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Dual-mode imaging guided multifunctional theranosomes with mitochondria targeting for photothermally controlled and enhanced photodynamic therapy in vitro and in vivo Siyu Wang, Fang Guo, Yanhui Ji, Meng Yu, Jinping Wang, and Nan Li Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00351 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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Molecular Pharmaceutics

Dual-mode imaging guided multifunctional theranosomes with mitochondria targeting for photothermally controlled and enhanced photodynamic therapy in vitro and in vivo Siyu Wang1#, Fang Guo1#, Yanhui Ji2, Meng Yu1 Jinping Wang1, Nan Li*1 (1 Tianjin Key Laboratory of Drug Delivery ＆ High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, 300072, Tianjin, PR China.) (2 Department of Nuclear Medicine, Tianjin Medical University General Hospital, 300052, Tianjin, PR China)

*(Li N.) Corresponding author at: School of Pharmaceutical Science and Technology, Tianjin University, 300072, Tianjin, PR China. E-mail address: [email protected]

#Author contribution These authors contributed equally.

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Abstract The photodynamic therapy (PDT) is commonly restricted by the inefficient tumor selectivity during clinical study. Hence, a mitochondria-targeting multifunctional nanocarrier “theranosome (TNS)” was developed for near-infrared fluorescent (NIRF) imaging and photoacoustic (PA) imaging. What’s more, the TNS can also enhance PDT efficacy. In this work, chlorin e6 (Ce6) undertakes reactive oxygen generation and fluorescence emission. Ce6 was quenched when being encapsulated into TNS together with IR780 iodide. When exposed under 808 nm NIR light, IR780 from the TNS can be photobleached, thus the photo-toxicity of Ce6 can be activated. The IR780 induced hyperthermia damaged tumor cells to perform photothermal therapy (PTT) effect. Then lysosomes disruption under PTT facilitated PDT effect induced by Ce6 through enhanced cytoplasmic delivery. Moreover, in vitro subcellular uptake experiments showed that triphenylphosphonium (TPP) group attached to the IR780/Ce6 TNS (ICT) could promote mitochondria targeting capacity. It can lead to PDT induced oxidizing damage to the mitochondria by mitochondrial membrane potential decreasing and cell apoptosis eventually. In in vivo antitumor studies, the TPP/IR780/Ce6 TNS (TICT) substantially repressed tumor growth in nude mice. Besides, we did not found any obvious side effects to normal tissues and organs. The results suggested the TICT conjugate provided a dual NIRF/PA tumor imaging modalities with spatial resolution and superior imaging contrast. This study offered an improved phototherapy for potential theranostic application. Keywords: Theranosome, Mitochondria targeting, NIRF/PA imaging, PTT/PDT synergistic treatment.


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1. Introduction Photodynamic therapy (PDT) is known as a novel strategy for its minimally invasiveness in the treatment of tumor.1-4 PDT contains the exciting light, molecular oxygen and photosensitizer (PS). PDT relies on the accumulation of PS in cancer cells and its activation (production of key cytotoxic agent singlet oxygen) within the tumor upon appropriate wavelength irradiation.5−8 Nevertheless, severe drawbacks still exist in PDT. On one hand, the primary drawback in PDT application is the inefficient tumor selectivity of PS. PS can accumulate in both the cancer cells and the healthy cells, thus causing the indiscriminate damage to the whole tissue. To overcome this drawback, nanocarriers are designed to selectively target to the tumor tissue. What’s more, an organelle (PS hypersensitive subcellular sites) targeted strategy after tumor cellular uptake was supposed. The possibility of targeting PS/nanocarriers to the desired organelle like mitochondria has gained much attention recently. Mitochondria plays a crucial role for cell growth

owing

to

its

special

function

in

energy

generation

and

executing

apoptosis-mediated cell death.9-14 For the PDT, considering mitochondria’s capacity of initiating cells apoptosis, it might be realized to control the PS’s subcellular localization to straightly act on mitochondria. This project is aim to design high-efficiency PDT.15-17 Nowadays, strategies indicated that the conveyance of modified PS to target the mitochondria is more effective compared to the non-targeted strategy. 15,16 In fact, various approaches

were

pushed

out

to

realize

mitochondria-targeting

purpose.

Triphenylphosphonium (TPP), a cation full of delocalized positive charge shows adequate lipophilicity. Therefore, it could easily permeate lipid bilayers and facilitate efficient in



agent accumulation within the mitochondria.18-20 On the other hand, when the normal tissue exposures to the exciting light, PS can be activated to generate nonspecific singlet oxygen. Among different kinds of photosensitizers, chlorin e6 (Ce6) is recommended for PDT. With a 660 nm wavelength NIR light for excitation, Ce6 could achieve a deep-tissue-activation.21-25 However, Ce6 could also produce singlet oxygen under sunlight and subsequently result in side effect such as sunburn and pain.26 That becomes the drawback caused by the undesired Ce6 activation. To overcome this limitation, some strategies have been designed. For example, the PS was supposed to be quenched

before taking action but be “switched on” after

accumulating in the target site by different stimuli including NIR laser irradiation, enzyme, redox or pH change.27-30 NIR laser irradiation has drawn much interest owing to multiple advantages such as noninvasive treatment and deeper tissue penetration.31 The NIR laser sensitive heptamethine dye IR780 iodide (IR780) is a stable and effective photothermal agent, which showed low aggregation, good imaging sensitivity, and preferential accumulation in multiple tumor cells.32,33 In addition, the IR780 dyes also exhibited remarkable properties in NIRF/PA imaging. It performs clear spatial resolution and superior imaging contrast, respectively.32 Upon the absorption spectra, an overlap can be find around 405 nm between Ce6 and IR780. Since the adjacent position of Ce6 and IR780, an electronically excited Ce6 (donor) could transfer its energy to IR780 (acceptor) through fluorescence resonance energy transfer (FRET) effect. In that way, the fluorescence of Ce6 is quenched. However, with the exciting light, Ce6 was “switched on” (fluorescent and high SOG) because of IR780’s photobleaching. More interesting, the NIR


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dye was able to produce rigorous hyperthermia to destroy cancer cells. Meanwhile, it could also lead to the lysosomal membrane disruption during the light irradiation.,34 These discoveries provide potential chances for enhanced cytoplasmic delivery. Herein, Ce6 and IR780 were embedded into the liposomes at the same time. This carrier was named “theranosomes” (TNS) in regard to the combination of multimodal therapeutic and dual imaging properties. TPP ligand was attached to the TNS via direct chemical conjugation to PEG-chain (TICT). The schematic of this system was shown in Scheme 1. After in vitro and in vivo antitumor evaluation, we demonstrated this system was successfully developed for multimodal tumor therapy, realizing tumor imaging property, PTT and multi-enhanced PDT efficacy. Firstly, IR780 induced hyperthermia could destroy cells to produce a unique PTT efficacy and the disruption of lysosomes under PTT improved cytoplasmic delivery of Ce6. And then the TPP group could facilitate Ce6 release and accumulation in mitochondria, enhanced photodynamic activity of Ce6 under subsequent PDT. Besides, Ce6 status (“quenched” or “on”) controlled by NIR irradiation and IR780 facilitated the targeted release of singlet oxygen, which resulted in further PDT efficacy enhancement. 2. Materials and methods 2.1. Experimental

Materials

Chlorin e6 (Ce6, J&K Scientific Ltd, Beijing, China), IR780 iodide (IR780, J&K Scientific

Ltd,

Beijing,

1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy

China), (polyethylene

glycol)-2000] (DSPE-PEG2000, Advanced Vehicle Technology Co., Ltd, Shanghai, China),



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Dipalmitoyl phosphatidylcholine (DPPC, Advanced Vehicle Technology Co., Ltd, Shanghai,

China),

1,2-Distearoyl-sn-glycero-3-phophoethanolamine-N-[amino(polyethylene ammonium]

(DSPE-PEG2000-NH2,

Laysan

Bio

Inc,

USA),

glycol)-2000

(3-carboxypropyl)

triphenyl-phosponium bromide (CTPP, Adamas, Swiss), cholesterol (Chol, Adamas, Swiss), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetrethylbenzimidazolylcarbocyanine iodide (JC-1, Invitrogen-life, USA), acridine orange (AO, Invitrogen-life, USA), Lysotracker Green DND-26 and Mitotracker Green FM (Invitrogen-life, USA ), Fetal bovine serum (FBS, Thermo Fisher Scientific Inc), Thiazolyl Blue (MTT, HEOWNS, Tianjin, China), 4', 6-diamidino-2-phenylindole (DAPI, HEOWNS, Tianjin, China). 2.2. Synthesis of DSPE-PEG2000-TPP DSPE-PEG2000-TPP was synthesized via amide coupling reaction between DSPE-PEG-NH2 (-NH2) and CTPP (-COOH). 1H-NMR analysis of DSPE-PEG2000-TPP polymer in deuterated chloroform (CDCl3) was performed by AVANCE III 600MHz spectrometer (Bruker, GER).35 2.3. Preparation and characterization of TNS Film hydration method was employed to synthesize TNS. Firstly, we dissolved the IR780, Ce6 and lipid mixture (See Table S1) in the methanol and chloroform combined solvent system. After that we utilized rotavapor to remove solvent, and then a lipid thin film was obtained. PBS (pH 7.4) was added to rehydrate the lipid film with sonication simultaneously. In membrane extrusion, we utilized different sizes of polycarbonate filters for sequential extrusion. At last, we removed free Ce6 and IR 780 through dialysis. To


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investigate the morphological characters, we employed JEM-100CXII transmission electron microscopy (TEM, JEOL, Japan) to observe the morphology of TNS. Malvern Zetasizer Nano (Malvern Instruments Ltd., U.K.) was utilized to measure the particle size distribution of TNS. UV-vis spectrum was examined by Cary 60 UV-vis spectrometer (Agilent, USA). The drug release behavior of Ce6 from the TNS was measured by dialysis. The control was free IR780/Ce6 (IC)solution. 808 nm NIR light exposure was the trigger to unleash Ce6. The process was completed in PBS (pH 7.4, 200 mM) at 37 °C constantly. UV-vis absorption of each sample was recorded. 2.4. Monitoring of SOG 1, 3-diaphenylisobenzofuran (DPBF) as the

1

O2 detector can react with

1

O2

inconvertibly. By monitoring the absorption of DPBF at 410 nm, we can evaluate the SOG simultaneously.1 DPBF (30 μL, 1.5 mg/mL) was added into TNS solution (Ce6 2 µg/ml, 2 mL), and further irradiated in periods. The absorbance was saved, respectively. Similarly, to prove SOG, the singlet oxygen sensor green (SOSG) kit was also utilized. 2.5. Photothermal effect measurement The ICT (10 µg/mL IR780) 0.5 mL was stored in glass vials. 808 nm NIR light (1W/cm2) was employed for irradiation. The thermal variation was recorded by thermocouple microprobe during 600s. The IR thermographic maps and maximum temperature was captured by Fluke Ti32 infrared (IR) thermal imaging camera 2.6. Cell culture and cytotoxicity HeLa cells (human cervical cancer cells) were incubated in DMEM culture medium (10% fetal bovine serum, penicillin/streptomycin 100 U/mL) at 37 °C with 5% CO2.



MTT assay was performed to test photocytotoxicity of free IC, ICT and TICT (CCe6=4 µg/mL) under 660 nm, 808 nm or 808nm plus 660 nm NIR light exposure, respectively. HeLa cells (1× 104cells per well) were cultured in the 96-well plates overnight. After 4 h’s incubation with TNS dispersion or free IC, the plates were exposed under NIR light. Each well was washed by PBS and replaced by fresh medium. MTT (5 mg/mL) was added and another 4 h’s incubation was given. In the end, DMSO (100 µL) was added to replace the incubation solution. A microplate reader (Thermo Scientific Varioskan Flash, USA) was utilized to record the absorption at 570 nm. To learn photocytotoxicity straightly, we gave the calcein-AM and PI co-staining to the cells. A confocal laser scanning microscope (CLSM, Olympus, Japan) was employed for observation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) study followed as: cells (1×105/mL) in control and experiment groups were plated in the cell culture dishes, cover-slipped, and incubated overnight. TNS was provided to the experiment group. The control group was saline. After incubated for 6 h, the experiment group was irradiated with NIR laser. The detection was based on the instruction of the TUNEL detection Kit (Roche, 11684817910) to analyze the apoptotic cells. 2.7. Intracellular ROS generation and co-localization imaging DCFH-DA was utilized to examine ROS generation in vitro. The fluorescent signal can be detected when ROS generated in cells, otherwise no signal can be found.36,37At first, cells (1× 10 5 cells/dish) were stabilized in confocal dishes for 12 h and then gave TNS treatment for 4 h’s incubation. After washing by PBS, DCFH-DA was added and then NIR light was irradiated. Fluorescence images were taken by CLSM (Ex=504 nm, Em=510-560


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nm). The cellular colocalization experiments were also performed using CLSM. Cells were incubated with TNS for 12 h and then washed with PBS for three times. After that cells were stained by Mitotracker Green (75 μm) or Lysotracker Green (100 μM) for half an hour. The results was observed by CLSM. To confirm ROS generation, we employed neutralization material tocopherol to quench ROS. Same as DCFH-DA operation. CLSM images were obtained, respectively. 2.8. Mitochondrial membrane potential JC-1 was employed to examine the variation of mitochondria membrane potential. Cells ( 1× 105 cells/dish) cultured with TNS systems in confocal dishes for 8 h (during which the cells were exposed to 808 nm NIR light for 1 min and 660 nm NIR light for 3 min) were stained by fresh JC-1 (1 μg/mL, 37 °C) for half an hour. After that cells were washed by PBS and observed by CLSM ( Ex = 488 nm, Em = 515-540 nm green signal, Em = 570-600 nm red signal). 2.9. Disruption of lysosomal membrane AO was utilized to assess the lysosomal membranes disruption in cells. As an acidic organelle integrity indicator, AO can perform variation by capturing proton. When cells were exposed under blue light, red signal in lysosomes showed the large densityof AO, however, in nuclei and cytosol it displayed greenish fluorescence.37 Cells were incubated with PBS and TNS in fresh medium for 12 h, and then exposed by 808 nm (1W/cm2) NIR light for 3 min. After washing by PBS, cells were stained by AO (5µg/mL, 37 °C) for 15 min. The results was observed by CLSM (Ex = 488 nm, Em = 515-545 nm green signal,



Em = 590-630 nm red signal). 2.10. In vivo study model 2.10.1. Animals and tumor model Female BALB/c nude mice (20 ± 2 g, 4-6 weeks old) were supplied by institute of radiation medicine Chinese academy of medical sciences. HeLa cells were implanted into the flank region of the mice (n=5) subcutaneously. Every other day, tumor size was measured by a caliper from two dimensions. Tumor volume calculation formula: Tumor volume = (tumor length) × (tumor width)2/2. 2.10.2. In vivo NIRF and PA imaging The non-invasive in vivo optical imaging technique was used to analyze the biodistribution and imaging. When the tumors growed up to 100-200 mm3, mice were treated with saline, free IC or TICT (0.5 mg IR780/kg body weight) by i.v. injection, respectively. The in vivo NIR fluorescence images were taken at 2, 12, 24 and 48 h post injection using the Kodak IVIS Spectrum (Ex = 704 nm, Em = 735 nm). After that the organs as heart, liver, spleen, lung, kidney and tumor were excised for analysis. In addition, for in vivo PA imaging, the mice were treated with saline, free IC or TICT (2.5 mg IR780/kg body weight) by i.v. injection, respectively. Then the PA imaging of mice was performed by a Vevo 2100 LAZR system (VisualSonics, Inc., Toronto, Canada) equipped with a 40 MHz, 256-element linear array transducer. ROIs were drawn over the tumor. Ultrasound transducer center frequency: 40 MHz; 2D gain: 18 dB; PA gain: 40 dB; frame rate: 25 fps; data acquisition time: 30 µs; laser wavelength: 808 nm. The average PA imaging signal intensity at tumor site at 0, 6 h, 12 h and 24 h post-injection


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was analyzed. 2.10.3. H&E staining and In vivo efficacy HeLa cells (2 × 106 cells/mouse) were subcutaneously implanted into the flanks of the mice. When the tumors growed up to 200 mm3, formulations as saline, ICT and TICT were injected via tail vein (at dose of 1.5 mg/kg IR780). After that, the tumors were exposed under NIR light. PDT treatment (5 min, 660nm, 0.5 W/cm2), initial PTT treatments (5 min, 808 nm, 1 W/cm2) and subsequent 5 min of PDT treatment (PTT/PDT treatment), respectively. In antitumor efficacy study, the tumor volumes are normalized against the initial volumes (0 day) to examine tumor growth.39 Besides, the organs and tumors were excised and fixed in formalin, embedded in paraffin, sectioned into slices and further stained with hematoxylin and eosin (H&E) to examine organ toxicity. 2.11. Statistical Analysis Data are reported as mean±SD. The differences were analyzed by one-way ANOVA analysis and Student’s t-test (p < 0.05 (*) significant, p < 0.01 (**) highly significantly). 3. Results and discussion 3.1. Synthesis of DSPE-PEG-TPP, Preparation and Characterization of TNS DSPE-PEG-NH2 (-NH2) and CTPP (-COOH) were conjugated by the amido bond. The PEG-chain was modified by mitochondria targeted functional group TPP. (Figure S1a). The proton NMR and UV-vis detection of DSPE-PEG2000-TPP (Figure S1b-c) validated the successful conjugation.35 The TNS were prepared via thin film hydration and membrane extrusion method. Through the UV-vis spectrum of TICT, (Figure 1a) two obvious peaks can be found at the wavelength of 660 nm and 780 nm. These two peaks are



originated from Ce6 and IR780, respectively. Based on the result, we can conclude the successfully embedded of Ce6 and IR780. TEM images and size distribution illustrated the morphological features of Ce6-TNS (CT), ICT, and TICT, respectively. The TNS performs remarkable monodispersity and spherical shape. (Figure 1c-d). The stability of TNS was also studied by measuring hydrodynamic diameter by DLS (Figure 1b). In PBS and FBS, no obvious variation could be found in the size of TNS over a period of 72 h, whereas the particle size increased from 132 to 256 nm after 72 h in the presence of H2O. Besides, the polydispersity index (PDI) as well as size increased on each subsequent measurement until 72 h when large aggregates were formed in H2O. The TNS in PBS and FBS maintained the initial particle size, without precipitation and aggregation at 72 h, suggesting a great stability of TNS. 3.2. PTT effect, singlet oxygen generation, photostability and cargo release To assess photo-thermal efficiency, free IR780/Ce6 (IC), ICT, TICT and PBS were irradiated under the NIR light (808 nm, 1 W/cm2) for 10 min, respectively. The temperature was saved at intervals of 60 s (Figure 2a). In 5 min, PBS could raise 8 °C. In addition, ICT and TICT presented proximate temperature raises (from 23 °C to 50 °C) after exposure, indicating that the TPP structure could not impact the photothermal feature of ICT. Compared to free IC, a higher temperature increase was observed in TICT, suggesting the improved photothermal efficiency of IR780. What’s more, the infrared thermal imager revealed the maximum temperature (Tmax) of PBS, free IC, ICT and TICT at 2 min after laser irradiation reached 34 °C, 52.1 °C, 58.2 °C, and 59.4 °C, respectively (Figure 2c).


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In order to further evaluate photothermal feature, we employed a repeated irradiation detection. In this part, NIR light was irradiated in three cycles. (Figure 2b). In these cycles, the raised temperature generated by TICT was significant higher than those generated by free IC, suggesting the better repeated heat production efficiency. The discovery indicated that liposomal encapsulation could enhance photothermal features of IR780. PTT and PDT efficiencies were significantly impacted by photostability. Thus, the further researches on the photostability of IR780/TNS were provided (Figure S2). UV-vis data revealed the free IC displayed sharply decline of absorbance under 808 nm laser exposure in 2 min, caused by photo-bleaching.40 However, IR780 in TICT presented tardily decline of the absorbance in a same condition. In this work, we discovered that IR780 could quench PDT effect and fluorescence emission induced by Ce6. The fluorescence of free IC, Ce6 TNS (CT) and ICT could be visualized with the fluorescence imaging system at Ex= 405 nm, Em= 550 nm-800 nm. The fluorescence quenching of Ce6 can be discover in ICT solution (Figure 3a). However, after 808 nm NIR exposure, the fluorescence intensity of Ce6 was dramatically increased in ICT solution.(Figure 3b). Furthermore, the photobleaching of IR780 in ICT under 808 nm NIR light exposure was confirmed by UV-vis absorbance (Figure S2a), manifested that 808 nm NIR light could not only trigger PTT effect but also IR780’s photobleaching. What's more, the light would not trigger photobleaching of Ce6 prior to PDT. To assess singlet oxygen generation (SOG) ability of ICT, we employed DPBF (a singlet oxygen capture reagent) to perform the evaluation. Under 660 nm NIR light exposure, the absorbance of DPBF at 410 nm was clearly declined within Ce6 TNS.



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However, within ICT, the variation of DPBF’s absorbance draws tardily. On the contrary, after two wavelengths of NIR light irradiation (5 min at 808 nm exposure and 3 min at 660 nm exposure), the SOG of ICT surpassed Ce6 TNS. Besides, the increase of fluorescence intensity and SOG was observed as follows: IR780/Ce6 TNS (2:1)

Dual-mode imaging guided multifunctional ... - ACS Publications

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