Elsevier

Acta Biomaterialia

Volume 118, December 2020, Pages 207-214
Acta Biomaterialia

Compositional inhomogeneity of drug delivery liposomes quantified at the single liposome level

https://doi.org/10.1016/j.actbio.2020.10.003Get rights and content

Abstract

Liposomes are the most used drug delivery vehicle and their therapeutic function is closely linked to their lipid composition. Since most liposome characterization is done using bulk techniques, providing only ensemble averages, the lipid composition of all liposomes within the same formulation are typically assumed to be identical. Here we image individual liposomes using confocal microscopy to quantify that liposomal drug delivery formulations, including multiple component mixtures mimicking Doxil, display more than 10-fold variation in their relative lipid composition. Since liposome function is tightly regulated by the physicochemical properties bestowed by the lipid composition, such significant variations could render only a fraction of liposomes therapeutically active. Additionally, we quantified how this degree of compositional inhomogeneity was modulated by liposome preparation method, the saturation state of the membrane lipid, and whether anti-fouling polyethylene glycol (PEG) conjugated lipids were added to the initial lipid mix or inserted after liposome formation. We believe the insights into the factors governing the degree of inhomogeneity offers the possibility for producing more uniform liposomal drug delivery systems, potentially increasing their therapeutic efficacy.

Introduction

Liposomes are the most common nanoscale carrier used for medical applications [1], [2], [3]. Characterizing the lipid composition of drug delivery liposomes is important, since their function and therapeutic efficacy are directly linked to the physicochemical properties bestowed by the lipids making up the liposome [4,5]. However, liposome characterization is typically done using bulk assays giving only an ensemble average read out, leading people to assume that all liposomes have identical lipid compositions. This concept has been challenged in recent years, with reports showing that liposomes in the same batch can display considerable inhomogeneities in their physicochemical properties [6], [7], [8], [9]. These earlier reports have been based on simple model membrane systems, with an attempt to understand the basic principles underlying the phenomena, including how such compositional inhomogeneities would influence protein binding experiments. Consequently, little is known about the compositional inhomogeneity of applied lipid membrane systems, in particular liposomal drug delivery vehicles. Also, how parameters such as their membrane lipid composition and preparation method affect the compositional inhomogeneity of clinically relevant drug delivery liposomes remains largely unexplored.

A compositional inhomogeneity that is too high could lead to a larger fraction of the liposome population harboring properties that renders them suboptimal as drug delivery vehicles [10,11]. This could be related to specific liposome compositions that might compromise e.g. liposome stability, targeting ability or circulation kinetics. Thus knowing how to reduce compositional inhomogeneity could potentially lead to more precise and controllable liposomal drug delivery systems, with increased therapeutic efficacy [12]. Here we have used a microscopy based single liposome assay to demonstrate that clinically relevant drug delivery liposomes display significant compositional inhomogeneity between individual liposomes. We studied how this inhomogeneity was affected by a number of liposomal structural features such as the saturation state of the membrane lipid and whether PEGylation was performed by adding PEG-lipids to the initial lipid mix or inserted after liposome formation. Also, we revealed that depending on the lipid composition, the liposome preparation method can significantly affect the compositional inhomogeneity. These findings highlight the importance of implementing the characterization of liposomal compositional inhomogeneity as a tool in the liposomal drug discovery process.

Section snippets

Materials

2-methyl-2-propanol (tert-Butanol) acquired from Honeywell Riedel-de Haën. Bovine Serum Albumin (BSA), Biotin labelled Bovine Serum Albumin (BSA-Biotin), Streptavidin (Strep), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonicacid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) acquired from Sigma-Aldrich (Søborg, Denmark). 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), L-α-phosphatidylcholine, hydrogenated Soy (HSPC), Cholesterol (Chol),

Single liposome assay for studying compositional inhomogeneity of drug delivery liposomes

To study the compositional inhomogeneity of drug delivery liposomes we used a previously described single liposome assay [7,16,18,28]. In brief, we labelled liposomes with pairs of various lipid fluorophores and immobilized single liposomes on a passivated glass surface (Fig. 1a). Using confocal microscopy we imaged hundreds of individual liposomes in parallel, in a high-throughput manner. Sequentially we imaged the two fluorescent channels before the integrated intensity of each channel was

Discussion

Despite the emergence of nanomedicine many decades ago, is has been argued that the number of nanomedicine based drugs that have reached the clinic is staggeringly low [37], [38], [39]. This translational gap has been proposed to be, at least in part, related to a lack of precision and sensitivity in the tools employed to characterize the physicochemical properties of drug delivery liposomes [10, 37]. Due to technical limitations, most characterization of liposome properties have traditionally

Conclusion

We have used a microscopy based single liposome assay to demonstrate that clinically relevant drug delivery liposomes display significant compositional inhomogeneity between individual liposomes. The DI was modulated by membrane lipid saturation state, the preparation method and the PEGylation strategy. As drug delivery systems are becoming more and more complex, but not necessarily more and more effective [48, 49], the concept of controlling compositional inhomogeneity will only be more

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supporting Information

Details on lipid composition, biophysical characterization of liposome systems and experimental error determination. Summary of all quantified DI values and statistical testing.

CRediT authorship contribution statement

Thomas Lars Andresen: Funding acquisition, Conceptualization, Supervision, Writing - review & editing. Jannik Bruun Larsen: Conceptualization, Methodology, Investigation, Writing - original draft.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgement

The work was supported by the Novo Nordisk Foundation (Grant No NNF16OC0022166). The authors would like to thank Fredrik Melander for operating the ICP-MS, Kim I. Mortensen (DTU Health Tech) for help with data analysis as well as Rasmus Münter (DTU Health Tech), Kasper Bendix Johnsen (DTU Health Tech) and Nanna Wichmann Larsen (DTU Health Tech) for critical feedback on the manuscript.

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