الفهرس 
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Abstract
The most common and second-deadliest cancer in women worldwide after lung cancer is breast cancer (BC). Cancer is a widespread, intricate, and diverse disease.The incidence of cancer is rising in both industrialized and developing nations, making rendering it a leading global source of death. Due to the intimate relationship between lifestyle variables and cancer, cancer prevention may be both more effective and less expensive. In addition finding novel and effective cancer therapies is a significant issue on a global scale. Natural remedies have been utilized to cure a variety of illnesses, and they are now a significant focus of drug development research. Numerous cascades, as 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 magnetic field assisted drug delivery of chlorella conjugated magnetite nanoparticle for dual sono-photo-dynamic and sono-photo-thermal cancer treatment in vitro and in vivo. An IR laser and US (in both puls.and cont. modes) were used as energy sources.
In the current study, 90 male Swiss albino mice, 60–65 days aged and 205 gm weighing were obtained. These mice and the human lung cancer cell line were used. Only after EAC cancer was implanted upon subcutaneous injection of Ehrlich ascites carcinoma cells 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 breast cancer cell line was kept in an environment free of drugs. group 1I: Breast cancer cell line was treated with chlorella conjugated magnetite nanoparticles (Chlorella-MNP) only. group III: Breast cancer cell line was exposed to IRL, for 3 min. group IV: Breast cancer cell line was treated with Chlorella-MNP and exposed to IRL for 3 min. group V: Breast cancer cell line was exposed to ultrasound for 3 min. group VI: Breast cancer cell line was treated with Chlorella-MNP and exposed to ultrasound for 3 min. group VII: Breast cancer cell line was exposed infra-red laser and ultrasound for 3 min. group VIII: Breast cancer cell line was treated with Chlorella-MNP, exposed laser and ultrasound for 3 min.
In vivo study groups were as follow; group I: 10 mice received only saline and be kept without treatment. group 1I: 10 mice weere received subcutaneous injection of 2 × 106 Ehrlich ascites carcinoma cells only EAC implantation and not receive any treatment. group III: 10 mice were subjected to same condition of group II and treated daily with (Chlorella-MNP) 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 (Chlorella-MNP), then irradiated tumor site with 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 ultrasound, for 3 min. for two weeks. group VII: 10 mice were subjected to same condition of group II and treated daily with (Chlorella-MNP), then irradiated tumor site with US at same conditions of group VI for two weeks. group VIII: 10 mice were subjected to same condition of group II and irradiated to infra-red laser followed by ultrasound, for 3 min. for two weeks. group IX: 10 mice were subjected to same condition of group II and treated daily with (Chlorella-MNP), then irradiated tumor site with IR laser followed by US 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 Chlorella-MNP nanocomposite in combination with laser and / or ultrasound on breast cancer cell line.
Biochemical examinations were applied to detect serum ALT, AST, urea and creatinine levels to detect effect of Chlorella-MNP 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 Chlorella-MNP 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 Chlorella-MNP nanocomposite on breast cancer cell line creatinine to detect the effect of Chlorella-MNP 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 Chlorella-MNP nanocomposite in combination with laser and / or ultrasound on EAC tissue.
Results of the study:
Chlorella-MNP sono-photosensitizer nanocomposite only without activation has little 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 Chlorella-MNP nanocomposite than using infrared laser alone.
Pulsed/continuous ultrasonic waves had a greater impact on the in vitro cell type and in vivo tumor than an IR laser did. Similar differences reported when using ultrasound exposure alone or with the Chlorella-MNP nanocomposite present, with the greatest impact encountered when using ultrasound with the Chlorella-MNP nanocomposite present.
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 Chlorella-MNP nanocomposite, with the combined use of the two having the greatest effects.
Using IR laser and pulsed/continuous ultrasound wave in combination with Chlorella-MNP nanocomposite was more efficent than using either IRL or pulsed/continuous US alone to treat the in vitro cell line and in vivo tumor.
The levels of MDA were significantly higher in the mice carrying the EAC tumor alone when compared to control healthy animals.
In the presence of Chlorella-MNP nanocomposite, the combination of photodynamic and sonodynamic therapy drastically reduced MDA levels.
All EAC cancer mouse groups displayed decreased SOD, CAT, GR, GST, and TAC enzyme antioxidant activity when compared to the healthy control group. In the groups getting a combined of PDT and SDT treatment, the addition of the Chlorella-MNP nanocomposite greatly boosted both the enzymatic and non-enzymatic antioxidant defenses.
It was found that Chlorella-MNP nanocomposite treatment improved a blood creatinine and urea level that is a sign of renal protection. This further supports the significance of Chlorella-MNP nanocomposite in protecting against renal toxicity. Additionally, Chlorella-MNP nanocomposite therapy prevented elevation in serum levels of ALT and AST that is a sign of the compound’s hepatoprotective properties. This further supports the importance of Chlorella-MNP 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 Chlorella-MNP nanocomposite on EAC cancer cell and detect the effect of Chlorella-MNP 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 EAC cancer showed necrosis and that all of the tumors contained highly aggressive cells. Compared to groups lacking Chlorella-MNP 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 EAC animals group that received a combination of PDT and SDT therapy in Chlorella-MNP nanocomposite presence.