**5.2 Surface morphology**

Electron microscopy can provide information on actual particle size, particle size distribution, surface morphology, and structure through its different techniques.

To study nanoparticles with Scanning electron microscopy (SEM), they must first be made into a dry powder, sprinkled on a sample holder, and coated with a layer of conductive metals such as gold, platinum, graphite, osmium, iridium, tungsten, osmium, chromium, or gold/palladium alloys, using a spray coating. Then, a highenergy electron beam is passed through the sample to generate various signals on the surface of the object samples [61]. The main limitation of SEM is the resolution of the images, which restricts its application to models of ~200 nm in size and does not provide any information on the internal structure of the particles [63].

Transmission electron microscopy (TEM) generally provides two-dimensional images of the internal structure of nanoparticles with a resolution of approximately 0.4 nm. The fraction of electrons transferred depends on the electron density of the sample. Therefore, components with differences in electron density appear as regions of different intensities in the image. In contrast, models are generally stained with phosphotungstic acid or uranyl acetate.

SLN formulations have low-melting lipids, and the electron gun used in electron microscopy can cause SLNs to melt, affecting their structure and integrity. These problems can be avoided using an improved technique called cryogenic field emission scanning electron microscopy (cryo-FESEM) [23].

It allows the examination of the nanomechanical properties of each molecule and particle under closer physiological conditions. Atomic Force Microscopy (AFM) is the most significant advantage over TEM/SEM in scanning nanocarriers without special sample preparation. It provides the opportunity for 3D visualization with qualitative and quantitative information on physical qualities such as the size and texture of the surface morphology and roughness. Additionally, a wide variety of particle sizes from 1 nm to 8 μm can be characterized in the same scan [61].

### **5.3 Determination of lipid polymorphism and crystallinity**

Differential Scanning Calorimetry (DSC) is an eminent method based on the measurement of structural modifications of materials in pharmaceutical research that are accompanied by heat exchanges such as heat absorption (involved in melting) or heat emission (involved in crystallization) [4, 61]. Depending on the device's sensitivity, the DSC can scan and account for even microscopic thermal activities in the material and recognize the temperature at which these problems occur. Still, it does not immediately reveal why trouble. Therefore, the exact nature of the thermal transition must be resolved by corresponding methods, such as thermogravimetry, microscopic observations, or X-ray diffraction (XRD) to distinguish the thermal shifts, polymorphic change, melting, loss of water from the hydrate or decomposition [20, 61].

X-ray diffraction (XRD) is a technique in which the scattering of X-rays by the atoms of a crystal creates an interference-effect. The resulting diffraction pattern helps identify and determine its various polymorphic forms' crystal structure and differentiation. According to Bragg's law, it detects fluctuations in electron density on a length scale. It is possible to determine the particle size, shape, crystalline structure of lipid nanoparticles, and changes in the crystalline order of the particles during production [63, 64].

Small Angle X-ray Scattering (SAXS) provides information on nanoparticles' size, shape, and size distribution and the internal structure of disordered and partially ordered systems. It is based on the analysis of the elastic scattering behavior of X-rays when traveling through the material, recording their scattering at small angles [26, 65].

In thermal gravimetric analysis (TGA), we can analyze the melting point, crystallinity, polymorphism, and endothermic and exothermic characteristics of the sample. In this technique, samples are heated at a controlled heating rate under different atmospheres such as nitrogen, oxygen, and argon [6].

Nuclear magnetic resonance (NMR) spectroscopy provides physical, chemical, electronic, and structural information about molecules that characterize their topology, dynamics, and three-dimensional structure in solution in the solid-state [66]. Proton nuclear magnetic resonance (1H NMR) is the most widely used and is particularly suitable for the structural characterization of liquid lipid domains in SLNs. 1H NMR can provide valuable information on the distribution of APIs in GS and the mobility of API molecules incorporated into lipid transporters [26, 67].

Infrared (IR) and Raman spectroscopy are used to explore the structural properties of lipids by relating the frequency of scattered radiation to functional groups of the molecules. Raman spectroscopy allows qualitative (measuring the frequency of scattered radiation) and quantitative (measuring the intensity of scattered radiation) analysis of SLNs [26]. Fourier transform IR spectroscopy (FTIR) is a type of IR spectroscopy commonly used to characterize nanoparticles. Modification of the surface of the particles can be controlled and confirmed using FTIR based on the functional groups present in the material [68].

#### **5.4 Load capacity and entrapment efficiency**

The loading capacity (DL) and entrapment efficiency (EE) are critical parameters related to the nanoparticles' dose regimen and efficiency. API loading capacity (DL) represents the relationship between the active ingredient in the particles and the total weight of the particles, while entrainment efficiency (EE), sometimes called encapsulation efficiency, is related to the number of active ingredients. Incorporated into the granules relative to the total amount of active ingredients present in the dispersion. The loading capacity (DL%) and the encapsulation efficiency (EE%) are determined using the following equations:

$$DL\left(\%\right) = \frac{amount\ of\ API\ determined\ experimentally\left(mg\right)}{total\ weight\ of\ SLN\left(mg\right)} \times 100\tag{1}$$

$$EE\left(\%\right) = \frac{amount\ of\ API\ determined\ experimentally\left(mg\right)}{amount\ of\ theoretical\ API\ in\ the\ formulation\left(mg\right)} \times 100\tag{2}$$

In general, the efficiency of trapping the active ingredients in lipid nanoparticles is usually greater than 70%. Unfortunately, drug-carrying capacity is not reported in most research papers, and reported values fluctuate between 0.1% and 80% [28].

The amount of drug encapsulated in the nanocarriers influences the release kinetics; therefore, it is essential to determine the encapsulation efficiency. EE is estimated after the removal of the free API. Free API is separated by ultracentrifugation, extensive dialysis, ultrafiltration, gel filtration, or centrifugal ultrafiltration. After removing free API, the amount of drug is estimated using a standard analytical technique such as UV spectroscopy or high-performance liquid chromatography (HPLC).

The main aspects that must be taken into account in the discussion on drug entrapment within SLNs are: (i) existence of supercooled melts; (ii) presence of various lipid modifications; (iii) form of lipid nano-dispersions; and (iv) gelation phenomena [66].

#### **5.5 Release of the active ingredient in vitro**

The drug release profile is performed analogously to encapsulation efficiency determination assays measured over time intervals to discover the release mechanism.

The release of trapped API from SLNs is governed by the following principles: (i) there is an inverse relationship between API release and drug partition coefficient; (ii) a smaller particle size promotes a greater surface area, which leads to a more significant release of API; (iii) the homogeneous dispersion of the drug in the lipid matrix causes the slow release of the drug; (iv) lipid crystallinity, and high API mobility lead to rapid drug release from SLNs.

In general, the drug release study is determined under controlled shaking and centrifugation. Essentially, drug release from nanomolecular systems occurs through five possible mechanisms for (a) dissociation of the API bound to the outer layer, (b) diffusion through the polymers matrix, (c) membrane-controlled diffusion, (d) erosion of the nanoparticle matrix, or (e) a combination of diffusion and erosion processes [69]. The kinetics of the nanocarrier release pattern could be evaluated using a bi-exponential equation:

$$\mathbf{C} = A\mathbf{e} - \alpha \mathbf{t} + \mathbf{B}\mathbf{e} - \beta \mathbf{t} \tag{3}$$

where C represents the drug levels within the nanocarriers at time t, A and B are constant; it depends on the properties of the matrix (A means for diffusion control and B for erosion control matrices) and, B is the rate constants that could be determined using a semi-logarithmic representation [61].

The release profile of API trapped in SLNs generally reveals a biphasic pattern with an initial burst effect followed by a prolonged release over several hours or days. Immediate removal of embedded drugs from solid lipid nanoparticles is based on diffusion from the surface of the external particles or matrix erosion induced by hydrolytic degradation. Thus, the distribution of the active substance is gradually released from the lipid core and promotes prolonged release and dissolution. The rate of freedom can be influenced by the nature and composition of the lipid substrate, the choice, and the concentration of surfactants, including technological parameters [70].

Drug release from nano-sized dosage forms can be evaluated using one of the following three categories, separate and sample (SS), continuous flow (CF), and dialysis membrane (DM).

#### **5.6 SLN storage stability**

The physical properties of SLNs during prolonged storage can be determined by monitoring changes in zeta potential, particle size, drug content, shape, and viscosity over time. External parameters such as temperature and light seem to be primary importance for long-term stability. Several factors can influence the physical strength of SLN, such as stress conditions (for example, high temperatures, exposure to light, and mechanical stress), the presence of liquid phase and electrolytes, contact with different surfaces, and high concentrations of particles. For example, storage temperatures at 4°C offer a more favorable environment; long-term storage at 20°C did not result in drug-laden SLN aggregation or drug release, while rapid particle size growth was observed at 50°C [29, 31, 71].

Spray drying and lyophilization are used in SLNs to increase or prolong stability, especially for preparations intended for intravenous administration. The aggregation of SLN can be decreased by adding cryoprotectants and obtaining a better redispersion of the dried product. Cryoprotectants favor the vitreous state of the frozen sample and reduce the osmotic activity of water and crystallization by avoiding contact between discrete lipid nanoparticles [72].

#### **5.7 In vitro evaluation of SLN**

A promising technique for in situ nanoparticle characterization is Fluorescence Correlation Spectroscopy (FCS). This technique is based on the measurement of fluctuations in the fluorescence intensity of the emissive species that diffuses through a low excitation focal volume. Autocorrelation analysis of the fluorescence intensity in the focal book provides information on the concentration, diffusion constant, and fluorescent particles' brightness. Furthermore, the fluorescence fluctuation spectroscopy analysis of fluorescence intensity fluctuations allows the quantitative analysis of

#### *Solid Lipid Nanoparticles (SLN) DOI: http://dx.doi.org/10.5772/intechopen.102536*

the brightness distribution, making it possible to characterize heterogeneous samples containing assembled molecules. FCS serves as a tool to measure the size and polydispersity of nanoparticles and evaluate their behavior in complex biological media and their stability. Also, in many reports, FCS has been used to characterize crown protein formation on the surface of nanoparticles or the interaction of human serum albumin with liposomes. However, only a few studies have reported the use of FCS to study charge release from nanoparticles [73].

In addition to the physical and chemical characterization of the nanocarriers, their biological responses are also measured in animal cell culture studies before the start of in vivo administration. This translocation of particles is determined mainly by flow cytometry (determines the amount of translocation) and confocal microscopy (determines the location).

The toxicity of nanoparticles is highly dependent on several factors, such as surface properties, coating, structure, size, and aggregation capacity, and these factors can be altered and manipulated in the manufacturing process. Nanoparticles with poor solubility have exhibited more pronounced toxicity [74]. Toxicological studies make it possible to evaluate its toxic effects, identify routes of exposure, and predict the risks of its synthesis or use.

Various in vitro cell culture techniques have been used to analyze nanotoxicity qualitatively and to study nanoparticle uptake (cell uptake assays), localization, and biodistribution. Cell culture assays for (i) cytotoxicity (altered metabolism, decreased growth, lytic or apoptotic cell death), (ii) genotoxicity, (iii) altered gene expression, and (iv) proliferation can be performed to rule out risks associated with nanotoxicity [35].

The cell viability study is the most widely used test for in vitro cytotoxicity evaluation. Other tests also evaluate cells' effect without necessarily leading to cell death. This includes oxidative stress: increased production of reactive oxygen species (ROS), lipid peroxidation, and alteration in the oxidized/reduced glutathione pool or DNA damage [26].

#### **5.8 In vivo evaluation of nanocarriers**

After nanocarriers reveal preliminary affectivity in vitro, these carriers further evaluate their toxicity and response profile in biological species. Some in vivo evaluations that can be carried out are: (i) the therapeutic dose-response study; (ii) the biodistribution of nanocarriers among the various organs of the body; (iii) acute and multidose efficacy studies and (iv) studies on safety parameters and pharmacokinetics (ADME processes). The ultimate goal of in vitro and in vivo evaluation is to match the physicochemical aspects of nanocarriers with their biological function [61].
