**2. Liposome preparation methods**

The manufactured liposome features are directly related to the preparation method. Although liposome formation may be spontaneous, often some mechanical agitation is required. In order to have control over the size and structure of the liposomes that are formed, increase the efficiency of entrapment of the desired molecules, and prevent subsequent leakage from the liposomes, different preparation methods have been devised.

There are a few parameters that should be considered during the method selection: 1) the physicochemical characteristics of the material to be entrapped and those of the liposomal ingredients, 2) the nature of the medium in which the liposomes are dispersed, 3) the effective concentration of the encapsulated material and its potential toxicity, 4) additional processes involved during application (delivery of the liposomes), 5) optimum size, polydispersity and shelf-life of the liposomes for the intended application and 6) batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products [24-26].

Liposome size is a crucial parameter in determining the circulation half-life of liposomes in drug delivery. The amount of encapsulated drug is also related with the size and the number of bilayers of the prepared liposome. According to the desired formulation, different liposome preparation methods can be employed. The main difference in these methods is their approach to overcome the low solubility of lipids in water. Accordingly, these methods can be classified as mechanical agitation, solvent evaporation, solvent injection, and detergent solubilization. In all the above mentioned methods, drug loading is passive.

#### **2.1. Mechanical agitation**

In this method, lipids are directly solubilized in water upon application of high mechanical agitation, through the use of probe sonication. It is one of the simplest methods of liposome preparation, however, yields small liposomes that are highly unstable in terms of their size and suffers from the drawback that it is impossible to remove completely the risk of lipid degradation by contact with the hot probe, and contamination with titanium from the probe. Its advantage is the exclusion of use of organic solvents as described in the following methods. However, for drug delivery applications, liposomes prepared with mechanical agitation are not suitable due to their size instability and high leakage of encapsulated drugs [27].

#### **2.2. Solvent evaporation**

such as stability, storage and sterilization are discussed along with the characterization techniques for size, charge, etc. Clinical applications of liposomes are a vast area of research where cancer therapy is the area of highest impact. Different clinical applications of liposomes and most recent advances in cancer therapy are summarized. New generation involving constituents other than conventional ones such as phospholipids prove to be a growing field in nanotechnology. A brief list of different types of new generation liposomes are given with

The manufactured liposome features are directly related to the preparation method. Although liposome formation may be spontaneous, often some mechanical agitation is required. In order to have control over the size and structure of the liposomes that are formed, increase the efficiency of entrapment of the desired molecules, and prevent subsequent leakage from the

There are a few parameters that should be considered during the method selection: 1) the physicochemical characteristics of the material to be entrapped and those of the liposomal ingredients, 2) the nature of the medium in which the liposomes are dispersed, 3) the effective concentration of the encapsulated material and its potential toxicity, 4) additional processes involved during application (delivery of the liposomes), 5) optimum size, polydispersity and shelf-life of the liposomes for the intended application and 6) batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products [24-26].

Liposome size is a crucial parameter in determining the circulation half-life of liposomes in drug delivery. The amount of encapsulated drug is also related with the size and the number of bilayers of the prepared liposome. According to the desired formulation, different liposome preparation methods can be employed. The main difference in these methods is their approach to overcome the low solubility of lipids in water. Accordingly, these methods can be classified as mechanical agitation, solvent evaporation, solvent injection, and detergent solubilization.

In this method, lipids are directly solubilized in water upon application of high mechanical agitation, through the use of probe sonication. It is one of the simplest methods of liposome preparation, however, yields small liposomes that are highly unstable in terms of their size and suffers from the drawback that it is impossible to remove completely the risk of lipid degradation by contact with the hot probe, and contamination with titanium from the probe. Its advantage is the exclusion of use of organic solvents as described in the following methods. However, for drug delivery applications, liposomes prepared with mechanical agitation are

not suitable due to their size instability and high leakage of encapsulated drugs [27].

short descriptions at the end of this chapter.

4 Application of Nanotechnology in Drug Delivery

**2. Liposome preparation methods**

liposomes, different preparation methods have been devised.

In all the above mentioned methods, drug loading is passive.

**2.1. Mechanical agitation**

In general this method consists of four major steps; first is the solubilization of the lipid (and a hydrophobic compound) in an organic solvent; second is solvent evaporation; third is hydration with a buffer (and the hydrophilic compound) and if need the fourth often involves obtaining unilamellar liposomes from the obtained multilamellar ones.

The aqueous volume enclosed within these lipid membranes is very small proportion of total volume used for preparation (5-10%). Consequently, large amount of water soluble drug is wasted during the preparation. On the other hand, lipid soluble drug can be encapsulated with 100% efficiency, providing that they are not present in quantities which overwhelm the structural components of the membrane [28]. The volume of entrapment can be significantly increased by the usage of negatively charged lipids in the membrane which tend to push the bilayers apart from each other. The same effect can also be achieved in the presence of neutral lipids by freezing and thawing repeatedly the obtained liposomes. 30% volume of entrapment can be achieved, which can further be increased at higher lipid concentrations [29]. The freezethaw protocol results in a dramatic change in liposome morphology followed by freezefracture electron micrographs. Before freeze-thawing, the samples exhibit the tightly packed "onions skin" arrangements of concentric bilayers normally associated with liposomal systems. After a few freeze-thaw steps, however, new structures are observed where the interlamellar spaces are much increased, and where closed lamellar systems can be intercalated between bilayers [30].

#### *2.2.1. Solubilization of the lipid*

The starting point of this liposome preparation method is to prepare an organic solution of membrane lipids in order to ensure complete and homogenous mixing of all the components as they are required in the final membrane preparation. Compounds to be incorporated which are lipid soluble will be added to the organic solution, while compounds to be entrapped in the aqueous compartment of liposomes will be dissolved in the aqueous environment. In this method, phospholipids are first dissolved in an organic solvent along with lipid soluble compounds (if any) to be incorporated in the liposome to ensure complete and homogenous mixing.

#### *2.2.2. Solvent evaporation*

The next step is the evaporation of the organic solvent. The simplest is to allow the solvent to evaporate in a glass container. A better method is evaporating the solvent using a rotary evaporator connected to a vacuum pump to obtain a thin film of the lipid on the walls of a round bottom flask. In order to increase encapsulation, it is recommended to start with a large volume round bottom flask so that the lipids will be dried down onto a large surface area possible to form a very thin film. The evaporator is detached from vacuum pump and introduced to nitrogen. The container is then removed from the evaporator and fixed to a lyophilizer or exposed to high vacuum overnight to remove the residual solvent.

An alternative method of dispersing the lipids in a finely-divided form before the addition of aqueous media is to freeze-dry the dissolved lipids in an organic solvent [31]. The important concept in this method is the choice of the organic solvent which should have a freezing point above the temperature of the condenser of the freeze-drying and also be inert with regard to rubber seals of commercial lyophilizers. When these restrictions are concerned, the most suitable organic solvent happens to be tertiary buthanol.

causing degradation. For these reasons, bath sonicators are the most widely used instrumen‐

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7

An even gentler method of reducing the size of the liposomes is to pass through a membrane filter of defined pore size [34]. This can be at much more lower pressure and can give popu‐ lations in which one can choose the upper size limit depending on the exact pore size of the filter used. This membrane extrusion technique can be used to process both LUVs and MLVs in which liposome contents are exchanged with the suspending medium during breaking and resealing of the phospholipid bilayers as they pass through the polycarbonate membrane. In order to achieve as high an entrapment as possible of water-soluble compounds, it is crucial to have these compounds present in the suspending medium during the extrusion. An almost completely unilamellar population can be produced after 5-10 repeated extrusions through

In freezing-thawing method, SUVs are rapidly frozen and thawed slowly. The short-lived sonication disperses aggregated materials to LUV. The creation of unilamellar vesicles is as a

In this type of preparation methods, lipids are first dissolved in an organic solvent and then brought into contact with the aqueous phase containing the materials to be encapsulated within the liposome. The lipids align themselves into a monolayer at the interface between the organic and aqueous phase which is an important step to form the bilayer of the liposome [29]. There are three categories in solvent dispersion method including; (i) a miscible organic solvent with the aqueous phase, (ii) an immiscible organic solvent with the aqueous phase that is used in excess, and (iii) an immiscible organic solvent used in excess with the aqueous phase.

In this method an ethanol solution of lipids is injected rapidly into an excess saline or other aqueous medium by a fine needle [38]. The injection force is usually sufficient to achieve complete mixing, so that ethanol is diluted in water, and lipids are dispersed evenly through‐ out the medium. This method yields a high proportion of SUVs. This method is extremely simple and it has a very low risk of degradation for sensitive lipids. Its major disadvantages are the limitation of solubility of lipids in ethanol and the volume of ethanol that can be introduced into the medium, which in turn limits the quantity of lipid dispersed, so that the resulting liposome solution is generally dilute. As a result, the percentage encapsulation for hydrophilic materials is very low. One last disadvantage for this method is the difficulty of the

This method [39, 40] involves injecting the immiscible organic solution very slowly into an aqueous phase through a narrow needle at a temperature that the organic solvent is removed by vaporization during the process. In this method, large vesicles are formed which might be

result of the fusion of SUV throughout the processes of freezing and thawing [35-37].

tation for SUV preparation.

two stacked membranes.

**2.3. Solvent injection**

*2.3.1. Ethanol injection method*

*2.3.2. Ether injection method*

removal of ethanol from the lipid membranes.

#### *2.2.3. Hydration*

Evaporation (or freeze-drying) of the solvent is followed by hydration of lipids with the aqueous medium. Often for hydration, a suitable buffer at a temperature above the phase transition temperature of the phospholipid is employed. The solution is swirled manually or mechanically (either with a bath sonicator or vortex mixer) until all the lipids have been incorporated into the solution. The resulting product is a milky suspension of lipids which is allowed to stand for a while for the complete swelling to give MLVs [28]. Further treatment is required for the preparation of ULVs, which will be discussed later in the text.

It is possible to obtain LUVs instead of MLVs during hydration by introducing an aqueous sucrose solution down the side of the flask by inclining the flask to one side and slowly returning the flask to the upright orientation, allowing the fluid to run gently over the lipid layer on the bottom of the flask. The swelling is carried out as usual without any shaking or agitation. The suspension is then centrifuged and the layer of MLVs floating on the surface is removed, leaving LUVs in solution.

#### *2.2.4. Obtaining SUVs from MLVs*

After preparation of MLVs by hydration of dried lipid, it is possible to continue processing the liposomes in order to modify their size and other characteristics. For many purposes, MLVs are too large of too heterogeneous population to work with. There are several methods devised to reduce their size. These include techniques such as micro-emulsification, extrusion, and ultrasonication. A second set of methods is designed to increase the entrapment volume of hydrated lipids, and/or reduce the lamellarity of the liposomes formed, and involves proce‐ dures such as freeze-drying, freeze-thawing or induction of vesiculation by ions or pH change.

Microemulsification of liposomes is performed with an equipment called micro fluidizer to prepare small vesicles from concentrated lipid suspension. This method can produce lipo‐ somes in 50-200 nm size range with the encapsulation efficiency of up to 75% [32].

Sonication [33] disrupts MLV suspensions by using sonic energy to produce SUVs with diameters in the range of 15-50 nm. There are two methods of sonication; bath sonication and probe sonication. The former method is used for large volumes of dilute lipids whereas the latter one is used for suspensions which require high energy, such as high concentration of lipid suspensions. The disadvantage of probe sonication is the contamination of preparation with metal from the tip of the probe which should be removed by centrifugation prior to use. Also, as a result of high energy, probe sonication suffers from overheating the lipid suspension causing degradation. For these reasons, bath sonicators are the most widely used instrumen‐ tation for SUV preparation.

An even gentler method of reducing the size of the liposomes is to pass through a membrane filter of defined pore size [34]. This can be at much more lower pressure and can give popu‐ lations in which one can choose the upper size limit depending on the exact pore size of the filter used. This membrane extrusion technique can be used to process both LUVs and MLVs in which liposome contents are exchanged with the suspending medium during breaking and resealing of the phospholipid bilayers as they pass through the polycarbonate membrane. In order to achieve as high an entrapment as possible of water-soluble compounds, it is crucial to have these compounds present in the suspending medium during the extrusion. An almost completely unilamellar population can be produced after 5-10 repeated extrusions through two stacked membranes.

In freezing-thawing method, SUVs are rapidly frozen and thawed slowly. The short-lived sonication disperses aggregated materials to LUV. The creation of unilamellar vesicles is as a result of the fusion of SUV throughout the processes of freezing and thawing [35-37].

#### **2.3. Solvent injection**

An alternative method of dispersing the lipids in a finely-divided form before the addition of aqueous media is to freeze-dry the dissolved lipids in an organic solvent [31]. The important concept in this method is the choice of the organic solvent which should have a freezing point above the temperature of the condenser of the freeze-drying and also be inert with regard to rubber seals of commercial lyophilizers. When these restrictions are concerned, the most

Evaporation (or freeze-drying) of the solvent is followed by hydration of lipids with the aqueous medium. Often for hydration, a suitable buffer at a temperature above the phase transition temperature of the phospholipid is employed. The solution is swirled manually or mechanically (either with a bath sonicator or vortex mixer) until all the lipids have been incorporated into the solution. The resulting product is a milky suspension of lipids which is allowed to stand for a while for the complete swelling to give MLVs [28]. Further treatment is

It is possible to obtain LUVs instead of MLVs during hydration by introducing an aqueous sucrose solution down the side of the flask by inclining the flask to one side and slowly returning the flask to the upright orientation, allowing the fluid to run gently over the lipid layer on the bottom of the flask. The swelling is carried out as usual without any shaking or agitation. The suspension is then centrifuged and the layer of MLVs floating on the surface is

After preparation of MLVs by hydration of dried lipid, it is possible to continue processing the liposomes in order to modify their size and other characteristics. For many purposes, MLVs are too large of too heterogeneous population to work with. There are several methods devised to reduce their size. These include techniques such as micro-emulsification, extrusion, and ultrasonication. A second set of methods is designed to increase the entrapment volume of hydrated lipids, and/or reduce the lamellarity of the liposomes formed, and involves proce‐ dures such as freeze-drying, freeze-thawing or induction of vesiculation by ions or pH change.

Microemulsification of liposomes is performed with an equipment called micro fluidizer to prepare small vesicles from concentrated lipid suspension. This method can produce lipo‐

Sonication [33] disrupts MLV suspensions by using sonic energy to produce SUVs with diameters in the range of 15-50 nm. There are two methods of sonication; bath sonication and probe sonication. The former method is used for large volumes of dilute lipids whereas the latter one is used for suspensions which require high energy, such as high concentration of lipid suspensions. The disadvantage of probe sonication is the contamination of preparation with metal from the tip of the probe which should be removed by centrifugation prior to use. Also, as a result of high energy, probe sonication suffers from overheating the lipid suspension

somes in 50-200 nm size range with the encapsulation efficiency of up to 75% [32].

required for the preparation of ULVs, which will be discussed later in the text.

suitable organic solvent happens to be tertiary buthanol.

*2.2.3. Hydration*

removed, leaving LUVs in solution.

6 Application of Nanotechnology in Drug Delivery

*2.2.4. Obtaining SUVs from MLVs*

In this type of preparation methods, lipids are first dissolved in an organic solvent and then brought into contact with the aqueous phase containing the materials to be encapsulated within the liposome. The lipids align themselves into a monolayer at the interface between the organic and aqueous phase which is an important step to form the bilayer of the liposome [29]. There are three categories in solvent dispersion method including; (i) a miscible organic solvent with the aqueous phase, (ii) an immiscible organic solvent with the aqueous phase that is used in excess, and (iii) an immiscible organic solvent used in excess with the aqueous phase.

#### *2.3.1. Ethanol injection method*

In this method an ethanol solution of lipids is injected rapidly into an excess saline or other aqueous medium by a fine needle [38]. The injection force is usually sufficient to achieve complete mixing, so that ethanol is diluted in water, and lipids are dispersed evenly through‐ out the medium. This method yields a high proportion of SUVs. This method is extremely simple and it has a very low risk of degradation for sensitive lipids. Its major disadvantages are the limitation of solubility of lipids in ethanol and the volume of ethanol that can be introduced into the medium, which in turn limits the quantity of lipid dispersed, so that the resulting liposome solution is generally dilute. As a result, the percentage encapsulation for hydrophilic materials is very low. One last disadvantage for this method is the difficulty of the removal of ethanol from the lipid membranes.

#### *2.3.2. Ether injection method*

This method [39, 40] involves injecting the immiscible organic solution very slowly into an aqueous phase through a narrow needle at a temperature that the organic solvent is removed by vaporization during the process. In this method, large vesicles are formed which might be due to the slow vaporization of solvent giving rise to an ether: water gradient extending on both sides of the interfacial lipid monolayer, resulting in the eventual formation of a bilayer sheet which folds in on to itself to form a sealed vesicle [29]. Ether injection treats sensitive lipids very gently and runs very little risk of causing oxidative degradation. Since the solvent is removed at the same rate as it is introduced, there is no limit to the final concentration of lipid which can be achieved, since the process can be run continuously for a long period of time, giving rise to a high percentage of the aqueous medium encapsulated within the liposomes. The major drawbacks of this method are the long time taken to produce a batch of liposomes and the need of careful control for the introduction of lipid solution.

removed. This purification is generally done by gel filtration column chromatography (Sephadex G-50, Pharmacia LKB) and dialysis (hollow fiber dialysis cartridge) on the basis of size differences between the liposomes and the non-encapsulated material. In the cases where DNA or proteins are being encapsulated, or where there is concern that non-encapsulated material may form large aggregates, techniques such as centrifugation can be employed due to the differences in the buoyant densities of liposomes and non-encapsulated material [42, 43]. A hydrophilic drug may not be encapsulated with high efficiency because the drug molecules can diffuse in and out of the lipid membrane. Thus, the drug would be difficult to retain inside the liposomes. However, compounds with ionizable groups and those that are both water and lipid soluble can be encapsulated with high efficiency (up to 90%) by the liposomes after the formation of membranes [44] by active loading. In this technique, the pH of the interior part of the liposome is such that the unionized drug which enters the liposome by passive loading is ionized inside the liposome, and ionized drug molecules lose their ability to diffuse through the lipid membrane. Therefore, high concentration of the ionized drug is obtained inside the liposome. For example, doxorubicin and epirubicin can be entrapped in preformed SUV with

Liposomes as Potential Drug Carrier Systems for Drug Delivery

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9

The pH difference can be brought about by encapsulating a non-permeating buffer ion such as glutamate inside the liposomes at low pH and replacing the extra-liposomal buffer with one which is iso-osmolar at pH 7.0. Alternatively, charged lipids may be incorporated into the membrane at low pH, followed by adjustment of the suspending medium to neutrality. A similar approach may be adopted by using a potassium gradient, in which the membrane is made selectively permeable to potassium ions entrapped inside the liposome by incorporation

Hydrophobic drugs are solubilized in the phospholipid bilayer of the liposomes that mainly provide a hydrophobic environment. Once trapped, they remain in the liposome bilayer as they have very low affinity towards the inner or outer aqueous regions of the liposomes. During the preparation of liposomes, hydrophobic drugs are solubilized in the organic solvent along with the phospholipids and during the subsequent hydration phase, they remain entrapped in the hydrophobic bilayer region. For example, the liposomal photosensitizer verteporfin (Visudyne) contains a hydrophobic drug that is rapidly transferred to blood proteins in vivo. Activation of the drug by targeting laser light to blood flowing though the eye causes its site-specific activity in the treatment of wet macular degeneration [49]. Ampho‐ tericin B and paclitaxel are the other most commonly investigated hydrophobic drugs in

Liposome stability can be explained by physical, chemical and biological means which are all interrelated. Generally, chemical (degradation of phospholipids structures) and physical

high efficiency through active loading [45, 46].

of valinomycin into the lipid membrane [47, 48].

*2.5.2. Encapsulation of hydrophobic drugs*

liposome formulations.

**3. Stability of liposomes**

#### **2.4. Surfactant (detergent) solubilization method**

In this method, the phospholipids are brought into contact with the aqueous phase via the intermediary of surfactants. Phospholipid molecules associate with surfactants and form mixed micelles. The basic feature of this method is the removal of the surfactant from preformed mixed micelles containing phospholipids, whereupon unilamellar liposomes form spontaneously. However, removal of surfactants is carried out using techniques such as, dialysis and column chromatography, inevitably remove other small water-soluble molecules, making this method not very efficient in terms of percentage encapsulation values attainable for water soluble compounds. On the other hand, surfactant solubilization method has the ability to vary the size of the liposomes by precise control of the conditions of surfactant removal and to obtain liposomes of very high size homogeneity [29].

The transfer from laboratory to industry was very important for liposomes, as it is for any biotechnological discipline. The first liposomal drug delivery experiments in humans were carried out by freshly prepared liposomes but in order to be a commercial product the liposome-drug formulation must have well-defined stability and a shelf life over a year. Of the several preparation methods described in the literature, only a few of them have the potential to be used in the large scale liposome manufacturing. The crucial problem is the presence of organic solvent residues, pyrogen control, stability, sterility, size and size distribution as well as batch to batch reproducibility.

In the parental administration the liposomes two important conditions involve being sterile and pyrogen free. In the case of animal experiments, the sufficient sterility can be obtained by the passage of the liposome preparations through the 400 nm pore size Millipore filters. In human experiments the sterilization depyrogenation techniques should be taken much more seriously starting from the raw materials, containers and working areas [41].

#### **2.5. Loading of drugs in liposome formulations**

#### *2.5.1. Encapsulation of hydrophilic drugs*

Once lipids are hydrated in the presence of hydrophilic drugs, a portion of the drug gets entrapped inside the liposome and another portion remains in the bulk, outside the aqueous core of the liposome. As only the entrapped drug is of interest, drug in the bulk should be removed. This purification is generally done by gel filtration column chromatography (Sephadex G-50, Pharmacia LKB) and dialysis (hollow fiber dialysis cartridge) on the basis of size differences between the liposomes and the non-encapsulated material. In the cases where DNA or proteins are being encapsulated, or where there is concern that non-encapsulated material may form large aggregates, techniques such as centrifugation can be employed due to the differences in the buoyant densities of liposomes and non-encapsulated material [42, 43].

A hydrophilic drug may not be encapsulated with high efficiency because the drug molecules can diffuse in and out of the lipid membrane. Thus, the drug would be difficult to retain inside the liposomes. However, compounds with ionizable groups and those that are both water and lipid soluble can be encapsulated with high efficiency (up to 90%) by the liposomes after the formation of membranes [44] by active loading. In this technique, the pH of the interior part of the liposome is such that the unionized drug which enters the liposome by passive loading is ionized inside the liposome, and ionized drug molecules lose their ability to diffuse through the lipid membrane. Therefore, high concentration of the ionized drug is obtained inside the liposome. For example, doxorubicin and epirubicin can be entrapped in preformed SUV with high efficiency through active loading [45, 46].

The pH difference can be brought about by encapsulating a non-permeating buffer ion such as glutamate inside the liposomes at low pH and replacing the extra-liposomal buffer with one which is iso-osmolar at pH 7.0. Alternatively, charged lipids may be incorporated into the membrane at low pH, followed by adjustment of the suspending medium to neutrality. A similar approach may be adopted by using a potassium gradient, in which the membrane is made selectively permeable to potassium ions entrapped inside the liposome by incorporation of valinomycin into the lipid membrane [47, 48].

#### *2.5.2. Encapsulation of hydrophobic drugs*

due to the slow vaporization of solvent giving rise to an ether: water gradient extending on both sides of the interfacial lipid monolayer, resulting in the eventual formation of a bilayer sheet which folds in on to itself to form a sealed vesicle [29]. Ether injection treats sensitive lipids very gently and runs very little risk of causing oxidative degradation. Since the solvent is removed at the same rate as it is introduced, there is no limit to the final concentration of lipid which can be achieved, since the process can be run continuously for a long period of time, giving rise to a high percentage of the aqueous medium encapsulated within the liposomes. The major drawbacks of this method are the long time taken to produce a batch of

In this method, the phospholipids are brought into contact with the aqueous phase via the intermediary of surfactants. Phospholipid molecules associate with surfactants and form mixed micelles. The basic feature of this method is the removal of the surfactant from preformed mixed micelles containing phospholipids, whereupon unilamellar liposomes form spontaneously. However, removal of surfactants is carried out using techniques such as, dialysis and column chromatography, inevitably remove other small water-soluble molecules, making this method not very efficient in terms of percentage encapsulation values attainable for water soluble compounds. On the other hand, surfactant solubilization method has the ability to vary the size of the liposomes by precise control of the conditions of surfactant

The transfer from laboratory to industry was very important for liposomes, as it is for any biotechnological discipline. The first liposomal drug delivery experiments in humans were carried out by freshly prepared liposomes but in order to be a commercial product the liposome-drug formulation must have well-defined stability and a shelf life over a year. Of the several preparation methods described in the literature, only a few of them have the potential to be used in the large scale liposome manufacturing. The crucial problem is the presence of organic solvent residues, pyrogen control, stability, sterility, size and size distribution as well

In the parental administration the liposomes two important conditions involve being sterile and pyrogen free. In the case of animal experiments, the sufficient sterility can be obtained by the passage of the liposome preparations through the 400 nm pore size Millipore filters. In human experiments the sterilization depyrogenation techniques should be taken much more

Once lipids are hydrated in the presence of hydrophilic drugs, a portion of the drug gets entrapped inside the liposome and another portion remains in the bulk, outside the aqueous core of the liposome. As only the entrapped drug is of interest, drug in the bulk should be

seriously starting from the raw materials, containers and working areas [41].

liposomes and the need of careful control for the introduction of lipid solution.

removal and to obtain liposomes of very high size homogeneity [29].

**2.4. Surfactant (detergent) solubilization method**

8 Application of Nanotechnology in Drug Delivery

as batch to batch reproducibility.

**2.5. Loading of drugs in liposome formulations**

*2.5.1. Encapsulation of hydrophilic drugs*

Hydrophobic drugs are solubilized in the phospholipid bilayer of the liposomes that mainly provide a hydrophobic environment. Once trapped, they remain in the liposome bilayer as they have very low affinity towards the inner or outer aqueous regions of the liposomes. During the preparation of liposomes, hydrophobic drugs are solubilized in the organic solvent along with the phospholipids and during the subsequent hydration phase, they remain entrapped in the hydrophobic bilayer region. For example, the liposomal photosensitizer verteporfin (Visudyne) contains a hydrophobic drug that is rapidly transferred to blood proteins in vivo. Activation of the drug by targeting laser light to blood flowing though the eye causes its site-specific activity in the treatment of wet macular degeneration [49]. Ampho‐ tericin B and paclitaxel are the other most commonly investigated hydrophobic drugs in liposome formulations.
