For photodynamic therapy (PDT), galactose – Lutetium (Lu)-phthalocyanine, as a novel photosensitizer, was administered by intratumoral injection in 8 outbred albino male mice with inoculated hepatoma in a dose of 2 mg/kg. Then, tumors were irradiated through the skin by 670 nm-diode laser source with irradiation dose 200 J /cm2 applied for 1000 sec. The withdrawal of the animals from the experiment and sampling of tissues for morphological study were performed before (n=4) and 72 hrs after PDT (n=4). Tissue of the tumor was taken for morphological examination and fixed in 10% formalin. Serial paraffin sections were stained with hematoxylin-eosin and with the use of immunohistochemical staining with antibodies to proliferation marker Ki-67 and apoptotic marker BAX (Abcam, UK). 3 days after PDT with galactose- Lu-phthalocyanine, pronounced necrotic changes and inflammation reaction were observed in central tumor area, necrosis fields occupied up to 80% of the area. The dystrophic changes, reduction of proliferation degree and development of apoptosis in tumor cells were observed on tumor periphery. The decreased expression of proliferation marker Ki-67 and the increased expression of apoptotic marker BAX were noted in tumor cells after PDT.
The goal of our research was to assess the vascularization degree of tumor to predict the efficiency of plasmonic photothermal therapy (PPT) and photodynamic therapy (PDT) in tumor-bearing rats. Before any treatment, 3D Doppler ultrasound imaging was used for assessment of the vascularization degree of transplanted rat cholangiocarcinoma. For PPT, the gold nanorods with aspect ratio of 4:1, functionalized with thiolated polyethylene glycol, were used. After multiple fractional intravenous (IV) injections in rats with cholangiocarcinoma, the tumours were irradiated through the skin by an 808-nm NIR diode laser at a power density of 2.3 W/cm2 for 15 min. For PDT, galactose – Luphthalocyanine, as a photosensitizer, was applied by intratumoural injection, in a dose of 2 mg/kg. Then, tumors were irradiated through the skin by 670 nm-diode laser source with power density 200 mW/cm2 applied for 1000 sec in three non-overlapping zones covering whole tumour surface to obtain a total irradiation dose of 200 J/cm2 for each lesion. The withdrawal of the animals from the experiment and sampling of tissues for morphological study were performed before and 72 hrs after PPT and PDT. The vascular microdensity in tumors was assessed on histological sections as vessel counts or vessel area per unit of assessed tumor area. It was shown that efficiency of PPT and PDT therapy was mostly due to the sufficient accumulation of photothermosensitizers in the tumor, therefore preliminary assessment of tumor vascularization degree was necessary before starting a therapy.
The target-specific drug release which is depending on the pH of the medium appears precious approach for increasing of drug selectivity and further efficacy in photodynamic diagnosis (PDD) and therapy (PDT) of cancer. A strongly hydrophobic Zn (II) phthalocyanine (ZnPc) along with its liposomal formulates belongs to the clinically approved phthalocyanines for cancer photodiagnosis PDD and PDT. Nevertheless closed to the ideal photo-physicochemical properties the planar molecular structure of ZnPc easily forms aggregates which finally limit the phototoxic effect. Different strategies are on hand, all aiming to minimize the formation of photo-inactive associates. At present time, the polymeric carrier systems are still under development as carrier systems for biomedical applications. The present work evaluated the efficiency of two recently proposed graft - copolymers namely polyimide (PI)-graft-polymethacrylic acid (PAT2 and PAT3) as vehicles of hydrophobic ZnPc. The potential of the proposed polymeric brushes with great number of carboxylic groups, for conformational changes in different pH media, can be used for ZnPc release as studied by fluorescence spectroscopy. Complexes developed were investigated in vivo on rat model system of gastric carcinoma lesions for photodiagnosis and photodynamic treatment of neoplasia and compared with the application of ZnPcs solely. The contrast achieved of the fluorescence detection using polymer brushes - phthalocyanine conjugate is about twice higher that standard applied photosensitizer using same protocol and doses of application. The photodynamic treatment efficacy was also significantly improved allowing to receive full response using doses of 0,5-1 mg/kg of the conjugates. |To obtain same efficiency for the ZnPc PDT in the gastric lesions treated drug doses of 1,5-2 mg/kg had to be applied. Initially detected fluorescence signal and photo-bleaching dynamics were used for the monitoring of the processes of accumulation and photodynamic treatment processes.
The new generations of photoactive compounds, which are derivatives of already well-established groups of photosensitizers, together with the modern light sources in the spectral region of their activation and in the PDT window range, are emerging as a reliable phototherapy approach for tumour treatment with higher efficiency. The substitution of a highly hydrophobic phthalocyanine with galactose units could improve the solubility and to decrease significant aggregation in polar solvents.
The control group of rats with transplanted cholangiocarcinoma without any treatment was used for comparison of PDT effectiveness. In the second group the white outbred male rats with cholangiocarcinoma lesions were treated using Zn-phthalocyanine, as a typical representative of phthalocyanine photosensitizers with known photodynamic properties and the third group of animals was treated with galactose – Lu-phthalocyanine, as a novel derivative from the same family with two specific differences – presence of galactose and replacing of Zn(II) with Lu(III) ion. To compare the PDT effectiveness of both compounds the same drug doses were applied - 2 mg/kg, applied by intratumoural injection. Diode laser source at 670 nm, on 50 mW output power with power density applied 200 mW/cm2 was applied for 1000 sec to obtain total irradiation dose of 200 J/cm2 for each lesion.
3 days after treatment, the animals were withdrawn from the experiment. Tissue from the central zone and periphery of the tumor was taken for morphological examination and fixed in 10% formalin. Serial paraffin sections were stained with hematoxilin-eosin and by immunohistochemical staining with antibodies to proliferation marker Ki-67 and apoptosis marker BAX. After photodynamic therapy, pronounced necrobiotic changes in combination with an inflammatory reaction were developed at the central zone of tumors. The dystrophic changes, reduction of proliferation and development of apoptosis in tumor cells were observed on tumor periphery as well.
Gastrointestinal cancerous lesions were induced in laboratorial animals using prolonged exposure to social (overpopulation) and chemical stress (nitrosamine diet), mimicking typical stress factors for humans in the big cities. Twenty laboratorial rats with developed stress-induced neoplasia in gastrointestinal tract were used. Three of them formed control group - without application of exogenous contrast fluorescent marker. Exogenous fluorescent spectroscopic measurements were used to evaluate gastrointestinal tract (GIT) neoplasia development noninvasively using excitation at 405 nm and emission was detected in the region of 500-850 nm using microspectrometer. We used as exogenous fluorescent marker protoporphyrin IX applying its precursor delta aminolevulinic acid in a dose of 20mg/kg, in laboratorial rats with stress-induced neoplasia. Spectroscopic analysis of primary gastric tumours and their metastases spreading in rats’ liver was carried out on animals in vivo, as excitation and emission light were delivered by fiberoptic probe 6+1 to the organs investigated during open surgery procedure. Biochemical indicators detected malignant alterations presence in GIT were also evaluated and used as complementary indices for lesions’ growth. Fluorescent observation using exogenous fluorophore addressed the exact area and borders of neoplastic lesions in stomach and liver. In some cases in inflammatory areas significant accumulation of delta-ALA had place, which lead to false-positive fluorescent signal of protoporphyrin IX in these tissues. Photodiagnostics accuracy of 93% was reached for cancerous cases and 87% for gastric pre-cancer lesions evaluation. Very good correlation between fluorescence data and histology examination of the lesions investigated was achieved as well.
In our study aluminium–phthalocyanine (AlPc) compounds were applied for exogenous fluorescent diagnostics of adenocarcinoma of stomach in laboratorial animals (male adult rats (n=50)) after application of an experimental model of adenocarcinoma formation with metastasis. The neoplastic lesions were developed under the influence of social stress, as well as chemical stress using nitrosamines during 9 months period of application. Significant fluorescence signal in the region of 670-700 nm was observed in the neoplastic lesions, which absent in the normal mucosa investigated, related to the fluorescence of accumulated in the tumour area phthalocyanines. Autofluorescence background covered region at 450-650 nm with a maximum of 480-520 nm and consists mainly of protein cross-links and co-enzymes - NADH and flavin signals. Endogenous porphyrins fluorescence was also observed in the lesions with a maximum at 630-640 nm. Other organs (liver, small bowel, lungs) were also investigated for a presence of metastases. Histology examination of the lesions was used as a “gold standard” for comparison of the spectral data. Visually, a presence of accumulation sites of AlPc in a form of bright pink patches after excitation at 405 nm was observed, vs. healthy tissue, which remained blue-violet due to autofluorescence signal. Thus allow to use the AlPc fluorescence discrimination not only in spectroscopic mode of detection, but for imaging of the lesions investigated, which is more preferable in the case of clinical applications during endoscopic observations in humans.
The main research objective is the development of innovative optical technologies for sensitive diagnosis of early stages of development of stomach cancer and monitoring of stress-induced appearance and development of tumors of the gastrointestinal tract by applying endogenous and exogenous fluorescence spectroscopy modalities.
Different mechanisms solely and in combination for evaluation of the joint impact of bioenvironmental factors (stress, Helicobacter pillory, exo-toxins in the food, water, soil and air) were applied to induce gastrointestinal tract (GIT) neoplasia in rats. The transformation of damaged areas of the stomach mucosa into malignancies in all parts of gastrointestinal tract were detected using exogenous fluorescence of photosensitizers - 5-aminolevulinic acid (5-ALA) and aluminum phthalocyanine (Al-Pc). Fluorescent mapping of different organs (liver, spleen, lungs, brain) also was developed – to evaluate the distribution of the photosensitizers in the whole body on the second hour after photosensitizer application by intravenous injection. Fiber-optic probe was used to measure the organs investigated. Fluorescence spectra were detected by microspectrometer USB4000 (OceanOptics Inc., USA), and FS405 LED source on 405 nm was used as excitation source for both types of photosensitizers applied.
Diagnostically-important parameters of oximetry, optical coherence tomography and speckle-imaging of the microcirculation of the stomach were also evaluated, to evaluate changes in the blood flow and vascular architecture, during the formation of the initial phases of the neoplasm development.
The light exposure on a daily basis has been well accepted as a competitive method for decontamination of wastewater. The catalytic properties of TiO2 offer a great potential to reduce the transmission of pathogens in the environment. Although the titanium dioxide shows high activity against pathogens, its general usage in water cleaning is limited due to the insufficient excitation natural light (about 3% of the solar spectrum). A hydrophobic dodecylpyridyloxy Zn(II)-phthalocyanine with four peripheral hydrocarbon chains of C12 (ZnPcDo) was immobilized on a photocatalyst TiO2 anatase (P25). The resulted greenish colored nanoparticles of phthalocyanine were characterized by the means of absorption, fluorescence and infrared spectroscopy. The laser scanning confocal fluorescence microscopy was used to visualize the phthalocyanine dye by the red fluorescence emission (650 – 740 nm). The intensive Q-band in the far red visible spectral region (~ 690 nm) suggested a monomeric state of phthalocyanine on TiO2 nanoparticles. Two pathogenic bacterial strains (methicillin-resistant Staphylococcus aureus - MRSA and Salmonella enteritidis) associated with wastewater were photoinactivated with the suspension of the particles. The effective photoinactivation was observed with 1 g.L-1 TiO2 anatase at irradiation with UVA 364 nm as with UVA 364 nm and LED 643 nm. The gram-negative Salmonella enteritidis was fully photoinactivated with ZnPcDo-TiO2 and TiO2 alone at UVA 346 nm and at irradiation with two light sources (364 nm + 643 nm). The proposed conjugate appears as an useful composite material for antibacterial disinfection.
Photodynamic therapy (PDT) is a clinically approved non-invasive and curative procedure for different
oncological and non-oncological applications. PDT is still under development due to several limitations which
lead to partially successful photodynamic response. The crucial steps in PDT procedure are binding of the
photosensitizer to outer cell membrane, its penetration and subcellular localization which envisage the target
sites of reactive oxygen species generated during irradiation. Since the surrounding normal cells are also exposed
to the photosensitizer and the ambient daylight can be harmful for healthy tissues after therapeutic light
application, the challenging task in PDT research is to optimize the procedure in a way to reach tumor cell
selectivity. The present study outlines the influence of a light exposure pre-treatment (prior therapeutic light)
with specific wavelengths (365 nm and 635 nm) on the uptake, the localization and further re-localization of
galactose-substituted Zn(II) phthalocyanines into MDA-MB-231 breast cancer cells. The in vitro photodynamic
effect towards tumor cells was studied in comparison to the normal cell line Balb/c 3T3 (clone 31) after pre-irradiation
with UV light (365 nm) and red LED (635 nm). The results suggest that the galactose functional
groups of Zn(II) phthalocyanine and the harmless UV light at 365 nm favor the selective PDT response.
The Gram-negative, oral bacterium Aggregatibacter actinomycetemcomitans has been implicated as the causative agent
of several forms of periodontal disease in humans. The new periodontal disease treatments are emergence in order to
prevent infection progression. Antimicrobial photodynamic therapy (a-PDT) can be a useful tool for this purpose. It
involves the use of light of specific wavelength to activate a nontoxic photosensitizing agent in the presence of oxygen
for eradication of target cells, and appears effective in photoinactivation of microorganisms. The phthalocyanine metal
complexes of Pd(II)- (PdPcC) and Al(III)- (AlPc1) were evaluated as photodynamic sensitizers towards a dental
pathogen A. actinomycetemcomitans in comparison to the known methylpyridyloxy-substituted Zn(II) phthalocyanine
(ZnPcMe). The planktonic and biofilm-cultivated species of A. actinomycetemcomitans were treated. The photophysical
results showed intensive and far-red absorbance with high tendency of aggregation for Pd(II)-phthalocyanine. The dark
toxicities of both photosensitizers were negligible at concentrations used (< 0.5 log decrease of viable cells). The
photodynamic response for planktonic cultured bacteria was full photoinactivation after a-PDT with ZnPcMe. In case of
the newly studied complexes, the effect was lower for PdPcC (4 log) as well as for AlPc1 (1.5-2 log). As it is known the
bacterial biofilms were more resistant to a-PDT, which was confirmed for A. actinomycetemcomitans biofilms with 3 log
reductions of viable cells after treatment with ZnPcMe and approximately 1 log reduction of biofilms after PdPcC and
AlPc1. The initial results suggest that a-PDT can be useful for effective inactivation of dental pathogen A.
actinomycetemcomitans.
Zn(II)-phthalocyanines with tetra-substitution of D-galactose group on non-peripheral (nGalPc) and peripheral (pGalPc)
positions have been studied as photodynamic sensitizers. The both complexes are water-soluble and highly aggregated in
water and cell culture medium. The non-peripheral galactose units attached to the phthalocyanine macrocycle (nGalPc)
lead to far red shift of absorbance maximum at 703 nm as compared to peripherally substituted pGalPc with maximum at
683 nm. The fluorescence maxima of the studied GalPcs were red shifted (8-14 nm) depending on the used solvent as
compared to the absorption maxima. The relatively low fluorescence quantum yields in dimethylsulfoxide (0.06 for
nGalPc and 0.21 for pGalPc) were determined. The singlet oxygen generation was determined with lower quantum yield
for pGalPc (0.21) as compared to nGalPc (0.38). The lack of dark toxicity towards breast cancer cell line (MCF-7) in
wide concentration range (0.125 - 10 μM) was observed. The uptake into the tumor cells and the subcellular localization
in MCF-7 cells were determined with higher accumulation for pGalPc, compared to nGalPc. The in vitro photodynamic
activity of GalPcs towards breast cancer cells was investigated for different dye concentrations and soft light parameters
of 635 nm irradiation. The antitumor activity of nGalPc was superior to the pGalPc-induced cytotoxicity, due to higher
generation of singlet oxygen and other reactive oxygen species.
The study presents the PDT with metal phthalocyanines on biofilms grown in root canals of ten representatives of the
Gram-positive and the Gram-negative bacterial species and a fungus Candida albicans which cause aqute teeth
infections in root canals..
The extracted human single-root teeth infected for 48 h with microorganisms in conditions to form biofilms of the above
pathogens were PDT treated. The stage of biofilm formation and PDT effect of the samples of the teeth were determined
by the scaning electron microscopy and with standard microbial tests. The PDT treating procedure included 10 min
incubation with the respected phthalocyanine and irradiated with 660 nm Diode laser for 10 min.
The most strongly antibacterial activity was achieved with zinc(II) phthalocyanine (ZnPc) against Enterococcus faecalis,
Staphylococcus aureus and Moraxella catarrhalis. The other Gram-negative bacteria and Candida albicans were 10-100
times more resistant than the Gram-positive species. The Gram-negative Moraxella catarrhalis and Acinetobacter
baumannii were more sensitive than the enterobacteria, but eradication of Pseudomonas aeruginosa in biofilm was
insignificant. The influence of the stage of biofilm formation and the initial conditions (bacterial density, photosensitizer
concentration and energy fluence of radiation) to the obtained level of inactivation of biofilms was investigated.
The PDT with ZnPc photosensitizers show a powerful antimicrobial activity against the most frequent pathogens in
endodontic infections and this method for inactivation of pathogens may be used with sucsses for treatment of the
bacterial biofilms in the root canals.
In the recent decade the applications of photodynamic therapy (PDT) rapidly increase in several topics and one of
areas where the PDT in the future will be play significant role is dentistry. The different photosensitizing complexes
with a good water solubility and with absorption with an intensive maximum in the red region (630-690 nm), which
makes them suitable for photodynamic treatments, were investigated. The photochemical properties of complexes for
singlet oxygen generation were investigated and were shown relations between uptake levels and light intensity to
achieve increase in photodynamic efficacy. Photodynamic efficacy against fungi Candida albicans and bacteria's E.
faecalis, MRSA and S. Mutans in planktonic media was evaluated. The high photodynamic efficacy was shown for
SiPc at very low concentrations (0.9 μM) and light doses of 50 J cm-2 by intensity of light 60 mW cm-2. The
photodynamic response for E. faecalis, MRSA and S. Mutans, after treatments with different photosensitizers show
strong dependence on concentrations of photsensitzers and micro organisms. The level of inactivation of the pathogen
bacteria's from 1-2 degree of initial concentration up to full inactivation was observed. The studied complexes were
compared to the recently studied Methylene blue, Haematoporphyrine and tetra-methylpirydiloxy Zn(II)-
phthalocyanines and experimental results show that some of them have a good potential for inactivation of
representative pathogenic bacterial strains.
Experimental results also indicate that photodynamic therapy appears an effective method for inactivation of oral
pathogenic bacterias and fungi.
New methods of control of tooth bleaching stages through simultaneous measurements of a reflected light and a
fluorescence signal are proposed. It is shown that the bleaching process leads to significant changes in the intensity of a
scattered signal and also in the shape and intensity of the fluorescence spectra. Experimental data illustrate that the
bleaching process causes essential changes in the teeth discoloration in short time as 8-10 min from the beginning of the
application procedure. The continuation of the treatment is not necessary moreover the probability of the enamel destroy
increases considerably. The proposed optical back control of tooth surface is a base for development of a practical set up
to control the duration of the bleaching procedure.
Phthalocyanines of gallium(III) and indium(III) (GaPc1 and InPc1) bearing four methylpyridyloxy groups on the
periphery of the phthalocyanine ring were synthesized. The both phthalocyanines were obtained with a good solubility
in water solutions, which make them suitable for application in the Photodynamic therapy (PDT). The absorbance in
the Uv-vis region of the complexes is typical for MPc with a highly intensive maximum in the far red spectra (681 nm
- 697 nm for GaPc1 and for InPc1, both in DMSO). The fluorescence maxima are red shifted (691 nm/716 nm). The
fluorescence quantum yields of the both complexes are lower than that for the unsubstituted MPcs with values of 0.25
for GaPc1 and much lower for InPc1 (0.012), which suggested a quenching from the substituents. The photochemical
properties of singlet oxygen generation show quenching curves of "Furane" test with a 1O2 formation that increase
significantly in the presence of the heavy atoms such as Ga(III) and especially In(III). Photodynamic efficacy against
C. albicans in planktonic media was evaluated with a high photodynamic effect for GaPc1 at low concentrations (0.5
μM, - 3 μM) at mild irradiation parameters (30-60 J cm-2 and 50 mW cm-2). The inactivation of the fungus cells with
InPc1 was insignificant even at strong treatment conditions (6.8 μM; 60 J cm-2). The water-soluble phthalocyanine
complexes of Ga(III) and In (III) were compared to the recently studied by us water-soluble Zn(II)-phthalocyanine,
which was shown to have a high potential for photodynamic inactivation of variety pathogenic bacterial strains.
Photodynamic therapy (PDT) today is an innovative and not yet widespread light-drug initiated treatment that is based
on the photoactive compound irradiated with proper light to produce oxygen species that are toxic to the pathogenic
biological objects- bacteria, viruses, tumor cells. The obstacles that limited the efficacy of PDT concern to the selectivity
and multi-drug resistance prolong time for cellular release and side effects of skin photosensitivity for commercial
porphyrin originated photosensitizers (PS). Now there are very intensive investigations for introducing in practice a new,
with a least side effects PSs for PDT. The usefulness of the more extended macromolecules structured with proper
substituents refers not only to the improved optical properties like far-red and with intensive absorption and emission
capacity, but mainly to the ability for selective delivery and adhesion to the target cells, such as bacteria or other
pathogens.
The present study focuses on the charge effect of photodynamic agent on the uptake capacity toward gram-negative
bacteria cells and their further photoinactivation. The multi-drug resistant microorganism Aeromanas hydrophilla, which
is causing diseases to fishes and humans, is treated. The new octa-cationic phthalocyanines are designed to compare
PDT efficacy to the efficacy of tetra-substituted derivatives with the same functional peripheral substituents. The higher
cellular accumulation to the bacteria cells as a result of the high number of positive charges of photosensitizer, leading to
the better adhesion to the cellular membranes and improved photoinactivation of bacteria causing superficial and intraorgan
infections.
These results set a base of a rationale design of covalently octa-substituted phthalocyanines with positive charge for a
successful treatment of microorganisms.
The phthalocyanine zinc(II) and aluminum (III) complexes were studied to photoinactivate the bacterial strains,
Staphylococcus aureus, methacillin-sensitive and methacillin-resistant, Pseudomonas aeruginosa and one yeast Candida albicans. The binding of phthalocyanines to bacteria and fungi cells was evaluated by the means of laserinduced
fluorescence technique. The fluorescent spectra of dyes (650 - 800 nm) after direct excitation (635 nm) were
measured as follows: 1. for the aqua supernatants obtained after 10 min cell incubation with the respected
phthalocyanines (1.6 &mgr;mol.l-1), 2. for the washed from the unbound dye cells, and 3. for the organic extracts from the
three times washed cells. Fluorescent intensities at the emission maximum (~690 nm) were compared to the spectra of
the phthalocyanines in organic solutions. The phthalocyanines uptake data for bacteria and fungi were determined at
different cell densities. Nevertheless the better fluorescence properties of AlPc (fluorescent quantum yield of 0.4
towards 0.3 for ZnPcs) the lower drug accumulation in microorganisms was obtained. PDI results indicated an intensive
lowering of the bacterial survival of both strains of S. aureus treated with cationic ZnPcMe followed by the anionic
ZnPcS, at irradiance of 100 mW cm-2 and fluence rate of 60 J cm-2. More resistant to phototreatment P. aeruginosa and
morphologically complicated yeast C. albicans were successfully inactivated only with cationic ZnPcMe. These data
indicate the promising future application of cationic phthalocyanine in photodynamic inactivation of pathogenic
microorganisms.
The easy and non-destructive fluorescence method for quantification of early changes in biological tissues improves the possibilities of the clinical research and diagnostics. Developments in this area are moving very rapidly in part because of advances in the technology and in part because of the numerous successful examples which are appearing. New family of photomultiplier tubes with a high detection sensitivity for near-infra red light (700-900 nm) were developed as a result of project IMPECABLE, which are valuable tools for early diagnosis of cutaneous pigmented melanoma using long-wave fluorescence dyes.
Several phthalocyanines that are promising fluorophores for photodiagnosis of cutaneous malignant melanoma have been studied in different solvents for concentrations from 10-5 to 10-15 mol. Argon pumped dye laser as an excitation source was used. Three different wavelengths (613, 633 and 660 nm) in the red region, corresponding to first absorption peak, minimum of the absorption and near to the Q-band maximum of Pcs were applied. Fluorescence signals in the region of 700 to 800 nm were detected using spectrometric systems (Perkin-Elmer, UK-with conventional PMT as a detector, and PC2000, Ocean Optics, USA-with CCD-array as a detector) and a newly developed red-sensitive PMT. Detectable signal from other spectrometric systems was obtain up to 10-8 mol concentrations, which could be used for significant reduction of concentrations applied for in vivo applications.
Fluorescence is a highly sensitive method of distinguishing between healthy and unhealthy tissue. The results demonstrate that extremely low concentrations of photosensitizers could be used to determine initial stages of melanoma. This application of PMT detectors will reduce extremely the negative side effects of higher concentrations of these drugs applied in the skin tissue. One can achieve high accuracy in the determination of pigmented malignant melanoma lesions with wide clinical applications.
In the present work the following silicon (IV) - phthalocyanines and -naphthalocyanines bearing methoxyethylene glycol or methoxypolyethylene glycol covalently bound at the silicon are investigated: SiPc[OCH2CH2OCH3]2 (SiPc1), SiNc[OCH2CH2OCH3]2 (SiNc), SiPc[(OCH2CH2)nOCH3] with n approximately 115 (SiPc2). The phototherapeutic effect was shown at Lewis lung carcinoma implanted in mice. SiPc2 is monomeric soluble in water whereas the other two compounds aggregated in this solvent. Therefore these compounds were dissolved monomer in in aqueous Cremophor solution before in vivo administration. Laser irradiation was applied 7 days after implantation and 24 h after drug administration at the following wavelength (eta) ext: 672 nm for SiPc1 and SiPc2, 782 nm for SiNc. In all cases a fluence rate of 370 mW/cm2 at fluence of 360 J/cm2 was used. The assessment criteria for the tumor response were the changes in the mean tumor diameter with time, regrowth delay and average survival time (AST). According to the first parameter the most promising result was obtained after treatment with SiPc1. For example the mean tumor diameter increases as follows: SiPc1 less than SiPc2 less than SiNc very much less than control group without photosensitizer. The regrowth delay showed the same trend. however, for AST another dependence was observed. AST was the longest for SiPc2 (26 days) and shortest for SiNc (22 days). Compared to the control group (without sensitizer and irradiation) the AST was 9 days longer after SiPc2 treatment. Comparing SiPc1 and SiPc2 the chain length of the substituents does not influence the phototherapeutic properties. The detected therapeutic results probably are connected with the long wavelength absorption of the photosensitizers. The relatively lower affectivity of SiNc may be due to a lower degree of tumor accumulation as it was observed in our preliminary pharmacokinetic studies. It is also possible that the shorter AST after treatment with SiNc is connected with a greater dark toxicity.
As it is known the tumor model Lewis lung carcinoma (LLC) is inclined to form metastases very early after its implantation. Although photodynamic therapy (PDT) of tumors is a local method, we studied its influence on the development of lung metastases in LLC bearing C57B1/6 mice after tumor implantation. The used photosensitizer was a newly synthesized Zn(II)-naphthalocyanine (tetrabenzylamidotetranaphtho (2, 3-b:2', 3'-g: 2', 3' - 1:2'', 3''-q) porphyrazinatozinc) incorporated into DPPC-liposomes and excited with monochromatic light at its absorption maximum (lambda) exc 774 nm). The appearance of metastases in the animal lungs was evaluated according to the method of Pal, et al. Fourteen days after LLC implantation the tumor diameter for the treated mice was 6 mm (a week after PDT) towards 13.6 mm for the untreated. The percentage of surviving animals was 100 for the treated towards 0 for the untreated (a month after the implantation). Fourteen days after LLC implantation only 25% from the treated animals developed metastases to 75% from the untreated. The summary metastasis volume was 4.5 times smaller for the treated than the untreated mice. In conclusion we consider that the used Zn(II)-naphthalocyanine could be an effective photosensitizer for LLC treatment and although the PDT-method is a local one it could influence the process of metastasis development.
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