**4. Sterilization of liposomes**

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

12 Application of Nanotechnology in Drug Delivery

that polymer coated liposomes showed less stability than non-coated ones [65, 70].

polyethylene glycol.

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

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‐ mal drug carrier formulations.

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 sterilization of liposome formulations is a major challenge.


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

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].

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