Ireneusz P. Grudzinski

A novel type of electrochemical sensor based on ferromagnetic carbon-encapsulated iron nanoparticles for direct determination of hemoglobin in blood samples

An effective, fast, facile and direct electrochemical method of determination of hemoglobin (Hb) in blood sample without any sample preparation is described. The method is accomplished by using the ferromagnetic electrode modifier (carbon-encapsulated iron nanoparticles) and an external magnetic field. The successful voltammetric determination of hemoglobin is achieved in PBS buffer as well as in the whole blood sample. The obtained results show the excellent electroactivity of Hb. The measurements are of high sensitivity and good reproducibility. The detection limit is estimated to be 0.7 pM. The electrochemical determination data were compared with the gravimetric data obtained with a quartz crystal microbalance. The agreement between these results is very good. The changes of the electrode surface morphology before and after Hb detection are monitored by electron microscopy. The functionality of the electrochemical sensor is tested with human and rat blood samples. The concentration of hemoglobin in the blood samples determined by using voltammetric/gravimetric detection is in perfect agreement with the data obtained from typical clinical analysis.

Non-Contrast-Enhanced Whole-Body Magnetic Resonance Imaging in the General Population: The Incidence of Abnormal Findings in Patients 50 Years Old and Younger Compared to Older Subjects

Purpose To assess and compare the incidence of abnormal findings detected during non-contrast-enhanced whole-body magnetic resonance imaging (WB-MRI) in the general population in two age groups: (1) 50 years old and younger; and (2) over 50 years old. Materials and Methods The analysis included 666 non-contrast-enhanced WB-MRIs performed on a 1.5-T scanner between December 2009 and June 2013 in a private hospital in 451 patients 50 years old and younger and 215 patients over 50 years old. The following images were obtained: T2-STIR (whole body-coronal plane), T2-STIR (whole spine-sagittal), T2-TSE with fat-saturation (neck and trunk-axial), T2-FLAIR (head-axial), 3D T1-GRE (thorax-coronal, axial), T2-TSE (abdomen-axial), chemical shift (abdomen-axial). Detected abnormalities were classified as: insignificant (type I), potentially significant, requiring medical attention (type II), significant, requiring treatment (type III). Results There were 3375 incidental findings depicted in 659 (98.9%) subjects: 2997 type I lesions (88.8%), 363 type II lesions (10.8%) and 15 type III lesions (0.4%), including malignant or possibly malignant lesions in seven subjects. The most differences in the prevalence of abnormalities on WB-MRI between patients 50 years old and younger and over 50 years old concerned: brain infarction (22.2%, 45.0% respectively), thyroid cysts/nodules (8.7%, 18.8%), pulmonary nodules (5.0%, 16.2%), significant degenerative disease of the spine (23.3%, 44.5%), extra-spinal degenerative disease (22.4%, 61.1%), hepatic steatosis (15.8%, 24.9%), liver cysts/hemangiomas (24%, 34.5%), renal cysts (16.9%, 40.6%), prostate enlargement (5.1% of males, 34.2% of males), uterine fibroids (16.3% of females, 37.9% of females). Conclusions Incidental findings were detected in almost all of the subjects. WB-MRI demonstrated that the prevalence of the vast majority of abnormalities increases with age.

Detection of active bile leak with Gd-EOB-DTPA enhanced MR cholangiography: Comparison of 20–25 min delayed and 60–180 min delayed images

OBJECTIVES The purpose of this study was to assess the value of contrast-enhanced magnetic resonance cholangiography (MRC) performed in different time delays after injection of gadoxetic acid disodium (Gd-EOB-DTPA) for the diagnosis of active bile leak. METHODS This retrospective analysis included Gd-EOB-DTPA enhanced MR images of 34 patients suspected of bile leak. Images were acquired 20-25 min after Gd-EOB-DTPA injection. If there was inadequate contrast in the bile ducts then delayed images after 60-90 min and 150-180 min were obtained. Results were correlated with intraoperative findings, ERCP results, clinical data, laboratory tests, and follow-up examinations. RESULTS Gd-EOB-DTPA enhanced MRC yielded an overall sensitivity of 96.4%, specificity of 100% and accuracy of 97.1% for the diagnosis of an active bile leak. The sensitivity of 20-25 min delayed MR images was 42.9%, of combined 20-25 min and 60-90 min delayed images was 92.9% and of combined 20-25 min, 60-90 min and 150-180 min delayed images was 96.4%. CONCLUSIONS Gd-EOB-DTPA enhanced MRC utilizing delayed phase images was effective for detecting the presence and location of active bile leaks. The images acquired 60-180 min post-injection enabled identification of bile leaks even in patients with a dilated biliary system or moderate liver dysfunction.

Comparative cytotoxicity studies of carbon-encapsulated iron nanoparticles in murine glioma cells.

Carbon-encapsulated iron nanoparticles (CEINs) have recently emerged as a new class of magnetic nanomaterials with a great potential for an increasing number of biomedical applications. To address the current deficient knowledge of cellular responses due to CEIN exposures, we focused on the investigation of internalization profile and resulting cytotoxic effects of CEINs (0.0001-100 μg/ml) in murine glioma cells (GL261) in vitro. The studied CEIN samples were characterized (TEM, FT-IR, Zeta potential, Boehm titration) and examined as raw and purified nanomaterials with various surface chemistry composition. Of the four type CEINs (the mean diameter 47-56 nm) studied here, the as-synthesized raw nanoparticles (Fe@C/Fe) exhibited high cytotoxic effects on the plasma cell membrane (LDH, Calcein AM/PI) and mitochondria (MTT, JC-1) causing some pro-apoptotic evens (Annexin V/PI) in glioma cells. The effects of the purified (Fe@C) and surface-modified (Fe@C-COOH and Fe@C-(CH2)2COOH) CEINs were found in quite similar patterns; however, most of these cytotoxic events were slightly diminished compared to those induced by Fe@C/Fe. The study showed that the surface-functionalized CEINs affected the cell cycle progression in both S and G2/M phases to a greater extent compared to that of the rest of nanoparticles studied to data. Taken all together, the present results highlight the importance of the rational design of CEINs as their physicochemical features such as morphology, hydrodynamic size, impurity profiles, and especially surface characteristics are critical determinants of different cytotoxic responses.

MR Imaging of Pulmonary Nodules: Detection Rate and Accuracy of Size Estimation in Comparison to Computed Tomography.

Objective The aims of this study were to assess the sensitivity of various magnetic resonance imaging (MRI) sequences for the diagnosis of pulmonary nodules and to estimate the accuracy of MRI for the measurement of lesion size, as compared to computed tomography (CT). Methods Fifty patients with 113 pulmonary nodules diagnosed by CT underwent lung MRI and CT. MRI studies were performed on 1.5T scanner using the following sequences: T2-TSE, T2-SPIR, T2-STIR, T2-HASTE, T1-VIBE, and T1-out-of-phase. CT and MRI data were analyzed independently by two radiologists. Results The overall sensitivity of MRI for the detection of pulmonary nodules was 80.5% and according to nodule size: 57.1% for nodules ≤4mm, 75% for nodules >4-6mm, 87.5% for nodules >6-8mm and 100% for nodules >8mm. MRI sequences yielded following sensitivities: 69% (T1-VIBE), 54.9% (T2-SPIR), 48.7% (T2-TSE), 48.7% (T1-out-of-phase), 45.1% (T2-STIR), 25.7% (T2-HASTE), respectively. There was very strong agreement between the maximum diameter of pulmonary nodules measured by CT and MRI (mean difference -0.02 mm; 95% CI –1.6–1.57 mm; Bland-Altman analysis). Conclusions MRI yielded high sensitivity for the detection of pulmonary nodules and enabled accurate assessment of their diameter. Therefore it may be considered an alternative to CT for follow-up of some lung lesions. However, due to significant number of false positive diagnoses, it is not ready to replace CT as a tool for lung nodule detection.

Assessing carbon-encapsulated iron nanoparticles cytotoxicity in Lewis lung carcinoma cells

Carbon‐encapsulated iron nanoparticles (CEINs) have been considered as attractive candidates for several biomedical applications. In the present study, we synthesized CEINs (the mean diameter 40–80 nm) using a carbon arc route, and the as‐synthesized CEINs were characterized (scanning and transmission electron microscopy, dynamic light scattering, turbidimetry, Zeta potential) and further tested as raw and purified nanomaterials containing the carbon surface modified with acidic groups. For cytotoxicity evaluation, we applied a battery of different methods (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide, lactate dehydrogenase, calcein AM/propidium iodide, annexin V/propidium iodide, JC‐1, cell cycle assay, Zeta potential, TEM and inductively coupled plasma mass spectrometry) to address the strategic cytotoxic endpoints of Lewis lung carcinoma cells due to CEIN (0.0001–100 µg ml–1) exposures in vitro. Our studies evidence that incubation of Lewis lung carcinoma cells with CEINs is accompanied in substantial changes of zeta potential in cells and these effects may result in different internalization profiles. The results show that CEINs increased the mitochondrial and cell membrane cytotoxicity; however, the raw CEIN material (Fe@C/Fe) produced higher toxicities than the rest of the CEINs studied to data. The study showed that non‐modified CEINs (Fe@C/Fe and Fe@C) elevated some pro‐apoptotic events to a greater extent compared to that of the surface‐modified CEINs (Fe@C‐COOH and Fe@C‐(CH2)2COOH). They also diminished the mitochondrial membrane potentials. In contrast to non‐modified CEINs, the surface‐functionalized nanoparticles caused the concentration‐ and time‐dependent arrest of the S phase in cells. Taken all together, our results shed new light on the rational design of CEINs, as their geometry, hydrodynamic and, in particular, surface characteristics are important features in selecting CEINs as future nanomaterials for nanomedicine applications. Copyright

Conjugation of polyethylenimine and its derivatives to carbon-encapsulated iron nanoparticles

Carbon-based nanomaterials functionalized by cationic polymers are interesting starting materials for the development of nanotheranostic systems. In this study, polyethylenimine (PEI) and its pre-synthesized derivatives were conjugated to carbon-encapsulated iron nanoparticles (CEINs). Branched PEIs of various molecular weight were derivatized. The aim of the polymer modification was to introduce carboxylic functionality to the PEI structure. Two different synthetic pathways were proposed: the amide-type reaction with succinic acid anhydride and reductive amination using p-formylbenzoic acid. The polyethylenimine derivatives were analyzed by means of spectroscopic methods (NMR and FT-IR). In order to determine the ratio of primary, secondary and tertiary amine groups in the modified polymers, inverted-gate 13C NMR spectroscopy was applied. Next, CEINs modified with two different surface carboxylic linkers were functionalized using pristine PEI and its derivatives. The conjugation of the polymer to the surface-modified nanoparticles was carried out using the carbodiimide–amine type reaction. The success of the conjugation process was confirmed by thermogravimetry and infrared spectroscopy. The morphological details were analyzed using transmission electron microscopy, whilst the surface zeta potential and the average particle size were determined by dynamic light scattering. It was found that the molecular weight of the polymer and the type of the surface linker were the key factors which crucially influenced the functionalization yield and the physicochemical features of the synthesized nanoplatforms. The best dispersion stability in aqueous media and the smallest mean hydrodynamic particle size was found for CEINs with the longer carboxylic linker.

Sulfhydrylated graphene-encapsulated iron nanoparticles directly aminated with polyethylenimine: a novel magnetic nanoplatform for bioconjugation of gamma globulins and polyclonal antibodies

This study presents for the first time the direct amination of graphene-encapsulated iron nanoparticles (GEINs) with polyethylenimine (PEI) via radical-type reaction. This work describes the first example of a direct addition of N-centered radical species onto the graphene layer. The pristine PEI and the PEI attached to GEINs have also been derivatized to introduce sulfhydryl functionalities. The proposed two-step protocol constitutes a novel, versatile and low cost method for the synthesis of polymer derivatives decorated with SH moieties. The derivatives of pristine polyethylenimine were analyzed by means of spectroscopic methods (NMR and IR), while the obtained carbon materials were studied by thermogravimetry, infrared spectroscopy, dynamic light scattering, and transmission electron microscopy. Finally, the concomitant part of this work focused on the bioconjugation type reactions of various biocompounds, including bovine gamma-globulins and human polyclonal antibodies of class IgG, with the as-obtained sulfhydrylated GEINs-PEI nanoplatform. The presence of immobilized molecules was confirmed by thermogravimetry, protein and fluorescence assays as well as confocal microscopy images.

Internalization and cytotoxicity effects of carbon-encapsulated iron nanoparticles in murine endothelial cells: Studies on internal dosages due to loaded mass agglomerates

Carbon-encapsulated iron nanoparticles (CEINs) qualified as metal-inorganic hybrid nanomaterials offer a potential scope for an increasing number of biomedical applications. In this study, we have focused on the investigation of cellular fate and resulting cytotoxic effects of CEINs synthesized using a carbon arc route and studied in murine endothelial (HECa-10) cells. The CEIN samples were characterized as pristine (the mean diameter between 47 and 56nm) and hydrodynamic (the mean diameter between 270 and 460nm) forms and tested using a battery of methods to determine the cell internalization extent and cytotoxicity effects upon to the exposures (0.0001-100μg/ml) in HECa-10 cells. Our studies evidenced that the incubation with CEINs for 24h is accompanied with substantial changes of Zeta potential in cells which can be considered as a key factor for affecting the membrane transport, cellular distribution and cytotoxicity of these nanoparticles. The results demonstrate that CEINs have entered the endothelial cell through the endocytic pathway rather than by passive diffusion and they were mainly loaded as agglomerates on the cell membrane and throughout the cytoplasm, mitochondria and nucleus. The studies show that CEINs induce the mitochondrial and cell membrane cytotoxicities in a dose-dependent manner resulting from the internal dosages due to CEIN agglomerates. Our results highlight the importance of the physicochemical characterization of CEINs in studying the magnetic nanoparticle-endothelial cell interactions because the CEIN mass agglomerates can sediment more or less rapidly in culture models.

New Insight into the Synthesis and Biological Activity of the Polymeric Materials Consisting of Folic Acid and β-Cyclodextrin

This work presents a very new look at folate targeting and is focused on synthesizing and assessing the biological activity of folic acid-targeted drug delivery materials based on β-cyclodextrin. Both folic acid and β-cyclodextrin have been covalently conjugated to branched polyethylenimine as the polymeric vector. Host-guest inclusion of folic acid into a β-cyclodextrin cavity, demonstrated by means of the spectroscopic methods (2-D NMR, IR, UV-Vis), is found to be of crucial importance for biological activity of nanotherapeutics. This paper describes the very first example of the versatile synthetic approach to create the polymeric biosystems, where folic acid activity is not limited by the inclusion phenomenon. Cytotoxicity of the obtained polymeric materials against Lewis lung carcinoma cells is determined by neutral red uptake assay. Folate receptor-binding studies reveal that the developed synthetic approach enables full exploitation of the potential of folic acid as a targeting ligand.