الفهرس 
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Abstract
In the present study, we prepared chitosan magnetic microspheres and study its effect on dynamic properties of blood. The prepared chitosan magnetic microspheres were characterized in terms of morphology by scanning electron microscope, Vibrating-sample magnetometer (VSM) to study the magnetic properties, Fourier-transform Infrared spectroscopy (FTIR) to investigate function groups, X-ray Diffraction (XRD) pattern in order to evaluate crystalline state and measurement of Thermogravimetric Analysis (TGA) to understand the behavior of the cross-linked chitosan magnetic microspheres on application of thermal energy and the chitosan magnetic microspheres thermal stability.
To study the effect of chitosan magnetic microspheres on dynamic properties of blood under the influence of magnetic field, two coils were designed in our laboratories. The first one was a solenoid with 100 t/cm in order to give magnetic field in the range of 0.016 to 0.6 mT to study the effect of magnetic field on blood samples viscosity with an without chitosan magnetic microspheres. The second coil was designed to creat strong magnetic field. The coil was wounded on iron core from 0.1 cm isolated copper wire with total length of 5 cm and cross sectional area of 10 cm2 with 100 t/cm. The coil was calibrated by teslameter found to have magnetic field in range of 1.5 to 10 mT, to study the erythrocytes aggregation.
Blood samples were obtained from healthy 20 volunteers; 10 ml from each was collected with EDTA as an anticoagulant. One ml of the blood samples were sent to hematology laboratory for the determination of RBC’s count, mean cell volume (MCV), hematocrit (HCT), mean corpuscle hemoglobin (MCH), mean corpuscle hemoglobin concentration (MCHC), hemoglobin (HGB), red cell distribution width (RDW) and serum cholesterol. The rest of the samples were divided into four parts for the measurement of blood viscosity, RBCs aggregation, osmotic fragility and Hemolysis. Part one used whole blood for viscosity measurements. For the left part separation of erythrocytes was obtained by centrifugation at 3000 rpm for 10 min. Plasma and Buffy coat were removed and the red blood cells were washed in isotonic phosphate-buffered saline solution (PBS)(PBS; pH=7.4; osmolality 290±3 mOsm/Kg) for three times. The left parts are classified into three parts. Part one used as 5% suspension for microscopic study of RBCs aggregation. Part two used for osmotic fragility. Part three used for blood Hemolysis measurements.
The blood samples were classified into four groups in order to study the blood viscosity, RBC’S aggregation, osmotic fragility and Hemolysis for blood samples before and after adding chitosan magnetic microphages. Also the effect of magnetic field in these samples was studied.
Group 1: The control group.
Group 2: The blood samples exposed to magnetic field of different values (0.16, 0.24, 0.3, 0.4, 0.5, 0.6 mT).
Group 3: The blood samples after adding the magnetic microspheres.
Group 4: The blood samples after adding the magnetic microspheres and exposed to magnetic fields of different voltage (0.016, 0.24, 0.3, 0.4, 0.5, 0.6 mT).
II- Conclusions
1- Preparation and characterization of chitosan magnetic microspheres
• The SEM photomicrographs of the chitosan magnetic microspheres said that the shapes of the dried microspheres were spherical, and the surface was rough, porous and unfolded. Furthermore the microspheres are not hollow. Mean particle size for magnetic microspheres was ranged from 50-300 µm.
• An x-ray diffraction profile of chitosan sample in dry state is being hydrated polymorphs (refers to the bound water, ~ 10 % present in chitosan sample, even after the material was dried), chitosan sample showed a strong reflection at 2Ə around 10―12o. In the hydrated crystals, the incorporation of bound water molecules into the crystal lattice gives rise to the more abundant polymorph, `Tendon` form of chitosan sample, having this reflection at 2Ə=11.3o (d= 7.76255Å), additionally a small peak recorded at around 2Ə=15―17o, due to anhydrous crystal lattice. Also the second characteristic peak with strong reflection appears at 2Ə=20.2o for chitosan was showed in sample at 2Ə=20.2o (d=4.37 Å). This peak corresponded to crystal forms amid II. An x-ray diffraction profile of chitosan magnetic microspheres in dry state is showed a strong reflection at 2Ə around 35o which could be due to the presence of Fe3O4 in the formed magnetic microspheres and also referring to the cart of XRD of magnetite (Fe3O4) ( Iron Oxide, Cart no: 88-0315, pcpdf WIN).Also the second characteristic peak with low intensity appears at 2Ə=20.1o for chitosan was showed in sample at 2Ə=20.1o (d=4.42 Å). This peak corresponded to crystal forms amid II which of low intensity in comparison with that appears at XRD of chitosan. The distinct difference in the diffraction patterns of chitosan sample and chitosan magnetic microspheres could be attributed to modification in the arrangement of molecules in crystal lattice due to the cross-linking between chitosan and magnetic fluid.
• Fourier-transform Infrared spectra of chitosan, Chitosan magnetic microspheres. For the IR spectrum of chitosan the characteristic absorption bands appeared at 3433 cm-1 indicate to hydroxyl group, 1641 cm-1 indicate to amide I, 1573 cm-1 indicate amide II, 1382 cm-1 indicate to amide III, band at 2920 cm-1 indicate to C-H stretching of polymer, band at 1029& 1080 cm-1 indicate to C-O stretching and band at 1622 cm-1 indicate to NH3 absorption of chitosan. Compared with the spectrum of chitosan magnetic microspheres the 3433 cm-1 peak of hydroxyl group shifted to 3409 cm-1 and a decrease in peak intensity of amid I (–NH2) group at 1566 cm−1 which indicated the ionic cross-linking between amid I (–NH2) group of chitosan and –C=O groups of glutraldehyde and new characteristic band appear at 1650 cm-1 indicate to C=N group which indicate to cross-linking.
• Thermal gravimetric Analysis showed that the chitosan powder showed a small peak at 26-106 0C due to the volatization of the solvent. Other strong transitions were found at 253-337 0C due to faster decomposition of chitosan powder. In case of chitosan magnetic microspheres the thermal gravimetric analysis shown a small peak at 26-79 0C due to the volatization of the solvent. Other strong peak at 210-298 0C and this may be due to the faster decomposition of chitosan magnetic microspheres. The last peak appeared at 412-495 0C due to glutraldehyde cross-linking. The difference in the thermal analysis between the two figures of chitosan powder and magnetic chitosan microspheres may be due to increase of thermal stability of magnetic microspheres than chitosan.
• The magnetic properties including hysteresis loop, saturation magnetization and coercivity of chitosan magnetic microspheres were measured in this research. In which the internal area of hysteresis loop represents the capability of magnetic energy storage of magnetic materials, which is an important parameter in electromagnetic absorption field. The hysteresis loop with great area brings on a large loss. The internal areas of hysteresis loops are great, which can be used as electromagnetic absorption materials. Moreover, the area of hysteresis loop becomes greater with the increase of the magnetic fluid. In the first fig. 48 the saturation magnetization were 8.065emu/g, coercivity were 76.05 Oe and retentivity were 1.057 emu/g. The second fig. 49 the saturation magnetization were 6.205 emu/g, coercivity were 61.81 Oe and retentivity were 1.099 emu/g. In which the maximum field were 5000 Oe and the same mass. from this data the first hysteresis loop represent the chitosan magnetic microspheres which have small particle size than the other one. Depending on that as the saturation magnetization and coercivity increase, the particle size decrease. The shape of the hysteresis loop tells a great deal about the material being magnetized. A material with narrow hysteresis loop has:
• Higher permeability
• Lower retentivity
• Lower coercivity
• Lower relucatance
• Lower residual magnetism.
• The particle size distribution curve shows sharp distribution range of microspheres, with 90%of spheres in size range of 1000-2600 nm with average particle size of 1800nm and only 10% were oversized.
3- In vitro study
• In our experiments, we studied the effect of magnetic chitosan microspheres with and without the application of AC magnetic field in the range (0.016, 0.24, 0.3, 0.4, 0.5, and 0.6) mT on viscosity of blood. Our results showed significant increase in viscosity of blood incubated with magnetic chitosan microspheres without application of magnetic field compared to control. On application of AC magnetic field, viscosity of blood showed higher increase compared to erythrocytes suspension incubated with magnetic chitosan microspheres without application of magnetic field, where the viscosity of blood increase as the magnetic field strength increase.
• The results concerning the effect of prepared microspheres on the osmotic fragility of RBCs showed a change in the fragility of RBCs exposed and not exposed to magnetic field. The results for RBCs not exposed to magnetic field showed that the mean corpuscular fragility (M.C.F.), which is the concentration of saline causing lysis at 50 %, was shifted from 0.49% NaCl for Normal RBCs with no microspheres to 0.5% NaCl for that of treated with magnetic microspheres. The results for RBC’s exposed to magnetic field shown that the mean corpuscular fragility (M.C.F.) was shifted from 0.52% NaCl for Normal RBC’s with no microspheres to 0.6% NaCl for that of treated with magnetic microspheres.
• Image analysis indicates a decrease in the aggregation shape parameters (ASP) with increasing of the aggregation size. RBCs treated by magnetic field with different strength (1.5, 2, 4, 6, 8, 10 mT) by no incubation with chitosan magnetic microspheres showed significant decrease on ASP. RBCs treated by magnetic field with different strength (1.5, 2, 4, 6, 8, 10 mT) by incubation with chitosan magnetic microspheres showed also a significant decrease on ASP but with low values. In which the incubation of chitosan magnetic microspheres with the RBCs has non significant effect on ASP.
• from the processed images of the aggregates, both the projected area of aggregates (PAA) and projected perimeter of aggregates (PPA) of various samples were calculated. The variation in the mean form factor (FF) of the normal RBCs suspension by no incubation with chitosan magnetic microspheres treated with magnetic field with different strength (1.5, 2, 4, 6, 8, 10 mT) has been studied and showed that there was increase in the mean form factor with increasing the magnetic field strength. The variation in the mean form factor (FF) of the normal RBCs suspension by incubation with chitosan magnetic microspheres treated with magnetic field with different strength (1.5, 2, 4, 6, 8, 10 mT) has been studied and showed that there is increase in the mean form factor with increasing the magnetic field strength. This means that the incubation of chitosan magnetic microspheres with RBCs has no singnficant effect on the mean form factor.
• The effect of magnetic field on RBCs hemolysis and no incubation with chitosan magnetic microspheres has been studied. Exposure of RBCs suspension to magnetic field at different strength (1.5, 2, 4, 6, 8, 10 mT) and no incubation with chitosan magnetic microspheres at different exposure time (3-15 min) showed increasing the hemolysis with increasing magnetic field strength at constsnt exposure time and increasing exposure time at constant magnetic field strength. Exposure of RBCs suspension to magnetic field at different strength (1.5, 2, 4, 6, 8, 10 mT) and incubation with chitosan magnetic microspheres at different exposure time (3-15 min) showed increasing the hemolysis with increasing magnetic field strength at constsnt exposure time and increasing exposure time at constant magnetic field strength. In which the incubation of chitosan magnetic microspheres showed non singnficant effect on the hemolysis of RBCs at constant magnetic field and exposure time in comparison with the control.