Development, Optimization and Characterization of Picroside-II loaded Proliposomes using Quality by Design (QbD) approach

Table 01 Factors for QbD

Table 02 Central Composite Design for two factors

Characterization of PK-II proliposomes

Entrapment efficiency

The entrapment efficiency (EE %) of Picroside-II in the proliposomes formulations was determined using the indirect centrifugation method. In this method, the accurately weighed 10 mg of proliposomes formulation was transferred into a microcentrifuge tube, followed by the addition of 2 mL of deionised water. The mixture was vortexed for 10 seconds to ensure complete hydration of the proliposomes. The hydrated dispersion was then centrifuged at 14,000 rpm for 20 minutes at room temperature. After centrifugation, 1 mL of the clear supernatant was carefully collected and suitably diluted with deionised water for UV spectrophotometric analysis at 218 nm to quantify the amount of unentrapped (free) drug. All measurements were performed in triplicate. The entrapment efficiency was calculated based on the difference between the initial amount of absolute Picroside-II present in the formulation and free Picroside-II amount detected in the aqueous supernatant. The following equation (Eq. 01) is used:

% drug entraped=total drug added-free drugtotal drug addedX 100

Particle size and zeta potential

Picroside-II liposomes were generated by reconstituting the proliposomes in deionized water. Proliposomes equivalent to 10 mg/mL of PK-II were dispersed in a small volume of deionized water and vortexed for 10 seconds to facilitate hydration and vesicle formation. The volume was then adjusted to 10 mL with deionized water to obtain a uniform liposomes dispersion. The resulting liposomes suspension was evaluated for particle size distribution, polydispersity index (PDI), and zeta potential using a dynamic light scattering (DLS) instrument (Horiba SZ-100).

SEM analysis

The surface morphology of the prepared Picroside-II proliposomes powder was examined using Scanning Electron Microscopy (SEM). SEM imaging was performed on a JSM-6510 (JEOL, Japan) high-resolution scanning electron microscope. A small quantity of sample (1–2 mg) was carefully placed onto an aluminium stub using double-sided conductive carbon tape. As the proliposomes powder is non-conductive, the mounted sample was sputter-coated with a thin layer of gold to enhance conductivity and improve image quality. Following vacuum stabilization of the instrument, the sample was loaded into the SEM chamber and evacuated to a pressure of 10⁻⁵–10⁻⁶ mbar. Imaging was carried out using an accelerating voltage in the range of 5–20 kV, selecting lower voltages to minimise charging effects. The working distance was maintained between 8–12 mm to obtain optimal focus and resolution. Micrographs were captured at 500× and 2000× magnifications to visualize both overall particle structure and fine surface details. Images were obtained from multiple fields to ensure representative observation. The acquired micrographs were evaluated for particle shape, surface texture, and uniformity of the proliposomes powder.

Liquid SEM analysis

The surface morphology of liposomes formed after hydration of the Picroside-II proliposomes powder was examined using a JSM-6510 (JEOL, Japan) SEM. Proliposomes powder equivalent to 10 mg of PK-II was hydrated with 1 mL distilled water and vortexed for 10 seconds to obtain a uniform liposomes dispersion. A 0.5 mL portion of the dispersion was mixed with an equal volume of 2.5% glutaraldehyde and fixed for 30 minutes at 4 °C. After gentle washing, a drop of the fixed sample was placed on an aluminium stub with conductive carbon tape and allowed to air-dry. The dried sample was sputter-coated with gold using a JEOL JFC-1600 coater. The stub was loaded into the SEM chamber and evacuated to 10⁻⁵–10⁻⁶ mbar. Images were captured at an accelerating voltage of 5–15 kV and magnifications of 500× and 2000×. The micrographs were analysed for particle shape, surface texture, and size uniformity of the liposomes

DSC analysis

The thermal behaviour of Picroside-II, its proliposomes formulation and blank proliposomes powder was evaluated using a Hitachi EXSTAR DSC7020 instrument. Approximately 3–5 mg of each sample (pure PK-II, proliposomes powder and blank proliposomes powder) was accurately weighed and sealed in aluminium DSC pans, with an empty sealed pan serving as the reference. The instrument was calibrated as per the manufacturer’s guidelines. Samples were scanned from 25°C to 300°C at a heating rate of 10°C/min, under a nitrogen purge of 50 mL/min to prevent oxidation. Thermograms were recorded and analysed to observe endothermic/exothermic transitions and assess any changes in the thermal behaviour of PK-II within the formulation.

FTIR analysis

FTIR spectroscopy was performed to identify functional groups and evaluate possible drug–excipient interactions in the Picroside-II proliposomes formulation. The analysis was carried out using a BRUKER FTIR Alpha spectrometer. Approximately 2–5 mg of proliposomes powder was mixed with 100–200 mg of dry, spectroscopic-grade KBr and compressed into a transparent pellet using a hydraulic press (5–10 tons). A background scan using a blank KBr pellet was recorded prior to sample analysis. The sample pellet was placed in the holder, and spectra were collected over a range of 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹, averaging 16–32 scans to improve signal quality. The obtained spectrum was analysed to identify characteristic peaks of PK-II and assess any potential interactions within the formulation.

Stability study

The stability of the Picroside-II proliposomes formulation was assessed following previously reported procedures. Proliposomes powder was stored in tightly closed glass vials wrapped in aluminium foil and kept at room temperature (~25°C) and refrigeration (4°C) for one month. After storage, samples were hydrated with distilled water and examined under an optical microscope to check for drug crystallization or any physical changes. The formulations were also evaluated for colour and entrapment efficiency to determine any stability-related variations.

RESULTS AND DISCUSSION

QbD based optimization

The QbD-based optimization of PK-II proliposomes using a Central Composite Design showed wide variation in entrapment efficiency (–7.29% to 35.24%), indicating a strong influence of the formulation variables. ANOVA confirmed that the quadratic model was significant, with both factors (phospholipid-to-cholesterol and phospholipid-to-carrier ratios), their interaction, and their quadratic terms contributing meaningfully to the response, while the non-significant lack-of-fit indicated good model suitability. Increasing the phospholipid-to-cholesterol ratio consistently improved entrapment, whereas the phospholipid-to-carrier ratio showed an optimum around the mid-level (1:10), beyond which EE% decreased. The response surface (Fig. 01) and contour plots (Fig. 02) showed clear curvature and strong interaction between the two factors, with the highest efficiency (35.24%) observed at a higher phospholipid-to-cholesterol ratio (1.7071) combined with a mid-level phospholipid-to-carrier ratio (10). Overall, the results identified the region around PL: CL ≈ 1.6–1.7 and PL: C ≈ 10 as the optimal design space for maximizing entrapment efficiency (Table 03) represent the runs and their response in the form of entrapment efficiency.

Fig. 02 Contour Plot representing the response of PL: CL and PL: C ratios on EE%

The entrapment efficiency of the developed Picroside-II proliposomes was assessed to determine the amount of drug incorporated within the lipid matrix. The optimized formulation, prepared using a phosphatidylcholine: cholesterol ratio of 1:1.7 and a phosphatidylcholine to carrier ratio of 1:10, demonstrated an entrapment efficiency of 35.24% (Table 03). This value reflects satisfactory drug loading in the proliposomes system. The entrapment efficiency was influenced by lipid composition and carrier ratio, where an appropriate balance between phosphatidylcholine and cholesterol facilitated stable vesicle formation and efficient encapsulation of Picroside-II.

Particle size and zeta potential

The particle size and zeta potential of the optimized PK-II proliposomes formulation were evaluated after hydration. The average liposomes size was found to be 104.4 nm, confirming the formation of nanosized vesicles (Fig. 03). Liposomes within the 1–200 nm range are classified as nanosized particles, which is advantageous for enhancing intestinal absorption, facilitating transport across biological membranes, and potentially improving the bioavailability of PK-II. The zeta potential of the optimized formulation was measured as ±20.2 mV, indicating good electrostatic stability (Fig. 04). Typically, particles with a zeta potential within ±30 mV range exhibit sufficient repulsion to prevent aggregation, suggesting that the developed liposomes dispersion is physically stable and less prone to settling over time. Overall, the nanoscale particle size and favourable zeta potential values support the suitability of the optimized PK-II proliposomes formulation for improved absorption and stability.

Fig. 03 Particle size of PK-II proliposomes graph

Fig. 04 Zeta potential of PK-II Proliposomes graph

The surface morphology of the Picroside-II proliposomes powder was evaluated using SEM. The micrographs showed irregular, porous particles characteristic of mannitol, which acts as the carrier matrix (Fig. 05). These particles appeared uniformly coated with a thin lipid layer, confirming successful deposition of phospholipids onto the carrier surface. As expected, liposomes vesicles were not visible in the dry proliposomes powder, since vesicle formation occurs only after hydration. The porous and irregular structure of mannitol is beneficial, as it promotes rapid hydration and efficient lipid dispersion, enabling quick liposome formation in aqueous media. Overall, SEM analysis verified the successful preparation of PK-II proliposomes, demonstrating effective lipid coating over the carrier. This morphology is expected to support improved hydration, stability, and eventual bioavailability of PK-II.

Fig. 05 Dry proliposomes SEM images from different magnification length

The surface morphology of the liposomes formed after hydration of the PK-II proliposomes powder was evaluated using SEM. The micrographs showed predominantly spherical vesicles with smooth, well-defined surfaces (Fig. 06), indicating efficient hydration and self-assembly of phospholipids into stable liposomes structures. The spherical shape and uniform surface suggest well-formed lipid bilayers, which are essential for maintaining vesicle integrity and providing an effective encapsulation environment for PK-II. Such morphology supports improved stability, drug protection, and controlled release behaviour. Overall, the SEM analysis confirmed successful transformation of the proliposomes powder into uniform liposomes vesicles upon hydration. The smooth and spherical structures are expected to contribute to enhanced solubility and bioavailability of PK-II.

Fig. 06 Liquid liposomes SEM images from different magnification length

DSC analysis

The thermal behaviour of Picroside-II, blank proliposomes, and the optimized PK-II proliposomes formulation was evaluated using DSC (Fig. 07), (Fig. 08), (Fig. 09). Pure PK-II exhibited a sharp endothermic peak at its characteristic melting point, confirming its crystalline nature. Blank proliposomes showed a broad endothermic peak corresponding to the lipid carrier and excipients. PK-II-loaded proliposomes displayed a thermogram similar to that of the blank, with the characteristic peak of PK-II absent. The absence of the drug’s melting peak in the loaded formulation indicates that PK-II is molecularly dispersed or encapsulated within the lipid matrix rather than existing in crystalline form. The similarity between blank and drug-loaded thermograms also suggests no significant chemical interaction between PK-II and the lipid excipients, confirming the thermal stability of the formulation.

Fig. 07 DSC of PK-II

Fig. 08 DSC of PK-II proliposomes

Fig. 09 DSC of PK-II blank proliposomes

The FTIR spectrum of pure PK-II (Fig. 10) exhibited characteristic absorption bands at 1015.52 cm⁻¹, 1265 cm⁻¹, and 796.93 cm⁻¹, confirming the presence of its functional groups. The blank proliposomes formulation spectrum (Fig. 12) showed prominent peaks at 1411.62 cm⁻¹, 1073.12 cm⁻¹, and 1010.07 cm⁻¹, corresponding to the phospholipid matrix and carrier components. The PK-II–loaded proliposomes (Fig. 11) demonstrated similar peaks at 1411.74 cm⁻¹, 1073.77 cm⁻¹, and 1010.54 cm⁻¹, with only minimal shifts compared to the blank formulation. The close alignment of peak positions between the blank and drug-loaded proliposomes indicates the absence of any significant chemical interaction between PK-II and the excipients. The slight variations observed are within the acceptable range and suggest physical encapsulation rather than chemical modification. These results confirm that PK-II was successfully incorporated into the phosphatidylcholine-coated mannitol matrix without structural incompatibility.

Fig. 10 FTIR of PK-II

Fig. 11 FTIR of PK-II proliposomes

Fig. 12 FTIR of blank Proliposomes

The optimized proliposomes batch was stored at 4°C and room temperature (~25°C) for one month. Samples were evaluated for entrapment efficiency, particle size and zeta potential. No significant changes were observed, and the entrapment efficiency remained largely unchanged, indicating that the formulation was physically stable under the tested conditions. The stability data are summarized in (Table 04).

Table 04 Stability study data