الفهرس 
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Abstract
Bladder cancer is the 11th commonest cancer in the world and the 4th commonest cancer in Egypt. The incidence of cancer is rising in both industrialized and developing nations, making it one of the worldwide major causes of death. 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. Shockwave-photodynamic therapy (SWPDT) 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 shockwave-photosenstiser, which is highly concentrated in the tumor area, which responds ideally to photonic energy and shockwaves, 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 shock wave assisted drug delivery of berries conjugated carbon dots particle for shockwave -photo-dynamic cancer treatment in vitro and in vivo. An infrared laser and shockwaves were used as energy sources.
In the current study, a total of 90 male Swiss albino mice, aged 60–65 days and weighing 205 gm were obtained. These mice and the bladder cancer cell line were used. Only after bladder cancer was induced with 0.00 % N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) 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 bladder cancer cell line was kept in an environment free of drugs. group 1I: Bladder cancer cell line was treated with berries conjugated carbon dots nanoparticles (Berries-CDs) only. group III: Bladder cancer cell line was exposed to laser, for 3 min. group IV: Bladder cancer cell line was treated with Berries-CDs and exposed to laser for 3 min. group V: Bladder cancer cell line was exposed to shockwaves for 3 min. group VI: Bladder cancer cell line was treated with Berries-CDs and exposed to shockwaves for 3 min. group VII: Bladder cancer cell line was exposed laser and shockwaves for 3 min. group VIII: Bladder cancer cell line was treated with Berries-CDs, exposed laser and shockwaves for 3 min.
In vivo study groups were as follow; group I: 10 mice received only water and be kept without treatment. group 1I: 10 mice weere treated with 0.00 % N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) only for inducing bladder cancer and not receive any treatment. group III: 10 mice were subjected to same condition of group II and treated daily with (Berries-CDs) only for two weeks. group IV: 10 mice were subjected to same condition of group II and exposed to laser, for 3 min. for two weeks. group V: 10 mice were subjected to same condition of group II and treated daily with (Berries-CDs), then tumor site
irradiated to laser at same conditions of group IV for two weeks. group VI: 10 mice were subjected to same condition of group II and exposed to shockwaves, for 3 min. for two weeks. group VII: 10 mice were subjected to same condition of group II and treated daily with (Berries-CDs), then tumor site irradiated to shockwaves at same conditions of group VI for two weeks. group VIII: 10 mice were subjected to same condition of group II and exposed to laser followed by shockwaves, for 3 min. for two weeks. group IX: 10 mice were subjected to same condition of group II and treated daily with (Berries-CDs), then tumor site irradiated to laser followed by shockwaves 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 Berries-CDs nanocomposite in combination with laser and / or shockwaves on bladder cancer cell line.
Biochemical examinations were applied to detect serum levels of ALT, AST, urea and creatinine to detect the effect of Berries-CDs nanocomposite in combination with laser and / or shockwaves 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 Berries-CDs nanocomposite in combination with laser and / or shockwaves 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 Berries-CDs nanocomposite on bladder cancer cell line creatinine to detect the effect of Berries-CDs nanocomposite in combination with laser and / or shockwaves 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 Berries-CDs nanocomposite in combination with laser and / or shockwaves on bladder tissue.
Results of the study:
Berries-CDs shockwave-photosensitizer nanocomposite only without activation has no effect.
The effect of exposing the in vitro cell line and in vivo tumor to laser as a PDT therapy elevated in the presence of the Berries-CDs nanocomposite than using infrared laser alone.
Shockwaves had a greater impact on the in vitro cell type and in vivo tumor than an IR laser did. Similar differences reported when using shockwaves exposure alone or with the Berries-CDs nanocomposite present, with the greatest impact encountered when using shockwaves with the Berries-CDs nanocomposite present.
Employing shockwaves had a greater impact on the in vitro cell line and in vivo tumor than did using a laser. Similar differences happened whether using shockwaves exposure alone or when it was combined with the Berries-CDs nanocomposite, with the combined use of the two having the greatest effects.
Using laser and shockwaves in combination with Berries-CDs nanocomposite was more effective than using either laser or shockwaves alone to treat the in vitro cell line and in vivo tumor.
The levels of MDA were significantly higher in the mice carrying the bladder tumor alone when compared to the healthy control animals.
In the presence of Berries-CDs nanocomposite, the combination of photodynamic and shockwaves sonodynamic therapy drastically reduced MDA levels.
All BCa 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 SWDT treatment, the addition of the Berries-CDs nanocomposite greatly boosted both the enzymatic and non-enzymatic antioxidant defenses.
It was found that Berries-CDs nanocomposite treatment improved blood creatinine and urea levels, which is a sign of renal protection. This further supports the significance of Berries-CDs nanocomposite in protecting against renal toxicity. Additionally, Berries-CDs nanocomposite therapy prevented an elevation in serum levels of ALT and AST, which is a sign of the compound’s hepatoprotective properties. This further supports the importance of Berries-CDs 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 Berries-CDs nanocomposite on bladder cancer cell and detect the effect of Berries-CDs nanocomposite in combination with laser and / or shockwaves 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 bladder cancer showed necrosis and that all of the tumors contained highly aggressive cells. Compared to groups lacking Berries-CDs nanocomposite as a shockwaves-photo-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 shockwaves sonodynamic therapy in the presence of Berries-CDs nanocomposite.