SOP for Formulation of Cationic Liposomes for DNA Delivery

Formulation of Cationic Liposomes for DNA Delivery

1) Purpose

The purpose of this SOP is to describe the procedure for the formulation of cationic liposomes for DNA delivery. Cationic liposomes are used for delivering genetic material such as plasmid DNA or siRNA into cells due to their positive charge, which facilitates interaction with negatively charged DNA and cell membranes. This SOP outlines the process for preparing cationic liposomes, ensuring efficient encapsulation and delivery of DNA.

2) Scope

This SOP applies to personnel involved in the formulation of cationic liposomes for gene therapy and research applications. The procedure includes instructions for lipid preparation, hydration, DNA encapsulation, size reduction, and quality control testing to ensure DNA encapsulation efficiency and liposome stability.

3) Responsibilities

Operators: Responsible for executing the procedure to ensure proper formulation and DNA encapsulation.
QA Team: Responsible for reviewing the batch records and ensuring compliance with SOP guidelines.
QC Team: Responsible for conducting quality control tests to assess the DNA encapsulation efficiency, liposome size, and stability.

4) Procedure

4.1 Equipment Setup

The equipment required for formulating cationic liposomes must be cleaned, calibrated, and set up before use. The following equipment is essential for the process:

4.1.1 Required Equipment

Rotary evaporator
Sonicator or extruder
Magnetic stirrer
pH meter
Temperature-controlled water bath
Dynamic light scattering (DLS) instrument

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 extruder is calibrated for producing uniform liposomes.
4.1.2.3 Calibrate the pH meter using standard buffer solutions (pH 4.0, 7.0, and 10.0).

4.2 Lipid Film Formation

The first step in the preparation of cationic liposomes is the formation of a lipid film using cationic lipids, such as DOTAP or DOTMA, dissolved in an organic solvent, followed by solvent evaporation under reduced pressure.

4.2.1 Lipid Dissolution

4.2.1.1 Weigh the required amounts of lipid components, including cationic lipids (e.g., DOTAP), phospholipids, and cholesterol, based on the formulation protocol.
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 Stir the solution with a magnetic stirrer to ensure complete dissolution of the lipids.

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 inner walls of the flask.
4.2.2.3 Once the solvent has evaporated, dry the lipid film under vacuum for an additional 30 minutes to ensure complete solvent removal.

4.3 Hydration of Lipid Film and DNA Encapsulation

The lipid film is hydrated with an aqueous phase containing the DNA to be delivered. This hydration process encapsulates the DNA within the cationic liposomes.

4.3.1 Preparation of the Aqueous Phase

4.3.1.1 Prepare the aqueous phase containing the DNA solution (e.g., plasmid DNA or siRNA) according to the formulation protocol. This solution should be sterile and prepared under aseptic conditions.
4.3.1.2 Adjust the pH of the aqueous phase to optimize liposome stability and DNA encapsulation.
4.3.1.3 Warm the aqueous phase to the required temperature (typically 37°C) in a temperature-controlled water bath.

4.3.2 Hydration Process

4.3.2.1 Add the warmed aqueous phase containing the DNA to the round-bottom flask containing the dried lipid film.
4.3.2.2 Stir the mixture gently for 30 minutes to allow complete hydration of the lipid film and formation of multilamellar vesicles (MLVs).
4.3.2.3 Allow the suspension to equilibrate for an additional 30 minutes to enhance DNA encapsulation efficiency.

4.4 Size Reduction of Cationic Liposomes

To achieve the desired liposome size for DNA delivery, the MLVs must be reduced in size using sonication or extrusion techniques.

4.4.1 Sonication Method

4.4.1.1 Transfer the MLV 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 prevent overheating, which could lead to DNA 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. The filter pore size (e.g., 100 nm or 200 nm) will determine the final liposome size.
4.4.2.2 Repeat the extrusion process 5 to 10 times to ensure uniform liposome size and encapsulation efficiency.

4.5 Quality Control of Cationic Liposomes

After DNA encapsulation and size reduction, the cationic liposomes must undergo quality control testing to confirm DNA encapsulation efficiency, liposome size, and stability.

4.5.1 Measure the particle size of the cationic liposomes using dynamic light scattering (DLS) to confirm size distribution.
4.5.2 Test the DNA encapsulation efficiency by quantifying the amount of DNA encapsulated within the liposomes using UV spectrophotometry or a similar method.
4.5.3 Assess the stability of the liposomes by storing them under specified conditions and monitoring their size, DNA release, and morphology over time.
4.5.4 Perform transfection efficiency tests to evaluate the ability of the cationic liposomes to deliver DNA into target cells.

4.6 Storage of Cationic Liposomes

The prepared cationic liposomes must be stored under conditions that preserve both the liposome structure and the encapsulated DNA. Store the liposomes in sterilized, airtight containers at 4°C or as specified in the formulation protocol. Label the storage containers with the batch number, preparation date, and storage conditions. Perform regular stability testing to assess the performance of the stored liposomes over time.

5) Abbreviations, if any

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

6) Documents, if any

Batch Manufacturing Record (BMR)
Particle Size Analysis Report
DNA Encapsulation Efficiency Report
Transfection Efficiency Test 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
    DNA Type
    Encapsulation Method
    Size Reduction Technique
    Operator Initials
    QA Signature
  
  
    Batch Number
    Lipid Name
    Weight in grams
    Plasmid/siRNA
    Hydration/Post-formation
    Sonication/Extrusion
    Operator Name
    QA Name

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