الفهرس 
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Abstract
The incidence of cancer is rising in both industrialized and developing nations, making it one of the major causes of death worldwide. Due to the intimate relationship between lifestyle variables and cancer, cancer prevention may be both more effective and less expensive. Natural remedies have been utilized to cure a variety of illnesses, and they are now a significant focus of drug development research. Numerous mechanisms, including cellular proliferation, differentiation, apoptosis, angiogenesis, and metastasis, are modulated by these products, notably phytochemicals, which have received significant research and have proven to have anti-carcinogenic properties. The majority of effective anti-cancer medications already on the market are phytochemicals or their equivalents, and some are even being tested on humans. Sono-photodynamic therapy (SPDT) is a breakthrough and a modern technology to eliminate tumor and affected tissues without affecting the healthy tissue adjacent to and away from the tumor. This requires the use of a so-called sonophotosenstiser, which is highly concentrated in the tumor area, which responds ideally to photonic energy and ultrasonic waves, which works to stop tumor growth and elimination, which is a promising technique to eliminate superficial and deep cancer tumors.
The present work aimed to study of ultrasound assisted drug delivery of chia, cress and flax conjugated hematite iron oxide nanoparticles for photothermal-photodynamic lung cancer treatment in vitro and in vivo. An infrared laser and ultrasound (in both pulsed and continuous wave modes) were used as energy sources.
In the current study, a total of 80 male Swiss albino mice, aged 60–65 days and weighing 205 g were obtained. These mice and the human lung cancer cell line were used. Only after lung cancer was induced with 100 mg/kg body weight of benzo[a]pyrene was the treatment trial launched. The Medical Research Institute at Alexandria University’s ethical criteria was followed when using experimental animals in the study methodology.
In vitro study groups were as follow; group I: As an untreated control, a lung cancer cell line was kept in an environment free of drugs. group 1I: Lung cancer cell line was treated with chia, cress and flax conjugated hematite iron oxide nanoparticles (CCF-HFe2O3) only. group III: Lung cancer cell line was exposed to IRL, for 3 min. group IV: Lung cancer cell line was treated with CCF-HFe2O3 and exposed to IRL for 3 min. group V: Lung cancer cell line was exposed to high frequency ultrasound for 3 min. group VI: Lung cancer cell line was treated with CCF-HFe2O3 and exposed to high frequency ultrasound for 3 min. group VII: Lung cancer cell line was exposed infra-red laser and high frequency ultrasound for 3 min. group VIII: Lung cancer cell line was treated with CCF-HFe2O3, exposed laser and high frequency ultrasound for 3 min.
In vivo study groups were as follow; group I: 10 mice received only corn oil and be kept without treatment. group 1I: 10 mice weere treated with 100 mg/ kg b.wt Benzo[a]pyrene only for inducing lung cancer (Magesh et al. 2009) and not receive any treatment. group III: 10 mice were subjected to same condition of group II and treated daily with (CCF- HFe2O3) only for two weeks. group IV: 10 mice were subjected to same condition of group II and exposed to IRL, for 3 min. for two weeks. group V: 10 mice were subjected to same condition of group II and treated daily with (CCF- HFe2O3), then tumor site irradiated to IRL at same conditions of group IV for two weeks. group VI: 10 mice were subjected to same condition of group II and exposed to high frequency ultrasound, for 3 min. for two weeks. group VII: 10 mice were subjected to same condition of group II and treated daily with (CCF-HFe2O3), then tumor site irradiated to high frequency ultrasound at same conditions of group VI for two weeks. group VIII: 10 mice were subjected to same condition of group II and exposed to infra-red laser followed by high frequency ultrasound, for 3 min. for two weeks. group IX: 10 mice were subjected to same condition of group II and treated daily with (CCF-HFe2O3), then tumor site irradiated to infra-red laser followed by high frequency ultrasound at same conditions of group VIII for two weeks.
The treatment effects evaluation:
• SRB cytotoxicity and cell viability was done to detect the effect of CCF-HFe2O3 nanocomposite in combination with laser and / or ultrasound on lung cancer cell line.
• Biochemical examinations were applied to detect serum levels of ALT, AST, urea and creatinine to detect the effect of CCF-HFe2O3 nanocomposite in combination with laser and / or ultrasound on liver and kidney functions.
• Activities of some antioxidants were measured, namely; (GST, SOD, GR, Cat, TAC) and (MDA) creatinine to detect the effect of CCF-HFe2O3 nanocomposite in combination with laser and / or ultrasound on antioxidant system and oxidative stress.
• Molecular detection of P53, Caspase 3,9, Bax, Bcl2, TNFα and VEGF gene expression using qRt-PCR to detect the effect of CCF-HFe2O3 nanocomposite on lung cancer cell line creatinine to detect the effect of CCF-HFe2O3 nanocomposite in combination with laser and / or ultrasound on pro and anti-apoptotic as well as necrosis and angiogensis.
• Evaluating the histological modifications in the tumour tissues after various therapies using Hematoxylin and Eosin (H&E) stain using light microscope to detect the effect of CCF-HFe2O3 nanocomposite in combination with laser and / or ultrasound on lung tissue.
Results of the study:
• CCF-HFe2O3 sono-photosensitizer nanocomposite only without activation has no effect.
• The effect of exposing the in vitro cell line and in vivo tumor to IRL as a PDT therapy elevated in the presence of the CCF-HFe2O3 nanocomposite than using infrared laser alone.
• The effect of exposure the in vitro cell line and in vivo tumor to pulsed/continuous ultrasound wave was more than that in case of using IR laser. Similar variations occurred in case of using ultrasound exposure only or in the presence of the CCF-HFe2O3 nanocomposite, with maximum effect occurred on using ultrasound in the presence of the CCF-HFe2O3 nanocomposite.
• Employing pulsed/continuous ultrasonic waves had a greater impact on the in vitro cell line and in vivo tumor than did using an IR laser. Similar differences happened whether using ultrasound exposure alone or when it was combined with the CCF-HFe2O3 nanocomposite, with the combined use of the two having the greatest effects.
• Using IR laser and pulsed/continuous ultrasound wave in combination with CCF-HFe2O3 nanocomposite was more effective than using either IR laser or pulsed/continuous ultrasound alone to treat the in vitro cell line and in vivo tumor.
• The levels of MDA were significantly higher in the mice carrying the lung tumor alone as compared to the healthy control animals.
• In the presence of CCF-HFe2O3 nanocomposite, the combination of photodynamic and sonodynamic therapy drastically reduced MDA levels.
• When compared to the normal control group, lower activity of the enzymatic antioxidants SOD, CAT, GR, GST, and TAC was seen in all lung cancer mouse groups. The inclusion of the CCF-HFe2O3 nanocomposite significantly increased both the enzymatic and non-enzymatic antioxidant defenses in the groups receiving a combination of photodynamic and sonodynamic therapy.
• It was found that CCF-HFe2O3 nanocomposite treatment improved blood creatinine and urea levels, which is a sign of renal protection. This further supports the significance of CCF-HFe2O3 nanocomposite in protecting against renal toxicity. Additionally, CCF-HFe2O3 nanocomposite therapy prevented an increase in serum levels of ALT and AST, which is a sign of the compound’s hepatoprotective properties. This further supports the importance of CCF-HFe2O3 nanocomposite in protecting against hepatotoxicity.
• Molecular detection of (P53, Caspase 3,9, Bax, and TNFα; positive) (Bcl2, and VEGF; negative) gene expression using qRt-PCR show the effect of CCF-HFe2O3 nanocomposite on lung cancer cell and detect the effect of CCF-HFe2O3 nanocomposite in combination with laser and / or ultrasound on pro and anti-apoptotic as well as necrosis and angiogensis.
• The histological analysis showed that none of the tumors from the group of mice with lung cancer showed necrosis and that all of the tumors contained highly aggressive cells. Compared to groups lacking CCF-HFe2O3 nanocomposite as a sonophoto- sensitizer, tumors removed from animals receiving therapy revealed appreciable areas of necrosis.
• Large foci of discrete necrosis zones were observed in the group of animals that received a combination of photodynamic and sonodynamic therapy in the presence of CCF-HFe2O3 nanocomposite.