Nanoassemblr benchtop instrument

All LNPs were prepared by combining appropriate volumes of cationic lipid (DODMA alone (DODMA-LNPs)or a combination of DODMA, DODAP and DOTAP lipids (Hydrid LNPs); 50 mol% unless otherwise stated), helper lipid (DOPE; 11.5 mol%), PEGylated lipid (PEG-DMPE; 1.0 mol%; unless otherwise stated), and cholesterol (37.5 mol%; unless otherwise stated) in anhydrous ethanol with plasmid DNA in 25 mM sodium acetate (pH 4.0) at a nitrogen to phosphorus (N/P) ratio of 6:1 using the NanoAssemblr Benchtop instrument and microfluidic cartridges (Precision Nanosystems, Vancouver, BC, Canada) set to a combined flow rate of 9 ml/min [flow rate ratio of 3 (DNA/aqueous) to 1 (ethanol/lipid)]. The total lipid concentration was maintained at 14.68 mM and the cholesterol content was varied to offset changes in cationic lipid or PEGylated lipid content. The LNP formulations were diluted with PBS (pH 7.4, without magnesium and calcium; Gibco) and subjected to three sequential diafiltration steps using Amicon Ultra centrifugal filters with a nominal molecular weight limit of 10 kDa (Millipore, Etobicoke, ON, Canada)^{5 (link)}.

The required amount of individual lipids according to the desired composition (Table 2) were dissolved in anhydrous ethanol and in a mixture of EtOH:DMSO (1:1) for composition 2,3,4 and 1, respectively at 4 mg/ml. The organic and aqueous phase (Milli-Q water) were rapidly mixed using the NanoAssemblr Benchtop instrument (Precision NanoSystems, Canada) at defined Flow Rate Ratios (FRR) 1:3 and Total Flow Rate (TFR) 7 ml/min to form unilamellar liposomes 1 mg/ml of lipid. During the mixing process, the temperature was controlled using NanoAssemblr Benchtop Heating Controller accessory (Precision NanoSystems, Canada). For preparation of liposomes containing DSPC, the temperature was set to 65 °C, respectively. NanoAssemblr instrument setting is presented at Fig. 9.Table 2

Lipid compositions of the liposome formulation.

lipid	composition (molar % of lipid)
	1	2	3	4	5
18:0 PE DTPA (Gd)	5	—	—	—	—
DMPC	—	—	—	—	100
SOPC	—	—	—	—	100
DSPC	—	—	(90–50)	—	—
EPC	65	70	—	(100–50)	—
Cholesterol	30	30	(10–50)	(0–50)	—

Figure 9

NanoAssemblr instrument settings with precise measurement of the temperature in the mixing cartridge and Zetasizer Ultra for size distribution measurement (A), Detail of microfluidic mixing “herring bone” structure in the NanoAssemblr Microfluidic Cartridge (B).

Ionizable LNPs were kindly provided by EnhancedBio Inc. (Seoul, South Korea). LNPs were prepared using the NanoAssemblr Benchtop Instrument (Precision Nanosystems Inc., Vancouver, Canada) according to a previously described method [28 (link)]. The lipid components (ionizable lipid, distearoylphosphatidylcholine, cholesterol, and polyethylene glycol lipid at a molar ratio of 42.5:13:43.5:1.0) were dissolved in ethanol, and HPV16 E6/E7 siRNAs (target sequence: 5’-GAC CGG UCG AUG UAU GUC UUG-3’) were dissolved in 50 mM sodium acetate. The final weight ratio of ionizable lipid to RNA was 7.5:1, and the final volume ratio was 1:3. LNPs were formulated by microfluidic mixing of the prepared solutions at a flow rate of 12 ml/min. The resulting LNPs were dialyzed against 1X phosphate-buffered saline (PBS) using dialysis cassettes with a 10,000 molecular weight cutoff (Life Technologies, CA, USA) for 16 hours to exchange the buffer. To characterize the prepared LNPs, dynamic light scattering was used to confirm their size, polydispersity index (PDI), and zeta potential. The encapsulation efficiency of RNAs was measured using the Quant-iTTM Ribogreen Assay (Life Technologies, CA, USA).

A NanoAssemblr Benchtop instrument (Precision NanoSystems) was used to prepare the O/W nanoemulsions. The disperse, organic phase was comprised of an ethanolic solution containing surfactants (Tween 80: Span 80; 2.3:1 v/v) with different concentrations of hempseed oil. Deionized water was used as a continuous aqueous phase. Instrument parameters total flow rate and flow rate ratio (aqueous: organic) were varied in the ranges of 2–12 mL/min and 1:1–4:1, respectively, in order to determine their effect on emulsion droplet size and stability. Ethanol was removed by dialysis (10 kDa MWCO dialysis bags) in deionized water at room temperature over 24 h and then samples were stored at 4 °C post dialysis. The displacement of organic and aqueous phases in a microfluidic mixer is shown schematically in Fig. 1.Figure 1

Controlled solvent displacement in a microfluidic mixer: (1) Organic (ethanol) and aqueous (PBS) phases enter the channel under laminar flow without mixing; (2) Microscopic features in the channel cause fluid streams to mix in a controlled fashion; (3) Intermingling of the phases increases through the mixer; (4) Fluids emerge, mixed as a stable emulsion.