الفهرس 
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Abstract
from the plethora of research lately, many novel drug delivery systems were
discovered, enhancing the drug’s skin deposition, retention, and permeation. Several
delivery systems have been exploited for such purpose, among which are the lipid
nanoparticles, such as the solid lipid nanoparticles (SLNs), the nanostructured lipid
carriers (NLCs), and the nano-emulsions, as well as vesicular systems, such as
liposomes, niosomes, ethosomes, transfersomes, and penetration enhancer vesicles
(PEVs). New delivery systems have emerged, such as aspasomes consisting of a
bilayer forming material ascorbyl palmitate and cholesterol as a stabilizing agent,
enclosing an aqueous environment, where they offer the advantage of having an antioxidant
property. Nanofibers is another delivery system consisting of fiber threads
with diameter in the nm range prepared by electrospinning technique, where their
morphology depends on three main process parameters; solution flow rate, distance,
and electrical voltage. Both systems offer many advantages in the topical delivery
of drugs.
Quercetin is a natural flavanol, which gained much interest in the last period
due to its tremendous number of actions regarding its anti-cancer, anti-bacterial
potential against P. acne, anti-inflammatory, and anti-oxidant potential, where it acts
as a topical anti-oxidant due to its ability to scavenge the free radicals. It also inhibits
the tumor necrosis factor alpha (TNF-α) production, responsible for chronic
inflammatory diseases such as acne. Therefore, the aim of this work was to
encapsulate quercetin as a bacteriostatic and anti-inflammatory herbal compound, in
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non-traditional nano-delivery systems such as aspasomes and nanofibers, to be
characterized and tested for their potential in the treatment of acne upon topical
application.
The work in this thesis was divided into three chapters:
Chapter I: Preparation and characterization of quercetin-loaded aspasomes
This chapter involved the formulation, and characterization of quercetinloaded
aspasomes. Aspasomes were prepared by the thin film hydration technique
and characterized for their particle size, zeta potential, entrapment efficiency (EE%),
stability when stored at a temperature of 4-8° C for three months, their ex-vivo skin
deposition/permeation, their anti-oxidant potential, their morphology using the
transmission electron microscope (TEM), and their thermal properties using
differential scanning calorimetry (DSC).
The work in this chapter included the following:
1. Preparation of the aspasomes by the thin film hydration technique, using
different concentrations of the lipidic mixture to formulate plain vesicles, where
they were first prepared without quercetin, nor any oils for optimization
purposes, then the optimized formula was selected for incorporation of the
quercetin and different types of oils namely tea tree oil, neem oil and cinnamon
oil at different amounts. Ingredients were dissolved in the organic solvent
mixture, which was evaporated till the formation of the thin film of dry lipid.
The dry film was then rehydrated with phosphate buffer saline (PBS pH 7.4),
and the dispersion was rotated to allow the rehydration and maturation of
vesicles. Finally, the aspasomal formulations were tested for further
investigations.
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2. characterization of the prepared aspasomes was done through the following
studies:
a) Particle size, PDI and surface charge using the Zeta-sizer Nano
b) Entrapment efficiency percentage (EE%) of the aspasomal formulae via an ultrafiltration
method using the viva-spin tubes.
c) Physical stability study on the selected aspasomal formulae by the assessment of
the effect of storage on the particle size, charge, PDI, and EE% of the
nanoparticles.
d) Ex vivo-deposition on rat skin using Franz diffusion apparatus
e) Measurement of the anti-oxidant potential of the selected aspasomal formulae
was carried out using theDiphenyl-1-picrylhydrazyl (DPPH) anti-oxidant assay
f) Morphology of the selected formula using the transmission electron microscope
(TEM).
g) Thermal analysis using the differential scanning calorimetry (DSC).
The results of this work revealed the following:
1. Quercetin was successfully loaded into aspasomes, using the thin film hydration
technique.
2. Aspasomes preparation necessitated the presence of three main components
ascorbyl palmitate, cholesterol, and dicetyl phosphate. Ascorbyl palmitate was
used as the bilayer vesicle forming material, while the cholesterol was used as
the main stabilizing component, and the dicetyl phosphate acted as a charge
inducer.
3. Particle size of the selected formulae (F10, F13, and F16) ranged from (125-184
nm) depending on the presence of cholesterol, the presence of neem oil, tea tree
oil, or their combination together. Some formulae were excluded due to their
micrometer size, and others were excluded due to the presence of oil droplets.
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4. All of the prepared aspasomes were charged, with charges ranging from (-79.9 to
-100 mV) suggesting that they are stable against vesicle aggregation and fusion.
5. The EE% of the selected formulae (F10, F13, and F16) ranged from (96.13-
99.81%) owing to the hydrophobicity of the drug, suggesting its successful
incorporation within the lipidic bilayers of the aspasomes.
6. Different EE% values were obtained for quercetin depending on the composition
of vesicles, the presence of cholesterol formulating them, and the presence of
neem oil, where the maximum entrapment was shown for F13 and F16 99.81%,
and 99.63% respectively.
7. Stability studies for the selected formulae suggested that minor changes in
particle size, PDI, zeta potential and EE% occurred, indicating adequate stability.
8. The Ex-vivo deposition of quercetin from the selected formulae (F13, and F16)
was 27.10% and 40.93% respectively, which showed that the aspasomes acted as
a good nanoparticulate system loading quercetin allowing its good skin retention,
deposition, and exhibiting low transdermal permeation rates.
9. DPPH assay suggested that quercetin showed an anti-oxidant activity when
loaded into aspasomes, where the quercetin solution, and the selected best
formulae (F13and F16) showed a better anti-oxidant activity compared to the
ascorbic acid reference.
10. Transmission electron microscope of the selected formula F16 demonstrated the
sealed spherical nature of quercetin vesicular system.
11. DSC charts showed the absence of the endothermic peak of quercetin, which
ensures the complete drug encapsulation within the lipid bilayers.
12. The selected formula F16 was selected for conduction of further experiments.
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Chapter II: Preparation and characterization of quercetin-loaded nanofibers
This chapter dealt with the formulation, and characterization of quercetinloaded
nanofibers, where preparation of both PVA/aspasomes composite NFs and
PVA/quercetin/essential oils NFs using PVA as a fiber forming polymer was
attempted. characterization of the nanofibers included visual examination,
morphological examination using the scanning electron microscope (SEM), ex-vivo
deposition/permeation using Franz diffusion apparatus, physical integrity of the
nanofibers using the water-retention test, the thermal properties by DSC and
chemical interaction using Fourier transform infrared spectroscopy (FT-IR).
The work in this chapter included the following:
1. Preparation of the nanofibers using a horizontal set up type electrospinner, where
10% PVA was used as the basic polymeric solution. Firstly, four trials for
preparation of PVA/aspasomes composite nanofibers were attempted by mixing
an aliquot of aspasomal formulation F16 with 10% PVA solution by different
ratios, followed by electrospinning. Secondly, another four trials were attempted,
by mixing different amounts of quercetin and neem/tea tree oils with the 10%
PVA solution, to yield PVA/QC/EOs nanofibers upon electrospinning.
2. characterization of the prepared nanofibers was done through the following
studies:
a) selection by visual examination
b) Morphological examination of the nanofibers using the scanning electron
microscope (SEM)
c) Ex-vivo deposition/permeation using Franz diffusion apparatus over a period of
6 hours
d) Fourier transform infrared spectroscopy (FT-IR) experiments to examine any
chemical interaction occurring within the nanofiber mats
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e) Thermal analysis using the differential scanning colorimetry (DSC)
f) Physical integrity (water-retention test) to ensure the mechanical strength of the
nanofibers
The results of this work revealed the following:
1. Quercetin was successfully loaded in the nanofibers, using the electrospinning
technique, operated by a horizontal type electrospinner.
2. The electrospinning process was found to be affected by many process
parameters which control the morphology and the average diameter for the
nanofibers. Those parameters are the electrical voltage, the solution flow rate,
and the distance.
3. The optimum parameters for the plain nanofibers P1 were a flow rate of 3 mL/hr,
distance of 10 cm, and 20 KV as voltage. As for the PVA/Asp composite
nanofibers (N1, N2, N3, and N4), they were excluded from further
characterization steps, owing to the high percentage of beads occurring as shown
in the SEM images, and the incidence of dropping.
4. SEM analysis showed that nanofibers were homogenously dispersed, where the
best formula H2 was homogenous and yielded an average fiber diameter of
313.08 nm, and it was considered beadless and showed no dropping.
5. The Ex-vivo deposition of quercetin from the selected nanofiber formulae H2 was
28.24%± 0.012, which showed that the nanofibers acted as a good nano-system
for the quercetin loading, allowing its good skin retention, deposition, and drug
accumulation on skin.
6. Permeation experiments showed very low in vitro permeated percent of quercetin
(less than 1%) which is favorable to produce its topical action on the acne
pimples.
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7. The FT-IR charts revealed broadening of the peaks which may be due to the
principle of superimposition which occurred between the PVA and the other
components, or a chemical interaction between different groups of the polymeric
PVA, and the quercetin and the two oils, and this is ascribed to the fact of having
a large number of hydroxyl groups in the composite nanofiber system.
8. DSC charts showed the absence of the endothermic peak for the quercetin, which
ensures the complete drug encapsulation within the nanofiber mat.
9. Water retention test revealed that the nanofiber mat H2 was proven to exhibit
high mechanical strength and good physical integrity, which hence qualifies it to
be used in topical drug delivery.
10. The selected nanofiber mat H2 was selected for further clinical investigations,
based on all the previous results.
Chapter III: Safety, efficacy and clinical applicability of the selected quercetin
loaded aspasomes and nanofibers formulations
The aim of the current chapter was to test the antibacterial properties and
safety of the selected aspasomes and nanofibers formulations, in addition to testing
their clinical efficacy on acne patients when compared to Panthenol® cream as
placebo.The work in this chapter included the following:
1. An anti-bacterial assay was conducted on the selected aspasomal and nanofiber
formulations to test their activity against Propionibacterium acne (P. acne).
2. The safety was evaluated by examining the viability percentage of the 3T3
CCL92 skin fibroblastic cells when both best formulae were applied, using the
neutral red cytotoxicity assay.
3. The clinical efficacy was evaluated where the study included 40 patients
suffering from mild or moderate acne vulgaris. group I patients were instructed
to apply a thin film of quercetin aspasomal formula F16, whereas, group II
patients were instructed to apply wet squared shaped quercetin nanofiber patches
(H2) of dimensions 1 cm x 1 cm, the placebo formulation was Panthenol®. The
treatment period continued up to 8 weeks, and patients were instructed to report
any discomfort or irritation encountered during the study. All patients were
photographed every 2 weeks and evaluated clinically after 8 weeks on both sides
of the face through the counting of comedones, inflammatory and total acne
lesions and a reduction percentage was calculated for all types of lesions.
The results of this work revealed the following:
1. The best aspasomal formula F16, and the nanofiber patch H2 were tested for their
anti-bacterial activity against Propionibacterium acne, their safety on the 3T3
CCL92 skin fibroblastic cells, and clinical applicability on different patients.
2. F16 displayed an average inhibition zone of 15±1.53 mm which was considered
significantly higher when compared to the quercetin alone (8.25± 2.08 mm), and
this was attributed to the formula ingredients; tea tree oil, neem oil, and the
bilayer forming material “ascorbyl palmitate”, showing synergistic antibacterial
activity with quercetin.
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3. H2 showed an average inhibition zone of 18± 0.01 mm which was significantly
larger when compared to the aspasomal formula F16 (15±1.53 mm), and the
quercetin (8.25± 2.08 mm).
4. The aspasomal and nanofibers formulations in addition to quercetin solution all
displayed considerable safety on 3T3 CCL92 skin fibroblastic cells, owing to the
safe nature of all the ingredients used in the formulae preparation.
5. The clinical applicability of the aspasomal formula F16, and the nanofiber patch
H2 was evaluated, where they displayed considerable percentages reduction for
inflammatory acne lesions and total acne lesions, which could be ascribed to the
presence of the anti-inflammatory drug quercetin, and the anti-inflammatory
ingredients tea tree oil, neem oil, and the ascorbyl palmitate.
Results of this thesis delineate that quercetin delivery systems adopted in the
current thesis (aspasomes and nanofibers) are both promising topical nanoformulations
which can be used in the treatment of acne vulgaris