Formulation and in-vitro Evaluation of Ethosomes using Anastrozole as a Modeling Drug

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Introduction
The key benefit of the transdermal drug delivery system is to overcome the difficulties through the oral route.The major advantages of Transdermal Drug Delivery System TDDS are improving bioavailability with controlled drug release, reduced side effects, and avoiding first-pass metabolism.TDDS documented itself as an essential portion of the Novel Drug Delivery System (NDDS) with the prescribed dosage forms.In TDDS, a drug is administered at relatively low amount to the surrounding area by a patch or other system, which circulates transversely through the skin barrier.Throughout the distribution course, the drug enters directly through the skin into the bloodstream; hence, a higher concentration is obtained in the blood (1).Ethosomes are transdermal drug delivery carriers consisting of phospholipid, cholesterol, alcohol, water, and propylene glycol with different concentrations.They are soft and flexible carriers, with size ranging from 30 nm to several microns that could penetrate the stratum corneum and enhance the permeation of various drugs (2).Greater flexibility is conferred by the Ethanol's presence in the vesicles compared to normal liposomes; this, together with the permeation-enhancing action of Ethanol itself that is attributed to the ability of Ethanol to fluidize the stratum corneum lipids, results in a higher quantity and deeper penetration of the medication through the skin.(3).Ethosomal vesicles are structurally composed of a phospholipid bilayer and an inner aqueous core containing a drug.Ethanol acts as an efficient penetration enhancer that makes the transportation of medicinal agents into a deeper skin layer and systemic circulation very easy and effective.They can entrap a wide variety of molecules, including hydrophilic, lipophilic, and high molecular weight substances.They can deliver the drug across the skin under occlusive and nonocclusive conditions (4).Anastrozole (ANZ) is a potent nonsteroidal aromatase Type II inhibitor (AI) that can selectively inhibit aromatase, this inhibition leads to lower estrogen levels, decreasing tumor mass or delaying the progression of tumor growth in some women (5).All optimal features needed for drug delivery across skin like dosage of 1 mg/day (low dosage), the solubility of 0.5 mg/ml, partition coefficient of 3.5, a molecular weight of 293.3 Daltons, and a half-life of 46.8h are in ANZ, therefore this drug has been used as a model drug for In vitro characterization of ethosomes (6).The research was characterized by use of only Ethanol and Phosphatidyl choline in preparation of ethosomes, while the previous studies used cholesterol and/or propylene glycol in addition to Ethanol and Phosphatidyl choline (7-9).The study of the effect ultrasonication pulse ratio on Vesicle size and PDI of ethosomes which is included in this research considered a characteristic study for the research The aim was formulation of ANZ-loaded ethosomes and characterization of the formulated ethosomes for particle size, PDI, entrapment efficiency and in vitro drug release in addition to P-XRD and FTIR.

Materials and Methods Materials
ANZ from (Baoji Guokang Bio-Technology Co., Limited, China) and Soy phosphatidyl choline were purchased from Hangzhou Hyper Chemicals Limited China, Franz diffusion cells and Probe sonicator (Biobase-China) HPLC method for determination of ANZ A modified USP-validated method was used for ANZ to be detected via HPLC (Knauer-Germany).Where the mobile phase composed of Acetonitrile: Water (60:40) and diluent same as the mobile phase were used, standard solution of 50µg/mL of ANZ powder in diluent used to determine the major peak and retention time.

Preparation of Ethosoms
Ethosomes were produced by the film hydration method, Briefly, the lipid mixture which composed of Soy phosphatidyl choline and ANZ powder was dissolved in 10 mL of Ethanol, which was then removed hot air oven at 40˚C for 24 hours, thus obtaining a thin film of dry lipid on the flask wall.The film was then reconstituted by addition of 10 ml of various percentage of Ethanol: Water mixture with stirring [9]; the probe sonicator (Biobase-China) was utilized for vesicle formation and size reduction for 10 min, with different pulse on/off ratios [10].Twenty-four formulas were prepared, as in Table (

Variables Affecting Particle size and PDI of ANZ-loaded Ethosomes
The parameters utilized to evaluate the ethosmoes and choose the appropriate formula for further research were the particle diameter and polydispersity index at the minimum level.

Effect of Phospholipid Percentage
Because they will affect the size, entrapment efficiency, stability, and penetration ability of the vesicles, the choice of phospholipid type and concentration for the formulation is crucial for the production of the ethosomal system.As in Table (1), different formulas (F1-F4) containing different Soy phosphatidyl choline percentages (0.5%-3%) were prepared (10).

Effect of Ethanol Concentration
Ethanol is an effective penetration booster.By giving the vesicles distinctive properties in terms of size, stability, entrapment efficiency, and increased skin permeability, it plays a crucial role in ethosomal systems.Formulas that contained different percentages of Ethanol (20%-50%) were prepared and tested for differences in particle size and PDI (F5-F8) (11).

Effect of Ultrasonication Pulse Ratio on/off
Different ultrasonication pulse ratio on/ off has been applied on ethosomal formula to examine its influence on particle size and PDI (F11-F12).

Fourier Transform InfraRed Spectroscopy
The solid powder could be obtained by crushing the membrane sample until finely ground solid powder is obtained.After that, the sample powder needs to be mixed with powdered potassium bromide (KBr) and pressing the mixture under high pressure.The ratio between sample powder and KBr is about 1:100 to form a very homogeneous KBr pellet.The resultant KBr pellet can be inserted into a holder in the FTIR spectrometer to get the FTIR spectrum (12).

X-Ray Diffraction
The sample crystallography was obtained using the random mounts method.In this method, particles were packed to a flat surface onto a sample holder to assume different crystallite orientations and ensure reflections from various planes, the angle for testing was (5-80) degrees (13).

Entrapment Efficiency
The dialysis centrifugation method was used to determine the drug-loaded ethosoms' entrapment efficiency.Briefly, 1mL of ANZ-loaded ethosoms was placed into a dialysis bag (molecular weight cutoff [MWCO] 8000-14,000 Da) and two ends of the dialysis bag were tied.This dialysis bag was then placed into a centrifuge tube and centrifuged for two hours at 6000 rpm at 25 C, the filtrate from dialysis bag has been collected and by the use of HPLC the ANZ content in the filtrate has been estimated which represent the free ANZ within formula (Wf).The total drug contained in the ANZloaded ethosoms was determined by disrupting the mixture with Methanol.It is done by diluting the formula trace in the dialysis bag with enough Methanol to disrupt the vesicle and again using HPLC the ANZ content within vesicle has been detected, the total drug contained within formula was the summation of free ANZ and ANZ entrapped within vesicle.(14) The following equation was used to compute the entrapment efficiency: EE %= (wtwf / wt) ×100%.------eq 1 Where wt is the total drug amount in the formula Wf is the amount of free drug in the formula

The In-vitro Release Studies
The precise volume, which comprised 1 mg of ANZ in the selected formulae was placed into dialysis membrane (2,000-8000MWT cut-off).The same was done to the hydroethanolic solution of the medicine as control.The fastened two ends membrane was placed in a beaker with 100 mL of pH 7.4 phosphate buffer and stirred at a speed of 60 rpm while the temperature was 37 °C.1mL of the medium was removed at distinct time intervals.To maintain a continuous sink condition, a 1 mL of new phosphate buffer pH 7.4 was replenished simultaneously.The concentration of the medication released from a tested sample was determined using HPLC (15)

Release Kinetics Modelling
To study the release kinetics, data obtained from in-vitro drug release study for ethosoms was fitted to the following kinetic models: zero order (cumulative drug amount released vs. time), first order (log of the cumulative drug percentage remaining vs. time), Higuchi ' s model (cumulative drug percentage released vs. square root of time) and Korsmeyer-Peppas (log of the cumulative drug percentage released vs. log time) as in equations 2-1, 2-2, 2-3and 2-4 respectively, to find the best-fitted line for predicting the drug release mechanism (16) Where: Qt is the drug amount released at time t, and Q0 is the initial drug amount in the ethosomes.k0, k1, kH, and kP are the release rate constants of zero order, first order, Higuchi and Korsmeyer-Peppas respectively.
While n is the release exponent indicating the release mechanism from spherical matrices.When n < 0.43, a Fickian diffusion drug release mechanism occurs, while if 0.43 < n < 0.85 a non-Fickian or anomalous diffusion is predominant.The Higuchi model (Eq.2-3) is related to a special case of Korsmeyer-Peppas (Eq.2-4)where n=0. 5 (17).

Statistical Analysis
To evaluate the difference between the results of studied formulations, the oneway analysis of variance (ANOVA) test using Microsoft Excel Add ins 2019.The significance level was specified at α = 0.05, and the difference between data with (P <0.05) was considered statistically significant while if the difference was (P˃0.05) then it will be considered as nonsignificant, All the results were illustrated as the mean values ±standard deviation (SD) in three replicates (n=3).

Drug detection by HPLC
The drug retention peak appeared at a time of 4.65 minutes of drug standard sample (20 µL) passage across a column with a peak width of 0.17 and intensity of 96 %,

Characterization of prepared ANZloaded ethosoms Characterization of particle size and PDI Variables Affecting Particle size and PDI of Ethosomes
Vesicle size is crucial for topical drug delivery systems since smaller vesicles transmit their contents more effectively across skin deeper layers (18).The index of polydispersity (PDI).The term "polydispersity" (or "dispersity") is used to describe the level of non-uniformity of the particles size distribution.Also referred as heterogeneity index.

Effect of Phospholipid Percentage
As (Figure 1 A and B) shows while the PDI declined with higher Soy phosphatidyl choline percentage, the rise in Soy phosphatidyl choline percentage results in a significant (p≥0.05)increase in particle size.This could be because as Soy phosphatidyl choline content raised, more Soy phosphatidyl choline molecules may have been available to serve as surfactants which stabilize and facilitate the production of more uniform vesicles (20).

Effect of Ultrasonication Power Amplitude
Ultrasonication is a widely used highenergy method to reduce the droplet size of nano preparation.In this method, mechanical vibrations from ultrasound waves (> 20 kHz) create sinusoidal pressure variation in the system.This processing leads to microjet and shockwave impacts and collisions between particles, resulting in particle-size reduction.Even though it is crucial to comprehend that the particle size once it reaches equilibrium during the ultrasonication process, it is vital to know the dynamic pathways to reduce the processing time and use optimum power, thus avoiding the over-supply of energy, which may result in larger particle size than expected (22) Sonication is a widely used technique for encapsulating nanoparticles.Large multilamellar vesicles (MLVs) can be prepared by hydrating dry phospholipid films and subjecting them to vigorous mixing.Sonication power can break up these vesicles into smaller, unilamellar vesicles (ULVs) of known size, which are more popular for encapsulation.Inadequate sonication will prevent MLVs from reducing to their ideal size, while excessive sonication will harm the vesicles by creating free radicals and causing aggregation (23).The sonication power affects the particle size and PDI of F5, F9, F10 (Figure 3 A and B).The changed sonication power showed a significant (p≤0.05)impact on particle size, with the best result obtained with the power of 60 % (300watt).When increasing ultrasonication power to 75%, the mean particle size showed a slight increase.This result might be justified by the possibility that, when the vibration amplitude increased, bubbles may grow so large that the time available in the adjacent rarefaction cycle will not be sufficient for them to collapse hence reducing cluster (24).

Effect of Ultrasonication Pulse Ratio on/off
The ultrasonication pulse ratio might be adjusted to keep the temperature of the formula at around 35 degrees Celsius.(24).The effect of pulse ratio changes on particle size and PDI in F5, F11, F12 was studied (Figure 4 A and B), Higher pulse ratio increases energy transfer to the medium, which increases medium temperature Sonication efficiency, however, increases when the medium temperature is low.When the temperature of the medium increases due to cavitation, the medium expands, leading to the production of less energetic shock waves from bubble implosion and hence less size reduction (25).The results showed the significant (p≤0.05)effect of pulse ratio on particle size and PDI.

Entrapment efficiency
Depending on the results from particle size and PDI characterization (F5, F6 and F7) which have the best particle size and PDI was chosen for further characterization.Table 3 shows the entrapment efficiency of ethosoms and drug contained within the formulas F5, F6 and F7.The relatively high entrapment of ANZ within the ethosomal vesicles is explained by multilamerality and the presence of Ethanol content (29).There was increase in the entrapped amount of drug was observed with an increase in Ethanol concentration, but when Ethanol concentration exceeded 30%, a decline in percent drug entrapment was observed.Improvement in aqueous solubility of ANZ was achieved with higher concentration of Ethanol, which could result from its co-solvent effect.Therefore, the more drug amount could be accommodated in the aqueous core of the vesicles however, drug leaked from the fluidized bilayer of the vesicle as the Additionally, the effectiveness of entrapment rises as lecithin concentration rises up to a certain limit (i.e 3%) above which drug permeability is reduced (18).

In-vitro ANZ Release Study
According to DLS, and entrapment efficiency results, F5 (which composed of 20% ethanol and 1% soy phosphatidyl choline and subjected to 300-Watt ultrasonication power with 1/3 sec ultrasonication pulse ratio on /off) shows the best particle size and PDI was chosen to be tested for the in-vitro drug release.In the case of hydroalcoholic drug solution, (99.8724 ± 1.089966) of the drug was released during 2-3 hours, whereas the maximum drug amount released from ethosomes takes 5-6 hours, demonstrating that the drug diffusion from ethosomal bilayers is the rate-limiting step in overall drug permeation through the cellophane membrane (31).There was no significant (p>0.05)difference in drug amount released between F5 and control.

Modeling of Ethosomes Release Kinetic
The selected formulas of ANZ-loaded ethosomes in-vitro release profile were fitted on various kinetic models; namely, first order, zero order, Higuchi square root of time, and Korsmeyer-Peppas models to calculate the rate constants (k0, k1, KH, kp, and n) and the regression coefficient (R2), the obtained fitting parameters are demonstrated in the Table (5).
In the Korsmeyer-Peppas model, fitting only the portion of the cumulative drug release curve below 60% was applied as described previously in literature to determine the (n) value.(17) The obtained data best fits the Korsmeyer-Peppas model with a higher regression coefficient (R2) of (0.9779), indicating that diffusion, matrix erosion, or relaxation contributes to the release mechanism.The formulation showing Korsmeyer (n) values greater than 0.43 (i.e., 0.43 < n < 0.85) indicates that an anomalous transport mechanism of drug release, which is a combination of Fickian diffusion combined with the non-Fickian mechanisms of matrix erosion and relaxation.(32) A comparison between the obtained release profile of formula F5 and the theoretical kinetic models of Korsmeyer-Peppas, kinetics is shown in the Figure 8.

Conclusion
From this work it has been concluded that film hydration method is a simple and productive method for preparation of ethosoms.Ethosom's vesicle size, PDI, and entrapment efficiency is affected by ethanol concentration, soy phosphatidyl choline concentration and ultrasonication power and mode, Ethosoms provide a good drug entrapment, and release when compared hydroalcoholic drug solution.

Figure ( 1 )
Figure (1) B: The effect of phospholipid concentration on PDIEffect of Ethanol ConcentrationAlthough Ethanol can increase the membrane fluidity of ethosoms and improve their stability, it has been demonstrated that Ethanol concentrations greater than 45% (w/w) reduce the value of entrapment efficiency.Perhaps because higher Ethanol contents may cause the membrane to leak, thus Ethanol concentrations in ethosoms should be kept within a certain range (21).The impact of

Figure ( 2 )Figure ( 2 )
Figure (2) A: The effect of Ethanol concentration on the particle diameter of the prepared ethosmal formula

Figure ( 3 )Figure ( 3 )
Figure (3)A:The effect of ultrasonication power on particle diameter of the prepared ethosmal formula

Figure ( 4 )
Figure (4)A: The effect of ultrasonication pulse ratio on particle diameter of the prepared ethosmal formula

Figure ( 5 )
Figure (5) FTIR spectra of A: ANZ, B: Soy phosphatidyl choline, C: physical mixture, D: F5 Powder X-ray diffraction Figure (6) represent the P-XRD for pure ANZ, it showed the sharpest diffraction peak at 18.5863, while the Soy phosphatidyl choline showed a characteristic diffraction broad peak at

B
Ethanol concentration rose above 30% as a result of Ethanol's fluidizing effect on lipid, which causes the lipid bilayer to leak (30).