SubscribeProtein delivery using SAINT-PhD is a versatile tool for studying the behavior of any protein or peptide in the cytoplasm of a cell.
There are many reasons why one wants to deliver a protein to the cytoplasm of a cell. For example, in metabolic diseases often an enzyme is not working or is totally absent. Then this enzyme needs to be delivered to the cells. Furthermore, to find out which bioactive peptide or protein is most suited for this kind of enzyme replacement therapy an efficient and reliable protein delivery system is needed.
The SAINT-PhD protein delivery system can be used to screen a range of proteins for the biological activity inside a cell. This offers great advantage over the traditional way of transfection of a plasmid construct. This method is indirect and protein expression is then dependent on transcription regulation of the plasmid DNA. However, by using SAINT-PhD the protein to be studied is delivered directly into the cytoplasm of a cell. This method offers a convenient way to study the behavior of proteins - even chemically modified ones - inside cells.
SAINT-PhD consists of a proprietary cationic pyridinium amphiphile and a helper lipid. Upon mixture of SAINT-PhD with the protein a particle of approximately 200nm in diameter is formed. In this particle the protein is enwrapped by at least one bilayer of lipids. Furthermore, in the complex formed only non-covalent interactions are present between SAINT-PhD and the protein.
The cationic amphiphiles on the surface of the particle have high affinity for the negatively charged cell surface. Upon fusion or entrapment of the particle the protein is released into the cytoplasm of the cell. The proteins delivered by SAINT-PhD are functional and unmodified.
A range of different cell types have been tested with SAINT-PhD. Protein delivery efficiencies of up to 100% of the cells have been reached. The cells that successfully have been tested with SAINT-PhD are listed in table 1.
Table 1: Cells successfully tested with SAINT-PhD
| Adherent cell lines |
| B16F10 |
| COS-7 |
| HEK-293 |
| SKOV-3 |
| U373 |
| Adherent primary cells |
| HUVEC |
| Suspension cell lines |
| Jurkat |
F(ab')2 Rabbit-anti-mouse IgG labeled with FITC is delivered efficiently into B16F10 cells by SAINT-PhD. Both in 24-well and in 96-well plates the delivery of this FITC labeled antibody has been successfully studied by FACS analysis. Figure 1 displays a typical experiment in the 24-well format showing that almost all cells are transduced when 2µg of FITC-labeled IgG was complexed with 10µL of SAINT-PhD reagent.
Figure 1: Intracellular delivery of FITC-labeled IgG with SAINT-PhD: 2µg of FITC-labeled IgG was complexed with 10µL of SAINT-PhD reagent during 5 minutes and subsequently the complex was added to B16F10 cells in a 24-well plate and incubated during 4 hours. FACS analysis reveals that 96.6% of the gated cells are positive for FITC. Controls without SAINT-PhD gave no fluorescence.
Figure 2: Linear correlation of delivered versus complexed IgG: Different amounts of FITC-labeled IgG in the range from 10ng to 300ng were complexed with 2.5µL of SAINT-PhD. FACS analysis shows a linear correlation between the total fluorescence intensity of the gated cells and the amount of FITC-labeled IgG that was used as input material to be delivered. The controls without 2.5µL of SAINT-PhD gave no fluorescence.
In figure 2 it is shown that there is a linear range for which the amount of protein that is delivered actually correlates with the amount of protein that is presented to the cells. This behavior is confirmed in a colorimetric assay of the delivery of β-galactosidase to B16F10 cells as shown in figure 3. This experiment shows that the enzyme is still active after intracellular delivery by SAINT-PhD.
Figure 3: Colorimetric assay of the delivery of β-galactosidase to B16F10 Cells: Different amounts of β-galactosidase in the range from 1ng to 1000ng were complexed with 2.5µL of SAINT-PhD. These complexes were delivered to B16F10 cells in a 96-well plate. After 24 hours of incubation the cells were washed twice and stained with X-gal. After 30 minutes color began to develop. And after 4 hours the color remained constant.
An important advantage of the SAINT-PhD protein delivery reagent is the ability to transduce proteins into cells in the presence of serum. The experiments described in this article were done using cells cultured in DMEM supplemented with 10% FBS. Therefore, the protocol for protein delivery by SAINT-PhD is very easy.
On the first day the cells are seeded. On the next day the protein of choice is complexed with SAINT-PhD and after just 5 minutes the complex was added to the cells without the need of changing the medium.
To summarize the results, proteins ranging from enzymes to antibodies can be delivered efficiently into the cytoplasm of various cell types. After only 4 hours of incubation the proteins that are delivered to the cells can be easily detected in the cytoplasm of the cells. Optimum delivery is protein dependent and varies between 4 and 24 hours of incubation.
The delivery efficiency is not influenced by the presence of serum. SAINT-PhD is a non-toxic protein delivery agent that is extremely easy to use. Therefore, it is an indispensable tool for anyone studying the intracellular function and behavior of proteins and peptides.