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In vivo CT/Photoacoustic imaging and NIR-Triggered chemophotothermal combined therapy based on a gold nanostar, mesoporous silica and thermo-sensitive liposomes composited nanoprobe Jie An, Xiao-Quan Yang, Kai Cheng, Xian-Lin Song, Lin Zhang, Cheng Li, Xiao-Shuai Zhang, Yang Xuan, Yuan-Yang Song, Bi-Yun Fang, Xiao-Lin Hou, Yuan-Di Zhao, and Bo Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15296 • Publication Date (Web): 10 Nov 2017 Downloaded from http://pubs.acs.org on November 11, 2017

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ACS Applied Materials & Interfaces

In vivo CT/Photoacoustic imaging and NIR-Triggered chemo-photothermal combined therapy based on a gold nanostar, mesoporous silica and thermo-sensitive liposomes composited nanoprobe

Jie An1‡, Xiao-Quan Yang1,2‡, Kai Cheng1, Xian-Lin Song2, Lin Zhang1, Cheng Li1, Xiao-Shuai Zhang1, Yang Xuan1, Yuan-Yang Song1, Bi-Yun Fang1, Xiao-Lin Hou1, Yuan-Di Zhao1,2*, Bo Liu1,2*

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Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics – Hubei Bioinformatics & Molecular Imaging Key Laboratory, Collaborative Innovation Center for Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, Hubei, P. R. China

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Key Laboratory of Biomedical Photonics (HUST), Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, Hubei, P. R. China

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ACS Paragon Plus Environment


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ABSTRACT Safe multifunctional nanoplatforms that have multiple therapeutic functions integrated with imaging capabilities are highly desired for biomedical applications. In this paper, targeted chemo-photothermal synergistic therapy and photoacoustic/CT

imaging

of

tumor

were

achieved

by

one

novel

multifunctional

nanoprobe

(GMS/DOX@SLB-FA), it was composed of gold nanostar core and doxorubicin (DOX) loading mesoporous silica shell (GMS), which was coated with folic acid (FA) modified thermosensitively supported lipid bilayer (FA-SLB) as gatekeeper. The multifunctional probe had perfect dispersion and stability, 2.1 nm mesoporous pore and 208 nm hydration particle sizes were obtained. In vitro studies indicated that drug-loading probe had excellent ability of controlling release of DOX with 71.98 ± 2.52% cumulative release after laser irradiation, which was significantly higher than that of unirradiation control group. 72.75 ± 4.37% survival rate of HeLa cells at 57.75 µg/mL probe also demonstrated low cytotoxicity of the targeted probe. Both in vitro and in vivo results showed that the probe could achieve targeted photoacoustic imaging of tumor due to the fact that FA modified probe could specifically recognize the overexpressed FA receptors on tumor cells; meanwhile the probe could also achieve the chemo-photothermal synergistic therapy of tumor through controlling the drug release from mesoporous channels by near infrared laser. Therefore, the probe had great potential in the early diagnosis and treatment of cancer.

Keywords: gold nanostar; drug release; photoacoustic imaging; CT imaging; chemo-photothermal combined therapy

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1. INTRODUCTION The unique physical and biochemical properties of nanomaterials provide great application potential for the diagnosis and therapy of disease. At present, researchers design various nanoprobes for early diagnosis and treatment of tumor as nanoparticles can gather at the site of the tumor by enhanced permeability and retention effect (EPR) and active targeting function.1,2 A variety of imaging techniques are used for early diagnosis, such as X-ray computed tomography (CT), photoacoustic imaging (PAI), magnetic resonance imaging (MRI), positron emission computed tomography (PET), fluorescence imaging (FI), ultrasound imaging (US) and so on.3 Among these techniques, CT is one of the most widely used imaging method in medicine diagnostic due to its relatively low cost and deep tissue penetration.4 However, it is unfavorable to discover early tumor because of its low sensitivity. As a new non-invasive and non-ionizing biomedical imaging method, PAI has attracted many researchers' interest. Compared with traditional imaging techniques, PAI combines the excellent contrast of optical imaging and high

penetration depth of ultrasound imaging, which reduces the effect of light scattering on imaging quality and breaks the soft limit of high resolution optical imaging depth, thus expects to obtain high resolution and high sensitive tissue image with a depth of 50 mm in vivo tissue imaging.5 Therefore, it is an ideal bimodal molecular imaging to combine PAI and CT, while the former provides the distribution and location of trace cancer related molecules in tissues and the latter provides deep tissue spatial information,6 providing image data for early diagnosis of tumor with complementary advantages. CT enhanced imaging relies on some small molecular iodine compounds. It is highly desired to explore new CT contrast due to the fact that iodine contrast agents with low molecular weight will be fast cleaned by kidneys, which is unfavorable for targeted tumor imaging.7 Recent studies suggest some metal nanoparticles present better CT contrast effect than iodine compounds8 owing to the relatively high atomic number (I, 53; Ta, 73; Au, 79; Bi, 83) and X ray attenuation coefficient (I, 1.94; Ta, 4.30; Au, 5.16; Bi, 5.74 cm2/g at 100 keV).9 Among these materials,

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gold nanoparticles have already broad application in the biomedical field, and a variety of gold nanoparticles with great potential have been developed, such as gold nanospere, gold nanorod, gold nanoshell, gold nanocage, and gold nanostar etc.10 More and more researchers take interest in gold nanostar (AuNS) due to its strongly plastic shape, good biocompatibility, and high thermal efficiency.11 The surface plasmon resonance (SPR) peak of AuNS can be conveniently tuned to near infrared (NIR) range (700 - 900 nm), so it can be used as dual modality imaging contrast agent for PAI and CT, and photosensitizer for photothermal therapy (PTT).12 In recent years, as an effective method, PTT has attracted extensive attention for tumor treatment. It is a hyperthermia therapeutic strategy that converts optical energy to heat by enriched photothermal agent at the tumor site, thereby kills cancer with little damage to the surrounding healthy tissues.13 Shi’s group reported an Au nanostar-coated, perfluorohexane-encapsulated hollow mesoporous silica nanocapsule (HMS) modified with poly(ethylene glycol) for tumor multimode US/CT/PAI/thermal imaging, and PTT.14 However, although NIR light can penetrate tissues up to several centimeters, its energy is gradually reduced due to light scattering and absorption of deeper tissues. Some tumor cells would inevitably receive suboptimal laser irradiation and may not be ablated.15 That is why many researchers shift from single PTT modality to combine chemo-photothermal therapy, with which enhanced therapeutic efficacies are reported.16 Current clinical studies show that PTT not only has toxic effect directly on tumor, but also can enhance the efficacy of chemotherapy and radiotherapy, improve immunity, and inhibit and prevent cancer recurrence and metastasis.17 The mainly reason is when PTT kills cancer cells, it helps chemotherapeutic drug into tumor tissue and increases the cytotoxicity of drug at the same time. This dual mode of combined therapy has drawn increasing attention from researchers. How to accurately deliver the drug molecules to tumor and selectively kill tumor cells has been the hotspot in the treatment of chemotherapeutic drug. Nanocarrier has become one of the most important methods to solve this problem. Mesoporous silica has broad application in drug delivery and controlled release system as its large

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specific surface area, conveniently controlled mesoporous aperture, and stable skeleton structure.18 However, one big problem is that the drug can easily leak from mesoporous silica during in vivo circulation. Therefore, it is necessary to control the release of drug and deliver the drug to the tumor. The responsive controlled drug delivery is an intelligent drug delivery system that can release the drug molecules in a controlled manner under stimulation of the external environment or body's own environmental factors. Common stimulation factors include pH, temperature, reducing substances, light, and enzymes, etc.19 For example, Cui’s group developed a mesoporous silica nanoparticle acting as the drug loading core, while a layer of copolymer−lipid including natural phospholipids and poly(N-isopropylacrylamide-methacrylic acid-octadecyl acrylate) copolymer served as the dual-responsive gating shell.20 And Yang’s group designed a multifunctional nanoplatforms which was composed of Fe3O4 magnetic nanoparticle as the core, mesoporous silica as the sandwiched layer, and thermo-sensitive P(NIPAM-co-NHMA) copolymer as the outer shell, while Zn(II) phthalocyanine tetrasulfonic acid (ZnPcS4) as a model drug.21 And some researchers have proposed using NIR light to precisely control the release of drug, because a number of nanomaterials have high efficient photothermal conversion under NIR irradiation.22 For example, Liu et al. designed a photothermal controlled drug delivery system, which was composed of gold nanorod-like core and mesoporous silica shell coated with 1-teradecanol as gatekeeper, it presented the ability to achieve drug release under NIR inducing.23 This kind of probe not only can control the drug release through an external light source, but also can generate heat energy to kill cancer cells by photothermal conversion, and thus has the functions of chemotherapy and PTT. To our knowledge, such study based on AuNS has not been reported. In this work, a novel multifunctional nanoprobe (GMS/DOX@SLB-FA) with targeted PAI and CT dual mode imaging and such chemo-photothermal combined therapy of tumor was designed. The probe was composed of AuNS core and mesoporous silica shell (GMS), which was coated with a thermosensitively supported lipid bilayers formed by folic acid (FA) modified PEG-phospholipid, dipalmitoyl phosphatidylcholine and distearoyl

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phosphatidylcholine mixture (SLB-FA), while doxorubicin (DOX) was loaded by electrostatic adsorption in the mesoporous channels. In vitro and in vivo results showed that GMS/DOX@SLB-FA could target specifically overexpressed FA receptors on tumor cells and achieve PAI and CT of tumor. At the same time, a precise drug controlled release from mesoporous channels was achieved by NIR laser due to the photothermal effect of AuNS core on thermosensitively supported lipid bilayer, and it provided promising opportunity for localized synergistic photothermal ablation and chemotherapy. Furthermore, compared with chemotherapy or phototherapy alone, the combined treatment showed a synergistic effect. Compared with references’ work, gold nanostars not only had been used for tumor CT/PA imaging and photothermal therapy, but also used to control the release of drug and achieved chemo-photothermal synergistic therapy of tumor, and it moved forward the study from references’ in vitro cell level to mouse and achieved the chemo-photothermal synergistic therapy in tumor-bearing mice in vivo. This work provided a new approach for the exploration of new integrative probe, and it was expected to provide evidence for early diagnosis and treatment of tumor (Figure 1).

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Figure 1. Schematic illustration of multifunctional probe GMS/DOX@SLB-FA preparation (A), targeted chemo-photothermal therapy and PAI/CT imaging of tumor (B).

2. RESULTS AND DISCUSSION As the first step of this work, AuNS was synthesized referring to Tuan Vo-Dinh method.24 The SPR peak of AuNS was tuned from 700 to 850 nm by adjusting the AgNO3 concentration (Figure S1A). With adding higher concentrations of AgNO3, the SPR peak of AuNS appeared redshift by forming longer, sharper, and more numerous branches (Figure S1B). As shown in TEM, the average diameter of AuNS was approximately 50 nm (Figure 2A) and consistent with the dynamic light scattering (DLS) (Figure 2F). After it was coated with mesoporous silica to obtain GMS, the layer of mesoporous silica was also tunable by adjusting the amount of tetraethyl orthosilicate

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(TEOS, Figure S2). Considering the size of nanoparticles, GMS with the layer of mesoporous silica 50 ~ 60 nm was chosen to obtain uniform spherical morphology with an average diameter of about 150 nm (Figure 2B, F), and high resolution TEM indicated that GMS had radial mesoporous silica as shell with unobstructed mesoporous (Figure 2C). As demonstrated by EDX spectra, Au and Si elements appeared in GMS (Figure 2E). Its wide-angle XRD pattern showed five well-resolved diffraction peaks in the range 20 - 90 °, which could be indexed to 111, 200, 220, 311 and 222 reflections of cubic metal gold (JCPDS 04-0784), further indicating crystalline structure of AuNS core. And their structure lacked long-range order due to weak diffraction shoulder peak appearing at 2.1° in low angle XRD pattern (Figure 2G). The nitrogen adsorption-desorption isotherms of GMS (Figure 2H) demonstrated representative type-IV curves with capillary condensation step at P/P0 = 0.2 - 0.5, which was typically associated with uniform mesoporous. Moreover, a hysteresis loop at higher pressure (P/P0 = 0.85 - 1.0) might reflect the interparticle packed pores. The BET surface area and total pore volume, analyzed by the nitrogen physisorption were 650.61 m2/g and 0.663 cm3/g respectively, with pore size distribution of approximately 2.1 nm.

Figure 2. TEM of AuNS (A), GMS (B, C), and GMS@SLB (D); EDX energy spectrum of GMS@SLB (E); hydrated particle size distribution of AuNS and GMS (F); wide-angle XRD pattern (G) and small-angle XRD pattern (inset); N2 sorption isotherms of GMS (H) and pore size distribution (inset).

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The UV-vis-NIR absorbance spectra showed GMS exhibited high NIR absorption at ~ 800 nm (Figure 3A). It was found there was slight redshift compared with AuNS because surface of the latter was coated with a layer of mesoporous silica. According to the methods reported in the literature,25 DSPE-PEG2000-NH2 was combined with FA by coupling agent DCC. Absorption spectra showed that coupling product had strong absorption at 300 nm and red shift compared with DSPE-PEG2000-NH2 (Figure 3B), which might be caused by condensation reaction. It could be confirmed that coupling product was DSPE-PEG2000-FA because there was no the absorption peak of FA in dialyzate even after several times dialysis. The surface potential of GMS was -31.96 ± 1.06 mV due to surface silanol groups, but it changed to -25.82 ± 0.67 mV after coating of SLB with positively charged amino groups (Figure 3C). If GMS was loaded with positive charge DOX and coated with SLB, zeta potential would increase to -3.34 ± 0.99 mV. However, if the package was SLB-FA, GMS/DOX@SLB-FA surface charge would only increase to -10.48 ± 0.85 mV due to the carboxyl group of FA (Figure 3C). At the same time, the mesoporous structure disappeared after GMS was coated with SLB (Figure 2D), indicating that the surface of mesoporous silica had been covered by SLB.26 Thermogravimetric analysis (TGA) was also conducted to investigate the weight percentage of the lipid bilayer and DOX in probe. Over temperature range from 50 to 500 °С, only 3.03 % weight loss was observed for GMS, indicating surfactant template had not been completely removed and moisture on the surface of GMS. But those qualities in case of GMS@SLB-FA and GMS/DOX@SLB-FA were 26.04% and 29.80 %, respectively. The former was likely due to further loss of SLB-FA on GMS surface (Figure 3F). Compared with GMS@SLB-FA, 3.76% mass loss of GMS/DOX@SLB-FA should be the mass of DOX in the channels. In the experiment, it was found that with the increase of DOX content, the drug loading rate of the probe did not improve significantly (Table S1). The encapsulation efficiency and drug loading rate reached up to 53.76 ± 4.71 % and 3.43

± 0.43 % respectively, and the results was in accordance with TGA. It could be concluded that DOX had been loaded in mesoporous channels and SLB-FA was successfully coated on GMS surface.

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AuNS had superior photothermal conversion efficiency, and this effect of GMS was also investigated. The results showed that the temperature of GMS with same concentration was increased obviously accompanied with the rise of laser intensity (Figure S3A, D). Furthermore, when GMS concentration was increased, temperature of the dispersion could be elevated up to higher temperature (Figure S3B, E) compared with PBS group and higher ∆T was obtained under the same laser intensity (Figure S3C). When concentration of GMS was low at 38.5 µg/mL and irradiated for 5 min, temperature could be up to 51.27 °С (Figure S3B), which was enough to kill tumor cells. The GMS/DOX@SLB-FA also exhibited highly stable photothermal conversion capability during 5 cycles of testing (Figure S4). Furthermore, the photothermal conversion efficiencies (η) of GMS/DOX@SLB-FA and GMS@SLB-FA were determined to be ∼29.94 % and 31.21 %, respectively (Figure S5). These results confirmed that GMS, even covered with mesoporous silica, DOX and SLB-FA, still had outstanding conversion efficiency and it was promised to reach the purpose of killing cancer.

Figure 3. UV−vis spectra of AuNS (38.5 µg/mL) and AuNS@mSiO2 (GMS, 38.5 µg/mL) (A), FA, DSPE-PEG2000-NH2, DSPE-PEG2000-FA and dialysate (B); zeta potentials of GMS, GMS@SLB, GMS/DOX@SLB and GMS/DOX@SLB-FA (C); DOX cumulative release from GMS/DOX@SLB-FA (0.24 mg/mL) at 25, 32, 37, 40, 45, 50, 55, and 65 °C after 60 h (D); DOX cumulative release from GMS/DOX@SLB-FA (0.24 mg/mL) under multiple NIR laser on/off cycle treatment after 60 h, while cumulative release at 25 °C as control (E); TGA curves of GMS, GMS@SLB-FA, and GMS/DOX@SLB-FA (F).

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In the designed probe, DOX was adsorbed into the pore of GMS through electrostatic interaction, and SLB-FA was used to block the pore to prevent the leakage of DOX. SLB-FA was prepared by mixing DPPC, DSPC and DSPE-PEG2000-FA at a molar ratio of 70 : 25 : 5. Previous studies had shown that phase transition temperature at this ratio was about 42 °С,27 which was suitable for our research on tumor PTT. The thermosensitive DOX release was performed in PBS buffer (pH 7.4), which was simulated as normal blood environment. The results indicated that DOX release rate was closely related to environmental temperature, and cumulative release of DOX after 60 h was increased with the rise of temperature. When temperature was up to 40 °С, cumulative release was increased significantly, indicating that the permeability of phospholipid membrane was increased after temperature tended to Tm of SLB, thus DOX could be released faster through SLB (Figure 3D). 40 °С was slight below the Tm (42 °С), this might be due to the fact that after SLB was encapsulated on the surface of mesoporous silica, the fluidity of phospholipids was enhanced, and thus Tm value was reduced28. DOX release reached stable after temperature was increased to 45 °С, indicating SLB had been unable to block the pore on account of over Tm value of SLB. The above results showed that DOX release from drug-loading probe could be effectively controlled by regulating the temperature. Moreover, taking the reversible phase-changing essence of SLB into consideration, it was also expected that multiple on/off treatment of NIR irradiation could also impart “on-demand” control over the drug release. To confirm this assumption, drug-loading probe was treated with NIR laser (808 nm, 1.0 W/ cm2) for on (10 min)/off (several min) alternating treatment, and drug release behavior before and after irradiation was monitored. The results authenticated that cumulative DOX release amount after laser irradiation was increased significantly after four times irradiation (Figure 3E), it reached 71.98 ± 2.52%, strikingly higher than the control group without laser irradiation (27.73 ± 1.64%). Therefore, drug-loading probe based on the thermosensitive characteristic of SLB could completely control the drug release by NIR laser.

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Figure 4. The photoacoustic response of different concentrations of GMS, insert: PAI (A); HU values as function of GMS concentrations, insert: CT imaging (B).

In addition to the photothermal effect, GMS also had the capabilities of PAI and CT. Subsequently, the photoacoustic and CT response of GMS were detected at different concentrations. It could be seen that accompanying with increasing GMS concentration, PA signal was obviously strengthened (Figure 4A). CT image and Hounsfield units (HU) value showed a sharp signal enhancement as the increase of GMS concentration (Figure 4B). These results stated clearly that GMS, covered with mesoporous silica, maintained excellent contrast ability. The stability of GMS/DOX@SLB-FA was further investigated. The results showed that probe diameter was about 208.14 ± 3.3 nm; size, PDI and zeta potential did not change significantly in water, PBS, DMEM, and DMEM + 10 % FBS solution at 4, 25 and 37 °С for 25 d (Figure S6), indicating temperature and solvent had little effect on GMS/DOX@SLB-FA during 4 - 37 °С. Furthermore, there was no significantly different between the TEM of GMS/DOX@SLB-FA before and after five laser on/off cycles irradiation (Figure S7). Therefore, GMS/DOX@SLB-FA presented outstanding stability.

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Figure 5. White light and CT results of different cells incubated with different probes (A) and photoacoustic response of different cells incubated with different probes (B). * : p < 0.05; ** : p < 0.01.

Nanoparticle could efficiently enter cells, which was the essential element for multifunctional drug-loading nanocarrier. The modification of targeted ligand on nanoparticle let probe recognize and bind receptors expressed on target cells, which triggered receptor mediated endocytosis, and increased probe uptake. Since FA receptors were highly expressed on surface of HeLa cells29and low expressed on A549 cells, 30 they were used as positive and negative cells for FA target recognition, respectively. GMS@SLB-FA was used as positive probe, and GMS@SLB as negative control probe. In experiments, GMS@SLB-FA and GMS@SLB were incubated with HeLa and A549 cells for 4 h respectively, CT and PAI of cells were performed after removing unbound probe. The results indicated that positive HeLa cells incubated with positive GMS@SLB-FA probe (Figure 5A, f) was remarkably darker in

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color than those incubated with negative probe (Figure 5A, e), and noticeably deeper than the other controls. CT results also showed that positive cells had the highest HU value under positive probe (Figure 5A). PAI results were consistent with CT results (Figure 5B). T-test showed that photoacoustic signal of HeLa treated by GMS@SLB-FA was significantly stronger than positive cells under negative probe (p < 0.05), and it also presented considerable difference compared with negative cells under positive probe and the others controls (p < 0.01). This proved that positive GMS@SLB-FA probe could specifically bind to HeLa rather than A549 cells, hence it had outstanding targeting ability. In order to verify the targeting of probe, HeLa and A549 cells incubated with different probes were observed by TEM. In contrast with blank cells, probe particles were observed in both HeLa and A549 cells, no matter what kind of probe incubating. But probe content of HeLa incubated with positive probe was apparently higher than the other groups (Figure S8). This was consistent with the results of CT and PAI. These results confirmed that GMS@SLB-FA probe had the potential to target tumor cells with high FA receptors expression, and presented CT and photoacoustic dual mode imaging feature.

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Figure 6. The cytotoxicity of GMS@SLB-FA (A); HeLa and A549 cells stained by AM and PI after incubation with positive GMS@SLB-FA probe or negative GMS@SLB probe, and then treated with or without NIR laser irradiation (2 W/cm2, 10 min), white dotted line: border of laser irradiation (B); the viability of HeLa cells after different treatments with different DOX concentrations (C). * : p < 0.05; ** : p < 0.01.

To study the cytotoxicity of GMS@SLB-FA, HeLa and A549 cells were incubated with different concentrations of GMS@SLB-FA for 24 h. MTT detection showed that when probe concentration reached 57.8 µg/mL, survival rate of HeLa cells was still as high as 72.75 ± 4.37 %, and that of A549 cells was 86.89± 4.81%. And the survival rate of HeLa cells was still over fifty percent at 154 µg/mL probe (Figure 6A). This was because the amount of GMS@SLB-FA in HeLa cells was higher than that in A549, resulting in relatively higher toxicity to HeLa than

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A549 cells. High survival rate suggested that GMS@SLB-FA had low cytotoxicity and perfect biocompatibility. It was clear that at temperature of 41 - 45 °C, tumor cells began to show apoptosis signal, while above 50 °C, it was associated with less apoptosis but more frank necrosis due to the thermo-induced protein denaturation.23 The excellent photothermal conversion capability of AuNS made probe be an ideal PTT probe. PTT effects of probes under different conditions were evaluated using PI and AM staining to distinguish dead and living cells (Figure 6B). The results showed that there was no dead cell in HeLa group after laser irradiation, and cells appeared green of AM, indicating that laser irradiation alone could not kill tumor cells. Despite the fact that negative probe could adsorb on the cell surface non-specifically (Figure S8E), same bright green fluorescence was observed after laser irradiation at cells incubated by GMS@SLB, that was because the amount of adsorbed negative probe was not sufficient to kill tumor cells under laser irradiation. After HeLa cells were incubated with GMS@SLB-FA and irradiated by NIR laser, it was observed that non-irradiated region presented green fluorescent and the irradiation area obviously appeared PI staining dead cells. The results also showed that only positive probe could effectively kill tumor cells after irradiation, indicating its perfect PTT effect. As for A549 with low FA receptors expression, cells showed green fluorescence no matter what kind of treatment condition (Figure 6B). The experiments corroborated that GMS@SLB-FA had excellent targeting ability to tumor with high FA receptors expression and outstanding PTT ability. The chemo-photothermal combined therapeutic effect of GMS/DOX@SLB-FA on HeLa was evaluated using MTT assay (Figure 6C). It should be pointed out that in order to keep DOX amount same in each concentration group, the amount of probe would increase accordingly because unit probe had a certain drug loading. The results showed that laser treatment had little effect on survival rate of cells. GMS@SLB-FA caused ~ 30% cells death in high concentration condition, but survival rate was sharply decreased after laser irradiation, due to the fact that GMS@SLB-FA had excellent photothermal conversion ability and it could effectively kill tumor cells by PTT

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under laser irradiation. The killing ability of DOX alone on tumor cells was only presented at high concentration, it was better than GMS/DOX@SLB-FA containing the same concentration of DOX without laser irradiation, and closed to the effect of DOX with laser irradiation. The main reason was that DOX loaded in GMS/DOX@SLB-FA was blocked by SLB-FA, and it could not be released to kill tumor cells. However, the killing effect of GMS/DOX@SLB-FA on tumor cells was obviously enhanced after laser irradiation, even at low concentration of DOX, there was significant difference compared with that of GMS@SLB-FA (p < 0.05). When the concentration of DOX was high, the effect of combined therapy was remarkably better than that of GMS@SLB-FA (p < 0.01). As the heat was produced by NIR irradiation, it improved the permeability of SLB-FA, which led to more DOX release from the pores, and tumor cells were killed more effectively under the combined action of drug and photothermal. These findings confirmed that the combination of chemotherapy and PTT was more effective than single mode treatment. In order to detect the toxicity of the probe, the potential toxicity of the probe in vivo was further performed. The blood routine examination, white blood cell (WBC) (Figure S9A), red blood cell (RBC) (Figure S9B) and hemoglobin (HGB) (Figure S9C) were measured after injection, it was found there was no significant difference compared with the control group; the platelet (PLT) in probe group was reduced after 0.6 h but returned to normal reference range compared with the control group at 3 d (Figure S9D). For the liver and kidney function markers examination, alanine aminotransferase (ALT) and aspartate transaminase (AST) were measured and fell well into the normal reference range compared with the control group (Figure S9E and F). Furthermore, heart, liver, spleen, lung, kidney, and small intestine from mice treated with or without GMS/DOX@SLB-FA were also collected for H&E staining (Figure S10) and CD68 staining (Figure S11). The results showed that no significant organ damage and inflammatory reaction observed in major organs. Therefore, those results indicated that GMS/DOX@SLB-FA did not present significant toxicity to the treated animals.

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Figure 7. PAI of tumor area on HeLa tumor-bearing nude mice after intravenous injection with probe under 744 nm laser, and PAI of the same site under 523nm laser excitation (A); CT image of mice before and after intratumoral injection with probe (B). PAI and CT contrast imaging of probe were then studied in nude mice with transplanted tumor due to its preeminent PAI and CT imaging capabilities. For in vivo PAI, tumor-bearing Balb/c nude mice were intravenous injected with targeting probe suspension, and PAI was performed at different time point on the same tumor site of nude mice by 744 nm laser (Figure 7A). Results showed that photoacoustic signal of tumor was not obvious after 1 h and 6 h of injection; and the quantitative results of PAI showed that there was a remarkable difference in the PA signal at the tumor site between 12 h and 6 h (Figure S12), indicating that probe had been gathered at the tumor site 12 h later. Furthermore, PAI of the same tumor site was performed using 523 nm laser because hemoglobin had strong photoacoustic response to 523 nm instead of 744 nm laser. The results demonstrated that tumor angiography results at these two wavelengths were consistent. It could be confirmed that photoacoustic signal of 744 nm at 12 h was derived from the targeting probe. The probe could enrich the tumor site and realize the imaging of tumor by EPR and active targeting to HeLa cells. For in vivo CT imaging, after probe was injected 2 h later via intratumoral injection, the tumor site had obvious tumor CT contrast effect compared with same site before injection (Figure 7B). To conclude our targeting probe had superior prospect in tumor contrast imaging.

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Figure 8. Infrared thermal images of HeLa tumor-bearing nude mice by intratumoral injection with PBS, DOX, GMS@SLB-FA (probe) and GMS/DOX@SLB-FA (probe + DOX) after exposure to NIR irradiation for 10 min (A); the temperature of tumor evolution curves over time (B); body weight curves (C) and tumor growth curves (D) after different treatments; the photos of tumor change during different treatments (E). PBS (I), PBS + Laser (II), DOX (III), DOX + Laser (IV), GMS@SLB-FA (V, probe), GMS@SLB-FA + Laser (VI, probe + laser), GMS/DOX@SLB-FA (VII, probe + DOX), and GMS/DOX@SLB-FA + Laser (VIII, probe + DOX + laser). n=5

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Encouraged by high NIR absorbance and strong in vitro PTT efficacy of probe, in vivo chemo-photothermal combined therapy of probe was then carried out further. After mice were intratumorally injected with PBS and DOX, exposed to 808 nm laser for 10 min, temperature at tumor site only increased 9 °C (Figure 8A, B), which was not enough to kill tumor cells. In comparison, mice were intratumorally injected with GMS@SLB-FA and GMS/DOX@SLB-FA, exposed to laser, it was found temperature at tumor site had gone up 34 °C and reached to 65 °C. This temperature was sufficient enough to kill tumor cells (Figure 8A, B). Tumor size and mice weight were measured every 2 d after treatment described above. During treatment and monitoring period, no death and no obvious weight loss were observed, illustrating that the injections did not produce serious toxicity and side effect to mice (Figure 8C). The tumor volume of nude mice injected with PBS or DOX, or DOX with irradiation, had been increasing during treatment period, indicating that chemotherapy alone or laser irradiation alone did not kill the tumor (Figure 8E). If mice were injected with GMS@SLB-FA or GMS/DOX@SLB-FA without laser irradiation, the tumor tissue had been still increasing. However, black scars were left at the initial tumor sites after PTT for probe (group VI) and drug-loading probe (group VIII), indicating the excellent PTT of AuNS in the probes. Unfortunately, after 8 - 12 d of treatment, tumor re-growth phenomena occurred in all 5 mice in group VI, and tumor volume continued to increase. In contrast, the tumor sites of group VIII underwent necrosis, scab and abscission until the skin healed naturally (Figure 8E). Only one in group VIII began to relapse after 12 d, indicating that only single PTT could not kill tumor cells completely, and the treatment effect was obviously improved after combination of the drug treatment. Several interesting phenomena could be seen by observed the changes in tumor volume after treatment with different methods (Figure 8D). 1) For PBS group, PBS group with irradiation, and non-drug-loading probe group without irradiation, the tumor growth was all very rapid and relatively similar. However, after treating with DOX, the tumor growth was significantly slower even without laser irradiation, no matter DOX was free or in the channels of probe (p < 0.01), this result showed that DOX had a certain therapeutic

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effect on tumor. 2) There was no significant difference in final tumor size between groups III (DOX) and IV (DOX + laser); however, the tumor growth of group VII (probe + DOX) was significantly higher than those of the former two groups (p < 0.05), indicating that SLB-FA blocked the mesoporous pore and led to the fact that DOX was unable to release out to kill the tumor. 3) It could be seen from group VI (probe + laser) and VIII (probe + DOX + laser), tumor growths were inhibited by laser irradiation. In comparison, the tumor in group VI began to recur after 8 d. Nevertheless, the growth rate was still lower than those in group III and IV after 22 d. 4) Only one of five mice began to relapse after 12 d in group VIII, and the average tumor volume was significantly lower than that of the group VI after 22 d (p

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