Evans Blue Derivative-Functionalized Gold Nanorods for

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Evans Blue Derivative Functionalized Gold Nanorods for Photothermal Therapy Enhanced Tumor Chemotherapy Xiangyu Wang, Shi Gao, Zainen Qin, Rui Tian, Guohao Wang, Xianzhong Zhang, Lei Zhu, and Xiaoyuan Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02195 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 13, 2018

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ACS Applied Materials & Interfaces

Evans Blue Derivative Functionalized Gold Nanorods for Photothermal Therapy Enhanced Tumor Chemotherapy Xiangyu Wang1, †, Shi Gao2, †, Zainen Qin3, Rui Tian4, *, Guohao Wang1, Xianzhong Zhang1,*, Lei Zhu1, *, Xiaoyuan Chen5 1. State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen 361005, China; 2. Department of Nuclear Medicine, China-Japan Union Hospital, Jilin University, Changchun, Jilin 130033, China; 3. Collaborative Innovation Center of Guangxi Biological Medicine and the Medical and Scientific Research Center Guangxi Medical University, Nanning, Guangxi 530000, China; 4. Department of Ophthalmology Second Hospital, Jilin University, Changchun, Jilin 130033, China; 5. Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, USA. †

These authors contributed equally to this manuscript.

* To whom correspondence should be addressed. E-mail: [email protected] (L.Z.), [email protected] (R.T.) and [email protected] (X.Z)

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Abstract: Chemotherapy is a standard care for cancer management, but the lack of tumor targeting and high dose-induced side effects still limit its utility in patients. Here, we report a chemotherapy combined with photothermal therapy (PTT) for enhanced cancer ablation by functionalization of gold nanorods (GNR) with a novel small molecule named truncated Evans Blue (tEB). Based on the high binding affinity of tEB with albumin, an Abraxane-like nanodrug, human serum albumin/hydroxycamptothecin (HSA/HCPT), was further complexed with GNR-tEB. This formed an HCPT/HSA/tEB-GNR (HHEG) with excellent biostability and biocompatibility. With photoacoustic and fluorescence imaging, we observed HHEG tumor targeting that mediated by enhanced permeability retention (EPR) effect. The accumulation of HHEG peaked in tumor at 12 h post-injection. Moreover, HHEG can effectively ablate tumor growth with laser illumination via chemo/thermal therapy after intravenous administration into SCC7 tumor. This combination is much better than chemotherapy or photothermal therapy alone. Collectively, we constructed a chemo/thermal therapy nanostructure based on a tEB-modified GNR for better tumor treatment effect. The use of tEB in gold nanoparticles can facilitate many new approaches to designing hybrid nanoparticles. Keywords: gold nanorod, Evans blue, chemotherapy, photothermal therapy, nanomedicine


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Introduction Cancer remains a public health concern worldwide1. Because many cancer patients are diagnosed at an advanced stage when tumors are surgically unresectable, chemotherapy is left as the only one of the few treatments regimens available

2-4

. Unfortunately, most traditional

chemotherapy drugs are water insoluble and lack of tumor targeting capabilities. This results in patient suffering during the treatment 5-6. With the development of nanotechnology, it is now possible to utilize nanovehicles carrying conventional chemotherapy drugs to alter their pharmacokinetic profiles, improve water solubility, increase tumor targeting, and reduce side effects

5, 7-9

. One common example is that human serum albumin (HSA) is used to carry

paclitaxel (PTX) to improve its water solubility

10-11

. An albumin-bound nanocomplex

(Abraxane®) got approved by the United States Food and Drug Administration (U.S. FDA) for the management of breast cancer, non-small cell lung cancer, and pancreatic cancer by itself or in combination with other medications 12. Despite the success, there are many significant side effects at high doses and a tendency to induce drug resistance of such chemotherapy agents. This encourages further improvements in the field 13-14. Combination therapy is an ideal approach to elevate therapeutic efficacy, overcome drug resistance, and reduce side effects

15-17

. In particular, photothermal therapy (PTT)

simultaneously delivered by nanoscale agents with chemotherapy has attracted tremendous attentions in recent years 18-20. Typically, PTT utilizes photothermal agents to transform photon energy into cytotoxic heat for diseases management including cancer without thermal damage to healthy tissues

21

. As compared to conventional cancer ablation methods, PTT is highly

localized and can be applied in regions where surgery is difficult. More importantly, it has been



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reported that in combination with chemotherapy, photothermal heating can simulate the cell internalization of chemotherapy drug, increase drug release from the nanocomplex and even improve drug extravasation from tumor microvasculature to elevate the cancer treatment effect in a synergistic way 22-24. Several types of nanocarriers have been engineered for cancer chemo/thermal therapeutics. For example, Chen et al. constructed a graphene oxide/carbon/silica nanocomplex for cancer treatment 25. The Liu group also reported an Abraxane-like nanodrug by encapsulation of ICG and PTX into albumin for primary and metastatic breast cancer ablation 26. More recently, our group reported an Evans Blue (EB)-dispersed carbon nanotube for cancer chemo/thermal therapy by complexing it with albumin/PTX complex

19

. In addition to these systems, gold

nanorod (GNR) has also gained much attention in applications of combined therapy because of the excellent light absorption and high photothermal conversion efficiency27-29. GNRs also offer efficient and easily scaled synthesis, facile functionalization, and colloidal stability. Thus, GNR is an ideal candidate for diseases chemo/thermal therapy28-30. However, biomedical applications of GNRs for chemo/thermal therapy is limited by the cytotoxic effects due to cetyltrimethylammonium bromide (CTAB)

27

that acts as a cationic stabilizer around the

surface of the GNRs. Many nanoscale encapsulation systems including polyethylene glycol (PEG), polymer, HSA, dendrimer, silica, chitosan, and DNAs have been developed to modify GNRs and can help manage cancer 31-33. Evans blue (EB) is a non-toxic azo dye that has been used in broad applications due to the high serum albumin binding affinity and excellent hydrophilicity

16, 21

. More recently, a

maleimide-modified truncated Evans Blue (tEB) was developed for a variety of bioconjugation


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applications in diseases diagnostic and treatment 34-39. Herein, we investigated the possibility of utilizing this truncated Evans Blue (tEB) to replace toxic CTAB and functionalize gold nanorods (GNRs) for combined cancer chemothermal therapy. GNRs are nanometer-sized gold particles with tunable near-infrared (NIR) absorption and scattering that minimizes tissue background. We hypothesized that tEBmodified

GNR

can

form

a

stable

nanocomposite

with

human

serum

albumin/hydroxycamptothecin (HSA/HCPT) complex after simple mixing (Figure 1). The resulting product was named HCPT/HSA/tEB-GNR (HHEG). In this system, HCPT can inhibit cancer cell proliferation by stabilizing the reversible covalent DNA-Topo-I complex 5, and GNR will kill cancer cells by converting photoenergy to heat. More importantly, we found that HCPT drug release efficiency in the system is increased under the local hyperthermia condition induced by NIR laser illumination. Therefore, it is believed that HHEG inhibits tumor growth via a synergistic mechanism whereby PTT effect simultaneously promotes drug delivery and increases the cytotoxicity. Indeed, an improved cancer cell ablation effect was found in vitro and in vivo with the existing of HHEG, and this is significantly better than either chemotherapy or PTT alone. Overall, we successfully developed a chemo/thermal therapy nanocomplex that contains tEB for an improved cancer chemo/thermal combined therapy. This tEB-mediated gold nanoparticle modification strategy can also be used with other antitumor agents for imaging-guided cancer combination therapy. Materials and methods: Reagents



Human

serum

albumin

(HSA)

is

obtained

from

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Abcam

(Cambridge,

MA),

Hydroxycamptothecin (HCPT), chloroauric acid (HAuCl4•3H2O), thiol-PEG-carboxyl (SHPEG-COOH), cystamine dihydrochloride, cetyltrimethyl ammonium bromide (CTAB), dimethylbenzidine, Boc anhydride, 1-amino-8-naphthol-2,4,-disulfonic acid monosodium salt, N-diisopropylethylamine (DIPEA), maleic anhydride, acetic anhydride, dichloromethane, acetonitrile, sodium borohydride (NaBH4), trifluoroacetic acid (TFA), ethylene mercaptan (EDT), thioanisole (TAS), methanol, ethanol, HCl, AgNO3, NaNO2, 3,6-di(O-acetyl)-4,5bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein, and L-ascorbic acid (AA) were bought from Sigma-Aldrich (MO, USA). Cell culture medium and dialysis bags were bought from Thermo Scientific (MA, USA). Human serum albumin (HSA) encapsulated HCPT Preparation HSA encapsulated HCPT was prepared following a reported procedure 16. Briefly, 12.5 mg of HCPT in 1 mL DMSO was mixed with 50 mg of HSA in 10 mL ultrapure water. The mixture was homogenized under high-pressure for 10 min in cold room. Then, the product was dialyzed against water to remove DMSO (Cutoff = 10 kDa) for 4 h. At last, the sample was freeze-dried, yielding a white powder. HCPT standard curve was generated according to high performance liquid chromatography (HPLC) analysis at 224 nm. Briefly, 1 mg HCPT in DMSO was diluted in a solution containing 50% methanol and 50% water to 250, 125, 62.5, 31.25, 15.62, and 7.81 µg/mL. After centrifugation (9,000 rpm, 15 min, 4°C), HCPT in the supernatant fraction was quantified. The standard curve is calculated as y=1.30x+16.66, according to which the efficiency of HCPT encapsulation into HSA was determined .


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Preparation of gold nanorods (GNRs) Gold nanorod (GNR) was prepared following a seed-mediated growth approach40. Briefly, 500 nM auric acid in 200 mM CTAB was reduced by 600 µL of cold sodium borohydride at concentration of 10 mM at 30°C to yield nanoparticles at about 5 nm in diameter, which was used as seed solution for next step. Then, 300 µL AgNO3 was added to a 50 mL 0.01 M auric acid in 0.1 M CTAB and was reduced by 320 µL 0.1M AA, during which the pale-yellow solution gradually changed to nearly colorless. At last, 250 µL seed solution was added to the above solution and stirred at 30°C overnight. After removing the remaining CTAB via repeated centrifugation at room temperature at speed of 6500 rpm, GNR was obtained. Preparation of maleimide-tEB The maleimide-tEB were obtained according to the method that was reported before34, 41. Briefly, 4.3 g tolidine and 4.4 g Boc-anhydride were dissolved with stir in 40 mL methylene chloride for 12 h. Silica gel column was used for purification desired product (3.2 g). 15 mL 0.3 M cooled HCl was introduced into 10 mL acetonitrile containing 0.147 M Boc-tolidine. 0.9 M cold NaNO2 was then dropped into the above solution and stirred in ice bath for 20 min to synthetize the diazonium salt. 0.59 g 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt was added into 20 mL cooled water in ice water containing 0.49 g NaHCO3, the diazonium salt solution was introduced into the above solution drop wise. Boc protecting group was deprotected with 10 mL TFA cocktail containing 80% TFA, 10% EDT and 10% TAS for 60 min. After deprotection, the solution was poured into iced water (50 mL) to obtain purple deposition. The above deposition was filtered and the residue was washed with HCl (pH=4.0) for three times. The residue was dried in the air to get the final product. Small amount of



product was purified with HPLC to give the desired product. 50 uL of DIPEA and 160 mg of maleic anhydride were added to a solution containing 30 mg EB in 4 mL of methanol at the room temperature with stir. The reaction takes 2 h. Then, 1 mL of acetic anhydride was added after removal of methanol and the system was kept at 105 °C for 30 min. HPLC was used for maleimide-tEB purification and the sample was lyophilized. Preparation of HCPT/HSA/tEB-GNR (HHEG) A cystamine dihydrochloride was used to replace CTAB by a ligand exchange process on GNR. Briefly, 20 times excess amount of cystamine dihydrochloride (74.5 mg, 0.33 mmol) was mixed with the washed GNR overnight. Then, excess cystamine was removed by ultrafiltration of the mixture (4000 rpm, 10 min). On the other hand, a short-chain SH-PEG-COOH (2 kDa, 66 mg, 0.033 mmol) was covalently conjugated to tEB-maleimide (20.6 mg, 0.033 mmol) at pH 6.5. tEB-PEG-COOH was finally modified on GNR by 1-ethyl-3-(3-dimethylaminopropyl) (EDC, 9.5 mg, 0.0495 mmol) and N-hydroxysuccinimide (NHS, 5.69 mg, 0.0495 mmol) at pH 8.5 and purified by ultrafiltration of the mixture at 4,000 rpm for 10 min. After that, 9 mg HSA/HCPT was mixed with 3 mg Au-EB, and then the HHEG was obtained through PD-10 column purification. At last, the desired product was lyophilized. The HCPT amount in HHEG analyzed and quantified by HPLC using the same way as described above. Characterization of HHEG Transmission electron microscopy (TEM) images of HHEG were captured by an electron microscopy. Ultraviolet-visible-near-infrared (UV-vis-NIR) spectrum of Mal-EB, HSA/HCPT, Au, HHEG and HCPT were obtained using a multi-scan spectrophotometer (Thermo Scientific). The hydrodynamic diameters were used to characterize the colloidal properties of


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the nanomaterials in PBS with dynamic light scattering (DLS, Malvern Zetasizer, UK). In Vitro PA imaging To investigate the PA signal generated by the GNR-tEB and HHEG, various concentrations (60, 80, 100, 150, 200, 400, 500 μg/mL) of GNR-tEB and HHEG were added to Eppendorf tubes (200 μL) and subjected to laser illumination in a PA imaging system (Endra Nexus128, Ann Arbor, MI). Laser was set at 780 nm. PA images for tEB were also taken is the same condition. In Vitro Photothermal effect of HHEG Various concentrations of the HHEG were illuminated with laser (808 nm, 0.5 W/cm2, 10 min) and thermal images were obtained by thermal camera (FLIR Systems Inc., Wilsonville, USA). HCPT in vitro release profile HCPT release from HHEG was studied in phosphate buffered saline (PBS) by dialysis (cutoff = 10 kDa) at 37°C and 50°C, respectively, for up to 72 h. At the selected time intervals, 70 μL aliquot of the 10 mL external solution was taken and the same amount of fresh medium was replenished. HCPT released from HHEG was determined by HPLC system combined with a separation module, a fluorescence detector, and a C-18 column. Flow speed was set as 1 mL/min and detection wavelength was set as 224 nm. Acetonitrile with 0.1% TFA as mobile phase increased from 5 to 65% versus distilled water containing 0.1% TFA over 30 min. The cumulative amount of HCPT released from the HHEG was calculated. For the stability test, HCPT was incubated at room temperature or heated at 50°C for 10 min before it was analyzed by HPLC and cell proliferation assay. Cell viability assays 5×104 human squamous cell cancer cells (SCC7) were passed in a 96-well plate and cultured



overnight. Various concentrations of HHEG (CHCPT = 0, 7.5, 15, 30, 60, 120, and 240 ng/mL) were added into the different plates, respectively. After incubated in the dark for another 24 h, 50 μg of MTT and 100 μL DMEM per well were added into the plates and incubated for 4 h in dark. Last, 100 μL dimethyl sulfoxide (DMSO) was added into the wells to dissolve the precipitates, the optical absorption of the wells was measured at 490 nm by a microplate reader (Thermo, Varioskan Flash). Cells in culture medium without treatment were used as a control. In Vitro Photothermal inhibition of SCC7 cells Different concentrations of HHEG (CHCPT = 0, 7.5, 15, 30, 60, 120 and 240 ng/mL) were added to SCC7 seeded wells. After 24 h culture, HHEG in medium was removed and the cells were subjected to laser irradiation for 10 min (808 nm 0.5 W/cm2). Cell viability was tested as above. Photoacoustic and Fluorescence Imaging of GNR-tEB in Vivo The animal experiments were performed following Institutional Animal Care and Use Committee guidelines and were approved by Xiamen University Laboratory Animal Center Ethics Committee. Mice (18-20 g) with SCC7 tumors were injected with tEB (1.5625 mg/kg per mouse, n=3) and GNR-tEB (equivalent of 1.5625 mg/kg EB and 3.125 mg/kg GNRs per mouse, n=3) intravenously (i.v.) as the tumor grew to 150-200 mm3. 1.5% isoflurane was used to anesthetize mice via a flexible tube. Next, PA images were obtained at 0, 2, 4, 8, 12, 24 and 48 h post-injection. Fluorescence imaging were taken by IVIS system with 580 nm excitation at 2, 4, 8, 12, 24, and 48 h after HHEG administration. For biodistribution assay, mice were sacrificed after imaging at 12 h time point. Tumor and the major organs including heart, liver, spleen, lungs and kidneys were collected before imaging and the average signal intensity was measured.


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In Vivo Chemo/Thermal Tumor Therapy As the tumor size reached about 100 mm3, 35 mice (18-20 g) with SCC7 tumors were randomly divided to following group: (1) HHEG containing 144.5 µg GNR and 50 µg HCPT w/ laser; (2) HHEG containing 144.5 µg GNR and 50 µg HCPT w/o laser; (3) GNR-tEB containing 144.5 µg GNR w/ laser; (4) GNR-tEB containing 144.5 µg GNR w/o laser; (5) PBS only; (6) HSA/HCPT containing 50 µg HCPT w/ laser; (7) HSA/HCPT containing 50 µg HCPT w/o laser. After 12 h injection, the photoirradiation was performed with laser illumination at power of 2 W/cm2 for 10 min. Tumor sizes (tumor length × (tumor width)2/2) and body weights were monitored every other day. Hematoxylin and Eosin (H&E) Staining After mice were sacrificed in each group, vital organs including heart, liver, spleen, lungs and kidneys as well as tumor were harvested and fixed in formalin. They were sectioned into 4 µm slices and stained with H&E before the images were captured with an optical microscope (Leica QWin). Statistical Analysis The bars stand for the average ± SEM for three repeated experiments. Statistical analysis was performed using one-way ANOVA. P < 0.05 was considered statistically significant. Results and Discussions Characterization of HHEG A number of chemical approaches have been explored to process metal into one dimensional (1 D) nanostructures. In this study, we took advantage of the tunable plasmon resonant absorption and scattering of GNRs in combination with chemotherapy to achieve an enhanced



and combined chemo/thermal cancer therapy. We used GNRs with 780 nm absorbance because this is where tissue has low absorption. First, GNR was synthesized using a seed-mediated route to yield a rod particle with uniform length at 30 ± 5 nm and width at 10 ± 3 nm (Figure 2 and Figure S1). Because the surface cetyltrimethyl ammonium bromide (CTAB) is known to be toxic to cells and animals, a 20-fold excess amount of cystamine dihydrochloride was used to replace CTAB through a ligand exchange reaction (Figure 1). At the same time, a structurally truncated Evans Blue (tEB) with a maleimide group was prepared following our previously publication34 and characterized (Figure S2 and S3). Subsequently, the hydrophilic tEB was conjugated onto GNR-cystamine complex via a PEG linker (Mw: 2000 Da) with -SH and -COOH on each end as shown in Figure 1 and Figure S4. This GNR-tEB complex is found to be stable under physiologically relevant conditions including water, PBS, and DMEM medium (Figure S5a). On the other hand, an Abraxane-like drug delivery system was constructed by encapsulation of hydroxycamptothecin (HCPT), a commonly used chemotherapy drug, into human serum albumin (HSA) as we reported before 5

. Different ratios (1:9, 1:4 and 2:3) of HCPT and HSA were studied to optimize the loading

efficiency. The loading is the highest with HCPT and HSA at a 1:4 ratio. This results in a HSA/HCPT complex with 19.66% HCPT encapsulation (Table 1). Next, HSA/HCPT was complexed with GNR-tEB in water (3:1, w/w) based on the strong binding of tEB to HSA. A UV-VIS-NIR spectrum of HHEG was collected to confirm the successful construction of the system. The absorbance peaks in the HHEG complex were found at 381, 542, and 780 nm (Figure 2a), which were characterized for HCPT, tEB, and GNR, respectively. To make sure GNR shape (Figure S1) is not affected during ligand exchange and


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modification, TEM of HHEG was taken (Figure 2a inserted). No obvious GNR morphology changes were found. The interactions between tEB and albumin were then studied by fluorescence spectrum and shown that HSA/tEB has strong fluorescence signals compared to the low fluorescence signal of tEB without HSA binding (Figure 2b). We did not find fluorescence quenching after binding GNR-tEB with HSA/HCPT, because the absorbance peak (around 780 nm) of GNR is far from the emission of HSA/tEB (ex/em: 580/670 nm). The diameter of the resulting HHEG is about 180 nm (Figure 2c) according to dynamic light scattering measurement (DLS). This size is reasonable for the enhanced permeability retention (EPR) effect mediated HHEG accumulation in tumor. HHEG stability was next evaluated by incubating HHEG with water, PBS, and DMEM medium. No precipitation or obvious absorbance spectral change was found for at least 72 h (Figure S5 a and b) indicating that HHEG is stable and can be used in vivo. The photoacoustic (PA) signals of HHEG were also tested, and we found that the strong PA signals of the GNR-tEB were not affected by complexation with HSA/HCPT (Figure 2 d and Figure S6) at 780 nm. As the concentration of HHEG increased from 60 μg/mL to 500 μg/mL, the intensity of PA signal increased and resulted in brighter images (Figure 2d). The PA signal and the nanoplatform concentration was found with linear relationship (R2=0.996; Figure S6), indicating that PA imaging can be used for analyzing the accumulation of HHEG at the tumor site. No PA signal was detected for tEB under identical conditions. These data suggested that HHEG can be successfully constructed with excellent biocompatibility stability and strong PA signals for further biomedical applications. Photothermal Effect of HHEG



Next, the photothermal conversion capability of HHEG was investigated in vitro. Figure 3 shows GNR, GNR-tEB, HSA/HCPT, and HHEG samples at 250 μg/mL irradiated by NIR laser. The temperature quickly reached 50 °C in less than 2 min for GNR, GNR-tEB, and HHEG groups (Figure 3b) indicating that the combination of HSA/HCPT with GNR does not affect the photothermal conversion efficiency. No obvious temperature changes were detected in HSA/HCPT group even with laser illumination. To verify the excellent PTT effect of HHEG, we gradient diluted HHEG from 250 μg/mL to 0.98 μg/mL and irradiated them in the same condition as above. The temperature of the HHEG solutions increased by 10, 19 and 30 °C, respectively, in 10 min as a function of increasing concentrations of HHEG (from 3.906 to 15.625 and to 250 μg/mL) as shown in Figure S7. A positive liner relationship between photothermal effect and the concentration of HHEG was observed. These data suggest that HHEG has a promising photothermal effect for tumor cell growth inhibition when accumulated in sufficient quantities. In Vitro HCPT Release The chemotherapy potency of HHEG was tested next. The cumulative release kinetics of HCPT at different temperatures (37 °C and 50 °C) were analyzed. HCPT released from HHEG slowly at 37 °C that only about 54% of HCPT was released within 48 h as calculated by the HCPT standard curve (Figure 4 and Figure S8). On the contrary, nearly 97% of HCPT was released from HHEG with a much faster release rate at higher temperatures (50 °C) in about 12 h. This illustrated that the relatively higher temperature improves the drug release rate. Specifically, when delivered in tumor, HHEG will generate heat with a NIR laser irradiation to promote HCPT drug release, which will in turn result in a better therapeutic response. To investigate the


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high temperature affection on HPCT, HCPTs incubated at room temperature and high temperature were analyzed by HPLC (Figure S9a). The retention times were found the same (13.28 min) for both HCPTs at room temperature and 50 °C. Additionally, the cytotoxicity of HCPT at different temperature was evaluated by cell proliferation assay. As Figure S9b shown, HCPTs after incubation at room temperature and 50°C presented a similar cancer cell growth inhibition, indicating that PTT effect of HHEG will only increase chemotherapy drug release without affection HPCT stability. In Vitro Cytotoxicity of HHEG Head and neck squamous cell carcinoma (HNSCC) is one of the most diagnosed solid tumor worldwide42. The exposure to tobacco smoke, alcohol and human papilloma virus (HPV) are the major reasons for HNSCC. Despite significant improvements in head and neck cancers management, long-term survival rates in patients with advanced-stage head and neck cancers have not increased significantly in the past 30 years. In our study, a mouse head and neck carcinoma cell line, SCC7, was chosen for evaluation of HHEG. Based on the outstanding photothermal effect and drug release profile, synergistic HHEG tumor cytotoxicity was first evaluated via a MTT assay. The HHEG showed a better SCC7 cell proliferation inhibition with laser irradiation than the same concentration of GNR-tEB, HSA/HCPT, and control group (30 ng/mL; Figure 5a) due to the combined chemo/thermal therapy. Here, 50.6 ± 6.2% cells were dead in the HHEG-treated group, while 67.57 ± 8.7% and 87.71 ± 5.9% cells were killed with GNR-tEB and HSA/HCPT groups after laser illumination, respectively. To further confirm the cytotoxicity of HHEG to tumor cells and present the advantages of combined therapy, different concentrations of HHEG (equal to 7.5, 15, 30, 60, 120, and 240



ng/mL of HCPT) were incubated with SCC7 cells for 24 h and illuminated with an laser. In another group, the same amount of HHEG was added to cells but were not illuminated with laser. This control only studied the chemotherapy effect. Here, 50.6 ± 6.2% of SCC7 cells died at 30 ng/mL in combination with photothermal therapy (PTT; Figure 5b). Only 19 ± 2.7% cells died at the same HCPT concentration without PTT. At 240 ng/mL HCPT with PTT, 89 ± 7.1% of the cells died, but more than 50% cells were still alive at the same concentration without PTT indicating that PTT and chemotherapy can be combined to achieve significantly improved tumor cell ablation. In Vivo Tumor Targetability of HHEG Before evaluating the HHEG tumor therapeutic efficacy in vivo, the tumor targetability of HHEG was studied via non-invasive photoacoustic (PA) and fluorescence (FL) imaging. PA imaging can offer increased imaging depth and spatial resolution compared to conventional fluorescence (FL) imaging, and FL imaging is preferable for whole body imaging studies in small animals because it offers overall in vivo distribution of contrast agents. Mice with SCC7 tumors were intravenously administrated with free tEB (31.25 μg) or GNR-tEB/HSA (containing 62.5 μg of GNR and 31.25 μg of tEB). At different time points (0, 2, 4, 8, 12, 24, 48 h) post-injection (p.i.), PA images were taken with 780 nm laser illumination, and PA images before injections were also taken. As shown in Figure 6, the PA signals for GNR-tEB/HSA group were getting stronger with time because of the GNR-tEB/HSA accumulation via EPR effect. The PA signal peaked at 12 h p.i., which is 2.5-fold higher than that before GNRtEB/HSA injection. Most GNR-tEB/HSA washed out at 48 h p.i. based on the weak PA signals in the tumor (Figure 6b). We did not notice PA signal changes in the tEB-injected group due


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to the weak tEB absorbance at 780 nm (Figure 2d). In the meantime, whole body FL imaging was also performed to study the in vivo biodistribution of GNR-tEB/has (ex/em: 580/600 nm). FL images were taken at 2, 4, 8, 12, 24, 48 h p.i.. As shown in Figure 6c, the tumor fluorescence signals increased because of the accumulation of GNR-tEB/HSA. Fluorescent signals peaked at 12 h, which is 2.15 ± 0.1 times higher than the background (Figure 6d). This result also uncovers that 12 h post-injection (p.i.) is an ideal time for initiating PTT because most HHEG accumulated in tumor at this time point. Although tEB also showed strong fluorescence signals after binding with HSA (Figure 2b), we did not detect much fluorescence signals in the tumor with tEB injections because tEB is lack of tumor targetability. To further confirm the in vivo distribution of GNR-tEB/HSA, the mice were euthanized at 12 h p.i. when GNR-tEB/HSA peaked in tumor. A strong fluorescence signal was observed in tumors with GNR-tEB/HSA administration due to the EPR effect (Figure S10), however, signals in tEB injected tumor were low and most free tEB was found in the liver, indicating that GNR-tEB/HSA is an ideal carrier for tumor targeted drug delivery. In Vivo Chemo/Thermal Combined Tumor Therapy of HHEG Finally, the in vivo anticancer activity of HHEG was assessed in an SCC7 tumor bearing mouse model. When the tumor volume grew to about 100 mm3, HHEG (containing 144.5 µg of GNR and 50 µg of HCPT), GNR-tEB (containing 144.5 µg of GNR), and HSA/HCPT (containing 50 µg of HCPT) were intravenously injected and the tumors were exposed to 808 nm laser irradiation (2 W/cm2, 10 min) at 12 h p.i.. The temperature in the tumor with HHEG injection quickly increased from 29°C to 55°C in less than 5 min with laser irradiation (Figure 7 a and



b). This temperature increase is sufficient to inhibit the cancer cells. Similarly, temperature of tumor with GNR-tEB administration also reached to 55°C with the same rate as HHEG, suggesting that the GNRs have excellent photothermal conversion efficiency for tumor inhibition. On the contrary, the HSA/HCPT treated tumor only increased about 4°C due to the lack of photothermal converting capability. Likewise, the tumors in the four other groups received the following treatments and were monitored: control without laser, HSA/HCPT (containing 50 µg of HCPT) without laser, GNR-tEB (containing 144.5 µg of GNR) without laser, and HHEG (containing 144.5 µg of GNR and 50 µg of HCPT) without laser. Figure 7 shows that the non-treated control mice tumor grew 8.98 ± 0.39 times larger after 14 days than the initial tumor volume. A similar growth rate (7.63 ± 2.93 times larger) was observed in the tumors that received GNR-tEB but without laser irradiation. On the contrary, the tumor size significantly decreased when laser irradiation was combined with the GNR-tEB treated group. The tumor was only 3.8 ± 2.6 times larger than the initial size. Of note, while PTT indeed effectively inhibit tumor growth at the beginning (from day 0 to day 10; Figure 7c), the tumor size suddenly began to increase fast indicating that PTT alone cannot efficiently kill tumor cells and might induce recurrence. In the HSA/HCPT treated group, we found that tumor shrank with time, but the tumor size remained constant after 8 days treatment regardless of the addition of laser suggesting that chemotherapy alone cannot remove tumors at this dose (2.5 mg/kg). In contrast, the tumor growth was totally ablated when chemotherapy and PTT were combined with HHEG treatment. The tumor shrank after 8 days. No tumors were seen after 2 weeks, suggesting that the combination therapy successfully ablated tumor growth. In addition, researchers have reported that cancer cell death induced by PTT can release the tumor antigens


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to the environment to promote the maturation of dendritic cells and produce antitumor cytokines for improve tumor therapy response43-45. Thus, immune response triggered by PTT might also be involved in our study for SCC7 tumor inhibition. Relevant studies on combined PTT/immunotherapy are under investigation in our group. During our study, obvious body weight changes were not found (Figure 7d). To further investigate the HHEG-mediated combination therapy, HSA/HCPT, GNR-tEB, and HHEG treated tumor and normal organs with and without laser irradiation were harvested and stained by H&E. Compared with control groups, clear tissue necrosis was seen in the laserirradiated HHEG group (Figure 7e) indicating that HHEG can effectively induce cancer cell death via combination chemo/thermal therapy. Little tissue destruction was seen in the other control groups due to insufficient toxicity to tumor cells. The normal organs were analyzed as well and showed no organ damage via H&E staining (Figure 8). Collectively, our results confirmed that HHEG-mediated therapy can efficiently ablate tumor cell growth by PA/fluorescent image-guided combination therapy. Conclusion: In summary, a structurally truncated EB (tEB) was successfully applied to functionalize GNRs for combination chemo/thermal therapy. An Abraxane-like drug complex, HSA/HCPT, was further complexed with GNR-tEB through the binding of tEB and albumin. The resulting nanosystem, HCPT/HSA-tEB/GNR (HHEG), has excellent tumor targetability, photothermal conversion efficiency, and biostability as confirmed by the in vitro characterization and in vivo imaging study. Our results also demonstrated HHEG-mediated chemo/thermal therapy is better than the mono-therapy. The tEB modification of GNRs facilitates new cancer treatment



nanoplatforms in which other anti-cancer drugs such as photosensitizers and functional genes can also be loaded onto the GNR-tEB system via HSA. Supporting Information. Chemical synthesis and characterization, GNR fabrication, stability test, TEM, photoacoustic effect, PTT effect, standard curve, HPLC, cytotoxicity and biodistribution are given in this section. Conflict of Interest: The authors declare no competing financial interest. Acknowledgements This work was supported by National Science Foundation of China (NSFC) (Grant No. 81571708, 81771869, 81501506, 51373144, and 81201129), the Research Fund of Science and Technology Department of Jilin Province (Grant No. 20150520154JH and 20160101001JC), the Foundation of National Health and Family Planning Commission of Jilin Province (Grant No.: 2015Q020), Health Service Capacity Building Program for Jilin Province (Grant No.: 3D517EC63429), the Department of Education of Jilin Province for Thirteen-Five Scientific Technique Research (Grant No.: [2016] 460), the Norman Bethune Program of Jilin University (Grant No. 2015219) and the Intramural Research program, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health.


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FIGURE LEGENDS Figure 1. Preparation of human serum albumin/hydroxycamptothecin (HSA/HCPT)fabricated tEB/GNR-based delivery system (HHEG). Figure 2. Characterization of HHEG. (a) UV-vis-NIR spectrum of HCPT, HSA/HCPT, GNR, maleimide-EB, and HHEG. Inserted: TEM image of HHEG. Scale bar equals to 100 nm. (b) Size measurement of GNR-tEB (35 nm), HSA/HCPT (150 nm), and HHEG (180 nm) in water. (c) and d) Photoacoustic (PA) effects and quantification of tEB, GNR-tEB, and HHEG. Strong PA signals were detected from HHEG and tEB. No PA signal was observed for tEB. The concentration of GNR-tEB and HHEG is correlated to PA signal. Figure 3. Photothermal conversion efficacy of HHEG. (a) Comparison of the photothermal effect of different samples including GNR, GNR-tEB, HSA/HCPT, and HHEG with laser illumination. (b) PTT effect of GNR, GNR-tEB, HSA/HCPT, and HHEG over time. Figure 4. HCPT releasing profile from HHEG complex at different temperatures (37°C and 50°C). Figure 5. Cell proliferation inhibition assay. (a) Normalized SCC7 cells viabilities after HSA/HCPT, GNR-tEB, and HHEG treatment with or without laser illumination. (b) Relative SCC7 cells viabilities after incubation with HHEG with or without laser irradiation. *, p < 0.05. Figure 6. Non-invasive FL and PA imaging of SCC7 tumors. (a) PA images of mice received tEB and GNR-tEB/HSA. (b) Quantification of signal intensity in tumors with tEB and GNRtEB/HSA treatment. (c) FL images of tEB and GNR-tEB/HSA accumulation in tumors. Tumor



is pointed out by arrows. (d) Quantification of tumors signal from tEB and GNR-tEB/HSA over time. Figure 7. Chemo/photothermal combined tumor inhibition. (a) Images of mice with laser illumination by thermal camera after intravenous injection of GNR-tEB, HSA/HCPT, and HHEG. (b) Temperature changes in tumor after different treatments. (c) Tumor sizes changes after different treatments. *, p < 0.05. (d) Mice body weight changes after different treatments. (e) Histological changes in murine tumors with H&E staining. Scale bars equals to 50 µm. Figure 8. H&E staining of normal organs after GNR-tEB, HSA/HCPT, and HHEG treatment with or without NIR laser illumination. Scale bars equals to 50 µm.

Figure 1.


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Figure 2.



Figure 3.

Figure 4.

100 Cumulative release (%)

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

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80 60 40

o 50 C

20

o 37 C

0

0

10

20 30 Time (h)


40

50

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Figure 5.

(a)

(b) 120 Relative cell viability (%)

120 100

Cell viability (%)

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


80 60 40 20

80 60 40

l Ctr

T er er CP las las w/ w/ A/H S B G H E E R-t HH GN

*

20 0

0

HHEG w/o laser AEHH w/o laser HHEG w/ laser AEHH w/ laser

100

0

7.5 15 30 60 120 240 Concentration of HCPT (ng/mL)

Figure 6.



Figure 7.


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Figure 8.

Table 1. HCPT Loading Efficiency HSA: HCPT Ratio (w/w)

Loading efficiency (%)

Loading contents (%)

3:2

83.95

33.58

4:1

98.30

19.66

9:1

64.50

6.45



REFERENCES 1. Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer Statistics, 2017. CA Cancer J Clin 2017, 67, 7-30. 2. Beatson, G.T. On the Treatment of Inoperable Cases of Carcinoma of the Mamma - Suggestions for a New Method of Treatment, with Illustrative Cases. Ca-a Cancer Journal for Clinicians 1983, 33, 108-121. 3. Eifel, P.J. Concurrent Chemotherapy and Radiation Therapy as the Standard of Care for Cervical Cancer. Nature Clinical Practice Oncology 2006, 3, 248-255. 4. Petru, E.; Schmahl, D. Cytotoxic Chemotherapy-Induced 2nd Primary Neoplasms - Clinical Aspects. Neoplasma 1991, 38, 147-155. 5. Chen, M.L.; Zhang, W.Q.; Yuan, K.; Bo, M.X.; Chen, B.; Li, L.; Ma, Q.J.; Zhu, L.; Gao, S. Preclinical Evaluation and Monitoring of the Therapeutic Response of a Dual Targeted Hyaluronic Acid Nanodrug. Contrast Media & Molecular Imaging 2017, 2017, 4972701. 6. Narvekar, M.; Xue, H.Y.; Eoh, J.Y.; Wong, H.L. Nanocarrier for Poorly Water-Soluble Anticancer Drugs-Barriers of Translation and Solutions. Aaps Pharmscitech 2014, 15, 822-833. 7. Wang, G.H.; Gao, S.; Tian, R.; Miller-Kleinhenz, J.; Qin, Z.N.; Liu, T.J.; Li, L.; Zhang, F.; Ma, Q.J.; Zhu, L. Theranostic Hyaluronic Acid-Iron Micellar Nanoparticles for Magnetic-Field-Enhanced Invivo Cancer Chemotherapy. Chemmedchem 2018, 13, 78-86. 8. Jabir, N.R.; Tabrez, S.; Ashraf, G.M.; Shakil, S.; Damanhouri, G.A.; Kamal, M.A. Nanotechnology-Based Approaches in Anticancer Research. Int J Nanomedicine 2012, 7, 4391-4408. 9. Gharpure, K.M.; Wu, S.Y.; Li, C.; Lopez-Berestein, G.; Sood, A.K. Nanotechnology: Future of Oncotherapy. Clinical Cancer Research 2015, 21, 3121-3130. 10. Miele, E.; Spinelli, G.P.; Miele, E.; Tomao, F.; Tomao, S. Albumin-Bound Formulation of Paclitaxel (Abraxane (R) Abi-007) in the Treatment of Breast Cancer. International Journal of Nanomedicine 2009, 4, 99-105. 11. Yin, L.M.; Yuvienco, C.; Montclare, J.K. Protein Based Therapeutic Delivery Agents: Contemporary Developments and Challenges. Biomaterials 2017, 134, 91-116. 12. Ma, P.; Mumper, R.J. Paclitaxel Nano-Delivery Systems: A Comprehensive Review. J Nanomed Nanotechnol 2013, 4, 1000164. 13. Zhao, M.Z.; Lei, C.N.; Yang, Y.D.; Bu, X.L.; Ma, H.L.; Gong, H.; Liu, J.; Fang, X.D.; Hu, Z.Y.; Fang, Q.J. Abraxane, the Nanoparticle Formulation of Paclitaxel Can Induce Drug Resistance by upRegulation of P-Gp. Plos One 2015, 10, e0131429. 14. Scripture, C.D.; Figg, W.D.; Sparreboom, A. Peripheral Neuropathy Induced by Paclitaxel: Recent Insights and Future Perspectives. Current Neuropharmacology 2006, 4, 165-172. 15. Dong, X.W.; Mumper, R.J. Nanomedicinal Strategies to Treat Multidrug-Resistant Tumors: Current Progress. Nanomedicine 2010, 5, 597-615. 16. Xie, L.S.; Wang, G.H.; Zhou, H.; Zhang, F.; Guo, Z.D.; Liu, C.; Zhang, X.Z.; Zhu, L. Functional Long Circulating Single Walled Carbon Nanotubes for Fluorescent/Photoacoustic Imaging-Guided Enhanced Phototherapy. Biomaterials 2016, 103, 219-228. 17. Wang, G.H.; Zhang, F.; Tian, R.; Zhang, L.W.; Fu, G.F.; Yang, L.L.; Zhu, L. Nanotubes-Embedded Indocyanine Green-Hyaluronic Acid Nanoparticles for Photoacoustic-Imaging-Guided Phototherapy. Acs Applied Materials & Interfaces 2016, 8, 5608-5617. 18. Song, X.R.; Wang, X.Y.; Yu, S.X.; Cao, J.B.; Li, S.H.; Li, J.; Liu, G.; Yang, H.H.; Chen, X.Y. Co9se8 Nanoplates as a New Theranostic Platform for Photoacoustic/Magnetic Resonance DualModal-Imaging-Guided Chemo-Photothermal Combination Therapy. Advanced Materials 2015, 27,


Page 28 of 31

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


3285-3291. 19. Zhang, L.W.; Rong, P.F.; Chen, M.L.; Gao, S.; Zhu, L. A Novel Single Walled Carbon Nanotube (Swcnt) Functionalization Agent Facilitating in Vivo Combined Chemo/Thermo Therapy. Nanoscale 2015, 7, 16204-16213. 20. Zha, Z.B.; Zhang, S.H.; Deng, Z.J.; Li, Y.Y.; Li, C.H.; Dai, Z.F. Enzyme-Responsive Copper Sulphide Nanoparticles for Combined Photoacoustic Imaging, Tumor-Selective Chemotherapy and Photothermal Therapy. Chemical Communications 2013, 49, 3455-3457. 21. Zhang, L.W.; Gao, S.; Zhang, F.; Yang, K.; Ma, Q.J.; Zhu, L. Activatable Hyaluronic Acid Nanoparticle as a Theranostic Agent for Optical/Photoacoustic Image-Guided Photothermal Therapy. Acs Nano 2014, 8, 12250-12258. 22. Browning, R.J.; Reardon, P.J.T.; Parhizkar, M.; Pedley, R.B.; Edirisinghe, M.; Knowles, J.C.; Stride, E. Drug Delivery Strategies for Platinum-Based Chemotherapy. Acs Nano 2017, 11, 8560-8578. 23. Ulbrich, K.; Hola, K.; Subr, V.; Bakandritsos, A.; Tucek, J.; Zboril, R. Targeted Drug Delivery with Polymers and Magnetic Nanoparticles: Covalent and Noncovalent Approaches, Release Control, and Clinical Studies. Chemical Reviews 2016, 116, 5338-5431. 24. Yuan, J.; Liu, J.L.; Song, Q.; Wang, D.; Xie, W.S.; Yan, H.; Zhou, J.F.; Wei, Y.; Sun, X.D.; Zhao, L.Y. Photoinduced Mild Hyperthermia and Synergistic Chemotherapy by One-Pot-Synthesized Docetaxel-Loaded Poly(Lactic-Co-Glycolic Acid)/Polypyrrole Nanocomposites. Acs Applied Materials & Interfaces 2016, 8, 24445-24454. 25. Chen, Y.W.; Chen, P.J.; Hu, S.H.; Chen, I.W.; Chen, S.Y. Nir-Triggered Synergic PhotoChemothermal Therapy Delivered by Reduced Graphene Oxide/Carbon/Mesoporous Silica Nanocookies. Advanced Functional Materials 2014, 24, 451-459. 26. Chen, Q.; Liang, C.; Wang, X.; He, J.K.; Li, Y.G.; Liu, Z. An Albumin-Based Theranostic NanoAgent for Dual-Modal Imaging Guided Photothermal Therapy to Inhibit Lymphatic Metastasis of Cancer Post Surgery. Biomaterials 2014, 35, 9355-9362. 27. Liao, J.F.; Li, W.T.; Peng, J.R.; Yang, Q.; Li, H.; Wei, Y.Q.; Zhang, X.N.; Qian, Z.Y. Combined Cancer Photothermal-Chemotherapy Based on Doxorubicin/Gold Nanorod-Loaded Polymersomes. Theranostics 2015, 5, 345-356. 28. Tong, X.; Wang, Z.; Sun, X.; Song, J.; Jacobson, O.; Niu, G.; Kiesewetter, D.O.; Chen, X. Size Dependent Kinetics of Gold Nanorods in Epr Mediated Tumor Delivery. Theranostics 2016, 6, 20392051. 29. Bao, C.C.; Conde, J.; Polo, E.; Del Pino, P.; Moros, M.; Baptista, P.V.; Grazu, V.; Cui, D.X.; De La Fuente, J.M. A Promising Road with Challenges: Where Are Gold Nanoparticles in Translational Research? Nanomedicine 2014, 9, 2353-2370. 30. Li, M.; Guan, Y.; Zhao, A.; Ren, J.; Qu, X. Using Multifunctional Peptide Conjugated Au Nanorods for Monitoring Beta-Amyloid Aggregation and Chemo-Photothermal Treatment of Alzheimer's Disease. Theranostics 2017, 7, 2996-3006. 31. Lim, Z.Z.J.; Li, J.E.J.; Ng, C.T.; Yung, L.Y.L.; Bay, B.H. Gold Nanoparticles in Cancer Therapy. Acta Pharmacologica Sinica 2011, 32, 983-990. 32. Duncan, B.; Kim, C.; Rotello, V.M. Gold Nanoparticle Platforms as Drug and Biomacromolecule Delivery Systems. Journal of Controlled Release 2010, 148, 122-127. 33. Rana, S.; Bajaj, A.; Mout, R.; Rotello, V.M. Monolayer Coated Gold Nanoparticles for Delivery Applications. Advanced Drug Delivery Reviews 2012, 64, 200-216. 34. Chen, H.J.; Wang, G.H.; Lang, L.X.; Jacobson, O.; Kiesewetter, D.O.; Liu, Y.; Ma, Y.; Zhang, X.Z.;



Wu, H.; Zhu, L.; Niu, G.; Chen, X.Y. Chemical Conjugation of Evans Blue Derivative: A Strategy to Develop Long-Acting Therapeutics through Albumin Binding. Theranostics 2016, 6, 243-253. 35. Liu, Y.; Wang, G.; Zhang, H.; Ma, Y.; Lang, L.; Jacobson, O.; Kiesewetter, D.O.; Zhu, L.; Gao, S.; Ma, Q.; Chen, X. Stable Evans Blue Derived Exendin-4 Peptide for Type 2 Diabetes Treatment. Bioconjug Chem 2016, 27, 54-58. 36. Chen, H.; Tong, X.; Lang, L.; Jacobson, O.; Yung, B.C.; Yang, X.; Bai, R.; Kiesewetter, D.O.; Ma, Y.; Wu, H.; Niu, G.; Chen, X. Quantification of Tumor Vascular Permeability and Blood Volume by Positron Emission Tomography. Theranostics 2017, 7, 2363-2376. 37. Tian, R.; Jacobson, O.; Niu, G.; Kiesewetter, D.O.; Wang, Z.; Zhu, G.; Ma, Y.; Liu, G.; Chen, X. Evans Blue Attachment Enhances Somatostatin Receptor Subtype-2 Imaging and Radiotherapy. Theranostics 2018, 8, 735-745. 38. Zhang, J.; Lang, L.; Zhu, Z.; Li, F.; Niu, G.; Chen, X. Clinical Translation of an Albumin-Binding Pet Radiotracer 68ga-Neb. J Nucl Med 2015, 56, 1609-1614. 39. Wang, Y.; Lang, L.; Huang, P.; Wang, Z.; Jacobson, O.; Kiesewetter, D.O.; Ali, I.U.; Teng, G.; Niu, G.; Chen, X. In Vivo Albumin Labeling and Lymphatic Imaging. Proc Natl Acad Sci U S A 2015, 112, 208-213. 40. Chen, H.Y.; Di, Y.F.; Chen, D.; Madrid, K.; Zhang, M.; Tian, C.P.; Tang, L.P.; Gu, Y.Q. Combined Chemo- and Photo-Thermal Therapy Delivered by Multifunctional Theranostic Gold Nanorod-Loaded Microcapsules. Nanoscale 2015, 7, 8884-8897. 41. Niu, G.; Lang, L.; Kiesewetter, D.O.; Ma, Y.; Sun, Z.; Guo, N.; Guo, J.; Wu, C.; Chen, X. In Vivo Labeling of Serum Albumin for Pet. J Nucl Med 2014, 55, 1150-1156. 42. Bozec, A.; Peyrade, F.; Milano, G. Molecular Targeted Therapies in the Management of Head and Neck Squamous Cell Carcinoma: Recent Developments and Perspectives. Anti-Cancer Agents in Medicinal Chemistry 2013, 13, 389-402. 43. Zou, L.L.; Wang, H.; He, B.; Zeng, L.J.; Tan, T.; Cao, H.Q.; He, X.Y.; Zhang, Z.W.; Guo, S.R.; Li, Y.P. Current Approaches of Photothermal Therapy in Treating Cancer Metastasis with Nanotherapeutics. Theranostics 2016, 6, 762-772. 44. Guo, L.R.; Yan, D.D.; Yang, D.F.; Li, Y.J.; Wang, X.D.; Zalewski, O.; Yan, B.F.; Lu, W. Combinatorial Photothermal and Immuno Cancer Therapy Using Chitosan-Coated Hollow Copper Sulfide Nanoparticles. Acs Nano 2014, 8, 5670-5681. 45. Bear, A.S.; Kennedy, L.C.; Young, J.K.; Perna, S.K.; Almeida, J.P.M.; Lin, A.Y.; Eckels, P.C.; Drezek, R.A.; Foster, A.E. Elimination of Metastatic Melanoma Using Gold Nanoshell-Enabled Photothermal Therapy and Adoptive T Cell Transfer. Plos One 2013, 8, e69073.


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TOC


Evans Blue Derivative-Functionalized Gold Nanorods for

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