*4.1.2.1 Microfluidic channel method*

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

medium within the micro-channels. The continuous axial mixing of the organic and aqueous solutions leads to liposomes formation because of local diffusion of phospholipids in aqueous phase, which encourages the self-assembly process. Among many others, the micro hydrodynamic focusing method represents the most commonly used microfluidic method for liposomes formulation. This method produces small and large unilamellar vesicles, 40–140 nm, with good size homogeneity (mono dispersed feature). The other microfluidic techniques include the microfluidic droplets and the pulsed jet flow microfluidic methods. The microfluidic droplets method involves dissolution of phospholipids in hexane for preparation of giant liposomes (4–20 μm). In the pulsed jet flow microfluidic method, the conventional film hydration method has been modified by drying the lipid solution in microtubes. The resultant lipid film is hydrated within the microtubes through a perfusion process that produces much larger vesicles, 200–534 μm, with remarkable encapsulation efficiency [4, 43]. As common advantages, the microfluidic methods offer the possibility for production of vesicles with desired size, due to the versatility and flexibility of the methods. The disadvantages of these methods include the imperative use of organic solvent and drastic agitation, as well as difficulty for large scale production [40].

## *4.1.2.2 Supercritical fluidic method*

While being considered as equivalent to the conventional reverse phase evaporation method, supercritical fluidic technique represents the most important novel liposome preparation methods that makes use of a supercritical fluid, such as carbon dioxide (CO2) maintained under supercritical conditions (supercritical temperature and pressure). In this state, CO2 is an excellent solvent for the lipids. The high-performance liquid chromatography (HPLC) pump provides a continuous flow of the aqueous phase into a view cell that contains the supercritical lipid solution, allowing phase transition of the dissolved phospholipids. Upon sudden decrease in pressure, CO2 gets completely removed and phospholipids self-assemble into a bilayered vesicular system. The supercritical fluidic method affords large unilamellar vesicles (100–1200 nm) with 5-fold higher encapsulation efficiency than the equivalent conventional method. Apart from being organic solvent-free methods, the supercritical fluidic method offers many other advantages such as the use of CO2, as a cheap and environmentally harmless solvent, possibility for controlling particle size, in situ sterilization and large-scale production in industrial settings. However, the disadvantages of the supercritical fluidic technique, including particularly its high cost, low yield and use of high pressures (200–350 bar) which require special infrastructures, restrict their universal applications for wider developments of liposomal technology [4, 40, 43].

#### *4.1.3 Post-preparation treatments*

#### *4.1.3.1 Freeze-thawing*

The freeze-thawing treatment involves freezing the liposomes dispersion in liquid nitrogen, and subsequently thawing it at the temperature above the phase transition temperature of the lipids used for formulation. Upon freeze-thawing, the liposomal vesicles are subjected to fusion since the lipid bilayers become fluid and highly permeable, allowing extensive diffusion of hydrophilic molecules, which leads to important cryoconcentration. These structural modifications encourage encapsulation of hydrophilic materials that are poorly loaded in liposomes when conventional methods are used. This underlines the reason why freeze-thawing

**41**

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

liposomes stability during and after freeze-drying treatment [6].

After production, liposome formulations are subjected to extensive characterization, evaluating the physicochemical properties of liposomes that affect their shelf stability and biological performance. The most routinely investigated parameters in liposome characterization include vesicle size and size distribution (or polydispersity), surface charge (or Zeta potential), shape and morphology, lamellarity, encapsulation efficiency, phase behavior (or polymorphism) and in vitro release profile [4, 46]. **Table 4** indicates the techniques used for evaluation of liposome characteristics. The most frequently used methods are briefly discussed

Also known as photon correlation spectroscopy or quasi-elastic light scattering, DLS represents the most commonly used method for determination of liposome size, size distribution (polydispersity) and Zeta potential (surface charge). DLS is done by an instrumental setting called Zetasizer Nano. The standard operational principle of DLS is based on continuous motion of dispersed particles due to their bombardment by solvent molecules (Brownian motion). This phenomenon causes remarkable scattering of the applied light. Since the extent of fluctuation in light intensity is associated with the diffusion rate of the suspended particles, which is related to particle diameter (smaller particles diffusing faster than the larger ones), the particle size is automatically deducted from the estimated amount of the scattered light. When addressing Zeta potential measurements, DLS allows surface charge determination by accessing changes in the scattered light intensity caused by particle motion due to the electric field applied. In other words, for surface

represents an important treatment in liposome technology. Amongst the key parameters to be considered for freeze-thawing optimization are the number and duration of freeze-thawing cycles. These can impact significantly not only the encapsulation efficiency but also structural characteristics, i.e., liposomes lamellar-

Commonly known as lyophilization, freeze-drying is a post-preparation treatment for liposomes that is applied in both laboratory and industrial settings to preserve the characteristics of liposomal products. Freeze-drying involves freezing of the aqueous samples and subsequent removal of ice by sublimation. Freeze-drying represents a very useful treatment for shelf stability of liposomal suspensions, since water molecules can trigger some chemical reactions and lead to modification of the cargo or excipients in the formulation. Freeze-drying appears to be of great interest when the prepared formulation contains thermos-sensitive materials such as proteins, nucleic acids, etc., which might undergo fast degradation when subjected to heat-drying. The use of freeze-drying has gained considerable attention in liposome technology due to improved storage stability of liposomal products. Because of potential leakage of liposomes during freeze-drying, addition of hydrophilic compounds, commonly called cryoprotective agents (such as carbohydrates), has been established to ensure good stability and quality of the final product. The cryoprotectants commonly used include mannitol, lactose, sucrose and trehalose. Among these, trehalose is the most reputed cryoprotecting agent since it preserves

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

ity and polydispersity [44, 45].

**4.2 Characterization techniques**

in the following paragraphs.

*4.2.1 Dynamic light scattering (DLS)*

*4.1.3.2 Freeze-drying*

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

represents an important treatment in liposome technology. Amongst the key parameters to be considered for freeze-thawing optimization are the number and duration of freeze-thawing cycles. These can impact significantly not only the encapsulation efficiency but also structural characteristics, i.e., liposomes lamellarity and polydispersity [44, 45].

### *4.1.3.2 Freeze-drying*

*Liposomes - Advances and Perspectives*

scale production [40].

*4.1.2.2 Supercritical fluidic method*

developments of liposomal technology [4, 40, 43].

*4.1.3 Post-preparation treatments*

*4.1.3.1 Freeze-thawing*

medium within the micro-channels. The continuous axial mixing of the organic and aqueous solutions leads to liposomes formation because of local diffusion of phospholipids in aqueous phase, which encourages the self-assembly process. Among many others, the micro hydrodynamic focusing method represents the most commonly used microfluidic method for liposomes formulation. This method produces small and large unilamellar vesicles, 40–140 nm, with good size homogeneity (mono dispersed feature). The other microfluidic techniques include the microfluidic droplets and the pulsed jet flow microfluidic methods. The microfluidic droplets method involves dissolution of phospholipids in hexane for preparation of giant liposomes (4–20 μm). In the pulsed jet flow microfluidic method, the conventional film hydration method has been modified by drying the lipid solution in microtubes. The resultant lipid film is hydrated within the microtubes through a perfusion process that produces much larger vesicles, 200–534 μm, with remarkable encapsulation efficiency [4, 43]. As common advantages, the microfluidic methods offer the possibility for production of vesicles with desired size, due to the versatility and flexibility of the methods. The disadvantages of these methods include the imperative use of organic solvent and drastic agitation, as well as difficulty for large

While being considered as equivalent to the conventional reverse phase evaporation method, supercritical fluidic technique represents the most important novel liposome preparation methods that makes use of a supercritical fluid, such as carbon dioxide (CO2) maintained under supercritical conditions (supercritical temperature and pressure). In this state, CO2 is an excellent solvent for the lipids. The high-performance liquid chromatography (HPLC) pump provides a continuous flow of the aqueous phase into a view cell that contains the supercritical lipid solution, allowing phase transition of the dissolved phospholipids. Upon sudden decrease in pressure, CO2 gets completely removed and phospholipids self-assemble into a bilayered vesicular system. The supercritical fluidic method affords large unilamellar vesicles (100–1200 nm) with 5-fold higher encapsulation efficiency than the equivalent conventional method. Apart from being organic solvent-free methods, the supercritical fluidic method offers many other advantages such as the use of CO2, as a cheap and environmentally harmless solvent, possibility for controlling particle size, in situ sterilization and large-scale production in industrial settings. However, the disadvantages of the supercritical fluidic technique, including particularly its high cost, low yield and use of high pressures (200–350 bar) which require special infrastructures, restrict their universal applications for wider

The freeze-thawing treatment involves freezing the liposomes dispersion in liquid nitrogen, and subsequently thawing it at the temperature above the phase transition temperature of the lipids used for formulation. Upon freeze-thawing, the liposomal vesicles are subjected to fusion since the lipid bilayers become fluid and highly permeable, allowing extensive diffusion of hydrophilic molecules, which leads to important cryoconcentration. These structural modifications encourage encapsulation of hydrophilic materials that are poorly loaded in liposomes when conventional methods are used. This underlines the reason why freeze-thawing

**40**

Commonly known as lyophilization, freeze-drying is a post-preparation treatment for liposomes that is applied in both laboratory and industrial settings to preserve the characteristics of liposomal products. Freeze-drying involves freezing of the aqueous samples and subsequent removal of ice by sublimation. Freeze-drying represents a very useful treatment for shelf stability of liposomal suspensions, since water molecules can trigger some chemical reactions and lead to modification of the cargo or excipients in the formulation. Freeze-drying appears to be of great interest when the prepared formulation contains thermos-sensitive materials such as proteins, nucleic acids, etc., which might undergo fast degradation when subjected to heat-drying. The use of freeze-drying has gained considerable attention in liposome technology due to improved storage stability of liposomal products. Because of potential leakage of liposomes during freeze-drying, addition of hydrophilic compounds, commonly called cryoprotective agents (such as carbohydrates), has been established to ensure good stability and quality of the final product. The cryoprotectants commonly used include mannitol, lactose, sucrose and trehalose. Among these, trehalose is the most reputed cryoprotecting agent since it preserves liposomes stability during and after freeze-drying treatment [6].

### **4.2 Characterization techniques**

After production, liposome formulations are subjected to extensive characterization, evaluating the physicochemical properties of liposomes that affect their shelf stability and biological performance. The most routinely investigated parameters in liposome characterization include vesicle size and size distribution (or polydispersity), surface charge (or Zeta potential), shape and morphology, lamellarity, encapsulation efficiency, phase behavior (or polymorphism) and in vitro release profile [4, 46]. **Table 4** indicates the techniques used for evaluation of liposome characteristics. The most frequently used methods are briefly discussed in the following paragraphs.

## *4.2.1 Dynamic light scattering (DLS)*

Also known as photon correlation spectroscopy or quasi-elastic light scattering, DLS represents the most commonly used method for determination of liposome size, size distribution (polydispersity) and Zeta potential (surface charge). DLS is done by an instrumental setting called Zetasizer Nano. The standard operational principle of DLS is based on continuous motion of dispersed particles due to their bombardment by solvent molecules (Brownian motion). This phenomenon causes remarkable scattering of the applied light. Since the extent of fluctuation in light intensity is associated with the diffusion rate of the suspended particles, which is related to particle diameter (smaller particles diffusing faster than the larger ones), the particle size is automatically deducted from the estimated amount of the scattered light. When addressing Zeta potential measurements, DLS allows surface charge determination by accessing changes in the scattered light intensity caused by particle motion due to the electric field applied. In other words, for surface


#### **Table 4.**

*Analytical methods commonly used for liposomes characterization.*

charge (Zeta potential) evaluation, changes in the intensity of the scattered light are governed by the applied electric field (which causes extensive motion of charged particles), in contrast to size measurements where Brownian motion is the key factor [4, 47]. Apart from being a simple, fast and reliable method for routine analyses, DLS offers many other advantages including the fact that the measurement is taken from a native environment, and a wide size range can be evaluated (from a few nanometers to several micrometers). However, DLS shows some limitations such as the difficulty of differentiating individual particles from aggregates and high sensitivity to contaminants [48]. In addition, DLS is technically unable to provide true particle size, but rather hydrodynamic diameter due to particle solvation. Water layers on particle surface lead to false readings of particle diameters in aqueous media [49].

#### *4.2.2 Transmission electron microscopy (TEM)*

The microscopic observation provides direct visualization of the liposomal vesicles as individual particles, which allows effective analysis of shape and morphology as well as a precise and reliable size reading. In this context, TEM techniques are commonly used in liposome technology for structural characterizations. In TEM experiments, the liquid sample is spotted onto a copper grid, and the solvent dried prior to the microscopic analysis. Under TEM instrument, liposomal vesicles mostly appear as black spherical particles on a white background. For a variant TEM technique like negative staining TEM, liposomes appear as bright spherical spots on a black background since the spotted sample is treated with uranyl acetate or phosphotungstic acid (as negative staining agent). Due to its effectiveness, TEM appears to be a powerful complementary technique to DLS for confirmation of the liposomal structure. Unlike DLS, TEM offers the advantage of differentiating individual vesicles from aggregates, allowing critical assessment of the liposome population. Nevertheless, TEM presents several limitations due to sample preparation. Apart from being time-consuming, sample pretreatments in TEM analyses may cause remarkable changes in liposomal shape or morphology: potential vesicle shrinkage, swelling or artifact formation [4, 50].

**43**

*4.2.4 Lamellarity assays*

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

*4.2.3 Differential scanning calorimetry (DSC) and X-ray diffraction (XRD)*

and production of both excipients and biologically active materials [57–59].

The lamellarity of liposomes is part of their structural characteristics that can have an impact on the intended applications. The number of lipid bilayers can be evaluated using chemically labeled or radiolabelled agents that can be distributed in the bilayer membranes. However, this technique is limited since these reagents might be distributed only on the outer lipid membrane and lead to false readings. To date, Cryo-TEM is the most commonly used technique for the determination of lamellarity by visualization [4]. The nuclear magnetic resonance spectroscopy of the 31-phosphorus (31P-NMR) is also being currently used to estimate the lamellarity of liposomes. This technique deals with the estimation of the ratio of phospholipid amount in the outer layers to that of the inner layers [60]. The 31P-NMR spectrum with a broad peak indicates the presence of MLV while a narrow peak

DSC and XRD are complementary techniques that evaluate the thermal behavior and crystallinity, respectively, and provide valuable information for characterization of loaded liposomes [46, 51, 52]. DSC evaluates the differences in heat flow (electric power) between a sample and a reference. In DSC experiments, the sample and the reference are subjected to a programmed heating, cooling or isothermal treatment in a controlled atmosphere (mostly saturated with nitrogen gas). The heating treatment is achieved either by the same heater (heat flux DSC) or by separate heaters (power compensated DSC). The experiment is conducted in specialized metal pans made of aluminum, tin, zinc or indium. Throughout the experiment, frequent electric power adjustments occur upon material phase transition (melting or crystallization), ensuring thermal equilibrium between the sample and the reference. This phenomenon is described and expressed by the plot of heat capacity against temperature or time (heat flow curve). The heat flow curve provides the respective transition temperature and enthalpy, which allows to identify the nature of thermal events: endo- or exothermic [53–55]. DSC represents the most useful thermal analysis technique in the study of lipid-based materials [46, 52, 54–56]. Unlike DSC where sample recovery is not possible, XRD is a non-destructive analytical tool that allows structural investigations of crystalline materials. XRD makes use of X-rays that deeply penetrate solid materials and provide useful information at atomic structure level. Though relatively expensive, an XRD instrument is an environmentally and user-friendly device that is easy to use. A wide range of materials such as powders, crystals and liquids can be quickly assessed by XRD. Its other advantages include high resolution, reliability, relatively cheap maintenance, and easy data collection, processing and interpretation. The phase transitions and polymorphism determined by XRD represent valuable information in pharmaceutical development

To overcome these limitations, Cryo-TEM was developed as a strategy to minimize liposome disruption by making use of a flash freezing treatment for direct particle visualization in solid-state (without solvent removal). Nowadays, Cryo-TEM is the most reliable technique for visual determination of liposome structure including lamellarity. However, Cryo-TEM appears to be sometimes limited since it works perfectly only with very small particles. This has led to the development of atomic force microscopy (AFM) for direct particle analysis in native environments. Although AFM offers the advantage of higher particle resolution at threedimensional level, the use of this technique is mostly limited by the high cost of the instrument, which compromises its universal availability and accessibility [3, 4].

*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*

To overcome these limitations, Cryo-TEM was developed as a strategy to minimize liposome disruption by making use of a flash freezing treatment for direct particle visualization in solid-state (without solvent removal). Nowadays, Cryo-TEM is the most reliable technique for visual determination of liposome structure including lamellarity. However, Cryo-TEM appears to be sometimes limited since it works perfectly only with very small particles. This has led to the development of atomic force microscopy (AFM) for direct particle analysis in native environments. Although AFM offers the advantage of higher particle resolution at threedimensional level, the use of this technique is mostly limited by the high cost of the instrument, which compromises its universal availability and accessibility [3, 4].

### *4.2.3 Differential scanning calorimetry (DSC) and X-ray diffraction (XRD)*

DSC and XRD are complementary techniques that evaluate the thermal behavior and crystallinity, respectively, and provide valuable information for characterization of loaded liposomes [46, 51, 52]. DSC evaluates the differences in heat flow (electric power) between a sample and a reference. In DSC experiments, the sample and the reference are subjected to a programmed heating, cooling or isothermal treatment in a controlled atmosphere (mostly saturated with nitrogen gas). The heating treatment is achieved either by the same heater (heat flux DSC) or by separate heaters (power compensated DSC). The experiment is conducted in specialized metal pans made of aluminum, tin, zinc or indium. Throughout the experiment, frequent electric power adjustments occur upon material phase transition (melting or crystallization), ensuring thermal equilibrium between the sample and the reference. This phenomenon is described and expressed by the plot of heat capacity against temperature or time (heat flow curve). The heat flow curve provides the respective transition temperature and enthalpy, which allows to identify the nature of thermal events: endo- or exothermic [53–55]. DSC represents the most useful thermal analysis technique in the study of lipid-based materials [46, 52, 54–56].

Unlike DSC where sample recovery is not possible, XRD is a non-destructive analytical tool that allows structural investigations of crystalline materials. XRD makes use of X-rays that deeply penetrate solid materials and provide useful information at atomic structure level. Though relatively expensive, an XRD instrument is an environmentally and user-friendly device that is easy to use. A wide range of materials such as powders, crystals and liquids can be quickly assessed by XRD. Its other advantages include high resolution, reliability, relatively cheap maintenance, and easy data collection, processing and interpretation. The phase transitions and polymorphism determined by XRD represent valuable information in pharmaceutical development and production of both excipients and biologically active materials [57–59].

#### *4.2.4 Lamellarity assays*

The lamellarity of liposomes is part of their structural characteristics that can have an impact on the intended applications. The number of lipid bilayers can be evaluated using chemically labeled or radiolabelled agents that can be distributed in the bilayer membranes. However, this technique is limited since these reagents might be distributed only on the outer lipid membrane and lead to false readings. To date, Cryo-TEM is the most commonly used technique for the determination of lamellarity by visualization [4]. The nuclear magnetic resonance spectroscopy of the 31-phosphorus (31P-NMR) is also being currently used to estimate the lamellarity of liposomes. This technique deals with the estimation of the ratio of phospholipid amount in the outer layers to that of the inner layers [60]. The 31P-NMR spectrum with a broad peak indicates the presence of MLV while a narrow peak

*Liposomes - Advances and Perspectives*

Zeta potential /Surface

charge

**Table 4.**

Particle shape / morphology

**Parameters Analytical techniques**

Lamellarity Cryo-TEM and 31P-NMR

*Analytical methods commonly used for liposomes characterization.*

charge (Zeta potential) evaluation, changes in the intensity of the scattered light are governed by the applied electric field (which causes extensive motion of charged particles), in contrast to size measurements where Brownian motion is the key factor [4, 47]. Apart from being a simple, fast and reliable method for routine analyses, DLS offers many other advantages including the fact that the measurement is taken from a native environment, and a wide size range can be evaluated (from a few nanometers to several micrometers). However, DLS shows some limitations such as the difficulty of differentiating individual particles from aggregates and high sensitivity to contaminants [48]. In addition, DLS is technically unable to provide true particle size, but rather hydrodynamic diameter due to particle solvation. Water layers on particle surface lead to false readings of particle diameters in aque-

Particle size Dynamic light scattering (DLS), size exclusion chromatography (SEC), field-flow

Microscopic techniques such as TEM, Cryo-TEM and AFM

Electrophoretic mobility, DLS

Phase behavior X-ray diffraction (XRD), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA)

by drug content determination

Encapsulation efficiency Centrifugation, dialysis or column separation for liposomes isolation, followed

Drug release Dialysis or centrifugation, followed by drug quantification using analytical

fractionation (FFF) and microscope technology: transmission electron microscopy (TEM), cryogenic-TEM (Cryo-TEM), and atomic force microscopy (AFM)

method, such as UV–Vis spectrophotometry, fluorescence spectrometry, enzyme- or protein-based assays, gel electrophoresis, HPLC, UPLC, LC-MS

The microscopic observation provides direct visualization of the liposomal vesicles as individual particles, which allows effective analysis of shape and morphology as well as a precise and reliable size reading. In this context, TEM techniques are commonly used in liposome technology for structural characterizations. In TEM experiments, the liquid sample is spotted onto a copper grid, and the solvent dried prior to the microscopic analysis. Under TEM instrument, liposomal vesicles mostly appear as black spherical particles on a white background. For a variant TEM technique like negative staining TEM, liposomes appear as bright spherical spots on a black background since the spotted sample is treated with uranyl acetate or phosphotungstic acid (as negative staining agent). Due to its effectiveness, TEM appears to be a powerful complementary technique to DLS for confirmation of the liposomal structure. Unlike DLS, TEM offers the advantage of differentiating individual vesicles from aggregates, allowing critical assessment of the liposome population. Nevertheless, TEM presents several limitations due to sample preparation. Apart from being time-consuming, sample pretreatments in TEM analyses may cause remarkable changes in liposomal shape or morphology: potential vesicle

**42**

ous media [49].

*4.2.2 Transmission electron microscopy (TEM)*

shrinkage, swelling or artifact formation [4, 50].

corresponds to SUV. The addition of paramagnetic ions such as Mn2+, Co2+, and Pr3+ shifts the respective peaks to either downfield or upfield because of ionic interactions with the phosphate backbone. By comparing the spectroscopic profile with and without the paramagnetic ion, the lamellarity of liposomes can be estimated. Some other techniques such as small-angle X-ray scattering (SAXS) and trapped volume determination can be used to estimate liposome lamellarity [4, 61].

#### *4.2.5 In vitro release assays*

The profile of release for the liposomal cargoes is commonly estimated in vitro using dialysis. This method implies trapping the liposomal dispersion into a dialysis bag of specific molecular weight cut off. The resultant tubing membrane is placed in a simulated physiological fluid (release medium) that is often a buffer maintained under well-defined conditions: specific temperature and speed of stirring/shaking. At predetermined time intervals, an aliquot is withdrawn from the release medium and an equal volume of the fresh buffer is replaced to maintain sink conditions. In the withdrawn sample aliquots, the released cargo is quantified using some routine analytical techniques such as UV-Vis spectrophotometry, HPLC, UPLC, etc., adapted to the molecular species under evaluation. The release profile is obtained by plotting the cumulative release percentage against the chosen time intervals [4]. Data from the in vitro release study are valuably considered as part of the rational development of formulations for controlled release, since they allow effective prediction of in vivo performance of the delivery systems [62].

### **5. Applications**

Liposomes have evolved so far from mere experimental tools of research to industrially established products for clinical and veterinary use. They have shown the ability to improve the physicochemical features of the cargoes and ferry them to the sites of interest. The concepts of liposomal encapsulation have been applied in several fields of life science. Liposomes are frequently used for the delivery of drug, gene, vaccine and diagnostic products; but other applications encompass encapsulation of food and cosmetic ingredients as well as routine analysis of chemical substances [2, 3]. The following paragraphs briefly present the current applications of liposomes.

#### **5.1 Application in drug delivery**

The use of liposomes in drug delivery aims at modifying the pharmacokinetics of drugs to improve the therapeutic efficacy while minimizing potential toxicity [6]. Liposomes can alter the spatial and temporal distribution of the entrapped drug molecules in vivo, leading to controlled delivery at the site of interest and reduced off-target adverse effects [63]. The liposomal systems have been extensively investigated for the delivery of existing and emerging drugs at various research levels, from basic stages related to research and development to preclinical and clinical applications. Nowadays, liposomes represent the most clinically established drug vehicles for human diseases [3, 5]. The efforts invested in liposomal technology have so far led to the development of several effective liposomal formulations that are currently used in clinics (**Table 5**).

Liposomal formulations have been used to address a wide range of pathological conditions through different administration routes, including dermal, transdermal, oral, pulmonary and parenteral routes. The clinical areas commonly explored in liposome research encompass skin disorders, cancers and infectious diseases [4].

**45**

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

Abelcet Amphotericin B Fungal infections AmBisome Amphotericin B Fungal infections Amphocil Amphotericin B Fungal infections

DepoDur Morphine sulfate Pain relief

Epaxal Inactivated hepatitis A virus Hepatitis A Evacet Doxorubicin Ovarian cancer

Inflexal V Inactivated hemagglutinin of influenza virus strains A and B

DaunoXome Daunorubicin Hematological malignancy DepoCyt Cytarabine Lymphomatous meningitis

Doxil Doxorubicin Kaposi's sarcoma and solid tumors

LipoDox Doxorubicin Kaposi's sarcoma and solid tumors Marqibo Vincristine sulfate Acute lymphoblastic leukemia Visudyne Verteporfin Photodynamic therapy

Influenza

**Drug name Therapeutic indications**

Amongst, cancer therapy appears to be in the forefront of liposome delivery, due to poor bioavailability and side effects of most of the anti-cancer drugs. However, several infectious diseases, most specially where the pathogen is hosted by the MNPS (i.e., tuberculosis, leishmaniasis, fungal infections), have been reported to be good candidates for liposome application, taking advantage of the spontaneous liposomes uptake by the cells of MNPS [2]. Apart from the nature and localization of the disease, the design and development of liposomes depend also on the intended administration route, since different anatomical and physiological characteristics

Furthermore, the application of liposomes in drug delivery is highly dependent on their colloidal and physiochemical features, i.e., vesicle size, surface charge and system stability [64]. For instance, small liposomes (SUV) are good candidates for Parkinson's and Alzheimer's diseases, due to the need for crossing the brain blood barrier to achieve brain targeted delivery. Meanwhile, large liposomes are preferred for macrophage targeted delivery of antimicrobials, when pathogens are located inside the MNPS cells (e.g., tuberculosis, leishmaniasis), taking advantage of the passive liposome cell uptake [2, 4, 46]. This underlines the need for thorough exploration of process and formulation parameters at early stages of products development to produce liposomes with desired characteristics, making the technology for liposomes manufacturing key to future therapeutic research and development.

Liposomes have been reported to achieve effective intracellular delivery of genes. These liposomes, also called lipoplexes, are generally made of cationic lipids, which allow for encapsulation of genetic materials via electrostatic interactions with the negatively charged phosphate backbones of nucleic acids. The positive charge on the surface of liposomes also influences their interactions with negatively charged cell membranes and promotes cell internalization [23, 65]. Cationic liposomes offer several advantages over viral gene vectors, including easy and safe production,

can be encountered from one route to another [46].

**5.2 Application in gene delivery**

*Clinically approved liposomal products [3, 4].*

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

**Branded product**

**Table 5.**

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


#### **Table 5.**

*Liposomes - Advances and Perspectives*

*4.2.5 In vitro release assays*

**5. Applications**

**5.1 Application in drug delivery**

that are currently used in clinics (**Table 5**).

corresponds to SUV. The addition of paramagnetic ions such as Mn2+, Co2+, and Pr3+ shifts the respective peaks to either downfield or upfield because of ionic interactions with the phosphate backbone. By comparing the spectroscopic profile with and without the paramagnetic ion, the lamellarity of liposomes can be estimated. Some other techniques such as small-angle X-ray scattering (SAXS) and trapped volume determination can be used to estimate liposome lamellarity [4, 61].

The profile of release for the liposomal cargoes is commonly estimated in vitro using dialysis. This method implies trapping the liposomal dispersion into a dialysis bag of specific molecular weight cut off. The resultant tubing membrane is placed in a simulated physiological fluid (release medium) that is often a buffer maintained under well-defined conditions: specific temperature and speed of stirring/shaking. At predetermined time intervals, an aliquot is withdrawn from the release medium and an equal volume of the fresh buffer is replaced to maintain sink conditions. In the withdrawn sample aliquots, the released cargo is quantified using some routine analytical techniques such as UV-Vis spectrophotometry, HPLC, UPLC, etc., adapted to the molecular species under evaluation. The release profile is obtained by plotting the cumulative release percentage against the chosen time intervals [4]. Data from the in vitro release study are valuably considered as part of the rational development of formulations for controlled release, since they allow effective

prediction of in vivo performance of the delivery systems [62].

Liposomes have evolved so far from mere experimental tools of research to industrially established products for clinical and veterinary use. They have shown the ability to improve the physicochemical features of the cargoes and ferry them to the sites of interest. The concepts of liposomal encapsulation have been applied in several fields of life science. Liposomes are frequently used for the delivery of drug, gene, vaccine and diagnostic products; but other applications encompass encapsulation of food and cosmetic ingredients as well as routine analysis of chemical substances [2, 3]. The following paragraphs briefly present the current applications of liposomes.

The use of liposomes in drug delivery aims at modifying the pharmacokinetics of drugs to improve the therapeutic efficacy while minimizing potential toxicity [6]. Liposomes can alter the spatial and temporal distribution of the entrapped drug molecules in vivo, leading to controlled delivery at the site of interest and reduced off-target adverse effects [63]. The liposomal systems have been extensively investigated for the delivery of existing and emerging drugs at various research levels, from basic stages related to research and development to preclinical and clinical applications. Nowadays, liposomes represent the most clinically established drug vehicles for human diseases [3, 5]. The efforts invested in liposomal technology have so far led to the development of several effective liposomal formulations

Liposomal formulations have been used to address a wide range of pathological conditions through different administration routes, including dermal, transdermal, oral, pulmonary and parenteral routes. The clinical areas commonly explored in liposome research encompass skin disorders, cancers and infectious diseases [4].

**44**

*Clinically approved liposomal products [3, 4].*

Amongst, cancer therapy appears to be in the forefront of liposome delivery, due to poor bioavailability and side effects of most of the anti-cancer drugs. However, several infectious diseases, most specially where the pathogen is hosted by the MNPS (i.e., tuberculosis, leishmaniasis, fungal infections), have been reported to be good candidates for liposome application, taking advantage of the spontaneous liposomes uptake by the cells of MNPS [2]. Apart from the nature and localization of the disease, the design and development of liposomes depend also on the intended administration route, since different anatomical and physiological characteristics can be encountered from one route to another [46].

Furthermore, the application of liposomes in drug delivery is highly dependent on their colloidal and physiochemical features, i.e., vesicle size, surface charge and system stability [64]. For instance, small liposomes (SUV) are good candidates for Parkinson's and Alzheimer's diseases, due to the need for crossing the brain blood barrier to achieve brain targeted delivery. Meanwhile, large liposomes are preferred for macrophage targeted delivery of antimicrobials, when pathogens are located inside the MNPS cells (e.g., tuberculosis, leishmaniasis), taking advantage of the passive liposome cell uptake [2, 4, 46]. This underlines the need for thorough exploration of process and formulation parameters at early stages of products development to produce liposomes with desired characteristics, making the technology for liposomes manufacturing key to future therapeutic research and development.

#### **5.2 Application in gene delivery**

Liposomes have been reported to achieve effective intracellular delivery of genes. These liposomes, also called lipoplexes, are generally made of cationic lipids, which allow for encapsulation of genetic materials via electrostatic interactions with the negatively charged phosphate backbones of nucleic acids. The positive charge on the surface of liposomes also influences their interactions with negatively charged cell membranes and promotes cell internalization [23, 65]. Cationic liposomes offer several advantages over viral gene vectors, including easy and safe production,

cost effectiveness, possibility of monitoring toxicity, biodegradability, biocompatibility and lack of dangerous immunogenicity. However, they are suffering from poor transfection efficiency due to their limited endosomal escaping ability, which exposes the genetic materials to enzymatic and acid degradation in lysosomes. This has been improved by incorporating fusogenic lipids, such as DOPE, in the liposomal formulation. These helper lipids facilitate endosomal escaping by membrane fusion with endosomes, leading to early cytoplasmic release of the gene. Though cationic liposomes have shown some dose-dependent toxicity, successful results have been obtained in cancer therapy when delivering genes encoding for tumor suppression proteins [66, 67]. He et al. have recently developed folate receptor alpha-targeted lipoplexes with therapeutic gene expression regulated by an hTERT promoter. These liposomes have shown some promise for the treatment of ovarian cancer [68].

#### **5.3 Application in vaccine delivery**

The use of specific lipids or molecules such as phosphatidylserine, DOTAP, fatty acids and monophosphoryl lipids can produce liposomes with attractive immune-stimulating activities. Liposomes carrying antigenic materials, either encapsulated in the aqueous core, grafted or coated on the surface; can stimulate immune responses on macrophage uptake. Following endolysosomal degradation, macrophages present the antigen to T-lymphocytes that initiate the production of cytokines and specific antibodies via activation of B-lymphocytes [4]. The immune response produced by liposomes can be influenced by their composition, lamellarity, size and surface charge [69]. Liposomes containing a glycolipid, trehalose 6,6'-dibehenate, and a cationic lipid, dimethyldioctadecylammonium, in a 1:5 mass ratio have demonstrated efficient delivery of the TB vaccine Ag85B-ESAT-6. This vaccine has shown prolonged immune response without any toxic effects [70]. Although a liposomal vaccine (namely Stimuvax®) targeting the major histocompatibility class I complex for lung carcinoma was not successfully implemented, some other vaccines such as Epaxal®, Inflexal® V and Mosquirix® have been clinically established for the treatment of hepatitis A, influenza virus infections and malaria, respectively. These vaccines, classified as virosomes, are liposomes generally made of reconstituted viral envelop supplemented with phosphatidylcholine. They offer the advantage of undergoing membrane fusion either with the cells or the endosomes, and thus leading to efficient cytosolic delivery [2, 9].

### **5.4 Application in diagnosis**

The use of liposomes for diagnostic purposes is one of current topics of great interest in biomedical applications. Liposomes with magnetic properties, also called magnetoliposomes, are made by entrapping superparamagnetic iron-based nanoparticles or iron oxides or gadolinium (III) chelates for magnetic resonance imaging. Functionalized liposomes labeled with radioisotopes can also be used as molecular probes in nuclear imaging. For instance, liposomes labeled with 64Cu, 18F, 89Zr or 52Mn have been reported in positron emission tomography while 99mTC, 111In or 67Ga labeled liposomes were applied in single photon emission computed tomography. Acoustic liposomes which are liposomes made of perfluoropropane gas can be used as contrast agents in ultrasound imaging technique. The encapsulation of quantum dots and fluorescent dyes into liposomes has also led to the development of attractive liposomal platforms for diagnosis. Additionally, liposomes have shown great potential for simultaneous accommodation of drugs and diagnostic agents such as radionuclides, magnetic or contrast substances. This can be achieved by encapsulation in the inner core, embedding in the lipid bilayer, chemically grafting

**47**

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

both the therapeutic effect and the diagnostic control [2, 71, 72].

between biologically active compounds and cell membranes [4].

or coating onto the surface of liposomes. A successful targeted co-delivery of these materials has given to liposomes the status of theranostic systems, as they provide

In analytical domains, liposome-based formulations can be usefully involved in immunoassays, biosensors analysis and liquid chromatography. Liposomes can be used to encapsulate, embed or conjugate the analytical entities with high loading capacity and huge surface area that can enhance the intensity of analytical signal. In a direct enzyme-linked immunosorbent assay (ELISA) like method, fluorophoreconjugated liposomes carrying a substantial amount of secondary antibody quickly bind to the antigen that has been fixed on the primary antibody. The addition of a colorimetric substrate leads to color development and allows for analytical estimation of the antigenic analyte. The use of liposomes has been deemed to lower the limits of detection of analytes and increase the sensitivity of immunological analysis technique [73]. In chromatography, the conjugation of liposomes to the stationary phase for gel permeation chromatography is useful for separation of drugs and proteins and for exploring possible molecular interactions on phospholipid membranes. Additionally, the cell-like appearance of liposomes makes them appropriate simulated cell models for studying and predicting the interactions

The use of liposomes in the field of cosmetics is based on the similarity between the lipid composition of the liposomal vesicle and that of the biological layers composing the skin. Because phospholipids can be subject to hydration, topical liposomes happen to contribute to the reduction of skin dryness. While playing the role of attractive vehicles for relevant cosmetic agents, liposomes provide a great source of skin ingredients, such as essential unsaturated fatty acids like linolenic acid. This has shown great potential for maintaining the skin and hair in good physiological standing, preventing the rise of some common topical diseases. Furthermore, the use of skin care formulations made of empty or hydrating agents loaded liposomes helps to reduce the transdermal water loss, which is a major cause of skin dryness [74, 75].

As versatile lipid-based systems, liposomes have shown some potential in the encapsulation of food ingredients. In this field, the use of liposomes aims to stabilize some nutraceutical or dietetic ingredients during the storage, to improve their organoleptic characters or to provide a controlled and targeted delivery of these substances in a specific tissue. The shelf life and efficacy of instable bioactive products such as vitamins, enzymes and anti-oxidative agents have been prolonged by their liposomal encapsulation. For example, proteinase and lipase loaded liposomes have been reported to improve the ripening of cheese notably in preventing proteolysis of casein [28, 76]. Yokota et al. [27] have successfully improved the taste and odor of casein hydrolysate in dietetic preparations by liposomal encapsulation. In addition to the encapsulation of dietetic compounds, liposomes have been recently used to encapsulate a cyanobacterium, namely Spirulina platensis, as a source of proteins [77]. Although the use of liposomes in food industry is still at the infant stages of development, advanced studies have been launched to investigate much more attractive applications, such as detection of food contaminants by means of stimuli-responsive liposomes for food safety [76].

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

**5.5 Application in analytical fields**

**5.6 Application in cosmetics**

**5.7 Application in food industry**

or coating onto the surface of liposomes. A successful targeted co-delivery of these materials has given to liposomes the status of theranostic systems, as they provide both the therapeutic effect and the diagnostic control [2, 71, 72].
