Liposomes represent a highly promising and adaptable class of drug carriers, offering a spectrum of advantages in the pharmaceutical industry, including encapsulating both hydrophobic and hydrophilic drugs [1], precise site-targeting capabilities while minimizing toxic side effects, controlled release mechanisms, and preventing degradation and rapid clearance [2], [3], [4]. These benefits have led to the successful approval and widespread clinical use of several liposomal drug products in recent decades [5], [6]. The brief lifespan of liposomes in the bloodstream constrains their usage, a challenge effectively addressed by linking them to polyethylene glycol (PEG), an FDA-approved polymer known for enhancing the solubility of conjugates in aqueous environments [7]. PEGylated liposomes enhance stability and circulation time in the bloodstream, making them a promising choice for drug delivery systems.

Previous studies have explored various methods for liposome synthesis, including ethanol injection [8], thin-film hydration [9], and reverse-phase evaporation [10] to generate multi- or unilamellar vesicles. All these methods produce liposomes with the self-assembly of lipids in the bulk phase, resulting in large, polydisperse, and multilamellar liposomes, necessitating numerous post-processing steps like sonication and extrusion for refinement. Various extrusion and sonication techniques can be employed to reduce the size of liposomes, facilitating size control and reproducibility. However, these methods often lead to significant product losses, posing a limitation for large-scale production [11].

In addition, these conventional methods have long struggled with inefficiency, susceptibility to sample contamination, limitations in scalability, and a lack of temperature control that heightens the risk of lipid degradation [12], [13]. All these challenges have limited the translation of liposome formulation from the research and development scale to the preclinical and clinical scales.

The microfluidic-based platform [12], [14], [15] offers an alternative approach to overcome these limitations, allowing for liposome production in a single step from its monomers, with remarkable speed and precise control over the process. Unlike the bulk methods, super-fast mixing of the lipids with the aqueous solution inside a micro-scale mixer results in the production of highly homogenous liposomes with the desired size for drug delivery in a continuous manner, which could be controlled by the operational parameter particularly total flow rate (TFR) and flow rate ratio (FRR) of two reagents [16]. This method enables not only the production of nanoparticles with low precursor volumes in the lab but also offers easy scalability to mass production, making it swiftly embraced by the pharmaceutical industry to increase throughput and reduce production costs and time [17].

There are two prevalent passive microfluidic platforms used for liposome production: hydrodynamic flow focusing [18], [19], [20], [21] and herringbone micromixers [22], [23], [24], [25]. In the former, two streams are introduced into a microchannel, where one stream flows at the center and is enveloped by another. This micromixer configuration relies on the diffusion mechanism to blend two reagents, resulting in a lower throughput of around a hundred microliters per minute. Although this microfluidic-based platform is a promising approach, there exists a scarcity of research concerning drug encapsulation on a chip. The sole existing studies focused on incorporating drugs such as β-carotene and hydrophobic molecules like Nile red into liposomes on a chip, resulting in an efficiency of 60% and a lower efficiency of 15%, respectively [26], [27].

On the other hand, the latter micromixer boasts a higher throughput of approximately a few millimeters per minute. Nevertheless, it employs a complex microstructure that poses challenges in fabrication. In addition, the majority of studies utilizing the herringbone micromixer rely on a commercialized chip that necessitates specialized equipment, notably the NanoAssemblr™ bench-top instrument (Precision NanoSystems Inc., Vancouver, Canada) [22], [23], [24]. This device is specifically tailored for the production of nanocarriers and is commercially accessible for research purposes. In addition, the efficiency of drug encapsulation varies within the ranges of 27–87% for hydrophobic drugs [28], [29] and 23–80% for hydrophilic drugs [30], [31].

A limited number of studies have explored alternative designs, exemplified by the periodic disturbance micromixer [32], which predominantly relies on molecular diffusion for mixing two reagents and exhibits a low throughput ranging from 5 to 20 ml/h. These research studies did not focus on micromixer design and their associated parameters, which are believed to exert a significant impact on liposome production. Additionally, none of these research endeavors incorporated drug encapsulation on a chip or conducted an extensive parametric examination of it.

This gap in knowledge presents an intriguing avenue for further exploration and development within this field to introduce a simple to fabricate platform capable of producing liposomes in tunable ranges and encapsulating drugs on a chip in a high throughput manner. Furthermore, there exists a significant gap concerning conducting a comprehensive parametric investigation of the microchip design, concurrently with the analysis of key operational parameters such as TFR and FRR.

Thus, to this end, in this study, a microfluidic platform was introduced that could produce highly reproducible and homogenous PEGylated liposomes with tunable size in a single step without any post-processing requirement. This platform produces liposomes in a wide range of 64–152 nm from small unilamellar vesicles (SUVs) to large unilamellar vesicles (LUVs) with PDI less than 0.2 with a single easy-to-fabricate microchip by manipulating operational parameters. A numerical model was developed in OpenFoam to demonstrate the fluid flow dynamics and mixing process within the described micromixer. A comprehensive exploration was conducted to investigate key parameters, such as the design of the chip, total flow rate, flow rate ratio, lipid concentration, and variations in buffer, and solvent composition. To demonstrate the platform's ability to load drugs within liposomes, NR was used as a stand-in for a hydrophobic drug. This platform excels not only in encapsulating hydrophobic drugs efficiently but also in generating fluorescently labeled liposomes for imaging applications. NR's fluorescent characteristics enable researchers to monitor and visualize how liposomes interact with cells or tissues, trace their movements, and evaluate how these liposomes are delivered to and taken up by cells.