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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
ACS Nano. Author manuscript; available in PMC 2011 December 28.
Published in final edited form as:
PMCID: PMC3246549
NIHMSID: NIHMS337883

In Vivo Quantitative Evaluation of the Transport Kinetics of Gold Nanocages in a Lymphatic System by Noninvasive Photoacoustic Tomography

Abstract

Sentinel lymph node (SLN) biopsy has emerged as a preferred method for axillary lymph node staging of breast cancer, and imaging the SLN in three-dimensional space is a prerequisite for the biopsy. Conventional SLN mapping techniques based on the injection of an organic dye or a suspension of radioactive colloids suffer from invasive surgical operation for visual detection of the dye or hazardous radioactive components and low spatial resolution of Geiger counters in detecting the radioactive colloids. This work systematically investigates the use of gold nanocages (AuNCs) as a novel class of optical tracers for noninvasive SLN imaging by photoacoustic (PA) tomography in a rat model. The transport of AuNCs in a lymphatic system and uptake by the sentinel lymph node were evaluated by PA tomography on the axillary region of a rat. Quantification of AuNCs accumulated in the lymph node was achieved by correlating the data from PA imaging with the results from inductively-coupled plasma mass spectrometry. Several parameters were systematically evaluated and optimized, including the concentration, size, and surface charge of the AuNCs. These results are critical to the further development of this AuNC-based PA tomography system for noninvasive SLN imaging, providing valuable information for metastatic cancer staging.

Keywords: gold nanocages, sentinel lymph node, photoacoustic tomography, metastatic cancer staging

The metastatic spread of a tumor occurs by invading the adjacent tissue and disseminating cancerous cells through the lymphatic system into the blood stream.1 The closest lymph node that receives the drainage from a tumor is known as the sentinel lymph node (SLN), which represents the most likely first location of metastatic spread. To reduce the side effects of axillary lymph node dissection,2 sentinel lymph node biopsy (SLNB) is widely performed and has become the standard for axillary staging in breast cancer patients.3 Although SLNB with blue dyes (such as lymphazurin or methylene blue) or radioactive colloidal tracers has an identification rate of more than 90%, these methods involve invasive surgical operations and use carcinogenic ionizing radiation. They may also fail to identify axillary diseases owing to a high false negative rate of 5–10%.4 Furthermore, they can pose a risk of postoperative complications, such as seroma formation, lymphedema, and motion limitation.5

Recently, ultrasound-guided fine needle aspiration biopsy (FNAB) has been clinically evaluated as a minimally invasive procedure.6 This technique requires accurate positioning of the SLN, which initiates studies to develop accurate, nonionizing, and noninvasive methods for SLN mapping. Photoacoustic (PA) tomography is such a technique with remarkable resolution, which is based on the optical absorption contrast mechanism.7,8 Since the spatial resolution of this technique is determined by ultrasound parameters, the imaging depth can be extended to the optical quasidiffusive or diffusive regime while maintaining high resolution. By using diffusive photons, the maximum PA imaging depth can be pushed up to 50 mm in biological tissues.9 Our previous work successfully demonstrated PA SLN imaging by using clinically available organic dyes.10,11 Although this dye-based PA imaging system has been demonstrated with a depth capability of ~31 mm, satisfactory spatial resolution, and clinical potential, the drawback of this system lies in that the dye molecules are rather small (<2 nm), and thus they can easily transport into the echelon lymph nodes, causing a high possibility of false positives.12

In contrast, gold nanocages (AuNCs), a novel class of nanostructures characterized by hollow interiors and ultrathin, porous walls, have proper size range (30–100 nm) to ensure sufficiently fast migration and sufficient duration of trapping in SLN for imaging.8 The localized surface plasmon resonance (LSPR) peak of AuNCs can also be precisely tuned to the near-infrared (NIR) region from 700 to 900 nm (the so-called transparent window), in which the attenuation of light by blood and soft tissue is relatively low.13 In addition, AuNCs can be easily bioconjugated with various types of ligands such as antibodies, peptides, and nucleic acids to target specific receptors,14,15 potentially eliminating the nee d for invasive axillary staging procedures in addition to providing noninvasive SLN mapping. Other attractive features of AuNCs include bio-inertness, large absorption cross sections (almost five orders of magnitude greater than those of conventional organic dyes),16 and the ability to encapsulate therapeutic drugs, which offers a great benefit for theranostic applications.17 In a previous publication, we have demonstrated a proof-of-concept use of AuNCs for SLN imaging by PA tomography, where an intradermal injection of 100 μL of 2 nM AuNC solution was performed on the forepaw pad of a rat, and the SLN was identified with enhanced contrast and good spatial resolution.18 Many parameters associated with the AuNCs, including the minimum requirement on the concentration and injected volume and the influence of particle size, shape, and surface characteristics on their transport kinetics and uptakes by lymph nodes, still need to be examined and optimized before thissystem can be further considered for potential clinical use.

Here we quantitatively evaluated the transport of AuNCs in the lymphatic system and the uptake by lymph nodes through PA imaging on a rat model. The influences of concentration, particle size, and surface charge were all systematically examined. The amounts of AuNCs accumulated in the SLNs were quantified by inductively-coupled plasma mass spectrometry (ICP-MS) analysis of the dissected lymph nodes after the rats had been euthanized. The results provide valuable information for further development of this AuNC-based PA imaging system for noninvasive lymph node mapping.

RESULTS AND DISCUSSION

The main objective of this study is to quantitatively investigate the PA contrast enhancement in SLNs provided by AuNCs with different concentrations, sizes, and surface characteristics. The influences of these parameters were examined by using suspensions of AuNCs with a range of concentrations, two different sizes (50 and 30 nm in edge length), and three different surface charges (negative, positive, and neutral). Figure 1, A and B shows typical transmission electron microscopy (TEM) images of AuNCs with an edge length of 50 and 30 nm, respectively. Figure 1C shows the UV-vis extinction spectra of 50- and 30-nm AuNCs. The locations of their LSPR peaks were slightly different, centered at 760 and 730 nm, respectively. During PA imaging, the laser wavelength was tuned to overlap with the corresponding absorption peak. Except for the experiments involving comparison of particle size, the AuNCs used in all studies were 50 nm in edge length. The surface of the as-synthesized AuNCs is typically covered with poly(vinyl pyrrolidone) (PVP, MW≈55,000), a biocompatible polymer. To investigate the effect of surface charges, the PVP layer was replaced by heterofunctional poly(ethylene glycol) (PEG, MW≈5,000) with one end terminated in the sulfhydryl group and the other terminated by the amine (-NH2), methoxy (-OMe), or carboxylic acid (-COOH) group, to generate positive, neutral, or negative surface charges, respectively. The as-prepared AuNCs covered by PVP were used in all experiments except the study of surface charges. The injection volume was kept the same at 100 μL for all experiments while the concentration of AuNCs was varied. PA imaging of the axillary region of a Sprague-Dawley rat (250–300 g) was performed at different time points after intradermal injection of an aqueous suspension of AuNCs in the left forepaw pad, and the amplitude change of PA signals was then monitored as a function of time.

Figure 1
TEM images of Au nanocages (AuNCs) with average edge lengths of (A) 50 nm and (B) 30 nm; (C) UV-vis extinction spectra of aqueous suspensions of AuNCs with edge lengths of 50 nm (solid line) and 30 nm (dashed line).

We first tested the ex vivo sensitivity of our current deep-reflection mode PA imaging system by using gelatin phantoms based on a mixture of gelatin and AuNCs.18,19 As shown in Figure 2, PA signals from the gelatin phantom containing AuNCs at a concentration as low as 10 pM could still be detected with a signal-to-noise ratio (SNR) of ~2.2 dB. This detection limit corresponds to ~2×105 (or 3.3×10−19 mol) AuNCs per imaging voxel. If we assume the typical volume of SLN in a rat is ~23.6 mm3 (the SLN is approximated as an ellipsoid of 3 mm × 5 mm × 3 mm), this detection sensitivity indicates that only ~1.3×108 (or 2.1×10−16 mol) AuNCs need to enter the lymph node to provide sufficient signals for imaging. The noise equivalent concentration (i.e., the concentration that provides a SNR of unity) is calculated to be ~4.5 pM. In addition, the ex vivo experiment showed that no structural change to the nanocages was observed after expossure to the laser (determined by the TEM image), indicating that the nanocages were stable during the PA imaging. The power density of the laser applied was 35 mW/cm2, and was kept at this level for all the in vivo studies.

Figure 2
PA signals recorded from gelatin phantoms containing AuNCs at various concentrations (R2=0.99), showing that the detection limit of AuNCs (50 nm in edge length) was less than 10 pM, or 2×105 (or 3.3×10−19 mol) AuNCs per imaging ...

To evaluate the in vivo sensitivity of our PA system and AuNCs for lymph node imaging, we intradermally injected an aqueous suspension of AuNCs at a specific, decreasing concentration (100, 50, and 20 pM) at a dose of 100 μL (this corresponds to an amount of 2.2×107, 1.1×107, and 0.55×107 AuNCs per gram of body weight, respectively) on the left forepaw pad of a rat and then the left axillary region of the rat was noninvasively imaged using the PA system. The field of view that possibly contains SLN was chosen based on our previous experience with lymph node imaging.10, 18, 20 Before the injection of AuNCs, PA images were acquired as control images (Fig. 3, A, D and G), revealing the vasculature within ~3 mm below the skin surface. After the injection of AuNCs, a series of PA images were obtained up to 120 min post-injection. It can be observed that the SLNs started to appear at 5 min after the injection of 100 pM (Fig. 3B) and 50 pM (Fig. 3E) AuNC suspensions while the SLN was not detected at 5 min after the injection of 20 pM AuNCs (Fig. 3H). As time elapsed, the PA signals gradually increased and the SLNs, including the case with the injection of 20 pM AuNCs, could be clearly observed at 120 min post-injection (Fig. 3, C, F and I). Note that the minimum concentration of 20 pM we used here for PA SLN imaging in a rat model was about 100 times less than the value (2 nM) reported in our previous work,18 greatly reducing the potential toxicity of AuNCs. It is also worth pointing out that the drainage of AuNCs to lymph nodes was much faster than that of Au nanorods, even at a relatively low injection concentration. In our previous study, it took more than 20 h for Au nanorods (10 nm in diameter and 41 nm in length) with an injection concentration of 1 μM to be observed at SLNs.21 These results suggest that AuNCs should serve as a better diagnostic imaging agent than Au nanorods for SLN imaging.

Figure 3
PA images of the axillary region of rats acquired at 0, 5, and 120 min after injection of the AuNCs with different concentrations. (A, D, G) PA images before injection of the AuNCs; (B, E, H) PA images 5 min after the injection of AuNCs at different concentrations. ...

We also performed in vivo PA SLN imaging in two additional rats at each concentration, following the same procedure. The PA signal enhancements in SLNs were summarized as a function of time (Fig. 4A). Each data point was normalized against the PA signal from the corresponding adjacent blood vessels (BV) acquired before the injection of AuNCs. This normalization method was also applied to the experiments described in other sections. We found that the PA signal enhancement increased with increasing injection concentration. The enhancement also increased with post-injection time, indicating the gradual accumulation of AuNCs in SLNs. For example, after the injection of 100 pM AuNCs, the enhancement was 182% ± 12% at t=5 min, and then gradually increased to 355% ± 24% at t=120 min post-injection. For the group with 20 pM AuNCs, the SLNs in two of the three rats were not even detected at t=120 min post-injection. This high false negative rate indicates that we had reached the in vivo detection limit of the PA system. Note that the standard errors for PA signal enhancement at 5 and 30 min post-injection in the group of 50 pM AuNCs injection were still relatively high compared to those with 100 pM sample, indicating that a 100 pM suspension of AuNCs at a dose of 100 μL (2.2×107 AuNCs per gram of body weight) seems to be the best candidate to start with for SLN imaging. We further quantified the number of AuNCs accumulated in the SLN by ICP-MS analysis of the excised node. The accumulated amount of AuNCs in SLN for the 100 pM sample was approximately 2 and 7.5 times higher than the 50 pM and 20 pM samples, respectively (Fig. 4B). The average numbers of AuNCs in SLN per mass at 120 min post-injection of 20, 50 and 100 pM of AuNCs were about (0.86±0.74)×1010, (3.25±1.07)×1010, and (6.43±0.94)×1010 AuNCs/g, respectively, which were qualitatively consistent with the trend revealed by PA enhancement shown in Figure 4A. According to our measurements, about 18% of the injected 50 pM and 100 pM AuNCs had been accumulated in the SLN at t=120 min post injection, while about 12% of the injected 20 pM AuNCs had been accumulated in the SLN. Based on the ICP-MS data, there were about twice as many AuNCs in the SLN for the 100 pM sample than for the 50 pM sample, while the PA signal enhancement for the 100 pM sample only showed a slight increase. This discrepancy can be attributed to two reasons: i) the AuNCs were actually not evenly distributed in the SLN. This phenomenon was observed in our previous report on SLN imaging.18 The edges of the SLN were found to contain more nanocages than the central portion of the SLN. Therefore, if some parts of the SLN contained a small amount of AuNCs that generated PA signals lower than the background, we would not be able to detect them; ii) the PA signal is sensitive to the imaging depth. If some parts of the SLN were away from the focus and the PA signal from AuNCs was lower than the background, the signal could not be detected either.

Figure 4
(A) PA signal enhancement in SLNs as a function of time after the injection of AuNCs at different concentrations (n=3 rats for each group): 100, 50, and 20 pM, respectively. Each data point was normalized against the PA signal from the corresponding adjacent ...

The SLNs of rats are located at ~2 mm below the skin surface. In humans, the mean depth of SLNs is ~12 mm underneath the skin.18 Therefore, it is necessary to demonstrate the minimal dose of AuNCs at the clinical depth by using the same PA imaging modality. Here, we used chicken breast tissues as biological phantoms. The previously reported penetration depth for 1/e decay in chicken breast tissue was ~0.94 cm at the wavelength of 760 nm,22 while the 1/e penetration depth at 785 nm in human breast was measured to be ~0.89 cm.23 Similar to the previous experiments, after the SLN of a rat in the group of 100 pM AuNCs was identified (Fig. 5A), a chicken breast tissue of 5 mm thick was placed on top of the axillary surface of the rat, and a PA image was acquired at 150 min post-injection. The SLN of the rat could be obviously identified from this image, and the total depth of the SLN was now ~7 mm (Fig. 5B). With the addition of a second layer of chicken breast tissue, an imaging depth of ~12 mm was achieved by acquiring a PA image at 180 min post-injection (Fig. 5C), and the SLN can still be imaged with a good contrast. In the B-scan image (Fig. 5D), the depth information of the SLN is clearly shown with ~7 dBSNR.

Figure 5
PAimaging of SLN in a rat after the injection of 100 pMAuNCs. The PA images were acquired (A) at 120 min post-injection with no chicken breast tissue added, (B) at 150 min post injection with one layer (thickness: 5 mm) of chicken breast tissue added, ...

An ideal mapping agent for lymphoscintigraphy should have proper size: on one hand, it should be small enough to rapidly drain to lymphatic vessels and then transport to lymph nodes; on the other hand, it should be large enough to stay within the lymph nodes during the imaging process. Particles smaller than 5 nm could easily leak into blood capillaries or migrate to echelon lymph nodes and thus cause false positives,24 while particles larger than 100 nm are believed to be trapped in the interstitial compartment for a long period of time, and are thus unable to transport to lymph nodes fast enough for lymphoscintigraphy.25 Therefore, it is critical to identify an optimal size range for the AuNCs in order to have sufficiently fast migration and sufficient duration of trapping for SLN imaging.

We compared the PA enhancements in SLNs as a function of time using AuNCs with edge lengths of 50 nm and 30 nm, respectively (Fig. 6A). We performed the in vivo PA imaging in 3 rats for each group. Note that the surface of the as-prepared AuNCs was covered with PVP, and thus the hydrodynamic diameter of the AuNCs measured by dynamic light scattering (DLS) was about 97.1 nm, so we did not choose AuNCs with an edge length larger than 50 nm. Additionally, nanoparticles with larger size (>50 nm) are known to exhibit short circulation times in blood,26 making them less useful in diagnostic imaging. The injection concentrations for both the 30-nm and 50-nm AuNCs were 200 pM (at a dose of 100 μL), as the SLN could hardly be detected with the injection of 30-nm AuNCs at a concentration of 100 pM. The amounts of Au injected for 30-nm and 50-nm AuNCs were calculated to be about 7.5 ng and 22.7 ng per gram of body weight, respectively. Both curves show that SLNs could be detected at 5 min post injection, and the PA signal enhancement kept increasing in the following 120 min. It can be seen that the 50-nm AuNCs showed higher signal enhancement (about 150% to 170%) than the 30-nm sample. This difference can be ascribed to the fact that the 30-nm AuNCs had a smaller optical absorption cross section than the 50-nm AuNCs, generating weaker PA signals. We have also measured the absorption cross sections of these AuNCs using a PA-based method developed in our groups.16 The absorption cross section of the 50-nm AuNCs at 760 nm was found to be about 2.1 times higher than that of the 30-nm AuNCs at 730 nm. Figure 6B shows a comparison of the accumulation of 50-nm and 30-nm AuNCs in SLNs as a function of post-injection time calculated from the PA signals. Since the PA signal is directly proportional to the absorption coefficient (μa) of the AuNCs,16 the accumulation of AuNCs can be calculated according to the equation: Nnanoa,nanoa,nano, where Nnano is the concentration of the AuNCs (number of particles per m3)and σ a,nano is the absorption cross section (m2) of the nanocages. Each data point was then normalized against the accumulation of 50-nm AuNCs in SLN at 5 min post-injection. It can be seen that the 30-nm AuNCs transported into the SLN faster than the 50-nm AuNCs and showed alarger amount of accumulation in the SLN. The ICP-MS data in Figure 6C indicate that the average number of 30-nm AuNCs in the excised SLN per mass at 120 min post-injection was about 1.8 times higher than that of the 50-nm AuNCs, (14.5 ± 1.0) × 1010 vs. (8.0 ± 0.6) × 1010 AuNCs/g, which is consistent with the trend of accumulation derived from the PA signals. The dose of Au accumulated in the SLN for 30-nm and 50-nm AuNCs at 120 min post-injection was about 2.5 × 10−5 g and 4.1 × 10−5 g per gram of SLN, respectively. Even though the 30-nm AuNCs showed a larger amount of accumulation in SLN based on the particle number, the amount of Au content in SLN for the 50-nm AuNCs was about 1.6 times higher than that for the 30-nm AuNCs.

Figure 6
(A) PA signal enhancement in SLNs after the injection of 50-nm and 30-nm AuNCs with the same concentration and dose (200 pM and 100 μL) as a function of post-injection time (n=3 rats for each group), respectively. (B) Accumulation of the 50-nm ...

Early studies on the transport kinetics and lymphatic uptake of particles showed that surface characteristics, such as charge and hydrophobicity, could affect the rate of particle drainage from the injection site to lymph nodes and their distribution within the lymphatic system.25 Therefore, it is important to study the transport behaviors of AuNCs with different surface charges. We replaced the PVP layer on the AuNCs with SH-PEG5000-X (X=OMe, NH2, or COOH). Table 1 summarized the hydrodynamic diameters and zeta-potentials of the PEGylated AuNCs. It can be observed that the zeta-potential of AuNCs covered with SH-PEG5000-OMe was about −7.2 mV, and thus the surface charge is considered as neutral. The zeta-potentials of AuNCs covered with SH-PEG5000-NH2 and SH-PEG5000-COOH were around +12.5 mV and −16.8 mV, giving a relatively positive and negative surface charge, respectively. Following the same procedure, we performed in vivo SLN imaging using AuNCs modified with different surface charges. For comparison, each group had 3 rats and the injection concentration was 200 pM instead of 100 pM for all 3 groups, because the SLN could be hardly detected at 60 min after the injection of AuNCs with positive charges at a concentration of 100 pM. Figure 7 shows typical PA images acquired before as well as 5 min and 60 min after the injection of AuNCs modified with different surface charges. Unlike the other two groups, AuNCs with negative charges accumulated not only in the SLN but also in the adjacent lymphatic vessels. The lymphatic vessels began to show up in PA images at 5 min post injection (Fig. 7B) and the AuNCs could be trapped there for 60 min (Fig. 7C). It is known that lymphatic vessels play an important role in lymphatic metastasis,27,28 providing crucial prognostic information. Cancer cells can exploit these vascular systems by expressing growth factors, altering the normal pattern of angiogenesis and lymphatic vessel growth, creating conduits for tumor metastasis. As a result, the ability to target and image aberrant drainage patterns is of clinical significance. However, studies on lymphatic vessels have been hindered by technical limitations.29 The AuNCs with negative charges can potentially be used as a good contrast agent for lymphangiogenesis imaging. In addition, AuNCs with positive charges also showed a unique feature. It took 30 to 60 min, a much longer time than the other two groups, to transport to SLNs and accumulate enough amounts for PA imaging (Fig. 7, D-F). Figure 8A shows PA signal enhancement in SLNs as a function of time after the injection of AuNCs with different surface charges. It can be seen that the signal enhancement originating from AuNCs with positive chargesis much lower than that from the neutral AuNCs, especially in the first 60 min post injection, indicating a much slower transport rate to lymph nodes. As for the PA enhancement from the AuNCs with negative charges, it was also lower (about 50% to 60%) than the neutral AuNCs, but higher (about 150% to 160%) than the positive AuNCs in the first 60 min post injection, and then leveled off. The ICP-MS data in Figure 8B show that the average number of AuNCs in the excised SLN per mass at 240 min post injection decreased in the order of neutral > positive > negative, which is consistent with the trend for PA signal enhancement shown in Figure 8A.

Figure 7
PA images acquired before, 5 min, and 60 min after the injection of AuNCs (200 pM, 100 μL) with different surface charges. The PVP layer on the nanocages surface was replaced with SH-PEG5000-X, X=OMe, NH2, or COOH to generate a neutral, positive, ...
Figure 8
(A) PA signal enhancement in SLNs as a function of time post injection of AuNCs with different surface charges: negative (blue line), positive (red line), and neutral (black line) (n=3 rats for each group). (B) Average number of AuNCs accumulated in the ...
Table 1
A sumamry of the hydrodynamic diameters and zeta-potentials of the AuNCs modified with different surface groups.

We also found that the AuNCs were not only able to transport to the SLN, but also further down to the second and third axillary lymph nodes of a rat. As shown in Figure 9, C and D, both in vivo and ex vivo PA images clearly show the accumulation of nanocages in all three lymph nodes of the rat after the injection of AuNCs at a relatively higher concentration (500 pM) at 72 h post injection (no transport to the second and third axillary lymph nodes had been observed by PA imaging after the injection of 100 pM or 200 pM nanocages). Taken together, these features can probably be used to design AuNCs with switchable surface charges for noninvasive axillary lymph node staging of breast cancer by taking advantage of their different transport kinetics in lymph nodes. For example, we can conjugate the surface of AuNCs with an enzyme-sensitive peptide which can be cleaved by enzymes (e.g., matrix metalloproteases, a recognized biomarker associated with cancer-cell invasion and metastasis) overexpressed by cancer cells in the metastatic lymph node.30 As such, the surface charges on the AuNCs will be different before and after the enzyme cleavage, and the transport rates of AuNCs between lymph nodes will be different for patients with different stages of metastatic cancer. In addition, we can also attach fluorescence dyes to the surface of AuNCs via an enzyme-cleavable peptide.31 The dye molecules will be cleaved and released from the surface of the nanocage in the presence of a protease, and thus fluorescence will be recovered. In practice, the distribution of AuNCs in lymph nodes can be mapped by PA imaging while the protease activity can be monitored by fluorescence spectroscopy, providing critical information for the diagnosis of breast cancer metastasis. Related researches are currently underway. With regard to the improvement for PA imaging, the system we used for this study employed a single ultrasonic transducer and a 10 Hz pulse-repetition-rate laser system, and thus the scanning time for a three-dimensional image was about 24 min with 0.2 mm and 0.4 mm step-sizes. For future studies, employing an ultrasonic array system and a higher pulse-repetition-frequency laser system can dramatically improve the temporal resolution. We are currently developing a handheld, array-based PA system for future clinical use.32,33

Figure 9
PA images acquired (A) before, (B) 1 h, and (C) 72 h after the injection of AuNCs (500 pM, 100 μL), showing the transport of AuNCs from SLN to the second lymph node (LN2) and third lymph node (LN3). (D) Ex vivo (i) PA image and (ii) photograph ...

CONCLUSION

We have quantitatively evaluated the transport of AuNCs in a lymphatic system and their uptakes by lymph nodes in a rat model through PA imaging. We systematically examined and optimized a number of experimental parameters including the concentration, size, as well as surface characteristics of the AuNCs. We reduced the concentration of injected AuNCs down to 100 pM (20 times lower than our previous proof-to-concept report, greatly reducing the potential toxicity of AuNCs) for SLN imaging at a depth of ~12 mm (the depth of SLN in human) while keeping a sufficient SNR. It was found that the 30-nm AuNCs exhibited a faster transport rate and a larger amount of accumulation in the SLN than the 50-nm AuNCs, but the latter generated stronger PA signals due to a larger optical absorption cross section. This result indicates that the 50-nm AuNCs seem to be a better contrast agent than the 30-nm AuNCs for SLN imaging by PA tomography. As for AuNCs with different surface charges, their transport rates to SLNs decreased in the order of neutral > negative > positive. In addition, AuNCs with negative charges could be trapped in lymphatic vessels, making them potentially useful as contrast agents for lymphangiogenesis by PA imaging. We also found that the AuNCs were not only able to transport to the SLN, but can also travel farther to the second and third axillary lymph nodes of a rat. These results can serve as guidelines for choosing optimal experimental parameters of AuNCs in metastatic lymph nodes mapping and other biomedical applications by noninvasive PA imaging.

METHODS

Synthesis and Surface Modification of AuNCs

The AuNCs were synthesized using the galvanic replacement reaction between Ag nanocubes and chloroauric acid in water according to our published protocol.34 In a typical synthesis, an aqueous solution of HAuCl4 (0.75 mM) was slowly titrated (at a rate of 45 mL/h using a syringe pump) into an aqueous suspension of Ag nanocubes placed in a three-neck flask at 90 °C. The extent of replacement could be readily monitored by taking UV-vis spectra of aliquots sampled from the solution. To obtain different surface charges, the nanocages were derivatized with SH-PEG5000-X (X=OMe, NH2, and COOH; all from Laysan Bio; Mw≈5,000). In a typical process, 0.1 mL of HS-PEG5000-X aqueous solution (0.1 mM) and 0.1 mL of AuNCs aqueous suspension (1 nM in terms of particles) were added to 2.8 mL deionized water. The mixture was kept stirring at 4°C overnight. After that, the mixture was centrifuged at 14,000 rpm for 15 min and the supernatant was decanted to remove the excess PEG. The PEGylated-AuNCs were then washed with water twice and re-suspended in water at a concentration of 200 pM (in terms of particles) for in vivostudies.

The PA Imaging System

A deep reflection-mode PA imaging system was used (Scheme 1 in ref. 19) for all PA experiments. A tunable Ti:sapphire laser (LT-2211A; Lotis TII, Minsk, Belarus) pumped by a Q-switched Nd:YAG (LS-2137; Lotis TII) laser was used for excitation, providing <15-ns pulse duration and a 10-Hz pulse repetition rate. A dark-field ring-shaped illumination was adopted to reduce the surface PA signal generation.35 The light energy on the sample surface was controlled to be less than the American National Standards Institute standard for maximum permissible exposure. Two wavelengths, 760 nm and 730 nm, were used for PA imaging to match the absorption peak positions of the 50-nm and 30-nm AuNCs, respectively. A 5-MHz central frequency, spherically focused ultrasonic transducer (V308; Panametrics-NDT, Waltham, MA, USA) was used to acquire the generated PA signals. The 5 MHz ultrasonic transducer yields axial and transverse resolutions of 150 and 560 μm, respectively. The signal was amplified by a low -noise amplifier (5072PR; Panametrics-NDT) and recorded using a digital oscilloscope (TDS 5054, Tektronix, Beaverton, OR). A photodiode (SM05PD1A, Thorlabs, Newton, NJ) was used to compensate for the energy instability of laser pulses. A linear translation stage (XY-6060, Danaher Motion, Radford, VA) was used for raster scanning to obtain three-dimensional (3D) PA data. The signal was not averaged for any image to shorten the data acquisition time. By measuring PA amplitudes according to the arrival times, one-dimensional depth-resolved images (A-lines) are acquired. Additional raster scanning along a transverse direction provides two-dimensional depth-resolved images (B-scans), consisting of multiple A-scans, and further scanning along the other traverse direction provides 3D images. The acquired 3D raw data can be processed as a maximum amplitude projection (MAP)—a projection of the maximum PA amplitude along each A-line onto the corresponding plane. Typical scanning values were as follows: field of view, 20 × 20 mm; voxel dimension, 0.2 × 0.4 mm; laser pulse repetition rate, 10 Hz; and acquisition time, ~24 min. The transducer was located inside a water container with an opening of 5 × 5 cm at the bottom, sealed beneath a photoacoustically transparent clear membrane. The rats or gelatin phantom samples made from a mixture of gelatin solution and AuNCs were placed under the membrane, and ultrasonic gel was used for ultrasound coupling.

Animal Handling

All animal experiments were in compliance with the Washington University Institutional Animal Care and Use Committee. Sprague–Dawley rats weighting 200–300 g (Harlan, Indianapolis, IN) were anesthetized with a mixture of ketamine (87 mg/kg) and xylazine (13 mg/kg) at a dose of 0.15 mL per 100 g body weight. The hair in the left axillary region was removed by gentle clipping and depilatory cream before imaging. PA imaging was acquired before and after intradermal injection of AuNC solution (0.1 mL) in the left forepaw pad. Full anesthesia of the animal was maintained throughout the experiment by using vaporized isoflurane (1 L/min of oxygen and 0.75% isoflurane) and vitals were monitored by a pulse oximeter (NOMIN Medical, 2000SA). The body temperatures of the rats were maintained with a water heating pad. After data acquisition, the rats were euthanized by overdosed pentobarbital and SLNs were dissected for ICP-MS measurements.

ICP-MS of AuNCs in Dissected Lymph Nodes

The excised lymph nodes were weighed and then were completely digested with 5 mL of aqua regia in a 50 mL beaker at boiling temperature. The solution was evaporated to 1 mL and subsequently diluted to 15 mL with deionized water. Samples were passed through a 0.45-mm filter to remove any undigested debris prior to ICP-MS measurement. The analysis of Au content was performed with ICP-MS (Perkin Elmer, Elan DRC II), and the concentration of Au ions was converted to the concentration of nanocages once the geometric dimensions of the nanocage had been determined from TEM images.

Acknowledgments

This work was supported in part by a 2006 NIH Director’s Pioneer Award (DP1 OD000798), a grant from the NCI (R01 CA13852701), and startup funds from Washington University in St. Louis to X.Y. It was also sponsored by other NIH grants (R01 EB000712, R01 EB008085, R01 CA134539, and U54 CA136398) and a grant from St. Louis Institute for Nanomedicine (SLIN) to L.V.W. Part of the research was performed at the Nano Research Facility, a member of the National Nanotechnology Infrastructure Network (NNIN), which is supported by the NSF under ECS-0335765. L.V.W. has a financial interest in Microphotoacoustics, Inc. and Endra, Inc., which, however, did not support this work.

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