PLGA Nanoparticle Synthesis Using 3D Microfluidic Hydrodynamic Focusing
Nanoparticles can be used as drug delivery systems suitable for most administration routes. A variety of natural and synthetic polymers have been explored for the preparation of nanoparticles. Poly lactide-co-glycolic acid (PLGA) has been extensively investigated because of its biocompatibility and biodegradability [1-2].
These have emerged as important vehicles in scientific and biomedical research thanks to their high biocompatibility and low toxicity. One of the main advantages of PLGA is the generation of lactic and glycolic acids upon hydrolysis, endogenous metabolites that can be degraded through the Krebs cycle. This results in minimal toxicity and establishes PLGA as one of the most easily metabolized and tolerated polymers by the body [1]. PLGA nanoparticles can encapsulate different APIs, from small hydrophobic and hydrophilic drugs to nucleic acids and proteins.
In this application note, PLGA nanoparticle synthesis with high monodispersity and in a wide range of particle sizes has been performed, using Secoya Technologies' RayDrop single emulsion chip and Fluigent's pressure-based flow controllers. The influence of the fluidic parameters on the size of the PLGA nanoparticle is also presented.
Secoya Technologies developed and manufactured the RayDrop used to perform this application note.
Key takeaways
- PLGA nanoparticles are biodegradable polymer carriers for drug delivery. PLGA hydrolyses into lactic and glycolic acids, endogenous metabolites the body degrades through the Krebs cycle [1].
- This application note produces the particles continuously by 3D microfluidic hydrodynamic focusing, using a RayDrop single emulsion chip and Fluigent pressure-based flow controllers.
- PLGA nanoparticles from 110 to 250 nm were generated, with a polydispersity index (PDI) between 0.05 and 0.1.
- The aqueous-to-organic flow rate ratio is the main control on particle size: increasing it decreases particle size, from 250 nm to 110 nm across the range tested.
- Focusing the PLGA–acetone stream in the centre of the channel isolates precipitating particles from the walls, minimising aggregation and clogging.
Why PLGA nanoparticles: drug delivery at the nanoscale
Polymers were first introduced three decades ago as bioresorbable surgical devices. Since then, polymer-based nanoparticles have been extensively studied. Nanocarriers formulated with biocompatible and biodegradable polymers approved by the US FDA (Food and Drug Administration) and EMA (European Medicines Agency) are being studied for the controlled delivery of various therapeutic agents [1].
Among the various polymers synthesized for formulating polymeric nanoparticles, poly(lactic-co-glycolic acid) (PLGA) is the most popular. PLGA nanoparticles have several beneficial properties such as controlled and sustained release, low cytotoxicity, long-standing biomedical applications, biocompatibility with tissues and cells, prolonged residence time and targeted delivery [3]. These characteristics have accelerated the PLGA nanoparticle synthesis for use as nano-drug delivery systems (nanoDDS) in a wide variety of diseases, including cardiovascular, neurodegenerative and inflammatory and immune system diseases, infection, cancer, regenerative medicine and the fields of theragnostic and vaccines [4].
When PLGA is used as an active pharmaceutical ingredient carrier, it’s important to produce highly monodispersed particles for drug release reproducibility. PLGA nanoparticle synthesis with different characteristics (size, size distribution, morphology, zeta potential) is also possible by controlling the parameters specific to the synthesis method employed [5].
Current methods of particle synthesis rely largely on batch stirred homogenizers (single emulsion, double emulsion, etc.). However, they generally tend to have low reproducibility and are not well controlled. Some have low encapsulation efficiency and low drug loading. As narrow distributions, small particle size, and controllable synthesis are required in the field of smart drug delivery, these do not provide a highly effective solution for the pharmaceutical industry [4].
Microfluidic methods and, especially the 3D hydrodynamic flow-mediated nanoparticle production strategy of the RayDrop™ make it possible to obtain a continuous PLGA nanoparticle synthesis with high monodispersity, high reproducibility and a wide range of nanoparticle size.
This note covers PLGA at the nanoscale. PLGA microspheres, formed by droplet templating at the microscale, are covered in the PLGA microparticles application note. Lipid carriers are covered in liposome nanoparticle synthesis and solid lipid nanoparticles. For the wider application context, see microfluidics in drug delivery. Polymers were first introduced three decades ago as bioresorbable surgical devices. Since then, polymer-based nanoparticles have been extensively studied. Nanocarriers formulated with biocompatible and biodegradable polymers approved by the US FDA (Food and Drug Administration) and EMA (European Medicines Agency) are being studied for the controlled delivery of various therapeutic agents [1].
Among the various polymers synthesized for formulating polymeric nanoparticles, poly(lactic-co-glycolic acid) (PLGA) is the most popular. PLGA nanoparticles have several beneficial properties such as controlled and sustained release, low cytotoxicity, long-standing biomedical applications, biocompatibility with tissues and cells, prolonged residence time and targeted delivery [3]. These characteristics have accelerated the PLGA nanoparticle synthesis for use as nano-drug delivery systems (nanoDDS) in a wide variety of diseases, including cardiovascular, neurodegenerative and inflammatory and immune system diseases, infection, cancer, regenerative medicine and the fields of theragnostic and vaccines [4].
When PLGA is used as an active pharmaceutical ingredient carrier, it’s important to produce highly monodispersed particles for drug release reproducibility. PLGA nanoparticle synthesis with different characteristics (size, size distribution, morphology, zeta potential) is also possible by controlling the parameters specific to the synthesis method employed [5].
Current methods of particle synthesis rely largely on batch stirred homogenizers (single emulsion, double emulsion, etc.). However, they generally tend to have low reproducibility and are not well controlled. Some have low encapsulation efficiency and low drug loading. As narrow distributions, small particle size, and controllable synthesis are required in the field of smart drug delivery, these do not provide a highly effective solution for the pharmaceutical industry [4].
Microfluidic methods and, especially the 3D hydrodynamic flow-mediated nanoparticle production strategy of the RayDrop™ make it possible to obtain a continuous PLGA nanoparticle synthesis with high monodispersity, high reproducibility and a wide range of nanoparticle size.
This note covers PLGA at the nanoscale. PLGA microspheres, formed by droplet templating at the microscale, are covered in the PLGA microparticles application note. Lipid carriers are covered in liposome nanoparticle synthesis and solid lipid nanoparticles. For the wider application context, see microfluidics in drug delivery.
PLGA nanoparticle synthesis: materials and methods
PLGA nanoparticle production has been performed with Fluigent and Secoya Technologies Nanoparticle Production Station, a robust and complete system for precise and long-term production of nanoparticles with flexible particle size range.
PLGA Nanoparticle production setup
Reagents
Continuous phase: deionized water and 1 % Polyvinyl alcohol Mw 9000 – 10000 80% hydrolyzed (Sigma Aldrich).
Inner phase: technical acetone, and PLGA Resomer 756 1 % (Sigma Aldrich).
Inner phase to initiate and clean : technical acetone.
Figure 1: Scheme of the fluidic setup
Figure 2: Picture of the Fluigent equipment
PLGA nanoparticle synthesis
In the microfluidic solvent diffusion method, nanoparticles are synthesized in a microchannel after mixing between PLGA-acetone solution and water, following a three-dimensional hydrodynamic flow focusing (3D MHF) strategy.
In this approach, flow focusing squeezes the PLGA in acetone stream between water streams fully surrounding the PLGA phase and resulting in rapid solvent exchange via diffusion and PLGA nanoparticles precipitation [6]. Particle formation takes place spontaneously at the nucleation spots that are distributed through the mixture (Figure 3) [7].
The reagents and precipitating NPs are isolated from the channel walls, minimizing aggregation and/or clogging. In addition, by constraining the sample stream in the center of the microchannel- where flow velocity reaches the maximum with less variation- the 3D focused sample stream is expected to have a uniform width and thus improve the uniformity of the solvent/non-solvent ratio. This allows a robust and predictable nanoparticle synthesis, and facilitates the production of highly uniform nanoscale PLGA nanoparticles [8,9,10].
Results: monodisperse PLGA nanoparticles with tunable size
Fluigent has generated PLGA nanoparticles of different sizes by varying parameters related to our microfluidic system, thus establishing a relation between the diameter of the nanoparticle, the stream diameter, the flow rate ratio and the total flow rate.
How flow rate ratio controls PLGA particle size
In 3D hydrodynamic focusing, the main parameter that sets particle size is the flow rate ratio (FRR) between the aqueous continuous phase and the PLGA–acetone inner phase. Increasing the aqueous-to-organic FRR decreases particle size, from 250 nm to 110 nm across the range tested (Figure 4).
The results above relate nanoparticle diameter to the diameter of the focused stream, the FRR and the total flow rate. The FRR sets how tightly the PLGA–acetone stream is focused. That focusing drives the rapid solvent exchange that precipitates the polymer, and size tunability by 3D flow focusing has also been reported in single-layer microchannels [10]. Across the conditions tested here, particles ranged from 110 to 250 nm with a PDI of 0.05 to 0.1. Size is a core design parameter for engineered nanoparticles for nanomedicine, and microfluidic-assisted preparation of PLGA delivery systems has been reviewed in detail [11]. The continuous phase matters as well: residual polyvinyl alcohol associated with PLGA particles has been shown to affect their physical properties and cellular uptake [12].
A stable ratio is what the instruments in the setup hold. Flow EZ pressure controllers drive the two phases, and flow sensors measure the flow rate of each phase. Together they keep the FRR set at the start of a run constant throughout production.
Figure 6 Schematic effect of the aqueous-to-organic flow rate ratio (FRR) on the focused PLGA–acetone stream and on particle size. Not to scale; measured diameters are shown in Figure 4.
Conclusion
PLGA nanoparticles as biocompatible nanocarriers represent one of the most innovative, non-invasive approaches for drug delivery applications. However, their targeting functions are largely affected by size. In the case of tumor targeting and drug delivery, currently the commonly recognized size range for PLGA nanoparticles is 100-300 nm, as it allows for the correct targeting of nanoparticles to the desired tissue.
PLGA nanoparticle size may be controlled by tuning the synthesis method and parameters of operation.
In this application note, we have demonstrated the PLGA nanoparticle synthesis using a microfluidic system (3D microfluidic hydrodynamic flow) consisting of pressure-based flow controllers and the RayDrop™ microfluidic device with standard configuration.
PLGA nanoparticles ranging from 110 to 250 nm were generated. This size range is optimal for various biological applications, such as tumor targeting, as it falls within the compatible size range. The Polydispersity Index (PDI) ranges from 0.05 to 0.1. Sizes can be adjusted by controlling the device flow input parameters, particularly the flow rate ratio (FRR). In this way, the ability to synthesize PLGA nanoparticles in a more controllable and reproducible way creates possibilities for custom tuning surface properties.
A full-featured, cost-effective and readily available platform for the on-demand production of monodisperse PLGA nanoparticles is now available. This allows for control of nanoparticle size and frequency by adjusting flow parameters.
Download the Complete Protocol
What is the difference between PLGA nanoparticles and PLGA microspheres?
What is droplet microfluidics used for?
The difference is size and formation method. PLGA nanoparticles, 110 to 250 nm in this note, form by nanoprecipitation as acetone diffuses out of a focused PLGA stream. PLGA microspheres form by droplet templating: polymer-solution droplets solidify as solvent diffuses out, giving beads of 15 to 50 µm in Fluigent’s microparticle work [13].
| PLGA nanoparticles (this note) | PLGA microspheres [13] | |
|---|---|---|
| Size regime | 110–250 nm | 15–50 µm |
| Formation | Nanoprecipitation in a 3D focused stream | Droplet templating, then solvent diffusion |
| Dispersity reported | PDI 0.05–0.1 | CV 2 % |
Batch routes to PLGA nanoparticles include solvent diffusion [14] and single or double emulsion methods [15]. The microscale protocol is detailed in the PLGA microparticle application note.
Are PLGA nanoparticles FDA approved?
PLGA, the polymer, has US FDA and European Medicines Agency approval in drug delivery systems for parenteral administration [2]. That approval concerns those specific drug delivery systems; PLGA nanoparticles prepared with this protocol are research materials, not approved drug products.
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