2.1.

PAI Setup

The photoacoustic experimental setup is given in Fig. 1. Laser pulses from an optical parametric oscillator (Vibrant B, Opotek) pumped by an Nd:YAG laser (Brilliant B, Bigsky) were used to illuminate the sample, either an ELISA well plate, as in Fig. 1a, or joints in a rat tail, as in Fig. 1b. The laser pulse repetition rate was 10 Hz, and each pulse had a width of 5.5 ns. The laser wavelength was tuned to 760 nm, the absorption peak of the GNRs. An optical diffuser was used to expand the laser beam to cover a well or a joint completely. The energy density of the laser pulses incident at the sample was adjusted to <10 mJ/cm², within the ANSI safety limit. Part of the laser beam was reflected off by a glass plate and measured by using a photodiode in order to take into account the incident light energy and the laser fluctuation. Neutral density filters were placed in the beam path to limit the maximum amount of light entering into the photodiode.

Fig. 1

(a) Schematic of the PAI setup, (b) scanning geometry on rat tail joints in situ, and (c) example photoacoustic signal from a rat tail joint.

For the experiment on the ELISA well plate, an unfocused ultrasonic transducer (XMS-310, Panametrics, Waltham, Massachusetts) with a central frequency of 10 MHz, –6 dB-bandwidth of 133% and a diameter of 2 mm was used to collect the photoacoustic signals from the illuminated well. Part of the well and the transducer were immersed in water for acoustic coupling. For the imaging of rat tail joints, a focused transducer (V312, Panametrics) with a central frequency of 10 MHz, –6-dB bandwidth of 130%, a diameter of 0.25 in., and a focal length of 0.75 in. was used to conduct a raster scan along the length of the tail. The –3-dB focal diameter of this transducer is 0.47 mm. Photoacoustic signals received by the transducer were amplified (PR5072, Panametrics) and sent into one channel of a digital oscilloscope (TDS 540B, Tektronics), which was triggered by the laser firing. The signal from the photodiode was sent into another channel of the oscilloscope. The signals received by the oscilloscope were digitized, collected, and stored in a computer for processing and analysis. The amplitude of the photoacoustic signal was divided by the photodiode signal amplitude to get the photoacoustic response.

2.2.

γ Imaging Setup

Radionuclide imaging was performed using the Gamma Imager (Biospace Lab, Paris). The planar imaging for ELISA experiments was performed with the Gamma Imager detection area facing upward, an ELISA well plate was placed directly on the parallel-hole collimator 1.3/0.2/20 (hole diameter/septum thickness/height in millimeters). Fifteen-minute duration image was acquired using an energy window of 15–70 KeV, and the image was then displayed and quantified using Gamma Vision +software (Version 3.0). Radioactivity was determined by drawing regions of interest and calculated based on a standard curve, which is generated by scanning a series of standards with known radioactivities.

2.3.

Micro–Single-Photon Emission Computed Tomography/Computed Tomography

SPECT/computed tomography (CT) was performed using a pair of collimators (1.3/0.2/35) that stand up to orient the detection area toward the animal (rat tail). The rat tail was fixed on a holder and aligned parallel to the face of detector. After a 36-min CT scan, a 30-min γ imaging was acquired and 2-D image was reconstructed using γ acquisition software. The reconstructed fusion image of SPECT/CT could be displayed and quantified using Amira software (Version 3.1).

2.4.

Synthesis of Gold Nanorods

GNRs are synthesized by a method modified from the literature.^{24, 25, 26, 27} Briefly, a seed solution is prepared by reducing Gold (III) Chloride (25 μL, 0.05 M) in hexadecyltrimethyl ammonium bromide (4.7 ml, 0.1 M) by addition of freshly prepared sodium borohydride (0.3 ml, 0.01 M) under vigorous stirring. An aliquot of seed solution 0.24 mL is added to the growth solution containing hexadecyltrimethyl ammonium bromide (100 mL, 0.1 M), gold (III) chloride (1.0 ml, 0.05 M), hydrochloric acid (2 mL, 1 M), ascorbic acid (0.8 ml, 0.1 M), and silver nitrate (1.2 mL, 0.01 M). The glass beaker is placed in a water bath maintained at 27°C for 3 h to complete the synthesis, and 50 mL aliquots of the synthesized rods are centrifuged at 10×10³ rpm for 1 h to obtain pellets of GNRs at the bottom of the tube. Supernatant is decanted, and the pellet is redispersed into 5 mL of deionized water to get a concentration of 10¹³ rods/mL. The concentration of GNRs were calculated by first estimating the number of gold atoms present in a single nanorod using an average dimension of GNRs from transmission electron microscopy (TEM) images, the size of the gold atom, and the lattice spacing of the gold atom in a GNR. Then, the standard absorption of atomic gold at 400 nm was measured on a ultraviolet-visible spectrophotometry (UV-vis) instrument by preparing a standard 0.5 mM gold salt solution. Finally, the absorption of prepared GNRs solution at 400 nm is also measured. Therefore, we can calculate the amount of gold atoms in the GNR solution by the standard absorption of gold at 400 nm and, from there, estimate the concentration of GNRs. GNRs with an average aspect ratio of 4 are obtained with transverse plasmon peak at 525 nm and longitudinal plasmon peak at ∼800 nm. The position of the longitudinal plasmon peak can be fine tuned fairly easily within the 600–900 nm range by varying the content of silver nitrate during synthesis.

2.5.

Bioconjugation of Gold Nanorods

Bioconjugation was performed by a slightly modified method previously published by our group. ^{18, 28, 29, 30, 31, 32, 33} A layer of thiol-PEG-carboxylic acid (Laysan Bio Inc.) is reacted onto the surface of the GNRs by adding 1.0 mL of 10 mg/mL thiol-PEG-carboxylic acid solution to 1 mL of the prepared GNRs solution. The mixture is stirred for 3 h, followed by centrifugation to remove excess thiol-PEG-carboxylic acid in the solution. The layer of thiol-PEG-carboxylic acid provides the –COOH functional group required for the conjugation. The thiol-PEG-carboxylic acid-coated GNRs are dispersed in 1 mL of deionized water followed by the addition of 100 μL of 0.2 M N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) and 100 μL of 0.2 M sulfo-N-hydroxy-succinimide (NHS). After waiting for 20 min, 5 μg of TNF-α antibody (R&D system) is injected to the reaction mixture. The EDC/NHS mixture forms an active ester intermediate with the GNRs, which undergoes an amidation reaction with the –NH2 group in the anti-TNF-α to yield the conjugate. The reaction mixture is stored at room temperature following centrifugation and redispersion in deionized water to remove the unconjugated antibody.

2.6.

Radiolabeling of Gold Nanorods with ¹²⁵I

[¹²⁵I]Radionuclide in 10⁻⁵ M NaOH (pH 8–11) purchased from PerkinElmer (100 mCi/mL) was diluted with deionized water. The concentration was determined on radioactivity. GNRs (either blank or conjugated with anti-TNF-α) dispersed in fresh deionized water (0.5 mL, 10¹³ rods/mL) was added to 0.5 mL diluted [¹²⁵I] sodium iodide. The mixture was shaken for 30 min at room temperature. The [¹²⁵I]-iodine labeled GNRs were then washed with deionized water by centrifugation and redispersion.

2.7.

ELISA Experiment

Two hundred microliters of 2 μg/mL human recombinant TNF-α (R&D system) in ELISA coating buffer (Immunochemistry Inc.) was added to each well of a 96-well plate and incubated for 12 h at room temperature. Then, the coating solution was aspirated out of the wells and the wells were washed twice by ELISA washing buffer (Immunochemistry Inc.). Then, 300 μL of blocking buffer was added to each well and incubated for 4 h. The blocking buffer was then aspirated out the well plates, and the plate was ready for the screening experiment. Then, 100 μL of GNRs conjugated with TNF-α antibody were added into each alternating well in row A; 100 μL GNRs conjugated with TNF-α antibody and radiolabeled (30 μCi) were added into each alternating well in row C; 100 μL radiolabeled (30 μCi) GNRs into each alternating well in row E; and 100 μL blank GNRs into each alternating well in row G. In each row, four alternating wells were used in order to average the results. After incubating the samples for 1 h, the 96-well plate was aspirated and washed with washing buffer three times. The dried plate was analyzed with PAI setup and Gamma Imager.

2.8.

Rat Tail Joint Model

The GNR concentration in the original solution was 10¹³ rods/mL. The original solution was diluted 40 times, first with distilled water and then 0.2 mL of solution was mixed with 0.5 mL of I-125 with radioactivity of 400 μCi for 30 min using a ROTAMIX rotater (Appropriate Technical Resources, Inc., Laurel, Maryland). The mixture was centrifuged for 4 min at a speed of 5000 cpm in a 1-mL tube. Then, the radiolabeled GNRs at the bottom of the tube were taken out and diluted again to 0.3 mL with distilled water (∼320 μCi). Therefore, in this solution (named agent 1), the GNR concentration was 10¹¹ rods/mL (i.e., 2×10⁻¹⁰ M or 200 pM). After agent 1 was made, 1 mL of solution was diluted 1:1 with distilled water, which resulted in agent 2, with a GNR concentration of 1×10⁻¹⁰ M (or 100 pM). After that, 1 mL of agent 2 was diluted further 1:1 with distilled water to get agent 3, with a GNR concentration of 5×10⁻¹¹ M (or 50 pM). As shown later in Fig. 4a, 0.02 mL of contrast agents 1, 2, and 3 were injected intra-articularly in every other joint in the rat tail, while same amount of water (i.e., 0.02 mL each) was injected in the other two joints as control. The estimated total amount of GNRs injected in the three joints were 2×10⁹, 1×10⁹ and 5×10⁸, respectively. Considering that some of the GNRs may stay in the upper medium in the tube after centrifuge, the actual number of GNRs injected should be slightly lower. Measured with a Capintec dose calibrator (CRC-543X, Capintec Inc., Ramsey, New Jersey), the total radioactivity of the GNRs injected into the three joints were 15.2, 7.4, and 3.6 μCi, (roughly at a ratio of 4:2:1)

This work conducted on rat tail joints is a part of our study on PAI and nuclear imaging of antirheumatic drug delivery aided by radiolabeled GNRs dual-modality contrast.^{34, 35, 36} Rat tails provide good samples to study the performance of imaging on human finger or toe joints, considering their morphological similarity. Rheumatic disease rat models, including those with inflammatory arthritis, have been researched extensively and provide the opportunity to evaluate antirheumatic drugs much more quickly than in humans. In this experiment, whole tails of euthanized rats were imaged. An optical beam with size of 1 cm diam and a wavelength of 760 nm illuminated the imaged rat tail and generated photoacoustic signals that were acquired with the focused V312 Panametric transducer. The distance between the transducer and the rat tail was ∼19 mm, which was also the transducer focal length. Considering an f-number of 3, the estimated lateral resolution was ∼0.55 mm; while the estimated axial resolution was ∼0.12 mm. An example A-line photoacoustic signal from a rat tail joint is shown in Fig. 1c, where the signals from the skin and the GNRs are marked. In order to acquire a 2-D image along a sagittal section of the tail, the tail immersed in water for ultrasound coupling was positioned through a computer-controlled stepper motor while the transducer and the laser beam were kept static. The scanning step was 0.6 mm (similar to the lateral resolution), and the scanning period was 60 mm (i.e., 100 steps). The total period for photoacoustic signal acquisition was ∼10 min when the signal at each scanning position was averaged over 50 laser pulses to achieve a better signal-to-noise ratio.

3.1.

Radiolabeling of Gold Nanorods with ¹²⁵I

The radiolabeling procedures are simple and reliable, with radiochemical yields of >80%. GNRs were found to be stable after binding with radioactive iodine. This agreed well with published data, which claim that there is high affinity and strong binding of iodide ions to the surface of GNRs.³⁷ Figure 2 shows the UV-vis absorption spectra of the GNRs conjugated with TNF-α antibody before and after the addition of radioactive iodine. The minor blueshift in the spectrum is due to the change in the surrounding of the GNR's surface due to the addition of iodine molecules. The radiolabeled GNR conjugate was found to be stable for a week when stored in a refrigerator at 4°C.

Fig. 2

UV-vis spectra of the GNRs solution before and after radiolabeling the anti-TNF-α conjugated GNRs with¹²⁵I

3.2.

ELISA Experiment

The ELISA experiment has been performed to evaluate the biological activity and the specific binding of the synthesized contrast agent carrying anti-TNF-α, which is an important therapeutic agent against arthritis. Row C with radiolabeled conjugated GNRs showed a strong signal in both the Gamma Imager and photoacoustic measurements (Fig. 3). The control in row E had a very weak photoacoustic amplitude signal and nearly zero radioactivity because the GNRs lacked the anti-TNF-α on their surface to cause specific binding on the well plate. Row A with GNRs conjugated with TNF-α antibody showed similar photoacoustic signal amplitude as row C but no radioactivity because it was not radiolabeled with ¹²⁵I. Another control of row G with blank GNRs also had a very weak photoacoustic signal and nearly zero radioactivity. The findings from the ELISA experiment conclude that the TNF-α antibody conjugated with ¹²⁵I labeled GNRs retains its biological activity and forms a highly specific system. The ratio of photoacoustic signal for specific (average photoacoustic amplitude from the area under row C) to nonspecific binding (average photoacoustic amplitude from the area under row E) for the targeted radiolabeled GNRs contrast agent was 6:1. The ratio of signal intensity in the γ-camera image for specific (average intensity from the area under row C) to nonspecific binding (average intensity from the area under row E) for the targeted radiolabeled GNRs contrast agent was 6.125:1. This also shows a strong one-to-one correspondence in results obtained respectively from the photoacoustic and nuclear imaging setups.

Fig. 3

(a) Mean ± standard deviation of photoacoustic signal intensity measured from each row of the ELISA plate. (b) γ-camera image of the ELISA plate. Row A contains {GNRs + anti-TNF-α}, row C contains {GNRs + anti-TNF-α + ¹²⁵I}, row E contains {GNRs + ¹²⁵I}, and row G contains {GNRs}. The photoacoustic signal and radioactive image correspond very well.

3.3.

Imaging Results on Rat Tail Joints

After the first photoacoustic image was taken [Fig. 4b], intra-articular injections of contrast agent and water were conducted. Then the second photoacoustic image was taken, as shown in Fig. 4c. The signal enhancement can be seen in the three joints with injected radiolabeled GNR contrast agent; while no noticeable change can be seen in the two joints with water injected. Because of the edge-enhancement phenomenon as a result of the limited bandwidth of the transducer,^{18, 38} the front contour of the volumetric contrast agent in the joint space presented the strongest signal intensity; while the photoacoustic signal from the rear contour was blocked mostly by bone. The maximum signal intensities in the three marked areas in Fig. 4c show a ratio of 1.16:0.60:0.27 that is close to ratio among the doses of injected contrast agent in the three joints (i.e., 4:2:1). After photoacoustic data were acquired, the rat tail was placed in our microSPECT/CT system. The 2-D images of SPECT and CT are shown in Figs. 4e, 4a, respectively. As shown in Fig. 4a, the acquired 2-D microCT image of the sagittal section of the rat tail clearly shows the bone structure. This microCT image was then fused with the photoacoustic image in Fig. 4c. In the microCT and photoacoustic combined image in Fig. 4d, the photoacoustic image presents the distribution of contrast agent and some soft tissue structures; while the microCT image describes detailed bone structure in the tail. With the morphological information presented by the microCT image, we can clearly see that the positions of signal enhancements in the photoacoustic image were at the intra-articular connective tissues in the three joints containing the contrast agent.

Fig. 4

In situ imaging of radiolabeled GNRs contrast agent in rat tail joints. (a) MicroCT image of a sagittal section of a rat tail with joint sections clearly presented. As marked in the image, intra-articular injection of contrast agent or water as control was conducted in each joint. Photoacoustic image of the sagittal section in the rat tail (b) before and (c) after intra-articular injection of contrast agent. The signal enhancements in the three joints with injected contrast agent are marked with dashed ellipses. (d) The photoacoustic image in (c) fused with the microCT image in (a). (e) SPECT image of the sagittal section in the rat tail presenting the radioactivities in the three joints with the injected contrast agent. (f) The SPECT image in (e) fused with the microCT image in (a).

As shown in Fig. 4e, when the GNRs concentration in the target tissue was on the order of 10 pM, the radioactivity from ¹²⁵I conjugated with the GNRs was sufficient to enable nuclear imaging with an excellent signal-to-noise ratio. The counted radioactivities in the three joints with injected contrast agent were 4939, 2702, and 1453 counts/min, respectively. The ratio among them is also close to that among the contrast agent doses in the three joints (i.e., 4:2:1). In Fig. 4f, nuclear imaging is fused with the microCT image presenting both the distribution of contrast agent and the morphological tissue structures.