Biomaterials 60 (2015) 31e41
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Multifunctional gold nanostar-based nanocomposite: Synthesis and application for noninvasive MR-SERS imaging-guided photothermal ablation Yongping Gao a, Yongsheng Li a, *, Jianzhuang Chen a, Shaojia Zhu b, Xiaohang Liu c, Liangping Zhou c, Ping Shi b, Dechao Niu a, Jinlou Gu a, Jianlin Shi a, d, * a
Lab of Low-Dimensional Materials Chemistry, Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237, China State Key Lab of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China c Department of Radiology, Shanghai Cancer Hospital, Fudan University, Shanghai 200032, China d Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2015 Accepted 3 May 2015 Available online
We report here the design and facile synthesis of multifunctional gold nanostars based nanocomposites (MGSNs) through direct organosilica coating onto anisotropic gold nanostars followed by the conjugation of Gd chelates. The as-synthesized MGSNs possess strong NIR absorbance, SERS signal and enhanced T1MR imaging capability with excellent dispersivity and uniform size, as well as great photothermal stability and Raman stability under photothermal conditions. Importantly, MGSNs present excellent performance in vivo after their intravenous injection for both MR and SERS imaging and the high efficiency for killing tumor cells through photothermal ablation with NIR irradiation. A combination of the high spatial resolution of MR and the exciting sub-cell-level sensitivity and resolution of SERS can provide comprehensive information about the tumor to achieve the optimized therapeutic outcome. Therefore, MGSNs are of great potential as a multifunctional nanoplatform for MR-SERS bimodal imaging-guided, focused photothermal tumor therapy. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Nanocomposite Gold MRI SERS Photothermal therapy
1. Introduction Various imaging technologies for early cancer diagnosis, such as magnetic resonance imaging (MRI) [1,2], computed X-ray tomography (CT) [3], ultrasound [4,5], positron emission tomography (PET) [6,7] and optical imaging (OI) [8e11] have been invented and clinically applied in the last decades. Apart from its own merits, each single imaging technique has drawbacks in terms of sensitivity, spatial resolution, data acquisition time and complexity, which make it difficult to obtain accurate and reliable information at the focal zone [12]. Therefore, multi-imaging modalities have been developed to overcome the shortcomings of individual imaging modality [13e17]. For example, MRI based on magnetite particles [18,19] is a non-invasive, biologically safe technique with high spatial resolution and no tissue penetration limit, but suffers
* Corresponding authors. E-mail addresses: [email protected] (Y. Li), [email protected] (J. Shi). http://dx.doi.org/10.1016/j.biomaterials.2015.05.004 0142-9612/© 2015 Elsevier Ltd. All rights reserved.
from low sensitivity. On the contrary, OI offers much higher sensitivity than MRI. Thus, MRI/OI dual-imaging has attracted tremendous attention based on the combination of the high spatial and temporal resolution of the former and the sensitivity of the latter [8,20e24]. In recent years, Surface-enhanced Raman Spectroscopy (SERS) has emerged as an alternative to fluorescencebased spectroscopy as a desirable modality in in vivo optical imaging with the advantages of high signal-to-noise ratio, nonphotobleaching feature, subpicomolar level sensitivity, excellent multiplexing capability and single photoexcitation [25]. Nie et al. [26] firstly developed a class of biocompatible and nontoxic gold nanoparticles for noninvasive tumor targeting SERS detection. SERS probes for multiplexed imaging in living subjects were also demonstrated [27e30]. These in vivo studies highlight the potential for SERS-based imaging to serve as an ultra-sensitive medical imaging modality to deliver accurate diagnosis from single cell level to subcutaneous tumors. When coupled with MRI, bimodal MR-SERS imaging can accomplish extremely high sensitivity and high spatial resolution simultaneously and provide more detailed anatomical or
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biological information about the target tumor. Medarova et al. [31] illustrated for the first time the design of a novel nanoprobe (AuMN-DTTC) as a bimodal contrast agent for in vivo MR imaging and Raman detection. Recently, a unique triple-modality MR-photoacoustic-SERS imaging nanocomposite has been fabricated to accurately help delineate the margins of brain tumors in living mice both preoperatively and intraoperatively [32,33]. The multimodal approach demonstrates its comprehensive imaging capabilities and great potentials for enabling more accurate cancer imaging. On the other hand, photothermal therapy (PTT) in cancer medicine has received great attention over the past decades thanks to its capability in locally ablating tumor. Currently, a variety of nanomaterials have been developed as PTT agents, such as different types of gold nanostructures [34,35], carbon nanoparticles [36,37], as well as other inorganic nanoparticles [38,39]. Among these nanoplatforms, gold nanostars are favorable for photothermal tumor ablation due to their tunable plasmon properties in the NIR tissue optical window and high absorption-to-scattering ratio [40,41]. To ensure the therapy efficiency, integrating imaging functions during PTT or, namely, imaging-guided therapy, has been proposed as a promising strategy for accurate therapy [42e44]. Gold nanostars (GNSs) feature multiple sharp branches, which could act as “hot spots” for SERS detection [45e47]. In addition, MR imaging property is able to be introduced through conjugation of gadolinium chelates to form multifunctional gold nanostars [48]. Thus, MR-SERS bimodal imaging-guided photothermal therapy would be particularly attractive since such a combination between MR and SERS imaging is expected to provide comprehensive information about the tumor location, size and shape, which is required to achieve the optimized therapeutic outcome and monitor the therapeutic response after treatment. However, to the best of our knowledge, multifunctional nanocomposite for in vivo MR-SERS imaging-guided photothermal therapy of cancer upon systemic administration has not yet been reported. In this work, multifunctional gold nanostar-based nanocomposites (MGSNs), i.e., gold nanostar@organosilica@Gd-PEG, with significantly enhanced MR/SERS imaging efficacy and photothermal ablation performance have been designed and fabricated. As shown in Fig. 1a, the inner cores of GNSs are mainly responsible for intensifying the Raman signal of the absorbed diethylthiatricarbocyanine iodide (DTTC) and photothermal heating, while the conjugated Gd chelates are employed as MRI contrast agent. The accompanied polyethylene glycol (PEG) modification is to prevent
the nanoparticles from aggregation, protect the Gd chelates and impart excellent water-solubility and biocompatibility. By organosilica coating onto DTTC-tagged gold nanostars with the following simultaneous conjugation of gadolinium chelates and PEG, MGSNs can be facilely synthesized. As organosilica rather than the widely used silica can be produced through a simple hydrolysis of 3mercaptopropyltriethoxysilane (MPTES) and the abundant thiol (-SH) groups in situ introduced by MPTES definitely facilitate and simplify the modification process, the synthetic approach is comparatively simple and controllable. More importantly, the resulting nanocomposites present strong NIR absorbance, excellent SERS signal and enhanced MR contrast imaging performance, demonstrating the promising potentials for imaging-guided photothermal cancer therapy.
2. Experimental section 2.1. Chemicals and characterization L-ascorbic acid and silver nitrate (AgNO3) were purchased from Sinopharm Chemical Reagent Co., Ltd. Sodium hydroxide (NaOH, S96%). Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4$3H2O), 3,3-diethylthiatricarbocyanine iodide (DTTC), 2-(N-morpholino) ethanesulfonic acid (MES) and gadolinium chloride (GdCl3) were purchased from SigmaeAldrich. 3-Mercaptopropyltriethoxysilane (MPTES) was from Alfa Aesar. Maleimide-DOTA was obtained from Macrocyclics. Maleimide-PEG and SH-PEG-COOH were purchased from JENKEM TECHNOLOGY CO., LTD. Ultrapure water (18.2 MU cm) was used in all experiments. All chemicals were used as received without further purification. Morphology and structure were observed on JEM 2100F electron microscope (TEM). UVeviseNIR spectra were recorded on a UV3600 Shimadzu spectroscope. Size distributions of the samples were measured using a Nicomp TM 380 ZLS zeta-potential/particle sizer (PSS Nicomp particle size system, USA). SERS spectra were obtained on a Renishaw inVia Raman Microscope configured with a 785 nm excitation laser line. Triplicate measurements with integration times of 10 s were performed directly on the suspensions in a 1 cm path length quartz cuvette. Laser power at the sample was measured to be 2 mW. The thermal images were obtained by the Infrared camera of FLIR T425. And confocal microscope images were taken by Fluoview FV1000 Confocal Microscope (Olympus).
Fig. 1. a) Schematic diagram of the structure design of MGSNs. A DTTC-tagged GNS is coated by an organosilica layer with abundant free thiol groups on the outer surface. The strong covalent bonding between eSH and maleimide facilitates the simultaneous conjugation of Gd chelates and PEG onto the outer surface of organosilica layer, forming the final MGSNs. b) Transmission electron microscopic images (TEM) of MGSNs. The Inset is the magnified TEM image.
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2.2. Synthesis of MGSNs Gold nanostars were synthesized following a previous paper [49]. Briefly, 20 ml 0.25 mM HAuCl4 solution, 10 ml of 1 M HCl and 200 ml of 12 nm citrate gold seeds were added followed by the simultaneous addition of 300 ml of 2 mM AgNO3 and 100 ml of 100 mM L-ascorbic acid under vigorous stirring. After 30 s, 300 ml SH-PEG-COOH (1 mgml1) was added into the above blue green solution under gentle stirring for 15 min. Then, DTTC (0.1 mM) in methanol was added and allowed to stir for 6 h. The DTTC-tagged nanostars were centrifuged (4200 rpm for 23 min) to remove excess DTTC and resuspended in 16 ml water. Organosilica coating was conducted by mixing 25 ml MPTES and 150 ml ammonia (28%) to the above 16 ml suspension and shaking the mixture for 90 s using IKA MS3 minishaker at a speed of 2000 rpm. The mixture was incubated at room temperature for another 12 h to obtain DTTCtagged gold nanostars@organosilica. The DTTC-tagged gold nanostars@organosilica was centrifuged and resuspended in 10 ml of 10 mM MES buffer, pH 7.0. Then, 1 mg Maleimide-DOTA and 3 mg Maleimide-PEG was added into the solution, reacted for 3 h at room temperature. The solution was centrifuged and redispersed in 10 ml MES buffer (pH ¼ 6.25), followed by the addition of 300 ml GdCl3 (2 mg/ml). The above solution was heated to 50 C for 2 h and then washed three times to obtain final MGSNs. Gd-DOTA was prepared by mixing Maleimide-DOTA and GdCl3 at the molar ratio of 1.2:1 in 10 ml MES buffer (pH ¼ 6.25). The above solution was heated to 50 C for 2 h to obtain Gd-DOTA. GNS@organosilica@Au multilayered coreeshell nanostructures were synthesized according to the proposed synthetic strategy based on one-pot, in-situ attachment developed by our group [50]. 2.3. Investigation of photothermal effect of MGSNs To study the photothermal effect induced by the NIR plasmonic resonance, the aqueous solution (1 ml) of the MGSNs at the extinction intensity of 1 was irradiated using an NIR laser (808 nm, 0.5 W, Hps3200, B&A technology Co., Ltd) for 10 min. The temperature of the solutions was recorded every 10 s by a digital thermometer with a thermocouple probe. Photothermal cell toxicity of MGSNs was evaluated on MDAMB-231 cells. For qualitative analysis, MDA-MB-231 cells (2 105 cells per well) were incubated in a humidified atmosphere containing 5% CO2 for 24 h. After incubated with MGS suspensions (2.0 ml per well, 20 mgml1 Au concentration) for another 4 h, cells were then exposed to NIR laser (0.5 W) for 5 min and stained with both calcein AM (calceinacetoxymethyl ester) and PI (propidiumiodide) to evaluate the photothermal effect on cancer cells (where green fluorescence from calcein and red fluorescence from PI indicate live and dead cells, respectively). In vitro cytoviability assay of MGSNs was then carried out on MDA-MB-231 cells and L-02 cells. MDA-MB-231 cells and L-02 cells (104 cells per well) were incubated in 96-well plates at 37 C for 24 h. Different amounts (0.1 ml per well, Au concentration from 0 to 400 mgml1) of MGS suspensions were added and incubated with cells for 24 h. The cell viabilities were determined by MTT assay. In vitro cytoviability assay of MGSNs combined with laser irradiation was also conducted on MDA-MB-231 cells. MDA-MB-231 cells (104 cells per well) were incubated in 96-well plates at 37 C for 24 h. Different amounts (0.1 ml per well, Au concentration from 0 to 20 mgml1) of MGS suspensions were added and incubated with cells for 4 h. Then, the cells were irradiated by NIR laser (0.5 W) for 5 min and then incubated for another 20 h. Finally, the cell viabilities were determined by MTT assay. For in vivo study, mice bearing MDA-MB-231 at 30 min post i.v. injection with MGSNs (100 ml, 4 mgml1) were exposed to NIR
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808 nm laser (0.5 W) for 5 min. For control groups, mice were either treated with the same volume of saline before laser irradiation, or injected with MGSNs but without laser exposure. Thermal images were taken every minute using Infrared camera of FLIR T425. The tumor size were measured by a caliper every other say and calculated by (length of tumor) (width of tumor)2/2. Relative tumor volumes were calculated as V/V0 (V0 is the tumor volume when the treatment was initiated). All animal experiments were performed in compliance with the Guidelines for the Care and Use of Research Animals established by the East China University of Science and Technology Animal Studies Committee. 2.4. In vivo distribution of samples in MDA-MB-231 tumor-bearing mice MGSNs at a dose of 20 mg/kg were intravenously injected into three groups (n ¼ 3 per group) of MDA-MB-231 tumor-bearing mice separately. To measure the concentration distribution of MGSNs, different organs (tumors, liver, spleen, lung, heart and kidney) were collected and weighted in different time interval (0.5 h, 6 h and 24 h) post-injection of the samples. And each organ section was digested by the mixed solution of HNO3 and HClO4 (3:1 in volume ratio) at 120 C. Then, the Au element concentration was measured by inductively coupled plasma atomic emission spectrometer (ICP-AES, Agilent). 2.5. Magnetic resonance imaging (MRI) For T1 MRI test, the Gd content of the MGSNs in water was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). Solutions of MGSNs containing Gd at concentration of 3, 18, 36, 72, 150, 300 mM were prepared in pure water. Relaxation values of r1 were calculated through the curve fitting of 1/T1 relaxation time (s1) versus the Gd concentration (mM). For in vivo MRI study, MR images were taken before and after the intravenously injection of MGSNs at a dose of 20 mgkg1. MRI was performed on a 3.0-T clinical MRI instrument (GE Signa 3.0-T), and the pulse sequence used was a T1-weighted FSE-XL/90 sequence with the following parameters: TR/TE ¼ 560/16.2, FOV ¼ 8 4.8, slice thickness ¼ 2, space ¼ 0.5, nex ¼ 2, matrix ¼ 384 256. After acquiring T1-weighted MR images, the signal intensity (SI) was measured within a manually drawn region of interest for each mouse. The enhanced values of relative T1 signal were calculated using SI measurements before (SIpre) and after (SIpost) the i.v. injection of contrast agents, using the formula: [(SIpost SIpre)/ SIpre] 100%. 2.6. In vivo SERS imaging Raman imaging experiments were performed on Renishaw inVia Reflex equipped with Streamline rapid Raman imaging system. The MDA-MB-231 tumor-bearing mice treated with intravenously injecting saline or MGSNs (100 ml, 4 mgml1) were subjected to point-scanning using 50 mW of 785 nm laser with 10 acquisitions of 1 s (Fig. S1). Streamline Raman imaging were then obtained with an integration of 5 s, a step size of 1.1 mm and 50 mW of 785 laser power. 2.7. In vivo systematic toxicity A total of 12 healthy Balb/c mice were allocated 4 groups, receiving single intravenous injection of saline (control, 100 ml) or MGSNs (100 ml for each mouse, 4 mgml1) followed by dissection in 3, 15, 30 days postinjection. Blood samples were collected from mice and analyzed and tissues recovered from the necropsy were
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fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for histological examination using standard techniques. After hematoxylin eosin staining, the slides were observed and photos were taken using an optical microscope. 3. Results and discussion 3.1. Characterization of MGSNs The formation of the designed coreeshell structured MGSNs was characterized by various techniques. As shown in the transmission electron microscopy (TEM) images (Fig. 1b and Figs. S2, S3 in the Supporting Information), monodispersed MGSNs have been obtained after coating DTTC-tagged GNSs with organosilica layer and the following modification with Maleimide-DOTA-Gd and Maleimide-PEG via covalent bonding. Obviously, the excellent dispersivity and uniformity in both morphology and dimension of MGSNs are not affected by the modifying steps. Moreover, the coreeshell structure composed of 60 nm gold nanostar and 20 nmthick organosilica layer is clearly exhibited in the highmagnification TEM image in the inset of Fig. 1b, in accordance with DLS result (Fig. S4). Along with the energy dispersive X-ray spectroscopy (EDS) detection (Fig. S5), the co-existence of gold, organosilica and Gd in one nanoparticle can be well confirmed. As far as we know, this is the first report on the functional combination of MR-SERS imaging and photothermal ablation properties into one unit, which could be simply implemented through the direct organosilica coating onto anisotropic gold nanoparticles with abundant thiol groups on the outer surface for facilely performing further modifications. Besides, through the one-pot and in-situ process developed by our group [21], multilayered coreeshell structured GNS@organosilica@Au nanoparticles (Fig. 2) have been successfully fabricated, further demonstrating the versatility of the proposed synthetic strategy [50]. Following the successful fabrication of the coreeshell structure, properties of MGSNs were measured accordingly. As shown in the UVeviseNIR extinction spectra in Fig. 3a, a 54 nm red-shift of MGSNs, relative to GNSs, was recorded due to the higher refraction index of organosilica layer than water [51]. The MGSNs exhibit a strong extinction band at approximately 800 nm, making it highly promising in photothermal therapy with 808 nm laser. In Fig. 3b, the temperature changes of pure water and aqueous solutions loaded with MGSNs are presented, which were recorded at the extinction intensity of 1 as a function of time under continuous
irradiation of 808 nm (0.5 W). It can be found that the temperature of the aqueous solutions loaded with MGSNs increases to 68 C, while no obvious temperature change is detectable for pure water. On the other hand, strong SERS signal from DTTC inside MGSNs can be clearly detected, as shown in Fig. 3c, while neither MGSNs without DTTC nor pure DTTC solution exhibit distinguishable SERS signals, indicating the significant Raman enhancement effect of the unique particle structure. For simplicity, in this paper, 507 cm1 SERS peak of DTTC was employed for SERS intensity determination. Furthermore, the capability of MGSNs to act as an MRI contrast agent was evaluated in terms of r1 relaxivity. The calculated r1value is as high as 8.40 mM1 s1 for MGSNs, much higher than that of 1.41 mM1 s1 for Gd-DOTA, clearly demonstrating the enhanced relaxivity of Gd chelates after their conjugating onto the outer surface of the organosilica layer (Fig. 3d). This observed ~6 times of enhancement in relaxivity is probably due to the restricted molecular tumbling and therefore increased tR rotational correlation time of the Gd chelates on the surface under the protection of PEG chain [23,52]. All these results confirm the successful fabrication of the coreeshell nanostructure which endows MGSNs with significantly enhanced SERS and T1-weighted MR imaging performances, as well as the excellent photothermal conversion capability. To ensure successful photothermal therapy and structurerelated imaging applications, such as SERS, the photothermal stability under NIR laser excitation of MGSNs was tested. After five cycles of laser heating, the thermal conversion efficiency of the assynthesized MGSNs was found to keep constant, while remarkable decrease was recorded for bare GNSs (Fig. 4a). By carefully analyzing the TEM images and UVevis spectra before and after repeated laser irradiation, no changes can be found on the morphology and dispersivity of MGSNs though bare GNSs lost the majority of the NIR absorbance owing to the morphology change (Fig. S6 and 4b). This verifies that the unique coreeshell structure is crucial to maintain the morphology and consequently the superior photothermal conversion efficiency. Surprisingly, PEG could also render GNSs good photothermal stability as PEGylated GNSs did not show any noticeable difference in photothermal conversion, optical properties and TEM images after repeated photo-induced local heating (Fig. 4a, b and S6ced), in contrast to the previous report [53]. However, the Raman stability study shows that the signal of DTTC (at 507 cm1) for PEGylated DTTC-tagged GNSs decreases dramatically in spite of the good photothermal stability (Fig. 4c), while in contrast, SERS signal remains essentially unchanged for MGSNs (Fig. 4d). These results evidence that the DTTC reporters have detached from the surface of PEGylated GNSs under
Fig. 2. TEM images of a) gold seeds grafted GNS@organosilica and b) GNS@organosilica@Au multilayered gold nanoshells.
Y. Gao et al. / Biomaterials 60 (2015) 31e41
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Fig. 3. a) UVeviseNIR extinction spectra for the GNSs and MGSNs (bands at 746 nm and 800 nm respectively). b) Temperature rise profiles of pure water and the aqueous solution of MGSNs as a function of heating time under the irradiation by an 808 nm laser (OD808nm ¼ 1, 0.5 W). c) Raman spectra of MGSNs (red line), MGSNs without DTTC (black line) and pure DTTC (blue line) collected under 785 nm excitation at a power of 2 mW and integration time of 10 s. Spectra are offset for clarity and have had their backgrounds removed manually. Samples were dispersed in water. The 507 cm1 SERS peak (pointed by the arrow) was chosen for the SERS intensity measurement. Scale bar: 2000 counts. d) Plot of the relaxation rate (T1 1 ) against Gd concentration for Gd-DOTA and MGSNs. The inset shows T1-weighted MR images of the solutions containing Gd-DOTA and MGSNs at concentrations ranging from 0.003 mM to 0.3 mM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
repeated laser exposure, resulting in significant decrease of SERS signal. However, the coating organosilica layer could protect the reporters from detaching, thus endowing the MGSNs with great Raman stability. These results demonstrate the superiority of organosilica coating and thus the high structural and SERS stabilities of the as-synthesized MGSNs under photothermal conditions.
the concentrations employed (Fig. 5b). Upon laser irradiation, the cell viability significantly decreased under the treatments with MGSNs, only about 10% of 231 cells remaining viable at Au concentration of 20 mgml1 (Fig. 5c). These verify that MGSNs could be used as an effective photothermal ablation agent. 3.3. In vivo MR-SERS imaging and photothermal therapy
3.2. In vitro cell experiments Encouraged by the excellent photothermal conversion capability and stability, the localized photothermal effect in vitro of MGSNs was further investigated by choosing 231 (human breast adenocarcinoma cell line) cells as a cell model. Calcein AM (green) and propidiumiodide (PI, red) co-staining was used to differentiate live and dead cells after photothermal therapy. As shown in the confocal microscopic images (Fig. 5a), a distinct demarcation between the dead (red) and live cell (green (in the web version)) regions can be observed with the presence of both MGSNs and laser exposure. It is obvious that MGSNs could kill the cancer cells only through the photothermal effect induced by NIR laser excitation, while neither the MGSNs nor the laser excitation alone can lead to significant cell death. Furthermore, MTT assay was carried out to quantitatively verify the cytotoxicity and photothermal therapy efficacy of MGSNs. After incubated with normal cells (L-02) and tumor cells (MDA-MB-231) for 24 h without laser irradiation, MGSNs exhibited negligible toxicity to these two types of cells at all
Based on the enhanced r1 relaxivity and strong SERS signal of MGSNs, in vivo bimodal imaging attempts on tumor-bearing mice were carried out. Mice bearing MDA-MB-231 tumor were i.v. injected with MGSNs (100 ml, 4 mgml1) and imaged under a 3.0-T clinical MR imaging system (Fig. 6a). Compared with the image of pre-injection, dramatic brightening effect is detected at the tumor area 30 min after injection of MGSNs. Quantitative analysis of MR signals revealed T1 signal intensity enhancement by 36% (Fig. 6c), suggesting high tumor accumulation of those nanoparticles after systemic administration. These imply that the present MGSNs would be a promising candidate as a contrast agent in MR imaging for cancers. Moreover, MGSNs could also serve as an in vivo contrast agent in noninvasive SERS imaging, which is based on the greatly enhanced Raman signal of DTTC by gold nanostars and offers remarkable sensitivity and resolution compared with traditional optical imaging techniques. As shown in Fig. 6c, distinguishable signal of DTTC at 507 cm1 is detected at different sites of the tumor in 30 min post i.v. injection of MGSNs, while the skin near the
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Fig. 4. a) Cyclic temperature profiles of the suspensions of MGSNs, GNSs-PEG and bare GNSs under irradiation by a 808 nm laser being turned on and off for five repeated cycles. b) UVevis spectra for MGSNs, GNSs-PEG and bare GNSs before and after five cycles of laser heating. Raman spectra of c) DTTC-tagged GNSs-PEG and d) MGSNs after 0e5 cycles of laser heating. Backgrounds of the spectra have been removed manually.
Fig. 5. a) Confocal microscopic images of differently treated MDA-MB-231 cells stained with calcein AM and PI: control; laser irradiation only; MGSNs only; both MGSNs and laser irradiation. b) Relative viabilities of L-02 and MDA-MB-231 cells after incubation with MGSNs for 24 h. c) MDA-MB-231 cells viabilities incubated with MGSNs at different Au concentrations without and with laser irradiation (808 nm, 0.5 W, and 5 min).
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Fig. 6. a) In vivo T1 weighted MR images of a tumor site before and 30 min after intravenous injection of MGSNs (4 mgml1, 100 ml). The tumor sites are marked with the red circles. b) The quantification of average T1-MR signal in the tumor by manual drawn region of interest (red circles in a)). Data represent mean ± SD ***P < 0.001. c) Raman spectra of tumor region after injected intravenously with MGSNs, saline solution, and skin near the tumor under 785 nm excitation at 50 mW of laser power and 10 acquisitions of 1 s (up-right). Scale bar: 2500 counts. d) SERS images at the two different sites of the injected tumor in c), produced by using the baseline corrected intensity of the 507 cm1 Raman band of DTTC with StreamLine ultrafast Raman imaging system. The images were obtained with an integration time of 5 s, a step size of 1.1 mm and 50 mW of 785 laser power. Scale bar: 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
injected tumor and the tumor with saline injection show no distinct Raman signals. Furthermore, Raman images were constructed based on the band of 507 cm1 with StreamLine ultrafast Raman imaging system at the two injected tumor sites, which clearly display the distributions of the MGSNs nanoparticles (Fig. 6d). It is thus confirmed that the as-synthesized MGSNs could enable effective noninvasive SERS imaging in large area over cancer, illustrating the excellent sensitivity and sub-cell-level resolution in vivo. As whole-body MR imaging is able to determine the overall uptake of nanoparticles in the tumor, SERS imaging could vividly display the heterogeneous intratumoral distribution of nanoparticles, beneficial for better planning of therapies for optimized treatment efficacy. The hydrophilic PEG molecules have been used to reduce phagocytic capture of nanoparticles by the immune system, leading to extended circulation and subsequent accumulation in tumors as a consequence of the enhanced permeability and retention (EPR) effect due to leaky vasculature and poor lymphatic drainage in tumors [54e57]. Thanks to their small particle size (
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Multifunctional gold nanostar-based nanocomposite: Synthesis and application for noninvasive MR-SERS imaging-guided photothermal ablation Yongping Gao a, Yongsheng Li a, *, Jianzhuang Chen a, Shaojia Zhu b, Xiaohang Liu c, Liangping Zhou c, Ping Shi b, Dechao Niu a, Jinlou Gu a, Jianlin Shi a, d, * a
Lab of Low-Dimensional Materials Chemistry, Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237, China State Key Lab of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China c Department of Radiology, Shanghai Cancer Hospital, Fudan University, Shanghai 200032, China d Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2015 Accepted 3 May 2015 Available online
We report here the design and facile synthesis of multifunctional gold nanostars based nanocomposites (MGSNs) through direct organosilica coating onto anisotropic gold nanostars followed by the conjugation of Gd chelates. The as-synthesized MGSNs possess strong NIR absorbance, SERS signal and enhanced T1MR imaging capability with excellent dispersivity and uniform size, as well as great photothermal stability and Raman stability under photothermal conditions. Importantly, MGSNs present excellent performance in vivo after their intravenous injection for both MR and SERS imaging and the high efficiency for killing tumor cells through photothermal ablation with NIR irradiation. A combination of the high spatial resolution of MR and the exciting sub-cell-level sensitivity and resolution of SERS can provide comprehensive information about the tumor to achieve the optimized therapeutic outcome. Therefore, MGSNs are of great potential as a multifunctional nanoplatform for MR-SERS bimodal imaging-guided, focused photothermal tumor therapy. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Nanocomposite Gold MRI SERS Photothermal therapy
1. Introduction Various imaging technologies for early cancer diagnosis, such as magnetic resonance imaging (MRI) [1,2], computed X-ray tomography (CT) [3], ultrasound [4,5], positron emission tomography (PET) [6,7] and optical imaging (OI) [8e11] have been invented and clinically applied in the last decades. Apart from its own merits, each single imaging technique has drawbacks in terms of sensitivity, spatial resolution, data acquisition time and complexity, which make it difficult to obtain accurate and reliable information at the focal zone [12]. Therefore, multi-imaging modalities have been developed to overcome the shortcomings of individual imaging modality [13e17]. For example, MRI based on magnetite particles [18,19] is a non-invasive, biologically safe technique with high spatial resolution and no tissue penetration limit, but suffers
* Corresponding authors. E-mail addresses: [email protected] (Y. Li), [email protected] (J. Shi). http://dx.doi.org/10.1016/j.biomaterials.2015.05.004 0142-9612/© 2015 Elsevier Ltd. All rights reserved.
from low sensitivity. On the contrary, OI offers much higher sensitivity than MRI. Thus, MRI/OI dual-imaging has attracted tremendous attention based on the combination of the high spatial and temporal resolution of the former and the sensitivity of the latter [8,20e24]. In recent years, Surface-enhanced Raman Spectroscopy (SERS) has emerged as an alternative to fluorescencebased spectroscopy as a desirable modality in in vivo optical imaging with the advantages of high signal-to-noise ratio, nonphotobleaching feature, subpicomolar level sensitivity, excellent multiplexing capability and single photoexcitation [25]. Nie et al. [26] firstly developed a class of biocompatible and nontoxic gold nanoparticles for noninvasive tumor targeting SERS detection. SERS probes for multiplexed imaging in living subjects were also demonstrated [27e30]. These in vivo studies highlight the potential for SERS-based imaging to serve as an ultra-sensitive medical imaging modality to deliver accurate diagnosis from single cell level to subcutaneous tumors. When coupled with MRI, bimodal MR-SERS imaging can accomplish extremely high sensitivity and high spatial resolution simultaneously and provide more detailed anatomical or
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biological information about the target tumor. Medarova et al. [31] illustrated for the first time the design of a novel nanoprobe (AuMN-DTTC) as a bimodal contrast agent for in vivo MR imaging and Raman detection. Recently, a unique triple-modality MR-photoacoustic-SERS imaging nanocomposite has been fabricated to accurately help delineate the margins of brain tumors in living mice both preoperatively and intraoperatively [32,33]. The multimodal approach demonstrates its comprehensive imaging capabilities and great potentials for enabling more accurate cancer imaging. On the other hand, photothermal therapy (PTT) in cancer medicine has received great attention over the past decades thanks to its capability in locally ablating tumor. Currently, a variety of nanomaterials have been developed as PTT agents, such as different types of gold nanostructures [34,35], carbon nanoparticles [36,37], as well as other inorganic nanoparticles [38,39]. Among these nanoplatforms, gold nanostars are favorable for photothermal tumor ablation due to their tunable plasmon properties in the NIR tissue optical window and high absorption-to-scattering ratio [40,41]. To ensure the therapy efficiency, integrating imaging functions during PTT or, namely, imaging-guided therapy, has been proposed as a promising strategy for accurate therapy [42e44]. Gold nanostars (GNSs) feature multiple sharp branches, which could act as “hot spots” for SERS detection [45e47]. In addition, MR imaging property is able to be introduced through conjugation of gadolinium chelates to form multifunctional gold nanostars [48]. Thus, MR-SERS bimodal imaging-guided photothermal therapy would be particularly attractive since such a combination between MR and SERS imaging is expected to provide comprehensive information about the tumor location, size and shape, which is required to achieve the optimized therapeutic outcome and monitor the therapeutic response after treatment. However, to the best of our knowledge, multifunctional nanocomposite for in vivo MR-SERS imaging-guided photothermal therapy of cancer upon systemic administration has not yet been reported. In this work, multifunctional gold nanostar-based nanocomposites (MGSNs), i.e., gold nanostar@organosilica@Gd-PEG, with significantly enhanced MR/SERS imaging efficacy and photothermal ablation performance have been designed and fabricated. As shown in Fig. 1a, the inner cores of GNSs are mainly responsible for intensifying the Raman signal of the absorbed diethylthiatricarbocyanine iodide (DTTC) and photothermal heating, while the conjugated Gd chelates are employed as MRI contrast agent. The accompanied polyethylene glycol (PEG) modification is to prevent
the nanoparticles from aggregation, protect the Gd chelates and impart excellent water-solubility and biocompatibility. By organosilica coating onto DTTC-tagged gold nanostars with the following simultaneous conjugation of gadolinium chelates and PEG, MGSNs can be facilely synthesized. As organosilica rather than the widely used silica can be produced through a simple hydrolysis of 3mercaptopropyltriethoxysilane (MPTES) and the abundant thiol (-SH) groups in situ introduced by MPTES definitely facilitate and simplify the modification process, the synthetic approach is comparatively simple and controllable. More importantly, the resulting nanocomposites present strong NIR absorbance, excellent SERS signal and enhanced MR contrast imaging performance, demonstrating the promising potentials for imaging-guided photothermal cancer therapy.
2. Experimental section 2.1. Chemicals and characterization L-ascorbic acid and silver nitrate (AgNO3) were purchased from Sinopharm Chemical Reagent Co., Ltd. Sodium hydroxide (NaOH, S96%). Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4$3H2O), 3,3-diethylthiatricarbocyanine iodide (DTTC), 2-(N-morpholino) ethanesulfonic acid (MES) and gadolinium chloride (GdCl3) were purchased from SigmaeAldrich. 3-Mercaptopropyltriethoxysilane (MPTES) was from Alfa Aesar. Maleimide-DOTA was obtained from Macrocyclics. Maleimide-PEG and SH-PEG-COOH were purchased from JENKEM TECHNOLOGY CO., LTD. Ultrapure water (18.2 MU cm) was used in all experiments. All chemicals were used as received without further purification. Morphology and structure were observed on JEM 2100F electron microscope (TEM). UVeviseNIR spectra were recorded on a UV3600 Shimadzu spectroscope. Size distributions of the samples were measured using a Nicomp TM 380 ZLS zeta-potential/particle sizer (PSS Nicomp particle size system, USA). SERS spectra were obtained on a Renishaw inVia Raman Microscope configured with a 785 nm excitation laser line. Triplicate measurements with integration times of 10 s were performed directly on the suspensions in a 1 cm path length quartz cuvette. Laser power at the sample was measured to be 2 mW. The thermal images were obtained by the Infrared camera of FLIR T425. And confocal microscope images were taken by Fluoview FV1000 Confocal Microscope (Olympus).
Fig. 1. a) Schematic diagram of the structure design of MGSNs. A DTTC-tagged GNS is coated by an organosilica layer with abundant free thiol groups on the outer surface. The strong covalent bonding between eSH and maleimide facilitates the simultaneous conjugation of Gd chelates and PEG onto the outer surface of organosilica layer, forming the final MGSNs. b) Transmission electron microscopic images (TEM) of MGSNs. The Inset is the magnified TEM image.
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2.2. Synthesis of MGSNs Gold nanostars were synthesized following a previous paper [49]. Briefly, 20 ml 0.25 mM HAuCl4 solution, 10 ml of 1 M HCl and 200 ml of 12 nm citrate gold seeds were added followed by the simultaneous addition of 300 ml of 2 mM AgNO3 and 100 ml of 100 mM L-ascorbic acid under vigorous stirring. After 30 s, 300 ml SH-PEG-COOH (1 mgml1) was added into the above blue green solution under gentle stirring for 15 min. Then, DTTC (0.1 mM) in methanol was added and allowed to stir for 6 h. The DTTC-tagged nanostars were centrifuged (4200 rpm for 23 min) to remove excess DTTC and resuspended in 16 ml water. Organosilica coating was conducted by mixing 25 ml MPTES and 150 ml ammonia (28%) to the above 16 ml suspension and shaking the mixture for 90 s using IKA MS3 minishaker at a speed of 2000 rpm. The mixture was incubated at room temperature for another 12 h to obtain DTTCtagged gold nanostars@organosilica. The DTTC-tagged gold nanostars@organosilica was centrifuged and resuspended in 10 ml of 10 mM MES buffer, pH 7.0. Then, 1 mg Maleimide-DOTA and 3 mg Maleimide-PEG was added into the solution, reacted for 3 h at room temperature. The solution was centrifuged and redispersed in 10 ml MES buffer (pH ¼ 6.25), followed by the addition of 300 ml GdCl3 (2 mg/ml). The above solution was heated to 50 C for 2 h and then washed three times to obtain final MGSNs. Gd-DOTA was prepared by mixing Maleimide-DOTA and GdCl3 at the molar ratio of 1.2:1 in 10 ml MES buffer (pH ¼ 6.25). The above solution was heated to 50 C for 2 h to obtain Gd-DOTA. GNS@organosilica@Au multilayered coreeshell nanostructures were synthesized according to the proposed synthetic strategy based on one-pot, in-situ attachment developed by our group [50]. 2.3. Investigation of photothermal effect of MGSNs To study the photothermal effect induced by the NIR plasmonic resonance, the aqueous solution (1 ml) of the MGSNs at the extinction intensity of 1 was irradiated using an NIR laser (808 nm, 0.5 W, Hps3200, B&A technology Co., Ltd) for 10 min. The temperature of the solutions was recorded every 10 s by a digital thermometer with a thermocouple probe. Photothermal cell toxicity of MGSNs was evaluated on MDAMB-231 cells. For qualitative analysis, MDA-MB-231 cells (2 105 cells per well) were incubated in a humidified atmosphere containing 5% CO2 for 24 h. After incubated with MGS suspensions (2.0 ml per well, 20 mgml1 Au concentration) for another 4 h, cells were then exposed to NIR laser (0.5 W) for 5 min and stained with both calcein AM (calceinacetoxymethyl ester) and PI (propidiumiodide) to evaluate the photothermal effect on cancer cells (where green fluorescence from calcein and red fluorescence from PI indicate live and dead cells, respectively). In vitro cytoviability assay of MGSNs was then carried out on MDA-MB-231 cells and L-02 cells. MDA-MB-231 cells and L-02 cells (104 cells per well) were incubated in 96-well plates at 37 C for 24 h. Different amounts (0.1 ml per well, Au concentration from 0 to 400 mgml1) of MGS suspensions were added and incubated with cells for 24 h. The cell viabilities were determined by MTT assay. In vitro cytoviability assay of MGSNs combined with laser irradiation was also conducted on MDA-MB-231 cells. MDA-MB-231 cells (104 cells per well) were incubated in 96-well plates at 37 C for 24 h. Different amounts (0.1 ml per well, Au concentration from 0 to 20 mgml1) of MGS suspensions were added and incubated with cells for 4 h. Then, the cells were irradiated by NIR laser (0.5 W) for 5 min and then incubated for another 20 h. Finally, the cell viabilities were determined by MTT assay. For in vivo study, mice bearing MDA-MB-231 at 30 min post i.v. injection with MGSNs (100 ml, 4 mgml1) were exposed to NIR
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808 nm laser (0.5 W) for 5 min. For control groups, mice were either treated with the same volume of saline before laser irradiation, or injected with MGSNs but without laser exposure. Thermal images were taken every minute using Infrared camera of FLIR T425. The tumor size were measured by a caliper every other say and calculated by (length of tumor) (width of tumor)2/2. Relative tumor volumes were calculated as V/V0 (V0 is the tumor volume when the treatment was initiated). All animal experiments were performed in compliance with the Guidelines for the Care and Use of Research Animals established by the East China University of Science and Technology Animal Studies Committee. 2.4. In vivo distribution of samples in MDA-MB-231 tumor-bearing mice MGSNs at a dose of 20 mg/kg were intravenously injected into three groups (n ¼ 3 per group) of MDA-MB-231 tumor-bearing mice separately. To measure the concentration distribution of MGSNs, different organs (tumors, liver, spleen, lung, heart and kidney) were collected and weighted in different time interval (0.5 h, 6 h and 24 h) post-injection of the samples. And each organ section was digested by the mixed solution of HNO3 and HClO4 (3:1 in volume ratio) at 120 C. Then, the Au element concentration was measured by inductively coupled plasma atomic emission spectrometer (ICP-AES, Agilent). 2.5. Magnetic resonance imaging (MRI) For T1 MRI test, the Gd content of the MGSNs in water was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). Solutions of MGSNs containing Gd at concentration of 3, 18, 36, 72, 150, 300 mM were prepared in pure water. Relaxation values of r1 were calculated through the curve fitting of 1/T1 relaxation time (s1) versus the Gd concentration (mM). For in vivo MRI study, MR images were taken before and after the intravenously injection of MGSNs at a dose of 20 mgkg1. MRI was performed on a 3.0-T clinical MRI instrument (GE Signa 3.0-T), and the pulse sequence used was a T1-weighted FSE-XL/90 sequence with the following parameters: TR/TE ¼ 560/16.2, FOV ¼ 8 4.8, slice thickness ¼ 2, space ¼ 0.5, nex ¼ 2, matrix ¼ 384 256. After acquiring T1-weighted MR images, the signal intensity (SI) was measured within a manually drawn region of interest for each mouse. The enhanced values of relative T1 signal were calculated using SI measurements before (SIpre) and after (SIpost) the i.v. injection of contrast agents, using the formula: [(SIpost SIpre)/ SIpre] 100%. 2.6. In vivo SERS imaging Raman imaging experiments were performed on Renishaw inVia Reflex equipped with Streamline rapid Raman imaging system. The MDA-MB-231 tumor-bearing mice treated with intravenously injecting saline or MGSNs (100 ml, 4 mgml1) were subjected to point-scanning using 50 mW of 785 nm laser with 10 acquisitions of 1 s (Fig. S1). Streamline Raman imaging were then obtained with an integration of 5 s, a step size of 1.1 mm and 50 mW of 785 laser power. 2.7. In vivo systematic toxicity A total of 12 healthy Balb/c mice were allocated 4 groups, receiving single intravenous injection of saline (control, 100 ml) or MGSNs (100 ml for each mouse, 4 mgml1) followed by dissection in 3, 15, 30 days postinjection. Blood samples were collected from mice and analyzed and tissues recovered from the necropsy were
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fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for histological examination using standard techniques. After hematoxylin eosin staining, the slides were observed and photos were taken using an optical microscope. 3. Results and discussion 3.1. Characterization of MGSNs The formation of the designed coreeshell structured MGSNs was characterized by various techniques. As shown in the transmission electron microscopy (TEM) images (Fig. 1b and Figs. S2, S3 in the Supporting Information), monodispersed MGSNs have been obtained after coating DTTC-tagged GNSs with organosilica layer and the following modification with Maleimide-DOTA-Gd and Maleimide-PEG via covalent bonding. Obviously, the excellent dispersivity and uniformity in both morphology and dimension of MGSNs are not affected by the modifying steps. Moreover, the coreeshell structure composed of 60 nm gold nanostar and 20 nmthick organosilica layer is clearly exhibited in the highmagnification TEM image in the inset of Fig. 1b, in accordance with DLS result (Fig. S4). Along with the energy dispersive X-ray spectroscopy (EDS) detection (Fig. S5), the co-existence of gold, organosilica and Gd in one nanoparticle can be well confirmed. As far as we know, this is the first report on the functional combination of MR-SERS imaging and photothermal ablation properties into one unit, which could be simply implemented through the direct organosilica coating onto anisotropic gold nanoparticles with abundant thiol groups on the outer surface for facilely performing further modifications. Besides, through the one-pot and in-situ process developed by our group [21], multilayered coreeshell structured GNS@organosilica@Au nanoparticles (Fig. 2) have been successfully fabricated, further demonstrating the versatility of the proposed synthetic strategy [50]. Following the successful fabrication of the coreeshell structure, properties of MGSNs were measured accordingly. As shown in the UVeviseNIR extinction spectra in Fig. 3a, a 54 nm red-shift of MGSNs, relative to GNSs, was recorded due to the higher refraction index of organosilica layer than water [51]. The MGSNs exhibit a strong extinction band at approximately 800 nm, making it highly promising in photothermal therapy with 808 nm laser. In Fig. 3b, the temperature changes of pure water and aqueous solutions loaded with MGSNs are presented, which were recorded at the extinction intensity of 1 as a function of time under continuous
irradiation of 808 nm (0.5 W). It can be found that the temperature of the aqueous solutions loaded with MGSNs increases to 68 C, while no obvious temperature change is detectable for pure water. On the other hand, strong SERS signal from DTTC inside MGSNs can be clearly detected, as shown in Fig. 3c, while neither MGSNs without DTTC nor pure DTTC solution exhibit distinguishable SERS signals, indicating the significant Raman enhancement effect of the unique particle structure. For simplicity, in this paper, 507 cm1 SERS peak of DTTC was employed for SERS intensity determination. Furthermore, the capability of MGSNs to act as an MRI contrast agent was evaluated in terms of r1 relaxivity. The calculated r1value is as high as 8.40 mM1 s1 for MGSNs, much higher than that of 1.41 mM1 s1 for Gd-DOTA, clearly demonstrating the enhanced relaxivity of Gd chelates after their conjugating onto the outer surface of the organosilica layer (Fig. 3d). This observed ~6 times of enhancement in relaxivity is probably due to the restricted molecular tumbling and therefore increased tR rotational correlation time of the Gd chelates on the surface under the protection of PEG chain [23,52]. All these results confirm the successful fabrication of the coreeshell nanostructure which endows MGSNs with significantly enhanced SERS and T1-weighted MR imaging performances, as well as the excellent photothermal conversion capability. To ensure successful photothermal therapy and structurerelated imaging applications, such as SERS, the photothermal stability under NIR laser excitation of MGSNs was tested. After five cycles of laser heating, the thermal conversion efficiency of the assynthesized MGSNs was found to keep constant, while remarkable decrease was recorded for bare GNSs (Fig. 4a). By carefully analyzing the TEM images and UVevis spectra before and after repeated laser irradiation, no changes can be found on the morphology and dispersivity of MGSNs though bare GNSs lost the majority of the NIR absorbance owing to the morphology change (Fig. S6 and 4b). This verifies that the unique coreeshell structure is crucial to maintain the morphology and consequently the superior photothermal conversion efficiency. Surprisingly, PEG could also render GNSs good photothermal stability as PEGylated GNSs did not show any noticeable difference in photothermal conversion, optical properties and TEM images after repeated photo-induced local heating (Fig. 4a, b and S6ced), in contrast to the previous report [53]. However, the Raman stability study shows that the signal of DTTC (at 507 cm1) for PEGylated DTTC-tagged GNSs decreases dramatically in spite of the good photothermal stability (Fig. 4c), while in contrast, SERS signal remains essentially unchanged for MGSNs (Fig. 4d). These results evidence that the DTTC reporters have detached from the surface of PEGylated GNSs under
Fig. 2. TEM images of a) gold seeds grafted GNS@organosilica and b) GNS@organosilica@Au multilayered gold nanoshells.
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Fig. 3. a) UVeviseNIR extinction spectra for the GNSs and MGSNs (bands at 746 nm and 800 nm respectively). b) Temperature rise profiles of pure water and the aqueous solution of MGSNs as a function of heating time under the irradiation by an 808 nm laser (OD808nm ¼ 1, 0.5 W). c) Raman spectra of MGSNs (red line), MGSNs without DTTC (black line) and pure DTTC (blue line) collected under 785 nm excitation at a power of 2 mW and integration time of 10 s. Spectra are offset for clarity and have had their backgrounds removed manually. Samples were dispersed in water. The 507 cm1 SERS peak (pointed by the arrow) was chosen for the SERS intensity measurement. Scale bar: 2000 counts. d) Plot of the relaxation rate (T1 1 ) against Gd concentration for Gd-DOTA and MGSNs. The inset shows T1-weighted MR images of the solutions containing Gd-DOTA and MGSNs at concentrations ranging from 0.003 mM to 0.3 mM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
repeated laser exposure, resulting in significant decrease of SERS signal. However, the coating organosilica layer could protect the reporters from detaching, thus endowing the MGSNs with great Raman stability. These results demonstrate the superiority of organosilica coating and thus the high structural and SERS stabilities of the as-synthesized MGSNs under photothermal conditions.
the concentrations employed (Fig. 5b). Upon laser irradiation, the cell viability significantly decreased under the treatments with MGSNs, only about 10% of 231 cells remaining viable at Au concentration of 20 mgml1 (Fig. 5c). These verify that MGSNs could be used as an effective photothermal ablation agent. 3.3. In vivo MR-SERS imaging and photothermal therapy
3.2. In vitro cell experiments Encouraged by the excellent photothermal conversion capability and stability, the localized photothermal effect in vitro of MGSNs was further investigated by choosing 231 (human breast adenocarcinoma cell line) cells as a cell model. Calcein AM (green) and propidiumiodide (PI, red) co-staining was used to differentiate live and dead cells after photothermal therapy. As shown in the confocal microscopic images (Fig. 5a), a distinct demarcation between the dead (red) and live cell (green (in the web version)) regions can be observed with the presence of both MGSNs and laser exposure. It is obvious that MGSNs could kill the cancer cells only through the photothermal effect induced by NIR laser excitation, while neither the MGSNs nor the laser excitation alone can lead to significant cell death. Furthermore, MTT assay was carried out to quantitatively verify the cytotoxicity and photothermal therapy efficacy of MGSNs. After incubated with normal cells (L-02) and tumor cells (MDA-MB-231) for 24 h without laser irradiation, MGSNs exhibited negligible toxicity to these two types of cells at all
Based on the enhanced r1 relaxivity and strong SERS signal of MGSNs, in vivo bimodal imaging attempts on tumor-bearing mice were carried out. Mice bearing MDA-MB-231 tumor were i.v. injected with MGSNs (100 ml, 4 mgml1) and imaged under a 3.0-T clinical MR imaging system (Fig. 6a). Compared with the image of pre-injection, dramatic brightening effect is detected at the tumor area 30 min after injection of MGSNs. Quantitative analysis of MR signals revealed T1 signal intensity enhancement by 36% (Fig. 6c), suggesting high tumor accumulation of those nanoparticles after systemic administration. These imply that the present MGSNs would be a promising candidate as a contrast agent in MR imaging for cancers. Moreover, MGSNs could also serve as an in vivo contrast agent in noninvasive SERS imaging, which is based on the greatly enhanced Raman signal of DTTC by gold nanostars and offers remarkable sensitivity and resolution compared with traditional optical imaging techniques. As shown in Fig. 6c, distinguishable signal of DTTC at 507 cm1 is detected at different sites of the tumor in 30 min post i.v. injection of MGSNs, while the skin near the
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Fig. 4. a) Cyclic temperature profiles of the suspensions of MGSNs, GNSs-PEG and bare GNSs under irradiation by a 808 nm laser being turned on and off for five repeated cycles. b) UVevis spectra for MGSNs, GNSs-PEG and bare GNSs before and after five cycles of laser heating. Raman spectra of c) DTTC-tagged GNSs-PEG and d) MGSNs after 0e5 cycles of laser heating. Backgrounds of the spectra have been removed manually.
Fig. 5. a) Confocal microscopic images of differently treated MDA-MB-231 cells stained with calcein AM and PI: control; laser irradiation only; MGSNs only; both MGSNs and laser irradiation. b) Relative viabilities of L-02 and MDA-MB-231 cells after incubation with MGSNs for 24 h. c) MDA-MB-231 cells viabilities incubated with MGSNs at different Au concentrations without and with laser irradiation (808 nm, 0.5 W, and 5 min).
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Fig. 6. a) In vivo T1 weighted MR images of a tumor site before and 30 min after intravenous injection of MGSNs (4 mgml1, 100 ml). The tumor sites are marked with the red circles. b) The quantification of average T1-MR signal in the tumor by manual drawn region of interest (red circles in a)). Data represent mean ± SD ***P < 0.001. c) Raman spectra of tumor region after injected intravenously with MGSNs, saline solution, and skin near the tumor under 785 nm excitation at 50 mW of laser power and 10 acquisitions of 1 s (up-right). Scale bar: 2500 counts. d) SERS images at the two different sites of the injected tumor in c), produced by using the baseline corrected intensity of the 507 cm1 Raman band of DTTC with StreamLine ultrafast Raman imaging system. The images were obtained with an integration time of 5 s, a step size of 1.1 mm and 50 mW of 785 laser power. Scale bar: 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
injected tumor and the tumor with saline injection show no distinct Raman signals. Furthermore, Raman images were constructed based on the band of 507 cm1 with StreamLine ultrafast Raman imaging system at the two injected tumor sites, which clearly display the distributions of the MGSNs nanoparticles (Fig. 6d). It is thus confirmed that the as-synthesized MGSNs could enable effective noninvasive SERS imaging in large area over cancer, illustrating the excellent sensitivity and sub-cell-level resolution in vivo. As whole-body MR imaging is able to determine the overall uptake of nanoparticles in the tumor, SERS imaging could vividly display the heterogeneous intratumoral distribution of nanoparticles, beneficial for better planning of therapies for optimized treatment efficacy. The hydrophilic PEG molecules have been used to reduce phagocytic capture of nanoparticles by the immune system, leading to extended circulation and subsequent accumulation in tumors as a consequence of the enhanced permeability and retention (EPR) effect due to leaky vasculature and poor lymphatic drainage in tumors [54e57]. Thanks to their small particle size (
