Interactive luminescent gold nanocluster embedded dsDNA and cisplatin as model nanoparticles for cancer theranostics

Abstract

Plasmid DNA embedded with luminescent gold nanoclusters were reacted with cisplatin to form composite nanoparticles (composite NPs) for theranostic applications. The composite NPs delivered the cisplatin to cervical cancer HeLa cells in vitro, inducing apoptosis mediated cell death. The luminescent gold nanoclusters were simultaneously used for cellular imaging.

---

The advent of theranostic agents promises to enhance the potential of targeted drug delivery with real time tracking of the vehicle.¹ In this regard, biocompatible synthetic polymers have proven to be important hosts to organic and inorganic drugs and probes. The ease of synthesis and functionalization has helped improve their potential as carriers.² There are also micellar,³ liposomal² and virosomal carriers,⁴ which can efficiently host a myriad of payloads that may include DNA, drugs, siRNA, and imaging molecules and have been used for their delivery in vitro. These carriers can also be made multifunctional through incorporation of individual entities in the same vehicle.⁵ However, their equilibrium structures are subject to environmental variations and thus may release drugs on the way to the target.⁶ Also, their (micron and sub-micron scale) dimensions prevent them from becoming the perfect choice for drug delivery.⁷ The current trends in research indicate that multifunctional single component nanoscale carriers may become dominant players in this regard.⁸ The key to success may lie in having a base structure, which can be engineered with ease and be able to host moieties with varying physicochemical properties.

A prominent candidate satisfying majority of the above mentioned criteria for the fabrication of nanoscale delivery vehicle is DNA. This is all the more exciting due to the availability of programmed synthetic DNA structures, in addition to their naturally occurring counter parts. That the nanostructures could be designed “ab initio” has opened new vistas in the form of DNA origami⁹ with 2D and 3D architectures in the nanoscale regime. With deterministic structures, DNA origami has also been employed for designing biosensors, theranostic agents, and nanorobots for biological applications.¹⁰ The excellent biocompatibility,¹¹ flexibility¹² and significant stability in cell lysates¹³ make the DNA-based structures preferred choices for developing customized nanocarriers. Besides, the possibility of functionalization^12,14 at the molecular level has helped design nanostructures, which facilitate targeted delivery of drugs and perform imaging simultaneously. For example, engineered nanovehicles having DNA as the core constituent are known to deliver small molecular drugs,^9,14 antibodies¹⁰ and siRNA.¹⁵ Also, self-assembled oligonucleotide nanotubes have been used for delivery of cy3 to KB cells efficiently.¹⁶ There is a report of M 13 dsDNA being used for delivery of doxorubicin to MCF 7 cells; however, it failed to cause significant cytotoxicity, while similar approach with the engineered form of DNA (via origami) proved more efficient.⁹ The other option for construction of such structures originates from the natural forms of DNA.

Interestingly, majority of the aforementioned structures rely on Watson–Crick pairing and thus require cumbersome procedures for fabrication of the constituent structures, which is necessary for the assembly of complex structures.¹⁷ This may be one of the reasons for rather limited growth of the field. Moreover, due to the negative surface charge of the plasma membrane bare DNA molecule – either single stranded (ssDNA) – or double-stranded (dsDNA) – or its derivative structure, which is also negatively charged, compromises transport across the membrane. On the other hand, coupling with nanoparticles has been reported to have increased the efficiency of transfection of DNA.¹⁸

In order to increase the theranostic potential of nanostructures based on DNA, conjugation of the probe is of vital importance. An optical probe – especially a photoluminescence based one – incorporated in the nanostructure would confer an external handle in monitoring the entry of the vehicle in target tissues and in addition, release of the drug into the cellular environment. An important candidate in this regard could be the luminescent atomic clusters of gold. This becomes even more relevant when the recent surge in DNA based synthesis of noble metal atomic clusters is considered.¹⁹ Significant photoluminescence quantum yield, large Stokes-shifted emission, high photostability, substantial two-photon excitation cross-section and biocompatibility make Au nanocluster an important candidate for theranostic applications. In an ideal scenario, the DNA, Au clusters and the drug ought to form a composite nanostructure, which would be sufficiently stable to provide long circulation lifetime and deliver the drug at the target at least passively through enhanced permeation and retention (EPR) effect.

We herein report the generation of theranostic nanoparticles consisting of luminescent Au nanocluster embedded DNA and anticancer drug cisplatin (Scheme 1). The Au nanoclusters of average size 1.38 ± 0.54 nm were synthesized on plasmid DNA (pDNA), which was isolated from Escherichia coli. When the cluster embedded DNA was treated with cisplatin, nanoscale particles were generated. The particles were luminescent and they efficiently delivered cisplatin to HeLa cells (cervical cancer cells), leading to apoptotic cell death. The delivery of the particles to the cells could be followed from the luminescence of the clusters. The formation of nanoscale particles, due to interaction between Au nanocluster embedded DNA and cisplatin, with the retention of anticancer activity and luminescence makes it unique theranostic system.

Scheme 1 A schematic depiction of the formation of composite NPs consisting of Au nanocluster studded ds-DNA and cisplatin, thereafter its uptake by the cervical cancer HeLa cells, which eventually culminated in cell apoptosis. The photoluminescence of the clusters was used concurrently for imaging the cells.

Experimentally, the Au nanoclusters of size 1.38 ± 0.54 nm (Fig. S1‡) were synthesized in the presence of plasmid DNA extracted from Escherichia coli. Briefly, an aqueous mixture of plasmid DNA, HAuCl₄ and mercapto propionic acid (MPA) were subjected to a thermal cycle. The product so obtained was then brought to room temperature. The UV-vis spectrum of the product medium consisted of an absorption at 260 nm (Fig. S2‡), which corresponds to absorption by the DNA molecule. However, there was no peak in the visible region thus discounting the possibility of formation of surface plasmon resonance active Au nanoparticles in the reaction medium. On the other hand, the medium – when excited by 320 nm light – exhibited an emission peak at 580 nm (Fig. 1a), which indicated the formation of Au nanoclusters. Owing of their size, which is comparable to Fermi wavelength of electron, nanocluster displays a discretization of its energy levels with high luminescence quantum yield.²⁰ The formation of Au nanoclusters was further confirmed by TEM study (Fig. S3‡) and was corroborated by AFM (Fig. S4‡). Besides, DNA based fluorescent gold nanoclusters are established in the literature.²¹ The quantum yield of the synthesised nanoclusters was measured to be 5% (Fig. S5‡), with the emission of quinine sulfate being taken as the reference.

Fig. 1 Characterization of the composite NPs. (a) Luminescence emission of the DNA-gold nanocluster (DNA Au NCs), that when interacted with cisplatin, and only DNA–MPA mixture. (b) TEM image of the composite NPs, with a magnified image (inset) showing the presence of Au NCs. (c and d) AFM image of the composite NPs and its 3D view. (e) Zeta potential distribution graph of the Au nanoclusters (DNA Au NCs). (f) Zeta potential distribution graph of the composite NPs.

Interestingly, when the so-synthesized Au nanoclusters in DNA were incubated with varying concentration of cisplatin in the dark for 1 h, it resulted in the loss of luminescence of the clusters. This indicated interaction between cisplatin and the cluster embedded DNA. The luminescence intensity decreased monotonically with the concentration of cisplatin. The variation of intensity with [cisplatin]/[base pair] followed a second order polynomial relationship (Fig. S6‡). For the further experiments, a ratio of cisplatin/base pair 0.55 was chosen as it yielded product with sufficient luminescence required for imaging. Transmission electron microscopy (TEM) investigation of cisplatin treated DNA containing Au nanoclusters revealed the formation of spherical nanoparticles with an average diameter of 90 ± 9 nm (Fig. 1b); the corresponding size distribution is given in the Fig. S7.‡ The uniformity in particle size and shape indicated the robustness of the structure based on the interaction between the cluster containing DNA and cisplatin. High resolution TEM images (Fig. 1b inset) indicated the presence of smaller particles (of sizes less than 2 nm) in each of the bigger particles, which could well be the Au nanoclusters. That the composite NPs were nearly spherical was further confirmed by atomic force microscopy (AFM) (Fig. 1c, d and S8‡). AFM images also revealed the formation of particles with average size 106 + 39 nm (Fig. S9‡). The small difference in the particle sized measured by AFM and TEM could be due to compactness of the particles when under vacuum (in TEM) as opposed to ambient condition evaporated arrays of particles on two-dimensional glass slide (for AFM measurement). The presence of Au and Pt in the composite was confirmed from energy dispersive X-ray (EDX) spectrum, which was recorded along with the TEM analysis of the same sample (Fig. S10‡). The interaction of cisplatin with the DNA containing nanoclusters and formation of the composite NPs were further confirmed by the zeta potential measurements. Thus while DNA with the clusters exhibited a value of −27.8 mV (Fig. 1e), the same upon addition with cisplatin resulted in a value of −17.7 mV (Fig. 1f). Further, the hydrodynamic diameter of the composite NPs was measured to be 280 nm (Fig. S11‡). The above results imply that the cisplatin not only was bound with the purine (A and G) bases (as is known from the literature)²² but also possibly interacted with the nanoclusters present in the DNA, which might have assisted in the formation of the spherical nanoparticle. Apparently, the drug molecule here played both structural and functional roles as it assisted in the formation of nano-structures while at the same time would serve the purpose of anticancer activity. Further, the above size suggests that the cisplatin containing luminescent nanoparticles could be ideal carriers for the delivery of the drug to leaky cancer tissues through EPR effect.²³ The loading efficiency of the composite NPs was found to be 86 ± 4% when the ratio of [cisplatin]/[base pair] was 0.55. Further experiments were carried out with the same ratio of [cisplatin]/[base pair].

Fluorescence microscopy was performed to assess the imaging potential of the composite NPs. HeLa cells were incubated for 3 h with the composite NPs and they were then probed by fluorescence microscopy. The cells exhibited bright yellow fluorescence due to Au NCs (Fig. 2a, b and S12‡). The results indicated that the composite NPs were readily taken up by the cells. The uptake of the composite NPs was further established by z-stacking (Fig. 2c), where the green fluorescence indicated the up-taken composite NPs. Observations were similar with HeLa cells when treated with only DNA Au NCs (Fig. S13‡). However, the control cells lacked any fluorescence (Fig. 2d and e). The ready uptake of the composite NPs can be attributed to its compact nature and size, rendering it capable of penetrating through the cellular membrane without the use of any transfecting agent.²⁴

Fig. 2 Fluorescence images of HeLa cell treated with the composite NPs. (a) Fluorescence image of the HeLa cell showing characteristic yellow emission of the DNA Au NCs and (b) merged image (with bright field and fluorescence images merged together) of the same cell. (c) A 12 µm z-stack projection of the treated cell showing the uptake of the composite NPs; here the scale depicted shows the distance of the entities from the cover slip. (d) Fluorescence image of the control cells showing no fluorescence and (e) merged image (with bright field and fluorescence images merged together) of the control cells.

In vitro release of cisplatin from the composite NPs was evaluated at both acidic condition and at physiological pH. The composite exhibited a spurt in cisplatin release in 2 h (Fig. 3a), when added to acetate buffer (pH 4.5). About 76% of the encapsulated drug was released by that time and no significant increase could be observed thereafter. On the other hand, a release of 26% by 2 h in phosphate buffer (pH 7.4), emulating the physiological pH condition (Fig. 3a), was observed. The observations demonstrated a pH sensitive release of the drug from the composite. The pH dependence of the release profile ensured that majority of the drug was released at acidic pH only. Now if the composite NPs were to be delivered in vivo, the required pH for drug release may be attained when the composite NPs would be inside the cells (perhaps the lysosomal compartment).¹⁴ It also ensures that if not taken up the by cells, the release of the cisplatin preferably would occur at tumor microenvironment, where pH can dip down to 5.8,²³ thus avoiding killing of normal cells. As the cisplatin has already been reported to be stable at low pH²⁵ so any structural change that may lead to loss of functionality can't be anticipated. The release can be attributed to the unwinding of the plasmid DNA itself at low pH and disassembling of the whole composite. Stability of the composite NPs was checked in the PBS buffer pH (7.4) using fluorescence. Fluorescence intensity of the as synthesized composite NPs was substantially retained even after 48 h in PBS buffer, which implied that composite NPs were stable and suitable for cellular uptakes in vivo. The observed loss in fluorescence is in compliance with the release data, which also suggested that some amount of the composite NPs was destabilized and thus released the drug in the PBS.

Fig. 3 Drug release and cytotoxicity. (a) Release profile of cisplatin from the composite NPs in PBS buffer (pH 7.4) and acetate buffer (pH 4.5); (b) MTT assay results. The values are represented as mean ± SD of results from three individual experiments. Statistical test was carried out to find out the statistical significance for each concentration between cisplatin and composite NPs. The statistical significance is denoted by ★ (p < 0.05), ★★ (p < 0.005), and ★★★ (p < 0.001); (c) summary of the cell cycle study stating the statistically significant improved cytotoxicity of the composite NPs over the free drug. The statistical significance is denoted by ★ (p < 0.05), ★★ (p < 0.005), and ★★★ (p < 0.001). (d and e) FESEM images of the control and treated cells, where the treated cells showed the presence of apoptotic bodies whereas the control cells lacked it.

That the luminescent composite NPs delivered the drug cisplatin to the cancer cells was further tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (a cell viability assay). The cell viability was thus measured following treatment of the HeLa cells for 48 h, with free cisplatin, nanocluster containing DNA and the composite NPs (consisting of DNA, Au nanoclusters and cisplatin) at various concentrations. The results as shown in Fig. 3b indicated that DNA with the nanocluster was non-cytotoxic, while cisplatin and the composite NPs led to the killing of cells conspicuously. Importantly, IC₅₀ value of the only drug was found to be 6.57 µg mL⁻¹, whereas the same for the drug encapsulated in the composite NPs was measured to be 3.8 µg mL⁻¹. MTT assay was also performed on the HEK 293 cells to check the effect of the composite NPs on normal cells. Here also the composite NPs displayed an improved cytotoxicity than the drug itself. The IC₅₀ value for the composite NPs was 3.8 µg mL⁻¹ compared to 4.4 µg mL⁻¹ with only drug (Fig. S15‡). This lowering of drug IC₅₀ concentration when encapsulated in the composite NPs is important for its potential in vivo applications. Further, that the DNA containing Au nanoclusters were found to be non-cytotoxic is valuable for the use of the composite as a drug delivery vehicle.

Cisplatin is a conventional anti-cancer drug; it is known to cause DNA damage, which finally leads to apoptosis-mediated cell death.²² To probe the mechanism of cell death induced by the current composite NPs, flow cytometry based cell cycle analysis was performed. For this, cells were incubated with the composite NPs for the aforementioned period at the IC₅₀ dose and then were checked for the cell cycle analysis by using propidium iodide staining method. Results (Fig. 3c) demonstrated that all the stages of cell cycle remained almost unaltered except sub-G0 population, which corresponds to apoptotic cells. It was also found that 14.35% of the HeLa cells treated with the composite NPs had sub-G0 apoptotic population. On the other hand, the cells treated with free drug (only cisplatin) had 8.96% of sub-G0 apoptotic population which is significantly lower than that obtained using the composite NPs. Additionally, the Au nanocluster containing DNA (only) showed a feeble 1% sub-G0 population, which was comparable to the control samples (Fig. S16‡). Overall, the above results reaffirmed that the composite NPs delivered the drug (cisplatin) to the cancer cells efficiently, which eventually led to apoptotic cell death. The aforementioned observations were validated by the FESEM imaging of the treated cells. The results as shown in (Fig. 3d and e) clearly indicated lack of any anomaly on the control cells; however, the formation of the apoptotic bodies and membrane blebbing were observed in treated cells, which are the key indicators of the apoptosis. Thus the results provided sufficient evidence for the composite NPs mediated delivery of cisplatin and consequent induction of apoptosis with high efficiency at a lower dose of the drug.

It is important to mention here that further experiments suggested that enhanced caspase 3 formation, which is an essential indicator of apoptosis, was induced in the composite NPs treated cells. Cells were treated with the drug and composite NPs at its IC₅₀ value and then they were checked for the caspase 3 level using flow cytometry analysis. Cells treated with the composite NPs showed the highest quantum of the caspase 3 (Fig. 4d), which was followed by the free drug (Fig. 4c); whereas a low level of expression in the cells treated with the DNA Au NCs (Fig. 4b), quite similar to the control was observed (Fig. 4a). Recent studies indicate the generation of mitochondrial reactive oxygen species (ROS) when the cells are treated with cisplatin.²⁶ In the current context, the treated cells displayed a high level of ROS generation at IC₅₀ value with the composite NPs and the same with a lower value in case of the free drug (Fig. 4e). Enhanced level of the ROS generation with composite NPs highlights the possibility of better transport and delivery of the drug (in the form of the composite NPs) as the prime reason for the augmented cytotoxicity of the composite NPs vis-à-vis the free drug.

Fig. 4 Caspase study involving (a) control cells and those treated with (b) DNA - Au NC; (c) cisplatin; (d) composite NPs. The results showed higher quanta of caspase positive cells in case of composite NPs as compared to free cisplatin treated cells. (e) ROS profile with the prominent shift observed in case of composite NPs and cisplatin in comparison to the control cells.

Conclusions

In essence, we have developed a novel DNA – Au-nanocluster – cisplatin composite NPs that served the dual purpose of cellular imaging and drug delivery. The composite NPs showed augmented cellular cytotoxicity in comparison to free drug and provided an option for pH triggered release. Simultaneously, the composite also proved to be a possible imaging agent. With the DNA as the basic platform – in conjunction with Au nanoclusters, the composite is expected to be biocompatible. The sizes and shapes of the nanoparticles, the ease of their formation and efficient incorporation and delivery of the drug cisplatin to cancer cells provide a new platform for drug delivery using natural form of DNA as the primary vehicular component, which would have a high potential for clinical translation.

Funding sources

We thank the Department of Electronics and Information Technology, Government of India for financial support (No. 5(9)/2012-NANO (Vol. II)).

Acknowledgements

Assistance from Central Instruments Facility (CIF), IIT Guwahati for FESEM analysis is highly appreciated.

References

1. S. S. Kelkar and T. M. Reineke, Bioconjugate Chem., 2011, 22, 1879–1903.
2. N. Kamaly, Z. Xiao, P. M. Valencia, A. F. Radovic-Moreno and O. C. Farokhzad, Chem. Soc. Rev., 2012, 41, 2971–3010.
3. J. S. Guthi, S.-G. Yang, G. Huang, S. Li, C. Khemtong, C. W. Kessinger, M. Peyton, J. D. Minna, J. Brown, C. Kathlynn and J. Gao, Mol. Pharm., 2010, 7, 32–40.
4. J.-W. Yoo, D. J. Irvine, D. E. Discher and S. Mitragotri, Nat. Rev. Drug Discovery, 2011, 10, 521–535.
5. J. T. Cole and N. B. Holland, Drug Delivery Transl. Res., 2015, 5, 295–309.
6. S. Mura, J. Nicolas and P. Couvreur, Nat. Mater., 2013, 12, 991–1003.
7. B. Felice, M. P. Prabhakaran, A. P. Rodríguez and S. Ramakrishna, Mater. Sci. Eng., C, 2014, 41, 178–195.
8. J. Xie, K. Chen, J. Huang, S. Lee, J. Wang, J. Gao, X. Li and X. Chen, Biomaterials, 2010, 31, 3016–3022.
9. Q. Jiang, C. Song, J. Nangreave, X. Liu, L. Lin, D. Qiu, Z. G. Wang, G. Zou, X. Liang, H. Yan and B. Ding, J. Am. Chem. Soc., 2012, 134, 13396–13403.
10. H. Pei, X. Zuo, D. Zhu, Q. Huang and C. Fan, Acc. Chem. Res., 2014, 47, 550–559.
11. V. J. Schüller, S. Heidegger, N. Sandholzer, P. C. Nickels, N. A. Suhartha, S. Endres, C. Bourquin and T. Liedl, ACS Nano, 2011, 5, 9696–9702.
12. J. Mikkilä, E. H. Eskelinen, A.-P. Niemelä, V. Linko, M. J. Frilander, P. Törmä and M. A. Kostiainen, Nano Lett., 2014, 14, 2196–2200.
13. Q. Mei, X. Wei, F. Su, Y. Liu, C. Youngbull, R. Johnson, S. Lindsay, H. Yan and D. Meldrum, Nano Lett., 2011, 11, 1477–1482.
14. M. Chang, C. Yang and D. Huang, ACS Nano, 2011, 5, 1–10.
15. H. Lee, A. K. R. Lytton-Jean, Y. Chen, K. T. Love, A. I. Park, E. D. Karagiannis, A. Sehgal, W. Querbes, C. S. Zurenko, M. Jayaraman, C. G. Peng, K. Charisse, A. Borodovsky, M. Manoharan, J. S. Donahoe, J. Truelove, M. Nahrendorf, R. Langer and D. G. Anderson, Nat. Nanotechnol., 2012, 7, 389–393.
16. S. H. Ko, H. Liu, Y. Chen and C. Mao, Biomacromolecules, 2008, 9, 3039–3043.
17. G. Zhu, R. Hu, Z. Zhao, Z. Chen, X. Zhang and W. Tan, J. Am. Chem. Soc., 2013, 135, 16438–16445.
18. Y. Ding, Z. Jiang, K. Saha, C. S. Kim, S. T. Kim, R. F. Landis and V. M. Rotello, Mol. Ther., 2014, 22, 1075–1083.
19. Z. Yuan, Y.-C. Chen, H.-W. Li and H.-T. Chang, Chem. Commun., 2014, 50, 9800–9815.
20. J. Zheng, P. R. Nicovich and R. M. Dickson, Annu. Rev. Phys. Chem., 2008, 22, 4109.
21. L. Y. Chen, C. W. Wang, Z. Yuan and H. T. Chang, Anal. Chem., 2015, 87, 216–229.
22. Z. H. Siddik, Oncogene, 2003, 22, 7265–7279.
23. V. Torchilin, Adv. Drug Delivery Rev., 2011, 63, 131–135.
24. D. A. Giljohann, D. S. Seferos, P. C. Patel, J. E. Millstone, N. L. Rosi and C. A. Mirkin, Nano Lett., 2007, 7, 3818–3821.
25. A. Karbownik, E. Szaełk, H. Urjasz, A. Głęboka, E. Mierzwa and E. Grzeskowiak, Wspolczesna Onkol., 2012, 16, 435–439.
26. R. Marullo, E. Werner, N. Degtyareva, B. Moore, G. Altavilla, S. S. Ramalingam and P. W. Doetsch, PLoS One, 2013, 8, 1–15.

---

Footnotes

† An Indian patent application has been filed and international patent filing is under consideration. This is related to the method of synthesis of gold nanoclusters on dsDNA using a single PCR based heating and cooling cycle.

‡ Electronic supplementary information (ESI) available: Detailed experimental procedure, size distribution, UV-vis absorption spectrum, quantum yield calculation, EDX, zeta potential, hydrodynamic diameter, Epi-fluorescence microscopy image and cell cycle analysis. See DOI: 10.1039/c6ra24325h

---