الفهرس 
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Abstract
Photodynamic Therapy (PDT) is a non-invasive promising treatment of localized tumors. PDT has been approved by the U.S. Food and Drug Administration for the treatment of tumors located in the lung, esophagus, colon, peritoneum, pleura, genitourinary tract, brain, eye, and also in dermatology for the treatment of melanoma and non-melanoma skin cancers. PDT is typically a two-step process that involves the administration of a photosensitizer (photosensitive dye, PS) leading to selective uptake and retention in the tumor cells. Following this, the PS that is in the ground non-excited state (S0) is activated by exposure to light of an appropriate wavelength and thus shifting first to an electronically excited singlet state (S1) and then, via intersystem crossing, to the excited triplet state (T1). The energy transfer via so-called photochemical reactions leads to the generation of reactive oxygen species (ROS) in cancer cells. Singlet oxygen (1O2) is the main and the primary phototoxic species generated by the most of the PSs, its accumulation leads to the oxidative stress of tumor cells which results, through the sequence of specific cellular pathways, in necrotic and/or apoptotic cell death. 1O2 also damages the vasculature of the tumor and surrounding healthy vessels, resulting in indirect tumor death via the induction of hypoxia and starvation. PDT also has been shown to cause DNA base oxidative damage, strand breaks and cross-links.
Most studies performed in experimental animal species bearing different tumor models have shown that the delivery systems provide a heightened tumor accumulation of PS thus enhancing their therapeutic potential. So, the choice of the suitable delivery system for appropriate PS and tumor type turns out to be extremely important since the mode of delivery of PS affects the biodistribution and pharmacokinetics. Conjugating the PSs to antibodies, directed against antigens or ligands that are specifically overexpressed on cancer cells is called photoimmunotherapy (PIT). This approach aims at the selective delivery of PSs to tumor cells thus the problem of cutaneous phototoxicity will be reduced.
The ideal PS should not only be preferentially accumulate in the target tumor cells, but it is also preferred to absorb light at wavelengths that fall within the red region of the spectrum (650 and 800 nm), known as a therapeutic window, where maximal penetration of light into tissues is observed. 4,5 Benzoindotricarbocyanine (Indocyanine green, ICG) is a tricarbocyanine dye, which has been approved by the United States Food and Drug Administration for medical diagnostic studies, and is widely used for the evaluation of cardiac output, liver function and visualization of the retinal and choroidal vasculatures. The recent interest in using ICG as a PS in PDT comes from the fact that this dye has a strong absorption band (between 700–800 nm) allowing deeper tissue penetration without causing significant heating.
The main two disadvantages of using ICG in PDT are that ICG easily binds to lipoprotein, which leads to the drug’s rapid clearance from the body (a plasmatic half-life of around 2–4 min) and the in vitro and in vivo instability of ICG (aqueous-instability, photo-degradation and thermal-degradation). In aqueous solutions, ICG undergoes physicochemical transformations such as aggregation and irreversible degradation. Such changes result in discoloration, decreased light absorption, decreased fluorescence, and a shift in the wavelength of maximum absorption. Thus, an injectable ICG product with a satisfactory stability in aqueous solutions, having long circulation times in the body and ability to be targeted at the desired site of action will be of great interest.
One of the novel approaches to achieve this can be a biocompatible and biodegradable nanoparticulate system entrapping ICG. In recent years, polymeric nanoparticles have received considerable attention as promising colloidal drug carrier. Polymeric nanoparticles provide efficient aqueous-stability, photo-stability and thermal stability to ICG. The protective effect of nanoparticles seemed to be due to the polymeric-envelop, which protected the entrapped ICG by isolating it from the surrounding environment. The ICG in nanoparticles showed a small shift in its wavelength of peak fluorescence and a decrease in its peak fluorescence intensity.
Recent publications present a dual-function, nanosize agent for both early-stage cancer detection by photoacoustic imaging and localized cancer treatment by PDT. The agent is designed by encapsulating ICG dye in a biocompatible matrix. These nanoparticles were developed based on PEBBLE (photonic explorers for biomedical use by biologically localized embedding) technology, using organically modified silicate (ormosil) as the matrix.
Overexpression of the EGFR by malignant cells is associated with poor prognosis and resistance to therapy. The majority of squamous cell carcinomas have high EGFR expression, thus conjugation of a PS to the Anti-EGFR antibody can be used for increasing the tumor selectivity of this PS. The polymeric nanoparticles containing ICG (ICG-PEBBLE) can be modified superficially using special targeting moieties (such as antibodies) for targeting and site specific action. ICG containing nanoparticles can be coated by a single layer of Anti-EGFR antibody, resulting in nanocapsules that can bind specifically to EGFR overexpress tumor cells.
In this study, we first investigated the characteristics and photodynamic activity of ICG entrapped in polymeric nanoparticles forming (ICG-PEBBLE) comparing to free ICG on two different cell lines; human breast adenocarcinoma cells (MCF-7) and hepatocellular carcinoma cells (HepG2). Then investigated the photodynamic therapeutic effect of both (ICG-PEBBLE) and (ICG-PEBBLE-Anti-EGFR) on two stage mouse carcinogenesis model.
ICG-PEBBLE nanoparticles were synthesized based on sol-gel chemistry and the yield was monodispersed nanoparticles with a diameter ranging from (200 – 300 nm), only negligible difference in the mean particles size was observed between PEBBLE nanoparticles without ICG and PEBBLE nanoparticles loaded with ICG dye. The enhanced stability of ICG-PEBBLE in aqueous media comparing to free ICG dye and the negligible dye leaching observed in PEBBLE filtrate support the notion that the ICG molecules are present in monomer form in the nanoparticles and the observed absorption spectrum corresponds to ICG molecules encapsulated in PEBBLE nanoparticles, rather than by free ICG dye in the solution, due to dye leaching.
The fluorescence emission of DPIBF was found to be decreased with either free ICG dye or ICG- PEBBLE using different concentrations, and this is an indication for the release of 1O2 from both free ICG dye and ICG-PEBBLE after laser exposure present. This measurement confirms the potential of ICG- PEBBLE as a PS releasing free 1O2 upon laser exposure.
The metabolic cytotoxicity MTT experiments revealed no dark toxicity in any ICG or ICG- PEBBLE concentrations used since treatment of both cell lines (MCF-7) and (HepG2) by the two drugs on its own (with no laser exposure) did not result in any significant decrease of cell viability. However, cell viability after 24h from laser exposure exhibited a behavior inversely proportional to the used ICG or ICG-PEBBLE concentrations. This phototoxic effect can be explained by the photodynamic activity of ICG or ICG- PEBBLE due to the production of reactive oxidative species upon exposure to laser irradiation as noticed from the photosensitivity assay.
According to the trypan blue cell viability assay, laser exposure for 20 min for MCF-7 or HepG2 cells treated with a fixed concentration of ICG or ICG- PEBBLE resulted in a dramatic membrane damage led to cell death. In contrast, incubation of the treated cells for the same time intervals without laser exposure resulted in normal percentage of cell damage.
To investigate the mode of cell death induced by ICG and ICG- PEBBLE, the fluorescence microscopic study demonstrated that necrosis preferentially occurred following PDT with ICG in MCF-7 and HepG2 cell lines, while PDT with ICG- PEBBLE enhanced the apoptotic mode of cell death in both cell lines. Since the dose of both drugs used was the same and the treatment condition was also similar in the two cell lines, so this change in the mode of cell death could be attributed to the subcellular localization of each PS.
The comet assay is an applicable method for the screening of DNA damage after PDT. In the present study the comet assay showed a significant DNA damage on both cell lines (as indicated from tail moment) after PDT using ICG or ICG-PEBBLE. The mechanisms of DNA damage induced by PDT are not well understood, PDT can cause base oxidation, cross-linking of DNA strands or sister chromatid exchange. The mediators of this damage are most likely the reactive oxygen species generated by type I and type II mechanisms. It was also obvious that the extent of DNA damage as represented by the tail moment was lower in the cell lines treated with ICG-PEBBLE followed by laser than the cells treated with ICG followed by laser, and this could be an indication that using ICG-PEBBLE in PDT can cause less oxidative damage to DNA unlike PDT with ICG, and this is an advantage for ICG-PEBBLE over ICG.
from the previous studies it was concluded that encapsulating ICG dye in a biocompatible matrix forming ICG-PEBBLE and using it in PDT can overcome the disadvantages of using free ICG dye and in the same time it has the same phototodynamic effect of ICG with better efficacy.
To investigate the photodynamic therapeutic effect of both (ICG-PEBBLE) and (ICG-PEBBLE-Anti-EGFR) on tumor cells in vivo, squamous cell carcinomas was induced in CD-1 by the two stage mouse carcinogenesis model. Treatment process had begun after the induction of tumor and proceeded for 4 weeks, where tumor bearing mice either treated with Laser alone, (ICG - PEBBLE + Laser) or (ICG- PEBBLE-Anti-EGFR + Laser). The DMBA/TPA group (positive control) was in parallel with all these treated groups.
The post treatment eye observations for the tumor morphology revealed that tumor bearing mice that were intratumorally injected with ICG- PEBBLE or ICG-PEBBLE-Anti-EGFR and then exposed to laser irradiation revealed in general that the tumor growth appeared to slow down (comparing to DMBA/TPA group) after the first session and some mice showed decrease in tumor size, mostly in (ICG- PEBBLE + Laser) group, but after the second session a significant decrease in tumors size in both groups was observed, in some cases remission was detected in both groups (often the self-mutilation physically removed part or most of the necrotic tumor tissue of treated tumor).
The histopathology of tumor sections from DMBA/TPA control and (DMBA/TPA + Laser) group suggested invasive squamous cell carcinoma comparing to the normal histology of the skin sections from normal control groups (Untreated, Acetone, ICG-PEBBLE, and Laser groups). The histopathological examination of sections from the PDT-tumors treated groups showed marked dermal suppurative inflammation and massive dead and living polymorphnuclear cells infiltration as a result of apoptosis and necrosis. from these histopathological features it can be concluded that intratumoral injection of ICG-PEBBLE or ICG-PEBBLE-Anti-EGFR followed by laser exposure induced tumor cell death and resulted in inhibition of further growth and promotion of skin tumor cells.
Cancer development originating from chronic inflammation, in our case as a result of TPA/DMBA application, may be driven by inflammatory cells and a variety of mediators, which together establish an inflammatory microenvironment. According to the results obtained in the present study, the plasma inflammatory mediators TNF-α and nitrite (as an index to the generation of nitric oxide) and the inflammatory mediators measured in skin tissue homogenate COX-2 and 5-LOX they all found to be significantly elevated in tumor groups (DMBA/TPA group and DMBA/TPA + Laser group) comparing to the Untreated group which can be an indication of the elevated inflammation, but treatment of tumor bearing mice with either ICG-PEBBLE or ICG-PEBBLE-Anti-EGFR followed by laser exposure showed significant decrease in the levels of these inflammatory mediators comparing to (DMBA/TPA) group, an indication of less inflammation levels. There were no significance differences in the levels of inflammatory mediators between the two treated groups, which can be an indication that PDT using ICG-PEBBLE-Anti-EGFR has no advantage concerning reduction in the levels of inflammatory mediators comparing to ICG-PEBBLE.
The formation of new blood vessels (angiogenesis) is critical for cancer progression since the growth potential of cells is limited by availability of nutrients. Vascular Endothelial Growth Factor (VEGF) is the most potent tumor angiogenic factor identified. In the two-stage chemical carcinogenesis protocol with DMBA and TPA, Ha-ras gene mutation (the critical event for papilloma development) has been shown to increase the expression of VEGF and this was obvious in the present study where a significant elevation in VEGF level was observed in tumor groups (DMBA/TPA group and DMBA/TPA + Laser group) comparing to the Untreated group which can be an indication of the extent of angiogenesis process induced through tumor progression. VEGF level in skin tissue homogenate of tumor bearing mice treated by PDT using either ICG-PEBBLE or ICG-PEBBLE-Anti-EGFR was found to be significantly lower comparing to DMBA/TPA group, which can be an indication that the angiogenesis process is somehow affected (damaged) by PDT. It was also found that VEGF level in tumor bearing mice treated by PDT using ICG-PEBBLE-Anti-EGFR was significantly lower than that using ICG-PEBBLE, this is an indication that conjugating EGFR antibody to the photosensitizer ICG-PEBBLE could potentially target tumor vasculature and thus enhance damaging the angiogenesis process, which means that ICG-PEBBLE-Anti-EGFR conjugate have a higher anti-angiogenic effect than the unconjugated ICG-PEBBLE.
PDT is reported to be a strong inducer of apoptosis. In many apoptosis pathways, activation of the effector Caspases is considered the final step. Among the spectrum of various Caspases, Caspase-3 and 7 are considered the major executioner Caspases and they are activated in most cases of PDT induced apoptosis with a number of different PSs. According to the results of the present study, Caspase-3 was significantly increased only in tumor bearing mice treated with ICG-PEBBLE-Anti-EGFR and then exposed to laser irradiation, level of Caspase-3 in this group was significantly higher comparing to the Untreated group or (DMBA/TPA) group, this can be indication that using ICG-PEBBLE-Anti-EGFR in PDT can enhance apoptotic mode of cell death. Concerning to Caspase-7 level no significant change was observed between all groups.
Another important marker for the detection of the apoptotic pathway is estimation of histone deacetylase (HDAC) activity. According to the results obtained from the present study, a significant elevation was observed in the activity of HDAC activity in tumor bearing groups comparing to untreated group, which can be an indication of the abnormal HDAC activity. An elevation in HDAC activity leads to an increase in the positive charge of histone tails and thus encouraging high-affinity binding between the histones and DNA backbone. The increased DNA binding condenses DNA structure and preventing transcription of specific tumor suppressor genes, thus contributing to tumor formation. The significant decrease in HDAC activity observed in tumor bearing mice treated with Laser only or (ICG-PEBBLE + Laser) or (ICG-PEBBLE-Anti-EGFR + Laser) comparing to (DMBA/TPA) group is an indication that tumor cell apoptosis observed in these groups is mediated by regulating histone function and subsequently gene transcription. This is an indication that PDT using ICG-PEBBLE or ICG-PEBBLE-Anti-EGFR can induce tumor death through apoptotic pathways.
The assessment of proliferating cell populations by immunohistochemistry has been used to aid in the differentiation of benign from malignant neoplasms, and it has been reported that assessment of proliferation markers and oncogenic determinants holds information regarding prognosis. In the present study, cell proliferation was detected by immunostaining of proliferating cell nuclear antigen (PCNA) in formalin-fixed, paraffin-embedded tissue sections, using anti-PCNA monoclonal antibody. Normal epidermis served as a positive control and reference.
Few PCNA-positive cells were detected in skin tissue sections from all of the control groups (Untreated, Acetone, ICG-PEBBLE and Laser groups), and there was no significant difference in the intensity for PCNA staining between these groups. In contrast, the expression of PCNA was very high in the tumor induced groups; (DMBA/TPA) and (DMBA/TPA + Laser). This higher PCNA positivity in these groups is randomly extended throughout the basal layer and the dermis, and is an indication of the hyperpoliferative and aggressive nature of this induced tumor.
A significant DROP in PCNA positivity was detected in tissue sections from tumor bearing mice treated by PDT using either ICG-PEBBLE or ICG-PEBBLE-Anti-EGFR, which could be an indication that tumor cells responded well to PDT leading to less hyperproliferation of tumor cells and increased necrosis, apoptosis and fibrosis of the cells after the PDT. This result is in parallel with the other mentioned results that revealed good response to PDT; this is an indication that PCNA expression may be useful for predicting tumor response to therapy.
To determine which mode of cell death (apoptosis or necrosis) is predominant in PDT using ICG-PEBBLE or ICG-PEBBLE-Anti-EGFR, morphological changes of the cells using Acridine orange / Ethidium bromide staining were examined. In the present study, most of the cells in the normal control groups; (Untreate, Acetone, ICG-PEBBLE and Laser) and in tumor groups; (DMBA/TPA group and DMBA/TPA + Laser group), are considered to be viable cells since they have uniform green nuclei with organized structure and to a lesser extent (in the normal level) there were observed sporadic bright green cells indication of early apoptotic and sporadic cells with yellow to orange chromatin indication of cells going through late apoptosis or necrosis.
On the other hand, tissue sections from tumor groups treated with (ICG-PEBBLE) or (ICG-PEBBLE-Anti-EGFR) showed a high incidence of early apoptosis (bright green cells), late apoptosis (cells display condensed and fragmented yellow chromatin) and necrosis (cells have a uniformly orange to red nuclei with organized structure). There was no difference between these two groups in the prevalence of a mode of cell death than other, suggesting that using (ICG-PEBBLE) or (ICG-PEBBLE-Anti-EGFR) in PDT for the treatment of in vivo induced tumor (under the same conditions of Ps dose and Laser influence rate) can initiate both mode of cell death (apoptosis and necrosis) with no prevalence of one mode on the other, although in the in vitro study PDT using ICG-PEBBLE enhanced apoptotic mode of cell death in both cell lines MCF-7 and HepG-2. This can be attributed to the PDT conditions used in vitro and in vivo. Low fluence rate of laser was used in the in vitro study, but in vivo high fluence rate of laser was used.
from the above in vitro and in vivo results it can be concluded that encapsulating ICG dye in a biocompatible matrix forming ICG- PEBBLE and using it in PDT can overcome the disadvantages of using free ICG dye and in the same time it has the same phototodynamic effect of ICG with better efficacy.
Using the synthesized photosensitizer ICG-PEBBLE in PDT showed a good photodynamic therapeutic effect against tumor cells and conjugating this ICG-PEBBLE to Anti-EGFR antibody as a trial for targeting a larger proportion of the tumor cells, only showed improved effect in decreasing the level of VEGF (anti-angiogenic effect) and increasing the level of caspase-3 comparing to ICG-PEBBLE alone, but there were no other preferential effects in the rest of the parameters and both ICG-PEBBLE and (ICG-PEBBLE- Anti-EGFR antibody) seemed to have the same photodynamic effect. This limitation could be due to the large weight of the Anti-EGFR antibody used for conjugation thus limiting their capability for penetrating more tumor cells. At final, it should be noted that the results presented in this study are only the short term results, up to 4 weeks. How to avoid the tumor recurrence by modifying this PDT protocol will ultimately decide the efficacy of this special modality.