Liposomes, as nanoscale drug carriers, are widely used in various medical applications due to their ability to encapsulate diverse drug compounds and protect them from degradation. Liposome size significantly impacts their efficacy and toxicity; smaller liposomes generally exhibit better bioavailability and enhanced permeability and retention (EPR) effects, leading to increased tumor accumulation. However, achieving precise size control and high throughput during liposome synthesis remains a challenge. Traditional batch methods, such as solvent injection, detergent removal, and extrusion, offer limited size control and high polydispersity. Continuous-flow microfluidic techniques offer improved control but often face throughput limitations. This research introduces MVF as a solution to this problem by combining hydrodynamic focusing and vortex-enhanced mixing to achieve high throughput and precise size control.

Existing liposome synthesis methods, including batch processes (solvent injection, detergent removal, extrusion) and continuous microfluidic techniques (flow focusing, rapid mixing), each present limitations. Batch methods lack precise size control and result in high polydispersity. Microfluidic flow focusing improves size control but struggles with scaling up for high throughput. Rapid micromixers, while simpler, yield liposomes with higher size variance and a more limited size range. Modifications to these methods to increase throughput have generally resulted in compromises in size control and polydispersity. This paper addresses these limitations by introducing a novel microfluidic approach.

The study introduces microfluidic vortex focusing (MVF) using an axisymmetric hydrocyclone flow cell. Aqueous buffer is tangentially injected to create a vortical flow, while lipid solution is introduced axially, creating a focused stream sheathed by the buffer. Liposome self-assembly occurs through hydrodynamic focusing and rapid mixing within the vortex. The device geometry was optimized through numerical simulations to achieve rapid mixing and focusing, ensuring the formation of smaller liposomes. The devices were fabricated using high-resolution additive manufacturing via stereolithography (SLA) with a digital light processor (DLP). Numerical modeling using COMSOL Multiphysics was employed to simulate solvent transport and mixing within the device under different operating conditions. Experiments involved liposome synthesis with varying buffer:lipid flow rate ratios (FRR) and total flow rates (TFR) using different lipid compositions (DMPC:cholesterol:DCP and DMPC:cholesterol:PEG2k-PE). Liposome size and polydispersity were characterized using dynamic light scattering (DLS) and cryo-transmission electron microscopy (cryo-TEM). Long-term stability was assessed by storing selected samples at 4°C for 99 days and re-measuring their size distributions.

The MVF device successfully synthesized monodisperse liposomes with tunable size control. Increasing the FRR resulted in smaller liposomes, with a minimum diameter of 61 nm achieved without PEG-lipids and 27 nm with PEG-lipids. Importantly, polydispersity remained low (PDI ~0.04 without PEG and 0.14 with PEG), regardless of FRR. Increasing the TFR also resulted in smaller liposomes and lower PDI. Higher lipid concentrations allowed for increased mass production rates, reaching 7.2 g/h for the DCP-based liposomes and exceeding 20 g/h for the PEGylated liposomes. Cryo-TEM imaging confirmed the DLS results. Long-term storage (99 days at 4°C) showed no significant change in liposome size or polydispersity. The incorporation of PEG-conjugated lipids significantly reduced liposome size, achieving a minimum diameter of 27 nm. The MVF platform demonstrated significantly higher production figures of merit compared to existing microfluidic methods.

The MVF technique successfully addresses the limitations of previous methods by combining the benefits of hydrodynamic focusing and vortex mixing. The rapid mixing induced by the vortex, coupled with the spatial confinement provided by focusing, reduces mixing times and diffusion lengths, leading to smaller and more uniform liposomes. The high throughput achievable with MVF makes it a promising candidate for production-scale liposome synthesis. The ability to control liposome size and polydispersity, combined with the high throughput, makes this technology a significant advancement in liposomal nanomedicine.

Microfluidic vortex focusing provides a novel and effective method for synthesizing size-tunable liposomes with high throughput. The combination of hydrodynamic focusing and vortex mixing leads to precise size control and low polydispersity, while high TFR and lipid concentration enable mass production rates exceeding 20 g/h. The straightforward device fabrication and reliable operation make MVF a promising technology for large-scale liposome production, advancing the field of liposomal drug delivery. Further research could investigate the impact of MVF on drug encapsulation efficiency and explore the application of this technique to other nanoparticle synthesis processes.

The current study primarily focuses on empty liposomes, and further investigation is needed to fully understand the impact of MVF on drug encapsulation efficiency for both hydrophilic and hydrophobic drugs. While the device fabrication is relatively simple, the yield was approximately 50%, with clogging a primary concern. Optimization of the 3D printing process and material selection could further improve the yield. The maximum flow rate is limited by the need to maintain laminar flow, potentially limiting the ultimate throughput. Further studies examining the scalability and long-term stability with encapsulated drugs are necessary for clinical translation.