liposomes & lipid nanoparticles.€¦ · liposomes arise partly from poor stability under shelf...

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Liposomes & Lipid nanoparticles.

Dave Palmer, Alex Kerr, Sam Trotter & Dai Hayward.

Since their discovery in 1965, by Alec D. Bangham, liposomes have been recognised as the drug delivery vehicle of choice. Their biocompatibility results in minimal adverse reactions. Their amphiphilic structure allows encapsulation of both hydrophilic and hydrophobic active pharmaceutical ingredients (APIs). More recently the liposome’s analogous cousin, the lipid nanoparticle, has gained prominence because of its ability to deliver therapeutic payloads, including DNA and mRNA for vaccines. They can both deliver their payload very precisely through treating their surface with proteins allowing highly specific binding to a target cell type.

Liposomes Many conventional drugs exhibit poor pharmacokinetics, limited bioavailability and a high toxicity, all of which restrain their use. To overcome these issues and improve the therapeutic indices of the drug, the field of nanomedicine has made significant progress in detection, diagnosis and treatment of several diseases1. Thanks to nanoparticles and liposomes, it has been possible to decrease the toxicity and improve the pharmacokinetic parameters.

Liposomes are nanoscale drug delivery vehicles that can be used to enhance the effectiveness of APIs. They are formed from biological ingredients and have similar structures to materials found in the human body. The amphiphilic phospholipid bilayer of liposomes has close resemblance to the mammalian cell membrane, enabling efficient interactions between liposomes and cell membrane and, subsequently, effective cellular uptake. They can, thus, shield a drug from detection by the immune system, allowing additional time to get to the point of need without triggering an immune response.

Drug loading can be achieved either actively (after liposome formation) or passively (the drug encapsulation during liposome formation) or actively • Hydrophobic drugs can be directly combined into liposomes during vesicle formation. The uptake amount and retention

are governed by drug-lipid interactions. Trapping of 100% is often achievable but is dependent on the solubility of the drug in the liposome membrane.

• Passive encapsulation of water-soluble drugs depends on the ability of liposomes to trap aqueous buffer containing a dissolved drug during vesicle formation. Trapping effectiveness is limited by the trapped internal volume of the liposomes together with drug solubility in the aqueous buffer.

Liposomes have become among the most applied technologies for the encapsulation and delivery of bioactive agents and many different compounds in biological, pharmaceutical, medical and nutritional applications2 since the first drug product, Doxil, was approved in 1995.

Lipid nanoparticles In the 1990s a need was identified for alternate approaches for nanoparticles based on lipid components other than phospholipids. Solid Lipid Nanoparticles (SLNs) represent a relatively new colloidal drug delivery system, composed of physiological lipids that remain in a solid state at both room and body temperature3. The solid lipid used forms a matrix material for drug encapsulation and includes mono-, di- or triglycerides, fatty acids and complex glyceride mixtures. SLNs have significant advantages, such as physical stability over a long period, possibility of controlled release of both lipophilic and hydrophilic drugs; protection of labile drugs; low cost; relative ease of preparation. Furthermore, SLNs have exceptionally low toxicity4. SLNs also have generally recognised disadvantages, such as moderate drug-loading capacity and drug expulsion due to the crystallization process under manufacturing and storage conditions.

1 Beltran-Gracia et al. 2019, ‘Nanomedicine review: clinical developments in liposomal applications’, Cancer Nano 10:11 2 Linder et al 2011, ‘Liposomes: A Review of Manufacturing Techniques and Targeting Strategies’, Current Nanoscience 7(3). 3 Garcia-Pinel et al 2019, ‘Lipid-Based Nanoparticles: Application and Recent Advances in Cancer Treatment’, Nanomaterials (Basel), Apr 9(4) 4 Mydin et al. 2019. ‘Nanoparticles in Nanomedicine Application: Lipid-Based Nanoparticles and Their Safety Concerns’. In Nanotechnology: Applications in

Energy, Drug and Food, Springer Link.

Fig1: Range of liposome configurations (Hua et al, 2015)


Nanostructured Lipid Carriers (NLCs) represent a second generation of lipid-based nanocarriers, developed from SLN, which comprise a combination of solid and liquid lipids5. This system has been developed in order to overcome the limitations of SLNs. NLCs have higher drug loading capacity and can also avoid drug expulsion during storage by avoiding lipid crystallization.

Lipid nanoparticles (LNPs), in conjunction with adjuvants to increase the immune response, enable mRNA encapsulation for protection from enzymatic degradation. This is of particular interest for vaccines as a complement to, or even replacement of, traditional vaccines6.

Liposome & lipid nanoparticle manufacture Typically, liposomes are manufactured as sterile injectables for delivery to the bloodstream, and release of the drug takes place when the lipid envelope breaks down. Having delivered the liposome to the desired location, controlled release of the API can be achieved by natural breakdown of the liposome, or it can be designed to react to a change in temperature, pH or shear stress.

Liposome properties differ considerably with lipid composition, surface charge, size, and the method of preparation. Furthermore, the choice of bilayer components determines the ‘rigidity’ or ‘fluidity’ and the charge of the bilayer. For instance, unsaturated phosphatidylcholine species from natural sources give much more permeable and less stable bilayers, whereas the saturated phospholipids with long acyl chains form a rigid, rather impermeable bilayer structure7.

Although liposomes have shown some success in drug product approvals, the limitations identified in the technology have remained almost stagnant over decades and have slowed its market penetration. The most common disadvantages of liposomes arise partly from poor stability under shelf and in-vivo conditions. This is mostly due to potential lipid oxidation and hydrolysis, leakage and loss of hydrophilic cargoes, as well as particle fission and fusion8.

There are numerous lab scale, and a few large-scale, techniques for liposome production, allowing for size control, from around 20nm up to several microns, and composing of one or more bilayers. As the self-assembly of liposomes is based on an interaction between phospholipids and water molecules, control of the interaction between them is key to making structures with the desired morphology, encapsulation efficiency and stability.

For liposomes the current state of the manufacturing art is characterised thus:

Liposomes produced by different methods have varying physicochemical characteristics, which leads to differences in their in vitro (sterilisation and shelf life) and in vivo (disposition) performances. Rapid, precise and reproducible quality control tests are required for characterising the liposomes after their formulation and upon storage for a predictable in vitro and in vivo behavior of the liposomal drug product. A liposomal drug product can be characterized for some of the parameters that are discussed below.

When liposomes are intended for inhalation or parenteral administration size distribution is of primary consideration since it influences the in-vivo fate of liposomes along with the encapsulated API molecule. Various techniques for determining the size of the vesicles include microscopy, diffraction and scattering techniques, and hydrodynamic techniques.

Column chromatography can be used to determine the percentage of API encapsulation in liposomes. After removal of any free API, the liposomes containing the encapsulated API are lysed. This exposed drug is assayed by a suitable technique which gives the percent drug encapsulated from which encapsulation efficiency can be calculated. A calculation of

trapped volume per lipid weight can also give the percent API encapsulated in a liposome vesicle. Determination of this can be

5 Garcia-Pinel et al 2019, Ibid 6 lankschtein et al. 2016. ‘mRNA vaccine delivery using lipid nanoparticles’, Ther. Deliv. 7(5), 319–334 7 Yadav et al. 2018 ‘A comprehensive review on novel drug delivery via unique property of liposome’. European Journal of Biomedical and Pharmaceutical

Sciences, Volume 5, Issue 02 184-192 8 Krause et al. 2019. ‘General Perception of Liposomes: Formation, Manufacturing and Applications’; in ‘Liposomes Advances and Perspectives’, IntechOpen,

Fig2: Adapted from Krause et al; 2019

Method Advantages Disadvantages

Film hydration (Bangham method)

Straightforward process

Use of organic solvent and mechanical agitation, production of large particles with no control on size, poor encapsulation efficiencies of hydrophilic materials, time consuming, sterilization issue

Reverse phase evaporation

Simple design, suitable encapsulation efficiency

Not applicable to fragile cargoes, use of large quantity of organic solvent, time consuming, sterilization issue

Solvent injection

Straightforward approach

Trace of organic solvent as residue, possible nozzle blockage in ether system, time consuming, sterilization issue

Detergent removal

Simple design, homogenous product, control of particle size

Presence of organic solvent, detergent residue, time consuming, low entrapment efficiency, poor yield, sterilization required

Heating method

Simple and fast process, organic solvent free, no need for sterilization, possible up-scale production

The need for high temperature


carried out by radioactive markers, fluorescent markers and spectroscopically inert fluids.

In general, LNPs are similarly formed by condensing lipids from an ethanol solution in water. However, the method by which they are synthesized is critical, because it directly affects both the LNP size and encapsulation efficiency.

The properties of individual LNPs strongly depends on local, microscopic, mixing rates, where diffusive transport effects can lead to LNPs with variable compositions. Therefore, rapid mixing of the ethanol–lipid phase with excess water is key for the synthesis of small, uniform LNPs.

A note of caution must be added. Many of the techniques also have the potential to degrade the efficacy of APIs due to the application of mechanical stresses (e.g. high shear, sonication, high pressure, etc.), unsuitable chemicals (e.g. volatile organic solvents, etc.), or extremes of pH. This is particularly true in the case of commercially available high temperature methods in which the lipids are heated to around 80oC.

The Micropore difference

Micropore Technologies already provides aseptic membrane devices for the formation PLGA microspheres. Recent work has pointed to other ways of using the membrane technology, as refined micro-mixers, mixing two miscible liquids together in a controlled manner.

Micropore’s alma mater, Loughborough University, has investigated the making of liposomes using membrane emulsification using Micropore’s LDC-19. Micropore has subsequently developed this initial work through its UK labs to engineer a scalable process.

Micropore’s approach to both liposomes and lipid nanoparticles is, at room temperature or below, to inject a dispersion of phospholipids and API in a suitable solvent which can be introduced in a controlled manner into a continuously flowing continuous phase. The resulting self-assembly is moderated without high temperature, pressure or shear. As droplets form on the surface of the membrane, gentle shear, provided by the flow of the continuous phase, deforms and detaches the droplet, meanwhile the water miscible solvent partitions into the aqueous phase, leaving the liposomes to form in its wake. In drug delivery applications using lipid-based carriers, such as liposome and nanoliposome formulations, a Polydispersity Index (PDI) of 0.3 and below is considered to be acceptable and indicates a homogenous population of phospholipid vesicles. The last edition of the FDA’s “Guidance for Industry” concerning liposome drug products emphasises the importance of size and size distribution as “critical quality attributes (CQAs)”, as well as essential components of stability studies of these products.

Micropore’s process has several advantages over the most common commercial processes.

1. A PDI of 0.16 is well within the FDA’s required PDI. 2. Tuneable particle size between 80 – 250 nm 3. Fully, easily and economically scalable from lab to full scale. 4. GMP compliant manufacturing equipment. 5. Minimal product degradation from low temperature, low shear operation. 6. No toxic lysolipid formation as a consequence of Micropore’s low temperature operation.

We’re ready to help you with your liposome & lipid nanoparticle formulations.

9 Vladisavljevic ́et al. 2013, ‘Production of liposomes using microengineered membrane and co-flow microfluidic device’, Colloids and Surfaces A:

Physicochem. Eng. Aspects 458 (2014) 168–177

Fig3: Micropore’s liposome manufacturing method

Fig4: Comparison of liposomal PDIs: Blue trace: Micropore method (d10: 149 nm; d50: 183 nm; d90 226 nm) vs. Green trace: Homogenisation (d10: 63 nm; d50: 104 nm; d90 147 nm)

liposomes & lipid nanoparticles.€¦ · liposomes arise partly from poor stability under shelf...

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