Ping Dong‡
,
Jianyu Xin‡,
Xiao Yang,
Jin Jia,
Wei Wu and
Jianshu Li*
College of Polymer Science and Engineering, Sichuan University, Chengdu, 610065, China. E-mail: jianshu_li@scu.edu.cn; Fax: +86-28-85405402; Tel: +86-28-85466755
First published on 12th September 2014
Worm-like carboxylated 3.5 generation dendronized poly(amido amine) (DPG3.5-COOH) was applied to synthesize gold nanoparticles (AuNPs), in which process DPG3.5-COOH played the roles of reducing agent and template. The morphology of AuNPs, e.g., nanorods and nanoparticles, could also be controlled by the interior-template function or the exterior-template function of DPG3.5-COOH at different pH values. The obtained AuNPs had low cytotoxicity, effective cellular uptake and strong photothermal therapy capability for HeLa cancer cells. Therefore, the AuNPs prepared using the worm-like dendronized polymer provide a new and effective approach for photothermal therapy.
In order to prepare AuNPs in a greener way and with controllable properties, researchers try to choose multifunctional and nontoxic reactants for the reduction and stabilization of AuNPs. Natural compounds such as proteins, DNA and glucan have been used to synthesize AuNPs in one step, which have exhibited pretty good results.17–20 Natural compounds like proteins are nontoxic and the natural configuration of the compounds can act not only as stabilizer but also template. For example, it is interesting to see that egg white proteins can be applied to synthesize highly fluorescent AuNPs. The gold ions can interact strongly with the amino acid residues and the following alkaline environment can result in the reduction of Au ions by tyrosine and tryptophan amino acid residues. It is believed that the limited vibrational motion and the bulkiness of proteins restrict the growth of particles, thus AuNPs with small size can be obtained.20
Compared with natural compounds, polymers can be designed with multiple functional groups and with various topologies, thus quite a few polymers have been designed to synthesize AuNPs.21 For this purpose, the most frequently used two kinds of polymers are block copolymers22–25 and dendrimers.6,10,26–32 For example, the PEO–PPO–PEO block copolymer with proper hydrophobicity can form micelles and helps in stabilizing AuNPs.33 Block copolymers can also be designed into different topologies and show great template effect. Gohy et al. applied a type of 5-arm star block copolymer as template in synthesizing AuNPs with tuneable hydrophilicity. The polymeric core can be swelled with Au iron in solvent and AuNPs can be formed in the core, thus the star polymer template can effectively control the size of obtained AuNPs.34 Meanwhile, dendrimers with well-defined and monodisperse structure have been demonstrated as ideal templates for AuNPs. For instance, poly(propyleneimine) (PPI) dendrimer can simultaneously act as the reducing agent and protective agent, thus the dendrimer-protected AuNPs can be facilely prepared by one-step heating.10 Poly(amido amine) PAMAM dendrimers have also been widely used in synthesizing AuNPs. The gold iron can enter into the space around the braches and the AuNPs are formed in these spaces, thus the size of AuNPs can be easily controlled by the PAMAM dendrimers of different generations or with different modified structures.6,26–32
It is noted that dendronized polymers, which are derived from traditional dendrimers, have hyperbranched architectures and worm-like morphologies resembling that of rod-like viruses.35–38 They have linear polymeric main chains and branched dendrons, and are the largest but still precise synthetic structures comparable to biological molecules.37 Due to their confined inner space, functional groups on the dendrons and intrinsic worm-like morphologies, they have been demonstrated as effective templates for Cds nanoparticles.39,40 However, to the best of our knowledge, neither their possibility in preparing AuNPs nor applications in biomedical field have been reported so far.
Herein, we applied carboxylated 3.5 generation dendronized poly(amido amine) (DPG3.5-COOH), which has a polymeric main chain attached with a number of dendrons, to synthesize AuNPs. DPG3.5-COOH is hyperbranched and has a one dimensional rod-like morphology. In the process, DPG3.5-COOH acts not only as stabilizer but also as reducing agent and template. The multifunctional DPG3.5-COOH simplify the method and the whole process is green and nontoxic as demonstrated by the cytotoxicity test. The influence parameters and the template effect of DPG3.5-COOH are investigated. In addition, since AuNPs are widely used in cancer therapy, we also investigated the photothermal therapy capability of the obtained particles.
The effect of HAuCl4 concentration on the synthesis process was also investigated while the concentration of DPG3.5-COOH was fixed at 0.5 mM. The results are compared by UV-vis spectroscopy as shown in Fig. 1. As can be seen, when the concentration of HAuCl4 is in the range of 0.5 to 1.5 mM, the surface plasmon resonance (SPR) of AuNPs, which appears around 535 nm, is increased with the increase of HAuCl4 concentration. It is noted that the SPR of AuNPs is not obvious at 0.5 mM. In the range of the investigated concentrations, the highest SPR absorbance is appeared at 1.5 mM, which should be the optimized concentration of HAuCl4. When the HAuCl4 concentration is increased to 2.0 and 2.5 mM, there is almost no obvious SPR absorbance. This should due to the aggregation of AuNPs: when the Au3+/DPG3.5-COOH ratio is too high, the stabilizing function of DPG3.5-COOH is not that significant. The results shown in Fig. 2 and S1–S3† also confirm this speculation. Since DPG3.5-COOH acts as the template and the stabilizer at the same time, the hydrodynamic diameters measured by DLS should be of all the AuNPs within each DPG3.5-COOH instead of individual AuNPs. When the HAuCl4 concentration increase from 0.5 to 1.5 mM, the diameters of obtained AuNPs increase gradually from 18 to 32 nm. However, the diameter of AuNPs increases dramatically to 130 nm when HAuCl4 concentration reaches 2.0 mM, which confirms our speculation that the nanoparticles have been aggregated to some extent. The aggregation of AuNPs was also observed in this experiment: the reaction was fast as it turned purple very quick when the HAuCl4 concentration was 2.0 mM; there was also many precipitates in the final solution. Thus 1.5 mM is chosen as the optimized HAuCl4 concentration in this work.
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Fig. 1 UV-vis spectra of AuNPs prepared with HAuCl4 of different concentrations (70 °C, pH 6.5, DPG3.5-COOH: 0.5 mM). |
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Fig. 2 DLS data of AuNPs prepared with HAuCl4 of different concentrations: (a) 0.5 mM; (b) 1.0 mM; (c) 1.5 mM; (d) 2.0 mM (70 °C, pH 6.5, DPG3.5-COOH: 0.5 mM). |
In the synthesis process of AuNPs, the reaction pH is an important influence factor. In this work, different types of AuNPs are formed at different pH values. As shown in Fig. 3, the SPR of AuNPs around 535 nm is obvious when the reaction pH is equal or higher than 6.5. Meanwhile, the height of the SPR peak decreases gradually with the increase of pH from 6.5 to 9.0, indicating that pH 6.5 is the optimized reaction pH. TEM and DLS were used to observe the morphology difference and the diameter of the AuNPs obtained at pH 6.5 and 6.3 (Fig. 4). When the reaction pH is 6.3, the obtained AuNPs are irregular and aggregated. The DLS result also shows that the diameter of the aggregated AuNPs is around 140 nm. When the reaction pH reaches 6.5, the hydrodynamic diameter of the AuNPs are between 20–30 nm, which is larger than that shown in TEM images as commonly observed. TEM images show that the AuNPs are most sphere-like and there is no obvious aggregation with homogeneously distributed diameter.
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Fig. 3 UV-vis spectra of AuNPs prepared at different pH values (70 °C, DPG3.5-COOH: 0.5 mM, HAuCl4: 1.5 mM). |
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Fig. 4 TEM images and DLS results of AuNPs prepared at different pH values. (a and b): pH 6.5; (c and d) pH 6.3 (70 °C, DPG3.5-COOH: 0.5 mM, HAuCl4: 1.5 mM). |
Thus, the using of DPG3.5-COOH to synthesize gold nanoparticles is a facile method without additional reducing agent. The optimized condition is 0.5 mM of DPG3.5-COOH, 1.5 mM of HAuCl4 and react at 70 °C under pH 6.5. The obtained AuNPs are around 30 nm and can be stably stored at room temperature for at least 6 month.
Usually, the pH of reaction solution influence the complex form of Au3+ in the solution,12 thus it can result in AuNPs with different morphologies. However, in our experiment, besides the different form of Au3+, the different structures of DPG3.5-COOH at different pH values also play important roles for the different morphologies of the AuNPs. In order to investigate how DPG3.5-COOH can influence the morphology of AuNPs, the synthesis was carried out following the optimized condition (70 °C, DPG3.5-COOH: 0.5 mM, HAuCl4: 1.5 mM) but the reaction pH was intentionally adjusted to 6.3. The samples was taken out at different reaction time points for TEM and UV-Vis spectroscopy characterizations, to investigate the developing process of AuNPs.
As shown in Fig. 5, all absorbance lines do not show obvious SPR peak. But with the reaction time going by, the lines in the Fig. 5 gradually grow up. The absorbance between 500 nm and 700 nm has clearly become higher, which is due to the generation of some spherical AuNPs.
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Fig. 5 UV-vis absorption spectra of AuNPs versus time (70 °C, DPG3.5-COOH: 0.5 mM, HAuCl4: 1.5 mM, pH 6.3). |
In order to determine what has been exactly generated, TEM was used to examine the morphology of different products at different reaction time points (Fig. 6). It is interesting to see there are a lot of one-dimensional gold nanorods generated at the earlier stage of reaction (2 and 5 min). When the reaction time is 2 min which is the first time that the colour of the reaction solution becomes darker, there are already lots of well-distributed nanorods. The length of a single gold nanorod is around 30 nm, which is close to the length of a single DPG3.5-COOH molecule.40 But the gold nanorods are tending to form aggregates which are larger than 100 nm. When the reaction time is 5 min, there are still lots of gold nanorods, suggests that the gold nanorods can exist stably for a short time. When the reaction time is 10 min, we can see a lot of irregular gold nanoparticles and the gold nanorods have almost disappeared.
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Fig. 6 TEM images of AuNPs prepared at 70 °C, DPG3.5-COOH: 0.5 mM, HAuCl4: 1.5 mM, pH = 6.3. (a) 2 min; (b) 5 min; (c) 10 min. |
Thus, there are obvious two types of final products, and the critical pH for them is between 6.3 and 6.5, which matches the pKa value of DPG3.5-COOH (data not shown). When the reaction pH is lower than the critical pH, the gold nanorods of 30 nm is produced first, then the gold nanorods tend to assemble into the further aggregates with irregular morphology. When the reaction pH is higher than the critical pH, the sphere-like gold nanoparticles of 30 nm can be directly synthesized (Fig. 4).
The coincidence that the critical pH matches the pKa value of DPG3.5-COOH suggests that the worm-like dendronized dendrimer is the main reason leads to the different morphologies of AuNPs. The functions of worm-like DPG3.5-COOH in this experiment are not only the reducing agent, but also the template. And its template function is obviously dependent on the pH of the reaction solution. The template function of DPG3.5-COOH can divide into two parts: the interior-template function and the exterior-template function. The interior-template function is mainly based on the structure inside the dendronized molecule, and the functional groups on the branches such as tertiary amine groups also help to exert this function. The exterior-template function is mainly based on the functional groups on the peripheral of the molecule and also the self-assemble ability of DPG3.5-COOH. Thus the pH of the reaction solution directly change the morphology of DGP3.5-COOH, and then influences its template function. Specifically, when the pH of the reaction solution is below the critical pH, the tertiary amine groups on the polymer branches are protonized, and this leads the inner part of DPG3.5-COOH molecule positively charged. This process makes the electrostatic repulsion force inside the polymer molecule increase, and the worm-like polymer molecule become more rigid. In this situation, the molecule of the DPG3.5-COOH is stretched, and the space inside the branches becomes lager which makes the Au3+ much easier to get into the molecule. In this way the DPG3.5-COOH shows a very strong exterior-template function, and passes its own one-dimensional worm-like morphology to the AuNPs, making the intermediate product to be nanorods. The length of a single nanorod matches the length of a single DPG3.5-COOH molecule. But because of the self-assemble ability of the template DPG3.5-COOH and the unstable surface of the nanorods, the nanorods tend to assemble into parallel bunches. And the unstable bunches are tending to aggregate. Thus, at pH 6.3, the irregular nanoparticles are synthesized of which the diameter is over 100 nm. When the pH of the solution is higher than the critical pH, the tertiary amine groups are mostly deprotonized, and the molecule of the DPG3.5-COOH curls up. In this situation, the interior-template function of the PAMAM becomes weaker. At the same time, the carboxyl groups on the periphery the polymer are also deprotonized which leads to the increase of the electrostatic repulsion force between DPG3.5-COOH molecules, which demonstrating the exterior-template function. At last, the AuNPs of 30 nm are synthesized based on the strong exterior-template function (illustrated in Fig. 7).
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Fig. 7 Schematic illustration of the template effect of DPG3.5-COOH in the synthesis process of AuNPs. |
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Fig. 8 CCK-8 assay for cytotoxicity of AuNPs. Error bars represent means ± standard error for n = 6. |
In order to kill diseased cells, deliver therapeutic drugs or alter cellular functions, AuNPs should have a good cellular uptake capability.42,43 The TEM image of the HeLa cells incubated with 60 μM AuNPs for 6 h is shown in Fig. 9. As indicated by the arrow, the gold nanoparticles have entered the cells and contained within cell vesicles within 6 h. It is also noted that the AuNPs are aggregated within the cell vesicles, which should be essential for them to exhibit the photothermal cell destroying effect as reported in previous work.5 The successful cell uptake of AuNPs prepared by DGP3.5-COOH can support their further intracellular biomedical functions.
In cancer therapy, the mainstream treatments such as surgery, chemotherapy and radiotherapy all have serious drawbacks. The SPR absorption of AuNPs can rapidly and efficiently covert the absorbed light into localized heat, thus they can be applied for the selective photothermal therapy of cancer.1 In this work, AuNPs with a diameter of 30 nm and spherical morphology (60 μM) are used to test the photothermal ability to kill the HeLa cell after being incubated for 6 h and then irradiated by a 535 nm laser for 30 min. As shown in Fig. 10, the AuNPs prepared by DPG3.5-COOH have obvious photothermal therapy effect. The cells in the control sample are still alive after laser irradiation. On the contrary, most of the HeLa cancer cells are died after laser irradiation for the sample incubated with AuNPs. Thus, the AuNPs prepared by DPG3.5-COOH can still have significant phototermal therapy effect.
Footnotes |
† Electronic supplementary information (ESI) available: Including additional TEM images. See DOI: 10.1039/c4ra08475f |
‡ These authors contributed equally to this work. |
This journal is © The Royal Society of Chemistry 2014 |