Poly (D,L-lactide-co-glycolic acid) – PLGA Nanoparticles

At nanoComposix, we offer customized encapsulation services and made-to-order PLGA nanoparticle fabrication for applications in drug or gene delivery, theranostics, and tailored to the specific needs of the customer. For common injectable applications, the desirable size range for in vivo delivery vehicles is 100‒300 nm. Our team can encapsulate your desired compound (drug, protein, peptide, nucleic acid) or modify the surface of PLGA particles to facilitate attachment of ligands, antigens, and antibodies in order to meet custom requirements for targeted applications. This article provides an overview of PLGA Nanoparticle Applications, Synthesis, and Manufacture at nanoComposix.

Applications of PLGA Nanoparticles

Poly (D,L-lactide-co-glycolic acid) (PLGA) is a biodegradable polymer commonly used to make particles for in vivo studies and is generally regarded as safe. PLGA is approved by both the US Food and Drug Administration (FDA) and European Medicine Agency (EMA) for use in vaccines, drug delivery, and tissue engineering.^1,2 PLGA particles can be loaded with either hydrophobic drugs or hydrophilic peptides/proteins, encapsulating the active compound and protecting it from proteolytic degradation. Moreover, the surface of the nanoparticles can be chemically modified for specific tropism or molecule release, or to evade the reticuloendothelial system (RES). Successful development of PLGA nanoparticles as a delivery system requires precise loading and encapsulation of active ingredients, followed by their controlled release.

PLGA is used in various research areas, including controlled release of encapsulated drugs, tissue engineering, healing of bone defects, and in vaccine development. Several PLGA-based products for controlled release of encapsulated proteins or peptides are already on the market. Since the approval of prostate cancer treatments Zoladex and Lupron Depot in 1989, hybrid poly (D,L-lactic acid) (PLA)/PLGA-based delivery systems have enabled development of extended-release formulations that can reduce dosing frequency and minimize drug side effects. To date, the FDA has approved 16 different PLA/PLGA-based products, 12 of them formulated as microspheres. Many of these products have become leading medicines in their respective categories due to enhanced safety, efficacy, and dosing profiles.

Methods to Formulate PLGA Nanoparticles

Various procedures are employed for the preparation of PLGA nanoparticles, from the classic emulsification-solvent evaporation technique to nanoprecipitation, or even the use of microfluidic devices. Here we discuss some of the common methods to fabricate these materials. Regardless of the synthetic approach, active ingredients can be encapsulated inside the core of the nanoparticle, trapped among polymer chains, or otherwise adsorbed on the surface of the nanoparticle.

Emulsification-evaporation (oil-water or water-oil-water emulsion)

Emulsification-evaporation is the most common method for the preparation of PLGA nanoparticles. This technique allows encapsulation of both hydrophobic and hydrophilic drugs on the micro- or nanoscale. Briefly, PLGA is dissolved into an organic phase (oil) that is emulsified with a surfactant or stabilizer in an oil immiscible phase (usually water). Hydrophobic drugs are added directly to the oil phase, whereas hydrophilic drugs may be first emulsified with the polymer solution prior to particle formation. High intensity sonication bursts can be employed to facilitate formation of small polymer droplets. The resulting emulsion is added to a larger aqueous phase and stirred for several hours, allowing the solvent to evaporate. The polymer precipitates as the solvent is removed, and hardened nanoparticles are collected and washed by centrifugation prior to lyophilization and long-term storage. PLGA possesses many advantages for drug delivery, but reproducible formation of nanoparticles can be challenging; considerable variability in particle size and encapsulation efficiency are introduced by using different equipment, precursor reagent batches, and the precise method of emulsification. Moreover, the evaporation step requires use of heat and vacuum, which can introduce additional variability in the resultant particles. The figure below provides an overview of the double emulsion procedure (water/oil/water, w1/o/w2) to obtain PLGA micro-/nanoparticles.

By adjusting the conditions of synthesis (stabilizers, solvents, and mixing procedure) it is possible to obtain micro-/nanospheres with a uniform matrix or micro-/nanocapsules with core-shell structures. Immunoparticles used for directed delivery can be obtained by attaching specific antibody molecules to the particle surfaces.

 

Nanoprecipitation

Also called interfacial deposition, nanoprecipitation is another method employed to prepare PLGA nanoparticles. In this approach, the polymer and the drug are dissolved in an organic solvent (such as acetone or DMSO) and then added dropwise to water. The organic solvent is evaporated, and the particles are collected as a pellet using centrifugation. This method is a straightforward, single-step process with high reproducibility and was initially applied for hydrophobic drugs.

Below are TEM images of PLGA nanoparticles prepared at nanoComposix via a single-step nanoprecipitation self-assembly method.

DLS measurements: Z-Ave = 233.3 nm, Std. Dev. = 3.413, PDI = 0.062
TEM results: Ave. Diam. = 193.3 nm; Std. Dev. = 63.7; CV = 32.9%

Nanoprecipitation as a synthetic method has its pros and cons. Unfortunately, particle size distributions are highly sensitive to the effectiveness of mixing during synthesis. This method can also yield particles that suffer from reportedly low drug loading, especially when hydrophilic components such protein and peptides are encapsulated.³ At nanoComposix, encapsulation efficiency can be drastically improved through adjustments such as varying pH,⁴ incorporating salt additives or oil solutions,⁵ and even employing a modified nanoprecipitation method.⁶ Moreover, studies demonstrate more efficient encapsulation using nanoprecipitation than emulsion-based methods.⁷

The figure below shows TEM images of PLGA nanoparticles loaded with human serum albumin (HSA) prepared via a modified nanoprecipitation method at nanoComposix.

DLS measurements: Z-Ave = 172.3 nm, Std. Dev. = 3.413, PDI = 0.076
TEM results: Ave. Diam. = 112 nm; Std. Dev. = 31.1; CV = 27.8 %

Flash nanoprecipitation

Flash nanoprecipitation is a method recently developed to generate polymeric nanoparticles using rapid micro-mixing. Developed as an effort to enhance the throughput of the nanoprecipitation synthetic approach, flash nanoprecipitation is performed using specially designed flow geometries, such as a confined impinging jet mixer or a multi-inlet vortex mixer. The images and diagrams below (adapted from Ref. 8) illustrate the design of a vortex mixer. The mixer (pictured, left) employs four syringes to supply the fluid to be mixed. The vortex mixer consists of four ports in a radial configuration and can process up to 100 mL of material per minute.

In the vortex mixer, the streams of fluid containing the different components come into contact under very turbulent high shear conditions and the change in solubility afforded by the solvent mixture induces rapid coprecipitation of the nanoparticles and cargo. Flash nanoprecipitation is an accessible lab-scale screening tool and a scalable approach to nanoparticle production for translational research. Flash nanoprecipitation can be employed for polymeric nanoparticles comprised of either a hydrophobic or a hydrophilic core. Under normal operating conditions, more than 95% of the core material is typically encapsulated at high mass fraction in the particle. Processing larger batch sizes simply requires running the process for longer durations of time, avoiding the mixing problems associated with traditional nanoprecipitation methods and enabling ready scale-up of lead formulations using syringe pumps or flow controllers.

Microfluidics-assisted synthesis

This technique can produce uniform, reproducible nanoparticles, but is normally limited to production on the milligram scale. Further scale-up typically entails automatic parallelization at high capital cost. In the microfluidics-assisted approach, an intimate mixture of PLGA and the cargo are suspended in a non-solvent and forced at very high pressure through a small pore above the glass transition temperature of the PLGA. The high shear forces generated in such a configuration reduce the size of the PLGA particles. Subsequent addition of a coating such as polyvinyl alcohol (PVA) traps the particle at a particular size and prevents particle agglomeration.

The image above from Microfluidics International Corporation depicts high-pressure pneumatic lab equipment for high shear fluid processing.

Characterizing & Manufacturing PLGA at nanoComposix

Analytical Capabilities

• Particle morphology can be characterized using scanning electron microscopy (SEM) and/or transmission electron microscopy (TEM). For optimal visualization by TEM, PLGA particles are negatively stained with Nano-W from Nanoprobes or ammonium molybdate. Size distribution, polydispersity index, and charge (zeta potential) are measured using a Zetasizer Nano-ZS from Malvern Analytical. Particle concentrations are quantified with nanoparticle tracking analysis (NTA).
• Chemical structure is confirmed by Fourier-transform infrared (FTIR) spectroscopy.
• Sterility is characterized using sensitive endotoxin detection methods with a limit of detection of 0.001 EU/mL in diluted samples.
• Encapsulation efficiency & drug loading is determined spectrophotometrically using UV-Vis or fluorescence, or by employing specific assays.
• Reaction yields are determined by mass balance measurements.
• Residual PVA associated with the PLGA nanoparticles is quantified spectrophotometrically, using an iodine assay developed at nanoComposix.
• Release kinetics are characterized in phosphate-buffered saline (PBS) or any other solvent or buffer that the customer requires.

A complete list of our analytical instruments can be viewed at the following link: Facilities & Equipment.

Process Optimization to Facilitate Scale-Up

nanoComposix has developed modifications to the methods described to improve their scalability, as many of the techniques are not amenable to large-scale operation (namely vortexing, evaporation, centrifugation, and sonication).

1. To satisfy the need for homogenization, the mixing process can be switched to an impinging flow mixer. For example, single or double emulsion can be replaced with nanoprecipitation using an impinging flow mixer.
2. At large scale, simple evaporation of solvent can be impractical and inefficient. nanoComposix is equipped with multiple rotary evaporators to facilitate rapid solvent removal for both small- and large-scale synthesis.
3. Centrifugation for particle purification is difficult to scale, especially when it involves spinning at high speeds using an ultracentrifugation. nanoComposix has developed proprietary methods for high-throughput nanoparticle processing to wash and remove remaining solvent and unreacted precursors.
4. Uneven energy exposure can introduce challenges for bath sonication at scale. Alternatively, the sonication step can be replaced with high shear mixing, Pasteur probe, or micro-fluidization to generate the high shear force and facilitate final dispersion. If necessary, a larger scale flow-through sonicator can be employed.
5. Sterile filtration is performed on the final nanoparticle product. Other sterilization methods utilize E-beam or gamma radiation. Finally, endotoxin levels are tested as routine characterization procedure.
6. nanoComposix lyophilizes the colloidal suspensions to facilitate particle stability and storage. The freeze drying/lyophilization process is performed in the presence of sugars as a cryoprotectant to avoid particle aggregation.

PLGA Nanoparticle Production under GMP

nanoComposix offers services to produce PLGA particles at scale to supply material for pre-clinical and Phase I/Phase II clinical trials with GMP and ISO13485 compliance. This includes the following capabilities:

• Scale-up of synthesis to produce lot sizes required for clinical trials or other customer needs.
• Sterility will be achieved by formulating the particles under aseptic conditions. Otherwise, electronic beam or gamma radiation can be applied subsequent to synthesis, as long as the process will not ionize the active ingredients in the active pharmaceutical ingredient (API). Particles are tested before and after exposure to radiation to ensure that performance is not compromised by the sterilization procedure.
• Particles optimized for performance and manufacturability to a design freeze, including initiation of design control.
• Quality system documentation associated with synthesis and characterization of the particles will be prepared. This includes particle synthesis with GMP controls and internal auditing for quality control.

We estimate a timeline of 6–18 months for the production of particles under GMP control. See more about our production capabilities on our Nanomedicine Manufacturing Services page and contact us if you have questions.

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References Cited

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2. Allahyari, M., Mohit, E. Peptide/protein vaccine delivery system based on PLGA particles. Hum. Vaccines Immunother. 2016, 12 (3), 806-828. doi.org/10.1080/21645515.2015.1102804
3. Rezvantalab S.; Drude, N. I.; Moraveji, M. K.; Güvener, N.; Koons, E. K.; Shi, Y.; Lammers, T.; Kiessling, F. PLGA-Based Nanoparticles in Cancer Treatment. Front Pharmacol.2018, 9, 1260. doi.org/10.3389/fphar.2018.01260.
4. Govender, T.; Stolnik, S.; Garnett, M. C.; Illum, L.; Davis, S. S. PLGA nanoparticles prepared by nanoprecipitation: drug loading and release studies of a water soluble drug. J. Control Release 1999, 57 (2), 171–185. doi.org/10.1016/S0168-3659(98)00116-3.
5. Dalpiaz, A.; Vighi, E.; Pavan, B.; Leo, E. Fabrication via a nonaqueous nanoprecipitation method, characterization and in vitro biological behavior of N⁶‐cyclopentyladenosine‐loaded nanoparticles. J. Pharm. Sci. 2009, 35, 1375-1383. doi.org/10.1002/jps.21710
6. Niu, X.; Zou, W.; Liu, C.; Zhang, N.; Fu, C. Modified nanoprecipitation method to fabricate DNA-loaded PLGA nanoparticles. Drug Dev. Ind. Pharm. 2009, 35 (11), 1375-1383. doi.org/10.3109/03639040902939221.
7. Alshamsan, A. Nanoprecipitation is more efficient than emulsion solvent evaporation method to encapsulate cucurbitacin I in PLGA nanoparticles. Saudi Pharm. J. 2009, 201422, 219–222. doi.org/10.1016/j.jsps.2013.12.002.
8. Markwalter, C. E.; Pagels, R. F.; Wilson, B. K.; Ristroph, K. D.; Prud’homme, R. K. Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles. Jove-J Vis Exp 2019, 143, e58757. doi.org/10.3791/58757.