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-

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

* Corresponding author.
E-mail address: [email protected] (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. Colloids and Surfaces B: Biointerfaces 206 (2021) 111967

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

Scheme 1. In vivo Mechanism of Redox-Sensitive IR-Loaded Liposomes.

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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

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

irinotecan. 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
2.5. Evaluation of redox-sensitivity wash and detach cells. After centrifugation (1500 rpm, 10 min), cells
were collected and resuspended in buffer. Then, PI (2 μL) and Annexin
TEM and DLS were used to studied the redox sensitivity of IR/SS-LP. V-FITC (2 μL) reagents were added to stain cells. Ultimately, the samples
IR/SS30-LP was exposure to reducing condition of PBS containing 10 were measured by flow cytometer.
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 2.10. Pharmacokinetic study
described in section 2.4 [25].
The pharmacokinetic study of IR/SS-LP in SD rats was evaluated by
2.6. In vitro drug release intravenous injection of IR/SS-LPs with equivalent irinotecan dose of 10
mg/kg using free irinotecan as a control. Blood samples were collected
The In vitro release of irinotecan from IR/SS-LP was researched using from the orbital vein at specific time intervals and stored in heparin
high performance liquid chromatography (HPLC). PBS solution (pH 7.4) sodium tubes. Blood samples were centrifuged at 3000 rpm (10 min, 4
with 1 mM or 0.1 mM GSH was used as the release medium. Meanwhile,
◦
C) to obtain plasma. After that, 200 μL methanol was added to the
free irinotecan and IR/LP (ONIVYDE®-like) were used as controls. The supernatant to extract the drug by centrifuged at 10,000 rpm (10 min, 4
release of IR/SS-LP was carried out in a regenerated cellulose dialysis
◦
C). Then, the supernatant was absorbed, dried with nitrogen, and then
bag (MWCO 8000), which was completely immersed in 200 mL of dissolved in methanol (100 μL). The concentration of SN38, the main
release medium while properly stirring at 37 ◦ C. 1 mL of the external metabolite product of IR, was determined by HPLC at 368 nm. Finally,
release medium was collected at predetermined time point while an pharmacokinetic parameters of groups were calculated by DAS software.
equal volume of fresh release medium was supplemented. The release
rate was calculated by the data which was derived from sample 2.11. Anti-tumor activity
measured by HPLC.
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
2.7. Cytotoxicity test
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
First, human breast adenocarcinoma cell line (MCF-7) and human
injected at an equivalent IR dose of 5 mg/kg every 3 days. Tumor vol­
lung carcinoma cell line (A549) were used to evaluate the cytotoxicity of
ume and body weight were measured every two days. After 21 days, all
SS-PC. 96-well plates were used to seed cells at density of 1 × 104 cells/
mice were sacrificed while the final tumors were weighed and
well. Then, cells were incubated in media containing control DSPC li­
cryopreserved.
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
2.12. Statistical analysis
atmosphere for 1, 4, or 7 days while culture fluid was refreshed every 2
days.
Statistical analyses were performed using SPSS (Statistics 25, IBM).
Secondly, the in vitro cytotoxicity of IR/SS-LPs was tested by using
All results were presented as means ± standard deviations (SD). One-
A549 or MCF-7 cell lines. Free irinotecan and IR/LP were used as con­
way analysis of variance (ANOVA) was employed in data comparisons.
trols. The cells seeded in 96-well plates were incubated in the media
P < 0.05 means a statistically significant difference and P < 0.01 means
containing different irinotecan liposomes. After that, the medium was
a statistically high significant difference.
replaced by 20 μL of MTT solution with a 4 h incubation. Ultimately
MTT was replaced with 100 μL of DMSO. Spectrophotometer (Thermo
3. Results and discussion
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
3.1. Preparation and characterization of SS-LP
according to the following formula:

Cell viability(%)= (ODA-ODB) / (ODC-ODB) ×100% Disulfide phosphatidylcholine (SS-PC) with the structure as shown in
Fig. 1a was used in the formulation of the liposomes. It’s phase transition
where, ODA is the OD value of free irinotecan, IR/LP and IR/SS-LPs temperature (Tc = 58 ◦ C) is close to that of DSPC (55 ◦ C). The SS-PC
treated cells, ODB is the OD value of DMSO while ODC is the OD value based liposomes (SS-LP) were prepared using an ethanol injection
of blank medium cultured cells without any treatment. method followed by mini extrusion. After that, irinotecan (IR) was
actively loaded into the liposomes by triethylammonium sucrose octa­
2.8. Cellular uptake analysis 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.
Confocal laser scanning microscope (CLSM) was used to check DLS was used to measure the size and zeta potential of the liposomes.
cellular uptake. Briefly, MCF-7 cells were seeded in 12-well (3 × 105 As listed in Table 2, the IR-loaded liposomes have an average size of
cells/well) and incubated for 24 h. After that, the cells incubated in about 120 nm. The PDI is about 0.1, which confirms the liposomes are
media containing IR, IR/LP, or IR/SS30-LP with the concentration of relatively uniform with a narrow distribution (Fig. 1c.). The Zeta po­
irinotecan 5 mM for 6 h. The cells were fixed with 4 % para­ tentials are all negative, which indicates the liposomes have strong
formaldehyde for 30 min after washed with cold PBS buffer twice. The stability. The liposomal morphology of IR/SS30-LP was characterized by
cells were observed by CLSM (Olympus, Japan) after washed three times using cryo-TEM and TEM. According to Fig. 1c, they display a uni­
with PBS. lamellar vesicle structure (ULV) with a particle size of about 100 nm.
DLS was further used to demonstrate the excellent storage stability of
2.9. Apoptosis analysis the liposomes since no significant change of the size was detected after
storage for 28 days at 4 ◦ C (Fig. 1d.).
The apoptosis of MCF-7 cells was investigated by using flow The EE and DL capacity of IR/SS30-LP and IR/LP (ONIVYDE®-like)
cytometry (BD Accuri C6, Bedford, MA). In brief, cells were seeded into were detected using size exclusion chromatography method. The EE and
6-well plates at density of 2 × 105 cells/well. Next, cells incubated in DL of IR/LP were calculated to be 31.7 ± 0.1 % and 98.1 ± 0.8 %, while

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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

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

The high concentration of reductive substances in tumor sites is 3.5. Analysis of cellular apoptosis
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 Cell apoptosis was detected by flow cytometry to investigate the
report, the intracellular reduction environment was simulated by 10 mM cytotoxic mechanism of the liposomes. Annexin V-FITC/PI kit was used
GSH solution. And, DLS and TEM was used for checking the to stain and identify apoptotic MCF-7 cells. As shown in Fig. 2b,
redox-responsive phenomenon of IR/SS-LP. apoptotic cells were found at Q3 and Q2 phases, while normal cells were
Obviously, the evidence of the destruction of IR/SS-LP after GSH found at Q4. Accordingly, the total percentage of apoptotic cells in IR/
treatment for 2 h was obtained by transmission electron microscopy SS30-LP group was 49.7 %, and 38.0 % in IR/LP group, while the free
(TEM) (Fig. 1f). Compared with before treatment, IR/SS-LP lost the IR group has a value of 23.8 %. The results indicate that both liposomal
spherical morphology and presented irregular black block structure formulations have significantly improved cell apoptosis compared to
(Fig. 1g). These black blocks may be a mixture of aggregates including free IR. This finding may be explained by the fact that the liposome
lipids and residual irinotecan-SOS gels. In addition, the particle size structure prevents active structure of irinotecan from destroyed in the
measured by using DLS also shown dramatically change. According to low pH microenvironment of tumor cells. Therefore, in both the
Fig. 1e, size distribution converted from a single peak (before GSH apoptosis assays and cytotoxicity, IR/SS30-LP has displayed the best,
treatment) to multiple individual peaks after GSH treatment (Fig. 1e). mainly owing to the disulfide breakage induced liposomal structure
The peak of big size shown in Fig. 1e (~1000 nm) may be ascribed to the disassociation.
large black lump that appears in the TEM image (Fig. 1g). The released
irinotecan gels aggregate to form larger, noncompact clusters. While the 3.6. In vitro cytotoxicity
signal peak less than 100 nm may be derived from the debris of the
destroyed liposomes. In summary, IR/SS-LP was specifically manifested Firstly, we tested the toxicity of SS-PC as a component of SS-LP li­
as redox-responsive release carrier of irinotecan in a reduced microen­ posomes by MTT assay. Cytotoxicity of SS30-LP was checked against
vironment due to the disulfide breakage of SS-PC inducing gradual A549 cells and MCF-7 cells. As a comparison, GSH pretreated cancer
collapse followed by complete destruction of liposomal structure. 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
3.3. In vitro release of irinotecan 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 drug release from IR/SS-LP with different SS-PC content was The data proved that SS-LP as well as its degradation products had no
monitored by dialysis. As shown in Fig. 1h, only 18.2 % irinotecan was toxic effect on cells.
released from IR/SS100-LP within 24 h in PBS at pH 7.4, while 19.3 % After IR loading, the in vitro cytotoxicity of IR/SS-LP with different
irinotecan was released from IR/LP. The results indicated that the sta­ SS-PC content was detected against A549 and MCF-7 cell lines for 24 h.
bility of IR/SS-LP in PBS is comparable to that of IR/LP. Under the As can be seen from the Fig. 3c-d, the cytotoxicity of the liposomes was
condition of 1 mM GSH of PBS (pH 7.4), the final release rates of IR/SS- significantly enhanced with the increasing of SS-PC ratio. While the IR
LP with 0%, 15 %, 30 %, 50 % and 100 % SS-PC content were 18.2 %, concentration was 100 μg/mL, the cytotoxicity of IR/SS100-LP against
30.3 %, 60.2 %, 67.8 % and 77.1 %, respectively. Apparently, with the MCF-7 and A549 reached 69.54 % and 64.11 %, compared to 53.22 %
increase of SS-PC content, the release rate of IR increased significantly. and 49.8 % of IR/LP. Notably, the cytotoxicity of IR/SS50-LP against
Therefore, the release of irinotecan from IR/SS-LP was attributed to the MCF-7 and A549 reached 62.1 % and 61.86 % respectively, compared to
redox-reaction breakage of SS-PC inducing liposome destruction. And, 59.36 % and 58.0 % of IR/SS30-LP. But, there was no significant dif­
the increase of SS-PC content accelerated the drug release of IR/SS-LP in ference between the latter two groups (P > 0.05). In other words, both
a dose-dependent manner with an appropriate release curve at 30 %. IR/SS50-LP and IR/SS30-LP could have similar in vitro anti-tumor

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

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

6
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 CRediT authorship contribution statement
(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 Xinsong Li, Tao Wang, Wei He and Yawei Du designed the study.
the three experimental groups was lower than that of the normal saline Tao Wang designed and performed the main experiment. The manu­
group (2.33 ± 0.33 g). An extremely significant difference was observed script was written by Tao Wang, Yawei Du and Xinsong Li. Tao Wang,
between the IR/LP group and the free IR group (P < 0.01), while there Wei He, Yawei Du, and Ji Wang participated in the data analysis and
was distinct difference between IR/LP and IR/SS30-LP group (P < 0.05). manuscript writing.
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 Declaration of Competing Interest
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 The authors report no declarations of interest.
tumor-bearing tumors (Fig. 4d) make these results more visual. There­
fore, IR/SS30-LP had improved anti-tumor efficiency in vivo compared Acknowledgments
with free IR and IR/LP.
This research was supported by the Major National Science and
4. Conclusion Technology Program of China for Innovative Drug (2017ZX09101002-
001-004).
In summary, the irinotecan loaded liposomes based on SS-PC were
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