A novel microfluidic-based approach to formulate size-tuneable large unilamellar cationic liposomes: Formulation, cellular uptake and biodistribution investigations

Abstract

Extensive research has been undertaken to investigate the effect of liposome size in vitro and in vivo.However, it is often difficult to generate liposomes in different size ranges that offer similar low polydispersity and lamellarity. Conventional methods used in the preparation of liposomes, such as lipid film hydration or reverse phase evaporation, generally give rise to liposomal suspensions displaying broad, multimodal size distribution combined with uncontrolled degree of lamellarity. In contrast, microfluidics allows highly homogeneous liposome dispersions to be produced and adjustment of microfluidic operating parameters (flow rate ratio (FRR) and total flow rate (TFR)) can offer size-tuning of liposomes (up to 300 nm, depending on the formulation). Herein, we demonstrate a novel method which allows the production of highly monodisperse, cationic liposomes over a wide particle size range (up to 750 nm in size). This is achieved through controlling the concentration of the aqueous buffer during production. Using this method, liposomes composed of 1,2-dioleoyl-sn-3phosphoethanolamine (DOPE) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or dimethyldioctadecylammonium (DDA) -DOPE:DOTAP and DOPE:DDA liposomes -of up to 750 nm were prepared and investigated. Investigating these formulations in vitro demonstrates that cellular uptake of small (40 nm) and large (>500 nm) liposomes in bone marrow-derived macrophages (BMDM) is similar terms of percentage of liposome + cells and mean fluorescence intensity (MFI). However, significant differences are observed in BMDM uptake when represented in terms of number of liposomes, liposome surface area or liposome internal volume. In vivo biodistribution studies in mice show that by creating small (<50 nm) liposomes we can modify the clearance rates of these liposomes from the injection site and increase accumulation to the draining lymphatics.

Manufacturing Considerations for the Development of Lipid Nanoparticles Using Microfluidics

“…It is hypothesised that the reactive amine group within the TRIS structure becomes protonated, resulting in cationic zeta potentials. Moreover, the effect that buffer type and concentration have on liposomal systems produced using microfluidics has been previously explained by Lou et al [39]. Lou et al demonstrated that DSPC:Chol:DOTAP liposomes were larger (approx.…”

“…100 nm) when prepared using citrate buffer pH 6 compared to TRIS buffer pH 7.4 (approx. 60 nm) even at the same buffer molarity [39]. Particle size was dependent on buffer type, concentration and cationic lipid content.…”

The Impact of Solvent Selection: Strategies to Guide the Manufacturing of Liposomes Using Microfluidics

“…We also show that alcohol mixtures can be used to fine-tune the liposome size. We have previously shown that buffer concentration can be used to offer size control [18]. Therefore, liposome size is under the influence of alcohol and the salt concentration of the aqueous buffer and both are key process parameters that should be considered in the development of microfluidic manufacturing processes.…”

“…Using this method, we have demonstrated the ability to entrap small molecules [10,11], nucleic acids [12] and proteins [13,14] within liposomes. We have also demonstrated the scale-independent production of liposomal adjuvants using this method [15][16][17][18].…”

Barriers and Strategies of Cationic Liposomes for Cancer Gene Therapy

“…Lipid composition and complexation with pDNA could influence the size of CLs and zeta potential and hence biodistribution and cellular entry. 158 , 159 , 160 Particles smaller than 5 or 50 nm are easily excreted by urine or lodged in the liver, respectively. 90 However, particles larger than 50 nm are difficult to penetrate deep into the tumor and thus reduce cellular uptake.…”