Oral Administration of PLGA Nanoparticles to Deliver Antisense Oligonucleotides to Inflammatory Lesions in the Gastrointestinal Tract.

In this study, we prepared antisense oligonucleotide (ASO)-encapsulated nanoparticles (NPs) with a suitable profile for oral administration for the treatment of inflammatory bowel disease (IBD). We chose a water-in-oil-in-water (w/o/w) method to prepare the NPs using poly(lactide-co-glycolide) as a matrix and Pluronic as a stabilizer. The obtained NPs had a suitable diameter (158 nm) for the penetration of the mucus layer, endocytic uptake by enterocytes, and accumulation in inflammatory lesions in the intestine. The amount of ASOs in the NPs was relatively large (6.41% (w/w)). When the NPs were stably dispersed in solutions that mimicked gastrointestinal (GI) juice, minimal leakage of ASOs was demonstrated over the required period. The NPs were administered orally to mice with colitis induced by dextran sodium sulfate, which reduced target gene expression in the colons and rectums of the mice, whereas naked ASO administration caused no reduction in gene expression. Thus, the NPs have the potential of promising oral carriers of ASOs for the treatment of IBD that specifically target inflammatory lesions in the GI tract, thereby reducing the non-specific toxic effects of ASOs.

Determination of Region-Specific Roles of the M3 Muscarinic Acetylcholine Receptor in Gastrointestinal Motility.

BACKGROUND: The specific role of the M3 muscarinic acetylcholine receptor in gastrointestinal motility under physiological conditions is unclear, due to a lack of subtype-selective compounds. AIMS: The objective of this study was to determine the region-specific role of the M3 receptor in gastrointestinal motility. METHODS: We developed a novel positive allosteric modulator (PAM) for the M3 receptor, PAM-369. The effects of PAM-369 on the carbachol-induced contractile response of porcine esophageal smooth muscle and mouse colonic smooth muscle (ex vivo) and on the transit in mouse small intestine and rat colon (in vivo) were examined. RESULTS: PAM-369 selectively potentiated the M3 receptor under the stimulation of its orthosteric ligands without agonistic or antagonistic activity. Half-maximal effective concentrations of PAM activity for human, mouse, and rat M3 receptors were 0.253, 0.345, and 0.127 µM, respectively. PAM-369 enhanced carbachol-induced contraction in porcine esophageal smooth muscle and mouse colonic smooth muscle without causing any contractile responses by itself. The oral administration of 30 mg/kg PAM-369 increased the small intestinal transit in both normal motility and loperamide-induced intestinal dysmotility mice but had no effects on the colonic transit, although the M3 receptor mRNA expression is higher in the colon than in the small intestine. CONCLUSIONS: This study provided the first direct evidence that the M3 receptor has different region-specific roles in the motility function between the small intestine and colon in physiological and pathophysiological contexts. Selective PAMs designed for targeted subtypes of muscarinic receptors are useful for elucidating the subtype-specific function.

Stability of DNA/Td3717 complexes for gene transfection, defined by the size and polydispersity of the complex.

The amphiphilic alpha-helical peptide, Td3717, is a bi-functional synthetic peptide that acts as both a polycation for DNA binding and a ligand for targeted delivery to tumor cells. Td3717 forms a stable complex with plasmid DNA, and the complex maintained high transfection efficiency after storage at 4 degrees C for six months and after four freeze/thaw cycles. During the storage and freeze/thaw cycling, the particle size of the DNA/Td3717 complex remained less than 100nm. The size of the complex is an important factor for its internalization into cells via the endocytosis pathway; therefore, the stability of the particles will strongly contribute to high transfection efficiencies after storage and repeated freezing/thawing.

Improvement of peptide vectors for gene delivery with active targeting profiles for phosphatidylserine.

A cationic peptide, Td3701, which was derived from factor VIII that has affinity with phosphatidylserine (PS), showed efficient transfection ability for cells that express PS on the cell surface. PS is exposed on tumor cell surfaces therefore we have focused on PS as the target molecule for tumor specific gene delivery. In this article, to improve transfection efficiency and specificity in targeting tumor cells, some amino acid residues of Td3701 were replaced. The resulting peptide, Td3717, shows higher transfection efficiency (more than 30 times that of Td3701). The transfection efficiency was dependent on the amount of PS on the cell surface, suggesting that Td3717 bound with plasmid DNA could recognize PS on the cell surface. Td3717 is expected to be useful as an efficient gene carrier molecule specific to PS-presenting tumor cells.

Peptide vector for gene delivery with high affinity for phosphatidylserine.

Since phosphatidylserine (PS) is known to translocate to the external face of the plasma membrane when the cell membrane becomes disordered, we decided to focus our attention on PS as a target molecule for gene delivery. In this paper, the novel peptide Td3701 was designed, synthesized, and characterized for its physico-chemico-biological properties. Td3701 simultaneously exhibited both characters as a DNA carrier and a sensor probe for active targeting, which seemed to be triggered by structural changes in the presence of PS. This is a very unique character among nonviral vectors, and it is believed that Td3701 could be used for selective gene delivery.

An analysis of the chest wall motions using the dynamic MRI in healthy elder subjects.

The objective of this study was to compare respiratory motions of the chest wall in the healthy elder subjects (N = 5; mean age, 71 years old) with those in healthy young subjects (n = 9; mean age, 29 years old). Thirty sequential images (scanning time, 0.4s per image) of dynamic MRI on sagittal and coronal planes were obtained while the subjects were deeply breathing. Lung volume change was simultaneously measured with pneumotachometer. In the elder subjects, dimensions of the middle and posterior parts of the diaphragm were linearly related to instantaneous lung volume. There were poor correlation between motion of the anterior diaphragm and transverse motions of the upper rib-cage and lower rib-cage. The contribution of individual part of the chest wall motion to a unit lung volume change, assessed by slope of the linear regression line, in elder subjects were not significantly different from those in young subjects. Either in the elder or young subjects, the middle and posterior parts of the diaphragm moved coordinately. We conclude that chest wall motion in the healthy elder subjects is not different from that in healthy young people and that middle and posterior parts of the diaphragm act as one functional unit during deep breathing.