Nucleic acid circulating

Nowadays the genomics-based techniques mainly employed include microarrays [31], serial analysis of gene expression (SAGE) [32] and circulating nucleic acids isolated from plasma or serum (CNAPS) [33]. 

C. Circulating Nucleic Acids Isolated from Plasma or Serum (CNAPS)... 

Fleischacker M, The 2nd International Symposium on Circulating Nucleic Acids in Plasma and Serum (CNAPS-2), Hong Kong, February 20-21, 2001, Eur. J. Med. Res., 6 364-368, 2001. 

The presence of tumor-derived DNA in the plasma of cancer patients has inspired researchers to look for other types of circulating nucleic acids. In particular, the similarity between the placenta and a malignant neoplasm has led to the discovery of cell-free fetal DNA in the plasma and serum of pregnant women. Fetal DNA has been shown to be present in the plasma of almost all pregnant women, from the early first trimester onward, and to be present in increasing... 

A highly stable and shielded polyplex should circulate in the blood stream without undesired interactions until it reaches the target cell. At that location, specific interactions with the cell surface should trigger intracellular uptake. While lipid membrane interaction is undesired at the cell surface, it should happen subsequently within the endosomal vesicle and mediate polyplex delivery into the cytosol. During or after intracellular transport to the site of action, the polyplex stability should be weakened to an extent that the nucleic acid is accessible to exert its function. 

Both approaches are simple and allow efficient encapsulation of nucleic acid-based molecules such as oligonucleotides (9,10) and pDNA (8,10,12) in liposomes that are small in size (about lOOnm diameter) and stable in circulation, protecting the cargo from degradation. In the sections to follow, we will provide a brief overview of these methods. 

Serum albumin circulates in the blood stream transporting essential nutrients such as fatty acids to peripheral tissue. Transported molecules, called ligands, often have a special affinity for selected binding sites on proteins and nucleic acids. In this experiment, the dynamics of ligand-protein interactions will be explored with the binding of the dye phenol red to bovine serum albumin. The technique of gel filtration will be used to separate the dye-protein complex. Data will be analyzed in order to construct binding curves. 

As previously discussed, the protection of pDNA against degrading enzymes is a critical parameter for a non-viral carrier. Such ability is needed for the polyplex to protect the nucleic acid for an extended period of time in the blood while the polyplex circulates and distributes. Research conducted in 1999 by Richardson and coworkers [101] to study the ability of chitosan to protect against DNase degradation revealed that incubation of polyplexes prepared at NIP ratio of 3/1 in the presence of DNase I (8 U, 1 h incubation) protected pDNA from degradation. Other studies of chitosans as gene delivery vehicles confirm that the NIP ratio has to be at least 3/1 to 5/1 in order to provide a sufficient protective effect against DNases. 

This is particularly important when considering the disposition of biotech drugs such as peptides, proteins or nucleic acids, which have in many instances been shown to distribute to specific tissue sites, redistribute slowly back into the systemic circulation compartment, and are prone to a much greater proportion of elimination from the tissue sites rather than from the systemic circulation (i. e.,... 

In case of systemic delivery of nucleic acid drugs via the oral route only comparatively small therapeutic agents such as oligonucleotides seem to reach the systemic circulation in significant quantities via the paracellular route of uptake. 

After administration, the drug circulates in the blood, primarily as the chloride (for cisplatin), or as another rather inert form (such as the biscar-boxylate in carboplatin). In the blood, also reactions with proteins and rescue agents can take place. Upon passing through cell walls (either actively or passively), intracellular reactions with peptides and proteins may take place, presumably followed by transfer to nucleic acids. Given the strong (kinetic) preference of Pt compounds to react with class-B donor atoms (such as those from thiolates and thioethers), binding to nucleic-acid bases (a thermodynamic end product) must at least occur partially via labile intermediates. 

Ideally, drags used in the treatment of diseases affecting the CNS would be delivered directly to the site of action. However, drags generally do not readily enter the brain from the circulating blood. Access to the brain is particularly difficult for the new biotherapeutics such as peptide, protein and nucleic-acid based biopharmaceuticals. 

---