Triple-modal imaging-guided chemo-photothermal synergistic therapy

Subscriber access provided by Université de Strasbourg - Service Commun de la Documentation

Biological and Medical Applications of Materials and Interfaces

Triple-modal imaging-guided chemo-photothermal synergistic therapy for breast cancer with magnetically targeted phase-shifted nanoparticles Long Wang, Sijie Chen, Yun Zhu, Meixiang Zhang, Shixiong Tang, Jingyi Li, Wenjing Pei, Biying Huang, and Chengcheng Niu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b16323 • Publication Date (Web): 15 Nov 2018 Downloaded from http://pubs.acs.org on November 15, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 44 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

ACS Applied Materials & Interfaces

Triple-modal imaging-guided chemo-photothermal synergistic therapy for breast cancer with magnetically targeted phase-shifted nanoparticles Long Wang,† Sijie Chen,‡ Yun Zhu,‡ Meixiang Zhang,‡ Shixiong Tang,§ Jingyi Li,† Wenjing Pei,‡ Biying Huang,‡ and Chengcheng Niu*,‡ †

Department of Orthopedics, Xiangya Hospital, Central South University, Changsha, Hunan

410008, China ‡

Department of Ultrasound Diagnosis, The Second Xiangya Hospital, Central South University,

Changsha, Hunan 410011, China §

Department of Radiology, The Second Xiangya Hospital, Central South University, Changsha,

Hunan 410011, China Keywords phase shift, magnetic target, triple-modal imaging, photothermal therapy, synergistic therapy Address all correspondence to: Chengcheng Niu, Department of Ultrasound Diagnosis, The Second Xiangya Hospital, Central South University, Changsha, Hunan,China, 410011; E-mail: [email protected]

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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

Abstract Current nanodrug-based cancer therapy is susceptible to the problems of rapid clearance from circulation and limited therapeutic efficacy. Herein, we report a magnetically targeted and photothermal-triggered drug release nanotheranostics system based on superparamagnetic iron oxide (Fe3O4), IR780, doxorubicin (DOX) and perfluoropentane (PFP) entrapped poly-lactide-co-glycolide (PLGA) nanoparticles (IR780/Fe3O4@PLGA/PFP/DOX NPs) for triple-modal imaging guided synergistic therapy of breast cancer. In this work, IR780 and Fe3O4 convert light into heat, which triggers DOX release from IR780/Fe3O4@PLGA/PFP/DOX NPs and a phase-shift thermoelastic expansion of PFP; this procedure further accelerates the DOX release and tissue extrusion deformation. Fe3O4 NPs also serve as the target moiety by an external magnet direct to the tumor. Specifically, the IR780/Fe3O4@PLGA/PFP/DOX NPs can be used for triple-modal imaging, including NIR fluorescence, magnetic resonance and ultrasound. Furthermore, the antitumor therapy studies reveal the extraordinary performance of IR780/Fe3O4@PLGA/PFP/DOX NPs in magnetically targeted synergistic chemo-photothermal therapy of cancer. Therefore, the multifunctional IR780/Fe3O4@PLGA/PFP/DOX NPs guided by the magnetic field shows a great potential for cancer theranostics. 1. Introduction Theranostics has recently attracted tremendous attention for cancer treatment due to its integration of the diagnosis and therapy into one system1-3. Due to its less invasiveness and efficient therapeutic effect, photothermal therapy (PTT) with imaging guided has shown superior characteristics compared to conventional chemotherapy, radiotherapy and surgery3-5. Lots of theranostic nanomaterials were used as PTT agents for its outstanding optical absorption properties, including Au nanomaterials6-9, carbon nanotubes10-12, iron oxide nanoparticles13-16,


Page 2 of 44

Page 3 of 44 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


copper sulfide nanoparticles17 and near infrared (NIR) dye-based nanocarriers18-20, which could convert light-to-heat energy, leading to thermal damage to tumor tissues. However, some of these nanomaterials have potential toxicity due to poor biodegradability and weak biocompatibility, which will unavoidably hinder their future clinical application. Among diverse theranostic nanomaterials, magnetic iron oxide nanoparticles are extremely fascinating because of their combination of outstanding magnetic properties, excellent magnetic resonance (MR) imaging and low toxicity21-22. As a physical targeting moiety, magnetic iron oxide nanoparticles are widely extensively used for magnetic targeting of theranostic agents by an magnet without the help of chemical conjugation of targeting ligands 4, 21, 23. This magnetic targeting theranostic system can induce the theranostic agents’ accumulation at the target tumor, thus reducing toxic effects to normal tissues and lowering the waste of theranostic agents. However, magnetic iron oxide nanoparticles are not the ideal PTT agent due to the disappointing optical to heat energy conversion efficiency and the low amount of accumulation at target tissues with an unmodified surface, and because the reticuloendothelial system (RES) system can easily uptake the nanoparticles, they are quickly cleared from the blood circulation, thereby hindering the PTT efficacy. To solve the rapid clearance issue, various polymers have been designed to decorate magnetic iron oxide nanoparticles22, 24-27. Poly-lactide-co-glycolide (PLGA) was extensively used for its wonderful biodegradability and fantastic biocompatibility. Due to its attractive loading capacity, PLGA has been investigated as a nanocarrier for versatile theranostic agents28-30. IR780 iodide is a lipophilic NIR dye, which possess a high NIR fluorescence (NIRF) intensity and can generate heat after laser irradiation31-32. However, the poor aqueous stability and



potential biocompatibility of IR780 iodide can severely hamper further clinical applications33-34. Some studies have been contributed to encapsulating IR780 into various nanomaterials to overcome these defects33-35. In this work, we propose to encapsulate IR780 into PLGA nanoparticles to improve the aqueous stability and biocompatibility for cancer NIRF imaging and PTT. Because of its core-shell construction, PLGA has been reported to coencapsulate various hydrophobic or hydrophilic materials to enhance antitumor efficacy through combination therapy36-37. Recently, liquid perfluorocarbon (PFC) has excited the curiosity of researchers due to its acoustic-responsive and optical-responsive vaporization properties, which could employed for ultrasound (US) imaging and cancer therapy28,

38-40.

Natalya Rapoport et al. reported that

doxorubicin (DOX) and perfluoropentane (PFP) coencapsulated polymeric nanoparticles for combining US imaging and US-mediated drug delivery38. In our previous work, we have successfully incorporated DOX or perfluoropentane (PFP) in the PLGA nanoparticles to improve antitumor efficacy through chemotherapy or PTT24-25. However, to the best of our knowledge, magnetically targeted and photothermal-triggered PLGA nanoparticles as a nanotheranostics platform to multimodal imaging, chemotherapy and PTT synergistic therapy of cancer have been little reported so far. Herein, we report a magnetically targeted and photothermal-triggered drug release nanotheranostics system based on superparamagnetic iron oxide (Fe3O4), IR780, DOX and PFP entrapped PLGA nanoparticles (IR780/Fe3O4@PLGA/PFP/DOX NPs) for triple-modal imaging and combination therapy of breast cancer (Scheme 1). Fe3O4 NPs serve as a target moiety directed to the tumor by a magnet. In the NPs, both IR780 and Fe3O4 could absorb and convert NIR light energy into heat, leading to DOX release and thermoelastic expansion of PFP, the latter further


Page 4 of 44

Page 5 of 44 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


accelerating DOX release and tissue extrusion deformation. In particular, these IR780/Fe3O4@PLGA/PFP/DOX NPs could be exploited as a triple-modal probe for NIRF/MR/US imaging-guided therapy of cancer. Due to the outstanding magnetically targeted ability, amazing photothermal conversion property and high DOX loading efficiency, IR780/Fe3O4@PLGA/PFP/DOX NPs present an excellent synergistic chemo-photothermal therapy of cancer. Therefore, these multifunctional IR780/Fe3O4@PLGA/PFP/DOX NPs will be promising in cancer theranostic nanomedicine. 2. Experiment Section 2.1 Materials IR-780 iodide, PLGA and poly vinyl alcohol (PVA) were all obtained from Sigma-Aldrich (USA). Fe3O4 nanoparticles treated with oleic acid were purchased from Ocean Nanotech Co. Ltd. (USA). Doxorubicin hydrochloride (DOX) was obtained from Solarbio Co. Ltd. (China). Liquid perfluoropentane (PFP) was obtained from Alfa Aesar (UK). 2.2 Preparation of IR780/Fe3O4@PLGA/PFP/DOX NPs Briefly, 100 mg PLGA and 3 mL chloroform were mixed together. After stirring well, 1 mg IR780, 0.2 mL Fe3O4 NPs suspension (31 mg Fe/mL), 0.2 mL liquid PFP, 0.2 mL DOX solution (20 mg/mL) and 15 mL 5% w/v cold PVA solution were added in order and emulsified for 2 min with an ultrasonic processor. The resulting emulsion was mixed in 20 mL deionized water and stirred until chloroform volatilization. Last, the resulting NPs were washed 3 times with deionized water (13 000 rpm, 20 min) and kept at 4°C. All procedures were operated in an ice bath in the dark. 2.3 Characterization



An NIR 808 nm laser (T808F2W, Minghui Optoelectronic Technology, China) was used to irradiate IR780/Fe3O4@PLGA/PFP/DOX NPs. Before and after laser irradiation, the morphologies of NPs were obtained by a Hitachi H-7600 transmission electron microscopy (TEM). Then, size distributions were analyzed using a Malven size analyzer (Malvern Nano ZS, UK). A fluorescence spectrophotometer (F-4600 FL, Hitachi, Japan) was used to identify the existence of DOX in the NPs. The atomic absorption spectrometry was used to measure the encapsulated iron amount in the NPs. A vibrating sample magnetometer (VSM, Lake Shore 7410-S) was applied to measure the hysteresis curve of free Fe3O4 and NPs. A magnet was placed nearby a glass vial filled with NPs solution to explore the magnetic feature of NPs. The IR780 loading was obtained by a Cary 5000 UV-Vis-NIR spectrophotometer (USA) and calculated according to the following equation24: Encapsulation efficiency (%) ＝ WE/ WT × 100％

(Eq 1)

where WE is the encapsulated IR780 amount in NPs, and WT is the total added IR780 amount. 2.4 In vitro PTT effect An infrared thermal imaging camera (FLIR C2, USA) was applied to monitor the temperature profile of IR780/Fe3O4@PLGA/PFP/DOX NPs. One mL of IR780/Fe3O4@PLGA/PFP/DOX NPs, IR780@PLGA/PFP/DOX NPs, IR780, Fe3O4, mixture of IR780 and Fe3O4, phosphate-buffered saline (PBS) or indocyanine green (ICG) was suspended in an Eppendorf tube and irradiated by the NIR 808 nm laser. The irradiation intensity was 1.0 W/cm2 and the irradiation time was 5 min. The concentration of NPs were 2 mg/mL, PBS and ICG were set as a negative or positive control. The amount of IR780 was equivalent in free IR780 and IR780/Fe3O4@PLGA/PFP/DOX NPs (11


Page 6 of 44

Page 7 of 44 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


μg/mL). The amount of Fe was 22.5 μg/mL, which is the same in Fe3O4 NPs and IR780/Fe3O4@PLGA/PFP/DOX NPs. The molar volume of ICG (0.016 μmol/mL) was equivalent to IR780 in this experiment. Then, different concentrations of IR780/Fe3O4@PLGA/PFP/DOX NPs (0, 0.5, 1.0, 2.0 mg/mL, 1 mL each concentration) were irradiated by the laser to measure photothermal effect. The temperature of all solutions was measured in 30 s intervals. One mL of 2 mg/mL IR780/Fe3O4@PLGA/PFP/DOX NPs (with IR780 concentration at 0.016 μmol/mL) or ICG (0.016 μmol/mL) was irradiated with the laser for ON/OFF cycle irradiation to study the photostability of NPs, which was repeated 4 times by laser irradiated (ON, 3 min) and naturally cooled down (OFF, 10 min). 2.5 Drug loading and characterization To quantify the drug loading capacity, DOX at various concentrations (5, 10, 20 mg/mL) and PFP at different volumes (0, 0.1, 0.2 mL) were loaded into IR780/Fe3O4@PLGA/PFP/DOX NPs following a similar procedure. Then, the DOX content was obtained by a spectrophotometer and calculated according to the following equations24: Encapsulation efficiency (%) ＝ WE/ WT × 100% = (WT - WU) / WT × 100% Loading efficiency (%) ＝ WE/ WNP × 100% = (WT - WU) / WNP × 100%

(Eq 2)

(Eq 3)

where WE is the encapsulated DOX amount in NPs, WT is the total added DOX amount, WU is the amount of unencapsulated DOX, and WNP is the weight of NPs. 2.6 Photo-triggered DOX release from IR780/Fe3O4@

/PFP/DOX NPs PLGA

To monitor the drug release from IR780/Fe3O4@PLGA/PFP/DOX NPs and further confirm the effect of PFP in drug release experiment, IR780/Fe3O4@PLGA/DOX NPs without containing PFP were used as controls. First, IR780/Fe3O4@PLGA/PFP/DOX NPs (25 mg, the DOX loading



efficiency was 2.39 % in weight) or IR780/Fe3O4@PLGA/DOX NPs (25 mg, the DOX loading efficiency was 2.39 % in weight) was reconstituted in 2 mL of Tris-HCl buffer. Then, each of them was transferred to dialysis bags (Mw = 8,000 Da), put in a reservoir with 20 mL of Tris-HCl buffer, and irradiated by the laser (1.0 W/cm2). The control groups were performed similarly, but without NIR irradiation. At desired time points, 1 mL of dialysate was received and then 1 mL of fresh buffer was added back to the reservoir. Each DOX concentration in dialysate was obtained by a spectrophotometer, and the accumulative released DOX was calculated. 2.7 Cell experiments The 4T1 mouse breast cancer cells were obtained from the Second Xiangya Hospital, Central South University (China) and cultured as recommended. The magnetic field strength of magnet

was measured by a hand-hold magnetic field strength meter (TD8620, Tunkia Co. Ltd., Changsha, China). To evaluate the cytotoxicity of IR780/Fe3O4@PLGA/PFP/DOX NPs, cell counting kit (CCK-8) assays were examined. 4T1 cells were incubated in 96-well plates with 1 × 104 cells per well at 37 °C for 12 h. Then, 0.1 mL of different dosages of NPs suspension was added into each well for 6 h incubation, respectively; medium without NPs was used as a control. Four round magnets with the diameter of 1.0 mm (each maximum magnetic field strength = 6.6 Gs) were set beneath four wells on 96-well plate for 2 h, respectively (Figure S1a, Supporting Information). The laser intensity was 1.0 W/cm2 and the irradiation time was 5 min. The magnetic fields decay with distance. The thickness of 96-well plate is about 1 mm, the depth of liquid in each well is 3 mm. The distance of 1 mm from the magnet, the maximum magnetic field strength is 5.8 Gs. The distance of 4 mm from the magnet, the maximum magnetic field strength is 5.1 Gs. Cells without


Page 8 of 44

Page 9 of 44 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


magnetic targeting were set as controls. Then, cell viabilities were calculated. To better elevate the magnetic targeting efficacy of IR780/Fe3O4@PLGA/PFP/DOX NPs, the phagocytosis study was carried out by a Zeiss LSM 510 confocal laser scanning microscopy (CLSM). After 4T1 cells were incubated into culture dishes for 12 h, 0.1 mL of medium containing 0.2 mg/mL of NPs was added. A round magnet with the diameter of 1.0 mm (maximum magnetic field strength = 6.6 Gs) was placed under the center of dish for 2 h (Figure S1b, Supporting Information), the thickness of culture dish is about 1 mm, the depth of liquid in dish is 3 mm. The distance of 1 mm from the magnet, the maximum magnetic field strength is 5.8 Gs. The distance of 4 mm from the magnet, the maximum magnetic field strength is 5.1 Gs. and then washed with PBS extensively to remove unattached NPs. Cells treated without magnetic targeting were used as controls. Then, cells were stained with DAPI and imaged. To verify the PTT effect combined with chemotherapy of IR780/Fe3O4@PLGA/PFP/DOX NPs in cells, 4T1 cells were incubated in culture dishes and randomly divided into 6 groups: (I) laser irradiation only; (II) IR780/Fe3O4@PLGA/PFP NPs without containing DOX; (III) free DOX; (IV) IR780/Fe3O4@PLGA/PFP/DOX NPs only; (V) IR780/Fe3O4@PLGA/PFP/DOX NPs with laser irradiation; and (VI) IR780/Fe3O4@PLGA/PFP/DOX NPs with laser irradiation and a magnetic targeting. A round magnet with the diameter of 1.0 mm (maximum magnetic field strength = 6.6 Gs) was placed beneath the center of the dish for 2h. The concentration of all NPs was 0.2 mg/mL, the laser intensity was 1.0 W/cm2 and the irradiation time was 5 min. Then, cells were washed twice with PBS, stained with propidium iodide (PI) and Hoechst 33342, imaged by CLSM. 2.8 Fluorescence imaging



For in vitro fluorescence imaging, IR780/Fe3O4@PLGA/PFP/DOX NPs with different dosages of IR780 (5, 10, 20, 40 and 80 μg/mL) and Fe3O4@PLGA/PFP/DOX NPs (without containing IR780) were placed in the hole of the special holder separately, and the NIRF images (λex = 790 nm and λem = 810 nm) were obtained by an FMT 4000 fluorescence tomography (PerkinElmer, Waltham, MA). The FMT 4000 system software (True Quant v3.0, PerkinElmer, Waltham, MA) was applied to analyze fluorescence data. BALB/c mice (20 g, female) were obtained from the Laboratory Animal Center of Central South University (China) and maintained in accordance with the guidelines of the Department of Laboratory Animals, Central South University, China. All experiments were approved by the Ethics Committee of the Second Xiangya Hospital, Central South University, China. The breast tumor mice were established by subcutaneously injection of 4T1 cells (1 × 106) into right flank and the tumor volume reached 100 mm3 one week later. For in vivo fluorescence imaging, ten mice were used for imaging on a Bruker In-Vivo FX PRO fluorescence tomography (Switzerland) and randomly divided into two groups (n=5): (1) with magnetic targeting; (2) without magnetic targeting. In addition, 0.2 mL of 20 mg/mL NPs was intravenously injected into the mice. The magnet with the maximum magnetic field strength of 32.5 Gs was placed nearby the tumor for 2 h to examine the magnetic targeting effect of NPs in vivo (Figure S1c, Supporting Information). The magnetic fields decay with distance. The depth of tumor in each group is about 5-6 mm. The distance of 3 mm from the magnet, the maximum magnetic field strength is 24.6 Gs. The distance of 6 mm from the magnet, the maximum magnetic field strength is 22.0 Gs. Mice treated without magnetic targeting were used as controls. At the 24-hour postinjection time period, tumor imaging was performed. Subsequently, important organs


Page 10 of 44

Page 11 of 44 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


and tumors were harvested and imaged. Bruker molecular imaging software (v.7.0.0.19693, Bruker, Switzerland) was applied to analyze fluorescence data. 2.9 MRI assessment For in vitro MR imaging, IR780/Fe3O4@PLGA/PFP/DOX NPs without Fe containing (I) and IR780/Fe3O4@PLGA/PFP/DOX NPs with different concentrations of Fe were put in cryotubes and imaged on a 3.0T Siemens Skyra MRI scanner (Germany). T2-weighted turbo spin echo (TSE) MR images (repetition time = 72 ms, echo time = 9 ms, flip angle = 90°, slice thickness = 3 mm, and field of view = 180 mm) were obtained, signal intensity (SI) within the region of interest (ROI) was measured. For in vivo MR imaging, ten mice were used for imaging on a 3.0T MRI scanner and randomly divided into two groups (n=5): (1) with magnetic targeting; and (2) without magnetic targeting. Before IR780/Fe3O4@PLGA/PFP/DOX NPs injection, all tumors were imaged. Then, 0.2 mL of 20 mg/mL NPs was injected into the mice via tail vein. The magnet with the maximum magnetic field strength of 32.5 Gs was placed nearby the tumor for 2 h in the magnetic targeting group. Mice treated without magnetic targeting were used as controls. After 24 h, tumor imaging was performed and MR parameters were the same as that in vitro. The SI within the ROI was measured and compared in the tumor tissue before and after injection. 2.10 US imaging The suspension of 0.5 mL of IR780/Fe3O4@PLGA/PFP/DOX NPs (20 mg/mL) was transplanted into a transparent rubber tube (d = 2 mm) and then irradiated. The laser intensity was 1.0 W/cm2 and the irradiation time was 5 min. An Olympus IX71optical microscope (Japan) was used to monitor the phase transition change of NPs. Ultrasonography was performed using an US



Page 12 of 44

scanner (S3000, Siemens, Mountain View, CA) with a 9L4 transducer for US examinations in 1 min intervals to observe the phase transition of the NPs. For tumor US imaging, fifteen mice were used for imaging on a Siemens S3000 ultrasound scanner and randomly divided into three groups (n=5): (1) with laser irradiation and magnetic targeting;

(2)

with

laser

irradiation;

and

(3)

without

laser

irradiation.

Before

IR780/Fe3O4@PLGA/PFP/DOX NPs injection, all tumors were imaged for B-mode and CEUS-mode US. Subsequently, 0.2 mL of 20 mg/mL NPs was intravenously injected into the mice. The group (1) was placed with a magnet (maximum magnetic field strength = 32.5 Gs) nearby the tumor for 2 h. After NPs injection 24 h later, the groups (1) and (2) were irradiated with the 808 nm laser (1.0 W/cm2, 10 min). The group (3) treated without magnetic targeting or laser irradiation was used as a control. Then, tumor imaging was performed for B-mode and CEUS-mode US. 2.11 In vivo antitumor therapy Mice were randomly divided into six groups (n=5) when tumor volumes reached 100 mm3: (1) saline; (2) DOX; (3) IR780/Fe3O4@PLGA/PFP NPs with laser irradiation; (4) IR780/Fe3O4@PLGA/DOX NPs with laser irradiation; (5) IR780/Fe3O4@PLGA/PFP/DOX NPs with laser irradiation; and (6) IR780/Fe3O4@PLGA/PFP/DOX NPs with laser irradiation and magnetic targeting. A 0.2 mL amount of saline containing DOX (0.478 mg/mL), IR780/Fe3O4@PLGA/PFP NPs without containing DOX, IR780/Fe3O4@PLGA/DOX NPs or IR780/Fe3O4@PLGA/PFP/DOX NPs was intravenously injected into the mice. All NPs containg DOX was at a concentraion of 20 mg/mL, with DOX concentration of 0.478 mg/mL. The group (6) was placed with a magnet (maximum magnetic field strength = 32.5 Gs) nearby the tumor for 2 h.


Page 13 of 44 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


At 24 hours after injection, the tumors of mice in groups (3-6) were irradiated with the 808 nm laser (1.0 W/cm2, 5 min). The temperature changes of the tumors were monitored by the infrared thermal imaging camera. Body weight and tumor volume were recorded every other day for 14 day. One day later, important organs and tumor were harvested and staining was performed with H&E stain. The tumor cell proliferation and apoptosis were analyzed by tumor immunohistochemical staining with antibody against PCNA and TUNEL. 2.12 Statistical analysis All data were analyzed by ANOVA and Students’ test with SPSS 17.0 software. *p < 0.05 was considered as significant for the differences. 3. Results and discussion 3.1 Characterization TEM was used to confirm the structure of IR780/Fe3O4@PLGA/PFP/DOX NPs before and after NIR irradiation. A TEM image of NPs before NIR irradiation appeared spherical in shape and many iron particles were uniformly dispersed in the shells (Figure 1a). After NIR irradiation, NPs vaporized and increased in diameter approximately five times of the initial size, and the iron particles in the NPs were uneven distributed (Figure 1b). Figure 1c shows the size distributions of NPs before and after NIR irradiation. After NIR irradiation, the average diameter of NPs increased from 190 to 955 nm, accompanied by an increase in the polydisperse index from 0.096 to 0.337. However, zeta potential of NPs had no significant change (from −6.56 to −6.25 mV) before and after NIR irradiation. The particle size increased significantly after laser irradiation, which could enable the NPs to serve as an ultrasound contrast agent and promote drug release. In the NPs with concentration of 10 mg/mL, the amount of Fe was 112.25 ± 4.21 μg/mL, and IR780 encapsulation



Page 14 of 44

efficiency was 54.53 ± 2.16 %. Figure 1d represented the UV-Vis-NIR absorption spectra IR780/Fe3O4@PLGA/PFP/DOX NPs and their various components. The absorption spectrum of PLGA/PFP NPs showed no absorption intensity in the range of 400~900 nm, which showed it did not have the ability to absorb light itself. The absorption spectrum of Fe3O4 NPs exhibited absorption intensity but no obvious peak in the range of 400-900 nm, and DOX showed a peak absorption at approximately 480~510 nm, whereas that of free IR780 presented a peak at approximately 780~790 nm. The absorption spectrum of IR780/Fe3O4@PLGA/PFP/DOX NPs showed a peak at approximately 790~800 nm with an obvious redshift, which confirmed that IR780 was loaded in the NPs successfully and could be used as a good photoabsorbing agent. The typical

absorption

of

DOX

did

not

appear

in

the

absorption

spectrum

of

IR780/Fe3O4@PLGA/PFP/DOX NPs due to its embedding inside the interior of the NPs and the affect by other substances which loaded outside or inside the NPs. However, the typical fluorescence spectrum of DOX was observed in the NPs, confirming that the DOX was also encapsulated in the NPs successfully (Figure S2, Supporting Information). To confirm the magnetization of the magnetic NPs, the hysteresis curves and an external magnetic

field

were

used.

The

saturation

magnetization

value

of

Fe3O4

and

IR780/Fe3O4@PLGA/PFP/DOX NPs was 48.9 emu/g and 31.2 emu/g, respectively (Figure 2a), indicating a superparamagnetic nature of the NPs at room temperature. The latter was lower than the former, indicating that the encapsulation of Fe3O4 into NPs could weaken the magnetization of Fe3O4. Nevertheless, NPs still exhibited a good magnetic property. Moreover, under an external magnetic field, NPs could accumulate nearby the magnet direction in PBS after 30 min (Figure 2b), indicating that NPs showed excellent magnetic responsiveness.


Page 15 of 44 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


3.2 In vitro PTT effect To study the PTT effect of IR780/Fe3O4@PLGA/PFP/DOX NPs, the FDA-approved PTT agent ICG was selected as the control. The aqueous suspensions of IR780/Fe3O4@PLGA/PFP/DOX NPs, their various components, PBS and ICG were irradiated by the 808 nm laser. The temperature has no obvious change for PBS after 5 min NIR irradiation (Figure 3). By contrast, obvious temperature increases were obtained in IR780/Fe3O4@PLGA/PFP/DOX NPs and IR780@PLGA/PFP/DOX NPs under NIR irradiation. The maximum temperatures achieved in IR780/Fe3O4@PLGA/PFP/DOX NPs and IR780@PLGA/PFP/DOX NPs solutions were 54.0 ºC and 52.5 ºC, respectively. Furthermore, the temperature changes of IR780, Fe3O4, the IR780 and Fe3O4 mixture, and ICG were shown in Figure S3a (Supporting Information). Similarly, obvious temperature increases were found for IR780, the mixture of IR780 and Fe3O4, and ICG after irradiation, and temperature was increased to 51.0 ºC, 53.0 ºC and 48.7 ºC, respectively. While the temperature of Fe3O4 increased slowly and steadily up to 38.0 ºC after irradiation. Compared with the temperature profile of ICG, IR780 contributed more to the rapidly increasing temperature. When IR780 and Fe3O4 were mixed, the temperature increased more rapidly than that of the single IR780. Moreover, IR780/Fe3O4@PLGA/PFP/DOX NPs showed almost the same PTT efficiency as that of the mixture of IR780 and Fe3O4, indicating that PLGA encapsulation has no impact on the PTT effect of IR780 or Fe3O4. Meanwhile, the temperature changes of IR780/Fe3O4@PLGA/PFP/DOX NPs with differnet concentrations (0, 0.5, 1.0, 2.0 mg/mL) were shown in Figure S3b (Supporting Information), the maximum temperatures achieved in different concentrations of NPs (0, 0.5, 1.0, 2.0 mg/mL) solutions were 25.4 ºC, 35.0 ºC, 42.1 ºC and 54.0 ºC, respectively, indicating that



IR780/Fe3O4@PLGA/PFP/DOX NPs has obvious concentration-dependent photothermal performance. The photothermal conversion efficiency (η) of IR780/Fe3O4@PLGA/PFP/DOX NPs (1.0 mg/mL) was evaluated by irradiated with 808 nm laser (1.0 W/cm2) according to the previously reported (see the calculation equations in the Supporting Information)15-17.τ can be obtained by the linear regression curve between cooling time and –ln(θ) (Figure S4, Supporting Information), then the η value of IR780/Fe3O4@PLGA/PFP/DOX NPs was calculated to be 35.7 %. According to Wu et al.’s study, the photothermal conversion efficiency of magnetic hollow porous carbon nanoparticles is 36%15. In another research of Zhang et al., the photothermal conversion efficiency of their magnetofluoresent nanocapsules is almost 37 %16, both of them are close to our NPs. To evalute the photostability of IR780/Fe3O4@PLGA/PFP/DOX NPs, ICG was also selected as the control. After each of the four laser ON/OFF cycles, the temperature in the IR780/Fe3O4@PLGA/PFP/DOX NPs increased to 50.2 °C, 48.1 °C, 46.2 °C and 45.0°C, and those for ICG were 45.2°C, 35.5°C, 33.5°C and 31.5°C (Figure S5, Supporting Information). It was found that IR780/Fe3O4@PLGA/PFP/DOX NPs remain an effective PTT agent after four ON/OFF cycles of laser irradiation, while free ICG gradually weaken after multiple rounds of NIR irradiation. Therefore, the excellent PTT performance and outstanding photostability of IR780/Fe3O4@PLGA/PFP/DOX NPs make it a promising PTT agent for tumors. 3.3 DOX-loading efficiency and phototriggered release kinetics Due to the core-shell structure and hydrophobic feature, PLGA NPs are recognized as ideal carriers to encapsulate drugs. To confirm the quality of IR780/Fe3O4@PLGA/PFP/DOX NPs for drug loading, DOX encapsulation and loading efficiency were evaluated. With the increasing of


Page 16 of 44

Page 17 of 44 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


the total added DOX amount, the loading efficiencies of DOX showed a slight increase, and the encapsulation efficiencies of DOX displayed a slight decrease. However, the encapsulation efficiency and loading efficiency of DOX with different concentrations of DOX were increased obviously with the increasing of an added PFP amount (p0.05). With the increasing concentrations of NPs, the cell viabilities in the groups without laser irradiation were decreased, indicating that IR780/Fe3O4@PLGA/PFP/DOX NPs were cytotoxic (p

Triple-modal imaging-guided chemo-photothermal synergistic therapy

Recommend Documents