Compositional inhomogeneity of drug delivery liposomes quantified at the single liposome level
Graphical abstract
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.
References (49)
- et al.
Insights into composition/structure/function relationships of Doxil (R) gained from "high-sensitivity" differential scanning calorimetry
Eur. J. Pharm. Biopharm
(2016) - et al.
Tailoring the lipid composition of nanoparticles modulates their cellular uptake and affects the viability of triple negative breast cancer cells
J. Controlled Release
(2019) - et al.
A fluorescence-based technique to construct size distributions from single-object measurements: Application to the extrusion of lipid vesicles
Biophys. J
(2008) - et al.
Insertion of poly(ethylene glycol) derivatized phospholipid into pre-formed liposomes results in prolonged in vivo circulation time
FEBS Lett
(1996) - et al.
A unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteins
Semin. Cell Dev. Biol
(2010) - et al.
Membrane Curvature and Lipid Composition Synergize To Regulate N-Ras Anchor Recruitment
Biophys. J.
(2017) - et al.
A review on phospholipids and their main applications in drug delivery systems
Asian. J. Pharm. Sci
(2015) - et al.
Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes
Biochim. Biophys. Acta
(2012) - et al.
PEGylation as a strategy for improving nanoparticle-based drug and gene delivery
Adv. Drug Del. Rev
(2016) - et al.
Characterization of nanomedicines: A reflection on a field under construction needed for clinical translation success
J. Controlled Release
(2018)
Doxil (R) - The first FDA-approved nano-drug: Lessons learned
J. Controlled Release
Ordering effects of cholesterol and its analogues
Biochim. Biophys. Acta-Biomembr
Preparation of liposomes at large scale using the ethanol injection method: Effect of scale-up and injection devices
Chem. Eng. Res. Des
A novel strategy for the preparation of liposomes: rapid solvent exchange
Biochim. Biophys. Acta-Biomembr
Seeing spots: Complex phase behavior in simple membranes
Biochimica Et Biophysica Acta-Molecular Cell Research
The beginning of the end of the nanomedicine hype
J. Controlled Release
Advances and Challenges of Liposome Assisted Drug Delivery
Front. Pharmacol
The evolving landscape of drug products containing nanomaterials in the United States
Nat. Nanotechnol
Nanocarriers as an emerging platform for cancer therapy
Nat. Nanotechnol
Proton Permeation into Single Vesicles Occurs via a Sequential Two-Step Mechanism and Is Heterogeneous
J. Am. Chem. Soc.
Observation of Inhomogeneity in the Lipid Composition of Individual Nanoscale Liposomes
J. Am. Chem. Soc.
Influence of the Preparation Route on the Supramolecular Organization of Lipids in a Vesicular System
J. Am. Chem. Soc.
Encapsulation Efficiency Measured on Single Small Unilamellar Vesicles
J. Am. Chem. Soc.
Nanoparticle heterogeneity: an emerging structural parameter influencing particle fate in biological media?
Nanoscale
Cited by (13)
On the uptake of cationic liposomes by cells: From changes in elasticity to internalization
2023, Colloids and Surfaces B: BiointerfacesCitation Excerpt :Liposome-cell interactions are dependent on the composition, structure, and dynamics of each cell membrane. In brief, a variety of biophysical methods have been applied over the years to investigate the fusion mechanism or/and effectiveness of drug delivery by using membrane models with a specific lipid composition [13]. In an earlier study we investigated the fusion mechanism of liposomes using a simplified model of the HeLa membrane that mimicked the cell lipid membrane [14].
Nano-drug delivery system with enhanced tumour penetration and layered anti-tumour efficacy
2022, Nanomedicine: Nanotechnology, Biology, and MedicineCitation Excerpt :To achieve effective curative effects, there is an urgent need for a drug delivery system that can fully penetrate and easily accumulate in the tumour. Liposomes are one of the most widely studied nanocarriers, have good biocompatibility and safety, are biodegradable, and are ideal nanocarriers.11–13 In addition to the above characteristics, cationic liposomes (CLS) preferentially and selectively target cancerous endothelial cells going through angiogenesis, which is beneficial for cellular uptake; however, because of their positive surface charge, they are easily eliminated from the body.14–19
Efficient exosome extraction through the conjugation of superparamagnetic iron oxide nanoparticles for the targeted delivery in rat brain
2022, Materials Today ChemistryCitation Excerpt :In the past decades, owing to the remarkable advances and therapeutic potential of nanotechnology, enormous efforts in the design and construction of nanoformulated drugs have been attempted [1–4]. To fulfill different clinical demands of the treatment of diseases, various drug nanocarriers, including nano-micelles, liposomes, hydrogels, and metal-based nanoparticles, have been used [5–7]. However, toward the effective clinical practice of synthetic nanomaterials, it remains critical as their immune response, in vivo toxicity, and drug delivery efficiency in the complicated biological system have to be closely evaluated [8,9].
Quantifying the heterogeneity of enzymatic dePEGyaltion of liposomal nanocarrier systems
2022, European Journal of Pharmaceutics and BiopharmaceuticsCitation Excerpt :Despite the lack of multiple distributions in the PCL density histograms, the enzymatic dePEGylation could still lead to increased liposome-to-liposome PEG density variations. To evaluate if this was the case, we calculated the degree of PCL density inhomogeneity (DI) using the coefficient of variation, a statistical term that relates the width (SD) and the mean of the Gaussian function fit to the PCL surface density histograms (DI = SD/mean) [28,38]. A higher DI denotes a larger variation in the PCL surface densities between individual liposomes and thus a less uniform PCL surface density.