RGD specifically binds to the integrin receptors, which makes helps with the nanoparticle-cell conjugate. Even though I am not using any internalisation technique (like electroporation or etc.), I am not sure if my particles would stay at the outer membrane or will they be internalised (by phagocytose or caveolea mediated internalisation and etc.).
I want my particles to stay outside and keep binding to the outer membrane receptors for a couple of weeks. And how can I confirm this extracellular binding?
Simplest way would be fluorescence imaging after tagging your NPs with a fluorophore. You can stain your cell with another dye so that two different colors could help you to track your nanoparticle. I am not sure if your NPs can stick and stay in the membrane for a week because anything that comes in contact with the cell would be taken in by phago or endocytosis.
Simplest way would be fluorescence imaging after tagging your NPs with a fluorophore. You can stain your cell with another dye so that two different colors could help you to track your nanoparticle. I am not sure if your NPs can stick and stay in the membrane for a week because anything that comes in contact with the cell would be taken in by phago or endocytosis.
Best way to check for the attachment to the membrane will be through TIRF microscopy. Another indirect method will be to fluorescent label the NP with a FITC dye and do a flow cytometry. While processing the samples you should also quench the membrane signals with trypan blue. In the flow cytometer, the green channel will give you the percentage of internalized samples and red channel will give the quantity of nanoparticle on the membrane(refer to http://www.pnas.org/content/105/33/11613.full.pdf) . RGD moeities are mainly reported to help the nanoparticles internalize into the cell due the presence of positively charged arginine. However, my suggestion is that you can use a higher sized nanoparticle which will have difficulty in internalization but targeting will happen any which ways due to RGD moeity.
Dear Hanie,
A very good question and excellent answers by Ramya and Anusha. I just one add to their suggestions to do your entire incubation at 0 to 4 degrees*** in the fridge and do not include any permeabilizing agents such as Triton x-100. From my past experience I find that incubation in cold temperature (0 to 4 degrees, very important***) significantly reduces internalization of large molecules.
best wishes,
Refik
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