Rod in Tube: A Novel Nanoplatform for Highly Effective Chemo

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Biological and Medical Applications of Materials and Interfaces

Rod in Tube: A Novel Nanoplatform for Highly Effective Chemophotothermal Combination Therapy Towards Breast Cancer Jun Zhang, Xiang Luo, Yanping Wu, Fan Wu, Yi-Fang Li, Rongrong He, and Mingxian Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b17533 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

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ACS Applied Materials & Interfaces

Rod in Tube: A Novel Nanoplatform for Highly Effective Chemophotothermal Combination Therapy Towards Breast Cancer Jun Zhang,†§ Xiang Luo,‡§ Yan-Ping Wu,‡ǁ Fan Wu,† Yi-Fang Li,‡ǁ Rong-Rong He,*‡ǁ Mingxian Liu*† †Department

of Materials Science and Engineering, Jinan University, Guangzhou 510632, China

‡Guangdong

Engineering Research Center of Chinese Medicine & Disease Susceptibility, Jinan

University, Guangzhou 510632, China ǁGuangdong

Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs

Research, College of Pharmacy, Jinan University, Guangzhou 510632, China

ABSTRACT Gold nanorods (GNRs) and doxorubicin (DOX) were loaded into the lumen of halloysite nanotubes (HNTs) via a rapid synthesis process (2 min) and physical adsorption. The targeting molecules of folic acid (FA) are then conjugated to HNTs via reactions with bovine serum albumin (BSA). The formation of GNRs in HNTs was verified by different techniques. The Au-HNTs-DOX@BSA-FA shows maximum of 26.8 oC temperature rising after 8 min 808-nm laser irradiation under 0.8 W cm-2. The functionalized HNTs exhibited stronger chemotherapeutic effect under laser irradiation, since the laser could promote the release of DOX and temperature rising. Au-HNTs-DOX@BSA-FA treated MCF-7 cells exhibited survival rate of 7.4% after laser irradiation. Au-HNTs-DOX@BSA-FA treatment do not induce an obvious toxicity in blood biochemistry, liver and kidney function in normal mice. In vivo chemo-photothermal treatment towards 4T1-bearing mice suggested Au-HNTs-DOX@BSA-FA exhibited remarkable tumor-targeted efficiency and good controlled-release effect for DOX. Also, the nanoparticles exhibited a rapid photothermal performance and inhibiting ability of the growth of tumor. Due to the synergistic effect of chemical-photothermal therapy, the toxicity of DOX to normal tissues was reduced on the premise of ensuring the same curative effect with a low dosage of 0.32 mg kg-1. This novel chemo-photothermal therapy nanoplatform provided a safe, rapid, effective, and cheap choice for treatment of breast tumor both in vitro and in vivo. KEYWORDS: photothermal therapy, gold nanorods, halloysite nanotubes, doxorubicin, chemophotothermal therapy

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1. INTRODUCTION So far, chemotherapy and radiotherapy are the main therapies for treating tumors in the clinic.1 But they also face a series of key obstacles, such as damage to normal tissues and drug resistance.2 As an emerging cancer treatment strategy, photothermal treatment (PTT) has attracted considerable research attentions in the treatment of cancer. It is the treatment of tumors by converting near-infrared (NIR) light into heat to kill cancer cells by photothermal agents.3-6 Currently reported photothermal agents include organic materials,7-8 carbon materials,9-10 and inorganic materials.11-12 Each of them has its own advantages and drawbacks. Among them, gold nanoparticles (GNPs) are widely used in the PTT of cancer due to their unique size, excellent biocompatibility, and good light stability.12-13 The localized surface plasmon resonance (LSPR) absorption band of GNPs is in the range of 450-520 nm.12 In the PTT, the laser light of the wavelength in the NIR region has the strongest penetrability to the tissue, so it is necessary to move the LSPR absorption band to the NIR region. Therefore, many different GNPs, such as gold nanorods (GNRs),13 gold nanoshells (GNSs),14 and gold nanocages (GNCs),15 have been extensively studied. Specially, GNRs with different aspect ratio can be obtained by adjusting the amount of reductant in order to adjust the light absorption range to the NIR region, thereby achieving maximum photothermal conversion efficiency.16 Halloysite nanotubes (HNTs) are natural nanoclay whose chemical formula is Al2Si2O5(OH)4·nH2O. HNTs have a unique hollow tubular structure consisting mainly of external silanol (Si-OH) groups and internal aluminoxane (Al-OH) groups. The average size of HNTs varies from 200-1000 nm, and the outer diameter varies between 50-70 nm.17 HNTs have been used as drug carrier materials in recent years due to their cavities, high adsorption capacity, ease of dispersion in polymers, and compatibility with biological and environmental properties. To increase the antitumor effect of HNTs, different surface modification procedure and different drug has been explored.18-21 HNTs can be loaded with genetic drugs, chemical drugs and antibacterial agents for the treatment of diseases.22-24 For example, cation polymers grafted functionalized HNTs are found with enhanced anti-cancer efficacy for drug of curcumin and doxorubicin (DOX).25 HNTs with pH-responsive polymers can also constitute a drug delivery platform of atorvastatin and celecoxib for colon cancer therapy.26 In addition, metal nanoparticles can also incorporate into surfaces of HNTs to improve their functionals.27-29 For example,


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GNPs can be loaded on the outer walls of HNTs as a selective catalyst30 and tunable plasma platform.31 GNPs immobilized HNTs surface can be used for surface-enhanced Raman scattering substrates.32 GNRs and GNPs can also be loaded into the lumen of HNTs.33-34 Very recently, a rapid (≤2 minute) and highly efficient synthesis method was reported to regulate and control the growth of GNRs in the lumen of HNTs. The size of the GNPs is mainly regulated by changing the reaction time and the amount of ascorbic acid used.35 Other precious metals such as silver36-37 and platinum38-39 loaded with HNTs have also been extensively studied. However, to the best of our knowledge, no work has been focused on the photothermal therapy of GNPs decorated HNTs towards cancer. Combinations of various treatment modalities such as chemotherapy and PTT, gene therapy and PTT are explored to improve the ability of cancer treatment. There are many works in this area and the combination therapy is a hot topic in cancer therapy. For example, simultaneous chemo-photothermal therapy against cancer can be achieved by modifying polysaccharide–protein complexes on Te nanorods.40 Oxidized mesoporous carbon nanospheres covalently bonded to polyethyleneimine (PEI) was developed as a versatile photothermal-combined gene therapy platform. Therapeutic genes select for tumor inhibition by inhibiting cell proliferation and inhibiting tumor angiogenesis, while oxidizing mesoporous carbon nanospheres eliminates tumors by photothermal.41 The gold nanorods and mesoporous silica shells can regulate the ability of DOX to release. Laser irradiation can increase temperature to promote drug release, which leads to the high anticancer ability.42 In this study, GNRs were firstly loaded into the lumen of HNTs, and the chemotherapy drug DOX and bovine serum albumin (BSA) were then adsorbed on the surfaces of HNTs through the physical adsorption. Ethanol was employed to trigger the aggregation formation of the Au-HNTs-DOX@BSA complex. This is a fast, efficient and convenient synthesis method. To enhance the specific uptake of Au-HNTs-DOX@BSA by breast cancer cells, folic acid (FA) as a targeting ligand was conjugated to the surface of Au-HNTs-DOX@BSA to form Au-HNTs-DOX@BSA -FA. Finally, the designed chemotherapy and photothermal therapy platform are used to synergistically treat breast cancer. The photothermal effect and the accelerated DOX release behavior from the Au-HNTs-DOX@BSA-FA were investigated both in vitro and in vivo experiment. Au-HNTs-DOX@BSA-FA exhibited therapeutic effects toward breast tumor by targeting to the tumor tissue, releasing drug, and rising the



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temperature. This study uses natural clay nanotubes (HNTs) to efficiently combine chemotherapy and light therapy for the treatment of cancer. In vivo experiments have shown that Au-HNTs-DOX@BSAFA can achieve better anti-tumor effects and reduce toxicity to normal tissues. It shows good cytocompatibility and hemocompatibility. Meanwhile, the drug toxicity to normal tissues is reduced, while a satisfied therapeutic effect is maintained. The novel chemo-photothermal therapy nanoplatform of Au-HNTs-DOX@BSA-HNTs provided a safe, rapid, effective, and cheap choice for treatment of breast tumor both in vitro and in vivo. 2. RESULTS AND DISCUSSION 2.1 Characterization of Au-HNTs and Au-HNTs-DOX@BSA-FA Nanocomposites. It is feasible to synthesize GNRs in HNTs tubes by oil-phase synthesis method. Figure 1A illustrates the synthesis processes of Au-HNTs-DOX@BSA-FA. First, the chloroauric acid is reduced in the HNTs by ascorbic acid in an organic solvent to form a gold nanorod. Gold ions and other components can be quickly loaded into the wettable clay nanotubes by capillary force. After the ascorbic acid is added to the solution, the Au3+ is rapidly reduced to atoms, and GNPs are formed when the atomic concentration reaches the minimum concentration required for nucleation. When 200 mg of ascorbic acid was added, GNPs were formed in a large amount, and the lumen limits the GNPs to attach or aggregate to grow into GNRs. It should be noted that only GNPs are formed outside the tubes, and the tubes provides a template for the formation of GNRs. Next, HNTs are mixed with BSA and DOX by physical adsorption, then fixed with ethanol. Finally, FA is grafted onto the surfaces of the HNTs by the condensation reaction of -COOH of FA with -NH2 in BSA. GNRs and Au-HNTs-DOX@BSA-FA can be uniformly dispersed in PBS solution by ultrasound (Figure S1A). However, after 10 hours of standing, Au-HNTsDOX@BSA-FA can still be stably dispersed, but a large number of sedimentation occurs in GNRs (Figure S1B). Figure S1C shows that the Au-HNTs-DOX@BSA-FA nanoparticles are stable in the medium after standing at 10 h, which is attributed to the presence of the adsorbed BSA on surfaces of HNTs. Au-HNTs-DOX@BSA-FA is a kind of nanoparticles that can exist stably in biological environment. Au-HNTs are more stable in PBS than GNRs for practical application, which is one of the advantages of the Au-HNTs hybrid system. GNRs can be stably exist in the tubes of HNTs for a


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long time, and they are fixed in the lumen by capillary force in the tubes of halloysite even after repeated centrifugation. The deformation vibration peak of the N-H bond at 1560 cm-1 and stretching vibration of C-N bond at 1210 cm-1 appear in the IR spectrum of Au-HNTs-DOX@BSA-FA (Figure S2). This indicates the formation of primary amines and FA grafted on BSA. TEM image of Figure 1B shows that raw HNTs have a unique tubular structure. By the in situ synthesis of GNPs in presence of HNTs, the lumen of HNTs is filled with GNRs of varying sizes (Figure 1C). The diameter and aspect ratio of the GNRs in HNTs lumen varies from 9.7-16.2 nm and 5.5-19.8, respectively. GNRs begin to grow at both ends of the lumen, leading to the formation of rod-like structures inside the HNTs. It is also found the GNR can also be formed outside the HNTs, but the free GNRs have been removed by washed with toluene (Figure S3). Figure 1D shows the UV-vis absorption spectra of the GNRs, HNTs and Au-HNTs. GNRs show the characteristic absorption at 510 nm and 782 nm. Meanwhile, Au-HNTs have the same absorption band in the NIR region, which suggest the formation of GNRs in HNTs. XRD patterns of the Au-HNTs nanocomposites indicate the presence of peaks assigned to both HNTs and gold (Figure 1E). Compared with the raw HNTs, there are diffraction peak of (111), (200), and (220)43 assigned to gold in Au-HNTs.



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Figure 1．(A) Synthesis processes of Au-HNTs-DOX@BSA-FA. (B) TEM images of HNTs and (C) Au-HNTs. (D) UV-vis absorption spectra of different samples. (E) XRD pattern of different samples. (F) High-resolution scanning of Al, Si, Au, and O elements of HNTs and Au-HNTs.

Figure S4 shows X-ray photoelectron spectroscopy (XPS) curves of HNTs and Au-HNTs, and TEM element mapping images of HNTs and Au-HNTs. It demonstrates that GNRs can be formed in the lumen of HNTs. The unclearness of oxygen element is caused by the adhesion of organic matter used in the synthesis of GNRs to the surfaces of HNTs. Figure 1F shows the high resolution XPS spectra of aluminum, silicon, gold and oxygen elements of HNTs and Au-HNTs. Obviously, the gold content in Au-HNTs is significantly higher than that of the original HNTs. The increase in carbon content and decrease in oxygen may be attributed to the fact that the oleic acid (OAc) and oleylamine (OAm) are not completely removed by the washing process. Due to the interactions between the OAc and OAm and HNTs, it is hardly totally removed from the product. However, they have negligible influence on the following cytotoxicity. All these results show that GNRs were successfully loaded into the lumen of HNTs. Figure S5 compares the TGA curves of Au-HNTs-DOX@BSA-FA with the original HNTs. Au-HNTs-DOX@BSA-FA has less weight loss than the original HNTs at 200-700 °C. HNTs lose weight due to dehydration of hydroxyl groups, while the Au-HNTs-DOX@BSA-FA also has a gold residue at 700 °C. The gold content in Au-HNTs is calculated to be 4.58 wt.%. The ICP analysis of the gold element in the Au-HNTs-DOX@BSA-FA was also carried out, and the content of gold was calculated to be 4.8% ± 0.23%, which is consistent with the result of TGA. Moreover, the synthesized materials were analyzed by particle size and zeta-potential. The hydrodynamic diameter of HNTs, Au-HNTs, and Au-HNTs-DOX@BSA-FA is 336.5 ± 2.3 nm (PDI = 0.212), 367.2 ± 3.6 nm (PDI = 0.219), and 424 ± 2.8 nm (PDI = 0.223), respectively (Figure S6). Their size distribution is relatively narrow. The increase in particle size is attributed to incorporation of organic layer around the tubes and the possible aggregation of HNTs when fixed by ethanol. The zeta-potential of HNTs, Au-HNTs, and Au-HNTs-DOX@BSA-FA is -25.2 mV, -23.3 mV, and -19.6 mV, respectively. Adsorption of positively charged DOX and BSA reduces the zeta potential of HNTs.


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In order to evaluate the role of Au-HNTs as photothermal agent in aqueous solution, temperature changes of Au-HNTs irradiated with NIR light were recorded by an infrared camera. As displayed in Figure 2A-2C, pure water has a negligible temperature change after laser irradiation, but Au-HNTs have a much higher temperature rise. As the concentration of Au-HNTs and laser power increase, the final temperature and temperature rising rate of the laser-irradiated nanoparticle suspension increases accordingly. For example, when the concentration of Au-HNTs is 400 μg mL-1, the temperature rose 26.8 oC after 8 min irradiation. The photothermal effects of Au-HNTs can be reproduced with 808-nm laser switch-on or switch-off for several times as shown in Figure 2D. This indicates that the photothermal effect of Au-HNTs is stable and repeatable during the photothermal therapy. Adsorption of molecules such as DOX and BSA does not affect the photothermal properties of Au-HNTs, and the photothermal effect of Au-HNTs-DOX@BSA-FA is consistent with Au-HNTs (data not shown).

Figure 2. (A) Temperature images of Au-HNTs solution at different concentrations and different powers under irradiation of 808-nm were recorded using infrared camera. (B) Temperature curves of Au-HNTs (1 mL) solution with different concentrations irradiated by 808-nm laser (0.8 W cm−2). (C)



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Temperature curves of Au-HNTs (800 μg L−1, 1 mL) upon NIR light irradiation with various powers. (D) Au-HNTs (400 μg L−1, 1 mL) solution with 808-nm laser switch-on or switch-off for five times.

Au-HNTs-DOX@BSA-FA nanocomposites were prepared to improve the biological affinity of AuHNTs and the therapeutic effect of tumors. In this system, photothermal therapy is the mainstay, supplemented by chemical therapy. To increase the uptake of Au-HNTs-DOX@BSA by cancer cells, FA was conjugated to the surface of Au-HNTs-DOX@BSA as a targeting ligand. Figure 3A shows the hemolysis rates of different samples. Hemolysis of nanoparticles is related to porosity, geometry and surface functionality.44 The hemolysis of nanoparticles is related to their geometry and surface structure. HNTs cause red blood cell damage due to their sharp surface structure, which exhibits a high hemolysis rate of 84.3%. Decrease in hemolysis by surface modification of HNTs such as PEI and chitosan oligosaccharide grafting was found in previous literatures.22-23 In this work, the hemolytic rate of Au-HNTs-DOX@BSA-FA is only 1.9%, which suggests that Au-HNTs-DOX@BSA-FA possesses good biocompatibility and it is safe for intravenous injection. This is because the sharp shape of HNTs is partly covered after loading BSA and DOX. In the process of animal experiments, AuHNTs-DOX@BSA-FA does not coagulate in the blood of mice to form a thrombus. Au-HNTsDOX@BSA-FA has good anticoagulant ability while reducing hemolysis rate. The the length of AuHNTs is larger than that of a typical nanoscaled drug carrier, but it has a nanoscaled diameter and a low hemolysis rate, and is specifically endocytosed by binding to FA receptors when blood in the bodycirculates to tumor cells. DOX was then loaded onto the Au-HNTs by physical adsorption under stirring for 12 h. The loading efficiency of DOX is calculated as ∼1.8% (mass ratio of DOX to AuHNTs-DOX@BSA-FA). The release of DOX from PBS is extremely low in dark conditions, but the release is greatly increased after laser irradiation (Figure 3B). This suggests that DOX release is accelerated after laser irradiation. This is consistent with the results reported by Zheng et al.45 Laser irradiation also promotes the release of DOX in vivo.46 Because the heat after laser conversion dissociates the strong interactions between DOX and the nanocomposite, it is reasonable of releasing more DOX molecules.47


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Figure 3. (A) The hemolysis rate of different samples. (B) Drug release curves of Au-HNTsDOX@BSA-FA (1 mg mL−1) under dark and 808-nm laser irradiation for 8 min at pH 7.4. 2.2 In vitro cellular uptake and chemo-photothermal treatment. The cytotoxicity of HNTs, AuHNTs, and Au-HNTs-DOX@BSA-FA was evaluated in PBS (pH 7.4) with or without 808-nm laser irradiation (1 W cm-2, 8 min). Figure 4A shows low-level biological toxicity of Au-HNTs through culture with MCF-7 cells. Even at relatively high concentrations, the survival rate of HeLa cells and MCF-7 cells treated with Au-HNTs is greater than 85%. At the same time, cytocompatibility assay using another cancer cell (Hela cells) and normal tissue cell (HUVECs) also indicates that Au-HNTs have good biocompatibility (Figure S7). After NIR laser irradiation, the cells treated with Au-HNTs are killed. As the irradiation time and concentration increase, the cell survival rate decreases. Among them, the survival rate of cells at high concentrations is only 10.5% in Figure 4B. Figure 4C shows that raw HNTs does not cause cell death after laser irradiation, but the cell survival rate is only 52.6% in the Au-HNTs group which is irradiated with 808-nm laser for 8 min. Au-HNTs-DOX@BSA-FA also kills cells by releasing small amounts of DOX in PBS without laser irradiation, and FA-conjugated nanoparticles also play a supporting role in specifically targeting MCF-7 cells with a positive FA receptor.48 The cell survival rate is 73% in the Au-HNTs-DOX@BSA-FA group without irradiation. After 8 min irradiation with 808-nm laser Au-HNTs-DOX@BSA-FA increases the apoptosis rate of MCF-7 cells and the survival rate of cells was only 7.4%. Direct observation of cell death induced by Au-HNTs-DOX@BSA-FA in MCF-7 cells was conducted using fluorescence live/dead cell assay. As displayed in Figure 4D, in the cells treated with PBS and HNTs, green fluorescence is clearly observed, indicating that the cells are still alive in the well plate. In the light-irradiated area, almost all the cells are dead induced by Au-HNTs (red fluorescence). In



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contrast, green fluorescence is clearly observed without laser irradiation. A small amount of red fluorescence is observed in the wells to which Au-HNTs-DOX@BSA-FA is added but not irradiated with laser light, indicating a small amount of cell death. However, a significant amount of red fluorescence is observed after laser irradiation. These results suggest that both DOX and GNRs in AuHNTs-DOX@BSA-FA plays a critical role in the cells under light irradiation. When Au-HNTsDOX@BSA-FA is added to the medium, the loaded DOX is slowly released, causing a small number of cells to apoptosis and reducing the total amount of cells. The cells irradiated by the laser have a large amount of apoptosis due to the temperature rising to about 50 °C. Cells that are not irradiated with laser light cause apoptosis only due to the presence of DOX. To further confirm the potent apoptosis-inducing ability in tumor cells, flow cytometry was used to analyze the apoptosis and necrosis of MCF-7 cells. As shown in Figure 4E, HNTs cause only 3.53% of cells apoptosis rate after laser irradiation, but Au-HNTs cause 65.66% of cells apoptosis rate after light irradiation. Au-HNTsDOX@BSA-FA can cause 18.68% of cells apoptosis rate without laser irradiation. However, after laser irradiation Au-HNTs-DOX@BSA-FA increases the temperature of PBS after laser irradiation, which promotes the release of DOX and leads to 92.26% of cell apoptosis. Apoptosis of MCF-7 cells is caused by the synergistic effect of chemo-photothermal therapy. Figure S8 shows the in vitro inhibitory effect of 4T1 cells by Au-HNTs-DOX@BSA-FA. As the laser irradiation time and material concentration increases, the cell viability of 4T1 cells is greatly reduced (Figure S8A). Au-HNTsDOX@BSA-FA exhibits the strongest cell killing effect after laser irradiation and the viability of 4T1 cells is only 11.2% (Figure S8B). The cell live/dead staining photographs show that HNTs have a very low cytotoxicity towards 4T1, while Au-HNTs can kill the cells after irradiation (Figure S8C). In contrast, most of the 4T1 cells died after the irradiation in the presence of Au-HNTs-DOX@BSA-FA. The 4T1 cell experiment result is consistent with the toxicity and inhibitory effect of MCF-7 cells.


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Figure 4. (A) Relative survival rate of HeLa cells co-cultured with different concentrations of HNTs and Au-HNTs for 24 h. (B) The cell viability of MCF-7 cells which were pretreated to Au-HNTs for 12 h at 37 °C, and then were irradiated by 808-nm laser for 8 min. (C) The viability of MCF-7 cells were treated with different samples (400 μg mL-1) for 24 h and then irradiated with or without 808-nm laser (0.8 W cm-2) for 8 min. Data are shown as means ± SD (n = 4). (D) Fluorescence live/dead cell images of MCF-7 cells irradiated with or without 808-nm laser (0.8 W cm-2) for 8 min, after treatment with HNTs, Au-HNTs, and Au-HNTs-DOX@BSA-FA. Scale bar: 50 μm. (E) Flow cytometry analysis of apoptosis and necrosis of MCF-7 cells irradiated with or without 808-nm laser (0.8 W cm-2) for 8 min, after treatment with HNTs, Au-HNTs, and Au-HNTs-DOX@BSA-FA.

The cellular uptake of Au-HNTs-DOX@BSA-FA is evaluated using the MCF-7 cells. Figure 5 shows the laser scanning confocal microscope (LSCM) images of MCF-7 cells incubated with Au-HNTsDOX@BSA-FA at different time periods. The red fluorescence of DOX appears in cells in MCF-7 cells incubated with Au-HNTs-DOX@BSA-FA for 2 h, and the red fluorescence intensity becomes stronger as the incubation time increases. This indicates that the modified HNTs carrying DOX is enriched in cells. FA-grafting could significantly improve the delivery of nanoparticles into the cancer cells.49 As time goes on, more Au-HNTs-DOX@BSA-FA is uptake by the cells, and the red



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fluorescence appears in cytoplasm become darker. The part of the black spots appearing in the bright field is the aggregation of HNTs, and the other part is the cell debris produced by the cells during the cultivation. This is because some cells will disintegrate during dehydration, leaving cell debris. The aggregation of HNTs does not affect the activity of the cells. From the results of CCK-8, the halloysite is less toxic to the cells in the long-term culture as shown in Figure 4A.

Figure 5. Fluorescence images of MCF-7 cells treated with Au-HNTs-DOX@BSA-FA at different time intervals. Scale bar: 20 μm.

Atomic force microscope (AFM) of the MCF-7 cells is employed to further prove that the material is phagocytosed by the cells rather than only enriched on the cell surface. Figure S9A shows a control group of MCF-7 cells that were incubated in the medium for 12 h without any material. One can see the cell membrane sticks on the base after it collapses due to dehydration, and its nuclei are prominent. Figure S9B shows a partial enlargement of MCF-7 cells. It can be seen that no HNTs are found around the nucleus and cell membrane. Figure S9C is AFM image of MCF-7 cells incubated with Au-HNTsDOX@BSA-FA (200 μg mL-1) for 12 h. The nucleus cannot be identified and the whole skeleton is raised, accompanying uneven particles appear on the cell surface. Because the HNTs are concentrated around the cells, some of them are taken up into the cytoplasm by the cells to make the entire skeleton convex, so that the nucleus is recessed. Obviously, as shown in Figure S9D, a part of the tubular


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particles adhered to the cell membrane. However, some HNTs are phagocytized in the cell membrane (indicated by the red arrow). TEM techniques have also been used to observe the intracellular release of nanoparticles in different cell lines.17, 50 The results of AFM show that HNTs are able to enter cells through endocytosis. 2.3 In vivo hemocompatibility of Au-HNTs-DOX@BSA-FA. To study the hemocompatibility of Au-HNTs-DOX@BSA-FA, a hemocompatibility study was performed using two groups of BALB/c mice (three mice in each group). Mice were intravenously injected with normal saline (200 μL), AuHNTs-DOX@BSA-FA (20 mg kg-1) every three days for 7 days. Blood was harvested from the orbital for blood smear, serum biochemistry and complete blood panel assay. Photomicrographs of the blood smears of mice treated with normal saline (control) or Au-HNTs-DOX@BSA-FA are shown in Figure S10. The result of blood smears reveal that Au-HNTs-DOX@BSA-FA treatment does not change the number and shape of red blood cells, platelet and white blood cells when compared to the control mice. A variety of biochemistry parameters were focused on liver function index such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, globulin and total protein (Figure S10B and S10C). No significant hepatic toxicity is shown in Au-HNTs-DOX@BSAFA treated-mice. Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, globulin and total protein are considered as the important biochemical indexes which reflecting the liver function in clinic (Figure S10B and S10C). In our study, no obvious hepatic toxicity is shown in Au-HNTs-DOX@BSA-FA treated-mice. The urea levels in blood as one indicator of kidney functions are shown with normal levels and with no difference between control group and Au-HNTsDOX@BSA-FA group (Figure S10D). In order to conduct hematological assessment, the hematology markers were detected by automatic biochemical analyzer: white blood cells, red blood cells, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count and mean corpuscular hemoglobin (Figure S10E–S10L). The results show that all of these indicators are in the normal level and with no difference between control group and Au-HNTs-DOX@BSA-FA group. Taken together, Au-HNTs-DOX@BSA-FA treatment do not induce an obvious toxicity in blood biochemistry, liver and kidney function in normal mice, which indicates that Au-HNTs-DOX@BSA-FA is biosafety and biocompatibility.



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2.4 In vivo chemo-photothermal treatment. Chemotherapy is the first choice to treat cancer in the clinic, but chemotherapy drug such as DOX, lacks of selectivity to cancer cell which induces lots of side effects.51 More than 40% of tumor cells overexpressed folate receptors, including lung, breast, kidney, brain and ovarian cancers. Functionalizing nanoparticles with targeting ligand molecules such as FA can be designed to tumor-targeted efficacy, which can recognize and bind to cancer-specific receptors, thereby enabling specific recognition of tumor cells. 52-54 The cytotoxic sensitivity of MCF-7 cell and 4T1 cell to DOX has no significant difference in the reported researches.23,55-56 Both cell lines are considered as the folate receptor positive expressing cells and are suitable for the tumor-targeted study in vivo and in vitro.57-58 Howerver, tumor-bearing mice (with normal immune) can be established easily with 4T1 cell, but MCF-7 tumor-bearing mice should be nude mouse (with immunosuppression) with higher cost. Therefore, we evaluated the tumor-targeted efficacy, toxicity and anti-tumor effect of the Au-HNTs-DOX@BSA-FA in vivo using the 4T1 cell through establishing the tumor-bearing mice. In order to study the the tumor-targeted efficacy of Au-HNTs-DOX@BSA-FA, 4T1-bearing mice were intravenously injected with DOX (0.32 mg kg-1) and Au-HNTs-DOX@BSA-FA (0.32 mg DOX equiv kg-1), respectively. DOX fluorescence was monitored by fluorescence imaging system at specific time points (1, 4, 8, 12, and 24 h) from the main tissues (tumor, heart, liver, spleen, lung and kidney) excised from 4T1-bearing mice after intravenous injection. As shown in Figure 6A and 6B, the distribution and elimination of Au-HNTs-DOX@BSA-FA in mice was indicated by the changes of DOX fluorescence in the major tissues at different time points after intravenous injection. The accumulation of DOX fluorescence in tumor site was undoubtedly increased after Au-HNTsDOX@BSA-FA treatement and achieved the maximum at 8 h, while the fluorescence intensity of DOX treatment was maximum at 4 h. 2.72-fold increase in DOX fluorescence was achieved for AuHNTs-DOX@BSA-FA group compared with free DOX group. These data confirm that Au-HNTsDOX@BSA-FA in liver, spleen, and lung tissues was accumulated, and blood-circulation lifetime was prolonged, which increased the efficiency of Au-HNTs-DOX@BSA-FA targeting on the tumor site. In addition, DOX fluorescence intensity in the tumor tissues treated with Au-HNTs-DOX@BSA-FA is remarkable higher than that of DOX groups (P < 0.05). Moreover, mice treated with Au-HNTsDOX@BSA-FA show obvious higher DOX fluorescence than DOX group (P < 0.01) at 24 h. These


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data suggest that Au-HNTs-DOX@BSA-FA exhibit remarkable tumor-targeted efficiency and good controlled-release of DOX in tumor.

Figure 6. Tumor-targeted delivery of DOX by Au-HNTs-DOX@BSA-FA in 4T1-bearing mice. (A) Fluorescence images of major organs (1. Tumor, 2. Heart, 3. Liver, 4. Spleen, 5. Lung, 6. Kidney) of 4T1-bearing mice after intravenous injection of DOX (0.32 mg kg-1) and Au-HNTs-DOX@BSA-FA (0.32 mg DOX equiv kg-1) at specific time points (2, 4, 8, 12, and 24 h), respectively. (B) DOX fluorescence level of tumor tissues at specific time points (2, 4, 8, 12, and 24 h). The data are shown by as mean ± SD (n = 3). The data were analyzed by for t-test.*P < 0.05, **P < 0.01 vs DOX.

Since Au-HNTs@BSA-FA has an excellent photothermal performance in vitro, a further photothermal effect was carried out in 4T1-bearing mice and the images were recorded in real-time via an infrared thermal imaging camera. Mice were intravenously injected with normal saline (200 μL), AuHNTs@BSA-FA (20 mg kg-1) or Au-HNTs-DOX@BSA-FA (20 mg kg-1). Then, mice were



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anesthetized after 4 h injection and exposed to a 808-nm laser at the power density of 1 W cm-2. As shown in Figure 7A and B, the photothermal image and temperature changes in tumor of mice were captured and recorded by an infrared thermal camera at indicated time intervals (0, 2, 4, 6, and 8 min). The tumor temperature of mice treated with normal saline has no obvious fluctuation after irradiation for 8 min. It is noted that the temperature at local surface tumor of mice treated with Au-HNTs@BSAFA and Au-HNTs-DOX@BSA-FA are rapidly increased to 58.2 ± 0.7 °C and 59.0 ± 0.4 °C within 8 min, respectively. Several researches pointed out that Au has a unique property of surface plasmon resonance which can rapidly absorb and effectively transform NIR light to heat during irradiation.59-61 The photothermal effects with no significant differences between Au-HNTs@BSA-FA and Au-HNTsDOX@BSA-FA indicate that the photothermal effect is actually induced by Au-HNTs@BSA-FA and DOX cannot increase the photothermal effect of Au-HNTs@BSA-FA. It is reported that the cancer cells can be killed when the temperature of tumor reaches 41 °C, which is due to the poor supplement of blood.62 Therefore, these results suggest that Au-HNTs-DOX@BSA-FA have an outstanding photothermal performance and it may be a excellent nanomaterial for antitumor therapy.

Figure 7. In vivo photothermal imaging of Au-HNTs-DOX@BSA-FA in 4T1-bearing mice. 4T1bearing mice were intravenously injected with normal saline (200 μL), Au-HNTs@BSA-FA (20 mg kg-1) or Au-HNTs-DOX@BSA-FA (20 mg kg-1) and followed by 808-nm laser at a density of 1 W cm−2 for 8 min after 4 h injection. (A) Photothermal images of mice were captured by infrared thermal camera. (B) The temperature changes in tumor tissues of mice during laser irradiation were recorded.


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The data are shown by as mean ± SD (n = 3). The data were analyzed by two-way ANOVA with Tukey’s post hoc test. ***P < 0.001 vs Laser.

According to the results of tumor-targeted efficiency and photothermal imaging in vivo, Au-HNTsDOX@BSA-FA was speculated to have a prominent antitumor effect. To verify this assumption, the antitumor activity of Au-HNTs-DOX@BSA-FA in 4T1-bearing mice was further studied. As presented in Figure 8A, the body weight of 4T1-bearing mice treated with DOX significantly decreases (P < 0.001) compared with control group, while Au-HNTs@BSA-FA, Au-HNTs@BSA-FA + Laser and Au-HNTs-DOX@BSA-FA + Laser groups have no obvious influence. The decrease of body weight is generally resulting from the toxicity of DOX, since it shows certain toxic effects on mice. However, Au-HNTs-DOX@BSA-FA exhibit no toxicity to mice which indicates that Au-HNTs-DOX@BSA-FA can be considered as a superior nanosystem in cancer treatment. The tumor volume recorded and calculated every 3 days are showed in Figure 8B. At the end of experiment (on the 16th day), the tumor tissue was isolated, weighted and photographed as shown in Figure 8C and 8D. As expected, when compared to control group, both tumor volume and weight of 4T1-bearing mice in Au-HNTs@BSA-FA + Laser group are reduced significantly (P < 0.001), while laser or Au-HNTs@BSA-FA alone treatment have no significant change. This result indicates that AuHNTs @BSA-FA has a potential to inhibit the growth of tumor contributing to the high photothermal effect under irradiation, which agrees with the result in photothermal imaging experiment showing that Au-HNTs @BSA-FA is potential photothermal material. Compared with control group, both tumor volume and weight in 4T1-bearing mice treated with Au-HNTs-DOX@BSA-FA + Laser (0.32 mg kg-1 DOX) are decreased prominently (P < 0.001), much more significant than DOX (0.32 mg kg-1) treatment. As expected, mice in Au-HNTs-DOX@BSA-FA + Laser group exhibits more excellent antitumor effect than Au-HNTs@BSA-FA + Laser group (P < 0.05) in 4T1-bearing mice. In particularly, the average tumor volume of control group on 15th day is 243.85 ± 78.88 mm3, while the average tumor volume of Au-HNTs@BSA-FA + Laser and Au-HNTs-DOX@BSA-FA + Laser is 79.43 ± 14.97 mm3 and 51.22 ± 5.06 mm3, respectively. Au-HNTs-DOX@BSA-FA has a better antitumor effect than Au-HNTs@BSA-FA, which can be attributed to the tumor targeting effect



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combined with chemotherapy effect. In total, it should be noticed that DOX can cause a decrease in body weight with toxicity in mice at a low concentration of 0.32 mg kg-1. In contrast, Au-HNTsDOX@BSA-FA carried the same low-dose of DOX (0.32 mg kg-1) reduces the volume of tumors significantly, while no significant toxic side effects are found on mice. Au-HNTs-DOX@BSA-FA can relief the toxic effects of DOX and enhance the antitumor ability. These data indicate that Au-HNTsDOX@BSA-FA + Laser treatment has an excellent antitumor efficacy in vivo far exceeding the effects of photothermal therapy or chemotherapy alone, which can be explained by the synergic photothermal, tumor-targeted and chemotherapy effect of Au-HNTs-DOX@BSA-FA. Additionally, to further confirm the antitumor efficacy in vivo, TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was performed to detected the apoptosis index in tumor tissues (Figure 8E and 8F). The brown spots (the red arrows) indicate the apoptosis cells in tumor tissue. Compared with control group, Au-HNTs@BSA-FA + Laser (P < 0.001) treatment and Au-HNTs-DOX@BSA-FA + Laser (P < 0.001) treatment lead obvious apoptosis in tumor tissue. Furthermore, Au-HNTsDOX@BSA-FA + Laser treatment exhibits more remarkable apoptosis than Au-HNTs@BSA-FA + Laser treatment (P < 0.01). These results are consistent with the tumor volume and tumor weight shown in Figure 8B and 8C. These data further prove that Au-HNTs-DOX@BSA-FA leads to an excellent antitumor efficacy in 4T1-bearing mice.


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Figure 8. Anti-tumor performance of Au-HNTs-DOX@BSA-FA in 4T1-bearing mice. (A) Body weight and (B) Relative tumor volumes of mice from control group, Laser group, Au-HNTs@BSA-FA group (20 mg kg-1), DOX group (0.32 mg kg-1), Au-HNTs@BSA-FA (20 mg kg-1) + Laser group and Au-HNTs-DOX@BSA-FA (0.32 mg DOX equiv. kg-1) + Laser group. (C) Tumor weight and (D) photographs of excised 4T1 solid tumor on the 16th day. (E) Representative images of tumor sections detected by TUNEL assay. Brown spots indicate TUNEL-positive cell nuclei. (F) Apoptosis cells number calculated blindly in 5 randomly selected regions in different groups. Scale bar: 20 μm. The data are shown by mean ± SD (n = 3). The data of body weight and tumor volume were analyzed by two-way ANOVA with Tukey’s post hoc test. The data of tumor weight and apoptosis index were analyzed by one-way ANOVA with Tukey’s post hoc test. *P < 0.05, ***P < 0.001 vs Control, ###P

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