Self-stabilized Pt(IV) amphiphiles by precise regulation of branch length for enhanced chemotherapy

Article abstract of DOI:10.1016/j.ijpharm.2021.120923

A surge of platinum(IV) compounds are utilized or investigated in cancer treatment but their therapeutic outcomes have been greatly compromised by remaining adverse effects and limited antitumor performance, attributable to nonspecific distribution and insufficient activation in tumor site. Herein, we designed a series of disulfide bond introduced Pt(IV)-lipid prodrugs with different branch length, all of which are able to self-stabilize into nanomedicine and be activated by high intracellular glutathione (GSH) level. The impact of precise modification of these prodrugs on their assembly stability, pharmacokinetics and cytotoxicity was probed to establish a connection between chemical structure and antiproliferation efficiency. With optimal assembly manner and delivery efficacy, the longest axial branched Pt(IV) prodrug CSS18 exhibited the most impressive therapeutic outcome, providing a potential path to more efficient nanocarriers for chemotherapeutic agents by chemical modulation and, giving insights into the rational design of reduction responsive platinum delivery system.

Full text of DOI:10.1016/j.ijpharm.2021.120923

International Journal of Pharmaceutics 606 (2021) 120923
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Self-stabilized Pt(IV) amphiphiles by precise regulation of branch length for
enhanced chemotherapy
,
*
Xiao Kuanga, Yuting Hua, Dongxu Chia, Haolin Zhanga, Zhonggui Hea, Yiguo Jiangb
,
,
*
Yongjun Wanga
a Department of Pharmaceutics, Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, China
b Department of Pharmacy, The Affiliated Suzhou Science & Technology Town Hospital of Nanjing Medical University, Suzhou 215153, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A surge of platinum(IV) compounds are utilized or investigated in cancer treatment but their therapeutic out-
comes have been greatly compromised by remaining adverse effects and limited antitumor performance,
attributable to nonspecific distribution and insufficient activation in tumor site. Herein, we designed a series of
disulfide bond introduced Pt(IV)-lipid prodrugs with different branch length, all of which are able to self-stabilize
into nanomedicine and be activated by high intracellular glutathione (GSH) level. The impact of precise
modification of these prodrugs on their assembly stability, pharmacokinetics and cytotoxicity was probed to
establish a connection between chemical structure and antiproliferation efficiency. With optimal assembly
manner and delivery efficacy, the longest axial branched Pt(IV) prodrug CSS18 exhibited the most impressive
therapeutic outcome, providing a potential path to more efficient nanocarriers for chemotherapeutic agents by
chemical modulation and, giving insights into the rational design of reduction responsive platinum delivery
system.
Disulfide bond
Pt(IV) prodrug
Self-stabilization
Chemical modification
Antitumor performance
1. Introduction
most successful oral Pt drug in clinical trials, with a better toxicity
profile than cisplatin, showed impressive in vitro activity against
Platinum(IV) complexes have aroused great interests in the field of
chemotherapy for cancer treatment in the past few decades (Rottenberg
et al., 2021; Wang and Lippard, 2005; Zajac et al., 2020; Wang et al.,
2019). Compared with parent Pt(II) compounds, Pt(IV) prodrugs are
more kinetically inert to ligand substitution before reduction, making it
possible for platinum drugs to lower the rate of aquation and subsequent
coordination with DNA inside cells. These unique properties make it
possible for platinum candidates to improve their toxicology manners,
which helps to attenuate side effects (Rabik and Dolan, 2007; Hartmann
and Lipp, 2003; Pan et al., 2020). The alleviation of dose-limiting
toxicity also increases patient tolerance and is expected to exert better
antiproliferation efficacy against various tumor by virtue of elevated
dosage of Pt drugs. However, the outcomes of Pt(IV) conjugates involved
in clinical trials were far from satisfactory as none of them touched the
final phase and entered the market. Most of them did not show enhanced
antitumor efficiency over conventional Pt(II) drugs (cisplatin and car-
boplatin), presumably deriving from poor specific accumulation and
insufficient DNA-crosslinked activity. Among them, satraplatin, the
cisplatin-resistant tumor cells (Doshi et al., 2012; Choy et al., 2008).
Despite the relatively positive outcome of the Phase III trial, satraplatin
still failed to obtain FDA approval for no significantly improved overall
survival (Kelland, 2007). Selective tumor accumulation and activation
are believed to be the pivotal factors of rationality designing Pt(IV)
prodrugs to enable appreciable activity against malignancies.
Nanotechnology has been prevailing in application of drug delivery
and regenerative medicine, endowing chemotherapeutics with good
compliance, prolonged blood circulation and preferable accumulation in
tumor (Shi et al., 2010; Luo et al., 2014), which seems to be capable of
addressing the dilemma encountered by Pt(IV) prodrugs. Nonetheless,
deficiency in drug loading capacity (DLC) and drawbacks originated
from great utilization of excipients have greatly hindered the develop-
ment of nanomedicine (Shi et al., 2010). Self-assembled prodrug nano-
carriers take advantages of nanotech and achieve high DLC with
minimum excipient employment, providing new insights into rational
design of Pt delivery system. As previously investigated by us (Luo et al.,
2014; Kuang et al., 2021; Wang et al., 2014), self-assembled
* Corresponding authors.
E-mail addresses: jiangyiguo0515@126.com (Y. Jiang), wangyongjun@syphu.edu.cn (Y. Wang).
https://doi.org/10.1016/j.ijpharm.2021.120923
Received 23 April 2021; Received in revised form 4 July 2021; Accepted 20 July 2021
Available online 23 July 2021
0378-5173/© 2021 Elsevier B.V. All rights reserved.

X. Kuang et al.
International Journal of Pharmaceutics 606 (2021) 120923
Fig. 1. Schematic illustration of self-stabilized Pt(IV) NPs for specific delivery of carboplatin and suppression of tumors. Disulfide introduced Pt(IV)-lipid prodrugs
with different length of fatty tails can be activated by high intracellular GSH to enable antitumor performance of nanomedicine.
nanoparticles (NPs) are able to load much more therapeutic agents than
other kinds of nanomedicine characterized with drug entrapment in
hydrophobic cores, and some homodimeric prodrug nanoassemblies
hit>65% of DLC (Yang et al., 2020; Zuo et al., 2020). The reported self-
vessels (Lee et al., 2013). Selective activation in the targeted spot con-
ducted by responsive bond may be one answer to addressing the dis-
counted antitumor performance of Pt(IV) candidates (Reshetnikov et al.,
2018; Yang et al., 2020).
assembly mechanism of most prodrugs depend on
π-
π
stacking domi-
Chemical structure of self-assembled prodrug exercises a decisive
influence on assembly stability, consequent in vivo fate and resulting
antiproliferation performance (Wang et al., 2015). The interactions
between self-assembled nanomedicine and biocomponents such as
proteins, phospholipid bilayers, nucleic acids and organelles, are
necessary to be explored for awareness of drug disposal after adminis-
tration. The colloidal chemistry of nanoparticles has a great impact on
their destiny when encountering these biocomponents to form in-
terfaces. Probing these interfaces promotes the predictive relationships
between prodrug structure and therapeutic activity that are bridged by
surface properties such as shape, size and outer coatings. For common
nanomedicine, drug–carrier compatibility can be tuned by modifying
carrier materials (Shi et al., 2015). It is simplified for self-assembled NPs
here to focus on the rationality of prodrug design on the basis of phys-
icochemical interaction. The “structure-assembly-activity” profile need
to be established to endow self-assembled prodrug NPs with preferable
drug accumulation and selective activation.
nated van der Waals interaction (Cheetham et al., 2017), for which it is
challenging to design platinum-based assembled nanomedicine if no
π
e-
exists. Strong lipophobicity of platinum makes it difficult to construct
stable Pt encapsulated nanocarriers due to precipitation tendency be-
tween heavy atoms (Rottenberg et al., 2021; Wang et al., 2019). Some Pt
(IV)-lipid conjugates can transform into nanoparticles in aqueous solu-
tion but the procedure need the help of surfactants with large proportion
(>60% w/w) (Chen et al., 2018; Ling et al., 2019) which is more like
emulsification rather than self-assembly. The difficulty in preparation of
Pt(IV) self-stabilized NPs impedes implement of high platinum loading
capability. Stabilized and controllable assembly behavior is required for
Pt(IV) NPs to enable enough tumor accumulation.
Insufficient activation is another drawback compromising thera-
peutic outcomes of Pt(IV) prodrugs. Stimulus responsive nanomedicine
focus on the distinctive features of cancer cells or tumor microenvi-
ronment, augmenting activation on corresponding target and mini-
mizing undesired effects in normal tissues. A typical product approved
for sale is Mylotarg®, the first antibody-drug conjugate (ADC) on the
market, covering a reduction responsive linker of disulfide bond (Damle
and Frost, 2003). Disulfide cleavage is GSH-consuming and accelerated
in cytoplasm where GSH level is 1000-fold higher than that of plasma
(Schafer and Buettner, 2001; Brülisauer et al., 2014). The introduction
of disulfide bond to specific prodrug has been proved a trigger for
explosive drug release in tumor cells (Sun et al., 2018; Luo et al., 2016)
but a harness of chemotherapeutics for prevention from leakage inside
To deal with poor tumor accumulation and insufficient activation of
Pt(IV) prodrugs, a series of disulfide bond introduced Pt(IV)-lipid am-
phiphiles with various branch length were prepared in this research.
Carboplatin was chosen as model drug for relatively good water solu-
bility (17 mg/mL) and carbon chains with different length as axial li-
gands to yield Pt(IV) amphiphiles (CSS6, CS12, CSS18, C-18 and
mCSS18). These conjugates assembled stably in water with minimum
PEGylation (15%) to produce uniform spherical NPs, namely self-
stabilized Pt(IV) NPs, which rapidly disintegrated in high intracellular
2

X. Kuang et al.
International Journal of Pharmaceutics 606 (2021) 120923
GSH due to disulfide cleavage. (Fig. 1) The impacts of disulfide inserted
branch length on assembly stabilization, pharmacokinetic manner and
intracellular activation were probed to optimize drug–carrier compati-
bility for enhanced delivery efficacy. We found that the most hydro-
phobic conjugate of all, CSS18, exhibited the best self-stabilized
capacity, highest tumor accumulation and consequent antitumor effi-
ciency. Overall delivery benefits were ranked as: CSS18 > CSS12 >
mCSS18 (equivalent to CSS9) > CSS6 that showed evident correlation to
branch length. These results indicate a drug-carrier association that
connect precise chemical constitution and delivery efficacy, providing
insights into the rational design of Pt(IV) delivery system.
DMAP, followed by continuous stirring for 12 h. Then 1 mmol EDCI was
added to the reaction solution to activate carboxyl group for 20 min.
And 0.8 mmol 1-Octadecanol was added to the reaction mixture and the
solution was stirred overnight. The resulting solution was filtered,
evaporated, and added to 50 mL diethyl ether. The precipitate was
collected, washed with diethyl ether, and dried under vacuum. mCSS18
(yield: 55.2%).
2.3. Self-stabilization of Pt(IV) NPs
Moderate amount of Pt(IV) prodrug (CSS6, CSS12, CSS18, C-18 or
mCSS18) were dispersed in 0.2 mL 0.35% DSPE-PEG2000 ethanol solu-
tion, followed by pipetting this mixture of prodrug and DSPE-PEG2000
dropwise to stirring deionized water with agitation of 600 rpm. Then
residual ethanol of all formulations were removed through rotar-
y evaporation under vacuum after 20 min agitation to yield self-
stabilized Pt(IV) NPs. CSS6, CSS12, CSS18, C-18 and mCSS18 NPs at
final equivalent Pt concentration of 0.5, 1.0, 1.5 mg/mL were prepared
respectively and CSS18 NPs of 3 mg Pt/mL were also made with the
same procedure. All Pt(IV) formulations were characterized by particle
size, PDI and zeta potential with DLS. Typical photos of TEM were taken
to indicate the morphology of NPs. And drug loading capacity was
determined with inductively coupled plasma mass spectrometry (ICP-
MS). Methods of sample preparation before measurement by ICP-MS
were provided from Sci-Tech innovation Co. Ltd (Qingdao, China). We
also evaluate the formation of colloid system via nanoprecipitation
method in this process with a light beam passing through Pt(IV) NPs to
confirm Tyndall effect.
2. Materials and methods
2.1. Materials
Carboplatin (Car) was purchased from Meilun (Dalian, China). H2O2,
2,2′-disulfanediyldiacetic acid, acetic anhydride (Ac2O), adipic anhy-
dride, 1-Hexanol, 1-Dodecanol and 1-Octadecanol were purchased from
Aladdin (Shanghai, China). 1-Ethyl-3-(3-dimethylaminopropyl)-carbo-
diimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP)
were obtained from Chemlin Pharm Co. Ltd. (Nanjing, China). DSPE-
PEG2000 were obtained from AVT (Shanghai) Pharmaceutical Co., Ltd.
Glutathione (GSH), GSH and GSSG assay kit were procured from
Solarbio Science & Technology Co., Ltd (Beijing, China). ELISA kit and
BCA protein assay kit was purchased from Neobioscience Technology
Co, Ltd (Beijing, China).
2.2. Synthesis of Pt(IV) prodrugs
2.4. Colloidal stability of Pt(IV) NPs
GSH-sensitive octahedrally coordinated carboplatin were synthe-
sized by coupling oxidized carboplatin and aliphatic alcohol with di-
sulfide linkage. 1-Hexanol, 1-Dodecanol and 1-Octadecanol were chosen
as aliphatic branches of disulfide-linked Pt(IV) prodrugs (abbreviated as
CSS6, CSS12, CSS18 respectively). And 1-Octadecanol was also grafted
onto carboplatin with a hexanedioic acid-linkage to yield non-sensitive
Pt(IV) prodrug (donated as C-18). Monocoordinated GSH-responsive
carboplatin with 1-Octadecanol tethered was synthesized via similar
method and described as mCSS18. Synthesis route and all experiment
details were shown in support information. Chemical structures of all
yield compounds were confirmed by MS (mass spectrum) and 1H NMR
(nuclear magnetic resonance).
To probe the colloidal stability of all prodrug nanoparticles, 1 mL
NPs sample was added to 20 mL PBS 7.4, containing 10% of fetal bovine
serum (FBS). The mixtures were incubated at 37 ◦C with gentle shaking.
At prescriptive intervals (0, 2, 4, 6, 8, 12, 24, 36, 48 h), the particle size
was measured (n = 3 for each group). Meanwhile, 1 mL NPs was also
added to 20 mL 10 mM PBS 7.4 and size of NPs were measured by
Zetasizer (n = 3 for each group) within 7 days.
2.5. In vitro drug release
In vitro Pt release profile of various Pt(IV) NPs was investigated in 10
mM PBS 7.4 in presence of 1 mM and 10 mM GSH. Briefly, dialysis bag of
MWCO 1000 D containing 1 mL Pt(IV) NPs was submerged into 20 mL
release media which was placed under 37 ◦C and vibration at 100 rpm.
At pre-programmed intervals, 1, 2, 4, 8, 12 and 24 h, 1 mL media was
taken out to be analyzed by ICP-MS to quantify Pt content released from
NPs.
Briefly, CSS6, CSS12 and CSS18 were all prepared in a similar pro-
cess. Briefly, 0.5 mmol Car(IV)–2OH was dispersed in 20 mL DMF at
35 ◦C with addition of DMF solution of 1.2 mmol 1,4,5-oxadithiepane-
2,7-dione and 0.2 mmol DMAP, followed by continuous stirring for
12 h. Then 1 mmol EDCI was added to the reaction solution to activate
carboxyl group for 20 min. And 1.5 mmol appropriate aliphatic alcohol
(1-Hexanol, 1-Dodecanol or 1-Octadecanol) was added to the reaction
mixture and the solution was stirred overnight. The resulting solution
was filtered, evaporated to 2 mL, and added to 50 mL diethyl ether for
precipitation. The deposition was collected, washed with diethyl ether,
and dried under vacuum. The final products were weight for calculation
of yields (CSS6: 56.1%, CSS12: 37.2% and CSS18: 40.3%). C-18 was
synthesized by following procedures: 0.5 mmol Car(IV)–2OH was
dispersed in 20 mL DMF at 40 ◦C with addition of DMF solution of 1.2
mmol adipic anhydride and 0.2 mmol DMAP, followed by continuous
stirring for 12 h. Then 1 mmol EDCI was added to the reaction solution
to activate carboxyl group for 20 min. And 1.4 mmol 1-Octadecanol was
added to the reaction mixture and the solution was stirred overnight.
The resulting solution was filtered, evaporated, and added to diethyl
ether of large volume. The precipitate was collected, washed with
diethyl ether, and dried under vacuum to yield pale yellow solid of C-18
(yield: 59.6%). mCSS18 was prepared in following process: 0.5 mmol
Car(IV)–2OH was dispersed in 20 mL DMF at 35 ◦C with addition of DMF
solution of 0.6 mmol 1,4,5-oxadithiepane-2,7-dione and 0.2 mmol
2.6. Cell culture
Mouse breast cancer 4T1 cells, mouse melanoma B16-F10 cells,
human ovarian carcinoma A2780 cells, and human fetal hepatocyte LO2
cells were obtained from Cell Resource Center, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences. 4T1, A2780 and LO2
cells were cultured in routine medium consisted of 90% RPMI 1640,
10% FBS. B16-F10 cells were cultured in routine medium consisted of
90% DMEM, 10% FBS, penicillin (30 mg/mL) and streptomycin (100
μ
g/mL). All cells were cultured at 37 ◦C in 5% CO2 atmosphere.
2.7. Intracellular Pt accumulation
The level of cellular internalization was tested by treating 4T1 cells
with Pt(IV) prodrugs and nanoparticles at equivalent concentration of
10 μg Pt/ml. Briefly, 4T1 cells were cultured in 12-well plates with a
density of 1 × 105 cells/well for 24 h and then immersed with 10
μg Pt/
3

X. Kuang et al.
International Journal of Pharmaceutics 606 (2021) 120923
Fig. 2. (A) Synthesis route of disulfide bond bridged conjugate of carboplatin and different fatty chains. (a) 50 ℃, 3 h, H2O; (b) 25 ℃, 2 h; (c) EDCI, DMAP, 45 ℃, 24
h, DMF; (d) EDCI, DMAP, 30 ℃, 12 h, DMF. (B) Typical TEM images of CSS18 NPs. Scale bar: 200 nm. (C) The appearance of CSS18 NPs at 1.5 mg Pt/mL. (D) Particle
size distribution of CSS18, CSS12, CSS6, C-18 and mCSS18 NPs. (E) Typical images of CSS18 (1), CSS12 (2), CSS6 (3), C-18 (4), mCSS18 (5) NPs with concentration of
1.5 mg Pt/mL and CSS18 NPs at 3 mg Pt/mL (6). (F) Tyndall Effect occurred in Pt(IV) NPs prepared with nanoprecipitation method: CSS18 (1), CSS12 (2), CSS6 (3),
C-18 (4), mCSS18 (5).
mL carboplatin, CSS6, CSS12, CSS18, C-18 and mCSS18 NPs (or pro-
drugs) for 1 or 4 h. Ultimately, cells were dissociated and digested
overnight for Pt detection by ICP-MS. And cells quantitation were car-
ried out by BCA protein assay kit (Neobioscience Technology Co, Ltd.).
B16-F10 and A2780 cells were seeded in 96-well plates at a density of 2
× 103 cells/well (3 × 103 cells/well for LO2) for 12 h, followed by
treatment with serial dilution of CSS6, CSS12, CSS18, C-18, mCSS18 NPs
and free carboplatin. The cells and preparations were further co-
incubated for 48 h prior to the addition of 20 μl MTT solution (5 mg/
mL). After another 4 h incubation, cells were washed with PBS and 200
l DMSO was added for evaluation of cell viability by microplate reader
at 490 nm.
2.8. Cytotoxicity assay
μ
The In vitro Cytotoxicity of all Pt(IV) NPs against 4T1, B16-F10,
A2780 and LO2 cells were assessed by MTT assay. Typically, 4T1,
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X. Kuang et al.
International Journal of Pharmaceutics 606 (2021) 120923
2.9. DNA-Pt adduct
Table 1
Characterization of Pt(IV) NPs (0.5 mg Pt/mL) determined by dynamic light
scattering (DLS).
4T1 cells were cultured as above-mentioned in 6-well plates for 24 h,
then 100 μM Pt equivalence of different nanossemblies were added.
Pt(IV) NPs
Size (nm)
PDI
Zeta (mV)
After 12 h incubation, the nucleus DNA of cells were extracted by
mammalian genomic DNA extraction kit (NEST Biotechnology) and
digested with nitric acid at 80 ◦C overnight. The Pt content was deter-
mined by ICP-MS.
CSS6
150.4 ± 21.53
102.4 ± 3.108
84.39 ± 1.054
87.53 ± 4.805
117.3 ± 4.513
0.409 ± 0.030
0.113 ± 0.033
0.132 ± 0.022
0.193 ± 0.032
0.290 ± 0.024
ꢀ 19.1 ± 1.501
ꢀ 25.4 ± 1.992
–22.5 ± 1.768
ꢀ 29.7 ± 2.326
ꢀ 14.6 ± 1.147
CSS12
CSS18
C-18
mCSS18
2.10. Animals
2.14. Statistical analysis
All animals in this research were supplied by the Laboratory Animal
Center of Shenyang Pharmaceutical University (Shenyang, Liaoning,
China). All associated animal experiments were carried out according to
the Guidelines for the Care and Use of Laboratory Animals approved by the
Institutional Animal Ethical Care Committee (IAEC)ofShenyang Phar-
maceutical University.
All the quantitative data are described using the mean ± SD (stan-
dard deviations), and statistical analysis was performed with Student’s t-
test and one-way ANOVA. p < 0.05 was considered statistically signif-
icant (* p < 0.05, ** p < 0.01, *** p < 0.001.).
3. Results and discussion
2.11. In vivo pharmacokinetic
3.1. Synthesis of Pt(IV) prodrugs
Male Sprague ꢀ Dawley rats (200–250 g) were used to evaluate the
pharmacokinetic profiles of Pt(IV) NPs. Prior to the experiments, the rats
were fasted for 12 h. The animals were intravenously administered
carboplatin solution, CSS6, CSS12, CSS18, C-18 and mCSS18 NPs at 3
mg Pt/kg (n = 5 for each group). At the predetermined time points (15
min, 1, 4, 8, 12 h), blood samples were collected and then centrifuged to
obtain the plasma. Nitric acid (HNO3, 65%, suprapure) was added into
the plasma and allowed them to digest overnight. Then Pt content were
measured by ICP-MS.
We developed various carboplatin prodrugs with different axial
disulfide-based ligands with synthetic routes shown in Fig. 2A. Both
axial orientations of platinum atom were modified by symmetrical di-
sulfide bond-inserted aliphatic chains that differs from each other in
carbon chain length (CSS6. CSS12 and CSS18). No disulfide inserted Pt
(IV) prodrug (C-18) and asymmetrically monoaxial carboplatin prodrug
(mCSS18) were also prepared. The detailed synthetic approaches of all
these amphiphilic Pt(IV) compounds were shown in Support Informa-
tion. The precise structure of Pt(IV) prodrugs were confirmed by MS and
1H NMR as shown in Figure S1-S5.
2.12. In vivo biodistribution
The in vivo distribution profiles of Pt(IV) NPs into tumor and oth-
er organs were assessed in 4T1 tumor-bearing Balb/c mice (female, n =
3). Mice with subcutaneous tumors of approximate 300 mm3 were
subjected to tail vein injection of carboplatin solution, CSS6, CSS12,
CSS18, C-18 and mCSS18 NPs at 5 mg Pt/kg. At 4 and 12 h after dosing,
mice were euthanized followed by the organs (heart, liver, spleen, lung
and kidney) and tumors harvested. Then organs and tumors were
washed in the saline, weighted and sheared by a tissue homogenizer.
Then the tissue homogenates were digested by nitric acid (HNO3, 65%,
suprapure) overnight. Pt content in tissues and tumors were analyzed by
ICP-MS.
3.2. Self-stabilization of Pt(IV) NPs
Pt(IV) nanoparticles were fabricated with nanoprecipitation we
previously reported (Yang et al., 2020; Sun et al., 2019). Typical images
of all NPs showed spherical morphology in Fig. 2B and S6A-C. As shown
in Table 1 and Fig. 2D, contrary to free carboplatin (Car), all amphiphilic
Pt(IV) conjugates are able to assemble in H2O at the concentration of 0.5
mg Pt/mL. Beyond the similar assembly behavior, the size and PDI of
nanoparticles differ a lot, exhibiting an evident correlation to chain
length of Pt(IV) conjugates: CSS18 ≈ C-18 < CSS12 < CSS6. Compared
with CSS18, larger particle size and increased PDI of monoaxial mCSS18
was observed as well (CSS18 < mCSS18). When coming to Pt(IV) NPs at
1 mg Pt/mL and 1.5 mg Pt/mL, the relevance between nanoparticle
properties and length of aliphatic branches was more evident (Table S1).
Even precipitation occurred in the preparation process of CSS6 and
mCSS18 NPs at 1.5 mg Pt/mL while CSS18 NPs at 6 mg Pt/mL showed
uniform and transparent appearance, which is shown in Fig. 2E. In
Fig. 2F, Tyndall Effect was confirmed in all nanoparticles at 1.5 mg Pt/
mL by a laser beam except for CSS6 NPs, in which the white precipita-
tion presented at the bottom of vial. It could be concluded that CSS6 NPs
are incapable of stabilization at high concentration by the evidence of
largest particle size and highest PDI. As for mCSS18 NPs, dim light beam
was visible in supernatant but precipitation still existed, which indicates
poor stability and weak assembly property. Of all nanoparticles, CSS18
had the best physicochemical properties and even stabilized at 1.5 mg
Pt/mL with light blue and limpid outlook (Fig. 2C). These results suggest
aliphatic chain length and location play a crucial role on self-stabilized
manner of carboplatin prodrugs. Double axial and long lipid branches
impart better assembly performance to Pt(IV) prodrugs than monoaxial
and short branched ones. It could be also reasonably hypothesized that
enough hydrophobicity of Pt(IV) amphiphiles contributes to NP integ-
rity that might prevent prodrug against cleavage or attack in the blood
circulation.
2.13. In vivo antitumor efficacy
The female Balb/c mice model bearing 4T1 breast xenograft tumors
were applied to investigate the in vivo antitumor efficacy. All mice were
separated into seven groups (n = 5) randomly and inoculated with the
same batch of 4T1 cells (5 × 106 cells per 100
μl). Mice with tumor
volume of about 100 mm3 were intravenously injected with 5% glucose,
carboplatin dissolved with 5% glucose, CSS6, CSS12, CSS18, C-18 and
mCSS18 NPs at 5 mg Pt/kg body weight every other day with total 4
times injection. The tumor size, 0.5 × (long side) × (short side)2, and
body weight change of every mouse were recorded. After tumor moni-
toring, all mice were euthanized and their blood samples were collected
for hepatorenal function test (ALT, alanine aminotransferase; AST,
aspartate aminotransferase; BUN, Blood Urea Nitrogen and CREA,
Creatinine), tissues (heart, liver, spleen, lung, and kidney) separated for
H&E staining. B16-F10 xenograft tumor model in C57BL/6 female mice
was also established which underwent the same dosing regimen. Tumor
size and body weight of each mouse were also monitored every other
day. When tumor volume was up to 2000 mm3, diseased mice were
considered as dying from excessive tumor burden and subjected to
euthanasia.
5

X. Kuang et al.
International Journal of Pharmaceutics 606 (2021) 120923
Fig. 3. Cumulative Pt release profiles of different formulations determined by ICP-MS in 10 mM PBS containing 0 mM (A), 1 mM (B) and 10 mM GSH (C). Cell uptake
and intracellular drug accumulation of prodrug Car, CSS6, CSS12, CSS18, C-18, mCSS18 NPs (D) and their NPs (E) in 4 T1 cells which were determined by ICP-MS.
(*p < 0.05, ** p < 0.01, *** p < 0.001, n = 3).
3.3. Colloidal stability
the boosted release tendency over C-18 NPs was mitigated and final
release percent of all groups was lower than that of 10 mM GSH (Fig. 3A-
C). And C-18 NPs release just 25% platinum content in total after 24 h
incubation in 1 mM GSH (Fig. 3B). Carboplatin solution reached diffu-
sion balance quickly with > 90% released Pt content at 1 h in all me-
diums. While accumulate release was less than 10% of each group in 0
mM GSH buffer. With GSH level dependent release manners, it is
reasonably speculated that disulfide bond guided prodrug nano-
assemblies are much more GSH-sensitive than ordinary lipid-Pt(IV)
prodrug and might be applied for rational design of cargo delivery
with tumor selective response due to evaluated GSH level in tumor site.
According to foregoing results, the longest aliphatic branched prodrug
CSS18 with favorable self-stabilized behavior and good colloid stability,
displayed basically equal GSH responsive capacity to other disulfide
prodrugs. This enlightened that chemical structure adjustment of
disulfide-tethered carboplatin(IV) molecules to enable enhanced as-
sembly and stability dose not impair specific responsiveness triggered by
intracellular GSH.
The stability of nanoparticles is of great significance to its manu-
facture, storage and clinical applications. As illustrated in Figure S7A
and B, all NPs remained stable in size and PDI in 10 mM PBS 7.4 for one
week except that CSS6 NPs has a sharp increase in particle size and PDI,
which suggests uncertainty in stabilization control of nanoparticles
composed of short lipid chain Pt(IV) amphiphiles. And facing chemical
challenges from plasma proteins, which is simulated by 10% FBS, C-18,
CSS12 and CSS18 NPs remained stable for 48 h with no visible
appearance change and size fluctuation. CSS6 NPs and single aliphatic
chain prodrug, mCSS18 NPs, showed poor stability with increased par-
ticle size at 48 h. In concert with assembly manner assay, precipitations
were observed in CSS6 and mCSS18 NPs group even at low concentra-
tion (0.5 mg Pt/mL). Size growth, PDI increase and precipitation present
in incubation media might indicated unsatisfactory property of short
lipid chain Pt(IV) NPs when interacted with endogenous proteins.
3.4. In vitro drug release
3.5. Intracellular Pt accumulation
As reported, the average reduced GSH level is about 1–11 mM in the
cytoplasm which is much higher than that about 2–10
μ
M in the blood
The internalization of carboplatin is conducted mainly via copper
influx transporter (CTR1) and organic cation transporter 2 (OCT2)
(Holzer et al., 2011). Chemical modulation of Pt compound is reported
to be likely to change the pathway of entry into cells (Chen et al., 2018).
Axial aliphatic branched modification seem to make Pt(IV) molecules
more prone to pass through lipid bilayer and enter cells with high
intracellular accumulation (Ling et al., 2019; Li et al., 2018). The entry
pathway of nanoparticles into cells is considered to be endocytosis
mediated by functional proteins which differs from free molecules. With
high Pt loading efficiency, nanoasemblies were expected to elevate
platinum uptake which was investigated by cell uptake assay. As shown
in Fig. 3D, intracellular drug accumulation of CSS18 and C-18 was
higher than any other prodrug at 4 h but there were no significant dif-
ferences between all prodrugs in 1 h. Strategy of prolonging axial lipid
chain to regulate affinity with biofilm enhanced Pt(IV) complexes
cellular uptake as widely reported. CSS18 or C-18 may be more likely to
circulation and extracellular environment (Schafer and Buettner, 2001;
Brülisauer et al., 2014). The redox potential preserved by intracellular
GSH/GSSG (~-250 mV) is also much higher than the extracellular one
(~-140 mV) and GSH/GSSH potential gap across cell membrane is
broader than other thiol redox couples such as Cys/Cyss (Brülisauer
et al., 2014; Moriarty-Craige and Jones, 2004). Disulfide bond is easy to
be attacked or reduced by hydrophilic GSH via with thiol–disulfide ex-
change reaction which happens in the endogenous proteins to stabilize
or deconstruct its tertiary and quaternary structures. To picture release
profile of disulfide inserted Pt(IV) NPs, 10 mM GSH was used in the in
vitro release assay to simulate reduction potential of tumor site. As
illustrated in Fig. 3A, redox-sensitive NPs (CSS6, CSS12, CSS18 and
mCSS18 NPs) showed accelerated Pt release of initial 70% at 4 h and
final 80% at 24 h, which exceed over non-disulfide C-18 NPs at any time
point. For release behavior of redox-sensitive NPs in 1 mM GSH buffer,
6

X. Kuang et al.
International Journal of Pharmaceutics 606 (2021) 120923
of 4T1, B16-F10, A2780 and LO2 cells were shown in Figure S8A-D.
CSS6 NPs showed the weakest inhibition against all cell lines than any
other group, which had still inferior activity to non-sensitive C-18 NPs.
Of all self-stabilized nanoparticles, CSS18 NPs exhibited the most
powerful cytotoxicity in three cancer cell lines and even showed a close
IC50 with carboplatin solution, implying long lipid branched tactics to
deliver Car could gain antiproliferation benefits from elevated cellular
Pt internalization and fast drug release mediated by disulfide bond. It
deserved to be mentioned that all Pt(IV) NPs showed less potent cyto-
toxicity than free Car after 48 h incubation in LO2 cells, which makes it
possible for this GSH-responsive Pt(IV)-lipid prodrug strategies to ach-
ieve tumor selectivity and low toxic (or harmless) in normal tissues.
Table 2
IC50 value (μM) of different Pt formulations treating for 48 h in 4 T1, B16,
A2780, and LO2 cells.
Sample
4 T1
24.37
B16-F10
A2780
LO2
47.28
Car sol
2.242
155.6
1.882
2.624
47.28
28.62
206.5
>1000
35.62
8.875
403.2
186.5
CSS6 NPs
CSS12 NPs
CSS18 NPs
C-18 NPs
mCSS18 NPs
818.1
34.37
10.62
115.9
62.42
684.7
76.95
191.2
439.4
321.4
be inserted into the phospholipid bilayer due to the similar amphiphilic
structure and even become component of cell membrane. It might make
prodrug CSS18 or C-18 easy to sneak into cytoplasm via component
exchange between membrane and cytosol. It is interesting that CSS18
NPs displayed the highest Pt uptake as well which is shown in Fig. 3E.
PEGylated nano-formulations are generally believed to form hydration
shell to cover chemical diversity of components when getting in
touchwith surface phospholipid layer. The high intracellular Pt content
detected of CSS18 NPs might derive from excellent stability in 1640
medium. While short chain branched CSS6 and mCSS18 NPs were in-
clined to be precipitated before being internalized by cells owing to
initial larger size and lack of good stabilization.
3.7. Detection of nucleus DNA-Pt adduct
DNA is confirmed to be the major cellular target of platinum drugs
where adducts and crosslinks are generated to impair its normal func-
tions and further initiate apoptosis. As shown in Fig. 4A, Pt content of
DNA extracted from 4T1 cells after incubation with Pt(IV) NPs was
detected by ICP-MS. The overall amounts of adducts caused by double
axial chain linked prodrugs are still associated with chain length: CSS18
> CSS12 > CSS6 NPs. Despite high intracellular accumulation, non-
sensitive C-18 NPs had the lowest DNA-Pt adduct level probably
because of difficulty in releasing carboplatin rapidly and thus delayed
entry into nucleus or other DNA located organelle. While disulfide bond
linked nanoparticles rendered more adducts detected by ICP-MS, sug-
gesting rapid release of active Pt(II) molecules make great contributions
to efficiency of coordination with DNA. Among them, CSS18 NPs with
compelling stability in extracellular medium and swift Car release trig-
gered by intracellular GSH, remained the most pronounced DNA adduct
3.6. In vitro cytotoxicity assay
Inspired by accelerated release and enhanced internalization of long
branches octahedrally coordinated Pt(IV) prodrug NPs, in vitro anti-
tumor efficiency was examined on different cell lines. IC50 values were
listed in the Table 2 and Pt concentration dependent cytotoxicity curves
Fig. 4. (A) DNA binding Pt content in 4 T1 cells treated with carboplatin, CSS6, CSS12, CSS18, C-18, mCSS18 NPs for 12 h. (B) Mean plasma Pt concentration–time
profiles of carboplatin, CSS6, CSS12, CSS18, C-18, mCSS18 NPs after i.v. administration in SD rats and biodistribution in 4 T1 tumor-bearing Balb/c mice (n = 3) at 4
h (C) and 12 h (D) (ns: no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, n = 3).
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X. Kuang et al.
International Journal of Pharmaceutics 606 (2021) 120923
Fig. 5. The antitumor efficacy in vivo. (A) Tumor growth curve of each mouse from different groups of 4 T1 tumor-bearing Balb/c mice during treatment of 5%
glucose, carboplatin solution, CSS6, CSS12, CSS18, C-18 and mCSS18 NPs (n = 5). The total tumor volume (B), tumor burden (C) and body weight (D) of 4 T1 tumor-
bearing Balb/c mice (n = 5). (E) Tumor growth curve of each mouse from different groups of B16-F10 tumor-bearing C57BL/6 mice during therapy (n = 5). The total
tumor volume (F), tumor burden (G) and body weight (H) of B16-F10 tumor-bearing C57BL/6 mice (n = 5). The arrow indicates the day of dosing different
formulation via tail vein injection. (ns means no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, n = 5).
level. High DNA binding efficiency may be one of the most important
factors that lead to cytotoxicity of CSS18 NPs.
nanoassemblies. It also verify our assumptions that enough hydropho-
bicity of Pt(IV) amphiphiles might preclude them from cleavage or
attack in the blood circulation.
3.8. In vivo pharmacokinetic
3.9. In vivo biodistribution
Colloid stability of prodrug was tested by co-incubation with 10%
FBS and relatively long aliphatic chain grafted NPs (CSS12, CSS18 and
C-18 NPs) showed good stability in vitro. Herein we further investigated
the in vivo pharmacokinetic profiles of all Pt(IV) NPs in SD rats. As shown
in Fig. 4B and Table S2, CSS18 and C-18 NPs had the most evident
improved AUC of 36.11 mg⋅h⋅L-1 which was about 8-fold higher value
than that of free Car. However, CSS6 NPs showed rapid elimination in
plasma with just a little increased AUC (2.2-fold) and t1/2 in comparison
with Car. The overall prolonged circulation time and augmented AUC
benefited from pharmacokinetic manners of different groups were
ranked as: CSS18 ≈ C-18 > CSS12 ≈ mCSS18 > CSS6, which is inspir-
ingly consistent with the order of carbon chain length grafted in the axis
of platinum atom. It revealed that appropriate modification of amphi-
philic Pt(IV) prodrug give rise to favorable in vivo fate of its
The biodistribution of five kinds of prodrug and free carboplatin in
4T1 bearing Balb/c mice model was also detected by ICP-MS. As illus-
trated in Fig. 4C and D, intravenous injected carboplatin was quickly
eliminated in plasma compared with Pt(IV) NPs, exhibiting enriched
distribution in kidney probably because of high hydrophilicity. As a
primary organ vulnerable to platinum, renal toxicity is common during
Pt treatment in clinic. Unlike free Car, all Pt(IV) NPs showed decreased
Pt content in kidney, indicating an alleviated toxicity profile. While Pt
(IV) NPs mainly accumulated in liver and spleen after intravenous
administration due to elimination by reticuloendothelial system (RES).
Notably, all NPs especially CSS18 and C-18 nanoparticles were more
enriched in tumor site, attributable to enhanced permeability and
retention (EPR) effect. With the most prolonged Pt residence in plasma
8

X. Kuang et al.
International Journal of Pharmaceutics 606 (2021) 120923
Fig. 6. Histopathological section (H&E staining) of the hearts, livers, spleens, lungs and tumors of Balb/c mice after different formulations administration. scale bar:
100 m. Liver (ALT (B), AST (C)) and kidney (CERA (D), BUN (E)) functional parameters at day 14 after mice were sacrificed.
μ
according to pharmacokinetics, CSS18 NPs exhibited maximal drug
concentration detected in tumor with around 5-fold level of Car group.
As a prerequisite for implementation of tumor selectivity and anti-
proliferation efficiency, ideal pharmacokinetic behaviors make it
possible for enhanced tumor accumulation.
3.10. In vivo antitumor efficacy
To evaluate the therapeutic effect of solid tumor, we investigated the
efficacy of Pt(IV) NPs using 4T1 and B16-F10 xenograft model devel-
oped by subcutaneous inoculation of cancer cells. As illustrated in
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X. Kuang et al.
International Journal of Pharmaceutics 606 (2021) 120923
Fig. 5A-C, 4T1 bearing Balb/c mice injected with 5% glucose loaded the
largest tumor of about 1200 mm3. Both CSS6 and C-18 NPs showed
limited inhibition on tumor growth rate comparable to carboplatin so-
lution, which was due to poor stability in vivo and delayed release of Pt
chemotherapeutics, respectively. Mice receiving CSS18, CSS12 and
mCSS18 NPs developed tumors with restrained growth rate and mark-
edly inhibited volume (400, 700 and 900 mm3) at day 14, especially
CSS18 NPs enabling the slowest proliferation rate and the smallest
tumor volume. Fig. 5D showed carboplatin-induced weight loss (>20%)
at day 6, implying systemic toxicity occurred and resulted from
nonspecific distribution of platinum. While the self-stabilized NPs
treated groups exhibited negligible body weight changes and no other
obvious toxic signs. For consideration of therapeutic universality, a
much highly proliferative type, B16-F10 xenograft model in C57BL/6
mice, was used to evaluate antitumor efficiency of all formulations.
Fig. 5E and F revealed a sharply increased tumor volume in mice
receiving 5% glucose, hitting 2060 mm3 at day 8. C-18 NPs treated
groups follow the rapid enlargement tendency and reach an average
tumor burden of 2302 mm3, suggesting an inadequate efficacy of non-
sensitive Pt(IV) prodrug. Other groups had significantly distinguished
tumor volumes with no more than 2000 mm3 at day 12. Tumor weight of
each group showed consistent tendency to volume in Fig. 5G. The B16-
F10 proliferation inhibition effect of all was determined as: CSS18 >
CSS12 > mCSS18 > CSS6 ≈ Car > C-18 > 5% Glucose, which is basically
in concert with inhibition outcome in 4 T1 model (CSS18 > CSS12 >
mCSS18 > CSS6 ≈ Car ≈ C-18 > 5% Glucose). No weight loss was
observed of all mice and even a few put on weight before euthanasia as
shown in Fig. 5H, indicating no systemic toxicity. In particular, C-18
prodrug showed comparable self-stabilization capacity and plasma sta-
bility to CSS18 but had minimal antiproliferation effect of all assembled
NPs which was as weak as carboplatin. These results verify that disulfide
bond, as an essential linker of Pt(IV) prodrugs in this research, endows
these nanoassemblies with reduction-sensitive character activated by
intracellular GSH and thus, has superior antitumor performance to non-
sensitive linkage. On the basis of the above facts, we are astonished to
discover that a subtle relationship between carbon chain length of
disulfide-inserted prodrugs and overall cancer therapeutic outcome is
established: CSS18 > CSS12 > mCSS18 (equivalent to CSS9) > CSS6. As
shown in Fig. 6A-D, no dysfunction was observed in H&E staining pic-
tures of all groups and no noticeable abnormal values were found in
hepatorenal function assay.
insights into structure–activity relationship of Pt(IV) prodrug and open a
new path to biosafety chemotherapeutic nanocarriers.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This research was supported by the Career Development Program for
Young and Middle-aged Teachers in Shenyang Pharmaceutical Univer-
sity (Shenyang, China). This research was also supported by Suzhou
Science and Technology Development Project (SYSD2019171) and
High-tech zone health talents key talent category (No.2019).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijpharm.2021.120923.
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