**5. Characterization of liposomes**

After preparation and before application, liposomes have to be characterized in order to ensure their in vitro and in vivo performance. Liposomal properties that are commonly discussed include lamellarity (the number of bilayers present in liposomes), diameter and size distribu‐ tion, lipid composition and concentration determination, the encapsulant concentration and its encapsulation efficiency.

For the characterization of chemical properties, phospholipids can be quantitatively in terms of concentration either by Bartlett Assay or Stewart Assay. The phospholipid hydrolysis might be followed by HPLC where the column outflow can be monitored continuously by UV absorbance to obtain a quantitative record of the eluted components. Moreover, the phospho‐ lipid oxidation can also be followed by a number of techniques i.e., UV absorbance method, TBA method (2-thiobarbutiric acid) (for endoperoxides), iodometric method (for hydroper‐ oxides) and GLC (gas-liquid chromatography) method [93].

of preparation methods is done through gas chromatography (GC) [100-101] This is a very rapid and reliable method and most analytical and organic laboratories are equipped with a

**Figure 2.** Cryo-TEM pictures of Size Exclusion Chromatography fractions eluted at 90 minutes and prepared from egg-

Adopted from Holzer, M., Barnert, S., Momm, J., Schubert, R., 2009. Preparative size exclusion chromatography com‐ bined with detergent removal as a versatile tool to prepare unilamellar and spherical liposomes of highly uniform size

Liposomes as Potential Drug Carrier Systems for Drug Delivery

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15

An important feature of liposomes is the existence of a temperature dependant, reversible phase transition, where the hydrocarbon chains of the phospholipid structures undergo a transformation from an ordered gel state to a more disordered fluid, liquid crystalline, state. This transition temperature is important in optimizing the storage conditions (i.e Temperature) to minimize fusion and drug leakage. These changes have been monitored by freeze fracture electron microscopy and much more easily by differential scanning calorimetry (DSC)

Entrapped volume is a crucial parameter that governs the morphology of liposomes. This internal volume is defined as the aqueous entrapped volume per unit quantity of lipids. The most promising way to determine the internal volume is to measure the quantity of water by replacing external medium (water) with a spectrophotometrically inert fluid (i.e. deuterium

It is essential to measure the quantity of the encapsulated material inside liposomal structures before studying the behavior of this encapsulated material physically and biologically since the effects observed experimentally will be dose related. After the removal of the nonencapsulated material by the separation techniques the quantity of material remained can be assumed as 100% encapsulated. Minicolumn centrifugation and protamine aggregation

Methods for determining the amount of material encapsulated within the liposomes typically rely on the destruction of the lipid bilayer and subsequent quantification of the released material [105]. In these measurements, the signal due to intact liposomes is typically monitored

methods are the general separation procedures that are commonly used [93].

oxide) and then measuring water signal by NMR [93].

distribution. J. Chromatogr. A 1216, 5838–5848.

GC.

[102-104, 93].

phosphatidylcholine.

The most direct method for determination of liposome size is the electron microscopy due to the possibility of viewing the liposomes individually and obtaining the exact information about the liposome population over the whole range of sizes [94]. As liposomes do not naturally create a contrast to be visible by electron microscopy, either cryo-TEM (Figure 2) should be used or staining of the liposome sample is required. Either way, it is a very timeconsuming method and it requires equipments that may not always be immediately accessible. The other method for the determination of liposome size, dynamic light scattering [95, 96], is very simple and rapid to perform but it measures an average size of liposome bulk. More recently, atomic force microscopy is also used to determine the morphology, size and stability of liposomal structures. All these size determination methods are very expensive. If only an approximate size range is required, gel exclusion chromatography might be suitable.

Electrostatic stabilization of liposomes may be a desirable feature to prevent fusion. The surface charge on the liposomes is measured by zeta-potential measurements [98]. These measure‐ ments are useful in determination of the in vivo behavior of liposomes. Often zeta potential values <-25 mV or >+25 mV are considered stable [99]. However, as mentioned earlier, charged liposomes have the disadvantage of being unstable in biological conditions.

Residual solvent is very unacceptable for drug delivery applications, therefore residual solvent should be kept at a minimum in the formulations. Quantification of residual solvents as a result

Filtration sterilization is relatively time-consuming and not efficient for the removal of viruses [91]. Studies have shown that polycarbonate membranes are less effective than hydrophobic Fluoropore membrane and cellulose acetate/surfactant-free membrane filtration units [91]. Although the limitations of filtration provoked researches on other sterilization methods, all resulted in the formation of degradation products via the previously mentioned degradation pathways. Filtration and the other methods are summarized according to their applicability

After preparation and before application, liposomes have to be characterized in order to ensure their in vitro and in vivo performance. Liposomal properties that are commonly discussed include lamellarity (the number of bilayers present in liposomes), diameter and size distribu‐ tion, lipid composition and concentration determination, the encapsulant concentration and

For the characterization of chemical properties, phospholipids can be quantitatively in terms of concentration either by Bartlett Assay or Stewart Assay. The phospholipid hydrolysis might be followed by HPLC where the column outflow can be monitored continuously by UV absorbance to obtain a quantitative record of the eluted components. Moreover, the phospho‐ lipid oxidation can also be followed by a number of techniques i.e., UV absorbance method, TBA method (2-thiobarbutiric acid) (for endoperoxides), iodometric method (for hydroper‐

The most direct method for determination of liposome size is the electron microscopy due to the possibility of viewing the liposomes individually and obtaining the exact information about the liposome population over the whole range of sizes [94]. As liposomes do not naturally create a contrast to be visible by electron microscopy, either cryo-TEM (Figure 2) should be used or staining of the liposome sample is required. Either way, it is a very timeconsuming method and it requires equipments that may not always be immediately accessible. The other method for the determination of liposome size, dynamic light scattering [95, 96], is very simple and rapid to perform but it measures an average size of liposome bulk. More recently, atomic force microscopy is also used to determine the morphology, size and stability of liposomal structures. All these size determination methods are very expensive. If only an

approximate size range is required, gel exclusion chromatography might be suitable.

liposomes have the disadvantage of being unstable in biological conditions.

Electrostatic stabilization of liposomes may be a desirable feature to prevent fusion. The surface charge on the liposomes is measured by zeta-potential measurements [98]. These measure‐ ments are useful in determination of the in vivo behavior of liposomes. Often zeta potential values <-25 mV or >+25 mV are considered stable [99]. However, as mentioned earlier, charged

Residual solvent is very unacceptable for drug delivery applications, therefore residual solvent should be kept at a minimum in the formulations. Quantification of residual solvents as a result

on liposomal preparations in Table 1 [92], given above.

oxides) and GLC (gas-liquid chromatography) method [93].

**5. Characterization of liposomes**

14 Application of Nanotechnology in Drug Delivery

its encapsulation efficiency.

Adopted from Holzer, M., Barnert, S., Momm, J., Schubert, R., 2009. Preparative size exclusion chromatography com‐ bined with detergent removal as a versatile tool to prepare unilamellar and spherical liposomes of highly uniform size distribution. J. Chromatogr. A 1216, 5838–5848.

**Figure 2.** Cryo-TEM pictures of Size Exclusion Chromatography fractions eluted at 90 minutes and prepared from eggphosphatidylcholine.

of preparation methods is done through gas chromatography (GC) [100-101] This is a very rapid and reliable method and most analytical and organic laboratories are equipped with a GC.

An important feature of liposomes is the existence of a temperature dependant, reversible phase transition, where the hydrocarbon chains of the phospholipid structures undergo a transformation from an ordered gel state to a more disordered fluid, liquid crystalline, state. This transition temperature is important in optimizing the storage conditions (i.e Temperature) to minimize fusion and drug leakage. These changes have been monitored by freeze fracture electron microscopy and much more easily by differential scanning calorimetry (DSC) [102-104, 93].

Entrapped volume is a crucial parameter that governs the morphology of liposomes. This internal volume is defined as the aqueous entrapped volume per unit quantity of lipids. The most promising way to determine the internal volume is to measure the quantity of water by replacing external medium (water) with a spectrophotometrically inert fluid (i.e. deuterium oxide) and then measuring water signal by NMR [93].

It is essential to measure the quantity of the encapsulated material inside liposomal structures before studying the behavior of this encapsulated material physically and biologically since the effects observed experimentally will be dose related. After the removal of the nonencapsulated material by the separation techniques the quantity of material remained can be assumed as 100% encapsulated. Minicolumn centrifugation and protamine aggregation methods are the general separation procedures that are commonly used [93].

Methods for determining the amount of material encapsulated within the liposomes typically rely on the destruction of the lipid bilayer and subsequent quantification of the released material [105]. In these measurements, the signal due to intact liposomes is typically monitored prior to bilayer disruption. The techniques used for this quantification depend on the nature of the encapsulant and include spectrophotometry [106, 107], fluorescence spectroscopy [108], enzyme-based methods [109] and electrochemical techniques. If a separation technique such as HPLC of field-flow fractionation (FFF) is applied, the percent encapsulation can be ex‐ pressed as the ratio of the unencapsulated peak area to that of a reference standard of the same initial concentration [110, 111]. This method can be applied if the liposomes do not undergo any purification following preparation. Either technique serves to separate liposome encap‐ sulated materials from those that remain in the extravesicular solution and hence can also be used to monitor the storage stability in terms of leakage or the effect of various disruptive conditions o the retention of encapsulants. Some authors have combined the size distribution and encapsulation efficiency determination in one assay by using FFF-MALS (multi angled light scattering) coupled to a concentration detector suitable for the encapsulant [112].

carrier, as well as their biological interactions between liposomes and cells. Based on these liposome properties, several modes of drug delivery can be listed: the major ones are enhanced drug solubilization (e.g. amphotericin B, minoxidil), protection of sensitive drug molecules (e.g. cytosine arabinose, DNA, RNA, antisense olgionucleotides, ribozymes), enhanced intracellular uptake (all agents, including antineoplastic agents, antibiotics and antivirals) and

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Although lipid based formulations have advantages as drug carriers, drug-delivery systems based on unmodified liposomes are limited by their short blood circulation time, instability in vivo and lack of target selectivity [119, 120]. To increase accumulation of liposomal formula‐ tions in the desired cells and tissues, the use of targeted liposomes including surface-attached ligands such as; antibodies, folates, peptides and transferrin that are capable of recognizing and binding to the desired cells. Despite of some improvements in targeting efficiency by these immunoliposomes, the majority of these modified liposomes were still eliminated rapidly by the reticulo endothelial system, primarily in the liver [120]. Better target accumulations are

Schematic drawing of cytosolic delivery and organelle-specific targeting of drug loaded nanoparticles (i.e. most frequently liposomes) via receptor-mediated endocytosis is shown in

**Figure 3.** Schematic drawing of the cytosolic delivery and organelle-specific targeting of drug loaded nanoparticles via receptor-mediated endocytosis. After receptor mediated cell association with nanoparticles, the nanoparticles are engulfed in a vesicle known as an early endosome. Nanoparticles formulated with an endosome disrupting property disrupt the endosomes followed by cytoplasmic delivery. On the other hand, if nanoparticles are captured in early en‐ dosomes, theymaymake theirway to lysosomes as late endosomes where their degradation takes place. Only fraction of non-degraded drug released in the cytoplasm interacts with cellular organelles in a random fashion. However, cyto‐ solic delivery of a fraction of organelle-targeted nanoparticles via endosomal escape or from lysosomes travel to the

targeting organelles to deliver their therapeutic cargo [121].

altered pharmacokinetics and biodistribution of the encapsulated drug.

expected if liposomes can be made to remain in the circulation long enough.

Figure 3.

Since techniques used to separate free materials from liposome-encapsulated contents can potentially cause leakage of contents and, in some cases, ambiguity in the extent of separation, research using methods that do not rely on separation are of interest. Reported methods have included 1H NMR where free markers exhibited pH sensitive resonance shifts in the external medium versus encapsulated markers [113]; diffusion ordered 2D NMR which relied on differences in diffusion coefficients of entrapped and free marker molecules [114]; fluorescence methods where the signal from unencapsulated fluorophores was quenched by substances present in the external solution [115]; electron pin resonance (ESR) methods which rely on the signal broadening of unencapsulated markers by the addition of a membrane-impermeable agent [116, 117].

The drug release from liposomes can be followed by the usage of a well calibrated in vitro diffusion cell in order to predict pharmacokinetics and bioavailability of drug before expensive and time-consuming in vivo studies. For the determination of pharmacokinetic performance of liposomal formulations, dilution-induced drug release in buffer and plasma was employed and for the determination of drug bioavailability, another procedure is followed which involves the liposome degradation in the presence of mouse-liver lysosome lysate [93].
