Intravenous iron preparations transiently generate non-transferrin-bound iron from two proposed pathways.

Intravenous iron-carbohydrate complex preparations (IVIPs) are non-interchangeable pro-drugs: their pharmacokinetics (PK) varies determined by semi-crystalline iron core and carbohydrate shell structures, influences pharmacodynamics (PD) and thus efficacy and safety. Examining PK/PD relationships of 3 IVIPs we identify a two-pathway model of transient NTBI generation following single dose administration. 28 hypoferremic non-anemic patients randomized to 200mg iron as ferric carboxymaltose (Fe-carboxymaltose), iron sucrose (Fe-sucrose), iron isomaltoside 1000 (Fe-isomaltoside-1000), n=8/arm, or placebo, n=4, on a 2-week PK/PD study, had samples analysed for total serum iron, IVIP-iron, transferrin-bound iron (TBI) by HPLC-ICP-MS, transferrin saturation (TSAT), serum ferritin (s-Ferritin) by standard methods, non-TBI (NTBI) and hepcidin as published before. IVIP-dependent increases in these parameters returned to baseline in 48-150h, except for s-Ferritin and TSAT. NTBI was low with Fe-isomaltoside-1000 (0.13µM at 8h), rapidly increased with Fe-sucrose (0.8µM at 2h, 1.25µM at 4h), and delayed for Fe-carboxymaltose (0.57µM at 24h). NTBI AUCs were 7-fold greater for Fe-carboxymaltose and Fe-sucrose than for Fe-isomaltoside-1000. Hepcidin peak time varied, but not AUC or mean levels. s-Ferritin levels and AUC were highest for Fe-carboxymaltose and greater than placebo for all IVIPs. We propose 2 mechanisms for the observed NTBI kinetics: rapid and delayed NTBI appearance consistent with direct (circulating IVIP-to-plasma) and indirect (IVIP-to-macrophage-to-plasma) iron release based on IVIP plasma half-life and s-Ferritin dynamics. IVIPs generate different, broadly stability- and PK-dependent, NTBI and s-Ferritin signatures, which may influence iron bioavailability, efficacy and safety. Longer-term studies should link NTBI exposure to subsequent safety and efficacy parameters and potential clinical consequences.

Optimizing hepcidin measurement with a proficiency test framework and standardization improvement.

Objectives: Hepcidin measurement advances insights in pathophysiology, diagnosis, and treatment of iron disorders, but requires analytically sound and standardized measurement procedures (MPs). Recent development of a two-level secondary reference material (sRM) for hepcidin assays allows worldwide standardization. However, no proficiency testing (PT) schemes to ensure external quality assurance (EQA) exist and the absence of a high calibrator in the sRM set precludes optimal standardization. Methods: We developed a pilot PT together with the Dutch EQA organization Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek (SKML) that included 16 international hepcidin MPs. The design included 12 human serum samples that allowed us to evaluate accuracy, linearity, precision and standardization potential. We manufactured, value-assigned, and validated a high-level calibrator in a similar manner to the existing low- and middle-level sRM. Results: The pilot PT confirmed logistical feasibility of an annual scheme. Most MPs demonstrated linearity (R2>0.99) and precision (duplicate CV>12.2%), although the need for EQA was shown by large variability in accuracy. The high-level calibrator proved effective, reducing the inter-assay CV from 42.0% (unstandardized) to 14.0%, compared to 17.6% with the two-leveled set. The calibrator passed international homogeneity criteria and was assigned a value of 9.07±0.24 nmol/L. Conclusions: We established a framework for future PT to enable laboratory accreditation, which is essential to ensure quality of hepcidin measurement and its use in patient care. Additionally, we showed optimized standardization is possible by extending the current sRM with a third high calibrator, although international implementation of the sRM is a prerequisite for its success.

Temporin L and aurein 2.5 have identical conformations but subtly distinct membrane and antibacterial activities.

Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and such properties are commonly ascribed to their ability to form secondary amphipathic, α-helix conformations in membrane mimicking milieu. Nevertheless, despite the high similarity in physical properties and preference for adopting such conformations, the spectrum of activity and potency of AMPs often varies considerably. Hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of primary-sequence-specific antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, α-helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes, notably their differing conformational flexibility at the N-terminus, ability to form higher order aggregates and the characteristics of induced ion conductance. Taken together, these differences provide an explanation of the greater potency and broader antibacterial spectrum of activity of temporin L over aurein 2.5. Consequently, while the secondary amphipathic, α-helix conformation is a key determinant of the ability of a cationic AMP to penetrate and disrupt the bacterial plasma membrane, the exact mechanism, potency and spectrum of activity is determined by precise structural and dynamic contributions from specific residues in each AMP sequence.

Provisional standardization of hepcidin assays: creating a traceability chain with a primary reference material, candidate reference method and a commutable secondary reference material.

Background Hepcidin concentrations measured by various methods differ considerably, complicating interpretation. Here, a previously identified plasma-based candidate secondary reference material (csRM) was modified into a serum-based two-leveled sRM. We validated its functionality to increase the equivalence between methods for international standardization. Methods We applied technical procedures developed by the International Consortium for Harmonization of Clinical Laboratory Results. The sRM, consisting of lyophilized serum with cryolyoprotectant, appeared commutable among nine different measurement procedures using 16 native human serum samples in a first round robin (RR1). Harmonization potential of the sRM was simulated in RR1 and evaluated in practice in RR2 among 11 measurement procedures using three native human plasma samples. Comprehensive purity analysis of a candidate primary RM (cpRM) was performed by state of the art procedures. The sRM was value assigned with an isotope dilution mass spectrometry-based candidate reference method calibrated using the certified pRM. Results The inter-assay CV without harmonization was 42.1% and 52.8% in RR1 and RR2, respectively. In RR1, simulation of harmonization with sRM resulted in an inter-assay CV of 11.0%, whereas in RR2 calibration with the material resulted in an inter-assay CV of 19.1%. Both the sRM and pRM passed international homogeneity criteria and showed long-term stability. We assigned values to the low (0.95±0.11 nmol/L) and middle concentration (3.75±0.17 nmol/L) calibrators of the sRM. Conclusions Standardization of hepcidin is possible with our sRM, which value is assigned by a pRM. We propose the implementation of this material as an international calibrator for hepcidin.

Proanthocyanidin trimer gallate modulates lipid deposition and fatty acid desaturation in Caenorhabditis elegans.

The incidence of obesity is rising at an alarming rate. Despite its recognition as an urgent healthcare concern, obesity remains largely an unsolved medical problem. A comprehensive screen for functional dietary phytochemicals identified proanthocyanidins as putative targets to ameliorate obesity. A full-scale purification of oligomeric proanthocyanidins (OPCs) derived from grape seed extract yielded pure OPC dimer, trimer, tetramer, and their gallates (pOPCs). Forward chemical screening conducted in Caenorhabditis elegans suggested that pOPCs reduced the activity of lipase in vitro and triglyceride storage capacity in vivo Proanthocyanidin trimer gallate in particular modified lipid desaturation in C. elegans, revealed by hyperspectral coherent anti-Stokes Raman scattering microscopy. Exposure to trimer gallate resulted in the transcriptional down-regulation of nhr-49 (an ortholog of the human peroxisome proliferator-activated receptor-α), and a key regulator of fat metabolism, and 2 downstream genes: fat-5 and acs-2 A combination exposure of 2 or 3 pOPCs (dimer gallate, trimer and/or trimer gallate) suggested the absence of synergistic potential. By using the whole-organism C. elegans coupled with versatile biochemical, biophysical, and genetic tools, we provide an account of the composition and bioactivity of individual OPCs and more generally highlight the potential of traditional Chinese medicine-derived drug leads.-Nie, Y., Littleton, B., Kavanagh, T., Abbate, V., Bansal, S. S., Richards, D., Hylands, P., Sturzenbaum, S. R. Proanthocyanidin trimer gallate modulates lipid deposition and fatty acid desaturation in Caenorhabditis elegans.

Effects of intravenous iron on fibroblast growth factor 23 (FGF23) in haemodialysis patients: a randomized controlled trial.

BACKGROUND: Intravenous iron affects serum levels of intact fibroblast growth factor-23 (iFGF23) and its cleavage product c-terminal FGF23 (cFGF23) in iron-deficient people with normal renal function. We hypothesized that intravenous iron modulates iFGF23 and cFGF23 in haemodialysis patients differently according to the type of iron used. METHODS: Prevalent, stable haemodialysis patients requiring protocol-based intravenous iron therapy were randomized to a single 200 mg dose of either ferric carboxymaltose (FCM) or iron sucrose (IS). The primary outcome was change in iFGF23 and cFGF23 from pre-infusion to Day 2 post-infusion. Serum hepcidin, ferritin and phosphate were also measured. Pair-wise comparisons utilised the Wilcoxon rank sum test; linear mixed models with an interaction term for treatment and time evaluated between-group effects. RESULTS: Forty-two participants completed the study. In those randomized to FCM (n = 22), median (interquartile range) values pre-infusion and Day 2, respectively, were 843 pg/mL (313-1922) and 576 pg/mL (356-1296, p = 0.05) for iFGF23, 704RU/mL (475-1204) and 813RU/mL (267-1156, p = 0.04) for cFGF23, and 1.53 mmol/L (1.14-1.71) and 1.37 (1.05-1.67, p = 0.03) for phosphate. These parameters did not change following IS. Both serum ferritin (p < 0.001) and hepcidin (p < 0.001) increased in both groups, and the increase in hepcidin was greater in the FCM group (p = 0.03 for between-group difference). CONCLUSIONS: Contrary to iron-deficient people with normal renal function, haemodialysis patients given protocol-driven intravenous FCM demonstrated a fall in iFGF23 and a rise in cFGF23, changes not evident with IS. This suggests a differential effect of intravenous iron treatment according to both formulation and renal function. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Register ACTRN12614000548639 . Registered 22 May 2014 (retrospectively registered).

Doxorubicin enhances curcumin's cytotoxicity in human prostate cancer cells in vitro by enhancing its cellular uptake.

Doxorubicin (DOX) is a widely used drug in cancer treatment. Despite its popularity, it suffers from systemic side effects and susceptibility to drug resistance. Curcumin (CURC), on the other hand, is a drug that recently gained popularity due to its wide range of biological activities, including anti-inflammatory and anti-cancer activities. Limitations to its clinical translation include its poor water solubility and the need for administration of high doses. Combinatory anti-cancer therapy has been proposed as a common approach to overcome one or more of these challenges. In this work, we propose a combinatory DOX and CURC anti-cancer therapy of prostate cancer cells in vitro. DOX and CURC were administered in the free drug and nanocapsule form, respectively. Cell size and complexity, cytotoxicity and apoptosis were studied by flow cytometry, MTT assay and sub-G1 quantification, respectively. Cellular uptake of CURC nanocapsules (CURC NCs) was quantified by fluorescence microscopy and high-performance liquid chromatography fluorescence detection. Results showed that in vitro treatment with CURC NCs in the presence of subtherapeutic concentrations of DOX, led to significant increase in prostate cancer cells (PC3) apoptosis and death. This was likely due to significantly enhanced CURC uptake by the cells. The study presents a good rationale for pursuing combinatory CURC/DOX therapy in pre-clinical tumor animal models in the near future.

Hepcidin Response to Iron Therapy in Patients with Non-Dialysis Dependent CKD: An Analysis of the FIND-CKD Trial.

Hepcidin is the key regulator of iron homeostasis but data are limited regarding its temporal response to iron therapy, and response to intravenous versus oral iron. In the 56-week, open-label, multicenter, prospective, randomized FIND-CKD study, 626 anemic patients with non-dialysis dependent chronic kidney disease (ND-CKD) and iron deficiency not receiving an erythropoiesis stimulating agent were randomized (1:1:2) to intravenous ferric carboxymaltose (FCM), targeting higher (400-600µg/L) or lower (100-200µg/L) ferritin, or to oral iron. Serum hepcidin levels were measured centrally in a subset of 61 patients. Mean (SD) baseline hepcidin level was 4.0(3.5), 7.3(6.4) and 6.5(5.6) ng/mL in the high ferritin FCM (n = 17), low ferritin FCM (n = 16) and oral iron group (n = 28). The mean (SD) endpoint value (i.e. the last post-baseline value) was 26.0(9.1),15.7(7.7) and 16.3(11.0) ng/mL, respectively. The increase in hepcidin from baseline was significantly smaller with low ferritin FCM or oral iron vs high ferritin FCM at all time points up to week 52. Significant correlations were found between absolute hepcidin and ferritin values (r = 0.65, p<0.001) and between final post-baseline increases in both parameters (r = 0.70, p<0.001). The increase in hepcidin levels over the 12-month study generally mirrored the cumulative iron dose in each group. Hepcidin and transferrin saturation (TSAT) absolute values showed no correlation, although there was an association between final post-baseline increases (r = 0.42, p<0.001). Absolute values (r = 0.36, p = 0.004) and final post-baseline increases of hepcidin and hemoglobin (p = 0.30, p = 0.030) correlated weakly. Baseline hepcidin levels were not predictive of a hematopoietic response to iron therapy. In conclusion, hepcidin levels rose in response to either intravenous or oral iron therapy, but the speed and extent of the rise was greatest with intravenous iron targeting a higher ferritin level. However neither the baseline level nor the change in hepcidin was able to predict response to therapy in this cohort.

Toward Worldwide Hepcidin Assay Harmonization: Identification of a Commutable Secondary Reference Material.

BACKGROUND: Absolute plasma hepcidin concentrations measured by various procedures differ substantially, complicating interpretation of results and rendering reference intervals method dependent. We investigated the degree of equivalence achievable by harmonization and the identification of a commutable secondary reference material to accomplish this goal. METHODS: We applied technical procedures to achieve harmonization developed by the Consortium for Harmonization of Clinical Laboratory Results. Eleven plasma hepcidin measurement procedures (5 mass spectrometry based and 6 immunochemical based) quantified native individual plasma samples (n = 32) and native plasma pools (n = 8) to assess analytical performance and current and achievable equivalence. In addition, 8 types of candidate reference materials (3 concentrations each, n = 24) were assessed for their suitability, most notably in terms of commutability, to serve as secondary reference material. RESULTS: Absolute hepcidin values and reproducibility (intrameasurement procedure CVs 2.9%-8.7%) differed substantially between measurement procedures, but all were linear and correlated well. The current equivalence (intermeasurement procedure CV 28.6%) between the methods was mainly attributable to differences in calibration and could thus be improved by harmonization with a common calibrator. Linear regression analysis and standardized residuals showed that a candidate reference material consisting of native lyophilized plasma with cryolyoprotectant was commutable for all measurement procedures. Mathematically simulated harmonization with this calibrator resulted in a maximum achievable equivalence of 7.7%. CONCLUSIONS: The secondary reference material identified in this study has the potential to substantially improve equivalence between hepcidin measurement procedures and contributes to the establishment of a traceability chain that will ultimately allow standardization of hepcidin measurement results.

Copper(II) binding properties of hepcidin.

Hepcidin is a peptide hormone that regulates the homeostasis of iron metabolism. The N-terminal domain of hepcidin is conserved amongst a range of species and is capable of binding Cu(II) and Ni(II) through the amino terminal copper-nickel binding motif (ATCUN). It has been suggested that the binding of copper to hepcidin may have biological relevance. In this study we have investigated the binding of Cu(II) with model peptides containing the ATCUN motif, fluorescently labelled hepcidin and hepcidin using MALDI-TOF mass spectrometry. As with albumin, it was found that tetrapeptide models of hepcidin possessed a higher affinity for Cu(II) than that of native hepcidin. The log K 1 value of hepcidin for Cu(II) was determined as 7.7. Cu(II) binds to albumin more tightly than hepcidin (log K 1 = 12) and in view of the serum concentration difference of albumin and hepcidin, the bulk of kinetically labile Cu(II) present in blood will be bound to albumin. It is estimated that the concentration of Cu(II)-hepcidin will be less than one femtomolar in normal serum and thus the binding of copper to hepcidin is unlikely to play a role in iron homeostasis. As with albumin, small tri and tetra peptides are poor models for the metal binding properties of hepcidin.

Carbon nanotubes' surface chemistry determines their potency as vaccine nanocarriers in vitro and in vivo.

Carbon nanotubes (CNTs) have shown marked capabilities in enhancing antigen delivery to antigen presenting cells. However, proper understanding of how altering the physical properties of CNTs may influence antigen uptake by antigen presenting cells, such as dendritic cells (DCs), has not been established yet. We hypothesized that altering the physical properties of multi-walled CNTs (MWNTs)-antigen conjugates, e.g. length and surface charge, can affect the internalization of MWNT-antigen by DCs, hence the induced immune response potency. For this purpose, pristine MWNTs (p-MWNTs) were exposed to various chemical reactions to modify their physical properties then conjugated to ovalbumin (OVA), a model antigen. The yielded MWNTs-OVA conjugates were long MWNT-OVA (~386nm), bearing net positive charge (5.8mV), or short MWNTs-OVA (~122nm) of increasing negative charges (-23.4, -35.8 or -39mV). Compared to the short MWNTs-OVA bearing high negative charges, short MWNT-OVA with the lowest negative charge demonstrated better cellular uptake and OVA-specific immune response both in vitro and in vivo. However, long positively-charged MWNT-OVA showed limited cellular uptake and OVA specific immune response in contrast to short MWNT-OVA displaying the least negative charge. We suggest that reduction in charge negativity of MWNT-antigen conjugate enhances cellular uptake and thus the elicited immune response intensity. Nevertheless, length of MWNT-antigen conjugate might also affect the cellular uptake and immune response potency; highlighting the importance of physical properties as a consideration in designing a MWNT-based vaccine delivery system.

Translocation of LRP1 targeted carbon nanotubes of different diameters across the blood-brain barrier in vitro and in vivo.

Brain glioblastoma and neurodegenerative diseases are still largely untreated due to the inability of most drugs to cross the blood-brain barrier (BBB). Nanoparticles have emerged as promising tools for drug delivery applications to the brain; in particular carbon nanotubes (CNTs) that have shown an intrinsic ability to cross the BBB in vitro and in vivo. Angiopep-2 (ANG), a ligand for the low-density lipoprotein receptor-related protein-1 (LRP1), has also shown promising results as a targeting ligand for brain delivery using nanoparticles (NPs). Here, we investigate the ability of ANG-targeted chemically-functionalised multi-walled carbon nanotubes (f-MWNTs) to cross the BBB in vitro and in vivo. ANG was conjugated to wide and thin f-MWNTs creating w-MWNT-ANG and t-MWNT-ANG, respectively. All f-MWNTs were radiolabelled to facilitate quantitative analyses by γ-scintigraphy. ANG conjugation to f-MWNTs enhanced BBB transport of w- and t-MWNTs-ANG compared to their non-targeted equivalents using an in vitro co-cultured BBB model consisting of primary porcine brain endothelial cells (PBEC) and primary rat astrocytes. Additionally, following intravenous administration w-MWNTs-ANG showed significantly higher whole brain uptake than the non-targeted w-MWNT in vivo reaching ~2% injected dose per g of brain (%ID/g) within the first hour post-injection. Furthermore, using a syngeneic glioma model, w-MWNT-ANG showed enhanced uptake in glioma brain compared to normal brain at 24h post-injection. t-MWNTs-ANG, on the other hand, showed higher brain accumulation than w-MWNTs. However, no significant differences were observed between t-MWNT and t-MWNT-ANG indicating the importance of f-MWNTs diameter towards their brain accumulation. The inherent brain accumulation ability of f-MWNTs coupled with improved brain-targeting by ANG favours the future clinical applications of f-MWNT-ANG to deliver active therapeutics for brain glioma therapy.

Inadequate hepcidin serum concentrations predict incident type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is closely associated with elevated body iron stores. The hormone hepcidin is the key regulator of iron homeostasis. Inadequately low hepcidin levels were recently reported in subjects with manifest T2DM. We investigated whether alterations of hepcidin levels precede the manifestation of T2DM and predict T2DM development independently of established risk conditions. METHODS: This prospective population-based study included 675 subjects aged 50-89 years, 51.9% of whom were female. Hepcidin levels were measured by gold standard tandem mass spectrometry. Diabetes was diagnosed according to American Diabetes Association criteria, and incident diabetes was recorded between baseline in 2000 and 2010. RESULTS: The baseline hepcidin-to-ferritin ratio in subjects that subsequently developed diabetes during follow-up was reduced on average by 29.8% as compared with subjects with normal glucose tolerance (95% confidence interval, -50.7% to -0.2%; p = 0.049). After adjustment for age, sex, and serum ferritin, higher hepcidin levels were associated with reduced risk of incident diabetes (hazard ratio per 1-unit higher log2 hepcidin, 0.80; 95% confidence interval, 0.64-0.98; p = 0.035; 33 events). Additional adjustment for established diabetes risk factors and determinants of hepcidin concentration did not appreciably change these results (HR, 0.81; 95% CI, 0.66-0.99). Likewise, inadequately low hepcidin levels were also detected in subjects with prevalent T2DM (n = 76). CONCLUSIONS: Hepcidin levels that are inadequately low in relation to body iron stores are an independent predictor for incident T2DM and may contribute to diabetes-related tissue iron overload. Copyright © 2015 John Wiley & Sons, Ltd.

Correlates of serum hepcidin levels and its association with cardiovascular disease in an elderly general population.

BACKGROUND: The expression of the key iron regulatory hormone hepcidin is regulated by iron availability, inflammation, hormones, hypoxia, and anaemia. Increased serum concentrations of hepcidin have recently been linked to atherosclerosis. We studied demographic, haematologic, biochemical, and dietary correlates of serum hepcidin levels and its associations with incident cardiovascular disease and with carotid atherosclerosis. METHODS: Serum hepcidin concentrations were measured by tandem mass spectrometry in samples taken in 2000 from 675 infection-free participants of the prospective population-based Bruneck study (age, mean±standard deviation, 66.0±10.2; 48.1% male). Blood parameters were measured by standard methods. Dietary intakes of iron and alcohol were surveyed with a food frequency questionnaire. Carotid atherosclerosis (365 cases) was assessed by ultrasound and subjects were observed for incident stroke, myocardial infarction, or sudden cardiac death (91 events) until 2010. RESULTS: Median (interquartile range) hepcidin levels were 2.27 nM (0.86, 4.15). Most hepcidin correlates were in line with hepcidin as an indicator of iron stores. Independently of ferritin, hepcidin was related directly to physical activity (p=0.024) and fibrinogen (p<0.0001), and inversely to alcohol intake (p=0.006), haemoglobin (p=0.027), and γ-glutamyltransferase (p<0.0001). Hepcidin and hepcidin-to-ferritin ratio were not associated with prevalent carotid atherosclerosis (p=0.43 and p=0.79) or with incident cardiovascular disease (p=0.62 and p=0.33). CONCLUSIONS: In this random sample of the general community, fibrinogen and γ-glutamyltransferase were the most significant hepcidin correlates independent of iron stores, and hepcidin was related to neither atherosclerosis nor cardiovascular disease.

Design and synthesis of novel pegylated iron chelators with decreased metabolic rate.

BACKGROUND: Deferiprone has proved to be a successful iron selective chelator in a range of pathologies. However, its use is limited by rapid Phase II metabolism, necessitating the administration of large doses. In an attempt to modify metabolic rate of this class of compounds, a range of pegylated 3-hydroxypyridin-4-ones has been synthesized. EXPERIMENTAL: The synthetic route in which the polyethylene glycol counterparts are introduced to a protected pyran ring involves either a Williamson etherification reaction or direct addition leading to polyethylene glycol-containing precursors. RESULTS & DISCUSSION: The introduction of the pegylated substituent was found to lead to a relatively low rate of metabolism for some of the derivatives (6a, 6b, 8a and 8b), offering a possible improvement over deferiprone.

Synthesis of double-clickable functionalised graphene oxide for biological applications.

Azide- and alkyne-double functionalised graphene oxide (Click(2) GO) was synthesised and characterised with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), thermogravimetric analysis (TGA) and Raman spectroscopy. Fourteen-percentage increase in azide content was found, after pre-treatment of GO with meta-chloroperoxybenzoic acid (mCPBA), determined with elemental analysis. No effect on A549 cell viability was found, up to 100 µg mL(-1) and 72 h of incubation, determined with the modified lactate dehydrogenase (mLDH) assay. Two sequential copper(i) catalysed azide-alkyne cycloaddition (CuAAC) reactions were performed to conjugate the propargyl-modified blood-brain barrier targeting peptide Angiopep-2, and a bis-azide polyethylene glycol (MW = 3500), to the Click(2) GO. The final conjugate was characterised with ATR-FTIR and TGA.

Passively Targeted Curcumin-Loaded PEGylated PLGA Nanocapsules for Colon Cancer Therapy In Vivo.

Clinical applications of curcumin for the treatment of cancer and other chronic diseases have been mainly hindered by its short biological half-life and poor water solubility. Nanotechnology-based drug delivery systems have the potential to enhance the efficacy of poorly soluble drugs for systemic delivery. This study proposes the use of poly(lactic-co-glycolic acid) (PLGA)-based polymeric oil-cored nanocapsules (NCs) for curcumin loading and delivery to colon cancer in mice after systemic injection. Formulations of different oil compositions are prepared and characterized for their curcumin loading, physico-chemical properties, and shelf-life stability. The results indicate that castor oil-cored PLGA-based NC achieves high drug loading efficiency (≈18% w(drug)/w(polymer)%) compared to previously reported NCs. Curcumin-loaded NCs internalize more efficiently in CT26 cells than the free drug, and exert therapeutic activity in vitro, leading to apoptosis and blocking the cell cycle. In addition, the formulated NC exhibits an extended blood circulation profile compared to the non-PEGylated NC, and accumulates in the subcutaneous CT26-tumors in mice, after systemic administration. The results are confirmed by optical and single photon emission computed tomography/computed tomography (SPECT/CT) imaging. In vivo growth delay studies are performed, and significantly smaller tumor volumes are achieved compared to empty NC injected animals. This study shows the great potential of the formulated NC for treating colon cancer.

Mechanisms of plasma non-transferrin bound iron generation: insights from comparing transfused diamond blackfan anaemia with sickle cell and thalassaemia patients.

In transfusional iron overload, extra-hepatic iron distribution differs, depending on the underlying condition. Relative mechanisms of plasma non-transferrin bound iron (NTBI) generation may account for these differences. Markers of iron metabolism (plasma NTBI, labile iron, hepcidin, transferrin, monocyte SLC40A1 [ferroportin]), erythropoiesis (growth differentiation factor 15, soluble transferrin receptor) and tissue hypoxia (erythropoietin) were compared in patients with Thalassaemia Major (TM), Sickle Cell Disease and Diamond-Blackfan Anaemia (DBA), with matched transfusion histories. The most striking differences between these conditions were relationships of NTBI to erythropoietic markers, leading us to propose three mechanisms of NTBI generation: iron overload (all), ineffective erythropoiesis (predominantly TM) and low transferrin-iron utilization (DBA).

Obesity does not influence hepcidin and hemojuvelin levels in hemodialysis patients.

BACKGROUND/AIMS: Clinical studies have shown increased levels of hepcidin causing functional iron deficiency in obese individuals. This study examined whether obesity contributes to increased hepcidin and hemojuvelin levels in adult hemodialysis patients. METHODS: In a case-control design, 37 obese [body mass index (BMI) >30 kg/m(2)] stable hemodialysis patients and 37 patients with normal BMI (20-25 kg/m(2)), matched for age, gender and race, who fulfilled a strict set of inclusion and exclusion criteria were included in the study. Serum hepcidin and hemojuvelin, markers of iron status and inflammation, and routine hematological and biochemical variables were measured on samples obtained prior to the midweek hemodialysis session. RESULTS: Obese and nonobese patients (BMI 35.1 ± 3.4 vs. 22.8 ± 1.4 kg/m(2); p < 0.001) were similar with regard to basic comorbidities and use of erythropoietin and iron. Levels of hemoglobin, hypochromic red cells and reticulocytes were similar in the two groups. Serum iron and transferrin saturation levels were on the low side and not different between obese and lean individuals; total iron-binding capacity showed a trend towards higher levels in obese patients (48.4 ± 8.3 vs. 44.9 ± 7.4 µmol/l; p = 0.065). Levels of serum ferritin (651 ± 302 vs. 705 ± 327 µg/l; p = 0.46), hepcidin (118.3 ± 67.7 vs. 119.3 ± 78.0 ng/ml; p = 0.95) and hemojuvelin (1.90 ± 1.11 vs. 1.94 ± 1.24 mg/l; p = 0.90) were high but similar between the two groups. Serum hepcidin showed a significant correlation only with ferritin (r = 0.287, p = 0.013). CONCLUSIONS: Hepcidin and hemojuvelin levels are already considerably elevated in dialysis patients, but obesity does not have an additional impact. Further studies should examine whether increased weight contributes towards hepcidin elevation in predialysis individuals, in whom there is a lesser burden of systemic inflammation.

Manipulating the pH response of 2,3-diaminopropionic acid rich peptides to mediate highly effective gene silencing with low-toxicity.

Cationic amphipathic pH responsive peptides possess high in vitro and in vivo nucleic acid delivery capabilities and function by forming a non-covalent complex with cargo, protecting it from nucleases, facilitating uptake via endocytosis and responding to endosomal acidification by being released from the complex and inserting into and disordering endosomal membranes. We have designed and synthesised peptides to show how Coulombic interactions between ionizable 2,3-diaminopropionic acid (Dap) side chains can be manipulated to tune the functional pH response of the peptides to afford optimal nucleic acid transfer and have modified the hydrogen bonding capabilities of the Dap side chains in order to reduce cytotoxicity. When compared with benchmark delivery compounds, the peptides are shown to have low toxicity and are highly effective at mediating gene silencing in adherent MCF-7 and A549 cell lines, primary human umbilical vein endothelial cells and both differentiated macrophage-like and suspension monocyte-like THP-1 cells.