Photodynamic Drug Delivery

65 Photodynamic Drug Delivery Julie Tzu-Wen Wang University College London

Josephine H. Woodhams University College London

Alexander J. MacRobert University College London

Stephen G. Bown University College London

Kristian Berg Norwegian Radium Hospital

65.1 Introduction.....................................................................................1529 65.2 Mechanisms of Action....................................................................1530 Photosensitizers and Their Subcellular Localization • Light- Induced Macromolecule Delivery

65.3 In Vitro Applications in Macromolecule Delivery.....................1532 Peptides and Proteins • Targeted Macromolecules: Immunotoxins • Multiple Drug Resistance • Oligonucleotides • Gene Therapy

65.4 In Vivo PCI Studies..........................................................................1535 65.5 Concluding Remarks and Clinical Applications........................1537 References.....................................................................................................1537

65.1 Introduction Photodynamic therapy (PDT) is considered to be a site-specific cancer therapy because the therapeutic effects only take place in the area exposed to light. With the rapid expansion of PDT, many new drugs either chemically or endogenously synthesized have been developed and exploited in various ways. Among them, there is a group of photosensitizers with a unique intracellular distribution pattern observed under fluorescence microscopy. These photosensitizers enter cells through endocytosis and localize at the membrane of endocytic vesicles instead of being diffusely localized in the cytosol. Upon irradiation, the generated cytotoxic agents destroy the endocytic membranes and release the content inside these organelles (e.g., lysosomal enzymes) [1,2]. Moreover, the redistribution of these sensitizers (i.e., an increase of fluorescence intensity and the change of distribution phenomenon to become diffuse) was also observed. This particular lysosomal photosensitization and the membrane disruption accompanying the release of content led to the concept of photochemical internalization (PCI)—a novel photochemical technology for inducing the release of macromolecules from endocytic vesicles [3]. Macromolecules generally refer to oligo- and polynucleotides, peptides, proteins, and polymers whose sizes are normally larger than 1 kDa. They can serve as alternatives to traditional drugs (e.g., low-molecular-weight chemotherapeutics) but their bioavailabilities are usually limited [4]. First of all, the cell membrane is normally impermeable to the aforementioned large molecular complexes, whereas small molecules such as glucose, ions, or amino acids can quickly diffuse into cells. Secondly, even

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though macromolecules still can enter cells through various active internalization mechanisms called endocytosis, escaping from endocytic vesicles to reach the intracellular targets is another problem [5]. Thirdly, degradation of these macromolecules within acidic lysosomes, which contain proteolytic enzymes, is a further factor that limits efficient delivery. There is therefore growing awareness of the need to develop drug delivery systems, which can efficiently release therapeutics accumulated in endosomes and lysosomes. The fact that macromolecules retain in intracellular vesicles and remain inactive is not ultimately a hindrance. If their biological effects can be manipulated by a delivery system, these macromolecular drugs will be superior to traditional chemotherapeutics in terms of specificity. In this regard, PCI that causes the rupture of endo/lysosomal membranes during photosensitization can provide a site-specifically light-induced delivery of macromolecules. The aim of PCI is to relocalize the therapeutics—escape from the barrier of endo/lysosomal membrane and ability to reach their targets. In other words, the principal considerations are the drug delivery effects and the cytotoxicity from macromolecules after PCI. The light and sensitizer doses required for PCI are much lower than the ones for PDT treatment whose ultimate goal is to kill the whole cell. Moreover, because the therapeutics used for PCI are normally potent drugs but not bioavailable without efficient delivery system, the drug dose can be reduced to extremely small (compared to its LD50 dose) using PCI technology. For these reasons, synergistic effects are expected for PCI strategy where individual treatment (i.e., PDT and drug alone) exhibits minor effect but an enhanced killing effect can be achieved when these macromolecules are activated after photosensitization. This is different from most of the combination therapies, which combines PDT with another treatment modality (e.g., hyperthermia, radiation therapy, antibody therapy, or chemotherapy). Although a synergistic response can be obtained if one mechanism influences the other, additive effects are often the results of these combined strategies where treatments are working independently. PCI has been established to enhance the intracellular delivery of a large variety of macromolecules including peptides, protein toxins, and genes [6]. It has been shown to be effective for release of these therapeutics both in vitro and in vivo [7]. However, some details of PCI remain to be investigated such as the exact mechanism of membrane rupture at molecular scale and the optimization of PCI in terms of the balance of photochemical treatment and drug toxicity.

65.2 Mechanisms of Action 65.2.1 Photosensitizers and Their Subcellular Localization To date, meso-tetraphenylporphine (TPPS2a) and aluminum phthalocyanine disulfonate (AlPcS2a) are the most efficient photosensitizers for PCI with two sulfonate groups on adjacent phthalate/phenyl rings (Figure 65.1). After being taken up by cells through endocytosis, these sensitizers localize specifically at the membrane of endo/lysosomes, with a hydrophobic part inserted into the membranes and a hydrophilic part free inside the vesicle matrix. This amphiphilic feature ensures their membrane localization and inhibits further penetration. It is not expected that all endo/lysosome-localized photosensitizers are suitable to be used for PCI. The selection of PCI sensitizers is based on the property of “membrane” localization at these endocytic vesicles. The reason for this requisition is simply to limit the photochemical destruction to the membrane and therefore minimize the possible photo-damage to the delivered macromolecules in the lumen compartment. As the reactive oxygen species, particularly singlet oxygen, generated from photochemical reactions have a short diffusion distance [8,9], amphiphilic sensitizers are desirable to fulfill this certain criteria. Evidence has also been shown that the exact lysosomal membrane localization is crucial to an efficient PCI effect [10]. To study the impact of intracellular localization of photosensitizers for PCI, several photosensitizers were examined for their ability to induce polylysine-mediated gene transfection.

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Photodynamic Drug Delivery

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FIGURE 65.1 The structures and molecular weights of three PCI photosensitizers—(from the left) TPPS2a, AlPcS2a, and TPCS2a.

This study showed that photosensitizers that are localized at the endo/lysosomal membrane (e.g., AlPcS2a and TPPS2a) have the greatest effects on the transfection of plasmid encoding enhanced green fluorescent protein (EGFP). In contrast, transfection was not significantly stimulated after photoactivation of non-lysosomally localized photosensitizers (e.g., 3THPP and 5-ALA). Moreover, other hydrophilic sensitizers, such as TPPS4 and Nile blue A, which localize within endocytic vesicles displayed only weak or negligible EGFP expression after exposure to light. As a result, the physical property of photosensitizers determines their intracellular localizations and amphiphilic sensitizers are the best photosensitizing compounds for PCI applications. Amphiphilic sensitizers also exhibit some advantageous photodynamic properties [11]. The cell uptake of amphiphilic photosensitizers was detected as good as hydrophobic ones but the tendency to aggregate was lower. This is an important feature as dimerized photosensitizers are less effective in inducing photochemical reactions. The absorbance spectrum should also be considered, especially in clinical practice, since nearinfrared is preferable for deeper tissue penetration of light. Phthalocyanines have a porphyrin-based macrocycle with four benzo rings on the pyrrol units, which results in a strong absorption in the farred region of spectrum (>670 nm) [12]. In this regard, AlPcS2a may be preferable to TPPS2a for in vivo or in clinic utilization. Tetraphenylchlorin disulfonate adjacent (TPCS2a, Amphinex®) is a new chlorin-based amphiphilic photosensitizer (PCI Biotech, Norway), which is designed for PCI (Figure 65.1). The chemical synthesis of Amphinex is rather simpler than AlPcS2a, and it is proposed to be as efficient as the well-known chlorin sensitizer, m-THPC, regarding the photodynamic ability.

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65.2.2 Light-Induced Macromolecule Delivery Figure 65.2 depicts the principle mechanism of PCI. It contains three processes: the uptake of photosensitizer and macromolecules, the illumination and induced membrane rupture, and finally the release of macromolecules. After diffusing into the cytosol, macromolecules can thereby perform their pharmacological action when reaching their targets. The key step is the photochemically induced membrane rupture. As PCI also utilizes the same photochemical reactions as PDT, the fundamental requirements, such as the presence of oxygen and sufficient photosensitizers, are also essential to PCI. It should be noted that the whole PCI process is dynamic, and therefore, the efficacy is highly dependent on the timing of drug (both sensitizers and macromolecules) administration and light exposure. Ideally, illumination should be carried out when sensitizers are sufficiently localized at the biomembranes, and the macromolecules have been internalized in endocytic vesicles but before being degraded

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Light

FIGURE 65.2 The principle of PCI. S: photosensitizer; G: molecules to be delivered. (From Hogset, A. et al., Drug Deliv. Rev., 56, 95, 2004.)

Light

FIGURE 65.3 Possible mechanism for “light before” PCI. S: photosensitizer; G: molecules to be delivered. (Modified from Hogset, A. et al., Drug Deliv. Rev., 56, 95, 2004.)

by the lysosomal enzymes. As a result, understanding the kinetics of the endocytosis of both sensitizers and macromolecules is very important. In contrast to the conventional concept of PCI as described earlier, another strategy called “light before” PCI has been found to be as effective as or even better than “light after PCI” in some cases in vitro [13]. The possible mechanism is shown in Figure 65.3. Irradiation is performed after photosensitizers have been localized at the membrane of endocytic vesicles but before the administration of the macromolecules. It is suggested that the molecules will be taken up through endocytosis as well but can leak out immediately once the vesicles fuse with the ones damaged by light treatment. The foremost advantage is that the possible photochemical effects on the therapeutics should therefore be diminished since those drugs are delivered after illumination.

65.3 In Vitro Applications in Macromolecule Delivery 65.3.1 Peptides and Proteins PCI has been employed to release some peptides or small proteins efficiently. It was reported that more than 60% of endocytosed horseradish peroxidase (HRP) can be released into cytosol using TPPS2a PCI. Similar findings were also obtained when it was shown that fluorescein-labeled ras peptide (p21ras), a cancer-specific peptide, was colocalized granularly with AlPcS2a in cells but became diffuse in cytosol after light exposure [3]. The studies of PCI-induced delivery of protein have mainly focused on ribosome-inactivating proteins (RIPs), a group of protein toxins that inhibit protein synthesis by damaging ribosomes

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catalytically. RIPs from plants are usually divided into two groups: Type II RIPs consist of an enzymatically active A chain (approximately 30 kDa) linked to a B chain (approximately 35 kDa), which contains a binding domain. Type I RIPs, however, which only consist of the A chain, are hardly take up by cells. The absence of a B chain with a binding domain indeed limits the entry of type I RIPs into cells and justifies their very low toxicity to cells and to animals compared to type II RIPs, with LD50 for mice of 40–75 mg/kg [14]. However, if they are delivered into cells, they can induce efficient inhibition of protein synthesis. Several strategies have been employed to improve the cellular delivery of type I RIPs including enclosing them in drug carriers [15], subjecting cells to shock waves [16], and conjugating to antibodies [17]. These protein toxins are ideal model proteins to evaluate and demonstrate PCI effects. Gelonin and saporin are two type I RIP toxins, which are mostly used in PCI strategy. The effect of PCI-induced delivery of gelonin has been investigated using several photosensitizers in a large number of cell lines of various origins [18]. The best results were found using TPPS2a PCI of gelonin in NHIK 3025 cells where protein synthesis was significantly reduced by more than a factor of 300 compared to each treatment alone [3]. Saporin is another type I RIP, which exerts a higher toxicity than gelonin [19]. Lai et al. investigated the PCI effect using AlPcS2a in combination of saporin in vitro [20]. In addition, the introduction of polyamidoamine (PAMAM) dendrimer to saporin showed an increased cellular uptake and cytotoxicity using PCI. Bleomycins are a family of water-soluble glycopeptidic antibiotics with at least four functional domains mediating DNA breakage, ion binding, and O2 activation [21]. Since first isolated by Umezawa in 1966, bleomycins have been used in combination with several anticancer agents for a number of cancer chemotherapies, notably squamous cell carcinomas and malignant lymphomas [22]. With a very high cytotoxicity, as few as 500 bleomycin molecules translocated to cytosol are sufficient to kill a cell in combination with electroporation in vitro [23,24]. However, due to its hydrophilic property, bleomycin penetrates membrane structures poorly and therefore accumulates in endocytic vesicles. The sensitivity of tumor cells to this drug is accordingly variable. Although many fundamental properties of bleomycin are still unknown, its therapeutic potential could be activated if delivered and exported sufficiently into cytosol. PCI has demonstrated enhanced cell growth inhibition in combination with bleomycin in vitro using different cell lines [25]. Further in vivo studies of PCI of bleomycin will be described later.

65.3.2 Targeted Macromolecules: Immunotoxins The targeting strategy was developed due to the demand of specific and efficient drug delivery system, especially in the in vivo studies. Immunotoxins are one of the designs that link antibodies with specific binding function to therapeutics which are mainly bacteria or plant protein toxins. The aims of combining targeting ligands are to achieve tumor specificity and a favorable biodistribution between normal and tumor tissues. The conjugates should meet several criteria to be used in clinical practice for cancer treatment: Because the number of receptors presenting on the cell surface is usually less than 105 molecules per cell, it is important to have very high toxicity to kill targeted cells efficiently. Moreover, most toxins enter the cell through the endocytic pathway; therefore, the toxin must be able to survive from the acidic compartments of endo/lysosomes and the proteolytic process [26]. Since cooperation of PCI and RIPs has inactivated protein synthesis efficiently, the use of immunotoxins—monoclonal antibody conjugated type I RIP—is believed to be able to increase the drug uptake and specificity. The first studies of PCI in combination with immuno-RIP conjugates were introduced by Selbo et al. coupling of monoclonal antibody MOC31 with gelonin [27,28]. This conjugation enables gelonin to directly bind to epithelial glycoprotein-2 (EGP-2), which is expressed on most carcinoma cells, resulting in increased uptake of the conjugates. The cell viability was significantly reduced using

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TPPS2a, AlPcS2a, and ALA PCI compared to immunotoxins alone. Moreover, the results also show that there was no difference between immunotoxin and toxin alone in the case of using PCI in a EGP-2 negative cell line. The specificity might be very useful for in vivo application. Epidermal growth factor receptor (EGFR) involves in regulating cell proliferation, metastasis, angiogenesis and is overexpressed in a variety of tumor cell lines [29], and targeting EGFR is one of the most common strategies to achieve specific cancer therapies. The EGFR inhibitor, C225, has been used as an anticancer drug in Phase-I to Phase-III trials for several malignant diseases and is approved for treatment of head and neck squamous cell carcinoma and colorectal cancer. It may therefore be a good combination employing EGFR-targeting system to PCI. Weyergang et al. established an EGF–saporin affinity toxin (biotin–streptavidin linkage) to investigate the PCI effects [30]. PCI of EGF–saporin enhanced the cytotoxicity about 1000-fold in EGFR overexpressing cells. Researchers from the same group later demonstrated increased cytotoxicity using PCI in combination with saporin-C225 complex (via the same linking method) in EGFR-positive cells [31]. The efficacy and selectivity of this combination of PCI and EGFR-targeting system indicate it can be of benefit for cancer therapies in clinic.

65.3.3 Multiple Drug Resistance Since PCI has been employed for enhancing the cellular uptake of drugs, which have difficulties in entering the cell, it is predicted that PCI might have the potential to deal with multiple drug resistance, which is a serious problem affecting many chemotherapy drugs. Two main mechanisms for MDR: The overexpression of p-glycoprotein (p-gp) efflux pumps leads to the extrusion of drugs from cytosol. The entrapment of anticancer drugs in endosome/lysosomes induces the degradation of drugs by lysosomal enzymes [32]. It was firstly established by Lou et al. that successful reversal of drug resistance can be induced by PCI [33]. Doxorubicin, a weak base chemotherapeutics, is often trapped in endocytic vesicles due to the increased acidification of these intracellular compartments. After TPPS2a PCI treatment, doxorubicin (administered after irradiation) was observed in the nuclei of MCF-7/ADR cells (a breast cancer cell line which is resistant to doxorubicin), and the drug dose required for 50% inhibition of cell survival was reduced to 10 times lower. Another study using similar concepts also indicated the PCI effects on the improvement of drug resistance. The cytotoxicity of mitoxantrone (a chemotherapy agent) against its drug-resistant cancer cells sensitized with hypericin (a photosensitizer) was enhanced after illumination [34]. The mobilization of mitoxantrone to cell nuclei was also found after light exposure. As pointed out earlier, there are many different phenotypes of drug resistance and some mechanisms may not be affected by PCI treatment. Selbo et al. reported that TPPS2a PCI has no influence in reducing the level of resistance to doxorubicin for MES-SA/Dx5 cells, a MDR cell line [35]. However, PCI of gelonin successfully reduced the cell survival. The results suggest that PCI may not be able to adjust the drug sensitivity for some MDR cells, but it still can be useful as a cancer treatment when in combination with other macromolecules.

65.3.4 Oligonucleotides Oligonucleotides are a group of macromolecules with recognized therapeutic potential. Some of these molecules exhibit antisense effects, resulting in site-specific gene silencing in the host cell through steric blocking of gene expression or degradation of mRNA. Peptide nucleic acids (PNAs) are examples of the former action, which are DNA mimics with a pseudopeptide backbone [36]. Small interfering RNA (siRNA) molecules are the example for the latter mechanism, which are able to control gene expression through the process of RNA interference [37]. The inefficient cellular uptake is the major obstacle for both therapeutic agents [38] and, as a result, several strategies, including PCI, have been explored to enhance the drug delivery.

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Recent studies have clearly shown that PCI represents an efficient delivery system for naked PNA conjugated with the catalytic component of human telomerase reverse transcriptase (hTERT-PNA) [39] and various cell-penetrating peptides (CPPs-PNA) [40]. As for siRNA molecules, substantial gene silencing was induced by PCI-mediated gene transfection of siRNA molecules against S100A4, a protein associated with the invasive and metastatic phenotype of cancer cells [41]. These promising in vitro results open the future applications using PCI technology for activating the biological antisense effects of oligonucleotides on animals or humans.

65.3.5 Gene Therapy The basic principle of gene therapy is that insertion of corrective genetic material into cells alleviates the symptoms of disease. However, there are some obstacles, and consequently few successes in gene therapy. One of the main problems of gene therapy is the lack of efficient delivery systems [42] and PCI, as a novel technology for releasing macromolecules from endosomes to cytosol could provide efficient delivery for gene therapy. Recent studies have shown encouraging results in gene transfection via either viral or nonviral vectors. AlPcS2a-based PCI-induced EGFP/polylysine complex delivery was tested and the transfection efficiency reached above 50% of surviving cells [43]. PCI using different photosensitizers has also been examined to induce EGFP transfection, and a light-dependent transfect efficacy was found [10]. In addition, various delivery agents have been evaluated in PCI-mediated gene transfection where transfection can be enhanced using polycationic vectors [e.g., polylysine or polyethylenimine (PEI)], but was inhibited by cationic lipid vector-mediated transfection [e.g., dioleoyltrimethylammoniumpropane (DOTAP)] [44]. One possible explanation suggested was that the photochemical treatment might perturb the dissociation of DNA/DOTAP complex and cause DNA inactivation. PCI-mediated delivery requires administration of both photosensitizers and candidate drugs or genes. There is a new design devised a complex composed of a core DNA packaged with cationic peptides and enveloped in the anionic dendrimer phthalocyanine (DPc) [45]. The concept was to reduce the administration of photosensitizers and drugs into one and was thought to be more suitable for in vivo application due to the integration of photosensitizers and DNA/polymer complex. After irradiation, an enhancement in transfection but with lowered photocytotoxicity was established in vitro compared to the results using unconjugated AlPcS2a. This study is also the first success in PCI-mediated gene delivery in vivo with transgene expression in conjunctival tissues of rats. Gene delivery through viral vectors is usually regarded as a very efficient process because of the natural occurrence of virus infections [46]. However, after being taken up into cells through endocytosis (mostly) [47], virus–DNA complex may still be trapped inside the endocytic system in cases of some virus vectors or in certain cell lines [48]. PCI-mediated gene transfection by adenoviral vectors was firstly introduced by Hogset et al. using β-galactosidase-encoding adenovirus [49]. Pronounced transduction efficiency was obtained, and the amount of virus dose was significantly lowered to achieve the same results using PCI compared with conventional adenovirus transfection (no illumination). A later study investigated the transfection efficacy of PCI-induced transduction of polycation-complexed adenovirus using several cell lines [50]. The efficacy was found to be cell type dependent but positive transgene expression was observed in all cell lines including cells with low or without the receptors to adenovirus on the cell surface.

65.4 In Vivo PCI Studies Most of the in vivo PCI studies have been carried out using AlPcS2a in transplanted xenograft tumor models in nude mice. AlPcS2a PCI of gelonin delayed the tumor growth significantly compared to animals treated with PDT alone [51,52].

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Bleomycin has been employed for in vivo PCI with AlPcS2a in four different xenograft tumors in mice [25,53]. A synergistic delay in the tumor growth was found in all models. The histological examination on the samples 7 days after surgery showed that the tumor regrowth in the peripheral zone was inhibited by PCI treatment. A further study also indicated that PCI treatment could act as an adjunct to surgery, resulting in the delay of tumor growth [54]. It is thus suggested that PCI may be suitable for treating residual tumor cells after incomplete surgical resection from the clinical point of view. Another combination strategy was investigated using AlPcS2a PCI of bleomycin and radiotherapy. The time to tumor progression was increased when ionizing radiation was carried out after PCI compared to PDT or bleomycin combined with radiotherapy [55]. None of these studies quantified the effects of PCI compared with PDT other than by tumor growth delay. However, this has now been done in a normal tissue, liver. The therapeutic efficacies between AlS2Pc PDT and PCI of gelonin were compared by measuring the necrosis induced in healthy rat liver in three dimensions [56]. The influence of drug doses and drug administration intervals to light delivery was investigated: the greatest enhancement in tissue damage was observed when gelonin was injected 1 h before irradiation and the effect was independent of the gelonin dose under a range of 5–50 μg/kg. In other words, significant treatment response can be induced with a very low dose of gelonin (5 μg/kg, 10,000 times lower than the LD50). This study demonstrated the dependence of the drug–light intervals on the treatment effects and emphasized the importance of optimization of the treatment conditions. In addition, the PCI-induced relocalization of gelonin in vivo was established for the first time using immunohistochemistry (Figure 65.4). Another study further compared the PCI effects of different drugs on different tissues. Experiments were undertaken using TPCS2a PCI in combination with saporin and bleomycin in normal rat liver/ colon and a transplanted syngeneic rat fibrosarcoma model (Wang et al., unpublished). Significantly enhanced necrosis was induced in combination with saporin in liver and tumor but not colon, and with bleomycin in each case. The lack of enhanced tissue damage in colon after PCI with saporin is due to the fact that saporin does not accumulate significantly in colon, in contrast to liver and tumor. This study demonstrates that the uptake of the individual drugs in different organs can strongly affect the treatment response to PCI. Altogether, the consistency between in vitro and in vivo experiments indicates the benefits of the PCI approach, which enhances PDT treatments and displays synergistic effects in combination with other therapeutic agents.

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FIGURE 65.4 (See color insert.) PCI-induced relocalization of gelonin in rat liver. Tissues were stained with the primary antibody against gelonin and counterstained with hematoxylin. Animals were treated with 25 J light delivered 48 h after 1 mg/kg AlS2Pc and 1 h after 500 μg/kg gelonin, examined 30 min after irradiation. (A) Area of liver not exposed to light (inset white arrows: distribution of gelonin in liver). (B) Areas exposed to light (inset black arrows: redistribution of gelonin).

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65.5 Concluding Remarks and Clinical Applications PCI is attracting interest as a novel technology for improving macromolecule delivery to cells. The key advantage of PCI is the site specificity due to the light-dependent activation of a photosensitizer, which is coadministered with the macromolecule. Additionally, PCI has the potential to approach different cancer treatments when in combination with different therapeutic agents since various macromolecules have been shown to be released by PCI and their size can range from small peptides to genetic materials. The combination with drugs coupled with specific targeting ligands may also improve the specificity of PCI treatment in vivo by directing drugs to the tumor cells. A recently published study demonstrated the use of a fusion toxin for PCI, which was recombinant fusion protein composed of gelonin and a single-chain Fv antibody fragment against a marker protein associated with a large number of cancer cells [57]. This construct was able to inhibit tumor growth significantly. The uptake of photosensitizers and macromolecules varies between cell lines and so does the light sensitivity [6]. In fact, these are all factors, which relate to the optimization of PCI treatment conditions. The killing effect originating from the photochemical reaction is beneficial to cancer therapy where the final goal is to kill the malignant cells, whereas it might be a disadvantage for gene therapy of other diseases. Toward this point, optimizing the treatment conditions should therefore be emphasized again. Collectively, PCI is a promising technology to improve a cytosolic delivery of macromolecules in vitro and in vivo. Despite the unclear mechanism, the significant release of large particles such as viral vectors indicates that PCI provides a remarkable drug delivery platform. A successful PCI should be well planned, and the photochemical reaction has to be efficient, precise, and well timed, in respect to the incorporated therapeutics. It is important to point out that PCI is in the stage of its first clinical trial using TPCS2a PCI in combination with bleomycin for head and neck cancers in University College London Hospital. As a cancer treatment, the challenges will be the same as what PDT faces: the benefit of its therapeutic effects against the conventional well-established therapies, such as chemotherapy, radiation therapy, and surgery. The fact that PCI seems flexible for delivering a variety of anticancer drugs certainly broadens the future applications of PCI in clinic to deal with different cancers.

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31. Yip, W. L., A. Weyergang, K. Berg, H. H. Tonnesen, and P. K. Selbo (2007) Targeted delivery and enhanced cytotoxicity of cetuximab–saporin by photochemical internalization in EGFR-positive cancer cells. Mol. Pharm. 4, 241–251. 32. Gottesman, M. M., T. Fojo, and S. E. Bates (2002) Multidrug resistance in cancer: Role of ATPdependent transporters. Nat. Rev. Cancer 2, 48–58. 33. Lou, P. J., P. S. Lai, M. J. Shieh, A. J. Macrobert, K. Berg, and S. G. Bown (2006) Reversal of doxorubicin resistance in breast cancer cells by photochemical internalization. Int. J. Cancer 119, 2692–2698. 34. Adigbli, D. K., D. G. Wilson, N. Farooqui, E. Sousi, P. Risley, I. Taylor, A. J. Macrobert, and M. Loizidou (2007) Photochemical internalisation of chemotherapy potentiates killing of multidrug-resistant breast and bladder cancer cells. Br. J. Cancer 97, 502–512. 35. Selbo, P. K., A. Weyergang, A. Bonsted, S. G. Bown, and K. Berg (2006) Photochemical internalization of therapeutic macromolecular agents: A novel strategy to kill multidrug-resistant cancer cells. J. Pharmacol. Exp. Ther. 319, 604–612. 36. Pooga, M., T. Land, T. Bartfai, and U. Langel (2001) PNA oligomers as tools for specific modulation of gene expression. Biomol. Eng. 17, 183–192. 37. Dykxhoorn, D. M., C. D. Novina, and P. A. Sharp (2003) Killing the messenger: Short RNAs that silence gene expression. Nat. Rev. Mol. Cell Biol. 4, 457–467. 38. Koppelhus, U., S. K. Awasthi, V. Zachar, H. U. Holst, P. Ebbesen, and P. E. Nielsen (2002) Cell-dependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates. Antisense Nucleic Acid Drug Dev. 12, 51–63. 39. Folini, M., K. Berg, E. Millo, R. Villa, L. Prasmickaite, M. G. Daidone, U. Benatti, and N. Zaffaroni (2003) Photochemical internalization of a peptide nucleic acid targeting the catalytic subunit of human telomerase. Cancer Res. 63, 3490–3494. 40. Shiraishi, T. and P. E. Nielsen (2006) Photochemically enhanced cellular delivery of cell penetrating peptide-PNA conjugates. FEBS Lett. 580, 1451–1456. 41. Boe, S., A. S. Longva, and E. Hovig (2007) Photochemically induced gene silencing using small interfering RNA molecules in combination with lipid carriers. Oligonucleotides 17, 166–173. 42. Verma, I. M. and N. Somia (1997) Gene therapy—Promises, problems and prospects. Nature 389, 239–242. 43. Hogset, A., L. Prasmickaite, T. E. Tjelle, and K. Berg (2000) Photochemical transfection: A new technology for light-induced, site-directed gene delivery. Hum. Gene Ther. 11, 869–880. 44. Prasmickaite, L., A. Hogset, T. E. Tjelle, V. M. Olsen, and K. Berg (2000) Role of endosomes in gene transfection mediated by photochemical internalisation (PCI). J. Gene Med. 2, 477–488. 45. Nishiyama, N., A. Iriyama, W. D. Jang, K. Miyata, K. Itaka, Y. Inoue, H. Takahashi, Y. Yanagi, Y. Tamaki, H. Koyama, and K. Kataoka (2005) Light-induced gene transfer from packaged DNA enveloped in a dendrimeric photosensitizer. Nat. Mater. 4, 934–941. 46. Anderson, W. F. (1998) Human gene therapy. Nature 392, 25–30. 47. Greber, U. F., M. Willetts, P. Webster, and A. Helenius (1993) Stepwise dismantling of adenovirus 2 during entry into cells. Cell 75, 477–486. 48. Hansen, J., K. Qing, H. J. Kwon, C. Mah, and A. Srivastava (2000) Impaired intracellular trafficking of adeno-associated virus type 2 vectors limits efficient transduction of murine fibroblasts. J. Virol. 74, 992–996. 49. Hogset, A., B. O. Engesaeter, L. Prasmickaite, K. Berg, O. Fodstad, and G. M. Maelandsmo (2002) Light-induced adenovirus gene transfer, an efficient and specific gene delivery technology for cancer gene therapy. Cancer Gene Ther. 9, 365–371. 50. Bonsted, A., B. O. Engesaeter, A. Hogset, G. M. Maelandsmo, L. Prasmickaite, O. Kaalhus, and K. Berg (2004) Transgene expression is increased by photochemically mediated transduction of polycation-complexed adenoviruses. Gene Ther. 11, 152–160. 51. Dietze, A., Q. Peng, P. K. Selbo, O. Kaalhus, C. Muller, S. Bown, and K. Berg (2005) Enhanced photodynamic destruction of a transplantable fibrosarcoma using photochemical internalisation of gelonin. Br. J. Cancer 92, 2004–2009.

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AQ3


52. Selbo, P. K., G. Sivam, O. Fodstad, K. Sandvig, and K. Berg (2001) In vivo documentation of photochemical internalization, a novel approach to site specific cancer therapy. Int. J. Cancer 92, 761–766. 53. Norum, O. J., J. V. Gaustad, E. Angell-Petersen, E. K. Rofstad, Q. Peng, K. E. Giercksky, and K. Berg (2009) Photochemical internalization of bleomycin is superior to photodynamic therapy due to the therapeutic effect in the tumor periphery. Photochem. Photobiol. 85, 740–749. 54. Norum, O. J., K. E. Giercksky, and K. Berg (2009) Photochemical internalization as an adjunct to marginal surgery in a human sarcoma model. Photochem. Photobiol. Sci. 8, 758–762. 55. Norum, O. J., O. S. Bruland, L. Gorunova, and K. Berg (2009) Photochemical internalization of bleomycin before external-beam radiotherapy improves locoregional control in a human sarcoma model. Int. J. Radiat. Oncol. Biol. Phys. 75, 878–885. 56. Woodhams, J. H., P. J. Lou, P. K. Selbo, C. A. Mosse, D. Oukrif, A. J. Macrobert, M. Novelli, Q. Peng, K. Berg, and S. Bown (2010) Intracellular relocalisation by photochemical internalisation enhances the cytotoxic effect of gelonin—Quantitative studies in normal rat liver. J. Control. Release 142(3), 347–353. 57. Selbo, P. K., M. G. Rosenblum, L. H. Cheung, W. Zhang, and K. Berg (2009) Multi-modality therapeutics with potent anti-tumor effects: Photochemical internalization enhances delivery of the fusion toxin scFvMEL/rGel. PLoS One 4, e6691.

Author Queries [AQ1] Please provide the expansion for “ALA,” “THPC,” “NHIK,” “EGF,” “MDR,” “MCF,” “ADR,” “MES-SA,” and “hTERT-PNA,” if appropriate. [AQ2] Please suggest whether “pyrrol” should be changed to “pyrrole.” [AQ3] Please check whether the added information (year, volume, and page range) to Ref. [56] is OK.

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