Fluorescent supramolecular micelles for imaging-guided cancer therapy.

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ARTICLE Fluorescent Supramolecular Micelles for Imaging-Guided Cancer Therapy Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x www.rsc.org/

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Mengmeng Sun , Wenyan Yin Xinghua Dong , Wantai Yang , Yuliang Zhao , and Meizhen Yin

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A novel smart fluorescent drug delivery system composed of a perylene diimide (PDI) core and block copolymer poly(D,Llactide)-b-poly(ethyl ethylene phosphate) is developed and named as PDI-star-(PLA-b-PEEP)8. The biodegradable PDI-star(PLA-b-PEEP)8 is a unimolecular micelle and can self-assemble into supramolecular micelles, called as fluorescent supramolecular micelles (FSMs), in aqueous media. An insoluble drug camptothecin (CPT) can be effectively loaded into the FSMs and exhibits pH-responsive release. Moreover, the FSMs with good biocompatibility can also be employed as a remarkable fluorescent probe for cell labelling because the maximum emission of PDI is benefit for bio-imaging. The flow cytometry and confocal laser scanning microscopy analysis demonstrate that the micelles are easily endocytosed by cancer cells. In vitro and in vivo tumor growth-inhibitory studies reveal a better therapeutic effect of FSMs after CPT encapsulation when compared with the free CPT drug. The multifunctional FSMs nanomedicine platform as nanovehicle has great potential for fluorescent imaging-guided cancer therapy.

Introduction Although many efforts have been made to develop effective treatment methods for cancers in the past decades, it still remains serious threat and a leading cause of morbidity and mortality to human life. Chemotherapy is a traditional treatment and one of the three pillars of cancer therapy 1 together with surgical treatment and radiation therapy. However, several limitations such as weak water-solubility, poor bioavailability and low therapeutic efficiency of conventional anticancer drug delivery systems should be 2-4 urgently resolved. With the rapid progress of nanotherapeutics for cancers in nanomedicine, a variety of 5 6-9 nanocarriers such as dendrimers, nanomicelles, polymer10-12 13 drug conjugates, liposomes, and inorganic 14, 15 nanoparticles have been extensively explored to increase drug solubility and bioavailability, as well as decrease the side effects of anticancer drugs. Among these carriers, nanomicelles, especially supramolecular micelles are one of the most widely used and promising nanocarrier, which are 16, 17 formed by amphiphilic polymers or dendrimers. The nanomicelle drug delivery systems possess several unique characteristics including efficient encapsulation of hydrophobic anticancer drugs, enhanced solubility in aqueous

solution, reduced systemic side effects of drugs, and improved preferential accumulation at the tumor site via enhanced 18-21 permeability and retention (EPR) effect. Therefore, we have witnessed an explosive development of supramolecular nanomicelle platform in the field of nanotherapeutics for cancers. Moreover, cancer nanotherapeutic systems incorporating not only therapeutic agents but also diagnostic imaging technology are emerging, which will provide a constructive biomedical platform for simultaneous therapy and imaging. Besides therapy, fluorescence imaging technique has also been widely used for the visualization of biological processes 22-24 due to the high selectivity and sensitivity. If the fluorescence probe owns unique advantages including high solubility in water, photostability and biocompatibility, it will be more beneficial for bioimaging. Especially, when absorption and emission wavelengths of the probe are above 500 nm, it can effectively reduce auto-fluorescence induced by bio25 tissues. Based on these investigations, perylene-3,4,9,10tetracarboxylic acid diimides (perylene diimides, PDIs) have attracted great interests due to outstanding chemical and photophysical stability, high extinction coefficients and high fluorescence quantum yields. However, due to the intrinsic π-π 26, 27 stacking interaction between perylene backbones, PDIs tend to form aggregation and exhibit poor solubility and weak fluorescence in aqueous solution, which results in the limitation of their application in the biomedical field. Therefore, to resolve these problems, many kinds of attempts have been performed by attaching small or bulky hydrophilic substituents 28-31 to the bay-region of PDIs. For example, Müllen and coworkers have incorporated small ionic moieties such as carboxylic, sulfonic, phosphonic acids, pyridinium, and

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Scheme 1. Schematic illustration of the formation of CPT-loaded PDI-star-(PLA15-b-PEEP25)8 nanomicelles (FSMs) and the fluorescent imaging-guided intracellular drug release.

quaternary ammonium groups into the bay-regions or ortho32, 33 positions of PDIs for cell labelling. Our group has also reported functional and water-soluble PDIs that are baysubstituted with small ionic moieties, star polymers or dendrimers, and can be applied as specific labelling and gene 34-38 delivery in vitro and in vivo. Importantly, multifunctional nanomicelles that combine diagnostic imaging with therapeutic drug delivery are emerging as the next generation of nanomedicines to improve the therapeutic outcome of drugs. Previously, fluorescent core-shell macromolecules with multiple carboxylic acids and amine groups have been synthesized and used as gene/drug carriers with cell labelling 5, 39, 40 by our group. However, biocompatible polylactide and polyphosphoesters have not yet been introduced to modify

PDI core. Therefore, in the present study, we designed and synthesized a novel PDI-cored star block copolymer poly(D,Llactide)-b-poly(ethyl ethylene phosphate) that is named as PDI-star-(PLA-b-PEEP)8. The star block copolymer actually is a unimolecular micelle and can self-assemble into supermolecular micelles as fluorescent supramolecular micelles (FSMs) with controllable sizes in aqueous solution. The PDI core with inherent fluorescence property has the 31 function as bio-tag for cell tracking. Especially, the polymer arms such as PLA and PEEP are biodegradable and the PEEP 41-43 can improve the water solubility of nanomicelles. Camptothecin (CPT), as one of the potent hydrophobic anticancer drugs, was chosen as model drug. The hydrophobic polymer arms of PLA can provide the capability to effectively encapsulate CPT and to form FSMs@CPT for effective drug

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delivery (Scheme 1). FSMs@CPT can offer pH-stimulated drug release and EPR-induced accumulation in tumor sites, which have been proven by ex vivo fluorescent imaging, thus exerting a significantly inhibitory effect on the growth of tumors. As a result, the FSMs nanomedicine platform as a potential multifunctional and biodegradable nanovehicle will provide exciting opportunities for fluorescent imaging-guided cancer therapy.

Experimental Section Materials D,L-lactide (LA, 99%) was purchased from Alfa Aesar Co., Ltd. (Shanghai, China) and recrystallized from dry ethyl acetate before use. 2-ethoxy-2-oxo-1,3,2-dioxaphospholane (Ethyl ethylene phosphate, EEP) was synthesized as previously 44 described in references. Isopropylidene-2,2-bis (oxymethyl)propionic anhydride was synthesized as previous 45 literature. N,N´-Bis-(2,6-diisopropylphenyl)-1,6,7,12-tetra-[4(2-hydroxyethyl)phenoxy]perylene-3,4,9,10-tetracarboxylic acid diimide (PDI-4OH) was prepared according to the previous 46 reports. Triethylamine and ethanol were refluxed with calcium hydride and distilled before use. 4-(Dimethylamino) pyridine (DMAP, 99%) was purchased from Alfa Aesar Co., Ltd. (Shanghai, China) and used as received. DMAP•HOSO2CF3 was 47 prepared according to the previous reports. 1,8diazabicyclo[5.4.0]-undec-7-ene (DBU) was purchased from Alfa Aesar Co., Ltd. and used as received. 2,2,5-Trimethyl-1,3dioxane-5-carboxylic acid was purchased from Nanjing Chemlin Chemical Industry Co., Ltd. and used as received. Tetrahydrofuran and dichloromethane were refluxed with sodium under nitrogen and distilled just before use. All the other reagents and solvents were purchased from the domestic suppliers and used without further purification. Characterizations Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV-III 400 MHz spectrometer with CDCl3 as solvents at room temperature. Gel penetration chromatograph (GPC) (Agilent 2600 Series) with dimethyl formamide (DMF) as the mobile phase. The column model was PLgel 5 μm 104 Å, the flow rate of mobile phase was set to be 1 mL/min, and the temperature for both column and detector was 25°C. The standard curve was determined by a series of narrow distribution polystyrene standard samples. Fluorescence spectra (FL) measurements were recorded on a FluoroMax-4 NIR spectrofluorometer (Horiba Jobin Yvon, USA). UV-vis absorption spectra were recorded on a spectrophotometer (Cintra 20, GBC, and Australia). Transmission electron 2 microscopy (TEM) images were obtained with a FEI Tecnai G 20 S Twin transmission electron microscope operating at an acceleration voltage of 200 kV. Dynamic light scattering (DLS) were measured with a Zetasizer Nano ZS (Malvern Instruments Ltd., UK) particle size analyzer. Synthesis of PDI-ester

PDI-4OH (100 mg, 0.08 mmol) and triethylamine View (0.23 mL,Online 1.6 Article DOI: 10.1039/C6NR00450D mmol) were dissolved in 20 mL dichloromethane. And then, DMAP (42 mg, 0.32 mmol) was added. A solution of isopropylidene-2,2-bis(oxymethyl)propionic anhydride (1.06 g, 3.2 mmol) in 10 mL dichloromethane was added dropwise under stirring. The mixture was stirred at room temperature under a nitrogen atmosphere and monitored by thin layer chromatography (TLC). After 24 h, the mixture was diluted with dichloromethane and extracted with saturated NaHSO4 (3× 50 mL), saturated NaCl (3 × 50 mL) and H2O (1 × 50 mL). The organic phase was dried with MgSO4, filtered and the filtrate was evaporated under vacuum. The crude product was purified by silica column chromatography eluting with CH2Cl2/EtOAc (V/V = 3:1) to obtain red PDI-ester (135 mg, 90%). 1 H NMR (400 MHz, CDCl3): δ 8.19 (s, 4H, perylene), 7.41 (t, 2H, Ph-H), 7.27 (d, 4H, Ph-H), 7.13 (d, 8H, Ph-H), 6.89 (d, 8H, Ph-H), 4.30 (t, 8H, -CH2O), 3.80-3.60 (d, 16H, -CH2O), 2.95 (t, 8H, PhCH2), 2.67 (h, 4H, -CH isopropyl), 1.37 (s, 12H, -CH3), 1.15 (s, 13 24H, -CH3), 1.10 (d, 24H, -CH3 isopropyl). C NMR (400 MHz, CDCl3): δ 174.12, 163.12, 155.86, 154.04, 153.84, 145.60, 134.06, 133.14, 130.50, 123.92, 122.94, 120.70, 120.32, 119.94, 98.07, 67.71, 65.99, 65.16, 49.25, 41.84, 34.36, 30.94, 29.08, 24.75, 24.55, 24.02, 22.77, 22.58, 18.70, 17.20, 17.15. Synthesis of PDI-8OH PDI-ester (100 mg, 0.053 mmol) was dissolved in 20 mL methanol. And then, 2 mL H2SO4 (2%, v/v) was added. The mixture was stirred at room temperature and monitored by TLC. After the reaction, NH4OH in methanol (10 ml, 50:50 solutions) was added to neutralize the mixture under continuously stirring for 30 min. The as-obtained mixture was evaporated under vacuum, dissolved in dichloromethane and extracted with H2O until it is neutral. The organic phase was dried with MgSO4, filtered and the filtrate was evaporated 1 under vacuum to obtain PDI-8OH (87 mg, 95%). H NMR (400 MHz, CDCl3): δ 8.19 (s, 4H, perylene), 7.43 (t, 2H, Ph-H), 7.28 (d, 4H, Ph-H), 7.14 (d, 8H, Ph-H), 6.95 (s, 8H, Ph-H), 4.34 (t, 8H, -CH2O), 3.80-3.61 (d, 16H, -CH2O), 2.95 (t, 8H, Ph-CH2), 2.66 (h, 4H, -CH isopropyl), 1.26 (s, 12H, -CH3), 1.10 (d, 24H, -CH3 isopropyl). Synthesis of polymer PDI-star-PLA8 Star polymer PDI-star-PLA8 was synthesized through ringopening polymerization (ROP) of LA using PDI-8OH as an initiator, DMAP and DMAP•HOSO2CF3 as the catalyst. Typically, PDI-8OH (34.4 mg, 0.02 mmol), DMAP (19.55 mg, 0.16 mmol), DMAP·HOSO2CF3 (43.56 mg, 0.16 mmol), LA (461.2 mg, 3.2 mmol) were dissolved in 2 mL dichloromethane. After that, the mixture solution was stirred at room temperature under nitrogen for 48 h and precipitated in cold n-pentane three times. Finally, the precipitate was dried under vacuum to obtain polymer PDI-star-PLA8 (yield: 75%). Synthesis of polymer PDI-star-(PLA-b-PEEP)8 Block copolymer of PDI-star-(PLA-b-PEEP)8 was prepared by ROP of EEP using PDI-star-PLA8 as the macro-initiator and DBU as the catalyst. In a typical polymerization, PDI-star-PLA8 (100


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mg, 0.0053 mmol), EEP (300 mg, 1.97 mmol) and DBU (0.05mL) were dissolved in 0.3 mL of anhydrous dichloromethane in a fresh flamed and nitrogen purged tube. The mixture was stirred at 35°C for 15 h and precipitated into cold ethyl ether twice. The precipitate was dried under vacuum to a constant weight at room temperature (yield: 53.7%).

modified Eagle Medium (DMEM), and Modified Dulbecco's View Article Online 4 10.1039/C6NR00450D 4 3 DOI: Medium (IMDM) at the densities of 1×10 , 3×10 , and 1×10 cells/well, respectively, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Then, these cells were seeded into 96-well cell culture plates and incubated for o 24 h at 37 C in a 5% CO2 atmosphere. After washing each well with PBS (pH 7.4) buffer, FSMs with different concentrations of Preparation of fluorescent supramolecular micelles (FSMs) by 0, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg/mL diluted with using PDI-star-(PLA-b-PEEP)8 the three corresponding culture medium were added to the Supramolecular nanomicelles were prepared by a dialysis wells. The cells were subsequently incubated in the same method. In brief, 10 mg of PDI-star-(PLA-b-PEEP)8 was condition for 24 h. Finally, the culture medium solutions of dissolved in 2 mL DMF and stirred at room temperature for 0.5 CCK-8 were added to each well and the cells were incubated h. Then, 2 mL of deionized water was slowly added to the for 1.5-2 h. For the in vitro anticancer effects of the FSMs@CPT, solution and stirred for another 2 h. Subsequently, the solution the MCF-7, HeLa cells and HepG2 cells were cultured in media was dialyzed against deionized water for 24 h (molecular containing free CPT, FSMs@CPT with equivalent CPT o weight cutoff: MWCO = 8000 Da), and the deionized water was concentrations from 0.01, 0.1, 1, 2.5, 5, to 10 μg/mL at 37 C exchanged every other 4 h. for 24 h and 48 h, respectively. The cell viabilities were determined by the CCK-8 assay. The cell viability (%) was Preparation of CPT-loaded FSMs (FSMs@CPT) evaluated by the absorbance at 450 nm using a microplate CPT was loaded into micelles by a dialysis method. In brief, 10 reader (SpectraMax M2 MDC, USA). Asterisks (*) denote mg of PDI-star-(PLA-b-PEEP)8 and 2 mg of CPT were dissolved statistically significant differences (*p < 0.05, ** p < 0.01 into 2 mL DMF and stirred at room temperature for 0.5 h. Then, compared with CPT group) calculated by t-test. 2 mL of deionized water was slowly added to the solution and stirred for another 2 h. Subsequently, the solution was FCS analysis of cell uptake of FSMs dialyzed against deionized water for 24 h (MWCO = 8000 Da), Flow cytometry analysis (FCS) was carried out to determine and the deionized water was exchanged every other 4 h. The cellular uptake at different time. HeLa cells were seeded in 65 solution was filtered through 0.45 mm filter to remove well plates at a density of 1×10 cells per well in DMEM. After unloaded CPT. Finally, this solution was freeze-dried and pink culturing the cells overnight, the culture medium was replaced powder was obtained. To determine the drug loading by serum-free DMEM containing 3 μg/mL of FSMs and the cells o parameters, the FSMs@CPT was dissolved in DMF. The were further incubated for 0.5, 5, 16, and 24 h at 37 C. After absorbance at 367 nm was measured to determine the CPT that, the cells were washed with cold PBS, treated with trypsin. concentration. The drug loading content (DLC) and drug Then, the cells were centrifuged, washed three times with PBS, loading efficiency (DLE) were calculated by the following and re-suspended in 1 mL PBS for FCS analyses using a flow equations: cytometer (Accuri c6, DB, USA). Weight of CPT loaded in micelles DLC(%) = × 100 Intracellular localization of FSMs and FSMs@CPT Weight of CPT loaded micelles Weight of CPT loaded in micelles Intracellular localization of FSMs or FSMs@CPT was DLE(%) = × 100 Weight of CPT used for drug loading determined using confocal laser scanning microscopy (CLSM, In vitro CPT release from FSMs@CPT The FSMs@CPT was suspended in phosphate buffered saline (PBS) with different pH values (pH 7.4 or 5.0). Then, the solution (3 mL) was transferred into the dialysis membrane tubing (MWCO = 8000 Da). And, the tubing was immersed into PBS buffer (27 mL) at 37 °C in a shaking water bath. At predetermined time points, the external buffer was collected, and the fresh PBS with equal volume was added. The concentration of CPT was determined by a UV-Vis spectrophotometer measured at 367 nm. In vitro cytotoxicity of FSMs and antitumor effects of FSMs@CPT The in vitro cytotoxic effects of FSMs were evaluated by a standard Cell Counting Kit-8 (CCK-8) colorimetric assay. Briefly, MCF-7 (a human breast adenocarcinoma cell line), HeLa (a human cervical carcinoma cell line), and HepG2 (a human liver hepatocellular carcinoma cell line) cells were used. MCF-7, HeLa, and HepG2 cells were grown in RPMI 1640, Dulbecco’s

UltraVIEW VOX Confocal System, PerkinElmer Co.). The HeLa cells were placed onto glass bottom Petri dishes at a density of 4 7×10 cells per dish and then cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at o 37 C in 5% CO2. After that, the HeLa cells were treated with FSMs at the concentration of 3 μg/mL for 0.5, 5, 16, and 24 h, respectively. For the sample of FSMs@CPT, the HeLa cells were treated with FSMs@CPT at various concentrations of 3, 6, and 9 μg/mL, respectively, for 5 h to ensure sufficient cellular uptake. Then, the cells were washed with PBS for three times while SYTO Green (KenGen BioTECH, China) was used to stain nucleus for 10 min. Finally, the SYTO signal was observed from the emission wavelength in the range of 521-580 nm. The FSMs signal can be obtained with the emission from 605 to 700 nm. Moreover, the fluorescence signal of the CPT moieties was observed with the emission channel of 415-470 nm.

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Toxicity assessment of FSMs in vivo FSMs at a total dose of 10 mg/kg were intravenously (i.v) injected into BALB/c mice (male, 5 weeks old) mice (n=4 for each group) through tail vein. The mice without any injection of the FSMs were acted as the control group. The body weights of the mice were recorded every other day during the 16 day. To further identify the biocompatibility, the major organs of the mice including heart, liver, spleen, kidney, and lung before and post i.v. injection of FSMs for 2, 7, and 16 days were collected, respectively, fixed in 10% neutral buffered formalin, processed routinely into paraffin, stained with hematoxylin and eosin (H&E). The pathology slices were examined by a digital microscope. Moreover, at the 16 day, blood chemistry analyses of the mice after administrated with the FSMs were investigated. The hematology data of the mice including hepatic function markers alanine aminotransferase (ALT), aspartate aminotransferase (AST), and renal function markers urea (UREA), and so on, were measured. Animals and tumor xenograft model Male BALB/c nude mice (5 weeks old) were purchased from Cancer Institute & Hospital, Chinese Academy of Medical Sciences, and used under protocols approved by the Institute’s Animal Care and Use Committee. To set up the tumor model, 6 1×10 human breast tumor cells (MDA-MB-231) suspended in ≈ 100 μL of serum-free RPMI 1640 medium were subcutaneously injected into the backside of the mice. The tumor volumes (V) were determined by measuring length (A) 2 and width (B) and calculated as V=AB /2. In/ex vivo fluorescence imaging and tissue biodistribution The nude mice bearing MDA-MB-231 tumor were used for fluorescence imaging when the tumor volumes approached 3 to100 mm . Before imaging, the tumor bearing mouse was i.v. injected with 200 μL of FSMs@CPT SP1 (1 mg/mL) while the control group of the mouse was i.v. injected with 200 μL saline. Firstly, the mice were placed onto the warmed stage inside of an IVIS light-tight chamber and anesthesia was maintained with 2.5% isoflurane. All the images acquisition were performed with a Maestra 3.0 in vivo fluorescence imaging system equipped with an excitation bandpass filter at 550 nm and an emission at 622 nm when the mice were anesthetized at 0 h, 1 h, and 4 h post-injection. Then, the mice were sacrificed after imaging. The major organs including the liver, lung, spleen, kidney, heart, and tumor were dissected and imaged with the fluorescence imaging system as describe above. The average signal intensities of these organs were analyzed using the Maestra image software.

FSMs@CPT (equivalent CPT dose of 5.0 mg/kg).View The tumor Article Online DOI: volumes were measured and calculated by10.1039/C6NR00450D a vernier caliper every other day. The weights of the mice were recorded during the whole experiments. Then, the mice were sacrificed at the th 24 day, the tumors were dissected and weighted, and the tumor inhibiting rates were calculated to evaluate the therapeutic efficacy. The dissected tumors of the groups (I) saline, (II) FSMs, (III) free CPT, and (IV) FSMs@CPT were embedded in paraffin and cryosectioned into 4 μm slices. Furthermore, the frozen slides were stained with H&E to further characterize therapeutical effect. The slices were imaged under an inverted fluorescence microscope.

Results and Discussions Synthesis and characterization of PDI-star-(PLA-b-PEEP)8 The detailed synthetic routes of PDI-star-(PLA-b-PEEP)8 are shown in Scheme 1a-c. PDI derivatives with 8 terminal hydroxyls (PDI-8OH) were prepared by esterification of isopropylidene-2,2-bis(oxymethyl)propionic anhydride and PDI-4OH and subsequent de-protection in the mixture of methanol and H2SO4 solution. The purified PDI-8OH was used as an initiator to initiate the ring-opening polymerization (ROP) of D,L-lactide to produce the intermediate PDI-star-PLA8 in dichloromethane at room temperature for 48 h. Then, the intermediate PDI-star-PLA8 and 1,8-diazabicyclo[5.4.0]-undec7-ene were used as a macroinitiator and catalyst to initiate the ROP of EEP monomer to produce amphiphilic block copolymer PDI-star-(PLA-b-PEEP)8 in dichloromethane at 35 C for 15 h. A series of star copolymers (Samples SP1, SP2, and SP3) with different arm lengths and molecular weights were synthesized and the results were shown in Table 1. It was found that the arm lengths of PLA and PEEP can be controlled easily by adjusting the molar ratio of LA/hydroxyl and EEP/hydroxyl in ROP. The total molecular weight of the copolymers increased with the molar ratio of monomer EEP and LA to the initiator. The chemical structures of PDI-star-PLA8 and PDI-star-(PLAb-PEEP)8 were characterized by NMR. As shown in Fig. 1a, the signals at 6.8-7.5 and 8.18 ppm are assigned to the protons of PDI core. In addition, the signals at 1.55 and 5.16 ppm are assigned to the protons of -COCH(CH3)O- and -COCH(CH3)O- in the PLA segment, respectively. The NMR results demonstrated that PDI-8OH successfully initiated the ROP of LA. Compared 1 with the H NMR spectrum of PDI-star-PLA8, three new signals Table 1 The characterization of fluorescent copolymer PDI-star-(PLA-b-PEEP)8 samples (SP1, SP2, and SP3) and the corresponding nanomicelles (FSMs SP1, SP2, SP3).

Sample

Therapeutical evaluation of FSMs@CPT for tumor bearing mice The BALB/c MDA-MB-231 tumor-bearing mice were randomly divided into four groups (n= 3 for each group). After the tumor 3 size reached about 100 mm , the mice were i.v. injected every four days with group (I): saline (200 μL), group (II): FSMs (10 mg/kg), group (III): free CPT (5.0 mg/kg), and group (IV):

WPLA/WPEEPa

Mna

Mnb

Mw/Mnb

Diameter (nm)

Pdic

c

SP1

15/25

49400

65800

1.04

250

0.317

SP2

15/23

46900

61400

1.04

200

0.383

SP3

15/20

43300

59200

1.13

150

0.476

a

Measured by 1H NMR spectra. b Measured by GPC. c Corresponding nanomicelles (FSMs SP1, SP2, and SP3) analyzed by DLS. Particle dispersion index was abbreviated as Pdi.


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Fig. 1 (a) 1H NMR spectrum of PDI-star-PLA 8. (b) 1H NMR and (c) 31P NMR spectra of PDI-star-(PLA-b-PEEP)8. (d) UV-visible absorption and fluorescence spectra of PDI-star-(PLA-b-PEEP) 8 in aqueous solution. 1

at 1.35, 4.15, and 4.26 ppm in the H NMR spectrum of PDIstar-(PLA-b-PEEP)8 were observed, which are assigned to the protons of -POCH2CH3, -POCH2CH3 and -POCH2CH2O- in the PEEP segment, respectively (Fig. 1b). This phenomenon reveals that macroinitiator PDI-star-PLA8 initiates the ROP of EEP 31 successfully. The P NMR spectrum of PDI-star-(PLA-b-PEEP)8 (Fig. 1c) further demonstrated the successful preparation of amphiphilic block copolymer PDI-star-(PLA-b-PEEP)8. The molecular weights and molecular weight distribution were 1 calculated by H NMR analysis and GPC, and corresponding data were summarized in Table 1. These star polymers showed a unimodel molecular weight distribution. The UV-visible absorption and fluorescence spectra of PDI-star-(PLA-b-PEEP)8 in aqueous solution were shown in Fig. 1d. The absorption bands at 450, 540 and 580 nm were similar to the previously reported PDI-cored dendritic macromolecules. The fluorescence spectrum at an excitation of 550 nm was also explored. The emission peak was located at the red light region and the maximum characteristic emission peak was centered at 622 nm, which can minimize the interference from cell auto-fluorescence. Micellization of PDI-star-(PLA-b-PEEP)8 and characterization of FSMs The amphiphilic nature and three-dimensional architecture of PDI-star-(PLA-b-PEEP)8 provide an opportunity to selfassemble into supermolecular micelles in aqueous environment. Typically, after dissolved in DMF and dialyzed against water, the unimolecular micelle of PDI-star-(PLA-bPEEP)8 was able to spontaneously self-assemble into supramolecular micelles as FSMs driven by strong hydrophobic/hydrophilic interaction. The morphology and size distribution of FSMs were characterized by TEM images. As expected, Fig. 2a-c reveals the coexistence of small particles and large particles 16, 48 (supramolecular micelles). Additionally, PDI-star-(PLA-b-

Fig. 2 TEM images of PDI-star-(PLA-b-PEEP)8 nanomicelles corresponding to the samples (a) SP1, (b) SP2, and (c) SP3 in Table 1. (d) In vitro cumulative release of CPT from CPT-loaded FSMs in PBS buffer at different pH conditions. (e) Photographs of FSM dispersion at a concentration of 50 μg/mL in water, PBS, saline, cell culture medium DMEM, and FBS.

PEEP)8 aggregated into spherical FSMs and the corresponding mean diameters of FSMs were approximately 180 nm, 160 nm, and 100 nm, respectively, for SP1, SP2, and SP3, depending on the block length of the copolymer. The size distribution of FSMs was further determined by DLS measurement (Table 1). As shown in Table 1, the hydrodynamic diameters of these nanomicelles in aqueous solution were approximately 250, 200, and 150 nm, respectively, for SP1, SP2, and SP3. The particle dispersion indexes of FSMs were 0.26, 0.31 and 0.20, respectively, for SP1, SP2, and SP3. Moreover, the nanomicelles have excellent stability in various physiological solutions including water, PBS, saline, DMEM, and FBS over 1 week, thereby implying their potential for biomedical application (Fig. 2e). CPT loading and release from PDI-star-(PLA-b-PEEP)8 FSMs CPT was loaded into PDI-star-(PLA-b-PEEP)8 FSMs using dialysis method. The drug loading content (DLC) and drug loading efficiency (DLE) of CPT were quantified to be as high as 17.5% and 70%, 18.6% and 74.4%, 18.0% and 72%, respectively, for SP1, SP2, and SP3. A critical point for drug delivery systems is the maintenance of drug encapsulation in micelles before reaching tumor sites, and the rapid release once entering into tumor cells. Therefore, we chose one of typical FSMs (SP1) formed from PDI-star-(PLA15-b-PEEP25)8 to explore the release behavior. The release behavior of CPT-loaded FSM (FSMs@CPT)

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system as a function of release time was carried out at different pH levels (pH 7.4 and 5.0) of PBS buffer at 37 °C to simulate the extracellular and intracellular conditions of cancer cells. As presented in Fig. 2d, after 100 h of incubation, the cumulative CPT release from FSMs@CPT was more than 80% in PBS at acidic pH 5.0, suggesting a relatively higher release rate. In marked contrast, cumulative CPT was only 30% at pH 7.4 during the same period. Therefore, the drug release rate may be affected by the degradation rate of supramolecular nanomicelles at different pH conditions. This result implied that the nanomicelles could rapidly release CPT under acidic endosome or lysosome after internalization into cancer cells, but the release of CPT within the hydrophobic core revealed a decreasing trend under the neutral condition.

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In vitro cytotoxicity and cellular uptake of FSMs In order to apply FSMs in the biomedical field, the biocompatibility of nanomicelles is of primary concern. We used CCK-8 assay to evaluate the potential cytotoxicity of FSMs assembled by PDI-star-(PLA15-b-PEEP25)8 in HepG2, MCF-7, and HeLa cells. Fig. 3a-c showed the cell viabilities after 24 h and 48 h incubation with FSMs SP1 nanomicelles at different concentrations. Even if the concentration of FSMs reached up -1 to 200 μg•mL , the cell viabilities of HepG2, MCF-7 and HeLa cells were still higher than 80%. Furthermore, we also measured cytotoxicity of the FSMs SP2 and FSMs SP3 on the HeLa cells after 24 h of co-incubation. As showin in Fig. S1, with the increasing concentrations of FSMs SP2 and FSMs SP3, neither cell viability nor the proliferation in HeLa cells was hindered even at the highest tested dosage. These studies indicated that FSMs with different polydispersity process low cytotoxicity and high biocompatibility to cancer cells. Sequentially, the cellular uptake of FSMs was also determined. HeLa cells were incubated with FSMs for 0.5, 5, 16, and 24 h, respectively. The cellular uptake rate of FSMs SP1 was analyzed with flow cytometry (FCS) by determining the fluorescence emitted from the PDI core in PDI-star-(PLA-bPEEP)8. As shown in Fig. 3d, more nanomicelles were taken up by HeLa cells when compared with the control group, and the cellular uptake rate of FSMs revealed a gradual increase with the prolonged incubation time.

Fig. 3 Cell viabilities of (a) HepG2, (b) MCF-7, and (c) HeLa cells against FSMs at various concentrations after culture for 24 and 48 h. (d) FCS analysis of HeLa cells after incubation with FSMs at different time points, the HeLa cells without treatment were used as the control group.

In vitro cytotoxicity of FSMs@CPT Furthermore, the in vitro cytotoxic effect of CPT drug delivered by FSMs was evaluated by CCK-8 assay in HepG2, MCF-7 and HeLa cells. Fig. 4a-f exhibited dose-dependent cytotoxicity to tested cells after 24 h and 48 h of incubation with free CPT and FSMs@CPT SP1 at various concentrations. With the prolonged incubation time from 24 h to 48 h, FSMs@CPT-induced cell death increased obviously when compared with that of free CPT at the tested concentrations exceeding 1.0 µg/mL. For instance, when the cells were cultured with FSMs@CPT at the concentration of 1.0 μg/mL, the cell viabilities were approximately 20.1%, 15%, and 11% for HepG2, MCF-7 and HeLa cells after 48 h incubation. It is important to note that the cytotoxicities of FSMs@CPT to three cancer cells were significantly higher than those of free CPT at higher

Fig. 4 The assays of in vitro cancer cells killing capability. Free CPT and FSMs@CPT SP1 nanomicelles were incubated with (a-b) HepG2, (c-d) MCF-7, and (e-f) HeLa cells for 24 and 48 h, respectively. The data were expressed as mean ± standard error of the mean (SEM) (M ± SEM) from six independent experiments.

concentrations (>1.0 µg/mL), which may be due to efficient cellular uptake and release of CPT from the micelles in an acidic intracellular environment. Intracellular localization of FSMs and FSMs@CPT nanomicelles Considering that PDI backbones of FSMs are fluorescent and can be used directly to measure cellular uptake, intracellular distribution and localization of FSMs fabricated from PDI-star(PLA15-b-PEEP25)8 in HeLa cells were observed by confocal laser


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Fig. 6 CLSM images of HeLa cells incubated with FSMs@CPT at (a) 3 µg/mL, (b) 6 µg/mL and (c) 9 µg/mL for 5 h.

for effective intracellular bioimaging, drug delivery and cancer therapy. In vivo pathological analysis with hematoxylin and eosin (H&E) staining In order to further evaluate in vivo biosafety of FSMs, toxic assessment was carried out in BALB/c mice. The FSMs dispersed in physiological saline (1 mg/mL) were intravenously (i.v.) injected at several time points and the body weights of scanning microscopy (CLSM). The cell nuclei were stained with the mice were monitored within 16 day. Compared with the SYTO. In CLSM studies, HeLa cells were incubated with FSMs at control group, the mice injected with FSMs showed no the concentration of 3 μg/mL for 0.5, 5, 16, and 24 h, abnormal behavior in appetite and fur color. Moreover, the respectively. As shown in Fig. 5, the bright red signal of PDIbody weights of the mice exhibited similar increase with that star-(PLA-b-PEEP)8 was observed in cells after 5 h of incubation, of the control group (Fig. S2), implying no detectable toxic and revealed an obvious increase as the prolonged incubation effects of FSMs in vivo. Moreover, histological assessment of time. The merged images showed enhanced yellow the mice organs including heart, liver, spleen, lung, and kidney fluorescence in cell nuclei, indicating that FSMs can not only be were conducted to determine whether FSMs could cause effectively internalized into cytoplasm but also into cell nuclei. tissue damage, inflammation or lesions. The organs of the As reported, antitumor mechanism of CPT can be ascribed mice with and without FSMs treatments were excised for to the inhibitory role in DNA replication and RNA histological assessment at 2, 7, and 16 days after i.v. injection. 49 transcription. Thus, it is critical to deliver CPT into cell As shown in Fig. 7, no obvious inflammation was observed in nucleus for improving the effect of cancer therapy. The cothese tissues at different time points. In comparison to the localization among CPT molecules (blue channel), PDI-starcontrol group, there was no necrosis in any groups. Moreover, (PLA-b-PEEP)8 polymer backbones (red channel), and cell barely significant mutative morphology and structures in these nuclei (green channel) were analyzed to probe the intracellular organs were observed in the test group. Importantly, serum location of CPT released from FSMs@CPT micelles. Typically, biochemical assays were used to quantitatively examine the HeLa cells were treated with FSMs@CPT at 3, 6, and 9 μg/mL impact on FSMs-treated mice. Especially, the potential impacts for 5 h to ensure sufficient cellular uptake. Meanwhile, when of FSMs on liver and kidney functions of mice were FSMs@CPT was at the condition with higher concentration investigated. As shown in Fig. S3 and S4, among all the (FSMs: 9 μg/mL, CPT: 1.5 μg/mL), partial degradation of FSMs measured factors, the important indicators for hepatic micelles was observed from the image within 5 h (Fig. 6a). function and renal function markers such as ALT, AST, and Besides, the majority of fluorescence signals of CPT lighted up th UREA were in the normal range at the 16 day when cytoplasm and cell nuclei, owning to the effective degradation compared with the control group, thus implying no noticeable of FSMs and intracellular release of CPT from FSMs even if inflammatory expression and hepatic and renal disorder. The FSMs@CPT was at lower concentration of 3 μg/mL, which complete blood panels of the treated group were similar to suggested strong and evenly distributed fluorescent signals those of the control group. Therefore, FSMs are highly (Fig. 6b-c). Interestingly, the overlay of three fluorescence biocompatible in vivo, and can be used as a potential images from FSMs, SYTO, and CPT presented obvious white nanovehicle for cancer therapy. color in the selected concentration range. These results indicated that CPT released from FSMs@CPT was successfully Ex/in vivo fluorescence imaging distributed into nuclei in the HeLa cells. Consequently, it can The inherent fluorescence of the PDI core in FSMs allows for be concluded that the nanomicelles are of great importance

Fig. 5 CLSM images of HeLa cells incubated with FSMs for 0.5 h, 5 h, 16 h, and 24 h, respectively.

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Fig. 8 (a) Ex vivo fluorescence imaging after i.v injection with saline and FSMs@CPT for 4 h. (b) Average fluorescence signal intensity of the corresponding organs from FSMs@CPT-injected mice.

Fig. 7 H&E stained tissue sections from the mice injected with FSMs nanomicelles for 2, 7, and 16 day. The mice without post-injection were used as the control group.

In vivo tumor inhibition efficiency

Motivated by these observations, in vivo tumor inhibition efficiency was evaluated in MDA-MB-231 tumor-bearing mice. The mice were divided into four groups: group (I) with the tracking the distribution of FSMs using ex/in vivo fluorescence injection of saline, group (II) with the injection of FSMs, group imaging. Based on the unique properties of FSMs, we (III) with the injection of free CPT, and group (IV) with the investigated and verified the biodistribution of FSMs@CPT SP1. injection of FSMs@CPT. For tumor therapy, the mice were i.v. Typically, the MDA-MB-231 tumor-bearing nude mice were treated with the saline, FSMs, free CPT, and FSMs@CPT every injected with saline (control) and FSMs@CPT (experimental four days. As shown in Fig. 9a, no significant loss of body group). The in vivo fluorescent imaging of the mice within 4 h weight of the mice for all groups was observed, indicating the of i.v. injection was recorded. As shown Fig. S5, the in vivo low toxicity of these groups. The average tumor weight of the fluorescence imaging had a limited tissue penetration depth to group treated by FSMs@CPT was significantly lower than that live tissues. Then, the mice were sacrificed and the ex vivo of the control group (Fig. 9b). Moreover, the average tumor fluorescent imaging for major organs were detected (Fig. 8a). inhibition rate can reach up to 77%, which was greatly higher In Fig. 8a, compared with the control group without than that of free CPT group (22%, Fig. S8). In Fig. 9d, fluorescence, strong fluorescence of FSMs@CPT localized in intravenous injection of FSMs@CPT showed a significant liver was determined after i.v. injection of FSMs@CPT for 4 h. suppression of the tumor growth at the end of the experiment Importantly, as shown in Fig. 8a-b, the fluorescence in the th (the 24 day), whereas the tumor volume reached up to 1000 tumor site testified by imaging can also be clearly observed 3 mm in the control group. However, the tumors of the saline after injection of FSMs@CPT for 4 h. The preferential group and FSMs group showed similar growth rate, suggesting accumulation of FSMs@CPT in tumor could be due to the that FSMs had no impact on tumor growth. The photographs enhanced permeability and retention efficiency (EPR) of the of the respective tumor-bearing mice (Fig. 9c) and the tumors nanomicelles in tumor tissues. (Fig. 9e) after 24 days’ treatment further indicated the Moreover, to further investigate whether or not the effective antitumor efficacy of FSMs@CPT because of the EPReffects of the excess CPT of the FSMs@CPT nanomicelles on induced accumulation of the nanocarrier in tumor tissues. the normal tissues lung, liver, kidney, and spleen of the mice Histological examination of tumor slices for each group further th after i.v. injection, the tissues were also dissected at the 24 confirmed the effective treatments of tumors by the day. Then, the tissues were fixed and stained with H&E. For chemotherapy of CPT-loaded FSMs (Fig. 9f). Based on H&E the FSMs@CPT treated group, these organs morphology were staining results, the typical characteristics for chemotherapynormal compared with those of the control group. For induced loss of nuclei, cell shrinkage and coagulation of tumor example, liver cells were regularly arrayed without other tissues treated with the free CPT group (III) and FSMs@CPT obvious changes. The results implied that the sample did not group (IV) can be clearly observed. Especially for the induced obvious toxicity within the test period (Fig. S6). The FSMs@CPT group, the loss of nuclei and cell shrinkage in high dose FSMs@CPT localized in the liver within 4 h could be tumors can be more obviously observed. This result suggests due to the partly aggregation of the sample in the blood that FSMs can serve as a powerful drug delivery nanovehicle protein which was proved by the DLS distribution (Fig. S7b). for in vivo chemotherapy of tumors. The low toxic to the liver and other normal tissues of the FSMs@CPT could be attributed to the advantages of the nanomicelles with well biocompatibility and slowly release of Conclusions CPT induced by the biodegradable PLA and PEEP of the FSMs, In summary, multifunctional biodegradable FSMs obtained which can avoid the direct damage to the normal tissues and from PDI-star-(PLA-b-PEEP)8 have been successfully cells. engineered as an effective drug delivery system. The supramolecular nanomicelles showed good biocompatibility in


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the Fundamental Research Funds for the CentralView Universities Article Online 10.1039/C6NR00450D (YS201402), and the Innovation and DOI: Promotion Project of Beijing University of Chemical Technology.

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