Blue-emitting and amphibious metal (Cu, Ni, Pt, Pd) nanodots prepared within supramolecular polymeric micelles for cellular imaging applications

Abstract

We propose a new method for the preparation of blue-emitting and amphibious metal (Cu, Ni, Pt, Pd) nanodots using supramolecular polymeric micelle nanoreactors. The supramolecular polymeric micelles were constructed by electrostatic interactions between hyperbranched poly(ethylenimine)s (HPEI) and palmitic acid (PA). After encapsulation of the metal ions and subsequent reduction by NaBH₄, blue-emitting metal nanodots in chloroform phase were formed. The resulting metal nanodots could be phase transferred from chloroform phase to aqueous phase by adding triethylamine, thus aqueous metal nanodots in the form of metal nanodot/HPEI could be obtained. The aqueous metal nanodot/HPEI exhibited preeminent fluorescence properties for bio-imaging. The fluorescent metal nanodot/HPEI integrates the fluorescence property of metal dots with the gene transfection character of HPEI, indicating an ideal fluorescence probe for tracking gene transfection behaviour.

---

1. Introduction

Metal nanodots comprise several or tens of metal atoms. In this size regime, the particles display interesting molecule-like properties such as strong size-dependent fluorescence from the ultraviolet to near-infrared region due to the spatial confinement of free electrons in the particles.^1–5 Different from semiconductor quantum dots and organic dyes, there is no concern of toxicity and photostability for metal nanodots.⁴ Fluorescent metal nanodots are suitable alternatives for bioimaging applications. To date, biomolecules (such as DNA,^6,7 proteins,⁸ amino acids,^9,10 peptides^11,12 and lysozymes¹³), dendritic polymers, ^14–16 linear polymers^17–19 and thiols^20–22 have been the common template or nanoreactor to generate metal nanodots such as Au, Ag and Cu nanodots. Among these types of nanodots, Au and Ag nanodots are easier to prepare compared with Cu nanodots owing to their redox properties. Furthermore, the ease of oxidation of Cu (E₀, 0.34 V) compared to that of Ag (E₀, 0.80 V) and Au (E₀, 1.50 V) has hindered developments in the synthesis of Cu nanodots, especially in an aqueous phase.¹³

Herein, we propose a new method for preparing fluorescent and amphibious metal (Cu, Ni, Pt, Pd) nanodots within supramolecular polymeric micelles. The supramolecular polymeric micelles were constructed by electrostatic interactions between hyperbranched poly(ethylenimine)s (HPEI) and palmitic acid (PA) in chloroform. By encapsulation of the aqueous metal ions (Cu²⁺, Ni²⁺, Pt⁴⁺, Pd²⁺) and further reduction by NaBH₄, blue-emitting metal (Cu, Ni, Pt, Pd) nanodots were prepared. Benefiting from the reversibility behaviour of supramolecular polymeric micelles, Cu, Ni, Pt and Pd nanodots can be phase transferred to an aqueous phase, thus water-soluble metal nanodots were obtained. The blue-emitting Cu nanodots were then used for bio-imaging in COS-7 cells and show good internalization by COS-7 cells.

2. Experimental

2.1 Materials

Hyperbranched poly(ethylenimine) [HPEI, the degree of branching (DB) = 60%, number average molecular weight (Mn) = 10 000 g mol⁻¹, polydispersity (PDI) = 2.5] and palmitic acid were purchased from Sigma-Aldrich. CuSO₄ (99.00%), H₂PtCl₆·6H₂O (Pt 37.5%), PdCl₂ (Pd 59–60%), NiSO₄·6H₂O (99.9%), NaBH₄ (98%) and CHCl₃ were from Sinopharm Chemical Reagent Company. Ultrapure water with 18.2 MΩ cm resistivity was used in all experiments.

2.2 Synthesis

Construction of supramolecular polymeric micelles based on electrostatic interactions and ion pairs. The 120 mg of amine-terminated HPEI was dissolved in 40 mL of chloroform and then 240 mg of palmitic acid (PA) was added. The molar ratio of PA to primary amines of HPEI was 1 : 1. After stirring overnight, the supramolecular micelles were obtained and were abbreviated as HPEI/PA.²³

Synthesis of luminescent Cu nanodots within supramolecular polymeric micelles. The 1 mL of CuSO₄ aqueous solution (30 mM) was added to the chloroform solution of HPEI/PA micelles. After vigorous stirring for 24 h at 5 °C, the color of the chloroform solution changed to blue due to the encapsulation of aqueous CuSO₄ by HPEI/PA micelles. The upper aqueous layer was removed, followed by slow addition of 1 equivalent of NaBH₄ (dissolved in 1 mL of ultrapure water, which was adjusted to pH 7.1) to the chloroform solution over 20 min. The mixture was stirred for another 24 h at 5 °C. Then, the upper aqueous layer was removed and the chloroform solution was vigorously stirred for more than 6 days at room temperature. Thus, an optically clear chloroform solution of Cu nanodots with a slight yellow color was obtained and was abbreviated as Cu–HPEI/PA. Blue fluorescence could be observed under UV light (365 nm).

Synthesis of luminescent Ni, Pd, and Pt nanodots within supramolecular polymeric micelles. A similar procedure was carried out for the synthesis of luminescent Ni, Pd, and Pt nanodots. 1 mL of NiSO₄·7H₂O (30 mM), PdCl₂ (30 mM) or H₂PtCl₆ (30 mM) were used as the raw materials for preparing luminescent Ni, Pd, or Pt nanodots, respectively. The PdCl₂ was dissolved in aqueous HCl solution (pH 2) and the solution was adjusted to pH 5.8–6 to prevent cleavage of HPEI/PA supramolecular micelles. The H₂PtCl₆ aqueous solution was also adjusted to pH 5.8–6 to prevent the possible breakup of HPEI/PA supramolecular micelles by acid.

Preparation of aqueous Cu nanodots by phase transfer from chloroform phase to aqueous phase. HPEI/PA is a type of supramolecular polymeric micelle based by electronic interactions. By adding enough acid or base, HPEI/PA supramolecular polymeric micelles dissociate and water-soluble HPEI transfers to the water phase. Herein, 10 fold (corresponding to the quantity of PA) of triethylamine was added to the Cu–HPEI/PA chloroform solution. After stirring overnight, isovolumetric ultrapure water was added and Cu/HPEI nanocomposites could be quickly transferred to the water phase, as proved by the blue fluorescence in the water phase.

2.3 Bio-imaging of aqueous Cu nanodots

The initial culture medium for COS-7 cells was replaced by the PBS solution of Cu/HPEI nanocomposites. The cell imaging was then characterized by fluorescence microscopy.

2.4 Measurements

Transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM) and selected area electronic diffractions (SAED) were performed on a JEOL 2010 microscope at an accelerating voltage of 200 kV. UV-vis spectra were obtained on a Varian Cary-50 UV-vis spectrometer. Photoluminescence spectra were obtained using a Varian Cary Eclipse spectrometer. Fourier transform infrared (FT-IR) spectra were obtained using a Varian 800 FT-IR spectrometer. Dynamic light scattering (DLS) measurements were carried out at 25 °C using a Zetasizer Nano-ZS.

3. Results and discussion

Chloroform-soluble, inverted micelle-like structures can be formed based on the formation of ion pairs between fatty acids (such as PA) and the terminal amine groups of dendritic polymers (such as HPEI). By this simple, noncovalent method, the HPEI exteriors were converted from hydrophilic (–NH₂) to hydrophobic and HPEI/PA supramolecular polymeric micelles were constructed, as proved by FT-IR and dynamic light scattering (DLS) tests in Fig. S1 and S2 in ESI.† These inverted micelle-like structures are especially well-suited for extracting hydrophilic guests such as Cu²⁺ ions from an aqueous phase to organic phase. After phase transfer, a blue chloroform solution was obtained due to the color of aqueous Cu²⁺. The Cu²⁺ encapsulated by HPEI/PA can be further reduced by 1 fold of NaBH₄ in aqueous solution (pH 7.1) or the amines of HPEI/PA. After stirring for more than 6 days, an organic solution with a slight yellow color under light and blue fluorescence under UV light was formed, as illustrated in Scheme 1a and b. Because the HPEI/PA inverted micelles were constructed by electrostatic interactions, acid (with pH lower than 1) or base (with pH higher than 10) could split the self-assemblies. They were pH responsive and endowed the copper nanodots obtained with pH response. Excess triethylamine was added to cleave the HPEI/PA self-assemblies and the Cu nanodots could then be transferred to the aqueous phase, as illustrated in Scheme 1b and c.

Scheme 1 Illustration for the preparation of metal nanodots based on the supramolecular polymeric micelles (a and b) and the phase transfer of metal nanodots from chloroform phase to water phase (b and c).

The emission peak of the as-synthesized fluorescent Cu, Ni, Pt, and Pd nanodots in chloroform was located at 454, 418/429, 441, and 435 nm, respectively (Fig. 1). The quantum yield (QY) was 5.1%, 6.0%, 2.0%, and 5.8% for fluorescent Cu, Ni, Pt and Pd nanodots, respectively (calibrated with coumarin 1). TEM images (Fig. 2) showed that the fluorescent metal (Cu, Ni, Pt, and Pd) nanodots in chloroform were all below 2 nm in size. The extremely small cavities of HPEI/PA limit the size increase of metal nanodots, resulting in metal nanodots of diameter smaller than 2 nm. Worth mentioning is that this simple synthetic protocol also could apply to other metallic (Ag, Au) and bimetallic (e.g., AuAg and AuPt) fluorescent nanodots.

Fig. 1 Photoemission spectra of the fluorescent Cu (a), Ni (b), Pt (c), and Pd (d) nanodots prepared within HPEI/PA supramolecular polymeric micelles in chloroform. The insets show the digital images of the metal nanodots under UV light (365 nm).

Fig. 2 TEM images of Cu (a), Ni (b), Pt (c) and Pd (d) nanodots.

An advantage of the present method is that the fluorescent metal nanodots in the organic phase can be easily shuttled back to the aqueous phase. For example, upon addition of triethylamine, a small organic molecule with amine groups, to the blue fluorescent metal nanodots in chloroform, triethylamine and PA formed a new electrostatic complex instead of the HPEI/PA electrostatic complex. After adding isopyknic ultrapure water, the metal nanodot/HPEI was transferred to aqueous phase (see Fig. S3 in ESI† for the FT-IR analysis of HPEI/PA, fluorescent Cu nanodots in chloroform and transferred to aqueous phase). The aqueous Cu, Ni, Pt and Pd nanodots were called Cu–HPEI, Ni–HPEI, Pt–HPEI and Pd–HPEI nanocomposites, respectively. After dialysis and rotary evaporation, metal nanodot/HPEI could re-disperse in chloroform solution containing PA. The reversible phase transfer of fluorescent metal nanodots between chloroform and aqueous solutions broadened their usability in both organic and aqueous environments.

Using Cu nanodots as an example, we demonstrated the phase transfer process from organic phase to an aqueous phase. Fig. 3A shows the absorption peak of Cu nanodots prepared within HPEI/PA located at 364 nm. After phase transfer to aqueous phase, a slight blue shift occurs and the absorption peak is at 350 nm, which can be observed in Fig. 3B. Fig. 4 presents the corresponding PL spectra of Cu nanodots. The PL peaks of Cu nanodots prepared within HPEI/PA self-assemblies and Cu nanodots transferred to an aqueous phase located at 454 and 444 nm, respectively. The inset shows the images of blue fluorescent Cu nanodots transferred to aqueous phase under UV light (365 nm).

Fig. 3 UV-vis spectra of Cu nanodots prepared within HPEI/PA supramolecular polymeric micelles (A) and Cu nanodots transferred to aqueous phase (B).

Fig. 4 PL spectra of Cu nanodots prepared within HPEI/PA supramolecular polymeric micelles (A) and Cu nanodots transferred to aqueous phase (B). The inset shows the image of blue fluorescent Cu nanodots transferred from chloroform phase to aqueous phase under UV light (365 nm).

Fig. 5 shows the TEM images of Cu nanodots prepared within HPEI/PA self-assemblies and Cu nanodots transferred to an aqueous phase. The fluorescent Cu nanodots are extremely smaller than 2 nm in size for both samples. The annular diffraction in the selected area electron diffraction (SAED) pattern for organic Cu nanodots implied the polycrystalline structure of the Cu nanodots, as shown in Fig. S4.†

Fig. 5 TEM images of Cu nanodots before (a) and after (b) phase transfer.

The cytotoxicity of nanodots is one of the key factors determining whether they can be applied in bioapplications. Herein, we evaluated the cytotoxicity of Cu–HPEI, Ni–HPEI, Pt–HPEI and Pd–HPEI nanocomposites in comparison with HPEI by MTT assay in the COS-7 cell line after 24 h incubation. Fig. 6 displays the cell viability after incubation with Cu–HPEI, Ni–HPEI, Pt–HPEI and Pd–HPEI nanocomposites at concentrations ranging from 0 to 50 μg mL⁻¹. HPEI exhibit concentration dependent cytotoxicity due to their high cationic charge, whereas the Cu–HPEI, Ni–HPEI, Pt–HPEI and Pd–HPEI nanocomposites all have lower cytotoxicity compared with HPEI. For the metal–HPEI nanocomposites, HPEI was incorporated in the metal nanodots though their amine groups, thus the positive charge of HPEI was shielded, resulting in the low cytotoxicity.

Fig. 6 In vitro cytotoxicity of HPEI, Cu–HPEI, Ni–HPEI, Pt–HPEI and Pd–HPEI nanocomposites in COS-7 cell culture determined by MTT assay. The cell viability was quantitated after adding samples to cells for 24 h. Results are means ± SD (n = 6).

To assess the potential application of blue-emitting Cu nanodots as a bioimaging probe, COS-7 cells were cultured in a medium containing 1 mg mL⁻¹ Cu–HPEI nanocomposites for 6 h and then evaluated under a fluorescence microscope. The COS-7 cells showed a blue color, indicating that the Cu/HPEI nanocomposites had been internalized into the cells through endocytosis, as shown in Fig. 7. Benefiting from HPEI, blue fluorescent Cu nanodots capped with HPEI could be endocytosed by the cells without another transfection reagent. The Cu/HPEI nanocomposites integrate the advantages of HPEI with those of Cu nanodots. With their excellent luminescence properties, the Cu nanodots endow the system with fluorescent imaging ability, whereas HPEI is one of the most promising gene vectors;^24–29 therefore, the fluorescent Cu/HPEI nanocomposites should be an ideal fluorescence probe for tracking gene transfection behaviour.

Fig. 7 Fluorescence micrograph of COS-7 cells incubated with Cu nanodots transferred to an aqueous phase. Left, before excitation; right, after excitation, showing the nanodots aggregating in the cells.

4. Conclusion

In summary, blue-emitting and amphibious metal (Cu, Ni, Pt and Pd) nanodots were prepared based on HPEI/PA supramolecular polymeric micelles. These types of metal nanodots could be transferred from chloroform phase to water phase by adding triethylamine to break the electrostatic interactions between HPEI and PA. The obtained water-soluble Cu nanodots show good results for bio-imaging in COS-7 cells. Benefiting from these attractive properties, the blue-emitting and amphibious metal (Cu, Ni, Pt and Pd) nanodots should be an ideal fluorescent probe for tracking gene transfection behaviour.

Acknowledgements

Y Shi thanks Jimin Du and Andrew M. Smith for their helpful discussions. This study is supported by the Joint Fund for Fostering Talents of National Natural Science Foundation of China and Henan Province (U1204213, U1304210, U1304504), the National Natural Science Foundation of China (21304001, 21401006), Program for International S&T Cooperation Projects of Henan Province (162102410001), Program for Science & Technology Innovation Talents in Universities of Henan Province (14HASTIT013) and Foundation for University Young Key Teacher by Henan Province (2014GGJS-107).

References

1. Y. Z. Lu and W. Chen, Chem. Soc. Rev., 2012, 41, 3594.
2. N. Goswami, K. Y. Zheng and J. P. Xie, Nanoscale, 2014, 6, 13328.
3. L. Shang, S. J. Dong and G. U. Nienhaus, Nano Today, 2011, 6, 401.
4. X. Yuan, Z. T. Luo, Q. B. Zhang, X. H. Zhang, Y. G. Zheng, J. Y. Lee and J. P. Xie, ACS Nano, 2011, 5, 8800.
5. S. Chandirasekar, C. Chandrasekaran, T. Muthukumarasamyvel, G. Sudhandiran and N. Rajendiran, ACS Appl. Mater. Interfaces, 2015, 7, 1422.
6. S. Chakraborty, S. Babanova, R. C. Rocha, A. Desireddy, K. Artyushkova, A. E. Boncella, P. Atanassov and J. S. Martinez, J. Am. Chem. Soc., 2015, 137, 11678.
7. C. I. Richards, S. Choi, J. C. Hsiang, Y. Antoku, T. Vosch, A. Bongiorno, Y. L. Tzeng and R. M. Dickson, J. Am. Chem. Soc., 2008, 130, 5038.
8. J. Xie, Y. Zheng and J. Y. Ying, J. Am. Chem. Soc., 2009, 131, 888.
9. X. Yang, M. M. Shi, R. J. Zhou, X. Q. Chen and H. Z. Chen, Nanoscale, 2011, 3, 2596.
10. X. D. Zhang, F. G. Wu, P. D. Liu, N. Gu and Z. Chen, Small, 2014, 10, 5170.
11. J. Yu, S. A. Patel and R. M. Dickson, Angew. Chem., Int. Ed., 2007, 46, 2028.
12. B. Adhikari and A. Banerjee, Chem.–Eur. J., 2010, 16, 13698.
13. R. Ghosh, A. K. Sahoo, S. S. Ghosh, A. Paul and A. Chattopadhyay, ACS Appl. Mater. Interfaces, 2014, 6, 3822.
14. J. Zheng, J. T. Petty and R. M. Dickson, J. Am. Chem. Soc., 2003, 125, 7780.
15. Y. P. Bao, C. Zhong, D. M. Vu, J. P. Temirov, R. B. Dyer and J. S. Mar-tinez, J. Phys. Chem. C, 2007, 111, 12194.
16. Y. C. Jao, M. K. Chen and S. Y. Lin, Chem. Commun., 2010, 46, 2626.
17. L. Shang and S. J. Dong, Chem. Commun., 2008, 1088.
18. S. Liu, F. Lu and J.-J. Zhu, Chem. Commun., 2011, 47, 2661.
19. M. J. Barthel, I. Angeloni, A. Petrelli, T. Avellini, A. Scarpellini, G. Bertoni, A. Armirotti, I. Moreels and T. Pellegrino, ACS Nano, 2015, 9, 11886.
20. Z. Wang, W. Cai and J. Sui, ChemPhysChem, 2009, 10, 2012.
21. L. Shang, R. M. Dörlich, S. Brandholt, R. Schneider, V. Trouillet, M. Bruns, D. Gerthsen and G. U. Nienhaus, Nanoscale, 2011, 3, 2009.
22. Z. N. Wu, J. L. Liu, Y. Gao, H. W. Liu, T. T. Li, H. Y. Zou, Z. G. Wang, K. Zhang, Y. Wang, H. Zhang and B. Yang, J. Am. Chem. Soc., 2015, 137, 12906.
23. Y. F. Shi, C. L. Tu, R. B. Wang, J. Y. Wu, X. Y. Zhu and D. Y. Yan, Langmuir, 2008, 24, 11955.
24. G. H. Chen, W. J. Chen, Z. Wu, R. X. Yuan, H. Li, J. M. Gao and X. T. Shuai, Biomaterials, 2009, 30, 1962.
25. M. Morille, C. Passirani, A. Vonarbourg, A. Clavreul and J. P. Benoit, Biomaterials, 2008, 29, 3477.
26. O. M. Merkel, M. Y. Zheng, H. Debus and T. Kissel, Bioconjugate Chem., 2012, 23, 3.
27. Y. F. Shi, J. M. Du, L. Z. Zhou, X. T. Li, Y. H. Zhou, L. L. Li and X. Y. Zhu, J. Mater. Chem., 2012, 22, 253.
28. Y. F. Shi, L. Z. Zhou, R. B. Wang, Y. Pang, W. C. Xiao, H. Q. Li, Y. Su, X. L. Wang, B. S. Zhu, X. Y. Zhu, D. Y. Yan and H. C. Gu, Nanotechnology, 2010, 21, 115103.
29. Y. F. Shi, D. F. Huang, L. Z. Zhou, X. F. Weng, F. S. Cai, B. J. Lv, X. Y. Hou, H. Li, J. H. Zhao, F. J. Ma, L. Liu and X. Y. Zhu, Sci. ANanotechnologydv. Mater., 2015, 7, 219.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra06116h

---