PEGylated Au@Pt Nanodendrites as Novel Theranostic Agents for

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PEGylated Au@Pt Nanodendrites as Novel Theranostic Agents for CT Imaging and Photothermal/Radiation Synergistic Therapy Xu Liu, Xing Zhang, Mo Zhu, Guanghui Lin, Jian Liu, Zhufa Zhou, Xin Tian, and Yue Pan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15183 • Publication Date (Web): 14 Dec 2016 Downloaded from http://pubs.acs.org on December 17, 2016

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ACS Applied Materials & Interfaces

PEGylated Au@Pt Nanodendrites as Novel Theranostic Agents for CT Imaging and Photothermal/Radiation Synergistic Therapy

Xu Liu,† Xing Zhang,¶ Mo Zhu,†† Guanghui Lin,‡ Jian Liu,‡‡ Zhufa Zhou,† Xin Tian,*,§ and Yue Pan*,†

†

State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, China. ††

Department of Radiology, The First Affiliated Hospital of Soochow University, 188, Shi Zi Road, Suzhou, 215006, China.

¶

Institute of Metal Research Chinese Academy of Sciences, Shenyang National Laboratory For Materials Science, Shenyang, 110016, China. ‡

Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, 215123, China.

‡‡

Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou, 215123, China.

§

School of Radiation Medicine and Protection, School for Radiological and Interdisciplinary

Sciences (RAD-X) & Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123, China.

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ABSTRACT The integration of photothermal therapy (PTT) with radiation therapy (RT) in a single nanoscale platform is believed to have a bright prospect for cancer therapy. In this work, the rationally designed PEGylated Au@Pt nanodendrites (NDs) have been developed as a novel X-ray computed tomography (CT) and PTT/RT enhanced theranostic agent for cancer therapy. The absorption of Au@Pt NDs was turned to near-infrared region with the growth of Pt nanobranches and thus enhances the efficacy of PTT. Furthermore, due to the high atomic number (high-Z) of Au as well as Pt, Au@Pt NDs significantly enhanced lethal effects of RT via inducing a highly localized radiation dose within cancer cells. More importantly, the combination of Au@Pt NDs-enhanced RT with PTT suppressed cancer cells growth more efficiently than that from RT or PTT alone, indicating a synergistic effect. Meanwhile, the Au@Pt NDs also possess significant CT imaging signal enhancement which has the potential to guide PTT/RT for cancers. The integrated strategy significantly improved CT and PTT/RT of cancer cells with mild laser and radiation. Thanks to these advantages, Au@Pt NDs have turned to be appealing and effective agents for cancer theranostic.

KEYWORDS: Au@Pt nanodendrites, computed tomography, photothermal therapy, radiotherapy, synergistic effect


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1. INTRODUCTION Due to its specific lesion destruction and favorable biosafety, tremendous attention has been aroused by near-infrared (NIR) laser-induced photothermal therapy (PTT) in biomedical field.1-5 As a minimally invasive therapeutic method, in order to kill cancer cells, PTT utilizes agents which can absorb NIR light to result in an increase in the temperature. Recently, many types of nanomaterials have been extensively explored and applied in PTT of cancers, functioning as powerful agents of NIR light absorption.6-11 However, utilizations of highly efficient photothermal nanomaterial agents still cannot eradicate tumors completely, especially for the deep-located tumors because of low absorption efficiency of the natural tissue absorbents.7 Recently, several studies have demonstrated combination of other therapeutic strategies may be effective for improving PTT efficiency.9, 12-15 As another minimally invasive therapy strategy, radiation therapy (RT) which is considered as one of the most common methods in clinical cancer therapy, employs radiation with great energy (such as X-rays, gamma rays, and charged particles) to cause fatal damage to cancer cells through breaking their DNA chains.16-18 Different from PTT, the destruction of cancer cells can be efficiently achieved in RT without depth restriction.18-20 Nevertheless, there are several disadvantages of RT that limit its applications. First of all, the utilization of RT to treat cancer leads to the exposure of normal tissues inevitably. High-energy radiation would cause severe damage to surrounding normal tissues when killing cancer cells.21 Moreover, numerous types of tumors are resistant to RT which need a higher amount of radiation in the therapy process.19-20, 22-23 A lot of efforts have been made to improve the efficiency of RT. Nanomaterials containing elements with a great atomic number (high-Z) have been explored to function as radiosensitizers to enhance the RT efficiency to cancer cells and reduce radio toxicity to normal tissues.24-26 In addition, a series of work demonstrates that the combination of PTT with RT has been regarded as a powerful and prospective strategy to facilitate therapeutic efficiency mainly due to the fact that the merits of both could be taken while the drawbacks of either could be avoided.27-29 3 ACS Paragon Plus Environment


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Several nanomaterials have been widely investigated as PTT/RT agents. For example, Zhao et al. found that WS2 quantum dots possess remarkable PTT/RT synergistic effect for cancer therapy.7 Recently, rare earth and bismuth nanomaterials with NIR light absorption and radio sensitization properties have been used as a multifunctional nanoscale platform for PTT and RT.27,

30

However, the concern about the

biocompatibility of rare earth and bismuth nanomaterials limits their further application in biomedical field. It has been proved that a higher regional radiation dose can be efficiently achieved with the application of Gold (Au) and platinum (Pt) nanomaterials. In this way, the amount of radiation and damage to normal tissues can be decreased.31-36 Additionally, in comparison with rare earth and bismuth nanomaterials, Au and Pt nanomaterials have a higher biocompatibility both in vitro and in vivo.37-39 Moreover, Au and Pt nanomaterials also exhibit good photostability and high photothermal conversion efficiency in PTT for cancers.37, 40 It has also been proved that hybrid bimetallic nanoparticles exhibit better optical and chemical properties than those of single element-containing nanoparticles.41-43 Therefore, we hypothesized that the combination of Au and Pt in one nanoscale platform could be a high-efficiency and low-toxicity

therapeutic

agent.

In

our

current

study,

PEGylated

Au@Pt

nanodendrites(NDs) are rationally designed and fabricated (Scheme 1). In this system, the Au nanocore and Pt nanobranches endow the nanostructures with a broad absorbance of NIR light and strong concentrate of X-ray, thus enhancing PTT as well as RT. It was found that a strong synergistic result can be achieved in the inhibition of cancer cells by means of adopting Au@Pt NDs to combine PTT and RT. Moreover, the Au@Pt NDs also possess significant computed tomography (CT) imaging signal enhancement, which shows the potential for image-guided tumor therapy in the future.


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Scheme 1.The illustration of the PEGylation and photothermal/radiation synergistic therapy application of the Au@Pt NDs.

2. EXPERIMENTAL SECTION 2.1. Synthesis of Au NPs. We first mixed 0.302 g HAuCl4•4H2O and 30 mL oleylamine in 100 mL three-necked flask. Then the mixture solution was heated to 80 °C and left at this temperature for 5 h before the reaction was cooled to room temperature. Ethanol was used to wash the product for at least three times and the product was then centrifuged at 8000 rpm for 10 min and finally dispersed in 3 mL n-hexane. 2.2. Synthesis of Au@Pt NDs. 1 mL Au NPs n-hexane solution, 0.501g Pt(acac)2 and 30 mL oleylamine were mixed in 100 mL three-necked flask and then sparged with nitrogen through a needle. After that hydrogen was introduced to the system. The mixture solution was heated to 90°C slowly and left at this temperature for 2 h. The products were washed at least three times with ethanol and centrifuged at 8000 rpm for 10 min and finally dispersed in n-hexane. 2.3. Synthesis of LA-PEG and Surface Modification of Au@Pt NDs. LA-PEG polymer which could help to impart stability in physiological solutions was synthesized following a reported protocol.44 50 mg of LA-PEG was dissolved in 2 mL of water in a flask (25 mL). Then, 10 mg of hydrophobic Au@Pt NDs dispersed in 1 5 ACS Paragon Plus Environment


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mL of hexane were added. The above mixture was sonicated with a bath sonicator for 15 min, and then left stirring overnight. Surface modified Au@Pt would slowly transfer to water phase. After blowing hexane away with nitrogen, excess LA-PEG was removed by centrifugation and washed with water. PEGylated Au@Pt NDs as the collected precipitates were then redispersed in 2 mL water. 2.4. Photothermal Experiments of Au@Pt NDs. To study the photothermal effect of the synthesized Au@Pt NDs, 1 mL aqueous solutions containing different concentrations (0, 25, 50, 75 µg/mL) of Au@Pt NDs were irradiated under 808 nm laser at a power density of 1 W/cm2 for 10 min. An IR thermal camera (Fotric 225-1) was used to record the temperature of the solution at each time point. 2.5. Cellular Uptake Assays. To determine the cellular uptake of Au@Pt NDs, the 4T1 cells were plated on a 6-well plate and cultured for 24 h. Next, Au@Pt NDs (50 µg/mL) were added into each well for incubation in culture medium. At a determined time, PBS was used to wash the cells for three times. A certain number of cells were collected to measure the cellular uptake of Au@Pt NDs by an ICP-MS instrument (Element-2, Thermo). The other cells were fixed with 4% paraformaldehyde and stained with nucleus dye Hoechst 33342 (Invitrogen). Then the cells were observed by dark-field microscopy (IX73, Olympus). To determine the Pt contents in cells by ICP-MS, PBS was used to wash the cells for three times. Then the cells were collected and aqua fortis (nitric acid/hydrochloric acid 3:1 volume ratio) was used for digestion. After adjusting the solution volume to 2 mL using 2% nitric acid and 1% hydrochloride acid (1:1), Pt assays were performed by ICP-MS measurement. Divided by the number of cells, the data of Pt contents per 104 cells were calculated. 2.6. Combined Photothermal and Radiotherapy. 4T1 cells were seeded into 96-well plates and incubated for a 24-hour period. Then 100 µL of culture medium with nanodendrites concentration of 50 µg/mL was co-incubated with 4T1 cells for 6 h. Next, 4T1 cells were exposed to 808 nm laser (1W/cm2) for 10 min and incubated for 30 min (PTT treatment), followed by 4 Gy of X-ray radiations (RT treatment) and incubation again for 20 h. MTT assay was performed to measure the cell viability. 6 ACS Paragon Plus Environment

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3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of PEGylated Au@Pt NDs. The multifunctional Au@Pt NDs were prepared according to a modified procedure. Briefly, they were synthesized via reduction of Pt(acac)2 on the surface of Au nanocore in oleylamine under hydrogen atmosphere. As shown in Figure S1, the average diameter of Au nanocore was about 8 nm. After Pt(acac)2 was added, the growth of Pt metal nanobranches on the surface of Au nanocore resulted in the hybrid core–branches bimetallic NDs. LA-PEG which was successfully synthesized and verified by 1H NMR (Figure S2) was finally grafted to impart water-dispersibility and biocompatibility. After the modification with hydrophilic LA-PEG, a stable dispersion of the PEGylated Au@Pt NDs was formed in water and PBS (Figure S3). The diameter of the NDs was about 30 nm (Figure 1A) as shown in transmission electron microscopy (TEM). The dendrite structure of NDs could be clearly observed through the TEM image (Figure 1B). ICP-MS was used to check the element composition of hybrid core–branches NDs (Table S1). Energy dispersive spectroscopic (EDS) element analysis was further performed to confirm the Au and Pt existence (Figure 1C). PEGylated Au@Pt NDs are successfully synthesized with a dendrite structure of Au nanocore and Pt nanobranches as indicated by above results. Au nanocore exhibited little absorbance at longer wavelengths; in contrast, the Au@Pt NDs exhibited an apparent broad photo absorption with the growth of Pt nanobranches (Figure S4), which benefited the penetration into deep tissues at NIR wavelengths. And with the increase of concentration, an obvious increase in absorption intensity was observed (Figure 1D). The reason for such a phenomenon is likely that the branch–structure of Au@Pt NDs benefits their light harvesting by reducing the light transmittance and reflection that usually occurred more easily in spherical particles.



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Figure 1. Characterization of Au@Pt NDs. (A) and (B) Representative TEM images of Au@Pt NDs at different magnifications; (C) EDS element analysis of Au@Pt NDs; (D) UV-Vis-NIR absorption spectra of the aqueous dispersion of PEGylated Au@Pt NDs.

3.2. Biocompatibility and Cellular Uptake of PEGylated Au@Pt NDs. The first concern of nanomaterials in biomedical application is biocompatibility and it ought to be investigated before functional experiments. Therefore, we first evaluated the effects of PEGylated Au@Pt NDs on cell viability. HUVEC (a normal cell line) as well as 4T1 cancer cells were incubated together with Au@Pt NDs for a 24-hour period. Results indicated that Au@Pt NDs exhibited no acute cytotoxicity to both HUVEC cells and 4T1 cells at the tested concentrations (Figure 2). Cell viability of both cell lines kept higher viability of >85% even at a high concentration (75 µg/mL) of Au@Pt NDs. This result indicates that Au@Pt NDs used in our current study present almost no cytotoxicity, which provides great advantage for further biomedical application. Since the Au@Pt NDs have no apparent toxicity, we further study the uptake of NDs by 4T1 cells. Dark-field microscopy is used to characterize the expression of scattered light from Au nanomaterials that can be utilized to visualize Au nanomaterials in living cells. To study the cellular uptake of the Au@Pt NDs, scattered light was used to assess the cellular uptake of NDs.45 Dark-field microscopy 8 ACS Paragon Plus Environment

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showed obvious scattered light signal in the cytoplasm after treatment with Au@Pt NDs for 3 h, suggesting the efficient cellular uptake and intracellular distribution of Au@Pt NDs (Figure 3A). Moreover, inductively coupled plasma mass spectroscopy (ICP-MS) was used to carry out quantitative measurements to detect the elemental Pt content of the cells treated with Au@Pt NDs. Results indicated that the uptake of Au@Pt NDs depended on the incubation time (Figure 3B).

Figure 2. Cytotoxicity of Au@Pt NDs on (A) HUVEC and (B) 4T1 cells. The error bars symbolize the standard deviation of three measurements (n = 3).



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Figure 3. Assessment of cellular uptake efficiency of Au@Pt NDs. (A) Dark-field microscopy images of 4T1 cells after treatment with PBS (control) or Au@Pt NDs for 3 h. Scale bar = 20 µM. (B) Pt contents in 4T1 cells after cells incubation with Au@Pt NDs for different times. The error bars symbolize the standard deviation of three measurements (n = 3).

3.3. PTT Effect of Au@Pt NDs. To demonstrate the potential of Au@Pt NDs as NIR PTT agent, their photothermal effect was investigated by monitoring the temperature increase upon 808 nm continuous laser (1W/cm2) for different times. As depicted in Figure 4A, pure deionization water did not show apparent response to the irradiation. In contrast, under the same experimental conditions the aqueous dispersion of the Au@Pt NDs manifested a drastic increase in temperature even at a lower concentration (25 µg/mL), which implied that Au@Pt NDs had helped to transform photo energy of the NIR laser into heat. The rate of temperature rise and the final temperature were proportional to the Au@Pt NDs concentration. Thus it is indicated that Au@Pt NDs would be able to function as PTT agents in cancer therapeutics.


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Figure 4. PTT effect of Au@Pt NDs. (A) The photothermal effects of Au@Pt NDs after laser irradiation (808 nm, 1 W/cm2, 10 min); (B) Viability of 4T1 cells after Au@Pt NDs treatment and laser irradiation. *p< 0.05 compared to untreated control cells; (C) Live/dead staining images of 4T1 cells after NDs treatment and laser irradiation: (a) without treatment; (b) NIR irradiation only (808 nm, 1 W/cm2, 10 min); (c) Au@Pt NDs only (75 µg/mL); (d) Au@Pt NDs only plus NIR irradiation. Live and dead cells were presented respectively in green (live cells) and red (dead cells) colors in above images. Scale bar = 100 µm. The error bars symbolize the standard deviation of three measurements (n = 3).

We next investigated the NIR laser-triggered photothermal tumor cell ablation facilitated by Au@Pt NDs. 4T1 cells and Au@Pt NDs were first incubated together for a 24-hour period in darkness and then treated with irradiation for 10 min using an 808 nm laser at a power density of 1 W/cm2 (Figure 4B). Test results indicated that the cell viability of 4T1 cells decreased gradually with the increase of the Au@Pt 11 ACS Paragon Plus Environment


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NDs concentration. And only about 45% of the 4T1 cells kept viable at an Au@Pt NDs concentration of 75 µg/mL after the photothermal ablation. Meanwhile, live/dead staining was adopted to assess the photothermal therapeutic capability of Au@Pt NDs on 4T1 cells. The results in Figure 4C revealed that after laser irradiation the number of living cells (stained green) is decreased significantly for 4T1 cells which were incubated with Au@Pt NDs at a concentration of 75 µg/mL. In contrast, from groups of 4T1 cells only, laser irradiation only and incubated with Au@Pt NDs only, very few dead cells were observed. These results indicated that our Au@Pt NDs can act as heat mediator for hyperthermia treatment of cancer cells. 3.4. Au@Pt NDs-Enhanced PTT/RT Synergistic Therapy. The presence of the high atomic number elements (Au and Pt), low toxicity and strong NIR absorption ability, made it possible for Au@Pt NDs to be used in synergistic PTT and RT of cancers cells. After different treatments, the MTT assay was operated to evaluate the cell viability of 4T1 cells. As indicated in Figure 5A, the cell viability was over 80% for cells treated with Au@Pt NDs alone at the concentration of 75µg/mL. However, the cell viability dropped to 45% after receiving irradiation of the NIR laser (808 nm, 1 W/cm2, 10 min). In a similar manner, it significantly declined to 32% after combinational treatments of Au@Pt NDs (75 µg/mL) and X-ray irradiation (4 Gy). Thus it was clearly indicated that Au@Pt NDs could work as photothermal agents and radiosensitizers. Next, we evaluated the synergistic effect of PTT/RT combinational therapy enhanced by Au@Pt NDs. 4T1 cells incubated with Au@Pt NDs at concentration of 50 µg/mL were first treated with laser irradiation (808 nm, 1 W/cm2, 10 min) and then X-ray irradiation (4 Gy). The cell viability was found to substantially decline to 30%, which was 42% lower than the result of Au@Pt NDs-enhanced PTT alone, 25% lower than the result of Au@Pt NDs-enhanced RT alone, and it was even 10% lower than the calculated projected additive value. The projected additive value was calculated by multiplying the cell viability of Au@Pt NDs-enhanced PPT group by the cell viability of the Au@Pt NDs-enhanced RT group.27 These data strongly confirmed the 12 ACS Paragon Plus Environment

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considerable Au@Pt NDs-enhanced PTT/RT synergistic effect for cancer therapy (Figure 5B).

Figure 5. Au@Pt NDs-enhanced PTT/RT increased apoptosis in 4T1 cells. (A) Quantitative analysis of cell viability with Au@Pt NDs at varied concentrations with or without NIR laser and RT. (B) Synergistic therapeutic effect on 4T1 cells that have taken up Au@Pt NDs (50 µg/mL) subjected to NIR, RT and the combined NIR/RT treatments. Same NIR condition (808 nm, 1W/cm2, 10 min) was used in all PTT experiments. And a dose of 4 Gy was used in all RT experiments. 3.5. CT Imaging of Au@Pt NDs. The X-ray attenuation agents normally contain high atomic number elements which enable them to generate contrasts in CT imaging.46 Thus we believe that the Au@Pt NDs can be employed as an outstanding CT imaging contrast agent. In order to attest our assumption, we carried out a CT imaging test. Figure 6A shows the CT image of Au@Pt NDs dispersions with different concentrations. It could be clearly observed that the CT signal was enhanced with the increase of concentrations. At the same time, the Hounsfield units (HU) 13 ACS Paragon Plus Environment


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values were linearly proportional to the concentrations of the Au@Pt NDs (Figure 6B), showing an X-ray absorption coefficient of 28.6 HU/mL/mg, largely higher than that of iopromide (16.4 HU/mL/mg), a widely used clinical CT contrast agent.47

Figure 6. (A) CT images of the Au@Pt NDs aqueous dispersion with different concentrations (mg/mL); (B) CT value (HU) of the Au@Pt NDs as a function of the concentration of NDs.

4. CONCLUSIONS In summary, a multifunctional nanoscale theranostic agent has been constructed for integrating nanomaterial-enhanced CT imaging and PTT/RT together. The results show that Au@Pt NDs exhibit broad absorbance in NIR region because of the dendritic structure and good radiosensitizing effect because of the composition of Au as well as Pt. Using Au@Pt NDs as an anticancer agent, cancer cells could be efficiently killed at relatively low NIR laser power and X-ray radiation dose through the synergistic effect of PTT and RT. Besides, the CT imaging exhibited by Au@Pt NDs has the potential to pinpoint the location of cancer and thus guides the PTT/RT. This study promises the use of Au@Pt NDs as a novel multifunctional theranostic agent for cancers. In the future, the integration of various functional materials is expected to provide more powerful theranostic nano-platform for cancer diagnosis and therapy.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Additional text with details on materials and experiment conditions. The composition of Au@Pt nanodendrites determined by ICP-MS, TEM image of the as-synthesized Au nanoparticles, 1H NMR spectrum of the LA-PEG, photos of the Au@Pt NDs in water and PBS and DLS data of Au@Pt NDs.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors acknowledge financial support from National Natural Science Foundation of China (51402203, 31400862), Natural Science Foundation of Jiangsu Province for Young

Scholars

(BK20140326),

China

Postdoctoral

Science

Foundation

(2015M571797), the Natural Science Foundation of Jiangsu Higher Education Institutions (14KJB430021), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions. The Project is also sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. We thank Mr.Yiheng Dai for his help in improving the English text.



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PEGylated Au@Pt Nanodendrites as Novel Theranostic Agents for

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