Bottom-Up Design and Synthesis of Limit Size Lipid Nanoparticle Systems with Aqueous and Triglyceride Cores Using Millisecond Microfluidic Mixing

Abstract

Limit size systems are defined as the smallest achievable aggregates compatible with the packing of the molecular constituents in a defined and energetically stable structure. Here we report the use of rapid microfluidic mixing for the controlled synthesis of two types of limit size lipid nanoparticle (LNP) systems, having either polar or nonpolar cores. Specifically, limit size LNP consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC), cholesterol and the triglyceride triolein were synthesized by mixing a stream of ethanol containing dissolved lipid with an aqueous stream, employing a staggered herringbone micromixer. Millisecond mixing of aqueous and ethanol streams at high flow rate ratios (FRR) was used to rapidly increase the polarity of the medium, driving bottom-up synthesis of limit size LNP systems by spontaneous assembly. For POPC/triolein systems the limit size structures consisted of a hydrophobic core of triolein surrounded by a monolayer of POPC where the diameter could be rationally engineered over the range 20-80 nm by varying the POPC/triolein ratio. In the case of POPC and POPC/cholesterol (55/45; mol/mol) the limit size systems achieved were bilayer vesicles of approximately 20 and 40 nm diameter, respectively. We further show that doxorubicin, a representative weak base drug, can be efficiently loaded and retained in limit size POPC LNP, establishing potential utility as drug delivery systems. To our knowledge this is the first report of stable triglyceride emulsions in the 20-50 nm size range, and the first time vesicular systems in the 20-50 nm size range have been generated by a scalable manufacturing method. These results establish microfluidic mixing as a powerful and general approach to access novel LNP systems, with both polar or nonpolar core structures, in the sub-100 nm size range.

The Niemann-Pick C1 Inhibitor NP3.47 Enhances Gene Silencing Potency of Lipid Nanoparticles Containing siRNA

“…7 Next, we investigated the effects of NP3.47 on LNP accumulation in a variety of cell lines. LNP siRNA systems containing the ionizable cationic lipid MC3 were formulated using the microfluidic mixing technique 11 and administered to cells in the presence and absence of NP3.47. The cell lines employed were NIH/3T3, HeLa, LNCaP, Raw 264.7, OVCAR-3, PC3, 22Rv1, and NPC1 knockout (NPC1 −/− ) cells which were incubated with DiIlabeled LNP-siRNA and NP3.47 at 0, 1 or 10 μmol/l for 24 hours.…”

“…LNP-siRNA formulations were manufactured using a microfluidic mixing method as previously described. 11,12 Briefly, MC3, DSPC, cholesterol, and PEG-DMG were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 with a final lipid concentration of 20 mmol/l; A solution of siRNA at a final concentration of 0.28 mg/ml was prepared in 25 mmol/l sodium acetate buffer at pH 4. To formulate LNPsiRNA, lipid and siRNA solutions were injected into a microfluidic mixer (Precision Nanosystems, Vancouver, Canada) at flow rates of 1.5 ml/min (siRNA) and 0.5 ml/min (lipids) respectively, using two syringes controlled by a syringe pump (PHD Ultra, Harvard Apparatus, Holliston, MA).…”

Metallosomes for biomedical applications by mixing molybdenum carbonyl metallosurfactants and phospholipids

“…23 This effect is a consequence of the drop in the final solvent concentration due to the FRR increase, which reduces liposome fusion. 24 Vesicle fusion, like many other alterations of membrane properties (such as permeability, surface tension, and area per molecule), 25 is driven by alcohol insertion into phospholipid bilayers. In this sense, MTS/SPC vesicular systems exhibit a behaviour analogous to that of liposomes, since any FFR increase, using either methanol or ethanol as solvent, causes a decrease in the vesicle size.…”

Drug Delivery Systems: Possibilities and Challenges

“…Furthermore, the advancement of microfluidic mixing techniques allowed scalable production of LUV in the range of 20-50 nm. 24 The first generation liposomes were rapidly cleared by the RES soon after administration. The first strategy implemented in order to generate long-circulating liposomes modified the characteristics of liposomes including composition and size.…”