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Synthesis, photophysical and biological evaluation of sulfated polyglycerol dendronized perylenebisimides (PBIs) – a promising platform for anti-inflammatory theranostic agents? Timm Heek, Christian Kuehne, Harald Depner, Katharina Achazi, Jens Dernedde, and Rainer Haag Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00683 • Publication Date (Web): 18 Feb 2016 Downloaded from http://pubs.acs.org on February 19, 2016

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Bioconjugate Chemistry

Synthesis, photophysical and biological evaluation of sulfated polyglycerol dendronized perylenebisimides (PBIs) – a promising platform for anti-inflammatory theranostic agents? T. Heek†, C. Kühne‡, H. Depner†, K. Achazi‡, J. Dernedde*,‡, R. Haag*,†

Abstract A set of four water-soluble perylene bisimides (PBI) based on sulfated polyglycerol (PGS) dendrons were developed, their photophysical properties determined via UV/Vis and fluorescence spectroscopy, and their performance as possible anti-inflammatory agents evaluated via biological in vitro studies. It could be shown that in contrast to charge neutral PG-PBIs the introduction of the additional electrostatic repulsion forces leads to a decrease in the dendron generation necessary for aggregation suppression, allowing the preparation of PBIs with fluorescence quantum yields of >95 % with a considerable decreased synthetic effort. Furthermore the values determined for L-selectin binding down to the nM range, their limited impact on blood coagulation and their minor activation of the complement system renders these systems ideal for anti-inflammatory purposes. Author present address † Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustraße 3, 14195 Berlin, Germany. Email: [email protected]. Tel: (+49) 30 838 52633. Fax: (+49) 30 838 53357. ‡ Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, CharitéUniversitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Email: [email protected]. Tel: (+49) 30 450 569 203. Fax: (+49) 30 450 569 900. Author contribution T. Heek and C. Kühne contributed equally to this work.

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Introduction Perylenebisimides (PBIs) possess a set of physical and chemical properties which made them an ideal material for a broad variety of different applications, for example as supramolecular building block, as organic semiconductor or absorber for fluorescent solar collectors.1-3 Due to the increasing importance of fluorescent dyes in the elucidation of biological processes within the last years there has been an increasing amount of reports which intent to create highly fluorescent water-soluble PBIs with the purpose of application e.g. as fluorescent label for biomolecules like proteins or site specific cell labeling agent.4-6 For most of these applications water-solubility is a mandatory prerequisite. Therefore different concepts of introducing water-solubilizing moieties onto the highly hydrophobic aromatic perylene core have been investigated which can be roughly categorized via the position of substitution (imide, bay, or ortho) and the type of solubilizing moiety (small molecule, polymeric, dendritic). A very successful approach was established from Müllen et al. where the introduction of small ionic aromatic moieties onto the bay region of the PBI cores led to highly watersoluble PBIs with nearly quantitative fluorescence quantum yields (FQY) due to the combination of electrostatic repulsion forces and a reduced aggregation tendency by a twist of the aromatic systems.7, 8 While the bay-substitution concept worked out quite well, the introduction of small ionic groups at the imide position usually does not inhibit aggregation efficiently, resulting in nearly quantitative fluorescence quenching due to H-aggregate formation.9 However the attachment of water-solubilizing moieties onto the imide positions would offer specific advantages over the bay substitution pathway. For example the photophysical properties of PBIs are mainly controlled via the substitution pattern at the bay sites, and therefore an optical tuning of imide water-soluble PBIs would be easily possible.10 For that reason others and we have recently shown, that the attachment of sterically demanding water-soluble dendrons at the imide positions is a feasible concept for the preparation of fluorescent PBIs in an aqueous environment covering the wavelength from 532-741 nm.11-14 It is worth noting that dendronization of PBIs at the bay positions also leads to highly water soluble PBIs.15 Besides working as an aggregation suppressor such dendritic substituted PBIs offer the opportunity for further functionalization with biological active moieties allowing to combine the good optical properties with the multivalent binding modes of such dendritic systems.16, 17 For example Wang et al. have prepared symmetric substituted dendritic PBIs end functionalized with mannose moieties showing extremely low IC50 values for Con A binding.18, 19 Another example are dendritic PAMAM substituted PBIs which worked as efficient siRNA transfection agents.20,

21

A

biomedical research field which dramatically benefits from multivalent interactions from the applied drugs is the development of anti-inflammatory agents.22,

23

In that context anionic, dendritic

polyglycerols (dPGs) have been intensively investigated as possible anti-inflammatory agents and it could be shown that within the tested anions dPG sulfates (dPGS) possesses the best antiACS Paragon Plus Environment

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inflammatory properties.24 The mechanism of their anti-inflammatory effect is based on reducing leukocyte extravasation by shielding the adhesion molecules either of the leukocytes (L-selectins) and/or the inflamed vascular endothelium (P-selectins) and inhibiting leukocyte chemotaxis by inhibiting the generation of anaphylatoxins due to binding to positively charged complement factors like C3 and C5. 24 As many diseases are often accompanied with inflammatory processes, like severe infections, stroke, arthritis and cancer, the extraordinary strong binding affinities down to the nM range render dPGS-based materials ideal for theranostic purposes. Therefore corresponding dPGSderivatives were functionalized with different visualisable mojeties like near-infrared (NIR)fluorophores allowing fluorescence imaging, with radiotracers like

64

Cu or 3H allowing positron

emission tomography (PET) or autoradiography, or with gold nanorods allowing multispectral optoacoustic tomography (MSOT).25-27 However, in all cases the studies were conducted with statistically labeled polydisperse polymers rendering a quantification and identification of the actual biological active species sometimes problematic, as it can lead to overestimation of specific effects, especially when there is a non-linear molecular weight dependent biological activity. Although these problems are minimized when investigating polymers with low PDIs, this problem is of intrinsic nature and the only way to circumvent it, is the use of macromolecules with precisely defined molecular structure and a defined amount of tracer moieties. Under these aspects dye cored dendrimers are ideal systems as one gains accurate control of molecular weight and degree of functionalization and therefore structure property relationships can be clearly revealed. Thus we have synthesized four different generations of PG-dendronized PBI-sulfates and investigated their anti-inflammatory potential as well as their photophysical properties. Results and discussion Synthesis The synthetic approach towards the targeted PBIs 3a-3b is illustrated in Scheme 1. Therefor literature known, hydroxyl terminated PG-dendronized PBIs 2a-2d were synthesized from PGdendrons and commercially available perylenebisanhydride (PBA) according to a slightly modified reaction sequence.12 Instead of coupling the hydroxyl terminated, amine cored PG-dendrons to the PBA core, isopropylindene protected dendrons were used resulting in organo-soluble PBI intermediates 1a-1d. This approach was chosen as these products are thought to be better starting materials for future core substitution reactions, which usually require a sufficient solubility in organic solvents. Thereby the same trend as observed for the direct coupling of deprotected PG-dendron amines was observed. While the coupling of [G1] - [G3] to the PBA-core gave reasonably high yields ~ 90% the [G4] coupling only resulted in a moderate yield of 45%. This can be attributed to the insufficient accessibility of the amine core which obviously is thoroughly shielded by the sterically ACS Paragon Plus Environment


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demanding PG-dendron. A subsequent acid catalyzed cleavage of the isopropylidene protecting groups using TFA led to the deprotected PBIs 2a-2d which were purified via ultrafiltration and dried in a high vacuum system before sulfation to remove residual traces of water. The sulfation was conducted using the standard procedure established for dPGS applying SO3/pyridine complex at moderate temperatures.28 After ion exchange by repeated ultrafiltration with aqueous NaCl-solution and water, the sulfated PBIs 3a-3d were obtained in high yields >92%. The degree of sulfation was determined via elemental analysis revealing a substitution between 88% and 97%. Gel electrophoresis on an agarose gel nicely showed the increasing molecular weight and the purity of the prepared products. Furthermore, a staining with cationic Alcian blue was easily achieved, demonstrating the anionic nature of the synthesized PBIs and the accessibility of the charged moieties (Figure S2, Supporting Information).

Scheme 1. Synthetic route towards sulfated PG-dendronized PBIs ([GX]-PBI-S) 3a-3d (top row) and chemical structures of the [G1] dendrons of compounds 1a-3a (bottom row). For chemical structures of higher generation dendrons refer supporting information Figure S1.

Optical Properties The optical properties of the sulfated PBIs 3a-3d were analyzed via UV/Vis and fluorescence spectroscopy in aqueous medium and compared with their uncharged analogues 2a-2d in order to determine the effect of the sulfation process. A list of the corresponding properties is given in Table 1


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and the absorption and fluorescence spectra are shown in Figure 1. Regarding these figures, it is obvious that the most striking differences were clearly evidenced upon comparison of the lower generation ([G1] and [G2]) substituted PBIs. While the uncharged species 2a showed an absorption pattern which results from strongly aggregated PBIs forming face to face stacked H-aggregates, with the maximum of absorption at 501 nm, the sulfated derivative 3a showed a fine vibronic absorption pattern typically resulting from monomeric PBIs with an absorption maximum at 535 nm. Similarly, in case of the sulfated [G2]-derivative 3b only monomeric fine resolved absorption spectra were detected in contrast to the unsulfated PBI 2b, which usually shows concentration dependent absorption spectra.

12

These differences were also reflected in the decreased A0-0/A0-1 ratios, which

are a common measure for the aggregation state of core unsubstiuted PBIs and which have been

Figure 1. UV/Vis spectra of (a) sulfated polyglycerol dendronized PBIs ([GX]-PBI-S, 3a-3d) and (b) neutral -6

dendronized PBIs ([GX]-PBI-OH) in water (c=10

M, T=25 °C). Steady-state fluorescence spectra of (c) -7

sulfated polyglycerol dendronized PBIs ([GX]-PBI-S, 3a-3d (c=10

M, T=25 °C, λex=495 nm) and (d)

-7

fluorescence quantum yields (c=10 M, T=25 °C, λex=495 nm) in water.

reported to be ~1.6 for monomeric and ≤0.7 for highly aggregated water-soluble PBIs.13,

29, 30

Obviously the changes result from the added electrostatic repulsion forces which are introduced through the ionic sulfate groups. On the other hand, the differences in the absorption spectrum from



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the sulfated [G3] and the [G4] derivatives 3c and 3d to the neutral PBIs 2c and 2d were only minor, which is in line with the assumption that these derivatives are already sterically well enough shielded by the uncharged PG-dendrons, therefore preventing aggregation. However, a small bathochromic shift of 5 and 3 nm respectively was observed when comparing the charged [G3] and [G4] derivatives with the uncharged analogues. Although core unsubstituted PBIs usually show only a minor positive solvatochromic behavior due to their low difference of dipole moments in the ground and the excited state this might be interpreted in terms of a slightly more polar local environment resulting from the close vicinity of the polysulfate groups. This would also explain why this effect decreased when increasing the generation from [G3] to [G4] where the sulfate groups are located further away from the PBI core and the local environment is more dominated via the polyether backbone, as it is the case for the unsulfated PBIs. It is interesting to note that no change in the absorption profiles could be detected up to a concentration of 0.1 mM irrespectively of the generation attached (Figure S3, Supporting Information,). This is in contrast to the behavior of newkome dendronized PBIs carrying either cationic or anionic end groups as water solubilizing moieties, which have been reported to strongly aggregate under neutral pH when attached to the smallest generation.29 Table 1. Properties of the synthesized sulfated PBIs ([GX-PBI-S, 3a-3d) and the corresponding reference substances

Sample

3a 3b 3c 3d 2a 2b 2c 2d [G4]dPGS [f] dPGS12kDa[f] Heparin [f]

MW,calc [kDa]

DS [%]

λmax,

λmax,

A0-0/A0-1 ratio[a] 1.59 1.52 1.61 1.60 0.53[d] 0.85[d] 1.59 1.58 -

0.95 0.92 0.95 0.98 0.33[e] 0.54[e] 0.74[e] 0.99[e] -

1350 250 74 11 300

100 98 175 489 400

CP activity [%][c] 100 91 55 43 42

1651 3060 5864 11500 835 1427 2613 4983 11000

92 88 93 97 98

535 536 536 534 501[c] 534[c] 532 531 -

552 552 552 549 549 549 547 545 -

Stokes shift [nm] 17 16 16 15 15 14 -

13400

93

-

-

-

-

-

2

223

22

15000 (peak)

65

-

-

-

-

-

12000

-[g]

72

[a] abs

em.

FQY [%][b]

IC50 [nM]

PTT [%] [c]

[a] Determined from the first and second vibronic peak maxima in the UV-Vis absorption spectra at ~535 nm -7 -6 and ~495 nm; [b] Measured at c= 10 M, T= 25°C and λex= 495 nm ; [c] Measured at c = 10 M ; [d] -6

Concentration dependent, value given at c = 10 M; [e] Data taken from ref. [12]; [f] Data taken from ref. [32]; [g] no coagulation.

Also in the fluorescence properties the generation dependent differences were observed. All sulfated PBIs 3a-d showed a mirror image fluorescence signal with the maximum located between 549-552 ACS Paragon Plus Environment

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nm (Figure 1c). The fluorescence quantum yields determined ranged all well above 90% while the uncharged analogues have shown generation dependent FQYs between 33% and 99% depending on the aggregation state (Figure 1d).12 Combined with the changes observed in the absorption spectra, these results underline the assumption that in case of the sulfated PBIs no aggregation is taking place and therefore to the best of our knowledge the [G1]-PBI-S 3a represents the smallest completely non-aggregated water-soluble PBI reported so far. Hence summing up, the following effects of sulfation were evidenced: it decreased the synthetic efforts necessary to obtain completely monomerized PBIs in water from [G4] in case for the uncharged PBIs to [G1] and (ii) due to the high pKa-value of the sulfate end groups in contrast to weakly acidic end groups like carboxylates the same amount of end groups on the dendrons lead to a better individualization.

Biological evaluation L-selectin binding efficacy L-selectin binding of polysulfates is one of the major pathways of their mode of operation as antiinflammatory agent and can be taken as a strong indication therefore. Here the binding affinities of the prepared PBI-sulfates 3a-3d were determined using a competitive SPR assay which allows determination of IC50 values over a large concentration range (pM-mM).31 In brief, L-selectin coated gold nanoparticles were passed over the ligand immobilized on the sensor chip and the resulting resonance value was set to 100% binding. A preincubation step of functionalized particles and inhibitors that bind to L-selectin reduced the binding signal dose-dependent. The results are shown in Figure 2.



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Figure 2. In vitro L-selectin binding studies of sulfated, dendronized PBIs ([GX]-PBI-S 3a-3d) in comparison with a dendritic polyglycerol sulfate (dPGS-12kDa), a perfect polyglycerol dendrimer sulfate ([G4]-dPGS) and heparin. (The values for the latter three compounds were taken from ref. [32])

A clear generation dependent trend of the IC50 values was observed which spans over two orders of magnitude. The lowest generation sulfated [G1]-PBI 3a showed the highest IC50 value with 1350 nM whilst the [G4]-derivative 3d showed the lowest one with 10 nM. This value is comparable with IC50 values which have been reported for dPGS with a comparable amount of sulfate groups and molecular weight (see dPGS-12kDa in Figure 2).32 However upon comparison with the reported IC50 values for a sulfated [G4]-PG-dendrimer with the same amount of sulfate groups and comparable molecular weight, which just lacks the PBI core, this value is lower by a factor of 30 (see [G4]-dPGS in Figure 2).32 This implies that the PBI core itself has a significant effect on the binding affinities. A possible explanation might be that the PBI core serves as spacer for the two attached dendrons so that the overall structure becomes more flexible and ellipsoidal in shape in comparison with the completely globular shaped perfect [G4]-dendrimer, which in the end allows more sulfate groups to participate in the multivalent binding mode. This would also be in line with the explanation for the extreme low binding constant of dPGS.32 Another explanation could be additional π-π-interactions from the PBI core with accessible aromatic residues at the L-selectin ligand binding interface leading to higher affinity. However more in depth studies have to be performed to clarify this. Blood coagulation assay


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Figure 3. Effect of sulfated PBIs ([GX]-PBI-S, 3a-d) on blood coagulation time (PTT pathway) at different inhibitor concentrations in comparison with reported values for sulfated PG-Dendrimer ([G4]dPGS),dendritic PG sulfate (dPGS-12kDa) and heparin (UHF). (The values for the latter three compounds were taken from ref. [32])

As the ability to influence blood coagulation time is an important aspect of polysulfated species in terms of applicability as anti-inflammatory agent it is important to also investigate anti-coagulant activity. In principle an ideal system for anti-inflammatory purposes should possess a low value to avoid possible side effects like bleeding. Therefore, the partial thromboplastin time (PTT) was determined which measures blood coagulation via the intrinsic pathway. Again a clear generation dependent trend was observed (Figure 3). While the lower generation substituted PBIs 3a-3b showed no significant change upon increasing the concentration from 50 nM up to 1000 nM, the PTT of the [G3]-derivative 3c slightly increased up to 175% and the [G4]-derivative 3d showed the strongest increase up to 490% which is comparable to the value reported for the sulfated PG-dendrimer of the same generation, indicating that the PBI core does not have a significant effect here.32 Although this seems to be a comparable high value, it is still substantially lower than for heparin, which is a commonly referred system for anti-inflammatory performance of polysulfated species. In fact, in direct comparison of the PTT values at a concentration of 100 nM, heparin showed a value of 433% and the [G4]-PBI sulfate 3d a value of 123%, which would in principle allow a threefold higher dosage of the PBI-sulfate.



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Complement Activity Assay The classical pathway (CP) of complement activation is triggered by binding of C1q to an antibodyantigen complex and finally results in the formation of a pore, the membrane attack complex (MAC), but also generates the anaphylatoxins C3a and C5a that direct neutrophils to the site of inflammation. Although complement activation ensures pathogen elimination, unbalanced activation of this pathway contributes to further tissue damage in several chronic diseases like arthritis, macular degeneration, and autoimmune disorders. Therefore, an intermittent therapy that dampens the complement driven inflammatory reaction would allow for self-healing. Within the investigated concentration range from 50 to 1000 nM major differences were observed upon comparison of the sulfated PBIs 3a-3d (Figure 4). While the [G1] derivative 3a showed no impact on the CP activity at all, a slight decrease was observed for the [G2] derivative 3b with increasing concentration. This trend became more apparent in the case of [G3] and [G4] derivatives 3c and 3d, whereas the [G4] derivative 3d showed the highest decrease down to 43% at a 1000 nM concentration. Similar as in the PTT-studies this value is comparable to the sulfated perfect [G4]dendrimer (42%)32, indicating that the CP activity is mainly affected by the PG-sulfate shell, rather than the core. In comparison to heparin (71.9%) the higher anti-complement activity by a factor of ~1.7 reduces the essential concentration to obtain the same effect.

Figure 4. Concentration dependence of the inhibitory effect of sulfated PBIs ([GX]-PBI-S, 3a-d) on the complement activity (determined via quantification of the membrane attack complex C5b-9) in


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comparison with reported values for sulfated PG-dendrimer ([G4]-dPGS), dendritic PG sulfate (dPGS12kDa) and heparin (UHF).

Cellular uptake studies To get a first impression of the interaction of sulfated PBIs in a biological system we investigated the cellular uptake of the sulfated PBIs dyes into A549 cells in live cell imaging experiments via confocal laser scanning microscopy (CLSM) and compared it with the uptake of fluorescently labeled dPGS12kDa. However as only the [G3] and the [G4] derivatives 3c and 3d showed promising antiinflammatory properties we did not investigate the lower generations. In all three cases nearly no uptake was determined after 4 h of incubation with a 1 μM solution of the corresponding compounds (Figure S4, Supporting Information). After 24h a clear uptake in small vesicular structures throughout the cells was observed (Figure 5). The time frame of cellular uptake in combination with the localization strongly indicate an active uptake mechanism, most probably via endocytosis. Moreover, the identical uptake pattern of the sulfated PBIs and the sulfated hyperbranched PG also imply that neither the amount of sulfate groups nor the PBI core do play a significant role on cellular uptake pathways. Furthermore for both PBI derivatives no changes in morphology or granularity of the cells were observed which indicates no cytotoxic effects for the applied concentrations. This was further proven via an MTS-assay analysis where up to 100 µM concentrations a cell viability of >90% was observed (Figure S5, Supporting Information).



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(a)

(b)

(c)

(d)

Figure 5. Live cell CLSM cellular uptake studies of [G4]-PBI-S 3d ((a) + (c)) and dPGS-12kDa ((b) + (d)) into A549 cells after 24 h incubation (red). Co-staining was either performed with only Hoechst 33342 (blue, nuclei) ((a) and (b)) or with Hoechst 33342 (blue, nuclei) and wheat germ agglutinin (WGA) conjugated with Alexa Fluor 594 (green, plasma membrane) ((b) + (d)). Both compounds show a strong uptake into vesicular cellular structures.

Influence of L-selectin binding on optical properties As it is known that the binding of fluorescent molecules to biomolecules like proteins can lead to a decrease in fluorescence intensities the behavior of the higher [G3] and [G4] derivatives 3c and 3d after target binding was also investigated. Therefor samples were prepared which contained 50 nM PBI and 1 µM L-selectin and the fluorescence intensities were compared to corresponding samples of pure dye. According to the determined IC50 values at this L-selectin concentration a full saturation of all PBIs can be assumed. As shown in Figure 6 in both cases a drop in fluorescence intensities of ~76 ACS Paragon Plus Environment

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% for 3c and 60 % for 3d was observed which correlates to a decrease in FQY from 0.95 to 0.34 and from 0.98 to 0.39 respectively. This is in line with the better shielding of the PBI core in case of the higher dendron generation leading to a decreased susceptibility towards quenching mechanisms like electron transfer processes or aggregation.

Figure 6. Normalized fluorescence spectra obtained from L-selectin binding experiments of [GX]-PBI-S (a) 3c and (b) 3d. The stronger decrease for 3c (76%) than for 3d (60%) indicates a better shielding of the PBI core by the bulkier dendron generation.

Conclusion A set of different generations of sulfated polyglycerol dendronized PBIs were successfully synthesized by a straight forwards approach. Major differences in their photophysical properties were observed upon comparison with their corresponding uncharged analogues mainly arising from the added electrostatic repulsion forces, which effectively inhibit dye aggregation even for the lowest [G1]dendron generation. Hence an increase in FQY from 33% for the uncharged up to 95% for the sulfated PBI was observed. This is a useful finding, as it shows that a completely disaggregation of purely imide substituted PBIs in water is possible with only limited effort in chemical synthesis. Furthermore, a biological evaluation of the dyes as potential anti-inflammatory agents was conducted. Therefore, the major important values (L-selectin binding, complement activity and anticoagulation time) were determined and compared with similar sulfated polyether systems. Overall an increase in performance with increasing dendron generation was observed, for the [G4] derivative, with an IC50 value of 10 nM for L-selectin binding and an inhibition of CP activity down to 43%, but a 5-fold increase in coagulation (PTT). An interesting observation was made upon comparison with the behavior of a comparable sulfated [G4]-PG-dendrimer without the PBI core with the same amount of sulfate groups and a comparable molecular weight. The L-selectin binding was increased by a factor of 30 due to the presence of the aromatic scaffold. Although the reasons for that are not fully understood and need further investigations, this finding demonstrates that the ACS Paragon Plus Environment


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incorporation of planar aromatic scaffolds is a versatile concept of increasing the performance of sulfated anti-inflammatory agents. Subsequent cellular uptake studies and cytotoxic evaluation with the higher generation dendronized PBIs showed uptake time-dependent intracellular accumulation after 24 h and no significant cytotoxic effects. A final examination of the impact of L-selectin binding on the optical properties revealed a decrease in FQY of up to 76%. However due to the extremely high FQYs of the unbound dyes, even in the bound state the remaining fluorescence is still in a range which can be easily detected via common fluorescence microscopes.

Experimental Part Materials All solvents and reagents were purchased from commercial sources and used as received without further purification, unless otherwise stated. The solvents for spectroscopic studies were of spectroscopic grade and used as received. The synthesis of the amine cored PG-dendrons and the cyanine labeled dPGS-12kDa were performed according to literature.

12, 32

Ultrafiltration was

performed in solvent resistant cells (Millipore, USA) with Ultracel regenerated cellulose membranes (MWCO = 1 kDa, Millipore, USA). Methods

1

H-NMR spectra were recorded on a Jeol EXC400 (400 MHz) at 25°C and calibrated against residual

solvent peaks as internal standard. NMR data was reported as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet), coupling constants(s) in Hertz (Hz) and integration. Multiplets (m) were reported over the range (ppm) at which they appear at the indicated field strength. UV/Vis spectra were measured on a Perkin Elmer Lambda 950 spectrophotometer using 1 cm Quartz glass cuvettes. For high concentration measurements 0,1 cm or 0,1 mm quartz glass cuvettes were used. The steady state fluorescence spectra were measured on a Jasco FP-6500 spectrofluorometer equipped with a Hamamatsu R928 Photomultiplier (corrected against photomultiplier and lamp intensity) using 1 cm quartz glass cuvettes. Relative fluorescence quantum yields

have

been

calculated

using =

with

N,N‘-bis(1-hexylheptyl)-3,4:9,10-

perylene(bisdicarboximide) in DCM ( =1.00) as reference.33 The optical density was kept below 0,05 at the absorption maximum. Elemental analysis was performed on a VARIO EL instrument (Elementar, Germany). Separation of polyanions by molecular weight and charge was performed in a 1.5% agarose gel in a Tris-acetate-EDTA buffer system at pH 8.0 (40 mM Tris base, 20 mM acetic acid,


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1 mM EDTA). Gel staining for anionic groups was conducted with 1% of the cationic dye Alcian blue 8GX (Sigma-Aldrich GmbH, Taufkirchen,Germany) in a 3% acetic acid solution for 30 min, destaining was performed in an aqueous solution of 20% ethanol and 10% acetic acid until contrast appearance.

Surface plasmon resonance measurements (Detection of L-Selectin Inhibition)

Competitive SPR measurements were performed on a BIAcore X instrument (GE Healthcare) at 25 °C. The general procedure based on a competitive selectin binding assay has been previously described.31 The gold nanoparticles (AuNP) (d=15 nm, Biotrend Chemikalien GmbH, Cologne, Germany) coated with L-selectin-IgG chimera (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany) basically resembled the leukocyte. The particles were then passed over the surface of an SPR sensor chip which presented common binding motifs for L-selectin. The surface of the sensor chip was modified with a fully synthetic, poly(acrylamide) (PAA) based L-selectin model ligand (SiaLex(20 mol%)-PAA-sTyr-(5 mol%)) on one track, and as a reference N-acetyllactosamine-PAA on a second parallel track (both compounds from Lectinity Holdings, Inc., Moscow, Russia). The selectin AuNP were dispersed in 20 mM HEPES buffer (pH 7.4, with 150 mM NaCl and 1 mM CaCl2) and a constant flowrate of 20 μL min-1 was applied. The resulting binding signal given in response units and corrected for the non-specific interactions detected from the second reference/ control track was set to 100% L-selectin binding to the model ligand on the chip surface. Defined concentrations of the investigated inhibitors as well as of control compounds (heparin) were preincubated with selectin AuNP and individually passed over the sensor chip surface. The reduced binding signals were recorded and calculated as X% binding of the untreated control L-selectin AuNP´s. The inhibitor concentration which reduced L-selectin mediated ligand binding by 50% was referred to as the IC50 value. Each concentration was measured at least as triplicates.

Complement Activation Assay

Normal pooled human serum from six donors was obtained by centrifugation (20 min, 4 °C, 3400 x g) of coagulated blood samples. The supernatant was stored in 100 µL aliquots at –20 °C until use and then thawed for one minute at 37 °C. Complement activation was tested for the classical pathway with an ELISA-based assay (Euro Diagnostica, Malmö, Sweden). Briefly, a multi-well plate setup with IgM coated wells was used for activation of the classical complement pathway. Therefore, 100 μL of freshly thawed and diluted (1:101) serum samples were incubated (60 min, 37 °C) with 2 μL test compound (final concentrations 0–1000 μM). The formation of the specific complement membrane attack complex (MAC) with components C5b-9 was detected with an alkaline phosphatase (AP)-



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labeled antibody and substrate para-nitrophenylphosphate (PNPP). Absorbance at 405 nm was recorded with a SpectraMax 340PC (Molecular Devices, Biberach an der Riss, Germany). The stated values for any concentration are the mean of three independent measurements.

Blood coagulation assay

Human blood was collected from at least four individual donors. Citrated plasma was obtained by centrifugation (15 min, 25 °C, 2400 x g) of blood-filled Vacutainer citrate tubes (BD, Heidelberg, Germany). 5 mL aliquots were stored at –20 °C. Before use, plasma was thawed for 6 min at 37 °C, mixed gently and reequilibrated for 15 min to room temperature (r.t.). The clotting assay was performed for partial thromboplastin time (PTT) at individual concentrations in duplicates using an Amelung coagulometer (Type 410A4MD, Lemgo, Germany). The measurements refer to the clotting time [s] of the untreated control (PBS) which was set to 100%. To determine the PTT, 100 μL plasma and 100 μL Actin FS (Siemens Healthcare, Erlangen, Germany) were mixed and incubated (3 min, 37 °C) with 4 μL test compound (final concentrations 0 – 1000 μM). The reaction was started by the addition of 100 μL of pre-warmed (37 °C) clotting activator CaCl2.

Cellular uptake studies

Confocal images of the equatorial plane of A549 cells cultured in DMEM - Dulbecco’s Modified Eagle Medium (Gibco, Life Technologies GmbH, Darmstadt, Germany) supplemented with 10% fetal bovine serum (BioChrom KG, Berlin, Germany), 100 U/mL penicillin-streptomycin (Gibco, Life Technologies GmbH, Darmstadt, Germany) were taken with an inverted confocal laser scanning microscope Leica DMI6000CSB SP8 (Leica, Wetzlar, Germany) with a 63x/1.4 HC PL APO CS2 oil immersion objective at 37 °C using the manufacture provided LAS X software. Therefore A549 cells (10.000 cells/ml DMEM, w/o phenol red) were seeded in 2 ibidi µ-Slides 8 Well (270 µl/well) and incubated (37 °C, 5% CO2) overnight. After 24h 30 µl of a 10 µM solution of the corresponding compound in 1x DPBS Dulbecco's Phosphate-Buffered Saline (Gibco, Life Technologies GmbH, Darmstadt, Germany) was added into two wells per slide resulting in a final concentration of 1 µM of the test substance. After additional 4 h and 24 h respectively every slide was incubated with either only with Hoechst 33342 (Life Technologies GmbH, Darmstadt, Germany) or with Hoechst and Alexa Fluor 594 wheat germ agglutinin (Life Technologies GmbH, Darmstadt, Germany) for additional 30 minutes. Afterwards excess dye was removed via two washing steps with DMEM medium prior to investigation. Sulfated PBIs 3c and 3d were excited at 488 nm using an argon gas-laser and emission was recorded with a hybrid detector in the range between 552 – 600 nm. DAPI was excited at 405 nm using a UV diode


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laser and detected in the range between 415-500 nm. Alexa Fluor 594 wheat germ agglutinin was excited at 561 nm using a diode laser and detected in the range between 571-644 nm. Cyanine labeled dPGS-12kDa was excited at 633 nm using a HeNe laser and detected in the range between 648-778 nm. MTS-assay Cytotoxicity of the was evaluated on A549 cells by the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) from Promega (Mannheim Germany) according to the manufacturer’s instructions. In short, A549 cells were seeded in a 96-well plate (4000 cells/well in 90 µl) and cultured over night at 37 °C before adding the sample substances (10 µl) in serial dilutions (100, 10, 1, 0.1, 0.01 and 0.001 µM). The surfactant SDS (0.01%) and non-treated cells served as controls. For background subtraction, also wells containing no cells but only sample were used. Cells were incubated for another day at 37 °C before the MTS solution (20 µl) was added. After 2:30 hours of incubation absorbance was measured at a measurement wavelength of 490 nm and a reference wavelength of 630 nm with a Tecan plate reader (Infinite pro200, TECAN-reader Tecan Group Ltd., Männedorf, Switzerland). Measurements were done in triplicates and repeated twice. The cell viability was calculated by setting the non-treated control to 100% and the non-cell control to 0% after subtracting the background using Microsoft Excel 2013 and GraphPad Prism (5.01). General synthetic protocol for protected PBIs 1a-1d 200 mg [GX]-NH2 (2.2 eq), the corresponding amount of perylene-3,4,9,10-tetracarboxylic dianhydride (1 eq.) and 100 mg Imidazole were added into a 10 ml Schlenkflask which was evaporated to remove residual traces of oxygen and charged with argon. Afterwards the reaction mixture was heated at 140 °C for 4h. After cooling to rt the solid mixture was dissolved in DCM, washed two times with H2O and dried over MgSO4. After evaporation of the solvent at the rotary evaporator the remaining crude product was purified via column chromatography on silica (DCM:MeOH, gradient from 99.5:0.5 up to 98.5:1.5). The products were obtained either in form of a dark red solid (1a, 258 mg, yield 87%) or as red honey like liquids (1b, 222 mg, yield 93%; 1c, 197 mg, yield 92%; 1d, 91 mg, yield 45%). General synthetic protocol for deprotected PBIs 2a-2d 50 mg of the corresponding PBI 1a-1d were dissolved in 2 ml DMSO and 1 ml H2O was added. Afterwards a drop of TFA was added to the solution and the mixture was stirred for 16 h at rt. The remaining mixture was diluted in 50 ml H2O and purified via ultrafiltration against water. After evaporation of the remaining water at the rotary evaporator the desired products 2a-2d were ACS Paragon Plus Environment


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obtained (2a, 37 mg, yield 88%; 2b, 38 mg, yield 92%; 2c, 36 mg, yield 90%, 2d, 38 mg, yield 95%). The analytical data corresponded to those published. 12 General synthetic protocol for sulfated PBIs 3a-3d 30 mg of the corresponding, thoroughly dried, hydroxyl terminated PBI 2a-2d were dissolved in 3 ml dry DMF in a 10 ml Schlenktube under argon atmosphere. Afterwards SO3/pyridine complex was added to the solution (2.5 eq. per OH-group) and the mixture heated to 50 °C and stirred for 16 h. After cooling to rt H2O (5ml) was carefully added, the mixture further diluted by adding it into a beaker with 100 ml H2O, and neutralized with saturated Na2CO3-solution to pH = 7.2. Further workup was conducted via ultrafiltration against a half-saturated aqueous NaCl-solution and three times against pure water. After evaporation of the remaining H2O at the rotary evaporator and subsequent freeze drying the products were obtained as fluffy red to orange powders (3a, 52 mg, yield 96%; 3b, 59 mg, yield 92%; 3c, 65 mg, yield 97%, 3d, 65 mg, yield 94%). 1a [G1]-PBI 1

H-NMR (CD2Cl2, 400 MHz): δ (ppm) = 8.51 (br s, 4H), 8.42 (m, 4H), 5.63 (m, 2H), 4.15 (m, 8H), 3.91

(m, 8H), 3.64-3.42 (m, 12H), 1.27 (br s, 6H), 1.25 (br s, 6H), 1.21 (br s, 12H). UV/Vis (CD2Cl2): λmax (Erel) = 525 (1.00), 489 (0.60), 457 (0.22) nm. Fluorescence (CD2Cl2): λmax (Erel) = 538 (1.00), 579 (0.59), 626 (0.14) nm. Elemental analysis calcd. for C54H62N2O16 for C, C 65.18, H 6.28, N 2.82 found C 64.02, H 6.37, N 2.77. 1b [G2]-PBI 1

H-NMR (Acetone-D6, 400 MHz): δ (ppm) = 8.44 (br s, 8H), 5.67 (m, 2H), 4.38-3.28 (m, 68H), 1.34-1.14

(m, 48H). UV/Vis (CD2Cl2): λmax (Erel) = 525 (1.00), 489 (0.60), 457 (0.22) nm. Fluorescence (CD2Cl2): λmax (Erel) = 538 (1.00), 579 (0.59), 626 (0.14) nm. Elemental analysis calcd. for C90H126N2O32 C 61.84, H 7.27, N 1.60 found C 60.12, H 7.12, N 1.50. 1c [G3]-PBI 1

H-NMR (CD2Cl2, 400 MHz): δ (ppm) = 8.68 (br s, 8H), 5.55 (m, 2H), 4.24-3.86 (m, 40H), 3.69-3.26 (m,

108H), 1.37-1.23 (m, 96H). UV/Vis (CD2Cl2): λmax (Erel) = 525 (1.00), 489 (0.60), 457 (0.22) nm. Fluorescence (CD2Cl2): λmax (Erel) = 538 (1.00), 579 (0.59), 626 (0.14) nm. Elemental analysis calcd. for C162H254N2O64 C 59.80, H 7.87, N 0.86 found C 58.11, H 7.74, N 0.83. 1d [G4]-PBI


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1

H-NMR (CD2Cl2, 400 MHz): δ (ppm) = 8.88 (br s, 8H), 5.60 (m, 2H), 4.24-3.87 (m, 64H), 3.70-3.27 (m,

244H), 1.38-1.28 (m, 192H). UV/Vis (CD2Cl2): λmax (Erel) = 525 (1.00), 489 (0.60), 457 (0.22) nm. Fluorescence (CD2Cl2): λmax (Erel) = 538 (1.00), 579 (0.59), 626 (0.14) nm. Elemental analysis calcd. for C306H510N2O128 C 58.66, H 8.21, N 0.45 found C 57.74, H 8.14, N 0.44. 3a [G1]-PBI-S 1

H-NMR (D2O, 400 MHz): δ (ppm) = 8.88 (br s, 4H), 8.70 (br s, 4H), 5.72 (m, 2H), 4.61 (br s, 4H), 4.33

(br s, 4H), 4.23-3.64 (m, 16H). UV/Vis (H2O): λmax (Erel) = 535 (1.00), 498 (0.63), 468 (0.26) nm. Fluorescence (H2O): λmax (Erel) = 552 (1.00), 596 (0.55) nm. Elemental analysis calcd. for C42H38N2Na8O40S8 C 30.55, H 2..32, N 1.70 S 15.53 found C 31.42, H 2.57, N 1.78, S 15.02. 3b [G2]-PBI-S 1

H-NMR (D2O, 400 MHz): δ (ppm) = 8.92 (br s, 4H), 8.73 (br s, 4H), 5.68 (br s, 2H), 4.62-3.40 (m, 68H).

UV/Vis (H2O): λmax (Erel) = 536 (1.00), 499 (0.66), 470 (0.29) nm. Fluorescence (H2O): λmax (Erel) = 552 (1.00), 597 (0.55) nm. Elemental analysis calcd. for C66H78N2Na16O80S16, C 25.91, H 2.57, N 0.92, S 16.76 found C 27.88, H 2.90, N 0.98, S15.72. 3c [G3]-PBI-S 1

H-NMR (D2O, 400 MHz): δ (ppm) = 9.03 (br s, 4H), 8.85 (br s, 2H), 8.79 (br s, 2H), 5.62 (br s, 2H), 4.69

(br s, 8H), 4.56 (br s, 8H), 4.38-4.02 (m, 40H), 3.78-3.42 (m, 92H). UV/Vis (H2O): λmax (Erel) = 536 (1.00), 498 (0.62), 468 (0.24) nm. Fluorescence (H2O): λmax (Erel) = 552 (1.00), 596 (0.55) nm. Elemental analysis calcd. for C114H158N2Na32O160S32 C 23.29, H 2.71, N 0.48, S 17.46 found C 24.08, H 2.97, N 0.51, S 16.91. 3d [G4]-PBI-S 1

H-NMR (D2O, 400 MHz): δ (ppm) = 9.08 (br s, 4H), 8.90 (br s, 2H), 8.82 (br s, 2H), 5.59 (br s, 2H), 4.69

(br s, 16H), 4.56 (br s, 16H), 4.36-4.04 (m, 80H), 3.84-2.26 (m, 196H). UV/Vis (H2O): λmax (Erel) = 534 (1.00), 496 (0.63), 466 (0.26) nm. Fluorescence (H2O): λmax (Erel) = 549 (1.00), 591 (0.55) nm. Elemental analysis calcd. for C210H318N2Na64O320S64 C 21.91, H 2.78, N 0.24, S 17.82 found C 21.98, H 2.81, N 0.25, S 17.72.

Acknowledgements The authors thank the Helmholtz Association for funding of this work through the HelmholtzPortfolio Topic “Technologie und Medizin – Multimodale Bildgebung zur Aufklärung des In-vivoACS Paragon Plus Environment


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Verhaltens von polymeren Biomaterialien” and also acknowledge support from the Deutsche Forschungsgemeinschaft (SFB 765) and Paul Hillmann for his assistance in the biological experiments.

Supporting Information Additional information about chemical structures, gel electrophoresis, concentration dependent UVVis behavior, cellular uptake and cytotoxicity is available in the supporting information.

(1) (2) (3) (4)

(5)

(6) (7) (8) (9)

(10)

(11) (12) (13) (14)

(15)

Debije, M. G., and Verbunt, P. P. C. (2012) Thirty Years of Luminescent Solar Concentrator Research: Solar Energy for the Built Environment. Adv. Energy Mater. 2, 12-35. Wurthner, F. (2004) Perylene bisimide dyes as versatile building blocks for functional supramolecular architectures. Chem Commun., 1564-79. Zhan, X., Facchetti, A., Barlow, S., Marks, T. J., Ratner, M. A., Wasielewski, M. R., and Marder, S. R. (2011) Rylene and related diimides for organic electronics. Adv. Mater. 23, 268-84. Peneva, K., Mihov, G., Herrmann, A., Zarrabi, N., Borsch, M., Duncan, T. M., and Mullen, K. (2008) Exploiting the nitrilotriacetic acid moiety for biolabeling with ultrastable perylene dyes. J. Am. Chem. Soc. 130, 5398-9. Peneva, K., Mihov, G., Nolde, F., Rocha, S., Hotta, J., Braeckmans, K., Hofkens, J., Uji-i, H., Herrmann, A., and Mullen, K. (2008) Water-soluble monofunctional perylene and terrylene dyes: powerful labels for single-enzyme tracking. Angew. Chem. Int. Ed. Engl. 47, 3372-5. Yin, M., Shen, J., Pflugfelder, G. O., and Mullen, K. (2008) A fluorescent core-shell dendritic macromolecule specifically stains the extracellular matrix. J. Am. Chem. Soc. 130, 7806-7. Kohl, C., Weil, T., Qu, J., and Mullen, K. (2004) Towards highly fluorescent and water-soluble perylene dyes. Chem. Eur. J. 10, 5297-310. Qu, J., Kohl, C., Pottek, M., and Mullen, K. (2004) Ionic perylenetetracarboxdiimides: highly fluorescent and water-soluble dyes for biolabeling. Angew. Chem. Int. Ed. Engl. 43, 1528-31. Ford, W. E. (1987) Photochemistry of 3,4,9,10-perylenetetracarboxylic dianhydride dyes: visible absorption and fluorescence of the di(glycyl)imide derivative monomer and dimer in basic aqueous solutions. J. Photochem. 37, 189-204. Huang, C., Barlow, S., and Marder, S. R. (2011) Perylene-3,4,9,10-tetracarboxylic acid diimides: synthesis, physical properties, and use in organic electronics. J. Org. Chem. 76, 2386-407. Gao, B., Li, H., Liu, H., Zhang, L., Bai, Q., and Ba, X. (2011) Water-soluble and fluorescent dendritic perylene bisimides for live-cell imaging. Chem. Commun. 47, 3894-6. Heek, T., Fasting, C., Rest, C., Zhang, X., Wurthner, F., and Haag, R. (2010) Highly fluorescent water-soluble polyglycerol-dendronized perylene bisimide dyes. Chem. Commun.46, 1884-6. Heek, T., Wurthner, F., and Haag, R. (2013) Synthesis and optical properties of water-soluble polyglycerol-dendronized rylene bisimide dyes. Chem. Eur. J. 19, 10911-21. Schmidt, C. D., Böttcher, C., and Hirsch, A. (2007) Synthesis and Aggregation Properties of Water-Soluble Newkome-Dendronized Perylenetetracarboxdiimides. Eur. J. Org. Chem., 5497-5505. Yang, S. K., Shi, X., Park, S, Doganay, S., Ha, T. and Zimmerman, S. (2011) Monovalent, Clickable, Uncharged, Water-Soluble Perylenediimide-Cored Dendrimers for Target-Specific Fluorescent Biolabeling, J. Am. Chem. Soc., 133, 9964-9967.


Page 20 of 22

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(26)

(27)

(28) (29)

(30)

(31)

(32)

Fasting, C., Schalley, C. A., Weber, M., Seitz, O., Hecht, S., Koksch, B., Dernedde, J., Graf, C., Knapp, E. W., and Haag, R. (2012) Multivalency as a chemical organization and action principle. Angew. Chem. Int. Ed. Engl. 51, 10472-98. Mintzer, M. A., Dane, E. L., O'Toole, G. A., and Grinstaff, M. W. (2012) Exploiting dendrimer multivalency to combat emerging and re-emerging infectious diseases. Mol. Pharm. 9, 34254. Wang, K. R., An, H. W., Rong, R. X., Cao, Z. R., and Li, X. L. (2014) Synthesis of biocompatible glycodendrimer based on fluorescent perylene bisimides and its bioimaging. Macromol. Rapid Commun. 35, 727-34. Wang, K.-R., An, H.-W., Qian, F., Wang, Y.-Q., Zhang, J.-C., and Li, X.-L. (2013) Synthesis, optical properties and binding interactions of a multivalent glycocluster based on a fluorescent perylene bisimide derivative. RSC Advances 3, 23190. Jiang, L., Ding, L., He, B., Shen, J., Xu, Z., Yin, M., and Zhang, X. (2014) Systemic gene silencing in plants triggered by fluorescent nanoparticle-delivered double-stranded RNA. Nanoscale 6, 9965-9. Xu, Z., He, B., Wei, W., Liu, K., Yin, M., Yang, W., and Shen, J. (2014) Highly water-soluble perylenediimide-cored poly(amido amine) vector for efficient gene transfection. J. Mater. Chem. B 2, 3079. Lamanna, G., Russier, J., Dumortier, H., and Bianco, A. (2012) Enhancement of antiinflammatory drug activity by multivalent adamantane-based dendrons. Biomaterials 33, 5610-7. Toppila, S., Lauronen, J., Mattila, P., Turunen, J. P., Penttila, L., Paavonen, T., Renkonen, O., and Renkonen, R. (1997) L-selectin ligands in rat high endothelium: multivalent sialyl Lewis x glycans are high-affinity inhibitors of lymphocyte adhesion. Eur. J. Immunol. 27, 1360-5. Weinhart, M., Groger, 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. Biffi, S., Dal Monego, S., Dullin, C., Garrovo, C., Bosnjak, B., Licha, K., Welker, P., Epstein, M. M., and Alves, F. (2013) Dendritic polyglycerolsulfate near infrared fluorescent (NIRF) dye conjugate for non-invasively monitoring of inflammation in an allergic asthma mouse model. PLoS One 8, e57150. Pant, K., Groger, D., Bergmann, R., Pietzsch, J., Steinbach, J., Graham, B., Spiccia, L., Berthon, F., Czarny, B., Devel, L. et al (2015) Synthesis and biodistribution studies of (3)h- and (64)culabeled dendritic polyglycerol and dendritic polyglycerol sulfate. Bioconjugate Chem. 26, 90618. Vonnemann, J., Beziere, N., Bottcher, C., Riese, S. B., Kuehne, C., Dernedde, J., Licha, K., von Schacky, C., Kosanke, Y., Kimm, M. et al (2014) Polyglycerolsulfate functionalized gold nanorods as optoacoustic signal nanoamplifiers for in vivo bioimaging of rheumatoid arthritis. Theranostics 4, 629-41. Turk, 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. Backes, C., Schunk, T., Hauke, F., and Hirsch, A. (2011) Counterion effect on the aggregation of anionic perylene dyes and the influence on carbon nanotube dispersion efficiencies. J. Mater. Chem. 21, 3554. Rehm, S., Stepanenko, V., Zhang, X., Rehm, T. H., and Wurthner, F. (2010) Sperminefunctionalized perylene bisimide dyes--highly fluorescent bola-amphiphiles in water. Chem. Eur. J. 16, 3372-82. Enders, S., Bernhard, G., Zakrzewicz, A., and Tauber, R. (2007) Inhibition of L-selectin binding by polyacrylamide-based conjugates under defined flow conditions. Biochim. Biophys. Acta 1770, 1441-9. Paulus, F., Schulze, R., Steinhilber, D., Zieringer, M., Steinke, I., Welker, P., Licha, K., Wedepohl, S., Dernedde, J., and Haag, R. (2014) The effect of polyglycerol sulfate branching on inflammatory processes. Macromol. Biosci. 14, 643-54. ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(33)

Langhals, H., Karolin, J., and Johansson, L. B. Å. (1998) Spectroscopic properties of new and convenient standards for measuring fluorescence quantum yields. J. Chem. Soc Farad. T. 94, 2919-2922.


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