Fluoresecent pH-dependent Lipobeads in vivo

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

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!

Fluorescence

Vibration relaxation

Fluo

resc

ence

Non

-rad

iativ

e re

laxa

tion

1. 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 3. Fluorescence

• molecule returns to ground state by emitting a photon

4. Non-radiative relaxation• molecule returns to ground

state but does not emit radiation

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

Structures of pH-sensitive Dyes

carboxy SNARF-1Orgeon Green

Fluorescein

Highlighted groups changed with pH

pH Insensitive Dyes

pH insensitive dyes, such as tetramethylrhodamine, are used as a control

Tetramethylrhodamine

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.

Liposome containing Fluorescein

Typical Liposome

Dye Delivery • Fluorescent dyes can be encapsulated

by phospholipid bilayer 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

Cellular data

• Mice macrophages were incubated with fluorescent pH-sensing lipobeads– Lipobead is a membrane on a polystyrene bead– Lipobeads filled with fluorescein and tetramethylrhodamine

• Dyes allowed to interact endocytosed allowing dyes to interact with intracellular environment

• Cells were analyzed with fluorescence-imaging microscopy– Exposure to detection light

• Intracellular pH is determined from emission peaks

Cellular Data

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

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.

Advantages & Disadvantages

• Advantages– No leaking like other methods (polymer matrix)– High chemical stability in solution– Protection of dye from quenching species

• Disadvantges– Biocompatability/Cytotoxicity