Induction of EAAD and treatment schedule.

<p>Schematic depiction of the experimental protocol used for the induction of EAAD, the application of treatments and the optical imaging performed.</p

Time course of NIRFlabeled anti-Siglec-F distribution in the lung.

<p><i>In vivo</i> lung scans of EAAD, control as well as dexamethasone and beta-escin treated animals before (prescan) and at 6 h, 24 h, 48 h and 72 h after antibody administration. Fluorescence intensity distribution is displayed in normalized counts (NC). In contrast to control mice (A, lower panel, n = 6), OVA-immunized mice have a marked accumulation of anti-SiglecF-750 within the lungs from 24 h, which decreases at 72 h (A, upper panel, n = 8). Anti-SiglecF-680 also reveals significant differences between EAAD (B, upper panel, n = 5) and control (B, lower panel, n = 4) fluorescence intensities derived from the lung. EAAD mice treated with either dexamethasone (C, upper panel, n = 5) or beta-escin (C, lower panel, n = 5) have low intensities over the lung, similar to healthy control mice (A and B, lower panels) at all scan times.</p

Anti-SiglecF-680 binds to eosinophils and macrophages.

<p>Fluorescence microscopy of cryosections from lungs of EAAD mice injected with anti-SiglecF-680 (A), confirms the binding of anti-SiglecF-680 (in green) to eosinophils (EMBP-positive, arrows in merge) and more weakly to macrophages, which were counterstained with anti-CD68 (magenta, arrow heads in merge). Lungs from healthy controls injected with anti-SiglecF-680 (B) have a low number of Siglec-F positive cells, which are all CD68-positive and therefore most probably represent macrophages. Nuclei are stained blue with DAPI. Scale bars = 20 µm.</p

Quantification of <i>in vivo</i> imaging results.

<p>Box plot of average fluorescence intensities over the lung area for all groups at 48-labeled anti-Siglec-F antibody injection. Lung intensities of EAAD mice are significantly higher (represented by asterisk *) compared with control mice and treated mice at 48 h and 72 h after antibody application; A = EAAD, C = control, AD = EAAD, dexamethasone treated, AE = EAAD, beta-escin treated.</p

<i>Ex vivo</i> imaging results.

<p>(A) Representative images of fluorescence intensities of explanted lungs, livers, kidneys and spleens of EAAD (upper panel) and control mice (lower panel). (B) Bar graph of average fluorescence intensities of explanted lungs from mice injected with anti-SiglecF-680 (left panel) or anti-SiglecF-750 (right panel). <i>Ex vivo</i> lung scans demonstrate a significant difference between signal intensities of EAAD lungs and healthy lungs (A and B), while liver, spleen and kidneys show low intensities in both EAAD and control mice (A). NC = normalized counts.</p

Time course of NIRF-labeled anti-Siglec-F distribution in the body.

<p><i>In vivo</i> representative full body scans of EAAD (upper panel, n = 8) and control (middle panel, n = 6) mice injected with 12 µg of anti-SiglecF-750, as well as EAAD mice injected with 12 µg of Alexa 750-labeled anti-IgG2a isotype control antibody (lower panel, n = 5) at the given time points. Fluorescence intensity distribution is displayed in normalized counts (NC). Excess anti-SiglecF-750 antibody accumulates within the liver (red elipse) and is excreted via the bladder (black arrows) within the first few hours after antibody administration. 24 h –48 h after anti-SiglecF-750 injection, EAAD mice, in contrast to all control animals, accumulate the Siglec-F-antibody in their lungs (yellow triangle).</p

Expression pattern of Siglec-F.

<p>Immunohistochemistry and immunofluorescence Siglec-F staining of lung sections and BAL cytospins of mice with EAAD (A - D, upper panels) and controls (A – D, lower panels). (A)– (B) represent sections of cryofrozen lungs stained with anti-Siglec-F antibody. (C) Representative images of cytospins from BAL stained with anti-Siglec-F antibody and (D) of cytospins from BAL fluid co-stained with anti-SiglecF-680 and anti-CD68. In EAAD lungs, Siglec-F is highly expressed in eosinophils surrounding the blood vessels (b/v) and airways (a/w) (A, upper panel), while control lungs are almost free of Siglec-F staining (A, lower panel), indicating the lack of immune cell infiltration. Higher magnification of EAAD lung sections demonstrates Siglec-F staining on eosinophils (arrows, bilobed nucleus) and macrophages (arrow heads) (B, upper panel). In cytospins, eosinophils (bilobed nucleus) from EAAD animals (C and D upper panel, arrows) demonstrate strong positive Siglec-F staining, whereas macrophages from both, EAAD and control animals (C, arrow heads and D, positive CD68 staining) show a variety of Siglec-F expression levels. Scale bars in A: 2.5 mm; in B–D: 5 µm.</p

Additional file 1: Figure S1. of Next-Generation Theranostic Agents Based on Polyelectrolyte Microcapsules Encoded with Semiconductor Nanocrystals: Development and Functional Characterization

Schematic diagram of a theranostic agent based on polyelectrolyte microcapsules. Figure S2. Size distributions of calcium carbonate microparticles obtained at stirring rates of 250 (a), 500 (b), and 750-rpm (c). The stirring duration was 30 s in all cases. The size distribution diagrams are based on the measurements of individual microparticles (n = 350). The differences between samples a, b, and c are significant (p < < 0.05). Figure S3. Size distributions of calcium carbonate microparticles obtained at stirring durations of 15 (a), 30 (b), and 60 s (c). The stirring rate was 250 rpm in all cases. The size distribution diagrams are based on the measurements of individual microparticles (n = 350). The differences between samples a, b, and c are significant (p < < 0.05). Figure S4. Size distribution of the solubilized quantum dots as estimated by the volume occupied by the particles (a), the number of the particles (b), or the light scattering intensity (c). (DOCX 457 kb

Multifunctional Phosphate-Based Inorganic–Organic Hybrid Nanoparticles

Phosphate-based inorganic–organic hybrid nanoparticles (IOH-NPs) with the general composition [<i>M</i>]²⁺[<i>R</i>_{<i>function</i>}(O)­PO₃]^2– (<i>M</i> = ZrO, Mg₂O; <i>R</i> = functional organic group) show multipurpose and multifunctional properties. If [<i>R</i>_{<i>function</i>}(O)­PO₃]^2– is a fluorescent dye anion ([<i>R</i>_<i>dye</i>OPO₃]^2–), the IOH-NPs show blue, green, red, and near-infrared fluorescence. This is shown for [ZrO]²⁺[PUP]^2–, [ZrO]²⁺[MFP]^2–, [ZrO]²⁺[RRP]^2–, and [ZrO]²⁺[DUT]^2– (PUP = phenylumbelliferon phosphate, MFP = methylfluorescein phosphate, RRP = resorufin phosphate, DUT = Dyomics-647 uridine triphosphate). With pharmaceutical agents as functional anions ([<i>R</i>_<i>drug</i>OPO₃]^2–), drug transport and release of anti-inflammatory ([ZrO]²⁺[BMP]^2–) and antitumor agents ([ZrO]²⁺[FdUMP]^2–) with an up to 80% load of active drug is possible (BMP = betamethason phosphate, FdUMP = 5′-fluoro-2′-deoxyuridine 5′-monophosphate). A combination of fluorescent dye and drug anions is possible as well and shown for [ZrO]²⁺[BMP]^2–_0.996[DUT]^2–_0.004. Merging of functional anions, in general, results in [ZrO]²⁺([<i>R</i>_<i>drug</i>OPO₃]_1–<i>x</i>[<i>R</i>_<i>dye</i>OPO₃]_<i>x</i>)^2– nanoparticles and is highly relevant for theranostics. Amine-based functional anions in [MgO]²⁺[<i>R</i>_<i>amine</i>PO₃]^2– IOH-NPs, finally, show CO₂ sorption (up to 180 mg g^–1) and can be used for CO₂/N₂ separation (selectivity up to α = 23). This includes aminomethyl phosphonate [AMP]^2–, 1-aminoethyl phosphonate [1AEP]^2–, 2-aminoethyl phosphonate [2AEP]^2–, aminopropyl phosphonate [APP]^2–, and aminobutyl phosphonate [ABP]^2–. All [<i>M</i>]²⁺[<i>R</i>_{<i>function</i>}(O)­PO₃]^2– IOH-NPs are prepared via noncomplex synthesis in water, which facilitates practical handling and which is optimal for biomedical application. In sum, all IOH-NPs have very similar chemical compositions but can address a variety of different functions, including fluorescence, drug delivery, and CO₂ sorption

Quantification of in vivo imaging results of dPGS-NIRF and pure dye.

<p>Box plots of ratios of average fluorescence intensity over the lung area compared with the mean value of each control group respectively are reported for asthmatic and healthy mice. Mice treated with free dye 4 hrs post injection showed a slight increase in fluorescence signal in asthmatic mice (n = 5) when compared to healthy mice (n = 5; increase in average ∼11%, p-value = 0.047, panel A). Mice treated with dPGS-NIRF probe 4 hrs post injection (healthy n = 6, asthmatic n = 6) showed an increased fluorescence signal in the thorax in asthmatic mice (increase in average ∼44% with p-value = 0.004, panel B left side). At 24 hrs post injection fluorescence signals over the lung areas of healthy (n = 5) and asthmatic mice (n = 10) shown no difference (difference ∼8%, p-value = 0.162, panel B right side). Both control dye and dPGS-NIRF probe were injected 72 hrs after last aerosol challenge. Note, intensity ratios were used to compare probes with different brightness, therefore the box plots are depicted in the same scale.</p