Characterization Techniques for Nanoparticles Enabled Medicinal Products

The number of innovative nanoparticles enabled medicinal products (NEMPs) being developed has been continually increasing.^[66] Multiple regulatory bodies^[67-71] implicitly demand product assessment in their quality, safety and efficacy in order to support their decision-making process, allowing effective translation toward a clinical application and subsequent commercialization.^[67-71]

With their small sizes, nanoparticles (NPs) present unique opportunities and challenges in both manufacturing and analysis:^[72]

If compared with ‘classical’ small molecule drugs, the assessment for NEMPs demands the investigation of many additional physico-chemical (PC) properties, including particle size distribution and polydispersity, dispersion stability, drug loading and drug release, particle shape, surface charge, surface coating, among many others.

To complicate matters, each NEMP is unique, imposing different methodological approaches to characterize its PC properties and to determine the critical quality attributes, which could impact its immunological effects, biodistribution, pharmacokinetics, metabolism and safety profile.

For example, measurements of particle size and zeta potential are routinely performed on the same instrument by dynamic light scattering (DLS) and laser Doppler electrophoresis (LDE), but require multiple experiments, and are challenged by polydisperse samples, for example, exosomes isolated from body fluids.

In this article, we will focus on three such characterization techniques for:

• Particle Size Distribution
• Surface Charge
• Surface Coating

Figure 1.  Antibody conjugated PLGA Nanoparticle

Properties of Nanoparticles

The reason why nanoparticles (NPs) are attractive for medical purposes is based on their important and unique features:

• Nanosize
• The small particle diameter allows the whole material to reach homogeneous equilibrium with respect to diffusion in a very short time.
• Small drugs may diffuse through the capillary walls into the tissues. Otherwise nanoparticles transport occurs through compromised endothelial barrier or mediated by specific transport systems.
• The small size not only makes them needle-injectable, but also enhance penetration through tissue barriers.
• Large area/volume ratio
• The high surface area of a material in nanoparticle form allows heat, molecules, and ions to diffuse into or out of the particles at very large rates.
• Offers a large surface area for bioconjugation and effortless natural clearance
• Ability to absorb and carry other compounds
• Drugs can be conjugated to nanoparticles surfaces via ionic or covalent bonding and physical absorption and NPs can deliver them and control their release through biological stimuli or other activations (see Figure 1).
• More subject to the brownian motion
• They usually do not sediment, like colloidal particles that conversely are usually understood to range from 1 to 1000 nm.
• Can easily pass through common filters (e.g., common ceramic candles)
• Separation from liquids requires special nanofiltration techniques.
• Cannot be seen with optical microscopes
• Being much smaller than the wavelengths of visible light (400-700 nm), nanoparticles require the use of electron microscopes or microscopes with laser.

With consistent efforts, researchers have strived to engineer these NPs by modifying their physical and chemical properties, referring to them as ‘smart’ or ‘intelligent’ NPs since they respond to external stimuli like temperature, pH, light, magnetic and electric fields, ionic strength, or enzymatic environment.

Particle Size Distribution

The particle size distribution (PSD) and the stability of NEMPs in complex biological environments are key attributes to assess their quality, safety and efficacy.

Nanoparticle's chemical, electronic, optical, magnetic and catalytic, properties, and self-assembly, inherently depend on their size and composition.^[73-79]

There are different methods to measure the nanoparticle size:

1. Dynamic light scattering
• Despite its low resolution, DLS is the most common sizing technique
2. Electron microscopy
3. Field flow fractionation coupled to online sizing detectors
4. Centrifugal techniques
• Ultracentrifugation, one of the most commonly used concentration methods, is time consuming and suffers from contamination.^[80,81]
• Size-based filtration through membranes can result in deformations or even break the particles.^[80,82]
5. Particle tracking analysis
6. Nanoparticle tracking analysis
7. Tunable Resistive Pulse Sensing

In a perspective article,^[65] it proposes a multi-step approach of incremental complexity to measure particle size distribution and size stability of NEMPs, consisting of:

1. A quick preliminary step to assess sample integrity and stability by low resolution techniques (pre-screening)
2. Followed by the combination of complementary high resolution sizing measurements performed both in simple buffers and in complex biological media

Surface Charge

The surface charge of nanoparticles is important as it determines its

• Stability
• Propensity to aggregate in the bloodstream
• Interacting with the cell membranes
• It has been revealed that cellular internalization is faster for nanogels of positive zeta potential or with high aspect ratios (for example, those with a rod-like shape).^[28]

Zeta potential is a measure of the surface charge of a nanoparticle. The magnitude of the zeta potential provides information about particle stability:

In a sense, the zeta potential represents an index for particle stability. For charged particles, as the zeta potential increases the repulsive interactions becomes larger, leading to stable particles, likely to have a more uniform size distribution.

The zeta potential of particles is typically measured by Electrophoretic Light Scattering (ELS).^[89] In contrast to streaming potential measurements no movement of the liquid is generated, but the movement of the particles is induced. Therefore, an electric field is applied and the electrophoretic mobility of particles is used to calculate the zeta potential. Due to the electric field particles will move at different speeds: highly charged particles will move faster than less charged particles. 

Figure 2. Core-shell nanoparticles: perspectives towards drug delivery applications (source: [88]).
In the review article,^[88] it explores state-of-the-art developments and advances in core–shell nanoparticle systems, the desired structure–property relationships, newly generated properties, the effects of parameter control, surface modification, and functionalization, and, last but not least, their promising applications in the fields of drug delivery, biomedical applications, and tissue engineering.

Surface Coating

Nanosystems have shown encouraging outcomes and substantial progress in the areas of drug delivery and biomedical applications. In this regard, core–shell type nanoparticles are promising nanocarrier systems for controlled and targeted drug delivery applications.

These functional nanoparticles are emerging as a particular class of nanosystems because of their unique advantages, including high surface area, and easy surface modification and functionalization. Such unique advantages can facilitate the use of core–shell nanoparticles for the selective mingling of two or more different functional properties in a single nanosystem to achieve the desired physicochemical properties that are essential for effective targeted drug delivery.

Several types of core–shell nanoparticles, such as metallic, magnetic, silica-based, upconversion, and carbon-based core–shell nanoparticles, have been designed and developed for drug delivery applications (see Figure 2).

Nanoparticles often develop or receive coatings of other substances, distinct from both the particle's material and of the surrounding medium. Even when only a single molecule thick, these coatings can radically change the particles' properties, such as and chemical reactivity, catalytic activity, and stability in suspension.

Many properties of nanoparticles, notably stability, solubility, and chemical or biological activity, can be radically altered by coating them with various substances — a process called functionalization.

For biological applications,

• Coatings that mimic those of red blood cells can help nanoparticles evade the immune system.^[86,90]
• Coating should be polar to give high aqueous solubility and prevent nanoparticle aggregation.
• In serum or on the cell surface, highly charged coatings promote non-specific binding, whereas polyethylene glycol linked to terminal hydroxyl or methoxy groups repel non-specific interactions.^[83,84]
• Coatings can be used to prevent agglomeration
• Which can keep the particles in colloidal suspension including various polymers like polyethylene glycol (PEG), poly(vinylpyrrolidone) (PVP) etc, natural polymers like dextran, chitosan, pullulan etc, and surfactants like sodium oleate, dodecylamine etc.^[85]

By consideration of surface energy, it is shown that these particles are expected to possess distinctly differing coating structures, with the polystyrene coating being incomplete. A comprehensive characterization of these systems can be done using a selection of complementary techniques including:

• Scanning electron microscopy (SEM)
• Scanning transmission electron microscopy (STEM)
• Thermal Gravimetric Analysis (TGA)
• Dynamic light scattering (DLS)
• Differential centrifugal sedimentation
• X-ray photoelectron spectroscopy

By combining the results provided by these techniques, it is possible to achieve superior characterization and understanding of the particle structure than could be obtained by considering results separately.^[87]

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