Cocktail of lipophilic and hydrophilic chemotherapeutics in high-load core@shell nanocarriers to treat pancreatic tumours.

ITC/Toc@Gd2(FLP)3 core@shell nanocarriers with a chemotherapeutic cocktail of lipophilic irinotecan (ITC) as the particle core and hydrophilic fludarabine phosphate (FLP) in the particle shell are realized. They are prepared via a microemulsion approach with ITC dissolved in tocopherol (Toc) as droplet phase and stabilized by water-insoluble Gd2(FLP)3. The synthesis can be followed by zeta-potential analysis. X-ray powder diffraction, infrared spectroscopy, elemental analysis, thermogravimetry, and photometry show a drug load of 49 µg per mL ITC and 317 µg per mL FLP at a nanocarrier concentration of 1.5 mg mL-1. Size and structure are evidenced by electron microscopy, resulting in a total diameter of 45 ± 16 nm, an inner core of 40 ± 17 nm, and a shell of 3-8 nm. In vitro studies with different cancer cell lines (i.e., human melanoma/SK-Mel-28, cervical cancer/HeLa, mouse pancreatic cancer/Panc02 and KPC as well as human pancreatic cancer/Capan-1 cells) prove efficient nanocarrier uptake and promising cytostatic efficacy. Specifically for KPC cells, ITC/Toc@Gd2(FLP)3 nanocarriers show an increased efficacy, with half maximal inhibitory concentration (IC50: 4.2 µM) > 10 times lower than the free drugs (IC50: ITC: 47.7 µM, FLP: 143 µM). This points to the synergistic effect of the ITC/FLP drug cocktail in the nanocarriers and may result in a promising strategy to treat pancreatic ductal adenocarcinoma (PDAC).

High-Load Gemcitabine Inorganic-Organic Hybrid Nanoparticles as an Image-Guided Tumor-Selective Drug-Delivery System to Treat Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a devastating prognosis without effective treatment options. Thus, there is an urgent need for more effective and safe therapies. Here, inorganic-organic hybrid nanoparticles (GMP-IOH-NPs) are presented as a novel drug-delivery system for the selective delivery of extraordinarily high concentrations of gemcitabine monophosphate (GMP), not only to the primary tumor but also to metastatic sites. GMP-IOH-NPs have a composition of [ZrO]2+ [GMP]2 - with GMP as drug anion (76% of total IOH-NP mass). Multiscale fluorescence imaging confirms an efficient uptake in tumor cells, independent of the activity of the human-equilibrative-nucleoside transporter (hENT1), being responsible for gemcitabine (GEM) transport into cells and a key factor for GEM resistance. Delivering already phosphorylated GMP via GMP-IOH-NPs into tumor cells also allows the cellular resistance induced by the downregulation of deoxycytidine kinase to be overcome. GMP-IOH-NPs show high accumulation in tumor lesions and only minor liver trapping when given intraperitoneally. GMP-IOH-NPs result in a higher antitumor efficacy compared to free GEM, which is further enhanced applying cetuximab-functionalized GMP-CTX-IOH-NPs. By maximizing the therapeutic benefits with high drug load, tumor-specific delivery, minimizing undesired side effects, overcoming mechanisms of chemoresistance, and preventing systemic GEM inactivation, GMP-IOH-NPs are anticipated to have a high chance to significantly improve current PDAC-patient outcome.

Theranostic inorganic-organic hybrid nanoparticles with a cocktail of chemotherapeutic and cytostatic drugs.

Theranostic inorganic-organic hybrid nanoparticles (IOH-NPs) with a cocktail of chemotherapeutic and cytostatic drugs and a composition Gd23+[(PMX)0.5(EMP)0.5]32-, [Gd(OH)]2+[(PMX)0.74(AlPCS4)0.13]2-, or [Gd(OH)]2+[(PMX)0.70(TPPS4)0.15]2- (PMX: pemetrexed, EMP: estramustine phosphate, AlPCS4: aluminum(III) chlorido phthalocyanine tetrasulfonate, TPPS4: tetraphenylporphine sulfonate) are presented for the first time. These IOH-NPs are prepared in water (40-60 nm in size) and have a non-complex composition with outstanding drug loading (71-82% of total nanoparticle mass) of at least two chemotherapeutic or a mixture of cytostatic and photosensitizing agents. All IOH-NPs show red to deep-red emission (650-800 nm) to enable optical imaging. The superior performance of the IOH-NPs with a chemotherapeutic/cytostatic cocktail is validated based on cell-viability assays and angiogenesis studies with human umbilical vein endothelial cells (HUVEC). The synergistic anti-cancer effect of the IOH-NPs with a chemotherapeutic cocktail is shown in a murine breast-cancer cell line (pH8N8) and a human pancreatic cancer cell line (AsPC1), whereas the synergistic cytotoxic and phototoxic efficacy is verified in response to illumination of HeLa-GFP cancer cells, MTT assays with human colon cancer cells (HCT116), and normal human dermal fibroblasts (NHDF). HepG2 spheroids as 3D cell cultures prove the effective uptake of the IOH-NPs with high uniform distribution and the release of the chemotherapeutic drugs with the strong synergistic effect of the cocktail of drugs.

Crosstalk between tumor acidosis, p53 and extracellular matrix regulates pancreatic cancer aggressiveness.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy with minimal treatment options and a global rise in prevalence. PDAC is characterized by frequent driver mutations including KRAS and TP53 (p53), and a dense, acidic tumor microenvironment (TME). The relation between genotype and TME in PDAC development is unknown. Strikingly, when wild type (WT) Panc02 PDAC cells were adapted to growth in an acidic TME and returned to normal pH to mimic invasive cells escaping acidic regions, they displayed a strong increase of aggressive traits such as increased growth in 3-dimensional (3D) culture, adhesion-independent colony formation and invasive outgrowth. This pattern of acidosis-induced aggressiveness was observed in 3D spheroid culture as well as upon organotypic growth in matrigel, collagen-I and combination thereof, mimicking early and later stages of PDAC development. Acid-adaptation-induced gain of cancerous traits was further increased by p53 knockout (KO), but only in specific extracellular matrix (ECM) compositions. Akt- and Transforming growth factor-ß (TGFß) signaling, as well as expression of the Na+ /H+ exchanger NHE1, were increased by acid adaptation. Whereas Akt inhibition decreased spheroid growth regardless of treatment and genotype, stimulation with TGFßI increased growth of WT control spheroids, and inhibition of TGFß signaling tended to limit growth under acidic conditions only. Our results indicate that a complex crosstalk between tumor acidosis, ECM composition and genotype contributes to PDAC development. The findings may guide future strategies for acidosis-targeted therapies.

Monitoring nanoparticle dissolution via fluorescence-colour shift.

[La(OH)]2+[ICG]-2 and [La(OH)]2+2[PTC]4- inorganic-organic hybrid nanoparticles (IOH-NPs) with indocyanine green (ICG) and perylene-3,4,9,10-tetracarboxylate (PTC) as fluorescent dye anions are used for emission-based monitoring of the dissolution of nanoparticles. Whereas ICG shows a deep red emission in the solid [La(OH)]2+[ICG]-2 IOH-NPs, the emission of PTC in the solid [La(OH)]2+2[PTC]4- IOH-NPs is completely quenched due to π-stacking. After nanoparticle dissolution, the emission of freely dissolved ICG is weak, whereas freely dissolved PTC shows intense green emission. We report on the synthesis of IOH-NPs and nanoparticle characterization as well as on the fluorescence properties and how to avoid undesirable energy transfer between different fluorescent dyes. The emission shift from red (intact solid nanoparticles) to green (freely dissolved dye anions), indicating nanoparticle dissolution, is shown for aqueous systems and verified in vitro. Based on this first proof-of-the-concept, the IOH-NP marker system can be interesting to monitor nanoparticle dissolution in cells and tissues of small animals and to evaluate cell processes and/or drug-delivery strategies.

pH-Dependent fluorescence of [La(OH)2]+[ARS]- hybrid nanoparticles for intracellular pH-sensing.

Saline inorganic-organic hybrid nanoparticles (IOH-NPs) [La(OH)2]+[ARS]- (ARS: alizarin red S) are prepared in water as a new compound (particle size: 47 ± 7 nm, ARS load: 65 wt%). The IOH-NPs not only show a pH-dependent absorption colour but also a pH-dependent fluorescence with green emission at pH 5.0-9.0 and red emission at pH < 4.5. According to first in vitro studies, the pH-dependend fluorescence can be used to monitor nanoparticle internalization in cells as well as the respective intracellular pH.

Decreased PRC2 activity supports the survival of basal-like breast cancer cells to cytotoxic treatments.

Breast cancer (BC) is the most common cancer occurring in women but also rarely develops in men. Recent advances in early diagnosis and development of targeted therapies have greatly improved the survival rate of BC patients. However, the basal-like BC subtype (BLBC), largely overlapping with the triple-negative BC subtype (TNBC), lacks such drug targets and conventional cytotoxic chemotherapies often remain the only treatment option. Thus, the development of resistance to cytotoxic therapies has fatal consequences. To assess the involvement of epigenetic mechanisms and their therapeutic potential increasing cytotoxic drug efficiency, we combined high-throughput RNA- and ChIP-sequencing analyses in BLBC cells. Tumor cells surviving chemotherapy upregulated transcriptional programs of epithelial-to-mesenchymal transition (EMT) and stemness. To our surprise, the same cells showed a pronounced reduction of polycomb repressive complex 2 (PRC2) activity via downregulation of its subunits Ezh2, Suz12, Rbbp7 and Mtf2. Mechanistically, loss of PRC2 activity leads to the de-repression of a set of genes through an epigenetic switch from repressive H3K27me3 to activating H3K27ac mark at regulatory regions. We identified Nfatc1 as an upregulated gene upon loss of PRC2 activity and directly implicated in the transcriptional changes happening upon survival to chemotherapy. Blocking NFATc1 activation reduced epithelial-to-mesenchymal transition, aggressiveness, and therapy resistance of BLBC cells. Our data demonstrate a previously unknown function of PRC2 maintaining low Nfatc1 expression levels and thereby repressing aggressiveness and therapy resistance in BLBC.

Optical and Optoacoustic Imaging.

The spatiotemporal determination of molecular events and cells is important for understanding disease processes, especially in oncology, and thus for the development of novel treatments. Equally important is the knowledge of the biodistribution, localization, and targeted accumulation of novel therapies as well as monitoring of tumor growth and therapeutic response. Optical imaging provides an ideal versatile platform for imaging of all these problems and questions.

Therapeutic Fluorescent Hybrid Nanoparticles for Traceable Delivery of Glucocorticoids to Inflammatory Sites.

Treatment of inflammatory disorders with glucocorticoids (GCs) is often accompanied by severe adverse effects. Application of GCs via nanoparticles (NPs), especially those using simple formulations, could possibly improve their delivery to sites of inflammation and therefore their efficacy, minimising the required dose and thus reducing side effects. Here, we present the evaluation of NPs composed of GC betamethasone phosphate (BMP) and the fluorescent dye DY-647 (BMP-IOH-NPs) for improved treatment of inflammation with simultaneous in vivo monitoring of NP delivery. Methods: BMP-IOH-NP uptake by MH-S macrophages was analysed by fluorescence and electron microscopy. Lipopolysaccharide (LPS)-stimulated cells were treated for 48 h with BMP-IOH-NPs (1×10-5-1×10-9 M), BMP or dexamethasone (Dexa). Drug efficacy was assessed by measurement of interleukin 6. Mice with Zymosan-A-induced paw inflammation were intraperitoneally treated with BMP-IOH-NPs (10 mg/kg) and mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI) were treated intranasally with BMP-IOH-NPs, BMP or Dexa (each 2.5 mg/kg). Efficacy was assessed in vivo by paw volume measurements with µCT and ex vivo by measurement of paw weight for Zymosan-A-treated mice, or in the AAI model by in vivo x-ray-based lung function assessment and by cell counts in the bronchoalveolar lavage (BAL) fluid and histology. Delivery of BMP-IOH-NPs to the lungs of AAI mice was monitored by in vivo optical imaging and by fluorescence microscopy. Results: Uptake of BMP-IOH-NPs by MH-S cells was observed during the first 10 min of incubation, with the NP load increasing over time. The anti-inflammatory effect of BMP-IOH-NPs in vitro was dose dependent and higher than that of Dexa or free BMP, confirming efficient release of the drug. In vivo, Zymosan-A-induced paw inflammation was significantly reduced in mice treated with BMP-IOH-NPs. AAI mice that received BMP-IOH-NPs or Dexa but not BMP revealed significantly decreased eosinophil numbers in BALs and reduced immune cell infiltration in lungs. Correspondingly, lung function parameters, which were strongly affected in non-treated AAI mice, were unaffected in AAI mice treated with BMP-IOH-NPs and resembled those of healthy animals. Accumulation of BMP-IOH-NPs within the lungs of AAI mice was detectable by optical imaging for at least 4 h in vivo, where they were preferentially taken up by peribronchial and alveolar M2 macrophages. Conclusion: Our results show that BMP-IOH-NPs can effectively be applied in therapy of inflammatory diseases with at least equal efficacy as the gold standard Dexa, while their delivery can be simultaneously tracked in vivo by fluorescence imaging. BMP-IOH-NPs thus have the potential to reach clinical applications.

Fluorescence- and multispectral optoacoustic imaging for an optimized detection of deeply located tumors in an orthotopic mouse model of pancreatic carcinoma.

A crucial point for the management of pancreatic ductal adenocarcinoma (PDAC) is the decrease of R1 resections. Our aim was to evaluate the combination of multispectral optoacoustic tomography (MSOT) with fluorescence guided surgery (FGS) for diagnosis and perioperative detection of tumor nodules and resection margins in a xenotransplant mouse model of human pancreatic cancer. The peptide cRGD, conjugated with the near infrared fluorescent (NIRF) dye IRDye800CW and with a trans-cyclooctene (TCO) tag for future click chemistry (cRGD-800CW-TCO), was applied to PDAC bearing immunodeficient nude mice; 27 days after orthotopic transplantation of human AsPC-1 cells into the head of the pancreas, mice were injected with cRGD-800CW-TCO and imaged with fluorescence- and optoacoustic devices before and 2, 6 and 24 hr after injection, before they were sacrificed and dissected with a guidance of FGS imaging system. Fluorescence imaging of cRGD-800CW-TCO allowed detection of the tumor area but without information about the depth, whereas MSOT allowed high resolution 3 D identification of the tumor area, in particular of small tumor nodules. Highly sensitive delineation of tumor burden was achieved during FGS in all mice. Imaging of whole-mouse cryosections, histopathological analysis and NIRF microscopy confirmed the localization of cRGD-800CW-TCO within the tumor tissue. In principle, all imaging modalities applied here were able to detect PDAC in vivo. However, the combination of MSOT and FGS provided detailed spatial information of the signal and achieved a complete overview of the distribution and localization of cRGD-800CW-TCO within the tumor before and during surgical intervention.

Influence of Label and Charge Density on the Association of the Therapeutic Monoclonal Antibodies Trastuzumab and Cetuximab Conjugated to Anionic Fluorophores.

The design of bright and functional dye-protein conjugates requires hydrophilic and stable fluorophores with high molar absorption coefficients and high fluorescence quantum yields, which must not be prone to dimerization, as well as conservation of protein function and suppression of protein association. Although many synthetic dyes meet these needs, the influence of dye charge on bioconjugate performance is commonly neglected. This encouraged us to assess the spectroscopic properties, antibody functionality, binding behavior, folding, and association of conjugates of the therapeutic antibodies trastuzumab and cetuximab with the red cyanine dyes S0586, S2381, and 6SIDCC (bearing two, three, and six sulfonate groups, respectively). Our results demonstrate a negligible effect of dye labeling on antibody folding, yet a strong influence of label charge and density on antibody isoelectric points and association. Especially 6SIDCC decreased strongly the isoelectric points of both antibodies and their heavy or light chains even at low labeling degrees, thus favoring protein association. Although an increasingly negative dye charge reduces antigen affinity as shown in a competitive immunoassay, all conjugates still bound to cells overexpressing the target of the respective antibody. Obviously, dyes that cause minimum dimerization with a small number of charged groups are best for conjugate brightness, minimum protein association, and strong target binding. This underlines the need to consider dye charge for the rational design of conjugates with optimum performance.

Targeting of the P2X7 receptor in pancreatic cancer and stellate cells.

The ATP-gated receptor P2X7 (P2X7R) is involved in regulation of cell survival and has been of interest in cancer field. Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer and new markers and therapeutic targets are needed. PDAC is characterized by a complex tumour microenvironment, which includes cancer and pancreatic stellate cells (PSCs), and potentially high nucleotide/side turnover. Our aim was to determine P2X7R expression and function in human pancreatic cancer cells in vitro as well as to perform in vivo efficacy study applying P2X7R inhibitor in an orthotopic xenograft mouse model of PDAC. In the in vitro studies we show that human PDAC cells with luciferase gene (PancTu-1 Luc cells) express high levels of P2X7R protein. Allosteric P2X7R antagonist AZ10606120 inhibited cell proliferation in basal conditions, indicating that P2X7R was tonically active. Extracellular ATP and BzATP, to which the P2X7R is more sensitive, further affected cell survival and confirmed complex functionality of P2X7R. PancTu-1 Luc migration and invasion was reduced by AZ10606120, and it was stimulated by PSCs, but not by PSCs from P2X7(-/-) animals. PancTu-1 Luc cells were orthotopically transplanted into nude mice and tumour growth was followed noninvasively by bioluminescence imaging. AZ10606120-treated mice showed reduced bioluminescence compared to saline-treated mice. Immunohistochemical analysis confirmed P2X7R expression in cancer and PSC cells, and in metaplastic/neoplastic acinar and duct structures. PSCs number/activity and collagen deposition was reduced in AZ10606120-treated tumours.

Preclinical evaluation of near-infrared (NIR) fluorescently labeled cetuximab as a potential tool for fluorescence-guided surgery.

The high rate of recurrence in patients with pancreatic ductal adenocarcinoma (PDAC) could be reduced by supporting the surgeons in discriminating healthy from diseased tissues with intraoperative fluorescence-guidance. Here, we studied the suitability of Cetuximab, a therapeutic monoclonal antibody targeting the human epidermal growth factor receptor (EGFR), near-infrared (NIR) fluorescently labeled as a new tool for fluorescence-guided surgery. Distribution and binding of systemically injected Cetuximab Alexa Fluor 647 conjugate (Cetux-Alexa-647) and the co-injected control human IgG Alexa Fluor 750 conjugate (hIgG-Alexa-750) was studied over 48 h by NIR fluorescence imaging in mice bearing human orthotopic AsPC-1 and MIA PaCa-2 PDAC tumors. Cetux-Alexa-647, but not the control hIgG-Alexa-750 fluorescence, was specifically detected in vivo in both primary pancreatic tumors with maximum fluorescence intensities at 24 h, and in metastases of AsPC-1 tumors as small as 1 mm. Lifetime analysis and NIR fluorescence microscopy of tumor sections confirmed the binding specificity of Cetux-Alexa-647 to PDAC cells. Comparable results were obtained with Cetuximab conjugated to Alexa Fluor 750 dye (Cetux-Alexa-750). Fluorescence-guided dissection, performed 24 h after injection of Cetuximab conjugated to IRDye 800CW (Cetux-800CW), enabled a real-time delineation of AsPC-1 tumor margins, and small metastases. Odyssey scans revealed that only the vital part of the tumor, but not the necrotic part was stained with Cetux-800CW. NIR fluorescently labeled Cetuximab may be a promising tool that can be applied for fluorescence-guided surgery to visualize tumor margins and metastatic sites in order to allow a precise surgical resection.

In vivo imaging of tumour xenografts with an antibody targeting the potassium channel Kv10.1.

The Kv10.1 (Eag1) voltage-gated potassium channel represents a promising molecular target for novel cancer therapies or diagnostic purposes. Physiologically, it is only expressed in the brain, but it was found overexpressed in more than 70 % of tumours of diverse origin. Furthermore, as a plasma membrane protein, it is easily accessible to extracellular interventions. In this study we analysed the feasibility of the anti-Kv10.1 monoclonal antibody mAb62 to target tumour cells in vitro and in vivo and to deliver therapeutics to the tumour. Using time-domain near infrared fluorescence (NIRF) imaging in a subcutaneous MDA-MB-435S tumour model in nude mice, we showed that mAb62-Cy5.5 specifically accumulates at the tumour for at least 1 week in vivo with a maximum intensity at 48 h. Blocking experiments with an excess of unlabelled mAb62 and application of the free Cy5.5 fluorophore demonstrate specific binding to the tumour. Ex vivo NIRF imaging of whole tumours as well as NIRF imaging and microscopy of tumour slices confirmed the accumulation of the mAb62-Cy5.5 in tumours but not in brain tissue. Moreover, mAb62 was conjugated to the prodrug-activating enzyme ß-D-galactosidase (ß-gal; mAb62-ß-gal). The ß-gal activity of the mAb62-ß-gal conjugate was analysed in vitro on Kv10.1-expressing MDA-MB-435S cells in comparison to control AsPC-1 cells. We show that the mAb62-ß-gal conjugate possesses high ß-gal activity when bound to Kv10.1-expressing MDA-MB-435S cells. Moreover, using the ß-gal activatable NIRF probe DDAOG, we detected mAb62-ß-gal activity in vivo over the tumour area. In summary, we could show that the anti-Kv10.1 antibody is a promising tool for the development of novel concepts of targeted cancer therapy.

Tracking of Inhaled Near-Infrared Fluorescent Nanoparticles in Lungs of SKH-1 Mice with Allergic Airway Inflammation.

Molecular imaging of inflammatory lung diseases, such as asthma, has been limited to date. The recruitment of innate immune cells to the airways is central to the inflammation process. This study exploits these cells for imaging purposes within the lung, using inhaled polystyrene nanoparticles loaded with the near-infrared fluorescence dye Itrybe (Itrybe-NPs). By means of in vivo and ex vivo fluorescence reflectance imaging of an ovalbumin-based allergic airway inflammation (AAI) model in hairless SKH-1 mice, we show that subsequent to intranasal application of Itrybe-NPs, AAI lungs display fluorescence intensities significantly higher than those in lungs of control mice for at least 24 h. Ex vivo immunofluorescence analysis of lung tissue demonstrates the uptake of Itrybe-NPs predominantly by CD68(+)CD11c(+)ECF-L(+)MHCII(low) cells, identifying them as alveolar M2 macrophages in the peribronchial and alveolar areas. The in vivo results were validated by confocal microscopy, overlapping tile analysis, and flow cytometry, showing an amount of Itrybe-NP-containing macrophages in lungs of AAI mice significantly larger than that in controls. A small percentage of NP-containing cells were identified as dendritic cells. Flow cytometry of tracheobronchial lymph nodes showed that Itrybe-NPs were negligible in lung draining lymph nodes 24 h after inhalation. This imaging approach may advance preclinical monitoring of AAI in vivo over time and aid the investigation of the role that macrophages play during lung inflammation. Furthermore, it allows for tracking of inhaled nanoparticles and can hence be utilized for studies of the fate of potential new nanotherapeutics.

Multifunctional phosphate-based inorganic-organic hybrid nanoparticles.

Phosphate-based inorganic-organic hybrid nanoparticles (IOH-NPs) with the general composition [M](2+)[Rfunction(O)PO3](2-) (M = ZrO, Mg2O; R = functional organic group) show multipurpose and multifunctional properties. If [Rfunction(O)PO3](2-) is a fluorescent dye anion ([RdyeOPO3](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 ([RdrugOPO3](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+)([RdrugOPO3]1-x[RdyeOPO3]x)(2-) nanoparticles and is highly relevant for theranostics. Amine-based functional anions in [MgO](2+)[RaminePO3](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 [M](2+)[Rfunction(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.

Functionalized synchrotron in-line phase-contrast computed tomography: a novel approach for simultaneous quantification of structural alterations and localization of barium-labelled alveolar macrophages within mouse lung samples.

Functionalized computed tomography (CT) in combination with labelled cells is virtually non-existent due to the limited sensitivity of X-ray-absorption-based imaging, but would be highly desirable to realise cell tracking studies in entire organisms. In this study we applied in-line free propagation X-ray phase-contrast CT (XPCT) in an allergic asthma mouse model to assess structural changes as well as the biodistribution of barium-labelled macrophages in lung tissue. Alveolar macrophages that were barium-sulfate-loaded and fluorescent-labelled were instilled intratracheally into asthmatic and control mice. Mice were sacrificed after 24 h, lungs were kept in situ, inflated with air and scanned utilizing XPCT at the SYRMEP beamline (Elettra Synchrotron Light Source, Italy). Single-distance phase retrieval was used to generate data sets with ten times greater contrast-to-noise ratio than absorption-based CT (in our setup), thus allowing to depict and quantify structural hallmarks of asthmatic lungs such as reduced air volume, obstruction of airways and increased soft-tissue content. Furthermore, we found a higher concentration as well as a specific accumulation of the barium-labelled macrophages in asthmatic lung tissue. It is believe that XPCT will be beneficial in preclinical asthma research for both the assessment of therapeutic response as well as the analysis of the role of the recruitment of macrophages to inflammatory sites.

Target-specific nanoparticles containing a broad band emissive NIR dye for the sensitive detection and characterization of tumor development.

Current optical probes including engineered nanoparticles (NPs) are constructed from near infrared (NIR)-emissive organic dyes with narrow absorption and emission bands and small Stokes shifts prone to aggregation-induced self-quenching. Here, we present the new asymmetric cyanine Itrybe with broad, almost environment-insensitive absorption and emission bands in the diagnostic window, offering a unique flexibility of the choice of excitation and detection wavelengths compared to common NIR dyes. This strongly emissive dye was spectroscopically studied in different solvents and encapsulated into differently sized (15, 25, 100 nm) amino-modified polystyrene NPs (PSNPs) via a one-step staining procedure. As proof-of-concept for its potential for pre-/clinical imaging applications, Itrybe-loaded NPs were surface-functionalized with polyethylene glycol (PEG) and the tumor-targeting antibody Herceptin and their binding specificity to the tumor-specific biomarker HER2 was systematically assessed. Itrybe-loaded NPs display strong fluorescence signals in vitro and in vivo and Herceptin-conjugated NPs bind specifically to HER2 as demonstrated in immunoassays as well as on tumor cells and sections from mouse tumor xenografts in vitro. This demonstrates that our design strategy exploiting broad band-absorbing and -emitting dyes yields versatile and bright NIR probes with a high potential for e.g. the sensitive detection and characterization of tumor development and progression.

Knockdown of prolyl-4-hydroxylase domain 2 inhibits tumor growth of human breast cancer MDA-MB-231 cells by affecting TGF-ß1 processing.

The prolyl-4-hydroxylase domain 1-3 (PHD1-3) enzymes are regulating the protein stability of the α-subunit of the hypoxia-inducible factor-1 (HIF-1), which mediates oxygen-dependent gene expression. PHD2 is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. In human cancers, HIF-1α is overexpressed as a result of intratumoral hypoxia which in turn promotes tumor progression. The role of PHD2 for tumor progression is in contrast far from being thoroughly understood. Therefore, we established PHD2 knockdown clones of MDA-MB-231 breast cancer cells and analyzed their tumor-forming potential in a SCID mouse model. Tumor progression was significantly impaired in the PHD2 knockdown MDA-MB-231 cells, which could be partially rescued by re-establishing PHD2 expression. In a RNA profile screen, we identified the secreted phosphoprotein 1 (SPP1) as one target, which is differentially regulated as a consequence of the PHD2 knockdown. Knockdown of PHD2 drastically reduced the SPP1 expression in MDA-MB-231 cells. A correlation of SPP1 and PHD2 expression was additionally verified in 294 invasive breast cancer biopsies. In subsequent analyses, we identified that PHD2 alters the processing of transforming growth factor (TGF)-ß1, which is highly involved in SPP1 expression. The altered processing capacity was associated with a dislocation of the pro-protein convertase furin. Thus, our data demonstrate that in MDA-MB-231 cells PHD2 might affect tumor-relevant TGF-ß1 target gene expression by altering the TGF-ß1 processing capacity.

High-sensitivity detection of breast tumors in vivo by use of a pH-sensitive near-infrared fluorescence probe.

We investigated the potential of the pH-sensitive dye, CypHer5E, conjugated to Herceptin (pH-Her) for the sensitive detection of breast tumors in mice using noninvasive time-domain near-infrared fluorescence imaging and different methods of data analysis. First, the fluorescence properties of pH-Her were analyzed as function of pH and/or dye-to-protein ratio, and binding specificity was confirmed in cell-based assays. Subsequently, the performance of pH-Her in nude mice bearing orthotopic HER2-positive (KPL-4) and HER2-negative (MDA-MB-231) breast carcinoma xenografts was compared to that of an always-on fluorescent conjugate Alexa Fluor 647-Herceptin (Alexa-Her). Subtraction of autofluorescence and lifetime (LT)-gated image analyses were performed for background fluorescence suppression. In mice bearing HER2-positive tumors, autofluorescence subtraction together with the selective fluorescence enhancement of pH-Her solely in the tumor's acidic environment provided high contrast-to-noise ratios (CNRs). This led to an improved sensitivity of tumor detection compared to Alexa-Her. In contrast, LT-gated imaging using LTs determined in model systems did not improve tumor-detection sensitivity in vivo for either probe. In conclusion, pH-Her is suitable for sensitive in vivo monitoring of HER2-expressing breast tumors with imaging in the intensity domain and represents a promising tool for detection of weak fluorescent signals deriving from small tumors or metastases.