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NIR Light-activated Drug Release for Synergetic Chemo-photothermal Therapy Ahu Yuan, Wei Huan, Xiang Liu, Zhicheng Zhang, Yifan Zhang, Jinhui Wu, and Yiqiao Hu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00820 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 7, 2016

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Molecular Pharmaceutics

NIR Light-activated Drug Release for Synergetic Chemo-photothermal Therapy

a,b,c,1

Ahu Yuan

, Wei Huan

a,1

a

a

a

, Xiang Liu , Zhicheng Zhang , Yifan Zhang , Jinhui Wu Yiqiao Hu

a,b,c,*

,

a,b,c,*

Affiliations: a

State Key Laboratory of Pharmaceutical Biotechnology, Medical School of Nanjing

University, Nanjing 210093, China b

c

Institute of Drug R&D, Medical School of Nanjing University, Nanjing 210093, China Jiangsu R&D platform for controlled & targeted drug delivery, Nanjing University, Nanjing

210093, China 1

These authors contributed equally

*Author for correspondence: Jinhui Wu, Ph.D. Address: 22 Hankou Road, Nanjing 210093, China Phone: +86-13913026062; Fax: +86-25-83596143 E-mail: [email protected] Yiqiao Hu, Ph.D. Address: 22 Hankou Road, Nanjing 210093, China Phone: +86-13601402829; Fax: +86-25-83596143 E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract: Nanocarriers like PEGylated liposomes have achieved enhanced drug accumulation in tumors and reduced systemic side effects, but failed to actively release the carried drug into cancer cells. To obtain improved therapeutic efficacy, we designed a novel liposome that was inserted by the amphiphilic agent PEG-IR780-C13 (PIC-Lipo) and encapsulated therapeutic agent doxorubicin (DOX), termed as DOX@PIC-Lipo. Upon NIR laser irradiation, the novel liposomes could generate hyperthermia and facilitate the release of encapsulated DOX from PIC-Lipo, which were confirmed by photothermal curves and the DOX release assay in vitro, respectively. In addition, the enhanced DOX release and sufficient hyperthermia have performed synergetic therapeutic efficacy both in vitro and in vivo. Therefore, DOX@PIC-Lipo might provide an active strategy to release the loaded drug for synergetic chemo-photothermal combined therapy.

Keywords: NIR light, activatable, doxorubicin, liposome, chemo-photothermal therapy


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1. Introduction Most chemotherapeutics used clinically are often restricted to insufficient delivery to tumor tissue and high toxicity on normal organs.1, 2 Currently, broad application of nanocarriers has profoundly changed that phenomenon. Chemotherapeutic drugs are usually incorporated into nanocarriers to achieve lower side effects and better tumor targeting.3 For example, doxorubicin has been proved to be an effective drug against various tumors. But its toxicity and side effects, especially the cumulative dose-dependent cardiotoxicity limit its further clinical application. The emergence of PEGylated liposomes has partially overcome these problems.4-6 PEGylated liposomes of doxorubicin with a highly hydrated and protected surface, prolong its blood-circulation time via inhibiting plasma protein adsorption and opsonization, and enhance its tumor accumulation via the enhanced permeability and retention (EPR) effect. Simultaneously, encapsulating doxorubicin into liposomes also significantly reduced its cardiotoxicity.6, 7 However, the better accumulation and biocompatibility of nanoparticles did not lead to an obvious improvement of drug therapeutic effect. As many researches reported, PEGylated liposomes could not significantly heighten the antitumor efficacy of doxorubicin. The main reasons were as follows: (1) The superficial PEGylated modification prevented the liposomes from entering into the tumor cells even though they have accumulated in tumor sites.8 Thus lots of PEGylated liposomal doxorubicin still existed in extracellular matrix. (2) The PEGylated liposomes were extremely stable and the rate of doxorubicin release from the liposomes were too slow.9 These aspects limit the therapeutic efficacy of liposomal doxorubicin. For better therapeutic efficacy, nanoscale systems responsible to specific stimuli (including temperature, light, pH, ultrasound, enzyme or reduction/oxidation) have been delicately designed.10-13 Among those stimuli, near infrared (NIR) light emerges as an intriguing external stimulus and the development of NIR light-activated drug delivery systems has garnered ever-increasing interest recently, because it is safe, deep penetrating and realizes controllable drug release both spatially and temporally.14, 15 For example, docetaxel-loaded PLGA nanoparticles coated with gold layer were engineered by Zhang et al. and the release of docetaxel could be accelerated from the broken nanoplatforms under the NIR laser irradiation.16 Chen et al. also fabricated AuSiO2-DOX and employed NIR laser to dissociate the strong interactions between silica and DOX for enhanced chemotherapy.17 In addition, Zhao et al. demonstrated a novel



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strategy that used upconversion nanoparticles and o-nitrobenzyl groups to realize the NIR light-responsive dissociation of block copolymer micelles and release their “payloads”.18 Although these researches have proved that NIR light could exhibit great potentials for clinical applications, the biodegradability of these inorganic materials involved in light-responsive nanoparticles limited their potential use in clinic.3, 10, 19 In our previous work, the amphiphilic and fluorescent molecule PEG-IR780-C13 (PIC) was demonstrated as an excellent photothermal agent for imaging and tumor phototherapy. Moreover, we have also confirmed that there was no significant toxicity to mice after intravenous injection with high dose of PIC.20 Considering the structural similarity between PIC and DSPE-PEG (component of traditional liposome) (Sfig.1), we assumed that PIC could replace the DSPE-PEG to insert into lipid bilayer and then DOX was encapsulated into liposomes to obtain light-responsive drug-loading liposomes (DOX@PIC-Lipo). When exposed to the NIR light, the degradation and heat generation of PIC might augment the lipid bilayer fluidity and permeability to facilitate the release of DOX. Compared to the previous PIC micelles, our DOX@PIC-Lipo would achieve NIR light-activated drug release and synergetic chemo-photothermal therapy, thus exert more powerful cancer therapeutics. In this study, we prepared a therapeutic liposome that was inserted by an amphiphilic agent PIC and encapsulated antitumor agent doxorubicin to pursue synergistic chemo-photothermal therapy (Fig.1). To confirm this hypothesis, we detected the DOX release and photothermal property of DOX@PIC-Lipo. Interestingly, the results revealed that DOX@PIC-Lipo not only possessed high efficiency of heat conversion but also released much more doxorubicin with the NIR laser irradiation. We also examined the size distribution, shape, and other optical properties, cellular location, cytotoxicity of DOX@PIC-Lipo. Furthermore, DOX@PIC-Lipo was injected intravenously and exhibited sufficient accumulation in tumor region. The antitumor efficacy of DOX@PIC-Lipo with NIR laser irradiation in vivo was superior to a single strategy on ectopic tumor xenografts.


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Fig.1. Schematic diagram of DOX@PIC-Lipo structure and synergetic chemo-photothermal therapy to eliminate tumor cells. PEG-IR780-C13 (PIC) was inserted into lipid bilayer and DOX was loaded into the aqueous phase core. After intravenous injection, DOX@PIC-Lipo was passively accumulated in tumor region. Then with the NIR laser irradiation, DOX@PIC-Lipo could generate hyperthermia, and simultaneously release DOX in the tumor region to achieve synergetic chemo-photothermal therapy.

2. Experimental section 2.1. Materials The components of liposome were obtained from Aladdin Corporation (Shanghai, China): lecithin, cholesterol. PEG-IR780-C13 (PIC) was synthesized according to the methods we previously reported.20 Doxorubicin (DOX) was purchased from MeiLun Biotech (Dalian, China). Cell Counting Kit-8 (CCK-8, Dojindo, Japan) was attained to detect cytotoxicity. The laser was manufactured by RuiLan Laser Corporation (Wuhan, China). The diameter of beam was 6mm and the beam was continuous wave. MCF-7 were cultured with high-glucose DMEM medium supplemented with 10% FCS (Wisent Corporation, Nanjing, China) and 1% 200mM L-glutamine (Thermo Fisher Scientific, USA). CT26 cells were cultured with RPMI 1640 supplemented with 10% FCS and 1% 200mM L-glutamine. Male BALB/c mice aged 6-8 weeks old were obtained



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from Medical Centre of Yangzhou University (Jiangsu, China). 2.2. Preparation of DOX@PIC-Lipo 40mg Lecithin, 12mg Cholesterol and 4mg PEG-IR780-C13 (PIC) was weighed into a 50ml round flask and moderate dichloromethane was added to fully dissolve the mixture. After removing the organic solvent by rotary evaporation, a thin lipid film was formed and hydrated with 4ml 200mM ammonium sulfate via 5min sonication. In order to acquire nanoscale and homogeneous liposomes, the solution was sequentially extruded through 400, 200 and 100 nm polycarbonate films for eight cycles. The free ammonium sulfate was further removed through dialysis in 800ml 0.9% NaCl solution for 6h (buffer exchanges every 2h). Then 2.8mg DOX was mixed with the liposome suspension and incubated at 45°C for 30min. The unloaded DOX was removed by using Amicon Ultra-4 centrifugal filter (Cutoff: 3KD, Millipore, USA). Then we used dimethyl sulfoxide to destruct the liposomes and detected the absorbance of PIC and DOX. Compared to the standard curves of free PIC and DOX dissolved in dimethyl sulfoxide, we calculated the concentration of PIC and DOX in DOX@PIC-Lipo. The DOX encapsulation and loading efficiency were calculated as follows: Encapsulation Efficiency (EE) was the ratio of the weight of DOX in DOX@PIC-Lipo / weight of initially added DOX. Loading Efficiency (LE) was the ratio of the weight of DOX in DOX@PIC-Lipo / weight of DOX@PIC-Lipo. 2.3. Characterization of DOX@PIC-Lipo The size distribution and zeta potential of DOX@PIC-Lipo and PIC-Lipo were measured by Particle Size Analyzer (90Plus zeta, Brookhaven, USA). For the morphology of DOX@PIC-Lipo, 2µl diluted samples were dropped on the copper grid overnight and then observed using TEM (Hitachi, Japan) at 80kV. Meanwhile, mean diameters of DOX@PIC-Lipo in 5% glucose, 0.9% NaCl and FBS at 25°C were investigated for 5d to evaluate the stability of nanoparticles. The UV-vis spectra of DOX@PIC-Lipo were also recorded by spectrophotometer (UV2450, Shimadzu Corporation). 2.4. Photothermal properties of DOX@PIC-Lipo 200µl of DOX@PIC-Lipo, PIC-Lipo, free PIC, free DOX and water were added into 96-well plate and irradiated with 808nm laser (1W/cm2) for 5min. The concentration of PIC in all groups was set as 400µg/ml. Each group contained three samples. A thermal probe (Fluke VT02, USA) was used to detect the temperature of solution every 20 second. In addition, the absorption spectra


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of DOX@PIC-Lipo irradiated by NIR laser irradiation were determined at every minute using the UV–vis spectrophotometer. To assess the stability of liposomes, the DOX@PIC-Lipo was also stored at 4°C in dark and the UV-vis absorption spectra were detected every two days. 2.5. Drug release profiles from DOX@PIC-Lipo Drug release profiles were acquired according to the following steps. Briefly, 500µl DOX@PIC-Lipo (DOX: 800µg/ml) aqueous solution was irradiated with 808nm laser (1W/cm2) for 0 and 5min, respectively, ant kept at 37°C in water bath. At 0.5, 2, 4, 6, 8, 10, 24h post NIR irradiation, these irradiated samples were transferred into Ultra-0.5 centrifugal filter tubes (Cutoff: 3KDa) and centrifuged (7000rpm) for 30min. The released DOX of the collected filtrate at corresponding time was determined by High Performance Liquid Chromatography (HPLC). Besides, DOX@PIC-Lipo aqueous solution was irradiated by 808nm laser (1W/cm2) for 0 and 5min, respectively. Then the emission fluorescence spectrum of DOX (Ex: 480nm) was measured by a TECAN Safire multifunctional microplate reader after irradiation with NIR laser (808nm, 1W/cm2) for 0 and 5min. 2.6. In vitro tumor cellular uptake 5 × 104 MCF-7 or CT26 cells were added into 6-well plates per well and cultured at 37 °C overnight for attachment. Then DOX@PIC-Lipo containing 100µg/mL DOX was irradiated with 808nm laser (1 W/cm2) for 0 or 5min and transferred into 6-well plates for cellular uptake. After 4h incubation, the media were discarded and cells were washed twice with PBS. Then MCF-7 or CT26 cells were collected using flow cytometry (FACS, BD Corp.) to analyze the DOX fluorescence (FL2 channel). Similarly, MCF-7 or CT26 cells were seeded onto glass coated 24-well plates with a density of 1× 104 per well. After overnight culturing at 37 °C, media were replaced by irradiated DOX@PIC-Lipo (5min, 808 nm, 1 W/cm2) or non-irradiated DOX@PIC-Lipo (containing 100 µg/mL of DOX). After 4 h incubation, 4% paraformaldehyde was added to fix the cells for 10min and the cells were stained by Hoechst 33342 (Invitrogen, USA) for 15min. Then confocal fluorescence microscope (Olympus FV1000) captured fluorescence images of tumor cells immediately. 2.7. Synergetic chemo-photothermal therapy in vitro 4 x 103 MCF-7 or CT26 cells were seeded into 96-well plate per well and cultured overnight for attachment. The cells were incubated with DOX@PIC-Lipo or PIC-Lipo at different



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concentrations for 2 hours and then irradiated with 808nm laser (1 W/cm2) for 0 or 5 min, respectively. After further incubation for 20 h, the cells were washed with PBS twice and each well was added with CCK-8 solution. Then cells were incubated for 2 h in a Thermo incubator (37°C, 5%CO2). In the end, multifunctional microplate reader was used to detect the absorbance at 450nm. The group without any treatment was set as Control, and cytotoxicity of cells treated by NIR irradiation alone was also conducted. 2.8. Tumor imaging and pharmacokinetics of DOX@PIC-Lipo 1x107 CT26 cells were subcutaneously implanted into the right flank of male Balb/c mice (aged 6-8 weeks). After 2-week growth, the volume of tumor reached 200mm3. Then the tumor imaging and temperature measurement were performed. First, 200µl DOX@PIC-Lipo (0.75mg/kg PIC) was intravenously injected into Balb/c mice bearing CT-26 tumor. At 0, 12, 24 and 48h, the mice were anesthetized and detected the NIR fluorescent signal using IVIS Lumina imaging system (Xenogen Corporation, USA) with 704nm excitation and 745nm emission. Exposure time was set to 1s. To further investigate the biodistribution of DOX@PIC-Lipo in mice, the tumors and major tissues were dissected and imaged using IVIS Lumina imaging system at 48h. The data were analyzed by IVIS Living Imaging Software. DOX@PIC-Lipo (DOX 8mg/kg, PIC 6.2mg/kg) was also intravenously injected into normal male Balb/c mice aged 8-10 weeks. Blood was collected by retro-orbital bleeding at 1min, 5min, 15min, 30min, 1h, 2h, 5h, 8h, 12h, 24h and 48h. Then the plasma was collected by centrifugal separation at 5000rpm for 5min. The concentration of DOX and PIC in plasma were detected by Nano Drop 2000 (Thermo Scientific, USA) according to the standard curves. 2.9. Tumor temperature measurement For tumor temperature measurement, mice bearing tumor were divided into two groups and each group has 3 mice. 200µl DOX@PIC-Lipo (7.5mg/kg PIC) and saline were injected into these mice via tail vein, respectively. Based on the results about tumor accumulation of DOX@PIC-Lipo, the tumors were irradiated with 808nm laser (1W/cm2) for 6min at 48h. The temperature was detected using Visual IR thermometer (VT02, Fluke, USA) and recorded per 1min. 2.10. Synergetic chemo-photothermal therapy in vivo To evaluate the synergetic chemo-photothermal therapy of DOX@PIC-Lipo, Balb/c mice


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bearing CT26 tumors was divided into six groups when the tumor volume reached 50mm3. The six groups were separately injected 200µl saline, saline + NIR, free Dox (8mg/kg), PIC-Lipo + NIR (6.2mg/kg PIC), DOX@PIC-Lipo, DOX@PIC-Lipo + NIR (8mg/kg DOX, 6.2mg/kg PIC) and each group has six mice. Then NIR laser irradiation (808nm, 1W/cm2, and 3min) was carried out at 48h after intravenous injection. The length (L) and width (W) of tumors were measured by Vernier Caliper every day. And tumor volume was calculated following the formula: Tumor Volume=L x W2/2. Relative tumor volume was the ratio between the tumor volumes at day X to the initial tumor volume. Meanwhile, the body weight of all mice were recorded every day. H&E staining was also performed to detect pathological changes of six groups with different treatments. Mice bearing tumors were divided into six groups and intravenously injected with various treatments then irradiated (808nm, 1W/cm2, and 3min) at 48h. The tumors were harvested at 72h, fixed in formalin, embedded into paraffin, sectioned, stained by hematoxylin/eosin (H&E) and observed by a microscope (Nikon ECLIPSE Ti). The pathological changes of livers were also detected by hematoxylin/eosin (H&E) in healthy mice. The healthy mice were divided into three groups and separately injected with Saline, PIC-Lipo (6.2mg/kg PIC) and DOX@PIC-Lipo (8mg/kg DOX, 6.2mg/kg PIC). After a week, the livers were harvested and fixed with formalin, embedded into paraffin, sectioned, stained by hematoxylin/eosin (H&E) and observed by a microscope (Nikon ECLIPSE Ti). 2.11. Statistical analysis Statistical analysis was performed via two-sided Student’s t-test, and p

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