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Action spectra of phthalocyanines with respect to photosensitization of cells
Abstract
Human carcinoma cells (NHIK 3025 cells) and Chinese hamster cells (V79 cells) were incubated with AlPcS1, AlPcS2 and AlPcS4, phthalocyanines with different lipophilicity but with similar photochemical properties when in monomeric solutions. The absorption- and fluorescence spectra of the dyes in the cells were recorded as well as their action spectra with respect to sensitizing cells to photoinactivation. These spectra show that under the present conditions AlPcS1 is strongly aggregated in both cell lines; AlPcS2 is aggregated in V79 cells but much less so in NHIK 3025 cells. A main finding is that the shapes of the action spectra are similar to that of the fluorescence excitation spectra, but not to the absorption spectra, indicating that the photosensitizing effects of the dyes are mainly due to their monomeric fraction in the cells. AlPcS2 and AlPcS4 localize intracellularly mainly in lysosomes while AlPcS1 was found to be more diffusely distributed in cells. As measured per quantum of fluorescence emitted, AlPcS1 and AlPcS2 are more efficient sensitizers than AlPcS4. The difference in efficiency between AlPcS2 and AlPcS4 is supposedly due to a different localization pattern on the suborganelle level.
... A number of second generation photosensitizers of different chemical families were synthesized in the 1980s. Sulfonated tetraphenylporphines (TPPS n ) [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33], aluminum and zinc phthalocyanines [34][35][36][37][38][39][40][41], meso-tetra(hydroxyphenyl)chlorin (m-THPC) [31,33,[42][43][44][45][46][47][48], chlorin e6 [16,31,49], meso-tetra(3-hydroxyphenyl)porphine (m-THPP) [31,33,43,50,51], merocyanine 540 (MC 540) [16,52], hyper- ...
... Furthermore, they found that only cell bound photosensitizers, and not those present in the fluid around the cell, were efficient [73]. The action spectra for HpD, Photofrin II, 3-THPP, chlorin e6, aluminium phthalocyanine tetrasulfonate (AlPcTs), benzoporphyrin derivative monoacid ring A (BPD-MA), zinc phthalocyanine tetrasulfonate (ZnPcS 4 ), AlPcS 2 , ZnPc, etiopurpurin were determined in vitro or in vivo [37,[74][75][76]. These spectra were found to have the same shape as the fluorescence excitation spectrum of the photosensitizer, indicating that primarily nonaggregated molecules generate 1 O 2 [37,[74][75][76]. ...
... The action spectra for HpD, Photofrin II, 3-THPP, chlorin e6, aluminium phthalocyanine tetrasulfonate (AlPcTs), benzoporphyrin derivative monoacid ring A (BPD-MA), zinc phthalocyanine tetrasulfonate (ZnPcS 4 ), AlPcS 2 , ZnPc, etiopurpurin were determined in vitro or in vivo [37,[74][75][76]. These spectra were found to have the same shape as the fluorescence excitation spectrum of the photosensitizer, indicating that primarily nonaggregated molecules generate 1 O 2 [37,[74][75][76]. Thus, only the fluorescent fraction of a photosensitizer is efficient. ...
Photodynamic therapy (PDT) is now an established treatment of malignant and premalignant dysplasias. A number of first and second generation photosensitizers have been studied in Norway. The aim has been to improve PDT efficiency and applicability. Many critical details regarding the mechanisms of PDT were elucidated by researchers in Norway. In this review we focus on the most important findings related to these basic mechanisms, such as generation of singlet oxygen, estimations of its lifetime, the oxygen effect itself, the subcellular localization of photosensitizers with different properties, their photodegradation during PDT and their tumour selectivity.
... FLIM is also useful for providing some information about the localization and distribution of PSs inside the cell. The localization and distribution of the PS is a critical issue to consider while understanding the mechanism of cell death by PDT [26,27], which can occur via programmed (apoptotic) or non-programmed mode (necrotic or autophagic) pathways [28]. In general, photosensitizers that have been preferentially located at nucleus, Golgi complex [36], and mitochondria [30] act by apoptosis, the type-I mode of cell death in PDT [6,29], although they should not be localized in the nucleus to avoid damaging the DNA and raise some mutagenesis effect of PDT. ...
... In parallel, it is known that designed structural modifications can be used to target specific cell organelles. Previous studies have shown that AlPcS 2 localizes mainly in lysosomes, however, AlPcS 4 is randomly distributed in the cytoplasm [27]. In this way, new AlClPc species targeting specific organelles in combination therapy may give rise to smart strategies for improving PDT efficacy. ...
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Current limited understanding of the intracellular self-aggregation process of several photosensitizers is one of the obstacles in the progress of Photodynamic Therapy (PDT) as a clinically viable option to photomodulate the biological responses. This is further intensified by the higher in vitro experimental complexity as compared to the experiments carried out in simple solution. This study describes the monomer(photoactive)/aggregate(non-active) equilibrium of aluminum chloride phthalocyanine (AlClPc) within the human breast adenocarcinoma cell line, MCF-7. Static fluorescence and fluorescence lifetime imaging microscopy demonstrated that there was an upper limit of the amount of monomeric AlClPc inside cells, which might be influenced by the high water content inside the biological tissue. In this process, high AlClPc concentrations triggered the aggregation process while lowering its monomeric active form. The dynamic monomer/aggregates equilibrium can be modulated even inside cell typically through cell replication process, which decreases the AlClPc content inside each cellular unit. The results underline the need for new strategies in order to enhance the AlClPc monomerization within cell, especially in key organelles for PDT including mitochondria and lysosome membrane. This study demonstrates a rational approach for the study of the state of aggregation of photosensitizers inside cells, which is a critical issue to be considered for PDT efficacy.
... The activity of a dye as a photosensitiser depends on the efficiency of formation of the triplet state, therefore, the observed enhancement of fluorescence quenching via energy migration will lead to lowering of its photosensitizing potential. Furthermore, quenching of the triplet state T 1 by ground state molecules has been observed previously [101]. At high dye concentrations quenching by ground state molecules will compete with quenching by oxygen, and the result will be a lower singlet oxygen quantum yield, further reducing the photosensitizing efficiency. ...
... The reasons are the tendency of AlPcS n to aggregate at high concentrations, which can be reached locally in cells and the possibility of fluorescence quenching. Although aggregates are not fluorescent and the action spectra of AlPcS n show that they are not photodynamically active [101], photoinduced changes in the cell can lead to disaggregation, and therefore generation of more active species. ...
In this review, we have shown that the dimerisation of phthalocyanine compounds, notably here the sulphonated aluminium phthalocyanines, is dependent upon concentration, on the medium in which the dye is dissolved, and upon pH. Complex equilibria between various monomer and dimer species are observed as a function of pH, and the probable structures of the dimers elucidated by semi-empirical and ab initio calculations. The formation of a red-shifted dimer leads to the quenching of monomer singlet state in concentrated solution, in reverse micelles, and in lipid vesicles, and this behaviour can account for the fluorescence intensity distributions and decay characteristics of phthalocyanine dyes in living cells as a function of irradiation time.
... The majority of them are more efficient PSs than porphyrinic PSs (HPD, Photofrin). Their efficiency and action mechanism depend on the degree of sulfonation [10][11][12][13]. AlPcS 1 and AlPcS 2 were shown to be more efficient PSs than AlPcS 4 ; the difference in efficiency between AlPcS 2 and AlPcS 4 is supposedly due to different localization patterns at the suborganelle level. ...
... AlPcS 1 and AlPcS 2 were shown to be more efficient PSs than AlPcS 4 ; the difference in efficiency between AlPcS 2 and AlPcS 4 is supposedly due to different localization patterns at the suborganelle level. Human carcinoma cells (NHIK 3025 cells) and Chinese hamster cells (V79 cells) were incubated with AlPcS 1 , AlPcS 2 and AlPcS 4 possessing different lipophilicities but similar photochemical properties in monomeric solution [12]. AlPcS 1 is strongly aggregated in both cell lines; AlPcS 2 is aggregated in V79 cells but much less in NHIK 3025 cells. ...
The application of phthalocyanine derivatives in medicine as photosensitizers for photodynamic therapy of cancer diseases is reviewed. The emphasis is on the work of Russian authors, which is less covered in the scientific literature.
... Irradiation of cells sensitized with TPPS 4 results in photooxidation of matrix components including the hydrolytic enzymes. 35,36 Because of the short half-life and diffusion limit of singlet oxygen in biological environments, 40 and perhaps as a result of the polysaccharide coating of the inner surface of the lysosomes, lysosomal enzymes are typically photoinactivated before the endocytic membranes are targeted in TPPS 4 -PDT protocols (Fig. 10). Photodamage to the latter is expected to result in loss of membrane integrity and release of the contents of the endocytic vesicles into the cytoplasm (Fig. 10). ...
... In particular, depending upon the specific lysosomal photosensitizer employed and the light dose applied, lysosomal luminal proteases may undergo irreversible inactivation without profound effects on lysosomal membrane permeability. 35,36 Alternatively, lysosomal membranes may be damaged and permeabilized, leading to the release of proteases and alkalinization of the organelle. 21 Such effects are expected to markedly affect either autophagosome-lysosome fusion, and/or degradation of the cargo contained in the autolysosomes. ...
Photodynamic therapy (PDT) is a procedure that has applications in the selective eradication of neoplasia where sites of malignant lesions are clearly delineated. It is a two-step process whereby cells are first sensitized to light and then photoirradiated. This results in the formation of singlet molecular oxygen and other reactive oxygen species that can cause photodamage at sites where the photosensitizing agent has localized. Photosensitizers found to be clinically useful show affinity for the endoplasmic reticulum (ER), mitochondria, lysosomes, or combinations of these sites. The induction of apoptosis and/or autophagy in photosensitized cells is a common outcome of PDT. This report explores the following issues: (1) Does the induction of autophagy in PDT protocols occur independent of, or in association with, apoptosis? (2) Does the resulting autophagy play a prosurvival or prodeath role? (3) Do photosensitizers damage/inactivate specific proteins that are components of, or that modulate the autophagic process? (4) Can an autophagic response be mounted in cells in which lysosomes are specifically photodamaged? In brief, autophagy can occur independently of apoptosis in PDT protocols, and appears to play a prosurvival role in apoptosis competent cells, and a prodeath role in apoptosis incompetent cells. Mitochondrial and ER-localized sensitizers cause selective photodamage to some (i.e., Bcl-2, Bcl-x(L), mTOR) proteins involved in the apoptotic/autophagic process. Finally, an aborted autophagic response occurs in cells with photodamaged lysosomes. Whereas autophagosomes form, digestion of their cargo is compromised because of the absence of functional lysosomes.
... Nowadays, several papers have evaluated the PDT antitumor effect using various LED light wavelengths applied to in vitro and in vivo studies (Babilas et al 2006, Dias Ribeiro et al 2010, Hatakeyama et al 2013, Helander et al 2014, Novak et al 2016, Jamali et al 2018. Other previous papers in which non-LED sources were employed have proposed a methodology for choosing the appropriate illumination wavelength based on the action spectrum of the photosensitizer (Moan and Sommer 1984, Moan et al 1989, Moan et al 1992. Moreover, green light has been employed in clinical PDT with δ-aminolevulinic acid sensitization of facial keratoses and compared with red light (Fritsch et al 1997). ...
Light of different wavelengths can be used to obtain a more profitable outcome of photodynamic therapy (PDT), according to the absorption bands of the photosensitizer (PS). Low-grade cervical intraepithelial neoplasias (CINs) are superficial lesions that can be treated with light of shorter wavelength than red because a large light penetration depth in tissue is not necessary. We report a comparative investigation performed to evaluate the efficacy of light-emitting diodes (LEDs) of different wavelengths in the photodynamic treatment applied to both 2D and 3D HeLa cell spheroid cultures. The spheroids are utilized as a PDT dosage model, and cell viability is evaluated at different sections of the spheroids by confocal microscopy. Cells incubated with m-tetrahydroxyphenyl chlorin are illuminated with LED systems working in the low fluence range, emitting in the violet (390 - 415 nm), blue (440-470 nm), red (620 - 645 nm) and dark red (640 - 670 nm) regions of the light spectrum at various exposures times (tI) comprised between 0.5 and 30 min. PDT experiments performed on both 2D and 3D cell cultures indicate that the PDT treatment outcome is more efficient with violet light followed by red light. Dynamic data from the front displacement velocity of large 2D-quasi-radial colonies generated from cell spheroids adhered to the Petri dish bottom as well as the evolution of the 3D growth give further insight about the effect of PDT at each condition. Results from 3D cultures indicate that the penetration of the violet light is appropriate to kill HeLa cells several layers below, showing cell damage and death not only in the outer rim of the illuminated spheroids, where a PS accumulation exists, but also in the more internal region. Results indicate that violet LED light could be useful to treat CINs involving superficial dysplasia.
... It is a very important issue, as localization of photosensitizer (PS) in a biosubstrate and photoirradiation conditions eventually determine the cell death mechanism. For example, aluminum sulphophthalocyanine predominantly localizes in lysosomes; and during photoirradiation, the organelle is destroyed; and releasing of all lysosomal enzymes and hydrolases, which are discharged from cancer cells, later on results in necrosis of the adjacent tissues [2,3]. Cationic metal-free porphyrins are bound mainly with the plasma membrane of mitochondria [4,5]; the membrane oxidation brings some changes in its permeability and, as a result, gives some content leakage, causing necrosis as well. ...
This study was focused on obtaining polymer complex of 5-(4′-N-tert-butyloxycarbonylglicinaminophenyl)-10,15,20-triphenylporphine and 5-(4′-amonophenyl)-10,15,20-triphenylporphine with chitosan. The polymer complexes were characterized spectrally and calorimetrically. The existence of a substituent capable of forming hydrogenic complexes with proteins and other biopolymers in porphyrins provides an effective immobilization of a photosensitizer. Binding with porphyrins was found to result in twisting the chitosan globule and encapsulation of porphyrins. Coating of polymer complex with bovine serum albumin promotes neutralization of zeta potential and formation of 180-nm capsules. As the result of the study, a new universal strategy of water insoluble preparation encapsulation in biopolymers for targeted delivery of drugs and their further release was suggested.
Graphical abstractᅟ
... Connelly et al. [94]. A short-lived component of 1 ns was also found and could be attributed to the quenching from non-fluorescence aggregates, which agreed with the cell measurement performed by Moan et al. [95]. ...
Photodynamic therapy (PDT) has been used clinically for treating various diseases including malignant tumors. The main advantages of PDT over traditional cancer treatments are attributed to the localized effects of the photochemical reactions by selective illumination, which then generate reactive oxygen species and singlet oxygen molecules that lead to cell death. To date, over- or under-treatment still remains one of the major challenges in PDT due to the lack of robust real-time dose monitoring techniques. Time-resolved fluorescence (TRF) provides fluorescence lifetime profiles of the targeted fluorophores. It has been demonstrated that TRF offers supplementary information in drug-molecular interactions and cell responses compared to steady-state intensity acquisition. Moreover, fluorescence lifetime itself is independent of the light path; thus it overcomes the artifacts given by diffused light propagation and detection geometries. TRF in PDT is an emerging approach, and relevant studies to date are scattered. Therefore, this review mainly focuses on summarizing up-to-date TRF studies in PDT, and the effects of PDT dosimetric factors on the measured TRF parameters. From there, potential gaps for clinical translation are also discussed.
... PSs taken up into the cell by endocytosis overall localize in the structures above mentioned, targeting the photosensitization upon irradiation [143]. Endosomally/lysosomally PSs, e.g., mono-L-aspartyl chlorin e6 (Npe6) [144], Meso-tetra-(p-sulphophenyl) porphine (TPPS 1 4 ) [116], AlPcS 2 4 [145], can localize to the target nducing membrane damage or lysosomal enzymes photoinactivation respectively. The lysosomal membrane breakdown affects the autophagic flux; the release of proteolytic enzimes triggers mitochondrial apoptosis by Bid cleavage [144]. ...
Autophagy is an important cellular program with a "double face" role, since it promotes either cell survival or cell death, also in cancer therapies. Its survival role occurs by recycling cell components during starvation or removing stressed organelles; when damage becomes extensive, autophagy provides another programmed cell death pathway, known as Autophagic Cell Death (ACD). The induction of autophagy is a common outcome in PhotoDynamic Therapy (PDT), a two-step process involving the irradiation of photosensitizer (PS)-loaded cancer cells. Upon tissue oxygen interaction, PS provokes immediate and direct Reactive Oxygen Species (ROS)-induced damage to Endoplasmic Reticulum (ER), mitochondria, plasma membrane, and/or lysosomes. The main biological effects carried out in cancer PDT are direct cytotoxicity to tumor cells, vasculature damage and induction of inflammatory reactions stimulating immunological responses. The question about the role of autophagy in PDT and its putative immunological impact is hotly controversial and largely studied in recent times. This review deals with the induction of autophagy in PDT protocols and its dual role, also considering its interrelationship with apoptosis, the preferential cell death program triggered in the photodynamic process.
... It should be noted that a mixture of differently sulfonated aluminum phthalocyanines termed Photosens has been developed as the drug for PDT in Russia and is used now in hospital [5]. The degree of sulfonation, isomeric composition and the nature of the central metal ion affect lipophilicity and aggregation of Pc dyes in water [6][7][8], while their photosensitizing effect is caused mainly by their photoactive monomer forms [9]. It was found that PDT efficiency of a series of sulfonated Pcʼs can be ordered as follows: AlPcS 2 ∼ ZnPcS 1 > AlPcS 1 > AlPcS 4 > ZnPcS 2 > ZnPcS 4 [10]. ...
The influence of sulfonation degree (n) and central atom nature on singlet oxygen quantum yield (ΦΔ) were studied for a series of sulfonated phthalocyanine metal complexes MPcSnmix. It was found that in DMSO, where the studied dyes exist as monomers, ΦΔ values are independent of number and position of the sulfogroups and are equal to 0.11 ± 0.02, 0.19 ± 0.03, 0.37 ± 0.05, 0.38 ± 0.05 and 0.68 ± 0.10 for the metal-free and Mg, Gd, Al and Zn complexes, correspondingly. However, in aqueous solutions, aggregation of the dyes determines their photochemical activity. Substitution in adjacent to macrocycle 3 and 6 benzene positions interferes with aggregation compared to 4 and 5 positions, presumably due to enhanced steric hindrances in the former case. In the ZnPcSnmix series (n varied from 2 to 4), as an example, the linear relationship between degree of aggregation and ΦΔ indicates that only the monomer fraction of the dye is responsible for singlet oxygen production. For less sulfonated samples the order of activity of the dyes in water is as follows: H2PcSnmix < MIIPcSnmix < MIIIPcSnmix which is in contrast to their tendencies to form aggregates.
... When the photosensitizer forms higher order aggregates or relocates in the cell over time, one cannot expect that their action spectra for photoinactivation of cells and sensitization of tumors parallel their monomeric absorption spectra. The influence of aggregated formations on the activation spectrum of sulfonated aluminum phthalocyanines is discussed by Moan et al. [11]. Excitation of the photosensitizer by low light levels is found to cause a redistribution of tetrasulfonated aluminum phthalocyanine (AlPcS 4 ) in the cell [12]. ...
Background and Objective
Variations in the optical coefficients in tissue and the photosensitizer during photodynamic therapy (PDT) will require adjustment of the light dose during the course of therapy. We have studied the dynamics using light transmission spectra for two different tumor models when tetrasulfonated aluminum phthalocyanine (AlPcS4) was used as photosensitizer.Study Design/Materials and Methods
Spectra were measured noninvasively in the EMT6/Ed murine tumor model, and with interstitially implanted source and probe fibers in the Dunning R3327-AT rat tumor model. Measurements were performed in the range 600–840 nm, using a tunable dye laser, a diode laser, and a Ti:Sapphire laser. AlPcS4 has absorption in the range 600–700 nm with an absorption peak at 670 nm in saline.ResultsThe in vivo spectrum of AlPcS4 both in the EMT6/Ed tumor model and the Dunning R3327-AT tumor model differs from the spectrum of AlPcS4 in saline. The absorption at 670 nm was reduced, whereas the absorption at 640 nm increased. Exposure of phototherapeutic levels of light caused reduced light absorption by the photosensitizer and further spectral shift.Conclusion
We found that the AlPcS4 absorption spectrum changes in a biological environment, and we also observed increased light transmission at the treatment wavelength during PDT in both tumor models. Instability in the absorption spectrum of the photosensitizer may influence the effectiveness of PDT. Lasers Surg. Med. 21:124–133, 1997. © 1997 Wiley-Liss, Inc.
... The local concentration of sensitizer in the highly fluorescent region shown in figure 4(a) must, therefore, be significantly greater than 1 mM if non-radiative quenching processes induce the observed quenching of the monomer fluorescence. It has been reported [40] that AlPcS2 can aggregate in V79-4 cells following 18 h incubation at 10 µM, although in this work a shorter incubation period was used. Nevertheless we presume that aggregated species are present under our conditions. ...
The potential for the application of fluorescence lifetime imaging (FLIM) microscopy to studies of photosensitization mechanisms in photodynamic therapy (PDT) has been investigated. The fluorescence microscope incorporates a standard inverted optical microscope, a picosecond pulsed dye-laser excitation source, and an intensified CCD camera detector capable of being gated on a sub-nanosecond timescale. Fluorescence lifetime images resulting from multi-component analysis of sub-nanosecond gated fluorescence images of monolayer V79-4 Chinese hamster lung fibroblasts stained with disulphonated aluminium phthalocyanine (AlPcS2), a photosensitizer used in PDT, are presented. The results of these measurements are discussed in terms of the intracellular localization of the sensitizer. Preliminary results from multi-component FLIM of V79-4 cells multiply stained with AlPcS2 and a potential intracellular pH lifetime probe, 5(+6)-carboxynaphthofluorescein, are also presented.
... The length and number of carbon side chains control the subcellular localization of porphyrins [33] and pyropheophorbides [29,34], and play an important role in the sequestration into intracellular vesicles [35], uptake mechanism [36] and PS' aggregation capacity [29]. In phthalocyanines, lipophilicity can be modulated by changing their degree of sulfonation [37,38], which induces changes not only in the intracellular localization but also in the specific organelle structure [39]. The effect of hydrophobicity on subcellular localization has also been recently reported in bacteriochlorins, accompanied with molecular charge dependency [40]. ...
Photodynamic therapy (PDT) is a promising modality for the treatment of tumours based on the combined action of a photosensitiser (PS), visible light and molecular oxygen, which generates a local oxidative damage that leads to cell death. The site where the primary photodynamic effect takes place depends on the subcellular localization of the PS and affects the mode of action and efficacy of PDT. It is therefore of prime interest to develop structure-subcellular localization prediction models for a PS from its molecular structure and physicochemical properties. Here we describe such a prediction method for the localization of macrocyclic PSs into cell organelles based on a wide set of physicochemical properties and processed through an artificial neural network (ANN). 128 2D-molecular descriptors related to lipophilicity/hydrophilicity, charge and structural features were calculated, then reduced to 76 by using Pearson's correlation coefficient, and finally to 5 using Guyon and Elisseeff's algorithm. The localization of 61 PSs was compiled from literature and distributed into 3 possible cell structures (mitochondria, lysosomes and "other organelles"). A non-linear ANN algorithm was used to process the information as a decision tree in order to solve PS-organelle assignment: first to identify PSs with mitochondrial and/or lysosomal localization from the rest, and to classify them in a second stage. This sequential ANN classification method has permitted to distinguish PSs located into two of the most important cell targets: lysosomes and mitochondria. The absence of false negatives in this assignation, combined with the rate of success in predicting PS localization in these organelles, permits the use of this ANN method to perform virtual screenings of drug candidates for PDT.
... This agrees with the findings of Moan et al., who mentioned that aluminum phthalocyanine tetrasulfonate exhibits high molar absorption at 672 nm, a wavelength that is not absorbed or dispersed by endogenous tissue components. 14 Cytotoxicity of AlS 4 Pc-Cl was studied within a range of concentrations and results showed that T 24 cells incubated for 24 hours with AlS 4 Pc-Cl ranging from 100 μM up to 500 μM had no significant effect on cell viability in non-irradiated cultures (Fig. 2). Therefore, sulfonated aluminum phthalocyanine has no dark toxicity. ...
Photodynamic therapy (PDT) has been the subject of several clinical studies. Evidence to date suggests that direct cell death may involve apoptosis. T(24) cells (bladder cancer cells, ATCC-Nr. HTB-4) were subjected to PDT with aluminum phthalocyanine tetrasulfonate chloride (AlS(4)Pc-Cl) and red laser light at 670 nm. Morphological changes after PDT were visualized under confocal microscopy. Raman microspectroscopy is considered as one of the newly established methods used for the detection of cytochrome c as an apoptotic marker. Results showed that PDT treated T(24) cells seem to undergo apoptosis after irradiation with 3 J cm(-2). Cytochrome c could not be detected from cells incubated with AlS(4)Pc-Cl using Raman spectroscopy whereas AlS(4)Pc-Cl seems to interfere with the Raman spectrum of cytochrome c.
... This requirement is difficult to achieve because the tumours are highly optically inhomogeneous, and temporal variations of optical properties may occur over the course of therapy. The absorption spectrum of the photosensitizer may change due to aggregation of the photosensitizer (1,2) or relocation in the cells (3), or due to photobleaching when exposed to therapeutic levels of light. The in vivo activation spectrum of the photosensitizer has been used to observe spectral changes in two previous studies (4,5). ...
Temporal and illumination-induced variations in the in vivo light transmission spectrum of the photosensitizer will influence light dosimetry for photodynamic therapy (PDT). The present authors have studied the in vivo spectra of four photosensitizers in the EMT6/Ed murine tumour model in Balb/c mice. The following photosensitizers were used: bis(dimethylthexylsiloxy)silicon 2,3-naphthalocyanine (SiNc 8), benzoporphyrin-derivative monoacid ring A (BPD Verteporfin), Photofrin and ethanolamined hypocrellin B (HBEA-R2). Spectra were measured non-invasively in the EMT6/Ed murine tumour model in the spectral range 600-840 nm, using a diode laser, a dye laser and a Ti:sapphire laser. Red-shift and broadening of the SiNc 8 absorption band was observed at 790 nm, and a slight red-shift was observed in the BPD, HBEA-R2 and Photofrin in vivo absorption spectrum. Exposure to 300 J of light at the peak absorption wavelength caused complete photobleaching of BPD at 690 nm, and a reduced absorption by SiNc 8 at 780 nm, Photofrin at 626 nm, and HBEA-R2 at 656 nm.
... When the photosensitizer forms higher order aggregates or relocates in the cell over time, one cannot expect that their action spectra for photoinactivation of cells and sensitization of tumors parallel their monomeric absorption spectra. The influence of aggregated formations on the activation spectrum of sulfonated aluminum phthalocyanines is discussed by Moan et al. [11]. Excitation of the photosensitizer by low light levels is found to cause a redistribution of tetrasulfonated aluminum phthalocyanine (AlPcS 4 ) in the cell [12]. ...
The goal of this study is to determine if flat cleaved fiber probes are appropriate for interstitial measurements of radiance in tissue. Flat cleaved probes have the advantage of high responsivity, and they are easy to insert into tissue. Owing to the non-isotropic response of flat cleaved probes, a calibration function is required, taking the anisotropy in the radiance in tissue into account.
The method used to determine this function consists of radiance measurements in tissue, performed with a flat cleaved fiber probe mounted on a stereotactic stage for insertion into the tissue from different directions. Interstitial irradiation at 630 nm was delivered by a spherical source.
We found that the degree of anisotropy in the radiance decreases with increasing distance from the interstitially implanted source in two different tissue phantoms and in the Dunning R3327-AT and R3327-H rat tumor models.
A position-dependent calibration function is required for interstitially implanted flat cleaved fiber probes. An anisotropy function is presented, which modifies the measurements of radiance with a flat cleaved probe, to account for the change in anisotropy in the radiance. The anisotropy functions for the two tumor models differ substantially.
... When the photosensitizer forms higher order aggregates or relocates in the cell over time, one cannot expect that their action spectra for photoinactivation of cells and sensitization of tumors parallel their monomeric absorption spectra. The influence of aggregated formations on the activation spectrum of sulfonated aluminum phthalocyanines is discussed by Moan et al. [11]. Excitation of the photosensitizer by low light levels is found to cause a redistribution of tetrasulfonated aluminum phthalocyanine (AlPcS 4 ) in the cell [12]. ...
Variations in the optical coefficients in tissue and the photosensitizer during photodynamic therapy (PDT) will require adjustment of the light dose during the course of therapy. We have studied the dynamics using light transmission spectra for two different tumor models when tetrasulfonated aluminum phthalocyanine (AlPcS4) was used as photosensitizer.
Spectra were measured noninvasively in the EMT6/Ed murine tumor model, and with interstitially implanted source and probe fibers in the Dunning R3327-AT rat tumor model. Measurements were performed in the range 600-840 nm, using a tunable dye laser, a diode laser, and a Ti:Sapphire laser. AlPcS4 has absorption in the range 600-700 nm with an absorption peak at 670 nm in saline.
The in vivo spectrum of AlPcS4 both in the EMT6/Ed tumor model and the Dunning R3327-AT tumor model differs from the spectrum of AlPcS4 in saline. The absorption at 670 nm was reduced, whereas the absorption at 640 nm increased. Exposure of phototherapeutic levels of light caused reduced light absorption by the photosensitizer and further spectral shift.
We found that the AIPcS4 absorption spectrum changes in a biological environment, and we also observed increased light transmission at the treatment wavelength during PDT in both tumor models. Instability in the absorption spectrum of the photosensitizer may influence the effectiveness of PDT.
... AlPcS4 was selected for its hydrophilic nature and because it features a high molar absorption coefficient at 672 nm, a wavelength of tissue penetrating light. 26 ...
Photodynamic therapy has attracted increasing interest over the last few years, whereby the activation of photosensitizers by light causes the production of reactive oxygen species (ROS), such as singlet oxygen, which are cytotoxic. The goal of our study was to enhance the photodynamic activity of the photosensitizer aluminum phthalocyanine tetrasulfonate (AlPcS4) through its specific delivery to tumor cells. Since many tumor cells, among which are HeLa cells, overexpress the transferrin receptor, we synthesized transferrin conjugated PEG-liposomes that contained AlPcS4 that could be internalized by receptor mediated endocytosis. The antiproliferative activity of the targeted liposomes was evaluated and compared to the native AlPcS4 and the non-targeted liposome. These findings were supplemented with data on intracellular concentration of the photo-active compounds. The accumulation together with ROS production after irradiation was visualized by using confocal microscopy to confirm the data found in the antiproliferative and accumulation assay. Tf-Lip-AlPcS4 was 10 times more photocytotoxic (IC(50), 0.63 microM) than free AlPcS4 at a light dose of 45 kJ/m whereas Lip-AlPcS4 displayed no photocytotoxicity at all. The high photocytotoxicity of Tf-Lip-AlPcS4 was shown to be the result of a high intracellular concentration (136.5 microM) in HeLa cells, which could be lowered dramatically by incubating the conjugate with a competing transferrin concentration. The images of intracellular accumulation and ROS production matched the accumulation and photocytotoxicity profile of the different photo-active compounds. The photodynamic activity of the Tf-Lip-AlPcS4 conjugate on HeLa cells is much more potent than free AlPcS4 as a result of selective transferrin receptor mediated uptake.
Photodynamic therapy (PDT) is a minimally invasive clinical protocol that combines a nontoxic photosensitizer (PS), appropriate visible light, and molecular oxygen for cancer treatment. This triad generates reactive oxygen species (ROS) in situ, leading to different cell death pathways and limiting the arrival of nutrients by irreversible destruction of the tumor vascular system. Despite the number of formulations and applications available, the advancement of therapy is hindered by some characteristics such as the hypoxic condition of solid tumors and the limited energy density (light fluence) that reaches the target. As a result, the use of PDT as a definitive monotherapy for cancer is generally restricted to pretumor lesions or neoplastic tissue of approximately 1 cm in size. To expand this limitation, researchers have synthesized functional nanoparticles (NPs) capable of carrying classical photosensitizers with self-supplying oxygen as well as targeting specific organelles such as mitochondria and lysosomes. This has improved outcomes in vitro and in vivo. This review highlights the basis of PDT, many of the most commonly used strategies of functionalization of smart NPs, and their potential to break the current limits of the classical protocol of PDT against cancer. The application and future perspectives of the multifunctional nanoparticles in PDT are also discussed in some detail.
Most nonviral gene transfection vectors deliver transfecting DNA into cells through the endocytic pathway (1,2). Poor escape from endocytic vesicles in many cases constitutes a major barrier for delivery of a functional gene, since the endocytosed transfecting DNA is unable to reach the cytosol and be further transported to the nucleus, but rather is trapped in endocytic vesicles and finally degraded in lysosomes (3). Therefore, the development of endosome-disruptive strategies is of great importance for the further progress of gene transfection. We have developed a new technology, termed photochemical internalization (PCI), to achieve light-inducible permeabilization of endocytic vesicles (4–8). The technology is based on photochemical reactions initiated by photosensitizers localized in endocytic vesicles and inducing rupture of these vesicles upon light exposure (4). This leads to the release of endocytosed macromolecules such as transfecting DNA from endocytic vesicles into the cytosol (Fig. 1). As a light-dependent treatment, PCI-mediated transfection (photochemical transfection) allows the possibility of directing the gene delivery to a desired site, e.g., achieving tumor-specific expression of a therapeutic gene in gene therapy in vivo. Fig. 1. Principle of photochemical internalization (PCI). I, endocytosis of the pho-tosensitizer (S) and the transfecting gene (G); II, localization of the photosensitizer and the transgene in the same endocytic vesicles; III, rupture of endosomal membrane upon light exposure and subsequent release of the transfecting gene into the cytosol.
Photodynamic therapy (PDT) is a clinical modality used to treat cancer and infectious diseases. The main agent is the photosensitizer (PS), which is excited by light and converted to a triplet excited state. This latter species leads to the formation of singlet oxygen and radicals that oxidize biomolecules. The main motivation for this review is to suggest alternatives for achieving high-efficiency PDT protocols, by taking advantage of knowledge on the chemical and biological processes taking place during and after photosensitization. We defend that in order to obtain specific mechanisms of cell death and maximize PDT efficiency, PSes should oxidize specific molecular targets. We consider the role of subcellular localization, how PS photochemistry and photophysics can change according to its nanoenvironment, and how can all these trigger specific cell death mechanisms. We propose that in order to develop PSes that will cause a breakthrough enhancement in the efficiency of PDT, researchers should first consider tissue and intracellular localization, instead of trying to maximize singlet oxygen quantum yields in in vitro tests. In addition to this, we also indicate many open questions and challenges remaining in this field, hoping to encourage future research.
Photochemical internalization (PCI) is a method for releasing macromolecules from endosomal and lysosomal compartments. The PCI approach uses a photosensitizer that localizes to endosomal and lysosomal compartments, and a light source with appropriate light spectra for excitation of the photosensitizer. Upon photosensitizer excitation, endosomal and lysosomal membranes are destroyed, due to the formation of reactive oxygen species, followed by release of the endocytosed material. PCI has been demonstrated to enhance and control (site- and time-specific) delivery of various macromolecules such as viruses, proteins, chemotherapeutics, nucleic acid, and so on. In this Review we present past and current studies of PCI-controlled delivery of natural and artificial nucleic acids, such as peptide nucleic acids, siRNA molecules, mRNA molecules and plasmids. We also discuss critical aspects to further the possibilities for successful gene targeting in space and time.
Photodynamic therapy (PDT) is a clinical treatment utilized in the therapy of cancer and
others diseases. In this therapy, ligth-sensitive-molecules, called photosensitizers, are
activated by visible ligth generating reactive species of oxygen. These reactive forms
initiate a sequence of oxidative events resulting in tumor cell death by apoptosis and/or
necrosis. The purpose of this article is to present the basic principles of PDT as well as
its potential of application on this modality of medical therapy.
A water-soluble phthalocyanine derivative with 16 poly-oxyethylene side chains which effectively prevent aggregation is reported. The first demonstration of luminescence in a water-soluble, non-ionic phthalocyanine in aqueous solution is discussed an important results from the development of effective manomeric dyes for use in phototherapy.
Confocal fluorescence microscopy, using a newly constructed laser line-scanning confocal microscope, was applied to an investigation of the early stages of photoinduced destruction of V79–4Chinese hamster fibroblasts using aluminum and zinc phthalocyanines as photosensitize. Results obtained in this work show that aluminum and zinc phthalocyanines, once internalized, localize in perinuclear sites that are disrupted upon light exposure resulting in fluorescence redistribution. The combination of laser-line scanning with charge-coupled device detection used in the confocal microscope developed in this work can enable rapid high-resolution sequential imaging, which is ideal for studying photoinduced intracellular fluorescence dynamics.
Steady state and time resolved confocal fluorescence microscopy, using a point scanning system, is applied to an investigation of the early stages of photo-induced changes in 3T3-L1 murine fibroblasts using di-sulphonated aluminum phthalocyanine (AlPcS2) as a photosensitizer. A comparison is made with data obtained using a line scan system and V79-4 Chinese hamster fibroblasts. The steady state data obtained in this work demonstrate that intracellular AlPcS2 fluorescence intensity increases progressively on photoirradiation. Time-resolved studies indicate that this could result from a progressive decrease in the concentration of the self-quenched membrane-associated form of AlPcS2 following its conversion into the fluorescent monomeric form.
The effect of photodynamic treatment on the yeast Kluyveromyces marxianus with aluminum-phthalocyanines has been studied. It was found that the nonsulfonated sensitizer caused light-dependent loss of colony-forming capacity, whereas the mono- and tetrasulfonated forms did not induce loss of clonogenicity. The effect of the nonsulfonated sensitizer increased with longer preincubation periods of cells with the dye. Formation of cellattached, mostly intracellularly localized monomelic sensitizer also increased with time. The amount of cell-bound multimeric nonsulfonated phthalocyanine did not vary with time. Experiments designed to specifically increase the amount of cell-attached monomers led also to an increased photoinactivation of the cells. It is therefore concluded that the photodynamic effect of the nonsulfonated Al-phthalocyanine is mediated by the monomeric form of the dye.
The results of a study of the effect of pH on the photophysics and photochemistry of di-sulphonated aluminum phthalocyanine (AlPcS2) in aqueous solution are presented. The pH dependence of the triplet quantum yield, fluorescence quantum yield, singlet-oxygen quantum yield, triplet lifetime, fluorescence lifetime and apparent dimerization constants is investigated and the results interpreted in terms of the pH dependence of the nature of the axial ligands. Evidence that the aluminum–axial ligand bond strength, rather than dimer binding energy that determines the extent of dimerization is provided by semi-empirical and ab initio calculations. Possible dimer structures obtained using ab initio calculations are discussed.
Purpose:
Pc4 is a silicone phthalocyanine photosensitizing agent that is entering clinical trials. Studies were undertaken in mice to develop a suitable formulation and analytical methodology for use in pharmacokinetic studies and to define the plasma pharmacokinetics, tissue distribution, and urinary excretion of Pc4 after i.v. delivery.
Methods:
An HPLC method suitable for separation and quantification of Pc4 was developed and validated for use in mouse plasma, tissues, and urine. The stability of Pc4 was characterized in a variety of formulations as well as in mouse plasma. Before pursuing pharmacokinetic studies, preliminary toxicity studies were undertaken. These studies utilized Pc4 formulated in diluent 12:0. 154 M NaCl (1:3, v:v). Pharmacokinetic studies involved Pc4 doses of 40 mg/kg, 10 mg/kg and 2 mg/kg administered as i.v. boluses to female, CD2F1 mice. Doses of 40 mg/kg, 10 mg/kg, and 2 mg/kg were studied with drug formulated in diluent 12:0.154 M NaCl (1:3, v:v). Doses of 10 mg/kg and 2 mg/kg were also studied with drug formulated in a vehicle consisting of polyethylene glycol:Tween 80:0. 01 M sodium phosphate buffer, pH 7.0 (40:0.2:59.8, v:v:v). Compartmental and non-compartmental analyses were applied to the plasma concentration-versus-time data. Concentrations of Pc4 were also determined in a variety of tissues, including brain, lung, liver, kidney, skeletal muscle, skin, heart, spleen, and abdominal fat. Urine was collected from animals treated with each of the doses of Pc4 mentioned above, and daily, as well as cumulative drug excretion was calculated until 168 h after treatment.
Results:
At a dose of 80 mg/kg, two of five male and two of five female mice were dead by 24 h after injection. Pathologic examination revealed gross findings of blue discoloration affecting many tissues, with lungs that were grossly hemorrhagic and very blue-black. Microscopic examination of the lungs revealed mild acute interstitial pneumonia, with perivascular edema and inflammation, and a detectable margination of neutrophils around larger pulmonary blood vessels. Animals sacrificed 14 days after treatment showed mild granulomatous pneumonia, characterized by clusters of multi-nucleated giant cells, with fewer macrophages and neutrophils. The giant cells frequently contained phagocytized particles, which were clear and relatively fusiform. All mice treated with 40 mg/kg or 20 mg/kg survived and returned to pretreatment weight during the 14 days after treatment. Intravenous bolus delivery of Pc4, at a dose of 40 mg/kg, produced "peak" plasma Pc4 concentrations between 7.81 and 8.92 microg/ml in mice killed at 5 min after injection (the earliest time studied after drug delivery). Sequential reduction of the Pc4 dose to 10 mg/kg in diluent 12:0.154 M NaCl (1:3, v:v), 10 mg/kg in polyethylene glycol:Tween 80:sodium phosphate buffer (40:0.2:59.8, v:v:v), 2 mg/kg in diluent 12:0.154 M NaCl (1:3, v:v), and, finally, 2 mg/kg in polyethylene glycol:Tween 80:sodium phosphate buffer (40:0.2:59.8, v:v:v) resulted in "peak" plasma Pc4 concentrations between 2.07 and 3.24, 0.68 and 0.98 microg/ml, and 0.29 and 0.41 microg/ml, respectively. Pc4 persisted in plasma for prolonged periods of time (72-168 h). Non-compartmental analysis of plasma Pc4 concentration-versus-time data showed an increase in area under the plasma Pc4 concentration-versus-time curve (AUC) when the dose of Pc4 increased from 2 mg/kg to 40 mg/kg. Across the 20-fold range of doses studied, total body clearance (CL(tb)) varied from 376 to 1106 ml h(-1) kg(-1). Compartmental modeling of plasma Pc4 concentration versus time data showed the data to be fit best by a two-compartment, open, linear model. Minimal amounts of Pc4 were detected in the urine of mice. After i.v. bolus delivery to mice, Pc4 distributed rapidly to all tissues and persisted in most tissues for the duration of each pharmacokinetic study. Tissue exposure, as measured by AUC, increased in a dose-dependent fash
The photosensitizing effects of di-sulphonated aluminium phthalocyanine (AlS2Pc) on proliferation of normal human epidermal keratinocytes of the established line UP were studied in cultures in 96-well
microtitre plates using the MTT-microculture tetrazolium assay as a method of assessing viable cell number. It was found that
the cytotoxic effects of AlS2Pc were dose-dependent. A cooled slow-scan CCD (charge-coupled device) imaging system with computerized image processing was
used for fluorescence measurements in cell cultures after administration of 25 μg ml−1 AlS2Pc. Fluorescence was at a peak at 24 h and this time was therefore considered to be the most appropriate for light sensitization.
Treatment with 25 μg ml−1 AlS2Pc for 24 h followed by exposure to total red light (660–700 nm) energy dose of 0.6 J cm−2 reduced cell survival by 100%. It seems that the photokilling capacity of AlS2Pc on keratinocytes is very effective and this may make it particularly suitable for the treatment of surface epithelial tumours.
Tetra(4-sulfonatophenyl)porphine (TPPS4) sensitizes cells to photoinactivation mainly formation of singlet oxygen. In human cervix carcinoma cells of the line NHIK 3025 TPPS4 localizes to a large extent in lysosomes as previously shown by fluorescence microscopical and spectroscopical techniques. In the present study photodamage to lysosomes was investigated. This was accomplished by measuring the activity of the lysosomal marker enzyme β-acetyl-D-glucosaminidase (β-AGA) after photochemical treatment (PCT). β-AGA activity was highly sensitive to light exposure in the presence of TPPS4. The enzymatic activity was reduced by ∼ 70% by non-lethal doses of photochemical treatment, indicating that inactivation of lysosomal hydrolases is not likely to contribute significantly to the cytotoxic effects of PCT. Centrifugation studies showed that TPPS4, but not β-AGA activity, was released from lysosomes after light exposure, 20–30% of the total β-AGA activity was resistant to the photochemical treatment. This was due to β-AGA activity in Golgi-derived vesicles (4–5%) and in vesicles with similar density as lysosomes but not containing TPPS4. The present results indicate that lysosomal hydrolases are inactivated by photochemical treatment before they eventually escape the lysosomal compartment.
We study the porphyrin S1→S0 fluorescence and the photosensitized singlet oxygen phosphorescence, both originating from absorption of photons with energy less than the porphyrin S0→S1 transition energy. By measuring the excitation intensity dependence of fluorescence at lowered sample temperatures, we are able to discriminate between two parallel processes of one-photon hot-band absorption (HBA) and simultaneous two-photon absorption (TPA). When the HBA and TPA contributions are comparable in magnitude, we use this new method to determine absolute TPA cross-section. We also demonstrate for the first time a singlet oxygen photosensitization via HBA in porphyrin.
A number of potentially therapeutical drugs, which are membrane impermeant, are captured and degraded in the lysosomes after cellular uptake, and are thereby prevented from executing their biological function. However, in the present thesis a technology is presented that makes it possible to overcome this substantial barrier for the cellular delivery of functional macromolecules. The technology, photochemical internalisation (PCI), is a unique procedure for light-induced release of several types of membrane impermeant molecules from endocytic vesicles to the cytosol of the target cells. The PCI technology can be utilised as a new efficient and site-specific method for drug delivery in the therapy of cancer and other diseases, and it can also be applied as research tool for macromolecule delivery both in vitro and in vivo.
A major barrier within the field of non-viral gene therapy toward therapeutic strategies, e.g., tumor therapy, has been lack of appropriate specific delivery strategies to the intended target tissues or cells. In this chapter, we describe a protocol for light-directed delivery of nucleic acids through the use of photochemical internalization (PCI) technology. PCI is based on a photosensitizing compound that localizes to endocytic membranes. Upon illumination, the photosensitizing compound induces damage to the endocytic membranes, resulting in release of endocytosed material, i.e., nucleic acids into cytosol. The main benefit of the strategy described is the possibility for site-specific delivery of nucleic acids to a place of interest.
Photochemical internalization (PCI) is under development for clinical use in treatment of soft tissue sarcomas and other solid tumors. PCI may release endocytosed bleomycin (BLM) into the cytosol by photochemical rupture of the endocytic vesicles. In this study, the human fibrosarcoma xenograft HT1080 was transplanted into the leg muscle of athymic mice. The photosensitizer disulfonated aluminum phthalocyanine (AlPcS(2a)) and BLM were systemically administrated 48 h and 30 min, respectively, prior to light exposure at 670 nm (30 J cm(-2)). The purposes of this study were to evaluate the treatment response to AlPcS(2a)-photodynamic therapy (PDT) and AlPcS(2a)-PDT in combination with BLM (i.e. PCI of BLM) in an orthotopic, invasive and clinically relevant tumor model and to explore the underlying response mechanisms caused by PDT and PCI of BLM. The treatment response was evaluated by measuring tumor growth, contrast-enhanced magnetic resonance imaging (CE-MRI), histology and fluorescence microscopy. The results show that PCI of BLM is superior to PDT in inducing tumor growth retardation and acts synergistically as compared to the individual treatment modalities. The CE-MRI analyses 2 h after AlPcS(2a)-PDT and PCI of BLM identified a treatment-induced nonperfused central zone of the tumor and a well-perfused peripheral zone. While there were no differences in the vascular response between PDT and PCI, the histological analyses showed that PDT caused necrosis in the tumor center and viable tumor cells were found in the tumor periphery. PCI caused larger necrotic areas and the regrowth in the peripheral zone was almost completely inhibited after PCI. The results indicate that PDT is less efficient in the tumor periphery than in the tumor center and that the treatment effect of PCI is superior to PDT in the tumor periphery.
The photodynamic therapy (PDT) activity of zinc phthalocyanine tetrasulphonic acid in a rodent tumour model was shown to be critically dependent on the wavelength of the excitation laser light over a relatively small wavelength range. Thus the sensitizer showed a doubling of the PDT activity with fibrosarcoma LSBD1 in BDIX rats when the wavelength of the illuminant was displaced from 680 to 692 nm. Under these conditions, the sensitizer is approximately three times more effective than polyhaematoporphyrin, whereas previously it has been considered to be of low PDT activity. This wavelength effect is attributed to a red shift of the absorption spectrum of the sensitizer in cells compared with that in solution. Fluorescence excitation studies with sensitizer absorbed in mouse 3T3 fibroblast cells are consistent with such a red shift.
V79 cells incubated with di- or tetrasulfonated aluminium phthalocyanines (AlPcS2 or AlPcS4) showed a granular fluorescence pattern. Co-staining with the lysosomotropic dye acridine orange (AO) indicated that the granules that were stained by these photoactive phthalocyanines were identical to lysosomes. Small light exposures made the lysosomes permeable to the dyes without inactivating the cells. Also, the lysosomal enzymes beta-AGA and cathepsin (L+B) were inactivated by small light exposures when AlPcS4 was present. Such small and almost nontoxic light exposures caused a redistribution of the dyes in the cells that was accompanied by a more than 10-fold increase in the fluorescence quantum yields of the dyes. Surprisingly, this redistribution and increase in fluorescence did not result in any significant increase in the photosensitivity of the cells.
Spectroscopic studies were carried out on the photosensitizer disulphonated aluminium phthalocyanine (AlS2Pc) which has prospective applications in photodynamic therapy. The fluorescence lifetimes of AlS2Pc were measured in a range of model systems and cultured leukaemic cells using laser excitation and time-correlated, single-photon-counting detection. In an investigation of non-covalent protein binding, we studied AlS2Pc in the presence of human serum albumin (HSA) in 0.1 M phosphate-buffered saline at pH 7.4. On addition of excess concentrations of HSA, small red shifts in the fluorescence and absorbance spectra were observed, together with an increase in fluorescence polarization anisotropy, consistent with binding of the phthalocyanine. Fluorescence decays could be resolved into two lifetimes for bound AlS2Pc with a dominant component of 5.5 ns and a minor component of 1 ns. Fluorescence imaging and time-resolved microfluorometry were carried out on intracellular AlS2Pc using leukaemic K562 cells. Microscopic imaging with a charge-coupled device (CCD) camera revealed that AlS2Pc fluorescence predominated in a discrete perinuclear region which was then probed selectively by a focused laser spot for fluorescence lifetime measurements. Bi-exponential decays with lifetime components of 6.1 and 2.2 ns were observed. On irradiation at 633 nm, the fluorescence intensity increased initially and subsequently declined due to photodegradation.
The uptake and biological activity of porphyrins and phthalocyanines in tumours were correlated with the geometrical features of the photosensitizer molecules. The data suggest that a critical distance of approximately 1.2 nm between oxygen atoms (originating in SO3-, COO- or OH substituents) characterizes a biologically active photosensitizer for photodynamic therapy. We propose that tubulin, which is available in large amounts during mitosis, is the main receptor molecule which binds these photosensitizers. Basic amino acid residues or tightly bound cations in tubulin or homologous proteins may act as binding sites on the receptor molecule.
The light-activated drugs AlPcS4 and T4MPyP were studied in a pancreatic carcinoma cell line for their effects on DNA integrity, cell division, proliferation, and survival. The micronucleus assay measured nuclear changes and also the number of actively dividing cells while, under similar conditions, the MTT assay measured cell survival. When tumour cells were exposed to light, pre-treatment with AlPcS4 induced more micronuclei than did T4MPyP at the same levels of cell division and survival. Both drugs showed a correlation between phototoxicity and changes to DNA integrity so establishing micronuclei formation as an important indicator of photodynamic drug action on tumour cells.
Radioiodinated zinc phthalocyanine including [125I]ZnPcI4 and differently sulfonated [65Zn]ZnPcS (ZnPcS4, ZnPcS3, ZnPcS2 and ZnPcS1.75, a mixture of adjacent di and 25% mono) were prepared in order to study cell uptake and release kinetics in EMT-6 cells. The same compounds were evaluated for their in vitro phototoxicity and the biological parameters were compared to partition coefficients to arrive at quantitative structure-activity relationships (QSAR). At 1 microM in 1% serum, at 37 degrees C, all dyes showed rapid cell uptake during the first hour followed by a slow accumulation phase. After 24 h, the highest cellular concentration was observed with the lipophilic ZnPcI4, followed by the amphiphilic ZnPcS2 and ZnPcS1.75. The hydrophilic ZnPcS4 and ZnPcS3 showed lower uptake. Dye release from dye-loaded cells during incubation in dye-free medium could reach up to 60% and was shown to depend mainly on the amount of drug incorporated rather than the type of compound. These results suggest that care should be taken in interpreting dye toxicity data, which involve in vitro cell manipulations in dye-free medium, particularly during in vitro-in vivo protocols. The EMT-6 cell survival after 1 h or 24 h incubation with 1 microM dye in 1% serum followed by exposure to red light was assessed by means of the colorimetric 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide (MTT) assay. Photocytotoxicities correlated inversely with the tendencies of the dyes to aggregate. Increased dye uptake by the cells also correlated with their activities, except for the lipophilic ZnPcI4, which showed the highest cell uptake but little phototoxicity. The QSAR between phototoxicity and the log of the partition coefficients (phosphate-buffered saline and n-octanol) gave a parabola with optimal partition values corresponding to the adjacent sulfonated ZnPcS2.
The absorption spectrum of aluminum phthalocyanine with an average disulphonation of 2.1 (hereafter called disulphonated aluminum phthalocyanine, A1S2Pc) was measured in vivo in a murine tumour model by means of time-resolved reflectance. Mice bearing the L1210 leukaemia were administered 2.5 or 5 mg/kg body weight (b.w.) of A1S2Pc intraperitoneally. Reflectance measurements were performed in the 650-695 nm range before and 1, 4 and 7 h after the drug administration. Fitting of the data with the diffusion theory allowed us to assess the absorption coefficient in both conditions (i.e. before and after). As a difference between the latter and the former data, the in vivo absorption spectrum of A1S2Pc was evaluated. 1 h after the administration of 2.5 mg/kg b.w. A1S2Pc, the absorption peak was centred at 685 nm, red-shifted about 15 nm with respect to the spectrum in aqueous solution. For the lower dose, the absorption line shapes 4 and 7 h after the administration remained very similar. The red shift of the absorption spectrum is consistent with the therapeutic efficacy of the photodynamic therapy which was measured at 672, 685 and 695 nm, and proved to be maximum at 685 nm for both the L1210 leukaemia and the MS-2 fibrosarcoma. With the higher drug dose, the absorption spectra taken from different animals showed significant differences. In particular, in some mice the line shape was similar to that measured with 2.5 mg/kg b.w., while in other subjects it showed a broadening or a second peak at shorter wavelengths. Measurements on some animals were performed also 18 and 24 h after the injection of 5 mg/kg b.w., leading to no time evolution or to a progressive line shape narrowing.
Confocal fluorescence microscopy, using a newly constructed laser line-scanning confocal microscope, was applied to an investigation of the early stages of photoinduced destruction of V79-4 Chinese hamster fibroblasts using aluminum and zinc phthalocyanines as photosensitizers. Results obtained in this work show that aluminum and zinc phthalocyanines, once internalized, localize in perinuclear sites that are disrupted upon light exposure resulting in fluorescence redistribution. The combination of laser-line scanning with charge-coupled device detection used in the confocal microscope developed in this work can enable rapid high-resolution sequential imaging, which is ideal for studying photoinduced intracellular fluorescence dynamics.
Phthalocyanines are useful sensitizers for photodynamic sterilization of red cell concentrates. Various lipid-enveloped viruses can be inactivated with only limited red cell damage. Because white cells are involved in the immunomodulatory effects of blood transfusions, the study of the effect of photodynamic treatment on these cells is imperative.
White cell-enriched red cell suspensions were photodynamically treated with either the hydrophobic Pc4 (HOSiPcOSi-(CH3)2(CH2)3N(CH3)2) or water-soluble aluminum phthalocyanine tetrasulfonate (AIPCS4) under virucidal conditions. Viability of white cell subpopulations on Days 0, 1, and 4 after treatment was determined by fluorescence-activated cell sorting by flow cytometric analysis of propidium iodide uptake. Apoptosis induction was studied by DNA ladder formation and staining for an early marker of apoptosis (annexin V).
Treatment with Pc4 causes a significant decrease in cell viability of all white cells, as shown by prodidium iodide uptake. Monocytes and granulocytes are the most sensitive, and lymphocytes are relatively more resistant. Some of the cells die by apoptosis, which is induced within 30 minutes after treatment. Treatment with AIPCS4 damages only monocytes; other cell populations are not affected.
Physicochemical properties of the photosensitizers partly determine their effect on white cells. Differences in intracellular localization are likely to be responsible for the effects observed.
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.
Most synthetic gene delivery vectors are taken up in the cell by endocytosis, and inefficient escape of the transgene from endocytic vesicles often is a major barrier for gene transfer by such vectors. To improve endosomal release we have developed a new technology, named photochemical internalization (PCI). PCI is based on photochemical reactions initiated by photosensitizing compounds localized in endocytic vesicles, inducing rupture of these vesicles upon light exposure. PCI constitutes an efficient light-inducible gene transfer method in vivo, which potentially can be developed into a site-specific method for gene delivery in in vivo gene therapy. In this paper the principle behind the PCI technology and the effect of PCI on transfection with different synthetic gene delivery vectors are reviewed. PCI treatment by the photosensitizer aluminum phthalocyanine (AlPcS2a
) strongly improves transfection mediated by cationic polymers (e.g., poly-L-lysine and polyethylenimine), while the effect on transfection with cationic lipids is more variable. The timing of the light treatment relative to the transfection period was also important, indicating that release of the DNA from early endosomes is important for the outcome of PCI-induced transfection. The possibilities of using PCI as a technology for efficient, site-specific gene delivery in in vivo gene therapy is discussed.
Aluminum sulfonated phthalocyanine has potential as a suitable photosensitizer for use in the photodynamic therapy of cancer. In the present study, cellular uptake and retention of the individual mono-, di-, tri-, and tetrasulfonated derivatives (AlS1-4Pc) were examined in tissue culture and in normal and neoplastic tissue of tumor-bearing mice. Uptake and retention of the various derivatives by cells in tissue culture correlated inversely with the degree of sulfonation. Accordingly, Colo 26 cells in monolayer culture, 24 h after addition of 10 microM of appropriate photosensitizer, had accumulated approximately 25-fold more AlS1Pc than AlS3Pc and retained this species longer than more sulfonated derivatives. In contrast to these in vitro results, it was found that Colo 26 growing s.c. in BALB/c mice accumulated photosensitizer to a greater extent when the degree of sulfonation increased, such that A1S4Pc greater than AlS3Pc greater than AlS2Pc greater than AlS1Pc. By 24-48 h after the i.v. injection of 0.1 ml 2.27 mM solution of individual photosensitizer, the relative ratios of tumor:adjacent tissue varied from greater than 10:1 to greater than 2:1, showing that selective tumor uptake may be affected profoundly by the composition of the phthalocyanine compound. The livers and spleens of both normal and tumor-bearing mice, unlike other normal tissue, took up the sulfonated derivatives in an order that provided a mirror image of that observed in neoplastic tissue. These complex in vivo distribution and retention characteristics appear to be a consequence of relative hydrophilicity/hydrophobicity properties of the sulfonated species and indicate the extent to which these characteristics may influence photosensitizer distribution and accumulation.
Recently, a number of new photosensitizers have been proposed for use in photodynamic cancer therapy (PDT). Three of the most promising ones of these sensitizers are tetra(3-hydroxyphenyl)porphyrin (3THPP), chlorin e6(Chl e6) and aluminium phthalocyanine tetrasulphonate (AlPCTS). 3THPP was recently shown to be a better tumorlocalizer and a more potent and selective tumor photosensitizer than the until now most widely used drugs HpD and DHE (Berenbaum et al., 1986; Peng et al., 1987; Moan et al., 1987). Chl e6has a similar structure as porphyrins and a significantly stronger absorbance of red light. AlPCTS and other phthalocyanines also have high absorbance of red light, are efficient photosensitizers and have been launched as future PDT sensitizers (see review by Spikes, 1986). In order to choose the most optimal wavelength for therapy it is necessary to know the action spectrum for cell inactivation. All these dyes tend to aggregate in aqueous media and one can therefore not expect that their action spectra for photoinactivation of cells and sensitization of tumors parallel their absorption spectra.
Tetrasulphophthalocyanine and the ZnII and CuII complexes have intense absorption in the near i.r. region but the compounds aggregate in aqueous solution. Aggregation is hindered by addition of organic solvents, such as pyridine, and here H2PcS4– and ZnPcS4– show strong fluorescence. Photophysical data have been collected for both compounds and for the CuII complex and the observed values related to the ideal properties of a chromophore for a three-component [chromophore/donor/methyl viologen (MV2+)] H2-producing system. The ZnII complex appears as the most attractive chromophore but irradiation in the presence of EDTA, MV2+ and colloidal Pt gives no H2 production due to efficient reverse electron-transfer. The yield of H2 is increased in the absence of MV2+ but the sulphonated phthalocyanines compare unfavourably with metalloporphyrins.
Abstract The 83 μM hematoporphyrin (HP)-sensitized photooxidation of 0.1 mM tryptophan in aqueous solution buffered at pH 7.4 or in binary mixtures of phosphate buffer and organic solvents of higher (formamide) or lower (N,N-dimethylformamide, methanol, ethanol, tetrahydrofuran) polarity proceeds by a pure singlet oxygen (1O2) mechanism as suggested by azide quenching experiments, the rate-enhancing action of deuterated solvents, and the lack of any significant reaction between triplet HP and tryptophan. Both the first-order rate constant of the photoprocess and the photooxidation quantum yield (φ= 0.011 in phosphate buffer at pH 7.4) increase when the medium polarity is increased (e.g. φ= 0.024 in 90% formamide); this results mainly from the greater quantum yield of 1O2 generation and the longer lifetime of 1O2. The intrinsic reactivity of 1O2 with tryptophan is independent of formamide concentration. A moderate decrease in the medium polarity (e.g. in the range 0-30% methanol) enhances the efficiency of tryptophan photooxidation (φ= 0.014 in 30% methanol) as a result of the enhanced quantum yields of triplet HP and 1O2 formation. In contrast, the overall photooxidation rate is depressed at high concentrations of low-polarity organic solvents (e.g. φ= 0.0039 in 90% methanol) due to a 5.5-fold drop of the rate constant for the 1O2-tryptophan reaction which counteracts the enhancement of the lifetime and quantum yield of triplet HP and 1O2. The solvent composition also affects the equilibria between monomeric and multimeric forms of HP. However, under our experimental conditions, the aggregation state of HP appears to exert only a minor influence on the efficiency of tryptophan photooxidation.
Abstract —Human cells of the line NHIK 3025 were labelled with hematoporphyrin derivative (Hpd) or Photofrin II (PII). Absorption and fluorescence excitation spectra of cell-bound porphyrins were recorded. Furthermore, the corresponding action spectra for photoinactivation of the cells were determined in the spectral region 350–650 nm. The cell-bound porphyrins were also analysed by means of HPLC. The following conclusions are drawn:
(a) Porphyrins with low polarity are selectively bound to the cells.
(b) Aggregated as well as unaggregated porphyrins are bound to the cells.
(c) The action spectra indicate that the aggregated porphyrins do not contribute significantly to the photosensitivity of the cells.
(d) The action spectra closely resemble the fluorescence excitation spectra of porphyrins bound to proteins.
The additional optical absorption in tissue resulting from the uptake of exogenous photosensitizers increases the effective attenuation of photoactivating light. This may be significant for the irradiation of solid tumours in photodynamic therapy, since it reduces the depth or volume of tissue treated. The effect has been studied in vitro by using dihaematoporphyrin ether (DHE) and 630 nm light in tissues representing a wide range of absorption and scattering conditions. While the attenuation may be markedly changed by small concentrations of DHE in pure scattering media, tissues with significant inherent light absorption are little affected by the additional absorption of DHE at concentrations relevant to clinical photodynamic therapy. However, it is shown that for other potential photosensitizers such as the phthalocyanines, which have substantially greater absorption at the treatment wavelength than DHE, the penetration of light in tissues may be significantly reduced.
The intracellular localization of meso-tetraphenylporphines sulfonated to different degrees (TPPSn), in a human cervix carcinoma cell line (NHIK 3025), was studied by fluorescence microscopy and fluorescence spectroscopy. After an 18 h incubation, TPPS4, TPPS2a and TPPS2o were localized in extranuclear granules. Studies of cells stained with both TPPS4, and acridine orange, which is known to fluoresce red in lysosomes, indicated that these granules were lysosomes. In addition, a fraction of the cellbound TPPS4, TPPS2a and TPPS2o seems to be associated with the plasma membrane. Fluorescence quenching studies of cells doublestained with acridine orange and TPPS4 indicated that TPPS4 is also localized in the nucleus and in the extralysosomal cytoplasm. The intracellular location of TPPS1 differed from that of the other TPPSns studied: In 6 out of 9 experiments fluorescing extranuclear granules were found. A diffuse fluorescence extending from the perinuclear area was also observed.
Photodynamic therapy (PDT) is the treatment of malignant lesions with visible light following the systemic administration of a tumor-localizing photosensitizer. Pharmacological and photochemical properties of the photosensitizer are combined with precise delivery of laser-generated light to produce a treatment which can offer selective tumoricidal action. Hematoporphyrin derivative (HD) and a purified component called Photofrin II are currently being used in clinical PDT. Initial patient results have been encouraging, and considerable interest has developed in the synthesis and evaluation of new photosensitizers with improved photochemical and pharmacological characteristics. In addition, there has been a gradual increase in knowledge related to in vitro and in vivo mechanisms of action of PDT. This report provides an overview of the properties and applications of PDT. Information and data related to drug development, photochemistry, subcellular targets, in vivo responses, and clinical trials of PDT are presented.
Cellular uptake of aluminum phthalocyanine sulfonated to different degree was studied by means of fluorescence measurements and HPLC chromatography. These results were correlated to the lipophilic property of each drug measured as the distribution of the drug between a lipophilic phase (Triton X-114) and an aqueous phase. All the sulfonated aluminum phthalocyanines were taken up into cells to a higher extent than porphyrins of a similar lipophilicity. The cellular uptake of monosulfonated aluminum phthalocyanine was 10-fold higher than the cellular uptake of tetrasulfonated aluminum phthalocyanine and at least 50% higher than tetra(3-hydroxy-phenyl)porphin which is so far the porphyrin shown to be taken up into cells to the highest extent.
The cellular photosensitivity caused by aluminum phthalocyanines sulfonated to different degrees (AlPcSn) has been investigated. The phototoxic effect increased with decreasing number of sulfonate groups on the macrocycle, with the exception of AlPcS1 which was less phototoxic than AlPcS2 but more phototoxic than AlPcS3 and AlPcS4. The tendency of the AlPcSns to aggregate in our cellular system increased with increasing lipophilicity of the sensitizers. The aggregates had little or no photosensitizing activity. The low efficiency of cell inactivation caused by AlPcS1 can be explained by the highly aggregated state of this sensitizer in the cells. AlPcS2 and AlPcS3 induced a lower degree of cell inactivation per fluorescing quantum and per quantum absorbed by monomeric species than did AlPcS2 and AlPcS1. AlPcS4 and AlPcS3 are therefore suggested to be in different intracellular locations than AlPcS2 and AlPcS1.
Several parameters of the following dyes, all relevant as sensitizers for photochemotherapy of cancer, have been studied: Photofrin II (PII), hematoporphyrin (HP)-di-hexyl-ether, HP-di-ethyl-ether, tetra (3-hydroxyphenyl) porphyrin, (3THPP), tetraphenyl porphine tetrasulphonate (TPPS4) aluminium phthalocyanine tetrasulfonate (A1PCTS), aluminium phthalocyanine (A1PC), chlorin e, (Chi e6) and merocyanine 540 (MC 540). The following parameters and features of these dyes were studied: (1) Tumor uptake in C3H mouse mammary carcinomas. (2) Skin/tumor concentration ratio in the same animal system. (3) Triton X-114/H20 partition coefficients at different pH-values. (4) Uptake of the dyes by human cells of the line NHIK 3025. (5) Relative fluorescence quantum yields of the dyes bound to cells. (6) Absorption-, fluorescence-excitation- and fluorescence-emission spectra of the cell-bound dyes. (7) Relative quantum yields for photoinactivation of cells after 18 h incubation with the dyes. (8) Relative quantum yields of photodegradation of the singlet oxygen trap 1,3-diphenylisobenzofuran (DPBF) in cells after 18 h incubation with the dyes.
Chloroaluminum phthalocyanine (CAPC) was recently shown to sensitize the inactivation of cultured Chinese hamster cells by visible light. Several factors affecting the photodynamic action of CAPC have been defined in the present study. Thus the photosensitized inactivation of Chinese hamster cells is not affected by superoxide dismutase, suggesting that O-2 radicals are not involved in the process. Postillumination treatments with D2O or heat (42 degrees C, 90 min) enhanced CAPC-induced photosensitivity, indicating the existence of a repair mechanism for photodamage. Preillumination treatments with sodium salicylate and 5-bromodeoxyuridine also enhanced photosensitivity. The later observation suggests that CAPC-induced DNA damage is potentially lethal. However, 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) synthesis which is involved in repair of DNA strand breakage, had no effect on the photosensitivity. Photosensitized inactivation by CAPC is dependent on the pH value of the medium during irradiation. Thus, in the range of pH values 6-8, the sensitivity was increased at the lower values.
The porphyrin content of cells labelled with hematoporhyrin derivative (Hpd) and tumours of mice injected with Hpd was analysed by means of high pressure liquid chromatography (HPLC). The components of Hpd may be classified in 3 groups: (A) Components with a high fluorescence quantum yield and with sharp peaks in the HPLC chromatogram. These are monomers. (B) Components with a lower fluorescence quantum yield and with sharp peaks in the HPLC chromatogram. These are probably dimers or oligomers. (C) Components with a low fluorescence quantum yield, with a short retention time on a P-10 column and with a broad and unresolved peak in the HPLC chromatogram. These are probably large aggregates. Components of group A are rapidly accumulated by cells but are easily removed by washing the cells with medium containing serum. Porphyrins of group B are significantly more concentrated by cells in vitro than porphyrins of group A and B and accumulate over a time interval of about 18 h. Porphyrins of group B gradually migrate to sites in the cells where they are more strongly retained. Tumors in mice behave differently from tumor cells in vitro since they mainly contain porphyrins of group C after an i.p. injection of Hpd in the mice.
99Tc-labeled-tetrasulfophthalocyanine was prepared by the condensing of sulfophthalic acid and pertechnetate in the presence of a reducing agent. Reaction products were purified in various chromatographic systems and characterized by combustion, specific activity and spectral analyses. The tissue distribution pattern of the major product was studied in tumor-bearing rats. Most of the activity accumulated in the liver, kidneys, ovaries and uterus, whereas tumor uptake mainly occurred in the exterior cell layers. The in vivo stability of the complex was evidenced by the absence of 99Tc accumulation in the thyroid and the stomach.