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Realizing a Record Photothermal Conversion Efficiency of Spiky Gold Nanoparticles in the Second NearInfrared Window by Structure-based Rational Design Cuixia Bi, Jin Chen, Ying Chen, Yahui Song, Anran Li, Shuzhou Li, Zhengwei Mao, Changyou Gao, Dayang Wang, Helmuth Möhwald, and Haibing Xia Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b00312 • Publication Date (Web): 29 Mar 2018 Downloaded from http://pubs.acs.org on March 30, 2018
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Chemistry of Materials
Realizing a Record Photothermal Conversion Efficiency of Spiky Gold Nanoparticles in the Second Near-Infrared Window by Structure-based Rational Design Cuixia Bi,† Jin Chen,‡ Ying Chen,‡ Yahui Song,† Anran Li,¶ Shuzhou Li,ξ Zhengwei Mao,‡,* Changyou Gao,‡ Dayang Wang,§ Helmuth Möhwaldς and Haibing Xia† * †State
Key Laboratory of Crystal Materials, Shandong University, Jinan, 250100, P. R China; Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China; ¶Key Laboratory of Bio-Inspired Smart Interfacial Science and Technology, Ministry of Education, School of Chemistry, Beihang University, Beijing 100191, P. R. China; ξSchool of Materials Science and Engineering, Nanyang Technological University, Singapore 639798; §State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, Changchun, 130012, P. R. China; ‡MOE
ς
Max Planck Institute of Colloids and Interfaces, Potsdam-Golm Science Park, 14476 Potsdam, Germany.
ABSTRACT: The current technical dilemma for gold nanoparticles as photothermal (PT) transducers in cancer therapy is that strong absorption in the second near-infrared (NIR) window is accompanied by strong scattering of the NIR light, which then overrides the absorption, thus significantly weakening the light-to-heat conversion efficiency. Here we successfully prepared spiky gold nanoparticles (spiky Au NPs) with a controlled number of spikes, designed according to our simulations and experimentally verified. Their overall sizes and the numbers, lengths and widths of the spikes were judiciously adjusted to locate their surface plasmon resonance peaks in the second NIR window and also to achieve a higher absorption to extinction ratio. As a result, the spiky Au NPs with optimal size and 6 spikes exhibited a record light-to-heat conversion efficiency (78.8%) under irradiation by 980 nm light. After surface PEGylation and conjugation with a lactoferrin (LF) ligand on the resulting spiky Au NPs, they in vivo displayed long circulation time (blood circulation half-life of ~ 300 min) and high tumor accumulation due to their larger surface-to-volume ratio. Therefore, spiky Au NPs allowed complete ablation of tumors without recurrence merely after 3 min light irradiation at 980 nm, opening up promising prospects of cancer photothermal therapy.
INTRODUCTION Photothermal (PT) therapy has been intensively developed as a minimally invasive approach for cancer treatment.1-3 Among the PT transducers developed thus far,4-14 spiky gold nanoparticles (Au NPs), namely nanoparticles with noticeable branches, are of particular interest,6,15-21 since their sharp tips and the high surface-to-volume ratios can significantly enhance light-to-heat conversion compared to those with smooth surfaces.22-26 Up to date, most currently accessible spiky Au NPs exhibit high efficiency for conversion of near infrared (NIR) light in the wavelength range of 700 – 900 nm (known as the first NIR window), into heat.27-29 Although spiky Au NPs can be produced to exhibit a profound surface plasmon resonance
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(SPR) absorption in the NIR range of 900 – 1400 nm, known as the second NIR (NIR-II) window,27 desirable for weak autofluorescence and deep penetration into tissues, these NPs generally tend to strongly scatter light, resulting in significant reduction in light adsorption and in turn reduced light-to-heat conversion efficiency. This is mainly because the ratio of absorption/scattering cross-sections depends on the spike number (m) as well as the core sizes,23 when the center position of their maximum SPR peak (λpeak) is shifted to the NIR-II window (Scheme 1) by increasing the aspect ratio (ratio of length to width, AR) of the spike.30 Theoretically, when the spiky Au NP is simplified as needles on a spherical core (Equations 1 to 6), its cross-section of scattering (Qsca) is proportional to its total volume squared (V𝑆𝑆𝑆𝑆ℎ𝑒𝑒𝑒𝑒𝑒𝑒 + m × V𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 ), which is equal to
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tinction spectrum of one spike Au NP is composed of absorption and scattering; the center position of its maximum SPR peak is denoted as λpeak.
the sum of the volumes of the spherical core (πD3 /6) and spikes (m × πW 2 L/4) while its cross-section of absorption (Qabs) exhibits linear growth to its total volume (V𝑆𝑆𝑆𝑆ℎ𝑒𝑒𝑒𝑒𝑒𝑒 + m × V𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 ).
< 𝑄𝑄abs >𝑆𝑆𝑆𝑆ℎ𝑒𝑒𝑒𝑒𝑒𝑒 = < 𝑄𝑄abs >𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 =
Herein, we have developed a new strategy for synthesis of spiky Au NPs with controlled number of long spikes (with large aspect ratios) on small cores via seed-mediated growth by using uniformly short, glutathione (GSH)functionalized, Au nanorods (NRs) as seeds in the presence of cetyltrimethylammonium chloride (CTAC) in the growth solution. Among these, the resulting six-spiked Au NPs can bear strong absorption in the NIR-II window and thus exhibit an ultrahigh light-to-heat conversion efficiency (78.8%) under irradiation of 980 nm light, the highest reported to the best of our knowledge. Meanwhile, the larger surface-to-volume ratio of well-defined spiky Au NPs may further favor the tumor targeting effect after surface modification,33 possibly due to stronger interaction between spiky Au NPs and the receptors on the cell membrane.34,35 Lactoferrin (LF), a mammalian cationic iron-binding glycoprotein belonging to the transferrin family, has strong interaction with various tumor cells, was chosen to modify the surface of spiky Au NPs.33 Taking the two advantages together, functionalized spiky Au NPs (spiky Au@PEG-LF NPs) were further prepared by successive surface PEGylation and conjugation with LF as ligand, which are stable in vitro and in vivo. The spiky Au NPs allow complete ablation of tumors without recurrence after light irradiation at 980 nm, exhibiting promising successful cancer PT therapy in the NIR-II window.
(1)
(2)
k𝑣𝑣 27 � � 𝜖𝜖 ′′ 3 (𝜖𝜖 ′ + 2)2 + 𝜖𝜖 ′′2
k𝑣𝑣 8 � + 1� 𝜖𝜖 ′′ 3 (𝜖𝜖 ′ + 1)2 + 𝜖𝜖 ′′2
< 𝑄𝑄sca >𝑆𝑆𝑆𝑆ℎ𝑒𝑒𝑒𝑒𝑒𝑒 = < 𝑄𝑄sca >𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 =
πD3 V𝑆𝑆𝑆𝑆ℎ𝑒𝑒𝑒𝑒𝑒𝑒 = 6 πW 2 L V𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 = 4
k 4 𝑣𝑣 2 27 |𝜖𝜖 − 1|2 � ′ � (𝜖𝜖 + 2)2 + 𝜖𝜖 ′′2 18𝜋𝜋
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(3) (4) (5)
k 4 𝑣𝑣 2 8 |𝜖𝜖 − 1|2 � ′ + 1� (6) (𝜖𝜖 + 1)2 + 𝜖𝜖 ′′2 18𝜋𝜋
Accordingly, the amplitude of Qsca should increase much faster than that of Qabs with the increasing spike number (m) at a given core size. Moreover, the spike number of the spiky Au NPs should have an optimal value, which leads to proper Qabs/Qsca in the NIR-II window and thus simultaneously bears strong absorption and highest light-to-heat conversion efficiency under irradiation of light in the NIRII window. Recent simulation studies have revealed that for PT therapy in the NIR-I window, spiky Au NPs with 6 long spikes and small cores might exhibit a large absorption to extinction cross-section (Qabs/Qext) ratios.31,32 In addition, our numerical simulations also indicate that the light scattering of spiky Au NPs significantly increases with the spike number in the NIR-II window if the number of the spikes is larger than 10 (Figure S1), leading to decreasing Qabs/Qsca ratios and thus poor light-to-heat conversion. Thus, it is necessary to synthesize spiky Au NPs with controlled spike numbers to verify the relationship between the structure and spectrum of spiky Au NPs, and to promote application in cancer PT therapy as a transducer with high light-to-heat conversion efficiency in the NIRII window.
RESULTS AND DISCUSSION
Scheme 1. (a) Geometrical model of one spiky Au NP and its corresponding structure parameters (aspect ratio of the spike, spike number, and the diameter of spherical core). The aspect ratio, length and width of the spike are represented as AR, L, and W, respectively; the spike number and the diameter of spherical core are represented as m and D, respectively. (b) The ex2
In order to grow sharp spikes on spiky Au NPs, CTAC was utilized instead of cetyltrimethylammonium bromide (CTAB) in the growth solution during seed-mediated NP growth, which is known to be able to accelerate the anisotropic growth of Au NPs, as the redox potential of AuCl4higher than that of AuBr4- (1.002 and 0.854 V).36,37 In addition, the adsorption ability of Cl- ions on the crystal facets of Au NPs is weaker than that of Br- ions. Note that the utilization of Au NRs with aspect ratios (ARs) of about 3 is also crucial. When spherical Au NPs were used as the seeds (Figure S2), multi-branched Au NPs were obtained (Figure S3), which are good agreement with previous works. Moreover, the number, the lengths and widths of the spikes decreased with increasing AR of Au NRs used, but the core sizes slight increased (Figure S4, S5, Table S1 and S2), thus allowing us to reduce spike number to 4. In a typical procedure for synthesis of spiky Au NPs, asprepared Au NRs were firstly functionalized by GSH ligands and then introduced into the growth solution containing CTAC, HAuCl4, AgNO3, and ascorbic acid (AA). As mentioned above, the use of CTAC solution was essential for the formation of spiky Au NPs; otherwise conventional hyperbranched Au NPs were formed when CTAB was used (Figure S6) under the same reaction conditions. The use of GSH is also essential for the formation of spikes during the overgrowth of Au NRs into spiky Au NPs (Figure S7). However, the numbers, the lengths and widths of the spikes decreased with increasing concentration of silver
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Chemistry of Materials porting Information). After TEM 3D reconstruction, the visualization of the whole spiky Au NPs can be achieved. For instance, the missing spikes can be observed by rotation as well as rather small spikes (fail to grow into big ones). A set of TEM images of individual spiky Au NPs was recorded at different tilting angles with x as the rotation axis from -55° to 60° at intervals of 5° (Figure S12), which further unveiled 6 sets of spikes on each spiky NP. Thus, the observation of just 5 spikes with different dimensions on individual spiky Au NPs is due to the tilting of the spiky Au NPs on the copper grids. Figure 2 shows a typical TEM image of one spiky Au NP with six spikes (Figure 2a). The edge regions of different spikes on the NP, highlighted by boxes in Figure 2a, were further investigated by high resolution TEM (HRTEM) imaging (Figure 2b to 2d). The lattice fringe spacings were about 0.144 nm, 0.236 nm and 0.204 nm, consistent with the spacings between the {110} planes, between the {111} planes, and between the {110} planes of fcc gold, respectively.
ions (Figure S8) and increased with the GSH concentration (Figure S7). At fixed other reaction conditions, the number, lengths and widths of the spikes and overall NP sizes decreased with increasing the amount of Au NRs (Figure S9). As a consequence, spiky Au NPs with defined number, length and width of the spikes and overall NP sizes were produced by judicious adjustment of the concentration of GSH, silver ions, Au NRs and HAuCl4 in the presence of CTAC in the growth solution (Table S3).
Figure 1. TEM images of as-prepared spiky Au4 NPs (a), spiky Au6 NPs (b), spiky Au7 NPs (c), and spiky Au8 NPs (d), where subscript number represents the number of spikes. High magnification TEM image (e) and 3D reconstruction images (f to h) of single spiky Au6 NP viewed from distinct angles, reconstructed by TEM tomography.
The core sizes and the number, length and width of spikes of as-prepared spiky Au NPs (Figure 1a to 1d and Table S4) can be adjusted by the concentration of HAuCl4 in the growth solution at optimal concentrations of GSH ligands and silver ions in the presence of CTAC in the growth solution. When the HAuCl4 concentration increased from 0.083 to 0.42 mM, the core sizes of as-prepared spiky Au NPs hardly increase (from 52 ± 5 to 57 ± 5 nm). The NP spikes, obtained at the HAuCl4 concentration of 0.083 mM (Figure 1a), are noticeably distorted, which is rather close to particles prepared via conventional methods reported in literature.38,39 The spike length of the resulting spiky Au NPs increases from 35 ± 10 to 58 ± 10, 77 ± 10, and 93 ± 10 nm, and their spike width increases from 13 ± 5 to 18 ± 5, 24 ± 5, and 29 ± 5 nm, respectively, with the HAuCl4 concentration increasing from 0.083 to 0.17, 0.24, and 0.42 mM (Figure 1a to 1d and Table S4). Note that when the HAuCl4 concentration increased to 1.7 mM, hyper-spiked Au NPs would be obtained while the small spikes are formed on the big spikes (Figure S10a). In addition, the ratio of light scattering of hyper-spiked Au NPs in the extinction spectrum (Figure S10b) would be rather high due to their so many spikes, on the basis of simulated formula (Equation 1 to 6) and our preliminary results (Figure S1f). Accordingly, their performance of PT therapy would be lower than those with fewer spikes. Their XRD pattern reveals well resolved peaks of {111}, {200} and {220}, indicative of the typical face-centeredcubic (fcc) structure of phase–pure Au (Figure S11). A typical two-dimensional (2D) image of single spiky Au NP with 6 spikes is shown in Figure 1e, while its 3D tomography images with front, 45° direction, and back views, are shown in Figure 1f to 1h (also see movie S1 in the Sup-
Figure 2. TEM image (a) and HRTEM images (b to d) of single spiky Au6 NP. HRTEM image (b) taken from the edge area of the spike of one spiky Au6 NP (a), viewed along the [011] direction. HRTEM images of the spikes (c and d) indicating the growth of the spikes along and direction. The insets in (b to d) are the corresponding FFT images.
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On the basis of the corresponding fast Fourier transformation (FFT) images (Insets in Figure 2c and 2d), the spikes of as-prepared spiky Au NP were aligned along the [011] zone axis. Thus, the results indicate that the spikes of as-prepared spiky Au NPs are grown along or direction from the cores, respectively. It is well-known that the extinction spectra of Au NPs are sensitive to their sizes and shapes. Figure S13 and S14A show that the SPR bands of as-prepared Au NPs shift from the NIR-I to the NIR-II window, when their shapes are changed from nanorod to spiky NPs. The original Au NRs exhibit a longitudinal SPR band at 717 nm and a
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transverse one at 514 nm (Figure S13), while the former is more narrow and stronger than the latter, indicative of the uniform nanorod shape.40,41 The spiky Au4 NPs with small spikes exhibit a weaker longitudinal SPR band centered at 1020 nm and a transverse one at 550 nm (black curve in Figure S14A). If the larger spikes are produced at higher HAuCl4 concentration (in Figure 1b to 1d), their longitudinal SPR bands gradually red-shift to 1250 nm (Figure S14A) due to the presence of spikes.16,42-44 Overall, the resulting spiky Au NPs exhibit a strong absorption at 980 nm, which renders them as good candidates for PT therapy in the NIR-II window. Figure S14B shows results of numerical simulations of the spectra of as-prepared spiky Au NPs with different number of spikes and cores of comparable size (Table S4). The absorption cross-section (Qabs) of the resulting spiky Aum NPs is larger than the scattering cross-section (Qsca) when the spike number is less than 7, while Qsca is gradually dominating when the spike size and number increase up to 8 (Figure S14B).
their Qabs/Qext ratios (R) of spiky Aum NP are plotted and shown in Figure 3b, based on numerical simulations of their spectra (Table S5). One can clearly see that Qabs of the resulting spiky Aum NP mainly increases with spike number and dimension (black curve in Figure 3b). However, the Qabs/Qext ratios decrease mainly with increasing spike number as well as dimension (red curve in Figure 3b) due to the increasing Qsca in its extinction spectrum. Obviously, there exists one crossing point between its Qabs and the Qabs/Qext ratio. Thus, there should be one optimum spike number for highest light-to-heat conversion efficiency. In addition, it is found that spiky Au6 NPs have the maximum net Qabs (in the order of 1019 nm2 g-1) after mass normalization (Figure 3c, Table S4 and S5). For practical application, the light-to-heat conversion efficiency of the resulting spiky Au NPs has to be further evaluated to account for their dependence on spike number under 980 nm light irradiation.
Figure 4. Temperatures of the solutions containing hyperbranched Au NPs of comparable size prepared in CTAB solution (Figure S1f), spiky Au6 NPs prepared in CTAC (Figure 1b) and water under 980 nm light irradiation (1.0 W/cm2) as a function of irradiation time. The concentrations of all Au NPs used are 200 μg/mL. Both Au NPs have comparable size.
Figure 3. (a) Relationship between average spike number of single spiky Aum NP and its corresponding absorption crosssection (Qabs), and scattering cross-section (Qsca) at maximum SPR peak, versus number of spikes. (b) Relationship between the average spike number and its corresponding absorption cross-section (Qabs), and the absorption-to-extinction ratio (R = Qabs/Qext) of single spiky Aum NP. (c) Relationship between average spike number and net Qabs of the corresponding spiky Aum NPs after mass normalization. (d) Plot of temperature of the aqueous dispersions of spiky Aum NPs (Figure 1a to d, 200 μg/mL) versus irradiation time under 980 nm light irradiation for 45 s every cycle (1.0 W/cm2).
Note that the length and width of the spikes on the spiky Aum NPs are not totally uniform, different from the model of spiky Au NPs used for simulation. Thus, their maximum SPR peaks in the experimental extinction spectra are broader (Figure S14A) than those in simulated extinction spectra (Figure S14B). As expected, the amplitude of its Qsca increase much faster than that of its Qabs with the increasing spike number. In addition, Qsca is rather close to Qabs, if the spike number is 8 (Figure 3a). Since light-to-heat conversion efficiency of Au NPs is greatly dependent on its Qabs ratio, the relationship between average spike number and corresponding Qabs of the SPR peak and
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In order to be applied in physiological environment,45 as-prepared spiky Au NPs were coated by thiol-terminated polyethylene glycol (HS-PEG-COOH, Mw~2kD) via ligand exchange, which caused no change in the morphology of the NPs and in their extinction spectra (Figure S15). As shown in Figure 3d, the spiky Au6 NPs (shown in Figure 1b) indeed provide the best PT performance on the basis of mass normalization. They are capable of warming up the aqueous solution to 66 oC after 45 s irradiation by 980 nm light, which is also better than that of conventional hyperbranched Au NPs with comparable sizes (Figure 4). The 980 nm light-to-heat conversion efficiency (η) of the spiky Au6 NPs is calculated to be 78.8% by a modified method proposed by Roper et al. (Figure S16),46 which is higher than that of all nanomaterials reported in literature (Table S6).9,17,18,47-51 It is worth noting that the light-to-heat conversion ability of the spiky Au NPs can be retained for several repeating cycles, underlining the excellent reusability and feasibility for multiple treatment. The effect of laser intensity on the light-to-heat conversion was also studied. As show in Figure S17, higher laser intensity leads to
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Chemistry of Materials sponding Au@PEG ones, leading to an almost double Au concentration inside the cells after 24 h incubation. Compared with the Au NRs and spiky Au NPs, hyperbranched Au NPs exhibited a higher cellular uptake rate and subsequently higher intracellular accumulation, especially when they were modified with LF on the surfaces. This might be attributed to the fact that hyperbranched Au NPs have rougher surfaces, higher surface areas, higher immobilized ligand amounts and subsequently stronger interaction with cells. The rougher surface morphology and LF conjugation are favourable for longer retention of the NP inside the cells, which may offer a longer operational window for PT therapy, as revealed in Figure 5b. In order to achieve localized heating, free NPs were carefully washed away, and the cells were incubated in fresh PBS prior to light irradiation. The death of cells was directly observed by fluorescence microscopy after fluorescein diacetate/prodium iodide (FDA/PI) staining, in which living cells emit green fluorescence while damaged cells show red fluorescence. As shown in Figure 5c, the cells with spiky Au6@PEG-LF NPs ingested inside are completely dead after 2 min irradiation with 980 nm light with a dose as low as 0.5 W/cm2, indicating the high PT efficiency. The cells treated with spiky Au6@PEG NPs and hyperbranched Au@PEG-LF NPs are partially dead, whereas the majority of the cells treated with hyperbranched Au@PEG NPs, Au@PEG-LF NRs, and Au@PEG NRs survived the exposure to the same dosage of NIR light irradiation. The spiky Au6@PEG-LF NPs have the best phototherapeutic effect due to the synergy of two effects: 1) the high tendency to be ingested by cells because of the surface conjugated LF ligand and special morphology with enhanced cell interaction area; 2) the highest PT conversion efficiency under 980 nm light. Furthermore, Au NRs with maximum SPR peak around 980nm were also synthesized and used as another control for comparison. As shown in Figure S27, the Au NRs have relatively lower photothermal conversion efficiency and cell uptake compared to spiky Au NPs, in turn resulted in reduced PT therapy efficiency in vitro at the same Au dosage. Apart from systemic toxicity, the anticancer drugs are always limited by the inherent or acquired resistance possessed by various cancers. Great efforts have been made to overcome drug resistance, but only limited achievement was gained in clinical applications. PT therapy is a perfect alternative for chemotherapy in combating against drug resistance. Encouraged by the excellent PT efficacy of the spiky Au NPs, we also tested the PT induced anti-tumor efficacy on resistant tumor model. Spiky Au6@PEG-LF NPs demonstrate best therapeutic efficiency on cisplatin resistant ovarian carcinoma (A2780CIS) in the presence of a NIR light irradiation (Figure S28).
higher temperature of irradiated solution. However, the light to heat conversion efficiency remains unchanged. In addition, higher laser intensity leads to higher cytotoxicity. All the cells are dead when the laser density is higher than 1.0 W/cm2 (show in Figure S18). Therefore, we choose light density of 0.5 W/cm2 for in vitro experiments to compare the PT therapy effect between different groups. PEG-capped, spiky Aum NPs (Figure S19 and S20) can be ingested by human hepatocellular carcinoma (HepG2) cells (Figure S21) without noticeable cytotoxicity (Figure S22). Their PT therapy effect positively correlates with light dosage at a given NP concentration (Figure S23 and S24), evidenced, as the area of dead cells increases with irradiation time. The spiky Au6 NPs show the best PT therapy effect due to their highest light-to-heat conversion efficiency (Figure 3d). Subsequently, as-prepared spiky Au6 NPs were chosen for the following study, while hyperbranched Au NPs with comparable size (Figure S1f) and Au NRs (about 37 nm in length and 10 nm in width, with a longitudinal absorption band around 808 nm) were used as controls.
Figure 5. (a) Amount of Au NPs ingested by HepG2 cells quantified by ICP-MS versus incubation time. (b) Percentage of remaining Au NPs in the cells after exposure to different Au NPs for 24 h. * indicates a significant difference at p < 0.05 level. (c) Fluorescence microscopy images of HepG2 cells irradiated with 980 nm NIR light (0.5 W/cm2) for 2 min, exposed to spiky Au6@PEG-LF NPs (Row 1), spiky Au6@PEG NPs (Row 2), hyperbranched Au@PEG-LF NPs (Row 3), hyperbranched Au@PEG NPs (Row 4), Au@PEG-LF NRs (Row 5), and Au@PEG NRs (Row 6). Green and red colors are representing viable and dead cells, respectively.
To improve the selectivity and enhance the uptake of spiky Au6 NPs in tumor cells, LF was further used to functionalize the spiky Au NPs because of its ability to interact with corresponding receptors on tumor cell membranes (Figure S25 and S26).33 The weight ratio of immobilized LF was 2.1 ± 0.4%, 3.2 ± 0.5% and 2.4 ± 0.3% on the spiky Au6 NPs, hyperbranched Au NPs and Au NRs respectively, quantified by conventional bicinchoninic acid (BCA) assay. All the Au NPs are ingested by HepG2 cells, and the NP concentrations in the cell significantly increase within the first 6 h during incubation and then slowly increase with the incubation time (Figure 5a). The Au@PEG-LF NPs are always taken up by the cells more efficiently than corre-
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damaging of healthy tissue and providing a longer operation window. No apparent histopathological abnormalities or lesions were observed in liver, spleen, heart, lung and kidney of the mice treated by Au@PEG NPs and Au@PEGLF NPs by haematoxylin and eosin (H&E) staining (Figure S30 and S31), confirming again excellent biocompatibility of the spiky Au6@PEG-LF NPs. Since the lower laser power density with shorter irradiation time is much desired in the invasive tumor therapy, the optimized 0.5 W/cm2 power density of laser with 3 min duration was chosen herein for PT therapy. After 3 min irradiation under 0.5 W/cm2 NIR light, the tumors containing the spiky Au6@PEG-LF NPs have maximum temperatures of 69.6 oC on their surface, which is above the threshold required to induce irreversible tissue and skin damage53,54 and completely destroy tumors without a relapse after 12 days (Figure 6c and Figure S32). The wounds generated by PT treatment are gradually healed and no remaining tumors are found 12 days after treatment, indicating a satisfactory PT therapy. In contrast, the tumors treated with the spiky Au6@PEG NPs, hyperbranched Au@PEG-LF NPs, hyperbranched Au@PEG NPs, have maximum temperatures of 56.0, 54.5 and 49.5 oC, respectively, leading to obvious skin burning and partial recession of the tumors. However, the tumors could continuously grow, because the heat generated was not sufficient to ablate all tumor cells/tissues (Figure 6c and Figure S32). Therefore, the spiky Au6@PEG-LF NPs caused a higher temperature increase inside the tumors and more severe burning than the other Au NPs under the same light dosage. This is ascribed to the higher NP concentration accumulated within tumors and to their higher light-toheat conversion efficiency. Histological analyses further support the results of tumor inhibition discussed above (Figure S33 and S34). The median survival time for mice treated with saline or light irradiation only was about 39 days, whereas spiky Au6@PEG-LF NPs administration followed by light irradiation greatly prolonged mice survival over 72 days without a single death (Figure 6d) and obvious body weight loss (Figure S35). Note that with spiky Au6@PEG NPs, hyperbranched Au@PEG-LF NPs, and hyperbranched Au@PEG NPs, PT therapy only prolongs the median survival time for mice to 60, 57 and 51 days respectively, while all mice were dead after 66 days. All these results demonstrate that the spiky Au6@PEG-LF NPs can act as effective PT agents for tumor ablation by single intravenous administration and one-time NIR-II light irradiation with limited local damage to normal tissues.
Figure 6. (a) Amount of different Au NPs in mice blood versus circulation time after intravenous injection. (b) Histograms of the tumor concentration of different Au NPs after various times post intravenous injection. ID stands for normalized injection dosage. (c) In vivo tumor growth inhibition curves for mice, that received different Au NPs and 3 min 980 nm light irradiation (0.5 W/cm2, n = 5 for each group). (d) KaplanMeier plots showing the percentage of animals remaining in the study as a function of time. The asterisk symbol (*) and (***) indicates significant difference at p
