Photochemical internalisation in drug and gene delivery

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Advanced Drug Delivery Reviews 56 (2004) 95–115

Photochemical internalisation in drug and gene delivery

Anders Høgseta,*, Lina Prasmickaiteb, Pal K. Selbob, Marit Hellumb,Birgit Ø. Engesæterb,c, Anette Bonstedb, Kristian Bergb

aPCI Biotech AS, Hoffsvn. 48, N-0377 Oslo, NorwaybDepartment of Biophysics, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway

cDepartment of Tumour Biology, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway

Received 14 April 2003; accepted 30 September 2003

Abstract

This article reviews a novel technology, named photochemical internalisation (PCI), for light-induced delivery of genes,

proteins and many other classes of therapeutic molecules. Degradation of macromolecules in endocytic vesicles after uptake

by endocytosis is a major intracellular barrier for the therapeutic application of macromolecules having intracellular targets of

action. PCI is based upon the light activation of a drug (a photosensitizer) specifically locating in the membrane of endocytic

vesicle inducing the rupture of this membrane upon illumination. Thereby endocytosed molecules can be released to reach

their target of action before being degraded in lysosomes. The fact that this effect is induced by illumination means that the

biological activity of the molecules can be activated at specific sites in the body, simply by illuminating the relevant region.

We have used the PCI strategy to obtain light-induced delivery of a variety of molecules, including proteins, peptides,

oligonucleotides, genes and low molecular weight drugs. In several cases, a >100-fold increase in biological activity has been

observed.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Photosensitiser; Endosomal release; Site-specific; Light-induced; Drug delivery; Gene therapy; Protein therapy; Oligonucleotide

delivery

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

2. PCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

2.1. Photochemically induced cytotoxicity and photodynamic therapy . . . . . . . . . . . . . . . . . . . . . . . . . . 97

2.2. Intracellular localisation of the photosensitisers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

2.3. The principle of photochemical internalisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

3. PCI-mediated drug delivery in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

3.1. Light dose-dependence of the PCI effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

3.2. Photosensitizers used in PCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

0169-409X/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.addr.2003.08.016

* Corresponding author. Tel.: +47-2325-4003; fax: +47-2325-4001.

E-mail address: [email protected] (A. Høgset).

A. Høgset et al. / Advanced Drug Delivery Reviews 56 (2004) 95–11596

3.3. PCI effects on different cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

3.4. PCI with different classes of molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

3.4.1. PCI-based protein delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

3.4.2. PCI with peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

3.4.3. PCI with oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

3.4.4. PCI with low molecular weight substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

3.4.5. PCI for gene delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

4. PCI in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

5. Advantages and limitations of PCI as a drug delivery technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

6. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

1. Introduction Internalisation can also be an issue for low molec-

The last years have seen a rapid increase in

research and development of macromolecular drugs,

both due to a steady improvement of production

technologies and due to an increasing understanding

of the premises for the design and use of such drugs.

As compared to more traditional drugs, macromolec-

ular drugs have the potential advantage of being

excellently specific for a given therapeutic target

and, at least in principle, of quite easy design of

molecules that could have such a specificity.

At the same time, there is also a rapidly increas-

ing interest in exploring and exploiting intracellular

drug targets, among other things because genomics

and proteomics research will lead to the identifica-

tion and validation of many very interesting such

targets, e.g. in intracellular signal transduction net-

works, in the regulation of gene expression, etc.

Thus, there will be an increasing need for therapeu-

tic molecules that are able to attack intracellular

drug targets and that consequently must be able to

be internalised into the cell. In addition, the emerg-

ing field of gene therapy relies entirely on ‘‘pro-

drugs’’ (genes) that must be internalised into cells in

order to be able to exert the desired biological

effect.

While macromolecular drugs and genes can quite

easily reach extracellular targets of action, it is a

severe limitation for the use of such molecules that

most classes of macromolecular drugs have great

difficulties in reaching intracellular targets. There-

fore, to fully exploit the potential of macromolecular

drugs, efficient and specific technologies for delivery

of such molecules into the target cells would be of

great value.

ular weight molecules. Although low molecular

weight drugs are often able to go into the cells, there

are also many drug candidates (e.g. hydrophilic sub-

stances) with excellent effects in cell-free systems that

do not readily pass the cell plasma membrane, and

thus will be unusable as drugs on their own. This has

hindered the realisation of the therapeutic potential of

many interesting classes of molecules, and delivery

technologies that could overcome the internalisation

barrier could have the potential to significantly extend

the spectrum of molecules that could be used for

therapeutic applications.

The unlucky fact that the above mentioned classes

of molecules are inactive as drugs in themselves

however also have a potential advantage, namely that,

given a specific drug delivery system, they could be

made into very specific therapeutics. Ideally, such

molecules should on their own have no ability to

cause adverse effect in non-target cells or tissues; their

biological effect would be totally dependent on the

delivery system. Thus, if such molecules could be

‘‘activated’’ by a specific delivery system, they could

have the potential to become more specific than most

drugs used today. And, as is well known, specificity in

many cases is of utmost importance for the therapeutic

outcome, exemplified by the low therapeutic index

usually found in cytotoxic cancer therapy.

A main reason for a failure to reach intracellular

targets is that the molecular structure of the molecules

in question makes them unable to pass directly

through the plasma membrane; thus, the only way

such molecules can get access to the interior of the

cell is through the process of endocytosis. Although

most molecules can be taken more or less efficiently

into the cell by endocytosis, such molecules will as a

A. Høgset et al. / Advanced Drug Delivery Reviews 56 (2004) 95–115 97

rule end up in endocytic vesicles such at late endo-

somes or lysosomes, in the end being degraded and

losing the biological activity. Since the therapeutic

targets are usually located outside endocytic vesicles

to exert a desired biological effect, the therapeutic

molecules will usually have to escape from the

vesicles before they are degraded. Thus, for such

molecules, the endosomal membrane constitutes a

severe barrier for the therapeutic use. In this chapter,

we will review the principle behind, and the results

obtained with, photochemical internalisation (PCI), a

novel photochemical technology for inducing the

release of molecules from endocytic vesicles. PCI is

a technology that can enhance the cellular biological

activity of many different classes of molecules, and,

since this effect is induced by illumination, PCI can be

used as a technology for site-specific drug or gene

delivery.

2. PCI

In photochemical internalisation, photosensitising

compounds (photosensitisers) are used for improving

endosomal release of endocytosed molecules. Photo-

sensitisers are compounds that make cells extraordi-

nary sensitive to illumination with visible light [1,2].

A photosensitiser in the ground state (PS) will absorb

the energy of a photon (hr) of a certain wavelength

and will thereby be converted into an excited singlet

state (1PS*). Then, 1PS* is quickly converted to an

excited triplet state (3PS*) that, by transferring the

absorbed energy to other molecules, can initiate

further photochemical reactions. With photosensitisers

like porphyrins and structurally related compounds,

the most important of these reactions proceeds via

singlet oxygen (1O2), a highly reactive form of oxy-

gen [3–5]. Schematically:

3PS* is thus converted back to PS, which is then

ready for further cycles of excitation and generation

of singlet oxygen [6,7]. Singlet oxygen is a very

powerful oxidizing agent that can oxidize many

different biomolecules [8] potentially inducing dam-

age into various cellular structures. However, since1O2 has a very short lifetime ( < 0.1 ms) and accord-

ingly a short range of action (10–20 nm) inside the

cell [7], only targets very close to the excited photo-

sensitiser will be oxidised by 1O2 upon light expo-

sure, while distant molecules will generally be left

unaffected.

2.1. Photochemically induced cytotoxicity and photo-

dynamic therapy

In general, the photochemical reactions induced by

photosensitisers induce cytotoxic effects, since impor-

tant intracellular structures may be damaged [5,9].

These cytotoxic effects can be exploited for cancer

treatment in photodynamic therapy (PDT), a treatment

modality where light exposure leads to photosensi-

tiser-induced killing of cancer cells [1,2,5].

PDT can also be used to treat non-oncologic

conditions, and is being explored in experimental

treatment of vascular diseases [10–13] and viral

infections [14–16], as well as in immunological

disorders such as rheumatoid arthritis [17–19] and

psoriasis [20,21]. Furthermore, PDT is widely used

for therapy of age-related macular degeneration with

choroidal neovascularisation disease [22–24].

2.2. Intracellular localisation of the photosensitisers

Photosensitisers can, depending on their physico-

chemical properties, be taken up by the cell both by

endocytosis and by active or passive transport

through the plasma membrane [25,26]. Partly as a

function of their mode of uptake, different photo-

sensitisers will localise differently inside the cell

[25–29]. For example, amphiphilic photosensitisers

such as TPPS2a (meso-tetraphenylporphine with two

sulfonate groups on adjacent phenyl rings) and

AlPcS2a (aluminium phthalocyanine with two sulfo-

nate groups on adjacent rings) (Fig. 1) will first

insert into the plasma membrane, thereafter being

taken in by endocytosis. Such photosensitisers end

up quite specifically in the membranes of endocytic

vesicles [30], with the hydrophobic part of the

photosensitisers inserted into the vesicle membrane

[31].

Fig. 1. Chemical structures of the photosensitisers AlPcS2a and


2.3. The principle of photochemical internalisation

The invention of the photochemical internalisation

technology was based on the finding that light expo-

sure of cells containing photosensitisers in their endo-

cytic vesicles causes a permeabilisation of the vesicles

and release of the photosensitiser into the cytosol

[30,32]. The same experiments showed that also

substantial amounts of lysosomal enzyme activities

could be found in the cytosol after light treatment,

indicating that in addition to the photosensitiser also

unrelated molecules located inside the lysosomes

TPPS2a.

Fig. 2. Light-induced cytosolic delivery by PCI. (A) Schematic present

Photosensitiser; S* excited photosensitiser. (B) The principle of PCI. (I)

molecule to be delivered (G) is invaginated into endocytic vesicles. (II) S

endosome membrane and lumen, respectively. (III) Illumination leads to

releasing G into the cytosol.

could be released into the cytosol. Moreover, this

photochemically induced release could occur without

inducing extensive cell death [30], and with mainte-

nance of the biological activity of the released mole-

cules. This is probably due to the short range of action

of the photochemically generated 1O2, since, with

photosensitisers mainly localising in the vesicle mem-

branes, molecules in the vesicle matrix should not be

very liable to the photochemical damage responsible

for destroying the membranes. Hence, the photochem-

ical treatment may be used to release endocytosed

molecules in a biologically active form from endo-

cytic vesicles, a principle we have named PCI. As

described in Fig. 2, the introduction of molecules into

the cytosol is achieved by first exposing the cells or

tissues to a photosensitising dye and the molecule

which one wants to deliver, both of which should

preferentially localise in endosomes and/or lysosomes

[33]. Secondly, the cells or tissues are exposed to light

of wavelengths inducing a photochemical reaction.

This reaction will lead to disruption of lysosomal and/

or endosomal membranes and the contents of the

endocytic vesicles will be released into the cytosol.

Detailed descriptions of practical aspects of the tech-

nology can be found in Berg et al. [34] and Prasmick-

aite et al. [35].

ation of the initiation of the photochemical reactions in PCI. (S)

The photosensitiser (S) localises to the plasma membrane, and the

and G are taken up into the cell by endocytosis, localising in the

photochemical damage and rupture of the endosome membranes,


The exact molecular mechanism of the photochem-

ically induced endocytic membrane rupture is not

known. There are indications from mitochondrial

[36] and erythrocyte [37] membranes that at least

for some photosensitisers damage to membrane pro-

teins may be more important for membrane rupture

than oxidation of membrane lipids. However, whether

this is also the case after photoactivation of photo-

sensitizers localised in endocytic membranes is not

known and requires further investigations.

Recently, we have also discovered another mode of

performing PCI. Quite unexpectedly, it was found that

PCI gives very good effects also if the photochemical

treatment is given before the molecule to be internal-

ised [38], i.e. the cells can be illuminated before they

even come into contact with the molecule to be

delivered. The mechanism behind this effect is still

unclear, but we have speculated that it is due to a

fusion between vesicles made leaky by the photo-

chemical treatment and newly incoming vesicles con-

taining the molecule to be delivered, causing the fused

vesicle to become leaky (Fig. 3). For drug delivery

purposes, the ‘‘light first’’ procedure may have several

important advantages that will be further mentioned

below.

Fig. 3. Possible mechanism for ‘‘light first’’ PCI. (I) The

photosensitiser (S) is taken op by endocytosis and localises in the

membrane of endocytic vesicles. (II) The cells are illuminated,

damaging endosomal membranes. (III) The cell is incubated with

the molecule to be delivered (G), which is taken up into novel

endocytic vesicles. (IV) The vesicles containing the molecule to be

delivered fuses with vesicles damaged by the photochemical

treatment. (V) The fused vesicles are leaky, so that the molecule

to be delivery will escape into the cytosol.

3. PCI-mediated drug delivery in vitro

In vitro, PCI has been shown to induce endosomal

release and, in many cases, also biological effects, of a

variety of molecules, such as plant protein toxins

[33,39], immunotoxins [40], peptides [33], ribozymes

and oligodeoxynucleotides [41] and genes delivered

by various vector systems, both non-viral [33,41–43]

and viral [44]. The relocalisation effects that can be

obtained by PCI are illustrated in Fig. 4, where it can

be seen that both photosensitisers (A, B), proteins (A)

and oligonucleotides (B) can be released from endo-

cytic vesicles, and that an endocytosed protein main-

tains its biological activity after the treatment (C). It

can also be seen that the PCI-induced endosomal

release is quite efficient, with about 60% of an

endocytosed protein being released upon the treatment

(Fig. 4C).

3.1. Light dose-dependence of the PCI effect

In order to induce the photochemical reactions that

in the end will lead to destruction of the endosomal

membrane, it is of course necessary to apply a light

dose that is above a certain threshold. Since the uptake

of photosensitiser, the concentration of photosensitiser

in the endosomal membranes and the sensitivity to the

cytotoxic effects of the photochemical treatment will

vary between different cell types, the optimal light

dose for PCI will also vary from cell type to cell type.

As discussed above, the photochemical treatment

will usually also give cytotoxic effects and, in many

cases, it can be important to balance the cytotoxic and

the drug delivery effects. This may be especially

important for gene therapy approaches where the goal

might be to induce the delivery of a gene that shall be

expressed for quite a long time after the treatment.

However, even in cases where the ultimate aim is to

kill the target cells, the balance between the (generally

unspecific) cytotoxic effects of the pure photochem-

ical treatment and the (potentially specific) effects of

the drug delivered by PCI may be very important, e.g.

in cases where diseased cells are interspersed between

normal cells that should not be harmed by the treat-

ment. Fig. 5 shows PCI effects and cell survival for

typical experiments with gene (A) and protein (B, C)

transfer. It can be seen that, while the PCI effect in

general seems to increase with the light dose (and

Fig. 4. PCI-induced endosomal release. (A) PCI-induced release of a protein. Fluorescence photomicrographs of AlPcS2a and Alexa-488-

labelled gelonin in THX melanoma cells. The cells were co-incubated with 30 Ag/ml Alexa-gelonin and 20 Ag/ml AlPcS2a for 18 h. After 4 h

chase in drug-free medium, the cells were exposed to 30 s microscopy light, which photochemically induced a release of both AlPcS2a and

Alexa-gelonin from endocytic vesicles. Both pictures were taken 1 min after light exposure. For details, see Ref. [40]. (B) PCI-induced

relocalisation of an oligodeoxynucleotide. THX melanoma cells were incubated for 18 h with 20 Ag/ml AlPcS2a. After further 4 h incubation

with a polylysine complex of a fluorescein-labelled oligodeoxynucleotide, the same cells were photographed before and after the exposure to 10

s microscopy light, a light dose sufficient for inducing the PCI effect. See Ref. [41], for details. (C) PCI-induced cytosolic delivery of functional

endocytosed horse radish peroxidase (HRP). NHIK 3025 cells were co-incubated with 3.2 Ag/ml TPPS2a and 1 mg/ml HRP for 18 h. The

medium was replaced with drug-free medium before the cells were exposed to the indicated light doses. HRP activity was measured in intact

cells (black bars) and in cytosol (white bars) separated from cytosol-free cell corpses by electropermeabilisation and a density centrifugation

technique (see Ref. [33], for details).


thereby with increasing cell death), there are very

substantial PCI effects also at light doses killing

relatively few cells. For example, a 15–20-fold in-

crease in gene transfection could be obtained at a light

dose killing about 15% of the cells (Fig. 5A) and, with

the highest dose of a protein toxin, a >100 times

enhancement of the toxin effect could be observed

with a light dose killing about 30% of the cells (Fig.

5C). The fact that a substantial PCI effect can be

achieved with light doses killing only a small fraction

of the cells have important implications for the ther-

apeutic use of the technology. Firstly, if the aim is to

kill all cells in a given area, such as e.g. in many kinds

of cancer therapy, PCI will allow the treatment of

thicker lesions than pure photodynamic therapy. The

light doses needed to confer the killing of all cells


with PCI (e.g. combined with a non-specific toxin) are

substantially lower than those needed in pure PDT;

thus, a therapeutic light dose can be achieved much

deeper into the tissue than what is possible with pure

Fig. 5. Effect of illumination on macromolecule delivery and cell

survival. (A) Transfection as a function of light dose. HCT 116 cell

were incubated with AlPcS2a over night and, after removal of the

sensitiser, the cells were further incubated with a pEGFP-N1/

polylysine complex for 4 h. The cells were illuminated with red

light. Forty-eight hours later, EGFP-expressing cells were scored by

flow cytometry and cell survival was measured by the MTT assay as

described earlier [41]. (n) EGFP-positive cells, (5) cell survival.

Error bars are SEM of three experiments. Reproduced with

permission [42]. (B and C) Cytotoxic effect of PCI with the protein

toxin gelonin in NHIK 3025 cells. The cells were treated with 3.2

Ag/ml TPPS2a and gelonin for 18 h, followed by 1 h in drug-free

medium before illumination. Protein synthesis was measured as

described previously (reproduced with permission [33]). (B) Protein

synthesis after treatment with TPPS2a and light in the absence (.) orpresence (o) of 0.2 Ag/ml gelonin. (C) Protein synthesis after

treatment with gelonin and 50 s of light (n) and gelonin and TPPS2ain combination with 50 s of light (o). Reproduced with permission

[33].

PDT. Secondly, in applications where it is desirable to

treat certain target cells that are interspersed with

normal cells, PCI could be used in combination with

a drug acting more or less specifically on the target

cells and a light dose that would in itself kill only a

low fraction of the cells, so that the main biological

effect would be that of the PCI-delivered (specific)

drug and not of the pure (unspecific) photochemical

treatment. The fact that in vivo relevant photosensi-

tisers often tend to accumulate preferentially in dis-

eased tissues (e.g. in tumours) may in many clinical

situations give additional specificity to the PCI-medi-

ated delivery as it can do for PDT effects [2].

3.2. Photosensitizers used in PCI

According to the proposed mechanism for PCI

photosensitizers to be used in PCI should fulfil certain

criteria: (i) they should localise in the endocytic

compartments. (ii) Within these compartment, they

should preferably localise to the membranes, to max-

imize the damaging effect on the membrane and to

diminish the possibility of photochemical destruction

of molecules in the lumen of the vesicles. (iii) Ag-

gregation of the photosensitizer should be kept low in

the cell, since aggregations reduce the ability of the

photosensitizer to transfer the energy of the excited

state to molecular oxygen, and hence reduce the

efficiency of the photochemical treatment.

To study the PCI efficiency with diverse photo-

sensitisers, sensitisers with different intracellular local-


isation were tested for their ability to enhance polyly-

sine-mediated transfection [29]. As expected, light-

activation of the non-lysosomally localised lipophilic

dye tetra(3-hydroxyphenyl)porphyrin (3THPP) and 5-

aminolevulinic acid (5-ALA)-induced protoporphyrin

IX did not significantly stimulate transfection. In con-

trast, the endosomally localised photosensitisers

AlPcS2a and TPPS2a had a strong stimulating effect

on transfection (Fig. 6). These photosensitisers have

two sulfonate groups on adjacent phthalate/phenyl

rings (Fig. 1), making the photosensitiser molecules

amphiphilic and able to insert into biological mem-

branes. The effect of the more hydrophilic tetrasulfo-

nated dye meso-tetraphenyl-porphine with four

sulfonate groups (TPPS4) was lower, maybe because

this sensitiser mainly localises in the lumen of endo-

Fig. 6. Effects of photochemical treatment with different photo-

sensitisers on polylysine-mediated transfection. THX cells were

treated with 20 Ag/ml AlPcS2a, 2 Ag/ml TPPS2a, 75 Ag/ml TPPS4,

30 Ag/ml p-TMPyPH2, 0.25 Ag/ml 3THPP or 2.5 Ag/ml Photofrin

for 18 h followed by a 6-h treatment with a pEGFP-N1/polylysine

complex in photosensitizer-free medium. With 5-ALA and Nile blue

A, the cells were incubated for 6 h with the pEGFP-N1/polylysine

complex in the presence of 1 mM 5-ALA or 4 mM Nile blue in

FCS-free or FCS-containing medium, respectively. For all sensi-

tisers, the cells were washed and transferred to complex-free

medium before being illuminated with a D50 dose light dose (i.e.

killing about 50% of the cells). Details can be found in Ref. [29].

Reproduced with permission [29].

cytic vesicles. Finally, the cationic hydrophilic dye

meso-tetra(N-methyl-4-pyridyl)porphine ( p-

TMPyPH2) and the lysosomotropic weak base Nile

blue A had very weak or no effects, although also

localising in endocytic vesicles. Thus, also among

photosensitisers localised in endocytic vesicles, differ-

ences in the PCI efficiency could be observed, indicat-

ing that amphiphilic photosensitisers expected to

localise mainly in the membranes of the endocytic

vesicles give the best effect [29].

For clinical use, other properties of the photosensi-

tiser will, of course, also be important: (i) The

photosensitizer should have favourable pharmacoki-

netics, preferably accumulating rapidly and preferen-

tially in diseased tissues. (ii) The photosensitizer

should not be toxic in non-illuminated regions and

should preferably have a rapid clearance from the

body, preventing the necessity of long time light

protection of the patient. (iii) A far red-light absor-

bance is preferable for most clinical uses, due to the

better tissue penetration of light in this region of the

spectrum.

The amphiphilic phthalocyanine photosensitizer

AlPcS2a (Fig. 1) meets most of the above criteria

[45] and should be well suited for in vivo PCI

applications. Phthalocyanines are based upon the

porphyrin macrocycle, which is extended with four

benzo rings on the pyrrol units. This results in an

enhanced absorption in the far-red region of the

spectrum. The chelation of aluminium with the four

central benzisoindole nitrogens leads to a stable

molecule, which is relatively easy to purify [46].

AlPcS2a absorbs light efficiently around 675 nm

where light tissue penetration is near its optimum.

In addition, AlPcS2a is relatively photostable and has

been shown to be a very efficient sensitizer [47]. For

in vitro work and other applications where deep

tissue penetration is not necessary or may even not

be desirable, the photosensitizer meso-tetraphenyl-

porphine with two sulfonate groups on adjacent

phenyl rings (TPPS2a) (Fig. 1) has been shown to

be equally efficient at its optimum wavelengths for

excitation (415–420 nm).

3.3. PCI effects on different cell lines

In Table 1 is shown a summary of the effects of

PCI on different cell lines. It can be seen that PCI can

Table 1

Cell lines tested for PCI effects with different macromolecules

Cell line Tissue Protein

transfer

Plasmid

transfer

Adenovirus

transfer

NHIK 3025 Cervix,

carcinoma

in situ

+

NCI-H146 Lung, small

cell carcinoma

+

WiDr Colon

adenocarcinoma

+ +

KM20L2 Colon

adenocarcinoma

+

Col115 Colon

adenocarcinoma

+

HCT116 Colon carcinoma + + +

T47D Breast, ductal

carcinoma

+

THX Skin, malignant

melanoma

+ + +

Malme-3 Skin fibroblast +

Malme-3M Lung metastasis,

malignant

melanoma

+

FM3 Malignant

melanoma

+ +

U87 Brain,

glioblastoma

+ +

D54 Brain,

glioblastoma

+

EB Immortalised

B cells

+

V79 Lung fibroblasts,

Chinese hamster

+

BL2-8G-E6 Mouse

fibroblastoma

+

COS-7 Kidney, green

monkey

+

HeLa Cervix

adenocarcinoma

+ +

COS-1 Kidney, green

monkey

+

A549 Lung carcinoma +

FEMXIII Malignant

melanoma

+

Neuro-2 Neuroblastoma +

DU 145 Prostate cancer +

BHK Kidney, baby

hamster

+

HFib Fibroblast +

Raji Burkitt’s

lymphoma

+


have positive effects on delivery of both proteins and

genes in a variety of cell, both tumour cell lines and

non-cancer cell lines. For two of the cell types, PCI

effects have also been documented in in vivo models

(see also below).

3.4. PCI with different classes of molecules

In the following, results of PCI for the delivery of

different classes of molecules will be discussed.

3.4.1. PCI-based protein delivery

For studying PCI-induced delivery of proteins, we

have focused mainly on the 30-kDa plant toxin

gelonin, a type I ribosome- inactivating protein

(RIP) [48], which possesses a number of attractive

properties. Gelonin consists of a single polypeptide

chain and has no domains for binding to the cell

surface or for facilitating endosomal release [49]. In

cell-free systems, gelonin is an extremely potent

inhibitor of protein synthesis working by a powerful

N-glycosidase enzymatic activity destroying the 28S

rRNA unit of eukaryotic ribosomes [50]. However,

due to its inability to be taken up and transferred into

the cytosol, gelonin has low toxicity on intact cells

and also very low in vivo toxicity, with LD50 for mice

of 40–75 mg/kg [51,52]. Free gelonin is supposed to

be taken up unspecifically by fluid phase endocytosis

and degraded by lysosomal hydrolases [39,53], and

gelonin would therefore be of low therapeutic interest

without a means for transferring the molecule to the

cell cytosol.

Based on these properties, gelonin should thus be

an ideal model protein to establish and demonstrate

the PCI technology. In fluorescence microscopy

studies, gelonin labelled with the dye Alexa-488

was shown to localise in the same intracellular

compartments as the photosensitizer AlPcS2a [39]

previously shown to localise in endocytic vesicles

both in vitro [27] and in vivo. Moreover, both Alexa-

gelonin and AlPcS2a was released from these vesicles

after illumination, clearly illustrating the PCI effect

(Fig. 4A).

By using the photosensitizers TPPS2a, TPPS4 or

AlPcS2a in combination with gelonin and light, we

have documented good effects of PCI in more than 20

different cell lines (Table 1). At best, a more than 300-

fold reduction of the protein synthesis has been

demonstrated by PCI of gelonin, as compared to the

application of toxin treatment alone or light and

photosensitizer alone [33].


The uptake and specificity of type I RIPs can be

increased substantially by conjugating or fusing the

toxin to a specific monoclonal antibody, generating an

immunotoxin (IT), e.g. designed to bind to specific

receptors on the surface of the target cells. ITs are

generally taken up by endocytosis, and lysosomal

degradation of ITs can in many cases be a major

obstacle for obtaining biological effects of such

molecules [54,55]. To explore the potential of PCI

in activating the cytotoxic effects of immunotoxins,

an immunoconjugate of gelonin with the antibody

MOC31 was used as a model [40]. The monoclonal

antibody MOC-31 recognises and targets the human

epithelial glycoprotein 2 (EGP-2), an antigen expressed

on nearly all types of carcinoma cells [56]. After PCI-

induced release of the immunotoxinMOC31-gelonin, a

>100-fold increase in cytotoxicity was achieved, as

compared to what was obtained with the ITwithout PCI

(Fig. 7) [40]. PCI with MOC31-gelonin was shown to

be very efficacious against several different carcinoma

cell lines (lung (H-146), breast (T47D) and colon

Fig. 7. Effect of MOC31-gelonin on protein synthesis in carcinoma

cells with and without PCI. Exponentially growing H146 cells were

exposed to different concentrations of the immunotoxin MOC31-

gelonin and 0.3 Ag/ml TPPS2a, and illuminated for 50 s. Protein

synthesis of the cells was evaluated 24 h after light exposure. Data

presented are the mean relative to control cells not given light.

Details can be found in Ref. [40].

(WiDr and KM20L2)), demonstrating that PCI with

ITs has a potential to become a potent anti-cancer

application [40].

3.4.2. PCI with peptides

Peptides could have several interesting applica-

tions as drugs acting in the intracellular environment.

Firstly, several naturally occurring peptides having

intracellular biological activity are known, e.g. en-

zyme inhibitors [57,58] or DNA-damaging agents

[59]. Secondly, phage display [60,61] and other

molecular diversity technologies can be used to

design peptides having a diversity of therapeutically

interesting biological activities, in many cases direct-

ed against intracellular drug targets. Thirdly, peptides

can be used as antigens for vaccination purposes.

Thus, for many peptides, access to the cytosol can be

crucial for achieving a desired biological activity;

however, most peptides will not be able to reach the

cytosol without the help of a delivery system. To

study the possibility of using PCI for internalisation

of peptides, we studied the effect of PCI on the

localisation of a fluorescently labelled antigenic pep-

tide. By microscopy, it could clearly be shown that

PCI could induce a relocalisation of the peptide from

endocytic vesicles into the cytosol [33], and we also

had indications that this led to an increased presenta-

tion of the antigenic peptide on the cell surface via the

MHC Class I pathway (T.E. Tjelle, unpublished).

3.4.3. PCI with oligonucleotides

Oligonucleotides are a class of molecules with a

well recognized therapeutic potential [62–64]. The

vast majority of therapeutic oligonucleotides have to

get into intracellular compartments to exert a biolog-

ical effect, e.g. antisense DNA, ribozymes, siRNA

and peptide nucleic acids will typically have their

biological action either in the cytosol or in the

nucleus, and ‘‘aptamer’’ oligonucleotides [65] can

have different sites of action depending on which

target structure they are directed against. Although

some oligonucleotides can be modified in such a way

that they can pass through the plasma membrane [66],

in many cases also this class of molecules will depend

more or less on endocytic uptake. Thus, PCI could

have a clear potential for improving the efficiency of

delivery of oligonucleotides, and also for site-direct-

ing the effect of such molecules, a possibility that in

Fig. 8. PCI effect on cytotoxicity of the anticancer drug bleomycin.

WiDr colon carcinoma cells were incubated with 5 Ag/ml AlPcS2afor 18 h. After washing, the cells were incubated in medium

containing 100 AM bleomycin (n) for 4 h; control cells (5) were

not treated with bleomycin. The cells were washed and, after

addition of 1 ml drug-free medium, they were illuminated. After 3

days of further incubation, cell survival was measured by a protein

synthesis assay measuring the incorporation of 3H-leucin.


many cases could be highly desirable. Our microsco-

py studies clearly show that PCI can relocalise

oligonucleotides from endocytic vesicles into the

cytosol (Fig. 4B) and in several cases even into the

nucleus (unpublished observations). Recent experi-

ments also clearly show that PCI can substantially

enhance the specific biological activity of a peptide

nucleic acid that could potentially be used in cancer

treatment [94].

3.4.4. PCI with low molecular weight substances

Many low molecular weight substances can have

excellent effects on interesting therapeutic target mol-

ecules, but anyway be of little use as therapeutic

compounds because of inability to get into the cell.

Such compounds will be obvious candidates for use

with PCI, not only because PCI can activate the

therapeutic potential of the compounds, but also

because such compounds, being membrane imperme-

ant, will generally not get into non-target cells and

should therefore only give very limited adverse effects

in non-target (i.e. non-illuminated) tissues. This is in

contrast to more traditional therapeutics that, because

of their ability to pass the cell membrane, will

generally be taken up into, and be active in, also

non-target cells, in many cases generating severe side

effects. The effect of the anticancer agent bleomycin is

known to be limited by poor uptake into the cell [59],

and it is known that bleomycin can be quite efficiently

taken up by endocytosis [67]. In vitro studies on PCI

with bleomycin have shown that photochemical treat-

ment can substantially enhance the biological effect of

this agent (Fig. 8). That this is a specific PCI effect is

indicated by the observation that bleomycin in the

doses used had no effect on cell survival without

illumination (Fig. 8, 0 min time point), and by

control experiments showing that the presence of a

photosensitiser is necessary to observe the light-

dependent increase in bleomycin cytotoxicity (data

not shown). Furthermore, preliminary experiments

indicate that PCI can enhance the effect of bleomycin

also in vivo (Høgset et al., in preparation), showing

that PCI has a clear potential to be used for site-

specific chemotherapy.

3.4.5. PCI for gene delivery

Gene therapy is a novel therapeutic modality

receiving great attention and being generally recog-

nised as having the potential to constitute treatment

for a lot of different diseases [68–70]. However,

although there are some successes [69,71,72], clinical

trials with gene therapy have hitherto largely given

quite disappointing results. An important reason for

this is that methods for efficient and specific delivery

of therapeutic genes in vivo is still lacking. With

most gene delivery systems, the therapeutic gene is

taken into the cell by endocytosis and, for many of

these systems, especially non-virus-based, the lack of

efficient mechanisms for translocating the gene out of

the endocytic vesicles constitutes a major hindrance

for realisation of the therapeutic potential of the

therapeutic gene.

Photochemical internalisation has been studied as a

gene delivery technology (reviewed in Ref. [73]) both

with several non-viral [29,33,41–43] and with ade-

noviral vectors [44], mainly by using reporter genes

such as genes encoding enhanced green fluorescent

protein (EGFP) or h-galactosidase. However, PCI-

mediated gene delivery has also been shown to induce

the delivery of therapeutic genes, such as the genes

encoding Herpes Simplex Virus thymidine kinase

(HSV-tk) (Prasmickaite, unpublished) and interleu-

kin-12 (IL-12) (Høgset, unpublished).

The effect of PCI on gene delivery can be illus-

trated by experiments with polylysine-mediated trans-


fection of AlPcS2a-treated HCT 116 human colon

carcinoma cells. In these experiments, a part of the

culture dish was covered by aluminium foil, while the

rest of the dish was illuminated. As can be seen from

Fig. 9A after PCI, many of the illuminated cells

exhibited visible EGFP-fluorescence, in contrast to

what was the case for the non-irradiated cells. Thus,

the light treatment strongly induces transfection in

HCT 116 cells. This experiment also indicates the

Fig. 9. PCI effects on gene delivery by a non-viral vector. (A) PCI-induce

HumanHCT 116 colon carcinoma cells were incubated with 20 Ag/ml AlPcS

for 6 h. After washing, the culture dish was partly covered by aluminium

treatment. Forty-eight hours later, the cells were analysed by fluoresce

fluorescence, and by phase contrast microscopy (right panel). Reproduced

cytometry. Human THXmelanoma cells were treated with 20 Ag/ml AlPcS2aThe cells were exposed to light (illumination times indicated on the figure)

flow cytometry as described in detail in Ref. [41]. The cells in the upper righ

that received no plasmid there were no cells in this quadrant (not shown). R

high degree of site-specificity that can be obtained,

reflected by the clear difference in transfection be-

tween the illuminated and the non-illuminated parts of

the culture dish.

The PCI effects of on transfection have been

studied in more detail by flow cytometry analysis of

the transfected cells. For example from the data

presented in Fig. 9B, it can be seen that the light

treatment induced an increase in transfection efficien-

d transfection of HCT 116 cells studied by fluorescence microscopy.

2a for 18 h, washed and treated with a pEGFP-N1/polylysine complex

foil (‘‘without light’’ region) before being subjected to 7 min light

nce microscopy for EGFP (left panel) or AlPcS2a (middle panel)

with permission [42]. (B) Light-induced transfection studied by flow

for 18 h and incubated with a pEGFP-N1/polylysine complex for 4 h.

, and red (AlPcS2a) and green (EGFP) fluorescence were analysed by

t quadrant were taken as positive for EGFP-expression, since for cells

eproduced with permission [41].


cy from about 1% EGFP-positive cells at 0 min of

light to about 50% positives after 5 min illumination

for THX melanoma cells, thus representing a light-

induced enhancement of transfection efficiency of

about 50 times. As previously discussed, the PCI

effect exhibits a clear light dose response; however,

the photochemical enhancement of transfection is

effective over a quite large range of light doses.

3.4.5.1. PCI with different gene delivery agents

Non-viral vectors. Whereas PCI in general has a

positive effect on transfection with polycations such as

polylysine and polyethylenimine (PEI), the effect on

transfection with cationic lipids is much more variable

[29,42]. While in some cell lines PCI seems to reduce

cationic lipid mediated transfection, in other cell lines,

PCI can have positive effects [74]. It also seems that

the effect of PCI depends strongly on the type of lipid

composition used for transfection. For example in

HCT 116 cells, PCI can enhance transfection mediated

by hAE-DMRIE/DOPE, while hAE-DMRIE-mediat-

ed transfection is not affected [74] (hAE-DMRIE: h-aminoethyl-dimyristoyl Rosenthal inhibitor ether;

DOPE: dioleoylphosphatidylethanolamine).

The observed differences in the PCI response

between the different transfection agents raise several

interesting questions. It seems logical to think that for

transfection agents where transfection is not stimulat-

ed by PCI, endosomal release is not a limiting factor

for ‘‘normal’’ transfection. The observation that trans-

fection by PEI complexes can be positively affected

by PCI [42,43] gives an indication that even if endo-

somal escape is induced by PEI; this process probably

is not always very effective. This may e.g. be related

to the size of the PEI/DNA complexes employed, as

pointed out by Ogris et al. [75]. With the proposed

mechanism of PEI enhancing endosomal release by

acting as a ‘‘proton sponge’’ [76], it would also be

necessary with a certain minimum amount of PEI

inside each endocytic vesicle in order to induce endo-

somal swelling and lysis. The observation that PCI

shows the greatest enhancement of PEI-mediated

transfection at low doses of DNA/PEI complex or at

low PEI/DNA ratios (Høgset, manuscript in prepara-

tion) is in accordance with this proposed mechanism,

since the positive effects of PCI should be expected to

increase with the decrease in the ability of the PEI to

effect endosomal release. This observation could also

have important implications for the use of PEI and

similar agents for in vivo gene therapy. With in vivo

delivery of DNA/PEI complexes, the amount of

complex reaching the target cells will often be very

limited and probably often well below the threshold

where PEI is able to induce efficient endosomal

escape. In these cases, PCI could be a very valuable

tool for increasing gene delivery specifically within

the target area for the therapy.

The negative effects of PCI seen with some lipidic

transfection agents might indicate that photochemi-

cally induced cytotoxicity may have some generally

inhibitory effect on transfection. If such effects play a

role, they may also affect transfection by polycationic

agents and may be reflected in a decrease in transfec-

tion by such agents that in some cases is observed at

higher light doses. PCI-induced transfection could

then be viewed as a balance between these general

negative effects and the positive effects caused by

increased endosomal release. Following this reason-

ing, for transfection agents that are very ineffective in

endosomal release, such as polylysine, the positive

effects would dominate, while for agents very efficient

in such release the negative effects would dominate.

To examine whether PCI would also have positive

effects on transfection when the DNA is delivered by

receptor-mediated endocytosis, we studied transfec-

tion with transferrin-polylysine. In HCT 116 cells,

PCI had an even better effect on transfection mediated

by transferrin-polylysine than on transfection with

unmodified polylysine [40,43], the same being true

for WiDr colon carcinoma cells (Olsen, unpublished

results). However, in THX melanoma cells, no trans-

fection with transferrin-polylysine could be observed,

neither with nor without PCI. Thus, PCI can work

very well also with receptor-mediated transfection

agents, but substantial cell line differences exist,

maybe related to the level of receptor expression in

the target cells.

PCI with adenoviral vectors. Adenovirus vectors

are known to be taken into the cell by endocytosis

and to be released from endosomes in a regulated

process. This endosomal release is usually regarded

as a very efficient process [77,78]. However, there

are still many cell types in which adenoviral gene

delivery is rather inefficient, and ineffective endo-

somal release might be important at least in some of

these cell lines. We have therefore investigated


whether gene delivery by adenoviral vectors might

also be amenable to improvement by PCI. Fig. 10

shows the result of an experiment where WiDr colon

carcinoma cells were subjected to infection by a h-galactosidase-encoding adenoviral vector at a low

virus dose. It can be seen that the PCI treatment

substantially increases transduction by this adenovi-

ral vector [44].

Further flow cytometric analysis showed that the

transduction efficiency in this cell line could be

increased up to about 30 times by the PCI treatment,

substantiating that also adenovirus-mediated gene

delivery can be strongly stimulated by PCI.

The finding that the effect of PCI seems to be

greatest at lower virus doses [44], may indicate that

adenoviral endosomal escape may be less efficient in

cases where there are relatively few viral particles in

the endocytic vesicles, although this remains to be

Fig. 10. PCI-induced adenovirus-mediated gene delivery. WiDr human col

h-galactosidase-encoding adenoviral vector at a multiplicity-of-infection (

incubation to allow for expression of the h-galactosidase transgene, the celin Ref. [44]. The cells were treated as follows: (A) no treatment, (B) aden

(E) AlPcS2a + adenovirus + 8 min light. Reproduced with permission [44]

proven. PCI with adenoviral vectors has now been

tested in 12 different cell lines, and in all cases a

positive effect on transduction has been observed

(Engesæter et al., submitted; [95]). However, the

magnitude of the effect seems to vary quite substan-

tially between different cell lines, maybe because of

different efficiency of uptake or endosomal release in

the different cell lines. The uptake mechanism [79–

82] and the subsequent intracellular trafficking of the

viral particles are probably also of importance for the

effect of the PCI treatment on adenovirus mediated

gene delivery (Engesæter et al., submitted).

Taken together, PCI has the potential of being a

very useful technology for in vivo gene delivery, both

because PCI can improve the delivery of genes in

general and because it does this in a light dependent

way, rendering the therapeutic gene active only in

illuminated sites of the body. Thus, by the employ-

on carcinoma cells were incubated with AlPcS2a (S), infected with a

MOI) of 5 and illuminated as indicated in the figure. After 2 days

ls were stained with X-gal and analysed by microscopy as described

ovirus only, (C) adenovirus + 8 min light, (D) AlPcS2a + 8 min light,

.


ment of PCI adverse effects due to expression in non-

target areas of e.g. a cytotoxic gene could largely be

avoided, making PCI especially attractive for gene

therapy of cancer and other localised diseases.

4. PCI in vivo

In vivo the effect of PCI-mediated therapy on

tumour treatment has been documented both with

the protein toxin gelonin [83] and with the cytostatic

Fig. 11. PCI in vivo. (A) Effect of gelonin-PCI on tumour growth. Kaplan–

WiDr adenocarcinomas subcutaneously growing in athymic mice. The end

have reached a tumour volume of >1000 mm3. 10 mg/kg AlPcS2a was injec

right hip. After 40 h, 50 Ag of gelonin was injected into the tumour and,

halogen lamp (Xenophot, HLX64640) filtered through a 580-nm long-pass

the tumour area, the animals were covered with aluminium foil. Further ex

(x) gelonin only; (.) AlPcS2a and light; (D) AlPcS2a, gelonin and light

treated with gelonin-PCI as described in the legend to panel A. Pictures

treatment (B) and 2 months after treatment (C).

drug bleomycin (Høgset et al., in preparation). In

these studies the photosensitiser AlPcS2a was admin-

istered by intraperitoneal injection, followed (48 h lat-

er) by a single intratumoral injection of gelonin or

bleomycin followed by illumination. In initial experi-

ments it was shown that with this mode of AlPcS2aadministration the photosensitiser localised in endo-

somes also in vivo, and could be relocalised by

illumination [83]. Furthermore, as shown in Fig.

11A and B, PCI with gelonin had a substantial effect

on tumour-growth. Thus, with this treatment regimen

Meier type plot of the effect of PCI with the protein toxin gelonin on

-point is the time after light exposure when the individual tumours

ted intraperitoneally in mice with 100 mm3 tumours growing on the

6 h later, the tumours were illuminated (135 J/cm2 from a 150-W

and a 700-nm short-pass filter, emitting 150 mW/cm2). Except above

perimental details can be found in Ref. [83]. (5) Untreated control;

(gelonin-PCI). (B) PCI-mediated tumour treatment. Animals were

of the tumour area were taken before treatment (A), 2 weeks after


67% of the mice receiving PCI became completely

tumour-free, while only 10% complete responses were

seen in animals treated with pure PDT, and none in

tumours treated with gelonin alone (data not shown).

The PCI treatment was found by Cox regression

analysis to be significantly different from the PDT

treatment ( p = 0.004).

There were no observations of toxic effects outside

the treated area and, despite some initial scarring, the

treated tissue seemed to regenerate extremely well

(Fig. 11B). This demonstrates that PCI is a highly

powerful and relevant technology for in situ delivery

and activation of molecules with a therapeutic poten-

tial [83]. The results obtained with bleomycin were

similar (Høgset et al., in preparation).

In further experiments, we have shown that PCI

also works very well in vivo when employing the

‘‘light first’’ procedure (Høgset et al., in preparation),

and also that the photosensitiser can be given as s

local injection, a fact that could make it possible to

substantially reduce to dose of photosensitiser and

thereby diminish the possibilities of side effects of the

photochemical treatment.

5. Advantages and limitations of PCI as a drug

delivery technology

There are several advantages of PCI for application

as a general drug delivery method. (i) Principally, there

are no restrictions on the size of the molecules that can

be effectively delivered, making PCI highly flexible

for a wide variety of molecules. Thus, the technology

has been shown to work very well with ‘‘molecules’’

of vastly different sizes, ranging from bleomycin

(MWc 1400) to adenoviral particles. (ii) Due to the

local and focused light-dependent activation, PCI is a

method with a high site-specificity limiting the bio-

logical effect to only illuminated areas, a property that

should lower potential systemic side effects of the

delivered drug. In addition, photodynamic therapy

has been established as an accepted cancer treatment

modality showing low or no systemic side effects [1].

(iii) PCI is a method with high efficiency for many

types of molecules and the dose of a drug may

therefore be reduced, resulting in reduced side effects.

(iv) In contrast to what is the case for radiation and

cytostatic therapy, therapy based on PCI may also be

efficient on non-dividing cells, which could be essen-

tial for the killing of resting malignant cells in cancer

therapy. (v) The photochemical treatment can induce

expression and secretion of cytokines e.g. in the

tumour parenchyma [84] resulting in a local response

that could give increased activity of inflammatory and

immune cells at the treatment site, possibly augment-

ing an anti-tumour activity of a PCI-based therapy. In

other treatment situations, this can, however, also be a

disadvantage. (vi) PCI is very well suited for combi-

nation with other modalities/strategies for targeted

drug delivery such as conjugation of drug molecules

with different ligands mediating target cell specific

receptor-mediated endocytosis.

Since the photosensitiser in PCI necessarily will

be located in quite close proximity to the molecule to

be internalised, an obvious potential disadvantage is

that the photochemical treatment will damage not

only the endosomal membrane, but also the molecule

to be internalised. It is well known that e.g. DNA

can be damaged by photochemically induced oxida-

tion [85,86], leading both to induction of mutations

and possibly to making the DNA unfunctional for

expression.

Thus, not unexpectedly, we have several indica-

tions that endocytosed molecules may be damaged by

the PCI procedure. For example, it can be seen from

Fig. 4C that the total enzymatic activity of the HRP

protein goes down at the higher light doses, indicating

photochemically induced damage to this protein. The

same phenomenon can in many cases be seen for PCI-

mediated gene transfer (see Fig. 5A and e.g. Refs. 73

and 43), indicating photochemical damage also to

transfecting DNA. Photochemical damage can also

explain the decrease in transfection seen after PCI

with some cationic lipid transfection agents [43,74].

This indicates that photochemical damage may be

more important for some transfection agents than,

for others, either because some transfection complexes

are located closer to the sensitiser (e.g. lipids could be

expected to localise nearer to the membrane contain-

ing the sensitiser than e.g. a polycationic transfection

agent), or because different complexing agents protect

the DNA from damage to a different extent.

The importance of photochemical damage for the

overall efficiency of PCI-mediated drug delivery is not

known and will probably vary substantially with the

molecule to be internalised, and maybe also with the


photosensitiser used. However, employment of the

‘‘light first’’ mode of PCI (see Section 2.3) should

potentially diminish photochemical damage since

many of the photochemically induced reactions should

be over at the time the molecule to be internalised is

introduced into the cell. Further discussions of these

issues can be found e.g. in Refs. 38 and 73.

Cytoplasmic drug delivery can also be achieved by

employing different peptides that confer membrane

permeability. Such peptides can in principle be used to

deliver a variety of different substances [87] and both

peptides inducing direct plasma membrane passage

[88] and peptides inducing endosomal release [89]

have been described. Due to differences in the exper-

imental systems, it is very difficult to make a quan-

titative comparison between literature data on the

effects obtained by such peptides and the effect

achieved by PCI. One important difference is, how-

ever, that such delivery peptides will usually exert

their effects in all regions of the body, in contrast to

the site-specificity inherent in the PCI technology.

Thus, it might seem that PCI could have advantages

for treating local disease, while the peptides could be

advantageous in cases where a systemic response is

desired.

As for photodynamic therapy, one important re-

striction of PCI in vivo is the limited penetration of

light into the tissue. However, although the limited

penetration depth for some applications obviously is

a disadvantage, it could in a sense also be viewed as

an advantage, since it makes it possible to quite

strictly confine the PCI effects to the desired region

of the body. In tissues, the light penetration decays

approximately exponentially (e� 1) for every 2–3

mm, with a theoretical maximum for PDT effects

of about 1 cm if a photosensitiser that absorbs in the

far-red region of the light spectrum is used [90,91].

For PCI, the penetration depth would be substantial-

ly larger, since very good PCI effects can be

achieved with light doses killing only a fraction of

the cells. Thus, from theoretical consideration, the

penetration depths of effective PCI could be expected

to something around 2 cm. Furthermore, recent

developments in fibre optics and laser technology

have also made it possible to illuminate many sites

inside the human body [1], e.g. in the gastrointestinal

tract, urogenital organs, lungs, brain and pancreas.

Thus, with the combination of a fibre-optic device

and a penetration depth of about 2 cm, most loca-

tions in the body could be reached by PCI-mediated

treatments.

PCI-mediated therapy could also be combined with

surgery. For example, for many localised cancers,

surgery will, of course, be the first option for treat-

ment. However, in many cases, local recurrence can

represent a serious problem. In such cases, local drug

or gene therapy killing off remaining undetected

tumour cells or inhibiting the proliferation of tumour

cells in the treated area could give substantial thera-

peutic benefit. Of course, when PCI is combined with

surgery illumination of the target area should repre-

sent no problem because of easy accessibility to the

lesions. Furthermore, employing the ‘‘light first’’

mode of PCI (see above) the whole treatment could

be done in one operation, with illumination being

performed directly after surgery, followed immediate-

ly by delivery of the therapeutic agent.

The fact that the photochemical treatment of cells

induces cytotoxicity will in many cases obviously be a

disadvantage, for example in several gene therapy

approaches. However, e.g. in cancer therapy where

the obvious goal is indeed to kill the tumour cells, the

cytotoxicity can also be viewed as an advantage.

Also for cardiovascular applications, the cytotox-

icity associated with the photochemical treatment

seems to be well tolerated and might have beneficial

effects. Thus, studies of photodynamic therapy of

restenosis and atherosclerosis indicate that the photo-

chemical treatment in itself may have substantial

clinical benefit without causing serious adverse effects

on the treated blood vessels [13,92,93].

A very important point also is that, although the

photochemical treatment induces cytotoxic effects,

these are generally restricted to illuminated areas of

the body [9], the photosensitisers in themselves usu-

ally have very little systemic toxicity. Thus, damage to

vital organs could generally be avoided. Also, there is

substantial clinical experience with relevant photo-

sensitisers, showing that they can be used safely in

humans [1].

6. Concluding remarks

PCI is a novel technology for specific delivery of

membrane impermeant molecules into the cytosol of


target cells. PCI is based on the use of photosensitising

compounds that can be used safely in humans and for

many of which there is considerable clinical experi-

ence. PCI’s main application is in the delivery of

molecules acting on intracellular drug targets, and in

delivery of genes for gene therapy. The PCI technology

can be used with a variety of ‘‘molecules’’, from low

molecular weight cytostatic drug to viral gene therapy

vectors. PCI-mediated drug delivery is induced by

illumination; thus, the technology represents a means

of achieving site-specific drug delivery that can be used

in all regions of the body where it is possible to deliver

light andwhere local activation of a drug is desirable. In

vitro PCI has been shown to work with important

classes of therapeutic molecules, such as proteins,

immunotoxins, peptides, oligonucleotides, genes and

a low molecular weight cytotoxic drug, and in vivo

very good effects on tumour treatment have been

demonstrated. In addition to the potential use with

macromolecules, PCI opens up the interesting possi-

bility of exploiting new classes of low molecular

weight therapeutic molecules that could be especially

advantageous for use with PCI. These would be mol-

ecules that would not to be able to reach their intracel-

lular target on their own. Since their activity would then

be totally dependent on the specific delivery technol-

ogy, the possibility of unwanted side effects should be

much smaller than for most current therapeutics. In

addition, PCI can also be used with many targeted

therapeutic agents, potentially adding further to the

specificity obtainable with such molecules. Altogether,

PCI should be a very valuable addition to the arsenal of

drug and gene delivery methods for in vivo therapy.

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