Precise Engineering of Lipid-Based Delivery Systems Using Microfluidics for Biomedical Applications

Abstract

Lipid-based delivery systems (LDS), including lipid nanoparticles (LNPs) and liposomes, have become indispensable tools in modern biomedicine owing to their biocompatibility, capacity to encapsulate diverse therapeutic agents, and potential for targeted delivery. Despite their clinical success, conventional batch-based manufacturing methods are hindered by variability, limited scalability, and complex processing steps, slowing their broader translation. Microfluidic technologies offer a transformative solution by enabling precise fluid handling, rapid mixing, and reproducible production of LDS with tunable physicochemical attributes such as particle size, lamellarity, and drug-loading efficiency. This review highlights advances in microfluidic design strategies, including hydrodynamic flow focusing, staggered herringbone mixers, and toroidal micromixers, and evaluates how critical parameters such as flow rate, solvent composition, and lipid concentration influence LDS performance. Furthermore, we discuss the application of microfluidics in drug delivery, nucleic acid therapeutics, and vaccine platforms, underscoring its role in improving scalability, quality control, and clinical translation. Finally, we examine current challenges, including throughput limitations and solvent handling, while outlining future directions for integrating emerging materials and additive manufacturing to optimize LDS fabrication. Collectively, microfluidic platforms provide a promising pathway for next-generation lipid nanomedicines with enhanced precision, reproducibility, and therapeutic efficacy.

1. Introduction

Lipid-based delivery systems (LDS) have emerged as highly versatile and clinically important platforms for the transport of therapeutic agents. Among these, lipid nanoparticles (LNPs) have gained prominence due to their capacity to efficiently encapsulate and deliver a wide range of cargos, from small-molecule drugs to complex biological macromolecules such as plasmid DNA, messenger RNA (mRNA), and small interfering RNA (siRNA). Their clinical success, highlighted by the approval of the siRNA therapeutic patisiran (Onpattro^®) and the widespread use of mRNA-based COVID-19 vaccines, demonstrates the transformative impact of LNP technology in modern medicine. Structurally, LNPs are non-bilayer, solid or amorphous lipid assemblies composed primarily of ionizable lipids, helper phospholipids, cholesterol, and PEG-lipids, forming a core–shell architecture optimized for nucleic acid complexation, protection, and intracellular delivery. In contrast, liposomes are classical phospholipid vesicles characterized by a bilayer membrane surrounding an aqueous core. This amphiphilic organization enables the simultaneous loading of hydrophilic and hydrophobic agents, with water-soluble molecules localized in the internal aqueous compartment and lipophilic compounds incorporated within the lipid bilayer. The formation, stability, and biocompatibility of liposomes are largely governed by the hydrophobic effect driving spontaneous phospholipid self-assembly in aqueous environments. Owing to these structural and physicochemical features, liposomes have been extensively utilized as delivery vehicles for a broad spectrum of therapeutics, including anticancer agents, antimicrobials, hormones, peptides, proteins, enzymes, vaccines, and nucleic acid-based drugs [1].

The clinical success of LNPs is attributed to several advantageous physicochemical features: controlled particle size, high drug loading capacity, low bilayer permeability, tunable surface charge, and the possibility of surface modification, such as PEGylation, to prolong circulation. These attributes collectively enhance therapeutic efficacy while reducing off-target toxicity. Despite such progress, the translation of LNPs from bench to bedside remains constrained by key challenges, including batch-to-batch variability, limited scalability of traditional manufacturing processes, and difficulties in predicting the in vivo behavior of engineered formulations in the complex human biological environment. To address these limitations, microfluidic technologies have gained traction as next-generation platforms for the scalable and reproducible production of LNP microfluidics enables rapid and homogeneous mixing of reagents within microscale channels, allowing precise control over particle size, uniformity, and encapsulation efficiency. These devices can be fabricated from diverse materials, such as polymers, silicon, and metals, and employ either active (e.g., electric, magnetic, thermal, or optical forces) or passive (e.g., flow rates and channel geometry) mechanisms to regulate fluid dynamics. Active control offers fine-tuned modulation of flow patterns, whereas passive control simplifies device design and minimizes risks such as heating or sample degradation [2]. Beyond nanoparticle synthesis, microfluidic devices have been applied to a broad spectrum of biomedical applications, including organ-on-a-chip systems, drug discovery, diagnostics, protein and nucleic acid analysis, and cell separation. Their ability to provide reproducible, size-controlled nanoparticle formulations with minimal reagent consumption has revolutionized the development pipeline for nanomedicines. In this review, we provide a comprehensive overview of LDS with a particular focus on microfluidic approaches for their design and production. We discuss the advantages and limitations of conventional manufacturing strategies, highlight the opportunities afforded by microfluidic technologies, and explore their expanding role in biomedical research and clinical translation.

2. Background and History of Microfluidics

Microfluidic technologies have attracted significant attention for the development of nanoscale drug delivery systems due to their ability to precisely manipulate fluids at the microscale. Over the past decades, extensive research efforts have been dedicated to employing microfluidic methods for nanoparticle fabrication, offering superior control over particle size, reproducibility, and encapsulation efficiency compared to conventional bulk techniques. Microfluidic chips can be fabricated from a variety of materials, including paper, silicon, glass, polytetrafluoroethylene (PTFE), and elastomeric polymers such as dimethylpolysiloxane/polydimethylsiloxane (PDMS). Among these, glass and PDMS are the most widely used materials [3,4]

Glass-based devices offer tunable surface properties, as the hydrophobicity or hydrophilicity of the surface can be modified to meet specific experimental requirements. PDMS, while biocompatible and optically transparent, requires more complex fabrication processes. The most commonly used method for manufacturing PDMS microfluidic devices is soft lithography, which allows the creation of microscale channel networks with high fidelity [5]. The microscale dimensions of these channels impart fluid dynamics that are markedly distinct from those observed in macroscale systems [6]. One of the defining parameters of microfluidics is the Reynolds number (Re), which represents the ratio of inertial to viscous forces in a fluid. In conventional systems, high Reynolds numbers (Re > 2000) correspond to turbulence with chaotic fluid motion, whereas low Reynolds numbers (Re < 2000) are associated with laminar flow dominated by viscous forces [7]. In microfluidic channels, the Reynolds number is typically much lower than 100, resulting in highly stable laminar flow. This property enables the precise control of fluid streams and the generation of monodisperse droplets, a key advantage for drug encapsulation applications [8].

Another important factor is the Péclet number (Pe), which describes the relative influence of convective transport and molecular diffusion. In microfluidic systems, low Pe indicates that molecular diffusion plays a significant role in governing solute distribution and transport behavior, whereas high Pe corresponds to convection-dominated regimes [9]. When immiscible fluids are introduced into laminar flow conditions, diffusion across liquid–liquid interfaces is minimal, maintaining distinct boundaries between phases [10]. For mutually miscible fluids, however, diffusion occurs gradually along lateral or longitudinal directions, allowing controlled mixing. This controlled interface and limited molecular transfer significantly enhance the encapsulation efficiency of therapeutic molecules in microfluidic-fabricated drug delivery systems [11]. Microfluidic devices are generally categorized into three primary geometries: T-junction, flow-focusing Y-junction, and coaxial flow designs. In T-junction devices, the dispersed phase flows perpendicularly from a vertical channel into a horizontal continuous phase channel, where it is sheared into monodisperse droplets or jets [12]. In flow-focusing Y-junction devices, the dispersed phase introduced through a central channel is stretched and fragmented by continuous phase streams entering from opposing side channels [13]. In coaxial flow devices, both phases are injected in parallel from the same direction, where the dispersed phase is elongated and broken into uniform droplets or jets within the continuous phase [14]. These monodisperse droplets serve as highly reproducible templates for the fabrication of nanoparticle-based drug delivery systems.

3. Effect of Physicochemical Parameters of Lipid-Based Systems on Microfluidic Processing

Microfluidic platforms have opened new opportunities for the reproducible and scalable manufacturing of LNPs. By enabling rapid and precisely controlled mixing of lipid-containing organic solvents with aqueous buffer systems, microfluidics facilitates the spontaneous self-assembly of lipid molecules into nanoscale structures such as small unilamellar vesicles. The polarity shift during solvent exchange drives this assembly process, resulting in well-defined LNP formulations with tunable characteristics [15]. The physicochemical attributes of LNPs, including particle size, morphology, rigidity, surface charge, and surface composition, play a pivotal role in determining their performance as drug delivery vehicles. These parameters directly influence formulation stability, circulation half-life, systemic toxicity, cellular internalization, biodistribution, and the ability to achieve targeted delivery [16]. Consequently, careful modulation of microfluidic process variables (such as flow rate ratio, total flow rate, and channel geometry) provides a powerful means of tailoring LNP properties to specific therapeutic applications (

Table 1. Key parameters influencing LNP size and polydispersity in microfluidic synthesis. Arrows indicate the direction of the effect of each parameter on lipid nanoparticle properties.

Parameter	Conditions	Effect on LNP Size	Effect on PDI	Mechanism/Explanation
Micromixer Type	HFF, SHM, TMM	SHM & TMM generally produce smaller and more uniform particles than HFF	SHM/TMM → lower PDI	Mixing efficiency and chaotic advection control nucleation and growth
Flow Rate Ratio (FRR)	Low → High aqueous: organic	Higher FRR → smaller particles	Higher FRR → lower PDI	Faster solvent dilution shortens lipid disc growth and produces uniform vesicles
Total Flow Rate (TFR)	Low → High	Minor effect on size (in many systems)	Usually minimal effect	Controls mixing time and production speed rather than nucleation
Lipid Concentration	Low → High	Higher concentration often increases size (until plateau)	Higher concentration may increase PDI	Increased lipid availability promotes vesicle growth and aggregation
Organic Solvent Type	Methanol vs. Ethanol vs. IPA	Methanol → smaller particles; IPA/Ethanol → larger	Variable	Solvent polarity influences lipid solubility and nanoprecipitation kinetics
Aqueous Buffer Composition	PBS, Tris, ionic strength	Can tune size (40–500 nm reported)	Moderate influence	Ionic strength and buffer composition affect self-assembly dynamics
Channel Geometry	45° vs. 90°, 2D vs. 3D focusing	Influences size and uniformity	Can improve PDI	Controls confinement and diffusion during nanoparticle formation
Temperature (if mentioned)	Higher temperature	Often reduces size slightly	Improves uniformity	Enhances lipid fluidity and diffusion
PEG-lipid content	Low → High	May slightly reduce size	Improves PDI	Steric stabilization prevents aggregation

). This review highlights advances in microfluidic strategies for controlling the physicochemical features of lipid-based delivery systems. We further discuss how these engineered attributes govern their biological behavior, shaping clinical outcomes in areas such as oncology, infectious disease, and genetic medicine.

3.1. Micromixer Design

Microfluidic systems used for lipid nanoparticle (LNP) production operate predominantly under laminar flow conditions characterized by low Reynolds numbers. Under these conditions, fluid behavior is governed by solvent velocity and viscosity, and mixing occurs primarily through molecular diffusion rather than turbulence. In contrast, macroscopic fluidic systems often exhibit turbulent flow, which promotes rapid and efficient mixing of solutes [17]. Although diffusion-driven mixing in microfluidics is highly controllable and reproducible, it can be relatively slow and may limit mixing efficiency under standard operating conditions [7]. To overcome these limitations, micromixer designs have been engineered to enhance mixing efficiency by modifying channel geometry and architecture. These innovations allow rapid and homogeneous mixing within short channels while maintaining high throughput [18]. In the context of LDS production, the residence time of fluids within the mixing channels, combined with device geometry, plays a critical role in controlling vesicle nucleation and growth. Thus, micromixer design directly influences particle size, uniformity, and efficiency. Several micromixer configurations have been developed and evaluated for liposome fabrication. Among the most widely studied are hydrodynamic flow focusing (HFF) (Figure 1A–C), staggered herringbone micromixers (SHM) (Figure 1D) and toroidal micromixers (TMM) (Figure 1E) [19]. Each design offers distinct advantages in promoting efficient mixing and controlling lipid self-assembly, thereby enabling the reproducible production of liposomal carriers with tailored physicochemical properties.

3.2. Hydrodynamic Flow Focusing (HFF)

HFF is one of the most widely applied strategies for generating liposomes with controlled physicochemical properties (Figure 2A). In this approach, three parallel inlet channels are used to inject aqueous and organic solvent streams at different velocities. Typically, a central stream containing lipids dissolved in ethanol is hydrodynamically compressed by two flanking aqueous streams, resulting in a narrow and highly confined ethanolic flow [20]. The degree of confinement and consequently the size of the resulting liposomes is primarily governed by the flow rate ratio (FRR) between the aqueous and organic phases. As diffusion occurs between the aqueous and ethanolic streams, the solubility of lipids decreases, driving their self-assembly into vesicles [21]. Jahn et al. were the first to demonstrate liposome synthesis via hydrodynamic flow focusing on a microfluidic device. They designed a cross-type glass microchannel (200 µm wide, 40 µm deep) incorporating silicone layers for mixing phosphate-buffered saline (PBS) and isopropanol (IPA). A lipid solution composed of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), cholesterol, and 1 wt% of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiIC18) in IPA (10 mM) was introduced into the system. By fixing the IPA flow rate at 2.4 mm/s and varying the PBS flow rate from 2.4 to 59.8 mm/s, they successfully controlled liposome sizes within the 100–300 nm range. Furthermore, comparative studies using devices with different chip geometries (45° vs. 90°) at FRR = 5 and a lipid concentration of 0.9 mg/mL revealed differences in particle size and polydispersity index (PDI). The 45° chip produced liposomes with a mean hydrodynamic diameter (MHD) of 92 nm and a broad PDI (0.35), while the 90° chip generated slightly larger particles (120 nm) but with improved uniformity (PDI = 0.27) [22]. Building upon these findings, John et al. designed two microfluidic devices to examine the combined effects of geometry and flow focusing on liposome synthesis. The first device featured 120 µm deep channels, with the central lipid inlet 42 µm wide and side channels 65 µm wide, leading into a 65 µm × 10 mm mixing channel. The second device was designed with smaller dimensions (36 µm deep, 10 µm wide) and a 10 mm mixing channel. Using a lipid solution of DMPC, cholesterol, and dihexadecyl phosphate (DCP) at 5 mM, and varying FRRs from 6 to 48, they observed that mean particle size distributions remained relatively consistent across both geometries [23].

Despite its advantages, HFF can be limited by lipid aggregation and vesicle formation along the channel walls, leading to particle size variability and potential channel clogging. To mitigate these issues, three-dimensional (3D) hydrodynamic flow-focusing designs have been developed. In such systems, the organic solvent stream is introduced through a central capillary surrounded by the aqueous stream, minimizing lipid deposition on channel walls and improving size control. Additionally, HFF platforms have been integrated with downstream processing steps such as on-chip microdialysis. This approach enables buffer exchange and the establishment of a pH gradient, facilitating active drug loading, for example, the encapsulation of doxorubicin, thereby allowing complete on-chip production of functional liposomal formulations [24].

3.3. Staggered Herringbone Mixers (SHM)

Chaotic advection is a widely used strategy to enhance fluid mixing in microfluidic systems, achieved by incorporating geometric patterns that generate transversal flow components, thereby stretching and folding fluid volumes across the channel cross-section (Figure 2B) [25]. The staggered herringbone mixer (SHM) leverages this principle by exponentially increasing the interfacial area between two fluids as they traverse the channel, which accelerates diffusional mixing compared to HFF at equivalent flow rate ratios. While mixing channels patterned with herringbone grooves on only one surface may show reduced mixing efficiency on the opposite face, incorporating herringbone features on multiple channel surfaces significantly improves mixing uniformity and enhances particle synthesis performance. In SHM systems, the increased contact area between aqueous and organic phases is achieved by carefully engineered microchannel configurations [26]. The design consists of asymmetric herringbone-shaped grooves embedded into the channel floor, which direct incoming fluid streams over a sequence of staggered protrusions. This arrangement induces chaotic flow patterns and transverse vortices that continuously shift due to the asymmetric geometry, leading to rapid and efficient mixing of solvent streams [27]. By controlling these dynamics, SHM platforms enable the reproducible production of LNPs with sizes tightly regulated by process parameters such as flow rate ratio, lipid concentration, and solvent composition [28,29]. Experimental studies have demonstrated that SHMs can generate liposomes with precisely controlled diameters. For example, at low lipid concentrations, SHM-based mixing successfully produced liposomes as small as 30 nm with narrow size distributions [30,31]. Beyond conventional liposome fabrication, SHMs have been employed for encapsulating a variety of therapeutic cargos, including doxorubicin [32] and siRNA [33]. More recently, Gustavo Lou et al. designed a Y-shaped SHM system for formulating self-amplifying mRNA vaccines using cationic liposomal carriers, showcasing the versatility of SHMs in the production of advanced nucleic acid delivery platforms [34].

3.4. Toroidal Micromixers (TMM)

Although staggered herringbone mixers (SHMs) have demonstrated strong mixing efficiency, their complex structural design presents certain limitations, particularly in terms of fabrication costs and throughput. To overcome these challenges, toroidal micromixers (TMMs) have been developed as an alternative design. In TMM systems, two inlet channels are employed, one delivering the aqueous phase and the other the organic solvent. The fluid streams are directed through circular structures integrated within the flow path, which sustain laminar flow even at high velocities. These toroidal geometries generate chaotic advection by creating centrifugal forces and multiple vortices, thereby significantly enhancing mixing efficiency and enabling higher throughput. A major advantage of TMMs is their scalability. By maintaining consistent operating parameters, these devices allow lipid nanoparticle (LNP) production to transition seamlessly from bench-scale experiments to good manufacturing practice (GMP)-level production. This scale-independent approach ensures reproducibility across manufacturing settings without the need for extensive process optimization [28]. Cameron Webb et al. performed a comparative study of SHM and TMM devices, marking the first demonstration of scaling microfluidic production from laboratory scale (12 mL/min) to GMP-compatible continuous production at 20 L/h using standardized operating parameters. Tangential flow filtration (TFF) was employed for downstream purification, ensuring high product recovery and quality. Both anionic and neutral liposomal formulations were synthesized using SHM and TMM at an aqueous-to-organic flow rate ratio (FRR) of 3:1 and a total flow velocity of 15 mL/min, followed by purification with TFF. Cationic liposomal formulations were prepared at FRR = 1:1 and a total flow rate of 10 mL/min, with purification performed using a 1/10 dilution in Tris buffer. Physicochemical analyses, including hydrodynamic diameter, surface charge, polydispersity index (PDI), and drug encapsulation efficiency, demonstrated no significant differences between formulations produced by SHM and TMM devices [29].

These findings highlight the robustness and scalability of TMM-based platforms, which enable efficient, reproducible, and scale-independent liposome production. By allowing the same parameter set points, operating ranges, and acceptable ranges to be applied from small-scale laboratory experiments to large-scale continuous manufacturing, TMMs reduce translational risk and streamline the path from bench to approved nanomedicine products.

3.5. Flow Rate (FR)

Flow rate (FR) is a critical parameter in microfluidic manufacturing that influences both nanoparticle formation and production efficiency. Although several reports suggest that the operating FR has minimal direct impact on liposomal size, it remains an important determinant of production speed, as lower flow rates can prolong manufacturing time. Conversely, other studies highlight that under specific conditions, both total flow rate (TFR) and flow rate ratio (FRR) can significantly affect the physicochemical attributes of LNPs [35,36]. For example, Sedighi et al. systematically evaluated the effects of TFR and FRR using a staggered herringbone mixer. Their findings indicated that FRR exerted a dominant influence on particle size and distribution, while vesicle characteristics remained largely unchanged at flow rates above 8 mL/min [27]. Similarly, Webb et al. compared SHM- and TMM-based production systems at flow rates ranging from 12 to 200 mL/min. Their results showed no significant differences in particle diameter, polydispersity index (PDI), or protein loading efficiency across this wide range, reinforcing the robustness of microfluidic designs for scale-up [29]. However, in cationic formulations composed of dimethyldioctadecylammonium bromide (DDAB) and trehalose 6,6′-dibehenate (TDB), both FRR and TFR demonstrated a measurable impact on vesicle size. Specifically, increasing FRR and TFR reduced particle diameters from approximately 1000 nm at FRR = 1:1 and 5 mL/min to around 160 nm at FRR = 5:1 and 20 mL/min [37]. Likewise, investigations of hydrogenated soy phosphatidylcholine (HSPC)- and distearoyl phosphatidylcholine (DSPC)-based LNPs across flow rates between 10 and 20 mL/min revealed no statistically significant changes (p > 0.05) in vesicle size, confirming that production speed can be increased without compromising physicochemical integrity [38].

Together, these findings suggest that while FR alone may not consistently dictate liposomal size, its interplay with FRR and formulation composition is critical. Optimizing these parameters allows for accelerated production while maintaining the desired stability and functional attributes of LNPs, thereby supporting both laboratory-scale studies and large-scale manufacturing.

3.6. Aqueous: Organic Flow Rate Ratio (FRR)

The aqueous-to-organic flow rate ratio (FRR) is one of the most influential parameters in microfluidic synthesis of LNPs. During liposome formation, the mixing of aqueous and alcohol phases drives the self-assembly of lipids into planar bilayers, which subsequently bend and close into spherical vesicles in order to shield their hydrophobic acyl chains from the aqueous environment [39,40]. The FRR directly affects this process by determining the rate of solvent exchange: higher FRRs accelerate the dilution of alcohol, shorten lipid disk stabilization time, and favor the production of smaller LNPs [41].

Experimental evidence consistently demonstrates the impact of FRR on vesicle size and polydispersity. For instance, varying the aqueous-to-solvent ratio (PBS: methanol) from 1:1 to 5:1, while operating at total flow rates of 5–15 mL/min, resulted in marked reductions in hydrodynamic diameter (HDD). At 1:1 ratios, vesicles were relatively large (200–300 nm) with broad PDI values (0.38–0.67). Increasing the FRR to 3:1 decreased vesicle size to ~120–130 nm, while further increasing to 5:1 produced uniform vesicles of ~80–90 nm with significantly reduced PDI (0.11–0.22). In all cases, the zeta potential remained near neutral, consistent with the expected physicochemical profile of such formulations [26]. Carla B. Roces et al. extended these findings by demonstrating that FRR modulation enables fine-tuning of vesicle size using both HSPC- and DSPC-based formulations containing cholesterol and PEG-DSPE. By increasing the FRR from 1:1 to 5:1, vesicle size was reduced from ~120 nm to ~45 nm, while intermediate ratios allowed size adjustments within the 70–120 nm range. Similarly, Neil Forbes et al. reported that FRR strongly influenced not only particle size but also protein loading and release profiles. Their study highlighted that microfluidics-based methods achieved substantially higher protein encapsulation efficiencies (20–35%) compared with traditional approaches such as sonication or extrusion (<5%) [41]. Additional investigations further confirm the inverse relationship between FRR and liposome size. Kastner et al. observed that decreasing ethanol concentration from 50% to 17% reduced particle size from 200 nm to 50 nm in formulations containing DOPE: DOTAP and PC: Chol. In cationic formulations of DDAB: TDB, increasing FRR from 1:1 to 5:1 reduced vesicle diameters from ~1000 nm to ~160 nm, while higher FRRs also improved polydispersity, achieving a PDI of ~0.3 at FRR 3:1 and TFR 10 mL/min [37]. Zhigaltsev et al. further demonstrated that increasing FRR from 1 to 9 reduced POPC- and POPC: Chol-based vesicle sizes from ~140 nm to ~40 nm [33]. Mechanistically, the dependence of vesicle size on FRR has been explained by the dynamics of lipid disk growth and closure. At lower FRRs, a wider organic stream introduces higher alcohol concentrations, which maintain conditions above the critical alcohol threshold for longer channel lengths. This favors repeated cycles of disk assembly, disassembly, and reassembly, leading to larger and more polydisperse vesicles. By contrast, at higher FRRs, alcohol dilution occurs more rapidly, resulting in shorter lipid disk growth and closure times, which produce smaller, more uniform particles [23,42]. This principle has also been applied to advanced formulations. Cheung et al. showed that PEGylated DOPC/DSPC: Chol: DSPE-PEG2000 vesicles were most strongly influenced by FRR, as solvent mixing ratios-controlled nanoprecipitation kinetics and bilayer formation [43]. Likewise, Rui Ran et al. demonstrated tunable particle sizes in folic acid- and TAT-modified multifunctional liposomes, where increasing FRR from 3:1 to 16:1 decreased vesicle diameter from ~180 nm to ~60 nm while maintaining low PDI and stable surface potentials [44]. These studies establish FRR as a dominant parameter in microfluidic liposome production. By governing solvent exchange dynamics and lipid disk assembly kinetics, FRR enables precise control over vesicle size, distribution, and encapsulation performance, making it a critical design factor in the scalable manufacturing of LNP-based therapeutics.

3.7. Solvent Effect

The choice of aqueous and organic solvents is a critical determinant of lipid nanoparticle (LNP) physicochemical properties, lipid solubility, and scalability of manufacturing processes. Solvent composition not only influences vesicle size, homogeneity, and drug loading but also impacts downstream translational performance and feasibility for large-scale production. Studies have shown that modulation of aqueous buffer conditions can serve as a powerful strategy to control LNP size. For example, Gustavo Lou et al. demonstrated that adjusting the Tris buffer concentration resulted in a marked size increase in cationic liposomes (from ~40 to 600 nm), while neutral liposomes remained largely unchanged. In vivo biodistribution experiments further revealed that smaller LNPs (<50 nm) exhibited faster clearance from the injection site and greater accumulation in draining lymph nodes, suggesting potential utility for vaccine and immunotherapy applications [45]. The selection of the organic solvent is equally important, as it governs lipid solubility, particle formation kinetics, and scalability. Reducing solvent polarity by, for instance, replacing methanol with isopropanol was shown to increase vesicle size and decrease drug encapsulation efficiency, although short-term stability and release profiles remained unaffected [46]. Similarly, Joshi et al. investigated liposomes composed of phosphatidylcholine (PC), DMPC, DPPC, or DSPC mixed with cholesterol (2:1 mass ratio) and found that solvent choice markedly influenced vesicle size. Using methanol with PBS consistently produced small, uniform particles (~70–100 nm), whereas DSPC: Chol formulations were highly sensitive to solvent selection. When ethanol replaced methanol, or Tris buffer replaced PBS, DSPC-based vesicles increased dramatically in size (>400 nm), accompanied by broader polydispersity indices (PDI). Methanol–PBS combinations yielded the most homogeneous LNPs with narrow size distributions [26]. Further evidence of solvent influence was provided by Roces et al., who studied HSPC- and DSPC-based formulations with cholesterol and PEG-DSPE (3:1:1 molar ratio) using a SHM microfluidic platform. Both methanol and ethanol were tested as organic solvents. At a 1.5:1 aqueous-to-organic FRR, liposome size significantly increased from ~60 nm (methanol) to ~90 nm (ethanol), although PDI (≤0.2) and zeta potential (~−12 mV) remained comparable. At higher FRR values (3:1), solvent choice did not significantly alter vesicle characteristics, with sizes stabilizing at ~47–50 nm and low PDI (<0.2) [47]. Importantly, while methanol often produced smaller vesicles, ethanol is generally preferred for large-scale GMP production due to its more favorable safety and regulatory profile. Collectively, these findings underscore the importance of solvent selection in microfluidic LNP production. Both the aqueous buffer composition and organic solvent type directly influence vesicle size, homogeneity, and encapsulation performance, with certain lipid chemistries (e.g., DSPC: Chol) displaying heightened sensitivity. Methanol may provide greater control at laboratory scale, but ethanol remains the solvent of choice for clinical translation due to safety considerations.

At the molecular level, lipid self-assembly in microfluidic systems is governed by the interplay between solvent exchange kinetics, bilayer curvature elasticity, entropic forces, and energetic barriers associated with nucleation and growth. Rapid dilution of the organic solvent into the aqueous phase reduces lipid solubility, driving spontaneous aggregation of lipid molecules into intermediate structures such as lipid disks or micellar assemblies [48]. The subsequent transition into stable vesicular or nanoparticulate architectures is influenced by bilayer curvature stress, which depends on lipid composition, molecular packing, and interfacial tension. Entropic contributions arising from solvent reorganization and hydrophobic interactions further promote lipid segregation and structural stabilization [49]. In addition, the height of kinetic energy barriers determines the rate of lipid nucleation, fusion, and rearrangement, thereby controlling particle size, uniformity, and lamellarity. Under microfluidic conditions, the precisely regulated mixing environment minimizes stochastic fluctuations and enables reproducible control over these molecular processes, ultimately allowing fine-tuning of nanoparticle physicochemical properties for specific therapeutic applications [50].

3.8. Effect of Lipid Concentration

In microfluidic-mediated synthesis of LNPs, the concentration of lipids is a key determinant of vesicle formation kinetics and physicochemical characteristics. Lipid concentration directly affects particle nucleation, growth, and stabilization, thereby influencing attributes such as particle size, polydispersity, and surface charge. Microfluidic platforms enable precise control of precursor concentrations, allowing size-tunable and reproducible LNP formulations. Forbes et al. investigated four neutral liposomal formulations PC: Chol, DMPC: Chol, DSPC: Chol, and DPPC: Chol with increasing hydrocarbon chain lengths. Their study showed that raising the initial lipid concentration from 0.3 mg/mL to 2 mg/mL decreased particle size, whereas at concentrations above 4 mg/mL the particle size plateaued, suggesting a saturation effect [41]. Similarly, Mijajlovic et al. examined POPC-based LNPs across a range of 2–20 mM in isopropanol at fixed flow rate ratios. They observed an increase in hydrodynamic diameter (HDD) with lipid concentration, except at very low concentrations, where particle formation was less efficient [51]. The direct relationship between lipid concentration and particle size was further highlighted in studies showing that reducing lipid concentration to 0.9 mg/mL produced small vesicles, while increasing concentration up to 90 mg/mL significantly enlarged vesicle diameter. Alessandra Zizzari et al. confirmed this trend, reporting a gradual increase in mean HDD from ~80 nm to ~190 nm when lipid concentrations increased from 0.9 to 90 mg/mL, accompanied by rising PDI values (0.18–0.26), indicating broader size distributions at higher concentrations [33]. Carla B. Roces et al. explored the effect of lipid concentration on cationic adjuvant-based formulations using a fixed 8:1 molar ratio of DDAB:TDB at concentrations from 0.3 to 24 mg/mL under constant microfluidic conditions (TFR = 10 mL/min, FRR = 3:1). At lower lipid concentrations (0.3 mg/mL), particle sizes were approximately 120 nm. In contrast, at concentrations between 1 and 24 mg/mL, particle sizes stabilized in the 250–350 nm range with PDI values between 0.2 and 0.4. Across all concentrations tested, zeta potential remained highly cationic (+60 to +75 mV), indicating consistent surface properties regardless of concentration [37]. These studies demonstrate that lipid concentration exerts a profound yet formulation-dependent effect on LDS size and polydispersity. While low concentrations favor the generation of smaller vesicles, higher concentrations often lead to increased particle sizes and broader distributions until a plateau is reached. Importantly, microfluidic control enables systematic optimization of lipid concentration to balance particle size, homogeneity, and surface charge for specific therapeutic applications.

4. Scale-Up Methods by Microfluidics

Conventional methods for manufacturing liposomal formulations at an industrial scale typically rely on multi-step, non-standardized procedures that are time-intensive and difficult to optimize. These processes often depend on specific manufacturing conditions and are prone to compromising the stability of biologically active molecules such as nucleic acids (siRNA, mRNA, pDNA), proteins, and peptides, due to prolonged handling and harsh processing steps [52,53]. Consequently, the translation of liposomal therapeutics from bench to bedside has historically been hindered by challenges in scalability and reproducibility (

Table 2. Comparison of microfluidic and conventional liposome/LNP preparation methods.

Parameter	Microfluidic Methods	Extrusion Methods	Sonication Methods
Reproducibility	Excellent batch-to-batch consistency due to controlled laminar mixing and precise flow regulation	Moderate; depends on membrane quality and manual processing	Variable; sensitive to operator technique and energy input
Particle Size Control	Highly tunable (typically 20–150 nm) with narrow size distribution (low PDI)	Good size control but often requires multiple extrusion cycles	Limited control; often produces heterogeneous populations
Encapsulation Efficiency	High; efficient and rapid nanoparticle self-assembly during controlled solvent exchange	Moderate; drug loss can occur during repeated extrusion steps	Low to moderate; possible cargo degradation due to heat and shear
Scalability	Easily scalable via continuous-flow and parallelized microfluidic systems	Limited scalability; batch-based and labor intensive	Poor scalability; difficult to standardize for large-scale production
Production Mode	Continuous, automated, and highly controllable	Batch process	Batch process
Reagent Consumption	Low due to microscale fluid handling	Moderate to high	Moderate
Processing Time	Fast and reproducible	Time-consuming (multiple passes required)	Fast but less controlled
Cost Consideration	Higher initial equipment cost; long-term cost efficiency with reduced waste and automation	Lower equipment cost but higher labor and variability costs	Low equipment cost but inconsistent performance
Thermal/Mechanical Stress on Cargo	Minimal; gentle self-assembly conditions	Low to moderate	High; risk of biomolecule degradation
Suitability for Clinical Translation	High; supports GMP, continuous manufacturing, and reproducibility	Moderate; widely used but limited by scalability	Limited; mainly used for small-scale laboratory preparation

).

Microfluidic-based manufacturing provides an innovative alternative that addresses many of these limitations, offering precise control over formulation parameters and enabling efficient, reproducible, and scalable production of LNPs. Compared to conventional methods, microfluidics reduces complexity, enhances automation, lowers costs, and allows consistent control over particle size and distribution across production scales [54,55]. The ability to support scale-independent and continuous operation makes this approach particularly attractive for clinical and industrial applications [33,56]. Microfluidic devices leverage micrometer-scale channels and small fluid volumes to facilitate rapid and controlled mixing of solvents, which enables the generation of uniform LNPs. Production can be seamlessly scaled from nanoliter to milliliter volumes and integrated with established purification techniques and real-time monitoring systems for quality control [57]. This ensures reproducibility and homogeneity, which are critical for regulatory approval and clinical translation. Recent advances have further demonstrated the feasibility of large-scale microfluidic production. For example, the group led by Yvonne Perrie successfully scaled up LNP manufacturing from 12 mL/min to 20 L/h using standardized operating parameters. Their work compared two laminar flow cartridge designs—staggered herringbone and toroidal micromixers and showed consistent performance at both the bench and industrial scales [29]. In parallel, novel microvortex-based microfluidic methods operating at high Reynolds numbers (~150) have achieved productivity improvements of up to three orders of magnitude compared to earlier approaches [58,59]. Together, these innovations establish microfluidics as a transformative platform for the industrial-scale production of LNPs. By enabling reproducible, scalable, and cost-efficient manufacturing, microfluidics not only accelerates the clinical translation of liposomal medicines but also lays the groundwork for continuous manufacturing strategies in nanomedicine.

5. Biomedical Applications

Microfluidics has emerged as a transformative technology in biomedical research and healthcare, offering unprecedented precision, reproducibility, and efficiency compared to conventional methodologies. By exploiting microscale fluid dynamics, microfluidic systems enable the manipulation of small sample volumes with high accuracy, thereby reducing reagent consumption, minimizing assay variability, and accelerating experimental throughput. These advantages make microfluidics particularly well-suited for applications requiring fine control over the cellular and molecular microenvironment. Beyond operational efficiency, microfluidic platforms provide unique opportunities for integrating multiple experimental processes to a single chip, facilitating rapid diagnostics, real-time monitoring, and high-throughput screening. Their adaptability allows them to be tailored for diverse biomedical tasks such as organ-on-a-chip modeling, drug discovery and delivery, protein and nucleic acid analysis, single-cell profiling, and clinical diagnostics. Collectively, these advances position microfluidics as a powerful enabler of translational medicine and personalized healthcare, with the potential to significantly impact disease prevention, diagnosis, and treatment [60].

In this section, we highlight the major biomedical applications of microfluidic technologies, focusing on their role in advancing diagnostic tools, therapeutic delivery systems, and next-generation research platforms. By outlining these developments, we aim to provide a comprehensive understanding of how microfluidics is reshaping biomedical sciences and accelerating the path toward precision medicine.

5.1. Drug Delivery

LNPs and liposomal formulations have long been explored as carriers for anticancer therapeutics, with doxorubicin representing one of the most successful examples. Doxorubicin was initially incorporated into bilayer liposomal systems of ~80–100 nm diameter using a pH gradient strategy, leading to the development of Doxil, the first FDA-approved liposomal doxorubicin formulation, with a drug-to-lipid (w/w) ratio of 0.125. This product demonstrated improved pharmacokinetics and reduced systemic toxicity, establishing a benchmark for liposomal drug delivery. Expanding upon this foundation, Zhigaltsev et al. prepared POPC-based LNPs using microfluidics at an FRR of 3 with 300 mM ammonium sulfate as the aqueous phase. These particles exhibited diameters of 18 ± 7 nm and achieved near-complete doxorubicin encapsulation (~100%) at drug-to-lipid ratios of 0.2 mol/mol, with loading completed within 30 min and without any change in vesicle size compared to unloaded controls [32]. While Doxil remains clinically relevant, traditional manufacturing approaches such as thin-film hydration rely heavily on organic solvents, which present challenges in terms of safe handling, removal, and environmental disposal, particularly when halogenated solvents are used [51]. To address these concerns, Khadke et al. developed an organic solvent-free, microfluidizer-based process to produce doxorubicin-loaded PEGylated LNPs. This approach generated particles of 100–110 nm with PDI < 0.2 and ~98% drug loading, while maintaining equivalent performance in non-PEGylated formulations. Moreover, microfluidic post-processing allowed further size reduction without compromising encapsulation efficiency [61].

Microfluidics has also been instrumental in advancing nucleic acid-based therapies. The production of Onpattro^® (patisiran), the first FDA- and EMA-approved siRNA-based therapy, employed microfluidic mixing of lipids dissolved in ethanol with siRNA in acidic aqueous buffer (pH 4). This process facilitated rapid ethanol dilution and spontaneous nanoparticle assembly without additional size-reduction steps [62]. The industrial manufacturing process consists of five key stages: (1) preparation of lipid and siRNA solutions, (2) microfluidic mixing to form LNPs, (3) ultrafiltration with buffer exchange, (4) dilution and bioburden filtration, and (5) sterile filtration and filling. Cryo-TEM analysis suggested that these particles do not form classical bilayer vesicles but instead exhibit spherical nanoparticles with dense cores. Nevertheless, their characterization followed the FDA’s draft guidance for liposome drug products (2015), underscoring the translational significance of microfluidics. Roces et al. evaluated PEGylated LNPs composed of HSPC: Chol: DSPE-PEG2000 or DSPC: Chol: DSPE-PEG2000 using SHM-based microfluidics at a 1.5:1 aqueous-to-organic FRR. Ammonium sulfate buffer (pH 5.5) enabled active doxorubicin loading via a transmembrane pH gradient, resulting in 80–100 nm vesicles (PDI < 0.2) with >90% encapsulation efficiency, comparable to Doxil. The formulations were stable for at least six months, and similar strategies enabled efficient loading of vincristine and acridine orange, producing ~100 nm PEGylated LNPs with >90% encapsulation [47].

Microfluidics further allows precise modulation of liposome size, which directly impacts tumor penetration and retention. Smaller LNPs (~50 nm) have been shown to penetrate more deeply into tumor tissue compared to larger (~75 nm) vesicles, potentially enhancing therapeutic efficacy [63]. Beyond small molecules, microfluidics has also been applied to multifunctional formulations. For instance, liposomal vesicles encapsulating Alexa Fluor 488-labeled antibodies, Hoechst 33342, and Alexa Fluor 647 Phalloidin demonstrated enhanced tumor accumulation and prolonged retention in vivo, as confirmed by fluorescence imaging in mice [53]. Surface modification strategies have been seamlessly integrated with microfluidic platforms. Rui Ran et al. developed multifunctional PEGylated liposomes co-modified with folic acid (DSPE-PEG2000-FA) and TAT peptide (DSPE-PEG2000-TAT). Using microfluidics with DMPC: Chol as the lipid base, LNPs of ~70 nm were produced at FRR 8:1 and a total flow rate of 28.8 μL/min. These dual-ligand formulations demonstrated enhanced tumor accumulation and prolonged retention in animal models, underscoring the potential of microfluidics for generating libraries of multifunctional nanocarriers with tailored biological performance [44]. Notably, Zhang et al. reported a unique cholesterol: TWEEN 80 (5:1 molar ratio) liposomal formulation fabricated using SHM. These particles (~76 nm) achieved ~96% doxorubicin loading and displayed distinct pharmacokinetic and biodistribution (PK/BD) profiles compared to PEGylated liposomal doxorubicin prepared by thin-film hydration. Unlike conventional PEGylated formulations, which primarily accumulate in Kupffer cells, these Tween 80-based vesicles delivered doxorubicin uniformly to hepatocytes and other liver cells, highlighting the potential of non-phospholipid liposomes to achieve novel biodistribution profiles [64].

Together, these studies highlight the versatility of microfluidic platforms for drug delivery applications. By enabling precise size control, efficient encapsulation, and compatibility with diverse cargos from chemotherapeutics to nucleic acids and multifunctional ligands, microfluidics has established itself as a cornerstone for the next generation of clinically translatable nanomedicine.

5.2. Vaccine Delivery

LNPs have been extensively studied as vaccine delivery systems and adjuvants due to their ability to protect antigens, enhance immunogenicity, and enable controlled immune activation [65,66]. However, conventional preparation methods for liposomal adjuvants typically involve multi-step procedures that are both costly and labor-intensive, creating barriers for global vaccine accessibility. Given that cost remains a major determinant of vaccine distribution, particularly in low- and middle-income countries, scalable and affordable production technologies are essential to ensure widespread availability [67]. Streamlining manufacturing processes is therefore crucial for reducing costs while maintaining the quality and functionality of liposomal adjuvants [45].

Microfluidics offers a promising alternative to traditional methods by providing a scale-independent, continuous, and reproducible approach to liposomal vaccine production. Using this technology, critical formulation attributes including particle diameter, polydispersity, and encapsulation efficiency can be tightly controlled. In addition, microfluidic manufacturing has been shown to achieve higher antigen or antibody loading efficiencies than conventional methods such as extrusion or sonication [68]. Since the physicochemical characteristics of liposomal adjuvants, including size, surface potential, lipid composition, bilayer fluidity, and degree of PEGylation, directly influence immunogenicity, antigen presentation, and pharmacokinetics, the ability to fine-tune these parameters using microfluidics is highly advantageous [69,70,71]. Indeed, optimized physicochemical features can modulate antigen retention at the injection site, enhance recruitment of antigen-presenting cells (APCs), and improve lymphatic targeting, thereby shaping both pharmacokinetic and immunological responses [72]. Recent studies have highlighted the potential of microfluidics in next-generation vaccine delivery. Gustavo Lou et al. developed self-amplifying mRNA (SAM) vaccines encapsulated in cationic LNPs (SAM-cLNPs) using a Y-shaped staggered herringbone micromixer (300 µm width, 130 µm height). At lipid concentrations of 4–8 mg/mL, FRR of 3:1, and TFR of 5 mL/min, they formulated DOPE- or DSPC-based LNPs containing PEGylated lipids and cationic components. These systems not only protected SAM from degradation but also promoted efficient delivery and localized immunostimulant, thereby enhancing adaptive immune responses [34]. Chatzikleanthous et al. engineered a novel cationic LNP–protein conjugate complex (GBS67-CpGODN+L) comprising DSPC: Chol: DDA (10:40:50 molar ratio). Formulated in ethanol and Tris buffer (pH 7.4) using microfluidics (FRR = 1:1, TFR = 12 mL/min), the nanoparticles were incubated with protein–CpG conjugates. This multifunctional system enhanced antigen-specific immune responses at low antigen doses, significantly reducing the number of immunizations required [68]. Cationic LNPs composed of dimethyldioctadecylammonium bromide (DDAB) and trehalose 6,6′-dibehenate (TDB) represent another class of potent liposomal adjuvants. Traditionally prepared by thin-film hydration, these formulations suffer from scalability issues and batch-to-batch variability. Microfluidics has enabled scalable, continuous production of DDAB: TDB-based adjuvants while maintaining comparable biodistribution and immunogenicity profiles to those prepared by conventional methods. Building upon this, researchers integrated a biotin–avidin complexation strategy to enhance lymphatic targeting and antigen retention within lymph nodes. Although this approach improved pharmacokinetic control, it did not translate into enhanced vaccine efficacy, emphasizing the complexity of immune modulation by LNP-based adjuvants [37].

Overall, these findings underscore the potential of microfluidics in transforming vaccine delivery. By enabling scalable, cost-effective, and precise engineering of liposomal adjuvants, microfluidic platforms provide a pathway toward the next generation of affordable and globally accessible vaccines.

5.3. Nucleic Acid Delivery

The advent of LNPs has transformed the delivery of nucleic acids, creating new opportunities for gene therapy, vaccination, and molecular medicine [73]. By encapsulating and protecting fragile biomolecules such as DNA, RNA, and siRNA, LNPs overcome key barriers including enzymatic degradation, inefficient cellular uptake, and poor biodistribution. Their development requires careful optimization of formulation components, delivery mechanisms, and clinical translation strategies to ensure safety and efficacy [74].

The composition of LNPs is central to their performance. Ionizable lipids, which acquire positive charges in acidic endosomal environments, promote endosomal escape and cytoplasmic release of the nucleic acid payload. Phospholipids contribute to structural integrity, cholesterol enhances membrane fluidity and stability, while polyethylene glycol (PEG)-lipids prolong circulation and reduce immune clearance [75]. High encapsulation efficiency is essential for therapeutic success, achieved through techniques such as microfluidic mixing or ethanol injection [76]. Particle size and surface charge further dictate biological behavior: LNPs are typically engineered to be ~100 nm in diameter with near-neutral to slightly negative zeta potential, maximizing circulation time while minimizing opsonization and clearance [77]. Cellular uptake occurs predominantly via endocytosis, where ionizable lipids facilitate endosomal escape by becoming protonated in the acidic milieu, ensuring effective cytoplasmic delivery [78]. To enhance tissue specificity, surface modifications such as ligand or antibody conjugation are employed, thereby improving targeting precision, therapeutic efficacy, and minimizing off-target effects [79]. Controlled intracellular release is also critical and can be tuned through lipid composition and formulation strategies [80].

The clinical utility of nucleic acid-loaded LNPs spans a broad spectrum of therapeutic areas. They are under investigation for treating genetic disorders such as cystic fibrosis and hemophilia by delivering functional genetic material to replace or repair defective genes [81]. LNPs have proven indispensable in the rapid development of mRNA vaccines, most prominently for COVID-19, where mRNA encoding viral antigens is delivered for in situ protein expression and immune activation. Similarly, siRNA- and RNAi-based LNP formulations are being advanced for cancer therapy and antiviral interventions by silencing disease-associated genes [82]. Moreover, LNPs are increasingly applied in the delivery of CRISPR/Cas9 genome-editing components, providing the potential for curative treatments through precise modification of pathogenic mutations [83]. Despite their success, challenges remain in stability, immunogenicity, and large-scale manufacturing. Nucleic acid-loaded LNPs can be prone to degradation during storage and transportation, prompting efforts in lyophilization and novel preservation methods. Although generally well-tolerated, certain lipid formulations may trigger immune responses, underscoring the need for improved lipid chemistries and surface modifications to minimize immunogenicity [84]. Equally critical is the establishment of robust, scalable production methods capable of maintaining product quality and batch-to-batch consistency, which are essential for regulatory approval and clinical translation [85].

Overall, nucleic acid-loaded LNPs represent a major advance in biomedical science, providing efficient and versatile platforms for genetic medicine. Their ability to deliver therapeutic nucleic acids with precision and scalability has already reshaped the fields of vaccinology and gene therapy. Continued innovations in lipid chemistry, formulation design, and manufacturing processes will further enhance their therapeutic potential and expand their applications across diverse disease areas [85].

6. Cost Considerations and Practical Limitations of Microfluidic Platforms

Despite the significant advantages of microfluidic technologies in producing highly reproducible and size-controlled LNPs, the economic aspects of these systems remain an important consideration for widespread adoption. The initial investment required for microfluidic infrastructure, including precision syringe or pressure pumps, flow controllers, microfluidic mixers, temperature control modules, and downstream purification systems such as TFF, can be substantial, particularly for small research laboratories [86]. In addition to capital equipment, the recurring cost of microfluidic chips represents a practical limitation. Many commercially available microfluidic cartridges are designed for single use to prevent cross-contamination and ensure reproducibility, which can significantly increase experimental expenses, especially during formulation screening and process optimization where multiple conditions must be tested. The disposable nature of certain microfluidic devices may therefore restrict iterative experimentation and large-scale parameter exploration in resource-limited settings [87]. Furthermore, specialized fabrication materials, device customization, and maintenance requirements can add to the overall operational cost. However, it is important to note that microfluidic technologies offer long-term economic advantages through reduced reagent consumption, improved batch-to-batch consistency, automation capability, and scalable manufacturing [88]. Continuous-flow microfluidic production also minimizes process variability and reduces downstream processing requirements, which may ultimately offset initial costs in translational and industrial settings. From a clinical and industrial perspective, advances in reusable microfluidic chips, parallelized systems, and scalable continuous manufacturing platforms are progressively addressing cost barriers. These innovations are expected to lower production costs, enhance accessibility, and facilitate broader adoption of microfluidic technologies in nanomedicine development [89]. Therefore, while cost and device disposability currently represent practical challenges, ongoing technological improvements are likely to improve economic feasibility and accelerate the clinical translation of LNP-based therapeutics.

7. Conclusions and Future Outlook

Microfluidic technologies have emerged as powerful tools for the controlled and reproducible production of LDS. By enabling precise regulation of liposome diameter, size distribution, and lamellarity, microfluidics enhances drug loading efficiency and overall formulation performance. Unlike conventional manufacturing methods, which are often associated with batch-to-batch variability, microfluidic platforms provide consistency, scalability, and the potential for continuous production. Importantly, the design of microfluidic mixers and the careful adjustment of process parameters, including flow rate, solvent ratio, and lipid concentration, are critical for achieving homogeneous formulations with predictable physicochemical properties. Solvent selection influences lipid dissolution and vesicle stability, while lipid concentration directly affects particle size and encapsulation efficiency. Collectively, these parameters allow fine-tuning of nanocarrier characteristics to meet therapeutic requirements. The versatility of microfluidic systems has facilitated their application across a broad range of biomedical domains, including drug delivery, nucleic acid delivery, vaccine development, and diagnostic assays. Their ability to generate nanoparticles with high reproducibility and tailored properties has accelerated translational research and positioned microfluidics as a cornerstone technology in nanomedicine. Despite these advances, important challenges remain. The most significant limitation is the relatively low production volume of current microfluidic platforms, which complicates direct translation to industrial-scale manufacturing. Achieving large-scale production while maintaining product quality and uniformity remains an active area of investigation. In addition, the frequent use of organic solvents during nanoparticle synthesis raises safety concerns, underscoring the need for efficient solvent removal strategies to ensure clinical applicability. Efforts are underway to address these challenges through optimization of post-processing methods, integration of real-time quality monitoring, and the adoption of continuous flow manufacturing strategies.

Looking forward, opportunities lie in the integration of additive manufacturing and advanced fabrication techniques to further improve microfluidic device design and scalability. Innovations in materials engineering and platform miniaturization are expected to expand the versatility of these systems. With continued refinement, microfluidic approaches will not only optimize the production of lipid-based nanoparticles but also accelerate their translation into clinically approved therapeutics and vaccines. In summary, microfluidics offers a transformative pathway for the development of next-generation lipid nanomedicines. By uniting precision engineering with biomedical innovation, these platforms promise to streamline formulation development, enhance therapeutic performance, and ultimately enable broader clinical adoption of lipid-based drug delivery systems across diverse disease indications.