Sensitivity to antitubulin chemotherapeutics is potentiated by a photoactivable nanoliposome

Accepted Manuscript Sensitivity to antitubulin chemotherapeutics is potentiated by a photoactivable nanoliposome Xiaobing Wang, Xiufang Liu, Yixiang Li, Pan Wang, Xiaolan Feng, Quanhong Liu, Fei Yan, Hairong Zheng PII:

S0142-9612(17)30433-7

DOI:

10.1016/j.biomaterials.2017.06.034

Reference:

JBMT 18154

To appear in:

Biomaterials

Received Date: 1 February 2017 Revised Date:

2 June 2017

Accepted Date: 22 June 2017

Please cite this article as: Wang X, Liu X, Li Y, Wang P, Feng X, Liu Q, Yan F, Zheng H, Sensitivity to antitubulin chemotherapeutics is potentiated by a photoactivable nanoliposome, Biomaterials (2017), doi: 10.1016/j.biomaterials.2017.06.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Schematic diagram showing formation, light controlled PTX/DVDMS release and

AC C

EP

TE D

synergy of photochemotherapy.

ACCEPTED MANUSCRIPT

Title page Title: Sensitivity to antitubulin chemotherapeutics is potentiated by a photoactivable nanoliposome

Quanhong Liua, Fei Yanb* and Hairong Zhengb* *The corresponding authors: Fei Yan & Hairong Zheng

Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry,

SC

a

RI PT

Authors: Xiaobing Wanga, b, Xiufang Liua, b, Yixiang Lia, Pan Wanga, Xiaolan Fenga,

M AN U

Ministry of Education, College of Life Sciences, Shaanxi Normal University, Xi’an Shaanxi, China. b

Paul C. Lauterbur Research Center for Biomedical Imaging, Institute of

Biomedicaland Health Engineering, Shenzhen Institutes of Advanced Technology,

TE D

Chinese Academy of Sciences, Shenzhen, China.

AC C

EP

E-mail: Fei Yan, [email protected]; Hairong Zheng, [email protected].

ACCEPTED MANUSCRIPT Sensitivity to antitubulin chemotherapeutics is potentiated by a photoactivable nanoliposome ABSTRACT

RI PT

Anti-microtubule therapy represents one of the most strategic cancer therapeutics. Tublin inhibitor such as paclitaxel (PTX) is well known to disturb the dynamic nature of microtubules, being considered as the first-line drug for various malignancies.

SC

However, PTX does not show favorable clinical outcomes due to serious systemic

M AN U

toxicities and low selectivity. The development of PTX delivery systems and combinational therapies has been conducted to enhance PTX efficacy with poorly defined mechanisms. Herein, we introduced a reactive oxygen species producible composite liposome based on a new photosensitizer sinoporphyrin sodium (DVDMS)

TE D

to enhance the therapeutic effect of PTX through photochemical stimulation, and more importantly, the pivotal molecular regulation mechanisms were specifically explored. Compared with DVDMS-liposome (DL) or PTX-liposome (PL), the

EP

composite liposome DVDMS-PTX-liposome (PDL) exhibited a superior anti-tumor

AC C

advantage following laser irradiation against MCF-7 breast cancer. The localized PTX release after PDL administration greatly decreased the drug dosage and laser power required, leading to much higher safety and lower costs. In vitro, the combined treatment significantly suppressed cell viability and potentiated cell apoptosis. The apoptotic central regulator Mcl-1 as a favorable target, was evaluated in association with photochemically enhanced sensitivity to anti-tubulin chemotherapeutics. Phosphorylation of Mcl-1 led to its direct degradation with the proteasome system,

ACCEPTED MANUSCRIPT making it relatively unstable and potentiating cell death resulting from photochemical synergy via PDL plus laser irradiation. Further, a decrease in ATP production and glycolysis after PDL plus laser would prevent the possible energy-switch and

RI PT

apoptosis-escape by PTX alone treatment, thereby resulted in increased cell death in combinational therapy. Systemic administration of PDL followed by in vivo photochemotherapy achieved significantly improved therapeutic effects compared to

SC

either alone. And, the intrinsic fluorescence of DVDMS facilitated real-time imaging

M AN U

of PDL in tumors. Therefore, the present strategy with details at the molecular regulation could be a promising platform for antitublin chemotherapeutics. Keywords: Antitublin chemotherapeutics, photoactivable nanoliposomes, Mcl-1, energetic metablisim, mitochondrial apoptosis

TE D

1. Introduction

Anti-microtubule therapy represents one of the most strategic cancer therapeutics [1]. Microtubules participate in multiple cellular activities, such as cell division, cell

EP

motility, cell metabolism, etc. Microtubule targeting agents interfere with the

AC C

microtubule function and arrest cells in mitosis, eventually leading to cell death [2]. The tubulin inhibitors in particular taxanes, paclitaxel continue to be clinical focus for various cancers. Paclitaxel (PTX) is a well developed microtubule stabilizer, binding to tubulin protein to induce microtubule polymerization and cell cycle arrest [3]. Although prolonged cycle arrest leads to initiation of apoptosis, which often suffer from the lack of tumor specificity and drug resistence [4, 5]. Hence, the development of PTX delivery systems, including the use of liposomes, polymeric nanoparticles,

ACCEPTED MANUSCRIPT micellar

dispersions,

and

cyclodextrin

complexes,

have

attracted

the

biopharmaceutical experts for developing efficient tumor targeting [6, 7]. Liposomal formulations represent the optimization of PTX delivery, with Lipusu® (liposomal

RI PT

PTX approved by the State FDA of China) being recently used in clinical applications [8]. This conventional preparation is composed of phospatidylcholine and phosphatidylglycerol. Nevertheless, despite a considerable reduction in the side

SC

effects, the tumor cell killing efficacy of PTX was not equally improved [9].

M AN U

Therefore, the challenge still remains to enhance cancer cell sensitivity to PTX and simultaneously reduce undesirable side effects.

Nanoscale drug delivery systems (DDS) have been developed to improve targeting drug delivery, exploiting the inherent passive tumor accumulation and the

TE D

regulatable potential for drug release at the specific sites of interest [10, 11]. An appealing approach is the use of light responsive DDS [12-14], through which spatiotemporal drug release can be controlled in response to a wide range of light

EP

wavelengths; this can be achieved by intracellular optogenetics [15] or through the

AC C

external loading of light-sensitive compounds such as porphyrins [16]. The co-loading of a porphyrin without the use of genetic engineering or chemical reactions is a particularly safe and convenient route to externally activate drug release [17]. Previous studies have mainly focused on the site-specific light responsive drug release from various polymeric micelles as well as the synergistic combination of chemo- and phototherapy [18-21] leading to enhanced therapeutic effects with reduced drug dosages, suggesting a potential clinical cancer treatment platform. Pasparakis [22]

ACCEPTED MANUSCRIPT utilized the first generation photosensitizer hematoporphyrin entrapped in bio-degradable polymers to exert photo-induced photochemotherapy. Similar designs have been made by others to sensitize chemotherapeutics [23], athough the

RI PT

photochemical internalization and photolysis were proved, the molecular details regarding photochemotherapy remain unclear, which greatly restrict their further development.

SC

In fact, few papers have addressed how and in what ways the loaded anti-cancer

M AN U

agents would be sensitized in a light-controlled DDS. Antitubulin agents, such as PTX and vincristine, target microtubules and thus block mitotic progression [24]. Following a prolonged mitotic arrest, cells typically face two fates: they either die in mitosis via apoptosis, or they undergo a process known as slippage, whereby they exit

TE D

mitosis without division [25]. Identifying how the balance between apoptosis and slippage can be tipped in favor of death, thereby sensitizing cancer cells to antimitotic drugs, remains unaddressed. Bcl-2 familily proteins are crutial regulators of cell

EP

apoptosis [26]. Studies indicate that the anti-apoptotic members such as Bcl-2, Bcl-xl,

AC C

Mcl-1, may confer resistance to antitubulin agents in tumors and promote tumor cells to evade apoptotic pathway through cellular upregulation [27]. Recent advances indicate Mcl-1 is an important regulator of mitotic cell fate [28], and the different phosphorylation sites of Mcl-1 would result in a distinct cell fate. In the prolonged mitotic period induced by microtubule stabilizing compounds, increased Mcl-1 expression enables the cells to survive. Conversely, inhibition of Mcl-1 has been shown to induce apoptosis [29]. Although Mcl-1 is known to oppose cell death,

ACCEPTED MANUSCRIPT precisely how it functions in response to photodynamic therapy and combined photo/chemotherapy is poorly understood. In the present study, a new PEGylated liposome simultaneously co-loaded with PTX and an excellent sensitizer used in

RI PT

photodynamic therapy (PDT), sinoporphyrin sodium (DVDMS) [30, 31], was designed (named as PDL) (Fig.1a). We showed the photochemical catalysis provided potential to improve the efficacy of PTX through phosphorylation of Mcl-1 and

SC

subsequent degradation, thus enhancing mitochondrial-dependent cell apoptosis,

M AN U

which might be a useful strategy for overcoming the serious clinical problem of paclitaxel resistence in malignant tumors.

Moreover, studies also indicate that energy switching contributes to mitotic slippage and limits the success of mitotic-targeted therapies [32, 33]. Following

TE D

microtubule poison treatment, an AMPK-dependent increase in glucose uptake and generation of lactate was observed to support cell survival [32]. Mcl-1 is also involved in bioenergetics, which may link mitochondria oxidative phosphorylation

EP

and glycolysis besides its role in apoptosis regulation [34]. Yet, what possible relation

AC C

between cellular energy supply, mitochondria integrity, and Mcl-1 expression needs exploration in this light responsible DVDMS-PTX nanoliposomes. Therefore, we further investigated the functional relevance of mitochondrial damage by studying the rates of mitochondrial respiration (oxygen consumption rate, OCR) and glycolysis (extracellular acid release, ECAR), aiming to explore the metabolic alterations occurring after PDL with laser treatment. The findings may provide new insights into the synergy of photo-antitubulin therapies.

ACCEPTED MANUSCRIPT

2. Experimental Section 2.1. Materials 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy

(polyethylene-glycol)-2000

(DSPE-PEG-2000),

2-dipalmitoyl-sn-glycero-3-phosphocholine

1,

RI PT

1,

(DPPC),

1,

2

-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were purchased from Avanti Polar

SC

Lipids Inc. (Alabaster, AL, USA). PTX were obtained from Zhejiang HISUN

M AN U

Pharmaceutical Co., Ltd (Hangzhou, China). DVDMS (98.5 % purity) was kindly provided by Professor Qicheng Fang from the Chinese Academy of Medical Sciences (Beijing, China). 4′, 6-diamidino-2- phenylindole (DAPI), 2-deoxy-D-glucose and oligomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,

TE D

7-Dichlorodihydrofluo-rescein-diacetate (DCFH-DA) was from Molecular Probes Inc. (Eugene, OR, USA). Guava Viacount Reagent was supplied by Guava Technologies (Hayward, CA, USA). An Annexin V-FITC Apoptosis Detection Kit was obtained

EP

from keygen technology co., LTD (Nanjing, China). In situ cell death detection

AC C

(terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling, TUNEL) kit was purchased from Roche. MG132 was purchase from ApexBio (Houston, TX, USA). GAPDH antibody was obtained from EarthOX (San Francisco, CA, USA). Antibodies raised aganist Cleaved caspase-3, Cleaved poly (ADP-ribose) polymerase (PARP), β-tubulin, β-actin, Mcl-1, phosphorylation of Mcl-1 and Bcl-xL were purchased from Cell Signaling Technology (MA, USA). 2.2. Preparation of liposomes

ACCEPTED MANUSCRIPT Co-encapsulated liposomes of PTX and DVDMS (PDL) were prepared through the thin film-hydration method [35]. Briefly, PTX was added into the mixture of DPPC/DSPE-PEG-2000/cholesterol/DOPC (molar ratio of 58:5:35:2) dissolved in

RI PT

chloroform, which was removed under a nitrogen flow until a thin lipid film was formed. The lipid film was further dried for over 4 h under vacuum and sonicated (40 kHz) for 10 minutes by hydration at 65°C with phosphate-buffered saline (PBS, pH

SC

7.4) containing DVDMS to obtain a final total lipid concentration of 10 mg/mL. After

M AN U

hydration, liposomes were extruded through a polycarbonate membrane (pole size: 100 nm) using a mini-extruder (Avanti Polar Lipids, Alabaster, AL). The untrapped free PTX and DVDMS were removed by size exclusion chromatography using a Sephadex G-50 column. The final liposomes were stored in tight containers at 4°C in

TE D

the dark until further use. A similar method was used to prepare PTX liposome (PL) and DVDMS liposome (DL), as described above. 2.3. Characterization of liposomes

EP

The vesicles’ average diameter and zeta potential were determined using

AC C

dynamic light scattering (DLS) at 25°C by Delsa Nano C Size/Zeta Potential Analysis Instrument (Beckman Instruments, German). Samples were analyzed after appropriate dilution in filtered PBS. Data were expressed as mean ± standard deviation of at least three different batches of each liposomal formulation. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to observe the liposome morphology. For AFM, a sample droplet (5 µL) was deposited on a freshly cleaved mica surface, spread, and dried at room

ACCEPTED MANUSCRIPT temperature. The samples were imaged in air using V-type Si3N4 tip (Olympus OMCL-TR800PSA-1, cantilevers of 200 µm length and spring constants of 0.32 N/m) in an SPM-9500J3 AFM imaging system (SHIMADZU Corporation, Japan). The

RI PT

images were controlled in contact mode with a 0.5-Hz scanning rate. Dimensional analyses were carried out using the “section of analysis” application on the system. For TEM, samples were prepared by depositing a drop of the sample (10 µl) onto

SC

carbon-coated copper grids and dried at room temperature. Then, a small drop of

M AN U

phosphotungstic acid solution (2%wt in water, pH 7.4) was added to the copper grid. The grid was dried overnight in a desiccator prior to TEM observation using the Hitachi model H-600 TEM (Tokyo, Japan) operated at an accelerating voltage of 80 kV.

TE D

2.4. Encapsulation efficiency and stability of liposomes

The encapsulation percentage of DVDMS and PTX in liposomes was determined through a spectrophotometric method. The liposome bilayer was disrupted with 1%

EP

Triton X-100 to release the entrapped drugs. Then, the concentration of DVDMS and

AC C

PTX in the solution was determined with a microplate reader (SpectraMax M5, Molecular Device). The encapsulation efficiencies (EE) of DVDMS/PTX were calculated using the formula: EE% = (Wencap/Wtotal) × 100%. Wencap is the measured amount of drugs in the liposome suspensions after passing through the Sephadex G-50 column, and Wtotal is the measured amount of drugs in the liposome suspensions before passing through the Sephadex G-50 column. The storage stability of liposomes was assessed at 1–6 weeks after preparation.

ACCEPTED MANUSCRIPT The samples (n = 3 batches) were maintained at 4°C. The parameters evaluated included mean diameter, zeta potential, and drug leakage. The mean values of these parameters were compared with those obtained at time zero.

RI PT

2.5. Cell lines and cell culture The human breast cancer cell lines MCF-7 were obtained from the cell bank at the Chinese Academy of Science, which were cultured in Dulbecco’s Modified

SC

Eagle’s Medium (Gibco, Life Technologies, Carlsbad, CA,USA) containing 10% fetal

M AN U

bovine serum, 1% penicillin-streptomycin (penicillin 100 U/mL and streptomycin 100 µg/mL), and 1% glutamine. Cultures were maintained at 37°C with humidity and 5% CO2. Cells in the exponential growth phase with a viability of ≥98 % determined by trypan blue exclusion test were used for this study.

TE D

2.6. Experimental treatment procedure

For PDT stimulus, a home-made laser (excitation wavelength: 635 nm; manufacturer: NingJu photoelectric technology limited company, Xi 'an, China) was

EP

used as the source of excitation light. Laser irradiance was measured using a

AC C

radiometer system (NingJu photoelectric technology limited company). As for in vitro experiments, the laser was used with a power intensity of 30.25 mW/cm2 and an irradiation time of 132 s such that the final dose of light was 4 J/cm2. Briefly, MCF-7 cells in the exponential phase were collected, resuspended in complete culture medium at the required cell density (2×105 cells/mL), placed in 35 mm culture dishes (Corning Company, USA), and cultured up to 80% of confluence. Then, all samples were randomly divided into six groups: control group, PL group (50 ng/mL of PTX),

ACCEPTED MANUSCRIPT DL group (50 ng/mL of DVDMS), and PDL group (DVDMS/PTX at 10, 30, 50 ng/mL). Cells after incubation with different liposomes for 3 h in Opti-MEM (GIBCO), then exposed to laser irradiation and normally cultured for additional times

RI PT

as specified, and subjected to different assays. All experiments were carried out in triplicate. 2.7. In vitro cytotoxic analysis

SC

Cell viability was measured by regular MTT assay and Viacount assay. The MTT

M AN U

assay was based on the reduction of tetrazolium salt to formazan crystals by living cells [30]. Viacount assay using the Guava Viacount Reagent (Millipore, USA) can distinguish viable and non-viable cells based on differential permeability of two DNA-binding dyes. The assay was performed according to the manufacturer’s

TE D

instructions at 24 and 48 h post treatment by flow cytometry (Guava easyCyte 8HT, Millipore, USA). Colony Formation Assay is used to identify the cell proliferation. After treatment, cells (2×103) were seeded into 24-well plates and cultured for 5 days.

EP

Colonies containing more than 50 cells were counted. For imaging the colony

AC C

formation, a picture of each well was taken after staining cells with crystal violet. Then Crystal violet was dissolved using a 33% acetic acid, and the OD ratio at 570 nm was determined using ELx800 Microplate Absorbance Reader (Bio-Tek, USA). 2.8. Mitochondrial damage and apoptosis evaluation Flow cytometric tests of mitochondrial membrane potential changes were performed with a fluorescent dye (Rh123). In brief, the treated cells were harvested and centrifuged at 3500 rpm for 5 min. Pellets were resuspended in 500 µL of PBS

ACCEPTED MANUSCRIPT containing Rh123 (5 µg/mL) for 15 min at 37°C in the dark. Samples were measured by flow cytometry and data expressed as the percentage of cells with lower fluorescence intensity of Rh123 among 10,000 cells. Cell apoptosis was determined

RI PT

through Annexin V-FITC/PI double staining with flow cytometry (NovoCyteTM, ACEA, USA). 2.9. Western blot analysis

SC

Following treatment, whole cell lysates were prepared and analyzed using

M AN U

standard western blotting to examine the levels of apoptosis-related proteins, including cleaved PARP, cleaved-caspase 3, Bcl-xL, and Mcl-1. In Mcl-1 detection, cells triggered by distinct stimuli were cotreated with or without the proteasome inhibitor MG132 (1 µM), to evaluate the role of ubiquitin proteasome system in

TE D

protein degradation. Proteins were separated in SDS-PAGE gels and transferred onto the nitrocellulose membrane. After incubation with blocking buffer, the membranes were incubated overnight at 4°C with primary antibodies. The bound primary

EP

antibodies were then tagged with IRDye 680 Conjugated IgG at room temperature for

AC C

1 h. The infrared fluorescence was detected using the Odyssey infrared imaging system (LI-COR Biosciences, USA). Anti-β-actin and GAPDH were used to ensure equal loading. Significant differences between the control and treatment groups were analyzed with Quantity One analysis software (Bio-Rad Laboratories, USA). 2.10. Intracellular ROS detection and energy metabolism evaluation Intracellular ROS production was tested by flow cytometry with 2,7-dichloro dihydrofluorescein diacetate (DCFH-DA) as previously described [30, 31]. Data were

ACCEPTED MANUSCRIPT expressed as the percentage of cells with higher fluorescence intensity of DCF among 10,000 cells. The OCR and ECAR were measured in real time using a Seahorse Bioscience

RI PT

XFp extracellular flux analyzer to evaluate the cellular energy metabolism post treatment. MCF-7 cells with PL, DL, and PDL were seeded at 6 × 104 cells/well in an 8-well plate and equilibrated with DMEM lacking bicarbonate at 37°C for 1 h in an

SC

incubator lacking CO2. OCR and ECAR readings were taken using a 3 min mix, 1 min

M AN U

wait, and 2 min read cycling protocol. All the XFp data were reported as OCR or ECAR values normalized to cell counts. Measurements are reported in pmol/min for oxygen consumption and mpH/min for extracellular acidification rate. Each experiment was performed at least three times.

TE D

2.11. Confocal observation and fluorescence imaging

To observe intracellular uptake of PDL, we took Rh123 instead of PTX [36] to compose co-encapsulated liposome with DVDMS. MCF-7 were seeded on cover glass

EP

slides in culture dishes and incubated in an incubator for 24 h. Cells were then

AC C

incubated for 3 or 6 h in the presence of PDL with/without laser irradiation and then imaged using fluorescence microscopy. Immunofluorescent staining was performed to evaluate the changes of β-tubulin

at 48 h post treatment. Cells were fixed with 4% paraformaldehyde for 15 min, washed thrice with PBS and permeabilized with 0.5% Triton X-100, blocked with normal goat serum for 45 min at 37°C, and then incubated with primary rabbit antibody against β-tubulin at 4°C overnight. Subsequently, the cells were washed and

ACCEPTED MANUSCRIPT incubated with FITC-labeled goat anti-rabbit IgG. The immunofluorescence-labeled coverslips were imaged with confocal microscopy. 2.12. In vivo and ex vivo optical imaging studies

RI PT

All animal experiments were conducted in compliance with the relevant laws and institutional guidelines and approved by the local ethics committee. BALB/c nude mice were provided by the Department of Experimental Animals, Institute of Process

SC

Engineering Chinese Academy of Sciences (Beijing, China). All mice were kept under

M AN U

specific pathogen-free conditions with standard and sterilized water and food. MCF-7 tumor-bearing mice were prepared by hypodermic injection of 2 × 106 MCF-7 cells in BALB/c nude mice. When the tumors reached an approximate volume of 200–300 mm3, PDL was injected via the tail vein. Using the intrinsic red fluorescence of

TE D

DVDMS, in vivo PDL tumor uptake was imaged at 0.5, 4, 8, 12, 24, and 36 h after administration. At 12 h, three mice were sacrificed for ex vivo imaging of DVDMS fluorescence intensity in the tumor and major organs, including heart, liver, spleen,

EP

lung, and kidney.

AC C

2.13. In vivo efficacy studies

The tumor-bearing mice were randomly assigned to four experimental groups

(with 6 mice per group), namely saline, PL (PTX concentration at 1 mg/kg), DL (DVDMS concentration at 1 mg/kg), and PDL (PTX concentration at 1 mg/kg, DVDMS concentration at 1 mg/kg). When the tumor volume reached 80–100 mm3, the drug was intravenously administered via the tail vein. At 12 h post injection, the mice were exposed to the indicated dose of light irradiation (100 J/cm2). After

ACCEPTED MANUSCRIPT treatment, the body weight and tumor size were monitored every 2 days. The mice were sacrificed on day 17, and the tumors and other organs were immediately harvested for histological staining.

RI PT

For histological analysis, the tumors and major organs were fixed with 10% formalin and were then paraffin-embedded, sectioned, and stained with H&E. To evaluate cell apoptosis in vivo, the paraffin-embedded tumor tissue sections (5 µm)

SC

were examined with a terminal deoxynucleotidyl transferase-mediated dUTP nick-end

3. Results and Discussion

M AN U

labeling (TUNEL) assay kit according to the manufacturer’s instructions (Roche).

3.1. Synthesis and characterization of composite liposomes

Liposomes have been extensively investigated as DDS because of their versatile capacities,

efficient

tumor

accumulating

ability,

and

excellent

TE D

loading

biocompatibility [37]. Herein, the standard formulation of PEGylated liposomes was chosen as a drug loading platform for the photosensitizer DVDMS and the

EP

microtubule inhibitor PTX. DVDMS was highly water soluble and was encapsulated

AC C

within the hydrophilic core of liposomes. On the other hand, PTX was loaded into the hydrophobic liposomal lipid bilayer. The composite liposomes were prepared as previously reported [35], with few modifications (see the experimental section for details).

The average particle sizes of all obtained liposomes were approximately 100 nm as determined by DLS, which are favorable for passive targeting of tumors via the enhanced permeation and retention effect [12]. The diameters of the PTX liposomes

ACCEPTED MANUSCRIPT (PL), DVDMS liposomes (DL), and co-loaded PDL were 105.64±2.21, 108.23±2.26, and 110.45±2.46 nm, respectively (Fig.1c). The zeta potentials were approximately – 4.37, –4.24, and –5.67 mV for PL, DL and PDL, respectively (Supporting Table 1).

RI PT

No significant change in the diameter and zeta potential over 6 weeks could be observed (Fig.1d), suggesting DDS stability. The morphology of the liposomes was examined by TEM and AFM, confirming their nearly spherical shape, relatively

SC

uniform size distribution, and dispersity (Fig.1b). The drug content of the liposomes

M AN U

was calculated based on the linear absorption of DVDMS and PTX. As shown in Supporting Table 1, the loading efficiencies of PTX in PL and DVDMS in DL were 62.13 ± 3.24% and 73.28 ± 4.25% of their feeding amounts, respectively. In PDL, both PTX and DVDMS were incorporated at a roughly 1:1 weight ratio, with a

TE D

loading efficiency of approximately 52%. However, the entrapment efficiency of both PTX and DVDMS in the composite PDL was slightly lower than for either PL or DL alone, likely due to the interactions between drugs and lipid components, which

AC C

EP

affected their spatial arrangement and distribution.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig.1. Systhesis and characterization of composite liposomes. (a) Schematic illustration of the simple synthetic process of PDL liposomes. (b) TEM and AFM images of different liposomes, scale bar in TEM = 200 nm. (c) Dynamic light scattering analysis of liposomes showing an average size distribution around 100 nm. (d) Changes of diameters and zeta potential distributions

TE D

of liposomes after different storage times at 4°C. Data expressed as mean ± standard deviation of three batches. (e) The yield of singlet oxygen using SOSG test in samples containing free DVDMS, DVDMS-liposomes, and PTX-DVDMS-liposomes (PDL) after 4 J/cm2 laser irradiation. PL, PTX liposomes with 50 ng/mL PTX; DL, DVDMS liposomes with 50 ng/mL DVDMS; PDL,

EP

PTX/DVDMS composite liposomes with 50 ng/mL PTX and 50 ng/mL DVDMS.

AC C

To investigate the composite PDL’s potential in photolysis and phototherapy, singlet oxygen production following 635 nm laser irradiation was determined by SOSG sensor, with the results showing that DVDMS in DL and PDL exhibited a high singlet oxygen production ability (Fig.1e), suitable for further photo-triggering of drug release and phototherapy. We also examined the release profiles of PTX and DVDMS from different drug-loaded liposomes upon dialysis against PBS at room temperature. The drug penetration trends were similar, and the amount of both

ACCEPTED MANUSCRIPT DVDMS and PTX released from PL, DL, or PDL was lower than 20 % within 12 h in the absence of laser irradiation (Fig.2a, c). On the other hand, drug release was approximately 45 % and 75 % at 6 and 12 h, respectively, after laser irradiation in DL

RI PT

and PDL (Fig.2b, d); drug release in PL was unaffected by laser irradiation. The pronounced PTX leakage was induced by photosensitization of DVDMS and the oxidative stress enhancing permeability of liposome membranes, in agreement with

SC

previous reports on the photodynamically induced liposome permeabilization derived

AC C

EP

TE D

M AN U

from the oxidation of phospholipids and cholesterol [38, 39].

Fig.2. PTX/DVDMS release from composite liposomes in the absence (a, c) and presence (b, d) of laser irradiation (4 J/cm2). c and d indicate the statistical analysis of a and b, respectively. Data shown are mean ± S.D. (n=3 independnet replicates), asterisks in d denot significant PTX release beween PL with laser group and PDL with laser group. (**P < 0.01, ***P < 0.001 between the groups, one-way ANOVA with Turkey’s test). PL and PL with laser groups indicate PTX release from 50 ng/mL PL liposomes; DL and DL with laser groups indicate DVDMS release from 50 ng/mL DL liposomes; PDL-PTX and PDL-PTX with laser groups indicate PTX release from

ACCEPTED MANUSCRIPT 50 ng/mL PDL liposomes；PDL-DVDMS and PDL-DVDMS with laser indicate DVDMS release from 50 ng/mL PDL liposomes.

3.2. Effects of co-encapsulated liposomes in cultured cells To examine the photocytotoxicity of the composite liposomes, the MTT assay

RI PT

was performed following various treatments in human breast cancer MCF-7 cells. PTX alone or DVDMS+PDT (4 J/cm2) showed a dose-dependent cytotoxicity against

SC

MCF-7 cells, and there were no obvious cytotoxic effects following individual treatment when PTX was ≤ 0.8 µg/mL or DVDMS was ≤ 0.1 µg/mL (Supporting

M AN U

Fig.1a, b). Conversely, in the co-loaded PDL liposomes (weight ratio of encapsulated PTX:DVDMS, 1:1), cell viability decreased with increasing dosage, which declined to 35.43% (P < 0.01) at a PTX/DVDMS concentration of 50 ng/mL 48 h post laser treatment (Supporting Fig.1c). Comparative analysis confirmed that PDL plus laser

TE D

treatment significantly reduced the half maximal inhibitory dosages (IC50) of PTX and DVDMS by 94.7- and 25.8-fold, respectively. Colony formation assay was performed

EP

to evaluate the ability of proliferation and clonogenicity of single MCF-7 cell following PDL plus laser treatment. The relative colony formation rates of DL and PL

AC C

group were 91.56% and 89.56%, respectively; but in PDL groups, the rate decreased significantly with increasing doses, resulting in more significant inhibition of clonogenicity and long-term proliferation compared with DL (P < 0.01) and PL alone (P < 0.01) (Supporting Fig.1d). Viacount assay further confirmed that, compared with either PL or DL with laser, cell survival was greatly reduced in the co-loaded PDL liposomes post laser treatment (Fig.3a), which could be mainly ascribed to the photochemically enhanced chemotherapeutic effect. The combination index (CI) was

ACCEPTED MANUSCRIPT introduced to quantitatively define the distinct effects, namely of synergism (CI > 1.15), addition (CI, 0.85–1.15), or antagonism (CI < 0.85) [40], using the equation CI = EA+B / (EA+EB-EA×EB), where EA or EB is the effect of PL chemotherapy or DL plus

RI PT

laser irradiation-photodynamic therapy, and EA+B is the effect of the combined PDL and laser irradiation. As calculated, the CI of PDL in the presence of laser was far more than 1.15, indicating a strong synergistic effect of photochemotherapy.

SC

Subsequently, cellular apoptosis triggered by the composite liposome was

M AN U

evaluated. A quantitative analysis of apoptotic cells was determined through Annexin-V-FITC/PI staining with flow cytometry. Annexin V positive cells were defined as apoptotic cells, and the results (Fig.3b) demonstrated that PDL with laser irradiation significantly induced the cell apoptosis of MCF-7 cells to 34.15% and

TE D

61.78% by PDL30 and PDL50, respectively, compared with those treated with PL50 (19.39%) or DL50 plus laser irradiation (16.55%), at 48 h post treatment, indicating that PDL plus laser treatment promotes cellular apoptotic response in MCF-7 cells.

EP

Results from western blot further evidenced that PDL plus laser irradiation could

AC C

induce apoptotic cell death of MCF-7 cells. The cell apoptosis markers caspase-3 and PARP were typically cleaved and activated to exert the caspase cascade (Fig.3c), indicating that cell apoptosis was efficiently induced and would be the main cell death mode induced by the synergistic photochemotherapy. We know, recent advances expand cell death modes, eg. cell apoptosis and autophagy interplays well [41, 42], we do not exclude the participation of autophagy in MCF-7 cell death triggered by our treatment protocol.

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig.3. Cell toxicity and apoptosis induction of MCF-7 cells after PDL and laser irradiation (4

EP

J/cm2). Cells after incubation with different liposomes for 3 h in Opti-MEM (GIBCO), then exposed to laser irradiation and normally cultured for additional times as specified, and subjected

AC C

to different assays. (a) Detection of MCF-7 cell viability by Guava Viacount assay. (b) Dot plot graphs show viable cells (Annexin−/PI−), early apoptotic cells (Annexin+/PI−), late apoptotic cells (Annexin+/PI+), and necrotic cells (Annexin−/PI+). (c) Effect of PDL plus laser irradiation on apoptosis-associated protein expression in MCF-7 cells. Cells were treated with PDL30 and PDL50 for 6, 12, and 24 h, respectively, and the expression levels of cleaved caspase-3 and cleaved PARP were analyzed by western blotting. The representative bands are shown in the left, and the right colume graph shows the ratio of protein expression versus β-actin (Error bars represent the S.D. from three independent experiments). *P < 0.05, **P < 0.01 versus control; ##P < 0.01 between the groups. Control, no treatment; PL50, PL liposomes with 50 ng/mL PTX; DL50,

ACCEPTED MANUSCRIPT DL liposomes with 50 ng/mL DVDMS plus laser irradiation; PDL10, PDL30, and PDL50 indicate liposomes co-loaded with equal amounts of PTX and DVDMS, at 10, 30, and 50 ng/mL, respectively, in the presence of laser irradiation.

3.3. Intracellular ROS generation and mitochondrial damage

RI PT

Intracellular ROS generation was examined by flow cytometry using DCFH-DA staining, with the DCF green fluorescence indicating adequate cellular ROS level [30, 31]. Results showed that PL50 did not trigger cellular ROS generation (Fig.4a), while

SC

DL50 plus laser irradiation increased intracellular ROS production. Approximately

M AN U

40.56 % of cells showed high DCF fluorescence, suggesting that a PDT reaction occurred under a 4 J/cm2 laser dose and at 50 ng/mL of DL. We previously reported that DVDMS at low concentrations (50 ng/mL) would not cause obvious ROS generation [31]; here, DL50 caused a considerable increase in ROS, which may be

TE D

attributed to the enhanced cellular internalization of DVDMS following encapsulation into the liposomes. In addition, PDL plus laser treatment enhanced ROS generation in

EP

a dose-dependent manner, with the high DCF green fluorescence cells accounting for 31.10%, 55.62%, and 70.54% of cells in PDL10, PDL30, and PDL50 groups,

AC C

respectively. PDL50 was much more efficient than DL50 in inducing ROS generation under the same laser conditions. We speculated that PTX in PL would not directly contribute to ROS level, but that when PTX was closely co-loaded with DVDMS in PDL, laser irradiation would activate DVDMS to cause instability of liposomes and promote PTX release into the cytoplasm to inhibit microtubule depolymerization, thus facilitating the photochemical reaction. Indeed, it has been reported that some microtubule inhibitors cause cellular microtubule rearrangement, leading to

ACCEPTED MANUSCRIPT mitochondrial dysfunction and release of proapoptotic proteins from mitochondria, increasing ROS generation and decreasing ATP level [43]. We previously found that DVDMS mainly accumulated in the mitochondria of

RI PT

several tumor cells, with DVDMS-mediated photodynamic therapy resulting in serious mitochondrial dysfunction [31]. To investigate whether mitochondria were damaged in MCF-7 cells during PDL plus laser treatment, rhodamine 123 (Rh123) was

SC

used to measure alterations of mitochondrial membrane potential [44]. The data

M AN U

obtained from flow cytometry (Fig.4b) evidenced that the mean fluorescence intensity of rhodamine 123 in PDL with laser treatment significantly decreased (P< 0.01 versus other groups), suggesting mitochondria were affected in close relation with ROS generation. Once the integrity of mitochondria network was damaged, the

AC C

EP

TE D

biognergetics function would be greatly disturbed.

Fig.4. Intracellular ROS generation and mitochondria membrane potential detection. (a) Flow cytometry is used to examine intracellular ROS with DCFH-DA at 0.5 h after treatment in MCF-7 cells. (b) Mitochondrial membrane permeability was evaluated following the various treatments by employing Rh123 at 3 h post exposure. The illustrations of control, PL50, DL50, PDL10, PDL30, and PDL 50 were same as described in Fig.3.

3.4. Cellular energy consumption changes after composite liposomes and laser irradiation

ACCEPTED MANUSCRIPT Glycolysis and mitochondrial oxidation both produce ATP to support cellular energy expenditure. In cancer cells, elevated glycolysis plays a more prominent role in ATP supply than normal cells [45, 46]. Additionally, the two energy supply routes

RI PT

occur at different ratios in distinct tumor lines [47]. Several reports have shown that the tumor energy supply patterns are affected by various stimuli or therapies [48, 49]. The rate of mitochondrial oxygen consumption and glycolysis were assayed using a

SC

Seahorse XFp analyzer in MCF-7 cells as described in the Materials and methods

M AN U

section. The OCAR and ECAR reflect the ratio of mitochondrial and glycolytic flux. As shown in Fig.5a, in vitro cultured MCF-7 cells slightly responded to hypoxia (oxygen level reduced to 5%), but in the presence of 2-deoxy-D-glucose (2-DG; a glucose analogue that cannot be metabolized), cell viability decreased more evidently

TE D

in hypoxia conditions than in normoxia. Conversely, in the presence of oligomycin, an inhibitor of mitochondrial electron transport chain, cell viability decreased but no obvious differences were observed in cell viability changes between hypoxia and

EP

normoxia conditions (Fig.5b); these results suggested MCF-7 tumor cells mainly

AC C

depend on oxidation phosphorylation to supply energy, which also implied mitochondrial dysfunction would be critical for MCF-7 cells. However, when mitochondria are damaged, smart tumor cells are usually prone to glycolysis to promote the efficient conversion of glucose into the macromolecules required for survival with or without oxygen. Under the experimental stimuli applied herein, we observed a reduction in ATP levels in MCF-7 cells after PDL and laser treatment, reflected by a reduced glycolytic capacity (P<0.01) and mitochondrial respiration

ACCEPTED MANUSCRIPT reserve ability (P< 0.05) (Fig.5c-f). We observed glycolysis was somewhat decreased by DL treatment (P< 0.05), which may suppress the metabolism shift from oxidative phosphorylation to glycolysis post PTX treatment, thus aggravating energy deficiency.

RI PT

The blocking of mitotic progression has been proposed as an attractive therapeutic strategy to impair proliferation of tumor cells. However, cells strive to survive through a switching of their energetic requirements, thus extenuating the therapeutic efficacy.

SC

Oxidative respiration is replaced by glycolysis in response to prolonged mitotic arrest

M AN U

[32]. In the present study, the photodynamic therapy disrupted the possible energetic replenishment, thus sensitizing cell apoptosis by PTX induction. The declined ATP

AC C

EP

TE D

production determined cell viability, in agreement with the above cell toxicity effects.

Fig.5. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) after different treatment in MCF-7 cells. (a) Measurements of MCF-7 cell response to hypoxia (oxygen level reduced to 5%) and normoxia in the presence of 2-DG and (b) oligomycin, respectively. (c) OCR and (d) ECAR tests were performed using a Seahorse XFp instrument. (e) ATP levels and (f) glycolysis were also calculated after the various treatments. Data show mean ± S.D. for n=3. *P < 0.05, *P < 0.01 versus control. PL, PTX liposomes with 50 ng/mL PTX; DL, DVDMS liposomes with 50 ng/mL DVDMS and 4 J/cm2 laser irradiation; PDL, PTX/DVDMS

ACCEPTED MANUSCRIPT composite liposomes with 50 ng/mL PTX/DVDMS plus 4 J/cm2 laser irradiation.

3.5. The molecular mechanisms of synergy between PDL and laser irradiation According to the assumptions of this study, PTX and DVDMS were

RI PT

simultaneously encapsulated in liposomes, whereby PTX was located in the lipid bilayer and DVDMS was encapsulated in the inner chamber. Analysis of the intracellular uptake and distribution of PTX/DVDMS would help to better understand

SC

the damage mechanism of composite liposomes after laser irradiation.

It is widely accepted that endolysosomal compartments are involved in the

M AN U

cellular endocytosis of liposomes and this is closely associated with the limited therapeutic effects of liposomes since it leads to degradation of the encapsulated drugs [50]. The light-controlled DDS potentiates the therapeutic effects of liposomes

TE D

through photosensitizer-based membrane oxidation of endosomes [51]. In this study, we utilized a small amount of Rh123 to replace PTX to synthesize composite liposomes. Following co-incubation for 3 h, the green fluorescence signals of Rh123

EP

and the red fluorescence signals of DVDMS were co-localized well and mainly

AC C

distributed in the cytoplasm of MCF-7 cells before laser treatment (Fig.6a), and prolonged incubation time did not increase cellular drug accumulation. Following laser treatment, increased fluorescence signals were observed in MCF-7 cells, suggesting more internalization or retention of DVDMS/Rh123 gained by laser irradiation. We also observed a portion of Rh123 fluorescence separated from DVDMS signal and diffused into the cytoplasm post irradiation, which was caused by the photochemically induced PTX escape from membranous entrapment to facilitate its

ACCEPTED MANUSCRIPT interaction with microtubules. The microtubule stabilizer PTX blocked mitotic progression following efficient release

from

composite

liposomes

by laser

exposure.

After

fixing

and

RI PT

permeabilization, microtubules were observed by fluorescence microscopy using an antibody against β-tubulin (Fig.6b). MCF-7 cells in the control exhibited a well-organized microtubule network throughout the cells, and the cells treated by DL

SC

plus laser and PL were not obviously changed compared with control. In contrast,

M AN U

cells treated with PDL plus laser irradiation presented increasing microtubule disorder, in particular β-tubulin in cells treated by PDL50 showed the most apparent tubulin network influence with the appearance of tubulin bundles at the cell poles. Such disarrangement of the cellular microtubule skeleton was mainly caused by PTX in the

TE D

composite liposomes. By integrating the photo-damage of DVDMS and the

AC C

EP

cytotoxicity of PTX, cellular apoptotic response was potentiated.

Fig.6. Characteristic fluorescence images of MCF-7 cells with or without laser treatment. (a) By using a similar hydrophobicity of Rh123 instead of PTX, cellular PDL uptake with or without laser (4 J/cm2) irradiation was evaluated; scale bar = 100 µm. (b) Changes of β-tubulin cytoskeleton with laser confocal microscope after different treatment using DAPI counterstain.

ACCEPTED MANUSCRIPT The scale bar is 25 µm. Representative images are shown from three independent experiments. The illustrations of control, PL50, DL50, PDL10, PDL30, and PDL 50 were same as described in Fig.3.

Anti-mitotic agents will either cause cell death via apoptosis or increase cell

RI PT

survival via slippage; therefore, improvement of the efficacy of anti-mitotic drugs would be achieved by accelerating the accumulation of death signals and/or slowing down slippage [52, 53]. In this photochemotherapy study, we further explored the

SC

molecules involved in the PDT chemotherapeutic strategy. Several key regulatory

M AN U

steps are involved in triggering death during a prolonged mitosis, yet Mcl-1 has recently emerged as a central player [54]. Here, we showed that the pro-survival protein Mcl-1 declined significantly after PDT treatment with DVDMS liposomes after 24 h (Fig.7a), and that this treatment stimulated Mcl-1 phosphorylation at the

TE D

Ser-64 site but not at Ser-159/Thr-163. Different phosphorylation sites of Mcl-1 may exert distinct functions and trigger various cellular responses [55]. We presumed that

EP

the Ser-64 phosphorylation initiated the ubiquitin system with the assistance of some other factors to accelerate Mcl-1 degradation and relieve its antagonistic apoptosis.

AC C

Then, we measured Mcl-1 expression in the presence and absence of the proteasome inhibitor MG132 (a wide accepted proteasome inhibitor [56]), results (Supporting Fig.2) show that the decrease of Mcl-1 expression caused by DL50 and PDL50 was obviously alleviated by co-treatment with MG132 (1 µM), suggesting the changes of Mcl-1 level in MCF-7 cells after treatment with nanoliposomes in the present study is partially dependent on proteasome mediated degradation. Because of the complexity of proteasome degradation system and its reciprocal action with another intracellular

ACCEPTED MANUSCRIPT major protein degration system--lysosome-dependent autophagy [57, 58], the precise molecular mechanisms involving in Mcl-1 degradation need further deep investigation.

RI PT

Another important Bcl-2 family member, Bcl-xl, also showed decreased expression post DL and laser treatment (DVDMS-PDT) (Fig.7a). The decline of Mcl-1 and Bcl-xl thereby sensitized MCF-7 cells to PTX, as exhibited by

SC

phosphorylation of Ser-64 and a declined level of Mcl-1 after PDL with laser

M AN U

treatment (Fig.7b). Mcl-1 might be a critical determinant of PTX sensitivity in this study; treatment with DVDMS-PDT decreased Mcl-1 expression, facilitating PTX

AC C

EP

TE D

induced mitotic arrest and intrinsic apoptosis.

Fig.7. Expression levels of Bcl-2 members in DL (a) or PDL (b) plus laser treatment. The treatment protocol was specified in the methods. GAPDH was used as a loading control. The representative bands from three independent experiments are shown in the left, and the right channels indicate statistical analysis by Quantity One® after various treatments. P-Mcl-1 indicates phosphorylation of Mcl-1. (Error bars represent the S.D. for n=3). *P < 0.05, **P < 0.01 versus control; #P < 0.05, ##P < 0.01 between the denoted groups.

ACCEPTED MANUSCRIPT 3.6. In vivo and ex vivo biodistribution studies As is known, the tumor vasculature is much more leaky and usually lacks an effective lymphatic drainage system compared to normal tissues, thus permitting the

RI PT

passive targeting of the tumor site by nanoparticles based on the enhanced permeability and retention effect [16]. Liposomes have been clinically used as good DDS due to their high capacity and excellent biocompatibility. The synthesized

SC

composite liposomes in this study met the basic requirements for passive targeting of

M AN U

tumors, namely a good size, sufficient drug loading, and stable storage. By using the intrinsic red fluorescence of DVDMS, in vivo real time imaging was attained following composite liposome PDL administration in nude mice bearing MCF-7 tumor cells. Fig.8a shows that the intensity of DVDMS signals in the

TE D

xenografted MCF-7 tumor sites (black circle mark) of nude mice increased gradually and reached a maximum at 12 h post tail-vein injection, when the signals exhibited a clear region near the surrounding tissues. Therefore, 12 h post composite

EP

liposome injection was selected as the laser irradiation window. Moreover, to avoid

AC C

organ-organ overlap in whole body imaging analysis, a semi-quantitative biodistribution analysis of each excised organ was performed by using quantitative region-of-interest analysis at 12 h post liposome administration. Besides the tumor, some organs like liver, kidney and lung also presented obvious fluorescent signals, which may be due to the nanoliposomes metabolic pathways after injection into the body [59]. The fluorescent signal of DVDMS in the tumor site was relatively higher compared to other organs (Fig.8b), suggesting that effective passive tumor targeting

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

was attained in the designed system.

Fig.8. In vivo behavior of PDL liposomes after injection via tail vein in MCF-7 breast cancer-bearing mice. (a) By utilizing the intrinsic fluorescence of DVDMS, the real time biodistribution images were taken at different time points post PDL administration. BALB/c nude mice was subcutaneouly xenografted with MCF-7 tumor cells in the left-lower flank and PDL was

TE D

intravenously injected when the tumors were approximately 5~7 mm in length (b) Ex vivo fluorescence images of the major organs/tissues at 12 post injection. The right side panel shows quantitative analysis of ex vivo DVDMS fluorescence intensity in the organs/tissues as listed on

EP

the left. Representive images are shown, results are mean ± S.D. (n=3 mice per group) Asterisks (P < 0.01) denote significance compared to tumor tissues.

AC C

3.7. In vivo photochemotherapy efficacy Previous reports have suggested that PDT in combination with chemotherapy,

named photochemotherapy, has great potential for cancer treatment as it may permit low doses of anticancer drugs and photosensitizers and diminish undesirable side effects [20, 60]. In the present study, the design of composite PDL liposomes shared similar principles with photochemotherapy, namely the use of light to trigger drug release and induce a tumoricidal effect, with excellent potentials in cancer therapy. In

ACCEPTED MANUSCRIPT order to investigate the therapeutic efficacy of PDL after laser treatment, MCF-7 tumor-bearing mice were divided into four groups and were administered with individual liposomes with equal DVDMS or PTX (PL, DL, PDL). PL group mice

RI PT

treated with 1 mg/kg PTX alone exhibited tumor growth similar to that of control mice treated with saline (Fig.9a), indicating that tumor growth was not affected by PL injection. Tumor size decreased to some extent in the DL group with light exposure,

SC

which was induced by DVDMS-PDT (1 mg/kg DVDMS plus 100 J/cm2 light), in

M AN U

agreement with a previous report [30]. While using the same light exposure, tumor growth in the PDL group (1 mg/kg DVDMS, 1 mg/kg PTX) was greatly stagnated, indicating a much more obvious additional tumor inhibition. Histological analysis indicated tumor tissues were seriously damaged by PDL plus laser irradiation, and

TE D

TUNEL staining confirmed notable cell apoptosis was induced in the combined treatment group compared to either monotherapy (Fig.9b). Despite the inspiring therapeutic performance of the composite liposomes, the

EP

potential adverse effects should be evaluated. In this study, body weight was measured

AC C

as an indicator to evaluate the toxicity in all groups during treatment, with no major changes being observed following the various treatments under the therapeutic dosage (Fig.9c). Additionally, the toxicity was also evaluated by histological images in major organs through H&E staining (Fig.9d), indicating that there was no obvious abnormality in normal tissues, which might be attributed to the low concentration of PTX/DVDMS (1 mg/kg) utilized in the current study.

SC

RI PT

ACCEPTED MANUSCRIPT

M AN U

Fig.9. In vivo anti-tumor efficacy. (a) Tumor growth curves after the various treatments. MCF-7 tumor-bearing mice were injected with PL (1 mg/kg PTX), DL (1 mg/kg DVDMS), and PDL (1 mg/kg PTX and 1 mg/kg DVDMS), respectively, and then exposed to 100 J/cm2 laser irradiation. Results are mean ± S.D. (n=8 mice per group). Asterisks (P < 0.01) denote significance. (b) Microscopic observation of tumor sections by H&E staining and TUNEL staining at 24 h post

TE D

laser irradiation; scale bar = 200 µm. (c) Body weight changes of the tumor-bearing mice were measured during the antitumor studies. (d) H&E staining shows no obvious histopathological changes of major organs dissected from tumor-bearing mice after the various treatments; scale bar

EP

= 200 µm.

The findings above also suggested that the combination system established in

AC C

this study could serve as an ideal platform for cancer therapy. The synergy between PTX and DVDMS with laser irradiation must be multifaceted. DVDMS initiated photodynamic therapy to stimulate PTX spatiotemporal release, and sensitized cell apoptotic response to PTX through energy metabolism interruption and signal pathway regulation. Further, efficient PTX release might facilitate the reactivity of ROS for tumor destruction in response to photodynamic therapy. The pinpointed light irradiation after maximum PTX/DVDMS accumulation was also critical for the higher

ACCEPTED MANUSCRIPT therapeutic efficacy.

4. Conclusions In conclusion, we developed a dual-effect liposome with co-encapsulated an

RI PT

anti-mitotic agent (PTX) and a new photosensitizer (DVDMS). Both in vivo and in vitro studies confirmed the composite liposomes possess superior anticancer activity through the synergy of DVDMS-PDT and cytotoxicity of PTX, with minimal side

SC

effects. DVDMS served as a ROS production facilitator boosted entrapped PTX

M AN U

release and strengthened the chemotherapeutic efficiency through a decrease in the core molecule Mcl-1, thus activating cell apoptosis. DVDMS-PDT treatment decreased cellular glycolysis, thereby preventing the possible energy switch and struggling survival post PTX treatment. Therefore, PDL with laser irradiation

TE D

enhanced PTX sensitivity of cells would be attributable to apoptosis susceptibility and Mcl-1 played a pivotal role in the action. We hope that the potential therapeutic effects

applications.

EP

and molecular mechanism of this nanoplatform will facilitate its future clinical

AC C

Acknowledgements

This work was supported by the National Key Basic Research Program of China

(973 Program) (Grant No. 2015CB755500), the National Natural Science Foundation of China (Grant No. 81472846, 81571834, 81527901, 11534013), the project funded by China Postdoctoral Science Foundation (Grant No. 2016M600684), and the Fundamental Research Funds for the Central Universities (Grant No. GK201502009). Supporting Information

ACCEPTED MANUSCRIPT Supporting Information is shown as supplementary material. References [1] J. R. Jackson, D. R. Patrick, M. M. Dar, P. S. Huang, Targeted anti-mitotic therapies: can we improve on tubulin agents, Nat. Rev. Cancer 7 (2007) 107-117.

RI PT

[2] K. Nepali, R. Ojha, H.Y. Lee, J.P. Liou, Early investigational tubulin inhibitors as novel cancer therapeutics, Expert Opin Investig Drugs 25 (2016) 917-936.

[3] E. Nogales, S. G. Wolf, I. A. Khan, R. F. Luduena, K. H. Downing, Structure of tubulin at 6.5

SC

angstrom and location of the taxol binding site, Nature 375 (1995) 424-427.

[4] M. A. Jordan, L. Wilson, Microtubules as a target for anticancer drugs, Nat. Rev. Cancer 4

M AN U

(2004) 253-265.

[5] M. S. Surapaneni, S. K. Das, N. G. Das, Designing Paclitaxel drug delivery systems aimed at improved patient outcomes: current status and challenges, ISRN Pharmacol 2012 (2012) 623139. [6] T. Yang, M. K. Choi, F. D. Cui, S. J. Lee, S. J. Chung, C. K. Shim, et al., Antitumor effect of paclitaxel-loaded PEGylated immunoliposomes against human breast cancer cells, Pharm. Res. 24

TE D

(2007) 2402-2411.

[7] M. V. Barbosa, L. O. Monteiro, G. Carneiro, A. R. Malagutti, J. M. Vilela, M. S. Andrade, et al., Experimental design of a liposomal lipid system: A potential strategy for paclitaxel-based breast cancer treatment, Colloids Surf B Biointerfaces 136 (2015) 553-561.

EP

[8] X. Xu, L. Wang, H.Q. Xu, X.E. Huang, Y.D. Qian, J. Xiang, Clinical comparison between paclitaxel liposome (Lipusu®) and paclitaxel for treatment of patients with metastatic gastric

AC C

cancer, Asian Pac. J. Cancer Prev.14 (2013) 2591-2594. [9] S. K. Ramadass, N. V. Anantharaman, S. Subramanian, S. Sivasubramanian, B. Madhan, Paclitaxel/Epigallocatechin gallate coloaded liposome: A synergistic delivery to control the invasiveness of MDA-MB-231 breast cancer cells, Colloids Surf B Biointerfaces 125 (2015) 65-72. [10] R. K. Jain, T. Stylianopoulos, Delivering nanomedicine to solid tumors, Nat Rev Clin Oncol 7 (2010) 653-664. [11] X. Pang, Y. Jiang, Q. Xiao, A.W. Leung, H. Hua, C. Xu, pH-responsive polymer-drug conjugates: Design and progress, J Control Release 222 (2016) 116-129. [12] M. S. Shim, Y. J. Kwon, Stimuli-responsive polymers and nanomaterials for gene delivery and

ACCEPTED MANUSCRIPT imaging applications, Adv. Drug Deliv. Rev. 64 (2012) 1046-1058. [13] B.Q. Spring, R. Bryan Sears, L.Z. Zheng, Z. Mai, R, Watanabe, M.E. Sherwood, et al., A photoactivable multi-inhibitor nanoliposome for tumour control and simultaneous inhibition of treatment escape pathways, Nat Nanotechnol 11 (2016) 378-387.

RI PT

[14] D. Bai, C.M.N. Yow, Y. Tan, E.S.M. Chu, C. Xu, Photodynamic action of LED-activated nanoscale photosensitizer in nasopharyngeal carcinoma cells, Laser Phys 20 (2010) 544-550.

[15] M. Borowiak, W. Nahaboo, M. Reynders, K. Nekolla, P. Jalinot, J. Hasserodt, et al., Photoswitchable inhibitors of microtubule dynamics optically control mitosis and cell death., Cell

SC

162 (2015) 403-411.

[16] Q. Sun, You Q, X. Pang, X. Tan, J. Wang, L. Liu, et al., A photoresponsive and rod-shape

M AN U

nanocarrier: Single wavelength of light triggered photothermal and photodynamic therapy based on AuNRs-capped & Ce6-doped mesoporous silica nanorods, Biomaterials 122 (2017) 188-200. [17] N. Fomina, J. Sankaranarayanan, A. Almutairi, Photochemical mechanisms of light-triggered release from nanocarriers, Adv. Drug Deliv. Rev. 64 (2012) 1005-1020.

[18] Z. Li, H. Wang, Y. Chen, Y. Wang, H. Li, H. Han, et al., pH- and NIR light-responsive polymeric

TE D

prodrug micelles for hyperthermia-assisted site-specific chemotherapy to reverse drug resistance in cancer treatment, Small 12 (2016) 2731-2740.

[19] X. Feng, D. Jiang, T. Kang, J. Yao, Y. Jing, T. Jiang, et al., Tumor-homing and penetrating photosensitizer-conjugated

PEG-PLA

nanoparticles

for

EP

peptide-functionalized

chemo-photodynamic combination therapy of drug-resistant cancer, ACS Appl Mater Interfaces 8

AC C

(2016) 17817-17832.

[20] T. Wang, D. Wang, H. Yu, M. Wang, J. Liu, B. Feng, et al., Intracellularly acid-switchable multifunctional micelles for combinational photo/chemotherapy of the drug-resistant tumor, ACS Nano 10 (2016) 3496-3508. [21] C. S. Lee, W. Park, S. J. Park, K. Na, Endolysosomal environment-responsive photodynamic nanocarrier to enhance cytosolic drug delivery via photosensitizer-mediated membrane disruption, Biomaterials 34 (2013) 9227-9236. [22] G. Pasparakis, T. Manouras, M. Vamvakaki, P. Argitis, Harnessing photochemical internalization with dual degradable nanoparticles for combinatorial photo-chemotherapy, Nat Commun 5 (2014) 3623.

ACCEPTED MANUSCRIPT [23] P. Thapa, M. Li, M. Bio, P. Rajaputra, G. Nkepang, Y. Sun, et al., Far-Red light-activatable prodrug of paclitaxel for the combined effects of photodynamic therapy and site-specific paclitaxel chemotherapy, J. Med. Chem. 59 (2016) 3204-3214. [24] K. E. Gascoigne, S. S, Taylor. How do anti-mitotic drugs kill cancer cells, J. Cell. Sci. 122 (2009)

RI PT

2579-2585. [25] C. H. Topham, S. S. Taylor, Mitosis and apoptosis: how is the balance set, Curr. Opin. Cell Biol. 25 (2013) 780-785.

[26] R. J. Youle, A. Strasser, The BCL-2 protein family: opposing activities that mediate cell death,

SC

Nat. Rev. Mol. Cell Biol. 9 (2008) 47-59.

[27] A. Watanabe, S. Yasuhira, T. Inoue, S. Kasai, M. Shibazaki, K. Takahashi, et al., BCL2 and BCLxL

Dermatol. 22 (2013) 518-523.

M AN U

are key determinants of resistance to antitubulin chemotherapeutics in melanoma cells, Exp.

[28] I. E. Wertz, S. Kusam, C. Lam, T. Okamoto, W. Sandoval, D. J. Anderson, et al., Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7, Nature 471 (2011) 110-114. [29] J. Cui, W. J. Placzek, PTBP1 modulation of MCL1 expression regulates cellular apoptosis

TE D

induced by antitubulin chemotherapeutics, Cell Death Differ. 23 (2016) 1681-1690. [30] X. Wang, J. Hu, P. Wang, S. Zhang, Y. Liu, W. Xiong, et al., Analysis of the in vivo and in vitro effects of photodynamic therapy on breast cancer by using a sensitizer, sinoporphyrin sodium,

EP

Theranostics 5 (2015) 772-786.

[31] L. Wu, X. Wang, Q. Liu, A. W. Leung, P. Wang, C. Xu, Sinoporphyrin sodium mediated

AC C

photodynamic therapy inhibits the migration associated with collapse of F-actin filaments cytoskeleton in MDA-MB-231 cells, Photodiagnosis Photodyn Ther 13 (2016) 58-65. [32] E. Domenech, C. Maestre, L. Esteb An-Martinez, D. Partidal, R. Pascual, G. Fernandez-Miranda, et al., AMPK and PFKFB3 mediate glycolysis and survival in response to mitophagy during mitotic arrest. Nat. Cell Biol. 17 (2015)1304-1306. [33] E. Komlodi-Pasztor, D. L. Sackett, A. T. Fojo, Inhibitors targeting mitosis: tales of how great drugs against a promising target were brought down by a flawed rationale, Clin. Cancer Res. 18 (2012) 51-63. [34] R. M. Perciavalle, D. P. Stewart, B. Koss, J. Lynch, S. Milasta, M. Bathina, et al., Anti-apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochondrial fusion to respiration, Nat.

ACCEPTED MANUSCRIPT Cell Biol. 14 (2012) 575-583. [35] F. Yan, H. Wu, H. Liu, Z. Deng, H. Liu, W. Duan, et al., Molecular imaging-guided photothermal/photodynamic therapy against tumor by iRGD-modified indocyanine green nanoparticles, J Control Release 224 (2016) 217-228.

RI PT

[36] A. G. Assanhou, W. Li, L. Zhang, L. Xue, L. Kong, H. Sun, et al., Reversal of multidrug resistance by co-delivery of paclitaxel and lonidamine using a TPGS and hyaluronic acid dual-functionalized liposome for cancer treatment, Biomaterials 73 (2015) 284-295.

[37] D. Luo, N. Li, K. Carter, A. Razi, J. Geng, S. Shao, et al., Doxorubicin encapsulated in stealth

SC

liposomes conferred with light-triggered drug release, Biomaterials 75 (2016) 193-202.

[38] A. W. Girotti, Photosensitized oxidation of membrane lipids: reaction pathways, cytotoxic

M AN U

effects, and cytoprotective mechanisms, J. Photochem. Photobiol. B, Biol. 63 (2001) 103-113. [39] D. Luo, N. Li, K. Carter, C. Lin, J. Geng, S. Shao, et al., Rapid light-triggered drug release in liposomes containing small amounts of unsaturated and porphyrin-phospholipids, Small 12 (2016) 3039-3047.

[40] S. Yang, P. Wang, X. Wang, K. Zhang, X. Zhang, Q. Liu, Efficacy of combined therapy with

TE D

paclitaxel and low-level ultrasound in human chronic myelogenous leukemia cell line K562, J Drug Target 21 (2013) 874-884.

[41] H. Wang, D. Li, X. Li, X. Ou, S. Liu, Y. Zhang, et al., Mammalian target of rapamycin inhibitor

EP

RAD001 sensitizes endometrial cancer cells to paclitaxel-induced apoptosis via the induction of autophagy, Oncol Lett 2016, 12 (2016) 5029-5035.

AC C

[42] L. Xu, J.H. Liu, J. Zhang, N. Zhang, Z.H. Wang, Blockade of autophagy aggravates endoplasmic reticulum stress and improves Paclitaxel cytotoxicity in human cervical cancer cells, Cancer Res Treat 47 (2015) 313-321.

[43] S. Kumar, A. K. Chaudhary, R. Kumar, J. O'Malley, A. Dubrovska, X. Wang, et al., Combination therapy induces unfolded protein response and cytoskeletal rearrangement leading to mitochondrial apoptosis in prostate cancer, Mol Oncol 10 (2016) 949-965. [44] F. Ravar, E. Saadat, M. Gholami, P. Dehghankelishadi, M. Mahdavi, S. Azami, et al., Hyaluronic acid-coated liposomes for targeted delivery of paclitaxel, in-vitro characterization and in-vivo evaluation, J Control Release 229 (2016) 10-22. [45] R. A. Gatenby, R. J. Gillies, Why do cancers have high aerobic glycolysis, Nat. Rev. Cancer 4

ACCEPTED MANUSCRIPT (2004) 891-899. [46] N. N. Pavlova, C. B. Thompson, The emerging hallmarks of cancer metabolism, Cell Metab. 23 (2016) 27-47. [47] C. Jose, N. Bellance, R. Rossignol, Choosing between glycolysis and oxidative

RI PT

phosphorylation: A tumor's dilemma, Biochim. Biophys. Acta 1807 (2011) 552-561. [48] M. Goto, H. Miwa, K. Suganuma, N. Tsunekawa-Imai, M. Shikami, M. Mizutani, et al., Adaptation of leukemia cells to hypoxic condition through switching the energy metabolism or avoiding the oxidative stress, BMC Cancer 14 (2014) 76.

SC

[49] J. Zhang, Q. Gao, Y. Zhou, U. Dier, N. Hempel, S. N. Hochwald, Focal adhesion kinase-promoted tumor glucose metabolism is associated with a shift of mitochondrial

M AN U

respiration to glycolysis, Oncogene 35 (2016) 1926-1942.

[50] Y. H. Bae, K. Park, Targeted drug delivery to tumors: Myths, reality and possibility, J Control Release 153 (2011) 198-205.

[51] A. Pashkovskaya, E. Kotova, Y. Zorlu, F. Dumoulin, V. Ahsen, I. Agapov, et al., Light-triggered liposomal release: membrane permeabilization by photodynamic action. Langmuir 26 (2010)

TE D

5726-5733.

[52] K. E. Gascoigne, S. S. Taylor, Cancer cells display intra- and interline variation profound following prolonged exposure to antimitotic drugs, Cancer Cell 14 (2008) 111-122.

EP

[53] H. C. Huang, J. Shi, J. D. Orth, T. J. Mitchison, Evidence that mitotic exit is a better cancer therapeutic target than spindle assembly, Cancer Cell 16 (2009) 347-358.

AC C

[54] T. Song, Z. Wang, F. Ji, Y. Feng, Y. Fan, G. Chai, et al., Deactivation of Mcl-1 by dual-function small-molecule inhibitors targeting the Bcl-2 homology 3 domain and facilitating Mcl-1 ubiquitination, Angew. Chem. Int. Ed. Engl. 55 (2016) 14248-14254. [55] R. Chu, S. E. Alford, K. Hart, A. Kothari, S. G. Mackintosh, M. R. Kovak, et al., Mitotic arrest-induced phosphorylation of Mcl-1 revisited using two-dimensional gel electrophoresis and phosphoproteomics: nine phosphorylation sites identified, Oncotarget 7(2016) 78958-78970. [56] H.C. Zheng, H.Y. He, J.C. Wu, J. Li, S. Zhao, G.F. Zhao, et al., The suppressing effects of BTG3 expression on aggressive behaviors and phenotypes of colorectal cancer: An in vitro and vivo study, Oncotarget 8(2017)18322-18336. [57] P. Tsvetkov, N. Reuven, Y. Shaul, Ubiquitin-independent p53 proteasomal degradation, Cell

ACCEPTED MANUSCRIPT Death Differ. 17(2010) 103-108. [58] U.B. Pandey, Z. Nie, Y. Batlevi, B.A. McCray, G.P. Ritson, N.B. Nedelsky, et al., HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS, Nature 447 (2007) 859-863.

RI PT

[59] C. Zylberberg, S. Matosevic, Pharmaceutical liposomal drug delivery: a review of new delivery systems and a look at the regulatory landscape, Drug Deliv 23 (2016) 3319-3329.

[60] J. Zhong, S. Yang, L. Wen, D. Xing, Imaging-guided photoacoustic drug release and synergistic chemo-photoacoustic therapy with paclitaxel-containing nanoparticles, J Control Release 226

AC C

EP

TE D

M AN U

SC

(2016) 77-87.