(K-440) Stereolithography 3D Printed Microfluidic Device for Liposomal Synthesis

Introduction:: In recent years, nanoparticles have been highly investigated for their potential to deliver therapeutic and/or diagnostic agents in cancer. This can be attributed to their tunable physiochemical properties allowing for protection of these agents from biological barriers, improved circulation times, and site-specific targeting. Of the many forms of nanoparticles, liposomes in particular, are of gaining interest due to their innate biocompatibility and sustained/controlled drug release. Conventional methods for liposomal synthesis, such as thin-film hydration, rely on bulk particle production, which generates a heterogeneous population of liposomes. These particles then require additional sonication and filtering to create a more homogeneous liposomal size distribution. Microfluidic device (MFD) is an emerging technology for liposome synthesis [1]. MFD enables precise control over mixing processes that in turn allows for direct control over the resulting liposomal distribution. Furthermore, MFD allows for continuous flow production of liposomes and has been shown to enhance encapsulation efficiency of loaded therapeutic and diagnostic agents. However, MFD production using conventional methods including photolithography are complex, time consuming, and costly. In the present study, we have investigated the potential for stereolithography (SLA) 3D printing techniques to develop a MFD for liposomal synthesis.


Materials and Methods:: The MFD device was designed to have three inlet channels each 8mm in length. All inlet channels were connected to a single linear channel 40mm in length for hydrodynamic flow focusing, a common technique implemented in MFD based liposome production. Each channel was designed to have a square cross-section of 790 x 790 microns. 20mM lipid mixtures in ethanol were prepared at a molar ratio of DSPC:Cholesterol:DSPE-mPEG = 5.1:4:0.9. For thin-film hydration, 110uL of the lipid mixture was further diluted in 2mL of ethanol followed by solvent removal using a rotary evaporator. The resulting thin lipid film was then rehydrated using PBS followed by water bath sonication for 5 minutes and sonic probe sonication at 30W for another 2 minutes. For MFD production of liposomes, 110uL of the lipid mixture was further diluted with 390uL of ethanol. A syringe pump was used to control the lipid mixture through the central inlet channel at a flow rate of 0.25mL/min. Water was simultaneously injected into the two adjacent inlet channels at a flow rate of 12mL/min (total flow rate = 12.25mL/min; flow rate ratio = 48). All resulting particles from both techniques were then filtered with a 0.45um membrane. Each experiment was repeated in triplet and all samples were analyzed using dynamic light scattering (Malvern Zetasizer Nano ZS90) to determine size, polydispersity index (PDI), and zeta potential.


Results, Conclusions, and Discussions:: As shown in Figure 1, the size distribution of liposomes produced using the SLA 3D printed MFD and thin-film hydration were highly comparable. Liposomes produced by the MFD were found to be smaller on average compared to the conventional method but did not reach statistical significance (p=0.086). Furthermore, no significant difference was found for the zeta potential (p=0.111) and PDI (p=0.435) between the MFD and thin-film hydration methods.



In conclusion, the current study has shown that the SLA 3D printed MFD can produce liposomes highly comparable to conventional bulk methods. Further investigations of the impact of varying the total flow rate, flow rate ratios, and device dimensions in the MFD are underway. Similarly, investigations will be performed of the ability of the MFD to enhance liposomal encapsulation efficiency of both therapeutic and diagnostic agents.


Acknowledgements (Optional): : This research was supported in part by NIH/NCI 1R01CA264102-01.


References (Optional): : [1] Shepherd SJ et al. Biomaterials. 2021. 274:120826.