الفهرس 
Scope of work...Only 14 pages are availabe for public view
 |  | from | 494 |  |  |
| | | from | 494 | | |
Abstract
Summary
Melasma is often difficult to treat, and the condition may be refractory. Many strategies have been adopted for treatment of melasma; however, the topical therapy remains the standard of melasma treatment. Several delivery systems have been exploited for such purpose, among which are polymeric nanoparticles as chitosan nanoparticles (CSNPs) as well as vesicular systems as liposomes, niosomes, transfersomes, ethosomes, etc.
Polymeric based nanoparticles constituting natural polymers such as chitosan were prepared. On the other hand, three vesicular systems were prepared namely; liposomes (consisting of a bilayer of phospholipid enclosing an aqueous compartment), penetration enhancer containing vesicles (PEVs) (consisting of a bilayer of phospholipid together with penetration enhancers as labrasol and transcutol enclosing an aqueous compartment) and invasomes (consisting of a bilayer of phospholipid together with terpenes and ethanol serving as penetration enhancers enclosing an aqueous compartment). In addition, two functional additives namely; hyaluronic acid (HA) and collagen were incorporated to maximize the potential effect of these systems.
Alpha-arbutin (α-arbutin) is widely used as a topical whitening agent by inhibiting tyrosinase activity, which is the main reason for skin hyperpigmentary disorders. Therefore, the aim of this work was to prepare a polymeric based and vesicular nanosystems loading α-arbutin as a skin whitening agent for the treatment of melasma.
Summary
-----------------------------------------------------------------------------------------
353
The work in this thesis is divided into three chapters:
Chapter 1: Preparation and characterization of chitosan nanoparticle-formulations loaded with alpha-arbutin
The work in this chapter included the following:
1- Preparation of α-arbutin-CSNPs by ionic gelation method using two functional additives namely; HA and/or collagen. A 24 full factorial design was utilized to study the effect of the formulation independent variables on the product characteristics as dependent ones.
2- characterization of the prepared CSNPs through the following studies:
a) Particle size (P.S), polydispersity index (PDI) and zeta potential (ζ-potential) analysis of freshly prepared α-arbutin-CSNPs formulations were carried out using zeta sizer.
b) Determination of α-arbutin entrapment efficiency (EE%) in CSNPs by analyzing the free drug after filtration using nanoseps centrifuge tubes technique.
c) Physical stability study on all α-arbutin-CSNPs formulations was conducted over a storage period of three months at 2-8° C by the assessment of the effect of storage on the P.S, PDI, ζ-potential and EE% of the nanoparticles.
d) In vitro release experiments were carried out over a period of 24 hours on the selected formulae based on a desirability study.
e) The selected CSNPs formulation was examined for morphology by transmission electron microscopy (TEM).
f) Ex vivo deposition/permeation of the selected formulae on rat skin was carried out using Franz diffusion apparatus.
Summary
-----------------------------------------------------------------------------------------
354
g) Differential scanning calorimetry (DSC) and Fourier Transform Infrared (FT-IR) spectroscopy were carried out on the selected formulae in order to study the possible interaction of α-arbutin with the excipients used in the selected formulae.
The results of this chapter revealed the following:
1- α-arbutin loaded-CSNPs were successfully prepared using the ionic gelation method based on the results of the preliminary study.
2- Results of the full factorial design based on four chosen independent variables namely; concentration of chitosan (XA), concentration of TPP (XB), concentration of HA (XC) and concentration of collagen (XD) and their effect on four dependent variables (responses) namely; P.S (nm), PDI, ζ-potential (mV) and EE% revealed:
a) The TPP concentration (XB) was proven to exhibit a significant effect on the P.S of the formulae, in which upon increasing the concentration of TPP, the P.S of CSNPs significantly decreased.
b) The TPP concentration (XB) was proven to exhibit a significant effect on the PDI of the formulae. PDI values decreased significantly with increasing the TPP concentration of the prepared CSNPs.
c) Positively charged CSNPs, with ζ-potential ranging from +36.55 to +41.71 mV were obtained, indicating good stability and high dispersion quality. However, the ζ-potential of all of the prepared CSNPs formulae was not significantly affected by any of the independent factors.
d) EE% values for α-arbutin into CSNPs ranging from 63.65% to 88.45% were obtained. Among the four independent variables, the EE% of α-arbutin within CSNPs was only dependent on the concentration of
Summary
-----------------------------------------------------------------------------------------
355
chitosan (XA), in which by increasing the chitosan concentration from 0.15 to 0.30 % w/v, the EE% of the prepared CSNPs decreased significantly.
3-α-arbutin-CSNPs displayed reliable storage properties for 3 months as manifested by the insignificant changes in P.S, PDI, ζ-potential and EE% values.
4-The selected α-arbutin-CSNPs formulations showed sustained release of α-arbutin over 24 hours, where ≈ 100% release was achieved after this period of time compared to the free drug solution which showed 100 % diffusion in two hours.
5-α-arbutin loaded-CSNPs dispersions were incorporated into carbopol-based hydrogels, with superior organoleptic characters as well as good spreadability, acceptable pH and rheological properties, sustained release profile and good stability after three months storage.
6- Ex-vivo skin deposition experiments of the selected CSNPs dispersions and hydrogels demonstrated high potential in accumulating the drug into the deeper epidermal and dermal layers of the skin without transdermal delivery.
7-Transmission electron microscopy of the selected CSNPs formulation displayed almost spherical and mono-dispersed nanoparticles with a smooth surface either individually or in groups.
8-The DSC thermogram of the selected CSNPs formulation revealed the disappearance of α-arbutin melting endothermic peak suggesting the amorphous nature of the drug in the CSNPs and that the entire drug was successfully encapsulated inside the polymeric matrix after nanoparticle formulation.
9- The FT-IR data of the selected CSNPs formulation confirmed the
Summary
-----------------------------------------------------------------------------------------
356
encapsulation of α-arbutin within the polymeric matrix.
Chapter 2: Preparation and characterization of different vesicular dispersion-formulations loaded with alpha-arbutin
The work in this chapter included the following:
1-Preparation of α-arbutin-liposomes by the reversed phase evaporation (REV) method using two different amounts of phospholipids in presence of the two functional additives namely; HA and/or collagen. A 23 full factorial design was utilized to study the effect of the formulation independent variables namely; phospholipid amount (XA), concentration of HA (XB) and concentration of collagen (XC) and their effect on four dependent variables (responses) namely; P.S (nm), PDI, ζ-potential (mV) and EE%. The optimized liposomal formula was utilized for the preparation of PEVs and invasomes.
2-Characterization of the prepared vesicles was carried out as previously performed in Chapter I.
The results of this chapter revealed the following:
1- α-arbutin-loaded liposomes were successfully prepared using the reverse phase evaporation technique based on a conducted preliminary study.
2- The full factorial design based on three chosen independent variables comprised eight formulae. The effect of the studied factors on the characteristics of the liposomal dispersions revealed:
a) P.S values ranging from 414.25 to 672.75 nm. The P.S of the prepared liposomal formulae were affected by the three formulation variables; the phospholipid amount (XA), HA (XB) and collagen concentrations (XC),
Summary
-----------------------------------------------------------------------------------------
357
in which by increasing the amount of phospholipid, P.S increased significantly, while in the presence of HA or COL, P.S decreased significantly.
b) PDI values ranged from 0.356 to 0.493. The amount of phospholipid (XA) was the only formulation factor that significantly affected the PDI values, in which there was a significant increase in the PDI values upon increasing the phospholipid amount (XA).
c) The formed liposomes were negatively charged with ζ-potential values varying from -12.50 ± 0.85 to - 16.92 ± 1.42 mV. The phospholipid amount (XA) was the only formulation factor that significantly affected ζ-potential values but the observed effect was opposite to what was expected, in which by increasing the phospholipid amount at constant HA and collagen concentrations, the surface charge of the particles significantly decreased. d) EE% values ranged from 93.81% to 99.29%. Similar to PDI and ζ-potential models, the phospholipid amount (XA) was the only formulation factor that significantly affected the EE% values, in which the EE% significantly increased upon increasing the phospholipid amount at constant HA and collagen concentrations.
3-α-arbutin-liposomes displayed reliable storage properties for 3 months as manifested by the insignificant changes in P.S, PDI, ζ-potential and EE% values.
4-Based on a desirability study, the selected α-arbutin-loaded liposomes dispersions showed sustained release of α-arbutin over 24 h.
5-α-arbutin-loaded liposomal dispersions were incorporated into carbopol-based hydrogels, which had proved superior organoleptic
Summary
-----------------------------------------------------------------------------------------
358
characters as well as good spreadability, acceptable pH and rheological properties, sustained release profile and good stability after three months of storage.
6-Ex-vivo skin deposition experiments of the selected liposomal dispersions and their respective hydrogels demonstrated their high potential in accumulating the drug into epidermal and dermal layers of the skin without transdermal delivery.
7-Optimization showed that the liposomal formulation comprising the two functional additives (HA and collagen) and exhibiting the smallest particle size was suitable for further characterization studies as well as the preparation of another two ultra-deformable vesicular systems namely; PEVs and invasomes.
8- α-arbutin-loaded PEVs and invasomes were successfully prepared using the reverse phase evaporation technique.
9- The P.S of the prepared PEVs and invasomes were affected by both the concentration and type of the employed penetration enhancer. In case of PEVs, P.S values ranged from 161.40 to 457.90 nm. Upon increasing the concentration of transcutol or labrasol from 5% to 10%, particle size increased significantly. Whereas, PEVs containing transcutol exhibited significantly smaller particle size compared with the PEVs containing labrasol at equivalent concentration. Regarding invasomes, P.S values ranged from 151.95 nm to 507.05 nm. Likewise, upon increasing the concentration of terpenes, the P.S of the prepared invasomes increased significantly. However, invasomes prepared with cineole resulted in larger vesicles compared with limonene vesicles.
10- The PDI values of all prepared vesicles (PEVs and invasomes) did
Summary
-----------------------------------------------------------------------------------------
359
not exceed 0.4.
11- PEVs and invasomes were negatively charged. Both transcutol and labrasol formulations displayed comparable ζ-potential values ranging from -16.37 to -17.48 mV, which is probably attributed to the phospholipid, owing to the non-ionizable nature of both penetration enhancers. Regarding invasomal formulations, ζ-potential values were in the range of -26.70 to -28.20 mV with an insignificant difference between ζ-potential values of cineole and limonene formulations. The presence of ethanol and phospholipid offered synergistic effect and provided higher negative surface charge, thus preventing vesicle aggregation due to electrostatic repulsion.
12- PEVs and invasomes displayed high EE% values owing to α-arbutin hydrophilicity as well as the preparation method. Regarding PEVs formulations, EE% values ranged from 95.17% to 99.53%. Upon increasing the concentration of each of labrasol® or transcutol®, a significant decrease in α-arbutin EE% was observed. However, both types of penetration enhancers did not significantly differ in their effect on the EE% of PEVs. In case of the invasomal dispersions, the EE% values ranged from 80.59% to 91.90%. Upon increasing cineole concentration, a significant increase in α-arbutin EE% was observed. However, the increase in the concentration of limonene was coupled with a significant decrease in α-arbutin EE%. Moreover, the type of terpene significantly affected α-arbutin EE%, in which limonene-containing invasomes showed significantly lower EE% compared to cineole containing ones at equivalent concentration.
13- Regarding the physical stability of the eight vesicular systems after three months’ storage, PEVs formulations displayed significant increase
Summary
-----------------------------------------------------------------------------------------
360
in P.S, accompanied by a significant increase in the values of PDI. Whereas, transcutol containing PEVs showed very slight and insignificant changes in ζ-potential values, which came in contrast with labrasol containing PEVs, in which a significant increase in ζ-potential values was observed upon storage. Moreover, the four PEVs formulations displayed insignificant changes in EE% upon storage. For invasomes, P.S increased significantly after three months’ storage together with an insignificant increase in the values of PDI. On the other hand, ζ-potential values decreased significantly. Moreover, invasomal formulations showed insignificant decrease in EE% upon storage.
14-The eight vesicular formulations showed sustained release of α-arbutin over 24 h, where ≈ 100% release was achieved after this period of time compared to the free drug solution which showed 100 % diffusion in two h. In case of PEVs formulations, upon increasing the concentration of transcutol (from 5 to 10, %w/v), the % released of the drug significantly increased. Though, the type of penetration enhancer did not significantly differ in their effect on the release of α-arbutin from PEVs. In case of invasomes, upon increasing the concentration of cineole or limonene from 1.5 to 3 % w/v, the % release of the drug significantly decreased. Moreover, α-arbutin-loaded invasomes prepared using limonene exploited significantly lower % of α-arbutin release compared to cineole formulae.
15-α-arbutin-PEVs and invasomal dispersions were incorporated into carbopol-based hydrogels, with good spreadability, acceptable pH and rheological properties, sustained release profile and good stability after three months of storage.
16-Ex-vivo skin deposition experiments of PEVs and invasomal hydrogels demonstrated high potential in accumulating the drug into epidermal and
Summary
-----------------------------------------------------------------------------------------
361
dermal layers of the skin and further across the skin which indicates the possibility of reaching the blood compartment.
17-The selected vesicular systems exhibited a predominant spherical shape, with a smooth surface. .
18-The DSC thermogram of the selected vesicular formulations revealed the disappearance of α-arbutin melting endothermic peak in both α-arbutin-containing vesicular systems suggesting that the entire drug was completely solubilized inside the aqueous core of lipid matrix after vesicular formulation.
19-The FT-IR data of the selected vesicular formulations confirmed the interaction of α-arbutin with the matrix structure of the prepared vesicles that might account for the reasonable encapsulation of α-arbutin in these vesicles.
Chapter 3: Clinical evaluation of selected chitosan nanoparticles and vesicular hydrogel-formulations on patients suffering from melasma.
The work in this chapter included the following:
The clinical study was conducted on 40 female patients suffering from epidermal melasma. A comparative split-face study was conducted on both sides of the faces. Patients were divided into 4 groups; group I (10 patients) was treated with topically applied CSNPs hydrogels (formula C5) on the right side, group II (10 patients) was treated with topically applied CSNPs hydrogels (formula C8) on the right side, group III (10 patients) was treated with topically applied liposomal hydrogels (formula L4) on the right side and group IV (10 patients) was treated with topically applied PEVs hydrogels (formula V1) on the right side. The left side of the face of each group was treated with α-arbutin
Summary
-----------------------------------------------------------------------------------------
362
hydrogels (as control). In each group, all patients were instructed to apply the provided formula once daily at night over the affected areas for a total period of 2 months along with a daily application of sunscreen of SPF ≥ 50 in the morning for a period of 8 weeks. Assessment of patients’ response clinically using photography/mMASI scoring and histopathologically using morphometric measurement of melanin and immunohistochemical evaluation was done after treatment based on demographical and diagnostic data.
The results of this chapter revealed the following:
1- The performed comparative split-face study revealed that the formulations applied on the right side of the face displayed significantly better therapeutic outcome in all groups compared to the drug-hydrogel applied on the left side, delineating that the former ones are more promising for melasma treatment.
2- For the tested formulations applied on the right side of the face in post-treated patients, clinical assessment using photography/mMASI scoring and histopathologic assessment using morphometric measurement of melanin and immunohistochemical evaluation showed that groups IV and III patients treated with PEVs and liposomal hydrogel formulations, respectively displayed higher reduction in mMASI scores, MPSA and MART-1 positive cells than group II and group I patients treated with CSNPs hydrogel formulations. On the other hand, α-arbutin gel applied on the other side showed comparable poor therapeutic outcome in all groups.
3- PEVs hydrogel formulation was superior to liposomal hydrogel formulation in terms of MPSA reduction, while CSNPs formulation containing both functional additives was superior to its respective one lacking both additives in terms of mMASI score reduction.
4- The encapsulation of α-arbutin in vesicular systems namely; PEVs and
Summary
-----------------------------------------------------------------------------------------
363
liposomes and in polymer based-nanocarriers chitosan nanoparticles (CSNPs) allowed for maximization of its therapeutic potential in melasma, as could be deduced from the aforementioned assessment criteria. PEVs displayed overall better clinical therapeutic outcome compared to liposomes, and chitosan nanoparticles containing hyaluronic acid and collagen as function additives were superior to those lacking the additives.