ABSTRACT
In this paper we report on the synthesis of diversified linear polyamine architectures with different chain lengths and compositions and their interaction with phosphate groups of DNA/siRNA. The polyplex formation between model nucleotide (dsDNA) and these linear polyamines has been determined at different nitrogen to phosphorus (N/P) ratios using small-angle neutron scattering (SANS) and atomic force microscopy (AFM) techniques. AFM images showed that while linear poly(ethylene imine) (PEI)/DNA complex results in bigger spherical aggregates, poly(propylene imine)s forms torroid and cigar shaped structures upon complexation with DNA. The poly(butylene imine)s (LPBI)s form compact and soluble DNA complexes with a radii range of R(g) = 15-30 nm. Among the studied linear polyamines, the LPBIs did show the best transfection efficiency.
Subject(s)
DNA/chemistry , Polyamines/chemical synthesis , RNA, Small Interfering/chemistry , Transfection/methods , Macromolecular Substances/chemistry , Microscopy, Atomic Force , Nanoparticles/chemistry , Neutron Diffraction , Polyamines/chemistry , Polyamines/therapeutic use , Scattering, Small Angle , Structure-Activity RelationshipABSTRACT
To investigate their potential mechanisms of action, the nucleoside analogue ribavirin and a TLR9 agonist were compared. The CpG oligodeoxynucleotides (ODN) demonstrated strong TLR9-related Th1-type effects, and ribavirin appeared only to mediate signaling in TLR-transfected cells. CpG ODN represent a promising new type of therapeutic drug for hepatitis C or other infectious diseases.
Subject(s)
Adjuvants, Immunologic/pharmacology , Antiviral Agents/pharmacology , DNA-Binding Proteins/agonists , Immunity, Cellular/drug effects , Oligonucleotides/pharmacology , Receptors, Cell Surface/agonists , Ribavirin/pharmacology , Th1 Cells/drug effects , Th1 Cells/immunology , Cells, Cultured , Chemokines/biosynthesis , Cytokines/biosynthesis , Humans , NF-kappa B/drug effects , Signal Transduction/drug effects , Th1 Cells/metabolism , Th2 Cells/drug effects , Th2 Cells/metabolism , Toll-Like Receptor 9 , TransfectionABSTRACT
Synthetic oligodeoxynucleotides (ODNs) bearing CpG dinucleotides can mimic the immunostimulatory effects of bacterial DNA in vertebrates. Besides the known CpG motifs, no other sequence motif has been shown to have independent immunostimulatory effects. Several past investigators have demonstrated that the nucleotide content or the phosphorothioate (PS) backbone may have effects independently of the sequence. However, the effect of both nucleotide content and PS backbone to stimulate human leukocytes is not well understood. We investigated the immunostimulatory activity of 34 PS-ODNs with different nucleotide contents, lengths, and methylation status on human leukocytes. The thymidine content showed strong CpG-independent contribution to immunostimulation. In contrast, ODNs rich in other nucleotides (guanosine, cytosine, or adenosine) induced no or much lower levels of immunostimulation. The observed effects were highly dependent on the PS backbone chemistry. In addition to the base content and the backbone chemistry, the length of the PS-ODN was directly related to the magnitude of its stimulatory effects, especially on B cells. In addition, methylation of CpG dinucleotides did not always cause an abrogation of the immunostimulation. Immunostimulatory effects could be observed with methylated CpG ODNs, specifically as the ODN length was increased from 18 to 24 or more nucleotides (nt). In contrast, PS-ODNs with inverted CpG dinucleotides showed some but only weak immunostimulation. Our results demonstrate that non-CpG ODNs rich in thymidine or ODNs with methylated CpG motifs have length-dependent immunostimulatory effects. Such ODNs can induce effects similar to those seen with CpG ODNs but are much less efficient in stimulating human immune cells.
