Conjugated polymer nanoparticles for fluorescent labelling of live cells and delivery of biological molecules into plant cells

Conjugated Polymer Nanoparticles (CPNs) are intrinsically fluorescent amine polymers whose engineered dimensions are100 nm. CPNs possess advantageous photophysical properties such as high fluorescent quantum yield, large excitation coefficient and efficient signal transduction which make them useful imaging agents. CPNs are water soluble and their cationic nature makes them suitable to use as a carrier of negatively charged biological molecules. In this study CPNs were assessed for their ability to transfect Tobacco BY--2 cells and protoplasts by directly adding them into the cell culture medium. More than 50% fluorescent positive protoplasts were observed within 2 h of delivery to the culture medium using an epifluorescence microscope. CPN--internalization by protoplasts was confirmed by flow cytometry. When CPNs were delivered to intact BY--2 cells they remained bound to the cell wall and were not internalized. Thus CPNs are membrane permeable but not cell wall permeable. The cytotoxic effects of CPNs was evaluated by measuring protoplast viability following delivery of various CPN concentrations between 5 to 500 muM. Protoplasts did not show significant reduction in cell viability following treatment with CPNs concentrations ≤ 50 muM after 24 hours of incubation. However higher concentrations of CPNs caused viability to decline to 60% of untreated protoplasts. We examined the ability of CPNs to deliver small RNAs into protoplasts. CPNs delivered commercially available siGLO small RNAs into protoplasts after 2 hours of incubation. Cellulose synthase ( Ces A) gene plays an essential gene in cell wall biosynthesis in plants. We employed CPNs to deliver NtCesA--1 siRNA and found that cell wall regeneration was inhibited following siRNA delivery. These outcomes show that NtCesA--1 plays an essential role in cell wall regeneration. Therefore CPNs are valuable tools for delivery of siRNAs to study vital cellular pathways at cellular level. In order to deliver CPNs into whole plants, a protocol was developed based on particle mediated gene transfer technology known as diolistics. CPNs were successfully delivered to intact N. benthamiana leaves by coating them on gold particles followed by bombardment using the gene gun. CPNs were observed in single N. benthamiana leaf epidermal cells at 2h post bombardment. This application creates new opportunities to fluorescently label live plant tissues without detaching from the plants.