Oligonucleotides entrapment

Ethanol is required for entrapment to occur. Addition of increasing amounts of ethanol to 100 nm DSPC/Chol/DODAP liposomes leads to the formation of large lipid structures following oligonucleotide addition and a concomitant increase in oligonucleotide entrapment levels (Table 1). The... 

Brignole, C., Marimpietri, D., Pagnan, G., Di Paolo, D., Zancolli, M., Pistoia, V., Ponzoni, M., and Pastorino, F. (2005), Neuroblastoma targeting by c-myb-selective antisense oligonucleotides entrapped in anti-GD2 immunoliposome Immune cell-mediated antitumor activities, Cancer Lett., 228,181-186. 

Antisense therapy Aimed at inhibiting the production of specific proteins, antisense oligonucleotides entrapped in PLGA nanospheres disrupt growth regulation of vascular smooth muscle cells and prevent... 

The microspheres—synthesised via a two-step process (acid-catalysed hydrolysis and condensation of 3-mercaptopropyltrimethoxysilane (MPS) in aqueous solution, followed by condensation catalysed by triethanolamine)—have a narrow size distribution (Figure 5.16) and are considerably more stable than polystyrene divinylbenzene microspheres as shown in phosphoramidite oligonucleotide synthesis by the excellent retention of fluorescence intensity in each of the reagent steps involved in phosphoramidite DNA synthesis (Figure 5.17, in which the organo-silica microsphere free thiol groups are derivatized with ATTO 550 maleimide coupled to the entrapped dye). 

Table 1 Entrapment of Antisense Oligonucleotide in the Absence of Polyethyleneglycol-Ceramide ...

Morphology. Structural details were visualized by cryo-TEM. Figure 1A is a cryo-TEM image of a sample with an entrapped oligonucleotide-to-lipid ratio of 0.13 mg/mg. It confirms the coexistence of unilamellar liposomes with bi- and multilamellar liposomes. The membranes of the latter are in close contact. The inset of Figure 1A is an expanded view of a multilamellar... 

Figure 3 Plot of the entrapment efficiency expressed as the entrapped oligonucleotide-to-lipid ratio (full circles) and percent entrapment (open circles) as a function of the initial oligonucleotide-to-lipid ratio. The ratios are given in w/w. Abbreviation AS, antisense oligonucleotide.

We and others have demonstrated that Raf-1 protein serine/threonine kinase is a druggable signaling molecule in cancer therapy (1,13,17,21-25). Our laboratory has developed a novel cationic liposomal formulation for systemic delivery of intact raf ASO (LErafAON) to normal and tumor tissues in mice (13,17). The liposome-entrapped raf antisense oligonucleotide (LErafAON) is also the first liposomal ASO drug tested in humans (26,27). Systemically delivered cationic liposomal nanoparticles containing rafsiRNA (LErafsiRNA) also inhibit Raf-1 protein expression in tumor and most normal tissues in human prostate tumor (PC-3)-bearing athymic mice (Fig. 1 and Color Plate 1, see Color Plate Section). 

In addition to the untreated and BL-treated control groups, additional controls such as liposome-entrapped mismatch oligonucleotide and/or liposome-entrapped non-specific ASO should also be included to rale out the off-targef effects. 

Mewani, R.R., Tang, W., Rahman, A., Dritschilo, A., Ahmad, I., Kasid, U.N., and Gokhale, PC. (2004) Enhanced therapeutic effects of doxorubicin and paclitaxel in combination with liposome-entrapped ends-modified raf antisense oligonucleotide against human prostate, lung and breast tumor models. Int. J. Oncol. 24, 1181-1188. 

From 1984, when they were first developed, DRV liposomes have been used for liposomal encapsulation of various active substances which may be divided into three main categories (1) Low MW drug molecules (mainly hydrophilic drugs) (3-20) (2) Proteins or peptides and enzymes (21-26), and (3) DNA or oligonucleotides (26-32). From these categories, the last two are primarily used as liposomal vaccines. Some examples of substances entrapped in DRV liposomes from the last 10 year literature are presented in Table 1. 

Liposomes were filter sterilized (0.45- or 0.2- tm sterile filters) and diluted in BSS (balanced salt solution). Peptide encapsulation was estimated to range between 80 and 90% according to previous determinations (22). Non-entrapped peptides and CpG oligonucleotides can either be kept in the preparation or removed by dialysis. 

De Rosa G, et al. (2003). Long-term release and improved intracellular penetration of oligonucleotide-polyethylenimine complexes entrapped in biodegradable microspheres. Biomacromol. 4 529-536. 

Macromolecules such as genes, oligonucleotides (ODN), and peptides-proteins are entrapped by endosomal sequestration after they experience a fluid-phase uptake or receptor-mediated endocytosis. When the macromolecrdes complete their vesicular uptake, the internalized carriers face degrada-tive pathways that are a collection of hydrolases. The types of hydrolases include proteases (e.g. cathepsins), phosphatases, lipases, and glycosidases (120). Unless endosomes-lysosomes are target organelles, the success of the macromolecular therapeutics depends on the ability of such drugs to escape from the endosomal compartment into the cytosol (121). 

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