**2. Composition of SLN**

SLNs are composed of lipids, surfactants, co-surfactants, and the API to be encapsulated. In addition, coating materials, antioxidants, preservatives, adhesives, viscosity enhancers, absorption enhancing agents, and other excipients can also be used.

SLNs are composed of approximately 0.1–30 (% w/w) solid fat dispersed in an aqueous phase. Surfactants are used in approximately 0aboutentrations to improve stability [18, 19].

#### **2.1 Lipids**

Lipids are the main component of the formulation and determine the stability, release, encapsulation, and loading of any API. Therefore, the selection of lipids is the key to a successful formulation, biocompatible/physiological and biodegradable lipids, as well as those that are in the classification of generally recognized as safe (GRAS), with a melting point more significant than 40°C to guarantee a solid state at room temperature and also at body temperature, are the ones of choice for the formulation of SLNs, which reduces the danger of toxicity [20].

GRAS, for its acronym in English "Generally Recognized as Safe," is a designation of the Food and Drug Administration of the United States (FDA) that a chemical or substance added to food is considered safe by experts, therefore which is exempt from the Federal Food, Drug, and Cosmetic Act (FFDCA) food additive tolerance requirements [21].

The melting point of lipids depends on their chemical structure, degree of crystallinity, and polymorphic variation. For example, the melting temperatures of saturated fatty acids and triglycerides increase in proportion to the number of C atoms. In contrast, the melting temperatures of unsaturated fatty acids decrease with increasing carbon atoms and double bonds. The polymorphic variation of lipids also determines physical characteristics such as melting and solidifying properties, morphology, and aggregation of large fat crystals and emulsions.

Selection of a solid lipid or lipid blend relevant to SLN generally depends on several factors: (i) the ability to produce particles in the submicron range, (ii) biodegradability, (iii) biocompatibility, (iv) carrying the capacity adequate drug, and (v) storage stability [22].

One of the most important considerations is the polarity of the drug to be used. Ideally, it is recommended that the lipid phase be considerably lipophilic so that lipophilic drugs can easily be incorporated and solubilized in it. Another parameter, which plays a crucial role, is the lipid's viscosity and contact angle (or a mixture of lipids) with the aqueous solution. Highly viscous lipids are challenging to work with and require higher energy for sonication, which is used to form nanoparticles [23].

The use of relatively low melting point solid lipids and the increase in the oil content in the lipophilic phase of the nanoparticle dispersion reduce the viscosity of the molten droplets during homogenization; this is how nanoparticles are formed in smaller sizes [24]. The average particle size of the SLN dispersion increases when higher melting lipids are used. The main technical point behind this phenomenon is a higher viscosity of the dispersed phase [25].

The degree of crystallinity and polymorphic modification of lipids is another factor that influences the properties of a lipid nanoparticle system because they affect the incorporation of the drug (drug loading efficiency, state, and location of the drug in the nanoparticles) and dictate its releasing properties [26, 27].

#### *2.1.1 Polymorphism and crystallinity of lipids*

Polymorphism can exist in more than one crystalline form due to the different lattice arrangements of the molecules. For example, the low-fusion lipids used in the preparation of SLN can exist in various polymorphic forms. The differences between the polymorphic forms are due to the packing of the hydrocarbon chain, the inclination of the chains against the plane of the methyl end group, and the differences in the region of the methyl end group. The predominant forms of triglycerides are polymorphs α and polymorphs β. Polymorphs α tend to convert to β′ and finally to polymorphs β, with a narrow chain arrangement and packaging. Polymorph β is considered the most stable, whereas polymorph β′ is metastable.

The lipids that make up the inner layer of colloidal lipid systems exhibit complex crystalline behavior. The crystallization of these materials occurs during the cooling process and can continue in the storage stage. The crystalline behavior of the solid lipid in the particles is determined by the composition and the droplet size of the molten lipids. Therefore, depending on the design and the preparation process, the lipids of the internal structure of the particles can have various conformations, such as liquid crystals, gels, or crystalline lamellar phases [28].

To avoid drug shedding due to polymorphic transitions, the use of complex lipids, such as long fatty acid chains, is the ideal choice to improve long-term stability and increase the number of drugs that can be encapsulated. These chains increase the average size of the particle, so the lipid nanoparticle formulation consists of a combination of long and short-chain fatty acids.

Among the most used lipids are triglycerides such as tristearin, tripalmitin, trilaurin; greases such as the Witepsol® series; acylglycerols such as glyceryl behenate; waxes such as cetyl palmitate; fatty acids with different hydrocarbon chain lengths such as stearic or palmitic acid. Cationic lipids, such as stearylamine, can improve drug penetration since ionic interactions are created between positively charged parts of the molecule and negatively charged cells, promoting better cellular internalization [27–29]. This increases the residence time on the surface and improves the drug's bioavailability [27]. **Table 1** shows some of the lipids used in the preparation of SLNs.

#### **2.2 Surfactants**

In general, in the preparation of lipid nanoparticles, surfactants play two critical roles: the dispersion of the lipid melt in the aqueous phase and the stabilization of the lipid nanoparticles in dispersions after cooling [30]. The main aspects to consider when using surfactants in the preparation of SLN are their safety, compatibility with other excipients, ability to produce the desired size with the minimum amount consumed, and also provide sufficient stability to the SLNs, covering their surfaces [25]. The surfactants used to produce these carriers can improve epithelial permeability (e.g., disrupt the cell membrane) and, therefore, overcome limitations in drug absorption [26].

On the other hand, the surfactants that surround the particles, in addition to guaranteeing their steric stability in aqueous dispersion, induce specific surface chemical properties and can also modulate the biopharmaceutical profile [30]. For selecting the best surfactant, several parameters must be considered: hydrophilic-lipophilic balance (HLB) values, its effect on lipid polymorphism, and particle size. HLB values for stabilizing oil dispersions in water vary between 8 and 18. The correct choice of surfactant minimizes the risk of producing particle aggregates that can compromise


#### **Table 1.**

*Lipids used in the preparation of SLN.*

the stability of the distribution in vitro and its performance in vivo [20]. **Table 2** shows some of the surfactants used in the preparation of SLNs.

Surfactants can be classified into three classes according to their electrical charge: ionic, non-ionic, and amphoteric. Ionic surfactants confer electrostatic stability, while non-ionic surfactants confer steric repulsion stability; amphoteric surfactants have negative and positively charged functional groups, so they exhibit characteristics of a cationic and anionic surfactant under low and high pH conditions, respectively [27]. The toxicity of a surfactant is an important consideration, and not all surfactants can be used to prepare all types of SLNs. The surfactants arranged in decreasing order of toxicity are: cationic, anionic, non-ionic, amphoteric.

#### **2.3 Other agents**

In addition to lipids and surfactants, lipid nanoparticle formulations may also contain other ingredients, including cryoprotectants used in SLN drying techniques such as lyophilization, spray drying, and charge modifiers and the surface. Adapting the surface of lipid nanoparticles with surface modifiers such as hydrophilic polymers can reduce their absorption by the reticuloendothelial system (RES) [27]. The rapid absorption of SLNs can be prevented by coating with a biocompatible polymer such as poly (ethylene) glycol (PEG), which can increase blood circulation time [31]. In **Table 3**, some of the charge and surface modifiers used in the preparation of lipid nanoparticles are listed.
