Starburst Diblock Polyprodrugs: Reduction-Responsive Unimolecular

Jan 18, 2019 - Institute for Clean Energy and Advanced Materials, School of Materials and Energy, Southwest University , Chongqing , 400715 , People's...
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Starburst diblock polyprodrugs: reduction-responsive unimolecular micelles with high drug loading and robust micellar stability for programmed delivery of anticancer drugs Xiaoxiao Shi, Meili Hou, Xiaoqian Ma, Shuang Bai, Tian Zhang, Peng Xue, Xiaoli Zhang, Gang Liu, Yuejun Kang, and Zhigang Xu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01566 • Publication Date (Web): 18 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019

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Starburst diblock polyprodrugs: reduction-responsive unimolecular micelles with high drug loading and robust micellar stability for programmed delivery of anticancer drugs Xiaoxiao Shia, c, Meili Houa, c, Xiaoqian Maa, c, Shuang Baia, c, Tian Zhanga, c, Peng Xuea, c, Xiaoli Zhangb*, Gang Liud, Yuejun Kanga, c, Zhigang Xua,c* aInstitute

for Clean Energy and Advanced Materials, School of Materials and Energy, Southwest University, Chongqing, 400715, P. R. China

b

Department of Hematology and Oncology, Shenzhen Children's Hospital, Shenzhen, Guangdong 518038, P.R. China

cChongqing

Engineering Research Center for Micro-Nano Biomedical Materials and Devices, Chongqing 400715, P. R. China

dState

Key Laboratory of Molecular Vaccinology and Molecular Diagnostics and

Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen, 361102, China

Email: X. Zhang ([email protected]); Z. Xu ([email protected]).

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Abstract: Polymeric prodrug based on therapeutic nanomedicine has demonstrated great promise for effective tumor growth inhibition, however, the drawbacks of low drug-loading and weak micellar stability limit its application for clinical cancer therapy. Herein, a reduction-responsive starburst block copolymer prodrug CCP [β-cyclodextrin (β-CD)-PCPTXX-POEGMA, xx: SS or CC] has been developed for cancer therapy. And CCP is composed of β-CD-Br core with multiple reactive sites, as well as a diblock copolymer containing hydrophobic polymerized camptothecin (PCPT) prodrug chain and hydrophilic poly[(ethylene glycol) methyl ether methacrylate] (OEGMA) chain. A family of CCP polymeric prodrugs with different drug loading contents (up to 25%) and various sizes of unimolecular micelles (UMs) (around 30 nm) were obtained by adjusting the block ratio of PCPTXX and POEGMA. On account of the amphiphilic structure feature, CPP could take shape water-soluble UMs in aqueous medium with excellent micellar stability. Under imitatively reductive tumor microenvironment, anticancer drug CPT could rapidly escape from CCP UMs in terms of disulfide bond breakage. However, this behaviour is strongly refrained in the physiological environment. In vitro and in vivo outcome confirmed that CCP UMs showed excellent performance of sufficient tumor accumulation, high-efficiency tumor growth inhibition and low-toxicity for healthy tissues. Based on these gratifying therapeutic efficacy, it is believed that as-present starburst prodrug strategy can offer a brand-new insight for high-efficiency therapeutic nanoplatforms for chemotherapy application.

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Keywords: Starburst, Polymeric prodrug, Unimolecular micelles, Chemotherapy, Stimuli-responsive

1. Introduction Since the commercial introduction of Doxil®,1 myriads of nanoscale polymeric carriers including micelles, dendrimers and liposomes have been developed for delivering anticancer drugs to tumor sites in order to enhance the specific desired toxicity as well as to reduce the non-specific toxicity.2-5 Most of these polymeric drug delivery systems (PDDS) based on the physical encapsulation of drugs via the noncovalent hydrophobic interaction driven by the self-assembly of amphiphilic polymers or oligomers, given the fact of the potential premature leakage of drugs during the blood circulation and the caused side effects of toxicity.6-8 To address this issue, drug can be covalently introduced into PDDS and modified into a masked, inactive form to afford so-called “prodrug”, where the controllable release of drug can be achieved in response to specific physiological environment in tumor.9-12 One prevalent approach to develop prodrugs is to conjugate hydrophobic drug to hydrophilic moiety such as folic acid13-14, polyethylene glycol15-17, peptide18-21 and biomolecules22-24 to fabricate amphiphilic delivery vehicles, which enables selfassembly into well-defined nanostructures in aqueous solution. Among them, small molecular prodrugs show the advantage of precise drug content controlling through rational molecular design.25-27 Shen and coworkers constructed a small molecular 3

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prodrug by conjugating one or two camptothecin (CPT) to an oligoethylene glycol chain (OEG8), accomplishing a high drug loading content up to 58 wt%.28 Furthermore, the self-assembled nanocapsules were employed to achieve a synergetic cancer cell cytotoxicity with additional doxorubicin (DOX) loading. In contrast, macromolecular prodrug can be facilely driven to create various morphologies by varying hydrophilic/hydrophobic ratio, providing a possibility to investigate the influence of shape and size on cellular uptake, in vitro cytotoxicity and circulation time.29-32 For instance, a series of amphiphilic polymeric prodrug with polyethylene glycol (PEG) as hydrophilic block and CPT-decorated moiety as hydrophobic block have been prepared with CPT loading contents above 50 wt%, which could self-assemble into spherical micelles with different diameters ranging from 20 nm to 300 nm and other morphologies, including vesicles, disks, and staggered lamellae.33 Recently, biomacromolecules-drug conjugates have emerged as a promising alternative due to the polymeric entities such as polypeptide or DNA possess functions to avoid rapid clearance.34 Cheetham and coworkers reported a kind of CPT-based amphiphiles with β-sheet-forming peptide sequence which can spontaneously associate into supramolecular nanofibers and nanotubes.35 A remaining challenge in the supramolecular assemblies of aforementioned linear amphiphilic prodrugs is to eliminate the dissociation risk of supramolecular assemblies caused by biological environment change during circulation in human body.36 This is daunting because the fidelity of assembly would be affected when the hydrophobichydrophilic balance in amphiphilic molecules is disturbed.37 We are interested in the 4

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development of unimolecular micelles (UMs) based polyprodrugs as a novel alternative to tackle this problem, because there is no critical micellar concentration (CMC) for UMs, leading to their excellent structural integrity. To date, it is still rarely reported except that UM-based polyprodrugs composed of hyperbranched and star polymers have been explored via random copolymerization of monomers or post-conjugation of drugs.38 Although these strategies can be effective, their limited water solubility renders the drug loading content less than 20%. In this work, we designed a novel unimolecular micelle based polyprodrug which included amphiphilic starburst diblock copolymers with a high drug loading content up to 25%. Specifically, linear diblock copolymers with polymerized reduction-responsive CPT prodrug (PCPT) as hydrophobic block and poly[(ethylene glycol) methyl ether methacrylate] (POEGMA) as hydrophilic block were embellished on β-cyclodextrin (βCD) (Scheme 1), yielding the starburst polyprodrug (β-CD-PCPTSS-b-POEGMA, denoted as CCP(SS), where SS represents the disulfide linker between CPT and polymeric carrier). To graft PCPT-b-POEGMA from β-CD in a controllable manner via atom transfer radical polymerization (ATRP), initiator α-bromoisobutyryl bromide (BiBB), was conjugated to β-CD. As-presented individual polyprodrug can form unimolecular micelle in aqueous medium, with the collapsed PCPT moiety as core and the stretched POEGMA chain as corona. The latter provides a good colloidability, playing an important role in facilitating the UMs formation. Under physiologically reductive environment, CPT prodrug units could be triggered upon the reductive cleavage reaction of disulfide by thiols such as L-glutathione (GSH), which is more 5

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enriched in tumor cells than in normal cells, resulting in tumor-specific release of CPT drugs.39-41 The in vitro experiments confirmed that these unimolecular micelle based polyprodrugs showed higher cytotoxicity for tumor cells than that for normal cells. For comparison to CCP(SS), a reduction-nonresponsive polyprodrug CCP(CC) (β-CDPCPTCC-b-POEGMA) was synthesized by replacing the disulfide linker with carboncarbon bond, which exhibited negligible toxicity against tumor cells. Similar results were observed from in vivo experiment upon administration of CCP(SS) and CCP(CC) into tumor-bearing mice. We further utilized these polyprodrugs to encapsulate nearinfrared fluorescent dye Dir to investigate the biodistribustion based on fluorescence intensity of tumor and normal tissues. 2. Experimental section 2.1 Materials. Camptothecin (CPT) was obtained from Adamas-beta® (China). All biological reagents including phosphate buffered saline (PBS, 1×), penicillin /streptomycin mixture, TrypLE™ Express Enzyme (1×), fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), PrestoBlue cell viability reagent, Alexa Fluor® 633 phalloidin, Lyso Tracker®Red dye and 1,1'-Dioctadecyl-3,3,3',3'Tetramethylindotricarbocyanine Iodide (Dir) were purchased from Life Technologies (China). TUNEL Apoptosis Assay Kit was purchased from Beyotime Biotechnology. All chemical reagents were supplied by Sigma-Aldrich (USA) and used as received unless otherwise noted. 2.2 Characterization. 1H NMR spectra were acquired by a Bruker AV 400 or 600

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NMR nuclear magnetic resonance (NMR) spectrometer. The size distribution results were determined by dynamic light scattering (DLS) using a Malvern BI-200SM. The morphology of the micelles was characterized by a JEM-1230EX transmission electron microscopy (TEM). The Fourier transform infrared (FT-IR) spectra were acquired on a Thermo Nicolet 6700 FT-IR spectrophotometer (USA) using spectrum pure KBr as the medium. Molecular weights of targeted polymers were measured by Agilent 1260 gel permeation chromatography (GPC) system, which have an Agilent 1260 pump, a styragel®HT column and a refractive index detector. Tetrahydrofuran (THF) and polystyrene were served as the eluent (1.0 mL/min) and the standard for calibration, respectively. Fluorescence emission spectra were measured using a fluorescence spectrometer (Shimadzu RF-5301PC). The optical absorbance spectra were acquired on a UV-1800 spectrophotometer (Shimadzu, Japan). The confocal images of cells were proceeded by a Zeiss-800 microscopy. The cellular uptake efficiency and anticancer effect of drugs were studied by flow cytometry (NovoCyte 2060R, USA). The images of H&E stained tissue slice were observed under bright field of a fluorescence microscope (Olympus IX71). The in vivo biodistribution of samples were characterized on a NIR imaging system (PerkinElmer IVIS Lumina Kinetic Series III). The blood testing were carried out by a Mindray BC-2600Vet hematology analyzer. 2.3 Synthesis of starburst polyprodrug β-CD-PCPT. The synthesis of two CPT monomers and β-CD-Br macroinitiator was shown in Supporting Information, and the reactive sites was~20 for each β-CD core based on the 1H NMR results of β-CD-Br (Figure S2). The starburst polyprodrug chain of β-CD-P(MABHD-CPT) (β-CD7

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PCPTSS) or β-CD-P(MAHDO-CPT) (β-CD-PCPTCC) were synthesized by ATRP reaction using β-CD-Br as the macroinitiator. β-CD-Br (46 mg, 0.01 mmol), CuBr (30.4 mg, 0.21 mmol), MABHD-CPT (1.260 g, 2.1 mmol) and anhydrous DMSO (6 mL) were added into a Schlenk tube (25 mL) under Ar (g). Then the solution mixture was degassed two freeze-pump-thaw cycles. Later, Me6TREN (58.0 µL, 0.21 mmol) was injected, and the mixture was degassed with two freeze-pump-thaw cycles again. Finally, the polymerization was carried out at room temperature for 24 h and then was terminated using liquid nitrogen. The crude product was obtained via purification using a mixed solvent of dichloromethane and diethyl ether for three times, then the collected precipitate was dried at room temperature for 48 h, to give pure β-CD-PCPTSS as pale solid powder (450 mg, 68.7%). The polymer β-CD-PCPTCC was synthesized by similar method. 2.4 Synthesis of amphiphilic polyprodrug β-CD-PCPT-b-POEGMA. The synthetic procedure of starburst amphiphilic polyprodrugs of β-CD-PCPT-b-POEGMA were as follows: β-CD-PCPT (200 mg, 0.005 mmol), oligo(ethylene glycol) methyl ether methacrylate (OEGMA) (2.310 g, 4.2 mmol), CuBr (15.2 mg, 0.105 mmol) and anhydrous DMSO (3 mL) were transferred into Schlenk tube (25 mL) under Ar (g). After a degassing process using liquid nitrogen, Me6TREN (29.0 μL, 0.105 mmol) was added into the above solution. Finally, the polymerization was implemented at room temperature for 24 h and was terminated using liquid nitrogen. After removing catalysis by Al2O3 column, the product was obtained by precipitating into diethyl ether. After being dried at room temperature for 24 h, resulting in a viscous pale solid (515.0 mg, 8

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61.8 %). The GPC results β-CD-PCPT-b-POEGMA showed a Mn of 32.1 kDa and Mw/Mn of 1.7 (Figure S9) .1H NMR of β-CD-PCPT-b-POEGMA was shown in Figure 1a and Figure S10. The obtained CCP prodrug was denoted as β-CD-PCPT21-bPOEGMA36 (CCP-3(SS)). Other reduction-responsive polymeric prodrugs including βCD-PCPT11-b-POEGMA53 (CCP-1(SS)), β-CD-PCPT11-b-POEGMA21 (CCP-2(SS)) and the non-reduction-responsive β-CD-PCPT18-b-POEGMA31 (CCP-3(CC)) were synthesized and summarized in Table 1 and Table S1. 2.5 Preparation of CCP UMs in different solvents. 5.0 mg of CCP prodrug was firstly dissolved in DMF (5 mL) of with constant stirring for 2h to offer CCP UMs in organic solution (concentration: 1.0 mg/mL). Besides, CCP UMs or Dir-labelled CCP UMs in aqueous medium were obtained using the dialysis method: 5.0 mg of CCP prodrug with or without 1.0 mg Dir dye was fully dissolved in 1.0 mL of DMF, as-obtained DMF solution was dropwise added into 6.0 mL of deionized water under continuous stirring. Where after, the solution was dialyzed against water to remove the residual solvent. Thus, CCP UMs in water were obtained. 2.6 In vitro CPT release from CCP UMs. The drug release of CPT from CCP UMs was conducted through a dialysis method by employing PBS with different DTT concentration (0 mM, 2 µM and 10 mM) at 37±1 °C. Specifically, 1.0 mL of CCP UMs was transferred into a vessel containing 80 mL of PBS with different DTT concentration, and then external solution (1.0 mL) was taken out following a timing sequence. The cumulative release of CPT was calculated using a CPT fluorescence calibration curve. 2.7 In vitro cytotoxicity. Cell viability of CCP UMs was evaluated against MCF-7, 9

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HeLa and L929 cells lines by PrestoBlue assays. Briefly, 1×104 cells were pre-seeded on each well of 96-well plates. Then, these cells were treated with fresh culture medium containing known concentrations of free CPT or CCP UMs. After 24, 48 or 72 h incubation, the PrestoBlue DMEM solution were added into each well for another 1 h incubation, the absorbance at 570 nm and the reference wavelength at 600 nm were measured by a Tecan Spark-10 plate reader. Untreated cells were served as a control group. 2.8 In vitro fluorescence images. Firstly, HeLa cells were fostered in 8-well plates at a density of 2×104 cells per well at 37 °C. After 80% confluence, the nutrient solution was discarded and washed once with PBS (1×). Then free CPT or CCP UMs in DMEM with 30 μg/mL of CPT were added for different incubation time. The nutrient solution of each well was emptied out and was then washed with PBS (1×) for six times, and then formalin solution was added for 30 min incubation. Then Alexa Fluor®633 phalloidin (AF-633) and Lyso-Tracker®Red were used to stain the cell membrane and lysosome, respectively. The lasers of 405 nm, 633 nm and 631 nm were used to excite CPT, AF-633 and Lyso-Tracker®Red, respectively. The corresponding imaging was recorded using a confocal microscopy. 2.9 In vitro cellular uptake. Briefly, HeLa cells were fostered in 6-well plates at 37 °C, and then Nile Red labeled CCP UMs in DMEM medium with 5 μg/mL of Nile Red was added and incubated for different time. After that, every well was washed with PBS (1×) and was added with 1.0 mL TrypLE™ Express Enzyme to digest cells, and then the culture medium was used to stop trypsinization. After centrifuging for 3 min, the 10

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suspension liquid was used for flow cytometry. The fluorescence intensity of Nile Red labeled CCP UMs were measured using PE channel. The cells without drug treatment were regarded as a control. 2.10 In vitro cell apoptosis. Briefly, HeLa cells were added in the 12-well plates at 37 °C. After removing the old medium, free CPT and CCP UMs in DMEM medium with 5 μg/mL of CPT were severally added for 24 h incubation. The suspended and adherent cells were collected using a centrifugation with 5000 rpm for 5 min. Next, the cells were washed with PBS (1×) and were diluted by binding buffer (100 μL), and then Annexin V-FITC (5 μL) and PI (1μL) in binding buffer were orderly added. Lastly, all groups were measured by flow cytometry. For Live/Dead assay, HeLa cells were seeded into 12-well plates and incubated with free CPT or CCP UMs for 24 h. Thereafter, the cells were stained by Calcein-AM and propidium iodide (PI), and the results were obtained by a fluorescence microscope (Olympus, IX73). 2.11 Animal model. The animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of Southwest University. BALB/c nude mice (5-6 weeks, female, and 18-20 g) and Kunming (KM) mice (6-weeks old, 28-30 g) were purchased from Academy of Military Medical Sciences (Beijing, China). The tumor model in female nude mouse was established by injecting 5×106 MCF-7 cells in 125 μL DMEM medium into the right axillary of mouse by subcutaneous injection. After around 10 days, the tumor volumes of female nude mice were allowed to increase to about 100 mm3 for in vivo experiments. 2.12 In vivo blood testing. The biosafety of CCP UMs in vivo were invested by tail 11

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vein injection method. The injection quantity of the CCP UMs was 5 mg/kg. Kunming mice were firstly randomly assigned into three groups (n = 3 per group), and then 200 μL of PBS, CCP-3(CC) or CCP-3(SS) UMs (5 mg/kg, CPT equivalent) were injected into mice by tail vein, respectively. After 6 days, 500 μL blood from KM mice was gathered and measured by a hematology analyzer (Mindray BC-2600Vet). 2.13 In vivo and ex vivo fluorescence imaging. The xenograft MCF-7 tumor-bearing nude mice were injected with free Dir, Dir-labeled CCP-3(CC) and Dir-labeled CCP3(SS) solution (Dir dose of 1.0 mg/kg, Dir equivalent) by tail vein using a microsyringe. After 24 h, the whole body optical images were taken by a NIR imaging system (PerkinElmer IVIS Lumina Kinetic Series III). Then, the mice were dissected and the tumors and primary normal organs were taken out for ex vivo optical images. 2.14 In vivo therapeutic assay. The in vivo therapeutic efficacy of CCP UMs were investigated through monitoring the tumor volumes constantly for 15 days. The xenograft MCF-7 tumor-bearing mice (~ 20 g each) were randomly assigned into three groups (n = 5), and were marked as “PBS”, “CCP-3(CC)” and “CCP-3(SS)”. Next, 200 μL of PBS, CCP-3(CC) or CCP-3(SS) solution with 1.0 mg/mL of CPT concentration by tail vein injection every three days. During the therapeutic period, the body weights and tumor volumes of mouse were recorded every day. The tumor volume was counted by the following formula: Volume = (Length × Width2)/2. 2.15 Histological studies. After chemotherapy, the mice were dissected, and the tumor, spleen, liver, kidney, lung and heart were treated with 4% paraformaldehyde solution, paraffined, and sliced into 5 μm thickness. The collected tumor slices were then stained 12

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by hematoxylin and eosin (H&E) and TUNEL, and the fluorescent images were observed by a fluorescence microscope (Olympus, IX73). 2.16 Data analysis. The statistical significance of in vitro and in vivo results were measured using t-test analysis of variance, and all date were expressed as mean ± standard deviation (S.D.).

Scheme 1. (a) Schematic illustration of reduction-responsive starburst CCP prodrug and the formation process of CCP UMs; (b) the mechanism of EPR effect mediated uptake and intracellular reduction-activated drug release for cancer therapy. 3. Results and Discussion 13

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The sections below first present the synthesis and characterization of the target reduction-responsive amphiphilic starburst polyprodrugs and their self-assembly in aqueous media to form UMs. Then, the influence of the chain length of POEGMA and PCPT block to the drug loading and the size of micelles were studied, and the reductioninduced release of CPT drugs was studied. Finally, the performance of these UM-based polyprodrug in fluorescence-based imaging and therapeutic treatment of cancers was examined at both cellular and animal levels.

Figure 1. (a) Synthetic route for the amphiphilic diblock polyprodrug β-CD-PCPTXXb-POEGMA. (b) 1H NMR spectra of β-CD-PCPTSS and β-CD-PCPTSS-b-POEGMA prodrugs in CDCl3. 3.1. Synthesis of amphiphilic starburst polyprodrug β-CD-PCPTXX-b-POEGMA The synthetic route for prodrug β-CD-PCPTXX-b-POEGMA was described in Figure 1a. According to our previous work, we firstly prepared a macroinitiator (β-CD-Br) 14

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based on β-CD conjugated with α-bromoisobutyryl bromide (BiBB), which was used for inducing the polymerization of reduction-responsive or reduction-nonresponsive CPT prodrug monomers (denoted as MABHD-CPT or MAHDO-CPT, respectively) via atom transfer radical polymerization (ATRP). Finally, four β-CD-PCPT-b-POEGMA prodrugs with various PCPT and POEGMA chain lengths were obtained after the chain extended polymerization of hydrophilic monomers OEGMA as listed in Table 1. The 1H NMR results showed that, after the polymerization of CPT monomers, the chemical shift corresponding to protons of methyl groups near bromine atom shifted from 1.9 ppm to 1.1 ppm, accompanied with the appearance of signals at 8.25~7.22 ppm from aryl protons of CPT (Figure 1b). Moreover, the absence of alkene signal from CPT monomers indicates the high purity of β-CD-PCPT. With further chain extension through the introduction of hydrophilic moiety, new signals at 3.64 ppm and 3.38 ppm were observed, which could be assigned to the methylene and methyl group on ethylene glycol units, respectively (Figure 1b). In the FT-IR spectra of β-CD-PCPTSS-bPOEGMA, the C=O vibration of MABHD-CPT emerged at 1750 cm-1 and the C-O vibration at 1108 cm-1 corresponding to OEGMA, providing a supporting evidence for the successful synthesis of target polyprodrugs (Figure S11). Based on the 1H NMR results, the CPT loading content (LC) of CCP-1(SS), CCP-2(SS), and CCP-3 (SS) and CCP-3 (CC) were calculated to be 11.1 wt%, 22.5 wt%, 24.1 wt% and 25.1 wt%, respectively (Table 1). The gel permeation chromatography (GPC) results were achieved with moderate molecular weight distributions by taking polystyrenes as the standards (for CCP-1(SS), 15

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Mn = 34.5 kDa, Mw/Mn = 1.64; for CCP-2(SS), Mn = 8.9 kDa, Mw/Mn = 1.35; for CCP3(SS), Mn = 32.1 kDa, Mw/Mn = 1.7; for CCP-3(CC), Mn = 30.1 kDa, Mw/Mn = 1.59). These starburst copolymers depict a relatively broad PDI, and it might be due to the multisite polymerization and high molecular weight for these branched and starburst copolymers.43

Furthermore, the gel permeating chromatography (GPC) results was summarized in Figure S9 and Table 1. Table 1. Molecular weights of amphiphilic starburst polyprodrugs of β-CD-PCPT-b-POEGMA Sample

M n, GPC a

M w /M n b

DP

DP

(CPT) c

(OEGMA) d

Fhydrophobice

LC(wt%)f

CCP-1(SS)

34 500

1.64

11

53

18.9%

11.1

CCP-2(SS)

8 900

1.35

11

21

39.7%

22.5

CCP-3(SS)

32 100

1.70

21

36

41.2%

24.1

CCP-3(CC)

30 100

1.59

18

31

40.2%

25.1

aNumber

average molecular weight, Mn,GPC determined by GPC. bPolydispersity index (PDI) =

Mw/Mn, determined by GPC. cDPCPT and dDPOEGMA are the degree of polymerization of CPT monomer

and OEGMA, which are calculated from 1H NMR data. e, fThe hydrophobic ration (Fhydrophobic ) and loading content (LC) were determined by 1H NMR data.

3.2 Size and morphology of CCP UMs. The amphiphilic diblock starburst nature of these well-defined CCPs enables the formation of unimolecular micelles in aqueous medium. A typical method was used by preparing micelles from a solution mixture containing good/selective solvents followed by a simple dialysis process. The TEM and DLS techniques were used to measure the morphologies and size distribution of the CCP micelles (Figure 2, and Table S2). In the TEM image of CCP-1(SS) (Figure 2a), spherical micelles with average diameters of 23.9 ± 5.3 nm were observed. In contrast, 16

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micelles of CCP-2(SS) exhibited a smaller size of 15.2 ± 5.3 nm (Figure 2d), while CPP-3(SS) had a comparable size of 21.2 ± 4.3 nm (Figure 2g and Figure S12). Such a difference is due to the shorter hydrophilic chain of CCP-2(SS) with respect to CCP1(SS). We also found that the CCP micellar size was mainly controlled by the molecular weight of CCP polymer. Moreover, DLS results showed that the hydrodynamic diameters of CCP-1(SS), CCP-2(SS), CCP-3(SS) and CCP-3(CC) micelles in aqueous were 32.0 nm, 18.8 nm, 29.7 nm and 26.9nm, respectively (Figure 2 and Figure S13), which were higher than those results by TEM owing to the hydration effect of hydrophilic POEGMA block in water medium.43 The photophysical property of UV-absorption and fluorescence emission spectra for CCPs were further characterized. Both CCP-3(SS) in water and its prodrug precursor in DMF showed maximum absorption peaks at 365 nm (Figure 2k), which was consistent with that of free CPT. In addition, the emission bands of CCP-3(SS) in water and its prodrug precursor located around 429 nm (Figure 2l). As provides a supporting evidence for the presence of grafted CPT molecules in CCP polymers. To verify whether CPPs formed unimolecular micelles in aqueous medium, we further prepared the micelles of CPPs in DMF, in which both PCPT block and POEGMA block had a good solubility. Clearly, CPPs would form unimolecular micelles in DMF due to the high stretching of each macromolecule. These UMs of CCP1(SS), CCP-2(SS) and CCP-3(SS) in DMF showed average diameters of 33.2 nm, 22.1 nm and 31.6 nm, respectively, which were more or less the same as those in aqueous media. As results offered a forceful evidence for the absence of intermolecular 17

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aggregation of CCP macromolecules in water, further validating the unimolecular micelles feature of CCPs in water.43 The subtle difference between DLS results in DMF and water could be attributed to the collapse of hydrophobic CPT core in water. To investigate the micellar stability of CCP UMs, we monitored the variation of the DLS results of CCP-3(SS) within 14 days. As depicted in Figure 3a, the hydrodynamic diameter of CCP-3(SS) UMs collected at different timepoint kept unchanged of around 30 nm, suggesting an excellent micellar stability of CCP UMs in aqueous medium.

Figure 2. TEM images and DLS histogram of CCP-1(SS) (a-c), CCP-2(SS) (d-f) and CCP-3(SS) (g-i) UMs in aqueous media and in DMF, respectively; the zeta potential of CCP-1(SS), CCP-2(SS) and CCP-3(SS) UMs in water medium (j); the absorbance (k) and fluorescence (l) spectra of CPT, MABHD-CPT, β-CD-PCPT and CCP-3(SS) UMs. 3.3 In vitro release and cytotoxicity. It has been widely reported in many polyprodrug based delivery system that the drugs can be triggered and released by cleavage of 18

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disulfide bond in the presence of glutathione. However, their kinetics of drug reductiontriggered release varies from the different structures of polyprodrugs. For instance, Hu et al. reported one polyprodrug-gated crosslinked vesicles to achieve a reductionactivated combinational chemotherapy.44 While Cai et al. employed a linear polymeric prodrug to achieve the dual-targeted and combinational antitumor efficacy driven by the acid and reduction-responsive feature.45 Due to the unique diblock starburst structure of CPPs, the CPT release behavior was studied in PBS buffer with different DTT concentration (0 mM, 2 μM and 10 mM) at 37 °C. Specifically, as shown in Figure 3b, the CPT cumulative release from CCP3(SS) UMs rapidly increased to approximately 68% within 5 h of incubation with 10 mM DTT and reached 85% after 48 h. In the case of 0 mM DTT or 2 μM DTT, only less than 1% cumulative release of CPT was observed. These results indicated that CCP-3(SS) could only respond to a high level of reductive species such as DTT or glutathione and release most of CPT in a sustained and robust manner. The DLS results showed that upon incubation with DTT for 24 h, the hydrodynamic diameter of UMs slightly increased from 31.6 nm to 35.7 nm. Such a difference suggested that the blocks in the core layer of UMs became stretched due to the conversion from hydrophobicity to hydrophilicity after efficient cleavage of disulfide bond by DTT (Figure 3d). However, almost no drug release can be observed from CCP-3(CC) UMs at each concentration of DTT, which implies that the CPT release from CCP-3(SS) UMs is ascribed to the fracture of disulfide bond. Additionally, the cumulative CPT release from CCP-1(SS) and CCP-2(SS) reached to 37.3% and 66.7% within 48 h of incubation 19

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with 10 mM DTT (Figure S14). The obviously slower release rate of both CCP-1(SS) and CCP-2(SS) than that of CCP-3 might be due to the shorter PCPT blocks in CCP1(SS) and CCP-2(SS). With less ratio of hydrophobic block, the suppress ability in terms of CPT escaping from carrier would become much stronger than that with a higher ratio of hydrophobic block, due to the high hydration effect of POEGMA chain. Although both CCP-1(SS) and CCP-2(SS) were decorated with the same hydrophobic PCPT block, the cumulative release of CCP-1(SS) was less than that of CCP-2(SS), confirming a fact that the longer POEGMA chains would endow CCP-1(SS) with better hydrophilia to prevent the drug escape from prodrug carrier core. Thereby, an optimized balance between hydrophilia and hydrophobia can effectively realize the balance of high drug loading and high cumulative release, leading to lower side-effect and better therapeutic efficacy.

Figure 3. The mean diameters distribution of CCP-3(SS) UMs under different time incubation (a). In vitro release of CPT from CCP-3(SS) (b) and CCP-3(CC) (c) 20

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incubated with different DTT concentration (0 mM, 2μM and 10mM). The data were presented as mean ± SD (n = 3). (d) The DLS change of CCP-3(SS) UMs after treatment with 10 mM DTT for 24 h. (e) Reduction-mediated release mechanism of CPT from CCP prodrugs. Under a highly reductive environment in terms of tumor microenvironment where is rich in thiol agent, the disulfide bond could be effectively attacked in carrier and liberated the drug molecules from these prodrug matrix.46 Therefore, CCP prodrugs were expected to effectively kill tumor cells. To assess the cytotoxic performance of these CCP prodrugs, PrestoBlue assay was employed to confront against two kinds of tumor cell lines of human breast cancer MCF-7 cells and human cervical cancer HeLa cells, and a normal cell line of L929 cells. In Figure 4a-c, after 72 h incubation with CCP-1(SS), CCP-2(SS), CCP-3(SS) and CCP-3(CC) UMs with CPT (10 μg/mL) could pull the cell viability down to 46%, 32%, 15% and 91% against HeLa cells, as well as to 29%, 25%, 1% and 78% against MCF-7 cells, respectively. However, the cell viability against L929 cells was still high up to 55%, 55%, 60% and 82% with the same incubation time. Different from free CPT, CCP UMs showed high cytotoxicity to tumor cells but low cytotoxicity to normal cells, which might be caused by reduction-triggered drug release behavior and high micellar stability of CCP UMs. Furthermore, free CPT and CCP prodrug showed a similar in vitro cell viability, however, the group of free CPT showed an obvious cytotoxicity during the initial stage, which could result in a a higher sideeffect than that of CCP prodrug group. This significant difference between tumor cells and

normal cells might indicate the inherent targeting ability of CCP UMs to tumor cells, 21

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which would be beneficial to normal cell/tissue, resulting in low side effect and advantageous therapeutic efficacy for clinical application.47 Additionally, the cell viability of HeLa and MCF-7 tumor cells after incubation with four kinds of CCP UMs for 24 h and 48 h were also presented in Figure S15. Compared to the results of 72 h, CCP UMs showed a time-dependent cytotoxicity. Furthermore, the half maximal inhibitory concentration (IC50) results (Figure 2d and Table S3) were 0.07 µg/mL, 0.58 µg/mL, 0.49 µg/mL, 0.41 µg/mL, >10 µg/mL for free CPT, CCP-1(SS), CCP-2(SS), and CCP-3(SS) and CCP-3(CC) UMs with 72 h incubation against MCF-7 cells, indicating the lower half-inhibitory concentration in comparison of non-responsive CCP prodrug, which is in consistance with the above cytotoxicity results. After treatment with different samples, the live/dead cells were treated with a double-staining dyes (Calcein-AM and propidium idodide (PI)). As shown in Figure 4e, when treated with CCP-3(SS) UMs, fewer live cells were visualized when compared with other CCP prodrugs, which is consistent with the in vitro MTT results. Moreover, the cell apoptosis kit with Annexin V-FITC&PI was employed to record the apoptosis and necrosis of cells after incubation with prodrug by flow cytometry (Figure 4f and 4g). After 24 h drug administration, the counts of late stage apoptotic cells were evaluated as 10.76%, 12.89%, and 14.61% and 5.49% for CCP-1(SS), CCP-2(SS), and CCP-3(SS) and CCP-3(CC), respectively. The obvious difference of apoptotic counts for CCP3(SS) and CCP-3(CC) was ascribed to the high drug release of CCP-3(SS) but almost no drug release of CCP-3(CC). And the higher late stage apoptotic count for free CPT was due to the much smaller size of free CPT. As a non-reduction responsive prodrug, 22

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CCP-3(CC) had a small micellar size and also entered into cells by EPR effect, which also caused a certain cell apoptosis. Thus, the starburst prodrug construction and reduction-triggered property endow these CCP prodrugs to regulate the drug release and deliver drugs into tumor cells, which will bring exciting in vivo therapeutic performance.

Figure 4. The in vitro cell viability of HeLa cell (a), MCF-7 cell (b) and L929 cell (c) incubated with free CPT, CCP-1(SS), CCP-2(SS), CCP-3(SS) and CCP-3(CC) for 72 h. (d) IC50 values of different drugs. (e) Live (Calcein-AM (green))/Dead (PI (red)) assay of HeLa cells after incubation with free CPT, CCP-1(SS), CCP-2(SS), CCP-3(SS) 23

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and CCP-3(CC) (The final equivalent CPT concentration: 15 μg/mL). (f) Data summary of cell apoptosis. The data were presented as mean ± SD (n=3). (g) The cell apoptosis of HeLa cell incubated with free CPT, CCP-1(SS), CCP-2(SS), CCP-3(SS) and CCP3(CC) with CPT concentrations of 5 μg/mL for 24 h. 3.4 Cellular internalization and In vitro imaging with CCP UMs. The confocal laser scanning microscopy (CLSM) was used to track the cellular internalization of aforementioned nanocarrier including CCP-1(SS), CCP-2(SS), CCP-3(SS) and CCP3(CC) UMs. The CLSM images of CCP UMs were labeled by the fluorescence of CPT (blue) and AF-633 (Alexa Fluors®633 Phalloidin, red), respectively. As shown in Figure 5a and 5b, after 2 h incubation, the blue fluorescence signals for CCP-3(SS) was obviously visualized and enhanced gradually with the increase of drug cultivation time from 0.5 h to 6 h, indicating a time-dependent cellular internalization process which might be caused by the increasingly cumulative concentration of CCP micelles and the breakage of the disulfide linkage in HeLa cells.10, 44 However, the CPT fluorescence for CCP-3(CC) hold fairly weak even after 6 h incubation, which further indicated that the absence of free CPT molecules in tumor cells due to the non-reduction-triggered drug release feature of CCP-3(CC), and the observed fluorescence was mainly from CPP UMs only. Moreover, both CCP-1(SS) and CCP-2(SS) UMs also followed the regular pattern of cellular internalization, and the CPT fluorescence signals were continuously strengthened with the cultivation time increasing from 0.5 h to 6 h (Figure S16). Although CCP-1(SS), CCP-2(SS) and CCP-3(SS) showed a similar uptake rate, CCP1(SS) showed a lowest release amount than that of CCP-2(SS) and CCP-3(SS) duo to 24

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the shortest PCPT blocks (Figure S14), which would lead to a lower cytotoxicity (Figure 4) than that of CCP-2(SS) and CCP-3(SS). The above results clearly demonstrate that the CPT molecules were gradually escaped from reduction-responsive CCP UMs and subsequently permeated into nucleus. To further affirm the pathway of these CCP UMs entering tumor cells, we conducted the co-localization study by staining the lysosome using Lyso-Tracker@Red dye. As shown in Figure 5c-d and S17-S18, the CPT fluorescence intensity from CCP UMs except for CCP-3(CC) UMs constantly enhanced in lysosome along with an increasing incubation time, which is caused by the continuous accumulation of CCP UMs into lysosome. However, the CPT fluorescence of CCP-3(CC) UMs was weak, because the sluggish release of drug from CCP-3(CC) UMs. Thus, this phenomenon could explain that CCP UMs firstly entered lysosome and then permeated into the nucleus of tumor cells.48

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Figure 5. The CLSM images of HeLa cells after being treatment with CCP-3(CC) (a and c) and CCP-3(SS) (b and d) UMs at 30 μg CPT/mL for different time. The fluorescence of CPT and Lyso-Tracker@Red were marked as blue and red. Scale bars: 50μm. Flow cytometry analysis of the cellular uptake of Nile Red labeled CCP-3(CC) (e) and CCP-3(SS) (f) UMs with an equivalent Nile Red amount of 5 μg/mL at different time points; (g) The mean fluorescence intensity of Nile Red labeled CCP-3(CC) and CCP-3(SS) UMs at different time points. The data are showed as means ± SD (n = 3).

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Furthermore, we quantitatively assessed the phagocytosis rate of CCP UMs using Nile Red labeled CCP UMs by flow cytometry. As depicted in Figure 5e-f and Figure S16-17, after 0.5 h prodrug administration, the cellular uptake ratio of CCP-1(SS), CCP-2(SS), CCP-3(SS) and CCP-3(CC) UMs were topped up to 87.19%, 81.51%, 88.53% and 88.44%, which was mainly attributed to the ultra-small micellar size, leading to a rapid phagocytosis efficiency.49-50 After prolonging the time to 6 h, cellular phagocytosis rates of four CCP UMs were more than 96%. Furthermore, the stepwise increasing mean fluorescence intensity (MFI) value also confirmed this verdict (Figure 5g). Thus, the cytophagy results revealed the size-dependent distribution of CCP UMs, which could help to deliver drugs into tumor cells to offer a high bioavailability of drugs. 3.5 Complete blood study. Although enhanced cytotoxicity against various tumor or normal cells in vitro were obtained from these CCP prodrugs, complete blood assay is further carried out to investigate the potential in vivo cytotoxicity of these CCP prodrugs. Thereby, chemical blood analysis were processed by haematology analyzer using Kunming (KM) mouse by injecting CCP-3(SS) and CCP-3(CC) UMs into healthy KM rats via tail vein injection. Several complete blood indices including white blood cells (WBC), lymphocyte (LYM), hematocrit (HCT), corpuscular hemoglobin concentration (CHC), red cell distribution width (RDW), hemoglobin (HGB), platelets (PLT) and red blood cells (RBC) were measured (Figure 6a). The KM rats with saline injection were chosen as control. After 6-days treatment with CCP UMs, these test values maintained within a reasonable range in contrast to the control group,51-52 indicating the excellent biocompatibility and low side effect. 27

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Figure 6. (a) Complete blood test in terms of red blood cell, haem regulation and white blood cell count of healthy KM rats injected with CCP-3(SS), CCP-3(CC) UMs and saline for 6 days; (b) In vivo fluorescence imaging of nude mice with MCF-7 tumor upon injection of Dir-labeled CCP-3(SS) UMs, Dir-labeled CCP-3(CC) UMs and free Dir by tail vein injection for 24 h. (c) Ex vivo fluorescence imaging of major organs and tumor after 24 h injection. (d) The relative fluorescence intensity of tumors and major 28

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organs (**P < 0.01), (e) The fluorescence intensity of Dir from Dir-labeled CCP-3(SS) UMs and Dir-labeled CCP-3(CC) UMs after adding 10 mM DTT for 2 h. Each point represents the mean ± SD (n = 3). 3.6 In Vivo Fluorescent Imaging. The aforementioned experimental results regarding in vitro results and in vivo blood test encourage us to explore the in vivo biodistribution of CCP UMs. By employing a lipophilic carbocyanine of DiOC18(7) (Dir) with NIR fluorescence, Dir-labeled CCP UMs were prepared and used to track the location of CCP UMs in vivo by visualization of NIR fluorescence. Taking CCP-3(SS) and CCP3(CC) UMs as examples, the Dir-labeled CCP-3 UMs were injected by tail vein into nude mice with MCF-7 tumor-bearing under armpit. After 24 h injection, as shown in Figure 6b, the strong Dir fluorescence of Dir-labeled CCP-3(SS) UMs at tumor site was observed, indicating an effective accumulation in tumor cells. Interestingly, very weak Dir fluorescence could be captured from the samples of free Dir, demonstrating the excellent tumor tissue targeting ability owing to the enhanced permeability and retention (EPR) effect of CCP UMs.44 In addition, fluorescent images of tumor and major organs of Dir-labeled CCP UMs and free Dir were also obtained and presented in Figure 6c. Specifically, the fluorescence intensity of Dir-labeled CCP-3(SS) UMs in tumor position was almost 10fold higher than that of free Dir, indicating an excellent candidate which was caused by the reductive-responsive drug release model. Indeed, the obvious difference referring to the tumor fluorescence intensity of CCP-3(SS) and CCP-3(CC) UMs was possibly due to the dissociation of CCP-3(SS) UMs under the reductive tumor 29

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microenvironment. Herein, further evidence was obtained by tracking the fluorescence intensity of Dir for these UMs after treatment within DTT solution for 2 h. As depicted in Figure 6e, the Dir fluorescence intensity from Dir-labeled CCP-3(SS) was much higher than that from Dir-labeled CCP-3(CC), indicating the reduction-triggered dissociation process of CCP-3(SS) UMs. Thus, compared with free Dir, CCP UMs could

enter tumor easily and then inhibited the tumor growth. 3.7 In Vivo Therapeutic Assay of CCP UMs. The in vivo therapeutic efficacy of CCP UMs was carried out using MCF-7 cells tumor model. As a hydrophobic feature, the intravenous injection of free CPT is usually administered by dissolving the drugs in DMSO solvent and then diluting with water solution for in vivo experiment, which can bring additional side-effect. Therefore, we did not use free CPT for in vivo comparison in this study to avoid addition factors that might affect the evaluation of the therapeutic effect of CCP prodrugs.

Firstly, three-group (n = 5 per group) MCF-7 tumor-bearing nude mice were injected with PBS, CCP-3(SS) and CCP-3(CC) UMs at a CPT equivalent dose of 5 mg/kg every third day, respectively. At 15 days after first injection, both PBS and CCP-3(CC) injection group showed the fast tumor growth rate and the average tumor volume increased to 13 and 10 times in comparison to the initial volume. The CCP-3(CC) group was weak to inhibit the tumor growth, which might be caused by the least CPT drugs releasing from CCP-3(CC) UMs (Figure 7a). However, compared with PBS control and CCP-3(CC) UMs groups, the CCP-3(SS) group showed more significant tumor therapeutic efficacy with the average tumor volume increasing to 6 times of the initial volume only. This outstanding success were mainly attributed to the continuous tumor 30

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accumulation of free CPT drugs (Figure 7a) driven by the feature of reduction-triggered drug release for CCP-3(SS), leading to good in vivo therapeutic effect. Moreover, the body weight of nude mice was observed without any obvious change and hold a relatively stability for all three groups, demonstrating the inconspicuous systemic toxicity during the 15 days treatment. As presented in Fig 7c and 7d, after 5 times injection, the average tumor weight of CCP-3(SS) group was only about 35% of the PBS group and showed a prominent ability to inhibit the growth of tumor in comparison with the two other groups. Moreover, hematoxylin and eosin (H&E) and TUNEL staining of tumor sections were further carried out to assess the in vivo therapeutic process. As depicted in Figure 7e, we observed the severe tumor tissue damage and a large area of cell apoptosis for CCP-3(SS) group based on the histological analysis, while the PBS group hardly induced the apparent tumor tissue necrosis and CCP-3(CC) group only caused the slight tumor cell apoptosis. Furthermore, obvious apoptotic cells (green fluorescence) were present in CCP-3(SS) group than two other groups of PBS and CCP-3(CC), suggesting an outstanding in vivo antitumor efficacy (Figure 7f). Simultaneously, the H&E staining assays was further used to evaluate the biosafety of CCP UMs (Figure 7g). After treatment with these formulations, only a few tissue damages from CCP-3(SS) group as well as PBS group and CCP-3(CC) group were observed, which might be owing to the low CPT administration dose during treatment and the excellent biocompatibility of CCP UMs, suggesting a good performance for balancing the maximum therapeutic effect and minimal side effects towards clinical application. 31

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Figure 7. In vivo antitumor efficacy of CCP-3(CC) and CCP-3(SS) UMs in MCF-7 cells tumor model. (a) Tumor volume, (b) body weight variation, (c) excised tumor weight and (d) tumor inhibition ratios after different treatment of CCP-3(CC) and CCP3(SS) UMs (Each point represents the mean ± SD. (n = 5, *P < 0.05, **P < 0.01.), (e) 32

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H&E staining images of tumor sections, (f) the TUNEL images staining with TUNEL (green) and DAPI (nuclei, blue) in tumors from different mouse groups. (g) H&E staining images for major organs of MCF-7 tumor-bearing nude mice after different treatments for 15 days. 4. Conclusion In conclusion, we have reported a class of amphiphilic starburst polyprodrugs (CCP) prepared under a mild condition, which were featured with high drug loading, high micellar stability, improved therapeutic efficacy and minimal side-effect for in vitro and in vivo cancer therapy. The micellar size and drug loading were controlled by adjusting the ratio of hydrophilic and hydrophobic chain. The amphiphilic structure of CCP prodrug dominated the formation process of unimolecular micelles (UMs) in water solution with high micellar stability. When these UMs exposed to tumor reductive condition, the disulfide bond embedded in the core of CCP could be decomposed by the reductive tumor microenvironment, leading to a rapid CPT release from these prodrug micelles. The cytotoxicity results revealed that the reduction-responsive CCP prodrug presented high toxicity against tumor cells but low toxicity for normal cells, however, the reduction-non-responsive CCP-3(CC) showed the negligible cell toxicity for both tumor cells and normal cells. The in vivo performance including the fluorescence imaging, blood test, therapeutic efficacy, H&E and TUNEL staining assays, could further indicate the CCP produgs were capable of accumulating at tumor sites and finishing fast drug release but reducing the unnecessary systemic damage. The present starburst polyprodrugs strategy integrated various essences from starburst 33

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polymer and liner polymeric prodrug, resulting in a new formation of polyprodrugs, which offered a unique avenue towards the design of intelligent polymeric prodrug for effective cancer therapy. Supporting Information Details of the synthesis and characterization of starburst β-CD-Br and CPT monomer; additional 1H NMR spectra, 13C NMR, FTIR spectra, mass spectrum and GPC results; the DLS result of CCP-3(CC) unimolecular micelles in water; In vitro CPT cumulative release amount from the CCP-1(SS) and CCP-2(SS) UMs with different DTT concentrations; the additional in vitro cytotoxicity results, confocal fluorescence microscope images and flow cytometry results. Acknowledgements This study was financially supported by the National Key Research and Development Program of China (2017YFA0205201 and 2018YFA0107301), the National Natural Science Foundation of China (51703187 and 31671037), the Basic and Frontier Research Project of Chongqing (cstc2018jcyjAX0104), the Sichuan Science and Technology Program (2018JY0392), Science and Technology Project from the Science Technology and Innovation Committee of Shenzhen Municipality (grant no.JCYJ20170817170110940),

Sanming

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(SZSM201512033) and Shenzhen Public Service Platform of Molecular Medicine in Pediatric Hematology & Oncology. Notes and references [1] Barenholz, Y. Doxil® -The First FDA-approved Nano-Drug: Lessons Learned. J. 34

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Control. Release 2012, 160, 117−134. [2] Cai, Y.; Si, W.; Huang, W.; Chen, P.; Shao, J.; Dong, X. Organic Dyes Based Nanoparticles for Cancer Phototheranostics, Small, 2018, 14, 1704247. [3] Cabral, H.; Miyata, K.; Osada, K.; Kataoka, K. Block Copolymer Micelles in Nanomedicine Applications. Chem. Rev. 2018, 118, 6844−6892. [4] Miao, T.; Wang, J.; Zeng, Y.; Liu, G.; Chen, X. Polysaccharide-Based Controlled Release Systems for Therapeutics Delivery and Tissue Engineering: From Bench to Bedside. Adv. Sci. 2018, 1700513. [5] Xu, Z.; Ma, X.; Gao, Y.; Xue, P.; Li, C.; Kang, Y. Multifunctional Silica Nanoparticles as A Promising Theranostic Platform for Biomedical Applications, Mater. Chem. Front. 2017, 1, 1257–1272. [6] Xiao, W.; Wang, P.; Ou, C.; Huang, X; Tang, Y; Wu, M.; Si, W.; Shao, J.; Huang, W.; Dong, X. A 2-Pyridone-Functionalized Aza-BODIPY Photosensitizer for ImagingGuided Sustainable Phototherapy, Biomaterials 2018, 183, 1-9. [7] Zhang, P.; Zhang, L.; Qin, Z.; Hua, S.; Guo, Z.; Chu, C.; Lin, H.; Zhang, Y.; Li, W.; Zhang, X.; Chen, X.; Liu, G. Genetically Engineered Liposome-Like Nanovesicles as Active Targeted Transport Platform. Adv. Mater. 2018, 30, 201705350. [8] Xu, Z. G.; Liu, S. Y.; Liu, H.; Yang, C. J.; Kang, Y. J.; Wang, M. F. Unimolecular Micelles of Amphiphilic Cyclodextrin-Core Star-Like Block Copolymers for Anticancer Drug Delivery. Chem. Commun. 2015, 51, 15768-15771. [9] Li, J.; Li, Y.; Wang, Y.; Ke, W.; Chen, W.; Wang, W.; Ge, Z. Polymer Prodrugbased

Nanoreactors

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For Table of Contents Use Only Starburst diblock polyprodrugs: reduction-responsive unimolecular micelles with high drug loading and robust micellar stability for programmed delivery of anticancer drugs Xiaoxiao Shia, c, Meili Houa, c, Xiaoqian Maa, c, Shuang Baia, c, Tian Zhanga, c, Peng Xuea, c, Xiaoli Zhangb*, Gang Liud, Yuejun Kanga, c, Zhigang Xua,c*

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