Redox

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Biological and Medical Applications of Materials and Interfaces

Octreotide-conjugated Core Cross-linked Micelles with pH/ Redox Responsivity Loaded with Etoposide for Neuroendocrine Neoplasms Therapy and Bioimaging with Photoquenching Resistance Jian-an Bai, Ye Tian, Fangzhou Liu, Xiaolin Li, Yun Shao, Xintong Lu, Jintian Wang, Zhu Guoqin, Bingyan Xue, Min Liu, Ping Hu, Na He, and Qiyun Tang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01827 • Publication Date (Web): 22 Apr 2019 Downloaded from http://pubs.acs.org on April 22, 2019

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ACS Applied Materials & Interfaces

Octreotide-conjugated Core Cross-linked Micelles with pH/Redox Responsivity Loaded with Etoposide for Neuroendocrine Neoplasms Therapy and Bioimaging with Photoquenching Resistance Jianan Baia,1, Ye Tiana,1, Fangzhou Liub,1, Xiaolin Li a, Yun Shaoa, Xintong Lua, Jintian Wanga, Zhu Guoqina, Bingyan Xuea, Min Liua, Ping Hua, Na Hea, Qiyun Tanga,* aDepartment

of Geriatric gastroenterology, The First Affiliated Hospital with Nanjing

Medical University, Nanjing, China bDepartment

of Head & Neck Surgery, Jiangsu Cancer Hospital & Jiangsu Institute of

Cancer Research & The Affiliated Cancer Hospital of Nanjing Medical University, Nanjing, China 1These

three authors contributed equally to this study.

*Corresponding to: Qiyun Tang, MD, PhD, Department of Geriatric gastroenterology, The First Affiliated Hospital with Nanjing Medical University, 300 Guangzhou Rd, Nanjing

210029,

China

(e-mail:

[email protected]).

Telephone:

+86-025-

13770642446

ABSTRACT The study of multifunctional polymer micelles combined with chemotherapy due to reduced systemic toxicity and enhanced efficacy has attracted intensive attention. Herein, a multifunctional core cross-linked hybrid micelle system based on mPEG-bPGu(BA-TPE) and OCT-PEG-b-PGu(DA-TPE) with pH- and redox-triggered drug release and aggregation-induced emission (AIE) active imaging has been developed for active targeting of neuroendocrine neoplasms (NENs), especially neuroendocrine carcinomas (NECs) with poor prognosis. These micelles showed excellent biocompatibility and stability. After the formation of borate ester bonds, core crosslinked micelles (CCLMs) showed enhanced emission property. In addition, etoposide (ETO), one of the most important anticancer drugs of NECs, was loaded into the hydrophobic core of micelles by self-assembly with an average diameter of 274.6 nm and spherical morphology. Octreotide (OCT) conjugated onto the micelles enhanced cellular uptake by receptor-mediated endocytosis. ETO-loaded micelles demonstrated the dual-responsive triggered intracellular drug release and great tumor suppression ability in vitro. Compared with free ETO, ETO-loaded CCLMs exhibited considerable antitumor effect and significantly reduced side effects. Considering the active tumor

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targeting, dual-responsive drug release and AIE effect, the polymer micelle system will be a potential candidate for diagnosis and oncotherapy of NENs. Keywords: neuroendocrine carcinomas, core cross-linked micelles, dual-responsive, aggregation-induced emission, targeted drug delivery

1. Introduction Neuroendocrine carcinomas (NECs) is one of the most notorious malignant type of neuroendocrine neoplasms (NENs) which are a cluster of peculiar tumors originated from the diffuse neuroendocrine system with a roaring incidence to 6.98/100,000 person during the past few decades and sometimes secrete several hormones inducing severe clinical syndromes [1,2]. Difficulty in diagnosis and limited therapeutic strategies lead to inferior quality of life to individuals and more global burden to our society [3,4]. There are some medications, including everolimus and sunitinib, used in poorly differentiated NENs. However, low response rate, drug resistance and severe systemic toxicity remain troublesome [5]. Etoposide (ETO)-based chemotherapy is currently the first line choice with prolonged median overall survival (OS) of 7.6-13.4 months in colorectum, pancreas and stomach NECs [6]. However, direct administration of ETO might induce severe side effects including grade 3/4 myelosuppression, druginduced liver and renal injury [7]. Hence, a new strategy based on ETO-loaded nanocarriers should be developed and expected to maximize the therapeutic efficacy and minimize the side effects of ETO. Nanocarriers, as promising anticancer drug vehicles, have great advantages of improved biocompatibility, prolonged retention time and enhanced therapeutical efficacy [8-10]. Nanocarriers are known to target cancerous cells and tissues via passive accumulation named as the enhanced permeability and retention (EPR) effect [11]. To further enhance the accumulation of nanocarriers in target sites, active-targeting abilities were endowed by conjugating specific molecules, such as folic acid, polypeptide and so on [12-19]. It has been reported that a drug delivery platform coloaded with ETO and platinate based on P(MeOx-b-BuOx-b-MeOx) was developed for small cell and non-small cell lung cancer therapy [20]. Worm-like polymeric micelles were prepared and increased synergy effect was achieved for cancer treatment. A nanosized, lipid-stabilized indium-ETO complex with high PEGylation was studied and achieved simultaneous delivery of both an imaging agent and an anticancer drug with


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a single nanoparticle system as reported in the literature [21]. Besides, to deliver the loaded anticancer drugs more effectively, nanocarriers were modified with somatostatin analogues, like OCT, which was attributed to a strong binding affinity between somatostatin analogues and overexpressed somatostatin receptors (SSTRs) in cytomembranes of tumor cells [22-26]. However, the application of nanocarriers for NENs is still rare, especially on the multifunctionalization of nanocarriers. Recently, several polymeric nanocarries with active luminous effect based on tetraphenyl ethylene (TPE), a typical AIE chromophore, were developed and used in bioimaging [27-32]. As reported, theranostic PC-hyd-TPE-DOX micelles achieved pH-responsive drug release and in vitro AIE imaging [27]. Multifunctional polymeric nanomicelles combined with AIE bioimaging were developed based on mPEG-P(TPE-co-AEMA) copolymer for pH/redox dualresponsive drug release [32]. All these reports demonstrated that polymeric micelles with AIE effect showed great potential in bioimaging. However, core cross-linked polymeric nanomicelles based on AIE properties had not been reported. We expected to develop a core cross-linked micelle system, capable of controlled drug release and bioimaging, with the ability of active targeting and excellent stability. In this study, a core cross-linked hybrid micelle system with photoquenching resistance for bioimaging has been explored to realize active tumor targeting and pH/redox dual-responsive drug release at target sites (Scheme 1). Successful crosslinking into the core of micelles was achieved by the formation of borate binds. The physicochemical properties of hybrid micelles were investigated, such as size distribution, Zeta potential and morphology. Then, other characterizations associated with AIE effect, stability and stimuli-responsivity were characterized respectively. After loading with ETO, in vitro drug release was performed in different conditions. Cellular imaging and cytotoxicity of hybrid micelles against NCH-446, LCC-18 and BON-1 were investigated. At last, the in vivo imaging, biodistribution and antitumor effect were further investigated in detail.

2. Materials and methods 2.1. Materials Poly(ethylene glycol) methyl ether amine (mPEG5k-NH2) and poly(ethylene glycol) carboxyl amine (HOOC-PEG5k-NH2) were obtained from Sigma-Aldrich. γ-Benzyl-Lglutamate-N-carboxyanhydride (BLG-NCA), 3-aminobenzeneboronic acid, octreotide



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and 3, 4-dihydroxyphenethylamine were obtained from Bide Pharmatech. Ltd. Etoposide and fluorescein 6-isothiocyanate (FITC) were bought from Alladin. N-(3dimethylaminopropyl)-N'-ethylcarbodiimide

hydrochloride

(EDCI)

and

N-

hydroxysuccinimide (NHS) were bought from Shanghai Macklin Biochemical Co., Ltd. 4-(1, 2, 2-Triphenyl vinyl) benzoic NHS ester (TPE-NHS) was purchased from Henan PSAI chemical products Co., Ltd. Trifluoroacetic acid (TFA), methanesulfonic acid and thioanisole were bought from Energy Chemical Technology (Shanghai) Co., Ltd. Anhydrous N, N’-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and tetrahydrofuran (THF) were bought from Energy Chemical Technology (Shanghai) Co., Ltd. Dichloromethane (DCM), hexane and diethyl ether were bought from Shanghai Run Jie Chemical Reagent Co., Ltd. All these reagents were used as received. TPE-SSNH2 was synthesized and characterized by 1H NMR in Supporting Information as described in Scheme S1. 2.2. Synthesis of mPEG-b-PBLG and HOOC-PEG-b-PBLG Methoxy poly(ethylene glycol)-b-poly(γ-Benzyl-L-glutamate) (mPEG-b-PBLG) and carboxyl poly(ethylene glycol) amine-b-poly(γ-Benzyl-L-glutamate) (HOOC-PEG-bPBLG) were synthesized via ring opening polymerization (ROP) according to the report [33]. Briefly, mPEG5k-NH2 or HOOC-PEG5k-NH2 (1 g, 0.2 mmol) and BLGNCA (1.2 g, 4.6 mmol) were dissolved in anhydrous DMF (10 mL). The reactions were maintained at room temperature for 4 days under N2 protection. The block copolymers were obtained by precipitation into excess amount of ice diethyl ether for five times. 2.3. Synthesis of OCT-PEG-b-PBLG Octreotide was conjugated onto HOOC-PEG-b-PBLG via EDCI/NHS method. In breif, HOOC-PEG-b-PBLG (200 mg, 1 eq), EDCI (20 mg, 5 eq) and NHS (12 mg, 5 eq) were dissolved in 5 mL DMF. Octreotide (41 mg, 2 eq) was added into the mixture 2 h later. The reaction was maintained for another 24 h. OCT-PEG-b-PBLG was obtained by precipitation into excess amount of ice diethyl ether for five times. 2.4. Synthesis of mPEG-b-PGu(BA-TPE) and OCT-PEG-b-PGu(DA-TPE) MPEG-b-PGu(BA-TPE) and OCT-PEG-b-PGu(DA-TPE) were synthesized through two steps: 1) mPEG-b-PBLG was dissolved in TFA at ice bath with methanesulfonic acid/thioanisole (1/1, v/v). The reaction was maintained at ice bath for 1 h and precipitated into excess amount of ice diethyl ether. The deprotected copolymer mPEGb-PLG was obtained by dialysis and lyophilization. 2) MPEG-b-PLG (200 mg, 19 eq carboxyl) was dissolved in dry 10 mL DMSO with EDCI (488 mg, 95 eq). The reaction


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was performed under N2 at room temperature before addition of TPE-SS-NH2 (182 mg, 13 eq). After reaction of 24 h, excess 3-aminobenzeneboronic acid (70 mg, 19 eq) was added. The reaction mixture was maintained for another 24 h. MPEG-b-PGu(BA-TPE) was obtained by dialysis and lyophilization. OCT-PEG-b-PGu(DA-TPE) was synthesized based on OCT-PEG-b-PLG and 3, 4-dihydroxyphenethylamine by the similar method. 2.5. Characterization of polymers The chemical structure of synthesized copolymers was measured by 1H NMR on a Varian Mercury Plus 300 spectrometer in DMSO-d6. The critical micelle concentration (CMC) of OCT-PEG-b-PGu(DA-TPE) and mPEG-b-PGu(BA-TPE) was determined by detecting the emergence of the fluorescence (FL) of samples when copolymers started to aggregate. Briefly, a fixed amount of OCT-PEG-b-PGu(DA-TPE) or mPEGb-PGu(BA-TPE) was added into vials. Then, PBS (pH 7.4) was added to the vials with concentrations ranging from 0.024 to 50.0 μg/mL. The vials were warmed up to 37 oC with slight vibration overnight. At last, the FL intensity at a wavelength of 460 nm (excited at 330 nm) was measured using a fluorescence spectrophotometer (HITACHI F-4600). The CMC was calculated as the cross point of the tangents to the two linear portions. The average molecular weight (Mw) and polydispersity (PDI) of mPEG-b-PBLG and HOOC-PEG-b-PBLG were measured by gel permeation chromatography (GPC) (PLGPC50, Agilent) with poly(ethylene glyco) as standards. Before measurement, mPEGb-PBLG or HOOC-PEG-b-PBLG was dissolved in DMF and the solution was filtrated through 0.22 μm filter. GPC measurement was carried out with DMF as eluents with a flow rate of 1.0 mL/min. 2.6. Preparation and Characterization of micelles The hybrid micelles, as core cross-linked micelles (CCLMs), were prepared with nanoprecipitation. In brief, 5 mg OCT-PEG-b-PGu(DA-TPE) and 5 mg mPEG-bPGu(BA-TPE) were dissolved in 1 mL DMF completely. Then, the solution was added slowly into 10 mL deionized water with a mechanical microsyringe. The suspensions were stirred for 3 h and dialyzed against deionized water. As a contrast, uncross-linked micelles (UCLMs) were prepared based on mPEG-b-PGu(BA-TPE) with similar procedure. The average size, size distribution and zeta potential of CCLMs and UCLMs were determined by dynamic light scattering (DLS) equipped with a Malvern Nano Zeta



Sizer (Malvern Instruments, Worcestershire, UK). The morphology of these two kinks of micelles were analyzed by TEM (H-7100; Hitachi, Tokyo, Japan). A drop of CCLMs or UCLMs was placed on acopper grid with a carbon film for 10 min. Then, the rest micelle suspension was removed by filter paper suction. 2.7. Dilution of micelles The suspensions (0.1 mL, 1 mg/mL) of CCLMs and UCLMs were diluted with 0.9 mL DMSO. Photos were taken under UV-light irradition at 365 nm and the red light from a laser pointer. To further confirm the AIE effect of micelles, FL spectra of CCLMs and UCLMs in water or diluted with DMSO were recorded from 400 nm to 750 nm. Besides, the size distribution of diluted micelles was measured by DLS. 2.8. Stability of micelles The prepared SDS solution in PBS (7 mg/mL) was added into suspension of UCLMs and CCLMs (1 mg/mL), respectively. The final concentrations of SDS and micelles were 3.5 mg/mL and 0.5 mg/mL, respectively. Afterwards, these two suspensions of micelles were warmed up and shaken at 37oC. At predetermined time intervals, scattering light intensity of micelles was investigated by DLS. The photos of micelles with SDS addition were taken under UV-light irradition at 365 nm as compared with micelles at the same concentration without SDS addition. 2.9. pH Responsivity and redox sensitivity of micelles To investigate stimuli-responsivity of micelles, CCLMs were placed in different conditions: a phosphate buffer solution (pH 7.4, 0 GSH), an acetate buffer solution (pH 5.0, 0 GSH) and an acetate buffer solution (pH 5.0, 2 mM GSH). The average size and polydispersity index (PDI) of CCLMs in different conditions were recorded by DLS. 2.10. Photoquenching experiment UCLMs and CCLMs were exposed to UV-light irradition at 365 nm with FITC. At predetermined time intervals, the FL intensity of these Luminescent reagents was measured by microplate system. 2.11. Preparation and Characterization of ETO-loaded micelles ETO was loaded into micelles with nanoprecipitation. Breifly, 10 mg ETO, 50 mg OCT-PEG-b-PGu(DA-TPE) and 50 mg mPEG-b-PGu(BA-TPE) were dissolved in 10 mL DMF completely. Then, the solution was added slowly into 100 mL deionized water with a mechanical microsyringe. Then, the suspensions were stirred for 3 h and dialyzed against deionized water to remove DMF and free ETO. The size distribution and morphology of ETO-loaded CCLMs were characterized by DLS and TEM.


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The drug loading content (DLC) and drug loading efficiency (DLE) were measured by high-performance liquid chromatography (HPLC) according to previous report [34]. The DLC and DLE were calculated as bellows: DLC % = DLE % =

Amount of ETO entrapped in micelles Initial amount of ETO added Amount of ETO entrapped in micelles Total amount of micelles

× 100 %

(1)

× 100 %

(2)

2.12. Drug release The drug release from ETO-loaded micelles was investigated in different conditions, pH 7.4 with or without 20 μM GSH, pH 6.0 with or without 20 μM GSH and pH 5.0 with or without 2 mM GSH. Briefly, 1 mL of the ETO-loaded CCLMs (1 mg/mL) was transferred into a dialysis bag (MWCO 2000), which was then immersed into 40 mL buffer solution, respectively. At predetermined intervals, the buffer solution containing released ETO was removed to measure the amount of ETO by HPLC and replaced by 40 mL fresh buffer solution. All release experiments were carried out in triplicate. 2.13. Cell culture A549 and NCH-446 cells (Chinese Academy of Sciences, Shanghai), BON-1 and LCC-18 cells (iCell Bioscience Inc., Shanghai) were used for biological effect research. Cells were cultured in F12 or RPMI 1640 medium supplemented with 10% fetal calf serum under a humidified atmosphere containing 5% CO2 at 37 °C. 2.14. Cellular uptake To investigate the AIE imaging of micelles and receptor-mediated cellular uptake, BON-1, LCC-18 and NCH-446 cells were plated in 6-well plates with 2 × 105 cells/well. After incubation for 24 h, micelles were added with a concentration of 100 μg/mL. After another incubation for 1 h, cells were washed with PBS for three times before imaging by a fluorescence microscope. As comparison, cells were pretreated with OCT with a concentration of 20 μg/mL and incubated for 10 min before addition of micelles. A549 cells, negative expression of SSTR2, were used as another control group. 2.15. Cytotoxicity The cytotoxicity of copolymers and ETO-loaded CCLMs against BON-1, LCC-18 and NCH-446 cells was investigated by CCK-8 assay. Cells were seeded in 96-well plate at a density of 4000 cells/well. After incubation for 12 h at 37 oC in 5 % (v/v) CO2 atmosphere, new culture medium containing copolymers, ETO or ETO-loaded CCLMs was added. The concentration gradients of ETO were 1, 2, 5, 10, 25 and 50 μg/mL. After cultivation for 48 h, 10 μL CCK-8 reagent was added to each cell and incubated



for another 2 h. The absorbance at 450 nm was detected by a microplate reader. Cell viability was calculated by the following formula: ODsample - ODblank

Cell viability % = OD

control

- ODblank

× 100 %

(3)

2.16. Cell apoptosis and cell cycle Cell apoptosis was examined by Annexin V-FITC apoptosis detection kit (BD Bioscience, USA). Cells was seeded in a 6-well plate at a density of 3 × 105 cells/mL for 24 h, and then treated with OCT-PEG-b-PGu(DA-TPE), mPEG-b-PGu(BA-TPE), ETO and ETO-loaded CCLMs (10 μg/mL of free ETO equivalent) for 24 h. Cells were collected, washed with cold PBS, resuspended in Annexi-binding buffer and stained with PI and FITC Annexin V. After incubating in the dark for 15 min at room temperature, cell suspensions were diluted by Annexin-binding buffer and detected by flow cytometry (BD Bioscience, USA). For cell cycle analysis, cells were cultured for 24 h, then treated with OCT-PEG-bPGu(DA-TPE), mPEG-b-PGu(BA-TPE), ETO and ETO-loaded CCLMs for 12 h. Treated cells were harvested by trypsinization and fixed in 70 % ice-cold ethanol at 4 oC

overnight. After washing with PBS, cells were resupended in 100 μL RNase A at 37

oC

for 30 min, then added with 400 μL PI for 30 min, and finally analyzed with a flow

cytometer. 2.17. Animal model and drug treatment Four-week-old BALB/c mice were purchased from the Animal Center of Nanjing Medical University and maintained in standard conditions. The protocol was proved by the Committee on the Ethics of Animal Experiments of Nanjing Medical University. To evaluate the in vivo tumorigenic effects, BON-1 cells (2×107) were inoculated subcutaneously in the right flank of the mice. When the xenografts mice grew to an appropriate size successfully (about 4 weeks), the mice were treated with saline, ETO or ETO-loaded CCLMs at 5 mg/kg by tail vein injection every 2 days for 10 times. Tumor growth was evaluated every 2 days and tumor volumes were calculated as the product of length × width2 × 0.5. 2.18. Ex vivo FL imaging study ETO-loaded CCLMs were injected into xenograft mice at a dose of 5 mg/kg, saline was used as the control group. At predetermined time points (12 and 24 h), major tissues including heart, liver, spleen, lung, and kidney as well as tumor were excised. Ex vivo imaging was performed on a Maestro imaging system at excitation wavelength of 400


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nm (the lowest excitation wavelength of the machine). 2.19. Histological change The fixed samples embedded in paraffin were cut into 5 μm for H&E staining and immonohistochemical analysis. Following deparaffinization, dehydration, and antigen retrieval, one section for each sample was stained with H&E. The samples were incubated with anti-Ki-67 antibodies at 4°C overnight. The HRP-conjugated secondary antibody staining was performed at room temperature, visualized in 3, 30diaminobenzidine (DAB, Sigma) solution and counterstained with hematoxylin. Data analysis was performed by using the Image Software. 2.20. TUNEL assay Cell apoptosis of tumor tissues was measured by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay. The experiment was carried out with an in situ cell death detection kit (Roche Diagnostics, Basel, Switzerland) according to the directions. The result of cell apoptosis was observed under an optical microscope (Eclipse CI, Nikon). 2.21. Statistical analysis All data were expressed as mean ± SD, and the data analysis was performed by GraphPad Prism 6.0. Scheme 1 was drawn by Adobe Illustrator CS6. 3. Results and discussion 3.1. Synthesis and characterization of polymers The copolymers were synthesized successfully according to Scheme 2. Methoxy poly(ethylene glycol)-b-poly(γ-benzyl-L-glutamate) (mPEG-b-PBLG) and carboxyl poly(ethylene glycol) amine-b-poly(γ-benzyl-L-glutamate) (HOOC-PEG-b-PBLG) were synthesized by ring opening polymerization (ROP). OCT was conjugated to HOOC-PEG-b-PBLG to generate OCT-PEG-b-PBLG. OCT-PEG-b-PBLG and mPEG-b-PBLG were deprotected successfully to obtain mPEG-b-PLG and OCT-PEGb-PLG characterized by 1H NMR spectrum as shown in Fig. S1. After deprotection, mPEG-b-PLG and OCT-PEG-b-PLG were modified with TPE-SS-NH2 containing disulfide bonds, then further decorated with 3, 4-dihydroxyphenethylamine and 3aminobenzeneboronic acid, respectively. The chemical structure of the copolymers was measured by 1H NMR (Fig. 1). The chemical structures of mPEG-b-PBLG and HOOCPEG-b-PBLG were shown in Fig. 1a,b. By calculating the typical peak integrals of PEG



segments (-OCH2CH2O-, δ 3.53) and phenyl segments (C6H5-, δ 7.40 ~ δ 7.20), the polymerization degrees of PBLG block of mPEG-b-PBLG and HOOC-PEG-b-PBLG were 19 and 21, respectively. The molecular weight of mPEG-b-PBLG and HOOCPEG-b-PBLG was calculated as 9.2 kDa and 10.5 kDa compared with those measured by GPC as 11.1 kDa and 12.3 kDa, respectively (Table S1). OCT was conjugated successfully onto HOOC-PEG-b-PBLG termed as OCT-PEG-b-PBLG (Fig. 1c), which was proved by the new signals pervaded around δ 4.32 ~ δ 0.78 compared with Fig. 1b. The 1H NMR spectrums of mPEG-b-PGu(BA-TPE) and OCT-PEG-b-PGu(DA-TPE) were demonstrated and typical peaks of TPE were observed (Fig. 1d,e). The signal peaks of BA and DA were covered by peaks of TPE, which makes it difficult to calculate grafting ratios of TPE, BA and DA directly. Fortunately, there were characteristic signals (δ 7.67 ~ δ 7.58) of TPE that didn’t overlap with those of BA or DA, which could be used to calculate the grafting ratio of TPE. The signal peaks around δ 7.22 ~ δ 6.90 consisted of signals of BA or DA and TPE. The grafting ratio of BA or DA could be identified after TPE was determined. The integrals of signals around δ 3.58 ~ δ 3.45, δ 7.22 ~ δ 6.90 and δ 7.67 ~ δ 7.58 were marked as shown in Fig. 1d and Fig. 1e. The grafting ratios of BA or DA and TPE were calculated as shown in Table S2. Interestingly, even if BA and DA were feeded in access, there were still 15.7 % of carboxyl groups of mPEG-b-PGu(BA-TPE) and 17.9 % of carboxyl groups of OCT-PEG-b-PGu(DA-TPE) undecorated, which was mainly attributed to the steric hindrance of TPE. 3.2. Preparation and characterization of micelles As reported [30], the fluorescence (FL) intensities of TPE increased when the circumstance changed from well solvent to poor solvent, which could be utilized to measure the critical micelle concentration (CMC) of polymers containing TPE. The self-assembly behavior of mPEG-b-PGu(BA-TPE) and OCT-PEG-b-PGu(DA-TPE) was characterized by FL intensities of copolymers with different concentrations in water at 37 oC (Fig. S2a). The CMC was calculated as ca. 7.35 μg/mL for OCT-PEGb-PGu(DA-TPE) and 3.41 μg/mL for mPEG-b-PGu(BA-TPE) through the cross point of the curve. CCLMs were prepared by the formation of borate ester bonds, which was believed as the viable way to realize successful core cross-linking without any addition and unnecessary by-products. Uncross-linked micelles (UCLMs) were prepared as the control group based on mPEG-b-PGu(BA-TPE). The DLS results including Zeta potentials, size distribution and polydispersity index (PDI) of CCLMs and UCLMs


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were demonstrated in Fig. S2b. Zeta potentials of both CCLMs and UCLMs were less than -25 mV, which was mainly caused by unreacted carboxyl groups. CCLMs showed a narrow size distribution with the average size of 175.3 nm (PDI: 0.133), which was smaller than that of UCLMs with the average size of 235.4 nm (PDI: 0.225). Size decrease could be attributed to the formation of borate ester bonds leading to shrinkage of hydrophobic core. The morphology of CCLMs and UCLMs were shown in Fig. S2 c,d. Size decrease was still observed from TEM photos, which was in accordance with DLS results. Besides, the size from TEM results was slightly smaller than that from DLS results, which was caused by the shrinkage of hydrophilic shell when micelles were dried during the process of sample preparation in all probability. 3.3. Dilution of micelles To characterize the AIE effect and validate the success of core cross-linking of micelles, DMSO, as good solvent for mPEG-b-PGu(BA-TPE) and OCT-PEG-bPGu(DA-TPE), was utilized to dissolve CCLMs and UCLMs. Sample a represented UCLMs in water, sample b with UCLMs diluted by DMSO and sample c with CCLMs diluted by DMSO (Fig. 2a). Under UV-light irradition at 365 nm, the fluorescence generated by aggregated TPE was observed clearly in sample a and c, which indicated TPE chromophores in sample b exhibiting a free state. The fluorescence observed in sample b was deemed as the intrinsic fluorescence of cuvettes, which was further testified by the red light path from a laser pointer. Bright path of red light was observed in sample a and c, but not in sample b. Besides, the FL spectra of micelles were measured (Fig. 2b). UCLMs and CCLMs in water exhibited strong FL intensity. After dilution with DMSO, CCLMs exhibited decreased FL intensity, while the FL intensity of UCLMs diluted with DMSO was negligible. Significant size distribution of CCLMs diluted with DMSO was found whereas UCLMs diluted with DMSO exhibited no obvious signal (Fig. 2c). All these results indicated that CCLMs presented successful core cross-linking and could maintain the structural stability faced with the dilution of DMSO, which made them possess great potential for drug delivery and bioimaging. 3.4. Stability of micelles When micelles were administrated into blood, they encountered not only the infinite dilution of blood, but also the high shear force during long-term blood circulation [35]. SDS, capable of disintegrating micelles, was used to demonstrate the stability of micelles. The scattering light intensity (SLI) of micelles was regarded to be related to the concentration and size of particles. The SLI of UCLMs with SDS added exhibited



rapid decrease within 1 min, while CCLMs with SDS added exhibited little change in SLI (Fig. 2d). It might be concluded that the SDS destroyed the structure of UCLMs rapidly, while CCLMs could keep stable with the presence of SDS due to the formation of borate located in the core of micelles, which was further proved (Fig. 2e). 3.5. pH Responsivity and reduction sensitivity To confirm the stimuli-responsivity of micelles, the average size and PDI of CCLMs in different conditions were investigated by DLS (Fig. S3). In pH 7.4 with 0 GSH condition, CCLMs showed PDI less than 0.22 with the average size around 176.2 nm (Fig. S3a). In pH 5.0 with 0 GSH condition, the overall trend of average size of CCLMs was increased with small fluctuations of PDI (Fig. S3b), which could be explained by gradual fracture of borate ester bonds in these conditions. When up to pH 5.0 with 2 mM GSH, both average size and PDI exhibited large fluctuations (Fig. S3c). To be specific, the size and PDI gradually grew larger and eventually stabilized at a relatively high level. The representative size distributions of CCLMs, in pH 5.0 with 2 mM GSH condition at 2 h, 8 h and 24 h were demonstrated (Fig. S3d). Emergence of multiple peaks indicated the degradation of micelles caused by the breakage of borate ester bonds and disulfide bonds. 3.6. Photoquenching resistance experiment The hypothesis to explain the mechanism of AIE phenomenon has been proposed, containing the restriction of intramolecular rotation (RIR), E/Z isomerisation and Jaggregation formation [36]. Recently, increasing evidence had been reported to support the RIR hypothesis as the fundamental and dominant mechanism of AIE molecules [36,37]. In this study, the FL quenching of FITC, UCLMs and CCLMs was conducted under UV-light irradition at 365 nm. UCLMs exhibited better photoquenching resistance than FITC (Fig. S4), which was also studied by the report [28]. Interestingly, the relative FL intensity of CCLMs remained higher than that of both UCLMs and FITC, which indicated the enhanced photoquenching resistance of AIE micelles after the core cross-linking of micelles. This phenomenon could be explained by the formation of borate ester bonds among the core of micelles, resulting in further restriction of intramolecular rotation of TPE molecules. 3.7. Incorporation of ETO and drug release from micelles ETO, as a hydrophobic anticancer drug, was chosen to be loaded into micelles via nanoprecipitation and dialysis method. The size distribution and morphology of ETOloaded CCLMs was demonstrated in Fig. 3a,b. The average size of ETO-loaded CCLMs


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was 274.6 nm that was larger than that of blank CCLMs, which was explained by enlarged hydrophobic core caused by ETO loading. The DLC and DLE of ETO loaded micelles were calculated as 8.2 % and 83.1 %, respectively. The ETO, released from ETO-loaded CCLMs, was conducted in different conditions (Fig.3c,d). To explore the influence of pH value to micelles, drug release was simulated in pH 7.4, 6.0 and 5.0 without GSH. With the decrease of pH value, ETO release became faster, which was demonstrated by the cumulative release for 54 h: 24.6 % for pH 7.4, 33.5 % for pH 6.0 and 50.4 % for pH 5.0. To explore drug release profile under different GSH levels, ETO release from ETO-loaded CCLMs were supervised under 20 μM GSH and 2 mM GSH at pH 7.4, 6.0 and 5.0 individually. As shown in Fig. S5, ETO release was accelerated under 2 mM GSH condition compared with that under 20 μM GSH. Meanwhile, it should be noticed that the influence of GSH was increasingly enhanced with the decrease of pH values. Under conditions with GSH to simulate the physiological environment and tumor microenvironment, the cumulative release in pH 7.4 with 20 μM GSH and pH 6.0 with 20 μM GSH was similar to that without GSH. However, the drug release in pH 5.0 with 2 mM GSH was increased significantly with the amount of cumulative ETO release up to 84.1 %. All these results revealed that drug release from CCLMs was controlled by the low pH values and reduction conditions that were considered as the typical conditions of tumor cell lysosomes. 3.8. In vitro cellular uptake and imaging To confirm the enhanced SSTR-mediated endocytosis, the cellular uptake behaviors of micelles with or without OCT treated were detected in SSTR-overexpressed NENs cell lines (BON-1, LCC-18 and NCH-446). A549 cells, negatively expressed SSTRs, were used as control group. Cells were pre-treated with OCT to block SSTR2 and SSTR5 and exhibited weak fluorescent signals compared with non-OCT treated group (Fig. 4a), demonstrating that the endocytosis of CCLMs was mediated by SSTR2 and SSTR5. Collectively, OCT-conjugated micelles were endowed with enhanced ability of cellular uptake, which could promote SSTR-mediated endocytosis effectively. 3.9. Cytotoxicity of ETO-loaded CCLMs To investigate the cytotoxicity of micelles, we employed CCK-8 assays to evaluate the viability of NENs cells, which were treated with mPEG-b-PGu(BA-TPE), OCTPEG-b-PGu(DA-TPE), free ETO or ETO-loaded CCLMs, respectively. Cell viability was above 90% with mPEG-b-PGu(BA-TPE) and OCT-PEG-b-PGu(DA-TPE) treatment at the concentration of 500 μg/mL in all groups and no cytotoxicity was



observed, which suggested the great biocompatibility (Fig. 4b). ETO and ETO-loaded CCLMs exhibited certain inhibition on NENs cells. Interestingly, ETO-loaded CCLMs exhibited great anticancer effect compared with free ETO on BON-1 cells. However, it was shown that free ETO exhibited greater cytotoxicity than that of ETO-loaded CCLMs on LCC-18 and NCH-446 cells (Fig. 4b). Considering the mechanism of drugs passing through cell membranes and the difference of intracellular environment of tumor cells, there are two possible reasons, as follows: 1) differential permeability of cell membranes; 2) differential drug release from micelles caused by the different intracellular conditions. 3.9. ETO-loaded CCLMs did not affect cellular apoptosis and cycle The effects of ETO on cell apoptosis of BON-1, LCC-18 and NCH-46 cells were evaluated by the Annexin V-FITC staining method. It could be seen that both free ETO and ETO-CCLMs markedly increased the ratios of early apoptotic cells (Q3) and late apoptotic cells (Q2) compared with the control group (Fig. 5a,c). As shown in Figure 5c, after being treated by free ETO and ETO-CCLMs, the ratio of total apoptotic cells (Q2 + Q3) rose to 62 % and 61.6 % from 9.69 % of control in BON-1 cells, 90.9 % and 87.8 % from 16.75 % of control in LCC-18 cells, 42.45 % and 45.75 % from 11.73 % of control in NCH-446 cells, respectively. These results indicated that both free ETO and ETO-CCLMs could induce the cell apoptosis of NENs cell lines without significant difference, in accordance with the result of CCK-8 assay. As shown in Fig. 5b, both free ETO and ETO-CCLMs markedly reduced the propotion of BON-1 cells in S-phase and prevented entry into S phase, indicating that ETO inhibited the cell growth by hampering its DNA replication. The ratio of G1 phase arrested in BON-1 cells significantly increased from 47.5 % of control to 96.92 % and 96.08 % of free ETO and ETO-CCLMs (Fig. 5d). However, the cell cycle distribution of treated LCC-18 and NCH-446 were different with that of BON-1, the ratio of S phase arrested in LCC-18 cells rose from 29.48 % of control to 64.12 % and 68.98 % of free ETO and ETO-CCLMs, and the ratio of G2 phase arrested in LCC-18 cells rose from 25.85 % of control to 72.56 % and 72.88 % of free ETO and ETO-CCLMs, respectively. 3.10. In vivo imaging and tumor accumulation of ETO-loaded CCLMs Nanocarriers for drug delievery to tumor sites were considered to accumulate in tumor tissue via passive and active targeting. As demonstrated above, CCLMs with excellent colloidal stability and enhanced FL imaging were promising to have long blood circulating time and improved tumor aggregation. Therefore, the biodistribution of


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ETO-loaded CCLMs was essential to evaluate the anticancer effect and the side effects. In vivo FL imaging of isolated tissues were observed at 12 h and 24 h post-injection of ETO-loaded CCLMs as saline administration for negative control (Fig. 6a). FL intensity at 12 h was enhanced in tumor compared to control group, indicating the accumulated effective of ETO-loaded CCLMs (Fig. 6b). At 24 h, FL intensity of tumor was further increased. In addition, gradually increasing FL intensity in liver was the proof of blood circulation of ETO-loaded CCLMs. These results suggested that ETOloaded CCLMs could effectively accumulate in NENs xenografts and hardly accumulated in other organs except the liver. 3.11. The antitumor effect of ETO-loaded CCLMs in vivo Then, mice xenografts model was employed to investigate the tumor growth inhibition ability of ETO-loaded CCLMs. The control groups were treated with saline. Free ETO and ETO-loaded CCLMs significantly inhibited the tumor growth with tumor volume of 374 and 480 mm3 respectively, while it was ~1,000 mm3 in control groups (Fig. 6c). These results indicated that ETO and ETO-loaded CCLMs were effective in repressing tumor growth. The toxicity induced by ETO was examined by body weight loss of mice while the control group had no side effect on body weight loss (Fig. 6d). However, distinct side effects could be observed in free ETO-treated mice. By contrast, only a slight body weight loss could be observed for ETO-loaded CCLMs-treated mice, demonstrating lower systemic toxicity compared with free ETO. Hence, ETO-loaded CCLMs could be a potential selection for NENs treatment with considerable antitumor effect and decreased side effects. 3.12. Histological analysis To investigate the toxicity and antitumor effects of ETO-loaded CCLMs, histological tissue sections of tumors and major organs were prepared. The toxicity effects of ETO were mainly located on liver and kidney and no obvious abnormity in heart, spleen and lung by H&E staining (Fig. 6e). Nevertheless, less toxicity was observed in liver and kidney of mice treated with ETO-loaded CCLMs compared with free ETO. Notably, ETO-loaded CCLMs had better antitumor effects against NENs when compared to free ETO. These data revealed that ETO-loaded CCLMs might have the capability to improve tumor therapy efficacy with slight adverse effects. To further explore the anti-tumor effects of ETO-loaded CCLMs on NENs, immunohistochemistry and TUNEL assays were performed. Control groups exhibited higher protein expression of Ki-67, which was a vital cellular proliferation marker (Fig.



S6). ETO-loaded CCLMs treated groups demonstrated significant lower protein expression of Ki-67 than free ETO-treated ones, indicating that ETO-loaded CCLMs could inhibit tumor growth effectively. In addition, ETO-loaded CCLMs were more efficient in tumor growth inhibition than free ETO by TUNEL assay. Collectively, these results demonstrated that ETO-loaded CCLMs exerted improved antitumor effects against NENs in vivo.

4. Conclusions In summary, a multifunctional micelle system combined chemotherapy with bioimaging had been developed for NENs oncotherapy. Successful core cross-linking of micelles was easily achieved and stable. ETO-loaded CCLMs showed pH and reduction dual-sensitive drug release based on the reverse borate bonds and disulfide bonds. In vitro results revealed that ETO-loaded CCLMs could effectively be assimilated by NENs cells via SSTR-mediated endocytosis which could be detected by AIE imaging, inducing remarkable cytotoxicity to NENs cells. Furthermore, in vivo FL imaging revealed the increasing accumulation of ETO-loaded CCLMs in tumor sites, which endowed ETO-loaded CCLMs with considerable tumor growth suppression efficacy and reduced adverse effects compared with free ETO. This multifunctional polymeric micelle system showed enormous potential for NENs theranostics. ACKNOWLEDGEMENTS This study was supported by the National Natural Science Foundation of China (No.81470806), Medical Key Talents Project of Jiangsu Province (Grant No. ZDRCA2016008) and the “333” Project of Jiangsu Province (Grant No. BRA2017535). Conflict of Interest The authors declare no conflict of interest.

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Figures and Figure legends Scheme 1. Schematic illustration of ETO-loaded CCLMs with SSTRs-mediated endocytosis for pH- and redox-triggered ETO release. Scheme 2. Synthesis of mPEG-b-PGu(BA-TPE) and OCT-PEG-b-PGu(DA-TPE) Figure 1. 1H-NMR (500 MHz, DMSO-d6) of the mPEG-b-PBLG (a), HOOC-PEG-bPBLG (b), OCT-PEG-b-PBLG (c), mPEG-b-PGu(BA-TPE) (d) and OCT-PEG-bPGu(DA-TPE) (e). Figure 2. Dilution and Stability of micelles. AIE effect and Tyndall effect of CCLMs and UCLMs (a). Sample a represents the UCLMs in H2O, sample b represents the UCLMs in DMSO and sample c represents CCLMs in DMSO. The FL intensity of CCLMs and UCLMs in H2O or DMSO (b). The size distribution of CCLMs in DMSO (c). The SLI of CCLMs and UCLMs with SDS addition (d). AIE photos of CCLMs and UCLMs with SDS addition after incubation for 70 min at 37 oC (e). Figure 3. The size distribution (a) and morphology (b) of ETO-loaded CCLMs; ETO release from micelles in pH 7.4 with 0 GSH, pH 6.0 with 0 GSH and pH 5.0 with 0 GSH (c); ETO release from micelles in pH 7.4 with 20 μM GSH, pH 6.0 with 20 μM



GSH and pH 5.0 with 2 mM GSH (d). (All data were analyzed by Student’s t-test. *p < 0.05, **p < 0.01 and ***p < 0.001, n = 3). Figure 4. Fluorescence microscopy images of cells with or without OCT treated to block corresponding SSTRs before incubation with CCLMs for 1 h. Cell viability of NENs cells treated with mPEG-b-PGu(BA-TPE) and OCT-PEG-b-PGu(DA-TPE) at the concentration of 500 μg/mL (a); concentration-dependent cell viabilities of BON1, LCC-18 and NCH-446 incubated with free ETO and ETO-loaded CCLMs for 48 h (b). (All data were analyzed by Student’s t-test. *p < 0.05 and ***p < 0.001, n = 3; scale bar: 100 μm). Figure 5. Cell apoptosis and cell cycle for NENs cells. (a) FACS detection of apoptotic cells of BON-1, LCC-18 and NCH-446 treated with free ETO and ETO-CCLMs for 24 h. (b) FACS detection of cell cycle analysis of NENs cells treated with free ETO and ETO-CCLMs for 12 h. (c) Quantification of apoptotic cells. (d) Quantification of the G1, S and G2 phase. Figure 6. Fluorescence images (a) and FL intensity (b) of excised tissues and tumors at 12 h and 24 h post-injection of ETO-loaded CCLMs with saline administration for control group; changes of tumor volume (c) and mice body weight (d) treated with saline, free ETO, and ETO-loaded CCLMs. Histologic analysis of heart, liver, spleen, lung, kidney and tumor tissues with H-E staining in mice treated with saline, free ETO and ETO-loaded CCLMs (e). (All data were analyzed by Student’s t-test. **p < 0.01, ***p < 0.001 and ns: no significant difference, n= 3; scale bar: 2 cm).


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Schematic illustration of ETO-loaded CCLMs with SSTRs-mediated endocytosis for pH- and redox-triggered ETO release.



Synthesis of mPEG-b-PGu(BA-TPE) and OCT-PEG-b-PGu(DA-TPE) 189x237mm (300 x 300 DPI)


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1H-NMR (500 MHz, DMSO-d6) of the mPEG-b-PBLG (a), HOOC-PEG-b-PBLG (b), OCT-PEG-b-PBLG (c), mPEG-b-PGu(BA-TPE) (d) and OCT-PEG-b-PGu(DA-TPE) (e). 195x138mm (300 x 300 DPI)



Dilution and Stability of micelles. AIE effect and Tyndall effect of CCLMs and UCLMs (a). Sample a represents the UCLMs in H2O, sample b represents the UCLMs in DMSO and sample c represents CCLMs in DMSO. The FL intensity of CCLMs and UCLMs in H2O or DMSO (b). The size distribution of CCLMs in DMSO (c). The SLI of CCLMs and UCLMs with SDS addition (d). AIE photos of CCLMs and UCLMs with SDS addition after incubation for 70 min at 37 oC (e).


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The size distribution (a) and morphology (b) of ETO-loaded CCLMs; ETO release from micelles in pH 7.4 with 0 GSH, pH 6.0 with 0 GSH and pH 5.0 with 0 GSH (c); ETO release from micelles in pH 7.4 with 20 μM GSH, pH 6.0 with 20 μM GSH and pH 5.0 with 2 mM GSH (d). (All data were analyzed by Student’s t-test. *p < 0.05, **p < 0.01 and ***p < 0.001, n = 3).



Fluorescence microscopy images of cells with or without OCT treated to block corresponding SSTRs before incubation with CCLMs for 1 h. Cell viability of NENs cells treated with mPEG-b-PGu(BA-TPE) and OCT-PEGb-PGu(DA-TPE) at the concentration of 500 μg/mL (a); concentration-dependent cell viabilities of BON-1, LCC-18 and NCH-446 incubated with free ETO and ETO-loaded CCLMs for 48 h (b). (All data were analyzed by Student’s t-test. *p < 0.05 and ***p < 0.001, n = 3; scale bar: 100 μm).


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Cell apoptosis and cell cycle for NENs cells. (a) FACS detection of apoptotic cells of BON-1, LCC-18 and NCH446 treated with free ETO and ETO-CCLMs for 24 h. (b) FACS detection of cell cycle analysis of NENs cells treated with free ETO and ETO-CCLMs for 12 h. (c) Quantification of apoptotic cells. (d) Quantification of the G1, S and G2 phase.



Fluorescence images (a) and FL intensity (b) of excised tissues and tumors at 12 h and 24 h post-injection of ETO-loaded CCLMs with saline administration for control group; changes of tumor volume (c) and mice body weight (d) treated with saline, free ETO, and ETO-loaded CCLMs. Histologic analysis of heart, liver, spleen, lung, kidney and tumor tissues with H-E staining in mice treated with saline, free ETO and ETOloaded CCLMs (e). (All data were analyzed by Student’s t-test. **p < 0.01, ***p < 0.001 and ns: no significant difference, n= 3; scale bar: 2 cm). 156x170mm (300 x 300 DPI)


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Redox

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