Continuous flow scalable production of injectable size-monodisperse nanoliposomes in easy-fabrication milli-fluidic reactors

Highlights

• Cheap and easy-fabrication glass T-shaped and coaxial milli-reactors are reported.

• Unilamellar liposomes used in a known pharmaceutical product are synthesized.

• T-shaped milli-reactors improve production rates compared to microreactors.

• Coaxial milli-reactors also decrease size and polydispersity of liposomes.

• Efficient mixing at water–ethanol interfaces was promoted by viscosity contrasts.

Abstract

The production of reproducible batches of liposomes suitable for parenteral administration is a time-consuming, multi-step process requiring extensive workable area. Microfluidic Hydrodynamic Focusing (MHF) chips allow one-pot synthesis of injectable size liposomes, although the use of low height-to-width aspect ratio (AR) microreactors limits the production rates.

Herein, aiming at scaling up liposome production, while avoiding lithographic methods and clean room facilities, easy-fabrication glass MHF large channel (MHF-LC) chips of AR = 1, 3 with T-shaped and coaxial injection geometries are demonstrated. Narrowly distributed unilamellar nanoliposomes (unprecedented minimum average diameters 85 nm and polydispersity index of 0.13) with the formulation of a known pharmaceutical product (i.e., DOXIL®(CAELYX®)) are synthesized at production rates 15–20 times larger than T-MHF chips. The dependence of liposome size on the Reynolds numbers (in the range of 5–50) in the coaxial configuration is discussed as due to viscosity-induced mixing dynamics at the water–ethanol interface.

Introduction

Liposomes are biocompatible micro- or nano-sized bilayered spherical membranes consisting of nontoxic phospholipids, organized to create a confined aqueous habitat (Immordino et al., 2006, Pattni et al., 2015). Because of their particular structure, liposomes are able to incorporate both hydrophilic bioactive cargos (within their aqueous core) and lipophilic molecules (within their hydrophobic compartment) (Pattni et al., 2015). Biocompatibility makes liposomes suitable in the biomedical field as drug delivery systems or as cellular models (Maeki et al., 2018, van Swaay and deMello, 2013). Depending on the applicative purpose, diameter, size distribution and composition of liposomes have to be accurately controlled. For instance, for successful clinical and drug delivery applications, size-monodisperse nanoliposomes (mean diameter D_AV ranging from 50 to 250 nm) with prolonged blood circulation time are needed to circumvent their possible capture by the mononuclear phagocytic system (Immordino et al., 2006, Vuković et al., 2011).

The production of liposomes relies on the spontaneous assembly of amphiphilic lipids in aqueous media. The molecules, dissolved in an organic solvent, expose their hydrophilic parts to water and tend to close in spherical vesicles, with an aqueous core, upon increasing polarity of the surrounding fluid (Bangham and Horne, 1964).

Bulk-scale and multi-step synthesis methods can be challenging to control vesicle size distributions and their physicochemical properties as required in clinical applications (Carugo et al., 2016). Moreover, they may suffer from high operation costs (due to post-processing procedures of size homogenization) and difficulty with scaling up the laboratory procedure (Marre and Jensen, 2010, Murday et al., 2009).

Recently, microfluidic technologies based on hydrodynamic focusing of miscible fluids in microreactors have been demonstrated suitable to produce nanoliposomes for drug delivery applications, thus overcoming some limitations that come with the bulk methods (Brody et al., 1996, Carugo et al., 2016, Jahn et al., 2013, Jahn et al., 2004, Zacheo et al., 2015a, Zook and Vreeland, 2010) in a more safety and greener way (Tortoioli et al., 2020).

The basic principle of Microfluidic Hydrodynamic Focusing (MHF) relies on constraining, shaping and focusing a central stream (main flow) by two lateral streams (sheath flows) that converge at a properly designed junction (Jahn et al., 2004). Downstream of the junction, all fluids flow along a common outlet channel, hereafter termed the mixing channel. To achieve the formation of a focused stream, the control of its width and the effective diffusion-driven mixing across the fluids’ interfaces, it is required that the flow rate through the lateral inlet is larger than that of the mainstream (Jahn et al., 2004), as well as properly designed junction geometries.

A focused stream can be generated by using a T-junction configuration of the channels, whereby the junction angle between the main and the side flow channels is 90°, or coaxial microfluidic reactors, where the mainstream flow is driven along the same direction as the sheath flows inside concentric coaxial channels (Ansari et al., 2012, Ashar Sultan et al., 2012, Balarac et al., 2007, Damian et al., 2017, Forstall, 1951, Nasir et al., 2011, Soleymani et al., 2008, Sultan et al., 2019).

In both examples, the mainstream of alcohol-solvated lipids is confined by the lateral flows of an aqueous solution. Interdiffusion occurring at the interface between the miscible alcohol and aqueous phases causes a reduction of the solubility of the lipids that promotes the spontaneous self-assembly of existing planar disc-like lipid bilayers (formed at the water-alcohol interface) into liposomes (Zook and Vreeland, 2010). The temperature at which such self-assembly occurs, strongly influences the final size, because of the dependence of the stiffness of lipid bilayers on the phase transition temperature (Tc) (Zook and Vreeland, 2010). Beyond Tc, the combined effect of a few working parameters impacts on the compositional and morphological properties and on the production rate (R) of the liposome formulation (Carugo et al., 2016). In this respect, critical factors are:

• the flow parameters as the flow rate ratio (FRR), defined as volumetric flow rate ratio between the lateral fluids to the mainstream fluid, the total volumetric flow rate (Q_tot), defined as the sum of all the inlet volumetric flow rates, and the Reynolds number (Re) of the mixing channel given by Re = Q_tot D/vA, where v is the kinematic viscosity of the mixture, D and A are the diameter and the cross-sectional area, respectively, of the mixing channel (Carugo et al., 2016);

• the device geometry: for instance, T-junction or coaxial geometry junction, that are of interest in our study (Phapal and Sunthar, 2013), and channel aspect ratio (AR, height-to-width ratio) (Hood et al., 2014);

• the mixing regimes, whether laminar or turbulent;

• the lipids’ concentration (Zizzari et al., 2017).

About the flow regimes used in the mixing schemes for driving the liposome formation, hydrodynamically focusing junctions operate under low Re conditions (Re ≪ 100, laminar flows) while inertial convective mixing relies on the formation of a turbulent liquid jet under high Re conditions (100 < Re < 3000) (Costa et al., 2016, Jahn et al., 2010).

In a preliminary study, we discussed the applicability of T-MHF chips for producing nanoliposomes suitable for parenteral administration (Zizzari et al., 2017). However, extensive application is limited by some open issues as: i) the production of relatively-poor size monodisperse nanoliposomes (mean size larger than 100 nm and polydispersity index (PDI) larger than 0.2); ii) the low values of liposome production rates (R) obtained in low AR mixing microchannels; and iii) the high costs and time constraints characteristic of the lithographic fabrication methods (Becker and Heim, 2000, Hung et al., 2005).

Recently, to overcome the throughput limits of conventional MHF architectures, large channels MHF devices (hereafter referred to as MHF-LC) with exceptionally high AR (AR = 100) (Hood and DeVoe, 2015), termed Vertical Flow Focusing (VFF) devices, were exploited. In particular, MHF-LC chips were demonstrated to synthesize liposomes with mean size and dispersity comparable to the conventional MHF approaches at speeds up to 18 mL/min. On the other hand, VFF chips produced similar size and lower PDI liposome populations than MHF devices at a rate of 4.5 mL/min. Advantageously, liposomes of unforeseen size uniformity were demonstrated by a coaxial geometry flow focusing device at volumetric flow rates up to 5 mL/min (Hood et al., 2014).

In this work, in the view of industrial applications of the MHF approach, we extend our early experiments to a scalable production by means of T-junction and coaxial MHF-LC chips with different ARs (Carugo et al., 2016).

As a first aspect, we report on a simple fabrication method developed to produce millimeter-scale MHF-LC chips without using lithographic techniques and clean room facilities. Glass millimeter-sized reactors assembled by thermally-bonding pre-cut glass blocks are cheaper than conventional low-volume MHF devices produced via optical lithography and wet etching (Zizzari et al., 2017). Then, we adapted the MHF-LC technology to a well-established pharmaceutical industrial process to obtain, in a single step, liposomes with formulation identical to the well-known commercial product DOXIL®(CAELYX®) (Barenholz, 2012).

The productions of liposomes in millimeter-sized T-shaped or coaxial-annular geometry devices were compared under variable flow conditions in a laminar flow regime (Costa et al., 2016, Hood et al., 2014, Liu et al., 2016). In particular, in the example of coaxial configuration, we discuss on a physical basis the formation mechanism of liposomes in a wide range of Re (from 5 to 50), and point out the role of the viscosity-induced mixing dynamics occurring at the water–ethanol interface. The quality of the unilamellar liposomes was assessed by cryogenic Transmission Electron Microscopy (cryo-TEM).

Noteworthy, compared with polymeric VFF, MHF-LC and coaxial chips (Phapal and Sunthar, 2013, Hood et al., 2014) operating at comparable production rates (in the range of a few mL/min), our glass chips are easier to be fabricated and more versatile. Indeed, their high chemical resistance to aggressive solutions allows a wider range of synthetic processes with important outcomes for industrial applications.