Effect of end group modification of DNA-functionalized gold nanoparticles on cellular uptake in HepG2 cells.

Understanding the dynamics of the cellular uptake of nanoparticles in human derived (cancer) cells is crucial to the rational design of functional nanoprobes that can be used for the targeting and delivery of drugs. This study reports on the cellular uptake of gold nanoparticles (GNPs) that were functionalized with different oligonucleotide derivatives using HepG2 cancer cells as a model system. DNA oligomers, in which the end group was modified (NH3, PO3, OH, CH3, and SH groups) were introduced onto the GNP surface. Then, quantitative and qualitative analyses using each DNA-GNP complex were carried out via dark-field scattering microscopy and ICP-MS measurements. Visualization of microscopic images of single cells indicated that the uptake of DNA-GNPs was highly dependent on the type of functionality of the end group in the DNA-GNP complex; the functionality of CH3, and SH resulted in less cellular uptake than that for modifications with NH3, PO3, OH for the same incubation time. This result was reinforced by ICP-MS quantitative analysis. These results were also strongly supported by the events of a DNA-GNP/protein corona; the different association and dissociation rates of proteins around the GNPs was dependent on the functionality of the end group in the DNA-GNP complex, providing further evidence for the conclusion that the components on the surface of nanoparticles directly affected cellular uptake. The findings reported herein provide a basis for the understanding of the fate of GNP-based delivery and provide important insights into the rational design of nanoprobes for the effective treatment of various diseases.

Detection of Aspergillus fumigatus pulmonary fungal infections in mice with (99m)Tc-labeled MORF oligomers targeting ribosomal RNA.

PURPOSE: Invasive aspergillosis is a major cause of infectious morbidity and mortality in immunocompromised patients. The fungus Aspergillus fumigatus (A. fumigatus) is the primary causative agent of invasive aspergillosis. However, A. fumigatus infections remain difficult to diagnose particularly in the early stages due to the lack of a rapid, sensitive and specific diagnostic approach. In this study, we investigated (99m)Tc labeled MORF oligomers targeting fungal ribosomal RNA (rRNA) for the imaging detection of fungal infections. PROCEDURES: Three phosphorodiamidate morpholino (MORF) oligomer (a DNA analogue) probes were designed: AGEN, complementary to a sequence of the fungal 28S ribosomal RNA (rRNA) of Aspergillus, as a genus-specific probe; AFUM, complementary to the 28S rRNA sequence of A. fumigatus, as a fungus species-specific probe; and cMORF, irrelevant to all fungal species, as a control probe. The probes were conjugated with Alexa Fluor 633 carboxylic acid succinimidyl ester (AF633) for fluorescence imaging or with NHS-mercaptoacetyl triglycine (NHS-MAG3) for nuclear imaging with (99m)Tc and then evaluated in vitro and in vivo. RESULTS: The specific binding of AGEN and AFUM to fungal total RNA was confirmed by dot blot hybridization while specific binding of AGEN and AFUM in fixed and live A. fumigatus was demonstrated by both fluorescent in situ hybridization (FISH) analysis and accumulation in live cells. SPECT imaging of BALB/c mice with pulmonary A. fumigatus infections and administered (99m)Tc labeled AGEN and AFUM showed immediate and obvious accumulation in the infected lungs, while no significant accumulation of the control (99m)Tc-cMORF in the infected lung was observed. Compared to non-infected mice, with sacrifice at 1h, the accumulation of (99m)Tc-AGEN and (99m)Tc-AFUM in the lungs of mice infected with A. fumigatus was 2 and 2.7 fold higher respectively. CONCLUSIONS: In vivo targeting fungal ribosomal RNA with (99m)Tc labeled MORF probes AGEN and AFUM may be useful for A. fumigatus infection imaging and may provide a new strategy for the noninvasive diagnosis of invasive aspergillosis and other fungal infections.

Novel amino linkers enabling efficient labeling and convenient purification of amino-modified oligonucleotides.

We developed new amino linker reagents for an oligonucleotide (ONT) terminus. These reagents consist of an aminoethyl carbamate main linkage and a side-chain residue, which was a naphthylmethoxymethyl, methoxymethyl, or methyl group or a hydrogen atom. The primary amine was protected with a monomethoxytrityl (MMT) group. The chemical properties of ONTs containing these amino-modifications were investigated. The MMT group of these amino-modifications could be quite rapidly removed from the amine under very mild acidic conditions, which are not strong enough for the deprotection of a conventional aliphatic amine. This significant feature enabled the amino-modified ONTs to be conveniently purified with a reverse phase column. Furthermore, the amino-modifications efficiently reacted to active esters, as compared with other amino-modifications. We also found that the pK(a) values of the amino-modifications were lower than that of the aliphatic amine. All of the experimental results showed that these chemical properties are closely related to their structures. We report here the chemical properties and the availability of the new amino linker reagents.

Immunofluorescence analysis of antisense oligodeoxynucleotide-mediated 'knock-down' of the mouse delta opioid receptor in vitro and in vivo.

We have previously used antisense oligodeoxynucleotides (ODN) to the cloned delta opioid receptor (DOR) to inhibit the antinociceptive response to spinally administered delta opioid receptor selective agonists in mice. Here we have examined the effect of DOR antisense ODN treatment on the level of DOR expressed in NG 108-15 cells and the spinal cord, through immuno-fluorescence microscopy, to determine the efficiency and selectivity of the antisense ODN-mediated "knock-down' of the DOR in these tissues. Antisense ODN, but not mismatch control, treatment resulted in a significant reduction in DOR immunoreactivity (-ir) in NG 108-15 cells and spinal cord. Thus, the inhibition of antinociceptive response to intrathecal delta selective agonists by DOR antisense ODN correlates with the loss of DOR-ir in the superficial layers of the dorsal horn of the spinal cord.

Internalization of oligodeoxynucleotide antisense to type-1 plasminogen activator inhibitor mRNA in endothelial cells: a three-dimensional reconstruction by confocal microscopy.

A three-dimensional reconstruction analysis of localization of phosphodiester and phosphorothioate oligonucleotide antisense to type-1 plasminogen activator inhibitor (PAI-1) mRNA within endothelial cells is described. When EA.hy 926 cells were incubated with fluorescently labelled phosphodiester (PO-16) or phosphorothioate (PS-16) oligonucleotides at low, not cytotoxical concentrations, the relative brightness composition of the images of the particular samples was much higher for PS-16 than PO-16 and dependent upon the extracellular concentration and the incubation time. The 3-D reconstructions based on the series of optical sections of the samples, spaced every 1.5 microns, showed the punctuate accumulation of the oligonucleotides and a striking difference in a spatial distribution between PO-16 and PS-16 within the cytoplasm. Even after 24 h incubation of endothelial cells with 2.5 microM of PO-16 and PS-16 oligonucleotides, there was a predominant oligonucleotide localization within the cytoplasm and only traces of oligonucleotides could be seen in the cell nucleus and/or perinuclear organelles.

Effects of intrahypothalamic administration of antisense DNA for progesterone receptor mRNA on reproductive behavior and progesterone receptor immunoreactivity in female rat.

Since reproductive behaviors of female rats can be correlated with estrogen-induced increases in progestin binding by hypothalamic neurons, we hypothesized that specific progesterone receptor (PR) antisense DNA sequences might decrease these behaviors. Antisense oligonucleotides (15 bases), spanning the translation start site of rabbit PR mRNA, were microinjected directly among ventromedial hypothalamic neurons, and their behavioral effects were compared to control oligonucleotides composed of the same nucleotide bases in scrambled order. When applied 12 but not 24 hr after estradiol, the PR antisense treatment significantly reduced iordosis behavior, measured either as a reflex or in a mating behavior test. Notably, proceptive behaviors, which are strongly progesterone dependent, were greatly reduced in their occurrence (80% decrease). To see if PR protein was also reduced, antisense DNA was administered near the ventromedial hypothalamus on one side of the brain, while the other side received the scrambled control sequence or vehicle. The total number of PR-immunoreactive cells on the antisense side was significantly lower in the ventromedial nucleus, but not in control measurements from the medial preoptic area. Interrupting gene expression for PR, a transcription factor, in hypothalamic neurons, can have behavioral and immunocytochemical effects.