People involved
M.Sc. Katja Steiner (PhD fellow sponsored by the PRC) - University of Vienna, Austria
Abstract
In our project, we hypothesize that due to the known biocompatibility of phospholipid derivatives and their lack of groups with distinct photoactivity, phospholipids could be useful for the stabilization of fluid sunscreen emulsions. Since no literature data on this topic exist, we aim to evaluate the behavior of different lecithin-based emulsifiers in aqueous dispersion and fluid oil-in-water emulsions under photo-stress. We aim to compare their behavior to conventional anionic and nonionic surfactants currently used in marketed products (negative control) and a known photosensitizer (for example, diclofenac sodium as positive control) to exclude any phototoxic or irritation potential.
To this end, we will employ different established methods. In vitro phototoxicity studies will be conducted using human primary cells.{{1}} Ex vivo diffusion cell studies and biophysical analysis of skin parameters, for example by confocal Raman spectroscopy, will serve to evaluate the effect of different test compounds and UV-A on skin barrier function. In vivo studies on human volunteers will be conducted, and corneocyte morphology after treatment and UVA irradiation will be conducted using atomic force microscopy.{{2}} The obtained knowledge will be relevant for the research community focusing on formulation development in the pharmaceutical and cosmetic sector and may contribute to the development of sunscreen formulations based on sustainable raw materials.
Benefit for the community
Dermal preparations based on phospholipids have many applications. To successfully establish their use in sustainable sunscreens, knowledge of their behavior under photo-stress is essential and reference values to other common surfactants are of high interest to enable an informed choice in product development both in academia and industry.
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People involved
B.Sc. Philipp Toplak (master student sponsored by the PRC) - University of Graz, Austria
Abstract
Nanoemulsions (especially o/w nanoemulsions) are a very interesting field in pharmaceutics due to their diverse applications. Besides their application in parenteral nutrition and use as a matrix for lipophilic active pharmaceutical ingredients (APIs), they can be used to modify the pharmacokinetics and the biodistribution of their affiliated drugs, or to prolong the release of a drug in form of a sustained release system, or both. When biodistribution is specifically changed, the term ''targeted drug delivery systems'' is used. Therefore, a ligand/receptor interaction at the cells to be affected and a carrier containing the actual drug substance is required. However, due to stability problems of o/w nanoemulsions and the complexity in analysis of the different production steps their application as pharmaceuticals has been rather challenging.
To promote the use of targeted drug delivery systems, it would be of great advantage to develop a universal synthesis scheme to produce stable, ligand-coupled (e.g. peptide-coupled) o/w nanoemulsions. However, by now research is focusing either on the PEGylation of peptides with varying PEG chain lengths and with molecular weights ranging from 5-50 kDa, or the use of o/w nanoemulsions as matrices for lipophilic drug substances.
This project is about the production of stable o/w nanoemulsions using different egg-lecithins in order to find the most suitable one for this approach, the synthesis of long-acting DSPE-PEG(2000) Maleimide-coupled peptides, and the combination of both. Furthermore, each synthesis step is analyzed with different analytical methods (HPLC, SEC, NMR, MALDI-TOF, laser diffraction, dynamic light scattering, zeta potential) to document the changes in the molecules and drug delivery systems. To demonstrate the functionality of the targeted drug delivery system, cell studies using 3T3-L1 pre-adipocytes will be performed. This knowledge will thus be of great interest, e.g., to the pharmaceutical industry and pharmaceutical scientists as inspiration for novel synthetic strategies and the development of various targeted drug delivery systems using peptide-coupled o/w nanoemulsions.
Benefit for the community
With the help of this project, lipophilic and/or lipophilized active pharmaceutical ingredients could be incorporated into the oil droplets of the o/w nanoemulsions and then released directly into the desired tissue by targeted drug delivery. For example, the lipophilized uncoupling protein 3 (UCP3), which intervenes in the lipid metabolism, could be used for targeted action on adipocytes. While the systemic administration of UCP3 sometimes leads to severe side effects in areas of the body other than the adipocytes, adverse reactions could be reduced or prevented by peptide-coupled drug targeting.
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People involved
Kirsten Ullmann (PhD fellow sponsored by the PRC) - KIT, Germany (kirsten.ullmann@kit.edu)
Dr. Gero Leneweit - Carl Gustav Carus-Institute, Germany
Abstract
While the previous project [Production of liposomes by centrifugation of water in oil emulsions] addressed the characterization of the applied substances to produce liposomes from a water-in-oil (w/o) nano-emulsion, primary tensiometry and nano-emulsions,{{1}}{{2}}{{3}}{{4}}{{5}}{{6}} this research project focuses on the optimization of liposome production process.
The proof of concept of the flotation of aqueous droplets from a w/o nano-emulsion to an aqueous phase by centrifugation shows advantages in comparison to known liposome production methods: the use of solvents is redundant, and the encapsulation efficiency of hydrophilic model substances is higher than anything described in literature so far, with the potential to approach complete encapsulation. The feasibility of encapsulating several model substances will be investigated and the production process by addressing the influence of phospholipids and the utilized hydrophobic phase on the encapsulation efficiency will be evaluated. Additionally, the determination of the liposome size and stability after centrifugation and release of encapsulated substance is investigated including the influence of an isotonic buffer on the stability. As the development is driven by engineering aspects, process parameters such as the influence of temperature and centrifugal force are examined. The key elements of this study bear a strong potential for large scale industrial applications.
Benefit for the community
The following benefits are expected for researchers in the field of phospholipid science, industry and society:
- Determination and quantification of successful encapsulation of macromolecular model API, especially proteins and RNA, by establishing a method based on spectroscopy.
- Based on specific characteristics of the encapsulated substances, API and PL, recommendations about the possible interactions and required process parameters for optimized liposome production are given.
- Industrial application for a liposomal encapsulation with a successful method that is controllable.
- Novel drug delivery systems can open completely new application routes for emerging API classes such as RNA and proteins, offering chances for novel treatments of life-threatening diseases.
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People involved
Fabio Strati (PhD fellow sponsored by the PRC) – IADP Halle, Germany (fabio.strati@iadp.eu)
Abstract
Natural plant-based phospholipids (PLs) are interesting excipients for the formulation of macro- and micro-emulsions (ME) for dermatological and cosmetic use. Many natural PLs are available for topical formulations. Although the PLs are well described in terms of head group and fatty acid composition, their physical-chemical properties are not described in detail to enable a more rational selection for the effective design of topical formulations.
This project will characterize plant based hydrogenated PLs for dermato-pharmaceutical and cosmetic use.{{1}}{{2}} PLs from different plant sources as well as monoacyl-phosphatidylcholine (monoacyl-PC) will be physically-chemically characterized in selected model systems (2D monolayers, 3D dispersions, liposomes, emulsions) using HLD (hydrophilic-lipophilic deviation) and CCP (critical packaging parameter), solubility studies as well as thermodynamic (DSC and ITC), structural (SAXS, GIXD), and spectroscopic (TRXF, FTIR, Raman) methods for this purpose.
Benefit for the community
The hydrogenated phospholipid preparations can be developed to valuable excipients/surfactants for use in pharmacy and cosmetics.
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People involved
Mag. Pharm. Claudia Vater (PhD fellow sponsored by the PRC) - Department of Pharmaceutical Technology and Biopharmacy, University of Vienna, Austria (claudia.vater@univie.ac.at)
Abstract
Phospholipid-based formulations have gained major interest in the field of topical drug delivery due to their physical-chemical properties. As amphiphilic constituents of cellular membranes, phospholipids exhibit both high biocompatibility and great emulsifying power. Thus, plentiful phospholipid-based formulations and drug carriers such as liposomes, solid-lipid nanoparticles (SLNs), micelles, and organogels have been developed and investigated by different research groups.{{1}}{{2}}{{3}}{{4}}{{5}}{{6}}
In the project, a range of phospholipids will be used to prepare different formulations. Importantly, the focus of this project is the evaluation of the employed phospholipids and the developed formulations on the cell viability of viable human keratinocytes and fibroblasts as determined by cell culture toxicity tests. To this end, a specific dermal cell culture line will be established in the laboratory. In addition, the effect of the formulations on skin penetration and permeation of incorporated model drugs will be determined.
Benefit for the community
The results will contribute to the general knowledge in the field of cytotoxicity of dermal formulations and especially of phospholipids. Moreover, feedback will be collected from colleagues within this and related fields of research and this interdisciplinary exchange will stimulate new creative approaches towards scientific use of natural phospholipids in dermal formulations as well as for natural cosmetics.
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People involved
Kirsten Ullmann (PhD fellow sponsored by the PRC) – KIT, Germany (kirsten.ullmann@kit.edu)
Dr. Gero Leneweit – Carl Gustav Carus-Institute, Germany (gero.leneweit@carus-institut.de)
Abstract
Liposomes are promising carriers for active pharmaceutical ingredients (API) and can be used for multiple purpose in drug targeting, reductions of adverse effects, and diagnostics. The production of liposomes with high encapsulation efficiency for high molecular weight API, which are sensitive towards heat and aggressive conditions of pH, organic solvents or surfactants, are an unmet need of increasing interest. Novel processes using centrifugation of water in oil (w/o) emulsions were proposed, based on the use of phospholipids as emulsifiers, avoiding the use of aggressive treatments and allowing asymmetric membrane functionalities. However, severe limitations regarding emulsion stability and phase transfer of liquid droplets do not allow an industrial application so far. To enable industrial liposome production for a broad class of hydrophilic API, innovative processes are to be developed for the engineering and the stability of phospholipid w/o emulsions.{{1}}{{2}}{{3}}{{4}}{{5}}{{6}}
We propose to explore the feasibility of this innovative liposome production process, starting from the interfacial and molecular behavior of phospholipids, reaching to w/o emulsion stability and lipid gels, all in perspective to progressively develop a successful phase transfer and promote liposome production for pharmaceutical applications. The interfacial and molecular behavior of phospholipids of different head groups and fatty acids will be studied by tensiometry. w/o emulsion stability and the formation or avoidance of lipid gels will be tested by an analytical centrifuge, nuclear magnetic resonance (NMR) and photon correlation spectroscopy (PCS). The key elements of the proposed study cover fields which were hardly explored for phospholipids so far but bear a strong potential for large-scale application in pharmaceutical engineering.
Benefit for the community
The following benefits are expected for the scientific community of phospholipid research, industrial users and for society in the field of health care:
- Protocols to produce nanometer-sized w/o emulsions with pharmaceutical excipients including tested short and long-term stability, e. growth rates. The screening of phospholipid headgroups and fatty acids and experiences about natural phospholipid blends will enable industrial users to select phospholipids according to their physicochemical characteristics and the requirements of the application.
- Exploring the phase transfer process of w/o emulsions in a centrifugal system with in-situ monitoring will elucidate ongoing research activities of many research groups for new production processes of liposomes. High encapsulation efficiency is a key requirement for the advancement of liposomal drug vehicles and their expanded application in the pharmaceutical industry and will also trigger new research projects.
- The potential to design asymmetric lipid bilayers makes the process attractive and versatile for other scientific and technical applications on smaller or larger scales.
- Increasing knowledge about phospholipid/solvent interactions and their effect on w/o emulsion stability and gel formation will support the design of new products involving phospholipids.
Results/Outcome
The project started with the investigations of tensiometric behavior of different phospholipids as interfacial phenomena have a large influence on the final phase transfer for liposome production. The oil phase was carefully selected to avoid the formation of a gel phase at the interface as well as to meet the requirements for a pharmaceutical applicable substance. Fluorocarbons have recently been in the focus of researchers because of their inert characteristics which makes them good candidates for pharmaceuticals. By changing the hydrophobic phase from an organic oil (squalene) to a fluorocarbon, interfacial behavior had to be investigated as there are not many data on interfacial tensions between a fluorocarbon and a phospholipid suspension. With the fluorocarbon new challenges appeared – optical methods such as profile analysis tensiometry were unsuitable because of similar refractive index of both phases. Hence, during the first year the suitability of the Du Noüy ring was studied and the results compared to a second method, the spinning drop tensiometry. We were able to show that the Du Noüy ring measures interfacial tensions with equal precision as the spinning drop and presented tensiometric data between a fluorocarbon and phospholipid suspensions consisting of phospholipid with different chain lengths. Shorter chain lengths reduce the interfacial tensions further. We additionally investigated the influence of temperature on the interfacial behavior, as temperature may also have an influence on the transfer of droplets. Results reveal that above the respective transition temperature of the phospholipid interfacial tensions of different phodpholipids become equal.
During year two and three, droplet size and stabilization of w/o nano-emulsions prepared with different phospholipids (head groups, chain length, natural blends) were investigated. With the fluorocarbon as the hydrophobic phase, phospholipids were dispersed in the aqueous phase. Nano-emulsions were stable for several weeks with an average droplet size of 200 nm. Higher concentrations of phospholipids lead to smaller droplets, head groups have, except for DPPS, no significant influence on the droplet size. Furthermore, influencing effects such as gas bubbles in the hydrophobic phase were examined.
With the change of the hydrophobic phase to the fluorocarbon, a breakthrough could be achieved. In addition to minor changes of the transfer process, liposomes could be produced. We further characterized these liposomes using dynamic light scattering (DLS) and small-angle x-ray scattering (SAXS) for size determination. First experiments regarding the encapsulation efficiency were carried out and show that high encapsulation of up to 86 % is possible with the proposed centrifugation method.
To summarize, the process of liposome production was successfully established by systematically analyzing interfacial behavior between the chosen hydrophobic and hydrophilic phases, using the gained information about the area per molecule to produce nano-emulsion with the needed amount of phospholipids for stabilization. From characterization of nano-emulsions, the transfer of droplets led to the production of liposomes. A model substance was used for encapsulation and revealed an encapsulation efficiency of up to 86 %.
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People involved
Felix Otto (PhD Fellow sponsored by the PRC)
Abstract
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.
Figure 1. Structural concept for microemulsions.
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.
Results/Outcome
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 ÎĽm. 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.