Bifunctional nanocapsules for magnetic resonance imaging and photodynamic therapy

Abstract

Here we present the fabrication of bifunctional nanocapsules via cation (Ca²⁺/Gd³⁺) induced self-assembly of partially hydrolysed α-lactalbumin. The prepared nanospheres, encapsulated with photosensitizer (Rose Bengal) and Gd³⁺ ion, show great potentials for magnetic resonance imaging and photodynamic therapy with a target-specific manner.

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Introduction

Theranostic nanomedicine, a nanoplatform with diagnosis and therapeutic functions in one cargo, has attracted wide attention in recent years.^1–7 The imaging guided drug delivery systems are of great promise in developing personalized medicine as they helps to identify the target sites and meanwhile monitors the effective drug delivery.^8,9 Ideally, imaging and drug delivery shall be noninvasive and capable of remote control. Magnetic resonance imaging (MRI) is a noninvasive medical imaging technique specializing in visualizing soft tissues of the human body with advantages of high spatial resolution, no ionizing radiation and no penetration limitations.^10–12 Gd³⁺ chelates and other paramagnetic complexes have served as versatile MRI contrast agents which could decrease T₁, resulting in a brighter (or positive) region in the image. For example, Sessler et al. reported Gd-Tx containing polymer micelles for in vivo quantitative MRI.¹³ In another aspect, photodynamic therapy is an emerging therapeutic modality in cancer treatment, which combines light, photosensitizer and oxygen to eradicate the tumor cells.^14,15 Photosensitizers, the main factor of photodynamic therapy (PDT), can absorb the appropriate light and transfer the optical energy to oxygen molecules (³O₂), which could generate cytotoxic reactive oxygen species (ROS) such as oxygen (¹O₂) to damage tumor cells.^5,16,17 The photo-triggered theranostics own superior advantages in on-demand treatment. When in dark, the PDT exhibits minimal damage to healthy tissues, whereas it becomes effective upon light exposure.¹⁸ The combination of MRI and PDT would be one of the most promising ways for noninvasive imaging-guided therapy.

The rational choice and design of nanocarriers is the key point for delivery imaging agents and therapeutic components to the target sites. Up to now, liposome, nanoemulsions, micelles, polymers, or protein assemblies have been used as cargo for this purpose.^19–23 Among them, protein-based nanocarriers are of particular interest since they are low cytotoxic, biodegradable, and easy to scale up.^19,24 α-lactalbumin, a major component of serum proteins, is widely studied in food applications.²⁵ In recent years, α-lactalbumin (PHLA) by a specific serine protease from Bacillus licheniformis has shown to self-assemble into long nanotubes with diameter of 20 nm via a nucleation and growth mechanism.^26–28 Previous studies reported that the metal cations such as Ca²⁺, Al³⁺ and Ni²⁺ ions could induce the partially hydrolysed products to form nanotubes or aggregates at 45 or 50 °C under appropriate pH. However, it was rarely reported to employ them as nanocarriers for imaging and drug delivery. Herein, the bifunctional protein nanosphere encapsulating the photosensitizer (RB, Rose Bengal) was fabricated via Ca²⁺/Gd³⁺ induced self-assembly of PHLA, which was further explored for simultaneous MRI and PDT applications.

Results and discussion

The bifunctional nanospheres (BFNS), as shown in Scheme 1, were prepared simply by mixing the partially hydrolysed α-lactalbumin, RB, Gd³⁺, and Ca²⁺ together in Tris buffer solution at pH 7.5. Utilization of the paramagnetic cation Gd³⁺, in one aspect, could help to induce the formation of nanospheres and in another aspect, Gd³⁺ cation could be potentially used in MRI. Meanwhile, the employed nanospheres help to resolve poor solubility of photosensitizer and potential toxicity of cations. In addition, in order to achieve efficient targeting specificity, BFNS were coupled with Arg-Gly-Asp (RGD) peptide that has specific affinity towards the α_vβ₃-integrin, which is overexpressed in nascent endothelial cells during angiogenesis (formation of new blood vessels) in various tumor types and not in normal endothelial cells.^3,4 Under the guidance of RGD, the BFNS could be administrated into cells by endocytosis, where the Gd³⁺ complex and RBs serve as contrast and therapeutic agents, respectively, for achieving simultaneous magnetic resonance imaging and photodynamic therapy.

Scheme 1 Illustration for fabrication of BFNS and their applications in MRI and PDT.

These nanocapsules were prepared by self-assembly of PHLA with trigger of cations at 37 °C. The transmission electron microscopy (TEM) images (Fig. 1A) showed that they were spheres with an average size of ∼300 nm. The BFNS were also characterized by Fourier Transform Infrared (FTIR) with PHLA and free RB as comparison (Fig. 1B). The characteristic peaks of PHLA have good agreement with that of BFNS, indicating the successful formation of BFNS by self-assembly process. The vibration peak of –COOH (1770 cm⁻¹) was observed in free RB (Fig. 1B(a)), while not shown on the surface of BFNS, indicating that RB, PHLA and the metal cations were combined together. The other characteristic peaks of RB such as 1560, 1406, 862 and 706 cm⁻¹ are in accordance with those on BFNS (Fig. 1B(a)), further suggesting the combination of RB to BFNS. The formation of BFNS could be further confirmed by centrifugation. The BFNS can be well dispersed in water as shown in Fig. 1C(b) (left). After centrifugation, free RB is still well dispersed in water (Fig. 1C(a)) while BFNB encapsulated with RB is precipitated by centrifuging at 14 000 rpm for 15 min (Fig. 1C(b), right). Previous study claimed that formation of BFNS was affected by many factors such as the pH and the concentrations of Ca²⁺, Gd³⁺ and RB. Here the formation of BFNBs was carried out at different pH values. It was found that at pH 6.5 or 7.5, a net structure of BFNS could be obtained (Fig. S1A–C, ESI†). At pH 8.5, a sphere like structure could still be observed. However, when increasing pH to 9.5, it failed to form BFNS. The formation of metal hydroxide might account for this since it results in weak interaction between PLHA and metal cations. Meanwhile, the concentrations of Ca²⁺ and Gd³⁺ play an important role in the shapes of nanospheres (Fig. S2 and S3, ESI†). In the absence of Ca²⁺, it can hardly obtain nanospheres, while addition of Gd³⁺ could facilitate the formation of nanospheres. In light of increase of Gd³⁺ would enhance the MRI imaging, the optimized concentration for Ca²⁺ and Gd³⁺ are 0.4 mM and 1.0 mM, respectively. Additionally, it was also found that too much of RB would increase the overall size of the afforded nanospheres (Fig. S4, ESI†). Considering that low centration of photosensitizer would be enough for generation of singlet oxygen, 0.12 mM of RB was encapsulated in the BFNS.

Fig. 1 (A) TEM image of BFNS. (B) FTIR spectra of RB (a), BFNS (b), PHLA (c). (C) Photos of free RB (a) and BFNS (b) dispersed in water before (left) and after (right) centrifugation. [RB] = 0.12 mM.

Generally, relaxivity (r₁) of the contrast agent is considered as the main parameter for evaluating its ability for MRI application. Increase of r₁ values would induce significant MRI contrast-enhancement effects. The value of r₁ is reflected by the slope, which could be determined by the relaxation rate (R₁, 1/T₁) at various concentrations of contrast agents. As plotted in Fig. 2A, BFNS demonstrate the r₁ value of 4.39 mM⁻¹ s⁻¹ (400 MHz NMR), which is comparative to that of the second generation T₁ contrast agent of Gd³⁺DOTA (r₁ = 4.2 mM⁻¹ s⁻¹).⁴ The MRI images are shown in Fig. 2B, it is clearly found that the images become brighter as the concentration of Gd³⁺ increase, which also suggests that the BFNS could be utilized for MRI.

Fig. 2 Correlation between relaxation rates (1/T₁, S⁻¹) and gadolinium concentration (A) and T₁-weighted images of the BFNS (B). The measures were carried out at 298 K.

In terms of photodynamic therapy, it is singlet oxygen that is used for killing cancer cells. Therefore, the PDT performance depends on the singlet oxygen generated by photosensitizer which could accept photons from light and transfer the energy to oxygen. We use 1,3-diphenylisobenzofuran (DPBF) as a detector to assess the singlet oxygen production because DPBF could react with singlet oxygen which induces a decrease of absorption at 418 nm. Upon irradiation at 550 nm, the absorption of DPBF with BFNS decreases 60% for 16 min while the absorption with free RB reduces 80% at the same concentration (Fig. 3). The generation of singlet oxygen by RB in BFNS is less efficient than that of free RB, which might be ascribed to that the singlet oxygen generated by free RB has less barrier to access to DPBF. As a control, DPBF was incubated with BFNS without irradiation and the absorption of DPBF decreased less than 10%. These results implied that the RB encapsulated in BFNS could generate singlet oxygen effectively, which would be useful for photodynamic therapy.

Fig. 3 The absorption of DPBF with BFNS at 418 nm over time under 550 nm irradiation (the red circle) and in dark (the blue triangle). The other was the absorption of DPBF with free RB under 550 nm irradiation (the black square). [RB] = 0.02 mM.

In order to improve the targeting ability of BFNS toward tumor cell, the BFNS was further functionalized with RGD, termed as BFNS-RGD. The PDT performance of BFNS and BFNS-RGD was further evaluated by in vitro tests. The cell viability was carried out by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.^29,30 In order to verify the photodynamic efficiency, cell viability test was both performed with and without irradiation at 550 nm. The HepG2 cells were incubated in the presence of different concentrations of BFNS and BFNS-RGD suspension for 24 h, separately. Fig. 4 shows that both BFNS and BFNS-RGD demonstrate good biocompatibility over a wide range of concentrations (without irradiation). When under irradiation at 550 nm, the BFNS-RGD group has cell viability of 19%, however, it is 49% for that with BFNS. These results demonstrate that BFNS has a good photodynamic therapy effect especially with RGD targeting.

Fig. 4 Cell viability of HepG2 cells after being incubated with various concentrations of BFNS and BFNS-RGD with or without light irradiation for 24 h.

Conclusions

In a word, we demonstrate a facile one-step strategy to synthesize bifunctional protein nanocapsules for both magnetic resonance imaging and photodynamic therapy. We use metal cations (Ca²⁺ and Gd³⁺) to induce self-assembly of partially hydrolysed α-lactalbumin. During the fabrication of protein nanospheres, the photosensitizer RB is encapsulated. It is the first time to use α-lactalbumin as a nanocarrier for Gd³⁺ and photosensitizer RB. These as-synthesized nanospheres present a good T₁ enhanced MRI and efficient photodynamic effect with good biocompatibility. For the partially hydrolyzed α-lactalbumin, it is easy to obtain and scale up without tedious synthetic procedures. Also, the original material is cheap, indicating its great advantage for future commercial application. Hence, it will find great potentials in the fields of diagnosis and therapy.

Acknowledgements

The authors are grateful for the financial support from the University's scientific research project of Beijing University of Chinese Medicine (Grant No. 2013-JYBZZ-JS-198).

Notes and references

1. Z. J. Zhang, J. Wang, X. Nie, T. Wen, Y. L. Ji, X. C. Wu, Y. L. Zhao and C. Y. Chen, J. Am. Chem. Soc., 2014, 136, 7317–7326.
2. M. V. Yezhelyev, L. F. Qi, R. M. O'Regan, S. Nie and X. H. Gao, J. Am. Chem. Soc., 2008, 130, 9006–9012.
3. J. A. Park, J. J. Lee, J. C. Jung, D. Y. Yu, C. Oh, S. Ha, T. J. Kim and Y. M. Chang, ChemBioChem, 2008, 9, 2811–2813.
4. J. F. Jin, Z. H. Xu, Y. Zhang, Y. J. Gu, M. H. W. Lam and W. T. Wong, Adv. Healthcare Mater., 2013, 2, 1501–1512.
5. A. S. Yang, Y. C. Zheng, C. Y. Long, H. B. Chen, B. Liu, X. Li, J. L. Yuan and F. F. Cheng, Food Chem., 2014, 150, 73–79.
6. B. K. Wang, J. H. Wang, Q. Liu, H. Huang, M. Chen, K. Y. Li, C. Z. Li, X. F. Yu and P. K. Chu, Biomaterials, 2014, 35, 1954–1966.
7. L. Cheng, J. J. Liu, X. Gu, H. Gong, X. Z. Shi, T. Liu, C. Wang, X. Y. Wang, G. Liu, H. Y. Xing, W. B. Bu, B. Q. Sun and Z. Liu, Adv. Mater., 2014, 26, 1886–1893.
8. S. Bhuniya, S. Maiti, E. J. Kim, H. Lee, J. L. Sessler, K. S. Hong and J. S. Kim, Angew. Chem., Int. Ed., 2014, 53, 4469–4474.
9. Z. H. Li, K. Dong, S. Huang, E. G. Ju, Z. Liu, M. L. Yin, J. S. Ren and X. G. Qu, Adv. Funct. Mater., 2014, 24, 3612–3620.
10. L. Y. Wang, J. W. Bai, Y. J. Li and Y. Huang, Angew. Chem., Int. Ed., 2008, 47, 2439–2442.
11. S. Huang, W. Yan, G. F. Hu and L. Y. Wang, J. Phys. Chem. C, 2012, 116, 20558–20563.
12. S. Peng, W. Yan and L. Y. Wang, Mater. Res. Bull., 2013, 48, 4693–4698.
13. N. M. Barkey, C. Preihs, H. H. Cornnell, G. Martinez, A. Carie, J. Vagner, L. P. Xu, M. C. Lloyd, V. M. Lynch, V. J. Hruby, J. L. Sessler, K. N. Sill, R. J. Gillies and D. L. Morse, J. Med. Chem., 2013, 56, 6330–6338.
14. Y. Cheng, J. D. Meyers, A. M. Broome, M. E. Kenney, J. P. Basilion and C. Burda, J. Am. Chem. Soc., 2011, 133, 2583–2591.
15. A. Hakeem, R. X. Duan, F. Zahid, C. Dong, B. Y. Wang, F. Hong, X. W. Ou, Y. M. Jia, X. D. Lou and F. Xia, Chem. Commun., 2014, 50, 13268–13271.
16. K. Liu, X. M. Liu, Q. H. Zeng, Y. L. Zhang, L. P. Tu, T. Liu, X. G. Kong, Y. H. Wang, F. Cao, S. A. G. Lambrechts, M. C. G. Aalders and H. Zhang, ACS Nano, 2012, 6, 4054–4062.
17. E. Ju, K. Dong, Z. Chen, Z. Liu, C. Liu, Y. Huang, Z. Wang, F. Pu, J. Ren and X. Qu, Angew. Chem., Int. Ed., 2016, 55, 11467–11471.
18. Z. P. Zhen, W. Tang, C. L. Guo, H. M. Chen, X. Lin, G. Liu, B. W. Fei, X. Y. Chen, B. Q. Xu and J. Xie, ACS Nano, 2013, 7, 6988–6996.
19. G. Mikhaylov, U. Mikac, A. A. Magaeva, V. I. Itin, E. P. Naiden, I. Psakhye, L. Babes, T. Reinheckel, C. Peters, R. Zeiser, M. Bogyo, V. Turk, S. G. Psakhye, B. Turk and O. Vasiljeva, Nat. Nanotechnol., 2011, 6, 594–602.
20. S. Y. Han, Y. X. Liu, X. Nie, Q. Xu, F. Jiao, W. Li, Y. L. Zhao, Y. Wu and C. Y. Chen, Small, 2012, 8, 1596–1606.
21. S. J. Lee, K. H. Min, H. J. Lee, A. N. Koo, H. P. Rim, B. J. Jeon, S. Y. Jeong, J. S. Heo and S. C. Lee, Biomacromolecules, 2011, 12, 1224–1233.
22. Y. L. Dai, P. A. Ma, Z. Y. Cheng, X. J. Kang, X. Zhang, Z. Y. Hou, C. X. Li, D. M. Yang, X. F. Zhai and J. Lin, ACS Nano, 2012, 6, 3327–3338.
23. P. Moitra, K. Kumar, P. Kondaiah and S. Bhattacharya, Angew. Chem., Int. Ed., 2014, 53, 1113–1117.
24. G. A. Eggimann, E. Blattes, S. Buschor, R. Biswas, S. M. Kammer, T. Darbre and J. L. Reymond, Chem. Commun., 2014, 50, 7254–7257.
25. J. F. Graveland-Bikker and C. G. de Kruif, Trends Food Sci. Technol., 2006, 17, 196–203.
26. J. Otte, R. Ipsen, R. Bauer, M. J. Bjerrum and R. Waninge, Int. Dairy J., 2005, 15, 219–229.
27. Y. Fang, P. P. Pang and Z. Y. Lai, Chem. Lett., 2011, 40, 1074–1076.
28. R. Ipsen and J. Otte, Biotechnol. Adv., 2007, 25, 602–605.
29. S. Huang, M. Bai and L. Y. Wang, Sci. Rep., 2013, 3, 2023–2028.
30. M. Bai, X. L. Bai and L. Y. Wang, Anal. Chem., 2014, 86, 11196–11202.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra22831c

‡ Hecheng Zhang and Shan Peng contributed equally to this study.

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