We prepared near-infrared (NIR) type II quantum dots (QDs) (see Supplementary Fig. 1
online) with an oligomeric phosphine coating7
that rendered them soluble in aqueous buffers (see Methods
and Supplementary Fig. 1
online). When sterile-filtered and stored as a 2- to 4-μM stock solution in phosphate-buffered saline (PBS), NIR type II QDs remained stable for at least four weeks at room temperature. Transmission electron microscopy (TEM) of NIR type II QDs dispersed in water showed fairly spherical semiconductor particles 10 nm in diameter (). Using a model described earlier by our group1
, we chose NIR emission wavelengths that offset the competing effects of tissue photon penetration and charge-coupled device (CCD) camera quantum efficiency. In neutral aqueous buffer, the peak emission was tuned to 840–860 nm while preserving absorption cross-section ( and Supplementary Fig. 1
online). The full-width half-maximum of the emission was 90 nm. NIR QDs showed typical broadband absorption, with an increasing extinction coefficient at bluer wavelengths (). At the first absorption peak (775 nm), the extinction coefficient was 580,000 M−1
(), consistent with the large particle size. The quantum yield in PBS was 13% (data not shown). By gel filtration, NIR QDs ran equivalently to a protein of 440 kDa. This corresponds to a hydrodynamic diameter of approximately 15.8 nm (), which is well within the critical hydrodynamic diameter range of 5–50 nm needed for retention of QDs in the sentinel lymph node (SLN)8
. The hydrodynamic diameter measured by quasi-elastic light scattering (QELS) was 18.8 nm (data not shown). Our data suggest that polydentate phosphines add a minimal thickness to the QD as the hydrodynamic diameter measured by both gel filtration and QELS was only 5.8–8.8 nm larger than the diameter of the inorganic core and shell measured by TEM.
Figure 1 Physical and optical properties of aqueous-soluble, NIR type II QDs. (a) TEM image of water-dispersed NIR QDs. (b) Molar extinction coefficient (solid curve; left axis) and photoluminescence intensity (dashed curve; right axis) of NIR QDs in PBS, pH 7.4. (more ...)
Conventional NIR fluorophores, such as IRDye78-CA, dissolved in serum or aqueous buffer rapidly photobleach when exposed to fluence rates above their photobleaching threshold (; ref. 9
). In contrast, NIR QDs coated with oligomeric phosphines actually photobrighten slightly as fluence rate increases and show no deterioration of fluorescence emission even at fluence rates of 600 mW/cm2
(). Photobrightening effects have been observed previously in similar systems10,11
. The mechanism is postulated to be either a photoannealing effect or a photoneutralization effect. We further assessed the serum stability of NIR QDs by incubating them in 100% serum at 37 °C and recovering them by filtration. After 30 min (twice as long as a typical SLN mapping procedure), total fluorescence emission decreased by only 10%, indicating minimal aggregation and/or reduction of fluorescence quantum yield (). These data suggest that type II NIR QDs will perform well in biomedical applications, such as SLN mapping, under conditions of high fluence rate and prolonged exposure to bodily fluids at core body temperature.
The type II NIR QDs described in this study were engineered with a hydrodynamic diameter of 15–20 nm, a maximal absorption cross-section, fluorescence emission at 840–860 nm, a reasonable aqueous quantum yield and a stable organic coating. Because of their optimal size for retention in lymph nodes, we explored whether these QDs could be used for lymphatic mapping. When injected intradermally in the paw of a mouse (n
= 4), NIR QDs entered the lymphatics and migrated within minutes to an axillary location that could be detected using our previously described intraoperative imaging system12
(). Reinjection of the same site with the gold-standard SLN mapping agent isosulfan blue resulted in colocalization of the NIR fluorescence signal and the blue dye () in what was confirmed histologically to be the SLN (data not shown).
Figure 2 NIR QD sentinel lymph node mapping in the mouse and pig. (a) Images of mouse injected intradermally with 10 pmol of NIR QDs in the left paw. Left, pre-injection NIR autofluorescence image; middle, 5 min post-injection white light color video image; right, (more ...)
These mouse data are relevant to axillary SLN mapping for diseases such as breast cancer; however, we wanted to prove that NIR QDs could be used in large animals the size of humans. We found that when 400 pmol of NIR QDs were injected intradermally on the thigh of a 35 kg pig (n = 5), a surgeon was able to follow lymphatic flow towards the SLN in real time and to quickly identify the position of the SLN (). These real-time images included lymph channels that diverge from the injection site and then coalesce into the SLN (). Localization of the SLN required only 3–4 min, and the NIR QDs permitted image guidance throughout the procedure. Indeed, such guidance minimized the size of the incision needed to find the node, because its position could be determined beforehand, and as tissue was dissected, the intensity of the SLN increased exponentially (). This removed all ambiguity about SLN location and permitted identification of the SLN even when it was part of a large lymph node cluster (see below), as is common in pigs. As was seen in the mouse, coinjection of isosulfan blue resulted in colocalization of NIR fluorescence and dye (). The average depth of the femoral SLN in the pig was approximately 1 cm from the skin surface and, as determined by fluorescence, approximately 2–4% of the injected dose accumulated in the SLN (data not shown). After the procedure, the surgical site was inspected with high sensitivity to ensure complete resection of the SLN (). Analysis of resected tissue showed that NIR QDs were completely trapped in the SLN () and confined to the outermost rim of the node (). These data are consistent with the known sieving properties of the lymph node's subcapsular sinus.
Figure 3 Post-resection inspection of the surgical field and evaluation of NIR QD lymph node retention. (a) Post-resection evaluation of the surgical field. Shown are color video (left), NIR fluorescence (middle) and color-NIR merge (right) images. Arrows indicate (more ...)
We have demonstrated that type II NIR QDs with a polydentate phosphine coating can be used effectively for SLN mapping in both small and large animals. These data are significant for several reasons. First, NIR QDs eliminate the need for both a radioactive tracer and a blue dye, because lymph flow and the SLN can be identified optically, and in real time, using intraoperative NIR fluorescence imaging. Second, the surgeon is provided with image guidance, including imaging of underlying anatomy, throughout the procedure. At present, the learning curve for SLN mapping is several dozen cases. NIR QDs have the potential to markedly improve this learning curve. Third, after resection, the surgeon can inspect the surgical site with high sensitivity to ensure completeness of the procedure. Fourth, the pathologist can easily identify the SLN after resection, even if it is part of a large nodal chain () or matted to other bloody tissue, and can focus attention on the specific part of the SLN that is most likely to contain malignant cells. Fifth, unlike small molecules such as isosulfan blue, NIR QDs have an optimal size (and thus do not flow past the SLN), have a maximal absorption cross-section and do not photobleach.
It should be emphasized that our large-animal studies were performed with only 5 mW/cm2
of NIR excitation light and 400 pmol of NIR QDs. Despite this low fluence rate, we were able to identify SLNs approximately 1 cm below the skin surface using reflectance imaging. Our data suggest that even higher sensitivity can be achieved without concern for photobleaching. Optical guidance, even in bloody surgical fields, is likely to permit SLN mapping of other human cancers, such as colon cancer, which until now has been technically difficult. Additionally, frequency-domain photon migration techniques13,14
will likely extend depth detection to the 6- to 10-cm range.
The toxicity of NIR QDs has not yet been examined. In their elemental form, the three metals (cadmium, tellurium and selenium) have known acute and chronic toxicities. However, no data exist on the toxicity of precomplexed nanocrystals injected subdermally. Without such data, one can only speculate by extrapolating the known dose-effect relationships for oral exposure. For the pig experiments described above, 400 pmol of NIR QDs corresponds to approximately 9.9 μg/kg, 7.3 μg/kg, 2.4 μg/kg and 4.1 μg/kg of cadmium, telluride, selenide and alkyl phosphines, respectively. In the case of cadmium, for which the most data are available, this dose is approximately 300 times lower than the daily dose that causes renal toxicity in rats after 6 weeks of continuous exposure in drinking water15
. Moreover, a treatment for elemental cadmium poisoning in humans is the infusion of elemental selenium to produce less toxic cadmium selenide salts. It is possible, therefore, that despite being composed of potentially toxic materials, the low dose and chemical form of the materials are such that the overall toxicity is low. In the short term, at least, we saw no change in electrocardiographic and pulse oximetry measurements during large-animal surgery and for several hours thereafter.
Our data suggest simple strategies for minimizing the potential for toxicity. The first is simply to increase the fluence rate and proportionally lower the injected NIR QD dose. The photobleaching data suggest that at least a 100-fold lower dose could be used. The second is that type II structures permit combinations of nontoxic materials, which would otherwise emit in the visible light range, to emit in the NIR. Finally, during a typical SLN mapping procedure, the tumor (site of QD injection) and SLN are both resected, thus minimizing exposure to the QD materials. Future studies should address these important issues and the use of type II QD structures in other biomedical assay and imaging applications.
Because of their unique optical properties, type II NIR QDs provide the surgeon with direct visual guidance throughout the entire SLN mapping procedure, minimize incision and dissection inaccuracies, and permit real-time confirmation of complete resection. SLN mapping has already revolutionized cancer surgery. The technology described in this study has the potential to improve the technique even further.