PROCEEDINGS ARTICLE | July 13, 2007
Proc. SPIE. 6632, Therapeutic Laser Applications and Laser-Tissue Interactions III
KEYWORDS: Confocal microscopy, Photodynamic therapy, Data modeling, Particles, Luminescence, Oxygen, Scanning electron microscopy, Dynamic light scattering, Flow cytometry, Picosecond phenomena
A broadband frequency domain fluorescence lifetime system (from ns to ms time scale) has been developed to study the
photochemical and photodynamic behavior of model, well-controlled photosensitizer-encapsulating liposomes.
Liposomes are known to be efficient and selective photosensitizer (PS) drug delivery vesicles, however, their chemical
and physical effects on the photochemical properties of the photosensitizer have not been well characterized. The
liposomes employed in this study (both blank and photosensitizer-complexed) were characterized to determine their: a)
size distribution (dynamic light scattering), b) image (scanning electron microscope, confocal fluorescence microscopy),
c) concentration of particles (flow cytometry), d) temperature-dependant phase transition behavior (differential scanning
calorimetry, and e) spectrofluorescent spectrophotometric properties, e.g. aggregation, in the confined environment. The
fluorescence decay behavior of two families of encapsulated photosensitizers, di-and tetrasulfonated
metallophthalocyanines, and 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH), has been examined as a function
of the liposome's physical properties (size-scale, distribution and concentration of scatterer) and the impact of the
photosensitizer spatial confinement determined. It is found that the achievable size range and distribution of the PS-liposomes
is controlled by the chemical nature of the PS for large liposomes (1000 nm), and is PS independent for small
PS-liposomes (~140nm). The lifetime decay behavior was studied for all three photosensitizer-liposome systems and
compared before and after confinement. We found the nature of the decay to be similar before and after encapsulation
for the sulfonated phthalocyanines containing ionic moieties (primarily monoexponential) but not for HPPH. In the
latter, the decay transitioned from multi- to monoexponential decay upon localizing lypophilic HPPH to the liposomal
membrane. This behavior was confirmed by obtaining a similar change in lifetime response with an independent timedomain
system. We also varied the environment in temperature and oxygen content to examine the effects on the
fluorescent lifetimes of the liposomal complexes. The fluorescence decay of all three PS-containing liposomes showed
that the local spatial confinement of PS (dictated by the PS chemistry) into different domains within the liposome
directly controls the temperature-response. Membrane-bound photosensitizers were less sensitive to temperature effects
as illustrated by the decay dynamics observed in solu, that is, they developed a unique decay behavior that correlated
with the phase transition of the membrane. The fluorescent lifetime of PS-encapsulated liposomes in deoxygenated
environments, relevant to oxygen independent type I phototoxicity, was also probed in the frequency-domain revealing
that liposome-confined PS display very different trends than those observed in solu.
