One-Step in Situ Synthesis of Polypeptide–Gold Nanoparticles Hybrid

Research Article Cite This: ACS Sustainable Chem. Eng. 2017, 5, 9841-9847

pubs.acs.org/journal/ascecg

One-Step in Situ Synthesis of Polypeptide−Gold Nanoparticles Hybrid Nanogels and Their Application in Targeted Photoacoustic Imaging Rui-Mei Jin,†,§ Ming-Hao Yao,†,‡,§ Jie Yang,†,‡ Dong-Hui Zhao,† Yuan-Di Zhao,*,†,‡ and Bo Liu*,†,‡ †

Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics - Hubei Bioinformatics & Molecular Imaging Key Laboratory, Collaborative Innovation Center for Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P. R. China ‡ Key Laboratory of Biomedical Photonics (HUST), Ministry of Education, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P. R. China S Supporting Information *

ABSTRACT: Hybrid nanogels have been widely used as multifunctional drug delivery carriers and imaging probes for biomedical applications. Two triblock artificial polypeptides PC10A and PC10ARGD were biosynthesized to prepare hybrid nanogels. When the concentration of these polypeptides drops to less than 2% (w/w), they can form nanogels by selfassembly. The physical characteristics of nanogels, such as surface potential, size, and targeting domain are able to be tuned. Polypeptide−gold nanoparticles hybrid nanogels were in situ synthesized using PC10A(RGD) as templates and photoinitiator I-2959 under 365 nm UV light irradiation in one step. The results of the effect of gold ion concentration on synthesized gold nanoparticles in hybrid nanogels showed that the size and the concentration of gold nanoparticles in hybrid nanogel increased gradually with the increasing of gold ion concentration. The concentration of polypeptide has no obvious effect on the properties of gold nanoparticles in hybrid nanogels and only influences the size of the hybrid nanogels. The concentration of gold nanoparticles in hybrid nanogels increased with the increasing of irradiation time. In addition, the change of pH (3.0−7.0) did not affect the properties of the gold nanoparticles in the hybrid nanogels. Cytotoxicity results showed that hybrid nanogels were almost nontoxic to HeLa cells when the concentration of Au ion was below 0.72 mM. An arginine-glycineaspartic acid motif could be introduced into the PC10ARGD−gold nanoparticles hybrid nanogels to enhance efficient receptormediated endocytosis in αvβ3 overexpressing HeLa cells as analyzed by photoacoustic imaging. These results indicate that such hybrid nanogels are promising to be used in biomedical applications. KEYWORDS: Polypeptide−gold nanoparticles, Hybrid nanogels, Size-tunable, Targeting, Photoacoustic imaging

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modify GNPs to improve their stability and biocompatibility.9 Physical encapsulation mainly utilizes polymer to coat GNPs. Cormode and co-workers used high-density lipoproteins with a good biocompatibility to coat GNPs and obtained good encapsulating and CT imaging effects.10 In recent years, nanogels have been widely utilized as multifunctional drug delivery vehicles due to high water content, high loading capability, and biocompatibility.11,12 Up to now, two major types of nanogels were reported to encapsulate drugs or nanoparticles.13,14 One type is crosslinking of preformed polymers and in situ formed polymers.15 The other type is nanogels self-assembled from interactive polymers, for example, nanogels assembled from triblock

INTRODUCTION Gold nanoparticles (GNPs) have been widely applied in biomedicine due to their attractive properties, such as unique optical, electrical, and catalytic properties and excellent biocompatibility, which make them promising candidates as imaging probes and drug carriers.1−3 Compared with original GNPs, GNPs modified with biomolecules possess more diversified functions. The modification of GNPs not only have no effect on the excellent optical property and enormous interface of GNPs, but also can endow GNPs with targeting or delivery functions. GNPs modified with biomolecules for biomedical applications have drawn considerable attention.4−6 To date, many methods have been developed for the functional modification of GNPs, mainly including chemical coupling and physical encapsulation. Chemical coupling mainly involves the binding of sulfhydryl-containing polymers and GNPs through the Au−S bond.7,8 For example, PEG-SH is often used to © 2017 American Chemical Society

Received: June 5, 2017 Revised: September 24, 2017 Published: September 25, 2017 9841

DOI: 10.1021/acssuschemeng.7b01784 ACS Sustainable Chem. Eng. 2017, 5, 9841−9847

ACS Sustainable Chemistry & Engineering

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polymers containing two hydrophobic sections and one hydrophilic section.16 This type of nanogel contains both hydrophilic and hydrophobic regions, which allows both hydrophilic and hydrophobic drugs to be loaded in the corresponding regions.16 In addition, encapsulating with nanogels is beneficial to in situ prepare polymer−nanoparticles hybrid nanogels. Many polymers (dendritic polymers, hyperbranched polymers, dendrigraft polymers, arborescent polymers, proteins, polypeptides, or DNA) were used as templates for synthesizing functional polymers−GNPs.17−21 Among them, proteins are considered to be more convenient and efficient carrier systems because of their biocompatible nature.22,23 However, a complicated structure and composition of natural proteins hinder their applications, and functional parts, such as targeting ligands, binding domain, and enzyme cleavage sites, need to be introduced by the further chemical conjugation or modification, which limits their applications.24 To address these issues, we chose nanogels formed from artificial polypeptides to modify GNPs. Artificial polypeptides with definite structures can be synthesized by the recombinant DNA technology at the molecular level. Some biologically active sequences can be introduced into artificial polypeptides to avoid further conjugation of the biomolecules.25,26 Herein, we developed a simple method by the genetically engineered polypeptide PC10A(RGD) as templates to in situ prepare polypeptide−GNPs hybrid nanogels in one step for targeted photoacoustic imaging (Scheme 1). The physical

Research Article

EXPERIMENTAL SECTION

Materials. Restriction endonuclease SpeI, NheI, BamH I, and T4 DNA ligase were purchased from New England Biolabs Inc. (Beijing, China). Ni-NTA agarose was obtained from Qiagen China (Shanghai) Co., Ltd. 2-Hydroxy-1-(4-(hydroxyethoxy) phenyl)-2-methyl-1-propanone (I-2959) was purchased from Ciba Inc. (Tarrytown, NY). 3(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and chloroauric acid tetrahydrate (HAuCl4·4H2O) were obtained from Sigma-Aldrich China (Shanghai) Co., Ltd. Ultrapure water (18.2 MΩ) was prepared by Milli-Q water purification system (Millipore, Bedford, U.S.A.). All solutions were prepared with ultrapure water. Synthesis and Purification of PC10A and PC10ARGD. The pQE9PC10A plasmid was kindly provided by Prof. D. Tirrell (California Institute of Technology, Pasadena, CA). PC10A and PC10ARGD were prepared according to our previous reports.16 The polypeptides PC10A and PC10ARGD were characterized using a Bruker Reflex III reflectron MALDI-TOF mass spectrometer (Bremen, Germany). PC10A (MS: 20932.6 Da, molecular weight (MW) calculated by the sequence: 20858.5 Da), PC10ARGD (MS: 22371.4 Da, MW calculated by the sequence: 22295.9 Da). Preparation of Polypeptide−GNPs Hybrid Nanogels. Polypeptide (PC10A or PC10ARGD) was dissolved in 1% I-2959 solution. The size of the nanogels was tuned by changing the concentration of polypeptides. A series of concentrations of HAuCl4·4H2O (0.24, 0.48, and 0.96 mM) were added in the mixture of polypeptide and I-2959, and the pH of the mixture was adjusted to 3.0, 5.0, or 7.0. After stirring for 30 min, the mixture was exposed under the UV light of 365 nm for different times (20, 40, and 60 s). The UV−vis absorption spectra of polypeptide−GNPs hybrid nanogels were recorded by a UV−vis spectrophotometer (UV-2550, Shimadzu, Japan). The sizes of polypeptide nanogels and polypeptide−GNPs hybrid nanogels were examined using a Tecnai G2 20 U-Twin TEM. Stability of PC10ARGD Nanogels. The zeta potential and hydrodynamic size of PC10ARGD nanogels (0.1%, w/w) and PC10ARGD-GNPs nanogels (0.1%, w/w PC10ARGD and 0.72 mM Au ion) were measured daily on a ZS90 Nanosizer (Malvern, U.K.) at 25 °C by laser Doppler anemometry methods and dynamic light scattering (DLS) within 20 days. Preparation of Polypeptide-GNPs-DOX Hybrid Nanogels. Oil soluble doxorubicin (DOX) was prepared according to previously reported methods.27 DOX (100 μL, 3 mg mL−1) in chloroform solution was dropwise added into PC10ARGD (0.1%, w/w), and the mixtures were ultrasonically treated for 5 min. After evaporating at room temperature for 5 h, the mixtures were centrifuged at 5000g for 15 min. The supernatant was used to prepare PC10ARGD-GNPs-DOX hybrid nanogels through the same method in the above section. In Vitro Drug Release at Different pH. A total of 1 mL of PC10ARGD-GNPs-DOX hybrid nanogels (0.1% PC10ARGD, 0.72 mM Au ion, and 159.7 μg mL−1 DOX) was drawn into a dialysis bag (MW cutoff:10 kDa) and immersed in 30 mL of acetate buffer (pH 5.0) or phosphate buffered saline (PBS, pH 7.4) both containing 0.1% (w/w) Tween 80. The samples were stirred at 37 °C. At each time point, 1.0 mL of the external buffer was taken out and replaced with 1.0 mL of fresh buffer. The amounts of released DOX were measured by UV−vis absorption at 480 nm. In Vitro Cytotoxicity of PC10ARGD Nanogels and PC10ARGDGNPs. HeLa cells were seeded in 96-well plates (5000 cells per well) and cultured in a cell incubator (5% CO2, 37 °C) for 22 h. The medium was discarded. The cells were washed twice with PBS and incubated with a series of concentrations of PC10ARGD and PC10ARGD-GNPs for another 48 h. The cells were washed twice with PBS and incubated with MTT (20 μL, 5 mg mL−1) solution for 4 h. The MTT solution was discarded, and DMSO (150 μL) was added in each well to dissolve the insoluble purple formazan crystals. The absorbance value at 490 nm was determined by a microplate reader (BioTek ELX808 IU, U.S.A.). Cell viabilities were calculated by assuming the control with a viability of 100%.

Scheme 1. Schematic Illustration of Preparing the PC10ARGD-GNPs Nanogels (a) and the Sequences of PC10A and PC10ARGD (b)

properties of polypeptide−GNPs hybrid nanogels were measured by UV−vis absorption spectra and transmission electron microscopy (TEM). The concentration of GNPs within hybrid nanogels was able to be tuned by changing the concentration of gold ions, and the size of hybrid nanogels could be tuned by changing the concentration of polypeptides. Different illumination time and pH effects on the synthesis of hybrid nanogels were also investigated. Cytotoxicity results suggested that both PC10ARGD nanogels and PC10ARGDGNPs presented good biocompatibility. Finally, we evaluated the targeting photoacoustic imaging of PC10ARGD-GNPs in HeLa cells and MCF-7 cells. Therefore, these hybrid nanogels have a great potential in the field of targeted bioimaging. 9842

DOI: 10.1021/acssuschemeng.7b01784 ACS Sustainable Chem. Eng. 2017, 5, 9841−9847

Research Article

ACS Sustainable Chemistry & Engineering Photoacoustic Imaging. PC10ARGD-GNPs hybrid nanogels prepared with different Au ion concentrations (0.24, 0.48, 0.72, and 1.20 mM) were dispersed into 50 μL of 1% agarose, and the mixture was transferred into disassemble 96-well plates which were filled with 100 μL of 1% agarose at the bottom. Photoacoustic signal intensities of PC10ARGD-GNPs hybrid nanogels were recorded by a home-built photoacoustic microscope (OR-PAM) with a 50 MHz, 0.25 in.diameter acoustic transducer. The laser wavelength was 523 nm. MCF-7 cells and HeLa cells were seeded on 35 mm glass-bottomed culture dishes (MatTek Corp., MA, U.S.A.) in their respective media and cultured in a cell incubator (5% CO2, 37 °C) for 22 h. The adherent cells were washed twice with PBS. PC10ARGD-GNPs prepared with 0.72 mM Au ion in serum-free DMEM was added in each culture dish, and the cells were incubated in a cell incubator for 2 h. HeLa cells were preincubated with PC10ARGD (2.5 μM) for 1 h in the competition assay of free RGD peptide. After washing twice with PBS, PC10ARGD-GNPs (0.72 mM) were added, and the cells were cultured for another 2 h. The cells were washed with PBS for three times and treated with 0.05% trypsin-EDTA at 37 °C for 5 min. The cells were dispersed into 50 μL of 1% agarose, and the mixture was transferred into disassemble 96-well plates which were filled with 100 μL of 1% agarose at the bottom. Photoacoustic signal intensities of PC10ARGD-GNPs hybrid nanogels were recorded by a home-built photoacoustic microscope (OR-PAM) with a 50 MHz, 0.25 in.diameter acoustic transducer. The laser wavelength was 523 nm.

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Figure 1. TEM images of PC10ARGD nanogels. The concentrations in panels a−d are 0.5% w/w, 0.1% w/w, 0.05% w/w, and 0.01% w/w, respectively.

RESULTS AND DISCUSSION The triblock genetically engineered polypeptides PC10A and PC10ARGD used in this study were expressed in E. coli and purified according to previous reports.16 PC10A consists of two coiled-coil domains (a zipper domain A and an associative domain P) and a random coil midblock C10 (Scheme 1b). An integrin-targeted tripeptide arginine-glycine-aspartic acid (RGD) was introduced into the polypeptide PC10A through the recombinant DNA technology for specific imaging of integrin αvβ3 expression, indicating that other interesting peptide sequences may also be introduced in the polypeptide PC10A. PC10A can form a stable physical hydrogel through the self-assembly of helical coiled-coil end domains (P and A) when the concentration is above 2% (w/w), which the P domain assembles into a pentameric physical association and the A domain assembles into a tetrameric physical association.28 Does the polypeptide PC10ARGD also interact with each other when the concentration is below 2% (w/w)? Based on the hydrogelforming capacity of the polypeptides through self-assembly, we assumed that micro- or nanosized hydrogels could be formed by these polypeptides when the concentration was very low. To verify this hypothesis, the form of PC10ARGD with various concentrations (0.5%, 0.1%, 0.05%, and 0.01% w/w) were investigated. The TEM results of PC10ARGD nanogels were shown in Figure 1. The size of 0.5%, 0.1%, 0.05%, and 0.01% PC10ARGD nanogels are 200−300, 100−150, 50−100, and 20−50 nm, respectively. Obviously, the PC10ARGD nanogels were observed, and the size of the nanogels could be tuned easily through changing the concentration of PC10ARGD. These results demonstrated that the size of PC10ARGD nanogels was able to be tuned by polypeptide concentration. In addition, it can be seen from TEM images that the PC10ARGD nanogels are not a typical vesicle structure because P and A can assemble into a physical association, and P and A cannot assemble with each other.28 Therefore, the structure of the PC10ARGD nanogels should be similar to the threedimensional polymer network of the macro-size PC10ARGD hydrogel.

The stability of PC10ARGD nanogels was examined by monitoring its hydrodynamic diameter and zeta potential. The size and zeta potential of 0.1% w/w PC10ARGD nanogels (25 °C, pH 7.4) varied with time are presented in Table 1. The size of PC10ARGD nanogels became progressively smaller with the time. The size of PC10ARGD nanogels at day 20 (101.4 ± 10.5 nm) was only half of that at day 1 (238.8 ± 13.4 nm). This may be attributed to the fact that PC10ARGD on the surface of nanogels could be released gradually.25,28 In addition, the zeta potential of PC10ARGD nanogels decreased from −35.2 ± 3.1 mV of day 1 to −15.3 ± 1.9 mV of day 20, which may be explained by the decreased size of PC10ARGD nanogels resulting from decreased density of the electric charge. However, the size of PC10ARGD nanogels remained 101.4 ± 10.5 nm after incubation for 20 days, which indicates that such nanogels are stable and can be used as templates for the synthesis of hybrid nanoparticles for long-time bioimaging or drug sustained release in biomedical applications. The excellent stability of this physical nanogel is consistent with the previously reported stability of the macro-size PC10A hydrogel.28 Photochemical synthesis methods have been developed to rapidly prepare metal nanoparticles in recent years.29−31 Previous studies have shown that GNPs could be synthesized quickly by reduction of HAuCl4 through photochemical decomposition of photoinitiator I-2959. Upon UV light excitation, the reducing agents of ketyl radicals were obtained by the decomposition of I-2959 via Norrish-type-I α-cleavage to reduce Au3+ to Au0.29 Housin et al. used BSA as templates to in situ synthesize functional BSA-GNPs in one step, which this method avoided further modification.32 Polypeptide-GNPs nanogels are expected to be in situ prepared by photochemical method in one step. The sequences of genetically engineered polypeptides can be designed and adjusted, which is beneficial to the systematic study of the relationship between the composition and properties of engineered polypeptides. First, 9843

DOI: 10.1021/acssuschemeng.7b01784 ACS Sustainable Chem. Eng. 2017, 5, 9841−9847

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ACS Sustainable Chemistry & Engineering Table 1. Size and Zeta Potential of 0.1% w/w PC10ARGD Nanogels (25 °C, pH 7.4) PC10ARGD nanogels

day 1

day 5

day 10

day 20

size (nm) zeta (mV)

238.8 ± 13.4 −35.2 ± 3.1

209.8 ± 8.7 −26.6 ± 3.7

170.3 ± 14.4 −16.2 ± 2.2

101.4 ± 10.5 −15.3 ± 1.9

the positively charged gold ions were adsorbed within the negatively charged PC10ARGD nanogels. The color of the mixture solution changed from colorless to yellow, which is the same color of the HAuCl4 solution. After exposure under 365 nm UV light irradiation, the color of the solution changed from yellow to wine red, indicating that Au3+ was reduced to form Au0. TEM images showed that formed GNPs were encapsulated within the PC10ARGD nanogels (Figure 2).

Figure 3. Absorption spectra (a and c) and photographs (b and d) of PC10ARGD-GNPs hybrid nanogels prepared by different UV (365 nm) irradiation time (20, 40, and 60 s) and at different pH values (3.0, 5.0, and 7.0).

hybrid nanogels at different pH values, whereas the absorption maxima of GNPs at pH 7.0 showed a slight blue-shift compared with that at pH 3.0 or 5.0. The stability of PC10ARGD-GNPs hybrid nanogels was also examined by a similar method applied to PC10ARGD nanogels. From Table 2, the size and zeta potential of PC10ARGD-GNPs hybrid nanogels decreased from 264.8 ± 11.2 nm and −29.5 ± 3.3 mV on day 1 to 166.7 ± 8.7 nm and −22.4 ± 2.1 mV on day 20, respectively. However, compared with the stability of the PC10ARGD nanogels in Table 1, we found that the size and potential of PC10ARGD-GNPs hybrid nanogels decreased more slowly. This is most likely due to the fact that PC10ARGD in hybrid nanogels can be absorbed aound the GNPs to improve its stability. To evaluate the ability of PC10ARGD nanogels as drug carriers, oil soluble DOX as model drug was successfully loaded in PC10ARGD nanogels by ultrasonic treatment (Figure S2). The PC10ARGD nanogels had a DOX loading content of 13.7% (w/w) by determining UV absorption at 480 nm. Previous reports have shown that the PC10ARGD hydrogel is pH sensitive.16 Therefore, PC10ARGD-GNPs-DOX is expected to exhibit pH-responsive drug release. The DOX release behaviors of PC10ARGD-GNPs-DOX hybrid nanogels were assessed using a dialysis method at 37 °C in acetate buffer (pH 5.0) containing 0.1% (w/w) Tween 80 and PBS buffer (pH 7.4) containing 0.1% (w/w) Tween 80.33 As shown in Figure 4, a rapid increase in the DOX release eventually reaching a plateau was observed. The release amounts of the DOX from the PC10ARGD-GNPs-DOX hybrid nanogels at pH 5 were significantly faster than those at pH 7.4. The rapid DOX release at pH 5.0 is probably due to pH sensitive properties of the PC10ARGD polypeptide. Nanogels used for delivery vehicles should present good biocompatibility.34 MTT assay was used to evaluate the cytotoxicity of PC10ARGD nanogels. HeLa cells were incubated with varied concentrations (0.05%, 0.1%, 0.5%, and 1% w/w) of PC10ARGD nanogels for 48 h to investigate the cytotoxicity of

Figure 2. Characterization of one step in situ synthesis of PC10ARGDGNPs hybrid nanogels. TEM of PC10ARGD-GNPs hybrid nanogels: (a) 0.1% PC10ARGD + 0.24 mM Au3+, (b) 0.1% PC10ARGD + 0.48 mM Au3+; the UV−vis absorption spectra (c) and photographs (d) of hybrid nanogels prepared with different Au3+ concentrations (0.24, 0.48, and 0.96 mM).

The Au ion concentration effect on the characterization of polypeptide−GNPs was investigated. The results showed that the density of GNPs within the PC10ARGD-GNPs increased with the increasing of the concentration of Au ion (Figure 2a,b). However, the size of GNPs had no obvious change and was 9.5 ± 0.6 nm. The UV−vis absorption spectra also showed the same result. When the Au ion concentration increased from 0.24 to 0.96 mM, the absorption peak position of hybrid nanogels had no obvious change, whereas the intensity of UV− vis absorption increased gradually and the color changed from light red to wine red (Figure 2c,d). Additionally, altering of the PC10ARGD concentration only affected the size of hybrid nanogels and presented no obvious effects on the size or the density of GNPs within hybrid nanogels (Figure S1). The results of the experiments conducted using different irradiation times were shown in Figure 3a,b. With the increasing of the irradiation time, the color of the hybrid nanogels solution deepened, and the intensity of the absorption enlarged. These results indicated that the density of GNPs within hybrid nanogels was able to be tuned by the irradiation time. The effect of pH value on fabrication of hybrid nanogels was also studied, and the results are shown in Figure 3c,d. There were no obvious differences in the density of GNPs within 9844

DOI: 10.1021/acssuschemeng.7b01784 ACS Sustainable Chem. Eng. 2017, 5, 9841−9847

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ACS Sustainable Chemistry & Engineering

Table 2. Size and Zeta Potential of PC10ARGD-GNPs Nanogels (0.1% w/w PC10ARGD and 0.72 mM Au Ion) under the Condition of 25 °C and pH 7.4 PC10ARGD-GNPs nanogels

day 1

day 5

day 10

day 20

size (nm) zeta (mV)

264.8 ± 11.2 −29.5 ± 3.3

252.6 ± 9.2 −28.0 ± 4.3

208.0 ± 12.2 −23.1 ± 2.3

166.7 ± 8.7 −22.4 ± 2.1

sensitive to the absorption characteristics of the sample. Acoustic signals in photoacoustic imaging are generated by the transient variations in temperature which are caused by the absorpted light. To evaluate the photoacoustic imaging of PC10ARGD-GNPs, PC10ARGD-GNPs hybrid nanogels prepared with different Au ion concentrations (0.24, 0.48, 0.72, and 1.20 mM) were used to assess the photoacoustic signal intensity by a home-built photoacoustic microscope (ORPAM) with a 50 MHz, 0.25 in.-diameter acoustic transducer. The laser wavelength was 523 nm. PC10ARGD-GNPs hybrid nanogels exhibited a superior photoacoustic signal (Figure 6a,b), which possibly benefit from the matching of UV−vis absorbance peak and laser wavelength. The concentration of Au ion between photoacoustic signal intensity shows an excellent linear relationship, indicating that PC10ARGD-GNPs hybrid nanogels could be applied in photoacoustic imaging. Considering the cytotoxicity and the photoacoustic signal intensity, we chose PC 10ARGD-GNPs hybrid nanogels prepared with 0.72 mM Au ion to perform cellular targeted photoacoustic imaging experiment. To evaluate the integrin αvβ3 binding affinity of the PC10ARGD-GNPs hybrid nanogels in vitro, HeLa cells with overexpression of αvβ3 integrin receptors and MCF-7 cells with lower-expression of αvβ3 integrin receptors were chosen for the target-specific photoacoustic imaging. PC10ARGD-GNPs hybrid nanogels prepared with 0.72 mM Au ion were incubated with MCF-7 and HeLa cells for 2 h. The photoacoustic signal results are shown in Figure 6c,d. It is clear that the control group (cell only) displayed almost no photoacoustic signal intensity, and the photoacoustic signal intensity of the test group (HeLa, 0.278 ± 0.005) was obviously higher than that of negative control group (MCF-7, 0.089 ± 0.002), which probably attributed to the receptor-mediated endocytosis between αvβ3 integrin receptors and RGD.16,39 In addition, the intergrin receptor specificity of PC10ARGD-GNPs hybrid nanogels was further verified by a competition experiment with free RGD peptide in HeLa cells. The photoacoustic signal intensity of HeLa cells using 2.5 μM free PC10ARGD polypeptide competition was very weak (Figure 6c,d). Therefore, this kind of hybrid nanogel has promise as a contrast agent for targeted photoacoustic imaging.

Figure 4. In vitro DOX release profile from PC10ARGD-GNPs-DOX hybrid nanogels (0.1% PC10ARGD, 0.72 mM Au ion, and 159.7 μg mL−1 DOX) in acetate buffer containing 0.1% (w/w) Tween 80 (pH 5.0) and PBS buffer containing 0.1% (w/w) Tween 80 (pH 7.4) at 37 °C.

PC10ARGD nanogels. As shown in Figure 5a, the viabilities of cells incubated with PC10ARGD nanogels were above 90%,

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Figure 5. In vitro cell viability of HeLa cells incubated with different concentrations of PC10ARGD nanogels (a) and the PC10ARGD-GNPs hybrid nanogels at the Au ion concentrations of 0.24, 0.36, 0.48, and 0.72 mM (b).

CONCLUSIONS In conclusion, two triblock polypeptides PC 10 A and PC10ARGD were used to prepare nanogels at low concentration through self-assembly. The sizes of nanogels were able to be tuned by simply altering the concentration of the polypeptides. PC10ARGD-GNPs hybrid nanogels were in situ synthesized by the photochemical synthesis method in one step. With the increasing of Au ion, the density of GNPs within the hybrid nanogels increased. Changing of polypeptide concentration showed no obvious effect on the density of GNPs within the hybrid nanogels. The increasing of irradiation time can enlarge the density of GNPs within the hybrid nanogels, and the pH value change from 3.0 to 7.0 had a little effect on the preparation of hybrid nanogels. MTT results indicated that PC10ARGD and PC10ARGD-GNPs hybrid

indicating that these nanogels are nontoxic. Therefore, PC10ARGD nanogels satisfy the requirement of biosafety as the vehicle. In addition, the cytotoxicity of PC10ARGD-GNPs hybrid nanogels was also measured with MTT (Figure 5b). Within 48 h, the viabilities of cells incubated with PC10ARGDGNPs containing Au ion less than 0.72 mM were above 80%. These results indicated that such PC10ARGD-GNPs hybrid nanogels showed a good biocompatibility and were suitable for application in biomedical fields. Photoacoustic imaging is an innovative biomedical imaging modality offering high spatial resolution, high contrast, and deep penetration.35−38 Photoacoustic imaging is highly 9845

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Figure 6. Linear relationship between photoacoustic signal intensity and the concentration of Au ion (1:0 M; 2:0.24 mM; 3:0.48 mM; 4:0.72 mM; and 5:1.2 mM) for preparation of PC10ARGD-GNPs hybrid nanogels (a and b); photoacoustic signal intensity of control group (HeLa cells only), MCF-7, and HeLa cells incubating with PC10ARGD-GNPs hybrid nanogels prepared with 0.72 mM Au ion for 2 h, and HeLa cells incubating with PC10ARGD-GNPs in the presence of free PC10ARGD polypeptide (2.5 μM; c and d).

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 81771878, 81471697), the Fundamental Research Funds for the Central Universities (Hust, 2016YXMS253 and 2017KFXKJC002), the China Postdoctoral Science Foundation (2017M612462), Yellow Crane Talent (Science & Technology) Program of Wuhan City and Applied Basic Research Program of Wuhan City (2016060101010044), and the Natural Science Foundation of Hubei Province (2015CFC892). The authors thank Prof. H.-R. Li (Wuhan Donghu University) for the valuable discussion and are grateful for Prof. D. Tirrell for generously providing pQE9PC10A plasmid. We also thank the Analytical and Testing Center (HUST) for the help with measurements.

nanogels with Au ion below 0.72 mM presented almost no cytotoxicity to HeLa cells. The results of photoacoustic imaging showed that PC10ARGD-GNPs hybrid nanogels possessed excellent cellular targeted photoacoustic imaging. All of these results suggest that such hybrid nanogels have a great potential application in biomedicine.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01784. TEM images of PC10ARGD-GNPs hybrid nanogels (a) 0.1% PC10ARGD + 0.24 mM Au3+ and (b) 0.05% PC 10 ARGD + 0.24 mM Au 3+ . Photographs of PC10ARGD-DOX hybrid nanogels and PC10ARGDGNPs-DOX hybrid nanogels (a) and standard curve of DOX by measuring UV absorption at 480 nm (b). (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: +86 27 87792202; Tel: +86 27 87793863. *E-mail: [email protected]. ORCID

Yuan-Di Zhao: 0000-0002-4286-4275 Bo Liu: 0000-0002-5450-5528 Author Contributions §

R-M.J. and M.-H.Y. contributed equally to this work

Notes

The authors declare no competing financial interest. 9846

DOI: 10.1021/acssuschemeng.7b01784 ACS Sustainable Chem. Eng. 2017, 5, 9841−9847

Research Article

ACS Sustainable Chemistry & Engineering

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DOI: 10.1021/acssuschemeng.7b01784 ACS Sustainable Chem. Eng. 2017, 5, 9841−9847