Centrifugal and Pressure Ultrafiltration in the Nanotechnology Field: Nanoparticles and Other Nanostructures

Nanoparticle Purification and Production

Nano-sized particles (NPs) have evolved into important systems for bioimaging, biosensing, targeted drug delivery, and controlled release of therapeutic agents. Efficient collection and preparation of nanoparticles is critical in nanomaterial manufacturing. Ultrafiltration (UF) has been widely used in the preparation and purification of nanoparticles after synthesis (Figure 1) 1. Historically, filtration was performed using pressurized ultrafiltration devices (pUF). The introduction of centrifugal ultrafiltration (cUF) techniques enabled the ability to work with smaller volumes in nanoparticle preparation and purification.

Figure 1. Applications of ultrafiltration in nanoparticle preparation and purification.

Nanoparticles can be fabricated with organic or inorganic materials, polymers, and metals. Such surface functionalization imparts specific biofunctions and enhances biocompatibility for biological application 2. Magnetic nanoparticles, quantum dots, metal nanoparticles, silica nanoparticles, and polymeric nanoparticles have biomedical importance 3. Many nanoparticles cannot be used directly after fabrication. Aqueous matrix formulations may contain unbound drug that requires removal. Biomolecules, chemicals, and other contaminants from lysates, body fluids, serum, buffers, media, and other reagents may need to be separated further. Some pose harmful effects to their surrounding biological environment 3. Reliable methods to concentrate, enrich, wash, and purify nanoparticles are essential.

Ultrafiltration Techniques in Nanoparticle Separation, Purification, and Concentration

Centrifugal ultrafiltration devices can be used to purify, wash, and concentrate nanoparticles based on size. Amicon® Ultra and Centricon® Plus centrifugal ultrafiltration units, as well as the Amicon® Pro purification system which enables simultaneous washing and concentration, can be used for:

  • Nanoparticle separation and purification
  • Nanoparticle concentration
  • Nanoparticle enrichment
  • Desalting
  • Buffer exchange
  • Cleanup post-functionalization
  • Depletion of abundant proteins

For pressure-based ultrafiltration (pUF) of larger sample volumes, Amicon® stirred cells can be used in combination with an appropriate Ultracel® filtration disc membrane. Amicon® stirred cells provide a gentle method of ultrafiltration.

After primary filtration, nanoparticles can be further centrifuged for washing and buffer exchange, or re-concentrated. Retained or passing solutes may be collected and analyzed to determine nanoparticle purity, encapsulation efficiency, binding, and drug concentration.

Membrane Selection for Nanoparticle Applications

Separation and concentration of molecules during ultrafiltration is based on size exclusion. The great majority of biomolecules have a molecular weight less than 500,000 Da, and nanoparticles fit nicely into this category. Amicon® Ultra centrifugal ultrafiltration units, Centricon® Plus centrifugal ultrafiltration units for larger volumes, and Amicon® Pro purification systems for simultaneous washing and concentration have specified nominal molecular weight limits (NMWL) defined by their filter membrane, indicating a particle size cut-off where >90% of particulate is retained. These filter systems are offered with membrane NMWLs of 3,000, 10,000, 30,000, 50,000 and 100,000 Da. Selection of an appropriate membrane pore size for a specific nanoparticle size and application can be confusing, as evidenced by a survey of published articles (Figure 2).

Figure 2. Comparison of published membrane sizes by nanoparticle type. Membrane pore size should be selected based on both nanoparticle size and application (e.g., purification from complex sample, concentration, buffer exchange, desalting).

Factors to consider when selecting an ultrafiltration method and membrane pore size:

  1. Nanoparticle size: Size can be estimated from published sources, or by measurement techniques such as microscopy, laser diffraction, and dynamic light scattering.
  2. Size of key separation targets in solution: The size of proteins, antibodies, drugs, chemicals, and other particles that need to be separated will affect membrane size selection.
  3. Sample volume: Processing volumes ranging from ≤0.5 mL to 70 mL are compatible with centrifugal ultrafiltration (cUF) devices. Larger volumes may benefit from using pressurized ultrafiltration (pUF) devices.

To retain nanoparticles, the molecular weight cut-off of the filter membrane needs to be smaller than the nanoparticle (~2 times smaller than the molecular weight of the nanoparticle), but large enough to allow smaller components to filter through.

 

cUF or UF Membrane NMWL (Da)
Nominal Pore Size (nm)
Recommended UF Nominal Retaining Size Range
Diameter (nm) Molecular Weight (Da)
Minimum Maximum Minimum Maximum
3,000 0.3 1 1.5 6,000 20,000
5,000 0.5 2 3 10,000 30,000
10,000 1 3 9 20,000 90,000
30,000 3 9 15 60,000 180,000
50,000 5 15 30 100,000 300,000
100,000 10 30 90 200,000 900,000

Table 1. Selection of ultrafiltration membrane NMWL based on nanoparticle size.

Conclusions

Centrifugal ultrafiltration (cUF) and pressure ultrafiltration (pUF) play an important role in nanoparticle purification and preparation. Applications for ultrafiltration of nanoparticles include separation, concentration, buffer exchange, drug monitoring, and removal of dyes, enzymes, and unbound components from nanoparticle preparations. Many publications have cited the utility of Amicon® and Centricon® centrifugal ultrafiltration devices, as well as Amicon® stirred cells for pressure ultrafiltration, in nanoparticle and macromolecular purification and concentration. Because impurities may be ionic, molecular, or particulate, the optimal filter choice can make a significant difference in yields, reproducible results, and filtrate quality. Amicon® and Centricon® filter units contain an Ultracel® regenerated cellulose filter membrane, whose complex structure removes impurities from a sample that may affect critical biological and chemical determinations, downstream analysis, and assay performance. Ultrafiltration techniques perform the separation, cleanup, and enrichment steps for lab-scale nanoparticle preparations. Both centrifugal ultrafiltration (cUF) and pressure ultrafiltration (pUF) processes provide quick, simple, and efficient ways to separate nano-material from smaller constituents and fluid passing into the filtrate. Physical composition, size, and shape are important attributes that need to be considered in filter selection. The Amicon® portfolio offers an array of ultrafiltration membrane devices for nanoparticle purification and production.

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References

  1. R.H. Fang, S. A. Quick synthesis of lipid–polymer hybrid nanoparticles with low polydispersity using a single-step sonication method. Langmuir. 2010;26:16958-16962.
  2. L.H. Reddy, J. A. Magnetic nanoparticles: design and characterization, toxicity and biocompatibility pharmaceutical and biomedical applications. Exp Biol Med. 2009;234:1128-1139.
  3. Jacob Weingart, P. V.-L. Membrane mimetic surface functionalization of nanoparticles: Methods and applications. Advances in Colloid and Interface Science. 2013, Sep;197–198:68-84.