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A non-viral gene vector based on hydroxyethyl ultra-low molecular weight chitosan nanoparticles (HE-ULMWCh NPs) has been synthesized. The HE-ULMWCh is used to form nanoparticles with an enhanced green fluorescence protein (pEGFP) encoding plasmid that possesses diameters of 30–50 nm and a molecular weight of less than 2 kDa. The cytotoxicity assay shows that this new gene vector is significantly less cytotoxic than polyethylenimine whilst still retaining the characteristics of a cationic polyelectrolyte. Cellular uptake of HE-ULMWCh NP–pDNA nanocomplexes shows that HE-ULMWCh NPs can readily enter into cells via endocytosis and then be delivered to lysosomes. pDNA can be easily released from HE-ULMWCh NPs in the acidic vesicles of lysosomes, therefore resulting in a significant increase in the transfection efficiency in three different cell lines compared to naked pDNA. The in vitro results using the new gene vector are further confirmed by in vivo transfection in which HE-ULMWCh NPs provided 3.2 fold greater transfection efficiency than naked pDNA. The HE-ULMWCh NPs can therefore be concluded to be a promising delivery system for in vitro and in vivo gene transfection.

Gold nanoparticles are extensively investigated for their potential biomedical applications. Therefore, it is pertinent to thoroughly evaluate their biological effects at different levels and their underlying molecular mechanism. Frequently, there are discrepancies about the biological effects of various gold nanoparticles among the reports dealing with different models. Most of the studies focused on the different biological effects of various nano-properties of the nanomaterials. We hypothesize that the biological models with different metabolic processes would be taken into account to explain the observed discrepancies of biological effects of nanomaterials. Herein, by using mouse embryo fibroblast cell line (MEF-1) and human embryonal lung fibroblast cell line (MRC-5) as in vitro models, we studied the cellular effects of gold nanorods (AuNRs) coated with poly (diallyldimethyl ammonium chloride) (PDDAC), polyethylene glycol and polystyrene sulfonae (PSS). We found that all three AuNRs had no effects on cellular viability at the concentration of 1 nM; however, AuNRs that coated with PDDAC and PSS induced significant up-regulation of heme oxygenase-1 (HO-1) which was believed to be involved in cellular defense activities in MEF-1 but not in MRC-5 cells. Further study showed that the low fundamental expression of transcription factor Bach-1, the major regulator of HO-1 expression, in MEF-1 was responsible for the up-regulation of HO-1 induced by the AuNRs. Our results indicate that although AuNRs we used are non-cytotoxic, they cell-specifically induce change of gene expression, such as HO-1. Our current study provides a good example to explain the molecular mechanisms of differential biological effects of nanomaterials in different cellular models. This finding raises a concern on evaluation of cellular effects of nanoparticles where the cell models should be critically considered.

A novel photocatalyst was prepared by anchoring Au nanoparticles (NPs) onto one-dimensional potassium niobate (KNbO3) nanowires. Photocatalytic activity towards rhodamine B degradation over Au/KNbO3 appears to be much greater than that of KNbO3 nanowires, nanorods and commercial Alfa Aesar. In terms of reaction rate constant (k), ultraviolet excitation (λ = 365 nm) is higher than that of visible-light (λ > 420 nm) and increasing the size of Au NPs from 5 to 10 nm significantly improves the reactivity. Notably, Au NPs with a size of ca. 10 nm supported on KNbO3 nanowires display the greatest photoreactivity, with k exceeding that of commercial KNbO3 by a factor of 15. The mechanism responsible for the enhancement of photocatalytic activity was discussed, highlighting the crucial role of surface plasmon resonance as well as interband transitions on Au NPs. This study is potentially applicable to a range of low-dimensional niobate-based nanostructures combined with Au and other plasmonic NPs with promising applications in photocatalysis and relevant areas.

Abstract 4248 During erythroid differentiation, heme biosynthesis increases dramatically and this is accompanied by a marked increase in transferrin receptor 1 (TfR1) expression and iron uptake from transferrin. It has long been known that 5-aza-2′deoxycytidine (5-aza-CdR), a demethylating agent, is able to induce murine erythroid leukemia (MEL) cell differentiation. However, the detailed mechanism underlying this induction remains elusive. In the current study, we report that 5-aza-CdR induces erythroid differentiation in MEL cells and BFU-E-derived erythroblasts and that this is associated with the nuclear translocation of the transcription factor c-Myc and its binding to Max to form functional complex. As markers of erythroid differentiation, the expression levels of TfR1, ferrochelatase and ALAS2 were analyzed by quantitative real-time PCR and western blotting. TfR1 and ferrochelatase expression were increased 5-fold following 5-aza-CdR treatment. To determine whether these changes in gene expression reflected differential methylation, we analyzed 1.5 kb up- and downstream from the transcription start site (TS) of these genes. Although many potential methylation sites (CpG islands) were found in both the TfR1 and ferrochelatase genes, bisulfite sequencing revealed that few sites (< 5%) were methylated in these promoters in untreated MEL cells and that there was no difference following 5-aza-CdR treatment. This observation suggests that the increased expression of TfR1 and ferrochelatase is not directly mediated by 5-aza-CdR-induced DNA demethylation. EKLF and GATA1 are key transcription regulators in erythroid differentiation, and the iron regulatory proteins (IRPs) are important for the post-transcriptional regulation of TfR1 mRNA levels. Bioinformatic analysis showed that there were many potential methylation sites in the EKLF and IRP2 promoters, however, bisulfite sequencing showed that there was no effect of 5-aza-CdR treatment at these sites in MEL cells. Since c-Myc has been reported to stimulate TfR1 expression, we then investigated the role of c-Myc in erythroid differentiation. Levels of c-Myc mRNA and protein were not increased by 5-aza-CdR, however, we did observe a dramatic increase in the nuclear fraction of c-Myc. Furthermore, co-immunopricipitation studies showed that the amount of the Myc-Max complex, which is the form in which c-Myc activates downstream gene expression, increased in the nucleus after 5-aza-CdR treatment. Interestingly, using chromatin-immunoprecipitation we also observed very strong binding of c-Myc to the CACGTG sequence upstream of the TfR1 transcription start site following 5-aza-CdR treatment. Analogous studies using primary mouse erythroblasts derived from BFU-Es gave similar results, with 5-aza-CdR treatment leading to increased TfR1 and ferrochelatase expression, c-Myc translocation to the nucleus and increased c-Myc binding activity to TfR1 promoter. Taken together, these studies point to an important role for c-Myc in the regulation of TfR1 during erythroid differentiation induced by demethylation. Precisely how demethylation is linked to alterations in c-Myc activity is under investigating. Disclosures: No relevant conflicts of interest to declare.

Abstract 1994 Poster Board I-1016 Divalent metal transporter 1 (DMT1) and natural resistance-associated macrophage protein 1 (Nramp1) are iron transporters that localize, respectively, to the early and late endosomal compartments. DMT1 is ubiquitously expressed, while Nramp1 is found only within macrophages and neutrophils. Our previous studies have identified a role for Nramp1 during macrophage erythrophagocytosis; however, little is known about the function of DMT1 during this process. Wild-type RAW264.7 macrophages (Nramp1-/-), and those stably transfected with Nramp1 (Nramp1+/+) were treated with either DMT1-siRNA, or with ebselen, a selective inhibitor of DMT1. While macrophages lacking either functional DMT1 or Nramp1 experienced a moderate reduction in iron recycling efficiency, the ability of macrophages lacking both functional DMT1 and Nramp1 to recycle hemoglobin-derived iron was severely compromised. Compared to macrophages singly deficient in either DMT1 or Nramp1 transport ability, macrophages where DMT1 and Nramp1 were both compromised exhibited an abrogated increase in labile iron pool content, released less iron, and experienced diminished upregulation of ferroportin and heme-oxygenase 1 levels following erythrophagocytosis. These results suggest that while the loss of either Nramp1 or DMT1 transport ability results in minor impairment following erythrophagocytosis, the simultaneous loss of both Nramp1 and DMT1 iron transport activity is detrimental to the iron recycling capacity of the macrophage. Disclosures: No relevant conflicts of interest to declare.

Mitochondrial ferritin (MtFt) is a mitochondrial iron storage protein, whose function and regulation is largely unknown. Our previous results have shown that MtFt markedly affects intracellular iron distribution and homeostasis in mammalian cells (Blood105: 2161–2167, 2005). Using tumor xenografts, we examined the effects of expression MtFt on tumor iron metabolism and growth. H1299 parental or MtFt overexpressing cells were implanted into nude mice. As compared to control tumor xenografts, the expression of MtFt dramatically reduced the implanted tumor growth. A cytosolic iron starvation phenotype in MtFt expressing tumors was revealed by increased RNA-binding activity of iron regulatory proteins (IRPs) and, concomitantly, both an increase in transferrin receptor levels and a decrease in cytosolic ferritin. MtFt overexpression also led to a decrease in both total cellular heme content and heme oxygenase-1 levels. In addition, the expression of MtFt in tumors was associated with a decrease in aconitase activity and lower frataxin protein levels. Mitochondrial iron deposition in MtFt expressing tumors was directly observed by transmission electron microscopy. The pattern of iron accumulation in MtFt overexpressing tumor cells is remarkably similar to that observed in the mitochondria of sideroblastic anemia patients. In conclusion, our study shows that MtFt expression significantly affected tumor iron homeostasis by shunting iron into mitochondria; iron scarcity resulted in partial defects in heme and iron-sulfur cluster syntheses. It is likely that deprivation of iron in the cytosol is the cause of the significant inhibition of xenograft tumor growth.

We have previously described a case of severe hypochromic microcytic anemia caused by a homozygous mutation in the divalent metal transporter 1 ( DMT1 1285G>C ). This mutation encodes for an amino acid substitution (E399D) and causes preferential skipping of exon 12 during processing of the DMT1 mRNA. To examine the functional consequences of this mutation, full length DMT1 transcript with the patient’s point mutation or a DMT1 transcript with exon 12 deleted was expressed in Chinese hamster ovary (CHO) cells. Our results demonstrate that the E399D substitution has no effect on protein expression and function. In contrast, deletion of exon 12 led to a decreased expression of the protein and disruption of its subcellular localization and iron uptake activity. We hypothesize that the residual protein in hematopoietic cells represents the functional E399D DMT1 variant, but because of its quantitative reduction, the iron uptake activity of DMT1 in the patient’s erythroid cells is severely suppressed. Because of the positive effect of erythropoietin (EPO) on the growth of patient’s BFU-Es in our in vitro studies, we have treated the patient with recombinant human EPO. The dose of 1.2 μg/kg given once a week did not lead to any improvement in hemoglobin level. However, doubling the dose of EPO (2.4 μg/kg) led to an increase in hemoglobin level from 75 to 91 g/L and to an improvement in the patient’s clinical condition.

Cytosolic ferritin is well known to sequester and store iron and, consequently, protect cells against iron-mediated free radical damage. However, the function of the newly discovered mitochondrial ferritin (MtFt) is unknown. To examine the role of MtFt in cellular iron metabolism, we established a cell line that stably overexpresses the mouse MtFt under the control of tetracycline. The overexpression of MtFt caused a dose-dependent iron deficiency in the cytosol that was revealed by increased RNA-biding activity of iron regulatory proteins (IRPs) with a concomitant increase in transferrin receptor levels and a decrease in cytosolic ferritin. Consequently, the induction of MtFt resulted in a dramatic increase (2-fold) in cellular iron uptake from transferrin, most of which ended up in MtFt within mitochondria. Similarly, the induction of MtFt caused a shift of iron from cytosolic ferritin to MtFt. Moreover, the cell permeable iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) mobilized approximately twice as much iron from wild-type cells as compared to those overexpressing MtFt suggesting that iron in this protein is less accessible to SIH. Furthermore, the expression of MtFt was associated with a decrease in the activity of both mitochondrial and cytosolic aconitase activity, the latter being in agreement with the increase of IRP binding activity. In conclusion, our results indicate that the overexpression of MtFt causes a dramatic change in intracellular iron homeostasis and that the shunt of iron to mitochondrial storage protein likely limits its availability for functional iron proteins.

Green tea polyphenols have aroused considerable attention in recent years for preventing oxidative stress related diseases including cancer, cardiovascular disease, and degenerative disease. Neurodegenerative diseases are cellular redox status dysfunction related diseases. The present study investigated the different effects of the five main components of green tea polyphenols on 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells, the in vitro model of Parkinson's disease (PD). When the cells were treated with five catechins respectively for 30 min before exposure to 6-OHDA, (-)-epigallocatechins gallate (EGCG) and (-)-epicatechin gallate (ECG) in 50-200 microM had obvious concentration-dependent protective effects on cell viability, while (-)-epicatechin (EC), (+)-catechin ((+)-C), and (-)-epigallocatechin (EGC) had almost no protective effects. The five catechins also showed the same pattern described above of the different effects against 6-OHDA-induced cell apoptotic characteristics as analyzed by cell viability, fluorescence microscopy, flow cytometry, and DNA fragment electrophoresis methods. The present results indicated that 200 microM EGCG or ECG led to significant inhibition against typical apoptotic characteristics of PC12 cells, while other catechins had little protective effect against 6-OHDA-induced cell death. Therefore, the classified protective effects of the five catechins were in the order ECGor = EGCGECor = (+)-CEGC. The antiapoptotic activities appear to be structurally related to the 3-gallate group of green tea polyphenols. The present data indicate that EGCG and ECG might be potent neuroprotective agents for PD.

Malignant tumors are complex organs consisting of tumor cells and their microenvironment. Increasing evidence has shown that the tumor microenvironment is critical to the initiation and progression of tumors. Rational design of tumor therapies via targeting the tumor microenvironment to inhibit tumor growth is thus becoming a consensus strategy. Gd@C(82)(OH)(22) nanoparticles, as novel endohedral hydroxylated metallofullerenes, have been demonstrated to be a potent antitumor nanomedicine via targeting multiple factors in the tumor microenvironment. Gd@C(82)(OH)(22) nanoparticles possess excellent biocompatibility and remarkable antineoplastic activity, as a result not of direct tumor cytotoxicity but of their diverse biological effects, including antioxidation, immune activation, angiogenesis inhibition, imprisoning cancer cells, and reversal of drug-resistance. In this article, we summarize the unique nanoscale physiochemical properties and the antineoplastic activities of Gd@C(82)(OH)(22) nanoparticles, and focus on the mechanisms underlying their regulation of the tumor microenvironment.

Precisely selective interactions of peptides with their unique binding partners represent an out-standing starting point for designing novel therapeutics. It is well established that peptides with a variety of critical physiological functions and specific mechanisms of action offer distinct ad-vantages, including excellent safety and higher efficiency over traditional small molecule thera-peutics. Certain intrinsic weaknesses of naturally occurring peptides such as negligible plasma half-life, low bioavailability, and potential immunogenicity have limited their administration as medicines. Nanotechnology has expanded several promising strategies to address the limitations associated with therapeutic peptides. This review aims to perform a state-of-the-art summary of the strategies that are actively used to develop efficient formulations of nanosystem based pep-tide medicines. We first focus on the recent advances and updates on peptide-based nanomedi-cines. Then we indicate how nanosystems improved the functionality of therapeutic peptides and what the future opportunities and challenges of developments in the field of therapeutic pep-tides are. Potential noninvasive delivery platforms for peptide incorporated nanoparticles through alternative administration routes are also discussed.