Development of Carvedilol-Encapsulated Liposomal Nanoparticles Using the Thin Film Hydration Method: A Design Expert®-Based Approach

INTRODUCTION 
Carvedilol is a nonselective beta-adrenergic antagonist commonly prescribed for the chronic management of heart failure with reduced ejection fraction, hypertension, and impaired left ventricular function following myocardial infarction in clinically stable patients. Additionally, it is sometimes used off-label to treat stable angina, control atrial fibrillation, prevent oesophageal variceal bleeding in cirrhotic patients, and manage ventricular arrhythmias [1]. 
Carvedilol exhibits low oral bioavailability and is poorly soluble in water, which can result in frequent dosing and reduced patient adherence to long-term treatment [2]. Currently on the market, about 40% of medications are classified as poorly soluble in water. Furthermore, it’s believed that roughly 70% of recently created medication candidates have poor solubility in water [3].
In order to get around this problem for weakly water-soluble APIs, several techniques have been developed, including liposomes, nanoemulsions, melt extrusion, salt creation, solid dispersions, nanostructured lipid carriers, and micellar systems [4]. In 1961, Alec D. Bangham, in England, developed the first artificial liposomes. He discovered that phospholipids spontaneously assemble into spherical structures when mixed with water, as each molecule possesses a water-soluble end and a water-insoluble end [5].
Liposomes are small, spherical vesicles composed of the same material as the cell membrane. The term ‘liposome’ originates from two Greek words: ‘Lipos, meaning fat, and ‘soma, meaning body. Phospholipid molecules can spontaneously self-assemble into a bilayer structure, forming these tiny vesicles [6]. Liposomes can encapsulate both lipophilic and hydrophilic drugs within their lipid bilayer and internal aqueous compartment, respectively [7]. Liposomes offer several advantages as oral drug carriers, such as excellent biocompatibility, the ability to encapsulate various drugs, adaptable membrane composition, modifiable surfaces, potentially enhanced permeability, and tunable pharmacokinetic profiles [8].
The present study aimed to formulate and optimise liposomal formulations using Design Expert® software to minimise both time and cost.

MATERIALS AND METHODS
Materials
Carvedilol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghi, china). 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from Shanghai CarbonBond Chemical Co., Ltd. (Shanghai, China). Hydrogenated soy phosphatidylcholine (HSPC) was purchased from JYOUKI Pharmaceutical CO., Ltd. (Fushun, China). IsoporeTM Polycarbonate (PC) Membrane Filters with a pore size of 100 nm were purchased from Merck Millipore Ltd. (Cork, Ireland).

Methods 
Preparation of carvedilol liposomes
This research involved the preparation of liposomes through the thin-layer hydration method using a Heidolph rotary evaporator [9]. For the preparation of liposome formulations, various quantities of phospholipids (DPPC, HSPC) and cholesterol were used, as shown in Table 1. In a round-bottom flask, all lipids and the hydrophobic medication are dissolved in 2:3 methanol and chloroform mixture [10].  The organic solvent was then removed using a rotary evaporator operated under reduced pressure at a temperature above the lipid transition temperature, until a thin film was formed [11]. 
The film was then hydrated by adding 10ml of phosphate buffer. The flask was once again circulated on the rotary flask evaporator without vacuum above the phospholipid transition temperature until a white, milky suspension was achieved in order to disperse the film in the solution [7]. In order to decrease the liposomes’ size further, the dispersion was repeatedly extruded 10 times through a polycarbonate membrane with a pore size of 100 nm. 

Characterisation of carvedilol liposomes
Measurement of particle size, polydispersity index
Particle size and polydispersity index of liposomes were determined by the dynamic light scattering method by using ABT-9000 Nano Laser Particle Size Analyser. In essence, PDI shows how the sizes of populations are distributed throughout a sample. A PDI of 0.3 or less is regarded as appropriate in drug delivery applications utilising lipid-based carriers, such as liposome and nanoliposome formulations, and denotes a uniform population of phospholipid vesicles [12].

Measurement of zeta potential
Zeta potential of liposome formulations was measured using a Zetasizer (Malvern Panalytical, UK). After diluting each sample with distilled water to the appropriate concentration, analysis was carried out at 25 ºC with a detection angle of 90 ºC [13].
The charges that are present on the liposome surface are known as the zeta potential. All liposomes in the formulation must have a charge on their surface, as this prevents particle coagulation [14]. This charge causes the liposome particles to repel one another, preventing coagulation [15]. It is necessary to have a zeta potential value that falls between +30 and -30mV. These ranges stop liposomal particles from aggregating [6].

Entrapment Efficiency and Drug Loading
The entrapment efficiency and drug loading were determined by separating non-encapsulated carvedilol from the liposomal suspension by centrifugation [16]. A 2 ml of liposome suspension was centrifuged at 13000 rpm for 30 min at 4 °C.
The supernatant was removed, and liposomes were disrupted with methanol to release the entrapped drug. The quantity of the drug was measured by using UV spectroscopy at 285 nm [17]. The EE and LC were measured using Eqs. 1 and 2.

In vitro drug release
The in vitro release of carvedilol-loaded liposomes was evaluated using the dialysis bag diffusion method. A dialysis membrane with a molecular weight cutoff of 14,000 Da was pre-soaked in phosphate buffer (pH 6.8) for 24 hours prior to use to ensure complete wetting and removal of any preservatives. The release study was performed in phosphate buffer (pH 6.8) containing 0.1% Tween 80, maintained at 37 ± 0.5 °C under constant magnetic stirring at 100 rpm [3]. A 2 mL aliquot of the liposomal dispersion, equivalent to 2 mg of carvedilol, was placed into the dialysis bag, and the open end was securely tied with a thread before immersion in the release medium. At predetermined time intervals (1, 2, 3, 4, 5, 6, 7, 8, and 14 hours), 5 ml of the release medium was withdrawn and replaced with an equal volume of fresh medium to maintain sink conditions [18]. The amount of carvedilol released was quantified using UV–visible spectrophotometry.

Fourier transform infrared spectroscopy
Several FTIR spectra were obtained using an FTIR spectrophotometer (Shimadzu, Japan) for pure carvedilol powder, a physical mixture, blank liposomal nanoparticles (without drug), and the carvedilol-loaded liposomal formulation [19]. For each measurement, the sample was thoroughly mixed with potassium bromide (KBr) powder and subsequently compressed into a disc containing approximately 2% (w/w) of the sample. The spectra were recorded over the range of 4000–600 cm⁻¹ with a resolution of 4 cm⁻¹ [20].

Statistical analysis
A full factorial design was employed using Design-Expert® software version 13 (Stat-Ease Inc., USA) to systematically investigate the influence of three independent variables -DPPC, HSPC, and cholesterol on the physicochemical properties of carvedilol-loaded liposomes [21]. The factorial design enabled the evaluation of both the individual effects of each variable and interaction effects between them on key responses, including particle size, polydispersity index (PDI), entrapment efficiency (EE), drug loading (DL), and zeta potential [22]. Statistical significance was assessed using analysis of variance (ANOVA), and model adequacy was evaluated through diagnostic plots such as Pareto charts and response surface plots. This approach facilitated the identification of an optimal formulation by maximising desirability, ensuring efficient and cost-effective development of a stable liposomal system.

RESULT AND DISCUSSION
Particle size and PDI
The influence of formulation variables A-DPPC, B-HSPC and C-Cholesteryl on the particle size of carvedilol-loaded liposomes was analysed using a Pareto chart, as shown in Fig. 1. The chart revealed that HSPC had the most significant positive effect on particle size, followed by DPPC and cholesterol [23]. This indicates that increasing their concentrations leads to larger vesicle sizes. Interaction terms such as AB, AC, and BC showed minor effects and were statistically insignificant compared to the main effects.
HSPC is a saturated phospholipid with a high phase transition temperature and tight lipid packing, leading to increased membrane rigidity and particle size. The observed increase in liposome particle size with increased concentrations of DPPC and cholesterol is consistent with the findings reported by Lopez-Pinto et al. (2005) and Kumar et al. (2010), who also demonstrated that increases in DPPC and cholesterol contribute to increased membrane rigidity and particle size [10, 24].
All formulations exhibited polydispersity index (PDI) values below 0.1, indicating a uniform and monodisperse particle distribution. The use of extrusion contributed significantly to achieving these low PDI values across all formulations. These results are consistent with the findings of Kuntsche et al. (2010), who demonstrated that extrusion effectively produces monodisperse liposomal formulations by reducing particle size [25].

Entrapment Efficiency and Drug Loading
The composition of phospholipids and cholesterol significantly influenced the entrapment efficiency of carvedilol-loaded liposomes. The combination of DPPC and HSPC exhibited the strongest, most potent positive effect on drug entrapment, suggesting a synergistic interaction between the two saturated phospholipids. Increasing the concentration of cholesterol also contributed positively, likely due to its role in enhancing bilayer stability and reducing membrane permeability [22]. 
This finding agrees with the results reported by El-Nesr et al. (2010), who observed that increasing the cholesterol content in liposomal formulations significantly improved the entrapment efficiency of fluconazole, attributing the effect to the reduced membrane fluidity and increased rigidity of the lipid bilayer, which minimises drug leakage [26]. Individually, DPPC had a greater impact on entrapment efficiency compared to HSPC, likely due to differences in lipid bilayer properties and specific interactions with carvedilol. This observation is consistent with findings reported by Najlah et al. (2018), where DPPC-based liposomes achieved 35–92% paclitaxel (PX) entrapment, compared to 25–60% with HSPC-based formulations [27].
In addition to entrapment efficiency, drug loading (DL) is another key parameter in evaluating liposomal formulations [28]. While EE reflects the proportion of drug successfully encapsulated relative to the total amount used, DL indicates the amount of drug loaded per unit weight of lipid, which is critical for determining dosing efficiency and excipient burden. An increased drug-to-lipid ratio may reduce formulation costs and minimise lipid-associated toxicity by requiring less lipid to deliver an effective drug dose.
The entrapment efficiency (EE) and drug loading (DL) of the formulations range from 55.41% to 92.38% and from 6.52% to 13.58% respectively.

Measurement of zeta potential
The zeta potential measurements of the formulations varied between −0.3 mV and −12.4 mV, indicating a generally negative surface charge across all samples. These results align with those reported by Chen et al., (2014), who investigated brucine-incorporated liposomes prepared with HSPC and DPPC, which exhibited zeta potentials ranging from −5.96 to −3.20 mV [29]. Despite being within the moderate range of electrostatic stabilisations (±30 mV), the formulations demonstrated good physical stability, likely due to uniform particle size and lipid composition.

In vitro drug release
Five formulations were tested, including four liposomal preparations (F1 = F14, F6 = F7, F16 = F17, F4 = F3) and a pure drug suspension. The release profiles demonstrated a biphasic pattern for all formulations, characterised by an initial moderate release followed by a sustained release phase. 
Among the formulations, F16 = F17 exhibited the highest cumulative drug release (87.29% at 14 h), attributed to its low HSPC content, which possibly led to higher membrane permeability. In contrast, F6 = F7, with a high cholesterol content (8 mg), showed a more sustained release profile (77.44% at 14 h), confirming the role of cholesterol in stabilising the lipid bilayer and retarding drug diffusion. F1 = F14 demonstrated an intermediate release behaviour (81.3% at 14 h), while F4 = F3 showed relatively rapid release due to lower DPPC content. The pure carvedilol suspension exhibited a non-controlled release pattern, reaching only 64.48% at 14 hours, likely due to its limited aqueous solubility, even in the presence of Tween 80.
These findings suggest that the lipid composition, particularly the ratio of cholesterol and phospholipids, has a considerable influence on the drug release kinetics from liposomal carriers. these observations are supported by the data shown in Fig. 3.

Fourier transform infrared spectroscopy
The FTIR analysis was performed on carvedilol powder to assess the drug’s purity and to evaluate its compatibility with the phospholipids and cholesterol. The study revealed distinctive peaks at 3346.5 cm⁻¹, corresponding to the stretching vibrations of N–H and O–H groups, and at 1255.66 cm⁻¹, attributed to C–O and O–H bending vibrations. These findings are in agreement with Rehab et al., (2021), who reported that pure carvedilol exhibits peaks at 3344.66 cm⁻¹ and 1255.39 cm⁻¹, respectively [30]. The peak at 2924.09 cm⁻¹ corresponds to C–H stretching vibrations, while the peak at 1602.85 cm⁻¹ results from bending vibrations of the N–H group. These results are in agreement with Alves et al., (2016), who reported that carvedilol exhibits peaks at 2924 cm⁻¹ and 1598 cm⁻¹, respectively [20]. These characteristic peaks observed in the FTIR spectrum confirm the purity of the carvedilol powder, as shown in Fig. 3.
The FTIR compatibility test was conducted to confirm that carvedilol is compatible with the other liposomal ingredients and to identify any potential chemical interactions. This was achieved by comparing the FTIR spectrum of pure carvedilol to the spectra of the physical mixture and the carvedilol-loaded liposome formulation. The spectra revealed no significant shifts or disappearance of characteristic peaks, indicating that the functional groups remained essentially unchanged. These results suggest the absence of any chemical interaction between carvedilol and the excipients, thereby confirming their compatibility, as shown in Fig. 4.

CONCLUSION
In this study, carvedilol-loaded liposomal nanoparticles were successfully developed using the thin-film hydration method and then subjected to particle size reduction via extrusion. The findings demonstrate that careful selection and optimisation of phospholipid and cholesterol concentrations, optimised by Design Expert® software, can produce liposomes with desirable particle size, high encapsulation efficiency, and good stability properties that are essential for enhancing the solubility and potential bioavailability of poorly water-soluble drugs like carvedilol. This approach highlights the potential of liposomal nanocarriers as a practical strategy for improving drug delivery and therapeutic outcomes. 

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.