Disulfide Bond-Driven Oxidation- and Reduction-Responsive Prodrug

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Disulfide Bond-Driven Oxidation/Reduction-Responsive Prodrug Nanoassemblies for Cancer Therapy Bingjun Sun, Cong Luo, Han Yu, Xuanbo Zhang, Qin Chen, Wenqian Yang, Menglin Wang, Qiming Kan, Haotian Zhang, Yongjun Wang, Zhonggui He, and Jin Sun Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b00737 • Publication Date (Web): 04 May 2018 Downloaded from http://pubs.acs.org on May 6, 2018

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Disulfide Bond-Driven Oxidation/ReductionResponsive Prodrug Nanoassemblies for Cancer Therapy Bingjun Sun†,⊥, Cong Luo†,⊥, Han Yu†, Xuanbo Zhang†, Qin Chen‡, Wenqian Yang†, Menglin Wang†, Qiming Kan§, Haotian Zhang§, Yongjun Wang†, Zhonggui He*, †, Jin Sun*, † †

Department of Pharmaceutics, Wuya College of Innovation, Shenyang Pharmaceutical

University, Shenyang, 110016, P.R. China ‡

Department of Pharmacy, Cancer Hospital of China Medical University, Liaoning Cancer

Hospital & Institute, Shenyang, 110042, P. R. China §

School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang,

110016, P. R. China

Abstract: Disulfide bond has been widely used to develop reduction-responsive drug delivery system (DDS) for cancer therapy. But we propose that the disulfide bond might be also used as an oxidation-responsive linkage just like thioether bond, which can be oxidized to hydrophilic sulfoxide or sulphone in the presence of oxidation stimuli. To test our hypothesis, we design three novel paclitaxel-citronellol conjugates, linked via different lengths of disulfide bondcontaining carbon chain. The prodrugs can self-assemble into uniform size nanoparticles with impressive high drug loading (>55%). As expected, the disulfide bond-bridged prodrug NPs shows redox dual-responsive drug release. More interestingly, the position of disulfide bond in

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the carbon chain linkage has profound impacts on the redox dual-responsiveness, thereby affecting the drug release, cytotoxicity, pharmacokinetics, biodistribution and in vivo antitumor efficacy of prodrug nanoassemblies. The redox dual-responsive mechanism is elucidated, and how the position of disulfide bond in the carbon chain affects the redox dual-responsiveness and antitumor efficiency of prodrug nanoassemblies is also clarified. Our findings give new insight into the stimuli-responsiveness of disulfide bond and provide a good foundation for the development of novel redox dual-responsive DDS for cancer therapy.

Key words: Disulfide bond; Paclitaxel; Prodrug nanoassemblies; Redox dual-responsive; Cancer therapy Cancer is still the leading cause of death in the world today 1. Over the past decades, great efforts have been made to battle with cancer. Among them, chemotherapy is the most common and effective strategy, especially for those advanced and metastatic tumors 2. However, the current state of chemotherapy is greatly restricted by the inefficient drug delivery, results in serious side effects, narrow therapeutic window and limited clinical efficiency 3. Therefore, developing high-efficiency DDS of anticancer drugs has long been a focus in the cancer therapy research. Paclitaxel (PTX) has been widely utilized in the clinical treatment of multiple types of tumors 4

. However, due to the poor water solubility of PTX, the commercial injection (Taxol) is

prepared by dissolving PTX in Cremophor EL and ethanol, leading to serious excipientassociated side effects and limited therapeutic efficiency 5. To address these problems, a wide variety of drug delivery strategies have been developed, including prodrugs and nanoparticulate drug delivery systems (nano-DDS)

6, 7

. Prodrug strategy usually refers to the reversible

modification of parent drug into an inactive form with desirable physicochemical properties,


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which can be readily converted into the bioactive parent drugs in vivo 8. Rational design of prodrugs can effectively cope with the undesirable properties of the parent drugs, such as poor stability, low solubility and serious toxicity 9. Prodrug strategy has been widely used to improve the delivery efficiency of PTX

10-12

. In addition, nano-DDS have also experienced tremendous

development in the past few decades, with distinct advantages including prolonged circulation time, improved tumor accumulation, facilitated cellular uptake, and controlled drug release 13, 14. Furthermore, prodrug-based self-assembled nanoparticles (NPs) integrating multiple drug delivery technologies into one system, has been emerging as a promising platform for the highefficiency delivery of chemotherapeutics 6, 7. In this case, the prodrugs themselves act as both the carriers and the cargos, exhibiting impressively high drug loading and low excipient-associated side effects. Moreover, how to realize precise drug release at tumor sites is also of crucial importance for efficient cancer therapy

15-18

. Compared with normal cells, tumor cells simultaneously

overproduce reactive oxygen species (ROS) and glutathione (GSH), leading to a redoxheterogeneous intracellular environment 19, 20. The redox potential difference between normal cells and tumor cells has been well studied to achieve on-demand drug release. Among them, disulfide bond has been widely utilized as a typical reduction-sensitive linkage to design tumorspecific stimuli-responsive DDS 21-23. Our group also developed several disulfide bond-bridged reduction-sensitive DDS for cancer therapy 24-26. Despite its excellent reduction-responsiveness, we further hypothesized that disulfide bond might be also used as an oxidation-responsive linkage just like thioether bond, which was found to be oxidized into sulfoxide or sulphone in previous studies

19, 27

. The two sulfur atoms of disulfide bond would also be oxidized to

hydrophilic sulfoxide or sulphone, thereby promoting the hydrolysis of the neighbouring ester


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bond and facilitating the release of anticancer drugs in the presence of oxidation stimuli within tumor cells. Chart 1. Chemical structures of the disulfide bond-bridged PTX-CIT prodrugs: α-PTX-SS-CIT, β-PTX-SS-CIT, and γ-PTX-SS-CIT.

To test our hypothesis, three novel PTX prodrugs (Chart 1) were synthesized by conjugating PTX with citronellol (CIT), using various lengths of disulfide bond-containing carbon chain as linkages (dithiodiglycolic acid, 3,3'-dithiodipropionic acid and 4,4'-dithiodibutyric acid). The prodrugs were abbreviated as α-PTX-SS-CIT, β-PTX-SS-CIT, and γ-PTX-SS-CIT, with the sulfur atoms located in the α-, β-, or γ-position of the ester bond, respectively. As shown in Figure 1, α-PTX-SS-CIT, β-PTX-SS-CIT and γ-PTX-SS-CIT could self-assemble into uniform size NPs, with impressively high drug loading (>55%). As expected, the disulfide bond-bridged prodrugs nanoassemblies demonstrated distinct redox dual-responsive capability in the presence of two opposite stimuli (oxidation and reduction). Moreover, we found that the position of disulfide bond in the carbon chain not only significantly affects the redox dual-responsiveness, but also has profound impacts on the drug release, cytotoxicity, pharmacokinetics,


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biodistribution and in vivo antitumor efficacy of prodrug nanoassemblies. We elucidated the redox dual-responsive mechanism of disulfide bond, and how the position of disulfide bond in the carbon chain affects the redox dual-responsiveness and antitumor efficiency of prodrug nanoassemblies was also investigated.

Figure 1. Schematic representation of the disulfide bond-bridged prodrugs nanoassemblies and the redox dual-responsive drug release in tumor cells. The nanoassemblies were formed by PTX-SS-CIT prodrugs themselves, and DSPE-PEG2k was utilized to prepare PEGylated prodrug NPs. After prodrug NPs were delivered into tumor cells, on-demand drug release could be achieved through the redox dual-responsive capability of disulfide bond in the presence of overproduced ROS and GSH in tumor cells. As illustrated in Chart 1, three novel PTX-CIT prodrugs were synthesized using various lengths of disulfide bond-containing carbon chain as linkages (dithiodiglycolic acid, 3,3'-


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dithiodipropionic acid and 4,4'-dithiodibutyric acid), abbreviated as α-PTX-SS-CIT, β-PTX-SSCIT, and γ-PTX-SS-CIT, respectively. The synthetic route was summarized in Scheme S1. The chemical structures of these disulfide bond bridged PTX-CIT prodrugs were confirmed by mass spectrum (MS) and 1H NMR (Figure S1-S3). Prodrug nanoassemblies were prepared by one-step nano-precipitation method according to our previous study (Figure S4A) 26. After being dispersed into water, the hydrophobic prodrugs spontaneously assembled into uniform nanoassemblies. DSPE-PEG2K was used to improve the colloidal stability and prolong the circulation time of prodrugs nanoassemblies in vivo. In our previous studies, multiple mechanisms were found to be involved in the self-assembly process, including chemical bond insertion (disulfide bond), structural flexibility (CIT), and intermolecular π-π stacking (PTX)

24, 26

. The prepared NPs showed spherical-shaped structures

with mean diameter around 85 nm (Figure S4B-D and Table S1). Because the prodrugs themselves act as both the carriers and the cargos, the prepared nanoassemblies showed a higher drug loading capacity (over 55%, w/w) than the conventional PTX-encapsulated nanoformulations (usually less than 10%, Table S1). High drug loading is one of the most outstanding advantages of self-assembled prodrug NPs, leading to significantly improved drug delivery efficiency and reduced excipient-associated toxicity. Moreover, the prodrugs nanoassemblies demonstrated good colloidal stability in pH 7.4 PBS supplemented with 10% FBS at 37 °C (Figure S4E).


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Figure 2. In vitro redox dual-responsive drug release of prodrug nanoassemblies in the presence of various concentrations of DTT or H2O2 (n=3). (A): 1 mM DTT; (B): 5 mM DTT; (C): 10 mM DTT; (D): 1 mM H2O2; (E): 5 mM H2O2; (F): 10 mM H2O2. So far, disulfide bond has been only reported to be a reduction-responsive linkage. But we wonder whether the disulfide bond could be sensitive to oxidation stimuli with the same mechanisms as thioether bond 19. In this section, we investigated the redox-responsiveness of the disulfide bond-bridged prodrug nanoassemblies using dithiothreitol (DTT, a prevailing simulatant of GSH) and H2O2 (a prevailing simulatant of ROS). As shown in Figure 2A-C, the prepared prodrug nanoassemblies exhibited DTT-triggered drug release in a concentration- and time-dependent manner, and the position of disulfide bond in the carbon chain had a great influence on the responsiveness. About 80% of PTX were released from α-PTX-SS-CIT NPs and γ-PTX-SS-CIT NPs in the presence of 1 mM DTT within 8 h (Figure 2A). In comparison, the release of PTX from β-PTX-SS-CIT NPs was quite slow, with only 45% of PTX released within 24 h even in the presence of 10 mM DTT (Figure 2C). The DTT-triggered drug release


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mechanism of disulfide bond bridged prodrug nanoassemblies were illustrated in Figure 3A 25, 28. The disulfide bond inserted in prodrugs was degraded into thiol groups in the presence of DTT, and the generated hydrophilic thiol groups could facilitate the hydrolysis of the adjacent ester bond and the release of PTX from prodrugs. Based on this principle, the drug release rate was closely related to the distance of sulphur atom from the ester bond. As a result, α-PTX-SS-CIT prodrug, with the sulfur atoms located in the α-position of the ester bond, is the closest to the hydrophilic thiol, resulting in the fastest drug release profile. However, the release rate of PTX from γ-PTX-SS-CIT NPs was distinctly faster than that from β-PTX-SS-CIT NPs (Figure 2A-C). This would be due to that the generated thiol in γ-PTX-SS-CIT could form a five-member ring thiolactone through intramolecular nucleophilic acyl substitution on the ester moiety, and then such a performance facilitated the release of PTX (Figure 3A)

21, 22

. We also evaluated the

reduction-responsive drug release of prodrug nanoassemblies in GSH-containing medium. As shown in Figure S5, the disulfide bond-bridged prodrug nanoassemblies also exhibited GSHtriggered drug release. α-PTX-SS-CIT NPs and γ-PTX-SS-CIT NPs showed faster drug release than β-PTX-SS-CIT NPs, which are in good correspondence with the DTT-trigged drug release (Figure 2A-C). Although the reduction-responsive mechanism of disulfide bond is already clear, we further illuminate how the position of disulfide bond in the carbon chain (α-, β-, or γ) affects the reduction-responsive drug release of prodrug nanoassemblies.


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Figure 3. The mechanisms of redox dual-responsiveness of disulfide bond. (A): The mechanism of DTT-triggered drug release. (B) The mechanism of H2O2-triggered drug release. More interestingly, we found that the disulfide bond-bridged prodrug nanoassemblies also exhibited H2O2-triggered drug release property. As shown in Figure 2D-F, the oxidationresponsiveness of the prepared prodrug nanoassemblies followed the order of α-PTX-SS-CIT NPs > β-PTX-SS-CIT NPs > γ-PTX-SS-CIT NPs. α-PTX-SS-CIT NPs quickly released over 90% of PTX in the presence of 1 mM H2O2 within 6 h (Figure 2D). By contrast, the release of PTX from γ-PTX-SS-CIT NPs was quite slow, with only 35% of PTX released within 24 h even in the presence of 10 mM H2O2 (Figure 2F). To the best of our knowledge, this is the first time that the disulfide bond-bridged prodrugs are found to demonstrate oxidation-responsive drug


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release, and the mechanism is unclear. As shown in Figure 3B, we proposed that the mechanism of oxidation-responsiveness of disulfide bond was similar to that of thioether bond, through the oxidation of disulfide bond to hydrophilic sulfoxide or sulphone

19

. To confirm our hypothesis,

we investigated the change of high-precise molecular weight after incubating prodrug nanoassemblies with 10 mM H2O2-containing release media. The mass spectra showed that the molecular weight of β-PTX-SS-CIT changed from 1206.48810 [M+Na]+ to 1222.46333 [Mmonoxide+Na]+, 1238.48152 [Mdioxide+Na]+, and 1254.45497 [Mtrioxide+Na]+, confirming the formation of sulfoxide and sulphone (Figure S6B). The mass spectra of α-PTX-SS-CIT and γPTX-SS-CIT showed similar results, and the sulfoxide groups of prodrugs were found when incubated with 10 mM H2O2 (Figure S6A and S6C). The extremely hydrophilic sulfoxide or sulphone groups facilitated the hydrolysis of the adjacent ester bond. Therefore, the release rate of PTX was in inverse proportion to the distance of sulphur atom from the ester bond, and αPTX-SS-CIT NPs exhibited the fastest drug release profile (Figure 3B). The cytotoxicity of prodrug nanoassemblies were evaluated in human oral epidermoid carcinoma cells (KB), human pulmonary carcinoma cells (A549), and mouse breast cancer cells (4T1) using MTT assay. The half maximal inhibitory concentrations (IC50) values were calculated and summarized in Table S2. As shown in Figure 4A-B and S7, the cytotoxicities of β-PTX-SS-CIT NPs and γ-PTX-SS-CIT NPs were comparable but lower than that of Taxol, probably due to the delayed release of PTX from prodrug nanoassemblies. The similar cytotoxicities of β-PTX-SS-CIT NPs and γ-PTX-SS-CIT NPs could be attributed to their comparable drug release rate. By contrast, α-PTX-SS-CIT NPs exhibited a comparable cytotoxicity when compared with Taxol, which would be ascribed to the rapid drug release of PTX from α-PTX-SS-CIT NPs (Figure 4A-B). These results suggested that the in vitro


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cytotoxicity significantly depended on the release rate of PTX from the prodrug nanoassemblies, and faster drug release resulted in higher cytotoxicity. We then measured the released PTX from prodrug nanoassemblies after incubation with KB cells. As shown in Figure 4C-D and S8, much more PTX were released from α-PTX-SS-CIT NPs than β-PTX-SS-CIT NPs and γ-PTX-SS-CIT NPs at 24, 48 and 72 h when incubated with KB cells. This was well consistent with the in vitro release study, confirming that α-PTX-SS-CIT prodrug showed distinct advantage over β-PTXSS-CIT and γ-PTX-SS-CIT in terms of rapid redox dual-sensitive drug release property. To investigate the intracellular drug release mechanism, we measured the metabolic intermediates of α-PTX-SS-CIT prodrug in KB cells using high resolution MS. As shown in Figure S9, the ion peaks of released PTX, sulfoxide-containing monoxide and thiol-containing reduction intermediate of α-PTX-SS-CIT were found. These results further confirmed that both mechanisms (ROS-mediated oxidation and GSH-mediated reduction) were involved in the drug release of disulfide bond-bridged prodrugs in tumor cells.

Figure 4. Cytotoxicity assay and cellular uptake of prodrug nanoassemblies (n=3). Viability of KB cells after treated with various concentrations of Taxol and prodrug nanoassemblies for


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(A) 48 h and (B) 72 h. Free PTX released from prodrug nanoassemblies after incubation with KB cells for (C) 48 h and (D) 72 h. * P < 0.05, ** P < 0.01, *** P < 0.001. (E) Confocal laser scanning microscopy (CLSM) images of KB cells incubated with free coumarin-6 or coumarin6-labeled prodrug nanoassemblies for 2 h. (F) Cellular uptake in KB cells after incubation with free coumarin-6 or coumarin-6-labeled prodrug nanoassemblies for 0.5 and 2 h by flow cytometry. Difference from coumarin-6, ***P < 0.001. Furthermore, we investigated the cytotoxicity of Taxol and prodrug nanoassemblies against normal cells (mouse fibroblast 3T3 cells). As shown in Figure S7E-F and Table S2, both Taxol and prodrug nanoassemblies showed lower cytotoxicity on 3T3 cells when compared with tumor cells. The IC50 of Taxol against 3T3 cells was 40-fold higher than that against KB cells at 48 h. In comparison, the IC50 of prodrug nanoassemblies against 3T3 cells were 110-170-fold higher than that against KB cells. These results suggested that the disulfide bond-bridged prodrug nanoassemblies demonstrated more potent and selective cytotoxicity against tumor cells than normal cells, due to the overproduced GSH/ROS in tumor cells. The high selective cytotoxicity of disulfide bond-bridged prodrug NPs against tumor cells could reduce the adverse side effects. To investigate the cellular uptake of prodrug nanoassemblies, KB cells were incubated with free coumarin-6 or coumarin-6-labeled prodrug nanoassemblies for 0.5 h or 2 h. As shown in Figure 4E-F and S10, prodrug nanoassemblies treated cells exhibited significantly higher intracellular fluorescence intensity than that of free coumarin-6 treated cells. Therefore, the prepared prodrug nanoassemblies showed significantly higher cellular uptake efficiency than the free coumarin-6. Sprague-Dawley rats were used to evaluate the pharmacokinetic profiles of prodrug nanoassemblies following intravenous administration. The molar concentration-time curves of


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the prodrugs, the released PTX, and the sum of them were illustrated in Figure 5, and the pharmacokinetics parameters were calculated and summarized in Table S3. As shown in Figure 5C, PTX in Taxol was rapidly cleared from blood due to the short half-life. In comparison, the prodrug nanoassemblies displayed significantly prolonged circulation time than Taxol. The area under the curve (AUC) of α-PTX-SS-CIT NPs, β-PTX-SS-CIT NPs and γ-PTX-SS-CIT NPs was 8.4-fold, 11.9-fold, and 16.6-fold higher than that of Taxol, respectively (Table S3). Long systemic circulation could benefit the tumor accumulation of prodrug nanoassemblies through the well-known enhanced permeability and retention (EPR) effect. Furthermore, the position of disulfide bond (α, β or γ) had an influence on the pharmacokinetic behavior. γ-PTX-SS-CIT NPs showed a longer blood circulation time than α-PTX-SS-CIT NPs and β-PTX-SS-CIT NPs, but the AUC value of the released PTX from prodrug NPs followed the order of α-PTX-SS-CIT NPs > β-PTX-SS-CIT NPs > γ-PTX-SS-CIT NPs (Figure 5B). We further investigated the drug release of prodrug nanoassemblies in rat plasma. As shown in Figure S11, γ-PTX-SS-CIT NPs was most stable in rat plasma, with slower drug release rate compared with α-PTX-SS-CIT NPs and β-PTX-SS-CIT NPs.

Figure 5. Pharmacokinetic profiles of prodrug nanoassemblies (n=5). Molar concentrationtime curves of (A) the prodrugs, (B) the released PTX, and (C) the sum of them.


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DiR-labeled prodrug nanoassemblies were prepared to investigate the biodistribution of prodrug nanoassemblies in KB tumor bearing nude mice. As shown in Figure 6, free DiR showed high accumulation in lung, but negligible fluorescent signal was detected in tumor. In comparison, DiR-labeled prodrug nanoassemblies demonstrated significantly higher fluorescent intensity in tumor, and the fluorescent signals in tumors increased over time from 4 to 24 h. The increased tumor accumulation of prodrug nanoassemblies would be attributed to the long systemic circulation time and the EPR effect. In addition, the biodistribution of prodrug nanoassemblies were well in line with their pharmacokinetic behavior. γ-PTX-SS-CIT NPs and β-PTX-SS-CIT NPs, with longer circulation time, exhibited better tumor accumulation than αPTX-SS-CIT NPs.

Figure 6. Ex vivo biodistribution of DiR-labeled prodrug nanoassemblies (n=3). Fluorescent imaging at (A) 4 h and (C) 24 h. Quantitative results of relative organ and tumor accumulation at (B) 4 h and (D) 24 h.


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KB tumor bearing nude mice were utilized to evaluate the antitumor efficiency of prodrug nanoassemblies. As shown in Figure 7, Taxol exhibited a moderate tumor-inhibiting activity (~ 330 mm3) compared with the control group (saline, ~ 750 mm3). Notably, prodrug nanoassemblies showed more potent antitumor activity (< 200 mm3) than Taxol, with almost no growth in tumor volume. The improved antitumor activity of disulfide bond-bridged prodrug nanoassemblies should be attributed to the multiple therapeutic advantages, such as improved drug-loading, prolonged circulation time, enhanced tumor accumulation, facilitated cellular uptake, and efficient drug release in tumor site. Interestingly, α-PTX-SS-CIT NPs exhibited more potent tumor-inhibiting activity than β-PTX-SS-CIT NPs and γ-PTX-SS-CIT NPs, with significantly reduced tumor volume (~ 100 mm3). Although β-PTX-SS-CIT NPs and γ-PTX-SSCIT NPs displayed a little longer circulation time and better tumor accumulation, the superior redox dual-responsive drug release of α-PTX-SS-CIT NPs in tumor site led to a more potent chemotherapeutic efficacy. Therefore, the position of disulfide bond in the linkage significantly affected the antitumor performance of prodrug nanoassemblies, and α-disulfide bond demonstrated distinct advantage over β-disulfide and γ-disulfide in terms of tumor-specific ondemand drug release.


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Figure 7. In vivo antitumor efficacy of prodrug nanoassemblies against KB xenograft tumors. (A) Tumor growth profiles treated with different formulations. (B) Tumor burden after the last treatment. (C) Images of tumors after the last treatment. (D) Body weight changes. * P < 0.05, ** P < 0.01, *** P < 0.001. There was no significant change in body weight and hepatorenal function in all groups (Figure 7D and S12), and no obvious histological damages in H&E-stained tissue sections of major organs (heart, liver, spleen, lung, and kidney) were observed, suggesting that the disulfide bondbridged prodrug nanoassemblies showed good safety, with negligible nonspecific toxicity to major organs and tissues (Figure S13). However, the H&E staining tumor sections showed that


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prodrug nanoassemblies, especially the α-PTX-SS-CIT NPs group, had widespread apoptosis and necrosis of the cancer cells, suggesting a potent antitumor efficacy. To sum up, three novel PTX-CIT prodrugs were designed using different lengths of disulfide bond-containing carbon chain as linkages. The prepared prodrugs could self-assemble into uniform size NPs with impressive high drug loading (>55%). As we hypothesized, the disulfide bond-bridged prodrugs nanoassemblies demonstrated distinct redox dual-responsive capability. The reduction-responsive mechanism of disulfide bond is already clear, but the oxidationresponsiveness and relevant mechanism is unknown. We found that the sulphur atoms of disulfide bond could be oxidized to hydrophilic sulfoxide or sulphone in the presence of H2O2, therefore facilitating the oxidation-responsive drug release form the prodrug NPs. More interestingly, the position of disulfide bond in the carbon chain not only significantly affects the redox dual-responsiveness, but also has profound impacts on the drug release, cytotoxicity, pharmacokinetics, biodistribution and in vivo antitumor efficacy of prodrug nanoassemblies. αPTX-SS-CIT NPs, with higher redox dual-responsiveness and faster tumor-specific drug release, showed more potent antitumor activity than β-PTX-SS-CIT NPs and γ-PTX-SS-CIT NPs. This is the first time that disulfide bond bridged prodrugs were found to be redox dual-responsive, and how the position of disulfide bond in the carbon chain affects the redox dual-responsiveness and antitumor efficiency of prodrug nanoassemblies was also illuminated for the first time. Our findings give new insight into the stimuli-responsiveness of disulfide bond and provide possibilities for the development of novel redox dual-responsive DDS for cancer therapy.

ASSOCIATED CONTENT


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Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.; 14 figures and 3 tables; 1H NMR and MS spectra of new compounds. AUTHOR INFORMATION Corresponding Author * [email protected]; Tel: +86-024-23986321; Fax: +86-024-23986321. * [email protected]; Tel: +86-024-23986325; Fax: +86-024-23986325. Author Contributions ⊥

These authors contributed equally.

Notes There are no conflicts of interest to declare. ACKNOWLEDGMENT This work was financially supported by the National Basic Research Program of China (973 Program, no. 2015CB932100), National Natural Science Foundation of China (no. 81703451, 81573371, 81773656), and a China Postdoctoral Science Foundation Grant (no.2017M611269). ABBREVIATIONS DDS, drug delivery system; NPs, nanoparticles; PTX, paclitaxel; nano-DDS, nanoparticulate drug delivery systems; NPs, nanoparticles; ROS, reactive oxygen species; GSH, glutathione; CIT, citronellol; MS, mass spectrum; FBS, fetal bovine serum; DTT, dithiothreitol; KB cells, human oral epidermoid carcinoma cells; A549 cells, human pulmonary carcinoma cells; 4T1 cells,


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mouse breast cancer cells; IC50, half maximal inhibitory concentrations; AUC, area under the curve; EPR, enhanced permeability and retention; CLSM, confocal laser scanning microscopy; REFERENCES (1) Smith, R. A.; Andrews, K. S.; Brooks, D.; Fedewa, S. A.; Manassaram-Baptiste, D.; Saslow, D.; Brawley, O. W.; Wender, R. C. CA Cancer J. Clin. 2017, 67, 100-121. (2) Bocci, G.; Kerbel, R. S. Nat. Rev. Clin. Oncol. 2016, 13, 659-673. (3) Chen, Q.; Liu, G.; Liu, S.; Su, H.; Wang, Y.; Li, J.; Luo, C. Trends Pharmacol. Sci. 2018, 39, 59-74. (4) Howat, S.; Park, B.; Oh, I. S.; Jin, Y. W.; Lee, E. K.; Loake, G. J. N. Biotechnol. 2014, 31, 242-245. (5) Wang, L. Asian J. Pharm. Sci. 2017, 12, 470-477. (6) Sun, B.; Luo, C.; Cui, W.; Sun, J.; He, Z. J. Control. Release 2017, 264, 145-159. (7) Luo, C.; Sun, J.; Sun, B.; He, Z. Trends Pharmacol. Sci. 2014, 35, 556-566. (8) Walther, R.; Rautio, J.; Zelikin, A. N. Adv. Drug Deliv. Rev. 2017, 118, 65-77. (9) Cheetham, A. G.; Chakroun, R. W.; Ma, W.; Cui, H. Chem. Soc. Rev. 2017, 46, 6638-6663. (10) Mura, S.; Zouhiri, F.; Lerondel, S.; Maksimenko, A.; Mougin, J.; Gueutin, C.; Brambilla, D.; Caron, J.; Sliwinski, E.; Lepape, A.; Desmaele, D.; Couvreur, P. Bioconjug. Chem. 2013, 24, 1840-1849. (11) Dosio, F.; Reddy, L. H.; Ferrero, A.; Stella, B.; Cattel, L.; Couvreur, P. Bioconjug. Chem. 2010, 21, 1349-1361. (12) Bradley, M. O.; Webb, N. L.; Anthony, F. H.; Devanesan, P.; Witman, P. A.; Hemamalini, S.; Chander, M. C.; Baker, S. D.; He, L.; Horwitz, S. B.; Swindell, C. S. Clin. Cancer Res. 2001, 7, 3229-3238.


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(27) Xiao, C.; Ding, J.; Ma, L.; Yang, C.; Zhuang, X.; Chen, X. Polym. Chem. 2015, 6, 738-747. (28) Klis, W. A.; Sarver, J. G.; Erhardt, P. W. Tetrahedron Lett. 2001, 42, 7747-7750.


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Chemical structures of the disulfide bond-bridged PTX-CIT prodrugs: α-PTX-SS-CIT, β-PTX-SS-CIT, and γPTX-SS-CIT. 83x75mm (300 x 300 DPI)


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Schematic representation of the disulfide bond-bridged prodrugs nanoassemblies and the redox dualresponsive drug release in tumor cells. The nanoassemblies were formed by PTX-SS-CIT prodrugs themselves, and DSPE-PEG2k was utilized to prepare PEGylated prodrug NPs. After prodrug NPs were delivered into tumor cells, on-demand drug release could be achieved through the redox dual-responsive capability of disulfide bond in the presence of overproduced ROS and GSH in tumor cells. 170x111mm (300 x 300 DPI)


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In vitro redox dual-responsive drug release of prodrug nanoassemblies in the presence of various concentrations of DTT or H2O2 (n=3). (A): 1 mM DTT; (B): 5 mM DTT; (C): 10 mM DTT; (D): 1 mM H2O2; (E): 5 mM H2O2; (F): 10 mM H2O2. 150x85mm (300 x 300 DPI)


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The mechanisms of redox dual-responsiveness of disulfide bond. (A): The mechanism of DTT-triggered drug release. (B) The mechanism of H2O2-triggered drug release. 85x124mm (300 x 300 DPI)


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Cytotoxicity assay and cellular uptake of prodrug nanoassemblies (n=3). Viability of KB cells after treated with various concentrations of Taxol and prodrug nanoassemblies for (A) 48 h and (B) 72 h. Free PTX released from prodrug nanoassemblies after incubation with KB cells for (C) 48 h and (D) 72 h. * P < 0.05, ** P < 0.01, *** P < 0.001. (E) Confocal laser scanning microscopy (CLSM) images of KB cells incubated with free coumarin-6 or coumarin-6-labeled prodrug nanoassemblies for 2 h. (F) Cellular uptake in KB cells after incubation with free coumarin-6 or coumarin-6-labeled prodrug nanoassemblies for 0.5 and 2 h by flow cytometry. Difference from coumarin-6, ***P < 0.001. 150x80mm (300 x 300 DPI)


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Pharmacokinetic profiles of prodrug nanoassemblies (n=5). Molar concentration-time curves of (A) the prodrugs, (B) the released PTX, and (C) the sum of them. 150x39mm (300 x 300 DPI)


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Ex vivo biodistribution of DiR-labeled prodrug nanoassemblies (n=3). Fluorescent imaging at (A) 4 h and (C) 24 h. Quantitative results of relative organ and tumor accumulation at (B) 4 h and (D) 24 h. 140x99mm (300 x 300 DPI)


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In vivo antitumor efficacy of prodrug nanoassemblies against KB xenograft tumors. (A) Tumor growth profiles treated with different formulations. (B) Tumor burden after the last treatment. (C) Images of tumors after the last treatment. (D) Body weight changes. * P < 0.05, ** P < 0.01, *** P < 0.001. 150x119mm (300 x 300 DPI)


Disulfide Bond-Driven Oxidation- and Reduction-Responsive Prodrug

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