oxidation-responsive docetaxel prodrugs for the hyperselective treatment of breast cancer

Journal of Controlled Release 285 (2018) 187–199

Contents lists available at ScienceDirect

Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

Precisely albumin-hitchhiking tumor cell-activated reduction/oxidationresponsive docetaxel prodrugs for the hyperselective treatment of breast cancer

T

Wei Weia, Cong Luoa, Jincheng Yangb, Bingjun Suna, Dongyang Zhaoa, Yan Liua, Yingli Wanga, ⁎ ⁎ Wenqian Yanga, Qiming Kanc, Jin Suna, , Zhonggui Hea, a b c

Department of Pharmaceutics, Wuya College of Innovation, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, China Department of Pharmaceutical engineering, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, China Department of Pharmacology, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Albumin-hitchhiking Docetaxel-maleimide prodrugs Reduction-sensitive Oxidation-sensitive

The anticancer efficacy of chemotherapy is greatly limited by short blood circulation and poor tumor selectivity. Thus, anticancer prodrugs with prolonged systemic circulation, tumor-specific distribution and bioactivation, could significantly strengthen the chemotherapy efficacy. Herein, we design two novel tumor cell reduction/ oxidation-responsive docetaxel (DTX) prodrugs, DTX-maleimide conjugates with disulfide bond (DSSM) or thioether bond (DSM) linkages, to evaluate the roles of different sensitive linkages in drug release, pharmacokinetics and therapeutic efficacy. An ester bond-linkage prodrug (DM) is utilized as a non-sensitive control. DSSM and DSM show reduction- or oxidation-sensitive release behavior, respectively, and exhibit hyperselective bioactivation and cytotoxicities between cancerous and normal cells. They could instantly hitchhike blood circulating albumin after i.v. administration with albumin-binding half-lives as short as 1 min, resulting in prolonged systemic circulation, increased tumor accumulation. In response to the upregulated reduction/oxidation environment within tumor cells, DSSM and DSM exhibit selectively release capacity in tumor tissues, their TAITumor/Liver values are over 30-fold greater than DM. Combining the above delivery advantages into one, DSSM and DSM achieve enhanced antitumor efficacy of DTX. Such a uniquely developed strategy, integrating high albumin-binding capability and reduction/oxidation-sensitive drug superselective release in tumors, has great potential to be applied in clinical cancer therapy.

1. Introduction Cancer still is one of the leading diseases with high mortality worldwide, and chemotherapy represents one of the most common treatment strategies in cancer therapy [1, 2]. Nevertheless, the efficacy of chemotherapy is far from satisfactory due to off-target drug delivery [2, 3]. To overcome the limitations, great effort has been made to design the effective delivery systems for anticancer drugs, mainly focusing on cancer nanomedicines [4, 5]. Drug delivery nanosystems tend to accumulate in tumors by taking advantage of the particle size-based enhanced permeability and retention (EPR) effect [6, 7]. Moreover, the modification with polyethylene glycol (PEG) on a surface of nanoparticles could extend them blood-circulating time in vivo and therefore improve the tumor accumulation [8–10]. However, PEGylation could compromise the cellular uptake and endosomal escape, and leads to the

⁎

unexpected immunogenic response following multiple injections, accelerated blood clearance (ABC) phenomenon [11, 12]. Albumin, the most abundant protein in plasma (35–50 mg/mL), has been widely used for healthcare applications due to its native long circulation capacity (19 days), high stability, low immunogenicity and good tumor-homing ability [13–16]. The free thiol (HS) group of cysteine-34 in HSA accounts for > 80% of the total free thiols in blood and is the most reactive in the plasma because of the low pKa of Cys-34 in HSA [17]. Therefore, thiol-reactive molecules could selectively bind to the endogenous albumin when entered the blood. Since a covalentbinding (6-maleimidocaproyl) hydrazone derivative of doxorubicin (DOXO-EMCH) has been applied in clinical trials, the thiol-binding prodrug platform technology which exploits endogenous albumin as a drug carrier is promising for anticancer drug delivery [18–20]. The rational design of albumin-based prodrugs can be attributed to the

Corresponding authors. E-mail addresses: [email protected] (J. Sun), [email protected] (Z. He).

https://doi.org/10.1016/j.jconrel.2018.07.010 Received 19 February 2018; Received in revised form 12 June 2018; Accepted 3 July 2018 Available online 12 July 2018 0168-3659/ © 2018 Published by Elsevier B.V.

Journal of Controlled Release 285 (2018) 187–199

W. Wei et al.

Fig. 1. Schematic diagram of albumin-hitchhiking DTX-maleimide prodrugs delivery strategy.

time profiles of the parent drug paclitaxel. According to the literature studies, the albumin binding rates of diverse maleimide conjugates were completely different [17, 20], which would significantly affect the drug delivery efficiency and antitumor effect. Compared with paclitaxel, docetaxel had smaller steric hindrance and higher hydrophilicity, leading to a much higher binding affinity with tubulin. Based on these, we wondered whether docetaxel-maleimide prodrugs would have superiority over paclitaxel-maleimide prodrugs in terms of albumin binding. Therefore, the research on whether docetaxel-maleimide prodrugs could improve the albumin binding ability, pharmacokinetics, biodistribution and antitumor efficiency still needed further investigation. In this article, we synthesized a series of docetaxel-maleimide prodrugs, including two reduction/oxidation-sensitive DTX-maleimide conjugates with disulfide bond (DSSM) and thioether bond (DSM) as linkages, and an ester bond linker conjugate (DM) as a non-sensitive control. We investigated the binding capacity of the docetaxel-maleimide prodrugs to albumin and developed a new method to evaluate the pharmacokinetic behavior of DTX-maleimide prodrugs by measuring the free and total DTX concentrations in plasma, respectively. We also detected the released free DTX levels in tumors and other normal organs at different timepoints and firstly put forward the tumor activation index (TAI: AUCtumor/AUCnormal tissues) to describe the drug accumulation degree in tumor in vivo. Importantly, both DSSM and DSM could superselectively release free drug under tumor reduction/ oxidation upregulated environment, in sharp contrast to normal tissues (Fig. 1). Similarly, we found that DSSM and DSM could exhibit hyperselective bioactivation and cytotoxicities between cancerous and normal cells. Our findings give new insight into the albumin-based prodrug strategy, and provide a good foundation for the development of novel redox-responsive albumin-based DDS for cancer therapy.

following aspects: (i) to enhance the stability of anticancer drugs, the conjugation with endogenous albumin can protect the parent drug from being degraded in vivo; (ii) to extend blood circulation time in the albumin-prodrug conjugate form; (iii) to achieve favorable targeting effects to tumors, several studies have suggested albumin (radius, approximately 7.2 nm) could accumulate in the tumor tissues via albuminmediating EPR effect which is shown in macromolecules by the increased vascular permeability and lack of lymphatic drainage [21, 22]. In addition, gp60 and SPARC are involved in specific retention of albumin at tumor sites. Specifically, the albumin binding receptor gp60, a 43 kDa glycoprotein, has been shown to bind albumin and mediate transcytosis across endothelial cells. SPARC has been shown to be broadly expressed in many different tumors, and is a high affinity albumin-binding protein and facilitate albumin transport and accumulation in the cancer tissues [23, 24]; (iv) to minimize side effects towards other normal tissues because of low off-target drug exposure and high tumor accumulation. Compared to normal cells, there is an upregulated reduction-oxidation balanced state in tumor cells. Tumor cells usually overproduce reactive oxygen species, reaching to 100 μM, approximately 100 times higher than that in normal cells, mainly due to the chronic inflammation, oncogene mutation or mitochrondrial dysfunction [25, 26]. Simultaneously, to alleviate the oxidation stress, tumor cells enhance the level of glutathione (GSH) to 2–10 mM, about 5–10 times higher than normal cells [27–29]. Although reduction/oxidation responsive drug delivery systems have been reported, a majority of the systems were interested in the sensitivity of drug release but ignored the superselective release difference between tumor and normal cells. A nonselective drug delivery systems induce premature drug release in normal cells and blood circulation, which results in unexpected side effects. Therefore, the superselective reduction/oxidation responsive drug delivery system with minimal cleavage in blood circulation and normal cells but rapid release in tumor cells, may serve as a promising carrier. Disulfide bond and thioether bond have been widely used as reduction/oxidation- sensitive linkages to facilitate a rapid release of anticancer drugs in tumor cells [28, 30–32]. Inspired by these, we have previously prepared several paclitaxel-maleimide prodrugs in our earlier work to develop albumin-based drug delivery system [33]. However, the biodistribution of the paclitaxel-maleimide prodrugs was generally not ideal, exhibiting lower tumor accumulation compared with paclitaxel solution. And as a result, compared with the paclitaxel solution group, there was no obvious advantage in the anti-tumor effect of the prodrugs. Furthermore, we haven't studied the concentration-

2. Material and methods 2.1. Materials Docetaxel (DTX) and Paclitaxel (PTX) were obtained from Dalian Meilun Biotech Co., Ltd., China. Docetaxel injection (Taxotere) was purchased from Bristol-Myers Squibb (New York, USA). Bovine serum albumin was purchased from HarveyBioGene Technology Co., Ltd. (Beijing, China) and the protein contained approximately 40% free thiol groups as assessed with Ellman's test. 4-Dimethylaminopyridine (DMAP), N,N-dicyclohexylcarbodiimide(DCC), 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 4-Methylmorpholine 188

Journal of Controlled Release 285 (2018) 187–199

W. Wei et al.

then the filtrate was dried to produce a crude product, which was purified by silica gel column chromatography (dichloromethane/methanol (100:2) to obtain 270 mg of DSSM (Compound 7) as white solid (40% yield). 1H NMR (400 MHz, DMSO‑d6, ppm) of DSSM: δ 7.97–8.01 (2H,d), 7.85–7.88(1H,d), 7.7–7.73 (1H,t), 7.64–7.69 (2H,t), 7.37–7.42 (4H,m), 7.14–7.18 (1H,t), 7.03 (2H,S), 5.75–5.82 (1H,t), 5.38–5.41 (1H,d), 5.08–5.1 (3H,m), 5.0–5.02 (1H,d), 4.9–4.94 (2H,t), 4.42–4.45 (1H,S), 4.15–4.17 (2H,t), 4.01–4.03 (3H,S), 3.65–3.68 (1H,d), 3.6–3.65 (2H,t), 2.24–2.91 (12H,m), 0.97–1.90 (24H,m). ESI-MS (m/z): [M + Na]+ = 1145.5, [M + K]+ = 1161.4. Synthesis of DTX-S-Maleimide (DSM): Firstly, thiodipropionic acid (356 mg, 2 mmol) was dissolved in 20 ml acetic anhydride and stirred at 30 °C for 3 h. Excess solvent was removed with anhydrous toluene in high vacuo three times at 30 °C to prepare compound 8. Then this product (Compound 8) was dissolved in 15 ml anhydrous dichloromethane, Mal-OH (310.2 mg, 2.2 mmol) and DAMP (48.89 mg, 0.4 mmol) were added into the solution. The reaction proceeded overnight at 25 °C under nitrogen. TLC was utilized to monitor the reaction process. At the end of reaction, solvent was evaporated and the crude product was purified by silica gel column chromatography (dichloromethane/methanol (100:0.75) to give about 380 mg compound 9 as a yellow oily solid (60% yield). Next, a solution of compound 9 (198.7 mg, 0.66 mmol) in 15 ml anhydrous dichloromethane was activated by HATU (456.3 mg, 1.2 mmol) at 0 °C for 30 min. Then 4-methylmorpholine (NMM) (0.13 mL, 1.2 mmol) and DTX (484.7 mg, 0.6 mmol) in 10 ml anhydrous dichloromethane was added to the mixture, and further stirred under nitrogen at 25 °C for 12 h. TLC was utilized to monitor the reaction process. At the end of reaction, filtered to remove the insoluble solids, then the filtrate was dried to produce a crude product, which was purified by silica gel column chromatography (dichloromethane/methanol (100:2) to obtain about 320 mg of DSM (Compound 10) as white solid (50% yield). 1H NMR (400 MHz, DMSO‑d6, ppm) of DSM: δ 7.97–7.99 (2H,d), 7.87 (1H,d), 7.67–7.72 (1H,t), 7.61–7.65 (2H,t), 7.37–7.41 (4H,m), 7.13–7.19 (1H,t), 7.03 (2H,S), 5.75–5.82 (1H,t), 5.37–5.41 (1H,d), 5.03–5.1 (3H,m), 5.0–5.03 (1H,d), 4.87–4.93 (2H,t), 4.41–4.45 (1H,S), 4.12–4.17 (2H,t), 4.01–4.07 (3H,S), 3.65–3.68 (1H,d), 3.6–3.65 (2H,t), 2.24–2.72 (12H,m), 0.97–1.88 (24H,m). ESIMS (m/z): [M + Na]+ = 1113.7, [M + K]+ = 1129.6.

(NMM), Maleic anhydride, 6-aminocaproic acid, Dithiodipropionic acid and thiodipropionic acid were purchased from Aladdin Industrial Corporation (Shanghai,China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT), trypsin, Dulbecco's modified eagle medium (DMEM, high glucose) and Roswell Park Memorial Institute (RPMI-1640) were bought from Gibco (Beijing, China). All other reagents and solvents were of analytical or HPLC grade. 2.2. Synthesis of DTX-Maleimide prodrugs Synthesis of DTX-Maleimide (DM): The prodrug DM was established by a two-step reaction. Firstly, 6-maleimidocaproic acid was synthesized as described previously [18]. Then DM was prepared through conjugating EMC to the C2’-hydroxyl position of DTX via a simple ester bond. Briefly, DTX (403.9 mg, 0.5 mmol) and EMC (116.2 mg, 0.55 mmol) were dissolved in 10 ml anhydrous dichloromethane, then DCC (206.2 mg, 1 mmol) and DMAP (12.2 mg, 0.1 mmol) were added and stirred under nitrogen for 12 h at room temperature. Upon completion, filtered to remove the insoluble solids N, N-dicyclohexylurea (DCU). Then the filtrate was dried under vacuum. The crude product was further purified by a silica gel column (dichloromethane/methanol (100:1.5)), and obtain 313.4 mg white powder (mass, 62% yield). 1H NMR (400 MHz, DMSO-d6, ppm) of DM: δ 7.97–7.99 (2H,d), 7.83–7.87(1H,d), 7.67–7.73 (1H,t), 7.64–7.67 (2H,t), 7.34–7.41 (4H,m), 7.13–7.18 (1H,t), 7.0 (2H,S), 5.75–5.78 (1H,S), 5.38–5.40 (1H,d), 4.89–5.08 (6H,m), 4.39–4.42 (1H,S), 4.01–3.62 (6H,m), 2.36–2.39 (2H,t), 2.21–2.23 (4H,m), 0.98–1.78 (30H,m). ESI-MS (m/z): [M + Na]+ = 1023.5. Synthesis of N-(2-Hydroxyethyl) Maleimide (Mal-OH): N-(2-hydroxyethyl) maleimide (Mal-OH) was prepared according to a previously reported method [3, 34]. Firstly, 30.0 g maleic anhydride (306 mmol) was dissolved in toluene and 22.4 ml furan (309 mmol) was slowly injected into the solution via syringe. After the mixture stirred for 24 h at room temperature, the reaction was stopped and the white crystals were precipitated from the mixture, collected and washed with toluene. The white powder was dried in a vacuum oven to prepare furan-protected maleic anhydride. Secondly, the furan-protected maleic anhydride (4.98 g, 30 mmol) was suspended in methanol and stirred at 0 °C. Then 1.8 ml of ethanolamine (30 mmol) was added dropwise to the solution, stirred and refluxed at 80 °C for 24 h. Then the reaction mixture was cooled to 0 °C, the product was crystallized from the mixture and collected to obtain furan-protected N-(2-hydroxyethyl)-maleimide. Finally, furan-protected N-(2-hydroxyethyl)-maleimide (2.1 g, 10 mmol) in 10 ml toluene was stirred and refluxed under nitrogen for 12 h. The reaction mixture was hot filtered, and the filtrate was stored at 4 °C overnight. The product crystallized from the mixture, then collected, dried under vacuum for 24 h to obtain N-(2-hydroxyethyl)-maleimide (Mal-OH). Synthesis of DTX-SS-Maleimide (DSSM): Firstly, dithiodipropionic acid (420 mg, 2 mmol) was dissolved in 20 ml acetic anhydride and stirred at 30 °C for 3 h. Excess solvent was removed with anhydrous toluene in high vacuo three times at 30 °C to prepare compound 5. Then this product (Compound 5) was dissolved in 15 ml anhydrous dichloromethane, Mal-OH (310.2 mg, 2.2 mmol) and DAMP (48.89 mg, 0.4 mmol) were added into the solution. The reaction proceeded overnight at 25 °C under nitrogen. TLC was utilized to monitor the reaction process. At the end of reaction, solvent was evaporated and the crude product was purified by silica gel column chromatography (dichloromethane/methanol (100:0.75) to give about 430 mg compound 6 as a yellow oily solid (65% yield). Next, a solution of compound 6 (219.8 mg, 0.66 mmol) in 15 ml anhydrous dichloromethane was activated by HATU (456.3 mg, 1.2 mmol) at 0 °C for 30 min. Then 4-methylmorpholine (NMM) (0.13 mL, 1.2 mmol) and DTX (484.7 mg, 0.6 mmol) in 10 ml anhydrous dichloromethane was added to the mixture, and further stirred under nitrogen at 25 °C for 12 h. TLC was utilized to monitor the reaction process. At the end of reaction, filtered to remove the insoluble solids,

2.3. Incubation with bovine serum albumin and plasma serum Compound DM, DSM and DSSM (300 μM) were added to a solution of BSA (700 μM in PBS, pH 7.4) and incubated at 37 °C for albumin binding studies. Then 50 μL samples were directly analyzed using HPLC after an incubation time of 5 min, 30 min and 90 min. Similarly, blood plasma (EDTA-stabilized) was taken from healthy Sprague-Dawley rats. Then, DM, DSM and DSSM were added to plasma serum at 37 °C and were incubated for the same time. A 50 μL sample was analyzed by high-performance liquid chromatography (HPLC). In addition, for blocking study, plasma serum was preincubated with an excess amount of EMC for an hour before adding compound DM, DSM and DSSM. At last, compound DTX (300 μM), which is not functionalized with the maleimide group as a negative control, was also added to a solution of BSA (700 μM in PBS, pH 7.4) and incubated at 37 °C for albumin binding studies. All the samples were analyzed by an analytical HPLC using a Waters e2695 pump, a Waters 2489 UV detector (λ = 254 nm) and the Empower 3 software (Waters Corp.). Column: Waters Symmetry 300™ C18 (250 mm × 4.6 mm, 5 μm); column temperature: 35 °C; flow rate: 1.0 mL/min; mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid; mobile phase B: water containing 0.1% trifluoroacetic acid; gradient: 0–5 min, 30% mobile phase A; 5–10 min, increase to 40% mobile phase A; 10–15 min, 40% mobile phase A; 15–35 min, increase to 80% mobile phase A; 35-38 min, 80% mobile phase A; 3840 min, decrease to 30% mobile phase A; 40–45 min, 30% mobile phase A; injection volume: 10 μL. 189

Journal of Controlled Release 285 (2018) 187–199

W. Wei et al.

RPMI 1640 medium consisted of 10% FBS, penicillin (100 units/mL) and streptomycin (100 μg/mL). L02 cells were cultured with Dulbecco's modified eagle medium (DMEM) containing the same as described above. All cells were maintained in a humidified, 5% CO2 incubator at 37 °C.

2.4. Preparation of the albumin-prodrug conjugates (BSA-DM, BSA-DSM and BSA- DSSM) The preparation method of the BSA-prodrug conjugates was established consistently with the above albumin binding studies, just incubating compound DM, DSM and DSSM (300 μM) with BSA (700 μM) in PBS (pH 7.4) for 2 h until no free prodrugs could be detected by HPLC. Then the solutions were collected and freeze-dried directly to obtain BSA-prodrug conjugates as white powder. The samples were kept at −80 °C and thawed prior to use.

2.9. In vitro cytotoxicity and intracellular drug release The cytotoxicity of free DTX and DTX-prodrugs was evaluated by the MTT assay in KB, PC3, 4 T1 and L02 cells. Briefly, cells were seeded into 96-well plates at a density of 5000 cells per well for 24 h attachment, then cells were incubated with the medium containing different drugs at various concentrations for 48 h and 72 h. The cell viability was calculated by MTT method. The cells without any treatment were utilized as control. All experiments were carried out in triplicate. For quantitative determination of the intracellular DTX release from the prodrugs, the 4 T1 and L02 cells were seeded into 24-well plates at a density of 5000 cells per well for 24 h attachment. Then, the culture medium was replaced with the medium containing different drugs at various concentrations (DTX equivalent concentrations: 50, 100 and 200 ng/mL). After incubating at 37 °C for 6 h, 12 h, 24 h and 48 h, the cells together with the drug-containing culture media were collected, then the cells were broken by ultrasonication. The concentrations of free DTX in the supernatants were measured by UPLC-MS-MS on an ACQUITY UPLC system (Waters Corp.). Next we determined whether the free DTX could release from the prodrugs in the cell culture media. Firstly, we collected the 4 T1 and L02 cells culture medium. Then, the three prodrugs were incubated in 2 ml cell culture media at 37 °C. At pre-determined time intervals, 0.2 ml samples were taken for analysis by HPLC. Extraction and analysis methods were similar to those in the pharmacokinetic experiments. All the experiments were performed in triplicate and the results were expressed as the percentage of released DTX.

2.5. Fluorescence and circular dichroism spectra of the albumin-prodrug conjugates For fluorescence spectroscopic studies, samples (1 mg/mL) of BSA, BSA-DM, BSA-DSM and BSA-DSSM conjugates were taken in a cuvette with 1 cm path length and measurement was performed on Model 500 Microplate Reader (BioRed, USA). The spectrum was recorded in the range of 300–500 nm using excitation wavelength of 280 nm. For circular dichroism (CD) measurements, the circular dichroism spectra were recorded from 190 to 250 nm at 1 nm intervals on a BioLogic MOS450 CD spectrometer (French) at room temperature (25 °C). BSA and BSA-prodrug conjugates were prepared in phosphate buffer (pH 7.4) and diluted to 1 μM. 2.6. Stability of the albumin-prodrug conjugates in rat plasma The in vitro stabilities of the three BSA-prodrug conjugates in rat plasma were also investigated. Blood was collected from the SpragueDawley rats jugular vein, after centrifugation at 10000 rpm for 5 min, fresh plasma was obtained and frozen at −20 °C until use for stability study. The samples of BSA-DM, BSA-DSM and BSA-DSSM lyophilized powder were incubated in 2 ml rat plasma at 37 °C for 24 h with a final concentration of 100 μg/ml (DTX equivalent). Then 100 μL samples were taken for analysis by HPLC at 0.25, 1, 2, 6, 12 and 24 h. Extraction and analysis methods were similar to those in the pharmacokinetic experiments. All the experiments were performed in triplicate and the results were expressed as the percentage of released DTX.

2.10. Detection of intracellular ROS and GSH Intracellular ROS levels were determined by the fluorescence using Eclipse TieU inverted microscope (Nikon Corp., Tokyo, Japan) and flow cytometer (Becton Dickinson, USA). Briefly, cells (KB, PC3, 4 T1 or L02 cells) were seeded into 6-well plates at a density of 3 × 105 cells per well and incubated for 24 h. Then the medium was replaced, the cells were collected and suspended in serum-free medium containing 10 μM DCFH-DA for 20 min at 37 °C. Later, the cells were washed 3 times with PBS and were observed as soon as possible via Eclipse TieU inverted microscope. The intracellular ROS levels were also analyzed by flow cytometer. For detecting the intracellular GSH concentrations, the cells were seeded into 6-well plates with a density of 3 × 105 cells per well and cultured for 24 h. Then, the cells were washed with PBS, collected by centrifugation and adding the protein removal reagent S solution. Later, the intracellular GSH concentrations were measured using a Total Glutathione Assay Kit (Beyotime, Shanghai, China).

2.7. Drug release behaviors of the albumin-prodrug conjugates The in vitro releasing behaviors of DTX from albumin-prodrug conjugates were studied by utilizing ethanol-containing PBS (pH 7.4, 30% ethanol (v/v)) as release media. Typically, 1 ml PBS of three BSAprodrug conjugates lyophilized powder were transferred into the dialysis bag (MW 10,000), then incubated in 30 ml release media at 37 °C. At pre-determined time intervals, 2 ml solution was withdrawn and replenished equal volume of fresh media. The releasing behaviors in the presence of H2O2 or DTT were carried out similarly, except for adding redox agents to the release media. The concentration of released DTX was determined by high-performance liquid chromatography (HPLC) using a Waters e2695 pump, a Waters 2489 UV detector (λ = 230 nm) and the Empower 3 software (Waters Corp.). Column: ODS Thermo-C18 column (150 mm × 4.6 mm, 5 μm); column temperature: 30 °C; flow rate: 1.0 mL/min; mobile phase: acetonitrile/water (50:50, v/v). All the experiments were performed in triplicate and the results were expressed by mean ± SD.

2.11. Experimental animals Male Sprague-Dawley rats (200–250 g) and female BALB/C mice (18-22 g) were supplied by the Animal Centre of Shenyang Pharmaceutical University (Shenyang, China). All the animal experiments were carried out in compliance with guidelines for the Care and Use of Laboratory Animals of Shenyang Pharmaceutical University Committee.

2.8. Cell culture KB human epidermoid carcinoma cells were originally obtained from American Type Culture Collection (ATCC). PC3 human prostatic carcinoma cells, 4 T1 mouse breast carcinoma cells and L02 human normal liver cells were bought from the cell bank of Chinese Academy of Sciences (Beiijng, China). Fetal bovine serum (FBS) was bought from Foundation (Beijing, China). KB, PC3 and 4 T1 cells were cultured in

2.12. Pharmacokinetic studies The Sprague-Dawley rats weighing 200–250 g were used to investigate the DTX prodrugs pharmacokinetic studies. The rats were 190

Journal of Controlled Release 285 (2018) 187–199

W. Wei et al.

p < 0.05 was considered statistically significant.

randomly divided into four groups (five animals per group) and were intravenously administrated Taxotere, DM, DSM and DSSM at a dose equivalent to 5 mg/kg of DTX. About 300 μL blood samples were collected and centrifuged to obtain plasma at predetermined time intervals. The plasma samples were stored at −80 °C until analysis. The plasma concentration of total and free DTX was measured by UPLC-MS/ MS on an ACQUITY UPLC system (Waters Corp.). For plasma samples in the three prodrugs groups, there were two kinds of processing methods. The first one was directly determined the plasma concentrations of free DTX, which was the same as the Taxotere group. The other one was to determinate the total concentrations of DTX by destroying the ester bond of different prodrugs. Briefly, for free DTX analysis, 25 μL PTX solution as the internal standard was added into 50 μL plasma samples and vortex mixed for 3 min. Then adding 3 ml methyl tert-butyl ether, vortex mixed and centrifuged at 3500 rpm for 10 min to extract the analytes. Next removed the upper layer and dryed under nitrogen gas flow at 37 °C. Finally, 100 μL acetonitrile was applied to redissolve the dried extracts and centrifuged at 10000 rpm for 5 min. The supernatant was collected to analyze using UPLC-MS/ MS. For total DTX analysis, 50 μL plasma samples were firstly added with 25 μL 10% H2O2 (for DSM group), 0.5% tetramethylammonium hydroxide (TMAOH) (for DM group) and 10 mM DTT (for DSSM group), then vortex mixed for 5 min to catalyze hydrolysis of the ester bond. Next adding 25 μL internal standard solution of PTX. The following processes were in accordance with the free DTX method as described above.

3. Results and discussions 3.1. Synthesis of DTX-Maleimide prodrugs Two novel reduction/oxidation-sensitive DTX-maleimide conjugates (DSM and DSSM) were designed and synthesized by conjugating maleimide to DTX via thioether bond and disulfide bond as linkages, respectively. Additionally, a simple ester bond linked conjugate (DM) was utilized as non-sensitive control. The synthetic routes were shown in Fig. S1. The chemical structures of the three prodrugs (DM, DSM and DSSM) were characterized by MS and 1H NMR (Figs. S2–S7), and the above results indicated that the three novel DTX-maleimide prodrugs were successfully synthesized. 3.2. Incubation with bovine serum albumin and plasma serum Firstly, the binding capacity of the prodrugs to serum albumin was investigated based on a commercially available BSA. The three prodrugs were incubated with BSA solutions and analyzed by HPLC (Fig. 2). The decrease of the peak area of the unbound prodrugs was monitored in Fig. 2d and the HPLC chromatograms, 5–90 min after incubation with BSA, were shown in Fig. 2a–c. Binding process was observed by the decrease of the prodrug peak intensity and appearance of the broad conjugate peak. It was clear that all the three DTX-maleimide prodrugs could rapidly bind BSA, approximately 50% of prodrugs were bound to albumin within the first minute and the bioconjugation process was almost accomplished (about 90%) after 5 min incubation. Next the binding ability was further confirmed by incubating prodrugs with fresh rat plasma to simulate in vivo albumin-binding. The three DTX-maleimide prodrugs could rapidly bind to serum plasma (Fig. S8). However, after the thiols of serum albumin were blocked by preincubating the rat plasma with abundant 6-maleimidocaproic acid, vast residual prodrugs (90–95%) could be detected even after 30 min incubation. In addition, compound DTX, as a maleimide- nonfunctionalized negative control, was also incubated with BSA solutions and analyzed by HPLC (Fig. S9). However, contrary to the above prodrugs (DSSM, DSM and DM) studies, the vast majority of residual DTX (about 95%) could be detected even after 90 min incubation, showing negligible protein binding ability during 90 min, and proving the very high specificity of the maleimide moiety towards albumin. These results exactly proved the fact that the Michael addition reaction between the sulfhydryl group of albumin and the maleimide group of prodrugs led to the covalent binding process.

2.13. In vivo biodistribution The in vivo tumor accumulation and biodistribution of DTX solution and different prodrugs were performed by tumor-bearing BALB/C mice. 100 μL PBS of 4 T1 cells (1 × 106 cells per mouse) were transplanted subcutaneously into the right flank region of 6 week-old female BALB/C mice. When the tumor volume reached about 200 mm3, DTX and the three novel prodrugs solutions were injected intravenously via tail veil at a dose equivalent to 8 mg/kg of DTX. At the desired times, mice were sacrificed and their major organs were harvested, weighed and stored at −20 °C until analysis. The tissues concentration of total and free DTX was measured by UPLC-MS/MS on an ACQUITY UPLC system (Waters Corp.). 2.14. In vivo antitumor effect 100 μL PBS of 4 T1 cells (1 × 106 cells per mouse) were transplanted subcutaneously into the right flank of 6 week-old female BALB/C mice. When the tumor reached approximate 150–200 mm3, the tumor bearing mice were randomly divided into five groups (n = 6). Then the mice were treated with Saline (control), DTX, DM, DSM and DSSM solutions at a dose equivalent to 8 mg/kg of DTX five times every other day via tail vein. Body weight and tumor volume were recorded every day. At the last day of observation, the mice were sacrificed, the tumors were isolated and weighed. Meantime the blood was collected and centrifuged at 10000 rpm for 5 min to obtain the serum for hepatic and renal function analysis. The major organs (heart, liver, spleen, lung, and kidney) were harvested for histopathological analyzing via hematoxylin and eosin (H&E) staining. The tumor volume (V) and the inhibition rate of tumor growth (IR) were calculated as follows: V (mm3) = (a2 × b)/2 (where a and b represented the width and length of the tumors). IR (%) = [(a - b)/ a] × 100 (where a and b represented the average tumor weight of the control and the treatment group).

3.3. Preparation and characterization of the albumin-prodrug conjugates The DTX-maleimide prodrugs (DM, DSM and DSSM) could bind albumin rapidly in PBS at 37 °C. According to the above binding studies, we prepared the albumin-prodrug conjugates by incubating prodrugs with BSA solutions for subsequent experiments. The intrinsic fluorescence spectra of BSA-prodrugs were analyzed to determine whether the prodrugs conjugation altered the conformation of BSA around the tryptophan residue (Trp-214). Upon excitation at 280 nm, both native BSA and BSA-prodrug conjugates had the same maximum emission intensity at 340 nm and nearly identical emission profiles (Fig. S10), indicating no quench of the intrinsic fluorescence of Trp-214 residue. This demonstrated that bioconjugation with the prodrugs did not affect the conformation of the hydrophobic binding pocket in the second αhelix domain of BSA. Circular Dichroism (CD) has been extensively used to determine the conformational change of BSA structure. There are two negative bands at 208 nm and 220 nm which correspond to π-π* and n-π* transition and are characteristic features of α-helical structures of BSA. To explore

2.15. Statistical analysis All the quantitative data were expressed as mean ± SD. Comparison between groups was performed with student's t-test and 191

Journal of Controlled Release 285 (2018) 187–199

W. Wei et al.

Fig. 2. Albumin binding studies of DM, DSM and DSSM. HPLC chromatograms of different prodrugs (blue) and the incubation with bovine serum albumin for 5 min (red), 30 min (pink), 90 min (green) and only BSA are also shown. (a) DM, (b) DSM, (c) DSSM, (d) The albumin-binding rate of the different prodrugs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

BSA-prodrug conjugates. Therefore, BSA-DSSM and BSA-DSM conjugates were incubated in PBS (pH 7.4) with different concentrations of DTT (a prevailing GSH simulatant) and H2O2 (a prevailing ROS simulatant), respectively. BSA-DM was also incubated in PBS (pH 7.4) with 10 mM DTT and 10 mM H2O2 as a control. BSA-DM conjugate was very stable in PBS either with 10 mM DTT or H2O2 and released < 10% DTX over 24 h (Fig. 3c). In contrast, both oxidation-sensitive BSA-DSM and redox-responsive BSA-DSSM conjugates exhibited triggered drug release performances. With increasing concentrations of DTT, the percentage of DTX released from BSA-DSSM conjugate gradually increased (Fig. 3a). Similarly, approximate 40% of DSM was hydrolyzed in the presence of 10 mM H2O2 over 24 h (Fig. 3b). The results revealed that BSA-DSM and BSA-DSSM conjugates would be relatively stable under normal physiological conditions, such as bloodstream and extracellular matrix, but rapid release in the unregulated redox/oxidation environment of tumor cells. The differential and rapid drug release within tumor cells would be an effective strategy to enhance the antitumor activity.

if there were some effects on the secondary structure of BSA by prodrug conjugation, circular dichroism measurement was performed for native BSA and BSA-prodrug conjugates. As showed in Fig. S10, circular dichroism spectra of the BSA-prodrug conjugates did not indicate any obvious changes in the 190–250 nm regions, especially in the negative bands at about 208 nm and 220 nm, indicating that the prodrug conjugation had a minimal influence on the secondary structure of BSA. 3.4. Stability of the albumin-prodrug conjugates in rat plasma The stability of BSA-prodrug conjugates in rat plasma was also investigated. < 20% of DTX were released from the three BSA-prodrug conjugates even after 24 h incubation with rat plasma (Fig. 3d). All of the three DTX-maleimide prodrugs rapidly bound with serum albumin, and the steric-hindrance effect would prevent the ester bond hydrolysis of the prodrugs, so the conjugates were relatively stable in plasma. Among the three BSA-prodrug conjugates, the cumulative release of DTX from BSA-DSM was a little faster than BSA-DSSM, mainly because of high oxygen content in blood circulation to facilitate its hydrolysis.

3.6. Cytotoxicity and intracellular drug release 3.5. Drug release behavior of the albumin-prodrug conjugates The in vitro cytotoxicities of DTX and prodrugs in the three tumor cells (KB, PC3 and 4T1cells) and one normal cell (L02 cells) were evaluated by MTT assays. As shown in Fig. 4, S11 and Table S1,

The lyophilized powders of BSA-DM, BSA-DSM and BSA-DSSM were directly used to investigate the reduction/oxidation-responsivity of the 192

Journal of Controlled Release 285 (2018) 187–199

W. Wei et al.

Fig. 3. Cumulative release of DTX from (a) BSA-DSSM conjugate in the presence of different concentrations of DTT; (b) BSA-DSM conjugate in the presence of different concentrations of H2O2; (c) BSA-DSSM, BSA-DSM and BSA-DM conjugates in the presence of 10 mM DTT or H2O2; (d) BSA-DSSM, BSA-DSM and BSA-DM conjugates in rat plasma. All data are mean ± SD (n = 3).

released intracellularly and not in the cell culture media. Based on these findings, we further evaluated the levels of ROS and GSH in different cancer and normal cells (KB, PC3, 4 T1 and L02 cells). As revealed in Fig. S13, in comparison with L02 cells, markedly higher levels of ROS and GSH were observed in the three tumor cells. Meantime, we also found among the three tumor cells, both ROS and GSH were higher in the same cancer cells. Interestingly, there were significantly higher intracellular ROS and GSH levels in KB and PC3 cells than 4 T1 cell. The large amount of ROS and GSH could trigger DSM and DSSM release free drug rapidly, in well accordance with the above cytotoxicity and intracellular drug release results, further substantiating that the reduction/oxidation levels of tumor cells determined the release capacity of the reduction/oxidation-responsive prodrugs DSSM and DSM.

compared to DTX solutions, the prodrugs were less effective in killing tumor cells because of the delayed release of the active DTX molecule. Among these prodrugs, both DSM and DSSM showed higher cytotoxicity than DM due to the tumor cell-triggered rapid drug release. However, compared with DTX solutions and DM treatments, only DSM and DSSM did not show obvious cytotoxicity to L02 cells and the cell viability was almost above 80%, with the DTX concentration ranging from 10 to 200 ng/ml after 48 h incubation (Fig. 5), indicating that the active DTX molecules were probably released to a less extent in the normal L02 cells. Motivated by the differential cytotoxicities towards cancer cells and normal cells, we further compared the intracellular drug release behavior of the prodrugs in 4 T1 and L02 cells. As shown in Fig. 6 and Table S2, much more DTX molecules were released from DSM and DSSM than that from DM at all timepoints in 4 T1 cells. More interestingly, there were dramatically different drug release behavior between 4 T1 and L02 cells in DSM and DSSM treated groups, a significant faster release rate in tumor 4 T1 cells than in normal L02 cells. As shown in Table S3, the tumor activation index (TAI: AUC4T1cells/AUCL02 cells) of DSM and DSSM were17–18 fold higher than that DM at the concentration of 100 ng/ml. The intracellular release results were well consistent with the above cytotoxicity, further confirming that both DSSM and DSM had better tumor cell-triggered rapid release of active DTX molecule, especially hyperselectivity towards tumor and normal cells. Later, we also evaluated the prodrugs release capacity in the cell culture media. As shown in Fig. S12, all the three prodrugs were very stable in either 4 T1 or L02 cells culture media and released < 4% DTX over 48 h. Based on these, we vigorously certified that the free DTX was

3.7. Pharmacokinetic studies The pharmacokinetic profiles of DTX, DM, DSM and DSSM were compared after intravenous administration to SD rats at a dose of DTX equivalent to 5 mg/kg. For direct comparison of different prodrugs, the total and free DTX concentration-time curves and pharmacokinetic parameters were summarized in Fig. S14 and Table S4. DTX was rapidly eliminated from blood for DTX solutions, with t1/2 and AUC0–48 h values being 2.2 ± 0.2 h and 865.4 ± 245.5 μg/L*h, respectively. By contrast, DTX-maleimide prodrugs exhibited significantly prolonged retention in blood circulation and the AUC0–48 h values of total DTX in the prodrugs groups were about 160–260 times higher than DTX solutions. Worth noting, in all the three prodrugs groups, the AUC0–48 h values of total DTX were extraordinarily higher than those of the free DTX form, 193

Journal of Controlled Release 285 (2018) 187–199

W. Wei et al.

Fig. 4. Cell viability treated with various concentrations of DTX, DSM, DSSM and DM after 48 h and 72 h treatment. 4 T1 cell (a) and (b). L02 cell (c) and (d). All data are presented as mean ± SD (n = 3).

Fig. 5. In vitro cytotoxicity against tumor cells (KB, PC3 and 4 T1 cells) and normal cells (L02 cells) after being incubated with (a) DSM, (b) DSSM, (c) DM and (d) DTX for 48 h. All data are presented as mean ± SD (n = 3), n.s, not significant, *P < 0.05, **P < 0.01, and *** P < 0.001.

194

Journal of Controlled Release 285 (2018) 187–199

W. Wei et al.

Fig. 6. Free DTX level released from the three prodrugs when incubation with 4 T1 and L02 cells at different timepoints (a) 6 h (b) 12 h (c) 24 h (d) 48 h (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, as compared with DM incubation with 4 T1 cells at the same time-point; ^P < 0.05, ^^P < 0.01, as compared with the same prodrug incubation with L02 cells at the same time-point.

Fig. 7. Quantitative analysis of released free DTX level in 4 T1 tumor-bearing mice at different times after intravenous administration of DTX, DSSM, DSM and DM (DTX = 8 mg/kg). (a) 12 h (b) 24 h (c) 48 h (d) 72 h. All data are presented as mean ± SD (n = 3).

195

Journal of Controlled Release 285 (2018) 187–199

W. Wei et al.

indicating that the overwhelming majority of the DTX prodrugs in plasma were stored in the form of albumin-prodrug conjugates. The albumin-prodrugs conjugates, generally accepted as being stable, can in fact undergo retro-Michael reactions in the presence of other thiol compounds at physiological environment, which may result in the unwanted drug release. However, the concentration of low molecular weight sulfhydryl compounds in human blood plasma, such as: cysteine (~10–12 μM), cysteinylglycine (~3–4 μM), or glutathione (~4–5 μM), was comparatively low when compared to the serum albumin, which was 400–500 μM according to the literature [17, 35, 36]. And the free thiol at the cysteine-34 position of HSA accounts for the major amount (80–90%) of the total thiols in blood plasma. Moreover, according to the above pharmacokinetic and plasma stability studies, the majority of the prodrugs were conjugated with albumin in the form of albuminprodrug adducts and the released free DTX was relatively few, implying the retro-Michael reactions may be rare and the design of albuminbinding prodrugs was a promising method to improve the in vivo pharmacokinetic behavior of DTX.

Table 2 The tumor activation index (TAI) of prodrugs for different tissues (TAI: AUC of tumors/AUC of normal tissues). Prodrugs

DTX

DM

DSM

DSSM

TAI TAI TAI TAI TAI

0.65 0.79 0.50 0.44 0.89

1.23 2.53 0.71 0.96 1.82

17.85 36.55 6.60 6.98 10.19

15.36 24.70 8.07 9.79 18.62

Tumor/Heart Tumor/Liver Tumor/Spleen Tumor/Lung Tumor/Kidney

in tumors via passive targeting (EPR effect) and active targeting. In summary, we can see that the DTX prodrugs could instantly bind serum albumin to form the stable albumin-prodrug adducts, then the prodrugs could accumulated in tumor tissues due to the tumor-targeting of albumin. When the albumin-prodrug adducts were endocytosed into cells, there were three main pathways for albumin according to the literature [24]: recycling, transcytosis and degraded. It is known that albumin is recycled by an FcRn-mediated endosomal sorting pathway. However, though FcRn rescued its ligand albumin from intracellular degradation, their levels in serum were determined by the saturable nature of the intracellular FcRn interaction. Once FcRn became saturated, ligand albumin would be delivered to the lysosomes and degraded, this was how tumors trapped plasma proteins and utilized their degradation products for proliferation. According to these, we proposed when albumin-prodrug adducts were internalized, some of them fused with endosomes and eventually destined for lysosomal degradation, the parent drugs were then released through albumin degradation to reach the cytoplasm. Moreover, it has been demonstrated that except for a large number of hydrogen ions and various enzymes, the lysosomes also contained lots of reducing agents, such as GSH, which can cleave disulfide bonds [37]. So the albumin-prodrugs could directly release free DTX in lysosomes, and then the free drug entered the cytoplasm by passive diffusion. But our understandings of how the prodrugs escaped the endosome were still not clear, it remained to be investigated in the future.

3.8. In vivo biodistribution It is well accepted that albumin can accumulate in the tumors due to EPR (enhanced permeation and retention) effect and active targeting. As the newly designed prodrugs, DSSM, DSM and DM have the ability to rapidly hitchhike serum albumin, we were interested in the prodrugs distribution and their bioactivation behavior in vivo. To this end, female 4 T1-bearing BALB/C mice were treated with the novel prodrugs and DTX solutions to investigate the biodistribution behavior. Free DTX levels in the tissues after i.v. injection of different prodrugs was determined to assess the possible toxic effect against normal organs. Fig. 7a–d and Table 1 showed the comparison of released free DTX levels in tumor, heart, liver, kidney, spleen and lung at different timepoints. In DSM and DSSM treated groups, the tumor activation index (TAI: AUCtumor/AUCnormal tissues) was dramatically higher than DM and DTX solutions (Table 2). As shown in Table 2, TAITumor/Liver of DTX group was only 0.79, but 36.55 and 24.70 for DSM and DSSM. The 46-fold and 31-fold increase of TAITumor/Liver for DSM and DSSM clearly demonstrated that the tumor-triggered prodrugs (DSM and DSSM) possess a hyperselective drug release capacity in tumors and can prevent undesired DTX toxicity against normal organs (e.g., liver, kidney, etc.). Worth noting, the overwhelming majority of the administered DTX prodrugs were stored in the albumin-prodrug conjugates form in plasma, so we further quantified the total DTX amount of the three prodrugs in tumor tissue. As shown in Fig. 8a–c, only 0.15 ± 0.06% μg/g of DTX was accumulated in tumor at 12 h post-injection in DTX solution group mice, and DTX was rapidly vanished in tumor. Compared with this, the total DTX tumor concentration was slowly decreased with time in the three prodrugs groups, about 35-fold higher concentration than that of DTX solution group at 12 h, suggesting that the serum albumin-prodrug conjugates could successfully accumulate

3.9. In vivo antitumor effect Encouraged by the excellent in vitro cytotoxicity, high intra-tumor accumulation and hyperselective release of active drug molecular in vivo, these novel prodrugs were further investigated for tumor therapy in 4 T1 tumor-bearing mice models. The tumor volume and body weight were monitored every day. Compared with the saline group, the other four administrated groups exhibited different tumor inhibition degrees after five injections treatments. As depicted in Fig. 9a–c, the tumors treated with PBS grew quickly with time, with the average tumor volume being about 1300 mm3 on day 11. Compared with PBS group, DM and DTX groups showed a moderate tumor inhibiting ability (400 mm3 on day 11). But, mice treated with DSM and DSSM achieved the most potent antitumor activity, with almost no growth in tumor volume.

Table 1 The AUC(0-t) (μg/g*h) values of free and total DTX in various tissues of mice after i.v. injection of DTX or prodrugs solutions (n = 3). Prodrugs

Heart Liver Spleen Lung Kidney Tumor

Tissue AUC(0-t) (μg/g*h)

Free DTX Free DTX Free DTX Free DTX Free DTX Free DTX Total DTX

DTX

DM

DSM

DSSM

10.16 ± 2.78 8.28 ± 2.04 13.24 ± 0.79 14.92 ± 3.25 7.36 ± 0.97 6.58 ± 1.55 6.58 ± 1.55

7.54 ± 1.10 3.66 ± 1.70 12.96 ± 2.41 9.62 ± 1.62 5.09 ± 0.14 9.26 ± 2.85 121.86 ± 11.4

2.17 ± 1.09 1.06 ± 0.19 5.87 ± 1.44 5.55 ± 1.63 3.80 ± 1.88 38.74 ± 3.62 86.62 ± 5.83

3.20 ± 0.48 1.99 ± 0.69 6.09 ± 0.99 5.02 ± 2.56 2.64 ± 0.63 49.15 ± 5.14 79.14 ± 5.93

196

Journal of Controlled Release 285 (2018) 187–199

W. Wei et al.

Fig. 8. In vivo tumor concentration-time profiles of free and total DTX following a single i.v. administration of DTX, DM, DSM or DSSM at a DTX equivalent dose of 8 mg/kg. (a) DSM (b) DSSM (c) DM. All data are presented as mean ± SD (n = 3).

upregulated environment, in sharp contrast to normal tissues (Fig. 1), leading to significantly enhanced antitumor efficiency and reduced toxicity.

These results further revealed that the low release rate of DTX from ester bond prodrug DM greatly limited its chemotherapeutic efficacy despite the long blood circulation and high tumor accumulation, a rapid and hyperselective release of active DTX in tumors is of crucial importance. Additionally, an obvious weight loss treated with DTX was shown in Fig. 9d, but no significant change in body weight was observed in the prodrugs groups. As shown in Fig. S15, H&E-stained pathological section results showed that the obvious tumor metastases were observed in the liver and lung of the mice treated with PBS and DTX solution, but no metastatic lesion was found in the prodrugs groups. These results suggested that DTX-maleimide prodrugs not only showed potent antitumor effect against the primary breast tumor but also could efficiently inhibit metastasis of breast cancer. In addition, the serum biochemistry analyses were also evaluated (Fig. S12). Similar to the above results, the levels of AST and ALT were dramatically higher only in DTX group, and no noticeable change was observed for the three DTX-maleimide prodrugs groups. Evidently, the novel three prodrugs could display appreciable safety profiles as evidenced by the serum biochemistry analysis, the histological analysis and body weight recording. In general, we found that when replacing paclitaxel with docetaxel, the novel prodrugs could have better binding ability with plasma albumin. They could instantly hitchhike blood circulating albumin after i.v. administration with albumin-binding half-lives as short as 1 min to obtain stable albumin-prodrug conjugates, achieve long blood circulation and better tumor accumulation via the specific transport mechanism of albumin in vivo. In addition, DSSM and DSM had a superselective cytotoxicity between normal and tumor cells, which could selectively release free drug under tumor reduction/oxidation

4. Conclusions To develop advanced albumin-based prodrug platform for anticancer drug delivery, we herein report two novel tumor reduction/oxidation-responsive DTX-maleimide smart prodrugs based on disulfide bond and thioether bond, respectively. The ester bond linked prodrug (DM) showed slow drug release, non-selectivity towards tumor and normal tissues, resulting in poor antitumor efficacy. By contrast, both the tumor cell-triggered reduction/oxidation-sensitive release prodrugs (DSSM and DSM) presented notable advantages as follows. They could not only rapidly bind to the plasma albumin, achieve long blood circulation and high intra-tumor accumulation, but also show an efficient intra-tumor drug release property and significant antitumor activity. Moreover, both reduction-responsive and oxidation-responsive prodrugs are capable of achieving the hyperselective release performances towards tumor and normal cells. Exploiting such a uniquely engineered prodrug delivery system, the therapeutic efficacy of DTX was significantly improved by integrating albumin- hitchhiking strategy and tumor cell-triggered drug release into one system.

Conflicts of interest The authors declare no competing financial interest.

197

Journal of Controlled Release 285 (2018) 187–199

W. Wei et al.

Fig. 9. In vivo antitumor activity of DTX and different prodrugs solutions against 4 T1 xenograft tumors. (a) Tumor growth profiles treated with different formulations. (b) Images of tumors after the last treatment (from up to bottom: PBS, DTX, DM, DSM and DSSM). (c) Tumor weight after the last treatment. (d) Body weight changes of different formulations. (e) H&E staining of the major organs and tumors after treatments. (f) Hepatic and renal function indicators of mice bearing 4 T1 tumor xenografts after treatment. All data are presented as mean ± SD (n = 3), *P < 0.05.

Acknowledgment [8]

This work was financially supported by the National Basic Research Program of China (973 Program, No. 2015CB932100), the National Nature Science Foundation of China (No. 81373336, 81473164), and The Innovation Team Project of Education Department of Liaoning Province (LT 2014022).

[9]

[10]

Appendix A. Supplementary data

[11]

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jconrel.2018.07.010.

[12]

References

[13] [14]

[1] B. Seruga, A. Ocana, I.F. Tannock, Drug resistance in metastatic castration-resistant prostate cancer, Nat. Rev. Clin. Oncol. 8 (2011) 12–23. [2] S. Tang, Q. Meng, H. Sun, J. Su, Q. Yin, Z. Zhang, H. Yu, L. Chen, W. Gu, Y. Li, Dual pH-sensitive micelles with charge-switch for controlling cellular uptake and drug release to treat metastatic breast cancer, Biomaterials 114 (2017) 44–53. [3] Y. Jiang, S. Wong, F. Chen, T. Chang, H. Lu, M.H. Stenzel, Influencing selectivity to cancer cells with mixed nanoparticles prepared from albumin-polymer conjugates and block copolymers, Bioconjug. Chem. 28 (2017) 979–985. [4] S. Tong, B. Xiang, D. Dong, X. Qi, Enhanced antitumor efficacy and decreased toxicity by self-associated docetaxel in phospholipid-based micelles, Int. J. Pharm. 413 (1–2) (2012) 413–419. [5] T. Minko, L. Rodriguez-Rodriguez, V. Pozharov, Nanotechnology approaches for personalized treatment of multidrug resistant cancers, Adv. Drug Deliv. Rev. 65 (2013) 1880–1895. [6] H. Maeda, Vascular permeability in cancer and infection as related to macromolecular drug delivery, with emphasis on the EPR effect for tumor-selective drug targeting, Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 88 (2012) 53–71. [7] H. Maeda, Toward a full understanding of the EPR effect in primary and metastatic

[15] [16]

[17]

[18]

[19]

198

tumors as well as issues related to its heterogeneity, Adv. Drug Deliv. Rev. 91 (2015) 3–6. P. Mishra, B. Nayak, R.K. Dey, PEGylation in anti-cancer therapy: an overview, Asian J. Pharm. Sci. 11 (2016) 337–348. X. Han, Z. Li, J. Sun, C. Luo, L. Li, Y. Liu, Y. Du, S. Qiu, X. Ai, C. Wu, H. Lian, Z. He, Stealth CD44-targeted hyaluronic acid supramolecular nanoassemblies for doxorubicin delivery: probing the effect of uncovalent pegylation degree on cellular uptake and blood long circulation, J. Control. Release 197 (2015) 29–40. J.G. Mehtala, C. Kulczar, M. Lavan, G. Knipp, A. Wei, Cys34-PEGylated human serum albumin for drug binding and delivery, Bioconjug. Chem. 26 (2015) 941–949. A.S. Abu Lila, H. Kiwada, T. Ishida, The accelerated blood clearance (ABC) phenomenon: clinical challenge and approaches to manage, J. Control. Release 172 (2013) 38–47. S. Zhu, M. Hong, G. Tang, L. Qian, J. Lin, Y. Jiang, Y. Pei, Partly PEGylated polyamidoamine dendrimer for tumor-selective targeting of doxorubicin: the effects of PEGylation degree and drug conjugation style, Biomaterials 31 (2010) 1360–1371. F. Kratz, A clinical update of using albumin as a drug vehicle - a commentary, J. Control. Release 190 (2014) 331–336. H.P. Liu, K.D. Moynihan, Y.R. Zheng, G.L. Szeto, A.V. Li, B. Huang, D.S. Van Egeren, C. Park, D.J. Irvine, Structure-based programming of lymph-node targeting in molecular vaccines, Nature 507 (2014) 519–522. F. Kratz, Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles, J. Control. Release 132 (2008) 171–183. E. Miele, G.P. Spinelli, E. Miele, F. Tomao, S. Tomao, Albumin-bound formulation of paclitaxel (Abraxane (R) ABI-007) in the treatment of breast Cancer, Int. J. Nanomedicine 4 (2009) 99–105. F. Kratz, A. Warnecke, K. Scheuermann, et al., Probing the cysteine-34 position of endogenous serum albumin with thiol-binding doxorubicin derivatives. Improved efficacy of an acid-sensitive doxorubicin derivative with specific albumin-binding properties compared to that of the parent compound, J. Med. Chem. 45 (2002) 5523–5533. D. Zhao, H. Zhang, W. Tao, W. Wei, J. Sun, Z. He, A rapid albumin-binding 5fluorouracil prodrug with a prolonged circulation time and enhanced antitumor activity, Biomater. Sci. 5 (2017) 502–510. S.W. Chung, J.U. Choi, B.S. Lee, J. Byun, O.C. Jeon, S.W. Kim, I.S. Kim, S.Y. Kim, Y. Byun, Albumin-binding caspase-cleavable prodrug that is selectively activated in

Journal of Controlled Release 285 (2018) 187–199

W. Wei et al.

as potent chemotherapeutic nanomedicine, Small 12 (2016) 6353–6362. [29] C.L. Grek, K.D. Tew, Redox metabolism and malignancy, Curr. Opin. Pharmacol. 10 (2010) 362–368. [30] T. Yin, Q. Wu, L. Wang, L. Yin, J. Zhou, M. Huo, Well-defined redox-sensitive polyethene glycol-paclitaxel prodrug conjugate for tumor-specific delivery of paclitaxel using octreotide for tumor targeting, Mol. Pharm. 12 (2015) 3020–3031. [31] Y.W. Hu, Y.Z. Du, N. Liu, X. Liu, T.T. Meng, B.L. Cheng, J.B. He, J. You, H. Yuan, F.Q. Hu, Selective redox-responsive drug release in tumor cells mediated by chitosan based glycolipid-like nanocarrier, J. Control. Release 206 (2015) 91–100. [32] X. Chuan, Q. Song, J. Lin, X. Chen, H. Zhang, W. Dai, B. He, X. Wang, Q. Zhang, Novel free-paclitaxel-loaded redox-responsive nanoparticles based on a disulfidelinked poly(ethylene glycol)-drug conjugate for intracellular drug delivery: synthesis, characterization, and antitumor activity in vitro and in vivo, Mol. Pharm. 11 (2014) 3656–3670. [33] J. Yang, Q. Lv, W. Wei, Z. Yang, J. Dong, R. Zhang, Q. Kan, Z. He, Y. Xu, Bioresponsive albumin-conjugated paclitaxel prodrugs for cancer therapy, Drug Delivery 25 (2018) 807–814. [34] X. Ke, H. Liang, L. Xiong, S. Huang, M. Zhu, Synthesis, curing process and thermal reversible mechanism of UV curable polyurethane based on Diels-Alder structure, Progress in Organic Coatings 100 (2016) 63–69. [35] M.A. Mansoor, A.M. Svardal, P.M. Ueland, Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma, Anal. Biochem. 22 (1992) 218–229. [36] J. Martensson, The effect of fasting on leukocyte and plasma glutathione and sulfur amino acid concentrations, Metabolism 35 (1986) 118–121. [37] J. Cui, J.J. Richardson, M. Björnmalm, M. Faria, F. Caruso, Nanoengineered templated polymer particles: navigating the biological realm, Acc. Chem. Res. 49 (2016) 1139–1148.

radiation exposed local tumor, Biomaterials 94 (2016) 1–8. [20] J. Mayr, P. Heffeter, D. Groza, L. Galvez, G. Koellensperger, A. Roller, B. Alte, M. Haider, W. Berger, C.R. Kowol, B.K. Keppler, An albumin-based tumor-targeted oxaliplatin prodrug with distinctly improved anticancer activity in vivo, Chem. Sci. 8 (2017) 2241–2250. [21] H. Maeda, H. Nakamura, J. Fang, The EPR effect for macromolecular drug delivery to solid tumors: improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo, Adv. Drug Deliv. Rev. 65 (2013) 71–79. [22] V. Torchilin, Tumor delivery of macromolecular drugs based on the EPR effect, Adv. Drug Deliv. Rev. 63 (2011) 131–135. [23] N. Desai, V. Trieu, B. Damascelli, P. Soon-Shiong, SPARC expression correlates with tumor response to albumin-bound paclitaxel in head and neck cancer patients, Transl. Oncol. 2 (2009) 59–64. [24] B. Malin, M.K.S. Kine, N. Jeannette, et al., The role of albumin receptors in regulation of albumin homeostasis: implications for drug delivery, J. Control. Release 211( (2015) 144–162. [25] Y. Kang, X. Ju, L.S. Ding, S. Zhang, B.J. Li, Reactive oxygen species and glutathione dual redox-responsive supramolecular assemblies with controllable release capability, ACS Appl. Mater. Interfaces 9 (2017) 4475–4484. [26] C. Luo, J. Sun, D. Liu, B. Sun, L. Miao, S. Musetti, J. Li, X. Han, Y. Du, L. Li, L. Huang, Z. He, Self-assembled redox dual-responsive prodrug-nanosystem formed by single thioether-bridged paclitaxel-fatty acid conjugate for cancer chemotherapy, Nano Lett. 16 (2016) 5401–5408. [27] Y. Chi, X. Yin, K. Sun, S. Feng, J. Liu, D. Chen, C. Guo, Z. Wu, Redox-sensitive and hyaluronic acid functionalized liposomes for cytoplasmic drug delivery to osteosarcoma in animal models, J. Control. Release 261 (2017) 113–125. [28] C. Luo, J. Sun, B. Sun, D. Liu, L. Miao, T.J. Goodwin, L. Huang, Z. He, Facile fabrication of tumor redox-sensitive nanoassemblies of small-molecule oleate prodrug

199