Generation of Polypeptide-Templated Gold Nanoparticles using

Jun 20, 2013 - ABSTRACT: Ionizing radiation, including γ rays and X-rays, are high-energy electromagnetic radiation with diverse applications in nucl...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/Langmuir

Generation of Polypeptide-Templated Gold Nanoparticles using Ionizing Radiation Candace Rae Walker,†,⊥ Karthik Pushpavanam,‡,⊥ Divya Geetha Nair,§,⊥ Thrimoorthy Potta,‡ Caesario Sutiyoso,‡ Vikram D. Kodibagkar,† Stephen Sapareto,∥ John Chang,∥ and Kaushal Rege*,‡ †

School of Biological and Health Systems Engineering, Arizona State University, Tempe, Arizona 85287, United States Chemical Engineering, Arizona State University, Tempe, Arizona 85287-6106, United States § Materials Science and Engineering, Arizona State University, Tempe, Arizona 85287, United States ∥ Banner-MD Anderson Cancer Center, Gilbert, Arizona 85234, United States ‡

S Supporting Information *

ABSTRACT: Ionizing radiation, including γ rays and X-rays, are high-energy electromagnetic radiation with diverse applications in nuclear energy, astrophysics, and medicine. In this work, we describe the use of ionizing radiation and cysteine-containing elastin-like polypeptides (CnELPs, where n = 2 or 12 cysteines in the polypeptide sequence) for the generation of gold nanoparticles. In the presence of CnELPs, ionizing radiation doses higher than 175 Gy resulted in the formation of maroon-colored gold nanoparticle dispersions, with maximal absorbance at 520 nm, from colorless metal salts. Visible color changes were not observed in any of the control systems, indicating that ionizing radiation, gold salt solution, and CnELPs were all required for nanoparticle formation. The hydrodynamic diameters of nanoparticles, determined using dynamic light scattering, were in the range of 80−150 nm, while TEM imaging indicated the formation of gold cores 10−20 nm in diameter. Interestingly, C2ELPs formed 1−2 nm diameter gold nanoparticles in the absence of radiation. Our results describe a facile method of nanoparticle formation in which nanoparticle size can be tailored based on radiation dose and CnELP type. Further improvements in these polypeptide-based systems can lead to colorimetric detection of ionizing radiation in a variety of applications.

■

INTRODUCTION Ionizing radiation, including X-rays and γ-rays, has several applications in nuclear power, astrophysics, and medical imaging and therapy. The distinguishing feature between Xand γ-rays is their origin; X-rays arise from electronic interactions, while γ-rays arise from nuclear interaction.1 These high-energy beams can be both beneficial and harmful to humans. For example, radiation can be employed for ablation of tumors and imaging of diseased tissue,2−7 while the same radiation can damage DNA via strand breaks and nucleotide oxidation. Improper repair of these strand breaks in cells can result in chromosomal alterations and mutations, ultimately leading to carcinogenesis.8−11 The interaction of ionizing radiation with different materials has led to several detection methods, including those based on superheated droplets, nanophosphors, CdTe, CdZnTe, graphenes, inorganic−organic structures and fluorescence,12−18 which detect ionizing radiation by means of electronic energy change. Ionizing radiation also causes radiolysis of water, leading to the formation of the hydrogen radical (H·), hydroxyl radical (·OH), and hydrated electrons (eaq−). These reactive species can reduce ionic species to a zero valence state, resulting in formation of metal clusters and, subsequently, nanoparticles. Elastin-like polypeptides (ELPs) are analogs of mammalian elastin and are characterized by the amino acid sequence, © 2013 American Chemical Society

VPGXG, where V is valine, P is proline, G is glycine, and X is any guest amino acid except proline. ELPs demonstrate a phase transition characterized by reversible intramolecular contraction and intermolecular coacervation at their respective transition temperatures.19 As a result of their reversible thermal response, ELPs have been explored for a variety of applications in biotechnology and medicine.19−32 We have demonstrated that engineered ELPs that contain cysteines in the polypeptide sequence can be interfaced with gold nanoparticles, resulting in photothermally responsive nanoassemblies and nanocomposites for optical sensing, hyperthermic ablation of cancer cells, photothermally triggered drug delivery, and photothermal bonding of tissues.32−34 In this study, gold salts (colorless) were dissolved with cysteine-containing ELPs (CnELPs, where n = 2 or 12 cysteines in the polypeptide repeat sequence) and irradiated with different doses of X-ray radiation. Maroon-colored gold nanoparticle (GNP) dispersions were formed at X-ray doses starting at 175−250 Gy in the presence of ELPs, while nanoparticle formation was not observed in the absence of ELPs. The presence of scavenging agents, namely isopropanol Received: February 11, 2013 Revised: May 15, 2013 Published: June 20, 2013 10166

dx.doi.org/10.1021/la400567d | Langmuir 2013, 29, 10166−10173

Langmuir

Article

post irradiation, in order to allow for these two different maturation times for gold nanoparticle formation. The solvent (i.e., nanopure water) was used as the blank in all cases. The presence of an absorbance peak at ∼520 nm was used as an indicator of gold nanoparticle formation.37,38 Particle Size Determination. Hydrodynamic sizes of the irradiated samples were determined using dynamic light scattering (DLS), which was carried out with a DLS Particle Sizer (Corrvus Advanced Optical Instruments) in concert with Corcle software or with a Zetasizer Nano (for C2ELP samples). Samples (∼750 μL) were pipetted into a square 1 mL cuvette and placed into the instrument for analysis. The Corcle software was set up for sample measurements with autocorrelation. Data were collected for 1 min per sample, and the hydrodynamic diameter was determined based on the software readout. Transmission Electron Microscopy (TEM). Transmission electron microscopy was carried out at the Leroy Eyring Center for Solid State Sciences at ASU using a JEOL-JEM-2000FX, operating at 200 kV. Specimen samples for TEM were prepared by casting a drop of the gold nanoparticle dispersion onto a carbon film on a copper mesh grid, followed by drying in air. Air-dried samples were examined by TEM at 200 kV. SDS−PAGE Analysis. SDS−PAGE analysis was carried out to investigate degradation of C2ELP following treatment with ionizing radiation with doses of 0.5, 100, 268, 536, and 1072 Gy. C2ELP (25 μg, 25 μL), along with 5 μL of 6× loading buffer, was heated at 95 °C for 3 min and allowed to cool to room temperature. The sample was run on a 15% SDS−PAGE gel at 110 V for 90 min. After electrophoresis, the gel was rinsed with distilled water before staining with Coomassie for 6 h. The gel was then destained with water for 3 h in order to facilitate visualization of the C2ELP bands. The image was postprocessed in MATLAB using the “circular averaging filter”. Mass Spectrometry. Mass spectrometry analyses were carried out on select samples in order to further verify C2ELP degradation following treatment with X-ray radiation. Sinapinic acid matrix (6 μL) was loaded with 1 μL of the sample (e.g., C2ELP irradiated with a particular X-ray dose). One microliter of this mixture was air-dried and analyzed such that 500 laser shots were carried out, and the average of these was reported.

and acetone, resulted in enhancement of GNP formation. In addition to a facile method of polypeptide-templated GNP synthesis, our results indicate that the observed color change upon nanoparticle formation may be employed for the colorimetric detection of X-ray radiation.

■

EXPERIMENTAL SECTION

Materials. Gold(III) chloride trihydrate (HAuCl4·3H2O) and Lcysteine were purchased from Sigma-Aldrich. Isopropyl alcohol (IPA) (99.5%), acetone, and Reductacryl resin were purchased from British Drug Houses Chemicals (BDH), Lab Safety Supply Inc., and EMD, respectively. Acrylamide/bis solution (30%) was purchased from BioRad. Ammonium persulfate (98+%), sodium dodecyl sulfate, and EZRun Rec Protein Ladder (BP3602) were purchased from Fisher Scientific. All chemicals were used as received from the manufacturer without any additional purification. Synthesis, Expression, and Purification of Elastin-Like Polypeptides (ELPs). Elastin-like polypeptides, with the single-letter amino acid sequence, MVSACRGPG-(VG VPGVGVPGVGVPGVGVPGVGVPG)8-(VGVPGVG VPGVG VPGCG VPGVG VPG)n-WP, where n is equal to two or twelve, were generated via recursive directional ligation;35,36 the C2 or C12 in C2ELP or C12ELP indicate the presence of two or twelve cysteines in the ELP repeat sequence. Oligonucleotides encoding the ELPs were first cloned into pUC19, followed by cloning into a modified version of the pET25b + expression vector at the sfiI site. Escherichia coli BLR(DE3) (Novagen) was used as a bacterial host for polypeptide expression, which was followed by purification and lyophilization, and stored at 4 °C as described previously.36 Preparation of CnELP Samples. CnELP bulk samples for irradiation and other experiments were prepared by dissolving the polypeptide in nanopure water (18.2 MΩ cm resistivity) to a concentration of 1 mg/mL. Reductacryl resin was added to the CnELP solution (1 mg resin for 1 mg CnELP) in order to reduce the cysteines in the polypeptide chain. The samples were mixed for at least 30 min under constant rotation at 4 °C. After 30 min, the Reductacryl resin was removed from the dissolved ELP via centrifugation at 6000 rcf for 10 min. Reduced CnELP bulk samples were stored at 4 °C until used. Preparation of Samples for Ionizing Irradiation. Two sets of samples were prepared for irradiation studies. The first sample set comprised of HAuCl4 (1 × 10−3 M), CnELP, where n = 2 or n = 12, (0.1% w/v), isopropyl alcohol (IPA) (0.2 M), acetone (0.058 M), and the balance nanopure water (18.2 MΩ cm resistivity). The second sample set comprised of the same components as the first, except that it did not contain isopropyl alcohol and acetone (scavenging agents). In order to verify whether free cysteines could also template the formation of nanoparticles under these conditions, we carried out the above experiments in the presence of this amino acid instead of C2ELP. The L-cysteine concentrations employed in these studies were chosen such that the thiol concentration was the same as those in the case of the C2ELP studies. In all cases, samples were mixed immediately prior to irradiation. Sample Irradiation. All ionizing irradiation experiments were carried out at the Banner-MD Anderson Cancer Center in Gilbert, AZ. A Truebeamlinear accelerator irradiated the samples to different doses, at a dose rate of ∼15 Gy/minute. The radiation beam is a 6 MV photon beam produced by a linear accelerator. Electrons are accelerated to 6 MeV and strike a tungsten target to produce a spectrum of X-rays whose maximum energy is about 6 MeV and average energy is approximately 2 MeV. CnELP−gold salt solutions were irradiated at doses of 0.5, 2, 5, 10, 25, 50, 100, 175, 200, 268, 536, 804, and 1072 Gy. After irradiation, samples were transported back to ASU in Tempe, AZ, to allow for maturation of nanoparticle formation (1 h or 18 h) and subsequent characterization. Absorbance Spectroscopy. Absorbance of the irradiated samples was measured using a BioTek Synergy 2 plate reader in concert with Gen5 software. The absorbance values were read from 300 to 995 nm, with a step size of 5 nm, using 150 μL sample in each well of a 96-well plate. Absorbance measurements were carried out ∼1 h and ∼18 h

■

RESULTS AND DISCUSSION Gold nanoparticles have been investigated in a variety of different applications, including sensing, biomedical imaging, and therapeutics.39,40 In this study, we investigated the use of cysteine-containing elastin-like polypeptides (CnELPs; n = 2 or 12) for nucleating the formation of gold nanoparticles upon exposure to ionizing radiation (schematic shown in Figure 1). Exposure to high-energy X-ray radiation results in radiolysis of water, which in turn, results in the generation of free radicals (H· and ·OH) and aqueous electrons (eaq−). These species can reduce metal ions to zerovalent clusters, ultimately leading to the formation of nanoscale particles. In addition to these species, the presence of thiols in cysteine-containing ELPs can offer additional reducing sites for nanoparticle formation from metal salts. We, therefore, investigated nanoparticle formation upon exposure to different doses of X-ray radiation in the presence and absence of C2ELP or C12ELP. In addition, the role of scavenging agents, namely isopropanol and acetone, on nanoparticle formation was also investigated. The maximal absorption peak at 520 nm is characteristic of the plasmonic properties of spherical gold nanoparticles and was used as an indicator of gold nanoparticle formation in all cases. The peak at 520 nm is also reflective of the maroon color that is seen for gold nanoparticle dispersions. In the absence of X-ray radiation, neither C2ELP nor C12ELP was able to reduce chlorauric acid to nanoscale gold particles to the extent that an absorbance peak at 520 nm could be visualized (Figures S1−S4 10167

dx.doi.org/10.1021/la400567d | Langmuir 2013, 29, 10166−10173

Langmuir

Article

both X-ray radiation as well as CnELPs were necessary in order to facilitate nanoparticle formation in the presence of the radiation doses employed (0−1072 Gy). Two experimental systems were set up in order to investigate gold nanoparticle formation following the above observations. In the first case, CnELPs in the presence of gold salt, IPA, and acetone, were exposed to X-ray radiation. In the second case, CnELPs in the presence of gold salt, were subjected to X-ray radiation, but in the absence of the scavenging agents (acetone and IPA). Changes in color (from colorless/light yellow to maroon or purple color) and concomitant changes in the absorption spectra were observed 1 h and 18 h post-irradiation in order to investigate nanoparticle formation; the absorbance peak at ∼520 nm was used as primary conformation of gold nanoparticle formation. Different incubation times (1 h and 18 h) were employed in order to allow for different maturation times for nanoparticle formation following exposure to ionizing radiation. Effect of X-ray Radiation Dose on Nanoparticle Formation. The above experimental systems were subjected to different radiation doses to determine the minimum dose needed to facilitate a color change and gold nanoparticle formation. Radiation doses of 0.5, 2, 5, 10, 25, 50, 100, 175, 200, 268, 536, 804, and 1072 Gy were employed in the study, as described in the Experimental section. No color change was observed for at least two days when samples were irradiated with lower doses (i.e., from 0.5 to 100 Gy) of X-ray radiation. Absorbance spectra measurements also indicated the lack of significant gold nanoparticle formation under these conditions (Figures S6−S19 of the Supporting Information). However, a pink/maroon-colored film was observed on the sides of the sample tubes, first observed after 18 h, likely due to the precipitation of the small amount of gold nanoparticles on the surface of the tubes. The lowest X-ray radiation dose at which gold nanoparticle formation was observed was at 175 Gy; a slight color change to maroon was seen 18 h post-irradiation at this dose (Figure 3).

Figure 1. Schematic of proposed formation mechanism of gold nanoparticles in the presence of CnELPs and ionizing radiation.

of the Supporting Information). The lack of an absorbance peak and absence of maroon color indicated lack of significant nanoparticle formation in the absence of X-ray radiation. Further, in the absence of ionizing radiation, gold salts without CnELP (▲ in Figure 2) or CnELPs without gold salts (■ in Figure 2) did not demonstrate any change in spectra/dispersion color.

Figure 2. Absorbance spectra of control systems. Gold salt in the absence of X-ray radiation and absence of C2ELP (▲). C2ELP in the absence of gold salt and absence of X-ray radiation (■). Gold salt subjected to X-ray radiation in the absence of CnELPs (⧫). CnELPs exposed to X-ray radiation in the absence of gold salts (×). No color change was observed in any of these cases, which is also reflected by the lack of an absorbance peak at 520 nm.

Figure 3. (a−g) Gold nanoparticle (GNP) formation as a function of X-ray radiation dose in the presence of 1 mg/mL C2ELP and isopropanol/acetone (maturation time of 18 h following irradiation). Color change from colorless to dark maroon can be seen starting at 175 Gy.

In the presence of X-ray radiation, gold salt solutions that did not contain CnELPs did not demonstrate any change in color, indicating the lack of visible nanoparticle formation in this case (⧫ in Figure 2). Similarly, no spectral changes were observed in the case when CnELPs were subjected to ionizing radiation in the absence of gold salts, which was along the expected lines (× in Figure 2). Inclusion of scavenging agents, isopropanol and acetone, also did not induce formation of GNPs in any of these control cases (data not shown). In addition, presence of equivalent concentrates of free cysteine (i.e., in the amino acid form, and not as part of the ELP sequence) showed no visible gold nanoparticle formation under all doses of radiation investigated (Figure S5 of the Supporting Information). Taken together, these results indicate that the presence of

Dynamic light scattering (DLS) also confirmed the formation of particles that demonstrated a hydrodynamic diameter of approximately 145 nm (Table 1). Higher doses of X-ray radiation clearly resulted in nanoparticle formation as seen in the digital images shown in Figure 3. Absorbance spectra of these maroon-colored dispersions (Figure 4) confirmed the formation of gold nanoparticles as seen from the maximal peak at ca. 520 nm. An increase in the radiation dose beyond 300 Gy resulted in a deepening of the maroon color of nanoparticle dispersion (Figure 3). This is likely due to the generation of higher amounts of reactive reducing species at higher doses of X-ray radiation. This, in turn, can result in higher conversions of AuIII ions to gold nanoparticles, which is manifested as the 10168

dx.doi.org/10.1021/la400567d | Langmuir 2013, 29, 10166−10173

Langmuir

Article

Table 1. Hydrodynamic Diameters of Gold Nanoparticles Formed upon Treatment of Systems with X-ray radiation a none avg (nm) std deviation c none avg (nm) std deviation

b DLS

IPA and acetone

175 Gy

200 Gy

268 Gy

536 Gy

804 Gy

1072 Gy

143.7 33.1

126.5 20.5

153 10.4

121.3 8.1

79.2 26.3

85.3 5.7

175 Gy

200 Gy

268 Gy

536 Gy

804 Gy

1072 Gy

147 10.4

148.3 26.8

128.2 29.8

116.7 8.1

104.7 19.2

92.2 6.3

avg (nm) std deviation

DLS

IPA and acetone avg (nm) std deviation

DLS 175 Gy

200 Gy

268 Gy

536 Gy

804 Gy

1072 Gy

154.3 26.3

152 36.8

166.7 30.2

90.2 21.0

74.3 34.6

54.4 22.4 d

175 Gy

200 Gy

268 Gy

536 Gy

804 Gy

1072 Gy

171.7 11.0

204 47.1

153.3 21.4

178.7 91.2

101.1 21.6

92.2 6.3

DLS

(a) C2ELP with Au, nanopure water, and radiation, (b) C2ELP with Au, IPA, acetone, nanopure water, and radiation, (c) C12ELP with Au, nanopure water, and radiation, (d) C12ELP with Au, IPA, acetone, nanopure water, and radiation. n = 3 independent experiments for all cases except for 200 Gy samples, which was n = 2. Hydrodynamic diameters were determined using dynamic light scattering in all cases.

Effect of Polypeptide Type on Nanoparticle Formation. CnELPs present in the system can act as templating and/or capping agents for gold nanoparticles; gold−thiol interactions allow for the attachment of the CnELPs to the surface of the gold nanoparticles.36 Gold ions begin to nucleate to form gold nanospheres upon reduction to zerovalent states, following exposure to ionizing radiation. The CnELPs present in the system help stabilize the gold nanoparticles and prevent them from aggregating, as seen in Figure 1. Given that thiols reduce metal ions,46 it is likely that these moieties from cysteines in the CnELP sequence aid the reduction gold ions to nanoparticles and/or aid the maturation of clusters to nanoparticles over extended periods of time (i.e., 18 h). As seen in Figure 5, C2ELP resulted in the formation of darkercolored dispersions and therefore, higher absorbance values, 1 h post X-ray radiation, compared to C12ELP-based systems. However, C12ELP- and C2ELP-based systems demonstrated similar absorbance values 18 h post X-ray radiation (Figure 5). These results indicate that C12ELP-based systems need a longer time for facilitating formation of gold nanoparticles, presumably due to steric effects associated with the higher molecular weight of this polypeptide compared to that of C2ELP. Interestingly, irradiation in the presence of free cysteines did not result in nanoparticle formation, even at the highest doses (Figure S5 of the Supporting Information), indicating that the ELP played a role in nanoparticle formation and stabilization. Transmission Electron Microscopy (TEM) Imaging of C2ELP-Templated Gold Nanoparticles. Samples of dispersions treated with and without radiation exposure were characterized by TEM to further verify gold nanoparticle formation. Samples containing C2ELP, without radiation exposure or those exposed to lower radiation doses (0−100 Gy), resulted in the formation of gold nanoparticles with a diameter of 1−2 nm in the presence of IPA and acetone, following incubation at room temperature for 18 h. (Figure 7, panels A and B). Most gold nanoparticles formed were spherical in shape and well-separated from each other (Figure 7). It is important to remind the reader that no spectral peaks (at 520 nm) and/or color changes were observed in these systems (e.g., Figures S1 and S2 of the Supporting Information), which indicated that the size (1−2 nm) and/or yields of these nanoparticles were too small for visual/ colorimetric detection. In addition, given that 1−2 nm particles were formed in the presence of C2ELP in the absence of radiation exposure, it is likely that the lower doses of radiation (0−100 Gy) had minimal influence on nanoparticle formation,

deepening of the color, as well as a concomitant increase in optical density. Dynamic light scattering (DLS) studies indicated that the hydrodynamic diameters of these nanoparticles ranged from 80 to 150 nm (Table 1). Interestingly, higher radiation doses resulted in the formation of nanoparticles with the lowest sizes (ca. 80 nm in diameter). Similar trends were seen in a study by Naghavi et al., where colloidal silver nanoparticles were synthesized by γ-radiation induced reduction of a metal salt precursor with the use of a polymer to control growth to homogeneous sizes.41 The authors proposed that metal nanoparticles formed clusters and coalesced by direct reduction, especially at higher doses of radiation. In other words, it is likely that the rate of nucleation events is higher at higher doses, resulting in smaller nanoparticles. However, these nucleating events are likely lower in frequency at low radiation doses, which, in turn, leads to nanoparticles of larger sizes. DLS data of C2ELP in water (in the absence of gold salts) are shown in Figure S20 of the Supporting Information and are consistent with other observations in the literature.42 Taken together, these studies indicate that polypeptide-templated nanoparticle size can be controlled based on the dose of radiation employed. Effect of Scavenging Agents, Isopropanol (IPA) and Acetone, on Nanoparticle Formation. X-ray radiationinduced radiolysis of water results in the formation of H· and ·OH radicals and hydrated electrons (eaq−). Isopropanol (IPA) reacts with the H· and ·OH radicals and acetone reacts with eaq−, resulting in the formation of 2-propyl radicals.43 The 2propyl radical can reduce AuIII ions in the system to their zero valence state, Au0, which leads to the formation of gold nanoparticles. Inclusion of IPA and acetone with CnELPs and chloroauric acid resulted in the formation of darker colored dispersions compared to cases where the scavenging agents were not used, following treatments with similar X-ray radiation doses. This is reflected in the higher absorbance values seen in Figure 5, which is indicative of higher yields of nanoparticles in the presence of isopropanol and acetone. Compounds containing 2-propyl radicals have been shown to aid in reduction reactions. It is, therefore, likely that formation of 2propyl radicals engenders the formation of gold nanoparticles with greater efficacies and rates compared to hydrated electrons acting alone.44,45 Figure 6 shows the differences in optical densities of the nanoparticle dispersions formed upon X-ray radiation with 1072 Gy in the presence and absence of both IPA and acetone, indicating the advantages of using these scavenging agents. 10169

dx.doi.org/10.1021/la400567d | Langmuir 2013, 29, 10166−10173

Langmuir

Article

Figure 5. Effect of X-ray radiation dose on gold nanoparticle formation upon irradiation of chloroauric acid (gold salt) in the presence of C2ELP and C12ELP. The optical density, measured at 520 nm for all samples, is indicative of concentrations of gold nanoparticles formed at maturation times of (a) 1 h and (b) 18 h, following X-ray radiation. Lines connecting the data points are included for visualization only.

Figure 6. Absorbance values at 520 nm for different experimental systems at 1072 Gy. Statistical significance is determined as follows: * = p ≤ 0.05, ** = p ≤ 0.01 for n = 3. Presence of scavenging agents (isopropanol or IPA and acetone) enhances gold nanoparticle formation, following treatment with X-ray radiation. Figure 4. Absorbance spectra of different systems treated with X-ray radiation. (a) C2ELP with chloroauric acid (gold salt), nanopure water, and X-ray radiation, (b) C2ELP with chloroauric acid, IPA, acetone, nanopure water, and X-ray radiation, (c) C12ELP with chloroauric acid, nanopure water, and X-ray radiation, and (d) C12ELP with chloroauric acid, IPA, acetone, nanopure water, and X-ray radiation.

in the formation of approximately 10−20 nm diameter nanoparticle cores (Figure 7, panels C−F). These results, in combination with DLS data, indicate that in the case of radiation-induced nanoparticle formation, the gold “core” is approximately 10−20 nm, while the ELP “shell” likely makes for the bulk of the hydrodynamic particle size. However, the precise location and morphology of the ELP is not known. C12ELP did not template nanoparticle formation in the absence of radiation (Figure 7, panels G and H), unlike that

and that the nanoparticle templating activity was mainly due to the polypeptide alone. Exposure of C2ELP and gold salt solutions to higher radiation doses (175 and 1072 Gy) resulted 10170

dx.doi.org/10.1021/la400567d | Langmuir 2013, 29, 10166−10173

Langmuir

Article

Formation of triangular-shaped nanoparticles was also observed under higher dose radiation (1072 Gy). Polypeptide Integrity upon Irradiation. The integrity of C2ELP following treatment with radiation was investigated using SDS−PAGE analysis and mass spectrometry. SDS− PAGE analysis shows a decrease in C2ELP band (molecular weight ∼21 kDa) intensity with increasing dose of radiation, consistent with other reports in the literature.47−49 (Figure 8

Figure 8. SDS−PAGE results of C2ELP following exposure to different doses of X-ray radiation. The shown image is obtained after postprocessing as described in the Materials and Methods section; the original SDS−PAGE gel image is provided in Figure S21 of the Supporting Information. The same volume of C2ELP was loaded in each well. From the left: lane 1, protein molecular weight marker, C2ELP irradiated with lane 2, 0 Gy (no irradiation control); lane 3, 0.5 Gy; lane 4, 100 Gy; lane 5, 268 Gy; lane 6, 536 Gy; lane 7, 1072 Gy. Lanes to the right of lane 7 were not loaded with any sample. Decrease in C2ELP band intensity at higher radiation doses indicates degradation of the polypeptide.

and Figure S21 of the Supporting Information). The significant reduction in band intensity in lane 6 (536 Gy) and lane 7 (1072 Gy) indicates degradation of the parental polypeptide at these radiation doses. It is likely that the smaller peptide fragments are either too low in concentration to be visualized on the gel or “run off” the gel and are therefore not visualized. MALDITOF results confirmed these observations in that significant loss of the C2ELP peak was observed for X-ray irradiated polypeptide samples (Figures S23, panels a−d, of the Supporting Information) compared to those not subjected to the radiation (Figure S22 of the Supporting Information). It is important to point out that although the ion current/intensity is proportional to the concentration of a sample, quantitative yields are not easily established.50 It is also likely that the degradation of C2ELP is responsible for the smaller hydrodynamic diameters of the nanoparticles formed at higher doses of radiation (Table 1).

Figure 7. TEM images of gold nanoparticles obtained from C2ELP (A−F) and C12ELP (G−L) in the presence of IPA and acetone. TEM images of gold nanoparticles without radiation exposure (A and B), with radiation exposure (C and D- 175 Gy, E and F-1072 Gy). TEM images of gold nanoparticles without radiation exposure (G and H), with radiation exposure (I and J- 175 Gy, K and L-1072 Gy).

■

CONCLUSIONS In this study, we report radiation-activated gold nanoparticle formation, templated and stabilized by engineered cysteinecontaining elastin-like polypeptides. Nanoparticle formation in the presence of ionizing radiation is accompanied by a color change (from clear/pale yellow to maroon color). This system facilitates the visible/colorimetric detection and measurement of the radiation dose. To the best of our knowledge, this is the first engineered polypeptide-based system for templating nanoparticles as a colorimetric indicator of radiation. A

observed in case of C2ELPs (Figure 7, panels A and B). Instead, a sparse population of larger gold particles, approximately 0.5 μm in diameter, was observed. It is likely that the presence of a larger number of thiols (cysteines) in C12ELP result in interparticle bridging, leading to particles with bigger sizes in the absence of radiation. However in the presence of ionizing radiation, C12ELP resulted in the formation of nanoparticles approximately 10−20 nm in diameter (Figure 7, panels I−L). 10171

dx.doi.org/10.1021/la400567d | Langmuir 2013, 29, 10166−10173

Langmuir

■

summary of the different experimental conditions employed, and their respective nanoparticle forming efficacies, is shown in Table 2.

CnELP

radiation

+ + − − + +

+ + + + − −

+ + + + + +

IPA/ acetone

visible colorimetric indication of nanoparticle formation

+ − − + − +

yes yes no no no no

*Address: Chemical Engineering, 501 E. Tyler Mall, ECG 301, Arizona State University, Tempe, AZ 85287-6106, United States. E-mail: [email protected]. Tel: 480-727-8616. Fax: 480-727-9321. Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

degree of formation

■

*** **

ACKNOWLEDGMENTS This work was supported by the Defense Threat Reduction Agency (DTRA) Young Investigator Award (Grant HDTRA110-1-0109) to K.R. C.R.W. acknowledges partial support from the Dean’s fellowship from Ira A. Fulton Schools of Engineering at ASU. The authors acknowledge Dr. Jacob Elmer, Dr. David Taylor, and Mr. Taraka Sai Pavan Grandhi in the Rege lab at ASU for insightful discussions and technical help.

a

The asterisks in the degree of formation column are qualitative indicators of observed nanoparticle formation.

■

In this initial study, we identified several important factors that led to nanoparticle formation following irradiation with ionizing radiation. Cysteine-containing elastin-like polypeptides (CnELPs) were critical in facilitating nanoparticle formation, while free cysteines alone did not show this behavior. It is likely that secondary and tertiary structures of the polypeptide facilitate nanoparticle formation and stabilization, although further experiments will be necessary to elucidate optimal conditions and actual mechanisms. The size of the ELP also plays a role in nanoparticle formation, since short-chain ELP (C2ELP) resulted in accelerated nanoparticle formation compared to longer-chain ELP (C12ELP), likely due to reduced steric hindrance. Of note, C2ELP is capable of inducing nanoparticle formation even in the absence of radiation, although forming significantly smaller nanoparticles (1−2 nm versus 10−20 nm); no visible color was observed in these dispersions formed in the absence of radiation. Biasing the system to preferentially generate alkoxy radicals (isopropyl radicals) resulted in enhanced nanoparticle formation due to the ability of these radicals to reduce gold cations. These factors combined to lower the threshold of nanoparticle formation from tens of kilo Gy typically reported in previous literature to our current minimum of 175 Gy. However, further optimization of our system is necessary in order to detect lower doses of radiation. We anticipate that detection of lower doses of ionizing radiation is likely possible by further engineering the polypeptide and by the optimal use of appropriate metal salts, solvents, and free radical generators. These advancements are anticipated to have high impact in portable detection of radiation for applications in personnel protection in space and on the battlefield, in preventing nuclear accidents, as well as in disposable radiation dosimetry in medicine.

■

AUTHOR INFORMATION

Corresponding Author

Table 2. Summary of Materials and Different Combinations of Conditions Employed in the Current Study, And Accompanying Observations on Gold Nanoparticle Formationa gold salt

Article

REFERENCES

(1) Washington, C. M.; Leaver, D. Principles and Practice of Radiation Therapy, 3rd ed.; Mosby Elsevier: St. Louis, 2010. (2) Falzon, G.; Pearson, S.; Murison, R.; Hall, C.; Siu, K.; Evans, A.; Rogers, K.; Lewis, R. Wavelet-based feature extraction applied to small-angle x-ray scattering patterns from breast tissue: A tool for differentiating between tissue types. Phys. Med. Biol. 2006, 51 (10), 2465−2477. (3) Huang, W. Y.; Jen, Y. M.; Lee, M. S.; Chang, L. P.; Chen, C. M.; Ko, K. H.; Lin, K. T.; Lin, J. C.; Chao, H. L.; Lin, C. S.; Su, Y. F.; Fan, C. Y.; Chang, Y. W. Stereotactic Body Radiation Therapy in Recurrent Hepatocellular Carcinoma. Int. J. Radiat. Oncol., Biol., Phys. 2012, 84 (2), 355−361. (4) Kang, J. K.; Kim, M. S.; Cho, C. K.; Yang, K. M.; Yoo, H. J.; Kim, J. H.; Bae, S. H.; Jung, D. H.; Kim, K. B.; Lee, D. H.; Han, C. J.; Kim, J.; Park, S. C.; Kim, Y. H. Stereotactic body radiation therapy for inoperable hepatocellular carcinoma as a local salvage treatment after incomplete transarterial chemoembolization. Cancer 2012, 118 (21), 5424−5431. (5) Liu, C. L.; Yan, X. H.; Zhang, X. Y.; Yang, W. T.; Peng, W. J.; Shi, D. R.; Zhu, P. P.; Huang, W. X.; Yuan, Q. X. Evaluation of x-ray diffraction enhanced imaging in the diagnosis of breast cancer. Phys. Med. Biol. 2007, 52 (2), 419−427. (6) Piegay, F.; Girard, N.; Mornex, J. F. Stereotactic radiotherapy and radiofrequency ablation for early-stage lung cancer. Bull. Cancer 2012, 99 (11), 1077−1081. (7) Wu, J.; Takeda, T.; Lwin, T. T.; Momose, A.; Sunaguchi, N.; Fukami, T.; Yuasa, T.; Akatsuka, T. Imaging renal structures by X-ray phase-contrast microtomography. Kidney Int. 2009, 75 (9), 945−951. (8) Lahtz, C.; Bates, S. E.; Jiang, Y.; Li, A. X.; Wu, X.; Hahn, M. A.; Pfeifer, G. P. Gamma irradiation does not induce detectable changes in DNA methylation directly following exposure of human cells. PLOS One 2012, 7 (9), 1−8. (9) Magnander, K.; Elmroth, K. Biological consequences of formation and repair of complex DNA damage. Cancer Lett. 2012, 327 (1−2), 90−96. (10) Suzuki, K.; Yamashita, S. Low-dose radiation exposure and carcinogenesis. Jpn. J. Clin. Oncol. 2012, 42 (7), 563−568. (11) Tubiana, M. Prevention of cancer and the dose-effect relationship: The carcinogenic effects of ionizing radiations. Cancer Radiotherapie 2009, 13 (4), 238−258. (12) Foxe, M.; Lopez, G.; Childres, I.; Jalilian, R.; Patil, A.; Roecker, C.; Boguski, J.; Jovanovic, I.; Chen, Y. P. Graphene field-effect transistors on undoped semiconductor substrates for radiation detection. IEEE Trans. Nanotechnology 2012, 11 (3), 581−587.

ASSOCIATED CONTENT

S Supporting Information *

Absorbance spectra and a table of molecular weights calculated C2ELP and C12ELP amino acid sequence and hydrodynamic radii determined for C2ELP and C12ELP by DLS. This material is available free of charge via the Internet at http://pubs.acs.org. 10172

dx.doi.org/10.1021/la400567d | Langmuir 2013, 29, 10166−10173

Langmuir

Article

(13) Kang, S.; Jung, B.; Noh, S.; Cho, C.; Yoon, I.; Park, J. Feasibility study of direct-conversion x-ray detection using cadmium zinc telluride films. J. Instrum. 2012, 7, C01010. (14) Kortov, V. S. Nanophosphors and outlooks for their use in ionizing radiation detection. Radiat. Meas. 2010, 45 (3−6), 512−515. (15) Lawrence, W. G.; Thacker, S.; Palamakumbura, S.; Riley, K. J.; Nagarkar, V. V. Quantum Dot-Organic Polymer Composite Materials for Radiation Detection and Imaging. IEEE Trans. Nucl. Sci. 2012, 59 (1), 215−221. (16) Liu, Z.; Xue, Wangxin; et al. A facile and convenient fluorescence detection of gamma-ray radiation based on the aggregation-induced emission. J. Mater. Chem. 2011, 21, 14487− 14491. (17) Lobez, J. M.; Swager, T. M. Radiation detection: Resistivity responses in functional poly(olefin sulfone)/carbon nanotube composites. Angew. Chem., Int. Ed. 2010, 49 (1), 95−98. (18) Sarkar, R.; Chatterjee, B. K.; Roy, B.; Roy, S. C. Radiation detection by using superheated droplets. Radiat. Phys. Chem. 2006, 75 (12), 2186−2194. (19) Urry, D. W. Physical Chemistry of biological free energy transduction as demonstrated by elastic protein-based polymers. J. Phys. Chem. B 1997, 101 (51), 11007−11028. (20) Janorkar, A. V.; Rajagopalan, P.; Yarmush, M. L.; Megeed, Z. The use of elastin-like polypeptide-polyelectrolyte complexes to control hepatocyte morphology and function in vitro. Biomaterials 2008, 29 (6), 625−632. (21) Swierczewska, M.; Hajicharalambous, C. S.; Janorkar, A. V.; Megeed, Z.; Yarmush, M. L.; Rajagopalan, P. Cellular response to nanoscale elastin-like polypeptide polyelectrolyte multilayers. Acta Biomater. 2008, 4 (4), 827−837. (22) Kim, J. Y.; O’Malley, S.; Mulchandani, A.; Chen, W. Genetically engineered elastin-protein A fusion as a universal platform for homogeneous, phase-separation immunoassay. Anal. Chem. 2005, 77 (8), 2318−22. (23) Lao, U. L.; Chen, A.; Matsumoto, M. R.; Mulchandani, A.; Chen, W. Cadmium removal from contaminated soil by thermally responsive elastin (ELPEC20) biopolymers. Biotechnol. Bioeng. 2007, 98 (2), 349−55. (24) Lao, U. L.; Sun, M.; Matsumoto, M.; Mulchandani, A.; Chen, W. Genetic engineering of self-assembled protein hydrogel based on elastin-like sequences with metal binding functionality. Biomacromolecules 2007, 8 (12), 3736−9. (25) Banki, M. R.; Feng, L.; Wood, D. W. Simple bioseparations using self-cleaving elastin-like polypeptide tags. Nat. Methods 2005, 2 (9), 659−662. (26) Liu, W.; McDaniel, J.; Li, X.; Asai, D.; Quiroz, F. G.; Schaal, J.; Park, J. S.; Zalutsky, M.; Chilkoti, A. Brachytherapy using injectable seeds that are self-assembled from genetically encoded polypeptides in situ. Cancer Res. 2012, 72 (22), 5956−5965. (27) McDaniel, J. R.; MacEwan, S. R.; Dewhirst, M.; Chilkoti, A. Doxorubicin-conjugated chimeric polypeptide nanoparticles that respond to mild hyperthermia. J. Controlled Release 2012, 159 (3), 362−367. (28) Madan, B.; Chaudhary, G.; Cramer, S. M.; Chen, W. ELP-z and ELP-zz capturing scaffolds for the purification of immunoglobulins by affinity precipitation. J. Biotechnol. 2013, 163 (1), 10−16. (29) Nettles, D. L.; Chilkoti, A.; Setton, L. A. Applications of elastinlike polypeptides in tissue engineering. Adv. Drug Delivery Rev. 2010, 62 (15), 1479−1485. (30) Andrew MacKay, J.; Chen, M.; McDaniel, J. R.; Liu, W.; Simnick, A. J.; Chilkoti, A. Self-assembling chimeric polypeptidedoxorubicin conjugate nanoparticles that abolish tumours after a single injection. Nat. Mater. 2009, 8 (12), 993−999. (31) Amruthwar, S. S.; Janorkar, A. V. In vitro evaluation of elastinlike polypeptide, collagen composite scaffold for bone tissue engineering. Dental Materials 2013, 29 (2), 211−220. (32) Huang, H.-C.; Walker, C. R.; Nanda, A.; Rege, K. Laser welding of ruptured intestinal tissue using plasmonic polypeptide nanocomposite solders. ACS Nano 2013, 7 (4), 2988−2998.

(33) Huang, H.-C.; Nanda, A.; Rege, K. Investigation of phase separation behavior and formation of plasmonic nanocomposites from polypeptide-gold nanorod nanoassemblies. Langmuir 2012, 28 (16), 6645−6655. (34) Huang, H. C.; Yang, Y.; Nanda, A.; Koria, P.; Rege, K. Synergistic administration of photothermal therapy and chemotherapy to cancer cells using polypeptide-based degradable plasmonic matrices. Nanomedicine (London, U.K.) 2011, 6 (3), 459−73. (35) Meyer, D. E.; Chilkoti, A. Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: Examples from the elastinlike polypeptide system. Biomacromolecules 2002, 3 (2), 357−67. (36) Huang, H. C.; Koria, P.; Parker, S. M.; Selby, L.; Megeed, Z.; Rege, K. Optically responsive gold nanorod-polypeptide assemblies. Langmuir 2008, 24 (24), 14139−44. (37) Frederix, F.; Friedt, J. M.; Choi, K. H.; Laureyn, W.; Campitelli, A.; Mondelaers, D.; Maes, G.; Borghs, G. Biosensing based on light absorption of nanoscaled gold and silver particles. Anal. Chem. 2003, 75 (24), 6894−6900. (38) Lismont, M.; Dreesen, L. Comparative study of Ag and Au nanoparticles biosensors based on surface plasmon resonance phenomenon. Mater. Sci. Eng., C 2012, 32 (6), 1437−1442. (39) Huang, H.-C.; Ramos, J.; Grandhi, T. S. P.; Potta, T.; Rege, K. Gold Nanoparticles in Cancer Imaging and Therapeutics. Nano LIFE 2010, 01 (03n04), 289−307. (40) Huang, H. C.; Barua, S.; Sharma, G.; Dey, S. K.; Rege, K. Inorganic nanoparticles for cancer imaging and therapy. J. Controlled Release 2011, 155 (3), 344−57. (41) Naghavi, K.; Saion, E.; Rezaee, K.; Yunus, W. M. M. Influence of dose on particle size of colloidal silver nanoparticles synthesized by gamma radiation. Radiat. Phys. Chem. 2010, 79 (12), 1203−1208. (42) Fluegel, S.; Fischer, K.; McDaniel, J. R.; Chilkoti, A.; Schmidt, M. Chain Stiffness of Elastin-Like Polypeptides. Biomacromolecules 2010, 11, 3216−3218. (43) Misra, N.; Biswal, J. Gamma radiation induced synthesis of gold nanoparticles in aqueous polyvinyl pyrrolidone solution and its application for hydrogen peroxide estimation. Radiat. Phys. Chem. 2012, 81, 195−200. (44) Sharma, K. K.; O’Neill, P.; Oakes, J.; Batchelor, S. N.; Rao, B. S. M. One-electron oxidation and reduction of different tautomeric forms of azo dyes: A pulse radiolysis study. J. Phys. Chem. A 2003, 107 (38), 7619−7628. (45) Hunt, P.; Worrall, D. R.; Wilkinson, F.; Batchelor, S. N. Quantitative rate constants for the reaction of dyes and alkenes with alpha-hydroxyalkyl radicals, measured by laser flash photolysis. Photochem. Photobiol. Sci. 2003, 2 (5), 518−523. (46) Ramdon, D. C.; Dixon, D. A.; Dasgupta, T. P. Mechanistic studies of the reaction between thioglycolic acid and chromium(VI): Substitution, isomerisation, and electron transfer. Inorg. React. Mech. 2005, 5 (3), 197−211. (47) Miller, J. H.; Draper, L. R.; Kempner, E. S. Direct radiation damage is confined to a single polypeptide in rabbit immunoglobulin G. Biophys. J. 2003, 84 (4), 2781−2785. (48) Lee, Y.; Song, K. B. Effect of gamma-irradiation on the molecular properties of myoglobin. J. Biochem. Mol. Biol. 2002, 35 (6), 590−4. (49) Filali-Mouhim, A.; Audette, M.; St-Louis, M.; Thauvette, L.; Denoroy, L.; Penin, F.; Chen, X.; Rouleau, N.; Le Caer, J. P.; Rossier, J.; Potier, M.; Le Maire, M. Lysozyme fragmentation induced by gamma-radiolysis. Int. J. Radiat. Biol. 1997, 72 (1), 63−70. (50) Randolph, T. W.; Mitchell, B. L.; McLerran, D. F.; Lampe, P. D.; Feng, Z. Quantifying peptide signal in MALDI-TOF mass spectrometry data. Mol. Cell. Proteomics 2005, 4 (12), 1990−9.

10173

dx.doi.org/10.1021/la400567d | Langmuir 2013, 29, 10166−10173