Fluorescence Imaging with Multifunctional Polyglycerol Sulfates

Article pubs.acs.org/bc

Fluorescence Imaging with Multifunctional Polyglycerol Sulfates: Novel Polymeric near-IR Probes Targeting Inflammation Kai Licha,*,† Pia Welker,† Marie Weinhart,‡ Nicole Wegner,† Sylvia Kern,† Stefanie Reichert,‡ Ines Gemeinhardt,# Carmen Weissbach,§ Bernd Ebert,§ Rainer Haag,‡ and Michael Schirner† †

mivenion GmbH, Robert-Koch-Platz 4, 10115 Berlin, Germany Institut für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany # Institut für Radiologie, Charité-Universitätsmedizin, Charitéplatz 1, 10098 Berlin, Germany § Physikalisch-Technische Bundesanstalt, Abbestr. 2-12, 10587 Berlin, Germany ‡

S Supporting Information *

ABSTRACT: We present a highly selective approach for the targeting of inflammation with a multivalent polymeric probe. Dendritic polyglycerol was employed to synthesize a polyanionic macromolecular conjugate with a near-infrared fluorescent dye related to Indocyanine Green (ICG). On the basis of the dense assembly of sulfate groups which were generated from the polyol core, the resulting polyglycerol sulfate (molecular weight 12 kD with ∼70 sulfate groups) targets factors of inflammation (IC50 of 3−6 nM for inhibition of L-selectin binding) and is specifically transported into inflammatory cells. The in vivo accumulation studied by nearIR fluorescence imaging in an animal model of rheumatoid arthritis demonstrated fast and selective uptake which enabled the differentiation of diseased joints (score 1−3) with a 3.5-fold higher fluorescence level and a signal maximum at 60 min post injection. Localization in tissues using fluorescence histology showed that the conjugates are deposited in the inflammatory infiltrate in the synovial membrane, whereas nonsulfated control was not detected in association with disease. Hence, this type of polymeric imaging probe is an alternative to current bioconjugates and provides future options for targeted imaging and drug delivery.

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surface properties, e.g., through conjugation with targeting structures or the assembly of charged moieties. 3,4 Most recently, we demonstrated that polyanionic, dendritic polyglycerol sulfates (dPGS) exert strong binding affinity to cellular targets involved in the inflammatory process, such as L- and Pselectins, which are bound with nanomolar affinity as demonstrated by a concentration-dependent SPR assay in vitro.5,6 By inhibiting leucocyte infiltration in vivo, the substance exhibited efficient anti-inflammatory efficacy in a mouse dermatitis model.5 Furthermore, dPGS binding to selectins was shown to increase with molecular weight and number of sulfate groups.6 Polyanionic analogues employing polyglycerol sulfonates, carboxylates, or phosphates exert only weak binding to selectins (around μM), thereby revealing the particular specificity of dPGS.7 The underlying rationale of our studies described herein encompasses, on one hand, the evaluation of dPGS as carrier molecule for the targeting of diagnostic molecules to sites of

INTRODUCTION Macromolecular conjugates are ideal entities for the delivery of drugs or diagnostics to diseased organs, tissues, and cells, as they can be effectively optimized for uptake, binding, release, and tolerability. Today, novel targeted macromolecules encompass mostly proteins and antibodies, which are recombinantly produced and either operate directly as therapeutics or are employed as carrier molecules for the delivery of conjugated effector molecules. A fundamental challenge with targeting molecules of biological origin is their inability in multipathway inhibition often leading to insufficient efficacy in the complex in vivo situation of disease treatment.1 An alternative to biological targeting molecules is provided by fully synthetic nanosized polymeric entities, which are accessible by comparably simple and economical chemical procedures. Many approaches using nanoparticles, dendrimers, and self-assembling systems as carriers for therapeutic drugs and diagnostic effectors are currently being followed. 2 Particularly, dendritic macromolecular polymers offer interesting applications for drug delivery and targeting. The multifunctional surface of dendritic structures is ideal to modify the physiological properties of the macromolecule by changing the © 2011 American Chemical Society

Received: May 30, 2011 Revised: November 5, 2011 Published: November 17, 2011 2453

dx.doi.org/10.1021/bc2002727 | Bioconjugate Chem. 2011, 22, 2453−2460

Bioconjugate Chemistry

Article

inflammation and, on the other hand, to gain information on the in vivo distribution of such polymers. Thus, our aim was to generate a novel type of imaging probe involving near-IR dyes of the kind of Indocyanine Green (ICG). By visualizing the interaction of the polyanionic macromolecular conjugate within inflammatory processes, a novel molecular targeting mechanism is translated from the in vitro biological behavior to the performance as in vivo contrasting probe.

atmosphere and stirred for 5 h. Then, the reaction mixture was allowed to cool to room temperature and to react for further 24 h. The reaction was quenched by the addition of aqueous NaOH (1 M) until pH 8 was reached. The solvent was removed under reduced pressure and the crude product was dialyzed against water for 24 h while the solvent was replaced 3 times. After concentration under reduced pressure and drying in high vacuum, 8.1 g (92%) of the title compound was obtained as colorless solid. 1H (400 MHz, D2O) δ 4.58−4.17 (m, CH2−OSO3Na) 4.16−3.55 (m, dPG-backbone); 1.83− 1.62 (m, alkyl-CH2CH2O, alkyl-CH2CH2N3); 1.57−1.31 (m, 7×CH2-alkyl). 13C (101 MHz, D2O) δ 79.1, 78.0, 76.6, 71.0, 70.3, 69.1, 68.4, 67.7 (PG-backbone), 52.1 (CH2N3), 29.5, 29.4, 29.3, 29.2, 29.0, 28.9, 29.8, 26.8, 26.1 (CH 2-alkyl). IR (KBr) ν/ cm−1 3481 (m), 2929 (m), 2871 (m), 2099 (m), 1653 (s), 1472 (m), 1255 (s), 1074 (s), 1048 (s), 939 (m), 779 (m). Elemental analysis: calcd. C 21.94, H 3.14, N 0.29, S 17.37; found C 19.48, H 3.204, N 0.238, S 17.20. Meso-4-(2-carboxyethyl)phenyl NIR Dye from IR-820. A mixture of IR-820 (0.59 mmol, 0.5 g) and 4-(2-carboxyethyl)phenylboronic acid (1.06 mmol, 0.21 g) in water (15 mL) was heated to 90 °C for 48 h in the presence of Pd(PPh3)4 (0.09 mol, 0.1 g). The reaction mixture was poured into diethyl ether and the resulting precipitate isolated by centrifugation. Purification was achieved by RP-18 chromatography (CombiFlash) using water/methanol as eluent. Evaporation of methanol followed by lyophilization afforded 0.2 g (37%) of the product as green amorphous solid. 1H (400 MHz, DMSOd6) δ 1.41 (s, 12H), 1.66−1.86 (m, 10H), 1.98 (t, 2H), 2.65− 2.81(m, 8H), 3.10 (t, 2H), 4.24 (t, 4H), 6.28 (d, 2H), 7.23 (dd, 4H), 7.45 (t, 2H), 7.56 (dd, 4H), 7.71 (d, 2H), 8.01 (dd, 4H), 8.09 (d, 2H). MS (ESI-TOF) m/z calcd for C55H59N2NaO8S2 [M]+ 962.3611, found 962.3591; calcd [M+Na]+ 985.3508, found 985.3491; calcd [M-H+2Na] + 1007.3328, found 1007.3308. Meso-propargyl NIR Dye (4). To as solution of meso-4-(2carboxyethyl)phenyl heptamethine dye (0.1 mmol, 100 mg) and 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate (HATU; 0.18 mmol, 67 mg) in dry DMF (3 mL) was added diisopropylethylamine (0.2 mmol, 35 μL). After 30 min stirring at room temperature, a mixture of propargylamine (0.16 mmol, 10 μL) and diisopropylethylamine (0.3 mmol, 50 μL) was added and the reaction stirred at room temperature for 20 h. The product was precipitated with diethyl ether and isolated by centrifugation. Purification was achieved by RP-18 chromatography (CombiFlash) using water/methanol as eluent. Lyophilization afforded 55 mg (53%) of the product as green amorphous solid. 1H (400 MHz, DMSO-d6) δ 1.41 (s, 12H), 1.65−1.78 (m, 10H), 1.96 (t, 2H), 2.65−2.85 (m, 8H), 3.15 (t, 2H), 4.15−4.35 (m, 6H), 3.30 (t, 1H), 6.30 (d, 2H), 7.25 (dd, 4H), 7.45 (t, 2H), 7.58 (dd, 4H), 7.72 (d, 2H), 8.05 (dd, 4H), 8.10 (d, 2H). MS (ESI-TOF) m/z calcd for C58H61N3NaO7S2 [M]+ 999.2406, found 999.3692; calcd [M+Na] + 1022.2298, found 1022.3827; calcd [M+K] + 1038.3384, found 1038.3559. Polyglycerol Sulfate−NIR Dye Conjugate (6). 11-Azidoundecanyl-polyglycerolsulfate 3 (7.0 μmol, 100 mg) and propargyl dye 4 (21 μmol, 21 mg) were dissolved in a 1:1 mixture of PBS/ethanol (1.2 mL). To this solution was added CuSO4·5H2O (3.0 μmol, 100 μL of a stock solution of 7.8 mg in 1 mL PBS), sodium ascorbate (6.0 mmol, 100 μL of a stock solution of 12.4 mg in 1 mL PBS) and another 200 μL of ethanol. The mixture was allowed to react at 40 °C for 72 h.

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EXPERIMENTAL SECTION Materials. All chemicals used were reagent grade, used as received, and purchased from Acros Organics (Belgium) or Sigma-Aldrich (Germany) unless stated otherwise. 11-Bromo1-undecanol (>97%) was purchased from Fluka (Germany), and 4-(2-carboxyethyl)-phenylboronic acid from Alfa Aesar (Germany). Dendritic polyglycerol (dPG, compound 1) with a molecular weight of 6000 g/mol and a PDI < 1.6 was synthesized according to literature via an anionic multibranching ring-opening polymerization of glycidol and pentaerythritol as starter.8,9 The ICC propargyl dye 5 was synthesized as published,10 and is commercially available through IRIS Biotech, Germany. Dialysis was performed with regenerated cellulose tubings (MWCO 2000) purchased from Carl Roth. Reversed phase chromatographic purification was performed using RP-18 Redisep flash columns (ISCO CombiFlash Rf system). 1H NMR and 13C NMR spectra were recorded at 25 °C at concentrations of 100 g·L−1 on a JEOL ECX 400 spectrometer, operating at 400 and 101 MHz, respectively. NMR chemical shifts δ are reported in ppm and the deuterated solvent peak was used for calibration. Mass spectra were obtained from an ESI-Time-of-Flight LC/M mass spectrometer 6210 (Agilent Technologies) operating at a flow rate of 10 μL/min. Combustional analysis to determine degrees of sulfation was performed on a Vario EL III elemental analyzer. IC50 values were determined by SPR as published.5 Synthesis of Conjugates. 11-Azidoundecyl-polyglycerol (2). dPG 1 (0.83 mmol, 5 g) was dissolved in dry DMF (40 mL) and statistically deprotonated with sodium hydride (60% in mineral oil, 2.08 mmol, 83 mg, 2.5 equiv) at 80 °C under Ar atmosphere for 30 min. 11-Azido-1-undecanyl-tosylate11 (1.5 mmol, 0.54 g, 1.8 equiv) was added to the mixture and the reaction was kept for further 24 h at 80 °C. Subsequently, the solvent was removed under reduced pressure, the residue redissolved in aqua dest. (50 mL), and repeatedly extracted with DCM (3 × 50 mL). The aqueous phase was concentrated and further purified by dialysis in MeOH for 48 h, while the solvent was replaced three times. After concentration under reduced pressure and drying in high vacuum, 4.6 g (88%) of the title compound was obtained as a slightly yellow, viscous oil. 1 H (400 MHz, MeOH-d4) δ 4.03−3.40 (m, dPG-backbone); 1.70−1.50 (m, alkyl-CH2CH2O, alkyl-CH2CH2N3); 1.47−1.26 (m, 7×CH2-alkyl). 13C (101 MHz, MeOH-d4) δ 81.5, 81.3, 79.7, 73.9, 72.8, 72.4, 72.2, 70.9, 70.6, 64.4, 63.0, 62.7 (PGbackbone), 52.4 (CH2N3), 31.7, 31.0, 30.6, 30.3, 29.9, 29.0, 28.6, 27.8, 27.0 (alkyl CH2). IR (KBr) ν/cm−1 3365 (s), 2927 (m), 2871 (m), 2071 (vs), 1122 (s), 1098 (s), 977 (m), 822 (m). Elemental analysis: calcd. C 49.065, H 8.250, N 0.680; found C 47.37, H 8.632, N 0.528. 11-Azidoundecyl-polyglycerolsulfate (3). AzidoundecyldPG 2 (0.63 mmol, 4.0 g) was dissolved in dry DMF (25 mL) and a solution of sulfurtrioxide pyridine complex (0.05 mol, 8.0 g, 80 equiv) in dry DMF (50 mL) was slowly added to the solution via a dropping funnel at 60 °C under Ar 2454



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Figure 1. Chemical structures of conjugates and synthetic pathway: (a) NaH, 11-azido-1-undecanyltosylate, DMF, 80 °C, 24 h; (b) SO3-pyridine, DMF, 60 °C, 5 h; (c) CuSO4, Na-ascorbate, PBS/ethanol, 40 °C, 72 h.

which has a value of 13%.12 Dye-to-polymer ratio was estimated from the UV−vis spectrum of a 5 μM conjugate solution prepared with the initial assumption of a dye-to-polymer molar ratio of 1 and a molecular weight of 13 000 g/mol. Cell Culture. The epithelial human lung cancer cell line A549 was routinely propagated as follows: DMEM medium, with 10% fetal calf serum, 2% glutamine, and penicillin/ streptomycin (all from PAN Biotech) added. Cells were seeded into medium at 1 × 105 cells/mL, cultured at 37 °C with 5% CO2, and split 1:5 two times a week. Peripheral blood mononuclear cells (PBMC) were prepared from peripheral blood of different healthy donors by a sequence of differential centrifugation on Ficoll-Hypaque, adherence to polystyrene culture flasks for 2 h, and subsequent washing with RPMI containing 10% fetal calf serum (both from PAN Biotech) to remove nonadherent cells. The cells were stimulated with 100 ng/mL lipopolysaccharide (LPS; Sigma) for 24 h to prime proinflammatory processes.13 In Vivo Fluorescence Imaging. Animal Model. Animals in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institute of Health (NIH publication no. 85−23, revised 1996). All experiments were approved by the local animal welfare committee. For arthritis studies, we used female Lewis rats with a body weight of (150 ± 10) g on arrival

After evaporation of ethanol, the residue was stirred in methanol/ethanol 1:1 and the supernatant decanted after precipitating the nonsoluble product by centrifugation. Finally, the product was isolated by RP-18 chromatography (CombiFlash) eluting as first fraction with water as eluent. Lyophilization afforded 165 mg (77%) of 6 as green solid. 1 H (400 MHz, D2O/DMSO-d6 1:1) δ 8.40−6.20 (m, dye aromatic and polymethine groups, triazol group); 4.55−4.25 (m, CH2−OSO3Na); 4.20−3.60 (m, dPG-backbone); 2.20− 1.80 (m, dye methylene groups); 1.65−1.20 (m, alkylCH2CH2O, alkyl-CH2CH2N3, CH2-alkyl linker, dye methyl groups). Elemental analysis: calcd. C 25.04, H 3.35, N 0.55, S 16.66; found C 22.41, H 2.412, N 0.539, S 17.93. Polyglycerol Sulfate−ICC Dye Conjugate (7). 11-Azidoundecanyl-polyglycerolsulfate 3 (7.0 μmol, 100 mg) and propargyl ICC dye 5 (21 μmol, 15 mg) were reacted in a 1:1 mixture of PBS/ethanol (1.2 mL) and the product isolated as described for compound 6. Lyophilization afforded 180 mg (85%) of 7 as red solid. Photophysical Characterization. Absorption spectra were recorded on a Lambda 950 spectrophotometer (PerkinElmer), fluorescence spectra were measured with a JASCO FP6500 spectrofluorometer (150 W xenon lamp, R928 Hamamatsu photomultiplier). Fluorescence quantum yields are calculated relative to the standard dye ICG in DMSO, 2455



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(Charles River) fed a normal diet. Collagen-induced arthritis (CIA) was induced as described before.14−16 After fluorescence imaging, the animals were killed in deep anesthesia by intravenous injection of T61 ad us. vet. (Intervet) at a dose of 2 mL/animal. Clinical Scoring. Clinical symptoms of the ankle joints after induction of arthritis are disposed in scores from 0 to 3. The ankle joints were scored in the following way: 0 − no clinical symptoms, 1 − low grade swelling, 2 − middle grade swelling and reddening, and 3 − high grade swelling, reddening, and mobility reduction. Administration of Conjugates. The imaging probe 6 was dissolved in sterile PBS (PAN Biotech, Berlin, Germany) and administered i.v. via the tail vein at a dose of 4 mg/kg (corresponding to 0.15 μmol dye per kg). Animals included for later histology additionally received ICC conjugate 7 at a dose of 1 mg/kg. Imaging Device and Image Processing. A camera-based fluorescence imaging system (Camera: EMCCD-Camera, IXON BV887, Andor) was used for imaging of animals. As excitation source, a light emitting diode (LED) array with λmax of 750 nm illuminating an area of 20 cm × 20 cm and a short pass filter (λ = 750 nm, LCLL 750) were used. The following parameters were adjusted: exposure time, 100 ms; EM gain, 3000; delay, 18.8 ms; shift time, 0.9 ns/line; preamplifier, 4.6; detector temperature, −65 °C; binning, 2 pixel by 2 pixel; film, kinetic series with 3000 frames, kinetic cycle time, 200 ms, 1 accumulation; images, accumulation series with 100 frames, accumulation cycle time 200 ms. Two long pass filters (λ 50% = 800 nm, 5 OD, 800 LP, 800 ALP) were applied to suppress excitation light. The images were analyzed as published.16 Briefly, regions of interest (ROI) were set and illumination intensities corrected for background. Images are normalized to the mean intensity from an ROI placed over the reference standard. Mean and 90th percentile values are determined in ROI for the right and left ankles, the area of the whiskers, and the eye.16 Cytochemistry. In the present study, cells were seeded at 5 × 104 cells/mL in a 24-well culture plate on glass coverslips (Sigma), and cultured for 24 h at 37 °C. Thereafter, cells were cultured with medium containing 10−6 M 6, 7, and 10−5 M polyglycerol-ICC (nonsulfated control)17 for different times at 37 °C. Afterward, cells were fixed with cold acetone, rinsed, and covered with 4,6-diamidino-2-phenylindole (DAPI, Abcam, Cabridge, UK) for nuclear counterstain. Image acquisition was performed using a Leica DMRB microscope (Leica, Solms, Germany). Images were taken with a digital camera (Spot 32, Diagnostic Instruments). Histochemistry. Histochemical staining was performed on paraffin sections (10 μm) of tissue of liver fixed with formalin or cryosections (5 μm) of tibiotarsal articulations. Tissues were removed 3 h post injection (p.i.) of dPGS-ICC or polyglycerolICC from control animals or rats with CIA. Tissue sections were deparaffinated and fixed with acetone. Non-decalcified tissues of tibiotarsal articulation were shock-frozen using liquid nitrogen. Microtome sections were removed from the block with cellotape and then transferred to slides, dried overnight, and fixed with acetone for 30 s.18 DAPI was used for nuclear counterstain. Image acquisition was performed using a Leica DMRB microscope (Leica). Images were taken with a digital camera (Spot 32, Diagnostic Instruments).

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RESULTS AND DISCUSSION

Synthesis of Polyglycerol Sulfate Conjugates with Cyanine Dyes. The synthetic path involves the derivatization of dendritic polyglycerol (dPG) with a reactive linker in a fashion in which approximately only one dye molecule on average is conjugated to the polymer. Dye conjugation via “Click” chemistry after sulfation of the majority of hydroxyl groups leads to the desired near-IR imaging conjugate. The synthesis is illustrated in Figure 1. The synthesis of dPG (compound 1 in Figure 1) was achieved by a one-step anionic multibranching ring-opening polymerization of glycidol started on the initiator pentaerythritol. After purification by dialysis, a highly viscous oil was obtained with a typical PDI of 1.4−1.6 and a number average molecular weight Mn of approximately 6000 g/mol.8,9 Before the sulfation reaction to the desired polyglycerol sulfate, a small proportion of the hydroxyl groups was modified by reaction with 11-azidoundecanyl-1-tosylate forming an ether linkage between polymer and aliphatic chain. The appearance of the azide functionality in 2 was supported by IR (characteristic band at 2100 cm−1). According to 1H NMR signal integration (see Supporting Information), 60% of polymer carried a linker moiety on average. Subsequently, the material was sulfated with SO3·pyridine complex5,19 giving the azidoundecanyl-modified sulfated polyglycerol 3 in high yield. Elementary analysis revealed a high degree of sulfation of 92% (75 of theoretically 80 hydroxyl functions converted for Mn 6000 g/mol); thus, the Mn of the sulfated species is approximately 12 000 g/mol. Conjugation for near-infrared imaging in the spectral range of Indocyanine Green (ICG) was accomplished with the novel propargyl derivative 4. We prepared this fluorescence label from the commercial dye IR820 referring to a recently published procedure by Achilefu et al. who applied the Suzuki method of aryl-halide substitution to the central chloro function in the IR-820 structure.20 In our approach, we used 4-(2-carboxyethyl)phenylboronic acid as reaction component, thereby introducing a carboxyethyl linker symmetrically into the chromophore. Amidation with propargylamine under standard conditions led to the desired derivative 4 for conjugation to the polyglycerolsulfate 3. Coupling of 4 was achieved by copper-mediated 1,3-dipolar cycloaddition (“Click”-conjugation) between the azide group at the polymer and the propargyl residue at the dye. The resulting conjugate 6 was characterized by 1H NMR and by photooptical methods. The conjugation reaction yielded a dye-to-polymer ratio of approximately 0.66 estimated from the absorption spectra (see below). We included furthermore a VIS fluorophore into our studies and synthesized an analogous conjugate (7) employing the indocarbocyanine (ICC) propargyl moiety 5 already published in other studies.7,10 The influence of dye conjugation on target binding was determined by SPR (Biacore)5,6 yielding inhibition (IC50) values for Lselectin similar to nonconjugated dPGS of identical sulfation but without linker modification (conjugate 6, IC50 3 nM; conjugate 7, IC50 6 nM; nonconjugated dPGS, 4 nM). Hence, dye conjugation does not hamper target affinity of the resulting conjugate. Photophysical Properties. In order to characterize the conjugate 6 with respect to its optical properties, we were interested in the dye-to-polymer ratio and in the resulting fluorescence quantum yield. The data for conjugate 6 in comparison with the free dye are summarized in Table 1. First, absorption measurements were conducted in phosphate2456



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chromophore type21,23 and also unmodified ICG in nanoformulations,24 however, this level appears sufficient for further biological studies. For the free dye, a very high extinction coefficient in DMSO was measured at 195 000 M −1 cm−1 representing the theoretically optimal condition to determine optical properties due to the absence of aggregation phenomena. Assuming an extinction coefficient of 150 000 M−1 cm−1 from the spectral shape in serum or PBS/DMSO (Figure 2) to estimate the dyeto-polymer ratio in 6, a concentration of 3.3 μM, which corresponds to a dye loading of 66%, can be calculated. This is in approximate agreement with the maximal achievable loading based on 60% linker attachment (see above). The difficulty in accurately determining the ratio between a carrier unit and a dye is similar to observations with antibody conjugates with rather lipophilic heptamethine dyes, such as ICG.21 In fact, as discussed by Villaraza et al., the clear analysis of dye loading as well as supposedly successfully achieved separation of covalently attached dye from aggregated nonattached impurities is difficult, as in size exclusion chromatography (SEC) both specimens may elute similarly. Furthermore, our polymer carriers do not exhibit UV absorption, thus lacking a method to quantify the polymer concentration by spectrophotometry. Cellular Uptake. On the in vitro level, we have already demonstrated the ability of polyglycerol sulfates to inhibit Land P-selectin binding with high specificity over other anionic groups, such as sulfonate and carboxylate.7 Applying conjugates 6 and 7, both of which exhibit high binding to L-selectin with conjugated fluorophore (see above) allows the translation to the cellular level. We found that the polymer clearly localizes within tumor cells, as well as in activated mononuclear cells, as shown in cell culture and tissue specimens. Figure 3A illustrates that the NIR conjugate 6 appears comparably in A549 cells when setting up a microscope with excitation and detection technique capable of exciting and imaging the near-IR fluorophore permitting direct visualization of 6. Compound 7, employing ICC dye7,10 with typical fluorescence properties fitting the Cy3-filter (550 nm/580 nm), allows the same subcellular localization with a sharper image

Table 1. Photophysical Properties of Free Dye and Polyglycerolsulfate Conjugate 6 compound free dye

6

7

solventa DMSO DMSO/PBS (1:1) DMSO/PBS (1:1) PBS serum PBS serum

absorption max. (nm)b

fluorescence max. (nm)

FQc

806 800

836 823

0.052 0.097

728, 804, −

823

0.027

730, 792, 886 738, 806, 886 518, 553, − 526, 559, −

817 818 568 572

0.006 0.018 n.d. n.d.

a

Data for free dye in PBS and conjugate 6 in DMSO are not determined due to insolubility, and no reasonable spectra were obtainable. bSpectral data for 6 and 7 from left to right: dimer band, monomer band, J-band. cFluorescence quantum yield relative to ICG in DMSO (0.13).12

buffered saline and in serum as most relevant physiological media. The spectra depicted in Figure 2 illustrate that the conjugate exhibits a pronounced tendency to form aggregates. For comparison, the theoretically optimal absorbance profile of the fluorophore is obtained by measuring the free dye in DMSO. Unfortunately, 6 is not soluble in DMSO or methanol due to the extremely high anionic charge, so we had to exclude this solvent. In PBS, strong aggregation is apparent from the collapsing extinction of the absorbance maximum of 6 at 792 nm and growth of the dimer shoulder at 730 nm, together with sign of a J-band at 886 nm. Also, the fluorescence yield is rather low at 0.6% (excitation 720 nm). This behavior has been studied intensively for protein and antibody conjugates with lipophilic fluorescent labels, mainly dyes of cyanine type.21,22 In serum as the more physiologically relevant medium, we obtained a slightly restored main absorption band, accompanied by an increased fluorescence quantum yield of 1.8%. Despite insolubility in DMSO, a mixture of PBS/DMSO of 1:1 gave a similar spectrum and a quantum yield of 2.7%. Compared to other covalent conjugates of the ICG

Figure 2. Absorbance spectra of conjugate 6 in different solvents compared to free dye in PBS/DMSO 1:1. Fluorescence emission spectrum of 6 in serum (excitation: 760 nm). 2457



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impression (Figure 3B). As expected, no cellular signals after incubation with polyglycerol-ICC as control could be detected (Figure 3C). Due to the absence of technical routine solutions for microscopic imaging in the near-IR, we continued to employ compound 7 as standard probe for microscopic studies, hereby assured that 6 and 7 exhibit identical cellular uptake behavior as proven by the above-described bridging comparison. Accordingly, uptake of conjugate 7 could be demonstrated in vitro in human peripheral blood mononuclear cells stimulated with LPS to induce for inflammatory processes (Figure 3D). Moreover, intracellular localization after targeting to macrophages in the living organism was proven by studying liver tissue 3 h post i.v. injection of conjugate 7 into rats. As shown in Figure 3E, enrichment in rat liver macrophages (Kupffer cells) with a certain level of fluorescence signals also detectable in activated endothelial cells and hepatocytes can be followed ex vivo by fluorescence microscopy of liver tissue specimens. In Vivo Imaging. Conjugate 6 was employed as imaging probe for in vivo studies using the well-established arthritis rat model in which joint inflammation is induced by the injection of bovine type II collagen.15,16 After i.v. administration of the probe (dose: 4 mg/kg of polymer corresponding to 0.2 μmol/ kg fluorophore), fluorescence images were generated up to 24 h post injection. Images from a time series of a control and two CIA animals are shown in Figure 4. The false colors allow easy identification of the diseased joints. As is typical in the disease progression of this animal model, a certain number of joints remains either unaffected or at a low disease score. In the fluorescence imaging setting, we were able to differentiate dye probe uptake depending on the acuteness of the inflammation within the same animal. In Figure 4, the middle row impressively shows that the absence of clinical signs (score 0) does not lead to pronounced fluorescence enhancement. For further quantification, images were analyzed by applying appropriate regions of interest (ROIs) to the area of ankle joints, eyes, and a fluorescent reference cube. Accordingly, a total of 10 joints in control rats and 20 joints in arthritic animals were evaluated. The fluorescence intensity from the eyes is helpful as a basic signal indicating the pharmacokinetic behavior of the injected probe in the blood compartment.

Figure 3. Uptake of dPGS conjugated with fluorescence dyes (red): A549 lung cancer cells cultured for 4 h with 6 (A) or 7 (B). (C) Monocytes isolated from human peripheral blood stimulated with 100 ng/mL LPS (24 h) and cultured for 4 h with 7. (D) Control with polyglycerol-ICC on A549 lung cancer cells. (E) Paraffin-embedded tissue of rat 3 h p.i. of 7. M − Liver macrophages (Kupffer cells), E −endothelial cells, H − hepatocyte, CV − central venule. Nuclear staining with DAPI (blue). Objective lens 40× (A−D), 100× (E).

Figure 4. Comparison of fluorescence images in false colors (normalized to a fluorescence reference cube) of a control rat and rats with collageninduced rheumatoid arthritis (different clinical scores are indicated) after 10 min, 1 h, and 24 h post injection of 6 (4 mg/kg b.w.). One representative example of at least n = 5. 2458



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Histological Examination of Tissues. To enable a postmortem histological recovery of the dPGS conjugate and confirm the in vivo imaging data, we analyzed tissues of the tibiotarsal articulation by fluorescence microscopy using the 550 nm channel (Cy3) to excite the ICC dye (Figure 6). No

Figure 5 illustrates the resulting average signal kinetics of joints of healthy animals, and joints of treated animals, grouped into

Figure 5. Temporal behavior of means of normalized fluorescence intensity derived from 90th percentiles of ROIs covering the ankle joints and the opened eye of the rats post injection of conjugate 6. The error bars indicate the single standard deviation derived from the number of measured values n (individual joints as independent events).

score 0 and scores 1−3, as well as in the eye. The results show that conjugate 6 generates an almost 3.5-fold signal increase in the affected joint (score 1−3) at the time of maximal intensity at 60 min. Interestingly, for joints of the treated group with no clinical signs (score 0) a 2-fold signal enhancement in comparison to the healthy group can still be calculated (n = 3). Data of fluorescence intensity of ICG derived from ROIs over ankle joints at defined time intervals of each animal group were analyzed employing the Mann−Whitney test for independent samples. At 10 min after bolus injection, the significance of fluorescence intensity values between controls and diseased joints (score 1−3) was calculated (MedCalc, vers. 11.1.0.0) and the two tailored probability yielding p < 0.0001. The significance is even more pronounced at later time points, as can be seen from the difference of means in Figure 5. These findings indicate that the molecular targeting of the polyanionic macromolecule is effectively directed to early signs of the disease before clinical manifestation can be evidenced. In fact, the targeting of early signs in joint inflammation, involving the formation of autoantibodies activating macrophages, the release of proinflammatory cytokines, the activation of endothelial cells, and finally the infiltration of inflammatory cells in the articulation,25 remains a challenge in diagnosis and treatment before clinical manifestation (edema, destruction of cartilage, and bone) occurs.26 The fluorescence signal measured over the eye, as a rough estimate for the change of dye concentration in the blood, reaches almost its minimal level at the time of maximal signal in the joints. This allows an explanation of the high and early contrast in the images. Typically, macromolecular targeting for diagnostic imaging purposes might be hampered by longcirculating probe fractions and resulting high signal background, so that the required target-to-blood signal ratios are not sufficiently developed. By employing the eye fluorescence as a provisional solution to monitor blood kinetics, a roughly estimated blood half-life is shorter than 1 h, fast enough for reasonable contrast enhancement.

Figure 6. Conventional and fluorescence histology of tissues from tibiotarsal articulation of rats with collagen-induced rheumatoid arthritis (CIA) or healthy controls 3 hs post i.v. injection of dPGSICC (7) or nonsulfated PG-ICC.17 Arrowhead: synovial membrane. B: bone. C: cartilage. Nuclear staining with DAPI (blue), compounds in red.

fluorescence signals were detectable in the articulations of healthy animals 3 h post i.v. administration of 7. In the tissues of rats with CIA, conjugate 7 was enriched in the inflammatory infiltrate in the synovial membrane. The treatment of both control and CIA rats with nonsulfated polyglycerol−ICC conjugate17 as control did not lead to uptake into the cells of the synovial membrane, thus further supporting the specific targeting behavior of dPGS observed with the near-IR fluorescent version 6 as in vivo imaging probe.

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CONCLUSION We have demonstrated that dendritic polyglycerol sulfate (dPGS) acts as a novel type of synthetic nanocarrier suited for inflammation-specific molecular imaging. The polymer was readily synthesized by a polymerization and sulfation procedure and conjugated with either a near-infrared dye or a VIS dye to facilitate in vitro and in vivo detection in disease models. Both conjugates behave similarly regarding its cellular uptake into epithelial cells and macrophages. The specific intracellular localization is yet to be further elucidated with respect to the mechanism involved. Our ongoing work involves studies on uptake possibly via transporter proteins and scavenger receptors into inflammatory cells, as well as the impact of the type of anionic group on cellular interaction.7 Translation into the diagnostic application was accomplished by in vivo 2459



Article

(12) Benson, R. C., and Kues, H. A. (1978) Fluorescence properties of indocyanine green as related to angiography. Phys. Med. Biol. 23, 159−163. (13) Surette, M. E., Nadeau, M., Borgeat, P., and Gosselin, J. (1996) Priming of human peripheral blood mononuclear cells with lipopolysaccharides for enhanced arachidonic acid release and leukotriene synthesis. Journal of Leukocyte Biology 59, 709−715. (14) Trentham, D. E., Townes, A. S., and Kang, A. H. (1977) Autoimmunity to type II collagen an experimental model of arthritis. J. Exp. Med. 146, 857−68. (15) Fischer, T., Gemeinhardt, I., Wagner, S., Stieglitz, D. V., Schnorr, J., Hermann, K. G., Ebert, B., Petzelt, D., Macdonald, R., Licha, K., Schirner, M., Krenn, V., Kamradt, T., and Taupitz, M. (2006) Assessment of unspecific near-infrared dyes in laser-induced fluorescence imaging of experimental arthritis. Acad. Radiol. 13, 4−13. (16) Vollmer, S., Vater, A., Licha, K., Gemeinhardt, I., Gemeinhardt, O., Voigt, J., Ebert, B., Schnorr, J., Taupitz, M., Macdonald, R., and Schirner, M. (2009) Extra Domain B fibronectin as a target for nearinfrared fluorescence imaging of rheumatoid arthritis affected joints in vivo. Mol. Imaging 8, 330−340. (17) Reichert, S., Welker, P., Calderón, M., Khandare, J., Mangoldt, D., Licha, K., Kainthan, R. K., Brooks, D. E., and Haag, R. (2011) Sizedependant cellular uptake of dendritic polyglycerol. Small 7, 820−9. (18) Kuhne, M., Erben, U., Schulze-Tanzil, G., Köhler, D., Wu, P., Richter, F. J., John, T., Radbruch, A., Sieper, J., and Appel, H. (2009) HLA-B27-restricted antigen presentation by human chondrocytes to CD8+ T cells: potential contribution to local immunopathologic processes in ankylosing spondylitis. Arthritis Rheum. 60, 1635−46. (19) Türk, H., Haag, R., and Alban, S. (2004) Dendritic polyglycerol sulfates as new heparin analogues and potent inhibitors of the complement system. Bioconjugate Chem. 15, 162−7. (20) Lee, H., Mason, J. C., and Achilefu, S. (2008) Synthesis and spectral properties of near-infrared aminophenyl-, hydroxyphenyl-, and phenyl-substituted heptamethine cyanines. J. Org. Chem. 73, 723−5. (21) Villaraza, A. J., Milenic, D. E., and Brechbiel, M. W. (2010) Improved speciation characteristics of PEGylated indocyanine greenlabeled panitumumab: revisiting the solution and spectroscopic properties of a near-infrared emitting anti-HER1 Antibody for optical imaging of cancer. Bioconjugate Chem. 21, 2305−12. (22) Pauli, J., Vag, T., Spieles, M., Wenzel, M., Kaiser, W. A., ReschGenger, U., and Hilger, I. (2009) An in vitro characterization study of new near infrared dyes for molecular imaging. Eur. J. Med. Chem. 44, 3496−3503. (23) Ogawa, M., Kosaka, N., Choyke, P. L., and Kobayashi, H. (2009) In vivo molecular imaging of cancer with a quenching nearinfrared fluorescent probe using conjugates of monoclonal antibodies and indocyanine green. Cancer Res. 69, 1268−72. (24) Yu, J., Javier, D., Yaseen, M. A., Nitin, N., Richards-Kortum, R., Anvari, B., and Wong, M. S. (2010) Self-assembly synthesis, tumor cell targeting, and photothermal capabilities of antibody-coated indocyanine green nanocapsules. J. Am. Chem. Soc. 132, 1929−38. (25) Firestein, G. S. (2005) Immunologic mechanisms in the pathogenesis of rheumatoid arthritis. J. Clin. Rheumatol. 11 (3 Suppl), S39−44. (26) Brown, A. K. (2006) Remission in rheumatoid arthritis: time to reflect on methods of assessment. Int. J. Adv. Rheumatol. 3, 84−9.

fluorescence imaging in a rat arthritis model, demonstrating fast and selective targeting of tissue inflammation and permitting the differentiation of early onset of disease progression. We conclude that dendritic macromolecules provide interesting opportunities beyond the usually employed drug delivery systems with nanoparticles and other high molecular weight systems based on passive targeting. Here, we utilized highly dense anionic charge to impart specific interaction with factors of inflammation, ultimately generating carrier systems for targeted diagnostics and therapeutics.

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ASSOCIATED CONTENT S Supporting Information * Experimental procedures for the synthesis of 11-Azido-1undecanol and 11-Azido-1-undecanyl-tosylate. 1H NMR spectra for compounds 2, 3, and 6. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]; tel.: +49-30-688379230; fax: +49-30-688379299.

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ACKNOWLEDGMENTS This work was supported by the European Regional Development Fund (EFRE) and by the Investitionsbank Berlin (IBB) code: 10138863.

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REFERENCES

(1) Juliano, R. (2007) Challenges to macromolecular drug delivery. Biochem. Soc. Trans. 35 (Pt 1), 41−3. (2) Khandare, J., and Haag, R. (2010) Pharmaceutically used polymers: principles, structures, and applications of pharmaceutical delivery systems. Handb. Exp. Pharmacol. 197, 221−50. (3) Svenson, S., and Tomalia, D. A. (2005) Dendrimers in biomedical applicationsreflections on the field. Adv. Drug Delivery Rev. 57, 2106−29. (4) Calderón, M., Quadir, M. A., Sharma, S. K., and Haag, R. (2010) Dendritic polyglycerols for biomedical applications. Adv. Mater. 22, 190−228. (5) Dernedde, J., Rausch, A., Weinhart, M., Enders, S., Tauber, R., Licha, K., Schirner, M., Zügel, U., von Bonin, A., and Haag, R. (2010) Dendritic polyglycerol sulfates as multivalent inhibitors of inflammation. Proc. Natl. Acad. Sci. U. S. A. 10, 19679−84. (6) Weinhart, M., Gröger, M., Enders, S., Riese, S. B., Dernedde, J., Kainthan, R. K., Brooks, D. E., and Haag, R. (2011) The role of dimension in multivalent binding events: structure-activity relationship of dendritic polyglycerol sulfate binding to L-selectin in correlation with size and surface charge density. Macromol. Biosci. 11, 1088−98. (7) Weinhart., M., Gröger, D., Enders, S., Dernedde, J., and Haag, R. (2011) Synthesis of dendritic polyglycerol anions and their efficiency toward L-selectin inhibition. Biomacromolecules 12, 2502−11. (8) Sunder, A., Hanselmann, R., Frey, H., and Mulhaupt, R. (1999) Controlled synthesis of hyperbranched polyglycerols by ring-opening multibranching polymerization. Macromolecules 32, 4240−46. (9) Sunder, A., Mulhaupt, R., Haag, R., and Frey, H. (2000) Hyperbranched polyether polyols: a modular approach to complex polymer architectures. Adv. Mater. 12, 235−39. (10) Sisson, A. L., Steinhilber, D., Rossow, T., Welker, P., Licha, K., and Haag, R. (2009) Biocompatible functionalized polyglycerol microgels with cell penetrating properties. Angew. Chem., Int. Ed. Engl. 48, 7540−5. (11) Yang, J., Wang, Y., Rassat, A., Zhang, Y., and Sinaÿ, S. (2004) Synthesis of novel highly water-soluble 2:1 cyclodextrin/fullerene conjugates involving the secondary rim of β-cyclodextrin. Tetrahedron 60, 12163. 2460


Fluorescence Imaging with Multifunctional Polyglycerol Sulfates

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