Self-reduction and morphology control of gold nanoparticles by dendronized poly(amido amine)s for photothermal therapy

Abstract

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.

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1. Introduction

In recent years, gold nanoparticles (AuNPs) have been widely studied for biomedical applications such as cell imaging, drug delivery and cancer therapy due to their unique optical properties, facile surface chemistry, appropriate size scale, strong X-ray attenuation characteristics and other biological properties.^1–9 To be applied in biomedical fields, AuNPs need to be nontoxic and easily synthesized with controllable shape and size. The most widely used method to synthesize AuNPs is reducing metal salts in the solution environment. Traditionally, this method involves the salt providing the Au iron like HAuCl₄ and the reducing agent such as sodium borohydride, citrate, carbon monoxide, hydrogen and alcohol.^10–13 For example, hydroquinone has been used to synthesize stable urchin-like AuNPs with high reducibility.¹³ In order to control the shape and size of AuNPs, this method could be improved by adding stabilizers and templates.^8,14–16 The stabilizers, e.g., surfactant, can keep AuNPs from aggregation and preserve their quality. On the other hand, the templates are able to control the size and shape of produced AuNPs. However, the utilization of additional participants also adds difficulties on the control of the reaction.

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 copolymers^22–25 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.

2. Experimental

2.1 Materials

DPG3.5-COOH were synthesized according to our previous report.⁴⁰ Chloroauric acid (HAuCl₄) was purchased from Aladdin Industrial Inc, and was prepared into 4 mM water solution (stored at 4 °C). Dulbecco's modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific Inc. Penicillin G sodium salt, streptomycin sulfate and trypsin were purchased from Beijing Solarbio Science & Technology. Cell Counting Kit-8 (CCK-8) was purchased from Beyotime Institute of Biotechnology. Propidium iodide (PI) was purchased from Aladdin Industrial Inc. Lysotracker green was purchased from Life Technologies. Ultra-pure water was used throughout.

2.2 Syntheses of AuNPs using DPG3.5-COOH

Briefly, DPG3.5-COOH was dissolved in water and filtrated through a 220 nm microporous membrane to prepare a final solution of 0.5 mmol L⁻¹. HAuCl₄ aqueous solution (0.5–2.5 mmol L⁻¹ in a series of experiments) was added to the above solution with vigorous stirring. The pH of the mixing solution was adjusted using HCl or NaOH solution, and the volume of the solution was quantified to 5 mL. The mixed solution was then put into a 70 °C water bath under vigorous stirring and kept for 20 min to get the final AuNPs solution. The reaction temperature, pH and the concentration of the reactant have been optimized in a series of orthogonal experiments.

2.3 Characterizations of AuNPs

UV-vis absorption spectra were examined on a Mapada UV-1800PC UV/vis spectrophotometer by scanning the solutions of the samples in quartz cells. Transmission electron microscopy (TEM) was performed on a Tecnai G2 F20 S-TWIN microscope operated at 200 kV. Dynamic light scattering (DLS) was recorded on a Brookhaven BI-200SM wide angle laser light scattering instrument. The solution was filtrated through a 220 nm microporous membrane before test.

2.4 Cytotoxicity measurement

HeLa cells were used in this experiment. The culture medium used in this experiment was DMEM with 10% (v/v) fetal bovine serum (FBS), 100 units per mL of penicillin and 100 μg mL⁻¹ of streptomycin. And all the cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ in air. The cytotoxicity of AuNPs was measured by CCK-8 assay. HeLa cells were seeded at the density of 5 × 10⁴ cells per well in a 96-well dish with 100 μL culture medium. After 24 h, the dish was washed by phosphate buffered saline solution (PBS) for 3 times and then 100 μL new medium with 10 μL different concentrations of AuNPs (1.5, 3, 15, 30, 60 and 100 μM) was added to each well. The cells were then incubated for 6 h or 24 h. Later the cells were washed with PBS and cultured in 100 μL medium with 10 μL CCK8 solution for 2 h and were tested by a KHB ST-360 micro-plate reader at 450 nm (n = 6).

2.5 Cellular uptake of AuNPs

HeLa cells were cultured in a culture flask (75 cm²) and the medium was changed every 3 days until the cells were grown to approximate 80% confluence. Then the medium was changed by normal medium with 60 μM AuNPs and incubated for 6 h. After that, excess medium was removed, and the cells were thoroughly washed with PBS, trypsined, centrifuged and fixed using pentanedial solution. After being embedded in resin and sliced, the sections containing the cells were observed by TEM (Hitachi H-600 microscope, 75 kV).

2.6 Photothermal therapy test

HeLa cells were seeded at the density of 2 × 10⁵ cells per well in 6-well dish with 2 mL culture medium for 24 h. After washed by PBS solution for 3 time, each well was added in a new medium with 60 μM AuNPs. After 6 h's incubation, the medium was discarded and the dishes were washed by PBS solution for 3 times. Then 2 mL new culture medium was added into each well. After that, each well was irradiated by a 535 nm green laser light at a power density of 2 W cm⁻² for 30 min. Then the cells were further incubated for 3 h. After that, the cells were stained by lysotracker green solution (2 μg mL⁻¹) and PI solution (5 μM). The medium in the wells were discarded and the wells were washed by PBS solution before adding 1 mL staining solution. The dishes with staining solution were put in 37 °C for 20 min. Then the staining solution was discarded and the dishes were observed under an Olympus IX71 fluorescence microscope.

3. Results and discussion

3.1 Syntheses and optimization of AuNPs

AuNPs have been synthesized with the worm-like DPG3.5-COOH, and the reaction temperature, the concentrations of the reactants and the pH value of the synthesis process have been carefully adjusted to obtain well-proportioned AuNPs. When the reaction was performed at the room temperature (25 °C), the reaction could last for 3 days. Thus the slowness of the process made it difficult to observe and control the reaction. However, when the reaction temperature was higher than 80 °C, the reaction could be very fast as the solution colour changed within 2 min, and the obtained AuNPs also tended to aggregate. The optimized reaction temperature was 70 °C. At this reaction temperature, the reaction solution gradually turned purple in 20 min and the final AuNPs solution had no precipitates.

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.

Fig. 1 UV-vis spectra of AuNPs prepared with HAuCl₄ of different concentrations (70 °C, pH 6.5, DPG3.5-COOH: 0.5 mM).

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.

Fig. 3 UV-vis spectra of AuNPs prepared at different pH values (70 °C, DPG3.5-COOH: 0.5 mM, HAuCl₄: 1.5 mM).

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.

3.2 Self-reduction and template effect of DPG3.5-COOH

In the above synthesis process of AuNPs, the worm-like DPG3.5-COOH acts as reducing agent, stabilizer and template. The addition of HAuCl₄ to DPG3.5-COOH with tertiary amine groups resulted in a protonated dendrimer with AuCl₄⁻ counterions²⁶ and the formation of a polysalt between dendrimer nitrogens and AuCl₄⁻,⁴¹ thus it can facilitate the electron transfer from Au³⁺ to the tertiary amine groups of DPG3.5-COOH. Therefore, the synthesis of AuNPs with DPG3.5-COOH can be called self-reduction without additional reducing agents. This self-reduction effect has also been reported in several previous reports regarding AuNPs preparation by different dendrimers.^10,30

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.

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.

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 pK_a 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 pK_a 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).

Fig. 7 Schematic illustration of the template effect of DPG3.5-COOH in the synthesis process of AuNPs.

3.3 Cytotoxicity, cellular uptake and photothermal therapy capability of obtained AuNPs

The toxicity of AuNPs is essential for their potential biomedical applications such as biological imaging, drug delivery and cancer treatment.⁸ Therefore, the cell cytotoxicity of the AuNPs prepared by DPG3.5-COOH was evaluated by CCK8 assay using HeLa cells (Fig. 8). The solution of AuNPs synthesized at the optimized conditions was filtered through a 2 μm microporous membrane before being added into the DMEM. As can be seen, when the incubation time is 6 h, AuNPs do not show obvious cytotoxicity up to 15 μM. The AuNPs even have >90% cell viabilities when their concentrations are 30 and 60 μM. When the incubation time is 24 h, AuNPs do not show obvious cytotoxicities up to 60 μM as the cell viability is still over 90%. The good cell cytotoxicity of the AuNPs prepared by DPG3.5-COOH justify their further broad applications in biomedical fields.

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.

Fig. 9 TEM image of the HeLa cells incubated with 60 μM AuNPs for 6 h.

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.

Fig. 10 Photothermal damage of HeLa cancer cells by AuNPs. Alive and dead cells were stained with LG (a–d) and PI (e and f), respectively. HeLa cells (a) remained alive after irradiation (c and e); HeLa cells incubated with AuNPs (b) were almost died after irradiation (d and f).

4. Conclusions

In conclusion, AuNPs can be synthesized by worm-like DPG3.5-COOH with well-controlled structure at optimized conditions. DPG3.5-COOH can fulfil the functions of reducing agent and template in this process. The morphology of AuNPs 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 character and strong photothermal therapy capability to HeLa cancer cells, which are potential for photothermal therapy. Further, the unique hyperbranched morphology and multiple peripheral groups on the dendrons can provide additional possibility to do surface modification to introduce multifunctional groups, e.g., cancer cell targeting moiety, to improve cancer therapy efficiency.

Acknowledgements

Financial support from the National Natural Science Foundation of China (51322303) and Foundation of Sichuan Province (2012JQ0009) are gratefully acknowledged.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Including additional TEM images. See DOI: 10.1039/c4ra08475f

‡ These authors contributed equally to this work.

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