Biologically Inspired Polydopamine Capped Gold Nanorods for Drug

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Biologically Inspired Polydopamine Capped Gold Nanorods for Drug Delivery and Light-mediated Cancer Therapy Shaowei Wang, Xinyuan Zhao, Shaochuan Wang, Jun Qian, and Sailing He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05907 • Publication Date (Web): 26 Aug 2016 Downloaded from http://pubs.acs.org on August 27, 2016

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ACS Applied Materials & Interfaces

Biologically Inspired Polydopamine Capped Gold Nanorods for Drug Delivery and Light-mediated Cancer Therapy Shaowei Wang1, Xinyuan Zhao2, Shaochuan Wang1, Jun Qian1,*, Sailing He1,*

1

State Key Laboratory of Modern Optical Instrumentations, Centre for Optical and

Electromagnetic Research, Zhejiang University, Hangzhou, Zhejiang, China, 310058; 2

Bioelectromagnetics Laboratory, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China, 310058. *Corresponding authors e-mail addresses: [email protected] & [email protected].

ABSTRACT

Multifunctional drug delivery and combined multimodal therapy strategies are very promising in tumor theranostic applications. In this work, a simple and versatile nanoplatform based on biologically inspired polydopamine capped gold nanorods (GNR-PDA) is developed. Dopamine, a well-known neurotransmitter associated with many neuronal disorders, can undergo self-polymerization on the surface of GNRs to form a stable PDA shell. Its unique molecular adsorption property, as well as its high chemical stability and biocompatibility, facilitate GNR-PDA as an ideal candidate for drug delivery. Methylene blue (MB) and doxorubicin (DOX) are directly adsorbed on GNR-PDA via electrostatic and/or π-π stacking interactions, forming

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GNR-PDA-MB and GNR-PDA-DOX nanocomposites, respectively. The GNR-PDA-MB can generate reactive oxygen species (ROS, from MB) or hyperthermia (from GNR-PDA) with high efficiency under deep-red/NIR laser irradiation, while the GNR-PDA-DOX exhibits light-enhanced drug release under NIR laser irradiation. The combined dual-modal light-mediated therapy, by using GNR-PDA-MB [photodynamic/photothermal therapy (PDT/PTT)] and GNR-PDA-DOX (Chemo/PTT) is carried out and shows remarkable cancer cell killing efficiency in vitro and significant suppression of tumor growth in vivo, which are much more distinct than any single-modal therapy strategy. Our work illustrates that GNR-PDA could be a promising nanoplatform for multifunctional drug delivery and multimodal tumor theranostics in the future. KEYWORDS: polydopamine, gold nanorods, drug delivery, photodynamic, photothermal, chemotherapy, light-mediated therapy

INTRODUCTION

Light-mediated therapy is a promising approach for tumor treatment, as it possesses minimal invasiveness and side effects, as well as improved therapeutic efficacy.1-2 Photodynamic therapy (PDT), photothermal therapy (PTT) and light-enhanced chemotherapy are three typical light-mediated therapy methods. PDT employs a photosensitizer to transfer photon energy to neighboring oxygen molecules, producing reactive oxygen species (ROS, mainly singlet oxygen, 1

O2) to kill cancer cells.3 PTT usually utilizes photothermal agents, which have strong absorption


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in the near infrared (NIR) spectral region, to convert light energy into hyperthermia and then lead to the ablation of adjacent cancer cells.4 Light-enhanced chemotherapy relies on the light-induced release of chemotherapeutic drugs from the delivery vehicles.5-6 In recent years, the development of synergistic therapy modalities (e.g., combination of PDT with other therapeutic strategies), has attracted considerable attentions, due to its enhanced therapeutic performance over any single therapy modality.6-13 Advances in nanoscience and biotechnology have provided opportunities for developing multimodal nanoplatforms to realize synergistic light-mediated therapy. Nanoplatforms can facilitate the delivery of drugs (e.g., photosensitizer or chemotherapeutic drugs) and provide imaging contrast and photothermal capabilities simultaneously.2,14-15 In addition, the introduction of nanoplatforms can overcome many restrictions that exist in conventional treatment methods, such as poor water-solubility of therapeutic drugs, low accumulation of drugs in therapeutic sites (for tumors), light induced destruction of drug molecules and other side effects.16-20 So far, many types of nanoparticles have been developed as novel nanoplatforms for multimodal light-mediated therapy, including upconversion nanoparticles,21 Fe3O4 nanoparticles,22 carbon nanotubes,23 graphene oxide,24 and gold nanoparticles.6,9 Although therapeutic performances have been improved by these nanoplatforms, some of them had complicated and time-consuming synthesis procedures. Thus, developing simple and versatile nanoplatforms, which can serve as drug delivery vehicles, photothermal agents, and imaging agents, would be very helpful to synergistic light-mediated therapy.



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Gold nanorods (GNRs) are one of the more popular nanomaterials owing to their excellent physicochemical properties, including ease of preparation and surface functionalization, low toxicity and good biocompatibility, as well as their rich optical properties.25-27 They have been extensively investigated and utilized as drug delivery nanoplatforms for multimodal tumor treatment, such as chemotherapy,10 photodynamic therapy,28-29 and gene delivery/therapy.30-31 In addition, GNRs have a strong surface plasmon resonance (SPR) enhanced absorption band in the NIR spectral region, which can be easily tuned by adjusting its size and aspect ratio (length to diameter).27 Therefore, heperthermia can be generated by GNRs upon NIR light irradiation and utilized for PTT of cancer cells.32-33 The SPR-induced tunable absorption and scattering features also facilitate GNRs as contrast agents for in vitro and in vivo bioimaging, including dark-field scattering imaging,33 multiphoton luminescence imaging,34 optical coherence tomography (OCT),35 and photoacoustic tomography (PAT).36 Thus, GNRs hold great potential as a multifunctional nanoplatform, which can realize imaging-guided drug delivery and cancer therapy in one single system. Dopamine is a type of small molecule inspired from adhesive proteins of mussel, and it can undergo self-polymerization onto many solid substrates at alkaline pH conditions without pretreatment, forming robust adherent polydopamine (PDA) films.37-38 The surfaces of PDA films contain numerous amino and catechol groups, allowing for further conjugation with various bio-molecules.39-40 Recently, nanoparticles with PDA coating have attracted considerable interest in biological applications, including drug delivery, biosensing, bioimaging, and targeted


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photothermal therapy, due to their ease of formation, stable chemical properties and special molecular adsorption capability.41 Zeng et al. reported a lipid-gold nanoparticles@PDA nanohybrid as a multifunctional theranostic agent for targeted magnetic resonance and computed tomography (MRI/CT) dual-modal imaging and PTT.42 Cui et al. developed a pH-sensitive PDA capsule loaded with doxorubicin for drug delivery and achieved enhanced efficacy in eradicating cancer cells.43 In addition, PDA nanospheres have been used as therapeutic agents for in vivo PTT of tumors, as they possess strong absorption in the NIR spectral region as well as excellent photothermal conversion efficiency. Very recently, Zhang et al. designed a nanoplatform for PDT & PTT dual-modal therapy, where PDA nanospheres served as drug carriers and photothermal agents, and the anti-tumor efficiency was significantly enhanced via the combination therapy.44 Park et al. functionalized the surface of PLGA NPs with folate, cRGD molecules and a stealth polymer by using the dopamine polymerization method without pre-modification of PLGA.45 Lin et al. synthesized Fe3O4@PDA core-shell nanocomposites for intracellular mRNA detection and MRI/photoacoustic dual-modal imaging-guided PTT.46 Ju et al. developed a dual-mode nanoprobe by using PDA-capped and bio-molecule-conjugated hollow gold nanoparticles for MRI and Raman imaging.47 PDA-capped gold nanoparticles have also been utilized for long-term intracellular mRNA detection in differentiating stem cells.48 Moreover, PDA has been reported to be very biocompatible and stable in in vitro and biomedical applications.49-50 Although PDA capped GNRs have been reported for specific cell targeting and PTT,51 their applications in multifunctional drug delivery and combined multimodal therapy in vitro and in



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vivo have not been investigated until now. In this study, we developed a simple and versatile nanoplatform based on PDA-capped GNRs (GNR-PDA) for drug delivery and combined light-mediated cancer therapy, as shown in Scheme 1. PEG molecules were grafted on GNRs before PDA capping, which may improve the stability of nanoparticles and avoid aggregation during the PDA capping process.34 Dopamine molecules then self-polymerized onto the surface of the PEGylated GNRs under alkaline conditions to form a stable PDA shell, whose thickness could be easily tuned by adjusting the concentration of the dopamine solution.47,50 Two kinds of drugs, methylene blue (MB, a type of photosensitizer for PDT) and doxorubicin (DOX, a type of drug for chemotherapy), were directly adsorbed on GNR-PDA via electrostatic and/or π-π stacking interactions,45-46,52 forming GNR-PDA-MB and GNR-PDA-DOX nanocomposites with high drug loading efficiency, respectively. Upon 671 nm-laser irradiation, GNR-PDA-MB nanocomposites exhibited high ROS generation efficiency in aqueous dispersion, as well as in cells. When excited by an 808 nm-laser, enhanced drug release was induced from the GNR-PDA-DOX nanocomposites. Efficient cellular internalization of the GNR-PDA nanocomposites was determined by fluorescence and two-photon luminescence imaging, which also confirmed the excellent drug delivery capability of GNR-PDA nanocomposites. Remarkable killing efficiency of cancer cells in vitro and significant suppression of tumor growth in vivo, which were induced by the combined dual-modal therapy (PDT & PTT based on GNR-PDA-MB nanocomposites and Chemo & PTT based on GNR-PDA-DOX nanocomposites), were demonstrated. On the basis of these in vitro and in vivo studies, GNR-PDA nanocomplexes could


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be a promising nanoplatform for multifunctional drug delivery and multimodal tumor theranostics in the future.

Scheme 1. Illustration for the preparation of GNR-PDA and its applications for multifunctional drug delivery and light-mediated therapy. MB and DOX were adsorbed on GNR-PDA to form GNR-PDA-MB and GNR-PDA-DOX nanocomposites, respectively. The dual-modal PDT & PTT and chemotherapy & PTT were demonstrated in vitro and in vivo. RESULTS AND DISCUSSIONS

Synthesis and Characterizations of GNR-PDA CTAB-coated GNRs were prepared using a seed-mediated method.53 As shown in Figure 1a, the longitudinal localized surface plasmon resonance (LSPR) peak of the GNRs was tuned around 650 nm by adjusting the added amount of AgNO3 and L-ascorbic acid. Transmission electron



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microscopy (TEM) images showed that the average length and width of the GNRs were ~ 50 nm and ~ 20 nm, with an aspect ratio of ~ 2.5 (Figure 1c). The average hydrodynamic diameter of the GNRs was estimated to be 27.32 nm (Figure 1g), which was measured with a dynamic light scattering (DLS) method. It has been reported that CTAB-coated GNRs could be directly used for polydopamine capping.51 However, CTAB-coated GNRs are unstable and prone to forming large aggregates in alkaline conditions, which is essential for dopamine self-polymerization on the metal surface.26 In addition, CTAB-coated GNRs are toxic due to the presence of CTAB molecules, and are not suitable for direct biological applications.54 In our case, we first replaced the CTAB layer on the surfaces of the GNRs with polyethylene glycol (SH-PEG-CH3) molecules through strong Au-S binding. The PEGylated GNRs (GNR-PEG) were biocompatible and very stable in various acidic and alkaline conditions, as demonstrated in our previous report.34 The absorption spectrum of PEGylated GNRs was very similar with that of CTAB-coated GNRs, except for a tiny blue-shift of the longitudinal LSPR peak (Figure 1a). This is due to the variation in the refractive index surrounding the surface of the GNRs, and illustrates a successful PEGylation of GNRs without aggregation. PDA was formed on the surface of the GNRs through the self-polymerization of dopamine, and the core-shell GNR-PDA nanocomplex was obtained. The thickness of the PDA shell could be tuned by adjusting the concentration of the dopamine solution prior to the self-polymerization reaction. GNR-PDA with shell thicknesses of 15 nm, 20 nm and 30 nm could be obtained (Figure 1d, 1e and 1f), when the concentrations of dopamine solution were 0.1, 0.5 and 1 mg/mL (referred to as GNR-PDA0.1, GNR-PDA0.5 and


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GNR-PDA1), respectively. Figure 1b showed the absorption spectra of GNR-PDA nanocomplexes with various thicknesses, as well as PDA solutions obtained from the same concentrations of dopamine that were used for the formation of GNR-PDA. All three kinds of GNR-PDA nanocomplexes showed two characteristic peaks, which corresponded to the transverse and longitudinal LSPR of the GNRs. After PDA capping, the longitudinal LSPR peak of the GNRs was red-shifted due to the larger refractive index of PDA (surrounding GNRs). With the increasing thickness of the PDA shell, the wavelength red-shift could reach ~ 100 nm (for GNR-PDA1, Figure 1b). In addition, GNR-PDA showed higher absorbance compared with GNR-CTAB and GNR-PEG, which increased as the PDA shell became thicker. We attributed this to the additive absorbance of the PDA shell, since PDA solutions (without GNR) showed obvious absorption in the visible region (Figure 1b).44,46 Furthermore, the absorption spectra of GNR-PEG broadened after PDA capping, and became more obvious as the PDA shell got thicker. This may be due to easier aggregation of larger sized GNR-PDA nanocomplexes, or the fact that two or more GNRs were capped together by the thicker PDA shell. Zeta potential measurement was performed to check the variation in surface charge of various GNR samples (Figure S1, Supporting Information). GNR-PEG showed a nearly neutral charge (4.2 mV) compared to the positively charged GNR-CTAB (36.1 mV), which illustrated a successful displacement of CTAB molecules by the PEG polymer. The Zeta potential of GNR-PDA nanocomplexes decreased to a negative value (-18.7 mV), which was ascribed to the catechol and -OH groups on the PDA shells.40,55 The negatively charged GNR-PDA could be used to adsorb drug molecules with



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positive charges, and holds great potential in drug delivery applications.

Figure 1. Characterizations of polydopamine capped GNRs (GNR-DPA). (a) Absorption spectra of CTAB-coated GNRs (GNR-CTAB) and PEGylated GNRs (GNR-PEG). (b) Absorption spectra of GNR-PDA with shell thicknesses of 15 nm, 20 nm and 30 nm (GNR-PDA0.1, GNR-PDA0.5 and GNR-PDA1), and PDA solutions with dopamine concentrations of 0.1, 0.5 and 1 mg/mL (PDA0.1, PDA0.5 and PDA1). (c-f) Representative TEM images of GNR-CTAB (c), GNR-PDA0.1 (d), GNR-PDA0.5 (e) and GNR-PDA1 (f). (g) Hydrodynamic size distributions of GNR-CTAB and GNR-PDA1. (h) Digital photographs of GNR-PDA and


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GNR-PDA-PEG in three different dispersions [from left to right: water, PBS and DMEM (+10% FBS)] at 0 h and 24 h. Scale bars, 100 nm. Characterizations of GNR-PDA as Drug Nanocarriers In some previous reports, PDA spheres and PDA capped materials have been demonstrated to be efficient to adsorb organic dyes.56-58 Both MB and DOX are cationic drugs and can be adsorbed by the negatively charged GNR-PDA through electrostatic attraction and/or π-π stacking interactions. In this work, we used GNR-PDA nanocomplexes (GNR-PDA1) with shell thickness of 30 nm as the drug nanocarriers, and the thicker PDA shell is considered to be more efficient for drug adsorption and delivery. The average hydrodynamic diameter of the GNR-PDA1 was estimated to be 94.00 nm (Figure 1g). PEG molecules were grafted on GNR-PDA nanocomplexes prior to the adsorption of drug, in order to improve the stability of nanocarriers. Compared to the GNR-PDA, the PEGylated GNR-PDA (GNR-PDA-PEG) showed much better chemical stability in PBS and cell culture medium (DMEM with 10% FBS), as shown in Figure 1h. GNR-PDA-PEG samples (0.1 nM) were mixed with 5, 10, 20, 40 or 80 µM MB for adsorption. The obtained GNR-PDA-MB nanocomposites were collected by centrifugation. The supernatant containing un-adsorbed MB molecules was also collected in order to calculate the amount of MB adsorbed by the GNR-PDA. For MB with various concentrations, only 80 µM MB is excessive for 0.1 nM GNR-PDA-PEG, and the maximum adsorbed concentration of MB was 50 µM. As shown in Figure 2a, the absorption spectra of GNR-PDA-MB nanocomposites had four peaks located at around 520, 610, 670 and 750 nm. The two peaks at 520 and 750 nm were the LSPR



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peaks of GNR-PDA-PEG, while the peaks at 610 and 670 nm could be assigned to the dimer and monomer forms of the MB molecules (Figure S2, Supporting Information),52 respectively. With the increase in the concentration of MB, the peak intensities at 610 and 670 nm got higher, which indicated that more MB molecules have been adsorbed on the GNR-PDA-PEG nanocomplexes. On average, 5×105 MB molecules were adsorbed on a single GNR-PDA-PEG nanocomplex, according to the quantification that 50 µM MB interacted with 0.1 nM GNR-PDA-PEG and the MB loading efficiency was estimated to be around 42.1% (w/w). After the adsorption of the MB molecules, the color of the aqueous dispersion of GNR-PDA-PEG changed from light brown to blue green (inset digital images in Figure 2a).


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Figure 2. Characterizations of GNR-PDA-MB nanocomposites. (a) Absorption spectra of GNR-PDA-MB with different adsorbed concentrations of MB and digital images of the aqueous dispersions of GNR-PDA-PEG and GNR-PDA-MB (lower left inset). (b) Fluorescence spectra and images (upper right inset) of aqueous dispersion of GNR-PDA-MB nanocomposites and free MB solution (with equal concentration of MB, 50 µM) in the centrifuge tubes. Excitation wavelength, 635 nm. (c) Absorption spectra of ABDA after photo-degradation by ROS, which was generated by GNR-PDA-MB upon the irradiation of 671 nm-laser (30 mW/cm2). (d) Temperature variation curves of DI water, free MB (50 µM) and aqueous dispersions of GNR-PDA-MB nanocomposites with different concentrations, upon the irradiation of NIR laser (808 nm, 2 W/cm2). GNR-PDA-PEG samples (0.1 nM) were each mixed with either 5, 10, 20 or 30 µg/mL DOX for adsorption, and the maximum concentration of adsorbed DOX on GNR-PDA was 20 µg/mL. On average, 3.4×104 DOX molecules were adsorbed on a single GNR-PDA-PEG nanocomplex, according to the quantification that 20 µg/mL DOX interacted with 0.1 nM GNR-PDA-PEG and the DOX loading efficiency was estimated to be around 52.6% (w/w). When GNR-PDA-PEG was mixed with the DOX solution, three absorption peaks located at around 480, 520 and 750 nm could be observed (blue curve in Figure 3a). The peak at 480 nm was from DOX (pink curve in Figure 3a). After the interaction of DOX with GNR-PDA-PEG, the obtained GNR-PDA-DOX was collected by centrifugation and the absorption spectra were recorded. However, no obvious absorption peak around 480 nm could be found from GNR-PDA-DOX nanocomposites (Figure 3a, and Figure S3, Supporting Information). This may be ascribed to the high absorption intensity of the PDA shell on the GNR surface. As shown in the digital images (inset of Figure 3a), the aqueous dispersion of GNR-PDA-DOX was light red, indicating that DOX molecules were adsorbed on the GNR-PDA-PEG nanocomplex (light brown). The high drug loading



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efficiency of GNR-PDA-PEG could be mainly attributed to the excellent adhesive capacity of PDA and is higher than that of other reported GNR based drug delivery vehicles (7.9% to 25.2%).5,10,59

Figure 3. Characterizations of GNR-PDA-DOX nanocomposites. (a) Absorption spectra of GNR-PDA-PEG, GNR-PDA-DOX, GNR-PDA-PEG mixed with free DOX (GNR-PDA-PEG + DOX) and free DOX. Inset in lower left of (a) shows digital images of the aqueous dispersion of GNR-PDA-PEG and GNR-PDA-DOX. (b) Fluorescence spectra and images (upper right inset) of aqueous dispersion of GNR-PDA-DOX nanocomposites and free DOX solution (with equal concentration of DOX, 20 µg/mL) in the centrifuge tubes. Excitation wavelength, 532 nm. (c) DOX release profiles (as a function of time) from GNR-PDA-DOX nanocomposites in pH 5.0 and 7.4 solutions with and without laser irradiation (808 nm, 2 W/cm2), calculated according to the absorption intensity of DOX at 490 nm. (d) Temperature variation curves of DI water, free


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DOX (20 µg/mL) and aqueous dispersions of GNR-PDA-DOX nanocomposites with different concentrations, upon the irradiation of NIR light (808 nm, 2 W/cm2). The successful adsorption of MB and DOX on the GNR-PDA-PEG nanocomplexes was also confirmed by fluorescence imaging. After MB adsorption, the fluorescence of the GNR-PDA-MB nanocomposites was much weaker than that of free MB with the same concentration in water (Figure 2b), which was attributed to the fluorescence quenching effect of GNRs.27 The fluorescence of DOX in GNR-PDA-DOX was also quenched after adsorption (Figure 3b). Furthermore,

Zeta

potentials

of GNR-PDA-MB

and GNR-PDA-DOX

nanocomposites increased to -16.2 mV and 8.6 mV respectively, compared with -18.7 mV of GNR-PDA (Figure S1, Supporting Information). All of these results illustrated successful adsorption of MB and DOX molecules on GNR-PDA-PEG nanocomplexes. ROS Generation of GNR-PDA-MB To investigate the generation of ROS from GNR-PDA-MB nanocomposites, 9,10anthracenediyl-bis(methylene)dimalonic acid (ABDA) was used as an indicator. The intensities of ABDA absorption peaks should decrease when it reacts with ROS.44 As shown in Figure 2c, aqueous dispersion of GNR-PDA-MB (0.1 nM, equivalent concentration of MB, 50 µM) containing ABDA was irradiated by a 671 nm-laser (30 mW/cm2), and a sharp decline in the ABDA absorption peaks was observed, indicating that the 671 nm-laser induced rapid ROS generation of GNR-PDA-MB nanocomposites. Similarly to GNR-PDA-MB, free MB exhibited obvious ROS generation upon the 671 nm-laser irradiation (Figure S4 and S5, Supporting Information). However, GNR-PDA-PEG alone (without MB) generated negligible ROS (Figure



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S4 and Figure S6, Supporting Information). These results confirmed that GNR-PDA-MB nanocomposites possessed high ROS generation efficiency under laser irradiation, and that ROS was generated from MB molecules. DOX Release from GNR-PDA-DOX In chemotherapy processes, drug delivery is of great importance. GNR-PDA-DOX could deliver DOX into tumor cells as a kind of drug nanocarrier. On the other hand, the release efficacy of DOX from GNR-PDA-DOX is also significant. We examined the DOX release profile (as a function of time) from GNR-PDA-DOX (0.1 nM, equivalent concentration of DOX, 20 µg/mL) in pH 7.4 and pH 5.0 solutions, which were treated both without laser irradiation and with 808 nm-laser irradiation. After centrifugation at fixed time intervals, the amount of released DOX was quantified by measuring its absorption spectra in various GNR-PDA-DOX samples, and 20 µg/mL free DOX was used as the control. As shown in Figure 3c, the amount of DOX released from the GNR-PDA-DOX depended on the pH value of the solution and the treatment of laser irradiation, as well as the releasing time. 48 h after laser irradiation, the release efficacy of DOX from GNR-PDA-DOX reached 88% in the pH 5.0 solution and 52% in the pH 7.4 solution, respectively. In the absence of 808 nm-laser irradiation, the release efficiencies of DOX from the GNR-PDA-DOX were 60% in the pH 5.0 solution and 34% in the pH 7.4 solution after 48 h. The enhanced DOX release under 808 nm-laser irradiation could be attributed to the photothermal effect of the GNR-PDA-DOX nanocomposites, which would make the PDA shell defective and accelerate the DOX effuse out from the GNR-PDA-DOX nanocomposites. The


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experimental results demonstrated that low pH and NIR laser irradiation could effectively enhance the DOX release from the GNR-PDA-DOX nanocomposites, which was in accordance with previously published reports.5,10,60 In Vitro Photothermal Effects The photothermal effect of GNRs has been extensively investigated and utilized for in vitro and in vivo photothermal therapy, as GNRs have strong tunable absorption in the NIR spectral region.26,33 In addition, PDA nanospheres have also been used as photothermal agents, since they have obvious absorption in the visible spectral region.44,61 Liu et al.61 reported that the photothermal conversion efficiency (η) of PDA was estimated to be around 40%, while GNRs have a lower η value of 22%, indicating that PDA is highly superior for PTT. The GNR-PDA that combines the advantages of both GNR and PDA would be very promising for PTT. On one hand, the easy tunability of the LSPR absorption band of GNR will be very helpful to increase the absorption of the nanocomposites in NIR spectral region, where light can penetrate deep in biological tissues. Furthermore, the high photothermal conversion efficiency of PDA could further enhance the PTT effects of the nanocomposites. On the other hand, the PDA layer capped on the surface of GNR could protect the GNR from morphological change and absorption decrease under long time laser irradiation, while providing high PTT efficiency. As shown in Figure S7a in Supporting Information, no absorption decrease of the aqueous dispersion of GNR-PDA-PEG was observed after 1 h laser irradiation (808 nm, 2 W/cm2). In addition, as the TEM image shown in Figure S7b, no obvious morphological changes could be observed from



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GNR-PDA-PEG which was subjected to 1 h laser irradiation, indicating high photostability of the nanocomposites. The evaluations of the photothermal effects from GNR-PDA-MB and GNR-PDA-DOX nanocomposites at different concentrations (0.1, 0.2, 0.4 and 0.6 nM) were carried out by monitoring the temperature variation under 808 nm-laser irradiation (2 W/cm2). 50 µM free MB, 20 µg/mL free DOX and DI water were used as the control groups. As shown in Figure 2e and 3d, the temperatures of the aqueous dispersions of GNR-PDA-MB and GNR-PDA-DOX nanocomposites both increased rapidly under the laser irradiation, and showed a dose-dependent relationship. At the concentration of 0.6 nM, the temperature increased up to over 70°C after 120 s laser irradiation, which would be sufficient to kill cancer cells. For the control groups, the temperatures of the free drug (MB and DOX) solutions and water showed slight changes and slower increase, reaching saturation at less than 35°C after 240 s laser irradiation. This demonstrated that the photothermal effects of GNR-PDA-MB and GNR-PDA-DOX mainly come from GNR-PDA-PEG (Figure S8, Supporting Information). These results illustrated that GNR-PDA-MB and GNR-PDA-DOX nanocomposites exhibited excellent photothermal effects under NIR laser irradiation. Cellular Uptake and Imaging Efficient cellular uptake of nanocarriers is significant to ensure drug delivery and therapeutic efficacy. After incubation with HeLa cells, both GNR-PDA-MB and GNR-PDA-DOX nanocomposites showed time-dependent cellular internalization efficiency, according to the flow cytometry analysis (Figure S9 and S10, Supporting Information). Considering that GNRs possess


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strong plasmon-enhanced two-photon luminescence (2PL) under femtosecond (fs) laser excitation and have been used for in vitro and in vivo cell/tissue imaging, 2PL imaging was performed to evaluate the cellular uptake of both GNR-PDA-MB and GNR-PDA-DOX nanocomposites.34,62-63 In addition, confocal microscopy was carried out to determine the distribution of MB/DOX inside cells. As shown in Figure 4, (one-photon) fluorescence signals (from MB, indicated red) and 2PL signals (from GNRs, indicated yellow) inside HeLa cells could be visualized after cells were incubated with 5 pM GNR-PDA-MB nanocomposites, illustrating that GNR-PDA-MB nanocomposites could be effectively internalized into HeLa cells. In the MB fluorescence channel (red), some bright dots could also be clearly seen (Figure 4b and Figure S11, Supporting Information), which may be some aggregates of GNR-PDA-MB nanocomposites. For cells treated with GNR-PDA-PEG, only the 2PL signal could be observed while no obvious fluorescence signal could be detected. Furthermore, fluorescence signals from cells treated with free MB were relatively weak, and no 2PL signal could be detected, indicating that it was inefficient for MB molecules to be internalized into cells without the delivery of nanocomposites.



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Figure 4. Transmission and fluorescence images of HeLa cells, treated with GNR-PDA-MB nanocomposites (a-d), GNR-PDA-PEG (e-h) and free MB (i-l). Scale bars (if not indicated), 50 µm. (m-p) Fluorescence images of HeLa cells incubated with GNR-PDA-MB nanocomposites and treated both with (m, n) and without (o, p) 671 nm-laser irradiation. Excitation wavelength, 488 nm. Similarly, (one-photon) fluorescence signals (from DOX, indicated red) and 2PL signals (from GNRs, indicated yellow) inside HeLa cells illustrated effective internalization of nanocarriers


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into cells (Figure 5a-d). In addition, the two signals were distributed in the cytoplasm of cells, which may be attributed that the GNR-PDA-DOX nanocomposites taken up by the HeLa cells were delivered to cytoplasm through endocytosis, and the DOX molecules were then released from the nanocomposites as the acidic environment in the cytoplasm could accelerate this process (Figure 5b).5 In contrast, most DOX molecules were found to be localized in the nuclei of the cells treated with free DOX (Figure 5f), with the same incubation time as GNR-PDA-DOX treated cells. This could be ascribed to the rapid passive diffusion of small DOX molecules into the nucleus after their internalization into the cells.64 These results demonstrate that both GNR-PDA-MB and GNR-PDA-DOX nanocomposites could effectively deliver drugs into cells and would be promising in the subsequent therapy applications.

Figure 5. Transmission and fluorescence images of HeLa cells treated with GNR-PDA-DOX nanocomposites (a-d) and free DOX (e-h). Scale bars, 50 µm.



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Intracellular ROS Generation of GNR-PDA-MB We have demonstrated that GNR-PDA-MB nanocomposites could generate ROS efficiently under 671 nm-laser irradiation in an aqueous dispersion (Figure 2c and 2d). To investigate the ROS generation of GNR-PDA-MB nanocomposites in HeLa cells, an indicator called 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was adopted. DCFH-DA could react with ROS generated in cells and form 2′,7′-dichlorofluorescein (DCF), which exhibits strong green fluorescence under 488 nm-laser excitation.65 HeLa cells were incubated with GNR-PDA-MB nanocomposites for 24 h, followed by 671 nm-laser irradiation for 3 min. The original culture media was then replaced with fresh culture media containing DCFH-DA, and the cells were incubated for another 30 min. As shown in Figure 4m, strong green fluorescence could be observed in cells treated with GNR-PDA-MB under 488 nm-laser excitation, indicating that intracellular ROS has been effectively produced under 671 nm-laser irradiation. As for the control experiments, (i) cells treated with free MB under 671 nm-laser irradiation showed relatively weak green fluorescence; (ii) no obvious signals could be detected in the cells treated with GNR-PDA-PEG under 671 nm-laser irradiation; and (iii) in the absence of 671 nm-laser irradiation, cells treated with GNR-PDA-MB, free MB and GNR-PDA-PEG all showed no obvious green fluorescence signals (Figure 4o-p and Figure S12, Supporting Information). It is worth mentioning that the green fluorescence in cells treated with GNR-PDA-MB was stronger than that in cells treated with free MB (equivalent concentration of MB) under 671 nm-laser irradiation, which indicated that the former produced more ROS. This may be attributed to the


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high chemical stability and efficient drug delivery capability of GNR-PDA-MB nanocomposites. All the experimental results demonstrated that intracellular ROS could be more effectively generated in the cells treated with GNR-PDA-MB upon 671 nm-laser.

Figure 6. In vitro evaluation of PDT/PTT and PDT & PTT effects based on GNR-PDA-MB



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nanocomposites. (a) Viability of HeLa cells treated with various concentrations of GNR-PDA-MB, GNR-PDA-PEG and free MB, but without laser irradiation. (b) Viability of HeLa cells incubated with various concentrations of GNR-PDA-MB, GNR-PDA-PEG and free MB under PDT treatment (671 nm, 30 mW/cm2, 3 min), PTT treatment (808 nm, 2 W/cm2, 5 min), and combined PDT & PTT treatment. (c) Viability of HeLa cells incubated with 2 pM GNR-PDA-MB (equivalent concentration of MB, 1 µM) under PDT treatment (671 nm, 30 mW/cm2) with varying laser irradiation time. (d) Viability of HeLa cells incubated with 5 pM GNR-PDA-MB (equivalent concentration of MB, 2.5 µM) under PTT treatment (808 nm, 2 W/cm2) with varying laser irradiation time. (e) Bright field images of HeLa cells incubated with 2 pM GNR-PDA-MB (equivalent concentration of MB, 1 µM), under either no treatment, PDT treatment, PTT treatment, or combined PDT & PTT treatment. (f) Apoptosis of HeLa cells incubated with 2 pM GNR-PDA-MB nanocomposites (equivalent concentration of MB, 1 µM), under either no treatment, PDT treatment, PTT treatment, or combined PDT & PTT treatment. Scale bars, 100 µm. **p < 0.01. In Vitro Cytotoxicity and PDT & PTT of GNR-PDA-MB The high ROS generation efficacy and stable photothermal effects of GNR-PDA-MB nanocomposites encourage us to further study their capability as a PDT & PTT dual-modal agent for light-mediated cancer therapy. Herein, HeLa cells were selected as the cell model to evaluate the cytotoxicity of GNR-PDA-MB nanocomposites. As shown in Figure 6a, both GNR-PDA-MB and GNR-PDA-PEG nanocomposites showed negligible cytotoxicity on HeLa cells in the absence of laser irradiation. The viability of the cells remained over 90% after 48 h incubation even with a high concentration of nanocomposites (30 pM, equivalent concentration of MB in GNR-PDA-MB, 15 µM). However, cells treated with free MB showed a dose-dependent cytotoxicity, and the cell viability decreased to 65% for the 15 µM concentration, which likely arose from the dark toxicity of the free MB molecules. The results proved that GNR-PDA-MB


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nanocomposites were very biocompatible and could also reduce the dark toxicity of MB molecules. Furthermore, we studied the combined PDT and PTT effects on HeLa cells in vitro, under the irradiation of lasers at 671 nm and 808 nm. HeLa cells were placed in a 48-well plate and incubated with various concentrations of GNR-PDA-MB for 24 h, and then subjected to either 671 nm-laser irradiation (30 mW/cm2, 3 min, for PDT treatment), 808 nm-laser irradiation (2 W/cm2, 5 min, for PTT treatment), or 671 & 808 nm-laser irradiation (for PDT & PTT), respectively. For the control experiments, HeLa cells incubated with the same concentrations of GNR-PDA-PEG were subjected to PTT treatment, and cells incubated with the equivalent concentration of free MB were subjected to PDT treatment. The untreated cells were set as another control group. After the laser treatment, the cells were incubated for 24 h prior to the CCK-8 assay test. As shown in Figure 6b, GNR-PDA-MB nanocomposites showed a dose-dependent PDT effect under the 671 nm-laser irradiation, and the cell viability decreased to 29.8% at the GNR-PDA-MB concentration of 10 pM (equivalent concentration of MB, 5 µM), which was much lower than that of the cells treated with the same concentration of MB (49%). This result could be ascribed to the enhanced cellular uptake and ROS generation of GNR-PDA-MB nanocomposites, and was in accordance with our previous results in the intracellular ROS generation evaluation. In addition, the PTT effect of GNR-PDA-MB nanocomposites under the 808 nm-laser irradiation was also dose-dependent. Cell viability decreased to 47.7% for the 10 pM concentration of GNR-PDA-MB, which was a little lower than



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that of the cells treated with the same concentration of GNR-PDA-PEG (55.7%). Furthermore, the viability of cells treated with GNR-PDA-MB dramatically decreased after the combined PDT & PTT treatment. For the 10 pM concentration of GNR-PDA-MB, cell viability declined to 16.1%, illustrating excellent PDT & PTT treatment effects of the GNR-PDA-MB nanocomposites. We also investigated the PDT and PTT effects of GNR-PDA-MB under laser irradiation with a different time. As shown in Figure 6c and 6d, both the PDT and PTT effects were irradiation time-dependent, and cell viability continued decreasing with the increase in laser irradiation time. Meanwhile, bright field images of the cells after laser-treatments were taken to directly show the PDT and PTT effects of GNR-PDA-MB nanocomposites (Figure 6e). Obvious cell deformation, apoptosis and death could be visualized in the laser-irradiated groups. More significantly, the surviving cells dramatically decreased upon the combined PDT & PTT treatment. These results demonstrated that the GNR-PDA-MB nanocomposites were biocompatible and could reduce the dark toxicity of MB molecules. In addition, they showed excellent PDT and PTT effects. Cell Apoptosis Induced by PDT & PTT Effect of GNR-PDA-MB To further evaluate the cytotoxicity and cell death induced by the PDT & PTT treatment of GNR-PDA-MB nanocomposites, the Annexin V-FITC and PI dyes staining method was used to determine the cell apoptosis. HeLa cells were incubated with GNR-PDA-MB (2 pM, equivalent concentration of MB, 1 µM) for 24 h, and then subjected to PDT, PTT and combined PDT & PTT treatments. 24 h after laser irradiation, each group was stained with Annexin V-FITC/PI


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dyes, and the cell apoptosis was determined by flow cytometry. As shown in Figure 6f, the cells were divided into four subgroups, which were the viable group, the early apoptotic group, the late apoptotic group and the dead cells group, corresponding to the lower left (B3), lower right (B4), upper right (B2), and upper left quadrants (B1). In the control group, most of the cells (95.57%) remained viable and were localized in the B3 quadrant. However, the percentages of the viable cells (B3) dramatically decreased in the GNR-PDA-MB incubated groups with PDT, PTT and PDT & PTT treatments. They were 50.97%, 72.37%, and 46.92% for the PDT, PTT and PDT & PTT treated cells, respectively. The percentages of cells in the late apoptotic stage (B2) increased to 44.91%, 23.84%, 47.67% after the PDT, PTT and PDT & PTT treatments, respectively. These results indicated that cell apoptosis induced by the PDT and PTT effects of GNR-PDA-MB nanocomposites was mainly responsible for cell death.



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Figure 7. In vitro evaluation of chemotherapy/PTT and chemotherapy & PTT effects based on GNR-PDA-DOX nanocomposites. (a) Viability of HeLa cells incubated with various concentrations of free DOX, GNR-PDA-DOX without laser-irradiation, GNR-PDA-PEG with PTT treatment (808 nm, 2 W/cm2, 5 min), and GNR-PDA-DOX with PTT treatment (808 nm, 2 W/cm2, 5 min). (b) Viability of HeLa cells incubated with 2.5 pM GNR-PDA-DOX nanocomposites (equivalent concentration of DOX, 0.5 µg/mL) without laser-irradiation and under PTT treatment (808 nm, 2 W/cm2) with varying laser irradiation time. (c) Bright field images of HeLa cells incubated with free DOX (1 µg/mL), 5 pM GNR-PDA-DOX (equivalent concentration of DOX, 1 µg/mL) without laser-irradiation, and 5 pM GNR-PDA-DOX with PTT treatment (808 nm, 2 W/cm2, 5 min). Scale bars, 100 µm. In Vitro Cytotoxicity and Chemotherapy/PTT of GNR-PDA-DOX The cytotoxicity of GNR-PDA-DOX nanocomposites with and without laser irradiation was investigated and compared with that of free DOX. As shown in Figure 7a, the viability of all the treated cells showed an obvious decrease and was dose-dependent. The viability of GNR-PDA-DOX treated cells (without laser-irradiation) was a little higher than that of free DOX. This may be attributed to the rapid passive diffusion of small DOX molecules into the nuclei of free DOX treated cells, while some of the DOX molecules released from GNR-PDA-DOX were still distributed in the cytoplasm at the same incubation time. After the PTT treatment (808 nm-laser irradiation for 5 min, 2 W/cm2), GNR-PDA-DOX incubated cells showed a more dramatic decrease in viability, compared to GNR-PDA-PEG with laser irradiation and GNR-PDA-DOX incubated cells without laser irradiation and free DOX incubated cells, illustrating the excellent combined therapeutic effect of chemotherapy & PTT under NIR laser irradiation. According to the DOX release effect in aqueous dispersion of GNR-PDA-DOX, NIR laser irradiation could accelerate the release of DOX from GNR-PDA-DOX nanocomposites and


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further enhance its chemotherapeutic capability. In addition, we also investigated the combined therapeutic effect of chemotherapy & PTT under laser-irradiation for various time. As shown in Figure 7b, the combined therapeutic effect was time-dependent, and the cell viability continued decreasing with the increase in laser-irradiation time, indicating that long-lasting laser-irradiation could enhance the release of DOX drugs from GNR-PDA-DOX, as well as the photothermal effect of GNR-PDA-PEG. Furthermore, bright field images of the cells after laser treatments were taken to directly show the chemotherapy and PTT effect of GNR-PDA-DOX nanocomposites. As shown in Figure 7c, after incubation with free DOX and GNR-PDA-DOX, the cell number saw a dramatic decrease compared with the control group (cells without any treatment), which could be attributed to the chemotherapy effect of the DOX drug. Meanwhile, obvious cell deformation and death could also be visualized. For GNR-PDA-DOX treated cells with NIR laser irradiation, cell deformation became more severe, and the surviving cells dramatically decreased. These results demonstrated that NIR laser irradiation could also trigger the release of DOX and enhance the therapeutic efficacy of the GNR-PDA-DOX nanocomposites inside cells. In Vivo Toxicity of the Nanocomposites Before the in vivo studies, histological examination of major organs was used to evaluate the in vivo acute and long-term toxicity of the nanocomposites (GNR-PDA-MB and GNR-PDA-DOX). Major organs including liver, spleen, kidney, lung and heart were excised from mice euthanized at 24 h, 72 h, and 30 days after the administration of 200 μL of GNR-PDA-MB or



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GNR-PDA-DOX ( 1 nM in 1 × PBS) through the tail vein. The control mice were treated with 200 μl 1 × PBS. As shown in Figure S13 and S14 in Supporting Information, no obvious inflammation or abnormalities could be found in the major organs after 24 h, 72 h and 30 days post-administration, the nanocomposites had negligible in vivo toxicity. In Vivo Photothermal Effects The in vivo photothermal effects of the GNR-PDA-PEG upon 808 nm-laser irradiation was studied by using a thermal imaging infrared cameras. Typically, 100 μL GNR-PDA-PEG (1 nM) or 100 μL PBS (1 ×) were intratumorally administered to the tumor of mice. 4 h post injection, the tumor regions were subjected to the laser irradiation (2 W/cm2) and the thermal images of the whole mice were recorded at different time points. As shown in Figure 8a and 8b, the temperature of the tumor region injected with GNR-PDA-PEG exhibited a rapid increase and reached 50.1°C within 1 min-irradiation. After 5 min-irradiation, the temperature reached saturation of 54.6°C (ΔT = 18.3°C), which is sufficient to kill cancer cells. For the control mouse injected with PBS, the temperature of the tumor region showed a slow increase and reached 39.1°C within 1 min-irradiation. It reached saturation of 40.7°C (ΔT = 5.6°C) after laser irradiation for 5 min. The results indicated that the GNR-PDA-PEG could generate hyperthermia in vivo to kill cancer cells upon NIR laser irradiation.


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Figure 8. (a) In vivo thermal images of tumor-bearing mice injected with GNR-PDA-PEG and PBS, upon 808 nm-laser irradiation for different periods of time. (b) Temperature variation curves of tumor region as a function of laser-irradiation time. In Vivo Anti-tumor Effects of GNR-PDA-MB In nanoparticle-assisted tumor theranostic applications, the efficiency of drug delivery to tumor site is critical to therapeutic effects and is mainly dependent on the delivery of nanoparticles. As the intratumoral injection is a very effective method to delivery the nanoparticles to tumor and



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could reduce the systematic toxicity and adverse effects caused by the accumulation of nanocarriers in normal tissues,66-68 we investigated the therapeutic efficiency of our nanocomposites by intratumoral injection method. To evaluate the anti-tumor effect of PDT, PTT and combined PDT & PTT of GNR-PDA-MB nanocomposites, nude mice bearing tumors were injected intratumorally with PBS, free MB, and GNR-PDA-MB nanocomposites. 4 h post-injection, mice tumors injected with GNR-PDA-MB received either PDT (671 nm, 30 mW/cm2, 10 min), PTT (808 nm, 2 W/cm2, 10 min), or combined PDT & PTT. Mice tumors injected with free MB received only the PDT treatment. Mice tumors injected with PBS were set as a control group. After laser-irradiation, tumors in each group were monitored by measuring their volumes, and the body weights of the mice were also recorded. As shown in Figure 9a, mice injected with PBS showed a rapid growth of tumor volume. In contrast, mice that received laser treatment (injected with GNR-PDA-MB nanocomposites or free MB) exhibited obvious delays in tumor growth. In addition, enhanced anti-tumor growth effects were observed in the mice treated with GNR-PDA-MB, compared to the mice treated with free MB. In the three groups treated with GNR-PDA-MB, mice that received PTT treatment only showed a better reduction in tumor volumes than those that received PDT treatment only, which could be attributed to most tumor cells being destroyed by the ablation of light converted heat, as scar tissue could be observed in the tumor 14 days after PTT treatment (Figure 9c). For mice that received combined PDT & PTT treatment, a significant inhibition in tumor growth was displayed. Tumor volume showed distinct decreases after PDT & PTT treatment, and only a small piece of scar tissue could


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be seen from the tumor on day 14, indicating strong synergistic therapeutic efficacy of combined PDT & PTT treatment based on GNR-PDA-MB nanocomposites. Furthermore, the hematoxylin and eosin (H&E) staining method was used to evaluate the anti-tumor efficiency of the dual-modal therapy. As shown in Figure 9d, a significant tissue and cellular damage was obseved in the combined PDT & PTT group and other groups that received one-modal treatment showed some degree of damge, which was in accordance with the tumor volume curves (Figure 9a and 9c). During the treatment and monitoring periods, no death and no obvious weight loss were observed from mice injected with free MB and GNR-PDA-MB, illustrating that they did not produce serious toxicity and side effects towards the mice (Figure 9b). Herein, an excellent anti-tumor effect of combined PDT & PTT based on GNR-PDA-MB nanocomposites was demonstrated, and it was more efficient than any single modality therapy (PDT or PTT).



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Figure 9. In vivo anti-tumor effect of GNR-PDA-MB nanocomposites. (a) Relative tumor volume (normalized according to the initial volume) of mice after various treatments (as indicated in the figure) as time went by. (b) Body weights of mice after various treatments (as indicated in the figure) as time went by. (c) Representative digital images of mice, 14 days after treatment (as indicated in the figure). (d) Representative images of H&E stained tumor tissues collected from different groups at 14 days after treatment. Scale bars, 50 μm. Each group included 4 mice (tumors). In Vivo Anti-tumor Effect of GNR-PDA-DOX The anti-tumor effect of GNR-PDA-DOX nanocomposites was assessed using nude mice bearing with tumors, which were injected intratumorally with PBS, free DOX, GNR-PDA-PEG and GNR-PDA-DOX. 4 h post-injection, mice tumors injected with GNR-PDA-DOX and GNR-PDA-PEG nanocomposites received PTT treatment (808 nm, 2 W/cm2, 10 min). Mice


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tumors injected with PBS were set as a control group. After laser-irradiation treatments, tumors in mice were monitored by measuring their volumes. The body weights of mice were also recorded. As shown in Figure 10a, a very obvious reduction in tumor volume was observed in the mice treated with GNR-PDA-DOX and NIR light irradiation, indicating the most efficient anti-tumor activity (inhibiting tumor growth) was provided by the combined chemotherapy & PTT based on GNR-PDA-DOX nanocomposites. In addition, mice that were injected with GNR-PDA-PEG and received PTT treatment also showed significant inhibition of tumor growth (but not as distinct as that in the first case), which arose from the high photothermal conversion of GNR-PDA-PEG. Free DOX and GNR-PDA-DOX treated mice both displayed some suppression of tumor growth compared to the control group (PBS treated mice), which was due to the chemotherapeutic effect of the DOX drug. These results illustrated that the photothermal effect of GNR-PDA-PEG alone was efficient to suppress tumor growth, and DOX adsorbed on the GNR-PDA-PEG nanocomposites could be released in vivo for chemotherapy after intratumoral injection. The H&E stained tumor tissues from different groups also confirmed that combined chemotherapy & PTT was the most efficient strategy to damage the tumor tissue and cells (Figure 10d). In addition, no death and no obvious weight loss were observed in all the experimental groups throughout the period, indicating that GNR-PDA-PEG, GNR-PDA-DOX and free DOX did not produce serious toxicity and side effects towards the mice (Figure 10b). All these results demonstrated that GNR-PDA-DOX nanocomposites combined with NIR laser irradiation could serve as efficient chemo/photothermal therapeutic agents for anti-tumor therapy



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in vivo.

Figure 10. In vivo anti-tumor effect of GNR-PDA-DOX nanocomposites. (a) Relative tumor volume (normalized according to the initial volume) of mice after various treatments (as indicated in the figure) as time went by. (b) Body weights of mice after various treatments (as indicated in the figure) as time went by. (c) Representative digital images of mice, 14 days after various treatments (as indicated in the figure). (d) Representative images of H&E stained tumor tissues collected from different groups at 14 days after treatment. Scale bars, 50 μm. Each group included 4 mice (tumors).

CONCLUSIONS In summary, we developed a simple and versatile nanoplatform based on PDA-capped GNRs (GNR-PDA) for multifunctional drug delivery and multimodal light-mediated therapy. The


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self-polymerized PDA shell, with high adsorption capability of therapeutic drugs, was very biocompatible and stable. High loading efficiency of MB and DOX on the GNR-PDA, forming GNR-PDA-MB and GNR-PDA-DOX nanocomposites, respectively, were confirmed. The GNR-PDA-MB nanocomposites could generate ROS effectively under deep-red/NIR laser irradiation, while the GNR-PDA-DOX nanocomposites exhibited light-enhanced drug release under NIR laser excitation. Distinct cellular uptake of GNR-PDA-MB and GNR-PDA-DOX nanocomposites was observed via microscopic imaging, indicating that GNR-PDA could be used for drug delivery into cells. Moreover, due to the tunable LSPR band of GNRs in the NIR spectral region, the GNR-PDA nanocomplex can be employed as an excellent photothermal agent. We demonstrated that the light-mediated dual-modal therapy (PDT & PTT based on GNR-PDA-MB nanocomposites and Chemo & PTT based on GNR-PDA-DOX nanocomposites) showed remarkable in vitro cancer cell killing efficiency and significant suppression of tumor growth in vivo, which were better than any single therapy modality. Thus, the GNR-PDA nanocomplex could serve as a drug delivery vehicle, photothermal agent, and imaging contrast agent simultaneously. By carrying two or more therapeutic drugs, GNR-PDA nanocomplexes hold great promise for imaging-guided multifunctional drug delivery and multimodal cancer therapy. MATERIALS AND METHODS

Materials. All chemicals were purchased from commercial suppliers and used without further purification. Dopamine hydrochloride, cetyltrimethylammonium bromide (CTAB), hydrogen



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tetrachloroaurate (III) trihydrate (HAuCl4•3H2O), sodium borohydride (NaBH4), silver nitrate (AgNO3), L-ascorbic acid, 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA), and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich. SH-PEG-CH3 (Methoxy Polyethylene Thiol, MW = 5000) and SH-PEG-NH2 (MW = 5000) were purchased from JenKem Technology Co., Ltd. Methylene blue was purchased from Sinopharm Chemical Reagent Co., Ltd. Doxorubicin hydrochloride and Tris(hydroxymethyl)aminomethane (Tris) were purchased from Aladdin Industrial Inc. Deionized (DI) water was used in all experiments. Synthesis of GNRs. GNRs with a longitudinal LSPR peak at ~ 650 nm were synthesized via a seed-mediated method in an aqueous solution. Briefly, 0.6 mL of 10 mM ice-cold NaBH4 was injected into a 10 mL aqueous solution containing 0.1 M CTAB and 0.25 mM HAuCl 4, under vigorous stirring. Gold nanoparticles (as seeds) were obtained after 2 min-stirring, and the solution was kept open in the air for 30 min at room temperature in order to allow for the hydrolysis of NaBH4. The solution for GNR growth was prepared by adding 0.2 ml of 25 mM HAuCl4 to 10 mL of 0.1 M CTAB. Then, 40 μL of 16 mM AgNO3 and 90 μL of 80 mM L-ascorbic acid were separately added to the solution. The growth solution turned colorless after hand shaking, and 12 μl of the previously prepared gold seeds solution was injected into it. The solution was left undisturbed at 37 °C for 18 h to let the GNRs grow. The as-synthesized GNRs were then purified by centrifugation twice at 7200 rpm for 10 min and redispersed in DI water. Preparation of PEGylated GNRs. PEG modified GNRs were prepared according to our


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previously reported method. Typically, 50 mL as-synthesized GNRs were centrifuged twice at 7200 rpm for 10 min, and the pellet was dispersed in a 25 mL aqueous solution of SH-PEG-CH3 (2 mg/mL, MW = 5000). The mixed dispersion was immediately vortexed for 3 min, and then stirred for 16 h. The above procedure was performed again, and PEGylated GNRs were collected via centrifugation and washed twice with water. PDA Capping on PEGylated GNRs. The PEGylated GNRs were dispersed in 10 mM Tris buffer (pH ~ 8.5), and the dispersion was vortexed and sonicated for 5 min. A small amount of dopamine in aqueous solution was added into the dispersion, to make the final concentration of dopamine 1 mg/mL. This mixed solution was vortexed and sonicated for another 30 min to make the dopamine self-polymerize on the surface of the PEGylated GNRs. The obtained PDA-capped GNRs (GNR-PDA) were centrifuged and washed with DI water twice, and then resuspended into water for further use. The concentration of dopamine (in the mixed solution) was adjusted from 0.1 to 0.5 mg/mL, in order to obtain GNR-PDA with different PDA-shell thicknesses. Characterizations of Nanocomposites. The absorption spectra of nanocomposites were measured by a Shimadzu 2550 UV-vis scanning spectrophotometer. TEM images of nanocomposites were taken by a JEOL JEM-1200EX microscope operating at 80 kV. Zeta potentials and hydrodynamic size distribution of nanocomposites were measured on a Malvern Zetasizer Nano ZS-90. Preparation of GNR-PDA-MB Nanocomposites. 10 mL aqueous dispersion of previously prepared GNR-PDA (0.1 nM) was centrifuged, and the pellet was resuspended into 10 mL



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aqueous solution of SH-PEG-NH2 (1 mg/mL, MW = 5000). The pH value of the dispersion was adjusted to 12 by adding sodium hydroxide, and it was sonicated for 1 h and stirred magnetically for 12 h. The obtained GNR-PDA-PEG was centrifuged and redispersed into 10 mL aqueous solution of MB (5, 10, 20, 40 and 80 µM). The mixed solution was vortexed and sonicated for 30 min, and stirred magnetically in the dark at room temperature for 24 h. The obtained GNR-PDA-MB was purified by centrifugation twice. The amount of MB adsorbed on the GNR-PDA-PEG was calculated by subtracting the amount of MB in the supernatant after centrifugation from the amount of MB in the initial reaction solution. Preparation of GNR-PDA-DOX Nanocomposites. 10 mL aqueous dispersion of previously prepared GNR-PDA-PEG (0.1 nM) was centrifuged, and the pellet was redispersed into a 10 mL aqueous solution of DOX (5, 10, 20 and 30 µg/mL). The mixed solution was vortexed and sonicated for 30 min, and stirred in the dark at room temperature for 24 h. The obtained GNR-PDA-DOX was purified by centrifugation twice. The amount of DOX adsorbed on the GNR-PDA-PEG was calculated by subtracting the amount of DOX in the supernatant after centrifugation from the amount of DOX in the initial reaction solution. In Vitro ROS Detection from GNR-PDA-MB. ABDA was employed to evaluate the generation of ROS from GNR-PDA-MB nanocomposites. Briefly, a 2 mL aqueous dispersion of GNR-PDA-MB (0.1 nM, equivalent concentration of MB, 50 µM) was mixed with ABDA (final concentration, 0.1 mg/mL), and the mixture was irradiated with a 671 nm-laser (30 mW/cm2). The absorption spectra (300-500 nm) of the ABDA were recorded after the mixture was


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irradiated for various durations. As control experiments, a 2 mL aqueous dispersion of GNR-PDA-PEG (0.1 nM) and 2 mL aqueous solution of free MB (50 µM), which contain 0.1 mg/mL ABDA, were performed separately with the same procedure. Release of DOX from GNR-PDA-DOX. To investigate the laser-driven drug release behavior of GNR-PDA-DOX, 0.5 mL aqueous dispersion of GNR-PDA-DOX (0.1 nM, equivalent concentration of DOX, 20 µg/mL) was centrifuged and redispersed into either pH 7.4 or pH 5.0 solution. At the fixed time intervals, the solution was subjected to the irradiation of an 808 nm-laser (2 W/cm2) for 3 min. Afterwards, the dispersion was centrifuged and the supernatant was collected for absorption spectrum measurement to determine the amount of released DOX from the GNR-PDA-DOX. The same volume of buffers (pH 7.4 or pH 5.0) was added back to the residual solution. The same aqueous dispersion of GNR-PDA-DOX without the laser irradiation was set as the controls. 20 µg/mL aqueous solution of free DOX was set as a positive control for complete DOX release. In Vitro Photothermal Evaluation. To investigate the photothermal effects of GNR-PDA-MB and GNR-PDA-DOX nanocomposites, 0.5 mL of aqueous dispersions containing various concentrations of GNR-PDA-MB/GNR-PDA-DOX nanocomposites (0-0.6 nM) were irradiated by an 808 nm-laser (2 W/cm2). The temperatures of the dispersion were measured by a thermocouple thermometer after it was irradiated for various times. As the control experiments, 0.5 mL of 50 µM free MB, 0.5 mL of 20 µg/mL free DOX and 0.5 mL DI water were separately subjected to laser irradiation with the same procedure.



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Evaluation of Cellular Uptake with Nanocomposites by Flow Cytometry. HeLa cells were placed in a 35 mm cell well at a density of 2×105 cells per well for 24 h and grew to ~ 80% confluence. The original culture medium was then replaced with 2 mL of fresh culture medium containing 5 pM GNR-PDA-MB (equivalent concentration of MB, 2.5 µM) or 5 pM GNR-PDA-DOX (equivalent concentration of DOX, 1 µg/mL), and the cells were further incubated for either 2 h, 6 h, 12 h or 24 h. Afterwards, the culture medium was removed again, and the cells were washed three times with PBS. Finally, the cells were harvested and redispersed in PBS for fluorescence measurement by a FC500 flow cytometer (Beckman Coulter, USA), with an excitation wavelength of 633 nm for cells treated with GNR-PDA-MB and 488 nm for cells treated with GNR-PDA-DOX. Cellular Imaging. HeLa cells were placed in 35 mm cell wells at a density of 2×105 cells per well for 24 h and grew to ~ 80% confluence. The culture medium was then replaced with a fresh medium containing various samples, including GNR-PDA-MB (5 pM, equivalent concentration of MB, 2.5 µM), GNR-PDA-DOX (5 pM, equivalent concentration of DOX, 1 µg/mL), GNR-PDA-PEG (5 pM), free MB (2.5 µM) and free DOX (1 µg/mL), for 24 h. After incubation, the cells were washed three times with PBS to remove the excessive added samples. Fluorescence and two-photon fluorescence images were acquired by a laser scanning confocal microscope (FLUOVIEW FV1000, Olympus) equipped with a Ti: sapphire femtosecond (fs) laser. To determine the distribution of GNR-PDA-PEG in the cells, two-photon fluorescence of the sample-treated cells was excited by the fs laser (at 800 nm), and the signals were collected


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within the 495-540 nm and 575-630 nm spectral channels. To determine the distribution of MB in cells, cells were excited by a CW laser at 635 nm, and the signals were collected within the 655-755 nm channel. To determine the distribution of DOX in cells, cells were excited by a CW laser at 488 nm and the signals were collected within the 505-605 nm channel. Detection of Intracellular ROS Generation. HeLa cells were placed in a 35 mm cell well at a density of 2×105 cells per well for 24 h and grew to ~ 80% confluence. The culture medium was then replaced with a fresh medium containing different samples, including GNR-PDA-MB (5 pM, equivalent concentration of MB, 2.5 µM), GNR-PDA-PEG (5 pM) and free MB (2.5 µM). After 24 h incubation, the culture medium was replaced by a fresh medium, and the cells were irradiated by a 671 nm-laser for 2 min (30 mW/cm2). The medium was replaced again by a fresh culture medium containing 20 µM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), and the cells were incubated for another 30 min. After that, the cells were washed three times with PBS and used for fluorescence imaging under a laser scanning confocal microscope with laser excitation at 488 nm. In Vitro Cell Cytotoxicity Testing with CCK-8 kit. The cytotoxicities of GNR-PDA-MB, GNR-PDA-DOX, GNR-PDA-PEG, free MB and free DOX toward HeLa cells were evaluated following the instructions of a cell counting kit (CCK-8). HeLa cells were seeded in a 48-well plate with a density of 2×104 cells per well for 24 h and grew to ~ 80% confluence. 200 μL of fresh culture medium containing the aforementioned samples with various concentrations were added into each well to replace the original medium, and the cells were incubated for 24 h.



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Subsequently, various light-mediated treatments were performed on these cells, including PDT treatment, PTT treatment and combined PDT & PTT treatment (see details in the main text). The treated cells were incubated for an additional 24 h, and the culture medium was replaced with a fresh culture medium containing CCK-8. The cells were incubated for another 1 h, and the absorbance of the culture medium at 450 nm was measured by using a microplate reader (Thermo, USA) to determine the viability of cells inside. Evaluation of In Vitro Cell Apoptosis. HeLa cells were seeded in a 48-well plate with a density of 2×104 cells per well for 24 h and grew to ~ 80% confluence. Then, 200 μL of fresh culture medium containing 2 pM GNR-PDA-MB nanocomposites (equivalent concentration of MB, 1 µM) were added into each well to replace the original medium, and the cells were incubated for 24 h. After that, the cells were subjected to PDT treatment (671 nm-irradiation, 30 mW/cm2, 3 min), PTT treatment (808 nm-irradiation, 2 W/cm2, 5 min), and combined PDT & PTT treatment, separately. The cells were then incubated for an additional 24 h after the light-mediated treatment. They were washed with PBS, harvested and stained with Annexin-V and Propidium Iodide (PI) according to the manufacturer’s instructions. The stained cells were examined with a FC500 flow cytometer (Beckman Coulter, USA), and over 10000 cells were collected for each kind of sample. In Vivo Photothermal Evaluation. All the animal studies were performed strictly in accordance with guidelines of the Institutional Ethical Committee of Animal Experimentation of Zhejiang University. Female BALB/c nude mice (20-22 g, 5 weeks old) were obtained from the


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Laboratory Animal Center of Zhejiang University (Hangzhou, China). HeLa cells were implanted subcutaneously into the abdomen of nude mice with a dose of 2×106 cells in 100 μL PBS (1 ×). Tumor growth was monitored every two days until a tumor size of approximately 100-120 mm3 was observed. 100 μL GNR-PDA-PEG (1 nM) or 100 μL PBS (1 ×) were intratumorally injected into the tumor of the mice. 4 h post injection, mice were subjected to the laser irradiation (808 nm, 2 W/cm2) and an thermal imaging infrared camera (M7500 Thermal Imager, MIKRON INFRARED) was used to capture the thermographs of the mice. The data of temperature changes of the mice were analyzed by MikroSpec R/T Software. In Vivo Anti-tumor Study. For the anti-tumor study of GNR-PDA-MB nanocomposites, tumor-bearing mice were divided into 5 groups with 4 mice per group, and 100 μL GNR-PDA-MB (1 nM, equivalent concentration of MB, 500 μM) or 100 μL free MB (500 μM) or 100 μL PBS (1 ×) were intratumorally injected into the mice. At 4 h post-injection, mice in each group were subjected to the following treatments: (1) PBS injected without laser treatment, (2) free MB injected with PDT treatment, (3) GNR-PDA-MB injected with PDT treatment, (4) GNR-PDA-MB injected with PTT treatment, and (5) GNR-PDA-MB injected with combined PDT & PTT treatment. Herein, the PDT treatment indicates light irradiation by a 671 nm-laser with a power density of 30 mW/cm2 for 10 min, and the PTT treatment indicates light irradiation by an 808 nm-laser with a power density of 2 W/cm2 for 10 min. For the anti-tumor study of GNR-PDA-DOX nanocomposites, mice were divided into 5 groups with 4 mice per group, and 100 μL GNR-PDA-DOX (1 nM, equivalent concentration of DOX, 200 µg/mL), 100 μL



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GNR-PDA-PEG (1 nM), 100 μL free DOX (200 µg/mL) or 100 μL PBS (1 ×) were intratumorally injected into the mice. 4 h post-injection, mice in each group were subjected to the following treatments: (1) PBS injected without laser treatment, (2) free DOX injected without laser treatment, (3) GNR-PDA-DOX injected without laser treatment, (4) GNR-PDA-PEG injected with PTT treatment, and (5) GNR-PDA-DOX injected with PTT treatment. Herein the PTT indicates light irradiation by an 808 nm-laser with a power density of 2 W/cm2 for 10 min. After the treatment, the size of the tumors was monitored by a caliper every other day. The volumes of the tumors were calculated as length × (width)2 × 1/2, and the relative tumor volume was calculated by normalizing the volume value to its initial size. Histological Examinations. Mice were intravenously injected with 200 μL of GNR-PDA-MB or GNR-PDA-DOX (1 nM in 1 × PBS) and major organs including liver, spleen, kidney, lung and heart were collected and stained with hematoxylin and eosin (H&E). For the in vivo anti-tumor study, tumor tissues after various treatments were also collected and stained with H&E. The bright field images were taken by an inverted microscope.

ACKNOWLEDGMENT This work was supported by National Basic Research Program of China (973 Program, 2013CB834704), the National Natural Science Foundation of China (61275190, 61178062, 61008052 and 91233208), the Program of Zhejiang Leading Team of Science and Technology Innovation (2010R50007), the Fundamental Research Funds for the Central Universities and the


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Swedish Research Council. Shaowei Wang is grateful to Mr. Kai Wu for help with animal experiments. Supporting Information Available: includes Zeta potentials of modified GNRs, UV-vis absorption spectra of GNR-PDA-DOX, ROS generation evaluation of free MB and GNR-PDA in aqueous dispersion with ABDA, cellular uptake of GNR-PDA-MB and GNR-PDA-DOX nanocomposites determined by flow cytometry, fluorescence images of intracellular ROS evaluation and H&E stained tissues and tumor images. This material is available free of charge via the Internet at http://pubs.acs.org.

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Biologically Inspired Polydopamine Capped Gold Nanorods for Drug

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