Redox-Sensitive Hydroxyethyl StarchâDoxorubicin ... - ACS Publications

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Redox-sensitive hydroxyethyl starch-doxorubicin conjugate for tumor targeted drug delivery Hang Hu, Yihui Li, Qing Zhou, Yanxiao Ao, Chan Yu, Ying Wan, Hui-Bi Xu, Zifu Li, and Xiangliang Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11932 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on October 29, 2016

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ACS Applied Materials & Interfaces

Redox-sensitive hydroxyethyl starch-doxorubicin conjugate for tumor targeted drug delivery

Hang Hu1, Yihui Li1, Qing Zhou1, Yanxiao Ao1, Chan Yu1, Ying Wan1, Huibi Xu1, Zifu Li1, 2*, Xiangliang Yang1* 1. National Engineering Research Center for Nanomedicine, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, P. R. China 2. Wuhan Institute of Biotechnology, high tech road 666, East Lake high tech Zone, Wuhan, 430040, P. R. China

*Corresponding authors:

Professor Zifu Li Tel.: 86 27 87792234 Fax: 86 27 87792234 E-mail: [email protected]

Professor Xiangliang Yang Tel.: 86 27 87792147 Fax: 86 27 87792234 E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract Doxorubicin (DOX) is one of the most potent anticancer agents in cancer chemotherapy, but the clinical use of DOX is restricted by its severe side effects caused by nonspecific delivery. To alleviate the side effects and improve the antitumor efficacy of DOX, a novel redox-sensitive hydroxyethyl starch-doxorubicin conjugate, HES-SS-DOX, with diameter of 19.9 ± 0.4 nm was successfully prepared for tumor targeted drug delivery and GSH-mediated intracellular drug release. HES-SS-DOX was relatively stable under extracellular GSH level (～2 µM) but released DOX quickly under intracellular GSH level (2-10 mM). In vitro cell study confirmed the GSH-mediated cytotoxicity of HES-SS-DOX. HES-SS-DOX exhibited prolonged plasma half-life time and enhanced tumor accumulation in comparison to free DOX. As a consequence, HES-SS-DOX exhibited better antitumor efficacy and reduced toxicity compared with free DOX in in vivo antitumor activity study. The redox-sensitive HES-SS-DOX was proved to be a promising prodrug of DOX, with clinical potentials, to achieve tumor targeted drug delivery and timely intracellular drug release for effective and safe cancer chemotherapy.

KEYWORDS: Redox-sensitive, Conjugate, Hydroxyethyl starch, Doxorubicin, Tumor targeted drug delivery, Chemotherapy


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1. Introduction Doxorubicin (DOX) is one of the most potent anticancer agents in cancer chemotherapy, and it can be used to treat a broad spectrum of cancers, including cancer of liver, breast, bladder, esophagus, stomach, and endometrial.1 But the use of DOX is limited by its side effects, such as severe cardiotoxicity and nephrotoxicity.2-3 To alleviate the side effects of DOX, macromolecules based drug carriers, such as polysaccharides, synthetic copolymers, polymeric micelles, and polymersomes, have been explored for both controlled and localized drug delivery.4-9 Among these drug carriers, polysaccharides have received tremendous attention recently because of their outstanding physicochemical and physiological properties, including biocompatibility, biodegradability, and polyfunctionality for chemical modifications.10 Several polysaccharides, such as pullulan, chitosan, dextran, and hyaluronic acid, have been explored for DOX delivery, where DOX is either conjugated onto these polysaccharides

or

physically

encapsulated

inside

polysaccharides

based

nanoparticles.11-14 Compared with physical encapsulation, conjugation strategies would be more preferable because conjugating drugs onto polysaccharides can produce more stable prodrugs with only two ingredients, which can minimize the possible toxicity induced by other foreign materials. These conjugates can preferentially accumulate in solid tumors during circulation in blood vessels by use of the leaky and permeable tumor vasculature, which is well known as “enhanced permeability and retention effect” (EPR effect).15 The resulting therapeutic



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advantages include enhanced anti-tumor activity and reduced systematic toxicity.10, 12, 16

However, few studies involved polysaccharides-DOX conjugates have entered the

clinical trials.17 A dextran-DOX conjugate once entered phase I clinical trials.12 This dextran-DOX conjugate, however, still suffered from some problems. Dextran was expected to be the safest carrier. Nevertheless, modification of oxidized dextran with pendant groups to conjugate drugs results in a non-biodegradable and immunogenic polymer. As a consequence, the dextran-DOX conjugate (AD-70) with a molecular weight of 70,000 g mol-1 showed unexpected toxicities at a dose starting from 40 mg/m2 in a phase I clinical trials. Therefore, the development of polysaccharide-drug conjugates with clinical potential is still a pressing unmet need. Many studies have revealed that selective chemotherapy of macromolecular prodrugs can only be realized when the linkages between macromolecules and drugs could remain stable in the blood while allow fast drug release after accumulated in tumor sites and/or endocytosed by tumor cells.18-19 Disulfide bond have been studied extensively as a redox-sensitive linkage, and it can be degraded by GSH through a thiol-disulfide exchange reaction.20-21 It is known that the intracellular GSH concentration (2-10 mM) is substantially higher than extracellular GSH concentration (～2 µM).22 Besides, it has been reported that the intracellular GSH level of tumor cells can be several times higher than that of normal cells.23 So disulfide bond could be stable in the extracellular environment while be rapidly cleaved by intracellular high concentration of GSH once entering into tumor cells. Up to now, disulfide bond has been widely utilized for polymer-camptothecin and polymer-paclitaxel


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conjugation.24-27 However, disulfide linked polymer-DOX conjugates are seldom reported so far.28-29 Hydroxyethyl starch (HES) is a semi-synthetic polysaccharide and has wide clinical use as plasma volume expander.30-31 HES is normally synthesized from the starting material waxy maize which contains more than 95% of amylopectin. The produced HES is highly water soluble and keeps the branched structure of amylopectin. HES can be categorized into various classes based on its molecular weight, mole substitution of hydroxyethyl, and substitution pattern (C2/C6 ratio). These parameters affect the α-amylase-mediated degradation of HES in blood, thus determine the pharmacokinetics of HES, enabling very convenient ways to tailor the in vivo fates of HES by simply adjusting these parameters.31-37 In addition, it has been reported that HES exhibited lower disturbance of coagulation and clearly reduced incidence of serious anaphylactoid reactions compared with dextran when used for plasma volume expanding and hemodilution.38-40 The good manufacturing practice, high water solubility, tailorability, biocompatibility, biodegradability, well defined in vivo safeties, and wide clinical applications make HES a promising drug carrier which warrants clinical translation explorations.16, 41-43 In this study, we, for the first time, designed and synthesized a DOX prodrug HES-SS-DOX, by employing HES as a biodegradable and hydrophilic carrier and disulfide bond as a redox-sensitive linkage between HES and DOX. Redox insensitive HES-DOX was also synthesized as a control. The disulfide bonds of HES-SS-DOX can remain stable in blood circulation, so the massive premature drug release can be



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prevented. HES-SS-DOX can prolong the half-life time of DOX and preferentially accumulate in tumors by EPR effect. After uptake by tumor cells, the disulfide bonds between HES and DOX can be rapidly cleaved by intracellular high concentration of GSH (2-10 mM), triggering timely drug release and causing selective tumor cytotoxicity.


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2. Materials and method 2.1. Materials HES with average molecular weight (Mw) 200 kDa and molar substitution of hydroxylethyl 0.5 was a gift from Wuhan HUST life Sci & Tech Co., Ltd (Wuhan, China). Tween-80 (98%), 3, 3'-Dithiodipropionic acid (DTDPA, 99%), octanedioic acid (ODA, 99%), dicyclohexyl carbodiimide (DCC, 99%), 4-dimethylaminopyridine (DMAP, 99%), N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 98%) and N-hydroxysuccinimide (NHS, 98%) were purchased from Aladdin Reagent Inc. (Shanghai, China). Doxorubicin (99%) was purchased from Beijing Huafenglianbo Technology Co., Ltd (Beijing, China). Cyanine5.5 mono NHS ester (Cy5.5-NHS, 95%) was purchased from Lumiprobe (USA). All other chemicals were of analytical grade and used as received, except for dimethyl sulfoxide (DMSO) which was dried with 4 Å molecular sieves before use. 2.2. Synthesis and characterization of HES-SS-DOX, HES-DOX and HES-Cy5.5 2.2.1. Synthesis of semi-3, 3'-dithiodipropionyl ester of HES (HES-DTDPA) and semi-octanedionyl ester of HES (HES-ODA) HES-DTDPA and HES-ODA were synthesized by similar methods. Take HES-DTDPA for example, DTDPA (2.96 g, 14.08 mmol) was dissolved in 20 mL of dry DMSO. DCC (579.98 mg, 2.82 mmol) and DMAP (172.36 mg, 1.41 mmol) were added to the above solution and the resulting mixture was stirred at room temperature for 0.5 h. After that, pre-dried (105 oC, 2 h) HES (1.00 g) was added and the mixture was reacted at room temperature for 48 h. The precipitate (dicyclohexyl urea, DCU)



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formed in the reaction was removed by filtration, and the filtrate was poured into 200 mL of mix solvent (ethanol/ethyl ether = 1/1, V/V). The resulting precipitate was isolated and washed with 50 mL of the mix solvent for three times, and dried in air. The crude product was re-dissolved in DMSO and dialyzed against deionized water for 3 days (MWCO 3500Da), and freeze-dried at -50 ℃ using a lyophilizer. The structure of HES-DTDPA and HES-ODA were examined by 1H-NMR and FT-IR. The MS of DTDPA or ODA in the modified HES was defined as the number of attached DTDPA or ODA per 100 anhydrous glucose units. The MS of DTDPA (4%) and ODA (8%) in the synthesized HES-DTDPA and HES-ODA was determined by 1

H-NMR and calculated as ICH2 ) MS (%) = NCH 2 × 100% IC H ( 1 ) 1 (

(1)

Where I CH2 is the integral for the protons of methylene groups in DTDPA or ODA, N CH2 is the number of the protons of methylene groups per molecule of DTDPA or ODA, and I C1H is the integral for the protons linked to the C1 carbon atom of AGU between 5.0 and 6.0 ppm. After rearranging Eq. (1), MS of DTDPA and ODA can be calculated as MS (%) =

ICH 2 × 100% N CH 2 * I C 1 H

(2)

2.2.2. Synthesis of HES-SS-DOX and HES-DOX HES-SS-DOX and HES-DOX were synthesized by conjugating DOX onto HES-DTDPA and HES-ODA through the amide bonds. Take HES-SS-DOX for example, HES-DTDPA (850 mg) was firstly dissolved in 20 mL of DMSO. To the


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solution, EDCI (84.21 mg, 0.44 mmol), NHS (101.28 mg, 0.88 mmol)，DOX · HCl (127.58 mg, 0.22 mmol) and TEA (30 µL, 0.22 mmol) were added and the resulting mixture was vigorously stirred at room temperature for 48 h. Then, the reacting mixture was poured into 100 ml of methanol. The formed precipitate was collected by centrifuge at 8000 r/min for 10 min. The obtained precipitate was thoroughly washed by methanol to remove unreacted DOX and dried under vacuum. After that, the crude product was re-dissolved in PBS (6.7 mM, pH 7.4) and dialyzed against deionized water for 3 days (MWCO 3500Da), and lyophilized at -50 ℃ using a lyophilizer. The whole reaction process was shielded from light. The structure of HES-SS-DOX and HES-DOX were examined by 1HNMR, FT-IR, UV/VIS and fluorescence spectra. The amount of DOX conjugated to the drug conjugates was determined by UV/VIS spectrophotometry at 490 nm using a standard calibration curve. Drug loading (DL) of the conjugates was calculated according to the following formula: DL (%) =

Wt (drug) × 100% Wt (drug conjugates)

Where Wt (drug) is the weight of drug in the dug conjugates, and Wt (drug conjugates) is the weight of the drug conjugates.

2.2.3. Synthesis of HES-Cy5.5 HES-Cy5.5 was synthesized by conjugating Cy5.5-NHS ester onto HES through the ester bonds. Briefly, HES (300 mg) and DMAP (5mg) were dissolved in 10 mL of DMSO. To the solution, 50 µL of Cy5.5-NHS ester solution (5 mg/mL in DMSO) was added and the resulting solution was vigorously stirred at room temperature for 48 h. After that, the reaction mixture was dialyzed against deionized



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water for 3 days (MWCO 3500Da) and freeze dried at -50℃ using a lyophilizer.

2.2.4. Charactarization 1

HNMR spectra were recorded on a nuclear magnetic resonance spectrometer

(AscendTM 600 MHz, Bruker) using tetramethylsilane (TMS) as an internal reference. FT-IR spectra were recorded on a flourier transform infrared spectrometer (Vertex70, Bruker) with an attenuate total reflection accessory. UV/VIS spectra were recorded on a UV/VIS spectrophotometer (TU-1901, Beijing Purkinje General Instrument Co., Ltd). Fluorescence spectra were recorded on a fluorescence spectrophotometer (F-4500, Hitachi). The hydrodynamic diameter was measured by dynamic light scattering (DLS, Nano-ZS90, Malvern) at a concentration of 2 mg/mL.

2.3. In vitro drug release study The release profiles of the drug from HES-SS-DOX and HES-DOX were studied using a dialysis method at 37 ℃ in four different media, i.e., PBS buffer (10 mM, pH 7.4, Tween-80 0.5%) without GSH, PBS buffer (10 mM, pH 7.4, Tween-80 0.5%) with 2 µM GSH, PBS buffer (10 mM, pH 7.4, Tween-80 0.5%) with 2 mM GSH and PBS buffer (10 mM, pH 7.4, Tween-80 0.5%) with 10 mM GSH. Briefly, 1 mL of HES-SS-DOX or HES-DOX (0.45 mg) aqueous solution was placed in a dialysis tube (MWCO 3500 Da), and the tube was immersed in 30 mL of release media and shaken at a speed of 200 rpm at 37 ℃. At desired intervals, 3.0 mL of release media was taken out and replenished with equal volume of fresh media. The amount of DOX released was determined by fluorescence measurement (F-4500, Hitachi). The excitation wavelength was 483 nm. The monitoring emission


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wavelength was 556 nm. The release experiments were conducted in triplicate. The results presented are the average values with standard deviations.

2.4. In vitro cell study 2.4.1. Cell culture Murine hepatoma cells H22 were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 µg/mL streptomycin at 37 ℃ in 5% CO2 atmosphere. Human hepatoma cells HepG-2 and Bel-7402 were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 µg/mL streptomycin at 37 ℃ in 5% CO2 atmosphere.

2.4.2. In vitro anti-tumor activity study In vitro anti-tumor activity of free DOX, HES-SS-DOX and HES-DOX against H22 cells was evaluated using counting kit-8 (CCK-8) assays, since H22 cells were suspended cells. The cells harvested at the logarithmic growth phase were seeded on 96-well plates at a cell density of 5×103 cells per well. Every drug of each drug concentration was tested in four wells. After incubating the cells with free DOX, HES-SS-DOX and HES-DOX at various DOX concentrations from 0.01 to 10 µg/mL for 48 h, 20 µL of CCK-8 dye (5 mg/mL) was added to each well and incubated with the cells for another 4h. The absorbance was measured at 450/570 nm using a microplate reader (381C Microplate Reader). Cells incubated with culture medium alone were used as reference for 100% viability.

In vitro anti-tumor activity of free DOX, HES-SS-DOX and HES-DOX against



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HepG-2 and Bel-7402 cells was evaluated using MTT assays. The cells harvested at the logarithmic growth phase were seeded on 96-well plates at a cell density of 5×103 cells per well. Every drug of each drug concentration was tested in four wells. After incubating the cells with free DOX, HES-SS-DOX and HES-DOX at various DOX concentrations from 0.01 to 10 µg/mL for 48 h, 20 µL of MTT dye (5 mg/mL) was added to each well and incubated with the cells for another 4h. Then the medium was removed and 150 µL of DMSO was added to dissolve the formed crystals. The absorbance was determined at wavelength of 492 nm using a microplate reader (381C Microplate Reader). Cells incubated with culture medium alone were used as reference for 100% viability. To test the GSH-mediated cytotoxicity of HES-SS-DOX, the intracellular level of GSH was manipulated by pre-treatment with glutathione ethyl ester (GSH-OEt). HepG-2 and Bel-7402 cells were pre-incubated with DMEM medium containing 10 mM of GSH-OEt for 4 h. After that, the medium was removed and the cells were washed by PBS (pH 7.4, 6.7 mM) for three times before adding HES-SS-DOX and HES-DOX solutions (4 µg/mL as DOX). Each drug was tested in four wells. Cells added with culture media alone were used as control. After incubation for 48 h, the cytotoxicity of the drugs was evaluated by MTT assays as mentioned above.

2.4.3. In vitro cellular uptake study H22 cells were seeded on 12-well plates at a cell density of 1×105 cells per well. Each drug was tested in 3 wells. After incubated with free DOX, HES-SS-DOX and HES-DOX (4 µg/mL as DOX) for 6 h, the cell suspension was collected and


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centrifuged at 1500 rmp for 10 min to remove the medium. The obtained cells were washed with PBS (pH 7.4, 6.7 mM) for three times. Then, the cells were re-suspended in PBS (pH 7.4, 6.7 mM) for cell counting. After that, the cell suspension was centrifuged at 1500 rmp for 10 min to remove PBS buffer. As for free DOX treated group, 1 mL of Triton X-100 (1%) was added and incubated with cells at 37 ℃ for 24 h to disintegrate the cell membranes. As for HES-SS-DOX and HES-DOX treated groups, 1mL of Triton X-100 (1%) with dithiothreitol (DTT, 100 mM) and with glucoamylase (10 U/mL ) was added respectively, and incubated with cells at 37 ℃ for 24 h to disintegrate the cell membranes and recover the fluorescence of DOX simultaneously. Afterwards, 1 mL of methanol was added and the mixture was vortexed for 5 min. After centrifuged at 8000 rmp for 10 min, the supernatant was monitored by fluorescence measurement (F-4500, Hitachi). The excitation wavelength was 483 nm, and the detected emission wavelength was 556 nm. HepG-2 and Bel-7402 cells were seeded on 12-well plates at a cell density of 1×105 cells per well. Each drug was tested in 3 wells. After incubated with HES-DOX, HES-SS-DOX and free DOX (4 µg/mL as DOX) for 6h, the medium was removed and the cells were washed with PBS (pH 7.4, 6.7 mM) for three times. Then, the cells were tripsinized and diluted with PBS (pH 7.4, 6.7 mM) for cell counting. After that, the cell suspension was centrifuged at 1500 rmp for 10 min to remove PBS buffer. Then, the intracellular DOX content was determined using the same method described above.

2.5. In vivo animal study



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2.5.1. Animal subjects Male SD rats and BALB/c mice were purchased from Hubei Research Center of Experimental Animals. All experimental procedures and the animal use and care protocols were approved by the Animal Care and Use Committee of Huazhong University of Science and Technology.

2.5.2. Pharmacokinetic study Male SD rats weighting 250-280 g were randomly divided into three groups, with 5 rats in each group. Free DOX, HES-DOX and HES-SS-DOX were administrated to each group via the tail vein at dose of 4 mg DOX/kg body weight. At desired intervals (5, 15, 30 min, 1, 2, 4, 8, 12, 24, 48 h), 0.5 mL of blood samples were collected from the retro-orbital sinus and transferred to heparin-treated tubes. The blood samples were centrifuged at 3500 rmp for 10 min to obtain the plasma samples. As for free DOX treated group, the plasma samples were directly extracted with methanol. As for HES-SS-DOX and HES-DOX treated groups, the plasma samples were firstly incubated with 100 mM of DTT and with 100 U/mL of glucoamylase at 37 ℃ for 24 h to fully cleave the disulfide bonds and degrade HES respectively, and then extracted with methanol. The extracted samples were analyzed by high performance liquid chromatography in combination with mass spectrometry (HPLC-MS, 1100 LC/MSD Trap, Agilent). The mobile phase was methanol and ammonium acetate buffer (pH 4.5) at a ratio of 80:20 (V/V). The excitation wavelength was 490 nm. The monitoring emission wavelength was 563 nm. The pharmacokinetic data was analyzed using Drug and Statistic software version 2.0.


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2.5.3. In vivo and ex vivo imaging To evaluate the bio-distribution of HES-based conjugates in H22 tumor-bearing mice, Cy5.5 was used to substitute DOX for in vivo and ex vivo imaging. H22 tumor-bearing mice were randomly assigned to 2 groups, with 3 rats in each group. Free Cy5.5 and HES-Cy5.5 were administrated to each group via intravenous injection. At desired intervals, mice were anesthetised with 2% isoflurane in oxygen at a flow of 2 L/min and the fluorescence images were collected by using an in vivo imaging system (IVIS lumina XR, Caliper). At the end of the test, mice of each group were sacrificed and autopsied. The major organs including heart, liver, spleen, lung, kidney and tumors were obtained for ex vivo fluorescence imaging.

2.5.4. Drug tissue distribution study H22 tumor-bearing mice were randomly assigned to 3 groups, with 5 rats in each group. Free DOX, HES-SS-DOX and HES-DOX were administrated to each group by intravenous injection at dose of 4 mg DOX/kg body weight. Mice were sacrificed 48 h post administration and the major organs including heart, liver, spleen, lung, kidney and tumors were harvested and homogenized. As for free DOX treated group, the tissue homogenate were directly extracted with methanol. As for HES-SS-DOX and HES-DOX treated groups, the tissue homogenate were firstly incubated with 300 mM of DTT and with 100 U/mL of glucoamylase at 37℃ for 24 h to fully cleave the disulfide bonds and degrade HES respectively, and then extracted with methanol. The extracted samples were analyzed by HPLC-MS as described above.



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2.5.5. Acute toxicity study Healthy male BALB/c mice were randomly divided into 4 groups, with 5 rats in each group. Free DOX, HES-SS-DOX and HES-DOX were intravenously injected to different groups at dose of 20 mg DOX/kg body weight. Saline was injected as control. The body weight of mice was measured everyday. Mice were sacrificed 4 days post injection and the blood samples were harvested. After centrifuged at 3500 rmp for 10 min, the serum samples were collected for serological analysis. Creatinine kinase (CK) and blood urea nitrogen (BUN) were measured to evaluate the functions of heart and kidney, since free DOX usually induces toxicity to heart and kidney. The organs including heart, liver, spleen, lung and kidney were also harvested and analyzed by hematoxylin and eosin (H&E) staining.

2.5.6. In vivo anti-tumor activity study Male BALB/c mice (20-26 g) were inoculated subcutaneously with H22 ascite tumor cells (each with 1×105 H22 ascite tumor cells) on the right lower back. H22 bearing mice were randomly assigned to 4 groups, with 5 rats in each group. Free DOX, HES-SS-DOX and HES-DOX were administrated to different groups by intravenous injection at dose of 4 mg DOX/kg body weight. Saline was injected as control. Administrations were started when the tumor volumes reached about 0.12 cm3 and carried out every 4 days for a total of 3 doses. Tumor volume and body weight of mice were measured every 2 days. At the end of the test, mice were sacrificed and the harvested tumors were weighted，imaged and analyzed by TUNEL staining. The organs including heart, liver, spleen, lung and kidney were also


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harvested and analyzed by H&E staining.

2.6. Statistic analysis All data were presented as the mean value ± standard deviation (SD). Statistical analysis was performed by Statistical Product and Service Solutions (SPSS) with independent samples T-test. Statistic significance was established at p < 0.05. * p < 0.05，** p < 0.01, *** p < 0.001.



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3. Results and discussion

3.1. Synthesis and characterization of HES-SS-DOX and HES-DOX. Due to the slow renal clearance rate and excellent tolerability following repeated administrations, HES 200/0.5 is selected for DOX conjugation in this work.32 HES-SS-DOX and HES-DOX were synthesized by very convenient procedures. The synthetic routes of HES-SS-DOX are shown in Scheme 1 (A). HES was firstly modified with DTDPA to afford HES-DTDPA via esterification reactions between carboxyl groups of DTDPA and hydroxyl groups of HES. Then, DOX was conjugated onto HES-DTDPA to produce HES-SS-DOX by the formation of amide bonds. HES-DOX was also synthesized as its insensitive counterpart by similar methods, Scheme 1 (B). The successful synthesis of HES-SS-DOX and HES-DOX were verified by 1H-NMR and FT-IR spectra. Figure 1 (A) shows the 1H-NMR spectra of HES-SS-DOX. The shift at 2.7–3.0 ppm corresponds to the protons of the methylene groups (5, 6) of DTDPA linker.28 The peaks at 1.2, 7.6 and 7.9 ppm correspond to the protons of methyl group (1) and aromatic ring (2–4) of DOX.28 The 1H-NMR spectra of HES-DOX (Figure S2 (B)) show the signals of ODA linker (1–4) and DOX (5–8). Figure 1 (B) shows the FT-IR spectra of HES (a), HES-DTDPA (b) and HES-SS-DOX (c). Compared with HES, the appearance of the characteristic band at 1713 cm-1 of HES-DTDPA is associated with the stretch vibration of C=O of the ester bonds, suggesting successful conjugating PTPDA onto HES by the ester bonds. Compared with HES-DTDPA, the appearance of the characteristic bands at 1575 and 1286 cm-1 of HES-SS-DOX are related to the backbone vibration of aromatic rings of


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DOX and C-N stretch vibration of the amide bonds respectively, indicating the successful DOX conjugation by amide bonds. The FT-IR spectra of HES-ODA (Figure S2 (C)) and HES-DOX (Figure S2 (D)) also confirm the successful conjugating ODA onto HES by ester bonds (1716 cm-1) and DOX conjugation by amide bonds (1579 cm-1, 1288 cm-1). Both 1H-NMR and FT-IR results corroborate the successful synthesis of HES-SS-DOX and HES-DOX. After conjugating onto HES, the fluorescence of DOX is self-quenched significantly (Figure S3), which is due to the homo Förster resonance energy transfer.44-45 Yet, once DOX is cleaved from HES, its fluorescence is recovered, as revealed in Figure S3. These results robustly validate the successful coupling between HES and DOX for HES-SS-DOX and HES-DOX. HES-SS-DOX was incubated with 100 mM DTT at 37 ℃ for 24 h. DOX-SH was detected as the resulting product by HPLC-MS (Figure S4), strongly suggesting successful coupling of HES and DOX with disulfide bonds, which are easily cleaved by DTT, for HES-SS-DOX.46 The drug loading of HES-SS-DOX and HES-DOX were determined by UV/VIS spectrophotometry at 490 nm according to a standard calibration curve. It should be noted that the drug loading of HES-SS-DOX (6.7 %) and HES-DOX (6.2 %) is manipulated to be close. So, we can study the two prodrugs comparatively both in vitro and in vivo. The hydrodynamic diameter of HES 200/0.5, HES-SS-DOX and HES-DOX were measured by DLS and presented in Figure 1 (C). The hydrodynamic diameter of HES 200/0.5, HES-SS-DOX and HES-DOX are 17.6 ± 1.2, 19.9 ± 0.4 and 20.5 ± 2.7 nm respectively, illustrating a small size increase after DOX conjugation. The measured diameter of HES 200/0.5 (17.6 ± 1.2 nm) is



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Page 20 of 47

consistent with the results reported in the literature,47 although HES are from various sources. Taken together, HES-SS-DOX and HES-DOX have been successfully prepared and characterized. O

A

S HO

OR O

O OR HO

OR O

O HO OR

O

O

OR

OR O

O

OR O

O OR

+

HO

S

OH

S

DCC/DMAP

O

O ORHO

HO

O

O O

O O HO OR

O

OR

HO

S

OH O

OR O

O

OR

HO (DTDPA)

(HES)

(HES-DTDPA)

O

O O

O

DOX

OR O

O

O OR HO

HO

EDCI/NHS

S

O O

O

O

OR

O HO OR

DOX

S OR O

O O

S

≡

HO

OR

OR O

O

O

O ORHO

O OR

HO

HO

O HO

OR O

O O HO OR

O

H N

S

OH OH

O

O

OH

O O

O

OR

HO R=H, CH 2CH2OH

(HES-SS-DOX)

O

B

OH

HO

OR O

O ORHO

OR O

O

O

OR

O HO OR

OR O

O

OR O

O OR

+

HO OH

DCC/DMAP

O

O ORHO

HO

O

O O

O O HO OR

O

OR

HO

OR O

(HES-ODA)

(ODA)

O

O O

O

H N

DOX DOX EDCI/NHS

HO

O OR HO

O

OR

HO

(HES)

OR O

O

O

O O

O

OR

O O HO OR

OR O OR

O O

≡

HO

OR O

O ORHO

O

O O

O

OR

O O HO OR

OR O

O HO

OH OH

O HO O O

O

OR

HO

HO (HES-DOX)

R=H, CH 2CH2OH

Scheme 1. Synthetic scheme of HES-SS-DOX (A) and HES-DOX (B).


O

OH

A

3

2 O

OR HO

6

O

O

O OR HO

O O

O

O

OR

5

O HO OR

S

O

5 OR O OR

O

OH OH

O

6

S

H N O

B

4

O

HO

OH

O

HO

a O

O

1

HO R=H, CH2 CH 2OH

H2O DMSO

b c 1713 1575

5,6

3,4 2

1

-1

D

40 HES 200/0.5 HES-SS-DOX HES-DOX

30 20 10 0

1

10

100

1000

750

Wavenumber (cm )

Cumulative release (%)

C

1286

3750 3000 2250 1500

9 8 7 6 5 4 3 2 1 ppm

Number (%)

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


Transmittance

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HES-SS-DOX, GSH 10 mM HES-SS-DOX, GSH 2 mM HES-SS-DOX, GSH 2 µM HES-SS-DOX HES-DOX, GSH 10 mM

120 80 40 0 0

Diameter (nm)

12

24

36

48

Time (h)

Figure 1. Characterizations of HES-SS-DOX and HES-DOX. (A) 1H-NMR spectra of HES-SS-DOX. (B) FT-IR spectra of HES (a), HES-DTDPA (b), and HES-SS-DOX (c). (C) Size distribution of HES, HES-SS-DOX and HES-DOX measured by DLS. (D) In vitro drug release profiles of HES-SS-DOX and HES-DOX in PBS buffer (pH 7.4, 10 mmol/L) with and without GSH. Data represent the mean ± SD (n = 3).

3. 2. In vitro drug release The in vitro drug release of HES-SS-DOX and HES-DOX was carried out under both extracellular (～2 µM) and intracellular (2-10 mM) GSH levels. As shown



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in Figure 1 (D), the drug release rate of HES-SS-DOX increases with increasing GSH concentration and ranks as: PBS pH 7.4, GSH 10 mM > PBS pH 7.4, GSH 2 mM > PBS pH 7.4, GSH 2 µM ≈ PBS pH 7.4, suggesting the GSH-triggered drug release of HES-SS-DOX. The drug release rate of HES-SS-DOX in PBS pH 7.4 with 10 mM or 2 mM GSH is significantly higher than that in PBS pH 7.4 with 2 µM or without GSH. For example, 90% and 87% of DOX is released from HES-SS-DOX in PBS pH 7.4 with 10 mM and with 2 mM GSH within 46 h. In contrast, only 31% and 27% of DOX is released from HES-SS-DOX in PBS pH 7.4 with 2 µM and without GSH at the same time point. These results imply that the linkages between HES and DOX in HES-SS-DOX can be relatively stable in blood during in vivo circulation, whereas rapid drug release can be achieved in the intracellular reductive environment within tumor cells. It should be noted that incubation of HES-SS-DOX with 10 mM or 2 mM GSH led to firstly burst drug release (0-4 h), followed by slower but sustained drug release (4-46 h). This can be ascribed to the branched structure of HES. DOX covalently linked to the surface of HES can be rapidly cleaved by GSH, whereas it may be slow to cleave disulfide bonds concealed in the starch branches.16 The two-stage release behaviors are likely to increase the total quantity and prolong the action time of the active drug.48 The drug release rate of HES-DOX is still very low even in PBS pH 7.4 with 10 mM GSH and only 30% of the drug loading is released after 46 h, indicating the drug release from HES-DOX can not be triggered by GSH. The drug release of HES-SS-DOX with 2 µM GSH or without GSH and HES-DOX with 10 mM GSH in the first few hours may be caused by the hydrolysis of the ester


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bonds linked to the surface of HES. Figure 1 (D) clearly reveals the redox triggered drug release, the redox-responsiveness, of HES-SS-DOX.

3.3. In vitro antitumor activity study To test whether the redox-responsiveness of HES-SS-DOX can benefit its cytotoxicity against tumor cells, in vitro cytotoxicity of free DOX, HES-SS-DOX, and HES-DOX against H22, HepG-2 and Bel-7402 cells was studied. As shown in Figure 2 (A)-(C), free DOX, HES-SS-DOX, and HES-DOX exhibit cytotoxicity in a dose-dependent manner in all tested cell lines. The half maximum inhibition concentration (IC50) of free DOX, HES-SS-DOX, and HES-DOX are summarized in Table S1. The cytotoxicity of the three tested groups ranks as: free DOX > HES-SS-DOX > HES-DOX in all tested cell lines. Free DOX can be readily transported into cells by a passive diffusion manner, whereas modification of DOX with hydrophilic HES inhibits the cellular uptake of the conjugates (Figure S5).48 So, it seems reasonable that free DOX exhibits the highest cytotoxicity. Indeed, polymer-DOX conjugates usually exhibit lower cytotoxicity than that of free DOX due to their slow internalization process and/or slow intracellular drug release.49-50 On the contrary, 10-HCPT-HES conjugates exhibited higher cytotoxicity against Hep-3B and SMMC-7721 cells than that of free 10-HCPT. It was attributed to the enhanced stability of the lactone of 10-HCPT after conjugation with HES.16

Notably,

HES-SS-DOX exhibits higher tumor cell inhibition effect, with IC50 10.2, 4.9 and 3.8 times lower than those of HES-DOX in H22, HepG-2 and Bel-7402 cells, respectively (Table S1). The enhanced tumor cell inhibition effect of HES-SS-DOX can be



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attributed to its redox-responsiveness. After uptake by tumor cells, HES-SS-DOX can be rapidly cleaved by intracellular high concentration of GSH, inducing timely drug release and causing cytotoxicity, whereas the slow drug release of HES-DOX limits its efficacy. To verify that the enhanced cytotoxicity of HES-SS-DOX is caused by its redox-responsiveness, the cytotoxicity of HES-SS-DOX and HES-DOX was assessed at a higher intracellular level of GSH by pre-incubation cells with 10 mM GSH-OEt. It is well-documented that GSH-OEt can penetrate cellular membranes and rapidly convert to GSH in cytoplasm by hydrolysis.51 HepG-2 and Bel-7402 cells were pre-incubated with GSH-OEt for 4 h to elevate intracellular GSH concentration. After removing the GSH-OEt containing medium, the cells were further incubated with HES-SS-DOX and HES-DOX solutions (equivalent to 4 µg/mL DOX) for 48h. HES-SS-DOX with GSH-OEt pre-incubation significantly improves its cytotoxicity for both cell lines at 48 h, whereas HES-DOX with GSH-OEt pre-incubation does not affect its cytotoxicity, as shown in Figure 2 (D). Consistently, this result underlines the redox-responsiveness of HES-SS-DOX and thus validates GSH-mediated cytotoxicity of HES-SS-DOX.26,

48

Collectively, Figure 1 (D) and Figure 2

corroborate that HES-SS-DOX has redox responsiveness, which results in higher in

vitro antitumor activity than its counterpart, insensitive HES-DOX.


Page 24 of 47

DOX HES-SS-DOX HES-DOX

100 80 60 40 20 0


100 80 60 40 20

0.01 0.1 1 4 7 10 DOX concentration (µg/mL)


100 80 60 40 20 0


C 120

0

B 120 Cell viability (%)

Cell viability (%)

A 120

Cell viability (%)

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



D 120 Cell viability (%)

Page 25 of 47

HES-DOX HES-DOX with GSH-OEt HES-SS-DOX HES-SS-DOX with GSH-OEt

100 80 60

***

***

40 20 0

HepG-2

Bel-7402

Figure 2. In vitro cytotoxicity of free DOX, HES-SS-DOX, and HES-DOX against H22 (A), HepG-2 (B) and Bel-7402 (C) cells after incubation for 48 h. GSH-OEt (10 mM)-mediated cytotoxicity against HepG-2 and Bel-7402 cells after incubation with HES-SS-DOX and HES-DOX (4 µg/mL as DOX) for 48 h (D). *** P

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