Boosting the photodynamic therapy efficiency by using stimuli-responsive and AIE-featured nanoparticles

Journal Pre-proof Boosting the photodynamic therapy efficiency by using stimuli-responsive and AIEfeatured nanoparticles Youmei Li, Qian Wu, Miaomiao Kang, Nan Song, Dong Wang, Ben Zhong Tang PII:

S0142-9612(19)30867-1

DOI:

https://doi.org/10.1016/j.biomaterials.2019.119749

Reference:

JBMT 119749

To appear in:

Biomaterials

Received Date: 30 October 2019 Revised Date:

21 December 2019

Accepted Date: 28 December 2019

Please cite this article as: Li Y, Wu Q, Kang M, Song N, Wang D, Tang BZ, Boosting the photodynamic therapy efficiency by using stimuli-responsive and AIE-featured nanoparticles, Biomaterials (2020), doi: https://doi.org/10.1016/j.biomaterials.2019.119749. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphical abstract

Boosting

the

photodynamic

therapy

efficiency

by

using

stimuli-responsive and AIE-featured nanoparticles Youmei Lia,b,c, Qian Wua,b,c, Miaomiao Kanga,b,c, Nan Songa,b,c, Dong Wanga*, Ben Zhong Tangc*

a

Center for AIE Research, College of Materials Science and Engineering, Shenzhen University, Shenzhen 518060, China

b

Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China

c

Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Institute of Molecular Functional Materials, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

*Corresponding author: Dong Wang, Ben Zhong Tang; E-mail addresses: [email protected], [email protected]

Abstract Photosensitizers with aggregation-induced emission (AIE) characteristics are of great interest for cancer theranostics involving both fluorescence imaging and photodynamic therapy (PDT). However, in the purpose of clinical trials of PDT, the development of prominent drug delivery systems for boosting the PDT efficiency of AIE photosensitizers is highly desirable but still remain a challenging task. Herein, a novel strategy is designed and performed for boosting PDT effect based on stimuli-responsive nano-micelles as extraordinary carriers for an AIE photosensitizer, namely MeTTMN. Those presented stimuli-responsive nano-micelles loading MeTTMN exhibit good biocompatibility, excellent stability, appropriate nanoparticle size, high loading efficiency, outstanding imaging quality and significantly promoted PDT performance, eventually making them remarkably impressive and significantly superior to commercially available nano-micelles carried MeTTMN. This study thus offers an ideal template for fluorescence imaging-guided PDT, as well as a promising candidate for clinical trials.

Keywords Stimuli-responsive nanoparticles; Aggregation-induced emission; Photodynamic therapy; High loading capacity; High ROS generation efficiency

1. Introduction Exploring effective technologies for cancer therapy has been captivating much interest, considering the rapidly growing threat of cancer to human health [1-4]. Among numerous advances achieved over the last decade, photodynamic therapy (PDT) that is driven by cytotoxic reactive oxygen species (ROS) generated by photosensitizer (PS) upon light irradiation, is an emerging therapeutic modality with minimal invasiveness, in-situ workability and high spatiotemporal precision, thus representing one of the most effective strategies for cancer treatment, as well as great potential for clinical use [5-9]. Although the utilization of PDT for cancer therapy has achieved initial success, the current situation is still far from ideal. Firstly, conventional PSs such as Rose Bengal, methyleneblue and porphyrin derivatives generally suffer from low generation efficiency of ROS, which impedes the anticancer efficiency by means of PDT [10,11]. Secondly, these PSs exhibit intrinsically weak fluorescence, especially as aggregates [12,13]. The insufficient fluorescence emission is unfavorable to manipulate fluorescence imaging (FLI)-guided PDT, which has been realized to be a prominent protocol for cancer theranostics. Additionally, in the purpose of clinical trials of PDT, the development of drug delivery systems sharing these characteristics of high drug-loading content (DLC), excellent entrapment efficiency (EE), remarkable stability and extraordinary stimuli responsiveness, is highly desirable, but still remain a challenging task. Therefore, the design of versatile multi-functional materials with boosted efficiency of photodynamic therapy is urgently needed. In a related context, the emergence of PSs with aggregation-induced emission (AIE) features has indeed triggered state-of-the-art development of PDT. AIE refers to a unique photophysical phenomenon that a family of fluorophores are barely emissive as single molecules in solution but the emission is significantly intensified in aggregation state [14,15]. AIE luminogens (AIEgens) well perform in fluorescence bioimaging in terms of bio-compatibility, imaging contrast to the organism background and photostability [16-19]. Importantly, it has been demonstrated that AIEgens are able to efficiently generate ROS as aggregates, allowing us to implement high-performance FLI-guided PDT [20-23].

Most of AIE PSs possess high hydrophobicity, which severely limits their biological application in vivo. Utilization of drug delivery system could be a potentially promising strategy for loading and delivering AIE PSs into tumor, in these cases, AIE PSs were encapsulated as the core of nanoparticles through hydrophobic interaction [24-28]. It has been reported that both the dense packing of AIE PSs and the limited oxygen in the nanoparticles, however, could reduce ROS generation efficiency [29]. In order to solve this problem, Liu’s group designed and synthesized AIEsomes based on AIEgen-lipid conjugate, which showed better oxygen exposure, resulting in high ROS generation efficiency and excellent effect of PDT for in vivo study [29]. This work provided a distinctive protocol to boost the efficiency of PDT, nonetheless, the complicated and tricky synthetic procedures significantly restricted its universal applications. In addition, those previously developed drug delivery systems for AIE PSs have other respective and collective drawbacks including inferior DLC, low EE, unsatisfying stability and the lack of stimuli responsiveness towards tumor microenvironment (such as acid environment, and excessive glutathione (GSH) [6,25,27,30-32]. Aiming to perfectly address these issues, stimuli-responsive nano-micelle with special core-shell structure, enhanced permeability and retention (EPR) effect and stimuli-sensitivity, could be an ideal alternative, mainly benefiting from their good drug loading capacity, delivery capacity and controlled release behaviors [33-35]. Nevertheless, the investigation on stimuli-responsive nano-micelle carried AIE PSs achieving boosted PDT effect was rarely reported. Inspired by the intrinsic advantages of stimuli-responsive nano-micelles and AIE PSs in cancer treatment, in this contribution, we designed two kinds of stimuli-responsive nano-micelles carrying a far red-emissive AIE PS, namely MeTTMN [36], aiming to enhance its ROS generation efficiency and PDT effect. As illustrated in Scheme 1, pH-responsive polymers mPEG-Hyd-PCL-CIN (P-Hyd) and redox-responsive polymer mPEG-SS-PCL-CIN (P-SS) were prepared based on hydrophilic poly(ethylene glycol) (mPEG) and hydrophobic caprolactone (ε-CL), meanwhile non-responsive polymer mPEG-b-PCL-CIN (P-Control) was synthesized as control. All the prepared polymers could self-assemble into nano-micelles with core-shell

morphology in aqueous solution spontaneously. It was observed that these nano-micelles can successfully carry MeTTMN showing high-efficiency loading and remarkable stability, resulting from weak π-π interaction between CIN moiety (cinnamic acid) and MeTTMN. After efficient uptake of the MeTTMN-loaded nano-micelles (M-Hyd, M-SS, and M-Control) by cancer cells, the hydrazone bond in M-Hyd was cleaved at lysosome acid environment, and the disulfide bond in M-SS was damaged due to the high concentration of GSH in cancer cells, leading to the disassembly of nano-micelles, as well as the release of MeTTMN. The released MeTTMN exposed more oxygen than in the core of nano-micelles, resulting in much higher ROS generation efficiency and significantly promoted PDT performance.

2. Materials and methods 2.1. Materials Poly(ethylene glycol) (mPEG, Mw = 5000), caprolactone (ε-CL), trana-cinnamic acid, 4-formylbenzoic acid and N,N’-dicyclohexylcarbodiimide (DCC) were purchased from Aladdin. 2-Hydroxyethylhydrazine was obtained from J&K. 2-Hydroxyethyl disulfide was purchased from

Alfa

Aesar.

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium

bromide

(MTT),

2’,7’-dichlorofluorescin diacetate (DCFH-DA), Annexin V-FITC, RPMI-1640, phosphate buffered solution (PBS: pH 7.4), acetate buffered solution (ABS: pH 5.0) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific. MeTTMN was synthesized according to a literature method [33]. All chemicals were used as received without further purification. Cell culture: 4T1 cells were incubated in 1640 medium containing 10% FBS and 1% antibiotics (penicillin-streptomycin) at 37 ℃ under 5% CO2.

2.2. Synthesis of stimuli-responsive polymers mPEG-Hyd-PCL-CIN and mPEG-SS-PCL-CIN Firstly, mPEG-Hyd-OH containing pH-sensitive bond (hydrazone bond, -Hyd-) and mPEG-SS-OH containing redox-sensitive bond (disulfide bond, -SS-) were synthesized according to the reported literatures [37,38]. Secondly, mPEG-Hyd-PCL and mPEG-SS-PCL were obtained

via ring-opening polymerization (ROP) of ε-CL, using mPEG-Hyd-OH and mPEG-SS-OH as macroinitiator

in

the

presence

of

novozyme-435

catalyst.

In

detail,

mPEG-Hyd-OH/mPEG-SS-OH and ε-CL (EG: ε-CL molar feed ratio of 5:1) were added in bulk containing novozyme-435 (10% of ε-CL in weight amount) and anhydrous toluene. The bulk was sealed and placed in an oil bath at 80 ℃. After 4 h polymerization, the products were obtained after filtering, condensing, re-dissolving in CH2Cl2, dialysis and freezing lyophilization. Lastly, the stimuli-responsive polymers mPEG-Hyd-PCL-CIN (P-Hyd) and mPEG-SS-PCL-CIN (P-SS) were constructed by the further modification of mPEG-Hyd-PCL and mPEG-SS-PCL with cinnamic acid (CIN) through esterification reaction [39]. mPEG-Hyd-PCL/mPEG-SS-PCL (0.05 mM) and CIN (0.21 mM) were dissolved in CH2Cl2 (15 mL) containing 0.05 mM DMAP at 0 ℃ under N2 atmosphere. Then 1.0 mM DCC dissolved in CH2Cl2 was slowly added. After stirring for 48 h, the products mPEG-Hyd-PCL-CIN (P-Hyd)/mPEG-SS-PCL-CIN (P-SS) were obtained after further purification, which were confirmed by 1H NMR (AVANCE III 600 MHz, Bruker). Yield: 72%/75%. The control polymer mPEG-b-PCL-CIN (P-Control, without sensitivity) was also prepared according to the above methods.

2.3 Preparation of nano-micelles and characterization The polymer P-Hyd, P-SS and P-Control could self-assemble into nano-micelles in water spontaneously, which was evaluated by critical micelle concentration (CMC) determined by using pyrene as a fluorescence probe according to literature [40]. The mean sizes of nano-micelles were measured at 1.0 mg/mL by dynamic light scattering (DLS) (Nano-ZSP, Malvern Instruments, UK). The result of each sample was an average value of three repeated measurements at 25 ℃. The stimuli-sensitivity and stability were also confirmed by DLS through the changes of size at different incubation time.

2.4 Preparation of MeTTMN-loaded nano-micelles

The MeTTMN-loaded P-Hyd nano-micelles (M-Hyd), MeTTMN-loaded P-SS nano-micelles (M-SS), and MeTTMN-loaded P-Control nano-micelles (M-Control) were prepared by dialysis method, and the whole procedure was performed in the dark. Briefly, the polymers (20 mg) and MeTTMN (10 mg) were mixed in a flask with 4 mL DMF, stirring for 1 h at room temperature. Then the mixture was transferred into dialysis tube (MWCO: 3500 Da) and dialyzed against deionized water for 24 h. In order to remove DMF completely, the water was replaced by fresh water every 4 h. The obtained nano-micelles solution was filtered through 0.45 µm pore-sized syringe to remove residual MeTTMN, and then the purified solution was lyophilized. The commercial polymer DSPE-PEG was also used as control and the MeTTMN-loaded DSPE-PEG nano-micelles (MDSPE-PEG) were prepared in a similar way.

2.5 Characterization of MeTTMN-loaded nano-micelles The sizes and stability of MeTTMN-loaded nano-micelles (1.0 mg/mL) were studied by DLS. Transmission electron microscope (TEM) observations were performed on a JEM-2100 microscope to investigated the morphologies of MeTTMN-loaded nano-micelles. All samples were prepared by placing a drop of micellar solution (0.1 mg/mL) onto the copper grid after staining with phosphotungstic acid. The fluorescence intensity and absorbance of MeTTMN-loaded nano-micelles were measured by fluorospectro photometer (Ex: 507nm) (Edinburgh Instruments FS5) and UV-Vis spectrophotometer (PerkinElmer Lambda 950). The MeTTMN-loading content was measured by dissolving the MeTTMN-loaded nano-micelles in DMF and then determined by a UV-Vis spectrophotometer at 507 nm. Drug-loading content (DLC) and entrapment efficiency (EE) were calculated as blow formulas: DLCwt% = weight of loaded drug ÷ weight of drug−loaded micelles × 100% EE% = weight of loaded drug ÷ weight of drug in feed × 100%

2.6 MeTTMN-loaded nano-micelles phagocytosed by cancer cells The confocal laser scanning microscopy (CLSM, ZEISS-LSM880) was used to evaluate cell

uptake of M-Hyd, M-SS, M-Control and MDSPE-PEG. Firstly, 4T1 cells were seeded in microscope slides containing 1640 culture medium at 37 ℃ under 5% CO2 atmosphere. After 24 h incubation, a series of MeTTMN-loaded nano-micelles (5 µg/mL of MeTTMN) were added and further incubated for different time (2 h, 4 h, 6 h, 8 h), respectively. Then the medium was removed and the cells were gently washed with PBS for three times. Lastly, the cells were re-incubated with 1 mL medium and observed as soon as possible by CLSM. Red fluorescence (MeTTMN, Ex: 488 nm, Em: 550-800 nm), scale bar: 20 µm. The endocytosis inhibition was studied by CLSM. 4T1 cells were seeded in microscope slides and pretreated with three kinds of endocytosis inhibitors for 1 h (10 µg/mL chlorpromazine, 2.0 mM amiloride and 5 µM nystatin) before adding M-Hyd (MeTTMN: 5 µg/mL) containing endocytosis inhibitor. After incubation for another 4 h, the cells were washed with PBS and observed by CLSM as soon as possible. The cells treated with M-Hyd but without any endocytosis inhibitor were used as control.

2.7 ROS generation efficiency studies The ROS generation efficiency in solution was investigated by using DCFH-DA as indicator. The fluorescence intensity of DCFH-DA enhanced upon reaction with ROS. In brief, the activated DCFH-DA solution (DCFH) was added in EP tube containing medium (ABS or PBS with or without 10 mM DTT) and 3 µL MeTTMN-loaded nano-micelles (MeTTMN: 1 mM). Afterward, the mixed solution was irradiated with light (white light, 22.1 mW/cm2) and determined by fluorospectro photometer (Ex: 488 nm, Em: 525nm). The ROS generation efficiency (I/I0-1) was calculated using the maximal FL intensity (I) of every irradiation and the initial FL intensity (I0) without irradiation. The intracellular ROS generation efficiency was also studied in living 4T1 cells using DCFH-DA as indicator. Cells were incubated with M-Hyd, M-SS, M-Control and MDSPE-PEG (at the same concentration of MeTTMN 5 µg/mL) for 4 h, respectively. Then the cells were washed with PBS and incubated with 1 mL fresh medium containing 5 µM DCFH-DA for further

incubation 20 min (24.0 mW/cm2). The cells were washed with PBS again for three times and irradiated by white light for 30 s or 2 min. Lastly, the cells were observed by CLSM. Green fluorescence: DCFH-DA, Ex: 488 nm, Em: 505-540 nm. Scale bar: 20 µm.

2.8 Cytotoxicity study The biocompatibility and cytotoxicity of prepared polymers and MeTTMN-loaded nano-micelles were evaluated by MTT assay in 4T1 cells. Briefly, 6 × 104 cells were seeded in 96-well culture plate by adding 100 µL cells suspension and incubated for 24 h. Then all kinds of polymers and MeTTMN-loaded nano-micelles were added at a series of concentration, respectively. After 4 h incubation, the cells were treated with or without white light irradiation (24.0 mW/cm2, 5 min) and further incubated for another 24 h. 10 µL MTT solution (5 mg/mL in PBS) was added into each well. 4 h later, all the mediums were removed and 150 µL DMSO was added. After that, the absorbance of solvent at 570 nm was measured by microplate reader (BioTek). The relative cell viability was calculated according to the below formula: Asamples − A0 × 100% Acontrol − A0 Asamples, A0 and Acontrol represented the absorbance of samples, the absorbance of cells and Cell viability % =

the absorbance of MTT without samples.

2.9 PDT-mediated apoptosis assay The PDT-mediated apoptosis was studied by Annexin V-FITC assay using CLSM and FCM (flow cytometry, Accuri C6, BD). 4T1 cells were incubated in microscope slides or in 6-well culture plate with MeTTMN-loaded nano-micelles. After 4 h incubation, the cells were treated with or without white light irradiation (24.0 mW/cm2) for 5 min and incubated for another 12 h. Then the cells were washed with PBS and stained with Annexin V-FITC for 15 min before observed via CLSM or collected for FCM assay. Green fluorescence (Annexin V-FITC, Ex: 488 nm, Em: 505-540 nm). Quantitative analysis of green fluorescence of CLSM images was obtained by Image-Pro Plus.

3. Results and discussion 3.1 Synthesis of P-Hyd, P-SS and P-Control As depicted in Scheme 2, two stimuli-responsive polymers and a polymer as control were synthesized. In the preliminary step, the mPEG-Hyd-OH containing pH-sensitive bond and mPEG-SS-OH containing redox-sensitive bond were prepared by modifying mPEG with 2-hydroxyethylhydrazine and 3,3’-dithiodipropionic acid. The desired final polymers were constructed through two steps: enzyme ring opening polymerization (ROP) and esterification reaction. Polymers mPEG-R-PCL (R: Hyd, SS) with good biocompatibility and biodegradability were obtained by ROP of ε-CL using mPEG-R-OH as macroinitiator and novozyme-435 as catalyst. In order to facilitate AIEgens encapsulation via π-π interaction, mPEG-R-PCL was modified by CIN to produce polymers mPEG-R-PCL-CIN (P-Hyd, P-SS and P-Control). All prepared polymers were confirmed by 1H NMR (Fig. S1, S2 and S3). In addition, through the integration of PEG signals at 3.67 ppm and CL signals at 1.75 ppm, the block radio of CL in the amphiphilic copolymers was calculated to be 5.3:1.0, which was close to the feed ratios, suggesting precise control over the polymerization.

3.2 Self-assembly of P-Hyd, P-SS and P-Control and characterizations Due to the appropriate amphiphilicity, the prepared polymers P-Hyd, P-SS and P-Control could self-assemble into nano-micelles in aqueous solution spontaneously, with hydrophilic mPEG as shell and hydrophobic PCL-CIN as core. The formation of nano-micelles was confirmed by critical micelle concentration (CMC) assay. As shown in Table 1 and Fig. S4, the CMC values of P-Hyd, P-SS and P-Control were 7.1, 6.9 and 8.7 µg/mL, respectively, which were similar and quite low, revealing the excellent self-assembling ability of all prepared polymers. Additionally, the size and distribution of prepared nano-micelles were measured by DLS. The particle sizes of P-Hyd, P-SS and P-Control were 97.7, 96.3 and 99.8 nm, with narrow PDI 0.246, 0.211 and 0.216, respectively (Table 1 and Fig. S5A, 5B, 5C), which were

appropriate for cell uptake and biological application. The similar results of both CMC and size of all presented nano-micelles demonstrated that the introduction of sensitive bone (-Hyd-, -SS-) in polymers have negligible effect on the self-assembling ability, avoiding the possible influence on cell experiments. The stability and sensitivity of P-Hyd and P-SS to pH or DL-dithiotheitol (DTT) were evaluated by monitoring the changes of size in corresponding environment using DLS. It was well known that hydrazone bond could be cleaved in intracellular acid environment (lysosome), while kept stable at physiological environment. The disulfide bonds could be cleaved in intracellular reductive environment and kept stable at physiological environment. Therefore, the sensitivity and stability were investigated under the conditions of physiological environment (PBS), acid environment (ABS) and DTT. As illustrated in Fig. 1A and 1B, obvious size distribution changes of hydrazone bond-containing P-Hyd nano-micelles in ABS and disulfide bond-containing P-SS nano-micelles in DTT were observed with the appearance of multiple peaks, suggesting the disassembly of P-Hyd and P-SS nano-micelles with excellent sensitivity upon the corresponding stimuli. The larger particles with the size over 1000 nm could be attributed to the aggregation of hydrophobic PCL-CIN blocks. In contrast, negligible changes of size were determined for P-Control nano-micelles incubated in ABS, PBS, or DTT (Fig. S5F). On the other hand, the sizes of both P-Hyd and P-SS nano-micelles remained constant in PBS, solidly indicating their high stability in the absence of stimuli (Fig. S5D and S5E). All the results revealed that the stimuli-responsive nano-micelles P-Hyd and P-SS would exhibit high stability during long blood circulation and extraordinary sensitivity in cancer cell environment.

3.3 Preparation of MeTTMN-loaded nano-micelles Benefiting from the ingenious combination of strong electron donor-acceptor (D-A) interaction and extended π-conjugation in the structure, MeTTMN was determined to be far-red emissive. Moreover, MeTTMN possesses both various freely rotated molecular rotators and twisted conformation, which lead to weak emission in solution and blocked emission quenching

in aggregates, respectively, endowing it with AIE features [36]. Taking AIEgen MeTTMN as model drug, it was encapsulated into P-Hyd, P-SS and P-Control nano-micelles by dialysis method. In addition, a commercially available amphiphilic co-polymer DSPE-PEG that is one of the most widely used and reputable polymer for encapsulating AIEgens, was employed as comparison. MeTTMN loading was manipulated at 50% feed weight of polymers. Four kinds of MeTTMN-loaded micelles (M-Hyd, M-SS, M-Control and MDSPE-PEG) were obtained and characterized by size, DLC and EE (Table 1). It was shown that the DLC values of M-Hyd, M-SS and M-Control were 31.2%, 31.4% and 29.6%, respectively, which were much higher than that of MDSPE-PEG (9.3%). In addition, the EE values of M-Hyd, M-SS and M-Control were determined to be 85.6%, 85.2% and 84%, respectively, while the EE value of MDSPE-PEG is only 15.2%. The superior loading capability of mPEG-R-PCL-CIN can be attributed to the weak π-π interaction between CIN unit and AIE PS. DLC and EE were two significant factors for evaluating a drug carrier [41]. The higher DLC and EE imply the larger amount of MeTTMN would be delivered to cancer cell at low concentration of MeTTMN-loaded nano-micelles, which was beneficial for clinic application and could reduce the burden of patients to absorb or metabolize carriers. The size distribution of MeTTMN-loaded nano-micelles was measured by DLS. As depicted in Table 1, their size was all around 90 nm with narrow size distribution (Fig. 1C and Fig. S6), and the size was smaller than that of commercial MDSPE-PEG. In addition, TEM images displayed that M-Hyd, M-SS and M-Control have uniform and spherical structure (Fig. S7) and the average sizes were about 50 nm, which were smaller than DLS results. This phenomenon may be reasoned by the possible dehydration of polymer [41]. More importantly, by continuous observing the size of nano-micelles in PBS or 10% FBS, it was found that the size of M-Hyd, M-SS and M-Control did not display any changes over 7 days, revealing excellent stability, which was the prerequisite of nanoagent for long time storage and blood circulation in vivo application (Fig. S8). The stability and sensitivity of M-Hyd, M-SS and M-Control to pH or DTT were also evaluated by monitoring the changes of size in corresponding environment using DLS.

As shown in Fig. S9, obvious size distribution changes of M-Hyd in ABS and M-SS in DTT were observed, while negligible changes of size were determined for M-Control in ABS or DTT and M-Hyd/M-SS in the absence of stimuli, which were consistent with the results of stability for P-Hyd, P-SS and P-Control (Fig. 1 and Fig. S5). Meanwhile, both M-Hyd and M-SS showed the non-uniform morphology and size after incubation in ABS or with DTT (Fig. S10). The results suggested that the stimuli-responsive MeTTMN-loaded nano-micelles M-Hyd and M-SS, as well as P-Hyd and P-SS, would exhibit extraordinary sensitivity in cancer cell environment. As shown in Fig. 1D and 1E, the absorption and emission maxima of MeTTMN-loaded nano-micelles in water were peaked at 507 nm and 680 nm, respectively, similar to the free MeTTMN (absorption: 507 nm; emission maxima: 690 nm), which suggested the photo-properties of MeTTMN wouldn’t be affected after loaded in nano-micelles. At the same concentration of nano-micelles, the fluorescence intensity of four kinds of MeTTMN-loaded nano-micelles were also studied. The fluorescence intensity of M-Hyd, M-SS and M-Control were similar to each other, stronger than that of MDSPE-PEG (Fig. 1F), which may be resulted from the much lower DLC of MDSPE-PEG. Moreover, as shown in Table S1, the quantum yields (4.7% and 5.1%) of MeTTMN in M-Hyd and M-SS dramatically increased comparing with that in solution state (0.4%). The results revealed the nano-micelles with higher DLC would have stronger FL intensity, which was beneficial to in vivo imaging application.

3.4 Cellular uptake of MeTTMN-loaded nano-micelles The in vitro cellular uptake of MeTTMN-loaded nano-micelles in 4T1 cells was monitored by CLSM. As displayed in Fig. S11, weak red fluorescence was found in the cytoplasm after incubating 4T1 cells with M-SS for 2 h. With increasing incubation time to 4 h, the bright red fluorescence was observed, revealing more M-SS internalized by 4T1 cells. Further increasing incubation time from 4 to 8 h, there was no obvious difference of fluorescence intensity. Therefore, the cellular uptake of MeTTMN-loaded nano-micelles was evaluated after 4 h incubation (Fig. 2). The red fluorescence revealed all MeTTMN-loaded nano-micelles could be

internalized by 4T1 cells after 4 h incubation. Among the MeTTMN-loaded nano-micelles, M-Hyd, M-SS and M-Control exhibited stronger red fluorescence than MDSPE-PEG in CLSM images with the same concentration of MeTTMN, which may be explained by the difference of size and DLC. It has been reported that relatively smaller nanoparticles generally possess higher efficiency for being internalized by cancer cells [42]. It seems reasonable to infer that the contents of M-Hyd, M-SS and M-Control with smaller size in 4T1 cells were higher than that of MDSPE-PEG. Furthermore, owing to the higher DLC, more MeTTMN would be delivered into 4T1 cells with the assistance of M-Hyd, M-SS and M-Control during same incubation time. As depicted in Fig. S12, the fluorescence intensities of MeTTMN-loaded nano-micelles remained almost constant after 40 scans within 15 min irradiation, revealing their excellent photostability in 4T1 cells. The in vitro cellular uptake results revealed M-Hyd, M-SS and M-Control with higher DLC and smaller size have great potential in bio-imaging and cancer therapy. As previous literatures reported, nanoparticles entered cancer cells through cell endocytosis [43,44]. To further confirm it and understand the pathway of nano-micelles endocytosed by 4T1 cells, three kinds of inhibitors of endocytosis were added into the cell culture to study the cellular uptake. Chlorpromazine, amiloride and nystatin were used to inhibit clathrin-mediated endocytosis, micropinocytosis and caveolin-mediated endocytosis, respectively. Compared to control group (M-Hyd without inhibitors), the cells treated with chlorpromazine and amiloride showed weaker red fluorescence (Fig. S13). The average of fluorescence intensity in CLSM images was calculated by Image-Pro Plus. Among three kinds of inhibitors, the suppression of endocytosis by chlorpromazine was the strongest, revealing clathrin-mediated endocytosis played major role in the endocytosis of M-Hyd (Fig. 2E). These findings not only explained the pathway of endocytosis, but also confirmed M-Hyd internalized into 4T1 cells through endocytosis, revealing pH-responsive M-Hyd would be internalized into lysosome and damaged at acid lysosome environment, with MeTTMN releasing from M-Hyd.

3.5 Detection of ROS generation efficiency

Oxygen level is crucial for ROS generation and cancer treatment by means of PDT. Therefore, the release of PS from compact nanoparticles could be a promising protocol to promote the ROS generation efficiency and PDT effect of AIE PSs. DCFH-DA was used as ROS indicator to evaluate ROS generation efficiency by monitoring the change of fluorescence intensity at around 525 nm upon white light irradiation. DCFH-DA is non-emissive but the emission can be triggered by in-situ generated ROS. As shown in Fig. 3A and B, the fluorescence intensity of DCFH-DA alone did not significantly enhance with raising irradiation time at PBS. Impressive increase of fluorescence intensity was found upon incubating with pH-responsive M-Hyd at ABS and redox-responsive M-SS at PBS with 10 mM DTT, while much less increase of fluorescence intensity was determined in the presence of M-Control and MDSPE-PEG under the same conditions (Fig. S14B and C). After 10 min light irradiation, in the cases of M-Hyd and M-SS under the corresponding stimuli-responsive environment, the fluorescence intensity enhancements were around 6 fold as that of M-Control and MDSPE-PEG (Fig. 3C), suggesting the excellent performance of stimuli-responsive nano-micelles in term of ROS generation efficiency, which was beneficial for PDT applications. This phenomenon was probably contributed to the release of MeTTMN from destroyed nano-micelles in simulative cancer environment with exposing to more free oxygen. Furthermore,

the

intracellular

ROS

generation

efficiency

of

MeTTMN-loaded

nano-micelles was also assessed using DCFH-DA as ROS sensor in 4T1 cells. As shown in Fig. S15, the green fluorescence was started to be observed after 4 h-incubation of MeTTMN-loaded nano-micelles with 4T1 cells with white light irradiation for 30 s. With increasing the exposure time to white light from 30 s to 2 min, the fluorescence intensity was rapidly boosted, and cells can be clearly visualized with excellent image contrast to the cell background (Fig. 3D, 3E, 3F and 3G). On the contrary, neither DCFH-DA nor MeTTMN-loaded nano-micelles alone provided such a fluorescence change (Fig. 3H, 3I, 3J and 3K). In addition, it was clearly demonstrated that stimuli-responsive nano-micelles M-Hyd and M-SS showed much brighter green fluorescence than M-Control and MDSPE-PEG, which was consistent with the results of

ROS detection in solution (Fig. 3C). These results further confirmed the hypothesis that AIEgens released from nano-micelles could enhance ROS generation efficiency.

3.6 In vitro biocompatibility and photo-toxicity efficacy Inspiring by the remarkable performance of MeTTMN-loaded nano-micelles for ROS production, their biocompatibility and in vitro photo-toxicity efficacies were further investigated in 4T1 cells by MTT assay. In the preliminary study, all prepared amphiphilic polymers including P-Hyd, P-SS and P-Control were tested. It was observed that the cell viability was close to 100% at series of concentrations of each polymer, even up to 800 µg/mL with or without irradiation, indicating their negligible cytotoxicity and good biocompatibility in 4T1 cells (Fig. 4A and Fig. S16). As shown in Fig. 4B to 4E and Fig. S17, all MeTTMN-loaded nano-micelles displayed negligible toxicity to 4T1 cells and normal cells (3T3 and MDCK cells) after incubation 28 h in the dark. Upon white light irradiation for 5 min, a dose-dependent cytotoxicity was found for all of them. The IC50 values (inhibitory concentration of 50% cell death) of M-Hyd, M-SS, M-Control and MDSPE-PEG were determined to be 1.20, 1.25, 2.60 and 5.61 µg/mL (Fig. 4F), respectively. Evidently, the PDT efficiencies of stimuli-responsive M-Hyd and M-SS were indeed much higher than those of M-Control and MDSPE-PEG, and the results were in good accordance with the experimental data of ROS generation. The results of cytotoxicity further solidly confirmed the hypothesis that AIE PSs released from nanoparticles could enhance ROS generation efficiency and PDT effect. Hence, the stimuli-responsive nano-micelles have the great potential as carrier for delivering AIE PSs for boosted PDT effect.

3.7 PDT-mediated apoptosis In addition, the apoptosis of 4T1 cells treated with MeTTMN-loaded nano-micelles were analyzed using Annexin V-FITC as indicator, showing green fluorescence after binding to the phosphatidylserine translocate plasma membrane in apoptotic cells. The green fluorescence was observed in 4T1 cells treated with MeTTMN-loaded nano-micelles upon white 5 min-light

irradiation (Fig. 5A), while almost no green fluorescence could be found in cells without irradiation (Fig. S18), indicating excellent apoptosis of 4T1 cells under irradiation. More interestingly, the green fluorescence of cells treated with M-Hyd and M-SS were brighter than other groups, while the fluorescence intensity of cells treated with MDSPE-PEG was the weakest among all presented MeTTMN-loaded nano-micelles, indicating that there were more 4T1 cells suffered from apoptosis upon treatment with M-Hyd and M-SS. These results demonstrated the stimuli-responsive AIE NPs could boost PDT effect. Furthermore, the outcome of average green fluorescence intensity obtained from CLSM images also confirmed the result (Fig. 5C). The green fluorescence intensity of M-Hyd and M-SS were similar, and 1.6-fold and 3.1-fold higher than that of M-Control and MDSPE-PEG, respectively. These results were attributed to the excellent ROS generation efficiency of M-Hyd and M-SS, resulting from their high DLC and release behaviors of MeTTMN in cancer cells. The higher uptake efficiency of cells towards both M-Hyd and M-SS with smaller size could be another important reason, since a higher accumulation of PS in cells would improve the ROS content effectively. Additionally, flow cytometry analysis using Annexin V-FITC was utilized to determine cell apoptosis. As shown in Fig. 5B, the percentage of cells treated by M-Hyd, M-SS, M-Control and MDSPE-PEG in apoptotic region was 99.7%, 92.4%, 64.0% and 37.4%, respectively. M-Hyd and M-SS showed the greatest apoptosis-inducing capacity, which was consistent with the results of CLSM. The mean fluorescence intensity (MFI) measured by FCM showed the similar results (Fig. 5D). Therefore, we could draw a conclusion that the release behavior of MeTTMN from nano-micelles in cancer cell could enhance PDT effect in cancer treatment.

4. Conclusions In summary, we have developed a simple and excellent strategy to boost PDT efficiency of AIE PSs by preparing stimuli-responsive AIE NPs. Two kinds of stimuli-responsive polymer mPEG-Hyd-PCL-CIN (P-Hyd) and mPEG-SS-PCL-CIN (P-SS) were successfully synthesized, meanwhile, non-responsive polymer mPEG-b-PCL-CIN (P-Control) was designed as control.

The prepared polymers with good biocompatibility can self-assemble into nano-micelles spontaneously in aqueous solution, which are capable of loading an AIE PS MeTTMN with extremely high drug loading capacity, and show well-performed behavior for delivery of AIE PS. The drug-loading content and entrapment efficiency of these polymers towards MeTTMN are enhanced to around 3.2- and 5.5-fold, respectively, comparing with those of commercially available polymer DSPE-PEG. Moreover, the estimation of ROS generation demonstrates that the use of stimuli-responsive nano-micelles carriers provides remarkably promoted ROS generation efficiency at simulative cancer environment than that of control groups. PDT-mediated apoptosis studies by CLSM and FCM indicate that these MeTTMN-loaded stimuli-responsive nano-micelles exhibit high efficiency on inducing 4T1 cells apoptosis. Considering the great significance of AIE PSs, the findings in this study offer an extraordinary strategy for boosting PDT efficiency of AIE PS by a distinctive pathway, which indeed narrows the gap between AIE PS and clinical anticancer therapy.

Acknowledgment This work was partially supported by the National Natural Science Foundation of China (Grant No. 21801169) and Natural science foundation of Shenzhen University (2019004).

Appendix A. Supplementary data Supplementary data to this article can be found online at

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Scheme 1. Illustration of stimuli-responsive AIE NPs with high ROS generation efficiency and boosted PDT effect.

Scheme 2. Synthetic routes to mPEG-Hyd-OH (A), mPEG-SS-OH (B) and mPEG-R-PCL-CIN (mPEG-Hyd-PCL-CIN, mPEG-SS-PCL-CIN and mPEG-b-PCL-CIN) (C).

Table 1 Properties of amphiphilic copolymer/MeTTMN-loaded nano-micelles. Amphiphilic

CMC

Sizea

PDI by

MeTTMN-

Sizea

PDI by

copolymer

(µg/mL)

(nm)

DLSb

Loaded micelles

(nm)

DLSb

P-Hyd

7.1

97.7

0.246

M-Hyd

85.6

0.112

31.2

85.6

P-SS

6.9

96.3

0.211

M-SS

85.2

0.131

31.4

85.2

P-Control

8.7

99.8

0.216

M-Control

84.0

0.129

29.6

84.0

MDSPE-PEG

175.1

0.237

9.3

15.2

DSPE-PEG

DLCc EEc (%)

(%)

a Size of amphiphilic copolymer/MeTTMN-loaded nano-micelles in water shown as mean ± standard deviation (n=3). b Polydispersity index of amphiphilic copolymer/MeTTMN-loaded nano-micelles obtained from DLS measurement. c Measured by a UV spectrophotometer at 507 nm.

Fig. 1. Size change of P-Hyd (A) in ABS and P-SS (B) in PBS with 10 mM DTT at different time; C) DLS size distribution of M-Hyd in water, PBS and FBS (10%); D) Normalized absorption spectra of MeTTMN in DMSO/water mixtures and MeTTMN-loaded nano-micelles in water; E) Normalized PL spectra of MeTTMN-loaded nano-micelles in water and MeTTMN in DMSO/water mixtures; F) The PL spectra of M-Hyd, M-SS, M-Control and MDSPE-PEG at the same concentration of micelles.

Fig. 2. Confocal images of 4T1 cells after incubation with M-Hyd (A), M-SS (B), M-Control (C) and MDSPE-PEG (D) for 4 h. Concentration of MeTTMN: 5µg/mL. E) Relative uptake efficiency of M-Hyd in 4T1 cells in the presence of various endocytosis inhibitors. Ex: 488 nm (1% laser power). Scale bar = 20 µm.

Fig. 3. ROS generation upon light irradiation of MeTTMN-loaded micelles. Relative change in fluorescent intensity (I/I0-1) at 525 nm of DCFH with M-Hyd (A) and M-SS (B) in different condition (PBS with or without DTT, ABS) upon white light irradiation for different time. Concentrations: 1×10-6 M MeTTMN and 5×10-6 M DCFH. C) Relative change in fluorescent intensity (I/I0-1) at 525 nm of DCFH with MeTTMN-loaded micelles upon white light irradiation for 10 min. Detection of intracellular ROS production by DCFH in 4T1 cells after incubation with M-Hyd (D,H), M-SS (E,I), M-Control (F,J) and MDSPE-PEG (G,K), respectively, with (D,E,F,G) or without (H,I,J,K) light irradiation (white light for 2 min). Ex: 488 nm (0.3% laser power). Scale bar = 20 µm.

Fig. 4. 4T1 cells viability after incubation with different concentrations of P-Hyd (A), M-Hyd (B), M-SS (C), M-Control (D) and MDSPE-PEG (E) in the absence or presence of white light irradiation for 5 min. F) The IC50 values of MeTTMN-loaded micelles.

Fig. 5. PDT-mediated apoptosis assay in 4T1 cells using Annexin V-FITC incubated with M-Hyd, M-SS, M-Control and MDSPE-PEG under light irradiation for 5 min, respectively. (A) Cell apoptosis images by CLSM (Ex: 488 nm, 3% laser power). Scale bar = 20 µm. (B) Flow cytometry profiles of cell apoptosis. (C) Quantitative analysis of the fluorescence intensity by confocal using Image-Pro Plus (n = 3). (D) The MFI calculated from the fluorescence intensity by flow cytometry. Cells without any treatment were set as control.

Declaration of interests

√☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: