Enhanced targeting of triple-negative breast carcinoma and malignant melanoma by photochemical internalization of CSPG4-targeting immunotoxins.

Triple-negative breast cancer (TNBC) and malignant melanoma are highly aggressive cancers that widely express the cell surface chondroitin sulfate proteoglycan 4 (CSPG4/NG2). CSPG4 plays an important role in tumor cell growth and survival and promotes chemo- and radiotherapy resistance, suggesting that CSPG4 is an attractive target in cancer therapy. In the present work, we applied the drug delivery technology photochemical internalization (PCI) in combination with the novel CSPG4-targeting immunotoxin 225.28-saporin as an efficient and specific strategy to kill aggressive TNBC and amelanotic melanoma cells. Light-activation of the clinically relevant photosensitizer TPCS2a (fimaporfin) and 225.28-saporin was found to act in a synergistic manner, and was superior to both PCI of saporin and PCI-no-drug (TPCS2a + light only) in three TNBC cell lines (MDA-MB-231, MDA-MB-435 and SUM149) and two BRAFV600E mutated malignant melanoma cell lines (Melmet 1 and Melmet 5). The cytotoxic effect was highly dependent on the light dose and expression of CSPG4 since no enhanced cytotoxicity of PCI of 225.28-saporin compared to PCI of saporin was observed in the CSPG4-negative MCF-7 cells. The PCI of a smaller, and clinically relevant CSPG4-targeting toxin (scFvMEL-rGel) validated the CSPG4-targeting concept in vitro and induced a strong inhibition of tumor growth in the amelanotic melanoma xenograft A-375 model. In conclusion, the combination of the drug delivery technology PCI and CSPG4-targeting immunotoxins is an efficient, specific and light-controlled strategy for the elimination of aggressive cells of TNBC and malignant melanoma origin. This study lays the foundation for further preclinical evaluation of PCI in combination with CSPG4-targeting.

Solubilization of the photosensitizers TPCS(2a) and TPPS(2a) in aqueous media evaluated by time-resolved fluorescence analysis.

The pH-dependent aggregation of the novel photosensitizer TPCS(2a) is investigated at low concentration (c = 10(-6) M) in aqueous media by means of time-correlated single-photon counting, and compared to that of the chemically related photosensitizer TPPS(2a). The efficacy of selected solubilizers, i.e., various nonionic Pluronic block copolymers and the nonionic surfactant Tween 80, in inhibiting aggregation of the two photosensitizers is evaluated, which is important for the further formulation of TPCS(2a).

Evaluation of physicochemical properties and aggregation of the photosensitizers TPCS2a and TPPS2a in aqueous media.

Physicochemical properties of the novel photosensitizer meso-tetraphenyl chlorin disulphonate (TPCS2a) and the chemically related meso-tetraphenyl porphyrin disulphonate (TPPS2a) were investigated in aqueous solutions as part of pharmaceutical preformulation. Inflection points were calculated to be 3.9 for both compounds based on spectral shifts of aqueous solutions in the pH range 2-12, which likely correlate with indistinguishable pKa values of the imino nitrogens of the molecular cores. Accordingly, the fluorescence emission spectra showed pH dependent spectral shifts. Porphyrin-like compounds are known for aggregation in aqueous environments, and a small percentage of Tween 80 (0.006 % v/v = 4 x cmc) seemed to stabilize the aqueous samples of the two photosensitizers through hindrance of aggregation. The distribution coeffient of TPCS2a determined spectrophotometrically in 1-octanol/water is 0.4 (- 0.4 SD) and 1.5 (- 0.5 SD) for the reference TPPS2a. This confirms amphiphilicity which indicates preferred distribution and further restrain of diffusion in membranes, which is relevant for the use of TPCS2a as a photosensitizer in the process of photochemical internalization in vivo.

Physicochemical characterization of the photosensitizers TPCS2a and TPPS2a 1. Spectroscopic evaluation of drug--solvent interactions.

The spectroscopic properties of the patented photosensitizer meso-tetraphenyl chlorin disulphonate (TPCS2a), intended for use in photochemical internalization (PCI) technology, and the chemically related photosensitizer meso-tetraphenyl porphyrin disulphonate (TPPS2a) were characterized in 14 organic solvents of varying polarity and amphiprotic properties. Absorption spectra and fluorescence emission spectra were acquired, and Stokes' shifts and fluorescence quantum yields determined. These investigations yield information on the physicochemical interactions between the photosensitizers and their surroundings (i.e., the physiological environment in vivo or the vehicle in vitro), which is essential for the further development of drug formulations. TPPS2a and TPCS2a are rigid molecules, built up by conjugated ring systems which possess limited interactions with the surroundings in the ground state (So). Accordingly, only small spectral shifts were observed in the absorption spectra, as well as in the fluorescence emission spectra. TPPS2a is spatially more planar than TPCS2a, which is twisted as a result of reduction of a double bond in the core. However, the two compounds were quite similarly influenced by properties of the solvents, indicating that twisting has limited importance in the interactions of the two photosensitizers with their environment. Both compounds possess a high character of pi-pi* transition upon light exposure, supported by high molar absorption coefficients. The fluorescence quantum yields (phi(f)) were influenced by solvent properties to a larger extent than the spectral shifts. This might indicate that the reactivity of the first excited singlet state (S1*) significantly depends on the properties of the surroundings.

Photochemical internalization: a novel technology for delivery of macromolecules into cytosol.

The therapeutic usefulness of macromolecules, such as in gene therapy, is often limited by an inefficient transfer of the macromolecule to the cytosol and a lack of tissue-specific targeting. The possibility of photochemically releasing macromolecules from endosomes and lysosomes into the cytosol was examined. Endocytosed macromolecules and photosensitizer were exposed to light and intracellular localization and the expression of macomolecules in the cytosol was analyzed. This novel technology, named photochemical internalization (PCI), was found to efficiently deliver type I ribosome-inactivating proteins, horseradish peroxidase, a p21ras-derived peptide, and a plasmid encoding green fluorescent protein into cytosol in a light-dependent manner. The results presented here show that PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that PCI may have a variety of useful applications for site-specific drug delivery, e.g., in gene therapy, vaccination, and cancer treatment.

Photochemical transfection: a new technology for light-induced, site-directed gene delivery.

The development of methods for specific delivery of therapeutic genes into target tissues is an important issue for the further progress of in vivo gene therapy. In this article we report on a novel technology, named photochemical transfection, to use light to direct a precise delivery of therapeutic genes to a desired location. The technology makes use of photosensitizing compounds that localize mainly in the membranes of endosomes and lysosomes. On illumination these membrane structures will be destroyed, releasing endocytosed DNA into the cell cytosol. Using a green fluorescent protein gene as a model we show that illumination of photosensitizer-treated cells induces a substantial increase in the efficiency of transfection by DNA-poly-L-lysine complexes. Thus, in a human melanoma cell line the light treatment can increase the transfection efficiency more than 20-fold, reaching transfection levels of about 50% of the surviving cells. In this article various parameters of importance for the use of this technology are examined, and the potential use of the technology in gene therapy is discussed.

Efficient secretion of human parathyroid hormone by Saccharomyces cerevisiae.

A cDNA encoding mature human parathyroid hormone (hPTH) was expressed in Saccharomyces cerevisiae, after fusion to the prepro region of yeast mating factor alpha (MF alpha). Radioimmunoassay showed high levels of hPTH immunoreactive material in the growth medium (up to 10 micrograms/ml). More than 95% of the immunoreactive material was found extracellularly as multiple forms of hormone peptides. Three internal cleavage sites were identified in the hPTH molecule. The major cleavage site, after a pair of basic amino acids (aa) (Arg25Lys26 decreases Lys27), resembles that recognized by the KEX2 gene product on which the MF alpha expression-secretion system depends. The use of a protease-deficient yeast strain and the addition of high concentrations of aa to the growth medium, however, not only changed the peptide pattern, but also resulted in a significant increase in the yield of intact hPTH (1-84) (more than 20% of the total amount of immunoreactive material). The secreted hPTH (1-84) migrates like a hPTH standard in two different gel-electrophoretic systems, co-elutes with standard hPTH on reverse-phase high-performance liquid chromatography, reacts with two hPTH antibodies raised against different parts of the peptide, has a correct N-terminal aa sequence, and has full biological activity in a hormone-sensitive osteoblast adenylate cyclase assay.

Light directed gene transfer by photochemical internalisation.

Numerous gene therapy vectors, both viral and non-viral, are taken into the cell by endocytosis, and for efficient gene delivery the therapeutic genes carried by such vectors have to escape from endocytic vesicles so that the genes can further be translocated to the nucleus. Since endosomal escape is often an inefficient process, release of the transgene from endosomes represents one of the most important barriers for gene transfer by many such vectors. To improve endosomal escape we have developed a new technology, named photochemical internalisation (PCI). In this technology photochemical reactions are initiated by photosensitising compounds localised in endocytic vesicles, inducing rupture of these vesicles upon light exposure. The technology constitutes an efficient light-inducible gene transfer method in vitro, where light-induced increases in transfection or viral transduction of more than 100 and 30 times can be observed, respectively. The method can potentially be developed into a site-specific method for gene delivery in vivo. This article will review the background for the PCI technology, and several aspects of PCI induced gene delivery with synthetic and viral vectors will be discussed. Among these are: (i) The efficiency of the technology with different gene therapy vectors; (ii) use of PCI with targeted vectors; (iii) the timing of DNA delivery relative to the photochemical treatment. The prospects of using the technology for site-specific gene delivery in vivo will be thoroughly discussed, with special emphasis on the possibilities for clinical use. In this context our in vivo experience with the PCI technology as well as the clinical experience with photodynamic therapy will be treated, as this is highly relevant for the clinical use of PCI-mediated gene delivery. The use of photochemical treatments as a tool for understanding the more general mechanisms of transfection will also be discussed.

The mature form of interleukin-1 alpha is constitutively expressed in immature male germ cells from rat.

In the present study we show that immature germ cells from rat testis contain both interleukin-1 alpha (IL-1 alpha) mRNA and immunoreactive proteins. In contrast, in primary cultures of Sertoli cells and peritubular cells. IL-1 alpha mRNA and immunoreactive protein were below the levels of detection. Immunoblots of lysates from these germ cells showed the presence of strong 17 kDa bands (mature forms of IL-1 alpha) and a much weaker 33 kDa band (precursor form). The finding of cell-associated mature forms of IL-1 alpha in germ cells indicates that immature male germ cells are able to process IL-1 alpha independent of its secretion. Data from isolated cell fractions, as well as from whole testis tissue from rats of various ages, indicate that IL-1 alpha expression takes place in late pachytene spermatocytes and early round spermatids. Whether IL-1 alpha plays a role intracellularly in germ cells or exerts its effects on neighboring Sertoli cells remains to be shown.

Expression of human parathyroid hormone in mammalian cells, Escherichia coli and Saccharomyces cerevisiae.

The entire human parathyroid hormone (hPTH) cDNA gene with its natural signal and pro-region is expressed in transfected mouse mammary tumor cells (C127I cells) and Chinese hamster lung cells (DON cells) under control of the murine metallothioneine-1 promoter in a vector in which replication functions are provided by the entire genome of bovine papilloma virus type I (BPV-1). Authentic hPTH is efficiently produced by the non-endocrine cells and secreted to the growth medium without any abberant processing. Immunoblots from SDS-PAGE gels of concentrated growth medium reveal one band corresponding to intact, undegraded hPTH. Purification by reversed-phase HPLC results in a peptide with an amino acid content and N-terminal sequence identical to hPTH. For comparison, hPTH cDNA with deleted prepro-region is also expressed as secretory proteins in Escherichia coli and in Saccharomyces cerevisiae. In E. coli the vector construct is based on the staphylococcal protein A promoter employing protein A signal sequence. In S. cerevisiae a mating factor alpha expression system containing the factor alpha-signal sequence is employed. The results show that intact hPTH is secreted in addition to proteolytically cleaved fragments in both microorganisms. Thus, the signal sequences promote efficient secretion, and correct N-terminal processing of hPTH in both mammalian, bacterial and yeast cells. However, the folding characteristics of hPTH make it susceptible to internal proteolytical cleavage which appears to be species specific in yeast and E. coli.

Evaluation of different photosensitizers for use in photochemical gene transfection.

Many potentially therapeutic macromolecules, e.g. transgenes used in gene therapy, are taken into the cells by endocytosis, and have to be liberated from endocytic vesicles in order to express a therapeutic function. To achieve this we have developed a new technology, named photochemical internalization (PCI), based on photochemical reactions inducing rupture of endocytic vesicles. The aim of this study was to clarify which properties of photosensitizers are important for obtaining the PCI effect improving gene transfection. The photochemical effect on transfection of human melanoma THX cells has been studied employing photosensitizers with different physicochemical properties and using two gene delivery vectors: the cationic polypeptide polylysine and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Photochemical treatment by photosensitizers that do not localize in endocytic vesicles (tetra[3-hydroxyphenyl]porphyrin and 5-aminolevulinic acid-induced protoporphyrin IX) do not stimulate transfection, irrespective of the gene delivery vector. In contrast, photosensitizers localized in endocytic vesicles stimulate polylysine-mediated transfection, and amphiphilic photosensitizers (disulfonated aluminium phthalocyanine [AlPcS2a] and meso-tetraphenylporphynes) show the strongest positive effect, inducing approximately 10-fold increase in transfection efficiency. In contrast, DOTAP-mediated transfection is inhibited by all photochemical treatments irrespective of the photosensitizer used. Neither AlPcS2a nor Photofrin affects the uptake of the transfecting DNA over the plasma membrane, therefore photochemical permeabilization of endocytic vesicles seems to be the most likely mechanism responsible for the positive PCI effect on gene transfection.

Photochemical internalization enhances the cytotoxic effect of the protein toxin gelonin and transgene expression in sarcoma cells.

Further advantages in the treatment of soft-tissue sarcomas will only be achieved by tailoring the adjuvant therapy after surgery. The photochemically directed release of macro-molecules from endosomes and lysosomes into the cytosol is a novel technology, named photochemical internalization (PCI), that has been evaluated for treatment of sarcoma cells in vitro. Two human synovial sarcoma cell lines (SW 982 and CME-1) were treated with the photosensitizer meso-tetraphenylporphine with two sulfonate groups on adjacent phenyl rings (TPPS2a) and a plasmid encoding enhanced green fluorescent protein (EGFP) complexed to poly-L-lysine to investigate the influence of PCI on gene transfer and with 5 micrograms/mL gelonin to investigate PCI of a Type-I ribosome-inactivating protein toxin. In addition, both cell lines were transduced with an Adenovirus serotype 5 encoding the Escherichia coli lacZ gene (AdHCMV-lacZ, expressing beta-galactosidase) and treated with TPPS2a and light to evaluate the effect of PCI on the transduction rate. Photochemically induced transfection with the reporter gene EGFP in CME-1 cells increased from 0% of cells at no light to 40% of the cells after 60 s of light exposure. In contrast, the SW 982 cells showed no enhanced expression of the gene. The fraction of virally transduced cells was about doubled in both cell lines by means of PCI, although the transduction was more efficient in the CME-1 cells. Both cell lines became up to four-fold more sensitive to light when combining photochemical treatment with gelonin incubation. Our experiments showed that PCI induced the endocytic escape of therapeutic substances in cells derived from human soft-tissue sarcomas.

5-aminolevulinic acid, but not 5-aminolevulinic acid esters, is transported into adenocarcinoma cells by system BETA transporters.

5-aminolevulinic acid (5-ALA) and its ester derivatives are used in photodynamic therapy as precursors for the formation of photosensitizers. This study relates to the mechanisms by which 5-ALA is transported into cells. The transport of 5-ALA has been studied in a human adenocarcinoma cell line (WiDr) by means of [14C]-labeled 5-ALA. The rate of uptake was saturable following Michaelis-Menten kinetics (K(m) = 8-10 mM and Vmax = 18-20 nmol.(mg protein x h)-1), and Arrhenius plot of the temperature-dependent uptake of 5-ALA was characterized by a single discontinuity at 32 degrees C. The activation energy was 112 kJ.mol-1 in the temperature range 15 degrees-32 degrees C and 26 kJ.mol-1 above 32 degrees C. Transport of 5-ALA was Na+ and partly Cl(-)-dependent. Stoichiometric analysis revealed a Na+:5-ALA coupling ratio of 3:1. With the exception of valine, methionine and threonine, zwitterionic and basic amino acids inhibited the transport of 5-ALA. 5-ALA methyl ester was not an inhibitor of 5-ALA uptake. The transport was most efficiently inhibited, i.e. by 65-75%, by the beta-amino acids, beta-alanine and taurine and by gamma-aminobutyric acid (GABA). Accordingly, 5-ALA, but not 5-ALA methyl ester, was found to inhibit cellular uptake of [3H]-GABA and [14C]-beta-alanine. Protoporphyrin IX (PpIX) accumulation in the presence of 5-ALA (0.3 mM) was attenuated 85% in the presence of 10 mM beta-alanine, while PpIX formation in cells treated with 5-ALA methyl ester (0.3 mM) or 5-ALA hexyl ester (4 microM) was not significantly influenced by beta-alanine. Thus, 5-ALA, but not 5-ALA esters, is transported by beta-amino acid and GABA carriers in this cell line.

Solubilization of the novel anionic amphiphilic photosensitizer TPCS 2a by nonionic Pluronic block copolymers.

The influence of four nonionic Pluronic block copolymers (L44, F68, P123 and F127) on the solubilization and aggregation of the novel anionic amphiphilic photosensitizer TPCS(2a), intended for use in the technology of photochemical internalization (PCI), was evaluated in aqueous media as part of pharmaceutical preformulation studies. The evaluation was performed by use of UV-Vis spectroscopy for diluted samples of TPCS(2a) (3×10(-6)M), and capillary viscosimetry, freezing point depression measurements and atomic force microscopy (AFM) at pharmaceutical relevant concentrations (2; 10 or 30 mg/ml TPCS(2a)). The critical micelle concentration (CMC) of the Pluronics in presence of TPCS(2a) was determined spectrophotometrically. The Pluronic block copolymers solubilized the photosensitizer above CMC at ambient temperature by formation of vehicle-drug complexes apparently organized in networks of varying viscosity and morphology, which were sensitive towards the addition of neutral and charged excipients.

Prolonged gene silencing by combining siRNA nanogels and photochemical internalization.

Small interfering RNAs (siRNAs) show potential for the treatment of a wide variety of pathologies with a known genetic origin through sequence-specific gene silencing. However, siRNAs do not have favorable drug-like properties and need to be packaged into nanoscopic carriers that are designed to guide the siRNA to the cytoplasm of the target cell. In this report biodegradable cationic dextran nanogels are used to deliver siRNA across the intracellular barriers. For the majority of non-viral siRNA carriers studied so far, endosomal confinement is identified as the most prominent hurdle, limiting the full gene silencing potential. Thus, there is a major interest in methods that are able to enhance endosomal escape of siRNA to improve its intracellular bioavailability. Photochemical internalization (PCI) is a method that employs amphiphilic photosensitizers to destabilize endosomal vesicles. We show that applying PCI at a later time-point post-transfection significantly prolonged the knockdown of the target protein only in case the siRNA was carried by nanogels and not when a liposomal carrier was used. Combining siRNA nanogels and PCI creates new possibilities to prolong gene silencing by using intracellular vesicles as depots for siRNA and applying PCI at the time when maintaining the RNAi effect becomes critical.

Delivery of siRNA to the target cell cytoplasm: photochemical internalization facilitates endosomal escape and improves silencing efficiency, in vitro and in vivo.

The prospect of introducing siRNA in a cell, to induce silencing of the corresponding gene, has encouraged research into RNAi-based therapeutics as treatment for human diseases. At present, the siRNA molecules that are in a more advanced stage of clinical evaluation have a common factor: all are delivered locally at the site of the disease. Thus, the state of the art in delivery of siRNA appears to be the local administration. This can certainly be attributed to the characteristics of siRNA molecules, such as relatively high molecular weight, negative charge, and susceptibility to nuclease degradation, which make systemic application as a drug molecule difficult. When focusing on local administration, the main concerns for siRNA delivery can be restricted to the trafficking of siRNA molecules from the vicinity of the target cells, to the intracellular compartment where RNAi takes place, i.e. the cytoplasm. This contribution is focused on the barriers and challenges in trafficking of siRNA upon local delivery. First, an overview is given on the current state of the art for siRNA delivery in clinical trials. Second, recent successful preclinical studies, involving direct and local administration of siRNA, are reviewed. Third, emphasis is given to the endosomal escape. Some of our recent work is presented: the application of photochemical internalization (PCI) to improve the endosomal escape of siRNA lipoplexes in vivo. Finally, concluding remarks focus on the advantages of employing a technique such as PCI to enhance the endosomal escape of siRNA molecules.

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 photosensitizers 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.

Correlation between suppressed meiotic recombination and the lack of DNA strand-breaks in the rRNA genes of Saccharomyces cerevisiae.

We have examined whether the suppressed homologous meiotic recombination within the rDNA of S. cerevisiae is reflected by a lack of possibly recombination-initiating strand-breaks in this part of the genome. Our findings indicate that bulk DNA in the ds-break repair deficient mutant rad52/rad52 accumulates a limited number of both ss- and ds-breaks during meiosis as compared to a RAD+/rad52 heterozygote. The rDNA-containing chromosome is however protected against these breaks, and thus this may be an explanation for the suppression of recombination in the rDNA. The fact that ds-breaks seem to be involved gives indirect support to the ds-break-repair model for recombination.

A 64 kDa protein is a candidate for a thyrotropin-releasing hormone receptor in prolactin-producing rat pituitary tumor cells (GH4C1 cells).

A thyrotropin-releasing hormone (TRH) binding protein of 64 kDa has been identified by covalently crosslinking [3H]TRH to GH4C1 cells by ultraviolet illumination. The crosslinkage of [3H]TRH is UV-dose dependent and is inhibited by an excess of unlabeled TRH. A 64 kDa protein is also detected on immunoblots using an antiserum raised against GH4C1 cell surface epitopes. In a closely related cell line (GH12C1) which does not bind [3H]TRH, the 64 kDa protein cannot be demonstrated by [3H]TRH crosslinking nor by immunoblotting. These findings indicate that the 64 kDa protein is a candidate for a TRH-receptor protein in GH4C1 cells.