Green synthesis of bioactive polysaccharide-capped gold nanoparticles for lymph node CT imaging

Accepted Manuscript Title: Green synthesis of bioactive polysaccharide-capped gold nanoparticles for lymph node CT imaging Authors: Saji Uthaman, Hyeon Sik Kim, Vishnu Revuri, Jung-Joon Min, Yong-Kyu Lee, Kang Moo Huh, In-Kyu Park PII: DOI: Reference:

S0144-8617(17)31188-8 https://doi.org/10.1016/j.carbpol.2017.10.042 CARP 12891

To appear in: Received date: Revised date: Accepted date:

10-9-2017 5-10-2017 10-10-2017

Please cite this article as: Uthaman, Saji., Kim, Hyeon Sik., Revuri, Vishnu., Min, JungJoon., Lee, Yong-Kyu., Huh, Kang Moo., & Park, In-Kyu., Green synthesis of bioactive polysaccharide-capped gold nanoparticles for lymph node CT imaging.Carbohydrate Polymers https://doi.org/10.1016/j.carbpol.2017.10.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Green

synthesis

of

bioactive

polysaccharide-capped

gold

nanoparticles for lymph node CT imaging Saji Uthamana, d, #, Hyeon Sik Kimb, #, Vishnu Revuric, Jung-Joon Minb, Yong-Kyu Leec, Kang Moo Huhd*, In-Kyu Parka* a

Department of Biomedical Sciences, BK21 PLUS Centre for Creative Biomedical Scientists,

Chonnam National University Medical School, 160 Baekseo-ro, Gwangju 61469, Republic of Korea b

Department of Nuclear Medicine, Chonnam National University Medical School, Gwangju

61469, Republic of Korea c

Department of Green Bioengineering, Korea National University of Transportation, Chungju

27469, Republic of Korea d

Department of Polymer Science and Engineering, Chungnam National University, 99 Daehak-

ro, Yuseong-gu, Daejeon 34134, Republic of Korea *

Corresponding Author E-mail: [email protected]

*

Corresponding Author E-mail: [email protected]

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Graphical Abstract

Highlights  

M-GNPs as CT contrast agent for lymph node imaging M-GNPs were spherical nanoparticles with an average diameter of 9.18 ± 0.71 nm



M-GNPs exhibited mannose receptor mediated endocytosis in antigen presenting



cells

Local administration of M-GNPs led to significantly enhanced X-ray imaging of popliteal lymph nodes

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ABSTRACT

The development of biologically targeted contrast agents for X-ray computed tomography (CT) imaging remains a major challenge. Here, we investigated a green chemistry-based synthesis of lymph node-targeted mannan-capped gold nanoparticles (M-GNPs) as a CT contrast agent. In this study, mannan was used as a reducing and stabilizing agent for gold nanoparticles (AuNPs). M-GNPs were readily internalized by antigen-presenting cells (APCs) through mannose receptors-mediated endocytosis. The M-GNPs, which had a spherical morphology, had an average diameter of 9.18 ± 0.71 nm and surface plasmon resonance (SPR) absorption spectra with maximal absorption at 522 nm. The M-GNPs displayed a concentration-based X-ray attenuation property with a maximum Hounsfield unit (HU) value of 303.2 ± 10.83. The local administration of M-GNPs led to significantly enhanced X-ray contrast for the imaging of popliteal lymph nodes. These findings demonstrated that M-GNPs can be used as biologically targeted contrast agents for CT imaging.

Keywords: X-ray computed tomography, mannan, gold nanoparticles, contrast agent, green chemistry.

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1. Introduction. Lymph nodes (LNs) are distributed throughout the body and play an important role in the immune response. Lymphatic networks are one of the major routes through which cancer cells spread during metastasis, and lymph nodes are considered the first site where metastasis occurs (Hayashi, Nakamura, & Ishimura, 2013; Schroeder et al., 2012). To prevent metastasis and cancer recurrence, the LNs near tumors are surgically removed. However, such operative techniques are very difficult because LNs are very small and difficult to identify through gross sectioning. Therefore, clear visualization is desirable. Molecular imaging techniques, such as Xray computed tomography (CT), positron emission tomography (PET) and magnetic resonance imaging (MRI), are attractive due to their non-invasive nature, which enables the naked eye visualization of LNs. Among the various molecular imaging techniques, CT is one of the most extensively used non-invasive diagnostic tools in clinics due to its cost effectiveness, strong tissue penetration and high three-dimensional resolution and reconstruction possibilities (H. Liu et al., 2014; Lusic & Grinstaff, 2013; Peng et al., 2013; Peng et al., 2012; Shi et al., 2016). The quality of CT images is based on the capability for contrast agents to enhance contrast effects. The commonly used, iodine-based CT contrast agents are non-specific and have short imaging times and therefore, cannot be used to obtain high-resolution images of specific organs (Y. L. Liu et al., 2012). Recently, nanoparticle-based contrast agents have been effectively developed to overcome the drawbacks of iodine-based CT contrast agents by exhibiting increased X-ray absorption and longer circulation half-lives (Detappe et al., 2017; Evertsson et al., 2017; Jin et al., 2017; Kim et al., 2017; Liang, Fang, Li, Zhang, & Wang, 2017; Meir et al., 2017; Mesbahi, Famouri, Ahar, Ghaffari, & Ghavami, 2017; Rabin, Perez, Grimm, Wojtkiewicz, & Weissleder, 2006). Among

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the various developed nanoparticles, gold nanoparticles (AuNPs) have been recognized as ideal contrast agents for CT imaging due to their longer blood circulation times, greater biocompatibility, higher X-ray absorption coefficients at 80-100 keV, unique surface plasmon resonance (SPR) characteristics, ease in surface modification, and passive tumor targeting functions (Chien et al., 2012; Hainfeld, Slatkin, Focella, & Smilowitz, 2006; Xi et al., 2012). Despite significant advances, CT imaging is generally not suitable for the targeted imaging of specific tissues or organs and lacks detection sensitivity compared with that of PET or MRI (Cai & Chen, 2007). To develop CT-based targeted imaging of specific tissues or cells, a larger number of nanoparticles with higher CT contrast must be selectively delivered to the target site, raising the need to develop new and effectively targeted CT contrast agents capable of binding to specific receptors expressed by the cells in the targeted tissues. Mannan is a bioactive polysaccharide consisting of D-mannose residues and is found in the cell walls of plants and some fungi. It is a highly branched polysaccharide with an α-(1→6) linked mannopyranose backbone (Ferreira, Pereira, Sampaio, Coutinho, & Gama, 2011). In addition to the promising properties expected from biocompatible biopolymers, mannan-based systems have additional functional properties, such as easy recognition and uptake by antigenpresenting cells (APCs) through mannose receptors expressed on their surface, causing mannosemediated endocytosis and phagocytosis. In this work, we developed mannan-capped gold nanoparticles (M-GNPs) for the targeted CT imaging of LNs using green chemistry-based synthesis (Scheme 1), wherein the mannan acts as a reducing and stabilizing agent for synthesizing AuNPs. We hypothesized that the easily synthesized, non-toxic, and LN-targeted M-GNPs would be effective contrast agents for CT imaging. The overall synthesis did not involve any toxic organic chemicals or solvents. The M-

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GNPs showed excellent diffusion from the site of injection to the targeted LNs and could be effectively visualized through CT imaging.

Scheme 1: Schematic representation of the green chemistry-based synthesis of M-GNPs and CT imaging. 2. Materials and Methods. 2.1. Materials.

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Mannan produced from Saccharomyces cerevisiae and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) was obtained from Promega (Madison, WI, USA). Roswell Park Memorial Institute medium (RPMI-1640) was purchased from Thermo Scientific (Waltham, MA, USA). All chemical reagents and solvents used in this study were purchased from commercial suppliers unless otherwise specified. 2.2. Synthesis of AuNPs. 2.2.1 Synthesis of citrate-capped AuNPs (C-GNPs). Citrate-capped gold nanoparticles were synthesized as previously described with slight modifications (Fu, Zhou, & Xing, 2013). Briefly, 50 mL of 1 mM of HAuCl4·3H2O was mixed with distilled water and boiled at 100°C. When the temperature reached 100°C, 5 mL of (38.8 mM) tri-sodium citrate was rapidly added to the gold salt solution, resulting in a color change from light yellow to dark. After this color change, the solution was stirred for 15 min, slowly cooled to room temperature, and purified by centrifugation. 2.2.2 Synthesis of mannan-capped AuNPs (M-GNPs). Synthesis of M-GNPs is shown in schematic diagram (Scheme 1). Briefly, Mannan (5 mg mL-1) was dissolved in distilled water at room temperature and then held at a temperature of 80°C. After one hour, 400 µL of 1 M NaOH was added, and the solution was stirred for 10 min. Then, 2 mL of 10 mM of HAuCl4·3H2O was added, and the solution was stirred for 3 hrs at 50°C. Finally, the solution was cooled to room temperature and purified through centrifugation (15,000 rpm, 15 min). 2.3. Physicochemical characterization.

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The average hydrodynamic diameter and zeta potential of M-GNPs (1 mg mL-1) were analyzed with dynamic light scattering (DLS) using a Zetasizer Nano Z (Malvern Instruments, Malvern, UK). The surface morphology of M-GNPs was examined by field-emission transmission electron microscopy (FE-TEM) (JEM-2100F, USA). The UV-visible (UV-vis) spectra of the M-GNPs (0.5 mg mL-1) were measured on a spectrophotometer (UV-2700 Shimadzu, Japan). The amounts of mannan coated on the GNPs were characterized by Thermogravimetric analysis (TGA) analysis (Mettler-Toledo, SDT851, Columbus, OH, USA). 2.4. Cell culture experiments. Cells of the DC 2.4 cell line (murine dendritic cell line) and RAW 264.7 cell line (murine macrophage cell line) were cultured in RPMI medium with 10% fetal bovine serum (FBS) and 1% antibiotics at 37°C and 5% CO2. Upon reaching 80% confluency, the cells were harvested with 0.05% Trypsin-EDTA and reseeded in a T-75 flask. All cells used for experimental procedures were passaged two times after revival. 2.5. Cell viability. The in vitro cytotoxicity of M-GNPs was assessed using the DC 2.4 and RAW 264.7 cell lines. Briefly, cells were seeded at concentrations of 1×104 cells mL-1 and grown overnight at 37°C before treatment. After overnight incubation, the incubation medium was aspirated and replaced with M-GNPs at various concentrations in OPTI-MEM® and incubated at 37°C for 6 hrs. Afterward, the medium was aspirated and supplemented with 0.1 mL of medium and incubated for 24 hrs at 37°C. Subsequently, an MTS assay was performed per the manufacturer’s protocol to determine cell viability. 2.6. Mannose receptor-specific intracellular internalization of M-GNPs and mannose receptorspecific competitive inhibition study.

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To determine the intracellular internalization of M-GNPs, 1×105 cells per well of DC 2.4 and RAW 264.7 cells were seeded in Lab-Tek® Chamber Slides and incubated for 24 hrs at 37°C and 5% CO2. Subsequently, the medium was removed, and M-GNPs (15.5 g mL-1) were added and incubated for 6 hrs. Afterward, the medium was removed, and the cells were washed with 1× PBS (3 times) and subjected to fixation by treatment with 4% paraformaldehyde for 30 min. DAPI and gold anti-fade reagent were used for nuclear staining. Confocal laser scanning microscopy was employed to visualize the sample fluorescence. For the mannose receptorspecific competitive inhibition assay, the medium was aspirated and supplemented with DMannose (10 mg mL-1) to block the mannose receptors prior to sample treatment. To further quantify uptake, 1×106 cells per well of DC 2.4 and RAW 264.7 cells were incubated overnight with M-GNPs. The cells were harvested after incubation, washed 4 times with 1× PBS, and quantified via ICP-MS after being digested in aqua regia solution. 2.7. X-ray CT imaging of a phantom and lymph nodes. To determine the minimum gold concentration required for visualization in X-ray CT, three different concentration of M-GNPs (20 mg mL-1, 10 mg mL-1, and 5 mg mL-1) in PBS were tested using a micro X-ray CT scanner (InveonTM, Siemens Healthcare, Erlangen, Germany) with an 80 W, tungsten anode, 30 kV to 80 kV variable focus X-ray source and a 165-mm detector. Images were acquired at 2×2 binning, an exposure time of 200 ms per projection, low magnification, 75 kV anode voltage, and an effective pixel size of 112.88 m. Images were reconstructed without down-sampling using the Shapp-Logan reconstruction filter. For lymph node CT imaging, C57Bl/6 mice (5 weeks, female) (Orient Bio Inc., Seongnam-si, South Korea) were anesthetized and given subcutaneous injections in the hind leg footpad with 50 µL of M-

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GNPs or C-GNPs. The gold contents at the site of injection and the popliteal and inguinal lymph nodes were determined by ICP-MS after being digested in aqua regia solution. 2.8 Silver-eosin staining. After CT imaging, the mice were euthanized. The popliteal and inguinal lymph nodes were collected and fixed in 4% paraformaldehyde. The organs were then dried, embedded in paraffin, and finally cut into 5-µm sections. For silver-eosin staining, the sections were subjected to dewaxing and rehydration and subsequently, washed with distilled water to obtain pretreated sections. These pretreated sections were stained with a silver enhancement kit and counter stained with 1% eosin Y solution for 0.5 min. The sections were then washed with distilled water, air dried, dehydrated and mounted with mounting medium for light microscopy. 2.9. Statistical analysis. Data were analyzed using GraphPad Prism Software (San Diego, CA, USA). Statistical analysis was performed using one-way ANOVA, two-way ANOVA, and Mann-Whitney U-tests. The results are expressed as means ± standard deviation. Significance was attributed as follows: *p < 0.05 **p < 0.01 and ***p < 0.001. 3. Results and Discussion. 3.1 Synthesis and characterization of M-GNPs. A green chemistry synthesis method was used to prepare M-GNPs. Briefly, mannan, a bioactive polysaccharide with LN-targeting properties (Uthaman, Maya, Jayakumar, Cho, & Park, 2014; Vu-Quang et al., 2012), was employed as a reducing and stabilizing agent in the formation of AuNPs. The overall synthesis method did not require any toxic solvents. The large number of hydroxyl groups on mannan acted as active reaction centers for the reduction of Au3+ to Au0, resulting in the efficient formation of AuNPs embedded and stabilized within the

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polymeric matrices (Tagad et al., 2014). The DLS measurement indicated that the resulting MGNPs had an average hydrodynamic diameter of 21.61 ± 12.32 nm and a unimodal size distribution. The average zeta potential of the M-GNPs was measured to be -37.1 ± 9.92 mV, indicating that the nanoparticles had a highly negatively charged surface. Fe-TEM images (Figure 1 a) of the M-GNPs demonstrated a spherical morphology with an average diameter of 9.18 ± 0.71 nm. The nano-scaled size and highly negative surface charge of M-GNPs could make them more susceptible to uptake by the macrophages present in LNs. Negatively charged particles are known to be more easily taken up by macrophages than positively or neutrally charged nanoparticles because macrophages have inherently superior phagocytic activity toward bacteria, which have negatively charged cell surfaces (Frohlich, 2012; Hayashi et al., 2013; Khan, Mudassir, Mohtar, & Darwis, 2013). To determine the composition of M-GNPs, TGA analysis was performed. The results (Figure 1 b) showed that mannan made up a 3.4 weight percentage of the M-GNPs. Because the AuNPs were embedded within the polymeric matrices of mannan, when subjected to heat up to 700°C, the thermal decomposition of mannan occurred while the AuNPs remained stable. By comparing the losses in weight percentage between mannan and M-GNPs, the amount of mannan coated on the AuNPs was calculated. SPR absorption is one of the characteristic properties of AuNPs that contributes toward the absorption spectra in the VIS-NIR region (Y. Zhang et al., 2017; Y. X. Zhang et al., 2016). As shown in Figure 1 c, the M-GNPs exhibited strong absorption peak at 522 nm, which was attributed to the SPR band of AuNPs, thereby demonstrating the formation of AuNPs.

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Figure 1: Physicochemical characterization of M-GNPs: a) FE-TEM image (inset: higher magnification), b) TGA and c) absorption spectra in the VIS-NIR region. 3.2. Cell viability. Cell viability is an indicator of the biocompatibility of samples, which is one of the criteria for in vivo applications (Thrivikraman, Madras, & Basu, 2014). To evaluate the cell viability profile of M-GNPs in APCs, MTS assays were performed on DC 2.4 and RAW 264.7 cells treated with varying concentrations of M-GNPs (Figure 2). Cells without any treatment and cells treated with 0.1% Triton X-100 were used as the positive control (P.C.) and negative control (N.C.), respectively. In both cell lines, M-GNPs exhibited higher cellular viability (> 90%) upon treatment over a wide concentration range from 100 µg mL-1 to 0.4 µg mL-1. Thus, the M-GNPs did not seem to cause any acute or intrinsic cytotoxicity to the DC 2.4 and RAW 264.7 cells.

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Figure 2: Cell viability of M-GNPs determined by the MTS assay. Cells without any treatment were designated the positive control (P.C.), whereas 0.1% Triton X-100 treated cells were designated the negative control (N.C.). Viability is stated as a percentage of the P.C. Values are expressed as means ± SD. N = 3. 3.3 Intracellular internalization of M-GNPs and mannose receptor-specific competitive inhibition study. To investigate the cellular uptake of the M-GNPs, RAW 264.7 and DC 2.4 cells, which express abundant mannose receptors (Jiang et al., 2009; Layek, Lipp, & Singh, 2015), were incubated with M-GNPs. Upon treating RAW 264.7 and DC 2.4 cells with 15.5 g mL-1 M-GNPs, the MGNPs were readily internalized (Figures 3 a & b). The enhanced uptake of M-GNPs may have resulted from the presence of the mannose receptors predominantly expressed over the surface of macrophages, enabling receptor-mediated endocytosis (Kaur, Jain, & Tiwary, 2008). To

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investigate the mannose receptor-mediated endocytosis of M-GNPs, both cell lines were pretreated with high doses of D-mannose for the competitive inhibition of the mannose receptors. Upon treatment with M-GNPs, significant reductions in the cellular uptakes of M-GNPS were observed. To quantify the cellular uptake and receptor-mediated internalization of M-GNPs, ICP-MS analysis was performed. As shown in Figure 3 c & d, the cells treated with M-GNPs had much higher concentrations of internalized AuNPs. However, for the cells pretreated with Dmannose prior to M-GNP treatment, the concentrations of internalized AuNPs were significantly reduced. These results demonstrated that M-GNPs could be effectively internalized by cell lines that overexpress mannose receptors through receptor-mediated endocytosis.

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Figure 3: Cellular uptake of M-GNPs. Confocal microscopy analysis of M-GNPs in RAW 264.7 (a) and DC 2.4 (b) cells. Au concentrations determined by ICP-MS analyses of RAW 264.7 (c) and DC 2.4 (d) cells. Scale bars in 40 x magnification corresponds to 20 m and in 100 x magnification corresponds to 5 m. 3.4. Lymph node imaging by CT. To investigate the potential use of M-GNPs as contrast agents for CT imaging, CT phantom studies were performed (Figure 4). As shown in Figure 4, the M-GNPs showed concentrationbased CT signals with the highest signal observed at 20 mg mL-1 (a maximum Hounsfield unit (HU) value of 303.2 ±10.83 HU). We further investigated the feasibility of using M-GNPs for lymph node CT imaging. In the CT imaging of mice before injection (0 hrs), the lymph node regions showed inherent contrasts when compared with surrounding tissues. However, upon the subcutaneous injection of M-GNPs into the footpads of the mice, the contrast of popliteal lymph nodes (PLNs) for the group injected with M-GNPs was enhanced compared with that of the group injected with C-GNPs (Figures 5 a & b). The PLN contrasts for the M-GNP-injected group showed a time-dependent increase starting from 1 hr post injection. Quantitatively, the HU value in the PLN region improved significantly from 86 ± 17.59 HU to 618.33 ± 80.31 HU (Figure 5c). The higher HU value was likely due to the efficient diffusion of M-GNPs from the site of injection (Figure 5 d) and higher accumulation of M-GNPs into the PLNs as shown by silvereosin staining (Figure 5 e). Because macrophages (Hieu et al., 2011) and dendritic cells (Haddadi, Hamdy, Ghotbi, Samuel, & Lavasanifar, 2014) contain mannan receptors, the likely mechanism for the higher HU values for M-GNPs compared to those of C-GNPs would be the selective uptake of M-GNPs by macrophages and dendritic cells over a period of several hours

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and the subsequent accumulation of M-GNPs in lymph nodes, thereby increasing the local X-ray density.

Figure 4: CT image and average Hounsfield unit (HU) value of M-GNPs (5 mg mL-1, 10 mg mL1

, and 20 mg mL-1) in PBS.

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Figure 5: In vivo CT image of M-GNPs: a) CT image of C-GNPs, b) CT image of M-GNPs, c) Hounsfield (HU) value of PLNs and ILNs, d) percentage of injected dose at 24 hrs post injection determined via ICP-MS and e) silver-eosin staining of PLNs and ILNs at 24 hrs post injection, scale bar = 100m. 4. Conclusion. The synthesized M-GNPs demonstrated selective targeting of lymph nodes, were internalized into APCs via mannan receptor-mediated endocytosis and could effectively enhance the contrast of PLNs during CT imaging. The M-GNPs showed an average diameter of 9.18 ± 0.71 nm. The M-GNPs possessed excellent cytocompatibility and lymph node targeting and can be used as potential contrast agents for lymph node CT imaging. Author Contributions #

These authors contributed equally

The manuscript was written through contributions of all authors. All authors have given approval of the final version of the manuscript. Ethical Approval All animal experiments were carried out under the guidelines of the Chonnam National University Medical School and Chonnam National University Hospital (CNU IACUC-H-201547), South Korea, in accordance with the principles of laboratory animal care (NIH publication No. 80-23).

DISCLOSURES The authors declare no competing financial interests.

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ACKNOWLEDGEMENTS This work was financially supported by Basic Science Research Program (Nos. 2016R1A2B4011184 & 2015R1D1A1A09056741) and the Bio & Medical Technology Development Program (No. NRF-2017M3A9F5030940) through the National Research Foundation of Korea (NRF) funded by the Korean government, MSIP; and the Pioneer Research Center Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (2014M3C1A3053035). Additionally, IKP acknowledges the financial support from a grant (CRI16071-3) of the CNUH-GIST. KMH acknowledges the financial support from the Industrial Technology Innovation Program (10060059, Externally Actuatable Nanorobot System for Precise Targeting and Controlled Releasing of Drugs) funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea). AUTHOR INFORMATION Corresponding Authors *

E-mail: Kang Moo Huh: [email protected]

*

E-mail: In-Kyu Park: [email protected]

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