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Stability and cytocompatibility of silk fibroin-capped gold nanoparticles
Materials Science and Engineering C 43 (2014) 231–236
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Stability and cytocompatibility of silk fibroin-capped gold nanoparticles Lan Jia ⁎, Li Guo, Jingxin Zhu, Yanlong Ma Key Laboratory of Interface Science and Engineering in Advanced Materials, Ministry of Education, College of Material Science and Engineering, Taiyuan University of Technology, Taiyuan 030024, China
a r t i c l e
i n f o
Article history: Received 27 March 2014 Received in revised form 23 May 2014 Accepted 3 July 2014 Available online 11 July 2014 Keywords: Silk fibroin Gold nanoparticles Stability Cytocompatibility
a b s t r a c t Surface engineering is crucial in the colloidal stability and biocompatibility of nanoparticles (NPs). Protein silk fibroin (SF), which gained interest in biomaterial and regenerative medicine, was used in this study to stabilize gold (Au) NPs. Characterization results from UV–Vis spectroscopy revealed that SF-capped Au NPs (SF-Au NPs) possessed remarkable colloidal stabilities in the pH range of 2 to 11 and salt concentration range of 50 mM to 1000 mM. In addition, dried particle samples were resuspended after lyophilization without aggregation. The results indicated that the steric hindrance rather than the electrostatic repulsion of SF-Au NPs was essential for colloidal stability. The SF-Au NPs manifested improved cytocompatibility compared with bare Au NPs, which was attributed to the inherent non-cytotoxicity of SF and the good colloidal stability of the NPs. The proposed method was simpler, more efficient, and more cost effective than the conventional modification strategies for Au NPs; thus, SF-Au NPs can be potentially used in biomedical applications. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Gold nanoparticles (Au NPs) have gained considerable interest for potential applications in biological and medical fields, such as biosensors, drug delivery, and diagnostic assays, because of their unique physical and chemical properties [1–3]. The simplest and most commonly used preparation route for Au NPs is the aqueous reduction of Au salt by sodium citrate under reflux [4]. However, the prepared Au NPs are susceptible to aggregation at high ionic strengths or acidic conditions because of the changes in particle surface charge [5]. Although the aggregation of Au NPs is beneficial for certain events of bimolecular recognition, the particles should be resistant to aggregation caused by variation in pH or environments with high ionic strengths. The toxicity of Au NPs from a biological perspective is likewise critical [6]. Most biomedical applications recommend water-soluble Au NPs that exhibit stability and biocompatibility [7]. Modifying the surface of NPs with specific ligands is an effective approach for protecting against unselective particular aggregation. Previous studies have evaluated various surface ligands, such as surfactants [8], thiol compounds [7,9–11], dendrimers [12–14], polymers [15,16], and zwitterionic materials [17–19]. However, most of these ligands suffer from poor biocompatibility, requirement of multiple synthetic steps, or expensive reagents. Silk fibroin (SF) is secreted from silk glands of the silkworm Bombyx mori. SF proteins belong to a class of unique, block copolymerlike proteins with high molecular weights [20]. Raw and regenerated SF has been extensively used in biomaterials and regenerative medicine ⁎ Corresponding author. Tel.: +86 351 6010021. E-mail address: [email protected] (L. Jia).
http://dx.doi.org/10.1016/j.msec.2014.07.024 0928-4931/© 2014 Elsevier B.V. All rights reserved.
because of its biocompatibility and nontoxicity [21–23]. The incorporation of SF proteins with colloidal Au NPs might have potential bioactivities for biotechnological applications. In the present study, we used SF proteins to stabilize Au NPs (Scheme 1). The colloidal stabilities of SF-capped Au NPs (SF-Au NPs) were tested under different pH values and salt concentrations by UV– Vis spectroscopy. The resuspension property of dried samples after freeze-drying was also evaluated. The cytotoxicity of SF-Au NPs was analyzed by standard MTT assay. Our method was simpler and more cost effective compared with the current methods for the modification and conjugation of Au NPs; thus, our method was promising for various biomedical applications. 2. Experimental 2.1. Materials HAuCl4·3H2O was purchased from Beijing Chemical Co. (Beijing, China). Bovine serum albumin (BSA) was bought from Shanghai Sangon (Shanghai, China). Simulated body fluid (SBF; 1.5-fold) was prepared as previously described [24]; pH was adjusted to 7.4 with HCl. All other reagents were of analytical grade and used as received. All aqueous solutions were prepared with triple distilled water. 2.2. Preparation of SF-Au NPs Scheme 1 illustrates the preparation of aqueous SF solutions based on a previous report [25]. The SF solution was dialyzed against distilled water using Slide-a-Lyzer dialysis cassettes (MWCO 3500, Pierce) for 3 d (triple-distilled water for the last day) until the
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conductivity of the dialyzed water was close to that of distilled water. The molecular weight of the as-prepared SF was measured to be approximately 25 kDa to 86 kDa via SDS-PAGE (Fig. S1). The absorbance peak at 275 nm (A275) did not significantly change within pH 3 to 9 (Fig. S2). A275 gradually increased with the weight concentration of SF; a good linearity was observed between A275 and SF concentration below 2 mg·mL−1 (Fig. S3). Thus, the weight concentration of SF can be obtained from A275. Au NPs were synthesized by citrate reduction of HAuCl4 as previously described [26]. The diameter of the NPs based on TEM images was 13 ± 2 nm. The Au NP concentration (ca. 15 nM) was determined from Beer's law using an extinction coefficient of ca. 108 M−1·cm−1 at 520 nm [27]. SF-Au NPs were obtained by mixing 300 μL of as-prepared Au NP solution with SF solution at ambient temperature, followed by incubation for 10 min, the final volume was 1 mL and the pH of the mixture was about 7. The final concentrations of SF and Au NPs were 2 mg·mL− 1 and 4.5 nM, respectively. 2.3. Colloidal stabilities of SF-Au NPs to pH and salts To determine the stability of the Au NPs with respect to pH, the solutions of SF-Au NPs and Au NPs were added to 20 mM tris(hydroxymethyl)aminomethane–2-(N-morpholino)ethanesulfonic acid (Tris–MES) or 20 mM phosphate buffer (PB) at pH 2 to 11 (volume ratio, 1:1). The stability of the Au NPs with respect to various salt concentrations was determined by adding 20 mM PB solution at pH 7.4 to the solutions of SF-Au NPs and Au NPs; the PB contained twofold of the desired salt concentrations (volume ratio, 1:1). The stability of the Au NPs in the physical environment was probed by adding 1.5-fold SBF solutions (volume ratio, 1:2) to the solutions of SF-Au NPs and Au NPs. The final concentrations of SF and Au NPs in all the solutions were 2 mg·mL−1 and 4.5 nM, respectively. The stabilities of the SF-Au NPs were investigated via UV–Vis spectroscopy.
the cells were further cultured at 37 °C for 4 h. Dark blue formazan crystals generated by mitochondria dehydrogenase in live cells were dissolved in 150 mL of dimethyl sulfoxide (DMSO); the absorbance at 570 nm was measured by a microplate reader (MODEL 550, Bio Rad, USA). The relative cell viability is given as
Relative cell viability ð%Þ ¼ absorption of treated well=absorption of control well 100:
ð1Þ
Five replicates were obtained for each sample, and the mean value was used as the final result. All data were presented as mean ± SD. Statistical analysis of data was made by ANOVA or Student's t-test. It was considered significantly different if p b 0.05. The working solution was freshly prepared by adding 5 μL of fluorescein diacetate (FDA, 5 mg·mL−1 acetone) stock solution to 5 mL of PBS. Up to 20 μL of FDA working solution was added to each well and then incubated for 15 min. The wells were washed twice with PBS; confocal laser scanning microscopy (CLSM, BIORAD 2000) was carried out to observe the cell morphology. 2.5. Characterization UV–Vis spectra were obtained with a UV/Vis Shimadzu UV-2505 spectrometer. TEM was performed on a JEM-1200EX (JEOL, Japan). Samples for TEM measurements were prepared by placing a drop of colloidal dispersion on a carbon-coated copper grid, followed by evaporation of the solvent. Samples were freeze-dried completely before being grinded into powder and pressed into pellets with KBr for measuring infrared spectra with a Fourier transform infrared spectrophotometer (FTIR) (Bruker VERTEX80 V, Germany). 3. Results and discussion
2.4. Cytotoxicity assay and cell morphology observation 3.1. Characterization of citrate-capped Au NPs (cit-Au NPs) and SF-Au NPs Human umbilical vein endothelial cells (HUVECs) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U·mL−1 penicillin, and 100 mg·mL−1 streptomycin; the HUVECs were then cultured at 37 °C in 5% CO2 humidified environment. Cytotoxicity was assessed by standard MTT assay. The HUVECs were plated at a density of 5 × 103 cells/well in a 96-well plate and then cultured for 24 h. The medium was replaced with fresh medium containing different concentrations of the cit-Au NPs and SF-Au NPs; the Au atomic concentration was determined by inductively coupled plasma mass spectrometry (ICP-MS). The cells cultured in NP-free media were used as controls. The wells were washed with PBS and the medium was replaced with 100 mL of fresh medium after 24 h of treatment. Up to 20 mL of MTT (5 mg·mL−1) was added to each well;
A characteristic absorption peak at 520 nm was observed in the UV– Vis spectra of cit-Au NPs (diameter, ~ 13 nm). As shown in Fig. 1a, the aqueous solution of SF-Au NPs displayed another strong absorption band at approximately 275 nm after the addition of 2 mg·mL−1 SF to the solution of cit-Au NPs. This band was assigned to the π → π* electron transition of the tyrosine (Tyr) residue in the SF molecular chain. A 5 nm red shift was observed from 520 nm to 525 nm, and the peak absorbance of the Au NPs increased, indicating the adsorption of SF onto the surface of the Au NPs [28]. No statistical differences in the average particle diameter or size distribution of the Au NPs were detected from the TEM images (Figs. 1b and c). The SF-Au NPs remained well separated, which implied the absence of aggregation.
Scheme 1. Preparation and stability tests of SF capped Au NPs.
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Fig. 1. (a) UV–Vis spectra and TEM images of (b) Au NPs and (c) SF-Au NPs.
3.2. Stabilities of SF-Au NPs The stability of NPs is necessary under environmental conditions, such as high salt concentrations and extreme pH and buffer solutions, if the particles are used in biological and medical applications. pH [29] and metallic ions [30] influence the rheology of SF proteins. The stability of Au NPs can be monitored by UV–Vis spectroscopy because aggregation, precipitation, and decomposition lead to distinctive changes in the spectra. The effects of pH on the stability of the SF-Au NPs were initially investigated. MES–Tris buffer was used to minimize the probable effect of salt ions in accelerating the aggregation of SF. Fig. 2 shows the UV–Vis absorption spectra of the Au NPs before and after SF modification under different pH conditions. The spectra of SF-Au NPs exhibited no discernible changes in the intensity or position of the absorbance peak at 525 nm in the pH range of 2 to 11 (Fig. 2a). By contrast, the corresponding peak of the spectra of cit-Au NPs red shifted with decreasing acidity after 6 h (Fig. 2b); this shift was accompanied with a color change of the solution from red to blue. This result was in accordance with previous results on the precipitation of Au NPs obtained by borohydride or citrate reduction routes even upon the slightest change in pH [31]. Similar phenomena were observed in PB (Fig. S4). The cit-Au NPs aggregated under acidic conditions, whereas the SF-Au NPs remained stable in the entire pH region. The color changed within 10 min in PB, whereas the time was extended to approximately 6 h in Tris–MES buffer. This result may be attributed to the effect of salt ions. To analyze the mechanism of stabilization, BSA was selected as the control protein because of its similar isoelectric point (PI) with SF, cost efficiency, and availability. As shown in Fig. S5, the BSA-capped Au
NPs (BSA-Au NPs) were stable at basic conditions. The BSA-Au NPs agglomerated when the solution pH was below the PI of BSA (~ 5). This result may be attributed to the neutralization of the negative charge and the subsequent decrease in electrostatic repulsion between the particles. Changes in molecular conformation of SF are usually characterized by amide bands in FT-IR spectra. To clarify the interaction between Au NPs and silk fibroin, the FT-IR spectra of SF-Au NPs at pH 4 and 7 were studied, and the results are shown in Fig. S6. The conformations of SF could be random coil or β-sheet structure. As shown in Fig. S6, FTIR spectra of amide bands at around 1650, 1540 and 1235 cm− 1 are typical feature of random coil [32]. The peak positions and shapes of the amide bands of SF-Au NPs at pH 4 are almost the same as those of SF-Au NPs at pH 7, which indicate that the conformation of SF would not turn to β-sheet structure from random coil with the pH increase. It is reported that morphological transition of silk fibroin (0.5 wt.%) occurred due to the formation of β-sheet by reducing the pH from 6.8 to 4.8 [29]. However, the concentration of the silk fibroin (0.2 wt.%) may be too low to form β-sheet in our system. The stability of NPs in the solution was mainly dependent on the surface properties of NPs (e.g., surface charge and ligand structure). The increase in the electrostatic repulsion and/or steric hindrance of the NPs surfaces can significantly improve the stability of NPs [10]. In this case, the surface charges of the cit-Au NPs and BSA-Au NPs considerably decreased with pH; this decrease resulted in NP aggregation because of the reduction in NP electrostatic repulsion. Compared with BSA and citrate, SF is a block copolymer-like protein with a random coil conformation in our system. The surface of the SF-Au NPs was coated with random coil protein chains, which increased the steric
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Fig. 2. UV–Vis spectra of (a) SF-Au NPs and (b) cit-Au NPs in Tris–MES buffer in the pH range of 2 to 11 after 6 h.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
hindrance of the particles. Therefore, SF can stabilize the Au NPs under a wide pH range even below the PI (ca. 4.58 to 5.00) [33]. The results indicated that electrostatic repulsion may be the primary mechanism for the stabilization of cit-Au and BSA-Au NPs and that the steric effect of SF may be crucial in SF-Au NPs. We analyzed the effects of salt concentrations on the stability of the SF-Au NPs. The final concentration of NaCl ranged from 50 mM to 1000 mM. As shown in Fig. 3, no significant changes in absorption features were observed for the SF-Au NPs when the concentration of NaCl reached 1000 mM, which was much higher than that of physiological saline solution (~ 154 mM). By contrast, the native cit-Au NPs were sensitive to ionic strength and quickly aggregated upon the addition of NaCl. The red shift and peak broadening in the UV–Vis absorption spectra showed that the cit-Au NPs aggregated at a low NaCl concentration of 100 mM. Metallic ions (e.g., K+, Ca2+, and Mg2+) influence the rheology of SF protein [30]. Therefore, their effects on the stability of Au NPs were also investigated. The final concentrations of Ca2+ and Mg2+ ranged from 12.5 mM to 125 mM to yield an ionic strength equal to that of Na+. All metal ions exhibited the same trends (Figs. S7 and S8). The cit-Au NPs aggregated when the concentrations of KCl and CaCl2 were only 50 and 12.5 mM, respectively; meanwhile, the SF-Au NPs remained stable at high ionic strengths. The spectra of the Au NPs with MgCl2 were almost the same as those of the particles with CaCl2. The high stability of the SF-Au NPs might be attributed to the fact that the SF adsorbed on the Au NPs prevented the particles from aggregating via steric repulsion.
Fig. 3. UV–Vis spectra of (a) SF-Au NPs and (b) cit-Au NPs in Tris–MES buffer with NaCl concentrations ranging from 50 mM to 1000 mM after 10 min.
Simulated body fluid (SBF) and the human plasma have very similar ionic compositions. The SF-Au NPs were incubated in SBF solution to determine their stability in a physiological solution. As shown in Fig. 4, the cit-Au NPs aggregated in SBF, whereas the SF-Au NPs remained stable after 48 h. The result implied that the SF-Au NPs were stable in complex biological systems. Therefore, the steric hindrance rather than the electrostatic repulsion of the SF-Au NPs may be essential for colloidal stability.
Fig. 4. UV–Vis spectra of SF-Au NPs and cit-Au NPs in SBF solution.
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Fig. 5. UV–Vis spectra of (a) SF-Au NPs, (b) BSA-Au NPs, and (c) cit-Au NPs in aqueous solution before and after freeze-drying.
Observation of the dissolution property of dried Au NP samples showed that the SF chains adsorbed on the Au NPs surface improved the colloidal stability of the Au NPs. As shown in Fig. 5, the resultant suspension exhibited nearly identical absorption spectra as the SF-Au NPs prior to freeze-drying. By contrast, the absorbance markedly decreased for the BSA-Au NPs after freeze-drying because of the significant aggregation of the Au NPs during the process (Fig. 5b). Significant precipitation was observed when dried cit-Au NPs were dissolved in water. Bare Au NPs were unable to form their suspensions because of intensive aggregation during freeze-drying (Fig. 5c). This feature was beneficial for the storage of SF-Au NPs and for potential applications in tissue engineering; many scaffolds were prepared via lyophilization.
The low cytotoxicity of the SF-Au NPs was attributed to two aspects. One is the important function of the inherent biocompatibility of SF; the other is the function of good colloidal stability. Small Au NPs can be endocytosed by cells and form internal aggregates, which result in cytotoxicity [6]. The increased cytotoxicity of the Au NPs was caused by aggregation; therefore, preventing the aggregation of the particles in the internal environment of the cells can greatly decrease the toxicity of the NPs. To confirm that the SF-Au NPs remained stable in the cell culture medium, we also tested the stability of the SF-Au NPs after incubation with PBS, RPMI-1640 medium with 10% and 100% FBS. The digital images (Fig. S10) implied the absence of aggregation after 48 h of incubation. Hence, the colloidal stability of the SF-Au NPs might have decreased the toxicity of the particles.
3.3. Cytotoxicity of SF-Au NPs
4. Conclusion
Low cytotoxicity is a prerequisite for NPs; the preliminary cytotoxicity of the SF-Au NPs and cit-Au NPs were evaluated by MTT assay in a HUVEC cell line. As shown in Fig. 6, both SF-Au NPs and cit-Au NPs exhibited negligible cytotoxicity when the concentrations were below 4.2 mg·mL− 1. A decrease in cell viability was observed upon treatment with the unmodified Au NPs at concentrations higher than 4.2 mg·mL− 1. The viabilities of HUVEC cells after 24 h of incubation with 8.4 mg·mL−1, 16.8 mg·mL−1 and 33.6 mg·mL−1 of cit-Au NPs were 89.5% ± 1.7%, 82.1% ± 1.1% and 70.2% ± 1.3% respectively. Several groups have also found cytotoxic effects to emerge in a dose-dependent manner for cit-Au NPs [6,34]. The viability of HUVECs with 33.6 mg·mL−1 Au was 93.0 ± 1.6%, indicating no marked cytotoxicity of SF-Au NPs in all tested concentrations. The fluorescence microscopic images of HUVECs after 24 h of treatment are shown in Fig. S9. The cells exposed to the SF-Au NPs were well distributed and showed similar concentrations in all tested conditions.
We presented a novel method for preparing highly stable and cytocompatible Au NPs. The remarkable colloidal stability of the Au NPs under different pH values and salt concentrations was attributed to the natural SF. The results indicated that the steric effect was crucial to the excellent stability of the SF-Au NPs. The particles can be readily dissolved in water after freeze-drying without aggregation, which is beneficial for storage. The SF-Au NPs showed excellent cytocompatibility because of the absence of cytotoxity in SF and the good colloidal stability of the particles. The use of natural SF as a stabilizer in our method was simpler, more efficient, and more cost-effective compared with the conventional modification strategies for Au NPs. This study opened possibilities for using SF-Au NPs in biomaterial applications, such as scaffolds for 3D cell culture and tissue engineering. Acknowledgments We gratefully acknowledge the financial support from the Nature Science Foundation of China (Grant No. 51303124), Natural Science Foundation of Shanxi (Grant Nos. 2013021009-2 and 2013011012-2), and the Qualified Personnel Foundation of Taiyuan University of Technology (Grant No. tyutrc-201155a). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.07.024. References
Fig. 6. Cytotoxicity of HUVEC cells evaluated by MTT assay after incubation with SF-Au NPs and different Au concentrations for 24 h. *p b 0.05 compared with the control.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Stability and cytocompatibility of silk fibroin-capped gold nanoparticles Lan Jia ⁎, Li Guo, Jingxin Zhu, Yanlong Ma Key Laboratory of Interface Science and Engineering in Advanced Materials, Ministry of Education, College of Material Science and Engineering, Taiyuan University of Technology, Taiyuan 030024, China
a r t i c l e
i n f o
Article history: Received 27 March 2014 Received in revised form 23 May 2014 Accepted 3 July 2014 Available online 11 July 2014 Keywords: Silk fibroin Gold nanoparticles Stability Cytocompatibility
a b s t r a c t Surface engineering is crucial in the colloidal stability and biocompatibility of nanoparticles (NPs). Protein silk fibroin (SF), which gained interest in biomaterial and regenerative medicine, was used in this study to stabilize gold (Au) NPs. Characterization results from UV–Vis spectroscopy revealed that SF-capped Au NPs (SF-Au NPs) possessed remarkable colloidal stabilities in the pH range of 2 to 11 and salt concentration range of 50 mM to 1000 mM. In addition, dried particle samples were resuspended after lyophilization without aggregation. The results indicated that the steric hindrance rather than the electrostatic repulsion of SF-Au NPs was essential for colloidal stability. The SF-Au NPs manifested improved cytocompatibility compared with bare Au NPs, which was attributed to the inherent non-cytotoxicity of SF and the good colloidal stability of the NPs. The proposed method was simpler, more efficient, and more cost effective than the conventional modification strategies for Au NPs; thus, SF-Au NPs can be potentially used in biomedical applications. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Gold nanoparticles (Au NPs) have gained considerable interest for potential applications in biological and medical fields, such as biosensors, drug delivery, and diagnostic assays, because of their unique physical and chemical properties [1–3]. The simplest and most commonly used preparation route for Au NPs is the aqueous reduction of Au salt by sodium citrate under reflux [4]. However, the prepared Au NPs are susceptible to aggregation at high ionic strengths or acidic conditions because of the changes in particle surface charge [5]. Although the aggregation of Au NPs is beneficial for certain events of bimolecular recognition, the particles should be resistant to aggregation caused by variation in pH or environments with high ionic strengths. The toxicity of Au NPs from a biological perspective is likewise critical [6]. Most biomedical applications recommend water-soluble Au NPs that exhibit stability and biocompatibility [7]. Modifying the surface of NPs with specific ligands is an effective approach for protecting against unselective particular aggregation. Previous studies have evaluated various surface ligands, such as surfactants [8], thiol compounds [7,9–11], dendrimers [12–14], polymers [15,16], and zwitterionic materials [17–19]. However, most of these ligands suffer from poor biocompatibility, requirement of multiple synthetic steps, or expensive reagents. Silk fibroin (SF) is secreted from silk glands of the silkworm Bombyx mori. SF proteins belong to a class of unique, block copolymerlike proteins with high molecular weights [20]. Raw and regenerated SF has been extensively used in biomaterials and regenerative medicine ⁎ Corresponding author. Tel.: +86 351 6010021. E-mail address: [email protected] (L. Jia).
http://dx.doi.org/10.1016/j.msec.2014.07.024 0928-4931/© 2014 Elsevier B.V. All rights reserved.
because of its biocompatibility and nontoxicity [21–23]. The incorporation of SF proteins with colloidal Au NPs might have potential bioactivities for biotechnological applications. In the present study, we used SF proteins to stabilize Au NPs (Scheme 1). The colloidal stabilities of SF-capped Au NPs (SF-Au NPs) were tested under different pH values and salt concentrations by UV– Vis spectroscopy. The resuspension property of dried samples after freeze-drying was also evaluated. The cytotoxicity of SF-Au NPs was analyzed by standard MTT assay. Our method was simpler and more cost effective compared with the current methods for the modification and conjugation of Au NPs; thus, our method was promising for various biomedical applications. 2. Experimental 2.1. Materials HAuCl4·3H2O was purchased from Beijing Chemical Co. (Beijing, China). Bovine serum albumin (BSA) was bought from Shanghai Sangon (Shanghai, China). Simulated body fluid (SBF; 1.5-fold) was prepared as previously described [24]; pH was adjusted to 7.4 with HCl. All other reagents were of analytical grade and used as received. All aqueous solutions were prepared with triple distilled water. 2.2. Preparation of SF-Au NPs Scheme 1 illustrates the preparation of aqueous SF solutions based on a previous report [25]. The SF solution was dialyzed against distilled water using Slide-a-Lyzer dialysis cassettes (MWCO 3500, Pierce) for 3 d (triple-distilled water for the last day) until the
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conductivity of the dialyzed water was close to that of distilled water. The molecular weight of the as-prepared SF was measured to be approximately 25 kDa to 86 kDa via SDS-PAGE (Fig. S1). The absorbance peak at 275 nm (A275) did not significantly change within pH 3 to 9 (Fig. S2). A275 gradually increased with the weight concentration of SF; a good linearity was observed between A275 and SF concentration below 2 mg·mL−1 (Fig. S3). Thus, the weight concentration of SF can be obtained from A275. Au NPs were synthesized by citrate reduction of HAuCl4 as previously described [26]. The diameter of the NPs based on TEM images was 13 ± 2 nm. The Au NP concentration (ca. 15 nM) was determined from Beer's law using an extinction coefficient of ca. 108 M−1·cm−1 at 520 nm [27]. SF-Au NPs were obtained by mixing 300 μL of as-prepared Au NP solution with SF solution at ambient temperature, followed by incubation for 10 min, the final volume was 1 mL and the pH of the mixture was about 7. The final concentrations of SF and Au NPs were 2 mg·mL− 1 and 4.5 nM, respectively. 2.3. Colloidal stabilities of SF-Au NPs to pH and salts To determine the stability of the Au NPs with respect to pH, the solutions of SF-Au NPs and Au NPs were added to 20 mM tris(hydroxymethyl)aminomethane–2-(N-morpholino)ethanesulfonic acid (Tris–MES) or 20 mM phosphate buffer (PB) at pH 2 to 11 (volume ratio, 1:1). The stability of the Au NPs with respect to various salt concentrations was determined by adding 20 mM PB solution at pH 7.4 to the solutions of SF-Au NPs and Au NPs; the PB contained twofold of the desired salt concentrations (volume ratio, 1:1). The stability of the Au NPs in the physical environment was probed by adding 1.5-fold SBF solutions (volume ratio, 1:2) to the solutions of SF-Au NPs and Au NPs. The final concentrations of SF and Au NPs in all the solutions were 2 mg·mL−1 and 4.5 nM, respectively. The stabilities of the SF-Au NPs were investigated via UV–Vis spectroscopy.
the cells were further cultured at 37 °C for 4 h. Dark blue formazan crystals generated by mitochondria dehydrogenase in live cells were dissolved in 150 mL of dimethyl sulfoxide (DMSO); the absorbance at 570 nm was measured by a microplate reader (MODEL 550, Bio Rad, USA). The relative cell viability is given as
Relative cell viability ð%Þ ¼ absorption of treated well=absorption of control well 100:
ð1Þ
Five replicates were obtained for each sample, and the mean value was used as the final result. All data were presented as mean ± SD. Statistical analysis of data was made by ANOVA or Student's t-test. It was considered significantly different if p b 0.05. The working solution was freshly prepared by adding 5 μL of fluorescein diacetate (FDA, 5 mg·mL−1 acetone) stock solution to 5 mL of PBS. Up to 20 μL of FDA working solution was added to each well and then incubated for 15 min. The wells were washed twice with PBS; confocal laser scanning microscopy (CLSM, BIORAD 2000) was carried out to observe the cell morphology. 2.5. Characterization UV–Vis spectra were obtained with a UV/Vis Shimadzu UV-2505 spectrometer. TEM was performed on a JEM-1200EX (JEOL, Japan). Samples for TEM measurements were prepared by placing a drop of colloidal dispersion on a carbon-coated copper grid, followed by evaporation of the solvent. Samples were freeze-dried completely before being grinded into powder and pressed into pellets with KBr for measuring infrared spectra with a Fourier transform infrared spectrophotometer (FTIR) (Bruker VERTEX80 V, Germany). 3. Results and discussion
2.4. Cytotoxicity assay and cell morphology observation 3.1. Characterization of citrate-capped Au NPs (cit-Au NPs) and SF-Au NPs Human umbilical vein endothelial cells (HUVECs) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U·mL−1 penicillin, and 100 mg·mL−1 streptomycin; the HUVECs were then cultured at 37 °C in 5% CO2 humidified environment. Cytotoxicity was assessed by standard MTT assay. The HUVECs were plated at a density of 5 × 103 cells/well in a 96-well plate and then cultured for 24 h. The medium was replaced with fresh medium containing different concentrations of the cit-Au NPs and SF-Au NPs; the Au atomic concentration was determined by inductively coupled plasma mass spectrometry (ICP-MS). The cells cultured in NP-free media were used as controls. The wells were washed with PBS and the medium was replaced with 100 mL of fresh medium after 24 h of treatment. Up to 20 mL of MTT (5 mg·mL−1) was added to each well;
A characteristic absorption peak at 520 nm was observed in the UV– Vis spectra of cit-Au NPs (diameter, ~ 13 nm). As shown in Fig. 1a, the aqueous solution of SF-Au NPs displayed another strong absorption band at approximately 275 nm after the addition of 2 mg·mL−1 SF to the solution of cit-Au NPs. This band was assigned to the π → π* electron transition of the tyrosine (Tyr) residue in the SF molecular chain. A 5 nm red shift was observed from 520 nm to 525 nm, and the peak absorbance of the Au NPs increased, indicating the adsorption of SF onto the surface of the Au NPs [28]. No statistical differences in the average particle diameter or size distribution of the Au NPs were detected from the TEM images (Figs. 1b and c). The SF-Au NPs remained well separated, which implied the absence of aggregation.
Scheme 1. Preparation and stability tests of SF capped Au NPs.
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Fig. 1. (a) UV–Vis spectra and TEM images of (b) Au NPs and (c) SF-Au NPs.
3.2. Stabilities of SF-Au NPs The stability of NPs is necessary under environmental conditions, such as high salt concentrations and extreme pH and buffer solutions, if the particles are used in biological and medical applications. pH [29] and metallic ions [30] influence the rheology of SF proteins. The stability of Au NPs can be monitored by UV–Vis spectroscopy because aggregation, precipitation, and decomposition lead to distinctive changes in the spectra. The effects of pH on the stability of the SF-Au NPs were initially investigated. MES–Tris buffer was used to minimize the probable effect of salt ions in accelerating the aggregation of SF. Fig. 2 shows the UV–Vis absorption spectra of the Au NPs before and after SF modification under different pH conditions. The spectra of SF-Au NPs exhibited no discernible changes in the intensity or position of the absorbance peak at 525 nm in the pH range of 2 to 11 (Fig. 2a). By contrast, the corresponding peak of the spectra of cit-Au NPs red shifted with decreasing acidity after 6 h (Fig. 2b); this shift was accompanied with a color change of the solution from red to blue. This result was in accordance with previous results on the precipitation of Au NPs obtained by borohydride or citrate reduction routes even upon the slightest change in pH [31]. Similar phenomena were observed in PB (Fig. S4). The cit-Au NPs aggregated under acidic conditions, whereas the SF-Au NPs remained stable in the entire pH region. The color changed within 10 min in PB, whereas the time was extended to approximately 6 h in Tris–MES buffer. This result may be attributed to the effect of salt ions. To analyze the mechanism of stabilization, BSA was selected as the control protein because of its similar isoelectric point (PI) with SF, cost efficiency, and availability. As shown in Fig. S5, the BSA-capped Au
NPs (BSA-Au NPs) were stable at basic conditions. The BSA-Au NPs agglomerated when the solution pH was below the PI of BSA (~ 5). This result may be attributed to the neutralization of the negative charge and the subsequent decrease in electrostatic repulsion between the particles. Changes in molecular conformation of SF are usually characterized by amide bands in FT-IR spectra. To clarify the interaction between Au NPs and silk fibroin, the FT-IR spectra of SF-Au NPs at pH 4 and 7 were studied, and the results are shown in Fig. S6. The conformations of SF could be random coil or β-sheet structure. As shown in Fig. S6, FTIR spectra of amide bands at around 1650, 1540 and 1235 cm− 1 are typical feature of random coil [32]. The peak positions and shapes of the amide bands of SF-Au NPs at pH 4 are almost the same as those of SF-Au NPs at pH 7, which indicate that the conformation of SF would not turn to β-sheet structure from random coil with the pH increase. It is reported that morphological transition of silk fibroin (0.5 wt.%) occurred due to the formation of β-sheet by reducing the pH from 6.8 to 4.8 [29]. However, the concentration of the silk fibroin (0.2 wt.%) may be too low to form β-sheet in our system. The stability of NPs in the solution was mainly dependent on the surface properties of NPs (e.g., surface charge and ligand structure). The increase in the electrostatic repulsion and/or steric hindrance of the NPs surfaces can significantly improve the stability of NPs [10]. In this case, the surface charges of the cit-Au NPs and BSA-Au NPs considerably decreased with pH; this decrease resulted in NP aggregation because of the reduction in NP electrostatic repulsion. Compared with BSA and citrate, SF is a block copolymer-like protein with a random coil conformation in our system. The surface of the SF-Au NPs was coated with random coil protein chains, which increased the steric
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Fig. 2. UV–Vis spectra of (a) SF-Au NPs and (b) cit-Au NPs in Tris–MES buffer in the pH range of 2 to 11 after 6 h.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
hindrance of the particles. Therefore, SF can stabilize the Au NPs under a wide pH range even below the PI (ca. 4.58 to 5.00) [33]. The results indicated that electrostatic repulsion may be the primary mechanism for the stabilization of cit-Au and BSA-Au NPs and that the steric effect of SF may be crucial in SF-Au NPs. We analyzed the effects of salt concentrations on the stability of the SF-Au NPs. The final concentration of NaCl ranged from 50 mM to 1000 mM. As shown in Fig. 3, no significant changes in absorption features were observed for the SF-Au NPs when the concentration of NaCl reached 1000 mM, which was much higher than that of physiological saline solution (~ 154 mM). By contrast, the native cit-Au NPs were sensitive to ionic strength and quickly aggregated upon the addition of NaCl. The red shift and peak broadening in the UV–Vis absorption spectra showed that the cit-Au NPs aggregated at a low NaCl concentration of 100 mM. Metallic ions (e.g., K+, Ca2+, and Mg2+) influence the rheology of SF protein [30]. Therefore, their effects on the stability of Au NPs were also investigated. The final concentrations of Ca2+ and Mg2+ ranged from 12.5 mM to 125 mM to yield an ionic strength equal to that of Na+. All metal ions exhibited the same trends (Figs. S7 and S8). The cit-Au NPs aggregated when the concentrations of KCl and CaCl2 were only 50 and 12.5 mM, respectively; meanwhile, the SF-Au NPs remained stable at high ionic strengths. The spectra of the Au NPs with MgCl2 were almost the same as those of the particles with CaCl2. The high stability of the SF-Au NPs might be attributed to the fact that the SF adsorbed on the Au NPs prevented the particles from aggregating via steric repulsion.
Fig. 3. UV–Vis spectra of (a) SF-Au NPs and (b) cit-Au NPs in Tris–MES buffer with NaCl concentrations ranging from 50 mM to 1000 mM after 10 min.
Simulated body fluid (SBF) and the human plasma have very similar ionic compositions. The SF-Au NPs were incubated in SBF solution to determine their stability in a physiological solution. As shown in Fig. 4, the cit-Au NPs aggregated in SBF, whereas the SF-Au NPs remained stable after 48 h. The result implied that the SF-Au NPs were stable in complex biological systems. Therefore, the steric hindrance rather than the electrostatic repulsion of the SF-Au NPs may be essential for colloidal stability.
Fig. 4. UV–Vis spectra of SF-Au NPs and cit-Au NPs in SBF solution.
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Fig. 5. UV–Vis spectra of (a) SF-Au NPs, (b) BSA-Au NPs, and (c) cit-Au NPs in aqueous solution before and after freeze-drying.
Observation of the dissolution property of dried Au NP samples showed that the SF chains adsorbed on the Au NPs surface improved the colloidal stability of the Au NPs. As shown in Fig. 5, the resultant suspension exhibited nearly identical absorption spectra as the SF-Au NPs prior to freeze-drying. By contrast, the absorbance markedly decreased for the BSA-Au NPs after freeze-drying because of the significant aggregation of the Au NPs during the process (Fig. 5b). Significant precipitation was observed when dried cit-Au NPs were dissolved in water. Bare Au NPs were unable to form their suspensions because of intensive aggregation during freeze-drying (Fig. 5c). This feature was beneficial for the storage of SF-Au NPs and for potential applications in tissue engineering; many scaffolds were prepared via lyophilization.
The low cytotoxicity of the SF-Au NPs was attributed to two aspects. One is the important function of the inherent biocompatibility of SF; the other is the function of good colloidal stability. Small Au NPs can be endocytosed by cells and form internal aggregates, which result in cytotoxicity [6]. The increased cytotoxicity of the Au NPs was caused by aggregation; therefore, preventing the aggregation of the particles in the internal environment of the cells can greatly decrease the toxicity of the NPs. To confirm that the SF-Au NPs remained stable in the cell culture medium, we also tested the stability of the SF-Au NPs after incubation with PBS, RPMI-1640 medium with 10% and 100% FBS. The digital images (Fig. S10) implied the absence of aggregation after 48 h of incubation. Hence, the colloidal stability of the SF-Au NPs might have decreased the toxicity of the particles.
3.3. Cytotoxicity of SF-Au NPs
4. Conclusion
Low cytotoxicity is a prerequisite for NPs; the preliminary cytotoxicity of the SF-Au NPs and cit-Au NPs were evaluated by MTT assay in a HUVEC cell line. As shown in Fig. 6, both SF-Au NPs and cit-Au NPs exhibited negligible cytotoxicity when the concentrations were below 4.2 mg·mL− 1. A decrease in cell viability was observed upon treatment with the unmodified Au NPs at concentrations higher than 4.2 mg·mL− 1. The viabilities of HUVEC cells after 24 h of incubation with 8.4 mg·mL−1, 16.8 mg·mL−1 and 33.6 mg·mL−1 of cit-Au NPs were 89.5% ± 1.7%, 82.1% ± 1.1% and 70.2% ± 1.3% respectively. Several groups have also found cytotoxic effects to emerge in a dose-dependent manner for cit-Au NPs [6,34]. The viability of HUVECs with 33.6 mg·mL−1 Au was 93.0 ± 1.6%, indicating no marked cytotoxicity of SF-Au NPs in all tested concentrations. The fluorescence microscopic images of HUVECs after 24 h of treatment are shown in Fig. S9. The cells exposed to the SF-Au NPs were well distributed and showed similar concentrations in all tested conditions.
We presented a novel method for preparing highly stable and cytocompatible Au NPs. The remarkable colloidal stability of the Au NPs under different pH values and salt concentrations was attributed to the natural SF. The results indicated that the steric effect was crucial to the excellent stability of the SF-Au NPs. The particles can be readily dissolved in water after freeze-drying without aggregation, which is beneficial for storage. The SF-Au NPs showed excellent cytocompatibility because of the absence of cytotoxity in SF and the good colloidal stability of the particles. The use of natural SF as a stabilizer in our method was simpler, more efficient, and more cost-effective compared with the conventional modification strategies for Au NPs. This study opened possibilities for using SF-Au NPs in biomaterial applications, such as scaffolds for 3D cell culture and tissue engineering. Acknowledgments We gratefully acknowledge the financial support from the Nature Science Foundation of China (Grant No. 51303124), Natural Science Foundation of Shanxi (Grant Nos. 2013021009-2 and 2013011012-2), and the Qualified Personnel Foundation of Taiyuan University of Technology (Grant No. tyutrc-201155a). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.07.024. References
Fig. 6. Cytotoxicity of HUVEC cells evaluated by MTT assay after incubation with SF-Au NPs and different Au concentrations for 24 h. *p b 0.05 compared with the control.
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