Passive enclosure
As already mentioned, the first method for the enclosure of active substances in liposomes is the passive method. Hydrophilic drugs dissolved in a hydrating solution are assumed to be enclosed in the hydrophilic interior of the liposomes during the formation of vesicles, whereas hydrophobic substances added to the lipid film (or organic solution of lipids) are built into the bilayer during the formation of liposomes.
The lipid bilayer of liposomes is a natural barrier to many hydrophilic substances that are well-soluble in water (such as e.g. sugar, peptides, some antibiotics, etc.) so that they can be enclosed inside liposomes. The stability of the preparations obtained in this way depends largely on the lipid composition of the bilayer. One of the key parameters affecting the stability of the drug, i.e. its leakage from liposomes, is the fluidity of the lipid bilayer, which is largely dependent on the lipid composition. Lipids of which a bilayer is composed have an influence on the phase of order in which it is at a given temperature. Liposomes, which the membrane at low temperatures (up to 37 degrees C) occurs in the liquid crystalline phase, are characterised by a higher fluidity and permeability of the membrane for hydrophilic substances compared to the gel phase membrane. It has been shown that cholesterol addition to lipid composition of liposomes significantly reduces leakage of hydrophilic substances from their interior. Cholesterol reduces the possibility of free rotation of hydrocarbon chains of phospholipids, which in turn translates into bilayer molecular order, its fluidity and an increase in the phase transition temperature (Manojlovic et al., 2008). In case of some very hydrophilic drugs, such as e.g. cisplatin, addition of cholesterol to the binary layer was so effective in stopping the drug inside the liposomes that it could not be effectively released, which in turn resulted in a poor biological effect (Zamboni et al., 2004). Hydrophobic acyclic chains of phospholipids also have a very significant influence on the stability of liposome formulations. The length and degree of unsaturation of hydrocarbon chains affects the order of the membrane, which in turn translates into its liquidity, which in turn affects its barrier properties for substances contained inside liposomes. In general, the shorter and the more unsaturated the hydrocarbon chains are, the more liquid and permeable the lipid bilayer is, and the longer and more saturated the chains are, the more rigid and less permeable the membrane (Moghaddam et al., 2011).
Although the liposomal formulations of passively enclosed hydrophilic drugs are characterized by high stability in terms of leakage of the active substance from liposomes (and in addition, this parameter can be controlled in some way by means of lipid composition), they have one major disadvantage, namely they are characterized by low efficiency of enclosure of active substances. In the case of the simplest method of enclosing drug solutions in liposomes, namely the thin lipid film method, only about 5 to 20% of the substance is enclosed, which translates into a low drug to lipid ratio and significant losses of material (Governor, 2011). An important parameter influencing the amount of drug enclosed inside liposomes in a passive way is the volume of solution enclosed inside the vesicles. In order to increase it, it is necessary to reduce the lamellarity of liposomes in the first place, for which purpose a number of techniques are used (Xu, Khan, & Burgess, 2012). As described in section 1.2.1, FAT can be used to reduce the lamellar properties of liposomes and to increase the penetration of the drug into the liposomes. In his work Chapman et al. described a number of factors affecting the efficiency of the enclosure of active substances in liposomes, among which the FAT technique increased the efficiency of liposomal enclosure by approximately 10 to 50 times (Chapman, Erdahl, Taylor, & Pfeiffer, 1990). Another very important technique that can improve the enclosing efficiency of hydrophilic substances is the preparation of liposomes using REV method. As already described in the previous section, this method allows for the production of LUV-type liposomes with an enclosure efficiency of active substances of 60-65 % (Eloy J, 2014). In comparison with the FAT method, the REV technique allowed for statistically higher efficiency of drug enclosure in case of 5-fluorouracyl enclosure. However, the obtained liposomes were characterized by high heterogeneity in terms of size (Elorza, Elorza, Frutos, & Chantres, 1993). An undeniable disadvantage of the REV method is the residue of solvent used to dissolve lipids, which may discredit it for practical use (Governor, 2011). Dried reconstituted vesicles (DRV) is another method of increasing the efficiency of drug enclosure. This method allows for high drug/lipid ratio forumation by hydration of previously lyophilised liposomes immediately before use (Mugabe, Azghani, & Omri, 2006). In the case of vancomycin, this method allowed for the enclosure of a larger amount of the drug not only compared to the TFH method but also compared to the active enclosure method (Muppidi, Pumerantz, Wang, & Betageri, 2012). A parameter allowing to increase the enclosure efficiency of a substance may be the impact of a loaded membrane with a drug having a different load. This relationship was demonstrated, inter alia, for gancyklovir, where stearylamine-containing EPC:Chol liposomes had a significantly higher enclosure efficiency compared to unloaded liposomes (Kajivara et al., 2007).
In summary, passive drug enclosure inside liposomes is characterized by an unsatisfactory enclosing efficiency and a weak drug/lipid ratio. This makes passive enclosure a far cry from ideal, which has forced scientists to develop more effective enclosure methods for active substances.
Active enclosure
The use of active drug enclosure (loading) inside the liposomes meets the problems related to passive enclosure and allows to obtain stable liposome formulations characterized by a very high enclosure efficiency and a very good drug/lipid ratio. This method was described for the first time by Deamer and his colleagues who described the active enclosure of catecholamines inside liposomes (Nichols J. & Deamer D., 1976). The difference in pH on both sides of the lipid bilayer is a driving force in this method and can be generated either by raising the pH of the external buffer using the alkali or by a two-step process in which liposomes are prepared in a solution of a certain pH in the first step and an external solution is replaced in the second step (e.g. by gel filtration) by a buffer with different pH (Ishida, Takanashi, Doi, Yamamoto, & Kiwada, 2002; Lipka et al., 2013). The active drug loading method is based on the phenomenon of different permeability of a bilayer for lipophilic molecules depending on their ionization state. For weak alkalis in neutral pH, unloaded drug particles can diffuse freely across the lipid bilayer, while in the middle of the vesicle they are protonated at low pH, which prevents their leakage to the outside. For this process to occur, the active substances must meet certain structural requirements, such as: logP value at pH 7 should be in the range -2.5 to 2.0, and pKa ≤11 and the compound must be at least slightly soluble in water (Clerc & Barenholz, 1998; Zucker, Marcus, Barenholz, & Goldblum, 2009). As mentioned, such conditions are met, inter alia, by weak organic alcalis. These compounds at pH ÷ 7 occur both in the charged (protonated) and in neutral state, while with the decrease in pH the equilibrium starts to move towards charged particles, and in the case of an increase in pH towards neutral particles. For many weak alkalis, already at pH 4.0 most particles are charged, which means that they cannot diffuse freely across the membrane. Most anthracyclines, which are weak alkalis in aqueous solutions, occur in a balance between the protonated and the neutral form, and only the neutral form can penetrate the lipid bilayer. When it enters the interior of liposms at low pH, it is "trapped" inside, wherein on the outer side of the liposome, the balance of anthracycline dissociation is shifted towards a neutral form which, as a result of free flow across the bilayer, is being accumulated in the liposomes.
The first method for active drug enclosure, developed by Bally and his collaborators, was based on a gradient of 300 mM citrate buffer replaced by HEPES buffer with pH 7.4 (Bally et al., 1988; Mayer, Bally, Hope, & Cullis, 1985). This method allowed for accumulation of weak amine alkalis in liposomes, such as antibiotics from the anthracycline group such as doxorubicin, daunorubicin or idarubicin with a very high efficiency (EE was almost 100%) with a very favourable drug/lipid ratio (0.3 wt/wt). In addition, it has been observed that in the case of anthracyclines in the liposomes, the structures of poorly soluble drug citrate salts are formed, which give the liposomes a shape similar to the coffee beans ( coffee bean liposomes). The obtained liposome formulations were characterized by high in vitro stability, especially when high temperature phase transition lipid such as disterylphosphatidylcholine (DSPC) or hydrogenated lecithin (HSPC) was used in forming of liposomes. Citrate gradient has been successfully used in commercial liposome-based preparations such as Myocet® (Enzon Pharamaceuticals) containing Doxorubicin and DaunoXome® (Nextar Pharamaceuticals) (Batist, Barton, Chaikin, Swenson, & Welles, 2002; Forssen, 1997).
The next milestone associated with the active enclosure of drugs inside liposomes was the development by Barenholz of a method based on ammonium sulphate gradient. The driving force behind the enclosure of weak alkalis in this method is the difference in ammonium sulphate concentrations across the lipid bilayer. In practice, this means that there is a 300 mM ammonium sulphate solution with a pH of 5.5 inside the liposomes and a buffer with a pH of approximately 7.4 outside the liposomes. Ammonia molecules formed by ammonium ion hydrolysis can diffuse freely across the lipid bilayer according to the concentration gradient, leaving the proton that generates the pH gradient acidifying the environment inside the liposomes (the lipid bilayer is practically impermeable to sulphate ions and hydrogen ions see Fig. 1.7) The gradient generated in this way allows for very efficient enclosure of anthracyclines, and the efficiency of this process is proportional to the ratio of ammonium ion concentration outside liposomes to ammonium ion concentration inside liposomes [NH4+]outs./[NH4+]ins. Leakage of ammonia from liposomes causes acidification of their internal environment, which in turn shifts the hydrolysis reaction to ammonia to ammonium ions. On the other hand, penetration of weak alkalis into the liposomes without a load causes their protonation and an increase in pH, which in turn increases the pH inside the liposomes by shifting the balance of the reaction of hydrolysis of ammonium sulphate towards ammonia. Thanks to this phenomenon, this method allows for enclosure at a very high drug efficiency (EE>95%) with a drug/lipid ratio even 0.3 or even 0.4. The unquestionable advantage of this method is that there is no need to produce liposomes at low pH thanks to an intrinsically generated gradient (Haran, Cohen, Bar, & Barenholz, 1993). In this method, the drug is enclosed inside the liposomes not only because it is protonated, but also because a poorly solvable complex between sulphate and anthracyclines is formed. This process is schematically presented in Fig. 1.8. In the case of doxorubicin, the complex formed is approximately three times less soluble than the corresponding complex with citrate in the pH range approximately 4.0 to 7.5 (Fritze, Hens, Kimpfler, Schubert, & Peschka-Suss, 2006). Ammonium sulphate gradient doxorubicin formulations are characterized by excellent stability both in vitro and in vivo and their pharmacokinetic and pharmacodynamic parameters are incomparably better than those of a free drug. Moreover, the method of drug active enclosure based on ammonium sulphate allowed to develop Doxil® the first drug enclosed in nanoliposomes approved by FDA. The use of liposomes as a carrier in Doxil® allowed the development of a preparation with significantly lower side effects, such as, inter alia, cardiotoxicity in comparison with a free drug due to the use of passive direction resulting from the EPR effect (Barenholz, 2012). Unfortunately, apart from the reduction of side effects, Doxil® did not meet the expectations of increasing the therapeutic effect. Probably the biggest advantage of Doxil®, that is its stability, is also partly its disadvantage because it causes low availability of the drug. In his work on the history of Doxil's development, Barenholz postulates the thesis that only a monomeric drug interacts with DNA, whereas the release of a free drug from liposomes, in which it forms a poorly soluble complex, may be too slow, which unfortunately affects its availability and activity (Barenholz, 2012).
Active enclosure method of doxorubicin based on ammonium sulphate gradient. 2D-NHCl - doxorubicin hydrochloride; D-NH+ - protonated doxorubicin; D-N - neutral doxorubicin; (D-NH2)SO4 - poorly soluble complex of doxorubicin and ammonium sulphate