Nanotechnology continues to advance anticancer gene therapy

Nov 20 2006

Given that cancer is a disease in which genetic errors play a major role, it should come as no surprise that many experts envision a time when gene therapy will play an equally important role in the treatment of cancer. But before that day can come, researchers much overcome a major hurdle: safely delivering therapeutic genes and other nucleic acid-based regulatory agents into malignant cells. Enter nanotechnology.

With the ability to sequester a wide variety of molecules and deliver them in a targeted manner to tumors, nanoparticles could prove to be the ideal delivery vehicle for oligonucleotide-based drugs such as anticancer genes, antisense oligodeoxynucleotides, and small interfering RNAs. Recently, for example, a team of investigators led by Huixin He, Ph.D., of Rutgers University, and T.J. Thomas, Ph.D., of the Robert Wood Johnson Medical School, demonstrated that dendrimer-based nanoparticles can deliver antisense oligodeoxynucleotides into breast cancer cells.

The researchers, who reported their work in the journal Nanotechnology, formed the nanoparticles using a biocompatible dendrimer made of poly(propyleneimine) (PPI). This particular type of dendrimer belongs to a family of what are known as amine-terminated polymers, a class of compounds that other investigators have found promote gene uptake by cells. These dendrimers are also relatively easy to modify chemically, affording the option of adding tumor-targeting agents or additional anticancer drugs to the nanoparticle.

In this study, the investigators showed that simply mixing the dendrimer with antisense oligodeoxynucleotides triggered a self-assembly process that generated stable nanoparticles. Electron microscopy revealed that these nanoparticles were toroidal in shape, a finding that implies that the dendrimer and oligodeoxynucleotide first zip together to form a single structure that then wrap around themselves to create the final nanoparticle.

Using an antisense oligodeoxynucleotide that they labeled with a fluorescent dye, the investigators were then able to track uptake of this agent by breast cancer cells. Little, if any, native oligodeoxynucleotide entered breast cancer cells, but oligodeoxynucleotide trapped within the dendrimer nanoparticle accumulated rapidly in the cells. Confocal microscopy revealed that the oligodeoxynucleotide not only entered the cells but built up in the cells' nuclei.

In another recent study, published in the journal Biomaterials, Andreas Bernkop-Schnürch, Ph.D., and colleagues at the Leopold-Franzens University in Innsbruck, Austria, used the naturally occurring polymer chitosan as the starting material for a gene delivery nanoparticle. The researchers first modified chitosan, a polymer obtained from shrimp and crab shells, with a chemical that added multiple free sulfur-containing thiol groups to each molecule of chitosan. Thiol groups play an important role in stabilizing some proteins by linking to one another – under certain cellular conditions, two thiol groups that come close to each other will react to form a sulfur-sulfur bond. These so-called disulfide bonds can stabilize a protein's three-dimensional structure.

The investigators in this study took advantage of this process to form chitosan-DNA nanoparticles that are stable in blood. Mixing thiol-modified chitosan with DNA triggers a self-assembly process that creates a nanoparticle. As the chitosan chains fold up upon one another, some of the thiol groups come close to other thiol groups. Disulfide bonds form as a result, stabilizing the nanoparticle.

To test this stability, the investigators exposed the nanoparticles to artificial intestinal fluid. Over the course of 10 hours, the nanoparticles released a mere 10 percent of their DNA payload. However, when the investigators added the nanoparticles to a solution that more closely mimics the chemical conditions inside a cell, the nanoparticles started falling apart, releasing nearly all of their DNA payload over the same time period. Under the latter conditions, disulfide bonds break easily, allowing the chitosan chains to drift apart from one another and release any DNA entrapped within them. In a final experiment, the researchers showed that the thiol-modified chitosan-DNA was more effective at getting DNA into cells than unmodified chitosan nanoparticles.

The work with dendrimers, which was funded by the National Cancer Institute, is detailed in a paper titled, "Oligodeoxynucleotide nanostructure formation in the presence of polypropyleneimine dendrimers and their uptake in breast cancer cells." An abstract of this paper is available at the journal's website. View abstract.

The work with chitosan nanoparticles is detailed in a paper titled, "Development and in vitro evaluation of a thiomer-based nanoparticulate gene delivery system." This paper has been published online in advance of print publication. An abstract is available through PubMed. View abstract.