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

Authors: I.V. Zhigaltsev, N. Belliveau, I. Hafez, A.K. Leung, J. Huft, C. Hansen and P.R. Cullis

Journal: Langmuir

DOI: 10.1021/la204833h

Publication - Summary

February 21, 2012


The potency of drug delivery systems can heavily rely on their ability to penetrate poorly vascularized tissues such as tumors, following intravenous administration. The drug delivery vehicle’s size greatly impacts this phenomenon. It is speculated that long-circulating nanoparticles with sizes 10-15 nm are ideal for such purposes and can access more tissues in the body. Limit size lipid nanoparticles, defined as the “smallest achievable aggregates compatible with the packing of the molecular constituents in a defined and energetically stable structure”, can approach this size, depending on the formulation and thus be more efficacious, in penetrating tumors, lymphatic tissue and bone marrow and are a great point of interest in the drug delivery field. Achieving the limit size however, is extremely challenging by using conventional nanoparticle synthesis techniques such as extrusion, sonication and ethanol injection while stir-mixing. These methods are often limited by many factors including lack of reproducibility, sample contamination and lack of scalability. In this article, the Cullis group from the University of British Columbia, in collaboration with Precision NanoSystems have generated a reproducible and scalable method for producing limit size lipid nanoparticles (LNP) using microfluidic mixing. The particles described here are formulated by the NanoAssemblrTM device and match the theoretically calculated limit size and are stable for long periods of time.

In their article featured in Langmuir, 2012, the authors generated limit size LNPs made up of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPC/cholesterol or POPC/triolein. Based on characterization of their structure, the POPC or POPC/cholesterol LNPs were found to be liposomes with a single bilayer and an aqueous core whereas POPC/triolein formed an emulsion with a nonpolar lipid core stabilized by a POPC monolayer. The three groups of LNPs were generated by rapid mixing at total flow rates (TFR) of 2 ml/min or higher*. In this method, the lipids (dissolved in a water miscible solvent ie. ethanol) and buffer are injected through the NanoAssemblr microfluidic mixer simultaneously and the change in solvent polarity allows for nanoprecipitation of lipids and self-assembly of LNPs. The authors varied the aqueous: organic flow rate ratio (FRR) to achieve the limit size in all three groups as follows:  POPC particles reached the limit size of 20 nm diameter with FRRs greater than 3:1, POPC/cholesterol particles achieved limit size of 40 nm with FRRs greater than 2:1 and POPC/triolein particles had a limit size of 20 nm with FRRs greater than 3:1. The structure of all three particles were confirmed using 31P NMR and cryo-transmission electron microscopy (Cryo-TEM).  POPC/triolein had highly electron dense cores as opposed to the POPC or POPC/cholesterol particles that exhibited a high electron dense ring surrounding a lower electron dense aqueous interior.

Having successfully achieved the limit size LNPs, the authors then went on to encapsulate a model cancer drug, doxorubicin. While limit size can be beneficial in accessing more tissues for drug delivery, the very small size can potentially restrict efficient drug loading. The authors therefore tested the loading efficiency of doxorubicin into limit size POPC liposomes. It was observed that using the pH gradient loading method, 100% of doxorubicin was encapsulated at drug-to-lipid ratios of 0.15 (w/w) or lower. This encapsulation efficiency is comparable with the commercial doxorubicin formulation of 0.125 drug-to-lipid (w/w) indicating that the limit size particles can be therapeutically practical systems. Additionally, the particles retained doxorubicin at 4˚C for longer than 8 weeks and at 37˚C lost only between 10-25% of the drug depending on the initial drug-to-lipid ratio.

This paper demonstrates the power of microfluidic mixing technique in formulating limit size LNPs that can efficiently load and retain therapeutic agents. The technique highlighted here is reproducible, can generate homogenous particles and is easily scalable for large scale studies. To summarize, four key findings in this paper are: 1) Unilamellar, aqueous core liposomes were made using microfluidic mixing method. 2) Hydrophobic core lipid emulsions were generated using the same method. 3) The concept of “limit size” was defined and experimentally verified for lipid nanoparticles. 4) Doxorubicin was successfully remote loaded into limit size POPC LNPs and sufficiently retained.  Overall, it is anticipated that limit size LNPs will consist a new class of therapeutics that can be applied to treat many conditions including those involving poorly vascularized tissues and tumors.

* Current NanoAssemblr® devices operate at higher flow rates up to 18 mL/min. Limit size is typically achieved at FRR > 2:1 and TFR > 4 mL/min



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.

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