Protein Delivery in Living Cells
Oz Biosciences (www.ozbiosciences.com) has developed a lipid-based formulation designed to interact with proteins and introduce them inside the cytoplasm of living cells. Many molecules and formulations were tested; some were able to interact efficiently with the assayed proteins, but few were able to deliver them inside cells. SM181 allowed the efficient delivery of several proteins in different cell lines. In fact, proteins rapidly accumulated inside the cytosol in four to five hours and appear as a diffuse label (Figure 1A, B, C, D). The use of proteins without SM181 did not lead to any fluorescent signal inside the cells.
Surprisingly, the presence of serum does not interfere with the protein delivery assay but helps to obtain more reproducible results instead. It appears that complexes between proteins and SM181 are smaller when serum is present during the incubation with cells, which could facilitate uptake. Down the road this feature could allow in vivo applications, which is not foreseeable with existing reagents.
Additionally, the delivered protein is still functional since the enzymatic activity of the b-galactosidase is maintained upon delivery (Figure 1A). This was confirmed by the delivery of an active caspase-3 inside cells.
The goal of this experiment was to study the impact of delivery of an apoptosis-inducing protein on cell physiology. After six hours of incubation, most of the cells were detached from the culture dish or showed a nonphysiological round shape (Figure 1E and F). The annexin-V-FITC staining confirms that almost 50% of the cells were apoptotic (Figure 1G).
The formulation and composition of SM181 was further refined to improve antibody delivery. The resulting new lipid formulation (Ab-SM181) allows delivery of FITC-labeled antibodies in the cytosol (Figure 2A) with high efficiency. The vast majority of the cells were positive after only a few hours of incubation, and optimum delivery was reached at four to six hours (Figure 2D).
Two different specific antibodies were used in additional research to see if they were able to reach and recognize their target inside the cell. The first one was directed against the cytosolic domain of giantin, a large transmembrane protein localized to the Golgi apparatus. The second one was directed against some proteins of the nuclear pore complex (NPC). The antigiantin antibody localized to an area close to the nucleus as a punctuate stain (Figure 2B), showing it is functional and able to reach its intracellular target. This result has been confirmed by the use of the anti-NPC antibody, which localized to and stained the nuclear envelope (Figure 2C).
After two to three hours of incubation, both antibodies showed diffuse staining in the cytosol before they accumulated in the expected area. This accumulation is not only time dependent but also cell-type and protein-concentration dependent. Consequently, it will take a longer time to observe a specific staining if high amount of antibodies are delivered.
In another experiment, we verified that NLS-bearing antibodies were able to localize and accumulate inside the nucleus confirming that the proteins are freely released and fully functional inside cells (data not shown). The protocol is a fast three-step procedure.