Liposomes are vesicular structures with multiple concentric bilayer arrangement of phospholipids. They have been explored extensively in drug delivery through multiple routes of administration. Liposomes have been functionalized,modified and investigated to meet specific demands of drug delivery. A wide spectrum of chemicals, drugs, proteins, genetic material have been encapsulated in liposomes. However, its difficult scale up from lab to commercial level has paralyzed its success as an effective drug delivery technology. This article attempts to give the reader a brief overview of the manufacturing techniques and their amenability to scale-up with useful tips for successful scale-up.

General methods of manufacturing
There are number of manufacturing methods for liposomes but only a few are scalable which are discussed in this article (Fig. 1).

Hydration of dried lipid mixture:
Thin film hydration method
The most classical method of liposome preparation is the lipid film hydration method. The method involves the deposition of thin film of lipids on the surface of a round bottom flask by evaporation of organic phase followed by hydration of the lipid film in aqueous media. The hydration is done by slow and uniform shaking of the flask. The process generally leads to formation of multi-lamellar structures which are further extruded or sonicated for size reduction.

Spray drying & supercritical fluid technology
Recently, novel approaches have been utilized for the preparation of liposomes. Spray drying of lipid mixtures in organic solvents lead to formation of proliposomes which can be hydrated in a suitable aqueous media to convert them in liposomes. Spray dried proliposomes can be stored at deep-freezed condition and utilized when desired. The same phenomenon can be achieved by the process called super critical fluid particle formation. This method utilizes carbon dioxide in its super critical state to precipitate lipid mixture particles, which may be subsequently hydrated.

Tg of the phospholipid mixture containing drug molecule, cholesterol and other excipients varies as compared to Tg of individual lipids due to presence of drug and hence, the drug and its concentration has very pronounced influence on Tg of a particular composition of lipids. Thus it is important to estimate the exact Tg and carry out hydration process above the Tg.

Solvent injection technique
Ethanol Injection and Ether Injection technique
Solvent injection method is yet another widely used technique which can be distinguished as ethanol injection method and ether injection. The ethanolic solution of lipids is injected into an excess of aqueous media which forms multilamellar vesicles. But it suffers from the disadvantage that complete removal of ethanol is not possible and ethanol may degrade any bio-molecule encapsulated in liposomes. An ether injection technique is also very similar where diethyl ether is used instead of ethanol. Both of these methods give uniform size distribution and the vesicle population is very homogenous.

Novel modified injection method
Lipids are dissolved in chloroform, dichloromethane, methanol or mixture of these solvents and injected into hydration media at optimal stirring at temperature above Tg of lipid mixture. Organic phase is to be allowed to evaporate and liposomal suspension annealed for 1-2h for proper orientation of lipid vesicles.

Detergent dialysis method
Detergent dialysis method utilizes the property of detergents to solubilize lipid at their critical micelle concentration followed by dialysis to remove detergents. Although the size is homogeneous and reproducible but traces of detergent are difficult to remove. There are regulatory issues against use of detergents. Further formation of unstable mixed micelle and leakage of hydrophilic drug is also an issue with this method.

Reverse phase evaporation/ emulsification method:
This method involves solubilization of lipid mixtures into solvent or solvent mixtures viz. either diethyl ether isopropyl ether or mixtures of two solvents such as isopropyl ether and chloroform. Emulsification is achieved by adding aqueous phase to organic phase (Organic phase:aqueous Phase- 3:1). Organic phase is evaporated using rotary evaporator. Final trace removal of organic solvent renovates the phospholipid gel phase to large unilamellar liposomes. The crucial parameters in the method are use of large amount of organic solvent, removal of trace organic solvent.

Physical stability of liposomes
Liposomes are supplied in two different forms viz. Liquid dispersion form and Lyophilized form.

Liqiud dispersion
Some liposomal formulations are stable at liquid form for a specified period of shelf life e.g. Doxil, Caelyx.

Lyophilized form
Liposomes are generally not stable at liquid state; they can be lyophilized to convert them in solid form. During lyophilization cryoprotectants are added to resist change in physical and chemical properties like particle size, surface morphology, drug leakage, drug crystallization etc. e.g. Ambisome. Upon rehydration of lyophilisate, water molecules quickly replace the cryoprotectants and liposomes reseals before significant leakage.  

Though in market smart lyophilizer are available which can generate lyophilization cycle itself, some critical points to be considered when lyophilizing the liposomes are:

• Collapse temperature of liposomal suspension
• Freezing beyond collapse temperature
• Primary drying below collapse temperature (minimum below 5°C)
• Secondary drying up to minimal water content in the lyophilized cake
• Well designed lyophilization cycle
• Optimal concentration of cryoprotectant
• Reconstitution of lyophilized cake

Characterization of liposomes
Reproducible, precise and validated methods are important in characterization and ensuring the batch to batch uniformity of liposomes. Characterization parameters for liposomes are:

Vesicle size distribution
Suitable method to monitor the liposome vesicles size distribution is the dynamic light scattering technique. Freeze fracture electron microscopy may be used to ascertain the morpholgical characteristics and to elucidate bilayer property.

Determination of residual organic phase in phospholipid mixtures:
Organic solvents are frequently used in the preparation of liposomes and hence, an accurate method to quantify their remnants in the finished product is essential.

Drug encapsulation
Encapsulation of drug molecule inside liposomes can be determined by column chromatography technique using sephadex G-50 or G-25. This is also to ascertain the amount of free drug which is not entrapped and need to be removed from the bulk.

Phospholipid assay
Phospholipids and degradation products are determined using two-dimensional thinlayer chromatographic (TLC) system or in a high performance liquid chromatographic (HPLC) system. Total phosphorous in a sample can be quantitatively determined by a spectrophotometer.

Other characterization parameters which are to be included in the list are percent assay as well as related substances, pH measurement, bacterial endotoxin load, microbial limit test, sterility testing, stability testing, trapped volume, lamellarity of liposome etc.

Also, the in-vitro drug release pattern, plasma stability studies should be reported before releasing the batch into market. Animal studies which need to be conducted are determination of LD50 and efficacy in suitable animal model. Pharmacokinetic parameters should also be established.

Scale-up issues Trace organic solvent
There are some major issues at scale up (Fig. 2) which keep on perplexing the formulation development scientist like removal of trace organic solvents from the liposomal preparation. Organic solvents are commonly used for dissolving the lipid mixture. Since they are toxic in nature, keeping them in safe limits is a must. Further, their presence can also lead to stability concerns. Hence, selection of organic solvent becomes crucial.

Lipid peroxidation
The unsaturated lipids used in manufacturing of liposomes can easily undergo oxidative degradation. These reactions can occur during preparation, storage or actual use. Oxidation of phospholipids may be minimized by a number of ways, namely protecting them, by keeping under inert gases, by keeping in light resistant containers, by removal of heavy metals (add EDTA) or by adding antioxidants. Generally in industry, alpha-tocopherol is added to the phospholipids to protect them from oxidative degradation.

Endotoxin removal
Removal of endotoxins is a serious issue in liposome sterility. Endotoxins can be removed by ultrafiltration under aseptic pharmaceutical manufacturing. Serial filtration may be required for raw materials with high pyrogen load.  Liposome preparation using raw materials of high quality with low endotoxin levels and under aseptic conditions with pyrogen free equipments and vessels may address the problem. Validation of depyrogenation task has been simplified by the development of reliable and quantitative LAL assays and the availability of endotoxin standards.

Free drug removal
Complete removal of free drug is prime requirement for the use of liposomes as controlled delivery vehicle. Various approaches that have been attempted to remove the unentrapped drug molecules in lab as well as in industry include ion-exchange chromatography, ultrafiltration technique and size exclusion chromatography.  

Size distribution
Ensuring homogenous liposome size distribution may be achieved by extrusion through polycarbonate membranes as per the desired size range.  The extrusion may be performed under nitrogen pressure aseptically. Desired size distribution can also be achieved by passing the liposomal suspension through high pressure homogenizer.

Sterilization
Liposomal formulation can be sterilized by passing the suspension through 0.22 um filter membrane. Gamma irradiation was unsuccessful due to accelerated hydrolysis and peroxidation of lipids.  A solution to sterilization is combination of aseptic manufacturing and sterile filtration before vial filling. Sterility assurance of the product can be obtained by non-destructive test of filter integrity.

From Laboratory to Manufacturing Plant
When using liposomes for parenteral applications the requirements and facilities needed are same as other injectable preparations. Then also some specific requisites are listed below:

Lipid particle/Lipid film generation
Spray dryer - Prepared lipid particles can be stored at deep freeze condition. The second option for solvent stripping is use of rotary flask evaporator.

Injection ports
It can directly incorporate lipid solution into the hydration media. Generally set of 4-6 ports can be arranged in a continuous circular manner for efficient delivery of lipid solution. From the experiments we have strongly concluded that fast injection of lipid solution instead of slow infusion gives smaller and narrow size distribution.

Hydration Tank
It must be closed and jacketed to maintain temperature above Tg of lipid mixture and should have continuous stirring facility with speed adjustment.

Nitrogen supply and air supply with appropriate pressure.

Size reduction facility
High pressure homogenizer with cooling jacket and extruder assembly

Filtration unit
Liposomes used for parenteral are colloidal systems having average particle size around 150 nm. Filtration may generate pressure onto the filter membrane which necessitates a membrane to withstand the pressure during filtration.

Perspective
Most commercially viable manufacturing is getting evolved with increase in demand for liposomal drugs. It is still in its infancy and needs optimization of parameters at each stage of manufacturing process and simultaneously, it allows the industries and researchers to patent their efforts. Still, it is a long way to go before dreams are realised in a most optimal manner.