Polynorepinephrine Nanoparticles: A Novel Photothermal Nanoagent

Subscriber access provided by Bibliothèque de l'Université Paris-Sud

Biological and Medical Applications of Materials and Interfaces

Polynorepinephrine Nanoparticles: A Novel Photothermal Nanoagent for Chemo-Photothermal Cancer Therapy Xin Liu, Zhuo Xie, Wei Shi, Zi He, Yang Liu, Huling Su, Yanan Sun, and Dongtao Ge ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03458 • Publication Date (Web): 15 May 2019 Downloaded from http://pubs.acs.org on May 15, 2019

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 32 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

Polynorepinephrine Nanoparticles: A Novel Photothermal Nanoagent for Chemo-Photothermal Cancer Therapy

Xin Liu§, Zhuo Xie§, Wei Shi*, Zi He, Yang Liu, Huling Su, Yanan Sun, Dongtao Ge*

Key Laboratory of Biomedical Engineering of Fujian Province University/Research Center of Biomedical Engineering of Xiamen, Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005, China

* Corresponding author. Tel./fax: +86 592 2188502. E-mail addresses: [email protected] (W. Shi), [email protected] (D. Ge)

1

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

Page 2 of 32

ABSTRACT Novel photothermal nanoagents (PTNAs) with excellent photothermal performance, smart-responsive property and biocompatibility are in urgent need for precise chemo-photothermal cancer therapy. Herein, polynorepinephrine nanoparticles (PNE NPs) with high photothermal conversion efficiency () of 808 nm laser (67%), pH/thermal responsibility and little to no long-term toxicity were synthesized from an endogenic

neurotransmitter

norepinephrine.

Comparing

to

their

analogues

polydopamine (PDA) NPs, a widely used PTNA, PNE NPs exhibited higher  value (enhanced 1.63-folds) and better cellular uptake efficiency (enhanced 2.57-folds). After modifying with PEG and loading with doxorubicin (DOX), PNE-PEG@DOX could realize responsive release of DOX under either a cytolysosome pH microenvironment (pH 5.0) or an 808 nm laser irradiation, resulting in an enhanced chemotherapeutic efficacy of DOX. Besides, in vivo combination therapy leads to nearly complete ablation of tumor tissues, while no significant side effects were found in normal tissues. Hence, this intelligent and biocompatible nano-platform based on PNE NPs holds great potential in promoting the clinic transformation of precise chemo-photothermal cancer therapy.

KEYWORDS: polynorepinephrine, photothermal therapy, chemotherapy, intelligent, nanomedicine

2


Page 3 of 32 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


1. INTRODUCTION Chemotherapy as a widely used clinical cancer treatment destructs malignant cells via various chemotherapeutic drugs, while photothermal therapy (PTT) is a recent booming cancer treatment which induces the cell death by hyperthermia from light triggered photothermal nanoagents (PTNAs) 1-6. Although both methods exhibit certain efficacy versus cancer, the severe side effects (harming normal cells and the immune system, etc.) of chemotherapy and the helplessness of PTT in totally ablating the tumors still hinder their clinical implementation, thus more secure and efficacious cancer treatments are in urgent need

7-9.

Owing to the synergistic effects along with the

minimal invasiveness of PTT and drug delivery upon PTNAs, the combined chemophotothermal therapy improved therapeutic efficacy and reduced side effects, attracting widespread attention in the cancer treatment 10-15. Despite satisfactory therapeutic efficacy, there is still a long way to the clinical implementation of combined chemo-photothermal therapy, because of the concerns about the in vivo long-term safety of PTNAs

16-18.

The currently available PTNAs,

which are mainly composed of exogenous inorganic and organic compounds (such as Pd, Au, CuS, carbon-based nanomaterials and conjugated polymer NPs), are poorly biometabolized due to their nondegradability, thus inevitably induce many toxic responses such as metal-relative issues and pulmonary inflammation 19,20. To settle this problem, exploiting PTNAs from endogenic substances might be conductive to their clinical applications, owing to the low-risk of causing any immune response. However, to the best of our knowledge, only two types of such PTNAs, (porphyrin-lipid and polydopamine (PDA) NPs), have been explored 21,22. To better meet the complex demands of chemo-thermal therapy in future clinical application, it is necessary to develop more PTNAs that consist of naturally endogenic substances. Norepinephrine (NE), a small molecule catecholamine in the body, could act as a neurotransmitter for regulating the sense of stress and maintaining blood pressure 23. Similar to dopamine, NE is found easily polymerized in an alkaline solution with oxygen and could obtain PNE NPs with various size and high NIR absorbance, 3



which might be suitable for in vivo PTT

24,25.

Moreover, the particular adhesive

property and conjugated molecular structure of PNE make PNE NPs highly superior for loading chemotherapeutic agents with aromatic ring. Last but not least, the abundant amino and hydroxyl groups of PNE NPs are beneficial to surface modification for biofunctionalities, which could obtain multifunctional theranostic nanoplatforms for cancer therapy. Besides the concerns about the biosafety of PTNAs, the unsatisfied pharmacokinetics such as insufficient target drug release of majority nanomedicines is another obstacle for combined chemo-photothermal therapy 26,27. Developing stimuliresponsive nanomedicines in response to exogenous (temperature, light and magnetic field, etc.) or endogenous (pH, cytosolic GSH and enzyme, etc.) stimuli could realize temporal, spatial and dosage-controlled drug release, thus improving the therapeutic efficacy

28-32.

However, single stimulus could only respond to specific area of tumor.

To confront the complex environments in tumor, multiple stimuli-responsive nanomedicines are proved to be more efficient. Especially those based on simultaneous exogenous and endogenous stimuli have received dramatically research interest, due to their on-demand drug delivery manner 33-35. Herein, a novel PTNA based on PNE NPs was fabricated, for the first time, and further integrated with HOOC-PEG-OH and DOX for combined chemo-photothermal cancer therapy (Scheme 1). Similar to their analogues PDA NPs, PNE NPs seldom caused any immune response36 and were highly superior for in vivo PTT, because of their components of endogenic neurotransmitter norepinephrine and high NIR absorbance. More excitingly, compared with PDA NPs, higher photothermal conversion efficiency (67%) and improved cellular uptake efficiency (enhanced 2.57folds) were detected for PNE NPs. Besides, benefitting from pH and photothermal responsive of PNE NPs, PNE-PEG@DOX NPs could specifically release DOX under acid cytolysosome (pH 5.0) or 808 nm laser irradiation conditions. By virtue of the above features, excellent combined therapeutic efficacy and ignored side-effects were observed in both in vitro and in vivo experiments, demonstrating the high superiority of PNE-PEG@DOX NPs for combined chemo-photothermal therapy. 4


Page 4 of 32

Page 5 of 32 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


Scheme 1. Schematic diagram of preparation of PNE-PEG@DOX and its application in combined chemo-photothermal therapy.

2. EXPERIMENTAL SECTION 2.1. Materials Norepinephrine hydrochloride and HOOC-PEG-OH (5 kDa) were purchased from Sigma–Aldrich. NaOH and HCl (36%) were obtained from Sinopharm Chemical Reagent Co., Ltd.. Doxorubicin hydrochloride (DOX·HCl) was purchased from Shanghai HaoYun Chemical Technology Co., Ltd.. Dulbecco modified eagle medium (DMEM) and dialysis bags (MWCO≈1 kDa) were provided by Sangon Biotech Co., Ltd.. Fetal bovine serum (FBS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphe-nyl-2-Htetrazolium bromide (MTT), fluorescein isothiocyanate (FITC), 4',6-diamidino-2phenylindole (DAPI), Lyso-Tracker Red and penicillin-streptomycin were obtained from Beyotime Bio-technology Co., Ltd.. 5



2.2. Characterization Scanning electron microscopy (SEM) images were obtained by a Hitachi S4800. Transmission electron microscopy (TEM) images were obtained by a JEOL JEM-1400 TEM. UV-Vis-NIR spectra were recorded using a SHIMADZU UV-1750 spectrophotometer. The Zeta potential and size of as-prepared NPs were measured by Malvern Zeta sizer Nano ZS (Model: ZEN 3600). Confocal fluorescence images and flow cytometry data were obtained by a Zeiss LSM 880+Airyscan laser scanning confocal microscope and a BD Accuri C6 flow cytometer, respectively. The Fourier transform infrared (FTIR) spectra of as-prepared NPs were obtained by a Nicolet iS10 FTIR spectrometer (Thermo Scientific) through KBr pellet technique. The photothermal performance was recorded by an infrared thermal camera (FOTRIC 225).

2.3. Synthesis of PNE and PNE-PEG NPs PNE NPs were synthesized via a facile method. In brief, 180 mg of norepinephrine hydrochloride was dissolved in 90 mL of deionized (DI) water, followed by adding 760 L 1 M NaOH aqueous solution. Then the mixed solution was heating to 50 °C with mechanical stirring and further reacted for 5 h. When the color of the solution changed from pale yellow to dark brown, the NPs were collected by centrifugation (16000 rpm, 20 min) and washed with DI water for several times. Finally, the obtained PNE NPs were dispersed in DI water and further centrifuged at 3000 rpm for 20 min, to discard any large NPs. For PEG functionalization, 2 mL HOOC-PEG-OH aqueous solution (20 mg/mL) was mixed with 10 mL PNE NPs aqueous solution (2 mg/mL) under magnetic stirring, followed by adding 50 mg EDC and 50 mg NHS. After 12 h of reaction, the obtained PNE-PEG NPs were collected via ultrafiltration centrifugation using a centrifugal filter (MWCO≈5 kDa, Milipore) and stored at 4 °C in water.

2.4. Photothermal Performance of PNE NPs 6


Page 6 of 32

Page 7 of 32 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


To evaluate the photothermal conversion performance, 2 mL PNE NPs solution of various concentration (0, 25, 50, 100, 200 g/mL) were irradiated by an 808 nm laser of different power density (1, 1.5, 2 W/cm2) for 10 min. To evaluate the photothermal stability, 2 mL PNE NPs solution (200 g/mL) was irradiated by an 808 nm laser (2 W/cm2) for three laser on/off cycles. A FOTRIC 225 infrared thermal camera was used to record the temperature variation of the solution at different time intervals. To investigate the photothermal conversion efficiency , 1 mL size-matched PNE and PDA NPs solution (200 g/mL) in quartz cell were irradiated by an 808 nm laser (2 W/cm2) for 18 min, and then the temperature variation of the solutions during and after the illumination was record by a FOTRIC 225 infrared thermal camera. The  value of PNE and PDA NPs was calculated via equation 1 and 2:

 

hA 

hAΔTmax - Qs I (1 - 10 - A )

(1)

λ

mC Ks

(2)

where h was the heat-transfer coefficient; A was the surface area of the quartz cell; Δ Tmax was the temperature variation of solution at the maximum steady-state temperature; Qs was the heat associated with the light absorption of the solvent; I was the laser power; A was the absorbance of NPs solution at 808 nm; m and C were the mass and heat capacity of the solvent, respectively; Ks was time constant of the solution, which can be determined via the fitting curve of time vs –lnθ, obtained from the cooling period 37,38.

2.5. Drug Loading and Release 500 g PNE-PEG NPs were mixed with different amounts of DOX (0.1, 0.3, 0.5, 0.7, 0.9 and 1.1 mg) in 5 mL DI water, and the mixed solution was stirred for 12 h in the dark. The DOX-loaded PNE-PEG NPs (PNE-PEG@DOX NPs) were collected by centrifugation (16000 rpm, 20 min) and washed with DI water for several times till the supernatant became colorless. The amount of DOX loaded onto PNE-PEG NPs was determined by UV-Vis spectrophotometer. The drug loading efficiency (DLE) and drug 7



encapsulation efficiency (DEE) were calculated as following: DLE = (weight of initial added DOX － weight of DOX in supernatant)/(weight of PNE-PEG NPs), DEE = (weight of initial added DOX－weight of DOX in supernatant)/(weight of initial added DOX). For in vitro DOX release experiments, 1 mL PNE-PEG@DOX NPs solution (2.4 mg/mL) was sealed in a dialysis bag (MWCO≈1 kDa) and dialyzed in 5 mL PBS (pH 5.0 or pH 7.4) at 37 °C with shaking in a dark room. At predesigned time intervals, all the dialysate was replaced with 5 mL fresh corresponding PBS. In addition, to investigate the influence of illumination on the drug release of PNE-PEG@DOX NPs, an 808 nm laser (2 W/cm2) was used to irradiate the PNE-PEG@DOX NPs solution at predesigned time intervals during the in vitro drug release experiments. Meanwhile, all the dialysate was collected and replaced with fresh PBS (pH 5.0) of same volume. The release amount of DOX in the dialysate was determined by UV-Vis spectrophotometer at wavelength of 488 nm.

2.6. In Vitro Cell Studies Hela and HUVEC cells (American type culture collection, ATCC) were cultured in 10 × 10 cm petri dishes at 37 °C in DMEM-H with 10% FBS and 1% penicillinstreptomycin under a 5% CO2 humid atmosphere. After reaching 90% confluence, the cells were trypsinized and subcultured for 10 generations during the whole cell experiments. For cell uptake studies, FITC was used to label size-matched PNE-PEG and PDAPEG NPs via Schiff base reaction, to form NPs with fluorescence performance. The confocal microscopy images and the cell uptake efficiency of PNE-PEG-FITC NPs by Hela cells were obtained for assessing the cell uptake ability. For confocal microscopy observation, 1.0 × 10 4 Hela cells were seeded onto a coverslip and incubated in a 24well plate for 24 h. Then serum-supplemented medium (SSM) containing PNE-PEGFITC NPs was added and incubated for 0.5 and 2 h. Before the confocal microscopy observation, cells were washed with PBS, fixed with 4% (w/v) formaldehyde solution and stained with DAPI, successively. For cell uptake efficiency (UE) calculation, Hela 8


Page 8 of 32

Page 9 of 32 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


cells were seeded in a black plate (96-well) at a density of 1.0 × 10 4 per well and incubated for 12 h. Then 200 L fresh SSM with or without fluorescence NPs (PNEPEG-FITC or PDA-PEG-FITC NPs) were added as experimental group and control group, respectively. After incubating for different time intervals (2, 4 and 6 h), cells were washed with PBS thoroughly and broken by 200 L lysis buffer. The fluorescence intensity of each well was recorded by a microplate reader (Ex: 495 nm, Em: 519 nm) and the UE was calculated as equation 3:

UE 

FIS  FIC  100% FII

(3)

where FIS, FIC and FII were the fluorescence intensity of experimental group, control group and initial FITC-labelled NPs solution, respectively. For cytolysosome co-localization, Hela cells were co-cultured with PNE-PEGFITC NPs in SSM for 2 h, followed by incubating with Lyso-Traker Red and DAPI for cytolysosome and cell nucleus staining. Finally, the cells were washed with PBS thoroughly before observing on a confocal laser scanning microscopy (CLSM, Zeiss LSM 880+Airyscan). To investigate the intracellular release of DOX, 1.0 × 10 4 Hela cells were seeded onto a coverslip and incubated in a 24-well plate for 24 h, followed by adding fresh SSM containing size-matched PDA-PEG@DOX or PNE-PEG@DOX NPs at the same mass concentration and further incubating for another 6 h. Before the confocal microscopy observation, cells were washed with PBS, fixed with 4% (w/v) formaldehyde solution and stained with DAPI for 10 min. The intracellular fluorescence of DOX was measured by a BD Accuri C6 flow cytometer (Ex: 488 nm, Em: 565 ~ 605 nm). The in vitro cytotoxicity was evaluated using the MTT assay. Hela cells were seeded into a 96-well plate (1 × 104 cells per well) and incubated for 24 h. Then 200 L fresh SSM containing different concentrations of PNE-PEG NPs was added and cocultured for another 24 h. Then, cells were washed with PBS thoroughly and 200 L 10% MTT solution was added and incubated in the CO2 incubator for 4 h. Finally, cells were lysed by 100 L DMSO and the absorbance of each well at wavelength 570 nm 9



was recorded by a microplate reader for calculating the cell viability. For in vitro antitumor performance studies, Hela cells with a density of 1 × 104 cells per well were seeded into a 96-well plate and incubated for 24 h. Afterwards, the cells were treated with fresh SSM containing various concentrations of PNE-PEG, DOX and PNE-PEG@DOX NPs for another 24 h, respectively. After incubating for 6 h, the PTT groups received an extra irradiation by an 808 nm laser (2 W/cm2) for 5 min. Finally, all the cells were washed thoroughly with PBS and the cell viability of each group was determined by a standard MTT assay. For live and dead cells staining, 2.0 × 105 Hela cells were seeded into a 6 × 6 cm petri dish and incubated for 12 h. Then 1 mL fresh SSM containing PNE-PEG or PNEPEG@DOX was added and co-cultured with the cells for 6 h. Then the PTT groups were irradiated by an 808 nm laser (2 W/cm2) for 5 min. Before the fluorescence microscope observation, all the cells were washed thoroughly with PBS, followed by staining with PI/Calcein-AM for 30 min. The fluorescence microscope (Leica 6000BDM) was used for photographing.

2.7. Hemolytic Assay 1 mL EDTA-stabilized fresh mouse blood was centrifuged at 2000 rpm for 10 min to collect the Red blood cells (RBCs). Then the RBCs were washed and diluted ten times by PBS before usage. For hemolytic assay, 300 L diluted RBCs solution was added to 1 mL PBS as negative control, 1 mL DI water as positive control and 1 mL PBS with PNE-PEG@DOX NPs at different concentration as experimental groups, respectively. All the samples were kept at 37 °C for 12 h, followed by centrifuging at 2000 rpm for 10 min. Finally, the absorption of all the collected supernatant at 541 nm was measured by a microplate reader for determining the released hemoglobin and the hemolysis percentage was calculated as following: hemolysis percentage = (As−Aso−Anc)/(Apc−Anc) ×100%, where As, Aso, Anc and Apc were the absorption of samples with RBCs, samples without RBCs, negative and positive controls, respectively. 10


Page 10 of 32

Page 11 of 32 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


2.8. Animals and Tumor Model Building Female BALB/c nude mice (5-6 weeks old) were purchased from Shanghai Slac laboratory animal Co., Ltd. (China) and used in accordance with the guidelines of the Chinese National Science and Technology Committee. For tumor model building, 1 × 107 Hela cells suspended in 100 µL PBS were subcutaneously injected into the right back of the mice. And the in vivo therapeutic experiments were proceeded when the tumor volume reached about 100 mm3. The tumor volume was calculated as (tumor length) × (tumor width)2/2.

2.9. In Vivo Photothermal Imaging and Biodistribution of PNE-PEG@DOX NPs For in vivo photothermal imaging, three Hela tumor-bearing mice were intravenously injected with 100 L of PBS (control group), PNE-PEG PBS solution (2 mg/mL) and PNE-PEG@DOX PBS solution (2 mg/mL), respectively. After 12 h, an 808 nm laser at a power density of 1 W/cm2 was used to irradiate the tumor sites of the mice for 10 min. Meanwhile, the thermal images and temperature variation were recorded by a FOTRIC 225 infrared thermal camera. To investigate the in vivo distribution of PNE-PEG@DOX NPs, 100 g PNEPEG/DOX NPs in 100 L PBS was administrated intravenously into Hela tumorbearing mice. At predesigned time intervals (2, 6, 12, 24, 48 and 72 h), the main organs of mice were harvested for fluorescence imaging via an IVIS Lumina II small animal imaging systems (Ex: 465 nm, Em: 612 nm). Mice received equal dosage of DOX were regarded as control groups.

2.10. In Vivo Therapeutic Experiments 35 Hela tumor-bearing mice were dived into seven groups (PBS, PBS + NIR, PNEPEG, DOX, PNE-PEG@DOX, PNE-PEG + NIR and PNE-PEG@DOX + NIR) to receive intravenous injection of PBS, DOX, PNE-PEG and PNE-PEG@DOX, respectively. After 12 h, the mice in the laser treatment groups were exposed to an 808 nm laser (1 W/cm2) for 6 min every other day during 14 days’ treatment. The tumor volume, body weight and digital photos of mice in each group were recorded at 11



Page 12 of 32

designed time points. In addition, one mouse from each group was sacrificed after 3 days’ treatment for harvesting its tumor for H&E staining.

2.11. Biological safety evaluation To investigate the influence of the laser dose on the skin, eight healthy mice were irradiated by an 808 nm laser (1 W/cm2) for different times of illumination (6 min each time), respectively. The interval of irradiations was 48 hours. Then, the photographs and the H&E staining slices of the skins received illumination were record. To evaluate the in vivo biosafety of as-prepared NPs, mice received different treatments were sacrificed after 14 days’ treatment, and their blood and main organs (heart, liver, spleen, lung and kidney) were harvested for blood biochemistry test and H&E staining.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of PNE and PNE-PEG NPs PNE NPs were prepared via oxidative polymerization of norepinephrine hydrochloride by oxygen in NaOH solution at 50 °C under mechanical stirring. The existence of oxygen, as well as amount of NaOH in solution plays vital roles in determining the size and shape of obtained PNE NPs. For instance, under a nitrogen atmosphere negligible color change was observed in the reaction solution, indicating oxygen was essential for the spontaneous oxidative polymerization of norepinephrine. Moreover, as the addition amount of NaOH increased, the size of synthesized PNE NPs decreased till the mole ratio of norepinephrine to NaOH reached 1:1 (Figure S1a,b). Nevertheless, amorphous PNE NPs were obtained while the amount of NaOH exceeded the norepinephrine (Figure S1c). Herein, PNE NPs with diameter of ≈100 nm were chosen for the following experiments, owing to their suitable size for efficient tumor accumulation via enhanced permeability and retention (EPR) effect

39-41.

As revealed

by SEM and TEM, the resultant PNE NPs were spherical in shape, with excellent monodispersity and an average diameter of ≈100 nm (Figures 1a and S1d). To prolong 12


Page 13 of 32 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 circulation of PNE NPs in blood, HOOC-PEG-OH was successfully modified onto PNE NPs via EDC/NHS amidation reaction, which was verified by the slightly increased diameter (106 nm) and zeta potential (-12 mV) of PNE-PEG NPs (Figure 1c,b and S2). The stability of as-prepared NPs (PNE, PNE-PEG and PNE-PEG@DOX NPs) in PBS for various time (0, 12, 24, 48 h) was studied via DLS and UV analysis. As shown in Figure S3, no significant discrepancies were observed in the particle size distribution curves and UV-Vis-NIR absorption spectra obtained at different time intervals. In addition, as-prepared PNE-PEG NPs also showed excellent colloidal stability in various media (Figure S4), further indicating their potential as drug delivery candidates for in vivo applications. 3.2. Photothermal Performance Evaluation Potential PTNAs should exhibit strong absorption in near-infrared (NIR) light region, known as a desirable biological window for its deeper tissue penetration depth 42-44.

Thus, we recorded the UV-Vis-NIR absorption curves of PNE NPs aqueous

solutions of various concentration. An increasing absorption value was observed for PNE NPs aqueous solution at higher concentration (Figure S5a). For photothermal performance evaluation, the photothermal curves, photostability and  value of PNE NPs were investigated. As shown in Figure S5b and Figure 1d, the photothermal performance of PNE NPs was power density and concentration dependent. A more rapid heating rate was detected in groups under laser of higher power density and groups of higher concentration. Similar results could also be clearly visualized in corresponding infrared thermal images of PNE NPs solutions after illumination (Figure S5c,d). Noteworthy, a sufficient high temperature (>40 °C) for causing cell death could achieve easily by irradiating PNE NPs solution (50 g/mL) at a power density of 2 W/cm2 for 10 min

45,46.

Next, the photostability of PNE NPs was evaluated via

monitoring the temperature attenuation during three laser ON/OFF cycles. The unimpaired photothermal performance demonstrated the excellent photostability of PNE NPs (Figure 1e). To further evaluate their photothermal conversion performance, the  value of PNE NPs was measured and compared with their analogue polydopamine (PDA), which is widely used in PTT 47-49. Interestingly, a much higher  value (≈67%) 13



was detected for PNE relative to the  value (≈41%) of PDA (Figure S6). Moreover, this  value was higher than majority of common photothermal agents, such as Au NPs (22%) and polypyrrole NPs (45%) etc. 50-53. These all demonstrated as-prepared PNE NPs were excellent candidates for potential application in PTT.

3.3. Drug Loading and Controlled-Release Property A clinic chemotherapeutic drug DOX was loaded onto PNE-PEG NPs via electrostatic adsorption. The characteristic peak of DOX at 488 nm appeared in the absorption spectrum of PNE-PEG@DOX NPs, demonstrating the successful loading of DOX (Figure 1f). Furthermore, the new FTIR characteristic peaks at 1583 cm–1, 1212 cm–1 and 990 cm–1 corresponding to DOX showed up in PNE-PEG@DOX, which further indicated the loading of DOX (Figure 1g). To evaluate the drug loading property of PNE-PEG to DOX, equivalent PNE-PEG NPs were added to 5 mL DOX aqueous solution of various concentrations for drug adsorption (Figure 1h). The maximum DLE (67.7%) of PNE-PEG was achieved, which was greatly higher than that of PDA-PEG NPs (42%). This higher DLE can be ascribed to the lower Zeta-potentials of PNE-PEG NPs (Figure S7), which were conducive to electrostatic adsorption of drugs with positive charge (DOX). Besides excellent drug loading property, controlled-release behavior at different internal microenvironments is also required for an ideal drug carrier. Herein, PBS 7.4 and PBS 5.0 were chosen to mimic the microenvironments of normal tissue and cytolysosome. As shown in Figure 1i, only 14% of DOX was released from PNEPEG@DOX after immerging in PBS 7.4 for 24 h, indicating slight drug leakage during blood circulation. Interestingly, more DOX up to 43% was released in PBS 5.0 after 24 h of incubation, suggesting potential pH responsive release of DOX in an acidic cytolysosome microenvironment. It is probably due to the weakened electrostatic adsorption between DOX and PNE-PEG, stemming from partial protonation of the amidogen of PNE under an acid condition. Moreover, under an 808 nm laser illumination, the release amount of DOX in PBS 5.0 in 24 h was further increased (≈ 56%). This could be ascribed to the thermal expansion of PNE-PEG caused by 14


Page 14 of 32

Page 15 of 32 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


photothermal effects, which is benefit for accelerating the release of DOX. Thus, asprepared PNE-PEG@DOX could realize controlled-release of DOX under both endogenous (pH) and exogenous (light) stimuli, which is of great value for facing complicated demands on drug release in vivo.

Figure 1. (a) SEM image of PNE NPs. (b) Zeta-potential of PNE and PNE-PEG NPs. (c) DLS analysis of PNE and PNE-PEG NPs in H2O. (d) Heating curves of PNE NPs aqueous solutions at different concentrations. (e) Photostability of PNE NPs during three laser ON/OFF cycles. (f) UV-Vis-NIR absorption spectra of free DOX, PNE and PNE-PEG@DOX NPs in H2O. (g) FTIR spectra of free DOX, PNE, PNE-PEG and PEN-PEG@DOX NPs. (h) Drug loading efficiency and encapsulation efficiency of PNE-PEG NPs to DOX at various DOX/PNE-PEG mass ratio. (i) Release profiles of DOX from PNE-PEG@DOX NPs in pH 7.4 and pH 5.0 with or without illumination.

3.4. Cellular Uptake and Lysosome Localization of PNE NPs Besides excellent dispersity, controlled-release property and photothermal 15



performance, ideal PTNAs for fabricating synergistic nanomedicine should also possess good cellular uptake behavior. For cellular uptake experiment, a green fluorescence probe FITC was covalently grafted onto PNE-PEG NPs. The obtained fluorescence NPs (PNE-PEG-FITC) were then co-cultured with Hela cells for cellular uptake. As shown in Figure S8, PNE-PEG-FITC NPs were effectively internalized by Hela cells, and the cellular uptake behavior was time dependent. Next, the cellular UE of PNE was calculated and compared with its analogue PDA, a commonly used PTNA. Both PNE and PDA NPs were modified with HOOC-PEG-OH and labeled by FITC, to form size matched fluorescence NPs (Figure S9). Higher cellular UE about 2.57 folds versus PDA-PEG-FITC NPs treated group was detected in PNE-PEG-FITC NPs treated group after 6 h incubating (Figure 2c), which might be contributed to the stronger adhesion between PNE-PEG NPs and cells.54 Since PNE-PEG@DOX NPs could release more DOX under pH 5.0 condition, which is known to be the pH microenvironment of cytolysosome 55, the intracellular co-localization of PNE-PEG-FITC NPs in Hela cells stained by Lyso-Tracker Red was observed via CLSM. As shown in Figure 2a,b, the PNE-PEG-FITC NPs were mainly found in cytolysosome, demonstrating the NPs could selectively localize in cytolysosome after being internalized by Hela cells. To investigate the drug delivery property in vitro, Hela cells were co-cultured with size-matched PDA-PEG@DOX and PNE-PEG@DOX NPs for 6 h. And the intracellular distribution of DOX was evaluated via detecting the fluorescence of DOX by flow cytometry and CLSM, respectively. According to the flow cytometry results, the amount of cell internalized DOX in PNE-PEG@DOX NPs treated group was 1.89 folds of that in PDA-PEG@DOX NPs treated group (Figure S10). In addition, much stronger red fluorescence of DOX was visualized in the cell nucleus of PNEPEG@DOX NPs treated group relative to PDA-PEG@DOX NPs treated group (Figure 2e). These all demonstrated that the drug delivery property of PNE-PEG NPs was superior to PDA-PEG NPs, which might be ascribed to the better cellular UE and higher drug loading efficiency of PNE-PEG NPs. 16


Page 16 of 32

Page 17 of 32 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


Figure 2. (a) Intracellular localization of PNE-PEG-FITC NPs (green) with cytolysosome-specific probe Lyso-Tracker Red under CLSM. (b) Lysosome colocalization analysis of PNE-PEG-FITC NPs in Hela cells. (c) The ratio of cellular UE of PNE-PEG-FITC to PDA-PEG-FITC NPs at different incubation time intervals. (d) Flow cytometry analysis and (e) corresponding CLSM images of Hela cells incubated with PDA-PEG@DOX and PNE-PEG@DOX NPs for 6 h. Blue and red fluorescence represent DAPI-stained nuclei and DOX fluorescence respectively.

3.5. Cytotoxicity and In Vitro Therapeutic Effects 17



Before in vitro therapeutic experiments, the cytotoxicity of PNE-PEG NPs without illumination was investigated via standard MTT assays. After incubation with PNEPEG NPs at various concentration for 24 h, negligible cytotoxicity was found in both cell lines (Hela and HUVEC) even at a NPs concentration up to 500 g/mL, indicating excellent cytocompatibility of PNE-PEG NPs (Figure 3a). However, for in vitro therapeutic experiments, each group exhibited a concentration-dependent cytotoxicity. Noteworthy, an enhanced therapeutic effect was observed in PNE-PEG@DOX + NIR group, which might be ascribed to the synergistic effects of chemotherapy and PTT (Figure 3b). In addition, to directly visualize the therapeutic effects, cells received different treatments were incubated with CalceinAM/PI for dead (red) and live (green) cell staining. According to the CLSM images (Figure 3c), no apparently dead cells were found in control, laser and PNE NPs treated groups, indicating laser and PNE NPs alone could not cause cellular death. Whereas, PNE-PEG@DOX, PNE-PEG + NIR and PNE-PEG@DOX + NIR groups exhibited gradually enhanced cellular mortality, which was consistent with the results of MTT assays.

18


Page 18 of 32

Page 19 of 32 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


Figure 3. (a) Relative viability of Hela and HUVEC cells co-cultured with PNE-PEG NPs at different concentrations for 24 h in the dark. (b) Relative viability of Hela cells in DOX, PNE-PEG@DOX, PNE-PEG + NIR and PNE-PEG@DOX + NIR treatment groups. (c) Live and dead cells staining by Calcein-AM/PI for different in vitro treatment groups.

3.6. Hemolytic Property Evaluation As PNE-PEG@DOX NPs were injected via intravenous administration, their hemocompatibility need to be investigated prior to the in vivo experiments. According to the hemolytic percentage and corresponding digital photos, negligible hemolysis was observed even at a NP concentration up to 1 mg/mL (Figure S11). These indicated asprepared

PNE-PEG@DOX

NPs

were

ideal

nanomedicine

19


with

excellent


hemocompatibility, which could be transported safely in the blood fluid.

3.7. In Vivo Infrared Thermal Imaging and Biodistribution To evaluate the tumor targeting property and in vivo photothermal effects of PNEPEG@DOX NPs. Three Hela tumor bearing mice received intravenous injection of PBS, PNE-PEG and PNE-PEG@DOX NPs were irradiated by an 808 nm laser (1 W/cm2) for 10 min. The temperature variation of tumor sites during the illumination process was recorded by a FOTRIC 225 infrared thermal camera. As shown in Figure 4a,b, both PNE-PEG and PNE-PEG@DOX NPs treated mice showed significant temperature rising (≈30 °C and ≈23 °C) at the tumor sites. On the contrary, slight temperature rising ( ≈ 3 °C) was detected at the tumor site of PBS treated mouse. Noteworthy, though the introduction of DOX impaired the photothermal effects of PNE-PEG@DOX NPs, compared to equal mass of PNE-PEG NPs. The ultimate temperature of tumor site in PNE-PEG@DOX NPs treated mouse could rise up to ≈ 55 °C, which was high enough for killing the cancer cells. Moreover, the in vivo photothermal effects also demonstrated the effective tumor targeting of PNEPEG@DOX NPs via EPR effect. Next, to investigate the in vivo drug delivery property of PNE-PEG@DOX NPs, the biodistribution of DOX in mice received intravenous injection of equimolar free DOX and PNE-PEG@DOX NPs was studied via ex vivo fluorescence imaging. As shown in Figure 4c,d, both groups detected obvious fluorescence in liver and kidney, which are two main organs for the metabolism of small molecule drugs. The difference between the two groups was that a much slower drug clearance rate was found in PNEPEG@DOX NPs treated group. This could be ascribed to the control-release performance and prolonged blood circulation of PNE-PEG@DOX NPs. Additionally, stronger fluorescence signals were detected in the tumors of PNE-PEG@DOX NPs treated mice after administration for 12 h. This indicated much more DOX could be taken to the tumor sites by virtue of the EPR effect and drug delivery performance of PNE-PEG@DOX NPs. 20


Page 20 of 32

Page 21 of 32 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


Figure 4. (a) Infrared thermal images of Hela tumor-bearing mice received intravenous injection of PBS, PNE-PEG and PNE-PEG@DOX NPs under a 1 W/cm2 808 nm laser illumination within 10 min. (b) Corresponding heating curves of tumor sites in mice during illumination. (c) Average fluorescence intensity and (d) fluorescence images of DOX in excised organs and tumors obtained at different time points after administration.

3.8. In Vivo Therapeutic Effects Evaluation Encouraged by the excellent in vivo photothermal effects and drug delivery 21



performance of PNE-PEG@DOX NPs, the in vivo combined therapeutic efficacy was evaluated on Hela tumor bearing mice. As shown in Figure 5a,b, the tumor of the mice exposed to only laser irradiation or injected by the PNE-PEG without laser irradiation exhibited fast tumor growths similar to the growth of the control group of mice, indicating that NIR and PNE-PEG alone could not inhibit the growth of tumors. As to DOX and PNE-PEG@DOX treated mice, the latter showed enhanced tumor inhibition, suggesting that combining DOX to PNE-PEG NPs could improve its chemotherapeutic effects. In addition, though the PNE-PEG + NIR treated mice showed excellent tumor inhibition, a regrowth of tumors was found during the therapeutic process. Therefore, neither chemotherapy nor PTT alone can cure the tumor bearing mice. Delightedly, thoroughly ablation of tumors was observed in PNE-PEG@DOX + NIR treated mice after 14 day of treatment, which demonstrated the outstanding combined therapeutic effects of PNE-PEG@DOX NPs under illumination. The H&E staining of tumor slices obtained from different groups at the third day of treatment was observed for further evaluating the therapeutic effects. Negligible tumor cell damage (condensed nucleus and cellular morphology change) was found in PBS, PBS + NIR and PNE-PEG treated groups, while gradually enhanced cell damage emerged in DOX, PNE-PEG@DOX, PNE-PEG + NIR and PNE-PEG@DOX + NIR treated group in sequence (Figure 5d). These results further demonstrated the combined therapeutic effects of PNEPEG@DOX under illumination. To study whether skin burns would be caused by illumination alone (1 W/cm2), we recorded the temperature variation and photographs of mice during the illumination. As shown in Figure 4a, 5a and S12, a relative low temperature (35 °C) and no obvious black scab were detected in the mice during illumination. In addition, the corresponding H&E staining slices of skin received difference dose of illumination exhibited normal cell morphology under the microscopy, indicating no cell damage occurs with a dose of 7 times illumination (1 W/cm2 for 6 min each time) (Figure S12). Thus, we speculated the laser power density and illumination time we used in the in vivo study were in the scope of biosafety. 22


Page 22 of 32

Page 23 of 32 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


Figure 5. (a) Representative photographs of mice in various treatment groups within 14 days. (b) Relative volume variation of tumors in mice received various treatments within 14 days. P values were calculated by ANOVA with Tukey's post-test: **P < 0.01. (c) Body weight fluctuation of mice in various treatment groups during 14 days. N.S.S. represents no statistical significance. (d) H&E staining images of tumors obtained from mice received various treatments for 3 days.

3.9. In Vivo Toxicity Evaluation The blood biochemical test was performed to study the hepatorenal toxicity of asdesigned NPs. As shown in Figure S13, the blood biochemistry indexes of mice in 23



different groups were all within the normal ranges, indicating negligible hepatorenal toxicity of as-designed NPs. In addition, the body weight variation and H&E staining slices of main organs of mice in different groups were further studied for evaluating the long-term in vivo toxicity of PNE-PEG@DOX NPs. As shown in Figure 5c and Figure S14, no obvious body weight fluctuation of mice and cell damage of main organs were found in different treatment groups. Therefore, as-prepared PNE-PEG NPs were ideal drug carriers with little or no long-term in vivo toxicity 5.

4. CONCLUSIONS In summary, a novel PTNA based on PNE NPs with excellent  (67%) of 808 nm laser, pH/thermal smart responsibility and biocompatibility was successfully obtained, and modified with HOOC-PEG-OH and DOX for combined chemo-photothermal cancer therapy. Compared with their analogue PDA NPs, a commonly used PTNA, the PNE NPs not only showed enhanced  value (1.63 folds), but also possessed higher cellular uptake efficiency (2.57-folds). Moreover, a broad-spectrum anticancer drug DOX could be easily loaded in PNE-PEG NPs with a maximum DLE of 67.7% and specifically released under acid cytolysosome (pH 5.0) or 808 nm laser irradiation conditions, leading to site-specific drug accumulation in cancer cells. By virtue of these features, the cell and animal experiments showed excellent combined therapeutic efficacy, while no significant long-term toxicity was detected. Therefore, the newly developed PNE NPs presented great potential in combined chemo-thermal therapy.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: SEM and TEM images, DLS analysis, UV-Vis-NIR absorption spectra, heating curves, infrared thermal images, CLSM images, flow cytometry analysis, blood biochemistry indexes and H&E staining images (PDF) 24


Page 24 of 32

Page 25 of 32 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


AUTHOR INFORMATION Corresponding Authors *Email: [email protected]. *Email: [email protected]. ORCID Wei Shi: 0000-0002-6718-7952 Dongtao Ge: 0000-0002-7892-6925 Xin Liu: 0000-0001-5944-4296 Yanan Sun: 0000-0003-3678-6255 Author Contributions §

These authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Nature Science Foundation of China (81271689, 31271009, 31870986) and the Program for New Century Excellent Talents in University. Jingru Huang is acknowledged for confocal laser scanning microscopy analysis assistance.

REFERENCES [1] DeVita, V.T.; Chu, E. A History of Cancer Chemotherapy. Cancer Res. 2008, 68, 8643-8653. [2] Gradishar, W.J.; Tjulandin, S.; Davidson, N.; Shaw, H.; Desai, N.; Bhar, P.; Hawkins, M.; O'Shaughnessy, J. Phase III Trial of Nanoparticle Albumin-Bound Paclitaxel Compared with Polyethylated Castor Oil-Based Paclitaxel in Women with Breast Cancer. J. Clin. Oncol. 2005, 23, 7794-7803. [3] Thorn, C.F.; Oshiro, C.; Marsh, S.; Hernandez-Boussard, T.; McLeod, H.; Klein, T.E.; 25



Altman, R.B. Doxorubicin Pathways: Pharmacodynamics and Adverse Effects. Pharmacogenetics Genom. 2011, 21, 440-446. [4] Huang, X.; El-Sayed, I.H.; Qian, W.; EI-Sayed, M.A. Cancer Cell Imaging and Photothermal Therapy in the Near-Infrared Region by Using Gold Nanorods. J. Am. Chem. Soc. 2006, 128, 2115-2120. [5] Yang, K.; Zhang, S.; Zhang, G.; Sun, X.; Lee, S.T.; Liu, Z. Graphene in Mice: Ultrahigh In Vivo Tumor Uptake and Efficient Photothermal Therapy. Nano Lett. 2010, 10, 3318-3323. [6] Feng, L.; Gai, S.; Dai, Y.; He, F.; Sun, C.; Yang, P.; Lv, R.; Niu, N.; An, G.; Li, J. Controllable Generation of Free Radicals from Multifunctional HeatResponsive Nanoplatform for Targeted Cancer Therapy. Chem. Mater. 2018, 30, 526-539. [7] Carelle, N.; Piotto, E.; Bellanger, A.; Germanaud, J.; Thuillier, A.; Khayat, D. Changing Patient Perceptions of the Side Effects of Cancer Chemotherapy. Cancer 2002, 95, 155-163. [8] Boer, M.D.; Djontono, J.; Visser, B.; Stoter, G.; Verweij, J. Patient Perception of the Side Effects of Chemotherapy; the Influence of the Introduction of 5TH3 Antagonists. Eur. J. Cancer 1993, 29, S264. [9] Wang, Y.W.; Fu, Y.Y.; Peng, Q.; Guo, S.S.; Liu, G.; Li, J.; Yang, H.H.; Chen, G.N. Dye-Enhanced Graphene Oxide for Photothermal Therapy and Photoacoustic Imaging. J. Mater. Chem. B 2013, 1, 5762-5767. [10] Liu, H.; Chen, D.; Li, L.; Liu, T.; Tan, L.; Wu, X.; Tang, F. Multifunctional Gold Nanoshells on Silica Nanorattles: A Platform for the Combination of Photothermal Therapy and Chemotherapy with Low Systemic Toxicity. Angew. Chem. Int. Edit. 2011, 50, 891-895. [11] Zhang, W.; Guo, Z.; Huang, D.; Liu, Z.; Guo, X.; Zhong, H. Synergistic Effect of Chemo-Photothermal Therapy Using PEGylated Graphene Oxide. Biomaterials 2011, 32, 8555-8561. [12] Wang, Y.; Wang, K.; Zhao, J.; Liu, X.; Bu, J.; Yan, X.; Huang, R. Multifunctional Mesoporous Silica-Coated Graphene Nanosheet Used for Chemo-Photothermal Synergistic Targeted Therapy of Glioma. J. Am. Chem. Soc. 2013, 135, 4799-4804. 26


Page 26 of 32

Page 27 of 32 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


[13] Zheng, M.; Yue, C.; Ma, Y.; Gong, P.; Zhao, P.; Zheng, C.; Sheng, Z.; Zhang, P.; Wang, Z.; Cai, L. Single-Step Assembly of DOX/ICG Loaded Lipid-Polymer Nanoparticles for Highly Effective Chemo-Photothermal Combination Therapy. ACS Nano 2013, 7, 2056-2067. [14] Shen, S.; Tang, H.; Zhang, X.; Ren, J.; Pang, Z.; Wang, D.; Gao, H.; Qian, Y.; Jiang, X.; Yang, W. Targeting Mesoporous Silica-Encapsulated Gold Nanorods for ChemoPhotothermal Therapy with Near-Infrared Radiation. Biomaterials 2013, 34, 31503158. [15] Xu, J.; Gulzar, A.; Yang, P.; Bi, H.; Yang, D.; Gai, S.; He, F.; Lin, J.; Xing, B.; Jin, D. Recent advances in near-infrared emitting lanthanide-doped nanoconstructs: Mechanism, design and application for bioimaging. Coordination Chemistry Reviews 2019, 381, 104-134. [16] Ali, M.R.; Rahman, M.A.; Wu, Y.; Han, T.; Peng, X.; Mackey, M.A.; Wang, D.; Shin, H.J.; Chen, Z.G.; Xiao, H.; Wu, R.; Tang, Y.; Shin, D.M.; EI-Sayed, M.A. Efficacy, Long-Term Toxicity, and Mechanistic Studies of Gold Nanorods Photothermal Therapy of Cancer in Xenograft Mice. P. Natl. Acad. Sci. USA 2017, 114, E3110E3118. [17] Chang, Y.; Yang, S.T.; Liu, J.H.; Dong, E.; Wang, Y.; Cao, A.; Liu, Y.; Wang, H. In Vitro Toxicity Evaluation of Graphene Oxide on A549 Cells. Toxicol. Lett. 2011, 200, 201-210. [18] Kostarelos, K. The Long and Short of Carbon Nanotube Toxicity. Nat. Biotechnol. 2008, 26, 774-776. [19] Valsami-Jones, E.; Lynch, I. How Safe are Nanomaterials. Science 2015, 350, 388389. [20] Sharifi, S.; Behzadi, S.; Laurent, S.; Forrest, M.L.; Stroeve, P.; Mahmoudl, M. Toxicity of Nanomaterials. Chem. Soc. Rev. 2012, 41, 2323-2343. [21] Lovell, J.F.; Jin, C.S.; Huynh, E.; Jin, H.; Kim, C.; Rubinstein, J.L.; Chan, W.C.W.; Cao, W.; Wang, L.V.; Zheng, G. Porphysome Nanovesicles Generated by Porphyrin Bilayers for Use as Multimodal Biophotonic Contrast Agents. Nat. Mater. 2011, 10, 324-332. 27



[22] Li, Y.; Jiang, C.; Zhang, D.; Wang, Y.; Ren, X.; Ai, K.; Chen, X.; Lu, L. Targeted Polydopamine Nanoparticles Enable Photoacoustic Imaging Guided ChemoPhotothermal Synergistic Therapy of Tumor. Acta Biomaterialia 2017, 47, 124-134. [23] Tanaka, M.; Yoshida, M.; Emoto, H.; Ishii, H. Noradrenaline Systems in the Hypothalamus, Amygdala and Locus Coeruleus are Involved in the Provocation of Anxiety: Basic Studies. Eur. J. Pharmaco. 2000, 405, 397-406. [24] Hong, S.; Kim, J.; Na, Y.S.; Park, J.; Kim, S.; Singha, K.; Im, G.I.; Han, D.K.; Kim, W.J.; Lee, H. Poly(norepinephrine): Ultrasmooth Material-Independent Surface Chemistry and Nanodepot for Nitric Oxide. Angew. Chem. Int. Edit. 2013, 125, 93579361. [25] Iwasaki, T.; Tamai, Y.; Yamamoto, M.; Taniguchi, T.; Kishikawa, K.; Kohri, M. Melanin Precursor Influence on Structural Colors from Artificial Melanin Particles: Polydopa, Polydopamine, and Polynorepinephrine. Langmuir 2018, 34, 11814-11821. [26] Li, W.; Guo, Q.; Zhao, H.; Zhang, L.; Li, J.; Gao, J.; Qian, W.; Li, B.; Chen, H.; Wang, H.; Dai, J.; Guo, Y. Novel Dual-Control Poly(N-Isopropylacrylamide-CoChlorophyllin) Nanogels for Improving Drug Release. Nanomedicine 2012, 7, 383392. [27] Saw, P.E.; Yu, M.; Choi, M.; Lee, E.; Jon, S.; Farokhzad, O.C. Hyper-Cell-Permeable Micelles as a Drug Delivery Carrier for Effective Cancer Therapy. Biomaterials 2017, 123, 118-126. [28] Karimi, M.; Ghasemi, A.; Sahandi, Z.P.; Rahighi, R.; Moosavi, S.M.B.; Mirshekari, H.; Amiri, M.; Shafaei, P.Z.; Aslani, A.; Bozorgomid, M.; Ghosh, D.; Beyzavi, A.; Vaseghi, A.; Aref, A.R.; Haghani, L.; Bahrami, S.; Hamblin, M.R. Smart Micro/Nanoparticles in Stimulus-Responsive Drug/Gene Delivery Systems. Chem. Soc. Rev. 2016, 45, 1457-1501. [29] Croissant, J.; Zink, J.I. Nanovalve-Controlled Cargo Release Activated by Plasmonic Heating. J. Am. Chem. Soc. 2012, 134, 7628-7631. [30] Tian, F.; Yu, Y.; Wang, C.; Yang, S. Consecutive Morphological Transitions in Nanoaggregates Assembled from Amphiphilic Random Copolymer via Water-Driven Micellization and Light-Triggered Dissociation. Macromolecules 2008, 41, 338528


Page 28 of 32

Page 29 of 32 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


3388. [31] Liu, G.; Dong, C.M. Photoresponsive Poly(S-(O-Nitrobenzyl)-L-Cysteine)-B-PEO from a L-Cysteine N-Carboxyanhydride Monomer: Synthesis, Self-Assembly, and Phototriggered Drug Release. Biomacromolecules 2012, 13, 1573-1583. [32] Chen, B.; Dai, W.; He, B.; Zhang, H.; Wang, X.; Wang, Y.; Zhang, Q. Current Multistage Drug Delivery Systems Based on the Tumor Microenvironment. Theranostics 2017, 7, 538-558. [33] Wang, Y.; Wei, G.; Zhang, X.; Xu, F.; Xiong, X.; Zhou, S. A Step-By-Step Multiple Stimuli-Responsive Nanoplatform for Enhancing Combined Chemo-Photodynamic Therapy. Adv. Mater. 2017, 29, 1605357. [34] Lei, Z.; Hao, W.; Li, Y. Stimuli-Responsive Nanomedicines for Overcoming Cancer Multidrug Resistance. Theranostics 2018, 8, 1059-1074. [35] Zheng, T.; Li, G.G.; Zhou, F.; Wu, R.; Zhu, J.J.; Wang, H. Gold‐Nanosponge‐Based Multistimuli‐Responsive Drug Vehicles for Targeted Chemo‐Photothermal Therapy. Adv. Mater. 2016, 28, 8218-8226. [36] Liu, Y.; Zhou, G.Q.; Liu, Z.; Guo, M.Y.; Jiang, X.M.; Taskin, M.B.; Zhang, Z.Y.; Liu, J.; Tang, J.L.; Bai, R.; Besenbacher, F.; Chen, M.L.; Chen, C.Y. Mussel Inspired Polynorepinephrine Functionalized Electrospun Polycaprolactone Microfibers for Muscle Regeneration. Sci. Rep. 2017, 7, 8197. [37] Roper, D.K.; Ahn, W.; Hoepfner, M. Microscale Heat Transfer Transduced by Surface Plasmon Resonant Gold Nanoparticles. J. Phys. Chem. C 2007, 111, 36363641. [38] Tian, Q.; Jiang, F.; Zou, R.; Liu, Q.; Chen, Z.; Zhu, M.; Yang, S.; Wang, J.; Wang, J.; Hu, J. Hydrophilic Cu9S5 Nanocrystals: A Photothermal Agent with a 25.7% Heat Conversion Efficiency for Photothermal Ablation of Cancer Cells In Vivo. ACS Nano 2011, 5, 9761-9771. [39] Chen, Q.; Feng, L.; Liu, J.; Zhu, W.; Dong, Z.; Wu, Y.; Liu, Z. Intelligent AlbuminMnO2 Nanoparticles as pH-/H2O2-Responsive Dissociable Nanocarriers to Modulate Tumor Hypoxia for Effective Combination Therapy. Adv. Mater. 2016, 28, 71297136. 29



[40] Huang, K.; Ma, H.; Liu, J.; Huo, S.; Kumar, A.; Wei, T.; Zhang, X.; Jin, S.; Gant, Y.; Wang, P.C.; He, S.; Zhang, X.; Liang, X.J. Size-Dependent Localization and Penetration of Ultrasmall Gold Nanoparticles in Cancer Cells, Multicellular Spheroids, and Tumors In Vivo. ACS Nano 2012, 6, 4483-4493. [41] Maruyama, K. Intracellular Targeting Delivery of Liposomal Drugs to Solid Tumors Based on EPR Effects. Adv. Drug Deliver. Rev. 2011, 63, 161-169. [42] Yang, K.; H, Xu.; Cheng, L.; Sun, C.; Wang, J.; Liu, Z. In Vitro and In Vivo NearInfrared Photothermal Therapy of Cancer Using Polypyrrole Organic Nanoparticles. Adv. Mater. 2013, 25, 5586-5592. [43] Liu, X.; Su, H.; Shi, W.; Liu, Y.; Sun, Y.; Ge, D. Functionalized Poly(Pyrrole-3Carboxylic Acid) Nanoneedles for Dual-Imaging Guided PDT/PTT Combination Therapy. Biomaterials 2018, 167, 177-190. [44] Kam, N.W.S.; O'Connell, M.; Wisdom, J.A.; Dai, H. Carbon Nanotubes as Multifunctional Biological Transporters and Near-Infrared Agents for Selective Cancer Cell Destruction. P. Natl. Acad. Sci. USA 2005, 102, 11600-11605. [45] Habash, R.W.Y.; Bansal, R.; Krewski, D.; Alhafid, H.T. Thermal Therapy, Part 2: Hyperthermia Techniques. Crit. Rev. Biomed. Eng. 2006, 34, 491-542. [46] Rong, Y.; Mack, P. Apoptosis Induced by Hyperthermia in Dunn Osteosarcoma Cell Line In Vitro. Int. J. Hyperther. 2000, 16, 19-27. [47] Lin, L.S.; Cong, Z.X.; Cao, J.B.; Ke, K.M.; Peng, Q.L.; Gao, J.; Yang, H.H.; Liu, G.; Chen, X. Multifunctional Fe3O4@Polydopamine Core-Shell Nanocomposites for Intracellular mRNA Detection and Imaging-Guided Photothermal Therapy. ACS Nano 2014, 8, 3876-3883. [48] Hu, D.; Zhang, J.; Gao, G.; Sheng, Z.; Cui, H.; Cai, L. Indocyanine Green-Loaded Polydopamine-Reduced Graphene Oxide Nanocomposites with Amplifying Photoacoustic and Photothermal Effects for Cancer Theranostics. Theranostics 2016, 6, 1043-1052. [49] Zhang, R.; Su, S.; Hu, K.; Shao, L.; Deng, X.; Sheng, W.; Wu, Y. Smart Micelle@Polydopamine Core-Shell Nanoparticles for Highly Effective ChemoPhotothermal Combination Therapy. Nanoscale 2015, 7, 19722-19731. 30


Page 30 of 32

Page 31 of 32 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


[50] Wang, S.; Shang, L.; Li, L.; Yu, Y.; Chi, C.; Wang, K.; Zhang, J.; Shi, R.; Shen, H.; Waterhouse,

G.I.N.;

Liu,

Organic‐Framework‐Derived

S.;

Tian,

Mesoporous

J.;

Zhang,

Carbon

T.;

Liu,

Nanospheres

H.

Metal–

Containing

Porphyrin‐Like Metal Centers for Conformal Phototherapy. Adv. Mater. 2016, 28, 8318-8318. [51] Chen, M.; Fang, X.; Tang, S.; Zheng, N. Polypyrrole Nanoparticles for HighPerformance In Vivo Near-Infrared Photothermal Cancer Therapy. Chem. Commun. 2012, 48, 8934-8936. [52] Liu, Y.; Ai, K.; Liu, J.; Deng, M.; He, Y.; Lu, L. Dopamine-Melanin Colloidal Nanospheres: An Efficient Near-Infrared Photothermal Therapeutic Agent for In Vivo Cancer Therapy. Adv. Mater. 2013, 25, 1353-1359. [53] Wang, S.; Riedinger, A.; Li, H.; Fu, C.; Liu, H.; Li, L.; Liu, T.; Tan, L.; Barthel, M.J.; Pugliese, G.; Donato, F.D.; D'Abbusco, M.S.; Meng, X.; Manna, L.; Meng, H.; Pellegrino, T. Plasmonic Copper Sulfide Nanocrystals Exhibiting Near-Infrared Photothermal and Photodynamic Therapeutic Effects. ACS Nano 2015, 9, 1788-1800. [54] Jiang, X.; Li Y.; Liu Y.; Chen C.; Chen, M. Selective enhancement of human stem cell proliferation by mussel inspired surface coating. RSC Adv. 2016, 6, 60206-60214. [55] De Jesus, O.L.P.; Ihre, H.R.; Gagne, L.; J Frechet, J.M.J.; Szoka, F.C. Polyester Dendritic Systems for Drug Delivery Applications: In Vitro and In Vivo Evaluation. Bioconjugate Chem. 2002, 13, 453-461.

31



TOC graphic

32


Page 32 of 32

Polynorepinephrine Nanoparticles: A Novel Photothermal Nanoagent

Recommend Documents