thin-film hydration must be properly calibrated and cleaned before use. The following equipment is essential for the preparation of liposomes:

4.1.1 Required Equipment

• Rotary evaporator (for solvent removal)
• Round-bottom flask (for lipid film formation)
• Magnetic stirrer
• Vacuum pump
• Sonicator or extruder (for size reduction)
• Temperature-controlled water bath
• pH meter (for adjusting the aqueous phase)

4.1.2 Equipment Calibration

• 4.1.2.1 Ensure that the rotary evaporator is calibrated for temperature and vacuum pressure. Check the seals and tubing for leaks.
• 4.1.2.2 Verify the calibration of the pH meter using standard buffer solutions (pH 4.0, 7.0, and 10.0).
• 4.1.2.3 Confirm that the sonicator or extruder is functioning correctly and is capable of producing the desired liposome size.

4.2 Lipid Film Formation

Lipids are dissolved in an organic solvent to create a thin film that will later be hydrated to form liposomes. The following steps outline the procedure for lipid film formation:

4.2.1 Dissolving Lipids

• 4.2.1.1 Weigh the required amount of lipids according to the formulation protocol. Record the weights in the Batch Manufacturing Record (BMR).
• 4.2.1.2 Dissolve the lipids in an appropriate organic solvent (e.g., chloroform or methanol) in a round-bottom flask.
• 4.2.1.3 Use a magnetic stirrer to ensure complete dissolution of the lipids.

4.2.2 Solvent Evaporation

• 4.2.2.1 Place the round-bottom flask containing the lipid solution in the rotary evaporator.
• 4.2.2.2 Evaporate the solvent under reduced pressure to form a thin, even lipid film on the inner surface of the flask.
• 4.2.2.3 Rotate the flask continuously to ensure that the film forms uniformly. This process usually takes 1 to 2 hours, depending on the solvent used.
• 4.2.2.4 After evaporation is complete, allow the lipid film to dry further under a gentle stream of nitrogen gas or in a vacuum chamber for 30 minutes to remove any residual solvent.

4.3 Hydration of the Lipid Film

The lipid film is hydrated using an aqueous phase, such as a buffer solution or drug solution, to form liposomes.

4.3.1 Preparation of the Aqueous Phase

• 4.3.1.1 Prepare the aqueous phase according to the formulation protocol. This may include buffers (e.g., phosphate-buffered saline) or drug solutions.
• 4.3.1.2 Adjust the pH of the aqueous phase to the required value using a calibrated pH meter.
• 4.3.1.3 Warm the aqueous phase to a temperature above the lipid phase transition temperature (typically 37°C) in a temperature-controlled water bath.

4.3.2 Hydration Process

• 4.3.2.1 Add the warmed aqueous phase to the flask containing the dry lipid film.
• 4.3.2.2 Vortex the mixture or stir gently for 15 to 30 minutes to allow the lipid film to hydrate fully and form liposomes.
• 4.3.2.3 Ensure that the lipid film is fully hydrated and that no visible lipid aggregates remain. If aggregates are present, additional stirring may be required.

4.4 Liposome Size Reduction

After hydration, liposomes are typically multilamellar and heterogeneous in size. Size reduction techniques such as sonication or extrusion can be used to achieve uniform, small unilamellar vesicles (SUVs).

4.4.1 Sonication

• 4.4.1.1 Place the liposome suspension in a sonicator bath.
• 4.4.1.2 Sonicate the suspension for 5 to 30 minutes, depending on the desired liposome size. Monitor the temperature during sonication to avoid overheating, which can destabilize the liposomes.
• 4.4.1.3 After sonication, allow the liposome suspension to cool to room temperature.

4.4.2 Extrusion

• 4.4.2.1 Pass the liposome suspension through polycarbonate membrane filters using an extruder to achieve the desired size and uniformity.
• 4.4.2.2 Repeat the extrusion process multiple times (typically 5 to 10 passes) to ensure that all liposomes are reduced to the same size.

4.5 Quality Control of Liposomes

Quality control tests must be performed to ensure that the liposomes meet the required size, stability, and encapsulation efficiency.

• 4.5.1 Measure the size of the liposomes using dynamic light scattering (DLS) or other particle size analysis methods.
• 4.5.2 Test the stability of the liposomes by evaluating their size and morphology over time.
• 4.5.3 Analyze the encapsulation efficiency by measuring the concentration of the encapsulated drug or active ingredient in the liposomes.

5) Abbreviations, if any

• DLS: Dynamic Light Scattering
• SUV: Small Unilamellar Vesicles
• QA: Quality Assurance
• QC: Quality Control

6) Documents, if any

• Batch Manufacturing Record (BMR)
• Particle Size Analysis Report
• pH Calibration Log

7) References, if any

• FDA Guidelines for Liposomal Drug Products
• ICH Q7: Good Manufacturing Practice Guide

8) SOP Version

Version 1.0

Annexure

Annexure 1: Batch Manufacturing Record Template

Batch No.	Lipid Type	Weight	Solvent	Aqueous Phase	Sonication Time	Operator Initials	QA Signature
Batch Number	Lipid Name	Weight in grams	Solvent Name	Buffer/Drug Solution	Minutes	Operator Name	QA Name