Amelioration of the therapeutic efficacy of 5-Flurouracil loaded chitosan nanoparticles in experimentally induced Hepatocellular Carcinoma

Amelioration of the therapeutic efficacy of 5-Flurouracil loaded chitosan nanoparticles in experimentally induced Hepatocellular Carcinoma EL-Hassan Mokhamer, Mona M Yehia, Heba S Ramadan, and, Ola EL-Gendy Zoology Department, Faculty of Science, Damanhour University Histochemistry and Cell Biology Department, Medical Research Institute, Alexandria University Medical Biophysicus Department, Medical Research Institute, Alexandria University Applied Medical Chemistry Department, Medical Research Institute, Alexandria University 4 (Email: elhassan,gaber@sci.dmu.edu.eg), Received Jan, 15, 2019; Accepted March,1,2019 Abstract

1 Introduction Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide (El-Serag, 2002) and the third most common cause of cancer death. Despite advances in prevention techniques, screening, and new technologies in diagnosis and treatment, incidence and mortality continue to rise.
The most important risk factor for the development of HCC is cirrhosis regardless of etiology. Hepatitis B and C are independent risk factors for the development of cirrhosis. (Balogh et al., 2016). Liver Transplantation offers the benefit of removal of cancer as well as the harboring risk of de novo HCC in a cirrhotic liver (Kulik et al., 2018). The disadvantages of most anti-cancer drugs that are currently available include low bioavailability, poor selectivity because they can act on both tumor cells and healthy cells, and immunosuppression that can cause complications and even patient death (Shah et al., 1985).
Continuous oxidative stress, which results from the generation of reactive oxygen species (ROS) by environmental factors or cellular mitochondrial dysfunction, has recently been associated with hepatocarcinogenesis.
In liver disease, the infiltration of activated phagocytic cells provides an additional source of ROS production that promotes oxidative stress and damage to proteins, lipids, and DNA (Kaplowitz, 2000). ROS react with polyunsaturated fatty acids to induce the release of toxic and reactive aldehyde metabolites such as malondialdehyde (MDA) (Cheeseman, 1993, Saad et al., 2014. Malondialdehyde (MDA) is a marker for oxidative stress that is formed by lipid peroxidation and that was shown to accumulate in serum of chronic hepatitis patients (Nagoev et al., 2002).  is widely used in the treatment of cancer. Over the past 20 years, an increased understanding of the mechanism of action of 5-FU has led to the development of strategies that increase its anticancer activity. Despite these advances, drug resistance remains a significant limitation to the clinical use of 5-FU. Modulation strategies, such as co-treatment with leucovorin and methotrexate, have been developed to increase the anticancer activity of 5-FU. (Longley et al., 2003).
Chitosan, a natural cationic polysaccharide, is prepared industrially by the hydrolysis of the aminoacetyl groups of chitin, a naturally available marine polymer. Chitosan is a non-toxic, biocompatible and biodegradable polymer and has attracted considerable interest in a wide range of biomedical and pharmaceutical applications including drug delivery, cosmetics, and tissue engineering (Giri et al., 2012). Chitosan nanoparticles are a drug carrier with wide development potential and have the advantage of slow/controlled drug release, which improves drug solubility and stability, enhances efficacy, and reduces toxicity. Because of their small size, they are capable of passing through biological barriers in vivo (such as the blood-brain barrier) and delivering drugs to the lesion site to enhance efficacy (Shi and Fan, 2002).
A new method of drug targeting to a tumor is based on the localized release of drug at the tumor site by ultrasound focusing, the application of the external ultrasound to control drug delivery and to release from nanocarriers is a relatively novel approach. In the application ultrasound triggers the release of the drug from micelles as well as decomposing the membrane, thus enhancing the cellular uptake of both encapsulated and released drugs (Rapoport et al., 2003).

I. Preparation of hollow chitosan nanoparticles
Chitosan has the ability to gel spontaneously on contact with multivalent polyanions due to the formation of inter-and intramolecular cross-linkage mediated by these polyanions. Among some polyanions investigated, tripolyphosphate (TPP) is the most commonly used because of its non-toxic property and quick gelling ability (Nagarwal et al., 2011, Aydin andPulat, 2012) Procedure: Chitosan 0 (Mohanraj and Chen, 2006 At the end of incubation, 0.5 ml of 2, 4-dinitrophenyl hydrazine was added, and the aliquot left for 30 minutes at room temperature. 0.5 ml of 0.4 N NaOH was added, and the aliquot was again left for 30 minutes. Absorbance was then recorded at 505 nm against water blank (Reitman and Frankel, 1957).

Hepatic Alanine aminotransferase activity (ALT)
The enzymatic activity of ALT in tissue homogenate was estimated colorimetrically according to the method of Reitman and Frankel. (Reitman and Frankel, 1957).

Hepatic Malondialdehyde level (MDA)
The hepatic MDA was estimated according to the method of Ottolenghi. The formation of malonaldehyde is the basis for the well-known thiobarbituric acid (TBA) method used for evaluating the extent of lipid peroxidation. At low pH and high temperature (100°C), MDA binds TBA to form a red complex that can be measured at 534 nm (Ottolenghi, 1959).

E. Histopathological examination of liver
Part of the liver was kept in 10% formalin and processed for paraffin sections. Paraffin sections were mounted on clean slides, placed at 37°C oven.
Sections were deparaffinized in xylene, rehydrated in descending grades of alcohol to distilled water, stained with Hematoxylin, washed in tap water then stained with Eosin, dehydrated in ascending grades of alcohol and cleared in xylene. Coverslip applied by Canada balsam and examined under a light microscope (Drury and Wallington, 1980).

F. Statistical analysis
The statistical analysis was computed using the Mann -Whitney test. P values ≤ 0.05 were considered statistically significant.

Serum ALT activity
Feeding of DAB and PB caused a significant increase in serum ALT activities through the different intervals when compared to their corresponding control animals, Figure (1).

Hepatic ALT activity:
The same trend as serum ALT activity, hepatic ALT levels were noticeably increased under the effect of DAB and PB feeding when compared to their corresponding control animals at all intervals Figure (2).

Hepatic MDA level:
Hepatic MDA level was significantly increased in DAB and PB fed mice at all intervals when compared with the corresponding control animals, Figure (3).

D. Histopathological results due to the different therapeutic effects on HCC DAB and PB feeding group (group I)
The liver sections of the untreated group lost its architecture with the appearance of giant cells with large nuclei and central vein congestion (Fig.4a).
While the sections exposed to the ultrasonic waves (US) showed little follicular nodules of infiltrated lymphocytic cells with angiogenesis and appearance of regenerative hepatocytes, i.e. a mild recovery caused by the effect of ultrasonic exposure (Fig.7.b).

Free 5-FU treated group (group II):
After the administration of free 5-FU intraperitoneal injection, the sections had proliferation in the wall of the artery and bile duct, with an increasing number of proliferating lymphocytic cells and a decreased number of apoptotic cells, Figure (8.a). On the other hand, liver sections of this group exposed to the US showed some recovery in liver tissue characterized by proliferating lymphocytes around the bile duct, dilation and congestion of the portal tract, increased number of kupffer cells and regenerative hepatocytes, Figure  (

5-FU loaded Cs nanoparticles treated group (group III) :
The liver sections of this group of mice

Discussion
Carcinogenesis typically involves multiple steps. An initiating step that mutates DNA of the cell, the promotional stage that may involve evasion of apoptosis and uninhibited cell growth in the presence of endogenous or exogenous growth factors. As the neoplasm proceeds, the cells acquire self-sufficiency in growth and overcome inhibitory signals and immune surveillance, followed by angiogenesis and invasion of host tissue (De Minicis et al., 2013, Filler et al., 2007, Hanahan and Weinberg, 2000, Basu, 2018. Owing to the physiologic and genetic similarities between rodents and humans, the laboratory mouse is one of the best experimental systems in defining the pathogenesis of HCC due to the availability of gene targeting methods (Xin et al., 2017).
Chronic use of the azo dye DAB is known to act as an initiator of liver cancer. Phenobarbital (PB), is known to have a carcinogenic effect on humans, mice, and rats when administrated repeatedly. When PB is used in combination with the azo-dye, their effects are more pronounced which unfailingly produce liver tumors that ultimately become neoplastic (Bhattacharjee et al., 2009a). Therefore this induced liver carcinoma serves as a good model for the study of events during carcinogenesis as well as to evaluate the anti-cancerous effect of a drug  (Bakiri and Wagner, 2013 The elevation value of serum ALT has been widely used as a sensitive parameter for the assessment of liver injury degree (Kwo et al., 2017). However, its

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In addition, chronic intake of DAB and PB is known to induce a plethora of cytotoxic and genotoxic alterations (Biswas et al., 2008). It was demonstrated that feeding of carcinogenic azo dyes produced liver damage followed by regeneration of parenchymal cells, a proliferation of bile ducts and connective tissue, and at later stages tumors developed from liver parenchyma that ended up with neoplastic characteristics (Bhattacharjee et al., 2009b). In the present study, at day 15 after DAB and PB feeding, In general, beneficial and reversible cellular effects can be induced by the low-intensity US, in contrast to high US intensities, which are more likely to induce cellular death. Sonoporation is an emerging and promising physical method for drug and gene delivery enhancement in vitro and in vivo (Larina et al., 2005, Zolochevska et al., 2011.
Ultrasound can be used in combination with chemotherapy agents for several reasons. It has been shown to enhance the transport of drugs and other chemicals into cells and tissues. The cytotoxic efficiency of chemotherapeutic agents has been shown to increase under the action of ultrasound.
Since ultrasound increases the local temperature of the exposed tissues, hyperthermia can be used as an additional ultrasonic advantage (Figueiredo and Esenaliev, 2012) The