Nanomaterials are comparable in size to various biomolecules (1 to 100 nm), and have unique properties such as enhanced electrical conductivity, increased chemical reactivity, and novel optical properties that make them attractive candidates for various biomedical applications. Their comparable size and unique optical properties have been utilized to develop efficient tools for subcellular imaging and delivery of biomolecules. Traditional bimolecular delivery methods utilize plasmids, cationic polymers, lipids, and viruses that have inherent disadvantages such as degradation in physiological solutions, and the need for complex conjugation techniques.

Quantum dots (QDs) are nanometersized semiconductor crystals typically between 2 and 6 nm in diameter. QDs have high chemical and biochemical stability, and their small size and large surface area allow simultaneous conjugation with multiple biomolecules. They have several advantages over traditional fluorescent molecules (organic dyes), such as high resistance to photo bleaching and tunable emission wavelength based on their core diameter and composition, making them highly desirable for different biomedical applications as imaging agents and biomolecular delivery vehicles.

Typically, QDs are dispersed in aqueous buffer solutions, and have been successfully functionalized with various biomolecules like DNA, proteins, and antibodies for delivery applications. However, a major challenge is the efficient intracellular delivery of monodispersed QDs-bioconjugates freely dispersed in the cytoplasm. Several methods have been developed for the delivery of QDs into cells, including both biochemical methods such as endocytosis, pinocytosis, and lipids, and physical methods such as electroporation and micro/nanoinjection. Physical methods like electroporation have also been used to deliver large quantities of quantum dots into cells. These methods, though successful in delivery of QDs into the cellular cytoplasm, accomplish delivery of QDs as aggregates that are not monodispersed, limiting the utility of delivered QDs.

This work demonstrates a new technique for reversible permeabilization of live cells for delivery of monodispersed QDs. To minimize the effect of the endocytotic internalization pathway of QDs, experiments were performed at 4 ºC to minimize the energy available for endocytosis. This technique is based on creating a hypotonic environment outside the cells. Hypotonic exposure causes an influx of fluid into the cell due to the osmotic pressure gradient, which can be exploited to transport QDs along with the fluid into the cytoplasm of the cell. It has been previously demonstrated that osmosis-based methods can be used to transport fluids and other macromolecules into cells.

To enhance the QD delivery and dispersion in the cells, a cell permeabilization agent was used in conjunction with the hypotonic exposure. Saponin, a plantderived glycoside, has been used for permeabilization of cells and introduction of peptides into the cells. Saponins react with membranes rich in cholesterol, such as the plasma membrane, and differential permeabilization of cells has been demonstrated.

The approach described here is based on the use of Saponin at low concentrations. Saponin permeabilization, however, allows bidirectional transport into and out of the cell, which can compromise cellular processes via loss of vital intracellular molecular contents. To ensure unidirectional transport into the cell, Saponin was used in conjunction with hypotonic exposure to enhance osmosis-driven transport of QDs into the cell while minimizing leakage of intracellular contents out of the cell. This method was found to be extremely efficient in accomplishing endocytosis-free delivery of QDs into the cellular cytoplasm while maintaining cell viability.

This work was done by Krishna Medepalli, Sethu Palaniappan, and Bruce Alphenaar of the University of Louisville for Johnson Space Center. MSC-25621-1