By direct deposition of the drug at the local site of action, injectable depot formulations – intended for treatment of a local disease or for local intervention – are designed to limit the immediate exposure of the active principle at a systemic level and to reduce the frequency of administration. To overcome known drawbacks in the production of some marketed phospholipid-based depots, here we propose to manufacture drug-loaded negatively charged liposomes through conventional technologies and to control their aggregation mixing a solution of divalent cations prior to administration. We identified phosphatidylglycerol (PG) as the most suitable phospholipid for controlled aggregation of the liposomes and to modulate the release of the anesthetic bupivacaine (BUP) from liposomal depots. In vivo imaging of the fluorescently-labelled liposomes showed a significantly higher retention of the PG liposomes at the injection site with respect to zwitterionic ones. In situ mixing of PG liposomes with calcium salts significantly extended the area under the curve of BUP in plasma compared to the non-depot system. Overall, controlling the aggregation of negatively charged liposomes with divalent cations not only modulated the particle clearance from the injection site but also the release in vivo of a small amphipathic drug such as BUP.

Alternatively-activated, M2-like tumor-associated macrophages (TAM) strongly contribute to tumor growth, invasiveness and metastasis. Technologies to disable the pro-tumorigenic function of these TAMs are of high interest to immunotherapy research. Here we show that by designing engineered nanoliposomes bio-mimicking peroxidated phospholipids that are recognised and internalised by scavenger receptors, TAMs can be targeted. Incorporation of phospholipids possessing a terminal carboxylate group at the sn-2 position into nanoliposome bilayers drives their uptake by M2 macrophages with high specificity. Molecular dynamics simulation of the lipid bilayer predicts flipping of the sn-2 tail towards the aqueous phase, while molecular docking data indicates interaction of the tail with Scavenger Receptor Class B type 1 (SR-B1). In vivo, the engineered nanoliposomes are distributed specifically to M2-like macrophages and, upon delivery of the STAT6 inhibitor (AS1517499), zoledronic acid or muramyl tripeptide, these cells promote reduction of the premetastatic niche and/or tumor growth. Altogether, we demonstrate the efficiency and versatility of our engineered “tail-flipping” nanoliposomes in a pre-clinical model, which paves the way to their development as cancer immunotherapeutics in humans.

Although the incorporation of photo-activatable lipids into membranes potentially opens new avenues for studying interactions with peptides and proteins, the question of whether azide- or diazirine-modified lipids are suitable for such studies remains controversial. We have recently shown that diazirine-modified lipids can indeed form cross-links to membrane peptides after UV activation and that these cross-links can be precisely determined in their position by mass spectrometry (MS). However, we also observed an unexpected backfolding of the lipid's diazirine-containing stearoyl chain to the membrane interface challenging the potential application of this modified lipid for future cross-linking (XL)-MS studies of protein/lipid interactions. In this work, we compared an azide- (AzidoPC) and a diazirine-modified (DiazPC) membrane lipid regarding their self-assembly properties, their mixing behavior with saturated bilayer-forming phospholipids, and their reactivity upon UV activation using differential scanning calorimetry (DSC), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and MS. Mixtures of both modified lipids with DMPC were further used for photo-chemically induced XL experiments with a transmembrane model peptide (KLAW23) to elucidate similarities and differences between the azide and the diazirine moiety. We showed that both photo-reactive lipids can be used to study lipid/peptide and lipid/protein interactions. The AzidoPC proved easier to handle, whereas the DiazPC had fewer degradation products and a higher cross-linking yield. However, the problem of backfolding occurs in both lipids; thus, it seems to be a general phenomenon.

Synthetic single-chain bolalipids with symmetrical headgroups have shown potential in various pharmaceutical applications, such as the stabilization of liposome bilayers. Despite their amphiphilic character, synthetic bolalipids have not yet been investigated for their suitability as solubilizing agents for poorly soluble drug compounds. In this study, three synthetic single-chain bolalipids with increasing alkyl chain lengths (C22, C24 and C26) were investigated. All three bolalipids were able to achieve an increased solubility of the model drug, mefenamic acid, by approximately 180% in a pH 7.4 buffer compared to only a 102–105% increase achieved by sodium dodecyl sulfate (SDS) or the non-ionic surfactant pegylated hydroxystearate (PEG-HS). Subsequently, interfacial activity of bolalipids and their ability to destabilize liposomal bilayers were investigated. The C22 bolalipid exhibited a consistently lower interfacial activity, which was consistent with its significantly lower cytotoxicity in the macrophage-like cell line, J774. A1, compared to C24 and C26 counterparts. The mean IC50 values of the bolalipids tested (0.035–0.093 mM) were approximately 4–100-fold lower than that of SDS (0.401 mM) or PEG-HS (0.922 mM), with the mechanism of toxicity linked to increased cell membrane permeability, as is expected for surfactants. In summary, evidence from this study shows that decreasing the length of the bolalipid alkyl linker from C26 to C22 resulted in a significantly decreased cytotoxicity with no loss in drug solubilization efficiency.

Although incorporation of photo‐activatable lipids into membranes potentially opens up novel avenues for investigating interactions with proteins, the question of whether diazirine‐modified lipids are suitable for such studies, remains under debate. Focusing on the potential for studying lipid/peptide interactions by cross‐linking mass spectrometry (XL‐MS), we developed a diazirine‐modified lipid (DiazPC), and examined its behaviour in membranes incorporating the model α‐helical peptide LAVA20. We observed an unexpected backfolding of the diazirine‐containing stearoyl chain of the lipid. This surprising behaviour challenges the potential application of DiazPC for future XL‐MS studies of peptide and protein/lipid interactions. The observations made for DiazPC most likely represent a general phenomenon for any type of membrane lipids with a polar moiety incorporated into the alkyl chain. Our finding is therefore of importance for future protein/lipid interaction studies relying on modified lipid probes.

AbstractThis review describes the use of phospholipid excipients to make Pharmaceutical Form (Dosage Form) Line Extensions of existing drugs as part of a Product Life Cycle Management. Product examples and development candidates, which show the versatility of phospholipids as key excipients in formulations to develop line extension drug products for any administration route by reformulating existing products, are provided. The resulting patented products enable the application of a drug substance for another administration route or show an increased efficacy and/or reduced toxicity of the formulated drug substances, or enable a more convenient use, through reduction of dosing frequency, or adapt a product for regulatory requirements for specific patient populations. Parenteral line extensions for lipophilic drugs using non‐toxic phospholipid‐based solubilising formulations are clearly an alternative to products with solubilising synthetic detergents with the risk for allergic and anaphylactic reactions. This review draws the attention to academic and industrial formulation scientist to consider using phospholipid excipients to reformulate existing products to improve the product properties by an active product life cycle management. Phospholipids are suitable for this purpose because they are biocompatible, biodegradable, non‐toxic, and available at large scale and of pharmaceutical grade. Besides, they are well known to regulatory authorities.

How does a small change in the structure of a phospholipid affect its supramolecular assembly? In aqueous suspensions, the substitution of one ester linkage in DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) by an ether linkage alters its phase behaviour completely. To unravel the effect of replacing a phospholipid's ester linkage by an ether linkage in lipid monolayers, we characterized pure monolayers of the model lipid DPPC and its sn-2 ether analogue PHPC (1-palmitoyl-2-O-hexadecyl-sn-glycero-3-phosphocholine) as well as mixtures of both by measurements of surface pressure-molecular area (π-Amol) isotherms. In addition, we used infrared reflection absorption spectroscopy (IRRAS) to study lipid condensation, lipid chain orientation, headgroup hydration, and lipid miscibility in all samples. Mixed monolayers consisting of DPPC and PHPC were studied further using epifluorescence microscopy. Our results indicate a strong influence of the sn-2 ether linkage on headgroup hydration and ordering effects in the regions of the apolar chains and the headgroups. Both effects could originate from changes in glycerol conformation. Furthermore, we observed a second plateau in the π-Amol isotherms of DPPC/PHPC mixtures and analysis of the mixed π-Amol isotherms reveals a non-ideal mixing behaviour of both lipids which may be caused by conformational differences in their headgroups.

This review summarizes the research on phospholipids and their use for drug delivery related to the Phospholipid Research Center Heidelberg (PRC). The focus is on projects that have been approved by the PRC since 2017 and are currently still ongoing or have recently been completed. The different projects cover all facets of phospholipid research, from basic to applied research, including the use of phospholipids in different administration forms such as liposomes, mixed micelles, emulsions, and extrudates, up to industrial application-oriented research. These projects also include all routes of administration, namely parenteral, oral, and topical. With this review we would like to highlight possible future research directions, including a short introduction into the world of phospholipids.

In this study, we characterized monolayers of an azide-modified lipid at the air-water interface, pure and in its mixtures with the model lipid DPPC, with the aim of proving its potential to be applied for photo-cross-linking with other molecules. We chose a phospholipid bearing a terminal azide group in one of its hydrophobic tails to study its monolayer characteristics with the Langmuir film balance technique. Furthermore, we performed infrared reflection absorption spectroscopy (IRRAS) to get detailed insights into the organization of those monolayers as well as high-resolution mass spectrometry (HRMS) to see the effects of UV-irradiation on the lipids' chemical structure and organization. Our results suggest that in expanded monolayers of pure azide-modified membrane lipids, the azido-terminated chain folds back toward the air-water interface. Above the LE/LC (liquid-expanded/liquid-condensed) phase transition, the chains stretched, and thus, the azide group detaches from the interface. From temperature-dependent monolayer compressions, we evaluated all relevant thermodynamic parameters of the monolayers, such as the phase transition pressure, the critical temperature, and the triple point, and compare them to those of model lipids. For future applications, we studied the miscibility of the azide-modified lipid with DPPC in monolayers and found at least a certain miscibility over all investigated mixing ratios ranging from 10 to 75% of the azidolipid. Finally, we irradiated the azidolipid monolayer with UV light at 305 nm and measured photodissociation of the azide, leading to chemical cross-linking with other lipids, which shows the potential to be used as a cross-linking agent within self-assembled lipid or lipid/protein layers.

Natural phospholipid (PL) excipients are native, biocompatible and relatively inexpensive alternatives to synthetic emulsifiers. A well-known PL excipient is lecithin, which primarily contains phosphatidylcholine (PC) and (depending on the purity grade) also contains a well-defined mixture of other PLs with a fatty acid composition, which reflects their natural source. Since all of these lipid species are emulsifiers, natural PLs can be considered as a mixture of (co-) emulsifiers. Many different HLB values for lecithins are given in the literature, which is why this needs to be clarified. To assess this, HLB values of thirteen different plant derived PLs differing in PC content were determined using a centrifugation stress method to determine the relative stability of an emulsion with the respective emulsifier and different oil phases. It could be shown that the studied PLs can be characterized by a broad HLB range, which may be linked to the composition of the PLs and the oil used. In order to emphasize the results of the HLB determination, it could be demonstrated that stable emulsions could be prepared with a wide range of oils using the plant-based PLs and that the preparation method of the emulsions is important in order to obtain stable emulsions. Therefore, assigning a specific exact HLB value to natural PLs instead of a wider range is inappropriate. The broad HLB ranges indicate the suitability of the studied PL emulsifiers for the preparation of oil-in-water emulsions for a wide range of oils: It is recommended to experimentally evaluate the suitability of these natural emulsifiers for the preparation of stable emulsions and to benefit from the wide range of HLB values of these emulsifiers instead of relying on inaccurate and confusing HLB values in the literature.