Optical biosensing of markers of mucosal inflammation.

We report the design and adaptation of iron/iron oxide nanoparticle-based optical nanobiosensors for enzymes or cytokine/chemokines that are established biomarkers of lung diseases. These biomarkers comprise ADAM33, granzyme B, MMP-8, neutrophil elastase, arginase, chemokine (C-C motif) ligand 20 and interleukin-6. The synthesis of nanobiosensors for these seven biomarkers, their calibration with commercially available enzymes and cytokines/chemokines, as well as their validation using bronchoalveolar lavage (BAL) obtained from a mouse model of TLR3-mediated inflammation are discussed here. Exhaled Breath Condensate (EBC) is a minimally invasive approach for sampling airway fluid in the diagnosis and management of various lung diseases in humans (e.g., asthma, COPD and viral infections). We report the proof-of-concept of using human EBC in conjunction with nanobiosensors for diagnosis/monitoring airway inflammation. These findings suggest that, with nanosensor technology, human EBC can be utilized as a liquid biopsy to monitor inflammation/remodeling in lung disease.

Development of a Gene Delivery System Composed of a Cell-Penetrating Peptide and a Nontoxic Polymer.

Major concerns have arisen with respect to using viral vectors for gene therapies. Collateral effects include cancer resistance, development of new cancers, and even systemic deaths. For this reason, researchers have focused on the alternative of using nonviral nanocarriers for gene therapy. In this study, a gene delivery nanocarrier was developed, comprising a cell-penetrating peptide called WTAS as a primary nanocarrier and a poly(ß-amino ester) (PBAE) polymer as a secondary nanocarrier. Here, the PBAE polymer is used to protect the WTAS peptide from early degradation while further facilitating the transportation into cells. WTAS is a peptide that penetrates cell nuclei within a few minutes after exposure, which makes it an ideal candidate to transport genetic materials. The PBAE-WTAS nanocarrier was assembled and tested against three cell lines (NSC, B16F10, and GL26). Cytotoxic studies demonstrated the relatively low toxicity of the PBAE-WTAS nanocarrier and PBAE-WTAS loaded with green fluorescent protein (GFP) plasmid DNA (pDNA@PBAE-WTAS) against all three cell lines. Cell transfection experiments were carried out using GL26 cells. These studies demonstrated a very high transfection rate of PBAE-WTAS loaded with GFP plasmid DNA, leading to virtually complete transfection (> 90%). In conclusion, we report a very promising gene delivery nanocarrier, which can be further modified to transport a variety of genetic materials for targeted therapy of multiple diseases.

Peptide Nanosponges Designed for the Delivery of Perillyl Alcohol to Glioma Cells.

Peptide nanosponges of low polydispersity are spontaneously formed from trigonal supramolecular building blocks in aqueous buffers, which feature cationic and/or anionic oligopeptides (n = 5-20) and a hydrophobic unit. In contrast to classical liposomes/vesicles, nanosponges feature interwoven hydrophilic and hydrophobic nanodomains and are readily taken up by mammalian cells. Perillyl alcohol is known to be a simple, but effective small molecule drug against glioma multiforme. However, its efficacy is limited by a poor bioavailability. In order to make perillyl alcohol bioavailable, two nanosponges consisting of 10 aspartates, to which perillyl alcohol is attached by means of an ester bond, and 20 lysines or arginines (type (D-POH)10K20 and (D-POH)10R20) were synthesized, purified, and characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM). These nanosponges were then tested in cell cultures of murine glioma cells (GL26) and murine neural progenitor cells (NPC) because the latter was previously utilized in cell-based cancer therapy. The two nanosponges exhibited significantly different biophysical properties (size distribution and ζ potentials). Consequently, different efficacies in killing GL26 and NPC were observed in serum-containing culture media. The results from these experiments confirmed that the type (D-POH)10K20 nanosponge is a promising candidate for the (cell-mediated) cytotherapy of glioblastoma.

Early detection of pancreatic cancers in liquid biopsies by ultrasensitive fluorescence nanobiosensors.

Numerous proteases, such as matrix metalloproteinases (MMPs), cathepsins (CTS), and urokinase plasminogen activator (UpA), are dysfunctional (that is, over- or under-expressed) in solid tumors, when compared to healthy human subjects. This offers the opportunity to detect early tumors by liquid biopsies. This approach is of particular advantage for the early detection of pancreatic cancer, which is a "silent killer". We have developed fluorescence nanobiosensors for ultrasensitive (sub-femtomolar) arginase and protease detection, consisting of water-dispersible Fe/Fe3O4 core/shell nanoparticles and two tethered fluorescent dyes: TCPP (Tetrakis(4-carboxyphenyl)porphyrin) and cyanine 5.5. Upon posttranslational modification or enzymatic cleavage, the fluorescence of TCPP increases, which enables the detection of proteases at sub-femtomolar activities utilizing conventional plate readers. We have identified an enzymatic signature for the detection of pancreatic adenocarcinomas in serum, consisting of arginase, matrix metalloproteinase-1, -3, and - 9, cathepsin-B and -E, urokinase plasminogen activator, and neutrophil elastase, which is a potential game-changer.

Porcine Wharton's jelly cells distribute throughout the body after intraperitoneal injection.

BACKGROUND: Wharton's jelly cells (WJCs) have multiple differentiation potentials and are easily harvested in large numbers. WJCs are well tolerated in allogeneic environments and there is a growing list of their therapeutic effects. Most therapies require administering large numbers of cells and this is generally accomplished by intravenous injection. Here, we studied the locations of porcine WJCs in immune-competent, allogeneic hosts after intraperitoneal (IP) injection. METHODS: Male porcine WJCs were administered to female neonatal piglets by IP injection. The location of transplanted cells was examined at 6 h, 24 h, and 7 days after administration using confocal microscopy and polymerase chain reaction (PCR). Transplanted cells were also retrieved from the intestines of recipients and were cultured. Previously transplanted cells were identified by fluorescence in-situ hybridization (FISH) using a Y-chromosome probe. RESULTS: Allogeneic cells were identified in the small and large intestine, stomach, liver, spleen, diaphragm, omentum, kidney, pancreas, mesenteric lymph nodes, heart, lungs, uterus, bladder, and skeletal muscle. Male cells (SRY positive) were found in cultures of cells harvested from the intestinal mucosa 1 week after administration of male porcine WJCs. CONCLUSIONS: Our results show that porcine WJCs distribute widely to the organs in immunocompetent allogeneic hosts after IP administration. They may distribute through the lymphatics initially, and a prominent site of incorporation is the mucosa of the gastrointestinal tract. In that location they could function in the niche of endogenous stem cells and provide secretory products to cells in the tissue damaged by intestinal disease.

Developing a xenograft human tumor model in immunocompetent mice.

Animal models are essential to cancer research, but current xenograft models are limited in their utility especially due to the lack of an immune system. Here we demonstrate that a xenograft tumor model can be developed in immunocompetent mice by tolerizing murine fetuses to human tumor cells. A375 human melanoma cells were injected into day E14 fetuses and after birth mice were challenged with A375 cells to determine their ability to develop tumors. Intravenous injections of cells resulted in metastatic-like lung tumors, which were verified to be human in origin by immunohistochemistry and PCR. These results were replicated with several other human tumor types: BxPC3 (human pancreatic adenocarcinoma), MDA-MB-231 (human breast adenocarcinoma), M21 (human melanoma), and HeLa (human cervical adenocarcinoma). Development of an immunocompetent xenograft tumor model would allow the further elucidation of the interaction of the immune system with therapy in both preclinical research and patient derived xenografts.

An integrated platform for small-animal hyperthermia investigations under ultra-high-field MRI guidance.

PURPOSE: Integrating small-animal experimental hyperthermia instrumentation with magnetic resonance imaging (MRI) affords real-time monitoring of spatial temperature profiles. This study reports on the development and preliminary in vivo characterisation of a 2.45 GHz microwave hyperthermia system for pre-clinical small animal investigations, integrated within a 14 T ultra-high-field MRI scanner. MATERIALS AND METHODS: The presented system incorporates a 3.5 mm (OD) directional microwave hyperthermia antenna, positioned adjacent to the small-animal target, radiating microwave energy for localised heating of subcutaneous tumours. The applicator is integrated within the 30 mm bore of the MRI system. 3D electromagnetic and biothermal simulations were implemented to characterise hyperthermia profiles from the directional microwave antenna. Experiments in tissue mimicking phantoms were performed to assess hyperthermia profiles and validate MR thermometry against fibre-optic temperature measurements. The feasibility of delivering in vivo hyperthermia exposures to subcutaneous 4T1 tumours in experimental mice under simultaneous MR thermometry guidance was assessed. RESULTS: Simulations and experiments in tissue mimicking phantoms demonstrated the feasibility of heating 21-982 mm3 targets with 8-12 W input power. Minimal susceptibility and electrical artefacts introduced by the hyperthermia applicator were observed on MR imaging. MR thermometry was in excellent agreement with fibre-optic temperatures measurements (max. discrepancy ≤0.6 °C). Heating experiments with the reported system demonstrated the feasibility of heating subcutaneous tumours in vivo with simultaneous MR thermometry. CONCLUSIONS: A platform for small-animal hyperthermia investigations under ultra-high-field MR thermometry was developed and applied to heating subcutaneous tumours in vivo.

Peptide nanosponges designed for rapid uptake by leukocytes and neural stem cells.

The structure of novel binary nanosponges consisting of (cholesterol-(K/D) n DEVDGC)3-trimaleimide units possessing a trigonal maleimide linker, to which either lysine (K)20 or aspartic acid (D)20 are tethered, has been elucidated by means of TEM. A high degree of agreement between these findings and structure predictions through explicit solvent and then coarse-grained molecular dynamics (MD) simulations has been found. Based on the nanosponges' structure and dynamics, caspase-6 mediated release of the model drug 5(6)-carboxyfluorescein has been demonstrated. Furthermore, the binary (DK20) nanosponges have been found to be virtually non-toxic in cultures of neural progenitor cells. It is of a special importance for the future development of cell-based therapies that DK20 nanosponges were taken up efficiently by leucocytes (WBC) in peripheral blood within 3 h of exposure. The percentage of live cells among the WBC was not significantly decreased by the DK20 nanosponges. In contrast to stem cell or leucocyte cell cultures, which have to be matched to the patient, autologous cells are optimal for cell-mediated therapy. Therefore, the nanosponges hold great promise for effective cell-based tumor targeting.

Rationally designed peptide nanosponges for cell-based cancer therapy.

A novel type of supramolecular aggregate, named a "nanosponge" was synthesized through the interaction of novel supramolecular building blocks with trigonal geometry. The cholesterol-(K/D)nDEVDGC)3-trimaleimide unit consists of a trigonal maleimide linker to which homopeptides (either K or D) of variable lengths (n=5, 10, 15, 20) and a consensus sequence for executioner caspases (DEVDGC) are added via Michael addition. Upon mixing in aqueous buffer cholesterol-(K)nDEVDGC)3-trimaleimides and a 1:1 mixture of cholesterol-(K/D)nDEVDGC)3-trimaleimides form stable nanosponges, whereas cholesterol-(D)nDEVDGC)3-trimaleimide is unable to form supramolecular aggregates with itself. The structure of the novel nanosponges was investigated through explicit solvent and then coarse-grained molecular dynamics (MD) simulations. The nanosponges are between 80 nm and several micrometers in diameters and virtually non-toxic to monocyte/macrophage-like cells.

Synergy of Iron Chelators and Therapeutic Peptide Sequences Delivered via a Magnetic Nanocarrier.

Here, we report the synthesis, characterization, and efficacy study of Fe/Fe3O4-nanoparticles that were co-labeled with a tumor-homing and membrane-disrupting oligopeptide and the iron-chelator Dp44mT, which belongs to the group of the thiosemicarbazones. Dp44mT and the peptide sequence PLFAERL(D[KLAKLAKKLAKLAK])CGKRK were tethered to the surface of Fe/Fe3O4 core/shell nanoparticles by utilizing dopamine-anchors. The 26-mer contains two important sequences, which are the tumor targeting peptide CGKRK, and D[KLAKLAK]2, known to disrupt the mitochondrial cell walls and to initiate programmed cell death (apoptosis). It is noteworthy that Fe/Fe3O4 nanoparticles can also be used for MRI imaging purposes in live mammals. In a first step of this endeavor, the efficacy of this nanoplatform has been tested on the highly metastatic 4T1 breast cancer cell line. At the optimal ratio of PLFAERD[KLAKLAK]2CGKRK to Dp44mT of 1 to 3.2 at the surface of the dopamine-coated Fe/Fe3O4-nanocarrier, the IC50 value after 24 h of incubation was found to be 2.2 times lower for murine breast cancer cells (4T1) than for a murine fibroblast cell line used as control. Based on these encouraging results, the reported approach has the potential of leading to a new generation of nanoplatforms for cancer treatment with considerably enhanced selectivity towards tumor cells.

Associations between activity of arginase or matrix metalloproteinase-8 (MMP-8) and metritis in periparturient dairy cattle.

Metritis, a uterine disease caused by bacterial infection, is highly prevalent in dairy cattle after parturition. Uterine disease has negative effects on milk production and reproductive efficiency. Finding markers or indicators that can predict cows at greater risk for uterine disease could be beneficial to mitigating these deleterious effects. This study investigates the immune-derived enzymes arginase and matrix metalloproteinase-8 (MMP-8) as potential markers for development of metritis in dairy cows. In a retrospective matched case-control study, 53 lactating Holstein cows diagnosed with metritis were matched and paired to 53 lactating Holstein control cows. In addition to examining cows for diagnosis of metritis on d 4, 7, 10, and 14 after parturition, occurrence of retained fetal membranes, gender of the calf, and the event of a stillbirth were recorded. Blood samples were collected 7 ± 3 d before calving, on the day of calving, and 7 ± 3 d after calving and were assayed for activity of arginase and MMP-8. Associations between metritis and activity of arginase or MMP-8 were determined by conditional logistic regression at each individual sampling time point. An interaction between activity of arginase, before and on the day of parturition, and retained fetal membranes tended (P ≤ 0.13) to be associated with metritis. After parturition, activity of arginase and the interaction between activity of arginase and retained fetal membranes were not (P ≥ 0.22) associated with metritis. Activity of MMP-8 was not (P ≥ 0.20) associated with metritis in the periparturient period. Retained fetal membranes were associated with the odds of developing metritis. Activity of arginase before and at the time of parturition might be a potential marker for occurrence of metritis, especially in cows that develop retained fetal membranes. MMP-8 does not seem to be a potential indicator for metritis.

Membrane Fusion-Mediated Gold Nanoplating of Red Blood Cell: A Bioengineered CT-Contrast Agent.

Red blood cells (RBCs) are the natural resident of the vascular lumen, therefore delivery of any agents within the vascular lumen could benefit by unique natural transporting features of RBCs. RBCs continuously circulate for ∼100 days before being sequestered in the spleen, they only extravasate at sites of vascular hemorrhage. Taking advantages of these features, we engineered RBC as a carrier in order to design a unique delivery system capable of delivering X-ray computed tomography (CT) contrast agents, gold nanoparticles (AuNPs), thereby acting as CT-contrast agent. A strategic membrane fusion technique was used to engineer the surface of RBC with gold nanoparticles in this in vitro study without altering its shape, size, and surface properties.

A nanobiosensor for the detection of arginase activity.

A nanobiosensor for arginase detection was designed and synthesized. It features a central dopamine-coated iron/iron oxide nanoparticle to which sulfonated cyanine 7.0 is tethered via a stable amide bond. Cyanine 5.5 is linked to the N-terminal of the peptide sequence GRRRRRRRG. Arginine (R) reacts to ornithine (O) in the presence of arginase. Based on calibration with commercially obtained arginase II, the limit of detection (LOD) is picomolar. It is noteworthy that the nanobiosensor for arginase detection does not show a fluorescence increase when incubated with the enzyme NO-reductase, which also uses arginase as substrate, but is indicative of an inflammatory response by the host to cancer and infections. Arginase activity was determined in a syngeneic mouse model for aggressive breast cancer (4T1 tumors in BALB/c mice). It was found that the arginase activity is systemically enhanced, but especially pronounced in the active tumor regions.

Early breast cancer screening using iron/iron oxide-based nanoplatforms with sub-femtomolar limits of detection.

Proteases, including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins (CTS) exhibit numerous functions in tumor biology. Solid tumors are characterized by changes in protease expression levels by tumor and surrounding tissue. Therefore, monitoring protease levels in tissue samples and liquid biopsies is a vital strategy for early cancer detection. Water-dispersable Fe/Fe3O4-core/shell based nanoplatforms for protease detection are capable of detecting protease activity down to sub-femtomolar limits of detection. They feature one dye (tetrakis(carboxyphenyl)porphyrin (TCPP)) that is tethered to the central nanoparticle by means of a protease-cleavable consensus sequence and a second dye (Cy 5.5) that is directly linked. Based on the protease activities of urokinase plasminogen activator (uPA), MMPs 1, 2, 3, 7, 9, and 13, as well as CTS B and L, human breast cancer can be detected at stage I by means of a simple serum test. By monitoring CTS B and L stage 0 detection may be achieved. This initial study, comprised of 46 breast cancer patients and 20 apparently healthy human subjects, demonstrates the feasibility of protease-activity-based liquid biopsies for early cancer diagnosis.

Human umbilical cord matrix mesenchymal stem cells suppress the growth of breast cancer by expression of tumor suppressor genes.

Human and rat umbilical cord matrix mesenchymal stem cells (UCMSC) possess the ability to control the growth of breast carcinoma cells. Comparative analyses of two types of UCMSC suggest that rat UCMSC-dependent growth regulation is significantly stronger than that of human UCMSC. Their different tumoricidal abilities were clarified by analyzing gene expression profiles in the two types of UCMSC. Microarray analysis revealed differential gene expression between untreated naïve UCMSC and those co-cultured with species-matched breast carcinoma cells. The analyses screened 17 differentially expressed genes that are commonly detected in both human and rat UCMSC. The comparison between the two sets of gene expression profiles identified two tumor suppressor genes, adipose-differentiation related protein (ADRP) and follistatin (FST), that were specifically up-regulated in rat UCMSC, but down-regulated in human UCMSC when they were co-cultured with the corresponding species' breast carcinoma cells. Over-expression of FST, but not ADRP, in human UCMSC enhanced their ability to suppress the growth of MDA-231 cells. The growth of MDA-231 cells was also significantly lower when they were cultured in medium conditioned with FST, but not ADRP over-expressing human UCMSC. In the breast carcinoma lung metastasis model generated with MDA-231 cells, systemic treatment with FST-over-expressing human UCMSC significantly attenuated the tumor burden. These results suggest that FST may play an important role in exhibiting stronger tumoricidal ability in rat UCMSC than human UCMSC and also implies that human UCMSC can be transformed into stronger tumoricidal cells by enhancing tumor suppressor gene expression.

Cell Based Drug Delivery: Micrococcus luteus Loaded Neutrophils as Chlorhexidine Delivery Vehicles in a Mouse Model of Liver Abscesses in Cattle.

The recent WHO report on antibiotic resistances shows a dramatic increase of microbial resistance against antibiotics. With only a few new antibiotics in the pipeline, a different drug delivery approach is urgently needed. We have obtained evidence demonstrating the effectiveness of a cell based drug delivery system that utilizes the innate immune system as targeting carrier for antibacterial drugs. In this study we show the efficient loading of neutrophil granulocytes with chlorhexidine and the complete killing of E. coli as well as Fusobacterium necrophorum in in-vitro studies. Fusobacterium necrophorum causes hepatic abscesses in cattle fed high grain diets. We also show in a mouse model that this delivery system targets infections of F. necrophorum in the liver and reduces the bacterial burden by an order of magnitude from approximately 2•106 to 1•105.

Carbon dioxide hydrogenation to aromatic hydrocarbons by using an iron/iron oxide nanocatalyst.

The quest for renewable and cleaner energy sources to meet the rapid population and economic growth is more urgent than ever before. Being the most abundant carbon source in the atmosphere of Earth, CO2 can be used as an inexpensive C1 building block in the synthesis of aromatic fuels for internal combustion engines. We designed a process capable of synthesizing benzene, toluene, xylenes and mesitylene from CO2 and H2 at modest temperatures (T = 380 to 540 °C) employing Fe/Fe3O4 nanoparticles as catalyst. The synthesis of the catalyst and the mechanism of CO2-hydrogenation will be discussed, as well as further applications of Fe/Fe3O4 nanoparticles in catalysis.

Cells as delivery vehicles for cancer therapeutics.

Cell-based therapeutics have advanced significantly over the past decade and are poised to become a major pillar of modern medicine. Three cell types in particular have been studied in detail for their ability to home to tumors and to deliver a variety of different payloads. Neural stem cells, mesenchymal stem cells and monocytes have each been shown to have great potential as future delivery systems for cancer therapy. A variety of other cell types have also been studied. These results demonstrate that the field of cell-based therapeutics will only continue to grow.

Nanoplatforms for highly sensitive fluorescence detection of cancer-related proteases.

Numerous proteases are known to be necessary for cancer development and progression including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins. The goal of this research is to develop an Fe/Fe3O4 nanoparticle-based system for clinical diagnostics, which has the potential to measure the activity of cancer-associated proteases in biospecimens. Nanoparticle-based "light switches" for measuring protease activity consist of fluorescent cyanine dyes and porphyrins that are attached to Fe/Fe3O4 nanoparticles via consensus sequences. These consensus sequences can be cleaved in the presence of the correct protease, thus releasing a fluorescent dye from the Fe/Fe3O4 nanoparticle, resulting in highly sensitive (down to 1 × 10(-16) mol l(-1) for 12 proteases), selective, and fast nanoplatforms (required time: 60 min).

A nanosensor for ultrasensitive detection of oversulfated chondroitin sulfate contaminant in heparin.

Heparin has been extensively used as an anticoagulant for the last eight decades. Recently, the administration of a contaminated batch of heparin caused 149 deaths in several countries including USA, Germany, and Japan. The contaminant responsible for the adverse effects was identified as oversulfated chondroitin sulfate (OSCS). Here, we report a rapid, ultrasensitive method of detecting OSCS in heparin using a nanometal surface energy transfer (NSET) based gold-heparin-dye nanosensor. The sensor is an excellent substrate for heparitinase enzyme, as evidenced by ~70% recovery of fluorescence from the dye upon heparitinase treatment. However, the presence of OSCS results in diminished fluorescence recovery from the nanosensor upon heparitinase treatment, as the enzyme is inhibited by the contaminant. The newly designed nanosensor can detect as low as 1 × 10(-9) % (w/w) OSCS making it the most sensitive tool to date for the detection of trace amounts of OSCS in pharmaceutical heparins.