Localized Light-Au-Hyperthermia Treatment for Precise, Rapid, and

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Biological and Medical Applications of Materials and Interfaces

Localized light-Au-hyperthermia treatment for precise, rapid and drug-free blood clot lysis Lina Dong, Xiao Liu, Tian Wang, Bixing Fang, Jinghuang Chen, Chen Li, Xinxin Miao, Chaochao Wei, Fen Yu, Hong-Bo Xin, Kui Hong, Xingwei Ding, and Xiaolei Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20616 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 23, 2018

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ACS Applied Materials & Interfaces

Localized light-Au-hyperthermia treatment for precise, rapid and drug-free blood clot lysis Lina Dong, #1 Xiao Liu, #2,3 Tian Wang,1 Bixing Fang,4 Jinghuang Chen,1 Chen Li,5 Xinxin Miao,5 Chaochao Wei,1 Fen Yu,1 Hongbo Xin,1 Kui Hong, *2,3 Xingwei Ding, *1 Xiaolei Wang *1 1Institute

of Translational Medicine, Nanchang University, Nanchang, Jiangxi 330088,

China. 2Department

of Cardiovascular Medicine, The Second Affiliated Hospital of

Nanchang University, Nanchang, Jiangxi 330006, China. 3Jiangxi

Key Laboratory of Molecular Medicine, Nanchang, Jiangxi 330006, China

4Department

of Otolaryngology Head & Neck Surger, The Third Affiliated Hospital

of Sun Yat-sen University, Guangzhou, Guangdong, 510630. 5Department

of Orthopedic Surgery, The Second Affiliated Hospital of Nanchang

University, Nanchang, Jiangxi 330006, China.

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ABSTRACT: Thrombus diseases, induced by blood stasis or vascular embolization normally, are frequently occurred with high disability and mortalities worldwide. At current state, drug thrombolysis, a primary clinical therapy for blood clot lysis, could increase lethal risk for hemorrhage when thrombolysis agents are over-used in the whole body. Therefore, a novel and advanced therapy for blood clot lysis, based on the remoted physical signals, is helpful for assisting clinical therapy. Here, we used the localized light-Au-hyperthermia (LAH) treatment, induced by gold nanorods (Au NRs) irradiated with near-infrared light (808 nm), for precise, rapid and drug-free blood clot lysis. The LAH technology was firstly introduced in murine hematoma model and murine myocardial infarction model for blood clot lysis. Compared with traditional therapy, LAH was assured to shorten the time of detumescence in murine hematoma model owning to their precise and localized hyperthermia. Meanwhile, we also discovered that the LAH was benefit to vascular recanalization in murine myocardial infarction model. In addition, the Au NRs used in the LAH present ideal biocompatiability in murine model, which endows it to be suitable for blood clot lysis in vivo.

KEYWORDS: blood clot lysis, photothermal therapy, hematoma, myocardial infarction


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INTRODUCTION Blood stasis, a recognized risk factor for thrombosis, is one of the pathogenic products caused by disorder of the blood circulation and blood stasis syndrome. The hematoma, one of clinical blood stasis, would be formed when the blood is permeated and detained outside the damaged blood vessels, due to disease or trauma1-2. In addition, the activated platelet would be attached on the damaged blood vessels, resulting in the early thrombus. When the thrombus shed and entry hunman’s organs with blood, which could give rise to some lethal cardiovascular diseases, such as myocardial infarction3-4. To promote blood circulation and clear blood clots, some physical stimulations5 or antithrombotic drugs are taken to realize vascular recanalization as routine treatments. However, the heating source of traditional thermotherapy is always placed on the skin, such as heating pad, which transmits limit heat to the injured site resulting to a long recovery time. Besides, a high dose of antithrombotic drugs could increase the risks of some devastating and lethal side effects6,7. Therefore, a precise, rapid and drug-free blood clot lysis therapy with limited antithrombotic drugs is significant for thrombolytic therapy. Nanotechnology has permeated into our life widely and given rise to a sort of remarkable benefit for our health8-10. A lot of nanoparticles are chosen to be agents for disease diagnosis and therapy due to the beneficial physical properties11-13. In the previous our research, we found

that

the

localized

hyperthermia,

induced

by

light-thermal

effection

of

gold@mesoporous silica nanospheres, could enhance the thrombolytic effect of antithrombotic drugs in vitro and in vivo14. Therefore, we wonder that whether the pure heating source could be helpful to thrombus disease. Herein, we firstly presented a localized light-Au-hyperthermia (LAH) treatment based on gold nanorods (AuNRs) in murine hematoma model and murine myocardial infarction model. Theoretically, compared to traditional treatment for hematoma, LAH can generate heat at the injected site under irradiation of NIR laser more precisely, which could shorten the loss of heat and the 3 ACS Paragon Plus Environment


transmitted time (Figure 1A). Furthermore, the LAH is also hopeful to shorten the recovery time of vascular recanalization, which is beneficial to thrombolysis, resulting to a safer dose of antithrombotic drugs. Therefore, murine myocardial infarction model was built to assess the light-hyperthermia effect for assistant vascular recanalization. EXPERIMENTAL PROCEDURES Materials. Tetrachloroauric acid, AgNO3, Cetyltrimethyl ammonium bromide (CTAB), NaBH4, thrombin and agarose, were obtained from Macklin Co. (Shanghai, China). RPMI 1640 medium, fetal bovine serum, penicillin and streptomycin were purchased by Gibco Co. (USA). Cell counting assay kit-8, 2’,7’-Dichlorodihydrofluorescein diacetate, NO immunosorbent assay kit, p-nitrophenyl phosphate, BCA kit, paraffin and hematoxylin-eosin were supplied from ThermoFisher Co. (USA). Polylactic acid (PLA) was provided by PrintRite Unicom Image Products Co. (Zhuhai, China) Synthesis and characterization of AuNRs. Au NRs were synthesized according to a previously published procedure[14]. Transmission electron microscope (TEM, JEM-2010 type; Japan Electronics Co.) and scanning electron microscope (SEM, Zeiss/sigma 300) were used to measure the size and feature of AuNRs. Laser diffraction particle size analyzer (LA-950 HORIBA, Japan) was employed to analyze the size distribution of AuNRs. Near infrared ray laser (NIR, 808 nm, Hi-Tech Optoelectronics Co. Ltd) was used to heat the AuNRs, China and the distribution of temperature was determined by infrared thermal imager (FLUKE, VT02). Ultraviolet-visible spectrum was determined by UV-VIS spectrophotometer (UV2600, SHIMADZU, China). Effect of photothermal conversion. The effect of photothermal conversion was assessedaccording to a previously published procedure[14]. The assessment of NIR penetration in agarose blocks. Firstly, the agarose blocks were incubated in 4°C for 12 h. Secondly, we injected 200 μL (200 μg/mL) Au NRs at the same site in two cubes and the cubes were treated with NIR laser irradiation or heating pad 4 ACS Paragon Plus Environment

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respectively. Thirdly, the temperature of agarose blocks was measured via infrared thermal imager at desired time. Cell culture and cytotoxicity assay. Human umbilical vein endothelial cell (HUVEC) were cultured according to a previously published procedure[14]. The cells viability of HUVEC cultured with 200 μg/mL AuNRs was examined by a cell counting assay kit-8 (CCK-8) for 1d, 3 d and 7 d. Preparation of blood clots and thrombolysis assessment in vitro. Sprague-Dawley rats (180-220 g) were supplied from the Laboratory Animal Science of Nanchang University and the blood clots were prepared according to a previous report.[14] Murine hematoma model. All animal procedures were performed according to the protocol approved by the Institutional Animal Care and Use Committee of China. The rat hematoma model was built according to a previous report16 with a minor modification. A total of 42 male Sprague-Dawley rats (180-220 g) were anesthetized with sodium pentobarbital (50 mg/kg). We used impact device, to build the standardized soft-tissue trauma, which was induced in rats on the lateral compartment of the left hind limb. After 24 hours, the rats were divided randomly into five groups and treated with 1 mL saline, saline (NIR), AuNRs (200 μg/mL), AuNRs (NIR), 2.5 and 5 kU/kg uPA at five points of the left hind limb and traditional treatment (heating pad). The circumference of limb was measured every 6 hours after treatment17. Biodistribution and biosafety evaluation in vivo. After the entire experiment (1 d), half of the rats (n=6) were sacrificed, the major organs and leg muscle of mice were separated. The other rats were fed normally for other 6 days. After that, the rats (n=6) were sacrificed and get the major organs and leg muscle, this group was marked as 7 d. Then, ICP-MS and hematoxylin-eosin staining were used to evaluate the biodistibution and biosafety of Au NRs in organs and muscle and according to a previously report.

[14]

Another five mice were

anesthetized with sodium pentobarbital (50 mg/kg), 1 mL Au NRs (200 μg/mL) were injected 5 ACS Paragon Plus Environment


at five points of the left hind limb. Then, the blood and urine of mice were collected every 24 h and was determinated by ICP-MS. Preparation of 3D printed anklet. The 3D printed anklet was designed by 3D software CATIA. Two 3D printers (SLA Pegasus Touch, America and MakerBot Replicator Z18) was used to print the models of the implant scaffolds. Murine myocardial infarction model and surgical procedures. Adult, healthy male Sprague Dawley (SD) rats(180-230g) used in all experiments were provided by Shanghai Laboratory Animal Centre (Shanghai, CAS). All animal care and this investigation conform to the Guide for the Care and Use of Laboratory Animals. All animal studies were approved by the Animal and Experimentation Committee of Nanchang University. and conducted in accordance with the “Guide for the Care and Use of Laboratory animals” (revised 1996). Myocardial infarction (MI) surgery were performed as previously described[18]. MI was induced in 21 (7 rats each group) SD rats, the left anterior descending artery (LAD) was ligated using a 6-0 silk suture. MI was confirmed by the presence of ST elevation. Control rats underwent a sham operations. After 24 h, the silk suture was cut and ligation of the LAD was loosened. Then the Au NRs (NIR) (80 μL,200 μg/mL) and saline (NIR) (80 μL) were injected at two points in the periphery of infarction tissues which was exposed under NIR laser. All the rats were sacrificed three days after the surgery. Echocardiography. Two-dimensional echocardiography measurements were conducted in rat heart after 3-day MI. We used the M-mode echocardiography to measured the cardiac function by using the Vevo770 system equipped with a linear transducer(35-MHz). Rats were intraperitoneally anaesthetized with sodium pentobarbital (50 mg/kg). Measurements of cardiac function were obtained in the M-mode tracings and at last using 5 cardiac cycles. Cardiac myocyte apoptosis. Cell apoptosis was determined by TUNEL assay using cell death detection kit (G3250, Promega, Madison, WI, USA). TUNEL assay were performed as previously described[19] 6 ACS Paragon Plus Environment

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2,3,5-triphenyltetrazolium chloride (TTC) staining. TTC assay were performed as previously described[19]. Myocardial ischemic size was defined as the ratio of the infarct area/left ventricle using image-pro plus 6.0 software (Media Cybernetics, Silver Spring, USA). Statistics. All data were expressed as means ± standard deviations (SD). The statistical analysis was performed using Student’s T-test and one-way analysis of variance (ANOVA) at confidence levels of 95 and 99% (OriginPro version 7.5).

RESULTS AND DISCUSSION The simulation of localized hyperthermia in vitro. To imitate that Au NRs can generate the localized hyperthermia in the lesion due to the irradiation of NIR laser, we fabricated agar blocks to compare NIR thermotherapy and traditional thermotherapy. As shown in Figure 1B, the Au NRs (200 μg/mL, 200 μL) and red inks were injected into agar blocks with same size respectively, which had been pre-freezed at 4 °C. Then, the agar blocks were treated with NIR laser irradiation and heating pad respectively. As shown in Figure 1C, interestingly, though the surface of agar block could be heated by heating pad quickly, the heat could not be transmitted effectively to the injected site. However, the Au NRs located at the injected site could be stimulated by NIR laser, which give rise to a targeted and rapid heating. This precise and rapid heating could reduce the loss of heat conduction and the time of localized hyperthermia for thermotherapy. Therefore, we hypothesized that the LAH could be applied in vascular precise and rapid recanalization. Synthesis and characterization of Au NRs. We firstly synthesized Au NRs by seedmediated growth method20. The Au NRs were used as photothermal agent due to the excellent photothermal conversion efficiency. SEM image showed that the synthesized Au NRs had a good dispersion in distilled water (Figure 2A) and the synthesized Au NRs were assured via energy-dispersive spectrometry (Figure 2A inset & Figure S1). TEM image showed Au NRs 7 ACS Paragon Plus Environment


had relatively uniform size with length of 50 nm (Figure 2B). Through the laser diffraction particle size analyzer, the mean size of Au NRs was 53.28 nm (Figure 2C), the result was corresponded with TEM image. In addition, we also used UV analyzer to determinate the absorbance of Au NRs, the result is in line with the previous report (Figure 2D)21. Furthermore, as shown in Figure 2E&F, the elevated temperature was influenced by the concentration of Au NRs and current intensity of NIR laser. The photothermal conversion efficiency was calculated and shown in Table S1. The detumescence assessment of LAH in murine hematoma model. An ideal therapeutic agent should possess acceptable cytocompatibility. To investigate the cytocompatibility of Au NRs, we assessed the cytotoxicity by human umbilical vein endothelial cell. The results of cytotoxicity revealed that Au NRs presented good cytocompatibility (Figure S2), which was in line with the results of previous reports14. To confirm that Au NRs could be applied in eliminating hematoma, murine hematoma model was built according to the previous report22. As shown in Figure 3A&B, the hematoma was induced after the rats were suffered to strong impact at the left hind limb. After 24 h, the injured hind limbs were treated with different therapy, include saline, saline (NIR), Au NRs, Au NRs (NIR), 2.5 and 5 kU/kg uPA and traditional therapy (Figure S3). From the change of circumference, the results revealed that the group treated with Au NRs (NIR) showed a better therapeutic effect compare with other treatments (Figure 3C&D). This is owning to the localized hyperthermia induced by photothermal effect of Au NRs, which could eliminate hematoma rapidly. However, the groups treated with traditional treatment (heating pad, Figure S3B), blood thinner (uPA) and NIR laser lonely present a slight decrease circumference, the limited therapy effect is on account of the limited heat which is transported from the heating source (on the surface of skin) to lesions. Biodistribution and biosafety evaluation in vivo. As shown in Figure 3E & Table S2, most of Au NRs were found in muscle, kidney, liver and spleen at the first day, which indicated the 8 ACS Paragon Plus Environment

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Au NRs could be delivered in metabolic organs. Then, the content of Au NRs in murine blood and urine was determinated at 1, 2, 3, 5, 7 day after injecting Au NRs (Figure S4). The above results indicate Au NRs could be metabolized in vivo. To further evaluate toxic side effects of the Au NRs, the major organs were treated with H&E stain. In Figure 3F, no toxicity and inflammatory cells were observed in the major organs at 1 d and 7 d, it indicates that Au NRs do not cause organic damage in vivo. These results suggest that it is expectable for eliminating hematoma rapidly by LAH in vivo. Therefore, we designed a wearable ankle device model by 3D printed technology for hematoma therapy (Figure S5A inset). The device included NIR laser source and battery, it could supply a continual NIR irradiation after Au NR were injected the site of blood stasis theoretically (Figure S5A&B). Thrombolysis assessment of LAH in vitro. According to the above phenomenon, we assumed that the LAH could be helpful for lysing blood clot. Therefore, the thrombus dissolution capability of Au NRs was evaluated through the assessment of blood clot lysis in vitro. In Figure S6, compared with the group of saline at 37 °C, the weight of blood clot present a gradual lysis with 200 μg/mL Au NRs under NIR laser irradiation (2 W/cm2). It suggest that hyperthermia induced by photothermal effect is confirmed to be helpful for lysing blood clot in vitro. So, it is reasonable to assume that the assistant thrombolysis of Au NRs could partly reduce the dose of antithrombotic drugs in vivo. The vascular recanalization and biosafety assessment of LAH in murine MI model. MI is one of lethal cardiovascular disease owning to vascular occlusion due to blood clot in vascular vessel. To evaluate vascular recanalization by LAH, we built murine MI model and explored the cardiac function, MI area, and cardiomyocyte apoptosis. The surgical flow was shown in Figure 4A. Cardiac function was detected by echocardiography after treatment of Au NRs (NIR) or saline (NIR). As shown in Figure 4B, the rats of saline (NIR) group were found to have serve left ventricular dysfunction, with a significant decrease in ejection fraction (EF)% (85.0 ± 4.1% vs 32.0 ± 2.3%, p < 0.05), fractional shortening (FS)% (60.6 ± 2.9% vs 30.6 ± 9 ACS Paragon Plus Environment


2.9%, p < 0.05) compared with sham group. In contrast, Au NRs (NIR) treatment significantly blunted the reductions of EF% (58.0 ± 5.8% vs 32.0 ± 2.3%, p < 0.05), FS% (46.6 ± 4.1% vs 32.3 ± 2.3%, p < 0.05) compared with saline (NIR) group (Figure 4B, Video S1,S2&S3). Consistent with impaired cardiac function, we observed the group of Au NRs (NIR) substantially attenuated ischemia-induced MI. The percentage of MI area to total area in Au NRs (NIR) group was significantly smaller compared with that in the saline (NIR) group (34 ± 2.6% vs 59.6 ± 3.7%, p < 0.05) (Figure 4C&D). Cardiomyocyte apoptosis could be induced by MI. Apoptotic cardiomyocytes was detected in all groups through using TUNEL staining (Figure 4E). However, the number of TUNEL positive cardiac myocytes was significantly decreased in the Au NRs (NIR) group when compared with that in the saline (NIR) group (8.3 ± 1.1% vs 4.8 ± 0.8%, p < 0.05) (Figure 4F). Furthermore, in Figure S7A, the MI rats get remission after treatment. Besides, the major organs did not appear noticeable toxicity and inflammatory cells after treatment (Figure S7B). Taken together, we confirmed that the LAH could be helpful for vascular recanalization in MI.

CONCLUSION In summary, LAH were firstly confirmed to be benefit to blood clot lysis precisely and rapidly in murine hematoma model and murine MI model. In murine hematoma model, compared with traditional therapy, LAH presented a rapid detumescence of hematoma due to the localized hyperthermia. More importantly, cardioprotection was observed in murine MI model treatment with LAH. Besides, Au NRs are bio-degradable and have limited toxicity with convenience, give rise to a promising, precise, rapid and drug-free blood clot lysis. The ongoing research would be focused on the mechanism of blood clot lysis by LAH.

ASSOCIATED CONTENT Supporting Information 10 ACS Paragon Plus Environment

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The Supporting Information is available free of charge on the ACS Publications website. The element distribution of Au NRs (Figure S1), cell viability of vascular endothelial cell treated with Au NRs (Figure S2), photos of treatment by NIR laser irradiation and traditional therapy (Figure S3), the Au amount in the blood and urine of mice, the ultrasound cardiogram of control, saline (NIR) and Au NRs (NIR) group (Figure S4), 3D printed simulated wearable ankle device (Figure S5), thrombolysis of Au NRs in vitro (Figure S6), the electrocardiogram and H&E staining images (Figure S7), the ultrasound cardiogram of control group (Video S1), saline (NIR) group (Video S2) and Au NRs (NIR) group (Video S3), photothermal conversion efficiency (Table S1), Au amount in muscle and major organs of mice (Table S2) Author information Corresponding Authors: *E-mail: [email protected] (K.H) *E-mail: [email protected] (X.D) *E-mail: [email protected] (X.W) Author Contributions #Lina Dong and Xiao Liu contributed equally and should be considered co-first authors. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (81760641 to X.D.); National Natural Science Foundation of China (81530013 to K.H.); National Natural Science Foundation of China (21461015 to X.W.); National Natural Science Foundation of China (81270202 and 91339113 to H.X.); National Key Basic Research Program of China (2013CB531103 to H.X.); Natural Science Foundation of Jiangxi Province (20165BCB19002， KJLD14010, 20153BCB23035 and 20161ACB21002 to X.W.); Nanchang University Seed Grant for Biomedicine to X.W.

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Figure 1. (A) Schematic illustration of the mechanism of NIR thermotherapy and transditional thermotherapy; (B) The photo of agarose blocks for localized light-hyperthermia



assessment; (C) The thermal distribution change of agarose blocks treated by NIR and heating pad.

Figure 2. Characterization of Au NRs. (A) SEM image of Au NRs and (inset); (B) TEM image of Au NRs; (C) The size distribution of Au NRs in saline; (D) The ultraviolet-visible spectrum of Au NRs; (E) Heating curves of different concentration of Au NRs at 2 W/cm2; (F) Heating curves of 200 μg/mL Au NRs with different intensity of electricity (0.5, 0.6 and 0.7A).


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Figure 3. (A) Schematic illustration of murine hematoma model and treatment; (B) The treatment and measurement procedure. The Au NRs were injected after the model was built, then leg circumference was measured every 6 h; (C) The statistical circumference change of groups treated with Saline (NIR), Au NRs, AuNRs (NIR), heating pad, 2.5 and 5 kU/kg uPA, 6/group; (D) The photos of legs before and after different treatments; (E) The Au amount in muscle and major organs of mice treated with Au NRs at 1 d and 7 d, 6/group; (F) Representative H&E staining images of major organs at 1 d and 7 d (scale bar: 20 μm).



Figure 4. (A) A diagram of surgical procedures. (B) Representative M-mode images of echocardiography (left). EF % and FS % were found to be significantly higher in Au NRs (NIR) group as compared with saline (NIR) group (right). 7/group. (C) Representative images of myocardial infarct size. 7/group. (D) Infarct size of Au NRs(NIR) group was significant lower compared with saline (NIR) group. 7/group. (E) Representative photomicrographs of detection of apoptotic cardiomyocytes by TUNEL staining. Green fluorescence shows TUNEL-positive nuclei (red arrow); Blue fluorescence shows nuclei of total cardiomyocytes. 7/group. (F) Percentage of apoptotic cardiomyocytes in the in Au NRs (NIR) group were significant lower as compared with saline (NIR) group. 7/group. # P < 0.05 compared with sham group, * P < 0.05 compared with saline (NIR) group.


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For Table of Contents Only



Figure 1 170x130mm (300 x 300 DPI)


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Figure 2 90x104mm (300 x 300 DPI)



160x140mm (300 x 300 DPI)


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Figure 4


Localized Light-Au-Hyperthermia Treatment for Precise, Rapid, and

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