Design, Optimization, and Characterization of Atorvastatin-Loaded Lipid-based Carriers for Enhanced Oral Bioavailability

INTRODUCTION

Nanoliposome, a new type of formulation, are work well with human physiology. Compared to all other macromolecules for numerous pharmaceutical drugs with poor bioavailability in the previous years, lipid-based nanocarriers have been recognized as excellent carrier and delivery systems. Additionally, unique qualities will be demonstrated by nanocarriers such as high biocompatibility, inexpensive scale-up production, nominal toxicity, and loading efficiency of high drug. ^1,2,3 To encapsulate and deliver bioactive compounds, adaptable platforms were provided by lipid-based nanocarriers, along with overcoming associated challenges. These novel carriers can improve the stability, drug retention enhancement, solubility, and therapeutic efficacy ^4,5,6 of designing formulations, the bioavailability of nutrients is the key aspect to be considered when designing formulations. The amount and speed at which a drug remains the same and enters the bloodstream is called bioavailability. Through food meals when nutraceuticals are consumed, the compounds pass through the mouth, stomach, and intestine before reaching the bloodstream, as well as major barriers such as degradation and uncontrolled release in the stomach and small intestine, as well as poor bloodstream penetration from the intestinal epithelium. ^7,8 Bioavailability will increase if these complications are resolved. Furthermore, bioavailability will also increase when greater bioaccessibility of nutrients results from protection against the gastrointestinal trac (GIT) environment, such as pH, by enhancing greater absorption from epithelial cells. To progress bioactive compounds bioavailability, it has been showed that nanocarriers are a virtuous candidate. ^9,10,11,12 To increase bioavailability and therapeutic activities, nanomedicine is an auspicious tool. Usually, a large surface area is provided by nanocarriers, which have their own characteristics, merits, and demerits; in addition, it will be helpful in overcoming anatomic barriers. ^13,14 In clinical use or being developed, many drugs are insoluble in water and have low bioavailability. Liposomal delivery in the digestive system is considered a promising method for improving drug dissolution and absorption. This is because liposomes are biocompatible and can retain water-repelling molecules inside their fatty layers. However, there are some issues, such as liposomes not passing easily through the cells in the intestines and becoming unstable due to their larger size. Despite the development of parenteral liposomes, no liposomal formulations have been approved for oral use. In the last decade, with the rapid growth in available studies, liposomal oral delivery has resurged. ^15,16,17,18 Owing to their ability to solubilize water-insoluble medications into nanoscale structures and modify their in in vivo behavior to decrease toxicity, liposomes and other particulate delivery systems have gained much attention. One of the most comprehensively tried systems among the nanoparticles is liposomes, since they are biocompatible enough to be approved for parenteral administration. ^19,20,21 Water-insoluble drugs can be dissolved in the liposomal membrane lipid domain by liposomes, which are vesicles bound by phospholipid bilayers. ^22,23,24,25 The structural and compositional similarity of liposomes to bio-membranes, along with their solubilizing capacity and biocompatibility, has promoted their application in non-invasive oral delivery of poorly permeable medications. ²⁶ Liposomes can help to dissolve drugs that do not mix well with water. In the digestive system, they protect these drugs from breaking down and help them pass through cell walls in the lining of the stomach and intestines. All of these factors make it easier for the body to absorb drugs when taken orally. Thus, liposomes are a better option for delivering drugs that are not water soluble. However, even though liposomes have these benefits, they have not been very effective in improving the absorption of drugs, such as peptides and proteins. To improve drug absorption through the mouth, biocompatible carriers such as liposomes are used. Along with their many benefits, these nanocarriers are useful because their structures are similar to those of the cell membranes. They adhere well to biological membranes, making them suitable for uptake by the lymph system. Moreover, when mixed with bile salts, they can form complexes that help dissolve drugs that do not mix well with water. Improving the oral bioavailability of a variety of compounds, including hydrophilic and lipophilic drugs, peptides, and proteins they have been popular. The main apprehension for oral liposomes is their stability under GI conditions; however, to increase the stability of oral liposomes, promising approaches have been suggested. These include polymer coating, appropriate use of lipid compositions, double liposome preparation and proliposomes, addition of stabilizing lipids to liposomal structures, and some other innovative approaches. ²⁷ ATV a lipid-regulating drug, is widely used to decrease the cholesterol and triglyceride levels. Drug delivery approaches that can increase solubility and avoid hepatic effects must be developed owing to their poor water solubility and hepatic metabolism. For this purpose, atorvastatin (ATV) loaded liposomes were prepared and characterized. ²⁸ A novel approach was developed to improve ATV bioavailability and stability of ATV by constructing liposome with ATV. However, statins have some limitations such as poor oral absorption and low aqueous solubility, and when administered orally, they have limited bioavailability. Currently, nanotechnology is a well-developed field, and new methods provide systems with enhanced statin aqueous solubility, bioavailability, higher absorption, and controlled release of statin at the site of administration controlled release of the statin. ²⁹ Due to their considerable molecular weight and limited aqueous solubility, statins have low oral bioavailability. Furthermore, drug-related adverse effects can develop in a significant number of patients. The bioavailability of statins can be increased by effective delivery methods, which could decrease the toxicity and side effects associated with increased statin plasma concentrations. ³⁰

MATERIALS AND METHODS

Materials

Atorvastatin (ATV) was received as gift from Dr. Reddy's Lab. (Hyderabad, India). Soya-L-α-lecithin (SPC), cholesterol (CHL), chloroform, Tween 80, Acetonitrile were purchased from Merck (Mumbai, India). butylated hydroxy toluene (BHT) was procured from Qualigens Fine Chemicals (Mumbai, India). Marketed tablet of ATV (Lipitor) was purchased from local market.

Methods

Prepartion of Liposomes

Using the thin film hydration method, ATV-containing nanoiposomes were prepared. ATV, CHL, and SPC (among the ingredients' specific amounts). were mixed together and dissolved in 10 mL of chloroform in a 250 mL round-bottom flask. After thoroughly mixing the content, the solvent was removed at 40°C using an aspirator and a rotary vacuum evaporator (Rotavap, model: PBU-6). The resulting thin film was placed overnight in a vacuum desiccator. It was subsequently soaked in PBS (pH 7.4) at 60°C with stirring at 160 rpm for one hour, followed by an additional hour at a frequency of 30 ± 3 KHz with an ultrasonicator (Trans-o-Sonic, Mumbai, India). At 15,000 rpm the mixture was centrifuged for one hour at 4°C in a refrigerated centrifuge (3K30 Sigma Lab Centrifuge). To assess the optimal dry mass, the liposome mass was collected in a Petri dish and freeze-dried using a laboratory freeze-dryer (Laboratory-Freeze Dryer, Instrumentation India Ltd., Kolkata).

Experimental Animals

Swiss albino mice (9 males, 9 females; average weights: 25-30 g. A total of 18 mice, divided into three groups) were used for the bioavailability assay under the approval of NCPT/IAEC-028/2025 by the IAEC of NSHM Knowledge Campus (Animal House) in Kolkata. A single dose of 10 mg/kg was administered orally to mice for 24 hr. Male Albino mice were kept in a controlled environment at the NSHM Knowledge Campus for one week before the experiment started. The conditions were temperature 22 ± 3°C and relative humidity 50 ± 5%. Before the experiment, the mice were kept under a regular dark and light cycle and fasted overnight. Table 1 describes the optimization of the liposome preparation.

Preparation of Calibration curve of ATV

Using a UV/vis spectrophotometer, drug absorption maxima were found in various working solutions, including PBS with 0.5% (w/v) TWEEN and in ACN. Respective absorbance against concentrations was determined in PBS at pH 7.4 and ACN. The data were plotted to develop the respective calibration curves shown in respectively.

Drug-excipients interaction study

The preliminary drug-excipients interaction was assessed by Fourier transform infrared (FTIR) spectroscopy. The individual components of the experimental formulations viz, ATV, CHL and SPC, a mixture of ATV, CHL & SPC; and experimental formulation were run in FTIR spectrophotometer within 4000 cm-1 to 400 cm-1. The spectrums were recorded and compared with the principle peaks from each other to know any changes in the structure.

Vesicle size and zeta potential

The size of nanoparticles was measured using a Nano ZS90 Zetasizer. The effective electric charge on the nanoparticles was checked at a temperature of 25°C with a fixed angle of 173° to reduce multiple scattering.

Surface Topography By Field-Emission Scanning Electron Microscope (Fe-Sem) And Cryogenic-Transmission Electron Microscopy (Cryo-Tem) Study

High-resolution images of the experimental formulation was captured using a field-emission scanning electron microscope (Thermofisher Apreo 2S, India). The small quantity of proliposomes were placed on double-sided sticky carbon tapes, were coated with platinum for 2 min to ensure an even layer on the surface of about 5 nm. and then attached to FE-SEM sample holders and images were taken at different magnification. The surface morphology was further substantiated by Cryo-TEM (Titan Krios ,Thermo Fisher, India). The sample under testing was placed on a carbon coated copper grid, followed by removal of extra solvent by blotting and then the grid was subjected to vitrification (Treated the grid with liquid nitrogen. The vitrified grid was placed in Cryo TEM sample holder and images were recorded and analyzed.

In vitro drug release

For in vitro drug release experiments, Franz diffusion cells with dialysis membranes were used for in vitro drug release experiments. The optimized formulation was administered to the donor compartment, whereas a phosphate buffer solution (pH 6.8) was administered to the receptor compartment. Using a circulating water bath, both compartments were maintained at 37°C with a 0.5% tolerance. A 2 mL aliquot was taken and 2 mL of freshly prepared buffer was added to maintain sink conditions at different time intervals (0, 15, 30, 60, 360, and 720 min), filtered, and analyzed using UV Spectroscopy.

Further to establish the mechanism of drug release, the release data were fitted in the different kinetics of drug release and the release mechanism was established by observing the correlation coefficient (R2) value.

release mechanism was established by observing the correlation coefficient (R2) value.

Statistical Optimization

Optimization of ATV loaded lipid based nano carriers to improve bioavailability by Response Surface Methodology (RSM).

Analyzing data and verifying the optimization model

In the existing optimization study, numerous Response Surface Methodology (RSM) calculations were performed using State-Ease Inc. Design Expert Software (Model 13). Multiple Linear Regression Analysis (MLRA) was used to create polynomial fashions that blanketed interplay and quadratic phrases for each reaction variable. The following equation illustrates the overall shape of the MLRA model: $Y=\mathrm{\beta ₀}+\mathrm{\beta ₁X₁}+\mathrm{\beta ₂X₂}+\mathrm{\beta ₃X₁X₂}+\mathrm{\beta ₄X₁²}+\mathrm{\beta ₅X₂²}+\mathrm{\beta ₆X₁X₂²}+\mathrm{\beta ₇X₁²X₂}$ Within the over condition, β₀ speaks to the captured and demonstrates that the scientific cruel of all quantitative comes about obtained from the nine tests. The coefficients β₁ to β₇ are inferred from the test reaction values of Y, whereas X₁ and X₂ demonstrate the coded levels of the impartial variable. The terms X₁, X₂, and Xi² (i = 1 to 2) were compared to the interaction and quadratic conditions, respectively. The factual legitimacy of the polynomial conditions was based on an ANOVA investigation within the Plan Master Computer program. These plots are exceptionally valuable for observing the interaction effects between the factors on the reaction.

In vivo bioavailability study

For this study, a High Performance Liquid Chromatography (HPLC) system (Shimadzu Corp) was used. The mobile phase was made with a mixture of 30% phosphate buffer and 70% methanol, and it flowed at a speed of 1 mL per minute. The retro-orbital route was used to collect blood (0.5 mL) before dosing at 15, 30, 60, 360, and 720 min. Blood samples were centrifuged for 10 min at 5500 rpm. Blood plasma was stored and kept at 20°C. We mixed 0.1 mL of blood was mixed with acetonitrile (0.5 mL) to extract the drug and allowed to stand for 10 min. Then, 150 μL of plasma and 50 mg of Internal Standard (IS) were added to each 1.5 mL centrifuge tube. After adding 300 μL of acetonitrile, the samples were shaken for five minutes and spun again at 6600 rpm for 5 min to complete deproteinization. We used micropipettes to collect clear liquid from each sample. The liquid was then filtered through a 0.45 micron filter. Finally, 20 μL of pure top layer was injected into the HPLC system. For the bioavailability study, three groups of six animals each were used. One group was the control group, one received the marketed formulation, and the third received ATV-loaded liposomes as nano-carriers. Pharmacokinetic studies were conducted by measuring the drug concentration over time to determine parameters such as C_max, t_max, AUC_0-t, AUC₀-∞ , t_1/2, and K_el. These pharmacokinetic data helped estimate the ATV, and the results are presented as the mean ± standard deviation.

Click to enlarge

RESULTS

Determination of lambda max and Calibration Curve of ATV

At 245 nm, the maximum absorption of ATV in a PBS containing 0.5% (w/v) Tween at pH 7.4 was noted. The absorption maximum of ATV in ACN was detected at 246 nm.

The FTIR spectra of ATV (A), physical mixture (B), SPC (C), CHL(D), and experimental formulation (E) were analyzed to investigate the compatibility of the drug with the selected liposomal excipients. The FTIR spectrum of A) exhibited its characteristic absorption bands corresponding to O-H stretching (~3400 cm⁻¹), aliphatic C-H stretching (~2920-2850 cm⁻¹), carbonyl (C=O) stretching (~1700 cm⁻¹), aromatic C=C vibrations (~1600-1500 cm⁻¹), and C-O/C-F stretching vibrations in the fingerprint region (1200-1000 cm⁻¹), confirming the identity and structural integrity of the drug. Soya lecithin (C) showed characteristic C-H stretching bands at 2920-2850 cm⁻¹, ester carbonyl absorption near 1735 cm⁻¹, phosphate (P=O) stretching around 1240 cm⁻¹, and P-O-C stretching near 1060 cm⁻¹. Cholesterol (D) displayed characteristic O-H stretching around 3400 cm⁻¹, aliphatic C-H stretching at 2930-2860 cm⁻¹, and C-O stretching in the region of 1050-1100 cm⁻¹. The experimental batch (E) retained all the characteristic peaks of both components without the appearance of any additional bands or significant peak shifts, Similarly, the physical mixture of atorvastatin, cholesterol, and soya lecithin (B) showed the characteristic absorption peaks of atorvastatin, cholesterol, and soya lecithin with only slight variations in peak intensity and broadening. No new absorption bands, disappearance of characteristic peaks, or significant shifts in wavenumber were observed. These findings indicate the absence of any chemical interaction or incompatibility between atorvastatin and the lipid components.

The minor changes in peak intensity and slight broadening observed in the physical mixture are attributed to weak physical interactions, such as hydrogen bonding, van der Waals f orces, and hydrophobic interactions between atorvastatin and the phospholipid bilayer constituents. Such non-covalent interactions are expected during liposome preparation and facilitate the incorporation of the drug within the lipid bilayer without altering its chemical structure.

Overall, the FTIR results confirm that ATV is compatible with cholesterol and soya lecithin, demonstrating no evidence of chemical interaction among the formulation components. The observed physical interactions are desirable, as they promote drug entrapment and stabilization within the liposomal vesicles, thereby supporting the successful development of a stable ATV-loaded liposomal formulation.

Click to enlarge

Click to enlarge

Vesicle Size & zeta potential

The particle size of NL3 (optimized best formulation) is shown in Figures 2 and 3, which represent particle sizes of 110.3 ± 14.6 nm, PDI values of 0.28 ±0.03, and zeta potentials of -58.7 ± 7.1 mV (Figures 2 and 3).

FE-SEM replace please with FE-SEM AND CRYO-TEM STUDY

This image shows the surface morphology and topography of the samples at the nanometer scale with spherical shape and uniform distribution throughout the matrix of the experimental sample of proliposomes batch with size less than 100 nm.

Further, the particle morphology for shape, size was confirmed by by Cryo-TEM study of the optimized formulation as shown in Figure 4 and 5. The size is less than 100 nm with smooth surface and intact unilamellar vesicle were observed.

In vitro drug release

In vitro dissolution data of liposomes were suitable for various kinetic equations, the correlation coefficient (R²) value was calculated from different kinetics models, and the value of Korsmeyer-Peppas release was 0.999. Table 2 describes two different in in vitro studies of the three best formulations with different ratios of drug and excipients.

Statistical Optimization

Experimental Design by RSM

Formulation trials were carried out for the formulation, physicochemical characterization, and assessment of atorvastatin loaded lipid based nano carriers to improve bioavailability With SPC and RPM (independent variables) and loading, particle size, and zeta potential (dependent variables) at different ratio levels used as per the experimental design (Figures 6 and 7). Where;

Click to enlarge

Click to enlarge

(a) Drug Loading (%) = 1028.28 + 970.45+ 77.83B + 165.38AB + 1.28A2 + 0.5408B2 Where F value = 85.45, R² = 0.984,p < 0.05. (b) Particle Size (nm)= 479.78 + 451.83 + 27.95B + 1021.44AB + 2.52A2 + 0.9339B2 Where F value = 113.47, R² = 0.974,p < 0.05. (c) Zeta Potential (mV) = 1047.12+ 991.20+ 101.92B +121.77AB + 0.2812A2 + 0.0952B2 Where F value = 94.29, R² = 0.964,p < 0.05.

(a) Significance results for % of Drug Loading (b) Particle Size (c) Zeta potential. The given equations show how the process variables interact to produce the response and its quantifiable effect. To assess the significance of the model, an analysis of variance (model: ANOVA 13) was performed using Design Expert software as described below. At a significance level of 5%, a model is considered significant if the p-value is less than 0.05.

In vivo bioavailability study

A C_max of ATV following orally administered atorvastatin with liposome was 9.9±1.10 μg/mL and then it took few hours to reach t_max and atorvastatin liposome had C_max, and the t_max was 26.12.±1.5 μg hr/mL respectively. The AUC₀-∞ as significantly elevated in atorvastatin-loaded liposome, compared to marketed ATV ranging from 172.1±23.1 in μg hr/mL up to 610.2±12.3μg hr/mL. Table 3 describes the different evaluation parameters of in vivo studies with the optimized formulation.

DISCUSSION

Statistical optimization of the selected formula was performed to obtain the best ratio and the most optimized formula. Nine formulation variables, including the SPC-to-CHL ratio, were optimized using a factorial design. Statistical analysis of the data on drug loading (%), particle size, and zeta potential showed that two variables, X1 and X2, were highly important for the formulation. As shown in Figure 6, formulation NL3 was selected as an atorvastatin-loaded lipid-based nanocarrier to improve its bioavailability by SPC and CHL. The medicine ATV calcium is hydrophobic and artificial with low bioavailability. Liposomes are tiny round vesicles with one or more lipid bilayers that can be crafted from herbal or artificial lipids. To avoid first-pass metabolism, liposomes can protect hydrophobic medicines from metabolism in physiological fluids and increase their solubility and bioavailability. The goal was to create, characterize physicochemically, and evaluate atorvastatin-loaded liposomes to increase ATV's bioavailability of ATV. The current study aimed to enhance the solubility, dissolution rate, and bioavailability of poorly soluble ATV. The procedure entails creating an ATV calibration curve in Acetonitrile (ACN) and PBS with 0.5% Tween 80, conducting an FTIR analysis, and creating ATV-loaded NLs using the thin-film hydration process and DLS analysis. Using PBS containing Tween and ACN, the drug absorption maxima were observed at 246 and 245 nm, respectively. All excipients were compatible according to the FTIR results. Based on numerous observations, the NL3 formulation was found to be the optimal formulation for the final product yield, drug abuse, and loading efficiency rate in the process. This formulation was approved for additional in research in vitro and in vivo. The zeta potential was -58.7 ± 7.1 mV, PDI was 0.28 ± 0.03, and particle size was 110.3 ± 14.6 for NL3, Cryo-TEM Showed unilamiller that produce unform distribution and AUC of animal studies increased two-fold rather than marketed formulation.

Click to enlarge

Click to enlarge

CONCLUSION

Nanocarrier technology represents a breakthrough for the delivery of pharmaceutical drugs. Tests show that these carriers boost drug bioavailability fold, which solves a basic problem in medicine: getting drugs exactly where they need to go. Traditional delivery methods are nowhere nearly as effective, with less than half the drug reaching its intended target. These systems excel through precise targeting and smart release. They work exceptionally well across different applications, from better cellular uptake to stronger antitumor effects. Market numbers validate these clinical results and indicate a promising future for nano-based delivery systems. Patients can expect better outcomes with higher absorption rates, fewer side effects, and easier treatment plans. Our ongoing research builds foundations for better therapeutic solutions that can change patient care worldwide.

SUMMARY

Administration of drugs via Nano liposomes could be an effective method for enhancing the bioavailability of poorly water-soluble drugs. This study showed a two-fold higher bioavailability than that reported for atorvastatin. According to in vivo tests, the C_max of atorvastatin increased dramatically when utilizing liposomes loaded with atorvastatin (formulation) compared to marketed atorvastatin 10 mg tablets. The AUC determination from experimental rat blood plasma revealed that liposome-encapsulated atorvastatin had greater bioavailability than the marketed formulation and test sample.

• Atorvastatin-loaded nano-liposomes have been successfully developed to overcome poor solubility and extensive first-pass metabolism.
• The liposomal formulation resulted in nanosized vesicles (~110 nm) with a narrow size distribution and high colloidal stability.
• Optimized formulation showed high drug loading efficiency (>60%).
• Nano-liposomes enhanced the oral bioavailability of atorvastatin by approximately few-fold compared with that of conventional dosage forms.
• Liposomal delivery demonstrated potential for dose reduction and sustained therapeutic action in hyperlipidaemia management.