Theragnostic pH-Sensitive Gold Nanoparticles for the Selective

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Theragnostic pH-sensitive Gold Nanoparticles for the Selective Surface Enhanced Raman Scattering and Photothermal Cancer Therapy. Sungwook Jung, Jutaek Nam, Sekyu Hwang, Joonhyuck Park, Jaehyun Hur, Kyuhyun Im, Nokyoung Park, and Sungjee Kim Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac401390m • Publication Date (Web): 24 Jul 2013 Downloaded from http://pubs.acs.org on August 5, 2013

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Analytical Chemistry

Theragnostic pH-sensitive Gold Nanoparticles for the Selective Surface Enhanced Raman Scattering and Photothermal Cancer Therapy. AUTHOR ADDRESSES. Sungwook Jung,† Jutaek Nam,‡ Sekyu Hwang,‡ Joonhyuck Park,† Jaehyun Hur,§ Kyuhyun Im,§ Nokyoung Park,§ and Sungjee Kim*,†, ‡ †

School of Interdisciplinary Bioscience and Bioengineering and ‡Department of Chemistr,

Pohang University of Science & Technology (POSTECH), San 31, Hyojadong, Namgu, Pohang 790–784, Korea §

Frontier Research Laboratory, Samsung Advanced Institute of Technology, Samsung

Electronics, Yongin, Kyunggi-do 446-712, Korea Keywords: SERS, gold nanoparticle, photothermal therapy, aggregation, cancer theragnosis ABSTRACT: We report a nanoparticle-based probe that can be used for ‘turn-on’ theragnostic agent for simultaneous Raman imaging/diagnosis and photothermal therapy. The agent consists of 10 nm spherical gold nanoparticle (NP) with pH-responsive ligands and Raman probes on the surface. They are engineered to exhibit the surface with both positive and negative charges upon mildly acidic condition, which subsequently results in rapid aggregations of the gold NPs. This

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aggregation simultaneously provides hot spots for the SERS probe with the enhancement factor reaching 1.3×104 and shifts the absorption to far-red and near-infrared (which is optimal for deep tissue penetration) by the coupled plasmon resonances; this shift was successfully exploited for low-threshold photothermal therapy. The theragnostic gold NPs are cancer-specific because they aggregate rapidly and accumulate selectively in cancerous cells. As the result, both Raman imaging and photothermal efficacy were turned on under cancerous local environment. In addition, the relatively small hydrodynamic size can have the potential for better access to targeted delivery in vivo and facilitated excretion after therapy.

1. Introduction Gold Nanoparticles (NPs) show distinctive optical properties resulting from the surface plasmon resonance (SPR).1 Gold NPs can resonantly absorb and scatter incident light upon excitation of their surface plasmon oscillations with their absorption cross sections that are orders of magnitude larger than those of strongly-absorbing organic molecules.2 The SPR characteristics of gold NPs depend on their size, shape and structure, so their optical properties can be easily tuned.2-3 Gold NPs are also ‘non-toxic’, so they are considered relatively biocompatible. Gold NPs have applications in surface-enhanced Raman scattering (SERS) imaging4 and photothermal cancer therapy.5 SERS is a non-invasive vibrational spectroscopic technique that exploits intense Raman scattering on or near the surface of metal nanostructures.6 SERS enhancement originates from chemical and electromagnetic (EM) contributions; the EM enhancement is typically predominant.7 Gold NPs can induce large increase in SERS by the strong local EM field enhancement near their surfaces after excitation of the localized SPR.8 Gold NP-based SERS can allow sensitive optical imaging because of the robustness against photobleaching, narrow spectral bandwidth, and high signal-to-noise ratio in complex biological systems.4a, 4b In addition,


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gold NPs undergo rapid nonradiative thermal relaxation, which can efficiently convert absorbed photons into heat. Therefore, the strong absorption and efficient heat conversion of gold NPs suggest that they can be

highly potent photothermal therapeutic agents.9 For efficient

photothermal therapy in deep tissues, gold NPs should have SPR characteristics in near-infrared (NIR) wavelengths that can penetrate deeper into tissues than visible light. Various gold nanostructures that can absorb NIR, including gold nanorods,5c-e nanoshells5f, 5g, nanocages,5h, 5i and popcorns5j have been developed for photothermal therapy using NIR excitation. In a few cases, gold nanostructures were designed to combine the SERS and photothermal therapy capablities; this combination allows simultaneous detection and therapy of target sites.5c, 5j, 10 Bhatia et al. reported SERS-active gold nanorods where Raman signals from implanted tumors were found in mice and ablative elevated temperatures were obtained upon laser irradiation.5c Ray et al. demonstrated popcorn-shaped gold NPs conjugated with Raman dyes; these NPs showed enhanced Raman signals and simultaneous photothermal destruction of cancer cells. 5j Liu et al. reported use of single walled-carbon nanotubes conjugated with gold NPs for cancer cell imaging and photothermal effects.10 These theragnostic NPs have the potential to maximize their therapeutic actions by real-time monitoring of the therapeutic biodistributions and responses at the sites of interest. However, some difficulties must be overcome before such theragnostic use of NPs becomes clinically feasible. Typical gold nanostructures of which SPR occurs in the NIR range have a large hydrodynamic size of >100 nm, which critically limits NP biodistribution upon administration and may impede their excretion.11 They also typically possess anisotropic shapes with sharp tips or rough surfaces for the enhanced SERS effect, which makes them difficult for uniform synthesis or simple scale-up. In addition, such NPs tend to reshape into a spherical form upon repeated excitations; this shape lacks SERS and photothermal


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therapeutic capabilities.5j, 12 Moreover, SERS and the photothermal effect of NPs are not turn-on based and their selectivity can be best achieved

using targeting moiety (i.e., antibody)

conjugations. Here we report turn-on based selective SERS and photothermal theragnostic gold NPs which can overcome the limitations caused by agents that depend on affinity or on concentration gradients. Previously, we reported simple, spherical ‘smart’ gold NPs that were designed to selectively form aggregates under mildly acidic conditions such as occur within animal cells.13 The ‘smart’ gold NPs are 10 nm gold spheres with surface molecules that contain hydrolysissusceptible citraconic amide units (‘smart’ ligands). The NP surfaces were engineered to have both positive and negative charges which induce rapid aggregation of the NPs by electrostatic interactions under mildly acidic conditions. The pH-responsive formation of aggregates shifts the absorption to the NIR region due to the appearance of coupled plasmon modes. This shift can be successively utilized for selective and deep-tissue-penetrating photothermal therapy which has effectiveness that is comparable to that of other gold nanostructures such as nanorods,5d,

5e

nanoshells,5f, 5g and nanocages. 5h, 5i We report a ‘smart’ gold NP-based theragnostic platform that exploits the selective and efficient SERS and photothermal effect of the gold NP aggregates (Scheme 1).

2. Experimental section Materials and instruments All chemicals were purchased from Sigma-Aldrich and used as received without further purification. Water was triply distilled using a Millipore filtration system. The particle hydrodynamic (HD) size was measured using a Malvern zetasizer Z. TEM images were recorded


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using a JEOL JEM-1011 in POSTECH biotech center. UV-VIS absorption spectra were obtained using an Agilent 8453. Dark-field images were recorded using a Zeiss Axioplan 2 microscope with a highly numerical dark-field condenser (0.75-1.0) and a 100× /1.3 oil Iris objective (Zeiss). The dark-field pictures were taken using a Zeiss Axiocam HR camera. SERS measurements were carried using Bruker SENTERRA dispersive Raman microscope in POSCO Research Institute of Industrial Science & Technology. Raman mapped-images were obtained using a Witech alpha 300R Raman microscope.

Synthesis of MBA/SMART-AuNPs and MBA-AuNPs 4-(2-(6,8-dimercaptooctanamido)ethylamino)-3-methyl-4-oxobut-2-enoic acid (‘smart’ ligand) was synthesized as described previously.13 ‘Smart’ ligands (9 mg, 25 µmol) and NaBH4 (1.9 mg, 50 µmol) were added to 2.5 ml distilled water, then stirred for 1 h. Then, 20 mM aqueous solution of 4-mercaptobenzoic acid (4-MBA, 2.5 ml, 25 µmol) was added to the reduced ‘smart’ ligand solution, and stirred for 5 min then 100 nM citrate gold NPs (50 ml, 5 nmol) were added to the solution. After stirring overnight, the resultant AuNP solution was dialysed using Amicon ultra 50 KDa Mw cutoff centrifugal filters to eliminate ligands that had not attached to NPs. MBA-AuNPs was prepared using the same procedure except that ‘smart’ ligands were not added.

Solution Raman measurement MBA/SMART-AuNP solution (550 nM, 0.9 ml, 0.5 nmol) was mixed with 10 µl of 2 mM HCl to make a pH 5.5 solution, then 50 µl of MBA/SMART-AuNP solution was immediately dropped into a concave slide glass and covered with a cover glass. The solution color gradually changed to violet-blue, indicating pH-induced aggregation. The slide glass was placed on the


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Bruker SENTERRA dispersive Raman microscope and excited using 785 nm laser (10 mW, 5 s) with a 10× objective lens. Stokes-shifted Raman scatterings were collected in the range of 100 to 3700 cm-1 with 9 - 15 cm-1 resolution.

Cell experiments B16 F10 mouse melanoma cells were incubated in Dulbecco’s Modified Eagle Medium (DMEM, HyClone)

supplemented

with

10%

fetal

bovine

serum

(FBS,

GIBCO)

and

1%

penicillinstreptomycin (PS). Cells were grown on 12 mm glass coverslips (for dark-field imaging) or in 24-well plates at a density of 1×105 cells/well at 37 °C under 5% CO2 (for in vitro photothermal therapy). After 24 h, cells were incubated with MBA/SMART-AuNPs, MBAAuNPs (100 nM each), or in cell growth medium only. For dark-field imaging, coverslips were fixed using 4% formaldehyde and mounted on slide glass using aqueous mounting medium with anti-fading agent (Biomeda). Cells were rinsed with culture media (DMEM) and exposed to laser illumination for in vitro photothermal therapy. Then they were stained with 0.4% trypan blue for 5 min to test cell viability. Raman mapped-images were obtained under 532 nm laser irradiation (0.5 mW, 0.5 s) using a Witech alpha 300R Raman microscope. The laser light source passed through 50× objective and Raman scatterings were collected using a Peltier cooled CCD. Spectral range was from 500 cm-1 to 3500 cm-1 and the resolution was < 1 cm-1.

Simultaneous SERS-diagnosis and photothermal therapy B16 F10 cells were incubated in DMEM supplemented with 10% FBS and 1% PS. The cells were grown on a chambered slide glass (Lab-Tek, a 8-well plate) as a population of 2×104 cells/well at 37 ℃ and 5% CO2 condition for 24 h. The cells were additionally incubated with


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MBA/SMART-AuNPs or MBA-AuNPs (40 nM, or 100 nM). After 12 h or 24 h, a chamber was removed from the well plate and a cover glass was placed on the cell-incubated slide glass. The slide glass was putted on Brucker SENTERRA dispersive Raman microscope system and laser light (785 nm, 25 mW) irradiated the cells for 60 s or 5 s through a 10× or 40× objective lens. Stokes-shifted Raman scatterings were collected in the range of 100 to 3700 cm-1 with 9 – 15 cm1

resolution. Subsequently, the identical cells were exposed to 785 nm laser illumination with

19.5 W/cm2 power density through 10× objective lens for 10 m. Output power from the objective lens was measured with powermeter (Thorlabs, PM100) and the irradiated area was calculated from laser light images recorded on the CCD camera.

3. Results and discussion Gold NPs and the ‘smart’ ligand were synthesized as previously described.13 The mixed ligand exchange method was used to prepare gold NPs that had both ‘smart’ and 4-mercapto benzoic acid (4-MBA) ligands.14 The 4-MBA was used as a model Raman reporter surface molecule. A 1:1 mixture of 4-MBA and ‘smart’ ligand was introduced to 10 nm citrate gold NPs. The resultant gold NPs (MBA/SMART-AuNPs) retained hydrodynamic size similar to that of ‘smart’ gold NP (Figure S1 for details). We hypothesize that if the partially-covered ‘smart’ ligands can pH-responsively form gold NP aggregates, they may have many ‘hot spots’ for the Raman probes in the nanometer-sized gaps within their internal structures; such gaps can cause a huge local EM field enhancement.15 This Raman enhancement can be selectively induced by changing the pH. At the same time, the gold NP aggregates can serve as photothermal therapeutic agents; this therapy is also selective because the gold NPs only absorb NIR excitations after forming aggregates. Strategies to induce gold NP aggregation for SERS enhancement include: (i) salt-


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induced charge screening,16 (ii) electrostatic interactions between oppositely surface-charged NPs,17 (iii) hydrogen bonding between NP surfaces,15a (iv) covalent crosslinking of NP surfaces by DNA-supported photoligation,18 (v) direct multivalent assemblies with small or supramolecules,19 and (vi) in situ aggregate synthesis mediated by sulfur.20 Unlike previously reported strategies, our MBA/SAMRT-AuNP can form unique aggregations by electrostatic interactions between mixed surface-charged NPs, and the induction of aggregation can be programmed by the pH of their local environment. As a control to MBA/SMART-AuNPs, gold NPs surface-modified solely with 4-MBA (MBAAuNPs) were prepared. Average numbers of surface ligands on the MBA/SMART-AuNPs and MBA-AuNPs were determined by spectrophotometric measurements after dissolving the AuNPs using KCN (Figure S2 and S3). MBA/SMART-AuNP had an average of ~620 surface ligands, 60% with 4-MBA and the rest with the ‘smart’ ligand. MBA-AuNPs had an average of ~1400 surface ligands (Figure S3). To confirm whether the partial coverage of ‘smart’ ligands on MBA/SMART-AuNPs can effectively respond to pH change, aggregation tests were performed using buffer solutions. MBA/SMART-AuNPs were placed in 10 mM pH 5.5 acetate buffer (Figure 1A) or in 10 mM pH 7.4 phosphate buffer (Figure 1B), and their absorption spectra were monitored. Over time, the spectrum of the pH 5.5 sample gradual red-shifted and broadened; this is ascribed to the appearance of coupled plasmon modes and the inhomogeneity of the aggregates.21 The pH 7.4 sample showed no noticeable change in absorption wavelength up to 60 min. As a control, MBA-AuNPs were placed in the pH 5.5 buffer; no noticeable absorption change was observed (Figure 1C). These results confirm the pH-responsive aggregation capability of MBA/SMART-AuNPs. Hydrodynamic size was measured over time in the two pH 5.5 samples of MBA/SMART-AuNPs and the control MBA-AuNPs. The MBA/SMART-AuNP


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showed rapid increase in hydrodynamic size, reaching 450 nm at 60 min, and the solution color changed from pink-red to violet-blue (Figure 1D and 1E). In contrast, the control MBA-AuNPs retained the initial hydrodynamic size throughout the period, and the solution color remained pink-red (Figure 1D and 1E). Time evolution of the MBA/SMART-AuNP aggregates was further studied using dark-field optical microscopy and transmission electron microscopy (TEM) (Figure 1F). Under the dark-field microscope, the scatters sequentially appeared as green, orange, red, and white as the sizes of aggregates increased over time; this result coincides well with TEM measurements. The TEM measurements also confirmed that the aggregates contain many nanometer-sized internal gaps that can form SERS hot spots. The average gap distance was measured as 2.9 nm (Figure 1G). The gap distance histogram shows that the aggregates have many nano-sized gaps that suite well for the effective plasmon coupling between nearby gold NPs and for the large EM enhancement.22 These results clearly demonstrate that MBA/SMARTAuNPs show the pH-responsive aggregation capability and that the aggregates contain many nanometer-sized internal gaps that suite well for SERS enhancement. As pH-induced aggregation was confirmed for MBA/SMART-AuNPs, the SERS effect in the gold NP aggregates due to the internally created nano-sized gaps was studied. MBA/SMARTAuNP and control MBA-AuNP samples (500 nM) in pH 5.5 solution were excited using a 785 nm laser (10 mW, 5 s) with a focused spot diameter of 20 μm, and the Raman spectra were recorded over time. For controls, MBA/SMART-AuNP and MBA-AuNP in pH 7.4 solutions, and 1 M MBA solution were used. The MBA/SMART-AuNP sample rapidly showed the enhanced Raman spectrum as early as 1 min after dispersion in pH 5.5 solution and the enhancement increased slightly over time (Figure 2A, left). To the contrary, all control samples in pH 7.4, the MBA-AuNP sample in pH 5.5, and the pure MBA solution did not show


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noticeable Raman scatterings regardless of the elapsed time (Figure 2A, right). The MBA-AuNP samples failed to show Raman scattering under our optical setup because dispersed individual 10 nm gold NPs cannot effectively induce light concentration and field enhancement.15a,

17b, 23

However, the MBA/SMART-AuNP aggregates showed large SERS. It is noted that an identical kind of gold NP was used for both MBA/SMART and MBA-AuNP. The SERS effect is attributed to the enhanced Raman scattering cross-sections in the internal nano-sized gap hot spots. Raman peak shifts and enhancement factors (EFs) were determined over time for the MBA/SMART-AuNP pH 5.5 sample (Figure 2B and 2C). The ring breathing Raman modes appeared at 1077 and 1588 cm-1, which were down-shifted by 19 and 6 cm-1 respectively, compared with the free 4-MBA signals.24 This peak shift is attributed to electron donation from 4-MBA to gold atoms through the surface-anchored sulfur atom.25 SERS EFs were determined as ~ 1×104 with slight increase over time. This increase accords well with the TEM measurements, in which small aggregates formed rapidly, then grew over time. The average inter-NP gap size remained unchanged over time as the aggregates grew, and thus the enhancement characteristics of the hot spots also seemed to remain unchanged. As aggregate size increases over time, the number of hot spots may increase and as a result EF may increase slightly. The maximal EF was 1.3×104, which agrees well with other reports using gold NP aggregates.17b Since pH-induced aggregation of MBA/SMART-AuNPs and their subsequent SERS enhancement were demonstrated, we further applied them for cellular Raman imaging. Most cells, especially cancerous ones including B16 F10 mouse melanoma cells, internalize NPs.14 ‘Smart’ gold NPs show cancer specificity over normal cells because they accumulate more efficiently than for normal cells.13 Cancer cells are more phagocytic than the normal and as the result ‘smart’ gold NPs get rapidly accumulated as their exocytosis is effectively blocked by the


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increased size of the aggregates. MBA/SMART-AuNPs and control MBA-AuNPs (100 nM each) were co-incubated with B16 F10 cells for 24 h. Efficient accumulations of MBA/SMARTAuNPs were observed as strong scatters under a dark-field microscope (Figure 3A, first column). The aggregated MBA/SMART-AuNPs visualize the morphology of the cells well. In contrast, no meaningful cellular NP uptake was observed in the MBA-AuNP control. To confirm the colloidal stability in the cell medium, MBA/SMART-AuNPs were placed in cell medium (Dulbecco’s Modified Eagle Medium) at 37 ℃ in 5% CO2 and the hydrodynamic size was monitored for 24 h. No noticeable change in the hydrodynamic size was found, which confirms the colloidal stability and the pH-responsiveness of MBA/SMART-AuNP (Figure S4). Raman cellular imaging experiments were performed using reconstruction from integrative Raman signals (1550 – 1650 cm-1 range) for a sample area of 40 μm × 40 μm with 532 nm laser excitation (0.5 mW, 0.5 s) of a focused spot of 0.5 μm diameter. Cells co-incubated with MBA/SMART-AuNPs showed well-correlated bright-fields and Raman mapped images revealing the cell morphologies (Figure 3A, middle and right columns). The MBA-AuNP control sample did not show meaningful Raman-mapped images. Four different positions were taken for spectroscopic measurements (Figure 3A, middle column): (a) the background area outside and (b) center area of a cell treated with MBA/SMART-AuNPs, and (c) the background area outside and (d) center area of a cell treated with MBA-AuNPs (Figure 3B). Only position (b) showed Raman signals; this result suggests that the SERS signal originated from aggregated MBA/SMARTAuNPs in the cells. Averaged Raman spectra were also obtained by merging the entire signals from the Raman mapping in Figure 3A, and only MBA/SMART-AuNP sample showed the SERS effect of which spectrum matches well with that from position (b) in Figure 3A. This


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result clearly demonstrates that MBA/SMART-AuNPs can function as a ‘turn-on’ Raman probes that are selective for cancerous cells. For MBA/SMART-AuNPs to be applied as theragnostic agent, they must have therapeutic function as well as imaging/probing capability. As confirmed in Figure 1, MBA/SMART-AuNPs can respond to pH change and form aggregates which cause a shift in absorption wavelengths to far-red and NIR. We studied whether this pH-responsiveness of the MBA/SMART-AuNP can be effectively exploited for photothermal therapy which can potentially applied for deep tissues (Figure 4). MBA/SMART-AuNPs (100 nM) were co-incubated with B16 F10 cells for 24 h. As controls, cells were incubated with MBA-AuNPs or without any NPs (‘No Au NP’). A cw diode laser of 660 nm was used for irradiation. It is stressed that the gold NPs cannot absorb this excitation wavelength unless they form aggregates. Circles in Figure 4 represent the positions of laser spots that have a ∼500 μm radius. The cells were irradiated using the laser for 10 min at power densities of 8, 10.5, 13, or 15.5 W/cm2. Trypan blue was used to stain the mortality of cells as the distinctive blue color. Cells treated with MBA/SMART-AuNPs began to die at a laser power ≥ 10.5 mW/cm2, and the number of damaged cells in the irradiated area increased linearly with the irradiation power density. MBA/SMART-AuNPs showed threshold behavior for photothermal therapy with the threshold occurring between 8 and 10.5 W/cm2. No noticeable mortality occurred in cells outside of the laser spot regardless of laser fluence power, so the NPs were minimally cytotoxic under dark conditions. In contrast, MBA-AuNP and ‘no gold NP’ samples failed to induce cell mortality under irradiation conditions identical to those used for MBA/SMART-AuNPs. These results indicate that MBA/SMART-AuNPs can be potent photothermal agents that selectively target cancerous cells; furthermore, the threshold laser power at which MBA/SMART-AuNPs induce cytotoxicity is comparable to those of other


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reported NIR-absorbing gold nanostructures such as nanorods (10~26.4 W/cm2),5d, 5e nanoshells (35~80 W/cm2),5f,

5g

and nanocages (1.5~2.4 W/cm2).

5h, 5i

Our MBA/SMART-AuNPs are

considered to show high photothermal efficacy due to their efficient internalization by cancerous cells and the subsequent massive accumulation of NP aggregates. SERS detection for cancer cells and the photothermal therapy have been demonstrated by MBA/SMART-AuNP. We have further pursued if MBA/SMART-AuNPs can be useful for theragnostic applications such as ‘simulateous‘ diagnosis and therapy of cancer where Raman signal is used to guide to the tumor area and to focus in on an area to interrogate with a laser to induce phototherapy effect. B16 F10 cells were co-incubated with 40 nM MBA/SMART-AuNPs for 12 h. Control cells were also prepared using 40 nM MBA-AuNPs or with no AuNPs. Upon a microscope system equipped with 785 nm laser, Raman spectra were acquired for the samples. The MBA/SMART-AuNP sample showed the distinct Raman peaks, while the MBA-AuNP and no AuNP control samples showed no detectable peaks. This accords with the optical micrographs where aggregates of MBA/SMART-AuNPs were observed as black spots inside of the cells. Representitve spots in the MBA/SMART-AuNP sample and the control samples were irradiated for 10 min by the same 785 nm laser with 19.5 W/cm2 power density (Figure 5). For the MBA/SMART-AuNP sample spots, distortion in the cellular morphology was observed which is indicative of the cell mortality (a1, a2, and a3 in Figure 5A). No noticeable change was found for the control MBA-AuNP and no AuNP samples under identical laser irradiation. The experiment was repeated with higher NP concentraion (100 nM) and longer co-incubation time (24 h). MBA/SMART-AuNP sample showed selective response on the Raman signal and the cell mortality after the laser irradiation (Figure 5B). The response was more notable than the case of 40 nM and 12 h treatment because of the larger accumulations of MBA/SMART-AuNPs inside


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of the cells. A larger NP uptake is expected for the MBA/SMART-AuNPs versus MBA-AuNPs by the cells. The optical microscope images also suggest the larger uptake of MBA/SMARTAuNPs. The MBA/SMART-AuNP probe is designed to respond to pH change, assemble in mild acidic intracellular environment, and accumulate by effectively blocking the cellular exocytosis pathways. However, the ‘turn-on’ theragnostic function of our probe is mainly attributed to the selective assembly of the NPs rather than the simply enhanced cellular uptake level. The theragnostic function of our MBA/SMART-AuNP showcases the potential clinical applicability detecting/diagnosing and treating/curing the cancer as a concurrent procedure; this can lead to more accurate and efficient intervention of cancerous tumors.

4. Conclusions We have demonstrated a ‘turn-on’ MBA/SMART-AuNP probe that can be used for simultaneous Raman imaging/diagnosis and photothermal therapy. The NP surface was engineered with pH-responsive surface ligands and Raman probes. Under mildly acidic conditions, the MBA/SMART-AuNPs aggregate rapidly and accumulate selectively in cancerous cells. This aggregation simultaneously provides hot spots for SERS with the EF reaching 1.3×104 and shifts absorption to far-red and NIR; this shift was successfully exploited for low-threshold photothermal therapy. We stress that this MBA/SMART-AuNP has demonstrated utility as a theragnostic probe that turns on under specific environmental conditions. It can promise a versatile platform for many theragnostic applications because of its simple extendibility by simple surface ligand modification. In addition, the relatively small hydrodynamic size can have the potential for better access to targeted delivery in vivo and facilitated excretion after therapy.


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pH 7.4 pH 5.5, 1 min pH 5.5, 10 min pH 5.5, 30 min pH 5.5, 60 min

1

0 500

600

700

800

C

B Normalized absorbance

A Normalized absorbance

900

pH 7.4, 1 min pH 7.4, 10 min pH 7.4, 30 min pH 7.4, 60 min

1

0 500

600

Wavelength (nm)

700

800

pH 7.4 pH 5.5, 1 min pH 5.5, 10 min pH 5.5, 30 min pH 5.5, 60 min

1

0

900

500

600

Wavelength (nm)

D Hydrodynamic size (nm)

700

800

900

Wavelength (nm)

E MBA/SMART-AuNP MBA-AuNP

500

MBA/SMART-AuNP

MBA-AuNP

400 300 200 100 0 0

20

40

pH 7.4

60

pH 5.5

pH 7.4

pH 5.5

Time (min)

F

50 nm

0 min

1 min

200 nm

10 min

200 nm

30 min

60 min

500 nm

500 nm

0. 2 탆

G

0. 5 탆

0.2 탆 0. 5 탆

50 40

Frequency (%)

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


Normalized absorbance

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20 nm

5 nm

30 20 10 0

0

1

2

3

4

Gap distance (nm)

5

6

Figure 1. pH-induced aggregation of MBA/SMART-AuNPs. Time evolution of absorption for MBA/SMART-AuNPs in 10 mM pH 5.5 acetate buffer (A) and in 10 mM pH 7.4 phosphate buffer (B). MBA-AuNPs in 10 mM pH 5.5 acetate buffer (C); (D) Hydrodynamic sizes of samples in pH 5.5 acetate buffer at different elapsed time; (E) Photographs of samples after 60 min in pH 7.4 or pH 5.5 buffer; (F) Dark-field optical microscope images (top row, scale bar: 10 μm) and TEM images (bottom row) of MBA/SMART-AuNPs in pH 5.5 acetate buffer with


15


different elapsed times; (G) Magnified TEM image (left) of MBA/SMART-AuNP aggregates (middle image shows nanometer-sized gaps) and histogram (right) for interparticle gap distances after 60 min in pH 5.5 acetate buffer. The histograms and average gap distances were obtained by measuring 400 different gaps.

A

MBA/SMART-AuNP

MBA-AuNP 15 counts

90

60

1 M MBA pH 5.5, 60 m pH 5.5, 30 m pH 5.5, 10 m pH 5.5, 1 m pH 7.4

30

0

1000

1200

1400

1600

1000

Raman shift (cm-1)

1200

1400

1600

Raman shift (cm-1)

B

C

1600

-1

MBA (1595 cm-1) (1588 cm-1)

1590

MBA (1096 cm-1)

1090 1080 1070

(1077 cm-1)

0 10 20 30 40 50 60 70

Incubation time (min)

Enhancement factor

(X 104)

Raman shift (cm )

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

Page 16 of 26

-1

1077 cm -1 1588 cm

1.3 1.2

1.1 1.0

0

10 20 30 40 50 60

Incubation time (min)

Figure 2. SERS effect of MBA/SMART-AuNPs. (A) Time evolution of Raman scattering for MBA/SMART-AuNPs (left) and MBA-AuNPs (right) in pH 5.5. Control samples in pH 7.4 and 1 M MBA solution are used for comparison; (B) Raman peak shifts of the 4-MBA ring breathing modes for MBA/SMART-AuNP sample; (C) SERS enhancement factors for the MBA/SMARTAuNP sample measured at 1077 and 1588 cm-1.


16

Page 17 of 26

A MBA/SMARTAuNP

Dark-field

Raman mapping

(b) (a)

(X 102)

20 μm

10 μm

7 6 5 4 3 2 1

10 μm

MBA-AuNP

(c)

0

(d) 20 μm

10 μm

1000 900 800 700

1000

10 μmμm 10 10 μm

C

(a) (b) (c) (d)

1100

1200

1400

Raman shift (cm

1600

-1

)

Raman intensity (a.u.)

B Raman intensity (a.u.)

Bright-field

SERS intensity (a.u.)

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


1000 950

MBA-AuNP MBA/SMART-AuNP

900 850 800 1000

1200

1400

Raman shift (cm

1600 -1

)

Figure 3. Optical and SERS microscopic imaging of B16 F10 cells. (A) Dark-field (first column), bright-field (second column) optical microscope images and Raman-mapped images (third column) of B16 F10 cells incubated with MBA/SMART-AuNPs or MBA-AuNPs. Spots (a) and (c) represents background area outside of the cells, and spots (b) and (d) do area for cells; (B) Raman spectra obtained from different positions of the cell samples as indicated by arrows in the bright-field images; (C) Averaged Raman signals from the entire sample areas.


17


MBA-AuNP

No AuNP

13 W/cm2

10.5 W/cm2

8 W/cm2

MBA/SMART-AuNP

15.5 W/cm2

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

Page 18 of 26

Figure 4. Photothermal therapy. B16 F10 cells were co-incubated with MBA/SMART-AuNPs (left column), MBA-AuNPs (middle column), or with no NPs (right column); Laser fluence rates are 8, 10.5, 13, and 15.5 W/cm2 from top to bottom rows. Circles: position of laser spot. Scale bar: 100 μm.


18

Page 19 of 26

A

SERS diagnosis

Photothermal therapy

a1 a2 a3

2000 counts


MBA/SMART-AuNP

0 W/cm2 (a1)

19.5 W/cm2

(a3)

(a2)

1000

1200

1400

1600 -1

Raman shift (cm ) b1 b2 b3


MBA-AuNP

2000 counts

(b2)

(b1)

1000

1200

1400

(b3)

1600 -1

Raman shift (cm ) c1 c2 c3


No AuNP

2000 counts

(c1) (c2)

1000

1200

1400

1600

(c3)

-1

Raman shift (cm )

B

SERS diagnosis

Photothermal therapy 19.5 W/cm2

10 counts


MBA/SMART-AuNP

0 W/cm2

1100

1200

1300

1400

1500

-1

1600

Wavelength (cm ) 10 counts


MBA-AuNP

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


1100

1200

1300

1400

1500

-1

1600

Wavelength (cm )


19


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

Page 20 of 26

Figure 5. Theragnostic SERS-based diagnosis and photothermal therapy of cancer cells using MBA/SMART-AuNPs. B16 F10 cells were co-incubated with 40 nM (A) or 100 nM (B) of MBA/SMART-AuNPs for 12 h (A) or 24 h (B). Co-incubations with MBA-AuNPs were used for control (second row in A and B). Another control with no AuNP was also performed (third row in A). An identical 785 nm laser was used for both the Raman spectra acquisition (25 mW, 5 to 60 seconds for the exposure time) and photothermal therapy (19.5 W/cm2 power density for 10 min exposure time). Left columns in A and B show the Raman spectra for the pointed areas prior to the photothermal therapy, and right columns compare the bright-field microscope optical images before and after the photothermal therapy. Diameter of the focused laser spot was 17 μm. Scale bars show 50 μm in (A) and 10 μm (B), respectively.


20

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


Hot spot: Enhanced EM field -

-

SERS imaging

-

Laser -

-

-

-

-

+

-

-

+-

+

-

+

-

-

+

-

+ +-

-

+

-

N H

+

+

-

-

-

-

-

+

-

-

-

-

+

Cancer cell

-

-

Photothermal therapy

-

O S S H H

-

+

+

+

-

-:

+

+-

+

-

+-

+

- -

-

-

-

+

+

+

-

-

+

-

-

O

H N

-O

O

‘Smart’ ligand

: MBA/SMART-AuNP

O

+:

S S H H

N H

Hydrolyzed ‘smart’ ligand

NH+ 3

:

OH HS

C

-

O

4-mercaptobenzoic acid

: MBA/SMART-AuNP aggregate

Scheme 1. Schematic illustration of the working mechanism of MBA/SMART-AuNP in a cancer cell. Upon internalization by cell, MBA/SMART-AuNPs form gold NP aggregates as the result of surface charge conversions of the NPs. The gold NP aggregates can form surface enhanced Raman scattering imaging probes at the internally created hot spots where Raman scatterings are greatly enhanced. At the same time, they form photothermal therapeutic agents upon nearinfrared excitation of surface plasmons.


21


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Page 22 of 26

ASSOCIATED CONTENT Hydrodynamic sizes of gold NPs; Surface ligand coverage calculation using UV-VIS absorption spectroscopy; Hydrodynamic size time evolution of MBA/SMART-AuNPs dispersed in cell medium. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Phone: (+82) 054-279-2108. Fax: (+82) 054-279-1498. E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by a Korea Science and Engineering Foundation grant funded by Ministry of Science and Technology (20120006280), the Korea Health 21 R&D Project Ministry of Health & Welfare (A101626), the Priority Research Center Program through National Research Foundation of Korea (NRF) (2011-0031405 and 20110027727), NRF (20120005973), and the Basic Science Research Programs (20110027236). REFERENCES (1) Eustis, S.; El-Sayed, M. A., Chem. Soc. Rev. 2006, 35, 209-217 (2) Jain, P. K.; Lee, K. S.; El-Sayed, I. H.; El-Sayed, M. A., J. Phys. Chem. B 2006, 110, 72387248 (3) Huang, X.; El-Sayed, M. A., J. Adv. Res. 2010, 1, 13-28


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For Table of Contents Only


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Theragnostic pH-Sensitive Gold Nanoparticles for the Selective

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