guest assembly of gold nanoparticles triggered by ... | ResearchGate

ChemComm View Article Online

Published on 30 October 2015. Downloaded by Zhejiang University on 26/12/2015 08:29:47.

COMMUNICATION

Cite this: Chem. Commun., 2016, 52, 582

View Journal | View Issue

Intracellular host–guest assembly of gold nanoparticles triggered by glutathione† Yin Wang,‡ Huan Li,‡ Qiao Jin* and Jian Ji*

Received 31st August 2015, Accepted 30th October 2015 DOI: 10.1039/c5cc07195j www.rsc.org/chemcomm

A simple method to achieve host–guest assembly of gold nanoparticles triggered by intracellular glutathione was demonstrated. The increased size of nanoparticles not only enhanced their retention time within cancer cells, but also induced apoptosis. This strategy may open an avenue for the development of smart nanocarriers for intracellular diagnosis and therapy.

Self-assembly is a ubiquitous process occurring at different scales which not only plays an important role in nature,1 but is also a robust strategy for constructing biomaterials.2 In most previous studies, nanoplatforms are first self-assembled from precursors and then deliver therapeutic agents to target lesions, in which payloads can be released by specific stimuli.3 Recently, there has been increased recognition of the importance of intracellular signaling molecules, which can result in precise subcellular targeting and enhanced therapeutic outcomes.4 For instance, Xu5 and other researchers4e,f,6 exploited intracellular enzymes as specific triggers to construct nanostructures after synthetic organic molecules were internalized into cells. They found that the assembled nanostructures could be used to regulate the fate of cells. Therefore, the assembly in situ triggered by intracellular signals would be of great interest for precision diagnosis and therapy. In recent years, gold nanoparticles (GNPs) have been widely investigated in nanomedicine due to their superior optical, chemical, and biological properties.7 More importantly, the simple regulation of GNPs from dispersive to aggregative can shift them from biocompatible to cytotoxic under NIR irradiation.7,8 Although this photothermal property is well known, to the best of our knowledge, few studies have shed light on intracellular signalinduced formation of GNP aggregates and their influence on the fate of cells.

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, P. R. China. E-mail: [email protected], [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5cc07195j ‡ These authors contributed equally.

582 | Chem. Commun., 2016, 52, 582--585

Herein, we report that GNP aggregates were obtained from dispersive GNPs by host–guest interactions between Fc and b-CD, and triggered by intracellular glutathione (GSH) (Scheme 1). The formed aggregates were expected to be retained in cancer cells for a relatively long time and induce apoptosis of cells due to their increased size. Moreover, the intracellular assembly would ensure that the GNP aggregates inhibited the proliferation of cancer cells due to enhanced light absorption in the near infrared (NIR) region. Small sized GNPs were used as model nanoparticles in this study due to their biocompatible properties, ease of functionalization and good penetration capability.7a Citrate-capped GNPs were prepared following the method outlined in previous studies7b,c and simultaneously modified by mono-6-thio-b-cyclodextrin (b-CD-SH) and mercapto capped poly(ethylene glycol) (PEG-SH) in the mole ratio of 1 : 1. The as-prepared gold nanoparticles after purification were still known as GNPs for convenience. In order to form inclusions with b-CD on GNPs, ferrocene (Fc) capped PEG was synthesized (abbreviated as Fc-PEG-Fc). It is known that Fc forms an inclusion complex with b-CD, and detaches when it is in its ionic form (Fc+).9 Interestingly, the ferrocenium cation could be reduced to the parental Fc with the aid of GSH.10 Thus, the reversible change between Fc and Fc+ could be used as a driving force to regulate the assembly or disassembly of the inclusion complex. Moreover, GSH is overexpressed in most types of cancer cells.11 Consequently, we hypothesized that high concentrations of GSH could be used as a trigger to achieve the assembly of Fc and b-CD in cancer cells. To verify that gold nanoclusters could be assembled from dispersive GNPs in the presence of a reducing agent, Fc-PEG-Fc was first subjected to oxidization by anhydrous ferric chloride (FeCl3),10a offering Fc+-PEG-Fc+ (detailed experimental procedures can be found in the ESI†). Then 10 mM of dithiothreitol (DTT) was added to the freshly prepared GNP solution. It can be seen from Fig. 1A that the precipitates were formed after 3 h of incubation with DTT in the presence of Fc+-PEG-Fc+ (c). However, GNPs were dispersive only when Fc+-PEG-Fc+ or DTT was added to the solution (b and d, the color was still claret-red). That is, the co-existence of Fc+-PEG-Fc+ and DTT was essential for the

This journal is © The Royal Society of Chemistry 2016

View Article Online


Communication

Scheme 1

ChemComm

Illustration of intracellular host–guest assembly of gold nanoparticles triggered by GSH.

Fig. 1 Digital photos (A, upper represents samples at the beginning of the experiment, bottom represents samples after 3 h) and DLS plots (B) of samples. In Fig. 1A, from left to right are GNPs (a), GNPs/Fc+-PEG-Fc+ (b), GNPs/Fc+-PEG-Fc+/DTT (c) and GNPs/DTT (d). The concentration of GNPs was the same and the DTT solution was 10 mM. Representative TEM images of GNPs/Fc+-PEG-Fc+/DTT (C and D). D was the magnification of C in the red rectangle. Both scale bars were 200 nm.

formation of aggregates. The aggregation can be attributed to the host–guest assembly between b-CD and Fc which was reduced from Fc+ in the presence of DTT. Dynamic light scattering (DLS) was also used to characterize the size changes in the samples. In Fig. 1B, the size of GNPs incubated with Fc+-PEG-Fc+ and


DTT shifted to around 2000 nm while maintaining their size at about 16 nm in other cases. Although the size of GNPs increased to 25 nm in the presence of Fc+-PEG-Fc+, the size remained the same for one month (data not shown). This also supported the conclusion that both Fc+-PEG-Fc+ and DTT were

Chem. Commun., 2016, 52, 582--585 | 583

View Article Online


ChemComm

Communication

Fig. 3 A histogram of the residual percentage of gold in cells after incubation with fresh media for 2, 4 and 6 h. The concentration of GNPs in both samples was the same as at the beginning of the experiment. *p o 0.05.

Fig. 2 Representative TEM images of HepG2 cells after incubation with GNPs (A and B) or GNPs/Fc+-PEG-Fc+ (C and D) for 12 h. GNPs in cells are highlighted in red ellipses. The scale bars in A and C were 2 mm and in B and D were 0.5 mm.

essential for nanocluster formation. Transmission electron microscopy (TEM) was carried out to confirm the formation of aggregates. Densely populated aggregates can be clearly seen in Fig. 1C and D. Consequently, it can be concluded from the aforementioned discussions that gold nanoparticle clusters can easily be obtained by host–guest interactions triggered by DTT. Cell experiments were carried out to assess the intracellular assembly within cancer cells. After incubating human hepatocellular carcinoma cells (HepG2 cells) with GNPs or GNPs/Fc+PEG-Fc+ for 12 h, cell slices were prepared and observed by TEM. As shown in Fig. 2, GNPs internalized into cancer cells by endocytosis exhibited different forms. In particular, evenly distributed GNPs can be seen in Fig. 2A and B where the incubation media contained GNPs only, while most GNPs formed aggregates (Fig. 2C and D) when both GNPs and Fc+-PEG-Fc+ were added to the culture media. It is known that aggregation can endow gold nanospheres with NIR photothermal properties;7a thus the viability of cancer cells under NIR irradiation could be used as a clue for intracellular assembly. To verify the role of GSH in intracellular assembly, glutathione monoester (GSH-OEt) and buthionine sulfoximine (BSO) was added to the culture media, respectively, and untreated cells were used as controls. GSH-OEt and BSO are a promoter and inhibitor of GSH in cells, respectively.12 As shown in Fig. S10 (ESI†), HepG2 cells pretreated with GSH-OEt and BSO exhibited the highest (54%) and lowest inhibition efficiency (42%) under NIR irradiation, respectively, compared with the control cells. It should also be noted that BSO and GSH-OEt at the tested concentrations did not show cytotoxicity in HepG2 cells. That is, GSH was the trigger for intracellular host–guest assembly. The effect of GSH triggered aggregation on the retention of nanoparticles within cells was further evaluated. Specifically, HepG2

584 | Chem. Commun., 2016, 52, 582--585

cells were incubated with GNPs or a mixture of GNPs and Fc+-PEG-Fc+ for 5 h before replacement with fresh media. We normalized the percentage of gold to 100% for comparison as the concentration of GNPs in cancer cells was almost the same after incubation (Fig. S11, ESI†). Clearly, the percentage of gold in cells decreased as time increased for both samples in Fig. 3. However, the rate of the decrease in cells pretreated with GNPs/Fc+-PEG-Fc+ was lower than that in cells pretreated with GNPs only. In particular, in the first 2 h after medium replacement, about 85% of gold remained in the cells treated with GNPs/Fc+-PEG-Fc+, while the amount of gold quickly decreased to around 60% in the cells incubated with GNPs only and to 37% after 6 h, which was less than its counterpart. The reason for the different retention behavior can be attributed to the formation of aggregates from dispersive GNPs. Triggered by intracellular GSH, Fc+ groups changed to Fc and formed inclusions with b-CD on GNPs, hence bridging the GNPs to form clusters and increasing the overall size, which blocked exocytosis of cells. To examine extrinsic apoptosis with the concurrent formation of aggregates, the annexin V conjugate staining assay was performed. The sum of the two right quadrants represents apoptosis, thus it can be concluded from Fig. 4 that both GNPs (7.59%) and Fc+-PEG-Fc+ (11.45%) induced some apoptosis in HepG2 cells. However, these values were comparable to those of untreated cells (6.52%, Fig. S12, ESI†). In contrast, when the cell culture media contained both GNPs and Fc+-PEG-Fc+, the apoptosis of cancer cells was significantly enhanced, and reached 36.4%. These results indicated that large aggregates induced apoptosis. As discussed above, GNPs or Fc+-PEG-Fc+ alone were compatible with cancer cells, while it became cytotoxic after assembly. Therefore, assembly-induced aggregation was the main cause of apoptosis. Furthermore, it should be noted that the shift from biocompatible to cytotoxic only took place inside cancer cells, which could be promising in reducing the unexpected side effects induced by traditional chemotherapy. In summary, a simple method to achieve intracellular host– guest assembly of nanoparticles triggered by GSH is demonstrated.


View Article Online


Communication

ChemComm

Fig. 4 Apoptosis of HepG2 cells. Cells stained with annexin V-FITC and propidium iodide (PI) were characterized by flow cytometry. Untreated HepG2 cells were used as a control. Cells that were negative for both annexin V-FITC and PI staining were classified as viable, whereas cells that were positive for annexin V-FITC and negative for PI were classified as apoptotic. Cells that were positive for PI were classified as necrotic.

The results show that a size increase in the aggregated GNPs could be achieved in cancer cells, which can prolong the retention of nanoparticles and induce apoptosis of cancer cells. Moreover, the assembly of GNPs increases their potency for photothermal therapy under NIR irradiation. The strategy of intracellular signal triggered transformation from biocompatible to toxic may open an avenue for the development of smart nanocarriers for intracellular diagnosis and therapy. Financial support from the National Natural Science Foundation of China (21174126, 51303154, 21574114), the Key Science Technology Innovation Team of Zhejiang Province (No. 2013TD02), the Research Fund for the Doctoral Program of Higher Education of China (20130101120177), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry ([2015]311) are gratefully acknowledged. We greatly thank Tian Jiang for the helpful discussion of manuscript preparation, and Wei Yu for the apoptosis experiments.

Notes and references 1 G. M. Whitesides and B. Grzybowski, Science, 2002, 295, 2418. 2 (a) S. Kiyonaka, K. Sada, I. Yoshimura, S. Shinkai, N. Kato and I. Hamachi, Nat. Mater., 2004, 3, 58; (b) G. A. Silva, C. Czeisler, K. L. Niece, E. Beniash, D. A. Harrington, J. A. Kessler and S. I. Stupp, Science, 2004, 303, 1352; (c) H. P. Xu, W. Cao and X. Zhang, Acc. Chem. Res., 2013, 46, 1647. 3 (a) A. G. Cheetham, P. C. Zhang, Y. A. Lin, L. L. Lock and H. G. Cui, J. Am. Chem. Soc., 2013, 135, 2907; (b) Y. Wang, H. Wang, Y. Chen, X. Liu, Q. Jin and J. Ji, Chem. Commun., 2013, 49, 7123; (c) Y. Wang, D. Li, H. Wang, Y. Chen, H. Han, Q. Jin and J. Ji, Chem. Commun., 2014, 50, 9390; (d) Y. Wang, J. W. Du, Y. X. Wang, Q. Jin and J. Ji, Chem. Commun., 2015, 51, 2999; (e) Z. K. Zhang, R. J. Ma and L. Q. Shi, Acc. Chem. Res., 2014, 47, 1426; ( f ) D. Y. W. Ng, Y. Z. Wu, S. L. Kuan and T. Weil, Acc. Chem. Res., 2014, 47, 3471; ( g) R. Lin and H. Cui, Curr. Opin. Chem. Eng., 2015, 7, 75; (h) S. Yu,


4

5

6 7

8 9 10

11

12

Y. Zhang, X. Wang, X. Zhen, Z. Zhang, W. Wu and X. Jiang, Angew. Chem., 2013, 52, 7272. (a) J. G. Huang, T. Leshuk and F. X. Gu, Nano Today, 2011, 6, 478; (b) B. Yameen, W. I. Choi, C. Vilos, A. Swami, J. Shi and O. C. Farokhzad, J. Controlled Release, 2014, 190, 485; (c) M. Kanapathipillai, A. Brock and D. E. Ingber, Adv. Drug Delivery Rev., 2014, 79–80, 107; (d) Y. Yuan, S. Ge, H. Sun, X. Dong, H. Zhao, L. An, J. Zhang, J. Wang, B. Hu and G. Liang, ACS Nano, 2015, 9, 5117; (e) C. H. Ren, J. W. Zhang, M. S. Chen and Z. M. Yang, Chem. Soc. Rev., 2014, 43, 7257; ( f ) C. H. Ren, H. M. Wang, D. Mao, X. L. Zhang, Q. Q. Fengzhao, Y. Shi, D. Ding, D. L. Kong, L. Wang and Z. M. Yang, Angew. Chem., Int. Ed., 2015, 54, 4823; ( g) D. J. Ye, A. J. Shuhendler, L. N. Cui, L. Tong, S. S. Tee, G. Tikhomirov, D. W. Felsher and J. H. Rao, Nat. Chem., 2014, 6, 519; (h) D. Neri and C. T. Supuran, Nat. Rev. Drug Discovery, 2011, 10, 767; (i) X. X. Sun, Y. N. Zhao, V. S. Y. Lin, I. I. Slowing and B. G. Trewyn, J. Am. Chem. Soc., 2011, 133, 18554; ( j) J. Li, J. Chao, J. Y. Shi and C. H. Fan, ChemBioChem, 2015, 16, 39. (a) J. Zhou and B. Xu, Bioconjugate Chem., 2015, 26, 987; (b) Y. Gao, Y. Kuang, Z. F. Guo, Z. H. Guo, I. J. Krauss and B. Xu, J. Am. Chem. Soc., 2009, 131, 13576; (c) Z. Yang, G. Liang, Z. Guo, Z. Guo and B. Xu, Angew. Chem., 2007, 46, 8216. A. Tanaka, Y. Fukuoka, Y. Morimoto, T. Honjo, D. Koda, M. Goto and T. Maruyama, J. Am. Chem. Soc., 2015, 137, 770. (a) Z. Qin and J. C. Bischof, Chem. Soc. Rev., 2012, 41, 1191; (b) X. Liu, Y. Chen, H. Li, N. Huang, Q. Jin, K. Ren and J. Ji, ACS Nano, 2013, 7, 6244; (c) H. Li, X. Liu, N. Huang, K. Ren, Q. Jin and J. Ji, ACS Appl. Mater. Interfaces, 2014, 6, 18930. J. Nam, N. Won, H. Jin, H. Chung and S. Kim, J. Am. Chem. Soc., 2009, 131, 13639. L. Peng, A. Feng, M. Huo and J. Yuan, Chem. Commun., 2014, 50, 13005. (a) Y. Chang, K. Yang, P. Wei, S. Huang, Y. Pei, W. Zhao and Z. Pei, Angew. Chem., Int. Ed., 2014, 53, 13126; (b) M. Nakahata, Y. Takashima, H. Yamaguchi and A. Harada, Nat. Commun., 2011, 2, 511. (a) P. Kuppusamy, M. Afeworki, R. A. Shankar, D. Coffin, M. C. Krishna, S. M. Hahn, J. B. Mitchell and J. L. Zweier, Cancer Res., 1998, 58, 1562; (b) P. Kuppusamy, H. Q. Li, G. Ilangovan, A. J. Cardounel, J. L. Zweier, K. Yamada, M. C. Krishna and J. B. Mitchell, Cancer Res., 2002, 62, 307. A. P. Zhang, Z. Zhang, F. H. Shi, J. X. Ding, C. S. Xiao, X. L. Zhuang, C. L. He, L. Chen and X. S. Chen, Soft Matter, 2013, 9, 2224.

Chem. Commun., 2016, 52, 582--585 | 585