Stable and Biocompatible Mushroom β-Glucan Modified Gold

Article Cite This: J. Agric. Food Chem. 2017, 65, 9529-9536

pubs.acs.org/JAFC

Stable and Biocompatible Mushroom β‑Glucan Modified Gold Nanorods for Cancer Photothermal Therapy Xiaojie Li,† Jiajing Zhou,‡ Chaoran Liu,† Qirong Xiong,‡ Hongwei Duan,*,‡ and Peter C. K. Cheung*,† †

School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive 637457, Singapore

‡

ABSTRACT: Naturally occurring β-glucans have been widely regarded as a natural source for functional foods and pharmaceuticals due to their immunomodulatory property and antitumor activity. However, physicochemically stable and biocompatible β-glucans are rarely explored as a carrier for nanomaterials to overcome the problems of aggregation and nanotoxicity. Here, we developed highly stable and biocompatible mushroom β-glucan coated gold nanorods (AuNR-Glu) for cancer photothermal therapy by integrating Pleurotus tuber-regium sclerotial β-glucan (Glu) and plasmonic gold nanorods (AuNRs) possessing photothermal property in the second near-infrared (NIR-II) window. AuNR-Glu showed high colloidal stability in various biological media, even in simulated gastric fluid. Moreover, AuNR-Glu had low cytotoxicity and high photothermal stability, which are excellent characteristics for photothermal agents for cancer therapy. In vitro experiments showed that AuNR-Glu nanohybrid was effective against MCF-7 (only 4.5 ± 0.9% viability) at a low dose of 20 μg/mL under NIR-II at a safe laser power density (0.75 W/cm2). Natural mushroom β-glucans are potential functional polymers that can be used to fabricate nanohybrids for biomedical applications. KEYWORDS: mushroom β-glucan, gold nanorods, cancer photothermal therapy, cytotoxicity, colloidal stability

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INTRODUCTION Mushroom β-glucans are natural food polysaccharides consisting of β-D-glucose monomers linked by either β-(1,3)-, β-(1,4)-, or β-(1,6)-glycosidic bonds. They have been valued as functional food ingredients with multiple bioactivities, including immunomodulatory,1,2 antitumor,3 antimutagenic,4 and antidiabetic5 properties. In addition to these beneficial effects, structural stability and biocompatibility are the distinctive properties of mushroom β-glucans for them to serve as natural polymeric carriers for nanoparticles in biomedical applications. For example, biocompatible nanoparticle/β-glucan hybrids have been fabricated by a one-pot method in which metal precursors and β-glucans were simply mixed.6 The abundant hydroxyl groups in β-glucans facilitate the formation of nanoparticles in situ due to their affinity to various metal ions such as SeO32−7 and AuCl4−.8 The nanoparticles prepared in this way were encapsulated by mushroom β-glucans, which showed good biocompatibility. However, there was no fine control over the size, shape, or function of these nanoparticles, which significantly limits their potential biomedical applications. Traditional wet-chemical synthesis, on the other hand, has been used to generate a large collection of uniform functional nanoparticles with well-defined structure. However, these uniform synthetic nanoparticles have poor colloidal stability9 and considerable nanotoxicity.10 To overcome these problems, postsynthesis treatment using synthetic polymers has been widely exploited to modify the surface properties of the synthesized nanoparticles for better stability and lower cytotoxicity. Until now, many synthetic polymeric materials such as dendrimers11 and linear polymers12 have been developed for these purposes, but there are very few reports on using biocompatible natural polymers to modify the surface © 2017 American Chemical Society

properties of nanomaterials. Therefore, the surface modification of nanoparticles with better stability and safety for biological applications is an important yet challenging topic. Pleurotus tuber-regium (PTR) is a tropical mushroom from basidiomycetes, and both its sclerotium and fruit body are edible.13 Notably, PTR is a rich source of β-glucans with its sclerotium containing over 60% dry weight of β-glucan.14 In our previous work, PTR sclerotial β-glucans were found to be hyper-branched glucans with spherical architecture, which might provide protection to nanoparticles in a core−shell structure.15,16 To the best of our knowledge, there is no previous work reporting the use of mushroom sclerotial βglucans to improve the stability and decrease the cytotoxicity of nanoparticles in a postsynthesis approach. Plasmonic gold nanorods (AuNRs) of controllable localized surface plasmon resonance (LSPR) have attracted tremendous attention for their applications in cancer photothermal therapy (PTT) by converting absorbed light into heat during the plasmon relaxation process.17 Recently, light in the second near-infrared (NIR-II) window (1000−1400 nm) has attracted particular interest in clinical applications. This is because NIR-II can penetrate or propagate to the deeper tissue region with less scattering of photons compared to visible light (390−700 nm) and the conventional first near-infrared (NIR-I) window (750− 900 nm).18 Moreover, NIR-II lasers have a value of human maximum permissible exposure (1 W/cm2) higher than that of the NIR-I laser (0.33W/cm2) according to the American Received: Revised: Accepted: Published: 9529

August 21, 2017 October 5, 2017 October 10, 2017 October 10, 2017 DOI: 10.1021/acs.jafc.7b03895 J. Agric. Food Chem. 2017, 65, 9529−9536

Article

Journal of Agricultural and Food Chemistry National Standard Institute laser safety guide.19 Thus, cancer PTT using NIR-II light is a relatively safer spectral tool for clinical use. However, there are some challenges in applying AuNRs with LSPR in NIR-II for cancer therapy. One of these challenges is the preparation of NIR-II responsive AuNRs within 200 nm, which is the cutoff size of porous blood capillaries in tumors.20 Another challenge that hinders the application of AuNRs is the cytotoxicity caused by the residual positively charged cetyltrimethylammonium bromide (CTAB), which is the essential structural template and surface ligand for AuNRs.10 Hence, the surface modifications of AuNRs with natural polymeric materials such as mushroom β-glucans is envisioned to be a safe and low-cost strategy to design biocompatible and efficient PTT agents for cancer therapy. In this project, we applied PTR sclerotial β-glucans as biocompatible coatings to encapsulate individual NIR-IIresponsive AuNRs into nanohybrids to enhance the stability and overcome the cytotoxicity of AuNRs for cancer PTT. The colloidal and photothermal stability as well as the cytotoxicity of the nanohybrid formed by AuNRs coated with PTR βglucans (AuNR-Glu) were evaluated. The cancer PTT effectiveness of AuNR-Glu against MCF-7 cells in NIR-II at low nanoparticle dose and safe laser power density was also investigated. The results indicated that naturally occurring biocompatible β-glucans are attractive candidates in the design of biomaterial-based functional nanohybrid systems with high stability and low cytotoxicity.

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respectively, while the uronic acid content was estimated by the mhydroxydiphenyl−sulfuric acid method.23 Gas chromatography (6890N, Agilent Technology, United States) coupled to mass spectrometry (5973N, Agilent Technology, United States) was used for both the analysis of monosaccharides as their alditol acetate derivatives and for linkage positions between sugar residues as their partially methylated alditol acetate derivatives.24,25 The degree of branching (DB) value was calculated according the equation below:

DB =

NT + NB NT + NB + NL

where NT, NB, and NL are the number of terminal residues, branching residues, and liner residues, respectively. The infrared spectrum of Glu was recorded with a Fourier transform infrared spectrometer (FT-IR, Nicolet 670) in the range 4000−400 cm−1 using the KBr-disk method.26 Synthesis of AuNRs. AuNRs were synthesized by using seedmediated growth method with CTAB templates as previously reported.27,28 The gold seeds were prepared by adding HAuCl4 solution (5 mL, 0.5 mM) to CTAB (5 mL, 0.2 M). Then, NaBH4 (600 μL, 10 mM) was freshly prepared and added under vigorous stirring to the mixture with an immediate color change from yellow to brownish yellow. This solution was stored at 30 °C for 1 h and used as the seed solution for the synthesis of AuNRs. After that, a growth solution was prepared by adding HAuCl4 solution (10 mL, 1 mM) to CTAB (10 mL, 0.2 M), followed by adding AgNO3 (120 μL, 0.1 M) and then hydroquinone (600 μL, 0.1 M), leading to a color change from yellow to colorless. Then, 300 μL seed solution was added into the mixture thoroughly and stored at 30 °C overnight. The AuNR solution was centrifuged 3 times (7500g, 15 min) to remove excess CTAB from AuNRs dispersion. Preparation of AuNR-Glu. Glu (10 mg) was dissolved in 1 mL of DI water with vigorous stirring at 85 °C for 30 min. Then, 0.5 mL freshly prepared AuNRs were added rapidly into the solution. After 1 min, 90 μL of 2.5 M NaCl was added, and the suspension was kept at 85 °C for 1 min. Then, AuNR-Glu was obtained by washing with DI water (3 times) followed by centrifugation (7500g, 10 min). Gel electrophoresis was run on 0.15% agarose gel with 0.5 × TBE as running buffer at 110 V for 15 min. The ζ-potentials of the AuNRs and AuNR-Glu in DI water were analyzed by using Malvern NANO-ZS90 Zetasizer. The masses of the gold element in AuNRs and AuNR-Glu solutions were quantified by prodigy inductively coupled plasma spectrometry (ICP-OES, Teledyne Leeman Laboratories), and the concentration of AuNRs and AuNR-Glu solutions were represented in terms of gold amount. Determination of Colloidal Stability. One milliliter of AuNRs and AuNR-Glu with the same optical density (OD = 1) at 1064 nm were centrifuged (9000g, 10 min) and resuspended in the same volume of either PBS, serum-containing medium, or simulated gastric fluid (SGF). SGF consisted of 3.2 mg/mL pepsin in 0.03 M NaCl at pH 1−2, as previously described.29 The UV−vis−NIR spectra were recorded at different time points between 400 and 1400 nm using an UV−vis−NIR spectrophotometer. Measurement of Photothermal Effect. To measure the photothermal effect of AuNR-Glu, 1 mL of AuNR-Glu solution of different concentrations was added to a cuvette exposed to 1064 nm laser at different power densities for 5 min. The photothermal stability experiments were carried out using 1 mL of AuNR-Glu (20 μg/mL) placed in a cuvette. The sample was irradiated using 1064 nm laser with power density of 1.0 W/cm2 for 20 min until a steady temperature was reached, followed by cooling to room temperature at 24 °C. The temperature was recorded every 30 s by an IR camera. Cell Line and Cell Culture. MCF-7 cells were cultured in a high glucose DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics with 5% CO2 at 37 °C. Cytotoxicity Analysis. Cytotoxicity of AuNRs and AuNR-Glu was evaluated using a cell-counting kit (CCK-8 assay).30 Briefly, MCF-7 cells in the log phase of growth were seeded in a 96-well plate at a

MATERIALS AND METHODS

Chemicals. Analytical grade reagents, including ethyl acetate, acetone, sodium hydroxide (NaOH), sodium borohydride (NaBH4), cetyltrimethylammonium bromide (CTAB), silver nitrate (AgNO3), and hydroquinone were purchased from Sigma-Aldrich, United States. Gold(III) chloride trihydrate (HAuCl4·3H2O) was supplied by Alfa Aesar. The cell counting kit-8 (CCK-8) was purchased from DOJINDO, Japan. Fetal bovine serum (FBS), DMEM (high glucose), and propidium iodide (PI) were purchased from Thermo Fisher Scientific, United States. Ultrapure water (18.2 MΩ·cm) was purified using a Millipore Milli-Q purification system and used in all experiments. Microscopic Structural Characterization. To observe transmission electron microscopy (TEM) images, 5 μL of colloidal suspension was pipetted onto a TEM sample grid and dried at room temperature. The TEM images of AuNRs and AuNR-Glu were acquired on a Jeol JEM 2010 electron microscope at an acceleration voltage of 300 kV. UV−vis−NIR spectra were recorded using Agilent Cary 5000 UV−vis−NIR spectrophotometer. Imaging experiments were conducted on an Olympus LX71 inverted microscope. Preparation of Glu. Natural mushroom β-glucan (Glu) was prepared according to our pervious reports with slight modification.16 Briefly, dried powders of sclerotium of P. tuber-regium were defatted by ethyl acetate (2 h, 3 times) and acetone (2 h, 3 times), followed by extraction in boiling water (2 h, 2 times). The water-insoluble fraction was further extracted with 1 M NaOH at room temperature (12 h, 2 times), and the extracts were then neutralized to pH 7.0 with 1 M HCl to precipitate the alkali-soluble polysaccharides. The polysaccharides obtained were dialyzed in water for 2 days using tubings with a molecular weight cutoff of 8000 Da to remove low molecular weight impurities and then freeze-dried to give Glu. Chemical Composition and Linkage Analysis of Glu. The molecular weight (Mw) profile of Glu was measured by a Waters size exclusion chromatography system coupled with a refractive index (RI) detector (Waters 2414, Waters Inc., United States), and the molecular weight was calibrated with pullulan standards. The amounts of the total carbohydrate and proteins in Glu were determined using the phenol−sulfuric acid21 and bicinchoninic acid protein assay,22 9530

DOI: 10.1021/acs.jafc.7b03895 J. Agric. Food Chem. 2017, 65, 9529−9536

Article

Journal of Agricultural and Food Chemistry

2900 cm−1 was due to the C−H antisymmetrical stretching vibration.31 Methylation analysis of the glycosidic linkages in Glu had found that it had a mixed-linkage linear chain consisted of →1)D-Glcp-(4 → (17.53%), → 1)-D-Glcp-(6 → (7.83%) and →1)D-Glcp-(3 → (6.55%) with 32.94% nonreducing terminal glucose (Table 1). There were two kinds of branched sugar

density of 1 × 104 per well. After overnight incubation, cells were treated with various concentrations of AuNRs and AuNR-Glu (10, 20, 40, and 80 μg/mL) and then incubated for 24 h. Then, 10 μL of CCK8 solution was added to each well and incubated for another 4 h. In this assay, the amount of formazan dye, generated by the activities of dehydrogenases in cells by reduction of WST-8, is directly proportional to the number of living cells. Hence, the absorbance of each well measured at 450 nm by using a microplate reader was used to determine the cell viability. Photothermal Therapy of Cancer Cells. MCF-7 cells were first seeded in 96-well plates at a density of 1 × 104 per well and incubated for 24 h. Then, the cells were treated 20 μg/mL AuNR-Glu. After incubation for 2 h, each well was exposed to a 1064 nm laser with different power densities for 5 min. Cells without AuNRs were used as control. Subsequently, the culture medium was changed back to fresh medium and incubated at 37 °C for another 12 h. The cell viability was investigated by the CCK-8 assay. Live/Dead Cell Staining Assay. MCF-7 cells with green fluorescent protein (GFP) stably expressed on their tubulin were seeded in 96 well plates at a density of 1 × 104 per well and were allowed to grow for 24 h at 37 °C until nearly 90% confluent. After that, cells were incubated with 20 μg/mL AuNR-Glu for 2 h and then irradiated with a 1064 nm laser at different power densities for 5 min. The cells without AuNR-Glu treatment were used as the control. Then, the culture medium was replaced with fresh medium, and the cells were maintained under incubation for 12 h. Finally, all cells were treated with PI solution (10 μM) in PBS buffer and incubated for 20 min at 37 °C followed by washing three times with PBS. After all the washings, the cells were visualized by fluorescence microscopy (Olympus, IX71). Statistical Analysis. Numeric data are expressed in mean values ± standard deviation with N = 3. Statistical significance of the means was analyzed by one-way ANOVA followed by Dunnett’s multiple comparison or two-way ANOVA with Sidak’s multiple comparison, as appropriate.

Table 1. Peak Area and Molar Ratio of Glycosidic Linkages in Glu methylated sugars 2,3,4,6-Me4-Glc 2,4,6-Me3-Glc 2,3,6-Me3-Glc 2,3,4-Me3-Glc 2,4-Me2-Glc 2,3-Me2-Glc TU/BPc DB

linkage pattern b

T-Glcp 1,3-Glcp 1,4-Glcp 1,6-Glcp 1,3,6-Glcp 1,4,6-Glcp

peak area (%)a

molar ratio

32.9 6.55 17.5 7.83 29.6 5.56 0.94

5.92 1.18 3.15 1.41 5.32 1 0.68

a

Data are the mean of the triplicate. bGlcp, glucopyranose. cTU/BP = terminal units/branching points.

residues in Glu 1,3,6-Glcp (29.59%) and 1,4,6-Glcp (5.56%), having branching points at the O-4 and/or O-3 positions. These results suggested that Glu was a hyper-branched glucan with a high DB value of 0.68. The mixed (1 → 4), (1 → 6), and (1 → 3) linked glycosidic bonds and high DB value are the unique structural features of Glu compared with those of other mushroom β-glucans such as Lentinula edodes32 and Poria cocos33 which both have only a linear (1 → 3) backbone and less (1 → 6) branching in the case of L. edodes. Preparation and Characterization of AuNRs and AuNR-Glu. AuNRs with a longitudinal LSPR peak at 1059 nm in the NIR-II window were successfully synthesized according to the seed-mediated growth method after some modifications (Figure 2A).27,28 Transmission electron microscopy (TEM) images showed that the AuNRs had a uniform size of 135.5 × 22.2 nm (Figure 2B), offering suitable dimensions for cancer therapy. Afterward, the AuNRs were mixed with different amount of Glu (1, 5, 10, and 15 mg/mL) to prepare AuNR-Glu. The Glu coating on the surface of AuNRs was confirmed by electrophoresis experiments (Figure 2C). Interestingly, CTAB-capped AuNRs aggregated in the well with no band shift, whereas AuNR-Glu exhibited an increased mobility after being coated with Glu, probably due to the negatively charged uronic acids in Glu. The surface of AuNRs seemed to be fully coated by Glu at a concentration of 10 mg/ mL, as evidenced by gel mobility and water dispersibility, whereas obvious precipitate and sedimentation was observed at the bottom of the vial containing 1 and 5 mg/mL Glu, respectively (Figure 2C). In line with the electrophoresis results, the ζ-potentials changed notably from 43.4 ± 3.1 to −26.9 ± 1.7 mV after the AuNRs were coated with 10 mg/mL Glu, suggesting the complete removal of the positively charged CTAB by Glu. TEM observation further confirmed that the individual AuNRs were encapsulated by a dense layer of Glu without changing their original nanostructure (Figure 2D). These findings indicated that AuNRs were possible coated by Glu through electrostatic attraction34 even though hydrogen bonding might be involved.7 It is notable that the LSPR peak of AuNR underwent a slight red shift from 1059 to 1074 nm after

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RESULTS AND DISCUSSION Structural Analysis of Glu. After the alkali extraction and purification step by dialysis, the content of total carbohydrates in the PTR sclerotial polysaccharides (Glu) was 93.5 ± 2.2% with only small amount of uronic acids (1.49 ± 0.03%), while no protein was detected. Glu had a Mw of 1.8 × 106 Da and contained only glucose as the neutral sugar as determined by the alditol acetate derivatives. The FR-IR spectrum of Glu exhibited an absorption peak at ∼900 cm−1 (Figure 1), which was the characteristic absorption peak of β configuration of glucan.16 The intense peak around 3370 cm−1 was attributed to the hydroxyl groups stretching vibration, and the weak peak at

Figure 1. FT-IR spectrum of Glu. 9531

DOI: 10.1021/acs.jafc.7b03895 J. Agric. Food Chem. 2017, 65, 9529−9536

Article

Journal of Agricultural and Food Chemistry

Figure 2. (A) UV−vis−NIR absorption spectra of Glu (10 mg/mL), AuNRs, and AuNR-Glu. (B) TEM image of AuNRs. (C) AuNRs coated with different amount of Glu. Lane 1: AuNRs, 2: 1 mg/mL Glu, 3: 5 mg/mL Glu, 4: 10 mg/mL Glu, 5: 15 mg/mL Glu. Top panel: agarose gel electrophoresis. Bottom panel: photo appearance. (D) TEM image of AuNR-Glu coated with 10 mg/mL Glu.

Figure 3. Colloidal stability of AuNR and AuNR-Glu in different biological media. AuNRs and AuNR-Glu were suspended in the same volume of (A, D) PBS, (B, E) simulated gastric fluid, and (C, F) serum-containing medium, respectively.

solutions, we found that AuNRs were severely aggregated and the LSPR peak absorption was lost after 0.5 h of dispersion in PBS and the simulated gastric fluid (SGF) (Figures 3A and B, respectively). This should be due to the high salt concentration (∼150 mM) of PBS and low pH (1−2) of the SGF, which both could significantly reduce the electrostatic repulsion between CTAB-capped AuNRs.9 Interestingly, AuNRs displayed improved colloidal stability in serum-containing medium with

Glu coating due to the high refractive index of Glu when compared to that of water (Figure 2A). Colloidal Stability of AuNR-Glu Dispersed in Relevant Biological Media. The colloidal stability of AuNRs and AuNR-Glu was determined by the longitudinal LSPR peak absorbance in the NIR-II region, which is sensitive to aggregate formation of AuNRs and the refractive index of surrounding medium. From the UV−vis−NIR spectra in different medium 9532

DOI: 10.1021/acs.jafc.7b03895 J. Agric. Food Chem. 2017, 65, 9529−9536

Article

Journal of Agricultural and Food Chemistry

Figure 4. (A) Heating and cooling curves of AuNRs and AuNR-Glu solutions (OD = 1) during irradiation by a 1064 nm laser (1.0 W/cm2) for 20 min. (B) Heating curves of AuNR-Glu with different concentrations by a 1064 nm laser at 1.0 W/cm2. (C) Heating curves of 20 μg/mL AuNR-Glu by different laser power densities. (D) Photothermal stability of AuNR-Glu solution (OD = 1) irradiated for 20 min at 1.0 W/cm2 to reach equilibrium.

was used (40 μg/mL). In the case of a fixed AuNR-Glu concentration (20 μg/mL), different power densities were used to evaluate the photothermal performance of AuNR-Glu (Figure 4C). The temperature of AuNR-Glu elevated to 66.7 °C dramatically at a power density of 1.0 W/cm2. Within a safe NIR-II laser dose (