Dual-emissive quantum dots for multispectral intraoperative fluorescence imaging

Biomaterials 31 (2010) 6823e6832

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Dual-emissive quantum dots for multispectral intraoperative fluorescence imaging Patrick T.K. Chin a, Tessa Buckle a, Arantxa Aguirre de Miguel b, Stefan C.J. Meskers b, René A.J. Janssen b, Fijs W.B. van Leeuwen a, * a

Departments of Radiology and Nuclear Medicine, Division of Diagnostic Oncology at The Netherlands Cancer Institute e Antoni van Leeuwenhoek Hospital, 1066 CX Amsterdam, The Netherlands Molecular Materials and Nanosystems, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 April 2010 Accepted 17 May 2010 Available online 18 June 2010

Fluorescence molecular imaging is rapidly increasing its popularity in image guided surgery applications. To help develop its full surgical potential it remains a challenge to generate dual-emissive imaging agents that allow for combined visible assessment and sensitive camera based imaging. To this end, we now describe multispectral InP/ZnS quantum dots (QDs) that exhibit a bright visible green/yellow exciton emission combined with a long-lived far red defect emission. The intensity of the latter emission was enhanced by X-ray irradiation and allows for: 1) inverted QD density dependent defect emission intensity, showing improved efficacies at lower QD densities, and 2) detection without direct illumination and interference from autofluorescence. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Quantum dots Dual-emissive particles Multispectral fluorescence imaging Sentinel lymph node Prostate cancer

1. Introduction Visual assessment of disease is often one of the first diagnostic lines in medicine. Especially surgical interventions demand a “seeand-treat” approach. Hence it is common that surgical interventions based on non-invasive imaging technologies e.g. radioguided sentinel lymph node (SLN) dissections are performed in combination with superficial visual guidance using a dye such as patent blue [1]. Recent developments in surgical image guidance have resulted in the inclusion of fluorescent, or rather luminescent, dyes [2,3]. The requirement of only superficial optical detection in the SLN procedure warrants the use of visible dyes with limited tissue penetrating properties. Indeed, several fluorescent imaging agents that emit in the visible range (450e650 nm) have been described for their potential in imaging applications e.g. fluorescein (in clinical use) [4], 5-aminovulinic acid (in clinical use) [5], dendrimers [6], microspheres [7] and quantum dots (QDs) [8e10]. The latter (QDs) are not only brightly fluorescent, with a size tunable emission color, but also have proven to be a versatile platform for further functionalization with e.g. tumor targeting moieties [10]. Approaches using luminescent probes in general require an external optical excitation, resulting in background signals induced

* Corresponding author. Tel.: þ31 20 5126084; fax: þ31 20 5122934. E-mail address: [email protected] (Fijs W.B. van Leeuwen). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.05.030

by reflectance, scattering or autofluorescence of tissue [11]. Moreover, the tissue absorbance of light leads to strong signal attenuation of both the excitation and emission signals [11]. For this reason the mainstream focus is on the use of near infra red (NIR; >800 nm) in fluorescence-based image guidance [12]. As an alternative to NIR imaging agents, the luminescent signal to background ratio (SBR) at increased tissue depths can also be improved with probes that do not require real-time excitation and emit in the tissue transparent range above 650 nm [13]. Examples of such probes are “self-illuminating” [13] and “defect-emission”[14] based imaging agents. Light emissions >650 nm become rapidly invisible to the human eye [15] and require specialized camera based imaging. On the other hand, to enable visual detection during (complex) surgical procedures an imaging agent should absorb or emit light between 400 and 650 nm. Combining superficial visual and “deep” far red/NIR image guidance requires either “cocktails” of dyes, or dual-emissive luminescent imaging agents that have an emission between 400 and 650 nm and one above 650 nm. Multispectral imaging is a technical extension in fluorescence imaging that allows for the detection of multiple emissions in a single imaging session. Spectral splitting of signals is a.o. used to: visualize multiple dyes at once, increase the detection specificity, and enable more quantitative measurements [16]. Because of these properties it is also an upcoming imaging technology in surgical image guidance [17]. In multispectral imaging the emission

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detection is typically performed after excitation at a single excitation wavelength [18]. Hence, dedicated QD based imaging agents for multispectral imaging have been reported in the form of “large” (1.2 mm) polymer particle assemblies containing multiple QDs; [19] most commonly used QDs have a similar absorbance trend with increased absorption values <400 nm. However, size matters during the (bio) distribution and clearance of an imaging agent; Choi et al. have demonstrated that smaller particles have better clearance properties [20]. Grabolle et al. have stated that the multispectral imaging technology would also benefit significantly from the incorporation of lifetime dependant emissions [21]. The use of lifetime imaging may help filter out the short-lived autofluorescence. Hence, a combination of lifetime dependence and multispectral imaging would allow for signal discrimination using both the emission wavelength and the emission lifetime, improving the accuracy of fluorescence imaging even further. The ideal multispectral imaging agent for surgical guidance to the SLN should thus be a “small” particle with at least two emissions that have respective short and long emission lifetimes. Despite the obvious advantages of imaging guidance during surgical interventions there are still challenges to be met in the development of luminescent imaging agents that allow for combined visual (superficial) and deep tissue imaging. In this manuscript we present (lifetime dependant) multispectral InP/ZnS QDs that meet both these requirements and that are applied in SLN procedures. 2. Methods 2.1. Reagents Indium chloride, tris(trimethylsilyl)phosphine (TMS)3P, hexadecylamine (HDA), stearic acid, sulfur, dodecanethiol, and 1-octadecene (ODE) were obtained from Sigma Aldrich. Zinc undecanoate was synthesized following an adapted metal carboxylic acid method published by Pradhan et al. [22] using triethylamine as the base. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (18:0 PEG2 PE) was obtained from Avanti Polar Lipids. D-luciferine fire fly potassium salt was obtained from Biosynth.

2.4. Lipid coating of QDs For the in vivo studies a micellular polyethylene glycol (PEG) coating was applied to make the QDs water-soluble and biocompatible [24]. This micellular coating comprised a pegylated phospholipid, PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(poly(ethylene glycol))-2000]). The 6 mg InP/ZnS single washed QDs (3.2 mg InP/ZnS from TGA) were dissolved in 2 mL of chloroform and were mixed with 18 mL PEG-DSPE (174 mg) solution in chloroform. The chloroform solution was slowly added to 70 mL water (1.5 h) at 80  C under vigorous stirring and nitrogen flow. Empty micelles and micelles containing a QD were separated via ultra-centrifugation (1.5 h, 368,000g). Next, the lipid coated QDs were redispersed in 0.9% saline solution. Thermogravimetric analysis (TGA) showed that 52 mass % of the single washed quantum dot content consists of InP/ZnS whereas 48% of the mass can be related to organic surfactants. 2.5. Optical Spectroscopy UVevis spectra were recorded using a Perkin Elmer Lambda 900 or Lambda 3b spectrophotometer. Steady state fluorescence spectra were measured with an Edinburgh Instruments FS920 spectrophotometer. Time-resolved fluorescence spectra were measured with an Edinburgh Instruments LP920 spectrophotometer equipped with a Flash lamp pumped Q-switched Nd:YAG laser (350 nm excitation output). An intensified charge-coupled device (CCD) camera was used to record the emission spectra. The absolute intensities (counts) measured with the Edinburgh Instruments LP920 cannot be compared with the absolute values measured in tissue and solution using the Xenogen IVIS 200, due to the machine related differences e.g. detector, excitation source, and measurement settings. Long term decay of the InP/ZnS defect emission was recorded using the IVIS 200 (Xenogen Corp.) camera, applying the bioluminescence settings (open filters; standard bioluminescence setting). The narrow bandpass filter set of the IVIS 200 system was used to record the defect emission spectrum of InP/ZnS QDs (see Fig. 2c) in ODE (camera integration time ¼ 30 s; lex ¼ 366 nm, 4 W electrical power, UV light). All defect emission measurements were excited (lex ¼ 366 nm) at least 20 min prior to the measurement, followed by an additional excitation shortly before the measurement as indicated in the experimental sections. 2.6. Quantum yield Photoluminescence quantum yields (hF) were estimated using rhodamine 101 (in ethanol þ 0.01% HCl; hF ¼ 100%) [25]. All solutions had an optical density <0.1 at the excitation wavelength (lex ¼ 520 nm) to minimize re-absorption and avoid absorbance saturation. The quantum yield was derived from luminescence spectra, by correcting for the optical density and the refractive index of the solvents used for sample and reference [26]. 2.7. X-ray irradiation

2.2. Synthesis of QDs InP/ZnS quantum dots were synthesized under nitrogen flow following a method published by Xu et al. [23] with some modifications as outlined below. (TMS)3P (60 mg; 0.2 mmol) dissolved in ODE (1 mL) was swiftly injected into a reaction mixture of stearic acid (57 mg; 0.2 mmol), HDA (65 mg; 0.7 mmol), ODE (6 mL), zinc undecanoate (172 mg; 0.39 mmol) and indium chloride (44 mg, 0.2 mmol) at 280  C. After stirring for 20 min at 240  C, the reaction was allowed to cool down to room temperature, followed by the addition of 100 mg zinc undecanoate (0.23 mmol), 108 mg HDA (0.45 mmol) and 6 mL ODE. Elemental sulfur (15 mg; 0.47 mmol) dissolved in ODE (2 mL) was added dropwise during 20 min at 230  C, followed by an annealing step of 60 min at 200  C. After cooling to room temperature 2 g HDA (8.3 mmol) and 0.5 ml (2.1 mmol) dodecanethiol were added to create an improved organic surface stabilization and the mixture was stirred overnight. The core shell InP/ZnS crystals were subsequently isolated by dissolving the reaction mixture in chloroform (10 mL), followed by precipitation through addition of acetone (20 mL). The QDs were isolated by centrifugation and dissolved in cyclohexane. This was followed by an additional acetone precipitation step and isolation by centrifugation. Subsequently the QDs were dried overnight under vacuum and stored under nitrogen.

2.3. QD size selection The size distribution of the InP/ZnS QDs could be improved by size selective precipitation. Size selective precipitation is based on the gradual decrease in solubility of the QDs by the addition of a non-solvent (methanol) to a QD solution. This resulted in a faster decrease in solubility for the larger particles. This approach allows for the collection of fractions with a narrower size distribution. To this end 3 mg of purified InP/ZnS QDs were dissolved in z2 ml chloroform, subsequently methanol was added dropwise under stirring until the solution became slightly turbid. The fraction of precipitated QDs was collected by centrifugation (4 min, 4200 rpm). This procedure could be repeated several times to collect different fractions.

To study the influence of X-ray irradiation on the exciton emission and defect emission InP/ZnS QD dispersions were irradiated using a PANTAK pmc1000 X-ray machine operated at 250 kV, 12 mA with a dose rate of 1.08 Gy/min. To determine the best irradiation dose, the QDs were exposed to doses of 2, 4, 6, 8, and 10 Gy. For the in vivo experiments lipid coated InP/ZnS QD solutions (0.9% saline solution) were irradiated with 10 Gy. 2.8. Exciton emission, defect emission, and bioluminescence imaging in mice or tissue Tumor bearing mice were surgically dissected with a clinical grade surgical microscope (Zeiss OPMI 6-SD) the InP/ZnS QDs were illuminated with experimental LED’s; a standard 400 nm LED and a high intensity set-up with a 380 nm LED (Nichia) (see Supplementary information Fig. S1). After visual imaging the animals and/or the dissected tissues were placed in the temperature controlled (37  C) imaging chamber of an IVIS 200 (Xenogen Corp.). The exciton emission was measured with the “standard” GFP excitation and emission settings (camera integration time ¼ 5 s). The defect emission was imaged (>3 min) after LED excitation (lex ¼ 400 nm) with open filters (standard bioluminescence setting; camera integration time: 120 s), and bioluminescence imaging (open filters; standard bioluminescence setting; camera integration time: 30 s) was performed 10 min after i.p. luciferin injection (180 mg/kg) or tissue incubation in a similar luciferin solution. No defect emission experiments were performed in mice with luciferase expressing tumors cells, because of a background signal that was detected in luciferase expressing tissue even without recent addition of luciferin. The signal intensities (photons/cm2/s) were quantified with Living image 3D software (Xenogen Corp.) 2.9. Emission absorbance by pig tissue using QDs For the tissue penetration measurements capillaries were filled with 20 mL of irradiated (10 Gy) InP/ZnS QD solution (10 mg/mL QDs containing 5.2 mg/mL InP/ ZnS from TGA) and placed at different depths in a piece of pig tissue. (See Fig. 6 and Supplementary information Fig. S9). The exciton emission flux trough pig tissue is

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Fig. 1. Visible fluorescent properties of the InP/ZnS QDs during a surgical SLN procedure in mice with metastatic prostate cancer. (a) Visible detection of the injection site with QDs trough the skin using under UV (lex ¼ 366 nm) excitation. (b) The absorbance spectrum (black line) of human blood, and the InP/ZnS QD steady state emission spectrum (lex ¼ 400 nm) demonstrates that the exciton emission does not completly overlap with the human blood absorbance spectrum. (c) Intraoperative visual detection of InP/Zn QDs; regions with a high QD density (i.e. injection site 1) are yellow-orange and migration sites with a lower QD density (i.e. lumbar lymph nodes 3 and 4) are green (lex 380 nm). (d) Ex vivo imaging of the prostate with seminal vesicles (1), caudal lymph node (2), and lumbar lymph nodes (3 and 4): Exciton emission setting provide a measure for the difference in signal intensities (lex 445e490 nm, lem 515e575 nm) (intensity bar unit: counts), and (e) Bioluminescence imaging of the luciferase expressing PC-3 tumor cells functions as a reference for the tumor spread. (Intensity bar unit: counts) (f) Visual color difference for InP/ZnS QDs in chloroform dispersion. From left to right, (I) QD emission after precipitation with methanol, (II) 7 mg/mL QD density in solution, (III) a lower 0.07 mg/mL QD density (lex ¼ 366 nm; 4 W electrical power). obtained using the IVIS 200 GFP settings and is divided by the background to obtain the signal to background ratio (SBRex). Using the following formula: SBRex ¼ IQD

ex =Iex background

Where IQD_ex and Iex_background are, respectively, the exciton emission of the inserted QDs and the background signal from surrounding tissue at the exciton emission imaging settings. The relative signal intensity of the defect emission was corrected for fluctuations in pre-excitation intensity (lex ¼ 366 nm) between blank and tissue measurements. To do this the small drop of QDs in the outer capillary segment that was not inserted in the tissue (see Fig. S9a and b) and provides a reliable reference for non tissue related changes in defect emission signal intensity. Leading to a correction factor (Id_dfe_blank/Id_dfe_control) for non tissue related pre-excitation intensity fluctuations. Where Id_dfe_blank represents the defect emission intensity from the reference drop on top of a black background, Id_dfe_control is the defect emission intensity from the reference drop when the capillary is inserted in the tissue. This correction factor was subsequently multiplied with the defect emission signal through tissue (IQD_dfe) and divided by the defect emission background signal (Idfe_background) to obtain the relative defect emission SBR (SBRdfe): SBRaft ¼

Id dfe blank Id dfe control

!

!,  IQD

dfe

Idfe

background

Each individual point in Fig. 6b for both the exciton emission and defect emission penetration is an average of 4 measurements. Within 1e3 min prior to the defect emission measurement the capillaries were additionally excited with UV light (lex ¼ 366 nm). A schematic representation of the different emission intensities used in the equations is provided in the Supplementary information Fig. S9. 2.10. Emission absorbance by pig tissue using ICG and fluorescein For the optical tissue penetration measurements glass seeds (0.9 mm  7 mm) were filled with 0.5 mg/ml ICG or fluorescein solution in water. These seeds were

subsequently placed at different depths in a piece of pig tissue. The fluorescence flux trough pig tissue is obtained using the IVIS 200 GFP or ICG settings and is divided by the background to obtain the SBR, using the following formula: SBR ¼ Iflu =Ibackground Where Iflu and Ibackground are, respectively, the fluorescence of ICG or fluorescein and the background signal from surrounding tissue. 2.11. Animals Luciferase expressing PC-3 prostate tumor cells were orthotopically transplanted in the prostate of immune deficient Balb/c nude mice (10e15 weeks of age; n ¼ 5, see Supplementary Fig. S2). The tumor progression was non-invasively monitored using the bioluminescent signal emitted by the PC-3 tumor cells. Before PC-3 prostate tumor cell transplantation and the surgical/imaging procedures, the animals were anaesthetized with a 1:1:2 mixture of hypnorm (Vetapharma), dormicum (Roche) and water (5 mL/g i.p.). After tumor cell transplantation, the incision was closed and mice received an i.p. injection of 10 mL of Temgesic (0.3 mg/mL buprenorfin; Schering Plough) in 1 mL of 5% glucose/saline solution for post operative pain relief. For the SLN procedure, animals were injected intratumorally with 20 mL lipid coated InP/ZnS QDs (3.7 mg/mL; 0.9% saline solution). For the comparison in autofluorescence at the exciton emission and defect emission settings mice (n ¼ 4) of the same strain (without tumor) where used. All animal experiments were performed in accordance with Dutch animal welfare regulations and were approved by the local ethics committee.

3. Results 3.1. Generation of InP/ZnS QDs InP/ZnS QDs were prepared and characterized as described in the experimental section, adapted from the procedure of Xu et al.

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Fig. 2. Optical properties of InP/ZnS QDs with dodecanethiol capping. (a) Absorption spectrum (solid spheres) and PL spectra (green and black; open symbols) in the time range between 100 ns and 5 ms after 350 nm excitation of InP/ZnS QDs, (b) Defect emission spectra in the time range 10 mse100 ms, demonstrating the formation of an additional emission peak around 600 nm. (c) Normalized PL spectra of the exciton e (green) and defect emission (red and bar diagram) illustrating the w100 nm red shift between the respective spectra at 100 ns, 100 ms (red) and 3 min (bar diagram). The defect emission spectrum depicted in bars was recorded using the different IVIS 200 narrow bandpass filters 3 min excitation of the sample (between 700 and 800 nm no filters were available). (d) Normalized PL spectra of the exciton e (green) and defect emission (red) from larger, more NIRshifted InP/ZnS QDs, illustrating a similar red shift between the respective spectra at 80 ns and 80 ms. (e) Normalized PL spectra of the exciton emission of small w2.8 nm (orange, open spheres) and a large w5 nm (dark red, solid squares) size selected InP/ZnS QD samples. (f) Defect emission signal intensities during a time period of 30 min recorded for small (orange, open spheres) and a large (dark red, solid squares) size selected QD samples (lex ¼ 366 nm, 4 W electrical power, 3 min before t ¼ 0).

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[23]. The InP/ZnS QDs collected from the reaction mixture exhibit a fluorescence quantum yield (hF) of z70% in chloroform. To allow for optimal visual detection sensitivity (520e550 nm range), we tuned the exciton emission of the InP/ZnS QDs towards that of fluorescein (lmax ¼ 520 nm), a dye clinically used for visual image guidance during surgical procedures (see above). 3.2. Visual luminescence based image guidance The SLN application, which allows for the use of relative high local imaging agent quantities and does not require specific targeting, is an ideal starting point to explore the potential of the InP/ ZnS QDs in visual surgical guidance. For these (non-targeted) studies of the migration of the InP/ZnS QDs to the tumor draining SLNs, only a PEG-lipid coating had to be applied to make them water-soluble, resulting in an z7 nm increase in diameter [24]. For the challenging SLN procedures of the prostate we used male mice wherein highly metastatic human PC-3 prostate tumor cells were orthotopically transplanted (see Supporting information Fig. S2). The intratumorally injected QDs could be (Fig. 1a) noninvasively detected under UV illumination (366 nm, 4 W electrical power flashlight). The spectral overlap between the QD exciton emission and hemoglobin absorbance is not complete as shown in Fig. 1b, hence the orange/red part of the QD emission can still be visually detected trough a well (blood-) perfused section of skin. During the surgical resection the luminescence at superficial depth from the injection site (primary tumor; orange) and SLNs (green) was clearly visible by eye, under direct excitation by either 380 nm (LED; Fig. 1c) or 366 nm (4 W electrical power flashlight) light. Ex vivo luminescence measurements of these emissive tissues (Fig. 1d) indicates that the emission intensity was highest at the orange regions. Similar color changes can also be observed when the particle density of the InP/Zn QD solutions is altered by dilution or when clustering of the particles is forced (see Fig. 1f, further discussed in the supporting section). The luciferase expression of the PC-3 tumor cells was used to accurately diagnose the metastatic tumor spread (Fig. 1e). Combined this result demonstrates the potential of InP/ZnS QDs to provide visual intraoperative guidance during a SLN procedure. 3.3. Multispectral properties of InP/ZnS QDs In addition to the visible luminescence which is also known as the exciton emission, just below the optical band gap, InP QDs often show a red shifted defect related emission. To study the difference in lifetime dependence of both the exciton emission of the InP/ZnS QDs and the defect related emission, we recorded the luminescence spectra in the 100 nse100 ms time range. Shortly (100 ns) after the excitation pulse (350 nm) the emission spectrum of the InP/ZnS QDs shows a relatively narrow emission at 520 nm with a full width at half maximum (FWHM) of 115 nm (Fig. 2a). This emission is attributed to exciton recombination. Over time (100 nse100 ms) a significant peak broadening is observed (FWHM: 237 nm at 100 ms) and the peak maximum is shifted to 600 nm (Fig. 2b and c). After even longer luminescent lifetimes (100 mse100 ms) the maximum shifts further to the far red ending with a maximum at 650 nm and thus a red shift of w130 nm (see Fig. 2c). The intensity of the defect emission is only reduced by half at 80 min after excitation (see Fig. 3d). For particles with an exciton emission peak at 706 nm (at 80 ns) the defect emission peak is shifted to 762 nm (at 80 ms), which for a substantial part extends into the NIR (>800 nm; Fig. 2d). The InP/ZnS QDs steady state exciton emission is relatively broad with a FWHM of 117 nm, as result of the relative size distribution. The influence of the size distribution on the defect

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emission was studied using InP/ZnS QDs after size selection. The luminescence spectra in Fig. 2e are obtained using two size selected InP/ZnS QDs dispersions in chloroform. The steady state exciton emission spectra show peak maxima at 566 nm (FWHM of 86 nm, InP core size w2.8 nm), and at 709 nm (FWHM of 64 nm, InP core size w5 nm). Both size selected samples also show a clear defect emission and in both cases the decay behavior is nearly identical (see Fig. 2f). Only the position of the defect emission is influenced by the QD size. From various bulk crystals it is known that dislocations within the crystal lattice (Fig. 3a), often referred to as Freckle defects [27], can be induced upon exposure to “high-energy” electromagnetic radiation (g), or high-energy particles (e.g. electrons (b)) [27,28]. These defects can function as (emissive) energy traps and as such can enhance the defect emission properties of the InP/ZnS QDs. Therefore, we subjected InP (data not shown) and InP/ZnS QDs dispersed in chloroform to different X-ray doses, namely 0, 2, 4, 6, 8, and 10 Gy (Fig. 3b and c). Irradiation of the QDs clearly increases the defect emission signal intensity; 10 Gy induces an approximately eighteen-fold increase in the defect emission signal intensity (Fig. 3c). Conversely, the same irradiation dose results in 50% decrease of the exciton emission intensity (Fig. 3b). In the SLN studies (see Fig. 1c) the QDs showed a concentration or rather particle density dependant visible emission color. To investigate the effect of the particle density on the defect emission, we monitored both the exciton and defect emission relative signal intensities over a broad QD density range in solution (0.57e11.4 mg/mL; dodecene). While the exciton emission signal gives a steady increase in relative intensity, which is in line with the increase in particle density (Fig. 4a and c; values at 11.4 mg/mL have been set at 100%), the defect emission shows an interesting deviation from this pattern. Increasing the QD density results in a sharp drop in the relative defect emission signal intensity that reaches a trough at 2.8 mg/mL (see Fig. 4a and b). This behavior can be further clarified by plotting the ratio between the absolute (photons/s/cm2) defect and exciton emission intensity as function of the particle density. The absolute ratio between the defect and exciton emission (>1 min) signal intensities decreased rapidly between 0.57 mg/mL (w104) and 2.8 mg/mL (w105), which is in line with the drop in relative signal intensity (see Fig. 4a and d). At densities above 2.8 mg/mL the absolute ratio stabilizes (see Fig. 4d), allowing for the gradual increase observed for the relative defect emission signal intensity upon increasing the QD density (see Fig. 4a and b). 3.4. In vivo application dual-emissive properties The dual-emissive properties were also studied in SLN applications. Unfortunately we found that the luciferase expressing PC-3luc tumor cells can also create a “weak” background signal in the absence of luciferase. Because such a signal can be misleading during defect emission imaging, we only include these in vivo images in the Supporting information (Fig. S8). To avoid the possibility of this background signal a control experiment was performed where the QDs were injected in mice holding a PC-3 tumor of which the tumor cells were not transfected with luciferase. Fig. 5 illustrates the multispectral detection of both QDs luminescent signals in the SLN. Ex vivo analysis of organs (prostate/seminal vesicles, kidneys, spleen, heart/lung, and liver) after the injection of InP/ZnS QDs in the prostate of control mice provides a clear comparison between the background signals encountered during exciton and defect emission imaging (see Fig. 5c and b). At the exciton emission setting, but also in the far red and NIR range (see Supporting information Fig. S7), autofluorescence from organs such as the

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Fig. 3. Influence of X-ray radiation on the optical properties of InP/ZnS QDs. (a) Schematic illustration of X-ray induced crystal lattice defects in the InP/Zn QDs. (b) Relative decrease of the exciton emission (at 100 ns) as a function of the amount of X-ray irradiation. (c) Relative increase of the defect emission (>3 min) as a function of the amount of X-ray irradiation. (d) Defect emission signal intensities during a time period of 80 min measured in a QD dispersion with concentrations of 2.5 mg/mL (n ¼ 6; InP/Zn QDs were illuminated lex ¼ 366 nm, 4 W electrical power, 3 min before t ¼ 0).

gall bladder, spleen, liver, and kidneys may be easily mistaken for exciton emission from InP/ZnS QDs. Imaging of the pre-illuminated defect emission; no real-time excitation required, does not detect any autofluorescence. In Fig. 6a and b we demonstrate that the initially highly similar SBR (w5.5) of the excition emission and defect emission generated by capillaries filled with 20 mL InP/Zn QDs (5.2 mg/mL) reduces at increased tissue depths. The defect emission can be differentiated from the background signal up to 13 mm, while the exciton emission settings can only be differentiated from the background up to 3 mm. A similar trend between the signal penetration of the exciton emission and defect emission was observed in one of the main absorbing substances in the body, namely blood (see Supplementary information Fig. S6a). In addition, we demonstrated that the defect emission peak is positioned almost completely beyond the hemoglobin absorption peak (see Supplementary information Fig. S6b) and that the presence of warm (36  C) serum does not have a negative influence on the defect emission signal intensity of these QDs (see Supplementary information Fig. S6c). To allow for a direct comparison between the reported dualemissive QDs and two clinically applied dyes: indocyanine green (ICG: NIR) and fluorescein (visible), we also measured the penetration depth of the latter (Fig. 6c). Both the exciton emission from the QDs and fluorescein are only visible up to a shallow depth of 3e4 mm. The initial much higher SBR of fluorescein compared to that of the QD exciton emission is most likely caused by the fact that the IVIS 200 excitation and emission filter options (lex 445e490 nm, lem 515e575 nm) are optimal for fluorescein, but far from optimal for our QDs. Despite its relative low absolute signal intensity (see above), the defect emissions lack of requirement for real-time excitation and its w130 nm increase in the emission wavelength results in a tissue penetration that is comparable to the NIR-dye ICG (14 mm). This underlines the value of such a long lasting defect emission. 4. Discussion In general the fluorescent probe development focuses on NIRdyes that cannot be detected by eye as these give an increased tissue penetration because of their longer emission wavelength and reduced autofluorescence in vivo. For reference, the NIR

penetration does not exceed the (still superficial) cm range and thus does not compare to 3D imaging modalities such as e.g. positron emission tomography. Based on our own experience with the NIR-dye ICG (both in preclinical SLN studies and recently also in clinical trails) we reasoned that it would be ideal if it becomes possible to also visually detect a dye with NIR-like properties. Clearly, a visual dye cannot replace a NIR-dye for a use in (deep) in vivo imaging applications. However, as stated in the introduction, superficial optical imaging of visual dyes is widely applied in surgical interventions such as SLN imaging. With the use of our dual-emissive InP/ZnS QDs we have demonstrated the (theoretical) value of multispectral detection in the superficial guidance needed during an SLN application. 4.1. Properties InP/ZnS QDs To avoid the use of the intrinsically toxic cadmium and thus generate better QD candidates for future clinical translation [29], the QDs in this study are based on indium phosphide (InP) instead of the more commonly used cadmium selenide (CdSe). InP is a semiconductor with a size tunable band gap over the entire visible range and NIR [30]. The bulk band gap of InP (1.35 eV) [31] is w20% smaller than that of CdSe (1.71 eV) [32], which makes it possible to obtain the same fluorescent properties with smaller particles. This reduction in size may yield improved biological clearance and thus reduce unwanted accumulation in organs such as the liver [20]. We specifically focused on InP/ZnS QDs that have an exciton emission in the visible range (lmax ¼ 520 nm), rather than on one of our larger more NIR-shifted InP/ZnS QD particles (see Fig. 2). The ZnS shell growth on InP resulted in a strong increase in PL quantum yield from 10 to 20% for InP core QDs to z70% (chloroform), implying a strong reduction of surface defects; coating QDs with a lager band gap semiconductor shell (e.g. ZnS) is a common approach to reduce such a defect related emission [23]. The remaining presence of the defect emissions after shell growth, therefore, suggests that (at least in part) this defect emission is induced by crystal defects in the InP particle core. This assumption is underlined by the fact that the decay behavior of the defect emission is nearly identical (see Fig. 2f) for differently sized particles; only the lmax position of the defect emissions (Fig. 2d) is influenced by the QD size. Furthermore, the eighteen-fold increase

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Fig. 4. Inverse QD density dependent defect emission in solution. (a) Relative signal intensities of the exciton and defect emission as a function of the QD density. IVIS images of the QD dilutions in a well plate; defect emission (b) and exciton emission (c), clearly illustrate the different behavior in luminescence intensities at different QD densities. (d) QD density dependence of the ratio between absolute defect and exciton emission signal intensities as a function of the QD density. An in vivo example of the QD density dependence of the defect emission intensity (e) and the exciton emission intensity (f) is given to illustrate the ability of the defect emission to accurately visualize low QD density spots in close proximity to a high(er) QD density spot.

in the defect emission signal intensity after 10 Gy irradiation of the QDs (see Fig. 3c), suggests the formation of additional (crystal) defects sites [27,28]. The simultaneous decrease in the exciton emission intensity of QDs (see Fig. 3b) is a logical result of the quenching effect of such (defect) centers [33].

The InP/ZnS QDs clearly posses an additional defect emission, however, there is a difference in the absolute exciton and defect signal intensities (see Fig. 4d). Nevertheless, the relatively low defect emission signal intensity, provides an added value in tissue and in vivo studies (see Figs. 4e6).

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Fig. 5. Utilization of the luminescent properties of the InP/ZnS QDs in sentinel lymph node procedure in mice with prostate cancer (no luciferase expression). In vivo imaging of the inguinal lymph node by the (a) exciton emission (IVIS 200; GFP settings) and (b) defect emission (IVIS 200; open filter settings) in the SLN procedure. Ex vivo measurements at exciton emission settings (lex 445e490 nm, lem 515e575 nm) (c) and defect emission settings (d) illustrate the lack of autofluorescence using the defect emission settings. The different organs are numbered: (1) the prostate þ seminal vesicles, (2) kidneys, (3) spleen, (4) heart þ lungs, and (5) liver. (Intensity bar unit: counts).

4.2. Concentration dependence of the exciton and defect emissions During e.g. clinical radioscintigraphy based SLN procedures, it is a challenge to identify migration sites that lie in close proximity to the injection site. Exactly in this application luminescence imaging could provide an added value. During our preclinical SLN imaging procedures and after dilution/precipitation of the InP/ZnS solutions we observed a: i) density dependant color change and ii) density dependent defect emission intensity (see Fig. 1c,f and 4). The change in the visual emission color in solution and in the superficially located tissues (see Fig. 1c and f) appears to be caused by the QD size distribution which is not highly monodisperse (see shallow absorption onset Fig. 2a), rather than by color selective absorbance of hemoglobin (see e.g. Fig. 1a). Most likely, at higher QD densities self-absorbance of the blue shifted emissions by the larger QDs causes a red shift in the exciton emission color. In addition to the density dependence of the visible luminescence (see Fig. 1c and f) the relation between the exciton and defect emission may also help to differentiate low QD dense migration sites from high dense QD injection sites. The potential of this “defect-feature” in the detection of low dense sites near sites with

a higher density is demonstrated in Fig. 4e and f. Under the same conditions the low dense site is harder to differentiate from the high dense site using the exciton emission. A likely explanation for the decrease in the relative defect emission intensity at increased QD densities is the interaction of emitted defect emission photons with defect states of other QDs. Combined the dual-emissive QDs allow for the discrimination between the injection site and the more valuable site of lymphatic migration (SLN) using both visual color and the relation between the exciton and defect emission intensities. 4.3. Multispectral/lifetime imaging dual-emissive QDs in tissue Next to having different wavelengths, the two emissions also have different emission lifetimes. Where the exciton emission has a lifetime in the nano-second range, the defect emission can even be detected 90 min after illumination (see Fig. 3d). This property makes these InP/ZnS QDs ideal for multispectral/lifetime dependant imaging procedures. The green (lmax ¼ 520 nm) exciton emission is then only detectable after direct illumination, while the defect emission (lmax ¼ 660 nm) can also be detected after pre-

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Fig. 6. Comparison between tissue penetration of different luminescent emissions. (a) Photograph of the experimental set-up used to determine the tissue penetration: pig tissue with on top glass capillaries containing 20 mL of 5.2 mg/mL InP/Zn QD dispersion in octadecene (366 nm illumination; 4 W electrical power). The circles over the capillaries indicate the reference drop. (b) The SBRs of the exciton emission (green; open symbols) and the defect emission (red; solid symbols), of glass capillaries as function of the tissue depth, demonstrating the increase in tissue penetration obtained with the defect emission signal. (c) The SBRs of the fluorescein emission (green symbols) and the ICG emission (blue symbols).

illumination. Exciton emission based detection at increased tissue depths of InP/ZnS QDs, and perhaps QDs in general, would require the use of high power UV-lasers for excitation [34]. Indeed, we found that InP/ZnS QDs with an exciton emission maximum at 660 nm give a similar tissue penetration as the InP/ZnS QDs 520 nm emission (data not shown). Hence the lack of tissue penetration of the UV excitation light appears to be the limiting factor for these particles. The long lifetime of the pre-illuminated defect emission (lmax ¼ 660 nm), however, results in a significantly improved tissue penetration that is similar to that of a NIR-dye (see Fig. 6). Furthermore, the defect emission also does not suffer from autofluorescence (see Fig. 5), increasing its value in imaging applications even more. The dual-emissive particles combine the benefits of a visual dye (detectable by eye, but only very superficial) with those of an NIRdye (not detectable by eye, but increased tissue penetration). This dual-emissive property is currently being applied in e.g. signal depth estimation studies.

Acknowledgments This research is supported, in part, by the Technology Foundation, applied science division of NWO and the technology program of the Ministry of Economic Affairs (Grant No. STW BGT 7528 Veni; FvL), a KWF-translational research award (Grant No. PGF 20094344; FvL), the European Community’s Seventh Framework Program (FP7/2007-2013; TB), and the Marie-Curie Research Training Network NANOMATCH (Grant No. MRTN-CT-2006035884; AAdM). We gratefully acknowledge the Nichia cooperation for providing us with three high intensity LEDs and we thank Aldrik Velders (University of Twente) for his support. Appendix. Supplementary information Supporting Information Experimental details on the particle synthesis, characterization and in vivo/ex vivo investigation. Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biomaterials.2010.05.030.

5. Conclusion Appendix We have demonstrated the manufacture of InP/ZnS QDs with both a short-lived visible (520 nm) and an induced long-lived farred defect emission (650 nm). InP/ZnS QDs provide the possibility to exploit a number of exciting (optical) properties. First, X-ray irradiation can be used to enhance the defect emission signal intensity. Second, these particles have lifetime dependent emissions wherein the exciton emission has a “short” lifetime and the defect emission a “long” lifetime. Third, the QD density influences both the visual exciton emission color and the defect emission intensity. Fourth, the exciton emission provides (superficial) visible surgical guidance, while the defect emission (no real-time excitation required and no autofluorescence) provides “deep” tissue penetration. This combination makes these QDs a promising new class of chromophores. The ability to combine the above features in a single dual-emissive imaging agent adds visual guidance and lifetime dependence on top of an “NIR-like” like tissue penetration.

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