Design of experiment based formulation optimization of chitosan-coated nano-liposomes of progesterone for effective oral delivery

The aim of this research was to design and develop chitosan-coated nano-liposomes of progesterone for its safe and effective oral delivery through the vesicular system providing sustained drug release, enhanced drug stability in gastro-intestinal (GI) fluid and improved drug absorption leading to better patient compliance. The aqueous solubility of progesterone (poorly soluble drug) was enhanced by hydroxy-propyl-beta-cyclodextrin complexation and the drug-loaded liposomes were prepared by ethanol injection method followed by surface coating with chitosan. Design of experiment-based formulation optimization was performed using Box-Behnken design selecting lipid, cholesterol, and drug content as formulation factors (independent variables) and mean particle size (MPS), polydispersity index (PDI), zeta potential (ZP), entrapment efficiency (EE), drug loading (DL) and cumulative % drug release (CDR) as evaluation parameters (response variables). The optimized formulation was prepared and evaluated for all preferred critical quality attributes which showed 168.3 nm MPS, 0.307 PDI, 24 mV ZP, 53% EE, 7.2% DL, and 76.4% CDR at 24 hours. In-vitro GI drug stability of chitosan-coated liposomes was studied in simulated gastric fluid and simulated intestinal fluid which exhibited 2.12 and 77.3 fold extended half-life, respectively. The ex-vivo GI-drug absorption study demonstrated two-fold rise in progesterone absorption from liposomal formulation. The chitosan-coated liposomes of progesterone which showed sustained drug release following Higuchi model kinetics was found to be a better alternative for oral delivery of progesterone overcoming drawbacks of conventional dosage forms.


INTRODUCTION
Oral administration of therapeutic drugs is one of the oldest and most preferred approaches of medication because, it is non-invasive, inexpensive, self-administrable, and provides controlled dosing frequency resulting in high patient compliance and therefore proved to be a promising route of administration for both natural and synthetic drugs (Alqahtani et al., 2021). However, oral administration of conventional dosage forms such as tablet, capsule, syrup, suspension, etc. also encounters the problems like gastrointestinal (GI) instability of drugs, the effect of GI fluid enzymes, poor pharmacokinetic profile, and limited GI drug absorption leading to low oral bioavailability (Homayun et al., 2019). One of the major causes of low oral drug bioavailability is the drug's poor water solubility and rate of dissolution in GI/ biological fluid, hence their effective oral delivery remains a challenge because about 70% of new drugs are practically insoluble in water (Ghassemi et al., 2018). Therefore, novel drug carrier systems such as liposomes, niosomes, polymeric micelles, nanocrystals, nanoparticles as well as drug-cyclodextrin complex are being widely explored for effective drug delivery eliminating drawbacks associated with conventional dosage forms (Babadi et al., 2021;Cagdas et al., 2014;Torchilin, 2005). Among all these, liposomal nano-drug carriers are extensively being reported showing desired/controlled drug release profile, improved uptake across biological membranes including GI absorption, prolonged half-life, and drug action, hence enhanced drug bioavailability with reduced side effects (Torchilin, 2005).
Liposomes are defined as spherical-shaped vesicles containing drug-loaded aqueous compartment that is enclosed by one or more concentric lipidic bilayers of 25-2,500 nm size range (Akbarzadeh et al., 2013;Liu et al., 2019). The water-soluble drug entraps in the aqueous compartment and water-insoluble drug intercalates in the lipophilic bilayer (Stanczyk et al., 2013). The essential components of the liposomal vesicle are phospholipid and cholesterol and they are considered biocompatible and biodegradable due to natural occurrence of these components in the biological membrane (Large et al., 2021).
As per literature review and regulatory reports, there is no marketed oral liposomal product available due to the limitations associated with their oral administration, e.g., liposomal instability in the gastric/intestinal fluid and leakage of the encapsulated drug resulting in low oral bioavailability (Lee, 2020). Various novel formulation approaches in liposome development such as modification of lipid components, liposomal surface modification, inner bilayer thickening, and enhanced absorption by mucoadhesion have been investigated to overcome the limitations associated with oral administration of liposomes (He et al., 2019). As compared to conventional liposomes, the chitosan-coated nano-liposomal formulation is considered a better approach for effective and safe oral drug delivery because chitosan coating protects liposomes from destruction in gastric/ intestinal fluid and facilitates oral absorption of drugs having poor water-solubility. The chitosan-coated positive charged (cationic) liposomes readily interact with the negatively charged biological membrane and stabilizes the drug encapsulated in liposomes resulting into enhanced and safe drug permeation by opening tight junctions between the cells to promote drug transportation (Elsayad et al., 2021;Nguyen et al., 2014).
Progesterone is a female hormone and widely used in hormonal replacement therapy in conditions such as endometrium hyperplasia and dysmenorrhea. The bioavailability of progesterone on oral administration is less than 5% and unlike other drugs, micronized progesterone also has only 8.6% bioavailability (Simon et al., 1993) due to its low water-solubility which disfavors the oral administration of progesterone. Various formulation techniques have been used to enhance drug solubility such as inclusion-complex with cyclodextrins, pH adjustment, particle size reduction, etc. Inclusion-complex with cyclodextrins/ hydroxy-propyl-beta-cyclodextrin (HP-β-CD) has been widely reported for enhancing the solubility and stability of drugs along with reducing their toxicity (Jansook and Loftsson, 2009;Lahiani-Skiba et al., 2006). Modification of drugs with complexing agents has been widely reported to increase the encapsulation efficiency of water-soluble as well as water-insoluble drugs in the liposomes (Kulkarni et al., 1995). Further development of chitosan-coated nano-liposomes may prove to be a better alternative and remedy to many limitations of oral drug delivery as conventional dosage forms.
The current study reports the formulation designing and design of experiments (DoE) based optimization of chitosancoated nano-liposomes of progesterone with HP-β-CD complex.
The Box-Behnken experimental design was used selecting different formulation factors as independent variables and critical quality attributes (CQAs) of liposomal products as the response variables. The developed liposomal formulation was characterized for different physico-chemical properties and evaluated for different in-vitro and ex-vivo performance parameters.

Materials
Progesterone was procured as a free sample from M/s. Encube Ethicals Pvt. Ltd. (Mumbai, India). Hydrogenated soy phosphatidylcholine (HSPC) and cholesterol (CH) were purchased from Sigma-Aldrich (India). HP-β-CD was received from Ningbo Hi-Tech Biochemicals (China). Low molecular weight chitosan (CS) was purchased from Himedia (Mumbai, India). All other chemicals and solvents used in this work were of analytical grade.

Inclusion complexation of progesterone with HP-β-CD
The HP-β-CD complex of progesterone was prepared by simply dissolving progesterone in the aqueous solution of HP-β-CD. Accurately weighed 1 g of HP-β-CD was dissolved in 10 ml of purified water to prepare 0.07 M HP-β-CD solution. Accurately weighed progesterone (5 mg/ml) was dissolved in this solution by vortexing for 10 minutes. The stability of this inclusion complex as shown in Figure 1 was determined by phase solubility analysis of progesterone and HP-β-CD (Lahiani-Skiba et al., 2006;Soni and Saini, 2019).

Preparation of progesterone loaded nano-liposomes
Progesterone-loaded nano-liposomes were prepared by ethanol injection technique as schematically shown in Figure 2. The accurate amounts of HSPC and cholesterol were dissolved in ethanol and maintained at 50°C to form an ethanolic phase. Progesterone was dissolved in 0.07 M HP-β-CD solution and kept on a magnetic stirrer at 50°C to prepare the aqueous phase. The ethanolic phase was injected into the pre-heated aqueous phase kept stirring at 500 rpm resulting in translucent liposomal dispersion which was further stirred for 60 minutes for removal of ethanol. Resultant liposomal dispersion (large vesicles) was sonicated by a probe sonicator (Sonics, VCX 500) for 2 minutes for size reduction (Gouda et al., 2021;Jaafar-Maalej et al., 2010).

Chitosan coating of drug loaded nano-liposomes
Chitosan coating of liposomal vesicles was done using 0.2% (w/v) solution of low molecular weight chitosan in 0.1% (v/v) acetic acid. The prepared liposomal dispersion was added with the help of a dropper into an equal volume of chitosan solution

Statistical analysis
The 15 experimental runs (optimization batches) suggested by software with the proposed composition were prepared and evaluated for 12 response variables (R1-R12). The observations (response data) as shown in Table 2 were provided to the software for the statistical fitting into different models i.e., linear, 2FI, quadratic, and cubic. After statistical justification by analysis of variance, the software suggested the best fit linear model for MPS, PDI, ZP, % CDR at 1, 12, 24 hours; quadratic model for % EE, % DL, % CDR at 0.5 hours; and 2FI model for % CDR at 3, 6, and 9 hours as shown in Table 3. The p-value (<0.05) was regarded as statistically significant (Weng and Tong, 2020).

Response surface analysis and optimization standards
The effect of independent variables on each response variable was studied by plotting 3D response surface graphs for each response variable . For finding the optimal composition of chitosan-coated progesterone-loaded nanoliposomes, the optimization goals for independent variables were fixed as, HSPC content (F1) in range, cholesterol content (F2) in range, and progesterone content (F3) was targeted to 50, while the response variables were set to minimum MPS, minimum PDI, maximum ZP, maximum %EE, maximum %DL, maximum CDR at 0.5, 1, 3, 6, 9, 12 and 24 hours. The above-mentioned optimization goals for both independent and response variables as shown in Table 1 were fed into the software. Consequently, the software predicted an optimized composition having maximum desirability value out of many alternative compositions.

Particle size, PDI and ZP
The MPS, PDI, and ZP of all prepared batches of chitosan-coated nano-liposomes of progesterone were determined by Nanopartica SZ-100 (Horiba Scientific) particle size analyzer. The principle involved in the determination of MPS and PDI was dynamic light scattering, while in ZP measurement it was laser Doppler electrophoresis. Recorded observations are shown in Table 2.

%EE and %DL
The EE of chitosan-coated progesterone-loaded nanoliposome batches was determined by centrifugal ultra-filtration method (Soni and Saini, 2021b). Accurate 0.5 ml of each liposomal formulation was taken in the centrifugal concentrator tubes (Microcon ® Ultracel YM-100) and centrifuged for 45 minutes at 10,000 rpm using refrigerated centrifuge (Eppendorf, Germany). The concentrated liposomes remained on the upper part of filter and the filtrate (containing unentrapped drug) was collected in the bottom part tubes which were then suitably diluted with ethanol for estimation of free drug content by UV spectrophotometer (Shimadzu 1700, Japan) at 241 nm. The %EE was calculated using the given formula (Panwar et al., 2010).
Total drug added -Free drug * 100 Total drug added The concentrated liposomes retained on upper filter tube were carefully collected and volume was accurately measured. Drug-loaded liposomes were lysed by adequate amount of ethanol with vortexing. The solution was analyzed for entrapped drug at

In-vitro drug release study
In-vitro drug release of chitosan-coated nano-liposomes of progesterone was studied by dialysis method using dialysis membrane (Himedia, India) with 12,000-14,000 Da molecular weight cut off. Prior to use, the dialysis membranes were activated as per previously reported method (Soni and Saini, 2021a). An accurately measured 1 ml of liposomal dispersion was introduced in a dialysis membrane bag and then was closed in both ends using closure clips. It was then immersed into 250 ml volume of 3% w/v sodium lauryl sulfate solution in 0.1 N HCl kept at 37°C ± 0.5°C temperature and stirred at 75 rpm. At different time intervals, i.e., 0.5, 1, 3, 6, 9, 12, and 24 hours, the release media were taken out. The amount of drug release was estimated by a UV spectrophotometer (Shimadzu 1700) at 245 nm. The drug release was calculated and reported in Table 2 and graphically shown in Figure 11.

Optimized formulation of chitosan-coated nano-liposomes of progesterone
The design expert predicted the optimized chitosancoated nano-liposomes of progesterone containing, 205.6 mg of HSPC, 108.1 mg of cholesterol, and 50 mg of progesterone with a maximum desirability value of 0.775. The predicted highest desirability in the optimized of chitosan-coated nano-liposomes of progesterone has been represented in the 3D response plot and the 2D contour plot as shown in Figure 12. The optimized formulation was prepared and experimentally evaluated for each response variable to perform the validation of software prediction.

Microscopic evaluation
The microscopic examination of the optimized formulation was performed under the optical microscope (Leica DM 1000) at a magnification of 100× under the oil immersionlens. The microscopic view is depicted in Figure 13.

Particle size, PDI, and ZP study
The MPS, PDI, and ZP of optimized batch were analyzed using Nanopartica SZ-100 (Horiba Scientific) particle size analyzer. The observations of particle size analysis are recorded as data in Table 4 and presented graphically in Figure 14.

EE and DL study
%EE and %DL were determined by the centrifugal ultrafiltration technique as discussed in the previous sections and has been recorded in Table 4.

In-vitro drug release profile and drug release kinetics
In-vitro drug release of optimized chitosan-coated nanoliposomes of progesterone was studied according to previously described dialysis method and the amount of drug release was estimated by UV spectrophotometer (Shimadzu 1700) at 245 nm. The observed % drug release at different time intervals is reported in Table 4.
The drug release kinetics of optimized liposomal formulation was assessed by statistical fitting of drug release data in different kinetic models, like zero order, first order, Korsmeyer-Peppas, Higuchi, and Hixon-Crowel (Fig. 16).

DSC study
The lyophilized form of chitosan-coated progesterone loaded nano-liposomes, progesterone drug sample, HSPC, cholesterol, HP-β-CD, chitosan was analyzed for thermal property using DSC (Perkin Elmer 6000, Waltham, MA). Approximately, 3 mg weighed samples were individually placed and sealed in an aluminum pan and kept against a blank aluminum pan as the reference. The thermograms as shown in Figure 17 were recorded when it was further heated from 50°C to 300°C at of 40°C/minute heating rate under the purging of nitrogen (inert) gas with 20°C/minute flow rate (Sharma et al., 2017).

Ex-vivo drug permeation study
The drug permeation study of optimized formulation of progesterone-loaded liposomes and prepared suspension of marketed tablet product were performed using non-everted chicken intestine (ileum) segment. A freshly excised complete lower GI tract of a healthy chicken was procured from a nearby slaughter house. To perform the study, the ileum was isolated and cut into 6 cm pieces and then rinsed with phosphate buffer saline (PBS) pH 6.8. One end of ileum segment was tied and 1 ml of each formulation was filled in different ileum segments and then the other end was also closed using thread. The formulation holding intestinal segments were immersed into 250 ml of PBS pH 6.8 kept in a beaker at 37°C and stirred at 100 rpm. Then samples were taken out at pre-determined time intervals and the fresh buffer was replaced for maintaining the sink condition (Hasan et al., 2020;Ma et al., 2014). The % drug permeation was estimated by UV

Maximize
Maximize spectrophotometer at 247 nm and the graphical presentation is given in Figure 18. The apparent permeability coefficient (Papp) and steady-state drug permeation flux (Jss) of progesterone from optimized liposomal formulation was calculated by below given formula and it was compared with prepared suspension of marketed tablet product (drug content 2.5 mg/ml).
where dQ/dt is the equilibrium state permeation rate in the media; A is surface area of the intestinal segment; while C˳ expresses initial drug concentration. (Cylindrical-shaped ileum segments had a 6 cm length and 0.55 cm inner diameter and thus, calculated surface area was 10.84 cm 2 in each segment.)

In-vitro GI stability study
In-vitro GI stability of progesterone-loaded chitosancoated nano-liposomes was studied in simulated gastric fluid (SGF:pH 1.2) and simulated intestinal fluid (SIF:pH 6.8). Accurate 1 ml liposomal dispersion was added to the 20 ml of each biological fluid. The solutions were homogenized and vortexed for 15 minutes, then incubated at 37°C for 2 hours. The study samples were taken out at 5, 15, 30, 60, 90, and 120 minutes time intervals and centrifuged for 15 minutes duration at 10,000 rpm. Then the sample was diluted with each simulated fluid, and estimation of progesterone content was performed spectrophotometrically. The degradation rate constant (K) of progesterone and degradation half-life (t ½ ) for both GI conditions were calculated (Braga Emidio et al., 2021;Wang et al., 2017). A similar study was performed with progesterone drug solution for comparison.

Inclusion complexation of progesterone
Complex formation of the drug showed enhanced aqueous solubility of progesterone. The presence of hydrophobic inner cavity along with a hydrophilic external surface in cyclodextrins contributed to accelerate the aqueous solubility of the drug (Loftsson et al., 2005). The highest drug solubility was found in HP-β-CD, accordingly, it was selected from amongst the different types of cyclodextrins for complexation of progesterone. Non-covalent attraction between drug particles and inner hydrophobic cavity of cyclodextrin is responsible for complex generation (Shimpi et al., 2005). The optimum concentration of HP-β-CD (0.07 M) was finalized for further development to meet the desired solubility of progesterone in the formulation.

Preparation of chitosan-coated nano-liposomes of progesterone
Selection of HSPC as phospholipid was done considering its phase transition temperature (T c ), i.e., approximately 50°C and high stability in GI fluids because lipids having phase transition temperature less than 37°C, gets readily degraded in gastric fluids (He et al., 2019). Cholesterol was used  to improve the vesicular stability and avoid drug leakage from the vesicles (Vemuri and Rhodes, 1995). Aqueous and organic phase temperature of ethanol injection method was selected to be 50°C because cholesterol shows lowering effect on the phase transition temperature (Lombardo and Kiselev, 2022;Schwendener and Schott, 2010). As the ZP of plain (uncoated) liposomes was anionic (i.e., −1.82 mV), it was planned to impart a positive charge on liposomes by surface coating. The use of chitosan for liposomal coating was an important factor in their surface modification to enhance the drug stability in GI fluid (Nguyen et al., 2016) and therefore, 0.2% (w/v) concentration of chitosan solution was selected for surface modification of progesterone nano-liposomes.

Optimization of progesterone loaded nano-liposomes
Formulation optimization was required to achieve the desired quality attributes in the progesterone-loaded nanoliposomes. As a result of optimization studies, the final formulation was developed with all the desired CQAs such as minimized MPS (nm) and PDI and maximized ZP (mV), EE (%), DL (%), CDR (%). The effect of independent variables on each response variable was analyzed by response surface methodology and is as discussed below.

Formulation factors versus MPS
The optimization software using response surface methodology exhibited a correlation between independent variables (formulation factors) and MPS (R1) by following a linear process order equation. R1 = 208.55 + 62.24A + 24.45B + 3.99C where A is HSPC, B is cholesterol and C is drug content. In this equation, the positive value of factors signifies their direct proportionality to the response variable. The MPS of various optimization batches was found between 138.9 and 275 nm ( Table 2). The 3D surface plot (Fig. 3) represented the effect of lipid, cholesterol, and drug on the MPS which goes on increasing with a rise in lipid (HSPC) content and cholesterol amount, whereas, there was no significant effect of the drug observed on MPS.

Formulation factors versus PDI
The optimization software using response surface methodology exhibited a correlation between independent variables (formulation factors) and PDI (R2) by following a linear process order equation. R2 = 0.3891 + 0.1006A + 0.0361B + 0.0088C        The PDI of optimization batches was observed from 0.256 to 0.490 (Table 2). The 3D surface plots (Fig. 4) showed that when lipid (HSPC) content and cholesterol content were increased the PDI was also increased, whereas no remarkable effect of the drug was seen on PDI.

Formulation factors versus ZP
The optimization software using response surface methodology exhibited a correlation between independent variables (formulation factors) and ZP (R3) by following a linear process order equation. R3 = 23.10 − 3.80A − 1.09B − 0.2625C The negative value of independent variables here signifies the inverse proportionality to the ZP. The ZP of optimization batches was found between 18 and 28 mV ( Table 2). The 3D surface plots (Fig. 5) exhibited that as the lipid (HSPC) content and cholesterol content increases the ZP decreases because the surface charge of cholesterol is negative, whereas, no significant effect of the drug was seen on the value of the ZP.

Formulation factors versus % DL
The optimization software using response surface methodology exhibited a correlation between independent variables (formulation factors) and % DL (R5) by following a quadratic process order equation.
The % DL of optimization batches was found in the range of 5.4%-8.8% ( Table 2). The 3D surface plots (Fig. 7) confirmed that % DL noticeably increased with rise in drug content, whereas, it did not show a significant effect of lipid (HSPC) and cholesterol content on % DL.

Optimized formulation of chitosan-coated nano-liposomes of progesterone
After goal setting of different independent variables as HSPC (in range), cholesterol (in range) and drug (target = 50) whereas the response variables MPS and PDI (minimum) and rest all other responses (maximum), the design expert finally suggested composition of lipid content (HSPC), cholesterol and drug as 205.6, 108.1 and 50 mg, respectively for the predicted optimized batch with the maximum desirability (0.775). The 3D-response and 2D-contour graphs as shown in Figure 12 displayed the highest desirability value of the optimized batch. Optimized formulation was prepared as per suggested composition by software and evaluated for all response variables to validate the predicted values as recorded in Table 4.

Microscopic evaluation
The microscopic study of the optimized chitosan-coated nano-liposomal formulation shown in Figure 13 confirmed the uniform, homogenous, spherical-shaped liposomal structures. The liposomes illustrated a high volume of aqueous core encapsulated in liposomal bilayers with entrapped drug.

Particle size, PDI and ZP analysis
The particle size and PDI directly affect the drug diffusion through the biological membranes. It was reported that liposomal particles smaller than 200 nm readily crosses the GI mucosal barrier; whereas drug transportation through the mucin was limited for particles larger than 500 nm (Bajka et al., 2015;Luo et al., 2021). After statistical analysis, the software predicted MPS was 148.6 nm whereas, the practically observed value of the optimized batch was observed to be 168.3 nm, and was relatively very close to the expected value (Fig. 14).
Physicochemical properties including size distribution affect the accumulation of nano-vesicles in the target tissue hence it requires homogenous dispersion. Generally, the PDI value ranges from 0.0 (indicates perfect sample for acceptance) to 1.0 (indicates multiple-size distribution). For lipid-based nano-vesicles, PDI of 0.3 or <0.3 is considered monodispersed (Danaei et al., 2018). The PDI of the optimized batch was found in the range of 0.256-0.490 and then the goal was set to a minimum value. After the analysis by software, the predicted PDI was 0.289 whereas the practically observed PDI of the optimized batch was found to be 0.307 which was relatively close to the value expected.
The ZP of liposomes depends on the surface charge of the dispersant and affects the stability of the formulation. Generally,  it is considered that a value in the range of −30 to +30 mV is the most acceptable for nano-dispersions. As the ZP of optimization batches was found between 18 and 28 mV and so the goal was set to maximum. The software predicted ZP was 26.84 mV and the practically observed value was found to be 24 mV, which was quite close to the value expected.

EE and DL
As %EE of optimization batches was found in the range of 46.2%-72.2% and the goal was set to a maximum value, so the software on statistical analysis predicted the % EE to be 48.29%, whereas, the practically observed % EE of the optimized batch was found to be 53.0%. The drug:HP-β-CD complexation was proved to be an excellent method for increasing the EE of progesterone for its effective use.
The % DL of optimization batches was observed to be from 5.4% to 8.8% and the goal was set to maximum. After statistical analysis software predicted the % DL of optimized batch to be 5.95%, whereas, the practically observed value was found to be 7.0%, which was better than the value expected. Higher DL would facilitate in achieving desired therapeutic effect on the administration of lower (small) dose volume of the formulation (Sur et al., 2014).

Drug release kinetics study
The significant information related to desired drug release profile is provided by release kinetics (Weng and Tong, 2020). Different kinetic models, i.e., zero order, first order, Hixon-Crowel, Higuchi, and Korsmeyer-Peppas model were studied and graphs were plotted as shown in Figure 16. The regression coefficient values from the plotted model as shown in Table 5 were found to be 0.811, 0.917, 0.961, 0.884, and 0.678, respectively. It was concluded that Higuchi kinetic model was being followed on In-vitro drug release study as it exhibited the highest regression coefficient (R 2 ) as shown in Table 5.

Differential scanning calorimetry (DSC)
The DSC analysis was performed to study the nature of drug and excipients, and also study the drugexcipient interaction (Chadha and Bhandari, 2014). The DSC thermograms of progesterone, HSPC, cholesterol, HP-β-CD, chitosan, and lyophilized chitosan-coated progesterone-loaded nano-liposomes were plotted (Fig. 17). An intense endothermic peak of progesterone at 108.49°C and cholesterol at 152.80°C confirmed their crystalline nature (Jin et al., 2013;Sharma et al., 2017). Whereas the absence of sharp endothermic peaks in the case of other excipients indicates their amorphous nature (Zafar et al., 2021). The effective complexation of the drug with HP-β-CD and complete entrapment of progesterone in nano-liposomes was confirmed by the absence of a sharp endothermic peak in the case of liposomal formulation.

Ex-vivo drug permeation study
The drug permeation across non-everted chicken intestinal segment was studied for prediction of the in-vivo drug absorption. The cumulative % drug permeation data of optimized nano-liposomal formulation was compared to prepared suspension of marketed tablet and are depicted in Figure 18. The apparent permeability coefficient of progesterone in optimized nano-liposomes and suspension of marketed tablet was found to be 3.6 × 10 −2 and 1.8 × 10 −2 cm.minute −1 , respectively. Their respective steady-state drug permeation flux was found to be 4.6 × 10 −2 and 9 × 10 −2 mg.cm −2 .minute −1 as shown in Table 6. Results of ex-vivo permeation study are indicating the higher drug permeation from the developed nano-liposomes as compared to conventional formulation. Therefore, it was confirmed that the chitosan coating of liposomes showed a positive effect on the drug permeation by intimate contact and interaction with negatively charged GI mucosa and epithelium. Hixon-Crowel Q 0 1/3 -Q t 1/3 = kt 0.884 0.076 Korsmeyer-Peppas log (Q 0 -Q t ) = logk + nlogt 0.678 0.886 Where Q 0 = initial drug amount, Q t = remaining drug amount, k 0 = rate constant, t = time.   Table 6. Ex-vivo drug permeation data of progesterone loaded nano liposomes versus marketed product.

In-vitro GI stability study
The drug degradation rate constant and half-life of the developed formulation were calculated for both GI conditions, i.e., SGF and SIF. The In-vitro stability assessment of chitosancoated nano-liposomes was performed to estimate the in-vivo GI stability of progesterone. The degradation rate constant (K) and half-life (t 1/2 ) of developed chitosan-coated liposomes and plain drug solution were calculated and recorded in Table 7. The observed results evidently confirmed that the chitosan-coated nano-liposomes exhibited significantly low drug degradation rate as compared to plain drug solution resulting in 2.12 and 77.3 fold extended half-life in SGF and SIF, respectively.

CONCLUSION
Formulation development and optimization of chitosancoated nano-liposomes of progesterone were successfully accomplished by response surface methodology using BBD. The aqueous solubility of progesterone was enhanced by HP-β-CD complexation which also lead to high EE in liposomes. The developed liposomes possessed all the desired physico-chemical properties (CQAs) in the acceptable range. The drug release data confirmed that the developed formulation exhibits sustained drug release profile following Higuchi's kinetic model which would be helpful in prolonged therapeutic action. The ex-vivo drug permeation study exhibited approximately twofold higher drug permeation in developed formulation in comparison to prepared suspension of marketed tablet. The stability data in GI fluid, i.e., SGF and SIF also confirmed that the chitosan-coated nano-liposomes of progesterone had better GI stability showing 2.12 and 77.3 fold longer half-life in SGF and SIF, respectively. It can be concluded that the chitosancoated nano-liposomes of progesterone can be a better alternative for its effective and safe oral delivery even in the presence of GI fluids. The in-vitro and ex-vivo evaluation studies confirmed that the present formulation approach significantly enhanced the drug permeation/absorption which would lead to improved drug bioavailability and better progesterone hormonal therapy via oral route eliminating the limitation of conventional dosage forms and improving the patient compliance.