Summary:

Liposomes are lipid-based supramolecular assemblies that can be engineered into efficacious drug carriers. Typically described as bilayer systems with an aqueous core, liposomes can increase the therapeutic index of a given drug molecule by decreasing its toxicity and/ or increasing its efficacy through enhanced circulation and permeation. Several liposomal formulations are already clinically applied to treat conditions such as cancer; and while liposomes are traditionally made by extrusion or sonication, these methods can be costly, labour-intensive and prone to batch-to-batch variations. However, to make the transition from bench to clinic, liposome manufacturing methods need to be reproducible, scalable and low cost. For this purpose, microfluidic-based technologies introduce new formulation opportunities that in addition to being scalable, provide extensive control over the size optimization and manufacturing process. In this particular formulation technique, an organic solvent (bearing the lipids) and a buffer (usually containing the hydrophilic drugs) are rapidly mixed through a micromixer and liposomes self assemble due to a shift in solvent polarity (see here for more info). In microfluidic mixing techniques, many variables such as solvents of choice, total flow rate (TFR) and flow rate ratio (FRR) can affect liposome size and characteristics. In their article featured in International Journal of Pharmaceutics, 2016, the Perrie group from the University of Strathclyde, UK, use the NanoAssemblr^TM Benchtop instrument to generate liposomes of different sizes by varying formulation parameters such as solvent selection, FRR and lipid concentration. Additionally, they dual-loaded a hydrophilic and a lipophilic drug into microfluidic manufactured liposomes in a single step. Both drugs are used in treating type-2 diabetes while one is highly lipophilic (glipizide) and the other hydrophilic (metformin), making co-treatments challenging. By dual-loading the glipizide and metformin, treatments can hypothetically become more convenient and effective.

The authors first investigated the effect of solvent selection on particle size. Four liposome formulations composed of different phospholipids (egg PC, DMPC, DPPC or DSPC) each mixed with equimolar cholesterol were studied with either ethanol, methanol or isopropanol as organic solvents and either Tris buffer or PBS as the aqueous solvent. The combination of methanol and PBS led to the smallest size range in comparison to other combinations of organic and aqueous solvents. Particle homogeneity followed a similar trend: liposomes produced using methanol and PBS had the lowest polydispersity index (PDI). Additionally, increasing the initial lipid concentration to 3 mg/ mL or higher led to smaller liposomes.

Next, the authors looked at the effect of TFR and FRR on liposome size and concluded that the largest vesicles formed with the aqueous: organic (FRR) of 1:1 regardless of the TFR. Across the range tested, the smallest liposomes formed with FRR of 5:1 and no significant change was observed by varying the TFR. The authors then encapsulated two model drugs using the methods optimized in the steps mentioned above. Lipophilic glipizide, along with the lipid mix was dissolved in methanol and metformin was added to PBS. Using the NanoAssemblr Benchtop, the drugs were simultaneously co-loaded during liposomal formulation in one step. The dual-loading encapsulation rate remained the same as each drug being encapsulated individually (40% for glipizide and 20% for metformin). Interestingly, liposome size decreased when dual-loaded with both glipizide and metformin suggesting structural changes due to the presence of both drugs. Cryo-electron microscopy analysis of the drug-loaded liposomes indicated small unilamellar vesicles for empty liposomes as well as ones loaded with glipizide and/or metformin. In addition to size, drug release rates changed when liposomes were co-loaded with the two drugs as opposed to drugs incorporated individually. When co-loaded, the rate of glipizide and metformin increased to 12% and 64% from 3% and 35% respectively when individually encapsulated.

In conclusion, the Perrie group’s work is amongst the first that thoroughly investigate the effect of different formulation parameters such as buffers and solvents and flow rates in microfluidic liposomal formulations. Also, this article is the first example of simultaneous entrapment of a lipophilic and a hydrophilic drug using a scalable microfluidic formulation method. This study highlights the potential of microfluidics as a convenient, flexible and scalable system for manufacturing of drug loaded liposomes.

Abstract:

Despite the substantial body of research investigating the use of liposomes, niosomes and other bilayer vesicles for drug delivery, the translation of these systems into licensed products remains limited. Indeed, recent shortages in the supply of liposomal products demonstrate the need for new scalable production methods for liposomes. Therefore, the aim of our research has been to consider the application of microfluidics in the manufacture of liposomes containing either or both a water soluble and a lipid soluble drug to promote co-delivery of drugs. For the first time, we demonstrate the entrapment of a hydrophilic and a lipophilic drug (metformin and glipizide respectively) both individually, and in combination, using a scalable microfluidics manufacturing system. In terms of the operating parameters, the choice of solvents, lipid concentration and aqueous:solvent ratio all impact on liposome size with vesicle diameter ranging from ∼90 to 300 nm. In terms of drug loading, microfluidics production promoted high loading within ∼100 nm vesicles for both the water soluble drug (20–25% of initial amount added) and the bilayer embedded drug (40–42% of initial amount added) with co-loading of the drugs making no impact on entrapment efficacy. However, co-loading of glipizide and metformin within the same liposome formulation did impact on the drug release profiles; in both instances the presence of both drugs in the one formulation promoted faster (up to 2 fold) release compared to liposomes containing a single drug alone. Overall, these results demonstrate the application of microfluidics to prepare liposomal systems incorporating either or both an aqueous soluble drug and a bilayer loaded drug.