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Medical Laser Application 21 (2006) 239250 www.elsevier.de/mla

Photochemical internalization (PCI): A novel technology for activation of endocytosed therapeutic agents
Kristian Berga,, Anders Hgsetb, Lina Prasmickaitea, Anette Weyerganga, Anette Bonsteda, Andreas Dietzea,f, Pei-Jen Louc, Stephen Bownd, Ole-Jacob Noruma,e, ( rda, Pa ( l Kristian Selboa Hanne Mali Thesen Mllerga
Department of Radiation Biology, Inst. for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway b PCI Biotech AS, Hoffsvn. 48, 0375 Oslo, Norway c Department of Otolaryngology, National Taiwan University Hospital and College of Medicine, Taipei 10016, Taiwan d National Medical Laser Centre, University College London, London, UK e Department of Surgical Oncology, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway f Department of Orthopedic Surgery, Vestfold County Hospital, N-3103 Tnsberg, Norway Received 31 July 2006; accepted 8 August 2006
a

Abstract
The utilization of macromolecules in the therapy of cancer and other diseases is becoming increasingly important. Recent advances in molecular biology and biotechnology have made it possible to improve the targeting and design of cytotoxic agents, DNA complexes and other macromolecules for clinical applications. In most cases the targets of macromolecular therapeutics are intracellular. However, 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. Photochemical internalization (PCI) is a novel technology for the release of endocytosed macromolecules into the cytosol. The technology is based on the activation by light of photosensitizers located in endocytic vesicles to induce the release of macromolecules from the endocytic vesicles. Thereby endocytosed molecules can be released to reach their target of action before being degraded in lysosomes. PCI has been shown to stimulate intracellular delivery of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), RIP-based immunotoxins, DNA delivered as gene-encoding plasmids or by means of adenoviruses or adeno-associated viruses, peptidenucleic acids and chemotherapeutic agents such as bleomycin. The efcacy and specicity of PCI of macromolecular therapeutic agents have been improved by combining the macromolecules with targeting moieties, such as the epidermal growth factor. Several animal models have been used for in vivo documentation of the PCI principle. Recent results also indicate that PCI may reverse doxorubicin resistance or be utilized to circumvent multidrug resistance. In general, PCI can induce efcient

Corresponding author. Tel.: +47 22 93 42 60; fax: +47 22 93 42 70.

E-mail address: kristian.berg@labmed.uio.no (K. Berg). 1615-1615/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.mla.2006.08.004

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light-directed delivery of macromolecules into the cytosol, indicating that it may have a variety of useful applications for site-specic drug delivery, as for example in gene therapy, vaccination and cancer treatment. r 2006 Elsevier GmbH. All rights reserved.
Keywords: Photodynamic; Photochemical internalization; Photosensitizer; Macromolecule; Peptide nucleic acid; Gene therapy; Drug delivery; Protein toxin

Introduction
Macromolecular drugs have the potential advantage of exerting a higher therapeutic specicity, than is possible with most smaller molecular chemotherapeutic agents. A large number of macromolecules are today under development for use as medicines and a few have already been approved, e.g. a CD33-targeted immunoconjugate (Mylotarg), an HER2-targeted antibody (Herceptin), an epidermal growth factor receptor (EGFR)-targeted antibody (Cetuximab) and recombinant IL-2 truncated diphtheria toxin fusion protein (Ontak) [1]. Macromolecules with therapeutic potential include proteins such as ribosome-inactivating protein (RIP) toxins for treatment of cancer and other indications (see below), antibodies and growth factors for cell surface targeting, peptides and mRNA for vaccination, DNA for gene therapy, and antisense, ribozymes, peptide nucleic acids (PNAs) and siRNA for gene silencing [2]. There are many extracellular and intracellular barriers for these molecules to overcome before they can arrive at the target cells, enter the cells and reach their intracellular targets [3]. Intracellular barriers may include the plasma membrane, endocytic membranes and, in gene therapy, the nuclear membrane. Many of the delivery methods developed are based on improved endocytic uptake and release of the therapeutic molecule from the endocytic vesicles into the cytosol. However, major limitations in the use of macromolecular therapy are still the low rate of penetration through the membranes of endocytic vesicles and degradation of the macromolecules by lysosomal enzymes [4]. Photodynamic therapy (PDT) is a treatment modality that takes advantage of the phototoxic effects induced by a photosensitizer and light in the presence of oxygen [5]. PDT is approved for several cancer indications as well as age-related macular degeneration. Fluorescence diagnosis (FD), based upon the uorescing properties of photosensitizers, has been recently approved for detection of bladder dysplasia and cancer. A major limitation of PDT is the penetration of light through tissues and the depth of necrosis that can be induced within an acceptable duration of light exposure. The depth of necrosis induced after PDT depends on the tissue concentration of photosensitizer, the total light dose and how the light is delivered as well as on the optical

properties of the target tissue. Typically the depth of necrosis induced by PDT is 210 mm. The photosensitizers show some degree of preferential retention in tumours, usually with a 23 fold higher accumulation in tumour tissues as compared to the normal surrounding tissue [6]. This specicity is however not sufciently high to avoid normal tissue damages. In many cases, normal tissue regenerates well after PDT [7], although severe adverse effects may occasionally be seen, such as skin sensitivity to light that can last up to several weeks after photosensitizer administration [8,9]. In general, there is a need for improving PDT with respect to efcacy, i.e. reduced time of light exposure, increased depth of necrosis and greater tumour specicity. Photochemical internalization (PCI) is a novel technology that is under development for utilizing the properties of PDT to enhance the therapeutic effect of macromolecules. Pre-clinical results indicate that PCI may increase the depth of necrosis and reduce the total uence needed to obtain similar effects to those seen with PDT. The basic principles and present status of the pre-clinical development of PCI is presented in this review.

Endocytic membranes as primary targets in photochemical drug and gene delivery

Intracellular localization and cytotoxic effects of photosensitizers
PDT is a treatment modality where a photosensitizing compound is activated by light in an oxygen-dependent manner, leading to oxidation of biomolecules in the light-exposed tissue. Porphyrins and many other structurally related photosensitizing compounds have been utilized in PDT and shown to induce cytotoxic effects on cells and tissues. The cytotoxic effects of PDT are mediated through formation of reactive oxygen species (ROS), mainly singlet oxygen (1O2). This reactive intermediate has a very short lifetime in cells (o0.04 ms) [10,11]. Thus, the primary cytotoxic effect of PDT is executed during light exposure and very close to the sites of formation of 1O2 (o20 nm). It is well documented that a number of photosensitizers, including di- and tetrasulphonated aluminium phthalocyanine (AlPcSn), sulphonated tetraphenylporphines (TPPSn),

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NPe6, nile blue, chlorin e6 derivatives, uroporphyrin I, lutetium texaphyrin (LuTex), silicone phthalocyanine (Pc 4), tin ethyl etiopurpurin (SnET2) and acridine orange are partly or mainly located in endosomes and lysosomes, collectively described as endocytic vesicles, of cells in culture [12]. This is in most cases due to endocytic uptake, while in some cases the dye is a weak base and is trapped in lysosomes and acidied endosomes. Photoimmunoconjugates linking photosensitizers to antibodies, other targeting macromolecules or

~10 nm
SO3 SO3 AlPcS 2a -

1O 2

lifetime: 0.01-0.04 s diffusion length: 10-20 nm

Macromolecules

~0.5-1 m

Fig. 1. Photochemical targeting. The gure illustrates an endocytic vesicle within a cell. In PCI, the photosensitizer molecules (illustrated by AlPcS2a) and the macromolecules accumulate in endocytic vesicles as indicated. The most efcient photosensitizers for PCI are mainly located in the membrane of the vesicles. Upon illumination reactive oxygen species, mainly singlet oxygen, are formed, constituents of the membranes are destroyed and passage of macromolecules to the cytosol becomes possible.

synthetic polymers may improve the specicity and efcacy of PDT [1315]. These conjugates are expected to be endocytosed and the photosensitizer will accumulate in endocytic vesicles as long as the conjugate is not enzymatically degraded or the photosensitizer is not able to penetrate the endocytic membranes after release from its linkage to the polymer. Light exposure of cells containing photosensitizers in their endosomes or lysosomes leads to a permeabilization of these vesicles and release of the photosensitizers into the cytosol [16]. In some cases, e.g. by using TPPS2a and TPPS1, but not TPPS4, substantial levels of lysosomal enzyme activity have been found in the cytosol after PDT, indicating that the lysosomal contents can be released into the cytosol without losing their activity [17]. These properties of photosensitizing dyes may be used to release endocytosed molecules, such as therapeutic macromolecules, in a functionally intact form as described in Fig. 1. This way of improving the therapeutic effect of compounds trapped in endocytic vesicles has been named PCI and has been shown to increase the biological activity of a large variety of molecules [18]. Based on these and other results (e.g. [19] the preferential localization of sulphonated TPPs and AlPcs in endocytic vesicles is as described in Fig. 2. Amphiphilic disulphonated compounds are mainly located in the vesicular membranes while the tetrasulphonated compounds are mainly located in the matrix of the endocytic vesicle. TPPS2a and AlPcS2a are currently the preferred photosensitizers for use in PCI. It was initially thought that the photosensitizer and macromolecule to be released from the endocytic compartments had to be located in the same compartments at the time of light exposure. However, it has

TPPS2a

AlPcS2a Endosomes/lysosomes Al
SO3SO3-

SO3-

SO3-

Inside TPPS4
SO SO 33 -

SO3-SO3-

-SO SO 3 3

Fig. 2. Drawing of an endocytic vesicle showing the main location of a tetrasulphonated and amphiphilic disulphonated photosensitizers (not to scale). The chemical structures of the 3 photosensitizers most studied for PCI are shown on the left.

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I S S S

II Light S S S S S

III

and the macromolecule. The present status on the use of PCI for activation of various macromolecular structures is reviewed below. Photochemical delivery of proteins Protein toxins have been extensively evaluated, especially as part of immunotoxins, for use in cancer therapy as well as in graft versus host disease, bone marrow purging, autoimmune diseases and HIV infection [21]. These protein toxins usually kill cells by inhibiting protein synthesis, either by inactivating the elongation factor 2 (EF-2) or the EF-2 binding site on the 28 S ribosome subunit. The 28S RIPs are plant toxins that constitute either only one chain (A-chain, Type I toxins) with enzymatic activity or in addition another targeting chain (B-chain, Type II toxins, e.g. ricin) linked to the A-chain by disulphide bonds (Fig. 4) [22]. Both types of toxin are endocytosed, but while type I RIPs enter the cells through pinocytosis type II toxins enter the cells by the more efcient receptor-mediated endocytosis due to the B-chain binding to the cell surface. Type I RIPs nally enter mainly lysosomes where they are degraded while type II RIPs are partly transported retrogradely from endosomes to the trans-Golgi network, endoplasmic reticulum and by a not fully understood mechanism enter the cytosol where they can exert their enzymatic activity [23]. Type I RIPs therefore exert a relatively low cytotoxic effect on intact cells, while type II RIPs are highly cytotoxic compounds despite the fact that both types of RIPs have the same ability to inactivate ribosomes in cell-free systems. The literature indicates that only 110 RIP molecules located in the cytosol are sufcient to kill the cell [24]. Type I RIPs should therefore be good candidates for a highly specic PCI-based activation of macromolecules. PCI has been shown to release the type I RIP gelonin from endocytic vesicles in vitro and to activate its cytotoxic effect both in vitro and in vivo as illustrated in Fig. 4 [18,25]. Type I RIPs, such as gelonin and saporin, which have been frequently used in PCI are relatively small proteins with a molecular mass of about 30 kDa, and cannot easily be used unmodied for systemic administration due to leakage through the kidneys and lack of tissue specicity [22]. PCI of gelonin has been evaluated by injecting gelonin intratumorally into 68 mm tumours, which led to 6080% complete responses while similar photochemical treatment with intratumoral injection of saline induced complete responses in only 010% of the animals [25,26]. One may therefore assume that gelonin is able to move several millimetres in the tumor tissue within the time frame set by the experimental procedures. The time required for a molecule with a diffusion coefcient D to move by diffusion across distance L is approximately

S
S Fusion S S M M MM S S

M M

M MM

Fig. 3. An illustration of how macromolecules may enter cytosol when the macromolecules are delivered after the photochemical treatment. The photosensitizer (S) is endocytosed by the cells (I) and exposed to light (II) causing damage to the membranes of the endocytic vesicles. When the macromolecules (M) are delivered to the cells new vesicles are formed (II) which may fuse with photochemically treated vesicles and cause release of the macromolecules to the cytosol (III).

recently been found that the photochemical treatment can be performed up to 68 h prior to the delivery of the macromolecule without reducing the synergistic effect of the combined treatment [20]. The assumed mechanistic basis for such an observation is that newly formed vesicles are able to fuse with photochemically damaged vesicles and that the macromolecules are released into the cytosol after fusion of such vesicles (Fig. 3). Further studies are however required to fully document this hypothesis.

Photochemical delivery of macromolecules

Macromolecules for therapeutic purposes may be divided into 3 groups, i.e. proteins and peptides, nucleic acids and synthetic polymers for drug delivery and protection. For the PCI technology to induce increased translocation of therapeutic macromolecules to the cytosol the macromolecules need to be located in endocytic vesicles at some stage in the process. The hydrolytic environment of late endosomes and lysosomes may therefore set limits to the therapeutic effect of PCI. Furthermore, the ROS formed by the photochemical treatment may oxidize the macromolecular drugs for example by oxidizing the amino acids in proteins and peptides and guanine in the nucleotides. The quantum yield for inhibition of the therapeutic activity of the macromolecules will depend on the 3-D structure of the macromolecule, i.e. the access of ROS to amino acids or guanine of importance for biologic activity, as well as the sub-vesicular localization of the photosensitizer, i.e. distance between the photosensitizer

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Fig. 4. Principle structure of ribosome inactivation protein toxins (A) and their intracellular routing (B). Type 1 RIPs are shown to end up in lysosomes, where they are degraded, while a fraction of the type 2 RIPs are transported retrogradly (red arrows) through trans-Golgi network, the Golgi apparatus and endoplasmic reticulum (ER) to end up in the cytosol where they can target the ribosomes.

L2/4D [27]. For a molecule like gelonin with an expected diffusion constant of about 6 107 cm2/s in tumour tissue, to diffuse 2 and 3 mm may take about 4.5 and 10.5 h, respectively. This correlates well with the injection of gelonin 6 h prior to the photochemical treatment. In addition, the photochemical treatment may also be provided prior to gelonin injection suggesting that the radius of action for gelonin in such experiments is probably larger than 23 mm (Fig. 5). It should also be noted that intratumoral injection may induce local high hydrostatic pressure stimulating hydraulic conductivity, i.e. more rapid transport of RIPs in the tumour tissue. One may therefore speculate that PCI after simultaneous multiple intratumoral injections of type I or A-chain of type II RIPs could be an efcient way to eradicate large tumours. Preferential retention of relevant photosensitizers in neoplastic tissues (tumour-to-normal surrounding tissue ratio of about 3:1) and the site-directed delivery of light provide specicity for such a treatment scenario [28]. A major limitation of PDT is the penetration of light through tissues and the depth of necrosis that can be induced within acceptable time of light exposure. Cut tissue sections of a brosarcoma treated with systemically delivered AlPcS2a and exposed to light from an optical ber showed an increased depth and volume of necrosis when combined with gelonin administration [26]. Similar results were obtained by performing PCI of gelonin on rat liver after systemic administration of both the photosensitizer and gelonin (P.-J. Lou, S. Bown and co-workers, unpublished data). These results indicate

that by combining PDT with a potentially cytotoxic agent the depth of necrosis can be increased compared to PDT alone.

PCI for gene delivery Gene therapy is a novel therapeutic modality receiving great attention and being generally recognized as having the potential to constitute treatment for a wide range of diseases [29]. However, although there are some encouraging results clinical trials with gene therapy have hitherto largely given quite disappointing conclusions [30]. An important reason for this is that methods for efcient and specic delivery of therapeutic genes in vivo are lacking. With most gene delivery systems the therapeutic gene is taken into the cell by endocytosis, and for many of these systems, especially non-virusbased ones, the lack of efcient mechanisms for translocating the gene out of the endocytic vesicles constitutes a major hindrance for realization of the therapeutic potential of the gene. PCI has been studied as a gene delivery technology (reviewed in [31] both with several non-viral [3234] and viral vectors [35] mainly by using reporter genes such as genes encoding eGFP (enhanced green uorescent protein) or -galactosidase. However, PCI-mediated gene delivery has also been shown to induce the delivery of therapeutic genes, such as the genes encoding HSV-tk (Herpes Simplex Virus thymidine kinase) [36] p53 [37], TRAIL ([38] unpublished results) and IL-12 (interleukin-12) (unpublished results).

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100

Non-viral vector

Cationic polymers e.g. polyethyleneimine Cationic lipids Target PCI Cytosol

Nucleus

cell

80
% of tumors <1000 mm2

DNA
60
Control gelonin, light after AlPcS2a + gelonin

Endosomes

Viral vector

Lysosomes Targets

Therapeutic product

40

PDT (AlPcS2a + light) PCI, light after Light only

20

0 0 50 100 150 200 Time after light exposure (days)

Fig. 5. KaplanMeier plot of response to PCI of gelonin on WiDr colon adenocarinoma tumors, grown subcutaneously in Balb/C (nu/nu) mice using AlPcS2a (10 mg/kg) 48 h prior to light delivery. The treatments are described on the gure. In the PCI groups gelonin (50 mg) was intratumorally injected immediately after (PCI, light rst) or 6 h before (PCI, light after) the light exposure*. In some of the controls light was delivered in the absence of AlPcS2a, e.g. 6 h after gelonin injection (gelonin, light after) or immediately before gelonin injection (gelonin, light rst). Some control groups have not been shown to make the gure more readable. Except for the time of gelonin injection the treatment was performed as previously described [25]. The end-point is the time after light exposure when the individual tumours have reached a tumor volume of 1000 mm3. The results are based on 6 or more animals per group. *Note: It should be noted that gelonin injection immediately before the light exposure induced a moderate tumor growth delay that was not seen when light was administered 6 h before gelonin injection. This difference may be related to heat generated by the light source as observed in this study (approx. 4041 0C intratumorally). The apparently higher cure rate observed with PCI of gelonin administrated immediately after the light exposure than 6 h before light exposure may therefore not be a PCI-related effect.

Fig. 6. The role of PCI in gene therapy. The transgene is delivered as a plasmid and complexed to a non-viral vector or delivered as part of the genome of a viral vector. The vector facilitates cellular uptake of the vector/DNA complexes (e.g. interaction of the positive overall charge of the non-viral vector/DNA complexes or Coxsackie and adenoviral receptor binding for the adenovirus). The vector/DNA complexes enter the endocytic vesicles and are either degraded by lysomal enzymes or are released into the cytosol. Alternatively, PCI stimulates the release from the endocytic vesicles before degradation. The transgene will thus reach the nucleus and become expressed.

Delivery of genes to cells for therapeutic purposes requires in most cases that the DNA is protected from degradation and that the cellular uptake is facilitated (Fig. 6). The vehicles used for such purposes are named vectors and may be divided into chemical or non-viral and viral vectors. In addition, physical methods such as electroporation may be utilized. The vectors need in addition to facilitate escape from the endocytic vesicles into the cytosol for further transfer to the nucleus. Polyethylenimine (PEI) is an example of a non-viral vector, which in a concentration-dependent manner enhance transgene delivery into the cell cytosol. The proposed mechanism for the PEI enhanced endosomal

release of DNA is based on the proton sponge effect suggesting that the pK values of the amino groups of PEI are close to physiological pH and thereby cause increased accumulation of water in the acidic environment of the endocytic vesicles until they lyse. Despite the intrinsic abilities of PEI to lyse endosomes, PCI has been shown to further enhance transgene delivery of plasmid/ PEI polyplexes in vitro and in vivo [36]. Furthermore, a recent in vivo study shows that treatment of squamous cell carcinomas decient in active p53 with intratumoral injection of the photosensitizer AlPcS2a and a plasmid expressing p53 complexed to a glycosylated PEI caused a dramatic tumour regression in all the transfected animals [39]. In contrast, similar PDT or PEI-p53 treatments did not delay tumour growth. Recently, a highly sophisticated vector design was shown by Kataoka and colleagues [40]. Their design was based on a disulphide-extended cationic peptide complexed with the plasmid. This cationic-peptide complex was mixed with a negatively charged dendrimer containing 32 carboxyl groups at the surface and a phthalocyanine photosensitzer in the core. It was shown that this complex caused efcient transfection at doses of light causing hardly any cytotoxicity. When this vector was evaluated in the conjunctiva of rats, transgene expression was shown only in the irradiated area without any observable damage to the treated tissue. This opens up therapeutic possibilities for indications, such as monogenetic diseases, where it is important to avoid tissue damage. Adenovirus vectors are known to be taken into cells by endocytosis and to be released from endosomes by a regulated process. This endosomal release is usually regarded as a very efcient process [41]. It is therefore

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somewhat surprising that PCI is able to increase the number of adenoviral tranduced cells by up to 30-fold. Nevertheless, PCI with adenoviral vectors has been tested in 12 different cell lines, and in all cases a positive effect on transduction has been observed [42]. A major limitation for adenoviral gene therapy is the ubiquitous expression of the adenovirus primary receptor (Coxsackie- and adenovirus receptor, CAR) on most epithelial cells, causing a lack of specicity of gene transfer. Moreover, the expression of CAR in cancer cells is in many cases lower than in its normal counterparts, resulting in resistance to Ad5 infection of cancer cells. We have shown that PCI is efcient for enhancing gene transfer from adenoviral vectors, which infect cells through alternate receptors than CAR. Such retargeting of adenovirus to cell-specic receptors combined with a light-specic treatment to enhance gene transfer and expression presents an opportunity to obtain high and site-specic cell-infectivity levels with reduced viral doses. Adeno-associated viruses (AAV) derived from endemic non-patogenic parvoviruses have several properties that make them attractive as vectors in gene therapy [43]. These properties include the absence of host inammatory response, low cytotoxicity, a low cellmediated immune response, the ability to transduce non-dividing cells, the induction of long-term gene expression and a broad tropism. A major limitation is the packing constraints of about 5 kbs. PCI has recently been found to increase the transduction efcacy of a serotype 5 AAV in one of two glioblastoma cell lines tested. The non-responding cell line required a much lower number of viral particles to transduce the same number of cells and it was speculated that PCI might improve the transduction efcacy only in cells difcult to transduce by AAVs [44]. Overall, PCI has the potential of being a very useful technology for in vivo gene delivery, both because it 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. The possibility to use photochemically activated promoters may further add to the specicity of such treatment [45,46]. Thus, by the employment of PCI adverse effects due to expression in non-target areas of, for example a cytotoxic gene could largely be avoided, making PCI especially attractive for gene therapy of cancer and localized diseases. PCI of oligonucleotides Several oligonucleotides are currently under investigation for regulation of protein expression patterns in neoplastic as well as other cell types [47]. Oligonuleotides have been designed in several ways in order to obtain stability towards degradation in biological environments and penetration through membrane bar-

riers without losing biological activity. PNAs are oligonucleotides where the bases are linked together by peptide bonds, which are more stable towards degradation than phosphate ester bonds [48]. PCI has been shown to improve the biological activity of PNA towards telomerase (hTERT); this is even more efcient than Tat-linked PNA where Tat is expected to be an efcient aid in the transfer of macromolecules across biological membranes [49]. PCI of PNA can be further improved by linking targeting ligands to the PNA [50,51]. PCI of low-molecular weight substances A few chemotherapeutic agents accumulate in endocytic vesicles probably due to their size and/or charge. Bleomycin (MW$1400) is a widely used chemotherapeutic agent for several types of cancer, but is also known for accumulating in endocytic vesicles [52]. PCI of bleomycin has been shown to improve its cytotoxic effect both in vitro and in vivo [53], indicating that not only large macromolecules like proteins and DNA may prot from a PCI-based treatment, but also smaller molecules unable to penetrate the biological membranes efciently. This may make it possible to reduce the administrated dose of bleomycin, so reduing the side effects without losing clinical efcacy. In earlier in vivo studies subcutaneously growing xenografts have been utilized to document the PCI principle. More recently, PCI of bleomycin has been found to signicantly delay growth of human brosarcoma cells injected intramuscularly in athymic mice (Norum et al., unpublished results).

Targeting and specicity

The therapeutic applicability of macromolecules as well as small molecular chemotherapeutic agents is in most cases limited by the adverse reactions to the treatment. Reduction of adverse effects might be feasible through drug delivery modalities that would reduce the uptake of the drugs by non-target cells and selectively deliver the drug only to the target cells (and/or intracellular sites) at relatively low extracellular concentrations. A current approach to site-specic drug delivery is to attach the therapeutic agent to a carrier recognized only by the cells where the pharmacological action is desired [54]. Alternatively, in gene therapy specicity may be enhanced by tissue specic promoters for the therapeutic gene delivered to the target cells, the use of tissue specic replication of viruses or gene silencing molecules binding to genes specically expressed in the diseased tissue [55,56]. However, specicity is still a major hurdle in the medical treatment of many diseases.

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The use of immunotoxins for treatment of solid tumours has not been very successful. This is partly due to the tumour physiology such as the long distance from the vasculature to the target cells and lack of convection due to a high interstitial pressure [27]. Furthermore, the rate constant for transfer of immunotoxins from endocytic vesicles to the cytosol has in some cases been shown to be as much as 104-fold slower than its rate of endocytosis, indicating that release from endocytic vesicles may be a rate limiting step in the cytotoxicity of some immunotoxins [57]. Accordingly, the immunotoxin MOC31-gelonin (MOC31 binds to the EGP-2 antigen, also known as Ep-Cam and 17-1A, found on most carcinomas) was found suitable for combination treatment with PCI, since the immunotoxin itself did not show any cytotoxic effects [58]. There are, however, large variations in the cytotoxic effect of immunotoxins and it remains to be seen how the cytotoxicity of the immunotoxin itself is related to a synergistic effect of a combination with PCI. Preliminary results with PCI of a systemically delivered scFv-rGelonin immunotoxin, inducing tumour growth inhibition alone, suggest that there is indeed a synergistic effect (Selbo et al., unpublished results). A further improvement of PCI-based gene delivery may be obtained by using targeting vectors. EGFR is a very promising targeting moiety in cancer therapy due to its strong expression in a large variety of cancers [59]. PCI of EGF-coupled protein toxin signicantly enhanced cytotoxicity as compared to PCI of the unconjugated protein toxin and EGFcoupled PEG/PEI/plasmid complexes as well as EGFR-retargeted adenovirus improved transfection efcacy in vitro [6062]. Similar results have been obtained using a transferrin-linked vector [33], indicating that PCI can improve the efcacy of targeted gene therapy vectors as well as other molecules linked to targeting moieties. The vascular leakage and reduced lymphatic drainage typical of tumour tissues causes enhanced permeability and retention (EPR) on the systemic delivery of macromolecules such as polymer-drug and polymerprotein conjugates [63]. The polymers usually have no intrinsic specicity for tumour tissues, but due to the EPR effect increased accumulation of therapeutic compounds are observed. In addition, enhanced uptake by tumour cells may be observed due to nonspecic binding to the plasma membrane, reduced immunogenicity, prolonged plasma half-life and enhanced stability of the therapeutic compound. These factors have made polymer-conjugates, collectively named polymer therapeutics, promising as anticancer conjugates. It has recently been found that PCI of conjugates between polyamidoamine dendrimers and saporin as well as doxorubicin enhance the relocation of these conjugates to the cytosol and

enhance cytotoxicity in vitro (P.-J. Lou and coworkers, unpublished results).

PCI to circumvent or reverse multidrug resistance

One of the major challenges in chemotherapy of neoplastic diseases is the inherent or acquired resistance to single substances and especially co-resistance to apparently chemically and structurally different agents. Acquired multidrug resistance (MDR) is often caused by increased expression of plasma membrane transporters, extruding cytostatic agents out of cells and thereby reducing the therapeutic efcacy [64]. These transporters belong mainly to the ABC protein superfamily, but nonABC transporters such as the major vault protein may also contribute to the MDR phenotype. Although small molecular inhibitors of MDR pumps have been shown to be efcient in vitro clinical documentation of efcient and safe MDR inhibitors is still lacking. An alternative method to attenuate the activity of the MDR transporters is the gene therapy approach, especially by transfection with various oligonucleotide such as antisense, ribozymes and siRNA [65]. The aim is to inhibit expression of the MDR transporters and combine the gene therapy with cytostatic agent treatment. Many of the clinical trials are focused on drug resistance (4.9% (56 trials)) of all clinical gene therapy trials, but so far these are mainly Phase I studies (see http://www.wiley.co.uk/genetherapy/clinical/). We have recently initiated studies to circumvent the MDR phenotype by delivering macromolecules, such as genes and protein toxins, by the PCI principle (Fig. 7) [66]. Macromolecules are not substrates for the MDR transporters and cells with the MDR phenotype due to expression of these transporters should thereby not be refractory to PCI of macromolecules. This was indeed found to be the case in an uterine sarcoma cell line overexpressing the common MDR transporter p-gp. The MDR cell line was found to be as sensitive to PCI of gelonin as the parental cells although the doxorubicin sensitivity was reduced 100fold. Similarly transgene activation by PCI of adenoviral-based gene therapy was enhanced in both the MDR and the parental cells. Alternative MDR mechanisms such as the acidication of endocytic vesicles and increased cytosolic pH, have been documented, causing entrapment of weak base chemotherapeutic agents (e.g. doxorubicin) in the endocytic vesicles lowering their concentration in the target organelles (for example the nucleus) [67]. PCI has been found to reverse such a drug resistant mechanism by rupturing the endocytic vesicles and releasing doxorubicin into the cytosol, thereby increasing the nuclear accumulation of doxorubicin. (Pei-Jen Lou et al., Int.J.Cancer, In press)

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Conclusions
From pre-clinical studies, PCI seems to be a highly efcient method for internalization of macromolecules. Substantial site-direction of the treatment is obtained by the need for light activation and in some diseases, such as solid cancers, the partial preferential retention of the photosensitizer in the diseased areas. This dual selectivity will reduce damage to the normal surrounding tissue. Most technologies developed so far are useful for internalization of only one group of compounds, while PCI has been shown to act efciently in internalization of a large variety of macromolecules and may be useful for internalization of different macromolecules simultaneously. The method can be combined with most other means for generating site and tissue specicity. In

addition, PCI has been found to induce cytotoxicity in deeper tissue layers than the corresponding PDT, indicating that thicker lesions may be treated or lower light doses may be required for obtaining satisfactory clinical outcome. PCI shows to be promising as a technology for cancer treatment, but also for other indications where localized tissue ablation is a major goal (e.g. rheumatoid arthritis [68]) and in cases where it is important to avoid normal tissue damage, photosensitizers may be designed to meet such a requirement [40].

Zusammenfassung
Die photochemische Internalisierung (PCI): Eine neue Technologie zur Freisetzung von therapeutischen Wirkstoffen, die durch Endozytose aufgenommen werden Die Nutzung von Makromoleku len bei der Therapie von Tumoren und anderen Krankheiten gewinnt immer mehr an Bedeutung. Fortschritte in der Molekularbiologie und Biotechnologie haben es ermo glicht, den gezielten Einsatz und die Entwicklung von zytotoxischen Substanzen, DNA-Komplexen und anderen Makromoleku len fu r klinische Anwendungen zu verbessern. In den meisten Fa llen sind die Ziele der Makromoleku rer Natur. Der Abbau von le intrazellula Makromoleku len in endozytotischen Vesikeln nach Endozytose stellt jedoch ein betra chtliches Problem fu r die therapeutische Anwendung von Makromoleku len mit intrazellula rer Wirkung dar. Die photochemische Internalisierung (PCI) ist eine neue Technologie zur Freisetzung von Makromoleku len ins Zytosol, die u ber Endozytose aufgenommen wurden.
Fig. 7. PCI to circumvent or reverse multidrug resistance (MDR). PCI of macromolecules may be utilized to circumvent MDR (upper gure) or to reverse accumulation of lysosomotropic weak bases such as doxorubicin in acidic vesicles such as late endosomes or lysosomes (lower gure). Upper gure: (A) P-gp pumps doxorubicin (Dox) out of the cell, a major obstacle in chemotherapy of cancer. (B) The macromolecular drug (M) is taken up together with the photosensitizer (S) by endocytosis. The drugs co-localize and are entrapped in endocytic vesicles, another common drug resistance mechanism. M is subjected to enzymatic degradation (light green) in the lysosomes. (C) Release of M from endosomes or lysosomes to cytosol by PCI. M subsequently reaches its intracellular target (e.g. the target for 30 kDa gelonin is 28S rRNA). M is too large to be efuxed by P-gp (or other ABC transporters). Lower gure: (A) Dox penetrates the plasma membrane and the intracellular membranes in its neutral version, but is trapped in acidic vesicles in its protonated form. (B) In cells where reduced intravesicular pH cause increased drug accumulation and reduced therapeutic effects PCI may reverse drug resistance.

Doxorubicin

P-gp
ATP ADP

A
Cytosol

B
S M M M M M M S M M M M M S

S M S

Cytosol

M M S

Endosome

Lysosome

Endocytosis
S M M M S M M S S M S S M

Cytosol

P-gp

Dox-H +

H+

Dox
Cytosol

Dox Dox

H+ Dox-H +

Endocytosis

Early Endosome

Late Endosome Lysosomes Cytosol

+

Dox-H

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Das Verfahren beruht auf der Lichtaktivierung von Photosensibilisatoren in endozytotischen Vesikeln. Auf diese Weise soll die Freisetzung von Makromoleku len aus den endozytotischen Vesikeln induziert werden. Dadurch ko nnen diese ihr Ziel erreichen, ehe sie in Lysosomen abgebaut werden. Es hat sich erwiesen, dass PCI den intrazellula ren Transport einer Vielzahl von Makro- und anderen Moleku len stimuliert, die die Plasmamemban sonst schwer durchdringen. Dazu geho ren Ribosomen inaktivierende Proteine (RIPs) vom Typ I, RIP-basierte Immunotoxine, genkodierende Plasmide, Adenoviren oder Adenoviren nahestehende Viren, Peptid-Nucleinsa uren sowie chemotherapeutische Substanzen wie Bleomycin. Die Efzienz und Spezita t der PCI makromolekularer Therapeutika kann noch verbessert werden, indem man die Makromoleku le mit Antiko rpern, z.B. dem epidermalen Wachstumsfaktor, kombiniert. Zur In-vivo-Dokumentation des PCI-Prinzips wurden verschiedene Tiermodelle verwendet. Neuere Ergebnisse deuten auch darauf hin, dass die PCI die Doxorubicinresistenz umkehren kann und generell gegen die multiple Medikamentenresistenz genutzt werden ko nnte. Zusammenfassend ist die PCI ein Verfahren, das nutzbringend beim gezielten Medikamententransport, z.B. in der Gentherapie, bei Impfungen und in der Tumortherapie eingesetzt werden kann.
sselwo rter: Photodynamische Therapie; Photochemische Schlu internalisierung; Photosensibilisatoren; Makromoleku le; Peptid-Nukleinsa uren; Gentherapie; Medikamentengabe; Proteintoxine

culas o de mole culas que no son capaces de macromole tica, incluyendo prode penetrar la membrana plasma te nas inactivantes de ribosomas (RIPs) tipo I, inmunotoxinas basadas en RIP, ADN liberado mediante smidos, o por medio de adenovirus, virus adenopla cidos nucleicos pept dicos o agentes quiasociados, a uticos como la bleomicina. La eciencia y la mioterape uticos macroespecicidad de la PCI de agentes terape moleculares ha sido perfeccionada combinando estas culas con blancos espec cos, tales como el macromole rmico. Varios modelos Factor de Crecimiento Epide n in animales han sido utilizados para la documentacio s au vivo del principio de la PCI. Ma n, resultados recientes indican que la PCI podr a revertir la resistencia a la doxorubicina o ser utilizada para prevenir la resistencia multidroga. En general, dado que permite n eciente de macromole culas en el citosol, la la liberacio n fotoqu mica podr a ser utilizada en una internalizacio gran variedad de aplicaciones en donde se requiere la n sitio-espec ca de la droga, como en terapia liberacio nica, vacunacio n o en el tratamiento del ca ncer. ge
mica; Internalizacio n fotoqu mica; Palabras clave: Fotodina cula; Fotosensibilizador; Macromole Acidos nucleicos nica; Fiberacio n de drogas; Toxinas pept dicos; Terapia ge proteicas

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