- Open Access
Biosynthesis of flower-shaped Au nanoclusters with EGCG and their application for drug delivery
© The Author(s) 2018
- Received: 6 August 2018
- Accepted: 30 October 2018
- Published: 13 November 2018
In the last decade, the biosynthesis of metal nanoparticles using organisms have received more and more considerations. However, the complex composition of organisms adds up to a great barrier for the characterization of biomolecules involved in the synthesis process and their biological mechanisms.
In this research, we biosynthesized a kind of flower-shaped Au nanoclusters (Au NCs) using one definite component—epigallocatechin gallate (EGCG), which was the main biomolecules of green tea polyphenols. Possessing good stability for 6 weeks and a size of 50 nm, the Au NCs might be a successful candidate for drug delivery. Hence, both methotrexate (MTX) and doxorubicin (DOX) were conjugated to the Au NCs through a bridge of cysteine (Cys). The introduction of MTX provided good targeting property for the Au NCs, and the conjugation of DOX provided good synergistic effect. Then, a novel kind of dual-drug loaded, tumor-targeted and highly efficient drug delivery system (Au-Cys-MTX/DOX NCs) for combination therapy was successfully prepared. The TEM of HeLa cells incubated with Au-Cys-MTX/DOX NCs indicated that the Au-Cys-MTX/DOX NCs could indeed enter and kill cancer cells. The Au-Cys-MTX/DOX NCs also possessed good targeting effect to the FA-receptors-overpressed cancer cells both in vitro and in vivo. Importantly, the Au-Cys-MTX/DOX NCs resulted in an excellent anticancer activity in vivo with negligible side effects.
These results suggest that the biosynthesized Au-Cys-MTX/DOX NCs could be a potential carrier with highly efficient anticancer properties for tumor-targeted drug delivery.
- Au nanoclusters
- Drug delivery
- Dual drug
In recent years, metal nanoparticles (NPs) have received considerable research interest due to their potential applications in catalysis [1, 2], electronics [3, 4], chemical sensing , optics , and biology [7, 8]. Among the metal NPs, gold NPs, with the properties of optical effect , heating [10, 11], and biocompatibility [12, 13], are receiving the greatest attention, mainly because of their broader application in biomedical science [14, 15]. Particularly, monolayer-protected Au NPs have recently emerged as an attractive candidate for delivering various therapeutic agents such as drugs, peptides, proteins, and nucleic acids to their targets [16, 17]. Since the monolayer could range from small organic compounds to macromolecules, the function and the property of Au NPs, such as the stability, targeting ability and drug loading capacity, could be largely improved in many ways, which makes Au NPs one of the most promising candidate for cancer treatment [18–20].
Although the property of Au NPs could be optimized by the monolayer, the nature of Au NPs, which mostly depends on the synthetic procedure, is of equal importance to the Au-based drug delivery system . There are various synthetic methods of Au NPs, such as chemical reduction, biological synthesis, lithography, ultraviolet irradiation, and so on [22, 23]. In recent years, the biosynthesis of Au NPs using organisms, as an environmentally friendly method (green chemistry), has become a major focus of researchers [24, 25]. Without use of harsh, and toxic chemicals, the safety of Au NPs would be improved largely, which would increase their potential medical application, such as imaging, drug delivery, disinfection, and tissue repair . Especially, the Au NPs produced by plants have been proved to be more various in shape and size in comparison with those produced by other organisms . Although, the spherical morphology is still in the majority of the plants-synthetic Au NPs, a variety of non-sphere shapes began to appear. For example, triangular NPs have become another general Au NPs, which could be obtained with the reduction of Aloe vera , Camellia sinensis , Cymbopogon flexuosus , Murraya koenigii , pear fruit extract , and so on. In addition, hexagonal , rod-shaped , and meatball-like Au NPs  were also prepared successfully using plants extract. Unlike general chemical reduction, the plants extracts may act both as reducing and capping agents in the preparation process, which would make the Au NPs more stable. Moreover, the faster synthesis rate and lower cost make the plants-biosynthesis an ideal candidate for the large-scale production of Au NPs. Since the plants-synthetic Au NPs possess such a lot of pronounced properties, there must be extensive and promising applications in the future. However, mainly owing to the complexity and variety of the plants extract, the research about the plants-synthesis methods is still very limited, especially in regard to the detection and characterization of biomolecules involved in the synthesis process, which play a parameter role on understanding the biological mechanisms of the biosynthesis.
Tea extract has always been a nice candidate for the biosynthesis of metal NPs, who would possess good stability and uniformity [35–37]. Nevertheless, the biomolecules with the critical function are still not fully understood in the tea-synthesis process. It is commonly accepted that there might be at least two biomolecules participated in the process, which act as the reducing and capping agent, respectively. However, we found that epigallocatechin gallate (EGCG), the most abundant catechin in green tea, could play the role of reducing and capping agent at the same time. Hence, in this work, a kind of flower-shaped Au nanoclusters (Au NCs) with a size of 50 nm were biosynthesized using only one component—EGCG. It is amazing that the flower-shaped Au NCs possessed surprisingly good uniformity and stability, which could be kept for more than 6 weeks without aggregation. Since the NPs of 50 nm were proved to be internalized much more rapidly and efficiently by cancer cells than those of other sizes, we investigated the application of the flower-shaped Au NCs as drug deliver carriers. Both methotrexate (MTX) and doxorubicin (DOX) were conjugated to the Au NCs through a bridge of cysteine. With the targeting property of MTX and the synergistic effect of the both drugs, a novel kind of dual-drug loaded, tumor-targeted and high efficient drug delivery system (Au-Cys-MTX/DOX NCs) for combination therapy was successfully prepared. Then, the drug delivery property and the anticancer efficiency of the Au-Cys-MTX/DOX NCs were systematic evaluated in vitro and in vivo. The results illustrated that the Au-Cys-MTX/DOX NCs also showed excellent stability the same as that of Au NCs, which was one of the most essential properties for pharmaceutical application. With outstanding targeting property and enhanced anticancer efficiency, the Au-Cys-MTX/DOX NCs would become a promising anticancer drug delivery system for combination therapy.
Hydrogen tetra chloroaurate (III) trihydrate (HAuCl4·4H2O, 99.9%) was purchased from Sinopharm Chemical Reagent Co. Ltd. Epigallocatechin gallate (EGCG, purity > 99%) was purchased from Hangzhou Gosun Biotechnologies Co., Ltd. MTX (purity > 99%) was purchased from Bio Basic Inc. Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. DOX (purity > 99%), S-trityl-l-cysteine (TRCYS), trifluoroacetic acid (TFA), triisopropylsilane were purchased from Shanghai Macklin Biochemical Co., Ltd. DMEM/High glucose and fetal bovine serum (FBS) were from Gibco. All chemicals were of analytical grade and used as received without further purification. Ultrapure water (18.2 MΩ/cm) was used throughout the work.
Animals and cell cultures
HeLa cells and HepG2 cells were frozen by medical college of Xiamen University. The complete growth medium was DMEM supplemented with FBS (10%) and penicillin/streptomycin (1%). The cells were cultivated in an incubator (Thermo Scientific) in the presence of 5% CO2 at 37 °C.
The BALB/C nude mice (5–6 weeks, 16–20 g) and BALB/C mice (5–6 weeks, 18–22 g) were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences. The tumor models were set up by injecting 1 × 106 HeLa cells subcutaneously in the selected positions of the mice.
Synthesis of flower-shaped Au nanoclusters (Au NCs)
Au NCs were synthesized by a microwave method. 0.25 mL of chloroauric acid solution (1 wt%) was added to a conical flask, followed by the addition of 23.25 mL of water. Then, 1.5 mL of EGCG (2.67 mg/mL) was introduced fleetly into the aqueous solution with vigorous stirring. The resulting mixture was immediately irradiated in a microwave oven (80 W) for 90 s when the yellowish-green color of Au NCs were appeared.
Synthesis of TRCYS-MTX/DOX conjugations
MTX (45 mg), TEA (30 mg), DCC (10 mg) and NHS (5 mg) were added into DMF (2 mL) and stirred at rt for 1 h. Then TRCys (40 mg) was added into the solution and stirred for 6 h to obtain the TRCys-MTX conjugations. Next, DOX (54 mg) was added to the solution and stirred for another 6 h to obtain the TRCyts-MTX/DOX conjugations. While purifying, the mixture was treated with ultrapure water (6 mL) and acetic acid (0.01 M) until the pH reached 7.0, at which time the TRCys-MTX/DOX would precipitate out. Then the precipitate was washed with methanol for three times and lyophilized for 24 h to obtain the dry TRCys-MTX/DOX powders.
Synthesis of dual-drug loaded Au nanoclusters (Au-Cys-MTX/DOX NCs)
TRCys-MTX/DOX (10 mg) were dissolved in DMF (2 mL). Then, trifluoroacetic acid (250 µL) and triisopropylsilane (50 µL) were added into the solution and stirred at 50 °C for 8 h to free the sulfydryl of Cys. Afterwards, freshly prepared Au NCs (5% wt, 2 mL) were added into the solution and stirred at rt for 4 h. At last, the suspicion was centrifuged at 8000 rpm for 7 min, and the precipitate was lyophilized for 24 h to obtain the dry Au-Cys-MTX/DOX NCs powders.
The structure of TRCys-MTX and TRCys-MTX/DOX were analyzed by H1NMR (AVANCE III 600 MHz). Morphology of the Au-Cys-MTX/DOX NCs and Au NCs was examined by SEM (UV-70) at 10 kV and TEM (Tecnai G2 Spirit) at 20 kV. The energy spectrum analysis of Au-Cys-MTX/DOX NCs and Au NCs was also examined by UV-70. The Size and zeta-potential values were determined by a Malvern Zetasizer Nano-ZS machine (Malvern Instruments, Malvern). Three parallel measurements were carried out to determine the average values. The content of Cys-MTX/DOX in Au-Cys-MTX/DOX NCs was determined by thermogravimetry analysis (SDT_Q 600) and ultraviolet spectrophotometry (Cary5000).
In vitro drug release study
The in vitro drug release studies of Au-Cys-MTX/DOX NCs were performed using the dialysis technique. The Au-Cys-MTX/DOX NCs were dispersed in a PBS buffer solution (10 mL) and placed in a pre-swelled dialysis bag (MWCO = 3500 Da). The dialysis bag was then immersed in PBS (0.1 M, 150 mL, pH 7.4) and oscillated continuously in a shaker incubator (180 rpm) at 37 °C. All samples were assayed by high performance liquid chromatography (HPLC).
In vitro intracellular distribution
The in vitro intracellular distribution of Au-Cys-MTX/DOX NCs were performed using a TEM (Tecnai G2 Spirit). HeLa cells were incubated in a 6-well plate with a density of 1 × 106 cells per well. The cells were incubated at 37 °C and 5% CO2 for 24 h. The Au-Cys-MTX/DOX NCs (4 wt%) were added for 1 h, 2 h, 4 h, and 8 h. After incubation, the cells were washed six times with PBS and fixed with 4% paraformaldehyde for 2 h. And the cells were detached by trypsinization and rinsed with PBS for three times. Then, the samples were send to the school of life sciences of Xiamen University for further process. And the processed samples were examined by TEM (Tecnai G2 Spirit) at 20 kV.
Confocal imaging of cells
MTX was labelled with Cy 5.5 via an amido bond to function as a fluorescent probe. The confocal imaging of cells were performed using a Leica laser scanning confocal microscope. Imaging of MTX-Cy 5.5 was carried out under the 673 nm laser excitation, and the emission was collected in the range of 700–750 nm. Imaging of DOX was carried out under the 480 nm laser excitation, and the emission was collected in the range of 550–600.
HeLa cells were incubated in 6-well plates with a density of 1 × 106 cells per well. The cells were incubated with 5% CO2 at 37 °C for 24 h. The Au-Cys-MTX (-Cy 5.5)/DOX NCs, Au-Cys-MTX (-Cy 5.5) NCs, or Au-Cys-DOX NCs ([MTX] = 0.2 µmol/mL, [DOX] = 0.24 µmol/mL) were added to the cells for 4 h. After incubation, the cells were washed three times with PBS, fixed with 4% paraformaldehyde and stained with DAPI for 5 min. Subsequently, the cells were further washed thrice with PBS before confocal imaging.
Au-Cys-MTX (-Cy 5.5) NCs and Au-Cys-DOX NCs were synthesized via the same method with that of Au-Cys-MTX (-Cy 5.5)/DOX NCs without the use of DOX or MTX (-Cy 5.5).
The cytotoxicity of various particles mentioned before was determined by the WST-1 assay. Briefly, HeLa cells of exponential phase was plated in quintuplicate in a 96-well flat bottomed microplate at a density of 5000 cells per well and incubated for 24 h. Then, Au-Cys-MTX/DOX NCs, Au-Cys-MTX NCs, or Au-Cys-DOX NCs ([MTX] = 0.087, 0.154, 0.308, 0.616, 1.232, 2.464 µg/mL, [DOX] = 0.125, 0.25, 0.5, 1.0, 2.0, 4.0 µg/mL) were added to plate. After 24 h incubation, 10 µL per well WST-1 was added and kept for another 2 h, and the intensity of developed color was measured by micro plate reader (Dynastic MR 5000, USA) operating at 450 nm wavelength. Untreated cells were considered 100% viable.
In this analysis, synergy is defined when CI < 1. And the smaller the CI is, the stronger the synergy is.
For in vivo fluorescence imaging, Cy 5.5 was conjugated to the formations. MTX-Cy5.5, DOX-Cy5.5, Au-Cys-Cy 5.5 NCs, and Au-Cys-MTX/DOX-Cy5.5 NCs ([MTX] = 20 nmol/kg, [DOX] = 24 nmol/kg) were administered intravenously into the HeLa tumor-bearing nude mice via the tail veins. At predetermined time intervals, the mice were anesthetized with isoflurane (2.5%) and imaged with the Maestro in vivo imaging system (Cambridge Research & Instrumentation, Woburn, MA, USA). After 24 h, the mice were sacrificed, and the tumor and major organs (spleen, liver, kidney, lung, and heart) were excised, followed by washing the surface with 0.9% NaCl for the ex vivo imaging of Cy 5.5 fluorescence using a Maestro in vivo imaging system.
Tumor inhibition in vivo
When the tumor volume of the HeLa tumor-bearing mice was approximately 60 mm3, the mice were randomly divided into 4 groups (10 mice per group), and treated by intravenous injection of 0.9% NaCl, the bulk MTX and DOX mixture, the mixture of Au-Cys-MTX NCs and Au-Cys-DOX NCs, and Au-Cys-MTX/DOX NCs ([MTX] = 10 nmol/kg, [DOX] = 12 nmol/kg) every 3 days for four times. The tumor volume and body weight were monitored every 3 days. The tumor volume was calculated by the following formula: tumor volume = 0.5 × length × width2. The highest and lowest data were discarded at last.
After 18 days, the mice were sacrificed and the tumors were excised and weighed. Next, the tumors were fixed in 4% paraformaldehyde overnight at 4 °C, embedded in paraffin, sectioned (4 μm), stained with hematoxylin and eosin (H&E), and examined using a digital microscopy system.
The statistical significance of treatment outcomes was assessed using Student’s t-test (two-tailed); P < 0.05 was considered statistically significant in all analyses (95% confidence level).
Synthesis of flower-shaped Au nanoclusters (Au NCs)
Since the nanoparticles could greatly improve the suboptimal pharmaceutical characters of the chemotherapeutics, the nanoparticle drug delivery system have received more and more attentions. And the study on cellular uptake of the particles have received great progress, although the influences are still not fully understood. According to previous works [16, 40–43], the cellular uptake shows a clear trend: the uptake of particles increases with the particle size of less than 50 nm, and decreases once the particles become larger. With the size of 50 nm, the Au NCs might show a highly enhanced effect in cancer cellular uptake. On account of this, we planned to conjugate dual anticancer drugs with synergistic effect to Au NCs, which might optimize the efficiency of drug delivery, and eventually benefit the cancer therapy.
Synthesis of dual-drug loaded Au nanoclusters (Au-Cys-MTX/DOX NCs)
In Au-NPs-based drug delivery system, a sulfydryl-containing linker is always used to functionalize the Au NPs and then loaded with the model drug via acylation reactions [44–46]. However, the stabilizing EGCG on the surface of Au NCs might be destroyed by the acylation reaction, which would influence the stability of Au NCs. Hence, in this work, the dual-drug loaded, sulfydryl-containing group was firstly synthesized and then conjugated to Au NCs. As shown in Additional file 1: Formula S1, the dual-drug loaded Au NCs were synthesized in four steps: (1) TrCys-MTX synthesis: conjugation of MTX to TrCys via formation of an amido bond; (2) TrCys-MTX/DOX synthesis: conjugation of DOX to the TrCys-MTX via formation of another amido bond; (3) Cys-MTX/DOX synthesis: deprotection of the triphenylmethyl group; (4) Dual-drug loaded Au NCs (Au-Cys-MTX/DOX NCs): modification of Au NCs with Cys-MTX/DOX via Au–S bonds (the chemical equations are shown in Additional file 1: Formula S1). The H-nuclear magnetic resonance (H1NMR) was used to monitor the reaction process.
The formations of TrCys-MTX and TrCys-MTX/DOX were confirmed by the H1NMR spectrum, as shown in Additional file 1: Figure S4. While many peaks (8.58 and 4.79 ppm) corresponded with the group of MTX in the H1NMR spectrum of TrCys-MTX, the peak from 7.19 to 7.32 ppm was ascribed to the protons of triphenylmethyl groups (Additional file 1: Figure S4A). The result confirmed the formation of TrCys-MTX. In the H1NMR spectrum of TrCys-MTX/DOX (Additional file 1: Figure S4B), in addition to the characteristic peaks of TrCys-MTX, the peak at 3.90 ppm was observed due to the methoxyl protons of DOX. These results clearly indicated the formation of TrCys-MTX/DOX. Since the peaks at 4.79 ppm and 3.90 ppm ascribed to MTX and DOX, respectively, they were integrated to calculate the ratio of the dual drugs. The ratio of MTX to DOX was 1:1.2.
Since the H1NMR of Au-Cys-MTX/DOX NCs was very similar to that of Cys-MTX/DOX, the structure of Au-Cys-MTX/DOX NCs could not be confirmed by H1NMR. Hence, the energy dispersive spectrum (EDS) was employed to investigate the elementary compositions of Au NCs and Au-Cys-MTX/DOX NCs. Compared with the EDS of Au NCs, the appearance of elemental S and N in the EDS of Au-Cys-MTX/DOX NCs corroborated the existence of -Cys-MTX/DOX (Fig. 1I, J, Additional file 1: Tables S1, S2). This confirmed that the Au-Cys-MTX/DOX NCs was successfully synthesized.
To observe the morphology changes of Au NCs after modification, SEM and TEM were employed once again, and the results are shown in Fig. 1. The SEM and TEM images show that the Au-Cys-MTX/DOX NCs maintained the flower shape (Fig. 1C, D, G, H). However, when compared with Au NCs, there was an obvious change that a fine coating could be observed clearly on the surface of the Au-Cys-MTX/DOX NCs, which could be attributed to the conjugation of Cys-MTX/DOX on Au NCs. This also confirmed that the decoration of Au NCs was successful, which was in according with the result of EDS. Another evidence of the decoration was the slightly increased particle size and the decreased zeta potential (Additional file 1: Figures S2, S3).
Then the drug loading capacity of Au-Cys-MTX/DOX NCs was evaluated via thermogravimetric analysis (TGA) (Additional file 1: Figure S5). The conjugated drug loading ranged from 8.2 to 20.9 wt%, which were obtained by changing the ratio of Au NCs to Cys-MTX/DOX (Additional file 1: Table S3). Since the UV-absorption of Au NCs was much weaker than that of Cys-MTX/DOX, the UV-absorption course of Au-Cys-MTX/DOX NCs was similar to that of Cys-MTX/DOX. Meanwhile, the drug loading of Au-Cys-MTX/DOX NCs was verified via UV spectrophotometry at 290 nm (Additional file 1: Figure S6), and results were in according with those via TGA (Additional file 1: Table S3). Interestingly, the drug loading was proved to play a paramount role on the stability of Au-Cys-MTX/DOX NCs (Additional file 1: Table S3). While the Au NCs kept good stability for more than 6 weeks, the Au-Cys-MTX/DOX NCs with a drug loading of 8.2 wt% could only stabilize for less than 5 weeks. When the drug loading increased to 20.9 wt%, the Au-Cys-MTX/DOX NCs would aggregate in 3 days. And the decreased Zeta potential also predicted their increasingly weak stability (Additional file 1: Table S3). In consideration of the drug loading and the stability, the Au-Cys-MTX/DOX NCs with a drug loading of 13.4 wt% were chosen for the following evaluation.
In vitro drug release study
The amido bond in the Au-Cys-MTX/DOX NCs allows the sustained release of DOX and MTX. The in vitro release studies of the Au-Cys-MTX/DOX NCs (13.4% content) were performed using a dialysis technique, alongside with bulk DOX or MTX powders. All samples were assayed by high performance liquid chromatography (HPLC). The release profiles are shown in Additional file 1: Figure S7. The free DOX was almost completely released within 8 h, while the bulk MTX need 18 h. This was owing to the different solubility of the dual drugs. Without the existence of protease, only small amount of drugs could be released from the Au-Cys-MTX/DOX NCs, which could be ascribed to the firmness of amido bond. Hence, the released drug would increase with the addition of protease, which could break amido bond. Compared with the bulk drug, the release of MTX from Au-Cys-MTX/DOX NCs exhibited a sustained and prolonged profile over a period of 48 h, just the same as that of DOX. The result also suggested the same form of both drugs in Au-Cys-MTX/DOX NCs.
In vitro intracellular distribution
Confocal imaging of cells
The CI value of different formulations
MTX and DOX
0.48 ± 0.06
0.81 ± 0.11
Au-Cys-MTX NCs and Au-Cys-DOX NCs
0.34 ± 0.05
0.50 ± 0.07
0.17 ± 0.07
0.35 ± 0.06
Tumor inhibition in vivo
In summary, the current study presents an environmentally friendly method using EGCG to obtain a flower-shaped Au nanoclusters with pronounced stability. Then as a carrier, the Au NCs were loaded with MTX and DOX for cancer therapy. The sustained and prolonged drug release property of the dual drug loaded Au NCs increases the acting time of the drug in vivo. The targeting property of MTX and the efficient-cellular-uptake size allow for enhanced targeting property to cancer cells. Plus the good synergistic effect of both drugs, the Au-Cys-MTX/DOX NCs possess excellent anticancer effect both in vivo and in vitro. The current study highlights the feasibility of biosynthesized Au nanoparticles for drug delivery. Particularly interestingly, biosynthesis method with one component opens a door for eco-friendly technique for production of well-characterized nanoparticles and provide a foundation for understanding their biological mechanisms.
SW conceived and carried out experiments, analysed data and wrote the paper. XY, TW and JY designed the study, supervised the project, analysed data. MZ, XY, and PX assisted in the synthesis and characterizations of the NCs. MZ, XY, and FL assisted in the biological evaluations of the NPs. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Ethics approval and consent to participate
All procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethical and Welfare Committee of Xiamen University.
This project was supported by the China Postdoctoral Science Foundation (Nos. 2018M632584 and 2017M622080), the Fundamental Research Funds for the Central Universities (No. 20720180042), and the National Natural Science Foundation of China (Nos. 81773770 and 81472458).
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