was distinct difference between IR/LP and IR/SS30-LP group (P < 0.05).
As shown in Fig. 4b, the tumor growth inhibition rate of the IR/SS30-LP
group was the highest. And, the difference became more obvious over
time among three experimental groups. After 21 days, tumor volumes in
the IR/SS30-LP, free IR and IR/LP treatment groups were 0.57 ± 0.10
cm3, 1.69 ± 0.14 cm3, and 0.70 ± 0.11 cm3, respectively. Images of
tumor-bearing tumors (Fig. 4d) make these results more visual. There­
fore, IR/SS30-LP had improved anti-tumor efficiency in vivo compared
with free IR and IR/LP.
4. Conclusion
In summary, the irinotecan loaded liposomes based on SS-PC were
developed with reductive responsiveness and improved antitumor ac­
tivity. Triethylammonium sucrose octasulfate (TEA8-SOS) gradient
method was applied to have IR loaded with ultra-high dose and stability
in the form IR-SOS gel in the core of IR/SS-PC liposomes. It could be
triggered by GSH, leading to structural collapse and disintegrate to
release IR. Specifically, IR/SS30-LP had the best in vitro cytotoxicity,
apoptosis ratio and pharmacokinetics. Cell imaging test demonstrated
that IR/SS30-LP was more easily internalized and enriched into nucleus
than free IR, mainly ascribing to GSH induced disulfide breakage of SS-
PC and collapse of liposomal structure. The most important is that IR/
SS30-LP displayed effective tumor inhibition with no significant
adverse effect. Therefore, the IR/SS-PC liposomes with ultra-high
loading, strong reduction responsiveness and enhanced antitumor ac­
tivity could be a promising formulation for the delivery of IR.
CRediT authorship contribution statement
Xinsong Li, Tao Wang, Wei He and Yawei Du designed the study.
Tao Wang designed and performed the main experiment. The manu­
script was written by Tao Wang, Yawei Du and Xinsong Li. Tao Wang,
Wei He, Yawei Du, and Ji Wang participated in the data analysis and
manuscript writing.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
This research was supported by the Major National Science and
Technology Program of China for Innovative Drug (2017ZX09101002-
001-004).
References
[1] Z. Zhang, J. Yao, Preparation of irinotecan-loaded folate-targeted liposome for
tumor targeting delivery and its antitumor activity, AAPS Pharm. Sci. Tech. 13
(2012) 802–810.
[2] C.J.H. Gerrits, M.J.A. deJonge, J.H.M. Schellens, G. Stoter, J. Verweij,
Topoisomerase I inhibitors: the relevance of prolonged exposure for present clinical
development, Br. J. Cancer 76 (1997) 952–962.
[3] C. Yoo, J.Y. Hwang, J.E. Kim, T.W. Kim, J.S. Lee, D.H. Park, S.S. Lee, D.W. Seo, S.
K. Lee, M.H. Kim, D.J. Han, S.C. Kim, J.L. Lee, A randomised phase II study of
modified FOLFIRI.3 vs modified FOLFOX as second-line therapy in patients with
gemcitabine-refractory advanced pancreatic cancer, Br. J. Cancer 101 (2009)
1658–1663.
[4] H. Bleiberg, E. Cvitkovic, Characterisation and clinical management of CPT-11
(Irinotecan)-induced adverse events: the European perspective, Eur. J. Cancer 32
(1996) S18–S23.
[5] X.B. Lin, L.A. Dieleman, A. Ketabi, I. Bibova, M.B. Sawyer, H. Xue, C.J. Field, V.
E. Baracos, M.G. Ganzle, Irinotecan (CPT-11) chemotherapy alters intestinal
microbiota in tumour bearing rats, PLoS One 7 (2012), e39764.

8
T. Wang et al.
[6] W. Yang, Z. Yang, J. Fu, M. Guo, B. Sun, W. Wei, D. Liu, H. Liu, The influence of
trapping agents on the antitumor efficacy of irinotecan liposomes: head-to-head
comparison of ammonium sulfate, sulfobutylether-beta-cyclodextrin and sucrose
octasulfate, Biomater. Sci. 7 (2018) 419–428.
[7] J.A. Zhang, T. Xuan, M. Parmar, L. Ma, S. Ugwu, S. Ali, I. Ahmad, Development and
characterization of a novel liposome-based formulation of SN-38, Int. J. Pharm.
270 (2004) 93–107.
[8] L.T. Chen, J.T. Siveke, A. Wang-Gillam, C.P. Li, G. Bodoky, A.P. Dean, Y.S. Shan, G.
S. Jameson, T. Macarulla, K.H. Lee, D. Cunningham, J.F. Blanc, C.F. Chiu,
G. Schwartsmann, F.S. Braiteh, K. Mamlouk, B. Belanger, F.A. de Jong, R.
A. Hubner, Survival with nal-IRI (liposomal irinotecan) plus 5-fluorouracil and
leucovorin versus 5-fluorouracil and leucovorin in per-protocol and non-per-
protocol populations of NAPOLI-1: expanded analysis of a global phase 3 trial, Eur.
J. Cancer 105 (2018) 71–78.
[9] H. Zhang, Onivyde for the therapy of multiple solid tumors, Onco Targets Ther 9
(2016) 3001–3007.
[10] S. Tran, P.J. DeGiovanni, B. Piel, P. Rai, Cancer nanomedicine: a review of recent
success in drug delivery, Clin. Transl. Med. 6 (2017) 44.
[11] W. Woo, E.T. Carey, M. Choi, Spotlight on liposomal irinotecan for metastatic
pancreatic cancer: patient selection and perspectives, Onco Targets Ther 12 (2019)
1455–1463.
[12] F.C. Passero Jr., D. Grapsa, K.N. Syrigos, M.W. Saif, The safety and efficacy of
Onivyde (irinotecan liposome injection) for the treatment of metastatic pancreatic
cancer following gemcitabine-based therapy, Expert Rev. Anticancer Ther. 16
(2016) 697–703.
[13] A. Wang-Gillam, C.-P. Li, G. Bodoky, A. Dean, Y.-S. Shan, G. Jameson,
T. Macarulla, K.-H. Lee, D. Cunningham, J.F. Blanc, R.A. Hubner, C.-F. Chiu,
G. Schwartsmann, J.T. Siveke, F. Braiteh, V. Moyo, B. Belanger, N. Dhindsa,
E. Bayever, D.D. Von Hoff, L.-T. Chen, C. Adoo, T. Anderson, J. Asselah,
A. Azambuja, C. Bampton, C.H. Barrios, T. Bekaii-Saab, M. Bohuslav, D. Chang, J.-
S. Chen, Y.-C. Chen, H.J. Choi, I.J. Chung, V. Chung, T. Csoszi, A. Cubillo,
L. DeMarco, M. de Wit, T. Dragovich, W. Edenfield, L.E. Fein, F. Franke, M. Fuchs,
V. Gonzales-Cruz, A. Gozza, R.H. Fernando, R. Iaffaioli, J. Jakesova, Z. Kahan,
M. Karimi, J.S. Kim, E. Korbenfeld, I. Lang, F.-C. Lee, K.-D. Lee, L. Lipton, W.W. Ma,
L. Mangel, R. Mena, D. Palmer, S. Pant, J.O. Park, P. Piacentini, U. Pelzer, J.
G. Plazas, C. Prasad, K.-M. Rau, J.-L. Raoul, D. Richards, P. Ross, L. Schlittler,
M. Smakal, V. Stahalova, C. Sternberg, T. Seufferlein, N. Tebbutt, J.J. Vinholes,
R. Wadlow, M. Wenczl, M. Wong, Nanoliposomal irinotecan with fluorouracil and
folinic acid in metastatic pancreatic cancer after previous gemcitabine-based
therapy (NAPOLI-1): a global, randomised, open-label, phase 3 trial, Lancet 387
(2016) 545–557.
[14] X. Liu, J. Jiang, R. Chan, Y. Ji, J. Lu, Y.P. Liao, M. Okene, J. Lin, P. Lin, C.H. Chang,
X. Wang, I. Tang, E. Zheng, W. Qiu, Z.A. Wainberg, A.E. Nel, H. Meng, Improved
efficacy and reduced toxicity using a custom-designed irinotecan-delivering
silicasome for orthotopic colon cancer, ACS Nano 13 (2019) 38–53.
[15] M. Mashreghi, P. Zamani, S.A. Moosavian, M.R. Jaafari, Anti-epcam aptamer
(Syl3c)-Functionalized liposome for targeted delivery of doxorubicin: in vitro and
in vivo antitumor studies in mice bearing C26 Colon carcinoma, Nanoscale Res.
Lett. 15 (2020) 101.
[16] Z. Li, M. Wu, H. Bai, X. Liu, G. Tang, Light-enhanced hypoxia-responsive
nanoparticles for deep tumor penetration and combined chemo-photodynamic
therapy, Chem. Commun. (Camb.) 54 (2018) 13127–13130.
Colloids and Surfaces B: Biointerfaces 206 (2021) 111967
[17] M. Amin, M. Mansourian, G.A. Koning, A. Badiee, M.R. Jaafari, T.L.M. Ten Hagen,
Development of a novel cyclic RGD peptide for multiple targeting approaches of
liposomes to tumor region, J. Control. Release 220 (2015) 308–315.
[18] M. Faal Maleki, A. Jafari, E. Mirhadi, A. Askarizadeh, B. Golichenari, F. Hadizadeh,
S.M. Jalilzadeh Moghimi, R. Aryan, M. Mashreghi, M.R. Jaafari, Endogenous
stimuli-responsive linkers in nanoliposomal systems for cancer drug targeting, Int.
J. Pharm. 572 (2019), 118716.
[19] Y. Zhou, X. Chen, J. Cao, H. Gao, Overcoming the biological barriers in the tumor
microenvironment for improving drug delivery and efficacy, J. Mater Chem. B 8
(2020) 6765–6781.
[20] Y. Bae, K. Kataoka, Intelligent polymeric micelles from functional poly(ethylene
glycol)-poly(amino acid) block copolymers, Adv. Drug Deliv. Rev. 61 (2009)
768–784.
[21] Q. Zhang, N.R. Ko, J.K. Oh, Recent advances in stimuli-responsive degradable
block copolymer micelles: synthesis and controlled drug delivery applications,
Chem. Commun. (Camb.) 48 (2012) 7542–7552.
[22] Y.E. Gao, S. Bai, X. Shi, M. Hou, X. Ma, T. Zhang, B. Xiao, P. Xue, Y. Kang, Z. Xu,
Irinotecan delivery by unimolecular micelles composed of reduction-responsive
star-like polymeric prodrug with high drug loading for enhanced cancer therapy,
Colloids Surf. B Biointerfaces 170 (2018) 488–496.
[23] E. Mirhadi, M. Mashreghi, M. Faal Maleki, S.H. Alavizadeh, L. Arabi, A. Badiee, M.
R. Jaafari, Redox-sensitive nanoscale drug delivery systems for cancer treatment,
Int. J. Pharm. 589 (2020), 119882.
[24] Y. Du, W. He, W. Zhou, X. Li, Disulfide phosphatidylcholines: alternative
phospholipids for the preparation of functional liposomes, Chem. Commun.
(Camb.) 55 (2019) 8434–8437.
[25] Y. Du, Z. Wang, T. Wang, W. He, W. Zhou, M. Li, C. Yao, X. Li, Improved antitumor
activity of novel redox-responsive paclitaxel-encapsulated liposomes based on
disulfide phosphatidylcholine, Mol. Pharm. 17 (2020) 262–273.
[26] X. Peng, Q. Pan, J. Li, W. Zhu, N. Zhang, Y. Pu, K. Luo, B. He, Polymer-directed
supramolecular assembly of photosensitizers: evocation of photothermal effect and
highly efficient loading of disulfiram for chemo-phototherapy of cancer, Appl.
Mater. Today 22 (2021).
[27] Y. Du, W. He, Q. Xia, W. Zhou, C. Yao, X. Li, Thioether phosphatidylcholine
liposomes: a novel ROS-Responsive platform for drug delivery, ACS Appl. Mater.
Interfaces 11 (2019) 37411–37420.
[28] D.C. Drummond, C.O. Noble, Z. Guo, K. Hong, J.W. Park, D.B. Kirpotin,
Development of a highly active nanoliposomal irinotecan using a novel
intraliposomal stabilization strategy, Cancer Res. 66 (2006) 3271–3277.
[29] Z. Wang, D. Chi, X. Wu, Y. Wang, X. Lin, Z. Xu, H. Liu, J. Sun, Z. He, Y. Wang,
Tyrosine modified irinotecan-loaded liposomes capable of simultaneously targeting
LAT1 and ATB(0,+) for efficient tumor therapy, J. Control. Release 316 (2019)
22–33.
[30] J. Liu, D. Chi, S. Pan, L. Zhao, X. Wang, D. Wang, Y. Wang, Effective co-
encapsulation of doxorubicin and irinotecan for synergistic therapy using
liposomes prepared with triethylammonium sucrose octasulfate as drug trapping
agent, Int. J. Pharm. 557 (2019) 264–272.
[31] S. Mura, J. Nicolas, P. Couvreur, Stimuli-responsive nanocarriers for drug delivery,
Nat. Mater. 12 (2013) 991–1003.
[32] Q. Pan, X. Peng, J.-E. Cun, J. Li, Y. Pu, B. He, In-situ drug generation and
controllable loading: rational design of copper-based nanosystems for chemo-
photothermal cancer therapy, Chem. Eng. J. 409 (2021).