Physicochemical characterization of natural phospholipid excipients and development of colloidal carrier systems for active substances (micro-emulsions) on the basis of phospholipids for dermal application
Prof. Dr. R. Neubert1), University Halle (Saale)/Germany and Prof. Dr. G. Brezesinski1), Max-Planck-Institute for Colloid and Interface Research/Potsdam/Germany
Felix Otto (PhD Fellow sponsored by the PRC)
Natural phospholipids are interesting excipients for formulation of macro- and microemulsion for dermatological and cosmetic use. Many natural phospholipids are available for topical formulations. Although the phospholipids are well described in terms of chemical structure and fatty acid composition, their physicochemical properties are not studied in detail to enable a more rational selection for design of topical formulations.
Colloidal systems, often described as microemulsions in literature, typically consist of oil, water, surfactant, and co-surfactant.1) Essential characteristics of these formulations are their transparent or slightly opalescent appearance, optical isotropy, and low viscosity. The formation of microemulsions occurs spontaneously, without any energy input. By forming a multitude of very fine droplets, the interface expands considerably. Substantially significant here is the sharp reduction of interfacial tension between the hydrophilic and lipophilic phases, often requiring the addition of a co-surfactant to the surfactant. The resulting emulsions are thermodynamically stable, form highly dynamic colloidal structures, which are in a state of constant collapse and reformation based on the high flexibility of their interfaces. Dependent on the mass ratio between lipophilic and hydrophilic components, microemulsions can be classified: "Bicontinuous structures" have approximately equal quantities of lipophilic and hydrophilic components; watery and oily domains co-exist and penetrate each other. More important though are "oil-in-water" (O/W) and "water-in-oil" (W/O) microemulsions. Their structural concept, based on macroemulsions and (invers) micellar systems, is shown in Figure 1. With 10 to 50 nm, the hydrodynamic radii of the associates of the inner phase are within the colloidal range.
Because of their excellent solubility and solubilization properties, microemulsions are gaining more and more importance as carrier systems for various “problematic” bio-pharmaceuticals as well as for cosmetic actives. The benefit of these carrier systems as a way of controlled drug delivery has already been proven in various studies for oral, parental, and dermal applications. Research about dermal application has a different approach. The transport of the active substances, that need to reach systemic bioavailability after absorption, must be studied separately from the topical treatment of the diseased skin area. It is necessary for the substances to reach the targeted skin layers with the intended concentrations. In this respect, colloidal carrier systems have been proven suitable, since they are able to temporarily lower the barrier function of the stratum corneum by interacting with the main lipids. The above-mentioned O/W microemulsions are predestined for the delivery of lipophilic active substances, while W/O systems are advantageous for the application of hydrophilic substances and cosmetic actives such as peptides. Limits for the wide use of microemulsions in dermatology and cosmetics are set by the high content of surfactants and their irritative potential. Another focus of this project was therefore the research regarding mild and dermal compatible detergents as well as tests to reduce the concentration of the surfactants (< 25 %) in the carrier systems.
The first part of the project comprises the physicochemical characterization of phospholipids from different vegetable sources and monoacyl-phospholipids using e.g. functionality tests, thermodynamic (DSC and ITC), structural (SAXD) and spectroscopic (FTIR, Raman) methods for this purpose. Based on these findings, macro- (O/W and W/O) and microemulsions have been formulated using the phospholipids investigated. A further focus was set on colloidal carrier systems for active substances (microemulsions) which are superior in terms of penetration of active substances compared to conventional formulations in topical therapy. Colloidal stability of the microemulsions has been optimized using as much as possible phospholipids which may be suitable as co-surfactants. The stable colloidal microemulsions using various phospholipids will be eventually studied for their penetration capacity for hydrophilic and lipophilic model actives/drugs.
Benefit for the community
The characterization of natural phospholipid (PL) mixtures enables interested scientists in industry and scientific institutions to use such PL as excipients in basic research, pharmacy and cosmetics.
Characterization of natural phospholipids to enable rational design of semisolid and colloidal formulations for active substances
PLs with a lower degree of purification regarding the PC amount exhibit a different behavior in forming mono- and bilayers. The stability of emulsions and the encapsulation efficacy of liposomes prepared with such PLs depend on the characteristics of the formed layers. Uncovering the physical-chemical characteristics of the PL layers is important for practical applications in different fields.
The influence of the amount of charged species in plant-based PL has been investigated in 3D (aqueous dispersions and liposomes) and 2D (Langmuir monolayers) model systems. PL mixtures with a higher amount of anionic species show a higher negative surface charge leading to reduced aggregation of droplets and vesicles. Another consequence of the surface charge is the repulsion between formed bilayers as SAXS data suggests. Further reason for the increase in the repeating distance can be a condensation effect in the hydrophobic part of the layer (transformation from gauche to all-trans) due to the additional mixture components. The lateral organization in Langmuir layers of the plant-based PLs leads to reduced area per gram as the content of charged species is increased. This indicates a higher packing density in the layer which might lead to an increased thickness of the hydrophobic region. The observed condensation leads to increased monolayer rigidity which is contributing to the physical stability of potential formulations because coagulation of droplets occurs due to emulsifier displacement and desorption along the interface. Some studies have already shown increased stability of PL membranes incorporating naturally occurring substances (e.g. cholesterol) compared to membranes consisting just of pure PC. It is likely that the composition of cell membranes with different types of PL is not only necessary for signal transduction but is also improving the stability of the bilayer.
However, the increased stability is limited to a smaller range of electrolyte concentration and pH value compared to pure phosphatidylcholine (PC). As several authors have shown, PC is forming the most stable preparations compared to other pure PLs such as phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI). This can be explained by the zwitterionic nature of the head group inducing stability over a wide pH range. Interestingly, no influence of the cation valency on the area per molecule has been found in the experiments with S 45, a soybean lecithin with 45% PC. Monolayers of pure PC were shown to condense in the presence of divalent cations such as calcium or magnesium. It is suggested that divalent cations induce a higher order in PC systems due to dehydration in the phosphate moiety of the head group. The presence of additional charged species in the layer interface seems to diminish this effect.
The good miscibility of the components in the less purified PLs leads to a homogenous layer. No phase separation could be detected in Brewster angle microscopy (BAM) experiments. However, it should be noted that the optical resolution of the used BAM is limited to about 2 μm. Therefore, partial de-mixing in small areas cannot be completely excluded. This behavior can be explained by the natural origin of the PLs favoring miscibility. Natural PLs have some minor drawbacks regarding the use as liposomes. Main problem to be solved are the unsaturated fatty acids leading to oxidative instability. Another issue is the possible phase separation caused by very different components in the mixture. As mentioned above, no proof of segregation could be found in all prepared layers.
For the use as emulsifying agent in semisolid formulations, plant-based PL offer some advantageous properties compared to pure PC. The natural occurring zeta potential prevents droplet coagulation due to electrostatic repulsion. The new insights found in our research can help to overcome the sparse use of PL as emulsifiers particularly in the light of the growing public skepticism towards aggressive synthetic and semisynthetic surfactants.
Furthermore, the hydrophilic-lipophilic-balance (HLB) value has been experimentally determined for plant-based PL. It could be shown that the HLB values of the PLs used are different depending on the PL content in the materials applied. The PL products having a high content of PL show similar HLB values independent on the plant source. In contrast, the PL products having a low content of PLs show fluctuating HLB values. These PL products can act as surfactants/emulsifiers over a wide range of the HLB scale. Therefore, it appears to be important to consider the entire curve which was used to estimate the HLB value of these PL products which are complex mixtures. Furthermore, it has been shown, using the hen's egg test on chorioallantoic membrane (HET-CAM test), that the plant-based PLs have no potential concerning skin irritation.
Microemulsion-based media as novel drug delivery systems
Adv. Drug Del. Rev. 45, 89-121
Physicochemical characterization of natural phospholipid excipients with varying PC content
Colloids Surf. A 558, 291-296