ChemComm
Communication
59%, which is much higher than the cells incubated with PAP
(about 18%, as shown in Fig. S9, ESI†). To confirm the apoptosis
pathway of PAP-AuNPs, apoptosis associated proteins have been also
detected by western blot analysis. As shown in Fig. S8B (ESI†),
significant differences in the release of cytochrome c and expression
of caspase-3 were observed for the cells incubated with PAP or PAP-
AuNPs. PAP-AuNPs can promote the release of cytochrome c and
expression of caspase-3, thus resulting in cell apoptosis. Obviously,
the apoptotic pathway is typically mitochondria-mediated.
In summary, pro-apoptotic peptide functionalized AuNPs
were fabricated and PAP-AuNPs can amplify the cell apoptosis.
PAP-AuNPs can induce the mitochondrial membrane swelling
and burst of membrane with mitochondrial matrix leaking
out. By specifically damaging the organelle of mitochondria,
PAP-AuNPs can cause the dysfunction of mitochondria, and
trigger mitochondria-dependent programmed cancer cell death,
thus leading to complete cancer cell apoptosis. Interestingly, this
work provides new insights into anti-cancer strategy and it may
lead to advanced treatment to cancers.
Fig. 3 CLSM images of HeLa cells incubated with PAP-AuNPs. Nuclei were
stained by Hoechst 33258 and mitochondria were stained by JC-1 fluorescence
probe. The scale bar was 20 mm.
This work was supported by National Natural Science Founda-
completely disappeared (Fig. 3d2). Meanwhile, the green J-monomer tion of China (51003079, 51125014, 51233003) and National Key
fluorescence also faded (Fig. 3d1) and the nuclei become condensed Basic Research Program of China (2011CB606202).
(Fig. 3d4). In addition, as shown in DIC bright field, the cells collapse
and become ‘‘thinner’’ (Fig. 3c and d compared with a and b
respectively) since most of the mitochondria have been destroyed.
With the damage to the mitochondria, the membrane permeability
of the cells is enhanced, leading to the leakage of cytoplasm. In fact,
the permeable state of cell membrane can be observed directly.
As shown in Fig. S6 (ESI†), cell edges are ruptured and cytoplasm
has penetrated out.
The flow cytometry was used to quantitatively mark the cell
population with the low DCm and proved that PAP-AuNPs have
a stronger ability to induce cell apoptosis. As shown in Fig. S7
(ESI†), P2 represents the cell population with the low DCm,
indicating those cells are in the apoptotic state and represent-
ing the cell apoptosis rate. After 72 h of incubation, approxi-
mately 36.7% cells had low DCm for those incubated with PAP,
but there were 54.1% cells with low DCm for those incubated
with PAP-AuNPs, which is much higher. Flow cytometry results
described above quantitatively confirmed that the apoptosis of
the cells incubated with PAP-AuNPs is significantly enhanced.
PAP-AuNPs can specifically damage mitochondria and induce a
collapse of mitochondrial membrane potential.
Notes and references
1 V. P. Torchilin, Annu. Rev. Biomed. Eng., 2006, 8, 343.
2 C. S. Lim, Adv. Drug Delivery Rev., 2007, 59, 697.
¨
3 (a) L. Rajendran, H. J. KnOlker and K. Simons, Nat. Rev. Drug Discovery,
2010, 9, 29; (b) B. Kang, M. A. Mackey and M. A. EI-Sayed, J. Am. Chem.
Soc., 2010, 132, 1517; (c) S. V. Boddapati, G. G. M. D’Souza, S. Erdogan,
V. P. Torchilin and V. Weissig, Nano Lett., 2008, 8, 2559.
4 (a) A. Szewczyk and L. Wojtczak, Pharmacol. Rev., 2002, 54, 101;
(b) H. M. Ellerby, W. Arap, L. M. Ellerby, R. Kain, R. Andrusiak,
G. D. Rio, S. Krajewski, C. R. Lombardo, R. Rao, E. Ruoslahti,
D. E. Bredesen and R. Pasqualini, Nat. Med., 1999, 5, 1032.
5 (a) D. R. Green and J. C. Reed, Science, 1998, 281, 1309; (b) T. Xia,
C. S. Jiang, L. J. Li, C. H. Wu, Q. Chen and S. S. Liu, FEBS Lett., 2002,
510, 62; (c) G. Kroemer, L. Galluzzi and C. Brenner, Physiol. Rev.,
2007, 87, 99.
6 (a) E. Boisselier and D. Astruc, Chem. Rev., 2009, 109, 1759;
(b) R. A. Sperling, P. R. Gil, F. Zhang, M. Zenella and W. J. Parak,
Chem. Rev., 2008, 108, 1896; (c) G. F. Paciotti, D. G. I. Kingston and
L. Tamarkin, Drug Dev. Res., 2006, 67, 47.
7 (a) B. D. Chithrani, A. A. Ghazani and W. C. W. Chan, Nano Lett.,
2006, 6, 662; (b) H. K. Patra, S. Banerjee, U. Chaudhuri, P. Lahiri and
A. K. Dasgupta, Nanomed.: Nanotechnol., Biol. Med., 2007, 3, 111;
(c) E. C. Cho, Q. Zhang and Y. N. Xia, Nat. Nanotechnol., 2011, 6, 385.
8 (a) J. Turkevich, P. C. Stevenson and J. Hillier, Discuss. Faraday Soc.,
1951, 11, 55; (b) G. Frens, Nat. Phys. Sci., 1973, 241, 20.
9 K. Eugenii and I. Willner, Angew. Chem., Int. Ed., 2004, 43, 6042.
10 M. M. Javadpour, M. M. Juban, W.-C. J. Lo, S. M. Bishop,
J. B. Alberty, S. M. Cowell, C. L. Becker and M. L. Mclaughlin,
J. Med. Chem., 1996, 39, 3107.
To further study the anti-cancer mechanism of PAP-AuNPs,
we conducted an apoptosis assay of HeLa cells. As shown in
Fig. S8A (ESI†), the cell apoptosis presents in a time-dependent
manner. Particularly for cells incubated with PAP-AuNPs for 72 h,
the percentage of apoptotic and necrotic cells is approximately
11 P. Nativo, I. A. Prior and M. Brust, ACS Nano, 2008, 2, 1639.
12 S. W. Perry, J. P. Norman, J. Barbieri, E. B. Brown and H. A. Gelbard,
BioTechniques, 2011, 50, 98.
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 6403--6405 6405