Size and Size Distribution

Size is one of the critical parameters that dictate the absorption, biodistribution, and route of elimination for biomedical nanomaterials.20 Generally, nanoparticles with dimensions of less than 5-10 nm are rapidly cleared after systemic administration, while particles from 10 to 70 nm in diameter may penetrate capillary walls throughout the body.21,22 Larger particles 70-200 nm often remain in circulation for extended times.22,23 This general correlation of biodistribution and elimination with respect to size may vary greatly depending on nanoparticle surface characteristics.

Specifically in cancer applications, size is an important factor in the accumulation of therapeutic nanomaterials in tumors, usually as a result of enhanced permeation and retention (EPR), caused by local defects in the vasculature and poor lymphatic drainage.24 Particle size can be precisely tuned to take advantage of this phenomenon and passively target and deliver a therapeutic payload to tumors.25-27

Depending on the category of the nanomaterial, synthesis and scale-up can be problematic. Most biomedical nanomaterials for therapeutic and diagnostic applications are complex and involve some combination of molecular self-assembly, encapsulation, and/or the use of nano-sized metal or polymer cores, surfactants and/or proteins to impart solubility and functionality. Due to inherent variability in the manufacturing process, one rarely achieves a monodisperse, homogeneous product. It is therefore important to ascertain the precise size, size distribution, and polydispersity index (PDI) of the material. There are several techniques available to assess these parameters, including electron microscopy, AFM, and light scattering. Light scattering techniques can measure overall size and polydispersity of the particles. TEM is powerful in ascertaining the homogeneity of nanoparticles with encapsulated metals and in determining core size. With knowledge of nanoparticle geometry and size, surface area can also be estimated.

For biological applications, it is important to measure the physical characteristics of the nano-material in isotonic solution at physiological pH and temperature. The hydrodynamic size can be measured under these conditions using dynamic light scattering (DLS) (also known as photon correlation spectroscopy [PCS] and quasi elastic light scattering [QELS]) and analytical ultracen-trifugation (AU). In a DLS experiment, the effects of Brownian motion (particle movement caused by random collisions in solution) provide information on particle size and size distribution. The sample is illuminated with a laser, and the intensity fluctuations in the scattered light are analyzed and related to the size of the suspended particles. This technique is useful in determining whether the nanomaterial is monodisperse in size distribution. These data are influenced by the viscosity and the temperature of the medium, since Brownian motion depends on these factors. The pH of the medium and salt concentration may also affect the degree of agglomeration in some samples. With DLS, sample preparation is easy, the measurement is quick, and data are reproducible on larger sample volumes compared to microscopy techniques; however, better standardization of procedures, conditions, and data analysis tools will be required. Static light scattering provides information on molar mass and root-mean-squared (rms) radius for fractionated or monodisperse samples. One limitation of light scattering instruments is the inability to measure the size when the nanoparticles absorbs in the wavelength of the laser being used. Small-angle x-ray scattering (SAXS)28 and small-angle neutron scattering (SANS)29 can be used to measure the size, shape and orientation of components. Due to their cost and infrastructure requirements, there is limited availability of these instruments. For fluorescent nanomaterials such as quantum dots, size can be measured using fluorescence correlation spectroscopy (FCS).30

The hydrodynamic size of nanoparticles can also be measured with AU, which is traditionally used to measure the size of proteins.31 The instrument spins the protein sample solution under high vacuum at a controlled speed and temperature while recording concentration distribution at set times. Even though this technique is designed to measure the size of proteins in solution, it has potential applications in the measurement of the hydrodynamic size of nanoparticles samples that are stable under the experimental conditions. Fractionation using SEC separates stable polymers into individual components and helps in the determination of the PDI. In the case of unfractionated samples, batch mode measurement provides averaged quantities such as weight-averaged molar mass and z-average rms radius. This technique is especially useful when combined with a refractive index detector to obtain absolute molecular weight for very high molecular weight polymers where traditional MS methods fail.

In cases where the separation and fractionation of nanomaterial is not possible using a column with a stationary phase, such as when the nanomaterial may interact with the column packing material and render it unstable, asymmetric-flow field flow fractionation (AFFF) is useful.32 In AFFF, separation occurs when the sample passes through a narrow channel with a cross-flow through a porous semi-permeable membrane. The faster moving smaller particles rise to the top of the flow and come out first followed by larger particles that stay closer to the membrane and migrate more slowly. One advantage in this method is that there is no stationary phase in the separation: the sample injected comes out intact with little loss of material due to nonspecific binding. This feature is particularly useful for less stable nanoparticles such as liposomes, or for polymer- or protein-coated metal nanoparticles that would otherwise interfere with the performance of a traditional GPC column. The efficiency of separation for AFFF is not as good as with GPC, but there have been recent improvements in instrumentation that are closing the gap in performance. For both GPC and AFFF, the quantity and hydrodynamic size of the nanoparticles are detected in eluted peaks by measuring absorbance, refractive index, and light scattering.

In addition to size, the shape of a nanoparticle may affect its distribution and absorption in the body. Spherical, tubular, plate-like, or nano-porous materials of the same composition can vary significantly in their surface energy, biological activity, and access to different physiological structures, such as cell walls, capillary vessels, etc. Methods such as AFM, SEM, TEM, and STM can be used to determine the distribution of shape in a nanoparticle preparation.

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