Plasmonic, Targeted, and Dual Drugs-Loaded Polypeptide Composite

Subscriber access provided by West Virginia University | Libraries

Article

Plasmonic, Targeted, and Dual Drugs-Loaded Polypeptide Composite Nanoparticles for Synergistic Cocktail Chemotherapy with Photothermal Therapy Xingjie Wu, Linzhu Zhou, Yue Su, and Chang-Ming Dong Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00721 • Publication Date (Web): 16 Jun 2016 Downloaded from http://pubs.acs.org on June 21, 2016

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 free 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 accessible to all readers and 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.

Biomacromolecules 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 42

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

Biomacromolecules

Plasmonic, Targeted, and Dual Drugs-Loaded Polypeptide Composite Nanoparticles

for

Synergistic

Cocktail

Chemotherapy

with

Photothermal Therapy

Xingjie Wu1, Linzhu Zhou1, Yue Su1 and Chang-Ming Dong*1,2

1

Department of Polymer Science & Engineering, School of Chemistry & Chemical

Engineering, Shanghai Jiao Tong University, Shanghai 200240, P. R. China. 2

Shanghai Key Laboratory of Electrical Insulation and Thermal Aging, Shanghai Jiao

Tong University, Shanghai 200240, P. R. China.

Submitted as an Article to Biomacromolecules Address correspondence to:

Chang-Ming Dong, Ph.D. Professor of Polymer Science Department of Polymer Science & Engineering School of Chemistry and Chemical Engineering Shanghai Jiao Tong University, Shanghai 200240, P. R. China Phone: 86-21-54748916 Fax: 86-21-54741297 E-mail: [email protected]

1

ACS Paragon Plus Environment

Biomacromolecules

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

ABSTRACT To integrate cocktail chemotherapy with photothermal therapy into one biocompatible and biodegradable nanocarrier, the plasmonic, lactose-targeted, and dual anticancer drugs-loaded polypeptide composite nanoparticles were for the first time fabricated in mild conditions. The glyco-PEGylated polypeptide micelles that self-assembled from the lactose (LAC) and PEG grafted polycysteine terpolymer were used as templates to generate the plasmonic composite nanoparticles, as mainly characterized by DLS, TEM, SEM, and XPS. These composite nanoparticles showed a broad and strong near infrared (NIR) absorption at 650−1100 nm and increased the temperature of phosphate buffer solution by 30.1 oC upon a continuous-wave laser irradiation (808 nm, 5 min, 2 W·cm-2); while same dose of NIR-mediated heating completely killed HepG2 cancer cells in vitro, presenting excellent photothermal properties. Two anticancer drugs doxorubicin (DOX) and 6-mercaptopurine (6-MP) were loaded into the composite nanoparticles through physical interactions and Au-S bond, respectively. The dual drugs-loaded composite nanoparticles exhibited reduction-sensitive and NIR-triggered cocktail drugs release profiles and the trigger-enhanced cytotoxicity. As evidenced by flow cytometry, fluorescence microscopy, and MTT assay, the LAC-coated composite nanoparticles were more internalized by HepG2 than HeLa cell line, demonstrating a LAC-targeting enhanced cytotoxicity towards HepG2. The combination cocktail chemo-photothermal therapy produced a lower half maximal inhibitory concentration than cocktail chemotherapy or photothermal therapy alone, displaying a good synergistic antitumor effect. 2


Page 2 of 42

Page 3 of 42

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

Biomacromolecules

INTRODUCTION Chemotherapy plays an indispensable role in most cancer treatments; however, single drug-based chemotherapy often faces serious side effects, multidrug resistance, and tumor recurrence and metastasis. This situation can be improved to some extent by cocktail or combination chemotherapy (i.e., the simultaneous administration of two or multiple therapeutic agents or drugs), of which the preliminary trials include doxorubicin (DOX), paclitaxel, and cisplatin based chemotherapeutics.1-3 However, the

traditional

cocktail

administration

still

suffers

from

uncontrollable

pharmacokinetics among different drugs. On the other hand, the polymeric drug-loaded nanoparticles (i.e., the nanomedicine formulations) have presented distinct advantages including the enhancement of drug solubility, the prolongation of blood circulation time, the improvement of pharmacokinetics and pharmacodynamics, and especially the enhanced permeation and retention (EPR) effect in solid tumors compared to free drugs.4-6 In this case, stimuli-responsive polymers provide a solid platform for constructing different cocktail drugs loaded nanomedicines (e.g., hydrophobic and hydrophilic drugs), which exhibit “on-demand” drug release profiles and combination cytotoxicity.7-9 Therefore polymeric nanotechnology might further enhance antitumor efficacy of cocktail chemotherapy. As a minimally invasive strategy, photothermal therapy mediated by both nanoparticles (NPs) and near-infrared light (NIR, 650−1000 nm) has increasingly attracted much attention for cancer therapy.10-17 Owing to their excellent optical and bio-inert properties, high surface-volume ratio, and facile surface modifications, various gold NPs including gold nanoshells, gold nanorods, and gold nanocages have 3


Biomacromolecules

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

been widely investigated for photothermal cancer therapy and theranostics.13-17 Moreover, photothermal treatment can be precisely achieved at tumor lesion without damaging healthy tissue by utilizing spatial light irradiation (e.g., optical fiber).11-15 By utilizing the size-mediated EPR and/or specific biomolecular binding (e.g., sugar-lectin and antibody-antigen recognition) of nanoparticles, photothermal therapy can also attain passive and/or active targeting effect to cancer cells with minimizing side effects.18-20 Furthermore, the photothermal heating effect can enhance the accumulation and penetration of drug-loaded nanoparticles in tumor and the cellular permeability and uptake, which might further improve the chemo-therapeutic efficacy. Therefore, the combination of cocktail chemotherapy with photothermal therapy is expected to generate a synergistic and better therapeutic efficacy than a single therapy, holding great potentials for new generation anticancer nanotherapeutics.21-31 To enhance therapeutic effect of single photothermal therapy, researchers have made great efforts on the preparation of plasmonic polymeric gold composite nanoparticles and their applications for the combination chemo-photothermal therapy.23-27,31-34 Organic photothermal agent of indocyanine green and DOX co-loaded nanoparticles presented good chemo-photothermal effect on cancer cells.35-37 However, the studies on multifunctional anticancer nanotherapeutics with synergistic combination of photothermal therapy and cocktail chemotherapy are scarcely reported.

4


Page 4 of 42

Page 5 of 42

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

Biomacromolecules

Figure 1. The fabrication of plasmonic DOX and 6-MP dual anticancer drugs-loaded polypeptide/gold composite nanoparticles in mild conditions. Herein, we for the first time fabricated a kind of plasmonic, lactose-coated, and dual anticancer drugs-loaded polypeptide/gold composite nanoparticles in mild conditions (Fig. 1). We synthesized lactose (LAC) and PEG grafted polycysteine terpolymer (PC-g-PEG-LAC) by sequential UV photolysis and thiol-ene click chemistry and then fabricated the glyco-PEGylated polypeptide micelles according to our previous publications.38,39 The blank polypeptide micelles and/or their DOX-loaded counterparts were used as templates to generate the gold-embedded composite nanoparticles by forming multivalent Au-S bonds. Finally the DOX-loaded 5


Biomacromolecules

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

composite nanoparticles were tethered by the thiol-containing drug 6-mercaptopurine (6-MP) to produce the dual drugs-loaded composite counterparts. The as-prepared plasmonic, lactose-coated, and dual drugs-loaded polypeptide/gold composite nanoparticles possessed the following characteristics: (1) both photothermal therapy with two anticancer drugs induced cocktail chemotherapy were integrated into one biodegradable and biocompatible polymeric nanocarrier, which exhibited a synergistic cocktail chemo-photothermal cancer therapy (Fig. 2); (2) the composite nanoparticles presented reduction-sensitive and NIR-triggered cocktail drugs release profiles and the trigger-enhanced cytotoxicity; (3) the lactose moieties dangled on the surface of the composite nanoparticles endowed them a targeting effect towards HepG2 cell line.

Figure 2. The lactose (LAC)-targeted cellular uptake of the plasmonic dual drugs-loaded composite nanoparticles, triggered cocktail drug release, and the synergistic cocktail chemo-photothermal therapy by using NIR laser irradiation. 6


Page 6 of 42

Page 7 of 42

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

Biomacromolecules

EXPERIMENTAL SECTION Materials. Dimethylformamide (DMF, 99.5%) was distilled from calcium hydride and stored in a glove box. Hydrogen peroxide aqueous solution (30%) and tetrachloroauric acid hydrate were purchased from Shanghai Sinopharm Corp. Acridine orange (AO, Aldrich), 2-aminoethyl methacrylate hydrochloride (90%, J&K CHEMICA), buthionine sulfoximine (BSO, 99%, Aldrich), dimethylphenylphosphine (DMPP, 99%, Aldrich), D,L-dithiothreitol (DTT, ≥99%, Aldrich), doxorubicin (DOX) hydrochloride (Beijing Huafeng Corp.), ethidium bromide (EB, Aldrich), lactobionic acid (98%, J&K CHEMICA), 6-mercaptopurine (6-MP) monohydrate (98%, Adamas), and poly(ethylene glycol) (PEG) methyl ether acrylate (Mn = 480, Aldrich) were purchased and used directly. Lactobionamidoethyl methacrylate (LAMA) was synthesized according to our previous publications.19,20 Dulbecco’s modified eagle medium (DMEM, PAA Laboratories), fetal bovine serum (FBS, PAA Laboratories), and

hoechst33342

(Ultra-Pure,

Aldrich),

methylthiazolyldiphenyl-tetrazolium

bromide (MTT, Ultra-Pure, Aldrich) were used as received. L929 (a mouse fibroblastic cell line), HepG2 (a human liver hepatocellular carcinoma cell line), and HeLa (a human uterine cervix carcinoma cell line) were obtained from Shanghai Institute of Biochemistry and Cell Biology. Methods. Fourier transform infrared (FT-IR) spectra were recorded on a Perkin Elmer Spectrum 100 spectrometer at frequencies ranging from 400 to 4000 cm-1 at room temperature, and powder samples were thoroughly mixed with KBr crystal and pressed into pellet form. 1H NMR (400 MHz) spectroscopy was recorded on a Varian 7


Biomacromolecules

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 8 of 42

Mercury-400 spectrometer at room temperature and tetramethylsilane was used as an internal standard. Vis-NIR spectroscopy was recorded at room temperature using a Specturmlab 54 UV-visible instrument. The near infrared (NIR) continuous wave diode

laser

(FC-960-6000-MM;

wavelength:

808

nm)

with

a

fiber

(FC/PC/200UM/1M) was purchased from Shanghai SFOLT Corporation. The laser power can be adjusted within 0 ~ 1650 mW and the laser spot size can be accurately controlled by a fiber collimator. Dynamic light scattering (DLS) was performed at 25 °C on a Malvern ZS90 instrument. All of the DLS measurements were repeated three times and the average diameter and polydispersity index (PDI) were given as the mean value ± standard deviation. Field emission scanning electron microscope (SEM) was performed on a FEI Nova NanoSEM 450 instrument under an accelerating voltage of 5 kV. Samples were deposited onto the surface of silicon wafers and excess solution was removed in vacuum. Transmission electron microscopy (TEM) was performed without staining by using a JEM-2010 at 200 kV accelerating voltage. Samples were deposited onto the surface of 300 mesh Formvar-carbon film-coated copper grids and excess solution was removed in vacuum. X-ray photoelectron spectroscopy (XPS) was conducted with a VG ESCALAB MKII spectrometer. The XPS PEAK software (Version 4.1) was used to deconvolute the narrow-scan XPS spectra of the C1s, N1s and O1s of the samples, using neutral carbon peak C1s at 284.5 eV to calibrate the binding energy. Thermogravimetric analysis (TGA) was obtained using a Perkin-Elmer TGA 7 instrument under nitrogen flow (10 mL/min) from room temperature to 900

o

C at 20

o

C/min. High Pressure Liquid

8


Page 9 of 42

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

Biomacromolecules

Chromatography (HPLC) was performed on a Series 200 (Perkin Elmer, Inc., USA) equipped with a C18 column. The deionized

water

and

mobile

phase

methanol (v/v = 9:1) and

was a

a

flow

mixture rate

of

of 1.0

mL/min. The 6-MP drug was detected by UV detector at 327 nm and the retention time of 6-MP was 3.4 ± 0.4 min. Preparation of the polypeptide graft terpolymers PNBC-g-PEG-LAC and PC-g-PEG-LAC. According to our previous publications,38,39 the polypeptide graft copolymer poly(S-(o-nitrobenzyl)-D,L-cysteine)45-g-PEG5 (i.e., PNBC45-g-PEG5; the subscript denotes the number of repeating units and/or the PEG grafts) was first synthesized by sequential UV photolysis and thiol-ene click chemistry. Then, 20 mg of PNBC45-g-PEG5 was dissolved in 35 mL of mixture solvents DMSO/acetonitrile (v/v = 6:4), which was irradiated by a high-pressure mercury lamp (wavelength: 365 nm; power: 150 W) for about 11 min to photocleave another 10 mol% NB groups. Both 7.8 µL (10% in DMF) of DMPP (5.5 µmol) and 5.2 mg of LAMA (11.0 µmol) were added into the above solution and reacted overnight at 40 oC. The crude product was dialyzed

against deionized

water (MWCO: 3500) for 48 h, precipitated into

ether, and then dried in vacuum at 35 °C to graft

terpolymer

PNBC40-g-PEG5-LAC5

give (72.5

16.3 mg %

of yield).

the polypeptide 1

H

NMR

(DMSO-d6/CF3COOD, v: v = 3: 1): δ (ppm) = 8.10-7.10 (m, Ar, 160H), 4.98-4.30 (m, ArCH2SCH2CH, 50H), 4.25-3.70 (m, O=COCH2, ArCH2SCH2CH, LAC, 120H), 3.60-3.25 (m, O(CH2CH2O)n, LAC, 185H), 3.20-3.05(m –OCH3, LAC, 30H). FT-IR (KBr, cm-1): FT-IR (KBr, cm-1): 3296 (νN-H, PNBC), 2917 (νC-H), 1660 (νC=O, amide I), 9


Biomacromolecules

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 10 of 42

1526 (δN–H, amide II), 1346 (νs NO2), 1102 (νC–O–C), 1039 (νC-OH, LAC), 713 (νC–S).

Mn,GPC = 8190, Mw/Mn = 1.74. After 30 min of 365 nm UV irradiation for full photocleavage of the residual NB groups, PNBC40-g-PEG5-LAC5 was finally transformed into PC40-g-PEG5-LAC5 (87.7 % yield). 1H NMR (DMSO-d6/CF3COOD, v: v = 3: 1): δ (ppm) = 4.76-3.65 (m, ArCH2SCH2CH, O=COCH2, LAC), 3.57-3.26 (m, O(CH2CH2O)n, LAC), 3.18-3.05 (m, –OCH3, LAC). FT-IR (KBr, cm-1): 3285 (νN-H, PC), 2926 (νC-H), 1666 (νC=O, amide I), 1521 (δN–H, amide II), 1113 (νC–O–C), 1041 (νC-OH, LAC). Mn, GPC = 1290, Mw/Mn = 2.07. Preparation of the glyco-PEGylated polypeptide/gold composite nanoparticles and

their

dual

drugs-loaded

counterparts.

Both

the

glyco-PEGylated

polypeptide/gold composite nanoparticles and their dual drugs-loaded counterparts were mainly fabricated by two steps. In the first step, the widely-used dialysis method was used to fabricate the blank (non-drug-loaded) micelles or the DOX-loaded ones of PC40-g-PEG5-LAC5 by aqueous co-assembly process. PC40-g-PEG5-LAC5 (6.5 mg) was dissolved in 2 mL of DMF, with or without addition of 5 mg of DOX·HCl and 5 µL of trimethylamine, followed by gradual addition of 18 mL of distilled water under vigorous stirring, finally dialyzed against 4 × 1 L of distilled water for 24 h to produce the blank micelles or the DOX-loaded ones. After addition of 200 µL H2O2 (0.1 M) to the above solutions with stirring for 3 h at 37 oC, and dialysis against 4 × 1 L of PBS (0.01 M) for 24 h to remove excess H2O2, the blank and/or the DOX-loaded micelles solutions were finally obtained and stored at 4 oC for further use. Using UV-vis analysis at 500 nm, the DOX drug-loading capacity was calculated as the weight ratio 10


Page 11 of 42

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

Biomacromolecules

of actual drug to drug-loaded nanoparticles. M , 0.31 In the second step, 1 mL of the above micelles solution (denoted as LAC○

mg/mL) was added by 1 µL of H2O2 (9.79 nmol) under vigorous stirring at room temperature, and then different amounts of HAuCl4 (e.g., 20, 30, 40, 50 µL, 10 mg/mL; Au: S = 0.39 − 0.98, mol: mol) was gradually added and reacted overnight in dark. The resulting micelles templated gold composite nanoparticles solution (denoted M Au30 if added by 30 µL of HAuCl4, 0.44 mg/mL) was further dialyzed as LAC○

against PBS, and stored at 4 oC for further use. As for 6-MP coated composite M Au30, and then stirred nanoparticles, 1 mg of 6-MP was added into 2 mL of LAC○

overnight in dark and at room temperature. The resultant 6-MP coated composite nanoparticles solution was further dialyzed against PBS to remove free 6-MP M Au30@MP, 0.45 mg/mL). As for the dual drugs-loaded composite (denoted as LAC○

nanoparticles, the DOX-loaded micelles solution (0.32 mg/mL) was vigorously with HAuCl4 (10 mg/mL, 40 µL) to produce the DOX-loaded composite nanoparticles M Au40, 0.46 mg/mL). Finally 6-MP under the reduction of H2O2 (denoted as DOX○

was conjugated onto the gold surface to produce the dual drugs-loaded composite M Au40@MP, 0.47 mg/mL). The drug-loading nanoparticles (denoted as DOX ○

capacity of 6-MP was determined by HPLC. Photothermal properties of the glyco-PEGylated polypeptide/gold composite nanoparticles. Generally, 200 µL of the blank composite nanoparticles was added to a 96-well plate and then irradiated by a continuous-wave diode laser (808 nm, power intensity = 2 W·cm-2) for different times. The temperature change was recorded by a 11


Biomacromolecules

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 12 of 42

digital thermometer every 30 s. To study the photostability and photothermal conversion efficiency (), the temperature change was recorded for three cycles when the samples were heated by the NIR laser for 5 min and then allowed to cool naturally for 10 min, in which was calculated according to the previous publications11,30 By using double fluorescent staining (AO/EB) technique, the photothermal effect on

cancer

cells

was

observed

by

an

inverted

fluorescence

microscope

(Leica DMI6000 B). HepG2 cells seeded in a 96-well plate (104 cells per well) were incubated with blank composite nanoparticles for 4 h, and then irradiated by the NIR laser for 5 min (808 nm, 2 W·cm-2). The treated cells were further incubated for 12 h and then stained by AO/EB, finally observed by an inverted fluorescence microscopy. To give a quantified result of AO/EB analysis, HepG2 cells seeded in a 24-well plate (105 cells per well) were incubated with blank composite nanoparticles for 4 h, and then irradiated by the NIR laser (each irradiation spot for 5 min, 808 nm, 2 W·cm-2). The treated cells were further incubated for 12 h, stained by AO/EB, and then analyzed by flow cytometry (BD Accuri C6, US BD Corporation). In vitro cocktail drugs release study. As a typical example, the dual drugs-loaded composite nanoparticles solution (1 mL) was put into a dialysis bag, which was then put in 5 mL of PBS (10 mM) in a tube at 37 °C. The drug release solution was collected and changed periodically every 2 h, and the released DOX amount was measured by UV−vis at 500 nm. To analyze the released 6-MP amount, all collected samples were conducted by HPLC. The standard curve was concentration (µg/mL) = 2.45 × 10-5 (Area) and the concentration ranges from 1.0 to 20.0 µg/mL. For DTT 12


Page 13 of 42

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

Biomacromolecules

triggered drug release, 10 mM DTT was added into PBS. For NIR triggered drug release, the solution was periodically irradiated by the NIR laser (808 nm, 2 W·cm -2) for 5 min every 1 h. All release experiments were carried out in duplicate. Synergistic cocktail chemotherapy and photothermal therapy of the dual drugs-loaded composite nanoparticles. According to the detailed protocol in our previous publications,30,39 the cytotoxicity of the blank composite nanoparticles was evaluated by using three cell lines including a mouse fibroblastic cell line L929 and two human carcinoma cell lines (HepG2 and HeLa). Both HeLa and HepG2 cell lines were used to study the targeting effect of the LAC-coated nanoparticles. In a typical protocol, the cells were cultivated in DMEM containing 10% FBS and antibiotics (50 units/mL penicillin and 50 units/mL streptomycin) at 37 °C under a humidified atmosphere containing 5% CO2. 200 µL of cells suspension (1×104 cells/well) in DMEM was added to each well in a 96-well plate and cultured for 24 h. The blank (non-drug-loaded) and/or dual drugs-loaded composite nanoparticles with gradient concentrations were added separately into 96-well plate and further cultured for 48 h to determine the cytotoxicity of the blank ones or the drug-release induced chemo-toxicity. After incubation and being washed with PBS twice, 200 µL of the new culture medium containing MTT reagent (20 µL) was added to each well and incubated for 4 h to allow formation of formazan dye. After removal of the medium, the purple formazan product was dissolved in DMSO. Finally, the OD (optical density) value was measured at 490 nm by Microplate Reader (Elx800, BioTek Company). Cell viability = ODtreated/ODcontrol, where ODcontrol and ODtreated were obtained in the 13


Biomacromolecules

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 14 of 42

absence or the presence of nanoparticles, respectively. As for the photothermal therapy and/or the combination cocktail chemotherapy with photothermal therapy, different concentrations of the composite nanoparticles were added into the cell wells and then incubated for 4 h before being irradiated by the NIR laser for 5 min (808 nm, 2 W·cm-2). The treated cells were further incubated for 12 h before standard MTT assay while 48 h was used for the combination therapy. Half maximal inhibitory concentration (IC50) was calculated by GraphPad Prism 6 software using 8 samples. The combination index (CI) was calculated according to the equation40: CIcocktail = [IC50

(cocktail, DOX)/IC50 (DOX)]

cocktail)/IC50 (cocktail)]

+ [IC50

(cocktail, MP)/IC50 (MP)];

CIcombination = [IC50

(combined

+ [IC50 (combined phototherapy)/IC50 (phototherapy)], and the detailed results

were summarized in supporting information Table S1. As for chemotherapy and cocktail chemotherapy, IC50 was calculated based on drug concentration; as for phototherapy and combined phototherapy, IC50 was calculated based on the blank composite nanoparticles concentration. Cell internalization. Cell internalization of nanoparticles was monitored by ﬂow cytometry (BD Accuri C6, US BD Corporation) and inverted fluorescence microscopy, respectively. In brief, HepG2 (5.0 × 105 cells per well) was seeded in a 6-well tissue culture plate and incubated for 24 h at 37°C. The dual drugs-loaded nanoparticles (equivalent DOX concentration: 8 µg/mL) and free DOX (8 µg/mL) were added to different wells and then incubated for predetermined times. Data was finally analyzed by a FlowJo software. For inverted fluorescence microscopy, HepG2 (1.0 × 105 cells per well) was seeded on coverslips in a 6-well tissue culture plate and then incubated 14


Page 15 of 42

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

Biomacromolecules

for 24 h. The dual drugs-loaded nanoparticles and free DOX were added to different wells and the cells were incubated at 37 °C for predetermined times. The cells were ﬁxed by 4% formaldehyde for 30 min, stained by Hoechst33342 for 5 min, and then rinsed with PBS for three times. Note that both HeLa and HepG2 cell lines were used to study the targeting effect by flow cytometry and inverted fluorescence microscopy.

M ) and the composite NPs Figure 3. (A) DLS data for the precursor micelles (LAC○

M Au30, Au30 denotes 30 µL of HAuCl4); (B, with various HAuCl4 dosage (e.g., LAC○ M and LAC○ M Au30; and (D) SEM micrograph for C) TEM micrographs for LAC○

M Au30 and inset C and D for high magnification image of one nanoparticle. LAC○

15


Biomacromolecules

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 16 of 42

RESULTS AND DISCUSSION Fabrication

and

characterization

of

the

plasmonic

glyco-PEGylated

polypeptide/gold composite nanoparticles. Biomimetic glyco-polypeptides possess excellent biocompatibility and specific biomolecular interaction (i.e., sugar-lectin binding) and the related glyco-coated nanoparticles have been widely investigated for targeted drug delivery systems.41-43 The polycysteine (PC) graft terpolymer PC40-g-PEG5-LAC5 that contains 5 lactose (LAC) moieties and 5 PEG (Mn = 480) grafts was synthesized according to our previous publications (see supporting information Scheme S1 and Fig. S1-2).38,39 We then fabricated the glyco-PEGylated M ) by polypeptide micelles with a disulfide-bond-cross-linked core (i.e., LAC ○

aqueous self-assembly process of amphiphilic PC40-g-PEG5-LAC5 followed by H2O2 oxidation (Fig. 1). It is speculated that the glyco-PEGylated polypeptide micelles have the attributes: (1) the hydrophilic LAC and PEG moieties endow the micelles with a targeting and a stabilization properties; and (2) the micelles are fixed by the dynamic covalent disulfide-bonds (-S-S-), enabling them suitable for the template to load gold (Au0) via forming multivalent Au-S bonds.31-34, 44,45 As a mild reductant, H2O2 was used for reducing HAuCl4 to gold (Au0), and various gold NPs with different morphologies and sizes can be obtained by varying pH values and H2O2/Au ratios.46-48 Thus, the polypeptide micelles solution (0.31 mg/mL, 10 mM PBS) was added with a tiny amount of H2O2 (9.79 nmol) followed by addition of different amounts of HAuCl4 (e.g., 20, 30, 40, 50 µL, 10 mg/mL; Au: S = 0.39−0.98, mol: mol) under vigorous stirring overnight and at room temperature. The resulting solution 16


Page 17 of 42

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

Biomacromolecules

turned from pale yellow to dark blue, suggesting the formation of the micelles templated gold composite nanoparticles.23-26,31-34 However, without the addition of H2O2 reductant, the control sample did not change at room temperature even for 7 days (see supporting information Fig. S3). As evidenced by DLS (Fig. 3A), the as-synthesized composite nanoparticles increased with increasing amounts of HAuCl4, and the largest ones increased from 76 ± 8 nm to 112 ± 15 nm when 30 µL of HAuCl4 M Au30; Au: S = 0.59, mol: mol). This result implied that in situ was added (i.e., LAC○

reduced gold was simultaneously captured and chelated by the dynamic disulfide-bonds of the micellar core via forming multivalent Au-S bonds (see the following XPS analysis). Imaginably, the embedded gold made the micellar core larger with concomitant stretching the polypeptide chains more rigid, resulting in the nanoparticle size increase.25,26 This result was further evidenced by TEM. However, small gold nanoparticles with a diameter of 21 ± 5 nm (about 13.2 % determined by M Au50). DLS) were obviously observed when 50 µL of HAuCl4 was used (i.e., LAC○

These results indicate that an optimal amount of HAuCl4 is pivotal for the preparation of the micelles templated gold composite nanoparticles, and excess gold would result in the formation of small gold nanoparticles that are detrimental to the NIR absorption and photothermal properties (see the following section). Compared with the precursor micelles, the composite nanoparticles presented a smooth and spherical morphology with a clearer contrast and their size increased about 40 nm, as measured by TEM (Fig. 3B and 3C). The size increase was comparable with that determined by DLS (about 36 nm). Moreover, the composite nanoparticles can be directly observed by 17


Biomacromolecules

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

SEM (Fig. 3D); however, the precursor micelles themselves cannot be discerned without gold staining. The average diameter calculated by SEM (61 ± 9 nm) was in agreement with that measured by TEM (57 ± 10 nm). The size determined by both TEM and SEM was largely deviated from that determined by DLS, which was because the former were measured in a dried state compared with the latter in aqueous solution.

Figure 4. The energy dispersive spectroscopy (A), XPS (B; -SOxH, x = 2 or 3), (C) WAXD, and (D) TGA curves of the lyophilized powder. The energy dispersive spectroscopy verified the existence of gold element within the composite nanoparticles (Fig. 4A). As characterized by XPS (Fig. 4B), the binding energy of S-Au bond appeared at 161.8 eV, which confirmed that the polypeptide micelles indeed played a template role for loading the reduced gold via 18


Page 18 of 42

Page 19 of 42

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

Biomacromolecules

forming multivalent S-Au bonds.49-52 WAXD of the lyophilized powder showed three Bragg diffraction peaks at 38.2o, 44.5o, and 64.7o, which was typical of cubic gold phase having (111), (200), and (220) reflections, demonstrating that the gold in the composite nanoparticles existed in a crystalline state (Fig. 4C). Calculated by TGA measurement, the composite nanoparticles contained 31.2 wt% gold, which was consistent with 31.4 wt% in feed (Fig. 4D).

M and the composite Figure 5. (A) Vis-NIR spectra of the precursor micelles of LAC○

M 30); (B) the temperature change NPs with various amounts of gold (e.g., LAC○

curves of the blank composite NPs solution upon the NIR irradiation (808 nm, 2 M 30 over W·cm-2) as a function of time; (C) the temperature change curves of LAC○

repeated laser on/off cycles; (D) the temperature change curves of DOX-loaded and/or dual drugs-loaded NPs solution upon the NIR irradiation as a function of time. 19


Biomacromolecules

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 properties of the plasmonic glyco-PEGylated polypeptide/gold composite nanoparticles. Compared with the precursor micelles, the composite nanoparticles showed a strong and broad surface plasmon resonance absorption at 650−1100 nm in the Vis-NIR spectra (Fig. 5A). The NIR absorption gradually increased with increasing amount of HAuCl4 and reached a maximum with 30 µL of M Au30); however, it reversely decreased with an excess amount of HAuCl4 (i.e., LAC○

HAuCl4 (e.g., 40 and 50 µL). The excess gold could not be fully captured by the disulfide-bonds of micelles and formed small gold nanoparticles (13.2%), which was observed and calculated by DLS. To study the photothermal effect of the composite nanoparticles, the nanoparticles solution was irradiated by a continuous-wave diode laser (λ = 808 nm, power density = 2 W·cm-2). The temperature of the nanoparticles solution was quickly elevated over the irradiation time and the magnitude of the temperature change presented a HAuCl4 M Au30 sample (i.e., 30 µL of dose-dependent manner. The temperature of LAC○

HAuCl4) increased by 30.1 oC after 5 min of NIR irradiation; however, phosphate buffer saline (PBS) was elevated only by 4.0 oC (Fig. 5B). Moreover, both “laser-on” and “laser-off” cycle curves for heating and naturally cooling process were performed three times, presenting a good photostability for the composite nanoparticles.30 Notably, these composite nanoparticles have a higher photothermal conversion efficiency of about 35.8 % than the known gold nanorods (about 22 %) and the gold nanoshells (about 13%).11-14 To study the stability of the composite nanoparticles in physiological mimetic conditions, they were incubated in PBS containing 10% fetal 20


Page 20 of 42

Page 21 of 42

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

Biomacromolecules

bovine serum (FBS) at 37 oC and then monitored by DLS. These composite nanoparticles slightly increased and kept stable without occurrence of macroscopic aggregates (less than 250 nm) for 24 h (Fig. S4). Taken together, it can be concluded that (1) the plasmonic glyco-PEGylated polypeptide/gold composite nanoparticles were successfully prepared in mild conditions, by using the polypeptide micelles as templates to load in situ reduced gold via forming multivalent Au-S bonds, and (2) they presented strong NIR absorption and excellent photothermal properties including high conversion efficiency and good photostability. In vitro triggered cocktail drugs release of the dual drugs-loaded composite nanoparticles. To enhance the chemotherapy efficacy and minimize the side effects, the cocktail chemotherapy strategy has been intensively investigated for current cancer therapy.1-3 In this work, two commercial anticancer drugs including doxorubicin (DOX) and 6-mercaptopurine (6-MP) were loaded into the composite nanoparticles, respectively. First, DOX was physically encapsulated into the polypeptide micelles during the co-assembly process (Fig. 1). The resulting DOX-loaded micelles were then used as templates to prepare their gold composite nanoparticles. Finally 6-MP was conjugated onto the gold surface, via forming S-Au M Au40 bond, to produce the dual drugs-loaded composite nanoparticles (i.e., DOX○

M Au40@MP gave a @MP). As determined by UV-vis spectroscopy and HPLC, DOX○

drug-loaded capacity of 11.7 wt% for DOX and 12.2 wt% for 6-MP, respectively. Characterized by means of DLS and TEM, the dual drugs-loaded composite nanoparticles showed nearly spherical morphology with a diameter of 133 ± 10 nm 21


Biomacromolecules

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

(Fig. S5). Compared with the DOX-loaded ones, the dual drugs-loaded composite nanoparticles increased about 50 nm and the size increase determined by DLS was basically consistent with that measured by TEM. The temperature elevation of the DOX-loaded composite nanoparticles also showed a HAuCl4 dose-dependent manner M Au40 @MP) could be and the dual drugs-loaded composite nanoparticles (i.e., DOX○

elevated by 24.0 oC after same dose of NIR irradiation (Fig. 5D). Notably, the loaded drugs showed minor effect on the photothermal properties of the composite nanoparticles taking account of the weight fraction of the dual drugs. In addition, more HAuCl4 (40 µL) was used to fabricate the DOX-loaded composite nanoparticles with optimal photothermal properties than that (30 µL) for the blank (non-drug-loaded) composite counterparts. As the amino-containing DOX probably chelated with gold, more HAuCl4 was consumed during the reduction process.53 Therefore, both blank, single drug-loaded, and dual drugs-loaded composite nanoparticles with optimal M Au30, LAC○ M Au30@MP, DOX○ M Au40, and photothermal properties (i.e., LAC○

M Au40@MP) were used for the following comparative studies. DOX○

M Au40 Figure 6. In vitro drug release profiles of DOX (A) and 6-MP (B) from DOX○

@MP in the presence of 10 mM DTT or upon NIR irradiation. The data are presented 22


Page 22 of 42

Page 23 of 42

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

Biomacromolecules

as average ± standard error (n = 4). Tumor tissues are known to have relatively high concentration of glutathione (GSH) than normal ones, while there is a large concentration gradient of GSH between intracellular and extracellular milieu. So the disulfide-bond-containing nanocarriers have been studied intensively for tumor-environmental or intracellular GSH-triggered nanomedicines.54-57 To mimic a reductive intracellular milieu, the drug release profiles of the dual drugs-loaded nanoparticles were monitored in 10 mM PBS with addition of 10 mM D,L-dithiothreitol (DTT) and at 37 oC. The apparent drug-release rate increased about 50 % with DTT treatment and 33 wt% of DOX was released for 12 h (Fig. 6A). In contrast to DOX, 6-MP displayed faster drug-release profiles (Fig. 6B). After DTT treatment, about 99.7 wt% of 6-MP was released for 12 h and the apparent drug-release rate increased about 100 %. On the other hand, it is known that the NIR-mediated heat can to some extent accelerate the drug diffusion from nanoparticles.11-14 Upon NIR laser irradiation, the apparent drug-release rate increased about 34 % and 29.7 wt% of DOX was released for 12 h; however, no accelerating effect was observed for 6-MP. This is because DOX was mainly entrapped in the hydrophobic core of the composite nanoparticles and released by a diffusion mechanism.57 However, 6-MP that was dynamically conjugated on gold surface (monovalent S-Au bond) was released without the diffusion barrier, which made the heat-triggered effect negligible. Collectively, these results demonstrate that the dual drugs-loaded composite nanoparticles presented reduction-sensitive and NIR-triggered cocktail drugs release profiles. 23


Biomacromolecules

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 7. Flow cytometry histograms of HepG2 incubated with the dual drugs-loaded M Au40@MP and free DOX for different times. composite nanoparticle DOX○

Cell internalization and targeting effect to HepG2 cell line. The cellular internalization of nanoparticles is a key point for drug delivery systems and nanomedicines as most anticancer drugs play their roles inside cells.4-6 As free DOX molecules emit strong red fluorescence, the cell internalization process of DOX-loaded nanoparticles can be easily monitored by flow cytometry. The dual M Au40@MP were incubated with drugs-loaded composite nanoparticles of DOX○

HepG2 (a human liver hepatocellular carcinoma cell line) for different time intervals and the flow cytometry histograms were recorded to provide statistics on the cellular M Au40@MP uptake process (Fig. 7A). The median fluorescence intensity of DOX○

pretreated HepG2 gradually increased with the incubation time. Taking into account M Au40@MP was the mean fluorescence intensity at 4 h, 30 % and 77 % of DOX○

internalized by HepG2, respectively. However, much lower fluorescence intensity was M Au40@MP compared to free DOX with same dose (Fig. 7B). obtained for DOX○

This result was because the fluorescence of the encapsulated DOX was greatly quenched by the embedded gold within the core of the composite nanoparticles.58 24


Page 24 of 42

Page 25 of 42

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

Biomacromolecules

Figure 8. Flow cytometry histograms (A) and the median fluorescence intensity (B) of the dual drugs-loaded composite nanoparticles incubated with HepG2 and HeLa cell lines for 4 h (p ≤ 0.05 was considered as significantly different and denoted as *). Owing to their lower immunogenicity, easy synthesis and lower cost, multivalent 25


Biomacromolecules

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

carbohydrate ligand-pendant glycopolymers and sugar-coated nanoparticles that can specifically recognize cell-surface receptors (e.g., lectins) have been increasingly studied for targeted drug delivery systems.41-43 Do the LAC-coated nanoparticles have targeting effect to the mammalian cells? HepG2 has overexpressed asialoglycoprotein (ASGP) receptors compared to HeLa, so the ability of LAC-coated nanoparticles to enter into both cell lines was comparatively evaluated by flow cytometry. As expected, M Au40@MP pretreated HepG2 showed stronger fluorescence the LAC-coated DOX○

(52% increase) compared with HeLa after same dose of DOX treatment, while the non-LAC-coated nanoparticles pretreated HepG2 gave similar fluorescence with HeLa (Fig. 8). After 5 mM free LAC treatment with HepG2, however, the competition assay demonstrated that LAC- or non-LAC-coated nanoparticles displayed approximate fluorescence intensity. These results indicate that the LAC-coated composite nanoparticles exhibited lactose-mediated targeting effect to M Au40@MP by HepG2. We further investigate the cell internalization of DOX○

M Au40@MP can be clearly fluorescence microscopy. As shown in Fig. 9, DOX○

observed in the cytoplasm of HepG2 after 4 h incubation, and some DOX molecules M Au40@MP have entered into the nucleus. Compared with that for HeLa, more DOX○

nanoparticles were internalized by HepG2 and more DOX molecules were transported M Au40@MP by into the cells. These results confirmed the cellular uptake of DOX○

HepG2 and/or HeLa cell lines and that the LAC-coated composite nanoparticles exhibited a targeting effect to HepG2 cell line. This result was consistent with that analyzed by flow cytometry. 26


Page 26 of 42

Page 27 of 42

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

Biomacromolecules

Figure 9. Fluorescence microscope images of HeLa and HepG2 cell lines incubated with the dual drugs-loaded composite nanoparticles for 4 h and at 37 oC. Synergistic cocktail chemo-photothermal therapy. To evaluate the potential toxicity, the blank composite nanoparticles were incubated with a mouse fibroblastic cell line L929 and two human carcinoma cell lines (HepG2 and HeLa) for 48 h, respectively. By a standard MTT assay, all the cells’ viabilities kept above 90 %, demonstrating that the blank composite nanoparticles had little cytotoxicity to these healthy and carcinoma cell lines. All the drug-loaded nanoparticles were incubated with HepG2 cells for 48 h to measure the cell viability except that it was noted by HeLa cells. Do the

dual

drugs-loaded

composite

nanoparticles

produce

the

intracellular

reduction-enhanced drug release and cytotoxicity? As shown in Fig. 10, the cell viability gradually decreased and presented a drug concentration-dependent manner. M Au40@MP displayed a half maximal The dual drugs-loaded nanoparticles of DOX○

inhibitory concentration (IC50) of 2.09 µg/mL (Table S1). When HepG2 cells were 27


Biomacromolecules

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

treated with 0.1 mM buthionine sulfoximine (BSO, an inhibitor for the intracellular M Au40@MP, BSO synthesis of GSH) for 12 h and then incubated with DOX○

apparently produced an inhibition effect and IC50 increased to 3.84 µg/mL (about 84 % increase). This was because BSO lowered intracellular GSH concentration, resulting in an attenuated drug release rate from the dual drugs-loaded nanoparticles.54-56 Do the composite nanoparticles produce the LAC targeting effect mediated cytotoxicity when they were incubated with HepG2 and HeLa cell lines, respectively? The M Au40@MP gave a greatly decreased IC50 LAC-coated nanoparticles DOX ○

(0.23-fold) for HepG2 compared with that for HeLa (8.96 µg/mL); however, the non-LAC-coated counterparts showed a bigger IC50 (5.83 µg/mL, 0.48-fold) for HepG2 compared with that for HeLa (12.23 µg/mL). This result clearly demonstrated M Au40@MP presented a LAC-targeting enhanced cytotoxicity to HepG2 that DOX○

cell line although the surface chemistry (i.e., LAC vs non-LAC ligands) of these nanoparticles produced a little effect on cell viability. We further study the synergistic effect of cocktail chemotherapy of DOX and M 6-MP. After 48 h incubation with HepG2, the cocktail chemotherapy of DOX○

Au40@MP gave an IC50 of 2.09 µg/mL that consists of 1.02 µg/mL for DOX and 1.07 M Au40 and LAC○ M Au30@MP produced 6.10 µg/mL µg/mL for 6-MP, while DOX○

for single DOX chemotherapy and 6.48 µg/mL for single 6-MP chemotherapy, respectively. As for the cocktail chemotherapy (i.e., the combination formulation of different drugs) and the combination therapies (e.g., photothermal therapy and chemotherapy), the combination index (CI) is used to evaluate the synergistic effect of 28


Page 28 of 42

Page 29 of 42

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

Biomacromolecules

the combination therapies. If the CI value 1, which means that they act additively or antagonistically.30,40,59,60 The dual drugs-loaded composite M Au40@MP gave a CI of 0.33, demonstrating an excellent nanoparticles of DOX○

synergistic effect for the cocktail chemotherapy of DOX and 6-MP. In addition, the cocktail chemotherapy of DOX and 6-MP in free forms showed a good synergistic effect with a CI of 0.43 (Fig. S6 and Table S1). However, the combination therapy that was performed by the cocktail chemotherapy of both free drugs and the photothermal therapy of the blank composite nanoparticles gave a CI of 0.93, which was close to 1, suggesting a nearly additive effect for the combination therapy in free forms.

Figure 10. (A) Cytotoxicity of the blank composite nanoparticles incubated with three 29


Biomacromolecules

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

cell lines for 48 h; (B) cytotoxicity of the composite nanoparticles loaded with DOX or 6-MP alone or both DOX and 6-MP with/without the treatment of 0.1 mM BSO; (C) cytotoxicity of the blank or drug-loaded composite nanoparticles under different treatments; (D) cytotoxicity of the LAC- or non-LAC-coated dual drugs-loaded composite nanoparticles. The data are presented as average ± standard error (n = 6). To demonstrate the synergistic effect of the cocktail chemotherapy with M photothermal therapy, the dual drugs-loaded composite nanoparticles of DOX○

Au40@MP were incubated with HepG2 for 2 h, irradiated by the NIR laser (5min, 808 nm, 2 W·cm-2), and then incubated for 48 h. As shown in Table S1, the M Au40@MP gave an IC50 combination cocktail chemo-photothermal therapy of DOX○

of 6.14 µg/mL that consists of 1.47 µg/mL for cocktail chemotherapy and 4.67 µg/mL for photothermal therapy, while single cocktail chemotherapy and photothermal therapy alone produced an IC50 of 2.09 µg/mL and 75.69 µg/mL, respectively. The M Au40@MP gave a CI of combination cocktail chemo-photothermal therapy of DOX○

0.76, which was less than 0.93 for that of both free drugs with hyperthermia and exhibited a good synergistic effect. The relatively high CI value was probably due to the two facts. One is that the hyperthermia had little effect on 6-MP drug release as it was conjugated to the gold surface, thus the single 6-MP-loaded composite nanoparticles gave a CI of 0.91 (Table S1). The other is that the single DOX-loaded composite nanoparticles showed a moderate drug release increment with the action of hyperthermia, which gave a CI of 0.79. This result was also comparable with that (CI = 0.65) reported for the targeted N-(2-hydroxypropyl) methacrylamide (HPMA) 30


Page 30 of 42

Page 31 of 42

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

Biomacromolecules

copolymer–drug conjugate and the known gold nanorod induced hyperthermia.61 As the fluorescent dyes of AO and EB differentiate live and dead cells as green and red, respectively, the photothermal ablation of HepG2 was observed by fluorescence microscopy. After HepG2 was irradiated only by the NIR laser (5 min, 808 nm, 2 W·cm-2) or incubated with the blank composite nanoparticles (120 µg/mL), all the cells looked bright green. However, all HepG2 cells completely died or underwent apoptosis if treated with the blank composite nanoparticles plus same dose (600 J.cm-2) of NIR irradiation (Fig. 11). On the boundary of the laser spot (Fig. S7), only the cells within the laser irradiated area died, showing intense red fluorescence; however, the cells outside the boundary of laser irradiation are mainly alive, showing green fluorescence. Compared with the photothermal experiments using more dose of NIR irradiation

for the known gold nanoshells (> 720 J.cm-2) and gold nanorods (>

1000 J.cm-2),13 thus, the heating effect mediated by both the NIR laser and the blank composite nanoparticles exhibited a higher efficiency for killing cancer cells in vitro.

M 30 or NIR Figure 11. Fluorescence microscopy images of HepG2 treated with LAC○ M 30 plus NIR irradiation. irradiation (5 min, 808 nm, 2 W·cm-2) or LAC○

31


Biomacromolecules

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

M Au30 or NIR Figure 12. Flow cytometry analyses of HepG2 treated with LAC○ M Au30 plus NIR irradiation: Q1, Q2, irradiation (5 min, 808 nm, 2 W·cm-2) or LAC○

and Q3 represents the percentage of the cells undergoing necrosis, late apoptosis, and early apoptosis, respectively; and the lower left denotes the percentage of live cells. The data are representative of three independent experimental results. The qualitative AO/EB result was further validated by quantitative flow cytometry analysis (Figure 12). The HepG2 cells were treated with the blank composite M Au30 or the NIR irradiation (5 min, 808 nm, 2 W·cm-2) or nanoparticles of LAC○

M Au30 plus NIR irradiation, and then labeled with AO and EB, respectively. LAC○

32


Page 32 of 42

Page 33 of 42

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

Biomacromolecules

The cell viability kept about 95% if HepG2 was only irradiated by the NIR laser or M Au30 (120 µg/mL). However, 64.5% of HepG2 underwent late incubated with LAC○

M Au30 plus same dose of NIR irradiation. apoptosis and necrosis if treated with LAC○

This result was in agreement with that determined by MTT assay (62.8% of HepG2 was dead, Fig. 10C).

CONCLUSION We successfully fabricated a kind of plasmonic glyco-PEGylated polypeptide/gold composite nanoparticles and their dual anticancer drugs-loaded counterparts under mild conditions, which exhibited strong NIR absorption and excellent photothermal properties.

The

dual

drugs-loaded

composite

nanoparticles

presented

reduction-sensitive and NIR-stimulated cocktail drugs release profiles, the trigger-enhanced cytotoxicity, and the lactose-mediated targeting effect towards HepG2. Both the combination cocktail chemo-photothermal therapy and the cocktail chemotherapy between DOX and 6-MP produced a combination index of 0.76 and 0.33, respectively, displaying good synergistic antitumor effects. Consequently, this work not only establishes a facile strategy for the fabrication of plasmonic, sugar-targeted, and dual anticancer drugs-loaded polypeptide composite nanoparticles, but opens up a new avenue for developing multifunctional nanotherapeutics for synergistic cocktail chemotherapy and/or its combination therapies with photothermal therapy. Supporting Information 33


Biomacromolecules

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 S1, Figures for 1H NMR, GPC, FT-IR, Vis-NIR, DLS, TEM, fluorescence microscope image, MTT assay, and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org. Author Information *Corresponding author E-mail: [email protected] ACKNOWLEDGMENTS The authors are grateful for the financial support of National Natural Science Foundation of China (21474061 & 21274086) and Shanghai Leading Academic Discipline Project (B202). The assistance of Instrumental Analysis Center of SJTU is also appreciated.

REFERENCES (1) Hu, Q. Y.; Sun, W. J.; Wang, C.; Gu, Z. Recent advances of cocktail chemotherapy by combination drug delivery systems. Adv. Drug Deliv. Rev. 2016, 98, 19-34. (2) Kemp, J. A.; Shim, M. S.; Heo, C. Y.; Kwon, Y. J. “Combo” nanomedicine: co-delivery of multi-modal therapeutics for efficient, targeted, and safe cancer therapy. Adv. Drug. Deliv. Rev. 2016, 98, 3-18. (3) Mignani, S.; Bryszewska, M.; Klajnert-Maculewicz, B.; Zablocka, M.; Majoral, J.-P. Advances in combination therapies based on nanoparticles for efficacious cancer Treatment: an analytical report. Biomacromolecules 2015, 16, 1-27. (4) Cabral, H.; Nishiyama, N.; Kataoka, K. Supramolecular nanodevices: from design 34


Page 34 of 42

Page 35 of 42

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

Biomacromolecules

validation to theranostic nanomedicine. Acc. Chem. Res. 2011, 44, 999-1008. (5) Torchilin, V. P. Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery. Nature Rev. Drug Discovery 2014, 13, 813-827. (6) Sun, T.; Zhang, Y. S.; Pang, B.; Hyun, D. C.; Yang, M.; Xia, Y. Engineered nanoparticles for drug delivery in cancer therapy. Angew. Chem. Int. Ed. 2014, 53, 12320-12364. (7) Xiao, W.; Zeng, X.; Lin, H.; Han, K.; Jia, H.-Z.; Zhang, X.-Z. Dual stimuli-responsive multi-drug delivery system for the individually controlled release of anti-cancer drugs. Chem. Commun. 2015, 51, 1475-1478. (8) Lee, W. L.; Guo, W. M.; Ho, V. H. B.; Saha, A.; Chong, H. C.; Tan, N. S.; Tan, E. Y.; Loo, S. C. J. Delivery of doxorubicin and paclitaxel from double-layered microparticles: The effects of layer thickness and dual-drug vs. single-drug loading. Acta Biomaterialia 2015, 27, 53-65. (9) Ramasamy, T.; Kim, J. H.; Choi, J. Y.; Tran, T. H.; Choi, H. -G.; Yong, C. S.; Kim, J. O. pH sensitive polyelectrolyte complex micelles for highly effective combination chemotherapy. J. Mater. Chem. B 2014, 2, 6324-6333. (10) Zhang, Z.; Wang, J.; Chen, C. Near-Infrared Light-Mediated Nanoplatforms for Cancer Thermo-Chemotherapy and Optical Imaging. Adv. Mater. 2013, 25, 3869-3880. (11) Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z. Functional nanomaterials for phototherapies of cancer. Chem. Rev. 2014, 114, 10869-10939. (12) Shanmugam, V.; Selvakumar, S.; Yeh, C.-S. Near-infrared light-responsive 35


Biomacromolecules

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

nanomaterials in cancer therapeutics. Chem. Soc. Rev. 2014, 43, 6254-6287. (13) Dreaden, E. C.; Alkilany, A. M.; Huang, X.; Murphy, C. J.; El-Sayed, M. A. The golden age: gold nanoparticles for biomedicine. Chem. Soc. Rev. 2012, 41, 2740-2779 (14) Melancon, M. P.; Zhou, M.; Li, C. Cancer theranostics with near-infrared light-activatable multimodal nanoparticles. Acc. Chem. Res. 2011, 44, 947-956. (15) Cherukuri, P.; Glazer, E. S.; Curley, S. A. Targeted hyperthermia using metal nanoparticles. Adv. Drug. Deliv. Rev. 2010, 62, 339-345. (16) Xia, Y.; Li, W.; Cobley, C. M.; Chen, J.; Xia, X.; Zhang, Q.; Yang, M.; Cho, E. C.; Brown, P. K. Gold nanocages: From synthesis to theranostic applications. Acc. Chem. Res. 2011, 44, 914-924. (17) Lal, S.; Clare, S. E.; Halas, N. J. Nanoshell-enabled photothermal cancer therapy: impending clinical impact. Acc. Chem. Res. 2008, 41, 1842-1851. (18) Fomina, N.; Sankaranarayanan, J.; Almutairi, A. Photochemical mechanisms of light-triggered release from nanocarriers. Adv. Drug Deliv. Rev. 2012, 64, 1005-1020. (19) Sun, L.; Ma, X. F.; Dong, C. -M.; Zhu, B. S.; Zhu, X. Y. NIR-responsive and lectin-binding doxorubicin-loaded nanomedicine from Janus-type dendritic PAMAM amphiphiles. Biomacromolecules 2012, 13, 3581-3591. (20) Liu, G.; Zhou, L.; Su, Y.; Dong, C. -M. An NIR-responsive and sugar-targeted polypeptide composite nanomedicine for intracellular cancer therapy. Chem. Commun. 2014, 50, 12538-12541. (21) Shanmugam, V.; Chien, Y.-H.; Cheng, Y.-S.; Liu, T.-Y.; Huang, C.-C.; Su, C.-H.; Chen, Y.-S.; Kumar, U.; Hsu, H.-F.; Yeh, C.-S. Oligonucleotides-assembled Au 36


Page 36 of 42

Page 37 of 42

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

Biomacromolecules

nanorod-assisted cancer photothermal ablation and combination chemotherapy with targeted dual-drug delivery of doxorubicin and cisplatin prodrug. ACS Appl. Mater. Interfaces 2014, 6, 4382-4393. (22) Lee, S. M.; Park, H.; Choi, J. W.; Park, Y. N.; Yun, C. O.; Yoo, K. H. Multifunctional nanoparticles for targeted chemophotothermal treatment of cancer cells. Angew. Chem. Int. Ed. 2011, 50, 7581-7586. (23) You, J.-O.; Guo, P.; Auguste, D. T. Drug-delivery vehicle combining the targeting and thermal ablation of HER2+ breast-cancer cells with triggered drug release. Angew. Chem. Int. Ed. 2013, 52, 4141-4146. (24) Huang, Y. F.; Lu, S. C.; Huang, Y. C.; Jan, J. S. Cross-Linked, self-fluorescent gold nanoparticle/polypeptide nanocapsules comprising dityrosine for protein encapsulation and label-free imaging. Small 2014, 10, 1939-1944. (25) Deng, H.; Dai, F. Y.; Ma, G. H.; Zhang, X. Theranostic gold nanomicelles made from biocompatible comb-like polymers for thermochemotherapy and multifunctional imaging with rapid clearance. Adv. Mater. 2015, 27, 3645-3653. (26) Wang, L.; Yuan, Y. Y.; Lin, S. D.; Huang, J. S.; Dai, J.; Jiang, Q.; Cheng, D.; Shuai, X. T. Photothermo-chemotherapy of cancer employing drug leakage-free gold nanoshells. Biomaterials 2016, 78, 40-49. (27) Yang, J.; Lee, J.; Kang, J.; Oh, S. J.; Ko, H.-J.; Son, J.-H.; Lee, K.; Suh, J.-S.; Huh, Y.-M.; Haam, S. Smart drug-loaded polymer gold nanoshells for systemic and localized therapy of human epithelial cancer. Adv. Mater. 2009, 21, 4339-4342. (28) Lee, S.-M.; Park, H.; Yoo, K.-H. Synergistic cancer therapeutic effects of locally 37


Biomacromolecules

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

delivered drug and heat using multifunctional nanoparticles. Adv. Mater. 2010, 22, 4049-4053 (29) Ma, Y.; Liang, X. L.; Tong, S.; Bao, G.; Ren, Q. S.; Dai, Z. F. Gold nanoshell nanomicelles for potential magnetic resonance imaging, light-triggered drug release, and photothermal therapy. Adv. Funct. Mater. 2013, 23, 815-822. (30) Gao, Y.; Wu, X. J.; Zhou, L.; Su, Y.; Dong, C.-M. A sweet polydopamine nanoplatform for synergistic combination of targeted chemo-photothermal therapy. Macromol. Rapid Commun. 2015, 36, 916-922. (31) Song, J. B.; Huang, P.; Duan, H. W.; Chen, X. Y. Plasmonic vesicles of amphiphilic nanocrystals: optically active multifunctional platform for cancer diagnosis and therapy. Acc. Chem. Res. 2015, 48, 2506-2515. (32) Song, J. B.; Zhou, J. J.; Duan, H. W. Self-assembled plasmonic vesicles of SERS-encoded amphiphilic gold nanoparticles for cancer cell targeting and traceable intracellular drug delivery. J. Am. Chem. Soc. 2012, 134, 13458-13469. (33) He, J.; Huang, X. L.; Li, Y. C.; Liu, Y. J.; Babu, T.; Aronova, M. A.; Wang, S. J.; Lu, Z.; Chen, X. Y.; Nie, Z. H. Self-assembly of amphiphilic plasmonic micelle-like nanoparticles in selective solvents. J. Am. Chem. Soc. 2013, 135, 7974-7984 (34) Huang, P.; Lin, J.; Li, W. W.; Rong, P. F.; Wang, Z.; Wang, S. J.; Wang, X. P.; Sun, X. L.; Aronova, M.; Niu, G.; Leapman, R. D.; Nie, Z. H.; Chen, X. Y. Biodegradable gold nanovesicles with an ultrastrong plasmonic coupling effect for photoacoustic imaging and photothermal therapy. Angew. Chem. Int. Ed. 2013, 52, 14208-14214. (35) Sheng, Z. H.; Hu D. H.; Zheng M. B.; Zhao P. F.; Liu H. L.; Gao D. Y.; Gong P.; 38


Page 38 of 42

Page 39 of 42

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

Biomacromolecules

Gao G. H.; Zhang P. F.; Ma Y. F. and Cai L. T. Smart Human Serum Albumin-Indocyanine Green Nanoparticles Generated by Programmed Assembly for Dual-Modal Imaging-Guided Cancer Synergistic Phototherapy. ACS Nano 2014, 8, 12310-12322. (36) Zan M. H.; Li J. J.; Huang M. M.; Lin S. Q.; Luo D.; Luo S. Z. and Ge Z. S. Near-infrared

light-triggered

drug

release

nanogels

for

combined

photothermal-chemotherapy of cancer. Biomater. Sci. 2015, 3, 1147-1156. (37) Wan Z. H.; Mao H. J.; Guo M.; Li Y. L.; Zhu A. J.; Yang H.; He H.; Shen J. K.; Zhou L. J.; Jiang Z.; Ge C. C.; Chen X. Y.; Yang X. L.; Liu G. and Chen H. B. Highly Efficient Hierarchical Micelles Integrating Photothermal Therapy and Singlet Oxygen-Synergized Chemotherapy for Cancer Eradication. Theranostics 2014, 4, 399-411. (38) 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. (39) Wu, X. J.; Zhou, L.; Su, Y.; Dong, C.-M. Comb-like poly(L-cysteine) derivatives with different side groups: synthesis via photochemistry and click chemistry, multi-responsive nanostructures, triggered drug release and cytotoxicity. Polym. Chem. 2015, 6, 6857-6869. (40) Ma, L.; Kohli, M.; Smith, A. Nanoparticles for Combination Drug Therapy ACS Nano, 2013, 7, 9518-9525. (41) Fallon, R. J.; Schwartz, A. L. Receptor-mediated delivery of drugs to hepatocytes. 39


Biomacromolecules

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 40 of 42

Adv. Drug. Deliv. Rev. 1989, 4, 49-63. (42) Ting, S. R. T.; Chen, G.; Stenzel, M. H. Synthesis of glycopolymers and their multivalent recognitions with lectins. Polym. Chem. 2010, 1, 1392-1412. (43) Top, A.; kick, K. L. Multivalent protein polymers with controlled chemical and physical properties. Adv. Drug. Deliv. Rev. 2010, 62, 1530-1540. (44) Hu, J. M.; Wu, T.; Zhang, G. Y.; Liu, S. Y. Efficient synthesis of single gold nanoparticle

hybrid

amphiphilic

triblock

copolymers

and

their

controlled

self-assembly. J. Am. Chem. Soc. 2012, 134, 7624-7627 (45) Chen, Y.; Zheng, X. C.; Wang, X.; Wang, C. Z.; Ding, Y.; Jiang, X. Q. Near-infrared emitting gold cluster–poly(acrylic acid) hybrid nanogels. ACS Macro. Lett. 2014, 3, 74-76. (46) Liu, X. K.; Xu, H. L.; Xia, H. B.; Wang, D. Y. Rapid seeded growth of monodisperse, quasi-spherical, citrate-stabilized gold nanoparticles via H2O2 reduction. Langmuir 2012, 28, 13720-13726. (47) Rica, R. D. L.; Stevens, M. M. Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye. Nature Nanotech. 2012, 7, 821-824. (48) Gobbo, P.; Biondi, M. J.; Feldb J. J.; Workentin, M. S. Arresting the time-dependent H2O2 mediated synthesis of gold nanoparticles for analytical detection and preparative chemistry. J. Mater. Chem. B 2013, 1, 4048-4051. (49) Engelbrekt, C.; Jensen, P. S.; Sørensen, K. H.; Ulstrup, J.; Zhang, J. D. Complexity of gold nanoparticle formation disclosed by dynamics study. J. Phys. Chem. C. 2013, 117, 11818-11828. 40


Page 41 of 42

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

Biomacromolecules

(50) Barngrover, B. M.; Aikens, C. M. The golden pathway to thiolate-stabilized nanoparticles: following the formation of gold (I) thiolate from gold (III) chloride. J. Am. Chem. Soc. 2012, 134, 12590-12595. (51) Dietrich, P. M.; Horlacher, T.; Girard-Lauriault, P.-L.; Gross, T.; Lippitz, A.; Min, H.; Wirth, T.; Castelli, R.; Seeberger, P. H.; Unger, W. E. S. Adlayers of dimannoside thiols on gold: surface chemical analysis. Langmuir 2011, 27, 4808-4815. (52) Büttner, M.; Belser, T.; Oelhafen, P. Stability of thiol-passivated gold particles at elevated temperatures studied by X-ray photoelectron spectroscopy. J. Phys. Chem. B 2005, 109, 5464-5467. (53) Bleach, R.; Karagoz, B.; Prakash, S. M.; Davis, T. P.; Boyer, C. In situ formation of polymer-gold composite nanoparticles with tunable morphologies. ACS Macro Lett. 2014, 3, 591-596. (54) Zhang, Q.; Ko, N. R.; Oh, J. K. Recent advances of stimuli-responsive degradable block copolymer micelles: synthesis and controlled drug delivery applications. Chem. Commun. 2012, 48, 7542-7552. (55) Wei, H.; Zhuo, R. X.; Zhang, X. Z. Design and development of polymeric micelles with cleavable links for intracellular drug delivery. Prog. Polym. Sci. 2013, 38, 503-535. (56) Cheng, R.; Meng, F.; Deng, C.; Zhong, Z. Bioresponsive polymeric nanotherapeutics for targeted cancer chemotherapy. Nano Today 2015, 10, 656-670. (57) Wong, P. T.; Choi, S. K. Mechanisms of drug release in nanotherapeutic delivery systems. Chem. Rev. 2015, 115, 3388-3432. 41


Biomacromolecules

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 42 of 42

(58) Wang, X.; Cai, X.; Hu, J.; Shao, N.; Wang, F.; Zhang, Q.; Xiao, J.; Cheng, Y. Glutathione-triggered

“off−on”

release

of

anticancer

drugs

from

dendrimer-encapsulated gold nanoparticles. J. Am. Chem. Soc. 2013, 135, 9805-9810. (59) Wu, X. J.; Zhou, L.; Su, Y.; Dong, C. -M. An autoreduction method to prepare plasmonic gold-embedded polypeptide micelles for synergistic chemo-photothermal therapy. J. Mater. Chem. B 2016, 4, 2142-2152. (60) Zhao, Y., Chen, F., Pan Y.; Ma, X.; Liang, X. -J. Nanodrug Formed by Coassembly of Dual Anticancer Drugs to Inhibit Cancer Cell Drug Resistance. ACS Appl. Mater. Interfaces 2015, 7, 19295-19305. (61) Larson, N.; Gormley, A.; Frazier, N.; Ghandehari, H. Synergistic enhancement of cancer therapy using a combination of heat shock protein targeted HPMA copolymer– drug conjugates and gold nanorod induced hyperthermia. J. Control. Release 2013, 170, 41-50.

Toc used only

42


Plasmonic, Targeted, and Dual Drugs-Loaded Polypeptide Composite

Recommend Documents