Iron oxide nanoparticles (IONPs) have been increasingly used in biomedical applications, but the comprehensive understanding of their interactions with biological systems is relatively limited. In this study, we systematically investigated the in vitro cell uptake, cytotoxicity, in vivo distribution, clearance and toxicity of commercially available and well-characterized IONPs with different sizes and coatings. Polyethylenimine (PEI)-coated IONPs exhibited significantly higher uptake than PEGylated ones in both macrophages and cancer cells, and caused severe cytotoxicity through multiple mechanisms such as ROS production and apoptosis. 10 nm PEGylated IONPs showed higher cellular uptake than 30 nm ones, and were slightly cytotoxic only at high concentrations. Interestingly, PEGylated IONPs but not PEI-coated IONPs were able to induce autophagy, which may play a protective role against the cytotoxicity of IONPs. Biodistribution studies demonstrated that all the IONPs tended to distribute in the liver and spleen, and the biodegradation and clearance of PEGylated IONPs in these tissues were relatively slow (>2 weeks). Among them, 10 nm PEGylated IONPs achieved the highest tumor uptake. No obvious toxicity was found for PEGylated IONPs in BALB/c mice, whereas PEI-coated IONPs exhibited dose-dependent lethal toxicity. Therefore, it is crucial to consider the size and coating properties of IONPs in their applications.

Vasoactive intestinal peptide (VIP) and other members of the pituitary adenylyl cyclase-activating peptide (PACAP) and secretin neuroendocrine peptide family are recognized with specificity by related G protein-coupled receptors. We report here the cloning, characterization, and chromosomal location of the gene encoding the human type I VIP receptor (HVR1), also termed the type II PACAP receptor. The gene spans -22 kb and is composed of 13 exons ranging from 42 to 1400 bp and 12 introns ranging from 0.3 to 6.1 kb. Primer extension analysis with poly(A)+ RNA from human HT29 colonic adenocarcinoma cells indicated that the transcription initiation site is located at position -110 upstream of the first nucleotide (+1) of the translation start codon, and 75 nt downstream of a consensus CCAAT-box motif. The G+C-rich 5' flanking region contains potential binding sites for several nuclear factors, including Spl, AP2, ATF, interferon regulatory factor 1, NF-IL6, acute-phase response factor, and NF-KcB. The HVRI gene is expressed selectively in human tissues with a relative prevalence of lung > prostate > peripheral blood leukocytes, liver, brain, small intestine > colon, heart, spleen > placenta, kidney, thymus, testis. Fluorescence in situ hybridization localized the HVR1 gene to the short arm of human chromosome 3 (3p22), in a region associated with small-cell lung cancer.The 28-aa vasoactive intestinal peptide (VIP) is structurally related to other members of a family of peptide neuroendocrine mediators that include pituitary adenylyl cyclaseactivating peptide (PACAP), secretin, glucagon, calcitonin, and parathyroid hormone. VIP has potent relaxing effects on nonvascular and vascular smooth muscle, enhancing blood flow. It also regulates water and ion flux from lung and intestinal epithelia, promotes neuronal growth and survival, and modulates many immune functions (1-3).Specific high-affinity receptors for VIP are found on distinct subsets of neural, respiratory, gastrointestinal, and immune cells (1-3). We previously cloned a cDNA encoding a human high-affinity type I VIP receptor (HVR1), also termed type II PACAP receptor, from HT29 colonic adenocarcinoma cells and human lung tissue (4). The 3-kb cDNA insert encoded a seven-transmembrane-domain HVR1 protein of 457 aa, with a deduced molecular mass of 52 kDa, that was most homologous to other members of the PACAP and secretin G proteincoupled receptor family and had a similar binding affinity for both VIP and PACAP (4 Analysis of Introns. The size of introns in the HVR1 gene was determined by polymerase chain reaction (9) of genomic clones with specific primers derived from exon sequences, for 35 cycles of 30 sec at 94°C, 2 min at 55°C, and 6 min at 72°C, using Taq polymerase (GIBCO/BRL). The reaction products were analyzed by electrophoresis in 1% agarose gels.Primer Extension. Two micrograms of poly(A)+ RNA from HT29 cells was hybridized overnight with 50,000 cpm of a 32P-end-labeled primer (5'-ACTTGGCGGGCGCATG-GTCT-3') which spans the cDNA translation sta...

Background: Gold nanoparticles (AuNPs) have shown great promise in biomedical applications. However, the interaction of AuNPs with biological systems, its underlying mechanisms and influencing factors need to be further elucidated. Purpose: The aim of this study was to systematically investigate the effects of particle size on the uptake and cytotoxicity of AuNPs in normal cells and cancer cells as well as their biological distribution in vivo. Results: Our data demonstrated that the uptake of AuNPs increased in HepG2 cancer cells but decreased in L02 normal cells, with the increase of particle size (5-50 nm). In both cancer cells and normal cells, small (5 nm) AuNPs exhibited greater cytotoxicity than large ones (20 and 50 nm). Interestingly, 5 nm AuNPs induced both apoptosis and necrosis in HepG2 cells through the production of reactive oxygen species (ROS) and the activation of pro-caspase3, whereas it mainly induced necrosis in L02 cells through the overexpression of TLR2 and the release of IL-6 and IL-1a cytokines. Among them, 50 nm AuNPs showed the longest blood circulation and highest distribution in liver and spleen, and the treatment of 5 nm AuNPs but not 20 nm and 50 nm AuNPs resulted in the increase of neutrophils and slight hepatotoxicity in mice. Conclusion: Our results indicate that the particle size of AuNPs and target cell type are critical determinants of cellular uptake, cytotoxicity and underlying mechanisms, and biological distribution in vivo, which deserves careful consideration in the future biomedical applications.

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