Professor University of Maryland-college park, United States
Introduction: Lipid nanomedicines have emerged as crucial players in modern therapeutics, notably in the development of COVID-19, mRNA vaccines, and cancer treatments. These advanced drug delivery systems rely on lipid nanoparticles to encapsulate and protect therapeutic agents, ensuring targeted delivery and controlled release. The production of lipid nanomedicines benefits significantly from continuous flow systems, which offer enhanced efficiency and scalability in lipid preparation, drug loading, and purification. Central to this process is buffer exchange, with microdialysis standing out as a particularly effective method. This technique ensures the removal of unencapsulated drugs and by-products, thereby improving the purity and effectiveness of the final product.
One critical aspect of this production is the study of pH shifts, which is essential for the optimal loading of certain drugs into lipid nanoparticles. Understanding and controlling these pH changes can significantly impact the encapsulation efficiency and stability of the therapeutic agents. Post-loading purification is equally vital, as it ensures that the final nanomedicine formulations have the desired therapeutic properties. Despite these advancements, challenges remain in optimizing pH shift studies and scaling up purification processes. Addressing these challenges is essential to enhance the production and effectiveness of lipid nanomedicines, for more robust and widely accessible treatments.
This study presents a continuous-flow microfluidic-enabled microdialysis technology capable of scaling to arbitrarily high flow rates and nanoparticle throughput by leveraging a novel multilayer fabrication process. Our design overcomes several important limitations of current systems, offering high-throughput in-line buffer exchange for broad applications in lipid nanomedicine preparation and beyond.
Materials and
Methods: The microfluidic device comprises two cross channels with a 0.025 µm pore size hydrophilic mixed cellulose ester (MCE) membrane sandwiched between them. Each microdialysis layer includes pressure-sensitive adhesive and polycarbonate substrates containing microchannels (Fig1). Numerical simulations were studies using COMSOL Multiphysics 6 predicted ion concentration profiles during microdialysis. Dye-loaded liposomes were prepared by combining DMPC, cholesterol, and DCP in chloroform, forming a thin lipid film, which was vacuum dried and dissolved in dehydrated ethanol. Fluorescein sodium salt was dissolved in PBS, and dye-loaded liposomes were produced using a microfluidic vortex focusing technique. Citrate buffer (pH 2.5) and isosmotic HEPES buffer (adjusted to pH 7.4 and 9.4) were prepared for microdialysis, filtered through 0.22 μm filters. Multilayered microfluidic devices with 1, 2, and 4 layers (Fig. 1) were used to evaluate buffer exchange efficiency for pH shift in acidic buffer. Citrate sulfate (pH 2.5) and isosmotic HEPES (pH 7.4 or 9.4) were introduced, with flow velocities ranging from 7 μL min⁻¹ to 49 μL min⁻¹. Fluorescein-loaded liposomes were injected into the microdialysis chip with isosmotic HEPES (pH 7.4) for buffer exchange, and absorbance measurements were analyzed to determine purification efficiency using a fluorescence microscope (Nikon TE2000-S) and ImageJ software.
Results, Conclusions, and Discussions: Device Fabrication bonding quality of the multilayer microdialysis device, using pressure-sensitive adhesive and PC, ensures reliability under operational pressures and flow rates without leakage. The modular design allows straightforward scaling, as each additional layer uses the same bonding procedure, facilitating increased throughput or extended residence times with minimal complexity. (Fig2).
Numerical Simulations Two-dimensional numerical simulations using COMSOL Multiphysics 6 were conducted to predict citrate ion transport during microdialysis for a multilayer device. The model indicated a 30-fold reduction in citrate ion concentration with increasing the microdialysis layers. Higher flow rates in the sample and dialysate streams promote faster ion transport and reduce residence times. (Fig. 3).
Ion Exchange The device's ion exchange capabilities were evaluated by introducing buffers with varying pH values through the sample and counterflow inlets and the sample flow exhibited a rapid pH change. Increasing the number of microdialysis layers extended the residence time, leading to pH shift from 1 to 5. A maximum pH shift of 5 units was achieved with counterflow buffers of pH 7.4 and an 8 min residence time. (Fig. 4).
Lipid Nanoparticle Purification Fluorescein-loaded liposomes were prepared and injected into the microdialysis devices, and fluorescence intensity in each purified sample was measured to calculate dialysis efficiency. The 4-layer microdialysis device achieved approximately 80% purification efficiency, compared with 35% efficiency for a 1 layer microdialysis chip (Fig. 5).
• Discussion These results demonstrate the improved microdialysis performance that can be achieved by increasing the number of chip layers, thereby increasing sample residence time within the device for a given flow rate. Alternatively, the system can achieve higher throughput while maintaining the same performance, highlights the device's adaptability for different operational scales, making it a versatile tool for continuous-flow microdialysis.
• Conclusion By incorporating multiple layers of hydrophilic MCE membranes within a thermoplastic microfluidic flow cell, the presented continuous-flow microdialysis technology can enhance scalability and performance for lipid nanomedicine drug loading and purification. This advancement can open new avenues for applications in nanomedicine development, as well as wider use in pharmaceutical biochemical research and manufacturing where scalable in-line microdialysis is needed.
