Novel multi-stimuli responsive functionalized PEG-based co-delivery nanovehicles toward sustainable treatments of multidrug resistant tumor

Article abstract of DOI:10.1039/d0tb02192j

The efficacy of ongoing anticancer treatment is often compromised by some barriers, such as low drug content, nonspecific release of drug delivery system, and multidrug resistance (MDR) effect of tumors. Herein, in the research a novel functionalized PEG-based polymer cystine-(polyethylene glycol)₂-b-(poly(2-methacryloyloxyethyl ferrocenecarboxylate)₂) (Cys-(PEG₄₅)₂-b-(PMAOEFC)₂) with multi-stimuli sensitive mechanism was constructed, in which doxorubicin (DOX) was chemical bonded through Schiff base structure to provide acid labile DOX prodrug (DOX)₂-Cys-(PEG₄₅)₂-b-(PMAOEFC)₂. Afterwards, paclitaxel (PTX) and its diselenide bond linked PTX dimer were encapsulated into the prodrug through physical loading, to achieve pH and triple redox responsive (DOX)₂-Cys-(PEG₄₅)₂-b-(PMAOEFC)₂^@PTXand (DOX)₂-Cys-(PEG₄₅)₂-b-(PMAOEFC)₂^@PTX?dimerwith ultrahigh drugs content. The obtained nanovehicles could self-assemble into globular micelles with good stability based on fluorescence spectra and TEM observation. Moreover, there was a remarkable “reassembly-disassembly” behavior caused by phase transition of micelles under the mimic cancerous physiological environment. DOX and PTX could be on-demand released in acid and redox stress mode, respectively. Meanwhile,in vivoanticancer studies revealed the significant tumor inhibition of nanoformulas. This work offered facile strategies to fabricate drug nanaovehicles with tunable drug content and types, it has a profound significance in overcoming MDR effect, which provided more options for sustainable cancer treatment according to the desired drug dosage and the stage of tumor growth.

Full text of DOI:10.1039/d0tb02192j

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Materials Chemistry B
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Novel multi-stimuli responsive functionalized
PEG-based co-delivery nanovehicles toward
sustainable treatments of multidrug resistant
Cite this: J. Mater. Chem. B, 2021,
9, 1297
tumor†
b
Jingwen Xu, *a Xiangji Yan,b Xin Ge,c Mingzhen Zhang,
Xugang Dang,d
e
a
Yan Yang,a Feng Xu, *e Yanling Luo
and Guoliang Li
The efficacy of ongoing anticancer treatment is often compromised by some barriers, such as low drug
content, nonspecific release of drug delivery system, and multidrug resistance (MDR) effect of tumors.
Herein, in the research a novel functionalized PEG-based polymer cystine–(polyethylene glycol)2-b-
(poly(2-methacryloyloxyethyl ferrocenecarboxylate)2) (Cys–(PEG45)2-b-(PMAOEFC)2) with multi-stimuli
sensitive mechanism was constructed, in which doxorubicin (DOX) was chemical bonded through Schiff
base structure to provide acid labile DOX prodrug (DOX)2–Cys–(PEG45)2-b-(PMAOEFC)2. Afterwards,
paclitaxel (PTX) and its diselenide bond linked PTX dimer were encapsulated into the prodrug through
@PTX
physical loading, to achieve pH and triple redox responsive (DOX)2–Cys–(PEG45)2-b-(PMAOEFC)2
@PTX dimer
and (DOX)2–Cys–(PEG45)2-b-(PMAOEFC)2
with ultrahigh drugs content. The obtained
nanovehicles could self-assemble into globular micelles with good stability based on fluorescence
spectra and TEM observation. Moreover, there was a remarkable ‘‘reassembly–disassembly’’ behavior
caused by phase transition of micelles under the mimic cancerous physiological environment. DOX and
PTX could be on-demand released in acid and redox stress mode, respectively. Meanwhile, in vivo
anticancer studies revealed the significant tumor inhibition of nanoformulas. This work offered facile
strategies to fabricate drug nanaovehicles with tunable drug content and types, it has a profound
significance in overcoming MDR effect, which provided more options for sustainable cancer treatment
according to the desired drug dosage and the stage of tumor growth.
Received 10th September 2020,
Accepted 22nd December 2020
DOI: 10.1039/d0tb02192j
rsc.li/materials-b
segments,2 providing a loading platform for hydrophobic drugs
1. Introduction
via chemical bonding or physical loading.3–5 Even so, it still
Currently, chemotherapy, an effective therapeutic means for inevitably suffers from some drawbacks, including low drug
cancer, has been confined by water-insolubility, non-selectivity, content and the resulting insufficient drug concentration at
limited stability and poor targeting of chemotherapeutic cancerous site. To tackle these issues, although increasing drug
agents.1 In the past decade, the amphiphilic polymers have dosage or multiple injections is an effective method for elevating
been deeply investigated as a hopeful anticancer drug carriers local drug concentration, it could cause severe side effects and
due to its bearing both hydrophilic and hydrophobic multidrug resistance (MDR) effect due to long-term usage, and
ultimately compromise the treatment efficacy.6,7
a School of Food and Biological Engineering, Shaanxi University of Science and
Technology, Xi’an 710021, China. E-mail: xujingwen@sust.edu.cn
b Institute of Medical Engineering, School of Basic Medical Sciences,
Xi’an Jiaotong University, Xi’an, Shaanxi Province, China
c The First Affiliated Hospital of USTC, Division of Life Science and Medicine,
University of Science and Technology of China, Hefei, Anhui, 230001, China
d College of Bioresources Chemical and Materials Engineering,
Shaanxi University of Science & Technology, Xi’an 710021, China
e Key Laboratory of Macromolecular Science of Shaanxi Province,
School of Chemistry and Chemical Engineering, Shaanxi Normal University,
Xi’an 710062, China. E-mail: fengxu@snnu.edu.cn
Stimuli responsive drug delivery systems (DDSs) always
contain certain components and active bonds that respond to
specific physiological signals at tumor region, which is particularly
appealing for cancer treatment.8 Up to now, polymethacrylic acid
and poly(2-(dimethylamino)ethyl methacrylate) have been intro-
duced in the backbone of pH responsive polymeric DDSs to control
drug release at cancerous weak acidic site by ‘‘protonation–depro-
tonation’’ circulation.9–11 Disulfide or diselenide bond containing
DDSs can be reduced into mercaptan or selenol by 2–10 mM
glutathione (GSH), and the latter also can be oxidized into selenic
acid by 10–100 mM H2O2 ROS. Specially, the bonding energy of
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0tb02192j
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diselenide bond is lower than that of disulfide bond, leading to a overcome MDR via rapid intracellular drug delivery.39,40
smart redox responsive behavior and synchronous drug release Herein, a PEG-based doxorubicin (DOX) prodrug containing
when diselenide bonds contained carrier crack. After that, selenium hydrazone (doxorubicin)2–cystine–(polyethylene glycol)2-b-(poly
in carriers not only can be used to enrich the trace elements that (2-methacryloyloxyethyl ferrocenecarboxylate)2) ((DOX)2–Cys–
necessary to body, but also relieve multidrug-resistance (MDR) (PEG45)2-b-(PMAOEFC)2) was prepared. With respect to the
effect.6,12,13 As a kind of famous eletroactive component, ferrocene different therapeutic mechanism of both DOX and paclitaxel
is liable to oxidize into ferrocenium by H2O2, KMnO4, NaClO and (PTX),41–43 and the potential of dual drugs co-delivery system
FeCl3, and it is apt to be reduced into ferrocene by vitamin C for overcoming MDR effect, diselenide bond linked PTX (PTX
inversely.14,15 Based on few previous studies, the synergistic of dimer) was synthesized, followed by encapsulating into the
ferrocene contained DDSs responded to dual and even multiple obtained prodrug through hydrophobic, p–p stacking and
special physiological signals will be an effective strategy for hydrogen bonding interactions. This system can afford a dual drugs
realizing site-specific, controlled and targeted release.16
co-delivery system (DOX)2–Cys–(PEG45)2-b-(PMAOEFC)2
@PTX dimer
Polymeric DDSs constructed via physical loading are always with 39.3% of DOX and 49.1% of PTX, this value far exceeds
of low drug content than 20% and poor stability due to drug those reported elsewhere.23,44 It can self-assemble into globular
bound with polymer through p–p stacking, hydrophobic and micellar aggregates bearing DOX, hydrophobic core of DOX,
hydrogen bonding interactions, even though the prodrug can PTX, PMAOEFC and hydrophilic PEG shell in aqueous solution,
be a promising vehicle by chemical bonding between drug and and critical micellar concentration as low as 5.78 mg Lꢀ1 from
polymer.17,18 Meanwhile, the inadequate reactive sites of pro- fluorescence spectrum. Due to the presence of Schiff base
drugs in polymer backbone also restrict the increase of drug structure, the break of hydrazone bond and resulting pH
content.19–22 Sun et al. fabricated a disulfide bond linked induced DOX diffuse behavior were confirmed by 1H NMR,
camptothecin (CPT) prodrug PCPTSP-co-PEEP with CPT content dynamic light scattering (DLS) and fluorescence spectrums.
ranged from 7.8–27.2%.23 Generally, a feasible approach to Additionally, the micellar disassembly-reassembly process
prodrug functionalization is to introduce various of stimulus caused by triple redox responsiveness was investigated by
responsive linkages such as hydrazone, disulfide, diselenide DLS, TEM and cyclic voltammetry (CV). Results found that it
bond and so on,24–26 the resulting prodrugs can be broken is a valid strategy to develop DDS with high drug content. The
under their corresponding physiological stimulus. However, it prepared nanovehicles had good stability and satisfactory
is still a pendent problem that an uncertain induction process therapeutic effect by designing stimuli responsive polymer
retards the whole release behavior. DDSs with PEG shell are and loading hydrophobic drug dimer afterwards, which can
particularly hidden and can prevent aggregation,27,28 prolong also make up the deficiency of delayed drug release of prodrugs
circulation time, and avoid clearance through making DDSs and evade MDR effect, realizing a sustained, on-demand and
highly resistant to protein adsorption.29,30 In addition, drug loading targeted release. The research opens up a variety of treatment
and release behavior are optimized by providing a physico- modes to tumors at different stages.
chemical barrier to drug leak via PEG,31–33 and the electrostatic
and steric hindrances of PEG improve transport coefficients in
mucus, obtaining a satisfactory drug delivery efficacy.34–36
Based on the complications of cancer therapy and MDR
effect, in the research such novel polymeric carriers are
2. Materials and methods
2.1 Materials and reagents
expected to have three main advantages: (1) good stability (Boc–Cys–OH)2 (98%), trifluoroacetic acid (TFA, 99%), tetrabuty-
due to a reasonable hydrophobic/hydrophilic blocks balance, lammonium hexafluorophosphate (TBAHFP, 98%), glutathione
(2) adjustable drug types and content thanks to drug and its (GSH, 98%) and L-ascorbic acid (Vc, 499.0%) were provided by
dimer encapsulation through chemical bonding and physical Shanghai Macklin Biochemical Co., Ltd (Shanghai, China).
loading, and (3) desired release behavior triggered by cancerous Polyethylene glycol 2000 (PEG2000), 2-bromoisobutyryl bromide
physiological signals. For this aim, the combination of triple (BiBB, 98%), paclitaxel (PTX, 98%), doxorubicin hydrochloride
redox responsive mechanism including ferrocene, disulfide (DOXꢁHCl, 98%) were purchased from J&K Chemical Ltd
and diselenide groups has great significance for specific, (Beijing, China). Sodium borohydride (NaBH4, 96%), anhydrous
targeted and controlled drug release, which can conduce to sodium sulfate (Na2SO4, Z99.0%), dichloromethane (DCM),
enhance drug delivery efficacy through synergistic effect. Var- N,N0-dimethylformamide (DMF), tetrahydrofuran (THF) and
ious of evidences showed that the enhanced activity of efflux triethylamine (TEA) were supplied by Sinopharm Chmical
pumps is one of the most common mechanisms involved in Reagent Co. Ltd (Shanghai, China). DCM was pre-dried in
cancer processes. Meanwhile, the membrane transport proteins anhydrous Na2SO4, and then purified in CaH2 for 4–5 h to
(particularly P-glycoprotein) can able to recognize and expel remove moisture. For DMF, were also dried over CaH2 and
various of exogenous hydrophobic drug molecules (such as reduced pressure distilled prior to use. 3-(4,5-Dimethyl-thiazol-
PTX, DOX, docetaxel and so on) out of the cell. Previous 2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 98%) was provided
researches indicated that the selenium compounds have a by Sigma and without further treatment. Unless otherwise
significant inhibitory ability to reverse MDR.37,38 In addition, specified, all other chemicals were brought from commercial
a rich variety of drugs and high drug loading content can supplier and without additional purification.
1298 | J. Mater. Chem. B, 2021, 9, 1297ꢀ1314
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2.2 Cells and animals
The human cervical cancer cell line HeLa and murine breast
cancer cell line 4T1 were offered by Mingjin Biology (Shanghai,
China). The cells were cultured in RPMI 1640 supplemented
with 10% fetal calf serum (Gibco, America) under the humidi-
fied conditions containing 5% CO2 atmosphere. Digestion and
selection of cells were carried out during exponential growth
phase for in vitro MTT assay and in vivo antitumor experiments.
Male Balb/c mice (20 ꢂ 2 g) were purchased from Laboratory
Animal Center of Xi’an Jiaotong University and picked for
establishing 4T1 tumor bearing model. Animals were fed with
chow and water under the condition of 25 1C and 55% humid-
ity. All animal experiments were performed under the permis-
sion of the Experimental Animal Ethics Committee of Xi’an
Jiaotong University Health Center. The mice were devided
randomly into 7 groups (n = 5), 100 mL of 4T1 cell suspension
with 1 ꢃ 106 cells was inoculated subcutaneously at the armpit
of mice afterwards. All treatments were performed when the
tumor size was approximately 100 mm3 (tumor size = ab2/2,
wherein a and b denote the longest and shortest edge of tumor,
respectively).
2.3 Synthetic procedure
The Schiff based linked DOX prodrug (DOX)2-b-Cys-(PEG)2-b-
(PMAOEFC)2 was synthesized in line with the route shown in
Scheme 1.
2.3.1 Synthesis of (Boc)2–Cys–(PEG45)2. (Boc–Cys–OH)2
(0.30 g, 0.68 mmol) and PEG (3.26 g, 1.63 mmol) were dissolved
in anhydrous DCM (50 mL), then N,N0-dicyclohexycarbodiimide
(DCC, 0.42 g, 2.05 mmol) was added into the above solution
with nitrogen inletting. After stirring for 48 h at 25 1C, insoluable
N,N0-dicyclohexylurea (DCU) was removed by filtration, the
mixture was precipitated in excess ether thrice, collected
and vacuum-dried overnight at 25 1C to afford white solid. After-
wards, the crude product was purified by encasing in dialysis tube
(MWCO = 3500 Da) and dialyzed against 2000 mL of deionized
water for 72 h to remove the unreacted PEG, followed by collecting
Scheme 1 Synthetic route of Schiff base linked DOX prodrug (DOX)2–
Cys–(PEG)2-b-(PMAOEFC)2.
2.3.3 Synthesis of (Boc)2–Cys–(PEG45)2-b-(PMAOEFC)2. The
the dialysate, freeze drying, to afford (Boc)2–Cys–(PEG45)2. (Yield: synthesis procedure of ferrocene monomer 2-methacryloyloxyethyl
31%) (1H NMR: 300 MHz, CDCl3), d (ppm): 3.55–3.77 (s, 180H, ferrocenecarboxylate (MAOEFC) was depicted in ESI.† The
–CH2CH2OH in PEG2000), 3.18 (m, 2H, –SCH2CH(NHBoc)– in amphiphilic block copolymer (Boc)2–Cys–(PEG45)2-b-(PMAOEFC)2
ꢀ ꢀ ꢀ ꢀ
ꢀ ꢀ
(Boc–Cys–OH)2), 1.45 ppm (s, 9H, C(CH3)3OOC–); FTIR (film): n was synthesized through atom transfer radical polymerization
ꢀ ꢀ
(cmꢀ1): 1007–1214 (C–O stretching vibration), 1726 (ester –CQO), (ATRP). Specifically, macromolecular initiator (Boc)2–Cys–(PEG45)2–
2848–2981 (–C–H stretching vibration), 3080–3755 (–OH).
Br2 (0.30 g, 6.38 ꢃ 10ꢀ2 mmol) and monomer MAOEFC (1.75 g,
2.3.2 Synthesis of macromolecular initiator (Boc)2–Cys– 5.10 mmol) were dissolved in anhydrous DMF (6 mL), and then
(PEG45)2–Br2. (Boc)2–Cys–(PEG45)2 (1.00 g, 0.227 mmol) was transferred into a 25 mL of Schlenk flask. Subsequently, PMDETA
dissolved in anhydrous DCM (8 mL) with stirring. Dissolved (79.5 mL, 0.383 mmol) and CuCl (31.6 mg, 0.319 mmol) were added
BiBB (84.17 mL, 0.681 mmol) in DCM (3 mL) was dropwise swiftly under nitrogen atmosphere. After that, ‘‘freeze–pump–thaw’’
added in the above solution with nitrogen inletting at 0 1C. cycles were performed thrice to expel oxygen in the system. After the
Following TEA (126 mL, 0.908 mmol) in DCM (2 mL) was reaction proceeded at 90 1C for 48 h, the mixture was diluted with
dropwise added into the mixed liquor. The reaction was carried THF, and then passed through neutral aluminum column to
out at 25 1C for 24 h. The resulting mixture was filtrated, removed remove ligand and catalyst. The mixture was rotary evaporated
solvent by rotary evaporation, and precipitated using excess ether and repeatedly purified by precipitating in excess ice ether
for several times, dried at 25 1C for 24 h to afford white solid thrice, the final brown precipitate was vacuum dried at
(Boc)2–Cys–(PEG45)2–Br2. (Yield: 36%) (1H NMR: 300 MHz, 40 1C for 24 h. (Yield: 38%) (1H NMR: 300 MHz, CDCl3),
CDCl3), d (ppm): 1.87 (s, 6H, –C(CH3)2Br from BiBB residues).
d (ppm): 1.68 (56H, m, –CH2C(CH3) BrCOO– in main chains),
ꢀ ꢀ
ꢀ ꢀ
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1.09 (84H, m, –CH2C(CH3)BrCOO–), 4.40 (112H, m, –COOCH2CH2- The molecular weight and polydispersity index (PDI) of
ꢀ ꢀ
ꢀ ꢀ ꢀ ꢀ
OCO–), 4.04 (5H in ferrocenyl protons), 4.20 (2H, s, meta-H in Cp products were measured using a gel permeation chromatogra-
rings), 4.80 (2H, s, ortho-H in Cp rings); FTIR (film): n (cmꢀ1): 427– phy (GPC, Waters Corp., USA) system equipped with Waters
548 (Fe-Cp), 2864–2949 (–C–H stretching vibration), 1717 (ester 515 pump and 2410 differential refractive index detector, and
–CQO), 3099 (QC–H), 1638 (CQC), 1061–1211 (C–O stretching linear polystyrene was used as calibrated standards. Chromatogra-
vibration), 783–862 (bending vibration ofQC–H in Cp rings).
2.3.4 Synthesis of Cys–(PEG45)2-b-(PMAOEFC)2. Cys–(PEG45)2-
phically pure THF was served as eluent at flow rate of 1 mL minꢀ1
The critical micelle concentration (CMC) is a representative
.
b-(PMAOEFC)2 was achieved through the deprotection of measurement for self-assembly behavior investigation, which
(Boc)2–Cys–(PEG45)2-b-(PMAOEFC)2. Typically, (Boc)2–Cys–(PEG45)2- was performed on a FluoroMax-4 spectrofluorophotometer
b-(PMAOEFC)2 (0.98 g, 0.041 mmol) was dissolved in DCM–TFA (HORIBA Scientific, America) employing pyrene as probe.
mixed solvent (15 mL, vDCM/vTFA = 2/1), the reaction was Specifically, 4.8 mL of pyrene–acetone solution (5 ꢃ 10ꢀ4 M)
performed at 25 1C. After 3 hours, the mixture was concentrated was added in sample tube, and dried to remove solvent com-
via rotary evaporation, then precipitated in excess ether thrice, pletely, followed by adding the micelles with the concentration
and vacuum dried to afford black solid Cys–(PEG45)2-b- from 5 ꢃ 10ꢀ4 to 1 mg mLꢀ1 into the corresponding tube under
(PMAOEFC)2, which was represented as P hereinafter. (Yield: stirring overnight. The spectroscopy was recorded under the
85%) (1H NMR: 300 MHz, CDCl3), d (ppm): 4.20 (5H in condition of the excitation wavelength at 330, and the emission
ferrocenyl protons), 4.34 (2H, s, meta-H in Cp rings), 4.80 (2H, wavelength ranged 350–500 nm, respectively, All the slit width
s, ortho-H in Cp rings); FTIR (film): n (cmꢀ1): 436–556 (Fe-Cp), was 2 nm. The CMC value was calculated from the intersection
1082–1225 (C–O stretching vibration), 3299–3662 (–N–H stretching of two tangents according to the fitted curves of the ratio of
vibration), 1133 (C–N stretching vibration), 3074–3149 (QC–H).
fluorescence intensity I383/I372 versus the logarithm of micellar
2.3.5 Synthesis of Schiff base linked DOX prodrug (DOX)2– concentration (logC).
Cys–(PEG45)2-b-(PMAOEFC)2. The prodrug (DOX)2–Cys–(PEG45)2-
The hydrodynamic diameter (Dh) and polydispersity index
b-(PMAOEFC)2 was fabricated through Schiff base linking (PDI) of the fresh micelles (500 mg Lꢀ1) were monitored by a
between DOX and Cys–(PEG45)2-b-(PMAOEFC)2 backbone. dynamic light scattering (DLS, BI-90Plus, USA) instrument. To
Specifically, Cys–(PEG45)2-b-(PMAOEFC)2 (205 mg, 8.7
ꢃ
further observe the micellar morphologies and size dispersity, a
10ꢀ3 mmol) was dissolved in anhydrous DMF (20 mL), then JEM-2100 transmission electron microscopy (TEM, Electro-
DOXꢁHCl (252 mg, 0.435 mmol) and TEA (60.3 mL, 0.435 mmol) nCrop., Japan) was applied to capture micellar photographs
solution in dessicated DMF (10 mL) was dropwise added into the at an accelerating voltage of 200 keV. The samples were
above solution. The reaction was performed at 25 1C for 48 h in prepared by transferring 10 mL of micellar solution dropped
the presence of nitrogen, (DOX)2–Cys–(PEG45)2-b-(PMAOEFC)2 on carbon films, followed by air-drying to ensure the moisture
was isolated by extensive dialysis, followed by freeze drying, removed completely.
and denoted as P-DOX thereinafter. (Yield: 91%) (1H NMR:
The ultraviolet-visible (UV-vis) absorption spectra of micellar
300 MHz, CDCl3), d (ppm): 7.48–8.09 (aromatic protons in solution were recorded on an UV-vis spectrophotometer
DOX), 4.18 (5H in ferrocenyl protons), 4.36 (2H, s, meta-H in Cp (UV-6100S, Mapada, Shanghai).
rings), 4.80 (2H, s, ortho-H in Cp rings), 3.51 (–OCH2CH2O– in
CV detection was performed on a CHI 660E electrochemical
ꢀ ꢀ ꢀ ꢀ
PEG2000); FTIR (film): n (cmꢀ1): 3013–3149 (QC–H and aromatic workstation (Shanghai Chenhua Instrument Co., China) with a
–C–H stretching vibration), 1720 (–CQO), 427–527 (Fe-Cp), conventional three-electrode cell in 0.1M TBAHFP electrolyte at
1574 (benzene skeleton vibration), 1613 (–CQN).
a scan rate of 1–50 mV sꢀ1 to investigate the electrochemical
property of monomer MAOEFC and polymer, glassy carbon
electrode (GCE), saturated calomel electrode and platinum wire
2.4 Preparation of nanomicelles
Polymeric micelles were prepared by dialysis method in aqueous electrode were served as working electrode, reference electrode
solution, briefly, polymer P powder (20 mg) was dissolved in and counter electrode, respectively.
DMF (10 mL) with stirring at 25 1C, followed by extensive dialysis
2.6 Formation of PTX laden micelles
(MWCO = 8000) against deionized water for 72 h till the solution
became opaque. Thus, the polymeric micellar solution with the PTX was entrapped into polymer P through the hydrophobic
origin concentration of 2000 mg Lꢀ1 was collected in a 10 mL interactions, p–p stacking and hydrogen bonding between
volumetric flask for further measurements. The P-DOX micelles PMAOEFC and drugs. Specifically, P powder (30 mg) and PTX
were prepared in line with the same procedure as the above.
(10 mg) were dissolved in DMF (10 mL) with stirring overnight,
then extensive dialysis (MWCO = 3500 Da) was performed
against deionized water for 72 h to remove solvent. The
2.5 Characterization
1H NMR spectra of products were recorded on a 400 MHz unloaded PTX was removed through low speed centrifugation,
Bruker Avance spectrometer (Bruker, Germany) at 25 1C serving followed by lyophilization to obtain PTX laden P micelles
tetramethylsilane (TMS) as internal standard and CDCl3, (denote as P@PTX hereafter). Similarly, PTX laden P-DOX, dis-
DMSO-d6 as solvent, respectively. Fourier transform infrared elenide bond linked PTX dimer (PTX–SeSe–PTX, signify as PTX
(FTIR) spectra were detected by an EQUINX55 FTIR spectro- dimer, the synthetic details were presented in ESI†) laden P-DOX
photometer (Bruker Corp., Germany) in KBr squash method. micelles were formed in accordance with the above method,
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which were represented as P-DOX@PTX and P-DOX@PTX dimer
respectively.
,
10 min within 1 h. And the fluorescence emission spectroscopy
ranged from 525 to 700 nm was collected within 90 min when
The drug laden micelles were dissolved in DMF to estimate the excitation wavelength was 469 nm, and all slit width
the drug loading content (DLC) and drug encapsulation effi- was 5 nm.
ciency (DEE) in light of the following equations:
Furthermore, in vitro DOX release experiments of P-DOX
micelles were administrated to detect pH sensitivity quantita-
tively. The lyophilized P-DOX micelles (4 mg) were suspended
in pH 5.0 and 7.4 PBS at the concentration of 1.0 mg mLꢀ1 and
shifted in dialysis bag (MWCO = 3500 Da), then dipped in a
beaker containing 200 mL of the corresponding pH value PBS at
37 1C with gentle stirring (stirring rate: 100 rpm). At predeter-
mined time intervals within 60 h, 4 mL of outer medium was
extracted and supplemented with isometric fresh medium. The
concentration of DOX released was detected using UV-vis
spectrophotometer at 480 nm, and calculated via the calibra-
tion eqn (4) to assess the cumulative DOX release amount.
DLC (%) = [mass of PTX (dimer) in PTX laden micelles/
mass of PTX (dimer) laden micelles] ꢃ 100%
(1)
DEE (%) = [mass of PTX (dimer) in PTX laden micelles/
mass of PTX (dimer) added initially] ꢃ 100%
(2)
The calibration equation of free PTX was obtained by the
relation between the concentration of PTX–DMF standard
solution and their corresponding UV-vis absorption, which
was presented as the following formula:
C (mg Lꢀ1) = (A ꢀ 0.0128)/0.39082 (R2 = 0.9982)
(3)
2.9 Stimuli induced in vitro drug release
where C and A is the concentration and UV absorbance at
210 nm of PTX–DMF standard solution, respectively.
Similarly, the calibration equation of free DOX–DMSO
solution was fitted according to the same way, as shown below:
Stimuli triggered PTX and DOX release behavior was investigated
by dialysis method. 4 mL of drug contained micelles solution
(1 mg mLꢀ1) was encased in dialysis tube (MWCO = 5 kDa) and
extensive dialyzed against 200 mL of pH 5.0 PBS with 10 mM
GSH/100 mM H2O2/0.5% NaClO at 39 1C for 60 h, respectively.
4 mL of outer medium was extracted at specific time points and
replenished with isometric fresh medium to detect the cumula-
tive amount of drugs released. The same release procedure was
performed in pH 7.4 PBS at 37 1C for 60 h.
C (mg Lꢀ1) = (A + 0.03737)/0.01882 (R2 = 0.9949)
(4)
where C and A denote the concentration and UV-vis absorbance
at 480 nm of DOX–DMSO standard solution, separately.
2.7 Redox responsiveness of the micelles
2.10 In vitro cytotoxicity
The redox responsive property of micelles was studies by DLS,
TEM and UV-vis spectrometer under specific conditions. To
investigate oxidization behavior, 0.1 mL of 1.5, 5.0, 7.5% NaClO
and 0.2 mL of H2O2 with the same mass concentrations were
blended into each 5 mL of micellar solution, successively. The
resulting solution was agitated for 4 h at 25 1C. Afterwards,
0.1 mL of 0.1 M Vc solution was added into the corresponding
oxidized micellar solution by 7.5% NaClO and 7.5% H2O2 with
stirring for 4 h respectively. To obtain reduced micelles, GSH
(12.3 mg) was added into 4 mL of the prepared micellar
solutions with agitation at 25 1C for 2, 6, 24 and 48 h.
The mouse fibroblast cell line L929 and HeLa cell line were
respectively selected as representatives of normal and tumor
cells for assessing the cytotoxicity of the prepared micelles. The
cells were seeded in 96-well at the density of 5 ꢃ 103 cells per
well and incubated in DMEM containing 10% FBS for 24 h,
followed by the medium removal, cells were rinsed using PBS
and fresh medium supplementation. The HeLa cells were
treated with the 0.1–16 mg Lꢀ1 of free drugs (PTX and DOXꢁ
HCl), 0.1–500 mg Lꢀ1 of the P, P@PTX, P-DOX, P-DOX@PTX
,
P-DOX@PTX dimer micellar solution were used for culturing
L929 and HeLa cells. After 48 h, the medium was discarded
and added another 200 mL fresh medium containing 20 mL MTT
2.8 pH responsiveness of P-DOX micelles
The measurements of H NMR, fluorescence spectroscopy and solution (5 mg mLꢀ1), followed by incubation at 37 1C for 4 h.
Dh of P-DOX micelles containing Schiff base structure were 150 mL of DMSO was used to dissolve formazan with shaking
performed to monitor the pH responsiveness. The P-DOX lyo- for 10 min. The optical density (OD) was recorded on an
1
philized micelles (60 mg) was dissolved in DMSO (10 mg mLꢀ1
)
universal microplate reader (Model 680, UK) at 570 nm. As
entirely, and divided into two groups, then 1 mL of pH 5.0 and control, HeLa cells were cultured under the same condition
isometric pH 7.4 PBS were mixed into their corresponding without any drug and nanoformula addition. Cell viability was
groups under stirring for 24 h, respectively. The resulting liquor calculated according to the following equation:
was vacuum dried and redissolved in DMSO-d6 for 1H NMR
Cell viability (%) = ODsample/ODcontrol ꢃ 100%
(5)
determination.
The detection of micellar size (Dh) changes and DOX fluorescence where the ODsample and ODcontrol represent the optical density
intensity after DOX cleaving from P-DOX micelles in weak of samples and control group, respectively.
acidic environment within different time intervals using DLS
2.11 Apoptosis analysis
and fluorescence spectrophotometer, respectively. Lyophilized
P-DOX micelles was dissolved in pH 5.0 and pH 7.4 PBS To observe the uptake of DOX into cells, HCT116 and HCT116/ADR
at the concentration of 0.5 mg mLꢀ1 and detect the Dh every cells were seeded in 6-well plates with cell slides (1 ꢃ 105 cells per
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well) and incubated for 24 h. Then the cells were washed with PBS Product and Service Solutions (SPSS). *, # and & signify P o
and cultured with a medium containing free DOX, P-DOX, 0.05, P o 0.01 and P o 0.001, respectively.
P-DOX@PTX and P-DOX@PTX dimer at 1 mg mLꢀ1 DOX concentration
for 4 h, respectively. RPMI1640 treated cells as a control group.
After that, the cells were washed using PBS and digested using
trypsin, followed by resuspension in buffer and staining with
Annexin V-FITC and propidium iodide (PI). The growth inhibition
3. Results and discussion
3.1 Synthesis of DOX prodrug P-DOX
caused by the above nanoformulations was detected by flow Schiff base linked DOX prodrug P-DOX was prepared through
cytometry.
the following five-step process: (i) esterification between (Boc–
Cys–OH)2 and PEG; (ii) acylbromination of (Boc)2–Cys–(PEG45 2
)
2.12 Qualitative and quantitative analysis of cellular uptake
affording macromolecular initiator (Boc)2–Cys–(PEG45)2–Br2;
(iii) ATRP of monomer MAOEFC yielding (Boc)2–Cys–(PEG45)2-
b-(PMAOEFC)2; (iv) de-protection of (Boc)2–Cys–(PEG45)2-b-
(PMAOEFC)2 and (v) conjugation of DOX with P resulting
P-DOX. 1H NMR was applied to confirm the chemical structure
of products, as demonstrated in Fig. 1. The chemical signal of
methylene in PEG appears at 3.65 ppm in Fig. 1A, by contrast, a
new signal shows at 1.87 ppm belonging to methyl from BiBB
To quantitative analysis the celluar uptake of free DOX, P-DOX,
P-DOX@PTX and P-DOX@PTX dimer, HCT116 and HCT116/ADR
cells were seeded in 6-well plate with a cover slip (1 ꢃ 105 cells
per well) and cultured for 24 h. After removing the old medium,
the cells were cultured with new medium containing samples at
2 mg mLꢀ1 DOX concentration for 1 and 4 h. Then the cells were
fixed using 4% paraformaldehyde and washed with PBS thrice.
The cytoskeleton was labled with 50 nM phalloidin-FITC,
similarly, nucleus were stained with 300 nM 4-, 6-diamidino-2
phenylindole (DAPI) and observed through confocal laser scan-
ning microscope (CLSM, Nikon A1, Japan).
Flow cytometer was used to quantitatively analyze intracellu-
lar drugs content. The HCT116and HCT116/ADR cells cultured
with DOX and all nanoformulations for 4 h. RPMI1640 treated
cells as a control group. Afterwards, cells were washed with PBS
thrice, digested using trypsin solution, centrifuged and collected
cells. The intracellular fluorescence intensity of DOX was deter-
mined using flow cytometry.’’
1
residues in Fig. 1B. After polymerization, H NMR spectrum of
(Boc)2–Cys–(PEG45)2-b-(PMAOEFC)2 not only presents peaks at
1.68 and 1.09 ppm assigned to methylene and methyl in
PMAOEFC main chains respectively, but also shows the
chemical shifts of ferrocenyl protons, meta-H and ortho-H
emerge at 4.04, 4.20 and 4.80 ppm, successively, see in
Fig. 1C. The characteristic signal of t-butyl at 1.43 ppm dis-
appears after de-protection, as exhibited in Fig. 1D. In addition,
as shown in Fig. 1E and F, new peaks display at 7.48–8.09 ppm
are the characteristic signal of aromatic protons in DOX struc-
ture, indicating the formation of P-DOX with Schiff base
structure. FTIR was employed to further confirm the chemical
structure of products in Fig. 2A.
2.13 In vivo anticancer study
In Fig. 2A(a), the strong vibration bands located at 1007–1214,
2848–2981 and 3080–3755 cmꢀ1 are attributed to asymmetric
C–O–C, –C–H stretching vibration in PEG and terminated –OH
from (Boc)2–Cys–(PEG45)2, respectively. For (Boc)2–Cys–(PEG45)2-
b-(PMAOEFC)2, the absorption peak of QC–H, QC–H in ferro-
cene rings and Fe–C/Cp–Fe emerge at 3100, 770–850 and 427–
563 cmꢀ1, as demonstrated in Fig. 2A(b). After de-protection, it
can be seen that –N–H and –C–N stretching vibration bands are
located at 3229–3662 and 1133 cmꢀ1, respectively, demonstrat-
ing the generation of polymer P. In addition to the tagged
spectral features of PMAOEFC and PEG appearing in
Fig. 2A(d), some new wide peaks also appear at 3013–3149,
1574 and 1613 cmꢀ1, which are attributable to unsaturated
–CQH stretching vibration, benzene ring skeleton stretching
vibration and –CQN– in P-DOX.
4T1 tumor bearing Balb/c mice were treated using DOXꢁHCl, PTX,
P, P-DOX, P-DOX@PTX and P-DOX@PTX dimer micellar solutions at
PTX and DOX dosage of 8 and 4 mg kgꢀ1, respectively, PBS treated
group was served as blank control group. All nanoformulas were
injected every three days. 4T1 tumor volumes (V) were measured
using caliper on day 0, 3, 6, 9, 12, 15, and mice were weighed
at the same time intervals. Tumor suppression ratio (TSR%) =
[(VPBS ꢀ VNF)/VPBS] ꢃ 100% (here VPBS and VNF denote the 4T1
tumor volume treated by PBS and nanoformula, respectively).
2.14 Histological analysis
On day 15, one mouse from each cohort was sacrificed to excise
the major organs (heart, liver, spleen, lung and kidney). After
immersion in paraformaldehyde and embedding in paraffin,
the organs were sliced into 5 mm sheets. The obtained slices
were stained with hematoxylic and eosin (H&E) for further
observation under digital microscope (Leica QWin).
The experimental molecular weight of (Boc)2–Cys–(PEG45)2-
b-(PMAOEFC)2 was assessed using integration area ratio of
t-butyl peak in Boc group at 1.45 ppm to the shift of ortho-H
from PMAOEFC block at 4.80 ppm. There are approximately 56
MAOEFC repeating units polymerized in (Boc)2–Cys–(PEG45)2-b-
2.15 Statistical analysis
The results of drug release, in vitro cytotoxicity, and in vivo (PMAOEFC)2, viz., the composition can be denoted as (Boc)2–
antitumor experiments have been denoted as the average ꢂ Cys–(PEG45)2-b-(PMAOEFC28)2. The apparent molecular weight
standard deviation (average ꢂ S.D.), tests were carried out (Mn, Mw) and polydispersity index (PDI, Mw/Mn) of all polymers
quantitative, and taken the average values. The significance were also determined through GPC, the obtained GPC trace is
of any couples of differences was affirmed through Statistical displayed in Fig. 2B and the molecular weight was shown in
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Fig. 1 1H NMR spectra of (Boc)2–Cys–(PEG45 2
(E) and DOXꢁHCl (F).
) (A), (Boc)2–Cys–(PEG45)2–Br2 (B), (Boc)2–Cys–(PEG45)2-b-(PMAOEFC)2 (C), polymer P (D), P-DOX
Fig. 2 FTIR spectra (A) of (Boc)2–Cys–(PEG45)2 (a), (Boc)2–Cys–(PEG45)2-b-(PMAOEFC)2 (b), polymer P (c) and P-DOX (d); (B) GPC trace of all polymers.
Table 1. Obviously, there is no tailing and shoulder peak in molecular weight, suggesting that the polymers have well
symmetric GPC trace during the whole retention region of control and satisfactory purification over synthesis process.
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Table 1 Molecular weight of polymers from GPC
Name Mw
dehydration process before TEM observation. The P-DOX, P-
DOX@PTX and P-DOX@PTX dimer formed globular core–shell
micellar aggregates with average size of 200, 220 and 235 nm
compared to 138 nm for P micelles. All the prepared micelles are of
homogenous dispersion as PDI results from Fig. 3B. As drugs were
introduced into P-DOX, P-DOX@PTX and P-DOX@PTX dimer micelles,
the enhanced hydrophobic interaction and expanded core volumes
lead to the larger micelles aggregates formed.
Mn
PDI (Mw/Mn)
(Boc)2–Cys–(PEG)2
(Boc)2–Cys–(PEG)2-b-(PMAOEFC)2
Cys–(PEG)2-b-(PMAOEFC)2
—
—
P
17 893 16 018 1.117
88 410 56 384 1.568
67 322 48 784 1.380
(DOX)2–Cys–(PEG)2-b-(PMAOEFC)2 P-DOX 70 410 66 910 1.052
All polymers possess reasonable molecular weight and uniform
molecular weight dispersion.
3.3 Redox responsiveness of PEG based polymer P micelles
Based on the existence of redox sensitive ferrocene in Polymer P
micelles, it was selected to investigate redox behavior of P. Apart
3.2 Self-assembly behavior in aqueous solution
As illustrated in Scheme 2, micelles were prepared using from H2O2, moreover, NaClO also can oxidize PMAOEFC into
dialysis method. Fluorescence spectroscopy, DLS and TEM were poly(ferrocenium), their redox responsiveness was monitored
used to study the micellization behavior. The CMC assessed in through UV-vis spectroscopy. As shown in Fig. 4, the UV-vis
the presence of pyrene probe was showed in Fig. 3A, suggesting absorption peak of PMAOEFC in a reduction state appears
that P-DOX@PTX dimer micelles possess the minimum CMC of approximately at 450 nm. The peak is weak or fades away after
5.78 mg mLꢀ1 compared that of other prepared micelles blending oxidants (H2O2 and NaClO) with the mass concen-
(238.93 mg mLꢀ1 for P micelles, 12.09 and 12.07 mg mLꢀ1 for tration of 1.5%, 5.0% and 7.5%. Upon H2O2 addition, as
P-DOX and P-DOX@PTX micelles). This could be attributed to illustrated in Fig. 4A, the peak at 450 nm of ferrocene is almost
the abundant drug types and highest drug content of fades away at the H2O2 concentration of 5.0% due to the
P-DOX@PTX dimer micelles, the enhanced hydrophobicity from formation of poly(ferrocenium). The oxidization mechanism
p–p stacking, hydrophobic interaction, hydrogen bond inter- involved is the production of unstable intermediates hydroper-
action of among PMAOEFC, PTX and DOX, which caused a oxide anions HOOꢀ, which is subsequently decomposed to
more stable micelle.45,46 The limited amount of DOX contained HOꢄ.49 With respect to NaClO, the characteristic peak decreases
in P-DOX micelles can improve stability compared with that of with the increasing concentration of NaClO in Fig. 4B. It is
P micelles. The sharp decrease of CMC shows that P-DOX, noteworthy that the hydrolysate of NaClO solution containing
P-DOX@PTX and P-DOX@PTX dimer micelles can withstand the NaOH and HClO can destroy micelles, resulting in the oxidation
dilution of body liquids during blood circulation. Thus, they and aggregation of micelles.50
have the potential to be applied in cancer treatment.47,48 The
As a reductant, Vc was added in the oxidized micellar
monodisperse trend exhibited in Fig. 3B shows the average Dh solution to reduce the poly(ferrocenium), the characteristic
of 151.7, 383, 443.8, 422.9 nm and PDI of 0.297, 0.336, 0.363, peak at 450 nm is reemerged, which is ascribed to the transi-
0.285 for P, P-DOX, P-DOX@PTX and P-DOX@PTX dimer micelles, tion of Cp2Fe+ to Cp2Fe under reduced condition. These results
successively, somewhat larger than micellar size from TEM, suggest the reversibility of redox-triggered assembly and dis-
as displayed in Fig. 3C–F, which is mainly ascribed to the assembly behavior.16,51 Furthermore, the smart responsive
Scheme 2 Schematic illustration of preparation, blood circulation, internalization, specific drug release of P-DOX, P-DOX@PTX and P-DOX@PTX dimer
micelles.
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Fig. 3 (A) The fluorescence intensity ratio (I383/I372) versus the logarithm of P (a), P-DOX (b), P-DOX@PTX (c) and P-DOX@PTX dimer (d) micellar
concentrations; (B) particle size dispersion of P (a), P-DOX (b), P-DOX@PTX (c), P-DOX@PTX dimer (d) micelles; TEM photos of P (C), P-DOX
(D), P-DOX@PTX (E) and P-DOX@PTX dimer (F) micelles.
behavior to redox is expected to be a new channel for intelligent from 1 to 50 mV sꢀ1, the results are shown in Fig. 4D. The
drug delivery.51–53
oxidation of MAOEFC segments and the reduction of MAOEFC+
occurred in each scanning process at different scan rate, the
peak potential DE is increase due to scanning speed up, which
results in a weaken reversibility. To further reveal electrode
process mechanism, the relationship between the peak currents
3.4 Electrochemical responsiveness of PEG based polymer P
The redox responsiveness was studied using cyclic voltammetry
(CV). As exhibited in Fig. 4C, the ferrocene content affects the
redox potential directly. The anodic oxidation potential of
monomer MAOEFC is 0.865 V, in case of polymer P, the
presence of cystine and PEG lead to the anodic oxidation
potential reduce to 0.849 V. Additionally, the potential difference
between anodes and cathodes was considered as the peak
separation DE (DE = EP,anodic ꢀ EP,cathodic), the sample containing
ferrocene has a good redox reversibility when DE is less than
0.1 V. Compared with DE of ferrocene monomer (0.030 V), DE of
P reached 0.157 V due to more MAOEFC repeating units oxidized
and the resulting MAOEFC+ segments reduced in electrolyte,
reflecting that P is more difficult to be oxidized. In fact, the
increase of DE is attributable to high ferrocene contents which
result in mass diffusion and charge transfer at a lower speed.
Therefore, the reversibility of P can be adjusted by decreasing the
concentration less than 10 mg mLꢀ1 and DE o 0.1 V.
(Ip) and the square root of their corresponding scan rate (n1/2
was fitted in the inset of Fig. 4D. The Ip is proportional to n1/2
)
,
taking on good linear relations Ip,a = 0.49371n1/2 ꢀ 2.14902 (R2 =
0.95125) for anode and Ip,c = ꢀ0.62037n1/2 + 2.7336 (R2 = 0.9744)
for cathode, respectively, which signify that the mass diffusion of
oxidation and reduction is adjustable, and the electron transfer
process is sufficient to oxidize and reduce the whole MAOEFC
units from P in electrolyte. In fact, the diffusion controlled redox
electrode process also can be deduced from Fig. 4D, where the
DE enhanced as n1/2 increasing. The result is probably ascribed to
the slow electron transfer process at the electrode–P interface in
electrolyte solution and even between contiguous ferrocene units.54
3.5 pH responsiveness of P-DOX micelles with Schiff base structure
Schiff base structure can be maintained under normal physiological
The electrode process mechanism of P was also uncovered by environments (pH 7.4), but it can be cracked into amino, aldehyde,
CV in DCM containing 0.1 M TBAHFP at the scan rates ranged and ketone under low pH condition (BpH 5.0), the mechanism is
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Fig. 4 UV-vis spectra of P micellar aqueous solution before and after oxidization through (A) H2O2 and (B) NaClO with the mass concentration
from 0–7.5%, followed by Vc reduction. Cyclic voltammograms of monomer MAOEFC and polymer P with a concentration of 10 mg mLꢀ1 in
DCM containing 0.1 M TBAHFP at a scan rate of 50 mV sꢀ1 (C), cyclic voltammograms (D) and linear relations (the inset) of peak current Ip
versus the square root of scan rates of P (10 mg mLꢀ1) at different scan rates ranged 1–50 mV sꢀ1 in the same electrolyte at room temperature.
exhibited in Fig. 5A.55,56 According to 1H NMR, DLS and fluorescence pH 5.0 PBS. As observed in Fig. 6A, the mean diameter of
spectrophotometry, the results are shown in Fig. 5B–F. In terms of P-DOX@PTX dimer micelles has a sharply decrease from 235 to
P-DOX, Schiff base structure was destroyed and the fluorescent DOX 141 nm after incubation in 10 mM GSH for 24 h, which was
disengaged from P-DOX micelles at pH 5.0, causing the fluorescence caused by the reduction of diselenium and disulfide bond into
intensity increased and micellar diameter decreased. As shown in the enhanced hydrophilic selenol and mercaptan, respectively,
Fig. 5B, with the induction of weak acidic ambient, more free DOX and the follow-up micellar disassembly. Thus, Dh firstly
was released after Schiff base structure was destructed and produced increased and then decreased. Besides, diselenide bonds were
the stronger fluorescence. By contrast, the limited florescence oxidized into selenic acid by 100 mM H2O2, meanwhile, Cp2Fe+
intensity increased in Fig. 5C, which was up to the less DOX was also generated after Cp2Fe oxidation. The micellar size of
separation and the better stability of P-DOX in pH 7.4 PBS compared the oxidized micelles from 285 to 457 nm (average Dh: 395 nm)
with that in pH 5.0 PBS. Similarly, as shown in Fig. 5D, Dh of P-DOX in Fig. 6B was ascribed to the electrostatic repulsion between
micelles was decreased from 516.7 nm to 330.3 nm after incubation Cp2Fe+ units and the swelling effect of the resulting hydrophilic
in pH 5.0 PBS for 1 h, while a 50 nm of micellar size decreased in poly(ferrcenium).61 In addition, the oxidation of PMAOEFC is a
Fig. 5E when P-DOX micelles were immersed in pH 7.4 PBS. P-DOX relatively slow process, and most of PTX still remain in
was treated with pH 5.0 PBS and then dissolved in DMSO to monitor carrier.60 Therefore, the increase of micellar size is depended
1
the acidic hydrolysis behavior of Schiff base using H NMR. It is on the synergistic effect of swelling, electrostatic repulsion and
noteworthy that the chemical shifts of amino proton appear at drugs residues. There were some debris on micellar surface
approximately 2.0 ppm in Fig. 5F(a), and without specific chemical showed in Fig. 6B, clearly suggested the oxidation of micelles.
feature emerges in Fig. 5F(b) with respect to P-DOX micelles in These results were consistent with the Dh change trends of
pH 7.4 PBS. These evidence was further demonstrated that the P-DOX@PTX dimer micelles within different reduction and
P-DOX micelles had pH triggered property under extracellular micro- oxidization time intervals in Fig. S4 (ESI†). The disassembly
environment in solid tumor,57 especially in endosomes and performance of P-DOX@PTX dimer micelles was reflected by DOX
lysosomes of tumor tissue.58–60
release under weak acidic condition (pH 5.0 PBS) after 60 h
shown in Fig. 6C, innumerable spots with approximately 18 nm
of average diameter appeared on the surface of hollow micelles
with the size of 363 nm, which precisely indicated that DOX was
disconnected from P-DOX@PTX dimer micelles after Schiff base
structure destruction under pH 5.0 PBS. The empty core was
formed when Schiff base linkage cleaved and the DOX diffused,
which can attest the pH responsiveness of P-DOX@PTX dimer
micelles. It is noticeable that the P-DOX@PTX dimer micelles
3.6 Redox and pH-triggered properties of P-DOX@PTX dimer micelles
In view of the presence of the multiple redox sensitive sites
including diselenide bond, disulfide bond and PMAOEFC
blocks in P-DOX@PTX dimer micelles, TEM was selected to
observe the changes of morphology and micellar size of
P-DOX@PTX dimer micelles under various of mimic cancerous
physiological conditions such as 10 mM GSH, 100 mM H2O2 and
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Fig. 5 Schematic of Schiff base hydrolytic process (A); fluorescence spectra of P-DOX micelles treated with pH 5.0 PBS (B) and pH 7.4 PBS (C); micellar
size dispersion of P-DOX after incubation with pH 5.0 PBS (D) and pH 7.4 PBS (E) at different time intervals; 1H NMR spectra of P-DOX after pH 5.0 and
7.4 PBS treatment (F).
was dissociated entirely under the condition of pH 5.0 PBS drugs variety, improve drug delivery process and therapeutic
containing 10 mM GSH, this is mainly due to the reduction of effect, and circumvent MDR effect, PTX and its diselenide bond
diselenide and disulfide bonds located in micellar core region, linked dimer PTX–SeSe–PTX were encapsulated into P-DOX in
leading to the formation of PTX unimers, and PTX and the same way, obtaining the dual drugs co-delivery systems
DOX release afterwards. As displayed in Fig. 6D, the intrinsic P-DOX@PTX (13.2% of LC, 57.0% of EE) and P-DOX@PTX dimer
morphology of P-DOX@PTXdimer micelles disappeared completely (49.1% of LC, 97.0% of EE), respectively. In particular, more
and reversely replaced by spherical nanoparticles of free drugs PTX was pulled in P-DOX by diselenide bond, leading to
with the Dh of approximately 7–23 nm.
enhanced hydrophobic interaction with DOX and PMOAEFC
blocks. Above all, diselenide bonds were used as highly
entangled networks convolve the micellar core comprised of
3.7 Drug encapsulation
DOX and PTX were used as template drugs to construct dual DOX, PMAOEFC and large amount of PTX, which could avoid
drugs co-delivery system. As presented in Table 2, DOX was the pre-leak of drugs before reaching tumor site. Together
linked with P scaffold by Schiff base structure, affording P-DOX with 39.3% of DOX, the total content of dual drugs in
micelles with 39.3% of DOX content. This value was higher P-DOX@PTXdimer had up to 88.4%. DOX and PTX dual drugs co-
than 15.8% of DOX content calculated from feed molar ratio in delivery systems with favorable loading performance were pre-
line with reaction formula, which was due to both strong pared hereto, indicating that the variety of nanocarriers can be
hydrophobic, hydrogen bond and p–p stacking interactions in enriched by choosing chemical bonding/physical encapsulation
DOX moieties. For comparison, polymer P, as a carrier to load and loading drug unimer/dimer delivery.59
PTX mainly through hydrophobic interaction with PMAOEFC
segments, produced a single PTX delivery system P@PTX con-
3.8 pH, redox triggered in vitro drug release
taining 12.7% of PTX. Even so, low drug content and single Due to the abundant drugs containing in P-DOX@PTX dimer
drug type were still existed in P and P@PTX system. To enrich micelles, in vitro drug release behaviors are shown in Fig. 7.
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Fig. 6 TEM photographs of P-DOX@PTX dimer micelles reduced by 10 mM GSH for 24 h (A), oxidized by 100 mM H2O2 for 24 h (B), released in pH 5.0 PBS
after 60 h (C) and released in pH 5.0 PBS containing 10 mM GSH after 60 h (D). The inset shows the partial enlarged photograph of DOX released P-
DOX@PTX dimer micelles.
Table 2 Chemical compositions, PTX LC, PTX EE and drug content of the resulting nanoformulas
Chemical compositions
(DOX)2–Cys–(PEG45)2-b-(PMAOEFC28 2
Sample code
PTX LC (%)
PTX EE (%)
DOX content (%)
Drug total content (%)
)
P-DOX
0
0
39.3
0
39.3
39.3
39.3
12.7
52.5
88.4
@PTX
Cys–(PEG45)2-b-(PMAOEFC28 2
(DOX)2–Cys–(PEG45)2-b-(PMAOEFC28 2
)
P@PTX
12.7
13.2
49.1
48.9
57.0
97.0
P-DOX@PTX
P-DOX@PTX dimer
@PTX
)
@PTX dimer
(DOX)2–Cys–(PEG45)2-b-(PMAOEFC28 2
)
Only 23.58% of PTX was released in pH 7.4 PBS from Fig. 7A(a), the same time under a single stimulus. Remarkably, as illu-
suggesting that the carrier could retard PTX release and avoid strated in Fig. 7A(e), the PTX release amount was accumulated
the side effects to normal tissues.
greatly into 84.04% under the synergistic reduction of disulfide
Unlike physical loading, 18.19% of DOX was disconnected and diselenide bonds for 60 h, while 71.97% of DOX released is
from P-DOX@PTX dimer micelles, which was due to the good mainly attributable to the pH 5.0 surrounding from Fig. 7B(e).
stability of chemical conjugate containing Schiff base structure Under the condition of pH 5.0 PBS containing 100 mM H2O2,
at pH 7.4, as illustrated in Fig. 7B(a). Schiff base was hydrolysis PTX unimer and poly(ferrocenium) were formed and then
under pH 5.0, resulting in 63.80% of DOX separated and occurred micellar phase transition, in which produced
diffused to micellar surface, as displayed in Fig. 7B(b) and C. 42.28% of PTX was released, as shown in Fig. 7A(f). Compared
Approximately 29.08% of PTX was released under the same with 63.80% of DOX released from P-DOX@PTX dimer micelles in
condition regardless of acid labile mechanism, as shown in pH 5.0 PBS, approximately 56.90% of DOX was released from
Fig. 7A(b). The diselenide and disulfide bond were reduced into the same carrier in pH 5.0 PBS containing 100 mM H2O2, which
selenol and mercaptan by 10 mM GSH, and the follow-up was ascribed to the limited diffused channels occupied by PTX,
micellar disintegration. Eventually 72.65% of PTX was released as shown in Fig. 7B(b) and (f). All these results suggested that
from micelles in Fig. 7A(c), while DOX was not affected by GSH the dual drugs carrier P-DOX@PTX dimer micelles can realize
reduction, and 18.77% of DOX was detached from Fig. 7B(c). the appropriate drug release amount by controlling different
Besides, as exhibited in Fig. 7A(d), the release rate of PTX was response mechanisms. These prepared drug (co-)delivery
controlled by the oxidation of diselenide bond and ferrocence systems with single, dual and even multiple stimuli responsive-
based PMAOEFC blocks. Meanwhile, approximately 39.48% of ness provide more choices for cancer treatment according to the
PTX was released after incubation in medium containing required drug dosage,16 especially the synergistic effects between
100 mM H2O2. It was apparent that the DOX release rate was pH and redox responsiveness of P-DOX@PTX dimer micelles can
essentially close to the amount released at pH 7.4. Indeed, it is facilitate the drug accumulation with high concentration at
difficult to obtain good release performance of dual drugs at tumor site.59
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Fig. 7 PTX (A) and DOX (B) release plots of P-DOX@PTX dimer micelles in pH 7.4 PBS (a), pH 5.0 PBS (b), pH 7.4 PBS containing 10 mM GSH (c), pH 7.4
PBS containing 100 mM H2O2 (d), pH 5.0 PBS containing 10 mM GSH (e) and pH 5.0 PBS containing 100 mM H2O2 (f) for 60 h. PTX (C) release plots of
P-DOX@PTX dimer (e), P-DOX@PTX (g) and P@PTX (i) micelles in pH 5.0 PBS containing 10 mM GSH for 60 h, DOX (D) release plots of P-DOX@PTXdimer (e),
P-DOX@PTX (g) and P-DOX (h) micelles in pH 5.0 PBS containing 10 mM GSH for 60 h. *P o 0.05 and #P o 0.01.
Due to the presence of hydrazone, disulfide and diselenide release amount in Fig. S6(B) (ESI†) was almost consistent with
bonds in P-DOX@PTX micelles, approximately 81.28% of PTX Fig. 7B(f).
released dramatically in pH 5.0 PBS containing 10 mM GSH was
ascribed to simultaneous breakage of hydrazone, disulfide and
3.9 In vitro cytotoxicity
diselenide bonds, as shown in Fig. 7C(e), while about 70.71% of Cytotoxicity of nanovehicles is crucial for drug delivery. HeLa
DOX was isolated from P-DOX@PTX dimer as hydrazone bonds cells were treated using free PTX, DOXꢁHCl. Meanwhile, poly-
cracked in Fig. 7D(e). The similar PTX diffusion mechanism of mer P, P-DOX, P@PTX, P-DOX@PTX and P-DOX@PTX dimer micellar
P-DOX@PTX and P-DOX resulted in the analogue release trend in solutions with different concentrations in MTT assay can reflect
Fig. 7C(g) and Fig. 7C(i), and about 75% of PTX was discon- the cytotoxicity of all nanoformulations against L929 and HeLa
nected from the two nanovehicles mentioned above. The steady cells. The significant cytotoxicity of free drugs was depicted in
release process of DOX occurred, as depicted in Fig. 7D(h), Fig. S7 (ESI†), 1.64 and 3.13 mg Lꢀ1 of IC50 was attributed to
causing 48.92% of DOX dissociated. Actually, DOX and free DOXꢁHCl and PTX respectively, the higher IC50 of free PTX
PTX competed to occupy the limited diffuse channels, thus a than that of DOXꢁHCl was related to poor blood circulation of
portion of DOX was bounded by PTX to release eventually, hydrophobic PTX. L929 cells did not have abnormal physio-
resulting in the amount of DOX released from P-DOX@PTX dimer logical signals to induce drug release. Therefore, the cell
surpassed that of DOX from P-DOX@PTX, as revealed in viability was almost exceed 80% in Fig. 8(A). Especially, after
Fig. 7D(e) and (g).
treating with polymer P micelles for 48 h, cell viability of cells
Owing to the unresponsiveness of hydrazone to pH 7.4 PBS, were higher than 88%, which was ascribed to the absence of
there was 9.11% of DOX released from P-DOX@PTX micelles, drugs, showing the biocompatibility and potential of nano-
showing slightly less content of PTX released compared with formulations for drug delivery.58 Less than 20% of cell apoptosis
P-DOX@PTX dimer, that may be ascribed to a small number of was caused by a small amount of drug release, consistent with
PTX dragged by DOX through hydrophobic interaction and p–p in vitro drug release results at pH 7.4. On the contrary, the special
stacking. As for P@PTX micelles, 19.48% of PTX was released in physiological signals in HeLa cells lead to more drugs release
pH 7.4 PBS as the limited PTX and unoccupied diffuse channels from nanomicelles. As shown in Fig. 8(B), IC50 of P@PTX, P-DOX,
from Fig. S5 (ESI†). Since PMAOEFC could be oxidized by 0.5% P-DOX@PTX and P-DOX@PTX dimer, was 136.46, 49.32, 41.91 and
NaClO into poly(ferrocenium), PTX was release from 32.13 mg Lꢀ1, equal to 1.64, 0.996 and 2.840 mg of PTX
P-DOX@PTX dimer along with the phase transformation of contained in P@PTX, P-DOX@PTX and P-DOX@PTXdimer at IC50,
PMAOEFC. In view of a slow oxidize process of PMAOEFC, respectively. Similarly, 3.489, 3.395 and 2.272 mg of DOX was
there was only 47.15% of PTX released in Fig. S6A(a) (ESI†). contained in P-DOX, P-DOX@PTX and P-DOX@PTXdimer in
DOX was basically unaffected by the oxidation of NaClO, the sequence. The sharp decrease of cell viability under the
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Fig. 8 The cytotoxicity of P-DOX (a), P@PTX (b), P-DOX@PTX (c), P-DOX@PTX dimer (d) and polymer P (e) micelles against L929 (A) and HeLa (B) cells, &P o
0.001. Qualitative analysis of cell uptake of DOX by CLSM in HCT116 (C) and HCT116/ADR (D) cells after 4 h incubation, scale bar =10 mm; quantitative
analysis of cell uptake of DOX by FCM in HCT116 (E) and HCT116/ADR cells (F). Cell apoptosis of free DOX (a), P-DOX (b), P-DOX@PTX (c) and
P-DOX@PTX dimer (d) in HCT116 (G) and HCT116/ADR cells (H).
treatment using nanoformulations signified the powerful leth- HCT116/ADR cells after 1 h incubation was shown in Fig. S8
ality to HeLa cells, reflecting the great potential in cancer (ESI†). As displayed in Fig. 8(C), the red fluorescence was
treatment.
observed in cytoplasm and nucleus region, suggesting that
the free DOX and nanoformulations could enter cells by
diffusion and endocytosis, respectively. Compared with nano-
3.10 Cellular uptake and apoptosis
To assess the intracellular drug internalization, accumulation, formulation groups, the stronger intensity of nucleus of free DOX
dispersion and content, HCT116 and HCT116/ADR cells were may be caused by direct diffusion into cell without being released
incubated with free DOX, P-DOX, P-DOX@PTX and from carrier. As for HCT119/ADR cells, the red fluorescence
P-DOX@PTX dimer, then observed by confocal laser scanning intensity of free DOX exhibited a slightly decrease than that of
microscope. Celluar uptake of DOX into HCT116 and nanoformulations from Fig. 8(D), which is attributed to the
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introduction of PTX. The combination of PTX and DOX could
Furthermore, flow cytometry was utilized to quantitative
efficiently improve MDR effect through inhibiting P-gp efflux analysis the above results, as presented in Fig. 8(E) and (F).
effect, resulting in enhanced internalization and accumulation There was almost no difference of internalized DOX for HCT116
in MDR cells. Free DOX across the cell membrane was through cells after treatment using samples with or without PTX.
dispersion. A large number of DOX molecules were distributed However, the amount of internalized DOX increased according
around the cell nucleus, while the prepared nanocarriers can to the order of free DOX, P-DOX, P-DOX@PTX and P-DOX@PTX dimer
,
be uptaken mainly via endocytosis-lysosome approach.62,63 which was in accordance with the above results from CLSM.
Compared with cells treated with free DOX, the intensive red These results demonstrated that the prepared PTX and DOX
fluorescence emerged in P-DOX and P-DOX@PTX dimer groups, co-delivery system could inhibit P-gp efflux effect and improve
implying that the prepared nanoformulations could enahnce drugs internalization and accumulation in MDR cells.
the cellular accumulation of the exogenous molecules. Moreover,
The apoptosis of HCT116 and HCT116/ADR cells were
P-DOX@PTX dimer presented higher permeability and retention observed using flow cytometry after incubation with different
efficiency than that of other groups, this was ascribed to the pH samples for 4 h. As shown in Fig. 8(G), the apoptotic ratios of
targeting effect of Schiff base and triple redox responsiveness of free DOX, P-DOX, P-DOX@PTX and P-DOX@PTXdimer were 55.00%,
PMAOEFC, S–S bonds and Se–Se bonds,64–66 where the fast delivery 56.96%, 38.53%, and 39.38% for HCT116 cells, respectively. The
of DOX could be attributed to the acceleration during PTX release higher cell killing capacity of free DOX than nanoformulations is
process. These results were also consistent with the higher DOX ascribed to different internalize pathway and time-lag drug
release content of P-DOX@PTX dimer in drug release study.
release mode of carriers. By contrast, the slightly lower apoptotic
Fig. 9 Changes of tumor volume (A), body weight (B) of 4T1 bearing mice treated with DOXꢁHCl (a), PTX (b), PBS (c), P (d), P-DOX (e), P-DOX@PTX (f) and
P-DOX@PTX dimer (g) at PTX and DOX dosage of 8 and 4 mg kgꢀ1, respectively. &P o 0.001, #P o 0.01 and *P o 0.05. Photographs of excised tumor on day
15 after treatments (C) and histology H&E stain photograms of sliced major organs of mice on day 15 (200ꢃ) (D).
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ratios of HCT116/ADR cells treated with free DOX and P-DOX 4. Conclusion
from Fig. 8(H) were 20.52% and 50.31%, respectively. It is
In summary, the acid labile and multiple redox sensitive PEG-
based PTX and DOX co-delivery systems were prepared through
chemical bonding of DOX and physical loading of PTX (dimer).
In particular, 39.3% of DOX and 49.1% of PTX were contained
in P-DOX@PTX dimer nanovehicles, and EE of PTX had up to
97.0%. These nanocarriers can self-assemble into globular
micelles with hydrophobic core comprising of PTX, DOX and
PAMOEFC and hydrophilic shell including PEG and cystine.
The destruction of micelles is attributable to the induction of
cancerous physiological signals including pH 5.0, 10 mM GSH,
100 mM H2O2, etc., causing drugs on-demand release. These
nanoformulas not only circumvented the serious cardiotoxicity
of free drugs, but also exhibited significant anticancer efficacy.
To the potential for adjusting drugs content, improving stabi-
lity and water solubility, overcoming MDR effect of tumors,
these dual drugs co-delivery systems are expected to utilize in
drug-resistant cancers sustainable treatment.
noteworthy that the significantly enhanced cytotoxicity of DOX
occurs after PTX participation, leading to 49.43% of apoptotic
ratio for P-DOX@PTX and 54.11% for P-DOX@PTX dimer, respectively.
These results suggest that PTX could improve DOX cytotoxicity
bioavailability, meanwhile, the selenium containing DOX and
PTX co-delivery system could overcome MDR effect efficiently
by inhibiting P-gp overexpression.
3.11 In vivo antitumor efficacy
To assess the in vivo anticancer efficacy of a series of nano-
formulas (P, P-DOX, P-DOX@PTX, P-DOX@PTX dimer), free drugs
and PBS were injected into 4T1 bearing Balb/c mice every three
days. The changes of tumor size and mice body weight from all
groups are presented in Fig. 9. Although the tumor volumes of
all groups were increased considerably after treatment with
PBS, the ignorable tumor suppression effect was caused by the
limited water solubility of free drugs.9,60 For other groups, the
tumors of P treated group had continuously growth because
there was no anticancer drugs contained in P micelles, suggesting
the good biocompatibility and bio-safety of the P micelles, as
shown in Fig. 9A(d). Nanocarriers fabricated through chemical
bonding and physical loading could promote drug content and
improve stability of carriers, leading to a significant tumor
inhibition. Especially, the P-DOX@PTX dimer micelles containing
both DOX and PTX could enhance drug accumulation in solid
tumor via EPR effect, as illustrated in Fig. 9A(g). This result may
be ascribed to the prepared dual drugs co-delivery systems that
could circumvent MDR effect. The apparent tumor suppression of
P-DOX and P-DOX@PTX treated groups was observed in
Fig. 9A(e and f), providing a promising treatment option for
early stage tumors. The maximum TSR was approximately
79.57% for P-DOX@PTX dimer and 70.87% for P-DOX@PTX micelles,
higher than that of 61.90% for free DOXꢁHCl and 58.13% for free
PTX. Compared with the change trends of tumor size, body weight
of mice treated with P-DOX@PTX dimer nanoformula increased
miraculously, as displayed in Fig. 9B. It is noteworthy that the
survival rate of all mice was 100% during treatment. Fig. 9C
exhibited the isolated tumor photographs from all experimental
groups, finding that the tumor size of groups treated with
nanoformulas was reduced significantly and even almost all
tumors are smaller than those treated with free drugs. In
particular, P-DOX@PTX dimer presented a remarkable inhibition
effect. These results were further attest the tumor size after
15 days treatment, which was in accordance with the results in
Fig. 9A.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We greatly acknowledge the financial support from the Funda-
mental Research Funds for the Central Universities (No.
GK201601003), the National Natural Science Foundation of
China (No. 21572123) and the Natural Science Research Fund
of Shaanxi University of Science and Technology (No. 2019BJ-43).
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