Integrated microfluidic devices for the synthesis of nanoscale liposomes and lipoplexes

Highlights

• A microfluidic platform with two regions efficiently generated liposomes and lipoplexes.

• The microfluidic device produced lipoplexes with significant reduced number of steps.

• Microfluidic-obtained lipoplexes efficiently transfected mammalian cells.

Abstract

In this work, pDNA/cationic liposome (CL) lipoplexes for gene delivery were prepared in one-step using multiple hydrodynamic flow-focusing regions. The microfluidic platform was designed with two distinct regions for the synthesis of liposomes and the subsequent assembly with pDNA, forming lipoplexes. The obtained lipoplexes exhibited appropriate physicochemical characteristics for gene therapy applications under varying conditions of flow rate-ratio (FRR), total volumetric flow rate (Q_T) and pDNA content (molar charge ratio, R±). The CLs were able to condense and retain the pDNA in the vesicular structures with sizes ranging from 140 nm to 250 nm. In vitro transfection assays showed that the lipoplexes prepared in one step by the two-stage configuration achieved similar efficiencies as lipoplexes prepared by conventional bulk processes, in which each step comprises a series of manual operations. The integrated microfluidic platform generates lipoplexes with liposome formation combined in-line with lipoplex assembly, significantly reducing the number of steps usually required to form gene carrier systems.

Introduction

Gene therapy has long been investigated for the treatment of diverse diseases by introducing nucleic acids into cells. The first studies of gene therapy focused on specific genetic disorders, and over time gene delivery has enabled the treatment of different inherited and acquired diseases [1]. This technology is based on the introduction of engineered, foreign genetic material into specific cells to substitute missing genes, replace defective genes, or silence gene expression [2]. However, the naked nucleic acid itself is not able to successfully enter cells; it requires the assistance of suitable vectors. To this end, several engineered nanomaterials have been used as nonviral vectors, which are safer and simpler alternatives over viral vectors [3]. A variety of nanostructured materials, such as biopolymers [4], dendrimers [5], solid lipid nanoparticles [6], and cationic lipids [7] can be used to condense nucleic acids and protect them against enzymatic degradation when in contact with interstitial fluids [8]. Among the nonviral systems, the use of cationic liposomes (CLs) as gene carriers is still a popular strategy [9].

Liposomes are vesicular systems composed of (phospho)lipids containing at least one lipid bilayer surrounding an aqueous core. Because of their intrinsic properties, liposomes are capable of encapsulating aqueous solutes within the liposome lumen, retain hydrophobic compounds within the bilayer, and are suitable for tailored surface modifications for targeted delivery [10], [11]. Current processes for the preparation of liposomes generate large, polydisperse vesicles that require a post-processing unit operation step to reduce the size and size polydispersity of liposome populations [12]. An example is the conventional bulk process based on thin lipid film hydration followed by multiple extrusions through membranes. This is one of the most used techniques for liposome formation and comprises several discrete manual operations [7], [12].

For gene delivery applications, the use of cationic lipids in the lipid mixture enables the formation of CLs and subsequently lipoplexes through electrostatic interactions with negatively charged nucleic acids, allowing for the condensation and delivery of genes into cells [13]. Lipoplexes can present different physicochemical, structural and biological characteristics depending on several factors, such as lipid mixture composition, the ionic strength of the media, order and type of mixing, solution concentrations, and lipid/genetic material charge ratio. The standard process of lipoplex formation relies on the electrostatic assembly of the pre-formed CLs with the genetic material (DNA or RNA) by mixing the two species via manually up-and-down pipetting or vortexing. Under certain conditions, these methods usually have limited scalability and might generate lipoplexes with inappropriate characteristics for biological studies, mainly due to the heterogeneous and uncontrolled fluid flows [7], [14].

By advances in microfabrication, microfluidic platforms have gained substantial attention for their use in liposome synthesis and lipoplex assembly [15], [16]. Over the past years, microfluidics has been among the most rapidly growing fields of scientific and technological research [17], [18], [19]. Microfluidic devices handle minute volumes of fluid within micro-sized channels in which the mixing occurs mainly under laminar flow by molecular diffusion, providing constant and reproducible mass transfer environments [20], [21]. For the preparation of liposomes and lipoplexes, microfluidic platforms not only can generate monodisperse nanocarriers but also can integrate a variety of sequential steps needed for the formation of liposomes and electrostatic complexes with genetic material, significantly reducing the number of steps required in conventional processes. Platforms based on the microfluidic flow-focusing (MFF) technique have previously generated liposomes with tunable sizes and low polydispersity in one step and in a continuous-flow process for different applications [22], [23], [24], [25], [26]. Integrated microfluidic devices allow for synthesis of liposomal formulations with minimal reagent waste at or near the point of care. As an example, liposomes containing high concentrations of loaded drug compounds have been prepared by microfluidic platforms, combining in-line regions for liposome synthesis, microdialysis purification, and remote drug loading [27]. For gene therapy purposes, lipid- and/or polymer-based systems have been prepared by MFF devices for the successful delivery of pDNA and siRNA [28], [29], [30], [31], becoming a promising strategy to accelerate the translation of gene therapy studies to clinical trials.

In the present work, an integrated microfluidic platform is employed for the formation of gene nanocarriers by combining in-line regions for liposome synthesis and lipoplex assembly in a continuous flow. Lipoplexes prepared by conventional bulk processes usually require a series of laborious and time-consuming operations. Alternatively, a single microfluidic device was used to prepare lipoplexes with similar physicochemical characteristics and biological behavior, dramatically reducing the number of steps compared to conventional procedures.