Fluoresecent pH-dependent Lipobeads in vivo

1. Fluoresecent pH-dependent Lipobeads in vivo

2. pH changes in biology • pH changes are important to several biological processes: • muscle contraction, endocytosis, cell proliferation, apoptosis, ion transport • Biological enzymes function at an optimal pH • Folded protein is stabilized by the specific ions • Protein enzyme ATPase uses a proton gradient for energy production

3. Standard pH meter • pH electrode • glass electrode filled with electrolyte and Ag/AgCl reference electrode • thin glass membrane in contact with solution • Potential difference builds up over the thin glass membrane due to differences in H+ concentration • Potential measured against reference electrodes and pH calculated • Too big to be implanted in a cell!

4. Fluorescence • Absorption • excites the molecule to excited state • some molecules may be in vibrationally or rotationally excited states • 2. Vibrational relaxation • molecule transitions to lowest energy excited state • Fluorescence • molecule returns to ground state by emitting a photon • 4. Non-radiative relaxation • molecule returns to ground state but does not emit radiation Vibration relaxation Non-radiative relaxation Fluorescence

5. pH sensitive fluorescent dyes • Fluorophores are aromatic or conjugated with delocalized electrons • pH sensitive fluorophores • emission differs at different pH values • Fluorescein and tetramethylrhodamine (pH insensitive) are used frequently in cellular applications • high absorbance and emission wavelength in the visible light range

6. Structures of pH-sensitive Dyes Highlighted groups changed with pH carboxy SNARF-1 Orgeon Green Fluorescein

7. pH Insensitive Dyes pH insensitive dyes, such as tetramethylrhodamine, are used as a control Tetramethylrhodamine

8. Experimental Results Fluorescence spectra of individual lipobeads containing fluorescein at varying pH levels: (a )pH = 5, (b) pH = 6 (c) pH = 7, (d) pH = 8.

9. Dye Delivery • Fluorescent dyes can be encapsulated by phospholipidbilayer vesicles (liposomes). • Water soluble while retaining same reactivity • Protect dye from quenchers • Liposome vesicle fuses with cellular lipid bilayer and delivers dye inside cell for reactions • Also used for drug or gene delivery Typical Liposome Liposome containing Fluorescein

10. Cellular data • Mice macrophages were incubated with fluorescent pH-sensing lipobeads • Lipobead is a membrane on a polystyrene bead • Lipobeadsfilled with fluoresceinand tetramethylrhodamine • Dyes allowed to interact endocytosedallowing dyes to interact with intracellular environment • Cells were analyzed with fluorescence-imaging microscopy • Exposure to detection light • Intracellular pH is determined from emission peaks

11. Cellular Data Mice macrophages loaded with fluorescent lipobeads under bright field (left) and fluorescent imaging (right) under x40.

12. Cellular Data • pH change of a single liposome of fluorescent marker: • A sharp drop in fluorsecence is observed (t=9 sec) when the cell ingests the dye into a more acidic environment. • The more acidic environment causes fluorescein fluorescence to decrease.

13. Advantages & Disadvantages • Advantages • No leaking like other methods (polymer matrix) • High chemical stability in solution • Protection of dye from quenching species • Disadvantges • Biocompatability/Cytotoxicity