Within this research we have looked at the ability to de-risk the translation of liposomes from bench to the clinic. We have used microfluidics for the rapid and scale-independent manufacture of liposomes and have incorporated in-line purification and at-line monitoring of particle size. Using this process, we have manufactured a range of neutral and anionic liposomes incorporating protein. Factors investigated include the microfluidics operating parameters (flow rate ratio (FRR) and total flow rate (TFR)) and the liposome formulation. From these studies, we demonstrate that FRR is a key factor influencing liposome size, protein loading and release profiles. The liposome formulations produced by microfluidics offer high protein loading (20-35 %) compared to production by sonication or extrusion (< 5%). This high loading achieved by microfluidics results from the manufacturing process and is independent of lipid selection and concentration across the range tested. Using in-line purification and at-line size monitoring, we outline the normal operating range for effective production of size controlled (60 to 100 nm), homogenous (PDI <0.2) high load liposomes. This easy microfluidic process provides a translational manufacturing pathway for liposomes in a wide-range of applications.
Figure 1: The effect of liposomal formulation on physicochemical characteristics of liposomes produced by micofluidics. Four liposome formulations (PC:Chol, DMPC:Chol, DPPC:Chol, and DSPC:Chol) with increasing hydrocarbon tail length were manufactured using microfluidics at a 3:1 FRR, 15 mL/min TFR and purified using dialysis. The effect of PC lipid chain length on A) liposomes z-average size (d.nm) and B) PDI. DSPC:Chol liposomes were selected and the effect of cholesterol content and heating block temperature were investigated in regards to C) z-average particle size and D) PDI. Results represent mean ± SD, n=3 independent batches.
Figure 2: The effect of microfluidic parameters on neutral and anionic liposome attributes. The effect of the initial lipid concentration on average liposome size (d.nm; represented by bars) and PDI (represented by discrete points) for liposomal formulation DSPC:Chol (10:5 wt/wt) (10 mL/min TFR) at a flow rate ratio of A) 1:1, B) 3:1, and C) 5:1. D) The effect of increasing initial lipid concentration on z-average particle size (d.nm) for DSPC:Chol:PS (10:5:4 wt/wt) (3:1 FRR, 10 mL/min TFR). E) Investigating the effect total flow rate (mL/min) on z-average particle size (d.nm) and PDI for liposomal formulation DSPC:Chol (4 mg/mL initial lipid, 3:1 FRR). F) The effect of total flow rate (mL/min) for anionic formulation DSPC:Chol:PS (4 mg/mL initial lipid, 3:1 FRR). Results represent mean ± SD, n =3 of independent batches.
Figure 3: Purification of liposomes using tangential flow filtration (TFF). Liposomes (DPPC:Chol; FRR 3:1, 15 mL/min TFR) were prepared and characterised as follows: A) Residual solvent (methanol) remaining in liposomes after consecutive wash cycles, B) removal of non-incorporated protein via TFF, C) liposome attributes before and after purification via TFF and cryo-EM images of liposomes before and after TFF purification. Results represent mean ± SD, n =3 of independent batches.
Figure 4: Concentration of liposomal formulations using tangential flow filtration. DSPC:Chol (10:5 wt/wt) and DSPC:Chol:PS (10:5:4 wt/wt) were prepared at 4 mg/mL initial lipid concentration, 3:1 FRR, 15 mL/min TFR following microfluidics, followed by 1,2 and 4 fold concentration steps. Particle Size (Z-Avg; represented by bars) and PDI (represented by discrete points) for A) DSPC:Chol and B) DSPC:Chol:PS both prepared C) and D) are intensity plots for the same conditions. Results represent mean ± SD, n =3 of independent batches.
Figure 5. Manufacture of protein loaded liposomes using microfluidics. A) Ovalbumin loading and physicochemical comparison between microfluidics and lipid-film hydration followed by extrusion or sonication. DSPC:Chol (10:5 wt/wt) liposomes were made with 1 mg/mL final total lipid and 0.18 mg/mL ovalbumin. The encapsulation efficiency, size and PDI of the liposomes. B) Microfluidics was further tested with respect to changes in lipid hydrocarbon tail length and concentration. Protein encapsulation, size and PDI for PC:Chol, DMPC:Chol, DPPC:Chol and DSPC:Chol (4 mg/ mL initial lipid and 0.25 mg/ mL Ovalbumin) made using microfluidics (3:1 FRR and 15 mL/ min TFR). C) The structural integrity of ovalbumin loaded into the liposomes measured by circular dichroism. DSPC:Chol (10:5 wt/wt) liposomes were prepared with OVA (8 mg/mL initial total lipid and OVA, 3:1 FRR, 15 mL/min TFR) and purified via TFF. Spectra was measured across 180 – 260 nm D) Log-log plot of lipid concentrations (0.5- 10 mg/ mL) against encapsulation efficiency (0.25 mg/ mL ovalbumin). Results represent mean ± SD, n=3 independent batches.
Figure 6: The effect of protein concentration in aqueous phase on entrapment efficiency and liposomal physicochemical characteristics for a neutral liposomal formulation (DSPC:Chol; 10:5 wt/wt) (A to C) and anionic formulation (DSPC:Chol:PS; 10:5:4 wt/wt) (D to F) using initial total lipid concentration of 4 mg/mL, 3:1 flow rate ratio and 15 mL/min TFR. A) Entrapment efficiency and protein loading across initial ovalbumin concentrations for neutral liposomal formulation. B) Average particle size and PDI, and C) Zeta potential for the same formulation. D) Entrapment efficiency and protein loading across initial ovalbumin concentrations for anionic liposomal formulation, E) Average particle size and PDI and, F) Zeta potential for the same formulation. In B) and E) particle size is shown by bars and PDI is shown by discrete points. Results represent mean ± SD, n=3 of independent batches.
Figure 7: Microfluidic manufacture of neutral and anionic liposomes encapsulating protein. Both neutral (DSPC:Chol; 10:5 wt/wt) (A to D) and anionic (DSPC:Chol:PS; 10:5:4 wt/wt) (E to H) were tested: A) Entrapment efficiencies for flow rate ratios 3:1 and 5:1 for DSPC:Chol (final lipid and OVA concentrations matched at 1 mg/mL and 0.525 mg/mL respectively). B) Average particle size and PDI for the same formulation C) Entrapment efficiencies with varying on total flow rate (mL/min) (3:1 flow rate ratio, 4 mg/mL and 0.7 mg/mL initial total lipid and OVA respectively) and D) the resulting particle size (d.nm) and PDI with varying total flow rate. E) Effect of flow rate ratio on entrapment efficiency for DSPC:Chol:PS (final lipid and OVA concentrations matched at 1 mg/mL and 0.525 mg/mL respectively). F) Entrapment efficiencies with varying on total flow rate (mL/min) (3:1 flow rate ratio, 4 mg/mL and 0.7 mg/mL initial total lipid and OVA respectively) and H) the resulting particle size (d.nm) and PDI with varying total flow rate. Where appropriate liposome size is shown by bars and PDI is shown by discrete points. Results represent mean ± SD, n=3 of independent batches.
Figure 8: Proof-of-concept scale-out studies. DSPC:Chol liposomes were prepared at a FFR 3:1, TFR 15 mL/min, and a concentration of 10 mg/mL on both the NanoAssemblr (total volume 2 mL) and Blaze (total volume 20 mL). A) The protein loading, size and polydispersity index (PDI) of both batches and B) an overlay of the intensity plots for both batches.
Figure 9. Protein release from liposome formulations manufactured by microfluidics. A) The release of OVA from PC:Chol, DMPC:Chol, DPPC:Chol and DSPC:Chol liposomes produced at a 3:1 ratio (15 mL/mi TFR) over 120 hours. B) Ovalbumin release from DMPC:Chol liposomes produced at a 3:1 and 5:1 FRR (15 mL/min TFR.) C) Ovalbumin release from DSPC:Chol liposomes produced at a 3:1 and 5:1 FRR (15 mL/min TFR, matched at a final concentration of 1 mg/mL total lipid, 0.1875 mg/mL OVA) Liposome suspensions were kept at 37°C with agitation. At set times, the sample was collected the OVA remaining inside the liposomes quantified. The results represent the mean of three independent batches ± SD.
Figure 10: Liposome at-line particle size monitoring as part of a production train. DSPC:Chol liposomes were prepared at a FFR 3:1, TFR 15 mL/min, and a concentration of 10 mg/mL. The size and polydispersity index (PDI) of all formulations were measured (at a ratio of 1:10) off-line using the Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) or at-line as part of the automated continuous manufacturing process using the Zetasizer AT (Malvern Instruments, Malvern, UK). To characterise liposomes in real time, the Zetasizer AT measured liposome size and PDI at a 1:10 dilution (liposomes to buffer), with adjustments to the automated mixing possible. The buffer (5 mL/min) and liposome formulation (0.5 mL/min) are taken up by the instrument, and enter into the flow cell where the size and PDI was measured. A total of 1 mL was required for each size measurement. Liposome formulations were purified using the KrosFlo Research Iii TFF system.