Reactive Oxygen Species and Glutathione Dual Redox-Responsive

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Reactive oxygen species and glutathione dual redox-responsive supramolecular assemblies with controllable release capability Yang Kang, Xin Ju, Li-Sheng Ding, Sheng Zhang, and Bang-Jing Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14640 • Publication Date (Web): 19 Jan 2017 Downloaded from http://pubs.acs.org on January 22, 2017

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ACS Applied Materials & Interfaces

Reactive oxygen species and glutathione dual redox-responsive supramolecular assemblies with controllable release capability Yang Kang, † Xin Ju, ‡ Li-Sheng Ding, † Sheng Zhang‡,* and Bang-Jing Li, †,* †

Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, Sichuan, China; ‡

State Key Laboratory of Polymer Materials Engineering (Sichuan University), Polymer Research Institute of Sichuan University, Chengdu 610065, China.

KEYWORDS: Supramolecular; Redox-responsive; Self-assembly; Micelles; Drug carrier

ABSTRACT: A dual redox and bio-relevant triggered supramolecular system is developed through the non-covalent supramolecular inclusion interactions between the ferrocene (Fc) modified on camptothecin (CPT) and β-cyclodextrin (β-CD) at the end of methoxy polyethylene glycol (mPEG). With these two segments, a stable non-covalent supramolecular structure, i.e. mPEG-β-CD/Fc-CPT, can be formed, and then self-assembled into micellar structures in water. Interestingly, these 1 ACS Paragon Plus Environment


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supramolecular micelles showed uniform sphere structure, high and constant drug loading content, hyper-fast redox-responsive drug release, and exhibited equal cellular proliferation inhibition toward A549 cancer cells, while the cytotoxicity evaluation of mPEG-β-CD indicated good biocompatibility. In vivo results revealed the mPEG-β-CD/Fc-CPT nanoparticles a higher in vivo efficacy without side effects. It is anticipated that this supramolecular complex may serve as a kind of new promising alternative for drug delivery systems.

1. Introduction In the past 30 years, the use of self-assembled nanoparticles has been extensively explored in both scientific and industrial areas. The particularly interesting applications of these nanostructures are to use in controlled and targeted drug delivery,1 protein sensing and protein delivery,2 due to their ability to solvate therapeutic drug molecules, which are normally hydrophobic by encapsulating in the water

excluded

hydrophobic

core.

In

the

abovementioned

literatures,

environmentally-responsive polymeric micelles can respond to a variety of stimuli, e.g. protons and hydroxyl ion,3 light,4 heating,5 redox species,6, 7 proteases8 as well as ultrasound and magnetic feild.9 Due to their versatilities, these micelles are excellent host candidates for molecules with a diversity of shapes, sizes and functionalities. Polymeric nano-carriers possess superior features, such as good stability and drug solubility, well-defined surface properties, interaction prevention in trafficking, and controlled drug releasing behaviors, have shown great potential for targeting delivery

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of therapeutic molecules to tumors.10-12 However, amphiphilic block copolymers are limited in their utilities due to the cumbersome and even uncontrollable syntheses requiring extensive purifications, and probably causing side reactions. Preparing amphiphilic block copolymers that could accumulate at target tissue with long-circulation in the blood remains a challenge work.

Apart from conventional multi-sensitive micelles with tunable properties linked by cleavable covalent bonds, supramolecular micelles are constructed via non-covalent interactions, e.g. electrostatic interactions, host-guest and metallic coordination, π-π interactions, hydrogen bonding and so on.13-18 Supramolecular approach is superior in that functional ligands can be conveniently incorporated onto the surface of supramolecular micelles surface, which brings functionalities such as targeting and cell penetration to these nanoparticles, and meanwhile environmental responsive

bonds can be easily introduced. Overall, the superiority of

supramolecular

amphiphiles

brings

more

potential

regarding

conventional

amphiphiles and bridges the supramolecular and colloidal sciences.

Host-guest interaction can enable a block copolymer to be fabricated in a non-covalent and adaptive manner.19 The non-covalent interactions between CDs and some small organic molecules such as azobenzene (AZO), Fc and adamantine (ADA) are excellent candidates for the preparation of new kinds of non-covalently connected amphiphilic block copolymers, resulting in the formation of non-covalently connected micelles (NCCMs).14, 20, 21 In particular, the supramolecular micelles, which are based



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on the connection between CDs/small molecule inclusion complexes and interconnected via terminal host-guest inclusion, would go through a reversible effect of association and dissociation. This would finally induce a preferable assembly and disassembly of micelles when applying external stimuli, e.g. pH,22 light,23 temperature,24 molecules,25 and redox agents26 or voltage.27 However, to the best of our knowledge, CDs based dual redox and bio-relevant triggered supramolecular system NCCMs with anticancer agent as hydrophobic segment in NCCMs has not been reported yet.

Redox sensitive micellar assemblies are particularly interesting because of the higher glutathione (GSH) concentrations present in cancer cells. In tumor sites, extracellular GSH concentration is in the micromolar range whereas it is in the milimolar range at the intracellular environment, thereby facilitating the selective intracellular cleavage of disulfide bonds.28 Micelles based on disulfide bonds can be utilized for intracellular delivery of drug molecules by taking advantage of the high GSH concentration. Therefore, GSH can be used as bio-stimulus to facilitate deformation and rapid drug release from delivery systems containing disulfide links. Besides, it is well-known that a diversity of cancer cells would demonstrate heterogeneities of cellular redox status. As an example, higher level of ROS (such as H2O2) would be generated by cancer cells compared to normal celles, which is primarily attributed to chronic inflammation, oncogene mutation or mitochrondrial dysfunction.29 Hence, some H2O2 responsive smart materials, including nanoparticles and hydrogels, have been developed for drug releasing carriers. For instance, based on 4 ACS Paragon Plus Environment

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the reversible interation between β-CD and Fc, Zhu’s group developed a kind of redox-responsive cationic supramolecular polymers, which could be used as

gene

nano-carrier and has satisfactory in vitro transfection studies.7 However, the majority of the currently studied host-guest based polymeric stimuli-responsive systems respond only to a single bio-stimulus. Therefore, it would be more beneficial if these systems were designed to release in a controlled and systematic manner by responding to multiple bio-stimuli other than a single stimulus as predicted.

In this study, we developed a kind of dual redox and bio-relevant triggered supramolecular micellar system, from which stable micelles can be formed in aqueous environment. This system is of polymer-drug conjugate which is fixed and relative high in the content of loading drug. While in the tumor cells, this conjugate released active drugs, showing the cytotoxicity in vitro and in vivo. The hydrophilic component was chosen to be mPEG because the inherent properties of excellent biocompatibility, water solubility and non-adhesive to proteins for PEG. Scheme 1 illustrated the formation of mPEG-β-CD/Fc-CPT supramolecular complex micelles and it’s selectively release drugs in cancer cells. After endocytosis, a high GSH or H2O2 in cytoplasm, would rapidly destroy the disulfide bonds or the interation between β-CD and Fc, respectively. Consequently, this would result in a fast release of loading drugs with a pattern of chain-breakage, which eventually leads to the programmed death of tumor cells as well as effective tumor growth inhibition.

2. Materials and methods



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2.1 Materials

Poly(ethylene glycol) methyl ether acrylate (mPEG-A, Mw = 480, Sigma-Aldrich) was dried over P2O5 under vacuum at 40 ºC for 48 h before use. The purification of β-CD (Aladdin Reagent Co., Ltd) from water was accomplished by recrystallization before use. Ferrocene carboxylic acid (FcA), (S)-(+)-camptothecin (CPT), irinotecan hydrochloride, bis(trichloromethyl) carbonate, 2, 2'-dithiodiethanol and L-glutathione reduced were obtained from TCI Development Co., Ltd. (Shanghai, China) and used as received. p-Toluenesulfonyl chloride, thiourea were purchased from Chengdu Kelong Chemical Reagent Co., Ltd. (Chengdu, China) and used as received. N,N-Dimethylformamide (DMF), dichloromethane (CH2Cl2) and tetrahydrofuran (THF) were purchased from Beijing Chemical Reagents Co. Ltd and distilled from CaH2 under reduced pressure. Mono-6-Tos-β-CD (Tos-β-CD) and mono-6-thio-β-CD (β-CD-SH) were synthetize d according to our previous methods.26 Having been analytical grade, all other reagents were ready to use, with water and all solvents freshly redistilled. 2.2 Instruments Bruker Ascend 400MHz spectrometer (Switzerland) was used to record the 1H NMR spectra and 2D nuclear overhauser enhancement spectroscopy (2D NOESY) 1

HNMR spectra of the samples by using deuterated chloroform (CDCl3) or deuterated

dimethyl sulfoxide (DMSO-d6) as solvent with TMS as the internal reference. TU-1901 double beams UV-vis spectrophotometer (Beijing Purkine General 6 ACS Paragon Plus Environment

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Instrument Co. LTD., China) was used to record the UV absorbance of samples. Finnigan LCQDECA ESI-MS spectrometer (San Jose, CA, USA) was used for the mass spectrometry analysis with scanning scope of m/z 100-2000. The dynamic light scattering (DLS) results were recorded on BI-9000AT, BI-200SM, Brookhaven Instruments Co., USA. Surface tension was measured with the pendant drop method at room temperature by a contact angle meter (Dataphysics OCA40 Micro, Germany). The transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were performed at voltage of 80 KV on Jeol JEM-100CX and on Jeol JSM-7600F, respectively. Liquid chromatography (LC) was investigated on a Waters ACQUITY ultrahigh pressure liquid chromatograph (UPLC) (Acquity, Waters Corp., U.S.) equipped an Acquity UPLC BEH RP18 column (2.1 mm × 100 mm, 1.7µm). The column was maintained at 50 °C with the mobile phase ranging from 15% to 100% methanol/water containing 0.1% formic acid in 5 minutes at a flow rate of 0.4 mL·min-1. A volume of 1 µL sample acetonitrile solutions was directly injected at a constant concentration.

2.3 Preparation of the mPEG-β-CD and Fc-CPT

Synthesis of mPEG-β-CD. Poly(ethylene glycol) methyl ether acrylate (mPEG-A, Mw = 480, 0.43 g, 0.88 mmol), mono-6-thio-β-CD (0.96 g, 0.84 mmol) and a drop of triethylamine were added in a 50 mL round-bottomed flask, followed dissolved by 20 mL anhydrous DMF at room terperature. Then, the reaction mixture was stirred for another 24 h under Ar protection, followed by condensed in vacuum



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and purified by using Sephadex LH-20 column with methanol as eluent to obtain mPEG-β-CD as pale solid (1.15 g, yield 84.6%). 1H NMR (400 MHz, ppm, DMSO-d6): δ 5.80-5.69 (m, 14H, OH-2,3), 4.86-4.78 (br s, 7H, H-1), 4.49-4.43 (m, 6H, OH-6), 4.12 (t, 2H, β-CD-SCH2CH2OOCH2CH2CH2O-), 3.78 (t, 2H, β-CD-SCH2CH2OOCH2CH2CH2O-), 3.69-3.47 (m, 72H, H-3,5,6, -OCH2CH2O-), 3.46

(t,

2H,

β-CD-SCH2CH2OOCH2CH2CH2O-),

3.44

(t,

2H,

β-CD-SCH2CH2OOCH2CH2CH2O-), 3.43-3.21 (m, overlaps with HOD, OH-2,4), 3.23 (s, 3H, -CH3). ESI-MS: m/z 1668±44n [M+Cl]- (44 is the molecular weight of the repeated unit of mPEG, n is the number of the repeated unit of mPEG). Synthesis of Fc modified 2, 2'-dithiodiethanol (HSEFc). FcA (4.61 g, 2.01 mmol), 2, 2'-dithiodiethanol (3.09 g, 2.02 mmol) and DMAP (0.66 g, 0.54 mmol) were dissolved in anhydrous dichloromethane of 60 mL in a round-bottom flask of 100 mL, after which the solution was cooled to be 0 °C . Whereafter, DCC (1.49 g, 7.23 mmol) was added into the solution and the reaction was conducted for another 24 h under Ar protection at 25 °C. Followed by remove the insoluble by-product dicyclohexyl carbodiurea (DCU) using buchner funnel. The organic solution was condensed in vacuum and the followed by purification on silica column chromatography with

DCM/Acetone (5:1) as eluent to obtain the pure product

HSEFc as yellow solid (6.95 g, yield: 94.7%). 1H NMR (400 MHz, CDCl3): δ 4.79 (s, 2H, =CHC(COOH)CH=), 4.49 (t, 2H,-CH2OOC-Fc), 4.40 (s, 2H,

-CH=CH-), 4.18

(s, 5H, another cyclopentyl), 3.92 (q, 2H, -CH2CH2OH), 3.02 (t, 2H,


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-SCH2CH2OOC-Fc), 2.92 (t, 2H, -CH2CH2OH) , 2.31 (t, 1H, --CH2CH2OH). ESI-MS: m/z 389 [M+Na]+. Synthesis of Fc modified camptothecin (Fc-CPT). Camptothecin (CPT, 1.01 g, 2.87 mmol) and DMAP (1.06 g, 8.69 mmol) were added in a 100 mL round-bottomed flask, followed dissolved by 50 mL anhydrous CH2Cl2 at room terperature under Ar atmosphere. Subsequently, triphosgene (0.29 g, 0.96 mmol) was added to the solution and the reaction solution was stirred for another 30 min at 25 °C. A solution HSEFc (1.16 g, 3.16 mmol) in 15 mL anhydrous CH2Cl2 was added solwly via a constant pressure funnel. The reaction mixture was stirred overnight during which a white precipitate was formed. After filtration and evaporating all the solvents, the residues were diluted with diethyl acetate and washed with water, 1.0 M HCl, and brine for twice, respectively. The organic solution was condensed in vacuum and the followed by purification on silica column chromatography with

DCM/Acetone (5:1) as eluent

to give the pure product Fc-CPT as yellow solid (2.96 g, yield 91.2%). 1H NMR (400 MHz, ppm, CDCl3): δ 8.37 (s, 1H, -Ar), 8.22 (d, 1H, -Ar), 7.93 (d, 1H, -Ar), 7.82 (t, 1H, -Ar), 7.66 (t, 1H, -Ar), 7.33 (s,1H, -Ar), 5.67 (d, 1H, -NCH2Ar), 5.40 (d, 1H, -NCH2Ar), 5.26 (d, 2H, -COOCH2Ar), 4.74 (s, 2H, =CHC(COOH)CH=), 4.42 (t, 2H,-CH2OOC-Fc), 4.40 (s, 2H,

-CH=CH-), 4.38 (t, 2H,

-O(O=C)OCH2CH2S-),

4.15 (s, 5H, another cyclopentyl), 2.97-2.95 (m, 4H, -CH2S-S-CH2-), 2.30-2.12 (m, 2H, -CH2CH3), 1.01 (t, 3H, -CH2CH3). ESI-MS: m/z 763 [M+Na]+. 2.4 Micellar preparation



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30.52 mg mPEG-β-CD and an equivalent molar amount of Fc-CPT (13.85 mg) were mixed by 1.0 mL of THF, and then sonicated for 0.5 h to make sure adequate dissolution and mPEG-β-CD/Fc-CPT supramolecular inclusion complexation formation. During stirring, 20.0 mL deionized water was dropped into the THF solution through a constant pressure drop funnel slowly in 1 h, and stirred for another 24 h to enable the sufficient evaporation of THF and the formation of the micellar solution at room temperature.

2.5 Characterization of the mPEG-β-CD/Fc-CPT supramolecular micelles The

determination

of

the

critical

micelle

concentration

(CMC)

of

mPEG-β-CD/Fc-CPT supramolecular complex was achieved with the method as described previously.30 Briefly, The surface tension method was applied to determine the CMC of mPEG-β-CD/Fc-CPT supramolecular polymer with its concentration varied from 0.001 to 1 mg/mL. Data acquisition was made using the pendant drop method via a contact angle meter at room temperature. The micellar nano-structure size in water (50 µg/mL) were obtained using the method of DLS via an electronic source of coherent innove 304 laser with a wavelength of 532 nm and a scattering angle of 90 ° at 37 °C. Each sample was tested for three times with the mean diameter shown for three replicate samples. The micellar nano-structure morphology was inspected by using TEM and SEM. Sample solution with a concentration of 100 µg/mL was dropped onto copper


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grid for TEM or mica plate for SEM and dried at 37 °C, respectively. TEM inspections were made with an accelerating voltage of 80 kV.

2.6 The drug releasing behavior of the mPEG-β-CD/Fc-CPT supramolecular micelles

The drug release of the supramolecular micelles was investigated in the phosphate buffer (PBS, pH 7.4). In brief, 2 mL of mPEG-β-CD/Fc-CPT supramolecular complex micelles solution (10 mg/mL) was placed in a dialysis bag (MWCO 3500) at 37 °C for the dialysis experiment in PBS solution containing various GSH (0, 2 µM, 20 µM, 2 mM, 20 mM) or H2O2 (1 µM, 20 µM, 50 µM, 100 µM) concentrations for drug releasing. At certain time intervals, a 4 mL PBS medium was aspirated and another 4 mL fresh PBS was added. The amount of CPT or Fc-CPT in the solution outside the dialysis bag was determined by UPLC. Meanwhile, the behaviors of Fc-CPT convert to HSEFc and CPT under the condition of GSH were further investigated. Briefly, 2 mL of Fc-CPT DMSO solution (10 mg/mL) was placed in a dialysis bag (MWCO 3500) at 37 °C for the dialysis experiment in PBS solution containing GSH (20 mM) for drug releasing. At certain time intervals, 50µL the solution was withdrawn from the dialysis bag and tested by UPLC. 2.7 In vitro anticancer efficiency

Methyl tetrazolium (MTT) assay was carried out on the A549 lung cancer cell line and human lung fibroblast HLF cell line for the cytotoxicity evaluation experiments of the mPEG-β-CD, free CPT, Fc-CPT and mPEG-β-CD/Fc-CPT 11 ACS Paragon Plus Environment


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supramolecular complex in 96-well plates.31 In brief, 96-well culture plates were utilized to culture HLF and A549 cells in DMEM and RPMI 1640 medium, respectively, with a density of 8000 cells per well. 10 % fetal bovine serum was supplied into the culture medium in a humidified environment of 5 % CO2 at 37 °C for 24 h. All cell lines were further incubated with mPEG-β-CD, free CPT, Fc-CPT and mPEG-β-CD/Fc-CPT complex at varying of CPT equivalent concentrations of 0.05-10 µg/mL by RPMI 1640 medium for A549 cells and DMEM medium for HLF cells two days incubation in 5 % CO2 incubator at 37 °C, respectively. 24 hour after the culture medium containing drug was aspirated and replaced by a 100 µL fresh one which contains 10% fetal bovine serum, 5 µL MTT solution with a concentration of 15 mg/mL was supplemented into each well followed by another 4 hours incubation in 5 % CO2 incubator at 37 °C. The MTT solution was then removed and 150µL of DMSO was added to dissolve the crystals for the generation a homogeneous blue-purple solution. A microplate reader was then used to record the solution absorbance at a wavelength of 570 nm. By comparing this absorbance with that for control cells containing only cell culture medium recorded at the same wavelength, the relative cell viability (%) can be obtained. This test was repeated for six times with the average data shown.

2.8 In vivo anticancer efficiency

Male Chinese Kun Ming mice (25-30 g) were used for the in vivo anticancer efficiency of the mPEG-β-CD/Fc-CPT supramolecular micelles. The experiment of


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the mice complies with the protocols approved by the Laboratory Animal Center at Sichan University. 18 mice were equally divided into three groups and kept under sterile conditions with a light/dark cycle of 12 h in an environment with controlled temperature. S180 tumor cells were implanted into the subaxillary of the mice, and after 1 week, tumors were found to be well established along the subaxillary. Three groups (6 mice per group) was administered with 100 µL mPEG-β-CD/Fc-CPT complexes micelles (CPT equivalent 10 mg/kg), irinotecan (CPT equivalent 10 mg/kg) in PBS solution or pure PBS solution per day by using tail vein injection for five successive days, respectively. During the experiment, no significant side effect was observed for the mice. After 24 h, all the mice were executed, followed by tumors dissected and weighted for analysis.

3. Results and discussion

3.1 Synthesis of the mPEG-β-CD/Fc-CPT supramolecular complex

To synthesize mPEG-β-CD, mPEG was functionalized with β-CD by thiol-yne click reaction between mono-6-thio-β-CD (β-CD-SH) and mPEG acrylate in 86.7 % yield (Scheme 2). The Fc-CPT was designed and synthesized by conjugating Fc to CPT via dithioether bond as linkages in 82.5 % yield (Scheme 3).

As known, a 1:1 inclusion complex could be formed through the interaction between β-CD and Fc, while the inclusion complex including the oxidized Fc could not because the host and guest mismatch with each other.32 Meanwhile, it is known that Fc moiety could be easily embedded into the cavity of β-CD for the formation of 13 ACS Paragon Plus Environment


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a complexation, since the inclusion constant between β-CD and Fc is about 2500 M-1 and the inclusion constant between β-CD and CPT is only 266 M-1.33, 34 Hence, the interaction between CPT and β-CD would not influence the complexation of Fc and β-CD. As shown in Figure 1, in the 2D NOESY NMR result for mPEG-β-CD/Fc-CPT supramolecular complex, obvious cross-peaks can be spotted due to the dipolar interaction between the signals at 3.65-3.46 ppm, which are ascribed to the 3-, and 5-H protons on the rim of torus shaped structure of β-CD, and the signals at 4.79 and 4.43 ppm, which are attributed

to the aromatic rings of Fc moieties, clearly

indicating the deep embedding of Fc moieties

into the cavities of β-CD, attributed to

the successfully preparation of mPEG-β-CD/Fc-CPT supramolecular complex via β-CD/Fc supramolecular interactions.

3.2 Preparation of the mPEG-β-CD/Fc-CPT supramolecular inclusion micelles

For the purpose to obtain an amphiphilic supramolecular structure, mPEG was chosen to be served as the hydrophilic block, while the water insoluble CPT was chosen to be served as the hydrophobic block. The amphiphilic mPEG-β-CD/Fc-CPT supramolecular complex could be spontaneously assembled into a micellar like structure in water with CPT as hydrophobic inner core and mPEG as hydrophilic outer shell. To test our hypothesis, the CMC of the mPEG-β-CD/Fc-CPT was firstly studied using the surface tension method. As is known, the concentration of amphiphilic copolymer is above CMC with the polymeric micelles formed for the minimization of the interfacial energy. While a significant change of the tensile force


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occurs around CMC, the tensile force could be conducted for wide-ranging concentrations. In Figure 2A, it is shown the CMC of mPEG-β-CD/Fc-CPT supramolecular complex formed micelles investigated by the means of surface tension is about 30.2 µg/mL. Moreover, the light transmittance of mPEG-β-CD/Fc-CPT supramolecular complex with various concentrations in water was investigated at 700 nm. As suggested by the translucent colloidal solution formation in Figure S7, the CMC value was 29.5 µg/mL, which is almost the same as that obtained by the surface tension method. Furthermore, DLS was carried out to provide indirect evidence for the micellar structures formation of mPEG-β-CD/Fc-CPT supramolecular complex. As the result shown in Figure 2B, it could be concluded that the mPEG-β-CD/Fc-CPT supramolecular complexes aggregate together were particles around about 180 nm with a narrow size distribution (PDI=0.152), which indicating the diameter of the mPEG-β-CD/Fc-CPT supramolecular complex formed micelles is homogeneous. Furthermore, the inspected data by TEM and SEM showed that mPEG-β-CD/Fc-CPT aggregates were uniform spherical nanoparticles around 190 nm (Figure 2C and Figure 2D), which was in agreement with diameters detected by DLS. 3.3 In vitro triggered drug releasing studies

In contrast to the conventional amphiphilic copolymers, the polymer component is replaced by the water-insoluble drug CPT in this study, so the micelles formed by mPEG-β-CD/Fc-CPT

supramolecular

complex

showed

a

fixed

and

high

drug-encapsulation content. As could be calculated from following formula (Eq1), the fixed drug loading content was about 14.7 wt %, while the drug contents of the 15 ACS Paragon Plus Environment


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conventional nanoparticles and liposomes are usually lower than 10% and with batch-to-batch differences.35 Drug loading content (%) =

Wdrug in nanocapsules Wnanocapsules

× 100

(Eq1)

It should be noticed that one of the unique features of tumors is their endo-/lysosomes and the cytosol have reductive environments compared to the extracellular compartments due to the active accumulation of proteinogenic cysteine and the presence of reductive enzymes,36,

37

such as gamma-interferon inducible

lysosomal thiol reductase (GILT), in lysosomes and the higher GSH concentrations in the cytosol (the intracellular GSH concentration could even as high as 2-10 mM, about 100-1000 times than that in extracellular). Therefore, redox-sensitive polymeric nanoparticles that respond to reductive environments of endo-/lysosomes or cytosol provide a rationale for the intracellular delivery of pharmaceutical payloads. Accordingly, polymers containing disulfide groups have been intensively investigated to design redox responsive nano-scaled drug carriers with the ability to undergo reduction in the endocytotic pathway as well as in the cytosol.38-40 Another special characteristic of tumors is their over-expressed of ROS, especially H2O2, in tumor cells. As is well known, H2O2 plays a crucial part during cancer development, and several tumor cell lines even could constitutively produce large amounts of H2O2 during their growth process.41 So, it is important to note the use of the micellar delivery systems with redox-sensitive behavior that can result in a decrease in the cellular levels of H2O2 and release the antitumor drugs to kill the cancer cells while


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these systems are accumulated in the tumor tissues due to the enhanced permeability and retention (EPR) effect.12, 42-45 However, the use of GSH and H2O2 dual responsive particles for exhibition of drug release under appropriate concentrations in the treatment of tumor cells remains challenging.

Herein, both GSH and H2O2 were chosen as redox agents for the investigation of the drug releasing behaviors of mPEG-β-CD/Fc-CPT complexes to mimic the environment in tumor cells. CPT, a well-known topoisomerase I Inhibitor, broad-spectrum antineoplastic agent applied to a variety of cancers such as leukemia, colon cancer, liver cancers and lung cancers, was selected to be the model molecule to study the drug releasing performance of mPEG-β-CD/Fc-CPT supramolecular complex micelles. It is interesting that the release rate and release efficiency (RE) of encapsulated CPT were quite different under the effect of GSH or H2O2 under relevant concentrations of tumor cells. As shown in Figure 3A, in the absence of GSH or at a normal concentration of cell GSH (such as 2 mM), the CPT only released about 10 % in the first 24 h. In comparison, a hyper-fast release can be achieved for the constituent CPT via the redox-responsive elimination reaction at a GSH concentration of 10 mM, i.e. around 31.4 % CPT molecules were released in the first 2 h with almost all the other molecules released within 10 h thereafter. Moreover, a burst release of 52.5 % Fc-CPT molecules could be achieved in prior 15 h at a tumor cell GSH concentration (e.g., 100 µM), indicating the well tumor microenvironment responsive character of the micelles. By using H2O2 as redox agent, the oxidation of Fc and dissociation of β-CD/Fc inclusion took place at the micelle corona-core 17 ACS Paragon Plus Environment


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interface. The Fc-CPT releasing behaviors of mPEG-β-CD/Fc-CPT supramolecular complex micelles at different H2O2 concentrations at 37 ºC were further investigated. As shown in Figure 3B, after 48 h, the Fc-CPT release amount increase from 8.5 %, 13.2 %, 42.1 % to 78.9 % as the concentration of the H2O2 increased from 1 µM, 20 µM, 50 µM to 100 µM, respectively. It is shown that the Fc-CPT can be released in a controlled manner by adjusting the H2O2 concentration. In contrast, without H2O2 stimulus or at a normal cell H2O2 concentration (e.g., 1 µM), less than 20 % Fc-CPT molecules were released, which may be caused by the dissolution and diffusion of the Fc-CPT. Although the released drug was Fc-CPT under the condition of H2O2, it should be noticed that almost Fc-CPT molecules could be converted into CPT and Fc molecules within 30 min with 10 mM GSH (Figure 4).

Hence, we could equal the

Fc-CPT molecules released amount as CPT molecules released amount in the drug release studies, considering the characteristic high concentrations of ROS and GSH in tumor cells.

The drug releasing behaviors of mPEG-β-CD/Fc-CPT complexes were also investigated using DLS. Firstly, the redox-sensitive behavior of mPEG-β-CD/Fc-CPT supramolecular polymer formed micelles was conducted with 10 mM GSH. Figure S8A shows the hydrodynamic diameter (Dh) distribution of the micelles detected by DLS 10 h after the addition of GSH, the occurred micrometer-scaled nanoparticles would be attributed to the aggregation of the water-insoluble CPT, which is formed after the cleavage of disulfide bonds by GSH. The CPT and mPEG are linked by redox-sensitive

β-CD/Fc

inclusion

pair.

Furthermore,

we

investigated

the 18

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redox-triggered behavior of mPEG-β-CD/Fc-CPT supramolecular polymer formed micelles in the presence of 1 µM H2O2 by DLS. Figure S8B shows the Dh distribution of the micelles detected by DLS 10 h after the addition of H2O2, the micrometer-scaled nanoparticles which would be attributed to the aggregation of the water insoluble Fc-CPT that was formed after the dissociation of β-CD/Fc inclusion. Hence, the results in Figure S8 could further demonstrate the drug release behaviors of mPEG-β-CD/Fc-CPT supramolecular polymer formed micelles in tumor microenvironment, patterned by dual redox-responsive chain-breakage.

The diverse responsiveness to bio-relevant stimuli of mPEG-β-CD/Fc-CPT complexes micelles provided an opportunity to realize the fine-tuning of the release behaviors of loaded molecules at certain circumstances. The release of encapsulated molecules could proceed either quickly and completely in tumor cells at a high GSH concentration, or relatively slowly and partially in tumor cells at a high H2O2 concentration. In addition, two kinds of release behaviors could also be realized by combination the effect of GSH and H2O2 in tumor microenvironment, suggesting the possible use of the supramolecular complex as a desired drug nano-carrier for tumor treatment due to the smart, hyper-fast and diverse stimuli-responsive CPT release pattern.

The responsive micelles are good candidates for encapsulation and release of molecules. The primary purpose of our molecular design is to bring out multi-sensitive release properties under tumor microenvironment to a single micelle.



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3.5 In vitro anticancer efficiency

The

cytotoxicities

for

free

CPT,

Fc-CPT

and

mPEG-β-CD/Fc-CPT

supramolecular complex micelles have been examined using MTT assays in cell lines for A549 lung cancer and Human lung fibroblast HLF. Figure 5A shows that the effect of Fc-CPT and mPEG-β-CD/Fc-CPT supramolecular complex micelles were showed equal of that of CPT at concentrations from 0.05-10 µg/mL, which means the Fc-CPT and Fc-CPT supramolecular complex micelles kept the excellent anticancer activity of CPT due to the hyper-fast drug release with a pattern of redox-triggered chain-breakage in tumor microenvironment. In contrast, HLF cells were also exposed to the micelles of mPEG-β-CD/Fc-CPT supramolecular complex with a concentration ranging of 0.05-10 µg/mL. As a result shown in Figure 5B, the cellular toxicity of micelles of mPEG-β-CD/Fc-CPT supramolecular complex to HLF cells is significantly lower than that to A549 cells over a wide range of tested concentrations. These phenomena may be because the lower concentration of H2O2 and GSH in normal HLF cells could not greatly affect the micellar structure of the supramolecular complex. This avoids the hyper-fast drug release behaviors, patterned by redox-responsive chain-breakage, in normal physiological activities and causes a lower cytotoxicity. Moreover, the result showed in Figure 5C revealed that mPEG-β-CD for constructing the supramolecular complex micelles was almost nontoxic to the cells with a maximum test concentration of 60 µg/mL, which indicating the inherited excellent biocompatibility property of mPEG and β-CD.


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3.6 In vivo anticancer efficiency

The in vivo studies of mPEG-β-CD/Fc-CPT complexes micelles on S180 sarcoma tumor implanted mice were further investigated. As could be figured out in Figure 6, compared to the control group for PBS, about a 3-fold inhibition of the tumor growth can be observed in the treatment group, indicating the promises of the mPEG-β-CD/Fc-CPT complexes as an anticancer drug. It should be noted that an appropriate amount of organic solutions such as ethanol should be used to dissolve free CPT and Fc-CPT for i.v. administration. Serious allergies, however, would be induced after using an excess of organic solvent, leading to the quick death of mice. Therefore, irinotecan hydrochloride, a water soluble CPT derivative, was used as the reference drug to investigate the inhibition efficiency for tumor growth of the micelles of mPEG-β-CD/Fc-CPT supramolecular polymer. It was also found that the mPEG-β-CD/Fc-CPT can significantly reduce the tumor burden to be 3/5 of irinotecan, which is an extensively-used agent for cancer treatment and are clinically effective for a variety of tumors. This is attributed to an accumulation of the micelles of mPEG-β-CD/Fc-CPT supramolecular polymer in the cancerous tissues due to tumors’ EPR effect in their presence, and the smart drug releasing performances showing a pattern of redox-triggered chain-breakage in the tumor microenvironment. Meanwhile, significant loss of weight or acute toxicity of the micelles has not been spotted over the experiment period. Thus we believe such satisfactory result is worth of making further thorough in vivo research. The statistical significance of the test



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data was evaluated by the Student’s t-test (P < 0.05) and data is plotted in the format of means ± standard errors (SE).

4. Conclusions

To sum up, a kind of non-covalently connected copolymers has been designed and fabricated, in which mPEG-β-CD and Fc-CPT segments are connected through the redox-triggered β-CD/Fc inclusion pair. These mPEG-β-CD/Fc-CPT complexes could form stable micelles in water. As drug carrier, the mPEG-β-CD/ Fc-CPT complexes micelles showed a significantly hyper-fast CPT release at tumor high cell redox microenvironment than normal cells. The polymer mPEG-β-CD is nontoxic even at high concentration about 60 µg/mL and the activity of mPEG-β-CD/Fc-CPT supramolecular complex micelles were showed equal of that of CPT at various CPT equivalent test concentrations. This dual redox responsive feature enables the fine-tuning of the release behaviors of encapsulated molecules. The encapsulated molecules could be released through three models according to the demand of application: hyper-fast in the tumor cells. Fabrication of multi-sensitive systems with fine-tunable release properties is considered to be a very important future direction. The new designing concept of connecting sensitive components by reversible non-covalent bonds demonstrated here will facilitate the development of various new platforms for achieving dual ROS and GSH bio-relevant triggered anticancer drug delivery, which would make it possible to invent a novel and clinically-applicable drug delivery system.


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Figure 1 2D NOESY NMR spectrogram of mPEG-β-CD/Fc-CPT supramolecular complex in the absence of H2O2 (solvent: DMSO-d6).


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Figure 2 (A) Plot of the surface tension of mPEG-β-CD/Fc-CPT supramolecular polymer formed micelles solution with different concentration at 25 °C; (B) DLS result for mPEG-β-CD/Fc-CPT-formed micelles; (C) TEM images of the mPEG-β-CD/Fc-CPT-formed micelles (scale bar of 250 nm) and (D) SEM images of the mPEG-β-CD/Fc-CPT-formed micelles (scale bar of 1 µm).



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Figure 3 (A) Release profiles of CPT from the mPEG-β-CD/Fc-CPT supramolecular complex micelles at different GSH concentrations; (B) Release profiles of Fc-CPT from the mPEG-β-CD/Fc-CPT supramolecular complex micelles at different H2O2 concentrations.


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Figure 4 (A) Mechanism of GSH-triggered CPT releasing out from Fc-CPT. The rapid UPLC analytical performance for drug release monitoring at (B) 0 min; (C) 15 min; (D) 30 min.



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Figure 5 (A) The in vitro cytotoxicity of free CPT, Fc-CPT and mPEG-β-CD/Fc-CPT supramolecular complex micelles to A549 cell lines; (B) The in vitro cytotoxicity of mPEG-β-CD/Fc-CPT supramolecular complex micelles to HFL cells; (C) The cell viabilities of A549 cell lines as the function of mPEG-β-CD concentrations.


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Figure 6 In vivo antitumor efficiency of mPEG-β-CD/Fc-CPT complexes and irinotecan to KM mice inoculated with S180 sarcoma (*p < 0.05).



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Scheme 1 (A) Illustration of formation of mPEG-β-CD/Fc-CPT supramolecular complex micelle; (B) Schematic of mPEG-β-CD/Fc-CPT supramolecular complex micelles selectively release drugs in cancer cells.


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Scheme 2 Synthesis of mPEG-β-CD.



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Scheme 3 Synthesis of ferrocene decorated camptothecin (Fc-CPT).


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ASSOCIATED CONTENT

Supporting Information. Mass spectra and 1H NMR spectra of mPEG-β-CD, HSEFc and Fc-CPT are available in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]; [email protected].

ORCID Yang Kang: 0000-0003-4580-9316

Xin Ju: 0000-0003-2571-5857

Bang-Jing Li: 0000-0002-0405-6883

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

Funding Sources 33 ACS Paragon Plus Environment


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All authors received funding from National Natural Science Foundation of China (Grant Nos. 51573187, 51373174), West Light Foundation of CAS.

ABBREVIATIONS DCM, dichloromethane; THF, tetrahydrofuran ; TEM, transmission electron microscope; RPMI-1640 medium, Roswell Park Memorial Institute-1640 medium; DMEM medium, dulbecco's modified eagle medium; KM mice, Kunming

mice;

PBS,

phosphate-buffered

saline;

MTT,

Methylthiazolyldiphenyl-tetrazolium bromide.

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Graphical abstract


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303x239mm (72 x 72 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

179x189mm (300 x 300 DPI)


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


175x97mm (300 x 300 DPI)


Reactive Oxygen Species and Glutathione Dual Redox-Responsive

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