Bioconjugate Chem. 2007, 18, 1709–1712
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Novel Arylhydrazone-Conjugated Gold Nanoparticles with DNA-Cleaving Ability: The First DNA-Nicking Nanomaterial Ming-Hua Hsu,† Thainashmuthu Josephrajan,† Chen-Sheng Yeh,‡ Dar-Bin Shieh,§ Wu-Chou Su,| and Jih Ru Hwu*,† Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 300, Republic of China, and Department of Chemistry, Department of Dentistry and Institute of Oral Medicine, and Department of Internal Medicine, National Cheng Kung University, Tainan, Taiwan 701, Republic of China. Received June 20, 2007; Revised Manuscript Received September 20, 2007
Arylhydrazones were linked onto gold nanoparticles through the poly(ethylene glycol) spacer to produce a new type of photoinduced DNA-cleaving nanomaterials with great potency.
Scheme 1. Synthesis of Arylhydrazone-Conjugated Au-NPsa
INTRODUCTION Nanobioscience and technology possess enormous potential, as their development could lead to new materials beneficial to human life. Prominent progress has been observed in various fields, including drug delivery, DNA sequencing, biosensor, biomolecular separation, magnetic resonance imaging, and so forth (1–6). Among various nanoparticles (NPs), gold nanoparticles (Au-NPs) are most stable and, thus, suitable for biological systems. Their inert surface prevents undesired reactions from taking place with different functional groups of biomolecules (7); yet, Au-NPs can still be capped with water-soluble organic compounds (8, 9) or biomolecules through thiols (10). For example, Mirkin (11–13), Yeh (14), and co-workers functionalized Au-NPs with 3′- and/or 5′-thiol oligonucleotides. Russell et al. (15) used a thiolated ethylene glycol anchor chain with various lengths to link lactose derivatives to preformed AuNPs. We planned to develop nanoparticles that bear multi-DNA cleaving “warheads”. It is necessary for the warheads to be controlled by a trigger, which can be used to initiate the DNAnicking process. These new functional nanoparticles may possess the following features and advantages: 1. The DNA-cleaving potency could be enhanced dramatically because of the high local density of warheads, many of which are attached to one nanoparticle nucleus. 2. The warheads as “soft” moieties are attached onto the “hard” core of nanoparticles, which could serve as bullets of a gene gun (16–18). Consequently, the DNAcleaving warheads can be delivered to a specific location by mechanical force. 3. The solubility of the hybrids can be adjusted by spacers, which link the warheads to nanoparticles. Our research group has been developing new artificial agents for cleaving nucleic acids under control conditions (19–22). Among these agents, arylhydrazones were found stable and can nick DNA by use of UV light as the trigger (22). Herein, we report our success in the attachment of arylhydrazones onto gold nanoparticles to form a new class of DNA-cleaving materials. Being water-soluble, these hybrid nanomaterials were manipu* E-mail:
[email protected]. Fax: 886 35 721 594. Tel: 886 35 725 813. † National Tsing Hua University. ‡ Department of Chemistry, National Cheng Kung University. § Department of Dentistry and Institute of Oral Medicine, National Cheng Kung University. | Department of Internal Medicine, National Cheng Kung University.
a Reagents and conditions: (a) PBr3, 60 °C, 4.0 h; (b) thioacetic acid, NaOH, THF, 25 °C, 6.0 h; (c) NaOH, MeOH, 25 °C, 5.0 h.
lated without difficulty in aqueous solutions for nicking DNA upon UV irradiation.
RESULTS Our synthetic strategy for incorporation of an arylhydrazone onto Au-NPs is shown in Scheme 1. The starting material AuNPs with diameters of 13 ( 1.8 nm were prepared by reduction of HAuCl4 with sodium tricitrate according to the established procedure (23, 24); meanwhile, poly(ethylene glycol) (PEG) was used as the spacer. Thus, we treated tetraethylene glycol (1) with PBr3 in ether to give the corresponding dibromide 2. Protection of one terminal in 2 with a stoichiometric amount of thioacectic acid in the presence of NaOH in THF led to thioacetate 3. Coupling of 3 with p-hydroxybenzaldehyde in the presence of K2CO3 in acetone provided PEG-containing
10.1021/bc700222n CCC: $37.00 2007 American Chemical Society Published on Web 10/23/2007
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Figure 1. TEM images of gold nanoparticles conjugated with arylhydrazones through PEG spacers (i.e., 7).
benzaldehyde 4, in which the acetyl group was deprotected with methanolic NaOH under carefully controlled conditions (25). The desired free thiol 5 was isolated in 77% yield by flash chromatography packed with silica gel. Its characteristic SCH2 protons appeared at 2.72 ppm with J ) 7.2 Hz and the benzaldehydic singlet proton at 9.85 ppm in the 1H NMR spectrum. A downfield resonance for the benzylic carbon exhibited at 190.63 ppm in its 13C NMR spectrum. Furthermore, a strong absorption at 1692 cm-1 associated with the conjugated CdO stretch also appeared in its IR spectrum. Condensation of this benzaldehyde 5 with phenylhydrazine gave the corresponding phenylhydrazone 6 with m/z ) 404.1765. In its 13C NMR spectrum, eight peaks showed up between 112.85 and 159.39 ppm for the aromatic carbons; meanwhile, the newly generated benzylic NdC carbon shifted to 159.39 ppm. The NdCH proton resonated at 7.62 ppm in its 1H NMR spectrum. To obtain conjugated target materials, we treated the ∼13 nm Au-NPs with a large excess (500–1000 equiv) of phenylhydrazone 6 (2.0 mL, 10 mM) in water at 25 °C for 4.0 h. The desired hybrid 7 was produced and isolated by centrifuge; a red solution of its colloids in water was attributed to their surface plasmon resonance (26, 27). The centrifuge process assured the following DNA-cleaving results coming from hybrid nanoparticles 7 instead of the organic molecule 6. Because the Au-NPs were attached by the HS-PEG-arylhydrazone ligand through the sulfide terminal, we observed a hyperchromic and bathochromic shift by 5 nm in the UV spectra in situ. These results are consistent with reports on spectral surface plasmon resonance shifts for the thiols on Au-NPs (28). By using the displacement method with mercaptoethanol reported by Nie et al. (29), we determined the number of ligands 6 per particle as 55–60 on average. In this experiment, the concentration of displaced hydrazone-containing thiol 6 from hybrid 7 was compared with that from freshly synthesized 6 at λ 345 nm of UV spectrometry. Furthermore, the transmission electron microscopy (TEM) was used for determination of the nanoscale structures. We dropped a casting solution of nanoparticles onto carbon-coated copper grids. The distribution of gold cores of hybrid 7 is shown in Figure 1a, which indicates that the nanoparticles were welldispersed. The amplified TEM in Figure 1b reveals that spherically shaped particles were obtained; of which the diameter of 13 ( 1.9 nm as the average was estimated on the basis of 120 sampled arylhydrazone–PEG-capped nanoparticles (30, 31). To investigate the DNA-cleaving ability, we dissolved the water-soluble Au-PEG–arylhydrazone colloids 7 (2.0 nM) in a sodium phosphate buffer (pH 7.4) and 10% ethanol containing the supercoiled circular φX174 RFI DNA (form I; 50 µM/base pair). These solutions were irradiated with UV light (312 nm, 1.43 mW/cm2) under aerobic conditions at room temperature for 2.0 h. By analyzing the results from gel electrophoresis on 1.0% agarose gel with ethidium bromide staining, we found
Figure 2. (a) Single-strand cleavages of supercoiled circular φX 174 RFI DNA: lane 1, DNA alone; lane 2, 13 nm gold nanoparticles in the dark; lane 3, 13 nm gold nanoparticles under UV light; lane 4, AuNPs hybrid 7 in the dark; lanes 5–8, 2.0 nM, 500 pM, 100 pM, and 20 pM of Au-NPs hybrid 7 under UV light. (b) Percentages of form II/ form I for lanes 1–8.
that the hybrid 7 caused single-strand cleavages of DNA efficiently to give the relaxed circular DNA (i.e., form II; see lane 6 of Figure 2). Our results shown in lane 7 indicate that hybrid 7 was able to cleave DNA at a concentration as low as 1.00 × 102 pM. To seek for the essential factors of the DNA nicking, we performed two control experiments. The first was to carry out the experiment under the same conditions except in the dark (see lane 4). The second involved UV irradiation; however, we replaced Au-PEG–arylhydrazone hybrid (7) by Au-NPs (see lane 3). These results indicate that the DNA were not damaged; thus, both the UV light and the integrated Au-PEG–arylhydrazone were essential for DNA nicking. Moreover, the positive results shown in lanes 5 and 6 clearly indicate that the Au-NP cores did not significantly inhibit UV light to activate arylhydrazones on the shell, although they showed UV absorption at 520 nm.
DISCUSSION There are three reasons to tether arylhydrazones onto the shell of Au-NPs by use of the PEG strands. First, an alkoxy substitutent can enhance the DNA-cleaving ability of arylhydrazones, which generate aminyl (R2N) and iminyl (R2CdN) radicals upon UV irradiation (22). A terminal oxygen atom of triethylene glycol for its connection to the arylhydrazone serves this purpose. Moreover, the great enhancement of DNA-cleaving potency may also indicate that the oxygen-containing PEG spacers prevented the nitrogen-containing radicals absorbed by gold surface, as reported by Meisel et al. (32). Second, the citrate anions were coordinated to the AuI shell, although the core of our Au-NPs was Au0 (13). When we treated the Au-NPs with salts, aggregations often took place among particles, and color changed from red to dark blue. This problem was circumvented after we attached the PEG spacers onto Au-NPs. The stability came from the steric repulsion effect of the tethered PEG spacers, which improved the dispersion of the Au-NPs–PEG– arylhydrazone particles in aqueous media. Third, the PEG strands on the surface of the functionalized Au-NPs improved their hydrophilicity. This effect enabled the hybrid nanoparticles of 7 to approach and, thus, to cleave the DNA target with ease. Our previous findings indicate that the hydrophobic organic compound, p-methoxylbenzaldehyde p-methoxylphenylhydrazone (PMBH), can cleave DNA at a concentration as low as 50 µM (22). The current findings indicate that arylhydrazonecontaining Au-NPs 7 cleaved DNA at a much lower concentration (i.e., 2.0 nM). In comparison with PMBH, the efficacy of the hybrid 7 was improved by a factor of >104 despite each
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nanoparticle containing 55–60 warheads. Although the total warhead number on each nanoparticle could be increased, we tried not to keep it overcrowded. This design is on the basis of our speculation that, otherwise, coupling reactions might take place among the radical warheads after their generation upon UV irradiation. Our results shown in lanes 5–7 of Figure 2 indicate that only form II DNA (open-circular product) was generated through single-strand breaks; meanwhile, the formation of form III DNA (i.e., linear product) resulting from double-strand breaks was not observed. The generation of form III DNA has to come from two separate single-stand cleavage events in close proximity. Therefore, we consider that the high efficiency of DNA single-strand breaks may be derived from the nanoparticles bearing multiradical warheads toward all directions on their spherical surface. When these hydrophilic “maces” with a high local density of radicals bumped into DNA, the chance for nicking a single strand would be much higher than individual organic radicals, such as the aminyl (R2N) and the iminyl (R2CdN) radicals. The efficiency resulting from the mace, however, may not be necessarily extendable to the double-strand break event. Further works will be in due course in order to have good insight into this intriguing issue. Recently, Mirkin et al. (33) reported a novel strategy for intracellular gene regulation with oligonucleotide-modified AuNPs. They demonstrate that nanoparticle-bound antisense oligodexoyonucleotides can be degraded by DNase. On the other hand, Liu and Lu (34) developed colorimetric biosensors on the basis of DNAzyme-assembled Au-NPs. In the presence of Pb2+, the DNAzyme catalyzes specific hydrolytic cleavage of the substrate strand, which disrupts the formation of the nanoparticle assembly. Up to the present date, these two approaches are likely to be the only published examples that involve cleavage of nanoparticles conjugated with DNA or oligonucleotides. In both examples, the cleaving “warheads” are not attached to the nanoparticles. In contrast, the currently developed conjugate materials 7 possessed warheads on their shells and can be regarded as a new type of DNase. In conclusion, gold nanoparticles conjugated with arylhydrazones through thiol-based PEG ligands (i.e., 7) were successfully synthesized. The diameters of these new nanoparticles with spherical shape were 13 ( 1.9 nm as observed by TEM. Upon triggering by 312-nm UV light, hybrid 7 cleaved supercoiled DNA to give a relaxed circular shape at a concentration as low as 2.0 nM. To the best of our knowledge, this is the first example of nanoparticles with the DNA-cleaving property.
ACKNOWLEDGMENT This work was supported by the National Science Council of Republic of China. Supporting Information Available: Details of synthetic and experimental procedures, and spectroscopic data of compounds. This material is available free of charge via the Internet at http:// pubs.acs.org.
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