Anette Weyergang

Porphyrin‐related photosensitizers for cancer imaging and therapeutic applications

A photosensitizer is defined as a chemical entity, which upon absorption of light induces a chemical or physical alteration of another chemical entity. Some photosensitizers are utilized therapeutically such as in photodynamic therapy (PDT) and for diagnosis of cancer (fluorescence diagnosis, FD). PDT is approved for several cancer indications and FD has recently been approved for diagnosis of bladder cancer. The photosensitizers used are in most cases based on the porphyrin structure. These photosensitizers generally accumulate in cancer tissues to a higher extent than in the surrounding tissues and their fluorescing properties may be utilized for cancer detection. The photosensitizers may be chemically synthesized or induced endogenously by an intermediate in heme synthesis, 5‐aminolevulinic acid (5‐ALA) or 5‐ALA esters. The therapeutic effect is based on the formation of reactive oxygen species (ROS) upon activation of the photosensitizer by light. Singlet oxygen is assumed to be the most important ROS for the therapeutic outcome. The fluorescing properties of the photosenisitizers can be used to evaluate their intracellular localization and treatment effects. Some photosensitizers localize intracellularly in endocytic vesicles and upon light exposure induce a release of the contents of these vesicles, including externally added macromolecules, into the cytosol. This is the basis for a novel method for macromolecule activation, named photochemical internalization (PCI). PCI has been shown to potentiate the biological activity of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome‐inactivating proteins, immunotoxins, gene‐encoding plasmids, adenovirus, peptide‐nucleic acids and the chemotherapeutic drug bleomycin. The background and present status of PDT, FD and PCI are reviewed.

Photochemical internalization provides time- and space-controlled endolysosomal escape of therapeutic molecules.

A successful cure of cancer by biopharmaceuticals with intracellular targets is dependent on both specific and sufficient delivery of the drug to the cytosol or nuclei of malignant cells. However, cytosolic delivery and efficacy of membrane-impermeable cancer therapeutics are often hampered by the sequestration and degradation of the drugs in the endolysosomal compartments. Hence, we developed photochemical internalization (PCI) as a site-specific drug delivery technology, which bursts the membrane of endocytic vesicles inducing release of entrapped drugs to the cytosol of light exposed cells. The principle of PCI has been demonstrated in >80 different cell lines and 10 different xenograft models of various cancers in different laboratories demonstrating its broad application potential. PCI-induced endosomal escape of protein- or nucleic acid-based therapeutics and some chemotherapeutics will be presented in this review. With a joint effort by life scientists the PCI technology is currently in a Phase I/II clinical trial with very promising initial results in the treatment of solid tumors.

Photochemical Internalization: A New Tool for Drug Delivery

The utilisation 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 targeting and design of cytotoxic agents, DNA complexes and other macromolecules for clinical applications. In many 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 internalisation (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), DNA delivered as gene-encoding plasmids or by means of adenovirus or adeno-associated virus, peptide nucleic acids (PNAs) and chemotherapeutic agents such as bleomycin and in some cases doxorubicin. PCI of PNA may be of particular importance due to the low therapeutic efficacy of PNA in the absence of an efficient delivery technology and the 10-100-fold increased efficacy in combination with PCI. The efficacy and specificity of PCI of macromolecular therapeutics has been improved by combining the macromolecules with targeting moieties, such as the epidermal growth factor. In general, PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that it may have a variety of useful applications for site-specific drug delivery as for example in gene therapy, vaccination and cancer treatment.

Photochemical internalization (PCI) in cancer therapy; from bench towards bedside medicine

PDT in cancer therapy has been reviewed several times recently and many published reports have been showing promising results. The clinical approvals for PDT include curative treatment of early or superficial cancers and palliative treatment of more advanced disease. Still PDT has yet to become a widely used cancer treatment. This may partly be due to limitations in current PDT regimens and partly due to effective alternative treatment modalities. If the specificity and selectivity of PDT could be improved, PDT would probably make substantial progress and comprise an even more competitive alternative in cancer treatment. The PCI technology is based on the same principles as PDT, the activation of a photosensitizer by light and subsequently followed by formation of reactive oxygen species. Unlike PDT, the photosensitizer used in PCI has to be located in the endocytic vesicles of the targeted cells and will, upon activation of light, induce a release of endocytosed therapeutic agents after a photochemically induced rupture of the endocytic vesicles. The endocytosed therapeutic agent will then be released and may reach their intracellular target of action before being degraded in lysosomes. This site-specific drug delivery induced by PCI will take place in addition to the well described cytotoxic, vascular and immunostimulatory effects of PDT. PCI has been shown to facilitate intracellular delivery of a large variety of macromolecules that do not otherwise readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), RIP-based immunotoxins, genes and some chemotherapeutic agents. Several animal models have been used for in vivo documentation of the PCI principle and more animal models of clinical relevance have recently been utilized for addressing clinical issues. This review will focus on the possibilities and limitations offered by PCI to overcome some of the challenges recognized in current PDT regimens in cancer treatment.

Photochemical internalization of tumor-targeted protein toxins

Photochemical internalization (PCI) is a method for intracellular delivery of hydrophilic macromolecular drugs with intracellular targets as well as other drugs with limited ability to penetrate cellular membranes. Such drugs enter cells by means of endocytosis and are to a large extent degraded by hydrolytic enzymes in the lysosomes unless they possess a mechanism for cytosolic translocation. PCI is based on photodynamic therapy (PDT) specifically targeting the endosomes and lysosomes of the cells, so that the drugs in these vesicles can escape into the cytosol from where they can reach their targets. The preferential retention of the photosensitizer (PS) in tumor tissue in combination with controlled light delivery makes PCI relatively selective for cancer tissue. The tumor specificity of PCI can be further increased by delivery of drugs that selectively target the tumors. Indeed, this has been shown by PCI delivery of several targeted protein toxins. Targeted protein toxins may be regarded as ideal drugs for PCI delivery, and may represent the clinical future for the PCI technology. Lasers Surg. Med. 43:721–733, 2011.

Photochemical Internalization of Therapeutic Macromolecular Agents: A Novel Strategy to Kill Multidrug-Resistant Cancer Cells

Drug resistance is a major problem for chemotherapy. Entrapment of anticancer drugs in endolysosomal compartments or active extrusions by plasma membrane proteins of the ATP-binding cassette (ABC) superfamily are important resistance mechanisms. This study evaluated photochemical internalization (PCI) of membrane-impermeable macromolecules that are not the target of ABC drug pumps for treating multidrug-resistant (MDR) cancer cells. We used the drug-sensitive uterine fibrosarcoma cell line MES-SA and its MDR, P-glycoprotein (P-gp)-overexpressing derivative MES-SA/Dx5 with the photosensitizer disulfonated meso-tetraphenylporphine (TPPS2a) and broad spectrum illumination. The PCI of doxorubicin, the ribosome-inactivating protein gelonin and adenoviral transduction were assessed in both cell lines, together with the uptake and excretion of TPPS2a and of two fluid phase markers easily detectable by fluorescence [lucifer yellow (LY) and fluorescein isothiocyanate (FITC)-dextran], as a model of gelonin uptake. Both cell lines were resistant to PCI of doxorubicin, but equally sensitive to PCI of gelonin, even though the endocytosis rates of LY and FITC-dextran were significantly lower in the MDR cells. In control studies, MES-SA/Dx5 cells were more resistant to photodynamic therapy (TPPS2a + light only). This was not mediated by P-gp, as there were no differences in the uptake and efflux of TPPS2a between the cell lines. After adenoviral infection, PCI enhanced gene delivery in both cell lines. In conclusion, PCI of macromolecular therapeutic agents that are not targets of P-gp is a novel therapeutic strategy to kill MDR cancer cells.

Photochemical Internalization (PCI): A Technology for Drug Delivery

The utilization of macromolecules in therapy of cancer and other diseases is becoming increasingly relevant. Recent advances in molecular biology and biotechnology have made it possible to improve targeting and design of cytotoxic agents, DNA complexes, and other macromolecules for clinical applications. To achieve the expected biological effect of these macromolecules, in many cases, internalization to the cell cytosol is crucial. At an intracellular level, the most fundamental obstruction for cytosolic release of the therapeutic molecule is the membrane-barrier of the endocytic vesicles. Photochemical internalization (PCI) is a novel technology for release of endocytosed macromolecules into the cytosol. The technology is based on the use of photosensitizers located in endocytic vesicles that upon activation by light induces a release of macromolecules from their compartmentalization in endocytic vesicles. PCI has been shown to potentiate the biological activity of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), gene-encoding plasmids, adenovirus, oligonucleotides, and the chemotherapeutic bleomycin. PCI has also been shown to enhance the treatment effect of targeted therapeutic macromolecules. The present protocol describes PCI of an epidermal growth factor receptor (EGFR)-targeted protein toxin (Cetuximab-saporin) linked via streptavidin-biotin for screening of targeted toxins as well as PCI of nonviral polyplex-based gene therapy. Although describing in detail PCI of targeted protein toxins and DNA polyplexes, the methodology presented in these protocols are also applicable for PCI of other gene therapy vectors (e.g., viral vectors), peptide nucleic acids (PNA), small interfering RNA (siRNA), polymers, nanoparticles, and some chemotherapeutic agents.

Strongly amphiphilic photosensitizers are not substrates of the cancer stem cell marker ABCG2 and provides specific and efficient light-triggered drug delivery of an EGFR-targeted cytotoxic drug.

A wide range of anti-cancer drugs are substrates of the ATP-binding cassette transporter ABCG2/CD338/BCRP/MXR, which is thought to play an important role in multi-drug resistance (MDR) and protection of cancer stem cells (CSC) against chemotherapeutics and photodynamic therapy (PDT). Hence, it is of importance to develop drugs that are not substrates of ABCG2. The aim of this study was to elucidate if photosensitizers utilized for the endo-lysosomal release drug delivery method photochemical internalization (PCI) are substrates for ABCG2. The breast carcinoma cell line MA11, with a Hoechst 33342 side population of >50% was used as an ABCG2high model. The photosensitizer Pheophorbide A (PhA) and Hoechst 33342 were used as positive control substrates of ABCG2. ABCG2-inhibition by fumitremorgin C (FTC) did neither induce an increased accumulation of three different PCI-photosensitizers (di-sulfonated meso-tetraphenylporphine (TPPS(2a)), di-sulfonated meso-tetraphenylchlorin (TPCS(2a)) and di-sulfonated aluminium phtalocyanine (AlPcS(2a))) nor enhanced the photosensitization (P=0.65 for TPCS(2a)-PDT) of these PCI-based photosensitizers in the MA11 cells. The same results were also obtained with TPPS(2a) in the malignant glioma cell line U87 having a SP of ~0.1%. In contrast, both uptake and PDT-induced cytotoxicity was strongly enhanced for PhA when combined with FTC (P<0.001)). Specific and efficient light-controlled killing of EGFR+/ABCG2+ MA11 cells was obtained by PCI of the targeting toxin EGF-saporin. The novel data obtained in this study demonstrates that strongly amphiphilic photosensitizers used for PCI-based drug delivery are not substrates of ABCG2. This important findings warrant further development of the PCI technology as a strategy for efficient and site-specific eradication of MDR cells and CSC.

Photodynamic therapy targets the mTOR signaling network in Vitro and in Vivo

Mammalian target of rapamycin (mTOR) is a regulator of cell growth and proliferation and its activity is altered in many human cancers. The main objective of this study was to evaluate in vitro and in vivo targeting of mTOR by photodynamic therapy (PDT), a treatment modality for cancer. The amphiphilic endolysosomal localizing photosensitizer AlPcS(2a) and the p53 mutated rapamycin-resistant colon adenocarcinoma cell line WiDr were used as models. AlPcS(2a)-PDT downregulated the levels of Ser(2448) phosphorylated mTOR (p-mTOR), total mTOR and phosphorylation of ribosomal S6 (p-S6) immediately after light exposure in a dose-dependent manner, indicating a direct targeting of the mTOR signaling network. Low-dose PDT attenuated the level of p-mTOR in a transient manner; approximately 35% reduction of p-mTOR was obtained 5 min after a LD(35) PDT dose, but returned to the basal level 24 h later. Treatment with the mTOR inhibitor rapamycin reduced the p-mTOR level by 25% after 4-24 h of incubation. Combination treatment of rapamycin and PDT in vitro resulted in synergistic cytotoxic effects when rapamycin was administered after PDT. However, antagonistic effects were obtained when rapamycin was incubated both before and after PDT. In vivo, activated mTOR in the WiDr-xenografts was downregulated by 35 and 75% 5 min and 24 h post PDT respectively as measured by immunoblotting. In contrast to untreated tumors where p-mTOR expression was found throughout the tumors, immunohistochemical staining revealed only expression of p-mTOR in the rim of the tumor at 24 and 48 h post PDT. In conclusion, AlPcS(2a)-PDT is a novel mTOR-targeted cancer therapy. Rapamycin synergistically enhances the cytotoxicity of PDT only when administered post light exposure.

Photodynamic therapy with an endocytically located photosensitizer cause a rapid activation of the mitogen-activated protein kinases extracellular signal-regulated kinase, p38, and c-Jun NH2 terminal kinase with opposing effects on cell survival

Photochemical internalization (PCI) is a method for release of endosomally/lysosomally trapped drugs into the cell cytosol. PCI is based on photosensitizers that accumulate in the membranes of endosomes and lysosomes. Light exposure generates reactive oxygen species that cause membrane rupture and subsequently drug release. PCI can be considered as a combination therapy of photodynamic therapy (PDT) and the administrated drug. The present work reports on mitogen-activated protein kinase signaling after PDT with the endocytically located photosensitizer TPPS2a (meso-tetraphenylporphine with two sulfonate groups on adjacent phenyl rings) as used for PCI in two cancer cell lines: NuTu-19 and WiDr. Both extracellular signal-regulated kinase (ERK) and p38 were activated immediately after PDT. The photochemically induced ERK phosphorylation was enhanced by epidermal growth factor stimulation to a level above that obtainable with epidermal growth factor alone. Expression of the ERK phosphatase, MAPK phosphatase-1, was increased 2 h after PDT but was not the cause of ERK dephosphorylation observed simultaneously. A transient activation of c-Jun NH2 terminal kinase was also observed after PDT but only in the NuTu-19 cells. Using suitable inhibitors, it is shown here that the p38 signal is a death signal, whereas c-Jun NH2 terminal kinase rescues cells after PDT. No direct connection was observed between PDT-induced ERK activation and toxicity of the treatment. The present results document the importance of the mitogen-activated protein kinases in TPPS2a-PDT-induced cytotoxicity. [Mol Cancer Ther 2008;7(6):1740–50]