A. Thomas, A. Brown, S.M. Garg, K. Ou, J. Singh, S. Chang, M. Ma, S. Sidhu, B. Versteeg, M. Assadian, A. Armstead, G. Heuck, S. Ip, T.J. Leaver, A.W. Wild, R. Lockard, R.J. Taylor, E.C. Ramsay
Precision NanoSystems Inc.,
Keywords: Liposomes, Lipids, Verteporfin, Hydrophobic Drugs, Hydrophilic Drugs, Scale Up
Summary:Purpose: Conventional methods for the liposome production are labor-intensive and pose challenges to size-control, scale-up, and reproducibility. The NanoAssemblr™ platform is a microfluidic technology that eliminates user variability and is capable of rapid, reproducible, and scalable manufacture of nanoparticles.1,2. We describe the microfluidic manufacture and in situ loading of the photodynamic therapy drug (PDT) verteporfin in liposomes composed mainly of natural egg and soy phosphatidylcholines (PC). Verteporfin has been explored as a sensitizer for cancer PDT. System parameters (mixing speed and mixing ratio), and formulation parameters were systematically explored to optimize size and drug loading at bench scale. Scale-up production was demonstrated by direct transfer of optimized conditions to the NanoAssemblr™ Blaze™ for preclinical scale-up, and the NanoAssemblr™ Scaleup System (with 8 parallel microfluidic mixers) designed for the cGMP environment. Methods: Liposomes were manufactured using the NanoAssemblr Benchtop, Blaze and Scaleup systems (Precision NanoSystems Inc., Vancouver, Canada). Lipids of varying compositions and concentrations in ethanol were nanoprecipitated by mixing with aqueous buffer in proprietary microfluidic mixers. The PDT drug verteporfin was loaded in situ, and comparisons between formulations containing soy- and egg- PC were compared to those containing POPC and DSPC. Results: Microfluidic mixing enabled rapid and consistent manufacturing of liposomes having diameters ranging from 25 – 75 nm depending on parameters such as aqueous:organic flow rate ratio, and lipid composition (Figure 1). Verteporfin was loaded in situ, with different liposome compositions. Figure 2 shows encapsulation efficiencies achieved using natural and synthetic PCs as the main component. Ninety percent (90%) encapsulation efficiency was achieved with soy-PC liposomes. Ongoing studies are further characterizing the encapsulation of hydrophobic and hydrophilic model drugs into liposomes as a function of particle size. Liposome formulations produced on the Blaze and 8x Scale-up systems were identical in size and PDI (< 0.1) to Benchtop batches demonstrating seamless scale up of liposome formulations and overcoming challenges associated with conventional liposome production. Conclusions: Herein, we have demonstrated the optimization of verteporfin-loaded egg- and soy-PC liposome formulations of defined size and drug encapsulation efficiencies of 90% using the NanoAssemblr™ Benchtop, and proof of concept of streamlined scale-up of liposome formulations. Liposomes manufactured using this platform have the potential to be used as delivery vehicles for small molecule therapeutics.