Lipid Nanoparticles For mRNA Delivery
Liposome Production Services

Liposome Production Services

Seattle Genova has spent years developing liposome technologies, and our extensive experience qualifies us as experts in liposome preparation and manufacturing. Our liposome platform's goal is to provide you with high-quality liposome services ranging from custom liposome production, analysis, and characterization to application.

Liposomes, which are artificially prepared vesicles, have become important tools for improving the delivery of a wide range of drugs, including antimicrobial agents, cancer drugs, antifungal drugs, peptide hormones, enzymes, vaccines, and genetic materials. Liposomes are classified based on their lamellarity, size, charge, and application due to differences in preparation methods and lipid compositions. Regardless of their solubility properties, their flexibility in behaviour can be used for drug delivery via various routes of administration.

The encapsulation of drugs in liposomes has provided an opportunity to improve the therapeutic indices of many drugs, primarily by altering their biodistribution and targeting the drug to specific tissues. The role of liposomes as a drug delivery system is to deliver drugs in a controlled manner, reducing undesirable side effects and improving in vitro and in vivo activity, as well as lowering drug toxicity and increasing encapsulated drug efficacy.


The primary goal of an ideal liposome formulation method is to achieve efficient drug entrapment, narrow particle size distribution, and long-term liposome product stability. The general procedure for all liposome preparation methods involves hydrating the lipid, then sizing the particles and removing the non-encapsulated drug.

There are two kinds utilized for the preparation of liposomes:

1.       Passive loading mechanical dispersion Methods

The drug is encapsulated in the passive loading method by introducing an aqueous phase of a water-soluble drug or an organic phase of a lipid-soluble drug prior to or during the preparation of the liposomes. The passive loading method for lipid-soluble drugs with a high affinity to the lipid membrane can achieve high drug encapsulation efficiency.

2.       Active loading methods

The drugs can be loaded using the active loading method by creating diffusion gradients for the ions or drugs across the external and internal aqueous phases.


The most common used methods in the preparation for liposomes are following:

1.       Thin-Film Hydration Method

The most popular and straightforward way for making MLV is through the thin-film hydration process, which involves dissolving phospholipids in organic solvents such dichloromethane, chloroform, ethanol, and chloroform-methanol mixture (2:1, 9:1, and 3:1, respectively). When solvent evaporates under vacuum at a temperature between 45 and 60 oC, a thin, homogenous lipid layer is created. In order to totally eliminate the remaining solvent, nitrogen gas is used. In the hydration process, a mixture of distilled water, phosphate buffer, phosphate saline buffer with a pH of 7.4, and regular saline buffer is utilised. At a temperature of 60–70 oC, the hydration process took between one and two hours. The liposomal suspension must spend the night at 4 oC in order to fully hydrate the lipids.

The major drawbacks of the method are associated to low encapsulation, difficulty of scaling up and the size distribution is heterogeneous.

2.       Injection Methods

          I.            Ether Injection Method

A lipid solution is dissolved in ether or a diethyl ether/methanol mixture, which is slowly injected into an aqueous solution of the substance to be encapsulated, in the ether injection method. Liposomes are created when the organic solvent is subsequently removed at lower pressure.

The main drawback of the technique is heterogeneous population and the exposure of compounds to be encapsulated to organic solvents or high temperature.

        II.            Ethanol Injection Method

In the ethanol injection method, a large amount of preheated distilled water or TRIS-HCl buffer is rapidly injected with an ethanolic lipid solution. The hydrophilic/hydrophobic nature of the substance determines whether it will be incorporated into the liposomal vesicle. Compared to 5-fluorouracil, which migrates to the exterior aqueous phase, nimesulide, a lipid soluble component, integrates better in liposomes.

The main benefit of ethanol injection method is including of non harmful solvent as ethanol, as well as easy scale up of the technique. The possibility of formation of azeotrope with water lessens its applicability.

3.       Sonication Method

The sonication technique is established on size transformation and involves the subsequent sonication of MLVs formulated by thin-film hydration method, utilizing sonic energy usually under an inert atmosphere including nitrogen or argon. The sonication method facilitates homogenous dispersion of small vesicles utilizing bath type or probe type sonicator with a potential for greater tissue penetration. The probe tip sonicator provides high energy to the lipid suspension. The possibility of overheating of the lipid suspension induces degradation. Sonication tips manage to release titanium particles into the lipid suspension which must be removed by centrifugation prior to utilize. The bath sonicators are the greatly widely utilized instrumentation for preparation of SUV. They are seed for big volume of dilute lipids.

The oxidation of unsaturated bonds in the fatty acid chains of phospholipids and hydrolysis to lysophospholipids and free fatty acids, as well as denaturation of thermolabile substances and very low encapsulation efficiency of internal volume are the main disadvantages of the technique.

4.       High-Pressure Extrusion Method

MLVs prepared by thin-film hydration technique are frequently passed through filters polycarbonate membranes reducing the liposome size in high-pressure extrusion procedure. The liposomes are prepared utilizing thin-film hydration method subsequently utilizing an extruder for ten cycles to receive extruded liposomes with uniform diameters.

5.       Reverse-Phase Evaporation Method

The reverse-phase evaporation method is utilized with the organic solvents such as diethyl ether/isopropyl ether or mixture of diethyl ether and chloroform (1:1 v/v)5 and a mixture of chloroform-methanol (2:1 v/v)21 including phospholipids. The organic phase should be immiscible with aqueous phase, thus an oil/water emulsion is developed. Phosphate buffer saline or citric-Na2HPO4 buffer3 is added to aqueous phase with aim to enhance the efficiency of liposome formulations. The formation of liposomes is permitted by continued rotary evaporation of the organic solvents under vacuum.

The major advantage of the technique is a very high encapsulation rate. The main disadvantage of the technique is the possibility of remaining the solvent in the formulation and it has problems to scale up.

6.       Calcium-Induced Fusion Method

The calcium-induced approach relies on calcium being added to the SUV. Fusion leads to the creation of multilamellar vesicles. LUV liposomes are created when ethylenediaminetetraacetic acid (EDTA) is added to the preparations. Only acidic phospholipids can be used in the creation of LUV liposomes.

7.       Dehydration-Rehydration Method

The process of dehydration and rehydration is also employed to prepare liposomes. By using the sonication approach, the tiny unilamellar vesicles made of phosphatidylcholine, 1,2-dioleoyl-3-(trimethylammonium) propane, cholesterol, and plasmid DNA are created. The final mixture is frozen and allowed to freeze-dry for the entire day. A controlled rehydration of the dry powders causes the creation of multilamellar dehydration-rehydration vesicles with DNA in their structure due to the binding of the cationic charges of the inner bilayers.

8.       Freeze-Thaw Method

To increase the confined volume of liposomal preparations, the freezing and thawing technique is introduced. The ionic strength of the medium and the concentration of phospholipids both affect the freeze-thaw procedure. It alters the lamellar structure physically, which results in the production of unilamellar vesicles. The unilamellar vesicles undergo rapid freezing, sluggish thawing, and multiple cycles of freezing and thawing. Using phospholipids dimyristoylphosphatidylcholine and distearoylphosphatidylcholine in phosphate buffered saline buffer, six freeze-thaw cycles are used to create the liposomal propranolol formulation.

9.       Microfluidization

For the large-scale production of liposomes, a microfluidization, or microemulsification, approach is used. Boltic et al. presented the process for making antimicrobial liposomes by thin-layer hydration, followed by sonication using a bath-type sonicator and microfluidization to produce partial homogenization. Microfluidization produces liposomes with good aqueous phase encapsulation and is reproducible.

10.   Supercritical Fluids (SCF) in the Preparation of Liposomes

In order to address the issues with current conventional procedures, such as the need for a large number of hazardous organic solvents and the limitation of laboratory scale production, supercritical fluids are introduced in the synthesis of liposomes. Supercritical carbon dioxide is the most often used supercritical fluid in the pharmaceutical industry for the synthesis of liposomes. It offers a number of benefits, including being non-toxic, non-flammable, recyclable, simple to remove from the solvent, operating at moderate temperatures, and preventing product degradation in an inert environment. Acetone, ethanol, methanol, dichloromethane, and ethyl acetate are examples of modifier solvents that can be added as cosolvents to change the extraction conditions when using SCF.



1.       Patel SS, Patel MS, et al “Development and evaluation of liposomes for topical delivery of tacrolimus (Fk-506)” J Sci Res, 2010, 2 (3): 585-596.

2.       Achim M, Precup C, et al “Thermosensitive liposomes containing doxorubicin. Preparation and in vitro evaluation” Farmacia, 2009, 57 (6): 703-710.

3.       Bhatia A “Tamoxifen in topical liposomes: development,characterization and in vitro evaluation” J Pharm Pharmaceut Sci, 2004, 7 (2): 252-259.

4.       Da Costa CAM, Moraes AM “Encapsulation of 5-fluorouracil in liposomes for topical administration” Maringá, 2003, 25 (1): 53-61.

5.       Chanda H, Das P, et al “Development and evaluation of liposomes of fluconazole” JPBMS, 2011, 5 (27): 1-9.

6.       Devi M, Kumar MS, et al “Amphotericin-B loaded vesicular systems for the treatment of topical fungal infection”IJRAPR, 2011, 4: 37-46.

7.       Kumar R, Rana AC, et al “Formulation and evaluation of elastic liposomes of clortimazole” Int J Drug Dev & Res, 2012, 4 (3): 348-355.

8.       Nidhal KM, Athmar DH “Preparation and evaluation of salbutamol liposomal suspension using chloroform film method” Mustansiriya Medical Journal, 2012, 11 (2): 39-44.

9.       Ramana LN, Sethuraman S, et al “Development of a liposomal nanodelivery system for nevirapine” J Biomed Sci, 2010, 17: 1-9.

Liposome Production with Thin-Film Hydration
Our liposome platform's goal is to provide you with high-quality liposome services ranging from custom liposome production, analysis, and characterization to application.
Liposomes Production with Injection Technologies
Our liposome platform's goal is to provide you with high-quality liposome services ranging from custom liposome production, analysis, and characterization to application.
Liposome Production by Sonication Method
Our liposome platform's goal is to provide you with high-quality liposome services ranging from custom liposome production, analysis, and characterization to application.
Liposome Production by High-Pressure Extrusion Method
High pressure Extrusion is a a method where the liposome suspension is passed through a membrane filter of specified pore size.
Liposome Production by Reverse-Phase Evaporation Method
The reverse-phase evaporation method is utilized with the organic solvents such as diethyl ether/isopropyl ether or mixture of diethyl ether and chloroform (1:1 v/v)5 and a mixture of chloroform-methanol (2:1 v/v)21 including phospholipids.
Liposome Production by Calcium-Induced Fusion Method
Calcium-induced interaction of liposomes formulated of pure phosphatidylserine (PS) has been researched utilizing a rapid-mixing, rapid-freeze device.
Liposome Production by Dehydration-Rehydration Method
The method is based on induction of the fusion of preformed vesicles by means of dehydration and controlled rehydration.
Liposomes Production by Freeze-Thaw Method
Freeze-thawing technique is usually used to improve the encapsulation capacity of MLV by reducing its lamellarity. Freeze-thawing method is conducted by submerging liposome sample under water and changing temperatures with large gradients.
Liposomes Production by Microfluidization Method
Microfluidization is an emerging technology for liposome synthesis, because it facilitates precise control of the lipid hydration procedure.
Liposomes Production by Supercritical Fluids (SCF)
The supercritical fluid can function as a solvent or a cosolvent for the lipids material which are next put in connection with an aqueous solution before or during the depressurization.
Ferritin-based Protein-nanoparticle Production Service
With the help of our scientists’ expertise and vast knowledgeable creativity, Seattle Genova has developed ferritin-based protein-nanoparticles after the successful services of lipids, and polymer based nanoparticles.
Exosomes for mRNA Delivery
With years of experience, our scientists at Seattle Genova offer services for large-scale exosome production and purification, appropriate for the requirements of commercial exosome production.

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