Disulfide-crosslinked reduction-responsive Prodrug Micelles for On-demand Paclitaxel Release

Article abstract of DOI:10.1016/j.jddst.2019.101168

In this report, disulfide-crosslinked paclitaxel (PTX) prodrug micelles were developed through covalently conjugating PTX onto water soluble poly(ethylene glycol)-dihydrolipoic acid (MeO-PEG_2k-DHLA) via a disulfide linkage and studied their perspectives for chemotherapic antitumor capacity. The polymer-PTX prodrug (MeO-PEG_2k-SS-PTX), structurally determined by ¹H NMR, possessed high PTX content up to 26 wt% and exhibited sharp reduction-responsive behavior. MeO-PEG_2k-SS-PTX micelles were then subjected to crosslinking by oxidization of thiol (-SH) groups in the core of micellar particles after self-assembly of the amphimictic prodrug. The resultant crosslinked micelles were stable with spherical morphology having the uniform size of 87.67 nm in phosphate buffer (PBS, pH 7.4), as confirmed by transmission electron microscope (TEM) and dynamic light scattering (DLS). The cross-linked micelles based on the redox-sensitive MeO-PEG_2k-SS-PTX prodrug exhibited an obvious glutathione (GSH)-dependant manner of fast PTX release with appropriate 65.6% in 12 h and 92.6% in 72 h after incubation in PBS medium containing 10 mM GSH. MTT assay together with apoptosis analysis showed that cross-linked MeO-PEG_2k-SS-PTX micelles worked the higher antitumor activity against MCF-7, A549, BEL-7402 and 4T1 cells, using free PTX as a positive control. More importantly, such micelles were found to hold a longer circulation time in bloodstream and be able to passively targeting accumulate in tumor sites of 4T1-transplanted BALB/c mice model.

Full text of DOI:10.1016/j.jddst.2019.101168

JournalofDrugDeliveryScienceandTechnology53(2019)101168
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology
journal homepage: www.elsevier.com/locate/jddst
Disulfide-crosslinked reduction-responsive Prodrug Micelles for On-demand
Paclitaxel Release
Zhi Wanga,1,*, Longbing Lingb,1, Qing Xiab, Xinsong Lib,*
a Department of Plastic Surgery, Peking Union Medical College Hospital, Beijing, 100730, PR China
b 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:
Paclitaxel
Micelles
Reduction-responsiveness
Loading efficiency
Antitumor activity
In this report, disulfide-crosslinked paclitaxel (PTX) prodrug micelles were developed through covalently con-
jugating PTX onto water soluble poly(ethylene glycol)-dihydrolipoic acid (MeO-PEG2k-DHLA) via a disulfide
linkage and studied their perspectives for chemotherapic antitumor capacity. The polymer-PTX prodrug (MeO-
PEG2k-SS-PTX), structurally determined by 1H NMR, possessed high PTX content up to 26 wt% and exhibited
sharp reduction-responsive behavior. MeO-PEG2k-SS-PTX micelles were then subjected to crosslinking by oxi-
dization of thiol (-SH) groups in the core of micellar particles after self-assembly of the amphimictic prodrug. The
resultant crosslinked micelles were stable with spherical morphology having the uniform size of 87.67 nm in
phosphate buffer (PBS, pH 7.4), as confirmed by transmission electron microscope (TEM) and dynamic light
scattering (DLS). The cross-linked micelles based on the redox-sensitive MeO-PEG2k-SS-PTX prodrug exhibited
an obvious glutathione (GSH)-dependant manner of fast PTX release with appropriate 65.6% in 12 h and 92.6%
in 72 h after incubation in PBS medium containing 10 mM GSH. MTT assay together with apoptosis analysis
showed that cross-linked MeO-PEG2k-SS-PTX micelles worked the higher antitumor activity against MCF-7,
A549, BEL-7402 and 4T1 cells, using free PTX as a positive control. More importantly, such micelles were found
to hold a longer circulation time in bloodstream and be able to passively targeting accumulate in tumor sites of
4T1-transplanted BALB/c mice model.
1. Introduction
devoid of CrEL [14,15] has been introduced to overcome these limita-
tions of Taxol®, but it also possesses many inadequacies, including high-
Paclitaxel (PTX), a natural diterpenoid isolated from the bark of
Taxus Brevifolia, has displayed substantial anticancer effects induced
by stabilizing microtubules, preventing mitosis, and prompting cellular
apoptosis [1,2]. As the first-line antitumor drug, PTX has been broadly
applied in the medication of various solid tumors, such as breast,
ovarian, and prostate cancers [3–5]. In addition, PTX has also been
realized to be successful in combating versus lung, non-small cell lung
carcinoma and AIDS-related Kaposi's sarcoma [6,7]. However, its ap-
plications in cancer chemotherapy are hampered by its poor solubility,
prominent toxicity, lower bioavailability and non-specificity [8–10]. A
formulation of PTX containing CremophorEL (CrEL) under the trade-
name Taxol® has been permitted by U.S. Food and Drug Administration
to improve its solubility and to cure patients having diverse malig-
nancies as mentioned above [10]. Unfortunately, severe side effects
were observed in Taxol®, such as hypersensitivity, nephrotoxicity and
peripheral neuropathy [11–13]. A novel PTX formulation (Abraxane®)
cost, short half-life and feeble antitumor effect compared to Taxol®.
Therefore, alternative formulations of the currently available com-
mercialized PTX are highly required in chemotherapeutic treatment.
To meet the challenge of associated toxicity of the commercial PTX
formulation, intelligent polymer-based drug delivery systems (DDS) of
PTX lacking CrEL as an additive have been developed to overcome such
kind of limitations [16–19]. Except for the lower toxic effects, poly-
meric formulations of PTX have various advantages over the standard
current chemotherapy, including increased aqueous solubility of con-
jugated drugs, along with nanoscaled characteristics, which enables
their effective tumor-targeted delivery into the tumor site via enhanced
permeability and retention (EPR) effect [20–22]. Recently, polymeric
prodrugs attract much attention in the community of drug delivery
[23]. Conjugation of the antitumor drugs with polymeric backbones
like polysaccharide (TPS) [24] or polyethylene glycol (PEG) [25–27],
potentially escape the reticuloendothelial system (RES) in normal cells/
* Corresponding authors.
E-mail addresses: wangzh@pumch.cn (Z. Wang), lixs@seu.edu.cn (X. Li).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.jddst.2019.101168
Received 15 April 2019; Received in revised form 18 July 2019; Accepted 18 July 2019
Availableonline19July2019
1773-2247/©2019ElsevierB.V.Allrightsreserved.

Z. Wang, et al.
JournalofDrugDeliveryScienceandTechnology53(2019)101168
Scheme 1. The detailed synthetic pathway for disulfide-linked PTX–SS–Pyr conjugate. Regents and conditions: (a) imidazole, TBDMSCl, DMF, r.t., 24 h; (b) p-
nitrophenyl chloroformate, DMAP, CH2Cl2, r.t., 12 h; (c) 2-(pyridin-2-yldisulfanyl)ethanol, CHCl3, 70 °C, 12 h; (d) TBAF, THF, r.t., 2 h.
tissues, thereby minimizing the drugs associated adverse side effects.
Such modification of PTX into nanoparticles also improves the phar-
macokinetic profile of PTX by increasing its half-life in bloodstream and
tumor accumulation. Along with the mentioned polymeric conjugates,
several other polymer-based nano-formulation approaches to deliver
PTX have been explored, including PTX-loaded micelles, emulsion [28]
and cyclodextrins modified complexes [29,30], however only few of
them was approved by the Food and Drug Administration (FDA). This is
possibly because of their lower drug loading, batch-to-batch dissim-
ilarities in its bio-physicochemical characteristics, inadequate bio-
compatibility, higher toxicity, and poor in vivo instability of these na-
nosystems [31,32].
release the payload under extremely reductive milieu in tumor. In this
context, Page and coworkers developed camptothecin (CPT)-conjugated
cross-linked micelles containing –SS– linkage, which demonstrated
potentially declined the premature diffusion of CPT from the micellar
core, when compared with physically entrapped CPT. The presence of
–SS– bonds in the prodrug micelles facilitated CPT release under re-
ducing environment, which potentially induced the deaths of MCF-
7 cells.
The rationale of the current study is to design stimuli responsive
core-cross linked micelles of covalently conjugated PTX having a –SS–
linker and to explore their stability, sustained release, antitumor effi-
cacy, as well as provide an evidence of their prolonged circulation in
bloodstream. For the purpose, core-cross linked micelles based on the
PTX-conjugated MeO-PEG2k-DHLA prodrug (MeO-PEG2k-SS-PTX) was
developed. In the designing of this prodrug, pyridyldithio-modified PTX
was covalently conjugated with MeO-PEG2k-DHLA block through a
cleavable –SS– bond and self-assembled into cross-linked micelles by
oxidization. With the PEGylated shell and disulfide crosslinking in the
core, these micelles exhibited substantial stability in physiological
condition but quickly liberate PTX at the rich GSH milieu of tumor sites
due to disulfide cleavage (Scheme 3). Besides, in vitro cytotoxicity,
apoptosis analysis and in vivo pharmacokinetics of cross-linked micelles
were investigated in detail.
The in vivo instability of the conventional micellar nanomedicines is
mainly linked to their early crumbling in the systemic circulation.
Dilution in the blood together with interactions of the micellar building
blocks with plasma proteins cause this premature disintegration, that
actively transfers the micellar equilibrium to the unimer status after
intravenous administration [33,34]. This phenomenon results in the
trivial developments in the circulation periods, targeted accumulation
at tumor site and therapeutic efficiency of the substantial number of
pre-clinically applied micellar nanomedicines [35]. Introduction of
crosslinking is an easy and straightforward approach to decline the
premature disintegration of polymer-based nanosystems to ensure ex-
tended circulation in bloodstream, as well as effective EPR-facilitated
targeted accumulation. Using this strategy, the assembly of unimers is
further stabilized through the construction of crosslinks (covalent, hy-
drogen bonding or π-π stacking) [36–38] both in the core or corona to
develop robust cross-linked micellar systems. However, the physically
entrapped drugs rapidly leaked out from the micellar core, nearly ir-
respective of core-crosslinking, thereby quickly eliminated from the
blood circulation. And the physically entrapped drug candidates re-
leased from the micellar systems in a non-controlled fashion, which is
inadequate for assuring sustained and tailored release kinetics.
One way to counteract such premature diffusion or controlled the
release of the incorporated drugs from the micellar systems at higher
level, is to develop covalently co-crosslinked drug micelles employing
various biodegradable linkers. Covalently cross-linking significantly
improved the pharmacokinetic properties of polymeric micelles versus
the physically (non-covalently) encapsulated drugs [39,40]. Specifi-
cally, the covalently attached stimuli-responsive core-cross-linked mi-
celles containing disulfide (–SS–) linkages could not only guarantee the
retaining of drug molecules in the micelles throughout the systemic
circulation, but also ensure its effective release after accumulating at
the target site [41]. Such core cross-linked micelles uphold higher
stability in bloodstream/extracellular environment with lower glu-
tathione(GSH) level [41], but could quickly break the –SS– bonds to
2. Materials and methods
2.1. Materials
Paclitaxel (PTX) with the purity of 98.5% was obtained from Aikon
Biopharmaceutical R&D Co., Ltd. Nanjing, China. Poly(ethylene glycol)
methyl ether (MeO-PEG-OH, Mw 2000 Da), succinic anhydride (SA),
serinol, lipoic acid (LA) and sodium borohydride (NaBH4) were pur-
chased from Sinopharm Chemical Reagent Co., Ltd. Shanghai, China. 4-
(dimethylamino) pyridine (DMAP), p-nitrophenyl chloroformate, so-
dium dodecyl sulfate (SDS), imidazole, tert-butyldimethylsilyl chloride
(TBDMSCl), dicyclohexylcarbodiimide (DCC) and 2,2′-dithiodipyridine
were purchased from J&K Scientific Ltd. Shanghai, China. Cell culture
reagents including completed RPMI-1640 medium, fetal bovine serum
(FBS) and trypsin-EDTA were purchased from Key Gen Biotech Co., Ltd.
Nanjing, China. All other chemicals and reagents were analytical grade
and used without further purification.
MCF-7 cells, A549 cells and BEL-7402 cells were provided by Public
Health Institute of Southeast University. Cells were incubated in com-
pleted RPMI-1640 medium supplemented with 10% FBS and main-
tained in a humidified atmosphere of 5% CO2 at 37 °C.
Female BALB/c mice (5-week-old, 18–20 g) were acquired from
2

Z. Wang, et al.
JournalofDrugDeliveryScienceandTechnology53(2019)101168
Scheme 2. The detailed synthetic pathway for disulfide-linked MeO-PEG2k-SS-PTX prodrug. Regents and conditions: (a) SA, DMAP, Et3N, CH2Cl2, reflux, 12 h; (b)
serinol, DCC/HOBt, CH2Cl2, r.t., 8 h; (c) LA, DCC/DMAP, DMF, r.t., 12 h; (d) NaBH4, MeOH/H2O, 0 °C, 4 h; (e) PTX–SS–Pyr, Et3N, DMF, r.t., 12 h.
white solid (1.52 g, yield 85.7%) after purification via silica gel column
chromatography (EtOAc:Hexane 1:2). MS m/z: calculated for
C
60H68N2O18Si [M+Na]+, 1155.42; found 1155.41 [M+Na]+
.
3rd step: PTX-4-nitrophenyl carbonate (1.5 g, 1.33 mmol), 2-(pyr-
idin-2-yldisulfanyl) ethanol (0.49 g, 2.66 mmol) and DMAP (0.46 g,
3.99 mmol) dissolved in 40 mL of anhydrous chloroform, reflux at 80 °C
for 12 h. After cooled, washed with distilled water (100 mL) and dried
by Na2SO4, removal of solvents and purified using silica column chro-
matography (EtOAc:Hexane 1:2) to obtain the product of TBS-
PTX–SS–Pyr as a white solid (1.26 g, yield 80.3%). MS m/z: calculated
for C61H72N2O16S2Si [M+Na]+, 1203.41; found 1203.39 [M+Na]+
.
4th step: The acquired TBS-PTX–SS–Pyr was further subjected to
deprotection of TBS group by solution to achieve the final product of
PTX–SS–Pyr. Briefly, to
a solution of TBS-PTX–SS–Pyr (1.18 g,
1.0 mmol) in 5 mL of THF, μL of 1 M tetra-butylammonium fluoride
(TBAF) in THF was added and allowed to maintain at r.t. for 2 h.
Removal of solvent followed by purification on silica column (1:1
EtOAc:hexane) provided PTX–SS–Pyr as a white solid (1.07 g, yield
73.5%). 1H NMR (500 MHz, CDCl3): δ 1.19–1.24 (m, 6H, –CH3), 1.77 (s,
3H, –CH3), 1.84 (s, 3H, –CH3), 2.30 (d, J = 9.7 Hz, 3H, –COCH3), 3.06
(d, J = 6.9 Hz, 2H, –CH2-), 3.84 (d, J = 6.7 Hz, 1H, H3), 4.08 (ddd,
J = 21.4, 11.4, 5.4 Hz, 2H, –CH2-), 4.24 (d, J = 8.3 Hz, 2H, H20), 4.36
(m, 2H, H19), 4.73 (m, 1H, H7), 4.88 (d, J = 7.9 Hz, 1H, H5), 5.37 (m,
1H, H3’), 5.60 (d, J = 6.8 Hz, 1H, H11), 5.72 (m, 2H, H2), 6.15–6.24
(m, 4H, H10, H13), 7.02–8.49 (m, –C6H6) ppm. 13C NMR (500 MHz,
CDCl3): δ 13.63, 19.66, 21.53, 25.55, 32.36, 34.53, 35.72, 42.22, 45.94,
53.93, 55.12, 64.93, 71.23, 74.24, 74.60, 75.40, 75.79, 76.00, 76.22,
77.52, 79.97, 82.80, 114.62, 118.79, 125.12, 127.36, 127.73, 127.99,
129.14, 131.01, 132.07, 132.57, 132.78, 136.16, 136.93, 139.46,
148.56, 152.89, 165.83, 166.16, 201.68 ppm. MS m/z: calculated for
Scheme 3. Schematic illustration of GSH-triggered PTX release (A) and the
proposed reduction-responsive mechanism (B) from cross-linked MeO-PEG2k
-
SS-PTX micelles in vitro.
Animal Core Facility of Nanjing Medical University and held under
sterile conditions. All animal's experiments were strictly conducted in
accordance with the guidelines set by the Institutional Animal Care and
Use Committee of Southeast University.
2.2. Synthesis of PTX–SS–Pyr conjugate
Scheme
1 gives the synthetic pathway of disulfide-linked
PTX–SS–Pyr conjugate using a four-step conjugation approach. The
synthesis and structural characterizations are detailed as follows:
1st step: 2′-TBS-PTX that 2′-OH of reactive PTX was protected by
TBDMSCl agent was synthesized as we previously reported [42]. The
final yield of 2′-TBS-PTX is appropriate 98.6%.
C
55H58N2O16S2 [M+Na]+, 1098.32; found 1098.39 [M+Na]+
.
2.3. Synthesis of MeO-PEG2k-SS-PTX prodrug
2nd step: To a mixture of 2′-TBS-PTX (1.5 g, 1.55 mmol) and 4-ni-
trophenyl chloroformate (0.62 g, 3.12 mmol) dissolved in 20 mL of
dried dichloromethane at 0 °C, DMAP (1.14 g, 9.30 mmol) was
dropwisely added and stirred for overnight. After that, the mixture was
washed by 0.1 M HCl (150 mL), dried by anhydrous Na2SO4 and solvent
was removed in vacuo. PTX-4-nitrophenyl carbonate was obtained as a
MeO-PEG2k-SS-PTX prodrug with a disulfide linkage was synthe-
sized by a five-step facial conjugation method as illustrated in Scheme
2. The carboxyl terminated PEG monomethyl ether (MeO-PEG2k
-
COOH) was first synthesized with SA (4 eq.) and DMAP (1 eq.) in
pyridine through an esterification reaction [43]. After that, serinol (3
3

Z. Wang, et al.
JournalofDrugDeliveryScienceandTechnology53(2019)101168
eq.) was attached to the MeO-PEG2k-COOH (1 eq.) using DCC (2 eq.)/
HOBt (2 eq.) as the coupling agents in chloroform overnight. The
polymer of MeO-PEG2k-Serinol was precipitated in ice-cold ether three
times and concentrated under vacuum. Furthermore, MeO-PEG2k-Ser-
inol, lipoic acid (LA), DCC and DMAP with the molar ratio of 1:4:4.5:1
were dissolved in anhydrous DMF and left to proceed at r.t. for 24 h.
The solution was filter and isolated as a light yellow solid of MeO-
PEG2k-LA polymer after precipitation in ice-cold ether. Afterward,
MeO-PEG2k-LA was completely reduced to MeO-PEG2k-DHLA in the free
thiol form (-SH) and the above-acquired PTX–SS–Pyr conjugate (4 eq.)
was finally coupled onto the host polymer of MeO-PEG2k-DHLA (1 eq.)
in triethylamine (Et3N) through a thiol–disulfide exchange reaction.
was 1 mg/mL. Typically, 1 mL of micelles was precisely injected into
dialysis cartridges (Sigma-Aldrich Co., MS, USA) and dialyzed against
100 mL of dissolution medium containing 0.05% Tween-80. The con-
centration of released PTX at various time points was analyzed by using
HPLC (Agilent, MA, USA). The release tests were carried out in tripli-
cate and values presented were the means with standard deviations
(M
SD).
2.6. MTT assay
The in vitro cytotoxicity of cross-linked MeO-SS-PTX micelles was
studied using MCF-7 cells, A549 cells and BEL-7402 cells by MTT assay
[45]. In brief, appropriate of 5 × 103 cells/well in 96-well plates was
incubated in 100 μL of RPMI-1640 medium with 10% FBS (37 °C, 5%
CO2) 24 h prior to the treatment. The micelles with the prescribed
concentration of PTX (0.156 μM–10 μM) were added, while free PTX
was used as positive control. After 48 h incubation, the microplate-680
reader (ThermoFisher Scientific, MA, USA) was conducted to check the
optical density (OD) of each sample after the addition of MTT (20 μL,
5 mg/mL) was dissolved in analytical DMSO at 570 nm. Cell viability
(%) of tested groups against cells was determined in comparition to the
blank controls containing the cells only and the results were shown as
the average data [(ODtested-ODblank)/(ODpositive-ODblank) × 100%] of
sextuplicate wells.
The reactants were stirred at room temperature for 12 h. MeO-PEG2k
-
SS-PTX prodrug was obtained by extensive dialysis (MWCO 1000)
against distilled water followed by lyophilization.
2.4. Preparation and characterization of cross-linked MeO-PEG2k-SS-PTX
micelles
Cross-linked MeO-PEG2k-SS-PTX micelles were fabricated by a DMF
dissolution of polymer MeO-PEG2k-SS-PTX prodrug dropwise into the
phosphate buffer saline (PBS, pH 7.4) under stirring at room tempera-
ture. To facilitate the intermolecular disulfide (–SS–) crosslinking, the
solution was oxidized by purging air continuously for 24 h. The loss of
free thiol (-SH) groups was checked using Ellman's test [44]. The mi-
celles obtained were then filter thrice via a syringe filter (0.11 μm,
Corning Incorporated, NY) and sonicated for 20 min, which were used
for the following characterization at a reserved concentration of 1 mg
PTX/mL.
2.7. Apoptosis analysis
For the analysis of cell apoptosis in vitro, representative MCF-7 cells
were treated with different formulations of free PTX and micelles (PTX
concentration of 2 μM) for 48 h at 37 °C, 5% CO2 atmosphere.
Afterward, 2 × 104 cells were collected and re-suspended in 0.5 mL of
binding buffer. Following the instruments of apoptosis detection kit
(KeyGEN BioTECH, Nanjing, China), 10 μL PI and 5 μL Annexin V-FITC
were added into the cells for 30 min staining in the dark. Finally, flow
cytometry (FACScan, BD Accuri, NJ) was used to record the fluores-
cence of double stained cells and data analysis was operated on FlowJo
software (version 7.6, Tree Star, Inc., OR).
Critical micelle concentration (CMC) of cross-linked MeO-PEG2k-SS-
PTX micelles was determined by a dye solubilization method using
pyrene. Sample suspensions of pyrene in acetone were prepared and
evaporated the solvent to
a final concentration of pyrene at
6 × 10−7 M. After that, different concentrations (3 mL) of MeO-PEG2k
-
SS-PTX prodrug were added to each tube for another 24 h equilibration
between micelles and pyrene. The fluorescence emission spectra were
measured at the excitation wavelength of 335 nm by a LS-50B spec-
trometer (Perkin Elmer, MA, USA). The intensity ratio of I3/I1 (third
and first highest energy bands) was plotted the logarithm of con-
centrations (Log C) to obtain CMC value.
2.8. Hemolysis study
The amount of conjugated PTX in the micelles was determined by
HPLC instrument (Agilent, MA, USA) after releasing PTX from MeO-
PEG2k-SS-PTX micelles by adding excessive GSH. The loading content
was calculated according to the standard calibration curve that is at-
tained between HPLC area values and different concentrations of PTX
drug in methanol. The definition of drug loading capacity (DLC) is
presented as the maximum PTX content accomplished by the micelles,
while the drug conjugation efficiency (CE) is defined as the ratio of
weight of conjugated drug to the initial drug input.
The size and size distribution of MeO-PEG2k-SS-PTX micelles after
crosslinking were monitored at 25 °C by dynamic light scattering (DLS,
Malvern, Worchestershire, UK). 1 mg/mL of micellar solution were
filtered through a 0.22 μm syringe filter before test and measurements
were performed by the Zetasizer Nano-ZS equipped with a 633 nm
He–Ne laser. Transmission electron microscopy (TEM) for morpholo-
gical observation was operated on the Tecnai G220 TEM (FEI company,
OH, USA) at an acceleration voltage of 200 kV. The samples were
prepared by placing 20 μL of 1 mg/mL micelles on the 200-mech copper
grid, followed by air-dry and stain with 2% (w/v) phosphotungstic acid.
Blood compatibility of cross-linked MeO-PEG2k-SS-PTX micelles was
evaluated by hemolysis assay, following the procedure as stated as
previously published literatures [46]. Human blood was obtained from
Department of Blood Transfusion of Affiliated Zhongda Hospital of
Southeast University (Nanjing, China). The red blood cells (RBCs) were
isolated by centrifugation at 1000 rpm for 30 min and prepared to a
final concentration of 2% (v/v) in phosphate solution (PBS, pH 7.4).
100 μL of RBCs suspension was mixed with MeO-PEG2k-SS-PTX micelles
at different concentrations for 37 °C, 4 h incubation. After that, the
solution was centrifuged at 1000 rpm for 15 min and the collected su-
pernatant was subjected to analysis by a micro-plate reader (Thermo-
Fisher Scientific, MA, USA) at 540 nm. RBCs treated with deionized
water (100% hemolysis) and PBS (pH 7.4, 0% hemolysis) were set as
positive and negative controls, respectively. Less than 5% of percent
hemolysis ratio (HR%) was regarded as nontoxic and HR% was calcu-
lated according to the following formula:
ODsample − ODnegative
HR(%) =
× 100
ODpositive − ODnegative
ODsample is the optical density of tested samples, while ODnegative and
ODpositive are the negative control and positive control, respectively.
2.5. GSH-triggered release of PTX from MeO-PEG2k-SS-PTX micelles
In vitro release profile of PTX from cross-linked micelles as well as
uncross-linked micelles was investigated by a dialysis method (MWCO
1000 Da) at 37 °C in two different media, i.e. PBS (pH 7.4) only and PBS
(pH 7.4) with 10 mM GSH. The initial PTX concentration of micelles
2.9. In vivo pharmacokinetics and biodistribution
Female BALB/c mice (5-week-old, 18–20 g) were injected with
4

Z. Wang, et al.
JournalofDrugDeliveryScienceandTechnology53(2019)101168
0.1 mL suspension of 4T1 mammary carcinoma cells (1 × 107) in the
right axilla subcutaneously. When the tumor size grew to 80–100 mm3
for two weeks before testing, the mice were randomly divided into
Taxol® and cross-linked MeO-PEG2k-SS-PTX micelles groups (n = 3).
The aqueous solutions of two formulations were intravenously ad-
ministrated via tail vein at the dosage of 5 mg PTX/kg. At various time
point post-injection, 100 μL of plasma was collected after centrifugation
at 3000 rpm for 15 min. Subsequently, methanol (1 mL) for protein
precipitation and extraction of PTX was mixed with the plasma and
centrifuged at 11,000 rpm for 5 min to obtain the supernatant. An ali-
quot of 20 μL of Taxol® samples was directly measured by using HPLC
(Agilent, MA, USA), while samples of MeO-PEG2k-SS-PTX micelles were
checked after treatment with excessive GSH firstly. The amount of PTX
in blood was acquired by HPLC, which is according to the standard
curve previously analysis of blood samples containing known amounts
of free PTX.
To assess the effect of cross-linked MeO-PEG2k-SS-PTX micelles on
tissue distribution, 4T1-bearing BALB/c mice were assigned into 2
groups at random and the protocol for administration in tissue dis-
tribution study was the same as the above-described pharmacokinetic
analysis. At 1 h, 3 h, 6 h and 12 h after post-injection, all mice (n = 4)
were scarified and tissues including heart, liver, spleen, lung and kidney
were collected. Free PTX was then extracted from the homogenized
tissue samples using 2 mL of diethyl ether. After vortexing for 5 min, the
organic phase was gathered, air-dried and re-dissolved in methanol for
analysis. The drug concentrations were determined using HPLC, and the
corresponding PTX concentrations in tissues were calculated accord-
ingly.
hydrophobic PTX domains, MeO-PEG2k-SS-PTX prodrug is definitely
bound to self-assemble into micelles in aqueous medium (PBS, pH 7.4).
The typical solvent exchange method was used to prepare the uncross-
linked MeO-PEG2k-SS-PTX micelles with an average hydrodynamic size
of 85.62 nm (Fig. S4), following dropwise a DMF dissolution of prodrug
into PBS solution (pH 7.4). Moreover, the thiol-containing MeO-PEG2k
-
SS-PTX micelles (uncross-linked micelles) are able to spontaneously
cross-link forming disulfide bond (–SS–) in the presence of air atmo-
sphere. After bubbled with air for 24 h in uncross-linked micelles, the
content of disulfide formation was checked by Ellman's test [48], which
was quantitated according to a standard curve composed of series of
known cysteine concentrations at 412 nm. Similar to our previous re-
port, it was as-expectedly found that > 95% conversion of thiol group
(-SH) was achieved, significantly indicating the cross-link based on the
disulfide (–SS–) formation (Data not given).
The aggregation behavior of cross-linked MeO-PEG2k–SS–PTX mi-
celles was monitored by using pyrene fluorescence probe technique.
Fig. S5 showed the variation of I3/I1 intensity ratio versus the loga-
rithm of concentration (Log C), where CMC was determined as the
concentration at the point of intersection. It was found that the CMC
value of MeO-PEG2k–SS–PTX micelles was 19.95 μg/mL and such low
CMC value suggested that the micelles can be formed under highly
dilute condition in PBS (pH 7.4). The resultant cross-linked MeO-PEG2k
-
SS-PTX micelles were further characterized for size distribution using
DLS and for spherical morphology using TEM, respectively. DLS mea-
surements in Fig. 1A revealed that the average diameter of cross-linked
micelles in PBS (pH 7.4) was appropriate 87.67 nm with a narrow
distribution (PDI: 0.185), that is close to the uncross-linked individual.
Besides, to prove the sufficient crosslinking of micelles, the size and size
distribution of cross-liked prodrug dissolved in DMSO were further
measured by DLS. As shown in Fig. 1B, the hydrodynamic size of cross-
linked micelles in DMSO was 92.48 nm with a with a calculated PDI
value of 0.205. The results presented are in consistence with the size
that dispersed in PBS (pH 7.4), which convincingly verified the na-
nostructures of MeO-PEG2k-SS-PTX micelles maintains undamaged in
organic solvent, due to the core crosslinking. Fig. 1C exhibited the TEM
micrograph of cross-linked micelles. Spherical morphology of uniform
sizes was observed, thereby confirming the self-assembly of MeO-
PEG2k-SS-PTX prodrug in aqueous.
3. Statistical analysis
Statistical analysis between different groups was performed by one-
way ANOVA analysis using GraphPad Prism software (version 6,
GraphPad Software, Inc., CA). All the results were expressed as
mean
S.D. The value of *P < 0.05 considered significant and
**P < 0.01 highly significant.
4. Results and discussion
4.1. Synthesis and characterizations of micelles
4.2. Stability study of cross-linked micelles
The objective reduction-responsive MeO-PEG2k-SS-PTX prodrug was
synthesized by a thiol–disulfide exchange reaction between polymer
MeO-PEG2k-DHLA and PTX–SS–Pyr conjugate (Schemes 1 and 2). 1H
NMR spectrum of MeO-PEG2k-SS-PTX prodrug successfully displayed
with representative signals at 3.62 ppm and 7.0–8.5 ppm in Fig. S3,
which are attributable to the methylene protons of PEG and the phenyl
protons of PTX, respectively. In addition, the peaks assignable to
–CH2CH2- linkage were detected from 1.8 to 2.0 ppm, further con-
firming the conjugation of PEG and PTX–SS–Pyr.
PTX–SS–Pyr compound was readily conjugated into the thiol-func-
tionalized MeO-PEG2k-DHLA at the fixed molar ratio of 4:1 (PTX:
Polymer) in a mild condition and the content of PTX was analyzed by
HPLC instrument at the wavelength of 227 nm. As examined, the PTX
content in MeO-PEG2k-SS-PTX prodrug was up to 26 wt% as well as the
conjugation efficiency (CE) 20%. According to the data, approximate
one PTX molecule is conjugated in one MeO-PEG2k-DHLA macro-
molecule (Scheme 2). It was reported that most polymeric prodrugs
possess less than 20 wt% drug content, such as PHPMA-peptide-PTX
conjugates (5 wt % PTX, PNU166945) used in phase I clinical trials and
PHPMA-hydrazide-LEV-PTX (7.6 − 16.3 wt % PTX) [47], for which
could avoid the premature burst drug release in blood circulation.
Herein, these reduction-responsive MeO-PEG2k-SS-PTX prodrug sig-
nificantly improved drug loading without drug leakage, exhibiting a
great advantage in prodrug-based delivery of chemotherapeutic agents.
With the inherent amphiphilicity of hydrophilic PEG and
The storage stability of disulfide cross-linked micelles was found to
be excellent at 4 °C, showing no significant size change after one month
(Fig. S6). After that, in vitro stability of the cross-linked micelles was
studied against the harsh micelles-destruction conditions in SDS
(2.5 mg/mL). As reported previously, SDS, a powerful ionic detergent,
could disrupt the nanostructure of polymeric micelles effectively. The
micelles (1 mg/mL) were mixed with the SDS solution at the micelle-
destruction concentration of 2.5 mg SDS/mL, the stability in particle
changes was accordingly monitored at the determined time intervals by
DLS. As shown in Fig. 1D, the cross-linked micelles have a stable size in
the presence of SDS. On the contrary, the size of the uncross-linked
micelles decreased sharply, indicating SDS inducing micelle destruc-
tion.
The attractive target of MeO-PEG2k-SS-PTX prodrug was its in-
tracellular redox-sensitivity designed for solving the problem of slow
release of payload drug. It is well-known that the content of in-
tracellular glutathione (GSH, 10 mM) is substantially higher compared
with the extracellular level (2 μM) [49,50]. Thus, the reduction-sensi-
tivity of cross-linked micelles was evaluated by recording the size
change in response to the 10 mM GSH using DLS. The results are de-
picted in Fig. 1D and found the immediate decrease of size signal of
cross-linked micelles after 20 min, following the addition of 10 mM
GSH in medium containing 2.5 mg/mL SDS. The response of cross-
linked MeO-PEG2k-SS-PTX micelles to the reductive GSH supportively
5

Z. Wang, et al.
JournalofDrugDeliveryScienceandTechnology53(2019)101168
Fig. 1. (A) Size distribution of cross-linked MeO-
PEG2k-SS-PTX micelles in PBS (pH 7.4) and (B)
in DMSO as determined by DLS. (C) TEM image
of cross-linked MeO-PEG2k-SS-PTX micelles after
staining with 2% phosphotungstic acid. (D)
Stability in particle size of cross-linked and un-
cross-linked micelles incubated with 2.5 mg/mL
SDS. (E) TEM image of the uncross-linked mi-
celles in the presence of 2.5 mg/mL SDS. (F)
TEM image of the cross-linked micelles treated
with 10 mM GSH in the presence of 2.5 mg/mL
SDS.
indicated that a critical number of formed disulfide bonds was cleaved
resulting in the rapid destabilization and disruption of the micelles. The
morphology of uncross-linked and cross-linked micelles was further
observed by TEM in the end of stability study (Fig. 1E and F). It was
synergistically confirmed that the nanostructures of uncross-linked
micelles were destroyed in ionic detergent of SDS, while cross-linked
micelles retained the spherical particles in SDS but completely dis-
association in the presence of SDS and GSH.
inhibition of release between these two micelles is likely ascribed to the
disulfide crosslinking effect, sequestering PTX payload in the hydro-
phobic cores. As expected, the cross-linked micelles based on the redox-
sensitive MeO-PEG2k-SS-PTX prodrug exhibited an obvious GSH-de-
pendant manner of drug release. Appropriate 65.6% of PTX was re-
leased from the cross-linked micelles in 12 h and 92.6% in 72 h in-
cubated in PBS medium (pH 7.4, 0.05% Tween 80) containing 10 mM
GSH, which is in agreement with the level of intracellular redox mi-
croenvironment in tumor cells. These results highlighted that the ex-
istence of disulfide (–SS–) linkage in polymeric prodrugs plays a key
role in accelerating intracellular drug release. As reported in the lit-
erature [51], the release of PTX can be reasonably explained that the
cleavage of –SS– bonds in MeO-PEG2k-SS-PTX under the reductive agent
GSH generates HS-modificated PTX intermediate (PTX-SH) followed by
the reaction of nucleophilic substitution at the carbonate functionality
to release free parent PTX with a five-membered ring thiolactone de-
parturing (Scheme 3) as checked by HPLC technique in Fig. S8. Con-
clusively, the reduction-responsive cross-linked MeO-PEG2k-SS-PTX
micelles would undertake an enhanced intracellular PTX release but
maintain an inhibited release during circulation in blood, thereby
limiting the systemic cytotoxicity.
4.3. Drug release of the micelles
In vitro PTX release from the formulated micelles was investigated
by a dialysis method at 37 °C under two stimulated physiological en-
vironments: (i) 0.05% Tween/PBS, pH 7.4; (ii) 0.05% Tween/PBS (pH
7.4) + 10 mM GSH. The uncross-linked MeO-PEG2k-SS-PTX micelles
were used as a control. As a start, the stability of PTX in PBS containing
0.05% Tween 80 for was monitored to confirm the feasibility of in vitro
PTX release kinetics. Actually, about 90.3% of PTX was left after 72 h
incubation at 37 °C (Fig. S7), indicating its stability in Tween/PBS re-
lease medium satisfying long-time sampling. The cumulative release
profiles of the micelles was illustrated in Fig. 2. MeO-PEG2k-SS-PTX
micelles after crosslinking possessed higher colloidal stability than
uncross-lined counterparts in physiological condition (PBS, pH 7.4)
with < 15% of cumulative PTX release in 72 h, while the amount of
released PTX from uncross-linked micelles was up to 40.3%. The large
4.4. Cytotoxicity and apoptosis assay
MTT assay was conducted to examine the in vitro cytotoxicity of the
cross-linked MeO-PEG2k-DHLA and MeO-PEG2k-SS-PTX micelles against
MCF-7, A549 and BEL-7402 cells using free PTX as positive control and
the results were shown in Fig. 3A–C, 3G and Fig. S9. After 48 h in-
cubation, these blank MeO-PEG2k-DHLA micelles possessed nontoxic to
the tested cell lines (cell viabilities: 92.7%–98.1%) at the concentration
from 31.25 μg/mL to 2 mg/mL, indicating their excellent biocompat-
ibility. By contrast, the designed cross-linked MeO-PEG2k-SS-PTX mi-
celles induced favorable antitumor effect against these three tested cells
with a dose-dependent proliferation-inhibition manner. Notably, the
cell cytotoxicity of the cross-linked micelles was significantly stronger
than that of free PTX group, particularly at high concentration
(> 2.5 μM, *P < 0.05). It may be ascribed to the quick PTX release
from micelles in response to the reduction environment after inter-
nalized into tumor cells, while the low solubility of PTX in culture
medium also limits the cytotoxicity of free PTX group. IC50 (half max-
imal inhibitory concentration) was further calculated to quantitatively
assess their cytotoxicities. As showed in Fig. 3G, the IC50 values of the
Fig. 2. PTX release profiles of uncross-linked and cross-linked micelles in PBS
(pH 7.4) with or without 10 mM GSH at 37 °C. The value is reported as
average
S.D for triplicate samples.
6

Z. Wang, et al.
JournalofDrugDeliveryScienceandTechnology53(2019)101168
Fig. 3. In vitro cytotoxicity of (A) MCF-7, (B) A549 and (C) BEL-7402 cells after treatments with free PTX and cross-linked micelles for 48 h. Effect of incubation of (E)
free PTX and (F) micelles on cell apoptosis of MCF-7 (48 h, PTX concentration of 2 μM) and (D) the untreated cells were used as blank control, determined by flow
cytometry. (G) IC50 values of free PTX and micelles against the three cancer line. values are reported as average
S.D for triplicate samples and *P < 0.05 is set as
statistical significance.
cross-linked
0.351 0.072 μM for A549 cells and 0.325
7402 cells) were lower than those of free PTX (0.403
MCF-7 cells, 0.448 0.026 μM for A549 cells and 0.474
micelles
(0.353
0.069 μM
for
MCF-7 cells,
4.5. Pharmacokinetics and biodistribution
0.077 μM for BEL-
0.046 μM for
0.014 μM
Since nanoparticles were intravenously administrated into blood
circulation for drug delivery, destructive interactions between these
nanoparticles and blood components must be avoided [52,53]. Herein,
in vitro hemolysis analysis of MeO-PEG2k-SS-PTX prodrug-based mi-
celles was studied in detail and the degree of hemolysis incubated with
2% human blood for 4 h is illustrated in Fig. S11. As seen, the per-
centage of hemolysis ratio (HR) slightly increased as a function of in-
creasing mass MeO-PEG2k-SS-PTX concentrations with blood con-
stitutes. But for the overall HR% of each group, percentage less than 5%
indicated that the tested nanomaterials are safe for in vivo injections
without damage to red blood cells (RBCs).
for BEL-7402 cells), respectively. Furthermore, a mouse mammary
carcinoma cell line, 4T1, was used to evaluate the antitumor activity of
the MeO-PEG2k-SS-PTX crosslinked micelles. As indicated in Fig. S10,
the cross-linked micelles also exhibited higher antitumor activity than
that of free PTX. Based on the reduction-sensitive profile above and the
cytotoxicity results presented here, MeO-PEG2k-SS-PTX crosslinked
micelles could retain the antitumor efficiency of PTX, emphasizing the
contribution of GSH-triggered intracellular drug release in tumor cells.
To further investigate the evaluated apoptosis of reduction-sensitive
cross-linked micelles towards MCF-7 cells, Annexin V-FITC/PI double
staining assay was utilized to measure their apoptosis-inducing cap-
ability. Similar results of quantitative analysis in Fig. 3D–F were
achieved as the provided MTT assay. The order of apoptosis ratio (AR,
Q2 + Q3) was cross-linked MeO-PEG2k-SS-PTX micelles > free PTX.
Precisely, the percentage of AR was established to be 16.02% for the
micelles while the group of free PTX was 13.04% at the same con-
centration of 2 μM. The data differences further demonstrated the
higher cytotoxicity of the developed micelles.
Accordingly, in vivo pharmacokinetics of cross-linked MeO-PEG2k
-
SS-PTX micelles was evaluated by administrating the respective PTX
dosage at 5 mg/kg to 4T1-bearing BALB/c mice. Considering that
Taxol®, as a clinical case, is formulated with the 1:1 (v/v) mixture of
Cremophor EL and dehydrated alcohol for enhancing the solubility of
PTX in vivo, Taxol® was chosen as the positive control in this content.
Fig. 4A shows the plasma concentration−time profiles of two for-
mulations and the corresponding pharmacokinetic parameters were
calculated in Table 1. Apparently, MeO-PEG2k-SS-PTX micelles possess
an extended blood circulation time compared with Taxol®, with a 1.46-
fold longer elimination half-life (t1/2), 1.53-fold higher mean residence
7

Z. Wang, et al.
JournalofDrugDeliveryScienceandTechnology53(2019)101168
Fig. 4. In vivo (A) pharmacokinetic profile and (B) tissue distribution of Taxol® and cross-linked MeO-PEG2k-SS-PTX micelles at the equivalent dosage of 5 mg PTX/kg
(M S. D, n = 3). The statistical significance level is **P < 0.01.
5. Conclusion
Table 1
Pharmacokinetic parameters of Taxol® and cross-linked MeO-PEG2k-SS-PTX
micelles in 4T1-bearing BALB/c mice after i.v. administration at the equivalent
PTX dosage of 5 mg/kg (n = 3).
In summary, a novel disulfide cross-linked micelle based on MeO-
PEG2k-SS-PTX prodrug was successfully developed, which possess the
characteristics of high drug loading efficiency, rapid reduction-sensitive
drug release, the EPR-specific PEG detachment, prolonged blood cir-
culation and passively accumulation of PTX to tumors in vivo. The re-
sults presented here drastically demonstrated these reduction-re-
sponsive prodrug micelles via crosslinking are able to maintain the
chemotherapic capacity of PTX in delivering system and inhibit tumor
cells growth effectively both in vitro and in vivo. These PTX prodrug
micelles show a great potential as versatile platform and may be further
studied for combined applications with other drugs or tumor-targeted
drug delivery.
aParameters (unit)
Taxol®
MeO-PEG2k-SS-PTX micelles
AUC0-t (mg/L·h)
MRT0-inf (h)
t1/2 (h)
80.76
3.69
2.62
0.16
0.029
23.56
0.5
152.17
5.63
3.83
Vz (L/kg)
0.22
CL (L/h/kg)
Cmax (μg/mL)
Tmax
0.006
26.46
0.5
a
Parameters: AUC, area under the curve from zero to time t; MRT0-inf, re-
sidence time; t1/2, elimination half-life; CL, plasma clearance; Vz, apparent
volume of distribution during elimination phase; Cmax, peak plasma con-
centration; Tmax, peak plasma time.
Conflicts of interest
We declare that we have no financial and personal relationships
with other people or organizations that can inappropriately influence
our work, there is no professional or other personal interest of any
nature or kind in any product, service and/or company that could be
construed as influencing the position presented in, or the review of this
paper.
time (MRT0-inf), 1.88-fold larger area under the curve (AUC0-t) and the
decreased value of plasma clearance (CL, 0.006 L/h/kg), which can
facilitate the accumulation of payload in micelles to tumor sites. The
better performance of MeO-PEG2k-SS-PTX micelles in vivo might be
ascribed to the following several aspects: (i) Nanoscaled character of
cross-linked micelles (< 100 nm) allows them significantly circumvent
the rapid uptake of reticular endothelial system (RES); (ii) The com-
bination of core crosslinking and surface modified by PEG segment
results in the strong stability in physiological environment without
disassembly as well as declining plasma elimination. These results were
in accordance with previously published data, where a manifold in-
crease in the plasma concentrations was observed after PEGylation.
In order to observe the passive targeting accumulation of cross-
linked micelles to 4T1-bearing BALB/c tumors, different tissue biodis-
tribution including tumor, heart, liver, spleen lung and kidney of PTX
was investigated at designed time intervals. As shown in Fig. 4B, PTX in
cross-linked micelles was rapidly distributed into the majority of tissues
after administration through tail vein. The maximum concentration of
PTX was recorded in liver, followed the order of kidney and spleen
within 1 h post-injection. Although MeO-PEG2k-SS-PTX micelles and
Taxol® held the similar effect of PTX biodistribution in mice, significant
enhanced tumor accumulation of micelles was acquired compared with
Taxol®. At 1 h time period, the amount of PTX checked by HPLC was
15.65 μg/g that is approximately equivalent to Taxol® group (15.02 μg/
g). Subsequently, its concentration passively targeted in tumor sites was
1.23, 1.45 and 1.47 times higher than that of Taxol® group at 3 h, 6 h
and 12 h, respectively. It was supported that the reduction-responsive
PEGylated PTX micelles can efficiently inhibit the fast clearance of RES
system but passively accumulate in tumor tissue owing to the EPR ef-
fects, offering a promising perspective for ideal evaluations of in vivo
antitumor activity.
Acknowledgement
This research was supported by the Major National Science and
Technology Program for Innovative Drug from the Ministry of Science
and Technology of China (2017ZX09101002-001-004).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jddst.2019.101168.
References
[1] P. Sapra, T. Allen, Ligand-targeted liposomal anticancer drugs, Prog. Lipid Res. 42
(5) (2003) 439–462.
[2] T. Kubo, T. Sugita, S. Shimose, et al., Targeted delivery of anticancer drugs with
intravenously administered magnetic liposomes in osteosarcoma-bearing hamsters,
Int. J. Oncol. 17 (2) (2000) 309–324.
[3] H. Cabral, K. Kataoka, Progress of drug-loaded polymeric micelles into clinical
studies, J. Control. Release 190 (2014) 465–476.
[4] E. Kadife, E. Chan, R. Luwor, et al., Paclitaxel-induced Src activation is inhibited by
dasatinib treatment, independently of cancer stem cell properties, in a mouse model
of ovarian cancer, Cancers 11 (2) (2019) 243.
[5] A. Potineni, D.M. Lynn, R. Langer, et al., Poly (ethylene oxide)-modified poly (β-
amino ester) nanoparticles as a pH-sensitive biodegradable system for paclitaxel
delivery, J. Control. Release 86 (2–3) (2003) 223–234.
[6] A. Dalla Pria, M. Bower, AIDS-related Malignant Disease, (2018) Medicine.
[7] C. Xu, H. Li, K. Zhang, et al., Photo-controlled release of paclitaxel and model drugs
from RNA pyramids, Nano Res. 12 (1) (2019) 41–48.
[8] S. Hussain, D.N. Anderson, M.E. Salvatti, et al., Onycholysis as a complication of
systemic chemotherapy: report of five cases associated with prolonged weekly
8

Z. Wang, et al.
JournalofDrugDeliveryScienceandTechnology53(2019)101168
[32] L.H. Reddy, J.L. Arias, J. Nicolas, et al., Magnetic nanoparticles: design and char-
acterization, toxicity and biocompatibility, pharmaceutical and biomedical appli-
cations, Chem. Rev. 112 (11) (2012) 5818–5878.
[33] A.N. Koo, H.J. Lee, S.E. Kim, et al., Disulfide-cross-linked PEG-poly (amino acid) s
copolymer micelles for glutathione-mediated intracellular drug delivery, Chem.
Commun. 48 (2008) 6570–6572.
[34] Y. Li, K. Xiao, W. Zhu, et al., Stimuli-responsive cross-linked micelles for on-demand
drug delivery against cancers, Adv. Drug Deliv. Rev. 66 (2014) 58–73.
[35] R.K. O'Reilly, C.J. Hawker, K.L. Wooley, Cross-linked block copolymer micelles:
functional nanostructures of great potential and versatility, Chem. Soc. Rev. 35 (11)
(2006) 1068–1083.
paclitaxel therapy and review of the literature, Cancer 88 (10) (2000) 2367–2371.
[9] A.J. Montero, B. Adams, C.M. Diaz-Montero, et al., Nab-paclitaxel in the treatment
of metastatic breast cancer: a comprehensive review, Expert Rev. Clin. Pharmacol. 4
(3) (2011) 329–334.
[10] T.B. Stage, T.K. Bergmann, D.L. Kroetz, Clinical pharmacokinetics of paclitaxel
monotherapy: an updated literature review, Clin. Pharmacokinet. 57 (1) (2018)
7–19.
[11] A. Kumar, H. Kaur, A. Singh, Neuropathic Pain models caused by damage to central
or peripheral nervous system, Pharmacol. Rep. 70 (2) (2018) 206–216.
[12] C.A. Rabik, M.E. Dolan, Molecular mechanisms of resistance and toxicity associated
with platinating agents, Cancer Treat Rev. 33 (1) (2007) 9–23.
[13] W. Xiao, H. Zheng, G. Bennett, Characterization of oxaliplatin-induced chronic
painful peripheral neuropathy in the rat and comparison with the neuropathy in-
duced by paclitaxel, Neuroscience 203 (2012) 194–206.
[36] Y. Che, A. Datar, K. Balakrishnan, et al., Ultralong nanobelts self-assembled from an
asymmetric perylene tetracarboxylic diimide, J. Am. Chem. Soc. 129 (23) (2007)
7234–7235.
[14] J. Bhattacharyya, J.J. Bellucci, I. Weitzhandler, et al., A paclitaxel-loaded re-
combinant polypeptide nanoparticle outperforms Abraxane in multiple murine
cancer models, Nat. Commun. 6 (2015) 7939.
[37] F. Li, Y. Zhu, B. You, et al., Smart hydrogels Co‐switched by hydrogen bonds and
π–π stacking for continuously regulated controlled‐release system, Adv. Funct.
Mater. 20 (4) (2010) 669–676.
[15] Q. Chen, C. Liang, C. Wang, et al., An imagable and photothermal “Abraxane‐like”
nanodrug for combination cancer therapy to treat subcutaneous and metastatic
breast tumors, Adv. Mater. 27 (5) (2015) 903–910.
[38] T. Suzuki, S. Shinkai, K. Sada, Supramolecular crosslinked linear poly (trimethylene
iminium trifluorosulfonimide) polymer gels sensitive to light and thermal stimuli,
Adv. Mater. 18 (8) (2006) 1043–1046.
[16] Y. Matsumura, K. Kataoka, Preclinical and clinical studies of anticancer agen-
t‐incorporating polymer micelles, Cancer Sci. 100 (4) (2009) 572–579.
[17] J. Nicolas, S. Mura, D. Brambilla, et al., Design, functionalization strategies and
biomedical applications of targeted biodegradable/biocompatible polymer-based
nanocarriers for drug delivery, Chem. Soc. Rev. 42 (3) (2013) 1147–1235.
[18] J. Thévenot, H. Oliveira, O. Sandre, et al., Magnetic responsive polymer composite
materials, Chem. Soc. Rev. 42 (17) (2013) 7099–7116.
[39] M. Talelli, M. Iman, A.K. Varkouhi, et al., Core-crosslinked polymeric micelles with
controlled release of covalently entrapped doxorubicin, Biomaterials 31 (30) (2010)
7797–7804.
[40] C.F. Van Nostrum, Covalently cross-linked amphiphilic block copolymer micelles,
Soft Matter 7 (7) (2011) 3246–3259.
[41] A.E. Ferentz, G.L. Verdine, Disulfide-crosslinked oligonucleotides, J. Am. Chem.
Soc. 113 (10) (1991) 4000–4002.
[19] A.Z. Wilczewska, K. Niemirowicz, K.H. Markiewicz, et al., Nanoparticles as drug
[42] L. Ling, Y. Du, M. Ismail, et al., Self-assembled liposomes of dual paclitaxel-phos-
pholipid prodrug for anticancer therapy, Int. J. Pharm. 526 (1–2) (2017) 11–22.
[43] X. Zhang, K. Liu, Y. Huang, et al., Reduction-sensitive dual functional nanomicelles
for improved delivery of paclitaxel, Bioconjug. Chem. 25 (9) (2014) 1689–1696.
[44] D. Fischer, Y. Li, B. Ahlemeyer, et al., In vitro cytotoxicity testing of polycations:
influence of polymer structure on cell viability and hemolysis, Biomaterials 24 (7)
(2003) 1121–1131.
delivery systems, Pharmacol. Rep. 64 (5) (2012) 1020–1037.
[20] T.M. Allen, P.R. Cullis, Drug delivery systems: entering the mainstream, Science 303
(5665) (2004) 1818–1822.
[21] H. Maeda, G. Bharate, J. Daruwalla, Polymeric drugs for efficient tumor-targeted
drug delivery based on EPR-effect, Eur. J. Pharm. Biopharm. 71 (3) (2009)
409–419.
[22] A. MaHam, Z. Tang, H. Wu, et al., Protein‐based nanomedicine platforms for drug
[45] G. Lv, D. Sun, J. Zhang, et al., Lx2-32c, a novel semi-synthetic taxane, exerts an-
titumor activity against prostate cancer cells in vitro and in vivo, Acta Pharm. Sin. B
7 (1) (2017) 52–58.
[46] N. Murthy, I. Chang, P. Stayton, et al., pH‐sensitive hemolysis by random copoly-
mers of alkyl acrylates and acrylic acid, Proceedings of the Macromolecular
Symposia, Wiley Online Library, 2001F.
delivery, Small 5 (15) (2009) 1706–1721.
[23] S. Li, X. Wang, W. Li, et al., Preparation and characterization of a novel conformed
bipolymer paclitaxel-nanoparticle using tea polysaccharides and zein, Carbohydr.
Polym. 146 (2016) 52–57.
[24] Z. Liu, Y. Jiao, Y. Wang, et al., Polysaccharides-based nanoparticles as drug delivery
systems, Adv. Drug Deliv. Rev. 60 (15) (2008) 1650–1662.
[47] Y. Gu, Y. Zhong, F. Meng, et al., Acetal-linked paclitaxel prodrug micellar nano-
particles as a versatile and potent platform for cancer therapy, Biomacromolecules
14 (8) (2013) 2772–2780.
[48] M. Moser, T. Behnke, C. Hamers-Allin, et al., Quantification of PEG-maleimide li-
gands and coupling efficiencies on nanoparticles with Ellman's reagent, Anal. Chem.
87 (18) (2015) 9376–9383.
[49] B.L. Hogan, E.S. Yeung, Determination of intracellular species at the level of a single
erythrocyte via capillary electrophoresis with direct and indirect fluorescence de-
tection, Anal. Chem. 64 (22) (1992) 2841–2845.
[50] L. Yan, W. Wu, W. Zhao, et al., Reduction-sensitive core-cross-linked mPEG–poly
(ester-carbonate) micelles for glutathione-triggered intracellular drug release,
Polym. Chem. 3 (9) (2012) 2403–2412.
[51] X. Hu, J. Hu, J. Tian, et al., Polyprodrug amphiphiles: hierarchical assemblies for
shape-regulated cellular internalization, trafficking, and drug delivery, J. Am.
Chem. Soc. 135 (46) (2013) 17617–17629.
[52] P. Aggarwal, J.B. Hall, C.B. McLeland, et al., Nanoparticle interaction with plasma
proteins as it relates to particle biodistribution, biocompatibility and therapeutic
efficacy, Adv. Drug Deliv. Rev. 61 (6) (2009) 428–437.
[25] Y. Bao, Y. Guo, X. Zhuang, et al., D-α-tocopherol polyethylene glycol succinate-
based redox-sensitive paclitaxel prodrug for overcoming multidrug resistance in
cancer cells, Mol. Pharm. 11 (9) (2014) 3196–3209.
[26] H.-J. Youk, E. Lee, M.-K. Choi, et al., Enhanced anticancer efficacy of α-tocopheryl
succinate by conjugation with polyethylene glycol, J. Control. Release 107 (1)
(2005) 43–52.
[27] Z. Zhang, S.-S. Feng, The drug encapsulation efficiency, in vitro drug release, cel-
lular uptake and cytotoxicity of paclitaxel-loaded poly (lactide)–tocopheryl poly-
ethylene glycol succinate nanoparticles, Biomaterials 27 (21) (2006) 4025–4033.
[28] H.S. Abandansari, M.R. Nabid, S.J.T. Rezaei, et al., pH-sensitive nanogels based on
Boltorn® H40 and poly (vinylpyridine) using mini-emulsion polymerization for
delivery of hydrophobic anticancer drugs, Polymer 55 (16) (2014) 3579–3590.
[29] M. Agüeros, V. Zabaleta, S. Espuelas, et al., Increased oral bioavailability of pacli-
taxel by its encapsulation through complex formation with cyclodextrins in poly
(anhydride) nanoparticles, J. Control. Release 145 (1) (2010) 2–8.
[30] M. Shah, V. Shah, A. Ghosh, et al., Molecular inclusion complexes of β-cyclodextrin
derivatives enhance aqueous solubility and cellular internalization of paclitaxel:
preformulation and in vitro assessments, J. Pharm. Pharmacol. 2 (2) (2015) 8.
[31] K.L. Aillon, Y. Xie, N. El-Gendy, et al., Effects of nanomaterial physicochemical
properties on in vivo toxicity, Adv. Drug Deliv. Rev. 61 (6) (2009) 457–466.
[53] Y. Yang, D. Guan, L. Lei, et al., H6, a novel hederagenin derivative, reverses mul-
tidrug resistance in vitro and in vivo, Toxicol. Appl. Pharmacol. 341 (2018) 98–105.
9