Enzyme and Redox Dual-Triggered Intracellular Release from Actively

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Enzyme and Redox Dual-Triggered Intracellular Release from Actively Targeted Polymeric Micelles Lei Zhang, Yi Wang, Xiaobin Zhang, Xiao Wei, Xiang Xiong, and Shaobing Zhou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14078 • Publication Date (Web): 10 Jan 2017 Downloaded from http://pubs.acs.org on January 11, 2017

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ACS Applied Materials & Interfaces

Enzyme and Redox Dual-Triggered Intracellular Release from Actively Targeted Polymeric Micelles

Lei Zhang, Yi Wang, Xiaobin Zhang, Xiao Wei, Xiang Xiong, Shaobing Zhou* Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China

Abstract Highly effective delivery of therapeutic agents into target cells using nanocarriers and subsequently rapid intracellular release are of great importance in cancer treatment. Here, we developed an enzyme and redox dual-responsive polymeric micelle with active targeting abilities to achieve rapid intracellular drug release. To overcome both its poor solubility in water and instability in the blood circulation, camptothecin (CPT) was chemically conjugated to monomethyl poly(ethylene glycol) (mPEG) via a redox-responsive linker to form polymeric prodrugs. The enzyme-responsive function is achieved by connecting hydrophobic polycaprolactone segments and hydrophilic PEG segments with azo bonds. Additionally, the end of the PEG segment was decorated with phenylboronic acid (PBA), endowing the nanocarriers with active targeting abilities. The dual-responsive targeting polymeric micelles can be generated by self-assembly of a mixture of the polymeric prodrug and enzyme-responsive copolymer. The in vitro drug release profile revealed that CPT was rapidly released

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from the micelles under a simulated condition similar to the tumor cell microenvironment. In vivo and ex vivo fluorescence imaging indicated that these micelles possess excellent specificity to target hepatoma carcinoma cells. The antitumor effect in mice liver cancer cells (H22) tumor-bearing Kunming (KM) mice demonstrated that this nanocarrier exhibits high therapeutic efficiency in artificial solid tumors and low toxicity to normal tissues, with a survival rate of approximately 100% after 160 days of treatment. Keywords: Enzyme-responsive; redox-responsive; nanocarrier; intracellular release; active-targeting

INTRODUCTION Chemotherapy has been widely used in cancer treatment; however, its serious side effects and low therapeutic efficiency set certain limits on the use of chemotherapeutics in clinical treatment. To overcome these shortcomings, nanoparticle (NP)-based drug delivery systems, which are capable of delivering their toxic cargos specifically into cancer cells, have drawn more and more attention.1-4 However, improving the accumulation of nanoparticles at the tumor site and subsequent rapid release of the drug cargo in tumor cells remain great challenges. Therapeutic agents are generally delivered using nanocarriers through two approaches: non-targeted delivery (passive targeting) and targeted delivery (active targeting). Passive targeting mainly takes advantage of the tumor blood vessels, which possess larger gaps between adjacent endothelial cells, and drug carriers less than 100


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nm are capable of passing through these spaces by the well-known enhanced permeation and retention (EPR) effect.5-8 In contrast, active targeting aims to take advantage of both the EPR effect in the tumor site and specific targeting of cancer cells. In general, active targeting requires grafting of targeting ligands onto the surface of nanocarriers for binding to a cell surface biomarker or receptor that is overexpressed in cancer cells. The ligands mainly consist of folic acid (FA), biotin, cyclic peptides including Arg-Gly-Asp (RGD) sequences and riboflavin.9-14 Here, phenylboronic acid (PBA) was selected as a targeting ligand and can recognize the sialic acid that is overexpressed on certain tumor cells such as hepatoma carcinoma cells.15-17 PBA-decorated nanocarriers can actively target tumor cells through multivalent binding between the nanocarrier surface and tumor cell membrane. After the nanocarriers enter tumor cells, the payloads must be rapidly released before the therapeutic agents can exhibit activity in tumor cells. To realize rapid release in the lesion, environment-responsive groups are frequently introduced into nanocarriers, which can cause the nanocarriers to disintegrate in a specially controlled manner upon the application of external stimuli.18-21 Among these stimuli, enzyme-responsiveness is of particular importance in drug delivery, owing to the fact that it provides both specificity and efficiency. Enzyme-responsive carriers have been widely used for delivering drugs.22 For example, matrix metalloproteinases can cleave extracellular matrices such as collagen, glycoproteins and proteoglycans,23 Phospholipases, cancer-associated proteases, kinases and acetyltransferases have also been studied for triggering drug release.24-26 However, the use of these enzymes still



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involves certain problems, including complexity, difficulty in synthesizing or extracting the substrate and resulting high costs, which greatly limits the application of enzyme-response nanocarriers. Azoreductase, which is an oxidation-reduction enzyme in hepatocellular microsomes, can cleave azo bonds in the presence of the coenzyme reduced nicotinamide adenine dinucleotide phosphate (NADPH).27-30 The azo bonds can be utilized as a functional group of the nanocarrier, which has a capacity of specifically responding to the azoreductase in the microsomes of hepatocellular carcinoma cells, and working as a trigger to induce the disintegration of the nanocarrier. Moreover, the synthesis of azobenzene compounds is very easy and cheap.31 Based on these thoughts, we design an amphiphilic azoreductase-specific enzyme-responsive copolymer with a combination of hydrophilic polyethylene glycol (PEG) and hydrophobic polycaprolactone (PCL) polymers connected via azo bonds. Accordingly, in this study, we developed an enzyme and redox dual-responsive polymeric micelle with active targeting abilities for achieving rapid intracellular drug release in cancer treatment. Camptothecin (CPT), which is an inhibitor of DNA topoisomerase I,32, 33 was used as a drug model. The clinical application of free CPT is often limited by the drug’s poor solubility in water and its low efficiency.34 In contrast to other types of hydrophobic drugs, it is still a challenge to physical encapsulation of CPT in the amphiphilic polymeric nanocarriers, because CPT has a intrinsically planar structure and moderate polarity that are both unfavorable to the encapsulation.35-37 To address this problem, CPT was chemically conjugated to mPEG via a redox-responsive linker to form polymeric prodrugs. Redox-responsive behavior


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is realized via the breakage of the disulfide bonds in the presence of a high concentration of glutathione (GSH).38-40 A great difference in the GSH concentration is found approximately 2-10 mM in the intracellular space and approximately 2-20 μM in extracellular space.18-21 The enzyme-responsive function is achieved by connecting the hydrophilic PCL segments and hydrophobic PEG segments with azo bonds. Additionally, PBA was grafted to the ends of the PEG segments, endowing the nanocarriers with active targeting abilities. The dual-responsive targeting polymeric micelle can be generated via self-assembly of a mixture of the redox-responsive prodrug, monomethyl poly(ethylene glycol)-ss-camptothecin (mPEG-ss-CPT) the

PBA-functionalized

enzyme-responsive

copolymer,

and

PBA-poly(ethylene

glycol)-4,4'-(diazene-1,2-diyl)benzoyl- poly(ε-caprolactone) (PBA-PEG-Azo-PCL) (Scheme 1). Via receptor-mediated endocytosis, the micelle can selectively enter tumor cells. Subsequently, the therapeutic agent can be rapidly released through the dual triggers of enzyme- and redox-responsiveness in the cytoplasm, and finally, both a high inhibitory effect on tumor cells and low side effects to normal tissues can be achieved. RESULTS AND DISCUSSION Characterization of the Multifunctional Polymers. The enzyme-responsive polymer PBA-PEG-Azo-PCL and the redox-responsive prodrug mPEG-ss-CPT were synthesized through a series of reactions shown in Figure 1A. The 1H NMR spectra of the intermediate products are shown in Figure S1-S3 in the Supporting Information (SI). The chemical structures of both PBA-PEG-Azo-PCL (Figure 1B) and



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mPEG-ss-CPT (Figure 1C) were confirmed by 1H NMR. The ratio of PEG and PCL in the PBA-PEG-Azo-PCL polymer was 0.67, which was obtained by calculating the number-average molecular weight (Mn) of PEG and PCL from the Figure 1B by integrating “peak l and f”. Based on these calculations, the number-average molecular weight (Mn) of PBA-PEG-Azo-PCL was found to be approximately 5.3 kDa, which is almost in agreement with the Mn value of 5.1 kDa from gel permeation chromatograph (GPC) measurement, with a polydispersion index (PDI) of 1.25 (Figure 2E). In addition, by integrating “peak a” and “peak c” which were the characteristic peaks of PBA, we obtained that the density of targeting ligand PBA on the polymeric micelles was 0.6. The density of PBA is not optimized, because when the density of the target group is more than 10%, the targeting effect would be effective.41-43 The grafting efficiency of CPT in the mPEG-ss-CPT was 87%, calculated from the 1H NMR results of Figure 1C. Characterization PBA-PEG-Azo-PCL

of

Micelles. and

The

blank

enzyme-responsive

dual-responsive

micelles micelles

PBA-PEG-Azo-PCL/mPEG-ss-CPT were prepared through the solvent evaporation method.44 The core-shell structure of these micelles was confirmed by 1H NMR in D2O. As shown in Figure 2A, C, the characteristic peaks of the hydrophobic PCL block and CPT disappeared; however, the peaks of both the active targeting ligand PBA and PEG still remained. This result indicates that the copolymers formed micelles with PCL and CPT in the core and PEG and PBA in the shell. The average size of both the blank enzyme-responsive micelles and dual-responsive micelles


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determined by dynamic light scattering measurement (DLS) was 127±5 nm (Figure 2B, D). The transmission electron microscopy images (TEM) in Figure 2E, F display that the micelles have well spherical shape with an average size of approximately 96 nm. According to the high-performance liquid chromatography (HPLC) results, the drug loading content was 7.0%, and the drug loading efficiency was 91.7%. The stability of the micelle solution was also investigated by testing the particle size change in phosphate buffer saline (PBS) at pH 7.4 for 7 days, and from the Figure S4 in SI, we can find that the particle size has little change, suggesting that the micelles can be stable in PBS for a long time. Dual-Responsive Behaviors and In Vitro Drug Release. To demonstrate whether azo bonds could be cleaved by the azoreductase and coenzyme NADPH, we incubated the enzyme-responsive micelles with NADPH (25 mmol/L) and liver microsomes (1 mg/mL) to mimic the tumor tissue microenvironment

29

for 5 h. Both

GPC and 1H NMR measurements were used to confirm the response process. As shown in the inserted photos in Figure 3A and B, the emulsion of the enzyme-responsive micelles was yellow at the initial stage. After liver microsomes and NADPH were added, the emulsion was gradually converted into a colorless, transparent solution, and some white flocculation was produced. 1H NMR analysis indicates that the white flocculation only contained a PCL characteristic peak (Figure 3B), while the original PBA-PEG-Azo-PCL not only exhibited a PCL characteristic peak but also had a PEG characteristic peak (Figure 3A). The results directly proved that the enzyme-responsive linker’s azo bonds were cleaved by the azoreductase and



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NADPH. The molecular weight of the remaining copolymer, as determined by GPC, also decreased sharply after exposure to azoreductase and NADPH (Figure 3D). To further study the dual-responsive behaviors of the micelles, the diameters of the micelles were determined by DLS. As shown in Figure 3C, the micelles possess an average diameter of 127 nm at the initial time, however, in the presence of only liver microsomes and NADPH, the average diameter was changed to 112 nm, which may be owing to the fact that the azo bonds in the PBA-PEG-Azo-PCL copolymers can be quickly cleaved as demonstrated in Figure 3A and B, leading to the formation of smaller micelles. These results suggested that the enzyme-responsive polymer exhibited highly sensitive tumor microenvironment-responsiveness. Additionally, when the micelles were placed in the presence of only GSH (10 mM), only the prodrug can be fractured due to the disulfide bonds, and the average diameter (205 nm) of micelles shows a slight change. Furthermore, in the presence of GSH (10 mM), liver microsomes and NADPH, the average size of the micelles obviously increased, and the size distribution changed to a bimodal model. These changes in the average micellar size directly prove the quick dual-response characteristics of the micelles. These responsive behaviors can be explained by a series of chemical reactions, as shown in Figure 2E. In the presence of azoreductase and NADPH, the azo bonds of PBA-PEG-Azo-PCL are cleaved to produce hydrophilic PEG and hydrophobic PCL.27-29 Simultaneously, the disulfide bonds of the mPEG-ss-CPT prodrug are cleaved with GSH attack to form sulfhydryl groups at the end of CPT, the sulfhydryl further attacks the carbonyl carbon due to its strong nucleophilicity. Thus, the


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resulting thiol undergoes an intramolecular nucleophilic substitution and in turn removes a five-membered ring thiolactone.45-47 Moreover, the released CPT molecules recover their original chemical structure, which is of great importance for the therapeutic agent to keep its activity. The in vitro CPT release behaviors of the micelles were studied at pH 7.4 using a dialysis approach (Figure 3F). The release profiles indicate that the amount of drug released within 48 h is less than 10% in both of the condition of 10 µM GSH similar with the blood circulation environment,48 and the condition of 10 µM GSH and azoreductase simultaneously. When the GSH concentration was increased to 10 mM, the amount of CPT release increase slightly to approximately 30%, which can be ascribed to the fact that the micellar structure is generally maintained due to intact azo bonds. However, in the presence of both 10 mM GSH and azoreductase, which is similar to the tumor cell microenvironment, the CPT release reached approximately 80% in 48 h because the azo bonds were ruptured and the micelles subsequently disassembled, resulting in the rapid drug release. Therefore, the in vitro release result indicates that these micelles can exhibit good stability in blood circulation but rapidly release their cargo inside tumor cells. In Vitro Cytotoxicity Assay. The in vitro cytotoxicity assay proved the safety of the materials. As Figure 4A shows, even when treated with 500 µg/mL of blank micelles, both the normal cells epithelial cell (EC) and tumor cells (HepG2) maintained a survival rate greater than 80%. Calcein dyeing further confirmed that the two cell types grew healthily after incubation with a series of different concentrations



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of micelles (Figure 4C). When HepG2 cells were cultured with different micelles for 48 h, the cell viability presented dose-dependent effects (Figure 4B). The half-maximal inhibitory concentration

(IC50)

values

for

PBA-PEG-Azo-PCL/mPEG-ss-CPT,

PBA-PEG-PCL/mPEG-ss-CPT and free CPT were 8.4, 20.7, and 5.1 µg/mL, respectively. Since the drug release profiles of these groups are different, and the diverse cytotoxicity levels were exhibited. For a single-response micelle, the amount of CPT released from the micelles is very low. Under the dual response conditions, the azo bonds of PBA-PEG-Azo-PCL/mPEG-ss-CPT are ruptured in response to enzymatic activity, leading to the disintegration of micelles, and the disulfide bonds are rapidly cleaved in response to GSH, which in turn results in the release of most of the CPT. Compared with the free CPT, dual-responsive micelles generated a higher IC50 due to the PBA-sialic-acid-mediated internalization which increased cellular uptake of the micelles. In Vitro Cellular Uptake of Dual-Responsive Micelles. In order to investigate the cellular internalization, HepG2 cells were co-culture with free CPT and dual-responsive micelles for different time intervals (1, 3, and 6 h). The behaviors were observed with confocal laser scanning microscopy (CLSM), as shown in Figure 5. Lysosomes were stained green, and the blue fluorescent areas were labeled by CPT due to its autofluorescence. Over 3 h, the fluorescence intensity increased in a time-dependent manner for all samples; moreover, the fluorescence in the PBA ligand dual-responsive micelle group was the strongest due to active targeting. At 6 h, the


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PBA ligand dual-responsive micelle group still exhibited the strongest fluorescence, but the fluorescence of the free CPT group was obviously decreased. This effect can be ascribed to the fact that the small molecular drugs were likely effluxed from the cells. According to the flow cytometry results (Figure 5D), at 1 h, the cellular uptake of the PBA ligand group was equal to that of the no PBA ligand group but less than that of the free CPT group. At 6 h, the cellular uptake of the PBA ligand group was greater than that of the no PBA ligand group. Therefore, micelles modified by PBA ligands can be concluded to effectively enhance cellular internalization. In Vivo and Ex Vivo Fluorescence Imaging. To visualize the in vivo delivery of the micelles directly, different groups of Nile red-loaded micelles were injected into nude mice bearing H22 tumors and then the fluorescence intensity of Nile red was monitored at different sites of the body at different times by ex vivo imaging. In Figure 6A, the PBA ligand-conjugated micelles exhibit stronger fluorescence at the tumor site at each time point, and at 6 h, only this group demonstrated tumor site fluorescence. Ex vivo imaging in Figure 6B shows that the tumor site fluorescence of the PBA ligand-conjugated micelles was also the strongest in all of these groups; moreover, little fluorescence can be found in any normal tissues. The average Nile red fluorescence intensity analysis performed using Maestro Ex Pro (CRI, USA) is shown in Figure 6C. The fluorescence intensity of the actively targeted micelle group at the tumor site was found to be 1.84-fold higher than that of the non-targeted micelle group and 3.56-fold that of the free Nile red group. In the liver, lung and kidney tissues, the fluorescence intensity of the free Nile red group was approximately twice



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as much as that of the other two groups.49, 50 All of these results indicate that the PBA-PEG-Azo-PCL/mPEG-ss-CPT micelles can accumulate at the tumor site instead of normal tissue through its active targeting function. In Vivo Anticancer Activity Evaluation and Histological Analysis. To further evaluate the anticancer efficacy of the dual-responsive micelles in vivo, H22-bearing KM mice were injected through the tail vein with saline, blank micelles, PBA-PEG-Azo-PCL/mPEG-ss-CPT, PBA-PEG-PCL/mPEG-ss-CPT or free CPT at an equivalent CPT dose of 5 mg/kg on days 0, 2, 4, and 6. Anticancer efficacy and toxicity were evaluated through tumor volume changes (Figure 7A, B), body weight changes (Figure 7C), survival rates (Figure 7D), and histological analyses (Figure 7E and Figure S5 in SI). The PBA-PEG-Azo-PCL/mPEG-ss-CPT group demonstrated the best therapeutic efficacy against the tumor and minimal side effects to normal tissues. As clearly observed in Figure 7A, B, the tumor volume at day 21 remains almost unchanged and is the smallest for the dual-responsive, actively targeted micelle group. The tumor inhibition rates calculated based on the tumor volume at day 21 for the dual-response micelle, free CPT and enzyme-responsive micelle group are approximately 85.3%, 57.9% and 44.2%, respectively, indicating that the dual-responsive micelles exhibited a good therapeutic effect on an artificial solid tumor. Even at 160 days post-treatment, the survival rate for this group remained approximately 100%, whereas almost all of the animals from the other groups died, further indicating that this micelle demonstrates a strong ability to selectively deliver CPT to tumor cells and simultaneously reduce its side effects on normal tissues.


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At day 14 after the first injection, the tumor tissue and main normal tissues (heart, liver, spleen, lung and kidney) were excised from the H22-bearing mice treated with blank micelles, saline and the other three CPT formulations for further histological analyses. From the hematoxylin and eosin (H&E) staining of the normal tissues in Figure S5 in SI, we observed that the free CPT group exhibited obvious renal toxicity, but for the PBA-PEG-Azo-PCL/mPEG-ss-CPT group, little toxicity to normal tissues was found because this micelle can reduce the side effects of CPT.51-52 As shown in the H&E staining of the tumor in Figure 7E and Figure S6, the tumor cells treated with any CPT formulation, but especially with PBA-PEG-Azo-PCL/mPEG-ss-CPT, became smaller and demonstrated nuclear lysis, compared with the saline and blank micelle groups, suggesting that the dual-responsive, actively targeted micelles generate a highly effective treatment response in tumors. TUNEL images were further employed to observe the apoptosis of the tumor cells, and the dark brown color represents apoptotic cells. From these images, we can clearly observe that the PBA-PEG-Azo-PCL/mPEG-ss-CPT group induced the highest rate of apoptosis. Moreover, the expression of Ki-67 (a marker of cell proliferation) was also examined to evaluate the anticancer effect of the various treatments. Positive expression of Ki-67 is indicated by a light yellow or tan color and negative expression by a blue color.53, 54 In the Ki-67 images, the PBA-PEG-Azo-PCL/mPEG-ss-CPT resulted in the lowest proliferation rate. CD31 immunohistochemical staining was also employed to investigate the formation of tumor vessels, and the result indicates that the tumor tissue treated with PBA-PEG-Azo-PCL/mPEG-ss-CPT contained fewer vessels.55, 56



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All of the results confirm that the dual-responsive, actively targeted micelles demonstrate the best treatment effect against tumor cells and minimal side effects on normal tissues. CONCLUSIONS In summary, we present an enzyme and redox dual-responsive polymeric micelle with active targeting abilities for achieving rapid intracellular drug release in cancer treatment. The poorly soluble anticancer drug CPT was highly effectively encapsulated into the polymeric micelles through PEGylation of CPT. The presence of active targeting ligands provided the micelles with the ability to be selectively taken up

by

tumor

cells.

These

micelles

exhibit

highly

sensitive

tumor

microenvironment-responsiveness in that the azo bonds in their polymer backbone are first cleaved in response to azoreductase, leading to micelle disintegration; subsequently, the polymeric prodrugs linked with disulfide bonds are transformed into therapeutic agents in the presence of a high concentration of glutathione, allowing rapid intracellular drug release. Both the in vitro and in vivo experiments confirm that this multifunctional nanocarrier can selectively deliver therapeutic agents to target cells and exhibits highly effective treatment of cancer and minimum side effects to normal tissues. Therefore, this nanocarrier provides a novel drug delivery platform for safe and highly effective cancer therapy. EXPERIMENTAL SECTION Materials.

Monomethyl

poly(ethylene

glycol)


(mPEG,

MW=2.0

kDa),

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poly(ethylene glycol) (PEG, Mw=2.0 kDa) was purchased from Sigma Aldrich (USA). Dicyclo-hexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), glutathione (GSH), 2, 2’-dithiodiethanol and ε-Caprolactone (CL) were all purchased from Adamas-beta. 3-aminobenzeneboronic acid (PBA) was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Camptothecin (CPT) was purchased from Lanbeizhihua (Chengdu, China). All the other chemicals were purchased from commercial supplier and used without further purification. Cell Culture and Animals. HepG2 cells were cultured in RPMI-1640 and EC cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), all the medium were mixed with 10% newborn calf serum. All the cells were cultured under fully humidified conditions at 37 ℃ and 5% CO2. H22 cells were harvested from the ascites of KM mouse, used PBS solution to prepare the cells suspension of 2×106 cells mL-1. Then, tumor cells suspension (0.15 mL) was underarm injected into female KM mice (purchased from Experimental Animal Center of Sichuan University), which were 25±3 g . After a week feeding, H22 cancer model was built. Animal experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University and all protocols for this animal study conformed to the Guide for the Care and Use of Laboratory Animals. Synthesis of 4,4’-(diazene-1,2-diyl) Dibenzoic Acid.31 p-Nitrobenzoic acid (3.25 g, 19.5 mmol), NaOH (12.5 g, 312.5 mmol) and 120 mL water were added to a 500-mL three-necked bottle. After adequate stirring, the temperature was raised to 50°C under nitrogen protection. Glucose (25 g, 113.75 mmol) was dissolved in 75 mL



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water, dripped into the three-necked bottle with a constant pressure funnel over 30 minutes and reacted for 8 h at 50°C. After being cooled to room temperature (RT), the reaction was neutralized to around pH=6 with dilute acetic acid. The solid was dissolved in hot potassium carbonate and was washed with acetic acid to acidification. The mixture was filtered, the filter residue dried under vacuum to obtain the palm red solid. Yield: 63%. 1H

NMR (400 MHz, D2O): δ 7.93 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H). Synthesis of N-Boc-ethylenediamine. Ethylenediamine (14 mL, 200 mmol) and

200 mL dichloromethane (DCM) were added to a 500-mL three-necked bottle. Di-tert-butyl pyrocarbonate (4.37 g, 20 mmol) in DCM (100 mL) was slowly added to the reaction over 3 h with a constant pressure drop funnel and stirred for 6 h in an ice bath at 0°C. Afterwards, filtered off, and used 50 mL saturated brine to wash the filtrate for 3 times. The organic layer was dried over by anhydrous MgSO4 and was subsequently evaporated to remove the organic solvent to obtain the colorless oily matter. Yield: 85%. 1H

NMR (400 MHz, CDCl3): δ 5.01 (s, 1H), 3.14 (dd, J = 11.0, 5.3 Hz, 2H), 2.76 (t,

J = 5.9 Hz, 2H), 1.42 (d, J = 9.6 Hz, 9H). Synthesis of ss-CPT.

57

CPT (1 g, 2.87 mmol), bis(trichloromethyl)carbonate

(0.32 g, 1.08 mmol) and DMAP (1.12 g, 9.2 mmol) were added to 100 mL of anhydrous

DCM

under

nitrogen

protection.

After

stirring

for

30

min,

2,2’-dithiodiethanol (4.43 g, 28.7 mmol) in anhydrous tetrahydrofuran (THF) (10 mL)


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was slowly added to the reaction over 30 min with a constant pressure drop funnel and then stirred overnight at RT. After Thin-layer Chromatography (TLC), to confirm whether the CPT was disappeared, the reaction solution was washed with 150 mL HCl (0.1 M) solution for 3 times, 50 mL saturated brine for 1 time and 50 mL distilled water for 1 time. Then, dried over by anhydrous MgSO4, filtered, and the filtrate was concentrated. The residue was purified by recrystallization from chloroform and methanol. Finally, the light yellow solid was obtained. Yield: 52%. 1H

NMR (400 MHz, CDCl3): 8.43 (s, 1H), 8.23 (d, J = 8.5 Hz, 1H), 7.96 (J = 8.2

Hz, 1H), 7.85 (tt, J = 9.1,4.6 Hz, 1H), 7.72-7.66 (m, 1H), 7.45 (s, 1H), 5.71 (d, J = 17.3 Hz, 1H), 5.43-5.28 (m, 3H), 4.37 (m, 2H), 3.92 (m, 2H), 2.91 (m, 4H), 2.23 (m, 2H), 1.02 (t, J = 7.5 Hz, 3 H). Synthesis

of

N-Boc-PCL.44

The

N-Boc-PCL

was

synthesized

by

N-Boc-ethylenediamine (0.5 g, 3.125 mmol), ε-CL (12 g, 107.14 mmol) monomer and SnCl2 (0.25 g, 2 wt%) at 140 °C for 8 h. The resultant polymer was dissolved in dichloromethane, used the excess cold ethanol to precipitate the products and collected by filtration. Then, the light white solid was dried. Yield: 82%. Synthesis

of

Carboxy-terminated

Poly(ethylene

glycol)

(COOH-PEG-COOH).58 10 g of PEG (5 mmol), 3 g of succinic anhydride (30 mmol) and 150 mL of chloroform were added to a 500-mL three-necked bottle, after stirring to dissolve, 0.6g of DMAP (5 mmol) added to the reaction, and then, heated to reflux under nitrogen protection for 48 h. The resultant solution was washed with



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saturated NH4Cl aqueous solution and brine, and then, used anhydrous MgSO4 to dry the organic layer. The solvent was removed in vacuo and precipitated in excess cold ether to obtain the white solid. Yield: 78%. Synthesis of PBA-PEG. COOH-PEG-COOH (8 g, 3.63 mmol), PBA (0.549 g, 4 mmol), 0.989 g of DCC (4.8 mmol), 0.443 g of DMAP (3.63 mmol) and 50 mL of DMF were added in to the bioreactor were dissolved in and reacted for 48 h at RT. Then, quenched the reaction by adding 5 mL of deionized water.

After that, filtered,

and evaporated the filtrate under reduced pressure. Finally, precipitated in cold ether to obtain the product. Yield: 75%. Synthesis of PBA-PEG-Azo. 6 g of PBA-PEG (2.6 mol), 0.74 g of DCC (3.6 mmol), N-Boc-ethylenediamine (0.483 mmol) and 0.318 g of DMAP (2.6 mmol) were dissolved in 50 mL of DCM. The reaction was reacted for 48 h at RT. Then, added 5 mL of deionized water to the solution to quench the reaction. Filtered, and the filtrate was evaporated by rotary evaporator and dissolved in 50 mL of THF. Later, 1 M HCl (5 mL) was added and reacted at RT for 3 h, after which trimethylamine (1 mL) was added to the resultant solution. Then, filtered, and concentrated of the filtrate in vacuo and dissolved residue in 50 mL of DMF. Afterwards, 2.16 g of 4,4’-(diazene-1,2-diyl) dibenzoic acid (8 mmol), 0.308 g of DMAP (2.6 mmol) and 0.74 g of DCC (3.6 mmol) were added to the mixture and reacted for 48 h at RT. Then, 5 mL of deionized water was added. Filtered, and the filtrate was concentrated by rotary evaporation. The concentrated viscous mixture was poured into cold ether to precipitate the palm red solid. The reaction was monitored by TLC. Yield: 61%.


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Synthesis of PBA-PEG-Azo-PCL. 8 g of N-Boc-PCL (2 mmol) was added into 50 mL of THF, then added 5 mL of HCl (1 M) to the reaction, the reaction continued to stir for 3 h at RT. Afterwards, trimethylamine (1 mL) was added to the resultant solution, then filtered, and the filtrate was concentrated in vacuo and precipitated in cold ethanol to obtain a white solid. Then, 3.5 g of the PBA-PEG-Azo (1.36 mmol), 0.167 g of DMAP (1.36 mmol), 4.68 g of NH2-PCL (1.2 mmol), and 0.412 g of DCC (2 mmol) were dissolved in 50 mL of DCM and stirred for 48 h at RT. Afterwards, quenched the reaction by 5 mL of deionized water. Finally, the solution was filtered, and the filtrate was concentrated by rotary evaporation to obtain a palm red solid. The reaction was monitored by TLC. Yield: 79%. Synthesis of mPEG-ss-CPT. 3 g of mPEG (1.5 mmol), 0.183 g of DMAP (1.5 mmol), 0.532 g of ss-CPT (1 mmol), 0.412 g of DCC (2 mmol) and 50 mL of DCM were all added to the round-bottom flask and stirred for 24 h under nitrogen protection. Next, 5 mL of deionized water was added. Filtered the reaction, and concentrated the filtrate in vacuo and precipitated in cold ether, then dried to yield a light yellow solid. The reaction was monitored by TLC. Yield: 87%. Characterizations. 1H nuclear magnetic resonance (1H NMR) spectra was collected using a Bruker AM 300 apparatus. The solvents were D2O and CDCl3, and the internal reference was tetramethylsilane (TMS). Gel permeation chromatography (GPC) with a Waters 2695 pump and a Styragel HT4DMF column was used to measure the polymer molecular weights. The particle size and size distribution of the micelles were determined by DLS (Zetasizer, Malvern Nano-ZS90, Malvern, U.K.).



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The morphology of the micelles was detected by TEM (JEOL 2010F instrument JEOL Ltd. Japan). High-performance liquid chromatography (HPLC) (Agilent 1260 Infinity, Agient, USA, chromatographic column C18 column (4.6× 100 mm, 3.5μm)) was used to determine the drug loading content, drug loading efficiency and drug release in vitro. Firstly, the standard curve of CPT was established by HPLC (acetonitrile/water = 30/70, v/v; 1.0 mL/min, 254 nm, 20 μm). The retention time of CPT was 12.85 min. Then 0.1 mg/mL drug-loaded micellar solution was detected by HPLC. According to the standard curve and formulas 1-1 and 1-2, the micellar drug loading content and drug loading efficiency can be calculated. Drug loading content % = The amount of the actual loaded drug / The total amount of the drug-loaded micelles×100% (1-1); Drug loading efficiency% = The amount of the actual loaded drug / The theoretical amount of the feeding drug×100% (1-2). Dual-Responsive Behaviors of the Micelles. The micelles were fabricated according to our previous report.44 PBA-PEG-Azo-PCL (5 mg) and mPEG-ss-CPT (5 mg) were added to ultrapurified water (10 mL) to fabricate the micelles. The enzyme-responsive behavior of the PBA-PEG-Azo-PCL micelles was analyzed as follows. The micelles were incubated with 0.15 mol/L Tris-HCl buffer (pH 7.4), 5 mmol/L MgCl2 and liver microsomes (1 g/L), which were shaken in a 37°C water bath for 5 min, and then 25 mmol/L NADPH was added with further oscillation for another 5 h at 37°C. Afterwards, the mixture was collected by centrifugation at 3000 rpm for 5 min. The centrifugal solid was washed three times with deionized water and


Page 20 of 42

Page 21 of 42

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dried in vacuo. The dried white solid was characterized by 1H NMR and GPC. The redox-responsive

and

enzyme-responsive

behaviors

of

the

PBA-PEG-Azo-PCL/mPEG-ss-CPT micelles were investigated as follows. The micelles were separately incubated with GSH (10 mM), liver microsomes (1 g/L) +MgCl2 (5 mmol/L) + NADPH (25 mmol/L) + 0.15 mol/L Tris-HCl buffer (pH 7.4) and GSH (10 mM) +liver microsomal (1 g/L) + MgCl2 (5 mmol/L) +NADPH (25 mmol/L) +0.15 mol/L Tris-HCl buffer (pH 7.4). Finally, the resultant products from the three conditions above were oscillated in a 37°C water bath for 5 h and were then analyzed by DLS. In Vitro Dual-Responsive Drug Release. The drug release experiment were also tested according to our previous report with slight change.44 2 mg of freeze-dried PBA-PEG-Azo-PCL/mPEG-ss-CPT micelle powder was dispersed in different media (2 mL), then transferred to an dialysis bag (MWCO 1000) in a tube (contained 30 mL release media). The release media contain GSH (10 mM), MgCl2 (5 mmol/L), NADPH (25 mmol/L) and liver microsomes (1 g/L) in a Tris-HCl buffer solution (pH 7.4). The shaking bed (100 rpm and 37°C) was used to incubate the release medium during the release study. At predetermined time intervals within 48 h, 1 mL of the solution in the tube was removed for HPLC detection, after that, replaced with 1 mL fresh medium. In Vitro Cytotoxicity Assays. The cytotoxicity were evaluated using an Alamar blue assay according to our previous report.44 EC and HepG2 cells were used in vitro cytotoxicity assays. The blank micelles PBA-PEG-Azo-PCL concentrations ranging



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Page 22 of 42

from 25 to 500 µg/mL. The cytotoxicity study of free CPT and dual-responsive micelles with HepG2 cells. The concentrations of free CPT and the CPT in dual-responsive micelles ranged from 0.1 to 25 µg/mL when free CPT and dual-responsive micelles were co-cultured with HepG2 cells for the cytotoxicity study. In Vitro Cellular Uptake. CLSM (Leica TCS SP8, Leica, Germany) was used to detect cellular uptake of free CPT and dual-responsive micelles by HepG2 cells in vitro. The cells were seeded onto a confocal dish (5×104 cells per well), cultured for 24

h.

Then,

free

CPT,

PBA-PEG-Azo-PCL/mPEG-ss-CPT

and

mPEG-PCL/mPEG-ss-CPT were added and incubated for 3 h. All the CPT concentrations were kept at 5 µg/mL. Afterwards, removed the medium in the dishes, and washed the cells with PBS three times. Fixed the cells with 2.5% glutaraldehyde for 30 min, finally, washed with PBS twice. Then, Lysotracker Green (Beyotime Biotech, China) was added and incubated with the cells for another 30 min. Finally, the cells were used for CLSM detection. Flow cytometry measurements were carried out as follows. The cells were seeded in 6-well plates (1×105 cells/well). After incubation for 1, 3 or 6 h, collected the cells by trypsinization and centrifuged at 2000 rpm for 3 min. Finally, the cells were suspended in PBS and analyzed for fluorescence intensity with a flow cytometer (FC500, Beckman, USA). In Vivo and Ex Vivo Fluorescence Imaging. Nile red was employed as a model drug to detect the fluorescence imaging in vivo and ex vivo. H22 (murine hepatic cancer cells) tumor-bearing nude mice at axilla were used as the cancer model mice.


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Injected the mice with Nile red-loaded micelles and free Nile red solution via the tail vein at a dosage of 5 mg Nile red/kg. At predetermined time intervals (1, 3, and 6 h), the mice were anesthetized and imaged using a Maestro Ex Pro instrument (CRI, USA). The emission fluorescence was 575 nm, and a 480-nm excitation filter was used. At 6 h post-injection, the normal organs (heart, liver, spleen, lung and kidney) and tumor tissues were excised for ex vivo imaging.59 In Vivo Antitumor Effect. H22 cells (2×106) were injected into the oxter of female KM mice (25±3 g). When the tumor volume reached to 50 mm3, treatments were started. The mice were randomly divided into five groups (n=5). Saline, blank micelles, PBA-PEG-Azo-PCL/mPEG-ss-CPT, PBA-PEG-PCL/mPEG-ss-CPT or free CPT were injected via the lateral vein at 2-day intervals (0, 2, 4, and 6 days) at a dosage of 5 mg CPT/kg body weight. The tumor size and body weight of the mice were measured at 1st, 3rd, 5th…21th day after the first treatment. Tumor volume was calculated

Used

the

following formula

to

calculate the tumor volume:

volume=0.5×a×b2, in which a and b represent the longest diameter and shortest diameter of the tumor, respectively. Histological assessment. At day 14 after the first injection, the mice were sacrificed, and the tumors were isolated, fixed with paraformaldehyde (4%), embedded in paraffin, sectioned into slices at a thickness of 5 mm and further stained with hematoxylin and eosin (H&E), terminal deoxynucleotidyltransferase mediated UTP end labeling (TUNEL), Ki-67 or platelet/endothelial cell adhesion molecule-1 (CD31). Images of all the samples were acquired by optical microscopy.



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Statistical analysis. All data were showed as means ± standard deviation (SD). The single factorial analysis of variance (ANOVA) was used to analyze the statistical significance of data.

#

P and

###

P both mean statistically significance,

##

P means

highly significant.

ASSOCIATED CONTENT Supporting Information 1

H

NMR

spectra

of

ss-CPT,

4,4'-(diazene-1,2-diyl)

dibenzoic

acid,

and

N-Boc-ethylenediamine; particle size change measured by DLS, Ex vivo histological analyses of the tumor sections, and the normal tissues including heart, liver, spleen, lung and kidney at 14 days after the first treatment, This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * (S.Z.) E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT L. Zhang and Y. Wang contributed equally to this work. This work was partially supported by National Basic Research Program of China (973 Program, 2012CB933600), National Natural Science Foundation of China (Nos. 51373138,


Page 25 of 42

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21574105) and the Sichuan Province Youth Science and Technology Innovation Team (Grant No.2016TD0026).

REFERENCES AND NOTES 1.

Ferrari, M. Cancer Nanotechnology: Opportunities and Challenges. Nat.Rev. Cancer 2005, 5, 161-171.

2. Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an Emerging Platform for Cancer Therapy. Nat.Nanotechnol. 2007, 2, 751-760. 3. Guo, X.; Wei, X.; Jing, Y.; Zhou, S. Size Changeable Nanocarriers with Nuclear Targeting for Effectively Overcoming Multidrug Resistance in Cancer Therapy, Adv. Mater. 2015, 27, 6450-6456. 4. Devadasu, V. R.; Vivekanand, B.; Kumar, M. N. Can Controversial Nanotechnology Promise Drug Delivey? Chem. Rev. 2013, 113, 1686-1735. 5.

Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. Tumor Vascular Permeability and the EPR Effect in Macromolecular Therapeutics: a Review. J. Controlled. Release 2000, 65, 271-284.

6.

Maeda, H. Tumor-Selective Delivery of Macromolecular Drugs via the EPR Effect: Background and Future Prospects. Bioconjugate Chem. 2010, 21, 797−802.

7.

Maeda,

H.

The Enhanced Permeability and Retention (EPR)

in Tumor Vasculature:

Effect

the Key Role of Tumor-Selective

Macromolecular Drug Targeting. Adv. Enzymol. Relat. 2001, 41, 189−207. 8. Hobbs, S. K.; Monsky, W. L.; Yuan, F.; Roberts, W. G.; Griffith, L.; Torchilin, V. P.; Jain, R. K. Regulation of Transport Pathways in Tumor Vessels: Role of Tumor Type and Microenvironment. Proc.Natl.Acad.Sci. U.S.A. 1998, 95, 46074612. 9. Lu, Y.; Low, P. S. Folate-Mediated Delivery of Macromolecular Anticancer Therapeutic Agents. Adv. Drug Delivery Rev. 2002, 54, 675−693.



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

Page 26 of 42

10. Thomas, T. P.; Huang, B.; Choi, S. K.; Silpe, J. E.; Kotlyar, A.; Desai, A. M.; Zong, H.; Gam, J.; Joice, M.; Baker, J. R. Polyvalent Dendrimer-Methotrexate as a Folate Receptor-Targeted Cancer Therapeutic. Mol Pharm. 2012, 9, 2669−2676. 11. Heo, D. N.; Yang, D. H.; Moon, H. J.; Lee, J. B.; Bae, M. S.; Lee, S. C.; Lee, W. J.; Sun, I. C.; Kwon, I. K. Gold Nanoparticles Surface-Functionalized with Paclitaxel Drug and Biotin Receptor as Theranostic Agents for Cancer Therapy. Biomaterials 2012, 33, 856−866. 12. Zempleni, J. Uptake, Localization, and Noncarboxylase Roles of Biotin. Annu. Rev.Nutr. 2005, 25, 175−196. 13. Xiao, Y.; Hong, H.; Matson, V. Z.; Javadi, A.; Xu, W.; Yang, Y.; Zhang, Y.; Engle,

J.

W.;

Nickles,

R.

J.;

Cai,

W.;

et

al.

Gold Nanorods Conjugated with Doxorubicin and cRGD for Combined Anticancer Drug Delivery and PET Imaging. Theranostics 2012, 2, 757−768. 14. Thomas, T. P.; Choi, S. K.; Li, M. H.; Kotlyar, A.; Baker, J. R. Design of Riboflavin-Presenting PAMAM Dendrimers as a New Nanoplatform for Cancer-Targeted Delivery. Bioorg.Med.Chem.Lett. 2010, 20, 5191−5194. 15. Deshayes, S.; Cabral, H.; Ishii, T.; Miura, Y.; Kobayashi, S.; Yamashita, T.; Matsumoto, A.; Miyahara, Y.; Nishiyama, N.; Kataoka, K. Phenylboronic Acid-Installed Polymeric Micelles for Targeting Sialylated Epitopes in Solid Tumors. J. Am. Chem. Soc. 2013, 135, 15501-15507. 16. Zhang, X.; Zhang, Z.; Su, X.; Cai, M.; Zhuo, R.; Zhong,Z. Phenylboronic Acid-Functionalized Polymeric Micelles with a HepG2 Cell Targetability. Biomaterials 2013, 34, 10296-10304. 17. Matsumoto, A.; Cabral, H.; Sato, N.; Kataoka, K.; Miyahara, Y. Assessment of Tumor Metastasis by the Direct Determination of Cell-Membrane Sialic Acid Expression. Angew. Chem. Int. Ed. 2015, 49, 5494-5497. 18. Zhou, K.; Liu, H.; Zhang, S.; Wang, X.; Huang, G.; Sumer, B. D.; Gao, J. Multicolored

pH-Tunable

and

Activatable

Fluorescence


Nanoplatform

Page 27 of 42

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


Responsive to Physiologic pH Stimuli. J. Am. Chem. Soc. 2012, 134, 7803-7811. 19. Wang, J.; Yang, G.; Guo, X.; Tang, Z.; Zhong, Z.; Zhou, S. Redox-Responsive Polyanhydride Micelles for Cancer Therapy. Biomaterials 2014, 35, 3080-3090. 20. Cheng,

R.;

Feng,

F.;

Meng,

F.;

Deng, C.;

Jan, F.;

Zhong,

Z.

Glutathione-Responsive Nano-Vehicles as a Promising Platform for Targeted Intracellular Drug and Gene Delivery. J. Control. Release 2011, 152, 2-12. 21. Guo, X.; Shi, C.; Yang, G.; Wang, J.; Cai Z.; Zhou, S. Dual-Responsive Polymer Micelles for Target-Cell-Specific Anticancer Drug Delivery. Chem. Mater. 2014, 26, 4405-4418. 22. Hu, Q.; Katti, P. S.; Gu, Z. Enzyme-Responsive Nanomaterials for Controlled Drug Delivery. Nanoscale 2014, 6, 12273−12286. 23. Nagase, H.; Woessner, J. F. Matrix Metalloproteinases, J. Biol. Chem. 1999, 274, 21491-21494. 24. Yamashita, S.; Yamashita, J.; Sakamoto, K.; Inada, K.; Nakashima, Y.; Murata K.; Saishoji, T.; Nomura, K. Increased Expression of Membrane-Associated Phospholipase A2 Shows Malignant Potential of Human Breast Cancer Cells. Cancer 1993, 71, 3058-3064. 25. Zhang, X.; Eden, H. S.; Chen, X. Peptides in Cancer Nanomedicine: Drug Carriers, Targeting Ligands and Protease Substrates. J. Controlled. Release 2012, 159. 2-13. 26. Gupta, S.; Andresen, H.; Stevens, M. M. Single-Step Kinase Inhibitor Screening Using a Peptide-Modified Gold Nanoparticle Platform. Chem. Commun. 2011, 47, 2249-2251. 27. Rao, J.; Khan, A. Enzyme Sensitive Synthetic Polymer Micelles Based on the Azobenzene Motif. J. Am. Chem. Soc. 2013, 135, 14056-14059. 28. Rao, J.; Hottinger, C.;Khan, A. Enzyme-Triggered Cascade Reactions and Assembly of Abiotic Block Copolymers into Micellar Nanostructures. J. Am. Chem. Soc. 2014, 136, 5872-5875. 29. Medina, S. H.; Chevliakov, M. V.; Tiruchinapally, G.; Durmaz, Y. Y.; Kuruvilla, S. P.; Elsayed, M. E.

Enzyme-Activated Nanoconjugates for Tunable Release

of Doxorubicin in Hepatic Cancer Cells. Biomaterials 2013, 34, 4655-4666.



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

30.

Page 28 of 42

Zbaida, S. Nitroreductases and Azoreductases. John Wiley & Sons, Ltd. 2002, 319-352.

31.

Metin, A.; Yildiz, H. B.; Toppare, L. Enzyme Immobilization in a Photosensitive Conducting Polymer Bearing Azobenzene in the Main Chain, Polym. Bull. 2014, 71, 1827-1841.

32.

Venditto, V. J.; Simanek, E. E. Cancer Therapies Utilizing the Camptothecins: a Review of the In Vivo Literature. Mol Pharm. 2010, 7, 307-349.

33.

Zhou, Z.; Ma, X.; Jin, E.; Tang, J.; Sui, M.; Shen, Y.; Van Kirk, E. A.; Murdoch, W.J.; Radosz, M.

Linear-Dendritic Drug Conjugates Forming

Long-Circulating Nanorods for Cancer-Drug Delivery. Biomaterials 2013, 34, 5722–5735. 34.

Ratain, M. J. Irinotecan Dosing: Does the CPT in CPT-11 Stand for "Can't Predict Toxicity"? J. Clin. Oncol. 2002, 20, 7–8.

35.

Kasai, H.; Murakami, T.; Ikuta, Y.; Koseki, Y.; Baba, K.; Oikawa, H.; Nakanishi, H.; Okada, M.; Shoji, M.; Ueda, M.; et al. Creation of Pure Nanodrugs and Their Anticancer Properties. Angew. Chem. Int. Ed. 2012, 51, 10315–10318.

36.

Liu, J.; Jiang, Z.; Zhang, S.; Saltzman, W. M. Poly(ω-Pentadecalactone-CoButylene-Co-Succinate)

Nanoparticles

as

Biodegradable

Carriers

for

Camptothecin Delivery. Biomaterials 2009, 30, 5707−5719. 37.

Wang, H.; Xie, H.; Wu, J.; Wei, X.; Zhou, L.; Xu, X.; Zheng, S. Structure-Based Rational Design of Prodrugs To Enable Their Combination with Polymeric

Nanoparticle

Delivery

Platforms

for

Enhanced

Antitumor

Efficacy. Angew. Chem. Int. Ed. 2014, 53, 11532-11537. 38. Salzano, G.; Costa, D. F.; Sarisozen, C.; Luther, E.; Mattheolabakis, G.; Dhargalkar, P.P.; Torchilin, V.P. Mixed Nanosized Polymeric Micelles as Promoter of Doxorubicin and miRNA-34a Co-Delivery Triggered by Dual Stimuli in Tumor Tissue. Small 2016, 12, 4837-4848. 39.

Zeng, L.; Zou, L.; Yu, H.; He, X.; Cao, H.; Zhang, Z.; Yin, Q.; Zhang, P.; Gu, W.; Chen, L.; Li, Y. Treatment of Malignant Brain Tumor by Tumor‐Triggered Programmed Wormlike Micelles with Precise Targeting and Deep Penetration. Adv. Func. Mater. 2016, 26, 4201-4212.


Page 29 of 42

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


40.

Liu, N.; Tan, Y.; Hu, Y.; Meng, T.; Wen, L.; Liu, J.; Cheng, B.; Yuan, H.; Huang, X.; Hu, F. A54 Peptide Modified and Redox-Responsive Glucolipid Conjugate Micelles for Intracellular Delivery of Doxorubicin in Hepatocarcinoma Therapy. ACS Appl. Mater. Interfaces 2016, 8, 33148-33156.

41. Xu, X; Wu, J; Liu, Y; Yu, M; Zhao, L; Zhu, X; Bhasin, X; Li, Q; Ha, E; Shi, J; Farokhzad, O. Ultra-pH-Responsive and Tumor-Penetrating Nanoplatform for Targeted siRNA Delivery with Robust Anti-Cancer Efficacy. Angew. Chem. Int. Ed. 2016, 55, 7091–7094. 42.

Zhong, Y; Yang, W; Sun, H; Cheng, R; Meng, F; Deng, C; Zhong, C. Ligand-Directed Reduction-Sensitive Shell-Sheddable Biodegradable Micelles Actively Deliver Doxorubicin into the Nuclei of Target Cancer Cells. Biomacromolecules 2013, 14, 3723−3730

43. Mohammadi, M; Li, Y; Abebe, D; Xie, Y; Kandil, R; Kraus, T; Gomez-Lopez, N; Fujiwara, T; Merkel, O. Folate Receptor Targeted Three-layered Micelles and Hydrogels for Gene Delivery to Activated Macrophages. J. Controlled Release 2016, in press. 44.

Qu, Q.; Wang, Y.; Zhang, L.; Zhang, X.; Zhou, S. A Nanoplatform with Precise Control Over Release of Cargo for Enhanced Cancer Therapy. Small 2016, 12, 1378-1390.

45.

Hu, X.; Hu, J.; Tian, J.; Ge, Z.; Zhang, G.; Luo, K.; Liu, S. Polyprodrug Amphiphiles:

Hierarchical

Assemblies

for

Shape-regulated

Cellular

Internalization, Trafficking, and Drug delivery. J. Am. Chem. Soc. 2013, 135, 17617-17629. 46. Hu, X.; Liu, G.; Li, Y.; Wang, X.; Liu, S. Cell-penetrating Hyperbranched Polyprodrug Amphiphiles for Synergistic Reductive Milieu-triggered Drug Release and Enhanced Magnetic Resonance Signals. J. Am. Chem. Soc. 2015, 137, 362-368. 47. Cai, K.; He, X.; Song, Z.; Yin, Q.; Zhang, Y.; Uckun, F. M., Jiang, C.; Cheng, J. Dimeric Drug Polymeric Nanoparticles with Exceptionally High Drug Loading and Quantitative Loading Efficiency. J. Am. Chem. Soc. 2015, 137, 3458-3461. 48.

Zhu, Y.; Zhang, J.; Meng, F.; Deng, C.; Cheng, R.; Feijen, J.; Zhong, Z. cRGD-Functionalized

Reduction-sensitive

Shell-sheddable

Bodegradable

Micelles Mediate Enhanced Doxorubicin Delivery to Human Glioma Xenografts in Vivo. J. Controlled Release 2016, 233, 29-38.



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

49.

Yang, W.; Guo, W.; Gong, X.; Zhang, B.; Wang, S.; Chen, N.; Yang, W.; Tu, Y.; Fang, X.; Chang, J. Facile Synthesis of Gd-Cu-In-S/ZnS Bimodal Quantum Dots with Optimized Properties for Tumor Targeted Fluorescence/MR in Vivo Imaging. ACS. Appl. Mater. Inter. 2015, 7, 18759-18768.

50.

Helle, M.; Rampazzo, E.; Monchanin, M.; Marchal, F.; Guillemin, F.; Bonacchi, S.; Salis, F.; Prodi, L.; Bezdetnaya, L. Surface Chemistry Architecture of Silica Nanoparticles Determine the Efficiency of in Vivo Fluorescence Lymph node Mapping. ACS Nano 2013, 7, 8645-8657.

51.

Bao, T.; Yin, W.; Zheng, X.; Zhang, X.; Yu, J.; Dong, X.; Yong, Y.; Gao, F.; Yan, L.; Gu, Z.; Zhao, Y. One-pot Synthesis of PEGylated Plasmonic MoO 3–x Hollow Nanospheres for Photoacoustic Imaging Guided Chemo-Photothermal Combinational Therapy of Cancer. Biomaterials 2015, 76, 11-24.

52.

Liu, Y. S.; Cheng, R. Y.; Lo, Y. L.; Hsu, C.; Chen, S. H.; Chiu, C. C.; Wang, L. F. Distinct CPT-induced Deaths in Lung Cancer Cells Caused by Clathrin-Mediated Internalization of CP Micelles. Nanoscale 2016, 8, 3510-3522.

53.

Gerdes, J.; Lemke, H.; Baisch, H.; Wacker, H. H.; Schwab, U.; Stein, H. Cell Cycle Analysis of a Cell Proliferation-Associated Human Nuclear Antigen Defined by the Monoclonal Antibody Ki-67. J. Immunol. 1984, 133, 1710-1715.

54.

Gobbo, A. D.; Pellegrinelli, A.; Gaudioso, G.; Castellani, M.; Marino, F. Z.; Franco, R.; Palleschi, A.; Nosotti, M.; Bosari, S.; Vaira, V.; et al. Analysis of NSCLC Tumour Heterogeneity, Proliferative and 18F-FDG PET Indices Reveals Ki67 Prognostic Role in Adenocarcinomas. Histopathology. 2016, 68, 746-751.

55.

Lutzky, V. P.; Carnevale, R. P.; Alvarez, M. J.; Maffia, P. C.; Zittermann, S. I.; Podhajcer, O. L.; Issekutz, A. C.; Chuluyan, H. E. Platelet-Endothelial Cell Adhesion Molecule-1 (CD31) Recycles and Induces Cell Growth Inhibition on Human Tumor Cell Lines. J. Cell. Biochem. 2006, 98, 1334-1350.

56.

Fong, D.; Hermann, M.; Untergasser, G.; Pirkebner, D.; Draxl, A.; Heitz, M.; Moser, P.; Margreiter, R.; Hengster, P.; Amberger, A. Dkk-3 Expression in the Tumor Endothelium: a Novel Prognostic Marker of Pancreatic Adenocarcinomas. Cancer Science 2009, 100, 1414-1420.

57.

Liu, J.; Liu, W.; Weitzhandler, I.; Bhattacharyya, J.; Li, X.; Wang, J.; Qi, Y.; Bhattacharjee, S.; Chilkoti, A. Ring-Opening Polymerization of Prodrugs: a


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Versatile Approach to Prepare Well-Defined Drug-Loaded Nanoparticles. Angew. Chem. Int. Ed. 2015, 54, 1002-1006. 58.

Paolo,

F.

Succinic Half-esters of Poly (Ethylene Glycol)s

and TheirBenzotriazole and Oligomeric Drug-Binding Matrices.

Imidazole Derivatives as J.

Macromol.

Chem.

1981,

182,

2183- 2192. 59.

Gupta, M. K.; Martin, J. R.; Werfel, T. A.; Shen, T.; Page, J. M.; Duvall, C. L. Cell Protective, ABC Triblock Polymer-Based Thermoresponsive Hydrogels with ROS-Triggered Degradation and Drug Release. J. Am. Chem. Soc. 2014, 136, 14896-14902.



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Figure captions: Scheme 1. Schematic design of enzyme and redox dual-triggered intracellular release from actively targeted polymeric micelles to enhance cancer treatment. Figure 1. Synthetic routes for the enzyme-responsive PBA-PEG-Azo-PCL polymer and

redox-responsive

mPEG-ss-CPT

prodrug

(A).

1

H

NMR

of

PBA-PEG-Azoo-PCL (B) and mPEG-ss-CPT (C) in CDCl3. Figure

2.

1

H

NMR

of

PBA-PEG-Azo-PCL

(A)

and

PBA-PEG-Azo-PCL/mPEG-ss-CPT (C) in D2O; particle size distribution of the PBA-PEG-Azo-PCL (B)

and

solutions

image

(D);

TEM

mPEG-ss-CPT/PBA-PEG-Azo-PCL micelle of

the

PBA-PEG-Azo-PCL(E)

and

mPEG-ss-CPT/PBA-PEG-Azo-PCL micelles (F). Figure 3. 1H NMR of PBA-PEG-Azo-PCL before (A) and after (B) treatment with azoreductase for 5 h; the inserted images correspond to micelle solutions; size distributions of the PBA-PEG-Azo-PCL/mPEG-ss-CPT micelles before and after separate treatment with GSH (10 mM), azoreductase or GSH (10 mM) plus azoreductase for 5 h (C); GPC curves of PBA-PEG-Azo-PCL before and after treatment with azoreductase for 5 h (D); the mechanisms of enzyme and redox dual-responsiveness

(E)

and

in

vitro

drug

release

from

PBA-PEG-Azo-PCL/mPEG-ss-CPT micelles (F). Figure 4. Cytotoxicity of PBA-PEG-Azo-PCL incubated with EC cells and HepG2 tumor cells. No significant toxicity was observed up to 500 µg/mL for 48 h (A), Viability of HepG2 tumor cells after exposure to polymeric micelles and CPT for 48 h (B). Fluorescence images (C) of HepG2 cells and EC cells after treatment with different concentrations of blank micelles for 48 h. The live cells were stained green, and the red indicates the dead cells. Scale bars: 50 μm. Figure 5. Confocal microscopy images of HepG2 cells after incubation with free CPT and various CPT-prodrug micelles for 1 h (A), 3 h (B) or 6 h (C). Scale bars = 25 μm. Flow cytometry analyses of HepG2 cells after incubation with free CPT and various CPT nanoformulations for 1, 3, or 6 h (D). Figure 6. In vivo Nile red fluorescence images of Nile red-loaded micelles after tail vein injection at 1 h, 3 h, or 6 h (A) and ex vivo Nile red distribution in isolated


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organs at 6 h post-injection (B). Nile red fluorescence intensity distribution in tissues after i.v. injection of Nile red solution and Nile red-loaded micelles at a dose of 1 mg Nile red equiv/kg after 6 h (C). Figure 7. In vivo antitumor effect and systemic toxicity of various CPT formulations injected into H22 tumor-bearing KM mice (n =6) at a CPT dose of 5 mg/kg. Excised H22 solid tumors at day 14 after the first treatment (A). Tumor volume changes (B), body weight changes (C), survival rate (D) and ex vivo histological analyses of tumor sections at 14 days after the first treatment with PBA-PEG-Azo-PCL/mPEG-ss-CPT (E). Scale bar =50 μm #P

Enzyme and Redox Dual-Triggered Intracellular Release from Actively

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