In vivo targeted imaging of early stage prostate cancer using a transferrin based near-infrared fluorescence probe

Abstract

Transferrin (Tf) stabilized Au nanoclusters were successfully applied in the near-infrared targeted imaging of prostate cancer overexpressing Tf receptor in vivo. The prepared Tf-Au NCs owned the merits of low cytotoxicity, good biocompatibility, and natural metabolism, excellent optical properties in the NIR region and receptor specific cancer targeting ability. In our study, serial imaging in vivo has been performed sequently. The results showed that targeted fluorescence imaging with Tf-Au NCs possessed an amazing capability of detecting the early-stage small tumours, and giving as accurate and reproducible measures of tumour size in mice as external calliper measurements. To our knowledge, this was the first time to study the biomedical application of Tf-Au NCs in vivo via intravenous administration rather than cell experiments or orthotopic injection. These remarkable data could provide direct and factual evidence that Tf-Au NCs are very promising nanoprobes which could be harmlessly and efficiently used for the targeted diagnosis of human tumours in vivo, indicating that the further clinical translation of Tf-Au NCs could be expected.

---

Introduction

Prostate cancer (PCa) is the most common cancer affecting men in the Western world. In 2012, it accounted for 28 000 deaths in the United States alone.¹ Among the patients, about 60% exhibit no symptoms at the early stage. Therefore, early detection and timely treatment play a pivotal role in the clinical outcome. Especially, if no appropriate therapeutic intervention is given, prostate cancer will inevitably aggravate and metastasize, subsequently leading to a poor clinical prognosis.² However, to date, this issue still remains a great challenge despite increasing availability of new imaging methods, such as CT, MRI, TRUS and PET which only depict alternations related to later manifestations of the diseases and are less sensitive for early lesions.^3,4 Nowadays, the advent of molecular imaging in 1999 unlocked a new-fangled era in early detection and targeted imaging of human tumours.⁵ As a promising noninvasive and high temporal resolution modality for cancer detection, optical molecular imaging has received immense attention because it can provide molecular information of the tumour metabolism and biochemistry before the change of organization of the structure appeared.⁶

Among the optical imaging technologies, near-infrared (NIR) fluorescence imaging, within the wavelength range of 700–1000 nm, is a promising strategy for detecting cancer in vivo due to its low absorption, little scattering effect and excellent tissue penetration.^7–9 In the past decades, numerous nanoprobes have been developed to apply in NIR fluorescence imaging.^10–15 But some inevitable shortcomings should be overcome before their successful translations into clinical purpose. For example, dyes-containing nanoparticles remain problematic for long-term molecular imaging secondary to their rapid photobleaching and quantum dots are limited by inherent toxicity and instability.^12,15 Compared with them, Au NCs are ultrasmall, stable and biocompatible, and as such, show great potential for cancer diagnosis in vivo by NIR fluorescent imaging.^16–23

Transferrin (Tf) is an iron-binding blood plasma glycoprotein that deliveries Fe³⁺ to vertebrate cells through receptor-mediated endocytosis. Tf receptor (TfR) is a cell transmembrane protein that is essential for the internalization process of iron-loaded Tf to all cells and regulates cells' growth. Compared with normal cells, cancer cells, which require large amounts of iron, are reported to overexpress about 100-fold TfR.²⁴ Various tumours like prostate cancer and breast tumour, have high expression level of TfR, indicating that Tf could be used as a biogenic and metabolic ligand that has high specialty and affinity to TfR overexpressing tumour cells.^25,26 Recently, Tf has been utilized to conjugate with various nanoparticles to aid in targeted diagnosis and treatment of glioma, hepatocellular carcinoma, ovarian carcinoma and acute myeloid leukemia.^27–31 As novel nanoprobes, Tf-Au NCs were first reported in 2013.¹⁸ Au NCs, synthesized inside human Tf protein, not only provide the targeted ability but also remain the naturally metabolic activity in the cell experiments. However, the applications of Tf coated Au nanoclusters in different cancers are in the starting stage. It is highly desirable to expand the use of such an excellent fluorescent nanoprobe.

In current work, Tf-Au NCs were systematically investigated in the application of prostate cancer-targeted imaging. The cytotoxicity and targeting efficacy of Tf-Au NCs in vitro were studied. Subsequently, the in vivo biocompatibility and tumour-specific fluorescent imaging after intravenous injection were also investigated. The experimental results indicated that the prepared Tf-Au NCs could be used as a kind of excellent prostate cancer-targeted imaging nanoprobes, which shows great potential in the application of the diagnosis of prostate cancer in clinical medicine.

Experimental section

Materials and chemicals

All chemicals and solvents used in the synthesis of Tf-Au NCs were of analytical grade and were used as-received without further purification. Ultrapure water (Hangzhou Wahaha Group Company Ltd, Hangzhou, China) was used throughout. Human transferrin was obtained from Sigma-Aldrich (St. Louis, MO, USA). Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂O) was purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Na₃PO₄·12H₂O was obtained from Guangfu Fine Chemical Research Institute (Tianjin, China). MTT stock solution was obtained from Amrseco (Solon, OH, USA).

Synthesis of Tf-Au NCs

Tf-Au NCs were synthesized according to the previous report.^18,23 Typically, the glassware was washed with aqua regia and rinsed with ultrapure water before use. 150 mg Tf was prepared in 6 mL of 0.1 M PBS, then HAuCl₄ (1.2 mL, 25 mM) was added to the above solution. After 5 min, the reaction was processed in dark at 37 °C for 12 h. The prepared Tf-Au NCs were washed five times via centrifugal filtration at 5000 rpm for 20 min. The collected Tf-Au NCs were stored at 4 °C for the following use.

Characterizations

The high resolution transmission electron microscopy (HRTEM) images of Tf-Au NCs were observed on a FEI Tecnai G2 F20 microscope with an acceleration voltage of 200 kV. Dynamic light scattering (DLS, Brookhaven Instruments Ltd, U.S.) was performed to detect the mean hydrodynamic particle size and size distribution at 25 °C. Fluorescence absorption and emission spectra of Tf-Au NCs were obtained on an F-4500 spectrofluorometer (Hitachi, Japan). UV-Vis-NIR absorption spectra were recorded using a UV-3600 UV-Vis-NIR spectrophotometer (Shimadzu, Japan) with a slit width of 2.0 nm and a time constant of 2.0 s. The concentration of the prepared Tf-Au NCs was determined after freeze-drying, and then the nanoparticles were dissolved in PBS. Au content in the stock solution was determined by the An X Series quadrupole inductively coupled plasma mass spectrometry instrument (ICP-MS, Thermo Elemental, Cheshire, U.K.). The stock solution was diluted to different concentrations and used in the following assays.

Cytotoxicity assay

To visualize the cytotoxicity of the prepared probe, PC-3 cells (TfR positive) growing in log phase were respectively seeded into a 96-well cell culture plate with RPMI 1640 containing 10% FBS (Thermo Fisher Scientific Inc., USA). All cells grew in a humidified atmosphere of 5% CO₂ at 37 °C for 24 h. The Tf-Au NCs solutions with a wide concentration range from 0 to 400 μg mL⁻¹ (0 μg mL⁻¹, 25 μg mL⁻¹, 50 μg mL⁻¹, 100 μg mL⁻¹, 200 μg mL⁻¹, 400 μg mL⁻¹) were subsequently added into the cells and cultured for another 24 h under the same conditions as above. The MTT stock solution (5 mg mL⁻¹) was added to each well and incubated for an additional 4 h. After the addition of DMSO (150 μL per well, Sigma-Aldrich, St. Louis, MO, USA), the assay plate was allowed to stand in shaker (QLBE, China) for 10 min. Finally, the absorbance of each solution was measured at 570 nm on a microplate reader (Thermo, Varioskan Flash).

In vivo toxicity assay

For further estimating the toxicity of Tf-Au NCs in vivo, 6 healthy Kunming mice (8–9 weeks) were randomly divided into two groups with 3 animals each. One group was injected with Tf-Au NCs (50 μg μL⁻¹, 0.33 mg Au for every kilogram of body weight) via tail veins while the other group was intravenously treated with the same amount of PBS buffer solution. The survival situation and body weights were continuously monitored during 2 weeks post injection, for finding if there were any significantly biological effects caused by Tf-Au NCs.

In vitro imaging

Prostate tumour PC-3 cells growing in log phase were seeded into a 24-well culture plate at a density of 5000 cells per well. After cultured in RPIM 1640 supplemented with 10% FBS at 37 °C under 5% CO₂ for 24 h, about 70% of the cells were attached to the wall. Afterwards, Tf-Au NCs at a concentration of 500 μg mL⁻¹ were added to the wells and the cells were further incubated for 4 h. Then, the cells were washed with PBS buffer (10 mM, pH 7.4) three times to remove the non-specific binding of Tf-Au NCs. Cell images were obtained by the fluorescent inverted microscope (Olympus, Japan).

Competition experiment

A competition experiment with unconjugated Tf was used to assess the binding specialty of Tf-Au NCs. PC-3 cells, incubated as described above, were seeded into a 24-well culture plate, treated with unconjugated Tf (2000 μg mL⁻¹) for 4 h, and washed three times with PBS. Then cells were incubated for another 4 h with Tf labelled Au NCs (500 μg mL⁻¹). After washing, fluorescent imaging was performed again.

Animal model

Male BALB/c nude mice (8–9 weeks) weighing 24.0 ± 0.5 g were used in the study. All the experiments were in accordance with the guidelines in the Second Hospital of Tianjin Medical University on the ethical use of animals. About 1 × 10⁶ PC-3 cells were subcutaneously transplanted into the left shoulder of BALB/c nude mice to generate tumour-bearing animal models. Animal experiments were performed when tumours grew to about 50 mm³ in volume (approximately 10 days post inoculation).

In vivo fluorescence imaging

Prior to imaging, normal mice and tumour-bearing mice were anesthetized with intraperitoneal injection of 5% chloral hydrate (7 mL kg⁻¹). Then, Tf-Au NCs at a concentration of 50 μg μL⁻¹ (0.33 mg kg⁻¹ Au) were administrated by tail vein injection. Dynamical fluorescence imaging was carried out over sequential time points after injection using an in vivo imaging system (NightOWL, Germany) with 485 nm excitation and 700 nm long-pass emission filters. The exposure time was set as 1 s.

Histology analysis

At 1 week post-injection, the mice were killed to dissect brain, spleen, kidney and liver. Meanwhile, untreated BALB/c nude mice were sacrificed to dissect the same organs as the control group. Subsequently these organs were fixed in 4% formalin for 48 h, washed three times in PBS for 30 min, embedded in paraffin, and sliced into 4 μm sections. Then tissue sections were mounted on glass slide and stained with hematoxylin/eosin for 5 min. Finally, morphological changes were observed by a microscope.

Results and discussions

Synthesis and characterization

Tf-Au NCs were synthesized using Tf as stabilizer, reducer and target molecule simultaneously, and the size and optical properties were characterized. As shown in Fig. 1A, HRTEM images revealed a spherical shape and good size uniformity with an average size of less than 3 nm. The average hydrodynamic diameter of Tf-Au NCs detected by DLS was 10.9 nm, larger than the size measured by HRTEM due to its hydrophilic property. Absorbance spectra of Tf-Au NCs showed the peak at 280 nm (Fig. 1B), which was the characteristic absorbance of the protein. In addition, no plasmon resonance of gold at 520 nm could be observed, indicating the absence of gold nanoparticles. The prepared Tf-Au NCs showed excellent colloidal stability due to the protection of Tf, and there was no precipitation in the Tf-Au NCs solution even after 3 months, which greatly facilitated the application in the biological environments in vitro and in vivo.

Fig. 1 (A) HRTEM image of the prepared Tf-Au NCs (100 nm), (B) absorption spectrum, (C) fluorescent excitation and emission spectra of the as-prepared Tf-Au NCs, (D) the photographs of Tf-Au NCs in aqueous solution under visible light (left) and UV light (right).

With different sizes and shapes, Au nanostructures have multiple biomedical applications for both cancer diagnosis and therapies. For example, spherical Au nanoparticles, which are composed of a metal element with high atomic number, have been used as computed tomography contrast agents due to their great X-ray attenuation effect.^32,33 Additionally, novel metal NCs have dimensions approaching the Fermi wavelength of electrons that make the continuous density of states break up into discrete energy levels, therefore dramatically different optical, electrical and chemical properties of such nanoparticles could be observed. In particularly, their attractive optical features, including strong photoluminescence, combined with good photostability, large Stokes shift, tunable emission colours in biocompatible scaffolds and high emission rates, establish them as a new class of ultrasmall, biocompatible fluorescent nanomaterials for applications in optical imaging-based early diagnosis of human cancers.^34,35

In this article, we fabricated clustered Au nanoparticles stabilized in transferrin. The prepared Tf-Au NCs showed excellent optical properties, emitting strong NIR fluorescence at the range of 650 nm to 800 nm with the maximum fluorescence emission at the wavelength of 700 nm (Fig. 1C). The fluorescence of the Tf-Au NCs solution could be clearly observed with an intense red emission under UV irradiation (Fig. 1D), which ensured the Tf-Au NCs could operate as potential nanoprobes for NIR fluorescent imaging.

In vitro assays

Optimal nanoprobes should be safe and nontoxic to cells and tissues. So we evaluated the cytotoxicity of the probe through MTT assays. The PC-3 cells were incubated without and with various concentrations of nanoparticles (25–400 μg mL⁻¹). As was shown in Fig. 2, no significant decrease was found in the viability of the PC-3 cells at the studied particle concentration compared with the control group, indicating that Tf-Au NCs had no obvious toxicity. The potential reason was that Au NCs synthesized inside Tf should be improving the bio-friendly nature of the probe.

Fig. 2 The result of MTT assay showed the viability of the PC-3 cells, which were incubated with various concentrations of Tf-Au NCs (0–400 μg mL⁻¹) for 24 h, had no significant decrease.

After a 4 h incubation with Tf-Au NCs, fluorescent signal was detected in PC-3 cells (Fig. 3). The Tf-Au NCs was uptaken by PC-3 cells and localized mainly in the cell through active targeting mechanisms. We further investigated the binding specificity of Tf-Au NCs to TfR positive tumours by a competition experiment. After incubation of a 4-fold excess of Tf-Au NCs unconjugated Tf after treatment with unconjugated Tf, no fluorescence was detected in the tumour cells. The result indicated that Tf-Au NCs could specially target to TfR overexpressing tumour cells.

Fig. 3 A–C: in vitro images of PC-3 tumour cells using fluorescent inverted microscope after treatment with Tf-Au NCs. Strong red fluorescence was found in the cytoplasm of TfR overexpressing PC-3 cells. D and E showed the results of competition experiments. Negligible signal of Tf-Au NCs was observed, indicating that fluorescence intensity was related to specific interaction between Tf-Au NCs and TfR. The scale bar was 100 μm.

In vivo experiments

The potential of Tf-Au NCs in vivo tumour imaging was tested by using BALB/c nude mice which were subcutaneously implanted with PC-3 tumour cells at the left shoulder. To the best of our knowledge, there existed no studies concerning the in vivo application of Tf-Au NCs through tail vein administration rather than orthotopic injection.

In our study, a series of fluorescence images were acquired by small animal in vivo imaging system. Fluorescence of Tf-Au NCs could be clearly visualized in tumour region of PC-3 bearing mice for an extended period of time after intravenous injection (Fig. 4 and 5A), permitting differentiation of the tumour from other tissues. Additionally, some slight signals from liver and other tissues appeared possibly resulting from nonspecific uptake of hepatic reticuloendothelial system or relative high level of TfR. In corresponding to cell experiment, in vivo imaging showed bright fluorescence signals in the tumour area as a result of enhanced permeability and retention effect as well as active targeting mechanism of Tf-Au NCs towards tumours, most of which overexpressed TfR.

Fig. 4 In vivo NIR fluorescence imaging of PC-3 tumour xenograft mice during an observation period of 12 h after Tf-Au NCs injection (8 μg Au per mouse). The arrows indicated the regions of PC-3 tumour. Fluorescence of Tf-Au NCs was clearly visualized in tumour region from 10 min post-injection and then increased progressively over the time with the maximum intensity at 2 h. Tumour fluorescence diminished gradually and reached background levels at 12 h post-injection.

Fig. 5 Fluorescence imaging (A), photograph of upper and lateral view of the xenograft mouse (B) and measurement of the isolated tumour (C) at 2 h post-injection of Tf-Au NCs (8 μg Au per mouse). Comparing to B, in vivo imaging (A) showed the tumour with significant enhancement had definite location to the actual tumour. Tumour size measured dependent on fluorescence imaging (A) was in close proximity to that of resected specimen (C).

Furthermore, metabolic process of Tf-Au NCs was continuously and dynamically monitored by fluorescence imaging until 12 h. As shown in Fig. 4, the obvious fluorescence of Tf-Au NCs could be observed in tumour region as early as 10 min after administration. Then, intensity of fluorescence signals increased progressively over the time and reached the maximum at 2 h post-injection secondary to constant accumulation of the probe. After 2 h, fluorescence in the tumour region gradually diminished with time and reached background level at 12 h post-injection. Therefore, we speculated that Au cluster didn't influence the bioactivities of Tf in the body and Tf-Au NCs could be metabolized in the normal way.

In addition, xenograft tumour was completely resected from the sacrificed mice (Fig. 5B). The actual size of isolated tumour measured ex vivo by calliper was 0.59 cm long and 0.37 cm wide (Fig. 5C). At 2 h post-injection when fluorescence intensity of the tumour reached the maximum, the tumour border could be differentiated clearly based on the fluorescent contrast between the tumour and its surrounding tissue. Tumour size was measured in vivo. The maximum length and width of hyperintense area were similar to that of specimen (as shown in Fig. 5A), respectively. Subsequently, these results suggested that targeted fluorescence imaging with Tf-Au NCs was a promising strategy of detecting the early-stage small tumour clearly, and even giving accurate and reproducible differentiation of tumour border in mice, which was very useful for radical resection and dynamic monitoring of tumour.

The toxicity of nanoparticles was one of the key factors to restrict the application in vivo. In toxicity assay in vivo, all mice in each group survived over 20 days. No obvious weight loss was found in Tf-Au NCs injection group, comparing to the control group during an observational period of 2 week post injection (as shown in Fig. S1†). In another addition experiment, one week after imaging, the mice were sacrificed and histological examination was used to investigate the potential toxicity to major organs. Hematoxylin/eosin (H&E) staining (Fig. 6) showed no obvious injury in cellular structures of major organs, indicating Tf-Au NCs did not cause apparent tissue damage in vivo. The good biocompatibility made Tf-Au NCs potentially applied in the early diagnosis of cancers.

Fig. 6 H&E staining analysis of vital organs (brain, kidney, spleen and liver) after 1 week post-injection of Tf-Au NCs (8 μg Au per mouse). No apparent cellular changes were observed in major organs, demonstrating the good biocompatibility of Tf-Au NCs and its potential use in vivo imaging. Untreated group was used as control. The magnifications were 20×.

Conclusions

In this work, we successfully applied Au NCs inside Tf in targeted fluorescence imaging of TfR overexpressing cancers like PCa. The prepared Tf-Au NCs presented low cytotoxicity to cells and excellent optical properties in the NIR region. In vivo imaging showed Tf-Au NCs could generate strong fluorescence in the tumour region. Meanwhile, Tf-Au NCs showed great tumour-targeting ability and biocompatibility, which were realized by maintaining the bio-function of Tf (i.e. high affinity toward TfR). With the analysis of the real-time fluorescence imaging, the probe achieved its maximum accumulation in tumour area at 2 h post-injection. At this point, targeted fluorescence imaging with Tf-Au NCs could give accurate information of tumour location and size which was the key to the early detection and diagnosis of neoplasms. No florescence was detected after 12 hours, indicating that the Tf-Au NCs might be naturally metabolized in vivo. The study firstly investigated in vivo application of Tf-Au NCs via intravenous administration, which could provide more direct evidence about introducing the nanoprobes into the clinic. In summary, Tf-Au NCs were ideal candidates for optical-imaging-based detection of PCa and other TfR overexpressing tumours at early stage.

Acknowledgements

We would like to acknowledge useful discussion and constructive proposals in the initial edition of the article received from Yunqing Zhao and Xizi Xin. Additionally, we would like to thank Enlong Zhang and Qi Zhang for their help in the characterization of Tf-Au NCs. This work was supported by the Nature Science Foundation of Tianjin Medical University (Grant 2013KYQ05).

Notes and references

1. R. Siegel, D. Naishadham and A. Jemal, C–Cancer J. Clin., 2012, 62, 283–298.
2. G. A. Lin, D. S. Aaronson, S. J. Knight, P. R. Carroll and R. A. Dudley, Ca–Cancer J. Clin., 2009, 59, 379–390.
3. B. Turkbey, P. S. Albert, K. Kurdziel and P. L. Choyke, AJR, Am. J. Roentgenol., 2009, 192, 1471–1480.
4. P. de Visschere, W. Oosterlinck, G. de Meerleer and G. Villeirs, J. Belge Radiol., 2010, 93, 62–70.
5. R. Weissleder, Radiology, 1999, 212, 609–614.
6. G. Choy, P. Choyke and S. K. Libutti, Mol. Imaging, 2003, 2, 303–312.
7. S. A. Hilderbrand and R. Weissleder, Curr. Opin. Chem. Biol., 2010, 14, 71–79.
8. M. A. Pysz, S. S. Gambhir and J. K. Willmann, Clin. Radiol., 2010, 65, 500–516.
9. J. V. Frangioni, Curr. Opin. Chem. Biol., 2003, 7, 626–634.
10. Y. Chen, M. Pullambhatla, S. R. Banerjee, Y. Byun, M. Stathis, C. Rojas, B. S. Slusher, R. C. Mease and M. G. Pomper, Bioconjugate Chem., 2012, 23, 2377–2385.
11. D. K. Chatterjee, M. K. Gnanasammandhan and Y. Zhang, Small, 2010, 6, 2781–2795.
12. F. Zhang, D. Yi, H. Sun and H. Zhang, J. Nanosci. Nanotechnol., 2014, 14, 1409–1424.
13. X. Wu, X. He, K. Wang, C. Xie, B. Zhou and Z. Qing, Nanoscale, 2010, 2, 2244–2249.
14. C. Wang, L. Xu, X. Xu, H. Cheng, H. Sun, Q. Lin and C. Zhang, J. Colloid Interface Sci., 2014, 416, 274–279.
15. V. Pansare, S. Hejazi, W. Faenza and R. K. Prud'Homme, Chem. Mater., 2012, 24, 812–827.
16. C. L. Liu, H. T. Wu, Y. H. Hsiao, C. W. Lai, C. W. Shih, Y. K. Peng, K. C. Tang, H. W. Chang, Y. C. Chien, J. K. Hsiao, J. T. Cheng and P. T. Chou, Angew. Chem., Int. Ed. Engl., 2011, 50, 7056–7060.
17. Y. Kong, J. Chen, F. Gao, R. Brydson, B. Johnson, G. Heath, Y. Zhang, L. Wu and D. Zhou, Nanoscale, 2013, 5, 1009–1017.
18. Y. Wang, J. T. Chen and X. P. Yan, Anal. Chem., 2013, 85, 2529–2535.
19. J. M. Liu, J. T. Chen and X. P. Yan, Anal. Chem., 2013, 85, 3238–3245.
20. A. Retnakumari, S. Setua, D. Menon, P. Ravindran, H. Muhammed, T. Pradeep, S. Nair and M. Koyakutty, Nanotechnology, 2010, 21, 055103.
21. A. Zhang, Y. Tu, S. Qin, Y. Li, J. Zhou, N. Chen, Q. Lu and B. Zhang, J. Colloid Interface Sci., 2012, 372, 239–244.
22. J. Zheng, P. R. Nicovich and R. M. Dickson, Annu. Rev. Phys. Chem., 2007, 58, 409–431.
23. S. K. Sun, L. X. Dong, Y. Cao, H. R. Sun and X. P. Yan, Anal. Chem., 2013, 85, 8436–8441.
24. L. Han, R. Huang, S. Liu, S. Huang and C. Jiang, Mol. Pharm., 2010, 7, 2156–2165.
25. H. N. Keer, J. M. Kozlowski, Y. C. Tsai, C. Lee, R. N. McEwan and J. T. Grayhack, J. Urol., 1990, 143, 381–385.
26. M. Singh, K. Mugler, D. W. Hailoo, S. Burke, B. Nemesure, K. Torkko and K. R. Shroyer, Appl. Immunohistochem. Mol. Morphol., 2011, 19, 417–423.
27. G. L. Malarvizhi, A. P. Retnakumari, S. Nair and M. Koyakutty, Nanomedicine, 2014, 10, 1649–1659.
28. Z. Yang, B. Yu, J. Zhu, X. Huang, J. Xie, S. Xu, X. Yang, X. Wang, B. C. Yung, L. J. Lee, R. J. Lee and L. Teng, Nanoscale, 2014, 6, 9742–9751.
29. R. Li, Q. Zhang, X. Y. Wang, X. G. Chen, Y. X. He, W. Y. Yang and X. Yang, Drug Res., 2014, 64, 541–547.
30. S. Dixit, T. Novak, K. Miller, Y. Zhu, M. E. Kenney and A. M. Broome, Nanoscale, 2015, 7, 1782–1790.
31. S. Tortorella and T. C. Karagiannis, J. Membr. Biol., 2014, 247, 291–307.
32. M. Shilo, T. Reuveni, M. Motiei and R. Popovtzer, Nanomedicine, 2012, 7, 257–269.
33. C. Xu, G. A. Tung and S. Sun, Chem. Mater., 2008, 20, 4167–4169.
34. J. Sun and Y. Jin, J. Mater. Chem. C, 2014, 2, 8000–8011.
35. S. Li, D. Shaojun and G. U. Nienhaus, Nano Today, 2011, 6, 401–418.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra06188a

---