Microfluidics for PLGA nanoparticle synthesis: a review

Written by Audrey Nsamela

Microfluidics for PLGA nanoparticle synthesis: an introduction

Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are of great interest for biomedical applications and especially for drug delivery. These polymeric nanoparticles are approved by the Food and Drug Administration (FDA) and valued for their biocompatibility and biodegradability. PLGA can be polymerized with different ratios of its monomers, lactic acid and glycolic acid which tune the hydrophobicity and the stability of the polymer. For example, a higher percentage of lactic acid leads to more hydrophobic PLGA which takes more time to degrade in water. For more complete information, the reader is advised to have a look at the article from Makadia & Siegel [1].

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Figure 1. Chemical formula of PLGA and its monomers (Source: Copyright Nanovex Biotechnologies)

Most commonly the PLGA nanoparticles are fabricated using two distinct methods: single or double emulsion method (also called solvent evaporation method) and nanoprecipitation method. With solvent evaporation methods, for example single emulsion preparation, the nanoparticles are created after emulsifying an organic phase containing polymeric chains into an aqueous solution and then evaporating the organic solvent.

Double emulsion methods are better suited to encapsulate hydrophilic drugs as they are based on a water-in-oil-in-water emulsion. For example, the nanoprecipitation method consists of injecting a water miscible solvent containing PLGA polymeric chains into an aqueous phase, usually a solution of water and surfactant.

In general, these methods produce mostly polydisperse spherical nanoparticles with a broad size distribution ranging typically from 150 – 300 nm. Two important parameters for drug delivery applications are the encapsulation efficiency and the drug loading degree. It has been shown that conventional methods result in a relatively low encapsulation efficiency (except the emulsion method) and low drug loading degree [2]. Microfluidics provide a very interesting alternative for polymeric micro/nanoparticle synthesis as well as inorganic nanoparticle synthesis. This review focuses on polymeric PLGA nanoparticles, and will present an overview on nanoparticle synthesis in microfluidics. Microfluidic systems act as microreactors and allow to precisely tune PLGA nanoparticle size, give narrower size distributions and reduce reagent consumption and waste production. Another advantage is that it can be less user dependent and decrease the variability from batch-to-batch. In microfluidics, the bulk techniques for PLGA nanoparticle synthesis can be adapted to droplet-based systems (mostly for emulsion methods) or to continuous laminar flow systems (mostly for nanoprecipitation method).

Microfluidics for PLGA nanoparticle synthesis: physics of nanoparticle formation

Figure 2. Stages in the nanoprecipitation process (Source: reproduced from [3])

To design the microfluidic chip for fabricating PLGA nanoparticles, one needs to understand the underlying principles of nanoparticle formation and the parameters that are involved in the reaction. In general, nanoparticle synthesis can be achieved using two different approaches: the “top-down” and the “bottom-up” approach. Both solvent evaporation and nanoprecipitation methods are bottom-up approaches because they start at the atomic/molecular level to form self-assembled nanoparticles. The precipitation or “self-assembly” of PLGA nanoparticles is illustrated in Figure 2. It is divided into three main stages: nucleation (I), growth (II) and nanoparticle formation (III). The nucleation starts with polymeric precursors concentrated in a solvent above a threshold called supersaturation. To reach this condition, the precursors in the organic solvent are placed into an aqueous phase and with intensive mixing (by diffusion and/or convection) the polymeric chains are getting close enough to each other to start precipitating. After the nucleation stage, more polymers are added to the nanoparticle seeds during the growth stage. It is very important that the growth is temporally separated from the nucleation to ensure a narrow size distribution. If some nanoparticles nucleate when others are already growing, they will have a different size when the reaction is stopped. It is worth noting that in microfluidics these two stages can also be separated spatially as the growth can happen further downstream in the chip than the nucleation point. The last step is reached when stable nanoparticles are formed and the growth phase is completed. This can be done by adding a reagent that will quench the reaction or when equilibrium is reached (in this case there is a risk of having agglomeration if the nanoparticles are unstable). PLGA nanoparticles need to be stabilized by adding surfactants, ligands or ions to the solution. This allows easy tuning of their surface chemistry. Alternatively, block copolymers (e.g. PLGA-PEG) with a hydrophilic end that act as a surfactant can be used [3-4].

Figure 3. Example of nanoprecipitation of PLGA in a microchannel (source: reproduced from [5])

The most critical parameter in this process is the mixing time t_mix. Mixing is usually enhanced by increasing the temperature and using a magnetic stirrer. The mixing will not be homogenous in the solution and will depend on the size of the stirrer and its position. In microfluidics, different mixing strategies or “micromixers” have been developed. In flow focusing devices, the mixing time can be calculated using the following equation:
Where w is the microchannel width, D the diffusivity of the organic solvent and R the ratio between the flow rate of the organic phase to the flow rate of the aqueous phase [6]. Apart from the chip geometry, the flow rate ratio plays a crucial role in determining the mixing time, and thus the size of the nanoparticles. The temperature will mainly influence the rate of diffusion but the importance of this parameter has not yet been reported for PLGA nanoparticle synthesis in microfluidics. Controlling the flow inside the microfluidic chip is therefore essential.

Microfluidics for PLGA nanoparticle synthesis: fabrication methods

Many different designs exist for PLGA nanoparticle fabrication on chip. In theory, any material can be used for the chip as long as it is chemically compatible with the solvent used. This review will not go into the details of chip fabrication, since a complete review is already available on materials for microfluidic device application. Instead, the focus will be on two strategies: hydrodynamic flow focusing and capillary devices. Droplet-based microfluidics are more suitable for polymeric microparticle production (ranging from 1-100 µm) or for emulsion based methods which will be briefly described in the capillary device section.

Figure 4. Design of a simple 2D hydrodynamic flow focusing device (Source : adapted from [6])

This is one of the most effective yet simplest ways to produce polymeric nanoparticles in continuous laminar flow systems. In this configuration, illustrated in Figures 4 and 5, PLGA polymers are dissolved in a water miscible organic solvent (acetone, acetonitrile, DMSO, etc…) along with drugs or inorganic components to be encapsulated in the nanoparticles. This solution is injected with pumps, such as syringe pumps or the OB1 flow controller, inside the central microchannel. At the cross-shaped junction, a solution of water and surfactant is injected into microchannels from both sides perpendicular to the central microchannel. With the flow rate of the water being greater than the flow rate of the organic phase, the latter is focused at the junction and the mixing time is highly reduced. As PLGA is not soluble in water, it will start precipitating as the organic phase diffuses in water.

Figure 5. Illustration of drug encapsulation in PLGA nanoparticles using a microfluidic device (source: adapted from [4])

So far, the 2D design of hydrodynamic flow focusing was described, but 3D configurations have also already been constructed. Their design, along with the use of PLGA-PEG polymers as precursors, allows the fabrication of very small PLGA nanoparticles (13 nm). Making polymeric nanoparticles smaller than 30 nm is very difficult compared to the fabrication of inorganic nanoparticles of the same size [7].

Capillary devices

Figure 6. Schematic of 3D coaxial capillary device (Source: reproduced from [8])

Capillary devices can be used for different purposes, from making a simple hydrodynamic flow focusing junction to complex double emulsion systems. Liu et al. reported a device made with glass capillaries to obtain 3D coaxial flows. This system, illustrated in Figure 6, ensures a better mass transfer due to microvortices created at the output of the inner capillary and thus reduces the mixing time. The authors used a mix of water and surfactant as the inner fluid and the polymeric precursors in organic phase as the outer fluid. They successfully obtained monodisperse polymeric nanoparticles (PLGA, chitosan and dextran-based) with high drug loading degree (~6%) and encapsulation efficiency (~90%) compared to conventional methods. This is mainly due to the fixed flow rate ratio between the precursors and water. They also reported an improved production rate of a couple hundreds of grams per day [8]. Another interesting study presented a microfluidic platform with coaxial glass capillaries to produce drug loaded PLGA nanoparticles from microdroplets. Here, the authors used a mix of 2 solvents, DMSO (water miscible) and DCM (water immiscible), as a dispersed phase to produce the droplets with PLGA polymers and drug encapsulated. After being flow focused at the end of the inner capillary, the DMSO diffused rapidly in the water phase of the outer fluid and the jet broke into droplets due to higher interfacial tension. The PLGA nanoparticles were then able to nucleate and grow in the “shrinking” droplets until all the DMSO and DCM remaining were diffused in the water and the nanoparticles were released [2].

Microfluidics for PLGA nanoparticle synthesis: applications for drug delivery

Microfluidics can be applied to a wide range of applications, especially in biomedicine. It can be a powerful tool for drug delivery applications as it can be used as a way to encapsulate high amounts of drugs inside PLGA nanoparticles. In order to be translated into the clinic, the nanoparticles need to fulfill certain criteria: they have to be biocompatible, biodegradable, produced in sufficiently large amounts in a short period of time (for in vivo studies and clinical trials), show low batch-to-batch variability and have appropriate drug loading/release behaviors. A big advantage with microfluidics is that it allows maintaining continuous reactions and thus overcoming the problem of reduced reproducibility between batches that occurs in bulk methods. Different drugs such as Paclitaxel [4], curcumin [6] or Doxorubicin [9] have been successfully encapsulated into PLGA nanoparticles using microfluidics. Since reaction times are greatly reduced in microfluidic systems compared to bulk methods, one can easily fabricate sets of nanoparticles with different surface chemistry (e.g. by using different surfactants) and find the optimal combination for a drug delivery application. A similar study has been made by Poller et al. to evaluate the influence of albumin on the synthesis of PLGA nanoparticles. Figure 7 shows the main differences in PLGA nanoparticles prepared in bulk or with microfluidics in a 3D coaxial flow configuration. A significant improvement in monodispersity, loading degree and encapsulation efficiency was found for PLGA nanoparticles produced with microfluidics.

Figure 7. Characterization of PLGA nanoparticles produced in bulk with microfluidic nanoprecipitation (source : reproduced from [8])

Microfluidics for PLGA nanoparticle synthesis: challenges and perspectives

Increasing the production rate of PLGA nanoparticles

One of the big remaining challenges before implementing microfluidic systems for effective production of nanoparticles is the low production rate. Indeed, with most microfluidic devices that efficiently produced PLGA nanoparticles, the amount produced was on the order of grams per day. Although that would be sufficient for in vitro studies, the number of nanoparticles needed for in vivo studies is far greater. Among the solutions suggested, one can either increase the total flow rate in the system (until a certain threshold dictated by the Reynolds number is reached), parallelize the devices or do both at the same time. For hydrodynamic flow focusing, a 2D cross-shaped design would be suitable for parallelisation of several devices and the parallelization of a 3D hydrodynamic flow focusing device has also been documented [7].

Decrease chip-to-chip variability

Currently, PDMS still represents the most comonly used material for microfluidic chip fabrication. This is due to its many advantages including optical transparency, flexibility and low cost. However, an important drawback for PDMS chip fabrication is that it involves manual and manufacturer dependent fabrication steps. This can be a source of variability from chip to chip. For example, the manual punching of the inlet holes can slightly differ each time and this is particularly problematic for 3D hydrodynamic flow focusing devices, where the lateral position of the inlet holes has a great influence on the flow pattern, and thus the nanoparticles outcome. To solve that problem, some groups have included SU-8 vertical posts for inlets and outlets in their mold fabrication, thus avoiding the manual holes punching [8].

Avoid PLGA nanoparticle adhesion on the channel walls

Another challenge when working with microfluidics and polymeric nanoparticles is to limit the hydrophobic interactions on the microchannels walls. This can lead to channel clogging, particle loss and make the chip out of use. That problem is solved with 3D hydrodynamic flow focusing devices, co-flow capillary devices and droplet-based systems. However, attention also needs to be given on the chip material. PDMS is incompatible with some organic solvents such as dichloromethane. It induces swelling of the PDMS and absorption of the nanoparticles inside the material. Since the pair solvent-polymer is an important factor in the outcome of the nanoparticles, the material should also be taken into account when optimizing the microfluidic platform.

Microfluidics for PLGA nanoparticle synthesis: conclusion

PLGA nanoparticles have been widely used as biocompatible nanocarriers for drugs, proteins or peptides. Conventional fabrication methods for these particles are suitable for mass production but show a lack of controllability and reproducibility. Therefore, the field of microfluidics has been investigated to produce PLGA nanoparticles with high throughput, monodispersity and reproducibility. Fairly simple designs of hydrodynamic flow focusing and capillary devices have been presented along with their application in the drug delivery field. Efforts are now made to improve microfluidic setups, to increase the production rate of nanoparticles and translate them into clinical studies.

References