Fucoidan

On the day of the experiment, THP-1 macrophages and hMDM were pretreated for 30 min at 37 °C with various concentrations of ligands, known to bind to the scavenger receptors class A, diluted in serum-free RPMI 1640 (Sigma-Aldrich, R8758) such as polyinosinic (poly(I), Sigma-Aldrich, P4154) and polyguanylic (poly(G), Sigma-Aldrich, P4404) acid, fucoidan (Sigma-Aldrich, F5631) and dextran sulfate (Sigma-Aldrich, D6001) or with their negative controls: polycytidylic acid (poly(C), Sigma-Aldrich, P4903), mannan (Sigma-Aldrich, M3640) and chondroitin sulfate (Sigma-Aldrich, C9819), respectively. Then, HFt-AF488 or positive control, AcLDL conjugated with Alexa Fluor 488 (AcLDL-AF488, Invitrogen, L23380) were added to cells to yield a final concentration of 100 or 5 µg/ml, respectively. Macrophages were incubated for another 30 min at 37 °C and then they were detached from wells using Accutase (Sigma-Aldrich, A6964), washed twice with PBS (Biowest, L0615) and finally resuspended in FACS buffer consisting of PBS (Biowest, L0615), 2% BCS (Hyclone, SH30072.03), 2 mM EDTA (Invitrogen, AM9260G) in the presence of DRAQ7 Dead Cell stain (BioLegend, 424001) for 10 min before flow cytometry analysis.

Materials. Bodipy™493/503 (4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene, Bodipy), LysoTracker™ Deep Red (LysoTracker), Cholera Toxin Subunit B - Alexa Fluor™647 Conjugate (CTB-A647) and CellMask™ Deep Red Plasma Membrane Stain (CellMask) were purchased from Thermo Fisher Scientific (Waltham, USA). 4′,6-diamidin-2-phenylindol (DAPI) was obtained from Dianova (Hamburg, Germany). Methyl-β-cyclodextrin (MβCD), genistein, wortmannin, fucoidan, polyinosinic acid (Poly I), and polycytidylic acid (Poly C) were obtained from Sigma Aldrich (München, Germany). Indocarbocyanine (ICC) was obtained from Mivenion (Berlin, Germany). A Caveolin-1-Alexa Fluor™488 (Cav-1-A488) conjugated antibody (reactivity: human, Clone# 7C8, Catalog# IC5736G) was obtained from R&D Systems (Minneapolis, Minnesota, USA). All other chemicals were of the highest purity available. 35 mm glass bottom cell culture dishes were purchased from Greiner Bio-One (Frickenhausen, Germany).
Core-multishell nanocarrier synthesis and Indocarbocyanine (ICC)-labeling. The ICC-labeled core-multishell nanocarrier (NC-ICC) was synthesized as described 41 (link). In short, hyperbranched polyglycerol amine (hPG-NH₂) with a molecular weight of 10 kDa and a degree of functionalization of amines of 70% was reacted with approx. 1 molecule of a NHS-ester of the ICC dye. Afterwards, the residual amines were reacted with the amphiphilic double shell, resulting in the empirical formula PG₁₀₀₀₀ (NH₂)_0.7(C₁₈mPEG_7.2)_0.98(ICC_0.02). The cargo Bodipy has a logP value of 3.50 ± 0.04 (octanol/water) 42 (link). Encapsulation by the nanocarriers was performed using a variation of the so-called film uptake method 41 (link). 1.2 mg of Bodipy was dissolved in ethanol, added into a vial and the solvent evaporated, leaving a film of the dye. The aqueous nanocarrier solution (1.5 mL, 5 g/L) was then added and the suspension stirred for 22 h at 1200 rpm and filtrated (regenerated cellulose, 450 nm pore size). The amount of encapsulated Bodipy was determined to 0.0027% (0.7 molecule Bodipy per molecule NC-ICC) by absorption spectroscopy after lyophilisation and redissolution of an aliquot in methanol using the extinction coefficient of Bodipy ε = 91000 M^-1cm^-1 at 493 nm (SI Figure S1).
Cell and tissue culture. Normal human keratinocytes and normal human dermal fibroblasts were isolated from juvenile foreskin of medically-indicated circumcisions of boys younger than 9 years old. Primary keratinocytes and fibroblasts (passage 3, pooled from three donors) were from therapeutically indicated circumcisions (ethical approval EA1/081/13, ethics vote from the Charité-Universitätsmedizin Berlin) after parents had signed the written informed consent. The SCC-25 cell line, passage 98-100, were obtained as a gift from Howard Green (Dana-Farber Cancer Institute, Boston, MA, USA) and were authenticated by single nucleotide polymorphism profiling (Multiplexion, Heidelberg, Germany). For all 2D live-cell FLIM experiments 2.5 x 10⁵ cells were seeded per compartment of glass bottom cell culture dishes and cultured in their respective medium for 2 days. For keratinocytes, Keratinocyte Growth Medium (KGM, Lonza, Köln, Germany) was used. Cell culture was performed according to standard operating procedures and referred to good cell culture practice.
SCC-25 were cultured in DMEM/F12, supplemented with 100 U/mL Penicillin, 100 µg/mL Streptomycin (Sigma Aldrich, München, Germany) and 2 mM L-Glutamine. Media and supplements were purchased from Sigma Aldrich, München, Germany; media were changed once on the 2^nd day.
3D skin tissue models were grown as described previously 43 (link), 44 (link).
2D and 3D uptake studies. Keratinocyte cells (2.5 x 10⁵ cells per compartment) were incubated for 15, 45, 90, 180, and 270 minutes at 37 °C and 5% CO₂ with NC-ICC/Bodipy at a concentration of 10 µg/mL in KGM. Directly before FLIM measurements (see below), cells were washed twice and KGM was exchanged with PBS (pH 7.4). Subsequently, the cells were stained with DAPI (28 µM) and CellMask (0.5 µg/mL) for 5-10 minutes. FLIM experiment series were performed four times. For each experiment series, and each time point two different images (usually full image and zoom) were recorded.
SCC25 cells were incubated in the respective growth medium (see above) for 15, 180, 270, 370 min. Cell uptake of NC-ICC was directly investigated by FLIM after two washing steps and medium exchange to PBS (pH 7.4).
For 3D skin model uptake studies, 30 µL/cm² of NC-ICC in PBS (pH 7.4) was applied onto the tissue surface for 6 and 22 h at 37°C, 5% CO₂. The incubated sample was placed top down (with the stratum corneum facing the bottom) into a 35 mm glass bottom cell culture dish and humidified by filter paper soaked with PBS before subjected to mpFLIM. For reference cryosections, the 3D skin models were snap frozen and sectioned into 7-µm slices (Leica CM 1510S, Wetzlar, Germany).
Fluorescence lifetime imaging microscopy. Fluorescence lifetime imaging microscopy (FLIM) was performed in a home-built setup 45 , 46 (link). The setup consists of an inverted microscope (IX71, Olympus, Shinjuku, Tokyo, Japan), a tunable ps-supercontinuum white light laser (SuperK Extreme EXU-3, NKT Photonics, Birkerød, Denmark), a ps-diode laser (BDL-405-SMN), a confocal scanning unit (DCS120), a hybrid PMT detector (HPM-100-40), and a time-correlated single photon counting (TCSPC) module (SPC160, all Becker & Hickl, Berlin, Germany). FLIM images were recorded by the SPCM software (Becker & Hickl, Germany) using a 60x objective (water, UPLSAPO60XW, Olympus, Japan) resulting in a total field of view with a side length of 300 µm. An acoustic-optical tunable filter (SELECT UV-VIS, NKT Photonics, Denmark) was used to select the individual fluorescence excitation wavelengths from the white light laser beam. The laser repetition rate was set to 19.5 MHz. Bodipy and Cav-1-A488 fluorescence were excited at 488 nm, NC-ICC fluorescence at 530 nm, and CellMask, CTB-A647 as well as LysoTracker fluorescence at 640 nm. DAPI fluorescence was exited at 405 nm by a ps-diode laser (BDL-405-SMN, Becker & Hickl, Germany) at a repetition rate of 20 MHz. Time-resolved fluorescence emission was spectrally selected by a band-pass filter (525/50 nm, Semrock, Rochester, USA) for Bodipy and Cav-1-A488, a combination of a long-pass filter (>545 nm, Chroma, Rockingham, USA) and a short-pass filter (<600 nm, Coherent, Santa Clara, USA) for CMS-ICC, a long-pass filter (>665 nm, Chroma, Rockingham, USA) for CellMask, LysoTracker, and CTB-A647 and a band-pass filter (452/45 nm, Semrock, USA) for DAPI. The TCSPC-module sorted the detected fluorescence photons into 1024 time channels with a channel width of 19.97 ps. The instrument response function of the system was less than 100 ps (FWHM). The acquisition time for Bodipy and NC-ICC was set to 300 s, for Cellmask and DAPI to at least 120 s. For live-cell applications, a temperature-controlled specimen holder was installed and adjusted to either 4°C or 37°C. Living cells were measured in glass bottom cell culture dishes (Greiner Bio-One, Germany).
Multiphoton fluorescence lifetime imaging microscopy. Multiphoton FLIM (mpFLIM) was conducted in a home-built setup 47 . A mode-locked pulsed femtosecond Ti:sapphire laser (Mira 900, Coherent, USA) is pumped by a diode-pumped solid state laser (Verdi V5, Coherent, USA) generating laser pulses shorter than 200 fs with a repetition rate of 76 MHz at a wavelength of 800 nm. An objective (60x water, UPSLAPO60XW, Olympus, Japan) focused and a scanning unit (DCS-120, Becker & Hickl, Germany) scanned the excitation laser beam over the sample placed on an inverted microscope (IX-73, Olympus, Japan). Fluorescence emission was separated from excitation by a dichroic mirror (H 643 LPXR superflat, AHF, Germany) and a short-pass filter (SP745 BrightLine HC, Semrock, USA). NC-ICC fluorescence was distinguished from fluorescence of other fluorescent species by a combination of a long-pass filter (>545 nm, Chroma, USA) and a short-pass filter (<600 nm, Coherent, USA) generating a spectral detection window of 545 to 600 nm. Fluorescence photons were collected in non-descanned detection mode by a hybrid detector (HPM-100-40, Becker & Hickl, Germany). The IRF of the system was below 120 ps (FWHM). Collected photons were sorted into 1024 time channels (width 9.7 ps) by a TCSPC module (SPC-160, Becker & Hickl, Germany). The same cluster-based FLIM analysis as for one-photon FLIM was applied.
Cluster-FLIM analysis. The temporal decay characteristics of multicomponent TCSPC data from a fluorescence decay matrix, i.e., the fluorescence decays in each pixel of an image, and the Poisson distributed signal noise restrict the extraction of the underlying fluorescence decay curves 35 (link). The underlying decay curves (FLSs) in the fluorescence decay matrix may originate from a multitude of fluorescing molecules and/or varying fluorescence decays of the same fluorophore depending on the local environment. The problem of determining the intrinsic unknown structure of the fluorescence decay data, when no information other than the recorded FLIM data is available, can be solved by grouping similar patterns (fluorescence decays) into clusters. The cluster can be discriminated from each other according to some similarity/dissimilarity measure used by a cluster algorithm, e.g., the Euclidian distance in k-means 48 . No a priori knowledge of patterns (i.e. FLSs) that belong to certain groups is necessary for this type of clustering. The raw FLIM data can be described as a set of patterns X' = [x'₁,…, x'_i,…, x'_N}, where x'_i represents the fluorescence decay histogram x'_i = (x'_i,1,…, x'_i,j,…, x'_i,b)^T constructed of b time bins in the single pixel i of the N pixels in a FLIM-image. The d dimensional feature space, with the individual features x_i,j, is generated and the Euclidian distance D_i,j for each cluster member x_i to a respective cluster center x_c is calculated by
and serves as a similarity/dissimilarity measure in the clustering. Using the distances D_i,j the fluorescence decay patterns of the individual pixels were partitioned into meaningful groups (i.e. clusters) by applying the k-means algorithm 48 . A validation of the method is shown in SI Figure S2. False-color images were generated by assigning a distinct color to all pixels containing a fluorescence decay trace that belonged to a certain cluster. The FLS of an identified cluster is generated through accumulation of all photons from image pixels belonging to this cluster. Subsequently, the FLSs could be fitted by deconvolution of a multi-exponential model function with a calculated IRF. After deconvolution of the fluorescence traces and the IRF, and taking the background counts into account, the time-dependent decay profile was fitted to a multi-exponential model function described by
with n the total number of decay components; α_i the amplitude and τ_i the fluorescence lifetime of the i-th component 49 (link)-52 . The mean (amplitude weighted) fluorescence lifetime τ_m,a was obtained from the following equation
and the component weighted mean fluorescence lifetime τ_m was calculated by
with β_i being the fractional amplitude of the i-th component with
A detailed account on the use of average fluorescence lifetime is given in 52 , 53 . FLIM data were analyzed with self-written routines in C++.
Analytical qualification of the Cluster-FLIM analysis tool was determined for sensitivity (hit rate) and specificity (correct rejection rate) in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines 54 :
Spatiotemporal development of the FLIM-Clusters in keratinocytes and kinetic model of NC-ICC internalization and transport. For analyzing the spatiotemporal development of the FLIM clusters in NHK cells we performed kinetic modelling using the relative cluster concentrations. The relative cluster concentrations were expressed as the fraction of the total NC-ICC intensity. The time-dependence of the concentration of the three FLIM-clusters (cyan (c), yellow (y), red (r)) in the plasma membrane (pm) and in the cytoplasm (cp) was fitted to a simple kinetic reaction model:
with [NC-R]_pm,c being NC-ICC bound to SR on the plasma membrane (cyan cluster). [NC-R]_pm,y describes NC-ICC bound to SR in lipid raft/caveolae within areas of the plasma membrane (yellow cluster). [NC-R-C]_cp,y is the fraction of NC-ICC bound to SR in caveolar vesicles in the cytoplasm (yellow cluster), while NC*_cp,r describes NC-ICC in lysosomal compartments (red cluster). We assume first order reactions and describe the kinetic model of NC-ICC internalization and transport by the following set of differential equations:
where τ₁ is the time constant of the transition of receptor bound NC-ICC in the plasma membrane to lipid raft/caveolae containing membrane areas. τ_-1 describes the time constant of the corresponding back transition. τ₂ is the time constant of the dissociation of NC-ICC loaded caveolar vesicles from the plasma membrane into the cytoplasm and τ₃ describes the time constant of the transport into lysosomes. The fitting procedure with the differential equations (Eq. 9-12) was conducted in Mathematica 11.0 (Wolfram Research, Champaign, IL, USA).
Concentration dependent NC-ICC uptake in monolayer experiments and displacement of NC-ICC from keratinocyte cell membranes by NC. To investigate the concentration dependence of the NC-ICC uptake, keratinocytes were incubated with NC-ICC for 4.5 h at concentrations ranging from 0 to 25 µM in KGM at 37°C. The uptake behavior was analyzed by the intracellular fluorescence intensities at the different NC-ICC concentrations. The observed non-linear uptake behavior was fitted by a Michaelis-Menten-like equation 55 (link):
with I_max being the saturation intensity, [NC-ICC] the concentration of NC-ICC, and K₅₀ the half-maximum uptake concentration.
To study NC-ICC displacement from the cell membrane by unlabeled NC, monolayer cultures were incubated with NC-ICC (0.5 µM) in KGM at 4 °C for 1 h. Afterwards, unlabeled NC was added in concentrations ranging from 10 to 10000 nM at 4 °C. The displacement of NC-ICC from the cell membrane was analyzed using the NC-ICC occupied cell membrane area. The decrease of occupied membrane areas with increasing NC concentrations was fitted by a modified Hill-function:
where A_max, A_min are the maximal and minimal area of NC-ICC membrane occupation, [NC] the NC concentration, K₅₀ the half-maximum binding constant (apparent binding affinity), and n the Hill-coefficient (cooperativity factor).
Inhibition studies of cellular uptake of NC-ICC towards primary keratinocytes. Uptake inhibition and receptor blocking experiments were performed by a 30 to 60-minute pre-incubation with the respective inhibitor and a subsequent co-incubation with NC-ICC/Bodipy without any washing or changing of KGM at 37°C. For ATP inhibition cells were incubated with azide (3 mg/mL). Cholesterol depletion experiments were performed by incubation with methyl-β-cyclodextrin (5 mg/mL). Phosphoinositide 3-kinase activity was inhibited by wortmannin (150 ng/mL). Clathrin-mediated endocytosis was inhibited by chlorpromazine (10 µg/mL). Caveolae-mediated cell uptake was blocked by genistein (27 µg/mL). Scavenger receptor (SR) binding was blocked by polyinosinic acid (50 µg/mL) or fucoidan (100 µg/mL). As a positive control, keratinocyte cells were incubated with polycytidylic acid, not a ligand of SR. Low temperature experiments were performed at 4°C. For investigating binding of unlabeled NC to SR of normal keratinocytes, cells were incubated at 4°C with NC at a concentration of 1000 µg/mL. Subsequently, cells were incubated for at least 180 minutes with NC-ICC/Bodipy at a concentration of 10 µg/ml in KGM.
Co-localization studies of NC-ICC with cholera toxin subunit B, caveolin-1 and LysoTracker. For co-localization with cholera toxin subunit B uptake pathways, normal keratinocytes were incubated for 15 min with NC-ICC/Bodipy (10 µg/mL) and CTB-A647 (5 µg/mL) at 37°C and 5 % CO₂ in KGM. For co-localization with caveolae, cells were incubated for 90 min with NC-ICC (10 µg/mL) and a Cav-1-A488 antibody at a 1:20 dilution. For lysosomal co-localization studies, cells were incubated for 600 min with NC-ICC/Bodipy (10 µg/mL) and during the last 45 minutes LysoTracker was co-incubated at a concentration of 50 nM. FLIM measurements were conducted without any washing or exchanging of KGM. Co-localization of NC-ICC and cholera toxin subunit B, Caveolin-1 and LysoTracker was quantified by Manders' co-localization coefficients M₁ and M₂, after an automatic threshold search as described in 56 (link). M₁ and M₂, representing the overlapping fraction of two spectral channels A and B with respect to the other, i.e., M₁ is the overlap of spectral channel A with spectral channel B and M₂ is the overlap of spectral channel B with spectral channel A. Manders' co-localization coefficients can take values between 0 (no co-localization) and 1 (co-localization). To obtain the coefficients we used the Coloc2 plugin integrated in the image-processing package FIJI 57 (link).

Four rat monoclonal antibodies (MAbs), designated BAM1 to BAM4, were derived subsequent to immunisation with a neoglycoprotein immunogen, prepared by coupling of fucoidan (Sigma-Aldrich F5631) to bovine serum albumin (BSA) by activation with 1-cyano-4-dimethylaminopyridium tetrafluroborate (CDAP) [36 (link)]. Two male Wistar rats were each injected with 250 μg fucoidan-BSA in complete Freund’s adjuvant administered subcutaneously on day 0, and the same was administered with incomplete Freund’s adjuvant on days 31, 59, 125 and 158. A pre-fusion boost of 100 μg fucoidan-BSA in 1 ml PBS was administered on day 215 prior to spleen removal. Hybridoma production and cloning procedures were performed as described [37 (link)].