ProtocolsIntegrated microfluidic devices for the synthesis of nanoscale liposomes and lipoplexes
Graphical abstract
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.
Section snippets
Microfluidic device
The microfluidic channels were fabricated in polydimethylsiloxane (PDMS) substrates via soft lithography techniques employing dry film photoresist molds, which use simple experimental procedures and avoids the use of a clean room [27]. The geometries of the microfluidic devices (Fig. 1) were designed using AutoCAD (Autodesk). To fabricate the microchannel molds, two layers of the dry film photoresist, each 35 μm to 40 μm, (Riston MM115i, DuPont, Research Triangle Park, NC) were laminated onto a
Results and discussion
Many studies have reported the development of high-efficacy gene delivery systems, but few have attempted to develop processes for lipoplex production with reduced number of steps. Usually, for lipoplex preparation via conventional bulk methods, the synthesis of liposomes requires a first step for the formation of vesicles that is followed by a second unit operation step for size reduction and homogenization. The pre-formed liposomes are then complexed with the genetic material to finally form
Conclusions
In this work, an integrated, continuous-flow microfluidic device with two distinct regions for the one-step preparation of plasmid DNA-containing lipoplexes has been demonstrated to show suitable physicochemical properties and biological activity for gene therapy applications. Process parameters were investigated to prepare liposomes and lipoplexes with higher lipid concentration and volumetric throughputs. It was possible to use a low-cost technique to fabricate microfluidic devices that
Acknowledgments
The authors gratefully acknowledge the financial support of the São Paulo Research Foundation − FAPESP, Brazil (Grants No. 2012/23143-9, 2013/05868-9 and 2013/14925-6). The microfluidic devices were partially constructed at the Microfabrication Laboratory (LMF) of the Brazilian Nanotechnology National Laboratory (LNNano). The authors would like to thank Meryem Ok for providing English language and editing services.
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Current address: Department of Mechanical Engineering, Pontifical Catholic University of Rio de Janeiro, PUC-Rio, Rio de Janeiro, RJ, 22451-900, Brazil.