Particle encapsulation – from particle synthesis to encapsulation methods

Find the ideal microfluidics technique for different particle encapsulation needs, and micro to nanoparticle synthesis.

Particle encapsulation help preserve the chemical, physical, and biological properties of active compounds – from drugs to nucleic acids, cells, and organoids. The technique has transformed the face of research in several sectors, such as electronic engineering, food industry, agriculture, biomedical and pharmaceutical research [1].

This article provides an overview of different particle encapsulation methods and explains some experimental techniques from the Elveflow library. The aim is to help you find the adequate technique and develop the ideal encapsulation protocol for your lab.

Nanoparticles

Because their size ranges from 10 to 100nm, nanoparticles are ideal for encapsulating nucleic acids, peptides, proteins, and protein complexes – such as CRISPR-Cas9 molecules. Nanoparticles are often used for drug delivery, gene therapy, genetic manipulation of cells and organisms, and food enhancement [2].

Whether lipid-based or polymers, nanoparticles can be produced by microfluidics in a reproducible and accurate manner. Their formulation is diverse and should be designed according to their use. 

Below, we give you a few examples and uses for different types of nanoparticles. 

Lipid nanoparticles and liposomes

Lipid-based nanoparticles are ideal for biological and biochemical applications, drug delivery, and targeted therapies. Most commonly composed of a phospholipid bilayer enclosing an aqueous solution, it can encapsulate hydrophilic or lipophilic molecules with good cell affinity and biodegradability [3]. Lipid nanoparticles have become more popular since the FDA approval of mRNA vaccines, and show extensive promise for several other fields in biomedicine. Microfluidics is currently the most reliable and accurate technique for producing lipid-based nanoparticles.

PLGA (Poly lactic-co-glycolic acid) nanoparticles

PLGA nanoparticles are quickly metabolized biodegradable polymers that can be designed for controlled drug release, making them ideal for drug delivery. Also, their hydrophobicity can be tuned, making them more or less water-resistant. Producing PLGA nanoparticles in microfluidics can be adapted to droplet-based systems (emulsion methods) or continuous laminar flow systems (nanoprecipitation method). These techniques provide  polydisperse spherical nanoparticles with sizes typically ranging from 150 – 300 nm.

Microparticles

The slightly bigger size of microparticles enables them to encapsulate cells and organoids. Microparticles are often used for cell culture, cell-based cytotoxic assays, probiotics encapsulation, and cosmetics.

Find some examples, uses, and formulations of microparticles below. 

Alginate microparticles

Alginate beads are often used as hydrogel-based delivery systems. These particles can be used for tissue engineering, cell encapsulation, regenerative medicine, and biomedical applications due to their low toxicity, high biocompatibility, and relatively low-cost production.  Because alginate gels can be assembled at neutral pH and mild temperatures, they can encapsulate and successfully deliver proteins and cells to specific locations [4]. Producing highly monodispersed alginate beads in your lab has become highly affordable and can be done by microfluidics droplet generation.

Thermoresponsive core-gap-shell microcapsules

These microparticles are stimuli-responsive so they release their encapsulated agent upon an external trigger. Thermoresponsive microparticles can act as sensors, catalysts, and actuators, and be used for targeted drug therapy.  These capsules can be loaded by diffusion or absorption with hydrophilic biomolecules for different engineering applications. Droplet-based microfluidics can produce these particles in a consistent and reproducible manner.

Using microfluidics to produce micro and nanoparticles

The manufacturing technique is chosen according to the physical properties of the particle used to encapsulate and the encapsulated agent. The selected method should provide high particle encapsulation efficiency and loading capacity of actives. Also, it is important that particles do not aggregate or adhere to the chip during the process. Accuracy, control, and reproducibility are essential features for successful particle encapsulation, and micro or nanoparticle synthesis.

All the described techniques below can be performed using the OB1 flow controller.

Producing Ca-alginate microparticles

Alginate microparticles are generated using an alginate/EDTA-Calcium solution to produce  microdroplets in a microfluidics setup. Next, a calcium compound is released into the formed droplet by a pH modifying agent. The calcium crosslinks with the alginate forming the Ca-alginate beads. Different experimental conditions have been tested to produce highly monodispersed droplets with sizes within the 50µm range. Other bead sizes can be achieved by using a similar setup.

Droplet generation and manipulation

The T junction and the flow-focusing techniques are the main methods used to produce droplets (Figure 1). Both techniques require an accurate flow control system to achieve the desired droplet parameters – size and frequency. The T junction system injects the two phases orthogonally, forming the droplet at the channels’ intersection. For the flow-focusing method, the middle phase is squeezed between two streams of a continuous phase. The equipment required to produce the drops and encapsulate particles may differ slightly according to the size of droplets needed.

Figure 1: The T junction system (left) injects two phases orthogonally, forming a droplet at the channels’ intersection. On the flow-focusing technique (right), the middle phase is squeezed between two streams of a continuous phase.

Producing droplets on demand

Producing droplets on demand facilitates droplet manipulation and gives an additional degree of control over the size and the formation frequency of micro-droplets. By setting the injection time, the droplet size is adjusted. The longer the injection time, the larger the droplet. Additionally, balancing water and oil pressure in the microfluidics setup generates a stable interface between water and oil. Adding a degree of negative pressure to the output of the droplet generator will induce droplet formation. By changing the magnitude of the negative pressure, one can control the size of the droplets generated in an accurate and on-demand manner. This additional feature is extremely useful for complex operations requiring a high degree of accuracy and extra control over drop parameters.

If you would like to develop better droplet size control with an automatic droplet size measurement and feedback system, watch our “Droplet size, uniformity control & detection” webinar by Remigijus Vasiliauskas, PhD.

Nanoparticle synthesis using microfluidics 

The two main microfluidics techniques for producing nanoparticles – single-phase or multiphase flow systems – use lab-on-chip technology with tubular designs and inner dimensions often smaller than 1mm. The fast mixing of the two phases (organic and aqueous) is an important part of the process. The single-phase flow method uses self-assembly and nanoprecipitation techniques, and is usually used to synthesize metal colloids, quantum dots, and liposomes. Multiple reactions can be carried out in parallel on the same chip to upscale production. This technique allows the subsequent addition of reagents and multi-step synthesis. The multiphase flow system uses similar microchannel structure with two or several fluids flowing in segmented phases. These can be liquid-liquid or gas-liquid (Figure 2). The mixing happens within each segment of the chip as it moves through the channels. Both systems offer high accuracy and reproducibility.

PLGA nanoparticles synthesis techniques 

PLGA nanoparticles are often synthesized by nanoprecipitation technique or emulsion methods – single and double. The nanoprecipitation technique uses an organic solution of the polymer that is emulsified in an aqueous solution, usually with a surfactant. The organic solvent is then removed by stirring, allowing nanoparticles to form. The single emulsion method consists in mixing a water-mixable solvent containing PLGA polymeric chains to an aqueous solution. As the solvent precipitates, the organic phase diffuses in water and the PLGA polymers come together, forming the nanoparticles. The double emulsion technique uses a water-in-oil-in-water emulsion, and so it is best fitted to encapsulate hydrophilic molecules.  All of these methods can potentially provide 150 – 300nm spherical nanoparticles with good polydispersity.

Fabrication of nanohydrogels  

Ideal for cosmetics and drug delivery applications, nanohydrogels may be synthesized in a split-and-recombine micromixer chip connected to a pressure controller and flow sensor. The sizes of the particles produced can be adjusted by setting the flow rate and ratio on the chip. The final polydispersity of the particles will depend on the polymer to salt solution flow ratio. 

Using microfluidics for particle encapsulation

Particle encapsulation can be introduced at different stages during the particle synthesis. Encapsulation efficiency is also very influenced by the method used, its accuracy, and the amount of control over pressure and fluid flow.

Below we briefly describe some existing methods and previously performed experiments. For more details, you may refer to our free access online library and resources.

Droplet encapsulation techniques

Cells can be encapsulated during or after droplet formation. By diluting the cells in the solution that forms the inner phase of the droplet, the cells are encapsulated during droplet synthesis. Although simple, this technique may generate many empty droplets, but with a high percentage of single-cell encapsulated per droplet. Injecting the cells – or any other agent – after droplet formation may increase number of droplets containing cells, provide more flexibility for droplet manipulation, but may be complicated and complex to set up. Nevertheless, two recently developed injection techniques – plug-based and picoinjection – provide good encapsulation efficiency.

PEGDA hydrogel generation and encapsulation

Continuous flow lithography is an ideal technique to accurately control the shape of polymeric particles – an essential aspect to successfully produce microgel and microdroplets. The technique forms polymerized hydrogels by projecting photolithography in a flow-based environment. Combining droplet-based microfluidics with continuous flow lithography can produce and encapsulate fibre Poly(ethylene glycol) diacrylate (PEGDA)-based hydrogel within the forming droplet.

Cell encapsulation for cytotoxicity assays

Confining cells into droplets for cytotoxicity assays can overcome the common spatial issues from standard bulk live imaging, and the temporal issues in flow cytometry assays. A flow-focusing junction can be used to produce droplets of different sizes and encapsulate cells simultaneously. By tuning the pressures applied to the oil and aqueous inlet channels, it is possible to form droplets of different diameters (∼105, 85, and 67 μm)5, a helpful feature when testing different cell types and designing new assays.

Encapsulating multivesicular vesicles 

Smaller vesicles can be added to the inner phase of a double emulsion solution to be encapsulated into bigger vesicles. First, the solution inside the water-in-oil-in-water droplet is pinched in the first junction of the chip by the oil phase. Next, the formed droplet travels until a second junction, where the oil phase is pinched by the outer aqueous solution, forming the water-in-oil-in-water droplet. Once the double emulsion is formed, the oil layer is spontaneously removed from the membrane, leaving only the lipid bilayer of the vesicles containing smaller vesicles inside.

Particle encapsulation and its many applications

Micro and nanoparticles can have different chemical and physical properties depending on the technique and materials used to design them. This flexibility enables a range of possible particle encapsulation applications and explains why this is a growing field in so many sectors – from biology to engineering.

Until recently, manufacturing and encapsulation methods were among the main challenges in the field. Recent microfluidics developments, however, have improved particle synthesis and encapsulation techniques, improving production, reproducibility and making it available to be used in several sectors.

Apart from the examples briefly described in this article, particle encapsulation techniques can also be applied to:

• NGS and antibody screening
• single spore or fungus encapsulation
• antifungal screening on-chip
• bacteria encapsulation in emulsion droplets
• single-cell encapsulation  
• single-cell omics, and spatial transcriptomics

The ability to control and manipulate liquids on the nano and microscale in a precise and reproducible manner, makes microfluidics ideal for particle encapsulation and particle synthesis. Droplets-based microfluidics, for instance, offers high monodispersity and encapsulation efficiency, and nanoparticles of different composition can be accurately produced in high throughput manner.

In biology, microfluidic encapsulation methods have been commonly used to capture cells in picolitre-scale, monodispersed droplets. The technique provides meaningful data on the interaction between cells in groups of controlled sizes as well as on single and isolated cells.

Recent developments also made microfluidics technology more accessible and easier to use for different levels of expertise. Whether you want to generate droplets or synthesize nanoparticles, predesigned packs can help you in a “plug-and-play” approach. 

When in doubt about how to set up your system, consult an expert in the field. They will advise you on the right solution based on your level of expertise and laboratory facilities.

This article was written by Thais Langer.

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