الفهرس 
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Abstract
SUMMARY
Epilepsy is one of the most common disabling, chronic and socially isolating CNS disorders that affects around 50 million people worldwide. It is a neurological condition in which individuals experience chronic abnormal bursts of electrical discharge in the brain, known as seizures.
The aims of management of epilepsy are to prevent seizures without causing major side effects in order to optimize the patient‟s quality of life. This is achieved by the use of AEDs.
AEDs are prescribed initially at low doses which are increased gradually to reach an effective therapeutic level. However, they can cause significant adverse events such as liver dysfunction, skin rashes, kidney stones, bone marrow suppression, and general CNS depression. OX is one of the most effective antiepileptics for the treatment of almost all types of epilepsy (is the 10 keto analogue of carbamazepine). It is a lipophilic compound and thus, very insoluble in water.
Its antiepileptic action is primarily due to the blockage of voltage sensitive sodium channels, resulting in stabilization of hyper-excited neural membranes and inhibition of repetitive neuronal firing. It also increases potassium conductance, reduces glutaminergic transmission and modulates calcium channel function.
OX is available on the market in conventional oral dosage forms which suffer from 40% plasma protein binding mainly to albumin. Therefore, an alternative route of drug delivery and a dosage form that can selectively target the drug directly into the brain is necessary, but, it
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will face the challenge of circumventing the main barrier to brain delivery, the BBB.
The BBB limits brain penetration of most CNS drugs due to its distinguishing features that cause a highly effective impediment for the entry of chemical compounds into CNS. Many strategies have been explored for circumventing the BBB, amongst them is the use of IN route for directly targeting the brain by direct nose-to-brain delivery across the trigeminal neural axis. Also, loading the drug on lipidic carrier to will allow the system to benefit from passive diffusion of lipid soluble molecules through the BBB. Many lipidic carriers have been investigated, such as liposomes, solid lipid nanoparticles and nanoemulsions. One of the most recent lipid nanoparticles that emerged are emulsomes, which are considered an intermediate between liposomes and emulsions.
It was also necessary to prolong the contact time between the drug loaded lipid nanoparticles and the nasal mucosa for optimal drug uptake, hence, two types of gels were fabricated, namely, cryogels and thermosensitive hydrogels, in order to achieve this goal.
The work in this thesis is divided into four chapters:
Chapter I
Preparation, characterization and optimization of oxcarbazepine-loaded emulsomes
This chapter involved the preparation of OX-emulsomes using the rotary evaporation method. The emulsomes consisted of soya PC sheath
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surrounding a TG. Four TGs were utilized in the preparation of emulsomes, namely, compritol, tristearin, tripalmitin and triolein, each in five PC:TG ratios (0.5:1, 1:1, 2:1 and 3:1 % w/w) and five total lipid amounts (15, 30, 45, 60 and 75 mg).
Emulsomes were characterized by determination of the entrapment efficiencies, particle sizes, zeta potential, vesicle morphologies and in vitro drug release. The effects of different: drug concentrations, TG type, total lipid amounts and PC to TG ratio on the size, charge and entrapment efficiency were studied. The effect of coating emulsomes using surface additives (chitosan and tween 80) in different concentrations was also evaluated on the sizes, charges and drug release. The stability study was conducted after storing the formulae for 3 months at refrigeration temperature. Changes in vesicle sizes, zeta potential and drug leakage from the vesicles were determined.
The results of this work revealed that:
1. An optimum drug concentration of 1% was obtained for maximal entrapment of OX.
2. Vesicle composition had a very strong influence on the entrapment of oxcarbazepine in emulsomes. For each PC to TG ratio, there was an optimum lipid amount in which all TGs had the highest EE%. This optmimum lipid amount was 60 mg for ratios 0.5:1 and 1:1 (except for TS), was 30 mg for the ratio 2:1 and 75 mg for the ratio 3:1 (except for TO). At each ratio, one of the TGs had highest EE% in all total lipid amounts, e.g. compritol was superior in ratio 0.5:1, TP in the ratio 1:1 and TO in the ratio 3:1, whereas, the ratio 2:1 didn‟t have a specific superior TG.
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3. As a general trend in all prepared emulsomes, increasing the content of PC, as reflected by an increase in PC to TG ratio, almost always resulted in an increase in EE%, making the ratio 3:1 ideal for all TGs, except for tristearin, in which the PC to TG of 2:1 was the best with highest EE%.
4. At each PC to TG ratio, increasing the total lipid amount from 15 to 75 mg upon utilizing the TGs C, TS and TP resulted in an increase the particle size of emulsomes. However, TO was an exception because an increase in total lipid amount resulted in a decrease in the particle size. In addition, all sizes obtained were between 120.4 and 567.8 nm.
5. All emulsomes exhibited a highly negative charge in the range of -28 to -67 mV.
6. In vitro drug release was conducted for C13, TO8, TO13, TO17 and TO18 because those were the formulations that had sizes below 200 nm with EE% above 60%. The study revealed that the least cumulative amount of OX released from emulsomes after 24 hrs was obtained from TO8 and the highest from TO17 and C13, with no significant variations between both.
7. Surface coating of the selected OX-emulsomal formulations with increasing concentrations of low molecular weight chitosan resulted in an increase in particle size and zeta potential. On the other hand, coating with increasing concentrations of Tween 80 resulted in an increase in zeta potential, a decrease in size, as well as, a marked increase in OX release.
8. The stability study revealed minute increases in size and zeta potential of emulsomes with high percentages in drug retained after storage for 3 months.
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9. The formulation of choice for subsequent loading into gels and biological studies was TO17-Tw emulsomes which consisted of 30 mg total lipid amount (PC + TO) in a ratio of 3:1. It had a size of 101.5 nm, a zeta potential of -6.7 mV and 81% drug release. It was proven morphologically by transmission electron microscopy to be spherical in shape with a surrounding PC bilayer.
Chapter II
Preparation, characterization and emulsomal-loading of Polyethylene glycol diacrylate (PEGDA) cryogels
This chapter involved the cryopolymerization of polyethylene glycol diacrylate (PEGDA) by redox initiated free radical polymerization of PEGDA monomer in the presence of ammomium per sulphate (APS) as an initiator and terta ethylmethyl ethylene diamine (TEMED) as an accelerator. Different monomer concentrations were tested (10, 5 and 2.5 % w/w) in the presence of different equimolar concentrations of APS/TEMED (1, 5 and 10 mM). The reaction mixtures were frozen for different time intervals (3, 6, 9 and 12 hrs).
The prepared cryogels were evaluated post thawing in terms of viscosity, swelling, water uptake capacity, mesh size and hydrolytic degradation. The cryogel formation was confirmed by fourier transform infrared spectroscopy and the macroporous structure was visualized by scanning electron microscopy. A factorial study was implemented in order to study in depth the influence of the varied factors on the viscosity of the cryogels. Finally, OX-loaded Emulsomes were sequestered within
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the chosen cryogel, with subsequent measurement of the emulsomal cryogel viscosity and in vitro drug release evaluation.
The results of this work revealed that:
1. Shear rate/shear stress plots showed that all cryogels exhibited shear thinning flow. Increasing PEGDA and APS/TEMED concentrations resulted in stiffer cryogels with higher shearing stress values. Cryogels B and C deformed at different shear rates, whereas, cryogels D to I were stiffer and did not deform up to shear rates of 400 min-1.
2. At each PEGDA concentration, raising APS/TEMED concentration and prolonging freezing time resulted in cryogels with higher viscosities. Increasing monomer concentration also had the same influence on viscosity, thus, I cryogels had the highest viscosity and B had the lowest (A cryogels did not polymerize). Subjecting the cryogels to higher rpm values caused marked reductions in their viscosities, with most pronounced declines occurring at specific rpms that differed between the cryogels.
3. Factorial analysis revealed that:
a. The studied factors (PEGDA concentration, APS/TEMED concentration and freezing time) at all their levels significantly affected the viscosity of cryogels (p<0.0001).
b. Significant two-way and three-way interactions between all factors were obtained with an overall increase in viscosity with an increase in all the tested factors.
4. After immersion of dried cryogels in water, they all started swelling after 5 min, with cryogel B9 have maximum swelling
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of 76.9 % in comparison to E9 and H9 that swelled up to 53.7 and 22.4 %, respectively. This proved that increasing PEGDA concentration reduced the swelling and water uptake capacity of cryogels.
5. Cryogel B9 had larger mesh size and molecular weight between cross links when compared to its counterparts, E9 and H9.
6. Cryogels with lower PEGDA concentration had higher degradation rates than stiffer ones prepared from higher PEGDA concentration. Hence, cryogel B9 completely dissolved after immersion in water for 4 weeks, while cryogels E9 and H9 retained 53 and 85.5 %, respectively, of their masses.
7. FTIR revealed the shifting and near attenuation of the characteristic carbonyl and vinyl groups of PEGDA indication successful cryopolymerization.
8. SEM of B9 cryogel confirmed its macroporous network structure.
9. Loading of TO17-Tw emulsomes in B9 cryogel resulted in a significant increase in its viscosity and a reduction in cumulative amount of oxcarbazepine released after 24 hrs.
10. Therefore, the formulation of choice for further biological studies was TO17-Tw/B9 emulsomal cryogel.
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Chapter III
Synthesis, characterization & emulsomal-loading of PLGA-PEG-PLGA thermosensitive triblock copolymer
This chapter involved the ring opening polymerization of lactic acid and glycolic acid in the presence of polyethylene glycol (PEG) as a macroinitiator in order to synthesis PLGA-PEG-PLGA triblock copolymer, which is a thermosensitive gel capable of gelling at a temperature below body temperature. Thermogels were prepared from different copolymer concentrations (5, 10, 20 and 30 % w/w) and Emulsomes were sequestered within the thermogel matrices in different concentrations (0, 10, 25 and 50 % v/v).
PLGA-PEG-PLGA copolymer was characterized by nuclear magnetic resonance and gel permeation chromatography in order to determine the polymer‟s molecular weight. Differential scanning colorimetry, dynamic light scattering and rheological studies were also conducted. Sol-gel transition temperature was measured for all the prepared thermogels by tube inversion method. Determination of the viscosities of the thermogels was also carried out. In vitro drug release and mucoadhesion studies were conducted on the chosen thermogel.
The results of this work revealed that:
1. The H1NMR spectrum of PLGA-PEG-PLGA copolymer revealed the characteristic peaks of lactide, glycolide and PEG protons, confirming successful polymerization. According to NMR, the copolymer had a number average molecular weight of 3380 Da with a formula of: (PEG)34 (lactide)36 (glycolide)10.
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2. GPC spectrum showed an earlier elution of PLGA-PEG-PLGA copolymer than PEG polymer, again confirming polymerization. According to GPC, the copolymer had a number average molecular weight of 4491 Da and a weight average molecular weight of 7610 Da.
3. The DSC thermograms revealed a sharp peak for PEG with a melting point of 49.2 ºC and Tg of the copolymer at -13.3 ºC.
4. Rheological studies revealed the following:
a. According to the temperature sweep test, increasing the temperature resulted in a continuous increase in viscosity until the critical gelation temperature was reached and full gelation achieved, after which, further rise in temperature caused a precipitation of the copolymer and the gel reverted back to sol.
b. According to the time sweep test, PLGA-PEG-PLGA thermogels required 900 sec to reach rheological stability.
c. According to the frequency sweep test, PLGA-PEG-PLGA polymer passed from a system in which G’ prevails to a system in which G” prevails with increasing frequencies, which is a behavior typical of visco-elastic solids.
5. DLS measurement ensured that the sizes of micelles formed upon dissolving PLGA-PEG-PLGA in water were not influenced by increases in temperature, until gelation was reached
6. Thermogel prepared from 5 and 10 % w/w did not gel completely. Sol-gel transition temperature of thermogels decreased upon increasing the copolymer concentration from 20 to 30 % w/w, but all were below body temperature.