Technology - Synthos CARE

Power of liposomes

Liposomes are structures which are spontaneously formed from phospholipids. These are in the form of vesicles sized 0.01-1 μm, filled with water (or an aqueous solution) and surrounded by a double lipid layer with a thickness of approx. 5 nm. The liposome envelope has the analogical structure of biological membranes. Liposomes are found in living organisms, e.g. in blood and are produced industrially.

Unique application

Artificially produced liposomes are used mainly in the pharmaceutical and cosmetic industries, as well as in scientific research, as models for biological membranes. Aqueous solutions or suspensions of various substances can be placed inside the liposomes, including medications or nucleic acids. This feature facilitates the use of liposomes as drug carriers, and the Synthos CARE research team is developing advanced systems of active substance carriers dedicated to cosmetics, dietary supplements and pharmaceuticals. We develop, manufacture, and sell advanced phospholipid carriers, which are revolutionising the effectiveness of supplying active substances to the human body.

In our modern laboratory we have created 100% biocompatible, biodegradable innovative carrier, which effectively improves the properties of the encapsulated substances - VECTICELL.

Our contribution to the development of the industry

VECTICELL is a modern system of active substance supply whose structure is based on phospholipids. Its high effectiveness is guaranteed by three features:

Lipid composition - VECTICELL carriers are made of pharmaceutical-standard phosphatidylcholine, thanks to which they perfectly build into cell membranes. It is a fully biodegradable and biocompatible molecule.
Flexible nano-carrier membrane – this double or triple encapsulation system, involving the covering of carrier structures with compounds regulating the flexibility of the lipid layer, guarantees high permeability between the cellular spaces,
Homogeneous Size - VECTICELL carrier calibration technology gives them a replicable and programmable size which has a direct effect on the increased effectiveness of reaching the skin cells.

Added value

VECTICELL is resistant to technological processes - homogenisation, detergent action, and high temperature. Tests confirm the high stability of VECTICELL in the formulation - the carriers are visible in a ready-made cream.

The use of VECTICELL creates the added value in the technological process:

As an aqueous solution with a high concentration of active substances, it is 100% miscible in water.
It can be added in the final phase of cold mixing of the cosmetic formulation, because the substances contained in it are not degradable.
The polydispersity of VECTICELL does not exceed 0.3 PDI, guaranteeing the replicability of the series.
No aggressive solvents, ethanol or parabens are used in the production of VECTICELL.
The entire production process is carried out under conditions in accordance with ISO 22716.

Additional information about liposomes

Liposoms
Methods for obtaining liposomes
Methods for the closure of active substances in liposomes

Liposomes were first described in England in 1960s by Bangham, who investigated the effect of phospholipids on blood clotting. He observed that in an aqueous solution phospholipids spontaneously form multilamaler vesicle-like structures that contain inside the aqueous solutions. He called the resulting structures liposomes (A. D. Bangham, Standish, & Watkins, 1965).

Lipid vesicle formation (liposomes) from phosphatidylcholine.

Liposomes are now defined as artificially obtained microscopic lipid vesicles made up of one or more lipid bilayers that contain an aqueous solution inside and that are mainly made up of phospholipids. The formation of liposomes in aqueous solutions is mainly due to the hydrophobic effect which organises the amphiphilic phospholipids in such a way as to minimise the entropic effect of hydrophobic fat chains on the surrounding aquatic environment. Additionally, this effect is stabilized by a number of electrostatic interactions, hydrogen bonds or Van der Waals interactions (Lasic & Papahadjopoulos, 1995). Due to its structure, liposomes can be used both as carriers for hydrophilic substances (which can be closed in a hydrophilic interior) and as hydrophobic in a lipid bilayer.

Schematic structure of liposome.

Liposomes can be classified according to their size and lamellarity (i.e. the number of bilayers of which liposomes are composed). Both of these parameters are crucial when we talk about liposomes as carriers of active substances, because they are responsible for the time of circulation of vesicles in the bloodstream and the amount and availability of the drug. The classification of liposomes in terms of size and lamellarity is as follows:
Small unilamellar vesicles - SUV :20-100nm
Large unilamellar vesicles - LUV :>100 nm
Giant unilamellar vesicles - GUV : >1000 nm
Mandarin oligolamellar vesicles - OLV : 100-500 nm
Multilamaler vesicles - MLV : >500 nm
In 2004, multivesicular liposomes (MVV multivesicular vesicles) were additionally described (Grant et al., 2004).

Classification of liposomes by size (Laouini A. et al., 2012).

As already mentioned, the main building material of liposomes are natural and/or synthetic/semi-synthetic phospholipids such as phosphatidylcholine, phosphatidyl ethanolamine or phosphatidilic glycerol. Distearylphosphatidylcholine and distearylphosphatidyl ethanylamine and their derivatives are currently among the most commonly used phospholipids in liposomal technologies. In addition to phospholipids, the liposome membrane may also contain other auxiliary lipids, e.g. cholesterol which affects the properties of the lipid bilayer by modulating its fluidity on the one hand and by reducing the permeability of the bilayer to water-soluble substances on the other hand. A number of polymer-lipid conjugates are also used to modify the properties of liposomes.

At present, there are many methods of producing liposomes which differ mainly in the way lipids are being deprived of organic solvent and in the way they are subsequently dispersed in an aqueous medium. The most important methods of obtaining liposomes are described below.

1) Thin Film Hydratation (TFH) method: this method is the oldest one to describe the production of liposomes and was originally used by Bangham for the first time. This method involves evaporation under vacuum (usually by means of a vacuum evaporator) of an organic solvent in which lipids were dissolved until a dry lipid film is obtained. The resulting lipid film is then hydrated with an aqueous solution, stirring the contents of the vessel at a temperature higher than the phase transition temperature of the lipids used (A.D. Bangham, de Gier, & Greville, 1967). This method is relatively simple and widely used; its biggest disadvantage is the production of liposomes (MLV) characterized by a very high polydispersion. Therefore, liposomes obtained by this technique often undergo subsequent treatment in order to reduce their size and polydispersion. The most commonly used methods to obtain smaller liposomes are sonication (to obtain SUV) or extrusion of MLV by polycarbonate filters to obtain LUV liposomes characterised by very low polydispersity (Olson F, 1979). In addition (in combination with extrusion) the use of the FAT (freeze and thaw) technique has been introduced, which depends on fast freezing of a suspension of liposomes, e.g. in liquid nitrogen, and then unfreezing them at a temperature of approx. 60ᵒC, which allows an increase in the water volume of liposomes and a reduction in the number of lipid layers. (Costa, Xu, & Burgess, 2014).

2) Reverse-phase Evaporation (REV) method: this method binds a solution of lipids dissolved in an organic solvent in an aqueous solution and then evaporates the organic solvent. This procedure allows to obtain LUV and OLV liposomes. The obtained liposomes are characterized by a large volume of aqueous solution enclosed (Laouini A et al., 2012).

3) Method of injecting the ethanol or ether solution: this method involves injecting an ethanol or ether solution of lipids into an aqueous solution, resulting in the formation of lipid vesicles. The method of injection of the ethanol solution was described in 1973 and allows to obtain liposomes characterized by a very low polydispersion and size of about 100 nm without any processing (Stano et al., 2004). The method of injecting the ether lipid solution differs from the method of injecting the ethanol solution in that the ether is not mixed with water and evaporates from it. This method involves injecting the lipid ether solution into a hot aqueous solution at a temperature higher than the boiling point of the ether, resulting in evaporation of the ether and the formation of unilamellar vesicles. The advantage of this method is that it gets rid of an organic solvent compared to an ethanol-based method, which enables a continuous process to be carried out and a concentrated liposomal solution to be obtained (Laouini A. et al., 2012). A number of new methods based on the ethanol injection method have now been developed, e.g. cross-flow injection technique or microfluidisation. In the cross-flow injection technique, Wagner et al. described how liposomes for pharmaceutical use were produced. This method is based on a cross linking of the lipid ethanol solution and an aqueous solution and continuous mixing of the two in order to obtain liposomes (Wagner .A, 2006). The microfluidisation was described by Jahn et al.; this method consists of injecting via microchannels of an aqueous and lipidic phase. By controlling the flow of both phases in the microchannels, liposomes of different sizes can be obtained.

4) Detergent dialysis method: this method makes it possible to obtain large unilamellar liposomes. In the first phase, mixed micelles consisting of detergent and phospholipids are formed. The detergent is then gradually removed from the solution by dialysis and the phospholipids form homogeneous liposomes. The disadvantage of this method is that liposomes are undoubtedly contaminated by detergent residues.

Liposomes are now one of the leading injectable drug delivery systems, and thanks to their design they can contain both hydrophilic substances in a hydrophilic water interior and hydrophobic substances in a hydrophobic phospholipid bilayer (see Fig. 1.5.). Generally, active substances inside liposomes can be enclosed in two ways: the first one, called passive, allows for encapsulation of hydrophilic and hydrophobic substances during the formation of lipid vesicles and the second one, called active, allows for enclosure of lipophilic substances containing weak ionizable groups in their chemical structure, such as e.g. amino or carboxyl group (Governor, 2011). The method of enclosing directly influences the properties of the preparation obtained, such as its stability, enclosing efficiency (EE) or drug/lipid ratio (D/L). All these parameters directly affect the pharmacokinetics of the drug, its stability and bioavailability, and thus the therapeutic effect of its formulation.

Methods of enclosure of active substances in liposomes

[Translate to English:] Zamykanie substancji wykres

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

In addition to ammonium sulphate, in 2006 Fritze et al. also studied the effect of other ammonium salts such as citrate, acetate and phosphoric acid on the enclosure and release of doxorubicin from liposomes. It showed that not only ammonium sulphate but also other ammonium salts allow for efficient enclosure of the drug with high efficiency (EE in case of using citrate = ~100%, phosphate 98.5%, sulphate 95% and acetate 77%). A very important achievement was also the demonstration that in the case of a drug enclosed with ammonium phosphate at low pH (5.5), the release of doxorubicin is significantly faster than in the case of formulation with ammonium sulphate. The solubility of the phosphate-doxorubicin complex at pH 5.0 was about twice as high as in the case of the sulphate-doxorubicin complex, which may indicate that there is a relationship between the availability of the drug enclosed in liposomes and its solubility. It is worth noting that ammonium phosphate formulations were characterized by very high stability during storage, which indicates that the drug leakage was stimulated in this case by a pH change (Fritze et al., 2006).

Another way to enclose weak alkalis in liposomes was to use transition metal ions gradient, i.e. sulphates and chlorides of manganese or copper. Abrahama et al. in 2004, a gradient of sulphate and manganese chloride in combination with a pH gradient with the previously described methods or with the method of citrate gradient and ammonium sulphate enclosing the topotekan in liposomes. All methods ensured drug accumulation in liposomes, however, in the case of a gradient based on manganese ions, a lower enclosure efficiency was observed, especially at a high drug:lipid ratio. Using a gradient based on copper ions in the form of sulphate and chloride salt Li et al. It enclosed myctoxantron, a highly hydrophilic anthracycline, in liposomes. The developed method allowed to enclose the drug with high efficiency, but the formulation based on copper (II) chloride was characterized by lower in vitro stability in comparison to the formulation based on copper (II) sulphate. What is very interesting, despite lower stability, the chloride based formulation was characterized by higher therapeutic activity, which was most probably related to the physical condition of the drug inside the liposomes. It is also interesting that in the case of mitoxantron no poorly soluble residue was observed inside liposomes and despite that the formulations were characterized by high stability (C. Li et al., 2008).

For low lipophilic anthroclines such as mitoxantron or medium lipophilic anthroclines such as, for example, doxorubicin or epirubicin, it appears that the formation of poorly soluble salts when enclosing the drug inside liposomes is not necessary and on the contrary it can sometimes reduce its bioavailability. In the case of the above mentioned anthracyclines, the lipid membrane itself, characterized by the high temperature phase transition, is a significant barrier for the drug. However, in the case of idarubicin, which is a strongly lipophilic anthracycline, the situation is quite the opposite. The use of a gradient of ammonium or citrate sulphate results in a very rapid accumulation of the drug inside the liposomes even below the phase transition temperature of the lipid membrane and, on the other hand, in a very rapid leak of the drug from the liposomes (Governor et al., 2010; Slifirski, Szelejewski, Grynkiewicz, Governor, & Kozubek, 2003). Additionally, interactions of the drug with cholesterol accelerate its leakage, destabilizing the whole system. Santos et al. developed Idarubicin formulations enclosed in liposomes that did not contain cholesterol and with less pegylated phosphatidylethanolamine, thus obtaining a formulation that achieves better pharmacokinetic properties than a formulation containing cholesterol, (Dos Santos et al., 2002). An innovative solution to obtain stable liposomal formulation of idarubicin was proposed by Governor et al. Using a gradient of ammonium edetate. The formula developed was based on the formation of hardly soluble salts between EDTA and idarubicin, much less soluble than sulphate or citrate salts. The formula developed was characterized not only by high in vitro stability, but also by significantly better pharmacokinetic parameters in comparison to the formula based on cholesterol-free liposomes (Governor et al., 2010). Edetate gradient has also been successfully used in less lipophilic anthroclines, such as epirubicin, obtaining stable formulations both in vitro and in vivo and with a very beneficial therapeutic effect (Governor et al., 2014).