Targeted Luminescent Near-Infrared Polymer-Nanoprobes for In Vivo

ARTICLE pubs.acs.org/ac

Targeted Luminescent Near-Infrared Polymer-Nanoprobes for In Vivo Imaging of Tumor Hypoxia Joanna Napp,†,‡ Thomas Behnke,§ Lorenz Fischer,^ Christian W€urth,§ Marieke Wottawa,z D€orthe M. Katschinski,z Frauke Alves,*,†,‡ Ute Resch-Genger,*,§ and Michael Sch€aferling*,^ †

Department of Hematology and Oncology, University Medical Center G€ottingen, Robert-Koch-Strasse 40, 37075 G€ottingen, Germany Department of Molecular Biology of Neuronal Signals, Max-Planck-Institute for Experimental Medicine, Hermann-Rein-Strasse 3, 37075, G€ottingen, Germany § BAM Federal Institute for Materials Research and Testing, Richard-Willstaetter-Strasse, 11, 12489 Berlin, Germany ^ Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, 93040 Regensburg, Germany z Department of Cardiovascular Physiology, University Medical Center G€ottingen, Humboldtallee 23, 37073 G€ottingen, Germany ‡

bS Supporting Information ABSTRACT: Polystyrene nanoparticles (PS-NPs) were doped with an oxygen-sensitive near-infrared (NIR)-emissive palladium meso-tetraphenylporphyrin and an inert reference dye which are both excitable at 635 nm. The nanosensors were characterized with special emphasis on fundamental parameters such as absolute photoluminescence quantum yield and fluorescence lifetime. The PS-NPs were employed for ratiometric dual-wavelength and lifetime-based photoluminescent oxygen sensing. They were efficiently taken up by cultured murine alveolar macrophages, yielding a characteristic and reversible change in ratiometric response with decreasing oxygen concentration. This correlated with the cellular hypoxic status verified by analysis of hypoxia inducible factor-1α (HIF-1α) accumulation. In addition, the surface of PS-NPs was functionalized with polyethylene glycol (PEG) and the monoclonal antibody herceptin, and their binding to HER2/neu-overexpressing tumor cells was confirmed in vitro. First experiments with tumor-bearing mouse revealed a distinctive ratiometric response within the tumor upon hypoxic condition induced by animal sacrifice. These results demonstrate the potential of these referenced NIR nanosensors for in vitro and in vivo imaging that present a new generation of optical probes for oncology.

T

he detection and monitoring of cancer-related pathological changes requires robust, cost-effective, and efficient tools and strategies in medical research and diagnostics.1
3 Fluorescence-based techniques for multiplexed biomarker analysis are particularly valuable because they enable the measurement of different parameters within a single scan.4
6 The ability to identify tumors in an early stage is of enormous importance for effective treatment of cancer.7,8 Basic characteristics of solid tumors such as hypoxia and acidic pH result from inadequate oxygen supply and abnormal tumor metabolism.5,7,8 Hypoxic tumor cells produce a number of factors which can stimulate growth of new vasculature and promote invasiveness and metastatic spread. Moreover, hypoxic regions of tumors are frequently treatment resistant, altogether resulting in overall poor clinical outcome.9 Therefore, the development of improved probes for the measurement and imaging of tissue oxygen supply in vivo and in real time is of considerable interest for tumor diagnostics and characterization as well as for the evaluation of improved therapies. Polymer nanoparticles (NPs) are promising tools, as they can be loaded with different sensor molecules that enable the simultaneous analysis of different disease-related parameters to increase the r 2011 American Chemical Society

information content gained within a single experiment. In addition, they can be equipped with a targeting unit such as an antibody, antibody fragment, or peptide, binding to a cancer-specific biomarker, e.g., a cell surface receptor, to ensure accumulation at specific sites thereby increasing the specificity. First approaches toward oxygen-sensitive probes for fluorescence imaging applications include oxygen-sensitive luminescent probes such as water-soluble platinum coproporphyrins,10,11 dendrimers with porphyrin cores,12
14 or iridium complexes15 that can be administered directly into cells or cancerous tissue. Lately, also probes consisting of an oxygen-responsive dye conjugated to cell-penetrating peptides have been reported.16 More advanced design strategies exploit biocompatible polymeric NPs equipped with a sensor function, i.e., loaded with a multitude of indicator dyes with oxygen-dependent emission properties.17,18 This not only represents a simple signal enhancement strategy19
21 but also improves dye photostability,22,23 minimizes potential toxicity effects, Received: July 25, 2011 Accepted: October 18, 2011 Published: October 18, 2011 9039

dx.doi.org/10.1021/ac201870b | Anal. Chem. 2011, 83, 9039–9046

Analytical Chemistry and reduces interferences, e.g., by pH changes or luminescence quenching by metal ions. Moreover, it circumvents nonspecific interactions of the dye molecules with abundant plasma proteins in the in vivo situation. A prerequisite for the reliable analyte quantification with such nanosensors is the simultaneous loading of the NPs with analyte-sensitive dyes and spectroscopically distinguishable, analyte-insensitive reference fluorophores to enable ratiometric dual wavelength measurements.24,25 Application of a reference dye provides the basis for measurements that are unaffected by nanoparticle concentration and fluctuations of the excitation light intensity and location within the tissue. Alternatively, oxygen determinations can be obtained by fluorescence lifetime imaging.26 The vast majority of oxygen-sensing dyes absorbs and emits with the visible wavelength region. Although such chromophores can be used for many in vitro imaging applications,27
29 in vivo imaging commonly requires near-infrared (NIR) fluorophores that absorb and emit in the biological window between 650 and 900 nm. This is mandatory for applications to deep tissue imaging30
33 and can be accomplished with new NIR-emissive dyes such as Pt(II) and Pd(II) porphyrin derivatives.11,12,16,17,34,35 First approaches to intracellular oxygen measurements using NIR-emitting porphyrins as indicator dyes and inert reference dyes embedded in silica core
shell or polyacrylamid nanoparticles have been reported.17,36 We present now an advanced nanosensor approach combining unique features. The oxygen sensitive dye and the reference dye can both be excited with a 635 nm laser diode, and their NIR emissions can be easily separated by optical filters. Referenced measurements can be achieved either by ratiometric dual wavelength or by luminescence lifetime measurements. Both responses have been calibrated thoroughly. The polystyrene matrix provides a high colloidal stability. Carboxylated polystyrene NPs (PS-NPs) have no or only minor effects on cell viability when applied in high concentrations.37 However, the cytoxocity of PS-NPs strongly depends on the particle size and surface charge.38 For instance, amino-modified PS showed a significant cell membrane damaging effect.39 We used Pd(II) mesotetraphenyl-tetrabenzoporphyrin (PdTPTBP) as oxygen-sensitive probe incorporated into 100 nm-sized PS-NPs using a previously described swelling procedure.40 Furthermore, we coembedded an oxygen-insensitive cyanine dye (DY-635). Targeted NPs were obtained by coating PS-NPs with maleimide-modified nontoxic polyethylene glycol (PEG)41 followed by functionalization of the surface with the clinically approved humanized IgG1 monoclonal antibody, herceptin, which targets the extracellular domain of the human epidermal growth factor receptor 2 (Her2/neu) overexpressed in many cancer types.1,42,43 The basic luminescent properties of the oxygen nanosensors have been studied, including absolute quantum yields, lifetimes, and oxygen response. In addition, we performed binding studies to estimate the potential of herceptin-functionalized PS-NPs to bind to a tumor-specific target on tumor cells. First in vivo experiments demonstrate an increase of the ratiometric fluorescence response within the tumor upon hypoxic condition.

’ MATERIALS AND METHODS Materials. Amino-functionalized latex beads (100 nm) were purchased from Micromod GmbH (Rostock-Warnemuende, Germany) and were ultrasonicated prior to use. 2-Iminothiolane (Traut’s reagent), 5,50 -dithio-bis(2-nitro-benzoic acid) (Ellman’s

ARTICLE

reagent), cysteine, sodium phosphate, ethylenediaminetetraacetic acid (EDTA), O-[N-(3-Maleimidopropionyl)aminoethyl]O0 -[3-(N-succinimidyloxy)-3-oxopropyl]heptacosaethylene glycol (NHS-PEG-Mal), sodium sulfite, and poly-L-lysine were purchased from Sigma-Aldrich (Germany). NaCl, EDTA, Tris/ HCl, and Triton X-100 were obtained from Carl Roth (Germany). DY-635 was obtained from Dyomics GmbH (Jena, Germany), and Pd(II)meso-tetraphenyl-tetrabenzoporphyrin (PdTPTBP) was from Sigma-Aldrich (Germany). PD-10 columns were purchased from GE Healthcare (Germany), and the BCA Protein Assay Reagent and Slide-A-Lyzer Dialysis Cassettes (20 000 MWCO, 0.1
0.5 mL) were from Pierce (Rockford, USA). Herceptin (Trastuzumab) and Mini Protease Inhibitor Cocktail Tablets were obtained from Roche Diagnostics (Germany). Rabbit antimouse Hypoxia inducible factor-1α (HIF-1α) antibody was obtained from Abcam (Germany), and mouse anti-ßactin was from Sigma (Germany). Horseradish-conjugated goat antirabbit or goat antimouse antibodies were acquired from Santa Cruz Biotechnology (Germany), and Bradford reagent was from Bio-Rad (Germany). Murine aveolar macrophages cell line, MH-S, was obtained from ATCC (Rockville, MD). All other used chemicals, solvents, and buffers are specified in the Supporting Information. Preparation of Oxygen Nanosensors (Ox-PS-NP). PS-NPs (100 nm-sized) were loaded with oxygen-sensitive and reference dyes using stock solutions of PdTPTBP in THF (c = 2 mmol L
1) and of DY-635 (Ox-PS-NP) in DMF (c = 1 mmol L
1, respectively). PS-NPs doped with only one type of dye were prepared for photolumincescence quantum yield measurements. Dye loading of the PS-NPs via a previously described swelling procedure40 was performed by addition of 330 μL of the PdTPTBP and 330 μL of the DY-635 solutions to 4 mL of an aqueous suspension containing 20 mg of either carboxylated or amino-modified PSNPs. After 30 min, 5 mL of water was added to the occasionally shaken suspension, which was then centrifuged. The resulting PS-NPs were washed twice with water. The amount of incorporated fluorophore was determined by dissolving the particles in DMF, followed by subsequent measurements of the absorption spectra of the DMF solutions. The average amount of incorporated dye was calculated from the absorbance at the dye’s longest wavelength absorption maximum, using the Beer
Lambert law and the molar absorption coefficient of the dye, previously determined in DMF in the presence of 0.1 wt % dissolved polystyrene. Preparation of PEGylated PS-NPs. Twenty milligrams of amino-modified Ox-PS-NPs was suspended in 1 mL of phosphate buffer (0.1 M, pH 8.0) and mixed with 200 μL of freshly prepared 50 mM NHS-PEG-Mal (1.5 kDa) in phosphate buffer. The mixture was shaken at 600 rpm for 2 h at room temperature. Subsequently, the PEG-Mal-modified PS-NPs were washed twice, each washing step including centrifugation, removal of supernatant, and resuspension of the PS-NPs in 1 mL of phosphate buffer. Preparation of Herceptin-Targeted PS-NPs. Targeted PSNPs were prepared from dye-stained PEGylated maleimide functionalized PS-NP reacted with thiolated herceptin.41 For details see Supporting Information. The PS-NPs were washed twice (each washing step including centrifugation, removal of the supernatant, and resuspension of the PS-NP in 1 mL of water) and characterized by DLS. The quantity of surface-bound herceptin molecules was determined with a BCA Protein Assay. Dynamic Light Scattering (DLS). DLS measurements were performed with a Zeta Nanosizer (Malvern) equipped with a 9040

dx.doi.org/10.1021/ac201870b |Anal. Chem. 2011, 83, 9039–9046

Analytical Chemistry 4 mW He
Ne laser (633 nm) that uses a scattering angle of 173. All particles were measured in Milli-Q water in concentrations of 0.1 wt % at 25 C. Transmission Electron Microscopy. The TEM images were recorded using a LEO 912AB electron microscope (Zeiss). Optical Spectroscopy. Absorption spectra were recorded on a Cary 5000 spectrometer (Varian Inc.), and fluorescence spectra were on a calibrated Spectronics Instruments 8100 spectrofluorometer44 in a 0/90 excitation
emission geometry using magic angle conditions. All measurements were performed with 1 cm-quartz cuvettes at 25 C. Absolute Photoluminescence Quantum Yields. The absolute photoluminescence quantum yields (Φf) of dyes and stained PS-NPs that equal the number of emitted photons (Nem) per absorbed photons (Nabs) were determined with a custom-designed integrating sphere setup (for details see, Supporting Information). The excitation wavelength was 620 nm. Calibration of Oxygen Response. PBS solutions (140 mM) with varying contents of dissolved oxygen were adjusted by addition of definite amounts of sodium sulfite (Na2SO3). The samples were prepared by adding 10 μL of a 1% (w/w) dispersion of the Ox-PS-NPs to 300 μL of these solutions in a half micro fluorescence cuvette. The cuvettes were sealed with 300 μL of paraffin oil and parafilm, and the spectra were measured with a Jasco FP-6300 fluorimeter. As the oxygen concentrations in Na2SO3-containing solution are not stable over time,45 the effective oxygen concentrations were determined in parallel with an OxoPlate oxygen sensor (PreSens, Germany; for details, see Supporting Information). The intensity ratio of the reference dye to the oxygen indicator was calculated by integrating the intensities in the interval from 795 to 805 nm (indicator; IPd) and 665 to 675 nm (reference; Iref) and determining the quotient Iref/IPd. Cell Culture. Murine alveolar macrophages, MH-S cells, were grown in RPMI 1640 medium containing 2-mercaptoethanol (0.05 mmol L
1) and supplemented with 1% L-glutamine. Human mammary carcinoma cells, SK-BR-3 cells (Her2/neu positive), were cultivated in RPMI 1640 medium. MDA-MB-231 cells (Her2/ neu negative) and human pancreatic adenocarcinoma AsPC1 cells were cultivated in DMEM medium. All media were supplemented with 10% FCS. The cells were cultivated in a humidified 5% CO2 and 95% air atmosphere at 37 C. The detailed procedures for intracellular oxygen imaging are available in the Supporting Information. Microscopy and Statistical Image Analysis. NIR microscopy was performed using a Zeiss Axiovert 200 M inverted microscope (Carl Zeiss, Germany) equipped with a xenon lamp and a high sensitivity ORCA-AG digital camera (Hamamatsu, Japan). For filter settings, see Supporting Information. Subsequent analyses were performed using ImageJ (available by ftp at zippy.nimh.nih.gov or at http://rsb.info.nih.gov/nih-imageJ; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD).46 Ratiometric responses of the Ox-PS-NP (IPd/Iref) were calculated for each acquired image and corrected for the image acquisition time and for the ratio in cell-free background. NIR Fluorescence Imaging. The Optix MX2 (ART, Advanced Research Technologies Inc.; Canada) was used for in vivo imaging (details see Supporting Information). First, in vitro measurements of the fluorescence response to differing oxygen concentrations were performed in solution. Ox-PS-NPs in concentration of 1 μg/μL in 100 μL of PBS containing 0%, 0.1%, or 1% (w/w) Na2SO3 were placed in black 96-well plates

ARTICLE

Figure 1. Luminescence emission spectra of Ox-PS-NPs (excitation at 635 nm) in deaerated (triangles) and oxygen-saturated (squares) aqueous solution; PdTPTBP: λmax = 800 nm; DY-635: λmax = 670 nm.

with transparent bottom, and the fluorescence intensity was measured immediately. In Vivo NIR Fluorescence Imaging. Female athymic nude NMRI-Fox1nu/nu mice (Winkelmann, Borchen, Germany) were used and handled according to German regulations for animal experimentations; the protocols were approved by the administration of Lower Saxony, Germany. For tumor xenograft, 1  106 human pancreatic tumor cells, AsPC-1, were injected subcutaneously into the right flank of the mouse. For imaging, either a tumor-free mouse or a mouse that developed a subcutaneous AsPC-1 tumor with a diameter of approximately 0.5 cm were anesthetized with 1.8
2% inhaled Isoflurane (Abbott; the Netherland). Mice were injected either subcutaneously with 200 μg (tumor-free mouse) or intravenously with 2000 μg (tumor-bearing mouse) of 100 nm Ox-PS-NPs in a total volume of 150 μL of 0.9% (w/w) NaCl. Imaging was performed with Optix MX2 using settings described in Supporting Information. The data were quantified as ratio of the average fluorescence intensities of PdTPTBP (IPd) and DY-635 (I ref) over the tumor area.

’ RESULTS AND DISCUSSION The incorporation of the NIR-emissive and oxygen-sensitive dye PdTPTBP and a reference fluorophore (DY-635) into PSNPs results in ratiometric oxygen nanosensors (Ox-PS-NP). Both dyes are excitable at 635 nm (see Supporting Information, Figure 1S). On average, we found 26.8 nmol of PdTPTBP and 5.0 nmol of DY-635 per 1 mg of Ox-PS-NP, equaling loading efficiencies of 81% and 30%. The colloidally stable Ox-PS-NPs show a narrow and homogeneous size-distribution as determined by TEM and DLS (Figure 2S, Supporting Information) with size and morphology being unaffected by dye loading. Coating with a 1.5 kDa PEG linker and herceptin led to an increase in the hydrodynamic diameter by about 20 nm. On average, 10 herceptin molecules were bound per nanoparticle. Spectroscopic Properties of Ox-PS-NPs. The emission spectra of Ox-PS-NP in an aqueous solution in the presence and absence of oxygen are summarized in Figure 1. While the phosphorescence of PdTPTBP is efficiently quenched in oxygen-saturated solution, the reference signals are not affected by oxygen. The emission of DY635 is spectrally well separated from the PdTPTBP signal. Measurement of absolute fluorescence quantum yields revealed values below 0.001 for PdTPTBP in air-saturated THF that rise to 0.005 upon 9041

dx.doi.org/10.1021/ac201870b |Anal. Chem. 2011, 83, 9039–9046

Analytical Chemistry

Figure 2. Calibration of the dual wavelength nanoprobe: Referenced luminescence intensity Iref (dye
635)/IPd (PdTPTBP) vs pO2 in PBS at 37 C with linear fit (R2 = 0.982).

encapsulation in PS-NPs due to the different concentrations of oxygen in both environments. Flushing with Argon led to a considerable fluorescence enhancement with Φf values of 0.007 and 0.02 for PdTPTBP in THF and in PS-NP, respectively. Calibration of Oxygen Response. The ratiometric response of the nanosensors was calibrated in PBS solution in the presence of oxygen scavenger, Na2SO3, to yield oxygen partial pressures ranging from 0 to 200 hPa of pO2 (Figure 2), thereby covering the physiological venous and arterial pO2, i.e., 52 hPa and 130 hPa, respectively. The referenced intensity ratio IPd/Iref was set to 1 at oxygen-saturated (pO2 ∼ 200 hPa) conditions to obtain a similar base level as in case of the in vitro and in vivo imaging experiments. It increased by a factor of 4 in oxygen-free (pO2 ∼ 0 hPa) solution. In addition, also the phosphorescence lifetime (τ) of PdTPTBP is strongly affected by pO2 (Figure 3S, Supporting Information). Lifetime measurements do not require further referencing. The lifetimes τ vary between approximately 135 μs at 20 hPa of pO2 and 30 μs at 200 hPa of pO2 at 37 C. They are obtained by means of a ratiometric time-resolved imaging method (Rapid Lifetime Determination).47,48 Details are available in the Supporting Information. Both referencing methods provide a good linear response in the range from 10 to 200 hPa of pO2 and show nearly identical signal changes. The ratiometric calibration is displayed as Iref/IPd for a better comparability to the Stern
Volmer plot of τ (Figure 3S, Supporting Information). The maximum pO2 in tissue is 130 hPa in a normoxic ventilated human at sea level based on the humidification and mixture of the inhaled air with the gases in the dead space. On the other hand, pO2 values of close to 0 hPa have been measured in tumor tissues. Thus, this linear range is significant for the in vivo situation. Cellular Uptake and Assessment of Oxygenation Status in Vitro. Cellular uptake of Ox-PS-NPs and the ratiometric response to oxygen in vitro were verified in cultured mouse alveolar macrophages. Ox-PS-NP were efficiently internalized by the cells within 4.5 h of incubation and showed no obvious effect on cell viability under all experimental conditions (20.9% of O2, 5% of O2, and 1% of O2, which mimic a pO2 of 193 hPa, 46.3 hPa, and 9.3 hPa, assuming a 100% humidification, respectively). A diminution in oxygen concentration in the ambient air led to an increase in PdTPTBP luminescence and, consequently, to an increase in the IPd/Iref ratio (Figure 3A,B). A narrow ratio distribution ranging from 0.64 to 1.79 with a mean value of 1.10 was calculated in cells incubated with Ox-PS-NPs under normoxic conditions (20.9% O2). Decreasing oxygen concentrations were accompanied by an increase in the mean IPd/Iref ratios,

ARTICLE

reaching values of 1.44 and 1.71 in the presence of 5% and 1% of O2, respectively. The distribution of the ratios is much wider upon reduced oxygen concentrations of 1% of O2, reaching values ranging from 0.76 up to 3.93, as not all counted cells obtain similarly hypoxic conditions (see histogram Figure 3C and Table 1S, Supporting Information). The ratiometric response of OxPS-NPs was reversible and decreased again after short (∼10 min) reoxygenation from both hypoxic states (1% or 5% of O 2 ), nearly reaching the value found for 20.9% of O2 again. The small deviation may be ascribed to a rapid activation of adaptive pathways like hypoxia-inducible signal transduction, which for example results in changes in oxygen consumption in hypoxia. An increased accumulation of HIF-1α was observed by Western-blot (see Supporting Information) analysis with decreasing O2 concentrations, which disappeared upon reoxygenation (Figure 3D). HIF-1α accumulation is a physiological well described adaptive mechanism in hypoxia at the cellular level. These results clearly confirm the potential of Ox-PS-NPs to act as reversible oxygen sensors of varying cellular oxygenation levels in the physiological range. Binding of Targeted PS-NPs to Tumor Cells in Vitro. We prepared Ox-PS-NPs coated with a PEG shell and functionalized the surface with herceptin to assess the principal potential of the herceptin-functionalized PS-NPs for tumor targeting. These were incubated with HER2/neu-overexpressing human mammary carcinoma cells, SK-BR-3, and with HER2/neu-negative human mammary carcinoma cells, MDA-MB-231 (Figure 4S, Supporting Information), for up to 24 h. Particle-cell interactions were analyzed by fluorescence microscopy. Specific binding of the targeted NPs to the SK-BR-3 cells was observed already within 1.5 h of incubation, with the fluorescence intensity increasing with longer incubation time. No staining of control MDA-MB-231 cells was observed within 4 h of incubation. In Vitro Oxygen Imaging. In further studies, we assessed the suitability of the Ox-PS-NPs to be applied in a commercially available in vivo imaging system, Optix MX2, equipped with a 635 nm laser diode, perfectly matching the longest wavelength absorption maximum of PdTPTBP and DY-635 (Figure 1S, Supporting Information). The use of suitable emission filters resulted in bright signals and enables the straightforward separation of the luminescence of PdTPTBP from that of the reference (Figure 4A). The ratio IPd/Iref of Ox-PS-NP measured under normal air
oxygen saturation was approximately 1.0 (Figure 4B) and increased up to 4.6 in oxygen-depleted solution (Na2SO3, 1% w/w), which is in good accordance to the spectrometric calibration. This indicates that with Ox-PS-NPs a new NIR-emissive referenced probe for oxygen imaging is available which can be applied in the physiologically relevant oxygen concentration range with commonly used optical in vivo imaging equipment. Proof of Concept for in Vivo Imaging. Finally, we applied the Ox-PS-NP nanosensors to nude mice and performed first proof-of-concept in vivo imaging experiments. In a first step, detection sensitivity was evaluated. The probe was subcutaneously injected into the right shoulder of a nude mouse (Figure 4C). Clear and strong fluorescence signals of both fluorophores were measured at the site of the injection 20 min after application. The oxygen supply was cut off by sacrificing the animal followed by scanning the mouse again postmortem to ensure the development of hypoxic conditions within the mouse. Consequently, the in vivo and postmortem fluorescence intensities were compared. The PdTPTBP-derived luminescence intensity 9042

dx.doi.org/10.1021/ac201870b |Anal. Chem. 2011, 83, 9039–9046

Analytical Chemistry

ARTICLE

Figure 3. In vitro evaluation of the ratiometric response to decreasing levels of oxygen concentration. (A) Representative fluorescence images of MH-S macrophages after 4.5 h incubation with Ox-PS-NPs in an ambient environment containing 20.9%, 5%, or 1% of O2 as well as images of the cells after reoxygenation from 5% to 20.9% of O2 (5% O2-reox) and from 1% to 20.9% of O2 (1% O2-reox), respectively. The blue color corresponds to the nucleic stain (DAPI), green to the reference fluorophore DY-635, and red to the PdTPTBP phosphorescence; all 3 channels are combined in the merged image. (B) Color-coded illustration of the ratiometric response (IPd/Iref) of the MH-S cells shown in (A). A strong increase in IPd/Iref ratio can be observed upon a decrease in the ambient oxygen concentration, which is reversible upon reoxygenation. (C) Normalized histograms summarizing ratiometric responses of the NPs internalized by MH-S macrophages grown at different oxygen concentrations. (D) Verification of the hypoxic status of the cells by detecting HIF1-α accumulation in Western blot analyses. Bar: 50 μm.

increased by a factor of 2.5 postmortem, ∼10 min after animal sacrifice. The emission of DY-635 remained constant. The resulting IPd/Iref intensity ratios were calculated to ∼2.1 in vivo and ∼5.0 postmortem under hypoxia-mimicking conditions. According to the spectrometric calibration, these would represent a pO2 in the

order of magnitude of 100 hPa and anaerobic conditions, respectively. In a second step, the biodistribution of Ox-PS-NPs as well as the accumulation of the probe in tumor tissue was studied. We applied the nanosensors intravenously to a mouse bearing a 9043

dx.doi.org/10.1021/ac201870b |Anal. Chem. 2011, 83, 9039–9046

Analytical Chemistry

ARTICLE

Figure 4. Evaluation of Ox-PS-NPs with the in vivo imaging system Optix MX2. (A) In vitro imaging of the representative luminescence intensities derived from DY-635 and PdTPTBP in PBS solution in the presence or absence of the oxygen-scavanger Na2SO3. (B) Summary of the data shown in (A). The IPd/Iref ratios increase in the presence of 0.1% and 1% sulfite. (C) In vivo and postmortem (∼10 min after animal sacrifice) scans of a nude mouse after subcutaneous application of Ox-PS-NPs. An IPd/Iref ratio of 2.1 was measured ca. 20 min after sensor application which increased by a factor of 2.5 upon sacrificing the animal.

subcutaneous measurements shown in Figure 5C. Also, in this case, shortly after sacrificing the mouse, the fluorescence intensity ratio increased almost 2-fold to 3.6 (Figure 5B). These results clearly demonstrate the suitability of the new Ox-PS-NP to determine and image varying partial oxygen pressures. Therefore, the targeted oxygen-sensitive probes represent promising tools as referenced real-time hypoxia nanosensors for in vivo applications. The NIR luminescence signals of the oxygen indicator and reference dyes can be recorded from tumorous tissue by means of a usual animal scanner. The ratiometric probes will be further tailored for target-oriented imaging by surface functionalization with different biomolecular recognition elements.

Figure 5. Proof-of-concept for in vivo imaging of tumor oxygenation with Ox-PS-NPs. (A) Fluorescence images obtained for a subcutaneous AsPC-1 tumor-bearing nude mouse 5 h after intravenous injection of Ox-PS-NPs in vivo and postmortem (∼10 min after animal sacrifice). (B) Diagram summarizing the average luminescence intensities derived from PdTPTBP and DY-635 measured over the tumor shown in (A). An almost 2-fold increase of the IPd/Iref ratio was measured upon induction of an artificial hypoxic tumor environment via sacrifice of the mouse. Bar: 1 cm.

subcutaneous pancreatic AsPC-1 adenocarcinoma and scanned it after ∼5 h. In vivo, a very strong fluorescence signal from the area of the liver was detected which presents, according to our experience, the major excretion pathway of 100 nm-sized PS-NPs (data not shown). In addition, when compared to the prescans of the mouse, we obtained a clear increase in the fluorescence intensity within the tumor area in vivo in both detection channels (Figure 5A). The calculated average fluorescence intensity ratio over the tumor area was 1.9, which is in good agreement with the

’ CONCLUSION AND OUTLOOK A synthetic procedure was developed that enables the preparation of NIR-emissive PS-NPs with sensor function suitable for ratiometric luminescence intensity imaging as well as for lifetime imaging of the oxygen distribution. The particles can be dispersed in sensor films or applied directly in cells or in vivo in animal models using conventional fluorescence microscopes or animal scanners equipped with a 635 nm laser diode. The NIRemissive metalloporphyrin PdTPTBP shows a nearly linear oxygen response of its phosphorescence intensity and lifetime in the bioanalytical relevant range of tissue and arterial blood which is in the range between 0 and 130 hPa. The referenced signal increases up to 4.5-fold from oxygen-saturated to oxygenfree conditions. The nanosensor can be easily adapted to various measurement systems and applications because PdTPTBP can be combined with different NIR-emissive reference dyes. Bright analyzable signals can be obtained from cells and from tumors because multiple fluorophores are incorporated into the PS-NPs. In vitro studies showed efficient uptake of the nanosensors by cultured MH-S cells or murine aveolar macrophages and their applicability to ratiometric and reversible oxygen sensing in a physiologically relevant concentration range. The selective uptake of PEG-coated and herceptin-functionalized Ox-PS-NPs was demonstrated by binding studies with cells overexpressing Her2/neu. Moreover, we provided a first in vivo
postmortem proof of concept of the general suitability of the targeted nanosensors for 9044

dx.doi.org/10.1021/ac201870b |Anal. Chem. 2011, 83, 9039–9046

Analytical Chemistry imaging of hypoxic conditions due to accumulation of the NPs in tumor tissue. In subsequent studies, we will assess other functionalization strategies to extend the in vivo applications of the nanosensor system to different targets. In this case, the PEGylated NPs will be of particular interest, as PEG is commonly used to improve biodistribution and pharmacokinetic properties of biological agents and is nontoxic when applied in clinical doses.49,50 This is also the case if fluorescent dyes are incorporated into PEGylated nanoparticles. Luminescence lifetime imaging in the microsecond time domain will also be optimized in vivo as an alternative to the dual wavelength measurements presented in this study.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*F.A.: e-mail, [email protected]; phone, ++49(0)551-3899-655; fax, ++49(0)551-3899-644. U.R.-G.: e-mail, [email protected]; phone, ++49(0)30-8104-1134; fax, ++49(0)30-8104-1159. M.S.: e-mail, [email protected]; phone, ++49-(0)941-9434015; fax, +49-(0)941/9434064.

’ ACKNOWLEDGMENT J.N. and T.B. contributed equally to this work. Financial support from the German Ministry of Education and Research (BMBF; Grant 13N8849) and from Deutsche Forschungsgemeinschaft (DFG), Schwerpunktprogramm SPP 1190 (Grant number: AL336/5-2) is gratefully acknowledged. We express our gratitude to R. Brehm, E.-M. Laux, and Dr. A. Hennig for help with the coupling of herceptin to the PS-NPs as well as to B. Heidrich and S. Greco for excellent technical assistance. ’ REFERENCES (1) Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L.; Urano, Y. Chem. Rev. 2010, 110, 2620. (2) Achilefu, S. Angew. Chem., Int. Ed. Engl. 2010, 49, 9816. (3) Weissleder, R. Science 2006, 312, 1168. (4) Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T. Nat. Methods 2008, 5, 763. (5) Berezin, M. Y.; Achilefu, S. Chem. Rev. 2010, 110, 2641. (6) Alford, R.; Ogawa, M.; Hassan, M.; Gandjbakhche, A. H.; Choyke, P. L.; Kobayashi, H. Contrast Media Mol. Imaging 2010, 5, 1. (7) Krohn, K. A.; Link, J. M.; Mason, R. P. J. Nucl. Med. 2008, 49, 129S. (8) Chitneni, S. K.; Palmer, G. M.; Zalutsky, M. R.; Dewhirst, M. W. J. Nucl. Med. 2010, 52, 165. (9) Hockel, M.; Vaupel, P. J. Natl. Cancer Inst. 2001, 93, 266. (10) O’Riordan, T. C.; Zhdanov, A. V.; Ponomarev, G. V.; Papkovsky, D. B. Anal. Chem. 2007, 79, 9414. (11) O’Riordan, T. C.; Fitzgerald, K.; Ponomarev, G. V.; Mackrill, J.; Hynes, J.; Taylor, C.; Papkovsky, D. B. Am. J. Physiol. Reg. Integr. Comp. Physiol. 2007, 292, R1613. (12) Rietveld, I. B.; Kim, E.; Vinogradov, S. A. Tetrahedron 2003, 59, 3821. (13) Lebedev, A. Y.; Cheprakov, A. V.; Sakadzic, S.; Boas, D. A.; Wilson, D. F.; Vinogradov, S. A. ACS Appl. Mater. Interfaces 2009, 1, 1292.

ARTICLE

(14) Sakadzic, S.; Roussakis, E.; Yaseen, M. A.; Mandeville, E. T.; Srinivasan, V. J.; Arai, K.; Ruvinskaya, S.; Devor, A.; Lo, E. H.; Vinogradov, S. A.; Boas, D. A. Nat. Methods 2010, 7, 755. (15) Zhang, S. J.; Hosaka, M.; Yoshihara, T.; Negishi, K.; Iida, Y.; Tobita, S.; Takeuchi, T. Cancer Res. 2010, 70, 4490. (16) Dmitriev, R. I.; Zhdanov, A. V.; Ponomarev, G. V.; Yashunski, D. V.; Papkovsky, D. B. Anal. Biochem. 2010, 398, 24. (17) Lee, Y. E. K.; Ulbrich, E. E.; Kim, G.; Hah, H.; Strollo, C.; Fan, W. Z.; Gurjar, R.; Koo, S. M.; Kopelman, R. Anal. Chem. 2010, 82, 8446. (18) Borisov, S. M.; Klimant, I. Microchim. Acta 2009, 164, 7. (19) Herz, E.; Ow, H.; Bonner, D.; Burns, A.; Wiesner, U. J. Mat. Chem. 2009, 19, 6341. (20) Wang, L.; Zhao, W. J.; Tan, W. H. Nano Res. 2008, 1, 99. (21) Muddana, H. S.; Morgan, T. T.; Adair, J. H.; Butler, P. J. Nano Lett. 2009, 9, 1559. (22) Miletto, I.; Gilardino, A.; Zamburlin, P.; Dalmazzo, S.; Lovisolo, D.; Caputo, G.; Viscardi, G.; Martra, G. Dyes Pigm. 2009, 84, 121. (23) Altinoglu, E. I.; Russin, T. J.; Kaiser, J. M.; Barth, B. M.; Eklund, P. C.; Kester, M.; Adair, J. H. ACS Nano 2008, 2, 2075. (24) Koo, Y. E.; Cao, Y.; Kopelman, R.; Koo, S. M.; Brasuel, M.; Philbert, M. A. Anal. Chem. 2004, 76, 2498. (25) Cao, Y.; Lee Koo, Y. E.; Kopelman, R. Analyst 2004, 129, 745. (26) Babilas, P.; Liebsch, G.; Schacht, V.; Klimant, I.; Wolfbeis, O. S.; Szeimies, R. M.; Abels, C. Microcirculation 2005, 12, 477. (27) Hama, Y.; Urano, Y.; Koyama, Y.; Bernardo, M.; Choyke, P. L.; Kobayashi, H. Bioconj. Chem. 2006, 17, 1426. (28) Longmire, M. R.; Ogawa, M.; Hama, Y.; Kosaka, N.; Regino, C. A. S.; Choyke, P. L.; Kobayashi, H. Bioconj. Chem. 2008, 19, 1735. (29) Ogawa, M.; Kosaka, N.; Choyke, P. L.; Kobayashi, H. ACS Chem. Biol. 2009, 4, 535. (30) Adams, K. E.; Ke, S.; Kwon, S.; Liang, F.; Fan, Z.; Lu, Y.; Hirschi, K.; Mawad, M. E.; Barry, M. A.; Sevick-Muraca, E. M. J. Biomed. Optics 2007, 12, 024017. (31) Kovar, J. L.; Simpson, M. A.; Schutz-Geschwender, A.; Olive, D. M. Anal. Biochem. 2007, 367, 1. (32) Ballou, B.; Ernst, L. A.; Waggoner, A. S. Curr. Med. Chem. 2005, 12, 795. (33) Mahmood, U.; Weissleder, R. Mol. Cancer Ther. 2003, 2, 489. (34) Borisov, S. M.; Zenkl, G.; Klimant, I. ACS Appl. Mater. Interfaces 2010, 2, 366. (35) Fercher, A.; Ponomarev, G. V.; Yashunski, D.; Papkovsky, D. Anal. Bioanal. Chem. 2010, 396, 1793. (36) Xu, H.; Buck, S. M.; Kopelman, R.; Philbert, M. A.; Brasuel, M.; Ross, B. D.; Rehemtulla, A. Isr. J. Chem. 2004, 44, 317. (37) Liu, Y.; Li, W.; Lao, F.; Wang, L.; Bai, R.; Zhao, Y.; Chen, C. Biomaterials 2011, 32, 8291. (38) Frohlich, E.; Samberger, C.; Kueznik, T.; Absenger, M.; Roblegg, E.; Zimmer, A.; Pieber, T. R. J. Toxicol. Sci. 2009, 34, 363. (39) Ruenraroengsak, P.; Novak, P.; Berhanu, D.; Thorley, A. J.; Valsami-Jones, E.; Gorelik, J.; Korchev, Y. E.; Tetley, T. D. Nanotoxicology, 2011, DOI:10.3109/17435390.2011.558643. (40) Behnke, T.; Wurth, C.; Hoffmann, K.; Hubner, M.; Panne, U.; Resch-Genger, U. J. Fluoresc. 2011, 21, 937. (41) Clift, M. J.; Rothen-Rutishauser, B.; Brown, D. M.; Duffin, R.; Donaldson, K.; Proudfoot, L.; Guy, K.; Stone, V. Toxicol. Appl. Pharmacol. 2008, 232, 418. (42) Scheuer, W.; Friess, T.; Burtscher, H.; Bossenmaier, B.; Endl, J.; Hasmann, M. Cancer Res. 2009, 69, 9330. (43) Kosaka, N.; Ogawa, M.; Paik, D. S.; Paik, C. H.; Choyke, P. L.; Kobayashi, H. Cancer Sci. 2010, 101, 820. (44) Resch-Genger, U.; Pfeifer, D.; Monte, C.; Pilz, W.; Hoffmann, A.; Spieles, M.; Rurack, K.; Hollandt, J.; Taubert, D.; Schonenberger, B.; Nording, P. J. Fluoresc. 2005, 15, 315. (45) Arain, S.; Weiss, S.; Heinzle, E.; John, G. T.; Krause, C.; Klimant, I. Biotechnol. Bioeng. 2005, 90, 271. (46) Collins, T. J. Biotechniques 2007, 43, 25. (47) Moore, C.; Chan, S. P.; Demas, J. N.; DeGraff, B. A. Appl. Spectrosc. 2004, 58, 603. 9045

dx.doi.org/10.1021/ac201870b |Anal. Chem. 2011, 83, 9039–9046

Analytical Chemistry

ARTICLE

(48) Fischer, L. H.; Stich, M. I. J.; Wolfbeis, O. S.; Tian, N.; Holder, E.; Schaferling, M. Chem.—Eur. J. 2009, 15, 10857. (49) Webster, R., Elliott, V., Park, B. K., Walker, D., Hankin, M., Taupin, P., Veronese, F. M., Eds. PEGylated Protein Drugs: Basic Science and Clinical Applications; Birkh€auser: Basel, 2009; p 127. (50) Sun, X.; Rossin, R.; Turner, J. L.; Becker, M. L.; Joralemon, M. J.; Welch, M. J.; Wooley, K. L. Biomacromolecules 2005, 6, 2541.

9046

dx.doi.org/10.1021/ac201870b |Anal. Chem. 2011, 83, 9039–9046