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Microfluidic PLGA Nanoparticles Synthesis

by NanoGenerator™ Microfluidic Mixing System

Nanoparticles are at the leading edge of the rapidly developing field of nanotechnology. Their unique size-dependent properties make these materials superior and indispensable in many areas. It has been used in many industries, such as pharmaceuticals, energy, and electronics. Nanoparticle synthesis is one of the key steps to enable nanoparticle applications. Since most applications utilize nanoparticle size properties, the size distribution, yield, and size reproducibility from batch to batch become very important target parameters for the evaluation of different nanoparticle synthesis methods. Traditional batch mode synthesis method (mixing in a bulk solution) has poor quality when the method is implemented in scaled-up production. The size distribution and reproducibility of nanoparticles are usually poor due to some uncontrollable factors such as aggregation, and heterogeneous mixing. 

The microfluidic technology-based miniaturized reactors enable the rapid mixing of reagents, the control of temperature, and the precise spatial-temporal manipulation of reactions. The controlled and homogeneous mixing in microfluidic synthesis methods results in smaller and uniform nanoparticles. The physicochemical properties of nanoparticles can be precisely controlled in a reproducible manner. The control of the reaction environment leads to improve quality of nanoparticle size distribution, better size reproducibility, and eventually improves the preparation process yield of nanoparticles.
 
NanoGenerator™ Flex provides a wide throughput range of 0.2-1 mL for Flex-S and 1-12 mL for Flex-M, which meets the needs from discovery and screening early pre-clinical study, and small-scale production while providing a compact and user-friendly platform. NanoGenerator™ Pro provides a higher throughput range of 2-200 mL, which is suitable for scaling up production and pre-clinical studies.

 Benefits:​

  • ​​​Compact and portable design

  • Tunable and controllable particle size

  • Fast response time

  • Low PDI

  • Low payload consumption (<0.2 mL output for Flex-S)

  • Wide application from early screening to pre-clinical studies

  • OEM service and module integration available

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NanoGenerator Flex System: Flex S (Left) and M (Right)

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NanoGenerator PRO System

System Setup:

Pro and Flex-M: In PLGA nanoparticle (PNP) synthesis, the PVA solution (“aqueous” phase) and PLGA in acetonitrile (“solvent” phase) are loaded in sample reservoirs. During experiments, pressure from the controller is applied to the reservoir kits. Solutions in the reservoir kits are pushed into the PTFE tubing connected to 3-way-valves. The pressure is automatically adjusted based on the readings from the flow sensors and the preset flow rates are reached and stabilized quickly. Before the stabilization of the flow rates, both solutions go to the waste bottle to avoid synthesized PNPs with non-uniform sizes. Once the flow rates have reached the preset values, the 3-way-valves switch to the ports connected to the microfluidic chip. Two phases are mixed in the microfluidic chip to form PNPs. The mixed solution is collected from the outlet reservoir connected to the microfluidic chip. Users can optimize the mixing ratio, target flow rates, and synthesis results by changing the flow rate settings using the pressure controller. The microfluidic herringbone mixing chip CHP-MIX-3 is used for PNP synthesis with these two instruments 

 

Flex-S: Reagent solutions are loaded in reservoir tanks. Preset pressures are directly applied to the reservoir tanks. The solutions are pushed into channels in the mixing chips. The pressures are set in such a way that the total flow rate and flow ratio match the user’s setting requirements. The microfluidic passive mixing chip CHP-MIX-4 is used for PNP synthesis with the Flex-S

Note that CHP-MIX-4 and 3 have different mixing efficiencies and flow rate ranges, CHP-MIX-3 compatible with higher flow rates.

NanoGenerator PRO System

NanoGenerator Flex-M System

NanoGenerator Flex-S System

System Performance:

PreciGenome Nanoparticle Synthesis System is used in a variety of applications and demonstrated synthesis of PLGA nanoparticle, which is widely used in pharmaceutical companies as drug delivery vehicles [1].

 

Flex-S module is for small volume synthesis (0.2–2 mL) for early screening purpose. Flex-M module is for large volume synthesis (1–12 mL) for animal studies. Pro is for bulk volume synthesis (2-200 mL) for scaling up production.

For this experiment, PVA aqueous solution (2% w/v) and PLGA acetonitrile solution (2% w/v) were used as “aqueous” and “solvent” phases correspondingly.

Due to the viscous nature of PVA solution, the Flex-M and S modules are compatible with separate chips with different mixing efficiencies and flow rate ranges. CHP-MIX-3 is suitable for the Flex-M and Pro, while CHP-MIX-4 is for the Flex-S.

PNP size tuning is controlled by the formulation parameters, the total flow rate and the flow rate ratio. 

Size Tuning by Total Flow Rate (TFR):

Adjusting the total flow rate (TFR) is the simplest method for size tuning of PNPs. In general, higher TFR results in faster mixing, which generates smaller nanoparticles. No significant size decrease was observed above 10 mL/min, while size changed more dramatically as TFR became very low. PDI remained within the range of 0.07 to 0.15 regardless of TFR. Flow rate ratio was fixed as 1:1

Size Tuning by Flow Rate Ratio (FRR):

Under the same TFR of 10 mL/min, PNP size was slightly affected by adjusting the flow rate ratio (FRR) of water to acetonitrile. From the most common FRR of 1:1, increases in FRR lead to slightly larger particles. No significant size increase was reported at higher FRR, while PDI remained within 0.10-0.15 except at very high FRR. 

Size Tuning by PLGA Content (% PLGA):

Formulation parameters such as PLGA concentration can greatly alter PNP size. Under the same TFR of 10 mL/min and FRR of 1:1 with PVA concentration fixed at 2% w/v, increases in PLGA content lead to much larger particles.  No significant difference was observed between 0.5% and 1% w/v PLGA, while PDI greatly increased from less than 0.10 to above 0.20 at high PLGA content. 

Size Tuning by PVA Content (% PVA):

PVA concentration is another formulation parameter that can alter PNP size. Under the same TFR of 10 mL/min and FRR of 1:1 with PLGA content fixed at 2% w/v, increases in PVA content lead to slightly smaller particles. No significant difference was observed between 2 and 4% w/v PVA, while PDI remained in a similar range regardless of PVA content. 

Batch-to-Batch Consistency (Flex-M):

Batch-to-batch variation is a major problem of conventional bulk preparation methods. Empowered by advanced microfluidics technology, the PreciGenome NanoGenerator system can generate highly consistent and reproducible results and reduce batch-to-batch variation. With formulation and synthesis parameters fixed, four individual samples from the Flex-M generated very similar results for PNP size and PDI, with approximately 5% variation in size and PDI around 0.15

Small Volume Synthesis Consistency (Flex-S)

Small-scale production may be desired for very early screening or to minimize wasted product. For this, the NanoGenerator Flex-S with MIX-4 chip can produce product volumes as low as 0.2 mL before inline dilution. With the formulation and synthesis parameters fixed for a 200 μL target volume with TFR 4 mL/min and FRR 1:1, batch-to-batch variation was low, with the average size of synthesized PNPs at 113.3 nm. This is similar to 8 mL/min on the Flex-M with MIX-3 chip. No significant difference was observed with a higher TFR, and PDI was similar to the Flex-M at around 0.15

Bulk Volume Consistency

Large volume synthesis presents some challenges in ensuring product consistency over long periods of operation. For applications which require even higher throughput than the range offered by the Flex system, Precigenome offers the NanoGenerator Pro that can achieve a max throughput of 200 mL. With the formulation and synthesis parameters fixed for a 50 mL target volume, each 5 mL fraction collected showed highly consistent PNP size and PDI. Across all fractions, the average size of synthesized PNPs was 102.8 nm and average PDI was 0.15 

References​

  1. Chiesa, E, et.al., The Microfluidic Technique and the Manufacturing of Polysacharide Nanoparticles, Pharmaceutics, 2018, 10:267-289

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