PHOTOSENSITIZATION OF SUBCELLULAR STRUCTURES
Statens stralevern, Norwegian Radiation Protection Authority (NRPA)
Boks 55, NO-1332 OSTERAS, Norway
When a cell responds to a photosensitizing compound combined with
optical radiation, the response may be of different types. The most
typical and best studied reactions are noxious to the cell; the cell
may die or become genetically altered, but in some situations the
cell may be stimulated to increase its synthesis of enzymes or its
rate of growth for a period after the irradiation.
The magnitude and type of reaction is determined by a number of
parameters, among others:
1. The light intensity.
2. The irradiation time.
3. The total dose of light, normally equal to the intensity x time.
4. The concentration of the photosensitizer.
5. The time of contact between the sensitizer and the cell.
6. The chemical structure of the sensitizer.
7. The transport of the sensitizer to the cell, and uptake in the cell.
8. The type of cell.
9. The physical and chemical characteristics of the environment,
extra- and intra-cellular.
10. The sub-cellular localization of the sensitizer.
In this module we will briefly describe the subcellular structures,
and how various photosensitizers are taken up by cells and
distributed among the different subcellular structures. When the
compounds are localised at different sites in the cell, they will
act as photosensitizers by a few basic biophysical processes, but it
will be shown that the biological effects on the cell will vary
The Inside of a Cell
The cell is composed of a multitude of specialized structures, which
may be grouped in:
• The outer cell membrane or plasma membrane. Plant cells and
bacteria have a cell wall composed of macromolecules
surrounding the plasma membrane.
• The cytosol, which is the liquid interior of the cell, "cell sap".
• The cytoskeleton that is composed of fibers supporting the
mechanically, and performing movements of the cell and its parts.
• The membrane bound organelles.
A more extensive review of cell structure can be found in some of
the Suggested Readings. Alberts, et al., and Becker, et al., are
both good sources.
Figure 1. Typical animal cell (left) and plant cell (right).
Where Can the Photosensitizers Come From and Where Will They End Up?
When we think about photosensitization, we often assume that the
effects are due to the addition of external sensitizers to a cell,
but a number of sensitizers originate from the interior of the cell
itself. Some amino acids and vitamins are essential to a cell's
function, and will in addition have photosensitising properties. The
effect of the photosensitization from natural substances coming from
the organism, itself, is observed typically under extreme
conditions. The synthesis of porphyrins in the cytosol and the
mitochondria is exceptional, since the control points in the body
can be circumvented causing a higher concentration of
photosensitizing porphyrins than normal.
The group of diseases called the porphyrias can in some cases cause photosensitivity of the skin by the over-production of naturally occurring porphyrins.
Water-soluble porphyrins cause increased skin fragility on
light-exposed areas such as hands, neck and face. On the other hand,
the less water soluble porphyrin, protoporphyrin IX, when present in
the skin in the porphyric disease erythropoietic protoporphyria
(EPP) will give rise to a prickling sensation in the skin, together
with an erythema. The severity of the symptoms usually increases
over 12-48 h after sun exposure. Chronic changes consist of circular
scars, and the development of a rough orange-peel appearance of the
From the above, it is clear that the properties of the
photosensitizer determine its biological effects. This is even more
marked when the photosensitizer is added to cells or injected. If
the substance stays outside the cell, its effect is normally weak,
since the local concentration of the sensitizer is low and the
distance between the exterior of the cell and vital structures that
can be altered by activated radiation products, such as singlet
oxygen or oxygen radicals, is normally too long (see below). Once
the photosensitizer is taken up, the effect increases.
Transport of photosensitizers from the outside of a cell to the
interior can take place by two main mechanisms: passive diffusion
and active uptake. A photosensitizer may diffuse through the lipid
bilayer of the plasma membrane by dissolving in the lipid phase.
Substances that are lipophilic will most readily dissolve in the
membrane. The partition coefficient between lipid phase and water will more or less determine the efficiency of the passive diffusion-mechanism. Amphiphilic photosensitizers represent a special case, since they may readily be associated with the lipid bilayer with the apolar region of the molecule, but transport through the bilayer is hindered by polar groups.
Certain molecules may be taken up in the cells by carrier-mediated
uptake, leading to a more efficient uptake than could be expected
from the partition coefficient. The carriers are specialized
structures that are designed to carry only molecules that fit the
carrier itself. The uptake of photosensitizers bound to antibodies
is an example of this mechanism. Common to both these mechanisms is
that there must be a concentration gradient that drives the
transport, and that no cellular energy is consumed in the process.
Active transport, on the other hand, requires a supply of energy
from the cell, and can be described as pumps or as transport by
vesicles that are shuttled into the cell. These vesicles are called
endosomes, and the transport connects the exterior of the cell with
the membrane bound compartments; lysosomes, endoplasmatic reticulum
and the Golgi apparatus.
Once inside the cell, the photosensitizer will distribute between
the compartments in the cell. Transport mechanisms between the
compartments are of the same general types as the ones mentioned above.
Transport of the protoporphyrin analogue, hematoporphyrin and its
Derivatives, is particularly well studied, because they are used in
photodynamic therapy of diseases. Some uptake mechanisms are slow
and some are rapid. With hematoporphyrin, the initial uptake is
fairly rapid, but a saturation of the uptake is obtained only after
extremely long incubation time. It has been shown that the portion
of hematoporphyrin that is bound rapidly to the cell is initially
present in the outer membrane, and this fraction can easily be
detached from the cell. At later times the photosensitizer becomes
redistributed to sites inside the cell, probably by association with
membrane structures and in organelles close to the nucleus. After a
longer time the sensitizer becomes more tightly bound to the cell,
and its photosensitizing efficiency increases.
As a result of the uptake and distribution in a cell, the various
photosensitizing substances can be found bound to more or less
specific structures in the cytoplasm or the nucleus. A good example
is the binding of psoralens, the plant-derived photosensitizers used
together with UV-A to treat psoriasis, between the DNA strands in
the double helix. Due to the close proximity to essential molecules,
the psoralen molecules will become covalently bound to the strands
of DNA as a result of absorption of UV-A radiation.
Redistribution of the photosensitizer in a cell may take place,
induced by a change in pH in the cell, or by metabolism of the
photosensitizer in the cell. Since photosensitizers are light
sensitive molecules, a brief light pulse can change the physical or
chemical properties of the sensitizer, and cause it to move from its
original location in the cell. The skin sensitivity of patients with
erythropoietic protoporphyria, mentioned above, involves a
redistribution of the photosensitizer protoporphyrin IX. The
protoporphyrin will detach from the primary binding sites in the red
blood cells, when a dose of light reaches the capillaries, and can be
transported to the skin, where the reactions that it causes are more
Effects of Irradiation
Type I and II.
Recall from the Basic Photosensitization Module that
photosensitization reactions can be classified as either Type I or
Type II. In a Type I reaction the photosensitizer transfers an
electron to another molecule. In a Type II reaction, the
photosensitizer transfers energy to an oxygen molecule, producing the
reactive species known as singlet oxygen.
Due to the close proximity between the psoralen molecule and the
DNA-strands, a Type I mechanism is likely to occur. A covalent bond
between one base and the psoralen is formed if a photon is absorbed
by the psoralen. Now the molecule has become glued to the DNA at one
end. If a second photon is absorbed, the other end of the molecule
may become attached to the opposite strand. The lesion, thus formed,
prevents further cell division, because it is hard to repair by
regular DNA-repair mechanisms, and the cell is likely to stop
dividing. This is exactly the purpose of treatment of psoriasis,
which is a disease characterized by hyper-proliferation of skin cells.
In real life, few things are as straightforward as the example
above, and psoralen photosensitization is no exception. This
traditional drug had been used for many years, it worked, and
everybody was happy about the simple and logical explanation of its
effect via a Type I mechanism. Eventually, however, it was shown
that the psoralens are also very efficient photodynamic (Type II)
sensitizers, affecting a number of targets by the production of
singlet oxygen. Psoralens are similar to porphyrins in the ability
to produce singlet oxygen, when the substances are present in
organelles in relatively dilute solutions, and when the sensitizer is
not bound in very close proximity to target molecules that may be
photosensitized by a Type I mechanism.
Singlet oxygen is a reactive oxygen species, but it reacts
efficiently with only particular biomolecules. The species will
diffuse in the cellular environment and collide with various
biomolecules until it hits a molecule with which it can react. After
all the lifetime of singlet oxygen is short; on the order of 106
(one microsecond), and during that time the reactive oxygen species
is able to diffuse only about 0.1 x 106
m (one tenth of a
micrometer). This distance can be compared to a typical diameter of
a cell, which is more than 100 times
larger. The lifetime of singlet oxygen is often higher in the lipid
phase of biological membranes, and membrane damage is often seen in
photosensitized reaction. But singlet oxygen can also cross interior
membranes in the cell, i.e., between the cytoplasm and the nucleus.
If the photosensitizer is confined within a membrane bound organelle
or vesicle in the cell, either dissolved in the interior or in the
membrane, the production of singlet oxygen will lead to disruption
of the vesicle. In the beginning of the era of research on
photodynamic action it was believed that the disruption of lysosomes
was the mechanism behind cell killing. The hypothesis was that the
cell was digested from within by release of hydrolytic enzymes
normally kept inside the lysosomes. Today it is known that this
hypothesis is only partly true. Cell death by apoptosis may occur
after treatment with photosensitizers and light and self
degeneration by lytic enzymes is a part of the apoptotic process. It
is interesting to note that uptake and targeted release of drugs and
other substances by light irradiation can be performed by exploiting
this well known mechanism.
Mitochondria are among the first organelles to show ultrastructural
changes after photosensitization. The function of these structures
is diverse, but the energy metabolism has been seen as their main
function in a cell. Other functions that take place in the
mitochondria, and are linked to photosensitization, are the synthesis
of natural porphyrins and the control of cell death by apoptosis. It
has been shown that cationic dyes can be rather selectively bound to
the inner membrane of the mitochondria, and that illumination then
can interfere with the respiratory functions known to take place at
that site. Localization of dye to the mitochondria can also
interfere with the distribution of calcium inside the cell, which in
turn can induce cell death under certain circumstances.
Organised arrays of macromolecules like DNA and intracellular
vesicles are not the only structures that can be photosensitised.
Molecules in the cytoplasm can also be targets. A good example of
this is the inhibition of the assembly of tubulin by
photosensitization with several sensitizers. Tubulin-fibers are
formed by the assembly of monomers during cell division, and are attached
to the chromosomes. The fibers pull the chromosomes to the two poles
of a mitotic cell, and distribute them evenly between the two
daughter cells (Figure 2).
Figure 2. Tubulin-mediated inhibition of mitosis. In normal
Mitosis, the chromosomes are pulled by microtubules, formed from
tubulin, towards the two centrioles, marking the location where the
nuclei of the two daughter cells will be formed. After that, the
cell membrane is pinched off, and the chromosomes decondense in the
newly formed cells. If the chromosomes are not separated by the
"ropes" formed from tubulin, the normal process of mitosis will not
be completed, and cell death may result.
An inhibition of the tubulin function leads to an arrest of the cells
in mitosis (Figure 3), and no further cell division as long as the chromosomes
are not transported to the two poles. A large number of cells have
been shown to die while they are arrested in this phase of cell
division. A mitotic arrest is the mechanism behind the function of
mitotic inhibitors commonly used in cancer therapy (drugs of the
group Vinca alkaloids).
Figure 3. Cell cycle block. Blocks in the progression through the
cell cycle may be formed at different points. A block at the end of
G1 is known to allow the cell to repair accumulated DNA-damage if
the cell has been irradiated. The figure shows a block in mitosis
that has been induced by photosensitization, and illustrates how an
arrest or severe inhibition in the regular progression will increase
the number of cells in the phase before the block, and reduce the
number of cells that can be found in the succeeding phase. It is not
known if mitotic inhibition can play any role in normal cell physiology.
Photosensitization of Extracellular Structures
The outside of a cell plays an essential function by transferring
signals, transporting nutrients, and attaching the cell to
neighboring cells or the extracellular matrix. Specialized proteins
mediate cell attachment, and these are critically important in
allowing cancer cells to grow and invade other tissues, for wound
healing, implantation of embryos in the uterus, etc. It has been
shown that photosensitizers can modify the outside of the cell and
the extracellular matrix probably by cross-linking and photooxidation
of proteins. Due to reduced binding of cells to the extracellular
matrix, photosensitization may reduce the risk of metastasis in
cancer therapy and inhibit the blockage of coronary arteries by
restenosis following angioplasty. The latter effect is important
because while it is true that recent medical advances have allowed
blocked or partially blocked arteries to be reopened, a process
called angioplasty, these areas often close down again, often within
months. Methods, such as photosensitization, that can delay this
secondary blockage, called restenosis, have tremendous therapeutic
Every structure of a cell can be photosensitized. Photosensitization
can influence vital functions of the cell. Different sensitizers
will be taken up differently by the cells, and cause different types
of damage after irradiation with light. The relative importance of
different types of damage is chiefly influenced by the distance
between the bound sensitizer, and the site where the damage can take
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of photodynamic sensitizers. Photochem Photobiol. 1996
Jori, G. Tumour photosensitizers: approaches to enhance the
selectivity and efficiency of photodynamic therapy. J Photochem
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Karger, Basel. Current Problems in Dermatology, vol 15, 1986 (Series
Editor: H. Hönigsmann, Volume 15: "Therapeutic Photomedicine" Volume
editors H. Hönigsmann and G. Stingl) Sections II,Therapeutic
principles and III, Molecular aspects.
Peng Q, Moan J, Nesland JM. Correlation of subcellular and
intratumoral photosensitizer localization with ultrastructural
features after photodynamic therapy. Ultrastruct Pathol. 1996
Spikes, John D., Photosensitization. In: The Science of
Photobiology, Kendric C. Smith, ed. Plenum Press, New York and
London, 1989, pp. 79-110.
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