Photoacoustic detection of follicular thyroid carcinoma using targeted Nano-Au-Tripods

Yang Gui,Kai Cheng,Ruojiao Wang,Sirui Liu,Chenyang Zhao,Rui Zhang,Ming Wang,Zhen Cheng,*,Meng Yang,,*

1 Department of Ultrasound,State Key Laboratory of Complex Severe and Rare Diseases,Pecking Union Medical College Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100730,China

2 Molecular Imaging Program at Stanford (MIPS),Department of Radiology and Bio-X Program,School of Medicine,Stanford University,Stanford CA 94305-5484,USA

Keywords:Affibody Follicular thyroid carcinoma Nano-Au-Tripods Nanoprobe Photoacoustic imaging

ABSTRACT Follicular thyroid carcinoma(FTC)is the second most common form of thyroid malignancy,and it is associated with more aggressive growth and worse long-term survival outcomes relative to papillary thyroid carcinoma (PTC).Reliable approaches to preoperative FTC detection,however,remain to be established.Herein,a targeted Affibody-Au-Tripod nanoprobe was developed and successfully utilized to facilitate the targeted photoacoustic imaging (PAI) of epidermal growth factor receptor (EGFR)-positive cells and tumors.These Affibody-Au-Tripods were found to be highly sensitive and specific for cells expressing EGFR when used as a PA contrast agent in vitro,and studies conducted in an FTC-133 subcutaneous tumor model system in mice further revealed that these Affibody-Au-Tripods were able to specifically target these EGFR-expressing tumors while providing a strong photoacoustic signal in vivo.Importantly,these nanoprobes exhibited negligible cytotoxicity and robust chemical and physical stability,making Affibody-Au-Tripods promising candidates for targeted PAI-based FTC diagnosis.In addition,these nanoprobes have the potential to facilitate the individualized treatment of patients harboring EGFRpositive tumors.

1.Introduction

Papillary thyroid carcinoma (PTC) is the most common form of thyroid malignancy,followed by follicular thyroid carcinoma(FTC).While FTC accounts for just 10%-15%of thyroid cancer cases[1],it is a more aggressive disease than PTC and is associated with poorer long-term patient survival[2,3],necessitating timely diagnosis and treatment.Pathological findings are necessary to definitively diagnose FTC at present.FTC tumors exhibit follicular cells which lack the atypical nuclei observed in PTC cells,instead exhibiting vascular invasion and capsular infiltration [4].As follicular thyroid adenoma (FTA) is another form of thyroid neoplasm composed of differentiated follicular cells,preoperative fine-needle aspiration cytology(FNAC)diagnoses often have difficulty reliably differentiating between FTA and FTC [5].This is true in part because carcinoma and adenoma cells exhibit similar morphological characteristics,and FNAC does not permit the evaluation of capsular or vascular invasion[6].Alternative imaging modalities including scintigraphy,computed tomography (CT),magnetic resonance imaging(MRI),and positron emission tomography(PET)also exhibit limited specificity as a means of differentiating between follicular nodes that are malignant and those that are benign[7–9].This inability to reliably identify benign neoplasms prior to surgical biopsy is associated with high rates of biopsy and patient overtreatment.

The ability to preoperatively diagnose FTC would reduce the need for unnecessary thyroidectomy procedures.Molecular biomarkers have the potential to aid in the differentiation between FTC and FTA.Epidermal growth factor receptor(EGFR)is a cell surface tyrosine kinase receptor that is responsive to extracellular growth factors,regulating key oncogenic processes including survival,proliferation,metabolic activity,and migration.EGFR dysregulation is a hallmark of many cancer types including head,neck,breast,lung,and pancreatic cancer [10],and it is thus a valuable biomarker that can help discriminate between malignant and benign lesions.Importantly,FTC nodules exhibit increase EGFR expression relative to FTA nodules [11].

Photoacoustic imaging(PAI)is an increasingly popular imaging modality that exhibits great promise in a range of preclinical and clinical settings [12–15].PAI is a noninvasive imaging modality that can be performed with or without exogenous contrast agent administration,yielding high-contrast images that are sensitive to tissue properties [16].A range of photoacoustic (PA) contrast agents has been explored to date in an effort to enhance PAI signal[17,18],and these agents can be readily translated to clinical settings to improve the utility of PAI [19].Gold tripods (Au-tripods)are nanoparticle-based PA contrast agents that have previously been reported to exhibit morphological stability,a small size,and a limited size distribution,in addition to exhibiting light absorption in the visible and near-infrared (NIRs) regions.Overall,these Au-tripods exhibit ideal physical and optical properties which make them excellent candidates for use as molecular PAI contrast agentsin vivo[20].

In light of the above results,we herein sought to develop and characterize anti-EGFR-modified Au-Tripod nanoparticles(Affibody-Au-tripods).These nanoprobes were composed of Autripods,which serve as an optimal PAI contrast agent,and an anti-EGFR Affibody (Ac-Cys-ZEGFR:1907),which is a small protein that can efficiently target and bind to EGFR [21].This study is the first to our knowledge to report the use of Au-tripods as a molecular probe for the specific PAI evaluation of EGFR-tumors.

2.Materials and Methods

2.1.Materials

Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC),N-hydroxysulfosuccinimide (sulfo-NHS),N-hydroxysuccinimide (NHS),and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide were ordered from Thermo Fisher Scientific.Hydrogen tetrachloroaurate(III) hydrate (HAuCl4)and platinum(II) acetylacetonate (Pt(acac)2) were obtained Strem Chemicals,Inc.Sigma-Aldrich was the source of all other chemicals,while Invitrogen was the source of all buffers and cell culture media.A Millipore Milli-DI Water Purification system was used to prepare deionized water.

2.2.Methods

2.2.1.Au-tripod surface modification

Au-Tripods were synthesized and purified as published in our previous study[20],and were subsequently modified with the Affibody derivative Ac-Cys-ZEGFR:1907which was 59 amino acids in length and harbored an N-terminal cysteine residue that was prepared as published previously[22,23].The molecular weight of this construct was approximately 6688.7 g?mol-1.

Au-Tripods were conjugated with Affibody ZEGFR:1907as discussed in our previous publications[22,24].Briefly,surface PEGylation of Au-Tripods was performed to render them water-soluble,after which they were suspended in PBS and incubated for 2 h with a cross-linker solution (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate,sulfo-SMCC).Excess sulfo-SMCC and associated byproducts were then eliminated,and the remaining thiol-active Au-Tripods were concentrated to a final 1.0 ml volume with a centrifugal-filter(Amicon centrifugal filter device,MWCO=30 kDa),after which they were combined for 24 h with an Affibody solution at 4 °C with constant stirring.Free Affibody and other byproducts were then removed with a PD-10 column (GE Healthcare,NJ,USA),yielding Affibody-Au-Tripods that were concentrated with a centrifugal-filter (MWCO=30 kDa).These nanoprobes were then stored at 4°C and were found to retain their targeting performance for at least 1 month without any detectable reduction.

2.2.2.Nanoprobe characterization

An FEI Tecnai G2 F20 X-TWIN transmission electron microscope(TEM)was used to assess Affibody-Au-Tripod size and morphology,with all TEM images being recorded at 200 kV.Zeta potential and hydrodynamic size values for these nanoprobes were establishedviadynamic light scattering (DLS) with a Malvern Zeta Sizer Nano S-90 DLS instrument (Malvern Instruments Ltd.).

To assess nanoprobe stability,the hydrodynamic sizes and UV–vis absorbance spectra of these nanoprobes were assessed as above following culture for 0–24 h in PBS containing 10% FB,with UV–Vis-NIR spectrum absorbance being measuredviaAgilent Cary 6000i UV–vis-NIR spectroscopy.

2.2.3.Cell culture and tumor xenograft model establishment

EGFR-positive FTC-133 cells were derived from an FTC metastasis were purchased from the European Collection of Cell Cultures(ECACC,Salisbury,UK),and were cultured in DMEM/F12 (Invitrogen) containing 10% FBS,1% penicillin,2 mmol?L-1L-glutamine,and 100 μg?ml-1streptomycin in a humidified 5% CO237 °C incubator.Media was exchanged every 24–48 h,and cells were collected using 0.5% Trypsin-EDTA as appropriate and used for downstream experimentation.

All animal studies were conducted by trained research staff in accordance with institutional protocols.Female nu/nu mice (5–7 weeks old) were purchased from Charles River Laboratories International,Inc.(Wilmington,MA),and were randomized into two groups (n=4 per group).FTC-133 cells (3 × 106cells in 100 μl of PBS)were subcutaneously implanted into the right flank of each mouse and allowed to grow to a size of 0.5–1.0 cm (3–4 weeks) prior to imaging.

2.2.4.Assessment of Affibody-Au-Tripod biocompatibility and cell uptake.

To assess nanoprobe uptake,FTC-133 cells were incubated with Au-Tripods or Affibody-Au-Tripods for 1–2 h.For blocking studies,Ac-Cys-ZEGFR:1907 was added to plated cells for 30 min prior to the addition of Affibody-Au-Tripods.After incubation for appropriate periods of time,cells were washed thrice with PBS,collected using trypsin,and counted with a hemocytometer.Affibody-Au-Tripod uptake was assessed by lysing cells with aqua regia and nitric acid,after which Au content in each sample was quantified with an inductively coupled plasma mass spectrometer (ICP-MS,Thermo Scientific Xseries 2 Quadrupole) as detailed in our prior study [12].

Affibody-Au-Tripod cytotoxicity was assessed using the NIH-3T3 murine fibroblast cell line.Briefly,these cells were added to 96-well plates and combined with a range of Affibody-Au-Tripod concentrations for 24 h,after which cell viability was assessedviaMTT assay (Sigma-Aldrich,MO,USA) based on provided directions.

2.2.5.Photoacoustic properties of Affibody-Au-Tripods

All PA signals for Affibody-Au-Tripods were recorded with a Nexus 128 photoacoustic instrument(Endra Inc.,Boston,MA)with an adjustable nanosecond pulsed laser.Ultrasound transducers were equipped in a bowl filled with water.Affibody-Au-Tripod solutions were loaded into a polyethylene capillary,which was subsequently immersed in water such that PA signal properties could be measured at continuous wavelengths from 680 to 950 nm across a range of concentrations.PAI data were evaluated with the Osirix software (Pixmeo SARL,Bernex,Switzerland) and an Amide’s a Medical Image Data Examiner (AMIDE).

2.2.6.In vivo photoacoustic imaging of Affibody-Au-Tripods

An Endra photoacoustic computed tomographic system was used to conduct PAI of tumor-bearing mice.First,mice were anesthetized,after which they were placed on an imaging tray such that the lesion was immersed in water.The lesion was then scanned using the PA system.

PAI of mice bearing FTC-133 tumors was conducted prior to injection,after which mice were separated into an Affibody-Au-Tripod group and a Receptor-Blocking group.Mice in the former of these groups were injected with 100–200 μl of PBS containing Affibody-Au-tripods (200 pmol?kg-1)viathe tail vein,while mice in the latter group were injected with both Affibody (Ac-Cys-ZEGFR:1907,25 μmol?kg-1)and Affibody-Au-Tripods(200 pmol?kg-1) in 100–200 μl PBSviathe tail vein.PAI was then performed at 0.5,1,and 2 h post-injection.

3.Results

3.1.Affibody-Au-Tripod synthesis and characterization.

The Affibody-Au-Tripod constructs reported herein were prepared in two primary steps.First,Au-Tripods with a bi-functional surface cross-linker were prepared as in prior reports (20),after which these nanoparticles were conjugated to Ac-Cys-ZEGFR:1907(Fig.1(a)).

Fig.1.Synthesis and characterization of Affibody-Au-Tripods.(a) Surface modification of Au-Tripods with Ac-Cys-ZEGFR:1907.(b) and (c) TEM images of Affibody-Au-Tripods in different magnifications.(d) Hydrodynamic size and (e) Zeta potential of water-soluble Au-tripods and Affibody-Au-Tripods.

After synthesis,we characterized the size and charge of the resultant nanoprobes.TEM imaging revealed these particles to be uniform in size and approximately 20 nm in diameter (Fig.1(b)and (c)).The hydrodynamic size and Zeta potentials of watersoluble Au-Tripods and Affibody-Au-Tripods were additionally assessed (Fig.1(d) and (e)).The size of Au-Tripods was(21.3 ± 0.8) nm,whereas Affibody-Au-Tripods were (30.1 ± 0.9)nm in size,consistent with their successful surface modification with the small Affibody protein.The respective Zeta potential values of Au-Tripods and Affibody-Au-Tripods were+(3.54±0.51)mV and (-13.9 ± 1.0) mV,with these differences being attributable to Affibody conjugation.These results additionally suggested that Affibody-Au-Tripods exhibited good stability.

The hydrodynamic size distribution of Affibody-Au-Tripods following incubation in a 10%FBS(in PBS)solution at 37for a range of times was next assessed(Fig.2(a)).These nanoprobes remained relatively stable from 0 to 24 h,with particles remaining(29.5±1.5)nm in size at the end of this incubation,with no significant changes in size distributions over this same period(Fig.2(b)).This further confirmed the excellent stability of these Affibody-Au-Tripods.Consistent with these findings,assessment of the Affibody-Au-Tripod UV–Vis spectrum absorbance over a 48 h period exhibited no significant changes in absorbance in the 300–700 nm range (Fig.2(c)).

3.2.Assessment of Affibody-Au-Tripod biocompatibility and cell uptake.

We next explored thein vitrobiocompatibility of these Affibody-Au-Tripods,assessing the viability of treated murine NIH-3T3 fibroblastsviaa colorimetric approach in response to a range of Affibody-Au-Tripod concentrations (0.5–200 nmol?L-1).These cells remained viable at all tested concentrations (Fig.3(a)),indicating that these nanoprobes exhibit minimal cytotoxicity.

We then assessed the ability of these Affibody-Au-Tripods to specifically target EGFR-positive tumor cells by incubating FTC-133 cells with Au-Tripods or Affibody-Au-Tripods for 4 h and then evaluating nanoprobe uptake by these cells in the presence or absence of a blocking Affibody (Fig.3(b)).This analysis revealed that FTC-133 cells harbored approximately 2300 Au-Tripods per cell,whereas there were approximately 8600 Affibody-Au-Tripods per cell.After blocking,however,this number fell to just 3300 per cell.Affibody-Au-tripods thus exhibited more robust and specific uptake than did Au-tripods,owing to the ability of the Affibody construct to target and bind to EGFR and to thereby facilitate nanoprobe uptake by these cancer cells.Blocking EGFR binding was sufficient to disrupt this targeting activity,thus reducing Affibody-Au-Tripod uptake by FTC-133 cells and confirming the selective targeting of EGFR-positive cells.

3.3.Assessment of Affibody-Au-Tripod PA properties.

The PA spectrum of prepared Affibody-Au-Tripods was next assessed (Fig.4(a)).This spectrum manifested as a wavelength function with a broad peak extending from the 680–950 nm region.The intensity of the PA signal associated with these nanoprobes exhibited excellent linearity with the Affibody-Au-Tripods input concentrations in the 3–50 μmol?L-1range (Fig.4(b)).As the threshold forin vitroPAI was set relative to the background signal level,the 3 μmol?L-1concentration level was identified as this threshold for these Affibody-Au-Tripods.

Fig.2.Stability of Affibody-Au-Tripods at 37 incubation.Hydrodynamic size(a,b)and UV–Vis spectrum absorbance(c)of Affibody-Au-Tripods measured at different time points of incubation in 10% FBS-PBS solution.

Fig.3.Cell study of Affibody-Au-Tripods.(a) Cytotoxicity of Affibody-Au-Tripods to NIH-3T3 cells.(b) Cellular uptake of PBS (labeled as control),Au-Tripods (labeled as Tripods),Affibody-Au-Tripods (labeled as Affibody-tripods) with or without blocking dose of the Affibody by FTC-133 cells after 4-hour incubation.

Fig.4.PA properties of Affibody-Au-Tripods.(a) PA spectrum of Affibody-Au-Tripods.(b) The linearity of the PA effect of Affibody-Au-Tripods.

Fig.5.Targeted PAI of EGFR-positive FTC-133 tumors in mice using Affibody-Au-Tripods.Mice (n=4 per group) were injected with the nanoprobe without (a) or with Affibody (b).

3.4.Nanoprobe-based targeted in vivo PAI of FTC-133 thyroid tumors

FTC-133 tumor-bearing mice (n=4 per group) were next used to assess the ability of Affibody-Au-Tripods to facilitate the PAI assessment of EGFR-positive lesions.Tumors were visualized prior to and after Affibody-Au-Tripod injectionviaPAI (Fig.5(a)).Affibody-Au-Tripod injection enhanced PA signal levels,with these signals being significantly higher at 2 h post-injection (Fig.5(b)).

4.Discussion

Nanotechnology is a rapidly advancing scientific field that can support cellular and molecular analyses and biological imaging of tumors and other disease states[25,26].PAI is a recently developed molecular imaging modality that leverages PA effects [27–30],with recent advances in PA contrast agent and imaging technology development having markedly improved its clinical utility.Many prior studies have reported on the promise of Au-based nanoparticles as tools for targeted PAI [16,17,31,32].

Our research group has previously reported the successful construction of Au-Tripods,which are highly biocompatible,stable,and small nanoparticles with a narrow size distribution in addition to exhibiting distinct patterns of absorption in the visible and NIR region of the spectrum [20].The further modification of these Au-Tripods has the potential to improve theirin vivoutility and targeting efficiency.As detailed previously,we have also prepared a small Affibody protein (ZEGFR:1907) and derivatives thereof [22,33–36].Following fluorescent labeling or radiolabeling,these Affibody constructs can serve as highly specific probes that can accumulate in EGFR-positive tumors,thereby permitting their specific imaging.Herein,we therefore developed Au-Tripods conjugated to Affibodies in order to improve their targeting efficiency,and we explored the utility of these targeted nanoprobes as a contrast agent when conducting PAIin vitroandin vivo.

The Affibody-Au-Tripods prepared in this study exhibit a number of properties consistent with their value for the PAI evaluation of EGFR-positive tumors.TEM images revealed that Afiibody-Au-Tripods prepared in this study exhibited desirable morphological characteristics,and subsequent analyses confirm the small size and uniformity of these nanoprobes.Importantly,these Affibody-Au-Tripods were also stable and biocompatible.We were then able to use these prepared Affibody-Au-Tripods to conductin vitroandin vivoPAI,with cell uptake experiments revealing that these nanoprobes were readily able to accumulate in cells positive for EGFR expression.Such high EGFR specificity suggests that these Affibody-modified nanoprobes are likely to efficiently target other EGFR-positive tumor cells in addition to the FTC-133 cells used herein.The binding of these nanoprobes to EGFR-expressing FTC-133 tumors was highly specific,as confirmedviaa blocking experiment,and a significantly more robust PA signal was detected following Affibody-Au-Tripod administrationin vivo.As such,these Affibody-Au-Tripods can be used to specifically and reliably conduct the targeted PAI of EGFR-positive lesionsin vivo.Subsequentin vitrocytotoxicity analyses further confirmed the biocompatibility of these Affibody-Au-Tripods,suggesting that they warrant future study as a potential tool for clinical diagnostic use.

The global understanding and practice of medicine have been shifted to precision medicine.During the process,molecular information is essential for the management of the tumors,not only for diagnosis but also for treatment.For thyroid cancer,FNAC is the primary diagnostic tool.While this method cannot distinguish between aggressive and subclinical lesions,as it is well known that thyroid cancer is over-diagnosed and treated recently.Because of the above reasons,molecular information is essential in the future.In this study,the application of PAI and Affibody-Au-Tripods is an attractive technique for molecular imaging of EGFR positive lesions and significantly crucial in oncologic studies,enabling the high potential to solve clinical questions with molecular information.

5.Conclusions

In summary,the Affibody-Au-Tripods developed in this study exhibited promising optical and physical properties.These nanoparticles were able to bind to EGFR-positive tumorsin vivoin mice and yielded strong PA signals.Importantly,these Affibody-Au-Tripods facilitate the extremely sensitive detection of EGFR-positive cellsin vivo,suggesting that these particles can be used as a diagnostic contrast agent when evaluating potential malignanciesviaPAI.Owing to the strong stability and biocompatibility of these Affibody-Au-Tripods,they may be amenable to modification to facilitate the customized treatment of FTC tumors,and the hold grade promise for use in future studies of the personalized treatment of an array of other tumor types known to be positive for EGFR expression.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81421004,81301268);Beijing Nova Program Interdisciplinary Cooperation Project xxjc201812);International S&T Cooperation Program of China(2015DFA30440);Beijing Nova Program(Z131107000413063);CAMS Innovation Fund for Medical Sciences (CIFMS 2020-I2M-C&T-B-035).