(W1230-02-10) Exploring Dual Centrifuge Wet Media Milling Process as an Organic Solvent Free Method to Prepare Liposomes with High Drug Loading

Purpose: Liposomes have gained popularity as an effective drug delivery system, owing to their ability to encapsulate various drugs and deliver them to targeted sites. It is amongst the most clinically approved and successful nanoformulation. However, Drug loading and large scale manufacturing with multi-steps processing pose significant challenges. Physicochemical properties of liposomes, including their size, shape, surface charge, and stability, are significantly influenced by the choice of phospholipids and method of preparation. Wet media milling, a widely used particle size reduction technique, has been successfully used to formulate nanoparticles. In this study, we aimed to develop and characterize liposomes using wet media milling with three chemically diverse water-insoluble drugs, Amphotericin B (AMB), Docetaxel (DTX), and Irinotecan (IRN) with different phospholipids (DOPC, DPPC, and HSPC) to evaluate their physicochemical properties. To the best of our knowledge, this is the first study to make liposome formulation for hydrophobic drug entrapment using wet media milling technique. This method can potentially offer several advantages, including rapid and sterile preparation of liposomes with very high drug loading and encapsulation efficiency. This study demonstrates feasibility of preparing liposomes of hydrophobic drugs by wet media milling and eliminates the additional steps of organic solvent removal and particle size reduction.

Methods: A free-flowing pro-liposome powder formulation of three different phospholipids (DOPC, DPPC, and HSPC) was prepared using modified solvent evaporation method (Fig. 1) with a molecularly dispersed mixture of phospholipids and cholesterol in a 3:1 molar ratio. Pro-liposome powder was then used to prepare AMB, IRN and DTX loaded liposomes using a dual centrifugation wet media milling method. Drug and pro-liposomal powder were mixed with water and immediately subjected to media milling. No organic solvent was used in processing. Each batch consisted of the lipid mixture, the aqueous solution, and 0.3 mm diameter zirconia beads, and milling process was carried out at 0 °C with 1 hour milling time at 1500 rpm/1.3 g milling beads. Various techniques were used to characterize the liposomes, including % Entrapment efficiency (%EE), % Drug loading (%DL), drug concentration (mg/mL), particle size, polydispersity index (PDI), and zeta potential. %EE for all three drugs was analyzed using a developed and validated HPLC method. Dynamic light scattering (DLS) was used to determine particle size, polydispersity index, and zeta potential of the liposome formulations. Cryo-TEM imaging was carried out to confirm the formation of liposomal structure as well as size. A graphical representation of study has been shown in Fig. 1.

Results: Physicochemical characterization, including particle size, polydispersity index (PDI), zeta potential, % Entrapment efficiency (%EE), % Drug loading (%DL), and drug concentration (mg/mL) of the liposome formulations were determined and presented in Fig. 2. Processing and preparation of all the drug molecules was very simple and time efficient. For AMB, the liposomes prepared using DPPC had smallest particle size (125.7 nm) and lowest PDI value (0.188), indicating good uniformity of liposome population. All three phospholipids showed high encapsulation efficiency, but DOPC had highest %EE (99.1%) and %DL (5.60%), as well as highest drug concentration (mg/mL) at 4.20 mg/mL compared to the other phospholipids. For DTX, liposomes prepared using DOPC showed smallest particle size (152.75 nm) and lowest PDI value (0.227), highest %EE of 99.1%. DOPC also had highest %DL of 6.9%, and highest drug concentration (mg/mL) was observed at 5.0 mg/mL. In contrast, liposomes prepared using DPPC had a larger particle size of 168.05 nm, and very low %EE (17.1%) and %DL (1.1%) among the other phospholipids (DOPC and HSPC). For IRN, the liposomes prepared using DOPC and DPPC showed smallest particle size 119.6 nm and 130.1 nm, respectively. Zeta potential was negative for all three phospholipids, with highest absolute value observed for liposomes prepared using DPPC (-9.78 mV). DPPC demonstrated highest drug encapsulation efficiency (%EE) of 98.1% and highest drug loading (%DL) of 6.40%. Furthermore, DPPC showed highest drug concentration of 4.8 mg/mL, among the different lipids tested. Formulations were stable for 1 months at 4 ˚C. Cryo-TEM micrographs showed the presence of small and large unilamellar vesicles, as well as multilamellar and multivesicular vesicles, in both AMB and IRN formulations, as shown in Fig. 3.

Conclusion: The study found that wet media milling is an effective technique for preparing liposomes with high %EE and drug loading for AMB, IRN, and DTX. Noteworthy, liposomal formulation of AMB and IRN are available commercially. Therefore, preparation of liposomes loaded with AMB and IRN using this novel method could be helpful in justifying the commercial viability of this technology. The choice of phospholipid had a significant impact on the %EE and drug loading of the liposomes. Overall, the results demonstrate that wet media milling is a promising method for the preparation of liposomal drug delivery systems, and the choice of phospholipid can be tailored to optimize drug encapsulation and delivery.

Graphical Representation for Liposome formulation development by wet media milling techniques

Illustrates the physicochemical characterization of liposomes using dynamic light scattering graphs to show particle size and polydispersity index (PDI) for three different lipids: (a) DOPC, (b) DPPC, and (c) HSPC. Graph (d) shows the ζ-potential of the liposome formulations, while (e) compares the entrapment efficiency and drug loading capacity of liposomes with the three different lipids.

Cryo-TEM images displaying the morphology of liposomes with different lamellarities. (A and B) DOPC-based liposomes encapsulating AMB, and (C and D) DPPC-based liposomes encapsulating IRN. Scale bar = 200 nm.