Stealth Particles

An attractive use of RGD-coated DNA particles would be targeting of the neovasculature of metastases following sys temic delivery (16). Indeed, resting polarized endothelial cells of normal blood vessels express matrix-binding molecules on their basolateral face, whereas growing and less differentiated blood vessel cells of metastases also express them on the lumi-nal side, hence the targeting. Intravenous injection experiments performed in our laboratory with RGD-PEI-DNA complexes, however, did not target tumors better than the passive accumulation of PEI/DNA or transferrin-PEI-DNA complexes through the leaky vasculature (21). This was presumably due to fast clearance of the complexes. The biodistribution of widely different types of particles has been shown to benefit from coating with a layer of polyethyleneglycol (PEG). This effect is due to the inert nature of the polyether backbone as well as to brush-type polymer crowding, which prevents capture by macrophages. However, the latter property also prevents proper DNA condensation (22,23) and may even interfere with the DNA exchange/release process in the cell. PEI-PEG diblock polymers (22-24-26) or PEG postgrafting strategies (27-29) are being explored as a means to avoid interference with condensation.

As an example of combination of this approach with the monomolecular DNA particle technology discussed above, we currently attempt targeting (30,31) of the high-affinity folic acid receptor, which is overexpressed on many cancer cells. Indeed, folic acid binding was shown to trigger internalization of the complexes (32). To this end, PEG (mw = 3400) was conjugated to a strong DNA-binding moiety one one end, and to folic acid on its distal end (Fig. 4).

Monomolecular plasmid DNA particles were formed with a tetradecane-cysteine-ornithine detergent (C14COrn) (S).

Figure 3 RGD peptide-mediated transfection of HeLa cells expressing avß5 integrin. Cells were transfected with pCMVluc plasmid complexed to PEI that was grafted with increasing amounts of RGD peptide. Luciferase expression (RLU/mg protein) was measured after 24 h incubation.

Figure 4 Stepwise assembly of stealth nanometric DNA particles. Plasmid DNA was condensed with a cationic cysteine detergent in aerobic conditions. The resulting gemini lipid/DNA particles were coated with a PEG-folate corona that was anchored to the DNA by a minor groove-binding moiety. See the color insert for a color version of this figure.

Figure 4 Stepwise assembly of stealth nanometric DNA particles. Plasmid DNA was condensed with a cationic cysteine detergent in aerobic conditions. The resulting gemini lipid/DNA particles were coated with a PEG-folate corona that was anchored to the DNA by a minor groove-binding moiety. See the color insert for a color version of this figure.

Aerobic disulfide bond formation led to stable (C14COrn)2-DNA complexes. Direct binding of PEG-fol to naked DNA as well as PEG-fol grafting onto the surface of the monomo-lecular DNA particles was monitored by gel electrophoresis (Fig. 5). Electron microscopy showed PEG-coated particles to be compact, monomolecular, and stable in physiological conditions. Finally, cytometry showed them to bind to KB cells (a human nasopharyngeal cancer cell line) when folate receptors are overexpressed (Fig. 6). Targeted gene delivery to cancer cells is being attempted with these complexes.

Was this article helpful?

0 0
10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook


Post a comment