الفهرس 
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Abstract
Cystic Echinococcosis is caused by the larva stage of the tapeworm Echinococcus granulosus and produce a long-lasting infection in humans and animals which is a serious public health problem worldwide
There has been a growing interest during the last decades regarding zoonotic infection. World Health organization has included echinococcosis as a neglected zoonosis subgroups in its 2008-2015 strategic plans for the control of neglected tropical diseases.
The wide distribution of Hydatid infection makes finding a safe and effective drugs a great succeess. The standard therapy is still hampered by sever adverse effects, current therapeutics ABZ do not decrease parasite infection and are often not satisfactory to patients.
In the search for new startegies that overcome the draw backs of previous drugs, nanomedicine finds its way to provide applicability for old drugs by providing their biodistribution, bioavailability and decrease toxicity.
Chitosan is widely distributed in nature as a component of bacterial cell walls and exoskeletons crustaceans and insects. Chitosan has been reported to possess immunological, antibacterial and wound healing properties, it has a biomedical application, cationic polymer with capacity for increasing penetration of drugs across barriers.
In the light of the above, the current work aimed to evaluate the activity of Albendazole, Albendazole Loaded Chitosan Naoparticles and Nitazoxanide against hydatid protoscoleces (In vivo studies)
In the current work, Cs NPs, ABZ, ABZ loaded Cs NPs, NTZ, NTZ loaded Cs NPs were prepared and were evaluated as a treatment of experimentally studied mice.
To fulfill the aim of the present study, the NPs were prepared by ionotropic gelation method and charactized determined by by using TEM, Zetasizer and spectrophotometer estimation of loading efficiency.
To study the drug efficacy on infected hydatidosis mice,170 male swiss Albino mice were infected intra petitoneal with 100 protoscoleces collected from naturally infected H.cysts from slaughtered camals.
Mice were divided into three group, control (20 mice), chemoprophylactically treated group (50 mice) and Therapeutic group (50 mice), another 50 mice infected and treated as chemoprophylactically treated (no 25), 25 mice of Therapeutic mice for study survival times and mortality rates, these groups were subdivided into subgroups as follows:
I. Chemoprophylactically treated Efficacy study group.
• The treatments were started 24 hours before the infection and were continued for a month in groups of ABZ, ABZ/Cs NPs and Cs NPs and 14 days in NTZ and NTZ/Cs NPs by intragastric administration by oesophagyl syringe (0.3 ml /mouse).
• Males infected mice (no=50) were allocated into five experimental subgroups and were treated as follows:
Subgroup 1: ABZ chemoprophylactically treated subgroup. (10 infected mice, treated with ABZ 0.3 ml contain 50mg/Kg body weight/mouse daily for one month)
Subgroup 2: ABZ loaded Cs NPs chemoprophylactically treated subgroup. (10 infected mice, treated with ABZ/Cs NPs 0.3 ml contain 50mg/Kg body weight /mouse daily for one month)
Subgroup 3: NTZ administered chemoprophylactically treated subgroup. (10 infected mice, treated with NTZ 0.3 ml contain 200 mg/kg body weight/mouse daily for 14 days)
Subgroup 4: NTZ loaded Cs NPs chemoprophylactically treated subgroup (10 infected mice, treated with NTZ/Cs NPs 0.3 ml contain 200 mg/kg body weight /mouse daily for 14 days)
Subgroup 5: Cs NPs administered infected subgroup (10 infected mice, treated with Cs NPs (30 mg/kg in 0.3 ml /mouse daily for one month)
All mice were sacrificed by cervical dislocation after 24 hrs of the end dose of treatments and necropsy was done to detect presence of Hydatid cysts in any organs (Liver, Lung and Spleen)
II. Therapeutic study group.
A three months of infection, 50 mice with suspected protoscoleces were allocated into the following experimental subgroups (10mice / each subgroup) and were treated as follows:
Subgroup a: Mice were treated with ABZ. (ABZ concentration was 50 mg/ kg body weight in 0.3 ml /mouse daily for one month)
Subgroup b: Mice received ABZ/Cs NPs. (ABZ/Cs NPs concentration was 50 mg/ kg body weight in 0.3 ml /mouse daily for one month)
Subgroup c: Infected Mice were treated with NTZ. (NTZ concentration was 200 mg / kg day weight in 0.3 ml /mouse daily for 14 days)
Subgroup d: infected Mice received a NTZ/Cs NPs. (NTZ/Cs NPs concentration was 200 mg / kg of mouse day weight in 0.3 ml /mouse daily for 14 days)
Subgroup e: Mice were treated with Cs NPs. (Cs NPs concentration was 30 mg / Kg in 0.3 ml /mouse daily for one month).
Experimental infected studied mice groups were repeated again by 25 mice to cover the all death and study the survival times and mortality rates.
III. Control group: (20 mice (.
It was further subdivided into two equal subgroups (10 /each subgroup).
Control subgroup a: normal uninfected control subgroup, this subgroup served for survival times, mortality rates of mice as well as the histopathological study.
Control subgroup b: infected untreated control subgroup, each mouse received 0.3 ml saline orally by oesophageal needle starting with treated subgroups as placebo served for survival times, mortality rates, histopathological study.
Used of experimental animals in the study was carried out in accordance with the Ethical Guidelines of the Medical Research Institute, Alexandria University (AU01219091511).
• Cs NPs: The dose of 30 mg/Kg body weight of mouse orally daily for four weeks
• ABZ: was used as a gold standard for treatment of hydatidosis at a dose of 50 mg/ kg body weight orally daily for four weeks.
• ABZ/Cs NPs: was used at doses of 50 mg/ kg body weight of mouse orally daily for four weeks.
• NTZ: was used in a dose of 200 mg / kg body weight of mouse orally daily for 14 days.
• NTZ/Cs NPs: was administered at dose of 200 mg/ kg body weight of mouse, orally daily for 14 days
Drug efficacy was assessed by parasitological Therapeutic observation and sub acute toxicity study of Cs NPs and drug loaded Cs NPs, Bioassay of Cs NPs and drugs loaded Cs NPs on mice of experimental subgroups (survival time and mortality rate) distribution of detected H.cysts, SEM morphology of isolated H.cysts, viability protoscoleces collected.
Histopathological study was performed for Hydatid cysts isolated from each subgroups and neighaboring tissue.They were stained with H&E stain for confirmation of infection.
The data were entered; verified and analyzed using SPSS softwere using appropriate statistical tests. differences and associations were considered statistically significant at p<0.05.
The results of physicochemical characterization of Cs NPs and drugs loaded Cs NPs showed that Cs NPs was microsphere consist of a number of crystallites with rutile structure, with a size range 2.57 - 14.62 µm. ABZ/Cs NPs ultra picture by TEM illustrate Albendazole successfully entrapped by CS NPs, increased spherical size, filled with Albendazole, their size range from 274.25 – 587.88 µm. NTZ/CS NPs ultra picture by TEM were with different shape from spherical polyclonal with enlarged size range from 50.81 – 113.15 µm.
The hydrodynamic particles size, Zeta potential and polydisparity index (PDI) were measured by Zetasizer instrument. The mean size Cs NPs, ABZ/Cs NPs and NTZ/Cs NPs
were 3351 nm, 5497 nm, and 2446 nm respectively, with PDI of 0.362, 0.082 and 0.616 respectively PDI was less than 0.5 which indicate medication homogeneous Mature, nanometer favourable particles size distribution..
The drug loading efficiency determined by spectrophotometric method, they were found to be 79.9% in ABZ/Cs NPs at 342 nm and 85.3% in NTZ/Cs NPs at 346 nm as calculated from the standerd curve.
In the current study, Assessment of drug efficacy on infected experimental mice carried out by parasitological studies, as Therapeutic behaviour, survival time, weight gained mortality rate, of all mice groups. All hydatidosis experimental mice treated with Cs NPs and those treated with drugs/Cs NPs showed normal health status through study periods without signs of toxicity in prophylactically treated group of mice, where as Therapeutic mice treated with drug alone (ABZ and NTZ) showed irritating observation after 5 days of drug administration, physical activity decreased after 7 days, and decreased in amount food feeding, aggrasive, loss their hairs, these features were increased until death.
Behaviour activity and appearance of mice subgroups treated with ABZ/Cs NPs were better followed by those treated with ABZ alone and those treated with NTZ/Cs NPs.
As regarding to Bioassay of Cs NPs and drugs loaded Cs NPs on experimental mice were studied, survival time in prophylactically treated mice group uninfected untreated control subgroup illustrated a mean survival time of 334.5 ± 58.38 days, Cs NPs alone, ABZ alone, ABZ/Cs NPs, NTZ alone and NTZ/Cs NPs subgroups mice were able to induce significant increase in the mean survival time in the infected mice (246.1 ± 34.97, 244.5 ± 39.42, 340.5 ± 47.27, 218 ± 40.17 and 253.1 ± 36.40 days respectively), in Therapeutic infected Hydatidosis mice subgroups mean survival times showed that Cs NPs alone, ABZ alone, ABZ/Cs NPs, NTZ alone and NTZ/Cs NPs subgroups mice (224.9 ± 37.73, 234.1 ± 41.57, 302.9 ± 44.09, 152 ± 34.81 and 189.7 ± 46.34 days respectively), were able to slightly increase with statistically unsignificant of survival time.
In the present study, the visual macroscopic collection of H.cysts from different organs of Therapeutic mice group as indicator for treatment loaded Cs NPs efficiency, liver, lung, spleen were appeared slightly larger than those organs in infected untreated control group. The statistically significant reduction was observed in the cyst numbers in liver, lung and spleen among all sub groups mice treated with Cs NPs and drugs loaded Cs NPs. liver act as the main infected organ followed by lung and the lastly spleen. This could be attributed to the degree of tissue elasticity and softness of organ.
All cysts collected from infected treated subgroups mice were decreased in number and degenerated less cyst weight in Cs NPs or drugs loaded Cs NPs subgroups, its were filled with fluid but when examined this fluid by eosin stained almost protoscoleces present in these cysts were dead absorb the pink colour of stain. This could be due to immune competence of the host (mice).
SEM ultra pictures of morphological study of isolated H.cysts collected from subgroups of infected treated mice normal untreated cysts (control group) with smooth surface rounded cyst. While those treated with Cs NPs alone showed a broken separated capsule of cysts from internal content. Cysts isolated from mice treated with ABZ/ Cs NPs
were illustrated by SEM with shrinkage appearance, larger in sizes and erosion their capsule surrounded the cyst. These mean adsorption and penetration of drugs/ Cs NPs. In case of treated mice with NTZ alone or NTZ/ Cs NPs, all cysts ultrastructured appeared as normal in size except some erosions or broken in capsule.
When, SEM were performed on protoscoleces collected from fluid of cysts from subgroups treated mice. They were delimiting anterior scolex with a stalk as posterior caudal body region attached to it with erosions and distortion loss in smooth surface among subgroups mice treated with Cs NPs or drugs loaded Cs NPs.
The current work of histopathological changes in liver and lung of all hydatidosis experimental mice in subgroups mice treated with Cs NPs or/and druges/Cs NPs illustrated, decreased the effect of pathological and cellular infiltration around the developed cyst comprised in tissue as compared to infected untreated mice control group.
In a subgroups mice infected hydatidosis treated with Cs NPs alone illustrate congested parenchyma loose of laminated layer with infiltration by mononuclear infiltration cells, when drugs loaded with Cs NPs the pathological changes are become less than the untreated subgroups, regenerative effect with degenerated H. cyst were noticed in the subgroups of ABZ loaded with Cs NPs