Advanced Microfluidics for Nanostructured Lipid Carriers Preparation

Microfluidics is a kind of science and technology mainly characterized by precise manipulation of fluids in micro and nano scale space, which has the ability to shrink the basic functions of biology, chemistry and other laboratories such as sample preparation, reaction, separation and detection to a few square centimeters on a chip, and its basic feature and biggest advantage is that a variety of unit technologies can be flexibly combined and integrated at scale in the overall controllable and tiny platform. Its basic feature and greatest advantage is the flexible combination and scale integration of multiple unit technologies on an overall controllable tiny platform. More specifically, microfluidics is the science and technology of systems that handle or manipulate very small amounts of fluid using tubing on the scale of tens to hundreds of micrometers. Additionally microfluidics allows for the design of sustainable processes with less capital requirements, while parallelization can be used to scale up to increase throughput. A range of nanostructured lipid carriers such as liposomes, lipoplexes, lipid nanoparticles, core-shell nanoparticles, and bionic nanovesicles can be synthesized by microfluidics.

What is Microfluidics?

Microfluidics is a field of science and technology that deals with the manipulation of fluids at the micrometer scale, typically within channels with diameters less than one millimeter. It allows for precise control of fluid flow, mixing, and reaction conditions, which is particularly beneficial for the fabrication of nanoparticles and other nanoscale materials.

Diagram of a microfluidic chip.

Microfluidic Technology

Microfluidic technology relies on the use of miniaturized devices and specialized equipment to manipulate small volumes of liquids, often in the nanoliter to microliter range. The core feature of microfluidics is the control over flow rates, mixing, and reaction conditions in real-time, providing the ability to generate highly uniform particles with consistent properties. The key components of a microfluidic system include microchips, which contain networks of channels that guide the flow of fluids, and various mixers and pumps to regulate fluid dynamics.

The advantage of microfluidic systems is their ability to scale up from laboratory-scale synthesis to large-scale production while maintaining precision and reproducibility. For the preparation of NLCs, microfluidic devices can provide fine control over the lipid-to-water ratio, emulsification techniques, and the encapsulation of active ingredients, leading to improved stability, drug release profiles, and bioavailability.

Microfluidic Methods

In general, lipid nanostructures can be formed by mixing two inlet streams containing lipids in a water-soluble solvent and another central inlet in an aqueous solution. When the fluids flow in parallel, the mixing process is initiated and the polarity changes, favoring the automatic aggregation of lipids in a reproducible manner. Here are the most commonly used microfluidic methods.

• Hydrodynamic Flow-Focusing (HFF): The HFF method uses a three-way cross-shaped inlet flow to form lipid nanoparticle structures through the diffusion between aqueous solutions and ethanol (containing lipids). It is suitable for producing small-sized lipid nanoparticles with narrow size distributions and is easy to operate. However, the flow rate limits its scalability and high-throughput application, especially for producing more complex structures like lipid nanoparticles (LNPs).
• Chaotic Advection Micromixers (CA-M): CA-M introduces obstacles inside the microchannel to generate chaotic advection, significantly improving the mixing efficiency for lipid nanoparticle production at higher flow rates. This method overcomes the efficiency limitations of HFF and is suitable for high-throughput production, but its complex design typically requires more expensive microfabrication processes.
• Droplet-Based Microfluidics: This method uses immiscible fluids (such as water/oil emulsions) to generate highly monodisperse droplets, suitable for producing micro-particles like giant unilamellar vesicles (GUVs) and microgels. By controlling the formation of droplets, droplet-based microfluidics enables the production of uniform and reproducible particles.

Nanostructured Lipid Carriers

Nanostructured lipid carriers (NLCs) are a class of drug delivery systems composed of solid lipid cores surrounded by a liquid lipid shell. These carriers offer several advantages over traditional drug delivery systems, including improved stability, bioavailability, and the ability to encapsulate both hydrophilic and hydrophobic drugs. The key types of NLCs include:

Liposomes

Liposomes are spherical vesicles consisting of lipid bilayers, which can encapsulate both hydrophilic and hydrophobic substances. Liposomes can be prepared using microfluidic systems by mixing lipid and aqueous phases under controlled conditions, resulting in uniform particles with improved drug encapsulation efficiency. Liposomes are commonly used for the delivery of anticancer drugs, gene therapies, and vaccines.

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Lipid Nanoparticles

Lipid nanoparticles (LNPs) are solid, spherical nanocarriers made from lipids. These carriers are ideal for delivering hydrophobic drugs and biologics such as RNA. Microfluidics enables the preparation of LNPs with precise control over particle size and encapsulation efficiency, making them suitable for high-value therapeutics such as mRNA vaccines and RNA-based therapeutics.

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Lipoplex

Lipoplexes are complexes formed by combining liposomes with nucleic acids, typically plasmid DNA or RNA. The electrostatic interactions between the positively charged liposome and negatively charged nucleic acids enable efficient encapsulation and protection. Microfluidic techniques can be employed to optimize the formation of lipoplexes, improving their stability and transfection efficiency for gene delivery applications.

Core-shell Nanoparticles

Core-shell nanoparticles consist of a central core surrounded by a shell of lipid or polymer material. These particles provide a platform for controlled drug release, where the drug is encapsulated in the core and released over time from the shell. Microfluidics can precisely control the formation of core-shell structures, allowing for high encapsulation efficiency and tailored release profiles for specific drugs.

Biomimetic Nanovesicle

Biomimetic nanovesicles are lipid-based structures designed to mimic biological membranes, offering the advantage of enhanced biocompatibility and the ability to interact with cellular membranes. These vesicles can be used for targeted drug delivery and intracellular trafficking. Microfluidic methods allow for the precise formation of biomimetic vesicles, enhancing their therapeutic potential in drug delivery applications.

Nanostructured Lipid Carriers Preparation Methods

The preparation of nanostructured lipid carriers typically involves several steps, including the mixing of lipid phases, the incorporation of the therapeutic agent, and the formation of nanoparticles. Traditional methods for NLC preparation include high-pressure homogenization, solvent evaporation, and emulsification. However, these methods often suffer from issues such as poor reproducibility and difficulty in scaling up production.

Microfluidic technology offers a more efficient and reproducible approach to NLC preparation. By manipulating the fluid dynamics within microchannels, researchers can achieve better control over nanoparticle size, morphology, and encapsulation efficiency. This results in the production of more stable and uniform NLCs, which are critical for their success in therapeutic applications.

Microfluidics in Nanostructured Lipid Carriers Preparation

Microfluidics have proven to be highly effective for the preparation of nanostructured lipid carriers. These methods enable the production of nanoparticles with narrow size distributions, high encapsulation efficiencies, and improved stability compared to traditional methods. By optimizing the flow rates, temperature, and other parameters, researchers can fine-tune the properties of NLCs to meet specific requirements for drug delivery, gene therapy, and other applications.

Advantages of Microfluidics in Nanostructured Lipid Carriers Preparation

• Uniformity: Microfluidic systems provide precise control over the size and morphology of NLCs, leading to uniform particles that are essential for consistent therapeutic performance.
• Scalability: Microfluidic systems can be scaled up for large-scale production without compromising particle quality or reproducibility.
• Encapsulation Efficiency: Microfluidic methods allow for higher encapsulation efficiencies, ensuring that the therapeutic agent is effectively delivered to the target site.
• Reproducibility: The precision of microfluidic systems ensures that each batch of NLCs has consistent properties, which is crucial for clinical and industrial applications.

Applications of Microfluidics in Nanostructured Lipid Carriers

Drug Delivery

In drug delivery, NLCs are favored due to their ability to transport a wide range of therapeutic agents, including small molecules, proteins, and nucleic acids. Microfluidic technology enhances the preparation of NLCs for drug delivery by enabling the generation of stable, homogeneous particles that provide controlled release profiles, reduce toxicity, and improve pharmacokinetics.

• Targeted Drug Delivery: Microfluidic systems enable the creation of NLCs with surface modifications, such as targeting ligands or antibodies, to specifically deliver drugs to target cells or tissues. This is particularly important in cancer therapies, where nanoparticles selectively target tumor cells while sparing healthy cells.
• Controlled Drug Release: Microfluidics also allows for the controlled release of drugs from NLCs over an extended period. By adjusting parameters like flow rates, mixing conditions, and lipid composition, researchers can optimize release rates, improving patient compliance and maintaining therapeutic drug levels in the bloodstream.

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Gene Therapy

Gene therapy aims to treat or prevent diseases by delivering genetic material such as DNA, RNA, or gene-editing tools like CRISPR/Cas9 to target cells. The challenge in gene therapy is the efficient and safe delivery of nucleic acids to cells, particularly to the nuclei, where genetic material can be expressed or modified. Nanostructured lipid carriers, especially lipid nanoparticles (LNPs), are widely used for RNA-based therapies, including mRNA vaccines and siRNA therapeutics. Microfluidics offers a precise method for formulating LNPs that encapsulate genetic material, ensuring efficient delivery and protection of the nucleic acids from degradation before they reach their target cells.

• mRNA Delivery: Microfluidic technology enables the preparation of LNPs that encapsulate mRNA, ensuring stability and effective delivery to target cells for protein expression. This technology was key in developing mRNA vaccines for COVID-19, protecting mRNA from degradation and enhancing its uptake by dendritic cells to trigger an immune response. This approach is also applied to cancer vaccines, delivering tumor antigen mRNA to stimulate immune responses.
• Gene Editing with CRISPR/Cas9: Microfluidics is used to deliver CRISPR/Cas9 components into cells via NLCs, ensuring safe and efficient gene editing. By optimizing lipid composition, microfluidics maintains the stability of CRISPR components, allowing them to reach the cell nucleus for successful editing.

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Organ on a Chip (OOC) Development

Microfluidic technology enhances cellular environment complexity, offering rapid response, portability, and low-cost processing. In drug/gene delivery, interactions occur within microenvironments, unlike traditional methods where cargo is randomly exposed. Microfluidic devices improve cell uptake by altering interactions between the payload and target cells, enabling studies on nanocarrier transport and drug resistance. Organ on a chip (OOC) platforms simulate tissue characteristics and dynamically manipulate fluid flow and mechanical signals. Over the past five years, microfluidic systems have advanced lipid-based nanocarrier delivery research, focusing on cell-nanocarrier interactions. Recent OOCs, such as blood-brain barrier and tumor chips, offer reliable platforms for preclinical evaluation.