**2. Liposomes formation and classification**

### **2.1 Liposomes formation**

The liposomal vesicles derive from hydration of phospholipids, which are amphiphilic molecules endowed with a hydrophilic head group and two hydrophobic acyl chains (**Figure 1**). In aqueous media, phospholipid molecules self-assemble

**Figure 1.**

*Chemical structure of a representative phospholipid molecule (distearoyl phosphatidylcholine).*

**33**

**Table 1.**

*General Perception of Liposomes: Formation, Manufacturing and Applications*

stabilized by high proportion of

phospholipids and ethanol (10%)

**Main components Illustrative application**

Pharmacosomes Conjugate of drug and phospholipid Pharmacosomes loaded with diclofenac for

Virosomes Viral glycoproteins Virosome containing HIV-1 gp41-subunit

Emulsomes loaded with Amphotericin B for the treatment of visceral leishmaniasis [9]

Encapsulation and delivery of superoxide dismutase for oxidative stress management [9]

Sphyngosomes loaded with vincristine (Marqibo®) for lymphoblastic leukemia

Transfersomes loaded with diclofenac for improved topical delivery/retention [10]

Mitoxantrone-loaded ethosomes for the

enhanced the bioavailability and reduced

antigens for protection against vaginal

PEG-lipid coated aquasomes containing interferon-α-2b for prolonged and enhanced

Bilosomes loaded with diphtheria toxoid for systemic and mucosal immunization [14]

Niosomes based formulation for enhanced oral bioavailability of candesartan cilexetil [15]

treatment of melanoma [11]

therapy [4]

toxicity [9]

simian-HIV [12]

cytotoxicity [13]

into a bilayered structure. Within the bilayer, phospholipid polar groups line up to form a water-attracting surface while their lipophilic chains face each other to yield a water-free zone. On mechanical shaking or heating, phospholipid bilayers continuously enclose the dispersing aqueous medium and form a vesicular system. In this system, hydrophilic groups of phospholipids are oriented towards the inner and outer aqueous phase, while their hydrophobic tails are centered within the bilayer [2, 4]. This architecture underlines the ability of liposomes to readily encapsulate hydrophilic and hydrophobic materials inside the inner aqueous core and the lipid

Depending on the particle size and number of bilayers forming the vesicles

(lamellarity), liposomes can be categorized in the following classes [4, 8]:

• Small unilamellar vesicles (SUV), size range 20–100 nm;

• Large unilamellar vesicles (LUV), size >100 nm;

• Giant unilamellar vesicles (GULV), size >1000 nm;

• Oligolamellar vesicles (OLV), size range 100–1000 nm;

bilayers, respectively (as illustrated in **Figure 2**).

**2.2 Liposomes classification**

*Presentation of liposome-type systems.*

*DOI: http://dx.doi.org/10.5772/intechopen.84255*

Emulsomes A mixture of fats and triglycerides

Enzymosomes Complexes of lipids and enzymatic proteins

Sphyngosomes Sphingolipids containing amide and ether bonds

Transfersomes A mixture of single chain surfactant,

Aquasomes Tin oxide, diamonds or brushite core

Bilosomes Bile salts and acids (deoxycholic acid)

Niosomes Non-ionic surfactants (span and tween)

covered with oligomeric film

Ethosomes Phospholipids and ethanol (20–40%)

lecithin

**Vesicle designation**

**Figure 2.**

*Flowchart illustrating liposome formation and encapsulation of drug molecules.*



#### **Table 1.**

*Liposomes - Advances and Perspectives*

liposome technology.

**2.1 Liposomes formation**

**2. Liposomes formation and classification**

*Flowchart illustrating liposome formation and encapsulation of drug molecules.*

the ability of performing surface modifications for targeted, extended and sustained release. Currently, there are several liposomal formulations that are clinically established for the treatment of various diseases, such as cancer, fungal and viral infections; and many more have reached advanced phases of clinical trials [4, 5]. Although liposomes have shown some success in drug product development, the limitations identified in liposomal technology have remained almost stagnant over decades. The most common disadvantages of liposomes arise partly from poor stability under shelf and in vivo conditions. This is mostly due to potential lipids oxidation and hydrolysis, leakage and loss of hydrophilic cargoes, as well as particles fission and fusion. To date, some of these problems can be circumvented by playing around formulation adjuvants, such as anti-oxidants, or post-preparation processing, such as freeze-drying [4, 6, 7]. While describing broadly the current perception of liposomes, regarding production, evaluation and applications; this chapter intends to highlight the longstanding bottlenecks that remained overlooked and challenging to product development and implementation. This would increase the understanding of the gaps in the field and provide future directions to new openings for improvements in

The liposomal vesicles derive from hydration of phospholipids, which are amphiphilic molecules endowed with a hydrophilic head group and two hydrophobic acyl chains (**Figure 1**). In aqueous media, phospholipid molecules self-assemble

*Chemical structure of a representative phospholipid molecule (distearoyl phosphatidylcholine).*

**32**

**Figure 2.**

**Figure 1.**

*Presentation of liposome-type systems.*

into a bilayered structure. Within the bilayer, phospholipid polar groups line up to form a water-attracting surface while their lipophilic chains face each other to yield a water-free zone. On mechanical shaking or heating, phospholipid bilayers continuously enclose the dispersing aqueous medium and form a vesicular system. In this system, hydrophilic groups of phospholipids are oriented towards the inner and outer aqueous phase, while their hydrophobic tails are centered within the bilayer [2, 4]. This architecture underlines the ability of liposomes to readily encapsulate hydrophilic and hydrophobic materials inside the inner aqueous core and the lipid bilayers, respectively (as illustrated in **Figure 2**).

#### **2.2 Liposomes classification**

Depending on the particle size and number of bilayers forming the vesicles (lamellarity), liposomes can be categorized in the following classes [4, 8]:


Based on their composition, liposomes can be classified as conventional, long circulating, cationic, stimuli-responsive and immunoliposomes. The differences between these categories will be highlighted later when discussing composition and evolution of liposomes.

Furthermore, there are many other vesicular systems considered as part of the liposome-type vesicles. These include emulsomes, enzymosomes, sphyngosomes, transfersomes, ethosomes, pharmacosomes and virosomes, which are lipid-based liposomes analogous. The non-lipid-based liposomes analogous are aquasomes, bilosomes and niosomes [8, 9]. All the liposome-type systems are briefly presented in **Table 1**.

## **3. Liposomes composition and evolution**

#### **3.1 Liposomes composition**

Liposomes are made of physiologically acceptable natural or synthetic phospholipids found in the lipid bilayer membranes of human cells. The most frequently used phospholipids for liposomes preparation are phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylserines (PS) and phosphatidylglycerols (PG) [16]. The molecular structures of these biocompatible lipids are shown in **Figure 3** and **Table 2**.

In liposomal technology, a considerable attention is given to the phase transition temperature (Tt) of these phospholipids. The Tt corresponds to the temperature above which phospholipids exist in liquid crystalline phase. In this fluid state, hydrophobic tails of phospholipids are randomly oriented but ready to form closely continuous bilayered vesicles (liposomes). Below the Tt, phospholipids exist in gel state, where the hydrophobic tails are completely expanded and well packed, thus not able to form liposomes [4, 17].

As most of the phospholipids used for liposomes formulation have Tt close to the physiological temperature (37°C), the addition of cholesterol has been adopted as a strategy to stabilize the liposomal vesicles in physiological media. This is especially for phospholipids that can undergo phase transition and leakage at room temperature, which can lead to premature release of the liposome cargo. In fact, due to its high hydrophobicity, cholesterol was found efficient in strengthening the packing of phospholipid bilayers, reducing therefore membrane permeability. Numerous

**35**

*General Perception of Liposomes: Formation, Manufacturing and Applications*

**Phospholipids PC PE PS PG**

studies have reported the ability of cholesterol to impact liposomes properties and functionality, including encapsulation efficiency and release characteristics [18– 20]. The work by Kirby et al. [21] demonstrated that increasing cholesterol content can prevent leakage and improve in vivo stability of liposome. Later, Lopez-Pinto et al. [22] observed a direct correlation between cholesterol content and liposome sizes. These observations have established cholesterol content to be a key parameter

Like cholesterol, there are many other ingredients that can affect liposomes behavior and afford the desired encapsulation or delivery profiles. Additive agents such as oleic acid and N-[1(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTAP) are useful for the preparation of negatively and positively charged liposomes, respectively. These charged liposomes offer the advantage of great liposomal stability during the storage, as charged particles repel each and reduce aggregation tendencies. While the cell internalization of positively charged liposomes (cationic liposomes) is promoted by their electrostatic interaction with cell membranes (which are negatively charged), liposomes bearing negative charges are subjective to poor cell internalization due to the corresponding repulsive forces. Cationic liposomes are used in gene therapy due to their ability to successfully

In addition, some special lipids such as cholesteryl hemisuccinate (CHEMS) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), have been widely used to prepare liposomes with pH-dependent release features. CHEMS can exhibit pH-sensitivity either alone or in the presence of other lipids. In ionized forms at basic or neutral pH, CHEMS stabilizes the lamellar form of DOPE in lipid-based vesicles. However, the protonated or molecular CHEMS formed at acidic pH promotes hexagonal phase of this lipid, which leads to the disruption of the vesicular systems and release of the encapsulated materials [24, 25]. Tocopherol hemisuccinate (THS) has also shown similar pH-responsiveness as CHEMS, due to their molecular similarity [26].

The composition of liposome appears to be a broad topic, but also very crucial for the desired product development. However, the nature and costs of the liposome

components used over decades viz., particularly the synthetic or highly purified natural phospholipids, have been reported to be part of the factors affecting negatively the universal implementation and affordability of liposome technology [16, 27]. The review by Machado et al. [28] discussed the feasibility of using crude soybean and rice lecithin for liposomal encapsulation of food ingredients. The authors demonstrated these naturally occurring phospholipid mixtures could be useful for liposomes preparation regardless of the intended area of application. Our group has recently investigated liposomal encapsulation of isoniazid using crude soybean lecithin. The formulated liposomes exhibited much better encapsulation efficiency than purified soybean lecithin [29]. This study proposed crude soybean lecithin for liposomal encapsulation of drug molecules. However, the complexity of this lipid mixture might be a bottleneck for some biomedical applications, where molecular architecture of the lipid bilayer is explored to get insights into potential

encapsulate nucleic acids by electrostatic forces [4, 23].

*DOI: http://dx.doi.org/10.5772/intechopen.84255*

*Description of –X moieties of different glycerol-phospholipids [16].*

in liposome formulation.

**Table 2.**

#### **Figure 3.**

*Structural representation of ester glycerol-phospholipid molecules, with R1 and R2 representing the hydrocarbon chains of different fatty acids.*

*General Perception of Liposomes: Formation, Manufacturing and Applications DOI: http://dx.doi.org/10.5772/intechopen.84255*

#### **Table 2.**

*Liposomes - Advances and Perspectives*

evolution of liposomes.

**3.1 Liposomes composition**

in **Figure 3** and **Table 2**.

not able to form liposomes [4, 17].

**3. Liposomes composition and evolution**

in **Table 1**.

• Multilamellar large vesicles (MLV), with size >500 nm;

• Multivesicular vesicles, size from 1000 nm to several thousand nanometers.

Based on their composition, liposomes can be classified as conventional, long circulating, cationic, stimuli-responsive and immunoliposomes. The differences between these categories will be highlighted later when discussing composition and

Furthermore, there are many other vesicular systems considered as part of the liposome-type vesicles. These include emulsomes, enzymosomes, sphyngosomes, transfersomes, ethosomes, pharmacosomes and virosomes, which are lipid-based liposomes analogous. The non-lipid-based liposomes analogous are aquasomes, bilosomes and niosomes [8, 9]. All the liposome-type systems are briefly presented

Liposomes are made of physiologically acceptable natural or synthetic phospholipids found in the lipid bilayer membranes of human cells. The most frequently used phospholipids for liposomes preparation are phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylserines (PS) and phosphatidylglycerols (PG) [16]. The molecular structures of these biocompatible lipids are shown

In liposomal technology, a considerable attention is given to the phase transition temperature (Tt) of these phospholipids. The Tt corresponds to the temperature above which phospholipids exist in liquid crystalline phase. In this fluid state, hydrophobic tails of phospholipids are randomly oriented but ready to form closely continuous bilayered vesicles (liposomes). Below the Tt, phospholipids exist in gel state, where the hydrophobic tails are completely expanded and well packed, thus

As most of the phospholipids used for liposomes formulation have Tt close to the physiological temperature (37°C), the addition of cholesterol has been adopted as a strategy to stabilize the liposomal vesicles in physiological media. This is especially for phospholipids that can undergo phase transition and leakage at room temperature, which can lead to premature release of the liposome cargo. In fact, due to its high hydrophobicity, cholesterol was found efficient in strengthening the packing of phospholipid bilayers, reducing therefore membrane permeability. Numerous

**34**

**Figure 3.**

*hydrocarbon chains of different fatty acids.*

*Structural representation of ester glycerol-phospholipid molecules, with R1 and R2 representing the* 

*Description of –X moieties of different glycerol-phospholipids [16].*

studies have reported the ability of cholesterol to impact liposomes properties and functionality, including encapsulation efficiency and release characteristics [18– 20]. The work by Kirby et al. [21] demonstrated that increasing cholesterol content can prevent leakage and improve in vivo stability of liposome. Later, Lopez-Pinto et al. [22] observed a direct correlation between cholesterol content and liposome sizes. These observations have established cholesterol content to be a key parameter in liposome formulation.

Like cholesterol, there are many other ingredients that can affect liposomes behavior and afford the desired encapsulation or delivery profiles. Additive agents such as oleic acid and N-[1(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTAP) are useful for the preparation of negatively and positively charged liposomes, respectively. These charged liposomes offer the advantage of great liposomal stability during the storage, as charged particles repel each and reduce aggregation tendencies. While the cell internalization of positively charged liposomes (cationic liposomes) is promoted by their electrostatic interaction with cell membranes (which are negatively charged), liposomes bearing negative charges are subjective to poor cell internalization due to the corresponding repulsive forces. Cationic liposomes are used in gene therapy due to their ability to successfully encapsulate nucleic acids by electrostatic forces [4, 23].

In addition, some special lipids such as cholesteryl hemisuccinate (CHEMS) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), have been widely used to prepare liposomes with pH-dependent release features. CHEMS can exhibit pH-sensitivity either alone or in the presence of other lipids. In ionized forms at basic or neutral pH, CHEMS stabilizes the lamellar form of DOPE in lipid-based vesicles. However, the protonated or molecular CHEMS formed at acidic pH promotes hexagonal phase of this lipid, which leads to the disruption of the vesicular systems and release of the encapsulated materials [24, 25]. Tocopherol hemisuccinate (THS) has also shown similar pH-responsiveness as CHEMS, due to their molecular similarity [26].

The composition of liposome appears to be a broad topic, but also very crucial for the desired product development. However, the nature and costs of the liposome components used over decades viz., particularly the synthetic or highly purified natural phospholipids, have been reported to be part of the factors affecting negatively the universal implementation and affordability of liposome technology [16, 27]. The review by Machado et al. [28] discussed the feasibility of using crude soybean and rice lecithin for liposomal encapsulation of food ingredients. The authors demonstrated these naturally occurring phospholipid mixtures could be useful for liposomes preparation regardless of the intended area of application. Our group has recently investigated liposomal encapsulation of isoniazid using crude soybean lecithin. The formulated liposomes exhibited much better encapsulation efficiency than purified soybean lecithin [29]. This study proposed crude soybean lecithin for liposomal encapsulation of drug molecules. However, the complexity of this lipid mixture might be a bottleneck for some biomedical applications, where molecular architecture of the lipid bilayer is explored to get insights into potential

cell membrane permeability. The versatility of crude soybean lecithin liposomes is therefore in question, considering the wide range of areas that the liposomal systems usually cover.

#### **3.2 Liposomes evolution**

Based on the composition, liposomal systems can be considered to have evolved from conventional, long circulating, targeted and immune-liposomes to stimuliresponsive liposomes. The liposomes composed purely of phospholipids with or without cholesterol (conventional liposomes) have shown some limitations due to their uptake by the cells of the mononuclear phagocytic system (MNPS), such as macrophages that ensure liposomes clearance through phagocytosis. This biological fate makes conventional liposomes appropriate vehicles for targeted drug delivery to infected MNPS cells, like the case of alveolar macrophages where *Mycobacterium tuberculosis* resides often. However, the uptake by the MNPS cells decreases liposomes half-life and exposes to high risk of therapeutic failure when the site of interest is beyond the MNPS [2].

Extensive studies conducted in liposome technology led to identification of some astute strategies for addressing the issue of MNPS' attack viz., liposome downsizing and surface modification. In this regard, it was observed that the physiological clearance of larger liposomes (MLV) was much quicker than that of smaller liposomes (SUL), which describe long-circulating profiles with increased half-lives [30]. The stealth strategy arising from surface modification involved grafting or coating hydrophilic polymers such as polyethylene glycol (PEG) and chitosan, which prevents detection of liposomes by the MNPS cells. While stealth behavior allows liposomes to achieve much longer circulation time (hence the name "long circulating liposomes"), this strategy comes along with poor targeting efficiency due to wider distribution of liposomes in the body. From this limitation, further developments have led to introduction of targeted liposomes. These liposomes are characterized by surface decoration with glycoproteins, polysaccharides or specific receptors ligands to achieve narrowed distribution and accumulation at the site of interest [9, 31]. The observation that ligand-decorated liposomes could provide selective drug accumulation inspired further design of antibody-functionalized liposomes (immunoliposomes) as well as stimuli-responsive liposomal systems for controlled drug delivery [32]. **Figure 4** shows the trend in the development of "intelligent" liposomes for site-specific delivery; from conventional liposomes, stealth liposome, targeted liposomes, immunoliposomes to stimuli-responsive liposomes.

Stimuli-responsive liposomes are smart liposomal systems that exhibit rapid release of the cargo upon changes in some physicochemical or biochemical stimuli, such as pH, temperature, redox potentials, enzymes concentrations, ultrasound, electric or magnetic fields [33, 34]. Among these stimuli, pH change is the most promising stimulus due to the existence of multiple pH gradients in the body [35]. In common practice, formulation of pH-sensitive liposomes involves incorporation of CHEMS and DOPE. Although CHEMS-DOPE-based liposomes have shown great promise for controlled delivery [24, 25, 36], the costly status of these lipids remains a deep concern for wider development and application of liposome products. To circumvent the use of such special lipids for pH-dependent delivery, our group has considered hydrazone derivatization of isoniazid (INH), as a small hydrophilic drug model, followed by encapsulation using crude soybean lecithin for cost-effective development [37]. Being poorly water soluble, the prepared conjugates were successfully embedded within the lipid membranes and INH release experiments were conducted in different pH media. The INH-conjugate loaded crude soybean lecithin

**37**

*General Perception of Liposomes: Formation, Manufacturing and Applications*

liposomes have demonstrated attractive nanoparticulate and stimuli-responsive characteristics for potential low-cost site-specific liposomal delivery [38]. However, the amounts of INH-derivatives loaded were found to be almost 10-folds lesser than the loading achieved when native INH was encapsulated in crude soybean lecithin liposomes, which was in turn associated with some burst release [29]. This is probably due to the fact that INH derivatives are encapsulated in the lipid bilayer, which offers limited space for loading [39], while the native INH was trapped within the aqueous core of liposomes. These limitations underline the need for further developments in liposome technology to achieve controlled release from cost-effective

*Schematic representation of the trend in liposome improvements for site specific delivery. (A) Conventional liposomes, (B) stealth liposome, (C) targeted liposomes, (D) immunoliposomes and (E) stimuli-responsive liposomes [32].*

liposomes, combining both use of cheap lipids and high drug loadings.

for up-scale production but require some special equipment [4].

Liposomes can be prepared using a wide range of methods that involve combination of lipids with aqueous media, and somehow affect liposomes characteristics, such as size, lamellarity and encapsulation efficiency (EE). The recently reported methods can be categorized as conventional, which mostly involve approaches that are easy to use at laboratory scale, and novel methods that appear to be more useful

The most commonly used methods for formulation of liposomes share the following fundamental stages: (i) lipids dissolution in organic solvents, (ii) drying of the resultant solution, (iii) hydration of dried lipid (using various aqueous media), (iv) isolation of the liposomal vesicles, and (v) quality control assays [6]. While sharing these basic stages, the conventional preparation methods gather different advantages and disadvantages that are comparatively presented in **Table 3**. The specific technological details of these methods are separately discussed in the

**4. Liposomes preparation and characterization**

**4.1 Preparation methods**

**Figure 4.**

*4.1.1 Conventional methods*

following paragraphs.

*DOI: http://dx.doi.org/10.5772/intechopen.84255*

*General Perception of Liposomes: Formation, Manufacturing and Applications DOI: http://dx.doi.org/10.5772/intechopen.84255*

#### **Figure 4.**

*Liposomes - Advances and Perspectives*

systems usually cover.

**3.2 Liposomes evolution**

interest is beyond the MNPS [2].

cell membrane permeability. The versatility of crude soybean lecithin liposomes is therefore in question, considering the wide range of areas that the liposomal

Based on the composition, liposomal systems can be considered to have evolved from conventional, long circulating, targeted and immune-liposomes to stimuliresponsive liposomes. The liposomes composed purely of phospholipids with or without cholesterol (conventional liposomes) have shown some limitations due to their uptake by the cells of the mononuclear phagocytic system (MNPS), such as macrophages that ensure liposomes clearance through phagocytosis. This biological fate makes conventional liposomes appropriate vehicles for targeted drug delivery to infected MNPS cells, like the case of alveolar macrophages where *Mycobacterium tuberculosis* resides often. However, the uptake by the MNPS cells decreases liposomes half-life and exposes to high risk of therapeutic failure when the site of

Extensive studies conducted in liposome technology led to identification of some astute strategies for addressing the issue of MNPS' attack viz., liposome downsizing and surface modification. In this regard, it was observed that the physiological clearance of larger liposomes (MLV) was much quicker than that of smaller liposomes (SUL), which describe long-circulating profiles with increased half-lives [30]. The stealth strategy arising from surface modification involved grafting or coating hydrophilic polymers such as polyethylene glycol (PEG) and chitosan, which prevents detection of liposomes by the MNPS cells. While stealth behavior allows liposomes to achieve much longer circulation time (hence the name "long circulating liposomes"), this strategy comes along with poor targeting efficiency due to wider distribution of liposomes in the body. From this limitation, further developments have led to introduction of targeted liposomes. These liposomes are characterized by surface decoration with glycoproteins, polysaccharides or specific receptors ligands to achieve narrowed distribution and accumulation at the site of interest [9, 31]. The observation that ligand-decorated liposomes could provide selective drug accumulation inspired further design of antibody-functionalized liposomes (immunoliposomes) as well as stimuli-responsive liposomal systems for controlled drug delivery [32]. **Figure 4** shows the trend in the development of "intelligent" liposomes for site-specific delivery; from conventional liposomes, stealth liposome, targeted liposomes, immunoliposomes to stimuli-responsive

Stimuli-responsive liposomes are smart liposomal systems that exhibit rapid release of the cargo upon changes in some physicochemical or biochemical stimuli, such as pH, temperature, redox potentials, enzymes concentrations, ultrasound, electric or magnetic fields [33, 34]. Among these stimuli, pH change is the most promising stimulus due to the existence of multiple pH gradients in the body [35]. In common practice, formulation of pH-sensitive liposomes involves incorporation of CHEMS and DOPE. Although CHEMS-DOPE-based liposomes have shown great promise for controlled delivery [24, 25, 36], the costly status of these lipids remains a deep concern for wider development and application of liposome products. To circumvent the use of such special lipids for pH-dependent delivery, our group has considered hydrazone derivatization of isoniazid (INH), as a small hydrophilic drug model, followed by encapsulation using crude soybean lecithin for cost-effective development [37]. Being poorly water soluble, the prepared conjugates were successfully embedded within the lipid membranes and INH release experiments were conducted in different pH media. The INH-conjugate loaded crude soybean lecithin

**36**

liposomes.

*Schematic representation of the trend in liposome improvements for site specific delivery. (A) Conventional liposomes, (B) stealth liposome, (C) targeted liposomes, (D) immunoliposomes and (E) stimuli-responsive liposomes [32].*

liposomes have demonstrated attractive nanoparticulate and stimuli-responsive characteristics for potential low-cost site-specific liposomal delivery [38]. However, the amounts of INH-derivatives loaded were found to be almost 10-folds lesser than the loading achieved when native INH was encapsulated in crude soybean lecithin liposomes, which was in turn associated with some burst release [29]. This is probably due to the fact that INH derivatives are encapsulated in the lipid bilayer, which offers limited space for loading [39], while the native INH was trapped within the aqueous core of liposomes. These limitations underline the need for further developments in liposome technology to achieve controlled release from cost-effective liposomes, combining both use of cheap lipids and high drug loadings.

#### **4. Liposomes preparation and characterization**

#### **4.1 Preparation methods**

Liposomes can be prepared using a wide range of methods that involve combination of lipids with aqueous media, and somehow affect liposomes characteristics, such as size, lamellarity and encapsulation efficiency (EE). The recently reported methods can be categorized as conventional, which mostly involve approaches that are easy to use at laboratory scale, and novel methods that appear to be more useful for up-scale production but require some special equipment [4].

#### *4.1.1 Conventional methods*

The most commonly used methods for formulation of liposomes share the following fundamental stages: (i) lipids dissolution in organic solvents, (ii) drying of the resultant solution, (iii) hydration of dried lipid (using various aqueous media), (iv) isolation of the liposomal vesicles, and (v) quality control assays [6]. While sharing these basic stages, the conventional preparation methods gather different advantages and disadvantages that are comparatively presented in **Table 3**. The specific technological details of these methods are separately discussed in the following paragraphs.


#### **Table 3.**

*Advantages and disadvantages of conventional methods.*

#### *4.1.1.1 Film hydration*

Also known Bangham method, film hydration represents the simplest and oldest method used in liposome technology. In this method, lipids are firstly dissolved in a suitable organic solvent, and dried down to yield a thin film at the bottom of the flask. The obtained lipid film is hydrated using an appropriate aqueous medium to produce liposomal dispersion. The structural organization of the formed vesicles can be affected by the hydration conditions. A gentle hydration of the lipid film forms giant unilamellar vesicles (GULV), whereas a hash hydration gives rise to multilamellar vesicles (MLV) with poor size homogeneity, which requires an additional downsizing step. The most commonly used sizing methods include probe and bath sonication that afford production of small unilamellar vesicles (SUV). Despite its higher effectiveness, probe sonication is often blamed for potential contamination (with titanium from the titanium-based nozzle used for mechanical agitation), and production of local heat that can affect lipids and drugs stability. Although the two sonication methods produce liposomes with identical characteristics, the use of bath sonication remains a better option due to easy control of operational parameters. Another technique used for liposome sizing includes consecutive extrusion of the liposomal formulation through polycarbonate filters of defined pore sizes. In this method, the number of extrusion cycles is the key parameter to control for effective homogenization [4, 6].

#### *4.1.1.2 Reverse phase evaporation*

Reverse phase evaporation is an alternative method to the film hydration that involves formation of water-in-oil emulsion between the aqueous phase (containing hydrophilic materials) and the organic phase (containing lipids and any hydrophobic materials). A brief sonication of this mixture is required for system homogenization. The removal of the organic phase under reduced pressure yields a milky gel

**39**

*General Perception of Liposomes: Formation, Manufacturing and Applications*

the stability and safety of the liposomal products [28, 40].

that turns subsequently into liposomal suspension. The liposomes can be isolated from the dispersion using centrifugation, dialysis or sepharose 24 column [28].

Solvent injection involves quick injection of the lipid solution (in ethanol or diethyl ether) into an aqueous medium. The experiment is performed either at room or at higher temperature (e.g., 60°C), depending on whether the organic solvent is water-miscible or not. The liposomes prepared by solvent injection process are mostly polydispersed and highly contaminated by organic solvents, especially ethanol due to formation of azeotrope mixture with water. As presented in **Table 3**, solvent injection suffers from several drawbacks including continuous exposure of the therapeutic agents to high temperature and organic solvent that might affect both

In the detergent removal method, phospholipids are dissolved in aqueous solution containing detergents at critical micelle concentrations (CMC). Upon detergent removal, the reaction medium frees individual phospholipid molecules that self-assemble into bilayered structures. Detergent removal is mostly achieved by means of a dialysis bag, polystyrene-based absorber beads or Sephadex columns (gel permeation chromatography). Dilution of the resultant mixture with some appropriate aqueous medium leads to restructuration of the formed micelles that

Among all the conventional methods, the heating method is known to be the most attractive method for liposomes preparation due to its organic solvent free characteristics. In the heating method, lipids are hydrated for 1 hour, and heated for another hour above the transition temperature of the phospholipids in the presence of a hydrating agent (glycerin or propylene glycol 3%). When cholesterol is part of the formulation, the reaction medium is heated up to 100°C because of its high melting point. Being prepared under heating conditions, the resultant liposomes can be readily used without any further sterilization treatments, which minimizes both formulation complexity and timing. In addition, there is no need for further removal of the hydrating agents employed, since these represent physiologically acceptable ingredients that are well-established for pharmaceutical applications. Moreover, the observation that these hydrating agents can prevent particle coagulation and sedimentation makes them much more attractive as stabilizer and isotonizing additives. The hydroxyl groups of these hydrating agents provide a cryoprotective effect that makes the heating method an efficient method for the

Microfluidic methods include all the novel techniques that make use of microscopic channels (in the size range of 5–500 μm). In this method, lipids are dissolved

in an appropriate organic solvent (ethanol or isopropanol) and the resultant solution is propelled perpendicularly or in the opposite direction to the aqueous

*DOI: http://dx.doi.org/10.5772/intechopen.84255*

*4.1.1.3 Solvent injection*

*4.1.1.4 Detergent removal*

evolve to liposomes [4, 6].

formulation of inhalable liposomes [41, 42].

*4.1.2 Novel preparation methods*

*4.1.2.1 Microfluidic channel method*

*4.1.1.5 Heating method*

that turns subsequently into liposomal suspension. The liposomes can be isolated from the dispersion using centrifugation, dialysis or sepharose 24 column [28].

## *4.1.1.3 Solvent injection*

*Liposomes - Advances and Perspectives*

**Method designation**

Film hydration (Bangham method)

Reverse phase evaporation

Detergent removal

*Adapted from [40].*

**Table 3.**

*4.1.1.1 Film hydration*

effective homogenization [4, 6].

*4.1.1.2 Reverse phase evaporation*

Also known Bangham method, film hydration represents the simplest and oldest method used in liposome technology. In this method, lipids are firstly dissolved in a suitable organic solvent, and dried down to yield a thin film at the bottom of the flask. The obtained lipid film is hydrated using an appropriate aqueous medium to produce liposomal dispersion. The structural organization of the formed vesicles can be affected by the hydration conditions. A gentle hydration of the lipid film forms giant unilamellar vesicles (GULV), whereas a hash hydration gives rise to multilamellar vesicles (MLV) with poor size homogeneity, which requires an additional downsizing step. The most commonly used sizing methods include probe and bath sonication that afford production of small unilamellar vesicles (SUV). Despite its higher effectiveness, probe sonication is often blamed for potential contamination (with titanium from the titanium-based nozzle used for mechanical agitation), and production of local heat that can affect lipids and drugs stability. Although the two sonication methods produce liposomes with identical characteristics, the use of bath sonication remains a better option due to easy control of operational parameters. Another technique used for liposome sizing includes consecutive extrusion of the liposomal formulation through polycarbonate filters of defined pore sizes. In this method, the number of extrusion cycles is the key parameter to control for

**Advantages Disadvantages**

Solvent injection Straightforward approach Trace of organic solvent as residue, possible

Simple design, suitable encapsulation efficiency

Simple design, homogenous product, control of particle size

organic solvent free, no need for sterilization, possible up-scale production

Heating method Simple and fast process,

*Advantages and disadvantages of conventional methods.*

Straightforward process Use of organic solvent and mechanical agitation,

sterilization issue

sterilization issue

sterilization issue

yield, sterilization required

The need for high temperature

production of large particles with no control on size, poor encapsulation efficiencies of hydrophilic materials, time consuming,

Not applicable to fragile cargoes, use of large quantity of organic solvent, time consuming,

nozzle blockage in ether system, time consuming,

Presence of organic solvent, detergent residue, time consuming, low entrapment efficiency, poor

Reverse phase evaporation is an alternative method to the film hydration that involves formation of water-in-oil emulsion between the aqueous phase (containing hydrophilic materials) and the organic phase (containing lipids and any hydrophobic materials). A brief sonication of this mixture is required for system homogenization. The removal of the organic phase under reduced pressure yields a milky gel

**38**

Solvent injection involves quick injection of the lipid solution (in ethanol or diethyl ether) into an aqueous medium. The experiment is performed either at room or at higher temperature (e.g., 60°C), depending on whether the organic solvent is water-miscible or not. The liposomes prepared by solvent injection process are mostly polydispersed and highly contaminated by organic solvents, especially ethanol due to formation of azeotrope mixture with water. As presented in **Table 3**, solvent injection suffers from several drawbacks including continuous exposure of the therapeutic agents to high temperature and organic solvent that might affect both the stability and safety of the liposomal products [28, 40].
