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. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: Magn Reson Med. 2021 Oct 31;87(3):1621–1637. doi: 10.1002/mrm.29039

OxyChip embedded with radio-opaque gold nanoparticles for anatomic registration and oximetry in tissues

Maciej M Kmiec 1, Kendra A Hebert 1, Dan Tse 1, Sassan Hodge 3, Benjamin B Williams 1,2,3, Philip E Schaner 2, Periannan Kuppusamy 1,2,3
PMCID: PMC8776570  NIHMSID: NIHMS1742535  PMID: 34719047

Abstract

Purpose:

Electron paramagnetic resonance (EPR) oximetry using the OxyChip as an implantable oxygen sensor can directly and repeatedly measure tissue oxygen levels (pO2). A Phase I, first-in-human clinical trial has established the safety and feasibility of using OxyChip for reliable and repeated pO2 measurements in a variety of tumors and treatment regimens. A limitation in these studies is the inability to easily locate and identify the implanted probes in the tissue, particularly in the long term, thus limiting spatial/anatomical registration of the implant for proper interpretation of the oxygen data. In this study, we have developed and evaluated an enhanced oxygen-sensing probe embedded with gold nanoparticles (GNP), called the OxyChip-GNP, to enable visualization of the sensor using routine clinical imaging modalities.

Methods:

In vitro characterization, imaging, and histopathology studies were carried out using tissue phantoms, excised tissues, and in vivo animal models (mice and rats).

Results:

The results demonstrated a substantial enhancement of ultrasound and computed tomography contrast using the OxyChip-GNP without compromising its EPR and oxygen-sensing properties or biocompatibility.

Conclusions:

The OxyChips embedded with gold nanoparticles (OxyChip-GNP) can be readily identified in soft tissues using standard clinical imaging modalities such as CT, CB-CT, or ultrasound imaging while maintaining its capability to make repeated in vivo measurements of tissue oxygen levels over the long term. This unique capability of the OxyChip-GNP facilitates precisely localized in vivo oxygen measurements in the clinical setting.

Keywords: OxyChip, Gold nanoparticles, EPR, Oximetry, Medical imaging, Fiducial

1. Introduction

EPR oximetry is a very effective method for measuring the absolute value of tissue oxygen concentration (pO2) directly and repeatedly (1). Our laboratory has developed a variety of EPR oxygen-sensing probes, collectively known as OxyChips, for an assortment of clinical applications (2-6). The OxyChip is fabricated by embedding oxygen-sensitive microcrystals of lithium octa-n-butoxy-naphthalocyanine (LiNc-BuO) (7,8) in polydimethylsiloxane (PDMS) polymer (3,9). Embedding the LiNc-BuO microcrystals in PDMS shields them from interaction with the biological milieu, preserves the localization and concentration of the crystals once implanted, and provides the structural integrity of the OxyChip (9,10). The probe is being evaluated for permanent implantation under an FDA investigational device exemption (IDE) and can be removed via surgical resection, e.g., during standard-of-care en bloc tumor resection (11). We have shown, in preclinical models and human subjects, that EPR oximetry using the OxyChip is a practical method for measuring tissue pO2 directly and repeatedly (3,6,12-23). A Phase I clinical study at Dartmouth-Hitchcock Medical Center has confirmed that this technique can be applied to human subjects successfully, thus meeting a currently important but unmet need—the ability to make direct, repeated measurements of oxygen in tissue at specific anatomical sites. (11) These studies also have provided useful information on some present limitations of EPR oximetry for clinical adaptation.

Reliable localization of the OxyChip to the area of interest (i.e., the tumor itself) is critical to the success of repeated hypoxia assessments and subsequent targeted treatments. It is clearly desirable to have a high degree of confidence that the OxyChip is placed within the tumor and that its location can be easily verified during and after placement. In a Phase I clinical study, patients who received surgery alone had the OxyChip placed within the tumor in 80% (n=5) and 73% (n=11) of the cases, with and without utilization of ultrasound imaging during implantation, respectively (11). For patients undergoing neoadjuvant chemotherapy or radiation therapy, the OxyChip appeared to be implanted within the tumor in all cases 100% (n=6) based on ultrasound imaging performed at the time of implantation. However, it was not possible to definitively assess whether the OxyChip stayed within the tumor during treatment due to treatment response, which makes analysis of repeated oxygen measurements throughout treatment difficult. The clinical relevance of this technique is dependent on successful placement and detection of the OxyChip throughout its residency in the tissue, not just at initial deployment and/or at resection. In addition, the position of the OxyChip relative to regions of the tumor responding to treatment identified by anatomical and functional imaging will be helpful in addressing the impacts of oxygen heterogeneity in the tumor. Currently, the OxyChip is not radio-opaque and is also difficult to visualize by ultrasound imaging. It is thus challenging to locate the OxyChip during EPR measurements or prior to or during surgery.

PDMS is often used as a biocompatible matrix to host different types of nanoparticles (24,25). Gold nanoparticles (GNP) have been widely used in modern medical and biology studies, including genomics, biosensors, immunoanalytics, clinical chemistry, detection and photo-thermolysis of microorganisms and cancer cells, targeted delivery of drugs, DNA, antigens, and optical bioimaging (26). In this study we utilized the unique ability of GNPs to cross-link with PDMS via covalent bonding, combined with their high attenuation to X-rays and biocompatibility, to provide increased contrast to the OxyChip for enhanced visualization using clinical imaging methods such as computerized tomography (CT) and ultrasound imaging. We report the preparation of the OxyChip embedded with GNPs (OxyChip-GNP) and the in vitro characterization, imaging, and histopathology results obtained from tissue phantoms, excised tissues, and in vivo animal models. The results demonstrated a substantial enhancement of ultrasound and CT contrast using the OxyChip-GNP without compromising its EPR and oxygen-sensing properties or biocompatibility. The enhanced probe, OxyChip-GNP, can function as a potential self-fiducial for anatomic registration of the implant and oximetry in tissues.

2. Methods

2.1. Fabrication of OxyChip

The OxyChip was fabricated by cast-molding polymerization of polydimethylsiloxane (PDMS) in the presence of LiNc-BuO microcrystals (3,7,9). Briefly, components of the PDMS polymer including medical-grade silicone elastomer MED-4210 (A-103), crosslinker (A-103C) and platinum accelerator (A-317) were obtained from Factor II, Inc. (Lakeside AZ). LiNc-BuO crystals were embedded in PDMS as reported with design specific modifications for OxyChip (3,6), which are uniquely related to the size and shape of the sensor.

2.1.1. Embedding of gold nanoparticles in OxyChip

GNPs were embedded into OxyChip by incubating the OxyChip in a 10% solution (w/v) of gold (III) chloride trihydrate (HAuCl4·3H2O, Sigma-Aldrich 520918) in 100% ethanol for variable time periods (24–120 hours). The probes were then removed, washed several times with deionized water, and stirred magnetically in deionized water for 24 hours to remove any surface residuals. After the washing process, they were dried in an oven at 80°C for 12 hours.

2.1.2. Sterilization and irradiation of OxyChip-GNP

The OxyChip-GNP probes were sterilized by autoclaving using gravity30/dry30 cycle – 121°C, 40 PSI in a steam sterilizer (Getinge Group, Model 522-CS). A small subset of the OxyChip-GNP probes were irradiated to a dose of 60 Gy using a Cesium-137 irradiator (Shepherd & Associates, USA) emitting 661 keV gamma radiation at 10 Gy/min rate to test for effects of irradiation on the calibration of the OxyChip-GNP.

2.1.3. UV-VIS spectroscopy of GNPs in PDMS

Thin (200 μm) PDMS films were prepared and cut into approximately 5x5 mm rectangular pieces for soaking in 10% solution (w/v) of gold (III) chloride trihydrate (HAuCl4·3H2O, Sigma Aldrich 520918) in 100% ethanol for 0, 24, 72, 120, and 144 hours. After soaking, each piece was washed in deionized water and dried at room temperature. Spectrophotometric measurements of the PDMS films were performed using JASCO V-630 UV-VIS spectrophotometer (Analitica, Spain) with self-masking, quartz, and 10-mm pathlength cuvette (Fisher Scientific) for absorbance. The PDMS films were attached to the side of the cuvette to cover the 3x3 mm light beam window. After each absorbance measurement, the attached films were removed, and the light window was cleaned, and new film was attached for measurement.

2.1.4. Scanning electron microscopy and energy dispersive spectroscopic analysis of OxyChip-GNP

Scanning electron microscopy (SEM) was performed using the Scios2 LoVac dual beam FEG/FIB SEM (Thermo Fisher Scientific) scanning electron microscope with a beam energy of 2-10 kV. Three different detectors were used: T1, Circular Backscatter Detector (CBS), Everhart-Thornley detector (ETD). OxyChip-GNP probes were inspected at magnifications up to 12000x. X-ray energy dispersive spectroscopy (EDS) analysis was performed on Helios SCX SEM (Oxford Instruments) with the Ultim® Extreme Silicon Drift Detector providing 10 nm spatial resolution. Data analysis was performed in Aztec EDS analysis suite (Oxford Instruments) for element analysis.

2.2. EPR measurements with OxyChip-GNP

In vitro and in vivo EPR measurements were performed to characterize the functionality, response to various oxygen concentrations, long-term stability in vivo, and effects of sterilization and irradiation on the performance of the OxyChip-GNP. All EPR measurements were performed using a custom-built continuous wave (CW) L-band (1.15 GHz) EPR spectrometer equipped with a surface-loop resonator (3,27) under optimized, non-saturated conditions of RF power and field-modulation amplitudes to prevent artificial spectral line broadening.

2.2.1. Oxygen sensitivity

Prior to being sterilized and implanted in tissues, all probes were calibrated using gases (Airgas) with known pO2 values (between 0 mmHg (0%) to ~160 mmHg (21%)). The measured linewidths (HWHM; G) were plotted against pO2 (mmHg) and fitted with a linear function to obtain oxygen sensitivity (G/mmHg). All calibrations were made at room temperature and ambient pressure. The time-response of OxyChip-GNPs to pO2 change were determined using pre-mixed gases switched between 0 to 2%, 0 to 5%, and 0 to 10% O2.

2.3. Animal preparation

All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Dartmouth College before the start of the study. Animals were maintained at the Dartmouth Animal Resource Center in a clean 12-h light/dark cycle room with free access to food and water. Four adult Fisher rats (Charles River Laboratories, MA, USA) were used for the following imaging procedures: ultrasound, micro-CT, clinical CT, clinical cone-beam CB-CT with OxyChip-GNP and clinically used fiducials. Adult female Balb/c mice (20–30g, Charles River Laboratories, MA, USA) were used to assess short term implantation effects of OxyChip-GNP. Two adult female Balb/c (20–30g, Charles River Laboratories, MA, USA) mice were used for muscle EPR pO2 tissue measurement with OxyChip and OxyChip-GNP and to assess for long-term (10 weeks) implantation effects of the OxyChip-GNP.

2.4. In vivo implantation of OxyChip, OxyChip-GNP, and clinical fiducials

The implantation site was shaved, and the skin was treated with a 70% alcohol scrub. The OxyChip-GNP, OxyChip, and clinical fiducials were implanted in the biceps femoris of rat and mice hind legs using an 18G needle.

2.5. In vivo EPR measurements

During all in vivo EPR measurements, animals were anesthetized using nosecone breathing with 2.5-3.0% isoflurane in 30% O2 for anesthesia induction and 1.5-2.0% isoflurane in 30% O2 for anesthesia maintenance. The temperature was maintained at 37±1°C using a small animal thermal pad with warm water recirculating pump. The location of the implanted probe was placed in the center of the magnet, and the surface-loop resonator was positioned over the implant site. As a control, animals with OxyChips were tested in the same conditions to compare in vivo OxyChip-GNP oxygen readouts. Post-implantation pO2 measurements in the muscle of mice were made for 10 weeks. The measurements were made continuously while the animals breathed 30% O2 (baseline) for 5 minutes and 100% O2 for 20 minutes (hyperoxygenation).

2.6. Pathology – short- and long-term implantation effects in living tissue

Animals were sacrificed and muscle tissue sections surrounding the OxyChip-GNP and OxyChip were recovered and submerged for 24 hours in formaldehyde before transferring into tissue cassettes. Five-micron thick tissue sections were H&E stained and evaluated.

2.7. Imaging of OxyChip-GNP and clinical fiducials

The OxyChip-GNP is designed to be visible during different clinical imaging modalities such as CT, CB-CT, ultrasound, and planar X-ray images. Four adult Fisher rats (Charles River Laboratories, MA, USA) were implanted with OxyChip-GNP and clinically used fiducials and the following imaging procedures were performed: ultrasound, micro-CT, clinical CT, clinical CB-CT. To compare the OxyChip-GNP with fiducials, the clinical standard of marking tissues of interest for imaging, four types of clinical fiducial markers were implanted alongside OxyChip-GNP in a tissue phantom and experimental animals (rats), and imaging procedures were performed. Clinical imaging was performed at Dartmouth-Hitchcock Medical Center, and preclinical imaging X-ray microtomography (micro-CT) and small animal ultrasound imaging procedures were performed using resources of the Dartmouth College Animal Resource Center.

2.7.1. Clinical fiducials

Two types of gold fiducials and one type of polymer-based fiducial marker were compared with OxyChip-GNP. The markers were bone fiducial marker - a 2-mm gold sphere (MTNW887805, CIVCO Orange City, IA, USA) and soft-tissue markers – a 0.8x3-mm gold rod, (MTNW887808, CIVCO, Orange City, IA, USA) and 0.8x3-mm PolyMark™ (PM-0.8-3, MTCTXPM0834, Portland, OR, USA). Sterile brachytherapy needles (18Gx5 cm) were used for tissue phantom and soft-tissue implantations.

2.7.2. Ultrasound imaging

Preclinical and clinical ultrasound imaging were performed on Fisher rats implanted with the OxyChip-GNP and the OxyChip in the hind-leg muscle at about 5 mm depth. Preclinical ultrasound imaging was performed using the VisualSonics VeVo 770 (Visual Sonics, London, ON, Canada) equipped with 700-series RMV (real-time micro visualization) scan heads optimized for small-animal research in automated or handheld mode. All images were acquired in B-mode. Clinical ultrasound imaging was performed using the General Electric LOGIQ e R6/R7 Series ultrasound system with the General Electric L4-12t-RS scan head.

2.7.3. Micro-CT imaging

Micro-computed tomography was performed on eXplore Locus Scanner GE Healthcare Biosciences (Canada). The eXplore Locus scanner uses cone beam volumetric tomography (CBVT), which allows the entire volume of the sample to be imaged in one cycle instead of scanning slice-by-slice. This method provides short image acquisition time, high image quality, and a good signal-to-noise ratio. Chicken meat phantoms (28) with implanted OxyChip-GNP, microChip-GNP, and gold and polymer-based fiducials were scanned with a 3D voxel resolution of 90 μm (0.0936 mm). All volumes were scanned with the same micro-CT scanner settings (60 kVp, 300 μA X-ray tube current, 100-msec exposure time).

2.7.4. Clinical CT and CB-CT imaging

Clinical imaging procedures were performed the Dartmouth-Hitchcock Medical Center using the clinical CT scanner (GE Medical Systems LightSpeed RT 16 CT scanner) and CB –CT scanner (Varian Medical Systems, On-Board Imaging (OBI) system, TrueBeam 1.0 scanner), which are used routinely for clinical treatments. Clinical scanning protocols were applied, utilizing helical acquisition, automated optimization of tube current, 120kVp, and slice thicknesses varying from 0.625 to 2.5 mm.

2.7.5. Image processing

All acquired images were saved in Digital Imaging and Communications in Medicine (DICOM) format. Volume rendering, segmentation, and 3D visualization were performed in 3D Slicer ver. 4.11.2. Image analysis was performer in 3D Slicer Quantification module (PerkLab Research).

2.8. Statistical analysis

Significant differences between the means of baseline and hyperoxygen pO2 values were assessed using a two-tailed unpaired t-test. Significant differences between repeated measurements of means of baseline and hyperoxygen pO2 values were assessed using a two-tailed paired t-test. For all tests, a P value of <0.05 was considered statistically significant. Unless otherwise mentioned, the error bars represent standard error of the mean (SEM).

3. Results

3.1. Preparation of OxyChips embedded with GNP

The embedding of GNPs in PDMS was studied using preformed PDMS film incubated with HAuCl4 solution in 100% ethanol (10% w/v) for 0–144 hours. HAuCl4 is known to diffuse into the PDMS polymer and get reduced by the free Si-H groups in PDMS followed by clustering and cross-linking to form GNP covalently embedded in PDMS (29). Fig. 1a shows photographic images of the 200-μm PDMS films as a function of incubation time. A progressive uptake and formation of GNP in PDMS was visually observed. Visible absorption spectra of PDMS showed a broad absorption peak in the range of 550–555 nm, which intensified as a function of incubation time (Fig. 1b). The peak amplitude at 552 nm (Fig. 1c) exhibited a saturation at ≥72 hours of incubation. Based on the visible spectral data, we chose 72 hours as the optimum incubation time. The incubation of pre-made OxyChips (LiNc-BuO microcrystals embedded in PDMS) with HAuCl4 solution in 100% ethanol for 72 hours to obtain OxyChip-GNP is schematically illustrated in Fig. 1d. The OxyChip-GNP are formed into a cylindrical fiber with a diameter of 0.6 mm and length of 5 mm, similar to the clinical OxyChip that we have used for the Phase I clinical trial in patients (11). All subsequent characterizations including in vitro and in vivo evaluations were carried out using the OxyChip-GNP of these dimensions.

Fig. 1. Preparation of OxyChips embedded with GNP.

Fig. 1

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The GNP were embedded in PDMS by incubation of cured PDMS film in HAuCl4 solution in 100% ethanol. a Photograph images of 200-μm-thick PDMS film showing time-dependent formation of GNP in PDMS for incubation periods 0–144 hours. b Visible absorption spectra of PDMS shown as a function of GNP-incubation time exhibiting an absorbance peak at about λ=552 nm. c Peak amplitude values (after baseline subtraction) at λ=552 nm corresponding to the different incubation periods. d Illustration of the procedure for making pre-formed OxyChip embedded with GNP (OxyChip-GNP).

3.2. Scanning electron microscope and X-ray spectroscopy analysis of OxyChip-GNP

The OxyChip-GNP probes were studied by SEM to analyze the surface element composition of GNP. The SEM images of the outer surface and cross-sectionally cut surface of OxyChip-GNP at 2500x and 350x magnification are shown Fig. 2a-b respectively. The images exhibited a sparse but homogeneous distribution of GNP on the outer surface, while the cut cross-section showed an intense but depth-dependent distribution of GNP in the bulk material. We used X-ray energy-dispersive spectroscopy (XEDS) to scan and characterize elemental content at the cross-sectional surface. The XEDS scans of two regions (outer and inner) in the OxyChip-GNP exhibited peaks corresponding to carbon (C), oxygen (O), silicon (Si), gold (Au) and chlorine (Cl) (Fig. 2c). Line-scanning of gold (Au) content across the cross-sectional surface showed a higher Au content at the edge (up to about 100-micron depth) followed by a progressive decrease to the center (Fig. 2d) suggesting a diffusion-limited distribution of GNP inside the OxyChip.

Fig. 2. SEM and XEDS analysis of GNP embedded in OxyChip.

Fig. 2

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OxyChip-GNP was prepared by incubating preformed OxyChips for 72 hours in a 10% solution (w/v) of HAuCl4 in 100% ethanol. Panels (a) and (b) show SEM images of the outer surface and cross-sectionally cut surface of OxyChip-GNP at 2500x and 350x magnification respectively. The brighter pixels represent gold particles. c X-ray energy-dispersive spectroscopy (XEDS) showing the content of carbon (C), oxygen (O), silicon (Si), gold (Au) and chlorine (Cl) in the OxyChip-GNP at two selected areas (outer and inner) in the cross-section. d X-ray energy-dispersive spectroscopy (XEDS) of gold (Au) along a line-scan in the middle of cross-sectional surface of OxyChip-GNP as shown in the inset. The red arrow indicates the position and direction of X-ray analysis. The data indicate a progressively decreasing level of gold with the highest density at a approximately 100-micron depth.

3.3. Effect of GNP on the oxygen sensitivity of the probe

We next evaluated the effect of GNP-embedding on the EPR properties of the OxyChip, particularly its oxygen sensitivity and stability. OxyChips were embedded with GNPs by incubating pre-made OxyChips in HAuCl4 solution (10% w/v in 100% ethanol) for 0–120 hours. EPR spectral width of OxyChip-GNP equilibrated with gases with different oxygen content (pO2, range 0–160 mmHg) was measured for each incubation time. All preparations exhibited a linear dependence of EPR width with pO2 (Fig. 3a); however, the oxygen sensitivity decreased in a linear fashion with incubation time, e.g., from 0.013 G/mmHg (Control; OxyChip) to 0.009 G/mmHg (Experimental; OxyChip-GNP with 120-hour incubation) (Fig. 3b). Measurement of response time of OxyChip-GNP (72-hour incubation) to dynamic changes of oxygen level (Fig. 3c) demonstrated a rapid response of ~30 sec) which is similar to OxyChip without GNP (6) suggesting that oxygen diffusion rate into PDMS was not affected by the GNP. The oxygen-sensing ability of the OxyChip-GNP was unaffected by exposure to 60 Gy high-energy radiation or sterilization by autoclaving (Fig. 3d). Evaluation of the OxyChip-GNP implanted in the leg muscle of rats for 30 days revealed no changes to the calibration parameters (Fig. 3e). Finally, 24-hour incubation of OxyChip in 100% ethanol, which we have used in the preparation of OxyChip-GNP, did not show any effect on the oxygen sensitivity when compared to OxyChip without ethanol incubation procedure (Fig. 3f) suggesting that the observed decrease in oxygen sensitivity is not due to incubation with ethanol solvent. Overall, the results established that embedding of GNP in OxyChip did not have any significant effect on the properties of the oxygen sensing implant except a decrease in oxygen sensitivity.

Fig. 3. Effect of GNP embedding on the oxygen-sensitivity of OxyChip.

Fig. 3

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a OxyChip-GNP were prepared by incubation of pre-made OxyChips in HAuCl4 solution (10% w/v in 100% ethanol) for 0–120 hours. Values of EPR spectral width of OxyChip-GNP equilibrated with gases with different oxygen levels (pO2) are shown for each incubation time. All probes show a linear dependence of EPR linewidth with pO2 in the range 0–160 mmHg (0 to 21% O2). b The oxygen response of the OxyChip-GNP (slope of linear fit in a) decreases with incubation time, e.g., from 0.013 G/mmHg (Control; OxyChip) to 0.009 G/mmHg (Experimental; OxyChip-GNP with 120-h incubation). c Response time of the OxyChip-GNP to changes of oxygen level. pO2 values were measured using 3-sec sampling frequency. In each case, the OxyChip-GNP was kept at 0 pO2 (anoxic, 100% N2 gas) for 70–75 sec, followed by manually switching to gas mixture with higher oxygen content, 7.6–76 mmHg at the time shown by the arrow. The results show a rapid response (~20 sec) of the chip to dynamic change in oxygen before reaching equilibrium. d Effect of autoclaving procedure and radiation exposure on the oxygen sensitivity of OxyChip-GNP. EPR spectral width of OxyChip-GNP equilibrated with different oxygen (pO2) levels. The calibration data show a linear response to pO2 in the range 0–160 mmHg (0 to 21% O2) and further that the oxygen response is not affected by autoclave procedure or exposure to 60-Gy 661 keV isotope 137Cs gamma radiation. e Calibration of OxyChip-GNP before and after implantation in tissue. EPR spectral widths of OxyChip-GNP before (pre-implantation) and the same chip removed after implantation in live rat leg muscle for 30 days are shown as a function of pO2. The data show that the calibration was stable and unaffected in tissue. f Effect of 24-h incubation of OxyChip in 100% ethanol did not show any effect on the oxygen sensitivity when compared to OxyChip not incubated in ethanol (Control).

3.4. Effect of GNP-embedding on ultrasound and micro-CT images of OxyChip

We implanted OxyChips (with and without GNP) in chicken meat phantom (28) to evaluate the possible contrast-enhancement for ultrasound imaging. The ultrasound images of OxyChip and OxyChip-GNP obtained using a small-animal ultrasound imager revealed a substantially higher level of contrast with OxyChip-GNP compared to OxyChip (Fig. 4a-b). The ultrasound images of OxyChip-GNP in the hind-leg muscle of a rat using a clinical ultrasound device demonstrated a clear visualization of OxyChip-GNP in the tissue (Fig. 4c-d). We further evaluated the CT contrast of OxyChip-GNP using micro-CT imaging. The OxyChips (with and without GNP) and standard clinical fiducials including a solid gold sphere (bone fiducial), a solid gold rod (soft tissue fiducial), and a PolyMark™ fiducial (soft tissue fiducial) were implanted in a chicken meat phantom and imaged using a micro-CT scanner. The micro-CT images demonstrated significant contrast enhancement in the OxyChip-GNP compared to the bare OxyChip (Fig. 4e). A 2.5-fold contrast enhancement was achieved by GNP conjugation to the OxyChip (Fig. 4f).

Fig. 4. Ultrasound (US) and micro-CT images of OxyChip-GNP.

Fig. 4

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Small-animal ultrasound images of OxyChip (a) and OxyChip-GNP (b) in a chicken meat phantom. The images (indicated by arrow) reveal a substantially higher level of contrast in OxyChip-GNP compared to the bare OxyChip. Clinical ultrasound images of OxyChip-GNP (c-d) in the hind-leg muscle of live rats. The images reveal the presence of the OxyChip-GNP (indicated by the arrow) in the tissue. The black area under the OxyChip is an acoustic shadow (ultrasound artifact) that occurs at boundaries between significantly different tissue impedances resulting in signal loss and a dark visual appearance. e Micro-CT images comparing OxyChips/GNP and clinical fiducials. The OxyChips/GNP and fiducials were implanted in a chicken meat phantom and imaged using a micro-CT scanner eXplore Locus Scanner GE Healthcare. Legend: (1) Solid gold sphere (bone fiducial); (2) Solid gold rod (soft tissue fiducial); (3) PolyMark™ fiducial (soft tissue fiducial); (4) OxyChip-GNP; (5) OxyChip (without GNP). f Quantitative assessment of CT contrast to noise ratio (CNR). Data show a 2.5-fold CT contrast enhancement in OxyChip-GNP (4) compared to the regular OxyChip (5); however, it is far less than the clinical fiducials.

3.5. Micro-CT images of OxyChip-GNP in comparison with OxyChip.

Micro-CT images were obtained on the same rat for comparison of their detection sensitivity. Micro-CT image obtained with two OxyChip-GNPs and one OxyChip implanted in the rat leg muscle showed a clear cross-sectional view of all three implants, although the OxyChip was barely visible (Fig. 5a). The micro-CT image showed the cross-sectional view of OxyChip-GNP, but the OxyChip could barely be detected (Fig. 5b). Intensity tracing through the probes in the images clearly demonstrated the enhanced contrast of OxyChip-GNP in the micro-CT image compared to OxyChip (Fig. 5c-d).

Fig. 5. Micro-CT images of OxyChip-GNP in comparison with OxyChip.

Fig. 5

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Two OxyChip-GNPs (one on each leg) and one OxyChip as control (on the right leg adjacent to the OxyChip-GNP) were implanted in the rat leg muscle. Micro-CT images were obtained on the same rat for comparison of their detection sensitivity. a Micro-CT image shows a cross-sectional view of all three implants, although the OxyChip is barely visible. b The same micro-CT image is shown at 2.4x expanded scale to compare the contrast between OxyChip-GNP and OxyChip. The panels (c) and (d) show the intensity along the line tracing through the probes as indicated in (a) and (b), respectively, demonstrate the enhanced contrast of OxyChip-GNP compared to the OxyChip.

3.6. Clinical CT and CB-CT images of OxyChip-GNP compared with clinical fiducials

We next compared the clinical CT and CB-CT images of OxyChip-GNP with clinical fiducials implanted in rat muscle as shown in (Fig. 6a). A torso phantom was included to simulate clinical conditions, including tube-current and beam-hardening effects. The images were obtained using a clinical CT scanner and CB-CT scanner (Fig. 6b-c). The reconstructed CT and CB-CT images (Fig. 6d-e) and quantitative analyses of the contrast-to-noise ratio (CNR) and signal-to-noise ratio (SNR) and intensity profile (Fig. 6f-g) clearly revealed the OxyChip-GNP along with the clinical fiducials demonstrating that the OxyChip-GNP can be visualized and localized by CT and CB-CT in the clinical setting.

Fig. 6. Clinical CT and CB-CT images comparing OxyChip-GNP with fiducials.

Fig. 6

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Two OxyChip-GNPs and two clinical fiducials (PolyMark, solid gold) were implanted in the rat leg muscle as shown in the drawing (a). The images were obtained using a clinical CT scanner (GE Medical Systems; LightSpeed RT16) (b) and CB-CT scanner (Varian Medical System - True Beam) (c) as shown in the photos. The clinical CT (d) and CB-CT (e) images reveal both the OxyChip-GNPs along with the clinical fiducials. f Quantitative assessment of CT contrast to noise ratio (CNR) and signal-to-noise ratio (SNR) of OxyChip-GNP in the CT image as a function of slice thickness. g Signal intensity (in HU) of the OxyChip-GNP and fiducials along the dashed lines shown in (e).

3.7. Planar X-ray image of OxyChip-GNP in rat muscle

A planar X-ray (also called scout image) is often performed in the radiation therapy clinic to aid patient positioning for treatment and may be followed by volumetric CB-CT imaging. In order to evaluate the visibility of the probe in a planar X-ray two OxyChip-GNP were implanted in rat muscle (Fig. 7a) and imaged using a clinical CB-CT scanner. The image, shown in (Fig. 7b-c), clearly revealed the presence of the implanted OxyChip-GNP in the muscle. We also had a third implant, an OxyChip without GNP, on the right leg muscle adjacent to the OxyChip-GNP, which was not revealed in the planar image.

Fig. 7. Planar X-Ray image of OxyChip-GNP in a rat muscle.

Fig. 7

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The planar X-ray image was obtained using a clinical CB-CT scanner (Varian Medical Systems, TrueBeam 1.0 scanner) with two OxyChip-GNP probes implanted in a rat muscle (a). The image clearly reveals the presence of the implanted OxyChips in the muscle (b). It should be noted that a third implant, which was an OxyChip without GNP, on the right leg muscle adjacent to the OxyChip-GNP, as in Fig. 5a, is not revealed in the planar image. Panels (c) and (d) show a zoom-in of the image (b) and intensity tracing (in HU) along the green line, respectively.

3.8. In vivo EPR measurement of pO2 in the muscle

We next evaluated the utility of the OxyChip-GNP for long-term pO2 measurements in the hind leg muscle of mice. The measurements were repeated on the same mice biweekly for 10 weeks. During each session, the measurements occurred continuously while the animals breathe 30% O2 (baseline) for 5 minutes followed by breathing 100% O2 (hyperoxygen) for 20 minutes. Representative EPR spectra of the OxyChip and the OxyChip-GNP (obtained during baseline and hyperoxygen breathing) are shown in Fig. 8a-b. Baseline and hyperoxygen pO2 values obtained repeatedly (biweekly) for 10 weeks from the OxyChip and the OxyChip-GNP are shown in Fig. 8c. A two-tailed paired t-test analysis of the OxyChip and the OxyChip-GNP pO2 data did not show any significant difference between the baseline (P=0.2948) or hyperoxygen (P=0.0978) values measured over the 10-week period suggesting that GNP embedding has no significant effect on the in vivo oxygen measurement capability.

Fig. 8. In vivo EPR measurement of pO2 in the rat leg muscle.

Fig. 8

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pO2 measurements in the hind leg muscle of mice were repeatedly made using the OxyChip and OxyChip-GNP for 10 weeks. The measurements were made continuously while the animals breathed room air (baseline) for 5 minutes followed by breathing 100% O2 for 20 minutes. Representative EPR spectra of OxyChip (a) and OxyChip-GNP (b) in the muscle of mice obtained 10 weeks after implantation. The spectra during room-air breathing and hyperoxygen breathing are shown as superimposed for each animal. (c) Mean pO2 values of baseline and hyperoxygen obtained biweekly from the OxyChip and OxyChip-GNP are shown for 2–10 weeks. The data represent mean±SEM, averaged from 6 data points each collected during baseline and at the end of hyperoxygen breathing. *P<0.05 - significantly higher compared to respective baseline value. A two-tailed paired t-test analysis of the OxyChip and OxyChip-GNP pO2 data measured did not show a significant difference among the baseline (P=0.2948) or hyperoxygen (P=0.0978) values measured over the 10-week period. Similar analysis on the baseline and hyperoxygen data of the OxyChip or OxyChip-GNP showed a significant increase of hyperoxygen pO2 compared to baseline (OxyChip, P=0.0018; OxyChip-GNP, P=0.0042) suggesting that GNP embedding has no significant effect on the in vivo oxygen measurement capability.

3.8. Histopathology of OxyChip-GNP by H&E staining

Both short-term (3 weeks) and long-term (3 months) histopathology of the OxyChip and the OxyChip-GNP implanted in mouse hind-leg muscle was analyzed using H&E staining. Fig. 9a-b show a representative staining of tissue at 3 weeks of implant duration of the OxyChip and the OxyChip-GNP, respectively. Fig. 9c-d show a representative staining of tissue at 3 months of implant duration of the OxyChip and the OxyChip-GNP, respectively. Histopathological examination showed moderate fibrosis surrounding the insertion track after 3 weeks, and no fibrosis or inflammatory reaction was found after 3 months.

Fig. 9. Histopathology of OxyChip implants.

Fig. 9

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H&E staining was used to analyze the effect of the implant in the mouse hind-leg for short-term (3 weeks; a, OxyChip; b, OxyChip-GNP) and long-term (3 months; c, OxyChip; d, OxyChip-GNP). The panels a’–d’ show a four-fold magnification of the implant location indicated by the rectangle in the respective a–d images. Histopathological examination showed moderate fibrosis surrounding the insertion track after 3 weeks and no fibrosis or inflammatory reaction was found after 3 months.

4. Discussion

The results of the present study demonstrate the utility of the OxyChip-GNP, a novel form of the OxyChip which is embedded with GNP. These data indicate that repeated, long term measurements of tissue oxygen levels using the OxyChip-GNP are comparable to the standard OxyChip. Incorporation of GNPs in preformed OxyChips did not compromise their EPR and oxygen-sensing properties or biocompatibility. The results also showed a substantial enhancement of ultrasound and CT contrast relative to the standard OxyChip, allowing for easy visualization of the OxyChip-GNP and demonstrating its potential for use facilitating anatomic registration of the implant. This development is highly significant in that repeated in vivo measurements of tissue pO2 with precise anatomic localization enhances our ability to accurately interpret the pO2 data obtained.

GNPs embedded in PDMS can be prepared in several different ways: (i) pre-made GNPs can be incorporated into PDMS during the polymer and crosslinker mixing and cured; (ii) the gold salt (HAuCl4) can be added during the polymer and cross linker mixing process and cured; or (iii) the GNPs can be synthesized in situ and linked in preformed PDMS (24,30-32). In this work we selected the third method since it allows us to use the existing FDA-approved Investigational Device Exemption (IDE) manufacturing process for the OxyChip without changing the procedure. GNP are incorporated into the PDMS polymer matrix by incubation in HAuCl4 solution. When the PDMS is in contact with the HAuCl4 solution, the anions, AuCl4−, slowly diffuse into the PDMS matrix and react with the residual (unpolymerized) Si-H groups, resulting in the formation of PDMS-GNP via covalent bonding (32). The residual (unpolymerized) Si-H group in PDMS is expected to reduce Au4+ and trigger a polymerization process of gold to form GNP (29).

Our preparation by incubating preformed OxyChip with HAuCl4 in ethanol may not be the most efficient approach. This method requires the permeation of HAuCl4 into the PDMS matrix and the availability of free -SH groups in PDMS for covalent bonding and cross-linking of gold to form GNP. As a matter of fact, there have been several other more effective methods reported for GNP-embedding in PDMS (24,30-32); however, as a first step toward achieving the formation of GNP inside PDMS, we wanted to use the preformed OxyChips that have IDE status from the FDA for clinical applications, and further that we have already been using for measurement of tumor pO2 in humans (11). Although it may not be the most effective way of embedding GNP, the results of our present approach suggest that GNP embedding is a promising way to achieve our goal of using the OxyChips as self-fiducials in clinical oximetry. Furthermore, the presented method is limited to diffusion of HAuCl4 into PDMS, which results in an inhomogeneous distribution of GNP in the probe; however, the data presented here indicate this is not a severe limitation for their identification using ultrasound or CT imaging methods.

Introduction of any metal into a paramagnetic probe for in vivo EPR detection may have some disadvantages, particularly causing electromagnetic perturbations and/or unwanted toxicity to the host tissue. The apparently sparse and inhomogeneous embedding of GNP does not seem to have any adverse effect on the EPR sensitivity; however, the response to oxygen, but not the anoxic line-width of the EPR spectrum, seems to have been affected by the incorporation of GNP in the probe. This may be due to GNPs acting as a barrier to oxygen diffusion and its interaction with the paramagnetic microcrystals in PDMS; however, this is only a speculation and needs to be verified, which is beyond the scope of this study. Nevertheless, the GNP content-dependent oxygen sensitivity of OxyChip-GNP is stable and does not seem to change with time or tissue microenvironment.

In recent decades, medical imaging techniques have vastly improved and have become essential for disease monitoring. Between 1997 and 2006, the use of ultrasound imaging increased by almost 40%, the number of CT procedures doubled, and magnetic resonance imaging (MRI) procedures almost tripled (33). CT has excellent resolution of X-ray absorbing anatomical elements, like bones, but cannot provide detailed images of soft tissue as well as MRI. Diagnostic ultrasound can produce real-time images of internal organs/soft tissues, but it is not a good clinical imaging modality to image bones. Image-guided radiation therapy after surgery and chemotherapy will be used in nearly 50% of cancer patients (34). The CT contrast enhancement of the OxyChip-GNP is adequate for its in vivo identification using a clinical CT scanner as well as a CB-CT. This is important, as it allows for the potential for daily visualization of the OxyChip-GNP during radiotherapy using daily CB-CT, thereby providing critical information about the anatomy being evaluated via tissue oximetry. In this regard, the OxyChip-GNP allows visualization of its location in addition to providing oxygen data at the site of implant. Visualization of the OxyChip-GNP may also allow it to serve as a traditional fiducial marker, enabling x-ray localization of soft-tissue structures during treatment or prior to surgical resection. Further optimizations including increased and more uniform distribution of the GNP in PDMS may be necessary to achieve higher contrast for ultrasound imaging.

Gold is used in some of the most stable and popular nanoparticles that are widely used in biomedical imaging and diagnostic tests (35). GNPs have extremely efficient optical absorption, with typically ~105-fold higher cross section than absorbing dyes, have been used in several diagnostic and imaging modalities (35). Furthermore, the unique optical absorption capabilities of GNP have been used in several therapeutic modalities including photothermal cancer therapy, radiofrequency (X-rays) ablation, antiangiogenic therapy, and targeted drug delivery in chemo-, immuno- and combined therapies (26,36,37). All these features and applications make GNP a highly desirable and biocompatible material for biomedical applications. A unique feature of the OxyChip-GNP formulation is that the GNPs are generated within PDMS in situ and covalently bound to PDMS so that the particles are stable and securely held within the probe enabling biocompatibility and CT contrast-enhancement for long-term applications.

During radiotherapy with megavoltage beams, the addition of high atomic number materials such as gold will lead to modestly increased radiation dose in tissue at the entrance-surface of the material due to backscatter and modestly decreased dose behind the material due to absorption. The impact is reduced for common treatments using opposed beams or arc therapy where these opposed effects partially balance. For solid gold fiducials, an overdose at the surface between 20-30% has been predicted (38). Given the small amount of gold contained in the GNP-OxyChips (relative to solid gold fiducials) and encapsulation of the GNPs within the PDMS volume, the dosimetric impact of the GNP-OxyChips in the neighboring tissue is predicted to be very small, at most on the order of a few percent. Relative to doses delivered during therapy, the dose from X-ray imaging will be negligible. Accordingly, dosimetric considerations do not pose a significant limitation for use of the GNP-OxyChip.

5. Conclusions

Preformed OxyChips embedded with gold nanoparticles (OxyChip-GNP) can be readily identified in soft tissues using standard clinical imaging modalities such as CT, CB-CT, or ultrasound imaging while maintaining its capability to make repeated in vivo measurements of tissue oxygen levels over the long term. This unique capability of the OxyChip-GNP facilitates precisely localized in vivo oxygen measurements in the clinical setting.

Acknowledgments

The study was supported by National Institutes of Health grants R01 EB004031 and P01 CA190193. We also acknowledge the support of Dr. Karen Moodie from the Animal Resource Center (ARC) for preclinical imaging measurements and the Division of Radiation Oncology and Department of Radiology at the Norris Cotton Cancer Center for providing the clinical imaging resources.

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