**5. SLN characterization**

The typical composition of the SLN identifies the following characteristics of great importance: (i) particle size and particle size distribution; (ii) surface morphology, functionalization, and zeta potential; (iii) structure, depending on the degree of crystallinity, lipid variability, drug binding and carrying capacity; (iv) drug release; (v) dynamic processes and the general existence of other nano and microstructures; and (vi) toxicity assessment according to the manufacturing process, lipids, and excipients used, their effect on drug toxicity and route of administration. In **Table 4**, some parameters and techniques used to characterize SLNs will be described later.

### **5.1 Particle size and surface charge**

The particle size range and concentrations reported for SLNs are wide. However, in vitro studies have shown that SLNs are acceptable at concentrations <1 mg/mL (total lipids). They can be less tolerated with a particle diameter > 500 nm, explained by their aggregation [20]. A decrease in the size of the nanoparticles provides easier absorption and leads to a significant increase in the rate of cellular absorption. For example, a particle size greater than 300 nm provides a sustained delivery of drugs; in this case, when the size range is 50–300 nm, rapid action is shown [24].

Particle size is a critical attribute of lipid nanocarriers, affecting stability, encapsulation efficiency, drug release profile, biodistribution, mucoadhesion, and cell uptake.

Regarding the particle size distribution characterization, the "polydispersity index" (PDI) defines the size range of lipid nanocarrier systems. The term "polydispersity" (or "dispersion" as recommended by IUPAC) is used to describe the degree of nonuniformity of a particle size distribution. Also known as the heterogeneity index, PDI is calculated from a two-parameter fit to the correlation data (cumulative analysis). This index is dimensionless and is scaled so that values less than 0.05 are mainly seen with highly monodisperse standards, while values greater than 0 [5].

The choices of a specific technique depend on several parameters, such as the expected size and distribution of the nanoparticles. Since the diameter of the particles affects the release of encapsulated agents, the smaller particles provide a larger surface area. Commonly used particle size analysis techniques include laser diffraction (LD), photo relation spectroscopy (PCS) or dynamic light scattering (DLS), colter counter, scanning ion occlusion sensing (SIOS), flow field fractionation (FFF), and nanoparticle tracking analysis (NTA).

On the other hand, the electrokinetic behavior defined by the zeta potential provides information on the magnitude of the charge on the surface of the particles in aqueous dispersion. It allows predicting the long-term physical stability of the formulations. For electrostatically stable nano-dispersion, a zeta potential value greater than 30 mV is the absolute value required. Still, if the stabilization combines steel and electrostatics, a minimum of 20 mV is enough.

The commonly reported zeta potential values for lipid particles to range from −20 to −40 mV. It should be noted that the zeta potential is determined by the nature of


#### **Table 4.**

*Main parameters for the characterization of solid lipid nanoparticles.*

the surface of the particles and is a function of the medium in which the nanoparticles reside and the concentration of the sample used to make the measurement. Therefore, the nature of the solvent, the pH, the ionic strength, and the nature and concentration of the electrolytes in the solution directly affect the magnitude of the zeta potential and, in many cases, the sign of the zeta potential [28].

The surface electrical potential of nanoparticles is essential, especially in formulation science, as it regulates interactions with neighboring particles (including adjacent nanoparticles) and biological systems. It is suggested that the determination of the potential of nanoparticles is carried out in suitable simulated physical solutions/environments where they interact with natural systems [61]. The zeta potential of nanoparticles influences the in vivo fate of SLNs; in general, it is seen that SLNs with positive zeta potential have a long-circulating half-life [23, 24]. In general, the zeta potential value of lipid nanoparticles is estimated from the determination of electrophoretic/electroacoustic mobility, and the LD and DLS techniques allow these determinations.

LD is a valuable technique covering a more comprehensive detection range (0.05–3500 μm) [22]. The results generated by the LD are used to estimate the respective spherical radii of the particles according to the Mie scattering solution (also known as "Mie theory") [26, 61].

The DLS is used to analyze the hydrodynamic diameter of nanoparticles with a size range of 20–600 nm [62]. However, many instruments have an operating range of 0.3 nm–10 μm [61]. The hydrodynamic or stokes diameter is the diameter of a sphere with the same translational diffusion coefficient as the particle being measured, assuming a hydration layer surrounding the particle or molecule [61]. DLS measurements also provide a polydispersity index (PDI).

SIOS is an advancement in the lattice counting method, is a recent tool to measure particle size. This methodology uses dynamically resizable nanopores to detect, quantify, and characterize individual particles in real-time. As the extent of the jam is proportional to the particle size, the exact size of the particles (40 nm–10 μm) can be evaluated after calibration with a known standard [23, 26].

FFF techniques can include simultaneous separation and measurement. These methods are based on the mobile phase's laminar flow action, ensuring separation and perpendicular force fields. FFF techniques are classified according to the type of force field applied, such as cross-flow (Fl), sedimentary (Sd), thermal (Th), electrical (El), magnetic (Mg), and dielectric (Dl). Flow field-flow fractionation (F4) is the most versatile FFF technique. This technique allows the separation of dispersed analytes over a wide range, from nano to micrometer analytes. Three variants of F4 differ in the design of the separation channel: (i) symmetric F4 (SF4), (ii) asymmetric F4 (AF4), and (iii) hollow fiber F4 (HF5). AF4 is the most used among the FFF techniques [26].

NTA is a practical, high-resolution method for measuring nanocarrier samples' size, size distribution, and concentration within a size range of 30–1000 nm. This method allows the scope of monodisperse and polydisperse pieces to be measured. Furthermore, it can calculate the surface charge of lipid-based carriers and detect their fluorescence signals [5].
