Multifunctional Hybrid Liposome as a Theranostic Platform for

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Imaging and Diagnostics

Multifunctional Hybrid Liposome as a Theranostic Platform for Magnetic Resonance Imaging Guided Photothermal Therapy Chunyang Zhang, Dan Wu, Liejing Lu, Xiaohui Duan, Jie Liu, Xiaoyan Xie, Xintao Shuai, Jun Shen, and Zhong Cao ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00176 • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 21, 2018

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ACS Biomaterials Science & Engineering

Multifunctional Hybrid Liposome as a Theranostic Platform for Magnetic Resonance Imaging Guided Photothermal Therapy Chunyang Zhang1‡, Dan Wu1‡, Liejing Lu2‡, Xiaohui Duan2, Jie Liu1, Xiaoyan Xie3, Xintao Shuai4, Jun Shen2, Zhong Cao1* 1

Department of Biomedical Engineering, School of Engineering, Sun Yat-sen University,

No.132, East Waihuan Road, Guangzhou Higher Education Mega Center, Guangzhou 510006, China. 2

Department of Radiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University. No.107

West Yanjiang Road, Guangzhou 510120, China. 3

Department of Medical Ultrasound, Institute of Diagnostic and Interventional Ultrasound, First

Affiliated Hospital, Sun Yat-Sen University. No.58 Zhongshan Road 2, Guangzhou, 510080, China. 4

PCFM Lab of Ministry of Education, School of Chemistry and Chemical Engineering, Sun Yat-

sen University. No. 135 West Xingang Road, Guangzhou, 510275, P.R. China. Corresponding Author E-mail: [email protected] KEYWORDS. Hybrid liposome, MR imaging, Photothermal therapy, Theranostic

ACS Paragon Plus Environment

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ABSTRACT. Photothermal therapy (PTT) is emerging modality for cancer treatment owing to its localized treatment nature and easy combination with other therapeutic approaches. An imaging guided tumor ablation will facilitate the implementation of the treatment to boost efficiency. A type of multifunctional hybrid liposome is synthesized by loading indocyanine green (ICG) in to a hybrid liposome based on a mixture of hybrid lipid and 1,2-Dimyristoyl-snglycero-3-phosphoethanolamine-diethylene

triamine

pentacetate

acid-gadopentetate

dimeglumine (DMPE-DTPA-Gd). The hybrid liposome exhibited high structure stability and narrow size distribution in aqueous media. According to magnetic resonance imaging (MRI), hybrid liposome after tail vein injection accumulated effectively in subcutaneous CT-26 tumor of mice. Moreover, photothermal therapy is able to ablate tumor effectively under MR imaging guidance. Thus, the MRI visible PTT agent-loaded theranostic nanoplatform is promising for effective cancer treatment.

INTRODUCTION Photothermal therapy (PTT) as localized cancer treatment method is received increasing interest because of high efficiency and low side effects. PTT use Near-Infrared (NIR) agents to convert optical energy to heat, which causes tumor cell death in the area of irradiation.[1-4] Nowadays, many efforts have been made in developing NIR-absorbing agents[5-7], leading to availability of various NIR-absorbing materials including organic compounds,[8-10] carbon nanomaterials,[11,12] metal nanoparticles[13-15] and so on. ICG, a tricarbocyanine NIR dye


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approved by FDA, is widely applied in imaging diagnosis.[16,17] Furthermore, ICG is useful for localized hyperthermia owing to its efficient conversion of optical energy to heat. [18] However, the use of this non-toxic optical probe in PTT remains restricted. Mainly because it may degrade quickly in aqueous solution and is promptly cleared from human body, showing very short halflife (t1/2 = 2~4 min).[19,20] Moreover, application of ICG is hampered by its intrinsic drawbacks such as fluorescence quenching, low photostability and thermal stability in aqueous solutions, non-tumor-specific distribution in vivo.[21] Thus, it would be of great interest to prepare ICGencapsulated nanoparticles which may improve the stability and tumor specificity of ICG. On the other hand, imaging-guided PTT is drawing great attention owing to its unique advantages, such as providing direct evidence for in vivo fate of nanomedicines (e.g. tumor accumulation) and monitoring therapeutic response of host at both tissue and cell levels.[22, 23] Although various imaging approaches have been proposed in designing multifunctional therapeutic nanoparticles. Integrating diagnostic and therapeutic functions are highly desirable for effective tumor ablation. MRI is a widely used clinical imaging diagnostic method with no limitation on detection depth and high spatial resolution.[24] However, the intrinsic drawbacks of MRI contrast agents clinically available (various gadolinium chelates), including short in vivo half-lives, low sensitivity and nonspecific distribution, impede their applications as tumor imaging probes.[25] Integration of gadolinium into nanocarriers with long blood circulation and passive tumor targeting properties has been an effective method for promoting multi-mode nanoprobes for biomedical applications.[26]


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Admittedly, liposomes represent a major group of nanoscale delivery systems. [27] However, their instability in aqueous media still remains a problem.[28,29] For instance, conventional liposomes tend to aggregate and lose drug in circulation via leakage. Thus, development of a new kind of delivery system with good stability is in an urgent need. [30] In recent years, a type of stable hybrid liposome called cerasome was synthesized from organokoxysilane-based lipid (Silipid) via a sol-gel process in combination with self-assembly process.[31] In our previous work, we prepared cerasome encapsulated anti-cancer drugs that exhibited controlled release behavior and excellent stability much better than those of conventional liposomes.[32].These nanohybrid liposome demonstrated great potential as a theranostic nanocarrier, which motivated us to further develop a theranostic nanoplatform showing good stability of cerasome carriers and high photothermal efficiency. Herein, a theranostic nanoplatform was developed by loading ICG into hybrid liposome based on a mixture of hybrid lipid and DMPE-DTPA-Gd, as schematically illustrated in Figure 1. The incorporation of DMPE-DTPA-Gd may render the hybrid liposome high T1 sensitivity, while the encapsulated ICG may act as NIR laser absorbing agent to generate hyperthermia for tumor ablation. Moreover, the MR imaging function would allow noninvasive monitoring of in vivo distribution of liposomes and thus could guide NIR laser-based tumor ablation at optimized post-injection time for desired photothermal efficiency. The mouse model bearing CT-26 colon tumor was adopted to demonstrate the theranostic potential of hybrid liposome for combined MR imaging and PTT.


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Figure 1. The schematic illustration for hybrid liposome as a theranostic nanoplatform.

EXPERIMENT SECTION Materials N-[N-(3-Triethoxysilyl) propylsuccinamoyl] dihexadecylamine (hybrid lipid) was prepared as previous reported.[31] 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) and DSPE-PEG-2000 was from A.V.T. Pharmaceutical Co.,Ltd. Company (Shanghai, China). Diethylene triamine pentacetate acid (DTPA), gadolinium (III) chloride were from J&K Science Ltd.

3-(3

dimethylaminopropyl)-1-ethylcarbodiimide

hydrochloride

(EDC)

and

N-

Hydroxysuccinimide (NHS) were purchased from Adamas Reagent, Ltd. The commercial


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gadopentetate dimeglumine was from Beijing Beilu pharmaceutical Co. Ltd. MTT, calcein-AM and propidium iodide were from Sigma-Aldrich.

Preparation of hybrid liposome.

Synthesis of DMPE-DTPA-Gd.

DMPE was dissolved in chloroform to prepare a 4 mL solution with a concentration of 0.1 mM. Triethylamine (30 µL) was added dropwise to a 20 mL solution of 1 mM diethyltriaminepentaacetic acid (DTPA) dissolved in dimethyl sulfoxide (DMSO). These two solutions were then mixed and incubated in an argon atmosphere at 25 °C for 3 h. The resulting product was dialyzed (MW 3500) against DMSO and deionized water respectively for 48 h at 25 °C. The prepared DMPE-DTPA was lyophilized and stored at -20 °C. Afterwards, 0.384 g of DMPE-DTPA was dissolved in sodium acetate buffer (pH= 5.8) before adding 3.1 mL gadolinium chloride solution (0.337 mM). The solution was adjusted to pH 6.5 with 1 M NaOH before heating at 50 °C for 5 h. Finally, the crude product obtained by centrifugation for 10 min, was washed using sodium acetate buffer (50 mM) for 2-3 times and freeze-dried for later use. Preparation of hybrid liposome.

3 mg of hybrid lipid, 1 mg of DMPE-DTPA-Gd and 1 mg of DSPE-PEG-2000 were dissolved in absolute ethanol and vortexed to full dissolution. Then, the ethanol solution was dispersed in the ICG solution (250 µg/mL) under ultrasonication (240 W, 2 min) by liquid


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injection, and then the mixture solution was sonicated under probe sonication for 30 s. Standing at room temperature for a period of time to allow crosslinking with the surface of the hybrid lipid, the resulting production was then dialyzed for 12 h against PBS (pH 7.4) solution to remove the organic solvent. Finally, the as-prepared ICG loaded hybrid liposome was obtained by removing free ICG with ultrafiltration centrifugation procedure using Microcon Centrifugal Filter Devices (Millipore, MW 100 kDa).

Characterization.

The hybrid liposome was observed under transmission electron microscopy (TEM, Hitachi, 7750, Japan). In brief, the freshly obtained hybrid liposome suspension were dropwise added into a 500 mesh copper grid coated with carbon, stained with 0.2% (w/v) phosphotungstic acid, then analyzed by TEM (Hitachi, 7750, Japan). The ICG loading efficiency was quantified by UV/Vis absorption by using with UV-Vis/NIR spectrophotometer (DU730, Beckman Co., USA). The size and zeta potential of synthesized hybrid liposome were determined with dynamic light scattering (DLS) using Malvern Zetasizer Nano ZS 90. The concentrations of gadolinium was quantified with ICP-MS (Thermo Jarrell Ash Co., USA). The polymerization degree of the hybrid liposome was detected with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Dithranol (Aldrich, 97%) was applied as matrix. Spectra were recorded on a BrukerUltrafleXtreme mass spectrometer with a 337 nm N2 laser.

ICG loading efficiency


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1 mg/mL mother liquor was prepared with accurately weighed ICG and ultrapure water as the solvent before using. In the measurement, the mother liquor was diluted with ultrapure water to different concentrations as standard solutions respectively. The prepared solution of hybrid liposme was added to 3 mL ultrafiltration centrifuge tube (MW100 kDa), centrifuged for 20 min (5000 rpm) and then the subnatant was took out. The absorbance (Abs) at 789 nm was measured on a UV-Vis/NIR spectrophotometer. The ICG concentration in solution was obtained from calibration curve, and then the drug loading (DL%) and encapsulation efficiency (EE%) were calculated. In vitro ICG release from hybrid and DPPC liposome Hybrid liposome and DPPC liposome (1 mL) were dialyzed against 10 mL of the PBS (pH 7.4, 0.01 M) at 37 °C in a shaker at 100 rpm. To investigate the effects of photothermal heating on ICG releasing behavior, equal amounts of liposomes were directly irradiated under an 808 nm laser (2 W/cm2，5 min) at 1 h interval. 3mL PBS was replaced with fresh phosphate buffer at predetermined time interval. Released ICG was measured by a UV-Vis/NIR spectrophotometer.

Stability studies. Certain volume of freshly prepared hybrid liposome solution was dispensed in equal volume of pH 7.4 PBS added with 10% fetal bovine serum. The solution was incubated on a shaker (37 °C, 100 rpm). The particle size of the solution was measured at various time points by DLS at 25 °C. The laser wavelength was 630 nm and the light scattering angle θ was 90 °.


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In vitro Photothermal Effect. Concentration gradients of hybrid liposome aqueous solution were irradiated (808 nm laser, 2 W/cm2, 10 min). A digital thermometer was applied to determine elevated solution temperature per 10 seconds. In order to investigate the relationship between hybrid liposome photothermal conversion efficiency and ICG concentration, we prepared various hybrid liposome solutions using deionized water. The near-infrared laser was irradiated (808 nm, 10 min, 2 W/cm2). The solution temperature was measured using a digital thermometer probe every 10 s. The stability of the temperature response of hybrid liposome photothermal conversion was further studied. 2 mL of hybrid liposome solution (ICG: 50 µg/mL) was irradiated (808 nm, 10 min, 2 W/cm2) and stopped for 20 min for one cycle. Five consecutive cycles were performed and temperature changes were recorded, in which the control group was free ICG solution (50 µg/mL). The photothermal transforming efficiency of hybrid liposome was calculated as reported (irradiation time=8 min).[17]

In vitro MR imaging. The concentration of gadopentetate dimeglumine (Gd) in hybrid liposmes and clinical used contrast agent, gadopentetate dimeglumine were diluted to the same concentration. In addition, the evaluation of hybrid liposome in vitro nuclear magnetic resonance imaging ability was performed by Philip 1.5T-C3 nuclear magnetic resonance imaging system. The T1 mappingweighted images was obtained using a T1-weighted sequence (MESE) and the enhanced imaging


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parameters based on our previous work.[26]

Hemolysis assay. The biocompatibility of hybrid liposome solution was studied by examing its effect on the red blood cells. The following methods were designed according to our previous work.[26] 2 mL whole blood was centrifuged (1500 rpm, 10 min), and the supernatant was removed. The product was washed with PBS and centrifuged several times until the supernatant has red color. The 5% erythrocyte suspension in PBS was prepared. The supernatant was diluted to different concentrations, and mixed with an equal volume of 5% erythrocyte suspension and then incubated at 37 ° C for 3 h. Afterwards, the mixed suspension was centrifuged for 10 min (1500 rpm), and the supernatant was measured at 541 nm on a microplate reader, and the hemolysis rate was calculated as follows:

Hemolysis rate (%) = (A sample - APBS) / (A water - APBS) × 100%

(1)

A sample, Awater and APBS were absorbance of sample group, deionized water and isotonic PBS group.

Cytotoxicity by MTT. The in vitro cytotoxicity was evaluated by MTT assay. The effects of ICG and hybrid liposome concentrations on cell viability were studied. CT-26 cells (4 × 103 cells per well) were incubated in the 1640 medium containing 10% fetal bovine serum (FBS) overnight in 96-well


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plates (37 °C, 5% CO2). The medium was replaced with fresh medium containing different concentrations of hybrid liposome, which was incubated for additional 24 h and 48 h. Then, MTT assay was performed. In vitro Photothermal Therapy. MTT and calcein-AM/PI assays were used to reveal the PTT efficiency of hybrid liposome at different concentrations when exposed to near-infrared laser irradiation. CT-26 cells seeded in 24-well plates were incubated overnight, and then incubated with hybrid liposome at various concentrations under irradiation (808 nm laser, 2 W/cm2, 5 min). Then the cell viability was evaluated by MTT assay. For calcein-AM/PI staining assay, CT-26 cells were incubated with 600 µg/mL hybrid liposome in 12 well plates (1×106 cells per well). Cells were incubated for 30 min after NIR laser illumination (2 W/cm2, 5 min), washed with PBS and stained with calcein-AM (2.0 mM) and PI (1.5 mM, PI). Finally, the fluorescent images were recorded on an inverted fluorescent microscope (LX71, Olympus Co., Japan).

In vivo MR Imaging. All animal experiments were in accordance with the Institutional Animal Ethical and Welfare Committee of Sun Yat-sen University. 5-6 weeks old Balb/c mice, were obtained through the laboratory animal center of Sun Yat-sen University. To establish the CT-26 tumor model, 5×105 CT-26 cells (100 µL cells in PBS) were subcutaneously injected into right flank


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area of mice. In vivo experiments were applied once tumor had grown to a size of 70–100 mm3. In vivo MR imaging experiment, hybrid liposome was intravenously injected into the mice (200 µL, 800 µg/mL). A series of T1 weighted Images of abdominal axial were obtained on a 3.0-T MRI scanner (Achieva, Philips Medical Systems, NL) using Axial and coronal fast spin echo sequence (FSE). Enhanced imaging parameters are based on our previous work.[26]

In vivo photothermal therapy.

For photothermal therapy, 4 groups with 5 tumor bearing mice in each group were included in the current study：(1) saline control; (2) saline with laser irradiation; (3) hybrid liposome (200 µL, 800 µg/mL) without laser irradiation; (4) hybrid liposome (200 µL, 800 µg/mL) with laser irradiation. NIR laser illumination was carried out 6 h after i.v. injection (2 W/cm2, 8 min). The thermographic maps of tumor tissues from group (2) and (4) were imaged using an infrared thermal camera (Ti27, Fluke Co., USA). After treatment, body weight and tumor size were recorded every other day.

Histology analysis. Fourteen days after injection of hybrid liposome, all mice were killed. Organs fixed in 10% formalin solution and embedded in paraffin were sectioned (5 mm), H&E stained, and observed under a microscope (BX53, Olympus Co., Japan).

RESULTS AND DISCUSSION


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The 1H-NMR measurement revealed the DTPA characteristic resonances at 4.09 ppm (CCONH) and DMPE at 0.86 ppm (-CH3) (Figure 2A), based on which the conjugation ratio of DTPA-g-DMPE was estimated to be around 90%. The hybrid liposome with uniform size distribution were fabricated by ethanol injection method. TEM image of hybrid liposome showed spherical shape with a diameter of 143.8 nm±18.1 nm (Figure 2B), in line with the DLS results (Figure 2C).

Figure 2. (A) 1H NMR spectra of DMPE-DTPA in D2O, Temperature 25°C. (B) TEM image of hybrid liposome. (C) Histograms with size distribution of hybrid liposome. (D) Stability of the hybrid liposome incubated in PBS with 10% FBS; data are represented as mean ±SD (n= 3).

Moreover, the zeta potential of hybrid liposome was measured to be -24.3 mV, owing to the


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polysilane presence on hybrid liposome. Hybrid liposome remained their initial size during incubation with PBS solution containing 10% FBS for 24 h, indicating high colloidal stability in bloodstream (Figure 2D). Obviously, the negatively charged surface should have contributed to the marked serum stability of hybrid liposome, which is critical for drug delivery to tumor site in vivo. As shown in Figure 3A-B, hybrid liposome produce an obvious positive contrast signal at a series of iron concentration. Furthermore, the transverse relaxivity (r1) of hybrid liposome was 28.9 mM-1 S-1, 8.5-fold higher than Gd-DTPA (commercial contrast agent, r1=3.4 mM-1 S-1). The relaxivity of hybrid liposome was higher because the linear characteristic of DSPE and GdDTPA-DSPE binding on the liposome, making the part of Gd-DTPA harder to rotate, which partly increase the relaxivity resulting from decrease of the rotational correlation time of the chelated part of the metal. Considering the potential application of hybrid liposome as a photothermal therapy agent, UV-Vis-NIR absorbance spectra of free ICG and hybrid liposome in aqueous solution were examined (Figure 3C). The hybrid liposome kept high absorption of ICG in the NIR region, where the tissues show minimum light absorbance.[33,34] Furthermore, the photothermal effect at different concentrations was detected under NIR irradiation (808 nm laser, 2W/cm2, 10 min). Temperature of hybrid liposome solution was rapidly increased to 52 °C from27 °C within 600 s, which demonstrated the effective conversion of NIR optical energy to thermal energy (Figure 3D). However, no much elevation in temperature was detected for deionized water exposed to 808 nm NIR laser, implying that the NIR laser is safe for normal


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tissue.

Figure 3. (A) T1-weighted MR images. (B) T1 relaxation rate against Gd concentration. (C) UV-vis spectra of free ICG and hybrid liposome; (D) Temperature changes of hybrid liposome at various concentrations under 808 nm NIR laser irradiation (2 W/cm2, 10 min).

Additionally, as illustrated in Figure 4A, the photothermal transforming efficiency of hybrid liposome was quantitatively calculated to be 37.17%, which is higher than some of ordinary photothermal agents.[17] Figure 4B showed the changes of free ICG and hybrid liposome solutions irradiated for five repeated cycles, which indicated that ICG encapsulated in lipid layers possessed higher photostability than that of free ICG. In other words, the hybrid liposome solution showed less attenuation in the reachable temperature during the repeated


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irradiations. The prolonged duration of the hybrid liposome was most likely due to the formation of surface siloxane framework which prevent the degradation of ICG under NIR irradiation. Moreover, in order to verify the stability of hybrid liposome, the release of ICG loaded in hybrid liposome and DPPC liposome were further investigate respectively. As demonstrated in Figure 4C, DPPC liposomes released 19.52% ICG and was increased to 33.97% after the NIR laser irradiation. As a contrast, hybrid liposome only released 5.67% ICG with or without laser irradiation. These results confirm the excellent stability of hybrid liposome.

Figure 4. (A) Photothermal profile of hybrid liposome aqueous solution (inset: τs = 255.37 s). (B) Temperature changes of hybrid liposome and free ICG over five laser on/off cycles. (C) In vitro ICG release profile of hybrid liposome and DPPC liposome with or without 808 nm laser irradiation (2 W/cm2, 5 min). (D) MALDITOF mass spectra of hybrid liposome and DSPE-PEG2000.


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MALDI-TOF-MS was used to verify the existence of the polyorganosiloxane on the hybrid liposome. As shown in Figure 4D, the oligomers of dimers (1374 m/z), trimers (2041 m/z) and higher-order oligomers (2709, 3375, 4041 and 4708 m/z) were detected in the sample. Moreover, the multiple peaks from 1508 to 3223 m/z corresponded to the presence of DSPE-PEG whose molecular weight is 2790.5 (the average MW due to polydispersity of PEG). These results directly confirm the formation of siloxane network with a high degree of polymerization on the surface, which substantially contributed to their high morphological stability. The enhanced stability of the nanomedicine was favorable for transportation in the bloodstream. The potential toxicity of free ICG and hybrid liposome was tested on CT-26 cells. No remarkable decrease of cell viability could be detected under the investigated concentration for 24 and 48 h, demonstrating biocompatibility of the hybrid liposome (Figure 5A-B). As illustrated in Figure 5C, positive and negative control groups were deionized water and PBS, respectively. Neither obvious erythrocyte agglutination nor much hemolysis was induced by the hybrid liposome, as shown in Figure 5D. Even at high concentration reaching 800 µg/mL, only approximately 4.6% hemolysis was detected over 3 h of incubation. Apparently, it can be concluded that hybrid liposome have negligible hemolytic toxicity.


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Figure 5. MTT assays of (A) hybrid liposome and (B) free ICG at various concentrations against CT-26 cells. (C) Microscopic photograph of the erythrocytes after incubation with PBS, deionized water and hybrid liposome (bar =100 µm). (D) Hemolytic percent of hybrid liposome with gradient concentrations. Data are shown as mean ± SD (n =3).

Laser triggered PTT effect at cellular level were investigated by MTT assay. As depicted in Figure 6A, cell viability in laser exposure group gradually decreased along with increasing concentration of hybrid liposome. In contrast, there was almost no cytotoxicity detected for the hybrid liposome group without laser irradiation or the only laser irradiation group. In addition, the photothermal effect was confirmed by Calcein-AM/PI assays. Compared to control group (Figure 6B-D), the red staining region (Figure 6E) relative to the green one suggested that cancer cells were killed upon incubation by hybrid liposome under irradiation (2W/cm2, 5 min ). In comparison, other groups showed only green stains, indicating that hybrid liposome


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incubation without laser irradiation or with a sole NIR irradiation caused no cell death, as shown in MTT results as well.

Figure 6. (A) Cell killing effect of hybrid liposome with or without NIR laser exposure. Photothermal destruction of CT-26 cells (B) control, (C) NIR laser alone, (D) hybrid liposome alone, (E) hybrid liposome combined NIR laser treatments (808 nm, 2 W/cm2).

To verify the MRI contrast enhancement capability of hybrid liposome in vivo, the T1weighted MR images were recorded before and after tail vein injection of hybrid liposome into Balb/c mice bearing CT-26 tumor. As shown in Figure 7, an obvious brightening signal in the field of tumor was detected, owing to the increased T1 relaxation rate brought by the paramagnetic Gd ion. The highest signal was obtained 6 h after injection, suggesting the maximal hybrid liposome accumulation for tumor photothermal therapy. The above results also implied that the hybrid liposome had good colloidal stability in bloodstream since it is essential for long blood circulation required for an effective tumor accumulation.


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Figure 7. (A) T1-weighted MR images and the pseudo color of T1-weighted MR images of CT-26 tumorbearing mice after intravenous injection. (B) Quantification of T1-weighted signals in tumor. (C) The contrast enhancement ratio of T1-weighted MR signals in tumor.

The hybrid liposome effectively accumulated in tumor site 6 h post injection. Tumor region was laser irradiated for 8 min at this time point. Temperature in tumor center was elevated rapidly to about 55 °C from 35 °C in the hybrid liposome treated animals, enough for ablating tumor cells (Figure 8). In contrast, the control group injected with saline only showed a slight temperature increment during 8 min NIR laser irradiation.


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Figure 8. (A) Infrared thermal photographs of CT-26 tumor-bearing mice injected with saline or hybrid liposome (6 h after i.v injection) exposed to NIR laser irradiation for 8 min. (B) Temperature elevations of CT26 tumor bearing mice.

Tumor volume was measured every two days after treatment. As shown in Figure 9A, the tumors in animals receiving hybrid liposome were effectively ablated after laser exposure, without tumor re-growth being detected within 14 days of the experiment. However, animals receiving free ICG only showed partial tumor growth inhibition after laser exposure. No obvious tumor regression was detected in the hybrid liposome, saline with laser, saline-alone groups during the experiment. No significant bodyweight loss upon treatment was observed for all of the five groups during the 14-day experiment period (Figure 9B), which indicated that all treatments were tolerable by mice bearing tumor. In order to investigate the long-term toxicity of hybrid liposome, mice were sacrificed at day 14 to examine potential side effects on major organs (Figure 9C). No appreciable abnormality was detected, indicating again low side effects of hybrid liposome. Moreover, as illustrated in Figure 9D, laser irradiation alone could not inhibit


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tumor growth, while the tumors in treatment group were utterly ablated, leaving black scars at day 14.

Figure 9. (A) Tumor volumes of mice bearing CT-26 tumors after treatments (n = 4). (B) Body weight changes of mice after treatments (n =4).(C) H&E stained histologic images of major organs obtained from mice on day 14. (D) Photographs of mice taken on day 0 and day 14 after NIR laser treatment.

CONCLUSION

A dual-functional theranostic agent (hybrid liposome) was successfully fabricated for MRI-


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guided photothermal therapy. The hybrid liposome possessed good colloidal stability, excellent photostability and high MR imaging sensitivity. Importantly, under the guidance of MR imaging, tumors growth was inhibited completely by photothermal therapy (PTT) assisted by NIR laser irradiation. Additionally, the photothermal treatments showed no obvious side effects in mice receiving the treatments at the experimental dosage of hybrid liposome. This study revealed hybrid liposome has the potential as a multifunctional theranostic agent promising for MRIguided photothermal therapy of solid tumors.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No.U1401242, 81530055, 81571739, 81701696 and 81671707), the Tip-top Scientific and Technical

Innovative

Youth

Talents

of

Guangdong

Special

Support

Program

(NO.2015TQ01R510), the project supported by Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2017), Natural Science Foundation of Guangdong, China (No. 2016A030311054, 2016A030313324) and Science and Technology Program of Guangzhou, China (No. 201605130856580). REFERENCES


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Page 24 of 31

Yang, T.; Liu, L.; Deng, Y.; Guo, Z.; Zhang, G.; Ge, Z.; Ke, H.; Chen, H., Ultrastable

Near-Infrared Conjugated-Polymer Nanoparticles for Dually Photoactive Tumor Inhibition. Adv. Mater. 2017, 29 (31), 1700487 (1-9). DOI: 10.1002/adma.201700487. 2.

Han, S.; Kwon, T.; Um, J. E.; Haam, S.; Kim, W. J., Highly Selective Photothermal

Therapy by a Phenoxylated-Dextran-Functionalized Smart Carbon Nanotube Platform. Adv. Healthcare. Mater. 2016, 5 (10), 1147-1156. DOI: 10.1002/adhm.201600015. 3.

Liu, J.; Zheng, X.; Yan, L.; Zhou, L.; Tian, G.; Yin, W.; Wang, L.; Liu, Y.; Hu, Z.; Gu,

Z.; Chen, C.; Zhao, Y., Bismuth Sulfide Nanorods as a Precision Nanomedicine for in Vivo Multimodal Imaging-Guided Photothermal Therapy of Tumor. ACS Nano 2015, 9 (1), 696707.DOI: 10.1021/nn506137n. 4.

Song, X.; Gong, H.; Yin, S.; Cheng, L.; Wang, C.; Li, Z.; Li, Y.; Wang, X.; Liu, G.; Liu,

Z., Ultra-Small Iron Oxide Doped Polypyrrole Nanoparticles for In Vivo Multimodal Imaging Guided Photothermal Therapy. Adv. Funct. Mater. 2014, 24 (9), 1194-1201. DOI: 10.1002/adfm.201302463. 5.

Wang, J.; Liu, Y.; Ma, Y.; Sun, C.; Tao, W.; Wang, Y.; Yang, X.; Wang, J., NIR-

Activated Supersensitive Drug Release Using Nanoparticles with a Flow Core. Adv. Funct. Mater. 2016, 26 (41), 7516-7525. DOI: 10.1002/adfm.201603195. 6.

Li, Y.; Deng, Y.; Tian, X.; Ke, H.; Guo, M.; Zhu, A.; Yang, T.; Guo, Z.; Ge, Z.; Yang,

X.; Chen, H., Multipronged Design of Light-Triggered Nanoparticles To Overcome Cisplatin Resistance for Efficient Ablation of Resistant Tumor. ACS Nano 2015, 9 (10), 9626-9637.


24

Page 25 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DOI:10.1021/acsnano.5b05097. 7.

Wang, X.; Li, F.; Yan, X.; Ma, Y.; Miao, Z.-H.; Dong, L.; Chen, H.; Lu, Y.; Zha, Z.,

Ambient Aqueous Synthesis of Ultrasmall Ni0.85Se Nanoparticles for Noninvasive Photoacoustic Imaging and Combined Photothermal-Chemotherapy of Cancer. ACS Appl. Mater. Interfaces 2017, 9 (48), 41782-41793. DOI: 10.1021/acsami.7b15780. 8.

Sheng, Z.; Hu, D.; Zheng, M.; Zhao, P.; Liu, H.; Gao, D.; Gong, P.; Gao, G.; Zhang, P.;

Ma, Y.; Cai, L., Smart Human Serum Albumin-Indocyanine Green Nanoparticles Generated by Programmed Assembly for Dual-Modal Imaging-Guided Cancer Synergistic Phototherapy. ACS Nano 2014, 8 (12), 12310-12322. DOI: 10.1021/nn5062386. 9.

You, C.; Wu, H.; Wang, M.; Zhang, Y.; Wang, J.; Luo, Y.; Zhai, L.; Sun, B.; Zhang, X.;

Zhu, J., Near-Infrared Light and pH Dual-Responsive Targeted Drug Carrier Based on CoreCrosslinked Polyaniline Nanoparticles for Intracellular Delivery of Cisplatin. Chem.-Eur. J. 2017, 23 (22), 5352-5360. DOI: 10.1002/chem.201700059. 10.

Liang, X.; Li, Y.; Li, X.; Jing, L.; Deng, Z.; Yue, X.; Li, C.; Dai, Z., PEGylated

Polypyrrole

Nanoparticles

Conjugating

Gadolinium

Chelates

for

Dual-Modal

MRI/Photoacoustic Imaging Guided Photothermal Therapy of Cancer. Adv. Funct. Mater. 2015, 25 (9), 1451-1462. DOI: 10.1002/adfm.201402338. 11.

Song, G.; Cheng, L.; Chao, Y.; Yang, K.; Liu, Z., Emerging Nanotechnology and

Advanced Materials for Cancer Radiation Therapy. Adv. Mater. 2017, 29 (32), 1700996 (1-26). DOI: 10.1002/adma.201700996.


25


12.

Page 26 of 31

Kim, Y. K.; Na, H. K.; Kim, S.; Jang, H.; Chang, S. J.; Min, D. H., One-Pot Synthesis of

Multifunctional Au@Graphene Oxide Nanocolloid Core@Shell Nanoparticles for Raman Bioimaging, Photothermal, and Photodynamic Therapy. Small 2015, 11 (21), 2527-2535. DOI: 10.1002/smll.201402269. 13.

Hu, Y.; Chi, C.; Wang, S.; Wang, L.; Liang, P.; Liu, F.; Shang, W.; Wang, W.; Zhang, F.;

Li, S.; Shen, H.; Yu, X.; Liu, H.; Tian, J., A Comparative Study of Clinical Intervention and Interventional Photothermal Therapy for Pancreatic Cancer. Adv. Mater. 2017, 29 (33), 1700449 (1-9). DOI: 10.1002/adma.201700448. 14.

Wu, P.; Gao, Y.; Zhang, H.; Cai, C., Aptamer-Guided Silver–Gold Bimetallic

Nanostructures with Highly Active Surface-Enhanced Raman Scattering for Specific Detection and Near-Infrared Photothermal Therapy of Human Breast Cancer Cells. Anal. Chem. 2012, 84 (18), 7692-7699. DOI: 10.1021/ac3015164. 15.

Xiao, J.-W.; Fan, S. X.; Wang, F.; Sun, L. D.; Zheng, X.-Y.; Yan, C. H., Porous Pd

nanoparticles with high photothermal conversion efficiency for efficient ablation of cancer cells. Nanoscale 2014, 6 (8), 4345-4351. DOI: 10.1039/c3nr06843a. 16.

Zheng, M.; Zhao, P.; Luo, Z.; Gong, P.; Zheng, C.; Zhang, P.; Yue, C.; Gao, D.; Ma, Y.;

Cai, L., Robust ICG Theranostic Nanoparticles for Folate Targeted Cancer Imaging and Highly Effective Photothermal Therapy. ACS Appl. Mater. Interfaces 2014, 6 (9), 6709-6716. DOI: 10.1021/am5004393. 17.

Ma, Y.; Wang, X.; Chen, H.; Miao, Z.; He, G.; Zhou, J.; Zha, Z., Polyacrylic Acid


26

Page 27 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Functionalized Co0.85Se Nanoparticles: An Ultrasmall pH-Responsive Nanocarrier for Synergistic Photothermal-Chemo Treatment of Cancer. ACS Biomater.Sci. Eng. 2017. DOI: 10.1021/acsbiomaterials.7b00878. 18.

Wu, L.; Fang, S.; Shi, S.; Deng, J.; Liu, B.; Cai, L., Hybrid Polypeptide Micelles Loading

Indocyanine Green for Tumor Imaging and Photothermal Effect Study. Biomacromolecules 2013, 14 (9), 3027-3033. DOI: 10.1021/bm400839b. 19.

Yaseen, M. A.; Yu, J.; Wong, M. S.; Anvari, B., Laser-Induced Heating of Dextran-

Coated Mesocapsules Containing Indocyanine Green. Anvari, Biotechnol. Prog. 2007, 23 (6), 1431-1440. DOI: 10.1021/bp0701618. 20.

Zheng, M.; Yue, C.; Ma, Y.; Gong, P.; Zhao, P.; Zheng, C.; Sheng, Z.; Zhang, P.; Wang,

Z.; Cai, L., Single-Step Assembly of DOX/ICG Loaded Lipid–Polymer Nanoparticles for Highly Effective Chemo-photothermal Combination Therapy. ACS Nano 2013, 7 (3), 2056-2067. DOI: 10.1021/nn400334y. 21.

Ma, Y.; Tong, S.; Bao, G.; Gao, C.; Dai, Z., Indocyanine green loaded SPIO

nanoparticles with phospholipid-PEG coating for dual-modal imaging and photothermal therapy. Biomaterials 2013, 34 (31), 7706-7714. DOI: 10.1016/j.biomaterials.2013.07.007. 22.

Cheng, L.; Liu, J.; Gu, X.; Gong, H.; Shi, X.; Liu, T.; Wang, C.; Wang, X.; Liu, G.; Xing,

H.; Bu, W.; Sun, B.; Liu, Z., PEGylated WS2 Nanosheets as a Multifunctional Theranostic Agent for in vivo Dual-Modal CT/Photoacoustic Imaging Guided Photothermal Therapy. Adv. Mater. 2014, 26 (12), 1886-1893. DOI: 10.1002/adma.201304497.


27


23.

Page 28 of 31

Zhang, J.; An, F.; Li, Y.; Zheng, C.; Yang, Y.; Zhang, X.; Zhang, X., Simultaneous

enhanced diagnosis and photodynamic therapy of photosensitizer-doped perylene nanoparticles via doping, fluorescence resonance energy transfer, and antenna effect. Chem. Commun. 2013, 49 (73), 8072-8074. DOI: 10.1039/c3cc43413c. 24.

Haris, M.; Yadav, S. K.; Rizwan, A.; Singh, A.; Wang, E.; Hariharan, H.; Reddy, R.;

Marincola, F. M., Molecular magnetic resonance imaging in cancer. J. Transl. Med. 2015, 13 (1), 313 (1-16). DOI: 10.1186/s12967-015-0659-x. 25.

Smith, C. E.; Shkumatov, A.; Withers, S. G.; Yang, B.; Glockner, J. F.; Misra, S.; Roy, E.

J.; Wong, C.-H.; Zimmerman, S. C.; Kong, H., A Polymeric Fastener Can Easily Functionalize Liposome Surfaces with Gadolinium for Enhanced Magnetic Resonance Imaging. ACS Nano 2013, 7 (11), 9599-9610. DOI: 10.1021/nn4026228. 26.

Zhang, C.; Zhang, F.; Wang, W.; Liu, J.; Xu, M.; Wu, D.; Shuai, X.; Shen, J.; Cao, Z.,

Chitosan coated gold nanorod chelating gadolinium for MRI-visible photothermal therapy of cancer. RSC Adv. 2016, 6 (112), 111337-111344. DOI: 10.1039/c6ra23769j. 27.

Moitra, P; Kumar, K; Sarkar, S; Kondaiah, P; Duan, W; Bhattacharya, S. New pH-

responsive gemini lipid derived co-liposomes for efficacious doxorubicin delivery to drug resistant

cancer

cells.

Chemical

Communications,

2017,

53(58),

8184-8187.

DOI:

10.1039/c7cc03320f. 28.

Choquet, C. G.; Patel, G. B.; Sprott, G. D.; Beveridge, T. J., Stability of pressure-

extruded liposomes made from archaeobacterial ether lipids. Appl. Microbiol Biotechno. 1994,


28

Page 29 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


42 (2), 375-384. DOI: 10.1007/s002530050266. 29.

Lasic, D. D., Novel applications of liposomes. Trends in Biotechnology 1998, 16 (7),

307-321. DOI: 10.1016/S0167-7799(98)01220-7 30.

Moitra, P; Kumar, K; Kondaiah, P; Bhattacharya, S Efficacious. Anticancer Drug

Delivery Mediated by a pH-Sensitive Self- Assembly of a Conserved Tripeptide Derived from Tyrosine Kinase NGF Receptor. Angew. Chem. Int. Ed. 2014, 53, 1113–1117. DOI: 10.1002/anie.201307247. 31

Katagiri, K.; Hamasaki, R.; Ariga, K.; Kikuchi, J., Layered Paving of Vesicular

Nanoparticles Formed with Cerasome as a Bioinspired Organic−Inorganic Hybrid. J. Am. Chem. Soc. 2002, 124 (27), 7892-7893. DOI: 10.1021/ja0259281. 32.

Cao, Z.; Yue, X.; Jin, Y.; Wu, X., Dai, Z., Modulation of release of paclitaxel from

composite

cerasomes.

Colloid.

Surface.

B.

2012,

98,

97-104.

DOI:

10.1016/j.colsurfb.2012.05.001. 33.

Hou,Z.; Deng, K.; Li,C.; Deng, X.; Lian, H.; Cheng, Z.; Jin, D.; Lin, J.; 808 nm Light-

triggered and hyaluronic acid-targeted dual-photosensitizers nanoplatform by fully utilizing Nd(3+)-sensitized upconversion emission with enhanced anti-tumor efficacy.Biomaterials, 2016, 101,32-46. DOI: 10.1016/j.biomaterials.2016.05.024. 34. Cerón, E.; Ortgies, D.; Rosal, B.; Ren, F.; Benayas, A.; Vetrone, F; Ma, D.; Sanz-Rodríguez, F.; Solé, J.; Jaque, D.; Rodríguez, E.; Hybrid Nanostructures for High ‐ Sensitivity Luminescence Nanothermometry in the Second Biological Window. Adv. Mater., 2015,


29


Page 30 of 31

27(32),4781-4787. DOI: 10.1002/adma.201501014.


30

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Multifunctional Hybrid Liposome as a Theranostic Platform for Magnetic Resonance Imaging Guided Photothermal Therapy Chunyang Zhang‡, Dan Wu‡, Liejing Lu‡, Xiaohui Duan, Jie Liu, Xiaoyan Xie, Xintao Shuai, Jun Shen, Zhong Cao*

Hybrid liposome as a theranostic nanoplatform for MRI guided photothermal therapy. 82x44mm (300 x 300 DPI)


1

Multifunctional Hybrid Liposome as a Theranostic Platform for

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