SOP for Preparation of Liposomes for Gene Delivery

Preparation of Liposomes for Gene Delivery

1) Purpose

The purpose of this SOP is to outline the procedure for preparing liposomes specifically designed for gene delivery applications. Liposomes are effective vehicles for delivering genetic material, such as DNA or RNA, into cells. This SOP will ensure proper encapsulation of nucleic acids and liposome size uniformity, which are crucial for gene transfection efficiency and stability.

2) Scope

This SOP applies to all personnel involved in the preparation of liposome formulations for gene delivery. It includes steps for the preparation of lipid films, encapsulation of nucleic acids, and size reduction to ensure uniformity in liposome size. This procedure is relevant for both research and production environments.

3) Responsibilities

Operators: Responsible for following the SOP procedures, ensuring correct encapsulation of nucleic acids, and documenting all processes.
QA Team: Responsible for reviewing batch records and ensuring compliance with SOP standards.
QC Team: Responsible for conducting quality control tests to verify liposome size, stability, and gene encapsulation efficiency.

4) Procedure

4.1 Equipment Setup

The equipment required for liposome preparation must be calibrated and cleaned before use. The following equipment is essential for this process:

4.1.1 Required Equipment

Rotary evaporator
High-pressure homogenizer or sonicator
Magnetic stirrer
Vacuum pump
pH meter
Temperature-controlled water bath
Particle size analyzer (DLS)

4.1.2 Equipment Calibration

4.1.2.1 Ensure that the rotary evaporator is calibrated for temperature and vacuum pressure.
4.1.2.2 Verify that the sonicator or homogenizer is functioning properly and is capable of reducing liposome size as required.
4.1.2.3 Calibrate the pH meter using standard buffer solutions (pH 4.0, 7.0, and 10.0).

4.2 Lipid Film Preparation

The first step in the preparation of liposomes for gene delivery is the creation of a lipid film, which will later be hydrated and loaded with nucleic acids. The following steps outline this process:

4.2.1 Lipid Dissolution

4.2.1.1 Weigh the required amount of lipid components (e.g., phospholipids, cholesterol, and cationic lipids such as DOTAP or DOTMA) based on the formulation protocol. Record the weights in the Batch Manufacturing Record (BMR).
4.2.1.2 Dissolve the lipids in an appropriate organic solvent, such as chloroform or ethanol, in a round-bottom flask.
4.2.1.3 Use a magnetic stirrer to ensure complete dissolution of the lipid components.

4.2.2 Solvent Evaporation

4.2.2.1 Attach the round-bottom flask to the rotary evaporator and set the water bath temperature slightly above the lipid phase transition temperature (e.g., 37°C).
4.2.2.2 Evaporate the solvent under reduced pressure to form a thin lipid film on the walls of the flask.
4.2.2.3 After the solvent has evaporated, allow the lipid film to dry under vacuum for an additional 30 minutes to ensure complete removal of any residual solvent.

4.3 Hydration of Lipid Film and Nucleic Acid Encapsulation

After the lipid film has been formed, it is hydrated with an aqueous phase containing the nucleic acid to be encapsulated, typically DNA, RNA, or siRNA. This process is critical for achieving high gene transfection efficiency.

4.3.1 Preparation of the Aqueous Phase

4.3.1.1 Prepare the aqueous phase containing the nucleic acid solution (e.g., plasmid DNA or RNA). This solution should be sterile and prepared under aseptic conditions.
4.3.1.2 Adjust the pH of the nucleic acid solution as required using a calibrated pH meter.
4.3.1.3 Warm the nucleic acid solution in a temperature-controlled water bath to approximately 37°C.

4.3.2 Hydration and Encapsulation

4.3.2.1 Add the warmed nucleic acid solution to the round-bottom flask containing the dried lipid film.
4.3.2.2 Vortex the mixture or stir gently for 30 minutes to allow the lipid film to hydrate and encapsulate the nucleic acid.
4.3.2.3 Allow the solution to incubate at room temperature for an additional 30 minutes to maximize encapsulation efficiency.

4.4 Size Reduction of Liposomes

After the nucleic acid has been encapsulated, the liposomes must undergo size reduction to ensure a uniform size distribution suitable for gene delivery. This can be done using sonication, extrusion, or high-pressure homogenization.

4.4.1 Sonication Method

4.4.1.1 Transfer the liposome-nucleic acid suspension to a sonicator and sonicate for 5 to 20 minutes, depending on the desired liposome size.
4.4.1.2 Monitor the temperature during sonication to ensure it does not exceed 40°C to prevent nucleic acid degradation.
4.4.1.3 After sonication, allow the suspension to cool to room temperature before further processing.

4.4.2 Extrusion Method

4.4.2.1 Pass the liposome suspension through polycarbonate membrane filters using an extruder. Choose the appropriate pore size (e.g., 100 nm or 200 nm) based on the desired liposome size.
4.4.2.2 Repeat the extrusion process 5 to 10 times to ensure uniform liposome size.

4.5 Quality Control of Gene Delivery Liposomes

The liposomes must undergo quality control testing to ensure proper encapsulation of nucleic acids, stability, and size uniformity. The following tests are recommended:

4.5.1 Measure the particle size using dynamic light scattering (DLS) to confirm the size distribution of the liposomes.
4.5.2 Test the encapsulation efficiency by quantifying the amount of nucleic acid encapsulated within the liposomes.
4.5.3 Evaluate the stability of the liposomes by monitoring their size and encapsulation efficiency over time under storage conditions.

5) Abbreviations, if any

DLS: Dynamic Light Scattering
DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane (cationic lipid)
QA: Quality Assurance
QC: Quality Control

6) Documents, if any

Batch Manufacturing Record (BMR)
Particle Size Analysis Report
Encapsulation Efficiency Report

7) References, if any

FDA Guidelines for Gene Therapy 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
    Nucleic Acid Type
    Encapsulation Method
    Size Reduction Technique
    Operator Initials
    QA Signature
  
  
    Batch Number
    Lipid Name
    Weight in grams
    DNA/RNA/siRNA
    Hydration/Post-formation
    Sonication/Extrusion
    Operator Name
    QA Name

Batch No.	Lipid Type	Weight	Nucleic Acid Type	Encapsulation Method	Size Reduction Technique	Operator Initials	QA Signature
Batch Number	Lipid Name	Weight in grams	DNA/RNA/siRNA	Hydration/Post-formation	Sonication/Extrusion	Operator Name	QA Name