Gold Nanorods Stabilized by Biocompatible and Multifunctional

Subscriber access provided by READING UNIV

Article

Gold Nanorods Stabilized by Biocompatible and Multifunctional Zwitterionic Copolymer for Synergistic Cancer Therapy Phim-on Khunsuk, Supatta Chawalitpong, Pritsana Sawutdeechaikul, Tanapat Palaga, and Voravee P. Hoven Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00780 • Publication Date (Web): 29 Nov 2017 Downloaded from http://pubs.acs.org on December 2, 2017

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.

Molecular Pharmaceutics 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 35 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

Molecular Pharmaceutics

Gold Nanorods Stabilized by Biocompatible and Multifunctional Zwitterionic Copolymer for Synergistic Cancer Therapy Phim-on Khunsuk1, Supatta Chawalitpong2, Pritsana Sawutdeechaikul3, Tanapat Palaga3,4, Voravee P. Hoven*1,4

1

Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road,

Pathumwan, Bangkok 10330, Thailand 2

Program in Biotechnology, Faculty of Science, Chulalongkorn University, Phayathai Road,


Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road,


Center of Excellence in Materials and Bio-interfaces, Chulalongkorn University, Phayathai

Road, Pathumwan Bangkok 10330, Thailand

*

E-mail: [email protected]; Tel.: +66 2218 7627; Fax: +66-2218-7598

KEYWORDS: MPC copolymer, gold nanorods, acid-labile linkage, drug-conjugated polymer, photothermal therapy

1 ACS Paragon Plus Environment

Molecular Pharmaceutics 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: A zwitterionic copolymer between methacryloyloxyethyl phosphorylcholine (MPC) and methacrylic acid (MA), PMAMPC is introduced as a potential versatile polymeric stabilizer for gold nanorods (AuNRs). The MA units in the copolymer serve as built-in feature for multiple functionalization, namely introducing additional thiol groups as active sites for binding with the AuNRs and conjugating with Doxorubicin (DOX), an anticancer drug via acidlabile hydrazone linkage. The MPC units, on the other hand, provide biocompatibility and antifouling characteristics. The chemically modified PMAMPC can act as an effective stabilizer for AuNRs yielding PMAMPC-DOX-AuNRs with a fairly uniform size and shape with good colloidal stability. In vitro cytotoxicity suggested that PMAMPC cannot only improve the AuNRs biocompatibility, but also decrease DOX toxicity to a certain extent. The PMAMPCDOX-AuNRs were efficiently internalized inside cancer cells and localized in lysosomes, where DOX was presumably acid-triggered released as monitored by confocal laser scanning microscopic analysis and flow cytometry. Furthermore, the combined photothermal-chemo treatment of cancer cells using PMAMPC-DOX-AuNRs exhibited a higher therapeutic efficacy than either single treatment alone. These results suggested that the PMAMPC-DOX-AuNRs could potentially be applied in pH-triggered drug delivery for synergistic cancer therapy.


Page 2 of 35

Page 3 of 35 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


INTRODUCTION Besides having a high surface area like many nanoparticles, gold nanorods (AuNRs) exhibit additional favorable characteristics, such as an enhanced permeability and retention effects1,2. They also possess unique optical properties with surface plasmon characteristics3,4. Specifically, AuNRs can be taken up into cells more efficiently than spherical gold nanoparticles (AuNPs), hollow, and core/shell silica/gold particles5,6, and are known to exhibit longitudinal surface plasmon resonance (SPR) absorption in the near-infrared (NIR) region, depending on their size and shape. They exhibit attractive optical properties and unique application potentials, especially in photothermal therapy7, 8, molecular imaging9,

10

and gene therapy11,

12

. When

AuNRs are applied as carriers for photothermal therapy, during NIR irradiation into the longitudinal SPR bands, the excited conduction band electrons decay to the ground state by releasing their energy as heat to the local environment. In other words, they can convert the absorbed light to heat, a phenomenon called the “photothermal effect”3,13. Given that NIR irradiation is in the 800–1100 nm region, it can deeply and non-invasively penetrate into tissues with minimal skin absorption11. Toxicity due to cetyltrimethylammonium bromide (CTAB), used as a template for AuNRs synthesis, severely limits their biomedical applications. To overcome this adverse effect, the adsorption of or exchange with a water soluble and biocompatible molecule is a common and effective approach. As driven by electrostatic interactions, poly(acrylic acid) (PAA) carrying negatively charged carboxylate groups can adsorb onto the positively charged quaternary ammonium groups of CTAB-coated AuNRs. Kirui, et al.7 demonstrated that the carboxyl groups on stable PAA-coated AuNRs were available for conjugating with A33scFv, a targeting agent for colorectal cancer cells. Although the simple coating with a biocompatible polymer via



electrostatic interactions can reduce the cytotoxicity, CTAB still remains between the surface of the AuNRs and the coated polymer. Another approach, which should permanently suppress CTAB toxicity, is to replace the CTAB bilayer on the AuNRs surface with biocompatible molecules by forming gold-thiol (AuS) linkages via a ligand exchange process. Thiol-terminated polymers, such as poly(ethylene glycol)s (PEGs)4,13,14,15 and DNAs11, have been shown to function effectively in providing steric stabilization to the AuNRs. Their performance is superior to small thiolated molecules that often lead to aggregation of AuNRs16. According to Liao and Hafner14, thiol-terminated PEGs can entirely replace the CTAB bilayer and yield highly stable and biocompatible PEGylated AuNRs, as evaluated by Raman spectroscopy. Li and co-workers have also demonstrated that polyamidoamine (PAMAM) dendrimer can be used to replace CTAB on the AuNRs. Upon conjugating with specific arginine-glycine-aspartic acid (RGD) peptides, the developed AuNRs exhibited potential applications for tumor targeting, imaging, as well as selective photothermal therapy upon NIR irradiation17. Zwitterionic molecules/polymers have recently been recognized as an alternative to PEG. Thioalkyl tetra(ethylene glycol)ated zwitterionic molecules have been used successfully as stabilizer for sub-10 nm AuNPs that are found to be primarily uptaken by cells via passive diffusion18. The zwitterionic AuNPs are non-toxic and also exhibited effective and selective antimicrobial activity19. In particular, poly(methacryloyloxyethyl phosphorylcholine) (PMPC), the design of which was inspired as a cell-membrane mimic structure18,19. Its excellent antifouling characteristic renders PMPC an attractive choice for the development of biomaterials and biomedical devices18,20,21. Chen et al.22 recently reported that the PMPC-containing copolymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-dihydrolipoic acid) (poly(MPC-


Page 4 of 35

Page 5 of 35 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


co-DHLA)) could be applied as polymeric stabilizer for AuNRs. The DHLA pendent groups of the copolymer provide multiple thiol entities for additional anchoring to the surface of AuNRs so that the resulting AuNRs are highly stable and can resist cyanide ion digestion. The copolymerstabilized AuNRs were found to be non-toxic to both cancerous and normal cells. Most impressively, this characteristic outperformed that of the PEGylated AuNRs. Using a polymeric stabilizer for AuNRs also gives the additional benefit that the active pendant groups of the polymer are available for further conjugation with the drug or target molecules. For cancer treatment, AuNRs stabilized with the desired anticancer drug-conjugated polymer should be able to provide a synergistic effect for cancer treatment via a combination of photothermal therapy upon NIR irradiation and chemotherapy once the conjugated drug is released. Since the tumor tissue environment is usually more acidic (pH 6.5) than blood and normal tissues (pH 7.4), and the pH values of the endosome and lysosome within the cell are even lower at ∼5.5−6.0 and ∼4.5−5.0, respectively23,24, drug conjugation via acid-labile or acid sensitive linkage is often a common strategy so that the drug release can be induced by acidtriggered mechanism24-28. A number of unavoidable drawbacks, such as a dose-limiting toxicity, short circulation time and low water solubility can be prevailed using this sustained release concept. Xiao et al.4 developed AuNRs as multifunctional drug carriers by conjugating doxorubicin (DOX) onto PEGylated AuNRs via an acid-labile hydrazone linkage for chemotherapy, while attaching the targeting molecule cyclo(Arg-Gly-Asp-D-Phe-Cys) peptides and 64Cu-chelators (1,4,7-triazacyclononane-N, N’, N’’-triacetic acid) allowed its use for positron emission tomography imaging. Li et al.19 synthesized AuNRs stabilized by PEG-attached PAMAM G4 dendrimers (PEG-PAMAM) and then conjugated these with DOX by acid-labile hydrazone linkage. The obtained PEG-DOX-PAMAM-AuNRs had an excellent colloidal



Page 6 of 35

stability and low cytotoxicity. In vitro drug release studies verified that DOX was negligibly released from the particles at pH 7.4, but its release was significantly enhanced in an acidic buffer (pH 5.0) with 88.2% release by 60 h. Moreover, in vitro and in vivo combined photothermal-chemo treatment of cancer cells exhibited a more effective therapeutic potency than either single treatment alone, suggesting that a combination of photothermal process with chemotherapy provided a synergistic effect on damaging the cancer cells and so enhancing the efficiency of cancer therapy. Our group have recently introduced a new platform for biosensing applications based on thiol-terminated

poly[(methacrylic

acid)-ran-(2-methacryloyloxyethyl

phosphorylcholine)]

(PMAMPC-SH), which was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization followed by aminolysis29. The carboxyl groups from the methacrylic acid (MA) units were attached with biotin. The presence of hydrophilic 2-methacryloyloxyethyl phosphorylcholine (MPC) units in the copolymer was found to be essential for suppressing unwanted non-specific adsorption and helped improve the detection limit of target analyte, avidin, in diluted blood plasma as monitored by surface plasmon resonance analysis. In light of this success, it was anticipated that this copolymer should be applicable for other biomedical applications. The MA units of the copolymer provide carboxyl groups as active sites for conjugation with the desired biomolecule or drug, whereas the MPC units should make the material more water soluble, biocompatible and anti-fouling. Therefore, the goal of this research was to introduce PMAMPC as zwitterionic and multifunctional polymeric stabilizer for AuNRs that can be developed into anticancer drug delivery system. PMAMPC-SH was first conjugated with DOX, a model anticancer drug commonly used in the treatment of a wide range of cancers30-32, by acid-labile hydrazone linkage


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


and then coated on the surface of the AuNRs via ligand exchange with CTAB-stabilized AuNRs in order to get PMAMPC-DOX-AuNRs. Investigation of the in vitro drug release and photothermal effect of the developed AuNRs was then performed. In vitro cytotoxicity and synergistic treatment against mammary gland adenocarcinoma (MDA-MB-231) cells were evaluated

using

the

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium (MTS) assay. Intracellular trafficking of PMAMPC-DOX-AuNRs and DOX release was then verified using confocal laser scanning microscopy (CLSM) and flow cytometry. The synergistic cancer therapy, through a combination of photothermal process and chemotherapy, via a pH-responsive drug release under the acidic environment in lysosomes and NIR irradiation is proposed in Scheme 1. It was anticipated that this investigation would open up a new possible application of MPC-based polymer in the drug-released area together with AuNRs, a format that has never been reported before.

Scheme 1. Schematic illustration of AuNRs stabilized with PMAMPC-DOX for synergistic cancer therapy.

EXPERIMENTAL SECTION



Materials. The MPC was purchased from NOF Corp. (Japan), MA was distilled under reduced pressure with added p-methoxyphenol (59 °C/13.5 mmHg) and DOX hydrochloride (DOX.HCl) was purchased from Chemieliva Pharmaceutical Co., Ltd. (Chongqing, China). 4,4′Azobis(4-cyanovaleric acid) (ACVA), 4-cyanopentanoic acid dithiobenzoate (CPD), hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O), CTAB (97%), L-ascorbic acid, hydrazine monohydrate, phosphate buffered saline pH 7.4 (PBS) and dialysis bag (cutoff molecular weight of 3500 g/mol) were purchased from Sigma-Aldrich

29

. Silver nitrate, sodium borohydride and

ethanol (EtOH) were purchased from Merck (Germany). 1-(3-(Dimethylamino)propyl)-3ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and cysteamine hydrochloride were purchased from Fluka

29

. The MTS assay reagent containing the electron

coupling agent phenazine ethosulfate was purchased from Promega 29. Synthesis of Thiol-terminated PMAMPC (PMAMPC-SH). The PMAMPC with a targeted degree of polymerization (DP) of 100 and a comonomer composition (MA:MPC) of 60:40 was synthesized by RAFT polymerization following the published procedure30. The MPC monomer (0.4 mg, 1.50 mmol) was completely dissolved in 1.87 mL of mixed solvent (1:1, EtOH: 10 mM PBS in Milli-Q water), and then MA monomer (190.3 µL, 2.20 mmol), ACVA (2.6 mg, 9.35 µmol) and CPD (10.5 mg, 37.41 µmol) were added to the solution. The RAFT polymerization was operated in a closed system under a nitrogen (N2) atmosphere by capping the reaction bottle with a septum and the solution was bubbled with N2 gas for 30 min and then put in an oil bath at 70 °C for 6 h. The polymer solution was purified by dialysis in deionized water (DW) for 4 d, filtered by Whatman® qualitative filter paper (Grade 1) and then lyophilized to yield an orange cotton-like material with a 61% yield. The composition of the copolymer was determined using 1H-NMR spectroscopy (See Supporting Information for detail). 1H-NMR


Page 8 of 35

Page 9 of 35 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


(D2O, 400 MHz): δ = 0.9 (s, 6H), 1.8 (br, 4H), 3.0 (s, 9H), 3.5 (s, 2H), 3.8-4.3 (m, 6H) and 7.4– 8.2 (m, 5H). Hydrazine (10–30 mole equiv. to dithiobenzoate group of the copolymer) was added to a 5 mM PMAMPC solution in Milli-Q water and then stirred at ambient temperature for 1 h or until the polymer solution became colorless. The solution was then added dropwise to 1.0 M HCl (aq) (10 mL) and the obtained PMAMPC-SH was purified by dialysis in HCl solution pH 3.0 and DW for 2 d each. The white cotton-like product of PMAMPC-SH was obtained after lyophilization. 1H-NMR (D2O, 400 MHz): δ = 0.9 (s, 6H), 1.8 (br, 4H), 3.0 (s, 9H), 3.5 (s, 2H) and 3.8–4.3 (m, 6H). Chemical Modification of PMAMPC-SH. The obtained PMAMPC-SH (120 mg, 0.313 mmol of MA unit) was dissolved in 2 mL of Milli-Q water and then EDC (0.2 M) and NHS (0.1 M) were added and stirred for 30 min to activate the carboxyl groups. After that, cysteamine (0.1 mol equiv to MA unit) was added and stirred overnight. The product was purified by dialysis in DW for 3 d. After lyophilization, a cotton-like product of PMAMPC-Cys was obtained, and 95 mg (0.25 mmol of MA unit) was dissolved in 1.2 mL of Milli-Q water and EDC (0.2 M) and NHS (0.1 M) were added and stirred for 30 min to activate the remaining carboxyl groups. Hydrazine (20 mol equiv to MA unit) was then added and stirred overnight. The product was purified by dialysis in DW for 3 d with the white solid of PMAMPC-Cys-Hy being obtained after lyophilization. Overall chemical modification of PMAMPC is outlined in Scheme 2 (Steps I and II). Conjugation of DOX onto the PMAMPC. The mixture of PMAMPC-Cys-Hy (4.0 mg, 0.01 mmol) and DOX.HCl (1:1 mol ratio of DOX:COOH group in PMAMPC) in 5 mL of MilliQ water was stirred in the dark at 60 °C for 2 d, subjected to dialysis in Milli-Q water for 4 d and



lyophilized to give the dark red cotton-like PMAMPC−DOX (Scheme 2 (Step III)). The DOX loading of the conjugate was calculated by UV-Vis absorbance at 485 nm, using the DOX calibration curve at 485 nm.

Scheme 2. Synthetic route employed for the preparation of PMAMPC-DOX.

Preparation of Polymer-Stabilized AuNRs via Ligand Exchange. All glassware was cleaned and rinsed with aqua regia (3:1 (v/v) HCl: HNO3) prior to use. The CTAB-capped AuNRs were synthesized by the seed-mediated growth method as reported22 and appeared as a brown-red solution. The Au element concentration was determined by inductively coupled plasma-optical emission spectrometry (ICP-OES) on an iCAP 6500 ICP-OES (Thermo Scientific, SciSpec CO., Ltd.) instrument, USA) using a HAuCl4 calibration curve. The CTABAuNRs solution (0.50 mL) was purified by two centrifugation-resuspension cycles (14 krpm, 10 min), where each centrifugation cycle was followed by dispersion in Milli-Q water to the same final volume. The washed CTAB-AuNRs solution was added dropwise to an aqueous solution of 10 ACS Paragon Plus Environment

Page 10 of 35

Page 11 of 35 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


PMAMPC-SH or PMAMPC−DOX (3.1 mg in 1 mL of 10 mM PBS) and the mixture was sonicated for 15 min, then incubated for 2 d at ambient temperature. The mixture was centrifugally washed as above but for three times to remove the unbound ligand and re-dispersed in 0.5 mL of 10 mM PBS. Characterization of the Copolymers and AuNRs. The PMAMPC, both before and after stepwise modification, was characterized by 1H NMR in D2O using a Varian, model Mercury400 nuclear magnetic resonance spectrometer 29 operating at 400 MHz, and by Fourier transform infrared spectroscopy (FT-IR) using a Nicolet Impact 6700 FT-IR spectrometer with 32 scans at a resolution of 4 cm-1 in the frequency range of 400–4000 cm-1. Samples were pressed into potassium bromide pellets. UV-Vis spectra were recorded on an Agilent 8453 UV-Vis spectrometer 29 in a quartz cell with a 1 cm path length. The zeta potential (ζ-potential) values of the AuNRs were evaluated using a Malvern Nano ZS90 Instruments Ltd., Worcestershire, UK equipped with a He-Ne laser beam at 658 nm) at a fixed scattering angle of 173° based on intensity. The dispersant viscosity and RI were set to 0.89 Ns.m2 and 1.33, respectively. The anhydrous morphology and size of AuNRs were analyzed by transmission electron microscopy (TEM) on a JEOL JEM-2010 (Japan) operating at 100 keV. The TEM samples were prepared by dropping approximately 10 µL of the concentrated AuNRs on a carbon-coated copper grid and drying in a desiccator before analysis. Negative staining was performed by dropping 1% (w/v) aqueous solution of phosphotungstic acid on the AuNRs deposited on the carbon-coated copper grid. The excess solution was blot-dried by filter paper. The sample was then air-dried in the dark overnight before subjected to TEM analysis. The average diameters of the observed AuNRs were reported from measurements of 50 random particles for each sample using the SemAfore software. The Au element concentration in digested AuNRs sample was determined by ICP-



OES. Determination of DOX Loading Content on AuNRs. The DOX loading content (DLC), defined as the weight percent (wt.%) of DOX on the AuNRs, was quantified by UV-Vis analysis after cyanide digestion. First, PMAMPC-DOX-AuNRs were washed twice by centrifugationredispersion cycles (14 krpm, 10 min/cycle) to remove the un-bound PMAMPC-DOX. After that, 1 mL of 12 mM KCN solution was added to the concentrated PMAMPC-DOX-AuNRs and incubated at ambient temperature for 2 d. The absorbance of DOX at 485 nm was measured to determine the DLC in the solution using a previously established calibration curve. The DLC measurements were performed in triplicate for each sample. The absorption of digested PMAMPC-Cys-AuNRs was defined as the blank. In vitro Drug Release Studies. The pH-responsive DOX release from PMAMPC-DOX was performed in acetate buffer (10 mM, pH 5.0) and phosphate buffer (10 mM, pH 7.4) using a dialysis method. An aliquot (1.0 mL) of PMAMPC−DOX (4.0 mg) was loaded in a dialysis bag and immediately placed in 20 mL of corresponding buffer at 37 °C. Periodically, 1 mL of the buffer solution outside the dialysis bag was taken out and replaced with an equal volume of fresh medium. The amount of DOX was quantified by measuring its absorbance at 485 nm against a standard curve. In vitro Photothermal Studies. The potential application of AuNRs for photothermal therapy was investigated by measuring the temperature rise of a PMAMPC-DOX-AuNRs solution upon irradiation by NIR laser. An aqueous suspension of PMAMPC-DOX-AuNRs (2.0 mL) of various AuNR concentrations (0, 2, 4, 6, 8 and 10 µg/mL) was prepared in a glass tube and irradiated with a NIR laser (2 W 800 nm Diode Laser; MDL-III-800-2000, Ultralasers, Inc., Canada) for 15 min. The temperature was detected with a ETI MicroTherma 2T thermometer at a


Page 12 of 35

Page 13 of 35 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


time interval of 1 min. Cell culture. Mammary gland adenocarcinoma MDA-MB-231 cells (ATCC® HTB-26TM) were cultured in CM (Minimum Essential Medium with Earle's Balanced Salts (GE Healthcare HyClone, USA) containing 1% (v/v) MEM Non-Essential Amino Acids (GE Healthcare HyClone, USA), 10% (v/v) fetal bovine serum (GIBCO, USA) and 1% (v/v) penicillinstreptomycin (GE Healthcare HyClone, USA)) at 37 °C under 5% (v/v) CO2 for 2 d to achieve approximately the same level of confluency (≈ 80%) before performing the experiments. In vitro Cytotoxicity Assay. The cytotoxicity of all modified AuNRs to MDA-MB-231 cells was examined using a colorimetric MTS cell viability assay. Cells were plated at 5,000 cells/well (96-well plates) in 100 µL CM/well, cultured for 24 h and then the media was replaced with 100 µL of various concentrations of PMAMPC-DOX-AuNRs, PMAMPC-Cys-AuNRs and DOX in CM and maintained at 37 °C under 5% (v/v) CO2 for 24 h. In addition, 100 µL of the same concentrations of PMAMPC-DOX-AuNRs, PMAMPC-Cys-AuNRs and DOX in CM without cells was used as the blank. After 24 h the MTS solution (10 µL) was added and incubated for 4 h before the absorbance was measured at 492 nm using a microplate reader (Biochrom® Anthos 2010, UK). The data were analyzed using the ADAP 2.0 Basic software. The absorbance of treated cell suspensions was subtracted from their respective blank and standardized to the untreated (control) wells (set at 100%) to deduce the relative cell viability. Cellular Uptake Study. MDA-MB-231 cells were seeded at 2.5×105 cells/well into an 8well Lab-Tek II Chamber Slide w/Cover RS Glass Slide in 500 µL of CM, and cultured for 24 h. The media was removed and a total of 500 µL of free DOX, PMAMPC-DOX or PMAMPCDOX-AuNRs solution (15 µg DOX/mL) was added immediately and incubated for 30 or 120 min, whereupon the cells were gently washed with 500 µL of PBS twice, followed by the



addition of 100 µL of 0.02 mg/mL 4,6-diamidino-2-phenylindole (DAPI) and incubated at ambient temperature in the dark for 5 min with shaking to stain the nucleus. After staining, cells were gently washed with 500 µL of PBS three times, the chamber was removed from the glass slide and the glass slide was allowed to dry. The anti-fade reagent, MOWIOL, was dropped on the slide and then closed with a cover slip. Next, CLSM analysis of these cells was performed on FluoView FV10i confocal microscope (OLYMPUS) and the images were analyzed using FV10ASW 4.2 Viewer software. In addition, the release of lysosome was observed using this technique, by staining the lysosome with LysoTracker red, except the DOX concentration was decreased to 7.5 µg/mL and the incubation time was increased to 6 h. Moreover, the cellular uptake of DOX was analyzed using flow cytometry. MDA-MB-231 cells were seeded at 5 × 105 cells/well (12-well culture plates) in 1.0 mL of CM and cultured for 24 h, whereupon the media was removed and a total of 1.0 mL of free DOX, PMAMPC-DOX or PMAMPC-DOX-AuNRs solution (7.5 µg DOX/mL) was added immediately and incubated for 6 h. Cells were then placed on ice and gently washed with 1.0 mL of PBS twice, resuspended in 200 µL of PBS and then the DOX uptake was analyzed by flow cytometry (FC500, Backman Coulter). Data analysis was performed using the FlowJo v10.2 software (FlowJo, LLC, USA). In vitro Synergistic Therapy. The efficacy of PMAMPC-DOX-AuNRs in synergistic therapy was examined using the colorimetric MTS cell viability assay. MDA-MB-231 cells (5,000 cells) were seeded at 5,000 cells/well (96-well plates) in 100 µL CM and cultured for 24 h. The media was then replaced with 100 µL of PMAMPC-DOX-AuNRs, PMAMPC-CysAuNRs or DOX (AuNRs concentration: 5 µg/mL) and incubated at 37 °C under 5% (v/v) CO2 for 24 h, while 100 µL of PMAMPC-DOX-AuNRs, PMAMPC-Cys-AuNRs and DOX at the same concentration in CM was used as a blank. The suspensions in selected wells were then


Page 14 of 35

Page 15 of 35 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


irradiated with a NIR laser (2 W 800 nm Diode Laser; MDL-III-800-2000, Ultralasers, Inc., Canada) at a 15 cm distance for 0 (control), 30, 45 and 60 s. The cells were then cultured for another 24 h and the cell viability was determined by the MTS assay as above to determine the relative cells viability (survival).

RESULTS AND DISCUSSION Synthesis and Chemical Modification of PMAMPC. PMAMPC was synthesized using RAFT polymerization in the presence of CPD and ACVA as the chain transfer agent (CTA) and radical initiator, respectively. The ratio of CTA/initiator or [CTA]/[I] was fixed at 4/1, as previously reported.29 From the 1H-NMR spectra of PMAMPC (Figure S1i, Supporting Information), the characteristic peaks of the MPC unit (-N(CH3)3 = 3.0 ppm, -CH2N (d) = 3.5 ppm, and –POCH2CH2N –COOCH2 – CH2CH2OP = 3.8-4.3 ppm) were clearly observed. The copolymer composition (MA:MPC) determined from the

1

H-NMR spectra (Figure S1i,

Supporting Information) was 50:50 with a DP of 110. The calculated molecular weight of PMAMPC was found to be 21,275 Da (targeted MW = 17,270 Da). The copolymer composition and molecular weight determined by 1H-NMR closely resembled the expected theoretical values, suggesting that the copolymerization was relatively well-controlled. The dithiobenzoate group at the chain end of PMAMPC was converted to a thiol group by reduction using hydrazine, and confirmed by the disappearance of the peak assigned to aromatic protons around δ 7.4−8.2 ppm in the 1H-NMR spectrum after reduction (Figure S1ii, Supporting Information) and so PMAMPC-SH with a thiol end was obtained. The success of the reduction was also supported by the vanishing UV absorbance at 306 nm (Figure S2, Supporting Information). Previously, we have attempted to prepare AuNRs stabilized by PMAMPC-SH via ligand



exchange of CTAB-stabilized AuNRs with PMAMPC-SH. It was found that the resulting AuNRs were not stable and aggregated (Figure 1), implying that the number of active thiol binding sites available only at the chain ends of PMAMPC-SH was insufficient to yield stable AuNRs. For this reason, we desired to add more thiol groups to the PMAMPC-SH prior to modification with hydrazine and DOX conjugation. As outlined in Scheme 2 (Step I-II), to introduce more thiol groups for Au-S formation, some carboxyl groups of PMAMPC-SH were modified with cysteamine via amide linkage using EDC/NHS as the coupling agent to yield PMAMPC-Cys. The remaining carboxyl groups were then covalently bonded with excess hydrazine to give PMAMPC-Cys-Hy that was further conjugated with DOX. The success of stepwise modification of PMAMPC-SH and subsequent DOX conjugation was verified by FT-IR analysis, with the data displayed in Figure 2.

Figure 1. TEM images of (a) CTAB-AuNRs, (b) PMAMPC-SH-AuNRs, (c) PMAMPC-CysAuNRs, (d) PMAMPC-DOX-AuNRs and (e) negative stained TEM image of PMAMPC-DOXAuNRs

From the FT-IR spectrum of PMAMPC-SH (Figure 2A, a), the characteristic peaks of the


Page 16 of 35

Page 17 of 35 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


MPC unit that appeared at 798, 970, 1085, 1176 and 1230 cm-1 were assigned to the stretching of C-O-P, N+(CH3)3, P-O, C-O and P=O groups, respectively. The C=O stretching of the carboxyl group in the MA unit appeared at 1720 cm-1. The relative intensity between the peak at 1631 cm1

, which was ascribed to the amide C=O/C=N stretching, and the peak at 1720 cm-1, which

corresponded to the C=O stretching of COOH groups, proportionally increased after each step of modification (Figure 2A, b-d). In particular, the former peak dominated the latter one after DOX conjugation. This verified the success of the stepwise chemical modification. Additional amide bonds were formed between the carboxyl groups of PMAMPC-SH and cysteamine and hydrazine. Besides, the presence of N-H bending of PMAMPC-Cys-Hy (Figure 2A, c) found at 1560 cm-1 can be used as an additional evidence of amino group availability to form hydrozone linkage with DOX. The signal from C=C stretching of the aromatic ring appeared in the spectrum of PMAMPC-DOX (Figure 2A, d), confirming that DOX had been incorporated in the copolymer. To prevent disulfide bond formation within or between the polymer molecules, all products obtained after stepwise modification were kept in solid form at -20°C. As evaluated by gel permeation chromatography (data not shown), the molecular weights of all products were unchanged with the molecular weight distribution remained as unimodal implying that there

was no disulfide coupling.



Figure 2. (A) FT-IR spectra of (a) PMAMPC-SH, (b) PMAMPC-Cys, (c) PMAMPC-Cys-Hy, (d) PMAMPC-DOX and (e) DOX; and the (B) UV-vis absorption spectra of CTAB-AuNRs, PMAMPC-Cys-AuNRs and PMAMPC-DOX-AuNRs suspension in 10 mM PBS.

PMAMPC-DOX exhibited the absorption of DOX with λmax ~485 nm (Figure S2, Supporting Information), which also confirmed the conjugation of DOX. The DOX loading level of the conjugate was calculated on the basis of UV-Vis absorbance at 485 nm, using the DOX calibration curve (Figure S3, Supporting Information) and revealed that the PMAMPCDOX contained a DOX loading level of 34.3 wt.%, which is relative high compared to previously reported work based on DOX-conjugated polymers (typically ∼7−22 wt.%)24,31,33,8. Preparation and Characterization of Polymer-stabilized AuNRs. From Figure 2B, the SPR band of the CTAB-AuNRs was typically split into two, corresponding to a transverse (short axis) band (λT) at 511 nm and a longitudinal (long axis) band (λL) at 790 nm in the NIR region, whose positions depended on the aspect ratio (length/width) of the AuNRs. As determined by TEM analysis, the anhydrous CTAB-AuNRs were quite uniform in size at 7.6 ± 3 nm wide and 26.7 ± 4 nm long with an aspect ratio of ~3.5 (Table 1). Having the aspect ratio close to 4, the 18 ACS Paragon Plus Environment

Page 18 of 35

Page 19 of 35 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


synthesized AuNRs should well absorb light in the NIR region3,9. There were also a few AuNPs, initially generated as seeds, remaining in the solution. As determined by ICP-OES, the synthesized CTAB-AuNRs contained an Au element concentration of about 22.8 ± 0.4 µg/mL (n = 3).

Table 1 Size and ζ-potential data of the developed AuNRs PMAMPC-SH- PMAMPC-CysPMAMPCAuNRs AuNRs DOX-AuNRs a Length (nm) 26.7 ± 4 25.9 ± 3 26.8 ± 3 26.9 ± 3 a Width (nm) 7.6 ± 3 7.6 ± 1 7.3 ± 1 7.4 ± 1 Aspect ratio 3.5 3.4 3.7 3.6 ζ-potential (mV) +24.1 ± 5 -22.3 ± 1 -19.5 ± 2 a Average ± 1 SD dimension of the observed AuNRs by TEM are reported from measurements of 50 random particles for each sample using the SemAfore software. CTAB-AuNRs

After PMAMPC-SH was coated onto the AuNRs surface through Au-S bond formation via ligand exchange, some aggregation of AuNRs occurred and was visualized by naked eye in terms of the color change of the AuNR suspension from brown-red to blue. To improve the colloidal stability, PMAMPC-SH was, therefore, further modified with cysteamine in order to increase the number of active binding sites for each PMAMPC chain before coating on the AuNRs surface. The fact that the λL of the PMAMPC-Cys-AuNRs appeared at 795 nm (Figure 2B) without broadening and tailing, in contrast to that of the as-synthesized CTAB-AuNRs suggested that the additional thiol groups introduced from cystamine to PMAMPC provided a greater number of active binding sites for anchoring to the AuNRs surface and so yielded PMAMPC-Cys-AuNRs with a good colloidal stability in 10 mM PBS. Likewise, upon ligand exchange, the obtained PMAMPC-DOX-AuNRs were quite stable, as seen from their UV-Vis absorption (Figure 2B) appearing at 820 nm, only slightly red-shifted from the 790 nm of the as19 ACS Paragon Plus Environment


synthesized CTAB-AuNRs. Neither broadening nor tailing of the SPR band of PMAMPC-DOXAuNRs was observed verifying that no aggregation of the particles occurred during this step19. A broad absorption band at around 485 nm confirmed the presence of PMAMPC-DOX on the PMAMPC-DOX-AuNRs. Morphological information, as evidenced from the TEM analysis (Figure 1), was in good agreement with the UV-Vis absorption results (Figure 2B), where PMAMPC-AuNRs showed some aggregation in the TEM images (Figure 1, b). Coating the AuNRs with PMAMPC-Cys and PMAMPC-DOX provided steric stabilization and improved the dispersibility of the AuNRs (Figure 1, c and d, respectively). Negative-stained TEM micrograph (Figure 1, e) confirmed the presence of thin polymeric layer (thickness ~ 2.6 nm) surrounding the PMAMPC-DOXAuNRs. As shown in Table 1, The PMAMPC-DOX-AuNRs were quite uniform in size at 7.4 ± 1 nm wide and 26.9 ± 3 nm long, and much better dispersed than the CTAB-AuNRs. The aspect ratio (~3.6) remained unchanged after ligand exchange, suggesting that polymer coating did not markedly affect the AuNRs morphology. The success of polymeric ligand exchange onto the AuNRs can be further verified by ζ-potential values displayed in Table 1. Apparently, the ζpotential turned from positive to negative upon the exchange of CTAB with PMAMPC-Cys and PMAMPC-DOX which are rich in carboxyl entities. It has been previously reported that the degradation of AuNRs coated with polymeric ligands would be slower than the CTAB-AuNRs which degrade rapidly in the presence 12 mM sodium cyanide22. The protecting effect by polymer coating that can retard the AuNRs degradation was also realized in this research. It took 48 h to completely digest the PMAMPCCys-AuNRs and PMAMPC-DOX-AuNRs. The progress of the cyanide digestion was monitored by UV-Vis spectroscopy (Figure S3, Supporting Information), where the diminishing SPR


Page 20 of 35

Page 21 of 35 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


band at 820 nm of the PMAMPC-DOX-AuNRs was observed and its disappearance was taken to indicate that the AuNRs were completely digested. After cyanide digestion, the DLC, defined as the DOX wt.% in the developed AuNRs, was approximately 19 wt.%, as quantified by UV-Vis analysis based on the calibration curve (Figure S4, Supporting Information). In vitro Drug Release. To demonstrate the pH-sensitive drug release behavior of PMAMPC-DOX-AuNRs, the in vitro DOX release was measured as a function of time at pH 7.4 and 5.0 (Figure 3). The release of DOX was accelerated under an acidic (pH 5.0) condition, a pH close to the acidic environment of lysosome compartments (∼4.5−5.0)23, with 60% accumulated release after 48 h compared to only 12% DOX released after the same period of time at pH 7.4. This demonstrates the potential efficacy of pH-sensitive drug release from PMAMPC-DOX-AuNRs in a physiologically relevant acidic environment.

Figure 3. In vitro DOX release profile from PMAMPC-DOX at pH 5.0 and pH 7.4. Data are shown as the mean ± 1 SD, derived from three independent trials.

In vitro Photothermal Studies. To determine the photothermal effect of AuNRs, aqueous suspensions of PMAMPC-DOX-AuNRs were exposed to NIR laser irradiation at 808 nm with a 21 ACS Paragon Plus Environment


power density of 2 W/cm2. Changes in temperature were detected using a thermometer. Both the temperature elevation rate and final temperature were dependent on the quantity of Au element (Figure S5, Supporting Information), where faster and greater temperature increases were observed for higher quantities of Au element. For example, 2 mL of PMAMPC-DOX-AuNRs suspension with 20 µg Au element could reach 70.1°C (increase of 39.7 °C) after 15 min irradiation, whereas the AuNRs-free solution (10 mM PBS) increased by only 4.4 °C. It is notable that the continuous laser irradiation did not cause any loss of the optical properties of AuNRs nor change the particle size/morphology (See TEM images in Figure S6, Supporting Information for detail), indicating that PMAMPC-DOX-AuNRs were stable under laser irradiation and temperature increase. In vitro Cytotoxicity. Prior to being exchanged onto AuNRs, in vitro cytotoxicity against MDA-MB-231 cells of PMAMPC-Cys and PMAMPC-DOX in comparison with free DOX was evaluated. As shown in Figure S7, Supporting Information, PMAMPC-Cys was non-toxic up to the maximum tested concentration of 10-4 M. The fact that the lowest concentration of PMAMPC-DOX that became toxic to the cells of 10-4 M is 2 orders of magnitude higher than that of the free DOX (10-6 M) strongly suggests that toxicity of DOX can be decreased once attaching to PMAMPC. PMAMPC-DOX-AuNRs were evaluated for their in vitro cytotoxicity against MDA-MB231 cells in tissue culture. Cells were incubated with CTAB-AuNRs, PMAMPC-Cys-AuNRs, PMAMPC-DOX-AuNRs and DOX at equivalent concentrations of Au element and DOX for 24 h, followed by cell viability determination using the MTS assay (Figure 4). Dose response curves showed an evident cytotoxicity of the CTAB-AuNRs at a concentration of 2.5 µg/mL, whereas PMAMPC-Cys-AuNRs were non-toxic to MDA-MB-231 cells over the entire evaluated


Page 22 of 35

Page 23 of 35 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


concentration range up to the maximum tested dose of 20 µg/mL, indicating that PMAMPC improved the biocompatibility of AuNRs. PMAMPC-DOX-AuNRs were tolerated by the cells to a slightly higher level than DOX suggesting that conjugation of DOX with PMAMPC can reduce the toxicity derived from DOX to some extent, with an estimated IC50 of 14.6 and 17.1 µg/mL for DOX and PMAMPC-DOX-AuNRs, respectively. From this data, an Au element concentration of 5 µg/mL was chosen for further study of potential synergistic therapy since this dose was well below the IC50 values of all the samples allowing enough cells to survive to be traced. It was also the lowest value investigated where the influence of each condition on the level of cell death could be distinguished.

Figure 4. Relative cell viability (%), as determined by the MTS assay, of MDA-MB-231 cells after incubation with increasing concentrations of: (a) PMAMPC-Cys-AuNRs, (b) PMAMPCDOX-AuNRs, (c) DOX and (d) CTAB-AuNRs. Data are shown as the mean ± 1SD obtained from three independent experiments.

Cellular Uptake. Due to its self-fluorescent property, the intracellular distribution of DOX can be directly followed by CLSM. Once conjugated with AuNRs, the fluorescence intensity of



DOX is quenched as a result of the nanosurface energy transfer (NSET) effect from the AuNRs19. After DOX release, under the acidic environment inside the cells, the fluorescence emission should be recovered. For uptake evaluation, MDA-MB-231 cells were incubated with 15 µM DOX with an equivalent concentration of the particles for 30 min and 2 h, then counterstained (in the nucleus) with DAPI, and observed by CLSM at a set camera exposure time. As expected, an obvious fluorescent emission of DOX derived from PMAMPC-DOXAuNRs was detected after 30 min of incubation (Figure 5). The extensive red fluorescence of DOX located inside the cells only slightly overlapped with blue fluorescence of the DAPIstained nucleus in the overlay image, implying that PMAMPC-DOX-AuNRs were localized in lysosomes and that DOX was released inside the lysosomes and partly penetrated into the nucleus. When the incubation time increased to 2 h, a greater amount of DOX overlapped with the DAPI-stained nucleus, seen as purple in the overlay image, indicating that the pH-triggered DOX release increased with time and increasingly trafficked into the nucleus (Figure S8, Supporting Information). These findings agreed well with the in vitro DOX release results at pH 5.0 observed during dialysis. In addition, PMAMPC-DOX alone did not penetrate the cells within a 2-h period, suggesting that the AuNRs enhanced the cell permeability so that PMAMPC-DOX could preferentially accumulate in the cancer cells. Based on these findings, it can be implied that the pH-responsive PMAMPC-DOX-AuNRs not only reduced the level of undesired drug release outside the cells during circulation but also enhanced and accelerated the intracellular drug uptake and release, which might elevate the potency of such cancer therapy.


Page 24 of 35

Page 25 of 35 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. Representative CLSM analysis of intracellular DOX release from PMAMPC-DOXAuNRs in comparison with DOX and PMAMPC-DOX. MDA-MB-231 cells were treated with PMAMPC-DOX-AuNRs, with 15 µg/mL of DOX, for 30 min, followed by CLSM observation with time. Blue fluorescence: nucleus staining dry, DAPI; red fluorescence: DOX; scale bar = 50 µm. Images shown are representative of those seen from at least 3 such fields of view per sample and 3 independent samples. 25 ACS Paragon Plus Environment


To verify whether the DOX release was induced in the lysosome, the incubation time was increased to 6 h and the DOX equivalent dose was decreased in half to 7.5 µg/mL. The dose reduction was applied to assure that enough cells survived the longer incubation time (6 h as opposed to 2 h used for incubation with 15 µg/mL DOX) so that the lysosomal release could be tracked by CLSM. The green fluorescence dot of lysosome was apparent in the LysoTracker channel of PMAMPC-DOX-AuNRs and also overlapped with the red fluorescence area of DOX, giving the yellow color, in the overlay image (Figure 6). This result strongly suggested that PMAMPC-DOX-AuNRs can induce lysosome formation and the particles localized in the lysosome can release that DOX inside the lysosome as opposed to the PMAMPC-DOX.

Figure 6. Representative CLSM analysis of intracellular DOX release from PMAMPC-DOXAuNRs in comparison with DOX and PMAMPC-DOX. MDA-MB-231 cells were treated with PMAMPC-DOX-AuNRs, with 7.5 µg/mL of DOX, for 6 h, followed by CLSM observation with 26 ACS Paragon Plus Environment

Page 26 of 35

Page 27 of 35 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


time. Blue fluorescence: nucleus staining dry, DAPI; red fluorescence: DOX; green fluorescence: LysoTracker; scale bar = 20 µm. Images shown are representative of those seen from at least 3 such fields of view per sample and 3 independent samples.

The level of cellular uptake of DOX was also be quantitatively monitored by flow cytometry. As shown in Figure 7A. The control sample showed a negligible level of autofluorescence intensity. After 6 h, the percentage of cells incubated with free DOX, PMAMPC-DOX and PMAMPC-DOX-AuNRs with DOX inside their cells, as calculated from their corresponding peak area, was 83.6 ± 6.8, 71.7 ± 7.8 and 87.1 ± 1.5 respectively. This set of data implied that compared to the free DOX the PMAMPC-DOX-AuNRs had an almost equivalent overall level of cell trafficking of DOX. Nevertheless, a greater number of cells were loaded with a higher DOX level in the latter case, as quantitatively evidenced from the mean fluorescence intensity (MFI) values (Figure 7B). The cells incubated with free DOX exhibited the highest MFI value, and this was about 2.4-fold higher than in the cells treated with PMAMPC-DOX-AuNRs. On the other hand, the PMAMPC-DOX group showed a low level of fluorescent signal indicating an only slight DOX uptake. The fact that the PMAMPC-DOXAuNRs gave a 2.5-fold higher MFI than the PMAMPC-DOX suggested that conjugating DOX to the AuNRs evidently increased the efficiency of intracellular uptake of the polymer-conjugated DOX.



Figure 7. Flow cytometric analysis of MDA-MB-231 cells treated with free DOX, PMAMPCDOX, PMAMPC-DOX-AuNRs (DOX concentration: 7.5 µg/mL), or medium alone (control) for 6 h. (A) Representative (of three repeats) histogram plots showing the DOX fluorescence following cellular uptake and (B) Summary data of flow cytometry results. Data are shown as the mean fluorescence intensity (MFI) values ± 1SD, obtained from three independent experiments.

In vitro Synergistic Therapy. The synergistic in vitro photothermal-chemotherapy effects of PMAMPC-DOX-AuNRs on MDA-MB-231 cells were evaluated using the MTS assay. MDAMB-231 cells were treated with PMAMPC-DOX-AuNRs at a fixed Au element concentration of 5 µg/mL for 24 h. After laser irradiation for various times (0 (control), 30, 45 and 60 s), the cells were further cultured in CM for another 24 h and then the cell viability was determined by the MTS assay. DOX-free PMAMPC-Cys-AuNRs was used as a control. No significant loss of cell viability occurred in PMAMPC-DOX-AuNRs-incubated cells without laser irradiation and in DOX-free PMAMPC-Cys-AuNRs-incubated cells after a 30 and 45 s laser irradiation (Figure 8). However, distinctly enhanced cell death was observed for PMAMPC-DOX-AuNRs-treated


Page 28 of 35

Page 29 of 35 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 after laser irradiation for only 30 sec. Photothermal treatment or chemotherapy alone showed no marked cell damage, and so the synergistic photothermal-chemotherapy was believed to promote the therapeutic effect. The evident reduction of cell viability after the combined photothermal-chemo treatment could potentially be explained that DOX released from PMAMPC-DOX-AuNRs weakened the cancer cells and then made them more vulnerable to the heat from the photothermal effect resulting in a greater proportion of dead cells. As the irradiation time increased to 60 s, the cells treated with PMAMPC-Cys-AuNRs showed a greater frequency of cell death, and those cells treated with PMAMPC-DOX-AuNRs showed 100% mortality. A reasonable explanation for the 100% mortality of PMAMPC-DOX-AuNRs-treated cells after 60 s irradiation was the additive hyperthermia effect from the longer photothermal treatment time, which resulted in lysosome membrane disruption and so a fast cell death19 In contrast, cell viability was slightly decreased from 44 to 37% after the DOX-treated cells were also irradiated by NIR for the same period of time suggesting that the photothermal effect is not quite significant in the absence of AuNRs.



Figure 8. In vitro cytotoxicity, as determined by the MTS assay, of PMAMPC-DOX-AuNRs, PMAMPC-Cys-AuNRs and free DOX with an Au element concentration of 5 µg/mL against MDA-MB-231 cells at various irradiation times. Data are shown as the mean value from three repeats, and numbers above each bar are the net average % relative cell viability.

This in vitro synergistic study demonstrated that the combined photothermal-chemo treatment against cancer cells using PMAMPC-DOX-AuNRs for synergistic hyperthermia ablation and chemotherapy was more effective than either single treatment alone, underlining the great potential of PMAMPC-DOX-AuNRs for cancer treatment.

CONCLUSION It has been demonstrated that the zwitterionic copolymer, PMAMPC, could serve as a platform for multiple and controllable chemical modification from introducing additional binding sites to the surface of AuNRs to anticancer drug conjugation. The chemically modified PMAMPC can act as an effective stabilizer for AuNRs yielding PMAMPC-DOX-AuNRs with a fairly uniform size and shape. In vitro cytotoxicity suggested that PMAMPC improved the AuNRs 30 ACS Paragon Plus Environment

Page 30 of 35

Page 31 of 35 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


biocompatibility. As monitored by CLSM and flow cytometry, PMAMPC-DOX-AuNRs were effectively uptaken into MDA-MB-231 cells and localized in lysosomes, where DOX was released (presumably acid-triggered) inside the lysosomes and some got into the nucleus. The fact that PMAMPC-DOX alone cannot penetrate the cells suggested that AuNRs enhanced the cell permeability, so that PMAMPC-DOX can preferentially accumulate inside the cancer cells. The combined hyperthermia ablation and chemotherapy using 5 µg/mL PMAMPC-DOX-AuNRs and 60 s of irradiation can cause 100% mortality, suggesting the potential of PMAMPC-DOXAuNRs for pH-triggered synergistic cancer therapy. This particular zwitterionic copolymer should also be applicable for gold nanoparticles having different shapes as well as other metal nanoparticles. The ability to conjugate with drug and/or targeting molecules also broaden its versatility.

ASSOCIATED CONTENT Supporting Information 1

H-NMR spectra of PMAMPC and PMAMPC-SH. Calculation of copolymer

composition and degree of polymerization, UV-vis absorption spectra of PMAMPC before and after reduction and PMAMPC-DOX. UV-vis absorption spectra of PMAMPC-DOX-AuNRs and PMAMPC-Cys-AuNRs both before and after digestion. Calibration curve of DOX. Temperature change of PMAMPC-DOX-AuNRs aqueous solutions having varied quantity of Au element during NIR laser irradiation. TEM images of PMAMPC-DOX-AuNRs before and after NIR laser irradiation. Relative cell viability of MDA-MB-231 cells after incubation with PMAMPC-Cys, PMAMPC-DOX, and DOX. CLSM analysis of intracellular DOX release from PMAMPC-



DOX-AuNRs inside MDA-MB-231 cells after 2 h incubation. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Tel.: +66-2218-7627; Fax: +66-2218-7598; E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Acknowledgements Financial support for this work was provided by the Thailand Research Fund (RSA5980071, DBG5580003) and Sci Super II, Ratchadapiseksomphot Endowment Fund under Outstanding Research Performance Program (GF_58_08_23_01). PK acknowledges the Development and Promotion of Science and Technology Talents Project (DPST) for a M.Sc. scholarship.

References 1. Tree-Udom, T.; Seemork, J.; Shyou, K.; Hamada, T.; Sangphech, N.; Palaga, T.; Insin, N.; Pan-In, P.; Wanichwecharungruang, S., Shape Effect on Particle-Lipid Bilayer Membrane Association, Cellular Uptake, and Cytotoxicity. ACS Appl. Mater. Interfaces 2015, 7, 2399324000. 2. Hauck, T. S.; Jennings, T. L.; Yatsenko, T.; Kumaradas, J. C.; Chan, W. C. W., Enhancing the Toxicity of Cancer Chemotherapeutics with Gold Nanorod Hyperthermia. Adv. Mater. 2008, 20, 3832-3838. 3. Alkilany, A. M.; Thompson, L. B.; Boulos, S. P.; Sisco, P. N.; Murphy, C. J., Gold nanorods: Their Potential for Photothermal Therapeutics and Drug Delivery, Tempered by the Complexity of their Biological Interactions. Adv. Drug Delivery Rev. 2012, 64, 190-199. 32 ACS Paragon Plus Environment

Page 32 of 35

Page 33 of 35 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


4. Xiao, Y. L.; Hong, H.; Matson, V. Z.; Javadi, A.; Xu, W.; Yang, Y. A.; Zhang, Y.; Engle, J. W.; Nickles, R. J.; Cai, W. B.; Steeber, D. A.; Gong, S. Q., Gold Nanorods Conjugated with Doxorubicin and cRGD for Combined Anticancer Drug Delivery and PET Imaging. Theranostics 2012, 2, 757-768. 5. Bartczak, D.; Muskens, O. L.; Nitti, S.; Sanchez‐Elsner, T.; Millar, T. M.; Kanaras, A. G., Interactions of Human Endothelial Cells with Gold Nanoparticles of Different Morphologies. Small 2012, 8, 122-130. 6. Li, W.; Zhang, X.; Hao, X.; Jie, J.; Tian, B.; Zhang, X., Shape Design of High Drug Payload Nanoparticles for More Effective Cancer Therapy. Chem. Commun. 2013, 49, 1098910991. 7. Kirui, D. K.; Krishnan, S.; Strickland, A. D.; Batt, C. A., PAA-Derived Gold Nanorods for Cellular Targeting and Photothermal Therapy. Macromol. Biosci. 2011, 11, 779-788. 8. Liao, J.; Li, W.; Peng, J.; Yang, Q.; Li, H.; Wei, Y.; Zhang, X.; Qian, Z., Combined Cancer Photothermal-Chemotherapy Based on Doxorubicin/Gold Nanorod-Loaded Polymersomes. Theranostics 2015, 5, 345-356. 9. Huang, X. H.; El-Sayed, I. H.; Qian, W.; El-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. 10. Choi, W. I.; Sahu, A.; Kim, Y. H.; Tae, G., Photothermal Cancer Therapy and Imaging Based on Gold Nanorods. Ann. Biomed. Eng. 2012, 40, 534-546. 11. Chen, C.-C.; Lin, Y.-P.; Wang, C.-W.; Tzeng, H.-C.; Wu, C.-H.; Chen, Y.-C.; Chen, C.P.; Chen, L.-C.; Wu, Y.-C., DNA−Gold Nanorod Conjugates for Remote Control of Localized Gene Expression by near Infrared Irradiation. J. Am. Chem. Soc. 2006, 128, 3709-3715. 12. Yan, P.; Zhao, N.; Hu, H.; Lin, X.; Liu, F.; Xu, F.-J., A Facile Strategy to Functionalize Gold Nanorods with Polycation Brushes for Biomedical Applications. Acta Biomater. 2014, 10, 3786-3794. 13. Huang, X.; Jain, P. K.; El-Sayed, I. H.; El-Sayed, M. A., Plasmonic Photothermal Therapy (PPTT) Using Gold Nanoparticles. Lasers Med Sci 2007, 23, 217-228. 14. Liao, H.; Hafner, J. H., Gold Nanorod Bioconjugates. Chem. Mater. 2005, 17, 46364641. 15. Niidome, T.; Yamagata, M.; Okamoto, Y.; Akiyama, Y.; Takahashi, H.; Kawano, T.; Katayama, Y.; Niidome, Y., PEG-modified Gold Nanorods with a Stealth Character for in Vivo Applications. J. Controlled Release 2006, 114, 343-347. 16. Chen, H.; Shao, L.; Li, Q.; Wang, J., Gold Nanorods and their Plasmonic Properties. Chem. Soc. Rev. 2013, 42, 2679-2724. 17. Li, Z. M.; Huang, P.; Zhang, X. J.; Lin, J.; Yang, S.; Liu, B.; Gao, F.; Xi, P.; Ren, Q. S.; Cui, D. X., RGD-Conjugated Dendrimer-Modified Gold Nanorods for in Vivo Tumor Targeting and Photothermal Therapy. Mol. Pharmaceutics 2010, 7, 94-104. 18. Jiang, Y.; Huo, S.; Mizuhara, T.; Das, R.; Lee, Y.-W.; Hou, S.; Moyano, D. F.; Duncan, B.; Liang, X.-J.; Rotello, V. M., The Interplay of Size and Surface Functionality on the Cellular Uptake of Sub-10 nm Gold Nanoparticles. ACS Nano 2015, 9, 9986-9993. 19. Huo, S.; Jiang, Y.; Gupta, A.; Jiang, Z.; Landis, R. F.; Hou, S.; Liang, X.-J.; Rotello, V. M., Fully Zwitterionic Nanoparticle Antimicrobial Agents through Tuning of Core Size and Ligand Structure. ACS Nano 2016, 10, 8732-8737.



20. Lewis, A. L.; Hughes, P. D.; Kirkwood, L. C.; Leppard, S. W.; Redman, R. P.; Tolhurst, L. A.; Stratford, P. W., Synthesis and Characterisation of Phosphorylcholine-based Polymers Useful for Coating Blood Filtration Devices. Biomaterials 2000, 21, 1847-1859. 21. Iwasaki, Y.; Ishihara, K., Cell Membrane-inspired Phospholipid Polymers for Developing Medical Devices with Excellent Biointerfaces. Sci. Technol. Adv. Mater. 2012, 13, 064101. 22. Chen, X. J.; Lawrence, J.; Parelkar, S.; Emrick, T., Novel Zwitterionic Copolymers with Dihydrolipoic Acid: Synthesis and Preparation of Nonfouling Nanorods. Macromolecules 2013, 46, 119-127. 23. Chen, X. J.; Parelkar, S. S.; Henchey, E.; Schneider, S.; Emrick, T., PolyMPCDoxorubicin Prodrugs. Bioconjugate Chem. 2012, 23, 1753-1763. 24. Du, J.-Z.; Du, X.-J.; Mao, C.-Q.; Wang, J., Tailor-Made Dual pH-Sensitive Polymer– Doxorubicin Nanoparticles for Efficient Anticancer Drug Delivery. J. Am. Chem. Soc. 2011, 133, 17560-17563. 25. Hou, W. X.; Zhao, X.; Qian, X. Q.; Pan, F.; Zhang, C. L.; Yang, Y. M.; de la Fuente, J. M.; Cui, D. X., pH-Sensitive Self-assembling Nanoparticles for Tumor Near-infrared Fluorescence Imaging and Chemo-photodynamic Combination therapy. Nanoscale 2016, 8, 104116. 26. Markovsky, E.; Baabur-Cohen, H.; Satchi-Fainaro, R., Anticancer Polymeric Nanomedicine Bearing Synergistic Drug Combination is Superior to a Mixture of Individuallyconjugated Drugs. J. Controlled Release 2014, 187, 145-157. 27. Rodrigues, P. C. A.; Beyer, U.; Schumacher, P.; Roth, T.; Fiebig, H. H.; Unger, C.; Messori, L.; Orioli, P.; Paper, D. H.; Mülhaupt, R.; Kratz, F., Acid-sensitive Polyethylene Glycol Conjugates of Doxorubicin: Preparation, in Vitro Efficacy and Intracellular Distribution. Bioorg. Med. Chem. 1999, 7, 2517-2524. 28. Ulbrich, K.; Etrych, T.; Chytil, P.; Pechar, M.; Jelinkova, M.; Rihova, B., Polymeric Anticancer Drugs with pH-controlled Activation. Int. J. Pharm. 2004, 277, 63-72. 29. Akkahat, P.; Kiatkamjornwong, S.; Yusa, S.-i.; Hoven, V. P.; Iwasaki, Y., Development of a Novel Antifouling Platform for Biosensing Probe Immobilization from Methacryloyloxyethyl Phosphorylcholine-Containing Copolymer Brushes. Langmuir 2012, 28, 5872-5881. 30. Venkatesan, R.; Pichaimani, A.; Hari, K.; Balasubramanian, P. K.; Kulandaivel, J.; Premkumar, K., Doxorubicin Conjugated Gold Nanorods: a Sustained Drug Delivery Carrier for Improved Anticancer Therapy. J. Mater. Chem. B 2013, 1, 1010-1018. 31. Pan, H.; Sima, M.; Yang, J.; Kopeček, J., Synthesis of Long-Circulating, Backbone Degradable HPMA Copolymer–Doxorubicin Conjugates and Evaluation of Molecular-WeightDependent Antitumor Efficacy. Macromol. Biosci. 2013, 13, 155-160. 32. Remant, B. K. C.; Thapa, B.; Xu, P., pH and Redox Dual Responsive Nanoparticle for Nuclear Targeted Drug Delivery. Mol. Pharmaceutics 2012, 9, 2719-2729. 33. Page, S. M.; Henchey, E.; Chen, X. J.; Schneider, S.; Emrick, T., Efficacy of PolyMPCDOX Prodrugs in 4T1 Tumor-Bearing Mice. Mol. Pharmaceutics 2014, 11, 1715-1720.


Page 34 of 35

Page 35 of 35 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


Graphic for Table of Contents


Gold Nanorods Stabilized by Biocompatible and Multifunctional

Recommend Documents