Application note: PLGA Microspheres
Poly(lactic-co-glycolic) acid copolymers (PLGA) are widely used as drug delivery systems due to excellent bioproperties including, biodegradation andbiocompatibility. Its use is approved by the US Food and Drug Administration as means for drug delivery in certain therapies. A key advantage to targeted and slow released drugs is the control over dose and direct delivery within the body. Both of these aspects are supported by the uniform and controlled size of the microparticles as well as the matrix structure of the particle. This is even more important for PLGA microparticles being used as “zero-order”, pulsatile or tandem release drug carriers. The use of microfluidic technology to create PLGA microparticles enables the requirements of uniform and accurately controlled size to be met. In addition to this, the proprietary technology to Tide Microfluidics is able to work at high production rates (>10kHz) while allowing the full control over particle sizes. This means Tide's technology as found in the MicroSphere Creator is able to produce PLGA particles at high production rates and sizes down into the nanometer range without any loss of product quality. This advantage of the MicroSphere Creator is due to microparticle production that is easy to control with pressure driven flows that allow precise process settings for accurate and reproductible production
Materials and methods
The MicroSphere Creator works using pressure regulators and an optical measurement & inspection system and is compatible with a variety of microfluidic chips for droplet and particle production, including flow focusing droplet generators. Microfluidic chips, especially, flow focusing geometries, allow greater precision and more controlled compared to traditional batch production methods as each particle is created individually and in controlled manner. The further advantages of the MicroSphere Creator for drug carrier production include the fully sterilisable components, an easy to assemble plug & play nature, as well as easy to use and control software and full CE certification.
How it works
The microfluidic chip allows two input fluids, one for the inner fluid, or dispersed phase, which eventually breaks-up into the microparticles and one for the outer phase, or continuous phase, which supports the microparticles. Using the pressure control of the MicroSphere Creator (maximum 6 bar) the solutions are “pushed” through the chip and focused into a microparticles. This is possible for a wide variety of geometries of microfluidic chips as can be seen below. However, the flow focusing design offers advantages in usability compared to the others for PLGA microparticle formulation. This application note covers only flow focusing produced PLGA microparticles.
The most common organic solvent for PLGA is Dichloromethane (DCM), this is a toxic solvent with limited solubility in water. Glass tubes and storage materials are needed as plastic dissolves readily in DCM, meaning processing materials need to be chosen carefully. Also, DCM requires elevated temperatures for a set time to ensure evaporation this is commonly done using a 37°C heated bath for 30 minutes. An alternative solvent for PLGA is Dimethyl carbonate (DMC), which is considerably less toxic, can be kept in plastic tubes and evaporates readily in water. The production as describe here was carried out using DMC to take advantage of these benefits.
To create PLGA microparticles using flow focusing techniques a 2% PVA in water solution is used as the continuous phase and the Dispersed phase of a 2% PLGA in DMC solution is used.
- A 2% PVA solution in ultrapure water is prepared and stored in a 15mL tube for connection to the MicroSphere Creator.
- A 2% PLGA in DMC is prepared and stored in a 15mL tube for connection to the MicroSphere Creator.
- The MicroSphere Creator is prepared with flow focusing microfludic chip inserted
- The 15mL ultrapure water tube is connected to the continuous phase inlet port of the MicroSphere Creator.
- The 15mL DMC tube is connected to the disperse phase inlet port of the MicroSphere Creator.
- Nitrogen gas is connected to drive both fluid supplies.
- The MicroSphere Creator software application is used to control the supply of water and DMC to the microfluidic chip. Once a stable stream of DMC droplets are formed the continuous phase is replaced by the PVA solution and the dispersed phase is replaced for the PLGA solution. The pressures used for the stable stream will be re-established for the PLGA and PVA solutions.
- Once the microfluidic process is running the microdroplets are collected at the outlet
- The system is cleaned using ultrapure water for the continuous phase and pure DMC for the dispersed phase.
|Dichloromethane (DCM)||Dimethyl carbonate (DMC)|
|Vapor pressure (kPa at 20°C)||47||47|
|Boiling point (°C)||39.6||39.6|
|Solubiligy in water (g/L at 20°C)||13||13|
|Molar mass (G/mol)||84.93||84.93|
|Viscosity (mPa's)||0.499 (15°C)||0.499 (15°C)|
|Toxicity (LD50, mg/kg)||1,600||1,600|
1: Characteristics of DCM and DMC.
For particle production three different settings were used, the results of these settings will be displayed on the next pages:
During the production of PLGA particles the visual inspection system of the MicroSphere Creator can be used to validate if droplets are created . In figure 3, different images taken during production are displayed. By changing the input pressures and varying the ratio between the input pressures different sized particles can be produced at different production frequencies. In figure 4 multiple examples of this are displayed.
- Setting #1:
Continuous phase pressure of 1000mbar,
Dispersed phase pressure of 650mbar.
- Setting #2:
Continuous phase pressure of 2000mbar,
Dispersed phase pressure of 1000mbar.
- Setting #3:
Continuous phase pressure of 800mbar,
Dispersed phase pressure of 400mbar.
Setting #1 (1000mbar/650mbar)
Setting #2 (2000mbar/1000mbar)
Setting #3 (800mbar/400mbar)
24 hour production test using setting #3
The MicroSphere Creator operated (unmanned) in production mode for 24 hours without problems. The results can be seen in figure 8.
Shrinking of the droplets
The shrinking of PLGA droplets after the evaporation of DMC has been observed. At the time of production (figure 8) the PLGA droplets are 15.35 μm in diameter. After production the DMC dissolves in water within 30 minutes, resulting in PLGA particles with a size of 3.94 μm. To estimate the size of the PLGA particles after evaporation of the DMC a predictive model was developed within Tide Microfluidics. Using this model the final size of the particles can be predicted accurately, as shown in figures 9 and 10. Using the model a final size prediction of 3.96 μm was made, only a 0.02 μm difference from the final result.
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