**3. Stability of liposomes**

Liposome stability can be explained by physical, chemical and biological means which are all interrelated. Generally, chemical (degradation of phospholipids structures) and physical (uniformity of size distribution and encapsulation efficiency) stability determine the shelf-life of liposomes. Once the liposomal formulations have been obtained, maintenance of the physical properties of these preparations can be difficult. Leakage of the encapsulated material due to the permeability of the membrane, change in the size distribution and stability problems due to the hydrolytic and oxidative degradation are the general problems upon storage. Methods are devised to overcome these instability problems, those designed to minimize the degradation processes and those which help liposomes to survive in the face of conditions which encourages these processes.

and their derivatives are employed [56]. Cycloprotectants, especially sucrose because of its high glass transition temperature, are believed to be effective to protect the liposome mem‐ branes against possible fracture and rapture that might cause a change in size distribution and a loss of the encapsulated material presumably by forming glasses under the typical freezing conditions used for lyophilization [57]. Lyophilization increases the shelf-life of the finished product by preserving in a relatively more stable dry state. Some liposome products on market or clinical trials are provided as lyophilized powder. For example, AmBisomeTM, a liposomal amphotericin, is the first liposome product to be marketed in several countries is supplied as a lyophilized powder to be reconstituted with sterile water injection. Additionally, paclitaxelliposome formulations have been developed which show good stability [58, 59]. These formulations once lyophilized can be stored at room temperature for extended time. On the other hand, once the preparation is reconstituted, it is not stable for more than a day in terms

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11

The physical degradation, leakage and fusion of liposomes, can occur as a result of the lattice defects in the membrane introduced during the manufacture, particularly in SUVs that are prepared below the membrane phase transition temperature. Annealing process, incubating the liposomes at a higher temperature than the phase transition temperature, can wipe out these defects by equalizing the differences in packing density between opposite sides of the bilayers. Even in annealed vesicles, aggregation and fusion can occur over a long period of time. In neutral liposomes, aggregation takes place because of the van der Waals interactions and because of the increased surface area it tends to be more pronounced in large liposomes. The simplest solution to overcome this aggregation is to add a small amount of negatively

SUVs have much more tendency to fusion when compared to large liposomes due to the presence of stress arising from the high curvature of the membrane. Since this can occur specifically at the transition temperature of the membrane, it would be better to store these liposomes at a temperature much lower than the transition temperature of the lipids. For example, SUVs should be stored above their transition temperature for no longer than 24 hours but LUVs can be stored for a longer period of time if the temperature of the solution is kept in a range of 4-8 0C for approximately 1 week before the leakage of the encapsulated material starts due to the hydrolytic degradation on the membrane structure [60]. Also, addition of cholesterol to the phospholipid mixture would be a solution to reduce or eliminate the transition. The presence of cholesterol prevents packing and aggregation by inducing orien‐ tation and more rigidity to the phospholipids. Other than cholesterol, peptide incorporation to the lipid membrane also enables the lipid membrane to be more rigid at physiological

Permeability of liposome membranes depends highly on the membrane lipid composition, as well as on the encapsulated material. Large polar or ionic molecules will be retained much more efficiently than low molecular weight lipophilic compounds. Generally, for both type of encapsulated material, a rigid, more saturated membrane with a higher ratio of cholesterol forms the most stable lipid membrane concerning the leakage of the encapsulated material.

charged phospholipid (e.g. 10% PA or PG) to the liposome composition [29]

of size.

temperature [61-63].

Two different types of chemical degradation can affect the performance of the phospholipids bilayers; hydrolysis of the ester bonds linking the fatty acids to the glycerol backbone and oxidation of the unsaturated acyl chains, if present. The level of oxidation can be kept to a minimum by taking some precautions like starting with freshly purified lipids and freshly distilled solvents, avoiding procedures involving high temperatures, carrying out the manu‐ facturing process in the absence of oxygen, deoxygenating the aqueous solutions by passing nitrogen, storing all liposome suspensions in an inert atmosphere and including an antioxidant, e.g. α-tocopherol [50], a common non-toxic dietary lipid, as a component of the lipids membrane. An alternative solution to the oxidation problem is to reduce the level of oxidizable lipids in the membrane by using saturated lipids instead of the unsaturated ones. Also, the mono-unsaturated ones have much less tendency of oxidation than the polyunsaturated ones. Thus; sphingomyelins, usually having only one double bond, are expected to degrade more slowly than other mammalian origin lipids. Entirely synthetic and saturated phospholipids; DMPC, DPPC and DSPC, can also be considered as a solution for the oxidative degradation of liposomes.

Hydrolysis type of chemical degradation of the ester linkages in the phospholipid structure occurs most slowly at pH values close to neutral. In general, the rate of hydrolysis has a "Vshaped" dependence, with a minimum at pH 6.5 and an increased rate at both higher and lower pH. In the active loading of drugs, as it is mentioned before, low pH levels are required which triggers the hydrolysis. This hydrolysis kind of chemical degradation is also very effective on the aqueous solutions of liposome due to the presence of water. Temperature also triggers the hydrolysis of the lipids which creates the need for refrigeration. In order to keep hydrolysis to a minimum during active loading, attention must be paid for the removal of residual solvent from the dried lipids. To avoid hydrolysis, instead of ester linked lipids, the usage of ether linkage containing lipids (e.g. found in the membrane of halophilic bacteria) would be an absolute solution [51]. Another chemical degradation, oxidation of the lipids in the liposome structures can be prevented by the addition of small amounts of antioxidants during the manufacturing steps.

The problems related to the lipid oxidation and hydrolysis during the shelf-life of the liposomal product can be reduced by the storage of liposomal dispersion in the dry state by freeze-drying (lyophilization), without compromising their physical state or encapsulation capacity [52]. However, freeze-drying of liposome systems without appropriate stabilizers will lead to fusion of vesicles, i.e. physical instability. To promote vesicle stability during the freeze-drying process, cycloprotectants [53-55], including saccharides (e.g. sucrose, trehalose, and lactose) and their derivatives are employed [56]. Cycloprotectants, especially sucrose because of its high glass transition temperature, are believed to be effective to protect the liposome mem‐ branes against possible fracture and rapture that might cause a change in size distribution and a loss of the encapsulated material presumably by forming glasses under the typical freezing conditions used for lyophilization [57]. Lyophilization increases the shelf-life of the finished product by preserving in a relatively more stable dry state. Some liposome products on market or clinical trials are provided as lyophilized powder. For example, AmBisomeTM, a liposomal amphotericin, is the first liposome product to be marketed in several countries is supplied as a lyophilized powder to be reconstituted with sterile water injection. Additionally, paclitaxelliposome formulations have been developed which show good stability [58, 59]. These formulations once lyophilized can be stored at room temperature for extended time. On the other hand, once the preparation is reconstituted, it is not stable for more than a day in terms of size.

(uniformity of size distribution and encapsulation efficiency) stability determine the shelf-life of liposomes. Once the liposomal formulations have been obtained, maintenance of the physical properties of these preparations can be difficult. Leakage of the encapsulated material due to the permeability of the membrane, change in the size distribution and stability problems due to the hydrolytic and oxidative degradation are the general problems upon storage. Methods are devised to overcome these instability problems, those designed to minimize the degradation processes and those which help liposomes to survive in the face of conditions

Two different types of chemical degradation can affect the performance of the phospholipids bilayers; hydrolysis of the ester bonds linking the fatty acids to the glycerol backbone and oxidation of the unsaturated acyl chains, if present. The level of oxidation can be kept to a minimum by taking some precautions like starting with freshly purified lipids and freshly distilled solvents, avoiding procedures involving high temperatures, carrying out the manu‐ facturing process in the absence of oxygen, deoxygenating the aqueous solutions by passing nitrogen, storing all liposome suspensions in an inert atmosphere and including an antioxidant, e.g. α-tocopherol [50], a common non-toxic dietary lipid, as a component of the lipids membrane. An alternative solution to the oxidation problem is to reduce the level of oxidizable lipids in the membrane by using saturated lipids instead of the unsaturated ones. Also, the mono-unsaturated ones have much less tendency of oxidation than the polyunsaturated ones. Thus; sphingomyelins, usually having only one double bond, are expected to degrade more slowly than other mammalian origin lipids. Entirely synthetic and saturated phospholipids; DMPC, DPPC and DSPC, can also be considered as a solution for the oxidative degradation

Hydrolysis type of chemical degradation of the ester linkages in the phospholipid structure occurs most slowly at pH values close to neutral. In general, the rate of hydrolysis has a "Vshaped" dependence, with a minimum at pH 6.5 and an increased rate at both higher and lower pH. In the active loading of drugs, as it is mentioned before, low pH levels are required which triggers the hydrolysis. This hydrolysis kind of chemical degradation is also very effective on the aqueous solutions of liposome due to the presence of water. Temperature also triggers the hydrolysis of the lipids which creates the need for refrigeration. In order to keep hydrolysis to a minimum during active loading, attention must be paid for the removal of residual solvent from the dried lipids. To avoid hydrolysis, instead of ester linked lipids, the usage of ether linkage containing lipids (e.g. found in the membrane of halophilic bacteria) would be an absolute solution [51]. Another chemical degradation, oxidation of the lipids in the liposome structures can be prevented by the addition of small amounts of antioxidants

The problems related to the lipid oxidation and hydrolysis during the shelf-life of the liposomal product can be reduced by the storage of liposomal dispersion in the dry state by freeze-drying (lyophilization), without compromising their physical state or encapsulation capacity [52]. However, freeze-drying of liposome systems without appropriate stabilizers will lead to fusion of vesicles, i.e. physical instability. To promote vesicle stability during the freeze-drying process, cycloprotectants [53-55], including saccharides (e.g. sucrose, trehalose, and lactose)

which encourages these processes.

10 Application of Nanotechnology in Drug Delivery

of liposomes.

during the manufacturing steps.

The physical degradation, leakage and fusion of liposomes, can occur as a result of the lattice defects in the membrane introduced during the manufacture, particularly in SUVs that are prepared below the membrane phase transition temperature. Annealing process, incubating the liposomes at a higher temperature than the phase transition temperature, can wipe out these defects by equalizing the differences in packing density between opposite sides of the bilayers. Even in annealed vesicles, aggregation and fusion can occur over a long period of time. In neutral liposomes, aggregation takes place because of the van der Waals interactions and because of the increased surface area it tends to be more pronounced in large liposomes. The simplest solution to overcome this aggregation is to add a small amount of negatively charged phospholipid (e.g. 10% PA or PG) to the liposome composition [29]

SUVs have much more tendency to fusion when compared to large liposomes due to the presence of stress arising from the high curvature of the membrane. Since this can occur specifically at the transition temperature of the membrane, it would be better to store these liposomes at a temperature much lower than the transition temperature of the lipids. For example, SUVs should be stored above their transition temperature for no longer than 24 hours but LUVs can be stored for a longer period of time if the temperature of the solution is kept in a range of 4-8 0C for approximately 1 week before the leakage of the encapsulated material starts due to the hydrolytic degradation on the membrane structure [60]. Also, addition of cholesterol to the phospholipid mixture would be a solution to reduce or eliminate the transition. The presence of cholesterol prevents packing and aggregation by inducing orien‐ tation and more rigidity to the phospholipids. Other than cholesterol, peptide incorporation to the lipid membrane also enables the lipid membrane to be more rigid at physiological temperature [61-63].

Permeability of liposome membranes depends highly on the membrane lipid composition, as well as on the encapsulated material. Large polar or ionic molecules will be retained much more efficiently than low molecular weight lipophilic compounds. Generally, for both type of encapsulated material, a rigid, more saturated membrane with a higher ratio of cholesterol forms the most stable lipid membrane concerning the leakage of the encapsulated material.

Many attempts have been made to enhance the physical stability of liposomes. Among these, surface modification of liposomes is an attractive method to improve liposomal stability both in vitro and in vivo. Some improvements in chemical and physical stability of polymer coated liposomes prepared with polysaccharide derivatives, such as mannan or amylopectin, have been demonstrated [64]. Several other substances also have been used for preparation of polymer coated liposomes such as poloxamer, polysorbate 80, carboxymethyl chitosan, and dextran derivatives [65-69]. While the possibility of coating liposomes with these polymers has been reported, few papers have dealt with the systematic evaluation of the physical stability of polymer coated liposomes. Moreover, contravening results have been also reported such as that polymer coated liposomes showed less stability than non-coated ones [65, 70].

**4. Sterilization of liposomes**

mal drug carrier formulations.

Filtration Low operation

γ-irradiation Moderate operation

temperature

temperature

Low operation temperature

reliability

Highest microbial death

UV-sterilization Low cost and convenient Poor penetration into products

**Table 1.** Summary of the Sterilization Techniques Applied on Liposomal Preparations.

**Sterilization Technique**

Final steam sterilization

Ethylene oxide

sterilization of liposome formulations is a major challenge.

Pharmaceutical industry in general differentiates between two principally different ap‐ proaches to ensure sterility of a parental product: terminal sterilization of the final product in its container (steam sterilization) and aseptical manufacturing. Terminal sterilization is the commonly used one because of its higher sterility assurance level achieved when compared with the aseptical methods. However, terminal sterilization is not applicable to many liposo‐

There are several sterilization methods; such as filtration, gamma irradiation, final steam sterilization, dry heat sterilization, ethylene oxide sterilization, and ultraviolet sterilization. Bearing in mind the susceptibility of liposomes to the previously mentioned physical and chemical degradation mechanisms, the conditions required in conventional sterilization techniques (except filtration) are rather concerning since they involve the usage of heat, radiation and/or chemical sterilizing agents. Therefore, identification of a suitable method for

**Advantage(s) Disadvantage(s) Convenience**

Operation under aseptic conditions

Risk of degradation of liposomes

Risk of degradation of liposomes

Filtration is the most suitable sterilization technique for the thermolabile liposomes since it does not include any form of heat or condition that can result in the degradation of liposomes or leakage of the encapsulated material. However, filtration has some drawbacks such as; being only applicable to the liposomes that are smaller than 200 nm in diameter and being an expensive method due to the equipment requiring to work under high pressure (25 kg/cm2 and above). Additionally, this technique must be performed under aseptic conditions [90].

diameter

Dry heat Low cost and convenient Risk of degradation of liposomes High

Large scale operation

Low cost and convenient Risk of degradation of liposomes High

Applicable to liposomes lower than 200 nm in

Possible carcinogenic residues Low

Low

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High

High

In vivo stability of liposomes is also dependent on their charge. In serum, there are several proteins that are both positively and negatively charged. Liposomes with neutral charge are found to be more stable as they have much less electrostatic affinity towards proteins. [71].

Biological liposome stability plays important roles at various stages of drug delivery. However, liposomes are somewhat biologically unstable as a parenteral drug delivery system owing to their rapid uptake and clearance from circulation by cells of the mononuclear phagocytic system (MPS) located mainly in the liver and spleen [72, 73]. Biological stability of liposomes is dependent on the presence of agents such as proteins that interact with liposomes upon application to the subject and the administration route. There have been many strategies to enhance the biological stability of liposomes that improve the liposomal drug delivery in vivo and increase the circulation time in blood stream [74]. The complexation between polymers and liposomes has been studied as a way to increase the long-term stability of liposomes. Grafting hydrophilic polymers onto the head groups of phospholipids, or the addition of water soluble polymers containing several hydrophobic groups has been shown to increase the circulation time in vivo, as well as to inhibit liposome fusion [75-77]. These kinds of liposomes are called stealth liposome [78] or sterically stabilized liposomes [79]. The steric repulsion of these liposomes stabilizes the liposomes against aggregation. One of the most popular and successful methods to obtain long-circulating biologically stable liposomes is to coat the surface of the liposome with poly(ethylene glycol), PEG [80-84]. Although the PEG chemistry is successful in coating the liposome surface, alternative sterically protecting polymers are also under research. The candidate polymers should be biocompatible, soluble, hydrophilic and have highly flexible main chain for drug delivery. Some of these polymers given in the literature are synthetic polymers of vinyl series i.e. poly(vinyl pyrrolidone) (PVP) and poly(acrylamide) (PAA) [85, 86]. PVP has a similar history on pharmaceutical application to PEG [87, 88]. It shows high degree of biocompatibility and also acts as efficient steric protector for liposomes. It was found that the liposomal bilayers containing lipids with covalently attached to polyethylene glycol by which the membrane surface steric inhibits protein and cellular interactions with liposomes drastically prolonging the blood circulation time when injected in animals [89]. Doxil® is the liposomal doxorubicin available in the market which is stable for more than 18 months in the liquid state due to being stabilized by the usage of polyethylene glycol.
