Layered drug nanoparticles can now be produced efficiently and at scale using a microfluidic method that eliminates purification and improves consistency.
(Nanowerk Spotlight) Precise control over drug delivery—timing, dosage, location within the body—has remained a central goal in pharmaceutical engineering. Nanoparticles have emerged as promising tools to meet this need, offering tunable size, surface chemistry, and the ability to encapsulate or conjugate therapeutic agents. Among the techniques for engineering nanoparticle surfaces, layer-by-layer (LbL) assembly stands out for its modularity. This method builds alternating layers of positively and negatively charged polymers around a particle core, creating tailored interfaces that influence drug release profiles, cell targeting, and stability in biological fluids.
Since its adaptation to nanoscale materials, LbL coating has enabled drug carriers with extended circulation time, resistance to premature clearance, and programmable interaction with specific cell types. However, despite its utility in laboratory settings, LbL synthesis for nanoparticles has been difficult to implement at scale. Traditional methods rely on excess polymers and repetitive purification cycles to remove unbound materials. These steps introduce inefficiencies, lower yield, and complicate reproducibility. Additionally, mixing techniques such as sonication or vortexing often lead to particle aggregation, especially in larger batches, limiting their applicability in clinical manufacturing.
Attempts to streamline LbL assembly have included fluidic systems and size-selective devices, but many of these have been restricted to larger microparticles or required heating steps that are incompatible with sensitive nanoparticle formulations. Methods based on magnetic separation, bed filtration, or thermal-assisted deposition remain scale-sensitive and often incompatible with the sub-200 nm particles used in drug delivery. These limitations have created a gap between promising laboratory designs and viable therapeutic formulations.
A new study published in Advanced Functional Materials (“High-Throughput Microfluidic-Mediated Assembly of Layer-By-Layer Nanoparticles”) by researchers at the Koch Institute for Integrative Cancer Research at MIT presents a microfluidic approach to LbL nanoparticle assembly that addresses these bottlenecks. The method allows nanoparticles to be coated efficiently without the use of excess polymer or the need for intermediate purification, providing a more direct route to clinical-scale production .
The process uses bifurcating microfluidic mixers—commercially available cartridges that split and recombine fluid streams repeatedly to ensure rapid, uniform mixing. The key innovation is the precise use of what the researchers define as the plateau onset point (POP), the minimum polymer-to-nanoparticle weight ratio required to fully reverse surface charge without leaving unbound polymer in solution. When particles are coated at this ratio, polymer deposition is complete and no excess remains, making purification unnecessary.
As a test case, the team focused on nanoparticles carrying interleukin-12 (IL-12), an immune-activating protein studied for cancer therapy. These were constructed using a liposomal core conjugated with IL-12 and layered with poly(L-arginine) and poly(L-glutamic acid), forming a surface designed to enhance tumor targeting and control retention at the cellular membrane.
Standard LbL methods require using more polymer than needed for coating, which prevents aggregation but necessitates washing steps. By precisely calibrating polymer input to the POP, the researchers showed that charge reversal could be achieved without using excess material. However, in static conditions, such precise ratios were insufficient at scale—large batches mixed without excess polymer tended to aggregate unless sonication and filtration were added. The microfluidic mixer, by contrast, allowed efficient polymer adsorption under flow, preventing aggregation even when operated at clinical-relevant batch sizes .
How polymer ratio affects nanoparticle stability during coating. Schematic illustration of how varying the amount of polymer added to nanoparticles influences surface charge and particle behavior. At low ratios, particles remain undercoated and unstable. Near the neutral charge point, they tend to clump together due to lack of repulsion. Only at the optimal ratio—known as the plateau onset point (POP)—is the surface fully coated, charge-reversed, and stabilized. This explains why conventional methods require excess polymer and purification, whereas precise mixing at POP enables stable assembly without those steps. (Image: Reprinted from DOI:10.1002/adfm.202503965, CC BY)
The system was configured in two stages. In the first, IL-12 liposomes were combined with poly(L-arginine) under controlled flow; after incubation, a second mixer introduced poly(L-glutamic acid). The resulting particles matched the size and surface charge of those produced via traditional methods. More importantly, they exhibited lower polydispersity and batch-to-batch variation. This improved uniformity suggests better reproducibility in downstream applications .
To test whether these microfluidically prepared nanoparticles retained their desired biological behavior, the researchers compared them with particles produced using their earlier tangential flow filtration (TFF) protocol. Both types showed similar uptake patterns in an ovarian cancer cell line. Notably, the LbL-coated particles remained on the cell surface after 24 hours, consistent with the design goal of maximizing local cytokine presence at the tumor interface. Particles without coating were instead internalized by the cells.
In vivo testing in a mouse model of metastatic ovarian cancer showed that both TFF- and microfluidic-derived particles slowed tumor progression and extended survival compared to uncoated nanoparticles or free IL-12. The similarity in outcomes confirmed that the streamlined process preserved the therapeutic properties of the formulation.
The flexibility of the microfluidic method was further demonstrated using alternative polyelectrolytes. Polymers such as hyaluronic acid, poly-L-aspartic acid, and polyacrylic acid each displayed distinct POP values, but the same principles applied—precise mixing at the POP allowed successful film formation without excess. The team also used synthetic latex particles of varying sizes to examine how surface area influenced coating efficiency. Smaller particles required higher polymer input due to their greater surface-to-volume ratios, and when tested for cell binding, the coated particles showed enhanced affinity compared to uncoated controls. Interestingly, the relative improvement in binding correlated with the available surface area, confirming a predictable relationship between nanoparticle geometry and coating performance.
Another practical consideration is compatibility with buffer systems. Many LbL formulations use pure water, but some therapeutic payloads, such as RNA, require salt-containing environments. The researchers tested their method in a buffer containing sodium chloride and HEPES and found that POP-based coating still functioned effectively. Microfluidic mixing maintained particle quality under these conditions, further supporting its generalizability across different formulations.
The significance of this work lies not in a new type of nanoparticle or therapeutic effect, but in the way these particles are manufactured. The microfluidic method reduces material waste by eliminating excess polymer use, avoids yield loss from purification, and can be scaled using existing good manufacturing practice (GMP)-compatible systems. The process improves uniformity, reduces hands-on time, and enables rapid generation of particle libraries with different coatings or sizes for screening purposes.
This approach addresses a persistent gap between small-scale nanoparticle research and the logistical demands of therapeutic development. By enabling consistent, reproducible, and purification-free assembly, microfluidic-based LbL synthesis may accelerate both the experimental evaluation of nanoparticle libraries and the clinical translation of nanoparticle-based treatments.
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