In the course of experiments for our FET open project Lumiblast, we set off to measure the excitation
of various photoactive drugs (photosensitizers, PS) by the luminescence emission of luminol. Luminol
(5-Amino-2,3-dihydrophthalazine-1,4-dione) is a chemical that interacts with reactive oxygen species
(ROS) in basic conditions, and in the presence of metal catalysts like Fe or Cu, gives out a characteristic
blue luminescence. When dissolved in organic solvents like DMSO, however, luminol only requires
the addition of bases like KOH, NaOH or potassium terbutoxide, to fulfil the conditions for
luminescence emission.
In the present work we employed a detection system based on a spectrograph coupled to a ccd camera
to register fluorescence (Fig 1B) or luminescence (Fig 1 A, C). In the case of characteristic fluorescence
registration (Fig. 1B), the PSs investigated were excited by a 532 nm laser with a variable power output.
We have documented the energy transfer from chemically induced luminol luminescence to a number
of PSs including rose bengal, erythrosin B, hypericin amongst others. In all cases both the luminol
emission and the luminol luminescence-induced PS fluorescence were registered as shown in the
example of luminol and erythrosine b in Fig. 1C.
We further attempted to register the generation of singlet oxygen from luminol-excited PSs. To achieve
this, we employed the near-infrared (NIR) photomultiplier tube (PMT) shown in Fig.1 E, with a cut-off
filter at 900nm and a bandpass filter at 1270±30 nm. This allowed only radiation within this spectral
region to reach the PMT, corresponding to the characteristic phosphorescence of singlet oxygen, spin
forbidden de-excitation to ground state triplet oxygen.
A characteristic steady state singlet oxygen registration can be seen in Fig. 1D, for erythrosine b which
has a high singlet oxygen quantum yield. The luminol luminescence was initiated by addition of
terbutoxide to the DMSO luminol solution, at which point we can see a rise of the signal at 1270 nm.
Upon addition of the singlet oxygen quencher, L-histidine, the signal dropped steeply to background
levels.
NOTE: Figures are not available.
Photochemical internalization (PCI) is a technology to enhance intracellular drug delivery by light-induced translocation of endocytosed therapeutics into the cytosol. The aim of this study was to explore the efficacy of PCI-based delivery of bleomycin and the impact on systemic anti-tumor immunity. Mouse colon carcinoma cells (CT26.CL25), stably expressing the bacterial β-galactosidase, were inoculated into the legs of athymic or immuno-competent BALB/c mice strains. The mice were injected with the photosensitizer AlPcS2a and bleomycin (BLM) prior to tumor light exposure from a 670 nm diode laser. Photochemical activation of BLM was found to induce synergistic inhibition of tumor growth as compared to the sum of the individual treatments. However, a curative effect was not observed in the athymic mice exposed to 30 J/cm2 of light while more than 90% of the thymic mice were cured after exposure to only 15 J/cm2 light. Cured thymic mice, re-challenged with CT26.CL25 tumor cells on the contralateral leg, rejected 57-100% of the tumor cells inoculated immediately and up to 2 months after the photochemical treatment. T-cells from the spleen of PCI-treated mice were found to inhibit the growth of CT26.CL25 cells in naïve thymic mice with a 60% rejection rate. The results show that treatment of CT26.CL25 tumors in thymic mice by PCI of BLM induces a systemic anti-tumor immunity.
The short lifetime and thereby the diffusion length of singlet oxygen as well as the correlation between photosensitizer (PS) fluorescence and treatment effects made it attractive in the 80-ties to analyze the intracellular localization of PSs to reveal the initial intracellular hit in PDT. Some PSs were found intracellularly in fluorescing granules that was found to be lysosomes and endosomes. Such vesicles were rupture upon exposure of the cells to light and the PS relocated to other compartments in the cells. About 40 different hydrolytic enzymes are found in late endosomes and lysosomes. Release of lysosomal hydrolases into cytosol has been documented to be a cytotoxic event. This suicide sac hypothesis originally postulated by De Duve suggests that released hydrolases lead to degradation of cellular constituents to such an extent that cellular functions are impaired and the cells subsequently die. However, it was found that a large fraction of the vesicles containing PSs could be ruptured without inducing substantial cytotoxicity. The scientific basis for this surprising observation as well as the potential utilization of the photochemical rupture of endosomes and lysosomes for intracellular delivery of various therapeutics, named photochemical internalization (PCI), will be discussed. During the last 20-30 years macromolecular therapeutics have become increasingly attractive for treatment of cancer due to their improved specificity and reduced side effects. Intracellular delivery of macromolecular therapeutics has however so far showed only limited success, a limitation that may be circumvented by PCI. The historic and scientific basis for PCI will be presented.
Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of
macromolecules and genes into the cell cytosol. Prodrug activating gene therapy (suicide gene therapy) employing
the transduction of the E. coli cytosine deaminase (CD) gene into tumor cells, is a promising method. Expression of
this gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-FC, to the toxic metabolite,
5-fluorouracil (5-FU). 5-FC may be particularly suitable for brain tumors, because it can readily cross the bloodbrain
barrier (BBB). In addition the bystander effect, where activated drug is exported from the transfected cancer
cells into the tumor microenvironment, plays an important role by inhibiting growth of adjacent tumor cells.
Tumor-associated macrophages (TAMs) are frequently found in and around glioblastomas. Monocytes or
macrophages (Ma) loaded with drugs, nanoparticles or photosensitizers could therefore be used to target tumors by
local synthesis of chemo attractive factors. The basic concept is to combine PCI, to enhance the ex vivo transfection
of a suicide gene into Ma, employing specially designed core/shell NP as gene carrier.
The overall objective of the research was to investigate the utility of photochemical internalization (PCI) for the enhanced nonviral transfection of genes into glioma cells. The PCI-mediated introduction of the tumor suppressor gene phosphatase and tensin homolog (PTEN) or the cytosine deaminase (CD) pro-drug activating gene into U87 or U251 glioma cell monolayers and multicell tumor spheroids were evaluated. In the study reported here, polyamine-DNA gene polyplexes were encapsulated in a nanoparticle (NP) with an acid degradable polyketal outer shell. These NP synthetically mimic the roles of viral capsid and envelope, which transport and release the gene, respectively. The effects of PCI-mediated suppressor and suicide genes transfection efficiency employing either “naked” polyplex cores alone or as NP-shelled cores were compared. PCI was performed with the photosensitizer AlPcS2a and λ=670-nm laser irradiance. The results clearly demonstrated that the PCI can enhance the delivery of both the PTEN or CD genes in human glioma cell monolayers and multicell tumor spheroids. The transfection efficiency, as measured by cell survival and inhibition of spheroid growth, was found to be significantly greater at suboptimal light and DNA levels for shelled NPs compared with polyamine-DNA polyplexes alone.
The overall objective of the research is to investigate the utility of photochemical
internalization for the enhanced nonviral transfection of genes into cells. We have examined,
in detail, the evaluation of photochemical internalization (PCI) as a method for the non-viral
introduction of the tumor suppressor gene PTEN and the PCI mediated transfection of the
cytosine deaminase (CD) pro drug activating gene into glioma cell monolayers and multi-cell
tumor spheroids. Expression of the CD gene within the target cell produces an enzyme that
converts the nontoxic prodrug, 5-fluorocytosine (5-FC), to the toxic metabolite, 5-fluorouracil
(5-FU).
Photochemical internalization (PCI) is a technique to improve the utilization of macromolecules (e.g.
chemotherapeutic agents) in cancer therapy in a site-specific manner. The concept is based on the use of
specially designed photosensitizers which localize preferentially in the membranes of endocytic vesicles.
Upon exposure to light the photosensitizers induce the formation of reactive oxygen species such as singlet
molecular oxygen. The photooxidation of the endocytic membranes leads to the release of the contents of
the vesicles into the cytosol. In this way, macromolecules encapsulated by the vesicles will reach the
cytosol and exert their biological activity instead of being degraded by lysosomal hydrolases.
The utility of PCI for the treatment of malignant gliomas was investigated in vitro using an F98 rat glioma
cell line. The cytotoxicity of 5-aminolevulinic acid (ALA) based PCI of bleomycin was compared to: (1)
ALA-PDT, and (2) bleomycin. In all cases, monolayers were incubated in ALA, bleomycin, or ALA +
bleomycin for 4 hours and were subsequently exposed to 635 nm light. Toxicity was evaluated using
colony formation assays.
F98 rat glioma cells in monolayer were found to be susceptible to the effects of both ALA-PDT and
bleomycin. ALA-PDT was found to be particularly effective when light was delivered at a low irradiance
of 5 mW cm-2. In this case, a radiant exposure of 20
J cm-2 resulted in only 4% survival. Bleomycin was
found to be toxic at relatively low concentrations, incubation of F98 cells in 10 &mgr;g ml-1 for 4 hours
resulted in 1% survival. The PCI effect was found to be negligible for the parameters investigated in the
F98 cell line suggesting that: (1) the incubation time was sub-optimal and/or (2) ALA was inappropriate for
this application.
Photodynamic therapy with 5-aminolevulinic acid (5-ALA) is a promising alternative treatment for several types of cancer. This work represents a first approach to characterize the transport system for 5-ALA in human cancer cells, using WiDr cells from a primary adenocarcinoma of the rectosigmoid colon as a model system. The transport of 5-ALA in WiDr was found to be dependent on pH and temperature, and partially inhibited by inhibitors of the energetic metabolism. Although WiDr was shown to express System A, a common transport system for small aliphatic amino acids, the transport of 5-ALA in WiDr was not found to be mediated by this system.
Photodynamic therapy (PDT)-induced kinetics of apoptosis were studied in V79 cells using several differently localized photosensitizing dyes, mostly tetraphenylporphine derivatives. Apoptotic fractions were quantified by flow cytometry after staining the samples by the terminal deoxynucleotidyl transferase (TdT)-assay. Methylene blue derivative (MBD), a new dye for PDT, and 5-aminolevulinic acid (ALA)-induced protoporphyrin IX that are both localized in mitochondria, induced apoptosis rapidly within hours after PDT. With MBD it was shown that rapid apoptosis was induced only with dye concentration above a certain threshold. With a lower dye concentration apoptosis was delayed more than one day and was induced due to inhibition of oxidative phosphorylation. After PDT with two membrane localized dyes, tetra(3- hydroxyphenyl)porphyrin (3THPP) and Photofrin, maximal induction of apoptosis took about 12 h. With two lysosomal localized sulfonated meso-tetraphenylporphines (TPPS2a and TPPS4) no apoptosis was induced until more than 12 h after PDT. The results are discussed in relation to evidence in the literature on the nature of possible pathways involved.
In the present study cellular uptake and intracellular localization of a liposomal formulation of Zn-PC, was studied in human cervix carcinoma cells of the line NHIK 3025. The cellular uptake of Zn-PC was found to be completed after 4 hours of incubation. Seventy to eighty percent of the dye taken up by the cells during 18 hours of incubation was retained after 1 hour of further incubation in sensitizer-free medium. The cellular uptake was attenuated by the presence of serum and at low temperature of the incubation medium. The uptake was not enhanced by decreasing the pH of the incubation medium. The present study indicated that Zn-PC is rapidly transferred from liposomes to lipoproteins. Analysis of inactivation of subcellular marker enzymes indicated that Zn- PC is located in the Golgi apparatus and mitochondria.
The present study is based on the use of vital microscopy and a charge couple device camera for localization of a porphyrin in normal and neoplastic tissue in nude athymic Balb/c nu/nu mice. Granular tissue was allowed to grow into the space between two optical widows, separated by a 40 micrometer thick spacer in a transparent chamber. WiDr cells derived from a primary adenocarcinoma of the rectosigmoid colon, were used as a tumor model. Tumor cells were grown in vitro as spheroids and transplanted to the tissue in the chamber. The photosensitizer, meso(tetra-phenyl)porphine with two sulfonate groups on adjacent phenylrings (TPPS2a), was injected i.p. to a concentration of 25 mg kg-1, and the porphyrin localization was evaluated in vivo and in situ by a fluorescence microscopical technique. A significant difference between normal and neoplastic tissue was observed, with a much faster uptake of the photosensitizer in tumor tissue than in normal tissue.
The topoisomerase I (Topo I) inhibitor camptothecin (CPT) has been combined with photodynamic treatment (PDT) in V79 and NHIK 3025 cells. Meso-tetra (N-methyl-4-pyridyl) porphine (TMPyPH2) was used as a photosensitizer. The dye has been shown to localize in granules in the cytoplasm of both cell lines. Some of the granularly located TMPyPH2 is relocated to the cytosol after exposure to a light dose inactivation 70% of the cells. The human NHIK 3025 carcinoma from cervix were more resistant to PDT (D50 approximately equals 0.33 J/cm2) and CPT (D50 approximately equals 320 nM) than Chinese hamster V79 lung fibroblasts (PDT, D20 approximately equals 0.3 J/cm2 and CPT, D20 approximately equals 55 nM). When the cells were treated with CPT for 18 hours before PDT, the combination of treatments led to slight synergistic effects for low CPT concentrations (up to 100 nM in NHIK 3025 cells) and high PDT doses (above 0.15 J/cm2). For higher CPT concentrations and lower PDT doses, the combination of treatment became additive.
Some key data concerning the pharmacokinetics of PCT photosensitizers are reviewed. The following topics are discussed: The binding of photosensitizers to serum proteins, and the significance of LDL binding for tumor localization, the distribution of sensitizers among different tissue compartments and the significance of extracellular proteins and other stromal elements, such as macrophages, low tumor pH, leaky vasculature and poor lymphatic drainage for tumor selectivity of drugs, the retention and excretion of sensitizers, and intracellular pharmacokinetics. Furthermore, the usefulness of fluorescence measurements in the study of sensitizer pharmacokinetics is briefly discussed. A key observation is that 1O2 has a short radius of action. Since practically all PCT sensitizers act via the 1O2 pathway, only targets with significant sensitizer concentrations can be damaged. A given number of 1O2 entities generated in different organelles (mitochondria, lysosomes, plasma membrane, etc.) may lead to widely different effects with respect to cell inactivation. Similarly, sensitizers localizing in different compartments of tissues may have different photosensitizing efficiencies even under conditions of a similar 1O2 yield.
Combination effects of photodynamic therapy (PDT) with meso-tetra (di-adjacent- sulfonatophenyl) porphine (TPPS2a) and the microtubule (MT) inhibitor, vincristine (VCR), were studied in the CaD2 mouse tumor model in mice. A synergistic effect was found when VCR, at an almost nontoxic dose (1 mg/kg), was injected i.p. into the mice 6 hr before PDT. The data on mitotic index show a 4 - 5 fold accumulation of the cells in mitosis 6 hr after injection of VCR into the mice. Cell cycle and ploidy distributions in tumor tissues were determined by means of image analysis with measurement of integrated optical density after Feulgen reaction on monolayers. Ploidy distribution of the tumors was not significantly changed 6 and 12 hr after administration of VCR only, while an increasing aneuploidy was observed 24 and 48 hr after VCR treatment. No prominent changes of the cell cycle and ploidy distributions were found in the tumor tissues after PDT or PDT combined with VCR.
The mechanisms of photoinactivation of NHIK 3025 cells in culture sensitized by tetrasulfonated phenylporphines (TPPS4) are described). Ultracentrifugation studies on postnuclear supernatants indicated that the intracellular distribution of TPPS4 resembles that of (beta) -N-acetyl-D-glucosaminidase ((beta) -AGA), a lysosomal marker enzyme, and that the cytosolic content of TPPS4 is below the detection limit of the ultracentrifugation method. Upon light exposure more than 90% of TPPS4 was lost from the lysosomal fractions, due to lysosomal rupture. The content of TPPS4 in the postnuclear supernatants was reduced by 30 - 40% upon exposure to light. This is most likely due to binding of TPPS4 to the nuclei, which were removed from the cell extracts before ultracentrifugation, after photochemical treatment. The unpolymerized form of tubulin seems to be an important target for the photochemical inactivation of NHIK 3025 cells. Since TPPS4 is mainly localized in lysosomes it was assumed that a dose of light disrupting a substantial number of lysosomes followed by microtubule depolymerization by nocodazole would enhance the sensitivity of the cells to photoinactivation. This was confirmed by using a colony-forming assay. The increased phototoxic effect exerted by such a treatment regime could be explained by an enhanced sensitivity of tubulin to light. Another cytosolic constituent, lactate dehydrogenase, was not photoinactivated by TPPS4 and light.
Cells of the line WiDr were incubated with media containing different concentrations of (delta) -aminolevulinic acid (ALA) and the amount of the produced protoporphyrin (Pp) IX was evaluated by means of steady-state fluorescence. The photomodification process of ALA- induced Pp IX was investigated by measuring spectroscopic properties of cells suspensions incubated with 0.2 mM and 1 mM of ALA. Light exposure of cells containing Pp IX resulted in a decrease of fluorescence intensities at 635 and 705 nm ((lambda) exc equals 413 nm) due to photobleaching and in an increase of fluorescence intensity at around 670 nm due to the formation of a photoproduct. These spectral changes arise from photochemical reactions resulting in the formation of photoprotoporphyrin and destruction of porphyrin ring. The fluorescence kinetics indicate that for ALA-induced Pp IX in cells the ring opening reaction prevails the photoprotoporphyrin formation. Singlet oxygen may be involved in both processes since the photobleaching is slowed down on bubbling N2 through the samples and photoprotoporphyrin formation is slightly faster in suspensions in phosphate buffered saline (PBS) made of D2O compared with suspensions in PBS made of H2O.
Five-Aminolevulinic acid (ALA) is being used in clinical photodynamic therapy. We have investigated if ALA has any modifying effects on x rays and found that at concentrations more than 1 mM it exerts a slight radiosensitizing effect on WiDr cells in culture.
The effect of sulfonations on the PDT efficiency of aluminum phthalocyanines and meso- tetraphenylporphines was studied in the CaD2 mouse mammary carcinoma. The PDT efficiencies were found to decrease in the following order: AlPCS2 > AlPCS4 > Photofrin > TPPS2a > AlPCS1 > TPPS4 > TPPS2o > TPPS1. Apparently, the degree of sulfonations of both families of dyes strongly affects the photobiological properties. It was found to be of great importance whether the sulfonate groups were located adjacently or opposite positions on the molecules.
The mechanisms of photoinactivation of NHIK 3025 cells in culture sensitized by sulfonated tetraphenyl porphines (TPPSn) are described. Di- and tetrasulfonated species are mainly located in lysosomes. TPPS1 is located diffusely in the extranuclear space, supposedly bound to endoplasmic reticulum as indicated by electron microscopical findings, and to some extent in lysosomes. After PDT TPPSn penetrates the lysosomal membrane. However, after exposure of cells to TPPS4 and light lysosomal enzymes are inactivated before they eventually can be released to the cytosol. In all cases electron microscopical studies show swollen secondary lysosomes after PDT. TPPSn and light induce accumulation of cells in mitosis. This is due to photochemical damage to the unpolymerized form of tubulin.
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