الفهرس 
Tacrolimus-loaded chitosan nanoparticles prepared using a modified ionic gelation techniqueOnly 14 pages are availabe for public view
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Abstract
Psoriasis is a chronic dermatological disease with original autoimmune pathogenesis. It is manifested as erythematous skin lesions covered by silvery scales, known as psoriatic plaques. This creates psychological burdens on patients’ lives. Moreover, the barrier function of the skin is disrupted by the appearance of these lesions. Therefore, physiological dysfunction is also associated with the disease.
Unfortunately, psoriasis is non-curable to-date. Therefore, the disease is managed by symptomatic treatment through chronic administration of anti-inflammatory and immune-suppressant agents, either topically or systemically. Systemic therapy is crucial for moderate to severe psoriasis.
Tacrolimus is a potent hydrophobic immune suppressant that has reported anti-psoriatic activity. It is used in an off-label scope for the management of psoriasis. Its immune suppressant action is reported to be superior to that of cyclosporine. However, systemic administration of tacrolimus, particularly for chronic use may lead to serious adverse effects such as: nephrotoxicity and hypertension.
Nanotechnology is an emerging field of science that achieves many benefits in drug delivery purposes. Polymeric nanoparticles, particularly charged ones, are reported to have higher affinity to the skin, especially upon incorporating hydrophobic agents. Another interesting finding in literature, was that polymeric nanoparticles have higher affinity to diseased skin rather than healthy one.
Chitosan is a natural cationic polymer that exhibits healing properties on burns and wounds. Therefore, it is often used for dermatological purposes.
Hybrid nanoparticle-systems are an emerging type of drug delivery systems that combine the advantageous properties of more than one nanoparticle type. Lecithin-chitosan hybrid was previously synthesized and was proved to have promising results for the incorporation of hydrophobic agents. It was also reported to achieve superior skin retention with minimal systemic absorption.
Gold nanoparticles exhibit anti-inflammatory and healing properties. Their potential success to synergize anti-psoriatic efficacy of herbal extracts as berry extracts and chemical anti-psoriatic agents such as: methotrexate is reported in literature.
The work in this thesis was divided into three chapters:
Chapter I: Tacrolimus-loaded chitosan nanoparticles prepared using a modified ionic gelation technique:
The work in this chapter involved the preparation of tacrolimus-loaded chitosan nanoparticles using a modified ionic gelation formulation technique. The purpose of developing this technique was to prepare the aforementioned particles using the simplest and most reproducible possible method with the elimination of using hazardous organic solvents. Hence, no emulsion containing preparation methods were used despite the fact that the drug is hydrophobic and the polymer is hydrophilic. The preparation technique involved adding an initial step to the conventional ionotropic gelation in which the drug was solubilized in a water-miscible organic solvent.
Several formulation variables were assessed, including; pH of Cs solution, ratio of Cs: TPP: co-solvent, type of co-solvent, Cs solution concentration and drug concentration. The selected formulation was further evaluated for in vitro drug release, ex-vivo skin permeation and retention, microscopic examination by TEM and SEM, FT-IR, X-ray diffraction and a three-months stability test. Moreover, an in vivo experiment was conducted on IMQ-induced mouse model to assess the anti-psoriatic efficacy in comparison to the marketed product.
The findings of this chapter revealed the following:
• Tacrolimus-loaded chitosan nanoparticles were successfully prepared using the novel formulation technique.
• The selected formulation (C9) was prepared using 0.1% chitosan solution (pH=4), 0.05% TPP solution, propylene glycol as co-solvent and 0.03% tacrolimus at a ratio; 5: 2: 3, respectively. It yielded a particle size ;140.23 nm ± 14.59, PDI 0.43 ± 0.11, surface charge; 22.2 mV± 4.06, EE%; 65.45% ± 1.3 and DL%; 39.27% ± 0.79.
• Microscopic examination by TEM and SEM confirmed the formation of spherical nanoparticles with no signs of aggregation and a particle size range consistent with the findings obtained by dynamic light scattering.
• In vitro drug test release showed a controlled release pattern that fitted the Higuchi model which denotes that the drug release took place by diffusion.
• The synthesized formulation achieved significantly higher skin deposition than the marketed product (82.07% vs. 34.07%) through a 24-hour time interval, particularly, to the stratum corneum and epidermis skin layers. Moreover, skin permeation was significantly lower than that of the marketed product (24.30% vs. 61.23%).
• Ft-IR analysis and X-ray diffraction confirmed the successful encapsulation of tacrolimus inside the synthesized chitosan nanoparticles.
• The synthesized nanoparticles showed acceptable stability of a 3-months period with no significant changes in particle size or PDI and minor change in EE%.
• The in vivo results on IMQ-mouse models revealed superior anti-psoriatic efficacy of the synthesized nanoparticles in comparison to the marketed product in terms of; visual observation and PASI score.
• The in vivo drug deposition showed superior skin deposition of the nanoparticles compared to the marketed product (54.65% vs. 13.4%). The results were in accordance with the findings obtained from the ex-vivo skin deposition test.
• Histopathological examination of skin samples confirmed the superior improvement of the group treated with tacrolimus-loaded chitosan nanoparticles compared to those treated with the marketed product.
Chapter II: Lecithin-Chitosan Hybrid nanoparticles incorporating tacrolimus using a modified formulation technique:
The work in this chapter involved the preparation of tacrolimus-loaded hybrid lecithin-chitosan nanoparticles using a suitable co-solvent to maximize the entrapment of the drug. A pre-liminary study was conducted to select a suitable co-solvent for further experimentation. Then, formulation studies were performed to assess different variables; L: Cs ratio, Cs solution concentration, co-solvent type and drug concentration. The selected formulations were further assessed by an in vitro drug release test. Further experimentation was conducted on the selected formulation by an ex-vivo skin permeation and retention test, microscopic examination by TEM, Ft-IR, DSC and a three-months stability test. Afterwards, an in vivo experiment was conducted on IMQ-induced mouse model to assess the anti-psoriatic efficacy in comparison to the marketed product.
The findings of this chapter revealed the following:
• Tacrolimus-loaded lecithin-chitosan nanoparticles were successfully prepared.
• Olive oil and Tween 80 are suitable co-solvent types for incorporation of tacrolimus into hybrid particles; Tween 80 allows higher drug release than olive oil.
• The selected formula (L11) was prepared using 0.5% Cs at a L:Cs ratio of 10:1, using Tween 80 as a co-solvent and 0.03% tacrolimus. It yielded a particle size; 118.7 nm ± 13.3, PDI; 0.43 ± 0.13, surface charge; 16.2 mV± 2.4, EE%; 66.72% ± 1.8 and DL%; 18.2% ± 0.5.
• In vitro drug release profile showed higher release from the formula synthesized with Tween 80 compared to that synthesized with olive oil. The release fitted zero order kinetic model.
• Microscopic examination by TEM confirmed the previous findings by showing the formation of spherical nanoparticles with a core and corona of different nature with no signs of aggregation.
• The synthesized formulation achieved significantly higher skin deposition than the marketed product (63.51% vs. 34.07%) through a 24-hour time interval, particularly, to the stratum corneum and epidermis skin layers. Moreover, skin permeation was significantly than that of the marketed product (40.83% vs. 61.23%).
• Ft-IR analysis and DSC confirmed the successful encapsulation of tacrolimus inside the synthesized lecithin-chitosan nanoparticles.
• The in vivo results on IMQ-mouse models revealed superior anti-psoriatic efficacy of the synthesized nanoparticles in comparison to the marketed product in terms of; visual observation and PASI score.
• The in vivo drug deposition showed superior skin deposition of the nanoparticles compared to the marketed product (74.9% vs. 13.4%). The results were in accordance with the findings obtained from the ex-vivo skin deposition test.
• Histopathological examination of skin samples confirmed the superior improvement of the skin condition of the group treated by tacrolimus-loaded lecithin-chitosan nanoparticles compared to the marketed product. The skin condition was comparable to that of the negative control group.
Chapter III: Gold nanoparticles hybridized with chitosan and lecithin-chitosan nanoparticles loaded with tacrolimus:
The work in this chapter involved the hybridization of the selected formulations from chapters I and II (C9 and L11) with negatively-charged gold nanoparticles. Confirmatory tests were conducted to identify on gold nanoparticles; XRD and elemental mapping by SAED using TEM. The hybrid products were characterized in terms of visual inspection of color change, particle size, PDI, ZP, TEM examination and XRD analysis and compared to sole-gold nanoparticles. The hybrid particles’ efficacy in psoriasis treatment was assessed in vivo on IMQ-induced mouse models and the findings were compared to those obtained by using the marketed product, C9 and L11 as treatment options. Skin samples of treatment groups on which gold-containing formulations was applied were examined using STEM microscope for further confirmation of skin deposition of the gold nanoparticles.
The findings of this chapter revealed the following:
• The gold nanoparticles conjugation with tacrolimus-loaded chitosan nanoparticles was unsuccessful and led to aggregation of both nano-systems; identified by the change of color of gold nanoparticles from wine red to purple and confirmed by DLS.
• The gold nanoparticles conjugation with tacrolimus-loaded lecithin-chitosan hybrid systems was successful with a particle size 195 ± 2.6 nm, PDI 0.53 ± 0.014 and surface charge -23.5 ± 4.24 mVand no color change for the gold nanoparticles.
• TEM examination confirmed the previous findings.
• The tacrolimus-loaded lecithin-chitosan and gold hybrid showed successful improvement of psoriatic plaques in vivo; in terms of visual observations, PASI score and histopathological examination of skin samples. However, it was indifferent from sole-tacrolimus-loaded lecithin-chitosan nanoparticles’ effect.
• Spleen to body weight ratio of mice revealed significantly lower values for the gold-containing formulations compared to all other treatment groups which confirms the anti-inflammatory effect exerted by the gold nanoparticles on the body.