Iron oxide nanoparticle-induced hematopoietic and immunological response in rats

The application and use of iron oxide nanoparticless (IONPs) in the biomedical field are steadily increasing, although it remains uncertain whether IONPs are safe or should be used with caution. In the present study, we investigated the toxicity profile of ultrafine IONPs in rats administered with 7.5, 15 and 30 mg IONPs/kg body wt intravenously once a week for 4 weeks. IONP treatment reduces bone marrow-mononuclear cell proliferation, increases free radical species and DNA damage leading to growth arrest and subsequently apoptosis induction at 15 and 30 mg doses. It also induces apoptosis in undifferentiated hematopoietic stem cells. IONP treatment significantly increased the pro-inflammatory cytokine (Interleukin (IL)-1β, TNF-α, and IL-6) level in serum. The induction in inflammation was likely mediated by splenic M1 macrophages (IL-6 and TNF-α secretion). IONP treatment induces splenocyte apoptosis and alteration in the immune system represented by reduced CD4+/CD8+ ratio and increased B cells. It also reduces innate defense represented by lower natural killer cell cytotoxicity. IONP administration markedly increased lipid peroxidation in the spleen, while the glutathione level was reduced. Similarly, superoxide dismutase activity was increased and catalase activity was reduced in the spleen of IONP-treated rats. At an organ level, IONP treatment did not cause any significant injury or structural alteration in the spleen. Collectively, our results suggest that a high dose of ultrafine IONPs may cause oxidative stress, cell death, and inflammation in a biological system.

The application and use of iron oxide nanoparticless (IONPs) in the biomedical field are steadily increasing, although it remains uncertain whether IONPs are safe or should be used with caution.     

Toward absolute quantification of iron oxide nanoparticles as well as cell internalized fraction using multiparametric MRI

Iron oxide nanoparticles (IONPs) are widely used as MR contrast agents because of their strong magnetic properties and broad range of applications. The contrast induced by IONPs typically depends on concentration, water accessibility, particle size and heterogeneity of IONP distribution within the microenvironment. Although the latter could be a tool to assess local physiological effects at the molecular level, it renders IONP quantification from relaxation measurements challenging. This study aims to quantify IONP concentration using susceptibility measurements. In addition, further analysis of relaxation data is proposed to extract quantitative information about the IONP spatial distribution. Mesenchymal stem cells were labeled with IONPs and the IONP concentration measured by mass spectroscopy. MR relaxation parameters (T₁, T₂, T₂*) as well as magnetic susceptibility of cylindrical samples containing serial dilutions of mixtures of free and cell‐internalized IONPs were measured and correlated with IONP concentration. Unlike relaxation data, magnetic susceptibility was independent of whether IONPs were free or internalized, making it an excellent candidate for IONP quantification. Using IONP concentration derived from mass spectroscopy and measured relaxation times, free and internalized IONP fractions were accurately calculated. Magnetic susceptibility was shown to be a robust technique to measure IONP concentration in this preliminary study. Novel imaging‐based susceptibility mapping techniques could prove to be valuable tools to quantify IONP concentration directly by MRI, for samples of arbitrary shape. Combined with relaxation time mapping techniques, especially T₂ and T₂*, this could be an efficient way to measure both IONP concentration and the internalized IONP fraction in vivo using MRI, to gain insight into tissue function and molecular imaging paradigms. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterization and evaluation of the efficacy of cationic complex mediated plasmid DNA delivery in human embryonic palatal mesenchyme cells

The purpose of this study was to develop and test a non‐viral gene delivery system that can be employed to deliver genes of interest into a pre‐osteoblastic cell line. Human embryonic palatal mesenchymal (HEPM 1486) cells were transfected with vector‐plasmid DNA (pDNA) complexes. We explored calcium phosphate and polyethylenimine (PEI) as non‐viral vectors and compared their respective in vitro transfection efficacies. Plasmid DNA encoding luciferase protein (LUC) was complexed with PEI (with differing N:P ratios) and calcium phosphate (with differing Ca:P ratios), using established protocols. The complexes prepared were then characterized for size and surface charge, using a Malvern Zetasizer Nano‐ZS. The transfection efficiency and cytotoxicity of the prepared complexes were evaluated in HEPM cells. The PEI–pDNA complexes over the whole range of N:P ratios were found to be < 160 nm in size, while the calcium phosphate–pDNA complexes were relatively bigger. The PEI–pDNA complexes prepared at a N:P ratio of 10 were found to have maximum transfection efficiency at 4 h of treatment, with minimal cytotoxicity. The highest transfection efficiency obtained with calcium phosphate–pDNA complexes (Ca:P 200) was nearly 12‐fold lower than that obtained with PEI–pDNA complexes (N:P 10). Following this, transgene expression in the HEPM cells treated with complexes prepared at a N:P ratio of 10 was further examined, using pDNA coding for enhanced green fluorescent protein (EGFP‐N1) or therapeutically relevant platelet‐derived growth factor B (PDGF‐B). In conclusion, PEI was a more effective vector for delivering genes of interest to pre‐osteoblasts than calcium phosphate. Copyright © 2014 John Wiley & Sons, Ltd.     

Biodistribution and transgene expression with nonviral cationic vector/DNA complexes in the lungs

Biodistribution of nonviral cationic vector/DNA complexes was studied after systemic or intratracheal administration to the lungs and correlated with transgene expression. Intravenous injection in C57Bl/6 mice gave maximal and significant luciferase expression in the lungs with the cationic polymer PEI 22K/DNA complexes at the highest ratios of positive/negative charges versus DNA alone. While DOTAP/DNA complexes with high charge ratio determined lower but still significant luciferase activity versus uncomplexed DNA, GL-67A and PEI 25K mediated negligible luciferase expression. Labelled PEI 22K and DOTAP complexes were evenly distributed in the alveolar region, where GFP expression was revealed, while PEI 25K and GL-67A complexes were not detected, suggesting a different interaction of these complexes with the plasma membrane of endothelial cells. Following an intratracheal injection, the highest and significant levels of transfection were obtained with slightly positive PEI complexes as compared with DNA alone, whereas cationic lipid-based vectors, DOTAP and GL-67A, gave not significant luciferase activity. Both types of polyplexes gave similar levels of lung luciferase expression by targeting different airway cell populations. PEI 25K complexes determined high levels of GFP in the bronchial cells, confirming confocal data on fluorescent complexes internalization. PEI 22K complexes gave mainly high GFP signal in the distal tract of the bronchial tree, where tagged complexes were recovered. Fluorescent lipid complexes were found in aggregates in the lumen of bronchi totally (DOTAP) or partially (GL-67A) co-localizing with surfactant protein A. Results indicated that cationic polymers could overcome the surfactant barrier which inhibited airway cell transfection mediated by cationic lipids.     

Nonlethal,attenuated, transfusion‐associated graft‐versus‐host disease in an immunocompromised child: case report and review of the literature

BACKGROUND: Transfusion‐associated graft‐versus‐host disease (TA‐GVHD) is a rare complication of transfusion of nonirradiated blood components. It usually affects children in high‐risk groups, including those who have primary immunodeficiencies (PIDs). It usually presents with skin, hepatic, digestive, and hematologic involvement and is normally fatal. CASE REPORT: We report the case of a nonlethal, attenuated, TA‐GVHD in a 7‐month‐old boy. The disease was marked by the presence of a severe rash but lacked all the other usual manifestations. We speculate that the unusually benign course of this disease, which has normally a fulminant course, was due to the fact that this child was under high‐dose corticotherapy at the time of the engraftment. This fortunate coincidence led to the survival of this child and allowed the diagnosis of a combined immunodeficiency. CONCLUSION: A high index of suspicion is required for the diagnosis and proper management of PID. The administration of nonirradiated blood components in the first year of life, sometimes before the clinical suspicion of a PID, is of great concern. TA‐GVHD may be more prevalent than reported in the literature and it is possibly a nonidentified cause of death in recipients with unexplained death and nondiagnosed PID.     

Encapsulation of rat bone marrow stromal cells using a poly‐ion complex gel of chitosan and succinylated poly(Pro–Hyp–Gly)

Encapsulation of stem cells into a three‐dimensional (3D) scaffold is necessary to achieve tissue regeneration. Prefabricated 3D scaffolds, such as fibres or porous sponges, have limitations regarding homogeneous cell distribution. Hydrogels that can encapsulate cells such as animal‐derived collagen gels need adjustment of the pH and/or temperature upon cell mixing. In this report, we fabricated a poly‐ion complex (PIC) hydrogel of chitosan and succinylated poly(Pro–Hyp–Gly) and assessed its effect on cell viability after encapsulation of rat bone marrow stromal cells. PIC hydrogels were obtained successfully with a concentration of each precursor as low as 3.0–3.8 mg/ml. The maximum gelation and swelling ratios were achieved with an equal molar ratio (1:1) of anionic and cationic groups. Using chitosan acetate as a cationic precursor produced a PIC hydrogel with both a significantly greater gelation ratio and a better swelling ratio than chitosan chloride. Ammonium succinylated poly(Pro–Hyp–Gly) as an anionic precursor gave similar gelation and swelling ratios to those of sodium succinylated poly(Pro–Hyp–Gly). Cell encapsulation was also achieved successfully by mixing rat bone marrow stromal cells with the PIC hydrogel simultaneously during its formation. The PIC hydrogel was maintained in the culture medium for 7 days at 37°C and the encapsulated cells survived and proliferated in it. Although it is necessary to improve its functionality, this PIC hydrogel has the potential to act as a 3D scaffold for cell encapsulation and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Tannic acid-stabilized gold nano-particles are superior to native tannic acid in inducing ROS-dependent mitochondrial apoptosis in colorectal carcinoma cells via the p53/AKT axis

Gold nanoparticle formulated tannic acid (AuNP-TA) was synthesized, and its anticancer activity was compared to that of free tannic acid (TA). The half maximal inhibitory concentration (IC₅₀) was reduced by half when cell lines were treated with AuNP-TA as compared to IC₅₀ values upon free TA treatment. Both showed better cytotoxic activity in HCT116 cell line as compared to MCF7 and HepG2. AuNP-TA induced death of HCT116 cells was associated with characteristic apoptotic changes. At the same treatment dose, AuNP-TA generated more ROS, caused a more extensive DNA damage and promoted higher expression of p53 and p21 than TA. Treatment with AuNP-TA regulated generation of p53 and ROS bi-directionally. Binding studies showed that TA lowered the expression of Akt, which inhibited the survival of colon cancer cells. Also, cell cycle arrest at the G2/M phase, enhanced expression of caspase-3/9, Bak, and Bax, loss of mitochondrial membrane potential, and enhanced level of cytosolic cytochrome c were observed in AuNP-TA treated HCT116 cells. Thus, AuNP-TA is more efficient than TA in inducing apoptotic cell death of HCT116 cells via the ROS/P53/Akt axis.

Tannic acid and AuNP-TA lead to death of colon cancer cells via the ROS/p53/Akt pathway, and AuNP-TA is more potent.     

Interaction between High Density and Low Density Lipoproteins during Uptake and Degradation by Cultured Human Fibroblasts

High density lipoprotein (HDL) inhibited the binding (trypsin-releasable radioactivity), internalization (cell-associated radioactivity after trypsinization), and degradation (TCA-soluble non-iodide radioactivity) of (125)I-low density lipoprotein ((125)I-LDL) by cultured normal human fibroblasts. At HDL:LDL molar ratios of 25:1 (protein ratios about 5:1), these parameters were reduced by about 25%. Unlabeled LDL was about 25 times more effective in reducing (125)I-LDL binding, implying that if HDL and LDL bind at common sites the affinity of HDL for these sites is very low or that the interaction is on some other basis. The fractional reduction in (125)I-LDL binding at a given HDL: (125)I-LDL ratio was independent of (125)I-LDL concentration and occurred equally with fibroblasts from a subject with homozygous familial hypercholesterolemia. Reciprocally, the binding, internalization, and degradation of (125)I-HDL were reduced by LDL. Preincubation of fibroblasts with HDL (or LDL) reduced the subsequent binding of (125)I-LDL (or (125)I-HDL) during a second incubation. In other studies HDL reduced the net increase in cell cholesterol content induced by incubation with LDL. HDL alone had no net effect on cell cholesterol content.These findings suggest that HDL reduces both the high affinity and the low affinity binding of LDL to human fibroblasts and that this in turn reduces the internalization and degradation of LDL. The effect of HDL on the LDL-induced changes in cell cholesterol content could be in part on this basis and in part on the basis of an HDL-stimulated release of cholesterol from the cells. These effects of HDL in vitro may be relevant to the negative correlations reported from in vivo studies between plasma HDL concentration and both body cholesterol pool size and the prevalence of clinically manifest atherosclerosis but further studies will be needed to establish this.     

Fluorescence lifetime imaging of activatable target specific molecular probes

In vivo optical imaging using fluorescently labeled self‐quenched monoclonal antibodies, activated through binding and internalization within target cells, results in excellent target‐to‐background ratios. We hypothesized that these molecular probes could be utilized to accurately report on cellular internalization with fluorescence lifetime imaging (FLI). Two imaging probes were synthesized, consisting of the antibody trastuzumab (targeting HER2/neu) conjugated to Alexa Fluor750 in ratios of either 1:8 or 1:1. Fluorescence intensity and lifetime of each conjugate were initially determined at endosomal pHs. Since the 1:8 conjugate is self‐quenched, the fluorescence lifetime of each probe was also determined after exposure to the known dequencher SDS. In vitro imaging experiments were performed using 3T3/HER2⁺ and BALB/3T3 (HER2^?) cell lines. Changes in fluorescence lifetime correlated with temperature‐ and time‐dependent cellular internalization. In vivo imaging studies in mice with dual flank tumors [3T3/HER2⁺ and BALB/3T3 (HER2^?)] detected a minimal difference in FLI. In conclusion, fluorescence lifetime imaging monitors the internalization of target‐specific activatable antibody–fluorophore conjugates in vitro. Challenges remain in adapting this methodology to in vivo imaging. Copyright © 2010 John Wiley & Sons, Ltd.     

Factors influencing inappropriate use of ED visits among type 2 diabetics in an evidence‐based management programme

Object This study analyses inappropriate use of emergency department (ED) services among type 2 diabetics under an evidence‐based management programme. Methods Using 1999‐2006 databases of Louisiana Health Care Services Division (HCSD) eight public hospitals ED visits among the uninsured and other patients in Louisiana, we termed urgent ED visits appropriate and less‐urgent visits inappropriate. Eliminating weekend ED visits, 17 458 urgent and 22 395 less‐urgent visits by 8596 patients were analysed, using generalized estimating equation methods. Results Caucasians were 0.82 times (95% CI: 0.751–0.889) less likely to use the ED inappropriately compared with African Americans. Patients with commercial insurance, Medicaid and Medicare used the ED more inappropriately than uninsured, with odds ratios of 1.28, 1.32 and 1.28, respectively. Patients hospitalized the prior year were 0.84 times (95% CI: 1.08–1.31) less likely for inappropriate. Patients in larger hospitals used the ED more inappropriately, with an odds ratio of 1.44 (95% CI: 1.32–1.56). Conclusions The study suggests that inappropriate use of the ED among diabetic patients in an evidence‐based management programme is more likely to occur among African American, patients with insurance coverage and those seeking care in larger hospitals. Reinforcing the regular use of clinic services for diabetes management, providing clinic access in off‐hours, and engaging the health plans in providing incentives for more appropriate use of the ED might reduce inappropriate ED visits. Notably, uninsured patients with diabetes from HCSD were more efficient users of the ED.     

Establishing an institutional therapeutic apheresis registry

Apheresis was first performed as a therapeutic procedure in the 1950s. The first national therapeutic apheresis (TA) registry was established in Canada in 1981 and other national registries followed, including two attempts at establishing an international TA registry. There is no national registry in the United States. Our large, academic, tertiary hospital has a very active TA service. We created a TA database to track all procedures performed by the apheresis service by transferring data from paper appointment logs and the electronic medical records into a Microsoft Access database. Retrospective data from each TA procedure performed at UAB from January 1, 2003 through December 31, 2012 were entered, including the type of procedure, indication, date, and patient demographics. Microsoft Excel was used for data analysis. During the 10‐year period, our TA service treated 1,060 patients and performed 11,718 procedures. Of these patients, 70% received therapeutic plasma exchange (TPE), 21% received extracorporeal photopheresis (ECP), 4.5% received red cell exchange (RCE), 4.2% received leukocytapheresis, and 0.6% underwent platelet depletion. Among the procedures, 54% were TPEs, 44% were ECPs, 1.3% were RCEs, 0.5% were leukocytaphereses, and 0.1% were platelet depletions. According to the current literature, national and international TA use is underreported. We believe that the UAB TA registry provides useful information about TA practices in our region and can serve as a model for other institutions. Furthermore, data from multiple institutional registries can be used for clinical research to increase the available evidence for the role of TA in various conditions. J. Clin. Apheresis 31:516–522, 2016. © 2015 Wiley Periodicals, Inc.     

Does early initiation of therapeutic plasma exchange improve outcome in pediatric stem cell transplant–associated thrombotic microangiopathy?

BACKGROUND: The use of therapeutic plasma exchange (TPE) in hematopoietic stem cell transplant–associated thrombotic microangiopathy (TA‐TMA) is controversial because the exact mechanism of injury in TA‐TMA is not yet understood. STUDY DESIGN AND METHODS: The study objective was to retrospectively review the outcome of children receiving TPE for TA‐TMA at our institution. We hypothesized that patients initiating TPE earlier in their disease course would receive a greater benefit than those starting later, regardless of the therapeutic mechanism. RESULTS: We identified 10 consecutive pediatric patients with TA‐TMA treated with TPE. Nine of these patients showed normalization of the laboratory variables associated with microangiopathy during their TPE course, but only five patients recovered renal function and survived TA‐TMA. The five survivors started TPE a median of 17 days (range, 4‐25 days) after TA‐TMA diagnosis while the five patients who died started TPE a median of 32 days (range, 17‐73 days) after TA‐TMA was diagnosed. Three of the five survivors had multiorgan failure at TA‐TMA diagnosis and completely recovered with early institution of TPE. These three survivors were able to discontinue renal replacement therapy, and all achieved a normal posttreatment creatinine. The five patients with later institution of TPE progressed to end‐stage renal disease and all died. There were no serious TPE‐related complications in either group. CONCLUSION: This is the first report evaluating TPE response in regard to procedure initiation time after TA‐TMA diagnosis. Our data suggests that early initiation of TPE might be beneficial even in patients with multiorgan failure due to TA‐TMA.     

Lactosylated poly-l-lysine targets a potential lactose receptor in cystic fibrosis and non-cystic fibrosis airway epithelial cells

Poly-l-lysine with 40% of the ε-amino groups substituted with lactosyl residues facilitated the internalization of lactosylated poly-l-lysine/cDNA complexes into cystic fibrosis (CF) and non-CF airway epithelial cells. It was previously shown that lactosylated poly-l-lysine enhanced the transfer of cDNA into the cell nucleus, resulting in transfection. The cell entry of lactosylated poly-l-lysine/cDNA complexes, however, has not been elucidated and we hypothesized that entry of the complex was by receptor-mediated endocytosis. It is shown here that binding of the vector/cDNA complexes to the cell membrane was inhibited by lactose but not N-acetyl glucosamine. Examination by electron microscopy revealed the complexes in clathrin-coated pits. Furthermore, the complexes colocalized with transferrin during cell entry and were shown in early endosomes. These results demonstrated that lactosylated poly-l-lysine/cDNA complexes enter airway epithelial cells via receptor-mediated endocytosis utilizing lactose-binding receptors, which employ the clathrin-coated pit for internalization. Taken together with the fact that nuclear translocation also is enhanced by lactose, these results demonstrate why lactosylated poly-l-lysine is an excellent vector for transfection of airway epithelial cells. Moreover, other carbohydrates covalently linked to poly-l-lysine for targeting other specific cell types, combined with lactosyl residues, can be designed for the development of other molecular conjugates for gene transfer.     

The pH sensitivity of –NH exchange in LnDOTA–tetraamide complexes varies with amide substituent

The amide proton exchange rates in various lanthanide(III) DOTA–tetraamide complexes were investigated by CEST as a function of variable chemical structures and charges on the amide substituents. Comparisons were made between YbDOTA–(gly)₄^? (Yb‐1), YbDOTA–(NHCH₂PO₃)₄^5? (Yb‐2) and YbDOTA–(NHCH₂PO₃Et₂)₄³⁺ (Yb‐3). The general shapes of the CEST vs pH profiles were similar for the three complexes but they showed maximum CEST intensities at different pH values, pH 8.3, 8.8 and 6.9 for Yb‐1, Yb‐2 and Yb‐3, respectively. This indicates that a more negatively charged substituent on the amide helps stabilize the partial positive charge on the amide nitrogen and consequently more base is required to catalyze proton exchange. The chemical shifts of the –NH protons in Yb‐1 and Yb‐2 were similar (?17 ppm) while the –NH proton in Yb‐3 was at ?13 ppm. This shows that the crystal field produced by the amide oxygen donor atoms in Yb‐3 is substantially weaker than that in the other two complexes. In an effort to expand the useful range of pH values that might be measured using these complexes as CEST agents, the shapes of the CEST vs pH curves were also determined for two thulium(III) complexes with much larger hyperfine shifted –NH proton resonances. The ratio of CEST from –NH exchange in Tm‐1 compared with CEST from –NH exchange in Tm‐3 was found to be linear over an extended pH range, from 6.3 to 7.4. This demonstrates a potential advantage of using mixtures of lanthanide(III) DOTA–tetraamides for mapping tissue pH by use of ratiometric CEST imaging. Copyright © 2011 John Wiley & Sons, Ltd.     

Absence of transfusion-associated microchimerism in pediatric and adult recipients of leukoreduced and gamma-irradiated blood components

BACKGROUND: Transfusion‐associated microchimerism (TA‐MC), the persistence of significant levels of donor white blood cells (WBCs) in blood recipients for prolonged periods, has been demonstrated after nonleukoreduced and leukoreduced transfusion to patients with severe traumatic injury. Development of TA‐MC has not been rigorously studied in settings that do not involve massive trauma where the blood is leukoreduced and irradiated. STUDY DESIGN AND METHODS: A cohort of 409 prospectively followed medical and surgical adult and pediatric female recipients of leukoreduced and mostly irradiated allogeneic red blood cell and platelet transfusions were evaluated to determine development of TA‐MC. Four‐ and 8‐weeks‐posttransfusion samples were analyzed using quantitative real‐time polymerase chain reaction for Y‐chromosome sequences in WBC DNA, the marker for microchimeric cells in female blood recipients. Repeat testing was performed on Y‐chromosome–positive samples to confirm microchimerism (MC), and subsequent posttransfusion samples were tested to investigate persistence of MC. RESULTS: On initial testing, 40 of 207 (19%) adult and 44 of 202 (22%) pediatric female blood recipients demonstrated low‐level MC. On repeat testing of these and additional specimens, 12 (3%) recipients demonstrated low‐level transient MC, but none had persistent TA‐MC similar to that seen in transfused trauma patients. CONCLUSION: Persistence of MC was not demonstrated in adult and pediatric recipients of leukoreduced and mostly irradiated blood components. The risk of TA‐MC appears to be dependent on the clinical setting and is rare other than in patients sustaining severe traumatic injury.     

Enhanced gene expression in mouse muscle by sustained release of plasmid DNA using PPE-EA as a carrier

Delivery of plasmid DNA by nanoparticles improves the DNA bioavailability, for instance in intramuscular administration, by localizing the DNA in the muscle tissue. Extracellular sustained release of the DNA may lead to more prolonged transgene expression. The present study describes a novel controlled gene delivery system based on a water soluble and biodegradable polyphosphoester, poly(2-aminoethyl propylene phosphate) (PPE-EA). The polymer degraded in PBS at 37 degrees C through the cleavage of the backbone phosphate bonds, and it was synthesized with a relative high molecular weight to ensure a suitable hydrolytic stability as a gene carrier. The tissue response and cytotoxicity study demonstrated a better tissue compatibility of PPE-EA in mouse muscle compared with commonly used polyethylenimine and poly-L-lysine. PPE-EA condensed DNA efficiently and protected DNA from nuclease and serum degradation. Sustained release of plasmid was achieved from PPE-EA/DNA complexes as a result of PPE-EA degradation. The DNA release profiles appear to be predominantly controlled by carrier degradation and the release rate of plasmid could be adjusted by varying the charge ratio of PPE-EA to DNA. At an N/P (amino to phosphate groups) ratio of 1, a 46% burst was observed for the first day, followed by about 4% release per day (24 microg DNA/day/mg of complex) for 12 days. Higher charge ratios reduced both the DNA release rate and the burst effect. The released DNA retained its structural and functional integrity. Intramuscular injection of PPE-EA-p43-LacZ complexes at N/P ratios of 0.5 and 1 resulted in enhanced beta-galactosidase expression in anterior tibialis muscle in Balb/c mice, as compared with naked DNA injections. Similarly, PPE-EA/IFN(alpha)2b DNA complexes generated an increased systemic level of interferon-alpha2b in mouse serum following intramuscular injection, as compared with naked DNA injection.     

At physiologic albumin/oleate concentrations oleate uptake by isolated hepatocytes, cardiac myocytes, and adipocytes is a saturable function of the unbound oleate concentration. Uptake kinetics are consistent with the conventional theory.

To reexamine the role of albumin in cellular uptake of long chain fatty acids, we measured [3H]oleate uptake by isolated hepatocytes, adipocytes, and cardiac myocytes from incubations containing oleate/albumin complexes at molar ratios from 0.01:1 to 2:1. For each ratio the uptake was studied over a wide range of albumin concentrations. In all three cell types and at any given oleate/albumin ratio, the uptake appeared saturable with increasing concentrations of oleate:albumin complexes despite the fact that the unbound oleate concentration for each molar ratio is essentially constant. However, the "Km" but not the "Vmax" of these pseudosaturation curves was influenced by substrate availability. At low albumin concentrations, uptake velocities did not correlate with unbound oleate concentrations. However, observed and expected uptake velocities coincided at albumin concentrations approaching physiologic levels and were a saturable function of the oleate/albumin ratios and the consequent unbound oleate concentrations employed. Hence, under the experimental conditions employed in this study using a variety of suspended cell types, oleate uptake kinetics were consistent with the conventional theory at physiologic concentrations of albumin.     

Assessment of Global Cardiac Uptake of Radiolabeled Iron Oxide Nanoparticles in Apolipoprotein-E-Deficient Mice: Implications for Imaging Cardiovascular Inflammation

Purpose

Atherosclerosis is a leading cause of death in industrialized countries and is characterized by the accumulation of lipids and inflammatory cells, including macrophages, in blood vessel walls. Therefore, the ability to image macrophages could help identify plaques that are precursors of acute thrombotic events. Previous research has shown that long-circulating nanoparticles could be used to detect macrophages within atherosclerotic plaques of the aorta. By conducting this study, we investigated whether global cardiac uptake of radiolabeled nanoparticles could allow assessment of total macrophage burden in the coronary arteries.

Procedures

Dextran-coated iron oxide nanoparticles (IONPs) were labeled with iodine-125 via Bolton–Hunter (sulfosuccinimidyl-3-[4-hydroxyphenyl]propionate) method. IONPs were characterized by means of dynamic light scattering and transmission electronic microscopy. Biodistribution studies were performed in healthy and atherosclerotic mice. Additionally, digital autoradiography of hearts from both healthy and atherosclerotic mice was performed to assess regional and global atherosclerotic burden.

Results

The [¹²⁵I]IONPs exhibited high radiolabel stability and long blood circulation, which eventually led to high heart uptake in apoE ?/? mice when compared with healthy controls. Furthermore, digital autoradiography showed substantially enhanced emission of signals from the hearts of atherosclerotic mice, while no or minimal cardiac signals were detected in healthy mice.

Conclusions

This preparation showed adequate physical-chemical properties for in vivo studies, such as small size (～30 nm), good radiolabel stability, and long circulation time. There was also significant accumulation in the heart of apoE?/? mice compared with that of healthy control animals. These findings suggest that radiolabeled dextran-coated iron oxide nanoparticles may have potential to become a useful tool to detect macrophages in the atherosclerosis plaques of coronary arteries; however, these preliminary findings should be confirmed by further studies in a larger scale in various atherosclerosis models.     

Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation

The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.     

Agar–gelatin hybrid sponge‐induced three‐dimensional in vitro ‘liver‐like’ HepG2 spheroids for the evaluation of drug cytotoxicity

Agar–gelatin hybrid sponges were used as scaffolds to induce the formation of three‐dimensional (3D) spheroids of HepG2 cells. Agar and gelatin in 2:1 ratio were used to make films and sponges. The cell adhesive properties of the films were evaluated by the attachment kinetics. The growth kinetics of HepG2 cells was studied using MTT assay and morphology of the 3D spheroids was observed through inverted optical microscopy. The liver cell‐specific functions of the 3D spheroids were evaluated in terms of albumin secretion and urea synthesis. Paracetamol was used as a model drug to investigate the use of these 3D spheroids in the preliminary cytotoxicity evaluation of drugs. The results showed that the agar–gelatin hybrid sponges induced the formation of 3D HepG2 spheroids with significant liver‐specific functions. These spheroids exhibited higher amounts of albumin and urea synthesis than the control monolayer culture. These 3D spheroids were found to be more sensitive to the drug (TCIC₅₀ value of 4.6 mM ) than the control monolayer (TCIC₅₀ value of 6.2 mM ). The study shows that agar–gelatin‐induced HepG2 3D spheroids can be used for the preliminary evaluation of the toxicity of drugs and chemicals. Copyright © 2009 John Wiley & Sons, Ltd.